Search PubMed⌕ Search

Biomedical subjects

X Yu

Publications and source records attributed to X Yu.

At least 397 records · Page 22Linked to original sources
PubMed1993

Mutations in the N-terminal region of human immunodeficiency virus type 1 matrix protein block intracellular transport of the Gag precursor.

The matrix domain of human immunodeficiency virus type 1 Gag polyprotein was studied for its role in virus assembly. Deletion and substitution mutations caused a dramatic reduction in virus production. Mutant Gag polyproteins were myristoylated and had a high affinity for membrane association. Immunofluorescence staining revealed a large accumulation of mutant Gag precursors in the cytoplasm, while wild-type Gag proteins were primarily associated with the cell surface membrane. These results suggest a defect in intracellular transport of the mutant Gag precursors. Thus, in addition to myristoylation, the N-terminal region of the matrix domain is involved in determining Gag protein transport to the plasma membrane. Wild-type Gag polyproteins interacted with and efficiently packaged mutant Gag into virions. This finding is consistent with the hypothesis that intermolecular interaction of Gag polyproteins might occur in the cytoplasm prior to being transported to the assembly site on the plasma membrane.

Amino Acid Sequence↗
PubMed1993

Vpx of simian immunodeficiency virus is localized primarily outside the virus core in mature virions.

Human immunodeficiency virus type 2 and the related simian immunodeficiency virus (SIV) contain a unique regulatory gene, vpx. The Vpx protein is packaged in mature virions and is required for efficient viral replication in peripheral blood lymphocytes and macrophages. To study the localization of Vpx in mature virions, conical and bar-shaped core structures of SIV from macaques (SIVmac) were purified. The SIVmac core has a density of approximately 1.25 g/cm3, compared with 1.16 g/cm3 for an intact virion. The relative proportions of major capsid protein (p27) and reverse transcriptase activity were similar for intact virions and core structures. The majority of matrix protein (p14) was removed from the purified core structure, suggesting its association with the viral membrane. Similarly, most of the Vpx protein was absent from the purified core structure. This result suggests that as with the matrix protein, the majority of Vpx proteins are localized outside the virus core. The localization of Vpx suggests that it may be involved in virus entry such as penetration or uncoating.

Blotting, Western↗
PubMed1993

Dentin bonding system shelf life and bond strength.

The purpose of this study was to assess the shelf life of dentin bonding systems by means of bond strength testing. The systems evaluated were Mirage-Bond, All-Bond and Tenure. Specimens were tested immediately, and 1, 2, 3, 4, 5 and 12 months after opening the kits. Flat dentin surfaces were created on 210 extracted human molars that had been stored in saline. Ten specimens were prepared for each dentin bonding system at each shelf time. Each material was prepared and placed following the manufacturer's instructions. Prisma MicroFine resin composite was then placed. Shear bond strength testing was conducted using a universal testing machine. A two-way ANOVA indicated a significant interaction between bonding system and storage time (F = 2.28, P = 0.01). Multiple comparison tests indicated statistically significant bond strength differences by storage time for All-Bond and Mirage-Bond. In addition, a comparison of bonding systems at each storage time revealed significant differences at three of the seven times. These results suggest that shelf life for two of the three dentin bonding systems may be significantly shorter than currently presumed.

Analysis of Variance↗
PubMed1993

Protective effect of Salmonella typhimurium Re-LPS antiserum.

There is increasing evidence that antiserum to LPS can reduce the morbidity and mortality of Gram-negative bacterial infections. We report that antiserum to S. typhimurium SL 1102 (Re mutant strain) has excellent cross-protective activity. Antisera to these bacteria and to their Re-LPS were prepared in rabbits immunized with heat-killed bacterial cells and with Re-LPS preparations. Re-LPS antibody titers were tested by immune hemagglutination (IHA) and by ELISA. These antisera were found to be capable of protecting ICR mice against lethal challenge with S-type S. typhimurium 50014 (100 LD50), E. coli 0111:B4 (32 LD50), Pseudomonas aeruginosa (8 LD50) and Klebsiella pneumonia (16 LD50). We used gastric mucin (5%) as a virulence enhancing agent for the bacterial challenges. The IHA titer of antibody to the homologous strain proved to be much higher than that of other strains. Protection by the sera was 75-100%, 25% and 0% when injected 24, 48 or 72 h before the challenge, respectively. The survival rate was more than 50% when the antiserum was injected 5-7 h after challenge with a ten-fold or higher lethal dose. No protection was observed against such high challenge when the serum was injected later. According to these results, Re-LPS antiserum provides better protection than S-type specific antisera.

