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INTRODUCTION: Radiological assessment of brain tumors is widely based on the Radiology Assessment of Neuro-Oncology (RANO) criteria that consider non-specific T1 and T2 weighted images. Limitation of the RANO criteria is that they do not include metabolic imaging techniques that have been reported to be helpful to differentiate treatment related changes from true tumor progression. In the current study, we assessed if the combined use of MRI and PET with hybrid 11C-MET PET/MRI can improve diagnostic accuracy and diagnostic confidence of the readers to differentiate treatment related changes from true progression in recurrent glioma. METHODS: Fifty consecutive patients with histopathologically proven glioma were prospectively enrolled for a hybrid 11C-MET PET/MRI to differentiate recurrent glioma from treatment induced changes. Sole MRI data were analyzed based on RANO. Sole PET data and in a third evaluation hybrid 11C-MET-PET/MRI data were assessed for metabolic respectively metabolic and morphologic glioma recurrence. Diagnostic performance and diagnostic confidence of the reader were calculated for the different modalities, and the McNemar test and Mann-Whitney U Test were applied for statistical analysis. RESULTS: Hybrid 11C-MET PET/MRI was successfully performed in all 50 patients. Glioma recurrence was diagnosed in 35 of the 50 patients (70%). Sensitivity and specificity were calculated for MRI (86.11% and 71.43%), for 11C-MET PET (96.77% and 73.68%), and for hybrid 11C-MET-PET/MRI (97.14% and 93.33%). For diagnostic accuracy hybrid 11C-MET-PET/MRI (96%) showed significantly higher values than MRI alone (82%), whereas no significant difference was found for 11C-MET PET (88%). Furthermore, by rating on a five-point Likert scale significantly higher scores were found for diagnostic confidence when comparing 11C-MET PET/MRI (4.26 ± 0,777) to either PET alone (3.44 ± 0.705) or MRI alone (3.56 ± 0.733). CONCLUSION: This feasibility study showed that hybrid PET/MRI might strengthen RANO classification by adding metabolic information to conventional MRI information. Future studies should evaluate the clinical utility of the combined use of 11C-MET PET/MRI in larger patient cohorts.
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Neoplasias Encefálicas/diagnóstico por imagem , Glioma/diagnóstico por imagem , Imageamento por Ressonância Magnética , Tomografia por Emissão de Pósitrons , Radioisótopos de Carbono , Humanos , Metionina/análogos & derivados , Recidiva Local de Neoplasia/diagnóstico por imagemRESUMO
A Brønsted acid-catalyzed addition of 2-alkylazaarenes to in situ generated N-sulfonylimines through selective C(sp(3))-H bond functionalization has been developed. This protocol provides an atom- and step-economic approach to α-substituted sulfonamides.
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OBJECTIVES: To analyze multiparametric MRI (mpMRI) characteristics of patients with International Society of Urological Pathology (ISUP) grade group (GG) 4 or 5 prostate cancer (PC) and to correlate MRI parameters with the occurrence of biochemical recurrence (BCR) after radical prostatectomy (RPE). METHODS: In this single-center cohort study consecutive patients with mpMRI and ISUP GG 4 or 5 PC were retrospectively analyzed. Clinical, MR-guided biopsy, and diagnostic mpMRI parameter were assessed. A subcohort of patients with RPE and follow-up was analyzed separately. A univariate and multivariate analyses were performed to determine parameters that are associated to patients with BCR after RPE. RESULTS: 145 patients (mean age 70y, median PSA 10.9 ng/ml) were analyzed. 99% had a PI-RADS classification of 4 or 5, 48% revealed MRI T3 stage, and median diameter of the MRI index lesion (IL) was 15 mm. IL showed a median ADC value of 668 ×10-6 mm2/s and exhibited contrast enhancement in 94% of the cases. For patients with follow-up after RPE (n = 82; mean follow-up time 68 ± 27 m), MRI parameters were significantly different for contact length of the IL to the pseudocapsule (LCC), MRI T3 stage, and IL localization (p < 0.05). Higher PSAD and MRI T3 stage were independent parameters for the risk of BCR when incorporating clinical, biopsy, and MRI parameters. CONCLUSION: ISUP GG 4 or 5 PC has distinctive characteristics on mpMRI and were detected on MRI in all cases. In addition, higher PSAD and MRI T3 stage were significant predictors for BCR after RPE.
