Analogs of diaminopimelic acid as inhibitors of meso-diaminopimelate decarboxylase from Bacillus sphaericus and wheat germ

Analogs (1----6) of diaminopimelic acid have been synthesized and tested for inhibition of meso-diaminopimelate decarboxylases from Bacillus sphaericus IFO 3525 and from wheat germ (Triticum vulgaris). Difluoromethyl diaminopimelate 1 does not irreversibly inactivate or strongly competitively inhibit either enzyme. Lanthionine sulfoxides (2ab, 2c, and 2d) are good competitive inhibitors (about 50% inhibition at 1 mM) of both decarboxylases. The meso and LL-isomers of lanthionine sulfone (3ab and 3c) and lanthionine (6ab and 6c) are weaker competitive inhibitors (about 50% inhibition at 10-20 mM). The corresponding DD-isomers (3d and 6d) are less effective. The N-modified analogs are the most potent competitive inhibitors. The inhibition constant (Ki) values for B. sphaericus and wheat germ decarboxylases with N-hydroxydiaminopimelate 4 (mixture of isomers) are 0.91 and 0.71 mM, respectively; for the N-aminodiaminopimelate 5 (mixture of isomers) the Ki values are 0.10 and 0.084 mM, respectively. These N-modified analogs do not effectively inhibit L-lysine decarboxylase. None of the compounds showed any time-dependent inactivation of the decarboxylases, in contrast to behavior of other pyridoxal phosphate-dependent enzymes with analogous substrate derivatives. Possible mechanisms of inhibition are discussed. In preliminary tests for antibiotic activity 4 and 5 both gave 75% growth inhibition of Bacillus megaterium at 20 micrograms/ml in defined media. Other analogs (1----3) showed essentially no antibacterial activity.     

Phosphatidyl glycerolphosphate serves as glycerolphosphate donor in polymer synthesis

Phosphatidyl glycerolphosphate was found to serve as the glycerolphosphate donor for polymer synthesis. When CDP-diglyceride and radiolabeled glycerolphosphate were incubated with the membrane enzyme prepared from Streptococcus sanguis, active syntheses of radiolabeled lipids and polymers were observed. The synthesis of polymer was not inhibited by low concentration of unlabeled phosphatidylglycerol. When [3H, 32P]glycerolphosphate was used, the polymer synthesized contained both 3H and 32P. The lipids formed were characterized as phosphatidylglycerol and phosphatidyl glycerolphosphate. The polymers formed from the latter were characterized as lipoteichoic acid like compounds by sodium dodecylsulfate-polyacrylamide gel electrophoresis.     

A comparison of techniques for isolation of the outer membrane proteins of Haemophilus influenzae type b

We compared several rapid techniques used for extraction of outer membrane proteins from gram-negative enteric bacteria to Haemophilus influenzae type b. After lysis of cells with a French press, the inner and outer membranes were separated by isopycnic centrifugation. Each membrane was identified by density, morphology, enzymatic activity, and susceptibility to solid-phase iodination of intact cells. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we identified 10 polypeptides which were enriched in the outer membrane band compared to the inner membrane band. Using these proteins, we compared the polypeptide pattern of outer membranes with that obtained by (1) selective solubilization with sodium dodecyl-beta-D-maltoside, octyl-beta-D-glucopyranoside, Triton X-100, sodium, or cholamidopropyl dimethylaminopropanesulfonate; (2) extraction with chaotropic agents and heat; and (3) differential centrifugation of vesicles shed during transition from log growth phase to stationary growth phase. There were definable differences between the polypeptide pattern of membranes obtained with each rapid technique compared to the polypeptide pattern of isolated outer membranes. The polypeptide pattern of lithium extracts and the Triton X-100 insoluble fractions of total membranes most closely approximated the polypeptide pattern of isopycnically isolated outer membranes. Depending on the outer membrane protein sought, one of these rapid techniques can be utilized when a rapid method of outer membrane protein isolation is required.     

Synthesis of cytoskeletal and contractile proteins by cultured IMR-90 fibroblasts

Models of the assembly of cytoskeletal and contractile proteins of eukaryotic cells require quantitative information about the rates of synthesis of individual component proteins. We applied the dual isotope technique of Clark and Zak (1981, J. Biol. Chem., 256:4863-4870) to measure the synthesis rates of cytoskeletal and contractile proteins in stationary and growing cultures of IMR-90 fibroblasts. Fibroblast proteins were labeled to equilibrium with [14C]leucine over several days, at the end of which there was a 4-h pulse with [3H]leucine. Fractional synthesis rates (percent per hour) were calculated from the 3H/14C ratio of cell protein extracts or protein purified by one- or two-dimensional polyacrylamide gel electrophoresis and the 3H/14C ratio of medium-free leucine. The average fractional synthesis rate for total, SDS- or urea-soluble; Triton-soluble; and cytoskeletal protein extracts in stationary cells each was approximately 4.0%/h. The range of values for the synthesis of individual proteins from total cell extracts or cytoskeletal extracts sliced from one-dimensional gels was similar, though this range was greater than that for major proteins of Triton-soluble protein extracts. Three specific cytoskeletal proteins--actin, vimentin, and tubulin--were synthesized at similar rates that were significantly slower than the average fractional synthesis rate for total protein. Myosin, on the other hand, was synthesized faster than average. Synthesis rates were the same for beta-and gamma-actin and polymerized (cytoskeletal extract) vs. Triton-soluble actin. The same was true for alpha- and beta-tubulin and two different forms of vimentin. Synthesis rates were uniformly higher in growing cells, though the same pattern of differential rates was observed as for stationary cells. Synthesis rates in growing cells were higher than the rate necessary to maintain the growth rate, even for those cytoskeletal proteins being synthesized slowly. Therefore, there appears to be some turnover of these cytoskeletal elements even during growth. We conclude that proteins in cytoskeletal extracts may have nonuniform rates of synthesis, but at least one important subclass of cytoskeletal proteins that comprise filament subunits have the same synthesis rates.     