Animals↗
PubMed1993

[Fermentation of engineered strain producing cholera toxin B subunit].

Studies indicate that the cholera B subunit could be synthesized efficiently in corn steep liquor by engineered E. coli strain MM2. We have also determined the optical density, pH and the yield of B subunit kinetically during fermentation. Corn steep liquor medium has advantages in low cost, simplicity in technological process and high yield of B subunit which can reaches 40 micrograms/ml in 50L fermentative tank.

Cholera Toxin↗
PubMed1993

Shear strength and microleakage of light-cured glass ionomers.

Bond to dentin and marginal integrity of three light activated glass ionomer cements were evaluated, and compared to a traditional chemical-cured glass ionomer cement. Results indicated no significant difference in shear bond strength among the glass ionomer cements tested. However, there was a significant difference among glass ionomer cements in bond strength variability, with coefficients of variation ranging from 55 to 102%. A discrepancy between manufacturers' instructions and optimum curing times is one possible explanation for these results. There were also no significant differences in microleakage among glass ionomer cements. Most samples exhibited minimal or no microleakage.

Analysis of Variance↗
PubMed1992

Structural data suggest that the active and inactive forms of the RecA filament are not simply interconvertible.

We have used electron microscopy to examine the two major conformational states of the helical filament formed by the RecA protein of Escherichia coli. The compressed filament, formed in the absence of a nucleotide cofactor either as a self-polymer or on a single-stranded DNA molecule, is characterized in solution by about 6.1 subunits per turn of a 76 A pitch helix, and appears to be inactive with respect to all RecA activity. The active state of the filament, formed with ATP or an ATP analog on either a single or double-stranded DNA substrate, has about 6.2 subunits per turn of a 94 A pitch helix. Measurements of the contour length of RecA-covered single-stranded DNA circles in ice, formed in the absence of nucleotide cofactor, indicate that each RecA subunit binds five bases, in contrast to the three bases or base-pairs per subunit in the active state. The different stoichiometries of DNA binding suggests that the two polymeric forms are not interconvertible, as has been suggested on biochemical grounds. A three-dimensional reconstruction of the inactive state shows the same general features as the 83 A pitch filament present in the RecA crystal. This structural similarity and the fact that the crystal does not contain ATP or DNA suggests that the crystal structure is more similar to the compressed filament than the active, extended filament.

Adenosine Triphosphate↗
PubMed1992

ATPase activity of partially purified P-glycoprotein from multidrug-resistant Chinese hamster ovary cells.

In vitro studies of multidrug-resistant cell lines have shown that a membrane protein, the P-glycoprotein, is responsible for resistance to a wide range of structurally and functionally dissimilar anti-cancer drugs. The amino-acid sequence of P-glycoprotein (Pgp) indicates two consensus sequences for ATP binding and the purified protein has been reported to possess a low level of ATPase activity. As part of our goal to further characterize the ATPase activity of P-glycoprotein, we have developed a procedure for rapid partial purification of the protein in a highly active form. Plasma membrane vesicles from multidrug-resistant CHRC5 Chinese hamster ovary cells were subjected to a two-step procedure involving selective extraction with different concentrations of the zwitterionic detergent CHAPS. The resulting extract was enriched in P-glycoprotein (around 30% pure) and displayed an ATPase activity (specific activity 543 nmol mg-1 min-1) that was not found in a similar preparation from drug-sensitive cells. The ATPase specific activity was over 10-fold higher than that previously reported for immunoprecipitated Pgp and 280-fold higher than that of immunoaffinity-purified Pgp. This ATPase activity could be distinguished from that of other ion-motive ATPases and membrane-associated phosphatases and is, thus, proposed to be directly attributable to P-glycoprotein. Optimal P-glycoprotein ATPase activity required Mg2+ at an ATP: Mg2+ molar ratio of 0.75:1 and the apparent Km for ATP was 0.88 mM. P-Glycoprotein ATPase could be completely inhibited by vanadate and by the sulfhydryl-modifying reagents N-ethylmaleimide, HgCl2 and p-chloromercuribenzenesulfonate. Certain drugs and chemosensitizers, including colchicine, progesterone, nifedipine, verapamil and trifluoperazine, produced up to 50% activation of P-glycoprotein ATPase activity.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

