Biomedical subjects

X Xu

Publications and source records attributed to X Xu.

At least 721 records · Page 40Linked to original sources

PubMed1996

The role of pyridine dinucleotides in regulating the permeability of the mitochondrial outer membrane.

Both NADH and NADPH reduce the permeability of the mitochondrial outer membrane to ADP. This is specific for the outer membrane and uncorrelated with the respiratory control ratio. This could result in a 7-fold difference between the concentration of ADP in the intermembrane space and that in the external environment (at 5 microM ADP). In both cases the permeability declines by a factor of 5, but NADH is more potent: KD = 86 microM for NADH versus 580 microM for NADPH. The lower apparent affinity for NADPH is partly explained by Mg2+-NADPH being the active species, and under our conditions only 30% of the NADPH is in this form. The corrected KD is 184 microM. Free NADH has the same charge as the Mg2+-NADPH complex, and thus both likely bind to the same site. The ability of NADH and NADPH to induce the closure of reconstituted VDAC channels is consistent with VDAC being the main pathway for metabolite flow across the outer membrane. Oncotic pressure, effective at inducing VDAC closure, also decreases the outer membrane permeability. Thus, in the presence of cytosolic colloidal osmotic pressure NAD(P)H may inhibit mitochondrial catabolic pathways and divert reducing equivalents to anabolic pathways.

Binding Sites↗

PubMed1996

Spacial compartmentalization of Ca2+ signaling complexes in pancreatic acini.

Imaging [Ca2+]i at high temporal resolution and measuring the properties of Ca2+ signaling in streptolysin O (SLO)-permeabilized cells were used to study the spacial organization of signaling complexes. Sequential stimulation of single cells within pancreatic acini with several Ca2+-mobilizing agonists revealed an agonist-specific pattern and propagation rate of Ca2+ waves in the same cells, with CCK8 stimulating the fastest and bombesin the slowest waves. More importantly, each agonist initiated the wave in a different region of the same cell. On the other hand, repetitive stimulation with the same agonist induced Ca2+ waves of the same pattern that were initiated from the same region of the cell. The agonist-specific Ca2+ signaling does not appear to be the result of coupling to different G proteins as infusion of an anti-Galphaq antibody into the cells through a patch pipette equally inhibited Ca2+ signaling by all agonists. Further evidence for compartmentalization of signaling complexes was developed in permeabilized cells. The time-dependent loss of Ca2+ signaling due to SLO permeabilization occurred in an agonist-specific manner in the sequence cabachol > bombesin > cholecystokinin. Signaling by all agonists could be completely restored with as low as 2 micro guanosine 5'-3-O-(thio)triphosphate (GTPgammaS). At this low concentration GTPgammaS recoupled inositol 1,4,5-trisphosphate production and Ca2+ release, rather than enhancing phospholipase C activity. Priming of Ca2+ signaling by GTPgammaS was agonist-specific. Guanosine 5'-O-(thio)diphosphate (GDPbetaS) uncoupled the ability of signaling complexes to release Ca2+ much better than stimulating inositol 1,4,5-trisphosphate production. The uncoupling of Ca2+ signaling by GDPbetaS was also agonist-specific. The combined findings of agonist-specific initiation sites of the Ca2+ wave and differential access of guanine nucleotides to signaling complexes suggest spacial compartmentalization of Ca2+ signaling complexes. Each complex must include a receptor, G protein, and phospholipase C that are coupled to a specific portion of the Ca2+ pool.

Acetylcholine↗

PubMed1996

Genetic variation in neuraminidase genes of influenza A (H3N2) viruses.

Nucleotide sequences of the neuraminidase (NA) genes of 33 influenza A (H3N2) epidemic strains isolated between 1968 and 1995 were analyzed to determine their evolutionary relationships. Phylogenetic analysis using the DNA maximum-likelihood method indicates that the NA genes of recent H3N2 field strains, like their hemagglutinin genes (HA), have evolved as two distinct lineages represented by the vaccine strains. A/Beijing/353/89 and A/Beijing/32/92 for A/Shanghai/24/ 90). Furthermore, genetic reassortment of NA genes between the two lineages occurred during their circulation. Genetic reassortants, which bear an A/Beijing/32/92-like HA and an A/Beijing/353/89-like NA, have circulated worldwide and are representative of current influenza A (H3N2) epidemic strains. The mutation rate of the NA gene was found to be 2.28 x 10(-3) per nucleotide site per year with 4.2% of the mutations resulting in amino acid substitutions. Thirty-five percent of the amino acid substitutions was located in sites previously suggested to be reactive to antibody. Amino acid residues involved in NA enzyme activity have been conserved. Seven potential glycosylation sites identified in the NA of A/Hong Kong/8/68 virus were conserved by the majority of isolates, with more recently circulating viruses having an additional glycosylation site. Comparison of the rate of amino acid substitutions in the NA stalk to that of entire NA revealed high variability in this region. These findings demonstrate the importance of closely monitoring both the HA and the NA genes of influenza viruses to aid vaccine strain selection.

