RESUMEN
HIF is a transcriptional complex that plays a central role in mammalian oxygen homeostasis. Recent studies have defined posttranslational modification by prolyl hydroxylation as a key regulatory event that targets HIF-alpha subunits for proteasomal destruction via the von Hippel-Lindau ubiquitylation complex. Here, we define a conserved HIF-VHL-prolyl hydroxylase pathway in C. elegans, and use a genetic approach to identify EGL-9 as a dioxygenase that regulates HIF by prolyl hydroxylation. In mammalian cells, we show that the HIF-prolyl hydroxylases are represented by a series of isoforms bearing a conserved 2-histidine-1-carboxylate iron coordination motif at the catalytic site. Direct modulation of recombinant enzyme activity by graded hypoxia, iron chelation, and cobaltous ions mirrors the characteristics of HIF induction in vivo, fulfilling requirements for these enzymes being oxygen sensors that regulate HIF.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans/fisiología , Proteínas de Unión al ADN/metabolismo , Proteínas del Helminto/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Supresoras de Tumor , Ubiquitina-Proteína Ligasas , 2,2'-Dipiridil/metabolismo , Secuencia de Aminoácidos , Animales , Cromatografía Líquida de Alta Presión , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica/genética , Células HeLa , Proteínas del Helminto/química , Proteínas del Helminto/genética , Homeostasis , Humanos , Hidroxilación , Factor 1 Inducible por Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia , Indicadores y Reactivos , Ligasas/metabolismo , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Oxígeno/metabolismo , Procolágeno-Prolina Dioxigenasa/metabolismo , Isoformas de Proteínas , Estructura Secundaria de Proteína , Ratas , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-LindauRESUMEN
Hypoxia-inducible factor (HIF) is a transcriptional complex that plays a central role in the regulation of gene expression by oxygen. In oxygenated and iron replete cells, HIF-alpha subunits are rapidly destroyed by a mechanism that involves ubiquitylation by the von Hippel-Lindau tumor suppressor (pVHL) E3 ligase complex. This process is suppressed by hypoxia and iron chelation, allowing transcriptional activation. Here we show that the interaction between human pVHL and a specific domain of the HIF-1alpha subunit is regulated through hydroxylation of a proline residue (HIF-1alpha P564) by an enzyme we have termed HIF-alpha prolyl-hydroxylase (HIF-PH). An absolute requirement for dioxygen as a cosubstrate and iron as cofactor suggests that HIF-PH functions directly as a cellular oxygen sensor.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Hidroxiprolina/metabolismo , Ligasas , Proteínas Nucleares/metabolismo , Oxígeno/fisiología , Procolágeno-Prolina Dioxigenasa/metabolismo , Proteínas/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor , Ubiquitina-Proteína Ligasas , Secuencia de Aminoácidos , Ácido Ascórbico/farmacología , Hipoxia de la Célula , Proteínas de Unión al ADN/química , Deferoxamina/farmacología , Compuestos Ferrosos/farmacología , Humanos , Hidroxilación , Factor 1 Inducible por Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia , Datos de Secuencia Molecular , Proteínas Nucleares/química , Mutación Puntual , Procolágeno-Prolina Dioxigenasa/antagonistas & inhibidores , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Factores de Transcripción/química , Células Tumorales Cultivadas , Ubiquitinas/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-LindauRESUMEN
Heparan sulfate proteoglycan (HSPG) has been found to be associated with amyloid deposits in a number of diseases including the cerebral amyloid plaques of Alzheimer's disease and the transmissible spongiform encephalopathies (TSEs). The role of HSPG in amyloid formation and the neurodegenerative pathology of these diseases have not been established. We have addressed these questions using a scrapie mouse model which exhibits both amyloid and nonamyloid deposition of abnormal PrP protein, the protein marker of TSE infection. The distribution of HSPG was examined throughout the course of the disease in the brains of experimentally infected mice and compared with the distribution of abnormal PrP. Abnormally high levels of HSPG were associated with most types of PrP pathology including all plaque types and diffuse neuroanatomically targeted forms. Scrapie-associated HSPG was present from 70 days after infection, the earliest time-point examined, in the same target areas as abnormal PrP. The association with amyloid plaques may indicate that HSPG is involved in amyloid plaque formation and/or persistence but involvement with early diffuse forms of PrP suggests a more fundamental role in scrapie pathogenesis.
