Biomedical subjects

Q Yao

Publications and source records attributed to Q Yao.

At least 73 records · Page 4Linked to original sources

PubMed1997

Fusion of phospholipid vesicles induced by the ribosome inactivating protein saporin.

The single chain ribosome-inactivating protein Saporin-S6 (SO-6) induces the fusion of acid phospholipid vesicles. The extent of fusion was measured by resonance energy transfer assay between the N-(7-nitro-2-1,3-benzoxadiazol-4-yl)-dimyristoylphosphatidyl lithanolamine (NBD-PE)(donor) and N-(lissamine rhodamine B sulphonyl)-diacylphoshaidylethanolamine (Rh-PE) (acceptor) incorporated in the vesicle. The saturated lipid/protein molar ratio is approx. 100:1. The time course of fusion of vesicles induced by the protein showed that the process was completed within 10 minutes, and the size of the particles in the medium was enlarged which conforms the occurrence of the fusion occurring. The fusion is temperature dependent and the liquid-crystalline state lipid is more apt to fuse than the gel phase lipid. The effect of SO-6 is also dependent on ionic strength and pH, high salt concentration and basic pH may abolish fusion, which suggests that both electrostatic and hydrophobic components may be involved in the process.

Hydrogen-Ion Concentration↗

PubMed1997

Association of the parainfluenza virus fusion and hemagglutinin-neuraminidase glycoproteins on cell surfaces.

We previously observed that cell fusion caused by human parainfluenza virus type 2 or type 3 requires the expression of both the fusion (F) and hemagglutinin-neuraminidase (HN) glycoproteins from the same virus type, indicating that a type-specific interaction between F and HN is needed for the induction of cell fusion. In the present study we have further investigated the fusion properties of F and HN proteins of parainfluenza virus type 1 (PI1), type 2 (PI2), and type 3 (PI3), Sendai virus (SN), and simian virus 5 (SV5) by expression of their glycoprotein genes in HeLa T4 cells using the vaccinia virus-T7 transient expression system. Consistent with previous results, cell fusion was observed in cells transfected with homotypic F/HN proteins; with one exception, coexpression of any combination of F and HN proteins from different viruses did not result in cell fusion. The only exception was found with the closely related PI1 HN and SN HN glycoproteins, either of which could interact with SN F to induce cell fusion upon coexpression as previously reported. By specific labeling and coprecipitation of proteins expressed on the cell surface, we observed that anti-PI2 HN antiserum coprecipitated PI2 F when the homotypic PI2 F and PI2 HN were coexpressed, but not the F proteins of other paramyxoviruses when heterotypic F genes were coexpressed with PI2 HN, suggesting that the homotypic F and HN proteins are physically associated with each other on cell surfaces. Furthermore, we observed that PI3 F was found to cocap with PI3 HN but not with PI2 HN, also indicating a specific association between the homotypic proteins. These results indicate that the homotypic F and HN glycoproteins are physically associated with each other on the cell surface and suggest that such association is crucial to cell fusion induced by paramyxoviruses.

Animals↗

PubMed1996

Flanking residues alter antigenicity and immunogenicity of multi-unit CTL epitopes.

CD8+ CTL responses constitute a critical component for vaccines developed to eliminate intracellular pathogens. One approach to achieve broad CTL diversity is based on genetically linking immunogenic peptides from multiple proteins to form poly-epitope Ags. To address the influence of flanking residues on class I Ag presentation, H-2d-restricted HIV-1 and mouse hepatitis virus CTL epitopes were linked via various spacer residues. The resulting 20 to 31 amino acid peptides were expressed using recombinant vaccinia viruses to monitor both CTL recognition and induction. Our data indicate that recognition is profoundly influenced by the nature of intervening residues forming carboxyl-terminal flanks for one and amino-terminal flanks for the other epitope. Flanking amino acids with aromatic (tyrosine), basic (lysine), and small aliphatic side chains (alanine) supported efficient CTL recognition of both epitopes. By contrast, acidic and helix breaking residues (glycine, proline) specifically inhibited recognition of the adjacent amino-terminal epitope. Flanking residues inhibitory for recognition were also detrimental for CTL induction, suggesting similar processing mechanisms in vitro and in vivo. The ratios of peptide-specific CTL precursors primed by the tandem epitopes varied up to 50-fold depending on molecular context. These data demonstrate a substantial role of carboxyl-flanking residues in governing the efficiency of class I Ag presentation both in vitro and in vivo. The dramatic influence of flanking residues on the hierarchy of CTL responses indicates that CTL induction by poly-epitope Ags can be optimized by strategically linking epitopes via selection of appropriate spacer residues.

