Presence of angiotensinogen messenger RNA in various cultured cell lines.

The presence of angiotensinogen messenger RNA (mRNA) was assessed in total RNA extracted from hepatoma, glioma, neuroblastoma, and glioma-neuroblastoma hybrid cell lines. Total RNA from 1 X 10(7) cells was extracted, transferred to a membrane, and hybridized with a 32P-labeled, full-length (1650-base pair) rat angiotensinogen complementary DNA (cDNA). Angiotensinogen RNA sequences could be definitively detected only in hepatoma cells. Steroids were used in an attempt to increase the angiotensinogen mRNA level. Dexamethasone (2 X 10(-6) M) or 17 beta-estradiol (1 X 10(-7) M) was added to the cultures 18 to 24 hours prior to harvest. Dexamethasone treatment of the hepatoma cells resulted in a large increase in angiotensinogen mRNA, whereas estradiol had no effect. Steroids failed to induce detectable levels of angiotensinogen mRNA in total RNA from the other cell lines. That the RNA was intact was ensured by hybridizing duplicate Northern blots to a 32P-labeled actin cDNA. Actin mRNA sequences were detected in all cell lines. Blot hybridization of poly(A)+RNA resulted in the visualization of a weak angiotensinogen mRNA signal for a glioma cell line and a glioma-neuroblastoma hybrid line. However, the ability to detect angiotensinogen mRNA in a cell may depend on the phenotype expressed, which can be governed by culture conditions.

Antimutagenic and antigenotoxic effects of vegetable matrices on the activity of pesticides.

Two in vitro tests, one to detect bacterial mutagenicity (Ames test) on Salmonella typhimurium TA98, TA100, and TA1535 and the other the primary DNA damage (SOS Chromotest) on Escherichia coli PQ37, were applied to determine the overall genotoxic activity of 12 pesticides (azinphos methyl, chlorothalonil, chlorphyriphos ethyl, chlorphyriphos methyl, lambda-cyhalothrin, cypermethrin, cyprodinil, fenazaquin, fludioxonil, indoxacarb, iprodione and penconazol). These were detected by gas chromatography (GC) analysis with electron capture (ECD) and nitrogen phosphorus detection (NPD) in 18 samples of vegetables. Some extracts of vegetables, found positive for pesticides with GC, were subjected to the Ames test and SOS Chromotest to evaluate the possible antimutagenic and/or antigenotoxic effects of vegetable matrices. The same bioassays were also performed on the mixtures of pesticides found in these samples to evaluate whether interactions could occur between pesticides and be responsible for the possible antimutagenic and/or antigenotoxic effects of the contaminated matrices. Experiments were also carried out to compare the results found for contaminated vegetables with their content of antioxidant components. Significant differences in mutagenicity and genotoxicity were found among the pesticides selected for this study. Of the 12 pesticides tested, only azinphos methyl, cyprodinil, fludioxonil and iprodione were found to be positive for both S. typhimurium and E. coli. No mutagenic/genotoxic activity was found in the extracts of vegetables contaminated by pesticides. S. typhimurium TA1535 showed a strong positive mutagenic effect for the mixtures of pesticides while they were not able to induce the SOS system. The data concerning the content of polyphenols and the total reducing activity of the contaminated vegetables indicated high amounts of antioxidants that could explain the inhibitory effect on the activity of pesticides shown by vegetables.

Development of a new fluorescent angiotensin II probe.

Angiotensin II (ANG II) was conjugated to polystyrene Latex fluorescent microspheres (0.5 or 0.05 micron diam) with carbodiimide. Biological activity of the ANG II-conjugated microspheres (ANG II microspheres) was assessed in dispersed hepatocytes. The 0.05 micron ANG II microspheres inhibited glucagon-stimulated adenosine 3',5'-cyclic monophosphate accumulation and stimulated phosphorylase activity in hepatocytes, whereas the 0.5 micron ANG II microspheres only stimulated phosphorylase activity. The biological activity of the ANG II microspheres was caused by the conjugated ANG II and not by "free" ANG II associated with the spheres or by trypsin-like activity of hepatocytes causing release of ANG II from the microspheres. Binding of 0.05 micron ANG II to hepatocytes was readily observed with fluorescence microscopy. Little, if any, binding was observed with microspheres without conjugated ANG II. This fluorescent preparation of ANG II may have great utility in the study of receptor behavior after binding in individual cells.

Actin and tropomyosin variants in smooth muscles. Dependence on tissue type.

Actin was found to be the major source of myofibrillar protein heterogeneity in smooth muscles. Three isoelectric variants, alpha-smooth muscle (alpha-SM), beta-non-muscle (beta-NM), and gamma-actins (gamma-SM and gamma-NM) were measured in 15 different smooth muscles, alpha-SM and gamma-actin contents displayed an inverse relationship in a given smooth muscle, some of which contained primarily alpha-SM actin while gamma-actins dominated in others. alpha-SM actin and gamma-actin distributions were tissue-specific, independent of species. A greater proportion of alpha-SM actin appears to be associated with tissues having a high degree of tonic activity. beta-Nonmuscle actin was a significant, and relatively constant, component of all smooth muscle tissues. The high NM-actin content of these tissues may reflect the importance of proliferative, synthetic, or secretory activities in smooth muscle, because the alpha-SM actin disappeared in tissue culture with a time course paralleling the modulation of phenotype from a contractile to a proliferative cell. Two tropomyosin subunits were present in approximately equal amounts in all smooth muscle tissues studied. One tropomyosin subunit exhibited identical mobility on two-dimensional gel electrophoresis, while the other was characterized by some species-specific variation which was unrelated to actin variant distribution. No variants of the 20,000-dalton regulatory light chain of myosin were observed. These results suggest that SM-specific actin variants are associated with functional diversity among smooth muscles.