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Biomedical subjects

X Qiu

Publications and source records attributed to X Qiu.

At least 91 records · Page 5Linked to original sources
PubMed1998

Antienvelope antibodies are protective against GBV-C reinfection: evidence from the liver transplant model.

An assay for the detection of antibody against the second envelope (E2) protein of GB virus type C (GBV-C) has been developed. Early reports suggested that this antibody was a marker of viral clearance, yet it is unknown whether anti-E2 is protective against further GBV-C infection. The primary aims were to determine (1) if posttransplantation immunosuppression alters the prevalence of anti-E2; and (2) if anti-E2 positivity pretransplantation protects against acquisition of GBV-C infection posttransplantation. Fifty-four recipients who underwent orthotopic liver transplantation for end-stage liver disease of nonviral etiologies were tested for GBV-C RNA using a PCR-based assay and anti-E2 antibodies by an enzyme-linked immunoassay. Anti-E2 was present in 35% and in 46% of patients pre- and posttransplantation, respectively. Anti-E2 positivity pretransplantation was strongly associated with anti-E2 positivity after transplantation (P < 0.001); 83% of patients with anti-E2 prior to transplantation remained anti-E2-positive after transplantation. A negative association between presence of GBV-C viremia and presence of anti-E2 was found in all patients tested either prior to or following transplantation (P=0.03). Acquisition of GBV-C infection was significantly lower in patients who were anti-E2-positive prior to transplantation (2/13) compared to those who were anti-E2-negative (12/26) (P=0.05). It is concluded that immunosuppression does not reduce the prevalence of anti-E2 after transplantation in those who are seroreactive prior to transplantation. Anti-E2 appears to be a neutralizing antibody whose presence at the time of liver transplantation protects against acquisition of GBV-C infection in the peritransplantation period.

Adult↗
PubMed1997

Active site cavity of herpesvirus proteases revealed by the crystal structure of herpes simplex virus protease/inhibitor complex.

Human herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) are responsible for herpes labialis (cold sores) and genital herpes, respectively. They encode a serine protease that is required for viral replication, and represent a viable target for therapeutic intervention. Here, we report the crystal structures of HSV-1 and HSV-2 proteases, the latter in the presence and absence of the covalently bound transition state analog inhibitor diisopropyl phosphate (DIP). The HSV-1 and HSV-2 protease structures show a fold that is neither like chymotrypsin nor like subtilisin, and has been seen only in the recently determined cytomegalovirus (CMV) and varicella-zoster virus (VZV) protease structures. HSV-1 and HSV-2 proteases share high sequence homology and have almost identical three-dimensional structures. However, structural differences are observed with the less homologous CMV protease, offering a structural basis for herpes virus protease ligand specificity. The bound inhibitor identifies the oxyanion hole of these enzymes and defines the active site cavity.

Binding Sites↗
PubMed1997

Transmembrane insertion of the colicin Ia hydrophobic hairpin.

Colicin Ia is a bactericidal protein that forms voltage-dependent, ion-conducting channels, both in the inner membrane of target bacteria and in planar bilayer membranes. Its amino acid sequence is rich in charged residues, except for a hydrophobic segment of 40 residues near the carboxyl terminus. In the crystal structure of colicin Ia and related colicins, this segment forms an alpha-helical hairpin. The hydrophobic segment is thought to be involved in the initial association of the colicin with the membrane and in the formation of the channel, but various orientations of the hairpin with respect to the membrane have been proposed. To address this issue, we attached biotin to a residue at the tip of the hydrophobic hairpin, and then probed its location with the biotin-binding protein streptavidin, added to one side or the other of a planar bilayer. Streptavidin added to the same side as the colicin prevented channel opening. Prior addition of streptavidin to the opposite side protected channels from this effect, and also increased the rate of channel opening; it produced these effects even before the first opening of the channels. These results suggest a model of membrane association in which the colicin first binds with the hydrophobic hairpin parallel to the membrane; next the hairpin inserts in a transmembrane orientation; and finally the channel opens. We also used streptavidin binding to obtain a stable population of colicin molecules in the membrane, suitable for the quantitative study of voltage-dependent gating. The effective gating charge thus determined is pH-independent and relatively small, compared with previous results for wild-type colicin Ia.