Animals↗

PubMed1996

Peptides corresponding to the heptad repeat sequence of human parainfluenza virus fusion protein are potent inhibitors of virus infection.

It has been suggested that a conserved heptad repeat region in paramyxovirus fusion (F) proteins is essential for viral fusion activity (Buckland et al., 1992; Sergel et al., 1994; Reitter et al., 1995) We have studied synthetic peptides containing the heptad repeat regions derived from the F proteins of human parainfluenza virus type 2 (Pl2) and type 3 (Pl3) for their function as potential inhibitors of virus-induced cell fusion as well as their effects on spread of viral infection. Two peptides containing sequences of heptad repeat B, adjacent to the transmembrane domain of the F protein, were synthesized for both Pl2 and Pl3 F proteins. We observed that the longer peptides [34 amino acids (a.a.) for Pl2F or 35 a.a. for Pl3F] which extend from heptad repeat B to the transmembrane domain showed complete inhibition of cell fusion induced by the respective virus as well as by the vaccinia-expressed F and HN proteins. The 50% effective concentration to inhibit virus-induced cell fusion was 2.1 microM for Pl2 and 1.2 microM for Pl3. Moreover, the inhibitory effects of each peptide on virus-induced cell fusion were found to be virus type-specific. These peptides were found to also inhibit viral entry and to prevent plaque formation when mixed with the virus inoculum. Furthermore, the peptides caused a reduction in virus yield when assayed 48 hr after low m.o.i. infection and in the size of viral plaques when added to the overlay. Shorter peptides (21 a.a. for Pl2F or 24 a.a. for Pl3F) which correspond to the partial sequence of heptad repeat B for Pl2F and the entire heptad repeat B for Pl3F showed partial inhibition of Pl2- or Pl3-induced cell fusion. These results indicate that peptides containing the heptad repeat B sequence have the potential to inhibit virus-induced cell fusion, virus entry, and spread of virus infection.

Amino Acid Sequence↗

PubMed1996

Purification of porcine phospholamban expressed in Escherichia coli.

Phospholamban (PLB) is a small hydrophobic protein that regulates contractility in the heart. This membrane protein expressed in bacterial cells is resistant to purification by conventional strategies that have been used to isolate expressed soluble proteins. Therefore, in order to obtain both wild-type and mutant PLB proteins, we have amplified the PLB gene by the polymerase chain reaction from genomic DNA of porcine heart and inserted it into the pGEX-2T plasmid expression vector. In this vector, the gene product fused to glutathione S-transferase has been expressed in JM109 Escherichia coli cells. The expressed fusion protein was found associated predominantly with insoluble cellular constituents. However, most of the fusion protein was readily extracted with SDS. PLB was subsequently purified by a simple procedure consisting of isolation of the fusion protein by preparative SDS-gel electrophoresis, followed by a second electrophoretic separation of PLB after its cleavage from the fusion protein by thrombin. This isolation method yields 3-4 mg of PLB per liter of cells, in a form which is capable of functional interaction with the Ca-ATPase in reconstituted proteoliposomes.

Animals↗

PubMed1996

The JHM strain of mouse hepatitis virus induces a spike protein-specific Db-restricted cytotoxic T cell response.

Cytotoxic T lymphocyte (CTL) activity specific for mouse hepatitis virus (MHV) JHM strain (JHMV or MHV-4) was examined using in vitro stimulated spleen cells derived from immunized C57BL/6 (H-2b) mice. Target cells infected with JHMV were specifically recognized; however, analysis of target cells expressing the virus structural proteins via recombinant vaccinia viruses showed no recognition of the viral nucleocapsid (N), membrane (M), small membrane (sM) or haemagglutinin-esterase (HE) proteins. Only target cells expressing the virus spike (S) protein were recognized. Furthermore, the majority of CTL activity was restricted to target cells expressing the MHC class I Db molecules. Analysis of truncations and deletions of the S protein expressed by recombinant vaccinia viruses and peptide coated targets identified a single antigenic epitope, aa 510-518, conforming to the Db binding motif. These amino acids are contained within a domain deleted from a number of strains of mouse hepatitis virus, suggesting a role for immune pressure. To determine the potential for CTL specific for an epitope(s) within a non-structural protein, 24 CTL lines were established and characterized. No evidence for the induction of non-specific CTL activity or virus-specific CTL restricted to an epitope in a non-structural protein was obtained. These data indicate that the predominant CTL activity in JHMV-infected C57BL/6 mice is Db restricted and specific for a single epitope contained within aa 510-518 of the S protein.

