RESUMO
AIM: To develop a screening tool for the detection of interstitial lung disease (ILD) patterns using a deep-learning method. MATERIALS AND METHODS: A fully convolutional network was used for semantic segmentation of several ILD patterns. Improved segmentation of ILD patterns was achieved using multi-scale feature extraction. Dilated convolution was used to maintain the resolution of feature maps and to enlarge the receptive field. The proposed method was evaluated on a publicly available ILD database (MedGIFT) and a private clinical research database. Several metrics, such as success rate, sensitivity, and false positives per section were used for quantitative evaluation of the proposed method. RESULTS: Sections with fibrosis and emphysema were detected with a similar success rate and sensitivity for both databases but the performance of detection was lower for consolidation compared to fibrosis and emphysema. CONCLUSION: Automatic identification of ILD patterns in a high-resolution computed tomography (CT) image was implemented using a deep-learning framework. Creation of a pre-trained model with natural images and subsequent transfer learning using a particular database gives acceptable results.
Assuntos
Aprendizado Profundo , Doenças Pulmonares Intersticiais/diagnóstico por imagem , Tomografia Computadorizada por Raios X/métodos , Conjuntos de Dados como Assunto , Humanos , Interpretação de Imagem Radiográfica Assistida por Computador , Sensibilidade e EspecificidadeRESUMO
A small subset of patients with antiphospholipid syndrome (APS) may develop widespread thrombotic disease with organ damage, referred to as catastrophic APS (CAPS) that is associated with a high mortality. Medical therapy typically involves a combination of anticoagulation, systemic glucocorticoids, plasmapheresis, and intravenous immune globulin (IVIG). There is currently no consensus for the management of refractory cases of CAPS. However, monoclonal antibodies such as rituximab and eculizumab have shown some benefits. Herein, we present a 29-year-old female with previous pulmonary embolism who presented with necrotic left toes and was eventually diagnosed with refractory CAPS, successfully treated with Plasmapheresis and Rituximab. With this case report, we hope to encourage the usage of Rituximab in the management of CAPS.
RESUMO
Diagnosing radiologically detectable lung lesions on the basis of cyto/histopathological staining often suffers from ambiguity, leading to faulty detection of lung diseases, especially cancer. Present study attempted to perform a multimodal characterzation of clinical samples from patients with radiologically detected lung lesions, in order to classify diseases with higher precision. The study included analysing nuclear morphometric and intensity based differences between benign and malignant lung lesions in a quantitative way. Simultaneous analysis of serum samples taken from similar sets of patients was performed by Fourier transform infra red (FTIR) spectroscopy which provided differential global biochemical insights of normal and diseased states. Both nuclear and serum biochemical features were helpful in accomplishing high classification accuracy in distinguishing benign and different malignant cases.
Assuntos
Pneumopatias , Diagnóstico Diferencial , Humanos , Neoplasias Pulmonares , Imagem MultimodalRESUMO
Open defecation is practised by over 600 million people in India and there is a strong political drive to eliminate this through the provision of on-site sanitation in rural areas. However, there are concerns that the subsequent leaching of excreta from subsurface storage could be adversely impacting underlying groundwater resources upon which rural populations are almost completely dependent for domestic water supply. We investigated this link in four villages undergoing sanitary interventions in Bihar State, India. A total of 150 supplies were sampled for thermotolerant (faecal) coliforms (TTC) and tryptophan-like fluorescence (TLF): an emerging real-time indicator of faecal contamination. Sanitary risk inspections were also performed at all sites, including whether a supply was located within 10 m of a toilet, the recommended minimum separation. Overall, 18% of water supplies contained TTCs, 91% of which were located within 10 m of a toilet, 58% had TLF above detection limit, and sanitary risk scores were high. Statistical analysis demonstrated TLF was an effective indicator of TTC presence-absence, with a possibility of TTCs only where TLF exceeded 0.4 µg/L dissolved tryptophan. Analysis also indicated proximity to a toilet was the only significant sanitary risk factor predicting TTC presence-absence and the most significant predictor of TLF. Faecal contamination was considered a result of individual water supply vulnerability rather than indicative of widespread leaching into the aquifer. Therefore, increasing faecal contamination of groundwater-derived potable supplies is inevitable across the country as uptake of on-site sanitation intensifies. Communities need to be aware of this link and implement suitable decentralised low-cost treatment of water prior to consumption and improve the construction and protection of new supplies.
