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X Xin

Publications and source records attributed to X Xin.

At least 55 records · Page 3Linked to original sources
PubMed1998

Identification of mouse CPX-2, a novel member of the metallocarboxypeptidase gene family: cDNA cloning, mRNA distribution, and protein expression and characterization.

A novel member of the metallocarboxypeptidase gene family was identified from its homology with carboxypeptidase E and has been designated CPX-2. The cDNA of 2500 nucleotides encodes a protein of 764 amino acids that contains an N-terminal signal peptide-like sequence, a 158-residue discoidin domain, and a 400-residue carboxypeptidase domain. The 400-residue metallocarboxypeptidase domain has 59% amino acid identity with a protein designated AEBP-1; 44% to 46% identity with carboxypeptidases E, N, and Z; and lower homology with other members of the metallocarboxypeptidase gene family. The discoidin domain of CPX-2 has 22% amino acid identity with the carbohydrate-binding domain of discoideum-I, 29% to 34% identity with the phospholipid-binding domain of human factors V and VIII, and 59% identity with the discoidin-like domain on AEBP-1. CPX-2 is missing several of the predicted active-site residues that are conserved in most other members of the metallocarboxypeptidase gene family and which are thought to be required for enzyme activity. Expression of CPX-2 using the baculovirus system produced several forms of protein, from 80 to 105 kDa, but no detectable activity toward a variety of carboxypeptidase substrates. A shorter 50-kDa form of CPX-2, which contains the carboxypeptidase domain but not the discoidin domain, was also inactive when expressed in the baculovirus system. CPX-2 is able to bind to Sepharose-Arg; this binding is blocked by 10 mM Arg. Northern blot analysis showed CPX-2 mRNA in mouse brain, liver, kidney, and lung. In situ hybridization analysis of brain revealed a broad distribution. Areas that are enriched in CPX-2 include the hippocampus, cerebral cortex, median eminence, and choroid plexus. Taken together, these data suggest a widespread function for CPX-2, possibly as a binding protein rather than an active carboxypeptidase.

Amino Acid Sequence↗
PubMed1997

Characterization of the alpha1B-adrenergic receptor gene promoter region and hypoxia regulatory elements in vascular smooth muscle.

We previously demonstrated that alpha1B-adrenergic receptor (AR) gene transcription, mRNA, and functionally coupled receptors increase during 3% O2 exposure in aorta, but not in vena cava smooth muscle cells (SMC). We report here that alpha1BAR mRNA also increases during hypoxia in liver and lung, but not heart and kidney. A single 2.7-kb alpha1BAR mRNA was detected in aorta and vena cava during normoxia and hypoxia. The alpha1BAR 5' flanking region was sequenced to -2,460 (relative to ATG +1). Transient transfection experiments identify the minimal promoter region between -270 and -143 and sequence between -270 and -248 that are required for transcription of the alpha1BAR gene in aorta and vena cava SMC during normoxia and hypoxia. An ATTAAA motif within this sequence specifically binds aorta, vena cava, and DDT1MF-2 nuclear proteins, and transcription primarily initiates downstream of this motif at approximately -160 in aorta SMC. Sequence between -837 and -273 conferred strong hypoxic induction of transcription in aorta, but not in vena cava SMC, whereas the cis-element for the transcription factor, hypoxia-inducible factor 1, conferred hypoxia-induced transcription in both aorta and vena cava SMC. These data identify sequence required for transcription of the alpha1BAR gene in vascular SMC and suggest the atypical TATA-box, ATTAAA, may mediate this transcription. Hypoxia-sensitive regions of the alpha1BAR gene also were identified that may confer the differential hypoxic increase in alpha1BAR gene transcription in aorta, but not in vena cava SMC.

Animals↗
PubMed1997

Cloning and sequence analysis of cDNA encoding rat carboxypeptidase D.

