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Biomedical subjects

J Xia

Publications and source records attributed to J Xia.

At least 109 records · Page 6Linked to original sources
PubMed1998

MinK-KvLQT1 fusion proteins, evidence for multiple stoichiometries of the assembled IsK channel.

IsK, a slowly activating delayed rectifier K+ current through channels formed by the assembly of two channel proteins KvLQT1 and MinK, modulates the repolarization of cardiac action potentials. Mutations that map to the KvLQT1 and minK genes account for more than 50% of an inherited cardiac disorder, the Long QT syndrome (Splawski, I., Tristani-Firouzi, M., Lehmann, M. H., Sanguinetti, M. C., and Keating, M. T. (1997) Nat. Genet. 17, 338-340). Despite the importance of these channels to human cardiac function, the molecular basis of their uniquely slow gating properties as well as the stoichiometry and interaction sites of these two subunits are still unclear. We have constructed several fusion channel proteins to begin investigating the stoichiometry of these two subunits and the role of voltage-dependent subunit assembly in channel gating. Functional properties of these constructs were measured using whole cell patch clamp recordings of transiently transfected Chinese hamster ovary cells. The constructs we tested are as follows: MK24 (C terminus of MinK linked to N terminus of KvLQT1); KK40 (a tandem homodimer of KvLQT1); and MKK44 (C terminus of MinK linked to N terminus of KK40). In control experiments (no DNA, control DNA, or only MinK), no time-dependent K+ current was observed. Expression of KvLQT1 or KK40 produced currents that activate and inactivate in a voltage-dependent manner as reported by others for KvLQT1. In contrast, expression of MK24 and MKK44 elicited current with activation kinetics and voltage dependence very similar to native IsK and identical to currents expressed by cells co-transfected with independent MinK and KvLQT1 cDNA. Expression of MK24 plus additional MinK significantly slows current kinetics. Our data raise the possibility 1) of multiple MinK/KvLQT1 stoichiometries and 2) indicate that uniquely slow kinetics of IsK channels is due to voltage-dependent conformational changes of the channel protein and not to assembly of channel subunits.

Animals↗
PubMed1998

[Molecular cloning and characterization of novel protein kinase gene DYRK3].

OBJECTIVE: To isolate full length cDNA of a novel protein kinase and to deduce the protein kinase's classification position and functions. METHODS: cDNA libraries gDNA library was screened with a partial cDNA clone which is homologous to human protein kinase DYRK2 as probe. FISH mapping was performed. RESULTS: Two full length cDNAs of a novel protein kinase from human muscle cDNA library and human testis cDNA library were isolated. The full length cDNA from muscle has an open reading frame which is predicted to encode a protein of 588 amino acid residues and the cDNA from testis to encode a protein of 568 amino acid residues. CONCLUSION: Because the sequence from the 27th codon to the 3' end of the cDNA from muscle is identical to that from the 7th codon to the 3' end of the cDNA from testis, they should be different transcripts of the same gene. As the gene is highly homologous to human protein kinase DYRK2, the present authors termed the gene DYRK3. DYRK3 is homologous to many serine/threonine protein kinases such as yeast Yak1, human Clk1, human Mnb, drosophila melanogaster Mnb and Cdk2. DYRK3 should belong to the Clk family in CMGC group of serine/threonine protein kinase. DYRK3 has been mapped to chromosome 1q32 by FISH.

Amino Acid Sequence↗
PubMed1998

Overexpression of nerve growth factor in the heart alters ion channel activity and beta-adrenergic signalling in an adult transgenic mouse.