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Neoplasias da Próstata , Masculino , Humanos , Idoso , Neoplasias da Próstata/diagnóstico por imagem , Neoplasias da Próstata/cirurgia , Imageamento por Ressonância Magnética , Estudos Retrospectivos , Seguimentos , Estudos de Coortes , Antígeno Prostático Específico , Biópsia Guiada por ImagemRESUMO
Human-human interactions are of central relevance for the success in professional and occupational environments, which also substantially influence quality of life. This is especially true in the case of individuals with high-functioning autism (HFA), who experience deficits in social cognition that often lead to social exclusion and unemployment. Despite good education and high motivation, individuals with HFA do not reach employment rates that are substantially higher than 50 %. This is an alarmingly high rate of unemployment considering that the United Nations have recently emphasized the inclusion of handicapped persons as a mandatory human right. To date, the specific needs of autistic persons with respect to their working environment are largely unexplored. It remains moreover an open question how support systems and activities, including newly developed communication devices for professional environments of individuals with HFA, should look like. The German health and social care systems are not adequately prepared for the proper support of this population. This leads us to suggest that supported employment programs should be developed for adults with HFA that specifically address their needs and requirements. Such programs should comprise (1) the adequate assessment of HFA, including a neuropsychological profile and an individual matching of persons' preferences with requirements of the working place, (2) on-the-job coaching activities that include systematic communication and interaction training, and (3) instruction of non-autistic peers, including colleagues and supervisors, about weaknesses and strengths of HFA.
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Transtorno Autístico/reabilitação , Readaptação ao Emprego/organização & administração , Transtorno Autístico/epidemiologia , Transtorno Autístico/psicologia , Alemanha/epidemiologia , Humanos , Terapia Ocupacional , Desemprego/estatística & dados numéricosRESUMO
AIM/PURPOSE: 18F-labeled PSMA ligands offer various advantages as PET tracers over 68Ga-labeled PSMA counterparts. Especially, an improved spatial resolution leads to improved detection rates of smaller prostate cancer (PCa) lesions. However, physiological PSMA uptake of ganglia of the sympathetic trunk can be quickly misinterpreted as possible PSMA-positive lymph node metastases. The aim of this retrospective study is to investigate [18F]PSMA-1007 uptake and its intra-individual reproducibility in ganglia of the sympathetic trunk. METHODS: We retrospectively included 28 consecutive patients (median age 69 ± 9 with a range of 49-90) with biochemical recurrence of PCa who underwent [18F]PSMA-1007 PET/CT scan and, accordingly, a follow-up examination between August 2018 and August 2021. Cervical, coeliac, and sacral ganglia were identified on the iterative PET reconstructions and correlated with CT component. Tracer uptake of ganglia was determined by measuring SUVmax and SUVmean values. Anatomical position of the ganglia in relation to adjacent vertebral bodies were noted. Statistical analyses were conducted using two-way repeated measures ANOVA and descriptive statistics. RESULTS: The highest [18F]PSMA-1007 uptake was found in coeliac ganglia followed by cervical and sacral ganglia. The SUVmax in coeliac ganglia was 3.13 ± 0.85 (follow-up scan 3.11 ± 0.93), in cervical ganglia 2.73 ± 0.69 (follow-up scan 2.67 ± 0.74), and in sacral ganglia 1.67 ± 0.50 (follow-up scan 1.64 ± 0.52). The SUVmean in coeliac ganglia was 2.28 ± 0.64 (follow-up scan 2.28 ± 0.66), in cervical ganglia 1.62 ± 0.43 (follow-up scan 1.61 ± 0.43) and in sacral ganglia 1.15 ± 0.33 (follow-up scan 1.12 ± 0.34). In a given ganglion station, there was no statistically significant difference of SUVmax or SUVmean values between baseline and follow-up scans. CONCLUSIONS: The first systematically described physiological [18F]PSMA-1007 uptake in ganglia of the sympathetic trunk showed a low variability of SUVmax or SUVmean and a good intra-individual reproducibility of [18F]PSMA-1007 uptake in follow-up scans. These findings might improve and guide the differentiation of ganglia from possible malignant lesions.
