Francisco J. Caballero

Degradation of p-nitrophenol by the phototrophic bacterium Rhodobacter capsulatus

Abstract The phototrophic bacterium Rhodobacter capsulatus detoxified p-nitrophenol and 4-nitrocatechol. The bacterium tolerated moderate concentrations of p-nitrophenol (up to 0.5 mM) and degraded it under light at an optimal O2 pressure of 20 kPa. The bacterium did not metabolize the xenobiotic in the dark or under strictly anoxic conditions or high O2 pressure. Bacterial growth with acetate in the presence of p-nitrophenol took place with the simultaneous release of nonstoichiometric amounts of 4-nitrocatechol, which can also be degraded by the bacterium. Crude extracts from R. capsulatus produced 4-nitrocatechol from p-nitrophenol upon the addition of NAD(P)H, although at a very low rate. A constitutive catechol 1,2-dioxygenase activity yielding cis,cis-muconate was also detected in crude extracts of R. capsulatus. Further degradation of 4-nitrocatechol included both nitrite- and CO2-releasing steps since: (1) a strain of R. capsulatus (B10) unable to assimilate nitrate and nitrite released nitrite into the medium when grown with p-nitrophenol or 4-nitrocatechol, and the nitrite concentration was stoichiometric with the 4-nitrocatechol degraded, and (2) cultures of R. capsulatus growing microaerobically produced low amounts of 14CO2 from radiolabeled p-nitrophenol. The radioactivity was also incorporated into cellular compounds from cells grown with uniformly labeled 14C-p-nitrophenol. From these results we concluded that the xenobiotic is used as a carbon source by R. capsulatus, but that only the strain able to assimilate nitrite (E1F1) can use p-nitrophenol as a nitrogen source.

Melatonin prevents experimental liver cirrhosis induced by thioacetamide in rats

Abstract:  Liver cirrhosis is a critical stage of chronic liver diseases that can produce liver failure, portal hypertension and hepatocarcinoma. Sustained oxidative stress plays a key role in cell damage and fibrosis induced during liver cirrhosis. We evaluated the effect of oxidative stress regulation by melatonin on the development of parenchymal destruction and stellate cell activation in experimental liver cirrhosis. Melatonin was administered to rats with liver cirrhosis induced by thioacetamide (TAA) for 1 or 3 months. Liver injury was assessed by serological analysis, as well as hematoxylin‐eosin staining and the in situ apoptosis detection assay in liver sections. Oxidative stress was evaluated by lipoperoxide and reduced glutathione levels, and by the measurement of catalase and superoxide dismutase activities in liver and serum respectively. The activation of stellate cells was evaluated by α‐smooth muscle actin expression in liver sections. Our results showed that TAA induced oxidative stress with extensive tissue damage and enhanced α‐smooth muscle actin expression in liver. Melatonin prevented the oxidative stress‐related changes associated with TAA toxicity. In conclusion, the study showed that melatonin prevents the tissue damage and fibrosis associated with TAA‐induced liver cirrhosis in rats.

Opposite effects of anandamide and n-arachidonoyl dopamine in the regulation of prostaglandin E2 and 8-iso-PGF2α formation in primary glial cells

It is widely accepted that neuroinflammation is a key player in various pathological events associated with brain injury. More specifically, glial activation and the subsequent release of pro‐inflammatory cytokines, reactive oxygen species (ROS), and prostaglandins play a role of paramount importance in cerebral damage. In this study, we examined the role of two endocannabinoids, anandamide (AEA) and N‐arachidonoyldopamine (NADA) in the regulation of prostaglandin E2 (PGE2) synthesis in primary glial cells. We show that NADA is a potent inhibitor of PGE2 synthesis in lipopolysaccharide (LPS) stimulated cells, without modifying the expression or enzymatic activity of COX‐2 and the production of prostaglandin D2. We also show that NADA has the ability to prevent the free radical formation in primary microglial cells. The key findings of this investigation are our observation that AEA and NADA have opposite effects on glial cells and, most importantly, the first description of NADA as a potential antioxidative and anti‐inflammatory agent acting through a mechanism that involves reduction in the synthesis of microsomal prostaglandin E synthase in LPS‐activated microglia. These findings provide new mechanistic insights into the anti‐inflammatory activities of NADA in the CNS and its potential to design novel therapeutic strategies to manage neuroinflammatory diseases.

