Alignment-free prediction of mycobacterial DNA promoters based on pseudo-folding lattice network or star-graph topological indices

The importance of the promoter sequences in the function regulation of several important mycobacterial pathogens creates the necessity to design simple and fast theoretical models that can predict them. This work proposes two DNA promoter QSAR models based on pseudo-folding lattice network (LN) and star-graphs (SG) topological indices. In addition, a comparative study with the previous RNA electrostatic parameters of thermodynamically-driven secondary structure folding representations has been carried out. The best model of this work was obtained with only two LN stochastic electrostatic potentials and it is characterized by accuracy, selectivity and specificity of 90.87%, 82.96% and 92.95%, respectively. In addition, we pointed out the SG result dependence on the DNA sequence codification and we proposed a QSAR model based on codons and only three SG spectral moments.     

Characterization of an Alcohol Dehydrogenase from the Cyanobacterium Synechocystis sp. Strain PCC 6803 That Responds to Environmental Stress Conditions via the Hik34-Rre1 Two-Component System

The slr1192 (adhA) gene from Synechocystis sp. strain PCC 6803 encodes a member of the medium-chain alcohol dehydrogenase/reductase family. The gene product AdhA exhibits NADP-dependent alcohol dehydrogenase activity, acting on a broad variety of aromatic and aliphatic primary alcohols and aldehydes but not on secondary alcohols or ketones. It exhibits superior catalytic efficiency for aldehyde reduction compared to that for alcohol oxidation. The enzyme is a cytosolic protein present in photoautotrophically grown Synechocystis cells. The expression of AdhA is enhanced upon the exposure of cells to different environmental stresses, although it is not essential for survival even under such stress conditions. The induction of the expression of the adhA gene is dependent on the Hik34-Rre1 two-component system, as it is severely impaired in mutant strains lacking either the histidine kinase Hik34 or the response regulator Rre1. In vitro DNA-protein interaction analysis reveals that the response regulator Rre1 binds specifically to the promoter region of the adhA gene.Medium-chain dehydrogenases/reductases (MDR) constitute a superfamily of alcohol dehydrogenases that catalyze the reversible NAD(P)-dependent oxidation of alcohols to aldehydes or ketones. It includes a large number of structurally related proteins, which catalyze several types of enzymatic activity (23, 41, 44). Screening of complete genome sequences has revealed that this family is widespread, complex, and of ancient origin (22, 44). MDR alcohol dehydrogenases are found in mammals, plants, fungi, and bacteria (52). The alcohol dehydrogenases fulfill an astonishing variety of functions in cell metabolism (21), also being a key enzyme in ethanol generation by Saccharomyces cerevisiae (6) and bacteria (10). Furthermore, the generation of biofuels by photoautotrophic microorganisms is of great biotechnological interest (43). Complementation of a cyanobacterium''s enzyme machinery with a specific exogenous gene(s) can result in the ability to generate bioethanol from photosynthetically fixed CO₂ (11). Notwithstanding, current knowledge of cyanobacterial alcohol dehydrogenases is rather limited. In the cyanobacterium Synechocystis sp. strain PCC 6803 (referred here as Synechocystis), the slr1192 gene encodes a putative MDR alcohol dehydrogenase. According to in silico analyses (38, 44), the slr1192 protein has similarity with two subfamilies of MDRs: the yeast ADH family (Y-ADH) and the cinnamyl ADH family (CADH). Y-ADH-related enzymes have catabolic functions and are involved mainly in the metabolism of ethanol or short-chain alcohols for which they exhibit broad substrate specificity. CADH and related enzymes, on the other hand, perform anabolic functions and participate in biosynthetic pathways in plants and bacteria (5, 25, 44).In Synechocystis, the expression of slr1192 is induced by osmotic (35) or salt (48) stress. In higher plants, alcohol dehydrogenase activity appears to be involved in aerobic metabolism under certain stress conditions (26, 56) such as low temperature, water deficit, or ozone exposure, but its function remains unknown. A temperature decrease seems to induce the accumulation of alcohol dehydrogenase mRNA in Arabidopsis thaliana (20), corn, and rice (9).In general, cyanobacteria perceive and respond to environmental changes by means of two-component regulatory systems, a ubiquitous signal transduction pathway that represents a prevalent signaling mechanism in bacteria (8, 61). Two-component systems consist of a histidine kinase (Hik) and a response regulator (Rre) and generally induce or repress the expression of specific genes in response to environmental stimuli. The histidine kinase autophosphorylates a conserved histidine residue in response to the environmental signal and then transfers the phosphate group to a conserved aspartate residue of the response regulator, which mediates the transfer of the signal. In Synechocystis, different Hik-Rre systems have been identified as being regulators of the response to different environmental stresses (37). A membrane-bound histidine kinase, Hik33, is involved in the perception of cold, salt, and osmotic stress (33, 35, 39, 48, 54). A cytosolic histidine kinase, Hik34, has been shown to be involved in the perception of salt and hyperosmotic stress (33, 39, 48) as well as heat shock (53). Specifically, the couples Hik33-Rre31, Hik10-Rre13, Hik16 Hik41-Rre17, Hik34-Rre1, and a putative Hik2-Rre1 have been identified as being elements involved in the perception and transduction of signals promoted by hyperosmotic and salt stress (39, 48).In the present work, a biochemical characterization of the slr1192 protein (designated AdhA here) from Synechocystis has been performed, revealing that the tetrameric 140-kDa enzyme is active toward linear and aromatic primary alcohols and that it preferentially reduces aldehydes rather than oxidizing alcohols. In addition, an extensive analysis of the expression of the adhA gene has verified its induction in response to heat shock, hyperosmotic stress, salt stress, and the addition of benzyl alcohol (BA). The Hik34-Rre1 two-component system has been shown to play a relevant role in the regulation of the expression of the adhA gene under these stress conditions. A specific interaction of Rre1 with the promoter region of the adhA gene has also been demonstrated. In light of this finding and additional information presented here, the physiological role of AdhA in Synechocystis is discussed.     

