Binding of lysophosphatidylcholine to the rat liver fatty acid binding protein

In this study, lysophosphatidylcholine (lysoPC) was shown to bind to a fatty acid binding protein isolated from rat liver. To demonstrate the binding, lysoPC was incorporated into multilamellar liposomes and incubated with protein. For comparison, binding of both lysoPC and fatty acid to liver fatty acid binding protein, albumin, and heart fatty acid binding protein were measured. At conditions where palmitic acid bound to liver fatty acid binding protein and albumin at ligand to protein molar ratios of 2:1 and 5:1, respectively, lysoPC binding occurred at molar ratios of 0.4:1 and 1:1. LysoPC did not bind to heart fatty acid binding protein under conditions where fatty acid bound at a molar ratio of 2:1. Competition experiments between lysoPC and fatty acid to liver fatty acid binding protein indicated separate binding sites for each ligand. An equilibrium dialysis cell was used to demonstrate that liver fatty acid binding protein was capable of transporting lysoPC from liposomes to rat liver microsomes, thereby facilitating its metabolism. These studies suggest that liver fatty acid binding protein may be involved in the intracellular metabolism of lysoPC as well as fatty acids, and that functional differences may exist between rat liver and heart fatty acid binding protein.     

Differentiation of Candida stellatoidea from C. albicans and C. tropicalis by temperature-dependent growth responses on defined media

C. stellatoidea differs from both C. albicans and C. tropicalis in its i) much greater growth differential on minimal and amino acid enriched media and ii) unique inability to grow on minimal medium containing glycerol as carbon source at 37C. The relative responses to amino acid enrichment occur on media containing either fermentative or oxidative carbon sources, at 25C or 37C. Under any given conditions of carbon source and temperature, different assortments of individual amino acids are stimulatory for each of the three species. All assortments include one or more members of the glutamic acid family. However, sulfur amino acids stimulate only C. stellatoidea on all three carbon sources. On minimal-glycerol medium, wild type strains of C. stellatoidea grow prototrophically at 25C but are auxotrophic for amino acids at 37C; the particular auxotrophies expressed vary from strain to strain. Slow growing, mycelial mutants, prototrophic on glycerol at 37C arise spontaneously in wild type strains at frequencies indicating nuclear gene mutation. Such mutants can be induced by both transition and frame shift mutagens. The implications of these observations for the taxonomic relationships between the three Candida species and for identification of C. stellatoidea in particular are discussed.     

Wirkungen von L-glutaminsäure auf grösse und puff-muster der Larvalen Speicheldrüsenchromosomen von Drosophila melanogaster in vivo

The ratio DNA: RNA in the dry substance of Drosophila melanogaster larvae changes when L-glutamic acid is added to the substratum. The number of cells in the salivary glands is not affected (Fahrig et al., 1967). The present paper deals with the effect of this altered ratio on the puffing pattern of the salivary gland chromosomes.Compared to controls of the same game age, glutamic acid fed prepupae younger than 15 min have five additional puffs. In all, 98 pairs of homologous puffs were studied. In 54 of these, size differences were tested statistically; in the glutamic acid series, 20 puffs were larger and 18 smaller than in the controls whereas 16 had the same size. Size reduction was stronger in the more prominent puffs. In prepupae reared at lower temperatures, chromosomes were significantly longer. Glutamic acid causes a significant increase in chromosome width. Combined measurements of both effects were made by determining the surface area of tested segments. Lowering the temperature adds higher size classes, whereas addition of glutamic acid causes a significant shift of the distributional peak towards the higher size classes. This excludes the possibility of an additional replication cycle. In salivary glands of glutamic acid fed prepupae the majority of nuclei have reached the highest degree of polyteny, which in controls is never found at 25° C but sometimes at 18° C. The agent causing both additional puffing and enhanced growth is effective only via the alimentary tract of the larva.     

Genlokalisation auf den larvalen speicheldrüsenchromosomen der stechmücke Culex pipiens L.

