Characterization of chloride transport pathways in cultured human keratinocytes.

In human keratinocytes, mediated transport of Cl- was found to occur mainly by two mechanisms: an anion exchange and an electrically conductive pathway. The contribution of the anion exchange, which accounted for about 50% of overall Cl- efflux, was assessed either by its sensitivity to inhibition by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), and by means of Cl- substitution experiments. The anion exchange exhibited a saturation behaviour over the range 10-135 mM Cl-; Cl- was more efficient than HCO3-, Br- and NO3- in increasing Cl- efflux rate, whereas SO4(2-) and I- inhibited Cl- efflux. The electrically conductive Cl- pathway, which accounted for about 40% of total Cl- efflux, was inhibited by the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and was at least partially sensitive to variation of the plasma membrane potential. The Cl- channel was insensitive to elevation in the intracellular concentration of either cyclic AMP and calcium ions. Indomethacin, an inhibitor of the cyclooxygenase, failed to reduce Cl- efflux, whereas nordihydroguaiaretic acid (NDGA), an inhibitor of the lipoxygenase, induced 50% inhibition of Cl- efflux. These results support the conclusion that endogenous production of lipoxygenase-derived arachidonic acid metabolite(s) might be responsible for high basal Cl- permeability in human keratinocytes.     

Characterization of pore-forming activity in liver mitochondria from Anguilla anguilla. Two porins in mitochondria?

A fast purification procedure for the isolation and purification of eukaryotic porin (De Pinto et al., (1987) Biochim. Biophys. Acta 905, 499-502) was applied to liver mitochondria of the fish Anguilla anguilla. A protein preparation was obtained which formed slightly anionically selective pores in reconstitution experiments with lipid bilayer membranes. The distribution of single-channel conductances had two maxima of 2.4 nS and 4.0 nS in 1 M KCl. Sodium dodecylsulfate electrophoretograms of the protein preparation showed the presence of two bands of very similar electrophoretic mobility (32 and 32.5 kDa). Both bands cross-reacted with antibodies raised against purified bovine heart porin and with antibodies raised against the 19 amino acids N-terminal end of human porin. No cross-reactivity was observed with antibodies against yeast porin. The peptide maps of the two bands showed slight differences. The possibility of the presence of two different porins in liver mitochondria of Anguilla anguilla is discussed. An extensive immunological comparison of different mitochondrial porins is presented.     

The use of genetic engineering to obtain efficient production of porcine pancreatic phospholipase A2 by Saccharomyces cerevisiae

We have developed an efficient production system for porcine pancreatic phospholipase A2 in Saccharomyces cerevisiae (baker's yeast). The cDNA encoding the prophospholipase A2 was expressed under the control of the galactose inducible GAL7 promotor, and secretion was directed by the secretion signals of yeast invertase. This construct yielded up to 6 mg prophospholipase A2 activity per 1 fermentation broth, secreted as a glycosylated invertase prophospholipase A2 hybrid protein. Upon genetically deleting the glycosylation site, the level of secretion decreased to 3.6 mg prophospholipase A2 per 1. Changing the invertase secretion signals for an invertase/alpha-mating factor prepro sequence-fusion increased the secretion level up to 8 mg per 1. The secreted non-glycosylated prophospholipase A2 species was correctly processed. Our results demonstrate the promises and limitations for rational design to obtain high level expression and secretion of heterologous proteins by S. cerevisiae.     

Influence of PMA and a low extracellular Ca2+ concentration on the development of the Na(+)-dependent hexose carrier in LLC-PK1 cells.

