Effect of lipid alkyl chain perturbations on the assembly of bacteriophage PM2

The lipid-containing bacteriophage PM2 can produce infectious virus in cultures infected at temperatures up to 31.5 °C, but not at 34 °C. Its host, Pseudomonas BAL-31, grows at 34 °C and cultures infected at that temperature undergo lysis. Sucrose-gradient analysis shows that 34 °C lysates contain no PM2-like particles. Temperature-shift experiments establish that the thermally sensitive process is late in infection when virus assembly is taking place.Adamantanone, a small hydrophobic molecule that perturbs membrane hydrocarbon zones prevents the production of infective virus. Concentrations which prevent virus production have no effect on host-cell growth or stability of mature virions. Adamantanone exerts its effects late in the infectious cycle, and lysates made in its presence contain no PM2-like particles. These experiments, carried out at 25 °C, indicate that adamantanone prevents the assembly of stable PM2 virus.Spin-label studies suggest that the lipid alkyl chains of the host-cell membrane are in an “ordered” state at temperatures below about 33 °C and undergo a transition to a “disordered” state above that temperature. Furthermore, the addition of adamantanone perturbs the hydrocarbon zones, producing a greater degree of disorder even below 25 °C. Our findings suggest that the cell membrane can function and grow with the lipid alkyl chains in either the “ordered” or “disordered” state, but that the “ordered” state must be maintained for PM2 assembly to occur.     

Effect of urethan on polyamine and DNA synthesis in the regenerating rat liver

When a single dose of urethan was injected into the peritoneal cavity of rats immediately after partial hepatectomy, DNA synthesis was delayed by 12 h. The induction of ornithine decarboxylase which was induced biphasically following partial hepatectomy was also reduced and delayed by 14–15 h by the administration of urethan. S-Adenosylmethionine decarboxylase activity in urethan-treated rat liver at 20 h and 29 h after operation was significantly lower than that of untreated animals. This enzyme activity was shown to increase thereafter, reaching a higher level than in untreated rats at 37–42 h. Hepatic spermidine content changed biphasically in a manner similar to DNA synthesis. These results suggest that the activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase may correlate with DNA synthesis and that an increase of spermidine concentration is necessary to DNA synthesis.     

Effect of acyclic polyisoprenoids on the biosynthesis of mannose-labeled glycolipids in rat liver microsomes

The effect of the acyclic polyisoprenoid (C₂₀); geranylgeranyl-acetone (GGA) and its derivatives on mannolipid formation from GDP-mannose in rat liver microsomes was studied. Remarkable increases in mannolipid biosynthesis (Rf 0.24 and/or Rf 0.50) were observed by $\text{in vitro}$ additions of GGA, its hydroxylated compound (GGA-OH) and phosphorylated material (GGA-OP). These results strongly suggest that GGA-OP is involved in mannolipid formation (Rf 0.24) as an acceptor, in analogy to retinylphosphate. On the other hand, the mechanism for increased formation of an endogenous mannolipid (Rf 0.50) by GGA and GGA-OH is attributable to the enhancement of mannosyltransferase activity.     

Effect of L-DOPA pretreatment on invivo protein synthesis in various rat brain regions

A single dose of L-dihydroxyphenylalanine (L-DOPA, 500 mg/kg, i.p.) that caused massive disaggregation of brain polysomes also suppressed the incorporation of [³H] lysine into trichloracetic acid (TCA)-precipitable proteins of cortex, cerebellum, hypothalamus, brainstem and striatum. The magnitude of this inhibition of [³H] protein synthesis was similar in all brain regions studied, and was not related to changes in the specific activity of the precursor amino acid.     

Effect of the arginine analog,canavanine, on the synthesis and secretion of procollagen

Canavanine was shown to competitively inhibit the activation of arginine when tested with tRNA and synthetases prepared from whole chick embryos. The canavanine has no effect when tested with other amino acids. The K_m for arginine was 2.5 μm and the K_i for canavanine was 35 μm. When fibroblasts from embryonic chick tendons were incubated with [³H]arginine and increasing concentrations of canavanine, there was a progressive decrease in the incorporation of [³H]arginine so that at 3 mm the incorporation into nondialyzable protein was only 14% of the control. A much smaller decrease in the incorporation of other radioactive amino acids was observed. Amino acid analysis of proteins isolated from cells incubated with canavanine showed conclusively that the analog was incorporated. When the cells were incubated with [¹⁴C]proline or [³H]glycine and 3 mm canavanine, the labeled procollagen containing the canavanine was secreted more slowly than normal and accumulated intracellularly. The retained procollagen chains were normally hydroxylated, disulfide linked, and triple helical. However, slab gel electrophoresis in sodium dodecyl sulfate demonstrated that they migrated with a lower mobility than control procollagen chains. We postulate that incorporation of canavanine inhibits normal proteolytic processing of signal sequences resulting in delayed secretion of the procollagen.     

