Biomedical subjects

S Zhang

Publications and source records attributed to S Zhang.

At least 757 records · Page 42Linked to original sources

PubMed1997

Mammalian homologs of seven in absentia regulate DCC via the ubiquitin-proteasome pathway.

DCC (deleted in colorectal cancer) is postulated to function as transmembrane receptor for the axon and cell guidance factor netrin-1. We report here that the DCC cytoplasmic domain binds to proteins encoded by mammalian homologs of the Drosophila seven in absentia (sina) gene, as well as Drosophila Sina. Sina has a critical role in R7 photoreceptor development and shows upward of 85% amino acid identity with its mammalian homologs (termed Siahs), but the function of the Sina/Siah proteins has not been defined. We sought, therefore, to characterize further their interaction with DCC. Immunofluorescence studies suggested the Sina/Siah proteins localized predominantly in the cytoplasm and in association with DCC. DCC was found to be ubiquitinated and the Sina/Siah proteins regulated its expression. Proteasome inhibitors blocked the effects of Sina/Siah on DCC, and the Sina/Siah proteins interacted with ubiquitin-conjugating enzymes (Ubcs). A mutant Siah protein lacking the amino-terminal Ubc-binding sequences complexed with DCC, but did not degrade it. The in vivo interaction between Sina/Siah and DCC was confirmed through studies of transgenic Drosophila lines in which DCC and Sina were ectopically expressed in the eye. Taken together, the data imply that the Sina/Siah proteins regulate DCC and perhaps other proteins via the ubiquitin-proteasome pathway.

Animals↗

PubMed1997

The coiled-coil region of the G protein beta subunit. Mutational analysis of Ggamma and effector interactions.

The beta and gamma subunits of the heterotrimeric G proteins remain tightly associated throughout the signaling cycle as the betagamma dimer interacts with Galpha, receptors, and effectors. A coiled-coil structure involving alpha-helical segments at the N termini of the beta and gamma subunits contributes to the dimerization interface and has been implicated in effector signaling in yeast. Scanning mutagenesis of the coiled-coil region of the mammalian beta1 subunit was performed to examine the effect of point mutations on betagamma assembly and effector signaling in COS cell cotransfection assays. In addition to the E10K mutation described previously, mutations A11E, L14E, and I18E in beta1 were found to block betagamma association, as evidenced by the failure of the Gbeta mutants to undergo cytosolic translocation with cotransfected nonisoprenylated Ggamma. Although none of 14 beta1 point mutations prevented the betagamma-dependent activation of the c-Jun N-terminal kinase (JNK) effector pathway, the D20K point mutation enhanced JNK but not phospholipase C-beta2 activation. These findings implicate the coiled-coil region of Gbeta in JNK signaling, provide further evidence that the structural features of the betagamma complex mediating effector regulation may differ among effectors, and identify single codons in the mammalian beta subunit where mutation might yield a phenotype of defective signal transduction.

Amino Acid Sequence↗

PubMed1997

Detection of proviral DNA of bovine immunodeficiency virus in bovine tissues by polymerase chain reaction (PCR) and PCR in situ hybridization.

In this study, experiments were designed to investigate the distribution of bovine immunodeficiency virus (BIV) proviral DNA in the tissues and cells of infected calves by solution-phase polymerase chain reaction (SP-PCR) and PCR in situ hybridization (PCR-ISH). Total DNA samples extracted from tissues of 10 BIV-infected and 5 uninfected calves were amplified by SP-PCR with the primers directed to the BIV conserved pol gene segment. The identity of the SP-PCR product was confirmed by Southern hybridization with a BIV pol gene cDNA probe. SP-PCR results demonstrated that BIV proviral DNA was present predominantly in neural tissues and some lymphoid tissues in BIV-infected calves. It also was detected frequently in other tissues including lung, heart, esophagus, and pancreas. Further investigation on cell location of BIV proviral DNA was performed by in situ amplification of DNA on formalin-fixed tissue sections. The amplified DNA was subjected to in situ hybridization with an internal biotinylated probe and detected with streptavidin-gold followed by silver enhancement. Specific BIV proviral DNA signals were observed in neurons, microglial cells, lymphocytes, septal macrophages, smooth muscle cells, and endothelial cells. On the basis of these results, we conclude that BIV replicates in a variety of bovine tissues in vivo and has a broad cell tropism.

