RESUMEN
Enterococcus faecalis is a natural inhabitant of the human gastrointestinal tract. This bacterial species is subdominant in a healthy physiological state of the gut microbiota (eubiosis) in adults, but can become dominant and cause infections when the intestinal homeostasis is disrupted (dysbiosis). The relatively high concentrations of bile acids deoxycholate (DCA) and taurocholate (TCA) hallmark eubiosis and dysbiosis, respectively. This study aimed to better understand how E. faecalis adapts to DCA and TCA. We showed that DCA impairs E. faecalis growth and possibly imposes a continuous adjustment in the expression of many essential genes, including a majority of ribosomal proteins. This may account for slow growth and low levels of E. faecalis in the gut. In contrast, TCA had no detectable growth effect. The evolving transcriptome upon TCA adaptation showed the early activation of an oligopeptide permease system (opp2) followed by the adjustment of amino acid and nucleotide metabolisms. We provide evidence that TCA favors the exploitation of oligopeptide resources to fuel amino acid needs in limiting oligopeptide conditions. Altogether, our data suggest that the combined effects of decreased DCA and increased TCA concentrations can contribute to the rise of E. faecalis population during dysbiosis.
Asunto(s)
Ácidos y Sales Biliares , Enterococcus faecalis , Aminoácidos/metabolismo , Ácidos y Sales Biliares/metabolismo , Ácido Desoxicólico/metabolismo , Ácido Desoxicólico/farmacología , Disbiosis , Enterococcus faecalis/genética , Humanos , Ácido Taurocólico/metabolismo , Ácido Taurocólico/farmacologíaRESUMEN
Ankyrin mediates the attachment of spectrin to transmembrane integral proteins in both erythroid and nonerythroid cells by binding to the beta-subunit of spectrin. Previous studies using enzymatic digestion, 2-nitro-5-thiocyanobenzoic acid cleavage, and rotary shadowing techniques have placed the spectrin-ankyrin binding site in the COOH-terminal third of beta-spectrin, but the precise site is not known. We have used a glutathione S-transferase prokaryotic expression system to prepare recombinant erythroid and nonerythroid beta-spectrin from cDNA encoding approximately the carboxy-terminal half of these proteins. Recombinant spectrin competed on an equimolar basis with 125I-labeled native spectrin for binding to erythrocyte membrane vesicles (IOVs), and also bound ankyrin in vitro as measured by sedimentation velocity experiments. Although full length beta-spectrin could inhibit all spectrin binding to IOVs, recombinant beta-spectrin encompassing the complete ankyrin binding domain but lacking the amino-terminal half of the molecule failed to inhibit about 25% of the binding capacity of the IOVs, suggesting that the ankyrin-independent spectrin membrane binding site must lie in the amino-terminal half of beta-spectrin. A nested set of shortened recombinants was generated by nuclease digestion of beta-spectrin cDNAs from ankyrin binding constructs. These defined the ankyrin binding domain as encompassing the 15th repeat unit in both erythroid and nonerythroid beta-spectrin, amino acid residues 1,768-1,898 in erythroid beta-spectrin. The ankyrin binding repeat unit is atypical in that it lacks the conserved tryptophan at position 45 (1,811) within the repeat and contains a nonhomologous 43 residue segment in the terminal third of the repeat. It also appears that the first 30 residues of this repeat, which are highly conserved between the erythroid and nonerythroid beta-spectrins, are critical for ankyrin binding activity. We hypothesize that ankyrin binds directly to the nonhomologous segment in the 15th repeat unit of both erythroid and nonerythroid beta-spectrin, but that this sequence must be presented in the context of a properly folded spectrin "repeat unit" structure. Future studies will identify which residues within the repeat unit are essential for activity, and which residues determine the specificity of various spectrins for different forms of ankyrin.
