RESUMEN
The external human ear is considered to be highly variable among individuals. Hence, forensic applications could be explored for human identification. This research compares the usefulness of Cameriere's ear identification method, in samples originating from six different countries (Brazil, India, Japan, Russia, South Africa and Turkey) in order to examine possible differences in their accuracy values. A sample of 2,225 photographs of the external human ear (1,134 left and 1,091 right ears) from 1,411 individuals (633 females and 778 males) was collected. The samples included healthy subjects with no systemic disorders and without any craniofacial trauma, maxillofacial abnormalities, auricular anomalies, ear diseases or previous auricular surgery. Cameriere's ear identification method was applied and measurements were performed on the images of each ear, considering four anatomic regions: helix, antihelix, concha, and lobe. The quantified measurement values were converted into a proposed coded number system. A search for identical codes was accomplished to find out the distinctiveness of the morphology of the human ear. The combined codes of left and right ears of each of the 814 subjects were not repeated in this multi-ethnic study sample. Dirichlet's distribution and the inherent study equation showed that the probability of two different individuals having the same code (false-positive identification) was found to be <0.0007. Because of the distinctive metrics of the ratios of external human ears, studies with Cameriere's ear identification method may be valuable for human identification. Studying the differences between the left and right ears of the same individual and across different ethnic groups could contribute to the development of supplementary tools for human identification.
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Oído Externo , Etnicidad , Masculino , Femenino , Humanos , Oído Externo/anatomía & histología , BrasilRESUMEN
The diagnostic accuracy of the I3M to assess the legal age of 18 years has already been tested in several specific-population samples. The left lower third molar has been extensively used for discriminating between minors and adults. This research aimed to compare the usefulness of lower third molar maturity indexes, from both left and right side (I3ML and I3MR), in samples originating from four distinct continents in order to examine possible differences in their accuracy values. For this purpose, a sample of 10,181 orthopantomograms (OPGs), from Europe, Africa, Asia and America, was analysed and previously scored in other studies. The samples included healthy subjects with no systemic disorders with both third molars and clear depicted root apices. Wilcoxon Signed Rank test for left and right asymmetry did not show any significant differences. Data about sensitivity, specificity, predictive values, likelihood ratio and accuracy were pooled together and showed similar results for I3ML and I3MR, respectively. In addition, all these quantities were high when only the I3MR was considered to discriminate between adults and minors. The present referable database was the first to pool third molar measurements using panoramic radiographs of subjects coming from different continents. The results highlighted that both I3ML and I3MR are reliable indicators for assessing the legal age of 18 years old in those jurisdictions where this legal threshold has been set as the age of majority.
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Determinación de la Edad por los Dientes/métodos , Tercer Molar/diagnóstico por imagen , Tercer Molar/crecimiento & desarrollo , Grupos Raciales , Etnicidad , Femenino , Humanos , Masculino , Mandíbula/diagnóstico por imagen , Mandíbula/crecimiento & desarrollo , Radiografía Panorámica , Sensibilidad y EspecificidadRESUMEN
To promote radiation protection and health promotion among returning residents (returnees) in coastal areas of Fukushima, eHealth principles were used to develop a new application tool (app) that can record radiation exposure and health status while providing comprehensive support to returnees. Intended users are returnees and health and welfare workers. After assessing their needs, a flowchart and prototype for operational logic were created using commercially available software tools. Professional developers will focus on improving the user interface and ensuring data security. The finished app will be compatible with mobile telephones and tablets. Utility and ease of use are paramount to serve returnees of all ages effectively.
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Accidente Nuclear de Fukushima , Exposición a la Radiación , Protección Radiológica , HumanosRESUMEN
The Bayesian approach is being a fundamental tool in forensic and legal field where inferences and decisions are made. In this study, a full Bayesian calibration model was developed to make probabilistic inferences about age estimation in a reference sample of 891 periapical X-rays of upper and lower canines. These teeth belonged to both deceased and living adult subjects, aged between 20 and 86 years, coming from five different countries (Turkey, Italy, Portugal, Japan and Mexico). For this purpose, the narrowing of pulp chamber due to the apposition of secondary dentine was analysed by means of the pulp/tooth area ratio. To determine the agreement of the method, intra- and inter-observer differences for measuring process were calculated by means of the intraclass correlation coefficient (ICC) analysis. Observer error tests showed excellent agreement between observers and between repeated assessments. According to the results of the ANCOVA, neither nationality nor sex was associated to the secondary dentine apposition while it is associated with individual's age. The results of the present study indicated that the concept of probability is intrinsically linked to the assessment of age in a forensic context, and the Bayesian approach could be considered a robust tool to overtake the bias generated by traditional regression models, thus helping the decision-making process in a legal framework.
