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Biomedical subjects

J Silver

Publications and source records attributed to J Silver.

At least 37 records · Page 2Linked to original sources
PubMed2000

Dynein light chain binding to a 3'-untranslated sequence mediates parathyroid hormone mRNA association with microtubules.

The 3'-untranslated region (UTR) of mRNAs binds proteins that determine mRNA stability and localization. The 3'-UTR of parathyroid hormone (PTH) mRNA specifically binds cytoplasmic proteins. We screened an expression library for proteins that bind the PTH mRNA 3'-UTR, and the sequence of 1 clone was identical to that of the dynein light chain LC8, a component of the dynein complexes that translocate cytoplasmic components along microtubules. Recombinant LC8 binds PTH mRNA 3'-UTR, as shown by RNA electrophoretic mobility shift assay. We showed that PTH mRNA colocalizes with microtubules in the parathyroid gland, as well as with a purified microtubule preparation from calf brain, and that this association was mediated by LC8. To our knowledge, this is the first report of a dynein complex protein binding an mRNA. The dynein complex may be the motor that is responsible for transporting mRNAs to specific locations in the cytoplasm and for the consequent is asymmetric distribution of translated proteins in the cell.

3' Untranslated Regions↗
PubMed2000

The adhesion signaling molecule p190 RhoGAP is required for morphogenetic processes in neural development.

Rho GTPases direct actin rearrangements in response to a variety of extracellular signals. P190 RhoGAP (GTPase activating protein) is a potent Rho regulator that mediates integrin-dependent adhesion signaling in cultured cells. We have determined that p190 RhoGAP is specifically expressed at high levels throughout the developing nervous system. Mice lacking functional p190 RhoGAP exhibit several defects in neural development that are reminiscent of those described in mice lacking certain mediators of neural cell adhesion. The defects reflect aberrant tissue morphogenesis and include abnormalities in forebrain hemisphere fusion, ventricle shape, optic cup formation, neural tube closure, and layering of the cerebral cortex. In cells of the neural tube floor plate of p190 RhoGAP mutant mice, polymerized actin accumulates excessively, suggesting a role for p190 RhoGAP in the regulation of +Rho-mediated actin assembly within the neuroepithelium. Significantly, several of the observed tissue fusion defects seen in the mutant mice are also found in mice lacking MARCKS, the major substrate of protein kinase C (PKC), and we have found that p190 RhoGAP is also a PKC substrate in vivo. Upon either direct activation of PKC or in response to integrin engagement, p190 RhoGAP is rapidly translocated to regions of membrane ruffling, where it colocalizes with polymerized actin. Together, these results suggest that upon activation of neural adhesion molecules, the action of PKC and p190 RhoGAP leads to a modulation of Rho GTPase activity to direct several actin-dependent morphogenetic processes required for normal neural development.

Alleles↗
PubMed1999

Controlled Formation of Low-Volume Liquid Pillars between Plates with a Lattice of Wetting Patches by Use of a Second Immiscible Fluid.

We describe a method for forming an array of microdroplets between two plates, at least one of which is patterned with a lattice of wetting patches, using a second immiscible fluid to control droplet formation. The method may be useful for performing multiple, small-volume biochemical reactions in parallel. We analyze the forces responsible for droplet formation, describe results of a computer simulation using Surface Evolver, and derive an analytic criterion for droplet formation in terms of the contact angles of the droplet:second fluid interface on the wetting patches and surrounding surface, the diameter of the wetting patches, the distance between wetting patches, and the distance between the plates. Copyright 1999 Academic Press.

Journal Article↗
PubMed1999

Cellular and molecular mechanisms of glial scarring and progressive cavitation: in vivo and in vitro analysis of inflammation-induced secondary injury after CNS trauma.

