Biomedical subjects

Z Huang

Publications and source records attributed to Z Huang.

At least 19 recordsLinked to original sources

PubMed1999

EC3, a novel heterodimeric disintegrin from Echis carinatus venom, inhibits alpha4 and alpha5 integrins in an RGD-independent manner.

EC3, a heterodimeric disintegrin (Mr = 14,762) isolated from Echis carinatus venom is a potent antagonist of alpha4 integrins. Two subunits called EC3A and EC3B were isolated from reduced and alkylated EC3 by reverse-phase high performance liquid chromatography. Each subunit contained 67 residues, including 10 cysteines, and displayed a high degree of homology to each other and to other disintegrins. EC3 inhibited adhesion of cells expressing alpha4beta1 and alpha4beta7 integrins to natural ligands vascular cell adhesion molecule 1 (VCAM-1) and mucosal addressin cell adhesion molecule 1 (MadCAM-1) with IC50 = 6-30 nM, adhesion of K562 cells (alpha5beta1) to fibronectin with IC50 = 150 nM, and adhesion of alphaIIbbeta3 Chinese hamster ovary cells to fibrinogen with IC50 = 500 nM; it did not inhibit adhesion of alphavbeta3 Chinese hamster ovary cells to vitronectin. Ethylpyridylethylated EC3B inhibited adhesion of Jurkat cells to immobilized VCAM-1 (IC50 = 6 microM), whereas EC3A was inactive in this system. The MLDG motif appeared to be essential for activity of EC3B. Linear MLDG peptide inhibited the adhesion of Jurkat to VCAM-1 in a dose-dependent manner (IC50 = 4 mM), whereas RGDS peptide was not active at the same concentration. MLDG partially inhibited adhesion of K562 cells to fibronectin (5-10 mM) in contrast to RGDS peptide (IC50 = 3 mM), inhibiting completely at 10 mM.

Amino Acid Sequence↗

PubMed1999

Expression of the transcriptional repressor protein Kid-1 leads to the disintegration of the nucleolus.

The rat Kid-1 gene codes for a 66-kDa protein with KRAB domains at the NH2 terminus and two Cys2His2-zinc finger clusters of four and nine zinc fingers at the COOH terminus. It was the first KRAB-zinc finger protein for which a transcriptional repressor activity was demonstrated. Subsequently, the KRAB-A domain was identified as a widespread transcriptional repressor motif. We now present a biochemical and functional analysis of the Kid-1 protein in transfected cells. The full-length Kid-1 protein is targeted to the nucleolus and adheres tightly to as yet undefined nucleolar structures, leading eventually to the disintegration of the nucleolus. The tight adherence and nucleolar distribution can be attributed to the larger zinc finger cluster, whereas the KRAB-A domain is responsible for the nucleolar fragmentation. Upon disintegration of the nucleolus, the nucleolar transcription factor upstream binding factor disappears from the nucleolar fragments. In the absence of Kid-1, the KRIP-1 protein, which represents the natural interacting partner of zinc finger proteins with a KRAB-A domain, is homogeneously distributed in the nucleus, whereas coexpression of Kid-1 leads to a shift of KRIP-1 into the nucleolus. Nucleolar run-ons demonstrate that rDNA transcription is shut off in the nucleolar fragments. Our data demonstrate the functional diversity of the KRAB and zinc finger domains of Kid-1 and provide new functional insights into the regulation of the nucleolar structure.

Animals↗

PubMed1999

Multisite autophosphorylation of p21-activated protein kinase gamma-PAK as a function of activation.

p21-activated protein kinase (PAK) is a family of serine/threonine kinases whose activity is stimulated by binding to small G-proteins such as Cdc42 and subsequent autophosphorylation. Focusing on the ubiquitous gamma-isoform of PAK in this study, baculovirus-infected insect cells were used to obtain recombinant gamma-PAK, while native gamma-PAK was isolated from rabbit reticulocytes. Two-dimensional gel electrophoresis of gamma-PAK followed by immunoblot analysis revealed a similar profile for native and recombinant gamma-PAK, both consisting of multiple protein spots. Following Cdc42-stimulated autophosphorylation, the two-dimensional profiles of native and recombinant gamma-PAK were characterized by a similar acidic shift, suggesting a common response to Cdc42. To understand the effect of differential phosphorylation on its activation status, gamma-PAK autophosphorylation was conducted in the presence or absence of activators such as Cdc42 and histone II-AS, followed by tryptic digestion and comparative two-dimensional phosphopeptide mapping. The major phosphopeptides were subjected to a combination of manual and automated amino acid sequencing. Overall, eight autophosphorylation sites were identified in Cdc42-activated gamma-PAK, six of which are in common with those previously reported in alpha-PAK, while Ser-19 and Ser-165 appear to be uniquely phosphorylated in the gamma-form. Further, the phosphorylation of Ser-141, Ser-165, and Thr-402 was found to correlate with gamma-PAK activation.

