Purification, characterization and substrate specificity of a trypsin from the Amazonian fish tambaqui (Colossoma macropomum)

An enzyme was purified from the pyloric caecum of tambaqui (Colossoma macropomum) through heat treatment, ammonium sulfate fractionation, Sephadex® G-75 and p-aminobenzamidine-agarose affinity chromatography. The enzyme had a molecular mass of 23.9 kDa, NH₂-terminal amino acid sequence of IVGGYECKAHSQPHVSLNI and substrate specificity for arginine at P1, efficiently hydrolizing substrates with leucine and lysine at P2 and serine and arginine at P1′. Using the substrate z-FR-MCA, the enzyme exhibited greatest activity at pH 9.0 and 50 °C, whereas, with BAPNA activity was higher in a pH range of 7.5-11.5 and at 70 °C. Moreover, the enzyme maintained ca. 60% of its activity after incubated for 3 h at 60 °C. The enzymatic activity significantly decreased in the presence of TLCK, benzamidine (trypsin inhibitors) and PMSF (serine protease inhibitor). This source of trypsin may be an attractive alternative for the detergent and food industry.     

A transposon toolkit for gene transfer and mutagenesis in protozoan parasites

Protozoan parasites affect millions of people around the world. Treatment and control of these diseases are complicated partly due to the intricate biology of these organisms. The interactions of species of Plasmodium, Leishmania and trypanosomes with their hosts are mediated by an unusual control of gene expression that is not fully understood. The availability of the genome sequence of these protozoa sets the stage for using more comprehensive, genome-wide strategies to study gene function. Transposons are effective tools for the systematic introduction of genetic alterations and different transposition systems have been adapted to study gene function in these human pathogens. A mariner transposon toolkit for use in vivo or in vitro in Leishmania parasites has been developed and can be used in a variety of applications. These modified mariner elements not only permit the inactivation of genes, but also mediate the rescue of translational gene fusions, bringing a major contribution to the investigation of Leishmania gene function. The piggyBac and Tn5 transposons have also been shown to mobilize across Plasmodium spp. genomes circumventing the current limitations in the genetic manipulation of these organisms.     

The ruthenium complex <Emphasis Type="Italic">cis</Emphasis>-(dichloro)tetrammineruthenium(III) chloride induces apoptosis and damages DNA in murine sarcoma 180 cells

Ruthenium(III) complexes are increasingly attracting the interest of researchers due to their promising pharmacological properties. Recently, we reported that the cis-(dichloro)tetrammineruthenium(III) chloride compound has cytotoxic effects on murine sarcoma 180 (S-180) cells. In an effort to understand the mechanism responsible for their cytotoxicity, study we investigated the genotoxicity, cell cycle distribution and induction of apoptosis caused by cis-(dichloro)tetrammineruthenium(III) chloride in S-180 tumour cells. cis-(dichloro)tetrammineruthenium(III) chloride treatment induced significant DNA damage in S-180 cells, as detected by the alkaline comet assay. In the cell cycle analysis, cis-(dichloro)tetrammineruthenium(III) chloride caused an increase in the number of cells in G1 phase, accompanied by a decrease in the S and G2 phases after 24 h of treatment. In contrast, the cell cycle distribution of S-180 cells treated with cis-(dichloro)tetrammineruthenium(III) chloride for 48 h showed a concentration-dependent increase in the sub-G1 phase (indicating apoptosis), with a corresponding decrease in cells in the G1, S and G2 phases. In addition, cis-(dichloro)tetrammineruthenium(III) chloride treatment induced apoptosis in a time-dependent manner, as observed by the increased numbers of annexin V-positive cells. Taken together, these findings strongly demonstrate that DNA damage, cell cycle changes and apoptosis may correlate with the cytotoxic effects of cis-(dichloro)tetrammineruthenium(III) chloride on S-180 cells.     