Bacterial Proteins↗
PubMed1997

Crystal structure of varicella-zoster virus protease.

Varicella-zoster virus (VZV), an alpha-herpes virus, is the causative agent of chickenpox, shingles, and postherpetic neuralgia. The three-dimensional crystal structure of the serine protease from VZV has been determined at 3.0-A resolution. The VZV protease is essential for the life cycle of the virus and is a potential target for therapeutic intervention. The structure reveals an overall fold that is similar to that recently reported for the serine protease from cytomegalovirus (CMV), a herpes virus of the beta subfamily. The VZV protease structure provides further evidence to support the finding that herpes virus proteases have a fold and active site distinct from other serine proteases. The VZV protease catalytic triad consists of a serine and two histidines. The distal histidine is proposed to properly orient the proximal histidine. The identification of an alpha-helical segment in the VZV protease that was mostly disordered in the CMV protease provides a better definition of the postulated active site cavity and reveals an elastase-like S' region. Structural differences between the VZV and CMV proteases also suggest potential differences in their oligomerization states.

Amino Acid Sequence↗
PubMed1997

Immunoassays to study prevalence of antibody against GB virus C in blood donors.

Immunoassays were developed to determine the seroprevalence of antibody against human GB virus C (GBV-C). The antigenic target in each assay was a 44.6-kDa glycosylated protein representing the first 315 amino acids encoded by the GBV-C E2 gene. Sera or plasma were assayed for E2 antibody using an anti-human EIA format in which antigen-coated polystyrene beads were reacted with sample, and bound antibody was detected by addition of enzyme labelled goat anti-human IgG. The presence of anti-E2 antibody was confirmed using a sandwich EIA format in which samples were reacted with antigen coated polystyrene beads, followed by addition of solution phase biotinylated antigen. Detection of antibody captured biotinylated E2 was accomplished by addition of enzyme-conjugated anti-biotin antibody. Antibody against the E2 antigen was detected in 7.4 and 7.8% of 500 sera and 500 plasma, respectively, from US volunteers donating to a Wisconsin blood center, and in approximately 10.7% of hepatitis and retrovirus marker-negative volunteer blood donors from a Missouri blood center. The rate in 1018 sera from US commercial donors at multiple US blood centers was 36.7%. These results indicated a relatively high prevalence of GBV-C exposure in US volunteer donors, and particularly in commercial donors. The clinical implication of the high exposure rate is unclear. These immunoassays are being combined with nucleic acid detection to assess prevalence of GBV-C world wide and to determine if GBV-C plays a role as an etiologic agent.

Animals↗
PubMed1997

[Effect of acetylcholine receptor antibody derived from myasthenia gravis on the rat central neuronal nicotinic acetylcholine receptor].

Immunoreaction between acetylcholine receptor antibody (AChRab) derived from myasthenia gravis (MG) and neuronal nicotinic acetylcholine receptor (AChR) and the action of AChRab on rat brain stem auditory transmission were investigated by immunocytochemistry and brain stem auditory evoked potential (BAEP) in a search for abnormalities indicating central nervous system (CNS) involvement in this disorder. 25 microliters of AChRab were injected into the ventricular system of rats once every 48 hours for periods of up to a week BAEP examination was taken before and after injection of AChRab. Immunocytochemical investigation of the rat brain sections provided evidence that the neuronal nicotinic AChR like immunoreactivity is widely distributed throughout the rat CNS such as cerebral cortex, hippocampus, motor neurons in brain stem, auditory nucleus cerebellar cortex, etc. Analysis of the immunocytochemical results showed that the distribution property of AChRab in rat CNS correlated well with known neuronal nicotinic AChR subtypes e.g., alpha 7 and alpha 4. BAEP inter peak latency was significantly prolonged in rats after injection of AChRab as compared with the control rats and the rats prior to injection of AChRab. The results indicated that cerebroventricular administration of AChRab not only cause dysfunction of brain stem auditory transmission but also leads to development of experimental autoimmune MG symptoms. It is concluded that AChRab influences the rat brain stem auditory transmission and it is suggested that the pathogenic effect of AChRab is not only limited at neuromuscular junction, but also involves the central neuronal nicotinic AChR.