Zusammenfassung Die Austauschhäufigkeiten zwischen cytologisch bekannten Bruchkontaktpunkten in den larvalen Speicheldrüsenchromosomen und Mutationen erlauben bei der Stechmücke Culex pipiens L. erstmals die Zuordnung von Genorten zu chromosomalen Strukturen oder Abschnitten. Die vorliegenden Kreuzungsergebnisse stimmen mit der bereits bekannten Zuordnung der drei Koppelungsgruppen zu den cytologisch sichtbaren Chromosomen überein.Um Kreuzungsergebnisse innerhalb eines Chromosoms jedoch sicher miteinander in Bezug setzen zu können, mußte das Problem der unterschiedlichen Austauschhäufigkeiten geklärt werden. Gründe dafür sind weder Alter noch Geschlecht, noch kleine chromosomale Aberrationen der heterozygoten Individuen. Die Höhe des Austausches zwischen zwei Faktoren ist jeweils eine Stamm-spezifische Eigenschaft, die vermutlich faktoriell bedingt ist. Dies konnte durch den Vergleich einer allelen Mutation in zwei Stämmen erarbeitet werden. Es sind deshalb nur solche Austauschwerte vergleichbar, die in ein und demselben Stamm gewonnen wurden, oder bei verschiedenen Stämmen, wenn diese in einen festen Bezug gebracht werden können.Im kleinen Chromosom I ist die Zuordnung des Geschlechts-bestimmenden Allelenpaares M bzw. m zu der heteromorphen Bande 10 C 3 in Arm I L durch Austausch-Analyse mit Bruchkontaktpunkten geschlechtsgekoppelter Translokationen bestätigt. Der Locus der Augenfarbmutation w liegt in unmittelbarer Nähe davon. Der Genort der Augenfarbmutation r ist in Arm I R, in Abschnitt 3 B/C gelegen.Im mittellangen Chromosom II werden zwei Genorte eingegrenzt: die Larvenfarbmutation d ist am distalen Ende des Armes II L gelegen; die Augenfarbmutation ru im zentralen Teil des Armes II R, zwischen den Abschnitten 29 A-21 A. Im langen Chromosom III ist der Genort der Männchen-begrenzten Palpenmutation kps dem Arm III L zugeordnet worden.Eine Zuordnung zu einer distinkten Bande oder Struktur war nur für den Geschlechtsfaktor möglich. Mit den vorliegenden Werten wird für Culex pipiens erstmals eine grobe cytologische Genkarte erstellt.

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Gene mapping on the salivary gland chromosomes of the mosquito Culex pipiens L.

Summary Crossing experiments were done with several mutations and aberrant lines of the mosquito Culex pipiens L. Gene loci and chromosomal structure could be correlated by comparing crossover rates of mutations with breakage points of chromosomal aberrations in the larval salivary gland chromosomes. This confirms linkage groups and their correlated chromosomes.Before comparing crossover rates in one chromosome in different experiments, the problem of different crossover rates between two distinct factors should be solved. The reason for these different rates is not sex, age or small chromosomal aberrations in the heterozygous individuals. It could be interstrain behaviour, characteristic for each strain. This was shown by comparing crossover rates of an allelomorph mutation in two different laboratory strains. Therefore, only results within one pure strain or between two strains with known correlation can be compared. In the small chromosome I, the correlation of the sex-determining allelomorphs M and m with the heteromorphic band 10 C 3 in arm I L was confirmed. This was done by crossover analysis of breakpoints in sex-linked aberrations. The locus of the eye colour mutation w is situated near this band. The eye colour mutation r is located in the segment 3 B/C in arm I R.In chromosome II, two gene loci are narrowed down: the larval colour mutation d is situated on the distal end of arm II L, the eye colour mutation ru in the central part of arm II R. In chromosome III, the male-limited mutation kps is located in arm III L.Hitherto only the sex-factor could be correlated which a distinct structure, i.e. the heteromorphic band 10 C 3 in arm I L. The results of the described experiments made it possible for the first time to establish a cytological gene map of Culex pipiens.

     

S-Adenosyl-Homocysteine Is a Weakly Bound Inhibitor for a Flaviviral Methyltransferase

The methyltransferase enzyme (MTase), which catalyzes the transfer of a methyl group from S-adenosyl-methionine (AdoMet) to viral RNA, and generates S-adenosyl-homocysteine (AdoHcy) as a by-product, is essential for the life cycle of many significant human pathogen flaviviruses. Here we investigated inhibition of the flavivirus MTase by several AdoHcy-derivatives. Unexpectedly we found that AdoHcy itself barely inhibits the flavivirus MTase activities, even at high concentrations. AdoHcy was also shown to not inhibit virus growth in cell-culture. Binding studies confirmed that AdoHcy has a much lower binding affinity for the MTase than either the AdoMet co-factor, or the natural AdoMet analog inhibitor sinefungin (SIN). While AdoMet is a positively charged molecule, SIN is similar to AdoHcy in being uncharged, and only has an additional amine group that can make extra electrostatic contacts with the MTase. Molecular Mechanics Poisson-Boltzmann Sovation Area analysis on AdoHcy and SIN binding to the MTase suggests that the stronger binding of SIN may not be directly due to interactions of this amine group, but due to distributed differences in SIN binding resulting from its presence. The results suggest that better MTase inhibitors could be designed by using SIN as a scaffold rather than AdoHcy.     

