Biomedical subjects

X Xu

Publications and source records attributed to X Xu.

At least 541 records · Page 30Linked to original sources

PubMed1998

Drosophila mef2 expression during mesoderm development is controlled by a complex array of cis-acting regulatory modules.

The function of the Drosophila mef2 gene, a member of the MADS box supergene family of transcription factors, is critical for terminal differentiation of the three major muscle cell types, namely somatic, visceral, and cardiac. During embryogenesis, mef2 undergoes multiple phases of expression, which are characterized by initial broad mesodermal expression, followed by restricted expression in the dorsal mesoderm, specific expression in muscle progenitors, and sustained expression in the differentiated musculatures. In this study, evidence is presented that temporally and spatially specific mef2 expression is controlled by a complex array of cis-acting regulatory modules that are responsive to different genetic signals. Functional testing of approximately 12 kb of 5' flanking region of the mef2 gene showed that the initial widespread mesodermal expression is achieved through a 280-bp twist-dependent enhancer. The subsequent dorsal mesoderm-restricted mef2 expression is mediated through a 460-bp dpp-responsive regulatory module, which involves the function of the Smad4 homolog Medea and contains several binding sites for Medea and Mad. The analysis also showed that regulated mef2 expression in the caudal and trunk visceral mesoderm, which give rise to longitudinal and circular gut musculatures, respectively, is under the control of distinct enhancer elements. In addition, mef2 expression in the cardioblasts of the heart is dependent upon at least two distinct enhancers, which are active at different periods during embryogenesis. Moreover, multiple regulatory elements are differentially activated for specific expression in presumptive muscle founders, prefusion myoblasts, and differentiated muscle fibers. Taken together, the presented data suggest that specific expression of the mef2 gene in myogenic lineages in the Drosophila embryo is the result of multiple genetic inputs that act in an additive manner upon distinct enhancers in the 5' flanking region.

Animals↗

PubMed1998

Functional interaction between InsP3 receptors and store-operated Htrp3 channels.

Calcium ions are released from intracellular stores in response to agonist-stimulated production of inositol 1,4,5-trisphosphate (InsP3), a second messenger generated at the cell membrane. Depletion of Ca2+ from internal stores triggers a capacitative influx of extracellular Ca2+ across the plasma membrane. The influx of Ca2+ can be recorded as store-operated channels (SOC) in the plasma membrane or as a current known as the Ca2+-release-activated current (I(crac)). A critical question in cell signalling is how SOC and I(crac) sense and respond to Ca2+-store depletion: in one model, a messenger molecule is generated that activates Ca2+ entry in response to store depletion; in an alternative model, InsP3 receptors in the stores are coupled to SOC and I(crac). The mammalian Htrp3 protein forms a well defined store-operated channel and so provides a suitable system for studying the effect of Ca2+-store depletion on SOC and I(crac). We show here that Htrp3 channels stably expressed in HEK293 cells are in a tight functional interaction with the InsP3 receptors. Htrp3 channels present in the same plasma membrane patch can be activated by Ca2+ mobilization in intact cells and by InsP3 in excised patches. This activation of Htrp3 by InsP3 is lost on extensive washing of excised patches but is restored by addition of native or recombinant InsP3-bound InsP3 receptors. Our results provide evidence for the coupling hypothesis, in which InsP3 receptors activated by InsP3 interact with SOC and regulate I(crac).

Animals↗

PubMed1998

Reduced nitric oxide production and altered myocardial metabolism during the decompensation of pacing-induced heart failure in the conscious dog.

