Biodegradation of tannins in oak (Quercus incana) leaves by Sporotrichum pulverulentum

Fermentation of oak leaves with Sporotrichum pulverulentum decreased the contents of total phenols and condensed tannins by 58 and 66% respectively in 10 d. The protein precipitation capacity decreased by 65%. Further increase of fermentation time up to 40 d did not increase substantially, the tannin degradation. Following fermentation, neutral detergent fibre, acid detergent fibre, hemicellulose, cellulose and lignin decreased by 12–16%, 8–10%, 31–51%, 0.3-3% and 14–15% respectively. The loss in dry matter was from 17–21%. The in sacco dry matter digestibility of the fermented leaves was 17% compared to 27% for the unfermented leaves.     

Protein-binding capacity of microquantities of tannins

The physiological effect of tannins is studied in terms of their protein-binding or precipitation capacity. A number of assays based on binding of hemoglobin or bovine serum albumin (BSA) and subsequent determination of unbound protein in supernatant or tannin in a protein-tannin complex are available but with various limitations. These methods are unable to estimate protein-binding capacity, if the quantity of tannin available is low. In the method reported here, tannins or other phenolics were applied on chromatography paper and reacted with BSA and unbound BSA was washed off. The protein in the tannin-protein complex was measured spectrophotometrically after staining with Ponceau S. It required microquantities of sample. Using this method the protein-binding capacity of total leaf extract and hydrolyzable and condensed tannins of Quercus incana, Q. semecarpofolia, and Q. dilatata was determined. The protein binding capacities of ellagic acid and quercetin (microgram BSA/mg) were 297.3 and 78.0, respectively.     

Molecular organization of peroxisomal enzymes: protein-protein interactions in the membrane and in the matrix

The beta-oxidation of fatty acids in peroxisomes produces hydrogen peroxide (H2O2), a toxic metabolite, as a bi-product. Fatty acids beta-oxidation activity is deficient in X-linked adrenoleukodystrophy (X-ALD) because of mutation in ALD-gene resulting in loss of very long chain acyl-CoA synthetase (VLCS) activity. It is also affected in disease with catalase negative peroxisomes as a result of inactivation by H2O2. Therefore, the following studies were undertaken to delineate the molecular interactions between both the ALD-gene product (adrenoleukodystrophy protein, ALDP) and VLCS as well as H2O2 degrading enzyme catalase and proteins of peroxisomal beta-oxidation. Studies using a yeast two hybrid system and surface plasmon resonance techniques indicate that ALDP, a peroxisomal membrane protein, physically interacts with VLCS. Loss of these interactions in X-ALD cells may result in a deficiency in VLCS activity. The yeast two-hybrid system studies also indicated that catalase physically interacts with L-bifunctional enzyme (L-BFE). Interactions between catalase and L-BFE were further supported by affinity purification, using a catalase-linked resin. The affinity bound 74-kDa protein, was identified as L-BFE by Western blot with specific antibodies and by proteomic analysis. Additional support for their interaction comes from immunoprecipitation of L-BFE with antibodies against catalase as a catalase- L-BFE complex. siRNA for L-BFE decreased the specific activity and protein levels of catalase without changing its subcellular distribution. These observations indicate that L-BFE might help in oligomerization and possibly in the localization of catalase at the site of H2O2 production in the peroxisomal beta-oxidation pathway.     

