Effect of tissue deterioration on postmortem BSE diagnosis by immunobiochemical detection of an abnormal isoform of prion protein

Surveillance for bovine spongiform encephalopathy (BSE) in fallen stock in Japan is conducted with a commercial enzyme-linked immunosorbent assay (ELISA) for mass screening, with Western blotting (WB) and immunohistochemistry performed for confirmation of the ELISA. All tests are based on immunological detection of an abnormal isoform of the prion protein (PrP(Sc)) in brain tissues, which have sometimes deteriorated by the time samples from fallen stock reach a diagnostic laboratory. To evaluate BSE surveillance procedures for fallen stock, we examined PrP(Sc) detection from artificially deteriorated BSE-affected bovine brain tissues with a commercial ELISA kit and compared the results with those of WB. The optical density (OD) values of the ELISA decreased with advancing deterioration of the tissues, whereas no reduction in the signal for PrP(Sc) was observed in WB, even when performed after 4 days of incubation at 37 degrees C. The progressive decrease in the OD values in the ELISA appear to be caused by a partial loss of the N-terminal moiety of PrP(Sc) due to digestion by endogeneous and/or contaminated microbial enzymes, and by the presence of ELISA inhibitors that are generated in deteriorated tissues. These results suggest that WB is the most reliable test for fallen stock, especially for cattle brains within decaying carcasses.     

Dietary Soy Peptide Enhances Thermotolerance and Survival of Juvenile Japanese Flounder,Paralichthys olivaceus

Soy peptide (SP), a soy protein enzymatic hydrolysate, contains bioactive substances that could be utilized as an immune‐stimulating feed ingredient. The experiment evaluated the efficacy of dietary SP on promoting growth, and enhancing tolerance and survival to heat stress in juvenile Japanese flounder, Paralichthys olivaceus. Four diets were incorporated with different levels of SP (0, 2, 5, and 10%) and a 6‐wk feeding trial ensued. Following the feeding trial, the experimental groups were subjected to heat stress to measure survival rate and heat shock protein 70s (HSP70s) in gill, liver, and skin. Fish fed diets with SP inclusion showed considerable decrease in percent weight gain. Significantly higher lethal time values to 50% mortality (LT₅₀) value were recorded for fish fed 10% SP. Moreover, LT₅₀ values of fish fed 2 and 5% SP were significantly higher compared with fish fed control diet. HSP70s produced in all the tissues were significantly highest in fish fed 10% SP. HSP70s values were significantly higher in fish fed 2 and 5% SP compared with fish fed control diet. A significant reduction in HSP70s among all groups during recovery period was also observed. These results suggest that SP can be used to enhance the immune response and survival of P. olivaceus under heat stress.     

Aluminum-26 in deep-sea sediment

The activity of (26)A1 in a North Pacific core was found to be 0.081 +/- 0.046 disintegration per minute per kilogram of dry sediment, which corresponds to a (26)A1/(10)Be ratio of 0.018 +/- 0.011 (or 0.019 +/- 0.012 when this ratio is corrected for decay). This ratio is in good agreement with that measured in Greenland ice, 0.017 +/- 0.008. These ratios are also in agreement with the calculated values for the production of these isotopes by cosmic rays in the atmosphere: 0.013 +/- 0.006. The contribution of cosmic dust bearing (26)Al seems small in comparison with the production of this nuclide in the atmosphere.     