C-Terminally PEGylated hGH-derivatives

A two-step strategy was used for the preparation of C-terminally PEGylated hGH-derivatives. In a first step a CPY-catalyzed transpeptidation was performed on hGH-Leu-Ala, introducing reaction handles, which were used in the second step for the ligation of PEG-moieties. Both oxime-ligation and copper(I) catalyzed [2+3]-cycloaddition reactions were used for the attachment of PEG-moieties. The biological data show a dependency of the potency of the hGH-derivatives on both size as well as shape of the PEG-group.     

Kinetic and structural analysis of a new group of Acyl-CoA carboxylases found in Streptomyces coelicolor A3(2)

Two acyl-CoA carboxylases from Streptomyces coelicolor have been successfully reconstituted from their purified components. Both complexes shared the same biotinylated alpha subunit, AccA2. The beta and the epsilon subunits were specific from each of the complexes; thus, for the propionyl-CoA carboxylase complex the beta and epsilon components are PccB and PccE, whereas for the acetyl-CoA carboxylase complex the components are AccB and AccE. The two complexes showed very low activity in the absence of the corresponding epsilon subunits; addition of PccE or AccE dramatically increased the specific activity of the enzymes. The kinetic properties of the two acyl-CoA carboxylases showed a clear difference in their substrate specificity. The acetyl-CoA carboxylase was able to carboxylate acetyl-, propionyl-, or butyryl-CoA with approximately the same specificity. The propionyl-CoA carboxylase could not recognize acetyl-CoA as a substrate, whereas the specificity constant for propionyl-CoA was 2-fold higher than for butyryl-CoA. For both enzymes the epsilon subunits were found to specifically interact with their carboxyltransferase component forming a beta-epsilon subcomplex; this appears to facilitate the further interaction of these subunits with the alpha component. The epsilon subunit has been found genetically linked to several carboxyltransferases of different Streptomyces species; we propose that this subunit reflects a distinctive characteristic of a new group of acyl-CoA carboxylases.     