Assuntos
Água Potável/microbiologia , Monitoramento Ambiental , Fluorescência , Saneamento/métodos , Triptofano/química , Abastecimento de Água , Fezes/microbiologia , Humanos , Índia , População RuralRESUMO
Plasmacytic infiltrates in renal allograft biopsies are uncommon and morphologically distinctive lesions that may represent variants of acute rejection. This study sought significant clinical and pathologic determinants that might have influenced development of these lesions and assessed their prognostic significance. Renal allograft biopsies (n = 19), from 19 patients, with tubulointerstitial inflammatory infiltrates containing abundant plasma cells, composing 32 +/- 8% of the infiltrating mononuclear cells, were classified using Banff '97 criteria. Clonality of the infiltrates was determined by immunoperoxidase staining for kappa and lambda light chains and polymerase chain reaction for immunoglobulin heavy-chain gene rearrangements, using V(H) gene framework 3 and JH consensus primers. In situ hybridization for Epstein-Barr virus encoded RNA (EBER) was performed in 17 cases. The clinical features, histology, and outcome of these cases were compared with kidney allograft biopsies (n = 17) matched for time posttransplantation and type of rejection by Banff '97 criteria, with few plasma cells (7 +/- 5%). Sixteen of 19 biopsies (84%) with plasmacytic infiltrates had EBER-negative (in 14 cases tested) polyclonal plasma cell infiltrates that were classifiable as acute rejection (types 1A [4], 1B [10], and 2A [2]). These biopsies were obtained between 10 and 112 months posttransplantation. Graft loss from acute and/or chronic rejection was 50% at 1 year and 63% at 3 years, and the median time to graft failure was 4.5 months after biopsy. There was no significant difference in overall survival or time to graft failure compared with the controls. Three of 19 biopsies (16%) had EBER-negative polyclonal plasmacytic hyperplasia, mixed monoclonal and polyclonal polymorphous B cell hyperplasia, and monoclonal plasmacytoma-like posttransplantation lymphoproliferative disease (PTLD) and were obtained at 17 months, 12 weeks, and 7 years after transplantation, respectively. Graft nephrectomies were performed at 1, 19, and 5 months after biopsy, respectively. Plasmacytic infiltrates in renal allografts comprise a spectrum of lesions from acute rejection to PTLD, with a generally poor prognosis for long-term graft survival.
Assuntos
Rejeição de Enxerto/patologia , Transplante de Rim/patologia , Transtornos Linfoproliferativos/patologia , Plasmócitos/patologia , Adulto , Biópsia , Contagem de Células , DNA/análise , Infecções por Vírus Epstein-Barr/complicações , Infecções por Vírus Epstein-Barr/patologia , Feminino , Rearranjo Gênico , Genes de Imunoglobulinas , Rejeição de Enxerto/terapia , Rejeição de Enxerto/virologia , Herpesvirus Humano 4/isolamento & purificação , Herpesvirus Humano 4/patogenicidade , Humanos , Técnicas Imunoenzimáticas , Imunossupressores/uso terapêutico , Hibridização In Situ , Transtornos Linfoproliferativos/virologia , Masculino , Pessoa de Meia-Idade , Nefrite Intersticial/patologia , Nefrite Intersticial/virologia , Complicações Pós-Operatórias , RNA Viral/análise , Transplante HomólogoRESUMO
Nowadays medical teachers use different audiovisual (AV) aids of teaching in their classes to make the subject more interesting and understandable. To assess the impact of three common lecture delivery methods, viz Blackboard (BB), Transparency and Over Head Projector (OHP) and Powerpoint Presentation (PP), a questionnaire based study was carried out among first year MBBS students of R.G.Kar Medical College, Kolkata. One hundred fouty students of academic session 2010-2011 were exposed to different aids of teaching, viz. Black Board (BB), Over Head Projector (OHP), power point presentation (PP) for ten months. They were taught Anatomy by different teachers who used all the three AV aids in their lectures. Then they were asked to respond to a questionnaire regarding these three AV aids of teaching. The students preferred Black Board teaching over OHP and result was statistically significant (p value < 0.0001). BB teaching was also preferred over PP presentations (p < 0.02). But in comparison to OHP, students preferred PP though the difference is not statistically significant (p < 0.10). Most of the students still prefer Black Board teaching to other modern AV aids like OHP and PP. For better understanding of a subject by students and improvement of their performance, a teacher should match the lectures with preferred AV aids and use the AV aids prudently.