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Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Neoplasias da Próstata , Masculino , Humanos , Pessoa de Meia-Idade , Idoso , Estudos Retrospectivos , Reprodutibilidade dos Testes , Radioisótopos de Gálio , Neoplasias da Próstata/patologia , Gânglios/patologia , Ácido EdéticoRESUMO
PURPOSE: This study investigates preoperative lymph node metastases (LNM) detection accuracy by MRI and CT in patients with prostate cancer (PCA). METHODS: All patients with preoperative MRI, CT or both and subsequent radical prostatectomy (RPE) and lymphadenectomy (LA) were included in this retrospective cohort study. Prostate specific antigen (PSA), PI-RADS, ISUP grade group, clinical and pathological tumor (T) stage was compared between negative and positive nodal (N) stage. LNM were assessed with size and localization and weather they were preoperatively detected or not. In patients with preoperative CT and MRI, the results were compared intermodally. The reference standard was the histopathological results after RPE and LA. RESULTS: A total of 228 patients were analysed including 24 patients with confirmed LNM (N1; 11%). PSA (median 9.7 vs. 14 ng/ml), PI-RADS (median 4 vs. 5), ISUP (median 2 vs. 4), and cT/pT-stage (median T2 vs. T3) was significantly higher in patients with LNM. No LNM were found in patients with ISUP-1-PCA. MRI was able to detect 67% of patients with LNM. Lymph node metastases presented on MRI predominantly small, round-shaped, located parailiacally with a minimum SAD of 4 mm (vs. CT SAD of 8 mm). In comparison, MRI was superior to CT in the detection of LNM (sensitivity 81% vs. 33%; specificity 99% vs. 97). CONCLUSION: LNM were very rare in patients with PSA < 10 ng/ml, PI-RADS ≤ 4, and ≤ cT2. MRI could detect LNM up to 4 mm with a moderate sensitivity and high specificity. Thus, MRI might optimise the preoperative diagnostic and therapy planning of patients with PCA, whereas CT was clearly limited for N-stage assessment.
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Imageamento por Ressonância Magnética Multiparamétrica , Neoplasias da Próstata , Humanos , Metástase Linfática/diagnóstico por imagem , Imageamento por Ressonância Magnética , Masculino , Prostatectomia , Neoplasias da Próstata/diagnóstico por imagem , Neoplasias da Próstata/cirurgia , Estudos Retrospectivos , Tomografia Computadorizada por Raios XRESUMO
Here, using a quantitative in vivo assay, we map three regions in the carboxy terminus of conventional kinesin that are involved in cargo association, folding and regulation, respectively. Using C-terminal and internal deletions, point mutations, localization studies, and an engineered 'minimal' kinesin, we identify five heptads of a coiled-coil domain in the kinesin tail that are necessary and sufficient for cargo association. Mutational analysis and in vitro ATPase assays highlight a conserved motif in the globular tail that is involved in regulation of the motor domain; a region preceding this motif participates in folding. Although these sites are spatially and functionally distinct, they probably cooperate during activation of the motor for cargo transport.
