Effect of protein structure on laccase-catalyzed protein oligomerization

Laccase-catalyzed oligomerization of proteins was studied using Trametes hirsuta laccase (ThL) and coactosin as a model system. The reaction mechanism was elucidated using free amino acids and the tripeptide Gly-Leu-Tyr as substrates. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and high-performance liquid chromatography (HPLC) as well as oxygen consumption measurements and SDS-PAGE were used to study the reactions. Of the 15 selected amino acids, ThL was found to oxidize tryptophan (Trp), tyrosine (Tyr), and cysteine (Cys), of which the reactions with Tyr and Cys have been described earlier. ThL was able to link four full-length coactosins, whereas coactosin that was truncated from its C-terminus remained unpolymerized. Of the four tyrosine residues present in coactosin, only the tyrosine in the C-terminus was found to be reactive. Polymerization between tyrosine side-chains was unambiguously shown using different oligomers of Gly-Leu-Tyr as parent ions in MALDI-TOF/TOF MS fragment ion analyses.     

Clenbuterol Affects the Expression of Messenger RNA for Interleukin 10 in Peripheral Leukocytes from Horses Challenged Intrabronchially with Lipopolysaccharides

On four occasions, four horses with heaves and four horses with small airway inflammatory diseases inhaled 0.9% saline based aerosol mixtures with or without lipopolysaccharides (LPS). Prior to the first saline and LPS inhalation, horses were untreated, while three and a half days prior to the third and forth inhalation horses had received 0.8 μ g/kg clenbuterol intravenously twice daily. The messenger RNA (mRNA) expression of tumour necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-4, IL-6, IL-8, IL-10 and interferon- γ (IFN- γ) was investigated by RT-PCR, all of which were expressed in the white blood cells of samples collected. Inhalation of LPS only changed the cytokine expression profile of IL-10, IL-4 and TNF-α mRNA which were higher after challenge with LPS. However in those horses that were treated with clenbuterol the LPS-induced IL-10 mRNA expression was shown to be suppressed. Further changes in IL-4 and TNF-α were not significant. Thus the results of this study indicated that clenbuterol can modulate the expression of IL-10 mRNA in peripheral white blood cells in those horses with small airway diseases that have been exposed to LPS. van den Hoven, R., Duvigneau, J.C., Hartl, R.T. and Gemeiner, M., 2006. Clenbuterol affects the expression of messenger RNA for interleukin 10 in peripheral leukocytes from horses challenged intrabronchially with lipopolysaccharides. Veterinary Research Communications, 30(8), 921–928     

Antimicrobial susceptibility patterns of respiratory tract pathogens from sheep and goats

OBJECTIVE: To determine the antimicrobial susceptibility of common respiratory tract pathogens from sheep and goats. DESIGN: Cross-sectional study. SAMPLE POPULATION: 41 respiratory tract isolates from sheep and 36 isolates from goats. PROCEDURES: Disk diffusion assay was used to determine antimicrobial susceptibility of isolates to amoxicillin-clavulanic acid, ceftiofur, ciprofloxacin, florfenicol, and tetracycline. Minimum inhibitory concentrations of florfenicol for these isolates were determined by use of the microbroth dilution technique. RESULTS: The most common isolates were Pasteurella multocida (n = 28) and Mannheimia haemolytica (39). All isolates were susceptible to amoxicillin-clavulanic acid, ceftiofur, ciprofloxacin, and florfenicol. Five percent (4/77) of isolates were resistant to tetracycline. CONCLUSIONS AND CLINICAL RELEVANCE: Susceptibility of respiratory tract pathogens isolated from sheep and goats to commonly used antimicrobial drugs in this study was high. Treatment of these species for bacterial respiratory tract disease is likely not complicated by antimicrobial resistance.     

Stallion spermatozoa selected by single layer centrifugation are capable of fertilization after storage for up to 96?h at 6°C prior to artificial insemination

Background

One of the challenges faced by equine breeders is ensuring delivery of good quality semen doses for artificial insemination when the mare is due to ovulate. Single Layer Centrifugation (SLC) has been shown to select morphologically normal spermatozoa with intact chromatin and good progressive motility from the rest of the ejaculate, and to prolong the life of these selected spermatozoa in vitro. The objective of the present study was a proof of concept, to determine whether fertilizing ability was retained in SLC-selected spermatozoa during prolonged storage.

