Calmodulin antagonist W-7 inhibits aggregation of human platelets induced by platelet activating factor

Experiments were done to test the hypothesis that aggregation of human platelets induced by platelet activating factor (PAF) may be mediated by calmodulin-dependent processes. W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide], a potent calmodulin antagonist, caused dose-dependent inhibition of PAF induced aggregation of human platelets in vitro. The ED50 for W-7 was 51.5 +/- 9.5 microM (mean +/- SEM). This concentration is known to be platelet calmodulin-specific. These data are consistent with the hypothesis.     

Suppression of tumor promoter phorbolmyristate acetate-induced chromosome breakage by antioxidants and inhibitors of arachidonic acid metabolism

To gain insight into the mechanism of formation of chromosomal aberrations by the tumor promoter phorbolmyristate acetate (PMA) in human lymphocytes, we investigated the effect of antioxidants and inhibitors of arachidonic acid metabolism. Among the antioxidants bovine erythrocyte CuZn superoxide dismutase, glutathione peroxidase, mannitol (a scavenger of hydroxyl radicals), butylated hydroxytoluene and butylated hydroxyanisole were anticlastogenic while catalase and dimethylfuran (a scavenger of singlet oxygen) were inactive. These results show that the induction of aberrations by PMA occurs via indirect action, i.e. the intermediacy of superoxide and hydroxyl radicals. The following inhibitors of arachidonic acid metabolism were strongly anticlastogenic: the cyclo-oxygenase inhibitors indomethacin and flufenamic acid and the lipoxygenase inhibitor BN1015. Imidazole, nordihydroguaiaretic acid BN 1048 and 5,8,11,14-eicosatetraynoic acid were moderately active. The inhibitor of phospholipase A₂, fluocinolone acetonide, was also anticlastogenic.

We conclude that the oxidative metabolism of arachidonic acid is involved in the induction of chromosomal aberrations by PMA in human lymphocytes. However, because of the limited selectivity of these drugs, it is not yet possible to identify unambiguously the step(s) in the arachidonic acid cascade responsible for PMA clastogenicity.     

Water deficit enhancement of proline and α-amino nitrogen accumulation in potato plants and its association with susceptibility to drought

Potato plants ( Solanum tuberosum L. cvs 'Up-to-Date', 'Desiree', 'Alpha', 'Spunta', 'Elvira' and 'Troubadour') were exposed to cycles of water stress and relief during growth. Severe water deficit induced increased proline content 6- to 7-fold in nonturgid leaves which just started to wilt, and 8- to 27-fold in fully wilted leaves of potatoes. However, proline content was not affected during the early stages of stress development over a range of osmotic potentials in the leaves. The rising proline content was related to turgor loss of leaves independent of changes in the osmotic potentials, which indicates that proline involvement in osmoregulation of potato leaves is unlikely.
Repeated cycles of water stress and relief resulted in increased proline and α-amino nitrogen content in the tuber tissue of some of the cultivars. The smallest increase in proline content was obtained in 'Alpha' tubers and the content of α-amino nitrogen remained unaffected by the water stress. Concomitantly, 'Alpha' was the most drought-tolerant cultivar, as determined by its capacity to accumulate dry matter in tubers under stress conditions. On the other hand, in tubers of cultivars which were more susceptible to drought, a marked increase in proline and α-amino nitrogen was observed in response to water stress. The possible association of these findings with tolerance of potatoes to repeated short periods of drought is discussed.     

Growth enhancement induced by prolonged L-dopa administration in rats

To follow growth of rats, in which growth hormone secretion has been chronically stimulated, L-Dopa (5 mg/kg) was injected subcutaneously twice daily for 70 days to growing rats. A control group, matched for sex and sibship, pair fed with the treatment group was given saline injections. At 10-day intervals, the rats were weighed and measured. At 90 days of age the rats were ether anesthesized, bled for growth hormone determination, and internal viscera weighed. Weight gain and length in the L-Dopa-treated group was found to be significantly greater. A mean weight gain of 6% and 12% in the male and female rats, respectively, and a mean length gain of 5% in both male and female rats was observed at 90 days of age. The thymus, thyroid, adrenals, uterus, and gonads all tended to be heavier in the L-Dopa-treated group. Significantly heavier kidneys were found in the L-Dopa group. Serum growth hormone was found to be 8.44 +/- 1.4(SE) ng/ml in the L-Dopa group and 4.6 +/- 0.9 ng/ml in the control group. It is concluded that the continuous administration of L-Dopa produces an increase of circulating serum growth hormone levels, and this in turn enhances growth.     

Differential inhibition of cellular and viral pp60src kinase by P1,P4-di(adenosine-5'')tetraphosphate.