Cysteine Oxidation Targets Peroxiredoxins 1 and 2 for Exosomal Release through a Novel Mechanism of Redox-Dependent Secretion

Nonclassical protein secretion is of major importance as a number of cytokines and inflammatory mediators are secreted via this route. Current evidence indicates that there are several mechanistically distinct methods of nonclassical secretion. We have shown recently that peroxiredoxin (Prdx) 1 and Prdx2 are released by various cells upon exposure to inflammatory stimuli such as lipopolysaccharide (LPS) or tumor necrosis factor alpha (TNF-α). The released Prdx then acts to induce production of inflammatory cytokines. However, Prdx1 and 2 do not have signal peptides and therefore must be secreted by alternative mechanisms, as has been postulated for the inflammatory mediators interleukin-1β (IL-1β) and high mobility group box-1 (HMGB1). We show here that circulating Prdx1 and 2 are present exclusively as disulfide-linked homodimers. Inflammatory stimuli also induce in vitro release of Prdx1 and 2 as disulfide-linked homodimers. Mutation of cysteines Cys51 or Cys172 (but not Cys70) in Prdx2, and Cys52 or Cys173 (but not Cys71 or Cys83) in Prdx1 prevented dimer formation and this was associated with inhibition of their TNF-α-induced release. Thus, the presence and oxidation of key cysteine residues in these proteins are a prerequisite for their secretion in response to TNF-α, and this release can be induced with an oxidant. By contrast, the secretion of the nuclear-associated danger signal HMGB1 is independent of cysteine oxidation, as shown by experiments with a cysteine-free HMGB1 mutant. Release of Prdx1 and 2 is not prevented by inhibitors of the classical secretory pathway, instead, both Prdx1 and 2 are released in exosomes from both human embryonic kidney (HEK) cells and monocytic cells. Serum Prdx1 and 2 also are associated with the exosomes. These results describe a novel pathway of protein secretion mediated by cysteine oxidation that underlines the importance of redox-dependent signaling mechanisms in inflammation.     

The extracellular microenvironment plays a key role in regulating the redox status of cell surface proteins in HIV-infected subjects

There is an overwhelming interest in the study of the redox status of the cell surface affecting redox signaling in the cells and also predicting the total redox status of the cells. Measuring the total surface thiols (cell surface molecule thiols, csm-SH) we have shown that the overall level of surface thiols is tightly controlled. In vitro, the total concentration of intracellular glutathione (iGSH) seems to play a regulatory role in determination of the amounts of reduced proteins on cells. In addition, short term exposure of the cell surface to glutathione disulfide (GSSG, oxidized GSH) seems to reduce the overall levels of csm-SH suggesting that the function of some cysteine containing proteins on the cell surface may be regulated by the amount of GSSG secreted from the cells or the GSSG available in the extracellular environment. Examination of peripheral blood mononuclear cells (PBMCs) from healthy or HIV-infected subjects failed to reveal a similar correlation between the intra- and extracellular thiol status of cells. Although there is a relatively wide variation between individuals in both csm-SH and iGSH there is no correlation between the iGSH and csm-SH levels measured for healthy and HIV-infected individuals. There are many reports suggesting different redox active proteins on the cell surface to be the key players in the total cell surface redox regulation. However, we suggest that the redox status of the cells is regulated through a complex and tightly regulated mechanism that needs further investigation. In the mean time, overall surface thiol measurements together with case specific protein determinations may offer the most informative approach. In this review, we discuss our own results as well as results from other laboratories to argue that the overall levels of surface thiols on the exofacial membrane are regulated primarily by redox status of the cell surface microenvironment.     

Acarbose attenuates experimental non-alcoholic steatohepatitis

The alpha-glucosidase inhibitor acarbose is beneficial in the prevention of type 2 diabetes. To determine whether it attenuates the commonly associated non-alcoholic steatohepatitis (NASH), we used an experimental NASH model. Rats were fed ad libitum a nutritionally adequate high fat diet (71% of calories as fat) with or without acarbose (200 mg/1000 calories) for 3 weeks. All rats given the high fat diet only developed typical NASH whereas acarbose attenuated several of the characteristic hepatic alterations of NASH: there was less steatosis and inflammation, with a significant reduction in the mRNA of the hepatic inflammatory cytokine TNF-alpha and of its protein. There was also a decrease in the CYP2E1 mRNA and in collagen, with similar trends for CYP2E1 protein and procollagen mRNA. Because acarbose attenuates many of the hepatic alterations associated with experimental NASH, it is now indicated to determine whether it exerts similar beneficial effects in patients afflicted by this disease.