Biomedical subjects

J Zheng

Publications and source records attributed to J Zheng.

At least 253 records · Page 14Linked to original sources

PubMed1999

Modification of isolation and culture of human retinal pigment epithelial cells.

PURPOSE: To modify the isolation of human retinal pigment epithelial (RPE) cells and to increase the purification and production of cultured RPE cells. METHODS: The human eyecups were fixed on a rubber holder. After digestion by trypsin, RPE cells were collected, then cultured and identified by morphology, immunohistochemistry and electron microscopy. RESULTS: The cultured RPE cells grew actively in the early stage with transparent nucleus and abundant melanin particles in cytoplasm. These cells were positive in DOPA oxidase reaction and in anti-pancytokeratin antibody staining. Cellular microvilli and tight junctions could be seen through transmission electron microscopy. CONCLUSION: We developed a rubber holder to fix the eyecup. Using this holder, more and purer cultured RPE cells can be obtained. These cultured RPE cells are similar to those in vivo in morphology and immunohistochemical staining.

Cell Separation↗

PubMed1999

Rapid diagnosis of chlamydial conjunctivitis in laboratory.

PURPOSE: To evaluate the techniques of rapid and accurate diagnosis of chlamydial conjunctivitis. METHODS: Total 100 conjunctivitis patients (200 eyes) were studied. Twenty-two of 100 cases were diagnosed as chlamydial conjunctivitis. The infected epithelia were scraped from tarsal conjunctiva of both eyes and stained separately with Giemsa (100 cases) and immunofluorescence (anti-chlamydial antigen monoclonal antibody, 100 cases). RESULT: In immunofluorescent staining, 38 cases were seen positive staining and 62 were negative. In Giemsa staining, 29 were positive, and 71 were negative. In 22 cases with clinical diagnosis of chlamydial conjunctivitis, 13 cases were confirmed, and 9 were excluded by immunofluorescent staining. Technically, immunofluorescent and Giemsa stain takes 45 and 40 minutes, respectively. CONCLUSION: Comparing to Giemsa stain, 38 of 100 scraping specimens were positive (38%) by immunofluorescent staining, 29 of 100 per cent were positive by Giemsa staining. Giemsa staining takes 5 minutes less than immunofluorescent staining (40 versus 45 minutes), however, the positive staining in immunofluorescent staining is much easier to be recognized than Giemsa staining.

Adolescent↗

PubMed1998

BIBP 3226 inhibition of nicotinic receptor mediated chromaffin cell secretion.

(R)-N 2-(diphenacetyl)-N-[(4-hydroxyphenyl)methyl]-argininamide (BIBP 3226) is a selective neuropeptide Y Y1 receptor antagonist with structural similarity to the C-terminal tripeptide of neuropeptide Y. Based on this similarity we questioned whether BIBP 3226 could act as an agonist. Incubation of BIBP 3226 with bovine chromaffin cells in culture results in the inhibition of nicotinic receptor-stimulated catecholamine secretion (IC50 = 2.4 microM). The effect of BIBP 3226 is independent of neuropeptide Y action since the presence of neuropeptide Y in the culture medium does not alter the effect of BIBP 3226. BIBP 3226 decreased the efficacy of the nicotinic receptor agonist, 1,1-dimethyl-4-phenylpiperizinium (DMPP), but did not change its potency suggesting non-competitive inhibition. BIBP 3226 has a similar effect on nicotinic receptor-stimulated 45Ca2+ influx. BIBP 3226 does not inhibit [3H]norepinephrine release induced by high K+ and its effect is not pertussis toxin-sensitive. We conclude that not only can BIBP 3226 act as a neuropeptide Y receptor antagonist in bovine chromaffin cells but also act as an agonist and inhibit catecholamine secretion.

Animals↗

PubMed1998

Cloning and characterization of a symbiosis-related gene from an ectomycorrhizal fungus Laccaria bicolor.

