scholarly journals A novel tau transcript in cultured human neuroblastoma cells expressing nuclear tau.

1993 ◽  
Vol 121 (2) ◽  
pp. 257-267 ◽  
Author(s):  
Y Wang ◽  
P A Loomis ◽  
R P Zinkowski ◽  
L I Binder
Keyword(s):  
Western Blot ◽  
Cell Line ◽  
Indirect Immunofluorescence ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Neuroblastoma Cells ◽  
Human Neuroblastoma Cell ◽  
Human Neuroblastoma Cells ◽  
Human Neuroblastoma

We previously reported the presence of the microtubule-associated protein, tau in the nuclei of primate cells in culture. The present study confirms the existence of nuclear tau in two human neuroblastoma cells lines by indirect immunofluorescence and Western blot using mAbs to tau. Northern blot analysis of poly A+ mRNA detects a novel 2-kb tau transcript coexpressed with the 6-kb message in cultured human cells and human frontal cortex. PCR and cDNA sequencing demonstrate that the 2-kb message contains the entire tau coding region. Furthermore, actinomycin D transcription inhibition experiments indicate that the 2-kb message is not derived from the 6-kb message, but instead arises from the original tau transcript. One of the human neuroblastoma cell lines examined contains both nuclear and cytoplasmic tau as assayed by both Western blot and indirect immunofluorescence. Northern blot analysis of this cell line indicates that copious amounts of the 2-kb message are present while little of the 6-kb transcript is obvious. Immunofluorescence analysis of this cell line demonstrates that the cytoplasmic tau is not localized to microtubules. Together, these results indicate that the 2-kb tau message in humans may specify tau for non-microtubule functions in both the cytoplasm and the nucleus. We hypothesize that this is accomplished via a message targeting mechanism mediated by the untranslated regions of the tau messages.

scholarly journals Tissue- and cell-specific expression of Ins(1,4,5)P3 3-kinase isoenzymes

1995 ◽  
Vol 306 (2) ◽  
pp. 429-435 ◽  
Author(s):  
V Vanweyenberg ◽  
D Communi ◽  
C S D'Santos ◽  
C Erneux
Keyword(s):  
Cell Lines ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Biological Data ◽  
Human Neuroblastoma Cell ◽  
Rat Tissues ◽  
Human Neuroblastoma ◽  
Mrna Species ◽  
Kinase A

The phosphorylation of Ins(1,4,5)P3 (InsP3) to Ins(1,3,4,5)P4 (InsP4) is catalysed by InsP3 3-kinase. Molecular-biological data have shown the presence of two human isoenzymes of InsP3 3-kinase, namely InsP3 3-kinases A and B. We have isolated from a rat thymus cDNA library a 2235 bp cDNA (clone B15) encoding rat InsP3 3-kinase B. Northern-blot analysis of mRNA isolated from rat tissues (thymus, testis, brain, spleen, liver, kidney, heart, lung and intestine) revealed that a rat InsP3 3-kinase B probe hybridized to a 6 kb mRNA in lung, thymus, testis, brain and heart. In contrast, Northern-blot analysis of the same tissues probed under stringent conditions with a rat InsP3 3-kinase A probe hybridized to a 2 kb mRNA only in brain and a 1.8-2.0 kb mRNA species in testis. Northern-blot analysis of three human cell lines (HL-60, SH-SY5Y and HTB-138) probed with a human InsP3 3-kinase B probe showed the presence of a 6 kb mRNA in all cell lines, except in the human neuroblastoma cell line (SH-SY5Y), where two mRNA species of 5.7 and 6 kb were detected. Using the same blot, no hybridization signal could be seen with a human InsP3 3-kinase A probe. Altogether, our data are consistent with the notion that the two InsP3 3-kinase isoenzymes, A and B, are specifically expressed in different tissues and cells.

scholarly journals Regulation of c-myb expression in human neuroblastoma cells during retinoic acid-induced differentiation.

