Purification and Characterization of Cathepsin B from the Muscle of Horse Mackerel Trachurus japonicus

An endogenous protease in fish muscle, cathepsin B, was partially purified and characterized from horse mackerel meat. On SDS-PAGE of the purified enzyme under reducing conditions, main protein bands were detected at 28 and 6 kDa and their respective N-terminal sequences showed high homology to heavy and light chains of cathepsin B from other species. This suggested that horse mackerel cathepsin B formed two-chain forms, similar to mammalian cathepsin Bs. Optimum pH and temperature of the enzyme were 5.0 and 50 °C, respectively. A partial cDNA encoding the amino acid sequence of 215 residues for horse mackerel cathepsin B was obtained by RT-PCR and cloned. The deduced amino acid sequence contains a part of light and heavy chains of cathepsin B. The active sites and an N-glycosylation site were conserved across species. We also confirmed that the modori phenomenon was avoided by CA-074, a specific inhibitor for cathepsin B. Therefore, our results suggest that natural cysteine protease inhibitor(s), such as oryzacystatin derived from rice, can apply to thermal-gel processing of horse mackerel to avoid the modori phenomenon. Meanwhile, this endogenous protease may be used for food processing, such as weaning meal and food for the elderly.     

Augmented Activity of the Pelvic Nerve Afferent Mediated by TRP Channels in Dextran Sulfate Sodium (DSS)-Induced Colitis of Rats

Enteritis has been recognized as a major symptom in domestic animals and human patients suffering from feed and food poisonings. The aim of the present study was to clarify the excitatory mechanism of the pelvic nerve afferent which may influence the occurrence of enteritis in response to nociceptive chemical stimuli of the colon in normal and abnormal rats with colitis induced by dextran sulfate sodium (DSS). The pelvic nerve afferent activity was markedly increased by colonic instillation of solution (0.5 ml) of acetic acid (5-25%) and capsaicin (100μg/ml). The nerve activity was augmented by colonic instillation of capsaicin to a greater extent in rats with DSS-induced colitis than in normal control rats. This augmented activity by capsaicin was more prominent at one day (DSS-1) than at 8 day (DSS-8) after the administration of DSS. The increased nerve activity caused by capsaicin in DSS-1 and DSS-8 was significantly inhibited by pretreatment with ruthenium red, which is a nonselective inhibitor of TRP channels of unmyelinated C-fibers (nociceptors). In conclusion, it was elucidated that the nociceptive function of the pelvic nerve was largely elevated at one day after DSS-induced colitis and such increased function was mostly mediated by TRP channels.     

Effect of steroid hormones on the innate immune response induced by Staphylococcus aureus in the goat mammary gland

The objective of this study was to compare the dynamics of innate immune components after intramammary infusion of Staphylococcus aureus (SA) under conditions of high oestrogen and high progesterone in goats. In one group (“E‐group”), controlled internal drug release (CIDR) devices were inserted intravaginally from days ?11 to ?4. Prostaglandin F2α was administered immediately after removal of the CIDR device at day ?3, and then oestradiol benzoate (E) was injected intramuscularly once a day from days ?2 to 3. Heat‐inactivated SA was then administered via intramammary infusion to the left udder at day 0, whilst only saline was infused to the right udder as a control. In a second group (“P‐group”), CIDR devices were inserted intravaginally from days ?3 to 7 and SA was infused at day 0 in the same way as in the E‐group. The milk yield and the concentration of innate immune components (somatic cell count (SCC), lactoferrin (LF), S100A7 and goat ß‐defensin 1 (GBD‐1)) in the milk were measured. Milk yield decreased drastically in both SA and control udders in the E‐group, whereas the P‐group exhibited increased milk yield in both SA and control udders. SCC increased after SA infusion in both E‐ and P‐groups, although it was higher in the E‐group than in the P‐group. There was no significant change in LF concentration in the E‐group, but a decrease was observed in the P‐group. Concentrations of S100A and GBD‐1 were significantly increased after SA infusion in the E‐group but not in the P‐group. These results suggest that E enhances the innate immune response induced by SA in the goat mammary gland. This effect may be due to the reduction in milk yield and upregulation of innate immune components.     

