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Cytoplasmic Fraction (cytoplasmic + fraction)
Selected AbstractsSequential activation of Notch family receptors during mouse spermatogenesisDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 1 2003Shintaro Mori The expression pattern of Notch family receptors during mouse spermatogenesis was examined by immunohistochemistry. The entire cytoplasm of spermatogonia, spermatocytes and spermatids showed staining with antibodies against extracellular domains of Notch1, 2 and 4. In contrast, the nuclei of spermatogonia showed staining with an antibody against the intracellular domain of Notch3, and the nuclei of spermatocytes and spermatids showed staining with antibodies against the intracellular domains of Notch1 and 4. During regeneration of spermatogonia in busulfan-treated mice, the nuclei of all proliferating cells showed staining for the intracellular domain of Notch3. Western blot analysis showed that the molecular weights of the intracellular domains of Notch1 and 3 localizing in the nuclear fraction were smaller than those in the cytoplasmic fraction. This was consistent with the theory that the intracellular domain of Notch was cleaved in the cytoplasm and translocated to the nucleus. These results suggest that different Notch signals are sequentially activated during mouse spermatogenesis and control the proliferation and differentiation of spermatogenic stem cells. [source] Differential proteomic profiling to study the mechanism of cardiac pharmacological preconditioning by resveratrolJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 4 2006Karel Bezstarosti Abstract Recent studies demonstrated that resveratrol, a grape-derived polyphenolic phytoalexin, provides pharmacological preconditioning of the heart through a NO-dependent mechanism. To further explore the molecular mechanisms involved in resveratrol-mediated cardioprotection, we monitored the effects of resveratrol treatment after ischemia-reperfusion on the protein profile by implementation of proteomic analysis. Two groups of rats were studied; one group of animals was fed resveratrol for 7 days, while the other group was given vehicle only. The rats were sacrificed for the isolated working heart preparation and for isolation of cytoplasmic fraction from left ventricle homogenates to carry out the proteomic as well as immunoblot at baseline and at the end of 30 min ischemia/2-h perfusion. The results demonstrate significant cardiopro-tection with resveratrol evidenced by improved ventricular recovery and reduced infarct size and cardiomyocyte apopto-sis. The left ventricular cytoplasmic fractions were separated by two-dimensional electrophoresis (2-DE). Differentially regulated proteins were detected with quantitative computer analysis of the Coomassie blue stained 2-DE images and identified by MALDI-TOF (MS) and nanoLC-ESI-Q-TOF mass spectrometry (MS/MS). Five redox-regulated and precondi-tioning-related proteins were identified that were all upregulated by resveratrol: MAPKK, two different aB-crystallin species, HSP 27 and PE binding protein. Another HSP27 species and aldose reductase were downregulated and peroxire-doxin-2 remained constant. The results of the immunoblot analysis of phosphorylated MAPKK, -HSP27 and -aB-crys-tallin and PE binding protein were consistent with the proteomic findings, but not with peroxiredoxin-2. The proteomic analysis showed also downregulation of some proteins in the mitochondrial respiratory chain and matrix and the myofila-ment regulating protein MLC kinase-2. The results of the present study demonstrate that proteomic profiling enables the identification of resveratrol induced preconditioning-associated proteins which reflects not only changes in their expression level but also isoforms, post-translational modifications and regulating binding or activating partner proteins. [source] Isoform- and subcellular fraction-specific differences in hippocampal 14-3-3 levels following experimentally evoked seizures and in human temporal lobe epilepsyJOURNAL OF NEUROCHEMISTRY, Issue 2 2006Clara K. Schindler Abstract 14-3-3 proteins are a family of signaling molecules involved in diverse cellular functions, which can mediate anti-apoptotic effects. Seizure-induced neuronal death may involve programmed (apoptotic) cell death pathways and is associated with a decline in brain 14-3-3 levels. Presently, we investigated the subcellular localization and effects of seizures on isoforms of 14-3-3 in rat hippocampus, and contrasted these to findings in human temporal lobe epilepsy (TLE). All brain isoforms of 14-3-3 were detected in the cytoplasmic compartment of rat hippocampus, while 14-3-3, and -, were also present in mitochondrial and microsome-enriched fractions. Focally evoked seizures in rats significantly reduced 14-3-3, levels within the microsome-enriched compartment at 4 h, with similar responses for 14-3-3,, while cytoplasm-localized 14-3-3,, -, and -, remained unchanged. Analysis of human autopsy control hippocampus revealed similar 14-3-3 