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Biomedical subjects

J Silver

Publications and source records attributed to J Silver.

At least 199 records · Page 11Linked to original sources
PubMed1993

Influence of HLA genes on T cell receptor V segment frequencies and expression levels in peripheral blood lymphocytes.

The effect of the HLA complex on the TCR repertoire in human peripheral blood was assessed by using nine V beta- and V alpha-specific mAb and the quantitative polymerase chain reaction specific for 22 V beta segments. Studies in randomly selected and unrelated individuals failed to show any influence of the HLA complex on the TCR repertoire. In contrast, studies in large families with multiple siblings showed a strong influence on the TCR repertoire by the HLA complex. In pairwise comparisons, HLA-identical sibs had more similar patterns of V segment frequencies, as measured with the nine V segment-specific mAb, as well as more similar expression levels of V beta-specific RNA, as measured by quantitative polymerase chain reaction, than totally mismatched or haplo-identical sibs. When the amount of V beta-specific RNA expressed in CD4+ and CD8+ T cells was compared, it was found that V beta 2, 5.1, 9, and 20 were skewed toward CD4+ T cells; on the other hand, V beta 7 and 14 showed a bias in expression for CD8+ T cells, suggesting that the former were positively selected predominantly by HLA class II gene products whereas the latter V beta segments were positively selected predominantly by HLA class I gene products. These studies unequivocally document the effects of HLA genes on TCR V segment frequencies and expression levels in peripheral blood T lymphocytes.

Antibodies, Monoclonal↗
PubMed1993

Immunocytochemical demonstration of early appearing astroglial structures that form boundaries and pathways along axon tracts in the fetal brain.

During normal development of the mammalian forebrain, the paired cerebral hemispheres are initially separated midsagittally by the connective tissue-filled longitudinal fissure. During subsequent stages, the hemispheres fuse as basal lamina is remodeled and fibroblasts are eliminated from the fissure to create new central nervous system (CNS) territory in the midline. Two axon pathways, the corpus callosum and dorsal callosal stria, eventually use this region as part of their pathway. In order to assess the possible role of glial cells in the fusion process and in the guidance of axons in this and several other areas of the forebrain, we have analyzed the developing brain in timed cat and mouse embryos with immunohistochemical and morphological techniques. With the use of astroglial-specific antibodies and electron microscopy, we have visualized two distinct, primitive astroglial structures associated with the cerebral midline, and seven more associated with other specific brain regions. The way in which one of these structures moves as a column along the hemispheric midline in synchrony with seam formation suggests the possibility that during morphogenesis of the telencephalon, astrocytes may aid in the fusion process. In addition, the compact assemblage, early appearance and location of this and the other glial structures in relation to well defined neuroanatomical landmarks or axon pathways suggest that they may transiently compartmentalize relatively large regions of the CNS and organize certain developing fiber systems by acting as guides or barriers at critical stages of ontogeny.

Animals↗
PubMed1993

Adhesion characteristics of chondrocytes cultured separately and in co-cultures with synovial fibroblasts.

The present study was designed to investigate the adherence mechanism(s) and behaviour of cultured chondrocytes under various culturing conditions, co-culturing with fibroblasts, or growth in the presence of conditioned medium either of fibroblasts or chondrocytes. The findings obtained indicate that chondrocyte time-adhesion curves and the final percentiles of attached cells to a plastic substrate are much slower and lower respectively than those of anchorage dependent cell types. The poorest adhesion occurs employing chondrocytes originated from suspension cultures, as compared to chondrocytes grown in monolayers. No interference with chondrocyte adhesion was found by inhibiting the production of proteoglycan (PG). Puromycin and to a lesser degree actinomycin but not cytosine arabinoside interfered with chondrocyte adhesion, suggesting the importance of protein synthesis in this process. The nature of proadhesion modifying molecules in synoviocytes conditioned media and antiadhesive agents in chondrocyte conditioned media suggests that both substances are heat labile, non-dialyzable, protein containing factors.

Animals↗
PubMed1993

Putative inhibitory extracellular matrix molecules at the dorsal root entry zone of the spinal cord during development and after root and sciatic nerve lesions.

