Biomedical subjects

Y Zhao

Publications and source records attributed to Y Zhao.

At least 541 records · Page 30Linked to original sources

PubMed1998

On-column sample preconcentration using sample matrix switching and field amplification for increased sensitivity of capillary electrophoretic analysis of physiological samples.

An on-line sample concentration method using sample matrix switching and field amplification peak stacking has been developed. A microbore LC guard column is used to slightly retain the analytes in order to switch from a high ionic strength sample matrix (the physiological fluid) to a low ionic strength matrix (the LC mobile phase). The eluted LC peak is then trapped in a CE system and preconcentrated by field amplification peak stacking. The concentrated sample peak is then analyzed by CE. Compared to normal hydrodynamic injection, the sensitivity was increased by more than 500-fold without loss in resolution. A limit of detection of less than 10 nM for a physiological sample was achieved using UV adsorption detection. This method can be used for negatively or positively charged analytes.

Bupivacaine↗

PubMed1998

Chromosome 5 aberrations and genetic predisposition to lung cancer.

In this study, we aimed to confirm the finding that chromosome 5 aberrations are predisposing factors for lung cancer. The study population consisted of 118 previously untreated lung cancer patients and 101 healthy controls. Lymphocytes were treated with bleomycin for 5 hr and then allowed to recover in a drug-free medium for 48 hr. The mean number of cells with chromosome 5 abnormalities among 100 cells examined was significantly higher in patients (9.12) than in controls (4.69) (p < 0.001). The most frequent aberration was a 5q deletion and the breakpoints clustered at the 5q13-5q31 region. We then dichotomized the number of induced chromosome 5 abnormalities in peripheral blood lymphocytes by the 75th percentile in that of the controls. 103 (87.3%), of the 118 patients, but only 31 (30.7%) of the 101 controls, exhibited induced breaks above this point. After adjustment for age, sex, ethnicity and smoking status, we found that the sensitive group was at 14.4-fold increased risk for lung cancer. There was also a significant (p < 0.01) gradient of increased risk for lung cancer with an increasing number of chromosome 5 lesions. Therefore, chromosome 5 lesions, especially those at 5q, may be a molecular target of carcinogens in the development of lung cancer.

Aged↗

PubMed1998

Induction of thymocyte apoptosis by Ca2+-independent protein kinase C (nPKC) activation and its regulation by calcineurin activation.

Glucocorticoids appear to participate in apoptosis of unselected CD4(+)CD8(+) thymocytes. Activation of Ca2+-independent novel protein kinase C (nPKC) precedes glucocorticoid-induced thymocyte apoptosis, while proper levels of Ca2+-dependent protein kinase C (cPKC) and calcineurin activities contribute to rescue thymocytes. To clarify the role of nPKC in thymocyte apoptosis, murine thymocytes were stimulated with the diterpene diester, ingenol 3, 20-dibenzoate (IDB). IDB induced selective translocation of nPKC-delta, -epsilon, and -theta and PKC-mu from the cytosolic fraction to the particulate fraction and induced morphologically typical apoptosis through de novo synthesis of macromolecules. The apoptosis was also induced by thymeleatoxin, a diterpene ester, at relatively high concentrations that induced translocation of cPKC, nPKC-theta, and PKC-mu. The IDB- or thymeleatoxin-induced death was inhibited by non-isoform-selective PKC inhibitors, but not by their structural analogs with weak PKC-inhibitory activity or the selective inhibitor of cPKC and PKC-mu, Gö 6976. The death was also inhibited by calcium ionophore ionomycin at concentrations within a narrow range. The range corresponded to the concentration range that contributes to the inhibition of glucocorticoid-induced apoptosis. The antiapoptotic effect was canceled by the immunosuppressant FK506 but not by rapamycin. These results indicate that activation of nPKC, especially nPKC-theta, induces apoptosis in thymocytes and that calcineurin activation regulates the apoptosis.

Animals↗

PubMed1998

A discordancy test approach to identify outliers of length of hospital stay.

A discordancy test approach is proposed to identify outliers of inpatient length of stay. This has implications not only for benchmarking service delivery but also for linking budget allocation procedures to efficiency of health service provisions. The effects of shifting trim point thresholds on hospital payments are assessed in a case study of a group of obstetrical patients.

Adolescent↗

PubMed1998

Molecular basis for substrate specificity of protein-tyrosine phosphatase 1B.

