Protease Inhibitors from Marine Venomous Animals and Their Counterparts in Terrestrial Venomous Animals

The Kunitz-type protease inhibitors are the best-characterized family of serine protease inhibitors, probably due to their abundance in several organisms. These inhibitors consist of a chain of ~60 amino acid residues stabilized by three disulfide bridges, and was first observed in the bovine pancreatic trypsin inhibitor (BPTI)-like protease inhibitors, which strongly inhibit trypsin and chymotrypsin. In this review we present the protease inhibitors (PIs) described to date from marine venomous animals, such as from sea anemone extracts and Conus venom, as well as their counterparts in terrestrial venomous animals, such as snakes, scorpions, spiders, Anurans, and Hymenopterans. More emphasis was given to the Kunitz-type inhibitors, once they are found in all these organisms. Their biological sources, specificity against different proteases, and other molecular blanks (being also K⁺ channel blockers) are presented, followed by their molecular diversity. Whereas sea anemone, snakes and other venomous animals present mainly Kunitz-type inhibitors, PIs from Anurans present the major variety in structure length and number of Cys residues, with at least six distinguishable classes. A representative alignment of PIs from these venomous animals shows that, despite eventual differences in Cys assignment, the key-residues for the protease inhibitory activity in all of them occupy similar positions in primary sequence. The key-residues for the K⁺ channel blocking activity was also compared.     

Hookworm dermatitis due to Uncinaria stenocephala in a dog from Saskatchewan

Uncinaria stenocephala hookworm dermatitis (uncinariosis) was diagnosed on fecal examination and macerated skin biopsy in a 1.5-year-old greyhound dog from Saskatchewan. This is the first reported case in Canada. Treatment with moxidectin cleared gastrointestinal and dermal infections.     

The base saturation in acidified Swiss forest soils on calcareous and noncalcareous parent material. A pH–base saturation anomaly

Long‐term soil acidification leads to lower pH values and to a concomitant decrease in base saturation (BS). The relationship between pH and base saturation (BS) in acidified forest soils can be disturbed by processes such as nutrient cycling by vegetation, temporary saturation by ground water that comes into contact with calcareous material, or by upward diffusion of base cations from deeper horizons. This paper examines the relationship between pH and BS in Swiss forest soils developed from calcareous and noncalcareous parent material and identifies some of the factors that can affect the BS in the decalcified parts of soils derived from calcareous parent material. The decalcified zone in the latter soils has a higher BS on average compared to soils from noncalcareous parent material, but their pH values are identical. In the pH range 4.0–4.5, the difference in BS may vary by a factor of three. The mean BS in the decalcified zone tends to decrease with increasing depth of the calcareous layer. The water regime also affects the BS in soils on calcareous parent material. In soils temporarily saturated by groundwater (gleysols), the BS in the decalcified zone is always high (85%–100%) because of the continuous contact between the soil water and the calcareous parent material. In addition, the inhibited drainage impedes the depletion of base cations in these soils. In contrast, soils that are temporarily saturated by rainwater are depleted in base cations due to the alternating wetting‐and‐drying regime and the associated leaching of dissolved ions. In such soils, the depletion of base cations is strongly related to the extent of hydromorphy. Stagnogleyic soils, with the longest period of water saturation, have the highest depletion levels. We conclude that in such soils, the diffusion of base cations from deeper zones is strongly compensated by leaching from the very acidic soil horizons. The pH–base saturation anomaly has consequences for some of the methods used to calculate the critical loads of acidity for forest soils in Switzerland, with many soils being less sensitive than previously reported.     

Influence of postharvest hot water treatment on nutritional and functional properties of kumquat (Fortunella japonica Lour. Swingle Cv. Ovale) fruit

The present study investigated the influence of a hot water dip (HWD) for 2 min at 50 degrees C, a standard and effective treatment for postharvest decay control of citrus fruit, on the nutritional and health-related properties of kumquats. The results show that most of the parameters examined, including titratable acidity, soluble solids content, maturity index, glucose, fructose, sucrose, ascorbic acid, dehydroascorbic acid, alpha- and gamma-tocopherols, beta-carotene, zeaxantin, rhoifolin, and antioxidant activity, were not significantly affected by treatment. The levels of beta-cryptoxanthin, narirutin, and total flavonoids increased after HWD, whereas lutein and total phenols decreased. The concentration of the essential oil and the relative percentage of the individual components of the essential oil were not affected by HWD except for the minor compound p-menta-1,5-dien-1-ol, which increased after HWD. After storage, lower levels of glucose, total sugars, beta-carotene, beta-cryptoxanthin and lutein were recorded in HWD fruit. A decrease in antioxidant activity and increases in alpha-tocopherol and total vitamin E were found both in control and HWD fruit. The influence of HWD at 50 degrees C for 2 min on individual nutraceuticals and health-related properties was thus generally low and may depend on storage conditions.     