The dorsal root entry zone (DREZ) of the spinal cord is the interface between the central and peripheral nervous systems and is the pathway through which sensory afferents enter the central nervous system during development. However, in the rat, the DREZ becomes a boundary to regenerating sensory axons after Postnatal Days 2-3. The cellular and molecular mechanisms that cause regenerative failure at the DREZ after the critical period for regeneration are unknown. Recent studies demonstrate that two extracellular matrix molecules, Cytotactin/tenascin (CT) and chondroitin 6-sulfate-containing proteoglycans (C-6S-PG) are present in normal boundary regions of the brain and spinal cord during development. In the present study we sought to visualize the expression of these two putative inhibitory molecules in the DREZ of normally developing and adult animals, and also in animals after injury. CT and C-6S-PG spread laterally from the midline to the DREZ by Postnatal Day 3, correlating exactly with the end of the critical period. The staining intensity for these two molecules increases further in the DREZ after root lesions, but not sciatic lesions, at ages when axons cannot regenerate into the spinal cord. Following root lesion CT and C-6S-PG were mostly present in association with reactive glia at the DREZ and in white matter, rather than with reactive glia in grey matter of the dorsal horn, suggesting that astroglia are heterogeneous in their response to root lesion. The coexpression of CT and C-6S-PG may create a molecular barrier which might channel or deflect axons at the DREZ during CNS development and inhibit their growth during regeneration.

Aging↗
PubMed1993

Chondroitin sulfate proteoglycans are associated with the lesions of Alzheimer's disease.

Chondroitin sulfate proteoglycans (CSPG) are extracellular matrix proteins inhibitory to neurite outgrowth in vitro and correlated with decreased neurite outgrowth after CNS injury. Previously, heparan sulfate proteoglycan and dermatan sulfate proteoglycan have been shown to be associated with senile plaques (SPs) and neurofibrillary tangles (NFTs) but CSPG was not. In an immunocytochemical study, three monoclonal antibodies to different sulfation states of the chondroitin glycosaminoglycan were used to localize CSPG in cases of Alzheimer's disease. Chondroitin 4-sulfate was found in both SPs and NFTs. An antibody to unsulfated chondroitin strongly immunostained intracellular NFTs and the dystrophic neurites of SPs. Chondroitin 6-sulfate was found in NFTs and the area around SPs. These results suggest that CSPG, in addition or as an alternative to beta-amyloid protein, could be responsible for the regression of neurites around senile plaques in Alzheimer's disease.

Aged↗
PubMed1993

Role of astroglial extracellular matrix in the formation of rat olfactory bulb glomeruli.

We describe the development of the olfactory bulb (OB) glomeruli, focusing on the role of purported astroglial-associated boundary molecules, as potential guidance factors in glomeruli formation. Glomeruli are unusual structures within the OB formed by extremely tight bundles of afferent fibers that fail to grow deeply into the brain. Using olfactory marker protein antibodies, we found that olfactory axons of stage E17-E18 rat embryos were organized into long strands at the outer-most edge of the bulb. By E20-E21 the axons clustered and formed small spheres and by PD1 they gathered into distinct glomeruli. Prior to and during the transformation of olfactory afferents into glomeruli, there was an enhanced expression of cytotactin/tenascin (CT) and chondroitin sulfate-containing proteoglycan (CS-PG) associated with VIM/GFAP-positive astroglial cell processes at the base of the glomeruli. During later stages of development (PD3-PD9), the GFAP-positive astroglial processes beneath and within the base of the glomeruli showed intensified GFAP staining while maintaining expression of CT and CS-PG. Some astroglial processes had extended and others went even further peripherally into the olfactory nerve layer (ONL) to encapsulate and fully infiltrate the glomeruli. By PD9, CS-PG and CT levels increased inside and outside the glomeruli. However, by PD15 both molecules had decreased inside the glomeruli but remained highly concentrated around the glomeruli to form rings. Thus, at later stages, axon/glia interactions which result in astroglial rearrangement and matrix alterations in relation to the glomeruli occur. Western blots of stage E20 bulb proteins revealed the presence of three proteoglycan core proteins each containing chondroitin-6-sulfate moieties. In addition, a type of keratan sulfate (KS) glycosaminoglycan was present in a core protein not associated with chondroitin-6-sulfate. Our data suggest that astroglial-associated ECM molecules CT and CS-PG, and perhaps KS-PG, present in the core of the early developing OB, could form a molecular "wall" that helps confine ingrowing olfactory axons within the ONL at the outer edge of the astroglial territory. We suggest that the astroglia do not precisely regulate the specific shape of the glomeruli, but rather play a fundamental role in directing the gross position at which formation of the synaptic glomeruli will occur.

Aging↗
PubMed1993

beta-Amyloid of Alzheimer's disease induces reactive gliosis that inhibits axonal outgrowth.