Amino Acid Sequence↗

PubMed1999

Protein folding at the air-water interface studied with x-ray reflectivity.

We report the results of x-ray reflectivity measurements of thin films formed by different water-soluble proteins at the air-aqueous solution interface. It is demonstrated that glucose oxidase, alcohol dehydrogenase, and urease molecules denaturate at the air-aqueous solution interface to form 8- to 14-A-thick peptide sheets. X-ray reflectivity data indicate that the spreading of a lipid monolayer at the aqueous solution surface before protein injection does not prevent proteins from unfolding. On the other hand, crosslinking of proteins results in intact enzyme layers at the subphase surface. A model that involves interaction of glucose oxidase molecules with a phospholipid monolayer is proposed. In this model, an observed decrease of the lipid electron density in the protein presence is explained in terms of "holes" in the monolayer film caused by protein molecule adsorption.

Animals↗

PubMed1999

Cloning and localization of Rab3 isoforms in bovine, rat, and human parathyroid glands.

Rab3 proteins are small GTP-binding proteins known to play a role in regulated exocytosis processes. This study examines the expression of Rab3 mRNA and protein in bovine, rat and human parathyroid glands. mRNAs of several Rab3 isoforms were detected in bovine (Rab3A, Rab3B and Rab3C) and rat (Rab3A, Rab3B and Rab3D) parathyroid glands by RT-PCR and sequencing. Rab3A protein was detected in the cytosolic extract from bovine parathyroid gland by Western blotting using a monoclonal antibody for Rab3A. Rab3A protein was localized to parathyroid hormone-containing chief cells by immunohistochemical staining. Subcellular localization of Rab3A protein by immunogold electron microscopy revealed that the majority of Rab3A protein was not associated with dense-core vesicles, but localized in the cytosol of the chief cells. Altogether, our results demonstrate that Rab3 isoforms are expressed in parathyroid chief cells, suggesting that they may play a role in regulated exocytosis in these cells.

Animals↗

PubMed1999

Investigation on process parameters involved in preparation of poly-DL-lactide-poly(ethylene glycol) microspheres containing Leptospira Interrogans antigens.

Block copolymer, poly-DL-lactide-poly(ethylene glycol) (PELA) with 11.5% of poly(ethylene glycol) (PEG) content was prepared by bulk ring-opening polymerization using stannous chloride as initiator. PELA microspheres with entrapped Leptospira Interrogans antigens, outer membrane protein (OMP) were elaborated by solvent extraction method based on the formation of multiple w/o/w emulsion, and the resulting microspheres were characterized with respect to particle size, OMP entrapment and morphology characteristics. The purpose of the present study is to perform the optimization of preparative parameters for OMP-loaded PELA micropsheres to control particle size and improve the OMP encapsulation efficiency. Of all the parameters investigated, the polymer concentration of organic phase and the external aqueous phase volume play major roles on particle size, while the organic phase volume, internal aqueous phase volume and the addition of surfactant into the internal aqueous phase display considerable effects on OMP loading efficiency. A small volume of internal aqueous phase and intermediate volumes of organic phase and external aqueous phase were favorable to achieve micropsheres with a size of 1-2 microns and high antigen encapsulation efficiency (70-80%). In vitro OMP release profiles from PELA microspheres consist of a small burst release followed by a gradual release phase. The OMP release rate shows some relations with the porous and water-swollen inner structure of the microspheres matrix. The presence of surfactant in microspheres accelerates OMP release, but the OMP entrapment within microspheres shows limited effects on the release profile.

Antigens, Bacterial↗

PubMed1999

Immunoregulatory role of CD8alpha in the veto effect.

