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Biomedical subjects

S Hu

Publications and source records attributed to S Hu.

At least 343 records · Page 19Linked to original sources
PubMed1994

The raspberry locus of Drosophila melanogaster includes an inosine monophosphate dehydrogenase like coding sequence.

DNA from the raspberry gene of Drosophila melanogaster has been cloned through P-element tagging of a dysgenically induced lethal allele. A transcribed DNA segment adjacent to the P insert encodes an amino acid sequence that is similar to known inosine monophosphate dehydrogenase (IMPDH) sequences. Further dysgenically induced lethals and all four known spontaneous ras eye-colour mutations have changes in the DNA either within or just upstream from the transcribed region. Identification of IMPDH as a probable product of the ras gene is compatible with the finding of an allele that requires dietary guanosine (GR), since the enzyme mediates the first of two biosynthetic steps leading from inosine monophosphate (IMP) to guanosine monophosphate (GMP). However, other auxotrophic mutants at the locus remain unexplained by the finding. The results further suggest that GMP synthesis from IMP is an essential process, despite the capacity of the fly for salvage synthesis of GMP from GR. Consideration of the phenotypes associated with mutants at the ras locus suggests that IMPDH activity is regulated in a tissue-specific manner.

Alleles↗
PubMed1994

Tumor necrosis factor-alpha potentiates glutamate neurotoxicity in human fetal brain cell cultures.

Cytokines may play a pathogenetic role in the brain. Using human fetal brain cell cultures, we investigated whether cytokines released during inflammation modulate neuronal injury. Exposure of human fetal neuronal cells to the excitatory amino acid neurotransmitter, glutamate, for 6 days resulted in a dose-dependent cell loss. Tumor necrosis factor (TNF)-alpha potentiated glutamate neurotoxicity. This TNF alpha-potentiated glutamate neurotoxicity was blocked by the glutamate receptor antagonists, 2-APV and MK-801, suggesting that the potentiating effect of TNF alpha is predominantly mediated by a glutamate receptor mechanism. Exposure of neuronal cultures to TNF alpha for 5 days resulted in a 27% decrease in astrocyte glutamine synthetase and in a 50% inhibition of 3H-glutamate uptake, suggesting that the effect of TNF alpha indirectly involves glutamate metabolism. These findings suggest that under pathologic conditions, TNF alpha may impair embryonic development of the brain by exacerbating excitotoxicity.

2-Amino-5-phosphonovalerate↗
PubMed1994

Priming effect of morphine on the production of tumor necrosis factor-alpha by microglia: implications in respiratory burst activity and human immunodeficiency virus-1 expression.

Opiates alter a variety of functional activities of the somatic immune system; within the central nervous system, however, their effects on immune responses are unknown. In the present study, we investigated the effect of morphine on the release of tumor necrosis factor (TNF)-alpha from murine neonatal microglia. Microglial cell cultures did not release TNF-alpha when incubated with morphine alone; however, an enhanced (P < .01) release of TNF-alpha was observed when cultures were first primed with morphine for 24 h and then stimulated with lipopolysaccharide. A bell-shaped dose-response curve was observed for the priming effect of morphine; maximal enhancement of TNF-alpha release (310 +/- 15% of control) was detected at a concentration of 10(-10) M morphine. Pretreatment of microglia for 30 min with opioid receptor antagonists (i.e. naloxone and beta-funaltrexamine) completely blocked the priming effect of morphine. In addition, morphine treatment amplified (P < .01) the priming effect of lipopolysaccharide on phorbol myristate acetate-triggered superoxide anion production by microglial cell cultures, and this effect was abrogated (P < .01) by anti-TNF-alpha antibody. Furthermore, culture supernatants derived from microglial cell cultures that had been treated with morphine before stimulation with lipopolysaccharide had an increased capacity to upregulate human immunodeficiency virus-1 expression in the latently infected promonocytic clone U1. This effect was also blocked by anti-TNF-alpha antibody. These findings suggest that morphine primes microglia for enhanced production of TNF-alpha which could alter several functional activities of these cells within the brain.

Acquired Immunodeficiency Syndrome↗
PubMed1993

Distinct heme active-site structure in lactoperoxidase revealed by resonance Raman spectroscopy.