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Determinación de la Edad por los Dientes/métodos , Diente Canino/diagnóstico por imagen , Pulpa Dental , Dentina Secundaria , Tejido Periapical/diagnóstico por imagen , Adulto , Anciano , Anciano de 80 o más Años , Teorema de Bayes , Femenino , Humanos , Masculino , Persona de Mediana Edad , Modelos Estadísticos , ProbabilidadRESUMEN
Previously, we have identified a gene encoding thrombospondin-related anonymous protein of Babesia gibsoni (BgTRAP), and have shown that the antisera raised against recombinant BgTRAP expressed in Escherichia coli inhibited the growth of parasites. In the present study, a recombinant vaccinia virus expressing the BgTRAP (VV/BgTRAP) was constructed. A specific band with a molecular mass of 80 kDa, which is similar to that of native BgTRAP on the merozoites of B. gibsoni, was detected in the supernatant of VV/ BgTRAP-infected RK13 cells. Mice inoculated with VV/BgTRAP produced a specific antiBgTRAP response. The antiserum against VV/BgTRAP showed reactivity against the native BgTRAP on parasites. These results indicated that the recombinant vaccinia virus expressing BgTRAP might be a vaccine candidate against canine B. gibsoni infection.
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Babesia/inmunología , Proteínas Protozoarias/inmunología , Vacunas Antiprotozoos/inmunología , Virus Vaccinia , Animales , Anticuerpos Antiprotozoarios , Femenino , Sueros Inmunes , Ratones , Ratones Endogámicos BALB C , Proteínas Recombinantes/inmunologíaRESUMEN
@#Previously, we have identified a gene encoding thrombospondin-related anonymous protein of Babesia gibsoni (BgTRAP), and have shown that the antisera raised against recombinant BgTRAP expressed in Escherichia coli inhibited the growth of parasites. In the present study, a recombinant vaccinia virus expressing the BgTRAP (VV/BgTRAP) was constructed. A specific band with a molecular mass of 80 kDa, which is similar to that of native BgTRAP on the merozoites of B. gibsoni, was detected in the supernatant of VV/ BgTRAP-infected RK13 cells. Mice inoculated with VV/BgTRAP produced a specific antiBgTRAP response. The antiserum against VV/BgTRAP showed reactivity against the native BgTRAP on parasites. These results indicated that the recombinant vaccinia virus expressing BgTRAP might be a vaccine candidate against canine B. gibsoni infection.
RESUMEN
Ichneumonidae (Hymenoptera) is one of the largest families of Insecta, but information on family diversity and distribution in Brazil is limited. The aim of the study was to assess the abundance, richness and seasonal distribution of Ichneumonidae in an urban secondary semideciduous montane forest. Insect specimens were captured in a Malaise trap placed within a restored sub-evergreen forest and sampling was performed every week during three non-consecutive 12-month periods. Of the 507 specimens collected, 338 were captured between May 1991 and May 1992, 95 between May 2000 and May 2001, and 74 between May 2007 and May 2008. Specimens were distributed among the subfamilies Pimplinae (n = 444), Anomaloninae (n = 42), Metopiinae (n = 16), Poemeniinae (n = 3) and Rhyssinae (n = 2). Species richness was highest in 1991-1992 with 33 rare and eight common species captured, followed by 2000-2001 with 31 rare and one common species captured, and 2007-2008 with 24 rare and one common species captured. The Shannon-Wiener diversity index (H') and Jackknife 1 species richness (S) values for the respective periods were 2.75/59.6, 3.15/35.8 and 2.83/35.8. In the 1991-1992 and 2000-2001 periods, parasitoid abundance was higher during the rainy season, while in 2007-2008 abundance was higher during the dry season. Colpotrochia mexicana (Cresson), Colpotrochia neblina Gauld & Sithole and Exochus izbus Gauld & Sithole were recorded for the first time in Brazil.