An in vitro system for a Laccaria bicolorxPinus resinosa interaction was used to identify and clone a symbiosis-regulated gene from L. bicolor employing the mRNA differential display technique (DDRT-PCR). The DDRT-PCR identified several cDNAs that are differentially expressed as early as 6h into the interaction. One such cDNA was used to screen a L. bicolor cDNA library enriched for mRNAs expressed during early interaction with red pine seedlings. Characterization of a cDNA clone, PF6.2, showed that it contained a 1551 bp insert coding for a protein of 433 amino acids. Sequence analysis of the PF6.2 cDNA revealed the presence of several evolving repeats in the protein. To confirm this, the gene corresponding to PF6.2 was isolated and sequenced. The PF6.2 gene consisted of seven exons interrupted by six relatively small introns. Although the amino-acid sequence of the PF6.2 did not show significant overall similarity to any previously characterized proteins, of several direct repeats it contained a feature similar to other proteins involved in signal transduction through protein-protein interaction. Northern analysis showed that the PF6.2 mRNA was detectable in the fungus 6h after interaction and continued to be expressed in established ectomycorrhizas, suggesting that it plays an important role in the formation and maintenance of the symbiosis.

Agaricales↗

PubMed1998

Inhibitor-1 interaction domain that mediates the inhibition of protein phosphatase-1.

Inhibitor-1 (I-1), a cyclic AMP-regulated phosphoprotein, inhibits protein phosphatase-1 (PP1) activity in response to hormones. The molecular mechanism for PP1 inhibition by I-1 remains unknown. Mutation of nine acidic residues lining a proposed I-1-binding channel in rabbit PP1alpha yielded one mutant (E256A) slightly impaired in its inhibition by I-1, with the IC50 increased by 3-fold, and one mutant (E275R) located in the beta12-beta13 loop that showed 4-fold enhanced inhibition by I-1. Substituting Tyr-272, a proposed binding site for the toxins okadaic acid and microcystin-LR, in the beta12-beta13 loop with Trp, Phe, Asp, Arg, or Ala impaired PP1alpha inhibition by I-1 by 8-10-fold. Chemical mutagenesis of the Saccharomyces cerevisiae PP1 gene (GLC7) yielded 20 point mutations in the PP1 coding region. Two-hybrid analyses and biochemical assays of these yeast enzymes identified four additional residues in the beta12-beta13 loop that were required for PP1 binding and inhibition by I-1. Ten-fold higher concentrations of I-1 were required to inhibit these mutants. Finally, deletion of the beta12-beta13 loop from PP1alpha maintained full enzyme activity, but attenuated inhibition by I-1 by >100-fold. These data identified the beta12-beta13 loop in the PP1 catalytic subunit as a domain that mediates binding and enzyme inhibition by I-1.

Binding Sites↗

PubMed1998

Inhibition of respiratory and bioenergetic mechanisms by hydrogen sulfide in mammalian brain.

The biochemical effects of hydrogen sulfide were investigated by treating enzyme homogenates and synaptosomes prepared from mammalian brain with sodium sulfide. Brain cytochrome c oxidase activity was highly sensitive to inhibition by sodium sulfide, as demonstrated by an IC50 of 0.13 microM. Sodium sulfide was also found to inhibit carbonic anhydrase activity in cerebellum, frontal cortex, and hippocampus. Synaptosomal oxygen consumption was significantly reduced as the concentration of sodium sulfide was increased from 20 to 100 microM; this was accompanied by a concentration-dependent depolarization of the synaptosomal mitochondrial membrane in situ and a reduction in synaptosomal ATP concentration. In other experiments using synaptosomes, sodium sulfide caused a significant calcium-independent increase in the extracellular accumulation of L-glutamate, inhibited Na+-dependent uptake of [3H]glutamate, but was unable to influence intrasynaptosomal free ionic Ca2+. Parallel studies conducted in vivo showed that rats exposed over a 5-d period to hydrogen sulfide (100 ppm for 3 h/d) had significantly higher concentrations of L-glutamate in the hippocampus compared to control animals. In summary, our results indicate that sulfide causes extensive disruption to respiratory and related mitochondrial functions in mammalian brain in vitro. The reduced capacity of nerve endings to take up L-glutamate may contribute to the raised L-glutamate levels observed in vivo.