Assuntos
Atitude do Pessoal de Saúde , Recursos Audiovisuais , Educação Médica/métodos , Estudantes de Medicina/psicologia , Feminino , Humanos , Índia , Masculino , Inquéritos e Questionários , Adulto JovemRESUMO
Zebrafish is an attractive model organism for studying apoptosis development because of its genetic accessibility. Here we describe the induction of clonally derived apoptosis in transgenic zebrafish expressing mouse caspase-3 (CASP3) under control of the zebrafish beta-actin promoter (betap). Visualization of apoptotic cells, expressing a chimeric transgene encoding CASP3 fused to green fluorescent protein (GFP) gene, revealed that apoptosis arose in the thymus, spread locally into gill arches and retro-orbital soft tissue, and then disseminated into abdominal organs like testis, kidney. This transgenic model provides a platform for over-expression of caspase-3 induced extensive apoptosis in embryos and adult.
Assuntos
Apoptose , Caspases/metabolismo , Peixe-Zebra/anatomia & histologia , Actinas/genética , Animais , Animais Geneticamente Modificados , Caspase 3 , Caspases/genética , Embrião não Mamífero/anormalidades , Embrião não Mamífero/anatomia & histologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Especificidade de Órgãos , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Peixe-Zebra/anormalidades , Peixe-Zebra/genéticaRESUMO
Analyses of sediment and water indicate the presence of heavy metal pollutants like lead, zinc, copper, mercury and cadmium of the river Damodar of India. These metals are responsible for causing morphological deformities of antennae and other parts of chironomid larvae. Percentage of deformity correlated positively with the concentrations of Pb in water and sediment (r > 0.6) at the confluence point. A new severity index, SISS((antenna)) has been proposed here to assess deformity at the family or subfamily level.
Assuntos
Chironomidae/efeitos dos fármacos , Monitoramento Ambiental/métodos , Metais Pesados/toxicidade , Órgãos dos Sentidos/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Animais , Chironomidae/crescimento & desenvolvimento , Relação Dose-Resposta a Droga , Índia , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Metais Pesados/análise , Rios , Órgãos dos Sentidos/anormalidades , Poluentes Químicos da Água/análiseRESUMO
The stoichiometry of Mn2+ binding to concanavalin A was found to be influenced by temperature, pH, and the presence or absence of saccharide. Demetalized concanavalin A binds one Mn2+ (S1 site) at 5 degrees C, pH 6.5, and two Mn2+ at 25 degrees C (S1 and S2 sites). The association constants for Mn2+ are 6.2 x 10(5) and 3.7 x 10(4) M-1 for the S1 and S2 sites, respectively, at 25 degrees C. Concanavalin A with one Mn2+ bound per monomer remains in an open conformation and exhibits a relatively high water proton relaxation rate. Concanavalin A with two Mn2+ ions remains in a closed conformation characterized by a lower relaxation rate. The rate of binding of the second Mn2+ to concanavalin A as determined by ESR and the rate of conversion of open form to closed form (folding over) as determined by proton relaxation rate measurements gave an identical rate constant of 80.0 +/- 5.8 M-1 h-1 at 17 degrees C. Ca2+, Sr2+, and high levels of methyl alpha-D-mannopyranoside also induce folding of concanavalin A. Ca2+ is not catalytic but stoichiometric in causing the folding. Mn2+ in the S1 site can be displaced by Ni2+, Co2+, and Zn2+, and Mn2+ in the S2 site can be displaced by Ca2+ and Sr2+. Concanavalin A with Ni2+, Co2+, Zn2+, or Mn2+ in the S1 site and Ca2+ or Sr2+ in the S2 site has a higher affinity for methylumbelliferyl alpha-D-mannopyranoside than Ni-Mn-, Co-Mn-, Zn-Mn-, and Cd-Cd-concanavalin A.
