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 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.

Hypoxia and expression of the proapoptotic regulator BNIP3 in cervical cancer

2006 ◽  
Vol 16 (3) ◽  
pp. 1314-1320 ◽  
Author(s):  
C. Leo ◽  
L. C. Horn ◽  
M. HÖCKEL
Keyword(s):  
Cervical Cancer ◽  
Cell Lines ◽  
Cancer Cell ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Cancer Cell Lines ◽  
Cervical Cancer Cell ◽  
Clinical Samples ◽  
Cancer Tissue

Hypoxia plays a major role in the malignant progression of tumors. Here, we investigate the expression of Bcl-2/adenovirus E1B 19 kd-interacting protein 3 (BNIP3), a proapoptotic Bcl-2 family member, and its relationship to hypoxia in cervical cancer cell lines and clinical samples of cervical cancer. Cervical cancer cell lines were grown under hypoxia or normoxia, and BNIP3 mRNA expression was examined by Northern blot analysis. In 50 patients with cervical cancer, intratumoral oxygen measurement with the Eppendorf electrode and needle biopsies of the tumor were performed. The obtained tissue was subsequently analyzed by immunohistochemistry with an anti-BNIP3 antibody. Cervical cancer tissue collected upon surgery was used for Northern blot analysis of in vivo BNIP3 mRNA expression. BNIP3 mRNA is strongly induced under hypoxic conditions in all cervical cancer cell lines investigated. Furthermore, Northern blot analysis revealed that BNIP3 mRNA is expressed in cervical cancer tissue. Using immunohistochemistry, we demonstrated that BNIP3 protein is expressed in 82% of the investigated cervical cancers and that more advanced tumor stages showed significantly stronger BNIP3 expression. However, we observed no correlation between BNIP3 expression and intratumoral hypoxia. In conclusion, BNIP3 is expressed in different cervical cancer cell lines as well as in clinical samples of cervical cancer. Although BNIP3 is clearly hypoxia-inducible in vitro, our results suggest additional mechanisms of BNIP3 regulation in vivo. Our findings therefore highlight a discrepancy between in vitro models of tumor hypoxia and the complexity of human cancer.

scholarly journals Expression of histidine decarboxylase and cellular histamine-like immunoreactivity in rat embryogenesis.

1995 ◽  
Vol 43 (12) ◽  
pp. 1241-1252 ◽  
Author(s):  
M J Nissinen ◽  
K Karlstedt ◽  
E Castrén ◽  
P Panula
Keyword(s):  
Mast Cells ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Striated Muscle ◽  
Histidine Decarboxylase ◽  
Muscle Cells ◽  
Nerve Fibers ◽  
Developmental Expression ◽  
Mrna Species

In this study we investigated the developmental expression of histidine decarboxylase (HDC) mRNA and the distribution of histamine-immunoreactive (histamine-ir) cells in the rat embryonic tissues. We applied Northern blot analysis, in situ hybridization with synthetic oligonucleotide probes complementary to the rat HDC cDNA, and indirect histamine immunocytochemistry. Northern blot analysis revealed the appearance of a major (2.6 KB) HDC mRNA species in liver on embryonic Day 14. Its hybridization level peaked on Day E18, when two minor (1.6 and 3.5 KB) mRNA species were also present. During the periparturition period, a rapid decrease in HDC RNA was apparent, as the 2.6 KB mRNA species was expressed at a low level on postnatal Day P1. The embryonic liver expressed HDC on days E14-E20. On days E18 and E20, the periosteum and the epiphyseal growth plates of the endochondrally ossificating bones, and some striated muscle cells, showed hybridization signal for HDC. Histamine immunoreactivity was detected in many epithelial and neuronal cell types during embryogenesis. An intense histamine immunoreaction appeared first in essentially all cells of the liver parenchyma on day E12. This parenchymal histamine immunoreactivity disappeared by birth, after which this immunofluorescence in liver was restricted to a few scattered mast cells until adulthood. Some neurons in the peripheral sensory, sympathetic and cranial nerve ganglia were histamine-immunoreactive from day E16 to birth. In addition, many immunoreactive nerve fibers were detected in the gastrointestinal muscularis externa, mesentery, salivary glands, kidney, lung, and muscle tissue. We conclude that during rat embryogenesis histamine is produced and stored transiently by cells in liver, developing bone, and a few striated muscle cells, in addition to previously reported neurons in rat brain. Many peripheral neurons, epithelial cells, and mast cells display histamine immunoreactivity during rat embryogenesis but are devoid of detectable HDC mRNA with the current method. It remains possible that histamine is formed by another enzyme or is taken up from the extracellular space. The results support the concept that a significant proportion of histamine is formed and stored by embryonic cells other than mast cells.

