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1.
bioRxiv ; 2023 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-38076833

RESUMO

In regions where reads don't align well to a reference, it is generally difficult to characterize structural variation using short read sequencing. Here, we utilize machine learning classifiers and short sequence reads to genotype structural variants in the alpha globin locus on chromosome 16, a medically-relevant region that is challenging to genotype in individuals. Using models trained only with simulated data, we accurately genotype two hard-to-distinguish deletions in two separate human cohorts. Furthermore, population allele frequencies produced by our methods across a wide set of ancestries agree more closely with previously-determined frequencies than those obtained using currently available genotyping software.

2.
J Mol Diagn ; 24(1): 18-32, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34656759

RESUMO

Neurotrophic tyrosine receptor kinase (NTRK1/2/3) fusions are oncogenic drivers in approximately 0.3% of solid tumors. High-quality testing to identify patients with NTRK fusion-positive tumors who could benefit from tropomyosin receptor kinase inhibitors is recommended, but the current NTRK testing landscape, including next-generation sequencing (NGS), is fragmented and availability of assays varies widely. The analytical and clinical performance of four commonly available RNA-based NGS assays, Archer's FusionPlex Lung panel (AFL), Illumina's TruSight Oncology 500 (TSO500), Thermo Fisher's Oncomine Precision Assay and Oncomine Focus Assay (OFA), were evaluated. Experiments were conducted using contrived samples [formalin-fixed, paraffin-embedded cell lines and SeraSeq formalin-fixed, paraffin-embedded reference material], NTRK fusion-negative clinical samples, and NTRK fusion-positive clinical samples, according to local assays. Estimated limit of detection varied across the four assays: 30 to 620 fusion copies for AFL (cell lines), versus approximately 30 to 290 copies for TSO500 and approximately 1 to 28 copies for OFA and Oncomine Precision Assay. All assays showed 100% specificity for NTRK fusions detection, but quality control pass rate was variable (AFL, 43%; TSO500, 77%; and OFA, 83%). The NTRK fusion detection rate in quality control-validated clinical samples was 100% for all assays. This comparison of the strengths and limitations of four RNA-based NGS assays will inform physicians and pathologists regarding optimal assay selection to identify patients with NTRK fusion-positive tumors.


Assuntos
Neoplasias , Proteínas de Fusão Oncogênica , Biomarcadores Tumorais , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias/genética , Proteínas de Fusão Oncogênica/genética , Oncogenes
3.
J Natl Cancer Inst ; 113(12): 1733-1743, 2021 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-34097070

RESUMO

BACKGROUND: In the phase III IMpassion130 study, atezolizumab plus nab-paclitaxel (A+nP) showed clinical benefit in advanced or metastatic triple-negative breast cancer patients who were programmed death-ligand 1 (PD-L1)+ (tumor-infiltrating immune cells [IC] ≥1%) using the SP142 immunohistochemistry assay. Here we evaluate 2 other PD-L1 assays for analytical concordance with SP142 and patient-associated clinical outcomes. METHODS: Samples from 614 patients (68.1% of intention-to-treat population) were centrally evaluated by immunohistochemistry for PD-L1 status on IC (VENTANA SP142, SP263, Dako 22C3) or as a combined positive score (CPS; 22C3). RESULTS: Using SP142, SP263, and 22C3 assays, PD-L1 IC ≥1% prevalence was 46.4% (95% confidence interval [CI] = 42.5% to 50.4%), 74.9% (95% CI = 71.5% to 78.3%), and 73.1% (95% CI = 69.6% to 76.6%), respectively; 80.9% were 22C3 CPS ≥1. At IC ≥1% (+), the analytical concordance between SP142 and SP263 and 22C3 was 69.2% and 68.7%, respectively. Almost all SP142+ cases were captured by other assays (double positive), but several SP263+ (29.6%) or 22C3+ (29.0%) cases were SP142- (single positive). A+nP clinical activity vs placebo+nP in SP263+ and 22C3+ patients (progression-free survival [PFS] hazard ratios [HRs] = 0.64 to 0.68; overall survival [OS] HRs = 0.75 to 0.79) was driven by double-positive cases (PFS HRs = 0.60 to 0.61; OS HRs = 0.71 to 0.75) rather than single-positive cases (PFS HRs = 0.68 to 0.81; OS HRs = 0.87 to 0.95). Concordance for harmonized cutoffs for SP263 (IC ≥4%) and 22C3 (CPS ≥10) to SP142 (IC ≥1%) was subpar (approximately 75%). CONCLUSIONS: 22C3 and SP263 assays identified more patients as PD-L1+ (IC ≥1%) than SP142. No inter-assay analytical equivalency was observed. Consistent improved A+nP efficacy was captured by the SP142 PD-L1 IC ≥1% subgroup nested within 22C3 and SP263 PD-L1+ (IC ≥1%) populations.


