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Biomedical subjects

C Zhang

Publications and source records attributed to C Zhang.

At least 523 records · Page 29Linked to original sources
PubMed1996

Expression of glutamate receptor subunit genes during development of the mouse retina.

The distribution of expression during retinal differentiation of nine AMPA and kainate glutamate receptor subunit genes was studied by in situ hybridization. Transcripts encoding all subunits were detected in the inner nuclear and ganglion cell layers of the developing mouse retina, but their time courses of expression differed. The earliest expression was detected at embryonic day 14 (E14) for GluR1, GluR4 and KA-2; all the other subunits were first detected at E16. Most subunits were expressed at higher levels during development than in the adult. This suggests they play a role in the processes of neuronal differentiation and synaptogenesis that occur during the early postnatal days in the rodent retina.

Animals↗
PubMed1996

Protective immunity to HIV-1 in SCID/beige mice reconstituted with peripheral blood lymphocytes of exposed but uninfected individuals.

Immunodeficiency typically appears many years after initial HIV infection. This long, essentially asymptomatic period contributes to the transmission of HIV in human populations. In rare instances, clearance of HIV-1 infection has been observed, particularly in infants. There are also reports of individuals who have been frequently exposed to HIV-1 but remain seronegative for the virus, and it has been hypothesized that these individuals are resistant to infection by HIV-1. However, little is known about the mechanism of immune clearance or protection against HIV-1 in these high-risk individuals because it is difficult to directly demonstrate in vivo protective immunity. Although most of these high-risk individuals show an HIV-1-specific cell-mediated immune response using in vitro assays, their peripheral blood lymphocytes (PBLs) are still susceptible to HIV infection in tissue culture. To study this further in vivo, we have established a humanized SCID mouse infection model whereby T-, B-, and natural killer-cell defective SCID/beige mice that have been reconstituted with normal human PBLs can be infected with HIV-1. When the SCID/beige mice were reconstituted with PBLs from two different multiply exposed HIV-1 seronegative individuals, the mice showed resistance to infection by two strains of HIV-1 (macrophage tropic and T cell tropic), although the same PBLs were easily infected in vitro. Mice reconstituted with PBLs from non-HIV-exposed controls were readily infected. When the same reconstituted mice were depleted of human CD8 T cells, however, they became susceptible to HIV-1 infection, indicating that the in vivo protection required CD8 T cells. This provides clear experimental evidence that some multiply exposed, HIV-1-negative individuals have in vivo protective immunity that is CD8 T cell-dependent. Understanding the mechanism of such protective immunity is critical to the design and testing of effective prophylactic vaccines and immunotherapeutic regimens.

Adoptive Transfer↗
PubMed1996

Expression of the retinoblastoma (RB) tumor suppressor gene inhibits tumor cell invasion in vitro.

To determine if replacement of the retinoblastoma (RB) tumor suppressor gene could inhibit invasion of RB-defective tumor cells, the capacity of tumor cells to migrate or invade was quantitated by the Boyden chamber assay. The studies were done in a diverse group of stable RB-reconstituted human tumor cell lines, including those derived from the osteosarcoma and carcinomas of the lung, breast and bladder. The expression of the exogenous wild-type RB protein in these tumor cell lines was driven by either a constitutively active promoter or an inducible promoter. It was found that significantly more tumor cells from the parental RB-defective cell lines and the RB revertants than from the RB-reconstituted RB+ cell lines penetrated through the Matrigel (P<0.001, two-tailed t-test), although both RB+ and RB- cells migrated at approximately the same rate on uncoated polycarbonate filters in the Boyden chambers. Of note, the inhibition of invasiveness of various RB-defective tumor cells by RB replacement was apparently well correlated with suppression of their tumorigenicity in vivo. In contrast, although either functional RB or p53 re-expression effectively suppressed tumor formation in nude mice of the RB-/p53null osteosarcoma cell line, Saos-2, replacement of the wild-type p53 gene had much less impact on their invasiveness as compared to the RB gene. These studies provided an insight into the broader biological basis of the RB-mediated tumor suppression in RB-defective tumor cells.

Animals↗
PubMed1996

Electron-dense granules in Desulfovibrio gigas do not consist of inorganic triphosphate but of a glucose pentakis(diphosphate).

