maT - A clade of transposons intermediate between mariner and Tc1

A group of transposons, named maT, with characteristics intermediate between mariner and Tc1 transposons, is described. Two defective genomic copies of MdmaT from the housefly Musca domestica, with 85% identity, were found flanking and imbedded in the MdalphaE7 esterase gene involved in organophosphate insecticide resistance. Two cDNA clones, with 99% identity to each other and 72%-89% identity to the genomic copies were also obtained, but both represented truncated versions of the putative open reading frame. A third incomplete genomic copy of MdmaT was also identified upstream of the putative M. domestica period gene. The MdmaT sequences showed high identity to the transposable element Bmmar1 from the silk-worm moth, Bombyx mori, and to previously unidentified sequences in the genome of Caenorhabditis elegans. A total of 16 copies of full-length maT sequences were identified in the C elegans genome, representing three variants of the transposon, with 34%-100% identity amongst them. Twelve of the copies, named CemaT1, were virtually identical, with eight of them encoding a putative full length, intact transposase. Secondary structure predictions and phylogenetic analyses confirm that maT elements belong to the mariner-Tc1 superfamily of transposons, but their intermediate sequence and predicted structural characteristics suggest that they belong to a unique clade, distinct from either mariner-like or Tc1-like elements.

Cyclopropyl fatty acids implicate a radical but not a cation as an intermediate in P450(BM3)-catalysed hydroxylations

Novel cyclopropyl containing fatty acids are good substrates for P450(BM3) catalysed hydroxylation and analysis of their oxidation products indicates the presence of a radical intermediate (maximum rebound rate 2.6x10(10) s(-1)) and the absence of any cationic intermediate.

Heterologous expression of an Aspergillus niveus xylanase GH11 in Aspergillus nidulans and its characterization and application

A xylanase was cloned from Aspergillus niveus and successfully expressed in Aspergillus nidulans (XAN). The full-length gene consisted of 890 bp and encoded 275 mature amino acids with a calculated mass of 31.3 kDa. The deduced amino acid sequence was highly homologous with the xylanase belonging to family 11 of the glycoside hydrolases. The recombinant protein was purified to electrophoretic homogeneity by anion-exchange chromatography and gel filtration. The optima of pH and temperature for the recombinant enzyme were 5.0 and 65 degrees C, respectively. The thermal stability of the recombinant xylanase was extremely improved by covalent immobilization on glyoxyl agarose with 91.4% of residual activity after 180 min at 60 degrees C, on the other hand, the free xylanase showed a half-life of 9.9 min at the same temperature. Affinity chromatography on Concanavalin A- and Jacalin-agarose columns followed by SDS-PAGE analyses showed that the XAN has O- and N-glycans. XAN promotes hydrolysis of xylan resulting in xylobiose, xylotriose and xylotetraose. Intermediate degradation of xylan resulting in xylo-oligomers is appealing for functional foods as the beneficial effect of oligosaccharides on gastrointestinal micro flora includes preventing proliferation of pathogenic intestinal bacteria and facilitates digestion and absorption of nutrients. (C) 2011 Elsevier Ltd. All rights reserved.

Production of beta-fructofuranosidases by Aspergillus niveus using agroindustrial residues as carbon sources: Characterization of an intracellular enzyme accumulated in the presence of glucose

The production of beta-fructofuranosidases by Aspergillus niveus, cultivated under submerged fermentation using agroindustrial residues, was investigated. The highest productivity of beta-fructofuranosidases was obtained in Khanna medium supplemented with sugar cane bagasse as carbon source. Glucose enhanced the production of the intracellular enzyme, whereas that of the extracellular one was decreased. The intracellular beta-fructofuranosidase was a trimeric protein of approximately 141 kDa (gel filtration) with 53.5% carbohydrate content, composed of 57 kDa monomers (SDS-PAGE). The optimum temperature and optimum pH were 60 degrees C and 4.5, respectively. The purified enzyme showed good thermal stability and exhibited a half-life of 53 min at 60 degrees C. beta-Fructofuranosidase activity was slightly activated by Cu(2+), Mn(2+), Mg(2+), and Na(+) at 1 mM concentration. The enzyme hydrolyzed sucrose, raffinose, and inulin, with K(d) values of 5.78 mM, 5.74 mM, and 1.74 mM, respectively. (C) 2008 Elsevier Ltd. All rights reserved.

