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Y Zhou

Publications and source records attributed to Y Zhou.

At least 595 records · Page 33Linked to original sources
PubMed1997

Temporal and spatial control of murine GATA-3 transcription by promoter-proximal regulatory elements.

GATA-3 is expressed in a temporally dynamic manner and fulfills vital functions during vertebrate fetal development. Homozygous mGATA-3 mutant embryos die at midgestation, thus complicating the analysis of its contribution to the development of specific cell fates in the many tissues where it is expressed during embryogenesis. We show here that the elements controlling GATA-3 regulation can be precisely refined, using transgenic mice, to discrete cis-acting domains: within 6 kb surrounding the transcriptional initiation site, separate sequences were found to control the expression of mGATA-3 in early muscle masses, in a subset of PNS neurons, in the genital tubercle, and in the branchial arches. The branchial arch regulatory element is particularly robust and was refined to a discrete enhancer sequence lying between nt -2832 and -2462 from the transcription initiation site. The enhancer contains potential binding sites for many well-characterized transcription factors, suggesting that mGATA-3 transcriptional activity may be regulated by these proteins (or related family members) in the mesenchyme of the arches that contribute to formation of the jaw. These studies show that discrete regulatory elements required for the elaboration of complex developmental programs can be individually localized, suggesting that the developmentally transient expression of individual transcription factors collaboratively contributes to the temporal and spatial pattern of cellular differentiation leading to the formation of adult anatomy.

Animals↗
PubMed1997

Expression analysis and chromosomal assignment of the human SFRS5/SRp40 gene.

Alternative splicing plays a major role in the regulation of gene expression. SFRS5/SRp40 is a member of the serine/arginine (SR) protein family of regulators of alternative pre-mRNA splicing. We cloned the human SFRS5 cDNA and observed two major SFRS5 transcripts, an approximately 1.8-kb short form and an approximately 3.3-kb long form, in both human and rat tissues. Both transcripts were detected in all human tissues examined, but there were notable tissue-specific differences in their relative abundance, the short form being most abundant in retina. Affinity-purified SFRS5 antisera recognized a single 40-kDa polypeptide in human and mouse retinal lysates. The abundant retinal expression of SFRS5 was not restricted to any specific cell type, since immunofluorescent labeling of human retinal sections identified the SFRS5 protein in nuclei of all three nuclear layers of the retina. The human SFRS5 gene was localized to human chromosome 14q24 by fluorescence in situ hybridization and PCR analysis of a human/hamster somatic cell hybrid panel.

Alternative Splicing↗
PubMed1997

Molecular cloning and characterization of a cDNA encoding the rat interleukin-8 receptor.

In this study we present the cloning, characterization and expression analysis of a cDNA encoding a rat interleukin-8 receptor (rIL-8R). A 1324 bp cDNA containing an open reading of 359 amino acids with an 86.1% overall identity with the previously characterized mouse IL-8R was isolated. Genomic DNA analysis using several restriction enzymes revealed a single band suggesting that the rIL-8R gene exists as a single-copy, which is in contrast to humans where there are two different IL-8Rs genes. Expression of rIL-8R mRNA was found in several tissues including spleen, heart, lung, liver, skeletal muscle and kidney. In brain and testis rIL-8R mRNA was not detectable. Rat IL-8R mRNA expression at the cellular level was studied in the spleen using RNA-RNA in situ hybridization and immunohistochemistry. IL-8R mRNA containing cells were predominately found in the mantle zone of the germinal center. These cells were identified as B lymphocytes using the OX-33 monoclonal antibody.

Amino Acid Sequence↗
PubMed1997

Expression and characterization of the small subunit of human DNA polymerase delta.

DNA polymerase delta is a heterodimer consisting of a catalytic subunit of 125 kDa and a small subunit of 50 kDa (p50). We have overexpressed p50 in Escherichia coli and have characterized the recombinant protein. p50 was readily overexpressed using the pET vector as an insoluble protein. A procedure was developed for its purification and renaturation. Examination of the physicochemical properties of renatured p50 showed that it is a monomeric protein with an apparent molecular weight of 60,000, a Stokes radius of 34 A, and a sedimentation coefficient of 4.1 S. Its physical properties were indistinguishable from p50 expressed as a soluble protein using the pTACTAC vector. Examination of the effects of recombinant p50 on the activity of DNA polymerase delta showed that p50 is able to slightly stimulate (about 2-fold) the activity of the recombinant 125-kDa catalytic subunit using poly(dA).oligo(dT) as a template in the absence of proliferating cell nuclear antigen. In the presence of proliferating cell nulear antigen, activity is stimulated about 5-fold. Seven stable hybridoma cell lines were established that produced monoclonal antibodies against p50. One of these antibodies (13D5) inhibited the activity of calf thymus DNA polymerase delta. This antibody, when coupled to a solid support, also was found to provide a method for the immunoafffinity purification of recombinant p50 and of DNA polymerase delta from calf thymus or HeLa extracts. Immunoprecipitation and enzyme-linked immunosorbent assays also confirmed that p50 interacts with the catalytic subunit of DNA polymerase delta.

