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8793779 | Morphology of the lumbar intertransverse process fusion mass in the rabbit model: a comparison between two bone graft materials--rhBMP-2 and autograft. | We puted tomography and the rabbit model to seek an explanation for the biomechanical superiority of binant human bone morphogenetic protein-2 (rhBMP-2) pared with autogenous iliac bone in lumbar intertransverse process fusions, i.e., the rhBMP-2 fusions demonstrated significantly higher strength and stiffness. The results show that the fusion masses using rhBMP-2 have higher volume, and higher minimum contact area of fusion mass attachment to the transverse process. For both bone graft materials there was a correlation between site of failure and the fusion mass quadrant that contained the minimum contact area of attachment to the transverse process. The ideal graft material would be one that produced strong, mature, early fusions that failed along their length at random positions, rather than failing with high frequency at the transverse process. The fusions using rhBMP-2 were nearer to the ideal than those using autograft: These were stronger, more mature, and only 6 of 9 rhBMP-2 failed in the quadrant that contained the minimum contact area of attachment to the transverse process, as opposed to 11 of 12 for autograft. |
8793780 | Do diurnal changes in loading affect the interpretation of MRI scans of the lumbar spine? | We tested the hypothesis that the visual interpretation of magnetic resonance imaging (MRI) scans of the asymptomatic lumbar spine are affected by diurnal changes in fluid exchange in the intervertebral discs. Ten male subjects carried a 20-kg backpack in the intervening 3-h period between two MRI scans of the lumbar spine. After the subjects arrived at the MRI center (within 45 min of awakening), they lay on a bed for 45 min. The first set of MRI scans were obtained. Each subject was then fitted with a 20-kg backpack. After they walked for 3 h, the pack was removed, and a second set of MRI scans were obtained immediately. The sets of MRI scans were reviewed by three radiologists: none of the three radiologists found differences in the interpretation of the MRI scans. On the basis of these results, diurnal changes in fluid exchange in the asymptomatic intervertebral disc (exaggerated in our case by the backpack) are undetectable by visual reading of MRI scans. |
8793781 | Pathomechanism of spontaneous regression of the herniated lumbar disc: histologic and immunohistochemical study. | To ascertain the pathomechanism of spontaneous regression of lumbar disc herniation, histological and immunohistochemical studies on 100 herniated discs were performed. Inflammatory findings such as cell infiltration, neovascularization, and granulation were observed in 16.9% of the protruded discs, 81.8% of the subligamentously extruded discs, 100% of the transligamentously extruded disks, and 80% of the sequestrated discs. The infiltrated cells posed mainly of macrophages and a small number of T lymphocytes. Furthermore, the cell infiltration was more prominent in the nucleus pulposus (NP) than in the annulus fibrosus (AF). There was no correlation between the occurrence rate of inflammatory response and the length of the period from onset to operation. Based on these findings, it is considered that an extruded or sequestrated disc has a potential to be resorbed by phagocytes. |
8793783 | Comparison of initial injury features in cervical spine trauma of C3-C7: predictive outcome with halo-vest management. | The purpose of this retrospective study was to examine specific patient variables and fracture morphologies to further elucidate the predictors of successful halo-vest treatment for cervical spine fractures (C3-C7). Eighty-seven cases of acute cervical spine injuries treated with halo-vest management were reviewed to assess initial injury features and radiographic es by measuring (a) subluxation and direction, (b) angulation, (c) facet abnormalities, and (d) vertebral body fracture patterns on plain radiographs puted tomography scans. The cases were divided into three groups: facet subluxations with fractures, facet subluxation without fractures, and fractures with no subluxation. Patients with facet subluxation and pression-flexion fractures (stages 4 or 5) were a distinct group when treated conservatively with a halo. Despite anatomic reduction, facet subluxations associated with pression-flexion fractures (stages 4-5), might be best treated surgically. Risk factors for late halo failure should include subluxations with pression-flexion fractures when treated conservatively. |
8793782 | Urodynamic and electrophysiologic study of the urinary disturbances caused by cervical myelopathy. | Urinary disturbance is one of the significant symptoms of cervical myelopathy. To make the diagnosis of urinary disturbance, preoperative urodynamic studies and evoked spinal cord potentials (ESCPs) recording were performed on 60 surgical patients with cervical myelopathy. Half (30) of this plained of urinary disturbance, and 22 (37%) were diagnosed as having neurogenic bladder. The presence of neurogenic bladder was closely correlated with severe limb symptoms and relatively slow ESCP velocity. It was confirmed that neurogenic bladder was caused by severe spinal cord damage. However, for 47% of the patients with plaints, findings of urodynamic examinations were negative; these patients probably had pathologic or psychosomatic factors other than neurogenic bladder due to cervical myelopathy. The prognosis of the neurogenic bladder appears to be influenced by irreversibility of the spinal cord lesions. |
8793784 | Validity and reliability of spinal cord monitoring in neuromuscular spinal deformity surgery. | Somatosensory evoked potentials (SSEPs) have e a standard of care in surgery for spinal deformity. Recent reports in the literature have suggested SSEP monitoring is not efficacious in surgeries for patients with neuromuscular disease. Electrophysiologic data were retrospectively analyzed from 74 patients with various neuromuscular disorders undergoing spinal-deformity surgery from 1989 through February 1995 at this medical center. The monitoring protocol included SSEPs recorded from multiple sites located cortically, subcortically, and peripherally. Neurogenic motor evoked potentials (NMEPs) were also employed. Anesthetic regimens were controlled patibility with evoked potential monitoring. Use of this intraoperative monitoring protocol resulted in reliable data for 95% of the patient population having baseline responses. Findings suggest that evoked potentials can be used effectively during surgery for neuromuscular spinal deformity. Use of a specific protocol allowed acquisition of reliable data intraoperatively, suggesting these methods are a valid means of monitoring neurologic status. |
8793785 | Efficacy of spinal cord monitoring in scoliosis surgery in patients with cerebral palsy. | Although spinal cord monitoring is mended during scoliosis surgery, a review from Rancho Los Amigos Medical Center stated that they were only able to obtain reproducible tracings in 53% of cerebral palsy patients. To ascertain that monitoring is both feasible and reliable in these patients, we reviewed the records of 34 consecutive patients with cerebral palsy who had scoliosis surgery at our institution. Spinal cord function was monitored by recording peripheral nerve, cervical/brainstem, and cortical somatosensory evoked potentials to posterior tibial nerve stimulation. Reproducible tracings were achieved in 31 of the 34 patients. Significant intraoperative changes were recorded in 12 of the 31 monitored patients, usually related to and requiring some modifications of the instrumentation. We conclude that with careful technique, spinal cord monitoring using cervical/brainstem somatosensory evoked potentials can be reliably achieved in most patients with cerebral palsy undergoing scoliosis surgery. |
8793786 | Effect of patient position on the sagittal-plane profile of the thoracolumbar spine. | Although the normal sagittal profile of the thoracolumbar spine has been described, this has been obtained primarily by using young individuals standing. We sought to describe the sagittal profile of the thoracolumbar spine in an older population in the supine cross-table lateral pared with that standing. We enrolled 50 volunteers with no history of back pain or spine deformity and 50 matched subjects with mechanical back pain (LBP) only. Lateral radiographs of the thoracolumbar spine (T10-S1) in both standing and cross-table supine positions were obtained. Lordosis from L1 to S1, kyphosis from T10 to L1, and the changes seen moving from the supine position to standing were calculated. There were few paring the two groups in either the standing or cross-table supine position, or when changing positions. Within each group, however, there were small, but significant, differences in the midlumbar and thoracolumbar spine paring supine versus standing. Both asymptomatic individuals and those with a history of LBP demonstrated similar small but statistically significant increases in lumbar lordosis and thoracolumbar kyphosis when standing versus supine. The clinical significance of these findings remains to be determined. |
8793787 | Prevalence of scoliosis in beta-thalassemia. | The objective of this study was to determine the prevalence and possible pathogenesis of scoliosis in beta-thalassemia in our country, and pare its characteristics to those of patients with idiopathic scoliosis from the same geographic area. Twenty-four [13 male and 11 female thalassemic patients aged 16 +/- 7 years (range 7-32 years)] of 115 examined patients with beta-thalassemia showed scoliosis of 14 degrees +/- 11 (range 10-65 degrees) radiologically. The prevalence of scoliosis in the thalassemic population was 21% in this series, whereas the overall prevalence of scoliosis in the general Greek population was 6% (Smyrnis PN, Valavanis J, Alexopoulos A, Siderakis G, Giannestras NJ: School screening for scoliosis in Athens, J Bone Joint Surg 61B:215-217, 1979). The scoliosis prevalence in the general population was significantly higher in the females (5%) than in the males (1%), whereas no difference in prevalence was found between the two sexes in the thalassemic population. The mon curve pattern in thalassemia was the left lumbar (38%) followed by the right lumbar (21%), whereas in patients with idiopathic scoliosis the left thoracolumbar monly appeared (25%) followed by the left lumbar (14%). No patient with thalassemia showed radiographic signs of congenital spinal deformities and spinal fractures, whereas all patients showed a significant retardation of their skeletal maturation. The age of the thalassemic patients with scoliosis was significantly (p = 0.0003) higher than in patients without scoliosis. The hematocrit of the thalassemic patients with scoliosis was significantly (p = 0.0012) lower than in those without scoliosis, whereas the rate of transfusions was not correlated with the magnitude of the scoliosis. The level of ferritin was significantly (p = 0.025) higher in the thalassemic patients with scoliosis than in those without scoliosis. The duration of Desferal treatment was significantly (p = 0.0357) longer in thalassemic patients with scoliosis pared with those without scoliosis. Thus, the prevalence, curve pattern, and etiology of scoliosis in beta-thalassemia differ from those of idiopathic scoliosis, indicating that the spinal deformities in thalassemia represent a distinct type of scoliosis. |
8793790 | Amlodipine increases cyclosporine levels in hypertensive renal transplant patients: results of a prospective study. | Calcium channel blockers (CCB) are considered the agents of choice to treat hypertension in cyclosporine (CsA)-treated renal transplant patients. Verapamil, diltiazem, and nicardipine, but not nifedipine or isradipine, can significantly increase CsA levels. The effect of a new CCB, amlodipine, has not been established. However, some hospitals are routinely switching patients to amlodipine from other CCB for reasons of cost. A case of a man with stable CsA levels who developed significantly increased CsA levels after being changed to amlodipine is presented along with a prospective trial to formally examine this issue. Eleven hypertensive, CsA-treated renal transplant patients were placed on amlodipine for an average of 6.9 wk and later withdrawn. Three measurements of CsA trough level, blood pressure, serum creatinine concentration, and BUN were obtained at baseline, during treatment with amlodipine, and after withdrawal of amlodipine. CsA levels on amlodipine increased an average of 40% above baseline (P = 0.003) and decreased to baseline (P = 0.001) after amlodipine was withdrawn, despite no significant change in CsA dose. Additionally, there was no change in serum creatinine, BUN, or mean arterial pressure values. Amlodipine can increase CsA levels |
8793791 | Urinary content of aquaporin 1 and 2 in nephrogenic diabetes insipidus. | Hereditary nephrogenic diabetes insipidus (NDI) is caused by mutations in either the X-chromosomal gene encoding the vasopressin V2-receptor or in the autosomal gene encoding aquaporin-2. Expressed in Xenopus oocytes, the AQP2 gene mutations found in NDl have been shown to reduce the stability of the encoded protein. This study investigated the in vivo stability of mutant and wild-type aquaporin-2 proteins by measuring their excretion in urine of NDl patients and healthy individuals. On immunoblots, the urine samples from healthy volunteers revealed clear aquaporin-1 and aquaporin-2 signals in antidiuretic but not diuretic states. In the urine of a female patient, whose NDl is explained by low expression of the wild-type V2-receptor gene, aquaporin-2 excretion was high parable with that in a healthy individual during antidiuresis. In the urine of a male patient with a non-sense mutation in the V2-receptor gene, a weak aquaporin-2 signal was detected. In NDl patients with mutations in the aquaporin-2 gene, aquaporin-2 could not be detected in urine, suggesting a low stability of mutant aquaporin-2 proteins. In four out of seven NDl patients, aquaporin-1 excretion was relatively high, which suggests pensatory increase in proximal reabsorption in NDl. |
8793792 | Vasopressin increases glomerular filtration rate in conscious rats through its antidiuretic action. | To evaluate the possible influence of chronic alterations in urine concentrating activity (CA) on renal hemodynamics, adult male Sprague-Dawley rats were submitted for 7 days to one of three different levels of CA. CA was either reduced by increasing water intake (mixing the food with a gel bringing 1.6 mL water per g food) (Low-CA), or increased by chronic intraperitoneal infusion of 1-desamino 8-D-arginine vasopressin (200 ng/day) (High-CA). Low-CA, High-CA, and control rats were housed in metabolic cages, ate the same quantity of dry food (the amount provided being slightly lower than the spontaneous intake), and had free access to drinking water. The only difference between groups thus concerned the water intake-vasopressin axis. Radiolabeled (14C)inulin was infused chronically by osmotic minipumps. Urine was collected during Days 5, 6, and 7, and blood samples were taken for determination of position (P), absolute and fractional (FE) urinary excretion, and clearance (C) of inulin, creatinine, urea, and main electrolytes. This protocol produced mean 24-h urine osmolality (Uosm) ranging from 500 to 3500 mosmol/kg H2O without inducing any disturbance in body fluids or plasma osmolality (Posm). Results show that GFR (Cinulin) was markedly and positively correlated with Uosm (r = 0.798, P < 0.001) and free water reabsorption (r = 0.819, P < 0.001). For Uosm = 2500 mosm/kg H2O, GFR was 47% higher than for Uosm = 500 mosm/kg H2O. Ccreat underestimated GFR in High-CA and overestimated it in Low-CA. FEurea was inversely related to Uosm, as expected from the increased reabsorption known to occur at low urine flows. It is tentatively proposed that the intrarenal recycling of urea, triggered by vasopressin and essential to the urinary concentrating mechanism, might influence GFR indirectly by modifying position of the tubular fluid at the macula densa and thus the intensity of the tubuloglomerular feedback control of GFR. Even if this mechanism remains to be confirmed, this study unequivocally demonstrates, in normal conscious rats, that the level of urinary concentrating activity has a major influence on basal GFR. |
