Biomedical subjects

T A Young

Publications and source records attributed to T A Young.

13 recordsLinked to original sources

PubMed2001

Chronic ethanol consumption alters the glutathione/glutathione peroxidase-1 system and protein oxidation status in rat liver.

BACKGROUND: Alcohol-induced liver damage is associated with oxidative stress, which might be linked to disturbances in liver antioxidant defense mechanisms. The effect of chronic ethanol consumption on the mitochondrial and cytosolic glutathione/glutathione peroxidase-1 (GSHPx-1) system and oxidative modification of proteins was therefore studied in the rat. METHODS: Male Sprague-Dawley rats were fed liquid diets that provided 36% total calories as ethanol for at least 31 days. Pair-fed controls received isocaloric diets with ethanol calories substituted with maltose-dextrins. Mitochondrial and cytosolic fractions were prepared from livers and assayed for GSHPx-1 and glutathione reductase activities and total and oxidized concentrations of glutathione. Catalase activity was measured in the postmitochondrial supernatant. Levels of GSHPx-1, lactate dehydrogenase, and the beta subunit of the F1 portion of the ATP synthase protein were determined by western blot analysis. Concentrations of mitochondrial and cytosolic protein carbonyls were measured to assess ethanol-induced oxidation of proteins. RESULTS: Chronic ethanol consumption significantly decreased cytosolic and mitochondrial GSHPx-1 activities by 40% and 30%, respectively. Levels of GSHPx-1 protein in cytosol were unaffected by ethanol feeding, whereas there was a small decrease in GSHPx-1 protein levels in mitochondria isolated from ethanol-fed rats. Glutathione reductase activities were increased in both intracellular compartments and catalase activity was increased as a consequence of ethanol exposure. Cytosolic total glutathione was mildly decreased, whereas ethanol feeding increased mitochondrial levels of total glutathione. Chronic ethanol feeding significantly increased both cytosolic and mitochondrial concentrations of protein carbonyls by 30% and 60%, respectively. CONCLUSIONS: This study demonstrates that chronic ethanol-induced alterations in the glutathione/GSHPx-1 antioxidant system might promote oxidative modification of liver proteins, namely those of the mitochondrion, which could contribute to the adverse effects of ethanol on the liver.

Alcoholism↗

PubMed2000

Alpha-synuclein immunoreactivity of huntingtin polyglutamine aggregates in striatum and cortex of Huntington's disease patients and transgenic mouse models.

Polyglutamine expansions in proteins are implicated in at least eight inherited neurodegenerative disorders, including Huntington's disease. These mutant proteins can form aggregates within the nucleus and processes of neurons possibly due to misfolding of the proteins. Polyglutamine aggregates are ubiquitinated and sequester molecular chaperone proteins and proteasome components. To investigate other protein components of polyglutamine aggregates, cerebral cortex and striata from patients with Huntington's disease and full-length cDNA transgenic mouse models for this disease were examined immunohistochemically for alpha-synuclein reactivity. Our findings demonstrate that alpha-synuclein can be used as a marker for huntingtin polyglutamine aggregates in both human and mice. Moreover in the HD transgenic mice, the intensity of immunoreactivity increases with age. The significance of recruitment of alpha-synuclein into huntingtin aggregates and its translocation away from the synapses remains to be determined. We propose that aberrant interaction of mutant huntingtin with other proteins, including alpha-synuclein, may influence disease progression.

Amino Acid Motifs↗

PubMed2000

Allosteric inhibitors of inducible nitric oxide synthase dimerization discovered via combinatorial chemistry.

Potent and selective inhibitors of inducible nitric oxide synthase (iNOS) (EC ) were identified in an encoded combinatorial chemical library that blocked human iNOS dimerization, and thereby NO production. In a cell-based iNOS assay (A-172 astrocytoma cells) the inhibitors had low-nanomolar IC(50) values and thus were >1,000-fold more potent than the substrate-based direct iNOS inhibitors 1400W and N-methyl-l-arginine. Biochemical studies confirmed that inhibitors caused accumulation of iNOS monomers in mouse macrophage RAW 264.7 cells. High affinity (K(d) approximately 3 nM) of inhibitors for isolated iNOS monomers was confirmed by using a radioligand binding assay. Inhibitors were >1,000-fold selective for iNOS versus endothelial NOS dimerization in a cell-based assay. The crystal structure of inhibitor bound to the monomeric iNOS oxygenase domain revealed inhibitor-heme coordination and substantial perturbation of the substrate binding site and the dimerization interface, indicating that this small molecule acts by allosterically disrupting protein-protein interactions at the dimer interface. These results provide a mechanism-based approach to highly selective iNOS inhibition. Inhibitors were active in vivo, with ED(50) values of <2 mg/kg in a rat model of endotoxin-induced systemic iNOS induction. Thus, this class of dimerization inhibitors has broad therapeutic potential in iNOS-mediated pathologies.