Carboxypeptidase D (CPD) is a recently described 180-kD enzyme with carboxypeptidase E-like enzymatic properties. CPD has been proposed to be present in the secretory pathway and to contribute to peptide hormone processing in the Cpe(fat)/Cpe(fat) mouse, which lacks functional CPE. Sequence analysis of cDNA clones encoding rat CPD show the protein to contain an amino-terminal signal peptide, three carboxypeptidase-like domains, a putative transmembrane domain, and a 60-amino-acid cytoplasmic tail. Whereas active site, substrate-binding, and metal-binding residues of other metallocarboxypeptidases are conserved in the first two domains of CPD, several of the critical residues are not conserved in the third domain; this third domain is not predicted to form an active carboxypeptidase. The overall homology between rat CPD and the duck homolog gp180 is high, with 75% amino acid identity. The three carboxypeptidase domains show 66%, 83%, and 82% amino acid identity between rat CPD and duck gp180. Homology is also high in the transmembrane domain (86%) and in the cytoplasmic tail (97%). The mouse Cpd gene maps to the medial portion of chromosome 11, approximately 45.5 cM distal to the centromere. Northern blot analysis of CPD mRNA shows major bands of approximately 8 and 4 kb in many rat tissues, and additional species ranging from 1.4 to 5 kb that are expressed in some tissues or cell lines. CPD mRNA is detectable in most tissues examined, and is most abundant in hippocampus, spinal cord, atrium of the heart, colon, testis, and ovaries. In situ hybridization of CPD mRNA shows a distribution in many cells in rat brain and other tissues, with high levels in hippocampus, olfactory bulb, and the intermediate pituitary. The broad distribution is consistent with a role for CPD in the processing of many peptides and proteins that transit the secretory pathway.

Amino Acid Sequence↗
PubMed1997

Alpha1D-adrenergic receptors and mitogen-activated protein kinase mediate increased protein synthesis by arterial smooth muscle.

Catecholamines may influence vascular smooth muscle cell (SMC) growth and vascular hypertrophic diseases. We previously demonstrated that stimulation of alpha1-adrenoceptors (AR) causes hypertrophy of vascular SMCs in vitro and in situ. Here, we used adult rat aorta SMCs that express alpha1D- and alpha1B-ARs (but not alpha1A-ARs) in vitro to examine the mechanisms and alpha1-AR subtypes involved. Norepinephrine (NE) increased protein synthesis and content in a time- and dose-dependent manner. To identify the responsible alpha1-AR subtype, we first documented the selectivity of two alpha1-AR subtype antagonists, BMY 7378 (alpha1D-AR antagonist) and chloroethylclonidine (CEC; alpha1B-AR antagonist), using Rat-1 fibroblasts stably transfected with the three different rodent alpha1-AR cDNAs. NE dose-dependently increased protein synthesis in each cell line. In alpha1D fibroblasts, BMY 7378 inhibited growth and protected alpha1D-ARs from CEC alkylation while having little blocking or protecting effect on the growth induced by stimulation of fibroblasts that express alpha1A- or alpha1B-ARs. In rat aorta SMCs, pretreatment with CEC in the presence of BMY 7378 to protect alpha1D-ARs had no effect on NE-induced protein synthesis. BMY 7378 inhibited the SMC growth response with a pKb of 8.4. NE caused rapid and transient p42-p44 mitogen-activated protein kinase (MAPK) activation that was alpha1D-AR dependent. Furthermore, NE caused tyrosine phosphorylation of multiple cellular proteins, phosphorylation of Raf-1, and stimulation of c-fos mRNA expression in aorta SMCs. The selective MAPK kinase inhibitor PD 98059 inhibited NE-induced protein synthesis and MAPK activation with IC50 values of 2.3 and 1.6 microM, respectively. These data demonstrate that SMC growth induced by NE is mediated by alpha1D-ARs that couple to activation of the MAPK cascade.

Animals↗
PubMed1997

In vivo sequence variability of human immunodeficiency virus type 1 envelope gp120: association of V2 extension with slow disease progression.

According to the rate of depletion of CD4 cell counts, we grouped 12 cases of human immunodeficiency virus type 1 (HIV-1) infection as 6 rapid (21.0 to 33.8 cells per microl per month) and 6 slow (0.9 to 7.9 cells per microl per month) progressors and determined the individual viral quasispecies patterns by sequencing the genome region encoding the V1, V2, and V3 loops of envelope protein. Although the quasispecies structures varied widely from one individual to another, a strong correlation was observed between a low rate of disease progression and a high degree of genetic diversity of HIV-1. Furthermore, the V2 loop extension was observed specifically in individuals with slow or no disease progression, whereas basic amino acid substitutions in V3 characteristic of a viral phenotype shift from non-syncytium inducing to syncytium inducing were observed in patients with advanced stages of disease regardless of their rate of disease progression. Studies with recombinant viruses suggested that elongation of V2 potentially restricts the capacity of HIV-1 to replicate in macrophages. Thus, our results suggest the association of distinct sequence features of both V3 and V2 with particular patterns of disease progression. Elongation of the V2 loop may be a good predictor of slow disease progression, while basic substitutions of V3 without elongation of V2 are characteristic of rapid progression.

