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Biomedical subjects

J Yang

Publications and source records attributed to J Yang.

At least 397 records · Page 22Linked to original sources
PubMed2000

Histomorphologically intact primary human breast lesions and cancers can be propagated in nude mice.

The natural history of various human breast lesions and cancer in patient is difficult to study since diagnosis is established only after surgical ablation and therefore the clinical course of the undisturbed lesion in vivo can no longer be followed. Development of an experimental system which can propagate and maintain human breast lesions and cancer, similar to those seen in surgical breast specimens, may be a first step in the analyses of these various entities. We report that histomorphologically intact human breast lesions and cancer can be recreated in an experimental system using athymic nude mice. When the dissociated cells from surgical breast cancer specimens are embedded in extracellular matrices (collagen gel, Matrigel, or a mixture of the two) and then transplanted into athymic nude mice, the transplanted cells undergo morphogenesis to reflect their original phenotype. A mixture of the two matrices most closely mirrored the breast tissue in situ when stained with Alcian blue-PAS which stains for mucins. In this mixture, not only is the histomorphology recreated, but well-established clinical molecular markers including the estrogen receptor, c-erbB-2 (HER-2/neu), and aberrant cell proliferation are maintained. With the use of an appropriate extracellular matrices, surgical specimens of human breast lesions and cancer can be analyzed in an in vivo experimental system.

Actins↗
PubMed2000

Fractal structure in the collision of vector solitons

We study the collision of two orthogonally polarized and equal-amplitude vector solitons in the nonintegrable coupled nonlinear Schrodinger equations. We show that the separation velocity versus collision velocity graph has a fractal structure. When we zoom into this graph, we get a structure qualitatively identical to the original one. In addition, collision dynamics in the zoomed-in windows is intimately related to that in the original graph. We explain this fractal dependence of the collision by a resonance mechanism between the translational motion of vector solitons and internal oscillations inside a vector soliton.

Journal Article↗
PubMed2000

Chromosome abnormalities in malignant melanoma: clinical significance of nonrandom chromosome abnormalities in 206 cases.

We report the cytogenetic abnormalities from a series of 206 primary malignant melanoma specimens referred to a single institution. A total of 169 out of 206 unique cases had chromosome breakpoints. A previously described statistical method was used to detect nonrandom distribution of chromosome breakpoints at the level of chromosome regions. Nonrandom occurrence of chromosome breakpoints (indicating that the observed number of breaks significantly exceeded the expected number of breaks) was detected in 28 regions, suggesting a hierarchy of genetic abnormalities in melanoma. Clinical variables and tumor characteristics were analyzed for associations with the presence of any nonrandom chromosome breakpoints; with individual, nonrandomly involved chromosome regions; and with paired, nonrandomly involved chromosome regions. No nonrandomly involved chromosome regions or pairs of regions appeared to significantly affect survival. These results identify recurring, nonrandom chromosome abnormalities in malignant melanoma. These results suggest that recurring, nonrandom chromosome alterations play a key role in the etiology and/or progression of malignant melanoma and identify targets within the genome for molecular genetic studies.

Chromosome Aberrations↗
PubMed2000

Regulation of the yeast transcriptional factor PHO2 activity by phosphorylation.

The induction of yeast Saccharomyces cerevisiae gene PHO5 expression is mediated by transcriptional factors PHO2 and PHO4. PHO4 protein has been reported to be phosphorylated and inactivated by a cyclin-CDK (cyclin-dependent kinase) complex, PHO80-PHO85. We report here that PHO2 can also be phosphorylated. A Ser-230 to Ala mutation in the consensus sequence (SPIK) recognized by cdc2/CDC28-related kinase in PHO2 protein led to complete loss of its ability to activate the transcription of PHO5 gene. Further investigation showed that the Pro-231 to Ser mutation inactivated PHO2 protein as well, whereas the Ser-230 to Asp mutation did not affect PHO2 activity. Since the PHO2 Asp-230 mutant mimics Ser-230-phosphorylated PHO2, we postulate that only phosphorylated PHO2 protein could activate the transcription of PHO5 gene. Two hybrid assays showed that yeast CDC28 could interact with PHO2. CDC28 immunoprecipitate derived from the YPH499 strain grown under low phosphate conditions phosphorylated GST-PHO2 in vitro. A phosphate switch regulates the transcriptional activation activity of PHO2, and mutations of the (SPIK) site affect the transcriptional activation activity of PHO2 and the interaction between PHO2 and PHO4. BIAcore(R) analysis indicated that the negative charge in residue 230 of PHO2 was sufficient to help PHO2 interact with PHO4 in vitro.

