Production of a xylose-stimulated beta-glucosidase and a cellulase-free thermostable xylanase by the thermophilic fungus Humicola brevis var. thermoidea under solid state fermentation

Humicola brevis var. thermoidea cultivated under solid state fermentation in wheat bran and water (1:2 w/v) was a good producer of beta-glucosidase and xylanase. After optimization using response surface methodology the level of xylanase reached 5,791.2 +/- A 411.2 U g(-1), while beta-glucosidase production was increased about 2.6-fold, reaching 20.7 +/- A 1.5 U g(-1). Cellulase levels were negligible. Biochemical characterization of H. brevis beta-glucosidase and xylanase activities showed that they were stable in a wide pH range. Optimum pH for beta-glucosidase and xylanase activities were 5.0 and 5.5, respectively, but the xylanase showed 80 % of maximal activity when assayed at pH 8.0. Both enzymes presented high thermal stability. The beta-glucosidase maintained about 95 % of its activity after 26 h in water at 55 A degrees C, with half-lives of 15.7 h at 60 A degrees C and 5.1 h at 65 A degrees C. The presence of xylose during heat treatment at 65 A degrees C protected beta-glucosidase against thermal inactivation. Xylanase maintained about 80 % of its activity after 200 h in water at 60 A degrees C. Xylose stimulated beta-glucosidase activity up to 1.7-fold, at 200 mmol L-1. The notable features of both xylanase and beta-glucosidase suggest that H. brevis crude culture extract may be useful to compose efficient enzymatic cocktails for lignocellulosic materials treatment or paper pulp biobleaching.

Generation of a Chinese Hamster Ovary Cell Line Producing Recombinant Human Glucocerebrosidase

Impaired activity of the lysosomal enzyme glucocerebrosidase (GCR) results in the inherited metabolic disorder known as Gaucher disease. Current treatment consists of enzyme replacement therapy by administration of exogenous GCR. Although effective, it is exceptionally expensive, and patients worldwide have a limited access to this medicine. In Brazil, the public healthcare system provides the drug free of charge for all Gaucher's patients, which reaches the order of $ 84million per year. However, the production of GCR by public institutions in Brazil would reduce significantly the therapy costs. Here, we describe a robust protocol for the generation of a cell line producing recombinant human GCR. The protein was expressed in CHO-DXB11 (dhfr(-)) cells after stable transfection and gene amplification with methotrexate. As expected, glycosylated GCR was detected by immunoblotting assay both as cell-associated (similar to 64 and 59 kDa) and secreted (63-69 kDa) form. Analysis of subclones allowed the selection of stable CHO cells producing a secreted functional enzyme, with a calculated productivity of 5.14 pg/cell/day for the highest producer. Although being laborious, traditionalmethods of screening high-producing recombinant cellsmay represent a valuable alternative to generate expensive biopharmaceuticals in countries with limited resources.

Transcriptional response to GAA deficiency (Pompe disease) in infantile-onset patients

Pompe disease is a genetic disorder resulting from a deficiency of lysosomal acid alpha-glucosidase (GAA) that manifests as a clinical spectrum with regard to symptom severity and rate of progression. In this study, we used microarrays to examine gene expression from the muscle of two cohorts of infantile-onset Pompe patients to identify transcriptional differences that may contribute to the disease phenotype. We found strong similarities among the gene expression profiles generated from biceps and quadriceps, and identified a number of signaling pathways altered in both cohorts. We also found that infantile-onset Pompe patient muscle had a gene expression pattern characteristic of immature or regenerating muscle, and exhibited many transcriptional markers of inflammation, despite having few overt signs of inflammatory infiltrate. Further, we identified genes exhibiting correlation between expression at baseline and response to therapy. This combined dataset can serve as a foundation for biological discovery and biomarker development to improve the treatment of Pompe disease. (C) 2012 Elsevier Inc. All rights reserved.

