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Biomedical subjects

W Xiao

Publications and source records attributed to W Xiao.

At least 19 recordsLinked to original sources
PubMed2006

Chemotherapy-evoked painful peripheral neuropathy: analgesic effects of gabapentin and effects on expression of the alpha-2-delta type-1 calcium channel subunit.

Chemotherapeutics in the taxane and vinca-alkaloid classes sometimes produce a painful peripheral neuropathy for which there is no validated treatment. Experiments with rat models of paclitaxel- and vincristine-evoked pain suggest that these conditions may not respond to all of the analgesics that have efficacy in other models of painful peripheral neuropathy. We tested gabapentin as a potential analgesic for paclitaxel- and vincristine-evoked pain. We used a repeated dosing paradigm because there are precedents showing that repeated drug exposure may be necessary to demonstrate analgesia in neuropathic pain models. Gabapentin is believed to work via binding to voltage-gated calcium channels that contain the alpha-2-delta type-1 (alpha(2)delta-1) subunit, and the expression of this subunit is known to be increased in some painful peripheral neuropathy models. Thus we also examined whether the paclitaxel-evoked pain syndrome was accompanied by an alpha(2)delta-1 increase, and whether gabapentin had any effect on subunit expression. We found that the paclitaxel- and vincristine-evoked mechano-allodynia and mechano-hyperalgesia were significantly reduced by gabapentin, but only with repeated dosing. Paclitaxel-evoked painful peripheral neuropathy was associated with an increased expression of the alpha(2)delta-1 subunit in the spinal dorsal horn, but not in the dorsal root ganglia. The spinal cord increase was normalized by repeated gabapentin injections. Together, these findings suggest that repeated dosing with gabapentin may be beneficial in patients with chemotherapy-evoked painful peripheral neuropathy and that gabapentin's mechanisms of action may include normalization of the nerve injury-evoked increase in calcium channel alpha(2)delta-1 subunit expression.

Amines↗
PubMed2006

Unscheduled CDK1 activity in G1 phase of the cell cycle triggers apoptosis in X-irradiated lymphocytic leukemia cells.

Cyclin-dependent kinase 1 (CDK1) is a major component of the cell cycle progression engine. Recently, several investigations provided evidence demonstrating that unscheduled CDK1 activation may also be involved in apoptosis in cancerous cells. In this article, we demonstrate that X-ray irradiation induced G1 arrest in MOLT-4 lymphocytic leukemia cells, the arrest being accompanied by reduction in the activity of CDK2, but increased CDK1 activity and cell apoptosis in the G1 phase. Interestingly, this increase in CDK1 and apoptosis by ionizing radiation was prevented by pretreatment with the CDK1 inhibitor, roscovitine, suggesting that CDK1 kinase activity is required for radiation-induced apoptotic cell death in this model system. Furthermore, cyclin B1 and CDK1 were detected co-localizing and associating in G1 phase MOLT-4 cells, with the cellular lysates from these cells revealing a genotoxic stress-induced increase in CDK1 phosphorylation (Thr-161) and dephosphorylation (Tyr-15), as analyzed by postsorting immunoprecipitation and immunoblotting. Finally, X-irradiation was found to increase Bcl-2 phosphorylation in G1 phase cells. Taken together, these novel findings suggest that CDK1 is activated by unscheduled accumulation of cyclin B1 in G1 phase cells exposed to X-ray, and that CDK1 activation, at the wrong time and in the wrong phase, may directly or indirectly trigger a Bcl-2-dependent signaling pathway leading to apoptotic cell death in MOLT-4 cells.

Apoptosis↗
PubMed2005

Concentration and detection of SARS coronavirus in sewage from Xiao Tang Shan Hospital and the 309th Hospital of the Chinese People's Liberation Army.