The aim of the present study was to determine whether cardiac nitric oxide (NO) production changes during the progression of pacing-induced heart failure and whether this occurs in association with alterations in myocardial metabolism. Dogs (n=8) were instrumented and the heart paced until left ventricular end-diastolic pressure reached 25 mm Hg and clinical signs of severe failure were evident. Every week, hemodynamic measurements were recorded and blood samples were withdrawn from the aorta and the coronary sinus for measurement of NO metabolites, O2 content, free fatty acids (FFAs), and lactate and glucose concentrations. Cardiac production of NO metabolites or consumption of O2 or utilization of substrates was calculated as coronary sinus-arterial difference times coronary flow. In end-stage failure, occurring at 29+/-1.6 days, left ventricular end-diastolic pressure was 25+/-1 mm Hg, left ventricular systolic pressure was 92+/-3 mm Hg, mean arterial pressure was 75+/-2.5 mm Hg, and dP/dtmax was 1219+/-73 mm Hg/s (all P<0.05). These changes in hemodynamics were associated with a fall of cardiac NO metabolite production from 0.37+/-0.16 to -0.28+/-0.13 nmol/beat (P<0.05). O2 consumption and lactate uptake did not change significantly from control, while FFA uptake decreased from 0.16+/-0.03 to 0.05+/-0.01 microEq/beat and glucose uptake increased from -2.3+/-7.0 to 41+/-10 microgram/beat (P<0.05). The cardiac respiratory quotient also increased significantly by 28%. In 14 normal dogs the same measurements were performed at control and 1 hour after we injected 30 mg/kg of nitro-L-arginine, a competitive inhibitor of NO synthase .O2 consumption increased from 0.05+/-0.002 mL/beat at control to 0.071+/-0.003 mL/beat after nitro-L-arginine, while FFA uptake decreased from 0.1+/-0.01 to 0.06+/-0.01 microEq/beat, lactate uptake increased from 0.15+/-0.04 to 0.31+/-0.03 micromol/beat, glucose uptake increased from 8.2+/-5.0 to 35.4+/-9.5 microgram/beat, and RQ increased by 23% (all P<0.05). Our results indicate that basal cardiac production of NO falls below normal levels during cardiac decompensation and that there are shifts in substrate utilization. This switch in myocardial substrate utilization also occurs after acute pharmacological blockade of NO production in normal dogs.

Animals↗

PubMed1998

Promiscuous coupling of receptors to Gq class alpha subunits and effector proteins in pancreatic and submandibular gland cells.

Mice with deficiencies in one or more Gq class alpha subunit genes were used to examine the role of the alpha subunit in regulating Ca2+ signaling in pancreatic and submandibular gland cells. Western blot analysis showed that these cells express three of the four Gq class subunits, Galphaq, Galpha11, and Galpha14 but not Galpha15. Surprisingly, all parameters of Ca2+ signaling were identical in cells from wild type and four lines of mutant mice: 1) Galpha11-/-, 2) Galpha11-/-/Galpha14-/-, 3) Galpha14-/-/Galpha15-/-, and 4) Galphaq-/-/Galpha15-/-. These parameters included the Kapp for several Gq class coupled receptors, induction of [Ca2+]i oscillations by weak stimulation, and a biphasic [Ca2+]i response by strong stimulation. Furthermore, Ca2+ release from internal stores and Ca2+ entry were not affected in cells from any of the mutant mice. We conclude that Galphaq, Galpha11, and Galpha14 promiscuously couple several receptors (m3 muscarinic, bombesin, cholecystokinin, and alpha1 adrenergic) to effector proteins that activate both Ca2+ release from internal stores and Ca2+ entry.

Animals↗

PubMed1998

Crystal structure of the complex of the cyclin D-dependent kinase Cdk6 bound to the cell-cycle inhibitor p19INK4d.

The crystal structure of the cyclin D-dependent kinase Cdk6 bound to the p19 INK4d protein has been determined at 1.9 A resolution. The results provide the first structural information for a cyclin D-dependent protein kinase and show how the INK4 family of CDK inhibitors bind. The structure indicates that the conformational changes induced by p19INK4d inhibit both productive binding of ATP and the cyclin-induced rearrangement of the kinase from an inactive to an active conformation. The structure also shows how binding of an INK4 inhibitor would prevent binding of p27Kip1, resulting in its redistribution to other CDKs. Identification of the critical residues involved in the interaction explains how mutations in Cdk4 and p16INK4a result in loss of kinase inhibition and cancer.