Findings

Sixteen mares were inseminated with SLC-selected sperm doses that had been cooled and stored at 6°C for 48 h, 72 h or 96 h. Embryos were identified in 11 mares by ultrasound examination 16–18 days after presumed ovulation.

Conclusion

SLC-selected stallion spermatozoa stored for up to 96 h are capable of fertilization.     

Primary ureteral giant cell sarcoma in a Pomeranian

An 8-year-old male neutered Pomeranian dog was presented to the Veterinary Teaching Hospital at Oregon State University for surgical treatment of hydronephrosis of the left kidney and a left cranial abdominal mass. A primary ureteral mass was found during exploratory surgery, and the mass was resected and ureteral anastomosis was performed. Cytologic evaluation of the mass revealed 3 distinct cell populations, including a large number of multinucleated giant cells, a moderate number of thin spindle-shaped cells, and cohesive clusters of transitional epithelial cells. The cytologic diagnosis was giant cell sarcoma. The diagnosis was confirmed by histologic examination, and immunohistochemical staining was performed. The spindle-shaped cells and multinucleated giant cells were both immunoreactive for vimentin and spindle-shaped cells for S-100. Tumor cells did not express wide-spectrum cytokeratin, broad-spectrum muscle actin, smooth muscle actin, sarcomeric actin, desmin, BLA36, Mac 387, synaptophysin, neuron-specific enolase, glial fibrillary acid protein, or von Willebrand factor. These findings are most consistent with an anaplastic sarcoma with giant cells. This is the first case report of a primary ureteral giant cell sarcoma in a dog.     

Increased anthocyanin content in purple pericarp × blue aleurone wheat crosses

Due to their antioxidant activity, anthocyanins are of increasing interest for nutritionists, food scientists and plant breeders. Anthocyanins in wheat grains are expressed in either the pericarp or aleurone layer. Previous studies revealed that different anthocyanins are present in wheat varieties carrying genes for either the purple pericarp or the blue aleurone trait. Progeny from crosses between red‐, purple‐ and blue‐grained wheat varieties were selected over several cycles for grain colour by visual scoring. Bulked F₅ grains were evaluated for their total anthocyanin content by UV‐VIS spectrophotometry and HPLC‐MS. The results demonstrate that it is possible to increase the anthocyanin content by the combination of the different genetic backgrounds for purple pericarp and blue aleurone, even though the majority of progeny were within the range of the purple‐ and blue‐grained check varieties. Visual scoring for grain colour is efficient, reliable and fast for selection in early breeding generations. Advanced breeding lines with high anthocyanin content can be identified by simple extraction methods and spectrophotometric measurements.     

A Conus regularis Conotoxin with a Novel Eight-Cysteine Framework Inhibits CaV2.2 Channels and Displays an Anti-Nociceptive Activity

A novel peptide, RsXXIVA, was isolated from the venom duct of Conus regularis, a worm-hunting species collected in the Sea of Cortez, México. Its primary structure was determined by mass spectrometry and confirmed by automated Edman degradation. This conotoxin contains 40 amino acids and exhibits a novel arrangement of eight cysteine residues (C-C-C-C-CC-CC). Surprisingly, two loops of the novel peptide are highly identical to the amino acids sequence of ω-MVIIA. The total length and disulfide pairing of both peptides are quite different, although the two most important residues for the described function of ω-MVIIA (Lys2 and Tyr13) are also present in the peptide reported here. Electrophysiological analysis using superior cervical ganglion (SCG) neurons indicates that RsXXIVA inhibits Ca_V2.2 channel current in a dose-dependent manner with an EC₅₀ of 2.8 μM, whose effect is partially reversed after washing. Furthermore, RsXXIVA was tested in hot-plate assays to measure the potential anti-nociceptive effect to an acute thermal stimulus, showing an analgesic effect in acute thermal pain at 30 and 45 min post-injection. Also, the toxin shows an anti-nociceptive effect in a formalin chronic pain test. However, the low affinity for Ca_V2.2 suggests that the primary target of the peptide could be different from that of ω-MVIIA.     