Amino Acid Sequence↗

PubMed1996

Aggregation of phospholipid vesicles induced by the ribosome inactivating protein saporin.

Saporin-S6(SO-6) is a single chain ribosome inactivating protein, which can inhibit protein synthesis by inactivating eukaryotic ribosomes. The interaction of SO-6 with phospholipid model systems was described. SO-6 can specifically interact with negatively-charged phospholipid vesicles and it induces the aggregation of the lipid vesicles. The kinetics of the vesicle aggregation induced by SO-6 was studied. The saturating protein/lipid molar ratio was determined to be 1:100 based on titration experiments. The aggregation is dependent on the temperature in a range that was many times higher than the phase transition temperature of the phospholipid. The effect of pH on the aggregation of the vesicles can not be explained by simple deprotonation of side chain amino groups of the protein, and may be related to conformational changes of the protein. The maintenance of physiological ionic strength was required for the aggregation of SO-6 with vesicles. Finally, the interaction was prompted by Ca2+ ions, and was totally inhibited by EDTA, which suggests that SO-6 may interact with phospholipid vesicles in a Ca(2+)-dependent manner.

Calcium↗

PubMed1995

Transcription and translation of proinflammatory cytokines following JHMV infection.

Infection with JHMV results in the transcriptional activation of two host cell genes encoding proinflammatory cytokines, tumor necrosis factor (TNF)-alpha and interleukin (IL)-1 beta. Analysis of irradiated mice showed that IL-1 beta mRNA accumulation in the central nervous system was predominantly derived from the mononuclear infiltrate. By contrast, accumulation of TNF-alpha mRNA was unaffected by immunosuppression, suggesting that resident cells were the source of this cytokine. Infected mice were treated with anti-TNF antibody to determine if TNF-alpha contributed to either the encephalomyelitis or demyelination associated with JHMV infection. Surprisingly, neither the cellular infiltrate nor demyelination were affected. In vitro analysis showed that IL-1 beta but not TNF was secreted from JHMV infected macrophages. The absence of TNF secretion is due to a block in translation of the TNF mRNA which accumulates during infection.

Animals↗

PubMed1995

Differences in the role of the cytoplasmic domain of human parainfluenza virus fusion proteins.

We have investigated the roles of the cytoplasmic domains of the human parainfluenza virus type 2 (PI2) and type 3 (PI3) fusion (F) proteins in protein transport and cell fusion activity. By using the vaccinia virus-T7 transient expression system, a series of F protein cytoplasmic tail truncation mutants was studied with respect to intracellular and surface expression and the ability to induce cell fusion when coexpressed with the corresponding hemagglutinin-neuraminidase (HN) proteins. All of the cytoplasmic tail truncation mutants of PI2F were expressed at high levels intracellularly or on cell surfaces as measured by immunoprecipitation and cell surface biotinylation assays. In addition, when coexpressed with PI2HN, these truncation mutants of PI2F were all found to be essentially unimpaired in the ability to induce cell fusion as measured by a quantitative cell fusion assay. In contrast, surface expression and cell fusion activity were found to be eliminated by a mutant of PI3F in which the entire cytoplasmic tail was deleted, and the mutant protein appeared to be unable to assemble into a high-molecular-weight oligomeric structure. To further investigate whether there is a specific sequence requirement in the cytoplasmic tail of PI3F, a chimeric protein consisting of the PI3F extracellular and transmembrane domains and the PI2F cytoplasmic tail was constructed. This chimeric protein was detected on the surface, and it was capable of inducing cell fusion when expressed together with PI3HN, although the fusogenic activity was reduced compared with that of wild-type PI3F. These results demonstrate that although PI2 and PI3 viruses belong to the same parainfluenza virus genus, these viruses show marked differences with respect to functional requirements for the cytoplasmic tail of the F glycoprotein.

Amino Acid Sequence↗

PubMed1995

Tumor necrosis factor expression during mouse hepatitis virus-induced demyelinating encephalomyelitis.

Neutralizing anti-tumor necrosis factor alpha (TNF-alpha) antibody treatment of mice infected with the neurotropic JHMV strain of mouse hepatitis virus showed no reduction of either virus-induced encephalomyelitis or central nervous system demyelination. TNF-alpha-positive cells were present in the central nervous system during infection; however, TNF-alpha could not be colocalized with JHMV-infected cells. In vitro, TNF-alpha mRNA rapidly accumulated following JHMV infection; however, no TNF-alpha was secreted because of inhibition of translation. Both live and UV-inactivated virus inhibited TNF-alpha secretion induced by lipopolysaccharide. These data show that TNF-alpha is not secreted from infected cells and indicate that if contributes to either JHMV-induced acute encephalomyelitis nor primary demyelination.