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Proteínas Sanguíneas/metabolismo , Proteínas de la Membrana/metabolismo , Espectrina/metabolismo , Secuencia de Aminoácidos , Ancirinas , Sitios de Unión , Análisis Mutacional de ADN , Escherichia coli , Humanos , Técnicas In Vitro , Datos de Secuencia Molecular , Estructura Molecular , Unión Proteica , Proteínas Recombinantes de Fusión , Alineación de Secuencia , Espectrina/química , Espectrina/inmunología , Relación Estructura-ActividadRESUMEN
Acquired resistance to conventional and targeted therapies is becoming a major hindrance in cancer management. It is increasingly clear that cancer cells are able to evolve and rewire canonical signalling pathways to their advantage, thus evading cell death and promoting cell invasion. The Axl receptor tyrosine kinase (RTK) has been shown to modulate acquired resistance to EGFR-targeted therapies in both breast and lung cancers. Glioblastoma multiforme (GBM) is a highly infiltrative and invasive form of brain tumour with little response to therapy. Both Axl and EGFR have been identified as major players in gliomagenesis and invasiveness. However, the mechanisms underlying a potential signalling crosstalk between EGFR and Axl RTKs are unknown. The purpose of this study was to investigate this novel and unconventional interaction among RTKs of different families in human GBM cells. With the use of western blotting, in vitro kinase activity, co-immunoprecipitation and bimolecular fluorescence complementation assays, we show that EGF stimulates activation of Axl kinase and that there is a hetero-interaction between the two RTKs. Through small interfering RNA knockdown and quantitative PCR screening, we identified distinct gene expression patterns in GBM cells that were specifically regulated by signalling from EGFR-EGFR, Axl-Axl and EGFR-Axl RTK parings. These included genes that promote invasion, which were activated only via the EGFR-Axl axis (MMP9), while EGFR-EGFR distinctly regulated the cell cycle and Axl-Axl regulated invasion. Our findings provide critical insights into the role of EGFR-Axl hetero-dimerisation in cancer cells and reveal regulation of cell invasion via Axl as a novel function of EGFR signalling.
RESUMEN
BACKGROUND: Unstable atherosclerotic lesions typically have an abundant inflammatory cell infiltrate, including activated T cells, macrophages, and mast cells, which may decrease plaque stability. The pathophysiology of inflammatory cell recruitment and activation in the human atheroma is incompletely described. METHODS AND RESULTS: We hypothesized that differential gene expression with DNA microarray technology would identify new genes that may participate in vascular inflammation. RNA isolated from cultured human aortic smooth muscle cells treated with tumor necrosis factor-alpha (TNF-alpha) was examined with a DNA microarray with 8600 genes. This experiment and subsequent Northern analyses demonstrated marked increases in steady-state eotaxin mRNA (>20 fold), a chemokine initially described as a chemotactic factor for eosinophils. Because eosinophils are rarely present in human atherosclerosis, we then studied tissue samples from 7 normal and 14 atherosclerotic arteries. Immunohistochemical analysis demonstrated overexpression of eotaxin protein and its receptor, CCR3, in the human atheroma, with negligible expression in normal vessels. Eotaxin was predominantly located in smooth muscle cells. The CCR3 receptor was localized primarily to macrophage-rich regions as defined by immunopositivity for CD 68; a minority of mast cells also demonstrated immunopositivity for the CCR3 receptor. CONCLUSIONS: Eotaxin and its receptor, CCR3, are overexpressed in human atherosclerosis, suggesting that eotaxin participates in vascular inflammation. These data demonstrate how genomic differential expression technology can identify novel genes that may participate in the stability of atherosclerotic lesions.
