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D Wang

Publications and source records attributed to D Wang.

At least 451 records · Page 25Linked to original sources
PubMed1999

Supersensitivity to allosteric GABA(A) receptor modulators and alcohol in mice lacking PKCepsilon.

Several of the actions of ethanol are mediated by gamma-aminobutyrate type A (GABA(A)) receptors. Here we demonstrated that mutant mice lacking protein kinase C epsilon (PKCepsilon) were more sensitive than wild-type littermates to the acute behavioral effects of ethanol and other drugs that allosterically activate GABA(A) receptors. GABA(A) receptors in membranes isolated from the frontal cortex of PKCepsilon null mice were also supersensitive to allosteric activation by ethanol and flunitrazepam. In addition, these mutant mice showed markedly reduced ethanol self-administration. These findings indicate that inhibition of PKCepsilon increases sensitivity of GABA(A) receptors to ethanol and allosteric modulators. Pharmacological agents that inhibit PKCepsilon may be useful for treatment of alcoholism and may provide a non-sedating alternative for enhancing GABA(A) receptor function to treat other disorders such as anxiety and epilepsy.

Allosteric Regulation↗
PubMed1999

Determination of ancestral alleles for human single-nucleotide polymorphisms using high-density oligonucleotide arrays.

Here we report the application of high-density oligonucleotide array (DNA chip)-based analysis to determine the distant history of single nucleotide polymorphisms (SNPs) in current human populations. We analysed orthologues for 397 human SNP sites (identified in CEPH pedigrees from Amish, Venezuelan and Utah populations) from 23 common chimpanzee, 19 pygmy chimpanzee and 11 gorilla genomic DNA samples. From this data we determined 214 proposed ancestral alleles (the sequence found in the last common ancestor of humans and chimpanzees). In a diverse human population set, we found that SNP alleles with higher frequencies were more likely to be ancestral than less frequently occurring alleles. There were, however, exceptions. We also found three shared human/pygmy chimpanzee polymorphisms, all involving CpG dinucleotides, and two shared human/gorilla polymorphisms, one involving a CpG dinucleotide. We demonstrate that microarray-based assays allow rapid comparative sequence analysis of intra- and interspecies genetic variation.

Alleles↗
PubMed1999

Efficient CFTR expression from AAV vectors packaged with promoters--the second generation.

Gene therapy studies of cystic fibrosis (CF) have shown that AAV-based vector was efficient in transferring but not in expressing the CFTR cDNA in the target cells. The levels of CFTR gene expression were limited by the small packaging capacity of AAV because it had been difficult to package the CFTR cDNA with an efficient promoter. In the present study we have developed a new generation of AAV/CFTR vectors which contain efficient short promoters to express the CFTR gene in target cells. To do so, we reduced the size of the CFTR cDNA by determining the minimal untranslated regions required for expression of CFTR cDNA. We also identified short and efficient promoters that could be packaged with the down-sized CFTR cDNA into a novel AAV vector that had a maximal packaging capacity. Functional analyses showed that the new vectors were packaged efficiently and expressed higher levels of CFTR than a vector in which the CFTR gene was driven by the ITR sequence of AAV. Transduction of airway epithelial cells containing [symbol: see text] 508 mutation with the new vectors demonstrated efficient expression of the wild-type CFTR and correction of the CF phenotype. In contrast, no significant CFTR expression was detected in cells infected with the vector that express the CFTR gene from the ITR. These findings support the notion that the AAV can be developed into an efficient vector to transduce the CFTR gene and vectors expressing higher levels of CFTR from an efficient promoter should provide better efficacy for gene therapy of cystic fibrosis.

Cystic Fibrosis↗
PubMed1999

Cytokines increase neonatal cardiac myocyte calcium concentrations: the involvement of nitric oxide and cyclic nucleotides.