Adenosine Triphosphate↗

PubMed1998

Prevention of DOI-mediated wet dog shakes by inhibitors of nitric oxide synthase.

The purpose of this study was to determine if (+/-)-1-(2, 5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI)-induced wet dog shakes (WDS) required the involvement of nitric oxide synthases (NOS). Systemic administration of the general NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME), but not its d-isomer, and the neuronal NOS (nNOS) inhibitor 7-nitroindazole completely blocked DOI-mediated WDS in a dose dependent manner. The data provides evidence that serotonin 5HT2 receptors are coupled to nNOS activation in the rat brain.

Amphetamines↗

PubMed1998

Human semaphorin K1 is glycosylphosphatidylinositol-linked and defines a new subfamily of viral-related semaphorins.

The semaphorin family contains a large number of secreted and transmembrane proteins, some of which are known to act as repulsive axon guidance cues during development or to be involved in immune function. We report here on the identification of semaphorin K1 (sema K1), the first semaphorin known to be associated with cell surfaces via a glycosylphosphatidylinositol linkage. Sema K1 is highly homologous to a viral semaphorin and can interact with specific immune cells, suggesting that like its viral counterpart, sema K1 could play an important role in regulating immune function. Sema K1 does not bind to neuropilin-1 or neuropilin-2, the two receptors implicated in mediating the repulsive action of several secreted semaphorins, and thus it likely acts through a novel receptor. In contrast to most previously described semaphorins, sema K1 is only weakly expressed during development but is present at high levels in postnatal and adult tissues, particularly brain and spinal cord.

Amino Acid Sequence↗

PubMed1998

Failure of egg cylinder elongation and mesoderm induction in mouse embryos lacking the tumor suppressor smad2.

smad genes constitute a family of nine members whose products serve as intracellular mediators of transforming growth factor beta signals. SMAD2, which is a tumor suppressor involved in colorectal and lung cancer, has been shown to induce dorsal mesoderm in Xenopus laevis in response to transforming growth factor beta and activins. The smad2 gene is expressed ubiquitously during murine embryogenesis and in many adult mouse tissues. Animals that lacked smad2 died before 8.5 days of development (E8.5). E6.5 homozygous mutants were smaller than controls, lacked the extraembryonic portion of the egg cylinder, and appeared strikingly similar to E6.5 smad4 mutants. This similarity was no longer evident at E7.5, however, because the smad2 mutants contained embryonic ectoderm within their interiors. Molecular analysis showed that smad2 mutant embryos did not undergo gastrulation or make mesoderm. The results demonstrate that smad2 is required for egg cylinder elongation, gastrulation, and mesoderm induction.

Animals↗

PubMed1998

Smad proteins act in combination with synergistic and antagonistic regulators to target Dpp responses to the Drosophila mesoderm.

Dorsal mesoderm induction in arthropods and ventral mesoderm induction in vertebrates are closely related processes that involve signals of the BMP family. In Drosophila, induction of visceral mesoderm, dorsal muscles, and the heart by Dpp is, at least in part, effected through the transcriptional activation and function of the homeobox gene tinman in dorsal mesodermal cells during early embryogenesis. Here we present a functional dissection of a tinman enhancer that mediates the Dpp response. We provide evidence that mesoderm-specific induction of tinman requires the binding of both activators and repressors. Screens for binding factors yielded Tinman itself and the Smad4 homolog Medea. We show that the binding and synergistic activities of Smad and Tinman proteins are critical for mesodermal tinman induction, whereas repressor binding sites prevent induction in the dorsal ectoderm and amnioserosa. Thus, integration of positive and negative regulators on enhancers of target genes appears to be an important mechanism in tissue-specific induction by TGF-beta molecules.

Amino Acid Sequence↗

PubMed1998

IFN-gamma induces cell growth inhibition by Fas-mediated apoptosis: requirement of STAT1 protein for up-regulation of Fas and FasL expression.