We have analyzed the development of Na(+)-dependent hexose transport during differentiation and during polarization of LLC-PK1, an established cell line with characteristics of the proximal tubule. When cell-cell contact was disturbed by a low extracellular Ca2+ concentration or by a phorbol myristate acetate (PMA) treatment, the development of Na(+)-dependent hexose transport was completely inhibited. The effect of PMA on the development of hexose transport could be uncoupled from its effect on the tight junctions. The PMA concentration needed for the latter effect was approx. 10-fold higher than for the former. As the primary cause of the PMA effect, an influence on the cytoskeleton is suggested. In contrast to PMA, the concentration dependence of both phenomena on the extracellular Ca2+ concentration was almost the same. Moreover, the incorporation of hexose carriers in the plasma membrane could be induced by changing the extracellular CA2+ concentration from low to normal. We conclude that there is a relation between the formation of tight junctions and the development of the Na(+)-dependent hexose carrier, possibly because Ca(2+)-dependent cell adhesion molecules play a role in both phenomena. However, a direct relation between Ca(2+)-dependent elements of the tight junctions and the insertion of the hexose carrier can not be excluded. The Ca(2+)-dependent development seems to be a common characteristic of apical membrane proteins in contrast to the development of the basolateral membrane protein, (Na(+)+K+)-ATPase.     

Phosphorylation of H+/K(+)-ATPase by inorganic phosphate. The role of K+ and SCH 28080.

The effects of K+ on the phosphorylation of H+/K(+)-ATPase with inorganic phosphate were studied using H+/K(+)-ATPase purified from porcine gastric mucosa. The phosphoenzyme formed by phosphorylation with Pi was identical with the phosphoenzyme formed with ATP. The maximal phosphorylation level obtained with Pi was equal to that obtained with ATP. The Pi phosphorylation reaction of H+/K(+)-ATPase was, like that of Na+/K(+)-ATPase, a relatively slow reaction. The rates of phosphorylation and dephosphorylation were both increased by low concentrations of K+, which resulted in hardly any effect on the phosphorylation level. A decrease of the steady-state phosphorylation level was caused by higher concentrations of K+ in a noncompetitive manner, whereas no further increase in the dephosphorylation rate was observed. The decreasing effect was caused by a slow binding of K+ to the enzyme. All above-mentioned K+ effects were abolished by the specific H+/K(+)-ATPase inhibitor SCH 28080 (2-methyl-8-[phenyl-methoxy]imidazo-[1-2-a]pyrine-3-acetonitrile). Additionally, SCH 28080 caused a 2-fold increase in the affinity of H+/K(+)-ATPase for Pi. A model for the reaction cycle of H+/K(+)-ATPase fitting the data is postulated.     

The activation of adenylate cyclase by pituitary adenylate cyclase activating polypeptide (PACAP) via helodermin-preferring VIP receptors in human SUP-T1 lymphoblastic membranes.

Competition binding curves, using [125I-acetyl-His1]PACAP-27 as radioligand and dose-effect curves of adenylate cyclase activation in human SUP-T1 lymphoblastic membranes showed that PACAP-27 and PACAP-38 stimulate the enzyme through a single class of helodermin-preferring VIP receptors with the following order of potency: helodermin = [acetyl-His1]PACAP-27 greater than PACAP-38 greater than PACAP-27 greater than VIP. PACAP (6-27) (Ki 0.5-0.8 microM) and [Des-His1, Asn3]PACAP-27 (Ki 1-2 microM) acted as competitive antagonists. Using a series of 13 PACAP-27 analogues and fragments and three VIP analogues, we identified positions 1, 2, 3, 9 and 13 in PACAP-27 as being of importance for high-affinity binding. Thus, we added further evidence for considering that the present helodermin-preferring VIP receptors, when compared to a majority of VIP receptors and PACAP receptors, exhibit an original specificity pattern.     

Fluorescence, CD, and NMR studies on spantide, a bombesin and substance P antagonist.

The conformational properties of spantide [(D-Arg, D-Trp, Leu) substance P] have been studied by fluorescence, CD, and nmr techniques. The fluorescence, CD, and nmr parameters in different solvents and in a micellar environment (SDS) are compared with the data collected for bombesin. A preliminary investigation on [D-Pro] spantide is also reported.     