1. The electrophysiological and pharmacological properties of cardiac myocytes from the hearts of adult transgenic mice engineered to overexpress nerve growth factor (NGF) in the heart were studied. 2. There was a 12% increase in the ventricular myocyte capacitance in NGF myocytes consistent with cardiac hypertrophy, and action potential duration at 90% repolarization (APD90) was prolonged by 142 % compared with wild-type (WT) myocytes. This was due, at least in part, to a decrease in the density of two K+ currents, Ito and IK(ur), which were significantly reduced in NGF mice with no change in their electrophysiological characteristics. We found no change in the current density or electrophysiological properties of the L-type Ca2+ current. 3. The effect on Ito and IK(ur) of TEA and 4-aminopyridine (4-AP) was not different in cells isolated from WT and NGF mice. The prolongation of APD observed in NGF cells was mimicked in WT cells by exposure to 1 mM 4-AP, which partially blocked Ito, completely blocked IK(ur) and increased APD90 by 157%. 4. The isoprenaline-induced increase in ICa was significantly smaller in NGF myocytes than in WT myocytes. This was not due to a decrease in beta-adrenergic receptor (beta-AR) density, as this was increased in NGF tissue by 55%. Analysis of beta-AR subtypes showed that this increase was entirely due to an increase in beta2-AR density with no change in beta1-ARs. 5. The response of the beta-AR-coupled adenylyl cyclase system to isoprenaline, Gpp(NH)p and forskolin was studied by measuring cAMP production. In NGF tissue, isoprenaline elicited a significantly smaller response than in WT myoyctes and this was not due to reduced adenylyl cyclase activity as the responses of NGF tissue to guanylylimidodiphosphate (Gpp(NH)p) and forskolin were unaffected. 6. In conclusion, the overexpression of NGF in the mouse heart resulted in a decrease in the current density of two K+ channels, which contributed to the prolongation of the cardiac action potential. Despite an increase in beta2-AR density in the hearts of the NGF mice, the response to isoprenaline was diminished, and this was due to an uncoupling of the beta-ARs from the intracellular signalling cascade. These potentially pathological changes may be involved in the occurrence of ventricular arrhythmias in cardiac hypertrophy and failure, and this mouse provides a novel model in which to study such changes.

Action Potentials↗
PubMed1998

[A technique of constructing human chromosomal band-specific probe pools using degenerate oligonucleotide primer].

OBJECTIVE: To establish a rapid and efficient technique of constructing human chromosomal band specific probe pools and their libraries. METHODS: A modified method of combining chromosome microdissection with degenerate oligonucleotide primed PCR(DOP-PCR) was used. 3p23-p26, 3q21-q22 and 4p12- p16 band from human chromosomes were microdissected and amplified as probe pools. The origins of the PCR products were determined by chromosome fluorescence in situ hybridization. The PCR products and pUC19 were digested by Xho I and Sal I respectively, and linke up. The DH5alpha were transformed by the recombinated vectors as the specific band libraries. The inserts were digested by EcoR I and Hind III, then measured by electrophoretic analysis. And the copies of inserts were identified by in situ bacterial colony hybridization with genomic DNA. RESULTS: All the three probe pools showed the special yellow-green signals in their microdissection responsible bands. The sizes of DOP-PCR products ranged from 300bp to 1800bp. 3q21-q22 probe pool generated about 1.2 x 10(4) clones. The average size of inserts was about 420bp by analysis of 30 positive clones. The rate of single-copy and low-repeated sequences was about 81%(178/220), while the rate of middle-repeated and high- repeated sequences was about 19%(42/220). CONCLUSION: The results proved that the modified microdissection combining DOP-PCR technique provided a simple and efficient method to construct the human chromosome band-specific probe pools and might contribute to gene cloning and complete sequencing of human genome.

Chromosome Banding↗
PubMed1998

Quantitation of MoMuLV envelope protein on the cell surface.

The envelope glycoprotein (Env) of Moloney murine leukemia virus (MoMuLV) is proteolytically processed and transported to the cell surface where it can be incorporated into budding virions. Cell surface Env is frequently detected using an indirect immunofluorescence assay and fluorescence-activated cell sorting (FACS). We found that the detection of Env in this manner requires the expression of the MoMuLV receptor (ATRC-1) on the cell surface, and the level of envelope protein detected correlates with the level of receptors expressed on the cell. In addition, Env detection corresponds to the Env protein's ability to bind to its receptor and can be competed out by the addition of a truncated form of the Env protein. These data suggest that Env detected on the cell surface by the FACS assay is protein that has rebound to its receptor after being secreted or shed, rather than actual surface-expressed protein. In contrast, a combined immunoprecipitation and biotinylation assay detected equal amounts of Env on the surface of both receptor-lacking and receptor-expressing cell lines. The immunoprecipitation-biotinylation assay is therefore a more appropriate method for detecting surface expression of the MoMuLV envelope protein.