The mechanism by which IFN-gamma inhibits tumor cell growth has not been fully understood. Here we report that IFN-gamma up-regulated the expression of Fas and Fas ligand (FasL) on HT29 cells, a human colon adenocarcinoma cell line, and subsequently induced apoptosis of these cells. The kinetics of cell death in IFN-gamma-treated HT29 cells paralleled the increase in the levels of Fas and FasL expression. We further show that IFN-gamma up-regulated the expression of Fas and FasL in STAT1-transfected U3A cells but not in STAT1-deficient U3A cells. Correspondingly, IFN-gamma induced cell death in STAT1-transfected U3A cells but not in STAT1-deficient U3A cells. IFN-gamma-induced cell death was inhibited by caspase-1 inhibitors. Our results suggest that cell growth inhibition by IFN-gamma is due to apoptosis mediated by Fas and FasL interaction.

Antineoplastic Agents↗

PubMed1998

Role of nitric oxide in the control of renal oxygen consumption and the regulation of chemical work in the kidney.

Inhibition of NO synthesis has recently been shown to increase oxygen extraction in vivo, and NO has been proposed to play a significant role in the regulation of oxygen consumption by both skeletal and cardiac muscle in vivo and in vitro. It was our aim to determine whether NO also has such a role in the kidney, a tissue with a relatively low basal oxygen extraction. In chronically instrumented conscious dogs, administration of an inhibitor of NO synthase, nitro-L-arginine (NLA, 30 mg/kg i.v.), caused a maintained increase in mean arterial pressure and renal vascular resistance and a decrease in heart rate (all P<0.05). At 60 minutes, urine flow rate and glomerular flow rate decreased by 44+/-12% and 45+/-7%, respectively; moreover, the amount of sodium reabsorbed fell from 16+/-1.7 to 8.5+/-1.1 mmol/min (all P<0.05). At this time, oxygen uptake and extraction increased markedly by 115+/-37% and 102+/-34%, respectively (P<0.05). Oxygen consumption also significantly increased from 4.5+/-0.6 to 7.1+/-0.9 mL O2/min. Most important, the ratio of oxygen consumption to sodium reabsorbed increased dramatically from 0.33+/-0.07 to 0.75+/-0.11 mL O2/mmol Na+ (P<0.05), suggesting a reduction in renal efficiency for transporting sodium. In vitro, both a NO-donating agent and the NO synthase-stimulating agonist bradykinin significantly decreased both cortical and medullary renal oxygen consumption. In conclusion, NO plays a role in maintaining a balance between oxygen consumption and sodium reabsorption, the major ATP-consuming process in the kidney, in conscious dogs, and NO can inhibit mitochondrial oxygen consumption in canine renal slices in vitro.

Animals↗

PubMed1998

Regulation of brain G-protein go by Alzheimer's disease gene presenilin-1.

To investigate a possible association between G-proteins and presenilin-1 (PS-1), a series of glutathione S-transferase-fusion proteins containing portions of PS-1 were prepared and used in vitro in binding experiments with tissue and recombinant G-proteins. The results demonstrate that the 39 C-terminal amino acids of PS-1 selectively bind the brain G-protein, Go. Addition of guanosine 5'-3-O-(thio)triphosphate promoted Go dissociation from PS-1, indicating that this domain mimics the function of G-protein-coupling domains found in receptors. The 39-amino acid synthetic polypeptide activated Go in a magnesium ion-dependent manner. Physical interaction of full-length PS-1 and Go was also demonstrated. Following transfection of Goalpha and N-terminally FLAG-tagged PS-1 in COS-7 cells, Go was immunoprecipitated by FLAG antibodies. In addition, endogenous PS-1 and Goalpha were colocalized immunocytochemically in human glioma cell lines. The results indicate that PS-1 regulates Go activities in living cells.

Alzheimer Disease↗

PubMed1998

Evidence that Meishan and large-white hybrid preovulatory follicles may differentially affect oocyte in vitro maturation and fertilization.

