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Y Yang

Publications and source records attributed to Y Yang.

At least 1,045 records · Page 58Linked to original sources
PubMed1996

Optimization of cross-linked lexitropsins.

In attempts to optimize the cross-linked lexitropsin design, a number of cross-linked dimers composed of two tris(N-methylpyrrolecarboxamide) strands were synthesized and their binding interactions with poly d(A).poly d(T) and poly d(A-T).poly d(A-T) were characterized by circular dichroism and ethidium fluorometry. While all alkanediyl-linked dimers showed a similar binding behavior to the homo AT polymer, particularly at low ligand concentrations, the decanediyl linker was found to be the optimal linker permitting the bidentate antiparallel side-by-side binding of the corresponding dimer to the alternating AT polymer. Thus, in comparison with the monomer, the decanediyl-linked dimer has a binding strength enhancement of about 1400 times in the 1:1 binding mode. Moreover, the hydrophilicity of the linker dimer has a significant effect on the bidentate binding strength. The (3,6)-dioxaoctanediyl-linked dimer has a further binding strength enhancement of 10 times over the decanediyl-linked dimer. Overall, the best optimized dimer has a binding strength enhancement of over 14,000 times in comparison with the monomer in the 1:1 binding mode. This binding enhancement parallels that observed in the best optimized bisintercalators. Distance-restrained molecular modeling provides support for the experimental results. Dimers of longer linkers can readily accommodate a bidentate antiparallel side-by-side binding mode but those of shorter linkers necessitate marked structural distortions in the bound ligand molecules. It is further observed that the binding strength enhancement to the alternating AT polymer is not always accompanied by the binding specificity improvement. Our analysis suggests that the non-specific appendage-DNA backbone interaction is a key factor that controls the specificity improvement.

Circular Dichroism↗
PubMed1996

A mutation in a Saccharomyces cerevisiae gene (RAD3) required for nucleotide excision repair and transcription increases the efficiency of mismatch correction.

RAD3 functions in DNA repair and transcription in Saccharomyces cerevisiae and particular rad3 alleles confer a mutator phenotype, possibly as a consequence of defective mismatch correction. We assessed the potential involvement of the Rad3 protein in mismatch correction by comparing heteroduplex repair in isogenic rad3-1 and wild-type strains. The rad3-1 allele increased the spontaneous mutation rate but did not prevent heteroduplex repair or bias its directionality. Instead, the efficiency of mismatch correction was enhanced in the rad3-1 strain. This surprising result prompted us to examine expression of yeast mismatch repair genes. We determined that MSH2, but not MLH1, is transcriptionally regulated during the cell-cycle like PMS1, and that rad3-1 does not increase the transcript levels for these genes in log phase cells. These observations suggest that the rad3-1 mutation gives rise to an enhanced efficiency of mismatch correction via a process that does not involve transcriptional regulation of mismatch repair. Interestingly, mismatch repair also was more efficient when error-editing by yeast DNA polymerase delta was eliminated. We discuss our results in relation to possible mechanisms that may link the rad3-1 mutation to mismatch correction efficiency.

Adenosine Triphosphatases↗
PubMed1996

Immunology of gene therapy with adenoviral vectors in mouse skeletal muscle.

Skeletal muscle is an attractive target for somatic gene transfer of both acquired and inherited disorders. Direct injection of adenoviral vectors in the skeletal muscle leads to recombinant gene expression in a large number of muscle fibers. Transgene expression has been transient in most organs and associated with substantial inflammation when experiments are performed in adult immune competent mice. In this report, we utilize a variety of in vivo and in vitro models of T and B cell function to characterize the nature of the immune response to adenoviral vectors injected into murine skeletal muscle. Cellular immunity dependent on CD4+ and CD8+ T cells contributes to the loss of recombinant gene expression and the development of localized inflammation. Antigen specific activation of T cells occurs to both viral proteins and the reporter gene beta-galactosidase. Systemic levels of neutralizing antibody to the capsid proteins of the vector are also generated. Destructive immune responses responsible for loss of transgene expression are largely directed against beta-galactosidase in that transgene expression was stable when beta-galactosidase was eliminated as a neoantigen in mice transgenic for lacZ. A strategy to prevent the cellular and humoral immunity to this therapy was developed based on transiently ablating CD4+ T cell activation at the time of vector delivery. Encouraging results were obtained when vector was administered with one of several immune modulating agents including cyclophosphamide, mAb to CD4+ cells, and mAb to CD40 ligand. These studies indicate that cellular and humoral immune responses are elicited in the context of gene therapy directed to skeletal muscle with adenoviral vectors. Transient ablation of CD4+ T cell activation prevents the effects responses of the CD8+ T and B cells.