8793794 | Impact of single use versus reuse of cellulose dialyzers on clinical parameters and indices of biocompatibility. | Hemodialysis with reprocessed dialyzers has been associated with an increased mortality in patients on chronic dialysis, but the causes for this increased mortality have not been identified thus far. The aim of this study was pare the qualitative and/or quantitative differences in activation of cellular and plasma elements, intradialytic signs and symptoms, adequacy of dialysis, and serum biochemistry and hematology in patients dialyzed with new or reprocessed cellulose dialyzers. This study measured the plasma levels and production of interleukin-1 receptor antagonist (IL-1Ra) by peripheral blood mononuclear cells (PBMC), indices of cytokine synthesis; plasma C3a levels, an index plement activation; plasma levels of lipopolysaccharide binding protein (LBP), an acute phase reactant; and plasma levels of bactericidal-permeability increasing factor (BPI), a neutrophil primary granule protein, in 37 patients on chronic hemodialysis with glutaraldehyde and bleach-reprocessed cellulose dialyzers after random assignment to 12 wk of dialysis with new (single use) or reprocessed (reuse) cellulose dialyzers. These indices were studied before dialysis, 15 min after the start of dialysis, and at the conclusion of dialysis in both groups. Intradialytic clinical symptoms and signs, urea reduction ratios, monthly blood chemistry, and hematology were also studied during the 12-wk period. Before randomization, clinical and laboratory characteristics and IL-1Ra production by PBMC were similar in the two groups. During the 12-wk study, the mean number of dialyzer reuses was 7 +/- 1 in the reuse group and there were no breaks in protocol in the single-use group. At the end of the study, plasma levels of IL-1Ra, cell content and production of IL-1Ra by unstimulated, endotoxin-stimulated, and lgG-stimulated PBMC among patients assigned to reuse were not significantly different from those in the single-use group either before dialysis, at 15 min, or at the conclusion of dialysis. Similarly, plasma levels of C3a, LBP, and BPl were not significantly different between groups at any of the three time points. During the 12-wk study, none of the patients in either arm of the study experienced chills, rigors, or fever, and there were no differences in the number of episodes of symptomatic hypotension in patients on reused dialyzers (11 +/- pared with patients on single-use dialyzers (8 +/- 2). The mean monthly urea reduction ratio during the 3 months of the study was 63 +/- 2% and 65 +/- 2% for reuse and single-use dialyzers, respectively (not significant). Similarly, the hematocrit, white blood cell count, serum calcium, phosphorus, cholesterol, triglycerides, total protein, and albumin levels were also not significantly different between the two groups at the end of the 12-wk study period. These results suggest that the reprocessing of cellulose dialyzers with glutaraldehyde and bleach does not affect indices of patibility, intradialytic symptoms and signs, adequacy of dialysis, or serum biochemistry and hematology. |
8793793 | Evidence for distinct vascular and tubular urea transporters in the rat kidney. | Facilitated urea transport has been demonstrated in several mammalian tissues, including those of the collecting ducts and red blood cells. Two urea transporters have been recently cloned: UT2, expressed in rabbit inner medullary collecting ducts, and HUT11, expressed in human erythrocytes. Because of significant identity (63%) between these two transporters, and because HUT11 is also expressed in the human kidney, they could represent the same transporter with species-related differences in their-sequences. In the study presented here, two different cDNA fragments, corresponding to the rat equivalents (rUT2 and rUT11) of the two previously cloned urea transporters, were isolated by reverse transcription-polymerase chain reaction. These rat probes were used for Northern analysis of RNA extracted from rat tissues. From the following findings, the results show that rUT2 and rUT11 are two distinct urea transporters: (1) The two cDNA fragments isolated in the rat exhibit different sequences; (2) The mRNA for rUT2 is found exclusively in the kidney, with two transcripts (3.2- and 4.4-kilobase (kb)), whereas rUT11 (only one transcript, 4.2 kb) is present in the brain, spleen, kidney, and testis; (3) in the kidney, the inner stripe of the outer medulla expresses rUT11 mRNA and the short transcript of rUT2, whereas the inner medulla expresses rUT11 and the two rUT2 transcripts; (4) In hydronephrotic kidneys that pletely lost their tubular epithelium but have intact vasculature, rUT2 transcripts are no longer expressed, whereas expression of rUT11 is intensified; (5) Experimental chronic alterations in urine concentrating activity induced different changes in the expression of rUT2 and rUT11. |
8793795 | A comparison of three brands of polysulfone membranes. | A prospective clinical crossover paring the functional performance and patibility of three brands of polysulfone membranes (Fresenius Polysultone (Fresenius Ag, Bad Homburg, Germany), Polyphen (Minntech Corp., Minneapolis, MN), and Biosulfane (WR Grace Inc., Danvers, MA)) incorporated in ethylene oxide-sterilized dialyzers parable surface area (1.3 to 1.35 m2) was undertaken. The clearance of small molecules by each membrane parable. Plasma levels of beta 2 microglobulin fell to 49.9% of pretreatment values by 210 min when using the Fresenius Polysulfone membrane, 60.2% with the Polyphen membrane, and 63.1% with the Biosulfane membrane. The reduction achieved by the Fresenius Polysulfone membrane was superior (P = 0.003). The plasma reductions were associated with the recovery of 195 mg beta 2 microglobulin from the dialysate for the Fresenius Polysulfone membrane and 158 mg for the Polyphen membrane, but no beta 2 microglobulin was recovered from the dialysate with the Biosulfane membrane. The dialysate collected with the Fresenius Polysulfone membrane also contained a mean of 6853 mg of total pared with 5490 mg with the Polyphen membrane and 8422 mg with the Biosulfane (P = 0.04) membrane. The neutropenia was slight and independent of membrane brand, as were the changes in C3a des arg and ponents. The reduction in platelet counts was higher for the Biosulfane membrane than for the other brands (P = 0.003). This study indicates that whereas the polymer base of the membrane is the same, its production and subsequent handling during dialyzer production induce changes that attain statistical significance, most notably in the way that the membrane removes beta 2 microglobulin and interacts with proteins. The differences observed are a consequence of the different alloying polymers used during manufacture and, consequently, the membranes cannot be considered equivalent. |
8793796 | Biofilm causes decreased production of interferon-gamma. | Interferon-gamma, a cytokine, is produced by lymphocytes when they are stimulated by cytokines from activated macrophages, and is essential for macrophage-mediated bactericidal operations. To investigate whether a strain of bacteria can activate macrophages and lymphocytes, the interferon-gamma levels may thus be measured. Current literature maintains that peritoneal dialysis patients with recurrent peritonitis have "unhealthy" macrophages and lymphocytes unable to produce interferon-gamma, but that the administration of interferon improves the rates of peritonitis. In an in vitro experiment, Staphylococcus epidermidis, in both its planktonic and biofilm forms, was added to a suspension of peritoneal dialysis effluents, macrophages, and healthy peripheral blood lymphocytes, which were incubated at 37 degrees C for 18 h and then centrifuged. Subsequent levels of interferon-gamma were measured in the supernatants. Three such experiments were done with peritoneal macrophages and dialysis effluents collected from each of the three different patients involved in the study. It was found that little or no interferon-gamma (0.42 +/- 0.17 U/mL) was produced when biofilm bacteria were tested, but significant amounts of interferon-gamma (9.25 +/- 4.63 U/mL) resulted in conjunction with the planktonic form of the same bacteria. To eliminate experimental errors, all conditions were left identical, appropriate control groups were added, and each of the three experiments was duplicated. These in vitro data therefore provide new insight in the role of biofilm in the pathogenesis of recurrent peritonitis in peritoneal dialysis patients. Further clinical studies are required. |
8793797 | Potential transfer of endotoxin across high-flux polysulfone membranes. | It has been postulated that synthetic membranes, such as polysulfone membranes, are rather impermeable for endotoxin or endotoxin fragments and can be used for sterile filtration of dialysate. It has never been investigated, however, whether endotoxin permeability may be different mercially available polysulfone membranes. In vitro, we found a significantly different permeability for endotoxin in two standard dialyzers and one test dialyzer with high-flux polysulfone membranes. In contrast to the F-60 dialyzer with a very low permeability for endotoxin, a stepwise increasing load of endotoxin concentration in the partment of the PN 1913 test dialyzer and Primus 1350 polysulfone dialyzer was followed by a stepwise increase of endotoxin in the partment. A significant transfer across the membranes was found when the endotoxin concentration in the partment was > 10 ng/mL in the PN 1913 and > 0.5 ng/mL in the Primus 1350. In the latter, about 0.5% of the endotoxin concentration of the partment was found in the partment. The data suggest that manufacturers have to evaluate the performance and other properties of their synthetic membranes in detail. |
8793798 | Risk factors for hospital utilization in chronic dialysis patients. Southeastern Kidney Council (Network 6). | It is not known if the risk factors for hospital utilization are similar to the risk factors for mortality in chronic dialysis patients. The risk factors associated with hospital days per year of patient risk were identified in a subset of patients in Network 6 (the states of North Carolina, South Carolina, and Georgia) who began dialysis in 1989. The demographic characteristics of this cohort of 1572 patients included a mean (+/- SD) age of 57.4 +/- 15.0 yr; 63.7% of the patients were African American, 52.4% were female, and 33.0% had diabetes mellitus as the primary cause of ESRD. The median number of hospital days per year of patient risk was 8.8, with 25th and 75th quartiles of 3.9 and 20.1, respectively. By using multiple regression analysis, the strongest predictors of the number of hospital days per year of patient risk included low serum albumin level (P = 0.0001), decreased activity level (P = 0.0006), diabetes mellitus as the primary cause of ESRD (P = 0.002), peripheral vascular disease (P = 0.004), white race (P = 0.01), increasing age (P = 0.03), the absence of hypertension (P = 0.03), and the presence of angina (P = 0.03), smoking (P = 0.03), and congestive heart failure (P = 0.045). These risk factors are similar to those reported for an increased risk of mortality in dialysis patients and some of them, such as smoking, are modifiable and may be amenable to interventional strategies. |
8793799 | Prostaglandin E1 reduces the glomerular mRNA expression of monocyte-chemoattractant protein 1 in anti-thymocyte antibody-induced glomerular injury. | To study whether prostaglandins (PG) can regulate the mRNA expression of monocyte-chemoattractant protein 1 (MCP-1) in glomerular immune injury, MCP-1 mRNA levels were evaluated in anti-thymocyte antibody (ATS) -induced glomerular injury by Northern blotting and reverse transcription-polymerase chain reaction. Immune injury was induced in vivo by the intravenous application of ATS to male Wistar rats and in vitro by the perfusion of isolated rat kidneys with ATS and rat serum. In vivo 3 h and 5 days after antibody application, glomerular mRNA expression of MCP-1 was markedly pared with controls. In the isolated perfused kidney, antibody plement also induced an increase in MCP-1 expression at 10 min and 60 min after antibody perfusion. When the rats were treated with PGE (250 micrograms, twice daily), the increase in MCP-1 expression was reduced. This was associated with a reduction of intraglomerular recruitment of monocytes/macrophages. In the isolated perfused kidneys, PGE1 (1 mg/L) prevented the antibody- and rat serum-stimulated increase in glomerular MCP-1 mRNA expression. These data demonstrate that PGE1 reduces glomerular MCP-1 mRNA expression in glomerulonephritis and in the isolated perfused rat kidney after induction of immune injury with antibody plement. The data suggest that prostaglandins might mediate MCP-1 effects in glomerular immune injuries. |
8793800 | Monocyte chemotactic peptide-1 expression in acute and chronic human nephritides: a pathogenetic role in interstitial monocytes recruitment. | Tubulointerstitial damage is mon histopathological feature of acute and chronic renal diseases and a prognostic indicator of renal function e. Monocytes infiltrating the interstitium, through the release of cytokines and/or growth factors, may play a key role in the pathogenesis of tubulointerstitial damage. Monocyte chemotactic peptide-1 (MCP-1) is a specific and powerful chemoattractant and activating factor for monocytes. This study investigated MCP-1 expression and its correlation with monocyte infiltration and tubulointerstitial damage in biopsies of patients with acute interstitial nephritis (AIN) and a chronic glomerulonephritis, namely immunoglobulin. A nephropathy (IgAN), often characterized by tubulointerstitial involvement. Six patients with AIN and 20 patients with IgAN, nine with mild (G1 to 2) and 11 with moderate to severe histologic lesions (G3 to 5), were studied. MCP-1 gene and protein expression were evaluated by in situ hybridization and immunohistochemistry. Infiltrating CD68-positive cells were identified as monocytes. MCP-1, weakly expressed in normal kidneys, was clearly upregulated in AIN biopsies. The gene and the protein expression were primarily localized in tubular and glomerular parietal epithelial cells, as well as in infiltrating mononuclear cells. In IgAN, a striking increase in MCP-1 mRNA and protein expression was observed only in the biopsies with moderate to severe lesions, with a pattern of expression similar to AIN. The MCP-1 expression strictly correlated with monocyte infiltrates and tubulointerstitial damage. In addition, the urinary excretion of this chemokine was studied in 17 IgAN patients. MCP-1 protein concentration was pared with healthy subjects, in IgAN patients, especially in the G3 to 5 group, and directly correlated with the renal MCP-1 gene expression. In conclusion, these data suggest that production of MCP-1 in the partment may play a key role in modulating monocytes influx and, consequently, tubulointerstitial damage. |
8793801 | Role of tumor necrosis factor-alpha on mesangial cell MCP-1 expression and monocyte migration: mechanisms mediated by signal transduction. | Monocyte chemotactic protein-1 (MCP-1), a specific chemoattractant for monocytes, has been thought to play an important role in the recruitment and accumulation of monocytes within the glomerulus seen in glomerular diseases. This study examined the role of tumor necrosis factor (TNF)-alpha-mediated cellular signal transduction pathways on mesangial cell MCP-1 gene expression and monocyte migration. Incubation of mesangial cells with TNF-alpha stimulated MCP-1 mRNA expression in a dose- and time-dependent manner. Phorbol myristate acetate (PMA), a protein kinase C (PKC) activator, increased MCP-1 message by mesangial cells while depleting PKC decreased MCP-1 gene expression to control levels. Activation of PKC-depleted mesangial cells with PMA but not with TNF-alpha inhibited MCP-1 mRNA expression. Similarly, calphostin C, a PKC inhibitor, failed to inhibit TNF-alpha-induced MCP-1 expression. The incubation of mesangial cells with various protein tyrosine kinase inhibitors (PTK, e.g., herbimycin, tyrphostin, genistein) blocked TNF-alpha-induced MCP-1 mRNA message. Additional experiments examining the role of cAMP on MCP-1 expression indicated that the preincubation of mesangial cells with various cAMP generating substances (pertussis toxin, isoproterenol, dbcAMP) did not induce mesangial cell MCP-1 mRNA transcripts. However, the coincubation of mesangial cells with TNF-alpha and pletely inhibited TNF-alpha-induced MCP-1 gene expression. Finally, TNF-alpha-activated mesangial cell media increased monocyte transmigration that could be blocked by neutralizing anti-MCP-1. These studies indicate that TNF-alpha facilitates monocyte transmigration into the glomerulus mediated by the increased expression of MCP-1 by mesangial cells. TNF-alpha-induced mesangial cell MCP-1 expression is regulated by signal transduction pathways involving PTK but not those dependent on PKC or cAMP. |
8793802 | Lupus nephritis in children: a longitudinal study of prognostic factors and therapy. | There are only a few studies in the pediatric literature that have analyzed risk factors for renal failure in childhood lupus nephritis. This study reviewed the e of 56 children (4 to 18 yr of age) with lupus nephritis seen at the authors' institution over a 27-yr period (1965 to 1992), in relation to risk factors and therapy. All children underwent percutaneous renal biopsy before the institution of therapy. From 1965 to 1987, treatment for Class III and IV lupus nephritis consisted of high-dose pulse methylprednisolone, 500 mg daily for 10 days, followed by oral prednisone. From 1987 to 1992, IV cyclophosphamide was given monthly for 6 months and then every 3 months for a period of 3 yr for patients with Class III and Class IV disease. Of 56 children, 42% had Class IV and 21% had Class III histology at onset. The mean follow-up period was 4 yr and ranged from 0.5 to 20.3 yr. Life-table analysis showed that the cumulative proportion of patients surviving was 82.8% at 5 yr and 67.7% at 10 yr. Renal survival was 44.4% at 5 yr and 29% at 10 yr, after the initial diagnosis of lupus nephritis was made. Age at diagnosis, race, sex, initial serum creatinine level, and the presence of proteinuria, hypertension, and DNA antibody titers were reviewed with respect to disease progression, as was the histological class at diagnosis. The effect of the different therapies was also examined. Univariate analysis revealed a significant association of progression to ESRD with an elevated serum creatinine level (P = 0.021), decreased plement (P = 0.024), hypertension (P = 0.053), and histological classification of Class IV lupus nephritis (P = 0.031). Multivariate analysis demonstrated that progression to ESRD was independently associated with an initial Class IV histology (relative risk, 1.78; P < 0.003), hypertension at presentation (relative risk, 1.67; P < 0.003), and a low plement level in conjuction with a high creatinine level (relative risk, 1.52; P < 0.028). Among children with lupus nephritis, those with Class IV disease, hypertension, high creatinine levels, and low plement levels at the time of diagnosis are at increased risk for ESRD. Initial histological classification of lupus nephritis was the most reliable prognostic factor for disease progression. This study was unable to detect a difference in e for the two treatment groups. |