DNA Polymerase III↗
PubMed1997

Evolution of paired domains: isolation and sequencing of jellyfish and hydra Pax genes related to Pax-5 and Pax-6.

Pax proteins are a family of transcription factors with a highly conserved paired domain; many members also contain a paired-type homeodomain and/or an octapeptide. Nine mammalian Pax genes are known and classified into four subgroups: Pax-1/9, Pax-2/5/8, Pax-3/7, and Pax-4/6. Most of these genes are involved in nervous system development. In particular, Pax-6 is a key regulator that controls eye development in vertebrates and Drosophila. Although the Pax-4/6 subgroup seems to be more closely related to Pax-2/5/8 than to Pax-3/7 or Pax-1/9, its evolutionary origin is unknown. We therefore searched for a Pax-6 homolog and related genes in Cnidaria, which is the lowest phylum of animals that possess a nervous system and eyes. A sea nettle (a jellyfish) genomic library was constructed and two pax genes (Pax-A and -B) were isolated and partially sequenced. Surprisingly, unlike most known Pax genes, the paired box in these two genes contains no intron. In addition, the complete cDNA sequences of hydra Pax-A and -B were obtained. Hydra Pax-B contains both the homeodomain and the octapeptide, whereas hydra Pax-A contains neither. DNA binding assays showed that sea nettle Pax-A and -B and hydra Pax-A paired domains bound to a Pax-5/6 site and a Pax-5 site, although hydra Pax-B paired domain bound neither. An alignment of all available paired domain sequences revealed two highly conserved regions, which cover the DNA binding contact positions. Phylogenetic analysis showed that Pax-A and especially Pax-B were more closely related to Pax-2/5/8 and Pax-4/6 than to Pax-1/9 or Pax-3/7 and that the Pax genes can be classified into two supergroups: Pax-A/Pax-B/Pax-2/5/8/4/6 and Pax-1/9/3/7. From this analysis and the gene structure, we propose that modern Pax-4/6 and Pax-2/5/8 genes evolved from an ancestral gene similar to cnidarian Pax-B, having both the homeodomain and the octapeptide.

Amino Acid Sequence↗
PubMed1997

Human cytotrophoblasts adopt a vascular phenotype as they differentiate. A strategy for successful endovascular invasion?

Establishment of the human placenta requires that fetal cytotrophoblast stem cells in anchoring chorionic villi become invasive. These cytotrophoblasts aggregate into cell columns and invade both the uterine interstitium and vasculature, anchoring the fetus to the mother and establishing blood flow to the placenta. Cytotrophoblasts colonizing spiral arterioles replace maternal endothelium as far as the first third of the myometrium. We show here that differentiating cytotrophoblasts transform their adhesion receptor phenotype so as to resemble the endothelial cells they replace. Cytotrophoblasts in cell columns show reduced E-cadherin staining and express VE-(endothelial) cadherin, platelet-endothelial adhesion molecule-1, vascular endothelial adhesion molecule-1, and alpha-4-integrins. Cytotrophoblasts in the uterine interstitium and maternal vasculature continue to express these receptors, and, like endothelial cells during angiogenesis, also stain for alphaVbeta3. In functional studies, alphaVbeta3 and VE-cadherin enhance, while E-cadherin restrains, cytotrophoblast invasiveness. Cytotrophoblasts expressing alpha4 integrins bound immobilized VCAM-1 in vitro, suggesting that this receptor-pair could mediate cytotrophoblast-endothelium or cytotrophoblast-cytotrophoblast interactions in vivo, during endovascular invasion. In the pregnancy disorder preeclampsia, in which endovascular invasion remains superficial, cytotrophoblasts fail to express most of these endothelial markers (Zhou et al., 1997. J. Clin. Invest. 99:2152-2164.), suggesting that this adhesion phenotype switch is required for successful endovascular invasion and normal placentation.

