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1.
Physiotherapy ; 101(2): 226-31, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25282386

RESUMO

OBJECTIVE: To investigate the perceptions of physiotherapists about their role in health promotion in an acute inpatient setting. DESIGN: Qualitative design using focus groups within a constructivist framework. Focus groups were recorded, transcribed verbatim and analysed using a constant comparative method. SETTING: An acute National Health Service (NHS) hospital trust. PARTICIPANTS: Three focus groups were conducted with a total of 22 physiotherapists who were recruited purposively from the medical and surgical unit of the study hospital. FINDINGS: Participants generally perceived health promotion to be within their scope of practice, particularly in relation to the promotion of physical activity and smoking cessation. Whilst some facilitators to engaging in health promotion were discussed, a large number of barriers to routine engagement in health promotion were identified. These included time constraints, the focus of acute settings on discharge, and scepticism of effectiveness. CONCLUSION: Pre- and postregistration physiotherapy education as well as mandatory training in NHS trusts should be reviewed to equip the workforce to engage in this crucial area of health care.


Assuntos
Atitude do Pessoal de Saúde , Promoção da Saúde/organização & administração , Percepção , Fisioterapeutas/psicologia , Papel Profissional , Adulto , Feminino , Grupos Focais , Humanos , Masculino , Pessoa de Meia-Idade , Pesquisa Qualitativa
2.
J Appl Microbiol ; 107(3): 1012-8, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19320952

RESUMO

AIMS: To examine the rate and the extent of spore formation in Anoxybacillus flavithermus biofilms and to test the effect of one key variable - temperature - on spore formation. METHODS AND RESULTS: A continuous flow laboratory reactor was used to grow biofilms of the typical dairy thermophile A. flavithermus (strain CM) in skim milk. The reactor was inoculated with either a washed culture or a spore suspension of A. flavithermus CM, and was run over an 8.5 h period at three different temperatures of 48, 55 and 60 degrees C. Change in impedance was used to determine the cell numbers in the milk and on the surface of the stainless steel reactor tubes. The biofilm developed at all three temperatures within 6-8 h. Spores formed at 55 and 60 degrees C and amounted to approx. 10-50% of the biofilm. No spores formed at 48 degrees C. CONCLUSIONS: The results suggest that both biofilm formation and spore formation of A. flavithermus can occur very rapidly and simultaneously. In addition, temperature variation has a considerable effect on the formation of spores. SIGNIFICANCE AND IMPACT OF THE STUDY: This information will provide direction for developing improved ways in which to manipulate conditions in milk powder manufacturing plants to control biofilms and spores of A. flavithermus.


Assuntos
Bacillaceae/crescimento & desenvolvimento , Biofilmes/crescimento & desenvolvimento , Reatores Biológicos/microbiologia , Leite/microbiologia , Esporos Bacterianos/crescimento & desenvolvimento , Animais , Bacillaceae/fisiologia , Contagem de Células , Contagem de Colônia Microbiana , Impedância Elétrica , Esporos Bacterianos/fisiologia , Aço Inoxidável , Temperatura
3.
J Agric Saf Health ; 13(1): 9-24, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17370910

RESUMO

With few established risk factors, cancer remains the second leading cause of death for children in the U.S. Agricultural pesticide use is one of many suspected factors that may contribute to the etiology of childhood cancer. This study tests the hypothesis that birth in Texas counties with moderate to intense agricultural activity increases childhood cancer risk. This case-control study analyzed 6974 cases and controls ages 0 to 14, identified through the Texas Cancer Registry and Texas birth records, respectively. Exposure data were obtained from the Census of Agriculture. Percent cropland in the county of birth and total county-specific pesticide exposure incorporating the EPA's carcinogenicity classification served as surrogates for pesticide exposure. Cancer sites examined include: all cancers, leukemia, lymphoma, CNS tumors, and several specific subsites. Elevated, although not statistically significant, ORs for the association between birth in counties with > or =50% cropland were produced for all CNS tumors (OR = 1.3, 95% CI = 0.9-1.8), astrocytoma (OR = 1.4, 95% CI = 0.8-2.2), and PNET (OR = 1.3, 95% CI = 0.7-2.5). A similar pattern was not observed using the index of total county-specific pesticide exposure. Although imprecise, these exposure assessment methods represent novel applications of agricultural census data. Although a pattern of increased risk was observed between percent cropland and CNS tumors, this study's results do not support an association between birth in Texas counties with moderate to intense agricultural activity and childhood cancer. Due to study limitations, such an association should not be ruled out. Future research should incorporate individual-level data from various sources to increase precision and decrease misclassification in the exposure assessment.