8793803 | Effect of duration of type I diabetes on the prevalence of stages of diabetic nephropathy defined by urinary albumin/creatinine ratio. | The objective of this study was to determine the prevalence of stages of diabetic nephropathy, defined by the albumin/creatinine ratio (AC ratio) in repeated measurements in random urine samples. Over a 30-month interval, 1613 patients with Type I diabetes (IDDM) (aged 15 to 44 yr, IDDM duration 1 to 39 yr), and 218 healthy control subjects provided multiple urine specimens. AC ratios measured in urine samples taken 5 months apart were highly reproducible (Spearman r = 0.83). A criterion for the boundary between normoalbuminuria and microalbuminuria was obtained by searching for a cutpoint that optimized agreement between serial specimens on individuals. The result was lower in men than women: 17 pared with 25 micrograms/mg. These two values corresponded to the 95th percentiles of the respective distributions of the AC ratio in healthy control subjects. Also these sex-specific cutpoints, when converted to albumin excretion rates, became almost equal: 30 and 31 micrograms/min. Microalbuminuria appeared early in the course of IDDM (6% of those with only 1 to 3 yr of diabetes) and then increased rapidly during two intervals, the first and third decades, before leveling off at 52%. By that time the cumulative risk of overt proteinuria had risen to 27%. Determinations of the AC ratio in random urine samples are easily obtained and are reliable indices of elevated urinary albumin excretion (microalbuminuria) in IDDM. The pattern of occurrence of microalbuminuria according to duration of IDDM suggests that there may be two subsets of diabetic nephropathy, one appearing early and the other late. Patients with microalbuminuria and 25 yr of postpubertal IDDM have low risk of progression to advanced diabetic nephropathy. |
8793804 | Expression of type IV collagen alpha 3 and alpha 4 chain mRNA in X-linked Alport syndrome. | X-Linked Alport syndrome is caused by mutations in the COL4A5 gene encoding the Type IV collagen alpha 5 chain (alpha 5(IV)). The authors' recent immunohistochemical study demonstrated abnormal expression of alpha 3(IV) and alpha 4(IV), as well as of alpha 5(IV), in patients with this syndrome, and a correlation between abnormal alpha 3(IV) and alpha 4(IV) expression and severity of the disease. The mechanism linking alpha 5(IV) mutations with abnormal alpha 3(IV) and alpha 4(IV) expression is unknown. To examine alpha 3(IV) and alpha 4(IV) mRNA expression in renal cortical tissues of patients with X-linked Alport syndrome, a nonradioisotopic, semiquantitative reverse transcription-polymerase chain reaction assay (alpha 3(IV) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), alpha 4(IV), and GAPDH coamplification) was performed. There were no significant differences among severely affected male (N = 3), mildly affected male (N = 2), and female (N = 1) X-linked Alport patients and control subjects (N = 2) with respect to alpha 3(IV) and alpha 4(IV) mRNA expression in renal cortical tissue. These findings indicate that alpha 3(IV) and alpha 4(IV) transcription is not turned off in X-linked Alport syndrome and suggest that abnormal expression of alpha 3(IV) and alpha 4(IV) proteins in this syndrome may be the result of failure of incorporation of alpha 3(IV) and alpha 4(IV) into the glomerular basement membrane. |
8793805 | In vivo ANA is a fixation artifact: nucleosome-complexed antinucleosome autoantibodies bind to the cell surface and are internalized. | It has been suggested that binding of anti-double-standed DNA antibodies to cell surfaces, followed by internalization and nuclear binding (so called in vivo ANA) is of pathophysiological significance for tissue damage in systemic lupus erythematosus. We have shown before that pathogenic antinuclear plexed to nucleosomal antigens can bind to heparan sulfate in the glomerular basement membrane in vivo. Because nucleosomes are also reported to bind to the cell surface, we hypothesized that in vivo ANA is a property of antinuclear antibodies bound to nucleosomal antigens. Therefore, we studied three antinucleosome monoclonal antibodies (mAb) that exhibit in vivo ANA as seen by immunofluorescence in mice inoculated intraperitoneally with the hybridoma producing the mAb. The same plexed to nucleosomal antigens after intravenous injection into mice induced in vivo ANA, in contrast to purified plexed mAb. To study this in more detail, we plexed mAb with various cell lines and found binding to the cell surface and subsequent internalization into cytoplasmic vesicles. However, no binding to the nucleus was observed by immunoelectron microscopy (IEM) and confocal laser microscopy. plexed mAb did not bind to the cell surface. Next, from mice bearing the hybridomas producing the mAb intraperitoneally, a small part of the kidney was snap frozen in liquid N2, fixed with acetone, and studied in immunofluorescence, whereas the remaining part of the kidney was fixed in vivo by renal perfusion with a mixture of 0.01 M sodium periodate, 0.075 M lysine HCl, 0.0375 M Na2HPO4, and 2% paraformaldehyde (PLP) and studied in both immunofluorescence and IEM. In the acetone-fixed kidney sections obtained without in vivo fixation we again observed in vivo ANA. However, after in vivo PLP perfusion fixation, no nuclear binding was found. In IEM, localization in cytoplasmic vesicles was seen. In conclusion, antinucleosome plexed to nucleosomal antigens can bind to the cell surface and are transported into the cytoplasm, but do not bind to the nucleus. The reported nuclear localization of antinuclear antibodies is caused by a fixation artifact. |
8793806 | Association of asialo-galactosyl beta 1-3N-acetylgalactosamine on the hinge with a conformational instability of Jacalin-reactive immunoglobulin A1 in immunoglobulin A nephropathy. | The O-glycans in Jacalin-binding immunoglobulin A1 (IgA1) were released by gas-phase hydrazinolysis and were fractionated by gel filtration and reversed-phase HPLC. Four peaks (P1 to P4) were obtained. There was a significant shift from Peak 2 (monosialylated Gal beta 1-3GalNAc) to Peak 4 (asialo-Gal beta 1-3GalNAc) in the IgA-nephropathy pared with the negative control group (P < 0.05). One of the functions of the carbohydrate side chains is to stabilize the three-dimensional structure of the glycopeptides. In order to evaluate the stability of the Jacalin-binding IgA1 molecule, the increase in turbidity as a consequence of the increase of the aggregated IgA1 level under the condition of high temperature (63 degrees C) was observed. The increase in the turbidity was significantly higher in the IgA nephropathy pared with the negative control group (21.7 vs. 5.9% at 150 min, P < 0.02). From a sample of IgA1 solution that had originated from a pooled normal serum, heat-tolerant (nonaggregated) and intolerant (aggregated) IgA1 molecules were separated by gel filtration. The heat-intolerant IgA1 had lower amounts of sialic acid (27.4 micrograms/mg IgA1) than the tolerant IgA1 (37.6 micrograms/mg IgA1). Further, the analysis of the O-glycans released from another IgA1 sample by the hydrazinolysis revealed that the ratio of the asialo-Gal beta 1-3GalNAc/total Gal beta 1-3GalNAc in the heat-in-tolerant IgA1 (27.2%) was pared with that in the heat-tolerant IgA1 (18.2%). From these results, it was suggested that unusual glycosylation on the hinge region of Jacalin-binding IgA1 induced an insufficient conformational stiffness to the hinge peptide, resulting in the aggregation of the IgA1 molecule in IgA nephropathy. |
8793808 | Access flow measurements in hemodialysis patients: in vivo validation of an ultrasound dilution technique. | In this study, access flow in hemodialysis patients with bridge grafts was measured by a newly developed device. The technique is based on the measurement of changes in the ultrasound characteristics of blood. These changes were initiated by an injection of isotonic saline and measured in the tubes of the extra-corporeal circuit. The access flow rate (mean +/- standard variation) was 880 +/- 440 mL/min (range, 166 to 1740) (N = 46). The mean coefficient of variation was 13.4 +/- 6.8% (median, 13.0%; range 3.5 to 29.4%). Measurements correlated well (r = 0.91, N = 22, P < 0.001) with flow rates determined by magnetic resonance angiography and by a technique based on intra-access flow-pressure curves (r = 0.84, N = 14, P < 0.001). In conclusion, access flow can be measured easily, noninvasively, and reliably by the ultrasound dilution device. |
8793807 | An application of electron paramagnetic resonance to evaluate nitric oxide and its quenchers. | Electron paramagnetic resonance (EPR) spectrometry has been used for detection of free radicals, including nitric oxide (NO). The method was applied to evaluate if lazaroids, nonspecific scavengers of oxygen-derived free radicals, can reduce another radical species, namely, NO. When solutions containing 0 to 10 microM lazaroids (U78517F, U83836E, and U74500A) were mixed with NO (approximately 8.0 microM), and with a spin-trap agent specific to NO (10 microM carboxy-2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3 oxide, c-PTIO), the spectrum of NO-c-PTIO determined by the EPR decreased with increasing concentrations of lazaroids. An identical study with 1 to 1000 microM methylprednisolone or dexamethasone, which are prototypes of lazaroids, failed to reduce NO. In a separate study, rat red blood cells preloaded with U78517F were exposed to NO. EPR spectrometry demonstrated that hemoglobin in these cells were less pared with the cells not preloaded with U78517F. An ability of lazaroids to quench NO in vivo was evaluated by an intravenous injection of sodium nitrite (4 mumol/kg body weight) into rats pretreated with 10 mg/kg lazaroids (U78517F and U74500A). The nitrosylation of RBC hemoglobin was markedly attenuated in rats pretreated with pared with those with vehicle. Thus, these pounds protected cells from NO-induced nitrosylation both in vitro and in vivo. Lazaroids may be applicable as anti-NO agents. |
8793809 | Interpretation of fracture follow-up roentgenograms: is a second opinion routinely necessary? | To determine whether a second interpretation of follow-up roentgenograms is necessary in fracture management, we analyzed 300 fracture follow-up visits to two orthopaedists with regard to the efficacy of obtaining a radiologist's interpretation of the follow-up films. Fracture management decisions were made at the time of the visit without the benefit of the radiologist's interpretation of the films. Both the clinic note and the radiologist's official report of the x-ray films were pared by two "blinded" physician observers. Major discrepancies were defined as those warranting an alteration of treatment plan. Only one major discrepancy was found, judged by both observers to be an incorrect interpretation by the radiologist. No major discrepancies were found in the treating physician's interpretation. The maximal prevalence of discrepancy was less than 1%. We conclude that the need for a second interpretation of routine fracture follow-up x-rays is questionable from the standpoint of quality of care. |
8793810 | Comparison of rear-entry (two-incision) and endoscopic techniques for reconstruction of the anterior cruciate ligament. | In this series of patients having reconstruction of the anterior cruciate ligament (ACL), the rear-entry (two-incision) technique with fixation from lateral incision into cortical bone pared with the endoscopic technique with fixation from inside to more cancellous bone. The rear-entry technique was used in 44 cases of ACL deficiency (8 chronic and 36 acute), and the endoscopic technique was used in 42 cases (19 chronic and 23 acute). Both groups had similar demographics. Objective and subjective criteria were evaluated at an average of 22 months postoperatively. Follow-up KT-1000 arthrometer findings, range of motion, and results of the Lachman and pivot shift tests pared. There were no significant differences between the two groups at 22 months' follow-up. Subjective assessment of intensity of pain, swelling, and giving way was done by visual analog scales (0 to 10 rating). None of these parameters showed any statistically significant differences between the two techniques. This study shows that both of these techniques for reconstruction of the ACL render satisfactory objective and subjective results. |
8793811 | Caveat arthroscopy: definition and guidelines for prevention. | Caveat arthroscopy is defined as an arthroscopy done with the intention of managing intra-articular nonneoplastic disease that suddenly escalates into the surgical treatment of an extra-articular neoplasm. The term caveat arthroscopy is used to describe a maloccurrence, not an act of negligence. The purpose of this retrospective review is to provide examples of this entity, examine its consequences, and suggest guidelines for prevention. We identified 13 individuals who had caveat arthroscopy over a 6-year period. All but one case involved the knee. Diagnoses included 5 soft tissue as, 5 benign bone tumors, 2 skeletal as, and 1 benign soft tissue tumor. In each case, there plications related to prereferral biopsy (ie, biopsy done before definitive management at a tertiary institution). Every subject had at least one untoward event consisting of partment contamination, inaccurate diagnosis, or delay in diagnosis. The best way to avoid plications of caveat arthroscopy is to avoid the tendency to biopsy extra-articular lesions. Instead, adequate imaging of a suspected neoplasm should be done, and referral before biopsy should be considered. |
8793812 | Osteonecrosis of the humeral head in sickle cell disease. | To determine the effects of sickle cell disease on the glenohumeral joint, 28 shoulders in 14 patients with SS sickle cell hemoglobinopathy were studied clinically and roentgenographically. patients were randomly selected; their mean age was 46 years (range, 22 to 63 years). Pain, stability, and function of the shoulders were assessed, and roentgenograms were evaluated for osteonecrosis. All 28 shoulders had some degree of pain with activity, but functional range of motion was maintained despite symptoms. Seventy-one percent of the patients had had total hip arthroplasty and 21% had had total knee arthroplasty for osteonecrosis; there was a mean of 1.5 previous joint implants per patient. Our study results show that, in patients with sickle cell hemoglobinopathy, symptoms of humeral head osteonecrosis are better tolerated than those of osteonecrosis in the lower extremities, delaying the need for surgical intervention. With severe pain and functional limitations, shoulder arthroplasty is the procedure of choice in this patient population. However, the risks are greater for patients with sickle cell disease than for other patients who have humeral head osteonecrosis, and thorough preoperative medical and anesthesia evaluations are necessary. These patients require perioperative transfusion or plasmapheresis and sufficient intraoperative hydration and oxygenation to avoid precipitating a sickle cell crisis; in addition, use of methyl methacrylate should be avoided. |
8793814 | Imaging of pelvic trauma: the role of CT with multiplanar and three-dimensional reconstruction. | Pelvic injury is a major cause of morbidity and mortality after trauma. Accurate and timely diagnosis is essential to the orthopaedic surgeon in choosing the proper management course. Traditional radiography is an important initial mode of evaluation. Computed tomography (CT) using multiplanar and three-dimensional reconstruction is a plementary modality. This article reviews the role of multiplanar and three-dimensional CT imaging in the assessment of pelvic injury. |
8793815 | Meniscal ossicle. | Meniscal ossicles are rare and may cause diagnostic difficulty on plain radiograph at presentation. Magnetic resonance imaging is useful in detecting meniscal ossicles because their characteristic appearance on MRI can differentiate them from other disease. Arthroscopy can also diagnose the meniscal ossicle, which in our patient appeared as a swelling in the meniscus. If the patient is asymptomatic before the time of diagnosis, removal of the ossicle may not be indicated and, as with our patient, normal function is possible with the ossicle retained. |
