Mannose-binding lectin from<Emphasis Type="Italic">Curcuma zedoaria</Emphasis> Rosc

A mannose-binding lectin was isolated from rhizomes of the medicinal plantCurcuma zedoaria. We used extraction with 20 mM phosphate buffer, ammonium sulfate precipitation, ion exchange chromatography on Q-Sepharose, gel filtration chromatography on Superdex 75, and reverse-phase HPLC. The purified lectin yielded a single band on SDS-PAGE that corresponded to a molecular mass of 13 kDa. This lectin exhibited hemagglutinating activity toward rabbit erythrocytes, which could be inhibited by mannose only. The lectin was digested with trypsin and its digests were analyzed using MALDI-TOF/TOF. Partial amino acid sequences were obtained from tandem mass spectra via automatedde novo sequencing, and were then identified by MS-BLAST homology searches to enable recognition of related proteins in other species. Inferred peptide sequences exhibited similarity to a mannose-binding lectin fromEpipactis helleborine, a member of the Orchidaceae.     

Cognitive functioning in opioid-dependent patients treated with buprenorphine,methadone, and other psychoactive medications: stability and correlates

Background

In many but not in all neuropsychological studies buprenorphine-treated opioid-dependent patients have shown fewer cognitive deficits than patients treated with methadone. In order to examine if hypothesized cognitive advantage of buprenorphine in relation to methadone is seen in clinical patients we did a neuropsychological follow-up study in unselected sample of buprenorphine- vs. methadone-treated patients.

Methods

In part I of the study fourteen buprenorphine-treated and 12 methadone-treated patients were tested by cognitive tests within two months (T1), 6-9 months (T2), and 12 - 17 months (T3) from the start of opioid substitution treatment. Fourteen healthy controls were examined at similar intervals. Benzodiazepine and other psychoactive comedications were common among the patients. Test results were analyzed with repeated measures analysis of variance and planned contrasts. In part II of the study the patient sample was extended to include 36 patients at T2 and T3. Correlations between cognitive functioning and medication, substance abuse, or demographic variables were then analyzed.

Results

In part I methadone patients were inferior to healthy controls tests in all tests measuring attention, working memory, or verbal memory. Buprenorphine patients were inferior to healthy controls in the first working memory task, the Paced Auditory Serial Addition Task and verbal memory. In the second working memory task, the Letter-Number Sequencing, their performance improved between T2 and T3. In part II only group membership (buprenorphine vs. methadone) correlated significantly with attention performance and improvement in the Letter-Number Sequencing. High frequency of substance abuse in the past month was associated with poor performance in the Letter-Number Sequencing.

Conclusions

The results underline the differences between non-randomized and randomized studies comparing cognitive performance in opioid substitution treated patients (fewer deficits in buprenorphine patients vs. no difference between buprenorphine and methadone patients, respectively). Possible reasons for this are discussed.

     

Low survival after release into the wild: assessing “the burden of captivity” on Mallard physiology and behaviour

Captive-reared animals used in reinforcement programs are generally less likely to survive than wild conspecifics. Digestion efficiency and naive behaviour are two likely reasons for this pattern. The Mallard is a species with high adaptability to its environment and in which massive reinforcement programs are carried out. We studied physiological and behavioural factors potentially affecting body condition and survival of captive-reared Mallards after being released. Digestive system morphology and an index of body condition were compared among three groups: captive-reared birds remaining in a farm (control), captive-reared birds released into the wild as juveniles (released) and wild-born birds (wild). We also compared behaviour and diet of released vs. wild Mallards. Finally, we conducted a 1-year survival analysis of captive-reared birds after release in a hunting-free area. Gizzard weight was lower in control Mallards, but the size of other organs did not differ between controls and wild birds. The difference in gizzard weight between released and wild birds disappeared after some time in the wild. Diet analyses suggest that released Mallards show a greater preference than wild for anthropogenic food (waste grain, bait). Despite similar time-budgets, released Mallards never attained the body condition of wild birds. As a consequence, survival probability in released Mallards was low, especially when food provisioning was stopped and during harsh winter periods. We argue that the low survival of released Mallards likely has a physiological rather than a behavioural (foraging) origin. In any case, extremely few released birds live long enough to potentially enter the breeding population, even without hunting. In the context of massive releases presently carried out for hunting purposes, our study indicates a low likelihood for genetic introgression by captive-reared birds into the wild population.     

