Methionine requirement of pigs between 5 and 20 kilograms body weight.

Four nursery experiments were conducted using a methionine (Met)-deficient feather meal-corn-soybean meal-dried whey basal diet (20% CP; 3,250 kcal of ME/kg, .11% choline, .19% Met, 1.00% cystine) supplemented with lysine, tryptophan, and histidine to determine the Met requirement of 5- to 10- and 10- to 20-kg pigs. Based on a true Met digestibility value of 81.6% estimated by a pig ileal digestibility assay, the Met-deficient basal diet contained .155% of digestible Met. A preliminary experiment (Exp. 1) indicated that pigs fed the Met-deficient basal diet when fortified adequately with Met could produce weight gains similar to those of pigs fed a 20% CP practical corn-soybean meal-dried whey diet. In Exp. 2 and 3, crossbred pigs weighing 5.8 kg initially were fed diets containing graded levels of digestible Met between .195 and .355%. Average daily gain increased quadratically (P less than .05) as the level of Met increased. When the data of Exp. 2 and 3 were examined together, the digestible Met requirement of 5- to 10-kg pigs was estimated to be .255% of the diet. In Exp. 4 and 5, crossbred pigs averaging 10 kg were fed digestible Met concentrations ranging from .155 to .315%. Average daily gain increased quadratically (P less than .05). The digestible Met requirement of 10- to 20-kg pigs was estimated at .255% for maximal weight gain, which was similar to that of 5- to 10-kg pigs. Assuming an 89% digestibility of Met in practical corn-soybean meal diets, the total Met level needed in practice would be .29%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Receptor-dependent and independent degradation of insulin by isolated swine adipocytes at 37 C

Insulin binding and degradation were measured at 37 C in isolated swine adipocytes. In preliminary experiments, binding decreased rapidly with increasing incubation time. This was associated with a marked increase in insulin degradation. Insulin binding was suppressed by some lots of bovine serum albumin (BSA), which suggests that some commercial preparations of BSA are contaminated with insulin-like molecules. In adipocyte suspensions, greater than 90% of the insulin degraded was due to a nonreceptor mediated process (i.e., insulin degrading activity present in the media). Despite the presence of insulin degrading activity in the media the cells were metabolically (as judged by lipogenic capacity and lactic dehydrogenase activity) and morphologically (greater than 98% excluded trypan blue) intact indicating that the cells were not leaking during the incubation. In subsequent experiments it was found that the specific step associated with transfer of cells during adipocyte isolation resulted in the release of insulin degrading activity. Implementation of a 30-min preincubation and washing sequence after adipocyte isolation removed the media insulin degrading activity, resulting in a marked reduction (approximately 70%) of insulin degradation by adipocyte suspensions. As a result of this modification, binding of tracer quantities of insulin attained steady-state binding conditions and maintained this for 2 h. These results demonstrate that techniques can be used to minimize nonreceptor mediated insulin degradation in adipocyte suspensions. As a result in vitro studies can be conducted that measure insulin binding and biological action in swine adipocytes at physiological temperatures.     

Comparison of the sensitivity of in vitro and in vivo tests for detection of the presence of a bovine viral diarrhoea virus type 1 strain

Veterinary vaccines are usually tested for the absence of contaminants. However, the quality control does not always imply that vaccines are not contaminated as, for example, illustrated by the bovine herpes virus 1 (BHV1) vaccine used in The Netherlands in 1999 that contained a small amount of bovine viral diarrhoea virus (BVDV1). Thousands of cows were vaccinated with BHV1 vaccine batches, and the question arose as to whether these small amounts of BVDV1, most likely not detected with in vitro tests, could have infected cattle. More in general, the question was whether the outcome of the in vitro tests, i.e. the in vitro infectivity, was indicative for the infectivity for cattle, i.e. the in vivo infectivity. We therefore carried out in vitro experiments to determine the sensitivity of a BVDV1 isolation assay. In addition, we performed two animal experiments, in which we estimated the lowest dose needed to infect calves with BVDV1. We extrapolated the experimental in vitro and in vivo results from a tissue culture infectious dose (TCID50) to a cattle infectious dose (CID50). We observed a partial response in the calves inoculated with this dose: four out of six calves turned out to be infected. In the tissue culture test, all 20 samples tested negative. The response in vivo, however, was not significantly higher than the in vitro response, which implies that no difference in susceptibility was observed between the animal test and the tissue culture test. Based on the results in our experiments, some cattle may have been infected with BVDV1 after the application of the contaminated BHV1 vaccine during the vaccination campaign. The question remains that how many cattle received contaminated vaccine, and became infected with BVDV1.     