CHEMICAL AND BIOCHEMICAL PROPERTIES OF ARAUCARIA ANGUSTIFOLIA (BERT.) KTZE. FOREST SOILS IN THE STATE OF SAO PAULO

Araucaria angustifolia, commonly named Araucaria, is a Brazilian native species that is intensively exploited due to its timber quality. Therefore, Araucaria is on the list of species threatened by extinction. Despite the importance of soil for forest production, little is known about the soil properties of the highly fragmented Araucaria forests. This study was designed to investigate the use of chemical and biological properties as indicators of conservation and anthropogenic disturbance of Araucaria forests in different sampling periods. The research was carried out in two State parks of Sao Paulo: Parque Estadual Turistico do Alto do Ribeira and Parque Estadual de Campos de Jordao. The biochemical properties carbon and nitrogen in microbial biomass (MB-C and MB-N), basal respiration (BR), the metabolic quotient (qCO(2)) and the following enzyme activities: beta-glucosidase, urease, and fluorescein diacetate hydrolysis (FDA) were evaluated. The sampling period (dry or rainy season) influenced the results of mainly MB-C, MB-N, BR, and qCO(2). The chemical and biochemical properties, except K content, were sensitive indicators of differences in the conservation and anthropogenic disturbance stages of Araucaria forests. Although these forests differ in biochemical and chemical properties, they are efficient in energy use and conservation, which is shown by their low qCO(2), suggesting an advanced stage of succession.

Effect of the interaction between food state and the action of estrogen on oxytocinergic system activity

Increased plasma osmolality by food intake evokes augmentation of plasma oxytocin (OT). Ovarian steroids may also influence the balance of body fluids by acting on OT neurones. Our aim was to determine if estrogen influences the activity of OT neurones in paraventricular nucleus (PVN) and supraoptic nucleus (SON) under different osmotic situations. Ovariectomized rats (OVX) were treated with either estradiol (E-2) or vehicle and were divided into three groups: group I was fed ad libitum, group II underwent 48 h of fasting, and group III was refed after 48 h of fasting. On the day of the experiment, blood samples were collected to determine the plasma osmolality and OT. The animals were subsequently perfused, and OT/FOS immunofluorescence analysis was conducted on neurones in the PVN and the SON. When compared to animals which were fasted or fed ad libitum, the plasma osmolality of refed animals was higher, regardless of whether they were treated with vehicle or E-2. We observed neural activation of OT cells in vehicle-or E-2-treated OVX rats refed after 48 h of fasting, but not in animals fed ad libitum or in animals that only underwent 48 h of fasting. Finally, the percentage of neurones that co-expressed OT and FOS was lower in both the PVN and the SON of animals treated with E-2 and refed, when compared to vehicle-treated animals. These results suggest that E-2 may have an inhibitory effect on OT neurones and may modulate the secretion of OT in response to the increase of osmolality induced by refeeding. Journal of Endocrinology (2012) 212, 129-138

Diurnal changes in storage carbohydrate metabolism in cotyledons of the tropical tree Hymenaea courbaril L. (Leguminosae)

(Diurnal changes in storage carbohydrate metabolism in cotyledons of the tropical tree Hymenaea courbaril L. (Leguminosae)). The cotyledons of Hymenaea courbaril store large amounts of xyloglucan, a cell wall polysaccharide that is believed to serve as storage for the period of seedling establishment. During storage mobilisation, xyloglucan seems to be degraded by a continuous process that starts right after radicle protrusion and follows up to the establishment of photosynthesis. Here we show evidence that events related to the hydrolases activities and production (alpha-xylosidase, beta-galactosidase, beta-glucosidase and xyloglucan endo-beta-transglucosilase) as well as auxin, showed changes that follow the diurnal cycle. The period of higher hydrolases activities was between 6pm and 6am, which is out of phase with photosynthesis. Among the enzymes, alpha-xilosidase seems to be more important than beta-glucosidase and beta-galactosidase in the xyloglucan disassembling mechanism. Likewise, the sugars related with sucrose metabolism followed the rhythm of the hydrolases, but starch levels were shown to be practically constant. A high level of auxin was observed during the night, what is compatible with the hypothesis that this hormone would be one of the regulators of the whole process. The probable biological meaning of the existence of such a complex control mechanism during storage mobilisation is likely to be related to a remarkably high level of efficiency of carbon usage by the growing seedling of Hymenaea courbaril, allowing the establishment of very vigorous seedlings in the tropical forest.

Induction of cellulase and hemicellulase activities of Thermoascus aurantiacus by xylan hydrolyzed products