Acid Phosphatase↗
PubMed2000

Activation of hepatocyte growth factor (HGF) by endogenous HGF activator is required for metanephric kidney morphogenesis in vitro.

The interaction of hepatocyte growth factor (HGF) with c-Met has been implicated in morphogenesis of the kidney, lung, mammary gland, liver, placenta, and limb bud. HGF is secreted as an inactive zymogen and must be cleaved by a serine protease to initiate Met signaling. We show here that a serine protease specific for HGF, HGF activator (HGFA), is expressed and activated by the ureteric bud of the developing kidney in vivo and in vitro. Inhibition of HGFA activity with serine protease inhibitors reduced ureteric bud branching and inhibited glomerulogenesis and nephrogenesis. Activated HGF rescued developing kidneys from the effects of inhibitors. HGFA was localized around the tips of the ureteric bud in developing kidneys, while HGF was expressed diffusely throughout the mesenchyme. These data show that expression of HGF is not sufficient for development, but that its activation is also required. The localization of HGFA to the ureteric bud and the mesenchyme immediately adjacent to it suggests that HGFA creates a gradient of HGF activity in the developing kidney. The creation and shape of gradients of activated HGF by the localized secretion of HGF activators could play an important role in pattern formation by HGF responsive tissues.

Amino Acid Sequence↗
PubMed2000

Midkine inhibits bradykinin-stimulated Ca(2+) signaling and nitric oxide production in endothelial cells.

Effects of the heparin-binding growth factor midkine (MK) were investigated on endothelial Ca(2+) signaling and nitric oxide (NO) production. Bradykinin (10 nM) and thapsigargin (1 microM) provoked large Ca(2+) influxes under fura-2/AM fluoroscopy. Pretreatment with human MK dose-dependently (1-500 ng/ml) inhibited the Ca(2+) response to bradykinin but not that to thapsigargin. Anti-MK antibody prevented this effect. In Ca(2+)-free medium, MK greatly inhibited intracellular Ca(2+) store release by bradykinin and not that by thapsigargin, which effect was prevented by the antibody. Bradykinin increased NO production by 6.7-fold, which was inhibited 6, 44, 79, and 90% by MK at 1, 10, 100, and 500 ng/ml, respectively. MK did not affect thapsigargin-induced NO production. Our data clearly indicate that MK inhibits bradykinin-induced Ca(2+) response and NO production from endothelial cells.

Animals↗
PubMed2000

Loss of signaling through the G protein, Gz, results in abnormal platelet activation and altered responses to psychoactive drugs.

Heterotrimeric G proteins mediate the earliest step in cell responses to external events by linking cell surface receptors to intracellular signaling pathways. G(z) is a member of the G(i) family of G proteins that is prominently expressed in platelets and brain. Here, we show that deletion of the alpha subunit of G(z) in mice: (i) impairs platelet aggregation by preventing the inhibition of cAMP formation normally seen at physiologic concentrations of epinephrine, and (ii) causes the mice to be more resistant to fatal thromboembolism. Loss of G(zalpha) also results in greatly exaggerated responses to cocaine, reduces the analgesic effects of morphine, and abolishes the effects of widely used antidepressant drugs that act as catecholamine reuptake inhibitors. These changes occur despite the presence of other G(ialpha) family members in the same cells and are not accompanied by detectable compensatory changes in the level of expression of other G protein subunits. Therefore, these results provide insights into receptor selectivity among G proteins and a model for understanding platelet function and the effects of psychoactive drugs.

Animals↗
PubMed2000

Interaction of inhibitor-2 with the catalytic subunit of type 1 protein phosphatase. Identification of a sequence analogous to the consensus type 1 protein phosphatase-binding motif.