Assuntos
Agricultura , Exposição Ambiental , Neoplasias/induzido quimicamente , Neoplasias/epidemiologia , Praguicidas/efeitos adversos , Adolescente , Estudos de Casos e Controles , Neoplasias do Sistema Nervoso Central/induzido quimicamente , Neoplasias do Sistema Nervoso Central/epidemiologia , Criança , Pré-Escolar , Intervalos de Confiança , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Razão de Chances , Saúde da População Rural , Texas/epidemiologia
4.
Arch Psychiatr Nurs ; 15(3): 140-7, 2001 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-11413505

RESUMO

Empathy is a concept deeply rooted in and central to professional nursing. Although viewed as an important concept, little consensus exists in the professional literature about either the definition or the application of the concept to nursing practice. This article will compare two theories of empathy, one borrowed from Kohut's self-psychology model and one derived from King's Interacting Systems nursing framework. The two theories are examined to clarify issues pertaining to the concept of empathy and to identify the contributions and limitations of borrowed theory as the basis for nursing practice. The article explores similarities and difference in the two views of empathy and highlights the necessity of developing nursing science from theory based in nursing which reflects the very unique nature of nursing practice. Nursing as a profession is distinct and unique, and borrowed theory must be questioned for its fit and applicability to the profession. The article concludes that empathy is a nursing phenomenon needing to be studied from a nursing perspective. The results of continued use of borrowed theory pertaining to empathy is discussed including the delay and misdirection of ongoing activity to develop the concept of empathy.


Assuntos
Empatia , Relações Enfermeiro-Paciente , Teoria de Enfermagem , Teoria Psicológica , Humanos , Pesquisa em Avaliação de Enfermagem , Enfermagem Psiquiátrica , Psicoterapia , Ciência , Psicologia do Self , Análise de Sistemas
5.
Syst Appl Microbiol ; 24(4): 539-48, 2001 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11876361

RESUMO

Molecular and biochemical assays were used to determine the identification of thermophilic bacilli isolated from New Zealand milk powder. One hundred and forty one isolates of thermophilic bacilli were classified into six species using biochemical profiles. Geobacillus stearothermophilus represented 56% of the isolates. All isolates were also analysed by randomly amplified polymorphic DNA (RAPD) analysis, with 45 types identified. Amplification of the 16S-23S rDNA internal spacer region produced two to eight amplification products per strain. The patterns from gel electrophoresis of the internal spacer region amplicons formed two major groupings suggesting the possibility of two distinct species. Partial sequences of 16S rDNA from representatives from each group were compared with sequences in GeneBank and were found to match the 16S rDNA sequences of B. flavothermus and G. thermoleovorans. Primers were designed for these species and used to screen an arbitrary selection of 59 of the dairy isolates. This enabled the identification of 28 isolates as B. flavothermus and 31 isolates as Geobacillus species and these appear to be the predominant isolates in the New Zealand milk powder samples examined. Comparison of the fragment pattern generated by amplification of the 16S-23S rDNA internal spacer region is a simple method to differentiate thermophilic Bacillus species associated with the dairy industry.


