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Biomedical subjects

H Xiao

Publications and source records attributed to H Xiao.

At least 91 records · Page 5Linked to original sources
PubMed1999

The Kaposi's sarcoma-related herpesvirus (KSHV)-encoded chemokine vMIP-I is a specific agonist for the CC chemokine receptor (CCR)8.

The Kaposi's sarcoma-related herpesvirus (KSHV), also designated human herpesvirus 8, is the presumed etiologic agent of Kaposi's sarcoma and certain lymphomas. Although KSHV encodes several chemokine homologues (viral macrophage inflammatory protein [vMIP]-I, -II, and -III), only vMIP-II has been functionally characterized. We report here that vMIP-I is a specific agonist for the CC chemokine receptor (CCR)8 that is preferentially expressed on Th2 T cells. Y3 cells transfected with CCR8 produced a calcium flux in response to vMIP-I and responded vigorously in in vitro chemotaxis assays. In competition binding experiments, the interaction of vMIP-I with CCR8 was shown to be specific and of high affinity. In contrast to its agonist activity at CCR8, vMIP-I did not interact with CCR5 or any of 11 other receptors examined. Furthermore, vMIP-I was unable to inhibit CCR5-mediated HIV infection. These findings suggest that expression of vMIP-I by KSHV may influence the Th1/Th2 balance of the host immune response.

Animals↗
PubMed1999

Both Sp1 and Sp3 are responsible for p21waf1 promoter activity induced by histone deacetylase inhibitor in NIH3T3 cells.

Histone deacetylase inhibitor-induced expression of p21WAF1 is p53 independent. In the present study, we provide evidence that trichostatin A (TSA), a specific inhibitor of histone deacetylase, can elevate H3 and H4 acetylation and p21WAF1 expression in NIH3T3 cells at first. To identify the transcription factor which is responsible for histone deacetylase inhibitor-induced expression of p21WAF1 and understand the potential events occurred during this process, we analyze the response of the mouse p21WAF1 promoter to TSA in detail. The region responsive to TSA treatment in the p21 promoter is located -100 bp upstream from transcription initiation site and contains a GC-box. The mutation introduced into this GC-box decreases most of the basal and TSA-induced promoter activity. The results from gel-shift assay show that Sp1 and Sp3 bind to this GC-rich region. Cotransfection with Sp1 and/or Sp3 expression constructs elevate both basal and induced promoter activity, and this elevation is dependent on the present of the GC-box. By contrast, cotransfection with reverse oriented Sp1 or Sp3 cDNA decreased basal and induced-promoter activity, as well as GC-box dependency. These findings provide physical and functional evidence which strongly indicated that both Sp1 and Sp3 are responsible for TSA-induced transactivation of the murine p21WAF1 promoter in NIH3T3 cells.

3T3 Cells↗
PubMed1999

Cooperative interaction of NF-kappaB and C/EBP binding sites is necessary for manganese superoxide dismutase gene transcription mediated by lipopolysaccharide and interferon-gamma.

Expression of the manganese superoxide dismutase (Mn-SOD) is induced by pro-inflammatory cytokines. We investigated the cis-acting elements within a tumor necrosis factor-responsive element (TNFRE) which was identified in the second intron of the murine Mn-SOD gene. Site-directed mutagenesis, reporter plasmid transfection studies and electrophoretic mobility shift assays demonstrated that inducible transcription factors enhanced the transcriptional activity of the Mn-SOD gene through the TNFRE. The cooperation between proteins binding to the newly identified NF-kappaB and C/EBP sites led to synergistic gene transcription. This report provides the first evidence that cooperation between two distinct cis-acting elements may be required for induction of Mn-SOD gene expression mediated by lipopolysaccharide and interferon-gamma.

3T3 Cells↗
PubMed1999

Cloning of a GADD34-like gene that interacts with the zinc-finger transcription factor which binds to the p21(WAF) promoter.

A histone deacetylase inhibitor has been shown to induce differentiation of many cancer cells and senescence-like state of human fibroblasts. Previously, our data suggested that the region responsive to trichostatin A (TSA), a specific inhibitor of histone deacetylase, treatment in the p21(WAF1) promoter is located -100 bp upstream from transcription initiation site and contains a GC-box where both Sp1 and Sp3 are responsible. Here we show that another zinc-finger transcription factor, BFCOL1, which binds to the proximal proalpha2(I) collagen promoter, could also bind to this GC-box of the p21 promoter. In addition, we cloned a gene whose product interacts with this factor by yeast two-hybrid method. The cloned gene was a variant of GADD34 and lacking one PEST region. We found that this cDNA product decreased the DNA binding activity of BFCOL1 to the GC-rich region of p21 minimal promoter.