Animals↗

PubMed1995

A new method to localize brain nuclei for surgery in extrapyramidal disease.

A CT localization method to be used with the Leksell stereotactic system was employed to locate the ventral lateral nucleus of the thalamus (VL) and globus pallidus (GP) for treating extrapyramidal disease (EPD). The study comprised 100 normal adults and 70 cases of EPD, and results in both populations were compared. The 70 cases of EPD were studied ventriculography, and 30 normal adults were studied my MRI. Results indicate the accuracy of localization of the primary targets was 94.25% with the CT localization method described herein and 88.9% in the control group. In conclusion, the new CT localization method is a valuable technique to locate brain nuclei and to replace ventriculography with stereotactic surgery in the treatment of EPD patients.

Adolescent↗

PubMed1995

Establishment and characterization of two cell lines derived from human transitional cell carcinoma.

Two continuous cell lines derived from the neoplastic urothelium had been maintained in culture for more than two years. The first cell line derived from the urothelium of a fusion papillocarcinoma on the left lateral wall of the bladder was designated as TBC-1 and grown in vitro for more than 150 generations. The second cell line derived from the urothelium of a papillocarcinoma in the left renal pelvis was designated as TPC-1 and grown in vitro for more than 100 generations. Characterization studies made on both cell lines showed that the cells had a rapid doubling time, exhibited multilayering and produced tumors in sc of BALB/c. Tumor nodules that produced sc of BALB/c kept similar cellular and pathological features to those of the primary biopsy specimens under light and electron microscopes. TPC-1 cell line exhibited a three-dimensional structure of transitional epithelium on the nylon-mesh disk which was coated with a layer of rat tail collagen. Both TBC-1 and TPC-1 cell lines formed colonies in soft agar. Their forming rates were 35% and 28%, respectively. The chromosome number of TBC-1 cells ranged from 17 to 84, with a modal number of 54; whereas that of TPC-1 cells ranged from 28 to 139, with a modal number of 49. The TBC-1 cells showed mutant p53 and ras p21 protein expression and expressed weakly ABH blood group isoantigens. Analysis of lactic dehydrogenase (LDH) isozymes showed the highest levels of LDH isozyme 4 sonicated cell lysates of TBC-1 and TPC-1 respectively.

Adult↗

PubMed1995

Fluorescence spectroscopic study of the interaction of adenine and nucleotide with trichosanthin.

Trichosanthin (TCS) is an N-glycosidase that can attack the 28s rRNA of the ribosome at a highly conserved adenine residue. The interactions of adenine and its derivative nucleotides with TCS are reported. The fluorescence of Trp 192 of TCS is sensitive to the proximity of adenine, and produces a marked red shift indicative of trytophan in a more hydrophilic environment. By contrast AMP and ATP quench the maximal emission at 328nm. The binding of the adenine and ATP with TCS result in lower tryptophan accessibility to the quencher acrylamide, but higher tryptophan accessibility to the quencher iodide, while AMP caused higher tryptophan accessibility to acrylamide, and lower tryptophan accessibility to iodide. Also, the binding of nucleotides induces tryptophan heterogeneity in the protein. These findings lead us to propose that binding of nucleotides and adenine base cause different microenvironmental changes of the tryptophan residue, and Trp 192 may be involved in the active site of TCS.

Adenine↗

PubMed1995

[The use of measuring serum soluble IL-2 receptor levels in cyclosporine-A treated lupus nephritis].

Twenty-one patients with Lupus Nephritis (LN) were treated with cyclosporine-A (CsA) for three months. Serum soluble interleukin-2 receptors (SIL-2R) were measured before and after treatment and correlated prospectively with serologic findings of LN activity. The patients had significantly higher IL-2R level before treatment (during LN flare) than after treatment (disease remission). A significant positive correlation was present between serum SIL-2R and proteinuria, and ANA as well, whereas the SIL-2R levels in LN patients were inversely correlated with serum levels of C3 and CH50. These observations suggest that serum SIL-2R level is an useful marker of disease activity in LN and may serve as a helpful adjunct in the management of this disorder.

Adult↗

PubMed1995

[Effect of tripchlorolide (T4) of Tripterygium wilfordii Hook on the production of endothelin-1 by endothelial cells of human umbilical vein].