Animals↗

PubMed1997

Selection of tumor antigens as targets for immune attack using immunohistochemistry: I. Focus on gangliosides.

Understanding the distribution of tumor-associated antigens on cancers and normal tissues is essential for selection of targets for cancer immunotherapy. Seven carbohydrate antigens, potential targets for immunotherapy, were studied using a panel of well-characterized MAbs by immunohistochemistry on cryostat-cut tissue sections of 13 types of cancers and 18 normal tissues. GD2 and GD3 were present on most cancers of neuroectodermal origin and GD2 was also present on B cell lymphomas. 9-O-acetyl-GD3 was detected only on melanoma while fucosyl GM1 was detected only on small cell lung cancers (SCLC). Surprisingly, GM2 was strongly expressed on all tested tumors, including cancers of neuroectodermal origin and cancers of epithelial origin. Polysialic acid was primarily expressed on SCLC and neuroblastomas. Globo H was present on most cancers of epithelial origin. These antigens were also identified in normal tissues. Fucosyl GM1 was not expressed significantly on any of the normal tissues analyzed. GD3, GD2, GM2 and polysialic acid were detected in normal brain to varying degrees. GM2 and Globo H were expressed on the luminal surface of epithelia of a variety of organs. The unexpected expression of GM2 on a broad range of cancers and normal epithelial tissues was confirmed by loss after methanol fixation and by immune thin layer chromatography.

Animals↗

PubMed1997

Selection of tumor antigens as targets for immune attack using immunohistochemistry: II. Blood group-related antigens.

Blood group-related antigens have been attractive targets for immunotherapy of cancer since their initial identification as cancer-related antigens. However, available information on the relative expression of most of these antigens on human malignant and normal tissues has been insufficient for selecting optimal antigens and tumors for immune attack. In this study, the distribution of the blood group-related antigens TF, Tn, sTn, Le(a), sialyl Le(a), Le(b), Le(x), sialyl Le(x), polyfucosyl Le(x) and Le(y) on 13 types of cancer and 16 normal tissues was compared. Our results show that sTn is strongly expressed on cancers of breast, colon, stomach, ovary, prostate and uterus; Tn on prostate cancer; TF on cancers of breast, colon, ovary, prostate and uterus; Le(y) on the cancers of colon, lung, pancreas and ovary; Le(a) and Le(x) on gastric cancer; and sialyl Le(a) and sialyl Le(x) on colon cancer. The complete absence of these antigens on cancers of neuroectodermal or mesodermal origin including melanoma, sarcoma, neuroblastoma and B cell lymphoma is as striking as their widespread presence on tumors of epithelial origin. Normal tissues were also tested. Tn and Le(b) were only detected on gastric and ovarian epithelia; sTn on Leydig cells of testis in addition to gastric and ovarian epithelia; Le(x) and sialyl Le(x) on polymorphonuclear leukocytes; and TF, Le(a), sialyl Le(a), Le(x), sialyl Le(x), polyfucosyl Le(x) and Le(y) on epithelia from a variety of tissues.

Animals↗

PubMed1997

Surface residue mutations which change the substrate specificity of Thermomonospora fusca endoglucanase E2.

