Physiological and morphological characteristics of stationary phase vibrio cells able to support phase growth

Growth of phase alpha 3a on stationary phase Vibrio cultures requires micro-aerophilic conditions and is inhibited by aeration. Since pre-conditioning of the bacteria by allowing them to stand for 24 h after shaking for 3 d is an important aspect of the stationary phase phage growth system, various physiological and morphological characteristics of the stationary phase cells during the transition from shaking to standing were investigated. Shaken stationary phase cells were less viable and more sensitive to ultraviolet irradiation and heat than standing stationary phase cells. During pre-conditioning the small, non-flagellated cells present in shaken stationary phase cultures underwent morphological changes and became large, flagellated rods which resembled exponential phase cells. The transition of stationary phase cells from shaking to standing was associated with a marked increase in total RNA synthesis but a rapid and large decrease in total protein synthesis. Intracellular concentrations of ATP in shaken stationary phase cells were 53% lower than those in standing stationary phase cells. Studies on leucine uptake indicated that its transport was inhibited by isoleucine and that the major part (90%) of the total leucine uptake was due to a shared system for uptake of both amino acids. Shaken stationary phase cells transported less leucine than standing stationary phase cells. Inhibition of phage growth in aerated stationary phase cultures was not due to the prevention of phase absorption by shaking. It is suggested that the observed differences between shaken and standing stationary phase cells could be due to aeration affecting the template specificity of the Vibrio RNA polymerase.     

Primary structure of rabbit alpha-lactalbumin.

Rabbit alpha-lactalbumin was purified from the milk of New Zealand White rabbits. It was found to exist predominantly as a glycoprotein, containing 5 mol of glucosamine per mol of protein, as well as other sugars. The amino acid sequence of the protein was determined by sequenator analysis and carboxypeptidase digestion. There are 122 amino acids in the protein and a single carbohydrate moiety, probably attached to an asparagine residue at position 45. The C terminus of rabbit alpha-lactalbumin is one residue shorter than that of the other alpha-lactalbumins. Sequence comparisons indicate that the alpha-lactalbumin gene has been undergoing more frequent mutation than had previously been thought. A new method of preparative peptide mapping using 2,5-diphenyloxazole (PPO) fluor to detect peptides is described.     

Density, distribution and mobility of surface anions on a normal/transformed cell pair

The distribution and mobility of cell surface anions was investigated on low passage cultures of secondary BALB/c embryonic fibroblasts and their SV40-transformed counterparts (VLM cells) using polycationized ferritin (PCF) as a label. While the absolute number of anions/nm² of membrane was equivalent on the two cell types, the topographical distribution and mobility of these anions was strikingly different. After pulse-labeling with PCF at 37 °C, anions on BALB/c fibroblasts occurred in large piled-up clusters separated by extensive areas of membrane ( = 0.47 μm) free of negative charges. Labeling at 4 °C reduced the degree of “piling up” within the clusters, but the intercluster spacing was maintained indicating that the anions have short-range mobility in the membrane and can be cross-linked into a tight lattice at 37 °C. These anions do not, however, demonstrate any long-range mobility during a 20 min post-label incubation in PBS. In contrast, anions on VLM cells are inherently present in a random configuration of microclusters and single anions with relatively small ( = 0.09 μm) intervening areas of low charge density. Short-range mobility of surface anions is not displayed, presumably as a result of the inter-site distance, but long-range mobility is indicated by the formation of large site clusters following a 20 min incubation in PBS after pulse-labeling. Very mild proteolysis of BALB/c fibroblasts induces a change in the topography of surface anions toward the random configuration typical of VLM cells. These data are discussed in relation to altered social interactions between tumor cells which may be influenced by cell-cell adhesion characteristics.     