Assuntos
Concanavalina A , Sítios de Ligação , Carboidratos , Cátions Bivalentes , Concentração de Íons de Hidrogênio , Cinética , Manganês , Metais , Conformação MolecularRESUMO
The mechanism of kinesin ATPase has been investigated by transient state kinetic analysis. The results satisfy the scheme [formula: see text] where T, D, and P(i) refer to nucleotide tri- and diphosphate and inorganic phosphate, respectively. The nucleotide-binding steps were measured by the fluorescence enhancement of mant (2'-(3')-O-(N-methylanthraniloyl)-ATP and mant-ADP. The initial rapid equilibrium binding steps (1) and (6) are followed by isomerizations (k2 = 170 +/- 30 s-1 at 20 degrees C, k-5 greater than 100 s-1). The increase in fluorescence is 20-25% larger for K.T** than K.D*. The rate constant of the hydrolysis step k3 is 6-7 s-1. The fluorescence decreases after formation of K.T** at a rate of 7-10 s-1. This change could occur in step 3 or in step 4 if k4 much greater than k3. The value of k4 is larger than 0.1 s-1. The steady state rate is 0.003 s-1 which agrees with the rate of ADP dissociation (k5). Step 5 is rate limiting in the scheme in agreement with the conclusion of Hackney (Hackney, D. D. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 6314-6318) that ADP dissociation is the rate-limiting step.
Assuntos
Adenosina Trifosfatases/metabolismo , Cinesinas/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Encéfalo/metabolismo , Bovinos , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Fluorescência , Hidrólise , Cinética , Suínos , Tubulina (Proteína)/metabolismoRESUMO
Exposure of mammalian oocytes to the protein phosphatase (PP)-1 (PP1) and PP2A inhibitor okadaic acid (OA) stimulates oocyte meiosis. However, treated oocytes do not develop beyond metaphase I (MI), and they display morphological aberrations. Experiments were conducted to define inhibitor treatment conditions for macaque oocytes that would result in germinal vesicle breakdown (GVB) stimulation and completion of meiosis without significant cytoplasmic abnormalities. As described above for OA, continual exposure of macaque oocytes to 50 nM calyculin-a (CL-A) significantly enhanced GVB at 24 h compared to that in controls, and the majority of the treated oocytes displayed cytoplasmic abnormalities. However, transient exposure (10 min) of rhesus macaque oocytes to either 50 nM CL-A or 1.0 microM OA enhanced GVB rates compared to that in controls and did not increase the incidence of cytoplasmic abnormalities. Meiotic maturation from germinal vesicle-intact oocytes to MII was enhanced following transient treatment with CL-A or OA compared to that in controls; however, development from MI to MII occurred at a similar frequency. In vitro-matured oocytes transiently exposed to OA and CL-A were capable of fertilization. In addition, ovarian immunohistochemical analysis revealed that both PP1 and PP2A were present in macaque oocytes. PP1 was localized throughout the cytoplasm with a predominance in the nucleus, whereas PP2A was evenly distributed throughout the cytoplasm with a reduction in the nuclear area. These results taken together-differential developmental responses to inhibitor treatment and intracellular enzyme localizations-may be indicative of multiple regulatory roles of PP1 and/or PP2A during meiosis.
