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Y Xu

Publications and source records attributed to Y Xu.

At least 973 records · Page 54Linked to original sources
PubMed1995

Location of the catalytic site for phosphoenolpyruvate formation within the primary structure of Clostridium symbiosum pyruvate phosphate dikinase. 2. Site-directed mutagenesis of an essential arginine contained within an apparent P-loop.

Pyruvate phosphate dikinase catalyzes the interconversion of adenosine 5'-triphosphate (ATP), orthophosphate (P(i)), and pyruvate with adenosine 5'-monophosphate (AMP), pyrophosphate (PP(i)), and phosphoenolpyruvate (PEP). The Arg 561 residue of Clostridium symbiosum PPDK is contained within a Gly-rich stretch of sequence spanning positions 553-563 (viz., GAEGIGLCRTE) located in the 35 kDa C-terminal domain of the enzyme. The possible role of this stretch of sequence as a phosphate binding loop participating in catalysis of the PEP/pyruvate partial reaction (viz., E+PEP<-->E-P+pyruvate, where E-P represents enzyme phosphorylated at the catalytic histidine) was deduced from the similarity of this sequence to other known phosphate binding loops and by its location in the 35 kDa PEP/pyruvate binding domain of PPDK. To test the proposed role of Arg 561, and hence, the signature sequence, in catalysis of the E+PEP<-->E-P+pyruvate partial reaction, the C. symbiosum PPDK site-directed mutants Arg 561-->Leu 561 and Arg 561-->Lys 561 were constructed and expressed in Escherichia coli JM101. Neither mutant catalyzed the full PPDK reaction, ATP+P(i)+pyruvate<-->AMP+PP(i)+PEP, but both catalyzed the E+ATP+P(i)<-->E-P+AMP+PP(i) partial reaction as efficiently as wild-type PPDK. Both mutants were shown to be unable to catalyze the PEP/pyruvate partial reaction. On the basis of these results it was proposed that Arg 561 and, possibly, the Gly-rich stretch of sequence spanning positions 553-563 are essential components of the active site of the PEP/pyruvate partial reaction.

Amino Acid Sequence↗
PubMed1995

Separate site catalysis by pyruvate phosphate dikinase as revealed by deletion mutants.

Previous studies had indicated that pyruvate phosphate dikinase (PPDK), an enzyme which catalyzes the interconversion of adenosine 5'-triphosphate (ATP), orthophosphate (P(i)), and pyruvate with adenosine 5'-monophosphate (AMP), pyrophosphate (PP(i)), and phosphoenolpyruvate (PEP), is made up of 25, 13, 18, and 35 kDa domains [Carroll, L. J., Xu, Y., Thrall, S. H., Martin, B. M. & Dunaway-Mariano, D. (1994) Biochemistry 33, 1134]. The catalytic histidine (which mediates the phosphoryl group transfers from ATP to P(i) and pyruvate) is located on the 18 kDa domain while the 25 and 13 kDa domains appear to contain the ATP binding site and the 35 kDa domain appears to contain the pyruvate binding site, respectively. The goal of this investigation was to examine functional interdependency of the putative ATP and pyruvate binding domains. Two truncated forms of PPDK were created by using recombinant DNA techniques. The 35 kDa (C-terminal) deletion mutant was found to catalyze the E+ATP+P(i)<-->E-P+AMP+PP(i) partial reaction but not the E-P+pyruvate<-->E+PEP partial reaction. The 25 kDa (N-terminal) deletion mutant was found to catalyze the E-P+pyruvate<-->E+PEP partial reaction but not the E+ATP+P(i)<-->E-P+AMP+PP(i) partial reaction. Neither mutant catalyzes the full ATP+P(i)+pyruvate<-->AMP+PP(i)+PEP reaction. These results are interpreted to mean that the ATP and pyruvate binding domains in PPDK are functionally independent, thus providing evidence for separate active sites for catalysis of the two partial reactions.

Base Sequence↗
PubMed1995

High-yield bacterial expression, purification, and functional reconstitution of the tricarboxylate transport protein from rat liver mitochondria.

