Biomedical subjects
Publications and source records attributed to H Zhao.
Puerariae isoflavone (500, 1000 mg/kg% x 7 d, p.o.) significantly lowered blood viscosity and platelet adhesion rate, inhibited thrombosis and ADP-induced platelet aggregation in rats, and showed obvious antagonism for platelet thrombosis in ADP-treated mice.
Protcective effect of paeonol was observed in the repeated cerebral ischemia-reperfusion model in rats. Paeonol (100, 50 mg/kg x 7 d i.p.) could inhibit the increase of Ca2+, the decrease of superoxide dimutase (SOD) activity and the content of malondialdehyde (MDA), improve Ca(2+)-ATPase activity in ischemia brain tissue. The results showed that paeonol could protect cerebral ischemia by inhibiting accumulation of Ca2+ and production of oxygen free radical.
The solid-phase enzyme immunoassay (EIA) was used for detecting anti-HEV immunoglobulin G (IgG) and M (IgM) by using a recombinant chimeric antigen (ORF3-2) from the open reading frame (ORF) 2 and 3 of hepatitis E virus gene. These sera were collected from the patients with sporadic acute viral hepatitis. The positive rate for anti-HEV IgG was 15.22% (7) among 46 patients with acute hepatitis, 5 of the 7 IgG positive sera were also positive (71.4%) for anti-HEV IgM. 91.8% (93) and 76.4% (78) were positive separately for anti-HEV IgG and IgM among 102 patients of suspected HEV infection (these patients were negative for anti-HAV, anti-HBV and anti-HCV), about 2/3 patients of HEV infection detected were simultaneously HEV-IgG and IgM positive. In control group of 98 normal persons, 4 (4.08%) were positive for anti-HEV IgG, but none were IgM positive. Paired sera of the patients in the acute phase and convalescent phase were detected, the titers of anti-HEV IgG with 4-16 fold decrease were seen in 6 cases and with 2-fold rise in 1 case.
The extreme discordant-sib-pair design has been found to be the most powerful, across most genetic models. In this paper, we address two of the most frequently asked questions related to this design. First, under the extreme discordant-sib-pair design, a large number of people have to be screened for the phenotype of interest, before the desired number of discordant sibs can be collected for genotyping and linkage analysis. When the phenotyping cost is not negligible compared with the genotyping cost, such methods might not be cost effective. The second question is how sensitive the cost is to the genetic model and allele frequency. In this paper, we compare the cost under different sampling strategies, different genetic models, and different phenotyping:genotyping cost ratios. Because our knowledge of the underlying genetic model for a trait is limited, the discordant-sib-pair design proves to be the most robust. When the cost for screening probands is not included, the design that genotypes sibs with one sib in the top 10% and the other sib in the bottom 30% of the population with respect to the trait of interest is, across most models studied, the optimum among the designs considered in this paper. The cost under this design, across different genetic models, appears to be relatively robust to allele frequency and model type, whether additive or dominant. If probands initially must be screened as well, then 25% appears to be the optimal portions of the upper and lower distributions to be studied.
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OBJECTIVES: To evaluate the effects of insulin-like growth factor-I (IGF-I) on the maturation, fertilization, and cleavage of murine oocytes. METHODS: Immature and mature murine oocytes were cultured alone or together with murine spermatozoa in vitro. Various concentrations of IGF-I were added to the media as experimental groups, and was not added as controls. Then the rates of maturation, fertilization and cleavage of murine oocytes were observed, and compared between the 2 groups. RESULTS: The rates of maturation, fertilization and cleavage of murine oocytes in experimental groups were significantly higher than those in the controls (P < 0.01 or P < 0.05). Unequal blastomeres and cell fragments were observed in both groups, but the differences during the same cultured period were not statistically significant (P > 0.05). CONCLUSIONS: IGF-I could induce murine oocyte maturation, promote its fertilization and cleavage in vitro, and do no harm to the fertilized oocytes and embryos.