A worldwide outbreak of severe acute respiratory syndrome (SARS) had been reported. Over 8439 SARS cases and 812 SARS-related deaths were reported to the World Health Organization from 32 countries around the world up to 5 July 2003. The mechanism of transmission of SARS-CoV has been limited only to close contacts with patients. Attention was focused on possible transmission by the sewage system because laboratory studies showed that patients excreted coronavirus RNA in their stools in Amoy Gardens in Hong Kong. To explore whether the stool of SARS patients or the sewage containing the stool of patients would transmit SARS-CoV or not, we used a style of electropositive filter media particle to concentrate the SARS-CoV from the sewage of two hospitals receiving SARS patients in Beijing, as well as cell culture, semi-nested RT-PCR and sequencing of genes to detect and identify the viruses from sewage. There was no live SARS-CoV detected in the sewage in these assays. The nucleic acid of SARS-CoV was found in the sewage before disinfection from both hospitals by PCR. After disinfection, SARS-CoV RNA could be detected from some samples from the 309th Hospital of the Chinese People's Liberation Army, but not from Xiao Tang Shan Hospital after disinfection. In this study, we found that the virus can survive for 14 days in sewage at 4 degrees C, 2 days at 20 degrees C, and its RNA can be detected for 8 days though the virus had been inactivated. In conclusion, this study demonstrates that the RNA of SARS-CoV could be detected from the concentrates of sewage of both hospitals receiving SARS patients before disinfection and occasionally after disinfection though there was no live SARS-CoV; thus much attention should be paid to the treatment of stools of patients and the sewage of hospitals receiving SARS patients.

Bacteriophages↗
PubMed2004

Acute wake-promoting actions of JNJ-5207852, a novel, diamine-based H3 antagonist.

1 1-[4-(3-piperidin-1-yl-propoxy)-benzyl]-piperidine (JNJ-5207852) is a novel, non-imidazole histamine H3 receptor antagonist, with high affinity at the rat (pKi=8.9) and human (pKi=9.24) H3 receptor. JNJ-5207852 is selective for the H3 receptor, with negligible binding to other receptors, transporters and ion channels at 1 microm. 2 JNJ-5207852 readily penetrates the brain tissue after subcutaneous (s.c.) administration, as determined by ex vivo autoradiography (ED50 of 0.13 mg kg(-1) in mice). In vitro autoradiography with 3H-JNJ-5207852 in mouse brain slices shows a binding pattern identical to that of 3H-R-alpha-methylhistamine, with high specific binding in the cortex, striatum and hypothalamus. No specific binding of 3H-JNJ-5207852 was observed in brains of H3 receptor knockout mice. 3 In mice and rats, JNJ-5207852 (1-10 mg kg(-1) s.c.) increases time spent awake and decreases REM sleep and slow-wave sleep, but fails to have an effect on wakefulness or sleep in H3 receptor knockout mice. No rebound hypersomnolence, as measured by slow-wave delta power, is observed. The wake-promoting effects of this H3 receptor antagonist are not associated with hypermotility. 4 A 4-week daily treatment of mice with JNJ-5207852 (10 mg kg(-1) i.p.) did not lead to a change in body weight, possibly due to the compound being a neutral antagonist at the H3 receptor. 5 JNJ-5207852 is extensively absorbed after oral administration and reaches high brain levels. 6 The data indicate that JNJ-5207852 is a novel, potent and selective H3 antagonist with good in vitro and in vivo efficacy, and confirm the wake-promoting effects of H3 receptor antagonists.

Administration, Oral↗
PubMed2003

High throughput creation of recombinant adenovirus vectors by direct cloning, green-white selection and I-Sce I-mediated rescue of circular adenovirus plasmids in 293 cells.

Ability of replication-defective adenovirus vectors to achieve efficient gene transfer in most of the mammalian cell types makes them useful vehicles for many gene transfer applications, including their use in assessing gene function. High throughput creation of recombinant adenovirus becomes a critical path to the expanding utility of adenovirus vector technology. Here, we report a process in which recombinant adenovirus vectors are isolated as single molecular clones through a convenient direct cloning and green-white selection procedure, and directly transfected into 293 cells where virus is rescued through an enzymatic reaction mediated by an intron-encoding rare endonuclease I-Sce I. This process of enzymatic rescue of circular molecular clones was at least 10-fold more efficient than that using linearized clones for transfection. This method will facilitate a high throughput creation of vectors as required for screening gene function.