Denbinobin, a naturally occurring 1,4-phenanthrenequinone, inhibits HIV-1 replication through an NF-κB-dependent pathway

Anthraquinones and structurally related compounds have been recently shown to exert antiviral activities and thus exhibit a therapeutic potential. In this study we report the isolation of the 1,4-phenanthrenequinone, denbinobin, from a variety of Cannabis sativa. Denbinobin does not affect the reverse transcription and integration steps of the viral cycle but prevents HIV-1 reactivation in Jurkat T cells activated by TNFalpha, mAbs anti-CD3/CD28 or PMA. In addition, denbinobin inhibits HIV-1-LTR activity at the level of transcription elongation and also TNFalpha-induced HIV-1-LTR transcriptional activity. We found that denbinobin prevents the binding of NF-kappaB to DNA and the phosphorylation and degradation of NF-kappaB inhibitory protein, IkappaBalpha, and inhibits the phosphorylation of the NF-kappaB p65 subunit in TNFalpha-stimulated cells. These results highlight the potential of the NF-kappaB transcription factor as a target for natural anti-HIV-1 compounds such as 1,4-phenanthrenequinones, which could serve as lead compounds for the development of an alternative therapeutic approach against AIDS.

Molecular and Regulatory Properties of the Nitrate Reducing Systems of Rhodobacter

Abstract. Phototrophic bacteria of the genus Rhodobacter possess several forms of nitrate reductase including assimilatory and dissimilatory enzymes. Assimilatory nitrate reductase from Rhodobacter capsulatus E1F1 is cytoplasmic, it uses NADH as the physiological electron donor and reduced viologens as artificial electron donors, and it is coupled to an ammonium-producing nitrite reductase. Nitrate reductase induction requires a high C/N balance and the presence of nitrate, nitrite, or nitroarenes. A periplasmic 47-kDa protein facilitates nitrate uptake, thus increasing nitrate reductase activity. Two types of dissimilatory nitrate reductases have been found in strains from Rhodobacter sphaeroides. One of them is coupled to a complete denitrifying pathway, and the other is a periplasmic protein whose physiological role seems to be the dissipation of excess reducing power, thus improving photoanaerobic growth. Periplasmic nitrate reductase does not use NADH as the physiological electron donor and is a 100-kDa heterodimeric hemoprotein that receives electrons through an electron transport chain spanning the plasma membrane. This nitrate reductase is regulated neither by the intracellular C/N balance nor by O2 pressure. The enzyme also exhibits chlorate reductase activity, and both reaction products, nitrite and chlorite, are released almost stoichiometrically into the medium; this accounts for the high resistance to chlorate or nitrite exhibited by this bacterium. Nitrate reductases from both strains seem to be coded by genes located on megaplasmids.

Nitrite uptake system in photosynthetic bacterium Rhodopseudomonas capsulata E1F1

Abstract Nitrite was taken up by Rhodopseudomonas capsulata E1F1 cells by means of an inducible energy-dependent system distinguishable from the enzymatic reduction process. Bacterial cells grown on ammonia or yeast extract were unable to consume nitrite, although there was nitrite reductase activity. The nitrite uptake system required either nitrate or nitrite in the light to be induced, depended on protein synthesis de novo, and was repressed by ammonia. Nitrite uptake showed saturation kinetics lacking diffusion component with an apparent K T lower than 1 μM and an activation energy of 1.75 kJ · mol −1 . 50 μM pHMB inhibited nitrite uptake, whereas nitrite reductase resulted unaffected. Uncouplers such as carbonyl cyanide m -chlorophenylhydrazone and dinitrophenol and the inhibitor of electron flow 2-thenoyltrifluoroacetone inhibited nitrite uptake without affecting enzymatic nitrite reduction. Nitrate completely suppressed nitrite uptake, whereas ammonia or l -methionine- dl -sulfoximine, which immediately stopped nitrate entrance, only inhibited partially nitrite consumption. On the basis of these and other published differences we conclude that in Rps. capsulata E1F1 nitrite is taken up by an inducible energy-dependent transport process different from the nitrate-transport system and distinguishable from enzymatic nitrite reduction.

Basiliolides, a class of tetracyclic C19 dilactones from Thapsia garganica, release Ca2+ from the endoplasmic reticulum and regulate the activity of the transcription factors nuclear factor of activated T cells, nuclear factor-kappa B, and activator protein 1 in T lymphocytes