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Arteriosclerosis/metabolismo , Quimiocinas CC , Citocinas/biosíntesis , Músculo Liso Vascular/metabolismo , Receptores de Quimiocina/biosíntesis , Vasculitis/metabolismo , Actinas/metabolismo , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Aorta Torácica/metabolismo , Aorta Torácica/patología , Arteriosclerosis/genética , Arteriosclerosis/patología , Northern Blotting , Arterias Carótidas/metabolismo , Arterias Carótidas/patología , Células Cultivadas , Quimiocina CCL11 , Citocinas/genética , Expresión Génica , Humanos , Inmunohistoquímica , Mastocitos/metabolismo , Mastocitos/patología , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/metabolismo , Receptores CCR3 , Receptores de Quimiocina/genética , Factor de Necrosis Tumoral alfa/farmacología , Vasculitis/genética , Vasculitis/patologíaRESUMEN
A specific soluble inositol phosphate 5-phosphomonoesterase has been purified approximately 2,700-fold from a 120,000g supernatant of rabbit neutrophil homogenate. The specific enzyme represented 25-50% of the total hydrolytic activity toward inositol, 1,4,5-trisphosphate (Ins-1,4,5-P3) with the remaining activity hydrolyzing both Ins-1,4,5-P3 as well as inositol 1,4-bisphosphate (Ins-1,4-P2). However, the enzyme could not be identified on sodium dodecyl sulfate-polyacrylamide gels stained with Coomassie blue, indicating that it represents a minor protein in the purified enzyme preparations. The purified enzyme has an apparent molecular mass of 43,000-47,000 daltons as determined by gel filtration and is free of other inositol phosphate phosphomonoesterases. The enzyme hydrolyzes Ins-1,4,5-P3 with an apparent Km of 18 microM and a Vmax of 1.2 mumol/min/mg. The 5-phosphomonoesterase requires Mg2+ for activity and is not affected by physiological concentrations of Ca2+ or calmodulin. The pH optimum for activity is 7.5. Inositol 1,3,4,5-tetrakisphosphate is a potent competitive inhibitor of Ins-1,4,5-P3 hydrolysis (Ki = microM), whereas Ins-1,4-P2 is a weak inhibitor (Ki = 173 microM). Ins-1,4,5-P3 hydrolysis is relatively unaffected by monophosphorylated substrates, however, bisphosphorylated substrates are potent inhibitors. Comparisons of neutrophil 5-phosphomonoesterase characteristics with those of platelet and rat brain enzymes support the idea that each 5-phosphomonoesterase may be a unique enzyme and play a different role dependent upon the cell or tissue in which it acts.
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Neutrófilos/enzimología , Monoéster Fosfórico Hidrolasas/sangre , Animales , Calcio/farmacología , Calmodulina/farmacología , Concentración de Iones de Hidrógeno , Magnesio/farmacología , Neutrófilos/citología , Cavidad Peritoneal/citología , Monoéster Fosfórico Hidrolasas/fisiología , Conejos , Especificidad por Sustrato/efectos de los fármacos , Especificidad por Sustrato/fisiologíaRESUMEN
OBJECTIVE: Adenosine receptor activation has been implicated in the mechanism of ischaemic preconditioning protection. Evidence suggests adenosine A1 receptor involvement, and possibly A3 receptor involvement in the rabbit. This study investigated the roles of these receptors in human preconditioning. Human A1- and A3-selective compounds were chosen based on Ki values for inhibition of N6-(4-amino-3-[125I]iodobenzyl)adenosine (125I-ABA) binding to stably expressed recombinant human A1 and A3 receptors. Cyclopentyladenosine (CPA), a 194-fold selective A1 agonist, and iodobenzylmethylcarboxamidoadenosine (IBMECA), a 10-fold selective A3 agonist were used alone and in combination with dipropylcyclopentylxanthine (DPCPX) a 62-fold selective A1 antagonist. METHODS: Human atrial trabeculae were superfused with oxygenated Tyrode's solution. After stabilisation, muscles underwent one of 8 protocols (n = 6 per group), followed by 90 min of simulated ischaemia and 120 min of reoxygenation. The experimental endpoint was recovery of contractile function, presented as percentage baseline function. RESULTS: 5 nM CPA (52.2 +/- 3.1%), 30 nM IBMECA (49.7 +/- 3.8%) and preconditioning (55.3 +/- 2.5%) produced similar functional recoveries at 120 min of reoxygenation; significantly different to controls (27.7 +/- 1.0%; P < 0.05, ANOVA). When DPCPX (200 nM) was added prior to 5 nM CPA, protection was lost (31.8 +/- 0.9%), but when added prior to 30 nM IBMECA, muscles continued to be significantly protected (41.5 +/- 2.3%). CONCLUSIONS: In human atrium both A1 and A3 receptor stimulation appears to mimic ischaemic preconditioning. This may represent the first evidence for A3 receptor involvement in 'pharmacological' preconditioning of human myocardium.