Under certain growth conditions the sulfate-reducing bacterium Desulfovibrio gigas forms electron-dense granules in the cells which had been claimed to consist of a magnesium triphosphate). We observed granules after cultivation in media with a low Fe2+ or NH4+ concentration and reinvestigated the nature of the electron-dense bodies. Energy-dispersive X-ray analysis of the granules in the cells showed that they contain large amounts of P, Mg, and K. Gel electrophoresis and chromatographic analyses of isolated granules which had been dissolved in 20 mM EDTA, however, revealed discrepancies with commercially available polyphosphates. 31P-NMR spectra also lacked the peaks in the -22-ppm region which are characteristic for inner phosphates of polyphosphates confirming that the phosphocompound as isolated from the electron-dense bodies of D. gigas did not consist of polyphosphates. Using multinuclear NMR spectroscopy we showed that the electron-dense bodies of D. gigas contained a novel metabolite which was identified as alpha-glucose 1,2,3,4,6-pentakis(diphosphate).

Ammonia↗
PubMed1996

CNS neuronal focal adhesion kinase forms clusters that co-localize with vinculin.

Focal adhesion kinase (FAK) is a non-receptor protein tyrosine kinase that appears to play a central role in integrin-mediated signal transduction in non-neuronal cells, linking the extracellular matrix to the actin-based cytoskeleton at focal adhesion contacts. Biochemical analysis has revealed the presence of FAK immunoreactivity in cells of neuronal lineage (Zhang et al., 1994) and in the CNS (Burgaya et al. 1995; Grant et al., 1995). In the current work, we have examined the immunodistribution of FAK in nerve cell cultures and tissue sections from the rat CNS. Cultures of B103 CNS neuroblastoma cells and primary cultures of hippocampal neurons both showed abundant FAK immunoreactivity in nerve cell bodies. Immunoreactivity also extended into neurites and growth cones. The most striking feature of FAK distribution was the presence of short, punctate clusters of high FAK concentration. These FAK clusters were maintained in triton-extracted cell ghosts, indicating association with the cytoskeleton. Double-label confocal imaging showed that clusters of FAK coincided with clusters of vinculin, another actin-associated signal transduction molecule implicated in control of growth cone motility. Data from hippocampal sections verified the presence of FAK in adult neurons where it was enriched in somato-dendritic domains and showed a non-uniform distribution. Quantitative FAK immunoprecipitation to compare adult with embryonic brain showed a 7-fold developmental down-regulation of FAK and a 21-fold down-regulation of FAK TyrP. The data suggest that neuronal FAK may participate in signal transduction complexes relevant to neuronal morphogenesis and plasticity.

Age Factors↗
PubMed1996

A mitochondrial membrane protein defined by a novel monoclonal antibody is preferentially detected in apoptotic cells.

Studies toward the biologic and molecular understanding of programmed cell death have been stimulated by the recent identification of genes and their products that regulate apoptosis. A panel of mAbs has been raised against dying cells in the present study by immunizing mice with apoptotic Jurkat cells. One of these Abs, anti-7A6, was found to react with apoptotic cells. Using ELISA or flow cytometry, little reactivity of anti-7A6 was observed in normal or digitonin-permeabilized human peripheral blood lymphocytes and a number of hemopoietic cell lines tested. The Ab, however, strongly reacted with these cells when they were induced to undergo apoptosis by irradiation or treatment with apoptosis-inducing agents. Cell sorting and DNA fragmentation experiments revealed that 7A6-positive cells, but not 7A6-negative cells, had apparent DNA fragments characteristic of cells undergoing apoptosis. By immunoblot, under reducing conditions, anti-7A6 detected a 38-kDa protein band in the cell lysates prepared from apoptotic cells. Immunoelectron microscopy showed the 7A6 Ag to be localized to the membrane of mitochondria in apoptotic Jurkat cells. These results indicate that anti-7A6 defines a novel epitope on the mitochondrial membrane protein that appears to be exposed on cells undergoing apoptosis, suggesting that the 7A6 molecule may be involved in the molecular cascade of apoptotic cell death.

Animals↗
PubMed1996

Do nitric oxide synthase, NMDA receptor subunit R1 and cytochrome oxidase co-localize in the rat central nervous system?