A protecting group-free synthesis of deazathiamine: a step toward inhibitor design

The discovery of 3-deazathiamine diphosphate (deazaThDP) as a potent inhibitor analog of the cofactor thiamine diphosphate (ThDP) has highlighted the need for an efficient and scalable synthesis of deazaThDP. Such a method would facilitate development of analogs with the ability to inhibit individual ThDP-dependent enzymes selectively. Toward the goal of developing selective inhibitors of the mycobacterial enzyme 2-hydroxy-3-oxoadipate synthase (HOAS), we report an improved synthesis of deazaThDP without use of protecting groups. Tribromo-3-methylthiophene served as a versatile starting material whose selective functionalization permitted access to deazaThDP in five steps, with potential to make other analogs accessible in substantial amounts.     

Crystal structure and molecular dynamics studies of human purine nucleoside phosphorylase complexed with 7-deazaguanine

In humans, purine nucleoside phosphorylase (HsPNP) is responsible for degradation of deoxyguanosine, and genetic deficiency of this enzyme leads to profound T-cell mediated immunosuppression. HsPNP is a target for inhibitor development aiming at T-cell immune response modulation. Here we report the crystal structure of HsPNP in complex with 7-deazaguanine (HsPNP:7DG) at 2.75 Å. Molecular dynamics simulations were employed to assess the structural features of HsPNP in both free form and in complex with 7DG. Our results show that some regions, responsible for entrance and exit of substrate, present a conformational variability, which is dissected by dynamics simulation analysis. Enzymatic assays were also carried out and revealed that 7-deazaguanine presents a lower inhibitory activity against HsPNP (K_i = 200 μM). The present structure may be employed in both structure-based design of PNP inhibitors and in development of specific empirical scoring functions.     

Long-Term CO2 Variability in Two Shallow Tropical Lakes Experiencing Episodic Eutrophication and Acidification Events

Here we report the long-term (13-year) dynamics of surface pCO₂ and its response to episodic eutrophication and acidification events in two contrasting tropical coastal lakes, one clear-water and the other humic. A short-term nutrient addition experiment was also conducted in mesocosms in the humic lake where in situ eutrophication was moderate. Our objective was to elucidate the response of pCO₂ to interannual changes in key limnological conditions, such as nutrient concentrations and pH. The humic waters showed a median pCO₂ almost ninefold higher across the 13-year study than the clear waters, supporting pCO₂ values about tenfold above atmospheric equilibrium. Eutrophication of the clear-water lake resulted in a decrease in pCO₂ to median values below atmospheric equilibrium, producing a strong sink for atmospheric CO₂. In contrast, pCO₂ increased by over tenfold in both lakes during the acidification phase, resulting in very large CO₂ emissions to the atmosphere. Experimental nutrient additions in the humic lake showed a strong persistence of high pCO₂. The extreme variability in pCO₂ observed here might be a characteristic of tropical lakes and may have important consequences for regional carbon budgets.     

Evolution of xyloglucan-related genes in green plants

Background  

The cell shape and morphology of plant tissues are intimately related to structural modifications in the primary cell wall that are associated with key processes in the regulation of cell growth and differentiation. The primary cell wall is composed mainly of cellulose immersed in a matrix of hemicellulose, pectin, lignin and some structural proteins. Xyloglucan is a hemicellulose polysaccharide present in the cell walls of all land plants (Embryophyta) and is the main hemicellulose in non-graminaceous angiosperms.     

Mutation accumulation in <Emphasis Type="Italic">Tetrahymena</Emphasis>

Background  

The rate and fitness effects of mutations are key in understanding the evolution of every species. Traditionally, these parameters are estimated in mutation accumulation experiments where replicate lines are propagated in conditions that allow mutations to randomly accumulate without the purging effect of natural selection. These experiments have been performed with many model organisms but we still lack empirical estimates of the rate and effects of mutation in the protists.     

Virulence-related <Emphasis Type="Italic">Mycobacterium avium</Emphasis> subsp <Emphasis Type="Italic">hominissuis</Emphasis> MAV_2928 gene is associated with vacuole remodeling in macrophages

Background  

Mycobacterium avium subsp hominissuis (previously Mycobacterium avium subsp avium) is an environmental organism associated with opportunistic infections in humans. Mycobacterium hominissuis infects and replicates within mononuclear phagocytes. Previous study characterized an attenuated mutant in which the PPE gene (MAV_2928) homologous to Rv1787 was inactivated. This mutant, in contrast to the wild-type bacterium, was shown both to have impaired the ability to replicate within macrophages and to have prevented phagosome/lysosome fusion.