Analysis of Physarum proteins throughout the cell cycle by two-dimensional PAGE

The accumulation of several hundred proteins during the nuclear division cycle of Physarum polycephalum was measured by digital image processing of silver-stained two-dimensional (2D) polyacrylamide gels. In contrast to previous studies, we have used an organism with a naturally synchronous cell cycle, so there are no uncertainties concerning synchronization artifacts or cell-sorting artifacts, and we have measured the specific amounts of each protein rather than the rate of synthesis. Since one-dimensional SDS-PAGE shows no significant fluctuations in the most abundant plasmodial proteins, we have loaded 2D gels so that proteins of low-to-moderate abundance appear in the linear range of the silver stain standard curve. Only five proteins showed reproducible, measureable fluctuations during the cell cycle. One of these proteins was tubulin. Full quantitative information was obtained by analysing the digital images of silver-stained gels by a general image processing system.     

Modular PH and C2 domains in membrane attachment and other functions.

The pleckstrin homology and C2 domains are modular protein structures involved in mediating intermolecular interactions. Although they represent distinct domains, there are several parallels regarding their function and type of interactions in which they participate. Both domains are stable structural entities that incorporate variable regions which, in different proteins, can be adapted to perform a specific function through binding to membrane phospholipids or specific protein ligands. A number of recent examples illustrate the function of some of these domains in regulated membrane attachment, with an important role in many cellular signalling pathways.     

Effect of dietary cis and trans fatty acids on serum lipoprotein[a] levels in humans.

Serum lipoprotein[a] (Lp[a]) is a strong risk factor for coronary heart disease. We therefore examined the effect of dietary fatty acid composition on serum Lp[a] levels in three strictly controlled experiments with healthy normocholesterolemic men and women. In Expt. I, 58 subjects consumed a control diet high in saturated fatty acids for 17 days. For the next 36 days, 6.5% of total energy intake from saturated fatty acids was replaced by monounsaturates plus polyunsaturates (monounsaturated fatty acid diet; n = 29) or by polyunsaturates alone (polyunsaturated fatty acid diet; n = 29). Both diets caused a slight, nonsignificant, increase in median Lp[a] levels, with no difference between diets. In Expt. II, 10% of energy from the cholesterol-raising saturated fatty acids (lauric, myristic, and palmitic acid) was replaced by oleic acid or by trans-monounsaturated fatty acids. Each of the 59 participants received each diet for 3 weeks in random order. The median level of Lp[a] was 26 mg/l on the saturated fatty acid diet; it increased to 32 mg/l (P less than 0.020) on the oleic acid diet and to 45 mg/l (P less than 0.001) on the trans-fatty acid diet. The difference in Lp[a] between the trans-fatty acid and the oleic acid diets was also highly significant (P less than 0.001). Expt. III involved 56 subjects; all received 8% of energy from stearic acid, from linoleic acid, or from trans-monounsaturates, for 3 weeks each. All other nutrients were equal.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification of phosphoinositide-specific phospholipase C from a particulate fraction of bovine brain

The coupling of various agonist receptors to the hydrolysis of phosphoinositides has generated much interest in the nature of the phospholipase C that is activated. Here we report the purification of a bovine brain phospholipase C derived from the particulate fraction. A 1000-fold purification was achieved by a combination of heparin-Sepharose, DEAE-cellulose and gel-permeation chromatography. The purified enzyme appears to be monomeric and under denaturing conditions shows a single staining major polypeptide of molecular mass 154 kDa in SDS gels. The enzyme is specific for phosphoinositides although it shows a marked preference for the polyphosphoinositides. With phosphatidylinositol 4,5-bisphosphate as substrate the enzyme expresses a specific activity of greater than 100 mumol min-1 mg-1. The phospholipase C is activated by Ca2+ (0.1-10 microM). The behaviour of this particulate enzyme is discussed in the context of a agonist-induced phosphatidylinositol hydrolysis.     

