Lipase of Candida albicans induces activation of NADPH oxidase and l-arginine pathways on resting and activated macrophages

Candida albicans secretes various hydrolytic enzymes which are considered to be an integral part in the pathogenesis. However, the role of lipases is far from being completely understood and the direct effects of these fungal enzymes during the host-pathogen interaction remain to be established. We recently isolated and characterized an extracellular C. albicans lipase (CaLIP), and demonstrated the ability of this fungal enzyme to interact directly with macrophages (M?) and hepatocytes and to operate as a virulence factor. Herein, we explored the effects of CaLIP on M? functions such as oxidative burst and l-arginine metabolism. The study was performed in cells with different activation status: normal-resting M?s and M?s primed in vivo or in vitro with C. albicans. The ability of this fungal factor to modulate the above-mentioned parameters was dependent on cells status, dose, and microenvironment, where the interaction took place. These results constitute a new finding in the biology of candidiasis and could illustrate an additional evolutive advantage for the fungus in the framework of the bidirectional host-pathogen interaction.     

Diazepam effects on carrageenan-induced inflammatory paw edema in rats: role of nitric oxide

High doses of diazepam (10.0-20.0 mg/kg) were shown to reduce the volume of acute inflammatory paw edema in rats as a response to carrageenan administration. This effect was attributed to an action of diazepam on the peripheral-type benzodiazepine receptor (PBR) present in the adrenal and/or immune/inflammatory cells. The present study was undertaken to analyze the involvement of nitric oxide (NO) on the effects of diazepam on carrageenan-induced paw edema in rats (CIPE) and to look for the presence of PBR and inducible/constitutive NO synthases (NOS) on slices taken from the inflamed paws of diazepam-treated rats. For that, an acute inhibition of NO biosynthesis was achieved using 50.0 mg/kg No mega-nitro-L-arginine (L-NAME), L-arginine (300.0 mg/kg), the true precursor of NO, and D-arginine (300.0 mg/kg), its false substrate, were also used. The following results were obtained: (1) diazepam (10.0 and 20.0 mg/kg) decreased CIPE values in a dose- and time-dependent way; (2) diazepam effects on CIPE were increased by L-NAME pretreatment; (3) treatment with L-arginine but not with D-arginine reverted at least in part the decrements of CIPE values observed after diazepam administration; (4) PBR were found in endothelial and inflammatory cells that migrated to the inflammatory site at the rat paw; (5) confocal microscopy showed the presence of both PBR and NOS in endothelial and inflammatory cells taken from inflamed paw tissues of rats treated with diazepam a finding not observed in tissues provided from rats treated with diazepam's control solution. These results suggest an important role for NO on the effects of diazepam on CIPE. Most probably, these effects reflect a direct action of diazepam on PBR present in the endothelium of the microvascular ambient and/or on immune/inflammatory cells. An action like that would lead, among other factors, to a decrease in NO, generated by NO synthase, and thus in the mechanisms responsible for CIPE.     

The effects on plasma L-arginine levels of combined oral L-citrulline and L-arginine supplementation in healthy males

We investigated the effects of combining 1 g of l-citrulline and 1 g of l-arginine as oral supplementation on plasma l-arginine levels in healthy males. Oral l-citrulline plus l-arginine supplementation more efficiently increased plasma l-arginine levels than 2 g of l-citrulline or l-arginine, suggesting that oral l-citrulline and l-arginine increase plasma l-arginine levels more effectively in humans when combined.     

A study of l-leucine, l-phenylalanine and l-alanine transport in the perfused rat mammary gland: possible involvement of LAT1 and LAT2

The transport of l-leucine, l-phenylalanine and l-alanine by the perfused lactating rat mammary gland has been examined using a rapid, paired-tracer dilution technique. The clearances of all three amino acids by the mammary gland consisted of a rising phase followed by a rapid fall-off, respectively, reflecting influx and efflux of the radiotracers. The peak clearance of l-leucine was inhibited by BCH (65%) and d-leucine (58%) but not by l-proline. The inhibition of l-leucine clearance by BCH and d-leucine was not additive. l-leucine inhibited the peak clearance of radiolabelled l-leucine by 78%. BCH also inhibited the peak clearance of l-phenylalanine (66%) and l-alanine (33%) by the perfused mammary gland. Lactating rat mammary tissue was found to express both LAT1 and LAT2 mRNA. The results suggest that system L is situated in the basolateral aspect of the lactating rat mammary epithelium and thus probably plays a central role in neutral amino acid uptake from blood. The finding that l-alanine uptake by the gland was inhibited by BCH suggests that LAT2 may make a significant contribution to neutral amino acid uptake by the mammary epithelium.     

