Application of empirical design methodologies to the study of the influence of reaction conditions and N-alpha-protecting group structure on the enzymatic X-Phe-Leu-NH(2) dipeptide synthesis in buffer/dimethylformamide solvents systems

The influence of five different N-terminal protecting groups (For, Ac, Boc, Z, and Fmoc) and reaction conditions (temperature and dimethylformamide content) on the alpha-chymotrypsin-catalyzed synthesis of the dipeptide derivative X-Phe-Leu-NH(2) was studied. Groups such as For, Ac, Boc, and Z always rendered good peptide yields (82% to 85%) at low reaction temperatures and DMF concentrations, which depended on the N-alpha protection choice. Boc and Z were the most reactive N-alpha groups and, in addition, the most suitable for peptide synthesis. On the other hand, the use of empirical design methodologies allowed, with minimal experimentation and by multiple regression, to deduce an equation, which correlates the logarithm of the first order kinetic constant (log k') with reaction temperature, DMF concentration, and hydrophobicity (log P values) of the different protecting groups. The predictive value of the equation was tested by comparing the performance of another protective group, such as Aloc, with the performance predicted by said equation. Experimental and calculated k' values were found to be in good agreement.     

Habitat specific growth rates and condition indices for the sympatric soles Solea solea (Linnaeus, 1758) and Solea senegalensis Kaup 1858, in the Tagus estuary, Portugal, based on otolith daily increments and RNA-DNA ratio

Habitat specific growth rates and condition indices were estimated for Solea solea and Solea senegalensis, in two nursery areas within the Tagus estuary, at the end of the estuarine colonization process, in 2005. While in the uppermost nursery area the two species of sole live in sympatry, in the lower nursery only S. senegalensis is present. Daily increments of left lapillar otoliths were used to estimate age (in days) and determine growth rates (mm per day). Condition indices were assessed through RNA‐DNA ratio in muscle samples. Growth rates were higher for S. senegalensis (0.970and 1.180 mm per day in nursery A and B, respectively) than for S. solea (0.767 mm per day in nursery A). Growth rates of S. senegalensis from the uppermost nursery area were lower when compared to those obtained for the other nursery. The RNA/DNA condition index followed the general trend given by the growth rate estimates, i.e. values were higher for S. senegalensis than for S. solea. However, no significant differences were detected in S. senegalensis from the two nurseries. Larger variations in salinity (10‰ amplitude in the uppermost nursery vs 0.2‰ in the lower nursery) and highest pollution loads may be important factors lowering the habitat quality of the uppermost nursery in comparison to the lower nursery. The use of growth rate estimates based on otolith readings and the RNA/DNA index as tools for habitat quality assessment was discussed.     

Worldwide Occurrence of Integrative Conjugative Element Encoding Multidrug Resistance Determinants in Epidemic Vibrio cholerae O1

In the last decades, there has been an increase of cholera epidemics caused by multidrug resistant strains. Particularly, the integrative and conjugative element (ICE) seems to play a major role in the emergence of multidrug resistant Vibrio cholerae. This study fully characterized, by whole genome sequencing, new ICEs carried by multidrug resistant V. cholerae O1 strains from Nigeria (2010) (ICEVchNig1) and Nepal (1994) (ICEVchNep1). The gene content and gene order of these two ICEs are the same, and identical to ICEVchInd5, ICEVchBan5 and ICEVchHai1 previously identified in multidrug resistant V. cholerae O1. This ICE is characterized by dfrA1, sul2, strAB and floR antimicrobial resistance genes, and by unique gene content in HS4 and HS5 ICE regions. Screening for ICEs, in publicly available V. cholerae genomes, revealed the occurrence and widespread distribution of this ICE among V. cholerae O1. Metagenomic analysis found segments of this ICE in marine environments far from the direct influence of the cholera epidemic. Therefore, this study revealed the epidemiology of a spatio-temporal prevalent ICE in V. cholerae O1. Its occurrence and dispersion in V. cholerae O1 strains from different continents throughout more than two decades can be indicative of its role in the fitness of the current pandemic lineage.     