Post-traumatic cystic cavitation, in which the size and severity of a CNS injury progress from a small area of direct trauma to a greatly enlarged secondary injury surrounded by glial scar tissue, is a poorly understood complication of damage to the brain and spinal cord. Using minimally invasive techniques to avoid primary physical injury, this study demonstrates in vivo that inflammatory processes alone initiate a cascade of secondary tissue damage, progressive cavitation, and glial scarring in the CNS. An in vitro model allowed us to test the hypothesis that specific molecules that stimulate macrophage inflammatory activation are an important step in initiating secondary neuropathology. Time-lapse video analyses of inflammation-induced cavitation in our in vitro model revealed that this process occurs primarily via a previously undescribed cellular mechanism involving dramatic astrocyte morphological changes and rapid migration. The physical process of cavitation leads to astrocyte abandonment of neuronal processes, neurite stretching, and secondary injury. The macrophage mannose receptor and the complement receptor type 3 beta2-integrin are implicated in the cascade that induces cavity and scar formation. We also demonstrate that anti-inflammatory agents modulating transcription via the nuclear hormone receptor peroxisome proliferator-activated receptor-gamma may be therapeutic in preventing progressive cavitation by limiting inflammation and subsequent secondary damage after CNS injury.

Animals↗
PubMed1999

Protein-RNA interactions determine the stability of the renal NaPi-2 cotransporter mRNA and its translation in hypophosphatemic rats.

Hypophosphatemia leads to an increase in type II Na(+)-dependent inorganic phosphate cotransporter (NaPi-2) mRNA and protein levels in the kidney and increases renal phosphate reabsorption. Nuclear transcript run-on experiments showed that the effect of a low phosphate diet was post-transcriptional. In an in vitro degradation assay, renal proteins from hypophosphatemic rats stabilized the NaPi-2 transcript 6-fold compared with control rats and this was dependent upon an intact NaPi-2 3'-untranslated region (UTR). To determine an effect of hypophosphatemia upon NaPi-2 protein synthesis, the incorporation of injected [(35)S]methionine into renal proteins was studied in vivo. Hypophosphatemia led to increased [(35)S]methionine incorporation only into NaPi-2 protein. The effect of hypophosphatemia on translation was studied in an in vitro translation assay, where hypophosphatemic renal proteins led to increased translation of NaPi-2 and other transcripts. NaPi-2 RNA interaction with cytosolic proteins was studied by UV cross-linking and Northwestern gels. Hypophosphatemic proteins led to increased binding of renal cytosolic proteins to the 5'-UTR of NaPi-2 mRNA. Therefore, hypophosphatemia increases NaPi-2 gene expression post-transcriptionally, which correlates with a more stable transcript mediated by the 3'-UTR, and an increase in NaPi-2 translation involving protein binding to the 5'-UTR. These findings show that phosphate regulates gene expression by affecting protein-RNA interactions in vivo.

3' Untranslated Regions↗
PubMed1999

Robust regeneration of adult sensory axons in degenerating white matter of the adult rat spinal cord.

We have recently reported that minimally disturbed adult CNS white matter can support regeneration of adult axons by using a novel microtransplantation technique to inject minute volumes of dissociated adult rat dorsal root ganglion neurons directly into adult rat CNS pathways (Davies et al., 1997). This atraumatic injection procedure minimized scarring and allowed considerable numbers of regenerating adult axons immediate access to the adult CNS glial terrain where they rapidly extended for long distances. A critical question remained as to whether degenerating white matter at acute and chronic stages (up to 3 months) after injury could still support regeneration. To investigate this, we have microtransplanted adult sensory neurons into degenerating white matter of the adult rat spinal cord several millimeters rostral to a severe lesion of the dorsal columns. Regeneration of donor sensory axons in both directions away from the site of transplantation was robust even within white matter undergoing fulminant Wallerian degeneration despite intimate contact with myelin. Along their route, the regrowing axons extended large numbers of collaterals into the adjacent dorsal horn. However, after entering the lesion, the rapidly extending growth cones stopped and became dystrophic within high concentrations of reactive glial matrix. Our results offer compelling evidence that the major environmental impediment to regeneration in the adult CNS is the molecular barrier that forms directly at the lesion site, and that degenerating white matter beyond the glial scar has a far greater intrinsic ability to support axon regeneration than previously thought possible.

Animals↗
PubMed1999

Positive selection of natural autoreactive B cells.

Lymphocyte development is critically influenced by self-antigens. T cells are subject to both positive and negative selection, depending on their degree of self-reactivity. Although B cells are subject to negative selection, it has been difficult to test whether self-antigen plays any positive role in B cell development. A murine model system of naturally generated autoreactive B cells with a germ line gene-encoded specificity for the Thy-1 (CD90) glycoprotein was developed, in which the presence of self-antigen promotes B cell accumulation and serum autoantibody secretion. Thus, B cells can be subject to positive selection, generated, and maintained on the basis of their autoreactivity.

