PubMed Health⌕ Search

Biomedical subjects

X Xu

Publications and source records attributed to X Xu.

At least 19 recordsLinked to original sources
PubMed1996

Cloning and characterization of a glucocorticoid-induced diacylglycerol kinase.

Diacylglycerol kinase (DGK) plays a key role in cellular processes by regulating the intracellular concentration of the second messenger diacylglycerol. We screened a hamster DDT1 smooth muscle cell library and isolated a unique, glucocorticoid-inducible cDNA with substantial homology to known DGKs. DGK activity was increased in lysates of insect cells infected with recombinant baculovirus containing this cDNA. Antibodies raised against expressed sequences recognized a glucocorticoid-inducible 130-140-kDa protein on immunoblots of DDT1 cell lysates. Thus, this sequence appears to be a new member of the DGK family that we refer to as DGKeta. Homology to other DGKs was apparent in domains that are thought to be important for DGK function including the cysteine-rich motifs and potential catalytic domains. DGKeta shares substantial homology with DGKdelta including the N-terminal pleckstrin homology domain. The tissue distribution of DGKeta message (determined by ribonuclease protection assays) and protein (determined by immunoblots) was broader than reported for other DGKs, indicating that DGKeta may play a more general role in regulating cellular DG levels than other DGKs. Heterogeneity among DGK family members indicates that individual DGKs may have unique functions.

Amino Acid Sequence↗
PubMed1996

Photopolymerization and Mass-Independent Sulfur Isotope Fractionations in Carbon Disulfide

Irradiation of gaseous carbon disulfide [CS2(g)] at 313 nanometers produces a dark brown aerosol of (CS2)x. Its thermal decomposition products include disulfur (S2), carbon monosulfide (CS), and (CS)x. The photopolymerization process is accompanied by a large mass-independent isotopic fractionation of sulfur (a 5 to 10 per mil sulfur-33 excess and a 61 to 84 per mil sulfur-36 deficit). Excess sulfur-33 has been observed in several classes of meteorites. Photochemical production of (CS2)x may be important in the origin and evolution of cosmochemical environments such as the presolar nebula, meteorites, asteroids, and planetary atmospheres.

Journal Article↗
PubMed1996

Cooperative DNA binding and sequence-selective recognition conferred by the STAT amino-terminal domain.

STAT proteins (signal transducers and activators of transcription) activate distinct target genes despite having similar DNA binding preferences. The transcriptional specificity of STAT proteins was investigated on natural STAT binding sites near the interferon-gamma gene. These sites are arranged in multiple copies and required cooperative interactions for STAT binding. The conserved amino-terminal domain of STAT proteins was required for cooperative DNA binding, although this domain was not essential for dimerization or binding to a single site. Cooperative binding interactions enabled the STAT proteins to recognize variations of the consensus site. These sites can be specific for the different STAT proteins and may function to direct selective transcriptional activation.

Animals↗
PubMed1996

Gbetagamma transduces [Ca2+]i oscillations and Galphaq a sustained response during stimulation of pancreatic acinar cells with [Ca2+]i-mobilizing agonists.

A central unresolved question in agonist-evoked [Ca2+]i signaling is the pathway by which [Ca2+]i oscillations and a sustained response are transduced. We show here that activation of Gbetagamma signal [Ca2+]i oscillations and activation of Galphaq signal a sustained response during stimulation by a number of Ca2+-mobilizing agonists. Thus, infusion of purified Gbetagamma into pancreatic acinar cells through a patch pipette evokes [Ca2+]i oscillations by Ca2+ release from internal stores, which were inhibited by two independent scavengers of Gbetagamma, the beta-adrenergic receptor kinase fragment, and a mutated Galphai1G203A. These proteins, as well as an inhibitory antibody against Galphaq/11, prevent [Ca2+]i oscillations and the sustained response when applied before cell stimulation, possibly by preventing the dissociation of Gq into its subunits. After cell stimulation and dissociation of Gq into Gbetagamma and Galphaq, scavenging Gbetagamma stabilized the sustained response and inhibited reassociation of the subunits on termination of cell stimulation with antagonist, whereas scavenging Galphaq inhibited the sustained response and uncovered the Gbetagamma-dependent oscillations. These findings provide a general mechanism by which Ca2+-mobilizing agonists can control the type of [Ca2+]i signal to be transduced to the cell interior.

