Areliable sex determination assay for bovine preimplantation embryos using the polymerase chain reaction

We have developed a polymerase chain reaction (PCR)-based method for accurate sex determination of preimplantation bovine embryos. The method utilizes three different sets of primers in the PCR. The first pair of primers recognizes the bovine-specific satellite sequence that is amplified in both females and males. In addition, two pairs of primers recognize bovine Y chromosome-specific sequences that are amplified in males only. Duplicate embryo extracts were used in the PCR; the first sample was run in the presence of bovine-specific as well as one set of the Y chromosome-specific primers; the second sample was run in the presence of the other male-specific primers. The method has been specifically designed for screening bovine embryos. Based upon examining blood cell DNA from adult males and females, the assay is extremely accurate, as no single incorrect result has occurred yet. Missing samples were easily detected by the absence of the bovine-specific signal. The method has been used for the transfer of bovine embryos on which sex determinations have been performed.     

Cytovillin, a microvillar Mr 75,000 protein. cDNA sequence, prokaryotic expression, and chromosomal localization

Cytovillin is a microvillar cytoplasmic peripheral membrane protein, with prominent expression in vivo in placental syncytiotrophoblasts and certain human tumors. Cytovillin cDNA was cloned from a human placental lambda gt11 library using affinity purified antibodies. The identity of cytovillin cDNA clones was confirmed by expression of cytovillin in Escherichia coli and using antibodies raised against the expressed fusion protein in comparison with antibodies against cytovillin purified from cultured human choriocarcinoma cells. In these cells Northern blotting analysis identified a major 3.5-kilobase cytovillin mRNA. The cDNA encodes a protein of 575 amino acids corresponding to a molecular weight of 68,084. According to secondary structure prediction, cytovillin is a hydrophilic protein with an extensive internal alpha-helical region ending in a sequence of 7 consecutive prolines. The predicted alpha-helical region showed limited homology to alpha-helical regions of cytoskeletal proteins and certain other proteins, but no extensive homologies were found in the cytovillin cDNA or the deduced amino acid sequence to other registered DNA or protein sequences. Southern blot analysis of a DNA panel of human mouse somatic cell hybrids localized the cytovillin gene to the end of the long arm of chromosome 6 (6q22-q27). Our results show that cytovillin is representative of a novel class of microvillar proteins.     

Enhanced activity of hyperthermostable Pyrococcus horikoshii endoglucanase in superbase ionic liquids

Biotechnology Letters - Ionic liquids (ILs) that dissolve biomass are harmful to the enzymes that degrade lignocellulose. Enzyme hyperthermostability promotes a tolerance to ILs. Therefore, the...     

Hyperosmolaric contrast agents in cartilage tomography may expose cartilage to overload-induced cell death

In clinical arthrographic examination, strong hypertonic contrast agents are injected directly into the joint space. This may reduce the stiffness of articular cartilage, which is further hypothesized to lead to overload-induced cell death. We investigated the cell death in articular cartilage while the tissue was compressed in situ in physiological saline solution and in full strength hypertonic X-ray contrast agent Hexabrix^TM. Samples were prepared from bovine patellae and stored in Dulbecco’s Modified Eagle’s Medium overnight. Further, impact tests with or without creep were conducted for the samples with contact stresses and creep times changing from 1 MPa to 10 MPa and from 0 min to 15 min, respectively. Finally, depth-dependent cell viability was assessed with a confocal microscope. In order to characterize changes in the biomechanical properties of cartilage as a result of the use of Hexabrix?, stress-relaxation tests were conducted for the samples immersed in Hexabrix? and phosphate buffered saline (PBS). Both dynamic and equilibrium modulus of the samples immersed in Hexabrix? were significantly (p<0.05) lower than those of the samples immersed in PBS. Cartilage samples immersed in physiological saline solution showed load-induced cell death primarily in the superficial and middle zones. However, under high 8–10 MPa contact stresses, the samples immersed in full strength Hexabrix? showed significantly (p<0.05) higher number of dead cells than the samples compressed in physiological saline, especially in the deep zone of cartilage. In conclusion, excessive loading stresses followed by tissue creep might increase the risk for chondrocyte death in articular cartilage when immersed in hypertonic X-ray contrast agent, especially in the deep zone of cartilage.     

CFD simulation of mass transfer and flow behaviour around a single particle in bioleaching process

The pathway to reach a certain target in many processes such as bioleaching, due to the complex and poorly understood hydrodynamics, reaction kinetics, and chemistry knowledge involved is not apparent. An investigation of the interactions between the parameters in bioleaching process can be applied to optimize the rate of metal extraction from sulphide minerals. Such investigations can be carried out with the aid of numerical simulations. In this study, a computational fluid dynamics (CFD) model was developed to better understand the mass transfer phenomenon and complex flow field around a single particle. The commercial software FLUENT 6.2 has been employed to solve the governing equations. Volume of fluid (VOF) method was used to predict the fluid volume fraction in a 3D geometry. The computational model has successfully captured the results observed in the experiments. Simulation results indicate that concentrations of species in a thin layer of liquid on the particle surface are much higher than their concentrations in the liquid bulk and significant gradients in the ion concentrations between the surface of the particle and the liquid bulk were observed.     