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Himenópteros , Animales , Brasil , Bosques , Himenópteros/anatomía & histología , Dinámica Poblacional , Estaciones del AñoRESUMEN
AIM: To determine the role of lymph node dissection (LND) in the treatment of urothelial carcinoma (UC) of the upper urinary tract (UUT). PATIENTS AND METHODS: [Study-1] A retrospective multi-institutional study evaluated 293 patients undergoing predominantly nephroureterectomy for UC of the UUT. Of 293 patients, 267 patients had pure UC and 26 demonstrated other histological components. Regarding the pathological node status, 130 patients had pN0 disease, 141 patients had pNx disease and 22 patients had pN+ disease. The sites of initial recurrence and time to first recurrence were reviewed. The sites of recurrence were classified as locoregional or distant recurrence. The relationship between node status and future recurrence was analyzed. [Study-2] Fifty-one patients treated by nephroureterectomy at Hokkaido University Hospital were included. All had LND and all LNs were negative on hematoxylin and eosin staining. We re-evaluated the presence of micrometastasis in LND specimens by anti-cytokeratin immunohistochemistory. RESULTS: [Study-1] Of 293 patients, 76 developed disease relapse. Regional lymph node recurrence was the most common site (34 patients). On multivariate analyses that adjusted for the effect of tumor stage and tumor grade, pNx (skipping LND) was an adverse factor not only for locoregional recurrence, but also for distant relapse. [Study-2] Immunohistochemistry identified micrometastases in 7 (14%) of 51 patients. Regarding survival, 5 of these 7 patients with micrometastases were alive at last follow-up. CONCLUSIONS: On relapse analysis, skipping LND was an adverse factor not only for locoregional recurrence, but also for distant relapse. Immunohistochemistry detected micrometastases in about 14% of patients previously diagnosed as pN0. These findings further support a potential therapeutic benefit of LND by eliminating micrometastases.
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Carcinoma/cirugía , Neoplasias Renales/cirugía , Escisión del Ganglio Linfático , Ganglios Linfáticos/patología , Nefrectomía/métodos , Neoplasias Ureterales/cirugía , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma/secundario , Femenino , Humanos , Inmunohistoquímica , Neoplasias Renales/patología , Metástasis Linfática , Masculino , Persona de Mediana Edad , Análisis Multivariante , Recurrencia Local de Neoplasia/patología , Recurrencia Local de Neoplasia/cirugía , Estadificación de Neoplasias , Recurrencia , Estudios Retrospectivos , Tamaño de la Muestra , Neoplasias Ureterales/patologíaRESUMEN
AIMS/HYPOTHESIS: Recently, various transgenic and knock-out mouse models have become available for studying the pathogenesis of diabetic retinopathy. At the same time, diabetes-induced retinal changes in the wild-type mice remain poorly characterised. The present study compared retinal biochemical changes in rats and mice with similar (6-week) durations of streptozotocin-induced diabetes. MATERIALS AND METHODS: The experiments were performed on Wistar rats and C57Bl6/J mice. Retinal glucose, sorbitol, fructose, lactate, pyruvate, glutamate, alpha-ketoglutarate and ammonia were measured spectrofluorometrically by enzymatic methods. Vascular endothelial growth factor (VEGF) protein was assessed by ELISA, and poly(ADP-ribosyl)ation by immunohistochemistry and western blot analysis. Free mitochondrial and cytosolic NAD(+)/NADH ratios were calculated from the glutamate and lactate dehydrogenase systems. RESULTS: Retinal glucose concentrations were similarly increased in diabetic rats and mice, vs controls. Diabetic rats manifested approximately 26- and 5-fold accumulation of retinal sorbitol and fructose, respectively, whereas elevation of both metabolites in diabetic mice was quite modest. Correspondingly, diabetic rats had (1) increased retinal malondialdehyde plus 4-hydroxyalkenal concentrations, (2) reduced superoxide dismutase (SOD), glutathione peroxidase, glutathione reductase and glutathione transferase activities, (3) slightly increased poly(ADP-ribose) immunoreactivity and poly(ADP-ribosyl)ated protein abundance, and (4) VEGF protein overexpression. Diabetic mice lacked these changes. SOD activity was 21-fold higher in murine than in rat retinas (the difference increased to 54-fold under diabetic conditions), whereas other antioxidative enzyme activities were 3- to 10-fold lower. With the exception of catalase, the key antioxidant defence enzyme activities were increased, rather than reduced, in diabetic mice. Diabetic rats had decreased free mitochondrial and cytosolic NAD(+)/NADH ratios, consistent with retinal hypoxia, whereas both ratios remained in the normal range in diabetic mice. CONCLUSIONS/INTERPRETATION: Mice with short-term streptozotocin-induced diabetes lack many biochemical changes that are clearly manifest in the retina of streptozotocin-diabetic rats. This should be considered when selecting animal models for studying early retinal pathology associated with diabetes.