Elevated expression of monocyte chemoattractant protein 1 by vascular smooth muscle cells in hypercholesterolemic primates.

Atherosclerosis is marked by an overt inflammatory infiltrate, with enhanced recruitment of monocytes/macrophages observed in both human and experimental atherosclerosis. We previously determined that monocyte chemoattractant protein 1 (MCP-1) accounts for virtually all of the chemotactic activity produced by vascular (aortic) smooth muscle cells in culture. We now report that arteries from a primate model of atherosclerosis with dietary-induced hypercholesterolemia exhibit increased levels of MCP-1 mRNA expression in vivo, whereas their normal counterparts demonstrate minimal MCP-1 expression. Furthermore, immunohistochemistry and in situ hybridization clearly indicate that the expression of MCP-1 protein and mRNA is in the smooth muscle cells of the medial layer of the artery and in monocyte-like and smooth muscle-like cells found in the overlying intimal lesion. These studies indicate that one of the responses to dietary hypercholesterolemia is the expression of MCP-1 by vascular smooth muscle cells. This expression, when augmented with other cellular and molecular factors, could significantly contribute to the recruitment of monocytes/macrophages to the vessel wall.

Animals↗
PubMed1992

Inhibition of GABA-gated chloride channel function by arachidonic acid.

The effects of arachidonic acid and its metabolites on gamma-aminobutyric acid (GABAA) receptor function were determined in rat cerebral cortical synaptoneurosomes. Incubation of synaptoneurosomes with phospholipase A2 decreased muscimol-induced 36Cl- uptake. Arachidonic acid, the major unsaturated fatty acid released by phospholipase A2, also inhibited muscimol-induced 36Cl uptake. Similar inhibition was obtained with other unsaturated fatty acids (docosahexaenoic, oleic) but not with saturated fatty acids (stearic, palmitic). The effect of arachidonic acid on muscimol responses was inhibited by bovine serum albumin (BSA), and BSA enhanced muscimol responses directly, indicating the generation of endogenous arachidonic acid in the synaptoneurosome preparation. The generation of endogenous arachidonic acid was also indicated by the ability of 2 inhibitors of arachidonic acid metabolism, indomethacin and nordihydroguaiaretic acid (NDGA), to inhibit muscimol-induced 36Cl uptake. We conclude that arachidonic acid probably has both direct and indirect actions on muscimol responses since both enzyme inhibitors inhibited muscimol responses but did not prevent the effect of exogenously added arachidonic acid. In additional experiments, arachidonic acid metabolites generated by cyclooxygenase, prostaglandins D2, E2 and F2 alpha, each decreased muscimol responses; prostaglandins F2 alpha was the most potent inhibitor. Since the unsaturated fatty acids and their metabolites are most susceptible to peroxidation, a generating system of superoxide radicals was tested on muscimol responses. A combination of xanthine and xanthine oxidase inhibited muscimol-induced 36Cl uptake in a concentration-dependent manner. We propose that the inhibition of GABAA neurotransmission by arachidonic acid and its metabolites can lead to increased neuronal excitability. This mechanism may play an important role in the development of neuronal damage following seizures or cerebral ischemia.

Animals↗
PubMed1992

Human papillomavirus (HPV) infection, HIV infection and cervical cancer in Tanzania, east Africa.