Two experiments were carried out to test the hypothesis that follicles recovered from Meishan animals may provide a more favourable environment for oocyte maturation in vitro than follicles recovered from Large-White hybrid animals. In Experiment 1, all follicles > or = 4 mm were recovered from six Meishan and seven Large-White hybrid gilts in the late follicular phase and healthy oocyte cumulus complexes recovered. Cumulus oocyte complexes were randomly divided into two groups, and each group cultured for 27 or 34 h (62 and 64; 56 and 56 for Meishan and Large-White hybrid, respectively) in defined medium in the presence of either of the two largest follicle shells per animal. Subsequent examination of oocyte nuclear maturation showed that although maturation did not differ significantly between the breeds after 27 h, more (P < 0.01) Meishan oocytes co-cultured with Meishan follicles developed to metaphase II stage than Large-White hybrid oocytes co-cultured with Large-White hybrid follicles after 34 h. The next eight largest follicles per animal were cultured for 34 h to produce conditioned media. In Experiment 2, oocytes recovered from the slaughterhouse were matured for 46 h in the presence of conditioned media from Meishan (612 oocytes) or Large-White hybrid (731 oocytes) follicles, or in fresh medium in the presence of a follicle shell from slaughterhouse ovaries. Oocytes were then inseminated and 12 h later examined for penetration and male pronuclear formation. A higher (P < 0.05) percentage of oocytes cultured in Meishan follicle conditioned medium underwent sperm penetration and male pronuclear formation than oocytes cultured in conditioned media from Large-White hybrid animals. Concentration of oestradiol and progesterone in the conditioned media did not differ between the breeds (P > 0.1). In conclusion, these results suggest that (1) Meishan oocytes have advanced maturational capacity when cultured with Meishan preovulatory follicle shells and (2) differences in follicle maturation in the Meishan compared to the Large-White hybrid pig may result in an improved ability of the follicles, via conditioned media, to support oocyte maturation and fertilization in vitro.

Animals↗

PubMed1998

Kinetic folding pathway of a three-disulfide mutant of bovine pancreatic ribonuclease A missing the [40-95] disulfide bond.

The oxidative refolding pathway of a three-disulfide mutant of bovine pancreatic ribonuclease A (RNase A) from the fully reduced unfolded form to the native state has been studied by using oxidized and reduced dithiothreitol as the redox reagents at pH 8.0 and 25 degrees C. This mutant was prepared by replacing Cys40 and Cys95 in RNase A with alanines while maintaining the other three native disulfide bonds to mimic one of the two major three-disulfide intermediates (des-[40-95]) observed in the regeneration of wild-type RNase A. The kinetics of refolding of this mutant were measured by quenching the regeneration reaction at various times with a rapid blocking reagent, 2-aminoethyl methanethiosulfonate (AEMTS), fractionating the disulfide intermediates by using cation-exchange HPLC, and analyzing the time course of each group of disulfide species. It was found that the disulfide intermediates formed during regeneration reach a steady-state distribution after a short period of preequilibration similar to that in the regeneration of wild-type RNase A. The experimental data acquired under different redox conditions were fit to a kinetic model with a steady-state treatment. The fitted results indicate that this mutant refolds through a rate-determining step which involves the oxidation of certain two-disulfide species to form a putative three-disulfide species which proceeds rapidly to the native protein. A rough estimation suggests that this pathway could constitute no more than 5% of the major pathway leading to the formation of des-[40-95] (the major three-disulfide intermediate formed) in the regeneration of wild-type RNase A. Several kinetic constants pertaining to the oxidation and reduction of various disulfide intermediates were compared with those obtained in the regeneration studies of wild-type RNase A to gain further understanding about the folding pathways of RNase A. Comparisons are also given for the oxidative refolding studies of several other three disulfide bond proteins, suggesting that the formation of a large number of disulfide-bonded intermediates during oxidative refolding is probably a common feature for most proteins.

Animals↗

PubMed1998

A tetrahedral zinc(II) complex of tris(2-pyridylethyl)amine.