The rat liver mitochondrial tricarboxylate transport protein has been overexpressed in E. coli. The expressed transporter, which contains a 21 amino acid N-terminal fusion sequence, accumulates in inclusion bodies. Subsequent extraction of the tricarboxylate transporter from isolated inclusion bodies yields approximately 90 mg of transport protein per liter of E. coli culture at a purity of greater than 90%. Upon incorporation into phospholipid vesicles the purified, overexpressed transporter catalyzes a 1,2,3-benezenetricarboxylate-sensitive citrate/citrate exchange (i.e., the defining reaction of the mitochondrial tricarboxylate transporter). Kinetic characterization of the reconstituted transporter indicates a Km of 0.37 mM and a Vmax of 101 nmol/min/mg protein. The substrate specificity of the reconstituted, expressed transporter is virtually identical to that of the native transporter. These studies represent the first overexpression of the rat liver mitochondrial tricarboxylate transporter. By providing a large amount of highly-purified, functionally competent transporter this system will now enable a variety of structural studies, including site-directed mutagenesis, which heretofore could not be performed.

Animals↗
PubMed1995

FLK-2/FLT-3 ligand regulates the growth of early myeloid progenitors isolated from human fetal liver.

The effects of the recently identified FLK-2/FLT-3 ligand (FL) on the growth of purified human fetal liver progenitors were investigated under serum-deprived culture conditions. FL alone was found to stimulate modest proliferation in short-term cultures of CD34++ CD38+ lineage (Lin)- light-density fetal liver (LDFL) cells and the more primitive CD34++ CD38- Lin- LDFL cells. However, the low levels of growth induced by FL were insufficient for colony formation in clonal cultures. Synergism between FL and either granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3) or KIT ligand (KL) was observed in promoting the growth of high-proliferative potential (HPP) colony-forming cells (CF) and/or low-proliferative potential (LPP)-CFC in cultures of CD34++ CD38+ Lin- and CD34++ CD38- Lin- LDFL-cells. FL, alone or in combination with other cytokines, was not found to affect the growth of CD34+ Lin- LDFL cells, the most mature subpopulation of fetal liver progenitors investigated. The growth of the most primitive subset of progenitors studied, CD34++ CD38- Lin- LDFL cells, required the interactions of at least two cytokines, because only very low levels of growth were observed in response to either FL, GM-CSF, IL-3 or KL alone. However, the results of delayed cytokine-addition experiments suggested that individually these cytokines did promote the survival of this early population of progenitors. Although two-factor combinations of FL, KL, and GM-CSF were observed to promote the growth of early progenitors in a synergistic manner, neither of these factors was found to make fetal liver progenitors more responsive to suboptimal concentrations of a second cytokine. Only myeloid cells were recovered from liquid cultures of CD34++ CD38- Lin- LDFL cells grown in the presence of combinations of FL, KL, and GM-CSF. These results indicate that FL is part of a network of growth factors that regulate the growth and survival of early hematopoietic progenitors.

ADP-ribosyl Cyclase↗
PubMed1995

Characterization of the proximal promoter region of the rat somatostatin receptor gene, SSTR4.

In the present study, the putative promoter region of rSSTR4 gene has been cloned and characterized. Primer extension and RNAase protection analysis reveals the existence of multiple transcriptional initiation sites, the five major ones mapping between -126 and -18 relative to the ATG initiation codon. Sequence analysis reveals that the region lacks TATA and CCAAT promoter elements, possesses a high GC content and contains a number of potential SP1 binding sites. In addition, potential AP-2, GCF, and PuF binding sites and an octimer motif were also identified. Thus, the promoter of rSSTR4 shows similar features to promoters of highly regulated growth factor receptors and oncogenes. The potential role these transcriptional binding sites play in regulating rSSTR4 expression is currently being investigated.

Animals↗
PubMed1995

Thrombin, phorbol ester, and cAMP regulate thrombin receptor protein and mRNA expression by different pathways.