8793816 | Selenium administration to a ten-year-old patient receiving long-term total parenteral nutrition (TPN)--changes in selenium concentration in the blood and hair. | Muscle pain in the lower limbs occurred in a child with short bowel syndrome who has been receiving longterm total parenteral nutrition (TPN). Biochemical parameters revealed that the plasma and erythrocyte selenium concentrations were below the normal range for children and intravenous injection of selenium prepared from selenious acid was started at a dose of 100 micrograms per day. Muscle pain in the lower limbs disappeared one month afterwards. At this point in time, the elevation of the plasma selenium concentration was noted but the erythrocyte selenium concentration remained low. When administration was suspended due to catheter-induced fever five months later, the whole blood selenium concentration decreased again and the symptoms recurred. Accordingly, the dose of selenium was increased to 200 micrograms/day. Subsequently, the blood selenium concentration recovered to the normal range for children. After the dose increase to 200 micrograms/day, concentrations in hair samples collected at every centimeter distance from the root end were determined. The selenium concentration at the root end was found to be higher than the normal range for children, indicating that this was an excessive dose case. Although the dose was decreased from 200 micrograms/day to 120 micrograms/day, the plasma and erythrocyte selenium levels did not go down. Furthermore, the selenium level in the hair reached a plateau, and no recurrence of symptoms was observed. The above results indicate the usefulness of monitoring the selenium concentration in hair in addition to determining the blood selenium level and GSH-Px activity in administering selenium to children undergoing TPN. |
8793817 | Determination of aluminium in samples from bone and liver of elderly Norwegians. | Recent reports associating aluminium with several skeletal and neurological disorders in humans suggest that exposure to aluminium may pose a health hazard. In connection with an epidemiological study on aluminium and Alzheimer's disease, information on the body burden of aluminium was needed. Aluminium is stored in the body mainly in bone and soft tissues such as the liver. Therefore, an analytical procedure was developed for the determination of aluminium in the head of femur and the liver using graphite furnace atomic absorption spectrometry. Problems in such analyses are associated with low levels of aluminium, risk of contamination during sample preparation, inhomogeneity of the sample tissues, plexity of the matrices. Bone samples gave poorer pared to liver samples and up to four replicate analyses were performed. In the analyses of the digested bone samples, severe chemical interference occurred when these either contained much dissolved bone mineral or had a high A1 concentration. It is possible that this interference is brought about by formation of aluminium phosphate in the graphite tube. The analytical results for bones are given relative to ash weight because the samples sometimes contained a lot of fat. The ash weight of the bone tissue varied between 8 and 69 per cent. The material consisted of 84 samples of head of femur and 95 liver samples from deceased elderly Norwegians. Two cases had high A1 levels in bone (16.8 and 18.0 mg/kg ash weight) and liver (10 and 22.7 mg/kg dry weight) tissues. The remaining cases showed about ten to fifteen-fold variation of the A1 level in both liver (0.4 - 5.7 mg/kg) and bone (0.5 - 5.8 mg/kg). There was no correlation between the level in liver and bone when the two cases with the highest levels were excluded. |
8793818 | Effect of selenium supplementation on biological constants and antioxidant status in rats. | Oxygen-derived free radicals are currently suspected to be widely involved in the aetiology of several clinical disorders. In animals as well as in man, antioxidant trials are often undertaken to prevent oxidative stress. Among antioxidant molecules selenium has been largely studied. This study shows that plasma Se level is not a good index of Se status in the organism, at least at high levels of selenium. Red blood cell Se seems to be a more reliable index of Se status and could replace plasma Se level in the supplementation trials both in animals and humans. Se supplementation did not result in a significant decrease in oxidative stress markers as evaluated by blood and tissue malondialdehyde contents in healthy animals. Furthermore, heart function was altered and plasma Alanine aminotransferase activity was significantly increased in the selenium-supplemented group, which could reflect a slight subtoxic effect of selenium supplementation at the level used here. In view of the results presented, the maximum selenium content in animal diet in selenium supplementation experiments should not be higher than 2 mg/kg. |
8793819 | Inorganic arsenic species in groundwater: a case study from Purbasthali (Burdwan), India. | A regional groundwater quality survey from 20 tube wells in the Purbasthali area of the Burdwan district of West Bengal province (India) identified arsenic pollution in this area. Arsenic was detected in 19 cases at a concentration level 0.5 to 135.9 micrograms/L. Speciation studies indicate that As(III) is present in only one sample and pounds have not been detected. Iron, antimony and pH of such water samples were also studied to see if there is any correlation of the presence of arsenic and these parameters. A high concentration of iron (0.3 to 10.7 mg/L) has been detected. Antimony is present in all these water samples (0.03 to 0.9 microgram/L). The pH value of the groundwater in this area shows that it is more or less neutral. |
8793820 | Calcium, magnesium, sodium, potassium and iron content of infant formulas and estimated daily intakes. | The calcium, magnesium, sodium, potassium and iron content of a total of 22 different infant formulas marketed in Spain were measured by atomic spectrometry, and the mineral intake of infants fed exclusively with these formulas was estimated. The contents (mg/100kJ) are in the following ranges: Ca, 14-24; Mg, 1.1-2.8; Na, 5.6-9.8; K, 19-35; Fe, 0.02-0.50. These values coincide with those mended by the Codex and European Society for Paediatric Gastroenterology and Nutrition (ESPGAN), and do not exceed the limits established by the European Union (EU). The mean values and ranges of estimated intakes for each formula type and period of infancy (0-1, 1-2, 2-3, 3-4 and 4-5 months) expressed in mg element/kg body weight are tabulated. The mean Ca, Mg, Na, K and Fe daily intakes of infants (0-5 months) fed with infant formulas meet the mended values (RDA), except for the iron intake when non-iron supplemented formulas were used. |
8793821 | Determination of selenium in the serum of healthy Swiss adults and correlation to dietary intake. | The serum selenium (Se) concentrations of apparently healthy 20-40 year old blood donors from different parts of Switzerland were determined by electrothermal atomic absorption spectrometry (GFAAS). Application of a rhodium/magnesium matrix-modifier resulted in improved performance parison with a palladium modifier. The method was validated by hydride ICP-MS and quality-controlled by independent analysis using GFAAS with palladium as matrix-modifier; no bias was detected. The serum Se concentrations for male (n = 387) and female (n = 243) subjects fell into a normal distribution with mean values and standard deviations of 96.0 +/- 13.3 micrograms/L (1.22 +/- 0.17 mumol/L) and 87.9 +/- 14.4 micrograms/L (1.11 +/- 0.18 mumol/L), respectively. These values corresponded well to the formerly estimated mean daily intakes. Small but significant differences in mean serum Se concentrations were found between genders, ethnic groups as well as geographic regions, whereas age had no influence. The overall Se status of the Swiss population is assessed as adequate, somewhat higher than in the countries adjacent to Switzerland, but lower than in the U.S.A. or Finland. There is no evidence that the Se status of the population has changed over the past 10 years. However, it appears that some Swiss population groups may have a borderline Se status. |
8793822 | Halothane anesthesia and serum electrolytes. | The disparate observations on the effect of halothane anesthesia on the serum electrolyte levels in humans prompted us to carry out this work. In this study the levels of sodium, potassium, magnesium, calcium, chloride and inorganic phosphorus were determined in serum of 25 male and 15 female patients, with an age range of 15 to 40 years, who had various pathologies requiring surgery and who were given halothane anesthesia. Significant difference were detected in the concentrations of sodium, potassium, calcium and inorganic phosphorus between presurgical and post-anesthesia induction samples. The truly striking finding in the present study was the significant increase in serum inorganic phosphorus in the intra-operative period. It is suspected that this increase is due to a defect in phosphorylating mechanisms which leads to a rapid hydrolysis of stored and preformed ATP. |
8793823 | Plasma zinc, copper and copper/zinc ratio in intrinsic asthma. | Plasma zinc and copper levels and copper/zinc ratio of 22 intrinsic asthma patients pared to that of 33 healthy control subjects. Five of the intrinsic asthma patients were aspirin (ASA) intolerant. The zinc content of plasma was found to be significantly lower in patients than in control individuals with the values being 0.80 +/- 0.01 mg/L versus 0.89 +/- 0.02 mg/L, while the plasma copper level and copper/zinc ratio were significantly higher in the asthma group than in the control group, with the values being 1.28 +/- 0.03 mg/L and 1.61 +/- 0.04 versus 1.06 +/- 0.02 mg/L and 1.21 +/- 0.02, respectively (mean +/- SE). The role of the essential trace elements zinc and copper and cytokines in the pathogenesis of asthma is discussed. |
8793825 | Detection of herpes simplex virus DNA in cerebrospinal fluid samples using the polymerase chain reaction and microplate hybridization. | As conventional polymerase chain reaction (PCR) procedures are time-consuming and laborious, we developed and evaluated a rapid semi-automatic microplate method to detect the amplified PCR products. The use of PCR, with subsequent hybridization in microplates, is described for the detection of herpes simplex virus (HSV) DNA in cerebrospinal fluid samples. The principle of the method is based on two phases. Firstly, the amplification of the viral DNA in the sample is undertaken using a pair of primers of which one is biotinylated. Secondly, the amplified viral genomic sequences are bound to the wells of streptavidin-coated microplates and hybridized with digoxigenin-labeled oligonucleotide probes which are then detected using anti-digoxigenin antibody enzyme conjugates and either a photometric, fluorometric or luminometric substrate and microplate reader. The method is highly sensitive allowing the detection of as few as five purified DNA molecules. Compared to conventional gel electrophoresis followed by Southern blotting the established microplate hybridization is also much less time-consuming and involves less manual work. The applicability of the method is described for use as a routine diagnostic procedure for detection of early central nervous system infections caused by HSV-1 and HSV-2. |
8793826 | Overexpression and simple purification of a truncated, immunologically reactive GST-HCV core (1-123) fusion protein. | A full-length and a truncated gene for the core protein of hepatitis C virus (HCV) were linked to the gene for glutathione S-transferase (GST), and the expression of each GST-HCV core fusion protein was analyzed. The truncated GST-HCV core (1-123) fusion protein was expressed as a mostly soluble and partly insoluble prising more than 50% of the total protein in Escherichia coli after induction by isopropylthio-beta-D-galactoside (IPTG), while the full length GST-HCV core (1-191) fusion protein was not expressed, suggesting that the hydrophobic carboxy terminal region in the core protein affects its expression. In addition, the GST-HCV core (1-123) fusion protein purified by GST-agarose chromatography reacted specifically with an anti-HCV serum from a patient. |
8793827 | Sensitivity and specificity of mu-capture ELISA for detection of enterovirus IgM. | The sensitivity and specificity of an in-house mu-capture enzyme linked immunosorbent assay (ELISA) for enterovirus IgM in routine use was determined by analysing the results of 77 serum samples from 55 enterovirus culture-positive patients with aseptic meningitis and single serum samples from 140 patients with other infections. In addition, sera from 10 laboratory staff pre- and post-polio virus vaccination and 20 rheumatoid factor positive sera were tested for specificity. On testing the first serum specimen received, only 21 of 55 patients (38%) with aseptic meningitis yielded a positive result, rising to 33 of 55 (60%) on testing a second sample, where available. Out of 14 patients from whom multiple serum samples were tested and negative results obtained with the first serum, 12 were positive with the second sample (86%). Only patients with acute hepatitis A produced a significant number of false positives by the enterovirus ELISA (12 out of 20), but the reverse was not true: patients with enterovirus IgM did not produce false positive results in tests for hepatitis A IgM. Excluding samples positive for hepatitis A IgM, the number of non-enterovirus infections correctly reported as negative was 118 out of 120--a specificity of 98%. This test is probably the most useful serological test available at present for diagnosing recent enterovirus infection, although the limited sensitivity needs to be borne in mind. |
8793828 | Capture and RT-PCR of hepatitis C virus RNA with safety primers. | The principle and practice of the polymerase chain reaction (PCR) has had a major impact on medical research. This is a powerful method but it does have its limitations, especially for clinical diagnostic work. We describe some improvements of hepatitis C virus (HCV) amplification such as simplification of specimen preparation, elimination of false negative reactions influenced by point mutations, and fluorimetric detection. The aim of the method is to make the procedure as easy and as inexpensive as possible for routine laboratories and for blood screening. After rapid chemical denaturation of the clinical specimen with guanidine thiocyanate and simultaneous hybridization of biotinylated primers to template HCV RNA, the product was fixed to streptavidin-coated magnetic beads and potential inhibitors were removed in easy washing steps. To eliminate the influence of point mutations within the primer binding sites, primer sets with different lengths at their 3'-end were developed for capture, reverse transcription, and amplification of genomic fragments by PCR. Positive results were identified by fluorescence staining. The low cost of the method allows the quantitation of templates by testing of dilution series as mon in microbiological laboratories. |
8793829 | A cytopathic infectivity assay of human immunodeficiency virus type 1 in human primary macrophages. | In addition to CD4+ T lymphocytes, cells of monocyte/macrophage lineage are a major target for human immunodeficiency virus type 1 (HIV-1) infection. In vitro studies of HIV-1 infection in human monocyte-derived macrophages can be undertaken by a reproducible cell-based assay. A macrophage-based infectivity assay was developed based on the semi-quantitative scoring of HIV-1 induced cytopathology in monolayer macrophage cultures. The assay exhibited dilution-dependent linearity with all three primary macrophage-tropic isolates tested. The end-point infectivity titers determined by this assay correlated with the results obtained by detecting viral p24 antigen in the culture supernatant. The applications of the assay in both neutralization and anti-viral protocols yielded identical results with the more time-consuming and costly p24 formats. Since the assay offers a simple and low-cost method of measuring HIV-1 infectivity in human primary macrophages, it can be used quite easily for large-scale screening or evaluation of candidate vaccines and anti-viral agents. |
8793830 | Infection and replication of a planthopper transmitted virus-rice stripe virus in rice protoplasts. | Rice stripe virus (RSV), a planthopper-transmitted virus, was inoculated into rice protoplasts, and a one-step growth curve was determined. The amount of virus in the protoplasts decreased following the inoculation, and then increased after 8 h. The replication of RSV reached its peak 20 h after inoculation. RSV replication in the inoculated protoplasts was further demonstrated by electron microscopy. |
8793831 | Heteroduplex mobility assay for subtyping HIV-1: improved methodology and comparison with phylogenetic analysis of sequence data. | The feasibility of using DNA heteroduplex mobility analysis (HMA) as a rapid and reproducible method for routine subtyping HIV-1 in clinical specimens was examined parison with subtype determination by sequencing in both the gag and env genes. The heteroduplexes formed were examined by conventional polyacrylamide gel electrophoresis (PAGE) and also by electrophoresis in the Pharmacia PhastSystem. The significance of the HMA results was determined by the Kruskal-Wallis test, a non-parametric one way analysis of variance. It was possible to obtain an HMA profile rapidly (1-2 days) using fast PCR conditions and the PhastSystem. The HMA bands were generally sharper and more satisfactory on the Phast gels than on conventional polyacrylamide gels and the use of Phast gels was an improvement over conventional PAGE. Non-B subtype viruses could be distinguished from B subtypes, but it was more difficult to distinguish between the non-B subtypes and to assign a subtype to them. Thus, HMA can be adapted to offer a rapid screening method for HIV-1 subtyping, but sequencing is still necessary to assign a definitive subtype. This reflects the empirical nature of the subtype definitions and the quasispecies nature of the HIV genome population. |
