Effects of either glucose or fructose and metabolic regulators on bovine embryo development and lipid accumulation in vitro

Our objective was to determine if replacing glucose with fructose would decrease cytoplasmic lipid accumulation during culture of embryos with or without regulators of metabolism. In vitro-produced bovine zygotes were cultured 60 hr in chemically defined medium-1 (CDM-1) plus 0.5% BSA and 0.5 mM fructose or glucose in Experiment 1, and glucose in Experiment 2. In both experiments, 8-cell embryos were next cultured 135 hr in CDM-2 plus 2 mM fructose or glucose in factorial combination with five treatments: (Experiment 1: control, 10% fetal calf serum (FCS), 0.3 microM phenazine ethosulfate (PES), 30 microM dinitrophenol (DNP), and PES + DNP), and (Experiment 2: control, PES, PES + DNP, and 1 and 3 microg/ml cerulenin (C1 and C3)). Day 7.5 blastocysts were stained with Sudan Black B to quantify cytoplasmic lipid droplets as small (SD, <2 microm), medium (MD, 2-6 microm), or large (LD, >6 microm). Blastocyst rates per oocyte were 22% (Experiment 1) and 15% (Experiment 2) higher (P < 0.05) for fructose than glucose. For Experiment 1, numbers of MD were lower for PES, DNP, and PES + DNP than control and FCS (P < 0.05). LD were lower for PES and DNP than control, and higher for FCS than all other treatments (P < 0.05). For Experiment 2, MD were lower (P < 0.05) for PES, and PES + DNP than C1, C3, and control. For LD, PES was lower (P < 0.05) than control, C1, and C3, but not different from PES + DNP. The only effect of hexose on lipids was that fructose resulted in fewer MD (P < 0.01) in Experiment 2. In conclusion, fructose produced more blastocysts than glucose, and PES reduced lipid accumulation.     

Reduced oxygen concentration improves the developmental competence of mouse oocytes following in vitro maturation

Reduced atmospheric oxygen concentration is beneficial to embryo development; however, optimal oxygen concentration for oocyte maturation remains undetermined. Likewise, there is no consensus of appropriate medium supplementation during maturation. The objective of this study was to determine whether oxygen tension (20% or 5% O2) and epidermal growth factor (EGF) affect oocyte metabolism and subsequent embryo development. Cumulus-oocyte complexes (COCs) were collected from 28-day-old equine chorionic gonadotropin (eCG) primed or unprimed F1 (C57BL/6xCBA) mice. COCs were matured in defined medium in one of four groups: 20% O2, 20% O2 + EGF, 5% O2, 5% O2 + EGF. In vivo matured COCs were also collected for analysis. COCs from unprimed mice, matured in 5% O2 +/- EGF or 20% O2 + EGF had higher metabolic rates than COCs matured in 20% O2 (P < 0.05). COCs from primed mice had higher metabolic rates when matured in the presence of EGF, regardless of oxygen tension (P < 0.01). Oxygen uptake and mitochondrial membrane potential were higher for in vivo matured oocytes and oocytes matured under 5% O2 compared to oocytes matured under 20% O2 (P < 0.05). Blastocyst formation was not different between maturation groups (primed or unprimed); however, embryo cell numbers were 20-45% significantly higher when COCs were matured at 5% O2 (P < 0.05). Results suggest that oocytes matured in physiological concentrations of oxygen have improved development and metabolic activity, more closely resembling in vivo maturation. These findings have implications for oocyte maturation in both clinical and research laboratories.     

CcpA mutants with differential activities in Bacillus subtilis

CcpA is the master regulator for carbon catabolite regulation in Bacillus subtilis and regulates more than 300 genes by repression or activation. To revealthe effects of different functional domains of CcpA on various regulatory modes, we compared the activities of CcpA point mutants in activation (alsS, ackA) and repression (xynP, gntR). CcpA variants mutated at residues in the HPrSerP-binding region without allosteric functions are inactive. On the other hand, CcpA variants mutated at residues that change their conformation upon HPrSerP or CrhP binding regulate only ackA. Another set of mutants with alterations in the corepressor-binding region show glucose-independent regulation of xynP. The data presented here demonstrate the involvement of HPrSerP and/or CrhP in activation of ackA and alsS by CcpA. Furthermore, these data also indicate that activation and repression mediated by CcpA may utilize different conformational changes of the protein.     

