Diurnal variation in 5′-nucleotidase activity in rat liver

Summary The diurnal variation of 5-nucleotidase activity in periportal and pericentral areas of rat liver parenchyma has been determined with quantitative histochemical means. 5-Nucleotidase activity was estimated using microdensitometry in cryostat sections after being incubated with a medium according to Wachstein and Meisel (1957). It appeared that 5-nucleotidase activity was significantly higher in pericentral areas than in periportal areas throughout the daily cycle and showed a maximum at the end of the light period. It was concluded that 5-nucleotidase activity may be related with the capacity to diminish messenger RNA resulting in protein breakdown.     

Reduced tumour necrosis factor-induced cytotoxicity by inhibitors of the arachidonic acid metabolism

The mechanism of tumour necrosis factor-mediated cytotoxicity was investigated by using various inhibitors of arachidonic acid metabolism. Phospholipase A2 inhibitors with different modes of action interfered with the cytotoxic action of TNF, whereas phospholipase C inhibitors did not. Neither cyclooxygenase nor lipoxygenase-blockers had a significant effect on TNF action. Experiments with scavengers of toxic oxygen radicals gave ambiguous results. The data obtained suggest the involvement of phospholipase A2 and arachidonic acid in the cytotoxic mechanism of TNF, but the exact role of these molecules is, however, still to be determined.     

Inhibition of human neutrophil collagenase by gold(I) salts used in chrysotherapy

Six gold(I) salts, some of which are used as drugs in chrysotherapy, are shown to be inhibitors of two forms of human neutrophil collagenase. The IC50 values vary over six orders of magnitude, the lowest being 3.5 nM for Myocrisin. Thus, inhibition is greatly affected by the identity of the ligands to the gold(I) atom. The inhibition of collagenase by these gold(I) salts may be a partial basis for their antiarthritic action.     

Kinetics and concentration dependency of cAMP-induced desensitization of a subpopulation of surface cAMP receptors in Dictyostelium discoideum

Extracellular cAMP induces the rapid activation of guanylate cyclase, which adapts within 10 s to constant cAMP concentrations. A new response can be induced either by a higher cAMP concentration or by the same cAMP concentration at some time (t1/2 = 90 s) after removal of the previous stimulus. Stimulation of guanylate cyclase is supposed to be mediated by a subpopulation of cell surface cAMP receptors (B-sites). These sites can exist in three states, BF, BS, and BSS, which interconvert in a cAMP and guanine nucleotide dependent manner. It has been proposed that the transition of BS to BSS represents the activation of a guanine nucleotide regulatory protein [Van Haastert, P.J.M., De Wit, R.J.W., Janssens, P.M.W., Kesbeke, F., & DeGoede, J. (1986) J. Biol. Chem. 261, 9604-9611]. Binding of [3H]cAMP to these sites was measured after a short preincubation with an identical concentration of nonradioactive cAMP. [3H]cAMP could still bind to BF and BS, but not to BSS, indicating that the transition of BS to BSS is blocked by the preincubation with cAMP. This blockade was rapid and showed first-order kinetics with t1/2 = 4 s. A half-maximal blockade was induced by 0.7 nM cAMP; at this concentration only 5% of the B-sites are occupied with cAMP. The blockade of the transition of BS to BSS was released by two conditions: (i) When the concentration of cAMP was increased, the blockade was released within a few seconds. (ii) When cAMP was removed, the blockade was released slowly with t1/2 = 90 s.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of amino acid transport system B0,+ in mouse blastocysts

The capacity of preimplantation mouse blastocysts to express the novel amino acid transport activity provisionally designated system B0,+ increased approximately 3-fold 1 day after administration of estrogen to their progesterone-primed, ovariectomized mothers. Nevertheless, blastocysts obtained 22-25 h after estrogen administration (implanting blastocysts) had to be incubated in vitro for about 20 min before they fully expressed their B0,+ activity. No similar increase in B0,+ activity was observed upon incubation of blastocysts obtained before estrogen administration (diapausing blastocysts). Rapid metabolic changes can be induced in the uterus by massaging it with a blunt instrument while it is receptive to implantation, and this treatment was found to increase the apparent B0,+ activity in implanting but not diapausing blastocysts. In contrast, the activity of an incompletely characterized, Na+-independent system, which accepts L-lysine as a substrate, decreased more than 2-fold when implanting blastocysts were incubated in vitro. No change in Na+-independent lysine uptake was detected during incubation of diapausing blastocysts. It is suggested that both uteri and blastocysts develop the capacity to change rapidly some of their metabolic processes near the time of implantation, and one of the processes which may be subject to rapid change in blastocysts is amino acid transport. These developmental events appear to coincide with and could be required for the decidual cell response and implantation of blastocysts in the uterus.     

