Improved Agar Bottle Plate for Isolation of Methanogens or Other Anaerobes in a Defined Gas Atmosphere

A bottle plate for the cultivation of methanogens or other organisms in a defined pressurized-gas atmosphere was developed. The bottle provides the convenience of an agar streak plate, solves the problem of the water exudate from agar medium, and provides a convenient way of adding or sampling a defined gas atmosphere.     

The alpha-glucosyltransferases of bacteriophages T2, T4 and T6. A comparison of their primary structures

Summary With the aim of comparing the primary structures of gene products coded for by T-even bacteriophages we constructed clone libraries of the DNAs of bacteriophages T2 and T6. Using hybrid M13 phages carrying the gene for the T4-coded -glucosyl transferase (gt) we isolated corresponding T2 and T6 clones. The nucleotide sequences of the three gt genes and the amino acid sequences derived were compared. The differences between the genes and their products are discussed in terms of structure, function and evolutionary aspects.Abbreviations bp base pair - gt glucosyl transferase - HMC 5-hydroxymethyl cytosine - orf open reading frame - Xgal 5-bromo-4-chloro-3-indolyl--d-galactoside     

Symplastic Transport in Ipomea tricolor Source Leaves : Demonstration of Functional Symplastic Connections from Mesophyll to Minor Veins by a Novel Dye-Tracer Method

A novel method for the delivery of the fluorescent dye Lucifer Yellow CH to the cytosol of a source leaf mesophyll cell was devised which utilized a preencapsulation of the dye in phospholipid vesicles (liposomes). The liposomes were easily injected into the vacuoles of leaf cells of Beta vulgaris or Ipomea tricolor, where fusion with the tonoplast resulted in the release of the dye into the cytosol. Subsequent cell-to-cell movement of the dye was readily followed by fluorescence microscopy. Using this liposome technique symplastic continuity from the the mesophyll to the minor veins of the source leaf of Ipomea tricolor was demonstrated. This agreed with ultrastructural studies which demonstrated the presence of plasmodesmata between all cells from the mesophyll to the minor veins. The symplastic movement of dye from the injected mesophyll cell to the minor veins was unaffected by pretreatment of the leaf tissues with 2 millimolar p-chloromercuribenzenesulfonic acid. Pretreatment of the leaf tissues at alkaline pH (3-[N-morpholino] propanesulfonic acid-KOH, pH 8.0) had no apparent effect on dye movement between adjacent mesophyll cells but inhibited the movement of dye into and along the minor veins. Thus, although there were no apparent barriers to symplastic solute movement in this leaf, symplastic barriers could be imposed by the experimental conditions used.     

Thyrotropin-releasing hormone activates a Ca2+-dependent polyphosphoinositide phosphodiesterase in permeable GH3 cells. GTP gamma S potentiation by a cholera and pertussis toxin-insensitive mechanism

Numerous hormones are known to rapidly activate polyphosphoinositide turnover in target cells by promoting phosphodiesteratic cleavage of the phospholipids; however, little is known about the enzymology of receptor-mediated phosphoinositide breakdown. In the present study, thyrotropin-releasing hormone (TRH) stimulation of polyphosphoinositide turnover has been characterized in electrically permeabilized, [3H]myoinositol-labeled GH3 cells. The permeable cells allow the influence of small molecular weight (Mr less than or equal to 1000) cofactors to be determined. We present evidence for the following: 1) TRH stimulates inositol phosphate generation in permeable cells; 2) optimal hormone-stimulated inositol phosphate generation requires Mg2+, ATP, and Ca2+; 3) Mg2+ and ATP requirements reflect polyphosphoinositide kinase reactions; 4) in the absence of MgATP, TRH stimulates the phosphodiesteratic breakdown of pre-existing polyphosphoinositides in a reaction which requires only low Ca2+ (10(-7) M); 5) hormone activation is potentiated in the presence of the stable guanine nucleotide, GTP gamma S; neither TRH-stimulated nor GTP gamma S-potentiated hydrolysis is inhibited by cholera or pertussis toxin treatment. These results demonstrate that hormone-induced phospholipid hydrolysis involves activation of a phosphoinositide phosphodiesterase; activation results in lowering the Ca2+ requirement of the phosphodiesterase such that maximal activity is observed at Ca2+ levels characteristic of a resting cell (10(-7) M). Furthermore, TRH regulation of polyphosphoinositide hydrolysis is modulated by guanine nucleotides; however, nucleotide regulation appears to involve a GTP-binding factor (Np) other than Ns or Ni.     

Gastrointestinal peptides and the adaptation to extrauterine nutrition

The adaptation to extrauterine nutrition involves complex physiological changes at birth which may be regulated by genetic endowment; enteral nutrients, secretions, and bacteria; and endogenous hormones and exogenous hormones in breast milk. The hypothesis is explored that enteral feeding after birth may trigger key adaptations in the gut and in metabolism partly through the mediation of gastrointestinal hormone secretion. Gut peptides are found in the early human fetal gut and by the second trimester some are found in high concentrations in the fetal circulation and amniotic fluid. Major plasma hormonal surges occur during the neonatal period in term and preterm infants: notably in enteroglucagon, gastrin, motilin, neurotensin, gastrointestinal peptide, and pancreatic polypeptide. These events do not occur in neonates deprived of enteral feeding. A progressive development of dynamic gut hormonal responses to feeding is observed. The pattern of gut endocrine changes after birth is influenced by the type and route of feeding. Potential pathophysiological effects of depriving high risk neonates of enteral feeding are considered. It is speculated that infants committed to prolonged total parenteral nutrition from birth may benefit from the biological effects of intraluminal nutrients used in subnutritional quantities.     

