A method for the production of D-tagatose using a recombinant Pichia pastoris strain secreting ß-D-galactosidase from Arthrobacter chlorophenolicus and a recombinant L-arabinose isomerase from

ABSTRACT: BACKGROUND: D-Tagatose is a natural monosaccharide which can be used as a low-calorie sugar substitute in food, beverages and pharmaceutical products. It is also currently being tested as an anti-diabetic and obesity control drug. D-Tagatose is a rare sugar, but it can be manufactured by the chemical or enzymatic isomerization of D-galactose obtained by a beta-D-galactosidase-catalyzed hydrolysis of milk sugar lactose and the separation of D-glucose and D-galactose. L-Arabinose isomerases catalyze in vitro the conversion of D-galactose to D-tagatose and are the most promising enzymes for the large-scale production of D-tagatose. RESULTS: In this study, the araA gene from psychrotolerant Antarctic bacterium Arthrobacter sp. 22c was isolated, cloned and expressed in Escherichia coli. The active form of recombinant Arthrobacter sp. 22c L-arabinose isomerase consists of six subunits with a combined molecular weight of approximately 335 kDa. The maximum activity of this enzyme towards D-galactose was determined as occurring at 52[DEGREE SIGN]C; however, it exhibited over 60% of maximum activity at 30[DEGREE SIGN]C. The recombinant Arthrobacter sp. 22c L-arabinose isomerase was optimally active at a broad pH range of 5 to 9. This enzyme is not dependent on divalent metal ions, since it was only marginally activated by Mg2+, Mn2+ or Ca2+ and slightly inhibited by Co2+ or Ni2+. The bioconversion yield of D-galactose to D-tagatose by the purified L-arabinose isomerase reached 30% after 36 h at 50[DEGREE SIGN]C. In this study, a recombinant Pichia pastoris yeast strain secreting beta-D-galactosidase Arthrobacter chlorophenolicus was also constructed. During cultivation of this strain in a whey permeate, lactose was hydrolyzed and D-glucose was metabolized, whereas D-galactose was accumulated in the medium. Moreover, cultivation of the P. pastoris strain secreting beta-D-galactosidase in a whey permeate supplemented with Arthrobacter sp. 22c L-arabinose isomerase resulted in a 90% yield of lactose hydrolysis, the complete utilization of D-glucose and a 30% conversion of D-galactose to D-tagatose. CONCLUSIONS: The method developed for the simultaneous hydrolysis of lactose, utilization of D-glucose and isomerization of D-galactose using a P. pastoris strain secreting beta-D-galactosidase and recombinant L-arabinose isomerase seems to offer an interesting alternative for the production of D-tagatose from lactose-containing feedstock.     

Bioactive conformations of two seminal delta opioid receptor penta‐peptides inferred from free‐energy profiles

Delta‐opioid (DOP) receptors are members of the G protein‐coupled receptor (GPCR) sub‐family of opioid receptors, and are evolutionarily related, with homology exceeding 70%, to cognate mu‐opioid (MOP), kappa‐opioid (KOP), and nociceptin opioid (NOP) receptors. DOP receptors are considered attractive drug targets for pain management because agonists at these receptors are reported to exhibit strong antinociceptive activity with relatively few side effects. Among the most potent analgesics targeting the DOP receptor are the linear and cyclic enkephalin analogs known as DADLE (Tyr‐D ‐Ala‐Gly‐Phe‐D ‐Leu) and DPDPE (Tyr‐D ‐Pen‐Gly‐Phe‐D ‐Pen), respectively. Several computational and experimental studies have been carried out over the years to characterize the conformational profile of these penta‐peptides with the ultimate goal of designing potent peptidomimetic agonists for the DOP receptor. The computational studies published to date, however, have investigated only a limited range of timescales and used over‐simplified representations of the solvent environment. We provide here a thorough exploration of the conformational space of DADLE and DPDPE in an explicit solvent, using microsecond‐scale molecular dynamics and bias‐exchange metadynamics simulations. Free‐energy profiles derived from these simulations point to a small number of DADLE and DPDPE conformational minima in solution, which are separated by relatively small energy barriers. Candidate bioactive forms of these peptides are selected from identified common spatial arrangements of key pharmacophoric points within all sampled conformations. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 21–27, 2014.     

Hematopoietic stem cell expansion: challenges and opportunities

Attempts to improve hematopoietic reconstitution and engraftment potential of ex vivo-expanded hematopoietic stem and progenitor cells (HSPCs) have been largely unsuccessful due to the inability to generate sufficient stem cell numbers and to excessive differentiation of the starting cell population. Although hematopoietic stem cells (HSCs) will rapidly expand after in vivo transplantation, experience from in vitro studies indicates that control of HSPC self-renewal and differentiation in culture remains difficult. Protocols that are based on hematopoietic cytokines have failed to support reliable amplification of immature stem cells in culture, suggesting that additional factors are required. In recent years, several novel factors, including developmental factors and chemical compounds, have been reported to affect HSC self-renewal and improve ex vivo stem cell expansion protocols. Here, we highlight early expansion attempts and review recent development in the extrinsic control of HSPC fate in vitro.     