Low-frequency resonance Raman spectra of the cyanide and carbon monoxide adducts of lactoperoxidase are obtained with Soret excitation. The nu(Fe-CN) and delta(Fe-C-N) modes are detected at 360 and 453 cm-1, respectively. Upon the isotopic substitution of 13C14N, 12C15N, and 13C15N, the band at 453 cm-1 in the natural abundance adduct shifts to 448, 452, and 445 cm-1, while the 360-cm-1 peak shifts to 358, 357, and 356 cm-1, respectively. The 360-cm-1 band is shifted to 355 cm-1 when the pH is changed from 7.0 to 10.5. On the basis of a previous normal-mode analysis of the cyanoferric adduct of myeloperoxidase, a bent Fe-C-N linkage is suggested for the cyanide adduct of lactoperoxidase. The nu(Fe-CN) (374 cm-1) and delta(Fe-C-N) (480 cm-1) modes are observed for the cyanide adduct of reduced lactoperoxidase. For the carbon monoxide adduct, the nu(Fe-CO) (533 cm-1) and delta(Fe-C-O) (578 cm-1) modes at pH 7.0 are observed to shift to 498 and 570 cm-1 as the pH is raised from 7.0 to 10.0. The strong intensity of delta(Fe-C-O) at both acid and alkaline pHs, along with a suggested bent structure of the Fe-C-N moiety, implies a narrow heme pocket for lactoperoxidase.

Animals↗
PubMed1993

Neuroprotective role of IL-4 against activated microglia.

Microglia have been proposed to play a pathogenetic role in immunologically mediated neurodegenerative diseases. In our study, using microglial/neuronal cell cocultures primed with IFN-gamma, we found that both LPS and TNF-alpha triggered neuronal cell injury (impairment of gamma-aminobutyric acid uptake and neuronal loss) via a nitric oxide mechanism. Pretreatment of cell cocultures with IL-4, an immunosuppressive cytokine, prevented, in a dose-dependent manner, neuronal cell injury induced by activated microglia. The mechanism by which IL-4 exerts its neuroprotective effect was found to involve the inhibition of IFN-gamma priming of microglia with a subsequent decrease in the production of TNF-alpha and nitric oxide.

Animals↗
PubMed1993

A linkage between DNA markers on the X chromosome and male sexual orientation.

The role of genetics in male sexual orientation was investigated by pedigree and linkage analyses on 114 families of homosexual men. Increased rates of same-sex orientation were found in the maternal uncles and male cousins of these subjects, but not in their fathers or paternal relatives, suggesting the possibility of sex-linked transmission in a portion of the population. DNA linkage analysis of a selected group of 40 families in which there were two gay brothers and no indication of nonmaternal transmission revealed a correlation between homosexual orientation and the inheritance of polymorphic markers on the X chromosome in approximately 64 percent of the sib-pairs tested. The linkage to markers on Xq28, the subtelomeric region of the long arm of the sex chromosome, had a multipoint lod score of 4.0 (P = 10(-5), indicating a statistical confidence level of more than 99 percent that at least one subtype of male sexual orientation is genetically influenced.

Female↗
PubMed1993

Glycosidase digestion, electrophoresis and chromatographic analysis of recombinant human granulocyte colony-stimulating factor glycoforms produced in Chinese hamster ovary cells.

Recombinant human granulocyte colony stimulating factor (G-CSF) produced in Chinese hamster ovary cells is glycosylated. The carbohydrate compositional analysis indicated that G-CSF molecule contains sialic acid, galactose and galactosamine. By isolation and characterization of the purified glycopeptides obtained from cleavages by Staphylococcal aureus V-8 protease and cyanogen bromide, the O-linked glycosylation site was confirmed to be a Thr residue at position 133. Neuraminidase and O-glycanase digestion followed by sodium dodecyl sulfate polyacrylamide and isoelectric focusing gel electrophoreses distinguished two possible carbohydrate structures attached at Thr-133: structure A, NeuNAc-Gal-beta(1,3)-GalNAc-O-Thr; and structure B, NeuNAc-Gal-beta(1,3)-[NeuNAc]-GalNAc-O-Thr. Different glycoforms, undigested or after glycosidase digestion, can also be separated by ion-exchange or reversed-phase high-performance liquid chromatography. The approach described in this report provides a simple and valuable procedure to characterize glycoprotein structures containing simple carbohydrate moieties.