8793832 | Detection and typing of subgroup F adenoviruses using the polymerase chain reaction. | A DNA amplification test was developed for the sensitive detection of the diarrhoea-associated subgroup F adenoviruses in clinical specimens. The test was made highly specific for serotypes 40 and 41 by using a region of the genome (the long-fiber gene) which is not significantly homologous to other human adenoviruses, but which is highly conserved between Ad40 and Ad41. A positive subgroup F adenovirus diagnosis was characterized by the presence of an amplification product of 152 base pairs, which could be digested into products of predictable length by restriction enzymes XbaI and SpeI. The viruses were typed as either Ad40 or Ad41 by digestion of the amplification product with a restriction enzyme which digested only Ad40 DNA. The specificity of the test was assessed using DNA from other adenoviruses, from human and simian cells, and from monly found in the human intestine. There was a strong correlation between results of typing obtained with PCR and restriction enzyme typing of Ad40 and Ad41, and also positivity using subgroup F specific probes in dot blot hybridizations. |
8793833 | Determination of the viremia threshold for dental cross-infection in a mouse model. | An animal model of dental virus transmission was developed using the lactate dehydrogenase-elevating virus (LDV) of mice to study cross infection. Mouse-to-mouse cross-infection was carried out by scaling the teeth of LDV-infected donor mice with dental instruments, immediately prior to using the contaminated instruments on the teeth of recipient indicator mice. The level of donor viremia was found to correlate with the rate of virus cross-infection, with a viremia threshold level of 10(7.5) ID50/ml observed for dental cross-infection. The blood volume transferred during dental cross-infection was approximately 10(-4) to 10(-5) ml, demonstrating the inefficiency of virus cross-infection, since deposition of about 1000 virions on dental instruments was associated with the threshold limit. Virus transferred during dental cross-infection rapidly entered the blood circulation, showing that dental cross-infection was not dependent on an oral infection. The results from these model studies predict the general inefficiency of dental instrument virus cross-infection, and a further reduced likelihood of dental cross-infection with appropriately cleaned instruments. |
8793834 | Immunoreactive core peptides of hepatitis C virus produced in Escherichia coli and in vitro DNA amplification-restricted transcription-translation system. | Three kinds of hepatitis C virus (HCV) core peptides were produced directly and efficiently in E. coli: 1-120 aa of the C region as NCC, 1-157 aa as NCCT and 1-190 aa as NCCL. These peptides were estimated to be 16, 22 and 24 kDa, respectively, by SDS-polyacrylamide gel electrophoresis. The processing to produce p22 core protein observed in insect cells and mammalian systems did not occur in E. coli. These peptides were similarly reactive with serum antibody from patients with hepatitis C. A mutant clone of NCC binant plasmid pKNCC4 was obtained, whose product, NCC4, was more stable in the E. coli lysate and was highly immunoreactive with sera of hepatitis C patients. This stable immunoreactive core peptide produced by pKNCC4 is useful for the detection of anti-HCV core antibody. Immunoreactive core peptides were also produced by DNA amplification-restricted transcription-translation. Five kinds of cDNA from C to E1 region were amplified and transcribed in vitro, and these five transcripts were then translated in vitro using rabbit reticulocyte lysate: 1-120 aa as 17 kDa of C1, 1-155 aa as 21 kDa of C2, 1-174 aa as 22 kDa of C3, 1-192 aa as 24 kDa of C4, and 1-213 aa as 26 kDa of C5. Cotranslational processing using microsomal membranes occurred in peptides C4 and C5 to produce p22 the same size as C3. These results indicate that the C-terminus of the mature core protein p22 may be generated at around aa 174 by cleavage with the signal peptidase. |
8793835 | Fluorescent focus reduction assay for the screening of antiadenoviral agents. | A method for screening of pounds against adenoviruses was established. pounds were diluted and plated in chamber slides for tissue culture. Drug-treated, virus-infected cultures were stained with fluorescein isothiocyanate conjugated rabbit antibodies against adenovirus hexon type 2 and fluorescent cells were counted by microscopy. This assay is more sensitive than the colorimetric method and requires smaller volumes pounds pared with the standard method using plaque assay. |
8793836 | Use of sequential immunoprecipitation to reveal discrete, separable populations of SV40 T-antigen binding to host cellular proteins. | Sequential immunoprecipitations were carried out to determine the usefulness of this method for separating subpopulations of SV40 plexed to binations of the cellular growth regulatory proteins pRB, p107, and p53. This approach was used successfully to separate discrete populations of SV40 T-antigen in a plex with pRB, p53, and p107, a plex with pRB and p107, and a plex with p107 and p53. This method was used as the first step towards isolating T-antigen for subsequent phosphopeptide mapping to address whether alterations in the overt phosphorylation of this viral oncoprotein is a major determinating factor to separation of T-antigen populations plexing with binations of cellular growth regulatory proteins. |
8793837 | A non-radioactive method for identifying enzyme-amplified products of the reticuloendotheliosis proviral env and LTR genes using psoralen-biotin labelled probes. | A novel polymerase chain reaction (PCR) system based on the env gene of reticuloendotheliosis virus (REV) strain REV-A for the detection of proviral DNA is described. The designed PCR product of 807 bp was identified using an internal probe of 278 bp produced by nested PCR from REV-infected DNA CEF. The env-gene PCR was pared with the previously described PCR for proviral REV-long terminal repeat and the PCR product served also as the probe. The probes were labelled with the psoralen-biotin system by photoactivation and the southern blot hybridization signal was detected colorimetricaly. The advantages of using a non-radioactive means of probe labelling were demonstrated clearly in that study, as well as the effective labeling of probes with psoralen-biotin and the simple colorimetric method of detection. The env-gene PCR detected all eleven REV strains used in the study. These included three REV prototype strains and eight Israeli REV isolates. Both PCR systems had similar levels of sensitivity. |
8793838 | Demonstration of specific detection of anti-HCV IgM core antibodies. | The specificity of IgM isotype response directed against the putative core of hepatitis C virus (HCV core IgM) was demonstrated in HCV IgM EIA reactive samples. No interference was noted when samples with increasing levels of rheumatoid factor (RF) alone, or bination with graded concentrations of anti-HCV IgG (HCV IgG), were tested. No deterioration in assay specificity was seen in 30 sera from patients wih monoclonal gammapathies (all isotypes). With Protein G affinity chromatography, RF/HCV IgG plexes and HCV core IgM antibodies displayed different binding characteristics, HCV core IgM appeared in the buffer eluate while HCV IgG/RF remained bound. With sucrose density gradient centrifugation HCV core IgM sedimented at 17-19S. |
8793839 | Green fluorescent protein as a tool for screening recombinant baculoviruses. | The gene encoding the green fluorescent protein (GFP) from the jellyfish Aequorea victoria, ligated to the honeybee melittin signal peptide-encoding sequence, was inserted under transcriptional control of the polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus and expressed in the Spodoptera frugiperda insect cell line Sf9 during viral infection. The binant green fluorescent protein was identified by SDS-PAGE gel electrophoresis followed by Coomassie blue staining of lysates from the binant baculovirus infected insect cells. Emission and excitation scanning of the binant baculovirus infected insect cells gave an emission maximum of 509 nm and excitation maximum of 398 nm. The GFP protein expressed was also detected in infected insect cells by a flow cytometer analysis. |
8793840 | Assaying the activity of HIV-1 integrase with DNA-coated plates. | Integration of reverse transcribed viral DNA of HIV into host chromosomes is mediated by the viral enzyme, integrase. This enzymatic activity can be monitored in vitro by integration of a small labeled DNA (donor) into a second unlabeled DNA (target). The methodology usually involves isotope labeling and gel electrophoresis. To simplify the measurement, a method mimicking enzyme-linked immunosorbent assay (ELISA) procedures was developed. Fragments of DNA were adsorbed directly on 96-well plates and used as the target DNA. The donor was a synthetic 21-bp DNA duplex of HIV-1 U5 LTR; biotin was incorporated into the 5' end of one strand whose two nucleotides at the 3' end were specifically removed during the integration. As a result of integration, the biotin-labeled donor DNA was joined with the target DNA and became immobilized on plates. These integration products were then measured by binding of avidin-alkaline phosphatase on plates. The method is simple and straightforward and can easily be adapted for high throughput screening of integrase inhibitors. |
8793841 | Lack of correlation between different hepatitis C virus screening and confirmatory assays. | Numerous 2nd and 3rd generation screening and confirmatory assays for the detection of anti-HCV antibodies have been introduced on the international market. The aim of the present study was pare the performance of five mercially available screening assays and four 'confirmatory' assays in a panel of serum samples that had tested positive or borderline with a 2nd generation EIA (Abbott HCV EIA 2nd generation). Considerable discrepancies were observed between the different screening assays and confirmatory tests. The antigens from the putative 'core' region of HCV were recognized most frequently by the confirmatory assays. By considering the reactivity to either NS5 (RIBA III and Inno-LIA) or E2/NS1 antigens (Inno-LIA Ab III) no sample could be identified as anti-HCV positive that would otherwise have been regarded as borderline or negative according to its banding pattern with core, NS3 and NS4 proteins. All 24 HCV-RT-PCR positive samples were anti-HCV reactive by the screening EIAs but only 18 and 21 samples were confirmed anti-HCV positive with the RIBA II and III, respectively. A clear association was observed between HCV-RNAemia in serum samples and index values (O.D. sample/O.D. cut-off) of the screening EIAs as well as with the number of reactive proteins in the confirmatory assays. In conclusion, the results of current screening and confirmatory assays are highly divergent. The additional diagnostic significance of the relatively expensive and labour-intensive immunoblots appears to be very limited. For the serological diagnosis of HCV infection and for blood donor screening, confirmatory assays should only be used if there is a borderline result by HCV EIA. The determination of infectivity by qualitative PCR and the follow-up of patients undergoing IFN therapy by HCV-RNA quantification appears to be much more useful. |
8793842 | A novel cytopathic microtitre plate assay for hepatitis A virus and anti-hepatitis A neutralizing antibodies. | The slow growth of hepatitis A virus (HAV) has made tissue culture assay for infectious virus difficult. Strains of the virus of greater cytopathogenicity have been selected for use in plaque cytopathic assays. However, in our hands, this assay has been difficult to reproduce consistently due to problems in maintaining intact cell monolayers over the long incubations involved. This report describes the development of a cytopathic TCID50 assay for HAV. From the results of repeated assay of one preparation of the virus, the coefficient of variation of the assay was calculated to be 4%. This assay has also been adapted to quantitate antibodies to HAV. Initial results of assaying the WHO standard immune serum globulin parable with the titre obtained by the radioimmunofocus inhibition test. Antibody titres in human and mouse sera could also be quantitated. The cytopathic TCID50 assay and the adapted inhibition assay described, may prove useful for the development and control of HAV vaccines and the validation of viral inactivation protocols. |
8793843 | Video imaging of firefly luciferase activity to identify and monitor herpesvirus infection in cell culture. | Expression of reporter genes incorporated plex viral genomes is being used increasingly to monitor virus infection in cell culture and in the organism. One of the most sensitive markers is luciferase from Photinus pyralis which catalyzes a luminescent reaction that can be traced in cell and organ extracts in luminometers. A novel method is described for monitoring Iuciferase activity after infection of cells with binant herpesvirus (pseudorabies virus) which carries stably and expresses luciferase using ultra high sensitive photon-counting enhanced video image acquisition. The data show that firefly luciferase activity in virus-infected cells can be monitored without destruction of the cells using video equipment for either macroscopic image acquisition or microscopy. Resolution down to a single-cell level can thereby be achieved. This method increases greatly the potential of monitoring virus infection in real time with a non-destructive highly sensitive method in cell culture and should also help to assay viral spread in the animal. |
8793844 | A major antigenic domain for the human humoral response to Puumala virus nucleocapsid protein is located at the amino-terminus. | Nephropathia epidemica (NE), the major form of hemorrhagic fever with renal syndrome in Europe, is caused by the hantavirus serotype Puumala (PUU). The PUU virus nucleocapsid protein (N) has been shown to be highly immunogenic both in laboratory animals and in man. We aimed to locate domains important in humoral immune reactivity and to use this information to develop a specific and sensitive enzyme-linked immunosorbent assay (ELISA) for serological diagnosis of NE. Escherichia coli poly-histidine fusion protein expression vectors containing over-lapping gene segments encoding the PUU virus N (PUU rN) were constructed. The resulting gene products were examined by immunoblots and ELISA with polyclonal and monoclonal antibodies. The dominating antigenic region of PUU rN was located between amino acids (aa) 7 and 94. A binant fusion protein containing aa 7-137 of PUU virus N (PUU rN delta 5) was used for the detection of specific IgG and IgM responses in NE. ELISA based on PUU rN delta 5 was found to have equal sensitivity and specificity pared to the full length binant PUU rN by ELISA, for both acute serological diagnosis of NE and for seroepidemiological screening purposes. Furthermore, this protein is easier to handle than full length PUU rN due to its higher solubility in aqueous solutions. |
8793845 | Nucleic acid binding by zinc finger-like motif of human papillomavirus type 16 E7 oncoprotein. | Human papillomavirus (HPV) types 16 and 6b E7 proteins and their chimeric or mutant proteins were analyzed for oligonucleotide-binding activity by surface plasmon resonance-based biomolecular interaction analysis. The results indicated that type 16 E7 protein has stronger nucleic acid-binding activity than that of type 6b E7 protein. In addition, the results also indicated that the zinc finger-like motif in the C-terminal region of the type 16 E7 protein plays an important role in this activity. |
8793846 | Production of the antigen and the antibody of the JC virus major capsid protein VP1. | The DNA of the major capsid protein VP1 of the human polyomavirus JC virus (JCV), Taiwan-3 strain, was generated from the urine of an autoimmune disease patient by polymerase chain reaction (PRC). The VP1 DNA was cloned into a prokaryotic expression vector, pGEX-4T-1, for expression in E. coli. The nucleotide sequences and the deduced amino acid sequences were determined pared with the JC virus prototype, Mad-1. Thirty nucleotides were different between these two strains. Six of the altered nucleotides affected amino acid coding and ten of them caused changes in endonuclease recognition sites. The binant VPI protein was purified and used to raise monospecific antiserum in rabbit. binant JCV VP1 protein and its monospecific antiserum are important clinical reagents and could possibly be developed as a subunit vaccine and as a serological diagnostic antigen in the future. In addition, the region between amino acid residues 40 and 80 of JCV VP1 is predicted to be an antigenic epitope on the basis of its hydropathy plot parison with the VP1 sequences of SV40 and BK virus. |
8793847 | Detection of stylet-borne and circulative potato viruses in aphids by duplex reverse transcription polymerase chain reaction. | A reverse transcription polymerase chain reaction (RT-PCR) assay was designed to amplify stylet-borne potato virus Yo (PVYo) in aphids using primers located in the viral capsid gene. A 480 bp long product was detected in aphids exposed to PVYo-infected potato plants. Approximately 40% of Myzus persicae and 15% of Aphis nasturtii exposed briefly to PVYo-infected plants acquired the virus. This rate of acquisition by both species of aphids was typical of our earlier observation of the virus transmission tests. No significant difference in virus detection was observed whether the aphids were tested immediately after exposure to virus sources or stored for up to 45 days in ethanol at room temperature. The addition of a second pair to primers located in the capsid gene of circulative potato leafroll virus (PLRV) allowed simultaneous amplification of two viruses (duplex RT-PCR) in single aphids. Acquisition of PVYo by the aphids already viruliferous with PLRV was significantly pared to aphids not carrying PLRV. Duplex RT-PCR for PVYo and PLRV could be applied to analyze aphids collected from the field to ascertain the relative presence of both viruses in a single test. |
