The role of residue Thr249 in modulating the catalytic efficiency and substrate specificity of catechol-2,3-dioxygenase from Pseudomonas stutzeri OX1

Bioremediation strategies use microorganisms to remove hazardous substances, such as aromatic molecules, from polluted sites. The applicability of these techniques would greatly benefit from the expansion of the catabolic ability of these bacteria in transforming a variety of aromatic compounds. Catechol-2,3-dioxygenase (C2,3O) from Pseudomonas stutzeri OX1 is a key enzyme in the catabolic pathway for aromatic molecules. Its specificity and regioselectivity control the range of molecules degraded through the catabolic pathway of the microorganism that is able to use aromatic hydrocarbons as growth substrates. We have used in silico substrate docking procedures to investigate the molecular determinants that direct the enzyme substrate specificity. In particular, we looked for a possible molecular explanation of the inability of catechol-2,3-dioxygenase to cleave 3,5-dimethylcatechol and 3,6-dimethylcatechol and of the efficient cleavage of 3,4-dimethylcatechol. The docking study suggested that reduction in the volume of the side chain of residue 249 could allow the binding of 3,5-dimethylcatechol and 3,6-dimethylcatechol. This information was used to prepare and characterize mutants at position 249. The kinetic and regiospecificity parameters of the mutants confirm the docking predictions, and indicate that this position controls the substrate specificity of catechol-2,3-dioxygenase. Moreover, our results suggest that Thr249 also plays a previously unsuspected role in the catalytic mechanism of substrate cleavage. The hypothesis is advanced that a water molecule bound between one of the hydroxyl groups of the substrate and the side chain of Thr249 favors the deprotonation/protonation of this hydroxyl group, thus assisting the final steps of the cleavage reaction.     

Hymenobacter perfusus sp. nov., Hymenobacter flocculans sp. nov. and Hymenobacter metalli sp. nov. three new species isolated from an uranium mine waste water treatment system

Three red-pink pigmented strains, designated A1-12(T), A2-50A(T) and A2-91(T), were recovered from two different sites in a uranium mine. For all strains, the optimum growth temperature was 25°C, the optimum pH was 6.0-6.5 and the DNA G+C contents were between 60 and 63.4 mol%. The major respiratory quinone was menaquinone 7 (MK-7) and the fatty acid profiles contained iso- and anteiso-branched C15 fatty acids, summed feature 3 (16:1 ω6c and/or ω7c and/or 15:0 iso 2-OH), summed feature 4 (17:1 anteiso B and/or iso I) and the unsaturated fatty acid 16:1 ω5c as the major components. Phylogenetic analysis of the 16S rRNA gene sequences showed that these organisms represented three distinct branches within the family Flexibacteraceae most closely related to the members of the genus Hymenobacter. Strain A1-12(T) formed a distinct phylogenetic line along with H. rigui KCTC 12533(T) and they shared approximately 98.9% 16S rRNA gene sequence similarity. However, these two strains shared only 14.7% pairwise similarity in their genomic DNA. Strains A2-50A(T) and A2-91(T) formed two distinct lineages, related to the species H. soli KCTC 12607(T), sharing about 95.5% 16S rRNA gene sequence similarity between themselves, and 88.3 and 92.0% with other members of the genus Hymenobacter. Based on the phylogenetic analysis and physiological and biochemical characteristics, these isolates were considered to represent three novel species for which we propose the names Hymenobacter perfusus for strain A1-12(T) (=CIP 110166=LMG 26000), Hymenobacter flocculans for strain A2-50A(T) (=CIP 110139=LMG 25699) and Hymenobacter metalli for strain A2-91(T) (=CIP 110140=LMG 25700).     

New ruthenium(II) mixed metallocene derived complexes: Synthesis, characterization by X-ray diffraction and evaluation on DNA interaction by atomic force microscopy

A new family of Ru(II) mixed metallocene complexes of the type [Ru(η⁵-C₅H₅)(η⁶-arene)][PF₆] has been synthesized and fully characterized by NMR and UV-Vis spectroscopy. X-ray analysis of single crystal was achieved for all the complexes and revealed the presence of two enantiomers expected for planar chirality originated by the η⁶ coordination of the arene prochiral ligands. Studies of interaction of the new complexes with pBR322 DNA by atomic force microscopy showed very strong and different types of interaction. Antiproliferative tests were examined on human leukemia cancer cells (HL-60) using the MTT assay, and the IC₅₀ values revealed low antiproliferative activity compared to cisplatin.     