Amino Acid Sequence↗
PubMed1993

Effects of cytokines on multiplication of Toxoplasma gondii in microglial cells.

Microglia may play a role in host defense against Toxoplasma gondii in the central nervous system. In our study, the antitoxoplasma activity of murine microglia was investigated. T. gondii multiplied readily in cultured microglial cells, and activation of these cells with IFN-gamma plus LPS suppressed (p < 0.01) intracellular growth of this parasite. This antitoxoplasma defense of activated microglia was significantly blocked by neutralizing antibodies against TNF-alpha and transforming growth factor-beta, suggesting that these cytokines play a role in the inhibitory process. Consistent with this hypothesis, activation of microglia with IFN-gamma plus TNF-alpha or transforming growth factor-beta inhibited, in a dose-dependent manner, T. gondii multiplication. Treatment with pentoxifylline or dexamethasone, drugs that suppress production of TNF-alpha, prevented (p < 0.01) the microglia from exerting an anti-T. gondii effect. These findings suggest that the host defense function of microglia against T. gondii is dependent primarily on the activating properties of IFN-gamma and TNF-alpha, and that agents that interfere with cytokine-mediated activation may promote the pathogenesis of this infection.

Animals↗
PubMed1993

Identification of a chemotactic domain of the pro-inflammatory S100 protein CP-10.

We previously reported the purification and partial amino acid sequence of a novel murine cytokine designated CP-10, which has chemotactic activity for murine polymorphonuclear cells (PMN) and macrophages. The complete cDNA encoding an 88-amino acid polypeptide has been isolated and the sequence is presented here. Transient transfection of CP-10 cDNA into CV-1 cells confirmed the chemotactic activity of rCP-10 for murine PMN. CP-10 has sequence homology with members of the S100 family of Ca(2+)-binding proteins with pronounced amino acid sequence similarities within the putative N- and C-terminal Ca(2+)-binding sites, but differences within their connecting hinge and C-terminal regions. We have confirmed the hypothesis of Kligman and Hilt that functional specificity of individual members of the S100 protein family may reside in the hinge region. A synthetic peptide corresponding to the hinge region of CP-10 (CP-10(42-55) was compared with native CP-10 in chemotaxis and skin test assays. Native CP-10 had potent activity for phagocytic cells, but not lymphocytes, in vitro (optimal activity, 10(-11) to 10(-13) M) and elicited a sustained recruitment of neutrophils and mononuclear cells over 24 h in vivo. The hinge-region peptide had strong chemotactic activity for murine phagocytic cells (optimal activity, 10(-10) - 10(-11) M) but elicited only a transient infiltration of neutrophils over 4 to 8 h after intradermal injection. Results indicate that although the hinge region contributes significantly to the functional specificity of the S100 protein CP-10, sustained cellular recruitment typical of a delayed type hypersensitivity response is apparently dependent on the structural integrity of the protein.

Amino Acid Sequence↗
PubMed1993

Heme active-site structural characterization of chloroperoxidase by resonance Raman spectroscopy.

Resonance Raman spectra are reported for the nitric oxide adducts of ferric and ferrous chloroperoxidase and carbon monoxide adducts of ferrous chloroperoxidase. The stretching, v(Fe-NO), and bending, delta(FeNO), modes are detected at 538 and 558 cm-1, respectively, for the ferric nitrosylchloroperoxidase. These two bands shift to 534 and 546 cm-1, respectively, upon substitution by 15N16O. The v(Fe-NO) mode of the nitric oxide adduct of ferrous chloroperoxidase is located at 542 cm-1, which shifts to 528 (15N16O), 540 (14N18O), and 524 (15N18O) cm-1 as the mass of the bound nitric oxide increases by 1 atomic unit. Two distinct states of the carbon monoxide adduct of chloroperoxidase, the acidic and alkaline forms, are found to undergo a reversible pH-induced transition. The v(Fe-CO) mode shifts from 484 to 492 cm-1 and the delta(FeCO) mode at 562 cm-1 disappears as the pH is reduced from 6.0 to 3.3. In addition, two low frequency modes at 382 and 420 cm-1, assignable to the delta(CbC1C2) bending of propionate and vinyl groups, respectively, also show pH sensitivity. The results suggest a peroxidase-like heme active-site environment for chloroperoxidase and indicate a facile conformational change of heme groups accompanying the acid-base transition.