Amino Acid Sequence↗
PubMed1997

CRE-like response element regulates expression of rat alpha 2D-adrenergic receptor gene in vascular smooth muscle.

Contractile and binding studies indicate that alpha 2-adrenergic receptors (ARs) are differentially expressed by vascular smooth muscle cells (SMCs) according to vascular segment (artery, arteriole, vein). In the present study, alpha 2D-AR mRNA was two- to threefold higher in vena cava than in aorta. To understand vascular regulation of alpha 2D-AR expression in these cells, we sequenced 2.8 kb of the 5' flanking region of the alpha 2D-AR gene. Notable features include two potential TATA boxes, an adenosine 3',5'-cyclic monophosphate response element (CRE)-like binding element, and an Sp1 element. Comparison of the rat and human genes revealed an overall homology of 74% over the 1.87-kb sequence 5' to the translation initiator methionine, including complete homology at the distal TATA, CRE-like, and Sp1 sites, alpha 2D-AR transcription starts from the guanine nucleotide 18 base pair downstream from the distal TATA box. Reporter gene constructs demonstrated strong alpha 2D-AR promoter activity, but with several differences in construct activity, in both rat aorta and vena cava SMCs. Analysis of an essential promoter fragment revealed two regions protected by aorta and vena cava SMC nuclear proteins. The core sequences of these protected regions are TGACGCTA and TATAA. The former CRE-like element conferred specific binding of both aorta and vena cava nuclear proteins. In addition, promoter activity was increased 300% by forskolin or 8-bromoadenosine 3',5'-cyclic monophosphate, indicating that the CRE-like element may regulate alpha 2D-AR expression in vascular tissue.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1997

Pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) stimulate interleukin-6 production through the third subtype of PACAP/VIP receptor in rat bone marrow-derived stromal cells.

Regulation of Interleukin-6 (IL-6) production in bone marrow (BM)-derived stromal cells by neuropeptides, pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP), was examined. Both forms of PACAP, PACAP-27 and PACAP-38, as well as VIP significantly increased IL-6 production by rat BM-derived stromal cells at physiological concentrations ranging from 10(-10)-10(-8) M. The three related peptides (PACAP-27, -38, and VIP) stimulated the production of both cAMP and inositol 1,4,5-trisphosphate (IP3) in rat BM-derived stromal cells with similar 50% effective concentrations. The stimulatory potency of the three related peptides for the production of IL-6, cAMP, and IP3 was almost consistent, suggesting that the dual signaling transduction pathways may be involved in PACAP/VIP-induced IL-6 production in rat BM-derived stromal cells. The messenger RNA (mRNA) for the third subtype of PACAP receptor (PVR3) was found to be abundantly expressed in both BM-derived stromal cells and the BM tissue, whereas little of the mRNA for type 1 (PVR1) nor type 2 (PVR2) was detected. Furthermore, the mRNAs for PACAP and VIP were detected in the BM tissue, suggesting that both PACAP/VIP and PVR3 are synthesized in vivo in the BM. The results shown in this paper suggest that PACAP/VIP and their receptor play an important role in the IL-6 production and perhaps in the hematopoiesis in the BM.

Animals↗
PubMed1996

Infectivities of human and other primate lentiviruses are activated by desialylation of the virion surface.

The envelope protein, gp120, of human immunodeficiency virus type 1 (HIV-1) is heavily glycosylated and sialylated. The heavy sialylation greatly affects the physical properties of the protein, as it resolves into a wide acidic pH range despite the basic pI value predicted for its polypeptide backbone (B. S. Stein and E. G. Engleman, J. Biol. Chem. 265:2640-2649, 1990). However, the functional significance of the heavy sialylation remains elusive. Here, we show that desialylation of HIV-1 with neuraminidase greatly augments the initial virus-cell interaction, leading to remarkably enhanced viral replication and cytopathogenicity. This enhancement appeared to be a direct result of the removal of negatively charged sialic acids but not of the exposure of galactose residues or complement activation. Complementing these results, studies with inhibitors of mannosidase I and mannosidase II showed that the processing of HIV-1 oligosaccharides into the complex type to acquire the terminal sialic acid residues impeded the full replication capacity of the virus and that its prevention also enhanced virus replication and cytopathogenicity. Enhancement of infection by desialylation was found widely, with HIV-1 laboratory strains of different cell tropisms and primary isolates as well as HIV-2 and simian immunodeficiency virus. Thus, the sialylation catalyzed by host cell pathways appeared to reduce the infectivity of human and nonhuman primate lentiviruses. Our results further suggested that desialylation would help increase the titers of HIV-based vectors.