Asunto(s)
Amiloide/análisis , Encéfalo/patología , Proteoglicanos de Heparán Sulfato/análisis , Priones/análisis , Priones/patogenicidad , Scrapie/patología , Alelos , Animales , Encéfalo/citología , Circulación Cerebrovascular , Endotelio Vascular/patología , Homocigoto , Inmunohistoquímica , Ratones , Ratones Endogámicos , Priones/genética , Valores de Referencia , Scrapie/genéticaRESUMEN
Increased levels of solar ultraviolet (290-320 nm) (UV-B) radiation could have profound effects on plant proteins because the aromatic amino acids in proteins absorb strongly in this spectral region. We have investigated the effects of UV-B radiation on plant proteins and have observed a novel 66-kD protein. This product was formed in vivo when Brassica napus L. plants grown for 21 d in 65 [mu]mol m-2 s-1 photosynthetically active radiation were subsequently exposed to 65 [mu]mol m-2 s-1 photosynthetically active radiation plus UV-B radiation (1.5 [mu]mol m-2 s-1). The protein appeared after 4 h of UV-B irradiation and accumulated during the next 16 h in UV-B. The 66-kD protein cross-reacted with an antiserum against the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) holoenzyme. Analysis of soluble leaf proteins revealed that the 66-kD product had a number of isoforms corresponding closely to those of the large subunit of Rubisco (LSU). Partial proteolytic digests of the LSU and the 66-kD protein resulted in an equivalent pattern of protein fragments, leading to the conclusion that the 66-kD protein was a photomodified form of the LSU. A similar high molecular mass variant of Rubisco was observed in soluble protein extracts from leaves of tomato (Lycopersicon esculentum), tobacco (Nicotiana tabacum), and pea (Pisum sativum L.) plants treated in vivo with UV-B, suggesting that it might be a common product, at least among C3 plants. It is interesting that the 66-kD product appears to be generated after incorporation of the LSU into holoenzyme complexes. This conclusion was drawn from two lines of evidence. First, the LSU variant co-purified with holoenzyme complexes isolated by nondenaturing polyacrylamide gel electrophoresis. Second, a UV-B-specific 66-kD protein did not accumulate in a tobacco mutant that synthesizes the Rubisco subunits but does not assemble them into normal holoenzyme complexes.
RESUMEN
Three general classes of photomorphogenic photoreceptors have been characterized in higher plants: phytochrome, a blue light/ultraviolet (UV)-A photoreceptor(s), and a UV-B sensory system(s). Although a great deal is known about phytochrome and the blue light/UV-A photoreceptor(s), little is known about UV-B detection processes. One reason for this is the lack of readily quantifiable morphogenic responses that are specifically induced by UV-B radiation. We have discovered a response to UV-B, upward curling of Brassica napus L. cotyledons, that may be useful for probing the mechanism of UV-B photoreception. The process was initially observed when B. napus seeds were germinated under visible light plus UV-B radiation, but did not occur under visible light alone or visible light plus UV-A. When 5-d-old seedlings grown in visible light were given relatively short exposures of UV-B (100 min of 5.5 [mu]mol m-2 s-1), the curling response was also observed. Development of curling was separated from the application of this UV-B pulse by a 14-h latent period. Pulses of red light, blue light, farred light, and UV-A (100 min of 5.5 [mu]mol m-2 s-1) did not induce curling, indicating UV-B specificity Additionally, these other spectral regions did not reverse or enhance the UV-B-triggered response. The degree of curling showed a log-linear dependence on UV-B fluence (6-40 mmol m-2) and reciprocity with respect to length of exposure and fluence rate. The data indicate that curling is photomorphogenic in nature and may be triggered by a single photoreceptor species.
RESUMEN
Cultures of Escherichia coli of urinary origin were examined after storage for 12 to 15 years by a combination of the techniques of biotyping, resistotyping, haemagglutinin typing, and colicin typing (multiple typing) which allowed strain identification to be made even when variation in one or more typing characters had occurred in vivo. Biotyping and resistotyping used in conjunction were sufficient to allow identification of as many as 106 of 110 pairs of E. coli examined. In the four pairs of isolates for which it was necessary to extend strain identification profiles to include data from other typing methods, probable strain identification was achieved for all but one pair. The multiple typing approach seems useful for retrospective analysis of stored cultures of E. coli.
