[Relevance of Surface-Exposed Lysine Residues Designed for Functionalization of Laccase].

Fungal laccases are oxidoreductases with low-specificity for substrates. The characterization of laccase's surface is a prerequisite used to obtain hybrid catalysts with new properties. Surface-exposed lysine residues are targets in immobilization reactions. In this work, LAC3-K0, an enzyme devoid of lysine, was used as a platform to detect potential surface-exposed sites suitable for replacement with a lysine residue. Seven sites were selected from a LAC3-K0 3-D model, and single lysine mutants (UNIKn, n = residue number) were obtained by site-directed mutagenesis. All mutants were expressed in Saccharomyces cerevisiae W303-1A and detected as functional secreted proteins by their ability to oxidize guaiacol or 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) on agar plates. All variants were active at acidic pH but presented no activity at neutral pH, as expected. Likewise, variants were stable a temperature between 15-55°C, and were completely inactivated at 70°C. Oxidation assays revealed that the replacement of one or two surface residues with lysine greatly affected enzyme activity and substrate specificity. The catalytic; parameters (KM^(app) and kcat^(app)) determined with ABTS were found to be different among the variants; Vmax^(app) was 1.5-2 fold higher in UNIK269 and triple mutant, with a KM^(app) of 0.27 and 0.30, respectively; kcat^(app )was 30.25 in UNIK238 and 32.34 in the triple mutant. The role of hydrophobic patches detected on the surface of LAC3-K0 was determined to be a favorable factor to be considered in the interaction of hybrid materials. All variants with uniquely surface located lysine created in this work can be in demand for obtaining laccases with a certain substrate specificity in the design of hybrid materials.

Removal of phenolic inhibitors from lignocellulose hydrolysates using laccases for the production of fuels and chemicals.

Lignocellulose is the most abundant biopolymer in the biosphere. It is inexpensive and therefore considered an attractive feedstock to produce biofuels and other biochemicals. Thermochemical and/or enzymatic pretreatment is used to release fermentable monomeric sugars. However, a variety of inhibitory by-products such as weak acids, furans, and phenolics that inhibit cell growth and fermentation are also released. Phenolic compounds are among the most toxic components in lignocellulosic hydrolysates and slurries derived from lignin decomposition, affecting overall fermentation processes and production yields and productivity. Ligninolytic enzymes have been shown to lower inhibitor concentrations in these hydrolysates, thereby enhancing their fermentability into valuable products. Among them, laccases, which are capable of oxidizing lignin and a variety of phenolic compounds in an environmentally benign manner, have been used for biomass delignification and detoxification of lignocellulose hydrolysates with promising results. This review discusses the state of the art of different enzymatic approaches to hydrolysate detoxification. In particular, laccases are used in separate or in situ detoxification steps, namely in free enzyme processes or immobilized by cell surface display technology to improve the efficiency of the fermentative process and consequently the production of second-generation biofuels and bio-based chemicals.

Autocrine modulation of glucose transporter SGLT2 by IL-6 and TNF-α in LLC-PK(1) cells.

We determined in cultured kidney epithelial cells (LLC-PK(1)) the effects of high glucose, interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) on mRNA and protein expression of the renal glucose transporters SGLT1 and SGLT2. Cultured monolayers were incubated with similar concentrations of IL-6 and TNF-α to those produced by LLC-PK(1) in the presence of 20 mM glucose. Confluent monolayers with either 5 (controls, C) or 20 mM glucose (high glucose, HG) were incubated in the presence of 5 mM glucose, 20 mM glucose, 10 pg/ml IL-6, or TNF-α alone or in combination. Separate groups with IL-6 and TNF-α were incubated with antibodies to their respective receptors. HG induced an increased SGLT1 mRNA at 48 h (p<0.05 vs. C) and protein expression in 120 h (p<0.05 vs. C). HG also induced an increased SGLT2 mRNA at 72 and 96 h (P<0.05 vs. C) and SGLT2 protein expression at 120 h (p<0.05 vs. C). In C, 10 pg/ml IL-6 or TNF-α did not modify SGLT1 mRNA (n.s vs. in the absence of cytokines). In contrast, cytokines induced an increased expression of SGLT1 protein at 120 h (p<0.05 vs. in the absence of cytokines), and SGLT2 mRNA and protein were increased at 96 and 120 h, respectively (p<0.05 vs. in absence of cytokines). No changes were observed when cells were incubated with cytokines and HG (n.s vs. C). In conclusion, this study showed that SGLT2 increased in the presence of IL-6 and TNF-α, indicating an autocrine modulation of the expression of this transporter by cytokines.

Comparative analysis of the renoprotective effects of pentoxifylline and vitamin E on streptozotocin-induced diabetes mellitus.