Surface immobilization and entrapping of enzymes on glutaraldehyde crosslinked gelatin particles

A mild and reproducible method has been developed for the surface-immobilization of enzymes on glutaraldehyde crosslinked gelatin beads. In this method glutaraldehyde is used in a dual capacity, as crosslinking agent and as the enzyme coupling agent. Glucoamylase (exo-α-1,4-d-glucosidase, EC 3.2.1.3), β-d-fructofuranosidase (invertase, EC 3.2.1.26) and β-d-glucoside (cellobiase, β-d-glucoside glucohydrolase, EC 3.2.1.21) have been successfully immobilized by this method, on the surface of the crosslinked gelatin particles. The method can be combined with the existing technology for the production of gelatin-entrapped enzymes. Thus, dual immobilized enzyme conjugates of glucoamylase and invertase have been prepared using this method, by entrapment of one enzyme in, and surface-binding of the other to, the gelatin matrix. The coupling of glucoamylase onto cross-linked gelatin particles by precipitation with poly(hexamethylenebiguanide hydrochloride) was also tested.     

The redox properties of (1,6-bis(benzimidazol-2-yl)-2,5-dithiahexane) chloro-copper(II) chloride as studied by cyclic voltammetry

Cyclic voltammetry in acetonitrile has been used to study the redox properties of the compound [Cu-(BBDH)Cl] Cl under various conditions. The nature of the species in solution was studied with the aid of ligand-field spectra and ESR spectra. It appears that in CH₃CN solution two Cu(II) species are predominating, viz. [Cu(BBDH)(CH₃CN)_x]²⁺ and Cu(BBDH)-Cl⁺. Minor species are dimeric in nature, such as [(BBDH)CuCl₂Cu(BBDH)]²⁺, as easily seen from ESR. The relative amount of the two major species was varied using added LiCl, allowing us to determine the nature of both species. The redox potential of the species [Cu(BBDH)Cl]⁺ appeared to be 0.62 V (against a normal hydrogen electrode), which is very high for a Cu(II) compound and in the same area as found for the blue copper proteins. Re-oxidation of Cu(BBDH)⁺ in the presence of LiCl shows that C$\text{l}$ slowly recoordinates after reoxidation.     

Reduced IgG anti-small nuclear ribonucleoprotein autoantibody production in systemic lupus erythematosus patients with positive IgM anti-cytomegalovirus antibodies

Introduction

In rheumatoid arthritis (RA), synovial fluid (SF) contains a large number of neutrophils that contribute to the inflammation and destruction of the joints. The SF also contains granulocyte-macrophage colony-stimulating factor (GM-CSF), which sustains viability of neutrophils and activates their functions. Using proteomic surveillance, we here tried to elucidate the effects of GM-CSF on neutrophils.

Methods

Neutrophils stimulated by GM-CSF were divided into four subcellular fractions: cytosol, membrane/organelle, nuclei, and cytoskeleton. Then, proteins were extracted from each fraction and digested by trypsin. The produced peptides were detected using matrix-assisted laser desorption ionisation-time-of-flight mass spectrometry (MALDI-TOF MS).

Results

We detected 33 peptide peaks whose expression was upregulated by more than 2.5-fold in GM-CSF stimulated neutrophils and identified 11 proteins out of the 33 peptides using MALDI-TOF/TOF MS analysis and protein database searches. One of the identified proteins was neutrophil gelatinase-associated lipocalin (NGAL). We confirmed that the level of NGAL in SF was significantly higher in patients with RA than in those with osteoarthritis. We next addressed possible roles of the increased NGAL in RA. We analysed proteome alteration of synoviocytes from patients with RA by treatment with NGAL in vitro. We found that, out of the detected protein spots (approximately 3,600 protein spots), the intensity of 21 protein spots increased by more than 1.5-fold and the intensity of 10 protein spots decreased by less than 1 to 1.5-fold as a result of the NGAL treatment. Among the 21 increased protein spots, we identified 9 proteins including transitional endoplasmic reticulum ATPase (TERA), cathepsin D, and transglutaminase 2 (TG2), which increased to 4.8-fold, 1.5-fold and 1.6-fold, respectively. Two-dimensional electrophoresis followed by western blot analysis confirmed the upregulation of TERA by the NGAL treatment and, moreover, the western blot analysis showed that the NGAL treatment changed the protein spots caused by post-translational modification of TERA. Furthermore, NGAL cancelled out the proliferative effects of fibroblast growth factor (FGF)-2 and epidermal growth factor (EGF) on chondrocytes from a patient with RA and proliferative effect of FGF-2 on chondrosarcoma cells.

