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Isolated Nuclei (isolated + nucleus)
Selected AbstractsProliferative activity and genetic changes in adrenal cortical tumors examined by flow cytometry, fluorescence in situ hybridization and immunohistochemistryINTERNATIONAL JOURNAL OF UROLOGY, Issue 2 2005KOUSUKE TAKEHARA Abstract Background: To determine differences in biological features among different adrenal tumors, we investigated the DNA ploidy, numerical chromosomal aberration and proliferative activity in human adrenal cortical neoplasms. Methods: Our study included six adrenal cortical adenomas with Cushing syndrome, 12 adenomas with hyperaldosteronism, three non-functioning adenomas and three adrenal cortical carcinomas. Isolated nuclei from frozen samples were used for fluorescence in situ hybridization (FISH) analysis, and formalin-fixed, paraffin-embedded tissues from the same materials were analyzed using flow cytometry (FCM) for DNA ploidy. Sections from paraffin blocks were stained immunohistochemically with antibodies against Ki-67 and p53. For FISH analysis, we used an ,-centromeric enumeration probe for chromosome 17. Results: The mean Ki-67 labeling index (LI) of adrenal cortical carcinomas was markedly higher than that of adrenal cortical adenomas (209.4 vs 8.7). In functional adrenal cortical adenomas, the LI was significantly lower in adenomas with hyperaldosteronism than in those with Cushing syndrome (P = 0.004), although FCM results indicated that tetraploid patterns were more frequently observed in the former type. Tumor size was significantly smaller in adenomas with hyperaldosteronism than in those with Cushing syndrome (P = 0.004). Chromosome 17 showed disomy in all adrenal cortical adenomas, whereas chromosome 17 abnormalities were found in two of three adrenal cortical carcinomas. Only the latter two cases strongly expressed p53 protein. Conclusions: Our study characterized various biological features of benign and malignant adrenal cortical tumors. The use of a combination of markers might provide additional information to assist our understanding of the clinical behavior of an individual adrenal cortical tumor. [source] Identification of a GM1/Sodium,Calcium exchanger complex in the nuclear envelope of non-neuronal cellsJOURNAL OF NEUROCHEMISTRY, Issue 2002X. Xie Our previous studies identified a Na,Ca exchanger (NCX) that is tightly associated with GM1 ganglioside and potentiated by it in the nuclear envelope (NE) of NG108-15 cells and primary neurons. The purpose of the present study was to explore whether this is a general phenomena or limited to neurons. Non-neuronal C6 (glioma), HeLa (Epithelial carcinoma) and NCTC (connective tissue) cell lines were used. Immunocytochemical staining with anti-NCX antibody and cholera toxin B subunit revealed that NCX and GM1 coexist in the nuclei from all 3 cell lines; in relation to plasma membrane, only HeLa cells showed staining for both NCX and GM1. Purified NE and non-nuclear membrane mixture (mainly plasma membrane) from the 3 cell lines were immunoprecipitated with a mouse monoclonal anti-NCX antibody and the precipitated proteins separated on SDS,PAGE. Analysis by immunoblot, showed that NCX is tightly associated with GM1 in the NE of all 3 cell lines. In contrast, NCX and the more loosely associated GM1 from plasma membrane of HeLa cells were separated by SDS,PAGE. Isolated nuclei from C6 cells were used for 45Ca2+ uptake experiments, which provided functional evidence that this exchanger protein is strongly potentiated by GM1. In similar experiments with Jurkat cells (T lymphocyte), no NCX was found. These results suggest a possible new and widely distributed mechanism for regulation of nuclear calcium by NCX in association with GM1. Acknowledgements:, supported by NIH grant NS 33912. [source] Regulation of early response genes in pancreatic acinar cells: external calcium and nuclear calcium signalling aspectsACTA PHYSIOLOGICA, Issue 1 2009N. Fedirko Abstract Nuclear calcium signalling has been an important topic of investigation for many years and some aspects have been the subject of debate. Our data from isolated nuclei suggest that the nuclear pore complexes (NPCs) are open even after depletion of the Ca2+ store in the nuclear envelope (NE). The NE contains ryanodine receptors (RyRs) and Ins(1,4,5)P3 receptors [Ins(1,4,5)P3Rs], most likely on both sides of the NE and these can be activated separately and independently: the RyRs by either NAADP or cADPR, and the Ins(1,4,5)P3Rs by Ins(1,4,5)P3. We have also investigated the possible consequences of nuclear calcium signals: the role of Ca2+ in the regulation of immediate early genes (IEG): c-fos, c-myc and c-jun in pancreatic acinar cells. Stimulation with Ca2+ -mobilizing agonists induced significant increases in levels of expression. Cholecystokinin (CCK) (10 nm) evoked a substantial rise in the expression levels, highly dependent on external Ca2+: the IEG expression level was lowest in Ca2+ -free solution, increased at the physiological level of 1 mm [Ca2+]o and was maximal at 10 mm [Ca2+]o, i.e.: 102 ± 22% and 163 ± 15% for c-fos; c-myc ,73 ± 13% and 106 ± 24%; c-jun ,49 ± 8% and 59 ± 9% at 1 and 10 mm of extracellular Ca2+ respectively. A low CCK concentration (10 pm) induced a small increase in expression. We conclude that extracellular Ca2+ together with nuclear Ca2+ signals induced by CCK play important roles in the induction of IEG expression. [source] Changes in the activities of protein phosphatase type 1 and type 2A in sea urchin embryos during early developmentDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 4 2000Manabu Kawamoto In the eggs and embryos of sea