Antigens, CD↗
PubMed1997

Preeclampsia is associated with failure of human cytotrophoblasts to mimic a vascular adhesion phenotype. One cause of defective endovascular invasion in this syndrome?

In human pregnancy, placental cytotrophoblasts that invade the uterus downregulate the expression of adhesion receptors that are characteristic of their epithelial origin, and upregulate the expression of adhesion receptors that are expressed by vascular cells. We suggest that this transformation could be critical to endovascular invasion, the process whereby cytotrophoblasts invade the uterine spiral arterioles and line their walls (Zhou et al. J. Clin. Invest. 1997. 99: 2139-2151.). To better understand the in vivo significance of these findings, we tested the hypothesis that in preeclampsia, an important disease of pregnancy in which endovascular invasion is abrogated, cytotrophoblasts fail to adopt a vascular adhesion phenotype. In experiments described here we stained placental bed biopsy specimens from age-matched control pregnancies and from those complicated by preeclampsia with antibodies that recognize adhesion molecules that are normally modulated during this transformation. In preeclampsia, differentiating/invading cytotrophoblasts fail to express properly many of these molecules, including integrin, cadherin, and Ig superfamily members. These results suggest that preeclampsia is associated with failure of cytotrophoblasts to mimic a vascular adhesion phenotype. The functional consequences of this abnormality are unknown, but are likely to affect negatively cytotrophoblast endovascular invasion and uterine arteriole remodeling, thereby compromising blood flow to the maternal-fetal interface.

Antigens, CD↗
PubMed1997

Bacterial scavengase p20 is structurally and functionally related to peroxiredoxins.

Scavengase p20 was recently identified as a novel family of bacterial antioxidant enzymes possessing thioredoxin-linked thiol peroxidase activity. In this study, the Escherichia coli gene coding for scavengase p20 was isolated from three different strains and the nucleotide sequence was determined. Multiple alignment of amino acid sequence revealed that a previously unidentified Cys-61 is most conserved among all bacterial p20 scavengases and corresponds to the active site in the well-characterized peroxiredoxins. Phylogenetic analysis further supported that scavengase p20 is a novel subfamily of peroxiredoxins. Site-directed mutagenesis studies demonstrated that Cys-61 is indispensable for the antioxidant activities of scavengase p20. Taken together, our findings strongly suggest that the p20 scavengases are structurally and functionally related to peroxiredoxins.

Amino Acid Sequence↗
PubMed1997

Scavengase p20: a novel family of bacterial antioxidant enzymes.

A novel antioxidant enzyme designated scavengase p20 was identified in various pathogenic bacteria through database searching for sequences strikingly homologous to a recently discovered Escherichia coli thiol peroxidase p20. The direct biochemical evidence for the existence of scavengase p20 in Haemophilus influenzae, Streptococcus pneumoniae and Helicobacter pylori was provided by protein microsequencing and by in vitro assays for antioxidant activities. Overlapping genes encoding scavengase p20 and superoxide dismutase were recognized in H. pylori and their functional implications are discussed.

Amino Acid Sequence↗
PubMed1997

Both the isomerase and chaperone activities of protein disulfide isomerase are required for the reactivation of reduced and denatured acidic phospholipase A2.

The spontaneous reactivation yield of acidic phospholipase A2 (APLA2), a protein containing seven disulfide bonds, after reduction and denaturation in guanidine hydrochloride is very low. Protein disulfide isomerase (PDI) markedly increases the reactivation yield and prevents the aggregation of APLA2 during refolding in a redox buffer containing GSH and GSSG. S-methylated PDI (mPDI), with no isomerase but as nearly full chaperone activity as native PDI, has no effect on either the reactivation or aggregation of APLA2. However, the simultaneous presence of PDI and mPDI in molar ratios to APLA2 of 0.1 and 0.9 respectively fully reactivates the denatured enzyme, as does PDI alone at a ratio of 1. At ratios of 0.1 and 0.15 respectively, they completely suppress APLA2 aggregation, as does PDI alone at a ratio of 0.25. Moreover, delayed addition of PDI to the refolding buffer greatly diminished the reactivation yield of APLA2, but this deteriorating effect can be alleviated markedly by the presence of mPDI in the refolding buffer. Without GSSG, mPDI prevents the aggregation of APLA2 during refolding. It is proposed that the in vitro action of PDI as a foldase consists of both isomerase and chaperone activities, and the latter activity can be fully replaced by mPDI.

