Herpesvirus saimiri oncogene STP-C488 encodes a phosphoprotein.

The STP-C488 open reading frame of herpesvirus saimiri encodes an oncoprotein that has transforming and tumor-inducing activities independent of the rest of the herpesvirus genome. STP-C488 protein has an unusual, membrane-associated, fibrous structure and is located primarily in perinuclear compartments. We now report that STP-C488 is phosphorylated in vivo. The phosphorylated form, which accounted for about 15% of STP-C488 in transformed cells, migrated slightly more slowly through sodium dodecyl sulfate-polyacrylamide gels than unphosphorylated STP-C488. A serine residue near the amino terminus was shown to be the site of phosphorylation. However, phosphorylation was not required for transformation of Rat-1 cells by STP-C488.     

Role of cytoskeleton in cell shaping of developing mesophyll of wheat (Triticum aestivum L.).

The effects of oryzalin and cytochalasin B (CB) on microtubule and actin microfilament arrays and on cell shaping were investigated in developing wheat mesophyll. Excised immature leaf sections capable of differentiating were incubated with the drugs. The behavior of the cytoskeleton was monitored by fluorescence microscopy after labeling with fluorescent dyes. Brief incubation with oryzalin (40 min, 10 microM) caused disassembly of microtubules. Recovery of microtubule arrays was comparatively slow after removal of the drug. Cells failed to establish transverse cortical bands of microtubules and transverse hoops of wall reinforcement. They expanded isodiametrically rather than longitudinally without forming lobes typical of wheat mesophyll cells. Brief treatment with CB (60 min, 20 micrograms ml-1) appeared to disrupt the microfilament arrays. Filaments recovered rapidly after removal of CB, and cells were able to shape in an apparently normal fashion. Continuous incubation at comparatively low concentration of CB (4 micrograms ml-1) appeared to cause selective loss of the fine transverse cortical microfilament arrays. Cortical transverse microtubule arrays persisted, but failed to form distinct bands in the majority of the cells. Cells were able to elongate in an almost normal fashion, but no lobes were formed.     

Alpha-helical small molecular size analogues of neuropeptide Y: structure-activity relationships.

C-terminal analogues of neuropeptide Y (NPY) of small molecular size have been synthesized. The influence of chain length, single or multiple amino acid substitution, and segment substitutions on receptor binding, pre- and postsynaptic biological activity, and conformational properties have been investigated. Receptor binding and in vivo assays revealed biological activity for NPY Ac-25-36 that increased with increasing alpha-helicity. In attempts to stabilize the alpha-helical content, three independent types of modified NPY Ac-25-36 analogues were synthesized. Strong agonistic activities could be detected in a series of discontinuous analogues, which are constructs of N-terminal parts linked via different spacer molecules to C-terminal segments. One of the most active molecules was NPY 1-4-Aca-25-36 (Aca, epsilon-aminocaproic acid). For the first time conformational properties of a series of small NPY analogues have been investigated by CD, and correlated with biological activity and receptor binding. A C-terminal dodecapeptide segment of NPY with an amount of 50% substitution to the native C-terminal sequence of NPY was found to exhibit significant receptor binding.     

Biophysical correlates of lysophosphatidylcholine- and ethanol-mediated shape transformation and hemolysis of human erythrocytes. Membrane viscoelasticity and NMR measurement

The effects of monopalmitoylphosphatidylcholine (MPPC or lysophosphatidylcholine) and a series of short-chain primary alcohols (ethanol, 1-butanol and 1-hexanol) on cell shape, hemolysis, viscoelastic properties and membrane lipid packing of human red blood cells (RBCs) were studied. For MPPC, the effective membrane concentration to induce the formation of stage 3 echinocytes (8 x 10(6) molecules per cell) was one order of magnitude lower than that needed to induce 50% hemolysis (7 x 10(7) molecules per cell). In contrast, short-chain alcohols induced both shape changes and hemolysis within close concentration range (2.5 x 10(8) to 3.5 x 10(8) molecules per cell). Viscoelastic properties of the RBCs were studied by micropipette aspiration and correlated with shape change. Ethanol-treated RBCs showed a decrease in membrane elastic modulus and an increase in membrane viscosity in the recovery phase at the early stage of shape change. MPPC-treated cells showed the same type of viscoelastic changes, but these were not observed until the formation of stage 2 echinocytes. High-resolution solid-state 13C nuclear magnetic resonance technique was applied to study membrane lipid packing in the ghost membrane by following the chemical shift of hydrocarbon chains. Both MPPC and ethanol caused the 13C-NMR chemical shift to move upfield, indicating that membrane lipids were expanded due to the intercalation of these exogenous molecules. Using data obtained from model compounds, we convert values of chemical shift into a lipid packing parameter, i.e., number of gauche bonds for fatty acyl hydrocarbon chains. Approximately 10(8) interacting molecules per cell are required to induce a detectable change of lipid packing by both MPPC and ethanol. The results indicate that homolysis occurs at a smaller surface area for MPPC- than ethanol-treated RBCs. Our findings suggest that progressive changes in the molecular packing in the membrane lead eventually to hemolysis, but the mode responsible for shape transformation varies with these amphipaths.     