Adenoviruses, Human↗
PubMed1996

Immunocytochemical localization of desmin in human fetal skeletal muscle.

The distribution of desmin filaments in the skeletal and cardiac muscles of experimental animals and cultured cells have been extensively studied. The purpose of this report is to identify the distribution of desmin filaments in vivo in human fetal skeletal muscle. Using 10nm gold-particle-labeling immunoelectron microscopy, desmin intermediate filaments were localized in skeletal muscles of 12, 20 and 29-week-old human fetuses. A number of desmin filaments were closely associated with the nascent nonstriated myofibrils. Nascent myofibrils were distributed in the subsarcolemmal space at all three developmental stages. Desmin, accompanying the nascent myofibrils, was most abundant after 12 weeks of gestation. An irregular network of desmin filaments was conspicuous in the subsarcolemmal space after 20 weeks of gestation. Desmin filaments penetrated the myofibrils and the Z-discs after 29 weeks of gestation. The developmental change in fine structural localization of desmin between 12, 20 and 29 weeks of gestation in human fetuses indicated that desmin filaments were involved in myofibril assembly in the subsarcolemmal space, and also in myofibrillogenesis in the inner space of the myofibrils.

Desmin↗
PubMed1996

Interleukin 4-induced proliferation in normal human keratinocytes is associated with c-myc gene expression and inhibited by genistein.

We studied the effect of IL-4 on the proliferation of cultured normal human keratinocytes. Keratinocyte proliferation was stimulated by IL-4 and inhibited by anti-IL-4 antibody in a concentration-dependent manner. Anti-IL-6 antibody did not inhibit normal human keratinocyte proliferation, suggesting that the IL-4 could directly induce proliferation of these cells. IL-4 significantly induced cell cycle G0/G1 to S phase progression. The keratinocyte proliferation by IL-4 was mediated through one of the growth control genes, c-myc protooncogene. The expression of c-myc mRNA was significantly increased after IL-4 treatment of the keratinocytes, suggesting that c-myc plays a key role in the control of proliferation. The signal transduction pathways induced by IL-4 in the keratinocytes were studied with inhibitors of signal transduction. Genistein, a tyrosine kinase inhibitor, suppressed the level of the induced c-myc mRNA expression, but H7, a serine/threonine kinase inhibitor, and okadaic acid, a protein phosphatase 1 and 2A inhibitor, did not block the induced c-myc gene expression. Taken together, these results suggest that IL-4 stimulates the proliferation of keratinocytes in vitro by promoting a transition from G0/G1 to S phase of the cell cycle. Induction of c-myc after IL-4 treatment could indicate an important role for c-myc in the proliferation of keratinocytes. Our observations also suggest that tyrosine kinases may be involved in IL-4-induced proliferation.

Base Sequence↗
PubMed1996

Kinetics of inhibition of aminoacylase activity by dithiothreitol or 2-mercaptoethanol.

The previously described kinetic method of the substrate reaction during irreversible inhibition of enzyme activity [Tsou (1988) Adv. Enzymol. Relat. Areas Mol. Biol. 61, 381-436] has been used to study the inactivation kinetics of aminoacylase by dithiothreitol (DTT) and 2-mercaptoethanol (MET). The results show that the inactivation of aminoacylase by DTT or MET is competitive slow-reversible inhibition. The microscopic rate constants for the inactivation reaction were determined. Removal of these inhibitors by dialysis can lead to complete recovery of enzymatic activity. The present results also show that the presence of equimolar Zn2+ to DTT gives complete protection of the enzyme against the inactivation by DTT. Moreover, addition of equimolar amounts of Zn2+ to DTT can induce recovery of the enzymatic activity of DTT-inactivated enzyme. It is known that aminoacylase from pig kidney contains no disulfide bonds. Therefore, it may be suggested that inactivation of aminoacylase by dithiothreitol or 2-mercaptoethanol is not due to the reduction of disulfide bonds, and is a competitive slow-reversible inhibition.