Allosteric Regulation↗

PubMed1999

Frequency-dependent modulation of inhibition in the rat olfactory bulb.

Active sniffing in rodents at theta frequency during exploratory behavior has been hypothesized to enhance odorant access to the receptor sheet and to evoke activity patterns in olfactory and limbic structures which facilitate induction of synaptic plasticity. The present results demonstrate a third potential consequence of theta frequency sniffing -- reversible enhancement of lateral/feedback inhibition in the rat olfactory bulb. Suppression of mitral/tufted cell single-unit spontaneous activity evoked by single lateral olfactory tract (LOT) shocks was examined during LOT stimulation at either 1 or 5 Hz (theta frequency). Theta frequency stimulation rapidly and reversibly enhanced LOT-evoked suppression compared with 1 Hz stimulation. This enhancement was not stimulus intensity dependent. Sniffing-induced modification of lateral/feedback inhibition may enhance odor processing during exploration.

Animals↗

PubMed1999

Crystal structures of zinc-free and -bound heme domain of human inducible nitric-oxide synthase. Implications for dimer stability and comparison with endothelial nitric-oxide synthase.

The crystal structures of the heme domain of human inducible nitric-oxide synthase (NOS-2) in zinc-free and -bound states have been solved. In the zinc-free structure, two symmetry-related cysteine residues form a disulfide bond. In the zinc-bound state, these same two cysteine residues form part of a zinc-tetrathiolate (ZnS(4)) center indistinguishable from that observed in the endothelial isoform (NOS-3). As in NOS-3, ZnS(4) plays a key role in stabilizing intersubunit contacts and in maintaining the integrity of the cofactor (tetrahydrobiopterin) binding site of NOS-2. A comparison of NOS-2 and NOS-3 structures illustrates the conservation of quaternary structure, tertiary topology, and substrate and cofactor binding sites, in addition to providing insights on isoform-specific inhibitor design. The structural comparison also reveals that pterin binding does not preferentially stabilize the dimer interface of NOS-2 over NOS-3.

Dimerization↗

PubMed1997

Platelet factor 4 binds to glycanated forms of thrombomodulin and to protein C. A potential mechanism for enhancing generation of activated protein C.

Platelet factor 4 (PF4) is an abundant platelet alpha-granule heparin-binding protein. We have previously shown that PF4 accelerates up to 25-fold the proteolytic conversion of protein C to activated protein C by the thrombin.thrombomodulin complex by increasing its affinity for protein C 30-fold. This stimulatory effect requires presence of the gamma-carboxyglutamic acid (Gla) domain in protein C and is enhanced by the presence of a chondroitin sulfate glycosaminoglycan (GAG) domain on thrombomodulin. We hypothesized that cationic PF4 binds to both protein C and thrombomodulin through these anionic domains. Qualitative SDS-polyacrylamide gel electrophoresis analysis of avidin extracts of solutions containing biotinylated PF4 and candidate ligands shows that PF4 binds to GAG+ but not GAG- forms of thrombomodulin and native but not Gla-domainless protein C. Quantitative analysis using the surface plasmon resonance-based BIAcoreTM biosensor system confirms the extremely high affinity of PF4 for heparin (KD = 4 nM) and shows that PF4 binds to GAG+ thrombomodulin with a KD of 31 nM and to protein C with a KD of 0.37 microM. In contrast, PF4 had no measurable interaction with GAG- thrombomodulin or Gla-domainless protein C. Western blot analysis of normal human plasma extracted with biotinylated PF4 demonstrates PF4 binding to protein C in a physiologic context. Thus, PF4 binds with relative specificity and high affinity to the GAG- domain of thrombomodulin and the Gla domain of protein C. These interactions may enhance the affinity of the thrombin.thrombomodulin complex for protein C and thereby promote the generation of activated protein C.