Protein-tyrosine phosphatases can exhibit stringent substrate specificity in vivo, although the molecular basis for this is not well understood. The three-dimensional structure of the catalytically inactive protein-tyrosine phosphate 1B (PTP1B)/C215S complexed with an optimal substrate, DADEpYL-NH2, reveals specific interactions between amino acid residues in the substrate and PTP1B. The goal of this work is to rigorously evaluate the functional significance of Tyr46, Arg47, Asp48, Phe182, and Gln262 in substrate binding and catalysis, using site-directed mutagenesis. Combined with structural information, kinetic analysis of the wild type and mutant PTP1B using p-nitrophenyl phosphate and phosphotyrosine-containing peptides has yielded further insight into PTP1B residues, which recognize general features, as well as specific properties, in peptide substrates. In addition, the kinetic results suggest roles of these residues in E-P hydrolysis, which are not obvious from the structure of PTP1B/peptide complex. Thus, Tyr46 and Asp48 recognize common features of peptide substrates and are important for peptide substrate binding and/or E-P formation. Arg47 acts as a determinant of substrate specificity and is responsible for the modest preference of PTP1B for acidic residues NH2-terminal to phosphotyrosine. Phe182 and the invariant Gln262 are not only important for substrate binding and/or E-P formation but also important for the E-P hydrolysis step.

Amino Acid Sequence↗

PubMed1998

Subunits of the yeast SWI/SNF complex are members of the actin-related protein (ARP) family.

The yeast SWI/SNF chromatin remodeling complex is comprised of 11 tightly associated polypeptides (SWI1, SWI2, SWI3, SNF5, SNF6, SNF11, SWP82, SWP73, SWP59, SWP61, and SWP29). We have used matrix-assisted laser desorption ionization time-of-flight mass spectrometry to identify the genes that encode the SWP59 and SWP61 subunits. Surprisingly, we find that SWP59 and SWP61 are encoded by the ARP9 and ARP7 genes, respectively, which encode members of the actin-related protein (ARP) family. Sequence analyses have shown that ARP9 and ARP7 are 24-26% identical (48-51% similar) to yeast actin and that they are likely to maintain the overall actin fold. Deletion of either the ARP9 or ARP7 gene causes typical swi/snf phenotypes, including growth defects on media containing galactose, glycerol, or sucrose as sole carbon sources. ARP9 and ARP7 are also required for expression of an HO-lacZ fusion gene and for full transcriptional enhancement by the GAL4 activator. The identification of two ARP family members as crucial subunits of the SWI/SNF complex suggests that the complex may contain a total of three different ATPase subunits; furthermore, the similarity of ARP7 and ARP9 to the HSP and HSC family of ATPases suggests the possibility that chromatin remodeling by SWI/SNF may involve chaperone-like activities.

Actins↗

PubMed1998

Structural changes in the heme proximal pocket induced by nitric oxide binding to soluble guanylate cyclase.

When expressed in Escherichia coli, the heme domain [beta1(1-385)] of rat lung soluble guanylate cyclase (sGC) is isolated with a stoichiometric amount of bound heme [Zhao, Y., and Marletta, M. A. (1997) Biochemistry 36, 15959-15964]. Nitric oxide (NO) binding to the heme in beta1(1-385) leads to cleavage of the Fe-His bond and formation of a five-coordinate NO-heme complex. Addition of imidazole to the five-coordinate NO complex shifts the Soret peak from 399 to 420 nm, which appears to result from the formation of a six-coordinate NO complex. Removal of the added imidazole by gel filtration results in formation of the five-coordinate NO complex once again. The EPR spectrum of the putative six-coordinate NO complex has nine distinct derivative-shaped lines (a triplet of triplets), which is the signature spectrum of a six-coordinate NO complex with two nitrogen atoms as the axial ligands. [15N]Imidazole simplifies the six-coordinate NO complex EPR spectrum to six distinct derivative-shaped lines (a triplet of doublets), indicating that the other axial ligand in the six-coordinate NO complex is an imidazole molecule. These results show that NO binding to sGC not only leads to the cleavage of the Fe-His bond but also induces a conformational change which opens the heme proximal pocket large enough to accommodate an exogenous imidazole molecule. These observations have important implications for determining the NO activation mechanism of sGC.

Animals↗

PubMed1998

Chemiluminescence-based detection: principles and analytical applications in flowing streams and in immunoassays.