BACKGROUND: Allogeneic bone marrow cell (allo-BMC) infusion induces tolerance to incompatible renal allografts in rhesus macaques after depletion of peripheral T lymphocytes with cytolytic anti-T cell antibodies. The tolerogenic effect of allo-BMC, ascribed to a veto mechanism, associates with specific functional deletion of antidonor cytotoxic T lymphocyte precursor (CTLp), and is dependent on a CD8+ donor BMC subset. In previous studies, the CD8 molecule was implicated by loss of suppression after blocking interaction between CD8 on allo-BMC and major histocompatibility complex class Ialpha3 domain on CTLp. CD8 cross-linking on BMC induced secretion of active transforming growth factor-beta1 (TGF-beta1), suggesting a regulatory mechanism(s) operating via a CD8-mediated signaling pathway. METHODS: CD8 on rhesus cells was cross-linked using IgG-conjugated beads, and TGF-beta1 mRNA and protein were quantified. CD8+ cells were tested for veto activity by mixed lymphocyte reaction (MLR)-induced cell-mediated lymphocytotoxicity (CML) assay. Activated rhesus T cells exposed to TGF-beta1 were examined for apoptosis by TdT-mediated end-labeling and annexin staining. RESULTS: CD8 cross-linking induces accumulation of TGF-beta1 mRNA and protein. Both CD3- CD8+CD16+ and CD3+ CD8+CD16- subsets of allo-BMC up-regulate TGF-beta1 mRNA after CD8 cross-linking, and exhibit veto activity. The CD3-CD8+CD16+ subset expresses more TGF-beta1 mRNA and increased veto activity at low BMC/CTLp ratios. Exposure of activated T cells to TGF-beta1 induces apoptosis. CONCLUSIONS: CD8+ allo-BMC are enriched for veto activity and activation via CD8 induces TGF-beta1 mRNA and protein accumulation. These results agree with the hypothesis that paracrine TGF-beta1 may be involved in peripheral deletion of alloreactive CTLp by CD8+ allo-BMC. We suggest that TGF-beta1 overexpression by donor lymphohematopoietic cells may enhance tolerance induction.

Animals↗

PubMed1999

Substituted furans as inhibitors of the PDE4 enzyme.

The synthesis and in vitro activity of a series of substituted furans as a novel structural class of PDE4 inhibitors is described. Comparison of emetic threshold with known PDE4 inhibitors is presented.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1999

Urease and hexadecylamine-urease films at the air-water interface: an x-ray reflection and grazing incidence x-ray diffraction study.

We report the results of surface x-ray scattering measurements performed on urease and hexadecylamine-urease films at the air-aqueous solution interface. It is demonstrated that although hexadecylamine does not form a stable monolayer on the pure aqueous surface, it does self-assemble into a stable, well-organized structure when spread on top of a urease film at the air-water interface. It is also likely that protein and hexadecylamine domains coexist at the interface.

Air↗

PubMed1999

Fathering pregnancies: marking health-risk behaviors in urban adolescents.

PURPOSE: To establish self-reported rates and associated correlates of fathering pregnancies in urban teen males, and to explore the possibility of using their pregnancy history as a marker for other health-risk behaviors. METHODS: A blinded, self-administered questionnaire was given to the predominantly African-American patients of an inner-city adolescent outpatient clinic. Urine was also collected in a blinded, anonymous fashion, matched to the questionnaires, and tested for five drugs of abuse. Males were classified as having or not having a pregnancy history (PH) according to a questionnaire response item. PH and non-PH participants were compared for eight risk factors using univariate and multivariate methods. RESULTS: A total of 24.2% reported a PH. These males were about 14 times more likely to report three or more sex partners in the last year, more than five times as likely to report a sexually transmitted disease history, more than three times as likely to test positive for drugs, and more than 2.5 times as likely to be inconsistent or nonusers of condoms as compared to males without a pregnancy history. An association between violent behavior and PH is unsupported. CONCLUSIONS: Pregnancy history can be a valuable marker for other risk factors among inner-city African-American males. With some patients, it may be easier for clinicians to discuss pregnancy history or fatherhood as opposed to drug abuse and other more sensitive risk factors. The topic can then be used as a gateway for discussion of other risk factors.

Adolescent↗

PubMed1999

Green fluorescent protein as a reporter to monitor gene expression and food colonization by Aspergillus flavus.

Transformants of Aspergillus flavus containing the Aequorea victoria gfp gene fused to a viral promoter or the promoter region and 483 bp of the coding region of A. flavus aflR expressed green fluorescence detectable without a microscope or filters. Expression of green fluorescent protein fluorescence was correlated with resistance to aflatoxin accumulation in five corn genotypes inoculated with these transformants.

Aspergillus flavus↗

PubMed1999

Role of signal transduction in internalization of the G protein-coupled receptor for parathyroid hormone (PTH) and PTH-related protein.