Assuntos
Antígeno B7-H1 , Neoplasias de Mama Triplo Negativas , Humanos , Biomarcadores Tumorais , Imuno-Histoquímica , Neoplasias de Mama Triplo Negativas/tratamento farmacológico
4.
Sultan Qaboos Univ Med J ; 15(3): e327-37, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26357552

RESUMO

OBJECTIVES: This study aimed to raise radiologists' awareness of skeletal muscle metastases (SMM) in renal cell carcinoma (RCC) cases and to clarify their imaging appearance. METHODS: A retrospective analysis was undertaken of 21 patients between 44-75 years old with 72 SMM treated from January 1990 to May 2009 at the MD Anderson Cancer Center in Houston, Texas, USA. Additionally, 37 patients with 44 SMM from a literature review were analysed. RESULTS: Among the 21 patients, the majority of SMM were asymptomatic and detected via computed tomography (CT). Mean metastasis size was 18.3 mm and the most common site was the trunk muscles (83.3%). The interval between discovery of the primary tumour and metastasis detection ranged up to 234 months. Peripheral enhancement (47.1%) was the most common post-contrast CT pattern and non-contrasted CT lesions were often isodense. Magnetic resonance imaging (MRI) characteristics were varied. Five lesions with available T1-weighted pre-contrast images were hyperintense to the surrounding muscle. Other organ metastases were present in 20 patients. Of the 44 SMM reported in the literature, the majority were symptomatic. Average metastasis size was 53.4 mm and only 20.5% of SMM were in trunk muscles. The average interval between tumour discovery and metastasis detection was 101 months. Other organ metastases were recorded in 17 out of 29 patients. CONCLUSION: SMM should always be considered in patients with RCC, even well after primary treatment. SMM from RCC may be invisible on CT without intravenous contrast; contrast-enhanced studies are therefore recommended. SMM are often hyperintense to the surrounding muscle on T1-weighted MRI scans.

5.
J Infect Dis ; 210(12): 1881-5, 2014 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-24907384

RESUMO

We measured interferon γ-induced protein 10 (IP-10) levels in 428 patients at baseline, week 1, and week 2 of all-oral treatment for hepatitis C virus (HCV) infection. An increased baseline IP-10 level was associated with a T allele in the IL28B gene, an increased alanine aminotransferase level in treatment-naive but not experienced patients, and an increased body mass index. At week 1, the mean decline in plasma IP-10 levels was the same in treatment-naive and treatment-experienced patients (-49%), whereas during week 2 the mean decline in IP-10 levels in treatment-naive patients (-14%) was significantly larger than in treatment-experienced patients (-2%; P = .0176). IP-10 thus may be a surrogate marker of the rate of intracellular viral replication complex decay.