8793848 | "Repair' of the chorionic somatomammotropin-A "enhancer' region reveals a novel functional element in the chorionic somatomammotropin-B enhancer. | Human chorionic somatomammotropin (CS) synthesis results from the independent expression of two homologous genes, CS-A and CS-B. A transcription enhancer factor-1 (TEF-1) element and an upstream 81 bp modulatory domain, containing repressor (RF-1) and derepressor (DF-1) activities, are important for efficient CS-B enhancer function in transfected placental JEG-3 cells. The equivalent region of the CS-A gene is not active. Although the TEF-1 element is conserved between the CS-A and CS-B genes, a single base substitution is present in the DF-1 element and two more are located between the RF-1 and DF-1 sites in a region we term AF-1. Repair of the DF-1 site increased CS-A enhancer function approximately 70-fold, but repair of previously uncharacterized AF-1 sequences was also required for full (CS-B like) enhancer activity. A 5 bp disruption of AF-1 sequences in the CS-B enhancer region, resulted in a 97% loss of stimulatory activity. The AF-1 sequences showed no intrinsic enhancer activity, however, they were able to significantly repress heterologous promoter activity stimulated by a TEF-1 enhancer element. A high affinity/specificity interaction between JEG-3 nuclear protein and AF-1 sequences was confirmed by gel mobility shift assay. parison to "wild type' AF-1 sequences, this interaction peted to a lesser extent by both RF-1 and DF-1 elements, but not by mutated AF-1 sequences. The major protein binding to AF-1 sequences was estimated to be 23 kDa by UV crosslinking. These data indicate that enhancer activity can be generated by modulating binding events proximal to the TEF-1 element in the CS-A "enhancer' region and that coordinated binding of AF-1 and DF-1 are required for efficient (CS-B) enhancer activity. |
8793849 | Sequence requirements for high affinity retinoid X receptor-alpha homodimer binding. | To better delineate the sequence requirements for high affinity binding of retinoid X receptor alpha (RXR alpha) homodimers, a selection protocol was used starting from a random pool of oligonucleotides. All recovered sequences contained at least two hexamers related to the consensus sequence for the thyroid/retinoid subfamily of nuclear receptors, A/GGGTCA. These hexamers were most frequently organised as direct repeats with one interspacing base pair (DR1) and as palindromic repeats without interspacing base pairs (PAL0), the established configurations for RXR response elements (RXREs). However, DR2 and DR6 configurations also appeared to bind RXR alpha homodimers with high affinity, as did elements consisting of three hexamers. Reporters containing single copies of these elements conferred 9-cis retinoic acid responsiveness to cells cotransfected with an RXR alpha expressing plasmid. The upstream hexamer of all recovered sites was preferentially preceded by a G and its consensus was GGGTCA. Based on position of the selected DR1 RXREs, and the functional and mutational analysis, the optimal DR1 RXRE consists of an upstream hexamer starting with A or G and preceded by A or G. The interspacing base can be either G, A or T but not C. The affinity of RXR alpha homodimers for a DR1 element is strongly reduced when the final position is taken by a C. The results of the present investigation indicate that RXR alpha homodimers may have broader DNA binding specificities than currently believed. The biological relevance of these alternative RXREs will need to be corroborated by the identification of natural elements of this kind. |
8793850 | Point mutations abolish 11 beta-hydroxysteroid dehydrogenase type II activity in three families with the congenital syndrome of apparent mineralocorticoid excess. | The 11 beta-hydroxysteroid dehydrogenase type II enzyme (11 beta HSD2) converts cortisol into mineralocorticoid receptor inactive cortisone, thus preventing occupation of the non-selective mineralocorticoid receptor by glucocorticoids in the kidney. Mutations generating inactive enzymes have been described in the HSD11B2 gene in the congenital syndrome of apparent mineralocorticoid excess (AME), although proof of mutant protein synthesis was not provided. In the present study we have examined the metabolism of cortisol in mammalian cells transfected with plasmids expressing the wild type and mutant enzymes from three additional families of patients with mutations in the HSD11B2 gene. These studies revealed that the mutants were enzymatically inactive in intact mammalian cells expressing significant levels of both full length and truncated proteins. This is the first study to definitively show that point mutations in the HSD11B2 gene abolish 11 beta HSD2 enzymatic activity in the syndrome of AME. |
8793851 | CRF stimulates expression of multiple fos and jun related genes in the AtT-20 corticotroph cell. | Recent studies have shown that corticotropin releasing factor (CRF) stimulates c-fos gene expression in the AtT-20 corticotroph cell line, and that overexpression of c-Fos results in activation of POMC gene transcription. Since transactivation by c-Fos requires dimerization with a Jun family member to form the active transcription factor AP-1, we have examined the expression of multiple fos and jun related genes and have correlated their expression with AP-1 DNA binding activity in AtT-20 nuclear extracts after stimulation with CRF. Although basal expression of c-fos mRNA was extremely low, it was rapidly and transiently stimulated in AtT-20 cells following administration of either constant or a single pulse of CRF. In contrast, basal expression of c-jun mRNA was slightly higher and underwent little or no change in response to CRF. Specific ribonuclease protection analysis showed that in addition to c-fos, mRNA transcripts encoding fos B and jun B were rapidly stimulated in response to CRF, though levels of induced fos B mRNA were 20-40 times lower than c-fos or jun B, respectively. Gel shift analysis demonstrated that CRF caused a sustained increase in AP-1 DNA binding to both a canonical AP-1 element as well as to the POMC exon-1 AP-1 site. Studies with specific antisera directed against c-Fos revealed that although no c-Fos could be detected in plexes in basal cell extracts, c-Fos became a ponent of AP-1 following CRF stimulation, reaching maximal levels by 4 h. Despite the fact that AP-1 DNA binding activity remained elevated for at least 24 h after CRF, c-Fos was most prominent during the early phase of the response. Similarly, JunB was shown to be a ponent of AP-1 DNA binding activity in CRF-stimulated AtT-20 nuclear extracts that persisted for at least 24h after stimulation. Despite the obvious induction of fos B mRNA in response to CRF, FosB protein was not detected in DNA bound plexes. These data demonstrate that CRF is a potent stimulus for corticotroph expression of c-fos, jun B and fos B, and suggest that the subsequent increase in AP-1 may play a role in activation of gene expression and/or as a modulator of glucocorticoid receptor function. |
8793852 | Molecular cloning and characterization of Japanese eel estrogen receptor cDNA. | A cDNA encoding the Japanese eel, Anguilla japonica, estrogen receptor (ER) was isolated and sequenced. This cDNA contains plete open reading frame encoding 573 amino acid residues, and the molecular weight of this protein is calculated to be 63417. The amino acid sequence of the eel ER shows high homology of the DNA binding (80%) and ligand binding (55%) domains with those of other species. However, the other domains show greatly reduced homology (10-20%). When the cDNA was ligated to the pSVL vector and transfected into COS7 cells, a protein was produced that had high affinity for estradiol-17 beta (E2) and specifically bound estrogens. Northern analysis showed that three ER mRNAs with lengths of 5.6, 3.8 and 1.2 kb were expressed in eel liver. Their expression was E2 inducible, with the 5.6 kb mRNA being strongly dependent on E2 stimulation. |
8793853 | Characterization of a nontumorigenic human breast epithelial cell line stably transfected with the human estrogen receptor (ER) cDNA. | Estrogens play an important role in breast cancer and the effect of estrogen on growth of breast cancer cells has been extensively studied. However, only little information is available about the response of normal breast epithelial cells to estrogen, mainly due to the difficulties in establishing estrogen receptor (ER)-positive human breast epithelial cells in culture. We have stably transfected the human estrogen receptor (hER) wt cDNA into the ER-negative, spontaneously immortalized human breast epithelial cell line, HMT-3522S1, in order to develop a model for studying the effect of estrogen on nonmalignant human breast epithelial cells. Characterization of the transfected clone F9 confirmed incorporation of the estrogen receptor gene in the genome, expression of hER mRNA and hER protein. However, proliferation of F9 cells was inhibited by both estradiol (E2) and tamoxifen, whereas the pure antiestrogen ICI 182,780 had no effect on cell proliferation. This seems paradoxical since E2 stimulated the expression of the endogenous genes, TGF-alpha, cathepsin D, and alpha1-antitrypsin. In breast cancer cell lines, high expression of these genes is correlated to estrogen-stimulated cell proliferation. The spontaneously immortalized HMT-3522S1 cells transfected with wt ER cDNA behave similarly to cell lines from nonmalignant breast tissue immortalized by carcinogens and transfected with mutated ER cDNA as described by others. The discrepancy between growth inhibition and induction of positive growth factors by E2 indicates that either ER-positive nonmalignant breast epithelial cells are growth-inhibited by E2 in contrast to malignant cells or that introduction of the ER into ER-negative cells is not sufficient for restoring "normal' estrogen responsiveness. |
8793854 | Molecular and cellular responses to DNA damage in a murine pituitary adenoma cell line. | Loss of cell cycle control and the inability of the cell to repair DNA at cell cycle checkpoints results in the propagation of genetic lesions which ultimately leads to cancer. To further our understanding of these pathways in pituitary tumorigenesis, we have investigated the effects of DNA damage by gamma radiation in a murine pituitary adenoma (AtT20) cell line with attention to cell cycle checkpoint responses, the induction of apoptosis, and the expression of known regulators of these processes. Irradiated cells exhibited characteristic morphologic changes of apoptosis beginning at 24 h, which included cell shrinkage, chromatin condensation, and cytoplasmic vacuolization, yet the ability to exclude trypan blue was retained for several days. DNA fragmentation could be demonstrated by ethidium bromide staining beginning at 24 h post-irradiation. By propidium iodide staining and flow cytometry, irradiated cells demonstrated G1 and G2 arrest at 24 h, followed at 48 h by a shift to a sub-G1 position of the apoptotic cell population. The G1 arrest coincided with an induction of p53 protein by Western blot analysis which peaked at 4 h post-radiation and persisted beyond 48 h. Expression of c-myc in irradiated cells was found to progressively decrease at 12, 24, and 48 h. Basal expression of the bcl-2 gene in AtT20 cells was found to be 15-fold higher than in normal mouse pituitary by RNase protection assay. Bcl-2 mRNA and protein levels, however, remained unchanged at 24 and 48 h following gamma-irradiation, suggesting that apoptosis occurs independently of bcl-2 gene expression in these cells following this stimulus, as reported in other cell types. We conclude that AtT20 cells undergo G1 and G2 arrest following DNA damage and that a significant proportion of cells then undergo apoptosis. The G1 arrest at 24 h is concurrent with a strong induction of p53 protein, while c-myc expression progressively diminishes. Bcl-2 is highly expressed in this cell line. The absence of variation in bcl-2 expression during apoptosis could be related to its high basal level in these cells. |
8793855 | Detection of aromatase cytochrome P450, 17 alpha-hydroxylase cytochrome P450 and NADPH:P450 reductase on the surface of cells in which they are expressed. | Using the membrane impermeant probe NHS-LC-biotin, we show in this report that a fraction of aromatase P450 (P450arom), the enzyme that catalyzes estrogen biosynthesis, is present at the surface of cells in which it is expressed, either endogenously or as a consequence of transfection. The same findings were obtained for a truncated form of P450arom lacking the putative membrane-spanning region, thus suggesting the presence of other membrane-spanning region(s) within its structure. P450arom is not unique in this regard as we find that a fraction of 17 alpha-hydroxylase P450 as well as NADPH:P450 reductase also are present at the cell surface. It is therefore possible that a number of microsomal P450s are expressed at the cell surface in this fashion. |
8793857 | Functional analysis of the human androgen receptor promoter. | Although androgen receptor levels vary widely during development, our previous studies have suggested that a single promoter is active in a number of human and rat tissues and cell lines. To examine the elements controlling androgen receptor expression, we performed deletion mapping and site-directed mutagenesis of the human androgen receptor promoter and assayed these promoter fusions in human cell lines that produce the androgen receptor as well as those deficient in the androgen receptor, both in the presence and absence of androgen. Our studies identify a region (-74 to +87) surrounding the site of transcription initiation that constitutes the core of the androgen receptor promoter. When assayed in T47D cells (an AR-expressing cell line), this segment was sufficient to direct the expression of reporter genes at levels similar to that observed for larger promoter fragments, and further deletions led to an appreciable loss of promoter activity. DNA sequences surrounding this region appear to modulate the activity of this minimal promoter in a cooperative fashion. Site-directed mutagenesis and mobility shift assays demonstrated the importance of regulatory regions upstream of the transcription initiation site including an SP1 binding site and two segments with similarities to consensus HLH protein binding sites (E-boxes). Androgen treatment did not cause a decrease in activity of any of the transiently transfected promoter fusions tested, suggesting that the promoter does not contain the information necessary for autoregulation or that a posttranscriptional or posttranslational mechanism is responsible for the regulation of AR mRNA by ligand. Furthermore, the androgen receptor promoter fusions displayed differing transcriptional activities when transfected into two cell lines deficient in androgen receptor expression suggesting heterogeneity in the mechanisms responsible for this deficiency. |
8793856 | Molecular cloning of c-jun and c-fos cDNAs from porcine anterior pituitary and their involvement in gonadotropin-releasing hormone stimulation. | The presence of the typical transcription factors c-Jun, c-Fos and cAMP-responsive element (CRE)-binding protein in the porcine anterior pituitary was examined by molecular cloning and their involvement in the membrane signal cascade, especially their roles in gonadotropin-releasing hormone (GnRH) stimulation, were studied. Several cDNA clones were isolated from a porcine anterior pituitary cDNA library using cDNA probes. They were identified as porcine c-jun and c-fos by determining their nucleotide sequences, but a homologue for CREB341 which is a member of CRE-binding protein was not detected in porcine anterior pituitary. Reverse transcription-polymerase chain reaction (RT-PCR) analysis was performed to estimate the c-jun and c-fos mRNA contents in GnRH-, forskolin- (cAMP activator) and tetradecanoyl phorbol acetate- (TPA; protein kinase C activator) treated primary cultures of porcine anterior pituitary cells. Densitometric quantification demonstrated that GnRH and TPA treatment increased c-jun and c-fos mRNA levels significantly, whereas forskolin reduced the levels of both. Therefore, c-Jun and c-Fos are definitely present in porcine anterior pituitary and their mRNAs differentially involved in the signal transduction pathway mediated by two kinases. In particular, GnRH might regulate gonadotropin expression by increasing of c-jun and c-fos levels. |
8793858 | Analysis of the functional state of T3 nuclear receptors expressed in thyroid cells. | T3 nuclear receptors (TR) are present in thyroid cells. We have analyzed the ability of thyroid TR to function as transcriptional regulators. Studies were performed on pig thyrocytes in primary culture. Messenger RNA corresponding to TR alpha 1, alpha 2 and beta were detected in pig thyrocytes by RT-PCR and Northern blot; the alpha 2 mRNA was more abundant than the alpha 1 mRNA. Thyrocytes were transiently transfected with different plasmids containing the CAT (chloramphenicol acetyl transferase) gene placed under the control of different promoters (delta MTV, TK or delta SV40) and bearing a thyroid hormone response element, TREp or TRE DR + 4. It was found that TSH induced a concentration-dependent increase of the transfection efficiency, an effect reproduced by (Bu)2cAMP and Forskolin. Cells transfected with either delta MTV-, TK- or delta SV40-TREp-CAT expressed similar basal CAT activities. Addition of T3 produced a 3-fold increase of CAT activity expressed from each of these vectors. In contrast, CAT activity expressed from a vector containing the TRE DR + 4 was decreased by about 50% by T3. Thus, TREp and TRE DR + 4 gave distinct responses. These data demonstrate that TR physiologically expressed in thyroid cells can act as transcriptional regulators in a T3-dependent manner. This finding directly substantiates the concept of autocrine regulatory actions of thyroid hormones. |