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Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Cinesinas/química , Cinesinas/metabolismo , Neurospora/enzimologia , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Sítios de Ligação , Sequência Conservada/genética , Imunofluorescência , Proteínas Fúngicas/genética , Teste de Complementação Genética , Cinesinas/genética , Cinética , Proteínas Motores Moleculares/química , Proteínas Motores Moleculares/genética , Proteínas Motores Moleculares/metabolismo , Dados de Sequência Molecular , Mutação , Neurospora/citologia , Neurospora/metabolismo , Fenótipo , Dobramento de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por SubstratoRESUMO
OBJECTIVES: To compare prostate specific membrane antigen (PSMA) Positron Emission Tomography/Computed Tomography (PET/CT) and computed tomography (CT) alone for the detection of biochemical recurrence of prostate cancer (PCa) and effect on treatment. METHODS: This retrospective study included 59 patients with recently recorded biochemical recurrence of PCa (mean PSA 1.96 ± 1.64 ng/mL) after radical prostatectomy. Patients received PET/CT with either 68Ga-PSMA-11 (n = 36) or 18F-PSMA-1007 (n = 23). PET/CT and CT images were evaluated separately in regard to PCa lesion count, type, and localisation by two physicians. Histopathology, follow-up imaging and PSA levels after salvage irradiation served as reference standard. A McNemar test was used to compare detection rates. Changes in therapeutic approaches based on staging differences between CT alone and PET/CT were assessed in a virtual multidisciplinary tumour board. RESULTS: There were 142 lesions in 50 of 59 patients. PSMA PET/CT detected 141 lesions (99.3 %) in 50 patients (84.7 %), while CT detected 72 lesions (50.7 %) in 29 patients (49.2 %). A significantly higher detection rate of PSMA PET/CT was observed on a lesion-based analysis (p < 0.0001) and on a patient based analysis (p < 0.0001). Herein, both 68Ga- and 18F-PSMA PET/CT performed significantly better than CT alone (p < 0.0001, respectively). In 9 patients (15.3 %) no relapse was detectable by either modality. All lesions detected by CT were also detected by PSMA PET/CT. In 38 patients PSMA PET/CT detected more lesions than CT alone, altering the treatment approach in 22 of these patients. CONCLUSION: PSMA PET/CT is superior to CT alone in detecting biochemical recurrence in PCa patients after radical prostatectomy and offered additional therapeutic options in a substantial number of patients.
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Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Neoplasias da Próstata , Humanos , Masculino , Recidiva Local de Neoplasia/diagnóstico por imagem , Antígeno Prostático Específico , Prostatectomia , Neoplasias da Próstata/diagnóstico por imagem , Neoplasias da Próstata/cirurgia , Recidiva , Estudos Retrospectivos , Tomografia Computadorizada por Raios XRESUMO
To identify novel genes that are involved in positive selection of thymocytes, we performed polymerase chain reaction (PCR)-based subtractive hybridization between selecting and nonselecting thymi. OT-1 T cell receptor (TCR) transgenic thymocytes on a recombination activating gene (RAG) null background are efficiently selected into the CD8 lineage in H-2(b) mice (RAG-2(-/-)OT-1, selecting thymi), but are not selected on a transporter associated with antigen processing (TAP) null background (RAG-2(-/-)TAP-1(-/-)OT-1, nonselecting thymi). We report here our studies of one gene, ITM2A, whose expression is dramatically higher in T cells in the selecting thymus. The expression pattern of ITM2A in thymocyte subsets correlates with upregulation during positive selection. In addition, ITM2A expression is higher in the thymus than in either the spleen or lymph nodes, but can be upregulated in peripheral T cells upon activation. ITM2A expression was also induced in RAG-2(-/-) thymocytes in vivo upon CD3 cross-linking. We demonstrate that ITM2A is a type II membrane glycoprotein that exists as two species with apparent M(r) of 45 and 43 kD and appears to localize primarily to large cytoplasmic vesicles and the Golgi apparatus, but is also expressed on the cell surface. Expression on the surface of EL4 cells increases with activation by phorbol myristate acetate (PMA) and ionomycin. Finally, overexpression of ITM2A under control of the lck proximal promoter in mice results in partial downregulation of CD8 in CD4(+)CD8(+) double positive (DP) thymocytes, and a corresponding increase in the number of CD4(+)CD8(lo) thymocytes. Possible roles for this novel activation marker in thymocyte development are discussed.