Inhibitor-2 (I-2) is the regulatory subunit of a cytosolic type 1 Ser/Thr protein phosphatase (PP1) and potently inhibits the activity of the free catalytic subunit (CS1). Previous work from the laboratory had proposed that the interaction of I-2 with CS1 involved multiple sites (Park, I. K., and DePaoli-Roach, A. A. (1994) J. Biol. Chem. 269, 28919-28928). The present study refines the earlier analysis and arrives at a more detailed model for the interaction between I-2 and CS1. Although the NH(2)-terminal I-2 regions containing residues 1-35 and 1-64 have no inhibitory activity on their own, they increase the IC(50) for I-2 by approximately 30-fold, indicating the presence of a CS1-interacting site. Based on several experimental approaches, we have also identified the sequence Lys(144)-Leu-His-Tyr(147) as a second site of interaction that corresponds to the RVXF motif present in many CS1-binding proteins. The peptide I-2(135-151) significantly increases the IC(50) for I-2 and attenuates CS1 inhibition. Replacement of Leu and Tyr with Ala abolishes the ability to counteract inhibition by I-2. The I-2(135-151) peptide, but not I-2(1-35), also antagonizes inhibition of CS1 by DARPP-32 in a pattern similar to that of I-2. Furthermore, a peptide derived from the glycogen-binding subunit, R(GL)/G(M)(61-80), which contains a consensus CS1-binding motif, completely counteracts CS1 inhibition by I-2 and DARPP-32. The NH(2)-terminal 35 residues of I-2 bind to CS1 at a site that is specific for I-2, whereas the KLHY sequence interacts with CS1 at a site shared with other interacting proteins. Other results suggest the presence of yet more sites of interaction. A model is presented in which multiple "anchoring interactions" serve to position a segment of I-2 such that it sterically occludes the catalytic pocket but need not make high affinity contacts itself.

Amino Acid Motifs↗
PubMed2000

Human mini-chromosomes with minimal centromeres.

We have introduced a 6.5 Mb human mini-chromosome with a complex centromere structure into DT40 cells and have used sequence targeting and telomere-directed chromosome breakage to dissect the sequence requirements for centromere function. These experiments proved that a vertebrate centromere with two blocks of functional alphoid DNA separated by 2.5 Mb can exist as a stable structure in some but not all vertebrate cells. Further experiments indicated that recovery of chromosomes with less than approximately 100 kb of alphoid DNA is very inefficient, suggesting that a functional centromere requires a minimum of approximately 100 kb of alphoid DNA. Mini-chromosomes with minimal centromeres segregate accurately in some but not all vertebrate cells and should be useful for the detection of sequence-specific factors required for vertebrate centromere maintenance.

Animals↗
PubMed2000

The origin of intermediary metabolism.

The core of intermediary metabolism in autotrophs is the citric acid cycle. In a certain group of chemoautotrophs, the reductive citric acid cycle is an engine of synthesis, taking in CO(2) and synthesizing the molecules of the cycle. We have examined the chemistry of a model system of C, H, and O that starts with carbon dioxide and reductants and uses redox couples as the energy source. To inquire into the reaction networks that might emerge, we start with the largest available database of organic molecules, Beilstein on-line, and prune by a set of physical and chemical constraints applicable to the model system. From the 3.5 million entries in Beilstein we emerge with 153 molecules that contain all 11 members of the reductive citric acid cycle. A small number of selection rules generates a very constrained subset, suggesting that this is the type of reaction model that will prove useful in the study of biogenesis. The model indicates that the metabolism shown in the universal chart of pathways may be central to the origin of life, is emergent from organic chemistry, and may be unique.

Biological Evolution↗
PubMed2000

Cellular mechanisms regulating protein phosphatase-1. A key functional interaction between inhibitor-2 and the type 1 protein phosphatase catalytic subunit.