Development and validation of a short questionnaire to assess sodium intake

OBJECTIVES: To develop and validate a short food-frequency questionnaire to assess habitual dietary salt intake in South Africans and to allow classification of individuals according to intakes above or below the maximum recommended intake of 6 g salt day-1. DESIGN: Cross-sectional validation study in 324 conveniently sampled men and women. METHODS: Repeated 24-hour urinary Na values and 24-hour dietary recalls were obtained on three occasions. Food items consumed by >5% of the sample and which contributed > or =50 mg Na serving-1 were included in the questionnaire in 42 categories. A scoring system was devised, based on Na content of one index food per category and frequency of consumption. RESULTS: Positive correlations were found between Na content of 35 of the 42 food categories in the questionnaire and total Na intake, calculated from 24-hour recall data. Total Na content of the questionnaire was associated with Na estimations from 24-hour recall data (r = 0.750; P < 0.0001; n = 328) and urinary Na (r = 0.152; P = 0.0105; n = 284). Urinary Na was higher for subjects in tertile 3 than tertile 1 of questionnaire Na content (P < 0.05). Questionnaire Na content of <2400 and > or =2400 mg day-1 equated to a reference cut-off score of 48 and corresponded to mean (standard deviation) urinary Na values of 145 (68) and 176 (99) mmol day-1, respectively (P < 0.05). Sensitivity and specificity against urinary Na > or =100 and <100 mmol day-1 was 12.4% and 93.9%, respectively. CONCLUSION: A 42-item food-frequency questionnaire has been shown to have content-, construct- and criterion-related validity, as well as internal consistency, with regard to categorising individuals according to their habitual salt intake; however, the devised scoring system needs to show improved sensitivity.     

National fortification of staple foods can make a significant contribution to micronutrient intake of South African adults

BACKGROUND: A national survey found that micronutrient deficiencies are prevalent in South African children, particularly calcium, iron, zinc, riboflavin, niacin, vitamin B6, folate, vitamin A, E and C. Mandatory fortification of maize meal and wheat flour were introduced in 2003 to combat some of the deficiencies found in children. To date however, there has not been a national survey on dietary intake in adults. OBJECTIVES: The main objectives of this study were to evaluate the micronutrient intake of the diet consumed by the average adult South African by means of secondary data analyses and secondly to evaluate the effects of fortification on selected nutrient intakes. STUDY DESIGN: Secondary data analysis was carried out with numerous dietary surveys on adults to create a database that included sampling (and weighting) according to ethnic/urban-rural residence in line with the population census, of which 79% were black Africans and the majority resided in rural areas. The effect of fortification was evaluated by substituting fortified foods in the diet for the unfortified products. SUBJECTS: The combined database used in this study comprised 3229 adults. RESULTS: Mean calcium, iron, folate and vitamin B6 intakes were very low particularly in women. Mean intakes of most micronutrients were lower in rural areas. Fortification of maize meal and wheat flour (bread) raised mean levels of thiamine, riboflavin, niacin, vitamin B6 and folate above the recommended nutrient intakes (RNIs). In women, despite fortification, mean iron intakes remained below the RNIs, as did calcium since it was not in the fortification mix. CONCLUSION: The average dietary intake of adults was of poor nutrient density, particularly in rural areas. Fortification of maize meal and wheat flour (bread) considerably improved mean vitamin B6, thiamine, riboflavin, niacin, folate and iron intakes as well as the overall mean adequacy ratio of the diet.     

Extraction of green labeled pectins and pectic oligosaccharides from plant byproducts

Green labeled pectins were extracted by an environmentally friendly way using proteases and cellulases being able to act on proteins and cellulose present in cell walls. Pectins were isolated from different plant byproducts, i.e., chicory roots, citrus peel, cauliflower florets and leaves, endive, and sugar beet pulps. Enzymatic extraction was performed at 50 degrees C for 4 h, in order to fulfill the conditions required for microbiological safety of extracted products. High methoxy (HM) pectins of high molar mass were extracted with three different enzyme mixtures. These pectins were subsequently demethylated with two pectin methyl esterases (PMEs), either the fungal PME from Aspergillus aculeatus or the orange PME. It was further demonstrated that high molar mass low methoxy (LM) pectins could also be extracted directly from cell walls by adding the fungal PME to the mixture of protease and cellulase. Moreover, health benefit pectic oligosaccharides, the so-called modified hairy regions, were obtained after enzymatic treatment of the residue recovered after pectin extraction. The enzymatic method demonstrates that it is possible to convert vegetable byproducts into high-added value compounds, such as pectins and pectic oligosaccharides, and thus considerably reduce the amount of these residues generated by food industries.