Melatonin-induced suppression of gonadotropin titers in male golden hamsters: Effect on gonadal feedback mechanisms

Daily subcutaneous injections of melatonin cause testicular regression and a decline in circulating gonadotropin levels in male hamsters maintained on long photoperiods. We examine here if a reduction in gonadotropin levels also occurs in castrates administered melatonin and if melatonin-regressed hamsters respond to castration with an increased release of pituitary gonadotropins — a typical “castration response.” Groups of intact and castrated male hamsters maintained on a photoperiod of LD 14:10 received subcutaneous injections of 15 ug melatonin/day. Controls received vehicle only. After 7 weeks of injections the intact males were castrated. All animals were sacrificed a few days later and serum was assayed for gonadotropin titers. Melatonin injections caused a marked decline in serum gonadotropins in castrates and in intact males also caused testicular regression. In the latter, no “castration response” was observed upon removal of the testes. Thus, daily injections of melatonin depress serum gonadotropins in castrate and intact males and block the castration-associated rise in circulating gonadotropins in the latter.     

Effect of 3'-deoxyadenosine (cordycepin) on the early development of the sand dollar, Dendraster excentricus

The embryos of the sand dollar Dendraster excentricus have been examined with regard to their ability to undergo the early events of larval development in the presence of cordycepin, a reported inhibitor of RNA adenylylation. It has been shown that all the morphogenetic events from hatching to prism are inhibited by cordycepin at a concentration of 25 μg/ml, while the aspects of development prior to hatching (cleavage, blastulation, and the formation of cilia) are not affected by cordycepin. The period of sensitivity of each developmental event to cordycepin has been determined; for the early developmental processes this period substantially precedes the event, while for later processes there is a closer temporal association. In embryos treated with cordycepin, DNA synthesis and cleavage are unaffected prior to hatching, but subsequently are blocked, whereas respiration and total RNA synthesis per cell remain unaffected.     

Trace metal modification of immunocompetence. I. Effect of trace metals in the cultures on in vitro transformation of B lymphocytes.

Lymphocytes obtained from the spleen of Balb/C mice have been subjected to transformation by LPS in the presence of varying concentrations of Pb²⁺, Cd²⁺, or Cr³⁺. Both DNA and protein turnover were followed. It was found that Pb²⁺ and Cr³⁺ are mitogenic over a broad range of concentrations, while Cd²⁺ is slightly mitogenic at very low (10^?6M) concentration and rapidly becomes inhibitory of both [³H]TdR and [³H]ALA uptake. Pb²⁺ appears to stimulate the action of LPS, while Cr³⁺ appears to inhibit. Each of the metals protects the lymphocytes from cell death arising from incubation with LPS. The mechanism of the observed changes is as yet obscure.     

Effect of heat shock on the synthesis of low molecular weight RNAs in drosophila: Accumulation of a novel form of 5S RNA

The synthesis and stability of low molecular weight RNAs following heat shock in Drosophila melanogaster cell cultures have been examined. When cultures are raised from 25°C to 37°C, the synthesis of tRNA and at least two other low molecular weight RNAs continues at the 25°C rate. 5.8S ribosomal RNA and most of the low molecular weight nuclear RNAs are not synthesized. The synthesis of 5S ribosomal RNA is greatly reduced. A large amount of an RNA of about 135 nucleotides in length accumulates at 37°C. Nucleotide sequence analysis reveals that this RNA is a novel form of 5S RNA with approximately 15 additional nucleotides at its 3′ end.     