Neonatal rat cardiac myocytes were treated with cytokines, with or without the nitric oxide synthase (NOS) inhibitors N-monomethyl-L-arginine (LNMMA) and N-nitro-L-arginine methyl ester (LNAME), and systolic and diastolic calcium levels were measured by fluorescence spectrophotometry and confocal microscopy. Time-dependent changes following interferon-gamma (IFN-gamma) treatment revealed a continuing increase in intracellular calcium, which was reduced with LNMMA, but not with LNAME. Increases in calcium also occurred with interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), but not to the extent seen with IFN-gamma. Increased cyclic guanosine monophosphate (cGMP) was involved in the results described with short-term (2 hr) TNF-alpha and long-term (18 hr) IFN-gamma treatments. Short-term exposure to IFN-gamma produced an increase in cyclic adenosine monophosphate (cAMP) and also an initial increase in the myocyte-bearing rate, with calcium levels either (i) subsequently returning to control levels while maintaining a fast beating rate or (ii), retaining a high systolic calcium level, but beating at control rates. Treatment with both IL-1beta and IFN-gamma stabilized the beating rate of the cells on some occasions. Shortening of myocytes increased with isoproterenol and following treatment with IFN-gamma, while isoproterenol stimulation of IFN-gamma-treated cells revealed increased contractile activity after short, but not long, treatment. LNMMA, but not reduced the increased contractile response with short-term IFN-gamma treatment. Our findings suggest that TNF-alpha acts via a cGMP-dependent pathway, whereas the actions of IFN-gamma involve adenylate cyclase, and possibly a NO-forming mechanism and cGMP pathway as well. It is also apparent that the two NO inhibitors function via different mechanisms or that LNMMA has a direct effect on the calcium-signaling pathway.

Analysis of Variance↗
PubMed1999

Espin contains an additional actin-binding site in its N terminus and is a major actin-bundling protein of the Sertoli cell-spermatid ectoplasmic specialization junctional plaque.

The espins are actin-binding and -bundling proteins localized to parallel actin bundles. The 837-amino-acid "espin" of Sertoli cell-spermatid junctions (ectoplasmic specializations) and the 253-amino-acid "small espin" of brush border microvilli are splice isoforms that share a C-terminal 116-amino-acid actin-bundling module but contain different N termini. To investigate the roles of espin and its extended N terminus, we examined the actin-binding and -bundling properties of espin constructs and the stoichiometry and developmental accumulation of espin within the ectoplasmic specialization. An espin construct bound to F-actin with an approximately threefold higher affinity (K(d) = approximately 70 nM) than small espin and was approximately 2.5 times more efficient at forming bundles. The increased affinity appeared to be due to an additional actin-binding site in the N terminus of espin. This additional actin-binding site bound to F-actin with a K(d) of approximately 1 microM, decorated actin stress fiber-like structures in transfected cells, and was mapped to a peptide between the two proline-rich peptides in the N terminus of espin. Espin was detected at approximately 4-5 x 10(6) copies per ectoplasmic specialization, or approximately 1 espin per 20 actin monomers and accumulated there coincident with the formation of parallel actin bundles during spermiogenesis. These results suggest that espin is a major actin-bundling protein of the Sertoli cell-spermatid ectoplasmic specialization.

Actins↗
PubMed1999

Spontaneous beta(2)-adrenergic signaling fails to modulate L-type Ca(2+) current in mouse ventricular myocytes.

A receptor can be activated either by specific ligand-directed changes in conformation or by intrinsic, spontaneous conformational change. In the beta(2)-adrenergic receptor (AR) overexpression transgenic (TG4) murine heart, spontaneously activated beta(2)AR (beta(2)-R*) in the absence of ligands has been evidenced by elevated basal adenylyl cyclase activity and cardiac function. In the present study, we determined whether the signaling mediated by beta(2)-R* differs from that of a ligand-elicited beta(2)AR activation (beta(2)-LR*). In ventricular myocytes from TG4 mice, the properties of L-type Ca(2+) current (I(Ca)), a major effector of beta(2)-LR* signaling, was unaltered, despite a 2.5-fold increase in the basal cAMP level and a 1.9-fold increase in baseline contraction amplitude as compared with that of wild-type (WT) cells. Although the contractile response to beta(2)-R* in TG4 cells was abolished by a beta(2)AR inverse agonist, ICI118,551 (5 x 10(-7) M), or an inhibitory cAMP analog, Rp-CPT-cAMPS (10(-4) M), no change was detected in the simultaneously recorded I(Ca). These results suggest that the increase in basal cAMP due to beta(2)-R*, while increasing contraction amplitude, does not affect I(Ca) characteristics. In contrast, the beta(2)AR agonist, zinterol elicited a substantial augmentation of I(Ca) in both TG4 and WT cells (pertussis toxin-treated), indicating that L-type Ca(2+) channel in these cells can respond to ligand-directed signaling. Furthermore, forskolin, an adenylyl cyclase activator, elicited similar dose-dependent increase in I(Ca) amplitude in WT and TG4 cells, suggesting that the sensitivity of L-type Ca(2+) channel to cAMP-dependent modulation remains intact in TG4 cells. Thus, we conclude that beta(2)-R* bypasses I(Ca) to modulate contraction, and that beta(2)-LR* and beta(2)-R* exhibit different intracellular signaling and target protein specificity.