The rat neu oncogene product encodes a 185 kDa receptor tyrosine kinase which is constitutively activated as a result of a single amino acid substitution (Val664-->Glu) within the transmembrane region. In this study, we show that the transforming activity of oncogenic p185neu (also termed Tneu) can be inhibited by co-expression of a truncated neu protein with a large cytoplasmic deletion (termed T691stop) which includes the tyrosine kinase domain. In cell lines co-expressing full-length and truncated neu proteins, we observed co-dimerization between full-length p185neu and truncated T691 stop, resulting in the formation of a kinase-inactive heteromeric complex. Phenotypic analysis of several different clones showed that the degree of inhibition of transformation in vitro and tumorigenicity in vivo was related to the ratio of full-length and truncated p185 proteins co-expressed in cells. These results provide evidence that expression of kinase-deficient neu proteins leads to co-dimerization that results in suppression of kinase activation and oncogenicity of associated p185neu-activated receptors. The mutant neu protein mediates inhibition in both transfected fibroblasts expressing oncogenic p185neu and mammalian cancer cells derived from a rat primary neuroglioblastoma expressing oncogenic p185neu. This truncated peptide may be important for the design of future therapies directed against erbB family oncoproteins.
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Human parainfluenza virus type 3 (HPIV3) genome RNA is transcribed and replicated by the virus-encoded RNA-dependent RNA polymerase, and specific cellular proteins play a regulatory role in these processes. To search for cellular proteins potentially interacting with HPIV3 cis-acting regulatory RNAs, a gel mobility shift assay was used. Two cellular proteins specifically interacted with the viral cis-acting RNAs containing the genomic 3'-noncoding region and the plus-sense leader sequence region. Surprisingly, by biochemical and immunological analyses, one of the cellular proteins was identified as the key glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The other protein was characterized as the autoantigen, LA protein. Both GAPDH and LA protein also interacted with the same cis-acting RNA sequences in vivo and were found to be associated with the HPIV3 ribonucleoprotein complex in the infected cells. By double immunofluorescent labeling, GAPDH was found to be co-localized with viral ribonucleoprotein in the perinuclear region. These observations strongly suggest that cellular GAPDH and LA Protein participate in the regulation of HPIV3 gene expression.
Zinc accumulation in Saccharomyces cerevisiae occurs through either of two uptake systems. A high affinity system is active in zinc-limited cells, and the ZRT1 gene encodes the transporter protein of this system. In this study, we characterized the low affinity system that is active in zinc-replete cells. The low affinity system is time-, temperature-, and concentration-dependent and prefers zinc over other metals as its substrate. Our results suggest that the ZRT2 gene encodes the transporter of this system. The amino acid sequence of Zrt2p is remarkably similar to those of Zrt1p and Irt1p, an Fe2+ transporter from Arabidopsis thaliana. Overexpressing ZRT2 increased low affinity uptake, whereas disrupting this gene eliminated that activity, but had little effect on the high affinity system. Therefore, the high and low affinity systems are separate uptake pathways. Analysis of the zinc levels required for growth of zrt2 mutant strains as well as the effects of the zrt2 mutation on the regulation of the high affinity system demonstrated that the low affinity system is a biologically relevant mechanism of zinc accumulation. Finally, a zrt1zrt2 mutant was viable, indicating the existence of additional zinc uptake pathways.
A series of azetidinone cholesterol absorption inhibitors related to SCH 48461 ((-)-6) has been prepared, and compounds were evaluated for their ability to inhibit hepatic cholesteryl ester formation in a cholesterol-fed hamster model. Although originally designed as acyl CoA: cholesterol acyltransferase (ACAT) inhibitors, comparison of in vivo potency with in vitro activity in a microsomal ACAT assay indicates no correlation between activity in these two models. The molecular mechanism by which these compounds inhibit cholesterol absorption is unknown. Despite this limitation, examination of the in vivo activity of a range of compounds has revealed clear structure-activity relationships consistent with a well-defined molecular target. The details of these structure-activity relationships and their implications on the nature of the putative pharmacophore are discussed.