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Diabetes Mellitus Experimental/fisiopatología , Retinopatía Diabética/fisiopatología , Retina/fisiopatología , Animales , Glucemia/metabolismo , Catalasa/metabolismo , Diabetes Mellitus Experimental/sangre , Ensayo de Inmunoadsorción Enzimática , Disulfuro de Glutatión/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratas , Ratas Wistar , Valores de Referencia , Especificidad de la Especie , Factor A de Crecimiento Endotelial Vascular/análisisRESUMEN
The intracellular levels of many proteins are regulated by ubiquitin-dependent proteolysis. One of the best-characterized enzymes that catalyzes the attachment of ubiquitin to proteins is a ubiquitin ligase complex, Skp1-Cullin-F box complex containing Hrt1 (SCF). We sought to artificially target a protein to the SCF complex for ubiquitination and degradation. To this end, we tested methionine aminopeptidase-2 (MetAP-2), which covalently binds the angiogenesis inhibitor ovalicin. A chimeric compound, protein-targeting chimeric molecule 1 (Protac-1), was synthesized to recruit MetAP-2 to SCF. One domain of Protac-1 contains the I kappa B alpha phosphopeptide that is recognized by the F-box protein beta-TRCP, whereas the other domain is composed of ovalicin. We show that MetAP-2 can be tethered to SCF(beta-TRCP), ubiquitinated, and degraded in a Protac-1-dependent manner. In the future, this approach may be useful for conditional inactivation of proteins, and for targeting disease-causing proteins for destruction.
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Aminopeptidasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas I-kappa B , Metaloendopeptidasas/metabolismo , Péptido Sintasas/metabolismo , Ubiquitinas/metabolismo , Animales , Extractos Celulares , Línea Celular Transformada , Humanos , Inhibidor NF-kappaB alfa , Óvulo/metabolismo , Unión Proteica , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Ligasas SKP Cullina F-box , Xenopus laevisRESUMEN
The ability of (-)-epicatechin (EC), (-)-epigallocatechin (EGC) and (-)-epigallocatechin gallate (EGCG) to inhibit the growth of HCT 116 colorectal and Hep G2 hepatocellular carcinoma cells was examined by MTT and clonogenic assays (CA). The respective catechins inhibited the growth of HCT 116 more strongly than Hep G2. In MTT assay, IC(50) values of EGC and EGCG against HCT 116 grew smaller on prolongation of the exposure times of the cells to the catechins. In CA, however, these two catechins had IC(50) values ranging between 7.6+/-0.4 and 11.2+/-0.5 microM against the same cells regardless of the exposure times. EC showed much weaker growth inhibitions relative to the two aforementioned catechins.