Adenylate Cyclase Toxin↗
PubMed1999

A heterogeneous nuclear ribonucleoprotein A/B-related protein binds to single-stranded DNA near the 5' end or within the genome of feline parvovirus and can modify virus replication.

Phage display of cDNA clones prepared from feline cells was used to identify host cell proteins that bound to DNA-containing feline panleukopenia virus (FPV) capsids but not to empty capsids. One gene found in several clones encoded a heterogeneous nuclear ribonucleoprotein (hnRNP)-related protein (DBP40) that was very similar in sequence to the A/B-type hnRNP proteins. DBP40 bound specifically to oligonucleotides representing a sequence near the 5' end of the genome which is exposed on the outside of the full capsid but did not bind most other terminal sequences. Adding purified DBP40 to an in vitro fill-in reaction using viral DNA as a template inhibited the production of the second strand after nucleotide (nt) 289 but prior to nt 469. DBP40 bound to various regions of the viral genome, including a region between nt 295 and 330 of the viral genome which has been associated with transcriptional attenuation of the parvovirus minute virus of mice, which is mediated by a stem-loop structure of the DNA and cellular proteins. Overexpression of the protein in feline cells from a plasmid vector made them largely resistant to FPV infection. Mutagenesis of the protein binding site within the 5' end viral genome did not affect replication of the virus.

Amino Acid Sequence↗
PubMed1999

Endogenous nitric oxide inhibits chloride transport in the thick ascending limb.

Nitric oxide (NO) inhibits transport in various nephron segments, and the thick ascending limb of the loop of Henle (TALH) expresses NO synthase (NOS). However, the effects of NO on TALH transport have not been extensively studied. We hypothesized that endogenously produced NO directly decreases NaCl transport by the TALH. We first determined the effect of exogenously added NO on net chloride flux (JCl). The NO donor spermine NONOate (SPM; 10 microM) decreased JCl from 101.2 +/- 9.6 to 65.0 +/- 7.7 pmol. mm-1. min-1, a reduction of 35.5 +/- 6.4%, whereas controls did not decrease over time. To determine whether endogenous NO affects cortical TALH transport, we measured the effect of adding the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME), the substrate L-arginine (L-Arg), or its enantiomer D-arginine (D-Arg) on JCl. L-NAME and D-Arg did not alter JCl; in contrast, addition of 0.5 mM L-Arg decreased JCl by 40.2 +/- 10.4% from control. The inhibition of chloride flux by 0.5 mM L-Arg was abolished by pretreatment with L-NAME, indicating that cortical TALH NOS is active, but production of NO is substrate-limited in our preparation. Furthermore, cortical TALH chloride flux increased following removal of 0.5 mM L-Arg from the bath, indicating that the reductions in chloride flux observed in response to L-Arg are not the result of NO-mediated cytotoxicity. We conclude that 1) exogenous NO decreases cortical TALH JCl; 2) cortical TALHs produce NO in the presence of L-Arg, which decreases JCl; and 3) the response of cortical TALHs to L-Arg is reversible in vitro. These data suggest an important role for locally produced NO, which may act via an autocrine mechanism to directly affect TALH sodium chloride transport. Thus TALH NO synthesis and inhibition of chloride transport may contribute to the diuretic and natriuretic effects of NO observed in vivo.

Absorption↗
PubMed1999

Expression of serotonin receptor 2c in rat type II pneumocytes.