Agkistrodon↗
PubMed1997

Regulation of the human long chain acyl-CoA dehydrogenase gene by nuclear hormone receptor transcription factors.

Mitochondrial fatty acid oxidation provides most of the energy required for myocardial function after birth. Long chain acyl-CoA dehydrogenase (LCAD) catalyzes the first step in the beta-oxidation spiral. Our objective was to define regulatory elements of the human LCAD gene required for high levels of expression in mature heart and to locate elements suppressing gene expression in the fetus. We characterized the human LCAD gene structure and used in vitro transfection into cardiomyocytes and hepatoma cells of LCAD genomic fragments fused to a reporter gene to examine the effects of putative regulatory elements on transcription. Binding of transcription factors to nuclear hormone receptor consensus DNA binding domains was studied by gel shift experiments. The 200 bp of the human LCAD gene immediately upstream of the transcription initiation site are sufficient to act as a minimal promoter for the gene and provide some tissue-specific positive regulatory elements. The region from -1800 bp to -250 bp contains elements which markedly suppress transcription, including nuclear hormone receptor response elements. The dominant interaction is with the repressor factor, chicken ovalbumin upstream promoter transcription factor. We conclude that the developmental and tissue-specific regulation of the human LCAD gene is mediated, in part, by these nuclear hormone receptor transcription factors.

3T3 Cells↗
PubMed1997

Homology modeling with internal coordinate mechanics: deformation zone mapping and improvements of models via conformational search.

Five models by homology containing insertions and deletions and ranging from 33% to 48% sequence identity to the known homologue, and one high sequence identity (85%) model were built for the CASP2 meeting. For all five low identity targets: (i) our starting models were improved by the Internal Coordinate Mechanics (ICM) energy optimization, (ii) the refined models were consistently better than those built with the automatic SWISS-MODEL program, and (iii) the refined models differed by less than 2% from the best model submitted, as judged by the residue contact area difference (CAD) measure [Abagyan, R.A., Totrov, M.J. Mol. Biol. 268:678-685, 1997]. The CAD measure is proposed for ranking models built by homology instead of global root-mean-square deviation, which is frequently dominated by insignificant yet large contributions from incorrectly predicted fragments or side chains. We demonstrate that the precise identification of regions of local backbone deviation is an independent and crucial step in the homology modeling procedure after alignment, since aligned fragments can strongly deviate from the template at various distances from the alignment gap or even in the ungapped parts of the alignment. We show that a local alignment score can be used as an indicator of such local deviation. While four short loops of the meeting targets were predicted by database search, the best loop 1 target T0028, for which the correct database fragment was not found, was predicted by Internal Coordinate Mechanics global energy optimization at 1.2 A accuracy. A classification scheme for errors in homology modeling is proposed.

Models, Molecular↗
PubMed1997

Quantitation of dopamine transporter mRNA in the rat brain: mapping, effects of "binge" cocaine administration and withdrawal.

Dopamine transporter (DAT) mRNA from selected brain regions of individual male Fischer rats was quantitated utilizing a sensitive solution hybridization assay in which the levels of RNase-protected 32P-labeled mRNA:cRNA hybrids were measured. DAT mRNA was detected in whole brain regions known to contain abundant DAT mRNA (mean picogram of DAT mRNA/microgram of total RNA +/- SEM): substantia nigra, 7.17 +/- 0.47; ventral tegmentum, 4.71 +/- 0.38. In regions known to contain low levels of DAT mRNA, these levels were detected: central grey, 0.39 +/- 0.06; hypothalamus, 0.14 +/- 0.03. In addition, DAT mRNA was detected in areas where it had not previously been identified: amygdala, 0.19 +/- 0.03; caudate-putamen, 0.15 +/- 0.03; nucleus accumbens, 0.13 +/- 0.01; pons/medulla, 0.12 +/- 0.02; globus pallidus, 0.09 +/- 0.04; pituitary 0.07 +/- 0.01; frontal cortex, 0.05 +/- 0.01. No DAT mRNA was detected in 150 micrograms of rat liver RNA. As cocaine binds to and inhibits the activity of the dopamine transporter, we sought to determine if there were differences in dopamine transporter mRNA levels between saline- and cocaine-injected rats or rats withdrawn from a chronic "binge" pattern (15 mg/kg per dose i.p.; three doses at 1 h intervals each day) cocaine injection. Using trichloroacetic acid precipitation of mRNA:cRNA hybrids from RNA extracted from whole brain regions, we found no significant differences in the substantia nigra or the ventral tegmentum following subacute (3 days) binge, chronic (14 days) binge or 10 days withdrawal from a chronic binge pattern cocaine or saline administration.