Additions to the cytologically investigated species of <Emphasis Type="Italic">Potentilla</Emphasis> L. (Rosaceae) from India

At present 14 species of Potentilla L. have been cytologically worked out from different geographical areas of Kashmir and Himachal Pradesh in the Western Himalayas. New chromosome numbers in nine species—Potentilla argyrophylla (n = 14), P. atrosanguinea (n = 7, 14), P. desertorum (n = 7), P. gerardiana (n = 14), P. indica (n = 14), P. micropetala (n = 14), P. nepalensis (n = 14), P. sibbaldia (n = 14) and P. thomsonii (n = 7)—have been reported on a worldwide basis for the first time. Additional chromosomal races of polyploid cytotypes for P. argyrophylla (n = 28) and P. desertorum (n = 14) along with a diploid cytotype for P. micropetala (n = 7) plus diploid cytotypes for the five species as P. fulgens (n = 7), P. gelida (n = 7), P. kleiniana (n = 7), P. sibbaldia (n = 7) and P. sundaica (n = 7) as well as a tetraploid cytotype for P. fruticosa (n = 14) all have been cytologically worked out from India for the first time. The course of meiosis varies from normal to abnormal in different populations of the majority of the species, such as P. argyrophylla, P. atrosanguinea, P. desertorum, P. fruticosa, P. fulgens, P. gelida, P. indica, P. nepalensis, P. sibbaldia and P. sundaica, except for normal meiosis observed in P. gerardiana, P. kleiniana, P. micropetala and P. thomsonii. The anomalous taxa are marked with meiotic abnormalities in the form of cytomixis, chromosomal stickiness, unoriented bivalents, formation of laggards and bridges resulting in abnormal microsporogenesis, and production of heterogenous-sized fertile pollen grains along with reduced pollen fertility. All the taxa with normal meiotic courses show nearly one hundred percent pollen fertility.     

Extracellular overexpression of recombinant <Emphasis Type="Italic">Thermobifida fusca</Emphasis> cutinase by alpha-hemolysin secretion system in <Emphasis Type="Italic">E. coli</Emphasis> BL21(DE3)

Background  

Extracellular expression of proteins has an absolute advantage in a large-scale industrial production. In our previous study, Thermobifida fusca cutinase, an enzyme mainly utilized in textile industry, was expressed via type II secretory system in Escherichia coli BL21(DE3), and it was found that parts of the expressed protein was accumulated in the periplasmic space. Due to the fact that alpha-hemolysin secretion system can export target proteins directly from cytoplasm across both cell membrane of E. coli to the culture medium, thus in the present study we investigated the expression of cutinase using this alpha-hemolysin secretion system.     

Apolipoprotein A-I mimetic peptide D-4F promotes human endothelial progenitor cell proliferation,migration, adhesion though eNOS/NO pathway

Circulating endothelial progenitor cells (EPCs) have a critical role in endothelial maintenance and repair. Apolipoprotein A-I mimetic peptide D-4F has been shown to posses anti-atherogenic properties via sequestration of oxidized phospholipids, induction of remodeling of high density lipoprotein and promotion of cholesterol efflux from macrophage-derived foam cells. In this study, we test the effects of D-4F on EPC biology. EPCs were isolated from the peripheral venous blood of healthy male volunteers and characterized by 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine-labeled acetylated LDL uptake and ulex europaeus agglutinin binding and flow cytometry. Cell proliferation, migration, adhesion, nitric oxide production and endothelial nitric oxide synthase (eNOS) expression in the absence and presence of D-4F or simvastatin (as a positive control), were assayed. We demonstrated that D-4F significantly enhanced EPC proliferation, migration and adhesion in a dose-dependent manner compared with vehicle. However, all of the favorable effects of D-4F on EPCs were dramatically attenuated by preincubation with NOS inhibitor L-NAME. Further, D-4F also increased nitric oxide production in culture supernatant and the levels of eNOS expression and phosphorylation. The stimulatory effects of D-4F (10 μg/ml) on EPC biology were comparable to 0.5 μM simvastatin. These results suggest that eNOS/NO pathway mediates the functional modulation of EPC biology in response to D-4F treatment and support the notion that the beneficial role of D-4F on EPCs may be one of the important components of its anti-atherogenic potential.     