Assuntos
Antivirais/uso terapêutico , Quimiocina CXCL10/sangue , Hepacivirus/imunologia , Hepatite C Crônica/tratamento farmacológico , Hepatite C Crônica/imunologia , Imunidade Inata , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Plasma/química
6.
AIDS ; 27(18): 2893-7, 2013 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-23842130

RESUMO

OBJECTIVES: To report the rates of grade IV adverse events and hepatitis C virus (HCV) treatment discontinuation associated with the use of telaprevir, pegylated interferon, and ribavirin. DESIGN: Retrospective cohort analysis. METHODS: The study included patients coinfected with HIV and HCV who underwent HCV treatment in a clinic-based setting with telaprevir, pegylated interferon, and ribavirin. The United States of America National Institutes of Health Division of AIDS grading system was used to rate severity of adverse events. RESULTS: Of the 24 consecutive patients treated for HCV using telaprevir/pegylated interferon/ribavirin, 50% (12/24) developed serious adverse events and 29% (7/24) discontinued HCV treatment due to adverse events, despite an intensive multidisciplinary monitoring approach. CONCLUSION: In this HIV clinic-based experience, a high rate of grade IV adverse events and treatment discontinuations were observed associated with HCV telaprevir-based treatment. Careful consideration of the risks and benefits of telaprevir-based therapy should be undertaken, given prospects for interferon-sparing therapy in the near future.


Assuntos
Antivirais/efeitos adversos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/epidemiologia , Infecções por HIV/complicações , Hepatite C Crônica/tratamento farmacológico , Oligopeptídeos/efeitos adversos , Adulto , Antivirais/uso terapêutico , Estudos de Coortes , Quimioterapia Combinada/efeitos adversos , Quimioterapia Combinada/métodos , Feminino , Humanos , Incidência , Interferon-alfa/efeitos adversos , Interferon-alfa/uso terapêutico , Masculino , Pessoa de Meia-Idade , Oligopeptídeos/uso terapêutico , Estudos Retrospectivos , Ribavirina/efeitos adversos , Ribavirina/uso terapêutico , Índice de Gravidade de Doença , Estados Unidos/epidemiologia , Suspensão de Tratamento
7.
J Infect Chemother ; 19(1): 42-9, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22797874

RESUMO

There are many limitations to the current antibiotics used for the treatment of severe methicillin-resistant Staphylococcus aureus (MRSA) infections. Ceftaroline is a new fifth-generation cephalosporin approved for the treatment of skin and soft tissue infections caused by MRSA and community-acquired pneumonia. We propose that ceftaroline can also be used successfully in more severe MRSA infections, including endocarditis. We conducted a retrospective chart review in a university-affiliated Department of Veterans Affairs hospital in San Diego, California (USA) of ten inpatients treated with ceftaroline for severe MRSA infection, including five cases of probable endocarditis (including two endocardial pacemaker infections), one case of pyomyositis with possible endocarditis, two cases of pneumonia (including one case of empyema), two cases of septic arthritis (including one case of prosthetic joint infection), and two cases of osteomyelitis. Seven of the 10 patients achieved microbiological cure. Six of the 10 patients achieved clinical cure. Seven patients were discharged from the hospital. Three patients were placed on comfort care and expired in the hospital; one achieved microbiological cure before death, and two remained bacteremic at time of death. In most patients, ceftaroline was effective for treatment of MRSA bacteremia and other severe MRSA infections. Adverse effects seen included rash, eosinophilia, pruritus, and Clostridium difficile infection. Ceftaroline can be a safe and effective drug for treatment of severe MRSA infections, and further comparative studies are warranted.


Assuntos
Antibacterianos/uso terapêutico , Bacteriemia/tratamento farmacológico , Cefalosporinas/uso terapêutico , Endocardite Bacteriana/tratamento farmacológico , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Pró-Fármacos/uso terapêutico , Infecções Estafilocócicas/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Artrite Infecciosa/tratamento farmacológico , Artrite Infecciosa/microbiologia , Bacteriemia/microbiologia , Endocardite Bacteriana/microbiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Osteomielite/tratamento farmacológico , Osteomielite/microbiologia , Pneumonia Estafilocócica/tratamento farmacológico , Pneumonia Estafilocócica/microbiologia , Estudos Retrospectivos , Infecções Estafilocócicas/microbiologia , Resultado do Tratamento , Ceftarolina
8.
Cell Stem Cell ; 10(2): 183-97, 2012 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-22305568

