Generation and purification of recombinant bovine pregnancy associated glycoprotein

Bovine pregnancy-associated glycoprotein-1 (bPAG-1) is predicted to play an essential role during pregnancy and is labelled as a potential biochemical marker of pregnancy in ungulates. We have compared the generation of the glycosylated form of recombinant bPAG-1 (rbPAG-1) by human embryonic kidney 293 (HEK 293) and Chinese hamster ovary (CHO) cells in attached cultures and evaluated the adaptation of the rbPAG-1 transfected cell line to suspension culture. The PAG cDNA was cloned from placental RNA obtained from a slaughtered cow on day 55 of pregnancy. The PAG-pRcRSV expression vector was transfected into HEK 293 and CHO cells. Western blot analysis showed that clonal HEK 293 cells expressed rbPAG-1 better than CHO cells in attached cultures. Transfected HEK 293 cells were adapted to suspension culture in spinner flasks and the rbPAG-1 purified to homogeneity using ion-exchange, pepstatin-sepharose affinity chromatographies and preparative SDS-PAGE. The expression of rbPAG-1 was immunocharacterised using a polyclonal antibody. Our findings indicated that 293 cells are suitable for production of glycosylated form of rbPAG-1 and that the availability of the recombinant glycoprotein will aid in further studies to elucidate the function and structure of the protein.     

Phospholipid-based microemulsions suitable for use in foods

The preparation of nonaqueous microemulsions using food-acceptable components is reported. The effect of oil on the formation of microemulsions stabilized by lecithin (Epikuron 200) and containing propylene glycol as immiscible solvent was investigated. When the triglycerides were used as oil, three types of phase behavior were noted, namely, a two-phase cloudy region (occurring at low lecithin concentrations), a liquid crystalline (LC) phase (occurring at high surfactant and low oil concentrations), and a clear monophasic microemulsion region. The extent of this clear one-phase region was found to be dependent upon the molecular volume of the oil being solubilized. Large molecular volume oils, such as soybean and sunflower oils, produced a small microemulsion region, whereas the smallest molecular volume triglyceride, tributyrin, produced a large, clear monophasic region. Use of the ethyl ester, ethyl oleate, as oil produced a clear, monophasic region of a size comparable to that seen with tributyrin. Substitution of some of the propylene glycol with water greatly reduced the extent of the clear one-phase region and increased the extent of the liquid crystalline region. In contrast, ethanol enhanced the clear, monophasic region by decreasing the LC phase. Replacement of some of the lecithin with the micelle-forming nonionic surfactant Tween 80 to produce mixed lecithin/Tween 80 mixtures of weight ratios (Km) 1:2 and 1:3 did not significantly alter the phase behavior, although there was a marginal increase in the area of the two-phase, cloudy region of the phase diagram. The use of the lower phosphatidylcholine content lecithin, Epikuron 170, in place of Epikuron 200 resulted in a reduction in the LC region for all of the systems investigated. In conclusion, these studies show that it is possible to prepare one-phase, clear lecithin-based microemulsions over a wide range of compositions using components that are food-acceptable.     

Adenosine-5'-triphosphate release by Mannheimia haemolytica, lipopolysaccharide, and interleukin-1 stimulated bovine pulmonary epithelial cells

Mannheimia haemolytica, one of the agents associated with bovine respiratory disease complex, can cause severe lung pathology including the leakage of vascular products into the airways and alveoli. Previous work by this laboratory has demonstrated that bovine lung endothelial and epithelial cells undergo dramatic permeability increases when exposed to adenosine-5'-triphosphate (ATP). Therefore, we wanted to determine if ATP levels were elevated in bronchoalveolar lavage (BAL) samples from calves experimentally infected with M. haemolytica. In addition, cultured bovine pulmonary epithelial (BPE) cells were stimulated with heat-killed and live M. haemolytica bacteria, lipopolysaccharide (LPS), lipoteichoic acid (LTA), interleukin-1 (IL-1), and zymosan activated plasma (ZAP) to determine whether they might release extracellular ATP during in vitro infection. Calves experimentally exposed to M. haemolytica had an approximately 2-fold higher level of ATP in their BAL samples compared to control. BPE cells exposed to increasing numbers of heat-killed or live M. haemolytica had significantly increased levels of ATP release as compared to time-matched controls. Finally, BPE cells treated with several concentrations of LPS and IL-1 had increases in ATP release as compared to time-matched controls. This increase appeared to be a result of active ATP secretion by the cells, as cell viability was similar between treated and non-treated cells. Neither ZAP nor LTA induced any ATP release by the cells. In conclusion, ATP levels are elevated in lung secretions from calves infected with M. haemolytica. In addition, lung epithelial cells can actively release ATP when exposed to heat-killed or live M. haemolytica, LPS or IL-1.     