We contrasted the protein kinase activities of pp60v-src, the transforming protein of Rous sarcoma virus, and its normal cellular homolog pp60c-src with respect to inhibition by P1,P4-di(adenosine-5')tetraphosphate by using the immune complex protein kinase assay. The concentration of P1,P4-di(adenosine-5')tetraphosphate required for 50% inhibition of pp60v-src kinase (1 microM) was found to be significantly lower than that required for inhibition of pp60c-src kinase (46 microM). Viral and cellular pp60src kinases differed to a lesser extent with respect to inhibition by adenosine-5'-tetraphosphate, di(guanosine-5')tetraphosphate, and ADP. No significant differences were found in the ATP Km values of pp60v-src (0.108 +/- 0.048 microM) and pp60c-src kinases (0.056 +/- 0.012 microM). These results demonstrate that the protein kinase activities of viral and cellular pp60src are functionally distinguishable, particularly on the basis of enhanced sensitivity of the viral enzyme to inhibition by P1,P4-di(adenosine-5')tetraphosphate. These functional differences are likely to be due to differences in the conformation of the active site and may be important for determining transformation potential.     

Highly Purified pp60src Induces the Actin Transformation in Microinjected Cells and Phosphorylates Selected Cytoskeletal Proteins In Vitro

The src gene product of Rous sarcoma virus (pp60(src)) was highly purified from a rat tumor cell line and shown to have physiological actin transformation activity in a cellular microinjection assay that measures the dissolution of actin microfilament bundles in vivo. The purified pp60(src) fraction consisted of two major proteins, seen on silver-stained sodium dodecyl sulfate-polyacrylamide gels: a 60,000-dalton (60K) protein, identified as pp60(src) by immunoprecipitation with tumor-bearing rabbit immunoglobulin G (IgG) and peptide mapping, and an unrelated 65K protein. There was no evidence for proteolytic cleavage of pp60(src). A 7,000-fold purification of the tyrosine-specific protein kinase activity of pp60(src) was achieved by this procedure. Purified pp60(src) phosphorylated tumor-bearing rabbit IgG heavy chains, casein, histones H1 and H2B, tubulin, and microtubule-associated proteins when assayed in vitro. When incubated with [gamma-(32)P]ATP in the absence of exogenous phosphoacceptor substrates, purified pp60(src) became labeled with (32)P at the tyrosine residues exclusively. Phosphatase and cyclic AMP-dependent protein kinase activities were undetectable in the purified fraction. Microinjection of highly purified pp60(src) into the cytoplasm of normal Swiss 3T3 mouse fibroblasts caused rapid and reversible dissolution of actin stress fibers, as visualized by indirect immunofluorescence with actin antibodies. The actin-disrupting activity was thermolabile and sensitive to inhibition by coinjection of tumor-bearing rabbit IgG, and purified to about the same extent (8,000-fold) as did the IgG kinase activity of pp60(src), thus implicating pp60(src) as the active agent. Examination of actin-associated proteins as substrates for the pp60(src) kinase in vitro showed that vinculin was phosphorylated directly by pp60(src), although to a small extent. Actin, myosin, and tropomyosin were not phosphorylated. Thus, pp60(src) purified by this procedure retains native functional properties and provides a useful probe for analyzing transformation-dependent changes in actin cytoarchitecture.     

R Factor Proteins Synthesized in Escherichia coli Minicells: Incorporation Studies with Different R Factors and Detection of Deoxyribonucleic Acid-Binding Proteins

Analysis of the protein synthesized by Escherichia coli minicells containing R factors demonstrated a variety of low- and high-molecular-weight polypeptides in sodium dodecyl sulfate (SDS)-polyacrylamide gels. Only half of this protein was released into a soluble fraction on lysis of these minicells. The other half remained associated with the minicell envelope. The efficiency of precursor incorporation into protein and the kinds of proteins synthesized changed with the age of the minicells at the time of harvest. About 1 to 2% of the soluble R factor-coded protein bound to calf thymus, E. coli, or R factor DNA-cellulose. Although most of these proteins were excluded from Sephadex G-100 columns, they migrated chiefly as low-molecular-weight-polypeptides (13,000 to 15,000) in SDS-polyacrylamide gels. Additional DNA-binding proteins that appeared to be higher-molecular-weight peptides were noted in extracts from younger minicells. At least one protein, identified as an SDS band, appeared to bind selectively to R factor DNA-cellulose. Minicells with R factors also contained DNA-binding proteins of cell origin, including the core RNA polymerase. No such binding proteins were found in R(-) minicells. These studies suggest that: (i) R factors code for proteins that may be involved in their own DNA metabolism; (ii) R factor DNA-binding proteins may be associated with larger host cell DNA-binding proteins or subunits of larger R factor proteins; and (iii) the age of the minicell influences the extent of protein synthesis and the kinds of proteins synthesized by R factors in minicells.