Assuntos
Inibidores Enzimáticos/farmacologia , Fertilização in vitro , Ácido Okadáico/farmacologia , Oócitos/fisiologia , Oxazóis/farmacologia , Fosfoproteínas Fosfatases/antagonistas & inibidores , Animais , Citoplasma/efeitos dos fármacos , Citoplasma/ultraestrutura , Feminino , Imuno-Histoquímica , Macaca mulatta , Masculino , Toxinas Marinhas , Meiose/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Oócitos/ultraestrutura , Folículo Ovariano/fisiologia , Fosfoproteínas Fosfatases/análiseRESUMO
Fibroblast growth factor-2 (FGF-2) stimulates proliferation and inhibits differentiated function of osteoblasts by suppressing synthesis of type I collagen and other proteins. However, little is known regarding the molecular mechanisms regulating the suppressive effects of FGF-2 on type I collagen synthesis in osteoblasts. The zinc finger transcription factor Egr-1 and the basic helix-loop-helix (bHLH) family of proteins have been implicated in the regulation of genes crucial to mesodermal cell growth and differentiation. The aim of this study was to determine whether Egr-1 and TWIST might be potential transcriptional regulators of the inhibitory effects of FGF-2 on alpha2(I) collagen expression in MC3T3-E1 osteoblasts which undergo a developmental sequence in vitro. Upon treatment of undifferentiated MC3T3-E1 cells with 1 nM FGF-2, Egr-1 mRNA increased with the effect maximal after 30-60 min. TWIST mRNA also increased with the effect maximal at 2 h. We analyzed the transcriptional control of alpha2(I) collagen gene expression by FGF-2 by transient transfection of an alpha2(I) collagen-luciferase construct (pH5) into undifferentiated MC3T3-E1 cells. The activity of the pH5 luciferase promoter decreased in a dose-dependent manner following treatment with.01 and 1 nM FGF-2. We identified putative Egr-1 and TWIST recognition sequences in the proximal region of the promoter for the murine alpha2(I) collagen gene and a putative Egr-1 site in the 5' region of the murine TWIST promoter. In gel mobility shift assays, potential Egr-1 response elements in the 5' region of the murine TWIST and alpha2(I) collagen genes demonstrated specific Egr-1 binding activity with bFGF-treated nuclear extracts obtained from MC3T3-E1 cells. These results indicate that Egr-1 and TWIST are expressed in undifferentiated MC3T3-E1 osteoblast-like cells following treatment with FGF-2 and they may be potential transcriptional regulators of FGF-2s negative effects on alpha2(I) collagen gene expression. J. Cell. Biochem. 80:550-559, 2001. Published 2001 Wiley-Liss, Inc.
Assuntos
Colágeno/biossíntese , Fator 2 de Crescimento de Fibroblastos/biossíntese , Regulação da Expressão Gênica , Proteínas Imediatamente Precoces , Transcrição Gênica , Animais , Sítios de Ligação , Ligação Competitiva , Northern Blotting , Diferenciação Celular , Divisão Celular , Núcleo Celular/metabolismo , Colágeno Tipo I , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta a Droga , Regulação para Baixo , Proteína 1 de Resposta de Crescimento Precoce , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Genes Reporter , Humanos , Luciferases/metabolismo , Camundongos , Proteínas Nucleares/metabolismo , Osteoblastos/metabolismo , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Fatores de Transcrição/metabolismo , Transfecção , Proteína 1 Relacionada a TwistRESUMO
Human immunoglobulin heavy chains are encoded by a highly complex locus, IGH, which encompasses many transcriptional units, including nine alternative constant region genes. Much of the constant region gene cluster was duplicated during hominoid evolution. In rodents, IGH is known to be regulated by multiple elements, including several enhancers 3' of the alpha chain-encoding A constant region gene, CA, the last transcribed region. Sequences downstream of the two human CA genes, possibly containing homologous enhancer elements, have not yet been reported. By long-range mapping of genomic DNA, a cluster of unmethylated rare restriction sites, representing a potential CpG island, was previously reported downstream of each CA gene, close to the 3' end of the duplicated region. Such potential CpG islands are candidates for additional IGH regulatory elements. We isolated bacteriophage clones containing these clusters of methylation-sensitive restriction sites, which lie within short CpG-rich stretches. Some of these sites showed tissue-specific methylation. 3' of the unmethylated CpG-rich sequences, clones derived from the 5' (telomeric) copy of the duplicated region, contained, in order, endogenous retroviral sequences, a processed ELK1 pseudogene, and the pseudogene IGHGP. Comparison with the presumed 3' (centromeric) copy of the duplicated region showed that similarity was lost exactly at the end of the retroviral long terminal repeat sequences. These results imply that an endogenous retroviral integration was present immediately 3' of IGH in the common hominoid ancestor and suggest models for the duplication of the region.
