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.     

Structural insight into the transmembrane segments 3 and 4 of the hERG potassium channel

The hERG (human ether‐a‐go‐go related gene) potassium channel is a voltage‐gated potassium channel containing an N‐terminal domain, a voltage‐sensor domain, a pore domain and a C‐terminal domain. The transmembrane segment 4 (S4) is important for sensing changes of membrane potentials through positively charge residues. A construct containing partial S2–S3 linker, S3, S4 and the S4–S5 linker of the hERG channel was purified into detergent micelles. This construct exhibits good quality NMR spectrum when it was purified in lyso‐myristoyl phosphatidylglycerol (LMPG) micelles. Structural study showed that S3 contains two short helices with a negatively charged surface. The S4 and S4–S5 linker adopt helical structures. The six positively charged residues in S4 localize at different sides, suggesting that they may have different functions in channel gating. Relaxation studies indicated that S3 is more flexible than S4. The boundaries of S3–S4 and S4–S4–S5 linker were identified. Our results provided structural information of the S3 and S4, which will be helpful to understand their roles in channel gating. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Salvaging Pyrococcus furiosus Protein Targets at SECSG

Proteins derived from the coding regions of Pyrococcus furiosus are targets for three-dimensional X-ray and NMR structure determination by the Southeast Collaboratory for Structural Genomics (SECSG). Of the 2200 open reading frames (ORFs) in this organism, 220 protein targets were cloned and expressed in a high-throughput (HT) recombinant system for crystallographic studies. However, only 96 of the expressed proteins could be crystallized and, of these, only 15 have led to structures. To address this issue, SECSG has recently developed a two-tier approach to protein production and crystallization. In this approach, tier-1 efforts are focused on producing protein for new Pfu(italics?) targets using a high-throughput approach. Tier-2 protein production efforts support tier-1 activities by (1) producing additional protein for further crystallization trials, (2) producing modified protein (further purification, methylation, tag removal, selenium labeling, etc) as required and (3) serving as a salvaging pathway for failed tier-1 proteins. In a recent study using this two-tiered approach, nine structures were determined from a set of 50 Pfu proteins, which failed to produce crystals suitable for X-ray diffraction analysis. These results validate this approach and suggest that it has application to other HT crystal structure determination applications.     

2,3-Diaminopyridine as a platform for designing structurally unique nonpeptide bradykinin B1 receptor antagonists

A novel class of 2,3-diaminopyridine bradykinin B1 receptor antagonists is disclosed. Structure-activity relationship studies (SARs) that led to compounds with significantly improved potency and pharmacokinetic properties relative to the lead compound are described.     

Acute thoracic aortic dissection presenting as sore throat: report of a case

Acute dissection of the aorta can be one of the most dramatic of cardiovascular emergencies. Its symptoms can occur abruptly and progress rapidly. Prompt recognition and appropriate intervention is crucial. However, not all aortic dissections present with classic symptoms of abrupt chest, back, or abdominal pain, and the diagnosis may be missed. Aortic dissection presenting as a sore throat is quite unusual. A 53-year-old man presented with sore throat as the early symptom of an acute thoracic aortic dissection. Unfortunately, the diagnosis was delayed, and the patient died. Given the high morbidity and mortality after delayed recognition or misdiagnosis, aortic dissection should be considered in the differential diagnosis of a patient presenting with sore throat and normal findings of neck and throat, even when there is no classic symptoms.     

Early development of reconstructed embryos after somatic cell nuclear transfer in a non-human primate

To improve efficiency and assess variation in nuclear transfer techniques in non-human primates, we investigated the following factors: type of donor cell, interval between enucleation and cell injection, activation after electrical pulsing and cytokinesis inhibitors. An average of 16.4 oocytes were recovered from 91 retrievals; however, 15 (14%) additional retrieval attempts yielded no oocytes due to a failure of follicular stimulation. Oocyte maturation rates at 36, 38 and 40 h post-hCG were 46.2, 52.6 and 61.2%, respectively. The MII spindle could be seen clearly using polarized microscopy in 89.1% (614/689) of oocytes. Nuclei were seen in 42% of the NT couplets, 53% of those cleaved to the 2-cell stage and 63% of the 2-cell embryos developed to the 8-cell stage by Day 3. There was no difference in the occurrence of nuclear formation between couplets created using fibroblasts or cumulus cells, although embryos were more reliably produced with fibroblasts. The interval (2, 3 and 4 h) between enucleation and cell injection did not affect NT efficiency. Ethanol treatment after electrical pulses yielded more 2-cell NT embryos than did treatment with ionomycin, but the frequency of nuclear formation and development to the 8-cell stage was not different. Treatment of couplets with cycloheximide and cytochalasin B for 5 h after activation had no impact on NT efficiency.     

