pp60c-src kinase activity in bovine coronary extracts is stimulated by ATP

pp60c-src kinase is believed to participate in regulating key cellular mechanisms including signal transduction and differentiation of smooth muscle during early embryogenesis. In this study, pp60c-src kinase activity was demonstrated in extracts from adult bovine coronary arterial smooth muscle. Activity, reflected by autophosphorylation of pp60c-src, phosphorylation of exogenous substrates, and phosphorylation of several endogenous substrates, was enhanced about 2 fold when added Mg2+ was replaced by Mn2+. Unexpectedly, activity was dramatically stimulated 20-50 fold by prior incubation with ATP. Such stimulation appears to be mediated through a novel mechanism which is independent of ATP-induced phosphorylation of reaction components. These new observations strongly suggest that a unique mechanism exists for regulation of coronary arterial pp60c-src kinase activity. Conceivably, this mechanism may serve important roles in modulating signal transduction and contractility of vascular smooth muscle.     

Effect of parathyroid hormone and forskolin on cytoskeletal protein synthesis in cultured mouse osteoblastic cells

Parathyroid hormone (PTH) has been shown to cause transient cell shape changes in bone cells. We have examined the effects of parathyroid hormone and forskolin on the organization and expression of cytoskeletal proteins in cultured mouse endosteal osteoblastic cells. Analysis of [35S]methionine-labeled cytoskeletal proteins isolated on two-dimensional gel electrophoresis showed that PTH treatment (24 h) stimulated the de novo biosynthesis of actin, vimentin and tubulins in confluent cells, whereas forskolin had a minor effect despite a huge stimulation of cAMP production. This PTH-induced stimulation was associated with cell respreading following a mild and transitory cell retraction. PTH increased the synthesis of monomeric subunits of actin and beta-tubulins in subconfluent bone cells, whereas both monomeric and polymeric levels of beta-tubulins were increased in confluent osteoblasts. Under conditions reducing cell spreading, osteoblastic cells had initially high levels of unpolymerized subunits. In these poorly spread cells, parathyroid hormone or forskolin had no effect on the de novo synthesis of cytoskeletal proteins despite a marked elevation in intracellular cAMP levels. It is concluded that PTH affects the biosynthesis of cytoskeletal proteins in osteoblastic cells and that cAMP production does not seem to be directly involved. In addition, the effect of PTH is modulated by cell spreading and by the initial pool of cytoskeletal subunits.     

Content of phenolic acids in callus culture of alfalfa (Medicago sativa): The effect of age and biochemical differentiation

Phenolic acids were separated into three fractions and determined by HPLC inMedicago sativa callus culture at the age of two, three and four weeks. The contents of free and especially of predominating ester-bound phenolic acids decreased with callus age to approx. 80 % while the content of phenolic acids nonextractable by methanol increased byca. 90 %. The proportion of benzoic acid derivatives rose from 15 to 21 % within four weeks. The determined difference in the contents of phenolic acids in the upper and lower parts of callus diminished with age. The content of bound forms was higher in the lower part regardless of the callus age. The content of free acids in two weeks old callus was half as high as in the upper part.     

Some factors affecting root formation onin vitro regenerated pea shoots

The rooting ability of 2 cm long shoots ofPisum sativum L., derived from differentin vitro shoot-tip cultures in two pea cultivars Bohatýr and Kleine Rheinländerin was evaluated. In three mutually independent experiments the full and half-strength Murashige-Skoog medium (containing full or half concentration of macro and microelements), with sucrose concentrations 10–30 g l-¹, and with various NAA and IAA combinations, was tested. The variant with half concentration of macro- and microelements, supplemented with 30 g l¹ sucrose, and with growth regulators in the quantity of 1 μM proved optimum.     

On the transposition of copia-like nomadic elements in cultured Drosophila cells

We studied the stability of the genomic distribution of six retrotransposon families in long-term and short-term cultures of Drosophila cells. In a subclone derived from Kc cells, no significant rearrangements were detected over an 8 year period. On the contrary, extensive reshuffling and amplification of transposon families were observed in recently established cell lines. These results show that in cultured Drosophila cells transposition appears to be restricted to the transition from the embryo to continuous cell lines.     

Single amino acid substitutions in the reactive site of antithrombin leading to thrombosis. Congenital substitution of arginine 393 to cysteine in antithrombin Northwick Park and to histidine in antithrombin Glasgow

Antithrombin Northwick Park and antithrombin Glasgow are functionally variant antithrombins with impaired abilities to interact with thrombin. Thrombosis is associated with their inheritance. Both of the purified, reduced, and S-carboxymethylated variant antithrombins were treated with cyanogen bromide and the major pools of each containing the amino acid sequence Gly339-Met423 were isolated. Following treatment of these pools with trypsin, fast atom bombardment mass spectrometry identified tryptic peptides (found also in normal antithrombin treated in the same way) that corresponded to amino acid sequences Gly339-Lys370 and Val400-Met423. The tryptic peptides, corresponding to amino acid sequences Ala371-Arg393 and Ser394-Arg399 were present in both variant preparations in greatly reduced amounts compared to a normal antithrombin preparation. However, two novel tryptic peptides of molecular mass (M + H)+ 2976 and 2952 were identified in the digests of antithrombin Northwick Park and Glasgow, respectively. Further analyses of these novel tryptic peptides were carried out by V8 protease treatment and sequential Edman degradation coupled with mass spectrometric analysis of the shortened peptides. This established that these peptides comprised the amino acid sequence Ala371-Arg399, but with single amino acid substitutions at the reactive site, Arg393 replaced by Cys (in antithrombin Northwick Park) and by His (in antithrombin Glasgow).     

Cyclic AMP inhibition of phosphoinositide turnover in human neutrophils

The effect of increased intracellular levels of cyclic AMP on phosphoinositide metabolism was studied in human neutrophils stimulated with fMet-Leu-Phe. Intracellular cyclic AMP was raised by preincubation either with dibutyryl cyclic AMP and theophylline or with prostaglandin E1. Concentrations of dibutyryl cyclic AMP and theophylline fully inhibitory for the metabolic responses inhibited phosphoinositide breakdown and phosphatidic acid formation to a large extent. The accumulation of the water-soluble inositol phosphates was also measured. In agreement with the data obtained on the phospholipids, inositol phosphate generation was found to be severely, though not completely, reduced. Treatment with dibutyryl cyclic AMP and theophylline also inhibited resynthesis of membrane inositol lipids. Treatment with prostaglandin E1 had a similar, though less, marked effect on inositol lipid turnover, which was parallel with a smaller inhibition of metabolic responses. We therefore suggest that the elevation of intracellular cyclic AMP mainly affects neutrophil responses by inhibiting the phosphoinositide cycle.     

The localisation of an Mr 74,000 major chromatin antigen on native salivary chromosomes of Drosophila melanogaster

An antigen making a major contribution to the immune response to Drosophila melanogaster chromatin resides primarily on a nonhistone charge-class family of proteins of Mr 74,000. Immunofluorescence detects this antigen at interbands, puffs and diffuse bands of D. melanogaster salivary chromosomes isolated without exposure to acid fixatives, and on nucleoplasmic ribonucleoprotein droplets. In the electron microscope, gold labelling reveals the binding of monoclonal antibodies specific for the antigen at chromosomal loci generally bearing putative ribonucleoprotein (RNP) particles. However, the locus 3C 11–12 is remarkable in that it bears putative RNP particles but is virtually unlabelled, suggesting protein specificity at different active loci.