Biochemical and structural characterization of a beta-1,3-1,4-glucanase from Bacillus subtilis 168

beta-1,3-1,4-Glucanases (E.C. 3.2.1.73) hydrolyze linked beta-D-glucans, such as lichenan and barley beta-glucan. Recombinant beta-1,3-1,4-glucanase from Bacillus subtilis expressed in Escherichia coil and purified by Ni-NTA chromatography exhibited optimum activity at 50 degrees C and pH 6.0. The catalytic half-life at 60 degrees C decreased from 90 to 5 min when the enzyme was incubated in the presence and absence of Ca(2+) respectively. The kinetic parameters of lichenan hydrolysis were 2695, 3.1 and 1220 for V(max) (mu mol/min/mg), K(m) (mg mL(-1)) and K(cat) (s(-1)), respectively. Analysis by DLS, AUC and SAXS demonstrated the enzyme is monomeric in solution. Chemical denaturation monitored by ITFE and far-UV CD yielded Delta G(H2O) values of 9.6 and 9.1 kcal/mol, respectively, showing that the enzyme has intermediate stability when compared with other Bacillus beta-1,3-1,4-glucanases. The crystal structure shows the anti-parallel jelly-roll beta-sheet conserved in all GH16 beta-1,3-1,4-glucanases, with the amino acid differences between Bacillus sp. enzymes that are likely determinants of stability being distributed throughout the protein. (C) 2011 Elsevier Ltd. All rights reserved.

Development of novel bioanodes for ethanol biofuel cell using PAMAM dendrimers as matrix for enzyme immobilization

This paper describes the preparation and application of a novel bioanode for use in ethanol/O(2) biofuel cells based upon immobilization of alcohol dehydrogenase (ADH) and polyamidoamine (PAMAM) dendrimers onto carbon cloth platforms. The power density measurements indicated a direct relationship between the amount of anchored ADH and the anode power values, which increased upon enzyme loading. The power density values ranged from 0.04 to 0.28 mW cm(-2), and the highest power density was achieved with the bioanode prepared with 28 U of ADH, which provided a power density of 0.28 mW cm(-2) at 0.3 V. The latter power output values were the maximum observed, even for higher enzyme concentrations. Stability of the bioanodes was quite satisfactory, since there was no appreciable reduction of enzymatic activity during the measurements. The method of bioanode preparation described here has proven to be very effective. The PAMAM dendrimer represents a friendly environment for the immobilization of enzymes, and it is stable and capable of generating high power density compared to other immobilization methods. (C) 2010 Elsevier B.V. All rights reserved.

Molecular View of the Interaction between iota-Carrageenan and a Phospholipid Film and Its Role in Enzyme Immobilization

Proteins incorporated into phospholipid Langmuir-Blodgett (LB) films are a good model system for biomembranes and enzyme immobilization studies. The specific fluidity of biomembranes, an important requisite for enzymatic activity, is naturally controlled by varying phospholipid compositions. In a model system, instead, LB film fluidity may be varied by covering the top layer with different substances able to interact simultaneously with the phospholipid and the protein to be immobilized. In this study, we immobilized a carbohydrate rich Neurospora crassa alkaline phosphatase (NCAP) in monolayers of the sodium salt of dihexadecylphosphoric acid (DHP), a synthetic phospholipid that provides very condensed Langmuir films. The binding of NCAP to DHP Langmuir-Blodgett (LB) films was mediated by the anionic polysaccharide iota-carrageenan (iota-car). Combining results from surface isotherms and the quartz crystal microbalance technique, we concluded that the polysaccharide was essential to promote the interaction between DHP and NCAP and also to increase the fluidity of the film. An estimate of DHP:iota-car ratio within the film also revealed that the polysaccharide binds to DHP LB film in an extended conformation. Furthermore, the investigation of the polysaccharide conformation at molecular level, using sum-frequency vibrational spectroscopy (SFG), indicated a preferential conformation of the carrageenan molecules with the sulfate groups oriented toward the phospholipid monolayer, and both the hydroxyl and ether groups interacting preferentially with the protein. These results demonstrate how interfacial electric fields can reorient and induce conformational changes in macromolecules, which may significantly affect intermolecular interactions at interfaces. This detailed knowledge of the interaction mechanism between the enzyme and the LB film is relevant to design strategies for enzyme immobilization when orientation and fluidity properties of the film provided by the matrix are important to improve enzymatic activity.