Laser raman studies of molecular interactions with phosphatidylcholine multilayers. I. Effect of iodine-iodide system

The interactions of iodine, iodide and triiodide with phosphatidylcholine multilayers have been investigated in order to find supporting data for conduction mechanisms of iodine-doped bimolecular lipid membranes. On the basis of order-disorder parameters it can be concluded that phospholipid multilayers interact with iodine rather than with its ionic forms. No evidence for the existence of triiodide chains in iodine-doped multilayers has been found. Consequently, the resistance-lowering effect of iodine must be based upon direct iodine-lipid interaction and iodine penetration rather than upon ion transport processes.     

Effects of lysolecithin on the surface properties of human erythrocytes

Human red blood cells (RBCs), transformed by incubation with the amphiphatic compound lysolecithin from their normal discocyte shape into echinocytes, have increased rates of agglutination in the presence of either poly- -lysine (PLL) or soybean agglutinin (SBA). Removal of lysolecithin by washing caused a reversal of shape back to the discocyte configuration and a lowering of agglutination rates. Methochlorpromazine, another amphiphatic echinocytogenic substance produced a similar increase in agglutination rates, suggesting that increased agglutinability may be a general property of echinocytes. Lysolecithin treatment of RBCs caused a decrease in the binding of cationized ferritin (CF) particles/μm² of RBC surface. The decrease in CF binding is due to a rearrangement of negative charge bearing molecules on the RBC surface rather than shedding of charged groups. These observations support the hypothesis that integral membrane proteins which bear negative charges and receptors are associated with a cytoskeleton within the red cell. Alterations in cell shape which result in distortion of the cytoskeleton may cause a redistribution of integral membrane proteins which bear charged groups at the RBC surface.     

The effect of gentamicin on calcium uptake by renal mitochondria

The effect of the nephrotoxic aminoglycoside antibiotic, gentamicin, on calcium uptake by renal cortical mitochondria was assessed in vitro. Gentamicin was found to be a competitive inhibitor of mitochondrial Ca⁺⁺ uptake. This effect displayed a dose response with a K_i of 233 μM and occurred at gentamicin concentrations below those that inhibit mitochondrial electron transport. These results further demonstrate the potential for gentamicin to alter membrane function and thereby contribute to toxic cell injury via its interactions with divalent cations.     

Effect of antinociceptive doses of oxotremorine on mouse brain acetylcholine turnover rate

The effect of antinociceptive doses of oxotremorine on the steady-state level and turnover rate of acetylcholine (ACh) was investigated in male Swiss-Webster mice. Oxotremorine produced dose-related increases in ACh levels which attained statistical significance (p ≥ 95; Dunnett's T) with the ED₈₄ antinociceptive dose in two sacrifice methods. The increased steady-state level of ACh was temporally correlated with the peak antinociceptive effect of oxotremorine. ACh turnover rate decreased with increasing doses of oxotremorine. The ACh turnover rate decreased from 11.06 ± 1.62 nmol/g/min to 5.38 ± 0.71 nmol/g/min by decapitation method and 30.20 ± 1.8 nmol/g/min to 19.99 ± 1.6 nmol/g/min by the microwave irradiation method (Ed₈₄ oxotremorine doses). The decrease in turnover rate of ACh produced by antinociceptive doses of oxotremorine is of a lesser magnitude than that produced by tremorogenic doses (1,2).     

Biochemical studies on a low molecular weight antiplasmin of human blood.

We previously reported a low molecular weight antiplasmin present in human platelets and plasma. This material as isolated by ultrafiltration was heterogeneous. The fraction showing maximum inhibition has been further purified by countercurrent distribution. The N-terminal residue of this purified inhibitor is alanine. Amino acid analysis showed glycine to be the predominant residue, and no basic amino acid was detected. Kinetic studies suggested a competitive inhibition of plasmin via enzyme-inhibitor complex formation. Carbohydrate and phosphate could not be detected. These data were similar for both platelet antiplasmin and serum antiplasmin. The relationship between the platelet and the serum inhibitor remains unclear. This low molecular weight antiplasmin may contribute to the consolidation of the thrombus.     

Comparative studies on myosin ATPase of a flying and nonflying bird.

Myosin was purified from the flight muscles of a flying (pigeon) and a nonflying (fowl) bird. Ki (ADP) of myosin ATPase of pigeon is higher, but the Km (ATP) is lower than that of fowl. The specific activity (mumole of Pi liberated/min/mg protein) is higher for the fowl. A0.5 (CaCl2) of myosin of both pigeon and fowl is similar. However, the two proteins differ in their interactions with ADP, ATP and p-chloromercuribenzoate. The two proteins have the same tyrosine, tryptophan and sulfhydryl contents. The electrophoretic patterns of the two myosins on SDS-polyacrylamide gels are different. These studies show significant molecular differences in the myosin derived from the flight muscles of a flying (pigeon) and a nonflying (fowl) bird.     