Transposon donor plasmids, based on ColIb-P9, for use in Pseudomonas putida and a variety of other gram negative bacteria

Summary The properties of pLG221, a derivative of the ColIb plasmid carrying the transposon Tn5 are described. This plasmid can be used to introduce Tn5 by conjugation from Escherichia coli into a variety of Gram negative bacteria outside the host range for maintenance of ColIb. Plasmid pLG221, and a similar plasmid pLG223 carrying Tn10 may be of general utility as vectors for transposon-mediated mutagenesis in a variety of Gram negative bacteria.     

Initial stages in the course of adventitious bud formation on embryos of Picea abies

Adventitious buds on embryos of Picea abies (L.) Karst. developed after a pulse treatment with 250 μ M benzyladenine (BA) of pH 5.5 for 2 h. Light and temperature regimes were not critical during the initial stages. Adventitious buds developed faster after a pulse treatment and the variation among different experiments was lower compared to when the embryos were cultured on media supplemented with BA. Various stages of the differentiation of adventitious buds were identified: stage 1 - appearance of meristematic centres (approximately the first two weeks); stage 2 - development of adventitious bud primordia (approximately the third week); stage 3 - adventitious bud development (from approximately the 4th to the 8th week). This system may be used for further studies on bud differentiation.     

Initiation and development of wound tissue and roots on hypocotyl cuttings of Pinus sylvestris in vitro

The rooting of hypocotyl cuttings from 20-day-old seedlings of Pinus sylvestris L. cultured in vitro is discussed. About 40% of the cuttings cultured on medium lacking activated charcoal produced roots during the first two months. When activated charcoal was added to the medium, either root formation (75% formed roots) or wound tissue growth (95% formed large wound tissues) was stimulated in different experiments. These large wound tissues did not develop any roots. The anatomical changes in the basal part of the cuttings were similar during the first two weeks in all the cuttings studied. A vascular cylinder composed of short tracheids with many pores developed. Thereafter the differentiation process became varied. The amount of wound tissue produced and the time for rooting differed among the cuttings. Tracheid nests which were in contact with the vascular system in the hypocotyl via short tracheids were observed after three weeks. Subsequently, roots developed from the tracheid nests. The longer root formation was delayed, the larger the wound tissue became.
Short tracheids were found close to the wound tissue surface. Their ability to adsorb nutrients and water is discussed.     

Immunochemical Characterization of Pyruvate Dehydrogenase Complex in Rat Brain

Pyruvate dehydrogenase complex (PDHC) in rat brain was studied immunochemically, using antibodies against the bovine kidney PDHC, by immunoblotting, immunoprecipitation, inhibition of enzyme activity, and enzyme-linked immunoabsorbent assay (ELISA). The immunoblots showed that the antibodies bound strongly to the alpha peptide of the pyruvate dehydrogenase (E1) component, and to the dihydrolipoyl transacetylase (E2) and the dihydrolipoyl dehydrogenase (E3) components of PDHC. A similar immunoblotting pattern was observed in all eight brain regions examined. On immunoblotting of the subcellular fractions, these PDHC peptides were observed in mitochondria and synaptosomes but not in the postmitochondrial supernatants. This agrees with other evidence that brain PDHC is localized in the mitochondria. These results, together with those from sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the immunoprecipitin, also showed that the alpha E1, beta E1, and E3 peptides of rat brain PDHC are very similar in sizes to those of the bovine kidney PDHC, being 42, 36, and 58 kD, respectively. The size of the E2 peptide, 66 kD, is different from that of bovine kidney E2, 73 kD. The relative abundance of PDHC protein in nonsynaptic mitochondria was compared by enzyme activity titration and ELISA. Both methods demonstrated that the amount of PDHC antigen in the mitochondria from cerebral cortex is greater than that in the olfactory bulb mitochondria. This is consistent with the results of the activity measurement. The ELISA also showed that the PDHCs in both mitochondrial populations are antigenically similar.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of a cold stimulus on the synthesis of uncoupling protein in brown adipose tissue of rats and newborn rabbits

Rats are known to respond to a cold stimulus by increasing the activity and amount of the uncoupling protein in brown adipose tissue. A 48 h cold stimulus was found to increase the synthesis of uncoupling protein 3.g-fold in 4–5 week old rats whereas no change was observed with newborn rabbits. The lack of response in the latter case may reflect a difference between rabbits and rats or that synthesis is already maximal in newborn rabbits.     

Inhibition of chlorophyll synthesis inHordeum vulgare by 3-amino 2,3-dihydrobenzoic acid (gabaculin)

Gabaculin (3-amino 2,3-dihydrobenzoic acid) is shown to be a very potent inhibitor of chlorophyll formation inHordeum vulgate. Exposure of leaf segments to 30/M gabaculin results in an 80% inhibition of chlorophyll synthesis, and this is paralleled by a decrease in carotenoid. Dual-inhibitor studies with dioxoheptanoic acid, which is an inhibitor of inolaevulinic acid dehydratase, show that gabaculin inhibits an earlier step than dioxoheptanoic acid and affects -aminolaevulinic acid synthesis rather than its subsequent metabolism.