The present paper provides the principles of chemiluminescence (CL) and its powerful applications in analytical chemistry, mainly in the area of flow injection analysis, column liquid chromatographic and capillary electrophoretic separating systems, and its potential in immunoassays. CL is light produced by a chemical reaction. The most common advantages of chemiluminescent reactions are the relatively simple instrumentation required, the very low detection limits and wide dynamic ranges, which have contributed to the interest of CL detection in flow injection analysis, high performance liquid chromatography, including miniaturized systems, and, most recently, the exploding area of capillary electrophoresis. The latter powerful microanalytical separation technique offers high numbers of theoretical plates and relatively short analysis times requiring only small sample volumes, the migrating system comprising aqueous buffer solutions. In non-isotopic immunoassays, covering a great variety of applications in human and veterinary medicine, forensic medicine, agriculture and food industry, the radioisotope is replaced by a fluorescence or chemiluminescent label. The use of CL as a detection principle permits quantitative determination of various compounds at low concentrations. Disadvantages of the CL-based technique may include lack of sufficient selectivity and sensitivity to various physicochemical factors.

Chemistry Techniques, Analytical↗

PubMed1998

Langmuir-Blodgett Film Formation of a Fullerene Dicarboxylic Acid Derivative C60(HOOCCHNHCHCOOH) and its Photocurrent Generation.

The Langmuir films of a fullerene dicarboxylic acid derivative I, C60(HOOCCHNH2CHCOOH)CF3SO3H (FDA), at the air/water interface have been investigated. FDA formed a stable monolayer on pure water when dilute solution (1.04 x 10(-5) mol/L) was used as the spreading solution. The monolayer formation on various subphases was investigated, including solutions containing different monovalent (Na+, Ag+) and divalent (Ca2+, Cd2+, Cu2+) cations. The introduction of these cations makes the monolayer more stable. The limiting molecular area is also dependent on the pH of the subphase. The surface pressure versus area isotherms at different temperature clearly indicate that FDA molecules easily aggregate at high temperature. The monolayer and multilayer LB films of FDA were deposited on quartz and glass substrates from Ca2+, Cd2+, Cu2+, or Cu2+/Phen solution subphase, and their UV-vis and XPS spectra were obtained. The data showed that for Cd2+ and Cu2+/Phen subphase the cation can be transferred onto the substrates together with FDA but not the other two. The photoelectrochemical behavior of FDA on an ITO electrode was investigated, which showed that the photocurrent is cathodic. Oxygen and negative bias are beneficial factors for the photocurrent generation. The external quantum yield is ca. 4%. Copyright 1998 Academic Press.

Journal Article↗

PubMed1998

Catalytic mechanism of the phospholipase D superfamily proceeds via a covalent phosphohistidine intermediate.

The phospholipase D (PLD) superfamily includes enzymes of phospholipid metabolism, nucleases, as well as ORFs of unknown function in viruses and pathogenic bacteria. These enzymes are characterized by the invariant sequence motif, H(X)K(X)4D. The endonuclease member Nuc of the PLD family was over-expressed in bacteria and purified to homogeneity. Mutation of the conserved histidine to an asparagine in the endonuclease reduced the kcat for hydrolysis by a factor of 10(5), suggesting that the histidine residue plays a key role in catalysis. In addition to catalyzing hydrolysis, a number of phosphohydrolases will catalyze a phosphate (oxygen)-water exchange reaction. We have taken advantage of this observation and demonstrate that a 32P-labeled protein could be trapped when the enzyme was incubated with 32P-labeled inorganic phosphate. The phosphoenzyme intermediate was stable in 1 M NaOH and labile in 1 M HCl and 1 M hydroxylamine, suggesting that the enzyme forms a phosphohistidine intermediate. The pH-stability profile of the phosphoenzyme intermediate was consistent with phosphohistidine and the only radioactive amino acid found after alkaline hydrolysis was phosphohistidine. These results suggest that the enzymes in the PLD superfamily use the conserved histidine for nucleophilic attack on the substrate phosphorus atom and most likely proceed via a common two-step catalytic mechanism.

Amino Acid Sequence↗

PubMed1998

Pig MHC mediates positive selection of mouse CD4+ T cells with a mouse MHC-restricted TCR in pig thymus grafts.

Remarkably normal immune function and specific T cell tolerance to discordant xenogeneic donors can be achieved by grafting fetal pig thymus and liver (FP THY/LIV) tissue to T cell and NK cell-depleted, thymectomized (ATX) mice. To determine whether or not host class II MHC molecules participate in the positive selection of mouse CD4+ T cells in FP THY/LIV grafts, we compared their development in ATX "AND" TCR-transgenic mice with positive selecting or nonselecting host MHC genotypes. Mouse TCR-transgenic CD4 single positive T cells repopulated the periphery significantly and to a similar extent in both T/NK cell-depleted, ATX AND mice with positive-selecting or nonselecting MHC backgrounds after grafting with FP THY/LIV. Therefore, MHC molecules from a widely disparate xenogeneic species can positively select T cells bearing a host class II MHC-restricted TCR without a contribution from the host MHC. These results, in combination with previous studies performed in this model, suggest that the T cell repertoire that is generated by the combination of positive selection on xenogeneic MHC and negative selection on both recipient and xenogeneic porcine MHC is tolerant of both donor and recipient and has sufficient cross-reactivity with host MHC/foreign peptide complexes to confer a high level of immunocompetence. The results have implications for the potential clinical applicability of xenogeneic thymic transplantation and also suggest a predominant role for the TCR recognition of species-conserved MHC residues in positive selection.

