Effect of veratryl alcohol and manganese (IV) oxide on ligninolytic activity in semi solid cultures of Phanerochaete chrysosporium

An inert carrier (nylon sponge), a non-inert carrier (barley straw) and the addition of veratryl alcohol or manganese (IV) oxide to the cultures were used to study the production of ligninolytic enzymes by Phanerochaete chrysosporium BKM-F-1767 (ATCC 24725) during semi solid state fermentation conditions. By supplementing the medium with these compounds we could stimulate the ligninolytic system of this fungus. The different carriers employed and the effect of adding veratryl alcohol or manganese (IV) oxide to the cultures were compared in order to determine the best system to produce high activities of ligninolytic enzymes. Lignin peroxidase (LiP) activities higher than 500 U/L and manganese-dependent peroxidase (MnP) activities about 1100 U/L were achieved.     

Evaluation of molecular typing techniques to assign genetic diversity among Saccharomyces cerevisiae strains.

Discrimination of strains within the species Saccharomyces cerevisiae was demonstrated by the use of four different techniques to type 15 strains isolated from spoiled wine and beer. Random amplified polymorphic DNA with specific oligonucleotides and PCR fingerprinting with the microsatellite oligonucleotide primers (GAC)5 and (GTG)5 enabled discrimination between the strains tested. Additionally, restriction enzyme analysis, with TaqI and MseI, of PCR-amplified fragments from the complete internal transcribed spacer and nontranscribed spacer, both present in the rRNA-encoding gene cluster, proved to be suitable for generating intraspecies-specific patterns. Random amplified polymorphic DNA with primers 24 and OPA-11 and PCR fingerprinting with primer (GTG)5 appeared to generate the highest degree of diversity. However, the results indicated that there was no single PCR-mediated typing technique enabling discrimination on the strain level. Discrimination of each individual strain was nevertheless possible by combining the results obtained with all typing techniques.     

Comparison of the mass and energy exchange of a pasture and a mature transitional tropical forest of the southern Amazon Basin during a seasonal transition

This research utilized tower‐based eddy covariance to quantify the trends in net ecosystem mass (CO₂ and H₂O vapor) and energy exchange of important land‐cover types of NW Mato Grosso during the March–December 2002 seasonal transition. Measurements were made in a mature transitional (ecotonal) tropical forest near Sinop, Mato Grosso, and a cattle pasture near Cotriguaçú, Mato Grosso, located 500 km WNW of Sinop. Pasture net ecosystem CO₂ exchange (NEE) was considerably more variable than the forest NEE over the seasonal transition, and the pasture had significantly higher rates of maximum gross primary production in every season except the dry–wet season transition (September–October). The pasture also had significantly higher rates of whole‐ecosystem dark respiration than the forest during the wetter times of the year. Average (±95% CI) rates of total daily NEE during the March–December 2002 measurement period were 26±15 mmol m^?2 day^?1 for the forest (positive values indicate net CO₂ loss by the ecosystem) and ?38±26 mmol m^?2 day^?1 for the pasture. While both ecosystems partitioned more net radiation (R_n) into latent heat flux (L_e), the forest had significantly higher rates of L_e and lower rates of sensible heat flux (H) than the pasture; a trend that became more extreme during the onset of the dry season. Large differences in pasture and forest mass and energy exchange occurred even though seasonal variations in micrometeorology (air temperature, humidity, and radiation) were relatively similar for both ecosystems. While the short measurement period and lack of spatial replication limit the ability to generalize these results to pasture and forest regions of the Amazon Basin, these results suggest important differences in the magnitude and seasonal variation of NEE and energy partitioning for pasture and transitional tropical forest.     

Dynamics of proteins and lipids in the stratum corneum: effects of percutaneous permeation enhancers

We have spin labeled the stratum corneum (SC) with a lysine specific reagent, succinimidyl-2,2,5,5-tetramethyl-3-pirroline-1-oxyl-carboxylate spin label (SSL), to assess the dynamics and hydration degree of SC proteins by electron paramagnetic resonance (EPR) spectroscopy taking measurements directly from the intact tissue. Treating the SC with two percutaneous penetration enhancers, 8 M urea or 20% (v/v) 1-methyl-2-pyrrolidone (1 MP), destabilizes the proteins thus promoting more mobile and solvent-exposed protein conformations. Upon SC lipid depletion the nitroxide side chain becomes more solvent exposed, suggesting that the removal of hygroscopic substances in the extraction process favors more hydrated protein conformations. On the other hand, the treatments with 8 M urea or 40% (v/v) 1 MP did not alter significantly the fluidity in the SC lipid domain as assessed by the probe 5-doxyl stearic acid; these permeation enhancers, specially 1 MP, seem to increase the probe solubility in the solvent leading to a considerable fraction of spin label to be removed from the lipid domain.     