Serotonin (5-hydroxytryptamine [5-HT]) is a multifunctional amine with wide occurrence in both neural and non-neural tissues, including the lung. The diverse responses to 5-HT are elicited through activation of different 5-HT receptor subtypes. We report the expression and localization of 5-HT receptor 2c subtype (5-HT2c-R) in rat lungs using reverse transcriptase-polymerase chain reaction, nonisotopic in situ hybridization (NISH), and immunohistochemistry. At the messenger RNA (mRNA) level, signal corresponding to approximately 430 base pairs was detected in whole-lung tissue extracts as well as in cultures of isolated alveolar type II cells from fetal and adult rat lung. Using antisense RNA probe for 5-HT2c-R, NISH showed strong positive signal in type II cells. The expression of mRNA signal differed between fetal and adult rat type II cells, with weak, predominantly perinuclear localization in the former and strong cytoplasmic localization in the latter. Immunohistochemistry, using specific monoclonal antibody against 5-HT2c-R, showed perinuclear localization in fetal type II cells; whereas in adult type II cells 5-HT2c-R immunoreactivity was confined mostly to the plasma membrane, as demonstrated by laser confocal microscopy. Identification of 5-HT2c-R expression in alveolar type II cells suggests an important role for this amine in modulating the function of these cells. The differences in cell domain localization between fetal and adult type II cells could indicate developmental regulation of 5-HT2c-R expression in the lung.

Animals↗
PubMed1999

Defining a novel 75-kDa phosphoprotein associated with SS-A/Ro and identification of distinct human autoantibodies.

Mothers of children with neonatal lupus erythematosus (NLE) and heart block, as well as patients with Sjögren's syndrome (SS) and systemic lupus erythematosus, have serum autoantibodies that recognize SS-A/Ro autoantigens including the 60-kDa ribonucleoprotein. By yeast 2-hybrid screening, we identified a novel 75-kDa protein (pp75) that interacts with the carboxyl 70% of 60-kDa SS-A/Ro. The specificity of interaction was confirmed using mammalian 2-hybrid and chemical crosslinking studies. Immunoprecipitation with radiolabeled HeLa cell extracts showed that pp75 was phosphorylated and associated with 2 other phosphoproteins of 64 kDa. In Northern blot analysis, pp75 was expressed in all tissues analyzed; the highest expression was in the human heart. Based on immunofluorescence of transfected HeLa cells, pp75 is localized predominantly in the cytoplasm, an observation confirmed by immunohistochemistry in untransfected cells. Based on Western blot and ELISA assays, sera from 14 of 84 mothers of children with NLE recognized pp75, including 1 mother in whom anti-SS-A/Ro antibodies were not detected. In addition, sera from 5 of 80 patients with SS were positive for anti-pp75 antibody. Identification of molecular partners is a first step toward elucidating the functions and possible involvement in pathogenesis of long-recognized autoantigens such as 60-kDa SS-A/Ro, which are at present poorly understood.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Effect of intravenous substance P on laryngeal adductor activity in young dogs.

Reflex laryngeal adduction is a component of both the laryngeal chemoreflex and the esophagolaryngeal adductor reflex, two life-threatening reflexes that occur in immature animals. These two reflex responses are also thought to exist in infants and may play a role in causing life-threatening laryngospastic events and perhaps sudden infant death syndrome. Identifying neurotransmitters that mediate laryngeal adduction is important to understanding the mechanism of reflex laryngeal responses and to identifying potential means of pharmacologic prevention. Substance P (SP), a tachykinin, putatively functions as a sensory neurotransmitter and may play a role in mediating laryngeal reflexes. Substance P-immunoreactive-like fibers and receptors are present in the subepithelial tissues of the larynx, the vagus nerves, the nodose and jugular ganglia, and the vagal brain stem nuclei. In this investigation, the effect of SP infusion on laryngeal motor activity in an in vivo model is reported. Substance P was infused intravenously into 8 puppies (20 to 133 days of age, mean 81.2), on a mean of 3.0 occasions (range 1 to 6). Cardiovascular, respiratory, arterial blood gas, and cricothyroid (CT), thyroarytenoid (TA), and genioglossus electromyographic (EMG) responses to infusion of the tachykinin were recorded and subsequently analyzed. The SP infusion induced a marked increase in CT or TA EMG activity in 23 of 24 studies, and the increase was typically apparent within 60 seconds of the infusion. An increase in genioglossus EMG activity did not occur. An immediate, profound decrease in mean arterial pressure and an increase in respiratory rate and depth of chest wall excursion accompanied the laryngeal response. Arterial blood gas values remained unchanged (p > .05). The laryngeal adductor response to SP infusion was blocked when animals were pretreated with a systemic SP antagonist (Pfizer CP-96,345). This study demonstrates that peripheral infusion of the tachykinin SP induces a marked increase in laryngeal adductor activity. The response may be blocked by pretreatment of animals with a systemic SP antagonist. Because SP is thought to act primarily as a sensory neurotransmitter, these findings may be important in understanding the mechanism of reflex laryngeal adductor responses.