Animals↗

PubMed1998

In utero gene therapy: transfer and long-term expression of the bacterial neo(r) gene in sheep after direct injection of retroviral vectors into preimmune fetuses.

We investigated whether directly injecting retroviral vectors into preimmune fetuses could result in the transfer and long-term expression of exogenous genes. Twenty-nine preimmune sheep fetuses were injected with helper-free retroviral vector preparations. Twenty-two fetuses survived to term, 4 of which were sacrificed at birth. Of the remaining 18 animals, 3 were controls and 15 had received vector preparations. Twelve of these 15 animals demonstrated transduction of hematopoietic cells when blood and marrow were analyzed by neo(r)-specific PCR. Eight experimental sheep have been followed for 5 years, during which time we have consistently observed proviral DNA and G418-resistant hematopoetic progenitors. The G418-resistant colonies were positive when analyzed by neo(r)-specific PCR. neo(r) gene expression was also demonstrated using several immunological and biochemical methods. The transduction of hematopoietic stem cells was confirmed when lambs transplanted with bone marrow from in utero-transduced sheep exhibited neo(r) activity in marrow and blood. Vector distribution was widespread in primary animals without pathology. PCR analysis indicates that the germ line was not altered. These studies demonstrate that direct injection of an engineered retrovirus is a feasible means of safely delivering a foreign gene to a developing fetus and achieving long-term expression without modifying the germ line of the recipient.

Animals↗

PubMed1998

Molecular cloning of a gene encoding phospholipase D from the pathogenic and dimorphic fungus, Candida albicans.

A phospholipase D gene (CaPLD) has been cloned from the Candida albicans genomic DNA library. The CaPLD is a member of a highly conserved gene family of PLD and has the highest homology to Saccharomyces cerevisiae PLD (SPO14) with an overall homology of 42%. Phylogenetic analysis indicated that fungus PLDs including CaPLD composed one of the three clusters of PLD genes.

Amino Acid Sequence↗

PubMed1998

Pleiotropic effects of HIV-1 protein R (Vpr) on morphogenesis and cell survival in fission yeast and antagonism by pentoxifylline.

Expression of HIV-1 Vpr causes cell cycle G2 arrest, change in cell shape, and cell death over a large evolutionary distance ranging from human to yeast cells. As a step toward understanding these highly conserved Vpr functions, we have examined the effect of Vpr on cytoskeletal elements and the viability of fission yeast. We demonstrate that the changes in cell morphology induced by Vpr in fission yeast are caused by several underlying cellular abnormalities, including increased biosynthesis of chitin in the cell wall, disruption of the actin cytoskeleton, and altered polarity for cell growth. The extent of these cellular alterations and cell survival correlates with the level of vpr expression. Accompanying cell death, Vpr induces aberrant nuclear morphologies in fission yeast which are similar to those found during the apoptosis induced by Vpr in mammalian cells. The Vpr-induced cytopathic effects and cell death can be suppressed by treatment with pentoxifylline, a compound that inhibits HIV-1 viral replication and suppresses Vpr-induced cell cycle G2 arrest in human and fission yeast cells. The results presented here suggest that pentoxifylline suppresses the effects of Vpr by blocking interactions of Vpr with cellular proteins. Given that pentoxifylline has potential therapeutic value in blocking the effects of Vpr in HIV-infected patients, understanding the molecular mechanisms by which pentoxifylline antagonizes Vpr may have general implications for HIV therapy.

Anti-HIV Agents↗

PubMed1998

Extrathymic T cell deletion and allogeneic stem cell engraftment induced with costimulatory blockade is followed by central T cell tolerance.