Binding Sites↗

PubMed1997

Teaching medical students to lie. The disturbing contradiction: medical ideals and the resident-selection process.

Although truthfulness and honesty have long been considered fundamental values within the medical profession, lying and deception have become standard practices within medicine's resident-selection process. Dishonesty is incorporated into and encouraged during this process, and there is little need for medical students and other participants to reflect upon their actions. This essay, which won the $1500 first prize in CMAJs 1996 Logie Medical Ethics Essay Contest, looks at the serious consequences of this lying and deception. Dr. Tara Young discusses the moral dilemma applicants for residencies face during their final year of undergraduate training.

Administrative Personnel↗

PubMed1997

Quantitative measurement for endothelial constitutive nitric oxide synthase in cultured human endothelial cells.

Constitutively expressed endothelial nitric oxide synthase (ecNOS) produces nitric oxide (NO) from L-arginine and is important for the maintenance of cardiovascular homeostasis. We report the development of a capture ELISA which is specific for ecNOS. The assay detection limit is 0.5 ng/ml ecNOS protein, allowing the measurement of ecNOS from as few as 6000 human endothelial cells cultured in 96-well microtiter plates. This ELISA has been used to measure a downregulation of ecNOS with 24-h TNFalpha treatment, consistent with results obtained by Western blot analysis. Quantitation of ecNOS in human endothelial cells showed a higher expression of ecNOS in human aortic endothelial cells (18.3+/-1.35 ng ecNOS per 10(6) cells, n = 3 donors) than in human umbilical vein endothelial cells (10.4+/-0.48 ng ecNOS per 10(6) cells, n = 3 donors). These studies demonstrate that this convenient, quantitative assay is currently the most sensitive method for investigating ecNOS protein regulation.

Amino Acid Sequence↗

PubMed1997

The afferent pupillary defect.

Examination of the pupils is an essential component of both the neurologic and ocular evaluation of a child in the acute care setting. The manifestation of an afferent pupillary defect has significant clinical implications for diagnosis and management. Correctly assessing the pupils for an afferent defect is an important clinical skill for the pediatric emergency physician. An appreciation of the anatomy and understanding of the mechanisms involved in abnormal pupil responses, knowledge of the proper technique of the swinging flashlight test, and an awareness of potential pitfalls in performing this test will aid the physician in the detection of an afferent pupillary defect.

Afferent Pathways↗

PubMed1996

The shape of thrombomodulin and interactions with thrombin as determined by electron microscopy.

Studies have been carried out to investigate aspects of the structure of thrombomodulin, an endothelial cell glycoprotein that binds thrombin and accelerates both the thrombin-dependent activation of protein C and the inhibition of antithrombin III. We have determined the shape of SolulinTM, a soluble recombinant form of human thrombomodulin missing the transmembrane and cytoplasmic domains, by electron microscopy of preparations rotary-shadowed with tungsten. Solulin appears to be an elongated molecule about 20 nm long that has a large nodule at one end and a smaller nodule near the other end from which extends a thin strand. About half of the molecules form bipolar dimers apparently via interactions between these thin strands. Electron microscopy of complexes formed between Solulin and human alpha-thrombin revealed that a single thrombin molecule appears to bind to the smaller nodule of Solulin, suggesting that this region contains the epidermal growth factor-like domains 5 and 6. Epidermal growth factor-like domains 1-4 comprise the connector between the small and large nodule, which is the lectin-like domain; the thin strand at the other end of the molecule is the carbohydrate-rich region. With chondroitin sulfate-containing soluble thrombomodulin produced from either human melanoma cells Bowes or Chinese hamster ovary cells, a higher percentage of molecules bound thrombin and, in some cases, two thrombin molecules were attached to one soluble thrombomodulin in approximately the same region. These structural studies provide insight into the structure of thrombomodulin and its interactions with thrombin as well as aspects of the mechanisms of its actions.

Animals↗

PubMed1996

Identification of genes whose expression is altered during mitosuppression in livers of ethinyl estradiol-treated female rats.