8793860 | Ovine 11 beta-hydroxysteroid dehydrogenase type 2 gene predicts a protein distinct from that deduced by the cloned kidney cDNA at the C-terminus. | The gene encoding ovine 11 beta-hydroxysteroid dehydrogenase type 2 (11 beta-HSD2) was cloned and characterized. This gene consists of five exons and is greater than 4 kb in length. It contains an open reading frame of 1215 bp, which encodes a protein of 404 amino acids with a predicted MW of 44 kDa. The deduced ovine 11 beta-HSD2 protein displays over 78% sequence identity to those of the human, rabbit, rat, and mouse. However, this differs from the published sequence of ovine kidney 11 beta-HSD2 cDNA which predicts a protein of 427 amino acids. Sequence alignment indicated that this discrepancy is attributed to two single nucleotide omissions in the published cDNA sequence which resulted in a shift in the open reading frame at the codon for residue 358. Therefore, the present results have provided conclusive evidence that the primary structure of 11 beta-HSD2 protein is well conserved between the sheep and the other four mammals. Moreover, Northern blot analysis of total RNA samples from 15 peripheral tissues and seven brain regions of the mature fetal sheep revealed that the expression of 11 beta-HSD2 gene is highly tissue-specific in that it is only expressed in the kidney and adrenal gland, and at a much lower abundance in the testis, colon and placenta. The cloning of the sheep 11 beta-HSD2 gene should facilitate future studies on the regulation of 11 beta-HSD2 gene expression during fetal development in a mammalian model. |
8793859 | Demonstration of an angiotensin II-induced negative feedback effect on aldosterone synthesis in isolated rat adrenal zona glomerulosa cells. | Although both angiotensin II (Ang II) and potassium ion (K+) induce marked elevations of cytosolic free calcium concentration, [Ca2+]c, in adrenal zona glomerulosa cells-an effect which is thought to trigger aldosterone synthesis-Ang II is also known to reduce the sustained [Ca2+]c rise induced by K+. We have examined whether this effect of Ang II on the calcium messenger system is reflected at the level of the final biological response, aldosterone synthesis. In superfused isolated rat glomerulosa cells, K+ (8 mM) induced a sustained, 60-fold increase in aldosterone production. In contrast, the maximal response to Ang II (10 nM) amounted to only 10 times the basal production. When added subsequent to K+ stimulation, Ang II provoked an immediate and dramatic drop in aldosterone synthesis, to levels obtained with Ang II alone. Under conditions of maximal K+ stimulation, this effect depended upon Ang II concentration, while the well-known synergistic effect was observed with submaximal concentrations of both agonists. The inhibitory effect of Ang II could be reproduced with dioctanoylglycerol, a selective activator of protein kinase C. By contrast, the aldosterone response to adrenocorticotropic hormone (ACTH) was not affected by Ang II. At submaximal concentrations of ACTH, the steroidogenic effect of Ang II was even additive to that of ACTH. Thus, we have shown that, under conditions of maximal stimulation, Ang II exerts a profound inhibition of steroidogenesis in K(+)-stimulated rat adrenal glomerulosa cells. This counter-regulatory mechanism may ensure adequate levels of aldosterone production in vivo. |
8793863 | Developmental and FGF-2-mediated regulation of syndecans (1-4) and glypican in oligodendrocytes. | Differentiating cells undergo developmentally regulated changes in cell-cell and cell-matrix adhesion that control migration through microenvironments, proliferation, and differentiation. The diversity of the patterns of expression of heparan sulfate proteoglycans (HSPGs), coupled with their interactions with extracellular matrix, cell adhesion molecules, and growth factors, has emphasized their critical importance in the regulation of these events. Syndecans (1-4), glypican, and cerebroglycan are membrane-associated HSPGs that have been implicated in these events in various tissues and several tumor cell lines. We have examined the developmental expression and FGF-2-mediated regulation of these HSPGs during differentiation within a specific lineage of primary cells, oligodendrocytes (OL). Northern analyses of highly purified, developmentally synchronized populations of OL-lineage cells at three stages of differentiation (early and late progenitors and mature OLs) showed that the expression of individual forms of these syndecans and glypican are developmentally regulated. Specifically, the level of expression of syndecan-2 and -4 and glypican mRNAs increased as the cells differentiated from proliferative late progenitors to postmitotic mature cells. The expression of syndecan-1 and -3 had the inverse developmental pattern. Therefore, these two sets of molecules may have different roles in regulating the onset of terminal differentiation in OLs. The levels of mRNA expression were regulated by FGF-2: in late progenitors, FGF-2 induced a doubling of the mRNA levels of syndecan-2, -3, and -4, while those for syndecan-1 and glypican remained unaffected; in mature OLs, the levels of syndecan-1 mRNA were up-regulated, the levels of syndecan-2 and -4 and glypican were down-regulated. These results suggest that the individual syndecan molecules have distinct functions during the differentiation process and that multiple levels of regulation must exist, leading to a changing repertoire of these molecules during OL lineage progression and myelinogenesis. |
8793864 | Intracellular compartmentalization of two differentially spliced s-rex/NSP mRNAs in neurons. | Using a subtractive hybridization technique directed to cloning transcripts partmentalized distributions within cerebral cortex neurons, we have isolated rat s-rex mRNAs that are analogues of the human neuroendocrine-specific NSP gene transcripts. Differential splicing produces two main s-rex mRNA that have different regional distributions in the developing and mature rat nervous system. In certain populations of adult brain neurons, most of s-rexs, mRNA and a substantial amount of s-rexb mRNA are localized to the axonal pole of the cell body. The localization of S-Rex/NSP proteins in these neurons suggests that s-rex partmentalization targets the encoded proteins to specific regions of the neuron. |
8793862 | Regulation of FGF receptors in the oligodendrocyte lineage. | Fibroblast growth factors (FGFs) affect a broad spectrum of developmentally regulated cellular responses involved in the control of growth and differentiation. To identify specific FGF receptor forms involved in these responses, we have characterized FGF receptor transcript expression, and its modulation by FGF-2, as enriched populations of oligodendrocyte progenitors differentiate into mature oligodendrocytes. The data demonstrate that the levels of mRNA expression for FGF high-affinity receptors-1, -2, and -3 are differentially regulated during lineage progression: FGF receptor-1 expression increases with lineage progression, FGF receptor-2 is predominantly expressed by terminally differentiated oligodendrocytes, and FGF receptor-3 reaches a peak level of expression in late progenitors and then declines upon further differentiation; FGF receptor-4 expression was not detected in oligodendrocytes. Distinct patterns of alternatively spliced variants of FGF receptor-1 and -2 transcripts are expressed: the predominant FGF receptor-1 transcripts contain three Ig-like domains (FGF receptor-1 alpha), whereas the FGF receptor-2 transcripts contain two Ig-like domains (FGF receptor-2 beta 2) and this form is up-regulated as oligodendrocytes differentiate. In addition, the expression of these receptors is differentially regulated by the ligand, FGF-2: FGF receptor-1 mRNA expression is up-regulated in early progenitors, and FGF receptor-2 mRNA expression is down-regulated in mature oligodendrocytes. Finally, astrocytes express FGF receptor-1, -2, and -3 transcripts, but at different levels and with different exon utilization (FGF receptor-1 beta, FGF receptor-2 beta 1/beta pared to oligodendrocytes. To our knowledge this is the first report that demonstrates that the mRNA expression of these three FGF receptor types is differentially regulated in primary cells as they differentiate along a lineage from progenitors to terminally differentiated cells. We propose that this pattern of expression provides a molecular basis for the developmentally varying response of cells to mon ligand. For example, according to this hypothesis, in response to FGF-2, FGF receptor-1 transduces signals that stimulate the prolonged proliferation and migration of early progenitors, FGF receptor-3 promotes the proliferation and arrest of differentiation of late progenitors, and FGF receptor-2 transduces signals for terminal differentiation, but not proliferation, in mature oligodendrocytes. |
8793865 | Clonal cell lines produced by infection of neocortical neuroblasts using multiple oncogenes transduced by retroviruses. | Toward understanding the early molecular development of neocortical neurons, we report the generation of two clonal murine cell lines derived by sequential oncogenic retroviral infection of neocortical neuronal precursors. The resulting cell lines stably express the telencephalon-specific gene BF-1 and a gene enriched in the neocortical ventricular zone, vzg-1. They also express early neuronal but not glial markers, possess neuron-like processes without chemical synapses by ultrastructure, and appear pyramidal or bipolar with processes resembling apical dendrites, bifurcating and beaded axons, or growth cones. Immortalization appears to have occurred through the expression of binations SV40 large T with vras or SV40 large T with both vsrc and vmyc. These cell lines may represent developing neocortical neuroblasts blocked plete differentiation, and they should be useful in the isolation and analysis of genes involved in early neocortical development. |
8793866 | Tenascin-C inhibits oligodendrocyte precursor cell migration by both adhesion-dependent and adhesion-independent mechanisms. | Tenascin-C is present within the developing central nervous system during oligodendrocyte precursor cell migration. Tenascin-C is antiadhesive for oligodendrocytes, suggesting a role in controlling the migration of oligodendrocyte precursors and hence the pattern of myelination. Here we show directly that tenascin-C is a repulsive (or antiadhesive) substrate for primary oligodendrocyte precursors and also inhibits their migration. The antimigratory effect of tenascin-C on oligodendroglia is mediated through two distinct mechanisms; reduced substrate adhesion and a direct inhibition of cell migration that is independent of adhesion. These two effects map to different domains of the tenascin-C molecule. The repulsive effect maps to the EGF-like repeats and the alternatively spliced FN III repeats while the direct migration-inhibiting effect maps to FN III repeats 7-8. Our results show tenascin-C to have the novel property of inhibiting migration by both adhesion-dependent and adhesion-independent mechanisms, with different regions of the same molecule responsible for the two effects. |
8793867 | Phage presentation. | There has recently been great interest in the use of the filamentous bacteriophage fd as a vehicle for the display of peptides and proteins. Phage libraries displaying random peptides up to 38 amino acids in length can be used (i) to select for ligands able to bind specific target molecules; (ii) to mimic non-proteinaceous ligands; and (iii) as a tool to map epitopes recognized by antibodies. The display of proteins or their functional domains provides a system for the analysis of structure-function relationships, and the potential to generate proteins with altered binding characteristics or novel catalytic properties. The display of short immunogenic determinants on fusion phage may provide a basis for the development of novel peptide vaccines, whilst the expression of libraries of antibody fragments may provide a method to by-pass hybridoma technology in the generation of monoclonal antibodies. |
8793868 | The sodium-driven polar flagellar motor of marine Vibrio as the mechanosensor that regulates lateral flagellar expression. | Certain marine Vibrio species swim in sea water, propelled by a polar flagellum, and swarm over surfaces using numerous lateral flagella. The polar and the lateral flagellar motors are powered by sodium- and proton-motive forces, respectively. The lateral flagella are produced in media of high viscosity, and the relevant viscosity sensor is the polar flagellum. The cell might monitor either the rotation rate of the flagellar motor or the mechanical force applied against the flagellum. To test these possibilities, we examined the effects of amiloride and its derivatives, which inhibit the rotation of the sodium-driven motor, on lateral flagellar gene (laf) expression in Vibrio parahaemolyticus. Phenamil, an amiloride analogue that inhibits swimming at micromolar concentrations, induced laf transcription in media devoid of viscous agents in a dose-dependent manner. The relationship between the average swimming speed and laf induction in the presence of various concentrations of phenamil was very similar to that observed when viscosity was changed. These results indicate that marine Vibrio sense a decrease in the rotation rate of (or the sodium influx through) the polar flagellar motor as a trigger for laf induction. Alternative mechanisms for laf induction are also discussed. |
8793869 | Participation of the molecular chaperone DnaK in intracellular growth of Brucella suis within U937-derived phagocytes. | In the intracellular bacterium Brucella suis, the molecular chaperone DnaK was induced under heat-shock conditions and at low pH. Insertional inactivation of dnaK and dnaJ within the dnaK/J locus led to the conclusion that DnaK, but not DnaJ, was required for growth at 37 degrees C in vitro. Viability of the dnaK null mutant was also greatly affected at low pH. Under conditions allowing intracellular multiplication, the infection of U937-derived phagocytes resulted in long-lasting DnaK induction in the wild-type bacteria. In infection experiments performed with both mutants at the reduced temperature of 30 degrees C, the dnaK mutant of B. suis survived but failed to multiply within U937 cells, whereas the wild-type strain and the dnaJ mutant multiplied normally. Complementation of the dnaK mutant with the cloned dnaK gene restored growth at 37 degrees C, increased resistance to acid pH, and increased intracellular multiplication. This is the first report of the effects of dnaK inactivation in a pathogenic species, and of the temperature-independent contribution of DnaK to intracellular multiplication of the pathogen B. suis. |
8793870 | Alternate promoters direct stress-induced transcription of the Bacillus subtilis clpC operon. | clpC of Bacillus subtilis is part of an operon containing six genes. Northern blot analysis suggested that all genes are co-transcribed and encode stress-inducible proteins. Two promoters (PA and PB) were mapped upstream of the first gene. PA resembles promoters recognized by the vegetative RNA polymerase E sigma A. The other promoter (PB) was shown to be dependent on sigma B, the general stress sigma factor in B. subtilis, suggesting that clpC, a potential chaperone, is expressed in a sigma B-dependent manner. This is the first evidence that sigma B in B. subtilis is involved in controlling the expression of a gene whose counterpart, clpB, is subject to regulation by sigma 32 in Escherichia coli, indicating a new function of sigma B-dependent general stress proteins. PB deviated from the consensus sequence of sigma B promoters and was only slightly induced by starvation conditions. Nevertheless, strong induction by heat, ethanol, and salt stress occurred at the sigma B-dependent promoter, whereas the vegetative promoter was only weakly induced under these conditions. However, in a sigB mutant, the sigma A-like promoter became inducible by heat and ethanol pensating for sigB deficiency. Only the downstream sigma A-like promoter was induced by certain stress conditions such as hydrogen peroxide or puromycin. These results suggest that novel stress-induction mechanisms are acting at a vegetative promoter. Involvement of additional elements in this mode of induction are discussed. |