Human mesangial cells have been used to study the regulation of thrombin receptor protein and mRNA expression during cross-talk between different signal transduction pathways. Persistent activation of thrombin receptor by thrombin led to homologous down-regulation of thrombin receptor protein. However, thrombin receptor mRNA expression was not affected, suggesting that increased receptor degradation is responsible for homologous down-regulation. Chronic activation of protein kinase C by phorbol 12-myristate 13-acetate (PMA) and of adenylylcyclase by prostaglandin E1 (PGE1) resulted in heterologous down-regulation of thrombin receptor protein. In contrast to thrombin, PMA and PGE1 reduced in parallel thrombin receptor mRNA levels to 51% and 24% of control, respectively, indicating that heterologous down-regulation of thrombin receptor protein is, at least in part, due to inhibition of receptor mRNA expression. The mechanisms of heterologous down-regulation of thrombin receptor protein have been studied in detail and compared to homologous down-regulation. PMA-induced down-regulation was completely blocked by GF 109 203 X, an inhibitor of protein kinase C. However, the loss of thrombin receptor induced by thrombin was not prevented by GF 109 203 X, indicating that homologous regulation is not dependent on protein kinase C activation. The heterologous effect of PGE1 was mimicked by 8-bromo-cAMP, isobutylmethylxanthine, and forskolin, suggesting that an increase in intracellular cAMP level is involved in heterologous regulation. Interestingly, heterologous down-regulation induced by PGE1 seems not to require previous internalization of thrombin receptor. These data indicate that thrombin receptor protein and mRNA expression can be regulated in homologous and heterologous ways by different mechanisms.

Cells, Cultured↗
PubMed1995

Regulation of somatostatin receptor mRNA expression.

The five somatostatin receptor mRNAs are expressed with distinct though overlapping patterns of distribution in the CNS and peripheral tissues. All receptor types are expressed in the anterior pituitary and hypothalamus and could therefore be modulated in states of growth hormone (GH) dysregulation. Metabolic perturbations such as food deprivation and diabetes mellitus lead to suppression of GH levels in the rat, in part due to increased somatostatin tone. In rats deprived of food, pituitary sstr1, 2 and 3 mRNAs were reduced by 80% compared to fed controls; sstr4 and sstr5 mRNAs were unchanged. Hypothalamic sstr mRNA expression was unaltered. In diabetic rats pituitary sstr1, 2 and 3 mRNAs were reduced by 50-80% with sstr1 mRNA restored in part by insulin therapy. Pituitary sstr4 mRNA and hypothalamic expression of these four types was unaffected. sstr5 mRNA is reduced by 70% in the pituitary and by 30% in the hypothalamus with restoration of both by insulin treatment. Altered pituitary sstr expression in food deprivation and diabetes could result from chronic exposure to increased plasma somatostatin. In rat GH3 pituitary tumour cells exposed to 1 microM somatostatin for up to 48 h, sstr1, 3, 4 and 5 mRNA increased dramatically while sstr2 mRNA exhibited a biphasic response. We observed a net increase in receptor binding associated with increased sstr mRNA. Somatostatin receptor expression is regulated in a tissue- and type-specific manner, adding further complexity to the action of the multifaceted peptide somatostatin.

Animals↗
PubMed1995

Effect of lysophospholipids on signaling in the human Jurkat T cell line.