Inhibitor-1 (I-1) and inhibitor-2 (I-2) selectively inhibit type 1 protein serine/threonine phosphatases (PP1). To define the molecular basis for PP1 inhibition by I-1 and I-2 charged-to-alanine substitutions in the Saccharomyces cerevisiae, PP1 catalytic subunit (GLC7), were analyzed. Two PP1 mutants, E53A/E55A and K165A/E166A/K167A, showed reduced sensitivity to I-2 when compared with wild-type PP1. Both mutants were effectively inhibited by I-1. Two-hybrid analysis and coprecipitation or pull-down assays established that wild-type and mutant PP1 catalytic subunits bound I-2 in an identical manner and suggested a role for the mutated amino acids in enzyme inhibition. Inhibition of wild-type and mutant PP1 enzymes by full-length I-2(1-204), I-2(1-114), and I-2(36-204) indicated that the mutant enzymes were impaired in their interaction with the N-terminal 35 amino acids of I-2. Site-directed mutagenesis of amino acids near the N terminus of I-2 and competition for PP1 binding by a synthetic peptide encompassing an I-2 N-terminal sequence suggested that a PP1 domain composed of amino acids Glu-53, Glu-55, Asp-165, Glu-166, and Lys-167 interacts with the N terminus of I-2. This defined a novel regulatory interaction between I-2 and PP1 that determines I-2 potency and perhaps selectivity as a PP1 inhibitor.

Amino Acid Sequence↗
PubMed2000

Three novel TBX5 mutations in Chinese patients with Holt-Oram syndrome.

Holt-Oram syndrome (HOS) is an autosomal dominant syndrome that comprises upper limb and cardiac defects. The gene responsible for HOS, TBX5, was isolated and many mutations have been identified in HOS patients. We analyzed 11 Chinese HOS patients (7 from three families and 4 sporadic cases) for TBX5 mutation by single strand conformation polymorphisms (SSCPs). Three SSCP changes were detected in two of the three familial cases and one sporadic case. Sequence analysis identified three novel, heterozygous mutations in TBX5: a frameshift mutation caused by one base deletion [C416del] in one family, a mis-sense mutation (Gln49Lys) induced by a base substitution (C145A) in another family, and the other mis-sense mutation (Ile54Thr) by T161C in one sporadic case. The patients with the frameshift mutations had severer clinical manifestations that involved aplasia/hypoplasia of the arm and thumbs, while those with the mis-sense mutations presented with milder anomalies such as absent or hypoplastic thumbs but without arm abnormalities. These observations may support a genotype-phenotype correlation in HOS patients with TBX5 mutation.

Amino Acid Substitution↗
PubMed2000

[Role of interleukin-6 and platelet-activating factor in delaying neutrophil apoptosis].

In the present study we investigated the role of platelet-activating factor (PAF) and interleukin-6 (IL-6) in delaying polymorphonuclear neutrophil (PMN) apoptosis. Isolation of PMN was performed by using the discontinuous plasma-Percoll gradient technique. PMN was cultured in enriched RPMI 1640 media at 2 x 10(7) PMN/ml for 24 h. Subgroups were treated with IL-6 or PAF or pretreated with PAF receptor antagonist BN52021 before IL-6 or PAF were added. Morphological assessment and quantitation of apoptosis were performed with acrodine/ethidium bromide stain and epifluorescent microscope. The results showed that both IL-6 and PAF suppressed PMN apoptosis. Pretreatment of PMN with BN52021 abrogated the effects of IL-6 and PAF. It is suggested that PAF may be a crucial cytokine in the suppression of PMN apoptosis. These observations may contribute to elucidating the mechanisms of IL-6 and PAF in mediating postinjury hyperinflammation and secondary organ dysfunction and provide a clue for the prevention and treatment of the conditions.

Apoptosis↗
PubMed2000

Primary 13C and beta-secondary 2H KIEs for trans-sialidase. A snapshot of nucleophilic participation during catalysis.

Trypanosoma cruzi trans-sialidase catalyzes a novel reaction that involves the transfer of sialic acid between host and parasite glycoconjugates. In this paper, we report kinetic isotope effect studies on recombinant trans-sialidase. beta-Dideuterium and primary 13C isotope effects were measured for a good substrate, sialyl-lactose, and a slow substrate, sialyl-galactose, in both acid-catalyzed solvolysis and enzymatic transfer reactions. The beta-dideuterium isotope effect for sialyl-lactose in the acid hydrolysis reaction was 1.113 +/- 0.012. The primary 13C isotope effects for hydrolysis of sialyl-lactose and sialyl-galactose were 1. 016 +/- 0.011 and 1.015 +/- 0.008, respectively. In the enzymatic transfer reactions, the beta-dideuterium and primary 13C effects for sialyl-galactose were 1.060 +/- 0.008 and 1.032 +/- 0.008, respectively. The isotope effects for hydrolysis describe a dissociative SN1-like mechanism, and these data are contrasted by the data for the enzyme-catalyzed reaction. The enzymatic deuterium isotope effects are lower by a factor of 2, but the primary carbon isotope effects are higher by a factor of 2. This pattern describes a mechanism involving nucleophilic participation in the rate-determining transition state.

