Studies on the functional significance of a C-terminal S-shaped motif in human arginase type I: essentiality for cooperative effects

The functional significance of a C-terminal S-shaped motif (residues 304-322) in human arginase I was explored by examining the kinetic properties of the R308A mutant and truncated species terminating in either Arg-308 or Ala-308. Replacement of Arg-308 with alanine, with or without truncation, yielded monomeric species. All mutants were kinetically indistinguishable from the wild-type enzyme at the optimum pH of 9.5. At the more physiological, pH 7.5, hyperbolic kinetics was observed for all the mutants, in contrast with the cooperative behavior exhibited by the wild-type species. In the presence of 2 mM guanidinium chloride (Gdn⁺), the single mutant R308A changed to a trimeric and kinetically cooperative form, whereas the other enzyme variants were not altered. The S-shaped motif is suggested as essential for the cooperative response of the enzyme to l-arginine at pH 7.5. Gdn⁺ is suggested to mimic the guanidine group of Arg-308 at the monomer-monomer interface.     

Expression and Physiological Role of Three Myxococcus xanthus Copper-Dependent P1B-Type ATPases during Bacterial Growth and Development

Myxococcus xanthus is a soil-dwelling bacterium that exhibits a complex life cycle comprising social behavior, morphogenesis, and differentiation. In order to successfully complete this life cycle, cells have to cope with changes in their environment, among which the presence of copper is remarkable. Copper is an essential transition metal for life, but an excess of copper provokes cellular damage by oxidative stress. This dual effect forces the cells to maintain a tight homeostasis. M. xanthus encodes a large number of genes with similarities to others reported previously to be involved in copper homeostasis, most of which are redundant. We have identified three genes that encode copper-translocating P_1B-ATPases (designated copA, copB, and copC) that exhibit the sequence motifs and modular organizations of those that extrude Cu⁺. The expression of the ATPase copC has not been detected, but copA and copB are differentially regulated by the addition of external copper. However, while copB expression peaks at 2 h, copA is expressed at higher levels, and the maximum is reached much later. The fact that these expression profiles are nearly identical to those exhibited by the multicopper oxidases cuoA and cuoB suggests that the pairs CuoB-CopB and CuoA-CopA sequentially function to detoxify the cell. The deletion of any ATPase alters the expression profiles of other genes involved in copper homeostasis, such as the remaining ATPases or the Cus systems, yielding cells that are more resistant to the metal.Copper is required as a cofactor for a number of enzymes involved in essential cellular processes. However, the two oxidation states of copper not only allow its participation in essential redox reactions but also form reactive oxygen species, leading to severe damage of cytoplasmic constituents (23). Copper homeostasis is a complicated process involving copper acquisition, sequestration, and efflux. These mechanisms are tightly regulated and are able to respond to changes in the extracellular bioavailability and intracellular demand for the metal. In order to prevent copper damage, sophisticated defense mechanisms have evolved, one of which is mediated by a group of P-type ATPases designated P_1B-type ATPases (1).P_1B-type ATPases catalyze the transport of transition metal or heavy metal ions across cell membranes, coupling ATP hydrolysis to the transport of cations through a catalytic cycle involving the autophosphorylation of an Asp residue within a highly conserved DKTGT motif. The range of transported substrates is wide, including monovalent (Cu⁺, Ag⁺, and Au⁺) as well as divalent (Cu²⁺, Co²⁺, Zn²⁺, Cd²⁺, Hg²⁺, Pb²⁺, and Mn²⁺) cations. Structural features and conserved sequence motifs of transition-metal P-type pumps suggest a division into several subgroups with distinct substrate specificities. Two subgroups of P_1B-ATPases export either Cu⁺ or Cu²⁺, which share a topological arrangement containing eight transmembrane domains (TMs) but differ in specific signature sequences in TMs 6, 7, and 8. Another structural characteristic of copper-dependent ATPases is the presence of cytoplasmic copper-binding domains in their N termini (1).Myxococcus xanthus is considered a model to study multicellularity and differentiation in prokaryotes due to its unique life cycle. Under nutrient-rich conditions, M. xanthus cells glide on soil in search of organic matter, including other microorganisms, on which they feed. Starvation triggers a developmentally regulated signaling cascade that induces aggregation and the formation of multicellular fruiting bodies filled with environmentally resistant spores, which germinate when conditions become favorable for growth again (33). Because M. xanthus resides on soils, this complex cycle must be carried out in the presence of a variety of changing elements (21). One of these elements is copper, which is present in soils in concentrations that range between 2 and 100 ppm.The elucidation of the copper response in M. xanthus has turned out to be interesting for several reasons. First, this metal induces the genes responsible for carotenogenesis (20). Second, the copper response can be studied during growth and development; in fact, growing cells exhibit around 15-fold-greater resistance to copper than do developing cells, while cells preadapted to this metal reach the same levels of resistance during both stages (27). Third, the concentration of metal required to induce the expression of some copper-dependent genes is around 10-fold lower during development than during growth (27).The M. xanthus genome holds a plethora of gene products with sequence similarities to proteins known to be involved in copper homeostasis in other Gram-negative bacteria, most of which are redundant (19). Although the abundance of paralogous genes in myxobacteria seems to correlate with their large genome sizes (9, 25, 28), it is surprising to find such a large number of genes involved in copper homeostasis in M. xanthus, because this bacterium is not especially resistant to this metal. In order to clarify the physiological roles during the complete life cycle and the advantage of maintaining copper-related duplicated genes during evolution, we have previously characterized two families of paralogs: three multicopper oxidases (MCOs) and six CBA-type heavy metal efflux systems (19, 27).In this work we have concentrated our efforts on the study of the three paralogs of the P_1B-type ATPase family, named CopA, CopB, and CopC. We have found that copA and copB are differentially regulated by this metal, contributing to the adaptive response that allows the completion of this bacterium life cycle in a copper-fluctuating environment. The complexity of M. xanthus copper homeostasis is further illustrated by the interplay among the large number of components that participate in this process.     

