BLOTTO-MF, an inexpensive and reliable hybridization solution in northern blot analysis using complementary RNA probes.
Explore the source record for details and available documents.
Blotting, Northern↗
Biomedical subjects
T Geijer
Publications and source records attributed to T Geijer.
Expression of preprodynorphin in human small cell lung carcinoma cell lines.
The expression of preprodynorphin has been studied using the Northern blot technique. Ten human cell lines, six small cell lung carcinoma (SCLC), one large cell carcinoma (LCC), two neuroblastoma and one lymphoblast-like cell line, were screened with a preprodynorphin cRNA-probe. Tryptic digestion followed by radioimmunoassay for Leu-enkephalin-Arg6 was used to detect possible translation of the preprodynorphin transcript. Of the ten cell lines investigated we found that all expressed preprodynorphin-mRNA to various degrees, and that this transcript is also translated. Two of the cell lines, neuroblastoma SK-N-MC and SCLC H69, also expressed preproenkephalin-mRNA. This set of cell lines provides a useful model of human origin in which the regulation of the preprodynorphin gene and the posttranslational processing of its products can be studied and compared.
Blotting, Northern↗
Expression of the cholecystokinin gene in a human (small-cell) lung carcinoma cell-line.
Expression of the cholecystokinin (CCK), gastrin and enkephalin A genes were studied by Northern blot analysis and a library of sequence-specific radioimmunoassays in human cell lines. The human small-cell lung carcinoma line (SCLC) U-1690 expressed moderate levels of CCK mRNA as compared to the human neuroepithelioma cell line SK-N-MC. Neither gastrin nor (pro)enkephalin A mRNAs were detectable in the U-1690 cell line. In contrast, the SCLC-line H-69 expressed Enk A but no CCK mRNA. The radioimmunoassays showed that the CCK mRNA transcript in the SCLC line U-1690 also is translated, and that preproCCK is processed into bioactive, carboxyamidated CCK peptides. Thus, the human small cell carcinoma cell line U-1690 is a useful model for studies of cell-specific CCK gene expression.
Animals↗
Procholecystokinin and proenkephalin A mRNA expression is modulated by cyclic AMP and noradrenaline.
Regulation of the expression of procholecystokinin (proCCK) and proenkephalin A mRNA was studied in the human neuroblastoma cell line SK-N-MC. Cells were treated with dibutyryl-3',5'-cyclic AMP (dbcAMP), noradrenaline or isoproterenol, a beta-adrenoceptor agonist. Levels of proCCK and proenkephalin A mRNA were determined by Northern blot analysis with proCCK- and proenkephalin A-specific cRNA hybridization probes 9 h after drug treatments. ProCCK and proenkephalin A mRNA were co-expressed in SK-N-MC cells. ProCCK mRNA levels were increased 1.5-2.5 times by dbcAMP, noradrenaline and isoproterenol when compared with controls. The level of proenkephalin A mRNA increased approximately two to three times under the same drug conditions, whereas the level of N-myc mRNA did not change significantly. These results suggest that expression of proCCK and proenkephalin A mRNA may be regulated by a similar cAMP-dependent mechanism in the SK-N-MC cell line.
Bucladesine↗
Modulation of proenkephalin A gene expression by cyclic AMP.
Regulation of proenkephalin A expression was studied in the human neuroblastoma SK-N-MC cell line with respect to mRNA-level, translation, posttranslational processing of the prohormone and secretion of the processed products into the culture medium. Cells were treated with either norepinephrine (NE), dexamethasone (DEX), dibutyryl-3',5'-cyclic AMP (dbcAMP) or the combination of NE and DEX. In an additional investigation, proenkephalin A mRNA levels were determined after 9 h of treatment with dbcAMP, NE, isoproterenol, NE + propranolol and dbcAMP + DEX. NE or dbcAMP for 1-48 h transiently elevated proenkephalin A mRNA 1.5-4.5 times compared to control. The effect of NE was partially blocked by the beta-adrenoceptor antagonist propranolol and was reproduced by the beta-adrenoceptor agonist isoproterenol, suggesting involvement of the beta-adrenoceptor. DEX alone had no significant effect. However it markedly antagonized the effect of NE but not that of dbcAMP suggesting an action on the beta-adrenoceptor. The intracellular content of Met-enkephalin-Arg6,Phe7 immunoreactivity was increased during drug treatment in parallel with changes in proenkephalin A mRNA. DEX gave no effect. No significant change in the ratio of low versus high molecular weight immunoreactive material could be detected in the cell extracts as determined at different time points. Secretion of immunoreactivity into the culture medium increased 5-fold after 18 h of treatment with NE, whereas dbcAMP gave a 2-fold increase. The proportion of low-molecular weight secreted material increased markedly. DEX alone did not induce any change but inhibited the effect of NE. Apparently, regulation of gene expression, prohormone processing and secretion are coordinated by a cAMP-dependent mechanism.
Cyclic AMP↗
A highly sensitive Northern blot assay detects multiple proenkephalin A-like mRNAs in human caudate nucleus and pheochromocytoma.
Total RNA from post mortem human caudate nucleus, cerebellum, cerebral cortex and pheochromocytoma tissues has been prepared. Northern blot analysis, using a single-stranded human proenkephalin A antisense probe (cRNA), revealed the existence of two different proenkephalin A-like sequences in the human caudate nucleus and pheochromocytoma RNA extracts of approximately 1400 and 1000 nucleotides in length respectively, whereas no specific RNA bands could be detected in the cortex and only the 1400 nucleotide band was present in the cerebellum. Under highly stringent hybridization conditions, the proenkephalin A-like RNA bands still appear, indicating that the detected RNA species have either identical or a closely related sequence to that of the well-characterized human proenkephalin A mRNA sequence.
Adrenal Gland Neoplasms↗
Expression of the proenkephalin gene in human neuroblastoma cell lines.
Several human tumour cell lines were screened for secretion of proenkephalin-derived peptides with an antiserum directed to its N-terminus, Met-enkephalin-Arg6,Phe7 and for proopiomelanocortin-derived peptides with an antiserum to beta-endorphin. The neuroblastoma SK-N-MC cell line secreted Met-enkephalin-Arg6,Phe7-immunoreactive peptides in relatively high amounts into the culture medium, although processing was not complete and there was no evidence for free Met-enkephalin-Arg6,Phe7. Gene expression was confirmed by the presence of proenkephalin mRNA and proenkephalin-derived polypeptides in extracts of the SK-N-MC cells and also in the neuroblastoma SH-SY5Y cell line. In the latter cells, however, the expression was approximately 3 times lower, there was less processing of proenkephalin and no evidence for secretion.
Adrenal Gland Neoplasms↗
A rapid and inexpensive method for preparing E. coli plasmid-DNA.
A simple, rapid and inexpensive scaled up miniprep procedure for preparing pure E. coli plasmid DNA is described. Bacterial cells were subjected to the boiling procedure and high molecular weight RNA was removed by LiCl-precipitation. Residual RNA and proteins were removed by subsequent treatment with RNase A and proteinase K/SDS respectively, followed by Sephadex G-50 and Sepharose 6B-Cl chromatography. The average yield from a 100 ml over-night bacterial suspension was 100 to 150 micrograms for pBR-322 DNA, and 250-500 micrograms for SP-6 derived recombinant plasmids. In addition, the described "scaled up" preparation does not require CsCl-ethidium bromide centrifugation; pure plasmid DNA can be prepared within 1 to 2 days.
Chromatography, Gel↗
Clinical experience of glaucoma therapy with tosmilen eye drops.
Explore the source record for details and available documents.
Adult↗