A reliable, nontoxic method of inducing transplantation tolerance is needed to overcome the problems of chronic organ graft rejection and immunosuppression-related toxicity. Treatment of mice with single injections of an anti-CD40 ligand antibody and CTLA4Ig, a low dose (3 Gy) of whole body irradiation, plus fully major histocompatibility complex-mismatched allogeneic bone marrow transplantation (BMT) reliably induced high levels (>40%) of stable (>8 mo) multilineage donor hematopoiesis. Chimeric mice permanently accepted donor skin grafts (>100 d), and rapidly rejected third party grafts. Progressive deletion of donor-reactive host T cells occurred among peripheral CD4(+) lymphocytes, beginning as early as 1 wk after bone marrow transplantation. Early deletion of peripheral donor-reactive host CD4 cells also occurred in thymectomized, similarly treated marrow recipients, demonstrating a role for peripheral clonal deletion of donor-reactive T cells after allogeneic BMT in the presence of costimulatory blockade. Central intrathymic deletion of newly developing T cells ensued after donor stem cell engraftment had occurred. Thus, we have shown that high levels of chimerism and systemic T cell tolerance can be reliably achieved without myeloablation or T cell depletion of the host. Chronic immunosuppression and rejection are avoided with this powerful, nontoxic approach to inducing tolerance.

Abatacept↗

PubMed1998

The major adult alpha-globin gene of antarctic teleosts and its remnants in the hemoglobinless icefishes. Calibration of the mutational clock for nuclear genes.

The icefishes of the Southern Ocean (family Channichthyidae, suborder Notothenioidei) are unique among vertebrates in their inability to synthesize hemoglobin. We have shown previously (Cocca, E., Ratnayake-Lecamwasam, M., Parker, S. K., Camardella, L., Ciaramella, M., di Prisco, G., and Detrich, H. W., III (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 1817-1821) that icefishes retain inactive genomic remnants of adult notothenioid alpha-globin genes but have lost the gene that encodes adult beta-globin. Here we demonstrate that loss of expression of the major adult alpha-globin, alpha1, in two species of icefish (Chaenocephalus aceratus and Chionodraco rastrospinosus) results from truncation of the 5' end of the notothenioid alpha1-globin gene. The wild-type, functional alpha1-globin gene of the Antarctic yellowbelly rockcod, Notothenia coriiceps, contains three exons and two A + T-rich introns, and its expression may be controlled by two or three distinct promoters. Retained in both icefish genomes are a portion of intron 2, exon 3, and the 3'-untranslated region of the notothenioid alpha1-globin gene. The residual, nonfunctional alpha-globin gene, no longer under positive selection pressure for expression, has apparently undergone random mutational drift at an estimated rate of 0.12-0.33%/million years. We propose that abrogation of hemoglobin synthesis in icefishes most likely resulted from a single mutational event in the ancestral channichthyid that deleted the entire beta-globin gene and the 5' end of the linked alpha1-globin gene.

Amino Acid Sequence↗

PubMed1998

[Role of CAAT/enhancer binding protein in IL-6 regulation of angiotensinogen gene expression].

OBJECTIVE: Angiotensinogen is the only known precursor of vasoactive angiotensin II and also one of the acute phase proteins. This study was intended to understand the regulation of angiotensinogen gene expression induced by IL-6. METHODS: Northern hybridization, electrophoretic mobility shift assay and transient transfections were conducted. RESULTS: Northern hybridization showed increase of angiotensinogen mRNA treated by IL-6 in Hep3B cells. Electrophoretic mobility shift assay further indicated the HAG IL-6RE homologous to IL-6 responsive element at -568 site of the angiotensinogen promoter binds C/EBP(CAAT/Enhancer Binding Protein). Consistent with this binding studies were the transient transfections of the expression vector in which 6 copies of HAG-IL-6RE linked to TK core promoter and fused to CAT reporter gene, revealing that C/EBPalpha was a transactivator under IL-6-induced condition. CONCLUSION: These observations suggest that C/EBP plays regulatory role in IL-6-induced angiotensinogen gene expression.

Angiotensinogen↗

PubMed1998

Crystal structure of a tyrosine phosphorylated STAT-1 dimer bound to DNA.

The crystal structure of the DNA complex of a STAT-1 homodimer has been determined at 2.9 A resolution. STAT-1 utilizes a DNA-binding domain with an immunoglobulin fold, similar to that of NFkappaB and the p53 tumor suppressor protein. The STAT-1 dimer forms a contiguous C-shaped clamp around DNA that is stabilized by reciprocal and highly specific interactions between the SH2 domain of one monomer and the C-terminal segment, phosphorylated on tyrosine, of the other. The phosphotyrosine-binding site of the SH2 domain in each monomer is coupled structurally to the DNA-binding domain, suggesting a potential role for the SH2-phosphotyrosine interaction in the stabilization of DNA interacting elements.

Amino Acid Sequence↗