The presence of HPV-DNA was determined in tumor biopsies of cervical-cancer patients and in cervical swabs of non-cancer patients from Tanzania, East Africa, by Southern blot hybridization and/or PCR. HPV types 16 and 18 were detected in 38% and 32%, respectively, of 50 cervical-carcinoma biopsies. A consensus primer PCR capable of detecting a broad spectrum of HPV types revealed the presence of HPV-DNA in 59% of 359 cervical swabs of non-cancer patients. Type-specific PCR showed that types 16 and 18 accounted for 13.2% and 17.5%, respectively, of all HPV infections. Therefore we concluded that HPV 18 is more prevalent in Tanzania than in any other geographical location so far reported. The strongest risk factors for the presence of any HPV-DNA in the 359 female non-cancer patients were young age and HIV infection. The epidemiology of HPV types 16 and 18 was found to differ from that of other HPV types, being associated in univariate analysis with trichomonas vaginalis infection, martial status (single/divorced), age at first intercourse, and young age at menarche. However, young age at menarche accounted for most of the effects of all other, variables in multivariate analysis. Of the non-cancer patients, 12.8% had antibodies against HIV I (no patient being severely symptomatic), and HIV infection was highly correlated with the presence of HPV-DNA, especially types 16 and 18. While HPV-DNA of any type was detectable 1.4-fold more often in HIV-positive patients than in HIV-negative patients, evidence of an infection with HPV types 16 or 18 was found 2.2-fold more often in the HIV-positive patients. The HIV-positive women did not show an increased rate of cervical cytological abnormalities as assessed by PAP staining of a single cervical smear, the overall rate of abnormalities being 2.8%. Furthermore, the age-adjusted prevalence of HIV antibodies was found to be considerably lower in 270 cervical-carcinoma patients (3% HIV-positive) in comparison with non-cancer patients. Thus there was no association observable between the prevalence of HIV infections and the frequency of cervical cytological abnormalities or cervical cancer in the setting of this cross-sectional study.

Cervix Uteri↗
PubMed1992

Direct visualization of dynamics and co-operative conformational changes within RecA filaments that appear to be associated with the hydrolysis of adenosine 5'-O-(3-thiotriphosphate).

Highly co-operative structural transitions and conformational changes can be directly observed in bundles of filaments formed by the RecA protein of Escherichia coli. These filaments have been formed with RecA protein, DNA and the ATP analog adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma-S). We show that while ATP-gamma-S has frequently been called non-hydrolyzable in the RecA literature, it is hydrolyzed by RecA with a kcat of about 0.01 to 0.005 min-1. This rate of ATP-gamma-S hydrolysis is significant to structural studies conducted on a time scale of hours. It has been shown that RecA subunits may be seen in different conformations within one particular form of RecA bundle. We now show that additional structural transitions take place within these bundles when they are allowed to incubate at 37 degrees C for several hours. This is the same time scale on which ATP-gamma-S is being hydrolyzed, and the suggestion that the observed structural transitions arise from the hydrolysis of ATP-gamma-S is supported by the fact that when the hydrolysis of ATP-gamma-S is inhibited (at 4 degrees C), the transitions are not observed. The transitions that occur are highly co-operative, with filaments as a whole changing their state over lengths of several thousand Angstroms. This shows that RecA filaments have an internal co-operativity, and we suggest that this is important to their function in vivo. The motions of subunits that we visualize appear to be mainly rotational, and this can be used to infer information about the motions of RecA subunits associated with the RecA ATPase that occurs during the DNA strand exchange reaction.

Adenosine Triphosphate↗
PubMed1992

The matrix protein of human immunodeficiency virus type 1 is required for incorporation of viral envelope protein into mature virions.

