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J Yang

Publications and source records attributed to J Yang.

At least 577 records · Page 32Linked to original sources
PubMed1999

Identification of the endonuclease domain encoded by R2 and other site-specific, non-long terminal repeat retrotransposable elements.

The non-long terminal repeat (LTR) retrotransposon, R2, encodes a sequence-specific endonuclease responsible for its insertion at a unique site in the 28S rRNA genes of arthropods. Although most non-LTR retrotransposons encode an apurinic-like endonuclease upstream of a common reverse transcriptase domain, R2 and many other site-specific non-LTR elements do not (CRE1 and 2, SLACS, CZAR, Dong, R4). Sequence comparison of these site-specific elements has revealed that the region downstream of their reverse transcriptase domain is conserved and shares sequence features with various prokaryotic restriction endonucleases. In particular, these non-LTR elements have a Lys/Arg-Pro-Asp-X12-14aa-Asp/Glu motif known to lie near the scissile phosphodiester bonds in the protein-DNA complexes of restriction enzymes. Site-directed mutagenesis of the R2 protein was used to provide evidence that this motif is also part of the active site of the endonuclease encoded by this element. Mutations of this motif eliminate both DNA-cleavage activities of the R2 protein: first-strand cleavage in which the exposed 3' end is used to prime reverse transcription of the RNA template and second-strand cleavage, which occurs after reverse transcription. The general organization of the R2 protein appears similar to the type IIS restriction enzyme, FokI, in which specific DNA binding is controlled by a separate domain located amino terminal to the cleavage domain. Previous phylogenetic analysis of their reverse transcriptase domains has indicated that the non-LTR elements identified here as containing restriction-like endonucleases are the oldest lineages of non-LTR elements, suggesting a scenario for the evolution of non-LTR elements.

Amino Acid Sequence↗
PubMed1999

Impaired ability of MHC class II-/- dendritic cells to provide tumor protection is rescued by CD40 ligation.

The contribution of CD4+ T cells to dendritic cell (DC) activation and to the induction of CD8+ T cell responses in vivo was investigated using a model of antitumor immune responses. Immunization with peptide-loaded MHC class II-deficient (MHC class II-/-) DC induced the activation of Ag-specific CD8+ T cells and their accumulation in the lymph nodes and spleens of immunized mice. The accumulation induced by MHC class II-/- DC immunization was lower than the accumulation observed after immunization with MHC class II+/+ DC. Similarly, immunization with peptide-loaded, MHC class II-/- DC induced some degree of protection against tumor challenge, but this protection was lower than the protection achieved after immunization with MHC class II+/+ DC. Incubation with a membrane-associated form of CD40 ligand resulted in the up-regulation of costimulatory molecules on MHC class II-/- DC and fully rescued their ability to induce antitumor immunity. We conclude that CD4+ T cells play a critical role in the generation of antitumor immune responses through their capacity to induce the activation of DC via CD40/CD40 ligand interaction, and thus maximize CD8+ T cell responses.

Animals↗
PubMed1999

LD78beta, a non-allelic variant of human MIP-1alpha (LD78alpha), has enhanced receptor interactions and potent HIV suppressive activity.

Chemokines play diverse roles in inflammatory and non-inflammatory situations via activation of heptahelical G-protein-coupled receptors. Also, many chemokine receptors can act as cofactors for cellular entry of human immunodeficiency virus (HIV) in vitro. CCR5, a receptor for chemokines MIP-1alpha (LD78alpha), MIP-1beta, RANTES, and MCP2, is of particular importance in vivo as polymorphisms in this gene affect HIV infection and rate of progression to AIDS. Moreover, the CCR5 ligands can prevent HIV entry through this receptor and likely contribute to the control of HIV infection. Here we show that a non-allelic isoform of human MIP-1alpha (LD78alpha), termed LD78beta or MIP-1alphaP, has enhanced receptor binding affinities to CCR5 (approximately 6-fold) and the promiscuous beta-chemokine receptor, D6 (approximately 15-20-fold). We demonstrate that a proline residue at position 2 of MIP-1alphaP is responsible for this enhanced activity. Moreover, MIP-1alphaP is by far the most potent natural CCR5 agonist described to date, and importantly, displays markedly higher HIV1 suppressive activity than all other human MIP-1alpha isoforms examined. In addition, while RANTES has been described as the most potent inhibitor of CCR5-mediated HIV entry, MIP-1alphaP was as potent as, if not more potent than, RANTES in HIV-1 suppressive assays. This property suggests that MIP-1alphaP may be of importance in controlling viral spread in HIV-infected individuals.