Animals↗
PubMed1999

Protease and protease inhibitor assays using biotinylated casein coated on a solid phase.

A new type of solid-phase assay for proteases and protease inhibitors has been developed using biotinylated casein. The assay involves coating of titer plate wells with biotinylated casein, hydrolysis of this substrate with a protease such as trypsin, reaction of the biotin from the unhydrolyzed substrate with an alkaline phosphatase-streptavidin complex, and finally quantification of the amount of casein remaining on the plate using alkaline phosphatase activity as the indicator. The activity of the bound indicator enzyme is oppositely related to the protease activity of the sample. In addition, the assay can be modified for quantitating the corresponding amount of protease inhibitor in the sample. The assay is simple, sensitive, accurate, inexpensive, and amenable to automation.

Biotinylation↗
PubMed1999

The human homologue of Drosophila TRF-proximal protein is associated with an RNA polymerase II-SRB complex.

Mammalian RNA polymerase II holoenzymes are large complexes that have been reported to contain, in addition to RNA polymerase II, homologues of several yeast SRBs, various general transcription factors, and other polypeptides. On the basis of its copurification with an SRB-containing RNA polymerase II complex by conventional chromatography procedures, we have identified a human homologue of Drosophila TRF-proximal protein, designated hTRFP, and isolated its cognate cDNA. Antibody specific for SRB7 can immunoprecipitate hTRFP and RNA polymerase II and, reciprocally, antibody specific for hTRFP can immunoprecipitate RNA polymerase II and SRB7. These data indicate that hTRFP is an integral component of an RNA polymerase II-SRB complex. Whereas the precise function of hTRFP remains to be determined, the hTRFP-containing RNA polymerase II-SRB complex supports basal level transcription and, relative to RNA polymerase II alone, enhances transcriptional activation by Gal4-VP16 in the presence of cofactor PC4. Thus, hTRFP may regulate transcription of class II genes through association with the RNA polymerase II-SRB complex.

Amino Acid Sequence↗
PubMed1999

The assignment of chemokine-chemokine receptor pairs: TARC and MIP-1 beta are not ligands for human CC-chemokine receptor 8.

Identification of chemokine receptors and their associated ligands is crucial to the understanding of most immune reactions. Three human chemokines [I-309, thymus and activation-regulated chemokine (TARC) and macrophage inflammatory protein-1beta (MIP-1beta)] have been reported to be ligands for CC-chemokine receptor 8 (CCR8). In this report, we present evidence that TARC and MIP-1beta did not bind to or induce chemotaxis through CCR8 on a stable transfected cell line (1D-21) and did not bind to CCR8 on in vitro differentiated human CD4(+) Th(2) cell cultures. Also, I-309-dependent calcium mobilization in 1D-21 cells and in Th(2) cells was desensitized by I-309 but not by MIP-1beta or TARC. These results provide strong evidence that, at physiologically relevant concentrations, I-309 is the only known human ligand for CCR8. These data also provide a framework for suggesting minimum requirements for the assignment of chemokine receptor-ligand pairs.

CD4 Antigens↗
PubMed1999

Development of a 96-well enzyme-linked solid-phase assay for beta-glucanase and xylanase.

An enzyme-linked sorbent assay (ELSA) for estimating beta-glucanase activity was developed on the basis of the use of biotinylated beta-glucan as a solid-phase substrate. The assay involves the coating of titer plate wells with biotinylated beta-glucan, the partial hydrolysis of this substrate with beta-glucanase, the reaction of the biotin from the unhydrolyzed substrate with an alkaline phosphatase-streptavidin complex, and quantitation of the remaining beta-glucan using alkaline phosphatase. The activity of the bound indicator enzyme, alkaline phosphatase, is proportionally related to the beta-glucanase activity in the sample. The ELSA is simple, can be readily adapted to the routine assay of a large number of samples (as many as 200 per person/day), and has good precision (CV = 4.0-6.4%) and high sensitivity (detects as low as 0. 001 mU of beta-glucanase/assay). A similar assay was developed for xylanase using biotinylated arabinoxylan. The ELSA provides a simple and sensitive estimate of beta-glucanase and xylanase activity.

Biotinylation↗
PubMed1999

Targeting human immunodeficiency virus (HIV) type 2 integrase protein into HIV type 1.