Asunto(s)
Bacteriuria/microbiología , Escherichia coli/clasificación , Adulto , Anciano , Antibacterianos/farmacología , Niño , Farmacorresistencia Microbiana , Escherichia coli/efectos de los fármacos , Escherichia coli/aislamiento & purificación , Femenino , Pruebas de Hemaglutinación , Humanos , Masculino , Persona de Mediana Edad , Preservación Biológica , SerotipificaciónRESUMEN
A series of 156 cultures of Escherichia coli isolated from sequential urines from 20 patients with urinary-tract infection was examined by biotyping, resistotyping, haemagglutinin typing, O serotyping and antibiogram typing. From 10 of the patients, we repeatedly isolated cultures of a single strain; from each of the other 10 patients, different strains were discriminated. All cultures were typable by biotyping and resistotyping, and those techniques should prove practicable for laboratories not able to perform complete serological analysis. Only a minority (43.6%) of the cultures was typed with a limited range of 24 commercial O sera. Maximum discrimination of strains was achieved by the combined use of several of these techniques.
Asunto(s)
Bacteriuria/microbiología , Escherichia coli/clasificación , Adolescente , Adulto , Anciano , Antibacterianos/farmacología , Niño , Preescolar , Farmacorresistencia Microbiana , Escherichia coli/aislamiento & purificación , Femenino , Variación Genética , Hemaglutininas/biosíntesis , Humanos , Lactante , Masculino , Manosa/farmacología , Persona de Mediana Edad , SerotipificaciónAsunto(s)
Maltrato a los Niños , Composición Familiar , Adolescente , Niño , Preescolar , Femenino , Humanos , Lactante , MasculinoRESUMEN
Both cultures of Escherichia coli were examined for mannose-sensitive (MS) haemagglutinin in rocked-tile tests with guinea-pig red cells at ambient temperature, and agar plate cultures were examined for mannose-resistant eluting (MRE) haemagglutinins against 14 species of red cells in tests mixed at 3--5 degrees C in the presence of .05% (w/v) D-mannose. Ox, sheep, human, pig, horse, guinea-pig, and fowl red cells were required to detect the various patterns of MRE haemagglutination with the different species of cells. Of 387 strains in 155 O serogroups, 95 formed both MS and MRE haemagglutinins (MS+/MRE+), 198 formed only MS (MS+/MRE-), 21 only MRE (MS+/MRE+), and 73 neither (MS-/MRE-). Strains of more than one of these types, and MRE+ strains with different cell specificities were found in many of the serogroups. Some strains in 144 O serogroups had MS haemagglutinin and some in 50 an MRE haemagglutinin. The presence of MS haemagglutinin in a culture was invariably associated with the presence of type-1 fimbriae on the bacteria. All MS+ strains shared a common antigen in their type-1 fimbriae and three groups of these strains possessed also a group-specific fimbrial antigen. The presence of certain kinds of MRE haemagglutinin in over half the MRE+ strains was associated with that of type-MRE fimbriae, but fimbriae were not detected in the other MRE+ strains. The antigens of the MRE haemagglutinins in different strains were heterogeneous and differed from those of the type-1 fimbriae of MS+ strains. Three series of strains from normal faeces, and from patients with infantile diarrhoea and urinary-tract infections each included a minority possessing neither type of haemagglutinin, but this observation did not preclude a role of the haemagglutinins in colonization or pathogenicity.
Asunto(s)
Aglutininas/análisis , Escherichia coli/inmunología , Hemaglutininas/análisis , Adulto , Animales , Pared Celular/inmunología , Pared Celular/ultraestructura , Niño , Eritrocitos/inmunología , Escherichia coli/ultraestructura , Infecciones por Escherichia coli/microbiología , Gastroenteritis/microbiología , Hemaglutinación , Hemaglutininas/inmunología , Humanos , Especificidad de la Especie , Infecciones Urinarias/microbiologíaRESUMEN
Observations of the behaviour of squirrel monkeys, including 8 opposite-sex pairs during daily 30-min social encounters and 2 mixed-sex permanent groups during daily 30-min observation sessions, over a 14-month period were used to determine the periodicity of the oestrous cycle and annual mating season. The median and modal length of the oestrous cycle was 8 days, within which copulations were limited to a 1--2 day period. In a cyclic female, plasma progesterone levels over a 24-day period dropped from 85--151 ng/ml to 25 ng/ml 2 days before oestrus. In non-cyclic females plasma progesterone values were less than 15-4 ng/ml. Males exhibited a 6-8--19-7 week 'season' of copulation and ejaculation. The onset of this 'mating season' in August coincided with the annual peak in male body weight (the 'fatted male' phenomenon).