Thermoascus aurantiacus is able to secrete most of the hemicellulolytic and cellulolytic enzymes. To establish the xylanase inducers of T. aurantiacus, the mycelia were first grown on glucose up until the end of the exponential growth phase, followed by washing and re-suspension in a basal medium without a carbon source. Pre-weighed amounts of xylose (final concentration of 3.5 mg/ml), xylobiose (7 mg/ml) and hydrolyzed xylan from sugarcane bagasse (HXSB) which contained xylose, xylobiose and xylotriose (6.8 mg/ml) were evaluated as inducers of xylanase. It was observed that xylose did not suppress enzyme induction of T. aurantiacus when used in low concentrations, regardless of whether it was inoculated with xylobiose. Xylobiose promoted fast enzyme production stopping after 10 h, even at a low consumption rate of the carbon source; therefore xylobiose appears to be the natural inducer of xylanase. In HXSB only a negligible xylanase activity was determined. Xylose present in HXSB was consumed within the first 10 h while xylobiose was partially hydrolyzed at a slow rate. The profile of alpha-arabinofuranosidase induction was very similar in media induced with xylobiose or HXSB, but induction with xylose showed some positive effects as well. The production profile for the xylanase was accompanied by low levels of cellulolytic activity. In comparison, growth in HXSB resulted in different profiles of both xylanase and cellulase production, excluding the possibility of xylanase acting as endoglucanases.

Mejillonesite, a new acid sodium, magnesium phosphate mineral, from Mejillones, Antofagasta, Chile

Mejillonesite, ideally NaMg(2)(PO(3)OH)(PO(4))(OH)center dot H(5)O(2), is a new mineral approved by the CNMNC (IMA 2010-068). It occurs as isolated crystal aggregates in thin zones in fine-grained opal-zeolite aggregate on the north slope of Cerro Mejillones, Antofagasta, Chile. Closely associated minerals are bobierrite, opal, clinoptilolite-Na, clinoptilolite-K, and gypsum. Mejillonesite forms orthorhombic, prismatic, and elongated thick tabular crystals up to 6 mm long, usually intergrown in radiating aggregates. The dominant form is pinacoid {100}. Prisms {hk0}, {h0l}, and {0kl} are also observed. The crystals are colorless, their streak is white, and the luster is vitreous. The mineral is transparent. It is non-fluorescent under ultraviolet light. Mohs' hardness is 4, tenacity is brittle. Cleavage is perfect on {100}, good on {010} and {001}, and fracture is stepped. The measured density is 2.36(1) g/cm(3); the calculated density is 2.367 g/cm(3). Mejillonesite is biaxial (-), alpha= 1.507(2), beta= 1.531(2), gamma= 1.531(2), 2V(meas) = 15(10)degrees, 2V(calc) = 0 degrees (589 nm). Orientation is X= a, Z= elongation direction. The mineral is non-pleochroic. Dispersion is r> v, medium. The IR spectrum contains characteristic bands of the Zundel cation (H(5)O(2)(+), or H(+)center dot 2H(2)O) and the groups P-OH and OH(-). The chemical composition is (by EDS, H(2)O by the Alimarin method, wt%): Na(2)O 9.19, MgO 26.82, P(2)O(5) 46.87, H(2)O 19, total 101.88. The empirical formula, based on 11 oxygen atoms, is Na(0.93)Mg(2.08)(PO(3)OH)(1.00) (PO(4)) (OH)(0.86) .0.95H(5)O(2) The strongest eight X-ray powder-diffraction lines [d in angstrom(I)(hkl)] are: 8.095(100)(200), 6.846(9) (210), 6.470(8)(111), 3.317(5)(302), 2.959(5)(132), 2.706(12)(113), 2.157(19)(333), and 2.153(9) (622). The crystal structure was solved on a single crystal (R = 0.055) and gave the following data: orthorhombic, Pbca, a = 16.295(1), b = 13.009(2), c = 8.434(1) angstrom, V= 1787.9(4) angstrom(3), Z = 8. The crystal structure of mejillonesite is based on a sheet (parallel to the b-c plane) formed by two types of MgO(6) octahedra, isolated tetrahedra PO(4) and PO(3)OH whose apical vertices have different orientation with respect to the sheet. The sheets are connected by interlayer, 5-coordinated sodium ions, proton hydration complexes, and hydroxyl groups. The structure of mejillonesite is related to that of angarfite, NaFe(5)(3+)(PO(4))(4)(OH)(4).4H(2)O and bakhchisaraitsevite, Na(2)Mg(5)(PO(4))(4)center dot 7H(2)O.