Integrase (IN) is the only retroviral enzyme necessary for the integration of retroviral cDNA into the host cell's chromosomes. The structure and function of IN is highly conserved. The human immunodeficiency virus type 2 (HIV-2) IN has been shown to efficiently support 3' processing and strand transfer of HIV-1 DNA substrate in vitro. To determine whether HIV-2 IN protein (IN(2)) could substitute for HIV-1 IN function in vivo, we used HIV-1 Vpr to deliver the IN(2) into IN mutant HIV-1 virions by expression in trans as a Vpr-IN fusion protein. Trans-complementation with IN(2) markedly increased the infectivity of IN-minus HIV-1. Compared with the homologous trans-IN protein, infectivity was increased to a level of 16%. Since IN has been found to play a role in reverse transcription (Wu et al., J. Virol. 73:2126-2135, 1999), cells infected with IN(2)-complemented HIV-1 were analyzed for DNA products of reverse transcription. DNA levels of approximately 18% of that of wild type were detected. The homologous trans-IN protein restored the synthesis of viral cDNA to approximately 86% of that of wild-type virus. By complementing integration-defective HIV-1 IN mutant viruses, which were not impaired in cDNA synthesis, the trans-IN(2) protein was shown to support integration up to a level of 55% compared with that of the homologous trans-IN protein. The delivery of heterologous IN protein into HIV-1 particles in trans offers a novel approach to understand IN protein function in vivo.

DNA, Complementary↗
PubMed1999

Human immunodeficiency virus type 1 integrase protein promotes reverse transcription through specific interactions with the nucleoprotein reverse transcription complex.

The human immunodeficiency virus type 1 (HIV-1) integrase protein (IN) is essential for integration of the viral DNA into host cell chromosomes. Since IN is expressed and assembled into virions as part of the 160-kDa Gag-Pol precursor polyprotein and catalyzes integration of the provirus in infected cells as a mature 32-kDa protein, mutations in IN are pleiotropic and may affect virus replication at different stages of the virus life cycle in addition to integration. Several different phenotypes have been observed for IN mutant viruses, including defects in virion morphology, protein composition, reverse transcription, nuclear import, and integration. Because the effects of mutations in the IN domain of Gag-Pol can not always be distinguished from those of mutations in the mature IN protein, there remains a significant gap in our understanding of IN function in vivo. To directly analyze the function of the mature IN protein itself, in the context of a replicating virus but independently from that of Gag-Pol, we used an approach developed in our laboratory for incorporating proteins into HIV virions by their expression in trans as fusion partners of either Vpr or Vpx. By providing IN in trans as a Vpr-IN fusion protein, our analysis revealed, for the first time, that the mature IN protein is essential for the efficient initiation of reverse transcription in infected cells and that this function does not require the IN protein to be enzymatically (integration) active. Our findings of a direct physical interaction between IN and reverse transcriptase and the failure of heterologous HIV-2 IN protein to efficiently support reverse transcription indicate that this novel function occurs through specific interactions with other viral components of the reverse transcription initiation complex. Studies involving complementation between integration- and DNA synthesis-defective IN mutants further support this conclusion and reveal that the highly conserved HHCC motif of IN is important for both activities. These findings provide important new insights into IN function and reverse transcription in the context of the nucleoprotein reverse transcription complex within the infected cell. Moreover, they validate a novel approach that obviates the need to mutate Gag-Pol in order to study the role of its individual mature components at the virus replication level.

DNA, Viral↗
PubMed1999

Protein-bound heparin/heparan sulfates in human adult and umbilical cord plasma.

We used thrombin times and a competitive radiometric assay to identify, quantitate and characterize endogenous heparin-like molecules in umbilical cord (n = 58) and normal adult (n = 25) plasma. Thrombin times for cord plasma (29.6+/-3.6 s) were significantly longer (p< or = 0.0005) than those for adult plasma (18. 9+/-2.3 s), suggesting increased endogenous heparins. A radiometric assay based on the displacement of (125)I-heparin from protamine-Sepharose revealed that protease-digested plasma contained heparin/heparan sulfate, and plasma that was not digested with protease appeared not to contain heparin/heparan sulfate. More heparin/heparan sulfate was identified in cord than in adult plasma (p< or =0.05), but heparinase digestion produced significantly (p< or =0.001) reduced concentrations of heparin/heparan sulfate in only 39% of the samples. The lack of heparinase sensitivity in 61% of the protease-digested samples apparently was due to low molecular weight (LMW) heparins, for control heparin fragments of 5 kD that did not extend thrombin times were also less affected by heparinase, but the same LMW heparins were detected by radiometric assay. Despite normal thrombin times in all samples, the amounts of endogenous heparin/heparan sulfate identified in protease-digested samples by radiometric assay were of sufficient concentrations to produce inordinately prolonged thrombin times when compared with the same concentrations of unfractionated heparin. Collectively, these findings suggest the presence of a plasma reservoir of endogenous heparin/heparan sulfates in normal cord and adult plasma. These endogenous heparin/heparan sulfates are bound to plasma proteins, and an as yet undetermined proportion of these bound heparin/heparans are most likely LMW molecules.

