Lipid Composition of Purified Vesicular Stomatitis Viruses

Methods are described for the production of vesicular stomatitis (VS) virus of sufficient purity for reliable chemical analysis. VS virions released from infected cells were concentrated and purified at least 150-fold by sequential steps of precipitation with polyethylene glycol, column chromatography, rate zonal centrifugation, and equilibrium centrifugation. The Indiana serotype (VS(Ind) virus) propagated in L-cells was found to contain 3% ribonucleic acid, 64% protein, 13% carbohydrate, and 20% lipid; the molar ratio of cholesterol to phospholipid was 0.6 or greater. Thin-layer chromatography revealed no unusual neutral lipids or phospholipids and gas-liquid chromatography revealed no unusual fatty acids incorporated into VS virions. The antigenically distinct New Jersey serotype (VS(NJ) virus) grown in L-cells showed a similar lipid profile except that the proportion of neutral lipids was larger than in VS(Ind) virus also grown in L-cells. This differences was less pronounced when the lipid composition of VS(Ind) and VS(NJ) viruses grown in chick embryo cells was compared, but VS(NJ) virus grown in either cell type always contained larger amounts of neutral lipids other than cholesterol than did VS(Ind) virus. The lipid composition of both VS(Ind) and VS(NJ) viruses grown in L-cells or chick embryo cells more closely resembled that of plasma membrane than of whole cells. A consistent finding was the relatively large amounts of phosphatidylethanolamine and sphingomyelin and the relatively small amounts of phosphatidylcholine in both VS viruses compared with uninfected whole L-cells and chick embryo cells or their plasma membranes. The methods available for isolation of plasma membranes were inadequate for conclusive comparison of the lipids of VS virions with the lipids of the plasma membranes of their host cells. Nevertheless, the data obtained are consistent with two hypotheses: (i) the lipid composition of VS viruses primarily reflects their membrane site of maturation, and (ii) the newly synthesized viral proteins inserted into cell membranes influence the proportions of phospholipids and neutral lipids selected for incorporation into the viral membrane.     

Immunological and Biophysical Separation of Dengue-2 Antigens

Antigenic compositions of slowly sedimenting dengue-2 hemagglutinin (SHA) and soluble complement-fixing antigen (SCF) were compared with the virion (rapidly sedimenting hemagglutinin, RHA) by radioimmune precipitation (RIP), RIP inhibition, kinetic neutralization, and neutralization blocking tests with the use of hyperimmune mouse ascitic fluids. RHA and SHA were unable to inhibit completely the RIP of each other by anti-RHA, and neutralization by anti-RHA was not blocked by SHA. This indicated that SHA is serologically related, but not identical, to RHA. SHA differed from RHA in that SHA lacked the “core” polypeptide but contained the two envelope polypeptides. In addition, SHA contained a polypeptide with a molecular weight of 16,500 daltons and a suggestion of several other proteins. These data, when considered with other evidence, suggest that SHA is a special form of “incomplete virus.” SCF was unable to inhibit the RIP of SHA or RHA or to block neutralizing antibodies. Further, anti-SCF did not neutralize RHA or precipitate significant levels of SHA or RHA. Polyacrylamide gel electrophoresis separated SCF from structural polypeptides by molecular size. This evidence suggests that SCF is a nonstructural antigen.     

Serological Relationships of Salmonella A Bacteriophages Isolated from Salmonellae in Different Kauffmann- White Groups

Six different Salmonella group A phages from salmonellae in Kauffmann- White groups B, C(1), C(2), and D were examined serologically. Those phages which were specific for a particular somatic antigen were found to be serologically very similar. Antiserum against a phage with one specificity was able to neutralize a different phage with the same specificity but unable to neutralize, in the normal way, a phage with a different specificity. Phages mixed with heterologous phage antiserum responded with an "inhibition response" in which there appeared to be a neutralization of the phage infectivity for the first 10 min, followed by a reversal of the neutralization until, by 20 or 25 min, there was no apparent neutralization. This response was interpreted to indicate that the adsorption antigens, probably situated on the tail fibers, were different for phages with different specificities but sufficiently similar so that heterologous antibodies could react with the antigens; but the antigen-antibody complex was quickly disassociated, resulting in a modification of the antibody molecules but no change in the specificity sites of the antigen. A subgrouping of the Salmonella A phages based on their antigenic specificity is suggested.     

Susceptibility of Differentiating Muscle Cells of the Fetal Mouse in Culture to Coxsackievirus A13

The interaction of coxsackievirus A13 with differentiating muscle cells, cultured from tissues of the fetal mouse, was studied. Cultures infected at that stage of myogenic differentiation characterized by the rapid formation of multinucleated myotubes produced maximum virus titers of over 10(7) plaque-forming units. Virus-induced cytopathic effect was characterized by a marked diminution in the number of multinucleated cells. The susceptibility of these cultures decreased appreciably when infection was initiated after the majority of the myotubes had formed. The demonstration of newly synthesized A13 virus antigen by immunofluorescence provided direct evidence that A13 virus replication occurred both in myoblasts and myotubes. The synthesis of A13 virus was markedly depressed in muscle cultures in which the formation of multinucleated cells was inhibited by BUDR or by fusion-inhibiting media. After reversal of this inhibition, the cultures acquired the increased susceptibility to A13 virus characteristic of cells undergoing myogenic differentiation. In contrast to the results obtained with coxsackievirus A13, the primary fetal mouse muscle cultures were resistant to poliovirus T1. It is suggested that changes in the surfaces of developing muscle cells may coincide with the formation and disappearance of specific virus receptors and thereby regulate the cell susceptibility to coxsackievirus A13.     

