Targeting and fusion in vesicular transport

Vesicular transport of proteins and lipids between distinct subcellular compartments is directly responsible for generating and maintaining the structure of the organelles of the secretory and endocytic pathways in eukaryotic cells. Rapid advances in a variety of experimental systems have resulted in the identification of molecules involved in late steps of the transport process. This article presents a general paradigm for vesicular fusion and reviews the available experimental evidence.     

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.     

Effects of t-butyl-4-hydroxyanisole and other phenolic antioxidants on tumoral cells and Trypanosoma parasites

The antioxidant food additives 2(3)-tert-butyl-4-hydroxyanisole (BHA), 2,6-di(tert-butyl)-p-cresol (BHT) and the methyl and propyl esters of gallic acid inhibited Trypanosoma cruzi culture growth and oxygen consumption. The I50 values for growth and oxygen uptake with BHA were 0.284 and 0.400 and for BHT 0.083 and 0.235 mM, respectively. Moreover, BHA inhibited the respiration of several tumor cells, as well as of the procyclic and bloodstream trypomastigote forms of T. brucei brucei, with I50 in the range 0.29-0.52 mM. Inhibition of the parasites' oxygen uptake by BHA was not of the pure Michaelis-Menten type, but may be of a mixed form. It is postulated that these compounds are inhibitors because they resemble ubiquinone.     

Effect of nitrogen form on the growth and nitrate concentration of lettuce

Summary A pot experiment with lettuce involving three N forms each at six application levels, showed that lettuce can be grown satisfactorily with a very low nitrate content when supplied with ammonium sulphate and a nitrification inhibitor. For plants growing on nitrate N, the optimum midrib sap nitrate concentration as maturity approached was about 1400 mg/1 NO₃-N. Large losses of mineral N were observed from the peat medium, even in the absence of plants. A relationship is presented which would enable a lettuce grower to estimate whole-shoot nitrate concentration from a quick test of midrib sapi.e. NO₃-N (mg/kg in fresh shoot) =0.14×NO₃-N (mg/l in sap). Tipburn was worst at intermediate levels of applied N, and was less serious with pure ammonium nutrition than with nitrate.