For G protein-coupled receptors, limited information is available on the role of agonist binding or of the second-messenger products of receptor signaling on receptor endocytosis. We explored this problem using the opossum PTH/PTH-related protein (PTHrP) receptor, a prototypical Class II G protein-coupled receptor, as a model. In one approach, we evaluated the endocytic properties of mutated forms of the opossum PTH/PTHrP receptor that we had previously shown to be impaired in their ability to initiate agonist-induced signaling when expressed in COS-7 cells. A point mutation in the third cytoplasmic loop (K382A) that severely impairs PTH/PTHrP receptor signaling significantly reduced internalization, whereas two mutant receptors that displayed only partial defects in signaling were internalized normally. To explore more directly the role of second-messenger pathways, we used a cleavable biotinylation method to assess endocytosis of the wild-type receptor stably expressed in human embryonic kidney (HEK) 293 cells. A low rate of constitutive internalization was detected (<5% over a 30-min incubation at 37 C); the rate of receptor internalization was enhanced about 10-fold by the receptor agonists PTH(1-34) or PTHrP(1-34), whereas the receptor antagonist PTH(7-34) had no effect. Forskolin treatment produced a minimal increase in constitutive receptor endocytosis, and the protein kinase (PK)-A inhibitor H-89 failed to block agonist-stimulated endocytosis. Similarly, activation of PK-C, by treatment with phorbol 12-myristate 13-acetate, elicited only a minimal increase in constitutive receptor endocytosis; and blockade of the PK-C pathway, by treatment with a bisindolylmaleimide, failed to inhibit agonist-induced receptor endocytosis. Immunofluorescence confocal microscopic studies of PTH/PTHrP receptor internalization confirmed the results using receptor biotinylation. These findings suggest that: 1) agonist binding is required for the efficient endocytosis of the PTH/PTHrP receptor; 2) receptor activation (agonist-induced receptor conformational change) and/or coupling to G proteins plays a critical role in receptor internalization; and 3) activation of PK-A and PK-C is neither necessary nor sufficient for agonist-stimulated receptor internalization.

Animals↗

PubMed1999

The snoring spectrum: acoustic assessment of snoring sound intensity in 1,139 individuals undergoing polysomnography.

STUDY OBJECTIVES: To quantify the snoring sound intensity levels generated by individuals during polysomnographic testing and to examine the relationships between acoustic, polysomnographic, and clinical variables. DESIGN: The prospective acquisition of acoustic and polysomnographic data with a retrospective medical chart review. SETTING: A sleep laboratory at a primary care hospital. PARTICIPANTS: All 1,139 of the patients referred to the sleep laboratory for polysomnographic testing from 1980 to 1994. INTERVENTIONS: The acoustic measurement of snoring sound intensity during sleep concurrent with polysomnographic testing. MEASUREMENTS AND RESULTS: Four decibel levels were derived from snoring sound intensity recordings. L1, L5, and L10 are measures of the sound pressure measurement in decibels employing the A-weighting network that yields the response of the human ear exceeded, respectively, for 1, 5, and 10% of the test period. The Leq is a measure of the A-weighted average intensity of a fluctuating acoustic signal over the total test period. L10 levels above 55 dBA were exceeded by 12.3% of the patients. The average levels of snoring sound intensity were significantly higher for men than for women. The levels of snoring sound intensity were associated significantly with the following: polysomnographic testing results, including the respiratory disturbance index (RDI), sleep latency, and the percentage of slow-wave sleep; demographic factors, including gender and body mass; and clinical factors, including snoring history, hypersomnolence, and breathing stoppage. Men with a body mass index of > 30 and an average snoring sound intensity of > 38 dBA were 4.1 times more likely to have an RDI of > 10. CONCLUSIONS: Snoring sound intensity levels are related to a number of demographic, clinical, and polysomnographic test results. Snoring sound intensity is closely related to apnea/hypopnea during sleep. The noise generated by snoring can disturb or disrupt a snorer's sleep, as well as the sleep of a bed partner.

Adult↗

PubMed1998

A retinal axon fascicle uses spitz, an EGF receptor ligand, to construct a synaptic cartridge in the brain of Drosophila.

Photoreceptor axons arriving in the Drosophila brain organize their postsynaptic target field into a precise array of five neuron "cartridge" ensembles. Here we show that Hedgehog, an initial inductive signal transported along retinal axons from the developing eye, induces postsynaptic precursor cells to express the Drosophila homolog of the epidermal growth factor receptor (EGFR). The EGFR ligand Spitz, a signal for ommatidial assembly in the compound eye, is transported to retinal axon termini in the brain where it acts as a local cue for the recruitment of five cells into a cartridge ensemble. Hedgehog and Spitz thus bring about the concerted assembly of ommatidial and synaptic cartridge units, imposing the "neurocrystalline" order of the compound eye on the postsynaptic target field.