Polyphenol-induced inhibition of the response of na(+)/glucose cotransporter expressed in Xenopus oocytes

To study the effects of polyphenols on the Na(+)/glucose cotransporter (SGLT1) response, SGLT1 was expressed in Xenopus oocytes by injecting cRNA synthesized from the cloned cDNA of the small intestine cotransporter of rats, and the electrical response elicited by glucose or galactose was measured by a voltage clamping method. Most phenol derivatives had no effect on the response. However, the polyphenols (+)-catechin, (-)-epicatechin gallate (ECg), and (-)-epigallocatechin gallate (EGCg), which are components of green tea, caused an inhibition of the response, which was almost independent of glucose concentration. The inhibition constants were estimated to be 2.3 mM for (+)-catechin and 0.45 mM for both ECg and EGCg, assuming the noncompetitive inhibition mechanism. Saponin prepared from tea seeds also inhibited the response significantly. Tannic acid and aqueous extracts of teas induced nonspecific electrical responses in both cRNA-injected and noninjected oocytes at lower concentrations than those that caused an inhibition of the SGLT1 response when their dose-dependent effects were examined. These results are possibly helpful in the development of a dietary supplement for diabetic patients.     

Effects of dietary phytol on tissue accumulation of phytanic acid and pristanic acid and on the tissue lipid profiles in mice

Recent in vitro evidence suggests that the phytol‐derived fatty acids, phytanic acid (PA) and pristanic acid (PrA), are components of animal products with the potential to cause both beneficial and harmful effects on human health. In this study, we investigated the in vivo tissue accumulation of PA and PrA and the changes in tissue lipid profiles, using mice fed a phytol‐containing diet. After 4 weeks of treatment with a diet containing 1.0% phytol, plasma, adipose tissue, liver, and brain were collected and their lipid profiles were biochemically and gas‐chromatographically determined. Dietary phytol caused PA and PrA accumulation in the adipose tissue and liver but not in the brain, and reduced plasma and liver triacylglycerol levels. Phytol intake also decreased the fatty acid concentrations in the adipose tissue, especially polyunsaturated fatty acids such as linoleic acid, but increased the concentrations of these fatty acids in the liver. However, dietary phytol had a low impact on the brain lipid profile. This study suggests that dietary phytol intake caused accumulation of PA and PrA and modified lipid profiles in the adipose tissue and liver, but that the brain is an insusceptible tissue to dietary phytol‐induced changes.     

Microbial hydrolytic enzyme activity in the sediments of a net cage aquaculture area

Potential microbial extracellular leucine-aminopeptidase (LAP) and β-glucosidase (βGlc) activity in the sediments of a red sea-bream (Pagrus major) aquaculture area were measured. Temporal variations in these enzymatic activities were low and stable from December 1999 to April 2000. They rose but fluctuated widely from May to November 2000. LAP activity in the sediments ranged from 334 to 4461 nmol/g per h in the 0–1-cm layer and from 106 to 258 nmol/g per h in the 4–5-cm layer. βGlc activity ranged from 221 to 4121 nmol/g per h in the 0–1-cm layer and from 18 to 238 nmol/g per h in the 4–5-cm layer. These values were higher than others reported and the organic matter load from the fish farms appeared to promote these enzymatic activities, although the relationship was not linear. The ratio of LAP to βGlc was smaller than other reported values in marine sediments or in water samples from the same aquaculture area, suggesting that not only quantitatively but qualitatively an aspect of organic matter degradation in the sediment might be changed by the heavy supply of organic materials from fish farms.     

A vital staining technique with fluorescein diacetate (FDA) and propidium iodide (PI) for the determination of viability of myxosporean and actinosporean spores

A vital staining technique with fluorescein diacetate (FDA) and propidium iodide (PI) was used for determination of viability of myxosporean stage spores of Myxobolus artus and actinosporean stage spores of M. cultus . Viable spores stained green with FDA and non-viable spores stained red with PI were clearly distinguishable in M. artus myxosporean spores released from live infected fish, while the spores collected from pseudocysts were not well stained with FDA. Immediately after release from the oligochaete, Branchiura sowerbyi , actinosporean spores of M. cultus , stained bright green in the sporoplasms and red in the spore processes. In vitro survivability tests revealed that the longevity of M. artus spores was 5 months at 25°C and 15 months at 18°C. Drying and ultraviolet irradiation (36 000 mW s cm^–2 for M. artus myxosporean spores, 600 mW s cm^–2 for M. cultus actinosporean spores) were most effective as sporicidal treatments.