Amidohydrolases↗
PubMed1996

A study of angiotensin II receptors after chronic inhibition of nitric oxide synthase in the spontaneously hypertensive rat.

1. Nitric oxide (NO) synthase inhibition, induces a sustained increase in blood pressure and amplifies the pressor response to infused angiotensin II (AngII). This study was designed to investigate the contribution of AngII receptors in the elevated blood pressure and enhanced pressor response to AngII in the spontaneously hypertensive rat (SHR) chronically treated with N(G)-nitro-L-arginine-methyl ester (L-NAME). 2. Two groups of 13 week old female SHR were housed four to a box. Group I rats received L-NAME for 7 days (2.5 mg/kg per day) in their drinking water. Group II rats received water only. Blood pressure was monitored daily by tail-cuff plethysmography. Plasma AngII was measured by radioimmunoassay. Aortic and uterine receptor binding was determined by saturation analysis using [125I]-Sar8, Ile1)AngII. Data was analysed using the computer program LIGAND. 3. Mean systolic blood pressure was significantly elevated in rats treated with L-NAME compared with the control group. Plasma AngII concentration was slightly decreased in rats treated L-NAME compared with control. Densities of both aortic and uterine AngII receptors increased significantly following NO synthase inhibition. Receptor affinity in the aorta decreased in the L-NAME group compared with control. However, uterine AngII receptor affinity was unchanged. 4. We conclude that the increased blood pressure and enhanced pressor responsiveness that occurs with chronic inhibition of NO synthesis may result partly from increased vascular AngII receptor expression.

Angiotensin II↗
PubMed1996

Urinary pregnanetriol-3-glucuronide in children: age-related change and application to the management of 21-hydroxylase deficiency.

Urinary concentrations of pregnanetriol-3-glucuronide (PT-3-G) were determined in 485 normal Japanese subjects (277 males and 208 females), aged 5 days to 20 years, using an enzyme-linked immunosorbent assay (ELISA). The usefulness of urinary PT-3-G concentrations before giving the morning dose of medications in monitoring the adequacy of glucocorticoid treatment was assessed in eight patients with 21-hydroxylase deficiency (21-OHD). The ratio of PT-3-G to excreted creatinine (PT-3-G/Cre ratio) increased significantly during the first month and did not change from age 1 month to 1 year of life. The ratio decreased to a nadir at age 3 or 4 years followed by continuous, significant increase until late adolescence. In the subjects treated with corticosteroids for 21-OHD, PT-3-G/Cre ratios at the 50th percentile or below suggested a risk of excessive treatment, as judged by the patients' growth. Measurement of the PT-3-G/Cre ratio enabled recognition of corticosteroid overtreatment, which was not demonstrated by determining the serum concentrations of 17 alpha-hydroxyprogesterone (17-OHP). On the other hand, ratios at the upper 95-99% tolerance limits seemed to be required for optimal control. The present study revealed the normal age-related changes in urinary excretion of PT-3-G and showed it to be a reliable marker for evaluating glucocorticoid treatment in young children with 21-OHD.

Adolescent↗
PubMed1996

Human trophoblast cell adhesion to extracellular matrix protein, entactin.

PROBLEM: Trophoblast interaction with endometrial extracellular matrix (ECM) is crucial during human embryo implantation and placentation. Entactin, a ubiquitous basement membrane glycoprotein, plays a central role in ECM assembly, cell attachment, and chemotaxis. The present study was conducted to examine the possible role of entactin in promoting human trophoblast adhesion. METHODS: Using an extended life span first trimester trophoblast cell line HTR-8/SVneo (HTR) and a cell adhesion assay, we measured the adherence of human first trimester trophoblasts to recombinant entactin and its domains. Also, we used flow cytometry and indirect immunofluorescence to detect the presence of integrins that may be involved in human trophoblast-entactin interaction; these methods were used to analyze HTR cells, as well as tissue sections and freshly isolated human trophoblasts from first trimester and term placenta. RESULTS: We found that first trimester trophoblast cells were highly adherent to entactin and its E and G2 domains but not to G1 or G3 domains. Using indirect immunofluorescence and flow cytometry, we found that both beta 1 and beta 3 integrin subunits were expressed on the surface of HTR trophoblast cells adhering to entactin; in contrast, beta 2 and beta 4 integrin subunits were not detected. In addition, we found that alpha v beta 3 was expressed on freshly isolated villous cytotrophoblasts and cytotrophoblast and syncytiotrophoblasts in tissue sections from term placenta. The beta 3 integrin subunit was expressed in cytotrophoblasts and syncytiotrophoblasts in villi of first trimester placental tissue sections. CONCLUSION: Recombinant entactin promotes human trophoblast cell adhesion through both its E and G2 domains and these specific adhesive interactions may be mediated by beta 1 and/or beta 3 class integrins.