Animals↗
PubMed1996

Induction of p21 mediated by reactive oxygen species formed during the metabolism of aziridinylbenzoquinones by HCT116 cells.

Aziridinylbenzoquinones are a group of antitumor agents that elicit cytotoxicity by generating either alkylating intermediates or reactive oxygen species. The mechanism of toxicity may not always, however, involve profound damage of cellular constituents, but may involve a cytostatic effect through interference with the cell cycle. In this context, we have examined the induction of the cell cycle inhibitor p21 (WAF1, CIP1, or sdi1), whose overexpression suppresses the growth of various tumor cells, in human tumor cells metabolizing 3,6-diaziridinyl-1,4-benzoquinone (DZQ) and its C2,C5-substituted derivatives: 2,5-bis-(carboethoxyamino) (AZQ) and 2, 5-bis-2(-hydroxyethylamino) (BZQ). Both DZQ and AZQ were effectively activated by HCT116 human colonic carcinoma cells; the activation of the former involved largely a dicoumarol-sensitive activity, whereas that of the latter appeared to be accomplished primarily by one-electron transfer reductases. BZQ was not a substrate for the dicoumarol-sensitive enzyme in HCT116 cells. Cellular activation of the first two quinones was associated with formation of oxygen-centered radicals as detected by EPR in conjunction with the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide. The redox transitions of DZQ involved hydroxyl radical formation and were strongly inhibited by catalase, whereas those of AZQ showed a strong superoxide anion component sensitive to superoxide dismutase. These signals were suppressed by N-acetylcysteine with concomitant production of a thiyl radical adduct. This suggests an effective electron transfer between the thiol and free radicals formed during the activation of these quinones. DZQ and AZQ induced significantly the expression of p21 in HCT116 cells, but a 10-fold higher concentration of AZQ was required to achieve the level of induction elicited by DZQ. BZQ had little effect on p21 expression. p21 induction at both mRNA and protein levels correlated with the inhibition of either cyclin-dependent kinase activity or cell proliferation. p21 induction elicited by the above quinones was inhibited by N-acetylcysteine, whereas the non-sulfur analog, N-acetylalanine, was without effect. Catalase and superoxide dismutase did not effect p21 induction by aziridinylbenzoquinones in HCT116 cells, thus suggesting that extracellular sources of oxygen radicals generated by plasma membrane reductases have no influence in the expression of this gene. Hydrogen peroxide, a product of quinone redox cycling, elicited an increase of p21 mRNA levels in HCT116 and K562 human chronic myelogenous leukemia cells. The latter lacks p53, one of the activators of p21 transcription, thus suggesting that p21 expression can be accomplished in a p53-independent manner in these cells. This study suggests that p21 induction is mediated by an increase in the cellular steady-state concentration of oxygen radicals and that the greater effectiveness in p21 induction by DZQ may be related to its efficient metabolism by NAD(P)H:quinone oxidoreductase activity in HCT116 cells.

Acetylcysteine↗
PubMed1996

Molecular basis for lack of expression of HLA class I antigens in human small-cell lung carcinoma cell lines.