The three dimensional structure of a T. fusca endoglucanase catalytic domain (E2cd) has been determined by X-ray crystallography at 1.0 A resolution (Wilson et al., 1995). The availability of a high resolution structure for E2cd allows us to initiate structure-based efforts to engineer cellulases with a high activity on native cellulose. The low activity on crystalline cellulose suggests that the entry of a cellulose molecule into the active site rather than catalysis may be the rate limiting step for hydrolysis of crystalline cellulose. Movement of a loop upon substrate binding has been proposed to play a crucial role in catalysis. A total of 15 surface mutants and 5 loop mutants were created by site-directed mutagenesis and their effects on activity and substrate specificity were determined. Circular dichroism spectra were used to monitor structural changes, and no major changes were found. The binding constants for two methyl umbelliferyl oligosaccharides and cellotriose were measured for some of the mutants and all of them showed binding similar to wild type E2. These results provide the first direct link between loop movement and catalysis by E2 and show that surface residues can affect its substrate specificity.

Cellulase↗

PubMed1997

Persistent echovirus infection of mouse cells expressing the viral receptor VLA-2.

Mouse cells are not susceptible to infection with echovirus 1 (EV-1) because they lack the viral receptor, human VLA-2. Two mouse fibroblast cell lines, L cells and 3T3 cells, were made susceptible to EV-1 infection after transformation with cDNAs of human VLA-2. After EV-1 infection, L cell transformants of human VLA-2 (alpha2beta1 L cells) develop cytopathic effect (CPE) as expected, while 3T3 cell transformants of human VLA-2 (alpha2beta1 3T3 cells) or the alpha2 subunit of human VLA-2 (alpha2 3T3 cells) become persistently infected. The distinct outcome is not a result of differential virus growth on these transformants because one-step growth curve analysis reveals little difference in EV-1 replication in both cell lines. In addition, 3T3 cell transformants expressing the poliovirus receptor (Pvr 3T3 cells) are lysed during poliovirus infection, suggesting that 3T3 cells are not intrinsically resistant to CPE caused by enterovirus infection. The results of limit dilution assays indicate that all EV-1-infected alpha2 3T3 cells produce infectious virus. All EV-1-infected alpha2 3T3 cells remain viable after EV-1 infection, and the kinetics of cell growth were not altered. FACS analysis reveals that receptor down-regulation is not involved in the establishment of persistent infection. Furthermore, inhibition of host protein synthesis was not observed in EV-1-infected alpha2 3T3 or alpha2beta1 L cells. Since alpha2beta1 L cells are lysed by EV-1 infection, these findings suggest that virus-induced translation inhibition is not a determinant of cell killing.

3T3 Cells↗

PubMed1997

Identification of two novel cis-elements in the promoter of the prostate-specific antigen gene that are required to enhance androgen receptor-mediated transactivation.

A monomeric androgen responsive element (ARE) is not sufficient to mediate significant androgen induction of the prostate-specific antigen (PSA) gene. Co-transfection experiments using a series of 5'deletion fragments of the proximal promoter region of the PSA gene linked to bacterial chloramphenicol acetyltransferase (CAT) as a reporter have identified two motif sequences which are indispensable for androgen receptor (AR)-mediated transactivation of the PSA promoter and have been designated as motifs A and B respectively. Of note, motif B alone has very little independent enhancer activity regardless of the presence or absence of androgen, whereas multi-copies of motif A exert androgenic inducibility for a heterologous promoter independent of the presence of ARE. Nucleotide substitutions in either motif significantly decrease the androgen inducibility and the nuclear protein binding ability. Furthermore, gel band shift experiments consistently demonstrate that nuclear proteins can bind these motifs, and they are non-receptor factors. Our data indicate that these two DNA motifs are novel cis -regulatory elements and exhibit different mechanisms in cooperation with ARE for AR-mediated transactivation.

Humans↗

PubMed1997

Inhibition of HIV type 1 RNA dimerization by antisense DNA corresponding to the 17-nucleotide sequence downstream from the splice donor site of HIV type 1 RNA.