1988 ◽  
Vol 8 (4) ◽  
pp. 1677-1683 ◽  
Author(s):  
C J Thiele ◽  
P S Cohen ◽  
M A Israel
Keyword(s):  
Retinoic Acid ◽  
Fetal Development ◽  
Neuroblastoma Cell ◽  
Neuroblastoma Cells ◽  
Human Neuroblastoma Cell ◽  
Human Neuroblastoma Cells ◽  
Human Neuroblastoma ◽  
Hematopoietic Lineage ◽  
Specific Manner ◽  
Neuroblastoma Cell Lines

We detected expression of the c-myb proto-oncogene, which was initially thought to be expressed in a tissue-specific manner in cells of hematopoietic lineage, in human tissues of neuronal origin. Since the level of c-myb expression declined during fetal development, we studied the regulation of its expression in human neuroblastoma cell lines induced to differentiate by retinoic acid. The expression of c-myb declined during the maturation of neuroblastoma cells, and this change was mediated by a decrease in c-myb transcription.

scholarly journals The Branch Point Enzyme of the Mevalonate Pathway for Protein Prenylation Is Overexpressed in theob/ob Mouse and Induced by Adipogenesis

2000 ◽  
Vol 20 (6) ◽  
pp. 2158-2166 ◽  
Author(s):  
David Vicent ◽  
Eleftheria Maratos-Flier ◽  
C. Ronald Kahn
Keyword(s):  
Skeletal Muscle ◽  
Western Blot ◽  
Branch Point ◽  
Northern Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Northern Blot Analysis ◽  
Geranylgeranyl Diphosphate ◽  
Protein Prenylation ◽  
Ggpp Synthase

ABSTRACT We have recently reported that skeletal muscle of theob/ob mouse, an animal model of genetic obesity with extreme insulin resistance, exhibits alterations in the expression of multiple genes. Analysis and cloning of a full-length cDNA of one of the overexpressed mRNAs revealed a 300-amino-acid protein that could be identified as the mouse geranylgeranyl diphosphate synthase (GGPP synthase) based on its homology to proteins cloned from yeast and fungus. GGPP synthase catalyzes the synthesis of all-trans-geranylgeranyl diphosphate (GGPP), an isoprenoid used for protein isoprenylation in animal cells, and is a branch point enzyme in the mevalonic acid pathway. Three mRNAs for GGPP synthase of 4.3, 3.2, and 1.7 kb were detected in Northern blot analysis. Western blot analysis of tissue homogenates using specific antipeptide antibodies revealed a single band of 34.8 kDa. Expression level of this protein in different tissues correlated with expression of the 4.3- and 3.2-kb mRNAs. GGPP synthase mRNA expression was increased 5- to 20-fold in skeletal muscle, liver, and fat of ob/obmice by Northern blot analysis. Western blot analysis also showed a twofold overexpression of the protein in muscle and fat but not in liver, where the dominant isoform is encoded by the 1.7-kb mRNA. Differentiation of 3T3-L1 fibroblasts into adipocytes induced GGPP synthase expression more than 20-fold. Using the immunoprecipitated protein, we found that mammalian GGPP synthase synthesizes not only GGPP but also its metabolic precursor farnesyl diphosphate. Thus, the expression of GGPP synthase is regulated in multiple tissues in obesity and is induced during adipocyte differentiation. Altered regulation in the synthesis of isoprenoids for protein prenylation in obesity might be a factor determining the ability of the cells to respond to hormonal stimulation requiring both Ras-related small GTPases and trimeric G protein-coupled receptors.

Gender and transcriptional regulation of NO synthase and ET-1 in porcine aortic endothelial cells

1997 ◽  
Vol 273 (4) ◽  
pp. H1962-H1967 ◽  
Author(s):  
Xiaofang Wang ◽  
Dustan A. Barber ◽  
Debra A. Lewis ◽  
Christopher G. A. McGregor ◽  
Gary C. Sieck ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Western Blot ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Ovarian Hormones ◽  
Intact Male ◽  
Male And Female ◽  
Aortic Endothelial Cells