In this study, our goal was to identify genes whose expression in liver is altered in female F-344 rats during mitosuppression induced by 42 days of ethinyl estradiol (EE) treatment (Yager et al., Carcinogenesis, 15, 2117-2123, 1994). Northern analysis demonstrated that the mRNA levels for transforming growth factor-beta1 (TGF-beta1) and the mannose 6-phosphate/insulin-like growth factor II receptor were significantly increased by EE treatment. Ten cDNA clones representing mRNAs whose expression was increased two- to four-fold in the mitosuppressed livers were identified by differential display. Sequence analysis revealed that one was homologous to the S-24 ribosomal protein and another to mitochondrial ATPase subunit e. The remaining clones showed no homology to known genes in GenBank. However, the expression of clones 15, 16 and 17 was increased in HepG2 cells following treatment with doxorubicin suggesting their induction by oxidative DNA damage. These results suggest that two independent but interrelated signalling pathways, one mediated through transforming growth factor-beta and the other through oxidative DNA damage, may contribute to hepatic mitosuppression caused by EE, perhaps through activation of cyclin-dependent kinase inhibitors.

Animals↗

PubMed1994

Modulation of glycosaminoglycan addition in naturally expressed and recombinant human thrombomodulin.

The two major glycoforms of full-length human thrombomodulin (TM), one with (TM(CS+)) and one without (TM(CS-)) chondroitin sulfate (CS) were analyzed on Western blots of primary and transformed cells and in cells expressing recombinant TM. TM on the surface of Chinese hamster ovary and COS-7 cells is solely TM(CS-). Primary arterial endothelial cells (HAEC and HPAEC) express a greater fraction of TM with CS attached than venous cells (HUVEC). Human lung carcinoma cells (A549) express more TM(CS+) than primary cells and recombinant TM on human melanoma cells (CHL-1) occurs in two very high molecular weight forms of TM(CS+). We explored this variation in TM(CS+) with soluble recombinant TM in several cell lines and analyzed the ambiguous CS addition site in human TM by site-directed mutagenesis. Mutation of Ser474 to Ala blocks CS addition in Chinese hamster ovary and COS-7 cells but not CHL-1 cells which add CS to Ser472 and Ser474. Structure of the O-link domain affects partitioning into TM(CS+) since substituting with the decorin CS addition sequence, substituting all Ser and Thr except Ser474 with Ala, and deleting around the potential beta-turn all increase the ratio of TM(CS+) to TM(CS-). A combination of the decorin substitution and deletion of the remaining O-link domain yields the most TM(CS+).

Amino Acid Sequence↗

PubMed1994

Activation of proglucagon gene transcription by protein kinase-A in a novel mouse enteroendocrine cell line.

The gene encoding proglucagon is expressed predominantly in the pancreas and intestine. The physiological importance of glucagon secreted from the islets of Langerhans has engendered considerable interest in the molecular control of proglucagon gene transcription in the endocrine pancreas. In contrast, little is known about the molecular control of proglucagon gene expression in the intestine. The recent demonstration that glucagon-like peptide-1 (GLP-1) secreted from the intestine is a potent regulator of insulin secretion and glucose homeostasis has stimulated renewed interest in the factors that control GLP-1 synthesis in the intestinal L-cell. To develop a model for the analysis of intestinal proglucagon gene expression, we have targeted expression of a proglucagon gene-simian virus-40 large T-antigen fusion gene to enteroendocrine cells in transgenic mice. These mice develop intestinal tumors that were used to derive a novel cell line, designated GLUTag, that expresses the proglucagon gene and secretes immunoreactive GLP-1 in vitro. GLUTag cells demonstrate morphological characteristics of enteroendocrine cells by electron microscopy and are plurihormonal, as shown by immunocytochemistry and RNA analyses. GLUTag cells express the proglucagon and cholecystokinin genes, consistent with the pattern of lineage-specific enteroendocrine differentiation described for mouse intestine. Proglucagon gene expression was induced by activators of the protein kinase-A pathway, and a combination of messenger RNA half-life and nuclear run-on experiments demonstrated that the protein kinase-A-induction is mediated by an increase in proglucagon gene transcription. In contrast, activators of protein kinase-C stimulated secretion, but not biosynthesis of the PGDPs in GLUTag cell cultures. Analysis of proglucagon processing in GLUTag cells demonstrated the liberation of glucagon, oxyntomodulin, glicentin, and multiple forms of GLP-1. These observations provide evidence for the direct induction of proglucagon gene transcription by a cAMP-dependent pathway and suggest that the GLUTag cell line represents a useful model for the analysis of the molecular determinants of enteroendocrine gene expression.

1-Methyl-3-isobutylxanthine↗