Improved cryopreservation of bovine preimplantation embryos cultured in chemically defined medium

The aim of this study was to examine the effects of modifications to a standard slow freezing protocol on the viability of in vitro produced bovine embryos. Bovine oocytes were matured, fertilized with frozen-thawed semen, and presumptive zygotes cultured in defined two-step culture media. The standard freezing medium was 1.5M ethylene glycol (EG), 0.1M sucrose, 10% fetal bovine serum (FBS) in Dulbecco's phosphate buffered saline (D-PBS). A preliminary trial showed that in vitro produced embryos cryopreserved in this medium had a survival rate of 74.6% at 24h and 53.5% at 48 h post-thaw. Experiment 1 studied the effects of omitting the sucrose supplement or replacing it with 0.1M xylose. In Experiment 2, the effects of partial (0%, 25% or 50%) or total (100%) replacement of sodium chloride with choline chloride in the cryopreservation medium were examined (the medium with 100% replacement was designated CJ1). The effects of replacing the 10% FBS with 0.4% BSA or 0.4% lipid-rich BSA (Albumax I) in CJ1 was studied in Experiment 3. In Experiment 4, pregnancy/calving rates following the post-thaw transfer of in vitro produced embryos cryopreserved in the standard freezing medium were compared with those of in vitro and in vivo produced embryos cryopreserved in the improved medium (Albumax I in CJ1). Supplementation of the cryopreservation medium with 0.1M sucrose resulted in higher post-thaw survival rates at 24 h (71.3% versus 53.5 and 51.7%; P<0.05), 48 h (51.1% versus 45.3 and 40.2%), and 72 h (34.0% versus 24.4 and 23.0%) than 0.1M xylose or no supplement, respectively, in Experiment 1. Experiment 2 showed that embryos cryopreserved in the standard medium had poorer survival rates at 24 h (72.8% versus 86.5%; P<0.05), 48 h (53.1% versus 66.3%) or 72 h (28.4% versus 44.9%) than those frozen in CJ1. The post-thaw survival rate of embryos frozen in medium supplemented with Albumax I was better than that for the FBS or BSA supplements at 24h (92.0% versus 90.7 and 87.3%), 48 h (87.3% versus 76.9 and 70.9%; P<0.05), and 72 h (70.4% versus 49.1 and 46 4%; P<0.05; Experiment 3). In Experiment 4, in vitro produced embryos cryopreserved in CJ1 medium supplemented with Albumax I resulted in higher pregnancy rates at Day 35 (31.9% versus 22.9%) and Day 60 (24.1% versus 14.3%) of gestation, and calving rates (22.6% versus 10.0%; P<0.05) than similar embryos frozen in the standard medium. However, in vivo produced embryos cryopreserved in Albumax I in CJ1 resulted in higher pregnancy rates at Day 35 (50.7%; P<0.05) and Day 60 (45.1%; P<0.05) of gestation, and calving rate (43.7%; P<0.05). It was concluded that modification of the freezing medium by addition of lipid-rich BSA and replacing sodium chloride with choline chloride improves the post-thaw survival of in vitro produced embryos, and their viability post-transfer.     

A complete library of amino acid alterations at R306 in Streptomyces clavuligerus deacetoxycephalosporin C synthase demonstrates its structural role in the ring-expansion activity

In a previous study, the conserved arginine residue at position 306 of Streptomyces clavuligerus deacetoxycephalsoporin C synthase (scDAOCS), when mutated to leucine, resulted in 191% increase in converting ampicillin to its expanded cephalosporin moiety compared with that of the wild-type enzyme. However, the role of this residue in eliciting the improved enzymatic activity is not well understood. In this study, probing the molecular basis of amino acid substitutions at position 306 has underscored its importance for engineering various improvements in the ring expansion activity. Structural modeling using SwissPdbViewer revealed that R306 is surrounded by a hydrophobic cleft formed by residues Y184, L186, W297, I298, and V303. Hence, the improved activity achieved by the R306L mutation was probably because of better hydrophobic packing in this region. To evaluate the role of amino acids at position 306 of scDAOCS and its influence on the molecular status of the enzyme at this locality, alteration to 18 other amino acids was done by site-directed mutagenesis. The effects of each substitution on the enzyme activity were determined by bioassay using penicillin substrates: ampicillin, penicillin G, phenethicillin, and carbenicillin. Results obtained showed a drastic reduction in enzyme activity when R306 was replaced with charged or polar residues, thus emphasizing the importance of hydrophobic packing around this site. The bioassay results also illustrated that apart from leucine, substitutions to nonpolar residues, isoleucine and methionine, were able to improve the ampicillin conversion activity of scDAOCS by 168 and 113% of the wild-type enzyme activity, respectively. Similar trend of effects from each mutation was also observed for penicillin G, phenethicillin, and carbenicillin conversions. The enhanced enzyme activities were supported by spectrophotometric assay indicating that all these mutants have lower K(m) values (R306L: 1.09 mM; R306I: 2.64 mM; R306M: 5.68 mM) than the wild-type enzyme (8.33 mM), resulting in improvement in the enzyme's substrate binding affinity. Hence, this mutational study of amino acids situated at 306 of scDAOCS has provided a better understanding of the significance of specific amino acid residues at this position which can improve its ring-expansion activity when given a plethora of beta-lactam substrates to generate corresponding, possibly new, cephalosporins.     