Fluorescence study of ligand binding to potato tuber pyrophosphate-dependent phosphofructokinase: evidence for competitive binding between fructose-1,6-bisphosphate and fructose-2,6-bisphosphate

The intrinsic fluorescence of potato tuber pyrophosphate:fructose-6-phosphate 1-phosphotransferase (PFP) was used as an indicator of conformational changes due to ligand binding. Binding of the substrates and the allosteric activator fructose-2,6-bisphosphate was quantitatively compared to their respective kinetic effects on enzymatic activity. PFP exhibited a relatively high affinity for its isolated substrates, relative to the enzyme's respective K(m) (substrate) values. There are two distinct types of fructose-1,6-bisphosphate interaction with PFP, corresponding to catalytic and activatory binding. Activatory fructose-1,6-bisphosphate binding shares several characteristics with fructose-2,6-bisphosphate binding, indicating that both ligands compete for the same allosteric activator site. Activation by fructose-1,6-bisphosphate or fructose-2,6-bisphosphate was exerted primarily on the forward (glycolytic) reaction by greatly increasing the enzyme's affinity for fructose-6-phosphate. Binding of substrates and effectors to PFP and PFP kinetic properties were markedly influenced by assay pH. Results indicate an increased glycolytic role for PFP during cytosolic acidification that accompanies anoxia stress.     

The organization of the microbial biodegradation network from a systems-biology perspective

Microbial biodegradation of environmental pollutants is a field of growing importance because of its potential use in bioremediation and biocatalysis. We have studied the characteristics of the global biodegradation network that is brought about by all the known chemical reactions that are implicated in this process, regardless of their microbial hosts. This combination produces an efficient and integrated suprametabolism, with properties similar to those that define metabolic networks in single organisms. The characteristics of this network support an evolutionary scenario in which the reactions evolved outwards from the central metabolism. The properties of the global biodegradation network have implications for predicting the fate of current and future environmental pollutants.     

The diversity and complexity of the cyanobacterial thioredoxin systems

Cyanobacteria perform oxygenic photosynthesis, which makes them unique among the prokaryotes, and this feature together with their abundance and worldwide distribution renders them a central ecological role. Cyanobacteria and chloroplasts of plants and algae are believed to share a common ancestor and the modern chloroplast would thus be the remnant of an endosymbiosis between a eukaryotic cell and an ancestral oxygenic photosynthetic prokaryote. Chloroplast metabolic processes are coordinated with those of the other cellular compartments and are strictly controlled by means of regulatory systems that commonly involve redox reactions. Disulphide/dithiol exchange catalysed by thioredoxin is a fundamental example of such regulation and represents the molecular mechanism for light-dependent redox control of an ever-increasing number of chloroplast enzymatic activities. In contrast to chloroplast thioredoxins, the functions of the cyanobacterial thioredoxins have long remained elusive, despite their common origin. The sequenced genomes of several cyanobacterial species together with novel experimental approaches involving proteomics have provided new tools for re-examining the roles of the thioredoxin systems in these organisms. Thus, each cyanobacterial genome encodes between one and eight thioredoxins and all components necessary for the reduction of thioredoxins. Screening for thioredoxin target proteins in cyanobacteria indicates that assimilation and storage of nutrients, as well as some central metabolic pathways, are regulated by mechanisms involving disulphide/dithiol exchange, which could be catalysed by thioredoxins or related thiol-containing proteins.     

Selecting thioredoxins for disulphide proteomics: target proteomes of three thioredoxins from the cyanobacterium Synechocystis sp. PCC 6803