Assuntos
Mapeamento Cromossômico , Ilhas de CpG/genética , Cadeias Pesadas de Imunoglobulinas/genética , Análise de Sequência de DNA , Sequência de Bases , Genoma Humano , Humanos , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
Okadaic acid (OA) enhances the resumption of meiosis in mouse oocytes, indicating that serine/threonine protein phosphatase-1 (PP1) and/or PP2A is involved. However, specific identification of PP1 and/or PP2A in mouse oocytes has not been reported. Here we demonstrate that fully grown germinal vesicle-intact (GVI) mouse oocytes contain mRNA corresponding to two isotypes of PP1, PP1alpha and PP1gamma. In addition, the transcript for PP2A was also present. At the protein level only PP1alpha and PP2A were recognized in fully grown GVI oocytes by Western blot analysis. Neither of the PP1gamma spliced variant proteins, PP1gamma1 and PP1gamma2, was detectable. Immunohistochemical analysis of ovarian tissue from gonadotropin-stimulated adult mice resulted in subcellular localization of both PP1alpha and PP2A, but not PP1gamma, in oocytes from all stages of folliculogenesis. In primordial oocytes, PP1alpha and PP2A were present in the cytoplasm. In more advanced stages of oogenesis, PP1alpha, although still present in the cytoplasm, was highly concentrated in the nucleus, whereas PP2A was predominantly cytoplasmic with a distinct reduction in the nuclear area. Both PP1alpha and PP2A were immunodetectable in oocytes during the prepubertal period. Eleven-day-old mouse oocytes, considered OA-insensitive and germinal vesicle breakdown (GVB)-incompetent, displayed both PP1alpha and PP2A predominantly in the cytoplasm. By 15 days of age mouse oocytes, which are beginning to acquire OA sensitivity and GVB competence, showed a relocation of PP1alpha into the nucleoplasm while PP2A remained predominantly cytoplasmic. This is the first specific identification of PP1alpha and PP2A in mouse oocytes. The differential localization of PP1alpha and PP2A, in addition to the relocation of PP1alpha during the acquisition of meiotic competence, suggests that these PPs have distinct regulatory roles during the resumption of meiosis.
Assuntos
Oócitos/enzimologia , Fosfoproteínas Fosfatases/análise , Animais , Sequência de Bases , Feminino , Imuno-Histoquímica , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Proteína Fosfatase 1RESUMO
The short-lived corpus luteum (CL) contributes to reproductive inefficiency during the postpartum period in beef cows. The cause for the early demise of the short-lived CL is not fully understood but is believed to involve a premature release of prostaglandin F2 alpha. The objectives of this study were to evaluate norgestomet-hCG-induced normal-lived CL and hCG-induced short-lived CL in postpartum cows with respect to serum progesterone (P4) and 13,14-dihydro-15-keto, prostaglandin F2 alpha (PGFM) concentrations and luteal LH receptor (LH-R) concentrations, LH-R mRNA levels, and vascularity. Although serum P4 profiles from the time of hCG administration (Day 0) until luteectomy (Day 6, 7, or 8) were similar between CL life span groups, PGFM concentrations were elevated (p < 0.05) on Day 8 in cows expected to have short-lived CL compared to normal-lived CL. The LH-R concentrations were similar between normal- and short-lived CL on all days measured. Irrespective of luteal life span and day of luteectomy, all CL possessed a 4.4-kb LH-R transcript. Actin-normalized LH-R mRNA levels were similar between normal- and short-lived CL on Days 6 and 7; however, Day 8 short-lived CL contained less (p < 0.05) LH-R mRNA than Day 8 normal-lived CL. Although the area of luteal tissue occupied by capillaries in normal- and short-lived CL was similar on Days 6 and 7, the area occupied by capillaries in short-lived CL was lower (p < 0.05) than that for normal-lived CL on Day 8. Collectively, these results indicate that there is a decrease in steady-state LH-R mRNA and a reduction in luteal vascularity in CL expected to be short-lived. These changes occur concomitantly with a rise in serum PGFM, but prior to a decline in serum P4.