Impact of L-arginine on hydrogen sulfide/cystathionine-gamma-lyase pathway in rats with high blood flow-induced pulmonary hypertension

The present study was designed to explore the possible effect of L-arginine on endogenous hydrogen sulfide/cystathionine-gamma-lyase (H(2)S/CSE) pathway in the pathogenesis of pulmonary hypertension and pulmonary vascular structural remodeling induced by high pulmonary blood flow. Thirty-two male Sprague-Dawley rats were randomly divided into control group (n=11), shunt group (n=11) and shunt with L-arginine group (n=10). Rats in the shunt and shunt with L-arginine group underwent an abdominal aorta-inferior cava vein shunt operation. After 11 weeks of shunting, the plasma level of H2S and lung tissue H2S production rate in the shunt with L-arginine group were much higher than those in the shunt group (P<0.01). Meanwhile, the expression of CSE mRNA in the lung tissues of rats in the shunt with L-arginine group was increased significantly (P<0.01), and in situ hybridization showed that CSE mRNA expression was obviously up-regulated in the smooth muscle cells (SMCs) of the pulmonary arteries of shunted rats treated with L-arginine when compared with shunted rats without the treatment of L-arginine (P<0.01). In conclusion, H2S/CSE pathway was up-regulated by L-arginine in pulmonary hypertension induced by high blood flow with the attenuation of pulmonary hypertension and pulmonary vascular structural remodeling.     

Interaction of L-arginine with dihexadecylphosphate unilamellar liposomes: the effect of the lipid phase organization

The interaction of L-arginine with unilamellar liposomes of dihexadecylphosphate sodium salt (DHP-Na) has been investigated using calorimetric, light scattering, fluorescence spectroscopy and zeta-potential techniques. Heating from room temperature, the bilayer exhibits a phase transition from a subgel (L(c)) to the gel (L(beta')) phase as well as a pre-transition (L(beta')-P(beta')), which is followed by the main lipid phase transition (P(beta')-L(alpha)). Direct studies of the interaction of L-arginine with the DHP-Na bilayers via isothermal titration calorimetry at 27 degrees C depict significant differences between samples in the L(c) and the L(beta') phases reflecting the effect of molecular organization of the lipids upon the interaction. While L-arginine has only a small impact upon the L(c) to L(beta') phase transition, it affects more significantly the transition temperature as well as the shape of the DSC peaks of the main lipid phase transition. Based on fluorescence and zeta-potential studies, the permeability of L-arginine through the liposomal membrane is higher within the temperature range of the main lipid phase transition. Encapsulated l-arginine obstructs the formation of the subgel phase.     

Production of L-xylose from L-xylulose using Escherichia coli L-fucose isomerase

l-Xylulose was used as a raw material for the production of l-xylose with a recombinantly produced Escherichia colil-fucose isomerase as the catalyst. The enzyme had a very alkaline pH optimum (over 10.5) and displayed Michaelis-Menten kinetics for l-xylulose with a K_m of 41 mM and a V_max of 0.23 μmol/(mg min). The half-lives determined for the enzyme at 35 °C and at 45 °C were 6 h 50 min and 1 h 31 min, respectively. The reaction equilibrium between l-xylulose and l-xylose was 15:85 at 35 °C and thus favored the formation of l-xylose. Contrary to the l-rhamnose isomerase catalyzed reaction described previously [14]l-lyxose was not detected in the reaction mixture with l-fucose isomerase. Although xylitol acted as an inhibitor of the reaction, even at a high ratio of xylitol to l-xylulose the inhibition did not reach 50%.     