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Antígenos CD4/metabolismo , Antígenos CD8/metabolismo , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Sequência de Bases , Diferenciação Celular , Primers do DNA/genética , Proteínas de Ligação a DNA/genética , Regulação para Baixo , Feminino , Ativação Linfocitária , Masculino , Proteínas de Membrana/química , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Subpopulações de Linfócitos T/citologiaRESUMO
PURPOSE: The aim of this study was to evaluate the detection rate of [68Ga]prostate-specific membrane antigen ([68Ga]PSMA-11) positron emission tomography (PET)/magnetic resonance imaging (MRI) and to compare it with [68Ga]PSMA-11 PET/X-ray computed tomography (CT) in patients with recurrent prostate cancer (PC) after radical prostatectomy. PROCEDURES: A total of 93 patients with biochemically recurrent prostate cancer underwent [68Ga]PSMA-11 PET/CT and subsequently a whole-body integrated PET/MRI examination. Board certified nuclear medicine physicians and radiologists evaluated PET/CT and PET/MRI datasets regarding identification of tumor lesions ((i) lymph nodes, (ii) bone lesions, (iii) local recurrence, and (iv) parenchymal lesions) based on maximum [68Ga]PSMA-11 uptake as well as morphological changes. Quality of PET images for both PET/CT and PET/MRI were rated using a 5-point scoring system by evaluating lesion homogeneity, contrast, contour, and delineation. Wilcoxon signed-rank tests were used to determine statistical differences. RESULTS: PC relapse was detected in 62/93 patients. PET/MRI detected 148 out of 150 lesions described in PET/CT. In addition, PET/MRI detected 11 lesions not detected in PET/CT (5 lymph nodes, 6 local recurrences). The exact McNemar statistical test (one-sided) showed significant difference between PET/CT and PET/MRI for diagnosis of local recurrence (p value = 0.031). Diagnostic confidence for (iii) was higher in PET/MRI compared with PET/CT (PET/CT = 1.1; PET/MRI = 4.9). Diagnostic confidence for (i) (PET/CT = 4.9; PET/MRI = 4.6), (ii) (PET/CT = 4.9; PET/MRI = 4.6), and (iv) (PET/CT = 4.6; PET/MRI = 4.8) was equivalent between PET/MRI and PET/CT. CONCLUSIONS: Integrated [68Ga]PSMA-11 PET/MRI provides a similarly high diagnostic performance for localization of recurrent PC as PET/CT. For the detection of local recurrences [68Ga]PSMA-11 PET/MRI is superior compared with [68Ga]PSMA-11 PET/CT.
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Antígenos de Superfície/metabolismo , Ácido Edético/análogos & derivados , Glutamato Carboxipeptidase II/metabolismo , Imageamento por Ressonância Magnética/métodos , Oligopeptídeos/farmacocinética , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada/métodos , Tomografia por Emissão de Pósitrons/métodos , Neoplasias da Próstata/patologia , Imagem Corporal Total/métodos , Idoso , Idoso de 80 Anos ou mais , Ácido Edético/química , Ácido Edético/farmacocinética , Isótopos de Gálio , Radioisótopos de Gálio , Humanos , Masculino , Pessoa de Meia-Idade , Imagem Multimodal/métodos , Recidiva Local de Neoplasia/diagnóstico por imagem , Recidiva Local de Neoplasia/metabolismo , Recidiva Local de Neoplasia/patologia , Recidiva Local de Neoplasia/cirurgia , Oligopeptídeos/química , Prostatectomia/métodos , Neoplasias da Próstata/diagnóstico por imagem , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/cirurgia , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/farmacocinética , Padrões de Referência , Distribuição TecidualRESUMO
Rhyolitic volcanic rock in the northern Black Hills has a potassium-argon isotopic age of 10.5 +/- 1.5 million years. This is considerably younger than any previously reported igneous activity in this or adjacent areas and indicates that the renewed uplift of the Black Hills, which occurred after the Oligocene epoch, was also accompanied by some volcanism.
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The yeast retroviruslike element Ty3 inserts at the transcription initiation sites of genes transcribed by RNA polymerase III (Pol III). An in vitro integration assay was developed with the use of Ty3 viruslike particles and a modified SUP2 tyrosine transfer RNA (tRNA(Tyr)) gene target. Integration was position-specific and required Ty3 integrase, Pol III transcription factor (TF) IIIB-, TFIIIC-, and Pol III-containing fractions showed that TFIIIB and TFIIIC, together, were sufficient for position-specific Ty3 integration, but not for transcription. This report demonstrates that in vitro integration of a retroelement can be targeted by cellular proteins.