Animals↗
PubMed1996

Constitutively activated JNK is associated with HTLV-1 mediated tumorigenesis.

Human T cell leukemia virus type I (HTLV-1) is the etiologic agent of adult T-cell leukemia (ATL) and HTLV-1 associated myelopathy, also called tropical spastic paraparesis (HAM/TSP). Both clinical and in vitro evidence have demonstrated that the virus or its transactivator Tax, are transforming. However, transformation appears to require additional, as yet poorly characterized, genetic changes in infected cells. JNK is a recently characterized member of the MAP kinase family. Its signaling cascade is distinct from other members and has been demonstrated to play an important role in T-cell activation, at least partially through its downstream targets, c-jun and ATF-2. Here we demonstrate constitutive activation of the JNK cascade in human lymphocytes transformed in vitro by HTLV-1 and also in Tax transformed murine fibroblasts. Such activation is not induced by Tax expression alone, and occurs only when infected lymphocytes become IL-2 independent or immortalized. Constitutive JNK activation was also found in leukocytes isolated from ATL patients. The acquisition of constitutive JNK activation may represent an important later event in HTLV-1 tumorigenesis.

Animals↗
PubMed1996

Nonrandom distribution of the one-disulfide intermediates in the regeneration of ribonuclease A.

The one-disulfide intermediates formed during the oxidative refolding of ribonuclease A (RNase A) have been characterized. This information is important for understanding the folding pathways of RNase A. The one-disulfide intermediates were blocked with 2-aminoethyl methanethiosulfonate, fractionated using ion-exchange chromatography, and digested with trypsin and chymotrypsin. The resulting peptide fragments were fractionated using reversed phase high-performance liquid chromatography, and identified using mass spectrometry. The relative population of each one-disulfide intermediate was determined from its disulfide bond concentration using a postcolumn disulfide detection system. A total of 24 out of 28 possible one-disulfide intermediates were found to be populated (greater than 0.3%) in the one-disulfide mixture. The population of one-disulfide intermediates displays a nonrandom distribution. All four native disulfide pairings have populations greater than those predicted by loop entropy calculations, suggesting the presence of enthalpic contributions stabilizing these species. The one-disulfide intermediate [65, 72], containing the disulfide bond between cysteines 65 and 72, comprises 40% of the entire one-disulfide population. The interactions that stabilize this intermediate may play an important role in the regeneration pathways of RNase A.

Animals↗
PubMed1996

Regulation of the inositol 1,4,5-trisphosphate-activated Ca2+ channel by activation of G proteins.

Streptolysin O-permeable pancreatic acini were used to study the regulation of the inositol 1,4,5-trisphosphate (IP3)-activated Ca2+ channel (IPACC) by agonists and antagonists. Measurements of the apparent affinity for IP3 (KappIP3) showed that the IPACC is dynamically controlled during cell stimulation and inhibition, i.e. agonists decreased and antagonists increased KappIP3. KappIP3 was also independently regulated by thimerosal, Ca2+ content of the stores, the incubation temperature, activation of protein kinases, and inhibition of protein phosphatases, but none of these mechanisms contributed to the regulation by agonists and antagonists. Incubating the cells with low concentration of GTPgammaS or AIF3 reproduced the effect of the agonist on KappIP3. Moreover, low [GTPgammaS] allowed activation of the IPACC by agonists at basal levels of IP3 and markedly impaired channel inactivation by antagonists. Channel sensitization by GTPgammaS also restored the ability of thimerosal to mobilize Ca2+ from internal stores with no change in cellular IP3 levels. The combination of low [GTPgammaS] and thimerosal locked the channel in an open, antagonist-insensitive state. All modulatory effects of GTPgammaS are independent of phospholipase C activation and IP3 production. We propose that the dynamic regulation of the IPACC by a G protein-dependent mechanism can play a major role in triggering and maintaining Ca2+ oscillations at low agonist concentrations when minimal or no changes in IP3 level take place.

Animals↗
PubMed1996

Sequence requirements of ATF2 and CREB binding to the human T-cell leukemia virus type 1 LTR R region.