Glyceraldehyde-3-phosphate, a glycolytic intermediate, plays a key role in controlling cell fate via inhibition of caspase activity

Glyceraldehyde-3-phosphate is a key intermediate in several central metabolic pathways of all organisms. Aldolase and glyceraldehyde-3-phosphate dehydrogenase are involved in the production or elimination of glyceraldehyde-3-phosphate during glycolysis or gluconeogenesis, and are differentially expressed under various physiological conditions, including cancer, hypoxia, and apoptosis. In this study, we examine the effects of glyceraldehyde-3-phosphate on cell survival and apoptosis. Overexpression of aldolase protected cells against apoptosis, and addition of glyceraldehyde-3-phosphate to cells delayed apoptosis. Additionally, delayed apoptotic phenomena were observed when glyceraldehyde-3-phosphate was added to a cell-free system, in which artificial apoptotic process was induced by adding dATP and cytochrome c. Surprisingly, glyceraldehyde-3-phosphate directly suppressed caspase-3 activity in a reversible noncompetitive mode, preventing caspase-dependent proteolysis. Based on these results, we suggest that glyceraldehyde-3-phosphate, a key molecule in several central metabolic pathways, functions as a molecule switch between cell survival and apoptosis.     

NSC-87877, inhibitor of SHP-1/2 PTPs, inhibits dual-specificity phosphatase 26 (DUSP26)

Protein phosphorylation plays critical roles in many regulatory mechanisms controlling cell activities and thus involved in various diseases. The cellular equilibrium of phosphorylation is regulated through the actions of protein kinases and phosphatases. Therefore, these regulatory proteins have emerged as promising targets for drug development. In this study, we screened protein tyrosine phosphatases (PTPs) by in vitro phosphatase assays to identify PTPs that are inhibited by 8-hydroxy-7-(6-sulfonaphthalen-2-yl)diazenyl-quinoline-5-sulfonic acid (NSC-87877), a potent inhibitor of SHP-1 and SHP-2 PTPs. Phosphatase activity of dual-specificity protein phosphatase 26 (DUSP26) was decreased by the inhibitor in a dose-dependent manner. Kinetic studies with NSC-87877 and DUSP26 revealed a competitive inhibition. NSC-87877 effectively inhibited DUSP26-mediated dephosphorylation of p38, a member of mitogen-activated protein kinase (MAPK) family. Since DUSP26 is involved in survival of anaplastic thyroid cancer (ATC) cells, NSC-87877 could be a therapeutic reagent for treating ATC.     

Trabeculae carneae as models of the ventricular walls: implications for the delivery of oxygen

Trabeculae carneae are the smallest naturally arising collections of linearly arranged myocytes in the heart. They are the preparation of choice for studies of function of intact myocardium in vitro. In vivo, trabeculae are unique in receiving oxygen from two independent sources: the coronary circulation and the surrounding ventricular blood. Because oxygen partial pressure (PO₂) in the coronary arterioles is identical in specimens from both ventricles, whereas that of ventricular blood is 2.5-fold higher in the left ventricle than in the right ventricle, trabeculae represent a “natural laboratory” in which to examine the influence of “extravascular” PO₂ on the extent of capillarization of myocardial tissue. We exploit this advantage to test four hypotheses. (1) In trabeculae from either ventricle, a peripheral annulus of cells is devoid of capillaries. (2) Hence, sufficiently small trabeculae from either ventricle are totally devoid of capillaries. (3) The capillary-to-myocyte ratios in specimens from either ventricle are identical to those of their respective walls. (4) Capillary-to-myocyte ratios are comparable in specimens from either ventricle, reflecting equivalent energy demands in vivo, driven by identical contractile frequencies and comparable wall stresses. We applied confocal fluorescent imaging to trabeculae in cross section, subsequently using semi-automated segmentation techniques to distinguish capillaries from myocytes. We quantified the capillary-to-myocyte ratios of trabeculae from both ventricles and compared them to those determined for the ventricular free walls and septum. Quantitative interpretation was furthered by mathematical modeling, using both the classical solution to the diffusion equation for elliptical cross sections, and a novel approach applicable to cross sections of arbitrary shape containing arbitrary disposition of capillaries and non-respiring collagen cords.     

Generation of red fluorescent protein transgenic dogs

Dogs (Canis familiaris) share many common genetic diseases with humans and development of disease models using a transgenic approach has long been awaited. However, due to the technical difficulty in obtaining fertilizable eggs and the unavailability of embryonic stem cells, no transgenic dog has been generated. Canine fetal fibroblasts were stably transfected with a red fluorescent protein (RFP) gene‐expressing construct using retrovirus gene delivery method. Somatic cell nuclear transfer was then employed to replace the nucleus of an oocyte with the nucleus of the RFP‐fibroblasts. Using this approach, we produced the first generation of transgenic dogs with four female and two male expressing RFP. genesis 47:314–322, 2009. © 2009 Wiley‐Liss, Inc.     

Shank2 associates with and regulates Na+/H+ exchanger 3

Na+/H+ exchanger 3 (NHE3) plays a pivotal role in transepithelial Na+ and HCO3(-) absorption across a wide range of epithelia in the digestive and renal-genitourinary systems. Accumulating evidence suggests that PDZ-based adaptor proteins play an important role in regulating the trafficking and activity of NHE3. A search for NHE3-binding modular proteins using yeast two-hybrid assays led us to the PDZ-based adaptor Shank2. The interaction between Shank2 and NHE3 was further confirmed by immunoprecipitation and surface plasmon resonance studies. When expressed in PS120/NHE3 cells, Shank2 increased the membrane expression and basal activity of NHE3 and attenuated the cAMP-dependent inhibition of NHE3 activity. Furthermore, knock-down of native Shank2 expression in Caco-2 epithelial cells by RNA interference decreased NHE3 protein expression as well as activity but amplified the inhibitory effect of cAMP on NHE3. These results indicate that Shank2 is a novel NHE3 interacting protein that is involved in the fine regulation of transepithelial salt and water transport through affecting NHE3 expression and activity.