Macrophyte and macroinvertebrate patterns in unimpacted mountain rivers of two European ecoregions

Hydrobiologia - The aim of the study was to compare the patterns of development of macrophytes and macroinvertebrates in different types of reference mountain rivers. The study is based on...     

Introduced Drosophila subobscura populations perform better than native populations during an oviposition choice task due to increased fecundity but similar learning ability

The success of invasive species is tightly linked to their fitness in a putatively novel environment. While quantitative components of fitness have been studied extensively in the context of invasive species, fewer studies have looked at qualitative components of fitness, such as behavioral plasticity, and their interaction with quantitative components, despite intuitive benefits over the course of an invasion. In particular, learning is a form of behavioral plasticity that makes it possible to finely tune behavior according to environmental conditions. Learning can be crucial for survival and reproduction of introduced organisms in novel areas, for example, for detecting new predators, or finding mates or oviposition sites. Here we explored how oviposition performance evolved in relation to both fecundity and learning during an invasion, using native and introduced Drosophila subobscura populations performing an ecologically relevant task. Our results indicated that, under comparable conditions, invasive populations performed better during our oviposition task than did native populations. This was because invasive populations had higher fecundity, together with similar cognitive performance when compared to native populations, and that there was no interaction between learning and fecundity. Unexpectedly, our study did not reveal an allocation trade‐off (i.e., a negative relationship) between learning and fecundity. On the contrary, the pattern we observed was more consistent with an acquisition trade‐off, meaning that fecundity could be limited by availability of resources, unlike cognitive ability. This pattern might be the consequence of escaping natural enemies and/or competitors during the introduction. The apparent lack of evolution of learning may indicate that the introduced population did not face novel cognitive challenges in the new environment (i.e., cognitive “pre‐adaptation”). Alternatively, the evolution of learning may have been transient and therefore not detected.     

The Light Skin Allele of SLC24A5 in South Asians and Europeans Shares Identity by Descent

Skin pigmentation is one of the most variable phenotypic traits in humans. A non-synonymous substitution (rs1426654) in the third exon of SLC24A5 accounts for lighter skin in Europeans but not in East Asians. A previous genome-wide association study carried out in a heterogeneous sample of UK immigrants of South Asian descent suggested that this gene also contributes significantly to skin pigmentation variation among South Asians. In the present study, we have quantitatively assessed skin pigmentation for a largely homogeneous cohort of 1228 individuals from the Southern region of the Indian subcontinent. Our data confirm significant association of rs1426654 SNP with skin pigmentation, explaining about 27% of total phenotypic variation in the cohort studied. Our extensive survey of the polymorphism in 1573 individuals from 54 ethnic populations across the Indian subcontinent reveals wide presence of the derived-A allele, although the frequencies vary substantially among populations. We also show that the geospatial pattern of this allele is complex, but most importantly, reflects strong influence of language, geography and demographic history of the populations. Sequencing 11.74 kb of SLC24A5 in 95 individuals worldwide reveals that the rs1426654-A alleles in South Asian and West Eurasian populations are monophyletic and occur on the background of a common haplotype that is characterized by low genetic diversity. We date the coalescence of the light skin associated allele at 22–28 KYA. Both our sequence and genome-wide genotype data confirm that this gene has been a target for positive selection among Europeans. However, the latter also shows additional evidence of selection in populations of the Middle East, Central Asia, Pakistan and North India but not in South India.     

Detection and Structural Characterization of Ether Glycerophosphoethanolamine from Cortical Lysosomes Following Traumatic Brain Injury Using UPLC‐HDMSE

The use of ultra performance liquid chromatography coupled to data independent tandem mass spectrometry with traveling wave ion mobility for detection and structural identification of ether‐linked glycerophosphoethanolamine is described. The experimental design generates 4D data (chromatographic retention time, precursor accurate mass, drift time with associated calculated collisional cross‐section, and time‐aligned accurate mass diagnostic product ions) for each ionization mode. Confident structure identification depends on satisfying 4D data confirmation in both positive and negative ion mode. Using this methodology, a number of ether‐linked glycerophosphoethanolamine lipids are structurally elucidated from mouse brain lysosomes. It is further determined that several ether‐linked glycerophosphoethanolamine structures are differentially abundant between lysosomes isolated from mouse cortex following traumatic brain injury as compared to that of sham animals. The combined effort of aligning multi‐dimensional mass spectrometry data with a well‐defined traumatic brain injury model lays the foundation for gaining mechanistic insight in the role lysosomal membrane damage plays in neuronal cell death following brain injury.