Mechanical comparison of materials used for extra-capsular stabilisation of the stifle joint in dogs

Objective The mechanical properties of three materials (No. 2 polypropylene, No. 5 polybutilate-coated multifilament polyester and 18, 27 and 36 kg test monofilament nylon leader material) commonly used for extra-capsular stabilisation of the stifle in dogs with cranial cruciate ligament insufficiency were determined. The ability of No. 5 polybutilate-coated multifilament polyester and 36 kg test monofilament nylon leader material, when placed as extra-capsular sutures, to mitigate cranial drawer was evaluated in hindlimbs of cadavers. Design An in vitro mechanical study. Animals Seven pairs of hindlimbs harvested from adult greyhound dogs recently euthanased for other reasons. Procedure Samples of each material, including samples of 27 kg test leader material that had been sterilised by one of three methods (ethylene oxide, one or five cycles in an autoclave), were loaded to determine tensile and stress relaxation properties. The effect of cyclic loading on a No. 5 polybutilate-coated multifilament polyester and 36 kg test leader material was also determined. Using the harvested hindlimbs, cranial drawer was measured before and after transection of the cranial cruciate ligament and on the first and twelfth cycle following extra-capsular stabilisation with either No. 5 polybu-tilate-coated multifilament suture or 36 kg test leader material. Results Leader material was found to have the most suitable mechanical characteristics for use as extracapsular stabilisation of the cranial cruciate ligament deficient stifle. Of the sterilisation methods, ethylene oxide was found to have the least detrimental effects on the handling and material characteristics of the leader material. Stifles stabilised with 36 kg test leader material had significantly less drawer than those stabilised with No. 5 polybutilate-coated multifilament polyester suture. Clinical implications Monofilament nylon leader material would appear to have suitable mechanical properties for extra-capsular stabilisation of the cranial cruciate ligament deficient stifle. If possible the material should be sterilised using ethylene oxide.     

A Chemoautotrophically Based Cave Ecosystem

Microbial mats discovered in a ground-water ecosystem in southern Romania contain chemoautotrophic bacteria that fix inorganic carbon, using hydrogen sulfide as an energy source. Analysis of stable carbon and nitrogen isotopes showed that this chemoautotrophic production is the food base for 48 species of cave-adapted terrestrial and aquatic invertebrates, 33 of which are endemic to this ecosystem. This is the only cave ecosystem known to be supported by in situ autotrophic production, and it contains the only terrestrial community known to be chemoautotrophically based.     

Counting low-copy number proteins in a single cell

We have designed a microfluidic device in which we can manipulate, lyse, label, separate, and quantify the protein contents of a single cell using single-molecule fluorescence counting. Generic labeling of proteins is achieved through fluorescent-antibody binding. The use of cylindrical optics enables high-efficiency (approximately 60%) counting of molecules in micrometer-sized channels. We used this microfluidic device to quantify beta2 adrenergic receptors expressed in insect cells (SF9). We also analyzed phycobiliprotein contents in individual cyanobacterial cells (Synechococcus sp. PCC 7942) and observed marked differences in the levels of specific complexes in cell populations that were grown under nitrogen-depleted conditions.     

Ameliorative Effects of Melatonin against Hydrogen Peroxide‐Induced Oxidative Stress on Boar Sperm Characteristics and Subsequent In Vitro Embryo Development

Melatonin, the major secretory product of the pineal gland, scavenges a variety of reactive oxygen and nitrogen species in vivo and in vitro, indicating that melatonin is a potent function as an antioxidant. The objective of this study was to investigate the effect of melatonin in the presence or absence of hydrogen peroxide (H₂O₂) on sperm characteristics (motility, viability, survival rate, membrane integrity, lipid peroxidation (LPO) and mitochondria activity) and also to examine the developmental rates to the blastocysts stage of porcine oocytes fertilized in vitro with semen treated with or without melatonin (100 nm ) in the presence or absence of H₂O₂ (250 μm ). The sperm were treated with melatonin in the presence or absence of H₂O₂ for 3, 6, 9 and 12 h at 37°C and then analysed for the sperm characteristics. The porcine embryos were produced by in vitro maturation and in vitro fertilization (IVM/IVF) using semen treated with or without melatonin (100 nm ) in the presence or absence of H₂O₂ (250 μm ) for 6 h. The semen characteristics, including motility, viability, survival rate, membrane integrity and mitochondria activity, were higher in the groups that were treated with melatonin in comparison to other groups, irrespective of incubation periods. Malondialdehyde levels in control, melatonin and melatonin + H₂O₂ groups were lower than H₂O₂ only group. A positive correlation was shown among motility, viability, survival rate and membrane integrity, but a negative correlation was observed between LPO and the other evaluation methods. The developmental rates to blastocysts of IVM/IVF porcine oocytes fertilized by semen treated with melatonin were significantly increased compared with any other groups, with the cell number of blastocysts shown to have a similar trend to the developmental rates. These results demonstrate that melatonin can improve the semen characteristics during in vitro storage and support the developmental ability of IVM/IVF embryos in pigs.     