RESUMO

Gene expression signatures relating mammary stem cell populations to breast cancers have focused on adult tissue. Here, we identify, isolate, and characterize the fetal mammary stem cell (fMaSC) state since the invasive and proliferative processes of mammogenesis resemble phases of cancer progression. fMaSC frequency peaks late in embryogenesis, enabling more extensive stem cell purification than achieved with adult tissue. fMaSCs are self-renewing, multipotent, and coexpress multiple mammary lineage markers. Gene expression, transplantation, and in vitro analyses reveal putative autocrine and paracrine regulatory mechanisms, including ErbB and FGF signaling pathways impinging on fMaSC growth. Expression profiles from fMaSCs and associated stroma exhibit significant similarities to basal-like and Her2+ intrinsic breast cancer subtypes. Our results reveal links between development and cancer and provide resources to identify new candidates for diagnosis, prognosis, and therapy.


Assuntos
Neoplasias da Mama/patologia , Carcinoma Basocelular/patologia , Células-Tronco Embrionárias/patologia , Glândulas Mamárias Humanas/embriologia , Glândulas Mamárias Humanas/patologia , Células-Tronco Neoplásicas/patologia , Células-Tronco Pluripotentes/patologia , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Carcinoma Basocelular/genética , Carcinoma Basocelular/metabolismo , Sobrevivência Celular , Transformação Celular Neoplásica , Células-Tronco Embrionárias/metabolismo , Feminino , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Regulação Neoplásica da Expressão Gênica , Humanos , Glândulas Mamárias Humanas/metabolismo , Camundongos , Camundongos SCID , Células-Tronco Neoplásicas/metabolismo , Proteínas Oncogênicas v-erbB/genética , Proteínas Oncogênicas v-erbB/metabolismo , Células-Tronco Pluripotentes/metabolismo , Transplante de Células-Tronco
9.
J Clin Microbiol ; 50(3): 922-6, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22219303

RESUMO

Human brucellosis is a common zoonosis worldwide. Here we present a case of focal vertebral brucellosis in a 71-year-old Mexican-American woman who contracted infection from unpasteurized goat milk. Standard agglutination serology was negative; the diagnosis was established by the isolation of Brucella melitensis from abscess fluid. A B. melitensis protein microarray comprised of nearly all proteins encoded by the bacterial genome was used to determine the kinetics of this patient's antibody responses to the complete collection of open reading frames existing in the genome (ORFeome). Three patterns of antibody responses against B. melitensis antigens were seen for serum samples obtained on days 0 (pretreatment), 14, 49, 100, and 180: (i) stable titers over time, (ii) a steady fall in titers, and (iii) an initial rise in titers followed by declining titers. Sera from this patient with chronic brucellosis recognized some of the same B. melitensis proteins as those recognized by sera from acute/subacute, blood culture-positive brucellosis patients but also recognized a distinct set of proteins. This study is the first to determine the kinetics of the human antibody responses to the complete repertoire of proteins encoded by a bacterial genome and demonstrates fundamentally different immunopathogenetic mechanisms between acute human brucellosis and chronic human brucellosis. While an extension of these findings to a larger patient population is necessary, these findings have important clinical and diagnostic implications and lead toward new insights into the fundamental immunopathogenesis of brucellosis.


Assuntos
Anticorpos Antibacterianos/sangue , Brucella melitensis/imunologia , Brucella melitensis/isolamento & purificação , Brucelose/imunologia , Espondilite/imunologia , Espondilite/microbiologia , Idoso , Animais , Antígenos de Bactérias/imunologia , Brucelose/diagnóstico , Brucelose/patologia , Feminino , Cabras , Humanos , Imageamento por Ressonância Magnética , Americanos Mexicanos , Leite , Radiografia , Soro/imunologia , Coluna Vertebral/diagnóstico por imagem , Espondilite/diagnóstico , Espondilite/patologia , Fatores de Tempo
10.
Cancer Res ; 71(17): 5616-20, 2011 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-21880602

RESUMO

The American Association for Cancer Research (AACR) held an exciting conference on Stem Cells, Development, and Cancer in Vancouver, British Columbia, Canada (March 3-6, 2011). The meeting was cochaired by Geoffrey Wahl, Connie Eaves, and Hans Clevers and was attended by 250 international researchers, 40% of whom were young investigators. Three key themes emerged: (i) heterogeneity in stem cells and cancer, (ii) solid tissue cancer stem cells, and (iii) lessons from development. The interdisciplinary foundation of this meeting was central to its success and appeal, underscoring the value of juxtaposing and interrelating work from the three topics addressed.