Assuntos
Bacillus/classificação , Microbiologia de Alimentos , Leite/microbiologia , Animais , Bacillus/genética , Bacillus/metabolismo , Sequência de Bases , Bovinos , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Espaçador Ribossômico/química , DNA Espaçador Ribossômico/genética , Dados de Sequência Molecular , Nova Zelândia , RNA Ribossômico 16S/química , RNA Ribossômico 16S/genética , Técnica de Amplificação ao Acaso de DNA Polimórfico , Homologia de Sequência do Ácido Nucleico
6.
Clin Orthop Relat Res ; (380): 220-5, 2000 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11064995

RESUMO

Few studies have examined the potential adverse effects of excess distraction and prolonged duration of external fixation for the treatment of distal radius fractures. In this study, 19 patients with distal radius fractures treated with external fixation and supplemental Kirschner wire fixation between August 1991 and November 1997 were studied retrospectively. Patients were evaluated by questionnaire, chart review, radiographs, and clinical examination an average of 161 weeks after injury. Although no significant correlation was found between amount of distraction, as measured by carpal height index, and scores for pain, function, radiographs, motion, grip, strength, and final result, a negative correlation was found of all categories with increasing carpal height index. A significant negative correlation was seen between duration of external fixation and scores for pain, motion, and total score, with motion scores being most affected. New York Orthopaedic Hospital grades of good or excellent were attained by 89% of the patients. The data suggest that external fixation with supplemental pin fixation is a satisfactory method of treating severe fractures of the distal radius. Outcome likely is improved with shorter duration of external fixation.


Assuntos
Fixação de Fratura , Osteogênese por Distração , Fraturas do Rádio/cirurgia , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Resultado do Tratamento
8.
Virology ; 259(1): 7-19, 1999 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-10364485

RESUMO

Thrombocytopenia is a consistent finding and one of the earliest hematological abnormalities in horses acutely infected with equine infectious anemia virus (EIAV), a lentivirus closely related to human immunodeficiency virus. Multifactorial mechanisms, including immune-mediated platelet destruction and impaired platelet production, are implicated in the pathogenesis of EIAV-associated thrombocytopenia. This study was undertaken to investigate whether regenerative thrombopoiesis and platelet destruction occurred in ponies acutely infected with EIAV. Circulating large, immature platelets were increased in ponies acutely infected with EIAV late in the infection when platelet count was at a nadir. Morphometric analysis of bone marrow from acutely infected ponies revealed significant increased in megakaryocyte area and megakaryocyte nuclear area. A trend toward increased numbers of megakaryocytes was also observed. Platelets from acutely infected ponies had increased surface-bound fibrinogen and ultrastructural changes consistent with in vivo platelet activation. Platelets also had hypofunctional aggregation responses to three agonists in vitro. We conclude that thrombocytopenia in ponies acutely infected with EIAV is regenerative and suggest that bone marrow platelet production is not severely compromised in these ponies. Our findings reveal that in vivo platelet activation occurs in ponies acutely infected with EIAV, and as a result platelets are hypofunctional in vitro. Activation of platelets in vivo may cause platelet degranulation or formation of platelet aggregates, which would result in removal of these damages platelets from circulation. This may represent a form of nonimmune-mediated platelet destruction in ponies acutely infected with EIAV.


Assuntos
Anemia Infecciosa Equina/sangue , Vírus da Anemia Infecciosa Equina/isolamento & purificação , Ativação Plaquetária , Trombocitopenia/sangue , Trombocitopenia/virologia , Animais , Plaquetas/patologia , Plaquetas/virologia , Anemia Infecciosa Equina/complicações , Anemia Infecciosa Equina/patologia , Humanos , Trombocitopenia/patologia
9.
Res Vet Sci ; 66(1): 77-80, 1999 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-10088717

RESUMO

Seven ponies were infected with the virulent wild-type Wyoming strain of equine infectious anaemia virus (EIAV). Infection status was monitored by serum reverse transcriptase activity, rectal temperature, and complete blood count. Preinfection serum and serum obtained during the initial febrile episode following infection were assayed for interleukin 6 (IL-6) activity. Postinfection IL-6 activity was significantly increased as compared to preinfection values. The magnitude of increase in IL-6 was positively correlated with reverse transcriptase activity (an indirect measure of viraemia) but was not correlated with rectal temperature. IL-6 production in response to EIAV infection may play a role in pathogenesis of disease, especially the hyperglobulinaemia and apparent polyclonal B cell activation in these horses.