Insertion/deletion polymorphism of the angiotensin converting enzyme gene and loss of the insertion allele in aldosterone-producing adenoma

The genetic mechanisms responsible for the formation of adrenocortical adenomas which autonomously produce aldosterone are largely unknown, The adrenal renin-angiotensin system has been implicated in the pathophysiology of these tumours, Angiotensin-converting enzyme (ACE) catalyses the generation of angiotensin II, and the insertion/deletion (I/D) polymorphism of the ACE gene regulates up to 50% of plasma and cellular ACE variability in humans. We therefore examined the genotypic and allelic frequency distributions of the ACE gene I/D polymorphism in 55 patients with aldosterone-producing adenoma, APA, (angiotensin-unresponsive APA n = 28, angiotensin-responsive APA n = 27), and 80 control subjects with no family history of hypertension, We also compared the ACE gene I/D polymorphism allelic pattern in matched tumour and peripheral blood DNA in the 55 patients with APA, The frequency of the D allele was 0.518 and 0.512 and the I allele was 0.482 and 0.488 in the APA and control subjects respectively, Genotypic and allelic frequency analysis found no significant differences between the groups, Examination of the matched tumour and peripheral blood DNA samples revealed the loss of the insertion allele in four of the 25 patients who were heterozygous for the ACE I/D genotype. The I/D polymorphism of the ACE gene does not appear to contribute to the biochemical and phenotypic characteristics of APA, however, the deletion of the insertion allele of the ACE gene I/D polymorphism in 16% of aldosterone-producing adenomas may represent the loss of a tumour suppressor gene/s or other genes on chromosome 17q which may contribute to tumorigenesis in APA.

Aerobic Exercise Training-Induced Left Ventricular Hypertrophy Involves Regulatory MicroRNAs, Decreased Angiotensin-Converting Enzyme-Angiotensin II, and Synergistic Regulation of Angiotensin-Converting Enzyme 2-Angiotensin (1-7)

Aerobic exercise training leads to a physiological, nonpathological left ventricular hypertrophy; however, the underlying biochemical and molecular mechanisms of physiological left ventricular hypertrophy are unknown. The role of microRNAs regulating the classic and the novel cardiac renin-angiotensin (Ang) system was studied in trained rats assigned to 3 groups: (1) sedentary; (2) swimming trained with protocol 1 (T1, moderate-volume training); and (3) protocol 2 (T2, high-volume training). Cardiac Ang I levels, Ang-converting enzyme (ACE) activity, and protein expression, as well as Ang II levels, were lower in T1 and T2; however, Ang II type 1 receptor mRNA levels (69% in T1 and 99% in T2) and protein expression (240% in T1 and 300% in T2) increased after training. Ang II type 2 receptor mRNA levels (220%) and protein expression (332%) were shown to be increased in T2. In addition, T1 and T2 were shown to increase ACE2 activity and protein expression and Ang (1-7) levels in the heart. Exercise increased microRNA-27a and 27b, targeting ACE and decreasing microRNA-143 targeting ACE2 in the heart. Left ventricular hypertrophy induced by aerobic training involves microRNA regulation and an increase in cardiac Ang II type 1 receptor without the participation of Ang II. Parallel to this, an increase in ACE2, Ang (1-7), and Ang II type 2 receptor in the heart by exercise suggests that this nonclassic cardiac renin-angiotensin system counteracts the classic cardiac renin-angiotensin system. These findings are consistent with a model in which exercise may induce left ventricular hypertrophy, at least in part, altering the expression of specific microRNAs targeting renin-angiotensin system genes. Together these effects might provide the additional aerobic capacity required by the exercised heart. (Hypertension. 2011;58:182-189.).