High-level expression of recombinant beta-galactosidases in Lactobacillus plantarum and Lactobacillus sakei using a Sakacin P-based expression system

This work presents the cloning and expression of the genes encoding heterodimeric beta-galactosidases from Lactobacillus reuteri L103, Lactobacillus acidophilus R22, Lactobacillus plantarum WCFS1, and Lactobacillus sakei Lb790. These enzymes consist of two subunits of approximately 73 and 35 kDa, which are encoded by two overlapping genes, lacL and lacM, respectively. We have cloned these genes into the lactobacillal expression vectors pSIP403 and pSIP409, which are based on the sakacin P operon of L. sakei ( S?rvig et al. Microbiology 2005, 151, 2439- 2449 ), and expressed them in the host strains L. plantarum WCFS1 and L. sakei Lb790. Results varied considerably, ranging from 2.23 to 61.1 U/mg of beta-galactosidase activity, depending on the origin of the lacLM genes, the host strain, and the expression vector used. Highest expression levels were obtained in a laboratory cultivation of L. plantarum WCFS1 harboring the plasmid pEH3R containing the lacLM gene from L. reuteri L103. These cultivations yielded approximately 23 000 U of beta-galactosidase activity per liter, corresponding to the formation of roughly 100 mg of recombinant protein per liter of fermentation medium, and beta-galactosidase levels amounted to 55% of the total intracellular protein of the host organism. To further verify the suitability of this expression system, recombinant beta-galactosidase from L. reuteri was purified to apparent homogeneity. The properties of the purified enzyme were essentially identical with the properties of purified native beta-galactosidase from L. reuteri L103. The presented results lead the way to efficient overproduction of beta-galactosidase in a food-grade expression system, which is of high interest for applications in food industry.     

Impact of the earthworm Lumbricus terrestris on the degradation of Fusarium-infected and deoxynivalenol-contaminated wheat straw

When conservation tillage is practised in agriculture, plant residues remain on the soil surface for soil protection purposes. These residues should be widely decomposed within the following vegetation period as microbial plant pathogens surviving on plant litter may endanger the currently cultivated crop. Important soil-borne fungal pathogens that preferably infect small grain cereals belong to the genus Fusarium. These pathogens produce the mycotoxin deoxynivalenol (DON), a cytotoxic agent, in infected cereal organs. This toxin frequently occurs in cereal residues like straw. So far it is unclear if DON degradation is affected by members of the soil food web within decomposing processes in the soil system. For this purpose, a microcosm study was conducted under controlled laboratory conditions to investigate the degradation activity of the earthworm species Lumbricus terrestris when exposed to Fusarium-infected wheat straw being contaminated with DON.Highly Fusarium-infected and DON-contaminated straw seemed to be more attractive to L. terrestris because it was incorporated faster into the soil compared with straw infected and contaminated at low levels. This is supported by a greater body weight gain (exposure time 5 weeks) and smaller body weight loss (exposure time 11 weeks) of L. terrestris, respectively, when highly contaminated straw was offered for different time periods.Furthermore, L. terrestris takes part in the efficient degradation of both Fusarium biomass and DON occurring in straw in close interaction with soil microorganisms. Consequently, earthworm activity contributes to the elimination of potentially infectious plant material from the soil surface.     

Quantification of heterocyclic aromatic amines in fried meat by HPTLC/UV-FLD and HPLC/UV-FLD: a comparison of two methods