Filtration assay for arachidonic acid release

Arachidonic acid (AA) release is a central message in cell signaling. Fatty acid release is generally assessed by manual sampling of radioactivity release from cells prelabeled with a radiolabeled fatty acid. The assay is laborious, time-consuming, and susceptible to high noise. Here we present a fast and reproducible method for 96-well filter plates and cells in suspension, a method that is best suited for agonist concentration-response studies and, thus, for ligand screening. The method offers tremendous time and effort savings and enables execution of large experimental series previously unattainable for AA release studies.     

Gnawing bones as enrichment for farmed blue foxes ( Vulpes lagopus)

According to present acts and regulations, farmed foxes shall have a gnawing or other enrichment object in their cages. However, research on the welfare effects of gnawing objects has been scarce. We assessed physiology and health, that is weight development, urinary cortisol-creatinine ratio, serum cortisol level after adrenocorticotropic hormone administration, internal organ masses and incidence of gastric ulcerations as well as dental and overall oral health, in pair-housed juvenile blue foxes that were housed either with or without a possibility to interact with bones (cattle femur) during their growing season (July to December). The results show that the physiological effects of the possibility to interact with bones were either non-significant or suggested that competition for bones may jeopardize the welfare of subordinate individuals. However, the results clearly show that gnawing bones are beneficial for the dental health of farmed foxes.     

Characterization of mutant xylanases using fourier transform ion cyclotron resonance mass spectrometry: stabilizing contributions of disulfide bridges and N-terminal extensions

Structural properties and thermal stability of Trichoderma reesei endo-1,4-beta-xylanase II (TRX II) and its three recombinant mutants were characterized using electrospray ionization Fourier transform ion cyclotron resonance (ESI FT-ICR) mass spectrometry and hydrogen/deuterium (H/D) exchange reactions. TRX II has been previously stabilized by a disulfide bridge C110-C154 and other site-directed mutations (TRX II mutants DS2 and DS5). Very recently, a highly thermostable mutant was introduced by combining mutations of DS5 with an N-terminal disulfide bridge C2-C28 (mutant DB1). Accurate mass measurements of TRX II, DS2, DS5, and DB1 verified the expected DNA-encoded protein sequences (average mass error 1.3 ppm) and allowed unequivocal assignment of the disulfides without chemical reduction and subsequent alkylation of the expected cross-links. Moreover, H/D exchange reactions provided means for the detection of a major heat-induced conformational change comprising two interconverting conformers of very different H/D exchange rates as well as allowed the apparent melting temperatures (T(m)) to be determined (62.6, 65.1, 68.0, and 82.2 degrees C for TRX II, DS2, DS5, and DB1, respectively). Residual activity measurements verified that the enzymes inactivated at significantly lower temperatures than expected on the basis of the apparent T(m) values, strongly suggesting that the inactivation takes place through minor conformational change other than observed by H/D exchange. ESI FT-ICR analyses also revealed molecular heterogeneity in DS5 and DB1 due to the propeptide incorporation. Resulting unintentional N-terminal extensions were observed to further improve the stability of the DB1 mutant. The extension of six amino acid residues upstream from the protein N-terminus increased stability by approximately 5 degrees C.     

Involvement of retinoid X receptor alpha in coenzyme Q metabolism

The nuclear retinoid X receptor alpha (RXRalpha) is the heterodimer partner in several nuclear receptors, some of them regulating lipid biosynthesis. Since coenzyme Q (CoQ) levels are greatly modified in aging and a number of diseases, we have investigated the involvement of RXRalpha in the biosynthetic regulation of this lipid by using a hepatocyte-specific RXRalpha-deficient mouse strain (RXRalpha-def). In the receptor-deficient liver, the amount of CoQ decreased to half of the control, and it was demonstrated that this decrease was caused by a significantly lowered rate of biosynthesis. On the other hand, induction of CoQ was extensive in both control and RXRalpha-def liver using the peroxisomal inducer di(2-ethylhexyl)phthalate (DEHP). Since the RXRalpha deficiency was specific to liver, no change in CoQ content or biosynthesis was observed in kidney. The other mevalonate pathway lipids, cholesterol and dolichol, were unchanged in the RXRalpha-def liver. Upon treatment with DEHP, cholesterol decreased in the control but remained unchanged in the receptor-deficient mice. In control mice, cold exposure elevated CoQ levels by 60%, but this induction did not occur in the liver of RXRalpha-def mice. In contrast, PPARalpha-null mice, which lack induction upon treatment with peroxisomal inducers, respond to cold exposure and CoQ content is increased. The amount of cholesterol decreased in both control and RXRalpha-def liver upon cold treatment. The results demonstrate that RXRalpha is required for CoQ biosynthesis and for its induction upon cold treatment, but does not appear to be involved in the basic synthesis of cholesterol and dolichol. The receptor is not involved in the elevated CoQ biosynthesis during peroxisomal induction.     

Regulatory aspects of coenzyme Q metabolism

A number of factors are involved in the regulation of the amount and distribution of coenzyme Q in cells and tissues. These factors modify preferentially the biosynthetic mechanism in order to keep up an optimal tissue concentration of the lipid. The amount of substrate provided by the mevalonate pathway is able to both up- and down-regulate the velocity of synthesis. At the translation level, regulation occurs by receptor-mediated ligand binding and appears most clearly upon treatment with hormones and peroxisomal inducers. There are a number of pathophysiological conditions when these mechanisms of regulation are modified and explain the decreased coenzyme Q tissue concentrations. It is of considerable interest to establish appropriate physiological, hormonal and drug-mediated conditions in order to counteract disturbed cellular functions caused by coenzyme Q deficiency.