Accumulation and localization of cesium in edible mushroom (Pleurotus ostreatus) mycelia

The characteristics of Cs accumulation and localization in edible mushrooms were examined using the mycelia of Pleurotus ostreatus-Y1. Scanning electron microscope images revealed the existence of white spots, and energy dispersive X-ray microanalyzer analysis indicated the presence of larger amounts of Cs and P in these spots in mycelia cultured on medium containing 25 mM CsCl. The (137)Cs activities in the mycelia were approximately 4-6 times higher than those in water used for (137)Cs elution. Higher Cs concentrations in the sediment fraction including vacuolar pellets were obtained compared to the upper fractions. It was observed that yellowish spots caused by the fluorescence of 4',6-diamidino-2-phenylindole (DAPI)-stained polyphosphate were localized in the mycelia. The higher fluorescence intensity of the yellowish-grained spots was measured in comparison with other regions in the mycelium. These results suggested that Cs in the mycelia was trapped by polyphosphate in vacuoles or other organelles.     

Changes in heart rate variability in horses during immersion in warm springwater

OBJECTIVE: To determine the effects of immersion in warm springwater (38 degrees to 40 degrees C) on autonomic nervous activity in horses. ANIMALS: 10 male Thoroughbreds. PROCEDURE: Electrocardiograms were recorded from horses for 15 minutes during a warm springwater bath after being recorded for 15 minutes during stall rest. Variations in heart rate (HR) were evaluated from the power spectrum in terms of low frequency (LF, 0.01 to 0.07 Hz) power and high frequency (HF, 0.07 to 0.6 Hz) power as indices of autonomic nervous activity. RESULTS: Mean (+/- SE) HR during stall rest and immersion in warm springwater was 31.1 +/- 1.7 and 30.3 +/- 1.0 beat/min, respectively. No significant difference was found between the HR recorded during stall rest and that recorded during immersion in warm springwater. The HF power significantly increased from 1,361 +/- 466 milliseconds2 during stall rest to 2,344 +/- 720 milliseconds2 during immersion in warm springwater. The LF power during stall rest and immersion in warm springwater was 3,847 +/- 663 and 5,120 +/- 1,094 milliseconds2, respectively, and were not significantly different from each other. Similarly, the LF:HF ratio did not change during immersion in warm springwater. The frequency of second-degree atrioventricular block, which was observed in 2 horses, increased during immersion in warm springwater, compared with during stall rest. CONCLUSIONS AND CLINICAL RELEVANCE: Increases in HF power indicates that the parasympathetic nervous activity in horses increases during immersion in warm springwater. Thus, immersion in warm springwater may provide a means of relaxation for horses.     

Development of a method of diagnosing ovarian disorders in sows and gilts using uterine ultrasonography

In ultrasonographic diagnosis of ovarian disorders and the estrous cycle in sows, transverse observation of the uterus yielded more characteristic findings than observation of sagittal sections. Transverse ultrasonography revealed that the low progesterone (P) type uterus showed a round structure, while the high P type uterus showed a flattened structure. These results corresponded well with rectal palpation findings: the low P type uterus had a hard, pipe-like structure and the high P type a soft, balloon-like structure. For gilts, we employed a minimum convex type transrectal prostate probe that had an approximately 18 cm insertion handle. The images of the uterus obtained thereby were a similar to those obtained from sows. The above results suggest that it should be possible to diagnose and treat many ovarian disorders in sows and gilts based only on the ultrasonograhic findings. In short, ultrasonograhic findings of a round structure of the uterine wall might be an indication for PMSG (pregnant mare serum gonadotropin) treatment, while findings of a flattened structure might be an indication for PGF(2) (alpha) administration.     

An angiotensin converting enzyme inhibitor,benazepril can be transformed to an active metabolite,benazeprilat, by the liver of dogs with ascitic pulmonary heartworm disease

To examine whether an angiotensin converting enzyme (ACE) inhibitor, benazepril, can be transformed to the active metabolite, benazeprilat, by severely injured liver of dogs with ascitic heartworm disease, benazepril hydrochloride was administered orally to dogs once daily for 7 consecutive days at a dose rate of 0.29 mg/kg to 0.63 mg/kg of body weight, and plasma benazepril and benazeprilat concentrations were determined on the 1st and 7th administration days. In 7 dogs with ascitic pulmonary heartworm disease, plasma benazeprilat concentrations tended to be higher than in 7 control dogs both on the 1st and 7th administration days. The peak concentration and area under the concentration-time curve tended to be greater in dogs of the ascites group than in control dogs, but the statistics could not detect significant differences in the time to peak concentration and t(1/2) between the control and ascites groups. Plasma ACE activities decreased after administration of benazepril. In dogs with ascitic heartworm disease, benazepril was readily transformed to benazeprilat by the liver, and was effective for suppression of plasma ACE activity.     