We have identified crucial transcription factor contact sites within the distal portion of the HTLV-1 LTR R region. Single base substitutions within this region greatly reduce formation of a complex which we previously described as a 70-kDa nuclear protein interacting with a CREB-like protein. Comparison of published sequences of HTLV-1 isolates obtained from scattered geographic locations revealed that clustered mutations in an 8-base segment near the U5 junction do, in fact, occur naturally. A single base substitution corresponding to a common naturally occurring mutation was introduced into the R region of an HTLV-1-LTR Cat construct. This resulted in derepression of the promoter in a cell line expressing high levels of the R region binding complex when compared to the wild-type LTR promoter. Affinity purification and electrophoretic mobility super-shift analysis identified a dominant 70-kDa DNA binding protein as ATF-2. Phosphorylated ATF-2 apparently interacts with CREB to form this downstream complex.

Activating Transcription Factor 2↗
PubMed1996

Binding and functional effects of transcriptional factor Sp1 on the murine interleukin-6 promotor.

The NF-kappaB and NF-IL6 elements have previously been shown to play an important role in regulation of both the mouse and human interleukin-6 gene. Between these two elements lies a G/C-rich sequence, which contains three repeats of the element CCACC, protein binding to which has not been previously characterized. In this study we demonstrate that the transcription factor Sp1 binds to these repeats and plays an important role in basal and in inducible expression of the murine interleukin-6 gene.

3T3 Cells↗
PubMed1996

Evidence for trans-cis isomerization of the p-coumaric acid chromophore as the photochemical basis of the photocycle of photoactive yellow protein.

Analysis of the chromophore p-coumaric acid, extracted from the ground state and the long-lived blue-shifted photocycle intermediate of photoactive yellow protein, shows that the chromophore is reversibly converted from the trans to the cis configuration, while progressing through the photocycle. The detection of the trans and cis isomers was carried out by high performance capillary zone electrophoresis and further substantiated by 1H NMR spectroscopy. The data presented here establish the photo-isomerization of the vinyl double bond in the chromophore as the photochemical basis for the photocycle of photoactive yellow protein, a eubacterial photosensory protein. A similar isomerization process occurs in the structurally very different sensory rhodopsins, offering an explanation for the strong spectroscopic similarities between photoactive yellow protein and the sensory rhodopsins. This is the first demonstration of light-induced isomerization of a chromophore double bond as the photochemical basis for photosensing in the domain of Bacteria.

Bacterial Proteins↗
PubMed1996

Consequences of functional expression of the plasma membrane Ca2+ pump isoform 1a.

The plasma membrane Ca2+-ATPase pump (PMCA) is an integral component of the Ca2+ signaling system which participates in signal transduction during agonist stimulated cell activation. To better understand the physiological function of the pump, isoform 1a (PMCA1a) was over-expressed in rat aortic endothelial cells using a stable transfection system under the control of a cytomegalovirus promoter. The cell lines selected after transfection with PMCA1a construct, expressed 3-4-fold increased pump protein which was mostly targeted to the plasma membrane as indicated by immunoperoxidase staining. Ca2+ uptake assays in a membrane preparation indicated a 3-4-fold increase in Ca2+ pumping activity in the transfected cells, and the expressed PMCA1a showed typical dependence on Ca2+ and calmodulin for stimulation of activity. Measurement of [Ca2+]i and [Ca2+]out showed that expression of PMCA1a had a profound effect on different aspects of the Ca2+ signal. The peak increase in [Ca2+]i evoked by ATP and/or thapsigargin was lower but the plateau phase was similar in the PMCA1a expressing cells. Accordingly, titration with ionomycin of Ca2+ content of internal stores, measurement of Ca2+ uptake into the thapsigargin- and oxalate-sensitive pool (endoplasmic reticulum) of isolated microsomes, Ca2+ uptake into streptolysin O-permeabilized cells, and analysis of SERCA mRNA and protein, showed that expression and activity of the SERCA pump was down-regulated in cells expressing PMCA1a pump. Expression of PMCA1a also down-regulated expression of the inositol 1,4,5-trisphosphate (IP3)-activated Ca2+ channel and the rate of IP3-mediated Ca2+ release in permeable cells, without affecting the affinity of the channel for IP3. On the other hand the rate of store depletion-dependent Ca2+ and Mn2+ influx (Ca2+ entry) into PMCA1a expressing cells was increased by about 2.6-fold. These changes prevented estimating the rate of pump-mediated Ca2+ efflux from changes in [Ca2+]i. Measurement of [Ca2+]out showed that the rate of Ca2+ efflux in cells expressing PMCA1a was about 1.45-fold higher than Neo controls, despite the 4-fold increase in the amount of functional pump protein. The overall study points to the flexibility, interdependence, and adaptability of the different components of the Ca2+ signaling systems to regulate the expression and activity of each component and maintain a nearly constant Ca2+ signal.