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.

scholarly journals Tissue distribution of rat glutathione transferase subunit 7, a hepatoma marker

1986 ◽  
Vol 240 (3) ◽  
pp. 885-889 ◽  
Author(s):  
S E Pemble ◽  
J B Taylor ◽  
B Ketterer
Keyword(s):  
Northern Blot ◽  
Blot Analysis ◽  
Genomic Dna ◽  
Southern Blot Analysis ◽  
Northern Blot Analysis ◽  
Glutathione Transferase ◽  
Rat Tissues ◽  
Polyadenylated Rna ◽  
Normal Rat ◽  
Rat Hepatoma

Polyadenylated RNA isolated from NN-dimethyl-4-aminoazobenzene-induced rat hepatoma was used to prepare a cDNA library in lambda gt10. Full-length clones complementary to mRNA coding for glutathione transferase subunit 7 were isolated and one of these clones (pGSTr7) was fully characterized. In Northern blot analysis, mRNA hybridizing to 32P-labelled pGSTr7 was found in poly(A)-containing RNA isolated from seven normal rat tissues but not from testis and liver. A similar hybridizing mRNA species was also detected in human placental mRNA. The same probe, used in a Southern blot analysis of genomic DNA, suggests the presence of a multigene family in the rat.

scholarly journals Expression and tissue distribution of rat sulfated glycoprotein-1 (prosaposin).

1996 ◽  
Vol 44 (4) ◽  
pp. 327-337 ◽  
Author(s):  
C R Morales ◽  
M El-Alfy ◽  
Q Zhao ◽  
S A Igdoura
Keyword(s):  
Epithelial Cells ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Biological Fluids ◽  
Cell Types ◽  
Rat Tissues ◽  
Fluid Compartments ◽  
Rat Tissue ◽  
Sulfated Glycoprotein

Sulfated glycoprotein-1 (SGP-1/prosaposin) exists as a sulfated secreted protein or as a lysosomal precursor of four smaller saposin molecules. The protein exhibits ubiquitous expression, evolutionary conservation, and diverse tissue inducibility. The lysosomal form of SGP-1 plays a role in the hydrolysis of glycolipids and sphingomyelin. The function of the secreted form of SGP-1 is still unclear. However, it could act as a glycolipid transfer protein, since several gangliosides (a series) were found to bind with high affinity to prosaposin. To identify cell types that produce SGP-1 mRNA, we constructed an SGP-1 cDNA and used for screening of different rat tissues by Northern blot analysis. To localize the translation product of SGP-1 transcripts, we immunostained the same tissues with an anti-SGP-1 antibody. The SGP-1 cDNA construct was generated by amplifying a rat testicular Zap cDNA library by PCR (polymerase chain reaction) with two synthetic oligonucleotide primers. A positive signal of 1.7 KB was isolated, subcloned into the pGEM-7Zf (+). Sequence analysis showed a near-identical nucleotide and amino acid similarity to a previous rat SGP-1 cDNA. The majority of the heterogeneites were conservative substitutions. Northern blot analysis demonstrated that all examined rat tissue and organs have SGP-1 mRNA. Immunocytochemistry identified two staining patterns in the cytoplasm of positive cells: (a) a granular reaction characteristic of lysosomes in the supranuclear and basal regions of epithelial cells and in the perinuclear region of neurons; and (b) a homogeneous reaction in the cytoplasm of Sertoli cells, Type II pneumocytes, macrophages, and epithelial cells lining the choroid plexus. The latter staining pattern could be characteristic of cells that exhibit a secretory routing of SGP- 1. The production of SGP-1 by a variety of specialized cells lining fluid compartments suggests that its secreted form has a role in the transport of lipids in biological fluids, possibly by the formation of soluble complexes with glycolipids. Similarly, the lysosomal form of SGP-1/prosaposin and their derived saposins also solubilizes certain glycolipids to promote their degradation by specific hydrolases.

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