Asunto(s)
Atrios Cardíacos/metabolismo , Isquemia Miocárdica/prevención & control , Receptores Purinérgicos P1/fisiología , Adenosina/análogos & derivados , Adenosina/farmacología , Fosfatos de Dinucleósidos/farmacología , Atrios Cardíacos/efectos de los fármacos , Humanos , Técnicas In Vitro , Precondicionamiento Isquémico Miocárdico , Modelos Biológicos , Contracción Miocárdica/efectos de los fármacos , Isquemia Miocárdica/metabolismo , Reperfusión Miocárdica , Agonistas del Receptor Purinérgico P1 , Antagonistas de Receptores Purinérgicos P1 , Receptor de Adenosina A3 , Xantinas/farmacologíaRESUMEN
OBJECTIVE: The aim of this study was to determine whether selective activation of the adenosine A3 receptor reduces infarct size in a Langendorff model of myocardial ischemia-reperfusion injury. METHODS: Buffer-perfused rabbit hearts were exposed to 30 min regional ischemia and 120 min of reperfusion. Infarct size was measured by tetrazolium staining and normalized for area-at-risk (IA/AAR). RESULTS: Preconditioning by 5 min global ischemia and 10 min reperfusion reduced infarct size (IA/AAR) to 19 +/- 4% (controls: 67 +/- 5%). Replacing global ischemia with 5 min perfusion of the rabbit A3-selective agonist, IB-MECA (A3 Ki: 2 nM; A1 Ki: 30 nM) elicited a concentration-dependent reduction in infarct size; 50 nM IB-MECA reduced IA/AAR to 24 +/- 4%. The A1-selective agonist, R-PIA (25 nM) reduced IA/AAR to a similar extent (21 +/- 6%). However, while the cardioprotective effect of R-PIA was significantly inhibited (54 +/- 7% IA/AAR) by the rabbit A1-selective antagonist, BWA1433 (50 nM), the IB-MECA-dependent cardioprotection was unaffected (28 +/- 6% IA/AAR). A non-selective (A1 vs. A3) concentration of BWA1433 (5 microM) significantly attenuated the IB-MECA-dependent cardioprotection (61 +/- 7% IA/AAR). CONCLUSIONS: These data clearly demonstrate that selective A3 receptor activation provides cardioprotection from ischemia-reperfusion injury in the rabbit heart. Furthermore, the degree of A3-dependent cardioprotection is similar to that provided by A1 receptor stimulation or ischemic preconditioning.
Asunto(s)
Adenosina/análogos & derivados , Isquemia Miocárdica/prevención & control , Fenilisopropiladenosina/uso terapéutico , Receptores Purinérgicos/efectos de los fármacos , Adenosina/uso terapéutico , Animales , Modelos Animales de Enfermedad , Masculino , Daño por Reperfusión Miocárdica/prevención & control , Conejos , Estimulación QuímicaRESUMEN
Discordant xenogeneic organ transplantation is a potential solution to the critical shortage of suitable donor organs. However, clinical application of xenotransplantation with physiologically suitable organs such as those from the pig, is currently limited by the lack of agents to prevent antibody and complement-mediated hyperacute rejection of the transplanted organ. We have used retrovirus-mediated gene transfer to express the terminal complement inhibitor protein, human CD59, in neonatal porcine aortic endothelial cells (nPAEC). Human CD59 was constitutively expressed in nPAECs at levels similar to that of native CD59 in human umbilical vein endothelial cells. The protein was tethered to the cell surface by a glycosyl-phosphatidylinositol anchor, as demonstrated by its removal following treatment with phosphatidylinositol-specific phospholipase C. In a model of antibody-dependent complement activation, nPAECs expressing human CD59 were protected from membrane pore formation and cell lysis by complement derived from either human or baboon sera. Conversely, nPAECs expressing CD59 were not protected from lysis by rabbit or dog complement, indicating that recombinant CD59 retained its species-restricted inhibitory activity. Additionally, CD59 expressed on nPAECs inhibited the C5b-9-dependent generation of membrane prothrombinase activity. Collectively, these data establish that stable expression of human CD59 on xenotypic (porcine) endothelial cells renders these cells resistant to both the cytolytic and procoagulant effects of human complement. We propose that expression of recombinant human CD59 on porcine donor organs may prevent complement-mediated lysis and activation of endothelial cells that leads to hyperacute rejection.