3T3 Cells↗
PubMed1998

Species-specific and sequence-specific recognition of the dG-rich strand of telomeres by yeast telomerase.

A gel mobility shift assay was developed to examine recognition of yeast telomeres by telomerase. An RNase-sensitive G-rich strand-specific binding activity can be detected in partially purified yeast telomerase fractions. The binding activity was attributed to telomerase, because it co-purifies with TLC1 RNA and telomerase activity over three different chromatographic steps and because the complex co-migrates with TLC1 RNA when subjected to electrophoresis through native gels. Analysis of the binding specificity of yeast telomerase indicates that it recognizes the G-rich strand of yeast telomeres with high affinity and specificity. The K d for the interaction is approximately 3 nM. Single-stranded G-rich telomeres from other species, such as human and Tetrahymena, though capable of being extended by yeast telomerase in polymerization assays at high concentrations, bind the enzyme with at least 100-fold lower affinities. The ability of a sequence to be bound tightly by yeast telomerase in vitro correlates with its ability to seed telomere formation in vivo. The implications of these findings for regulation of telomerase activity are discussed.

Animals↗
PubMed1998

Age differences in the expression of metabotropic glutamate receptor 1 and inositol 1,4,5-trisphosphate receptor in mouse cerebellum.

Age differences in the expression of cerebellar metabotropic glutamate receptor 1 (mGluR1) and inositol 1,4,5-trisphosphate receptor (IP3R) were investigated using male C57BL/6NNIA mice 5, 15 and 24 months of age. In situ hybridization for mGluR1 mRNA in the granule cell layer indicated significantly higher mRNA levels in the 24-month-old group as compared to the 5- and 15-month-old groups. However, mRNA levels of individual Purkinje neurons did not show age differences. Western blot analysis using antibody against the predominant isoform, mGluR1a, showed a decline in protein levels in the 24-month-old animals. In situ hybridization for IP3R type 1 mRNA in Purkinje neurons showed a slight but not significant decline in the 24-month-old group. Further assay of [3H]IP3 binding with cerebellar membranes showed significant reduction in Bmax values in the 15- and 24-month-old groups as compared to the 5-month-old group but Kd values were not changed. The decrease in mGluR1a receptor protein together with reduction in IP3R binding sites may play an important role in the decline in cerebellar functions with increasing age.

Aging↗
PubMed1998

Dietary supplementation of grape polyphenols and chronic ethanol administration on LDL oxidation and platelet function in rats.

Polyphenolic compounds have been implicated as the active ingredients for the cardiac protective effect in red wine. We tested the effects of dietary supplementation of polyphenols from grape (GP) and chronic ethanol administration on low-density-lipoprotein (LDL) oxidation and platelet function in rats. Four groups of young male Sprague-Dawley rats were fed the following diets for 2 months: (I) a high fat Lieber-DeCarli liquid diet with an isocaloric amount of maltose, (II) with 5% ethanol (w/v), (III) with 5 mg/dL of GP, and (IV) ethanol plus GP. Platelet aggregation was induced by thrombin and phorbol myristate acetate (PMA) and LDL oxidation was induced by Cu2+. Chronic ethanol administration resulted in a significant increase in LDL oxidation and this effect was partially protected by supplementation with GP. Although platelet number was not affected by either ethanol or GP administration, platelet aggregation induced by thrombin was reduced in ethanol, GP and ethanol plus GP groups as compared to controls. On the other hand, platelet aggregation induced by PMA was not altered in any groups, suggesting that protein kinase C was not a causal factor for the reduction of aggregatory response induced by thrombin. These results show similar effects of ethanol and GP on platelet aggregation but different effects on LDL oxidation. It can be concluded that dietary supplementation with GP may exert partial protection on oxidative insults such as those elicited by chronic ethanol ingestion.

Animals↗
PubMed1998

Changes in IP3R1 and SERCA2b mRNA levels in the gerbil brain after chronic ethanol administration and transient cerebral ischemia-reperfusion.