The zinc ion in the title complex, [N,N'-bis(2-pyridylethyl)-2-(2-pyridyl)ethylamine-kappa 4N]zinc(II) diperchlorate, [Zn(C21H24N4)](ClO4)2, displays a distorted tetrahedral configuration. The Zn-Npy distances range from 1.979 (5) to 1.999 (5) A, while the Zn-Namine distance is 2.028 (5) A. The Npy-Zn-Npy and Npy-Zn-Namine angles range from 111.9 (2) to 119.6 (2) degrees and from 102.3 (2) to 103.4 (2) degrees, respectively. This behavior contrasts with the five-coordinate geometry normally observed in tripodal ligands of similar structure, and can be ascribed to the presence of weakly-coordinating anions and the ligand-metal chelate-ring size.

Crystallography, X-Ray↗

PubMed1998

Detection of programmed cell death using fluorescence energy transfer.

Fluorescence energy transfer (FRET) can be generated when green fluorescent protein (GFP) and blue fluorescent protein (BFP) are covalently linked together by a short peptide. Cleavage of this linkage by protease completely eliminates FRET effect. Caspase-3 (CPP32) is an important cellular protease activated during programmed cell death. An 18 amino acid peptide containing CPP32 recognition sequence, DEVD, was used to link GFP and BFP together. CPP32 activation can be monitored by FRET assay during the apoptosis process.

Amino Acid Sequence↗

PubMed1998

Cloning and characterization of vascular endothelial growth factor (VEGF) from zebrafish, Danio rerio.

We have cloned and sequenced a zebrafish (Danio rerio) Vascular Endothelial Growth Factor (vegf) cDNA. It encodes a precursor protein of 188 amino acids with a putative 23 amino acids signal peptide. Sequence comparison analysis indicates that the zebrafish vegf cDNA corresponds to the human VEGF165 isoform and shows about 52% identity to human VEGF165 at the amino acid level. A 2.8 kb vegf message RNA was detected in adult zebrafish by Northern blot analysis. Expression of vegf165 is also detected by RT-PCR in adult fish and throughout the zebrafish embryonic development. Whole mount in situ hybridization of zebrafish embryos indicates strong expression in four areas of the 18-19 h post-fertilization (hpf) embryo: within the anterior central nervous system in the prospective optic stalk, in mesoderm overlapping the bilaterally located merging heart fields, in mesoderm underlying and flanking the hindbrain posterior to rhombomere 4, and in medial regions of the somites. The study of vegf function in zebrafish embryonic vascular development will contribute to our understanding of the mechanisms of vertebrate endothelial cell differentiation and vasculature formation.

Amino Acid Sequence↗

PubMed1998

The tumor suppressor SMAD4/DPC4 is essential for epiblast proliferation and mesoderm induction in mice.

Members of the transforming growth factor (TGF)-beta superfamily have been shown to play a variety of important roles in embryogenesis, including dorsal and ventral mesoderm induction. The tumor suppressor SMAD4, also known as DPC4, is believed to be an essential factor that mediates TGF-beta signals. To explore functions of SMAD4 in development, we have mutated it by truncating its functional C-domain. We show that Smad4 is expressed ubiquitously during murine embryogenesis. Mice heterozygous for the Smad4(ex8/+) mutation are developmentally normal, whereas homozygotes die between embryonic day 6.5 (E6.5) and 8.5. All Smad4(ex8/ex8) mutants are developmentally delayed at E6 and show little or no elongation in the extraembryonic portion of late egg cylinder stage embryos. Consistent with this, cultured Smad4(ex8/ex8) blastocyst outgrowths suffer cellular proliferation defects and fail to undergo endoderm differentiation. Although a portion of mutant embryos at E8.5 show an increase in the embryonic ectoderm and endoderm, morphological and molecular analyses indicate that they do not form mesoderm. Altogether, these data demonstrate that SMAD4-mediated signals are required for epiblast proliferation, egg cylinder formation, and mesoderm induction.

Animals↗