Solubilization of active green fluorescent protein from insoluble particles by guanidine and arginine

Expression of green fluorescent protein (GFP) in Escherichia coli (E. coli) resulted in only small amount of soluble and fluorescent GFP protein and hence most of the protein in insoluble particles. The expressed GFP in insoluble particles, however, was fluorescent, indicating that it is at least in part folded with an intact chromophore. The GFP in insoluble particles could not be solubilized by an aqueous (denaturant-free) buffer. Solubilization of active GFP from insoluble particles was then attempted with guanidine hydrochloride (GdnHCl), a strong protein-denaturant, or L-arginine, an aggregation suppressor. Solubilization from insoluble particles by 6M GdnHCl led to complete denaturation of the GFP existing in insoluble particles, while GdnHCl solution at lower concentration could solubilize fluorescent GFP. Solubilization of fluorescently active GFP from insoluble particles was also achieved by L-arginine. It is noteworthy that L-arginine was stronger in solubilizing insoluble GFP than GdnHCl below 2M. These results demonstrate that some proteins expressed in E. coli may form insoluble particles containing native conformation and L-arginine may be used to recover the proteins in the native form from such insoluble particles.     

Achacin induces cell death in HeLa cells through two different mechanisms

Achacin, which belongs to the L-amino acid oxidase group, oxidizes free amino acids and produces hydrogen peroxide in cell culture systems. Morphological changes in cells incubated with achacin were similar to those of cells incubated with H(2)O(2). In both cases, the end result was cell death. To examine the mechanism of achacin-associated cytotoxicity, the H(2)O(2) scavenger catalase was added to culture media. Features typical of apoptosis, including morphological changes, DNA fragmentation, and PARP cleavage, were observed when cells were incubated with achacin in the presence of catalase. Moreover, apoptosis was inhibited by Z-VAD-fmk, a broad-spectrum caspase inhibitor. Herein, we present evidence that two pathways are involved in achacin-induced cell death. One is direct generation of H(2)O(2) through the L-amino acid oxidase activity of achacin. The other is the caspase-mediated apoptotic pathway that is induced by depletion of L-amino acids by achacin.     

Nitric oxide and protein nitration in the cystic fibrosis airway

Cystic fibrosis (CF), characterized by chronic airway infection and inflammation, ultimately leads to respiratory failure. Exhaled nitric oxide (NO), elevated in most inflammatory airway diseases, is decreased in CF, suggesting either decreased production or accelerated metabolism of NO. The present studies performed on two groups of CF patients provide further support for a disordered NO airway metabolism in CF respiratory tract disease. Despite confirmation of subnormal NOS2 in the CF airway epithelium, alternative isoforms NOS1 and NOS3 were present, and inflammatory cells in the CF airway expressed abundant NOS2. Increased immunohistochemical staining for nitrotyrosine was demonstrated in lung tissues from patients with CF as compared to control. To our knowledge, this is the first report localizing nitrotyrosine in diseased CF lung tissue. While the relative NOS2 deficiency in CF respiratory tract epithelium may contribute to the lower expired NO levels, these results suggest that increased metabolism of NO is also present in advanced CF lung disease. The significance of altered NO metabolism and protein nitration in CF remains to be fully elucidated.     

Total synthesis and identification of two diastereoisomers of 1-methyl-2-[N-(p-tolylsulfonyl)-2-pyrrolidinyl]ethyl beta-L-fucopyranoside

The reaction of a racemic mixture of (2R,2'S)- and (2S,2'R)-N-(p-tolylsulfonyl)-2-pyrrolidinyl-2-propanol, prepared from (S)-proline, with 2,3,4-tri-O-acetyl-alpha-L-fucopyranosyl trichloroacetimidate led to both diastereoisomers of the title compound after O-deacetylation.     

Base catalysed isomerisation of aldoses of the arabino and lyxo series in the presence of aluminate

Base-catalysed isomerisation of aldoses of the arabino and lyxo series in aluminate solution has been investigated. L-Arabinose and D-galactose give L-erythro-2-pentulose (L-ribulose) and D-lyxo-2-hexulose (D-tagatose), respectively, in good yields, whereas lower reactivity is observed for 6-deoxy-D-galactose (D-fucose). From D-lyxose, D-mannose and 6-deoxy-L-mannose (L-rhamnose) are obtained mixtures of ketoses and C-2 epimeric aldoses. Small amounts of the 3-epimers of the ketoses were also formed. 6-Deoxy-L-arabino-2-hexulose (6-deoxy-L-fructose) and 6-deoxy-L-glucose (L-quinovose) were formed in low yields from 6-deoxy-L-mannose and isolated as their O-isopropylidene derivatives. Explanations of the differences in reactivity and course of the reaction have been suggested on the basis of steric effects.     