Acquisition of Serum Antibodies Reactive With Enterohemorrhagic Escherichia coli Virulence-Associated Factors by Healthy Brazilian Children and Adults

Background: Patients with hemorrhagic colitis or hemolytic uremic syndrome due to enterohemorrhagic Escherichia coli (EHEC) develop serum IgM and IgG response to lipopolysaccharide (LPS) and to virulence factors such as intimin. The small numbers of cases of diarrhea associated with EHEC strains in Brazil suggests a pre-existing immunity probably due to previous contact with diarrheagenic E. coli. Our aim was to evaluate the development of the serum antibody repertoire to EHEC virulence factors in Brazilian children and adults. Methods: Serum IgM and IgG antibodies were determined by enzyme-linked immunosorbent assay with LPS O111, LPS O26, and LPS O157 in 101 children between 2 months and 10 years of age and in 100 adult sera, by immunoblotting with protein membrane extracts and purified beta intimin; the ability of adult sera to neutralize Shiga toxin2 was also investigated. Results: Children older than 24 months had IgM concentrations reactive with the 3 LPS equivalent to those seen in the adult group, and significantly higher than the group of younger children (P < 0.05). Anti-O26 and anti-O157 LPS IgG concentrations were equivalent between the 2 groups of children and were significantly different from the adult group (P < 0.05). The anti-O111 LPS IgG levels in older children were intermediate between the younger group, and adults (P < 0.05). Immunoblotting revealed strong protein reactivity, including the conserved and variable regions of beta intimin and more than 50% of the adult samples neutralized Shiga toxin 2. Conclusions: Our results demonstrate an increasing anti-LPS and antiprotein antibody response with age, which could provide protection against EHEC infections.

Reprint of ""Dynamics of the expression of intermediate filaments vimentin and desmin during myofibroblast differentiation after corneal injury""

Previous studies have suggested that abnormal corneal wound healing in patients after photorefractive keratectomy (PRK) is associated with the appearance of myofibroblasts in the stroma between two and four weeks after surgery. The purpose of this study was to examine potential myofibroblast progenitor cells that might express other filament markers prior to completion of the differentiation pathway that yields alpha-smooth muscle actin (SMA)-expressing myofibroblasts associated with haze localized beneath the epithelial basement membrane after PRK. Twenty-four female rabbits that had -9 diopter PRK were sacrificed at 1 week, 2 weeks, 3 weeks or 4 weeks after surgery. Corneal rims were collected, frozen at -80 degrees C, and analyzed by immunocytochemistry using anti-vimentin, anti-desmin, and anti-SMA antibodies. Double immunostaining was performed for the co-localization of SMA with vimentin or desmin with SMA. An increase in vimentin expression in stromal cells is noted as early as 1 week after PRK in the rabbit cornea. As the healing response continues at two or three weeks after surgery, many stromal cells expressing vimentin also begin to express desmin and SMA. By 4 weeks after the surgery most, if not all, myofibroblasts express vimentin, desmin and SMA. Generalized least squares regression analysis showed that there was strong evidence that each of the marker groups differed in expression over time compared to the other two (p < 0.01). Intermediate filaments - vimentin and desmin co-exist in myofibroblasts along with SMA and may play an important role in corneal remodeling after photorefractive keratectomy. The earliest precursors of myofibroblasts destined to express SMA and desmin are detectible by staining for vimentin at 1 week after surgery. (C) 2009 Elsevier Ltd. All rights reserved.