Pathological lesions in the brains of patients with Alzheimer's disease (AD) are characterized by dense deposits of the protein beta-amyloid. The link between the deposition of beta-amyloid in senile plaques and AD-associated pathology is, at present, controversial since there have been conflicting reports on whether the 39-43 amino acid beta-amyloid sequence is toxic or trophic to neurons. In this report, we show that beta-amyloid peptide when presented as an insoluble substrate which mimics its conformation in vivo can induce cortical glial cells in vitro and in vivo to locally deposit chondroitin sulfate containing proteoglycan. In vitro the proteoglycan-containing matrix deposited by glia on beta-amyloid blocks the usual ability of the peptide to allow cortical neurons to adhere and grow. Chondroitin sulfate-containing proteoglycan was also found in senile plaques of human AD tissue. We suggest that an additional effect of beta-amyloid in the brain, which compounds the direct effects of beta-amyloid on neurons, is mediated by the stimulation of astroglia to become reactive. Once in the reactive state, glial cells deposit large amounts of growth-inhibitory molecules within the neuropil which could impair neuronal process survival and regeneration leading to neurite retraction and/or dystrophy around senile plaques in AD.

Alzheimer Disease↗
PubMed1993

Calcitonin gene regulation in vivo.

In view of the importance of the caliotropic hormones to calcium homeostasis and bone strength we have studied the regulation of the expression of the calcitonin gene in vivo in the rat. Normal rats were injected with 1,25-dihydroxyvitamin D3 (12.5-200 pmol/100 g body weight) and calcitonin mRNA levels measured. 1,25(OH)2D3 led to a marked decrease in calcitonin mRNA levels, which reached 4% of basal at 24 h. Nuclear transcript experiments showed that the effect was transcriptional. After large changes in serum calcium for periods from 6 h to 3 weeks there were no differences in calcitonin mRNA levels. Estrogens given to ovariectomized rats led to increases in calcitonin mRNA. The estrogen receptor was present in the thyroid C-cells. Therefore the calcitonin gene is regulated by vitamin D and estrogens, but not by calcium.

Animals↗
PubMed1993

Transgenic Fv-4 mice resistant to Friend virus.

Fv-4 is a mouse gene that confers resistance to infection with ecotropic retroviruses. A candidate Fv-4 gene was cloned previously and found to resemble the 3' half of a murine leukemia virus (MuLV). To study the effect of this gene in vivo, we generated two transgenic mouse strains carrying the Fv-4 env gene under control of its presumed natural promoter, a cellular sequence unrelated to retroviruses. Transgenic progeny expressed a 3-kb Fv-4 env RNA in all of the organs and tissues examined, as well as an Fv-4 envelope antigen on the surface of thymocytes and spleen cells, similar to mice carrying the natural Fv-4 gene. One of the two transgenic strains (designated Fv4-2) expressed three to nine times as much transgene RNA and protein as the other strain (Fv4-11). When challenged with a Friend virus complex containing up to 10(4) XC PFU of Friend MuLV, Fv4-2 mice were completely resistant to development of splenomegaly and had no detectable ecotropic virus in the spleen or blood, confirming that the cloned Fv-4 gene is responsible for resistance to ecotropic MuLV in vivo. In contrast, Fv4-11 mice were only partially resistant, developing viremia and splenomegaly at the highest inoculum dose but recovering from viremia several weeks after inoculation with 10-fold less virus. The phenotype of recovery from viremia in Fv4-11 mice was unexpected and suggests that low levels of expression of the Fv-4 gene enhance the effectiveness of the immune response.

Animals↗
PubMed1993

Molecular analysis of HLA-DR beta and DQ beta polymorphism in Chinese with rheumatoid arthritis.