Rat liver pyruvate kinase: influence of ligands on activity and fructose 1,6-bisphosphate binding

The ability for various ligands to modulate the binding of fructose 1,6-bisphosphate (Fru-1,6-P2) with purified rat liver pyruvate kinase was examined. Binding of Fru-1,6-P2 with pyruvate kinase exhibits positive cooperativity, with maximum binding of 4 mol Fru-1,6-P2 per enzyme tetramer. The Hill coefficient (nH), and the concentration of Fru-1,6-P2 giving half-maximal binding [FBP]1/2, are influenced by several factors. In 150 mM Tris-HCl, 70 mM KCl, 11 mM MgSO4 at pH 7.4, [FBP]1/2 is 2.6 microM and nH is 2.7. Phosphoenolpyruvate and pyruvate enhance the binding of Fru-1,6-P2 by decreasing [FBP]1/2. ADP and ATP alone had little influence on Fru-1,6-P2 binding. However, the nucleotides antagonize the response elicited by pyruvate or phosphoenolpyruvate, suggesting that the competent enzyme substrate complex does not favor Fru-1,6-P2 binding. Phosphorylation of pyruvate kinase or the inclusion of alanine in the medium, two actions which inhibit the enzyme activity, result in diminished binding of low concentrations of Fru-1,6-P2 with the enzyme. These effectors do not alter the maximum binding capacity of the enzyme but rather they raise the concentrations of Fru-1,6-P2 needed for maximum binding. Phosphorylation also decreased the nH for Fru-1,6-P2 binding from 2.7 to 1.7. Pyruvate kinase activity is dependent on a divalent metal ion. Substituting Mn2+ for Mg2+ results in a 60% decrease in the maximum catalytic activity for the enzyme and decreases the concentration of phosphoenolpyruvate needed for half-maximal activity from 1 to 0.1 mM. As a consequence, Mn2+ stimulates activity at subsaturating concentrations of phosphoenolpyruvate, but inhibits at saturating concentrations of the substrate or in the presence of Fru-1,6-P2. Both Mg2+ and Mn2+ diminish binding of low concentrations of Fru-1,6-P2; however, the concentrations of the metal ions needed to influence Fru-1,6-P2 binding exceed those needed to support catalytic activity.     

A novel effect of 6-azauridine on growth and protein synthesis in wheat embryonic axes

Growth and the rate of protein synthesis in germinating wheat embryonic axes are inhibited by the analog 6-azauridine via a mechanism which is independent of the usual effect of this compound as an inhibitor of de novo synthesis of UTP. The effects on growth and protein synthesis can be separated from that on UTP biosynthesis by analyses of the kinetics by which each effect is maximized following a 1.5-h pulse with 6-azauridine, and by saturation of the responses at different doses of the analog. The inhibitions of growth and protein synthesis are apparently not mediated through the rate of poly A(+) RNA synthesis (reduced as little as 8%), but rather by an effect on translation. Since cordycepin reduces the azauridine inhibitions of growth and protein synthesis, it is suggested that these latter effects of 6-azauridine may depend upon the synthesis of an inhibitory azauridyl-RNA.     

Effects of density-dependent inhibition of growth on phospholipid metabolism in monolayer cultures of animal cells.

The onset of density-dependent inhibition of growth is correlated with a change in the synthesis of the major phospholipids of either BHK-21 (hamster) and 3T3 (mouse) cells. The change consists of inhibition of phosphatidylcholine synthesis while the synthesis of phosphatidylethanolamine remains constant or increases. The incorporation of radioactive precursors reflects turnover rates, since no significant differences were observed in phospholipid composition. No major differences in fatty acyl chains of these same phospholipids were observed as a function of growth rate despite considerable individuality of relative patterns of polar lipids in both cell lines. No other density-dependent differences in phospholipid metabolism were observed which were common to both cell lines. These results suggest that major aspects of growth regulation may be metabolic rather than compositional and that regulation of the enzymes of lipid metabolism may be useful probes of effector mechanisms in growth control.