1-Deoxynojirimycin↗
PubMed1996

Mapping 638 STSs to regions of human chromosome 3.

The initial step of mapping STSs to chromosome 3 has been by mapping to a reference panel of 21 somatic cell hybrids containing fragments of chromosome 3. In this study we map 638 STSs to 23 bins on chromosome 3. The bin information greatly facilitates further mapping by radiation hybrids and YAC clones.

Chromosome Mapping↗
PubMed1995

Regulation of vascular smooth muscle growth by alpha 1-adrenoreceptor subtypes in vitro and in situ.

Rat aorta smooth muscle cells which express all three alpha 1-adrenoreceptors (alpha 1A, alpha 1B and alpha 1D) were used to determine the effect of stimulation of alpha 1-adrenergic receptor subtypes on cell growth. "Combined" alpha 1-adrenoreceptor subtype stimulation with norepinephrine alone caused a concentration-dependent, prazosin-sensitive increase in protein content and synthesis: 48 h of stimulation at 1 microM increased cell protein to 216 +/- 40% of time-matched controls (p = 0.008) and RNA to 140 +/- 13% (p = 0.03); protein synthesis increased to 167 +/- 13% (p < 0.01) after 24 h. Stimulation with norepinephrine plus the selective alpha 1A/alpha 1D antagonist 5-methylurapidil produced greater increases in alpha-actin mRNA (270 +/- 40% at 8 h; p = 0.007), total cell protein (220 +/- 45% at 24 h; p = 0.004), and RNA (135 +/- 8% at 24 h; p = 0.01). These effects were prevented by pretreatment with the selective alpha 1B antagonist chloroethylclonidine. Comparable results were obtained for intact aortae. Stimulation with norepinephrine plus 5-methylurapidil increased (p < 0.05) tissue protein, RNA, dry weight, and alpha-actin mRNA; and as in culture cells, combined stimulation with norepinephrine alone attenuated these responses. By comparison, adventitia (fibroblasts) was unaffected. Removal of endothelial cells had no effect. alpha 1B mRNA decreased by 42 +/- 12% (p = 0.01) in cultured cells during combined alpha 1-adrenoreceptor stimulation and by 23 +/- 8% (p = 0.03) for intact aorta. alpha 1D and beta-actin mRNA were unchanged in cultured cells, aorta media, and adventitia. These findings suggest that prolonged stimulation of chloroethylclonidine-sensitive, possibly alpha 1B-adrenoceptors induces hypertrophy of arterial smooth muscle cells and that stimulation of 5-methylurapidil-sensitive, non-alpha 1B-adrenoreceptors attenuates this growth response.

Actins↗
PubMed1995

Opposite actions of transforming growth factor-beta 1 on the gene expression of atrial natriuretic peptide biological and clearance receptors in a murine thymic stromal cell line.

The regulation of the gene expression of the atrial natriuretic peptide receptor (ANPR) subtypes, ANPR-A, ANPR-B, and ANPR-C, was investigated in a murine thymic stromal cell line, MRL 104.8a. When MRL 104.8a cells were cultured with transforming growth factor (TGF)-beta1, [125I]ANP binding sites increased with increasing dose of TGF-beta1. These binding sites were identified as ANPR-C by a displacement experiment with ANPR-C-specific ligand, C-ANF, and by the affinity cross-linking of the [125I]ANP binding sites with a chemical cross-linker to determine the molecular weight of the ANPR. This augmentation of the ANPR-C expression was elucidated to occur at the transcriptional level by Northern blot experiment, comparison of the relative amounts of mRNA by reverse transcription (RT)-PCR, and in vitro nuclear transcription assay. Conversely, the expression of the ANP biological receptors, ANPR-A and ANPR-B, was shown to be down-regulated by TGF-beta1. These data suggest that TGF-beta1 regulates the gene expression of ANPRs in the thymic stromal cells and that ANP and TGF-beta1 might affect the thymic stromal cell functions.

Animals↗
PubMed1995

Assignments of the genes for rat pituitary adenylate cyclase activating polypeptide (Adcyap1) and its receptor subtypes (Adcyap1r1, Adcyap1r2, and Adcyap1r3).

Chromosomal assignments of the genes for rat pituitary adenylate cyclase activating polypeptide (Adcyap1) and all of its receptor subtypes (Adcyap1r1, Adcyap1r2, and Adcyap1r3) were performed by PCR analysis of somatic cell hybrid DNAs. Adcyap1r1, Adcyap1r2, and Adcyap1r3 were localized to rat chromosomes 9, 4, 8, and 4, respectively.