Influence des héméropériodes très courtes sur la croissance de Lolium multiflorum,sa composition pigmentaire et l'ultrastructure chloroplastique

Summary We have described some characteristics of Lolium multiflorum cultivated under very short photoperiods (2 hours and 1 hour). The estimations of leaf growth were based on dry weight, surface measurements, and chlorophyll content. The pigment analyses were carried out by column chromatography; chloroplast ultrastructure was observed after chemical fixation.These measurements have permitted us to note a sharp drop in the growth curve of plants grown under different day-lengths: the limiting photoperiod lies between 1 hour and 2 hours of daily illumination.Pigment analyses and chloroplast ultrastructure observations show that there is a greater difference between plants cultivated under 1 hour and 2 hours of daily illumination than between plants cultivated under 2 hours and 12 hours.A decrease in day-length causes a deficit in the chlorophyll b content as well as a poor development of the grana.We have attempted to correlate these structural anomalies with the abnormal chlorophyll a/chlorophyll b ratio.     

Instability of the growth-limiting proteins of the Avena coleoptile and their pool size in relation to auxin

Summary The stability and pool size of the growth-limiting proteins (GLP) of the Avena coleoptile have been studied by measuring the time required for cycloheximide to inhibit the growth of auxin-treated segments. Inhibition of growth follows inhibition of protein synthesis by 20–25 min regardless of the growth rate. This indicates that the growth inhibition is due to inherent instability of the GLP rather than to exhaustion of the pool through growth. A study of the amount and rate of auxin-induced growth which occurs when cycloheximide is added just before or after the auxin indicates that the rate of elongation is determined by the size of the GLP pool, and that the pool of GLP is low in the absence of auxin, but rapidly expands and reaches a maximum 20–25 min after addition of auxin. Three ways in which auxin might expand the pool of GLP are discussed.     

Respiratory Chain of a Pathogenic Fungus, Microsporum gypseum: Effect of the Antifungal Agent Pyrrolnitrin

Pyrrolnitrin has been reported to inhibit Bacillus megaterium primarily by forming complexes with phospholipids and to block electron transfer of Saccharomyces cerevisiae between succinate or reduced nicotinamide adenine dinucleotide (NADH) and coenzyme Q. We found that pyrrolnitrin inhibited respiration of conidia of Microsporum gypseum. In mitochondrial preparations, pyrrolnitrin strongly inhibited respiration and the rotenone-sensitive NADH-cytochrome c reductase. The rotenone-insensitive NADH-cytochrome c reductase, the succinate-cytochrome c reductase, and the reduction of dichlorophenolindophenol by either NADH or succinate were inhibited to a lesser extent. However, the activity of cytochrome oxidase was not affected by pyrrolnitrin. The extent of reduction of flavoproteins by NADH and succinate, measured at 465 - 510 nm, was unaltered; however, the reduction of cytochrome b, measured at 560 - 575 nm, was partially inhibited by pyrrolnitrin. The level of totally reduced cytochrome b was restored with antimycin A. We, therefore, concluded that the primary site of action of this antifungal antibiotic is to block electron transfer between the flavoprotein of the NADH-dehydrogenase and cytochrome b segment of the respiratory chain of M. gypseum.     

Influence of Temperature of Incubation and Type of Growth Medium on Pigmentation in Serratia marcescens

Maximal amounts of prodigiosin were synthesized in either minimal or complete medium after incubation of cultures at 27 C for 7 days. Biosynthesis of prodigiosin began earlier and the range of temperature for formation was greater in complete medium. No prodigiosin was formed in either medium when cultures were incubated at 38 C; however, after a shift to 27 C, pigmentation ensued, provided the period of incubation at 38 C was not longer than 36 hr for minimal medium or 48 hr for complete medium. Washed, nonpigmented cells grown in either medium at 38 C for 72 hr could synthesize prodigiosin when suspended in saline at 27 C when casein hydrolysate was added. These suspensions produced less prodigiosin at a slower rate than did cultures growing in casein hydrolysate at 27 C without prior incubation at 38 C. Optimal concentration of casein hydrolysate for pigment formation by suspensions was 0.4%; optimal temperature was 27 C. Anaerobic incubation, shift back to 38 C, killing cells by heating, or chloramphenicol (25 mug/ml) inhibited pigmentation. Suspensions of washed cells forming pigment reached pH 8.0 to 8.3 rapidly and maintained this pH throughout incubation for 7 days. Measurements of viable count and of protein, plus other data, indicated that cellular multiplication did not occur in suspensions of washed cells during pigment formation. By this procedure utilizing a shift down in temperature, biosynthesis of prodigiosin by washed cells could be separated from multiplication of bacteria.