HLA class I molecules present antigenic peptides to cytotoxic T lymphocytes and thus play an important role in immune surveillance of cells infected with virus or altered by malignant transformation. Immunochemical studies have demonstrated a marked deficiency or lack of expression of class I molecules on the surface of many different types of tumor cells. It is likely that this allows these cells to escape immune surveillance. In the present study, we examined the molecular basis for lack of expression of class I antigens in small-cell lung carcinoma cell lines. Our results demonstrate that these cell lines also lacked products of MHC-encoded proteasome subunit LMP2 and the putative peptide transporter TAP1. In contrast, LMP7 and TAP2 genes were expressed in these cell lines. Pulse-chase experiments showed that class I molecules were unstable and thus not transported to the cell surface from endoplasmic reticulum. Our results suggest that antigenic peptides were not available for binding to class I alpha chains due to lack of TAP1 and LMP2 gene products. Investigations of the regulatory mechanisms of TAP1 and LMP2 genes showed that the tumor cells lacked trans -regulatory nuclear protein(s), which binds to the interferon-gamma (IFN-gamma) response element (ISRE) in the TAP1, LMP2 bidirectional intergenic promoter. Treatment of tumor cells with IFN-gamma induced ISRE-binding nuclear protein(s) and resulted in expression of TAP1 and LMP2 genes with a concomitant increase in cell-surface expression of class I molecules. Our data provide credence for a role of TAP and LMP genes in immune response.

Base Sequence↗
PubMed1996

High-resolution structure of the diphtheria toxin repressor complexed with cobalt and manganese reveals an SH3-like third domain and suggests a possible role of phosphate as co-corepressor.

The crystal structure of diphtheria toxin repressor (DtxR) in complex with the corepressor Co2+ has been determined at 2.0 A resolution and in complex with Mn2+ at 2.2 A resolution. The structure of the flexible third domain could be determined at this high resolution. It appears to contain five antiparallel strands exhibiting a fold very similar to the SH3 domain. A superposition of 46 equivalent C alpha atoms of DtxR and alpha-spectrin SH3 resulted in an rms deviation of 3.0 A. The sequence identity is only 7%. This third domain of DtxR appears to have no interactions with the DNA binding domain nor with the metal binding domain of the repressor. Yet, flexibility in the region between the second and the third domain allows in principle significant conformational changes such as might occur upon DNA binding. The two metal binding sites in the second domain have been unraveled in considerable detail. Metal binding site 1 was well occupied in both the cobalt and manganese structures and showed a surprising sulfate ion as ligand. The sulfate was proven beyond doubt by the high peak at its position in a selenate versus sulfate difference Fourier. The presence of the intriguing sulfate ion at such a crucial position near the metal corepressor suggests the possibility that under physiological conditions phosphate may act as a "co-corepressor" for this class of metal-regulated DNA binding proteins in Corynebacteria, Mycobacteria, and related organisms. The second metal binding site is significantly different in these two DtxR structures. In the 2.0 A cobalt structure, the site is not occupied by a metal ion. In the 2.2 A manganese structure the site is well occupied, at approximately the same position as observed previously in cadmium DtxR. The ligands are Glu105, His106, the carbonyl oxygen of Cys102, and a water molecule. The reasons for differential occupancy of this site in different structures are intriguing and require further investigations.

Amino Acid Sequence↗
PubMed1996

Unique fold and active site in cytomegalovirus protease.

Human herpesviruses are responsible for a variety of diseases. They are divided into three subfamilies: alpha includes herpes simplex viruses (HSV-1 and HSV-2) and varicella-zoster virus (VZV); beta includes cytomegalovirus (CMV) and human herpesvirus-6 (HHV-6); and gamma includes Epstein-Barr virus (EBV). Each virus encodes a serine protease that is essential for its replication and is a potential target for therapeutic intervention. Human CMV is a ubiquitous opportunistic pathogen that can result in life-threatening infections in congenitally infected infants, immunocompromised individuals and immunosuppressed cancer or transplant patients. Here we report the crystal structure of human CMV protease at 2.5 angstroms resolution. The structure reveals a fold that has not been reported for any other serine protease, and an active site consisting of a novel catalytic triad in which the third member is a histidine instead of an aspartic acid, or possibly a catalytic tetrad consisting of a serine, two histidines and an aspartic acid. An unusual dimer interface that is important to the protease activity has also been identified.

Amino Acid Sequence↗
PubMed1996

Identification and characterization of a C(K/R)TC motif as a common epitope present in all subtypes of hepatitis B surface antigen.

