Identification by immunoblotting of somatostatin proforms in a rat pancreatic cell line.

The immunoblotting technique is applied to the analysis of "somatostatin" compounds secreted by R.I.N. T3 cells. We can confirm that two proforms of 15,300 +/- 750 and 29,000 +/- 1,100 Da accumulate in the extracellular medium. The unexpected form of 29 kDa, probably a dimeric form, disappears in reducing conditions. However, the 15 kDa peptide is characterized by several antibodies directed against either the intramolecular cycle of somatostatin-14 or the N-terminal extension of somatostatin-28. The 15 kDa form presents the same electrophoretic mobility in SDS-PAGE than the prosomatostatin isolated from a hypothalamic extract. Furthermore, this compound corresponds to the calculated mass of 10,388 Da deduced from the cDNA sequence. The detection of an immunoreactive 6 kDa peptide in the gel filtration fractions suggests an intermediate step in the prosomatostatin processing in these cells.

Further characterization of an E. coli strain resistant to the toxic and mutagenic action of cis-diamminedichloroplatinum(II).

An increased resistance to the toxic and mutagenic activity of the antitumor drug cis-diamminedichloroplatinum(II) (cis-DDP) in the E. coli strain BS21 compared to its wild-type parent, F26, has been reported. This resistance was neither due to different binding of cis-DDP to DNA nor to adaptive DNA repair (Germanier et al., 1984). In the present work, we found that mutation of the uvrA, recA and polA genes did not abolish the resistance of BS21 to the toxic action of cis-DDP. The lower mutability of BS21 was not influenced by the polA mutation, while uvrA greatly reduced and recA eliminated the mutagenic activity of cis-DDP in both strains. Treatment of BS21 and F26 with equal doses of cis-DDP produced the same initial number of platinum-DNA lesions. Little excision repair was detected in vivo in either strain during 6-h post-treatment incubation, the F26 strain being the most efficient of the two for this process. In contrast, F26 and BS21 were transformed identically by pBR322 DNA which had been treated with cis-DDP in vitro. Analysis of the platinum-DNA adducts which were formed between cis-DDP and salmon sperm DNA in the buffer conditions of this experiment suggests that plasmid DNA contains 80% monofunctional adducts and 20% bifunctional bis-guanine adducts. These data indicate that the selective toxicity and mutagenicity of these two strains in vivo are neither a result of different numbers of Pt-DNA lesions nor of their repair. The selectivity disappeared when the two bacterial strains were transformed by pBR322 DNA containing identical platinum-DNA lesions, suggesting that the biochemical events which process platinum-DNA lesions are the same in both strains. Hence, it appears that cis-DDP may form qualitatively different platinum-DNA adducts in the BS21 and F26 strains which are responsible for the different toxicity and mutagenicity.

HPLC analysis of somatostatin peptides secreted by a rat pancreatic endocrine cell line (RINT3): stimulation studies.

A combination of anion exchange and reverse phase HPLC leading to the purification of a 15-kd proform of somatostatin from culture medium of the pancreatic tumoral endocrine RINT3 cell line is described. Elevation of extracellular calcium concentration causes a dose-dependent stimulation of somatostatin release; maximal stimulation (2.8-fold over basal) was reached with 5 mM Ca. Furthermore, 0.01 microM TPA induced a stimulation of about the same amplitude while forskolin had a low effect. These data suggest that secretion of somatostatin in this model can be regulated by the Ca/C protein-kinase pathway.