Amino Acid Sequence↗

PubMed1996

Transfection of C3H10T1/2 cells with the Harvey-ras oncogene reduces cytosolic phospholipase A2 function.

Several lines of evidence suggest that phospholipase A2 may play a role in the activated ras-mediated transformation process. In the present study, phospholipase A2 activity and expression were assessed in a murine fibroblast cell line (C3H10T1/2 cells) that was stably transfected with the Harvey ras oncogene, a cellular model used for studying multistage carcinogenesis. Reduced levels of fatty acids were released from the ras-transfected cells compared to untransfected controls. The in vitro phospholipase A2 activity apparent in the C3H10T1/2 showed preference for sn-2 arachidonyl phosphatidylcholine compared to dipalmitoyl phosphatidylcholine. The activity, as well as the cytosolic phospholipase A2 immunoreactive protein, was reduced by 50% in the ras-transfected cells compared to control cells. These results suggest that the cytosolic phospholipase A2 is the predominant form of this enzyme family expressed in C3H10T1/2 cells and that the activity and protein amount is reduced by 50% in these cells when stably transfected with the Harvey ras oncogene.

Animals↗

PubMed1996

Self-catalyzed insertion of proteins into phospholipid membranes.

We present evidence that the mitochondrial channel, VDAC, when reconstituted into a phospholipid membrane, can catalyze the insertion of other VDAC channels. This property called "auto-directed insertion" was first proposed by Zizi et al. (1995) to explain observations on asymmetric VDAC channels. We found that 2 urea or guanidinium chloride (GdmCl) caused a burst of insertions of VDAC channels when added to the same side as VDAC addition. More strikingly, when added to the opposite side they caused a 10-60-fold sustained yet reversible increase in insertion rate. Protein stabilization by sarcosine eliminated the effect of urea and GdmCl on VDAC insertion. Control experiments showed that water flow, ionic strength, osmotic force, phospholipid type, and membrane potential were not involved. Therefore, although both urea and GdmCl affect the properties of phospholipid membranes, it is more likely that these agents act either by changing the structure of the pre-inserted channels, allowing them to be more effective catalysts for VDAC insertion, or by flowing through the channels and acting on nearby VDAC channels inducing them to insert. Either way, insertion must be occurring next to pre-inserted channels. Urea and GdmCl may mimic chaperones by partially unfolding VDAC and keeping it in an insertion-competent state. "Auto-directed insertion" may ensure both correct targeting and orientation of nascent proteins in vivo.

Catalysis↗

PubMed1996

Vav in natural killer cells is tyrosine phosphorylated upon cross-linking of Fc gamma RIIIA and is constitutively associated with a serine/threonine kinase.

Cross-linking of Fc gamma RIIIA (CD16) receptor on natural killer (NK) cells induces receptor-associated tyrosine kinase activation and tyrosine phosphorylation of numerous intracellular proteins, including phospholipase C (PLC)-gamma 1, PLC-gamma 2 and the associated zeta chain. Here we report that Vav, a proto-oncogene, also became tyrosine phosphorylated upon stimulation of CD16 in interleukin 2-activated NK cells (LAK-NK) as well as in an NK cell line, NK3.3. In addition, we observed that in LAK-NK cells, Vav was associated with a 70 kDa protein that also became tyrosine phosphorylated upon CD16 cross-linking. The association of this 70 kDa protein with Vav was disrupted by ionic detergent treatment. Tyrosine phosphorylation of Vav was inhibited by herbimycin A, a specific tyrosine kinase inhibitor. In vitro kinase assays with Vav immunoprecipitates derived from NK3.3 cells or LAK-NK cells resulted in the appearance of a phosphorylated 58 kDa protein, suggesting the presence of a kinase within the Vav immunoprecipitates. Cross-linking of CD16 did not enhance this Vav-associated kinase activity. Phosphoamino acid analysis of the 58 kDa protein revealed that it was phosphorylated only on serine and threonine residues, indicating that an unidentified serine/threonine kinase is constitutively associated with Vav. These observations suggest that the downstream signalling events regulated by Vav and its associated proteins are complex involving both tyrosine kinases as well as the yet unidentified serine/threonine kinase in NK cells.