Biological activity of the antitumor protein neocarzinostatin coupled to a monoclonal antibody by N-succinimidyl 3-(2-pyridyldithio)-propionate

The chromophore free apoprotein of neocarzinostatin was coupled to monoclonal IgG₁ antibody using N-Succinimidyl 3-(2-pyridyldithio)-propionate as heterobifunctional reagent. After coupling active chromophore was reassociated with the apoprotein. We present here experimental evidence that the hybrid protein retains biological activity as measured by the degradation of T2-DNA and bacteriostatic action.     

Synergistic effects of lectins in the interaction of thrombin with human platelets

Several lectins have been studied for their effects on the interaction of thrombin with human platelets. Wheat germ agglutinin, concanavalin A and Ricinus communis lectin increased the number of high affinity sites for diisopropylphosphothrombin on washed platelets from 3000 to about 12 000 but the binding affinities were unchanged (Kd approx 4 nM). Two other lectins, Lens culinaris and Bandieria simplicifolia, were without effect. (2) Using formalinized platelets to avoid possible complications of the platelet release reaction, wheat germ agglutinin showed a marked increase (5-fold) in the binding of active thrombin, peanut agglutinin had no effect while Ricinus communis and :Bandieria simplicifolia showed marginal increases (2-fold). Thrombin binding was decreased to about one quarter with Lens culinaris, Phaseolus vulgaris and concanavalin A. (3) Wheat germ agglutinin caused a synergistic increase of platelet aggregation at low concentrations of thrombin (12.5 mU/ml) and ADP (1 microM), both in the absence and presence of added fibrinogen, but had no effect on ristocetin-induced aggregation.     

Manganese oxidation by an intracellular protein of a Pseudomonas species.

Cultures of a Pseudomonas sp. strain MnB 1 produce an intracellular, manganese oxidizing protein (abbrev. as Mn ox. protein) during the stationary phase of growth. This protein is heat labile, can be inactivated by protease and has a pH-optimum for manganese oxidation at pH 7.0. Mn2+ is oxidized only at concentrations below 3-10(-5) M. The occurrence of the protein is not dependent on the presence of Mn2+, but is clearly related to the cessation of growth after the end of the exponential growth phase. Oxygen, coenzymes, and low molecular weight components of the cell extract seem not to be involved in the reaction as electron acceptors for the oxidation of Mn2+. Continued manganese oxidation by Mn ox. protein results in a progressive decrease in activity which corresponds to the amount of formed manganese oxide.     

Role of catecholamines in hypotensive response to dieting.

The effect of dieting on blood pressure and catecholamine metabolism was assessed in 11 normotensive obese women by providing first a weight-maintenance regimen high in carbohydrate and then a low-energy diet. All dietary constituents other than carbohydrate were maintained constant throughout the 18-day study. The low-carbohydrate diet led within 48 hours to a 41% fall in the urinary output of 4-hydroxy-3-methoxy mandelate and a significant fall in systolic and diastolic blood pressure. Plasma noradrenaline concentrations also fell and the hypotensive effect of the diet continued despite a maintained total body sodium. Thus the fall in blood pressure appeared to be mediated by changes in catecholamine metabolism independent of sodium intake. This may explain both the usefulness of weight reduction in hypertensive patients and the fainting that occurs in some normotensive obese subjects taking slimming regimens low in carbohydrate.     

An improved enzymatic cycle for nicotinamide-adenine dinucleotide phosphate.

We describe the use of column chromatography on the nonpolar adsorbent. Amberlite XAD-2, and on silanized silica gel in the desalting and partial purification of cobalamins. These techniques are both simpler and more versatile than phenol extraction, without sacrificing efficiency. In addition, a solvent system for thin-layer chromatography on silanized silica gel is described which rapidly separates naturally occurring cobalamins.     

Electrophoretic studies of several hydrolytic enzymes in relation to the cold tolerance of alfalfa.

Two cultivars of alfalfa (Medicago sativa L.), cold-tolerant Vernal and cold-sensitive Sonora, were grown under summer, winter, and dehardening environments to determine the characteristics and relationships of several hydrolytic enzymes to cold tolerance.Soluble enzymatic proteins, extracted from lyophilized crown and root tissues with three different solvents, were separated by polyacrylamide disc-gel electrophoresis and evaluated on the basis of equal dry weights of tissue and equal quantities of protein.Gels assayed for amylases, acid phosphatases, esterases, leucine aminopeptidases, and adenosine triphosphatases exhibited mainly quantitative differences in isoenzymes depending upon extractant, cultivar, and environmental differences. The qualitative differences detected were generally due to differential solubilities of isoenzymes in the three extractants and, to a lesser extent, were related to environmental, cultivar, or stability differences.While activities of esterases, acid phosphatases, and leucine aminopeptidases increased in winter samples, as soluble protein increased, only slight decreases in these enzymes occurred during dehardening. Conversely, activities of amylases were slightly lower in winter samples than in the other samples, and adenosine triphosphatase activity decreased in the most coldtolerant sample.The measured levels of total nonstructural carbohydrate, total soluble sugar, and starch indicated differences between cultivars in starch-sugar conversion. Further, the differential heat stabilities of the isoamylases also provided some information as to the nature of “protected activity” of diastatic enzymes.Differential cryostabilities of peptidases and adenosine triphosphatases detected between cultivars and environments also demonstrated the influence of the extraction medium in maintaining enzyme activity, and these observations may be important to an understanding of cold tolerance in alfalfa. The obvious speculations regarding enzyme stability and the factors involved as related to the cold tolerance of alfalfa require further examination.