Asunto(s)
Antígenos CD/inmunología , Endotelio Vascular/inmunología , Glicoproteínas de Membrana/inmunología , Secuencia de Aminoácidos , Animales , Antígenos CD/metabolismo , Antígenos CD59 , Activación de Complemento , Citotoxicidad Inmunológica , Activación Enzimática , Glicosilfosfatidilinositoles , Humanos , Glicoproteínas de Membrana/metabolismo , Datos de Secuencia Molecular , Proteínas Recombinantes , Especificidad de la Especie , Porcinos , Tromboplastina/metabolismoRESUMEN
In this study we present a comprehensive evaluation of the molecular interactions between human T cells and porcine aortic endothelial cells (PAEC) that contribute to human T cell activation. Binding assays demonstrated that porcine erythrocytes (E) and PAEC express ligand(s) for the human T cell glycoprotein CD2. Prior incubation of human T cells with a blocking monoclonal antibody directed against CD2 (alpha CD2-BL) completely inhibited T cell/E and T cell/PAEC interaction. Xenogeneic mixed lymphocyte reactions (XMLR) revealed that human PBMC, or highly purified T cells were activated by PAEC in the absence of human antigen-presenting cells (APC). Addition of alpha CD2-BL or alpha LFA-1 to these assays inhibited PAEC-mediated human T cell activation. Furthermore, we demonstrated that highly purified human CD4+ and CD8+ T cells proliferated in response to PAEC and that this response was blocked by monoclonal antibodies directed against LFA-1 and CD2. Addition of alpha SLA class I blocked the proliferation of CD8+ but not CD4+ T cells, indicating direct presentation of SLA class I antigens to human T cells. We have recently shown that expression of the human complement inhibitor (CD59) on PAEC (PAEC-LXSNCD59) rendered these cells resistant to human complement-mediated activation and lysis, suggesting that human CD59 expression on PAEC could be an effective therapy for hyperacute rejection (HAR). However, recent studies have shown that in addition to its role as a complement inhibitor, CD59 binds human T cell CD2 and contributes to T cell activation. We therefore examined whether human CD59 expression on PAEC augmented the human antiporcine T cell response. We demonstrated that human T cells do not display increased binding to or activation by PAEC-LXSNCD59 relative to PAEC controls. Taken together, our data establish that PAEC directly stimulate human T cells in vitro and that interactions between the human accessory molecules CD2, LFA-1 and their PAEC surface ligands contribute to human T cell activation. In addition, the expression of human CD59 on porcine donor organs may confer resistance to human complement-mediated HAR without exacerbating the human antiporcine cellular response.
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Antígenos CD/inmunología , Antígenos de Diferenciación de Linfocitos T/inmunología , Endotelio Vascular/fisiología , Eritrocitos/fisiología , Activación de Linfocitos , Antígeno-1 Asociado a Función de Linfocito/inmunología , Receptores Inmunológicos/inmunología , Linfocitos T/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Antígenos de Superficie/inmunología , Aorta , Antígenos CD2 , Linfocitos T CD4-Positivos/inmunología , Antígenos CD8/inmunología , Células Cultivadas , Endotelio Vascular/inmunología , Eritrocitos/inmunología , Citometría de Flujo , Humanos , Ligandos , Mitomicina/farmacología , Formación de Roseta , Ovinos , Porcinos , Linfocitos T/efectos de los fármacosRESUMEN
Halobacterium species display a variety of responses to light, including phototrophic growth, phototactic behavior, and photoprotective mechanisms. The complete genome sequence of Halobacterium species NRC-1 (Proc Natl Acad Sci USA 97: 12176-12181, 2000), coupled with the availability of a battery of methods for its analysis makes this an ideal model system for studying photobiology among the archaea. Here, we review: (1) the structure of the 2.57 Mbp Halobacterium NRC-1 genome, including a large chromosome, two minichromosomes, and 91 transposable IS elements; (2) the purple membrane regulon, which programs the accumulation of large quantities of the light-driven proton pump, bacteriorhodopsin, and allows for a period of phototrophic growth; (3) components of the sophisticated pathways for color-sensitive phototaxis; (4) the gas vesicle gene cluster, which codes for cell buoyancy organelles; (5) pathways for the production of carotenoid pigments and retinal, (6) processes for the repair of DNA damage; and (7) putative homologs of circadian rhythm regulators. We conclude with a discussion of the power of systems biology for comprehensive understanding of Halobacterium NRC-1 photobiology.