Modeling growth and succinoglucan production by Agrobacterium radiobacter NCIB 9042 in batch cultures

Wild-type Agrobacterium radiobacter NCIB 9042 has been cultivated in batch cultures on a synthetic medium which was adapted for growth and succinoglucan production. Experiments were carried out in a 4-L stirred-tank aerated reactor. Glucose, biomass, polysaccharide, protein, and inorganic- and organic-nitrogen concentrations were measured, and oxygen consumption and CO(2) production rates were obtained by a gas-balance technique. Nitrogen balance shows that inorganic nitrogen is entirely recovered into proteins. The carbon balance is satisfied with in +/-5%. Stoichiometric equations for biomass growth and succinoglucan synthesis were established. The biosyntheticpolymer pathways including ATP and cofactor consumption were investigated. From previous studies, a (P/O) value of 1.66 is selected for oxygen sufficient cultures. The actual ATP requirements of 25.4 mmol ATP/g succinoglucan (38.5 mol ATP/mol succinoglucan), determined by a metabolic analysis, is 2.39 times the stoichiometric value. Experimental results were modeled by a system of differential equations. The exponential growth phase was described by a nitrogen-limited Monod equation. Subsequent succinoglucan synthesis followed a slightly modified Luedeking-Piret relation partitioning internal and external polysaccharide. Experimentally determined coefficients are compared with published results for continuous culture of A. radiobacter NCIB 11883.     

In vitro testing and the carcinogenic potential of several nitrosated indole compounds

4-chloro-methoxyindole is a naturally occurring compound in Vicia faba which can easily react with nitrite to form a N-nitroso compound. In this in vitro study, the potential genotoxic effects of nitrosated 4-chloro-6-methoxyindole and its structural analogue 4-chloroindole were evaluated for the first time by using both Salmonella and Chinese hamster V79 cells. Additionally, the inhibition of gap junctional intercellular communication in V79 cells by these compounds was determined; this is a validated parameter for tumor-promoting activity. Most assays were also performed with nitrosated indole-3-acetonitrile, a naturally occurring compound in brassicas. Both nitrosated chloroindoles were highly mutagenic to Salmonella typhimurium TA100 without the need of exogenous metabolic activation and were potent inducers of Sister Chromatid Exchanges. Nitrosated indole-3-acetonitrile generated the same effects, although at much higher concentrations. Equivocal results were obtained for the nitrosated chloroindoles in a forward mutation assay using the hypoxanthine guaninephosphoribosyltransferase locus. All nitrosated indole compounds significantly inhibited gap junctional intercellular communication. These results indicate that nitrosated chloroindoles and nitrosated indole-3-acetonitrile should be considered as mutagens and agents with potential tumor-promoting capacity.Abbreviations BrdU 5-bromo-2-deoxyuridine - 4Cl 4-chloroindole - 4C6MI 4-chloro-6-methoxy-indole - DMSO dimethyl sulfoxide - EBSS Earle's balanced salt solution - EMS ethyl methanesulfonate - GJIC gap junctional intercellular communication - HBSS Hanks balanced salt solution - HGPRT hypoxanthine guaninephosphoribosyl transferase - I₃A indole-3-acetonitrile - MNNG 1-methyl-1-nitroso-3-nitroguanidine - NOC N-nitroso compounds - NQO 4-nitroquinolone-N-oxide - SCE sister chromatid exchange - 6TG 6-thioguanine - TPA 12-O-tetradecanoylphorbol-13-acetate     

Extension of the range of recognition sequences for triple helix formation by oligonucleotides containing guanines and thymines.

Oligodeoxynucleotides containing G and T can bind to homopurine.homopyrimidine sequences on double-stranded DNA by forming C.G x G and T.A x T base triplets. The orientation of the third strand in such triple helices depends on the number of GpT and TpG steps. Therefore a single oligonucleotide can be designed to bind to two consecutive homopurine.homopyrimidine sequences where the two homopurine stretches alternate on the two strands of DNA. The oligonucleotide switches from one homopurine strand to the other at the junction between the two sequences. This result shows that it is possible to extend the range of DNA sequences that can be recognized by a single oligonucleotide.