Adenoviridae↗
PubMed2003

A single adeno-associated virus (AAV)-murine factor VIII vector partially corrects the hemophilia A phenotype.

A major obstacle for delivery of factor (F)VIII using adeno-associated virus (AAV) vectors is the large size of FVIII cDNA, which is well above the 5 kb packaging limit for AAV. Here we construct a < 5 kb FVIII-AAV vector using murine FVIII cDNA and a strong liver-specific albumin promoter. We assessed the efficacy of this vector using three different routes of administration, intraportal, intrasplenic and tail vein injection, in FVIII knockout (FVIII KO) mice. The peak level of FVIII observed was about 8% of normal mouse FVIII activity. Even at 9 months, post vector injection, 14 of 19 mice receiving FVIII-AAV demonstrated phenotypic correction and roughly 2% FVIII activity. The transgene copy number ranged from 0.001 to 0.1 copies per cell, depending upon the somatic tissue. The potential for germline transmission of AAV was assayed in 34 pups obtained from five pairs of treated, phenotypically corrected adult hemophilic mice. Although the parents harbored the transgene in liver, spleen, and gonads, none of the 34 offspring was positive for the transgene, suggesting that the risk of inadvertent germline transmission is low.

Animals↗
PubMed2003

Optimized production of high-titer recombinant adeno-associated virus in roller bottles.

Adeno-associated viral (AAV) vectors are used for in vivo gene transfer in a number of preclinical models of genetic diseases (including large-animal models) and are currently being tested in clinical trials for treatment of hemophilia B and cystic fibrosis. Protocols for production of AAV vectors in a helper virus-free system are available and are based on transient transfection of HEK-293 cells with multiple plasmids. Scale-up of vector production has been labor intensive and inefficient because of a lack of larger culture vessels suitable for growth of adherent cells, large-scale transfection, and vector production. Here we report efficient production of AAV vector in roller bottles, which represents a 10-fold scale-up from the conventional flask or plate method. Optimized production yielded greater than 10(13) vector genomes per bottle and was as cost effective as published protocols using plates. Successful vector production by this method was dependent on optimization of transfection by calcium phosphate precipitation, of monitoring of cell growth (by measurement of glucose consumption), of cell culture conditions, and CO2/air exchange with the culture vessel.

Cell Count↗
PubMed2002

Replication competent helper functions for recombinant AAV vector generation.

Adeno-associated virus (AAV) is a promising gene transfer vector tested in both animal studies and human clinical trials. However, current production methods are generally inefficient and require improvements to meet the increasing clinical need for economical, high titer and high quality rAAV vectors. The inefficiency of the current systems largely arises from the AAV helper function, which contains only the AAV coding region but lacks inverted terminal repeats. The terminal repeats were originally removed to prevent replication competent AAV contamination. Here we designed a novel and highly efficient rAAV helper function containing AAV terminal repeats. The new helper function not only mimics the wild-type AAV growth as it replicates along with the vector plasmid, but also restores the cis regulating function of the AAV terminal repeats. Addition of heterologous introns to the helper genome and use of a mutant AAV terminal repeat defective in packaging effectively controls the contamination of replication competent AAV particles. This new strategy also performs better in AAV producing cell lines than those based on non-replicating AAV rep and cap genome.

Cell Line↗
PubMed2001

Deletion of the MAG1 DNA glycosylase gene suppresses alkylation-induced killing and mutagenesis in yeast cells lacking AP endonucleases.