Flow,force, behaviour: assessment of a prototype hydraulic barrier for invasive fish

Hydrobiologia - Migration barriers being selective for invasive species could protect pristine upstream areas. We designed and tested a prototype protective barrier in a vertical slot fish pass....     

A hybrid process for chiral separation of compound‐forming systems

The resolution of chiral compound‐forming systems using hybrid processes was discussed recently. The concept is of large relevance as these systems form the majority of chiral substances. In this study, a novel hybrid process is presented, which combines pertraction and subsequent preferential crystallization and is applicable for the resolution of such systems. A supported liquid membrane applied in a pertraction process provides enantiomeric enrichment. This membrane contains a solution of a chiral compound acting as a selective carrier for one of the enantiomers. Screening of a large number of liquid membranes and potential carriers using the conductor‐like screening model for realistic solvation method led to the identification of several promising carriers, which were tested experimentally in several pertraction runs aiming to yield enriched (+)‐(S)‐mandelic acid (MA) solutions from racemic feed solutions. The most promising system consisted of tetrahydronaphthalene as liquid membrane and hydroquinine‐4‐methyl‐2‐quinolylether (HMQ) as chiral carrier achieving enantiomeric excesses of 15% in average. The successful production of (+)‐(S)‐MA with a purity above 96% from enriched solutions by subsequent preferential crystallization proved the applicability of the hybrid process. Chirality, 2011. © 2010 Wiley‐Liss, Inc.     

Dot1 and histone H3K79 methylation in natural telomeric and HM silencing

The expression of genes residing near telomeres is attenuated through telomere position-effect variegation (TPEV). By using a URA3 reporter located at TEL-VII-L of Saccharomyces cerevisiae, it was proposed that the disruptor of telomeric silencing-1 (Dot1) regulates TPEV by catalyzing H3K79 methylation. URA3 reporter assays also indicated that H3K79 methylation is required for HM silencing. Surprisingly, a genome-wide expression analysis of H3K79 methylation-defective mutants identified only a few telomeric genes, such as COS12 at TEL-VII-L, to be subject to H3K79 methylation-dependent natural silencing. Consistently, loss of Dot1 did not globally alter Sir2 or Sir3 occupancy in subtelomeric regions, but only led to some telomere-specific changes. Furthermore, H3K79 methylation by Dot1 did not play a role in the maintenance of natural HML silencing. Therefore, commonly used URA3 reporter assays may not report on natural PEV, and therefore, studies concerning the epigenetic mechanism of silencing in yeast should also employ assays reporting on natural gene expression patterns.     

Imaging Glycans in Zebrafish Embryos by Metabolic Labeling and Bioorthogonal Click Chemistry

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or alkyne-tagged monosaccharides^{1, 2}. The modified monosaccharides, processed by the glycan biosynthetic machinery, are incorporated into cell surface glycoconjugates. The bioorthogonal azide or alkyne tags then allow covalent conjugation with fluorescent probes for visualization, or with affinity probes for enrichment and glycoproteomic analysis. This protocol describes the procedures typically used for noninvasive imaging of fucosylated glycans in zebrafish embryos, including: 1) microinjection of one-cell stage embryos with GDP-5-alkynylfucose (GDP-FucAl), 2) labeling fucosylated glycans in the enveloping layer of zebrafish embryos with azide-conjugated fluorophores via biocompatible Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), and 3) imaging by confocal microscopy³. The method described here can be readily extended to visualize other classes of glycans, e.g. glycans containing sialic acid⁴ and N-acetylgalactosamine^{5, 6}, in developing zebrafish and in other living organisms.Download video file.^{(41M, mov)}