Optimization of growth conditions for the production of proteolytically-sensitive proteins in the periplasmic space of Escherichia coli

Summary The expression of many secreted recombinant proteins in Gram-negative bacteria is limited by degradation in the periplasmic space. We have previously shown that the production of protein A--lactamase, a secreted fusion protein highly sensitive to proteolysis in Escherichia coli, can be increased in mutant strains deficient in up to three cell-envelope-associated proteolytic activities. In this work we investigated the effect of fermentation conditions on suppressing any residual proteolytic activity in various protease-deficient strains. Optimal production of the fusion protein was observed in cells grown under mildly acidic conditions (5.5pH6.0) and at low temperatures. These conditios were shown to specifically decrease the rate of proteolysis. In addition, a further increase in production was observed in cultures supplemented with 0.5 to 0.75 mM zinc chloride. This may relate to the inhibition of a cell envelope protease by Zn²⁺ ions. Offsprint requests to: G. Georgiou     

Biotransformation of 2-acetylthiophene by micromycetes

Summary The biotransformation of 2-acetylthiophene by 800 strains of micromycetes has been investigated. Among them, 460 strains have been selected on solid media and cultivated in a liquid synthetic medium. Of these, 106 strains were able to completely deplete 2-acetylthiophene with or without production of intermediary metabolites. 2-Thienylglyoxylic acid was not detected but 72 strains produced 2-thiophenecarboxylic acid. Among them, eight strains have been selected for further optimization of this bioconversion.     

Expedient syntheses of neoglycoproteins using phase transfer catalysis and reductive amination as key reactions

Starting from peracetylated chloro- or bromo-glycosyl donors ofN-acetylneurmainic acid,N-acetylglucosamine, glucose and lactose, the correspondingp-formylphenyl glycosides were synthesized stereospecifically under phase transfer catalysed conditions at room temperature in yields of 38–67%. After Zemplén de-O-acetylation, the formyl groups were directly and chemoselectively coupled to the lysine residues of bovine serum albumin by reductive amination using sodium cyanoborohydride. The conjugation reactions were followed as a function of time and under a series of different molar ratios of the reactants to provide glycoconjugates of varying degree of antigenicities. Thus, carbohydrate protein conjugates were made readily available using essentially two key reactions.Presented in part at the 15th International Carbohydrate Symposium, Yokohama, Japan, August 12–17, 1990.     

Purification and characterization of G proteins from human brain: modification of GTPase activity upon phosphorylation

Summary Three G proteins from human brain membranes were purified to near homogeneity by conventional techniques including preparative electrophoresis. These G proteins were characterized by their ability to bind GTP, GDP and GTP analogs. Two of these proteins have molecular weights of 50,000 (G₅₀) and 36,000 (G₃₆), as determined on SDS-gels. G₃₆ was ADP-ribosylated by pertussis toxin. Thus, G₅₀ could represent a G_sα subunit, whereas G₃₆ could be G_iα or G_oα. G₅₀ was phosphorylated by cAMP dependent protein kinase and protein kinase C. G₃₆ was phosphorylated by a protein kinase independent of calcium and phospholipid, a proteolytic product of protein kinase C, analogous to protein kinase M. Phosphorylation of G₃₆ by this protein kinase induced a dramatic decrease in its GTPase activity. The third G protein, of molecular weight 22,000 probably belongs to the group of monomeric G proteins possessing functional similarities withras gene products. The regulation of G proteins involving calcium-dependent and independent pathways is delineated.     

Spectrum of phenylketonuria mutations in Western Europe and North Africa,and their relation to polymorphic DNA haplotypes at the phenylalanine hydroxylase locus

Summary A total of 252 chromosomes from 126 patients with phenylalanine hydroxylase (PAH) deficiencies were analyzed for both mutant genotypes and restriction fragment length polymorphism (RFLP) haplotypes at the PAH locus. The mutant genes studied originated either from Western Europe (116 alleles) or from Mediterranean countries (136 alleles). Only 27% of all mutant alleles were found to carry identified mutations, particularly mutations at codon 252 (2.3%), 261 (7.5%), 280 (6.3%), 408 (3.5%) and at the splice donor site of intron 12 (6.3%). The mutant genotypes were associated with RFLP haplotypes 7, 1, 38, 2 and 3 at the PAH locus respectively. Except for the splice mutation of intron 12, these associations were preferential, but not exclusive, since the other four mutations were found on the background of at least two RFLP haplotypes. These results, together with the observation that 85% of PAH deficient patients are heterozygotes for their mutant genotypes, emphasize the great heterogeneity of PAH deficiencies in Mediterranean countries and hamper systematic DNA testing for carrier status in this population.     