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Catequina/farmacología , Neoplasias del Colon/tratamiento farmacológico , Neoplasias Hepáticas/tratamiento farmacológico , Bebidas , Catequina/uso terapéutico , División Celular/efectos de los fármacos , Neoplasias del Colon/patología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Humanos , Neoplasias Hepáticas/patología , Células Tumorales CultivadasRESUMEN
Based on the prodrug concept as well as the combination of two different classes of anti-HIV agents, we designed and synthesized a series of anti-HIV double-drugs consisting of HIV protease inhibitors conjugated with a nucleoside reverse transcriptase inhibitor in an effort to enhance the antiviral activity. For the conjugation, a series of linkers that conjoins the two different classes of inhibitors has been investigated. Double-drugs using a succinyl amino acid linker were shown to release the parent drugs via spontaneous imide formation at a faster rate compared to compounds using a glutaryl amino acid linker, as expected from the energetically favorable cyclization to the five-membered ring. Among the double-drugs, KNI-1039 (3b) with a glutarylglycine linker exhibited extremely potent anti-HIV activity compared with that of the individual components. Double-drug 3b was relatively stable in culture medium, whereas it regenerated active species in cell homogenate. These results suggested that the synergistic enhancement of anti-HIV activities of 3b may be due to their ability to penetrate into the target cell and subsequent regeneration of two different classes of anti-HIV agents in the cytoplasm.
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Fármacos Anti-VIH/química , Fármacos Anti-VIH/farmacología , Inhibidores de la Proteasa del VIH/química , Inhibidores de la Proteasa del VIH/farmacología , Inhibidores de la Transcriptasa Inversa/química , Inhibidores de la Transcriptasa Inversa/farmacología , Animales , Fármacos Anti-VIH/síntesis química , Diseño de Fármacos , Evaluación Preclínica de Medicamentos/métodos , Inhibidores de la Proteasa del VIH/síntesis química , Humanos , Profármacos/química , Profármacos/farmacología , Inhibidores de la Transcriptasa Inversa/síntesis química , Relación Estructura-Actividad , Tiazoles/química , Tiazoles/farmacología , Células Tumorales Cultivadas/virología , Zidovudina/química , Zidovudina/farmacologíaRESUMEN
1. The pylorus plays an important role in the regulation of gastric emptying. In addition to the autonomic neuropathy associated with long-standing diabetes, acute hyperglycaemia per se has effects on gastric emptying. In this study, the role of the central nervous system in modulating the effects of hyperglycaemia on gastric distension-induced pyloric relaxation was investigated. 2. Gastric distension-induced pyloric relaxation was significantly reduced by subdiaphragmatic vagotomy, hexamethonium (20 mg kg(-1)) and N (G)-nitro-L-arginine methyl ester (L-NAME; 10 mg kg(-1)), a nitric oxide synthase (NOS) biosynthesis inhibitor, in anaesthetized rats. In contrast, neither splanchnectomy nor guanethidine (5 mg kg(-1)) had an effect. 3. An intravenous (I.V.) infusion of D-glucose (20 %) for 30 min, which increased blood glucose concentrations from 5.4 to 12.8 mM, significantly inhibited gastric distension-induced pyloric relaxation. 4. An intracerebroventricular (I.C.V.) injection of D-glucose (3 micromol) also significantly inhibited gastric distension-induced pyloric relaxation without affecting peripheral blood glucose concentrations. 5. I.V. infusion of D-glucose significantly elevated hypothalamic neuropeptide Y (NPY) concentrations. 6. Intracerebroventricular (I.C.V.) administration of NPY (0.03--3 nmol) and a Y1 receptor agonist, [leu(31), pro(34)] NPY (0.03--3 nmol), significantly inhibited gastric distension-induced pyloric relaxation in a dose-dependent manner. 7. I.C.V. administration of a Y1 receptor antagonist, BIBP 3226 (30 nmol), and of a NPY antibody (titre 1:24 000, 3 microl) abolished the inhibitory effects of hyperglycaemia on gastric distension-induced pyloric relaxation. 8. Taken together, these findings suggest that gastric distension-induced pyloric relaxation is mediated via a vago-vagal reflex and NO release. Acute hyperglycaemia stimulates hypothalamic NPY release, which, acting through the Y1 receptor, inhibits gastric distension-induced pyloric relaxation in rats exposed to acute elevations in blood glucose concentrations.