The a determinant of hepatitis B surface antigen (HBsAg) is the most critical determinant for both diagnosis and immunoprophylaxis of the hepatitis B virus. We have used synthetic peptides and an anti-a mAb to identify a peptide sequence corresponding to amino acid residues 117 to 128 of HBsAg as an antigenic epitope contributing to the a determinant. Compared to the native protein HBsAg, the cyclic form of the peptide (aa 117-128) is only 20-fold less effective, whereas the linear form of the peptide is 160-fold less effective in the inhibition of mAb binding to HBsAg. Based on these results, we have postulated a previously unidentified disulfide bond between residue Cysl21 and Cysl24. Individual substitution of amino acids in the peptide (aa 117-128) with alanine identified three residues Cys121, Thr123, and Cys124 as the most critical residues for mAb recognition. Substitution of alanine for any one of the three residues caused a substantial loss in binding free energy (greater than 4.5 kcal/mol). Sequence analysis indicated that the C(K/R)TC motif is highly conserved among 100 subtypes and mutants of HBsAg isolates. Collectively, these results show that the cyclic C(K/R)TC motif is an essential part of the a determinant of HBsAg. Synthetic peptides containing the C(K/R)TC motif are potentially useful as alternative hepatitis B vaccines and as diagnostic reagents for the detection of the hepatitis B virus.

Amino Acid Sequence↗
PubMed1996

PO149, a new member of pollen pectate lyase-like gene family from alfalfa.

PO149 is a low-copy-number gene expressed in the late stages of pollen development. The promoter region contains no similarities in DNA sequence to those of other pollen-specific genes, except for a tobacco sequence (AAATGA), which occurs four times in this alfalfa gene and much further upstream than in tobacco. Four distinct TATA boxes were detected in the promoter with the distal and proximal TATA boxes being separated by a spacer of 269 nucleotides. Hairpin loop structures were found in the 5'- and 3'-untranslated regions of PO149 mRNA. The coding region of PO149 is interrupted by two introns and encodes a putative prepeptide of 450 amino acids with homology to pollen pectate lyase-like proteins and pollen allergens. The coding region also contains sequences characteristic of both a signal peptide and a nuclear localization signal.

Allergens↗
PubMed1996

Polymorphism in both X and Y box motifs controls level of expression of HLA-DRB1 genes.

The HLA class II antigens of the human major histocompatibility complex play an important role in immune response. The quality of the immune response is determined not only by polymorphisms in their coding region, but also by the level of their cell-surface expression which affects, for example, the extent of T-cell activation. We have previously described allelic polymorphisms in the upstream regulatory regions of HLA-DRB genes, which affected DNA-protein interactions and resulted in significantly different promoter strengths. In the present study, we investigated the effect of polymorphisms in the X and Y box motifs on the transcriptional activity of DRB1 gene promoters in the DR1, DR51, and DR53 haplotype groups. We used normal, chimeric, and mutated DRB promoters and compared their relative abilities to initiate transcription of the CAT reporter gene in human B-cell lines. The results show that polymorphisms in both the X1 and Y box motifs play a dominant role in the promoter strength. In the gel mobility shift assay, we observed differential ability of nuclear proteins that bind to the polymorphic X1 and Y box elements. The results in the present study confirm earlier data in that the nucleotide variation in the X1 box affects the level of expression of DRB1 genes. In addition, the present data demonstrate that polymorphism in the Y box, which affects the inverted CCAAT sequence, also plays a dominant role in the transcriptional activity of DRB1 promoters.

Base Sequence↗
PubMed1996

Surgical treatment of bone tumors in conjunction with microwave-induced hyperthermia and adjuvant immunotherapy. A preliminary report.