Tauroursodeoxycholic Acid Affects PPAR gamma and TLR4 in Steatotic Liver Transplantation

Numerous steatotic livers are discarded for transplantation because of their poor tolerance to ischemia-reperfusion (I/R). We examined whether tauroursodeoxycholic acid (TUDCA), a known inhibitor of endoplasmic reticulum (ER) stress, protects steatotic and nonsteatotic liver grafts preserved during 6 h in University of Wisconsin (UW) solution and transplanted. The protective mechanisms of TUDCA were also examined. Neither unfolded protein response (UPR) induction nor ER stress was evidenced in steatotic and nonsteatotic liver grafts after 6 h in UW preservation solution. TUDCA only protected steatotic livers grafts and did so through a mechanism independent of ER stress. It reduced proliferator-activated receptor-gamma(PPAR gamma) and damage. When PPAR gamma was activated, TUDCA did not reduce damage. TUDCA, which inhibited PPAR gamma, and the PPAR gamma antagonist treatment up-regulated toll-like receptor 4 (TLR4), specifically the TIR domain-containing adaptor inducing IFN beta (TRIF) pathway. TLR4 agonist treatment reduced damage in steatotic liver grafts. When TLR4 action was inhibited, PPAR gamma antagonists did not protect steatotic liver grafts. In conclusion, TUDCA reduced PPAR gamma and this in turn up-regulated the TLR4 pathway, thus protecting steatotic liver grafts. TLR4 activating-based strategies could reduce the inherent risk of steatotic liver failure after transplantation.

Lack of beta(2)-AR improves exercise capacity and skeletal muscle oxidative phenotype in mice

beta(2)-adrenergic receptor (beta(2)-AR) agonists have been used as ergogenics by athletes involved in training for strength and power in order to increase the muscle mass. Even though anabolic effects of beta(2)-AR activation are highly recognized, less is known about the impact of beta(2)-AR in endurance capacity. We presently used mice lacking beta(2)-AR [beta(2)-knockout (beta(2) KO)] to investigate the role of beta(2)-AR on exercise capacity and skeletal muscle metabolism and phenotype. beta(2) KO mice and their wild-type controls (WT) were studied. Exercise tolerance, skeletal muscle fiber typing, capillary-to-fiber ratio, citrate synthase activity and glycogen content were evaluated. When compared with WT, beta 2KO mice displayed increased exercise capacity (61%) associated with higher percentage of oxidative fibers (21% and 129% of increase in soleus and plantaris muscles, respectively) and capillarity (31% and 20% of increase in soleus and plantaris muscles, respectively). In addition, beta 2KO mice presented increased skeletal muscle citrate synthase activity (10%) and succinate dehydrogenase staining. Likewise, glycogen content (53%) and periodic acid-Schiff staining (glycogen staining) were also increased in beta 2KO skeletal muscle. Altogether, these data provide evidence that disruption of beta(2)AR improves oxidative metabolism in skeletal muscle of beta 2KO mice and this is associated with increased exercise capacity.

Immbolization of uricase enzyme in Langmuir and Langmuir-Blodgett films of fatty acids: Possible use as a uric acid sensor

Preserving the enzyme structure in solid films is key for producing various bioelectronic devices, including biosensors, which has normally been performed with nanostructured films that allow for control of molecular architectures. In this paper, we investigate the adsorption of uricase onto Langmuir monolayers of stearic acid (SA), and their transfer to solid supports as Langmuir Blodgett (LB) films. Structuring of the enzyme in beta-sheets was preserved in the form of 1-layer LB film, which was corroborated with a higher catalytic activity than for other uricase-containing LB film architectures where the beta-sheets structuring was not preserved. The optimized architecture was also used to detect uric acid within a range covering typical concentrations in the human blood. The approach presented here not only allows for an optimized catalytic activity toward uric acid but also permits one to explain why some film architectures exhibit a superior performance. (C) 2011 Elsevier Inc. All rights reserved.

Kaurenoic Acid from Sphagneticola trilobata Inhibits Inflammatory Pain: Effect on Cytokine Production and Activation of the NO-Cyclic GMP-Protein Kinase G-ATP-Sensitive Potassium Channel Signaling Pathway

Kaurenoic acid [ent-kaur-16-en-19-oic acid (1)] is a diterpene present in several plants including Sphagneticola trilobata. The only documented evidence for its antinociceptive effect is that it inhibits the writhing response induced by acetic acid in mice. Therefore, the analgesic effect of 1 in different models of pain and its mechanisms in mice were investigated further. Intraperitoneal and oral treatment with 1 dose-dependently inhibited inflammatory nociception induced by acetic acid. Oral treatment with 1 also inhibited overt nociception-like behavior induced by phenyl-p-benzoquinone, complete Freund's adjuvant (CFA), and both phases of the formalin test. Compound 1 also inhibited acute carrageenin- and PGE(2)-induced and chronic CFA-induced inflammatory mechanical hyperalgesia. Mechanistically, 1 inhibited the production of the hyperalgesic cytokines TNF-alpha and IL-1 beta. Furthermore, the analgesic effect of 1 was inhibited by L-NAME, ODQ, KT5823, and glybenclamide treatment, demonstrating that such activity also depends on activation of the NO-cyclic GMP-protein kinase G-ATP-sensitive potassium channel signaling pathway, respectively. These results demonstrate that 1 exhibits an analgesic effect in a consistent manner and that its mechanisms involve the inhibition of cytokine production and activation of the NO-cyclic GMP-protein lcinase G-ATP-sensitive potassium channel signaling pathway.