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RNA Polimerase III/genética , Recombinação Genética , Retroelementos , Saccharomyces cerevisiae/genética , Fatores de Transcrição TFIII , Fatores de Transcrição/metabolismo , Integração Viral , DNA Nucleotidiltransferases/metabolismo , Integrases , Modelos Genéticos , Mutagênese Sítio-Dirigida , RNA de Transferência de Tirosina/genética , Fator de Transcrição TFIIIB , Transcrição GênicaRESUMO
RAD52 and RAD9 are required for the repair of double-strand breaks (DSBs) induced by physical and chemical DNA-damaging agents in Saccharomyces cerevisiae. Analysis of EcoRI endonuclease expression in vivo revealed that, in contrast to DSBs containing damaged or modified termini, chromosomal DSBs retaining complementary ends could be repaired in rad52 mutants and in G1-phase Rad+ cells. Continuous EcoRI-induced scission of chromosomal DNA blocked the growth of rad52 mutants, with most cells arrested in G2 phase. Surprisingly, rad52 mutants were not more sensitive to EcoRI-induced cell killing than wild-type strains. In contrast, endonuclease expression was lethal in cells deficient in Ku-mediated end joining. Checkpoint-defective rad9 mutants did not arrest cell cycling and lost viability rapidly when EcoRI was expressed. Synthesis of the endonuclease produced extensive breakage of nuclear DNA and stimulated interchromosomal recombination. These results and those of additional experiments indicate that cohesive ended DSBs in chromosomal DNA can be accurately repaired by RAD52-mediated recombination and by recombination-independent complementary end joining in yeast cells.
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Antígenos Nucleares , Proteínas de Ciclo Celular , DNA Helicases , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Desoxirribonuclease EcoRI/metabolismo , Proteínas Fúngicas/metabolismo , Recombinação Genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Reparo do DNA , Autoantígeno Ku , Proteínas Nucleares/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA , Saccharomyces cerevisiae/metabolismoRESUMO
We have identified a novel LIM gene encoding the thymus LIM protein (TLP), expressed specifically in the thymus in a subset of cortical epithelial cells. TLP was identified as a gene product which is upregulated in a thymus in which selection of T cells is occurring (Rag(-/-) OT-1) compared to its expression in a thymus in which selection is blocked at the CD4+ CD8+ stage of T-cell development (Rag(-/-) Tap(-/-) OT-1). TLP has an apparent molecular mass of 23 kDa and exists as two isomers (TLP-A and TLP-B), which are generated by alternative splicing of the message. The sequences of TLP-A and TLP-B are identical except for the C-terminal 19 or 20 amino acids. Based on protein sequence alignment, TLP is most closely related to the cysteine-rich proteins, a subclass of the family of LIM-only proteins. In both medullary and cortical thymic epithelial cell lines transduced with TLP, the protein localizes to the cytoplasm but does not appear to be strongly associated with actin. In immunohistochemical studies, TLP seems to be localized in a subset of epithelial cells in the cortex and is most abundant near the corticomedullary junction. We generated mice with a targeted disruption of the Tlp locus. In the absence of TLP, thymocyte development and thymus architecture appear to be normal but thymocyte cellularity is reduced by approximately 30%, with a proportional reduction in each subpopulation.
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Proteínas de Homeodomínio/química , Proteínas Quinases/biossíntese , Proteínas Quinases/química , Timo/metabolismo , Actinas/metabolismo , Sequência de Aminoácidos , Animais , Northern Blotting , Antígenos CD4/biossíntese , Antígenos CD8/biossíntese , Núcleo Celular/metabolismo , Clonagem Molecular , Citoplasma/metabolismo , DNA/metabolismo , DNA Complementar/metabolismo , Citometria de Fluxo , Glutationa Transferase/metabolismo , Proteínas de Homeodomínio/biossíntese , Immunoblotting , Imuno-Histoquímica , Proteínas com Domínio LIM , Quinases Lim , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Modelos Genéticos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , RNA/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Retroviridae/metabolismo , Homologia de Sequência de Aminoácidos , Timo/citologia , Regulação para CimaRESUMO
We have performed a systematic structure-function analysis of Saccharomyces cerevisiae TAF25, an evolutionarily conserved, single-copy essential gene which encodes the 206-amino-acid TAF25p protein. TAF25p is an integral subunit of both the 15-subunit general transcription factor TFIID and the multisubunit, chromatin-acetylating transcriptional coactivator SAGA. We used hydroxylamine mutagenesis, targeted deletion, alanine-scanning mutagenesis, high-copy suppression methods, and two-hybrid screening to dissect TAF25. Temperature-sensitive mutant strains generated were used for coimmunoprecipitation and transcription analyses to define the in vivo functions of TAF25p. The results of these analyses show that TAF25p is comprised of multiple mutable elements which contribute importantly to RNA polymerase II-mediated mRNA gene transcription.