Differential regulation of central vasopressin receptors in transgenic rats with low brain angiotensinogen

The consequences of permanent alteration to the brain renin-angiotensin system (RAS) on central vasopressinergic system was studied in transgenic rats with low brain angiotensinogen [TGR(ASrAOGEN)]. Levels of vasopressin (AVP) and V1a receptor mRNAs were measured by ribonuclease protection assay (RPA) and AVP by radioimmunoassay (RIA). AVP (100 pmol/50 nl) was microinjected into the nucleus tractus solitarii (NTS) of urethane-anesthetized TGR(ASrAOGEN) and Sprague-Dawley (SD) rats and the mean arterial pressure (MAP) and heart rate (HR) baroreflex induced by phenylephrine were evaluated. AVP but not its mRNA levels were significantly lower in the hypothalamus and hypophysis of TGR(ASrAOGEN) rats. Brainstem V1a mRNA levels were significantly higher in TGR(ASrAOGEN) in comparison to SD rats (5.2+/-0.4% vs. 3.3+/-0.2% of beta-actin mRNA, P<0.05). In contrast, the hypothalamic V1a mRNA levels in TGR(ASrAOGEN) were not different from those found in SD rats. AVP microinjections induced a greater decrease in MAP in TGR(ASrAOGEN) in comparison with SD rats (-19.9+/-5.2 vs. -7.5+/-0.7 mm Hg, P<0.01). The significantly higher baroreflex sensitivity observed in TGR compared to that of SD rats was normalized after AVP microinjection. The increased brainstem V1a mRNA levels and sensitivity to AVP in TGR(ASrAOGEN) rats indicates a functional upregulation of AVP receptors in the NTS. The fact that the hypothalamic V1a mRNA levels are not altered indicates that these receptors are differentially regulated in different brain regions. This study demonstrates that a permanent deficit in brain angiotensinogen synthesis can alter the functionality of central vasopressinergic system.     

Synthesis, in vitro and in vivo biological evaluation,COX-1/2 inhibition and molecular docking study of indole-N-acylhydrazone derivatives

The objective of this work was to obtain and evaluate anti-inflammatory in vitro, in vivo and in silico potential of novel indole-N-acylhydrazone derivatives. In total, 10 new compounds (3a–j) were synthesized in satisfactory yields, through a condensation reaction in a single synthesis step. In the lymphoproliferation assay, using mice splenocytes, 3a and 3b showed inhibition of lymphocyte proliferation of 62.7% (±3.5) and 50.7% (±2), respectively, while dexamethasone presented an inhibition of 74.6% (±2.4). Moreover, compound 3b induced higher Th2 cytokines production in mice splenocytes cultures. The results for COX inhibition assays showed that compound 3b is a selective COX-2 inhibitor, but with less potency when compared to celecoxib, and compound 3a not presented selectivity towards COX-2. The molecular docking results suggest compounds 3a and 3b interact with the active site of COX-2 in similar conformations, but not with the active site of COX-1, and this may be the main reason to the COX-2 selectivity of compound 3b. In vivo carrageenan-induced paw edema assays were adopted for the confirmation of the anti-inflammatory activity. Compound 3b showed better results in suppressing edema at all tested concentrations and was able to induce an edema inhibition of 100% after 5?h of carrageenan injection at the 30?mg?kg^?1 dosage, corroborating with the COX inhibition and lymphoproliferation results. I addition to our experimental results, in silico analysis suggest that compounds 3a and 3b present a well-balanced profile between pharmacodynamics and pharmacokinetics. Thus, our preliminary results revealed the potentiality of a new COX-2 selective derivative in the modulation of the inflammatory process.     

A yeast DNA repair gene partially complements defective excision repair in mammalian cells.