Isolation and characterization of a new compound from Prunus mume fruit that inhibits cancer cells

An active compound that inhibits cancer cells was isolated from the fruit of Prunus mume, and its structure and in vitro activities were characterized. The n-hexane fraction obtained from methanol extracts exhibited the strongest inhibitory effect on the growth of cancer cells. From the n-hexane fraction, a new compound named B-1 was purified through preparative thin-layer chromatography, ODS column chromatography, and reverse phase high-performance liquid chromatography and its structure was analyzed by fast atom bombardment mass spectrometry and 1H and 13C NMR. The molecular formula of B-1 was C19H22O6 {2-hydroxy-1-[(7-hydroxy-2-oxo-2H-chromen-6-yl)methyl]-2-methylpropyl-(2Z)-3-methyl-but-e-enoate:prunate}, and the IC50 value was in the range of 39-58 microg/mL in descending order of the cancer cell lines Hep-2, SW-156, HEC-1-B, and SK-OV-3. B-1 exhibited 81-96% inhibition at a concentration level of 100 microg/mL against all cells, based on an 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. However, B-1 showed little effect against normal cells with only 23% or less growth inhibition at 100 microg/mL. Thus, B-1 has a highly specific inhibitory effect against cancer cells but little effect against normal cells. When the cancer cell lines Hep-2 and SK-OV-3 were incubated with B-1 for 72 h, most of the tested cells suffered strong growth inhibition. The compound has the potential to be developed as a nutraceutical.     

Study on the porcinophilic foot-and-mouth disease virus I. production and characterization of monoclonal antibodies against VP1

Monoclonal antibodies (MAbs) reported here were produced against the porcinophilic foot-and-mouth disease virus (FMDV) that caused the devastating swine disease on 1997 in Taiwan. A panel (25) of MAbs were found to react with VP1 of O/Taiwan/97 (O/97) by ELISA with various potencies. The biological identities of these VP1 reacting MAbs, such as neutralization activity, isotype and capability to distinguish between two serotype O FMDVs, O/97 and O/Taiwan/KM1/99 (O/99), were further analyzed. Eleven out of the total eighteen O/97 neutralizing MAbs were able to neutralize heterologous O/99. Eight O/97 neutralizing and five non-neutralizing MAbs could differentiate two serotype O FMDVs by immunofluorescence assay (IFA) implied that these thirteen MAbs recognized O/97 specific epitope(s). Furthermore, reactivities of the VP1 reacting MAbs with a 29 amino acids synthetic peptide (P29) representing the betaG-betaH loop of VP1 were analyzed by ELISA and fourteen were found positive. MAb clone Q10E-3 reacting strongest with VP1 and P29, neutralizing both but not differentiating two serotype O viruses suggested that the antibody binding site might involve the RGD motif and its C terminal conserved region on betaG-betaH loop. MAbs with diverse characters presented in this study were the first raised against porcinophilic FMDV. The complete set of MAbs may be used for further studies of vaccine, diagnostic methods, prophylaxis, etiological and immunological researches on FMDV.     