Adult↗
PubMed1999

[Determination of risperidone and 9-hydroxyrisperidone in plasma by high performance liquid chromatography].

A reversed-phase HPLC method for the determination of risperidone and 9-hydroxyrisperidone in plasma was established. Risperidone and 9-hydroxyrisperidone can be separated on a Zorbax ODS C18 column with a mobile phase of MeOH-water-ammonium acetate (mol/L)-ammonia water(3 mol/L) (300:50:3:1, volume ratios) and detected at 280 nm. The flow rate was 0.8 mL/min. The linear range was 2-600 micrograms/L, r = 0.996 for risperidone, and 2-800 micrograms/L, r = 0.998 for 9-hydroxyrisperidone. The average recovery was (98.2 +/- 3.5)% for risperidone, and (97.8 +/- 3.8)% for 9-hydorxyrisperidone. The intra-day and inter-day coefficients of variation were 4.12% and 4.83% for risperidone respectively, 4.28% and 4.81% for 9-hydroxyrisperidone respectively. The method has been used to determine risperidone and 9-hydroxyrisperidone mass concentration in patient plasma. The data obtained showed that the sensitivity and selectivity of this method were adequate for drug monitoring in clinical research.

Antipsychotic Agents↗
PubMed1999

[Study of HSP70 gene upstream regulation element on expression efficiency of GST gene in M. smegmatis].

Four different expression vectors were constructed by cloning foreign gene which encode Schistosoma japonicum 26 kD antigen (Sj26GST) into Escherichia coli-Mycobacteria shuttle plasmid pBCG-2000 and investigated their expression efficiency in mycobacteria smegmatis. The plasmid which contains promoter of human mycobacterial tuberculosis heat shock protein 70 was firstly digested with Nco I and modified with two different ways to lead to two kinds of SD sequences, and ligated with Sj26GST encoding gene. Then, the DNA fragment contained HSP70 promoter and Sj26GST gene was obtained and cloned into E. coil-mycobacteria shuttle plasmid pBCG-2000, and finally four recombinant mycobacterial expression vectors that differenciated in SD sequence, orientation and copy number were selected. The expressed native recombinant Sj26GST(rSj26GST) was soluble and could be observed on SDS-PAGE about at the molecular weight of 26 kD obviously. Analysis with protein density scanning indicated: the expression efficiency that containing double-copy promoter-foreign gene vector was the highest and the expressed protein which was about 1.6 times than others was 28% of total protein of Mycobacteria smegmatis. The cloned direction and SD sequence had no significant effect on expression efficiency.

Animals↗
PubMed1998

Antagonistic effect of calcium, zinc and selenium against cadmium induced chromosomal aberrations and micronuclei in root cells of Hordeum vulgare.

The antagonistic effect of calcium (Ca2+), zinc (Zn2+) and selenium (Se4+) at different concentrations (10-2-10-6 M) against cadmium (Cd2+) induced genotoxic effects in root cells of Hordeum vulgare were studied. The results showed that 10-3-10-5 M could induce chromosomal aberrations and micronuclei formation. But in the treatment with 10-2-10-6 M of Ca2+, Zn2+ and Se4+ together with Cd2+ (10-3-10-5 M), respectively, the frequencies of chromosomal aberrations and micronuclei effectively decreased after 48 h of treatment. The treatment with 10-4-10-6 M of Ca2+ together with 10-4-10-5 M Cd2+, 10-6 M of Zn2+ together with 10-5 M Cd2+ and 10-6 M of Se4+ together with 10-5 M Cd2+ suggested rather obvious antagonistic effects. The order of the antagonisms of Ca2+, Se4+ and Zn2+ against Cd2+ toxicity was Ca2+>Se4+>Zn2+. The degree of antagonisms of Ca2+, Se4+ and Zn2+ against Cd2+ related to their concentration ratio.

Cadmium↗
PubMed1998

Activation of integrated provirus requires histone acetyltransferase. p300 and P/CAF are coactivators for HIV-1 Tat.