Animals↗

PubMed1998

Cleavage and activation of p21-activated protein kinase gamma-PAK by CPP32 (caspase 3). Effects of autophosphorylation on activity.

p21-activated protein kinase gamma-PAK (Pak2, PAK I) is cleaved by CPP32 (caspase 3) during apoptosis and plays a key role in regulation of cell death. In vitro, CPP32 cleaves recombinant gamma-PAK into two peptides; 1-212 contains the majority of the regulatory domain whereas 213-524 contains 34 amino acids of the regulatory domain plus the entire catalytic domain. Following cleavage, both peptides become autophosphorylated with [gamma-32P]ATP. Peptide 1-212 migrates at 27,000 daltons (p27) upon SDS-polyacrylamide gel electrophoresis and at 32,000 daltons following autophosphorylation on serine (p27P); the catalytic subunit migrates at 34,000 daltons (p34) before and after autophosphorylation on threonine. Following caspase cleavage, a significant lag (approximately 5 min) is observed before autophosphorylation and activity are detected. When gamma-PAK is autophosphorylated with ATP(Mg) alone and then cleaved, only p27 contains phosphate, and the enzyme is inactive with exogenous substrate. After autophosphorylation of gamma-PAK in the presence of Cdc42(GTPgammaS) or histone 4, both cleavage products contain phosphate and gamma-PAK is catalytically active. Mutation of the conserved Thr-402 to alanine greatly reduces autophosphorylation and protein kinase activity following cleavage. Thus activation of gamma-PAK via cleavage by CPP32 is a two-step mechanism wherein autophosphorylation of the regulatory domain is a priming step, and activation coincides with autophosphorylation of the catalytic domain.

Caspase 3↗

PubMed1998

Isolation and sequence of cDNA encoding the motilin precursor from monkey intestine. Demonstration of the motilin precursor in the monkey brain.

The motilin precursor cDNA has been isolated and sequenced from a cDNA library prepared from monkey small intestine. The sequence indicates a 345 bp open reading frame, a 63 bp 5' untranslated region and a 154 bp 3' untranslated region. The sequence encodes a 115 amino acid motilin precursor composed of a 25 amino acid signal peptide, the 22 amino acid motilin peptide and a 68 amino acid motilin associated peptide (MAP). Compared with the human motilin precursor cDNA, there are two amino acid substitutions in the signal peptide, one in motilin and four in the MAP. The presence of the motilin precursor in hypothalamus, hippocampus and cerebellum was demonstrated by RT-PCR.

Amino Acid Sequence↗

PubMed1998

Stroke protection by 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors mediated by endothelial nitric oxide synthase.

The treatment of ischemic strokes is limited to prophylactic agents that block the coagulation cascade. Here, we show that cholesterol-lowering agents, 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors, protect against cerebral injury by a previously unidentified mechanism involving the selective up-regulation of endothelial NO synthase (eNOS). Prophylactic treatment with HMG-CoA reductase inhibitors augments cerebral blood flow, reduces cerebral infarct size, and improves neurological function in normocholesterolemic mice. The up-regulation of eNOS by HMG-CoA reductase inhibitors is not associated with changes in serum cholesterol levels, but is reversed by cotreatment with L-mevalonate and by the downstream isoprenoid, geranylgeranyl pyrophosphate and not by farnesyl pyrophosphate. The blood flow and neuroprotective effects of HMG-CoA reductase inhibitors are completely absent in eNOS-deficient mice, indicating that enhanced eNOS activity by HMG-CoA reductase inhibitors is the predominant if not the only mechanism by which these agents protect against cerebral injury. Our results suggest that HMG-CoA reductase inhibitors provide a prophylactic treatment strategy for increasing blood flow and reducing brain injury during cerebral ischemia.

Animals↗

PubMed1998

Identification of the CD8 DE loop as a surface functional epitope. Implications for major histocompatibility complex class I binding and CD8 inhibitor design.

We used an approach of protein surface epitope mapping by synthetic peptides to analyze the surface structure-function relationship of the CD8 protein. Small synthetic peptide mimics of the CD8 DE loop were shown to effectively block CD8 binding to major histocompatibility complex (MHC) class I molecules and possess significant inhibitory activity on in vitro CD8(+) T cell function. These results suggested that the DE loop region of the CD8 protein is an important functional epitope mediating CD8-MHC class I interaction and the activation of CD8(+) T cells, a finding that is consistent with the recently reported crystal structure of the CD8-MHC class I complex. The structural basis for the biological activity of the DE loop peptide was further analyzed in a series of analogs containing alanine substitutions. This study provides support for the concept of bioactive peptide design based on protein surface epitopes and suggests that such an approach may be applicable to other protein-protein complexes, particularly those of immunoglobulin superfamily molecules.

Amino Acid Sequence↗