Despite epidemiological studies indicating a positive relationship between alcohol and stroke, little is known with regard to effect of chronic alcohol on neuronal injury after stroke. In this study, we examined the effect of chronic ethanol on mRNA levels of sarcoplasmic or endoplasmic Ca2+-ATPase (SERCA2b) and inositol 1,4, 5-triphosphate receptor (IP3R1) in gerbils subjected to global cerebral ischemia induced by ligation of both common carotid arteries. Gerbils were given daily by intragastric intubation either a liquid diet containing ethanol (4 g/kg) or the same diet with an isocaloric amount of sucrose for 35 days. They were subsequently subjected to a 5 min ischemic insult followed by reperfusion for 48 h. In agreement with other studies, ischemic insult caused significant decreases (P<0.05) in mRNA levels of both IP3R1 and SERCA2b in the hippocampal CA1 region but not in the dentate gyrus. Nevertheless, despite a significant (P<0.05) decrease in SERCA2b mRNA in the Purkinje neurons, chronic ethanol did not alter the expression of this mRNA species in the hippocampal CA1 neurons nor did it alter the decrease in SERCA2b mRNA due to cerebral ischemic insult. Since IP3R1 and SERCA2b are key mediators for regulation of intracellular Ca2+ stores, the decrease in SERCA2b mRNA but not IP3R1 mRNA in cerebellar neurons may be an important mechanism underlying alteration of calcium homeostasis and cerebellar degeneration upon chronic ethanol consumption.

Administration, Oral↗
PubMed1998

Interaction of the N-ethylmaleimide-sensitive factor with AMPA receptors.

Glutamate receptors mediate the majority of rapid excitatory synaptic transmission in the central nervous system (CNS) and play important roles in synaptic plasticity and neuronal development. Recently, protein-protein interactions with the C-terminal domain of glutamate receptor subunits have been shown to be involved in the modulation of receptor function and clustering at excitatory synapses. In this paper, we have found that the N-ethylmaleimide-sensitive factor (NSF), a protein involved in membrane fusion events, specifically interacts with the C terminus of the GluR2 and GluR4c subunits of AMPA receptors in vitro and in vivo. Moreover, intracellular perfusion of neurons with a synthetic peptide that competes with the interaction of NSF and AMPA receptor subunits rapidly decreases the amplitude of miniature excitatory postsynaptic currents (mEPSCs), suggesting that NSF regulates AMPA receptor function.

Amino Acid Sequence↗
PubMed1998

Camptothecin-20-PEG ester transport forms: the effect of spacer groups on antitumor activity.

An improved synthesis of the hindered PEG-camptothecin diester transport form has been achieved using the Mukaiyama reagent. We have also assessed the effect of changing the electronic configuration of the (d-position of PEG-camptothecin transport forms on the rates of hydrolysis of the pro-moiety, and attempted to correlate these differences to efficacy in two animal models. In addition to the simple substitution of N for O, other synthetic modifications of these atoms were accomplished by employing heterobifunctional linker groups. The half lives by disappearance (rates of hydrolysis) of the transport forms in buffer and rat plasma were determined. It was established that anchimeric assistance to hydrolytic breakdown of the pro-moiety occurs in a predictable manner for some of these compounds. Results for the new derivatives in a P388 murine leukemic model and HT-29 human colorectal xenograft study are also presented. The use of a glycine linker group was found to provide similar efficacy in rodent models to that of simple camptothecin 20-PEG ester, and displayed enhanced pharmacokinetics.

Animals↗
PubMed1998

Patterns of excitation-contraction coupling in arterioles: dependence on time and concentration.