Animals↗
PubMed1995

Endothelin-induced interleukin-6 production by rat aortic endothelial cells.

Endothelin (ET) produced by endothelial cells has recently been found to be a potent vasoconstricting hormone. In this paper, ET isopeptides, both ET-1 and ET-3, were shown to be potent stimulators of interleukin (IL)-6 production by a rat aortic endothelial cell clone, WAE-1. Semi-quantitative polymerase chain reaction analysis indicated that augmentation of IL-6 production is due to an increase in IL-6 messenger RNA level. Ligand binding assay indicated that most of the [125I]ET-1 binding sites correspond to ET receptor type A (ETAR). However, ET receptor type B (ETBR) was shown to be also present on this cell line by reverse transcription polymerase chain reaction using ETBR-specific primers and by ligand binding assay using [125I]ET-3, although the protein receptor level is much lower than that of ETAR. ET-1, but not ET-3, induced inositol 1,4,5-triphosphate production and an increase in intracellular Ca2+ concentration with similar dose response. These data suggest that ET-1 stimulates IL-6 production through ETAR-phospholipase C activation axis, whereas ET-3 stimulates IL-6 production through different signaling pathway. The results shown in this paper raise the possibility that ET plays a role in inducing inflammation in endothelium.

Animals↗
PubMed1995

The rat homologue of the Wilson's disease gene was partially deleted at the 3' end of its protein-coding region in Long-Evans Cinnamon mutant rats.

LEC mutant rats exhibit an abnormal hepatic copper accumulation, due to the defection of the rat homologue of the Wilson's disease gene. In this study, we found a definitive evidence that the rat Wilson's disease gene of LEC rats was partially deleted at the 3' end of its protein-coding region, by performing Southern blot analysis. Furthermore, in Northern blot analysis, we confirmed that expression of the rat Wilson's disease gene was deficient in the liver of LEC rats. The partial deletion of the rat Wilson's disease gene should be responsible for the deficient expression of that gene in LEC rats.

Animals↗
PubMed1994

The predominant form of fibroblast growth factor receptor expressed by proliferating human arterial smooth muscle cells in culture is type I.

Fibroblast growth factors (FGF) and their specific receptors (FGFR) have diverse roles, including induction of proliferation in smooth muscle cells which contributes to restenosis after coronary artery balloon angioplasty. The relative levels of expression of the four major types of FGFR were studied in 13 different human arterial smooth muscle cell isolates. Cell lines were established by the explant technique from intima/media tissue samples obtained from patients undergoing either coronary artery bypass surgery or cardiac transplantation procedures. Expression of FGFR isoforms was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) using primers for the conserved tyrosine kinase (TK) domain followed by Southern blotting with TK insert probes unique to each isoform. The data indicate that FGFR I is the major form of FGF receptor mRNA expressed by proliferating human arterial smooth muscle cells. This strongly suggests that it is this type of FGFR that mediates the signal transduction cascade associated with mitogenesis in proliferating human smooth muscle cells.

Arteries↗
PubMed1994

Stimulation of interleukin-6 production by endothelin in rat bone marrow-derived stromal cells.

Endothelin (ET) produced by endothelial cells has recently been found to be a potent vasoconstricting hormone. In this report, ET is shown to be a potent stimulator of interleukin-6 (IL-6) production by rat bone marrow (BM)-derived stromal cells. It was also shown that ET increased the level of mRNA for IL-6 in these cells. The two types of ET receptor (R), ETAR and ETBR, were shown to be expressed on both BM-derived stromal cells in culture and ex vivo in BM tissue, suggesting that ET works as a physiologic stimulator of IL-6 production in the BM. It was shown that ETAR is coupled to phospholipase C activation, leading to the production of inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DAG) as second messengers in BM-derived stromal cells. This was corroborated by data showing that IL-6 production in these cells was induced by combined stimulation with ionomycin and phorbol myristate acetate, thereby bypassing the effects of IP3 and DAG, respectively. This is the first report on the hormonal regulation of IL-6 production by BM stromal cells, indicating that hematopoiesis is subject to endocrinologic regulation under physiologic conditions. ET has recently been reported to be produced by macrophages in response to bacterial lipopolysaccharide and human immunodeficiency virus-1 glycoprotein 120. These facts, taken together with our findings, raise the possibility that ET shares the same role of IL-1 as a local cytokine, mediating an intercellular signal between macrophages and BM stromal cells in response to bacterial or viral stimulation.

Animals↗