Assuntos
Anemia Infecciosa Equina/sangue , Anemia Infecciosa Equina/imunologia , Interleucina-6/sangue , Animais , Temperatura Corporal , Cavalos , Lentivirus/isolamento & purificação , Lentivirus/patogenicidade , DNA Polimerase Dirigida por RNA/sangue , Fatores de Tempo , Virulência
10.
Genomics ; 62(3): 477-82, 1999 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-10644446

RESUMO

Based on searches of EST databases for signal sequences and amphipathic helices, we have identified and cloned an angiopoietin-like gene, ANGPTL3. Multiple tissue Northern blots show that ANGPTL3 is expressed principally in the liver. ANGPTL3 is expressed early during liver development, and expression is maintained in adult liver. Human ANGPTL3 is a 460-amino-acid polypeptide with the characteristic structure of angiopoietins: a signal peptide, an extended helical domain predicted to form dimeric or trimeric coiled-coils, a short linker peptide, and a globular fibrinogen homology domain (FHD). Murine ANGPTL3 is a 455-acid polypeptide encoded by seven exons on mouse chromosome 4, spanning about 11 kb of DNA. ANGPTL3 contains the four conserved cysteines implicated in the intramolecular disulfide bonds within the FHD, but it does not contain two other cysteines that are found within the FHD of angiopoietins 1, 2, and 4. ANGPTL3 also does not contain the characteristic calcium binding motif found in the other angiopoietins. By radiation hybrid mapping and the use of surrounding genes, human ANGPTL3 maps to the 1p31 region.


Assuntos
Angiopoietinas , Substâncias de Crescimento/genética , Peptídeos e Proteínas de Sinalização Intercelular , Fígado/metabolismo , Glicoproteínas de Membrana/genética , Proteínas/genética , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Angiopoietina-1 , Angiopoietina-2 , Proteína 3 Semelhante a Angiopoietina , Proteínas Semelhantes a Angiopoietina , Animais , Sequência de Bases , Sítios de Ligação/genética , Northern Blotting , Cromossomos Humanos Par 1/genética , Glicosilação , Substâncias de Crescimento/biossíntese , Humanos , Camundongos , Dados de Sequência Molecular , Especificidade de Órgãos , Mapeamento Físico do Cromossomo , RNA Mensageiro/biossíntese , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos
11.
Am J Vet Res ; 59(8): 1009-15, 1998 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-9706205

RESUMO

OBJECTIVE: To determine blood protein concentration, immunoglobulin concentration, and lymphocyte profiles in equine infectious anemia virus (EIAV) seropositive, naturally infected horses without clinical signs of disease. ANIMALS: 26 clinically normal seropositive horses, 6 febrile ponies with experimentally induced EIA, and 52 clinically normal seronegative horses and ponies. PROCEDURE: Serum and EDTA-anticoagulated blood were obtained from all horses and ponies, and total serum protein and albumin concentrations, immunoglobulin concentrations, and blood lymphocyte subset counts were determined. RESULTS: Compared with seronegative horses, EIAV seropositive inapparent carrier horses had no significant difference in serum reverse transcriptase activity, PCV, or platelet count. Inapparent carrier horses had increased plasma total solids and serum globulin concentrations and decreased serum albumin concentration and albumin-to-globulin ratio. Total serum immunoglobulin and serum IgM concentrations were increased. Inapparent carrier horses had significantly decreased percentages of CD5+ and CD4+ blood lymphocytes. CONCLUSIONS: Serum protein and lymphocyte subset changes in EIAV-infected inapparent carrier horses are consistent with immune activation or chronic inflammation, both of which may, in part, be the result of virus-induced polyclonal B-cell activation. CLINICAL RELEVANCE: EIAV seropositive horses have immune-related abnormalities consistent with ongoing viral activity regardless of the duration they have been infected, even when the usual signs of disease (anemia, fever, weight loss) are not apparent.