The RAD10 gene of Saccharomyces cerevisiae is required for nucleotide excision repair of DNA. Expression of RAD10 mRNA and Rad10 protein was demonstrated in Chinese hamster ovary (CHO) cells containing amplified copies of the gene, and RAD10 mRNA was also detected in stable transfectants without gene amplification. Following transfection with the RAD10 gene, three independently isolated excision repair-defective CHO cell lines from the same genetic complementation group (complementation group 2) showed partial complementation of sensitivity to killing by UV radiation and to the DNA cross-linking agent mitomycin C. These results were not observed when RAD10 was introduced into excision repair-defective CHO cell lines from other genetic complementation groups, nor when the yeast RAD3 gene was expressed in cells from genetic complementation group 2. Enhanced UV resistance in cells carrying the RAD10 gene was accompanied by partial reactivation of the plasmid-borne chloramphenicol acetyltransferase (cat) gene following its inactivation by UV radiation. The phenotype of CHO cells from genetic complementation group 2 is also specifically complemented by the human ERCC1 gene, and the ERCC1 and RAD10 genes have similar amino acid sequences. The present experiments therefore indicate that the structural homology between the yeast Rad10 and human Ercc1 polypeptides is reflected at a functional level, and suggest that nucleotide excision repair proteins are conserved in eukaryotes.     

Automatic Background Knowledge Selection for Matching Biomedical Ontologies

Ontology matching is a growing field of research that is of critical importance for the semantic web initiative. The use of background knowledge for ontology matching is often a key factor for success, particularly in complex and lexically rich domains such as the life sciences. However, in most ontology matching systems, the background knowledge sources are either predefined by the system or have to be provided by the user. In this paper, we present a novel methodology for automatically selecting background knowledge sources for any given ontologies to match. This methodology measures the usefulness of each background knowledge source by assessing the fraction of classes mapped through it over those mapped directly, which we call the mapping gain. We implemented this methodology in the AgreementMakerLight ontology matching framework, and evaluate it using the benchmark biomedical ontology matching tasks from the Ontology Alignment Evaluation Initiative (OAEI) 2013. In each matching problem, our methodology consistently identified the sources of background knowledge that led to the highest improvements over the baseline alignment (i.e., without background knowledge). Furthermore, our proposed mapping gain parameter is strongly correlated with the F-measure of the produced alignments, thus making it a good estimator for ontology matching techniques based on background knowledge.     

Bifunctional Homodimeric Triokinase/FMN Cyclase: CONTRIBUTION OF PROTEIN DOMAINS TO THE ACTIVITIES OF THE HUMAN ENZYME AND MOLECULAR DYNAMICS SIMULATION OF DOMAIN MOVEMENTS*

Mammalian triokinase, which phosphorylates exogenous dihydroxyacetone and fructose-derived glyceraldehyde, is neither molecularly identified nor firmly associated to an encoding gene. Human FMN cyclase, which splits FAD and other ribonucleoside diphosphate-X compounds to ribonucleoside monophosphate and cyclic X-phosphodiester, is identical to a DAK-encoded dihydroxyacetone kinase. This bifunctional protein was identified as triokinase. It was modeled as a homodimer of two-domain (K and L) subunits. Active centers lie between K1 and L2 or K2 and L1: dihydroxyacetone binds K and ATP binds L in different subunits too distant (≈14 Å) for phosphoryl transfer. FAD docked to the ATP site with ribityl 4′-OH in a possible near-attack conformation for cyclase activity. Reciprocal inhibition between kinase and cyclase reactants confirmed substrate site locations. The differential roles of protein domains were supported by their individual expression: K was inactive, and L displayed cyclase but not kinase activity. The importance of domain mobility for the kinase activity of dimeric triokinase was highlighted by molecular dynamics simulations: ATP approached dihydroxyacetone at distances below 5 Å in near-attack conformation. Based upon structure, docking, and molecular dynamics simulations, relevant residues were mutated to alanine, and k_cat and K_m were assayed whenever kinase and/or cyclase activity was conserved. The results supported the roles of Thr¹¹² (hydrogen bonding of ATP adenine to K in the closed active center), His²²¹ (covalent anchoring of dihydroxyacetone to K), Asp⁴⁰¹ and Asp⁴⁰³ (metal coordination to L), and Asp⁵⁵⁶ (hydrogen bonding of ATP or FAD ribose to L domain). Interestingly, the His²²¹ point mutant acted specifically as a cyclase without kinase activity.