Antioxidant evaluation and oxidative stability of structured lipids from extravirgin olive oil and conjugated linoleic acid

Structured lipid (SL) was synthesized from extravirgin olive oil (EVOO) and conjugated linoleic acid (CLA) via a lipase-catalyzed reaction. CLA provides a variety of health benefits, but it is not consumed in free fatty acid form. The synthesized SL olive oil contained 42.5 mol % CLA isomers, and the major isomers were cis-9,trans-11-CLA (16.9 mol %) and trans-10,cis-12-CLA (24.2 mol %). The antioxidant activity determined by the radical scavenging capacity with the 2,2-diphenyl-1-picrylhydrazyl radical was lower in SL olive oil than in EVOO. The oxidative stability was also lower in SL olive oil since it had a higher peroxide value, rho-anisidine value, and 2-thiobarbituric acid reactive substances values during 20 days of storage at 60 degrees C. This observation could be due to the reduction in the natural phenolic compounds (97%) and tocopherols (56%), and the incorporated CLA with two conjugated double bonds in the SL olive oil. The oxidative stability of SL olive oil was increased by added rosemary extracts at concentrations of 100, 200, and 300 ppm. The present study suggests that the SL olive oil may be a suitable way to incorporate or deliver CLA into human diets. However, the addition of a proper antioxidant would be required for improving its oxidative stability.     

Characterization of three Colletotrichum acutatum isolates from Capsicum spp.

Colletotrichum acutatum causes anthracnose on peppers (Capsicum spp.), resulting in severe yield losses in Taiwan. Fungal isolates Coll-153, Coll-365 and Coll-524 collected from diseased peppers were found to differ in pathogenicity. Pathogenicity assays on various index plants revealed that Coll-524 was highly virulent and Coll-153 was moderately virulent to three commercially available pepper cultivars. Both isolates induced anthracnose lesions and produced abundant conidia. Coll-365 was only weakly virulent on pepper fruit, where it caused small lesions and hardly produced conidia on pepper fruit. However, Coll-365 was highly pathogenic to tomato fruit and mango leaves, where it caused anthracnose lesions and formed acervuli and conidia. All three isolates showed similar abilities in the attachment and germination of conidia, formation of highly branched hyphae and appressoria, penetration of cuticles, and infection of epidermal cells on chili peppers. Coll-365 accumulated less turgor pressure in appressoria but produced higher levels of cutinase and protease activity than Coll-153 and Coll-524 did. All three isolates invaded the neighbouring cells through plasmodesmata in chili peppers and showed similar pectinase or cellulase activities in culture. However, the most virulent strain Coll-524 expressed stronger laccase activity and was more resistant to capsaicin compared to Coll-153 and Coll-365. The three isolates are different in numbers and sizes of double-stranded RNAs. Depending on the cultivar genotypes, cellular resistance of chili pepper to C. acutatum might rely on the ability to restrict penetration, colonization, or conidiation of the pathogen. We conclude that the differences in pathogenicity among the three C. acutatum isolates of pepper are attributed to their ability to colonize the host plant.     

Antiviral activity of Alpinia katsumadai extracts against rotaviruses

In vitro anti-rotavirus activity of Alpinia katsumadai (AK) extracts were evaluated against bovine G8P[7] and porcine G5P[7] rotaviruses in two different assay strategies, a mixed treatment assay and a post treatment assay. In the mixed treatment assay, six AK extracts [AK-1 (EtOH extract), AK-3 (H(2)O layer), AK-5 (40% methanol fraction), and AK-9-11 (H(2)O extract, polysaccharide fraction, supernatant fraction)] exhibited inhibitory activities against G5P[7] rotavirus with the EC(50) values ranging from 0.7±0.4 to 33.7±6.5 μg/mL. Extracts AK-1, AK-3, and AK-5 inhibited rotavirus infection against G8P[7] rotavirus, the with EC(50) values of 8.4±2.2 μg/mL, 6.5±0.8 μg/mL and 8.4±5.0 μg/mL, respectively. By hemagglutination inhibition (HI) assay, six AK extracts completely inhibited viral adsorption onto human RBCs in both strains of rotaviruses at less than 11 μg/mL. However, in the post treatment assay, there was no anti activity shown against both strains of rotaviruses. As a result, six AK extracts were attributed mainly to having a strong interaction with hemagglutinin protein on the outer surface of rotavirus, resulting to blockage of viral adsorption.