Assuntos
Portador Sadio/veterinária , Anemia Infecciosa Equina/imunologia , Doenças dos Cavalos/imunologia , Hipergamaglobulinemia/veterinária , Vírus da Anemia Infecciosa Equina/isolamento & purificação , Subpopulações de Linfócitos/imunologia , Animais , Proteínas Sanguíneas/análise , Portador Sadio/imunologia , Portador Sadio/virologia , Doenças dos Cavalos/sangue , Cavalos , Hipergamaglobulinemia/imunologia , Hipergamaglobulinemia/virologia , Imunoglobulinas/sangue , Valores de Referência , Albumina Sérica/análise
12.
Am J Vet Res ; 58(10): 1092-6, 1997 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-9328660

RESUMO

OBJECTIVE: To evaluate a method for detecting thiazole orange-positive (TO+, reticulated) platelets in equine blood, using flow cytometry. ANIMALS: 16 healthy, equine infectious anemia virus (EIAV)-negative horses and ponies; 9 thrombocytopenic, EIAV-positive horses and ponies; and 2 thrombocytopenic, EIAV-negative horses. PROCEDURE: Blood from healthy and thrombocytopenic horses was collected by jugular venipuncture. Appropriate sample requirement and incubation time for the assay were evaluated, using blood anticoagulated with EDTA or sodium citrate, or platelet-rich plasma in sodium citrate. The sample of blood or platelet-rich plasma was incubated with thiazole orange, and flow cytometric analysis was performed. Percentage of circulating TO+ platelets was determined from fluorescence (FL-1) logarithmic histograms. RESULTS: Healthy ponies (n = 9) had 1.28 to 2.83% (mean +/- SD, 2.03 +/- 0.50%) and horses (n = 7) had 0.9 to 3.44% (2.12 +/- 1.14%) TO+ platelets in circulation. Thrombocytopenic ponies (n = 7) had 11.14 to 48.41% (26.51 +/- 11.99%) and thrombocytopenic horses (n = 4) had 2.33 to 8.52% (6.19 +/- 2.68%) TO+ platelets in circulation. Mean platelet counts for the thrombocytopenic ponies and horses were 24,400 +/- 20,500 and 39,300 +/- 13,500 platelets/microliters, respectively (reference range, 94,000 to 232,000 platelets/ microliters). CONCLUSION: Thiazole orange-positive platelets can be detected in equine blood and percentages of TO+ platelets are increased in thrombocytopenic horses. CLINICAL RELEVANCE: Enumeration of TO+ platelets may prove to be a helpful noninvasive clinical measurement of bone marrow platelet production and aid in the assessment of platelet kinetics in thrombocytopenic horses.


Assuntos
Plaquetas/química , Plaquetas/patologia , Citometria de Fluxo/veterinária , Corantes Fluorescentes/análise , Doenças dos Cavalos/sangue , Tiazóis/análise , Trombocitopenia/veterinária , Animais , Anticoagulantes , Benzotiazóis , Medula Óssea/patologia , Citratos , Ácido Edético , Anemia Infecciosa Equina/sangue , Anemia Infecciosa Equina/complicações , Anemia Infecciosa Equina/patologia , Citometria de Fluxo/métodos , Doenças dos Cavalos/patologia , Cavalos , Quinolinas , Citrato de Sódio , Trombocitopenia/sangue , Trombocitopenia/complicações , Trombocitopenia/patologia
13.
Vet Microbiol ; 52(3-4): 209-21, 1996 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-8972047