Experiments were designed to determine whether normal fluctuations in sex steroid hormones alter gene transcription for endothelial nitric oxide synthase (NOS) and preproendothelin-1 (prepro-ET-1). Aortic endothelial cells were removed from adult, gonadally intact male and female or ovariectomized Yorkshire pigs. Endothelial cells were prepared for Northern blot analysis, Western blot analysis or enzyme activity. Nitric oxide products (NOx) and endothelin-1 (ET-1) in plasma were measured by chemiluminescence and radioimmunoassay, respectively. Northern blot analysis identified single bands corresponding to endothelial NOS and prepro-ET-1. Quantification of the blots showed an increase in expression of mRNA for both endothelial NOS and prepro-ET-1 in ovariectomized pigs compared with gonadally intact male and female pigs. There were no differences in amount of endothelial NOS protein identified by Western blot analysis among groups. On the contrary, plasma concentrations of NOx were significantly decreased in ovariectomized pigs, and there were no differences either in the concentrations of ET-1 in the plasma or extracts from the coronary arteries. These results suggest that expression of endothelial NOS and prepro-ET-1 may be regulated at transcriptional level by ovarian hormones. In addition, the ovarian hormones may regulate production of these endothelium-derived factors at the posttranscriptional level.

1,25-Dihydroxyvitamin D3 Induces Differentiation of a Retinoic Acid–Resistant Acute Promyelocytic Leukemia Cell Line (UF-1) Associated With Expression of p21WAF1/CIP1 and p27KIP1

Blood ◽  
1999 ◽  
Vol 93 (7) ◽  
pp. 2225-2233 ◽  
Author(s):  
Akihiro Muto ◽  
Masahiro Kizaki ◽  
Kenji Yamato ◽  
Yohko Kawai ◽  
Maiko Kamata-Matsushita ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Cell Line ◽  
Acute Promyelocytic Leukemia ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Promyelocytic Leukemia ◽  
G1 Arrest ◽  
Dihydroxyvitamin D3 ◽  
1,25 Dihydroxyvitamin D3

Retinoic acid (RA) resistance is a serious problem for patients with acute promyelocytic leukemia (APL) who are receiving all-transRA. However, the mechanisms and strategies to overcome RA resistance by APL cells are still unclear. The biologic effects of RA are mediated by two distinct families of transcriptional factors: RA receptors (RARs) and retinoid X receptors (RXRs). RXRs heterodimerize with 1,25-dihydroxyvitamin D3[1,25(OH)2D3] receptor (VDR), enabling their efficient transcriptional activation. The cyclin-dependent kinase (cdk) inhibitor p21WAF1/CIP1 has a vitamin D3–responsive element (VDRE) in its promoter, and 1,25(OH)2D3 enhances the expression of p21WAF1/CIP1 and induces differentiation of selected myeloid leukemic cell lines. We have recently established a novel APL cell line (UF-1) with features of RA resistance. 1,25(OH)2D3 can induce growth inhibition and G1 arrest of UF-1 cells, resulting in differentiation of these cells toward granulocytes. This 1,25(OH)2D3-induced G1 arrest is enhanced by all-trans RA. Also, 1,25(OH)2D3 (10−10 to 10−7 mol/L) in combination with RA markedly inhibits cellular proliferation in a dose- and time-dependent manner. Associated with these findings, the levels of p21WAF1/CIP1 and p27KIP1 mRNA and protein increased in these cells. Northern blot analysis showed that p21WAF1/CIP1 and p27KIP1 mRNA and protein increased in these cells. Northern blot analysis showed that p21WAF1/CIP1 and p27KIP1 transcripts were induced after 6 hours’ exposure to 1,25(OH)2D3 and then decreased to basal levels over 48 hours. Western blot experiments showed that p21WAF1/CIP1 protein levels increased and became detectable after 12 hours of 1,25(OH)2D3treatment and induction of p27KIP1 protein was much more gradual and sustained in UF-1 cells. Interestingly, the combination of 1,25(OH)2D3 and RA markedly enhanced the levels of p27KIP1 transcript and protein as compared with levels induced by 1,25(OH)2D3 alone. In addition, exogenous p27KIP1 expression can enhance the level of CD11b antigen in myeloid leukemic cells. In contrast, RA alone can induce G1 arrest of UF-1 cells; however, it did not result in an increase of p21WAF1/CIP1 and p27KIP1transcript and protein expression in RA-resistant cells. Taken together, we conclude that 1,25(OH)2D3 induces increased expression of cdk inhibitors, which mediates a G1 arrest, and this may be associated with differentiation of RA-resistant UF-1 cells toward mature granulocytes.