Credneramides A and B: Neuromodulatory Phenethylamine and Isopentylamine Derivatives of a Vinyl Chloride-Containing Fatty Acid from cf. Trichodesmium sp nov.

Credneramides A (1) and B (2), two vinyl chloride-containing metabolites, were isolated from a Papua New Guinea collection of cf. Trichodesmium sp. nov. and expand a recently described class of vinyl chloride-containing natural products. The precursor fatty acid, credneric acid (3), was isolated from both the aqueous and organic fractions of the parent fraction as well as from another geographically and phylogenetically distinct cyanobacterial collection (Panama). Credneramides A and B inhibited spontaneous calcium oscillations in murine cerebrocortical neurons at low micro-molar concentrations (1, IC50 4.0 mu M; 2, IC50 3.8 mu M).

Trans fatty acid intake is associated with insulin sensitivity but independently of inflammation

High saturated and trans fatty acid intake, the typical dietary pattern of Western populations, favors a proinflammatory status that contributes to generating insulin resistance (IR). We examined whether the consumption of these fatty acids was associated with IR and inflammatory markers. In this cross-sectional study, 127 non-diabetic individuals were allocated to a group without IR and 56 to another with IR, defined as homeostasis model assessment-IR (HOMA-IR) >2.71. Diet was assessed using 24-h food recalls. Multiple linear regression was employed to test independent associations with HOMA-IR. The IR group presented worse anthropometric, biochemical and inflammatory profiles. Energy intake was correlated with abdominal circumference and inversely with adiponectin concentrations (r = -0.227, P = 0.002), while saturated fat intake correlated with inflammatory markers and trans fat with HOMA-IR (r = 0.160, P = 0.030). Abdominal circumference was associated with HOMA-IR (r = 0.430, P < 0.001). In multiple analysis, HOMA-IR remained associated with trans fat intake (beta = 1.416, P = 0.039) and body mass index (beta = 0.390, P < 0.001), and was also inversely associated with adiponectin (beta = -1.637, P = 0.004). Inclusion of other nutrients (saturated fat and added sugar) or other inflammatory markers (IL-6 and CRP) into the models did not modify these associations. Our study supports that trans fat intake impairs insulin sensitivity. The hypothesis that its effect could depend on transcription factors, resulting in expression of proinflammatory genes, was not corroborated. We speculate that trans fat interferes predominantly with insulin signaling via intracellular kinases, which alter insulin receptor substrates.

Isolation and characterization of moojenin, an acid-active, anticoagulant metalloproteinase from Bothrops moojeni venom

A fibrinogenolytic metalloproteinase from Bothrops moojeni venom, named moojenin, was purified by a combination of ion-exchange chromatography on DEAE-Sephacel and gel filtration on Sephacryl S-300. SDS-PAGE analysis indicated that moojenin consists of a single polypeptide chain and has a molecular mass about 45 kDa. Sequencing of moojenin by Edman degradation revealed the amino acid sequence LGPDIVSPPVCGNELLEV-GEECDCGTPENCQNE, which showed strong identity with many other snake venom metalloproteinases (SVMPs). The enzyme cleaves the A alpha-chain of fibrinogen first, followed by the E beta-chain, and shows no effects on the gamma-chain. Moojenin showed a coagulant activity on bovine plasma about 3.1 fold lower than crude venom. The fibrinogenolytic and coagulant activities of the moojenin were abolished by preincubation with EDTA, 1,10-phenanthroline and beta-mercaptoethanol. Moojenin showed maximum activity at temperatures ranging from 30 to 40 degrees C and its optimal pH was 4.0. Its activity was completely lost at temperatures above 50 degrees C. Moojenin induced necrosis in liver and muscle, evidenced by morphological alterations, but did not cause histological alterations in mouse lungs, kidney or heart. Moojenin rendered the blood uncoagulatable when it was intraperitoneally administered into mice. This metalloproteinase may be of medical interest because of its anticoagulant activity. (C) 2012 Elsevier Ltd. All rights reserved.