Assuntos
Crescimento e Desenvolvimento/fisiologia , Neoplasias/patologia , Células-Tronco , Separação Celular/métodos , Análise Citogenética , Humanos , Células-Tronco/citologia , Células-Tronco/patologia , Células-Tronco/fisiologia
11.
Nat Genet ; 41(8): 931-5, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19597493

RESUMO

Tetralogy of Fallot (TOF), the most common severe congenital heart malformation, occurs sporadically, without other anomaly, and from unknown cause in 70% of cases. Through a genome-wide survey of 114 subjects with TOF and their unaffected parents, we identified 11 de novo copy number variants (CNVs) that were absent or extremely rare (<0.1%) in 2,265 controls. We then examined a second, independent TOF cohort (n = 398) for additional CNVs at these loci. We identified CNVs at chromosome 1q21.1 in 1% (5/512, P = 0.0002, OR = 22.3) of nonsyndromic sporadic TOF cases. We also identified recurrent CNVs at 3p25.1, 7p21.3 and 22q11.2. CNVs in a single subject with TOF occurred at six loci, two that encode known (NOTCH1, JAG1) disease-associated genes. Our findings predict that at least 10% (4.5-15.5%, 95% confidence interval) of sporadic nonsyndromic TOF cases result from de novo CNVs and suggest that mutations within these loci might be etiologic in other cases of TOF.


Assuntos
Dosagem de Genes , Variação Genética , Tetralogia de Fallot/genética , Pré-Escolar , Cromossomos Humanos Par 1/genética , Cromossomos Humanos Par 3/genética , Regulação da Expressão Gênica , Ventrículos do Coração/metabolismo , Humanos , Fenótipo , Tetralogia de Fallot/patologia
12.
Biochemistry ; 44(4): 1278-87, 2005 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-15667221

RESUMO

The alpha-factor receptor (Ste2p) stimulates mating of the yeast Saccharomyces cerevisiae. Ste2p belongs to the large family of G protein-coupled receptors that are characterized by seven transmembrane alpha-helices. Receptor activation is thought to involve changes in the packing of the transmembrane helix bundle. To identify residues that contribute to Ste2p activation, second-site suppressor mutations were isolated that restored function to defective receptors carrying either an F204S or Y266C substitution which affect residues at the extracellular ends of transmembrane domains 5 and 6, respectively. Thirty-five different suppressor mutations were identified. On their own, these mutations caused a range of phenotypes, including hypersensitivity, constitutive activity, altered ligand binding, and loss of function. The majority of the mutations affected residues in the transmembrane segments that are predicted to face the helix bundle. Many of the suppressor mutations caused constitutive receptor activity, suggesting they improved receptor function by partially restoring the balance between the active and inactive states. Analysis of mutations in transmembrane domain 7 implicated residues Ala281 and Thr282 in receptor activation. The A281T and T282A mutants were supersensitive to S. cerevisiae alpha-factor, but were defective in responding to a variant of alpha-factor produced by another species, Saccharomyces kluyveri. The A281T mutant also displayed 8.7-fold enhanced basal signaling. Interestingly, Ala281 and Thr282 are situated in approximately the same position as Lys296 in rhodopsin, which is covalently linked to retinal. These results suggest that transmembrane domain 7 plays a role in receptor activation in a wide range of G protein-coupled receptors from yeast to humans.