HIV-1 RNA dimerization involves at least two key regions, one located upstream from the splice donor (SD) site, and the other located downstream from the SD site. To determine the precise location and the mechanism of action of the downstream region, we constructed a model system using a synthetic HIV-1 RNA fragment (HXB2, 455-1146), which dimerized at relatively low salt concentrations (100 mM KCl, 1 mM MgCl2). We tested in this system antisense DNAs that are complementary to both the upstream and downstream regions of HIV-1 RNA for their possible inhibitory effects on dimerization. Antisense DNAs complementary to nucleotides 773-789 located downstream from the SD site effectively inhibited dimerization of HIV-1 RNA. These inhibitory antisense DNAs hybridized with the dimer form of HIV-1 RNA, and dissociated the dimer into monomers. However, antisense DNAs complementary to the region upstream from the SD site did not hybridize with the dimer, although they inhibited RNA dimerization and also dissociated the preformed dimer.

Base Sequence↗

PubMed1997

Expression of human prostate-specific glandular kallikrein protein (hK2) in the breast cancer cell line T47-D.

Human glandular kallikrein (hK2) protein, like prostate-specific antigen (PSA), is produced mainly in prostatic epithelium. It may be useful as a new diagnostic indicator for prostate cancer. Recently, a number of hK2-specific monoclonal antibodies have been developed that enable us to detect hK2 protein in human prostate tissue, seminal fluid, and sera. Whether hK2 can be expressed, like PSA, in nonprostatic cells is not known. In this study, we have characterized the presence of hK2 in an androgen-responsive breast cancer cell line T47-D at both the protein and mRNA levels with an immunoassay, Western blot analysis, Northern blot analysis, and the reverse transcription-PCR. Using a sensitive immunoassay with monoclonal antibodies to hK2, we found that T47-D cells could be induced with androgens, mineralocorticoids, glucocorticoids, and progestins to produce significantly more hK2 than PSA. Estrogens failed to mimic the effect of the other steroids, blocking instead the stimulatory effect of androgens. Androgen induction of hK2 in T47-D cells was dose dependent. More interestingly, we found that the hK2 in androgen-induced T47-D cell spent media appears to be the pro-form of hK2 rather than mature hK2. Our study demonstrates that hK2, a serine protease thought to be found only in prostate-related tissues and fluids, is also produced in a breast cancer cell line T47-D after steroid stimulation. This finding suggests that hK2 may have a potential role in breast cancer as well as prostatic cancer and will be the impetus for further studies of hK2 distribution and function.

Blotting, Northern↗

PubMed1997

Memory-related changes of nitric oxide synthase activity and nitrite level in rat brain.

The changes of nitric oxide synthase (NOS) activity and nitrite level in rat brain regions after spatial learning were investigated. NOS activity was assayed by conversion of [3H]L-arginine to [3H]L-citrulline, and a sensitive fluorometric assay for quantification of nitrite was used. Compared with sham-trained rats, NOS activity and nitrite level in hippocampus and cortex, and also the nitrite level in cerebellum, was elevated significantly one day after rats had learnt a water-rewarded spatial alteration task. These results suggest a spatial memory-related changes of endogenous NO in rat brain, and support the idea that NO participates in learning and memory processes.

Animals↗

PubMed1997

Long-term effects of neonatal axoplasmic transport attenuation on the organization of the rat's trigeminal system.

The current study examined the long-term effects of infraorbital nerve (ION) axoplasmic transport attenuation with vinblastine on the organization of trigeminal (V) primary afferents and central vibrissae-related patterns. Retrograde tracing and single unit recording were used to evaluate the innervation of vibrissae follicles in adult (P > 60) rats that sustained application of vinblastine to the ION at birth. Single units recorded from vinblastine-treated animals yielded responses to deflection of a single vibrissa, and a significantly (P < 0.001) higher percentage of these cells (85.7%) showed rapidly adapting responses compared with normal rats (42.2%). Retrograde tracing revealed a qualitatively and normal distribution of V ganglion cells innervating A-row and E-row vibrissae follicles in vinblastine-treated rats. Transganglionic tracing with horseradish peroxidase (HRP) demonstrated a qualitatively and quantitatively normal somatotopic organization of vibrissae follicle input to V nucleus principalis (PrV) and V subnucleus interpolaris (SpI) in the vinblastine-treated animals. Despite the nearly normal mapping of V ganglion cell axons onto the vibrissae follicles and brainstem, staining for either cytochrome oxidase (CO) or parvalbumin failed to reveal vibrissae-related patterns in PrV, SpI, or the magnocellular portion of V subnucleus caudalis in these animals. Labelling of thalamocortical afferents with HRP and staining for CO also failed to reveal a cortical vibrissae-related pattern in the vinblastine-treated rats. The present results indicate that although transient attenuation of axoplasmic transport with vinblastine has limited effects on the peripheral and central projections of surviving V primary afferents, it permanently disrupts the normal development and maintenance of central vibrissae-related patterns.