urchins, the activity of protein phosphatase type 2A (PP2A) increased during the developmental period between fertilization and the morula stage, decreased after the prehatching blastula stage and increased again after hatching. The PP2A activity changed keeping pace with alteration to the activities of cAMP-dependent protein kinase (A kinase), Ca2+/calmodulin-dependent protein kinase (CaM kinase) and casein kinase. Probably, PP2A contributes to the quick turning off of cellular signals because of protein phosphorylation. The activity of protein phosphatase type 1 (PP1) was not detectable up to the morula stage and appreciably increased thereafter. In the isolated nucleus fraction, specific activities of PP1 and PP2A were higher than in whole embryos at all stages in early development. Exponential increase in the number of nuclei because of egg cleavage probably makes PP1 activity detectable in whole embryos after the morula stage. In isolated nuclei, the activities of PP1 and PP2A appreciably decreased after hatching, whereas the activities of A kinase, Ca2+/phospholipid-dependent protein kinase (C kinase) and CaM kinase, as well as casein kinase, became higher. In nuclei, cellular signals caused by protein phosphorylation after hatching do not seem to be turned off by these protein kinases so quickly as before hatching. The PP1 and PP2A in nuclei also seem to contribute to the elimination of signal noise. [source] Simultaneous assessment of DNA ploidy and biomarker expression in paraffin-embedded tissue sectionsHISTOPATHOLOGY, Issue 1 2010Stijn J H M Fleskens Fleskens S J H M, Takes R P, Otte-Höller I, van Doesburg L, Smeets A, Speel E-J M, Slootweg P J & van der Laak J A W M (2010) Histopathology,57, 14,26 Simultaneous assessment of DNA ploidy and biomarker expression in paraffin-embedded tissue sections Aims:, Aneuploidy is a potential biomarker for predicting progression of premalignancies. Ploidy assessment is mostly performed on nuclei isolated from tissue sections. Ploidy assessment in situ in tissue sections may be a large improvement, enabling selective sampling of nuclei, thus allowing the correlation between ploidy and histology. Existing ploidy analysis methods in sections suffer from limited sensitivity. The aim was to reliably assess ploidy in sections, combined with simultaneous assessment of other markers at the individual cell level. Methods and results:, Ploidy was measured in 22 paraffin-embedded oral premalignancies. The DNA stoichiometric Feulgen procedure was used on isolated nuclei, as well as fluoresence in situ hybridization analysis. In tissue sections, Feulgen was combined with immunohistochemistry for Ki67 proliferation marker, enabling distinction between cycling euploid and aneuploid cells. Aneuploidy was reliably detected in tissue sections (sensitivity 100%, specificity 92%). One section in which aneuploidy was detected was misclassified in isolated nuclei analysis. Sections were also successfully analysed using our model combined with DNA double strand break marker ,-H2AX in fluorescence microscopy, underlining the power of biomarker evaluation on single cells in tissue sections. Conclusions:, The analysis model proposed in this study enables the combined analysis of histology, genotypic and phenotypic information. [source] Isolated plant nuclei as mechanical and thermal sensors involved in calcium signallingTHE PLANT JOURNAL, Issue 1 2004Tou Cheu Xiong Summary Calcium signals in the nucleus elicit downstream effects that are distinct from those of cytosolic calcium signals. In the present work, we have evaluated the ability of plant nuclei to sense stimuli directly and to convert them into calcium changes. We show that individual mechanical stimulation of isolated nuclei elicits a single calcium transient at acidic pHs, whereas a series of stimulations leads to oscillations whose frequency reflects that of the stimuli. Conversely, at alkaline pHs, nuclei respond to temperature but not to stretch. The stretch- and the temperature-activated processes differ by their sensitivity to pharmacological drugs known to affect ion channel activities in animal cells. Our data demonstrate that isolated nuclei are able to gauge physical parameters of their environment. This might have a profound influence on the functioning of calcium-dependent processes known to control a large array of molecular events in the nucleus. [source] Telomere looping in P. sativum (common garden pea)THE PLANT JOURNAL, Issue 2 2003Anthony J. Cesare Summary Telomeres vary greatly in size among plants and, in most higher plants, consist of a long array of 5,-TTTAGGG-3,/3,-AAATCCC-5, (TTTAGGG) repeats. Recently, telomeric DNA in human, mouse, oxytricha, and trypanosome chromosomes have been found arranged into loops (t-loops), proposed to sequester the telomere from unwanted repair events and prevent activation of DNA damage checkpoints. We have asked whether t-loops exist in the higher order plant Pisum sativum (garden pea). DNA was isolated from the shoots and root tips of germinating seeds. Analysis of the telomeric restriction fragments showed that DNA hybridizing to a (TTTAGGG)n probe migrated as a smear centering around 25 kb, and direct sequencing verified the repeat to be (TTTAGGG)n. Total DNA in isolated nuclei was photo-cross-linked, and the telomeric restriction fragments were purified by gel filtration. Electron microscopic (EM) analysis revealed DNA molecules arranged as t-loops with a size distribution consistent with that seen by gel electrophoresis. Some molecules had loops as large as 75 kb. These results show that the arrangement of telomeric DNA into loops occurs in higher plants. [source] | |||||||||||||