Searching for enzymes and other proteins which can be redox-regulated by dithiol/disulphide exchange is a rapidly expanding area of functional proteomics. Recently, several experimental approaches using thioredoxins have been developed for this purpose. Thioredoxins comprise a large family of redox-active enzymes capable of reducing protein disulphides to cysteines and of participating in a variety of processes, such as enzyme modulation, donation of reducing equivalents and signal transduction. In this study we screened the target proteomes of three different thioredoxins from the unicellular cyanobacterium Synechocystis sp. PCC 6803, using site-directed active-site cysteine-to-serine mutants of its m-, x- and y-type thioredoxins. The properties of a thioredoxin that determine the outcome of such analyses were found to be target-binding capacity, solubility and the presence of non-active-site cysteines. Thus, we explored how the choice of thioredoxin affects the target proteomes and we conclude that the m-type thioredoxin, TrxA, is by far the most useful for screening of disulphide proteomes. Furthermore, we improved the resolution of target proteins on non-reducing/reducing 2-DE, leading to the identification of 14 new potentially redox-regulated proteins in this organism. The presence of glycogen phosphorylase among the newly identified targets suggests that glycogen breakdown is redox-regulated in addition to glycogen synthesis.     

<Emphasis Type="Italic">In Vitro</Emphasis>, Melatonin Treatment Decreases Nitric Oxide Levels in Murine Splenocytes Cultured with the Venezuelan Equine Encephalomyelitis Virus

The purpose of this work was to determine the effect of melatonin on the nitric oxide levels in murine splenocytes cultured with the Venezuelan equine encephalomyelitis virus. After incubation, nitric oxide levels were measured by the diazotization assay. Those cultures with the Venezuelan equine encephalomyelitis virus increased nitric oxide levels. Splenocytes infected and treated with 100 and 150 μg/ml of melatonin, decreased significantly the nitric oxide levels when compared to infected and non-treated splenocytes. These findings show that splenocytes infected with the Venezuelan equine encephalomyelitis virus generate important amounts of nitric oxide and suggest that melatonin protects the mice infected with the Venezuelan equine encephalomyelitis virus by a mechanism involving the decreasing of nitric oxide concentrations in tissue.     

An ultrasensitive capture ELISA for detection of Fasciola hepatica coproantigens in sheep and cattle using a new monoclonal antibody (MM3)

A capture enzyme-linked immunosorbent assay (ELISA) using a new monoclonal antibody (mAb MM3) is reported for the detection of Fasciola hepatica excretory-secretory antigens (ESAs) in feces of infected hosts. The mAb MM3 was produced by immunization of mice with a 7- to 40-kDa purified and O-deglycosylated fraction of F. hepatica ESAs, which has previously been shown to be specific for the parasite. The specificity and sensitivity of the MM3 capture ELISA were assessed using feces from sheep and cattle. Sheep feces were obtained from a fluke-free herd (with most animals harboring other nematodes and cestodes), from lambs experimentally infected with 5-40 F. hepatica metacercariae and in some cases treated with triclabendazole at 14 wk postinfection (PI), and from uninfected control lambs. Cattle feces were collected at the slaughterhouse from adult cows naturally infected with known numbers of flukes (from 1 to 154) or free of F. hepatica infection (though in most cases harboring other helminths). The MM3 capture ELISA assay had detection limits of 0.3 (sheep) and 0.6 (cattle) ng of F. hepatica ESA per milliliter of fecal supernatant. The assay detected 100% of sheep with 1 fluke, 100% of cattle with 2 flukes, and 2 of 7 cattle with 1 fluke. The false-negative animals (5/7) were probably not detected because the F. hepatica individuals in these animals were immature (5-11 mm in length). As expected, coproantigen concentration correlated positively (r = 0.889; P < 0.001) with parasite burden and negatively (r = 0.712; P < 0.01) with the time after infection at which coproantigen was first detected. Nevertheless, even in animals with low fluke burdens (1-36 parasites), the first detection of F. hepatica-specific coproantigens by the MM3 capture ELISA preceded the first detection in egg count by 1-5 wk. In all sheep that were experimentally infected and then untreated, coproantigen remained detectable until at least 18 wk PI, whereas in sheep that were experimentally infected and then flukicide treated, coproantigen became undetectable from 1 to 3 wk after treatment. None of the fecal samples from sheep or cattle negative for fascioliasis but naturally infected with other parasites including Dicroelium dendriticum showed reactivity in the MM3 capture ELISA. These results indicate that this assay is a reliable and ultrasensitive method for detecting subnanogram amounts of F. hepatica antigens in feces from sheep and cattle, facilitating early diagnosis.