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Arginina/análogos & derivados , Sistema Nervioso Central/fisiopatología , Hiperglucemia/fisiopatología , Píloro/fisiopatología , Estómago/fisiopatología , Enfermedad Aguda , Animales , Arginina/farmacología , Cateterismo , Electrofisiología , Hipotálamo/metabolismo , Inyecciones Intraventriculares , Masculino , Relajación Muscular/efectos de los fármacos , Relajación Muscular/fisiología , Músculo Liso/fisiología , Neuropéptido Y/metabolismo , Neuropéptido Y/farmacología , Ratas , Ratas Sprague-Dawley , Receptores de Neuropéptido Y/antagonistas & inhibidores , Valores de ReferenciaRESUMEN
Insulin-like growth factor-1 (IGF-1) plays important roles in the developing and mature retina and in pathological states characterized by retinal neovascularization, such as diabetic retinopathy. The effects of IGF-1 on glucose transport and proliferation and the signal transduction pathways underlying these effects were studied in a primary bovine retinal endothelial cell (BREC) culture model. IGF-1 stimulated uptake of the glucose analog 2-deoxyglucose in a dose-dependent manner, with a maximal uptake at 25 ng/mL (3.3 nM) after 24 h. Increased transport occurred in the absence of an increase in total cellular GLUT1 transcript or protein. IGF-1 stimulated activity of both protein kinase C (PKC) and phosphatidylinositol-3 kinase (PI3 kinase), and both pathways were required for IGF-1-mediated BREC glucose transport and thymidine incorporation. Use of a selective inhibitor of the beta isoform of PKC, LY379196, revealed that IGF-1 stimulation of glucose transport was mediated by PKC-beta; however, inhibition of PKC-beta had no effect on BREC proliferation. Taken together, these data suggest that the actions of IGF-1 in retinal endothelial cells couple proliferation with delivery of glucose, an essential metabolic substrate. The present studies extend our general understanding of the effects of IGF-1 on vital cellular activities within the retina in normal physiology and in pathological states such as diabetic retinopathy.
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Endotelio Vascular/efectos de los fármacos , Glucosa/metabolismo , Factor I del Crecimiento Similar a la Insulina/farmacología , Vasos Retinianos/fisiología , 3-O-Metilglucosa/metabolismo , Animales , Transporte Biológico , Bovinos , División Celular/efectos de los fármacos , Células Cultivadas , Desoxiglucosa/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Inhibidores Enzimáticos/farmacología , Transportador de Glucosa de Tipo 1 , Indoles/farmacología , Maleimidas/farmacología , Proteínas de Transporte de Monosacáridos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteína Quinasa C/metabolismo , Vasos Retinianos/efectos de los fármacos , Timidina/metabolismoRESUMEN
The Fas-Fas ligand (L) system is one of the major signalling pathways to induce apoptosis in various cells and tissues. The aim of this study was to investigate the expression of the Fas-Fas L system in rat and human oocytes and preimplantation embryos. We determined the expression of Fas and Fas L mRNA of rat oocytes and embryos up to the blastocyst stage, and of human embryos at the 2- or 4-cell stage, using reverse transcription polymerase chain reaction (PCR) and nested PCR techniques. Moreover, we investigated the expression of Fas mRNA in human fragmented embryos. In rat embryos, Fas mRNA was expressed at the 2-cell stage only, whereas Fas L mRNA was expressed in oocytes, and at the pronuclear (1-cell) and 2-cell stages. In human embryos, Fas mRNA was expressed at the 4-cell stage only, whereas Fas L mRNA was expressed at both 2- and 4-cell stages. Human fragmented embryos expressed both Fas and Fas L mRNA. Because simultaneous expression of Fas and Fas L mRNA occurred in 2-cell rat embryos and in 4-cell human embryos, the Fas-Fas L system might be involved in the apoptotic pathway in the early embryos of these species.