Adenosine Triphosphate↗

PubMed1998

Aggrecan synthesis and secretion. A paradigm for molecular and cellular coordination of multiglobular protein folding and intracellular trafficking.

Each globular domain of exported multiglobular proteins putatively undergoes chaperone surveillance in the endoplasmic reticulum lumen. It is difficult to visualize how surveillance of multiple globular domains might be orchestrated and regulated. Aggrecan core protein has been used as a prototype for this problem by examining transfection of informative constructs into Chinese hamster ovary cells. The salient results are as follows: 1) aggrecan's N-terminal G1 domain is minimally secreted, and its flanking Golgi reporter sites are not decorated with glycsoaminoglycan chains; in contrast, its C-terminal G3 domain is readily secreted with flanking GAG chains, and G3 also facilitates G1 secretion; 2) G3 but not G1 can be intracellularly cross-linked to chaperone Hsp25; 3) G3 and Hsp25 remain noncovalently bound and are secreted together when G3 is situated N-terminal to its normal location; 4) exon 15, which encodes the center of G3's C-lectin subdomain, is necessary and sufficient for G3 secretion. A model is proposed in which Hsp25 piggybacks onto nascent G3 in the cytosol during a translocational pause and enters the ER lumen with G3, and once G3 properly folds, Hsp25 releases G3 and recycles to the nucleus while G3 continues to the Golgi stacks, providing passage for the entire core protein.

Aggrecans↗

PubMed1998

Structural and functional consequences of haloenol lactone inactivation of murine and human glutathione S-transferase.

Mass spectrometric analysis of proteolysis products of haloenol lactone-modified glutathione S-transferase isozyme mGSTP1 indicates that the haloenol lactone 3-cinnamyl-5(E)-bromomethylidenetetrahydro-2-furanone is covalently attached to the protein at Cys-47. Comparisons of the extent of adduct formation with losses in enzymatic activity indicate that mGSTP1 exhibits greatest reactivity toward the haloenol lactone, followed by mGSTM1 and mGSTA3. Activities of mGSTP1 and mGSTM1 decrease in inverse proportion to haloenol lactone concentration, whereas modification had no apparent effect on catalytic activity of mGSTA3. Decreases in activity agree with the extent of protein modification observed in ESI mass spectra for mGSTP1 and mGSTM1 but not for mGSTA3. Kinetic studies employing recombinant human proteins with replacement of cysteine by serine at Cys-47 and Cys-101 indicate that rapid inactivation (t1/2 = 2 min) occurs only when residue 47 is cysteine. Mass spectra of C47S-hGSTP1 incubated with haloenol lactone demonstrate covalent attachment of a haloenol lactone-glutathione conjugate and suggest that an ester forms between the lactone and Ser-47. Therefore, we propose that initial opening of the lactone ring is promoted by Cys-47 through thioester formation between the lactone carbonyl and the Cys-47 sulfhydryl. Enol-keto tautomerization and enzyme-mediated hydrolytic cleavage of the thioester produces a reactive alpha-bromoketone which reacts a second time with Cys-47 and inactivates the enzyme. These results suggest that Pi class GSTs have thioesterase activity and that haloenol lactone inactivation occurs through an enzyme-mediated process.

4-Butyrolactone↗

PubMed1998

Development of highly selective SH3 binding peptides for Crk and CRKL which disrupt Crk-complexes with DOCK180, SoS and C3G.