KNOCKDOWN OF THE IONOTROPIC γ‐AMINOBUTYRIC ACID RECEPTOR (GABAR) RDL GENE DECREASES FIPRONIL SUSCEPTIBILITY OF THE SMALL BROWN PLANTHOPPER,Laodelphax striatellus (HEMIPTERA: DELPHACIDAE)

Insect γ‐aminobutyric acid receptors (GABARs) are important molecular targets of cyclodiene and phenylpyrazole insecticides. Previously GABARs encoding rdl (resistant to dieldrin) genes responsible for dieldrin and fipronil resistance were identified in various economically important insect pests. In this study, we cloned the open reading frame cDNA sequence of rdl gene from fipronil‐susceptible and fipronil‐resistant strains of Laodelphax striatellus (Lsrdl). Sequence analysis confirmed the presence of a previously identified resistance‐conferring mutation. Different alternative splicing variants of Lsrdl were noted. Injection of dsLsrdl reduced the mRNA abundance of Lsrdl by 27–82%, and greatly decreased fipronil‐induced mortality of individuals from both susceptible and resistant strains. These data indicate that Lsrdl encodes a functional RDL subunit that mediates susceptibility to fipronil. Additionally, temporal and spatial expression analysis showed that Lsrdl was expressed at higher levels in eggs, fifth‐instar nymphs, and female adults than in third‐instar and fourth‐instar nymphs. Lsrdl was predominantly expressed in the heads of 2‐day‐old female adults. All these results provide useful background knowledge for better understanding of fipronil resistance related ionotropic GABA receptor rdl gene expressed variants and potential functional differences in insects.     

生物法生产2,3-丁二醇研究进展

2,3-丁二醇是一种重要的化工原料,可广泛应用于多个领域。二战期间由于合成橡胶需要大量1,3-丁二烯,2,3-丁二醇生产空前发展。近年来,由于聚对苯二甲酸丁烯树脂、γ-丁内酯,Spandex弹性纤维及其前体的需求增长,2,3-丁二醇的需求和产量也稳步增长。多年来,生物法生产2,3-丁二醇虽然得到了广泛的研究,但一直没有实现工业化。本文从产生2,3-丁二醇的菌种及2,3-丁二醇的生理意义、代谢途径、旋光异构体的形成机理、影响发酵的因素与产物的提纯等方面对生物法生产2,3-丁二醇进行了综述并提出了生物法生产2,3-丁二醇要解决的几个问题。     

Rubisco activase requires residues in the large subunit N terminus to remodel inhibited plant Rubisco

The photosynthetic CO₂ fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) forms dead-end inhibited complexes while binding multiple sugar phosphates, including its substrate ribulose 1,5-bisphosphate. Rubisco can be rescued from this inhibited form by molecular chaperones belonging to the ATPases associated with diverse cellular activities (AAA+ proteins) termed Rubisco activases (Rcas). The mechanism of green-type Rca found in higher plants has proved elusive, in part because until recently higher-plant Rubiscos could not be expressed recombinantly. Identifying the interaction sites between Rubisco and Rca is critical to formulate mechanistic hypotheses. Toward that end here we purify and characterize a suite of 33 Arabidopsis Rubisco mutants for their ability to be activated by Rca. Mutation of 17 surface-exposed large subunit residues did not yield variants that were perturbed in their interaction with Rca. In contrast, we find that Rca activity is highly sensitive to truncations and mutations in the conserved N terminus of the Rubisco large subunit. Large subunits lacking residues 1–4 are functional Rubiscos but cannot be activated. Both T5A and T7A substitutions result in functional carboxylases that are poorly activated by Rca, indicating the side chains of these residues form a critical interaction with the chaperone. Many other AAA+ proteins function by threading macromolecules through a central pore of a disc-shaped hexamer. Our results are consistent with a model in which Rca transiently threads the Rubisco large subunit N terminus through the axial pore of the AAA+ hexamer.