RESUMEN
The two diastereomeric sulphoxides and the sulphone derived from the formyl-methionyl tripeptide chemoattractant CHO-L-Met-L-Phe-OMe have been synthesized and fully characterized. The diastereomeric sulphoxide tripeptides have been separated at the stage of their N-tert-butyloxycarbonyl synthetic precursors. All of the oxidized sulphur derivatives induce secretion of granule enzymes with ED50s from 1-2 x 10(-9) M with no significant differences in activity among them. They are also active to the same relative extent in inducing chemotaxis. In parallel, a solution conformational analysis has been performed in solvents of widely different polarities and capabilities of H-bond formation using circular dichroism, infrared absorption and 1H nuclear magnetic resonance. No significant propensity for formation of intramolecularly (C = O...H-N) H-bonded folded forms has been detected in any of the four tripeptides. Intermolecular S = O...H-N interactions are postulated to tentatively explain the 1H nuclear magnetic resonance behavior of the Met and, particularly, Leu NH resonances of the two sulphoxide tripeptides in CDCl3 solution. The biological and conformational data agree with the recently proposed model of the chemotactic peptide receptor of rabbit neurotrophils, which involves the extended backbone of the integrity of the Met side-chain sulphide sulphur atom as a corollary point of ligand interaction.
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N-Formilmetionina Leucil-Fenilalanina/análogos & derivados , N-Formilmetionina Leucil-Fenilalanina/síntesis química , Dicroismo Circular , Espectroscopía de Resonancia Magnética , Oxidación-Reducción , Conformación Proteica , Estereoisomerismo , Relación Estructura-Actividad , Sulfonas , SulfóxidosRESUMEN
High resolution measurement in both exercised skeletal and cardiac tissue made radially outward from capillaries and longitudinally parallel to capillaries by Gayeski and Honig (1986, 1986a,b) and Honig and Gayeski (1987) indicate shallow variation of tissue PO2 and the absence of strong causal relation between the PO2 at a point and the proximity of that point to the nearest active capillary. Proposed as a model for the analysis of this tissue PO2 distribution, so contrary to the expectations of Krogh type models, are a class of multicellular tissue cylinder models. Each cylinder is penetrated by many parallel capillaries. In order to better represent the natural irregularities of the skeletal and cardiac tissue both with regard to radial placement and the stagger of the capillary inlets, the following types of models both with and without yoglobin are examined: regular square arrays where the capillary PO2 levels are random, uniform capillary PO2 levels but random capillary positions, and those with both the capillary PO2 levels and the positions are random. The results of the model calculations show that the superposition of the oxygen diffusion fields of all the capillaries produce a tissue PO2 distribution with the properties: (1) lower tissue PO2 levels than those predicted by Krogh theory, (2) significant non-local contributions to the PO2 at a point in the tissue which greatly reduces this correlation between PO2 at a point and its proximity to an active capillary, (3) shallow transcellular PO2 variation.