Assuntos
Corpo Lúteo/fisiologia , Endotélio Vascular/fisiologia , Receptores do LH/genética , Animais , Northern Blotting , Bovinos , Gonadotropina Coriônica/administração & dosagem , Corpo Lúteo/irrigação sanguínea , Corpo Lúteo/efeitos dos fármacos , Dinoprosta/análogos & derivados , Dinoprosta/sangue , Dinoprosta/metabolismo , Endotélio Vascular/efeitos dos fármacos , Feminino , Pregnenodionas/administração & dosagem , Progesterona/sangue , Progesterona/metabolismo , Congêneres da Progesterona/administração & dosagem , RNA Mensageiro/análise , RNA Mensageiro/genética , Receptores do LH/efeitos dos fármacos , Receptores do LH/metabolismo , Distribuição Tecidual/efeitos dos fármacos , Distribuição Tecidual/fisiologiaRESUMO
The gene BCL6 encodes a zinc finger protein with similarities to transcription factors. We previously reported that a number of viral genomes, including human immunodeficiency virus type I (HIV-1), contain sequences which are similar to the BCL6 DNA-binding consensus in their promoter regions. Electrophoretic mobility shift assays showed that the full-length BCL6 protein extracted from transfected COS cells and a bacterially expressed truncated protein containing the BCL6 zinc fingers can bind specifically to DNA from the U3 promoter/enhancer region of HIV-1. Transient transfections were performed to analyze the effects of the BCL6 protein on luciferase expression driven by the HIV-1 long terminal repeat (LTR) sequences. Full-length BCL6 significantly repressed luciferase activity compared with multiple controls. We conclude that the BCL6 protein can bind to the HIV-1 promoter-enhancer region and contains a domain upstream of its zinc fingers that can repress transcription from the HIV-1 LTR.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Regulação Viral da Expressão Gênica , Ampliador HIV , HIV-1/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/fisiologia , Fatores de Transcrição/fisiologia , Transcrição Gênica , Dedos de Zinco , Animais , Sequência de Bases , Sítios de Ligação , Western Blotting , Células COS , Sequência Consenso , DNA/metabolismo , DNA Complementar/química , Proteínas de Ligação a DNA/genética , Eletroforese em Gel de Poliacrilamida , Genes Reporter , Células HeLa , Humanos , Luciferases/genética , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-bcl-6 , Fatores de Transcrição/genéticaRESUMO
Chromosomal rearrangements of BCL6 are commonly associated with diffuse large-cell lymphomas. We set out to determine the DNA-binding site of a glutathione-S-transferase fusion protein containing the BCL6 zinc finger region by employing cyclic amplification and selection of targets (CASTing). From oligonucleotides containing 16 central random bases, sequences binding to the protein on glutathione-coated beads were repeatedly selected and amplified by polymerase chain reaction (PCR). The binding sites were cloned and sequenced. A consensus, TTTNNNGNNATNCTTT, was obtained. Protein binding studies of double-stranded oligomers containing point mutations within the 3' CTTT confirmed the binding specificity of this part of the consensus. In addition, evidence indicated that some of the base pairs held constant in the oligonucleotides used for CASTing also contributed to binding.