l-Lactate generates hydrogen peroxide in purified rat liver mitochondria due to the putative l-lactate oxidase localized in the intermembrane space

In order to ascertain whether and how mitochondria can produce hydrogen peroxide (H₂O₂) as a result of l-lactate addition, we monitored H₂O₂ generation in rat liver mitochondria and in submitochondrial fractions free of peroxisomal and cytosolic contamination. We found that H₂O₂ is produced independently on the respiratory chain with 1:1 stoichiometry with pyruvate, due to a putative flavine-dependent l-lactate oxidase restricted to the intermembrane space. The l-lactate oxidase reaction shows a hyperbolic dependence on l-lactate concentration and is inhibited by NAD⁺ in a competitive manner, being the enzyme different from the l-lactate dehydrogenase isoenzymes as shown by their pH profiles.     

L-Glutamate in the extracellular space regulates endogenous D-aspartate homeostasis in rat pheochromocytoma MPT1 cells

In previous studies [FEBS Lett. 434 (1998) 231, Arch. Biochem. Biophys. 404 (2002) 92], we demonstrated for the first time that D-aspartate (D-Asp) is synthesized in cultured mammalian cell lines, such as pheochromocytoma 12 (PC12) and its subclone, MPT1. Our current focus is analysis of the dynamics of D-Asp homeostasis in these cells. In this communication, we show that L-glutamate (Glu) and L-Glu transporter substrates in the extracellular space regulate the homeostasis of endogenous D-Asp in MPT1 cells. D-Asp is apparently in dynamic homeostasis, whereby endogenous D-Asp is constantly released into the extracellular space by an undefined mechanism, and continuously and intensively taken up into cells by an L-Glu transporter. Under these conditions, L-Glu and its transporter substrates in the medium may competitively inhibit the uptake of D-Asp via the transporter, resulting in accumulation of the amino acid in the extracellular space. We additionally demonstrate that DL-TBOA, a well-established L-Glu transporter inhibitor, is taken up by the transporter during long time intervals, but not on a short time-scale.     

Efficient synthesis of 2-deoxy-L-erythro-pentose (2-deoxy-L-ribose) from L-arabinose

An efficient and practical route for the large-scale synthesis of 2-deoxy-L-erythro-pentose (2-deoxy-L-ribose) starting from L-arabinose was developed using Barton-type free-radical deoxygenation reaction as a key step. The radical precursor, a phenoxythiocarbonyl ester, was prepared in situ, and the most efficient deoxygenation was achieved by slow addition of tributyltin hydride to the reaction mixture.     

L-Gln and L-Ser suppress the D-galactosamine-induced IL-18 expression and hepatitis

d-Galactosamine (GalN) induces acute hepatitis in experimental animals and this hepatitis has been shown to be suppressed by preceding ingestion of amino acids such as Gly, l-Ser, and l-Gln. However, little is known about the mechanism of its action. The present study shows for the first time that IL-18 reduction is involved in the suppressive actions of l-Gln and l-Ser on GalN-induced hepatitis. Elevation of IL-18 mRNA expression in liver and its concentration in serum in GalN-treated rats were found to be suppressed by preceding ingestion of 10% l-Gln- or 10% l-Ser diets, and resulted in the attenuation of the increase in serum transaminase (ALT and AST) activities, indexes of hepatic injury. These results suggest that suppressive effects of some dietary amino acids on the GalN-induced hepatitis are mediated by IL-18 reduction.     

Arginine antimetabolite L-canavanine induces apoptotic cell death in human Jurkat T cells via caspase-3 activation regulated by Bcl-2 or Bcl-xL

L-Canavanine, a natural L-arginine analog, is known to possess cytotoxicity to tumor cells in culture and experimental tumors in vivo. In this study, we first show that apoptotic cell death is associated with antitumor activity of L-canavanine against human acute leukemia Jurkat T cells. When Jurkat T cells were treated with 1.25-5.0mM L-canavanine for 36 h, apoptotic cell death accompanying several biochemical events such as caspase-3 activation, degradation of poly(ADP-ribose) polymerase (PARP), and apoptotic DNA fragmentation was induced in a dose-dependent manner; however, cytochrome c release from mitochondria was not detected. Under these conditions, the expression of Bcl-2 and its functional homolog Bcl-xL was markedly upregulated. The L-canavanine-induced caspase-3 activation, degradation of PARP, and apoptotic DNA fragmentation were suppressed by ectopic expression of Bcl-2 or Bcl-xL, both of which are known to play roles as anti-apoptotic regulators. These results demonstrate that the cytotoxic effect of L-canavanine on Jurkat T cells is attributable to the induced apoptosis and that L-canavanine-induced apoptosis is mediated by a cytochrome c-independent caspase-3 activation pathway that can be interrupted by Bcl-2 or Bcl-xL.     