DNA base excision repair (BER) is initiated by DNA glycosylases that recognize and remove damaged bases. The phosphate backbone adjacent to the resulting apurinic/apyrimidinic (AP) site is then cleaved by an AP endonuclease or glycosylase-associated AP lyase to invoke subsequent BER steps. We have used a genetic approach in Saccharomyces cerevisiae to address whether AP sites are blocks to DNA replication and the biological consequences if AP sites persist in the genome. We found that yeast cells deficient in the two AP endonucleases (apn1 apn2 double mutant) are extremely sensitive to killing by methyl methanesulfonate (MMS), a model DNA alkylating agent. Interestingly, this sensitivity can be reduced up to 2500-fold by deleting the MAG1 3-methyladenine DNA glycosylase gene, suggesting that Mag1 not only removes lethal base lesions, but also benign lesions and possibly normal bases, and that the resulting AP sites are highly toxic to the cells. This rescuing effect appears to be specific for DNA alkylation damage, since the mag1 mutation reduces killing effects of two other DNA alkylating agents, but does not alter the sensitivity of apn cells to killing by UV, gamma-ray or H(2)O(2). Our mutagenesis assays indicate that nearly half of spontaneous and almost all MMS-induced mutations in the AP endonuclease-deficient cells are due to Mag1 DNA glycosylase activity. Although the DNA replication apparatus appears to be incapable of replicating past AP sites, Polzeta-mediated translesion synthesis is able to bypass AP sites, and accounts for all spontaneous and MMS-induced mutagenesis in the AP endonuclease-deficient cells. These results allow us to delineate base lesion flow within the BER pathway and link AP sites to other DNA damage repair and tolerance pathways.

Alkylating Agents↗
PubMed2001

MMS1 protects against replication-dependent DNA damage in Saccharomyces cerevisiae.

A series of yeast mutants were isolated that are sensitive to killing by the monofunctional DNA-alkylating agent methyl methanesulfonate (MMS) but not by UV or X-radiation. We have cloned and characterized one of the corresponding genes, MMS1, and show that the mms1 Delta mutant is dramatically sensitive to killing by MMS and mildly sensitive to UV radiation. mms1 Delta mutants display an elevated level of spontaneous DNA damage and genomic instability. Furthermore, the mms1 Delta cells are sensitive to killing by conditions that induce replication-dependent double-strand breaks, such as treatment with camptothecin, and incubation of a cdc2-2 strain at the restrictive temperature. rad52 Delta is epistatic to mms1 Delta for MMS and camptothecin sensitivity, indicating that Mms1 acts in concert with Rad52. However, unlike mutants of the RAD52 group, mms1 Delta cells are not sensitive to gamma-rays, which induce double-strand breaks independently of DNA replication. Together these results suggest a role for an Mms1-dependent, Rad52-mediated, pathway in protecting cells against replication-dependent DNA damage.

Cloning, Molecular↗
PubMed2001

Interleukin-6 regulation of the human DNA methyltransferase (HDNMT) gene in human erythroleukemia cells.

Methylation of mammalian DNA by the DNA methyltransferase enzyme (dnmt-1) at CpG dinucleotide sequences has been recognized as an important epigenetic control mechanism in regulating the expression of cellular genes (Yen, R. W., Vertino, P. M., Nelkin, B. D., Yu, J. J., el-Deiry, W., Cumaraswamy, A., Lennon, G. G., Trask, B. J., Celano, P., and Baylin, S. B. (1992) Nucleic Acids Res. 20, 2287-2291; Ramchandani, S., Bigey, P., and Szyf, M. (1998) Biol. Chem. 379, 535-5401). Here we show that interleukin (IL)-6 regulates the methyltransferase promoter and resulting enzyme activity, which requires transcriptional activation by the Fli-1 transcription factor (Spyropoulos, D. D., Pharr, P. N., Lavenburg, K. R., Jackers, P., Papas, T. S., Ogawa, M., and Watson, D. K. (1998) Mol. Cell. Biol. 15, 5643-5652). The data suggest that inflammatory cytokines such as IL-6 may exert many epigenetic changes in cells via the regulation of the methyltransferase gene. Furthermore, IL-6 regulation of transcription factors like Fli-1, which can help to direct cells along opposing differentiation pathways, may in fact be reflected in part by their ability to regulate the methylation of cellular genes.

Cell Differentiation↗
PubMed2001

Noncovalent interaction between ubiquitin and the human DNA repair protein Mms2 is required for Ubc13-mediated polyubiquitination.