Animals↗
PubMed1999

GLUT1-deficiency: barbiturates potentiate haploinsufficiency in vitro.

Barbiturates are known to inhibit glucose transport mediated by the facilitative sugar transporter GLUTI. We have studied such inhibition in children with GLUT1-deficiency. Zero-trans influx of 14C-labeled 3-O-methyl glucose (3OMG) into erythrocytes of patients (n = 3) was 35% of controls (n = 6). Preincubation with 10 mM phenobarbital or pentobarbital reduced patients' 30MG influx to 17%. In patients and controls, preincubation with barbiturates significantly decreased Vmax in a dose-dependent manner (for pentobarbital, IC50 = 0.84 mM, patient 2). The apparent Km in individuals remained largely unchanged. Three-OMG influx without preincubation resulted in a stronger inhibition at lower barbiturate concentrations. The patients' data are discussed in the light of individual missense mutations (patient 1: R126L and K256V; patient 2: T310I; patient 3: S66F) in the GLUTI gene. In conclusion, in controls and patients with GLUT1-deficiency barbiturates interact with GLUT1, lowering its intrinsic activity. The use of barbiturates in this condition for anesthesia or as anticonvulsants could therefore potentially aggravate the existing glucose transport defect and may put these patients at increased risk.

Adolescent↗
PubMed1999

(Z)-3-hexenyl and trans-linalool 3,7-oxide beta-primeverosides isolated as aroma precursors from leaves of a green tea cultivar.

6-O-beta-D-Xylopyranosyl-beta-D-glucopyranosides (beta-primeverosides) of (Z)-3-hexenol and trans-linalool 3,7-oxide were newly isolated from fresh leaves of a tea cultivar (Camellia sinensis var. sinensis cv. Yabukita). In addition, the already identified beta-primeverosides of benzyl alcohol, methyl salicylate, and trans-linalool 3,6-oxide from an oolong tea cultivar were isolated from the Yabukita cultivar. It was confirmed that all aglycones of the linalool oxide glycosides isolated here were of the optically active S-form by chiral GC after enzymatic hydrolysis with glycosidase.

Acyclic Monoterpenes↗
PubMed1999

Isolation and characterization of MC3T3-E1 preosteoblast subclones with distinct in vitro and in vivo differentiation/mineralization potential.

A series of subclonal cell lines with high or low differentiation/mineralization potential after growth in the presence of ascorbic acid (AA) were derived from murine MC3T3-E1 cells. Subclones were characterized in terms of their ability to mineralize a collagenous extracellular matrix both in vitro and in vivo and express osteoblast-related genes. When compared with nonmineralizing cells, mineralizing subclones selectively expressed mRNAs for the osteoblast markers, bone sialoprotein (BSP), osteocalcin (OCN), and the parathyroid hormone (PTH)/parathyroid hormone-related protein (PTHrP) receptor. In contrast, alkaline phosphatase mRNA was present in certain nonmineralizing as well as mineralizing subclones, suggesting that its expression may be subject to different controls from other osteoblast markers. Only highly differentiating subclones exhibited strong AA-dependent induction of a transiently transfected OCN promoter-luciferase reporter gene, indicating that there was a good correlation between mRNA levels and transcriptional activity. Consistent with its postulated role in biomineralization, BSP as measured by Western blotting was only present in mineralizing subclones. After implantation into immunodeficient mice, highly differentiating subclones formed bone-like ossicles resembling woven bone, while poorly differentiating cells only produced fibrous tissue. Interestingly, subclones with both high and low differentiation potential produced similar amounts of collagen in culture and expressed comparable basal levels of mRNA encoding Osf2/Cbfa1, an osteoblast-related transcription factor. Although some strongly differentiating cells exhibited a modest AA-dependent up-regulation of Osf2/Cbfa1 mRNA, there was no clear relationship between levels of this message and induction of mRNAs for other differentiation markers. Thus, the mere presence of Osf2/Cbfa1 in a subclone was not sufficient for osteoblast differentiation. These subclones will be very useful for studying critical events in osteoblast differentiation and mineralization.