Amino Acid Sequence↗
PubMed1999

The C domain of HIV-1 gp41 binds the putative cellular receptor protein P62.

OBJECTIVE: To characterize the binding of gp41 with the putative receptor protein P62. DESIGN: HIV-1 gp41 binds several cellular proteins by two binding sites, one of which has been shown to bind to a putative receptor protein P45 (45 kDa). Based on this, an attempt was made to determine the relationship between the two binding sites and P62 (62 kDa). METHODS: Using surface plasmon resonance (SPR) measurement, the interaction was measured between recombinant soluble gp41 (rsgp41, Env aa539-684) and protein P62. Inhibition of this interaction was attempted by the use of synthetic peptides (P1, aa583-599; P2, aa646-674) corresponding to the two binding sites in gp41. In addition, the direct binding of P62 to peptide P2 was examined in an enzyme-linked immunosorbent assay. RESULTS: Using SPR measurement, the interaction between P62 and rsgp41 was confirmed, and the interaction was found to be inhibited by only the synthetic peptide P2 sequence that corresponds to the C domain of gp41; neither P1 nor a control peptide inhibited the interaction. Moreover, like rsgp41, P2 was able to bind P62 whereas P1 and another recombinant gp41 (aa567-648 that does not include the C domain) were not. CONCLUSIONS: P62 bound rsgp41 and the synthetic peptide P2. This interaction could be inhibited only by P2. These results indicate that the C domain of HIV-1 gp41 binds P62.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Isolation, characterization, and functional analysis of a novel cDNA clone encoding a small rubber particle protein from Hevea brasiliensis.

Biochemical evidence reported so far suggests that rubber synthesis takes place on the surface of rubber particles suspended in the latex of Hevea brasiliensis. We have isolated and characterized a cDNA clone that encodes a protein tightly bound on a small rubber particle. We named this protein small rubber particle protein (SRPP). Prior to this study, this protein was known as a latex allergen, and only its partial amino acid sequence was reported. Sequence analysis revealed that this protein is highly homologous to the rubber elongation factor and the Phaseolus vulgaris stress-related protein. Southern and Northern analyses indicate that the protein is encoded by a single gene and highly expressed in latex. An allergenicity test using the recombinant protein confirmed that the cloned cDNA encodes the known 24-kDa latex allergen. Neither ethylene stimulation nor wounding changed the transcript level of the SRPP gene in H. brasiliensis. An in vitro rubber assay showed that the protein plays a positive role in rubber biosynthesis. Therefore, it is likely that SRPP is a part of the rubber biosynthesis machinery, if not the rubber polymerase, along with the rubber elongation factor.

Allergens↗
PubMed1999

The chicken HPRT gene: a counter selectable marker for the DT40 cell line.

We describe the cloning, characterisation and chromosomal mapping of the chicken hprt gene together with the construction of two counter selectable hprt-/- DT40 derived cell lines. One of these cell lines contains a stably integrated gene encoding a conditionally active cre recombinase and thus allows efficient manipulation of targeted loci by site-specific recombination. These cell lines will enhance the utility of the hyper-recombinogenic DT40 cell line as a system for the genetic analysis of cell autonomous functions in vertebrates and as a tool for mammalian chromosome engineering.

Amino Acid Sequence↗
PubMed1999

Maintenance of G2 arrest in the Xenopus oocyte: a role for 14-3-3-mediated inhibition of Cdc25 nuclear import.

Cdc2-cyclin B1 in the G2-arrested Xenopus oocyte is held inactive by phosphorylation of Cdc2 at two negative regulatory sites, Thr14 and Tyr15. Upon treatment with progesterone, these sites are dephosphorylated by the dual specificity phosphatase, Cdc25, leading to Cdc2-cyclin B1 activation. Whereas maintenance of the G2 arrest depends upon preventing Cdc25-induced Cdc2 dephosphorylation, the mechanisms responsible for keeping Cdc25 in check in these cells have not yet been described. Here we report that Cdc25 in the G2-arrested oocyte is bound to 14-3-3 proteins and that progesterone treatment abrogates this binding. We demonstrate that Cdc25, apparently statically localized in the cytoplasm, is actually capable of shuttling in and out of the oocyte nucleus. Binding of 14-3-3 protein markedly reduces the nuclear import rate of Cdc25, allowing nuclear export mediated by a nuclear export sequence present in the N-terminus of Cdc25 to predominate. If 14-3-3 binding to Cdc25 is prevented while nuclear export is inhibited, the coordinate nuclear accumulation of Cdc25 and Cdc2-cyclin B1 facilitates their mutual activation, thereby promoting oocyte maturation.