Animals↗

PubMed1997

Domain structure of human nuclear DNA helicase II (RNA helicase A).

Full-length human nuclear DNA helicase II (NDH II) was cloned and overexpressed in a baculovirus-derived expression system. Recombinant NDH II unwound both DNA and RNA. Limited tryptic digestion produced active helicases with molecular masses of 130 and 100 kDa. The 130-kDa helicase missed a glycine-rich domain (RGG-box) at the carboxyl terminus, while the 100-kDa form missed both its double-stranded RNA binding domains (dsRBDs) at the amino terminus and its RGG-box. Hence, the dsRBDs and the RGG-box were dispensable for unwinding. On the other hand, the isolated DEXH core alone could neither hydrolyze ATP nor unwind nucleic acids. These enzymatic activities were not regained by fusing a complete COOH or NH2 terminus to the helicase core. Hence, an active helicase required part of the NH2 terminus, the DEXH core, and a C-terminal extension of the core. Both dsRBDs and the RGG-box were bacterially expressed as glutathione S-transferase fusion proteins. The two dsRBDs had a strong affinity to double-stranded RNA and cooperated upon RNA binding, while the RGG-box bound preferentially to single-stranded DNA. A model is suggested in which the flanking domains influence and regulate the unwinding properties of NDH II.

Adenosine Triphosphatases↗

PubMed1997

Altered hematopoiesis, behavior, and sexual function in mu opioid receptor-deficient mice.

The mu opioid receptor is thought to be the cellular target of opioid narcotics such as morphine and heroin, mediating their effects in both pain relief and euphoria. Its involvement is also implicated in a range of diverse biological processes. Using a mouse model in which the receptor gene was disrupted by targeted homologous recombination, we explored the involvement of this receptor in a number of physiological functions. Mice homozygous for the disrupted gene developed normally, but their motor function was altered. Drug-naive homozygotes displayed reduced locomotor activity, and morphine did not induce changes in locomotor activity observed in wild-type mice. Unexpectedly, lack of a functional receptor resulted in changes in both the host defense system and the reproductive system. We observed increased proliferation of granulocyte-macrophage, erythroid, and multipotential progenitor cells in both bone marrow and spleen, indicating a link between hematopoiesis and the opioid system, both of which are stress-responsive systems. Unexpected changes in sexual function in male homozygotes were also observed, as shown by reduced mating activity, a decrease in sperm count and motility, and smaller litter size. Taken together, these results suggest a novel role of the mu opioid receptor in hematopoiesis and reproductive physiology, in addition to its known involvement in pain relief.

Animals↗

PubMed1997

Involvement of the frontal ventrolateral orbital cortex in descending inhibition of nociception mediated by the periaqueductal gray in rats.

Our previous findings which indicated that electrical stimulation of ventrolateral orbital cortex (VLO) can depress the rat tail flick (TF) reflex and that the VLO-evoked inhibitory effect is blocked by electrolytic lesions of periaqueductal gray (PAG) suggest a role of the VLO in the modulation of nociception. To further investigate the involvement of VLO in this nociceptive modulatory pathway, we tested the effects of microinjections of glutamate (200 mM, 0.7 microliter) into the VLO on the TF reflex. An unilateral microinjection of glutamate into the VLO significantly depressed the TF reflex; and this effect was repeatable. Furthermore, bilateral microinjections of gamma-aminobutyric acid (GABA: 100 mM, 0.5 microliter on each side) into the ventrolateral parts of PAG could eliminate this VLO-evoked inhibition of the TF reflex. These results, along with our previous findings provide further support for a hypothesis that VLO, as a higher center in the frontal cortex, plays an important role in modulation of nociception, and this role is mediated by PAG leading to activation of the brainstem descending inhibitory system which depresses the nociceptive information at the spinal level.