Assuntos
Mutagênese , Peptídeos/química , Receptores Acoplados a Proteínas G/genética , Receptores de Peptídeos/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/metabolismo , Alanina/genética , Sequência de Aminoácidos , Cisteína/genética , Análise Mutacional de DNA , Genes Supressores , Testes Genéticos , Fator de Acasalamento , Dados de Sequência Molecular , Peptídeos/fisiologia , Fenilalanina/genética , Receptores Acoplados a Proteínas G/fisiologia , Receptores de Fator de Acasalamento , Receptores de Peptídeos/genética , Proteínas de Saccharomyces cerevisiae/fisiologia , Serina/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Treonina/genética , Fatores de Transcrição/genética , Tirosina/genética
13.
Radiology ; 233(3): 659-66, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15516606

RESUMO

PURPOSE: To compare three-dimensional (3D) mangafodipir trisodium-enhanced T1-weighted magnetic resonance (MR) cholangiography with conventional T2-weighted MR cholangiography for depiction and definition of intrahepatic biliary anatomy in liver transplant donor candidates. MATERIALS AND METHODS: One hundred eight healthy liver transplant donor candidates were examined with two MR cholangiographic methods. All candidates gave written informed consent, and the study was approved by the institutional review board. First, breath-hold transverse and coronal half-Fourier single-shot turbo spin-echo and breath-hold oblique coronal heavily T2-weighted turbo spin-echo sequences were performed. Second, mangafodipir trisodium-enhanced breath-hold fat-suppressed 3D gradient-echo sequences were performed through the ducts (oblique coronal plane) and through the entire liver (transverse plane). Interpretation of biliary anatomy findings, particularly variants affecting right liver lobe biliary drainage, and degree of interpretation confidence at both 3D mangafodipir trisodium-enhanced MR cholangiography and T2-weighted MR cholangiography were recorded and compared by using the Wilcoxon signed rank test. Then, consensus interpretations of both MR image sets together were performed. Intraoperative cholangiography was the reference-standard examination for 51 subjects who underwent right lobe hepatectomy. The McNemar test was used to compare the accuracies of the individual MR techniques with that of the consensus interpretation of both image sets together and to compare each technique with intraoperative cholangiography. RESULTS: Biliary anatomy was visualized with mangafodipir trisodium enhancement in all patients. Mangafodipir trisodium-enhanced image findings agreed with findings seen at combined interpretations significantly more often than did T2-weighted image findings (in 107 [99%] vs 88 [82%] of 108 donor candidates, P < .001). Confidence was significantly higher with the mangafodipir trisodium-enhanced images than with the T2-weighted images (mean confidence score, 4.5 vs 3.4; P < .001). In the 51 candidates who underwent intraoperative cholangiography, mangafodipir trisodium-enhanced imaging correctly depicted the biliary anatomy more often than did T2-weighted imaging (in 47 [92%] vs 43 [84%] donor candidates, P = .14), whereas the two MR imaging techniques combined correctly depicted the anatomy in 48 (94%) candidates. CONCLUSION: Mangafodipir trisodium-enhanced 3D MR cholangiography depicts intrahepatic biliary anatomy, especially right duct variants, more accurately than does conventional T2-weighted MR cholangiography.


Assuntos
Ductos Biliares Intra-Hepáticos/anatomia & histologia , Meios de Contraste , Ácido Edético/análogos & derivados , Transplante de Fígado , Doadores Vivos , Imageamento por Ressonância Magnética/métodos , Manganês , Fosfato de Piridoxal/análogos & derivados , Adolescente , Adulto , Ductos Biliares Intra-Hepáticos/diagnóstico por imagem , Colangiografia , Feminino , Hepatectomia , Humanos , Aumento da Imagem/métodos , Imageamento Tridimensional/métodos , Cuidados Intraoperatórios , Masculino , Pessoa de Meia-Idade , Radiografia Intervencionista , Estatísticas não Paramétricas
14.
Liver Transpl ; 10(8): 1049-54, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15390332