Animals↗
PubMed1999

Chromatin structure and nuclear remodeling.

Nuclear architecture is remodeled during interphase in response to changes in gene activity as well as to changing structural and functional requirements during cell division. Using the monoclonal antibody mAb2A, we have identified two proteins that appear to play important roles in these processes: JIL-1 is a tandem serine-threonine kinase implicated in the regulation of chromatin structure, whereas Skeletor is a novel protein participating in structural nuclear remodeling during the cell cycle. Antibody staining and live imaging of JIL-1-GFP transgenic flies show that JIL-1 localizes to the gene-rich interband regions of larval polytene chromosomes and is upregulated almost twofold on the hypertranscribed male X chromosome compared with autosomes. We propose that JIL-1 may play a role in transcriptional control potentially by regulating chromatin structure. The other mAb2A antigen, Skeletor, is distributed in a nuclear meshwork pattern that can be observed in stereo pair images to reorganize during the cell cycle to form a spindle-like structure at prometaphase that is distinct from the microtubule spindle apparatus. Taking advantage of the powerful molecular and genetic approaches offered in Drosophila, the study of these two proteins promises to yield new insight into what defines nuclear architecture at the molecular level and how its remodeling is regulated.

Amino Acid Sequence↗
PubMed1999

Elevated blood levels of soluble tumor necrosis factor receptors in nasopharyngeal carcinoma: correlation with humoral immune response to lytic replication of Epstein-Barr virus.

Nasopharyngeal carcinoma (NPC) is tightly associated with Epstein-Barr virus (EBV) infection and a heavy infiltration of lymphoid cells in the tumor tissue. Although various lines of evidence have shown that the immune systems of NPC patients have the potential to attack the tumor cells, it is not yet understood how this potential is blocked. In this study we determined the circulatory soluble tumor necrosis factor receptors (sTNFRI and sTNFRII), which are proven to be inhibitory to the anti-tumor effects of tumor necrosis factor-alpha (TNF-alpha), in NPC patients. The serum concentration of both sTNFRI and sTNFRII was determined with an ELISA method, and shown to be significantly higher in 28 NPC patients than in matched healthy controls. This elevation was found to be positively correlated with the serum titers of IgA against EBV early antigens and viral capsid antigens in NPC patients, suggesting that the increased serum concentration of sTNFRI and sTNFRII is possibly due to the EBV infection in NPC tumor cells. This is partly supported by FACS analysis of the circulatory T cells. Phenotypical expression of activation markers such as CD25, CD38, CD69 and CD71 in blood T cells was not significantly different between the NPC and control individuals, indicating the elevation of the sTNFRs is indeed derived from the local immune response in the tumor area. Based on these results, it seems that the increased sTNFRs may act as an inhibitor to decrease the host immune response towards tumor cells in NPC patients.

Adult↗
PubMed1999

Mutation analysis of the Smad3 gene in human ovarian cancers.

The Smad3 gene is a member of the Smad family, vertebrate homologues of Drosophila Mad, and its gene product is a cytoplasmic element in the transforming growth factor-beta (TGF-beta) signaling pathway. Mutations in TGF-beta receptors and their cytoplasmic elements of transduction signals commonly accompany various cancers. Using PCR-SSCP analysis we searched for the presence of Smad3 gene mutations in 36 human ovarian cancers, and found that 15 cases (41. 7%) had a polymorphism at codon 103. Because this mutation was not accompanied by amino acid replacement, the present results show that the mutations in the Smad3 gene are unlikely to be involved in human ovarian cancers.

Adolescent↗