Genotyping of feline MHC (FLA) class II DRB by PCR-RFLP method using group-specific primers

For genotyping of feline major histocompatibility complex (FLA) class II DRB, the polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method using group-specific primers was tried. Sixty-six DRB genes were classified into 8 groups according to differences in the first 5' amino acid sequences. The group-specific primers were designed as forward ones, which were specific for 5' base sequences of genes in each group. Three to 7 appropriate restricted enzymes were selected by computer analysis for RFLP typing of the genes divided into each group. In 6 out of 9 cats, the results of DRB typed by direct sequence method agreed with results of the PCR-RFLP method using group-specific primers. In the other 3 cats, the number of genes amplified by group-specific primers was I or 2 more than those detected by direct sequence method. The direct sequence method in 9 cats identified 5 new FLA-DRB genes. The PCR-RFLP method using group-specific primers could divide 66 genes into 37 genes and 10 subgroups from the RFLP pattern. One to 6 genes in each cat, and a total of 203 genes and subgroups were detected in 68 domestic cats. The genes detected might be biased to the subgroup G1-1a (28.8%), DRB*0501 (10.3%), G1-2a (9.4%) and G6b (7.4%). The PCR-RFLP method using group-specific primers may be useful in typing FLA class II DRB.     

Isolation and sequence analysis of the promoter and an allelic sequence of the iron-sulfur protein subunit gene from the white-rot fungusPleurotus ostreatus

We have isolated a structural gene ofsdil, which encodes the iron-sulfur protein (Ip) subunit of succinate dehydrogenase (EC 1.3.99.1), from a white-rot basidiomycete,Pleurotus ostreatus. Here we report isolation of the promoter region ofsdil and an allelic sequence encoding the second-type cDNA fragment isolated in the former experiments. The nucleotide sequence analysis of the promoter region revealed the existence of putative CAAT and TATA boxes, which permits us to develop an expression system in this species. The Southern blot analysis and the restriction fragment length polymorphism assay using monokaryotic strains demonstrated that no family genes tosdil exist in the haploid genome ofP. ostreatus. Moreover, a genetic analysis to detect a linkage between thesdil genotypes and flutolanil resistance in the mutantP. ostreatus strains was also developed.     

Rapid and convenient method for preparing aurapten-enriched product from hassaku peel oil: implications for cancer-preventive food additives

Aurapten (7-geranyloxycoumarin) has been reported to be an effective inhibitor of chemical carcinogenesis in some rodent models. In the present study, a method for preparing an aurapten-enriched agricultural product has been established. Out of 17 Rutaceae varieties, the aurapten content in hassaku (Citrus hassaku Hort ex Y. Tanaka) fruit peel was marked, as well as that in natsumikan (C. natsudaidai) and grapefruit (C. paradisi). The aurapten content in hassaku peel was most abundant in April. Hassaku fruit peel oil, which was dissolved by heating precipitates including aurapten which had formed after freezing the peel oil at -20 degrees C, was used. After adsorbing aurapten from peel oil onto synthetic adsorbent SP70, the adsorbent was washed with 40% (v/v) ethanol in water to remove essential oils and pigments remaining on the adsorbent. Aurapten was then eluted with 80% (v/v) ethanol. In a laboratory-scale test, the recovery rates of aurapten and total carotenoids from the eluates were 74.3 and 4.6%, respectively. In a pilot-scale test, the recovery rate of aurapten in the aurapten-enriched preparation from dissolved hassaku oil was 91.0%, and its concentration was 64.1% (w/w). When stored for 180 days under sunlight, aurapten in powder form remained at 88.0-89.0% of the initial level, but only 31.3-43.8% in ethanol. The stability of aurapten in the aurapten-enriched preparation was higher than that of purified aurapten. These results suggest that aurapten is readily recovered from hassaku peel oil using SP70, and thus may be used as a food additive.