Candidate gene analysis of GH1 for effects on growth and carcass composition of cattle

We present an approach to evaluate the support for candidate genes as quantitative trait loci (QTLs) within the context of genome-wide map-based cloning strategies. To establish candidacy, a bacterial artificial chromosome (BAC) clone containing a putative candidate gene is physically assigned to an anchored linkage map to localise the gone relative to an identified QTL effect. Microsatellite loci derived from BAC clones containing an established candidate gone are integrated into the linkage map facilitating the evaluation by interval analysis of the statistical support for QTL identity. Permutation analysis is employed to determine experiment-wise statistical support. The approach is illustrated for the growth hormone 1 ( GH1 ) gene and growth and carcass phenotypes in cattle. Polymerase chain reaction (PCR) primers which amplify a 441 bp fragment of GH1 were used to systematically screen a bovine BAC library comprising 60 000 clones and with a 95% probability of containing a single copy sequence. The presence of GH1 in BAC-110R2C3 was confirmed by sequence analysis of the PCR product from this clone and by the physical assignment of BAC110R2C3 to bovine chromosome 19 (BTA19) band 22 by fluorescence in situ hybridisation (FISH). Microsatellite KHGH1 was isolated from BAC110R2C3 and scored in 529 reciprocal backcross and F₂ fullsib progeny from 41 resource families derived from Angus ( Bos taurus ) and Brahman ( Bos indicus ). The microsatellite KHGH1 was incorporated into a framework genetic map of BTA19 comprising 12 microsatellite loci, the erythrocyte antigen T and a GH1-TaqI restriction fragment length polymorphism (RFLP). Interval analysis localised effects of taurus vs. indicus alleles on subcutaneous fat and the percentage of ether extractable fat from the longissimus dorsi muscle to the region of BTA19 harbouring GH1 .     

Characterization of pharmacogenetically relevant CYP2D6 and ABCB1 gene polymorphisms in a Portuguese population sample

Differences in metabolism of drugs can lead to severe toxicity or therapeutic failure. In addition to cytochrome P450 2D6, which plays a critical role in drug metabolism, ABCB1 encoded P‐glycoprotein (PGP) is also an important determinant in drug bioavailability. The genes encoding these molecules are highly variable among populations and, given their clinical importance in drug therapy, determining CYP2D6 and ABCB1 allele frequencies in specific populations is very important for useful application in clinical settings. In this study the frequency of the pharmacologically relevant CYP2D6*3, *4, *5, *6 allelic variants and gene duplication, and ABCB1 C1236T and C3435T gene polymorphisms and their haplotypes was determined in a population sample of 100 Portuguese healthy subjects. CYP2D6 allele frequencies were 1.4% (*3), 13.3% (*4), 2.8% (*5), 1.8% (*6) and 6.1% (gene duplication), with 5% of the individuals classified as PM and 8.4% as UM. The frequencies obtained for the non‐functional alleles and for the CYP2D6 gene duplication are in agreement with other South European populations, and reinforce the previously suggested south/north gradient of CYP2D6 duplications. Allelic frequencies for the ABCB1 polymorphisms were 52% (3435C) and 54% (1236C) and the most common haplotype (1236C‐3435C) occurred with a frequency of 45.5%. Although allele and haplotype frequency data for ABCB1 in Southern Europe is limited, some discrepancies were found with other European populations, with possible therapeutic implications for PGP substrate drugs. Copyright © 2009 John Wiley & Sons, Ltd.     

Enhanced extraction of proteins using cholinium‐based ionic liquids as phase‐forming components of aqueous biphasic systems

Aqueous biphasic systems (ABS) composed of ionic liquids (ILs) are promising platforms for the extraction and purification of proteins. In this work, a series of alternative and biocompatible ABS composed of cholinium‐based ILs and polypropylene glycol were investigated. The respective ternary phase diagrams, tie‐lines, tie‐line lengths and critical points were determined at 25°C. The extraction performance of these systems for commercial bovine serum albumin (BSA) was then evaluated. The stability of BSA at the IL‐rich phase was ascertained by size exclusion high‐performance liquid chromatography and Fourier transform infrared spectroscopy. Appropriate ILs lead to the complete extraction of BSA for the IL‐rich phase, in a single step, while maintaining the protein's native conformation. Furthermore, to evaluate the performance of these systems when applied to real matrices, the extraction of BSA from bovine serum was additionally carried out, revealing that the complete extraction of BSA was maintained and achieved in a single step. The remarkable extraction efficiencies obtained are far superior to those observed with typical polymer‐based ABS. Therefore, the proposed ABS may be envisaged as a more effective and biocompatible approach for the separation and purification of other value‐added proteins.     

Spatial and Temporal Variation in Reproduction of a Generalist Crocodilian,Caiman crocodilus yacare,in a Seasonally Flooded Wetland

We monitored the number of caiman (Caiman crocodilus yacare) nests in two ranches in the Brazilian Pantanal that cover an area of about 50.000 ha for 28 years (1987–2014). The number of nests was related to combinations of rainfall, water level, and number of days with temperature below 20°C, depending on the area. Most of the variation in number of nests could not be predicted by the environmental variables, but could be represented mathematically by a sine wave. We were not able to identify any external driver and suspect that the regular fluctuations may have resulted from an intrinsic population process. Presently, ranches are used as management units under the legislation for ranching Pantanal caimans. However, although some breeding females were recaptured in the area after periods of up to 21 years, most were not recaptured near nests or in general surveys of the area, suggesting that females are not strongly philopatric and that ranches do not represent isolated demographic units.     