BACKGROUND: Oxidative stress (OxS) induced by diabetes plays an important role in the development of diabetic nephropathy. Studies have shown that antioxidants are beneficial in its reduction. Vitamin E has been documented as providing the most improvement in the antioxidative status. Recently, pentoxifylline (PTX) has been proposed to have antioxidant properties. AIM: The aim of the study was to assess the ability of two antioxidants to reduce lipid peroxidation and renal hypertrophy in vivo. METHODS: Diabetes was induced by streptozotocin (STZ) in rats. Treatment groups were divided as follows: healthy (H), diabetic without treatment (STZ), PTX treated group (STZ+PTX), and vitamin E supplemented (STZ+E) group. At 8 weeks, kidneys were removed; one was homogenized to quantify lipoperoxide levels (LPOS), and the other was used to study the morphological changes by electron microscopy (EM). Additionally, plasma total antioxidant activity (TAA) was quantified. RESULTS: A reduction in LPOS was observed in both groups: PTX and vitamin E with regard to STZ group. PTX increased TAA compared to STZ+E, which restored it to its normal values. However, both treatments reduced the LPO/TAA ratio to lower basal levels; hence, similar results were obtained in terms of correcting functional parameters. Structural changes in STZ rats included a glomerular membrane thickening, podocyte flattening, as well as loss of fenestration in the endothelial layer. All these changes were less aggressive for treated rats. CONCLUSIONS: Vitamin E and PTX have potential therapeutic properties that may help to retard the rate of deterioration of diabetic kidneys.

Pentoxifylline diminishes the oxidative damage to renal tissue induced by streptozotocin in the rat.

Oxidative damage has been suggested to be a contributing factor in the development to diabetic nephropathy (DN). Recently, there has been evidence that pentoxifylline (PTX) has free radical-scavenging properties; thus, its anti-inflammatory and renoprotective effects may be related to a reduction in reactive oxygen species production. It is likely that the pharmacological effects of PTX include an antioxidant mechanism as shown in in vitro assays. The aim of this study was to evaluate whether the reported renoprotective effects of PTX could be the result of its antioxidant actions in streptozotocin (STZ)-induced DN in rats. The administration of PTX over a period of 8 weeks, in addition to displaying renoprotective effects, caused a significant reduction in lipoperoxide levels (LPOS) in the diabetic kidney (P < 0.05), compared to untreated rats. These levels were comparable to those in the healthy kidney of experimental animals (P > 0.05). All untreated STZ rats exhibited an increase in LPOS as opposed to healthy controls (H) (P < 0.001). The total antioxidant activity (TAA) in plasma was increased significantly already after 2 days of STZ (P < 0.05). When we examined the progression of TAA in STZ rats, there was a significant decrease over 8 weeks (P < 0.05). PTX treatment caused an increase in TAA when compared to untreated STZ rats (P < 0.05). Renal hypertrophy was less evident in PTX-treated STZ than in untreated STZ rats, evaluated by kidney weight/body weight ratio. These results indicate that PTX decreases the oxidative damage induced by these experimental procedures and may increase antioxidant defense mechanisms in STZ-induced diabetes in rats.

Chromosomal integration of heterologous DNA in Escherichia coli with precise removal of markers and replicons used during construction.

A set of vectors which facilitates the sequential integration of new functions into the Escherichia coli chromosome by homologous recombination has been developed. These vectors are based on plasmids described by Posfai et al. (J. Bacteriol. 179:4426-4428, 1997) which contain conditional replicons (pSC101 or R6K), a choice of three selectable markers (ampicillin, chloramphenicol, or kanamycin), and a single FRT site. The modified vectors contain two FRT sites which bracket a modified multiple cloning region for DNA insertion. After integration, a helper plasmid expressing the flippase (FLP) recombinase allows precise in vivo excision of the replicon and the marker used for selection. Sites are also available for temporary insertion of additional functions which can be subsequently deleted with the replicon. Only the DNA inserted into the multiple cloning sites (passenger genes and homologous fragment for targeting) and a single FRT site (68 bp) remain in the chromosome after excision. The utility of these vectors was demonstrated by integrating Zymomonas mobilis genes encoding the ethanol pathway behind the native chromosomal adhE gene in strains of E. coli K-12 and E. coli B. With these vectors, a single antibiotic selection system can be used repeatedly for the successive improvement of E. coli strains with precise deletion of extraneous genes used during construction.

[Crossed immunoelectrophoresis in the serological diagnosis of amebiasis]. / Estudio de la immunoelectroforésis cruzada en el diagnóstico serologico de la amibiásis

Sixty-two patients were divided into two groups, and their sera was analyzed. a) In the first series, a study was made of the sera of patients with hepatic abscess wherein tests were positive in 100% of the cases. b) In the second serie, a study was made of the sera of patients with evidence of amoebiasis wherein tests were negative in 100% of the cases. Evaluation and diagnoses of patients were performed on the basis of clinical, radiological, scintillogram, coprological and endoscopic examinations.

Autocrine modulation of glucose transporter SGLT2 by IL-6 and TNF-alpha in LLC-PK1 cells

We determined in cultured kidney epithelial cells (LLC-PK1) the effects of high glucose, interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha) on mRNA and protein expression of the renal glucose transporters SGLT1 and SGLT2. Cultured monolayers were incubated with similar concentrations of IL-6 and (..) (AU)