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Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/fisiologia , Proteínas Fúngicas/química , Proteínas Fúngicas/fisiologia , Regulação Fúngica da Expressão Gênica , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Fatores Associados à Proteína de Ligação a TATA , Fatores de Transcrição TFII/química , Sequência de Aminoácidos , Sequência Conservada , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Genes Fúngicos , Substâncias Macromoleculares , Dados de Sequência Molecular , Mutação , Subunidades Proteicas , RNA Polimerase II/fisiologia , RNA Fúngico/biossíntese , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Transativadores/química , Transativadores/metabolismo , Fator de Transcrição TFIID , Técnicas do Sistema de Duplo-HíbridoRESUMO
We describe a novel sequencing approach that combines non-gel-based signature sequencing with in vitro cloning of millions of templates on separate 5 microm diameter microbeads. After constructing a microbead library of DNA templates by in vitro cloning, we assembled a planar array of a million template-containing microbeads in a flow cell at a density greater than 3x10(6) microbeads/cm2. Sequences of the free ends of the cloned templates on each microbead were then simultaneously analyzed using a fluorescence-based signature sequencing method that does not require DNA fragment separation. Signature sequences of 16-20 bases were obtained by repeated cycles of enzymatic cleavage with a type IIs restriction endonuclease, adaptor ligation, and sequence interrogation by encoded hybridization probes. The approach was validated by sequencing over 269,000 signatures from two cDNA libraries constructed from a fully sequenced strain of Saccharomyces cerevisiae, and by measuring gene expression levels in the human cell line THP-1. The approach provides an unprecedented depth of analysis permitting application of powerful statistical techniques for discovery of functional relationships among genes, whether known or unknown beforehand, or whether expressed at high or very low levels.
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Hibridização de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , Análise de Sequência de DNA/métodos , Linhagem Celular , Clonagem Molecular , DNA Complementar/metabolismo , Etiquetas de Sequências Expressas , Biblioteca Gênica , Técnicas Genéticas , Humanos , Microesferas , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/genética , Análise de Sequência de DNA/instrumentaçãoRESUMO
Here we show that emb-30 is required for metaphase-to-anaphase transitions during meiosis and mitosis in Caenorhabditis elegans. Germline-specific emb-30 mutant alleles block the meiotic divisions. Mutant oocytes, fertilized by wild-type sperm, set up a meiotic spindle but do not progress to anaphase I. As a result, polar bodies are not produced, pronuclei fail to form, and cytokinesis does not occur. Severe-reduction-of-function emb-30 alleles (class I alleles) result in zygotic sterility and lead to germline and somatic defects that are consistent with an essential role in promoting the metaphase-to-anaphase transition during mitosis. Analysis of the vulval cell lineages in these emb-30(class I) mutant animals suggests that mitosis is lengthened and eventually arrested when maternally contributed emb-30 becomes limiting. By further reducing maternal emb-30 function contributed to class I mutant animals, we show that emb-30 is required for the metaphase-to-anaphase transition in many, if not all, cells. Metaphase arrest in emb-30 mutants is not due to activation of the spindle assembly checkpoint but rather reflects an essential emb-30 requirement for M-phase progression. A reduction in emb-30 activity can suppress the lethality and sterility caused by a null mutation in mdf-1, a component of the spindle assembly checkpoint machinery. This result suggests that delaying anaphase onset can bypass the spindle checkpoint requirement for normal development. Positional cloning established that emb-30 encodes the likely C. elegans orthologue of APC4/Lid1, a component of the anaphase-promoting complex/cyclosome, required for the metaphase-to-anaphase transition. Thus, the anaphase-promoting complex/cyclosome is likely to be required for all metaphase-to-anaphase transitions in a multicellular organism.