A unique aspect of the retrovirus life cycle is the obligatory integration of the provirus into host cell chromosomes. Unlike viruses that do not integrate, retroviruses must conserve an ability to activate transcription from a chromatin context. Human immunodeficiency virus (HIV)-1 encodes an unusual and an unusually potent transcriptional transactivator, Tat, which binds to a nascent viral leader RNA, TAR. The action of Tat has been well studied in various reductive model systems; however, the physiological mechanism through which Tat gains access to chromatin-associated proviral long terminal repeats (LTRs) is not understood. We show here that a nuclear histone acetyltransferase activity associates with Tat. Intracellularly, we found that Tat forms a ternary complex with p300 and P/CAF, two histone acetyltransferases (HATs). A murine cell defect in Tat transactivation of the HIV-1 LTR was linked to the reduced abundance of p300 and P/CAF. Thus, overexpression of p300 and P/CAF reconstituted Tat transactivation of the HIV-1 LTR in NIH3T3 cells to a level similar to that observed for human cells. By using transdominant p300 or P/CAF mutants that lack enzymatic activity, we delineated a requirement for the HAT component from the latter but not the former in Tat function. Finally, we observed that Tat-associated HAT is preferentially important for transactivation of integrated, but not unintegrated, HIV-1 LTR.

3T3 Cells↗
PubMed1998

Glutamine-rich domains activate transcription in yeast Saccharomyces cerevisiae.

Activation domains of eukaryotic transcription factors can be classified into at least three distinct types based on their amino acid composition: acidic, proline-rich, and glutamine-rich. Acidic activators, such as yeast GAL4 and GCN4 and herpes simplex virus VP16, have been shown to stimulate transcription in various higher and lower eukaryotic cells. Similarly, proline-rich activators also function in both mammalian and yeast cells. These activators are regarded to possess "universal" activating potentials. By contrast, several studies have suggested that glutamine-rich activators such as human Sp1 are active in higher (mammalian) but not lower (yeast) eukaryotic cells. One interpretation is that lower eukaryotic cells lack a critical co-factor necessary for a glutamine-rich domain. This reasoning is counter-intuitive because many native yeast activator proteins contain glutamine-rich domains. Here, we have investigated the activity of a glutamine-rich GAL4-Sp1 domain A (Sp1A) hybrid protein in yeast Saccharomyces cerevisiae. We show that GAL4-Sp1A activated a GAL1-lacZ reporter by more than 200-fold over basal when the reporter was carried on a 2mu vector. The generality of the Sp1A results is supported by our finding that yeast glutamine-rich domains from HAP2 and MCM1 are also transcriptionally active in S. cerevisiae. Interestingly, we found that glutamine-rich domains are considerably less potent when responsive promoters (i.e. GAL1-lacZ) are integrated into yeast chromosome. Thus our results segregate the inherent transcriptional activity of a glutamine-rich domain in yeast S. cerevisiae from its apparent lack of activity when assayed on chromosomally embedded promoters.

CCAAT-Binding Factor↗
PubMed1998

Human male germ cell tumor resistance to cisplatin is linked to TP53 gene mutation.

Male germ cell tumors (GCTs) are uniquely sensitive to cisplatin-based chemotherapy, with more than 90% of newly diagnosed cases cured. The underlying cause for resistance to treatment in 20-30% of metastatic lesions remains to be identified. Unlike other solid tumors, no mutations in the TP53 gene have been identified to date in random panels of GCT specimens, which could account for the exquisite sensitivity of these tumors to genotoxic insult. However, in a panel of resistant GCTs that did either not respond to cisplatin-based chemotherapy or subsequently relapsed and resulted in the death of the patient, we have now identified a subset of tumors to contain TP53 mutations within exons 6-9. A cell line derived from one of these tumors (228A) displayed the same TP53 mutation as the tumor specimen, expressed only mutant TP53 mRNA, and exhibited a relative resistance to cisplatin in vitro in comparison to a cell line (218A) derived from a responsive tumor with wild-type TP53. The resistant cell line displayed a much reduced apoptotic cell death and did not exhibit an induction of transcription of the p53-responsive genes WAF1 and MDM2 following cisplatin treatment, compared to that observed in the sensitive cell line. The levels of bax, an agonist of apoptosis, were found to be reduced in the resistant cell line. The simplest explanation for the resistance of this subset of GCTs that are resistant to cisplatin-based chemotherapy, is the inability of the cells to mount an apoptotic response following exposure due to a functionally inactivating mutation in the TP53 gene.

Antineoplastic Agents↗