14-3-3 Proteins↗
PubMed1999

Hormonal prevention of breast cancer: mimicking the protective effect of pregnancy.

Full term pregnancy early in life is the most effective natural protection against breast cancer in women. Rats treated with chemical carcinogen are similarly protected by a previous pregnancy from mammary carcinogenesis. Proliferation and differentiation of the mammary gland does not explain this phenomenon, as shown by the relative ineffectiveness of perphenazine, a potent mitogenic and differentiating agent. Here, we show that short term treatment of nulliparous rats with pregnancy levels of estradiol 17beta and progesterone has high efficacy in protecting them from chemical carcinogen induced mammary cancers. Because the mammary gland is exposed to the highest physiological concentrations of estradiol and progesterone during full term pregnancy, it is these elevated levels of hormones that likely induce protection from mammary cancer. Thus, it appears possible to mimic the protective effects of pregnancy against breast cancer in nulliparous rats by short term specific hormonal intervention.

Animals↗
PubMed1999

Heptameric structures of two alpha-hemolysin mutants imaged with in situ atomic force microscopy.

Atomic force microscopy has been used to study self-assembled structures of two alpha-hemolysin mutants. For a mutant (alphaHL-H5) that was locked into the prepore state on fluid phase egg-PC membranes, we visualized, for the first time, heptameric prepores and showed that the 7-fold axis in the prepore lies perpendicular to the membrane surface. For another mutant (TCM) with the transmembrane domain, the self-assembled oligomer that assumes the conformation of the fully assembled pore is also a heptamer. These results show that heptamers are the preferred oligomerization state of alpha-hemolysin.

Bacterial Toxins↗
PubMed1999

Self-glucosylation of glycogenin, the initiator of glycogen biosynthesis, involves an inter-subunit reaction.

Glycogenin is a dimeric self-glucosylating protein involved in the initiation phase of glycogen biosynthesis. As an enzyme, glycogenin has the unusual property of transferring glucose residues from UDP-glucose to itself, forming an alpha-1,4-glycan of around 10 residues attached to Tyr194. Whether this self-glucosylation reaction is inter- or intramolecular has been debated. We used site-directed mutagenesis of recombinant rabbit muscle glycogenin-1 to address this question. Mutation of highly conserved Lys85 to Gln generated a glycogenin mutant (K85Q) that had only 1-2% of the self-glucosylating activity of wild-type enzyme. Consistent with previous work, mutation of Tyr194 to Phe in a GST-fusion protein yielded a mutant, Y194F, that was catalytically active but incapable of self-glucosylation. The Y194F mutant was able to glucosylate the K85Q mutant. However, there was an initial lag in the self-glucosylation reaction that was abolished by preincubation of the two mutant proteins. The interaction between glycogenin subunits was relatively weak, with a dissociation constant inferred from kinetic experiments of around 2 microM. We propose a model for the glucosylation of K85Q by Y194F in which mixing of the proteins is followed by rate-limiting formation of a species containing both subunit types. The results provide the most direct evidence to date that the self-glucosylation of glycogenin involves an inter-subunit reaction.

Amino Acid Substitution↗
PubMed1999

Cell separation on microfabricated electrodes using dielectrophoretic/gravitational field-flow fractionation.

Dielectrophoretic/gravitational field-flow fractionation (DEP/G-FFF) was used to separate cultured human breast cancer MDA-435 cells from normal blood cells mixed together in a sucrose/dextrose medium. An array of microfabricated, interdigitated electrodes of 50 microns widths and spacings, and lining the bottom surface of a thin chamber (0.42 mm H x 25 mm W x 300 mm L), was used to generate DEP forces that levitated the cells. A 10-microL cell mixture sample containing approximately 50,000 cells was introduced into the chamber, and cancerous and normal blood cells were levitated to different heights according to the balance of DEP and gravitational forces. The cells at different heights were transported at different velocities under the influence of a parabolic flow profile that was established in the chamber and were thereby separated. Separation performance depended on the frequency and voltage of the applied DEP field and the fluid-flow rate. It took as little as 5 min to achieve cell separation. An analysis of the DEP/G-FFF results revealed that the separation exploited the difference in dielectric and density properties between cell populations. The DEP/G-FFF technique is potentially applicable to many biological and biomedical problems, especially those related to microfluidic systems.