Antigens, CD↗
PubMed1996

Effects of tumor necrosis factor-alpha on human trophoblast cell adhesion and motility.

PROBLEM: Adhesive interaction between trophoblast cells and uterine endometrial basement membrane is one of the critical processes in embryo implantation. This interaction is directly or indirectly regulated by hormones, growth factors, and cytokines. Since tumor necrosis factor-alpha (TNF-alpha) is synthesized by both decidual and trophoblast cells, we hypothesized that TNF-alpha may play a regulatory role in trophoblast cell invasion. To test this hypothesis, we have used in vitro models to determine the effect of TNF-alpha on human trophoblast cell adhesion and motility, two major steps in trophoblast invasion. METHODS: The effect of TNF-alpha on the motility of extended-lifespan first trimester trophoblasts (HTR) and JEG-3 choriocarcinoma cells was tested using the phagokinetic track motility assay. An in vitro adhesion assay was used to determine the effect of TNF-alpha on the adhesion of HTR and JEG-3 cells to laminin, a major basement membrane component. In addition, the effect of TNF-alpha on the surface expression of the laminin receptor beta 1 integrin subunit was examined using flow cytometry. RESULTS: HTR or JEG-3 cells strongly adherent to laminin which was not significantly altered by TNF-alpha treatment. We also measured the effect of TNF-alpha on the surface expression of beta 1 integrin on HTR and JEG-3 cells; no difference was observed between control and treatment groups. Interestingly, the motility of both HTR and choriocarcinoma JEG-3 cells was significantly inhibited by TNF-alpha. CONCLUSIONS: The role of TNF-alpha in human embryo implantation is currently unknown. Our data demonstrate that TNF-alpha does alter trophoblast cell adhesion to laminin, but significantly inhibits trophoblast cell motility in vitro, suggesting that TNF-alpha may play a regulatory role in trophoblast cell invasion.

Cell Adhesion↗
PubMed1996

Characterization of IMI-1 beta-lactamase, a class A carbapenem-hydrolyzing enzyme from Enterobacter cloacae.

In 1984, a year prior to the U.S. approval of imipenem for clinical use, a wound isolate and a bile isolate of Enterobacter cloacae were obtained from two patients in a California hospital. These isolates were resistant to imipenem, penicillins, and inhibitor combinations; early cephalosporins such as cephalothin, cefamandole, and cefoxitin; and cefoperazone. However, they were susceptible (MICs, < 4 micrograms/ml) to cefotaxime, ceftriaxone, ceftazidime, and moxalactam. Both strains produced an apparent TEM-1 beta-lactamase; an inducible NmcA-type imipenem-hydrolyzing beta-lactamase, IMI-1, with a pl of 7.05; and an inducible beta-lactamase with a pI of 8.1, typical of an E. cloacae AmpC beta-lactamase. Purified IMI-1 hydrolyzed imipenem and benzylpenicillin at modest rates, but more slowly than cephaloridine. The enzyme was inhibited by clavulanic acid and tazobactam. EDTA did not inhibit the cephaloridine-hydrolyzing activity. The beta-lactamase gene encoding IMI-1, imiA1, was cloned from E. cloacae 1413B. Sequence analysis identified the imiA1 gene as encoding a class A serine beta-lactamase. Both the imiA1 DNA and encoded amino acid sequences shared greater than 95% identity with the NmcA gene and its encoded protein. DNA sequence analysis also identified a gene upstream of imiA1 that shares > 95% identity with nmcR and that may encode a regulatory protein. In conclusion, IMI-1, a carbapenem-hydrolyzing beta-lactamase inhibited by clavulanic acid, was identified as a group 2f, class A, carbapenem-hydrolyzing cephalosporinase.