Aging↗
PubMed1999

Glial fibrillary acidic protein is necessary for mature astrocytes to react to beta-amyloid.

Upregulation of the glial fibrillary acidic protein (GFAP) in astrocytes is a hallmark of the phenomenon known as reactive gliosis and, yet, the function of GFAP in this process is largely unknown. Our previous studies have shown that mature astrocytes react vigorously to substrate bound beta-amyloid protein (BAP) in a variety of ways (i.e., increased GFAP, enhanced motility, unusual aggregation patterns, inhibitory ECM production). In order to uncover which, if any, of these phenomena are causally related to the function of GFAP, primary cortical astrocytes from transgenic mice lacking GFAP were cultured on BAP substrates at low or high density and at various lengths of time following in vitro maturation. Differences between mutant and control cells became progressively more obvious when cells were matured in vitro for two weeks or longer and especially in cultures that were at high density. Mature control astrocytes show a dramatic response to BAP by aggregating into a meshwork of rope-like structures that completely bridge over the peptide surface. In marked contrast, mature GFAP-null astrocytes initiate the response much more slowly and had a much reduced ability to aggregate tightly. Furthermore, we prepared hippocampal slice cultures from GFAP-/- and GFAP+/+ mice and compared their astrocytic responses to injected BAP. GFAP-/- astrocytes of hippocampal slice cultures failed to form a barrier-like structure around the edge of the BAP deposit as did GFAP+/+ astrocytes. Our data suggest that GFAP may be essential for mature astrocytes to constrain certain types of highly inflammatory lesions in the brain.

Amyloid beta-Peptides↗
PubMed1999

Reduced bacterial dissemination and liver injury in CD14-deficient mice following a chronic abscess-forming peritonitis induced by Bacteroides fragilis.

The CD14 myelomonocytic differentiation antigen plays a major role in acute Gram-negative infections with Escherichia coli; however, its role in chronic infections has not yet been analyzed. To address this question, we studied the role of CD14 in a chronic abscess-forming peritonitis, induced by Bacteroides fragilis. B. fragilis (3x10(8) CFU/ml) were resuspended in a liquid nutrient agar and injected into the peritoneal cavity of CD14-deficient (CD 14-/-) and normal C57BL/6J (CD 14+/+) mice, respectively. After 3 days there was a severe phlegmonous intra-abdominal inflammation in both groups. After 7 days an abscess-forming peritonitis developed and by 14 days the infectious foci were compartimentalized. These observations were indistinguishable between CD14-/- and CD14+/+ mice. Although no differences were seen in abscess formation, CD14-/- mice were able to clear B. fragilis more efficiently from the blood than CD14+/+ mice. After 3, 7, and 14 days blood cultures were B. fragilis positive in 11% (1/9), 20% (2/10), and 0% (0/9) in CD14-/-compared with 90% (9/10), 78% (7/9), and 20% (2/10) in CD14+/+ mice, respectively (P<0.05). Furthermore, although the infection resulted in hepatocellular necrosis and severe hepatitis in both groups, at day 14 the liver cell damage was more severe in CD14+/+ than in CD14-/- mice (P<0.05). These results show that the chronic abscess formation induced by B. fragilis capsular polysaccharides is CD14 independent; however, bacterial clearance and/or dissemination and liver cell damage are at least partially influenced by CD14-dependent mechanisms.

Abscess↗
PubMed1999

Paediatric community vision screening--a new model.

The purpose of this study was to establish if a community based model using a Hospital Optometrist and Community Orthoptist can provide a practical secondary vision screening service for children. These professionals working in an Inner London Health Centre, assessed children who had failed primary vision screening. In total 483 new patients were seen between April 1994 and March 1996 with the largest referral source being the school nurse screening programme. The majority were managed by the team with a total onward referral rate to the Hospital Eye Service of 14%. In 78% of these cases the consultant's diagnosis agreed with the reason for referral. Where the consultant's diagnosis differed the children were identified as normal or a variant of normal. This model of care provides a 'one stop service' where a child identified as having a potential visual problem at primary screening can be assessed, refracted and provided with spectacles in a local setting without hospital referral. Referrals to the Hospital Eye Service are considerably reduced and a convenient service is provided for parents and children.

Age Distribution↗
PubMed1999

Regulation of the parathyroid hormone gene by vitamin D, calcium and phosphate.