Algorithms↗
PubMed1999

Familial aggregation of blood pressure in a rural Chinese community.

This study investigated blood pressure in 1,183 Chinese nuclear families (mother, father, and first two children) via a cross-sectional 1994-1997 survey. The mother's, the father's, and the first sibling's blood pressures were each significantly and independently related to the second sibling's blood pressure after adjustment for sex, age, height, weight, education, smoking, and alcohol consumption. The association was consistent across the four age strata (6-10, 11-14, 15-19, and > or = 20 years). The rate of high systolic blood pressure in the second sibling was lowest (2.3%) when both parents and the first sibling were in the low blood pressure tertile (low-low group) and highest (26.0%) when these family members were in the high blood pressure tertile (high-high group). The rate was intermediate if only the parents (10.7%, high-low group) or the first sibling (8.4%, low-high group) was in the high blood pressure tertile. As compared with the low-low group, the odds ratios for the high-high, high-low, and low-high groups were 14.3 (95% confidence interval 4.3-48.2), 4.3 (95% confidence interval 1.2-15.6), and 3.9 (95% confidence interval 1.1-14.4), respectively. A similar pattern was found for diastolic blood pressure. The data indicate a strong familial aggregation of blood pressure in this population and show that such a familial influence on blood pressure can be detected from early childhood onward.

Adolescent↗
PubMed1999

Transcriptional regulation of alpha1-adrenoceptor gene in the rat liver during different phases of sepsis.

Changes in alpha1-adrenoceptor (alpha1AR) gene expression in the rat liver during different phases of sepsis were studied. Sepsis was induced by cecal ligation and puncture (CLP). Septic rats exhibit two metabolically distinct phases: an initial hyperglycemic phase (9 h after CLP, early sepsis) followed by a hypoglycemic phase (18 h after CLP; late sepsis). The [3H]prazosin binding studies show that the density of alpha1AR was increased by 30% during the early phase while it was decreased by 24% during the late phase of sepsis. Western blot analyses reveal that alpha1AR protein level was elevated by 48% during early sepsis but was decreased by 55% during late sepsis. Northern blot analyses depict that the steady-state level of alpha1bAR mRNA was enhanced by 21% during the early phase but was declined by 29% during the late phase of sepsis. Nuclear run-off assays show that the transcription rate of alpha1bAR gene transcript was increased by 76% during early sepsis while it was decreased by 29% during late sepsis. The actinomycin D pulse-chase studies indicate that the half-life of alpha1bAR mRNA remained unaffected during the early and the late phases of sepsis. These findings demonstrate that during the early phase of sepsis, the increase in the rate of transcription of alpha1bAR gene paralleled with the elevations in the alpha1bAR mRNA abundance and alpha1AR protein level, while during the late phase of sepsis, the decrease in the rate of transcription of alpha1bAR gene coincided with the declines in the alpha1bAR mRNA abundance and the alpha1AR protein level in the rat liver. These observations indicate that the altered expression of alpha1AR genes in the rat liver during the progression of sepsis was regulated transcriptionally.

Adrenergic alpha-Antagonists↗
PubMed1999

Molecular cloning and characterization of LKv1, a novel voltage-gated potassium channel in leech.

We have cloned a novel voltage-gated K channel, LKv1, in two species of leech. The properties of LKv1 expressed in transiently transfected HEK293 cells is that of a delayed rectifier current. LKv1 may be a major modulator of excitability in leech neurons, since antibody localization studies show that LKv1 is expressed in the soma and axons of all neurons in both the central and peripheral nervous systems. Comparison of the biophysical and pharmacological properties of LKv1 with native voltage-gated conductances in leech neurons suggests that LKv1 may correspond to the previously characterized delayed rectifier current, I(K). Phylogenetic analysis of LKv1 shows that it is related to the Shaker subfamily of voltage-gated K channels although it occupies a separate branch from that of the monophyletic Shaker clade composed of the flatworm, Aplysia, Drosophila, and mammalian Shaker homologs as well as from that of two recently identified Shaker-related K channels in jellyfish. Thus, this analysis indicates that this group of voltage-gated K channels contains several evolutionarily divergent lineages.

Amino Acid Sequence↗
PubMed1999

Nuclear import of Cdk/cyclin complexes: identification of distinct mechanisms for import of Cdk2/cyclin E and Cdc2/cyclin B1.