A recently developed HPTLC/UV-FLD method was compared to the routinely used HPLC/UV-FLD method for the quantification of heterocyclic aromatic amines (HAA) formed at trace levels during the heating process of meat. For formation of these process contaminants under normal cooking conditions, beef patties were fried in a double-contact grill at 230 degrees C for five different frying times and extracted by solid-phase extraction. The HAAs most frequently found, that is, 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP), 2-amino-3,8-dimethylimidazo[4,5- f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5- f]quinoxaline (4,8-DiMeIQx), 9 H-pyrido[3,4- b]indole (norharman), and 1-methyl-9 H-pyrido[3,4- b]indole (harman), were quantified by two chromatographic methods, which were orthogonal to each other (normal versus reversed phase system). Both methods showed a similar performance and good correlation of the results ( R (2) between 0.8875 and 0.9751). The comparison of running costs and run time in routine analysis proved HPTLC/UV-FLD to be more economical (factor of 3) and faster (factor of 4) due to its capability of parallel chromatography. The HAA findings calculated by standard addition increased with the heating time from <1 to 33 microg/kg related to 3-6 min of frying time. The precision (RSD) was between 7 and 49% (HPTLC) and between 5 and 38% (HPLC) at these very low HAA levels formed.     

Effects of dietary L‐arginine supplementation to early pregnant mares on conceptus diameter—Preliminary findings

The importance of the amino acid L‐arginine (ARG) for conceptus growth and litter size has been demonstrated in various species. L‐arginine is part of embryo‐derived polyamines, a substrate for nitric oxide synthase and stimulates protein synthesis by the embryo. In the present study, we have investigated whether dietary L‐arginine supplementation stimulates early conceptus growth in mares. Warmblood mares with singleton pregnancies received either an arginine‐supplemented diet (approximately 0.0125% of body weight, n = 12) or a control diet (n = 11) from days 15 to 45 after ovulation. Diameter of the embryonic vesicle (days 14, 17, 20 of pregnancy) and size of the embryo respective foetus (length and maximal diameter, days 25–45 of pregnancy at 5‐day intervals) were determined by transrectal ultrasound. At foaling, weight and size of the foal and the placenta were determined. Blood for determination of equine chorionic gonadotrophin (eCG) and progestin concentrations was collected repeatedly. Neither eCG nor progestin concentration in plasma of mares differed between groups at any time. No effects of arginine treatment on diameter of the embryonic vesicle between days 14 and 20 of pregnancy were detected. Diameter of the embryo/foetus on days 40 to 45 of pregnancy strongly tended to be enhanced by arginine supplementation (p = 0.06). Weight and size of neither the foal nor placenta at birth differed between groups. In conclusion, L‐arginine supplementation was without negative effects on early equine embryos and may support embryonic growth at the beginning of placentation.     

Einfluss von Unterlagen im Vergleich zu selbstbewurzelten Birnenbäumen auf vegetative und generative Parameter unter biologischen Anbaubedingungen in Ostösterreich

In spring 2006, in the research center of the University of Natural Resources and Life Sciences in Vienna, the pear cultivars ‘Bosc’s’, ‘Williams’ Christ’ and ‘Uta’ were planted on the rootstocks Kirchensaller Mostbirne (Seedling), Pyrodwarf, Farold 69, Quince Adams (with intercropping ‘Conference’) and own-rooted from in vitro culture, trained as spindles and tested for eight years under organic production rules. The own-rooted ‘Williams’- and ‘Bosc’s’-trees had many tree losses in the first year, they grew medium and started with fruiting later than the grafted trees. The trees on rootstock Quince Adams were growing very weakly in the high lime containing soil at the site, with the consequence of chlorosis, tree losses and small fruits, but also a higher brix-, vitamin C- and apple acid-content in the fruits. ‘Bosc’s’ showed low yields on all rootstocks in the observing period. ‘Williams’ on Farold 69 and Seedling had good yield and fruit quality, while the weakly growing cultivar ‘Uta’ showed low tree losses and high yields on Seedling. Pyrodwarf had very few tree losses and was similar in growth and yield compared to Farold 69 and Seedling, however the fruits of ‘Williams’ and ‘Bosc’s’ were smaller.     

Toxoplasma gondii and ovine toxoplasmosis: new aspects of an old story

Ovine toxoplasmosis, caused by the intracellular protozoan parasite Toxoplasma gondii, was first described in 1954 and while the incidence of ovine infection is difficult to define, it has been reported that in the UK it is responsible for between 1 and 2% of neonatal losses per annum. Recent reports have suggested that sheep persistently infected with T. gondii may pass infection to the fetus in subsequent pregnancies more readily than previously thought. These data show a high proportion of both successful and failed pregnancies in sheep to be positive by PCR for T. gondii with a tendency for samples from certain genetic lines of Charollais sheep more likely to be positive than samples from other lines, suggesting that some sheep have a particular genetic susceptibility to T. gondii.