Conclusions

Our results indicate that GM-CSF contributes to the pathogenesis of RA through upregulation of NGAL in neutrophils, followed by induction of TERA, cathepsin D and TG2 in synoviocytes. NGAL and the upregulated enzymes may therefore play an important role in RA.     

Kinetics and mechanism of the cutinase-catalyzed transesterification of oils in AOT reversed micellar system

The kinetics of the enzymatic transesterification between a mixture of triglycerides (oils) and methanol for biodiesel production in a bis(2-ethylhexyl) sodium sulfosuccinate (AOT)/isooctane reversed micellar system, using recombinant cutinase from Fusarium solani pisi as a catalyst, was investigated. In order to describe the results that were obtained, a mechanistic scheme was proposed, based on the literature and on the experimental data. This scheme includes the following reaction steps: the formation of the active enzyme–substrate complex, the addition of an alcohol molecule to the complex followed by the separation of a molecule of the fatty acid alkyl ester and a glycerol moiety, and release of the active enzyme. Enzyme inhibition and deactivation effects due to methanol and glycerol were incorporated in the model. This kinetic model was fitted to the concentration profiles of the fatty acid methyl esters (the components of biodiesel), tri-, di- and monoglycerides, obtained for a 24 h transesterification reaction performed in a stirred batch reactor under different reaction conditions of enzyme and initial substrates concentration.     

Chromosome evolution and phylogeny in Ronderosia (Orthoptera,Acrididae, Melanoplinae): clues of survivors to the challenge of sympatry?

In an attempt to unveil the origin of neo‐sex chromosomes in Ronderosia Cigliano grasshoppers, we performed a combined phylogenetic analysis based on morphological (external morphology and male genitalia) and molecular data (COI, COII, 16S and ITS2) to explore the chromosome evolution within the genus. We also analysed the distributional patterns of the various Ronderosia species and considered the possible role of chromosome rearrangements (CRs) in speciation processes within the genus in the light of ‘suppressed‐recombination’ models. We mapped the states of three chromosomal characters on the combined tree topology. The combined evidence supported Ronderosia as a monophyletic group. The cytogenetic analyses of the genus demonstrated the importance of rearranged karyotypes with single, complex and multiples neo‐sex chromosome determination systems in all species. The chromosome character optimisation suggests X‐autosome centric fusion as the mechanism responsible for neo‐sex chromosome formation in most Ronderosia species, except in R. dubia and R. bergii. Similar autosomes were involved in fusions with the ancestral X chromosome in Ronderosia, supporting previous hypotheses on the unique origin of X‐autosome fusion for the sex chromosome in the genus. As a source of chromosome variation, autosome‐autosome centric fusion played a secondary role in Ronderosia compared with other Dichroplini. Given the homogeneity in the morphological features, the sympatric distribution of closely related species and the intrinsic property of centric fusion as suppressors of the crossing over, we suggest that CRs may have played a key role during the speciation process within Ronderosia.     

Systematic study of high molecular weight compounds in Amazonian plants by high temperature gas chromatography-mass spectrometry

The fractions of hexane and dichloromethane extraction from marupá (Simaruba amara) and (Bertholletia excelsa) leaves were analyzed by HT-HRGC (high temperature high resolution gas chromatography) and HT-HRGC coupled to mass spectrometry (HT-HRGC-MS). Several compounds can be characterized including unusual high molecular weight compounds.