Ubiquitin-conjugating enzyme variants share significant sequence similarity with typical E2 (ubiquitin-conjugating) enzymes of the protein ubiquitination pathway but lack their characteristic active site cysteine residue. The MMS2 gene of Saccharomyces cerevisiae encodes one such ubiquitin-conjugating enzyme variant that is involved in the error-free DNA postreplicative repair pathway through its association with Ubc13, an E2. The Mms2-Ubc13 heterodimer is capable of linking ubiquitin molecules to one another through an isopeptide bond between the C terminus and Lys-63. Using highly purified components, we show here that the human forms of Mms2 and Ubc13 associate into a heterodimer that is stable over a range of conditions. The ubiquitin-thiol ester form of the heterodimer can be produced by the direct activation of its Ubc13 subunit with E1 (ubiquitin-activating enzyme) or by the association of Mms2 with the Ubc13-ubiquitin thiol ester. The activated heterodimer is capable of transferring its covalently bound ubiquitin to Lys-63 of an untethered ubiquitin molecule, resulting in diubiquitin as the predominant species. In (1)H (15)N HSQC ((1)H (15)N heteronuclear single quantum coherence) NMR experiments, we have mapped the surface determinants of tethered and untethered ubiquitin that interact with Mms2 and Ubc13 in both their monomeric and dimeric forms. These results have identified a surface of untethered ubiquitin that interacts with Mms2 in the monomeric and heterodimeric form. Furthermore, the C-terminal tail of ubiquitin does not participate in this interaction. These results suggest that the role of Mms2 is to correctly orient either a target-bound or untethered ubiquitin molecule such that its Lys-63 is placed proximally to the C terminus of the ubiquitin molecule that is linked to the active site of Ubc13.

Binding Sites↗
PubMed2001

DNA postreplication repair and mutagenesis in Saccharomyces cerevisiae.

DNA postreplication repair (PRR) is defined as an activity to convert DNA damage-induced single-stranded gaps into large molecular weight DNA without actually removing the replication-blocking lesions. In bacteria such as Escherichia coli, this activity requires RecA and the RecA-mediated SOS response and is accomplished by recombination and mutagenic translesion DNA synthesis. Eukaryotic cells appear to share similar DNA damage tolerance pathways; however, some enzymes required for PRR in eukaryotes are rather different from those of prokaryotes. In the yeast Saccharomyces cerevisiae, PRR is centrally controlled by RAD6 and RAD18, whose products form a stable complex with single-stranded DNA-binding, ATPase and ubiquitin-conjugating activities. PRR can be further divided into translesion DNA synthesis and error-free modes, the exact molecular events of which are largely unknown. This error-free PRR is analogous to DNA damage-avoidance as defined in mammalian cells, which relies on recombination processes. Two possible mechanisms by which recombination participate in PRR to resolve the stalled replication folk are discussed. Recombination and PRR are also genetically regulated by a DNA helicase and are coupled to the cell-cycle. The PRR processes appear to be highly conserved within eukaryotes, from yeast to human.

Animals↗
PubMed2001

Activation of estrogen receptor blocks interleukin-6-inducible cell growth of human multiple myeloma involving molecular cross-talk between estrogen receptor and STAT3 mediated by co-regulator PIAS3.

Estrogen receptors (ERs)(1) highly expressed by multiple myeloma (MM) cells and stimulation of estrogenic ligands leads to cell apoptosis. Interleukin (IL)-6 is a major growth factor in the pathogenesis of MM. However, little is known concerning the molecular consequences of ER activation on IL-6-regulated MM cell growth. Here we show that the ER agonist 17 beta-estradiol completely abolished IL-6-inducible MM cell proliferation. By contrast, the ER antagonist ICI 182,780 overcame the inhibitory effect of estrogen. Estrogen blocked STAT3 DNA binding and transactivation but failed to affect the mRNA expression of IL-6 receptor chains or activation of JAK2 and STAT3. Estrogen-activated ER did not associate directly with STAT3. Estrogen induced the mRNA expression of PIAS3 (protein inhibitor of activated STAT3) and increased PIAS3 physical association with STAT3, suggesting a possible mechanism of STAT3 inhibition requiring PIAS3 as a co-regulator modulating the cross-talk between ER and STAT3. These data directly demonstrate STAT3 to be a molecular participant in ER inhibition of the IL-6 signaling pathway in human MM cells and provides the molecular basis for the potential use of estrogenic ligands in the treatment of MM or other tumors where IL-6 has an autocrine or paracrine role.