Peptide gomesin triggers cell death through L-type channel calcium influx,MAPK/ERK,PKC and PI3K signaling and generation of reactive oxygen species

Gomesin is an antimicrobial peptide isolated from hemocytes of a common Brazilian tarantula spider named Acanthoscurria gomesiana. This peptide exerts antitumor activity in vitro and in vivo by an unknown mechanism. In this study, the cytotoxic mechanism of gomesin in human neuroblastoma SH-SY5Y and rat pheochromocytoma PC12 cells was investigated. Gomesin induced necrotic cell death and was cytotoxic to SH-SY5Y and PC12 cells. The peptide evoked a rapid and transient elevation of intracellular calcium levels in Fluo-4-AM loaded PC12 cells, which was inhibited by nimodipine, an L-type calcium channel blocker. Preincubation with nimodipine also inhibited cell death induced by gomesin in SH-SY5Y and PC12 cells. Gomesin-induced cell death was prevented by the pretreatment with MAPK/ERK, PKC or PI3K inhibitors, but not with PKA inhibitor. In addition, gomesin generated reactive oxygen species (ROS) in SH-SY5Y cells, which were blocked with nimodipine and MAPK/ERK, PKC or PI3K inhibitors. Taken together, these results suggest that gomesin could be a useful anticancer agent, which mechanism of cytotoxicity implicates calcium entry through L-type calcium channels, activation of MAPK/ERK, PKC and PI3K signaling as well as the generation of reactive oxygen species.     

Protective effect of anacardic acids from cashew (Anacardium occidentale) on ethanol-induced gastric damage in mice

Cashew nut-shell liquid and the contained anacardic acids (AAs) have been shown to possess antioxidant, lipoxygenase inhibitory, anti-Helicobacter pylori and antitumor properties. Despite these known effects, hitherto there were no published reports on their likely gastroprotective effects. The present study was designed to verify whether AAs afford gastroprotection against the ethanol-induced gastric damage and to examine the underlying mechanism(s). Gastric damage was induced by intragastric administration of 0.2 mL of ethanol (96%). Mice in groups were pretreated orally with AAs (10, 30 and 100 mg/kg), misoprostol (50 μg/kg), or vehicle (2% Tween 80 in saline, 10 mL/kg), 45 min before ethanol administration. They were sacrificed 30 min later, the stomachs excised, and the mucosal lesion area (mm²) measured by planimetry. Gastroprotection was assessed in relation to inhibition of gastric lesion area. To study the gastroprotective mechanism(s), its relations to capsaicin-sensitive fibers, endogenous prostaglandins, nitric oxide and ATP-sensitive potassium channels were analysed. Treatments effects on ethanol-associated oxidative stress markers GSH, MDA, catalase, SOD, and total nitrate/nitrite levels as an index of NO were measured in gastric tissue. Besides, the effects of AAs on gastric secretory volume and total acidity were analysed in 4-h pylorus-ligated rat. AAs afforded a dose-related gastroprotection against the ethanol damage and further prevented the ethanol-induced changes in the levels of GSH, MDA, catalase, SOD and nitrate/nitrite. However, they failed to modify the gastric secretion or the total acidity. It was observed that the gastroprotection by AAs was greatly reduced in animals pretreated with capsazepine, indomethacin, l-NAME or glibenclamide. These results suggest that AAs afford gastroprotection principally through an antioxidant mechanism. Other complementary mechanisms include the activation of capsaicin-sensitive gastric afferents, stimulation of endogenous prostaglandins and nitric oxide, and opening of K⁺_ATP channels. These combined effects are likely to be accompanied by an increase in gastric microcirculation.     