Animals↗

PubMed1997

Defining a functional androgen responsive element in the 5' far upstream flanking region of the prostate-specific antigen gene.

Previously, an androgen responsive element (ARE or promoter ARE) was identified in the proximal promoter region of the prostate-specific antigen (PSA) gene. The proximal promoter fragment could mediate androgen induction of expression of a heterologous reporter gene in androgen receptor (AR)-less cells with exogenous AR in co-transfection assays. However, it exerted little androgen inducibility in androgen sensitive human prostate cells, LNCaP, which produce PSA mRNA and protein. In this study, we have identified a second functional ARE (or upstream ARE) approximately 4 kb upstream of the cap site of the PSA transcript. Interestingly, although the AREs are necessary for androgen induction, the DNA sequences surrounding the upstream ARE are also required for androgen induction by the PSA promoter in LNCaP cells. The results indicated that the upstream DNA sequences can cooperate with either ARE to mediate androgen induced gene expression in LNCaP cells.

Cell Nucleus↗

PubMed1997

Protein kinase CK2 down-regulates glucose-activated expression of the acetyl-CoA carboxylase gene.

It has been suggested that, in pancreatic beta-cells, acetyl-CoA carboxylase (ACC) is a key enzyme in glucose signal transduction leading to glucose-induced insulin secretion. The PII promoter is the only active promoter for the ACC gene in the beta-cell. Here we report that, in the pancreatic beta-cell, high glucose levels (above 20 mm) activate Sp1 binding to the glucose response element of the PII promoter, which leads to a dose-dependent increase in PII transcription. The expression of a gene coding protein kinase CK2 (CK2) alpha subunit, or the presence of okadaic acid (a serine/threonine protein phosphatase inhibitor), partially blocks the glucose activation of PII transcription. The inhibitory effect of CK2 alpha, or okadaic acid, was not observed in the absence of glucose or at low glucose concentrations. Phosphorylation of Sp1 by CK2 alpha leads to the inactivation of Sp1 binding to PII. Dephosphorylation of the phosphorylated Sp1 by protein phosphatase 1 (PP1) activates the binding of Sp1 to PII. Inhibition of PP1-catalyzed Sp1 dephosphorylation by okadaic acid, or PP1 specific inhibitor 2, decreases Sp1 binding to PII. These results suggest that the phosphorylation/dephosphorylation of Sp1 by CK2/PP1 may be the underlying mechanism by which the expression of the PII promoter of ACC is controlled in the process of glucose-mediated insulin secretion in pancreatic beta-cells.

Acetyl-CoA Carboxylase↗

PubMed1997

Loss of p53 is an early event in induction of brain tumors in mice by transplacental carcinogen exposure.

Experimental carcinogenesis studies using p53-deficient mice have suggested that loss of function of this tumor suppressor gene is generally not an early event but is rather related to tumor progression. However, the biological functions of p53 and the accumulating evidence of alteration in human tumors imply a possible role for loss of p53 in the initial stages of tumorigenesis. Ethylnitrosourea administration to p53-heterozygous pregnant mice resulted in rapid development of primary brain tumors, which are extremely rare in mice, in 70% of the p53-null offspring. Brain tumors also developed later in 4% of heterozygous mice, but they had lost the wild-type allele. Thus, loss of normal p53 gene expression is of direct significance to early events in brain tumorigenesis, and this tumor suppressor gene may protect embryos from DNA damage in the brain induced by transplacental carcinogen exposure.

Animals↗