Reversible phosphorylation of nuclear proteins is required for both DNA replication and entry into mitosis. Consequently, most cyclin-dependent kinase (Cdk)/cyclin complexes are localized to the nucleus when active. Although our understanding of nuclear transport processes has been greatly enhanced by the recent identification of nuclear targeting sequences and soluble nuclear import factors with which they interact, the mechanisms used to target Cdk/cyclin complexes to the nucleus remain obscure; this is in part because these proteins lack obvious nuclear localization sequences. To elucidate the molecular mechanisms responsible for Cdk/cyclin transport, we examined nuclear import of fluorescent Cdk2/cyclin E and Cdc2/cyclin B1 complexes in digitonin-permeabilized mammalian cells and also examined potential physical interactions between these Cdks, cyclins, and soluble import factors. We found that the nuclear import machinery recognizes these Cdk/cyclin complexes through direct interactions with the cyclin component. Surprisingly, cyclins E and B1 are imported into nuclei via distinct mechanisms. Cyclin E behaves like a classical basic nuclear localization sequence-containing protein, binding to the alpha adaptor subunit of the importin-alpha/beta heterodimer. In contrast, cyclin B1 is imported via a direct interaction with a site in the NH2 terminus of importin-beta that is distinct from that used to bind importin-alpha.

Animals↗
PubMed1999

Tissue-specific and developmental stage-specific DNA binding by a mammalian SWI/SNF complex associated with human fetal-to-adult globin gene switching.

SWI/SNF complexes in yeast and higher eukaryotes are thought to facilitate gene activation and transcription factor binding by disrupting repressive chromatin structures. Little is known, however, about how these complexes target specific genes for activation. We now have purified a specialized SWI/SNF-related complex (PYR complex) from murine erythroleukemia (MEL) cell nuclear extract that binds pyrimidine-rich elements at the human and murine beta-globin loci. PYR complex DNA-binding activity is restricted to definitive hematopoietic cells and is both DNA sequence- and length-dependent. Mass spectrometric identification of purified peptides and antibody supershift assays indicate that PYR complex contains at least four known mammalian SWI/SNF subunits: BAF57, INI1, BAF60a, and BAF170. PYR complex broadly footprints a 250-bp pyrimidine-rich element between the human fetal and adult beta-globin genes. A short intergenic deletion that removes this element from a human globin locus cosmid construct results in delayed human fetal-to-adult globin gene switching in transgenic mice. Taken together, the data suggest that PYR complex may act through this intergenic element to facilitate human fetal-to-adult globin gene switching, presumably by opening the locus in the region of the adult genes to permit the binding of beta-globin transcriptional activators.

Animals↗
PubMed1999

Role of peroxide in AC electrical field exposure effects on friend murine erythroleukemia cells during dielectrophoretic manipulations.

The effects of AC field exposure on the viability and proliferation of mammalian cells under conditions appropriate for their dielectrophoretic manipulation and sorting were investigated using DS19 murine erythroleukemia cells as a model system. The frequency range 100 Hz-10 MHz and medium conductivities of 10 mS/m, 30 mS/m and 56 mS/m were studied for fields generated by applying signals of up to 7V peak to peak (p-p) to a parallel electrode array having equal electrode widths and gaps of 100 micrometer. Between 1 kHz and 10 MHz, cell viability after up to 40 min of field exposure was found to be above 95% and cells were able to proliferate. However, cell growth lag phase was extended with decreasing field frequency and with increasing voltage, medium conductivity and exposure duration. Modified growth behavior was not passed on to the next cell passage, indicating that field exposure did not cause permanent alterations in cell proliferation characteristics. Cell membrane potentials induced by field exposure were calculated and shown to be well below values typically associated with cell damage. Furthermore, medium treated by field exposure and then added to untreated cells produced the same modifications of growth as exposing cells directly, and these modifications occurred only when the electrode polarization voltage exceeded a threshold of approximately 0.4 V p-p. These findings suggested that electrochemical products generated during field exposure were responsible for the changes in cell growth. Finally, it was found that hydrogen peroxide was produced when sugar-containing media were exposed to fields and that normal cell growth could be restored by addition of catalase to the medium, whether or not field exposure occurred in the presence of cells. These results show that AC fields typically used for dielectrophoretic manipulation and sorting of cells do not damage DS19 cells and that cell alterations arising from electrochemical effects can be completely mitigated.

Animals↗