Many Src Homology 3 (SH3) domains function as molecular adhesives in intracellular signal transduction. Based on previous ultrastructural studies, short motifs which bind to the first SH3 domains of the adapters Crk and CRKL were selectively mutagenised to generate Crk/CRKL SH3-binding peptides of very high affinity and selectivity. Affinities were increased up to 20-fold compared to the best wildtype sequences, while the selectivity against a similar SH3 domain [Grb2SH3(N)] was not only retained, but sometimes increased. Blot techniques with GST-fusion peptides and in solution precipitation assays with biotinylated high affinity Crk binding peptides (HACBPs) were subsequently used to analyse the binding of these sequences to a large panel of SH3 domain-containing fusion proteins. Only those proteins which contained the CrkSH3(1) or CRKLSH3(1) domains bound efficiently to the HACBPs. A GST-HACBP fusion protein precipitated Crk and CRKL proteins out of 35S-labelled and unlabelled cell lysates. Very little binding of other cellular proteins to HACBP was detectable, indicative of a great preference for Crk and CRKL when compared to the wide variety of other endogenous cellular proteins. Moreover, HACBP disrupted in vitro preexisting Crk-complexes with DOCK180 and the exchange factors SoS and C3G, which are known targets of Crk adapters, in a concentration dependent manner. HACBP-based molecules should therefore be useful as highly selective inhibitors of intracellular signalling processes involving Crk and CRKL.

Adaptor Proteins, Signal Transducing↗

PubMed1998

[Detection of hemophilia A carriers by PCR analysis of hind III polymorphism in the factor VIII gene].

OBJECTIVE: To explore a scheme of using PCR analysis in the detection of carriers of Hind III polymorphism of factor VIII gene of hemophilia A. METHODS: Implicating intron 19 of the factor VIII gene of 6 patients with the hemophilia A and 207 unrelated X-chromosomes were amplified by PCR and were analysed by means of Amp-RFLPs of Hind III. RESULTS: The incidence of the polymorphic Hind III sites in the given population was found to be 0.29. The frequence of the Hind III heterozygotes in women calculated according to Hardy-Weinberg equation was 0.41, which proved to be informative enough for carrier detection and prenatal diagnosis of hemophilia A. 2 out of 6 families (33%)examined in this study were informative. CONCLUSION: The new scheme proved to be effective for hemophilia A carrier detection and prenatal diagnosis.

Deoxyribonuclease HindIII↗

PubMed1998

The solution structure and dynamics of the pleckstrin homology domain of G protein-coupled receptor kinase 2 (beta-adrenergic receptor kinase 1). A binding partner of Gbetagamma subunits.

The solution structure of an extended pleckstrin homology (PH) domain from the beta-adrenergic receptor kinase is obtained by high resolution NMR. The structure establishes that the beta-adrenergic receptor kinase extended PH domain has the same fold and topology as other PH domains, and there are several unique features, most notably an extended C-terminal alpha-helix that behaves as a molten helix, and a surface charge polarity that is extensively modified by positive residues in the extended alpha-helix and the C terminus. These observations complement biochemical evidence that the C-terminal portion of this PH domain participates in protein-protein interactions with Gbetagamma subunits. This suggests that the C-terminal segment of the PH domain may function to mediate protein-protein interactions with the targets of PH domains.

Amino Acid Sequence↗

PubMed1998

Physiological signals and oncogenesis mediated through Crk family adapter proteins.

The viral Crk oncogene (v-Crk) is known to induce sarcomas in chicken and its cellular homologs c-Crk I, c-Crk II, and Crk-like (CRKL) have been implicated in many signal transduction events. These include cell differentiation, cell migration, and the induced nonresponsiveness of T-cells to stimulation of the T-cell receptor (TCR), a state known as anergy. CRKL is also the most prominent substrate of the Bcr-Abl oncoprotein which causes human chronic myelogenous leukemias (CML). The modular composition of the Crk family adapters which largely consist of Src homology (SH2 and SH3) domains has prompted an intensive search for physiological and pathological upstream and downstream signalling partners which selectively bind to these adapters. Upstream proteins include various receptors and large multisite docking proteins, while several protein kinases and guanine nucleotide release proteins (GNRPs) have been suggested to function downstream of c-Crk and CRKL. Most Crk/CRKL SH2- and SH3-binding proteins contain several docking sites with considerable sequence similarity. Thus the binding requirements of Crk/CRKL SH2 and SH3 domains are now well defined, providing a basis for the design of small inhibitory molecules to block the function of these adapter proteins. The enzymatic cascades activated through Crk family adapters are only partially known, but stress kinases (SAPKs/JNKs) and the GTPase Rap1, as well as the B-Raf isoform of the Raf protein kinases, are affected in some systems. Several yet unidentified, highly selective Crk interacting proteins detectable in specific cell types remain to be studied. More detailed analyses of the enzymatic activities triggered through Crk-type adapters will also be crucial to fully define the signalling pathways controlled by this protein family.