We have investigated the IFN-mediated inhibition of human parainfluenza virus-3 (HPIV-3) replication in cultured human A549 cells. IFN-alpha inhibited the virus yield significantly with concomitant reduction of viral RNA accumulation by more than 90%. Further studies indicated that the inhibitory action of IFN was at the level of primary transcription of HPIV3 replication. Since the IFN-inducible protein, MxA, has been shown to inhibit virus replication in several RNA viruses, we examined the role of MxA in HPIV-3 replication using a stably transfected human glioblastoma cell line expressing MxA. In these cells HPIV-3 replication was decreased by more than 100-fold depending on the virus dosage used with concomitant inhibition of viral RNA synthesis by about 80%. However, the viral primary transcription was not affected in this MxA-producing cell line. In contrast, in the parental cell line IFN-mediated inhibition occurred at the primary transcription step of HPIV-3 replication. These data suggest that in addition to MxA, other IFN-inducible proteins are involved in the anti-HPIV-3 effect of IFN in both the cell lines used.
Comparative peptide mapping of a monoclonal antibody chimeric BR96 and corresponding doxorubicin (DOX) immunoconjugate was performed using capillary electrophoresis (CE) and capillary liquid chromatography (CLC). A unique, highly sensitive and selective approach combined with both UV absorbance and laser-induced fluorescence (LIF) detection has been developed and applied to studies including enzymatic digests of antibody and conjugate and related drug and conjugation linker substances. The analytical methodology has been established based on the unique characteristic of the anticancer drug DOX which yields native fluorescence. With an excitation wavelength of 488 nm from argon-ion laser, DOX conjugated to the monoclonal antibody using a hydrazone linker can be determined with a detection limit at the attomole level. Approaches were developed based on the successful conjugation and analysis of a model peptide conjugate. Enzymatic digests of the monoclonal antibody BR96 and its immunoconjugate were mapped by CE and CLC with on-line UV and LIF detection, which results in a unique fingerprint for structural analysis. With a two-dimensional LC-CE approach, conjugated peptide-DOX species from LC were further analyzed by CE with LIF detection. The drug-containing peptide fragments in the mixture were readily detected, which can be further characterized using other complementary analytical techniques.
The yeast Saccharomyces cerevisiae has two separate systems for zinc uptake. One system has high affinity for substrate and is induced in zinc-deficient cells. The second system has lower affinity and is not highly regulated by zinc status. The ZRT1 gene encodes the transporter for the high-affinity system, called Zrt1p. The predicted amino acid sequence of Zrt1p is similar to that of Irt1p, a probable Fe(II) transporter from Arabidopsis thaliana. Like Irt1p, Zrt1p contains eight potential transmembrane domains and a possible metal-binding domain. Consistent with the proposed role of ZRT1 in zinc uptake, overexpressing this gene increased high-affinity uptake activity, whereas disrupting it eliminated that activity and resulted in poor growth of the mutant in zinc-limited media. Furthermore, ZRT1 mRNA levels and uptake activity were closely correlated, as was zinc-limited induction of a ZRT1-lacZ fusion. These results suggest that ZRT1 is regulated at the transcriptional level by the intracellular concentration of zinc. ZRT1 is an additional member of a growing family of metal transport proteins.
1. To characterize [Ca2+]i signalling in salivary duct cells a procedure was developed for the rapid preparation and isolation of intralobular ducts, some of which had attached intercalated ducts. The isolated ducts retained agonist-induced Ca2+ signalling after permeabilization with streptolysin O (SLO). 2. The improved cell preparation technique was reflected in the repertoire and intensity of agonist responsiveness of the cells. Measurements of [Ca2+]i in intact cells showed that all agonists previously reported to affect electrolyte transport by the submandibular salivary gland (adrenaline, carbachol, isoprenaline and forskolin) mobilized Ca2+ from internal stores and increased Ca2+ influx across the plasma membrane. 3. The use of the SLO-permeabilized ducts showed that all agonists, including isoprenaline and forskolin, mobilized Ca2+ exclusively from the inositol 1,4,5 trisphosphate (IP3)-sensitive pool. However, in granular ducts only adrenaline mobilized the entire IP3-sensitive pool whereas all other agonists mobilized only part of the pool. 4. All regions of the duct responded to substance P and the luminally secreted agonist ATP. Interestingly, the intercalated duct was most responsive to ATP and demonstrated only a minimal response to all other agonists. The granular region of the same duct and the extralobular duct always responded best to stimulation by adrenaline. 5. The perfused extralobular duct was used to show that adrenaline and carbachol stimulated the duct through the basolateral membrane whereas the receptors for ATP were localized in the luminal membrane of the duct. This suggests the presence of an ATP-dependent positive feedback loop in salivary duct with decreased activity along the ductal tree.