Effect of Dietary l-Glutamine on the Hepatotoxic Action of d-Galactosamine in Rats

The protective effect of dietary l-glutamine against the hepatotoxic action of d-galactosamine (GalN) was investigated by model experiments with rats. Rats fed with 20% casein diets containing 10% free amino acids were injected with GalN, and the serum aspartate aminotransferase, alanine aminotransferase and lactate dehydrogenase activities and the hepatic glycogen content were assayed 20 hours after the injection. These enzyme activities in the group fed with the 10% l-glutamine diet for 8 days were lower than those in the groups fed with the control, 10% l-glutamic acid and 10% l-alanine diets for 8 days. The more prolonged the feeding period with the 10% l-glutamine diet was, the more the serum activity levels of such enzymes were decreased. Although neomycin also lowered these enzyme activities, its simultaneous ingestion with neomycin did not show any additive or synergistic effect. The hepatic glycogen content in the 10% glutamine group still remained high after the GalN treatment. It is therefore assumed that the effectiveness of glutamine intake would have been mediated by glycogen metabolism rather than by uridine metabolism.     

The influence of selected O-alkyl derivatives of cyclodextrins on the enzymatic decomposition of l-tryptophan by l-tryptophan indole-lyase

A series of O-alkyl derivatives of cyclodextrin: heksakis[2,3,6-tri-O-(2′-methoxyethyl)]-α-cyclodextrin; heksakis(2,3-di-O-methyl)-α-cyclodextrin; heptakis(2,3-di-O-methyl)-β-cyclodextrin; heksakis[2,3-di-O-methyl-6-O-(2′-methoxyethyl)]-α-cyclodextrin; heptakis[2,3-di-O-methyl-6-O-(2′-methoxyethyl)]-β-cyclodextrin; heksakis[2,3-di-O-(2′-methoxyethyl)]-α-cyclodextrin and heptakis[2,3-di-O-(2′-methoxyethyl)]-β-cyclodextrin have been synthesized. Purity and composition of the obtained substances were examined. The cyclodextrin derivatives listed above as well as (2-hydroxypropyl)-α-cyclodextrin and (2-hydroxypropyl)-β-cyclodextrin, the two commercially available ones, have been investigated as the additives in the course of enzymatic decomposition of l-tryptophan by l-tryptophan indole-lyase. It has been found that each of cyclodextrin derivatives causes the inhibition of enzymatic process, both competitive and non-competitive. The competitive inhibition is connected with the formation of inclusion complexes between cyclodextrins and l-tryptophan, related to the geometry of these complexes. The mechanism of the non-competitive inhibition is not so evident; it could be related to the formation of the cyclodextrin complexes on the surface of the enzyme, leading to the change in the flexibility of the enzyme molecule.     

Purification and Characterization,of Rice Bran Lipase II

A species of rice bran lipase (lipase II) was purified by ammonium sulfate precipitation, followed by successive chromatographies on DEAE-cellulose, Sephadex G–75 and CH-Sephadex C–50. Both polyacrylamide disc electrophoresis and ultracentrifugation demonstrated that the enzyme protein is homogeneous. The isoelectric point of the enzyme was 9.10 by ampholine electrophoresis. The sedimentation coefficient of the enzyme was evaluated to be 2.60 S, and the molecular weight to be 33,300 according to Archbald’s method. The enzyme showed the optimum pH between 7.5 and 8.0, and the optimum temperature at about 27°C. It was stable over the pH range from 5 to 9.5 and below 30°C. In substrate specificity, the enzyme exhibited a high specificity toward triglycerides having short-carbon chain fatty acids, although it was capable of hydrolyzing the ester bonds in the rice and olive oil.