8793871 | A gene product related to Tral is required for the mobilization of Bacteroides mobilizable transposons and plasmids. | The antibiotic-resistance transposon Tn4555 from Bacteroides can be transferred between strains by conjugation. The transposon is not self-transmissible and must be mobilized by resident chromosomal tetracycline-resistance elements. In the present report, the mechanism of transfer was examined at the genetic level by deletion analysis and nucleotide sequencing of clones that conferred a transmissible phenotype on a non-mobilizable plasmid. The results suggested that the product of mobATn was required for mobilization and it worked in concert with a cis-acting oriT-like sequence. This mechanism pared with the mobilization system of a cryptic Bacteroides plasmid, pBI143, and the two systems were found to share mon transfer strategy. The mobA gene products from both genetic elements were related and they had limited homology to the broad group of mobilization proteins (relaxases) typified by Tral of RP4. Phylogenetic analysis of MobA and several other mobilization proteins mensal gastro-intestinal tract organisms suggested that they formed a new subgroup of the Tral superfamily. The mobilization regions of both Tn4555 and pBI143 were located on discrete segments of DNA within the parent genetic element. These segments were delineated by regions of secondary structure, suggesting that they could be defined mobilization cassettes. |
8793872 | Multiple signalling pathways trigger the exquisite sensitivity of yeast gluconeogenic mRNAs to glucose. | The transcription of the yeast FBP1 and PCK1 genes, which encode the gluconeogenic enzymes fructose-1,6-bisphosphatase and phosphoenolpyruvate carboxykinase, is repressed by glucose. Here, we show that this repression is both very strong and exceptionally sensitive to glucose, being triggered by glucose at concentrations less than 0.005% (0.27 mM). This repression remains operative in yeast mutants carrying any one of the three hexose kinases, but is lost in a triple hxk1, hxk2, glk1 mutant. In addition, 2-deoxyglucose can trigger the repression, but 6-deoxyglucose cannot, suggesting that internalization and phosphorylation of the glucose is essential for repression to occur. While gluconeogenic gene transcription is subject to the Mig 1p-dependent pathway of glucose repression, the exquisite response to glucose is maintained in hxk2 and mig1 mutants, suggesting that this pathway is not essential for the response. The response can also be triggered by the addition of exogenous cAMP, suggesting that the Ras/cAMP pathway can mediate repression of the FPB1 and PCK1 mRNAs. However, the response is not dependent upon this pathway because it remains intact in Ras, adenyl cyclase and protein kinase A mutants. The data show that yeast cells can detect very low glucose concentrations in the environment, and suggest that several distinct signalling pathways operate to repress FPB1 and PCK1 transcription in the presence of glucose. |
8793873 | RAG3 gene and transcriptional regulation of the pyruvate decarboxylase gene in Kluyveromyces lactis. | The RAG3 gene has been cloned from a Kluyveromyces lactis genomic library plementation of the rag3 mutation, which shows impaired fermentative growth on glucose in the presence of respiratory inhibitors. From the nucleotide sequence of the cloned DNA, which contained an open reading frame of 765 codons, the predicted protein is 49.5% identical to the Pdc2 protein of Saccharomyces cerevisiae, a regulator of pyruvate decarboxylase in this yeast. Measurement of the pyruvate decarboxylase activity in the original rag3-1 mutant and in the null mutant confirmed that the RAG3 gene is involved in pyruvate decarboxylase synthesis in K. lactis. The effect is exerted at the mRNA level of the pyruvate decarboxylase structural gene KIPDCA. Despite analogies between the RAG3 gene of K. lactis and the PDC2 gene of S. cerevisiae, these genes were unable to plement. |
8793874 | Ste50p sustains mating pheromone-induced signal transduction in the yeast Saccharomyces cerevisiae. | In the yeast Saccharomyces cerevisiae, the heterotrimeric G protein transduces the mating pheromone signal from a cell-surface receptor. Free G beta gamma then activates a mitogen-activated protein (MAP) kinase cascade. STE50 has been shown to be involved in this pheromone signal-transduction pathway. In this study, we present a functional characterization of Ste50p, a protein that is required to sustain the pheromone-induced signal which leads cells to hormone-induced differentiation. Inactivation of STE50 leads to the attenuation of mating pheromone-induced signal transduction, and overexpression of STE50 intensifies the pheromone-induced signalling. By genetic analysis we have positioned the action of Ste50p downstream of the alpha-pheromone receptor (STE2), at the level of the heterotrimeric G protein, and upstream of STE5 and the kinase cascade of STE11 and STE7. In a two-hybrid assay Ste50p interacts weakly with the G protein and strongly with the MAPKKK Ste11p. The latter interaction is absent in the constitutive mutant Ste11pP279S. These data show that a ponent, Ste50p, determines the extent and the duration of signal transduction by acting between the G protein and the MAP plex in S. cerevisiae. |
8793876 | Genetic organization and functional analysis of the otn DNA essential for cell-wall polysaccharide synthesis in Vibrio cholerae O139. | In 1992 a new Vibrio cholerae strain, designated V. cholerae O139 Bengal, emerged which has been responsible for large outbreaks of cholera in India and Bangladesh. Previously, we have shown that this strain arose from a V. cholerae O1 strain by the acquisition of novel DNA. Sequence analysis revealed that the novel DNA is flanked by two genes, rfaD and rfbQRS, which are also found in O1 strains. The mosaic structure of rfaDVCO139 indicated that it was one of the regions involved in bination between donor and acceptor DNA. However, sequence divergence between the O1 and O139 rfbQRS genes indicated that the second bination site between donor and O1-acceptor DNA is probably located downstream of rfbDVCO139. The DNA region between rfaDVCO139 and rfbQRSVCO139, designated otn, contained seven open reading frames (ORFs). Two ORFs, otnA and otnB, showed homology with genes involved in cell-wall polysaccharide synthesis. Mutations in otnA and otnB indicated that they are required for capsule synthesis but not lipopolysaccharide synthesis. The otn DNA is also found in V. cholerae O69 and O141 strains, and the organization of this DNA was essentially identical to that in the O139 strain. However, sequence divergence of the otnAB genes indicated that the O139 otn DNA region was not derived from the O69 or O141 strains. No antigenic relationship was found between the different V. cholerae serotypes carrying otn DNA, so the genes determining the antigenic specificity of the O antigen or capsule must be located outside the otn DNA. The O139 otn DNA contained a JUMPstart sequence, which is associated with polysaccharide-synthetic genes in several bacterial species. Furthermore, a repeat motif was observed in extragenic regions. A number of observations suggest that these sequences may facilitate gene flow between V. cholerae strains and the assembly of clusters of functionally related genes. |
8793875 | A single substitution in the putative helix-turn-helix motif of the pleiotropic activator PrfA attenuates Listeria monocytogenes virulence. | PrfA, the regulator of virulence-gene expression in the pathogenic bacterium Listeria monocytogenes, displays sequence similarity to members of the CAP-FNR family of transcriptional regulators. To test the functional significance of this similarity, we constructed and analysed substitutions of two amino acids of PrfA predicted to contact DNA, i.e. Ser-184 and Ser-183. Substitution of Ser-184 by Ala reduced DNA binding and virulence-gene activation, and attenuated the virulence in a mouse model of infection. In contrast, substitution of Ser-183 by Ala had the opposite effect in these functional assays. A 17bp DNA sequence, which includes a putative PrfA site, was shown to be sufficient for target-site recognition by PrfA and PrfA-S183A. Our results strongly support the hypothesis that PrfA is a structural and functional homologue of CAP. In addition, they establish a clear correlation between DNA binding by PrfA, virulence-gene activation, and virulence. |
8793877 | Independent interaction of the acyltransferase HlyC with two maturation domains of the Escherichia coli toxin HlyA. | The apparently unique fatty acylation mechanism that underlies activation (maturation) of Escherichia coli haemolysin and related toxins is further clarified by investigation of the interaction of protoxin with the specific acyltransferase HlyC. Using deleted protoxin variants and protoxin peptides as substrates in an in vitro maturation reaction dependent upon HlyC and acyl-acyl carrier protein, two independent HlyC recognition domains were identified on the 1024-residue protoxin, proA, and they were shown to span the two target lysine residues K564 (KI) and K690 (KII) that are fatty acylated. Each domain required 15-30 amino acids for basal recognition and 50-80 amino acids for wild-type acylation. The two domains (FAI and peted with each other in cis and in trans for HlyC. The affinity of FAI for HlyC is approximately four times greater than that of FAII resulting in an overall 80% acylation at KI and 20% acylation at KII in both whole toxin and peptide derivatives. No other proA sequences were required for toxin maturation, and excess Ca2+ prevented acylation of both lysines. The lack of primary sequence identity between FAI and FAII domains in proA and among corresponding sites on related protoxins currently precludes an explanation of the basis of HlyC recognition by proA. |
8793878 | Protein engineering studies of A-chain loop 47-56 of Escherichia coli heat-labile enterotoxin point to a prominent role of this loop for cytotoxicity. | Heat-labile enterotoxin (LT), produced by enterotoxigenic Escherichia coli, is a close relative of cholera toxin (CT). These two toxins share approximately 80% sequence identity, and consists of one 240-residue A chain and five 103-residue B subunits. The B pentamer is responsible for GM1 receptor recognition, whereas the A subunit carries out an ADP-ribosylation of an arginine residue in the G protein, Gs alpha, in the epithelial target cell. This paper explores the importance of specific amino acids in loop 47-56 of the A subunit. This loop was observed to be highly mobile in the inactive R7K mutant of the A subunit. The position of the loop in wild-type protein is such that it might require considerable reorganization during substrate binding and is likely to have a crucial role in substrate binding. Five single-site substitutions have been made in the LT-A subunit 47-56 loop to investigate its possible role in the enzymatic activity and toxicity of LT and CT. The wild-type residues Thr-50 and Val-53 were replaced either by a glycine or by a proline. The glycine substitutions were intended to increase the mobility of this active-site loop, and the proline substitutions were intended to decrease the mobility of this same loop by restricting the accessible conformational space. Under the hypothesis that mobility of the loop is important for catalysis, the glycine-substitution mutants T50G and V53G would be expected to exhibit activity equal to or greater than that of the wild-type A subunit, while the proline substitution mutants T50P and T53P would be less active. Cytotoxicity assays showed, however, that all four of these mutants were considerably less active than wild-type LT. These results lend support for assignment of a prominent role to loop 47-56 in catalysis by LT and CT. |
8793879 | Variability of gene order in different Helicobacter pylori strains contributes to genome diversity. | Considerable genomic microdiversity has been reported previously among Helicobacter pylori isolates. We have constructed genome maps of four unrelated H. pylori strains (NCTC11637, NCTC11639, UA802 and UA861) using pulsed-field gel electrophoresis (PFGE) with NotI and NruI, hybridization with extracted PFGE DNA fragments and probing with 17 gene probes. These strains of H. pylori pared with a fifth unrelated H. pylori strain NCTC11638 mapped previously. Considerable diversity in gene arrangement was evident among the five H. pylori maps, and no consistent gene clustering was found. The association of only four genes, katA (catalase gene), vacA (vacuolating cytotoxin gene), hpaA (a putative adhesin gene), and pfr (bacterial ferritin gene) were generally conserved within approximately the same 25% of the genome; however, the order of these genes also varied. Our study demonstrates that macrodiversity, i.e. variability in gene order, in addition to microdiversity, is a characteristic of the H. pylori genome. |
8793880 | Interaction of CodY, a novel Bacillus subtilis DNA-binding protein, with the dpp promoter region. | The product of the codY gene is required for nutritional repression of the Bacillus subtillis dipeptide permease operon (dpp), an operon expressed at early stationary phase in nutrient-rich medium. Though unrelated to any known DNA-binding protein, CodY was shown to bind specifically to the dpp promoter region. DNase I footprinting experiments revealed that the CodY-protected region passes the dpp transcription start site and overlaps with the region protected by another regulatory protein, AbrB. CodY and AbrB were found pete, in vitro, for binding to the dpp promoter region. Binding of CodY was altered in mutants defective in nutritional regulation. |
8793881 | Characterization of an Escherichia coli mutant, feeA, displaying resistance to the calmodulin inhibitor 48/80 and reduced expression of the rare tRNA3Leu. | We previously described a mutation feeB1 conferring a temperature-sensitive filamentation phenotype and resistance to the calmodulin inhibitor 48/80 in Escherichia coli, which constitutes a single base change in the acceptor stem of the rare tRNA3Leu recognizing CUA codons. We now describe a second mutant, feeA1, unlinked to feeB, but displaying a similar phenotype, 48/80 resistance and a reduced growth rate at the permissive temperature, 30 degrees C, and temperature-sensitive, forming short filaments at 42 degrees C. In the feeA mutant, tRNA3Leu expression (but not that of tRNA1Leu) was reduced approximately fivefold relative to the wild type. We previously showed that the synthesis of beta-galactosidase, which unusually requires the translation of 6-CUA codons, was substantially reduced, particularly at 42 degrees C, in feeB mutants. The feeA mutant also shows drastically reduced synthesis of beta-galactosidase at the non-permissive temperature and reduced levels even at the permissive temperature. We also show that increased copy numbers of the abundant tRNA1Leu, which can also read CUA codons at low efficiency, suppressed the effects of feeA1 under some conditions, providing further evidence that the mutant was deficient in CUA translation. This, and the previous study, demonstrates that mutations which either reduce the activity of tRNA3Leu or the cellular amount of tRNA3Leu confer resistance to the drug 48/80, with itant inhibition of cell division at 42 degrees C. |
8793882 | Phase variation in Francisella tularensis affecting intracellular growth, lipopolysaccharide antigenicity and nitric oxide production. | Many microbial pathogens, such as Mycobacterium spp. and Salmonella spp., use macrophage intracellular growth or antigenic variation as mechanisms for avoiding the host immune system. In this work we present evidence to show that the intracellular pathogen Francisella tularensis uses phase variation to alter antigenicity and the host macrophage nitric oxide response simultaneously, thereby modulating its intracellular growth. The lipopolysaccharide (LPS) and lipid A of F. tularensis fails to stimulate production of significant levels of nitric oxide (NO) by rat macrophages. However, spontaneous variants of F. tularensis expressing an antigenically distinct LPS induce rat macrophages to produce increased levels of NO, thereby suppressing microbial intramacrophage growth. Similarly, lipid A isolated from these variants stimulates increased levels of NO production. A reverse phase shift can occur, which returns the LPS to the original antigenic form, reduces NO production, and restores intramacrophage growth. These findings represent the first demonstration of a phase-variation phenomenon which modulates intracellular growth and an innate immune response. Furthermore, these results suggest that a microbial pathogen can exploit macrophage NO production for its own benefit, perhaps by prolonging the host-pathogen association during the acute phase of disease or during the process of establishing a carrier state. |
8793883 | Involvement of the narJ and mob gene products in distinct steps in the biosynthesis of the molybdoenzyme nitrate reductase in Escherichia coli. | The Escherichia coli mob locus is required for synthesis of active molybdenum cofactor, molybdopterin guanine dinucleotide. The mobB gene is not essential for molybdenum cofactor biosynthesis because a deletion of both mob genes can be plemented by just mobA. Inactive nitrate reductase, purified from a mob strain, can be activated in vitro by incubation with protein FA (the mobA gene product), GTP, MgCl2, and a further protein fraction, factor X. Factor X activity is present in strains that lack MobB, indicating that it is not an ponent of factor X, but over-expression of MobB increases the level of factor X. MobB, therefore, can participate in nitrate reductase activation. The narJ protein is not ponent of mature nitrate reductase but narJ mutants cannot express active nitrate reductase A. Extracts from narJ strains are unable to support the in vitro activation of purified mob nitrate reductase: they lack factor X activity. Although the mob gene products are necessary for the biosynthesis of all E. coli molybdoenzymes as a result of their requirement for molybdopterin guanine dinucleotide, NarJ action is specific for nitrate reductase A. The inactive nitrate reductase A derivative in a narJ strain can be activated in vitro following incubation with cell extracts containing the narJ protein. NarJ acts to activate nitrate reductase after molybdenum cofactor biosynthesis plete. |