A novel inositol-phospholipid-specific phospholipase C. Rapid purification and characterization

A novel bovine brain inositol-phospholipid-specific phospholipase C has been identified on the basis of chromatographic behaviour and purified to apparent homogeneity by a rapid three-step procedure. The purified enzyme has a molecular mass of 85 kDa on SDS/polyacrylamide gel electrophoresis and a specific activity of 24 mumol.min-1.mg-1. The enzyme is dependent on Ca2+ and shows a marked preference for inositol phospholipid substrates. The unique nature of this polypeptide was confirmed through partial protein sequence analysis.     

Final height after growth hormone therapy in peripubertal boys with a subnormal integrated concentration of growth hormone.

The aim of this study was to test the effect of growth hormone (GH) therapy on final height in peripubertal boys with idiopathic short stature in whom a subnormal integrated concentration of GH (< 3.2 micrograms/l) was found. Twenty-eight peripubertal children were studied. Height was below 2 SD for age, growth velocity was < 4.5 cm/year, bone age was more than 2 SD below mean for age and GH response to provocative tests was more than 10 micrograms/l. Eleven subjects (group B) were treated with recombinant GH 0.75 unit/kg/week, divided into 3 weekly doses for 2 years, and then the same weekly dose divided into daily injections was administered until final height was attained. Seventeen untreated children (group A) who were followed until cessation of growth served as controls. The GH-treated patients reached their target heights (-2.1 +/- 0.5, mean +/- SD in SDS) and predicted heights (-1.8 +/- 0.8) determined by the Bayley and Pinneau method, while the final heights of the untreated patients were significantly lower than their target heights and their predicted final heights (-2.7 +/- 0.7, -1.8 +/- 1.0 and -2.7 +/- 0.7, respectively). The main effect of GH was observed during the 1st year of treatment when height velocity was significantly higher in the GH-treated group than in the untreated one (9.3 +/- 2.1 vs. 5.3 +/- 1.1, respectively, p < 0.001). The high cost of the treatment in this specific age group should be weighed against the results.     

Partial purification and characterization of a new phosphoprotein kinase from cells infected with pseudorabies virus

Cytoplasmic fractions from normal baby hamster kidney fibroblasts and from fibroblasts infected with pseudorabies virus were fractionated by DEAE-cellulose chromatography and fractions assayed for protein kinase activity. In preparations from uninfected and infected cells protein kinase activities identified as casein kinase I and II, the two isoforms of the cyclic-AMP-dependent protein kinase, protein kinase C, and a presumed proteolytic fragment of protein kinase C were present in comparable amounts. However in infected cells a new protein kinase activity was detected, appearing about 4 h after infection and increasing during the following 6 h at least. This new protein kinase was purified 100-fold by high-performance gel-permeation and ion-exchange chromatography, and characterized. It has an apparent relative molecular mass of 68 000 on the basis of gel-permeation chromatography, and a sedimentation coefficient of 4.3 S. It catalysed the phosphorylation of serine residues of basic proteins in vitro, with protamine a better substrate than mixed histones; and used ATP (apparent Km = 60 microM), but not GTP, as phosphoryl donor. Molecules that can serve as effectors for other protein kinases (cyclic AMP, cyclic GMP, Ca2+ + calmodulin, Ca2+ + phospholipid, double-stranded RNA, and heparin) did not significantly alter the activity of this enzyme. A distinguishing characteristic of the protein kinase was a high KCl concentration optimum with the persistence of activity up to 800 mM KCl, at least.     

Regression analysis of the spontaneous spike activity of neurons in snail ganglia

Regression analysis of the spontaneous spike activity of neurons in Helix pomatia was carried out with the aim to establish the statistical parameters of this activity under constant experimental conditions and during longer time intervals. The activity of 38 randomly chosen neurons in visceral and parietal ganglia, penetrated by microelectrodes and activated either endogenously by pacemaker potentials or by synaptic inputs, was recorded during time intervals lasting from 20 min to 3 h. The main results of the statistical analyses are presented in the table where the parameters of both cell types are listed. The validity of the regression analysis applied here is discussed from the point of the possibility it offers for carrying on the data processing quickly and without applying complex calculating means. The results are also considered regarding the current interest of our research group.