A review of the use of isotopic and nuclear techniques in animal production

Isotopic and nuclear techniques play an important role in food and agriculture, health and industry. This paper discusses the use of these techniques and highlights potential for their use in the area of Animal Production. These techniques are discussed in two parts: (1) Isotopic methods and (2) non-isotopic nuclear methods. The isotopic techniques discussed are: stable- (¹⁵N) and radio-isotope (³⁵S or ³²P) incorporation methods for measuring microbial mass in vitro and in vivo; ¹²⁵I-labelled bovine serum albumin and ¹⁴C-labelled polyethylene glycol assays for measuring tannin in feeds; a method based on the feeding of isotope-labelled protein (¹⁵N or ¹²⁵I) complexed with tannin for ranking different tannins for their abilities to release protein for digestion in vivo; ¹⁴C-uric acid and ¹⁴C-allantoin infusion methods for development of models describing excretion of purine derivatives in urine and microbial protein supply to ruminants, which permit assessment of nutritional status of animals and determination of nutritional quality of feed resources; a ¹⁵N isotope dilution technique using ¹⁵N-leucine to distinguish feed and endogenous secretions at the ileum, for determination of true digestibility of protein-rich tree leaves and aquatic plants in pigs; progesterone radioimmunoassay (RIA) for enhancing reproductive efficiency of ruminants, and RIA based leptin and insulin like growth factor assays for assessing the nutritional status of animals; feeding of ¹⁵N enriched plant material to generate ¹⁵N-labelled excreta for research on the fate of excreta N in the environment; ¹⁵N, ¹³C and ³⁴S isotopic methods for nutrient budgeting and for following the nutrient pathways in the soil–plant–animal continuum; ³²P- or ³³P-labelled fertilizers for estimating the efficiency of P utilization in legume leaf production used for livestock feeding; double labelled water (¹⁸O and ²H labelled) method for estimating energy expenditures of grazing animals, body composition, basal metabolic rate, and milk output in cows with calves; NaH¹³CO₃/NaH¹⁴CO₃ infusion for estimation of the carbon dioxide production, which in turn is used to estimate energy expenditure in free-ranging animals; ³H- or ¹⁴C-labelled methane and ¹⁴C-labelled volatile fatty acids dilution technique for direct and indirect (using stoichiometry of carbohydrate fermentation) for determination of methane emission from livestock; ¹⁵N dilution technique requiring labeling the soil with ¹⁵N fertilizer (¹⁵N-ammonium sulphate or ¹⁵N-urea) for estimation of nitrogen fixation by leguminous trees and pastures.

The non-isotopic nuclear techniques that have been used or have the potential for use are: dual energy X-ray absorptionmetry and computer tomography for body composition determination; nuclear magnetic resonance techniques, fast atom bombardment mass spectroscopy, and mass ionisation spectroscopy for identification and structure determination of bioactive moieties of plant origin having potential for rumen manipulation or controlling internal parasites; gamma irradiation for inactivating antinutrients such as protease inhibitors, lectin, phytic acid, non-starch polysaccharides and oligosaccharides in feeds; induced mutations with gamma radiation, electron beam and fast neutrons for producing useful mutants of forage plants with improved yield, nutrient profiles and uptake.     

Preliminary studies on tannin degradation by Aspergillus niger van Tieghem MTCC 2425

A tannin-degrading strain of Aspergillus niger van Tieghem was grown at pH 5·0 and 30°C in a defined medium where tannins were the sole source of carbon and energy. The fungus had variable growth in tannic acid- and quebracho tannin-medium and could tolerate these tannins even up to 150 g⁻¹ without showing any growth inhibition.     

Effect of dietary saponins from Quillaja saponaria L. on fatty acid composition and cholesterol content in muscle Longissimus dorsi of lambs

The purpose of this study was to evaluate the effects of increasing levels of saponins from Quillaja saponaria on fatty acid (FA) composition and cholesterol content in muscle Longissimus dorsi of lambs. A total of 24 Barbarine lambs were assigned to four dietary treatments: control diet (C) consisting of oat hay ad libitum and 400 g of concentrate (80% barley, 17.5% soybean meal and 2.5% vitamin and mineral supplement); C diet plus 30 ppm of Q. saponaria L. (QS30); C diet plus 60 ppm of Quillaja (QS60); C diet plus 90 ppm of Quillaja (QS90). Saponin supplementation reduced the concentration of C14:1 cis-9 (P = 0.001) and of its desaturation index (P = 0.002). None of the FA intermediates of ruminal biohydrogenation (BH) was affected by Quillaja saponin supplementation (P > 0.05). The concentration of C20:4n-6 was higher in the meat of animals receiving 60 ppm of Quillaja than C and QS30 groups. Supplementing 60 ppm of Quillaja reduced the ratio between α-linolenic and linoleic acids compared with the C group (P = 0.023). We did not find any significant effect of Quillaja saponins on muscle cholesterol level. Further investigations are necessary to assess the metabolic fate of saponins in the rumen and to understand whether there is an effect of saponin on Δ9-desaturase enzyme activity, ruminal BH and cholesterol metabolism in ruminants. Supplementing up to 90 ppm of Quillaja saponins did not produce detrimental effects on the overall meat FA profile.     