Pyruvate metabolism after in vivo exposure to oral arsenic

This study investigated altered pyruvate metabolism after prolonged oral arsenic exposure. Male rats were given access to deionized drinking water containing 0, 40 or 85 ppm sodium arsenate (As⁵⁺) for 3 weeks. Respiration studies with mitochondria isolated from treated animals indicated decreased state 3 respiration (with ADP) and decreased respiratory control ratios (RCR) for pyruvate/malate-mediated respiration, but not for succinate-mediated respiration, as compared to control respiration values. In addition, pyruvate dehydrogenase activity was measured, in both liver and intestine, before and after Mg-activation in vitro. After 3 weeks, the effects of arsenic at the highest dose level were pronounced on the basal pyruvate dehydrogenase activity (before activation) as well as the total pyruvate dehydrogenase (after activation). The inhibition of pyruvate dehydrogenase activity both before and after Mg-activation suggests an arsenic effect on mitochondrial pyruvate metabolism which, in part, involves inhibition of pyruvate decarboxylase. Evidence is also presented which may indicate an arsenic effect on the kinase and/or phosphatase which regulate pyruvate dehydrogenase activity.     

Interference by unstable nucleotides in the study of enzymes that cleave N-glycoside bonds in nucleic acids

The guanylation of tRNA involves the excision of a base from within the polynucleotide chain by cleavage of the N-glycoside bond. The excised base is replaced by guanine. During studies using chemically tritiated tRNA to study the guanylation reaction, spurious radioactivity was released from tRNA. We have identified the substance released as the hypermodified compound known as Y base. Our report (i) warns workers, who are studying enzymes that excise bases at the polynucleotide level, against this pitfall; (ii) indicates how to determine if counts released from RNA or DNA are spurious; and (iii) describes methods that avoid release of spurious counts while studying enzymes that cleave N-glycoside bonds in polynucleotides.     

PfkA locus of Escherichia coli.

pfkA was know, on the basis of three mutants, as the likely locus of phosphofructokinase in Escherichia coli, and the unlinked pfkB1 mutation suppressed these mutations by restoring some enzyme activity (Morrissey and Fraenkel, 1972). We now report a new search for the complete inactivation of pfkA (e.g., by deletion or amber mutation), done to assess whether the pfkB1 suppression is by an independent enzyme, phosphofructokinase activity 2 (Fraenkel, Kotlarz, and Buc, 1973). Ten new phosphofructokinase mutants all were at pfkA, rather than at pfkB or pfkC. One of them (pfkA9) gave temperature-sensitive reverants with heat-labile enzyme. Another (pfkA11) proved genetically to be a nonsense mutation, but showed no restored activity when suppressed by supF. However, even unsuppressed it was found to contain an enzyme related to phosphofructokinase activity 1 kinetically (more allosteric), physically (almot identical subunit), and antigenically. All the pfkA mutants apparently contained cross-reacting material to activity 1. All (including pfkA11) were suppressed by the pfkB1 mutation. Several results support the idea that pfkA is the structural gene for the main phosphofructokinase of E. coli (activity 1), but that there is some restriction to its complete inactivation.     

Ovary-autonomous maternal inheritance at a developmental locus in Drosophila

The sex-linked temperature-sensitive mutation shibire^ts of Drosophila melanogaster shows a maternal effect causing embryonic lethality at 29°C. The maternal influence is due to gene action autonomous to the ovary. Embryos carrying the paternally derived wild-type gene can survive at 29°C but only if heat pulses are begun at least 9 hr after oviposition. The paternal rescue is presumably due to zygotic gene action at this locus beginning part way through embryogenesis. A maternal wild-type genome, however, can produce shi embryos that have sufficient shi⁺ product to support embryogenesis up to the hatching stage even at 29°C.     

Unstable generalized transduction in Achromobacter.

Six auxotrophic markers of a halotolerant collagenolytic strain of Achromobacter were transduced by four alpha hages. Abortive transduction was also demonstrated. The generalized transduction system is unusual as the transductants were unstable, characteristic of transduction by lysogeny. The Achromobacter strain is a cryptic lysogen for alpha and purified transductants were either sensitive or resistant to alpha. Purified clones from four resistant transductants and one sensitive transductant liberated phage spontaneously. The host ranges of these spontaneous phage differed from that of the alpha phage used for the transduction experiment. Some initially resistant transductants became simi-sensitive to alpha (efficiency to plating) e.o.p. (10minus-1 to 10minus-2) after repeated cloning.