Binding Sites↗
PubMed1993

Activation of K+ channel in vascular smooth muscles by cytochrome P450 metabolites of arachidonic acid.

Arachidonic acid can be oxidatively metabolized by cytochrome P450 epoxygenase to four regioisomeric epoxyeicosatrienoic acids (5,6-; 8,9-; 11,12-; 14,15-EET), which exhibit vasorelaxant effects in vivo and in vitro with unknown mechanisms. In this study, the patch-clamp method was used to examine the effects of EETs on the Ca(2+)-activated K+ channel in cells from rabbit portal vein, rat caudal artery, guinea pig aorta and porcine coronary artery. In all four cell types, EETs in the bath activated the K+ channel in cell-attached patches by increasing the single channel open-state probability. Potencies of the four EETs did not differ significantly for each cell type. The concentrations for doubling open-state probability were 0.1 microM in portal vein and coronary artery, 0.3-1 microM in aorta and 1-3 microM in caudal artery. In caudal artery cells, K+ channel activation by 3 microM 5,6- and 1 microM 11,12-EET was blocked and reversed by glyburide at 0.5 microM. In aorta, coronary artery, and caudal artery cells, micromolar EETs induced a dose-dependent and reversible augmentation of whole-cell K+ current by 50-120% and a 5-12 mV hyperpolarization. EETs on the cytosolic side of inside-out patches produced little or no potentiation of K+ channels, implying an interaction of receptor-mediated nature. Thus, EETs may promote vasodilation by functioning as endogenous K+ channel openers.

Animals↗
PubMed1993

Activated microglia inhibit multiplication of Toxoplasma gondii via a nitric oxide mechanism.

The role of microglia in host defense against Toxoplasma gondii is unknown. In the present study, we investigated the multiplication of T. gondii tachyzoites in murine microglial cell cultures. T. gondii multiplied readily in these cells; multiplication was prevented when microglia were activated with interferon-gamma plus lipopolysaccharide, a treatment that also upregulates nitric oxide (NO) synthase activity. Simultaneous treatment of microglial cell cultures with activation signals and the NO synthase inhibitor NG-monomethyl-L-arginine (NGMA) prevented the antitoxoplasmic activity. Transmission electron microscopic analysis demonstrated degenerative tachyzoites in activated microglia but not in control or NGMA groups. These findings support the view that the host defense function of activated microglia against T. gondii involves generation of the free radical NO.

Animals↗
PubMed1993

Recombinant human transforming growth factor-beta 1: expression by Chinese hamster ovary cells, isolation, and characterization.

A partial cDNA clone encoding the gene for human transforming growth factor-beta 1 (TGF-beta 1) was isolated from a human bladder carcinoma cell line (5637) cDNA library. Following restriction enzyme processing and ligation of synthetic oligonucleotide linkers, the gene was inserted into a plasmid and transfected into Chinese hamster ovary cells. Clonal selection and growth conditions resulted in a method for production of recombinant human TGF-beta 1 at 7 mg/liter in conditioned cell medium. Through a combination of low pH treatment, cation-exchange chromatography, and salt precipitation, the recombinant human TGF-beta 1 was purified in milligram amounts to > 95% purity in a yield of about 36%. Purification to homogeneity was accomplished by chromatography on C18 silica gel. Amino acid analysis, N-terminal sequencing, and growth inhibition assays indicate identity with the molecule from human platelets.

Amino Acid Sequence↗
PubMed1993

Morphine inhibits the release of tumor necrosis factor in human peripheral blood mononuclear cell cultures.

Opiates modulate a variety of immune responses of peripheral blood mononuclear cells (PBMC). When PBMC were treated with morphine for 24 h, cells released less (P < 0.05) bioactive TNF, a cytokine important in host defense, in the following 24-h incubation period when stimulated with lipopolysaccharide and phytohemagglutinin. Morphine alone did not significantly alter the release of TNF from PBMC cultures. Pretreatment of PBMC cultures for 1 h with naloxone blocked (P < 0.05) the inhibitory effect of morphine on the release of TNF upon stimulation with phytohemagglutinin, but not with lipopolysaccharide, suggesting the involvement of an opioid receptor. The mechanism of morphine-induced suppression of TNF release appears to be counteracted by the effect of this opiate on the release of transforming growth factor (TGF)-beta, since antibodies to this immunoregulatory cytokine further enhanced morphine-related inhibition of TNF release. Taken together, these findings indicate that morphine suppresses the release of bioactive TNF from PBMC and that TGF-beta plays a modulatory role in this inhibitory process.