Tripchlorolide (T4), a single active ingredient of Tripterygium wilfordii Hook was shown to possess both anti-inflammatory and immunosuppressive actions in experimental animals. The paper reports the effect of T4 on the production of endothelin-1 (ET-1) by endothelial cells of human umbilical vein. The cells were incubated with different doses of T4 (2.5, 5, 10 and 20 ng/ml) for 24 hours. By radioimmunoassay the results showed that T4 at concentration of 5 ng/ml significantly inhibited the production of ET-1 by endothelial cell in a dose dependent trend. The significance of T4 action was discussed.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed1995

[Proliferation of PBMC in patients with Sjögren's syndrome via CD2 pathway].

Primary Sjögren's syndrome is an autoimmune disease whose etiology is unknown. Recent studies show that there is T cell abnormalities in addition to B cell hyperreactivity. In order to better understand the immunoregulatory abnormalities of primary Sjögren's syndrome, cellular immunology has become the main focus of recent studies. As such, the function of CD2 and CD3 pathways constitutes an integral part of these studies. The proliferation of PBMC, non-adhesive cells (mainly T cells) and adhesive cells (mainly B cells) has been investigated in patients with primary Sjögren's syndrome and normal controls; the results show that the proliferation of PMBC and non-adhesive cells in patients is much lower than that in normal controls (P < 0.05), whereas there is no difference in that of adhesive cells between these two groups (P > 0.05). It is also found that the non-adhesive cells' abnormality can not be adjusted by adding adhesive cells of normal controls. In addition, it seems that there is a relationship between the proliferation of PBMC via CD2 pathway in patients and the positivity of anti-SSA and anti-SSB antibodies. However, the underlying mechanism behind the pathogenesis of primary Sjögren's syndrome has yet to be fully understood. This study warrants further research into gaining a better concept of the CD2 pathway at molecular levels.

Autoantibodies↗

PubMed1994

Protective effect of epicatechin, epicatechin gallate, and quercetin on lipid peroxidation in phospholipid bilayers.

Antioxidative effect of (-)-epicatechin,(-)-epicatechin gallate, and quercetin was examined by measuring the inhibition of lipid peroxidation in large unilamellar liposomes composed of egg yolk phosphatidylcholine (PC). These catechol-type flavonoids were stable in the liposomal suspension. They retarded the accumulation of PC-hydroperoxides depending on their concentrations when the suspension was exposed to an water-soluble radical initiator, 2,2'-azobis(2-amidinopropane)hydrochloride (AAPH). Their inhibitory effects lasted longer than that of alpha-tocopherol. When each flavonoid and alpha-tocopherol were mixed in the liposomes, epicatechin and epicatechin gallate disappeared in favor of alpha-tocopherol. Quercetin also decreased faster than alpha-tocopherol in the initial stage of incubation. Kinetic studies of the inhibition of radical chain oxidation of methyl linoleate in solution demonstrated that the rate constants for the inhibition of oxidation by these flavonoids (kinh) were 5-20 times smaller than that by alpha-tocopherol. It is likely that the flavonoids are localized near the surface of phospholipid bilayers suitable for scavenging aqueous oxygen radicals and thereby they prevent the consumption of lipophilic alpha-to-copherol. Epicatechin and epicatecin gallate gave smaller kinh values than quercetin. Voltammograms of these compounds showed that electron-donating ability of catechins was lower than that of quercetin. However, antioxidative effects of catechins were comparable to that of quercetin in AAPH-initiated peroxidation of the liposomal suspension. It is concluded that catechins and quercetin serve as powerful antioxidants against lipid peroxidation when phospholipid bilayers are exposed to aqueous oxygen radicals.

Antioxidants↗

PubMed1994

[Effects of tripchlorolide (T4) of Tripterygium wilfordii Hook on the proliferation of peripheral blood mononuclear cells of rheumatoid arthritis patients].

Tripchlorolide (T4) is a single active ingredient recently isolated from Tripterygium wilfordii Hook. The effects of T4 on the proliferation of peripheral blood mononuclear cells (PBMC) of rheumatoid arthritis (RA) patients and normal subjects were studied, RA patients and sex- and age-matched normal subjects (each 8 cases) were selected. PBMC were incubated with T4 of various doses (10, 20, 30 and 40 ng/ml) in the presence or absence of PHA for 72 hours. The results were as follows: T4 remarkably inhibited the proliferation of both PHA-stimulated and unstimulated PBMC of normal subjects and PHA-stimulated PBMC of RA patients as measured by MTT colormetric method. However, T4 showed a biphasic reaction in unstimulated PBMC of 3 RA patients. These results indicates that T4 would be useful in the therapy of RA. The significance of biphasic reaction occurring in RA patients needs to be further investigated.

Adult↗