BACKGROUND: Simian virus 40 (SV40) and murine polyomavirus (polyoma) are non-enveloped DNA tumor viruses. Their structurally similar capsids, about 500 degrees in diameter, are formed by 72 pentamers of the major coat protein VP1. RESULTS: We describe in this paper the structure determination of SV40 and polyoma at 3.8 degree resolution, focusing particularly on methodological issues, and on a comparison of the overall molecular organization in the two related virus particles. Initial phases for SV40 were obtained by single isomorphous replacement at 6.5 degree. Phases were refined and the resolution extended to 3.8 degree by a combination of strict 5-fold and partial 30-fold electron-density averaging. The structure of polyoma was subsequently determined by systematically translating and rotating the individual VP1 pentamers, in order to find the maximum correlation between calculated and observed structure factors. The resolution was then extended to 3.8 degree, also by phase refinement through electron-density averaging. CONCLUSION: The strategies for density averaging and for molecular replacement, used to determine the SV40 and polyoma structures, are likely to be generally useful. The individual building blocks, the VP1 pentamers, are essentially identical in both cases, as are the local details of their interactions with neighboring pentamers. Nevertheless, the arrangement of the pentamers with respect to each other is somewhat different in the two viruses. Whereas SV40 is almost spherical, with all pentamers at identical radii, the pentamers in polyoma that lie on icosahedral fivefold axes are displaced outward by about 5 degree.
Bryostatin 1, a natural product anticancer agent isolated from a marine bryozoan, has been shown in tissue culture to activate protein kinase C. This agent has recently undergone Phase I testing in humans given either as a bolus i.v. injection or a continuous infusion. To understand how bryostatin 1 might be used best as an anticancer agent, a study of the pharmacokinetics, tissue distribution, metabolism, and elimination of bryostatin 1 in mice was undertaken, using [C26-3H]-labeled bryostatin 1. Following i.v. administration, the plasma disappearance curve for bryostatin 1 could be described by a two-compartment model, with half-lives of 1.05 and 22.97 h, respectively. In contrast, the plasma disappearance curve for bryostatin 1 administered i.p. was better described by a first order absorption one-compartment model, with an absorption half-life of 0.81 h and an elimination half-life of 28.76 h, respectively. The majority of radioactivity in plasma was associated with the intact drug for up to 24 h after dosing. In the first 12 h after i.v administration, urinary excretion represented the major pathway of elimination, with 23.0 +/- 1.9% (mean +/- SD) of the administered dose excreted. Within 72 h after i.v. administration, approximately equal amounts of radioactivity (40%) were excreted in feces compared to urine. Bryostatin 1 was widely distributed in many organs but concentrated in the lung, liver, gastrointestinal tract, and fatty tissue. The concentration in the gastrointestinal tract, along with the fecal excretion, suggests the possibility of enterohepatic circulation of this drug. In summary, this study demonstrates that bryostatin 1 is relatively stable in vivo, widely distributed but concentrated in some major tissues, and rapidly excreted first through urine and at later times through the feces. The data from this animal study should be useful in the design of future human trials with this anticancer drug.
Substance P (SP), a member of the tachykinin family of neuropeptides, is an important immunomodulator of lymphocyte and monocyte/macrophage function. We have examined the effects of SP on human immunodeficiency virus type 1 (HIV-1) infection of peripheral blood monocyte-derived macrophages (MDMs) in vitro. Human monocytes isolated by Ficoll gradient followed by adherence were maintained in vitro for 10 days and infected with HIV-1. The addition of SP resulted in a 2- to 8-fold-enhanced HIV-1 expression in the MDMs isolated from 7 of 13 healthy donors as determined by reverse transcriptase (RT) activity and p24 protein expression assays, as compared to control cultures incubated with HIV-1 alone. There was no correlation observed, however, between SP-stimulated TNF production and HIV-1 expression in MDMs obtained from a subset of these donors. These effects of SP on HIV-1 expression in MDMs in vitro may have in vivo implications relevant to modulation of monocyte/macrophage functions, to HIV-1 infection of monocytes/macrophages, and to the immunopathogenesis of HIV-1 infection.