Animals↗
PubMed1997

A new system for rapid measurement of ATP.

The paper introduces a new type instrument for rapid measuring ATP. The system consists of a micromodule ATP sensor and an instrument for measuring weak light transmitted by optic fiber. The micromodule ATP sensor mainly is composed of enzyme membrane, a probe and a bundle of optic fiber. The instrument measuring weak light consists of photomultiplier, high voltage power, pulse amplifier and counter. The instrument was characterized by simple structure, small size, rapid response time (< 5s), high sensitivity (10(-12) mol/L), stable performance (measuring the same sample for 50 times, CV < 5%), long enzyme storage time (> 3 months).

Adenosine Triphosphate↗
PubMed1997

Behavioral stereotypies induced by "binge' cocaine administration are independent of drug-induced increases in corticosterone levels.

Cocaine administration causes dramatic stereotypic behavior and elevation of circulating corticosterone levels in rodents. The present study tested the possible role of increased corticosterone in mediating stereotypic behavior caused by "binge' pattern cocaine administration. Animals were administered saline or cocaine intraperitoneally for 3 days, with or without pretreatment with a D1 (SCH 23390, 2 mg/kg) or D2 (sulpiride, 50 mg/kg) dopamine receptor antagonist. Three days of cocaine "binges' significantly increased corticosterone levels in vehicle pretreated rats (P < 0.01). Both SCH 23390 and sulpiride pretreatment daily significantly attenuated this increase (P < 0.01). Cocaine administration caused stereotypic behaviors in vehicle pretreatment rats (P < 0.01). These behavioral responses were blocked by the D1 dopamine receptor antagonist SCH 23390, but not by the D2 antagonist sulpiride. These findings reaffirm the dominant role of the D1 receptor in mediating behavioral stereotypy caused by elevations of extracellular dopamine in the synaptic cleft. The fact that the dose of sulpiride used in these studies prevented the elevation of plasma corticosterone caused by cocaine, without blocking the stereotypy caused by cocaine, indicates that this stereotypic behavior does not require drug-induced elevation in circulating levels of corticosterone.

Animals↗
PubMed1997

Upregulation of nicotinamide adenine dinucleotide phosphate-diaphorase reactivity in the ventral horn motoneurons of lumbosacral spinal cord after urethral obstruction in the guinea pig.

The role of nitric oxide (NO) in pathophysiology of urethral obstruction in male guinea pig was investigated by using nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry. In normal and sham-operated control animals, NADPH-d reactivity in the ventral horn motoneurons at L5-L6 and S1-S2 segments of spinal cord was barely detectable or virtually absent. In animals receiving urethral ligation and killed at 6 h after operation, NADPH-d reactivity in the ventral horn motoneurons was comparable to that of control animals. At 12 h, NADPH-d reactivity in the same cells began evident and was markedly enhanced in animals killed at 24 and 48 h. In order to verify whether the increased NADPH-d reactivity was linked to neuronal death, some sections of the lumbosacral spinal cord from urethral obstructed animals were stained in Nissl staining. There was no sign of cell death or atrophy of the ventral horn neurons. Present results suggest the plasticity of NADPH-d in ventral horn neurons which is readily upregulated by urethral ligation. The enhanced NADPH-d reactivity would imply increased nitric oxide synthase (NOS) activity and consequently generation of higher levels of NO in ventral horn neurons. Such alteration maybe involved in distension-induced urethral relaxation in the external urethral sphincter following urethral ligation.

Animals↗
PubMed1997

Prodynorphin, proenkephalin and kappa opioid receptor mRNA responses to acute "binge" cocaine.

Previous studies showed that preprodynorphin (ppDyn) mRNA increases in caudate-putamen while kappa opioid receptor (KOR) mRNA decreases in substantia nigra after 3 and 14 days "binge" cocaine. To further characterize opioid mRNA responses, rats were administered: saline; 1 day cocaine followed by 1 day saline; 1 day cocaine; or 2 days cocaine. ppDyn mRNA in caudate-putamen increased in both groups receiving cocaine on the final day compared to groups receiving saline. Preproenkephalin (ppEnk) mRNA in caudate-putamen increased, and KOR mRNA in substantia nigra decreased, after 2 days of cocaine. Thus ppDyn mRNA is elevated acutely by cocaine, while ppEnk and KOR mRNAs show a significant response only on the second day of "binge" cocaine.

Animals↗