RESUMO

Equine infectious anemia virus (EIAV) is a lentivirus that replicates predominantly in mature tissue macrophages. Viral expression is strongly influenced by the state of differentiation of the host cell. While blood monocytes can be infected, viral transcription is limited until the cell differentiates into a mature macrophage. Activation of mature macrophages infected with EIAV might also alter viral expression, presumably through binding of cellular transcription factors to viral nucleic acid sequences within the long terminal repeat (LTR). Using DNA amplification techniques, we compared LTR sequences of U.S. field strains of EIAV to sequences of a laboratory adapted strain of the virus. All field strain sequences were more closely related to Wyoming strain than to the Malmquist laboratory adapted strain or a previously sequenced infectious molecular clone of EIAV. Primary equine monocyte-derived macrophage cultures were infected with virulent and avirulent strains of EIAV and the effects of macrophage stimulation on EIAV expression were determined. Stimulation of macrophages with phorbol ester activated the cells to secrete tumor necrosis factor alpha (TNF alpha). This activation signal also resulted in a significant downregulation of viral expression as determined by supernatant reverse transcriptase activity. This effect occurred independent of the virulence of the virus strain used or the nucleic acid sequence of the viral LTR. This may represent an adaptive response of EIAV to evade the host immune response and establish a persistent infection.


Assuntos
Anemia Infecciosa Equina/virologia , Vírus da Anemia Infecciosa Equina/genética , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Animais , Sequência de Bases , Sobrevivência Celular , Células Cultivadas , Regulação Viral da Expressão Gênica , Cavalos , Vírus da Anemia Infecciosa Equina/fisiologia , Macrófagos/fisiologia , Macrófagos/virologia , Dados de Sequência Molecular , Sequências Repetitivas de Ácido Nucleico
14.
J Dairy Sci ; 79(6): 943-55, 1996 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-8827459

RESUMO

Six Lactobacillus acidophilus, 5 Bifidobacterium, and 6 Streptococcus thermophilus strains were studied for characteristics that are important to activity and stability in unfermented fluid milk products. Speciation, strain relatedness, frozen concentrate stability, bile sensitivity, and lactase activity were evaluated. The microbiological stability of a culture-containing fluid milk product was also determined. Two of the bifidobacteria cultures contained > 1 strain. Some strains were shown to be closely related or identical by pulsed-field gel electrophoresis of fragmented chromosomal DNA. Selective media that distinguished among all 3 added genera were identified. All lactobacilli and most of the bifidobacteria were resistant to bile concentrations varying from 1 to 3%, and all streptococci were sensitive to bile. Lactase activities were highest for S. thermophilus strains, supporting use of this species in fluid milk and dairy products to aid in the digestion of lactose by consumers. The experimental product evaluated in this study contained 10(7) cfu/ml of both L. Acidophilus and Bifidobacterium spp. and 5 x 10(7) cfu/ml of S. thermophilus. Lactic, but not psychrotrophic, populations were fairly stable during storage. The results suggest that milk formulated with high concentrations of three different genera of probiotic bacteria can be manufactured with commercial strains.


Assuntos
Bifidobacterium/fisiologia , Lactobacillus acidophilus/fisiologia , Leite/microbiologia , Streptococcus/fisiologia , Animais , Bifidobacterium/genética , Bile , Eletroforese em Gel de Campo Pulsado , Genótipo , Lactase , Lactobacillus acidophilus/genética , Sondas de Oligonucleotídeos , Streptococcus/genética , beta-Galactosidase/metabolismo
15.
J Virol ; 70(1): 590-4, 1996 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-8523576

RESUMO

Equine infectious anemia virus is a lentivirus that replicates in mature tissue macrophages of horses. Ponies were infected with equine infectious anemia virus. During febrile episodes, proviral DNA was detectable, but viral mRNA was not detectable. As cultured blood monocytes from these ponies differentiated into macrophages, viral expression was upregulated. In situ hybridization confirmed that viral transcription occurred in mature macrophages.


Assuntos
Anemia Infecciosa Equina/virologia , Vírus da Anemia Infecciosa Equina/fisiologia , Monócitos/virologia , Replicação Viral , Doença Aguda , Animais , Diferenciação Celular , DNA Viral , Anemia Infecciosa Equina/sangue , Cavalos , Vírus da Anemia Infecciosa Equina/isolamento & purificação , Monócitos/metabolismo , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Regulação para Cima
16.
Biotechnol Bioeng ; 46(1): 22-7, 1995 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-18623258

RESUMO

The secretion rate of activated protein C (APC) by BHK cells was increased 35-fold by increasing the cDNA copy number per cell from 50 to 240. In this range, the relation between APC secretion and cDNA copy number was not linear and the rate of APC secretion per cDNA copy increased sevenfold. This apparent cooperative effect of multiple cDNA copies could be related to their integration in tandem. For cDNA copy numbers higher than 240, the APC secreation rate per cDNA and per cell decreased dramatically. The gamma-carboxylation of glutamic acid residues, a posttranslational modification required for APC biological activity, was also investigated. The proportion of APC that was fully gamma-carboxylated decreased as the secretion rate of APC increased. (c) 1995 John Wiley & Sons, Inc.