Calcium concentration within the endoplasmic reticulum (ER) plays an essential role in cell physiology. We have investigated the effects of basiliolides, a novel class of C19 dilactones isolated from Thapsia garganica, on Ca2+ mobilization in T cells. Basiliolide A1 induced a rapid mobilization of intracellular Ca2+ in the leukemia T-cell line Jurkat. First, a rapid calcium peak was observed and inhibited by 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-acetoxymethyl ester. This initial calcium mobilization was followed by a sustained elevation, mediated by the entry of extracellular calcium through store-operated calcium release-activated Ca2+ (CRAC) channels and sensitive to inhibition by EGTA, and by the CRAC channel inhibitor N-{4-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl}-4-methyl-1,2,3-thiadiazole-5-carboxamide (BTP-2). Basiliolide A1 mobilized Ca2+ from ER stores, but in contrast to thapsigargin, it did not induce apoptosis. Basiliolide A1 induced nuclear factor of activated T cells 1 dephosphorylation and activation that was inhibited by BTP-2 and cyclosporine A. In addition, we found that basiliolide A1 alone did not mediate IκBα degradation or RelA phosphorylation (ser536), but it synergized with phorbol 12-myristate 13-acetate to induce a complete degradation of the nuclear factor-κB inhibitory protein and to activate the c-Jun NH2-terminal kinase. Moreover, basiliolide A1 regulated both interleukin-2 and tumor necrosis factor-α gene expression at the transcriptional level. In basiliolide B, oxidation of one of the two geminal methyls to a carboxymethyl group retained most of the activity of basiliolide A1. In contrast, basiliolide C, where the 15-carbon is oxidized to an acetoxymethine, was much less active. These findings qualify these compounds as new probes to investigate intracellular calcium homeostasis.

Immunoelectrophoretic approach to the metabolic regulation of glutamine synthetase in Rhodopseudomonas capsulata E1F1: role of glutamine

Anti-glutamine synthetase serum was raised in rabbits by injecting purified glutamine synthetase (GS) of the phototrophic bacterium Rhodopseudomonas capsulata E1F1. The antibodies were purified to monospecificity by immunoaffinity chromatography in GS-sepharose gel. These anti-GS antibodies were used to measure the antigen levels in crude extracts from bacteria, grown phototrophically with dinitrogen, nitrate, nitrite, ammonia, glutamate, glutamine or alanine as nitrogen sources. The amount of GS detected by rocket immunoelectrophoresis was proportional to Mn2+-dependent transferase activity measured in the crude extracts. Addition of GS inhibitor l-methionine-d,l-sulfoximine (MSX) to the actively growing cells promoted increased antigen levels, that were not found in the presence of glutamine or chloramphenicol. The ammonia-induced decrease in GS relative levels was reverted by MSX. GS levels remained constant when phototrophically growing cells were kept in the dark.

Effect of the C/N balance on the regulation of nitrogen fixation in Rhodobacter capsulatus E1F1

In Rhodobacter capsulatus E1F1, nitrogenase synthesis was repressed by l -glutamate, l -glutamine, l -alanine, l -arginine, l -aspartate, l -asparagine, l -cysteine and l -serine when used in the light as the sole nitrogen and carbon sources. With either d,l -malate or pyruvate as carbon source, nitrogenase repression by these l -amino acids was alleviated and in some cases prevented. Nitrogenase derepression was, also observed at high (3 or more) d,l -malate to l -glutamine external ratios. Ammonium and urea, but not methylammonium, repressed nitrogenase, l -ethionine- d,l -sulfoximine (MSX) prevented nitrogenase repression by ammonium or urea, whereas repression by l -glutamine did occur in the presence of this specific inhibitor of glutamine synthesis. The role of ammonium and l -glutamine in nitrogenase regulation was assessed by comparing half-life values of nitrogenase with and without MSX. We conclude that nitrogenase synthesis in Rb. capsulatus E1F1 is not regulated by ammonium per se or l -glutamine alone but rather by the intracellular C/N balance.

Regulation of reduced nitrogen assimilation in Rhodobacter capsulatus E1F1

The phototrophic bacterium Rhodobacter capsulatus E1F1 assimilates ammonia and other forms of reduced nitrogen either through the GS/GOGAT pathway or by the concerted action of l-alanine dehydrogenase and aminotransferases. These routes are light-independent and very responsive to the carbon and nitrogen sources used for cell growth. GS was most active in cells grown on nitrate or l-glutamate as nitrogen sources, whereas it was heavily adenylylated and siginificantly repressed by ammonium, glycine, l-alanine, l-aspartate, l-asparagine and l-glutamine, under which conditions specific aminotransferases were induced. GOGAT activity was kept at constitutive levels in cells grown on l-amino acids as nitrogen sources except on l-glutamine where it was significantly induced during the early phase of growth. In vitro, GOGAT activity was strongly inhibited by l-tyrosine and NADPH. In cells using l-asparagine or l-aspartate as nitrogen source, a concerted induction of l-aspartate aminotransferase and l-asparaginase was observed. Enzyme level enhancements in response to nitrogen source variation involved de novo protein synthesis and strongly correlated with the cell growth phase.