Chagas disease: Present status of pathogenic mechanisms and chemotherapy

There are approximately 7.8 million people in Latin America, including Chile, who suffer from Chagas disease and another 28 million who are at risk of contracting it. Chagas is caused by the flagellate protozoan Trypanosoma cruzi. It is a chronic disease, where 20%-30% of infected individuals develop severe cardiopathy, with heart failure and potentially fatal arrhythmias. Currently, Chagas disease treatment is more effective in the acute phase, but does not always produce complete parasite eradication during indeterminate and chronic phases. At present, only nifurtimox or benznidazole have been proven to be superior to new drugs being tested. Therefore, it is necessary to find alternative approaches to treatment of chronic Chagas. The current treatment may be rendered more effective by increasing the activity of anti-Chagasic drugs or by modifying the host's immune response. We have previously shown that glutathione synthesis inhibition increases nifurtimox and benznidazole activity. In addition, there is increasing evidence that cyclooxygenase inhibitors present an important effect on T. cruzi infection. Therefore, we found that aspirin reduced the intracellular infection in RAW 264.7 cells and, decreased myocarditis extension and mortality rates in mice. However, the long-term benefit of prostaglandin inhibition for Chagasic patients is still unknown.     

The biological role of pituitary adenylate cyclase‐activating polypeptide (PACAP) in growth and feeding behavior in juvenile fish

To date, many technologies have been developed to increase efficiency in aquaculture, but very few successful biotechnology molecules have arrived on the market. In this context, marine biotechnology has an opportunity to develop products to improve the output of fish in aquaculture. Published in vivo studies on the action of the pituitary adenylate cyclase‐activating polypeptide (PACAP) in fish are scarce. Recently, our group, for the first time, demonstrated the biological role of this neuropeptide administrated by immersion baths in the growth and development of larval fish. In this work, we have evaluated the effects of recombinant Clarias gariepinus PACAP administration by intraperitoneal injection on growth performance and feeding behavior in juvenile fish. Our results showed the physiological role of this peptide for growth control in fish, including the juvenile stage, and confirm that its biological functions are well conserved in fish, since C. gariepinus PACAP stimulated growth in juvenile tilapia Oreochromis niloticus. In addition, we have observed that the growth‐promoting effect of PACAP in juvenile tilapia was correlated with higher GH concentration in serum. With regard to the neuroendocrine regulation of growth control by PACAP, it was demonstrated that PACAP stimulates food intake in juvenile tilapia. In general, PACAP appears to act in the regulation of the growth control in juvenile fish. These findings propose that PACAP is a prominent target with the potential to stimulate fish growth in aquaculture. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Molecular cloning and characterization of the crustacean hyperglycemic hormone cDNA from Litopenaeus schmitti. Functional analysis by double-stranded RNA interference technique

The crustacean hyperglycemic hormone (CHH) plays an important role in the regulation of hemolymph glucose levels, but it is also involved in other functions such as growth, molting and reproduction. In the present study we describe the first CHH family gene isolated from the Atlantic Ocean shrimp Litopenaeus schmitti. Sequence analysis of the amplified cDNA fragment revealed a high nucleotide sequence identity with other CHHs. Northern blot analysis showed that the isolated CHH mRNA from L. schmitti is present in the eyestalk but not in muscle or stomach. We also investigated the ability of dsRNA to inhibit the CHH function in shrimps in vivo. Injection of CHH dsRNA into the abdominal hemolymph sinuses resulted in undetectable CHH mRNA levels within 24 h and a corresponding decrease in hemolymph glucose levels, suggesting that functional gene silencing had occurred. These findings are the first evidence that dsRNA technique is operative in adult shrimps in vivo.     