RESUMO

Our objective was to investigate the coexistence of vascular and biliary anatomic variants, the latter of which are known to increase the risk of biliary complications in living liver donor transplantation. A total of 108 consecutive liver donor candidates were examined by magnetic resonance (MR) imaging that included 2 MR cholangiography methods, T2-weighted MR cholangiography and mangofodipir-enhanced T1-weighted three-dimensional (3D) MR cholangiography, as well as gadolinium-enhanced MR angiography and venography of the liver. Images were interpreted by at least 2 investigators in consensus for definition of hepatic arterial, portal venous, and biliary anatomy. A subset of 51 subjects underwent laparotomy for right hepatectomy. Of the 108 subjects examined, 50 (46%) demonstrated normal hepatic artery, portal vein, and biliary anatomy. Variants of the hepatic artery were found in 27 of 108 (25%) subjects, of the portal vein in 12 of 108 (11%) subjects, and of the bile ducts in 30 of 108 (28%) subjects. Of the 27 subjects with hepatic arterial variants, 8 (30%) also had variant biliary anatomy. The association between hepatic arterial variants and biliary variants was not statistically significant (P >.5). However, of the 12 subjects with portal vein variants, 7 (58%) had biliary variants, and in 6 of 7 cases, the right posterior hepatic duct was anomalous. By chi-square analysis, the association between portal venous and biliary variants was significant (P =.012). In conclusion, over half of subjects with portal vein variants were found to have anomalous biliary anatomy, which always involved the hepatic ducts of the right lobe. The association between portal venous and biliary variants is statistically significant, while there is no significant association between hepatic arterial and biliary variants.


Assuntos
Sistema Biliar/anormalidades , Artéria Hepática/anormalidades , Transplante de Fígado/fisiologia , Fígado , Doadores Vivos , Seleção de Pacientes , Veia Porta/anormalidades , Hepatectomia , Humanos , Coleta de Tecidos e Órgãos
15.
Mol Cell Biol ; 24(5): 2041-51, 2004 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-14966283

RESUMO

The alpha-factor receptor (Ste2p) that promotes mating in Saccharomyces cerevisiae is similar to other G protein-coupled receptors (GPCRs) in that it contains seven transmembrane domains. Previous studies suggested that the extracellular ends of the transmembrane domains are important for Ste2p function, so a systematic scanning mutagenesis was carried out in which 46 residues near the ends of transmembrane domains 1, 2, 3, 4, and 7 were replaced with cysteine. These mutants complement mutations constructed previously near the ends of transmembrane domains 5 and 6 to analyze all the extracellular ends. Eight new mutants created in this study were partially defective in signaling (V45C, N46C, T50C, A52C, L102C, N105C, L277C, and A281C). Treatment with 2-([biotinoyl] amino) ethyl methanethiosulfonate, a thiol-specific reagent that reacts with accessible cysteine residues but not membrane-embedded cysteines, identified a drop in the level of reactivity over a consecutive series of residues that was inferred to be the membrane boundary. An unusual prolonged zone of intermediate reactivity near the extracellular end of transmembrane domain 2 suggests that this region may adopt a special structure. Interestingly, residues implicated in ligand binding were mainly accessible, whereas residues involved in the subsequent step of promoting receptor activation were mainly inaccessible. These results define a receptor microdomain that provides an important framework for interpreting the mechanisms by which functionally important residues contribute to ligand binding and activation of Ste2p and other GPCRs.


Assuntos
Estrutura Secundária de Proteína , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Peptídeos/metabolismo , Transdução de Sinais/fisiologia , Fatores de Transcrição , Sequência de Aminoácidos , Sítios de Ligação , Biotina/química , Biotina/metabolismo , Divisão Celular/fisiologia , Cisteína/metabolismo , Genes Reporter , Ligantes , Mesilatos/química , Mesilatos/metabolismo , Modelos Moleculares , Mutação , Fenótipo , Feromônios/metabolismo , Ligação Proteica , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Receptores de Fator de Acasalamento , Receptores de Peptídeos/química , Receptores de Peptídeos/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
16.
Biochemistry ; 42(2): 293-301, 2003 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-12525156