Validation of the Cardiosphere Method to Culture Cardiac Progenitor Cells from Myocardial Tissue

Background

At least four laboratories have shown that endogenous cardiac progenitor cells (CPCs) can be grown directly from adult heart tissue in primary culture, as cardiospheres or their progeny (cardiosphere-derived cells, CDCs). Indeed, CDCs are already being tested in a clinical trial for cardiac regeneration. Nevertheless, the validity of the cardiosphere strategy to generate CPCs has been called into question by reports based on variant methods. In those reports, cardiospheres are argued to be cardiomyogenic only because of retained cardiomyocytes, and stem cell activity has been proposed to reflect hematological contamination. We use a variety of approaches (including genetic lineage tracing) to show that neither artifact is applicable to cardiospheres and CDCs grown using established methods, and we further document the stem cell characteristics (namely, clonogenicity and multilineage potential) of CDCs.

Methodology/Principal Findings

CPCs were expanded from human endomyocardial biopsies (n = 160), adult bi-transgenic MerCreMer-Z/EG mice (n = 6), adult C57BL/6 mice (n = 18), adult GFP⁺ C57BL/6 transgenic mice (n = 3), Yucatan mini pigs (n = 67), adult SCID beige mice (n = 8), and adult Wistar-Kyoto rats (n = 80). Cellular yield was enhanced by collagenase digestion and process standardization; yield was reduced in altered media and in specific animal strains. Heparinization/retrograde organ perfusion did not alter the ability to generate outgrowth from myocardial sample. The initial outgrowth from myocardial samples was enriched for sub-populations of CPCs (c-Kit⁺), endothelial cells (CD31⁺, CD34⁺), and mesenchymal cells (CD90⁺). Lineage tracing using MerCreMer-Z/EG transgenic mice revealed that the presence of cardiomyocytes in the cellular outgrowth is not required for the generation of CPCs. Rat CDCs are shown to be clonogenic, and cloned CDCs exhibit spontaneous multineage potential.

Conclusions/Significance

This study demonstrates that direct culture and expansion of CPCs from myocardial tissue is simple, straightforward, and reproducible when appropriate techniques are used.     

Intracoronary beta-irradiation enhances balloon-injury-induced tissue factor expression in the porcine injury model

Intracoronary brachytherapy (ICBT) effectively reduces restenosis but is associated with late thrombosis. Since tissue factor (TF) is an important mediator of arterial thrombosis, we tested the hypothesis that ICBT results in persistently augmented TF expression. Coronary arteries from 12 pigs were randomized to: control (C; no injury), oversized balloon injury (BI), or BI followed by ICBT. Animals were sacrificed at 1, 7, 14, or 60 days postprocedure, and coronary arteries collected for expression analyses and immunostaining. ICBT-treated arteries had higher TF antigen and activity at all time-points compared to BI arteries (Western blot: 16 571 +/- 2090 vs 10 135 +/- 2939 densitometric units, p = 0.001; ELISA: 0.42 +/- 0.13 nM vs 0.25 +/- 0.14 nM, p = 0.001; TF activity assay: 0.303 +/- 0.11 nM vs 0.18 +/- 0.07 nM, p = 0.01; immunohistochemical staining: 30.6 +/- 6.6% vs 11.5% +/- 3.2%, p = 0.01). TF expression increased following BI, increased further following ICBT, and persisted for the duration of the study. We conclude that TF expression increases after BI, but is further increased and persists for a longer duration following ICBT, suggesting that a TF-mediated mechanism may play a role in late thrombosis following ICBT.