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Desarrollo Embrionario , Expresión Génica , Glicoproteínas de Membrana/genética , ARN Mensajero , Receptor fas/genética , Animales , Antígenos CD/genética , Desarrollo Embrionario y Fetal , Proteína Ligando Fas , Femenino , Humanos , Embarazo , Ratas , Ratas Wistar , Receptores del Factor de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis TumoralRESUMEN
Oncoprotein 18 (Op18) is a microtubule-destabilizing protein that is negatively regulated by phosphorylation. To evaluate the role of the three Op18 phosphorylation sites in Xenopus (Ser 16, 25, and 39), we added wild-type Op18, a nonphosphorylatable triple Ser to Ala mutant (Op18-AAA), and to mimic phosphorylation, a triple Ser to Glu mutant (Op18-EEE) to egg extracts and monitored spindle assembly. Op18-AAA dramatically decreased microtubule length and density, while Op18-EEE did not significantly affect spindle microtubules. Affinity chromatography with these proteins revealed that the microtubule-destabilizing activity correlated with the ability of Op18 to bind tubulin. Since hyperphosphorylation of Op18 is observed upon addition of mitotic chromatin to extracts, we reasoned that chromatin-associated proteins might play a role in Op18 regulation. We have performed a preliminary characterization of the chromatin proteins recruited to DNA beads, and identified the Xenopus polo-like kinase Plx1 as a chromatin-associated kinase that regulates Op18 phosphorylation. Depletion of Plx1 inhibits chromatin-induced Op18 hyperphosphorylation and spindle assembly in extracts. Therefore, Plx1 may promote microtubule stabilization and spindle assembly by inhibiting Op18.
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Proteínas de Microtúbulos , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Huso Acromático/fisiología , Proteínas de Xenopus , Animales , Sitios de Unión , Proteínas de Ciclo Celular , Extractos Celulares , Cromatina , Óvulo/fisiología , Fosfoproteínas/genética , Fosforilación , Estatmina , XenopusRESUMEN
Recent reports in the United States have found that fewer peptic ulcers are due to Helicobacter pylori than previously believed. The aim of this study is to determine if the declining prevalence of H. pylori infection in the general population can account for the apparent increase in the frequency of non-H. pylori ulcers. A total of 396 patients with peptic ulcer or ulcer scar were enrolled in this study. The pre-1950 population consisted of 149 patients with gastric ulcers and with 44 duodenal ulcers. The post-1950 population consisted of 96 patients with gastric ulcers and 107 with duodenal ulcers. The frequency of H. pylori-negative gastric ulcers was 5.4% in patients born before 1950 and 4.2% in patients born after 1950, and the frequency of H. pylori-negative duodenal ulcers was 0% and 1.9%, respectively. There are no statistical differences between the two populations in gastric and duodenal ulcers. H. pylori seropositivity was 74.9% in asymptomatic volunteers born before 1950 and 20.7% in those born after 1950 (P < 0.01) in the general population. The attributable risk of H. pylori infection in peptic ulcer diseases was not affected by the prevalence of H. pylori infection in the general population in Japan. This suggests that the apparent increase in frequency of non-H. pylori ulcers in the United States is not simply due to the declining prevalence of infection. Other explanations for non-H. pylori ulcers should be sought.
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Úlcera Duodenal/epidemiología , Úlcera Duodenal/microbiología , Infecciones por Helicobacter/complicaciones , Infecciones por Helicobacter/epidemiología , Helicobacter pylori , Úlcera Gástrica/epidemiología , Úlcera Gástrica/microbiología , Adulto , Distribución por Edad , Antiinflamatorios no Esteroideos/efectos adversos , Estudios de Casos y Controles , Úlcera Duodenal/etiología , Úlcera Duodenal/patología , Infecciones por Helicobacter/prevención & control , Humanos , Japón/epidemiología , Persona de Mediana Edad , Vigilancia de la Población , Prevalencia , Factores de Riesgo , Úlcera Gástrica/etiología , Úlcera Gástrica/patologíaRESUMEN
The ventromedial hypothalamus (VMH) has been proposed to be a glucose sensor within the brain and appears to play a critical role in initiating the counterregulatory response to hypoglycemia. Transport of glucose across the brain capillaries and into neurons in this region is mediated by different isoforms of the sodium-independent glucose transporter gene family. The objective of the present study was to identify the specific glucose transporter isoforms present, as well as their cellular localization, within the VMH. Immunohistochemistry was performed for GLUT1, GLUT2 and GLUT4 in frozen sections of hypothalami from normal rats. GLUT1 was present on the endothelial cells of the blood-brain barrier (BBB) of the VMH. GLUT2 immunoreactivity was seen in the ependymal cells of the third ventricle and in scattered cells in the arcuate and periventricular nuclei. There was no GLUT2 expression in the VMH. The insulin-sensitive GLUT4 isoform was localized to vascular structures within the VMH. Double-labeled immunohistochemistry demonstrated co-localization of GLUT4 with GLUT1 and with the tight junction protein ZO-1 in the VMH and suggested that VMH GLUT4 expression was restricted to the BBB. The role of GLUT4 in the brain and within the VMH is unknown, but given its location on the BBB, it may participate in brain sensing of blood glucose concentrations.