OBJECTIVES: Several studies have suggested that genetic predisposition to rheumatoid arthritis may be related to the presence of specific polymorphic HLA sequences that are often associated with HLA-DR4 haplotypes. This study was performed to determine if an association exists between Chinese with rheumatoid arthritis and a particular HLA-DR beta or DQ beta subtype. METHODS: This study used the polymerase chain reaction to amplify HLA-DR beta and DQ beta genes, and oligonucleotide probe hybridisation to examine the association of certain polymorphic sequences with rheumatoid arthritis in 23 Chinese patients from Shanghai. RESULTS: An HLA-DR4 associated sequence was significantly increased in the Chinese patients (43%) compared with healthy controls (14%) from the same location (relative risk = 4.6, 95% confidence limits 1.1 to 19.3). Analysis of the third hyperpolymorphic region of DR4 positive samples was performed to detect polymorphic sequences associated with Dw4, Dw10, Dw13, Dw14, Dw15, and KT2 cellular specificities. Examination of this region showed that 91% of patients had sequences encoding amino acids QRRAA (associated with Dw14 and Dw15) or QKRAA (associated with Dw4) compared with 64% of the DR4 positive controls. CONCLUSIONS: Rheumatoid arthritis in the Chinese is associated with HLA-DR4. There is a possible relationship between sequences within the third hyperpolymorphic region of the DRB allele and rheumatoid arthritis in the Chinese.

Amino Acid Sequence↗
PubMed1993

Effects of calcitriol, 22-oxacalcitriol, and calcipotriol on serum calcium and parathyroid hormone gene expression.

Calcitriol markedly decreases PTH gene transcription, but because of the concern about hypercalcemia, there is interest in nonhypercalcemic analogs. We have studied the effects of calcitriol, oxacalcitriol, and calcipotriol on serum calcium and PTH mRNA levels in vivo and in vitro. In vivo in rats, calcitriol was the most effective analog in decreasing PTH mRNA levels, with a maximal effect of about 70% at 25-100 pmol after 24 h. Only 100 pmol led to hypercalcemia. Oxacalcitriol led to a maximal decrease in PTH mRNA levels of 44% at 2 and 5 nmol, similar to 1 nmol calcipotriol. Oxacalcitriol at 200 and 500 pmol led to an increase in serum calcium at 3 h, but not at 6 and 24 h, unlike calcitriol (100 pmol) which caused an increase only at 24 h. In vitro, in primary cultures of bovine parathyroid cells, calcitriol and oxacalcitriol both decreased PTH mRNA levels at similar concentrations. Therefore, in vivo calcitriol is the most effective analog studied in decreasing PTH mRNA levels, including a range of doses that does not cause hypercalcemia. Oxacalcalcitriol and calcipotriol are less effective, but have a wider dose range where they do not cause hypercalcemia. The results in vitro confirm that oxacalcitriol and calcitriol both effectively decrease PTH mRNA levels at the same concentration. The marked activity of calcitriol analogs in vitro compared to in vivo probably reflects differences from calcitriol in their pharmacokinetics.

Animals↗
PubMed1993

Surprising lability of biotin-streptavidin bond during transcription of biotinylated DNA bound to paramagnetic streptavidin beads.

We investigated the use of immobilized DNA templates as substrates for bacteriophage RNA polymerases in order to develop a simple method for separating template DNA from synthesized RNA. Double-stranded DNA molecules with a T7 or T3 RNA polymerase promoter at one end and a single biotin moiety at the other end were attached to streptavidin-coated paramagnetic beads and used in transcription reactions. When the biotin was attached by a nucleotide base on the nontemplate strand, the DNA-bead complex was moderately stable and could be used for multiple rounds of RNA synthesis. However, when the biotin was attached through a phosphodiester bond on the template strand, the enzymatic activity of RNA polymerase reversibly dissociated up to 80% of biotinylated DNA from the streptavidin beads. Biotinylated DNA bound to streptavidin beads in this system with a binding constant on the order of 10(12) M-1. These results stress the need for careful evaluation of solid phase adaptations of standard solution reactions in molecular biology.

Bacterial Proteins↗
PubMed1993

Glia-neuron interactions at the midline of the developing mammalian brain and spinal cord.

The midline of the developing brain is a place where axon guidance decisions effect the basic wiring pattern related to laterality in both sensory and motor systems. A variety of glial structures that develop along the midline may play crucial roles in directing axons to project to the left or right side of the central nervous system. Several of these glial structures and their association with axons are described. The molecular mechanisms that underlie axons guidance at the midline, in particular, repulsive factors that provide guidance cues via turning signals, are highlighted.

Animals↗
PubMed1992

Chondroitin sulfate as a regulator of neuronal patterning in the retina.

Highly sulfated proteoglycans are correlated with axon boundaries in the developing central nervous system which suggests that these molecules affect neural pattern formation. In the developing mammalian retina, gradual regression of chondroitin sulfate may help control the onset of ganglion cell differentiation and initial direction of their axons. Changes induced by the removal of chondroitin sulfate from intact retinas in culture confirm the function of chondroitin sulfate in retinal histogenesis.

Animals↗