Stimulation by calcium and carbamoylcholine of the ouabain-sensitive uptake of 86Rb+ in isolated rat pancreatic acinar cells

The uptake of 86Rb+ was assayed in isolated rat pancreatic acinar cells to determine the effect of calcium and carbamoylcholine on the ouabain-sensitive and ouabain-insensitive components. The presence of calcium in the medium bathing the cells during the preincubation and the main incubation periods was needed to preserve in optimum conditions the uptake of 86Rb+, the stimulation by carbamoylcholine and the sensitivity to ouabain. In the presence of calcium, the ouabain-sensitive component of 86Rb+ uptake was higher than the ouabain-insensitive. The ouabain-sensitive component was 3-times lower in cells incubated in a medium lacking calcium and containing 1 mM EGTA, as compared to cells incubated in the presence of calcium. Carbamoylcholine, at 5 X 10(-4) M, stimulated the uptake of 86Rb+ and this effect depended on the presence of calcium in the bathing medium. Maximal stimulation by carbamoylcholine was reached at 0.2 mM calcium. The nett stimulation by carbamoylcholine was inhibited up to 85% by 1 mM ouabain. As judged by digitonin-disruption of plasma membrane, the above-indicated effects were limited to a cytoplasmic pool of 86Rb+ and a leaky plasma membrane could be ruled out. The results suggest that in rat pancreatic acinar cells, carbamoylcholine stimulated the ouabain-sensitive uptake of 86Rb+ and required the presence of calcium in the bathing medium.     

Isolation and characterization of butanol-resistant mutants of Clostridium acetobutylicum.

In a wild-type strain of Clostridium acetobutylicum isolated from soil, solvent production appeared limited by butanol toxicity. Butanol-resistant mutants have been obtained which produced significantly higher solvent concentrations (about 30%) than the wild-type strain. Some other physiological differences were observed between a selected resistant mutant and the wild-type strain at the level of solvent resistance and sporulation.     

Cytochemical study of macrophage lysosomal inorganic trimetaphosphatase and acid phosphatase

Cytochemical investigations have associated acid inorganic trimetaphosphatase (TMPase) activity with the lysosomes of certain cell types. We have used the modified staining technique of Berg to show that this enzyme activity is present in normal mononuclear phagocytes and macrophage cell lines. We have found this enzyme activity to be present in murine RAW264 macrophages, in human U937 macrophages, in normal human blood monocytes, and in guinea pig peritoneal macrophages. All of the RAW264 and U937 macrophages showed intense TMPase activity. Many of the human monocytes and most of the guinea pig macrophages were labeled by this method. The reaction product was associated with the lysosomes of these cell types. The lysosomal staining-pattern was similar to that of acid phosphatase. Differences with regard to Golgi staining were noted. This indicates that TMPase is a lysosomal enzyme of mammalian macrophages. The distinction between TMPase and acid phosphatase activity has been demonstrated by measuring the pH optimum of each enzyme. Using substrates identical to those of the ultrastructural cytochemistry, we show that the pH optimum of TMPase is 4.0 and that of acid phosphatase is 5.0. The enzymatic activities are therefore ultrastructurally and biochemically distinct. Following phagocytosis of latex, yeast (Saccharomyces cerevisiae), or Corynebacterium parvum, TMPase has been found to be associated with phagosomes. This enzyme may take part in the degradation of phagocytosed materials, particularly microorganisms which contain inorganic polyphosphates and metaphosphates.     

Zymomonas mobilis mutants blocked in fructose utilization

Summary A mutant ofZymomonas mobilis deficient in the utilization of fructose for growth and ethanol formation was shown to lack fructokinase activity. When grown in media which contained glucose+fructose or sucrose, both the mutant and wild type produced sorbitol in amounts up to 60 g·l^-1, depending on the initial concentrations of sugars. Sorbitol formation was accompanied by an accumulation of acetaldehyde, gluconate, and acetoin. A ferricyanide-dependent sorbitol dehydrogenase could be localized in the cell membrane; it thus resembles the sorbitol dehydrogenase ofGluconobacter suboxydans. Neither a NAD(P)H dependent reduction of fructose nor a NAD(P) dependent dehydrogenation of sorbitol could be detected in cell-free extracts. The use of fructose-negative mutants ofZ. mobilis for the enrichment of fructose in glucose+fructose mixtures is discussed.     

Nitrogenase activity in the non-heterocystous cyanobacterium Oscillatoria sp. grown under alternating light-dark cycles

The non-heterocystous cyanobacterium Oscillatoria sp. strain 23 fixes nitrogen under aerobic conditions. If nitrate-grown cultures were transferred to a medium free of combined nitrogen, nitrogenase was induced within about 1 day. The acetylene reduction showed a diurnal variation under conditions of continuous light. Maximum rates of acetylene reduction steadily increased during 8 successive days. When grown under alternating light-dark cycles, Oscillatoria sp. fixes nitrogen preferably in the dark period. For dark periods longer than 8 h, nitrogenase activity is only present during the dark period. For dark periods of 8 h and less, however, nitrogenase activity appears before the beginning of the dark period. This is most pronounced in cultures grown in a 20 h light – 4 h dark cycle. In that case, nitrogenase activity appears 3–4 h before the beginning of the dark period. According to the light-dark regime applied, nitrogenase activity was observed during 8–11 h. Oscillatoria sp. grown under 16 h light and 8 h dark cycle, also induced nitrogenase at the usual point of time, when suddenly transferred to conditions of continuous light. The activity appeared exactly at the point of time where the dark period used to begin. No nitrogenase activity was observed when chloramphenicol was added to the cultures 3 h before the onset of the dark period. This observation indicated that for each cycle, de novo nitrogenase synthesis is necessary.