Lysophospholipids have recently been demonstrated to induce activation and proliferation of fibroblasts and other cell lineages by interacting with high affinity cell surface receptors leading to specific intracellular signaling events. Platelet activation, likely at the site of injury or inflammation, results in increased production of lysophospholipids suggesting a possible source of lysophospholipids. We have recently demonstrated that high concentrations of lysophospholipids are present in ascites and plasma from ovarian cancer patients, suggesting that physiologically produced lysophospholipids could interact with cells present in these fluids, including lymphocytes, and alter their function. We demonstrate herein that lysophosphatidic acid (LPA), lysophosphatidylserine (LPS), and sphingosylphosphorylcholine (SPC) activate the Jurkat T cell line. Each of the lysophospholipids induced a transient increase in cytosolic free calcium ([Ca2+]i) in Jurkat cells. Increases in [Ca2+]i were cross-desensitized by LPA, LPS and SPC, suggesting that the lysophospholipids share the same receptor(s) or that their downstream signaling pathways converge or interact. Lysophosphatidylglycerol (LPG), a competitive inhibitor of the putative LPA receptor, inhibited the calcium releasing activity of LPA, but not that of LPS and SPC, suggesting that these lysophospholipids interact with different receptors and that desensitization is due to interactions in downstream signaling pathways. The ability of the lysophospholipids to induce increases in [Ca2+]i was attenuated, but not completely blocked, by increases in [Ca2+]i induced by activation of the thrombin receptor. In contrast, increases in [Ca2+]i induced by the lysophospholipids and cross-linking the CD3 component of the T cell receptor complex with the UCHT1 antibody did not undergo heterologous desensitization. Strikingly, LPA is sufficient to stimulate proliferation of Jurkat cells in serum-free medium or in synergy with low concentrations of fetal bovine serum. In addition, LPA also increased the production of the T cell growth factor, interleukin 2 (IL-2), by Jurkat cells treated with phorbol esters. LPS, in contrast, inhibited Jurkat proliferation while increasing IL-2 production and SPC inhibited both processes. Thus, although all three lysophospholipids were sufficient to induce a transient increase in [Ca2+]i in Jurkat cells, they induced markedly different physiological consequences.

Calcium↗
PubMed1995

Myocardial tagging with B1 insensitive adiabatic DANTE inversion sequences.

A new technique, based on adiabatic delays alternating with mutations for tailored excitation (DANTE) inversion sequences, is presented for generating uniform contrast tags across the myocardial wall even in the presence of B1 inhomogeneities. The utility of this pulse was demonstrated using a surface coil for both transmission and signal reception in phantom and animal heart tagging studies. The experimental data demonstrated uniform grid contrast over a sixfold variation of B1 magnitude, sharp tagging profiles, and the ability to follow the cardiac wall motion through the deformation of the fine rectangular tagging grid at different phases throughout the cardiac cycle.

Animals↗
PubMed1995

Diversity and modulation of plasminogen activator activity in human prostate carcinoma cell lines.

Baseline cellular plasminogen activator (PA) activity, and the cellular proteins responsible for variations in PA activity were evaluated in three human prostate carcinoma cell lines. Net PA activity in the cell lines PC-3, DU-145, and LNCaP was measured using a plasminogen-dependent fibrin lysis assay. These three cell lines were then analyzed to determine the specific protein(s) responsible for differences in PA activity. mRNA and protein levels of cellular urinary PA (uPA), tissue PA (tPA), PA inhibitor 1 (PAI1), PA inhibitor 2 (PAI2), and uPA receptor (uPAr) were measured using Northern analysis and ELISA assays. Net cellular PA activity in the three cell lines varied over a 3-fold range (PC3 > DU145 >> LNCaP). Net PA activity in the fibrinolysis assay demonstrated a direct correlation with mRNA transcript levels of uPA, tPA, PAI1, and uPAr (PC-3 > DU-145 > LNCaP). uPA protein was identified in both the PC-3 and the DU-145 lines. tPA, PAI1, and PAI2 proteins were identified only in PC-3 cells. In general, cellular protein levels correlated with mRNA levels. These findings demonstrate that prostate carcinoma cell lines vary in their net PA activity. This variability results from both qualitative and quantitative differences in the cellular expression of PA regulatory proteins.

Blotting, Northern↗
PubMed1995

Thrombin and phorbol ester induce internalization of thrombin receptor of human mesangial cells through different pathways.