Antioxidative Effects of Astaxanthin against Nitric Oxide‐Induced Oxidative Stress on Cell Viability and Gene Expression in Bovine Oviduct Epithelial Cell and the Developmental Competence of Bovine IVM/IVF Embryos

The aim of the present study was to elucidate the fundamental mechanism of bovine oviduct epithelial cell (BOEC) co‐culture on developmental capacity of bovine in vitro oocyte maturation/in vitro fertilization (IVM/IVF) embryos. We examined the effects of astaxanthin against nitric oxide‐induced oxidative stress on cell viability by MTT assay, lipid peroxidation (LPO) by using thiobarbituric acid (TBA) reaction for malondialdehyde (MDA) and the expression of antioxidant genes (CuZnSOD, MnSOD and Catalase) or apoptosis genes (Bcl‐2, Caspase‐3 and Bax) by RT‐PCR in BOEC. We also evaluated the developmental rates of bovine IVM/IVF embryos co‐cultured with BOEC pre‐treated with astaxanthin (500 μm ) in the presence or absence of sodium nitroprusside (SNP, 1000 μm ) for 24 h. Cell viability in BOEC treated with SNP (50–2000 μm ) lowered, while astaxanthin addition (50–500 μm ) increased it in a dose‐dependent manner. Cell viability in astaxanthin plus SNP (1000 μm ) gradually recovered according to the increase in astaxanthin additions (100–500 mm ). The LPO in astaxanthin group (50–500 μM) gradually decreased in a dose dependent manner and among SNP or astaxanthin plus SNP group, SNP alone and astaxanthin (50 μM) plus SNP shown a significant increase than other groups (p < 0.05). Expression of apoptosis or antioxidant genes was detected by RT‐PCR. Bcl‐2 and antioxidant genes were detected in astaxanthin or astaxanthin plus SNP group, and Caspase‐3 and Bax genes were only found in SNP group. When bovine IVM/IVF embryos were cultured for 6–7 days under co‐culture system such as BOEC treated with astaxanthin in the presence or absence of SNP, the developmental ability to blastocysts in 500 μm astaxanthin group was the highest of all groups. These results suggest that astaxanthin has a antioxidative effect on cell viability and LPO of BOEC, and development of bovine IVM/IVF embryos due to the induction of antioxidant genes and suppression of apoptosis genes.     

High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor

Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors constitute the largest family of eukaryotic signal transduction proteins that communicate across the membrane. We report the crystal structure of a human beta2-adrenergic receptor-T4 lysozyme fusion protein bound to the partial inverse agonist carazolol at 2.4 angstrom resolution. The structure provides a high-resolution view of a human G protein-coupled receptor bound to a diffusible ligand. Ligand-binding site accessibility is enabled by the second extracellular loop, which is held out of the binding cavity by a pair of closely spaced disulfide bridges and a short helical segment within the loop. Cholesterol, a necessary component for crystallization, mediates an intriguing parallel association of receptor molecules in the crystal lattice. Although the location of carazolol in the beta2-adrenergic receptor is very similar to that of retinal in rhodopsin, structural differences in the ligand-binding site and other regions highlight the challenges in using rhodopsin as a template model for this large receptor family.     

Myocyte adrenoceptor signaling pathways

Adrenoceptors (ARs), members of the G protein-coupled receptor superfamily, form the interface between the sympathetic nervous system and the cardiovascular system, with integral roles in the rapid regulation of myocardial function. However, in heart failure, chronic catecholamine stimulation of adrenoceptors has been linked to pathologic cardiac remodeling, including myocyte apoptosis and hypertrophy. In cardiac myocytes, activation of AR subtypes results in coupling to different G proteins and induction of specific signaling pathways, which is partly regulated by the subtype-specific distribution of receptors in plasma membrane compartments containing distinct complexes of signaling molecules. The Connections Maps of the Adrenergic and Myocyte Adrenergic Signaling Pathways bring into focus the specific signaling pathways of individual AR subtypes and their relevant functions in vivo.     

Assessment of Genetic Variability in the Coding Sequence of Melatonin Receptor Gene (MTNR1A) in Tropical Arid Sheep Breeds of India

Seasonal behaviour in sheep, which varies in tropical and temperate environmental conditions, is a matter of study, because it can provide a clue to address the problem of seasonality in sheep. Melatonin receptor is the membrane‐bound G‐coupled receptor, sensing the message of photoperiodic cues thorough melatonin. Restriction fragment length polymorphism (RFLP) studies were carried out to assess the variability of gene at G612A and C606T SNPs in MTNR1A gene, which have been studied to be markers for out‐of‐season breeding. Allelic frequency distribution corresponded to higher frequency of GG and CC genotype, in tropical arid sheep breed in comparison with temperate region sheep breed. PCR amplification of MTNR1A gene of 30 animals was performed and single nucleotide polymorphisms (SNPs) identification was carried out using Lasergene software. Seven SNPs/mutations were identified, but most of them were synonymous, except the one G706A, leading to substitution of valine by isoleucine. Polyphen‐2 analysis of G706A mutation revealed that it is a benign mutation. Two important SNPs C426T and G555A, which were identified in temperate sheep breeds, could not be traced in Magra and Marwari breeds of sheep. Thus, the Magra and Marwari breeds of tropical, arid region demonstrated the presence of both polymorphic SNPs markers G612A and C606T, associated with out‐of‐season breeding. GG and CC genotypes were having a higher prevalence in the studied population.