Secondary hyperparathyroidism is a frequent complication of chronic renal failure resulting in severe bone disease. Secondary hyperparathyroidism is composed of increased in parathyroid hormone (PTH) synthesis and secretion due to an increase in PTH gene expression and parathyroid cell proliferation. PTH gene expression is regulated by calcium, phosphate and 1,25-dihydroxy vitamin D (1,25(OH)2D). 1,25(OH)2D3 injected to rats leads to a dramatic decrease in PTH gene transcription without any increase in serum calcium. Hypocalcemia leads to a large increase in PTH mRNA levels which is post-transcriptional. Hypophosphatemia leads to a marked decrease in PTH gene expression that is also post-transcriptional. The mechanisms of the post-transcriptional effects of calcium and phosphate on the PTH gene have shown to be due to changes in protein-RNA interactions at the PTH mRNA 3'-UTR. Hypocalcemia leads to increased binding of parathyroid cytosolic proteins to the PTH mRNA 3'-UTR and hypophosphatemia to decreased binding of these proteins to the PTH mRNA 3'-UTR. The binding of the parathyroid proteins stabilizes the PTH RNA in an in vitro degradation assay. In rats with experimental uremia due to 5/6 nephrectomy, there is an increase in PTH mRNA levels due to a decrease in degradation of the PTH RNA as determined by this assay. The characterization of the parathyroid cytosolic proteins that interact with the PTH mRNA 3'-UTR may lead to a clearer understanding of how changes in serum calcium and phosphate result in secondary hyperparathyroidism.

3' Untranslated Regions↗
PubMed1999

Vitamin D and the parathyroid.

Vitamin D's biologically active metabolite, 1,25(OH)2D3, has important effects upon the parathyroid cell that are relevant to both the physiology of mineral metabolism and the regulation of the secondary hyperparathyroidism of chronic renal failure. 1,25(OH)2D3 markedly decreases parathyroid hormone (PTH) gene transcription and thus PTH synthesis and secretion. It also acts to decrease parathyroid cell proliferation. Nonhypercalemic analogs of 1,25(OH)2D3 are being developed that may have a wider therapeutic window than 1,25(OH)2D3 itself. In the situations of chronic hypocalcemia and hypophosphatemia, there are interesting interrelationships between 1,25(OH)2D3 and the post-transcriptional regulation of the PTH gene. In nodular secondary hyperparathyroidism, there is down-regulation of the vitamin D receptor in the parathyroid. Different vitamin D receptor genotypes may be associated with higher levels of serum PTH and a predisposition to autonomous hyperplasia.

Cell Division↗
PubMed1999

Mechanism of increased parathyroid hormone mRNA in experimental uremia: roles of protein RNA binding and RNA degradation.

Patients with chronic renal failure develop secondary hyperparathyroidism with increased synthesis and secretion of parathyroid hormone (PTH) resulting in severe skeletal complications. In rats with secondary hyperparathyroidism due to 5/6 nephrectomy, there are increased PTH mRNA levels, and this mechanism was studied. Parathyroid glands were microdissected from control and 5/6 nephrectomy rats and analyzed for PTH mRNA and control genes, and the nuclei were used for nuclear run-on experiments. The cytosolic proteins of the parathyroids were used to study PTH mRNA protein binding by ultraviolet cross-linking and the degradation of the PTH transcript in vitro. Nuclear run-ons showed that the increase in PTH mRNA levels was posttranscriptional. Protein binding to the PTH mRNA 3'-UTR determines PTH mRNA stability and levels. Parathyroid proteins from uremic rats bound PTH mRNA similar to control rats by ultraviolet cross-linking. To determine the effect of uremia on PTH mRNA stability, an in vitro RNA degradation assay was performed with parathyroid proteins from uremic rats. When parathyroid proteins from control rats were incubated with PTH mRNA, there was transcript degradation already at 30 min, reaching 50% at 60 min and 90% at 180 min. With uremic parathyroid proteins, the PTH mRNA was not degraded at all at 120 min and was moderately decreased at 180 min. This decrease in degradation by uremic parathyroid proteins suggests a decrease in parathyroid cytosolic endonuclease activity in uremia resulting in a more stable PTH transcript. The increased PTH mRNA levels would translate into increased PTH synthesis and serum PTH levels, which would lead to metabolic bone disease in many patients with chronic renal failure.

3' Untranslated Regions↗