Farnesyl diphosphate synthase is a cytosolic enzyme in Leishmania major promastigotes and its overexpression confers resistance to risedronate

Farnesyl diphosphate synthase is the most likely molecular target of aminobisphosphonates (e.g., risedronate), a set of compounds that have been shown to have antiprotozoal activity both in vitro and in vivo. This protein, together with other enzymes involved in isoprenoid biosynthesis, is an attractive drug target, yet little is known about the compartmentalization of the biosynthetic pathway. Here we show the intracellular localization of the enzyme in wild-type Leishmania major promastigote cells and in transfectants overexpressing farnesyl diphosphate synthase by using purified antibodies generated towards a homogenous recombinant Leishmania major farnesyl diphosphate synthase protein. Indirect immunofluorescence, together with immunoelectron microscopy, indicated that the enzyme is mainly located in the cytoplasm of both wild-type cells and transfectants. Digitonin titration experiments also confirmed this observation. Hence, while the initial step of isoprenoid biosynthesis catalyzed by 3-hydroxy-3-methylglutaryl-coenzyme A reductase is located in the mitochondrion, synthesis of farnesyl diphosphate by farnesyl diphosphate synthase is a cytosolic process. Leishmania major promastigote transfectants overexpressing farnesyl diphosphate synthase were highly resistant to risedronate, and the degree of resistance correlated with the increase in enzyme activity. Likewise, when resistance was induced by stepwise selection with the drug, the resulting resistant promastigotes exhibited increased levels of farnesyl diphosphate synthase. The overproduction of protein under different conditions of exposure to risedronate further supports the hypothesis that this enzyme is the main target of aminobisphosphonates in Leishmania cells.     

Mature-onset obesity and insulin resistance in mice deficient in the signaling adapter p62

Signaling cascades that control adipogenesis are essential in the regulation of body weight and obesity. The adaptor p62 controls pathways that modulate cell differentiation. We report here that p62(-/-) mice develop mature-onset obesity, leptin resistance, as well as impaired glucose and insulin intolerance. The metabolic rate was significantly reduced in p62(-/-) nonobese mice, which displayed increased mRNA levels of PPAR-gamma and reduced levels of UCP-1 in adipose tissue. Basal activity of ERK was enhanced in fat from nonobese mutant mice. Embryo fibroblasts from p62(-/-) mice differentiated better than the wild-type controls into adipocytes, which was abrogated by pharmacological inhibition of the ERK pathway. p62 is induced during adipocyte differentiation and inhibits ERK activation by direct interaction. We propose that p62 normally antagonizes basal ERK activity and adipocyte differentiation and that its loss leads to the hyperactivation of ERK that favors adipogenesis and obesity.     

NF-kappaB activation prevents apoptotic oxidative stress via an increase of both thioredoxin and MnSOD levels in TNFalpha-treated Ewing sarcoma cells

Repression of activation of c-Jun N-terminal kinase (JNK) participates in the anti-apoptotic effect of nuclear factor-kappaB (NF-kappaB) in TNFalpha-treated Ewing sarcoma cells. As oxidative stress is one of the most prominent activators of JNK, we investigated the relationship between TNFalpha-induced NF-kappaB activation and the control of oxidative stress. Inhibition of NF-kappaB activation resulted in an increase in TNFalpha-induced ROS production, lipid peroxidation and protein oxidation. Those ROS and lipid peroxides were both involved in TNFalpha-induced apoptosis, whereas only ROS elevation triggered sustained JNK activation. TNFalpha increased the level of two antioxidant enzymes, thioredoxin and manganese superoxide dismutase by an NF-kappaB-dependent mechanism. Inhibition of expression or activity of these enzymes sensitized cells to TNFalpha-induced apoptosis, indicating their functional role in protection from cell death. Thus, agents that inhibit activities of these enzymes may prove helpful in the treatment of Ewing tumors.