Amino Acid Sequence↗
PubMed1996

Biotransformation of chlorpromazine and methdilazine by Cunninghamella elegans.

When tested as a microbial model for mammalian drug metabolism, the filamentous fungus Cunninghamella elegans metabolized chlorpromazine and methdilazine within 72 h. The metabolites were extracted by chloroform, separated by high-performance liquid chromatography, and characterized by proton nuclear magnetic resonance, mass, and UV spectroscopic analyses. The major metabolites of chlorpromazine were chlorpromazine sulfoxide (36%), N-desmethylchlorpromazine (11%), N-desmethyl-7-hydroxychlorpromazine (6%), 7-hydroxychlorpromazine sulfoxide (36%), N-hydroxychlorpromazine (11%), 7-hydroxychlorpromazine sulfoxide (5%), and chlorpromazine N-oxide (2%), all of which have been found in animal studies. The major metabolites of methdilazine were 3-hydroxymethdilazine (3%). (18)O(2) labeling experiments indicated that the oxygen atoms in methdilazine sulfoxide, methdilazine N-oxide, and 3-hydroxymethdilazine were all derived from molecular oxygen. The production of methdilazine sulfoxide and 3-hydroxymethdilazine was inhibited by the cytochrome P-450 inhibitors metyrapone and proadifen. An enzyme activity for the sulfoxidation of methdilazine was found in microsomal preparations of C. elegans. These experiments suggest that the sulfoxidation and hydroxylation of methdilazine and chlorpromazine by C. elegans are catalyzed by cytochrome P-450.

Antipsychotic Agents↗
PubMed1996

The psa locus is responsible for thermoinducible binding of Yersinia pseudotuberculosis to cultured cells.

Yersinia pseudotuberculosis inv mutant strains cured of the virulence plasmid exhibit thermoinducible adhesion to cultured mammalian cells. To identify the genes responsible for this phenotype, Y. pseudotuberculosis homologs of the Y. enterocolitica ail and the Y. pestis psa loci were identified. Mutations in the Y. pseudotuberculosis ail and psa loci were constructed and tested for thermoinducible binding. Results of cellular binding assays indicated that only mutations in psa, not in ail, resulted in defects for thermoinducible binding, with inv yadA psa strains showing no detectable cell adhesion. In addition, an inv psa strain was defective for hemagglutination of sheep erythrocytes, in contrast to an inv psa+ strain which was fully competent for hemagglutination. The introduction of a plasmid containing a 6.7-kb KpnI-ClaI fragment of Y. pseudotuberculosis encompassing the psa locus was sufficient to complement both the cell adhesion and hemagglutination defects of the psa mutant. Results from subcloning and transposon mutagenesis indicated that the complete 6.7-kb region was required for thermoinducible binding and hemagglutination.

Amino Acid Sequence↗
PubMed1996

Recombinant adeno-associated virus-mediated high-efficiency, transient expression of the murine cationic amino acid transporter (ecotropic retroviral receptor) permits stable transduction of human HeLa cells by ecotropic retroviral vectors.

Adeno-associated virus has a broad host range, is nonpathogenic, and integrates into a preferred location on chromosome 19, features that have fostered development of recombinant adeno-associated viruses (rAAV) as gene transfer vectors for therapeutic applications. We have used an rAAV to transfer and express the murine cationic amino acid transporter which functions as the ecotropic retroviral receptor, thereby rendering human cells conditionally susceptible to infection by an ecotropic retroviral vector. The proportion of human HeLa cells expressing the receptor at 60 h varied as a function of the multiplicity of infection (MOI) with the rAAV. Cells expressing the ecotropic receptor were efficiently transduced with an ecotropic retroviral vector encoding a nucleus-localized form of beta-galactosidase. Cells coexpressing the ecotropic receptor and nucleus-localized beta-galactosidase were isolated by fluorescence-activated cell sorting, and cell lines were recovered by cloning at limiting dilution. After growth in culture, all clones contained the retroviral vector genome, but fewer than 10% (3 of 47) contained the rAAV genome and continued to express the ecotropic receptor. The ecotropic receptor coding sequences in the rAAV genome were under the control of a tetracycline-modulated promoter. In the presence of tetracycline, receptor expression was low and the proportion of cells transduced by the ecotropic retroviral vector was decreased. Modulation of receptor expression was achieved with both an episomal and an integrated form of the rAAV genome. These data establish that functional gene expression from an rAAV genome can occur transiently without genome integration.