Animals↗
PubMed1996

Roles of active site residues and the NH2-terminal domain in the catalysis and substrate binding of human Cdc25.

Human Cdc25 proteins are dual specific protein phosphatases that play important roles in cell cycle regulation. In this study, the catalytic mechanism and substrate binding specificity of human Cdc25A and -B proteins were investigated by site-directed and deletion mutagenesis methods. Mutations of the cysteine or the arginine residues in the active site motif abolished the Cdc25 phosphatase activity. However, the cysteine mutation in both Cdc25A and -B created enzymes that still retain the ability to bind their substrates. This allowed us to test the ability of Cdc25A and -B to bind various cyclin-Cdk complexes in vitro. While Cdc25A Cys --> Ser could interact with cyclin A-Cdk2, cyclin B-Cdc2, and cyclin E-Cdk2 strongly, Cdc25B mutant was only found to bind to cyclin A-Cdk2 at significant levels. We also identified Arg452 and Ser449 as two crucial residues that could be directly involved in the molecular interactions between Cdc25 and cyclin-Cdk proteins. Deletion mutagenesis data also indicate that the phosphatase catalytic domains of Cdc25A and -B proteins are located within their carboxyl terminus.

Amino Acid Sequence↗
PubMed1996

Identification of ligand binding residues in extracellular loops of the melanocortin 1 receptor.

To investigate whether residues in the extracellular domains of melanocortin 1 receptor (MC1R) are required for ligand binding, a number of mutants were constructed where charged residues were converted to alanine. The residues targeted for mutagenesis were Ser6, Glu102, Arg109, Asp184, Glu269, and Thr272. The mutant receptor DNAs were transiently expressed in COS-1 cells and their ability to bind [N1e4,D-Phe7]-alpha-MSH (NDP-MSH) was evaluated. Substitution of Asp184 by alanine completely abolished the binding of radiolabelled NDP-MSH as well as ACTH, even though the mutated receptor could be detected on cell surface using anti MC1R specific polyclonal antiserum. Mutations of Ser6, Glu269 and Thr272 resulted in a considerable loss of affinity for radiolabelled NDP-MSH as well as the ability of alpha-MSH to displace the bound radiolabelled NDP-MSH. The results demonstrate that the extracellular loops of human MC1R contain important ligand binding epitopes.

Affinity Labels↗
PubMed1996

Lack of correlation between Sendai virus P/C mRNA structure and its utilization of two AUG start sites from alternate reading frames: implications for viral bicistronic mRNAs.

The polycistronic P/C mRNA of Sendai virus encodes five proteins (C', P, C, Y1, and Y2) each of which initiates from a distinct start site. Two major proteins, P and C, are expressed in approximately equimolar amounts from two consecutive AUGs in overlapping reading frames. To better understand the mechanism of expression of the C protein from a downstream AUG, site-directed mutants of the P/C mRNA were created and expressed in COS1 cells. The secondary structure of the mRNA was examined to determine whether the mRNA structure played any role in the synthesis of the C protein. Our results ruled out any significant involvement of the 5' UTR, sequence contexts, secondary structure, distance between the start sites, and sequences downstream to the C-AUG. However, they are consistent with the concept that the synthesis of the C protein is primarily dependent on the orientation of its reading frame, i.e., +1 in relation to the upstream P reading frame. The downstream reading frame was translated poorly when it occurred in +2 orientation in relation to the upstream reading frame. Interestingly, all the known functional bicistronic mRNAs with overlapping reading frames from cytoplasmic RNA viruses have their downstream reading frame in +1 orientation relative to the upstream frame. We propose that the evolutionary conservation of the downstream reading frame in +1 orientation in these bicistronic mRNAs is important for its efficient translation.