Flocculation in ale brewing strains of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>: re-evaluation of the role of cell surface charge and hydrophobicity

Flocculation is an eco-friendly process of cell separation, which has been traditionally exploited by the brewing industry. Cell surface charge (CSC), cell surface hydrophobicity (CSH) and the presence of active flocculins, during the growth of two (NCYC 1195 and NCYC 1214) ale brewing flocculent strains, belonging to the NewFlo phenotype, were examined. Ale strains, in exponential phase of growth, were not flocculent and did not present active flocculent lectins on the cell surface; in contrast, the same strains, in stationary phase of growth, were highly flocculent (>98%) and presented a hydrophobicity of approximately three to seven times higher than in exponential phase. No relationship between growth phase, flocculation and CSC was observed. For comparative purposes, a constitutively flocculent strain (S646-1B) and its isogenic non-flocculent strain (S646-8D) were also used. The treatment of ale brewing and S646-1B strains with pronase E originated a loss of flocculation and a strong reduction of CSH; S646-1B pronase E-treated cells displayed a similar CSH as the non-treated S646-8D cells. The treatment of the S646-8D strain with protease did not reduce CSH. In conclusion, the increase of CSH observed at the onset of flocculation of ale strains is a consequence of the presence of flocculins on the yeast cell surface and not the cause of yeast flocculation. CSH and CSC play a minor role in the auto-aggregation of the ale strains since the degree of flocculation is defined, primarily, by the presence of active flocculins on the yeast cell wall.     

Neurogenesis of bone marrow-derived mesenchymal stem cells onto β-mercaptoethanol-loaded PLGA film

Bone marrow-derived mesenchymal stem cells (BMSCs) are of particular interest in the field of tissue engineering because of their potential to differentiate into osteoblasts, chondrocytes, and neuronal cells. In order to promote the differentiation of BMSCs into specific cell types, appropriate scaffold biomaterials and bioactive molecules that can support the differentiation of BMSCs into specific cell types are needed. We hypothesized that β-mercaptoethanol (BME), which has been reported to induce the differentiation of BMSCs into neural-like cells, promotes BMSCs to differentiate into neural-like cells when BME is added to polymeric scaffolds containing the BMSCs. We fabricated biocompatible film shaped scaffolds composed of poly(lacti-co-glycolic) acid (PLGA) and various concentrations of BME to confirm that BME-promoted differentiation of BMSCs is concentration-dependent. Cell proliferation increased as the BME concentration in the films increased at the early stage, and the proliferation rate remained similar on the PLGA films for 3 weeks following the BMSC seeding. The expression of neuronal markers in differentiated BMSCs was assessed by RT-PCR. At 2- and 3-week time-points, mRNA expression of neurofilament and neuron specific enolase was significantly increased in PLGA/BME films containing 400 μM BME compared to PLGA films. Thus, we have identified BMSC-seeded PLGA/BME films with 200 μM and 400 μM BME as potentially useful candidates for neural tissue engineering applications by promoting BMSC proliferation and differentiation towards neural-like cells.     

Prioritization of SNPs for genome-wide association studies using an interaction model of genetic variation,gene expression,and trait variation

The identification of true causal loci to unravel the statistical evidence of genotype-phenotype correlations and the biological relevance of selected single-nucleotide polymorphisms (SNPs) is a challenging issue in genome-wide association studies (GWAS). Here, we introduced a novel method for the prioritization of SNPs based on p-values from GWAS. The method uses functional evidence from populations, including phenotype-associated gene expressions. Based on the concept of genetic interactions, such as perturbation of gene expression by genetic variation, phenotype and gene expression related SNPs were prioritized by adjusting the p-values of SNPs. We applied our method to GWAS data related to drug-induced cytotoxicity. Then, we prioritized loci that potentially play a role in druginduced cytotoxicity. By generating an interaction model, our approach allowed us not only to identify causal loci, but also to find intermediate nodes that regulate the flow of information among causal loci, perturbed gene expression, and resulting phenotypic variation.