Amino Acid Transport Systems↗
PubMed1996

Role of viral antigens in destructive cellular immune responses to adenovirus vector-transduced cells in mouse lungs.

Adenoviruses missing E1 have been used as gene delivery vectors to the lungs for the treatment of cystic fibrosis. Transient expression of the recombinant gene and the development of inflammation have been two major limitations to the application of first-generation recombinant adenoviruses for gene therapy. Studies with mouse models of liver- and lung-directed gene therapy suggested that CD8+ cytotoxic T lymphocytes (CTLs) are effectors that contribute to extinction of transgene expression. The precise antigens responsible for activation of CTLs have not been identified. In this study, we examine the relative contributions of viral proteins versus the transgene product to the activation of CTLs which eliminate transgene-containing cells in mouse lungs. Instillation of a lacZ-expressing virus into the lungs of C57BL/6 mice elicited CTL responses to both viral proteins and the transgene product, beta-galactosidase, which collectively contribute to loss of trans-gene expression in mouse airways. Similar results were obtained in two experimental models in which the animals should be tolerant to the transgene, i.e., lacZ virus delivered to an animal transgenic for lacZ and a virus expressing the liver-specific enzyme ornithine transcarbamylase administered to the lungs of various strains of immune-competent mice. These data confirm the hypothesis that CTLs specific for viral antigens contribute to the problem of transgene instability in mouse lungs and indicate that CTLs specific for transgene product alone cannot account for the observed problem.

Adenoviridae↗
PubMed1996

Biology of adenovirus vectors with E1 and E4 deletions for liver-directed gene therapy.

Recombinant adenoviruses with E1 sequences deleted efficiently transfer genes into a wide variety of target cells. Antigen- and nonantigen-specific responses to the therapy lead to toxicity, loss of transgene expression, and difficulties with vector readministration. We have created new cell lines that allowed the isolation of more disabled adenovirus vectors that have both E1 and E4 deletions. Studies with murine models of liver-directed gene therapy indicated that the E1- and E4-deleted vector expresses fewer virus proteins and induces less apoptosis, leading to blunted host responses and an improved safety profile. The impact of the E4 deletion on the stability of vector expression was confounded by immune responses to the transgene product, which in this study was beta-galactosidase. When transgene responses were eliminated, the doubly deleted vector was substantially more stable in mouse liver than was the E1-deleted construct. These studies indicate that adenovirus vectors with both E1 and E4 deletions may have advantages in terms of safety and efficacy over first-generation constructs for liver-directed gene therapy.

Adenovirus E1 Proteins↗
PubMed1996

Transient subversion of CD40 ligand function diminishes immune responses to adenovirus vectors in mouse liver and lung tissues.

First-generation adenovirus vectors will have limited application in gene therapy for chronic diseases because of destructive host immune responses. Important immune effectors include CD8+ T cells, which mediate target cell destruction and ablate transgene expression, and B cells, which produce neutralizing antibodies that block effective readministration of vector. Previous studies indicated that activation of CD4+ T cells by virus capsid proteins is necessary for full realization of effector function of CD8+ T cells and B cells. In this paper, we present a strategy for preventing CD4+ T-cell activation by an adenovirus vector delivered to mouse liver and lung tissues which is based on interfering with T-cell priming via CD40 ligand-CD40 interactions. Adenovirus transgene expression was stabilized in mice genetically deficient in CD40 ligand (CD40L), and neutralizing antibody to adenovirus did not develop, allowing efficient readministration of vector. A transient blockade of T-cell activation with an antibody to CD40L infused into the animal at the time of adenovirus vector-mediated gene transfer led to stabilization of transgene expression and diminished production of neutralizing antibody, allowing readministration of vector. In vitro T-cell assays suggested that a block in the primary activation of CD4+ T cells was responsible for the lack of B-cell- and cytotoxic-T-cell-dependent responses. This suggests a strategy for improving the potential of adenovirus vectors based on administration of an antibody to CD40L at the time of vector administration.

Actins↗