Role of de novo protein synthesis in the interconversion of glucose transport systems in the yeast Pichia ohmeri

Glucose-repressed cells of the yeast Pichia ohmeri IGC 2879 transported glucose by facilitated diffusion. Derepression led to the formation of a glucose/proton symport and the simultaneous reduction of the facilitated diffusion capacity by about 70%. Cycloheximide prevented this interconversion indicating its dependence on de novo protein synthesis (proteosynthetic interconversion). In buffer with 2% glucose the glucose/proton symport suffered irreversible inactivation while the facilitated diffusion system was simultaneously restored. This reverse interconversion process did not require de novo protein synthesis as indicated by its lack of sensitivity to cycloheximide (degradative interconversion). Thus the glucose/proton symport system appeared to consist of about 70% of the facilitated diffusion proteins turned silent through association with additional protein(s) the latter being sensitive to glucose-induced repression and glucose-induced inactivation.     

Immobilization of microorganisms by adhesion: interplay of electrostatic and nonelectrostatic interactions

The adhesion of three microorganisms (Saccharomyces cerevisiae, Acetobacter aceti, and Moniliella pollinis) to different materials has been studied using various supports (glass, metals, plastics), some of which were treated by an Fe(III) solution. The surface properties of the cells were characterized by the zeta potential and an index of hydrophobicity; characterization of the supports involved surface chemical analysis (XPS) and contact angle measurements. Cell suspensions in pure water at a given pH were left to settle on plates; the latter were then rinsed and examined microscopically, Saccharomyces cerevisiae and A. aceti adhere to metals under certain pH conditions but do not adhere to any of the other materials tested unless it is previously treated by ferric ions; adhesion of these hydrophilic cells is essentially controlled by electrostatic interactions. Moniliella pollinis adhere spontaneously to glass and to polymeric materials, but its attachment is also influenced by cell-cell or cell-support electrostatic repulsions; near the cell isoelectric point, cell flocculation is competing with adhesion to a support.     

Migration of O-acetyl groups in N,O-acetylneuraminic acids

Highly purified N-acetyl-4-O-acetylneuraminic acid (Neu4,5Ac2), N-acetyl-7-O-acetylneuraminic acid (Neu5,7Ac2) and N-acetyl-7,9-di-O-acetylneuraminic acid (Neu5,7,9Ac3) were used to study spontaneous migrations of acetyl groups between hydroxyl groups. The techniques applied involved thin-layer chromatography, gas-liquid chromatography/mass spectrometry, high-performance liquid chromatography and 360-MHz 1H-NMR spectroscopy. It was found that at pH values at which no significant de-O-acetylation is observed: (a) Neu5,7Ac2 can easily be transformed into Neu5,9Ac2, (b) Neu5,7,9Ac3 yields an equilibrium of Neu5,7,9Ac3 and Neu5,8,9Ac3 in a molar ratio of approximately 1:1, and (c) Neu4,5Ac2 does not give rise to O-acetyl migrations. The importance of these findings is discussed in terms of the biosynthesis of O-acetylated sialic acids.     

Structural analysis of the carbohydrate moieties of α-l-fucosidase from human liver

Acid -l-fucosidase (EC 3.2.1.51) was obtained from human liver and purified to homogeneity. The enzyme consists of four subunits; each of these has a molecular mass of 50 kD_a and bears oneN-linked carbohydrate chain. The structures of these chains were studied at the glycopeptide level by methylation analysis and 500-MHz¹H-NMR spectroscopy. Oligomannoside-type chains andN-acetyllactosamine-type chains are present in an approximate ratio of 31. While the oligomannoside-type chains show some heterogeneity in size (Man_5–8GlcNAc₂), theN-acetyllactosaminetype chains are exclusively bi-(2–6)-sialyl, bi-antennary in their structure.These observations on the carbohydrate moieties of -l-fucosidase substantiate our hypothesis [Overdijket al. (1986) Glycoconjugate J 3:339–50] with respect to the relationship between the oligosaccharide structure of lysosomal enzymes and their residual intracellular activity in I-cell disease. For the series of enzymes examined so far, namely, -N-acetylhexosaminidase, -l-fucosidase and -galactosidase, the relative amount ofN-acetyllactosamine-type carbohydrate increases, while the residual intracellular activity in I-cell disease tissue decreases in this order. The system which is responsible for preferentially retaining hydrolases with (non-phosphorylated) oligomannoside-type chains both in I-cells and in normal cells has yet to be identified.