Gelatinolytic matrix metalloproteinases-2 and -9 in tracheobronchial lavage fluid obtained from calves with concurrent infections of Pasteurella multocida and Mycoplasma bovirhinis

OBJECTIVE: To study gelatinolytic matrix metalloproteinases (MMPs) in tracheobronchial lavage fluid (TBLF) obtained from clinically normal calves and calves with Pasteurella multocida infection. SAMPLE POPULATION: Samples of TBLF obtained from 11 calves with clinical signs of respiratory tract disease and growth of P multocida and Mycoplasma spp during culture of TBLF and samples of TBLF from 6 clinically normal calves with no bacterial growth during culture of TBLF. PROCEDURE: MMPs in TBLF were analyzed by use of gelatin zymography. Gelatinases were identified on the basis of molecular weights and inhibition by EDTA. RESULTS: The main gelatinolytic MMPs detected were the proform (90 to 110 kd) and active form (75 to 85 kd) of MMP-9 (gelatinase B) and the proform (67 to 75 kd) and active form (< 65 kd) of MMP-2 (gelatinase A). Increased amounts of active MMP-2 and MMP-9 were detected in TBLF of calves with respiratory tract disease, compared with amounts of active MMP-2 and MMP-9 in TBLF of clinically normal calves. Concurrent infection with Mycoplasma bovirhinis in calves with pneumonia attributable to P multocida was associated with higher concentrations of MMP-9. CONCLUSIONS AND CLINICAL RELEVANCE: The host response to P multocida includes increases in MMP-2 and MMP-9 concentrations in TBLF. Greater amounts of MMPs detected in calves with concurrent M bovirhinis and P multocida infection indicates synergism between these organisms.     

In utero infection with PRRS virus modulates cellular functions of blood monocytes and alveolar lung macrophages in piglets

The putative immunosuppressive effect of PRRS virus (PRRSV) on innate immune responses was studied in piglets infected in utero with PRRSV. Phagocytosis and oxidative burst capacities in 2-, 4- and 6-week-old in utero infected piglets were investigated and compared with age-matched control piglets. Phagocytic capacity of blood monocytes against Salmonella bacteria was investigated by flow cytometry. Oxidative burst in blood monocytes and in alveolar lung macrophages was investigated by luminol- and lucigenin-enhanced chemiluminescence, respectively. Decreased phagocytosis against Salmonella was found in blood monocytes from 4- and 6-week-old infected piglets compared to controls. In contrast, 2-week-old infected piglets showed phagocytic responses comparable to age matched control piglets. While oxidative burst capacity was increased in blood (PBMC) from in utero PRRSV infected piglets, the oxidative burst capacity of alveolar lung macrophages was decreased, especially in 2- and 4-week-old piglets, compared to age-matched control piglets. The present results indicate that in utero infection with PRRSV inhibits phagocytosis against Salmonella in blood monocytes as well as the oxidative burst capacity of alveolar macrophages. These observations indicate that PRRSV in utero infection induces at state of immunosuppression in piglets paving the way for enhanced secondary infections.     

Mg/Al ordering in layered double hydroxides revealed by multinuclear NMR spectroscopy

The anion-exchange ability of layered double hydroxides (LDHs) has been exploited to create materials for use in catalysis, drug delivery, and environmental remediation. The specific cation arrangements in the hydroxide layers of hydrotalcite-like LDHs, of general formula Mg2+(1-x)Al3+(x)OH2(Anion(n-)(x/n)).yH2O, have, however, remained elusive, and their elucidation could enhance the functional optimization of these materials. We applied rapid (60 kilohertz) magic angle spinning (MAS) to obtain high-resolution hydrogen-1 nuclear magnetic resonance (1H NMR) spectra and characterize the magnesium and aluminum distribution. These data, in combination with 1H-27Al double-resonance and 25Mg triple-quantum MAS NMR data, show that the cations are fully ordered for magnesium:aluminum ratios of 2:1 and that at lower aluminum content, a nonrandom distribution of cations persists, with no Al3+-Al3+ close contacts. The application of rapid MAS NMR methods to investigate proton distributions in a wide range of materials is readily envisaged.     

Modelling low molecular weight organic acid dynamics in forest soils

Low molecular weight organic acids such as citrate and oxalate have been hypothesized to play a key role in rhizosphere ecology and pedogenesis. A mathematical site-specific model, DYNLOW, was constructed to describe the temporal and spatial dynamics of these organic acids in coniferous forest soils using the modelling software STELLA^®. Experimentally derived values for biodegradation, adsorption, and daily values of soil temperature, moisture and hydrological flow were used to parameterize the model. The model describes the dynamics and downward movement of oxalate and citrate through the horizons (O, AE, E, Bhs, Bs) of three podzolic soil profiles in Sweden. After calibration, the model predicted average soil solution organic acid concentrations ranging from <1 to 90 μM, which was in agreement with experimental measurements (<1 to 116 μM). The model results indicated that microbial degradation of organic acids was in quantitative terms the biggest process regulating soil solution concentrations. Primary production rates of organic acid in the soil were predicted to be high (<1 to 1250 nmol g⁻¹ soil d⁻¹) in comparison to the amount present at steady state in the soil solution pool (<0.1 to 240 nmol g⁻¹ soil). The downward transfer of organic acids between soil horizons due to mass flow was predicted to be a small flux (<0.1 to 3% of the total loss) compared to that lost by microbial biodegradation. The model predicted that the amount of basal soil respiration that could be attributable to the microbial turnover of organic acids was on average 19±22% of the basal CO₂ production across all sites and horizons for citrate and 7±7% for oxalate. The model results are discussed in the context of pedogenesis, forest soil respiration and organic matter production.     