From water‐in‐oil to oil‐in‐water emulsions to optimize the production of fatty acids using ionic liquids in micellar systems

Biocatalysis is nowadays considered as one of the most important tools in green chemistry. The elimination of multiple steps involved in some of the most complex chemical synthesis, reducing the amounts of wastes and hazards, thus increasing the reaction yields and decreasing the intrinsic costs, are the major advantages of biocatalysis. This work aims at improving the enzymatic hydrolysis of olive oil to produce valuable fatty acids through emulsion systems formed by long alkyl chain ionic liquids (ILs). The optimization of the emulsion and the best conditions to maximize the production of fatty acids were investigated. The stability of the emulsion was characterized considering the effect of several parameters, namely, the IL and its concentration and different water/olive oil volumetric ratios. ILs from the imidazolium and phosphonium families were evaluated. The results suggest that the ILs effect on the hydrolysis performance varies with the water concentration and the emulsion system formed, that is, water‐in‐oil or oil‐in‐water emulsion. Although at low water concentrations, the presence of ILs does not present any advantages for the hydrolysis reaction, at high water contents (in oil‐in‐water emulsions), the imidazolium‐based IL acts as an enhancer of the lipase catalytic capacity, super‐activating 1.8 times the enzyme, and consequently promoting the complete hydrolysis of the olive oil for the highest water contents [85% (v/v)]. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1473–1480, 2015     

Sulfated polysaccharides from marine sponges: conspicuous distribution among different cell types and involvement on formation of in vitro cell aggregates

Marine sponges (Porifera) display an ancestral type of cell-cell adhesion, based on carbohydrate-carbohydrate interaction. The aim of the present work was to investigate further details of this adhesion by using, as a model, the in vitro aggregation of dissociated sponge cells. Our results showed the participation of sulfated polysaccharides in this cell-cell interaction, as based on the following observations: (1) a variety of sponge cells contained similar sulfated polysaccharides as surface-associated molecules and as intracellular inclusions; (2) ³⁵S-sulfate metabolic labeling of dissociated sponge cells revealed that the majority (two thirds) of the total sulfated polysaccharide occurred as a cell-surface-associated molecule; (3) the aggregation process of dissociated sponge cells demanded the active de novo synthesis of sulfated polysaccharides, which ceased as cell aggregation reached a plateau; (4) the typical well-organized aggregates of sponge cells, known as primmorphs, contained three cell types showing sulfated polysaccharides on their cell surface; (5) collagen fibrils were also produced by the primmorphs in order to fill the extracellular spaces of their inner portion and the external layer surrounding their entire surface. Our data have thus clarified the relevance of sulfated polysaccharides in this system of in vitro sponge cell aggregation. The molecular basis of this system has practical relevance, since the culture of sponge cells is necessary for the production of molecules with biotechnological applications.     

Molecular docking and 3D-QSAR studies of HIV-1 protease inhibitors

HIV-1 protease is an obligatory enzyme in the replication process of the HIV virus. The abundance of structural information on HIV-1PR has made the enzyme an attractive target for computer-aided drug design strategies. The daunting ability of the virus to rapidly generate resistant mutants suggests that there is an ongoing need for new HIV-1PR inhibitors with better efficacy profiles and reduced toxicity. In the present investigation, molecular modeling studies were performed on a series of 54 cyclic urea analogs with symmetric P2/P2′ substituents. The binding modes of these inhibitors were determined by docking. The docking results also provided a reliable conformational superimposition scheme for the 3D-QSAR studies. To gain insight into the steric, electrostatic, hydrophobic and hydrogen-bonding properties of these molecules and their influence on the inhibitory activity, comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) were performed. Two different alignment schemes viz. receptor-based and atom-fit alignment, were used in this study to build the QSAR models. The derived 3D-QSAR models were found to be robust with statistically significant r ² and r ² _pred values and have led to the identification of regions important for steric, hydrophobic and electronic interactions. The predictive ability of the models was assessed on a set of molecules that were not included in the training set. Superimposition of the 3D-contour maps generated from these models onto the active site of enzyme provided additional insight into the structural requirements of these inhibitors. The CoMFA and CoMSIA models were used to design some new inhibitors with improved binding affinity. Pharmacokinetic and toxicity predictions were also carried out for these molecules to gauge their ADME and safety profile. The computational results may open up new avenues for synthesis of potent HIV-1 protease inhibitors.     

Modulation of cellulosome composition in Clostridium cellulolyticum: Adaptation to the polysaccharide environment revealed by proteomic and carbohydrate‐active enzyme analyses

Clostridium cellulolyticum is a model mesophilic anaerobic bacterium that efficiently degrades plant cell walls. The recent genome release offers the opportunity to analyse its complete degradation system. A total of 148 putative carbohydrate‐active enzymes were identified, and their modular structures and activities were predicted. Among them, 62 dockerin‐containing proteins bear catalytic modules from numerous carbohydrate‐active enzymes' families and whose diversity reflects the chemical and structural complexity of the plant carbohydrate. The composition of the cellulosomes produced by C. cellulolyticum upon growth on different substrates (cellulose, xylan, and wheat straw) was investigated by LC MS/MS. The majority of the proteins encoded by the cip‐cel operon, essential for cellulose degradation, were detected in all cellulosome preparations. In the presence of wheat straw, the natural and most complex of the substrates studied, additional proteins predicted to be involved in hemicellulose degradation were produced. A 32‐kb gene cluster encodes the majority of these proteins, all harbouring carbohydrate‐binding module 6 or carbohydrate‐binding module 22 xylan‐binding modules along dockerins. This newly identified xyl‐doc gene cluster, specialised in hemicellulose degradation, comes in addition of the cip‐cel operon for plant cell wall degradation. Hydrolysis efficiencies determined on the different substrates corroborates the finding that cellulosome composition is adapted to the growth substrate.