17.
Biochemistry ; 30(2): 367-72, 1991 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-1988035

RESUMO

The human protein C precursor undergoes extensive co- and posttranslational modification during its biosynthesis in the liver. These modifications include glycosylation, gamma-carboxylation, and beta-hydroxylation of specific amino acids and endoproteolytic processing to remove the pre- and propeptides as well as the pair of basic amino acids which connect the light and heavy chains in the precursor. Previous studies with a recombinant mammalian expression system have indicated that the endopeptidase in several mammalian cell types which recognizes and cleaves this dibasic site has a substrate specificity for sites which also include a basic amino acid in the -4 position (Foster et al., 1990). Since the human protein C precursor has His154 in the -4 position, it is poorly and incompletely cleaved in BHK and several other mammalian cell lines and also apparently secreted from the liver as a mixed population of mature two-chain and precursor one-chain molecules. In the present study, a mammalian expression system has been used to study the effect of coexpressing the protein C precursor together with the yeast Kex2 endopeptidase which is known to recognize and process dibasic pairs within peptide precursors in yeast. Coexpression of the KEX2 gene resulted in complete conversion of the protein C precursor to the mature two-chain form. Amino-terminal sequencing of the cleavage products has indicated that the cleavage occurs in the correct location and that this site is preferentially recognized by the yeast endopeptidase within the context of the mammalian cell secretory pathway.


Assuntos
Pró-Proteína Convertases , Proteína C/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae , Serina Endopeptidases/metabolismo , Subtilisinas , Sequência de Aminoácidos , Animais , Células Cultivadas , Cricetinae , Análise Mutacional de DNA , Vetores Genéticos , Técnicas In Vitro , Dados de Sequência Molecular , Testes de Precipitina , Proteína C/imunologia , Proteína C/isolamento & purificação , Proteínas Recombinantes de Fusão , Transfecção
18.
Biochemistry ; 29(2): 347-54, 1990 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-2405901

RESUMO

The human protein C precursor undergoes extensive co- and posttranslational modification during its biosynthesis in the liver. These modifications include glycosylation, gamma-carboxylation and beta-hydroxylation of specific amino acids, and endoproteolytic processing to remove the pre- and propeptides and also to remove the pair of basic amino acids that connect the light and heavy chains in the precursor. Specific molecular signals have been elucidated which direct several of these modifications; however, the mechanism for cleavage and removal of the basic amino acid pair has not been established. In the present study, a recombinant mammalian expression system has been used to study the molecular signals that direct removal of this basic amino acid pair. Mutations were introduced by site-directed mutagenesis either to insert additional basic amino acids or to alter the sequence adjacent to the basic pair by point mutations. The mutant protein precursors were expressed and analyzed for the degree of processing to 2-chain form and also for the location of the cleavage site (by N-terminal sequencing) and subsequent removal of the basic amino acids from the newly formed C terminus of the light chain. These experiments have shown that human protein C can be readily synthesized and secreted in several mammalian cell lines. However, cell lines vary considerably in their capacity to remove the dibasic pair in the protein C precursor and, like the liver, secrete a mixed population of 1-chain and 2-chain forms of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)


Assuntos
Endopeptidases/metabolismo , Proteína C/metabolismo , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular , Clonagem Molecular , Cricetinae , Brometo de Cianogênio , Humanos , Técnicas de Imunoadsorção , Dados de Sequência Molecular , Mutação , Proteína C/genética , Especificidade por Substrato , Transfecção
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