Animals↗

PubMed1998

Modification of materials formed from poly(L-lactic acid) to enable covalent binding of biopolymers: application to high-density three-dimensional cell culture in foams with attached collagen.

We describe a method for increasing the hydrophilicity of materials formed from biodegradable polymers and introducing chemical functional groups on their surfaces. Poly(L-lactic acid) was blended with poly(epsilon-CBZ-L-lysine) at an 80:20 ratio. Films of the mixture were prepared and foams were made by solvent casting and salt leaching. Amino groups on the surface of the polymer mixture were deprotected by acid hydrolysis. As an example of the applicability of the technique for attachment of biomolecules, we covalently linked collagen to the deprotected amino groups, creating a surface capable of high density growth of a differentiated cell type (bovine adrenocortical cells). The method should be generally useful for surface modification of biodegradable polymer materials used in tissue engineering.

Adrenal Cortex↗

PubMed1998

Matrix metalloproteinases-2 and -9, and tissue inhibitor of metalloproteinases-1 of the sheep placenta during the last third of gestation.

Uteroplacental and fetoplacental blood flows increase dramatically during the last third of gestation. Therefore, angiogenesis and tissue remodelling may regulate placental growth. On days 110, 120, 130, and 142 (term= 145+/-3; n=5 ewes/day) of gestation, sheep placentae were separated into maternal (caruncular and intercaruncular) and fetal (cotyledonary and intercotyledonary) components; tissues were minced and cultured (24 h) and media were analysed for gelatinase activity and for tissue inhibitor of metalloproteinases-1 (TIMP-1) protein. Both the 72-kDa (matrix metalloproteinase-2; MMP-2) and 95-kDa (matrix metalloproteinase-9; MMP-9) gelatinases were detected by zymography. There was more (P<0.001) MMP-2 activity in the caruncle versus intercaruncle and intercotyledon, and all had greater MMP-2 activity (P<0.001) than the cotyledon. There was less (P<0.04) MMP-9 in the intercotyledon than in the other components. By Western analysis, there was more (P<0.004) TIMP-1 (30 kDa) in the caruncle-conditioned media compared to the other tissues, which did not differ from each other (P> or =0.38). The immunohistochemical staining for MMP-2, MMP-9, and TIMP-1 determined that staining was associated with the epithelial cells of the intercotyledon, the uterine glands, cells of the fetomaternal interface of the placentome (trophoblast and binucleate cells), and the endothelial cells of microvessels. Therefore, MMP-2, MMP-9, and TIMP-1 are present in the last third of gestation, suggesting that there is an ongoing tight regulation of tissue remodelling and growth throughout this critical time of pregnancy.

Animals↗

PubMed1998

Modified bilateral cleft lip repair with orbicularis reconstruction and prolabial self-elongation.

PURPOSE: This article describes the details of a modified method for bilateral cleft lip repair and presents some examples of patients whose condition was repaired by this technique. PATIENTS AND METHODS: Sixty-five patients ranging in age from 2 to 5 years were treated with this modified procedure. The method was characterized by prolabial lengthening and reconstruction of the orbicularis oris muscle in one stage. RESULTS: Satisfactory results were achieved with this method. The Cupid's bow and the central tubercle appeared more natural. None of the patients developed dehiscence of the wound. CONCLUSIONS: This modified procedure is of special value for complete bilateral cleft lip patients with a short prolabium. The long-term effect of muscle reconstruction on maxillary growth remains to be evaluated.

Child, Preschool↗

PubMed1998

Downregulation of human FGF8 activity by antisense constructs in murine fibroblastic and human prostatic carcinoma cell systems.