OBJECTIVE: To develop an alternative approach in conjunction with microwave-induced hyperthermia. PATIENTS AND METHODS: Thermotherapy with microwave intracorporeal irradiation was used to treat 73 patients with bone tumors. The series was composed of 58 patients with malignant tumors and 15 with benign tumors: most of tumors occurred about knee joints (53/73 = 72.6%). The surgical procedure included separating the tumor bearing segment from surrounding normal tissues with a safe margin, cooling the normal tissues including the neurovascular bundle and the intraarticular structures with a water circulation system, while heating the tumor with the antenna array of a microwave system and providing an adequate soft-tissue cover for the dead bone. Postoperatively, an immune therapy regimen was carried out regularly. The patients' immunologic functions were monitored by assay of the subpopulation of T cells, IL-2 and sIL-2 R (soluble IL-2 receptor). RESULTS: Follow-up varied from 3 to 38 months (mean 19 months). Excluding 3 patients with malignancy in the vertebrae treated for palliation, 70 were evaluated according to oncological and orthopedic criteria. Five patients had local recurrence and required amputation. The remaining 65 had excellent local control. In 6 of the 55 patients with malignancy of the extremities, lung metastasis occurred one to two years after surgery. The oncological results were similar to those obtained by other limb-saving procedures. Pathological fracture occurred at devitalized bone in 5 patients. In 72.5% of the patients (29 of 40 tumor-free cases followed more than one year), knee joints functioned well, being stable and painless with almost full range of motion. Single photon emission computered tomography (SPECT) for 16 patients revealed revascularization of the devitalized tumor bearing bone segment could accomplish in one year or more. The immune states were improved in various extends after thermotherapy plus immunotherapy in the majority of patients. CONCLUSION: These results show that the use of microwave hyperthermia and adjuvant immunotherapy in conjunction with the surgical treatment of bone tumors can be considered a definitive procedure, which is safe and well-tolerated. The oncological and orthopedic results are encouraging.

Adolescent↗
PubMed1996

Implantation of autologous skin fibroblast genetically modified to secrete clotting factor IX partially corrects the hemorrhagic tendencies in two hemophilia B patients.

OBJECTIVE: To examine the safety and effects of gene therapy for hemophilia B by implantation of autologous fibroblasts genetically modified to secrete clotting factor IX (hFIX). PATIENTS AND METHODS: Two hemophilia B patients LD and LW were selected from one family to accept gene transfer study. The hFIX protein of both patients were about 100 ng/ml plasma and hFIX activity was about 2%. The autologous skin fibroblasts of the two patients were genetically modified by retrovirus-mediated gene transfer with XL-IX and N2CMVIX vector (HBSF-IX). Human hFIX protein was measured by ELISA, hFIX activity was measured by one-stage clotting assay and barium citrate sorbent method. hFIX inhibitor was assayed by Bethesda methods. Human hFIX cDNA was detected by PCR. HBSF-IX cells were mixed with collagen for injection after safety assessments. RESULTS: The HBSF-IX cells from the two patients secreted hFIX at high levels in vitro. After implantation of autologous HBSF-IX cells, no treatment-related side effects were observed. Plasma hFIX protein in both patients increased over 2 folds after several injections of HBSF-IX cells and persisted for more than 420 days. Blood clotting activity increased significantly in both patients, hemorrhagic tendencies have been partially corrected after treatment. Further elevation of hFIX can be achieved by repeating the same treatment 420 days later in Patient LD. CONCLUSIONS: Implantation of autologous fibroblast genetically modified to secrete human hFIX offers a simple, safe and effective approach to gene therapy of hemophilia B.

Adolescent↗
PubMed1996

[Determination of puerarin in gegen ginlian tablets by RP-HPLC].

A quantitative method was developed for the determination of puerarin in Gegen Qinglian Tablets by reversed phase HPLC. Chromatographic conditions included column ODS-C18, column temperature: 35 C, UV detector: 250 nm, mobile phase: EtOH-H2O (23:77), flow rate: 1 ml/min. The number of theoretical plates calculated for puerarin peak was no less than 2000. The standard curve was linear in the concentration range of 5-80 micrograms/ml, and the correlation coefficient was 0.9999. The average recovery and the relative standard deviation were 97.6% and 1.8% respectivily.

Chromatography, High Pressure Liquid↗