Asunto(s)
Barrera Hematoencefálica , Proteínas de Transporte de Monosacáridos/análisis , Proteínas Musculares , Núcleo Hipotalámico Ventromedial/química , Animales , Capilares/química , Ventrículos Cerebrales/química , Técnica del Anticuerpo Fluorescente Indirecta , Glucosa/metabolismo , Transportador de Glucosa de Tipo 1 , Transportador de Glucosa de Tipo 4 , Insulina/metabolismo , Masculino , Músculo Esquelético/química , Especificidad de Órganos , Ratas , Ratas Sprague-Dawley , Sarcolema/química , Núcleo Hipotalámico Ventromedial/irrigación sanguíneaRESUMEN
Wee1 inactivates the Cdc2-cyclin B complex during interphase by phosphorylating Cdc2 on Tyr-15. The activity of Wee1 is highly regulated during the cell cycle. In frog egg extracts, it has been established previously that Xenopus Wee1 (Xwee1) is present in a hypophosphorylated, active form during interphase and undergoes down-regulation by extensive phosphorylation at M-phase. We report that Xwee1 is also regulated by association with 14-3-3 proteins. Binding of 14-3-3 to Xwee1 occurs during interphase, but not M-phase, and requires phosphorylation of Xwee1 on Ser-549. A mutant of Xwee1 (S549A) that cannot bind 14-3-3 is substantially less active than wild-type Xwee1 in its ability to phosphorylate Cdc2. This mutation also affects the intranuclear distribution of Xwee1. In cell-free kinase assays, Xchk1 phosphorylates Xwee1 on Ser-549. The results of experiments in which Xwee1, Xchk1, or both were immunodepleted from Xenopus egg extracts suggested that these two enzymes are involved in a common pathway in the DNA replication checkpoint response. Replacement of endogenous Xwee1 with recombinant Xwee1-S549A in egg extracts attenuated the cell cycle delay induced by addition of excess recombinant Xchk1. Taken together, these results suggest that Xchk1 and 14-3-3 proteins act together as positive regulators of Xwee1.
Asunto(s)
Proteínas de Ciclo Celular , Proteínas Nucleares , Proteínas Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Tirosina 3-Monooxigenasa/metabolismo , Proteínas 14-3-3 , Animales , Secuencia de Bases , Proteína Quinasa CDC2/metabolismo , Ciclo Celular , Línea Celular , Núcleo Celular/metabolismo , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Cartilla de ADN/genética , Replicación del ADN , Escherichia coli/genética , Femenino , Técnicas In Vitro , Mutación , Oocitos/metabolismo , Unión Proteica , Proteínas Quinasas/genética , Proteínas Tirosina Quinasas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Spodoptera , Tirosina 3-Monooxigenasa/genética , Xenopus , Proteínas de XenopusRESUMEN
We have identified Claspin, a novel protein that binds to Xenopus Chk1 (Xchk1). Binding of Claspin to Xchk1 is highly elevated in the presence of DNA templates that trigger a checkpoint arrest of the cell cycle in Xenopus egg extracts. Xchk1 becomes phosphorylated during a checkpoint response, and we demonstrate directly that this phosphorylation results in the activation of Xchk1. Immunodepletion of Claspin from egg extracts abolishes both the phosphorylation and activation of Xchk1. Furthermore, Claspin-depleted extracts are unable to arrest the cell cycle in response to DNA replication blocks. Taken together, these findings indicate that Claspin is an essential upstream regulator of Xchk1.