Accumulating evidence suggests that the matrix (MA) protein of retroviruses plays a key role in virus assembly by directing the intracellular transport and membrane association of the Gag polyprotein. In this report, we show that the MA protein of human immunodeficiency virus type 1 is also critical for the incorporation of viral Env proteins into mature virions. Several deletions introduced in the MA domain (p17) of human immunodeficiency virus type 1 Gag polyprotein did not greatly affect the synthesis and processing of the Gag polyprotein or the formation of virions. Analysis of the viral proteins revealed normal levels of Gag and Pol proteins in these mutant virions, but the Env proteins, gp120 and gp41, were hardly detectable in the mutant virions. Our data suggest that an interaction between the viral Env protein and the MA domain of the Gag polyprotein is required for the selective incorporation of Env proteins during virus assembly. Such an interaction appears to be very sensitive to conformational changes in the MA domain, as five small deletions in two separate regions of p17 equally inhibited viral Env protein incorporation. Mutant viruses were not infectious in T cells. When mutant and wild-type DNAs were cotransfected into T cells, the replication of wild-type virus was also hindered. These results suggest that the incorporation of viral Env protein is a critical step for replication of retroviruses and can be a target for the design of antiviral strategies.

Animals↗
PubMed1992

The C terminus of human immunodeficiency virus type 1 matrix protein is involved in early steps of the virus life cycle.

Deletion mutations at the C terminus of the matrix (MA) protein of human immunodeficiency virus type 1 (HIV-1) were generated by site-directed mutagenesis. The resultant mutant viruses had a severe defect in virus infectivity. This defect did not involve late steps of the virus life cycle, as the synthesis and processing of the Gag polyprotein and the assembly and release of mutant virions were not greatly affected. The incorporation of viral proteins and the viral RNA genome was similar for mutant and wild-type virions. In contrast, the early steps of the virus life cycle were severely affected, as the synthesis of viral DNA postinfection was dramatically reduced in mutant-virus-infected cells. One stretch of amino acids that was deleted in one of the mutants has significant homology with a region in VP1 of the picornavirus family. This region of VP1 is presumably involved in poliovirus penetration into cells. These results suggest that in addition to its functional role in virus assembly, the MA protein of HIV-1, and possibly of other retroviruses, plays an important role in virus entry.

Amino Acid Sequence↗
PubMed1992

Cellular regulation of the benzodiazepine/GABA receptor: arachidonic acid, calcium, and cerebral ischemia.

The effects of cellular mediators that contribute to ischemia-induced neuronal degeneration on gamma-aminobutyric acid (GABAA)-receptor function were studied. In vitro, phospholipase A2 (PLA2) inhibited muscimol-induced 36Cl- uptake in cerebral cortical synaptoneurosomes. The major hydrolysis product of PLA2 activity, arachidonic acid, also inhibited GABA-mediated 36Cl- uptake. The unsaturated nature of arachidonic acid makes it (and its metabolites) highly susceptible to peroxidation by oxygen radicals. Incubation of synaptoneurosomes with the superoxide radical-generating system, xanthine and xanthine oxidase, decreased muscimol-induced 36Cl- uptake, suggesting that the peroxidation of arachidonic acid and/or its metabolites interferes with GABAA-receptor function. Another factor involved in ischemia-induced neuronal degeneration is an increase in intracellular Ca2+. Calcium also inhibited GABA-mediated 36Cl- flux, consistent with its ability to activate PLA2. In contrast, Mg2+, which blocks Ca2+ channels, enhanced muscimol-induced 36Cl- uptake, consistent with its neuroprotective effects. Each of these cellular processes is activated during cerebral ischemia and can lead to neuronal degeneration. We used a model of transient forebrain ischemia in gerbils to determine if GABAA-receptor regulation is altered in vivo at a time when CA1 hippocampal cells have degenerated. Four days after a 5 minute bilateral carotid artery occlusion, receptor autoradiography was performed to measure the binding of [35S]t-butylbicyclophosphorothionate (TBPS) to the GABA-gated chloride channel. Significant decreases in TBPS binding were observed only in the dendritic layers (stratum oriens and lacunosem moleculare) of the CA1 hippocampus. The results suggest that ischemia-induced cellular processes that contribute to cell death can decrease GABA-gated chloride channels on dendrites of CA1 pyramidal cells, and that GABAA receptors may also reside on neurons afferent to or intrinsic to the dendritic layers of CA1 hippocampus.

Animals↗