Chimaereicella alkaliphila gen. nov., sp. nov., a Gram-negative alkaliphilic bacterium isolated from a nonsaline alkaline groundwater

A Gram-negative bacterium designated AC-74(T) was isolated from a highly alkaline groundwater environment (pH 11.4). This organism formed rod-shaped cells, is strictly aerobic, catalase and oxidase positive, tolerates up to 3.0% NaCl, has an optimum growth temperature of 30 degrees C, but no growth occurs at 10 or 40 degrees C, and an optimum pH value of 8.0, but no growth occurs at pH 7.0 or 11.3. The predominant fatty acids are iso-15:0, iso-17:1 omega9c and 16:1 omega7c and or iso-15:2OH. The G+C content of DNA was 43.5mol%. The phylogenetic analyses of the sequences of the 16s RNA genes indicated that strain AC-74(T) belongs to the family "Flexibacteriaceae" and is phylogenetically equidistant ( approximately 94.5%) from the majority of the species of the genus Algoriphagus and from the genus Hongiella. Based on the phylogenetic analyses and distinct phenotypic characteristics, we are of the opinion that strain AC-74(T), represents a new species of the novel genus for which we propose the name Chimaereicella alkaliphila gen. nov., sp. nov.     

Preliminar evaluation of cytokines in the hepatitis C-schistosomiasis co-infection

Evaluation of hepatic fibrosis is usually performed by histopathological examination of biopsies. However, this is an invasive and potentially dangerous procedure. Several studies have proposed serum biological markers of hepatic fibrosis. This communication evaluates the use of serum cytokines as markers of hepatic fibrosis in hepatitis C, schistosomiasis, and co-infection.     

Relationships among biological elements (macrophytes, macroinvertebrates and ichthyofauna) for different core river types across Europe at two different spatial scales

The objective of this study was to evaluate differences in correlations among Biological Elements and environmental parameters for different river types, analysed at two different spatial scales. A total of 82 sites, with at least good ecological status, were sampled across Europe, representing three core river types: Mountain rivers (26 sites); Lowland rivers (29 sites) and Mediterranean rivers (17 sites). At each site samples of macrophytes, macroinvertebrates and fishes were taken during spring, following the methodological procedures established by the European STAR project. Environmental parameters were also recorded, based on a site protocol developed by the European projects AQEM and STAR. Environmental parameters were divided into three categories: aquatic habitats (mesohabitat scale), global features (reach scale) and obligatory typology parameters of Water Framework Directive (WFD) (geographical scale). Data were analysed to evaluate at the two scales, first, relationships among biological elements, and second, relationships between biological elements and environmental parameters. Within each river type, correlation matrices (Bray–Curtis distance) were calculated separately for each biological element and for each category of environmental parameters. All biological elements were correlated (p<0.01) to the larger spatial scale: macrophytes and macroinvertebrates are more correlated in lowland and mountain rivers, while in Mediterranean rivers, fish and macrophytes presented higher correlations. These links tend to be consistent for different spatial scales, except if they are weak on a larger regional scale, obligatory parameters of WFD were, in most cases, significantly correlated with the three biological communities (p<0.05). Results at different spatial scales supported the hierarchical theory of river formation. Reach and mesohabitat environmental parameters tend to explain aquatic communities at a lower spatial scale, while geographical parameters tend to explain the communities at a major spatial scale.     

Colony internal conditions related to caste production in Melipona compressipes fasciculata (Apidae, Meliponini)

Caste fate conflict is expected in Melipona bees because queens and workers are the same size and are reared in identical sealed cells. Extrinsic and intrinsic colonial factors, however, seem to have limiting effects on queen production. The consequences of colonial conditions on both queen and worker production, particularly the effects of food storage in the colonies, are still poorly understood. Here, we investigated whether caste production in seven natural colonies of Melipona compressipes fasciculata was affected by food resources, seasonal factors, or internal factors. The results showed that, at the populational level, about 5% of the females developed into gynes; in the strongest colony, 12.7% of the females developed into gynes. Male production was verified only in stronger colonies. We suggest that caste ratio is primarily affected by intra-colonial conditions rather than by food resources. Received 1 November 2005; revised 30 January 2006; accepted 17 February 2006.