Base Sequence↗
PubMed1996

Chromosomal localization and 5' sequence of the human protein serine/threonine phosphatase 5' gene.

Protein phosphorylation plays a crucial role in the regulation of a wide array of proteins involved in many cellular processes. Protein phosphatase 5 (PP5) is a novel member of the protein serine/threonine phosphatase family. The majority of the cDNA sequence of PP5 has been reported recently. In our study, a sequence encoding the whole open reading frame of PP5 was cloned from a human fetal brain cDNA library. The protein phosphatase cDNA sequence of our clone is longer at the 5' end than the recently published sequence. It's likely that the extended sequence contains the start codon ATG, since a translation stop codon TAG is present upstream of the ATG codon in the same open reading frame. The mRNA of the PP5 gene was detected in all the human tissues examined. The PP5 gene was localized to human chromosomal region 19q13.3.

Amino Acid Sequence↗
PubMed1996

Increased micronucleus frequency in lymphocytes from smokers with lung cancer.

We investigated whether lung cancer was associated with an increased micronucleus (MN) frequency in lymphocytes in a case-control study. Epidemiological data were obtained by an interviewer-administered questionnaire and included information on smoking history, intake of dietary micronutrients, general medical history, environmental and occupational exposures to mutagens and carcinogens, and family history of cancer. A modified cytokinesis-block method was used to determine individual MN frequency. Polymorphisms in glutathione S-transferase class mu were determined by PCR analysis. Overall, 55 controls and 42 cases were studied. MN frequency in cases and controls was not associated with age, smoking, metabolic genetic polymorphisms, environmental and occupational exposures, or medical history. Female controls had a significantly higher MN frequency than male controls (p = 0.05). Overall, MN frequency was significantly higher in cases than in controls (p < 0.01). Twenty-four cases (57%) had an MN frequency higher than the upper 95% confidence interval of the mean value for controls (11.5 MNs/1000 binucleated cells). Further analysis showed that, cases who were current and former smokers had significantly higher MN frequencies than controls (p = 0.04); this difference was not seen in the group that had never smoked. The significantly higher MN frequency among cases with a history of smoking may be attributable to the presence of lung neoplasm per se or to the interaction of smoking with endogenous factors associated with the development of lung cancer.

Aged↗
PubMed1996

Effects of leflunomide and other immunosuppressive agents on T cell proliferation in vitro.

Leflunomide and its active metabolite, A771726, are structurally unrelated to immunosuppressive agents currently under investigation. Previous in vitro studies have revealed that leflunomide primarily inhibits interleukin-2-stimulated T cell proliferation. In the current study, we have extended our previous work and demonstrate that leflunomide prevents T cell progression induced by phytohemagglutinin into the S phase of the cell cycle. To discriminate further the action on T cells of leflunomide from other immunosuppressive agents, we performed kinetic studies where leflunomide was added either after the initiation of mixed lymphocyte cultures (MLC) or after interleukin-2 stimulation of CTLL-4 cell proliferation. These studies revealed that leflunomide acted comparably to rapamycin, but was distinct from brequinar sodium in the MLC, and from cyclosporine and mycophenolic acid in both MLC and CTLL-4. Although previous biochemical studies indicated that leflunomide can inhibit src-family tyrosine kinase activity, more recent studies have suggested that leflunomide can also inhibit pyrimidine synthesis. Our data demonstrate that the ability of leflunomide (25-100 microM) to inhibit MLC and CTLL-4 cell proliferation is partially antagonized by uridine (25-100 microM), and support the hypothesis that leflunomide inhibits pyrimidine synthesis in T cells. Unique molecular mechanisms of immunosuppression suggest that drug combinations may result in synergistic immunosuppression. Our in vitro studies revealed synergistic inhibition of T cell proliferation with the combinations of leflunomide with cyclosporine or with rapamycin. We have extended those studies to quantitate inhibition of MLC by the combinations of leflunomide and brequinar sodium or mycophenolic acid.

Cell Division↗