Base Sequence↗
PubMed2001

Molecular cloning and functional characterization of two murine cDNAs which encode Ubc variants involved in DNA repair and mutagenesis.

Ubiquitin-conjugating enzyme (Ubc) variants share structural similarity with Ubcs but lack the essential cysteine residue required to form a thioester bond with ubiquitin. Yeast Mms2 is a Ubc variant and plays an important role in error-free DNA postreplication repair to protect cells from killing by DNA damaging agents and mutagenesis. Ironically, one of two known Mms2 homologs, CROC1, has been linked to cell immortalization and tumorigenesis. To further investigate cellular roles played by mammalian Mms2 homologs, we report here the molecular cloning, tissue distribution and functional characterization of two mouse cDNAs encoding mMMS2 and mCROC1. Unlike human CROC1, the mCROC1 gene does not encode two alternative transcripts in most tissues. Instead, nonoverlapping sequences were found in two distinct cDNA clones that together would constitute a full-length open reading frame homologous to CROC1B. Both mMMS2 and the C-terminal mCROC1 core domain are able to complement the yeast mms2 mutant functionally and are able to interact with Ubc13 in a yeast two-hybrid assay, indicating that they are true yeast Mms2 homologs and may play a similar role in DNA postreplication repair. We propose several hypotheses to reconcile the seemingly contradictory observations regarding roles of the two mammalian Mms2 homologs in tumorigenesis and carcinogenesis.

Amino Acid Sequence↗
PubMed2001

CCAAT/enhancer-binding protein beta mediates interferon-gamma-induced p48 (ISGF3-gamma ) gene transcription in human monocytic cells.

Previous studies have identified a novel interferon-stimulated response element-like element, termed gamma-interferon-activating transcription element, within the interferon-stimulating gene factor-3gamma (p48) promoter region that is bound by novel transcription factors in response to stimulation with interferons (IFNs) (Weihua, X., Kolla, V., and Kalvakolanu, D. V. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 103-108). In the present study, we have identified CCAAT/enhancer-binding protein beta (C/EBP-beta) as one of the gamma-interferon-activating transcription element cognate transcription factors by screening a human monophage-derived cDNA library in a yeast one-hybrid system. Electrophoretic mobility shift assay studies suggest that C/EBP-beta dynamically regulates p48 gene expression upon IFN-gamma stimulation by undergoing changes in its heterodimerization partners. Transient transfection studies demonstrate that overexpression of C/EBP-beta strongly enhanced IFN-gamma-induced transcription from the p48 promoter. However, deletion mutants of C/EBP-beta that lack the N-terminal transactivation domain were unable to stimulate the p48 promoter. Western blotting revealed that C/EBP-beta is induced by IFN-gamma stimulation in THP-1-derived macrophages. Collectively, these results suggest that C/EBP-beta plays an important role in the human IFN-gamma signaling pathway by transcriptional regulation of p48 gene expression, an essential component in the IFN signaling pathway.

Base Sequence↗
PubMed2001

AMP deaminase inhibitors. 5. Design, synthesis, and SAR of a highly potent inhibitor series.

A highly potent AMP deaminase (AMPDA) inhibitor series was discovered by replacing the N3 substitutents of the two lead AMPDA inhibitor series with a conformationally restricted group. The most potent compound, 3-[2-(3-carboxy-4-bromo-5,6,7,8-tetrahydronaphthyl)ethyl]-3,6,7,8-tetrahydroimidazo[4,5-d][1,3]diazepin-8-ol (24b), represents a 10- to 250-fold enhancement in AMPDA inhibitory potency without loss in the enzyme specificity. The potency of the inhibitor 24b (AMPDA K(i) = 0.002 microM) is 10(5)-fold lower than the Km for the substrate AMP. It represents the most potent nonnucleotide AMPDA inhibitor known.

AMP Deaminase↗