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Modelos Teóricos , Músculos/metabolismo , Miocardio/metabolismo , Oxígeno/metabolismo , Animales , Tampones (Química) , Capilares/fisiología , Circulación Coronaria , Matemática , Músculos/irrigación sanguínea , Oxígeno/sangre , Presión Parcial , Flujo Sanguíneo RegionalRESUMEN
Our objective was to perform a retrospective analysis of breeding soundness evaluations (BSEs) as classified by the 1993 Society for Theriogenology (SFT) guidelines [Chenoweth et al., Guidelines for using the bull breeding soundness evaluation form, in: Theriogenology Handbook, 1993, pp. B-10]. Data included BSE information obtained from five performance-testing stations in South Carolina (SC1, SC2, SC3) and Tennessee (TN1, TN2) from 1986 through 1999 on 3648 Angus, Brangus, Charolais, Chianina, Gelbvieh, Limousin, Polled Hereford, Santa Gertrudis, Simbrah, and Simmental bulls. Analyses were simplified by classifying all bulls as either satisfactory or unsatisfactory potential breeders. Of the 3648 bulls evaluated, 76.2% were classified as satisfactory potential breeders. Of all bulls evaluated, 4.0% were unsatisfactory due to inadequate spermatozoal motility, 7.0% due to inadequate spermatozoal morphology and 2.6% due to a combination of inadequate motility and morphology. Unsatisfactory classifications due to non-spermatozoal parameters out of all bulls were 10.2%, with 7.1% for inadequate scrotal circumference and 3.1% for physical abnormalities. For satisfactory and unsatisfactory bulls, respectively, means and standard deviations were 35.8 +/- 2.7 and 33.0 +/- 4.1 cm (P < 0.001) for scrotal circumference, 63 +/- 18 and 35 +/- 24% (P < 0.001) for percent motility, and 86 +/- 7 and 63 +/- 21% (P < 0.001) for percent normal morphology.
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Cruzamiento/métodos , Bovinos , Animales , Bovinos/clasificación , Masculino , Escroto/anatomía & histología , Motilidad Espermática , Espermatozoides/anomalíasAsunto(s)
Síndrome de Inmunodeficiencia Adquirida/prevención & control , Folletos , Estudiantes , Universidades , Síndrome de Inmunodeficiencia Adquirida/psicología , Adulto , Femenino , Educación en Salud , Humanos , Masculino , Pennsylvania , Evaluación de Programas y Proyectos de Salud/métodos , Vergüenza , Estudiantes/psicologíaRESUMEN
Intact rabbit neutrophils were found to express phospholipase A2 activity against [14C]oleate-labeled autoclaved Escherichia coli. Cells obtained 12-14 h rather than 4 h after intraperitoneal injection of glycogen had approximately threefold higher activity. The higher activity was due neither to contaminating mono-nuclear cells nor to the degranulation associated with the prolonged inflammatory response in the peritoneum. Of the phospholipase A2 activity of the intact 12-hour cells, approximately one half remained cell-associated, but the other half was released from the neutrophils during incubation. The cell-associated activity was not due to phagocytosis of substrate and subsequent release of [14C]fatty acid. The cell-associated activity was inhibited by the membrane-impermeant diazonium salt of sulfanilic acid. The results indicate that the cell-associated activity of intact neutrophils is due to an ectophospholipase A2.
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Neutrófilos/enzimología , Fosfolipasas A/metabolismo , Fosfolipasas/metabolismo , Conejos/inmunología , Animales , Membrana Celular/metabolismo , Centrifugación por Gradiente de Densidad , Compuestos de Diazonio/farmacología , Escherichia coli/inmunología , Cavidad Peritoneal/citología , Fagocitosis , Fosfolipasas A/análisis , Fosfolipasas A2RESUMEN
Evidence is shown that vimentin, the intermediate filament protein, is a substrate for protein kinase C: (a) Purified vimentin from Chinese hamster ovary cells can be phosphorylated by protein kinase C prepared from rabbit peritoneal neutrophils. Tryptic peptic analysis reveals multiple sites of phosphorylation distinct from those phosphorylated by cAMP-dependent protein kinase. (b) phosphorylation of membrane associated vimentin is stimulated in phorbol 12-myristate 13-acetate treated neutrophil membranes, suggesting that vimentin can be a substrate for membrane associated protein kinase C and (c) phorbol 12-myristate 13-acetate also stimulates the phosphorylation of vimentin in 32P-labeled intact neutrophils.