Accurate determination of oosporein in fungal culture broth by differential pulse polarography

A simple and accurate differential pulse polarographic method has been developed for the determination of oosporein in the culture broth of the fungus Beauveria brongniartii. This hydroxybenzoquinone derivative is the only major secondary metabolite secreted by this entomopathogenic fungus, which is used as biological pest control agent (BCA) against Melolontha melolontha larvae. It can be found in the host organism as well as in the formulated product. The polarographic behavior of oosporein was examined in various buffer systems over the pH range 3-10. In Britton-Robinson buffer/methanol solution (3:7 v/v, pH 5.5) the differential pulse polarograms exhibited reproducible peaks at E(p) = -0.18 V vs silver/silver chloride/potassium chloride (3 M). Under these conditions, a plot of peak height vs concentration of oosporein was found to be linear over the range 5.9 x 10(-)(7) to 2.5 x 10(-)(5) M (0.18-7.74 microg mL(-)(1); r = 0.9998). The detection limit was calculated to be 54 ng mL(-)(1). To evaluate the concentration of oosporein, the standard addition method was applied. The analysis of oosporein in the culture broth led to a mean value of 524.9 microg mL(-)(1) broth with a relative standard deviation (S(rel)) of +/-2.6%. The proposed polarographic method is accurate, not time-consuming, and it is of low cost because no separation steps are necessary.     

Flavonoids in baby spinach (Spinacia oleracea L.): changes during plant growth and storage

The variation in flavonoid concentration and composition was investigated in baby spinach (Spinacia oleracea L.) cv. Emilia sown on three occasions, each harvested at three growth stages at 6-day intervals. After harvest, leaves were stored in polypropylene bags at 2 or 10 degrees C. Flavonoids were analyzed by reversed phase HPLC. Twelve flavonoid peaks were detected. The main flavonoid, making up on average 43% of the total flavonoid concentration, was identified as 5,3',4'-trihydroxy-3-methoxy-6:7-methylenedioxyflavone-4'-glucuronide. Four other flavonoids each contributed 7-12% of the total flavonoid content. Total flavonoid content was relatively stable during normal retail storage conditions, although some of the individual flavonoid compounds showed considerable variation. The youngest plants had the highest flavonoid concentration, indicating that by harvesting the baby spinach a few days earlier than the current commercial stage of harvest, the flavonoid concentration in the product may be increased and the content of potentially health-promoting compounds enhanced.     

Endosperm structure affects the malting quality of barley (Hordeum vulgare L.)

Twenty-seven barley (Hordeum vulgare L.) samples collected from growing sites in Scandinavia in 2001 and 2002 were examined to study the effect of endosperm structure on malting behavior. Samples were micromalted, and several malt characteristics were measured. Samples were classified as having a mealier or steelier endosperm on the basis of light transflectance (LTm). Because endosperm structure is greatly dependent on protein content, three barley sample pairs with similar protein contents were chosen for further analysis. During malting, the steelier barley samples produced less root mass, but showed higher respiration losses and higher activities of starch-hydrolyzing enzymes. Malts made from steelier barley had a less friable structure, with more urea-soluble D hordein and more free amino nitrogen and soluble protein. The reason for these differences may lie in the structure or localization of the hordeins as well as the possible effects of endosperm packing on water uptake and movement of enzymes.     

Screening of Natural Plant Volatiles to Control the Potato ( Solanum tuberosum ) Pathogens Helminthosporium solani , Fusarium solani , Phoma foveata and Rhizoctonia solani

The potential utility of natural volatiles in various essential oils (EOs) from plants as fumigants to control potato tuber (Solanum tuberosum L.) pathogens was assessed. The antifungal effects of the volatiles at various concentrations were studied at 10 °C both in vitro using conidial suspensions of Helminthosporium solani, Fusarium solani var. coeruleum and Phoma foveata plated on agar, and in vivo by inoculating potato tubers. The effects of the volatiles on the mycelial growth of Rhizoctonia solani were also studied, but only in vitro. Vapours of many of the EOs tested exhibited some fungicidal activity but volatiles of garlic, Allium sativum, were, with few exceptions, most effective on all four pathogens in all experiments. An exposure time of at least 2 weeks was usually required for good control of disease development in vivo. Vapours of A. sativum never stimulated conidial germination, as was observed with some other oils, or damaged tubers, but those of Armoracia rusticana caused tuber collapse. Volatiles from thyme (Thymus vulgaris) EO showed antifungal activity in vitro on all four pathogens, but did not control F. solani, P. foveata or H. solani in vivo. In contrast, sage (Salvia officinalis) EO was ineffective against P. foveata and H. solani in the in vitro system, but controlled disease development in vivo at similar doses. The sometimes conflicting results obtained in the two test systems show that screening in vitro only is insufficient for evaluation of potent antifungal substances to be used in practice.