Total biodegradation of 4-chlorobiphenyl (4 CB) by a two-membered bacterial culture

Summary Several bacterial strains that can oxidize mono- and dichlorinated biphenyls with one unsubstituted ring have already been described. The major route for this biodegradation leads ultimately to the corresponding chlorobenzoic acid, but several other minor chlorinated metabolites that might possibly be of concern for the environment have also been described previously. Since none of the bacterial strains that are able to oxidize these chlorinated biphenyls in pure culture are known to degrade chlorobenzoic acid, the oxidation of these substrates by axenic cultures always generates chlorobenzoates plus several other metabolites. In the present study, we have estimated the biodegradation of 4-chlorobiphenyl (4CB) by a two-membered bacterial culture containing one strain able to grow on 4CB and to transform it into 4-chlorobenzoate (4CBA) and one strain able to degrade 4CBA. The results were encouraging, since it was shown that the degradation of 4CB was more rapid and complete with the double bacterial culture.     

An immunocytochemical study of the optic ganglia of the crayfish Astacus leptodactylus (Nordmann 1842) with antisera against biologically active peptides of vertebrates and invertebrates

Summary The peptidergic system in the optic ganglia of Astacus leptodactylus is characterized by the immunocytochemical application of 15 antisera raised against biologically active peptides of vertebrates and invertebrates. Positive reactions were found with anti-FMRFamide, antiMSH, anti-vasotocin, anti-gastrin, anti-CCK, anti-oxytocin, anti-secretin, anti-glucagon and anti-GIP. Based on immunochemical reaction and localization it is possible to distinguish 30 cell groups. Only part of these cell groups is found in the known classical neurosecretory cell regions. This observation demonstrates a more extensive peptidergic system than formerly recognized. The morphology of this peptidergic system suggests that one part is neurohormonal and the other part neurotransmitter-like or neuromodulatory.     

Serological survey of T-lymphocyte differentiation antigens in wild mice

Allelic distributions of Thy-1, Ly-l, and Ly-2 antigens in wild mice are characteristic of each Mus musculus subspecies. Eastern mice (M.m.molossinus, M.mmusculus, M.m.castaneus, M.m.bactrianus) express the Thy-1.1 antigen, whereas Western mice (M.m. domesticus, M.m.brevirostris) express the Thy-1.2. All mice from wild populations examined in this survey express the Ly-1.2. The Ly-2.1 is distributed in Eastern mice and some Western mice, and the Ly-2.2 is found in the remaining Western mice. Allelic distributions of these antigens were also examined in two other species, Mus spretus and Mus spicilegus. Allelic constitutions of Thy-1 and Ly-1 in these species are similar to those of Eastern mice. Some M.spicilegus, however, express the Ly-1.1 antigen. This antigenic type is not found in M.musculus. Some Eastern mice related to M.m.castaneus react weakly to Ly-1.2-specific and Ly-2.1-specific monoclonal antibodies in both the complement-mediated cytotoxicity test and the absorption test. These results suggest that M.m.castaneus has unique alleles in the Ly-1 and Ly-2 loci.     

Ovarian and fat body protein concentrations in Ips sexdentatus (Coleoptera: Scolytidae) parasitized by nematodes

The protein concentrations of fat body and ovaries in Ips sexdentatus either uninfected or infected by Parasitaphelenchus sp., P. sexdentati, or Contortylenchus diplogaster were measured at various stages of insect development, from preswarming maturation to the first oviposition (24 hr after mating). Weight variations of the fat body and ovaries in insects infected by C. diplogaster show the same evolution as those observed in uninfected insects, but at a much lower level. Fat body proteins in uninfected insects reach their minimum level during swarming, but they remain fairly constant throughout the maturation of the first egg. After dropping shortly after swarming, the ovarian protein level in such insects increases in two stages during ovarian maturation. The first stage, which corresponds to a slow protein incorporation, takes place during the first 12 hr after mating. During the second stage, i.e., beyond 12 hr, a significant level of proteins is rapidly incorporated into the ovaries. In insects infected by Parasitaphelenchus fat body proteins are reduced and protein incorporation into the ovaries is reduced; Parasitaphelenchus would thus affect at least some proteins required for ovarian maturation in their host. Fat body protein levels are even more affected by C. diplogaster than by Parasitaphelenchus, while incorporation into ovaries seems to be less affected in spite of slower ovarian growth. C. diplogaster might thus essentially act both upon proteins which are not required for the ovarian maturation of their host and upon nonproteinaceous substances that are required for such maturation. Results are discussed in relation to the possible mode of action of parasitic nematodes.