Asunto(s)
Estro , Haplorrinos/fisiología , Saimiri/fisiología , Conducta Sexual Animal/fisiología , Animales , Copulación , Femenino , Masculino , Embarazo , Progesterona/sangre , Saimiri/sangre , Estaciones del AñoRESUMEN
Observations of six female squirrel monkeys, Saimiri sciureus, indicate that the appearance of the external genitalia is hormone-dependent. Estradiol benzoate induced opening of the vagina, swelling of the labia, enlargement of urethral papilla, and copious discharge of thick white vaginal secretion in ovariectomized females.
Asunto(s)
Estradiol/farmacología , Genitales Femeninos/efectos de los fármacos , Haplorrinos/anatomía & histología , Saimiri/anatomía & histología , Animales , Castración , Femenino , Saimiri/fisiología , Vagina/efectos de los fármacos , Vulva/efectos de los fármacosRESUMEN
Sixty-three girls with covert bacteriuria were included in a controlled trial of therapy. Recurrent infection in the treated group was common and was not significantly different from the rate of persistent infection in the untreated control group. Two children in each group developed clinical pyelonephritis; the others have remained healthy and all of them have a normal rate of growth. 2 years after diagnosis three of the thirty-four children in the control group and one of twenty-six children in the treated group have radiological evidence of new scars of pyelonephritis. These changes were relatively minor and in both groups of children renal growth was similar to that in normal children. It is suggested that for most of these children therapy is not essential, and that when renal changes occur they are of little or no significance. Prescriptive screening for cobert bacteriuria of childhood cannot be recommended at present.
Asunto(s)
Bacteriuria/tratamiento farmacológico , Ampicilina/uso terapéutico , Bacteriuria/complicaciones , Bacteriuria/diagnóstico , Bacteriuria/microbiología , Niño , Preescolar , Ensayos Clínicos como Asunto , Quimioterapia Combinada , Escherichia coli/aislamiento & purificación , Femenino , Estudios de Seguimiento , Humanos , Riñón/crecimiento & desarrollo , Klebsiella/aislamiento & purificación , Nitrofurantoína/uso terapéutico , Proteus/aislamiento & purificación , Pseudomonas/aislamiento & purificación , Pielonefritis/complicaciones , Pielonefritis/diagnóstico por imagen , Radiografía , Recurrencia , Sulfametoxazol/uso terapéutico , Factores de Tiempo , Trimetoprim/uso terapéutico , Reflujo Vesicoureteral/complicaciones , Reflujo Vesicoureteral/diagnóstico por imagenAsunto(s)
Ciproterona/farmacología , Conducta Sexual Animal/efectos de los fármacos , Animales , Castración , Citratos/metabolismo , Eyaculación , Estradiol/farmacología , Femenino , Fructosa/metabolismo , Macaca mulatta , Masculino , Semen/análisis , Motilidad Espermática , Espermatozoides/efectos de los fármacos , Testículo/anatomía & histología , Testículo/efectos de los fármacos , Testosterona/sangreAsunto(s)
Infecciones Bacterianas/epidemiología , Infecciones Urinarias/epidemiología , Adolescente , Bacteriuria/complicaciones , Sedimentación Sanguínea , Estatura , Peso Corporal , Niño , Femenino , Hemoglobinometría , Vivienda , Humanos , Leucocitos , Tamizaje Masivo , Pielonefritis/complicaciones , Pielonefritis/diagnóstico por imagen , Pielonefritis/epidemiología , Piuria/complicaciones , Radiografía , Escocia , Clase Social , Infecciones Urinarias/complicaciones , Reflujo Vesicoureteral/complicaciones , Reflujo Vesicoureteral/diagnóstico por imagen , Reflujo Vesicoureteral/epidemiologíaRESUMEN
During the statutory medical examination on entrance to primary school 943 5-year-old girls were screened for asymptomatic bacteriuria. A prevalence of 2.1% was found. None of the 20 children with asymptomatic bacteriuria was recognized by the parents as having a urinary infection, though 14 of them had symptoms of lower urinary tract infection. In 16 of the children with bacteriuria either the intravenous pyelogram or the micturating cystogram was abnormal. In 12 the height and weight were below the 25th percentile, and in this group the most severe radiological changes were found.Though the significance of asymptomatic bacteriuria is unknown, these results confirm that in this age group it is often associated with a urinary tract abnormality.