scholarly journals Ionizing Radiation-Induced Extracellular Vesicle Release Promotes AKT-Associated Survival Response in SH-SY5Y Neuroblastoma Cells

Cells ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 107
Author(s):  
Flavia Tortolici ◽  
Simone Vumbaca ◽  
Bernadette Incocciati ◽  
Renu Dayal ◽  
Katia Aquilano ◽  
...  
Keyword(s):  
Cell Line ◽  
Extracellular Vesicles ◽  
Neuroblastoma Cell ◽  
Neuroblastoma Cells ◽  
Protein Composition ◽  
Human Neuroblastoma Cell ◽  
X Rays ◽  
Human Neuroblastoma ◽  
Secondary Neoplasms ◽  
Mesenchymal Transition

Radiation therapy is one of the most effective methods of tumor eradication; however, in some forms of neuroblastoma, radiation can increase the risk of secondary neoplasms, due to the ability of irradiated cells to transmit pro-survival signals to non-irradiated cells through vesicle secretion. The aims of this study were to characterize the vesicles released by the human neuroblastoma cell line SH-SY5Y following X-ray radiations and their ability to increase invasiveness in non-irradiated SH-SY5Y cells. We first purified the extracellular vesicles released by the SH-SY5Y cells following X-rays, and then determined their total amount, dimensions, membrane protein composition, and cellular uptake. We also examined the effects of these extracellular vesicles on viability, migration, and DNA damage in recipient SH-SY5Y cells. We found that exposure to X-rays increased the release of extracellular vesicles and altered their protein composition. These vesicles were readily uptaken by non-irradiated cells, inducing an increase in viability, migration, and radio-resistance. The same results were obtained in an MYCN-amplified SK-N-BE cell line. Our study demonstrates that vesicles released from irradiated neuroblastoma cells stimulate proliferation and invasiveness that correlate with the epithelial to mesenchymal transition in non-irradiated cells. Moreover, our results suggest that, at least in neuroblastomas, targeting the extracellular vesicles may represent a novel therapeutic approach to counteract the side effects associated with radiotherapy.

scholarly journals A Spore Coat Protein, CotS, of Bacillus subtilis Is Synthesized under the Regulation of ςKand GerE during Development and Is Located in the Inner Coat Layer of Spores

1998 ◽  
Vol 180 (11) ◽  
pp. 2968-2974 ◽  
Author(s):  
Hiromu Takamatsu ◽  
Yukari Chikahiro ◽  
Takeko Kodama ◽  
Hidekatsu Koide ◽  
Satoshi Kozuka ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Western Blot ◽  
Northern Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Northern Blot Analysis ◽  
Spore Coat ◽  
Consensus Sequences ◽  
Coat Layer ◽  
Spore Coat Protein

ABSTRACT The spore coat of Bacillus subtilis has a unique morphology and consists of polypeptides of different sizes, whose synthesis and assembly are precisely regulated by a cascade of transcription factors and regulatory proteins. We examined the factors that regulate cotS gene expression and CotS assembly into the coat layer of B. subtilis by Northern blot and Western blot analysis. Transcription of cotS mRNA was not detected in sporulating cells of ςK and gerE mutants by Northern blot analysis. By Western blot analysis using anti-CotS antibody, CotS was first detected in protein samples solubilized from wild-type cells at 5 h after the start of sporulation. CotS was not detected in the vegetative cells and spores of a gerEmutant or in the spores of mutants deficient in ςE, ςF, ςG, or ςK. CotS was detected in the sporangium but not in the spores of a cotEmutant. The sequence of the promoter region of cotS was similar to the consensus sequences for binding of ςK and GerE. These results demonstrate that ςK and GerE are required for cotS expression and that CotE is essential for the assembly of CotS in the coat. Immunoelectron microscopic observation using anti-CotS antibody revealed that CotS is located within the spore coat, in particular in the inner coats of dormant spores.