Thrombin is a potent activator of human mesangial cells probably by activation of its functional receptor. Northern blot analysis demonstrates the presence of mRNA encoding the functional thrombin receptor in mesangial cells, and surface expression of thrombin receptor antigen has been confirmed by immunocytochemistry. Using 125I-labeled ATAP2, a monoclonal antibody against the functional thrombin receptor, we found that thrombin and thrombin receptor agonist peptide (TRAP) induce homologous internalization of thrombin receptor in a dose-dependent manner. Redistribution of thrombin receptor from the cell surface to vesicular structures in the cytoplasm has been followed by immunocytochemistry. Additionally, a dose-dependent loss of cell surface thrombin receptor is induced by phorbol 12-myristate 13-acetate (PMA), suggesting that thrombin receptor undergoes heterologous internalization in response to PMA. The time course of thrombin-induced receptor internalization is different from that observed with TRAP and PMA. Protein kinase C inhibitors, staurosporine and GF 109 203 X, do not affect thrombin receptor internalization induced by thrombin and TRAP but block receptor internalization stimulated by PMA. These data suggest that heterologous thrombin receptor internalization induced by PMA is mediated by protein kinase C. However, activation of protein kinase C is not responsible for homologous thrombin receptor internalization caused by thrombin and TRAP.

Alkaloids↗
PubMed1995

A global optimization method based on variable target functions for fitting of the experimental and calculated NOESY spectra.

A new method for structure refinement, based on optimization of the variable target function (Sugár and Xu, Prog. Biophys. Mol. Biol. 58, 61, 1992), is examined in order to explore the conditions of high-resolution polypeptide structure determination. The method of variable target function was used to analyze simulated NOESY spectra of different spectral resolutions, of different signal/noise ratios, and of different number of mixing times. The goodness of the results was assessed by comparing the known polypeptide conformation with the optimized ones, as well as by comparing the simulated experimental spectra with the spectra back-calculated from the optimized structures. According to the test calculations, in order to get high-resolution polypeptide structures, the number of constraints should be larger than the number of dihedral angles, and the constraints should be distributed over the dihedral angles more or less homogeneously. The success rate of the variable-target-function method in finding high-resolution structures was high until a critical signal/noise ratio, while below this threshold the success rate was low. The critical signal/noise ratio could be decreased, however, by increasing the number of mixing times. The test calculations show that the variable-target-function method is capable of high-resolution polypeptide structure determination with a success rate of 85-90% under common experimental conditions, such as 10 constraints/residue, 40% noise/signal ratio, and six mixing times.

Magnetic Resonance Spectroscopy↗
PubMed1995

Fine structure and function of the osmotin gene promoter.

The gene encoding osmotin, a tobacco pathogenesis-related protein, has been shown to be regulated by an array of hormonal and environmental signals. The osmotin promoter fragment -248 to -108 upstream of the transcription start site (fragment A), was sufficient to direct reporter gene expression when fused to a minimal CaMV 35S promoter in transient assays using microprojectile bombardment. This was consistent with previous 5'-deletion analyses of the osmotin promoter which showed that the promoter sequence from -248 to -108 is absolutely required for reporter gene activity. Nuclear protein factors from salt-adapted tobacco cells, ABA-treated unadapted cells, and young cultured tobacco leaves were shown to interact with fragment A by gel mobility-shift assays. DNase I footprinting revealed that three conserved promoter elements in fragment A interact specifically with nuclear factors. These elements are: (1) a cluster of G-box-like sequences (G sequence); (2) an AT-1 box-like sequence, 5'-AATTATTTTATG-3' (AT sequence); (3) a sequence highly conserved in ethylene-induced PR gene promoters, 5'-TAAGA/CGCCGCC-3' (PR sequence). Transient expression assays performed with fragment A deletions fused to GUS indicated that osmotin promoter activity correlated with the presence of these elements. UV cross-linking analysis showed that the protein complex bound to fragment A consisted of at least four individual proteins with approximate molecular masses of 28, 29, 40 and 42 kDa. One component of this protein complex, which was associated with the G sequence, was a 14-3-3 like protein.

Base Sequence↗
PubMed1995

Characterization of a rice gene family encoding root-specific proteins.

