NuMA Assembles Into an Extensive Filamentous Structure When Expressed in the Cell Cytoplasm

NuMA is a 236 kDa protein that participates in the organization of the mitotic spindle despite its strict localization in the nucleus during interphase. To test how cells progress through mitosis when NuMA is localized in the cytoplasm instead of the nucleus, we have deleted the nuclear localization sequence of NuMA using site-directed mutagenesis and transiently expressed this mutant protein (NuMA-DeltaNLS) in BHK-21 cells. During interphase, NuMA-DeltaNLS accumulates in the cytoplasm as a large mass approximately the same size as the cell nucleus. When cells enter mitosis, NuMA-DeltaNLS associates normally with the mitotic spindle without causing any apparent deleterious effects on the progression of mitosis. Examination of the cytoplasmic mass formed by NuMA-DeltaNLS using transmission electron microscopy (TEM) revealed an extensive network of approximately 5 nm filaments that are further organized by the presence of dynamic microtubules into a dense web of solid, approximately 23 nm cables. Using flow cytometry, we have isolated the intact filamentous mass formed by NuMA-DeltaNLS from lysates of transiently transfected cells. These isolated structures are constructed of networks of interconnected 5 nm filaments and are composed exclusively of NuMA. These data demonstrate that NuMA is capable of assembling into an extensive filamentous structure supporting the possibility that NuMA serves a structural function either in the nucleus during interphase or at the polar ends of the mitotic spindle

Phosphorylation Regulates the Assembly of Numa in a Mammalian Mitotic Extract

NuMA is a 236 kDa nuclear protein that is required for the organization of the mitotic spindle. To determine how NuMA redistributes in the cell during mitosis, we have examined the behavior of NuMA in a mammalian mitotic extract under conditions conducive to the reassembly of interphase nuclei. NuMA is a soluble protein in mitotic extracts prepared from synchronized cultured cells, but forms insoluble structures when the extract becomes non-mitotic (as judged by the inactivation of cdc2/cyclin B kinase and the disappearance of mpm-2-reactive antigens). These NuMA-containing structures are irregularly shaped particles of 1–2 microm in diameter and their assembly is specific because other nuclear components such as the lamins remain soluble in the extract under these conditions. NuMA is dephosphorylated during this assembly process, and the assembly of these NuMA-containing structures is catalyzed by protein dephosphorylation because protein kinase inhibitors enhance their formation and protein phosphatase inhibitors block their formation. Finally, immunodepletion demonstrates that NuMA is an essential structural component of these insoluble particles, and electron microscopy shows that the particles are composed of a complex interconnected network of foci. These results demonstrate that phosphorylation regulates the solubility of NuMA in a mammalian mitotic extract, and the spontaneous assembly of NuMA into extensive structures upon dephosphorylation supports the conclusion that NuMA serves a structural function

Libro: Manual de Parasitología : Rina Girard de Kaminsky. Año: 2014. Páginas 185.. Edición: 3a. Honduras

El contenido del Manual se ha agrupado en diferentes partes: Introducción al trabajo en laboratorios de parasitología de atención primaria, Microscopio y microscopía, Parásitos intestinales luminares y tisulares, Parásitos trasmitidos por vectores, Fotografías y microfotografias, Algoritmos y acceso a paginas web. Su objetivo fundamental es constituirse en un instrumento de trabajo, consulta y referencia para la ejecución de las técnicas necesarias en el diagnóstico parasitológico. Abarca desde la organización y registro de resultados hasta la utilización correcta de la aparatología necesaria, como el uso correcto del microscopio, su calibración, medición y cuidados del mismo.Asociación Parasitológica Argentin

Does needle calibre affect pain and complication rates in patients undergoing transperineal prostate biopsy? A prospective, randomized trial

Transperineal prostate biopsy is a procedure that can be used to obtain histological samples from the prostate. To improve both the quality of the biopsy core samples and prostate cancer detection, we are currently performing a prospective, randomized trial comparing prostate biopsy samples obtained using an 18 G-needle to those obtained using a 16 G needle. The aim of this preliminary study was to evaluate pain and complication rates in both groups in order to assess whether performing a prostate biopsy with a larger calibre needle is a feasible procedure. One hundred and eighty-seven patients undergoing transperineal prostate biopsy were prospectively evaluated and divided into two groups. The first group (94 patients, Group A) received a transperineal prostate biopsy using a 16 G-needle and the second group (93 patients, Group B) underwent transperineal prostate biopsy with an 18 G-needle. Anaesthesia was obtained with a single perineal injection at the prostatic apex in all subjects. A visual analogue scale (VAS) and facial expression scale (FES) were used to assess pain during multiple steps of the procedure in each group. A detailed questionnaire was used to obtain information about drug use because it could potentially influence the pain and complications that patients experienced. Two weeks after the procedure, early and late complications were evaluated. Statistical analysis was carried out using non-parametric tests. Prostate Specific Antigen (PSA) and drug use were similar at baseline between the two groups. Pain during prostate biopsy, which was measured with both the VAS and FES instruments, did not differ significantly between the 18- and 16 G-needle groups, and no significant differences were found in early or late complication rates between the groups. Transperineal prostate biopsy with a 16 G-needle is a feasible procedure in terms of pain and complication rates. Further studies with larger patient populations are required to assess whether or not this procedure can improve prostate cancer detection rates

Renal Cell Carcinoma with venous neoplastic thrombosis: A ten years review

Purpose: To review the 10-year experience of our urological unit in the surgical management of renal cell carcinoma (RCC) with neoplastic tumor thrombosis focusing on postoperative survival. Materials and Methods: We underwent a retrospective analysis of the patients treated for this pathology during the last decade 2002-2012, stratifying them by tumor thrombus level and histological subtype. Kaplan-Meyer curves were used to assess survival. Results: Overall, 67 patients underwent surgery for RCC with neoplastic tumoral thrombosis in the period under review. 60 were clear cell RCC, 4 were urothelial papillary tumors of the renal pelvis and 3 were rare histotypes, as a nefroblastoma, a spinocellular tumor of the renal pelvis and an unclassifiable renal carcinoma. Thrombus level was I in 40 cases, II in 17, III in 2 and IV in 8 patients. We report the main postoperative complications and our survival data, with mean follow up of 36 months. Tumor stage is the most important variable in predicting survival. Patients with N0M0 disease had 70% survival at 36 months, instead of 20% for those with primitive metastatic tumor. Conclusion: Our survival results fit with the main reports in literature and our surgical management was completely in keeping with international guidelines. We did not observe relevany post-operative complications, except of hemorrhagic ones that occurred in 6 patients (9% of total) and were always successfully managed. Eighteen patients (26.87% of total) underwent caval filter positioning, without evidence of complications during its positioning or removal. Life expectancy was particularly low for the cases of RCC without clear cell histotype (7 cases in our series, 10.4% of total) that always was less than one year from surgery