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans/citologia , Proteínas de Ciclo Celular/metabolismo , Ligases/genética , Proteínas de Saccharomyces cerevisiae , Complexos Ubiquitina-Proteína Ligase , Alelos , Sequência de Aminoácidos , Anáfase/fisiologia , Ciclossomo-Complexo Promotor de Anáfase , Animais , Subunidade Apc4 do Ciclossomo-Complexo Promotor de Anáfase , Caenorhabditis elegans/metabolismo , Proteínas Cdc20 , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Metáfase/fisiologia , Dados de Sequência Molecular , Mutação , Oócitos/fisiologia , Alinhamento de Sequência , Ubiquitina-Proteína LigasesRESUMO
Spontaneous renal rupture is a rare but nevertheless life-threatening complication in cases of benign and malignant space-occupying lesions in the kidneys. A precise preoperative differentiation often turns out to be difficult because of the formation of a hematoma. We report on a 50-year-old female patient with a retroperitoneal hematoma due to a spontaneous renal rupture for which an angiomyolipoma could only by diagnosed after the nephrectomy with histological work-up. In search of the cause, sonography, abdominal computed tomography, and digital subtraction angiography were conducted, which could provide findings suggestive of a space-occupying lesion, but because of the distinct hemorrhagic infarction could not determine whether it was benign or malignant. As shown in this case, when the radiological result is ambiguous, renal exposure is indicated from both a diagnostic and therapeutic standpoint.
Assuntos
Angiomiolipoma/diagnóstico , Hemorragia/diagnóstico , Nefropatias/diagnóstico , Neoplasias Renais/diagnóstico , Abdome Agudo/etiologia , Angiografia , Angiomiolipoma/patologia , Angiomiolipoma/cirurgia , Diagnóstico Diferencial , Feminino , Hemorragia/patologia , Hemorragia/cirurgia , Humanos , Rim/irrigação sanguínea , Rim/patologia , Nefropatias/patologia , Nefropatias/cirurgia , Neoplasias Renais/patologia , Neoplasias Renais/cirurgia , Pessoa de Meia-Idade , Necrose , Neovascularização Patológica/diagnóstico , Neovascularização Patológica/patologia , Neovascularização Patológica/cirurgia , Nefrectomia , Espaço Retroperitoneal , Ruptura Espontânea , Tomografia Computadorizada por Raios XRESUMO
The DNA polymerases delta and epsilon are the major replicative polymerases in the yeast Saccharomyces cerevisiae that possess 3' --> 5' exonuclease proofreading activity. Many errors arising during replication are corrected by these exonuclease activities. We have investigated the contributions of regions of Polepsilon other than the proofreading motifs to replication accuracy. An allele, pol2-C1089Y, was identified in a screen of Polepsilon mutants that in combination with an exonuclease I (exo1) mutation could cause a synergistic increase in mutations within homonucleotide runs. In contrast to other polymerase mutators, this allele specifically results in insertion frameshifts. When pol2-C1089Y was combined with deletions of EXO1 or RAD27 (homologue of human FEN1), mutation rates were increased for +1 frameshifts while there was almost no effect on -1 frameshifts. On the basis of genetic analysis, the pol2-C1089Y mutation did not cause a defect in proofreading. In combination with a deletion of the mismatch repair gene MSH2, the +1 frameshift mutation rate for a short homonucleotide run was increased nearly 100-fold whereas the -1 frameshift rate was unchanged. This suggests that the Pol2-C1089Y protein makes +1 frameshift errors during replication of homonucleotide runs and that these errors can be corrected by either mismatch repair (MMR) or proofreading (in short runs). This is the first report of a +1-specific mutator for homonucleotide runs in vivo. The pol2-C1089Y mutation defines a functionally important residue in Polepsilon.