Adult↗
PubMed1993

Intracellular survival and multiplication of Toxoplasma gondii in astrocytes.

Primary neonatal murine astrocyte cultures were used to investigate the role of these glial cells in host defense of the central nervous system (CNS) against Toxoplasma gondii. For comparison, neonatal murine microglial cells were also studied. Microscopic analyses revealed that uptake of T. gondii into astrocytes was parasite-driven and was followed by uniform intracellular survival and multiplication of tachyzoites. Treatment of astrocytes with interferon (IFN)-gamma and lipopolysaccharide (LPS) had no apparent effect on the survival or growth of T. gondii. Microglia, on the other hand, had both an intrinsic phagocytosis-associated antitoxoplasma activity and a nitric oxide-dependent inhibitory activity that was up-regulated by IFN-gamma and LPS. The results of this study suggest that in contrast to microglial cells, astrocytes may provide a safe harbor within the CNS for T. gondii.

Animals↗
PubMed1993

Coupling of endothelin receptors to ion channels in rat glomerular mesangial cells.

Endothelin (ET) induces depolarization and contraction of glomerular mesangial cells (MCs), thereby influencing intraglomerular hemodynamics and filtration rate. In an attempt to clarify the ionic mechanism by which ET regulates MC function, we examined, using the whole-cell configuration of the patch-clamp technique, the effects of ET-1 and its related peptides, ET-3, sarafotoxin 6c (S6c), and IRL 1620, on ion currents and membrane potential in the primary culture of rat MCs. The resting potential of MCs was -48.4 +/- 1.9 mV (n = 23). It depolarized in response to ET-1, ET-3, and IRL 1620 by 14 (n = 7), 8 (n = 5), and 13 mV (n = 9), respectively. Whole-cell recording in combination with ion substitution ascertained the coexistence of potassium (IK) and chloride (ICl) currents. ET-1 (0.01-100 nM), ET-3 (1-100 nM), IRL 1620 (0.1-100 nM), and S6c (0.01-10 nM) augmented ICl in a concentration-dependent fashion, with ET-1 and S6c being the most potent. These actions were blocked by IRL 1038, a selective ETB receptor antagonist, but not by 1 microM BQ 123 (a selective ETA receptor antagonist) or 0.1 microM nifedipine (an L-type Ca(2+)-channel blocker). These results suggest a close coupling of the ETB receptor to ICl. ET-1, IRL 1620, and SRTX-6c in a similar concentration range also caused suppression of IK. This action was partially blocked by IRL 1038 and minimally affected by BQ 123, indicating a contributory role for ETB receptors in the regulation of IK.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Sequence variation in the androgen receptor gene is not a common determinant of male sexual orientation.

To test the hypothesis that DNA sequence variation in the androgen receptor gene plays a causal role in the development of male sexual orientation, we have (1) measured the degree of concordance of androgen receptor alleles in 36 pairs of homosexual brothers, (2) compared the lengths of polyglutamine and polyglycine tracts in the amino-terminal domain of the androgen receptor in a sample of 197 homosexual males and 213 unselected subjects, and (3) screened the the entire androgen receptor coding region for sequence variation by PCR and denaturing gradient-gel electrophoresis (DGGE) and/or single-strand conformation polymorphism analysis in 20 homosexual males with homosexual or bisexual brothers and one homosexual male with no homosexual brothers, and screened the amino-terminal domain of the receptor for sequence variation in an additional 44 homosexual males, 37 of whom had one or more first- or second-degree male relatives who were either homosexual or bisexual. These analyses show that (1) homosexual brothers are as likely to be discordant as concordant for androgen receptor alleles; (2) there are no large-scale differences between the distributions of polyglycine or polyglutamine tract lengths in the homosexual and control groups; and (3) coding region sequence variation is not commonly found within the androgen receptor gene of homosexual men. The DGGE screen identified two rare amino acid substitutions, ser205-to-arg and glu793-to-asp, the biological significance of which is unknown.

Base Sequence↗