RESUMO

The alpha-factor receptor (STE2) stimulates a G protein signaling pathway that promotes mating of the yeast Saccharomyces cerevisiae. Previous random mutagenesis studies implicated residues in the regions near the extracellular ends of the transmembrane domains in ligand activation. In this study, systematic Cys scanning mutagenesis across the ends of transmembrane domains 5 and 6 identified two residues, Phe(204) and Tyr(266), that were important for receptor signaling. These residues play a specific role in responding to alpha-factor since the F204C and Y266C substituted receptors responded to an alternative agonist (novobiocin). To better define the structure of this region, the Cys-substituted mutant receptors were assayed for reactivity with a thiol-specific probe that does not react with membrane-imbedded residues. A drop in reactivity coincided with residues likely to be buried in the membrane. Interestingly, both Phe(204) and Tyr(266) are located very near the interface region. However, these assays predict that Phe(204) is accessible at the surface of the receptor, consistent with the strong defect in binding alpha-factor caused by mutating this residue. In contrast, Tyr(266) was not accessible. This correlates with the ability of Y266C mutant receptors to bind alpha-factor and suggests that this residue is involved in the subsequent triggering of receptor activation. These results highlight the role of aromatic residues near the ends of the transmembrane segments in the alpha-factor receptor, and suggest that similar aromatic residues may play an important role in other G protein-coupled receptors.


Assuntos
Espaço Extracelular/fisiologia , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Membrana/fisiologia , Fenilalanina/fisiologia , Receptores de Superfície Celular/metabolismo , Receptores de Peptídeos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição , Tirosina/fisiologia , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Cisteína/genética , Espaço Extracelular/genética , Ligantes , Proteínas de Membrana/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Novobiocina/metabolismo , Novobiocina/farmacologia , Fenótipo , Fenilalanina/genética , Estrutura Terciária de Proteína/genética , Receptores de Superfície Celular/agonistas , Receptores de Superfície Celular/genética , Receptores de Fator de Acasalamento , Receptores de Peptídeos/agonistas , Receptores de Peptídeos/genética , Proteínas de Saccharomyces cerevisiae/agonistas , Proteínas de Saccharomyces cerevisiae/genética , Tirosina/genética
17.
Dev Dyn ; 225(4): 499-510, 2002 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12454926

RESUMO

The frog Xenopus laevis has provided significant insights into developmental and cellular processes. However, X. laevis has an allotetraploid genome precluding its use in forward genetic analysis. Genetic analysis may be applicable to Xenopus (Silurana) tropicalis, which has a diploid genome and a shorter generation time. Here, we show that many tools for the study of X. laevis development can be applied to X. tropicalis. By using the developmental staging system of Nieuwkoop and Faber, we find that X. tropicalis embryos develop at similar rates to X. laevis, although they tolerate a narrower range of temperatures. We also show that many of the analytical reagents available for X. laevis can be effectively transferred to X. tropicalis. The X. laevis protocol for whole-mount in situ hybridization to mRNA transcripts can be successfully applied to X. tropicalis without alteration. Additionally, X. laevis probes often work in X. tropicalis--alleviating the immediate need to clone the X. tropicalis orthologs before initiating developmental studies. Antibodies that react against X. laevis proteins can effectively detect the X. tropicalis protein by using established immunohistochemistry procedures. Antisense morpholino oligonucleotides (MOs) offer a new alternative to study loss of gene activity during development. We show that MOs function in X. tropicalis. Finally, X. tropicalis offers the possibility for forward genetics and genomic analysis.


Assuntos
Biologia do Desenvolvimento/métodos , Xenopus/embriologia , Animais , Sequência de Bases , Proteínas do Citoesqueleto/metabolismo , DNA Complementar/metabolismo , Ectoderma/metabolismo , Endoderma/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Biblioteca Gênica , Imuno-Histoquímica , Hibridização In Situ , Mesoderma/metabolismo , Dados de Sequência Molecular , Oligonucleotídeos Antissenso/farmacologia , RNA Mensageiro/metabolismo , Fatores de Tempo , Transativadores/metabolismo , Proteínas de Xenopus , beta Catenina
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