We sought to understand the excitation-contraction coupling process in arterioles. KCl or phenylephrine (PE) was applied via the superfusion solution or by brief pulsatile ejections from a micropipette onto unpressurized arterioles (in vitro) from either the guinea pig small intestine or hamster cheek pouch. With either mode of application, KCl caused depolarizations that were tightly and predictably correlated with subsequent constrictions (electromechanical coupling). In contrast, the relationship between membrane potential and vasoconstriction in response to phenylephrine was dependent on both stimulus duration and agonist concentration. Application of short pulses of PE (< 1 s) produced mechanical responses that were dominated by pharmacomechanical coupling (i.e., they were not associated with changes in membrane potential). With longer PE stimuli, electromechanical coupling became more important and dominated microvessel responses. We conclude that adequate understanding of the signaling process in microvessels requires a consideration of both concentration and duration of application. Both the mode and duration of agonist application affect the relative degree of electromechanical or pharmacomechanical coupling in response to a vasomotor stimulus. These observations have important implications for intracellular and intercellular signaling.

Animals↗
PubMed1998

A common variant in PPP1R3 associated with insulin resistance and type 2 diabetes.

Selected candidate genes have been analyzed in the Pima Indians of Arizona based on evidence that insulin resistance and type 2 diabetes have significant genetic determinants. An amino acid substitution at codon 905 of the glycogen-targeting subunit of type 1 protein phosphatase that regulates skeletal muscle glycogenesis was recently reported to be associated with changes in insulin action in Danish subjects. In addition to the variant at 905, we report here a novel substitution at codon 883 and common variant of an "ATTTA" element in the 3'-untranslated region (UTR) of the corresponding gene (PPP1R3). The 3'-UTR variant resembled the mRNA-destabilizing AT(AU)-rich elements (AREs) and resulted in a 10-fold difference in reporter mRNA half-life, was correlated with PPP1R3 transcript and protein concentrations in vivo, and was associated with insulin resistance and type 2 diabetes in the Pimas. The variant is more common in Pimas (0.56) than in Caucasians (0.40). Because of its apparent effect on expression of PPP1R3, it may, in part, contribute to the higher prevalence of type 2 diabetes in this Native American population.

Adult↗
PubMed1998

[Molecular cloning of EXT2 and EXT4 gene].

Hereditary multiple exostose(EXT) is an autosomal dominant disorder of skeletal system. Three genetic loci have been identified at 8q24.1(EXT1), 11p11(EXT2) and 19p(EXT3) respectively. In this paper, EXT2 gene was cloned with positional cloning and homologous screening. SSCP and sequencing analysis have been done in 37 EXT patients who came from 20 EXT families, 2 mutations of insertion were tested in 2 patients. This confirmed that the gene cloned in this paper was EXT2 gene which locus at 11p11. Additionally EXT4 gene was cloned with homologous screening and located at 1p36.1 with FISH in this paper.

Amino Acid Sequence↗
PubMed1998

[Clinico-pathological significance of microvessel density and VEGF expression in primary liver cancer].

OBJECTIVE: To evulate the clinico-pathological singnificance of intratumoral microvessel density (MVD) and VEGF expression in primary liver cancer (PLC). METHODS: A retrospective study including 63 postoperative small PLC (diameter < 5 cm) patients was done. One group of 29 patients developed recurrence or metastasis within 2 years. The other group of 34 patients had no evidence of recurrence or metastasis within 2 years. Three PLC sections were taken from each patient, one for H. E. staining, the other two for VEGF and vascular endothelial cell immunohistochemical staining, respectively. MVD was counted by endothelial cells which were highlighted by Bio-UEA-I. RESULTS: The MVD in patients with cancer recurrence or metastasis was (49.6 +/- 29.7) significantly greater than the other group (22.7 +/- 28.2) (P < 0.01). The positive rate of VEGF in cancer recurrence group was 86.2% (25/29), being significantly higher than the other group (47.1%) (P < 0.01). The stage of the tumor, the positive rate of satellite nodules and portal vein embolus were all significantly different between the 2 groups. CONCLUSION: Besides tumor stage, satellite nodules and portal vein embolus, the MVD and VEGF expression are also of prognostic significance.

Carcinoma, Hepatocellular↗
PubMed1998

[Orthogonal design-direct analysis for PCR optimization].

Polymerase chain reaction (PCR) optimization is difficult but very important. It is hard to gain the optimal condition due to the confusion of too many factors influencing the experiment. We report a new method, orthogonal design-direct analysis, which has been confirmed to be scientific and easy to follow by many times of PCR.

Brain Neoplasms↗