Factors associated with failure in breeding soundness examination of Western USA rams

Breeding-soundness examination (BSE) and eradication of Brucella ovis infection in rams are critical components of flock-health programs. The aims of this retrospective, cross-sectional study were to describe the results of BSE in a large sample of rams in the Western USA and to determine the association between BSE outcome and the semen collection method (penis manually extended vs. retained in the preputial cavity), ram body-condition score (BCS), the presence of ulcerative posthitis, and the size of the flock of origin. We evaluated the first BSE in a given year for rams from Colorado, Wyoming, and Utah, USA, from 2000 through 2007. Breeding-soundness examination consisted of physical examination, scrotal circumference and BCS measurement, semen collection by electroejaculation, and microscopic examination of semen motility, morphology, and leukocyte concentration. We assigned a reason for failure to each failed BSE and used multivariable logistic and Poisson regressions to measure associations between ram and flock variables and the risk or reason for failure on BSE. A non-random, owner-selected subset of rams was tested for antibodies to B. ovis by serum indirect ELISA (iELISA). The Rogan-Gladen corrected B. ovis seroprevalence was measured. Of the 14,667 BSEs performed on 11,804 rams, 29.0% were classified as "failed;" the most common reason for failure was substandard semen parameters (43.8%). Breeding-soundness examinations were more likely to have been categorized as failure for inflammatory causes when performed on rams from medium-sized flocks (OR 1.6; 95% CI 1.1, 2.3) and large flocks (OR 1.4; 95% CI 1.0, 1.9) (P=0.02), suggesting that larger flocks are at higher risk of contagious diseases. The adjusted seroprevalence of B. ovis antibodies among tested rams in this study was 10.0%. Of 233 rams seropositive to B. ovis, 125 (53.6%) were subclinical, a finding that supports the importance of this test in ram BSE. We found that emaciation in rams was associated with an increased risk of BSE failure from substandard semen parameters (P<0.001), but ulcerative posthitis and the semen collection method were not (P=0.09 and 0.34, respectively). However, collection of semen with the penis retained in the preputial cavity resulted in greater odds of leukospermia relative to semen collection with the penis extended (OR 4.1; 95% CI 2.9, 5.9; P<0.001), presumably from contamination of the semen sample with preputial leukocytes. For ram BSE, therefore, semen collection with the penis manually extended from the sheath is recommended to limit leukocyte contamination of the sample.     

Bubbles in chapatti doughs

Traditionally, chapattis are flatbreads made from atta (wholemeal Indian wheat flour). Non-atta chapattis have not been popular due to substandard product quality. To investigate what makes atta special for making chapattis, products were made using atta, Australian wholemeal wheat flour, gluten-free lupin flour, and a blend of lupin and wheat flours. Doughs were characterised by measuring strain-hardening and elastic recovery in compression and also bubble structures via 3-D X-ray micro-tomography. A method was developed to identify and separate bran, which appears as bubbles, in scans of doughs.     

Biochemical and spectroscopic characterization of morning glory peroxidase from an invasive and hallucinogenic plant weed Ipomoea carnea

A novel heme peroxidase MGP from the latex of Ipomoea carnea subsp. fistulosa (morning glory) belonging to the Convolvulaceae family was purified to homogeneity using ammonium sulfate precipitation, anion exchange, hydrophobic interaction, and gel filtration chromatography. The enzyme is glycosylated and has a molecular mass of 42.06 kDa (MALDI-TOF) and an isoelectric point of pH 4.3. The enzyme has high yield, broad substrate specificity, and a high stability toward pH, temperature, chaotrophs, and organic solvents. The extinction coefficient (epsilon 280 (1%)) of the enzyme was estimated as 20.56 and it consists of 13 tryptophan, 9 tyrosine, and 8 cysteine residues forming 4 disulfide bridges. There is significant effect of inhibitors targeting S-S bridges (mercaptoethanol, l-cysteine, glutathione), as well as of inhibitors targeting heme (sodium azide and hydroxylamine) on peroxidase activity, whereas inhibition was not observed with ethylmaleinimide due to the absence of reduced cysteine in the enzyme. Polyclonal antibodies against the enzyme have been raised in rabbit, and immunodiffusion suggests that the antigenic determinants of MGP are unique. The N-terminal sequence of MGP (D-E-A-C-I-F-S-A-V-K-E-V-V-D-A) exhibited considerable similarity to the sequence of other known plant peroxidases. Spectroscopic studies (absorbance, fluorescence, and circular dichroism) reveal that MGP has secondary structural features with alpha/beta type with approximately 20% alpha-helicity.     

Mango explant browning: Effect of ontogenic age,mycorrhization and pre-treatments

The in vitro performance of mango shoot culture is delimited by several factors, of which explant necrosis and media browning are considered as major constraints for successful exploitation of such an important commercial crop. Our results showed that source of explants had a significant effect on the performance of in vitro cultures of mango. The variations in survival of explants collected from glasshouse grown seedlings and field-grown stock plants indicated towards differences between their physiological/ontogenic age. Though, chronologically, both are young, the glasshouse grown shoots are ontogenetically younger and; therefore, responded better over field-grown shoots. Improved performance of explants harvested from mycorrhizal plants suggests integration of mycorrhizal biotechnology with tissue culture biotechnology in order to achieve better results as mycorrhiza being a well-known stress alleviator may help explants mitigate in vitro stresses. Furthermore, shoot tip explants of field-grown ‘Amrapali’ mango was assayed for their browning propensity at pre- and post-culture stages. Status of different bio-chemicals such as in vivo phenol, in vitro phenol exudation, phenylalanine ammonia lyase, peroxidase and polyphenol oxidase as affected by various pre-treatments were estimated to establish the relationship of these bio-chemicals with necrosis of mango shoot cultures. The different pre-treatments comprised etiolation of newly emerged shoots (5–7 days old) of stock plants using black polythene for 7 days, etiolation treatment + agitation of explants in antioxidant solution (antioxidants: ascorbic acid at 100 mg l⁻¹ + citric acid at 50 mg l⁻¹), etiolation treatment + agitation of explants in polyvinylpyrrolidone solution (PVP) at 0.2% and non-etiolated control. Of these, explants treated with PVP proved to be superior to other treatments with regards to explant necrosis percentage as such explants exhibited least activities of phenols and oxidative enzymes. Correlation studies indicated existence of high positive correlation between explant necrosis and these bio-chemicals.