The activation of nitric oxide synthase (NOS) has been linked to excitatory input via NMDA receptors. We hypothesized that NOS-positive neurons that have NMDA receptors on their surface would have high levels of cytochrome oxidase (C.O.) as energy generator for membrane repolarization. In order to compare the distribution of these markers on the same section, we reacted rat brain sections for C.O. histochemistry followed by NOS immunogold silver staining (IGSS). Adjacent sections were reacted for NOS IGSS followed by indirect immunoperoxidase for NMDA receptor subunit R1 (NMDAR1). We found that the staining pattern varied among regions but were consistent within each region examined. There are three types of NOS immunoreactive (NOS-ir) cells: (1) NOS-ir neurons that had moderate to high levels of both NMDAR1 and C.O. staining, such as the pontine reticular nuclei, motor and mesencephalic nuclei of the trigeminal nerve, and some motor neurons in the spinal cord. (2) NOS-ir neurons that were immunoreactive for NMDAR1 (NMDAR1-ir) but had low levels of C.O. activity in thei- somata. Their dendrites, however, were both NMDAR1-ir and rich in C.O. Examples of this type include neurons in the caudate and putamen, and periglomerular cells in the olfactory bulb. (3) We also found that some NOS-ir neurons were not NMDAR1-ir and had low C.O. activity. In addition to postsynaptic neurons, C.O. and NOS levels were both high in the inner segments of retinal photoreceptor cells where energy-demanding active ion transport maintains the dark current and where NO presumably activates guanylate cyclase for the production of cGMP, which keeps the Na+ channels open in the dark. Our findings suggest that NMDA receptors are available for the majority of NOS-ir neurons, which comprise a heterogenous population with varying energy demands.

Animals↗
PubMed1996

Functional analysis of opioid receptor subtypes in the ventromedial hypothalamic nucleus of the rat.

Effects of [Met5]enkephalin and agonists selective for mu-, delta- and kappa-opioid receptors were tested in vitro on neurons of the hypothalamic ventromedial nucleus of ovariectomized, estrogen-primed rats. Brain slices were perfused with artificial cerebrospinal fluid and opioid drugs were applied by bolus injection into the perfusion line. Single unit activity was recorded extracellularly. The majority of ventromedial hypothalamic nucleus neurons tested exhibited marked inhibitory responses to [Met5]enkephalin. The inhibition was blocked by naloxone, by the selective delta-opioid receptor antagonist naltrindole and, to a lesser extent, by the mu-opioid receptor antagonist beta-funaltrexamine. The kappa-opioid receptor antagonist nor-binalmorphimine had virtually no effect on [Met5]enkephalin inhibition. Agonists selective for delta-([D-Pen2,D-Pen5]enkephalin, DPDPE) and for mu-([D-Ala2,MePhe4,Gly-ol5]enkephalin, DAGO) opioid receptors also potently inhibited the ventromedial hypothalamic nucleus neurons while the kappa-opioid receptors agonist U50,488 only produced a small inhibition in a smaller number of units. These results provide functional evidence that [Met5]enkephalin, a potential opioid transmitter in the ventromedial hypothalamic nucleus, can exert an inhibitory effect by acting on delta-and mu-opioid receptors.

Action Potentials↗
PubMed1996

Syk-dependent phosphorylation of Shc. A potential link between FcepsilonRI and the Ras/mitogen-activated protein kinase signaling pathway through SOS and Grb2.

Antigen receptors on T- and B-cells activate Ras through a signaling pathway that results in the tyrosine phosphorylation of Shc and the formation of a complex of Shc with the Grb2 adaptor protein. The high affinity receptor for immunoglobulin E (FcepsilonRI) in cultured mast (RBL-2H3) cells has been reported to function differently. Here we show to the contrary that engagement of FcepsilonRI with antigen leads to increased tyrosine phosphorylation of Shc and the association of Shc with Grb2 and other proteins (p120 and p140). Like the FcepsilonRI-mediated activation of the mitogen-activated protein kinase cascade, these responses are dependent on the tyrosine kinase Syk; they are enhanced by overexpression of Syk and are blocked by expression of dominant-negative Syk. Sos is constitutively associated with Grb2 in these cells but dissociates from Shc on stimulation with antigen. These reactions are rapid, reversible, and associated with the activation of Ras. Therefore, the Syk-dependent tyrosine phosphorylation of Shc and its association with Grb2 may provide a pathway through Sos for activation of Ras by FcepsilonRI.

Adaptor Proteins, Signal Transducing↗
PubMed1996

A beta peptide enhances focal adhesion kinase/Fyn association in a rat CNS nerve cell line.