Two cDNA clones (RCc2 and RCc3) corresponding to mRNAs highly expressed only in root tissues of rice (Oryza sativa L.) seedlings were characterized. Respectively, they encode polypeptides of 146 (14.5 kDa) and 133 amino acids (13.4 kDa) that share high (> 70%) sequence similarity with a polypeptide encoded by a cDNA (ZRP3) encoding an mRNA preferentially expressed in young maize roots. Genomic DNA blot analysis revealed that they are members of a small gene family and RCg2, the gene corresponding to RCc2, was isolated. A 1656 bp 5'-upstream sequence of RCg2 was translationally fused to a beta-glucuronidase (GUS) reporter gene and stable introduction of the chimeric construct into rice was confirmed by PCR and genomic DNA blot analyses. Histochemical analysis of transgenic rice plants containing the full-length chimeric gene showed high levels of GUS activity in mature cells and the elongation and maturation zones of primary and secondary roots, and in the root caps, but no GUS activity was detected in root meristematic regions. Surprisingly, high GUS activity was also detected in leaves of the same plants. This raises the possibility that the RCg2 5'-upstream element may not be sufficient for the proper spatial control of root specificity in transgenic rice.

Amino Acid Sequence↗
PubMed1995

A variable target intensity-restrained global optimization (VARTIGO) procedure for determining three-dimensional structures of polypeptides from NOESY data: application to gramicidin-S.

A global optimization method for intensity-restrained structure refinement, based on variable target function (VTF) analysis, is illustrated using experimental data on a model peptide, gramicidin-S (GS) dissolved in DMSO. The method (referred to as VARTIGO for variable target intensity-restrained global optimization) involves minimization of a target function in which the range of NOE contacts is gradually increased in successive cycles of optimization in dihedral angle space. Several different starting conformations (including all-trans) have been tested to establish the validity of the method. Not all optimizations were successful, but these were readily identifiable from their large NOE R-factors. We also show that it is possible to simultaneously optimize the rotational correlation time along with the dihedral angles. The structural features of GS thus obtained from the successful optimizations are in excellent agreement with the available experimental data. A comparison is made with structures generated from an intensity-restrained single target function (STF) analysis. The results on GS suggest that VARTIGO refinement is capable of yielding better quality structures. Our work also underscores the need for a simultaneous analysis of different NOE R-factors in judging the quality of optimized structures. The NOESY data on GS in DMSO appear to provide evidence for the presence of two orientations for the ornithine side chain, in fast exchange. The NOESY spectra for this case were analyzed using a relaxation rate matrix which is a weighted average of the relaxation rate matrices for the individual conformations.

Amino Acid Sequence↗
PubMed1995

Metabolism of cyclohexane carboxylic acid by the photosynthetic bacterium Rhodopseudomonas palustris.

Cyclohexane carboxylate supported relatively rapid growth (doubling times 7-8 h) of Rhodopseudomonas palustris under oxic or photosynthetic conditions, but did not serve as a substrate for either of the known aromatic CoA ligases. A CoA ligase that thioesterifies cyclohexane carboxylate was partially purified and did not cross react immunologically with the two CoA ligases purified previously from this bacterium. Crude extracts of R. palustris cells grown with a range of aromatic or alicyclic acids contained a dehydrogenase that reacted with cyclohexane carboxyl-CoA or cyclohex-1-ene carboxyl-CoA, using 2,6-dichlorophenolindophenol or ferricenium ion as electron carrier. This activity was not detected in extracts of adipate-, glutamate-, or succinate-grown cells. No oxidation or reduction of nonesterified cyclohexane carboxylate or cyclohexene carbocylate was detected in extracts of cells grown with aromatic or aliphatic substrates, neither aerobically nor anaerobically. A constitutively expressed thioesterase that hydrolyzed cyclohexane carboxyl-CoA and also some alicyclic and aliphatic CoA derivatives was purified and characterized. The enzyme had little or no activity on benzoyl-CoA or 4-hydroxybenzoyl-CoA. The presence of a thioesterase that effectively hydrolyzes cyclohexane carboxyl-CoA suggests that transient production of cyclohexane carboxylate is a physiological response to temporary excess of reductant during metabolism of aromatic compounds.

Amino Acid Sequence↗