isoform expression profiles. In TLE samples, the microsome-enriched fraction also showed differences, but here 14-3-3, and -, levels were higher than controls. TLE sample 14-3-3 isoform abundance within the cytoplasmic fraction was not different to controls. This study defines the subcellular localization of 14-3-3 isoforms in rat and human hippocampus and identifies the microsome-enriched fraction as the main site of altered 14-3-3 levels in response to acute prolonged and chronic recurrent seizures. [source] Activated Stat3 expression in gestational trophoblastic disease: correlation with clinicopathological parameters and apoptotic indicesHISTOPATHOLOGY, Issue 2 2008H Y Chan Aims:, To assess the expression profile of the activated form of signal transducer and activator of transcription (Stat)3 in gestational trophoblastic disease (GTD) and correlate the findings with clinicopathological parameters. Methods and results:, By immunohistochemistry, both cytoplasmic and nuclear expression of p-Stat3-Ser727 was demonstrated in 88 trophoblastic tissues, including placentas and GTD. Nuclear immunoreactivity of p-Stat3-Ser727 was significantly higher in hydatidiform mole (HM) (P < 0.001) and choriocarcinoma (P = 0.009) when compared with normal placentas. Placental site trophoblastic tumours (PSTT) and epithelioid trophoblastic tumours (ETT) also demonstrated higher nuclear p-Stat3-Ser727 expression than their normal trophoblast counterparts. Higher p-Stat3-Ser727 expression was confirmed in choriocarcinoma cell lines, JEG-3 and JAR, than in a normal trophoblast cell line, with both nuclear and cytoplasmic fractions demonstrated by immunoblotting. Spontaneously regressed HM showed significantly increased nuclear and cytoplasmic p-Stat3-Ser727 immunoreactivity over those that developed gestational trophoblastic neoplasia (GTN) (P = 0.013, P = 0.039). There was a significant positive and inverse correlation between nuclear p-Stat3-Ser727 immunoreactivity and apoptotic indices [terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end labelling and M30 CytoDeath antibody] (P = 0.001, P < 0.001, Spearman's , test) and Bcl-2 expression (P = 0.034), respectively. Conclusions:, p-Stat3-Ser727 plays a role in the pathogenesis of GTD, probably through the regulation of apoptosis. p-Stat3-Ser727 immunoreactivity is a potential marker in predicting GTN in HM. [source] Intracellular redistribution and modification of proteins of the Mre11/Rad50/Nbs1 DNA repair complex following irradiation and heat-shockJOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2004Joshua D. Seno Mre11, Rad50, and Nbs1form a tight complex which is homogeneously distributed throughout the nuclei of mammalian cells. However, after irradiation, the Mre11/Rad50/Nbs1 (M/R/N) complex rapidly migrates to sites of double strand breaks (DSBs), forming foci which remain until DSB repair is complete. Mre11 and Rad50 play direct roles in DSB repair, while Nbs1 appears to be involved in damage signaling. Hyperthermia sensitizes mammalian cells to ionizing radiation. Radiosensitization by heat shock is believed to be mediated by an inhibition of DSB repair. While the mechanism of inhibition of repair by heat shock remains to be elucidated, recent reports suggest that the M/R/N complex may be a target for inhibition of DSB repair and radiosensitization by heat. We now demonstrate that when human U-1 melanoma cells are heated at 42.5 or 45.5°C, Mre11, Rad50, and Nbs1 are rapidly translocated from the nucleus to the cytoplasm. Interestingly, when cells were exposed to ionizing radiation (12 Gy of X-rays) prior to heat treatment, the extent and kinetics of translocation were increased when nuclear and cytoplasmic fractions of protein were analyzed immediately after treatment. The kinetics of the translocation and subsequent relocalization back into the nucleus when cells were incubated at 37°C from 30 min to 7 h following treatment were different for each protein, which suggests that the proteins redistribute independently. However, a significant fraction of the translocated proteins exist as a triple complex in the cytoplasm. Treatment with leptomycin B (LMB) inhibits the translocation of Mre11, Rad50, and Nbs1 to the cytoplasm, leading us to speculate that the relocalization of the proteins to the cytoplasm occurs via CRM1-mediated nuclear export. In addition, while Nbs1 is rapidly phosphorylated in the nuclei of irradiated cells and is critical for a normal DNA damage response, we have found that Nbs1 is rapidly phosphorylated in the cytoplasm, but not in the nucleus, of heated irradiated cells. The phosphorylation of cytoplasmic Nbs1, which cannot be inhibited by wortmannin, appears to be a unique post-translational modification in heated, irradiated cells, and coupled with our novel observations that Mre11, Rad50, and Nbs1 translocate to the cytoplasm, lend further support for a role of the M/R/N complex in thermal radiosensitization and inhibition of DSB repair. J. Cell. Physiol. 199: 157,170, 2004© 2004 Wiley-Liss, Inc. [source] | |||||||||||||