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Proteína Quinasa C/metabolismo , Vimentina/metabolismo , Animales , Membrana Celular/enzimología , Núcleo Celular/enzimología , Cricetinae , AMP Cíclico/farmacología , Femenino , Neutrófilos/enzimología , Ovario/análisis , Fragmentos de Péptidos/metabolismo , Fosforilación , Proteínas Quinasas/metabolismo , Conejos , Acetato de Tetradecanoilforbol/farmacología , Tripsina/metabolismoRESUMEN
Transforming growth factor-beta 1 (TGF-beta 1) is thought to play a role in modulating vascular cell function in vivo. In vitro, it decreases endothelial cell proliferation and migration. We postulated that these biologic activities could be mediated through TGF-beta 1 modulation of specific gene expression. Therefore we differentially screened a human umbilical vein endothelial cell cDNA library with cDNAs prepared from both untreated and TGF-beta 1-treated bovine aortic endothelial cells. Using this technique, we isolated many TGF-beta 1-induced cDNA clones. Sequence analysis of these cDNAs showed that many of them corresponded to alternatively spliced fibronectin mRNAs. These fibronectin clones all contained the extradomain I (ED I) but three different forms of the type III connecting segment (IIICS). These different fibronectin cDNAs were expressed in bacteria and the recombinant proteins used to study the effects of IIICS alternative splicing on cell attachment, spreading, and migration in bovine aortic endothelial and smooth muscle cells and B16F10 melanoma cells. The results of these experiments show that attachment and spreading of bovine aortic endothelial and smooth muscle cells depend primarily on the presence of the Arg-Gly-Asp-Ser (RGDS) sequence in the recombinant fibronectin proteins. However attachment and spreading of bovine aortic endothelial cells are modulated by alternative splicing in the IIICS region. Specifically splicing of the IIICS region decreases spreading and increases migration rates of the endothelial cells. On the contrary, using a cell line (B16F10 melanoma cells) that is known not to require the RGDS sequence for adhesion confirmed previous findings that B16F10 melanoma cells do not require the presence of the RGDS sequence for attachment and spreading. Indeed B16F10 cells were able to attach and spread on two recombinant proteins that did not contain the RGDS sequence. However attachment and spreading of B16F10 were dramatically inhibited when a 75-base pair DNA fragment was removed from the 5' end of the IIICS region. These results suggest that various regions of the fibronectin molecule may be able to interact with different cell populations to promote cell attachment and spreading, and that alternative splicing may modulate this process.
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Aorta/metabolismo , Endotelio Vascular/metabolismo , Fibronectinas/genética , Empalme del ARN , ARN Mensajero/genética , Animales , Aorta/citología , Movimiento Celular , Clonación Molecular , ADN/genética , Endotelio Vascular/citología , Técnica del Anticuerpo Fluorescente , Biblioteca de Genes , Integrinas/metabolismo , Melanoma/patología , Músculo Liso Vascular/citología , Péptidos/metabolismo , Plásmidos , Proteínas Recombinantes , Factor de Crecimiento Transformador beta/farmacología , Células Tumorales CultivadasRESUMEN
We have observed that resonant Rayleigh scattering dominates the emission from poly(p-phenylene vinylene) excited with photons at energies below the threshold at which excitonic migration is reduced. The intensity of the resonant emission decays exponentially with a lifetime of up to 450 fs after pulsed excitation. The coherent nature of the emission was confirmed by angular variations in the far-field emission intensity-bright and dark speckles. Persistence of a coherent polarization was demonstrated by coherent control using phase-locked pulses.
RESUMEN
Rabbit peritoneal neutrophils, permeabilized with Triton X-100, contain inositol phosphate 5-phosphomonoesterase activity capable of converting [3H]inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) to [3H]inositol 1,4-bisphosphate. This activity is found predominantly associated with the soluble component of fractionated neutrophils. It is comprised of specific and nonspecific activities toward Ins-1,4,5-P3 which can be separated by cation exchange chromatography. Treatment of neutrophils with phorbol 12-myristate 13-acetate (PMA) prior to permeabilization does not affect the rate of Ins-1,4,5-P3 breakdown by these cells. In addition, activation of endogenous protein kinase C in a soluble fraction prepared from neutrophils does not affect the specific inositol phosphate 5-phosphomonoesterase activity of this fraction. Taken together, these results provide evidence that activation of protein kinase C in the neutrophil does not affect its 5-phosphomonoesterase activity. Unlike platelets, the phosphorylation of a 5-phosphomonoesterase, if it occurs, may not play a role in the inhibitory effects of PMA on neutrophil responsiveness.