Animals↗

PubMed1996

Two activities of the immunosuppressive metabolite of leflunomide, A77 1726. Inhibition of pyrimidine nucleotide synthesis and protein tyrosine phosphorylation.

Previous studies have demonstrated that the active metabolite of leflunomide, A77 1726 [N-(4-trifluoromethylphenyl-2-cyano-3-hydroxycrotoamide)], is capable of inhibiting the activities of tyrosine kinases and dihydroorotate dehydrogenase (DHO-DHase). In the present study, we define the relative contribution of these activities to the ability of A77 1726 to inhibit proliferation of the murine leukemia cell line LSTRA. A77 1726 inhibited LSTRA cell growth and proliferation (IC50 = 10-30 microM); this inhibition, however, could be reversed by the addition of exogenous uridine, suggesting that the anti-proliferative activity of A77 1726 may be due to inhibition of de novo pyrimidine nucleotide synthesis. Quantitation of nucleotide levels revealed that A77 1726, at an IC50 of about 10 microM, selectively inhibited pyrimidine nucleotide but not purine nucleotide synthesis. In vitro enzyme assays confirmed that A77 1726 directly inhibited the activity of DHO-DHase, the fourth enzyme in the de novo pathway of pyrimidine nucleotide synthesis (IC50 = 220 nM). LSTRA cells overexpress p56lck and have elevated levels of tyrosine phosphorylated intracellular proteins. A77 1726 reduced the intracellular levels of tyrosine phosphorylated proteins with relatively high IC50 values ranging from 50 to 100 microM. A77 1726 also inhibited p56lck activity in LSTRA membrane preparation and immunoprecipitates; the IC50 values for inhibition of immunoprecipitated p56lck autophosphorylation and exogenous substrate histone 2B were 80 and 40 microM, respectively. The anti-tyrosine phosphorylation activity of A77 1726 was not affected by uridine. These studies therefore demonstrate the two activities of A77 1726: inhibition of pyrimidine nucleotide synthesis and interference with tyrosine phosphorylation.

Animals↗

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

Genomic footprinting of HTLV type I and HIV type 1 in human T cell lines.

Genomic footprinting of integrated HTLV-I and HIV-1 confirmed many aspects of retroviral transcriptional regulation deduced from previous studies. However, many notable differences were seen. HTLV-I genomic protein-binding patterns corresponded more closely to elements defined by transient transfection expression studies than to those mapped by in vitro protein-binding studies. HIV-1 genomic footprinting showed activation-related binding to adjacent NF-KB/SP1 sites and a large (90 bp) region transversing the R/U5 boundary, but minimal protein binding to NFAT, NRE, LBP-1, and CTF/NF1 sites relative to previous in vitro footprinting studies.

Base Sequence↗

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

Characterization, localization and axial distribution of Ca2+ signalling receptors in the rat submandibular salivary gland ducts.

1. To characterize [Ca2+]i signalling in salivary duct cells a procedure was developed for the rapid preparation and isolation of intralobular ducts, some of which had attached intercalated ducts. The isolated ducts retained agonist-induced Ca2+ signalling after permeabilization with streptolysin O (SLO). 2. The improved cell preparation technique was reflected in the repertoire and intensity of agonist responsiveness of the cells. Measurements of [Ca2+]i in intact cells showed that all agonists previously reported to affect electrolyte transport by the submandibular salivary gland (adrenaline, carbachol, isoprenaline and forskolin) mobilized Ca2+ from internal stores and increased Ca2+ influx across the plasma membrane. 3. The use of the SLO-permeabilized ducts showed that all agonists, including isoprenaline and forskolin, mobilized Ca2+ exclusively from the inositol 1,4,5 trisphosphate (IP3)-sensitive pool. However, in granular ducts only adrenaline mobilized the entire IP3-sensitive pool whereas all other agonists mobilized only part of the pool. 4. All regions of the duct responded to substance P and the luminally secreted agonist ATP. Interestingly, the intercalated duct was most responsive to ATP and demonstrated only a minimal response to all other agonists. The granular region of the same duct and the extralobular duct always responded best to stimulation by adrenaline. 5. The perfused extralobular duct was used to show that adrenaline and carbachol stimulated the duct through the basolateral membrane whereas the receptors for ATP were localized in the luminal membrane of the duct. This suggests the presence of an ATP-dependent positive feedback loop in salivary duct with decreased activity along the ductal tree.

Adrenergic alpha-Antagonists↗