Neurotoxicity of the amyloid beta protein (A beta) is known to correlate with a selective change in protein tyrosine phosphorylation (Tyr(P)) of focal adhesion kinase (FAK) (Zhang et al., J. Biol. Chem., 269 (1994) 25247-25250). The current work has found that exposure of neuronal cells to A beta upregulates the stable association of FAK with Fyn, a neuronally-enriched protein tyrosine kinase of the Src-family. In cells incubated with aged A beta 1-42, the amount of immunoprecipitable FAK-Fyn complex increased approximately 280%. Equivalent results were obtained whether anti-FAK or anti-Fyn was used to precipitate the complex. Cells incubated with non-toxic A beta 17-42, which makes aggregates and attaches to cells but does not upregulate FAK Tyr(P), exhibited no increase in FAK-Fyn complex. Aberrant Fyn activity due to the A beta evoked association with FAK could play a role in neuronal degeneration and also cause anomalies in synaptic plasticity. These possibilities are of particular significance because of the reported increase in Fyn immunoreactivity in Alzheimer's-afflicted neurons (Shirazi and Wood, NeuroReport, 4 (1993) 435-437).

Amyloid beta-Protein Precursor↗
PubMed1996

Comparison of different quantitative PCR procedures in the analysis of the 4977-bp deletion in human mitochondrial DNA.

Four quantitative PCR procedures were compared to analysing the level of the 4977 bp deletion in human mitochondrial DNA. A single preparation of total cellular DNA from the heart of a 69-year-old female subject was used for all four methods. We estimated the deletion to represent 0.005% of total mtDNA molecules using the serial dilution procedure and two internal standard methods with two separate sets of reference recombinant plasmids. However, the value obtained with the kinetic PCR analysis was 0.5%, two orders of magnitude higher. The internal standard procedures are likely to be the most accurate among the four methods used, but the more technically convenient serial dilution method would also be an appropriate choice.

Aged↗
PubMed1996

Deafferentation leads to a down-regulation of nitric oxide synthase in the rat visual system.

To test the hypothesis that monocular enucleation can cause a decrease in nitric oxide synthase (NOS) in subcortical visual centers, serial sections of superior colliculi and lateral geniculate nuclei from normal and enucleated rats were processed for NOS immunohistochemistry and NADPH-diaphorase histochemistry. Adjacent sections were also reacted for cytochrome oxidase, a metabolic marker, to demonstrate the change in energy demands. We found that both visual centers normally contained moderate levels of NOS, and eye removal caused a consistent down-regulation of NOS, NADPH-diaphorase and cytochrome oxidase on the deprived contralateral side. Optical densitometric values supported these findings (P < 0.01). Thus, the level and the activity of NOS are regulated by neuronal activity in the rat subcortical visual centers.

Animals↗
PubMed1996

Protein kinase C and F-actin are essential for stimulation of neuronal FAK tyrosine phosphorylation by G-proteins and amyloid beta protein.

Focal adhesion kinase (FAK) is a protein tyrosine kinase implicated in signal transduction pathways for integrins, neuropeptides, and lysophosphatidic acid. FAK, first discovered in non-neuronal cells, recently has been reported to occur in neurons, where its tyrosine phosphorylation is upregulated by fibronectin and by the Alzheimer's Abeta peptide. The current work has elucidated molecular events leading to tyrosine phosphorylation of FAK in the rat B103 CNS nerve cell line. Activation of receptor-coupled G-proteins by Mas-7 was found to evoke rapid upregulation of FAK tyrosine phosphorylation (Tyr(P)). Upregulation by Mas-7 was blocked by GF109203X, a potent inhibitor of protein kinase C (PKC). Phorbol ester also upregulated FAK-YP, verifying a role for PKC in the transduction cascade. Upregulation of FAK-YP by activation of G-proteins and PKC was dependent upon intact F-actin, as cytochalasin D abolished stimulation by Mas-7 and by phorbol ester. The relatively slow increase in FAK-YP evoked by chronic exposure to Abeta also was abolished by GF109203X and by cytochalasin D. The results show that tyrosine phosphorylation of FAK in neurons is regulated positively by PKC, functioning down-stream from G-proteins through an F-actin-dependent mechanism. The Alzheimer's Abeta peptide is capable of activating elements of this same signal transduction pathway, via membrane events that remain to be determined.

Actins↗