Previously, we described cloning of three alternatively spliced mRNA forms of human FGF8, a, b, and e, of which the b form is the major expressed species in both normal and tumor prostatic epithelial cells. In this report, we describe construction and overexpression of sense and antisense sequences of either the full length FGF8b coding region (215-amino acids or 215aa), 103aa N-terminal part or a smaller N-terminal region (34aa), each including the 23aa putative signal peptide domain, via a retrovirus system. While the morphologic transforming activities of the sense 215aa and 103aa constructs were similar in NIH3T3 cells, 103aa displayed reduced soft agar clonogenic activity. The 34aa construct was practically inert in these assays, although its expression could mimic the ability of 215aa or 103aa in conferring cell growth under reduced serum condition. Overexpression of any of the three constructs in antisense orientation, however, was similarly effective in reversing the morphology and anchorage-independent growth property of FGF8b-transfected NIH3T3 cells. The expression of the antisense 215aa construct significantly reduced the growth rate of the human prostatic carcinoma DU145 cells and inhibited their soft agar clonogenic activity and in vivo tumorigenicity in nude mice. Taken together, these results identify N-terminal portions of FGF8 protein isoform for having the domains necessary for one or more of the biologic effects examined, and suggest that low levels of FGF8 expressed in prostatic epithelial cells may contribute significantly to their growth and tumorigenic properties.

3T3 Cells↗

PubMed1998

Intermediate conductances during deactivation of heteromultimeric Shaker potassium channels.

A previous study of the T442S mutant Shaker channel revealed activation-coupled subconductance levels that apparently represent kinetic intermediates in channel activation (Zheng, J., and F.J. Sigworth. 1997. J. Gen. Physiol. 110:101-117). We have now extended the study to heteromultimeric channels consisting of various numbers of mutant subunits as well as channels without mutant subunits, all in the background of a chimeric Shaker channel having increased conductance. It has been found that activation-coupled sublevels exist in all these channel types, and are traversed in at least 80% of all deactivation time courses. In symmetric K+ solutions, the currents in the two sublevels have a linear voltage dependence, being 23-44% and 54-70% of the fully open conductance. Sublevels in different channel types share similar voltage dependence of the mean lifetime and similar ion selectivity properties. However, the mean lifetime of each current level depends approximately geometrically on the number of mutant subunits in the channel, becoming shorter in channels having fewer mutant subunits. Each mutant subunit appears to stabilize all of the conducting states by approximately 0.5 kcal/mol. Consistent with previous results in the mutant channel, sublevels in channels with two or no mutant subunits also showed ion selectivities that differ from that of the fully open level, having relatively higher K+ than Rb+ conductances. A model is presented in which Shaker channels have two coupled activation gates, one associated with the selectivity filter and a second associated with the S6 helix bundle.

Animals↗

PubMed1998

Suppression of inflammatory neurotoxins by highly active antiretroviral therapy in human immunodeficiency virus-associated dementia.

A human immunodeficiency virus type 1 (HIV)-seropositive, antiretroviral-naive patient presented with significant cognitive dysfunction. Neuropsychologic, neuroradiologic, immunologic, and virologic studies confirmed HIV-associated dementia (HAD). After 12 weeks of highly active antiretroviral therapy (HAART) with ibuprofen, dramatic improvements were demonstrated in neurologic function and were sustained for > 1 year. HIV-1 RNA in cerebrospinal fluid (CSF) decreased from 10(5) to 10(4) copies/mL after 4 weeks. After 20 weeks of therapy, plasma viremia decreased from 10(6) copies/mL to undetectable (< 96 copies/mL). Assays of neurotoxins (tumor necrosis factor-alpha, quinolinic acid, and nitric oxide) in plasma and CSF were considerably elevated at presentation and significantly decreased after therapy. Baseline plasma and CSF demonstrated neurotoxic activities in vitro, which also reduced markedly. These data, taken together, support the notion that HAD is a reversible metabolic encephalopathy fueled by viral replication. HAART used with nonsteroidal antiinflammatory agents leads to the suppression of inflammatory neurotoxins and can markedly improve neurologic function in HAD.

AIDS Dementia Complex↗