Prolactin and growth hormone mRNA and protein characterization in SMtTW rat pituitary tumours

1995 ◽  
Vol 15 (3) ◽  
pp. 233-243 ◽  
Author(s):  
M Delhase ◽  
F Rajas ◽  
P Verdood ◽  
C Remy ◽  
P Chevallier ◽  
...  
Keyword(s):  
Western Blot ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Biological Significance ◽  
Protein Characterization ◽  
Pituitary Tumours ◽  
Rat Pituitary ◽  
Normal Rat

ABSTRACT We have combined different techniques to analyse passages of five different rat spontaneous pituitary tumours (SMtTW) that were transplanted under the kidney capsule. These tumours were secreting prolactin (PRL), GH or both hormones. RIA, immunocytochemistry (ICC) and Western blot analysis were applied to characterize the hormone(s) stored (ICC and Western blot) and secreted (RIA). mRNA content was analysed by PCR, Northern blot analysis and in situ hybridization. The data point not only to the reliability of the techniques used at both protein and RNA levels for each tumour studied but also to the complementarity of some techniques. For example, whereas Northern blot analysis demonstrates the presence and size of hormone mRNA, in situ hybridization indicates the percentage of cells expressing a given hormone mRNA and allows the presence of one population (or more) of cells in a given tumour to be identified. Moreover, the tumours were compared with normal rat pituitary. Although the PRL and GH mRNAs were identical in size, the amount of mRNA was lower in the tumours. At the protein level, the PRL and GH variants exhibited a different pattern of expression in tumours compared with the normal rat pituitary. The biological significance of these differences is discussed.

scholarly journals Absence of c-kit receptor and absent proliferative response to stem cell factor in childhood Burkitt's lymphoma cells

Blood ◽  
1995 ◽  
Vol 86 (4) ◽  
pp. 1469-1480 ◽  
Author(s):  
J Tomeczkowski ◽  
A Beilken ◽  
D Frick ◽  
B Wieland ◽  
A Konig ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Northern Blot ◽  
Blot Analysis ◽  
Stem Cell Factor ◽  
Northern Blot Analysis ◽  
Thymidine Incorporation ◽  
Rt Pcr ◽  
Low Level

The cytokine stem cell factor (SCF) synergizes with interleukin-7 (IL- 7) to enhance the proliferation of pre-B cells. To examine the role of SCF and its receptor, c-kit, in the pathogenesis of pediatric Burkitt's lymphomas (BL), we investigated the expression of SCF and c-kit in BL cells and the mitogenic activity of SCF on BL cells. A panel of 13 BL cell lines and 7 fresh biopsy tumors was investigated. BL cells were stimulated either by Epstein-Barr virus (EBV) infection or by different reagents and cytokines, and expression of SCF and c-kit was studied on the mRNA level by Northern blot analysis and reverse-transcriptase polymerase chain reaction (RT-PCR), followed by Southern blotting. c- kit expression was also studied by fluorescence-activated cell sorting and by crosslinking of digoxigenin-labeled recombinant human SCF to the cell surface. Proliferation of BL cell lines was measured by 3H- thymidine incorporation. Low-level expression of c-kit mRNA was detected in 2 of 13 unstimulated BL cell lines and in 1 fresh BL tumor. One cell line showed upregulation of c-kit mRNA with A23187 and downregulation with phorbol myristate acetate. Neither c-kit nor SCF could be detected in any other cell line under any condition of stimulation as analyzed by Northern blot analysis, RT-PCR followed by Southern blot analysis, crosslinking, and immunofluorescence. No response to SCF was seen in 3H-thymidine incorporation assays. We conclude that most BL cells express neither SCF nor c-kit and that the low-level expression of c-kit in some BL cells most likely has no biologic significance.

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