Animals↗
PubMed2000

Elastase mimics pancreatitis-induced hepatic injury via inflammatory mediators.

BACKGROUND: Recent evidence suggests that pancreatitis-associated hepatic injury is regulated by inflammatory mediator production. Our laboratory demonstrated in vitro that pancreatic elastase is a pancreatic enzyme that can induce inflammatory cell cytokine production. Therefore we now explore the in vivo effects of elastase on the liver. MATERIALS AND METHODS: Elastase (1.5 U) +/- CNI-1493, which attenuates mediator production through p38 MAP kinase inhibition, was administered intraperitoneally to mice while control animals received saline. Acute pancreatitis (AP) was induced with a choline-deficient, ethionine-supplemented (CDE) diet. Serum hepatic enzymes and hepatic neutrophil infiltration by myeloperoxidase (MPO) activity were measured as indicators of hepatic insult. Serum tumor necrosis factor (TNF) protein (ELISA), hepatic TNF mRNA (reverse transcription polymerase chain reaction), and hepatic activation of the transcription factor nuclear factor kappa B (electrophoretic mobility shift assay) were also determined. RESULTS: A significant increase in hepatic enzymes and MPO activity was induced by AP and mirrored by intraperitoneal elastase. Both types of liver injury resulted in near identical elevations in serum TNF protein and hepatic TNF mRNA. Elastase-treated animals with mediator production inhibited (CNI-1493) had attenuated hepatic enzymes, MPO activity, TNF protein, and TNF mRNA. Activation of nuclear factor kappa B occurred 30 min after elastase administration. CONCLUSION: Exposure of the liver to pancreatic elastase results in hepatic inflammation and injury which appears identical to that seen during severe AP. Prevention of inflammatory mediator production by intrahepatic leukocytes attenuates injury and supports recent adult respiratory distress syndrome and in vitro data suggesting that elastase is the principal factor that propagates pancreatic inflammation into a systemic illness through direct activation of systemic inflammatory cells.

Adjuvants, Immunologic↗
PubMed2000

Chronic insulin effects on insulin signalling and GLUT4 endocytosis are reversed by metformin.

Decreases in insulin-responsive glucose transport and associated levels of cell surface GLUT4 occur in rat adipocytes maintained in culture for 20 h under hyperinsulinaemic and hyperglycaemic conditions. We have investigated whether this defect is due to reduced signalling from the insulin receptor, GLUT4 expression or impaired GLUT4 trafficking. The effects of chronic insulin treatment on glucose transport and GLUT4 trafficking were ameliorated by inclusion of metformin in the culture medium. In comparison with the ic insulin treatment attenuated changes in signalling processes leading to glucose transport. These included insulin receptor tyrosine phosphorylation, phosphoinositide 3-kinase activity and Akt activity, which were all reduced by 60-70%. Inclusion of metformin in the culture medium prevented the effects of the chronic insulin treatment on these signalling processes. In comparison with cells maintained in culture without insulin, the total expression of GLUT4 protein was not significantly altered by chronic insulin treatment, although the level of GLUT1 expression was increased. Trafficking rate constants for wortmannin-induced cell-surface loss of GLUT4 and GLUT1 were assessed by 2-N-4-(1-azi-2, 2,2-trifluoroethyl)benzoyl-1,3-bis(D-mannose-4-yloxy)-2-propyla min e (ATB-BMPA) photolabelling. In comparison with cells acutely treated with insulin, chronic insulin treatment resulted in a doubling of the rate constants for GLUT4 endocytosis. These results suggest that the GLUT4 endocytosis process is very sensitive to the perturbations in signalling that occur under hyperinsulinaemic and hyperglycaemic conditions, and that the resulting elevation of endocytosis accounts for the reduced levels of net GLUT4 translocation observed.

Adipocytes↗