Northern Blot Analysis of Sepiapterin Reductase mRNA in Mammalian Cell Lines and Tissues

1993 ◽  
pp. 195-198 ◽  
Author(s):  
Josef Maier ◽  
Karin Schott ◽  
Thomas Werner ◽  
Adelbert Bacher ◽  
Irmgard Ziegler
Keyword(s):  
Cell Lines ◽  
Mammalian Cell ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Sepiapterin Reductase ◽  
Mammalian Cell Lines ◽  
Reductase Mrna

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 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- 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 Structure-guided selection of puromycin N-acetyltransferase mutants with enhanced selection stringency for deriving mammalian cell lines expressing recombinant proteins

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Alessandro T. Caputo ◽  
Oliver M. Eder ◽  
Hana Bereznakova ◽  
Heleen Pothuis ◽  
Albert Ardevol ◽  
...  
Keyword(s):  
Cell Lines ◽  
Mammalian Cell ◽  
Recombinant Proteins ◽  
Cultured Cells ◽  
Hek293 Cells ◽  
Reaction Products ◽  
Enzyme Form ◽  
X Ray Crystallography ◽  
Mammalian Cell Lines ◽  
Apparent Loss

AbstractPuromycin and the Streptomyces alboniger-derived puromycin N-acetyltransferase (PAC) enzyme form a commonly used system for selecting stably transfected cultured cells. The crystal structure of PAC has been solved using X-ray crystallography, revealing it to be a member of the GCN5-related N-acetyltransferase (GNAT) family of acetyltransferases. Based on structures in complex with acetyl-CoA or the reaction products CoA and acetylated puromycin, four classes of mutations in and around the catalytic site were designed and tested for activity. Single-residue mutations were identified that displayed a range of enzymatic activities, from complete ablation to enhanced activity relative to wild-type (WT) PAC. Cell pools of stably transfected HEK293 cells derived using two PAC mutants with attenuated activity, Y30F and A142D, were found to secrete up to three-fold higher levels of a soluble, recombinant target protein than corresponding pools derived with the WT enzyme. A third mutant, Y171F, appeared to stabilise the intracellular turnover of PAC, resulting in an apparent loss of selection stringency. Our results indicate that the structure-guided manipulation of PAC function can be utilised to enhance selection stringency for the derivation of mammalian cell lines secreting elevated levels of recombinant proteins.

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