Solubilization of type I and V collagens in Japanese flounder muscle during chilled storage

ABSTRACT:   The post-mortem changes of type I and V collagens in Japanese flounder muscle during chilled storage were examined. The muscle softened significantly within 12 h under chilled storage. Both type I and V collagens isolated by ion-exchange chromatographies showed no remarkable changes in band patterns even after 24 h of storage, suggesting that degraded collagen molecules may be removed from the collagen fraction by a conventional preparation method. In contrast, type I and V collagen molecules were detected by Western blotting with each specific antibody in a saline extract and gradually increased during chilled storage. These results suggest that both type I and V collagens may participate in the post-mortem softening of fish muscle.     

Time course of changes in antioxidant activity of milk from dairy cows fed a trehalose‐supplemented diet

This study was to investigate the time course of changes to the antioxidant activity of milk from cows fed a trehalose‐supplemented diet, and to determine possible underlying mechanisms for observed changes. Six Holstein cows were used, and subjected to two experimental feeding periods consisting of a 1% trehalose‐supplemented diet for 10 days, followed by a basal diet only (no trehalose) for 10 days. 1,1‐Diphenyl‐2‐picrylhydrazyl (DPPH) radical scavenging activities in milk were gradually increased during the trehalose supplementation period and were highest at the end of the second period. However, trehalose was not detected in the milk and plasma of dairy cows fed a diet supplemented with trehalose for 10 days, indicating that the increased antioxidant activity in the milk of trehalose‐fed cows is not due to the direct transfer of trehalose to the milk. Plasma DPPH activities exhibited a similar time course to that seen for milk. Relative superoxide dismutase (SOD) activities in the rumen were higher 3 days after the end of trehalose supplementation than at any other time during the experimental periods. These results suggested that the improved antioxidant activity in milk and plasma of cows fed a trehalose‐supplemented diet was due to improved ruminal relative SOD activity.     

Comparison of the activity of non‐steroidal ecdysone agonists between dipteran and lepidopteran insects,using cell‐based EcR reporter assays

BACKGROUND: Diacylhydrazine (DAH) analogues have been developed successfully as a new group of insect growth regulators, called ecdysone agonists or moulting accelerating compounds. These DAHs have been shown to manifest their toxicity via interaction with the ecdysone receptor (EcR) in susceptible insects, as does the natural insect moulting hormone 20‐hydroxyecdysone (20E). A notable feature is their high activity and specificity, particularly against lepidopteran insects, raising the question as to whether non‐lepidopteran‐specific analogues can be isolated. However, for the discovery of ecdysone agonists that target other important insect groups such as Diptera, efficient screening systems that are based on the activation of the EcR are needed. RESULTS: In this study, a dipteran‐specific reporter‐based screening system with transfected S2 cells of Drosophila melanogaster Meig. was developed in order to discover and evaluate compounds that have ecdysone agonistic or antagonistic activity. A library of non‐steroidal ecdysone agonists containing different mother structures with DAH and other related analogues such as acylaminoketone (AAK) and tetrahydroquinoline (THQ) was tested. None of the compounds tested was as active as 20E. This is in contrast to the very high activity of several DAH and AAK congeners in lepidopteran cells (Bombyx mori L.‐derived Bm5 cells). The latter agrees with a successful docking of a DAH, tebufenozide, in the binding pocket of the lepidopteran EcR (B. mori), while this was not the case with the dipteran EcR (D. melanogaster). Of note was the identification of two THQ compounds with activity in S2 but not in Bm5 cells. Although marked differences in activity exist with respect to the activation of EcR between dipterans and lepidopterans, there exists a positive correlation (R = 0.724) between the pLC₅₀ values in S2 and Bm5 cells. In addition, it was found through protein modelling that a second lobe was present in the ligand‐binding pocket of lepidopteran BmEcR but was lacking in the dipteran DmEcR protein, suggesting that this difference in structure of the binding pocket is a major factor for preferential activation of the lepidopteran over the dipteran receptors by DAH ligands. CONCLUSIONS: The present study confirmed the marked specificity of DAH and AAK analogues towards EcRs from lepidopteran insects. THQ compounds did not show this specificity, indicating that dipteran‐specific ecdysone‐agonist‐based insecticides based on the THQ mother structure can be developed. The differences in activity of ecdysone agonists in dipteran and lepidopteran ecdysone‐reporter‐based screening systems are discussed. Copyright © 2010 Society of Chemical Industry     

Multiple copies of <Emphasis Type="Italic">AMT2</Emphasis> are prerequisite for the apple pathotype of <Emphasis Type="Italic">Alternaria alternata</Emphasis> to produce enough AM-toxin for expressing pathogenicity

The apple pathotype of Alternaria alternata produces the cyclic depsipeptide AM-toxin and causes Alternaria blotch of apple. Previously, we cloned AMT2 from the apple pathotype as an orthologue of AFTS1, which is required for biosynthesis of the decatrienoic acid ester AF-toxin I of the strawberry pathotype. These genes were predicted to encode aldo-keto reductases involved in biosynthesis of a common precursor, 2-hydroxy-isovaleric acid, of AF-toxin I and AM-toxin. In this study, we analyzed the function of AMT2 in AM-toxin biosynthesis in the apple pathotype. DNA gel blot analysis of the apple pathotype strain IFO8984 with five restriction enzymes suggested that this strain has a single copy of AMT2 in the genome. However, gene disruption experiments showed that IFO8984 probably has three copies of AMT2. We made mutants having one or two copies of AMT2 disrupted. The single-copy mutants produced less AM-toxin than did the wild type and were still as pathogenic as the wild type. The two-copy mutants produced trace or undetectable amounts of AM-toxin and were markedly reduced in pathogenicity. Thus, AMT2 was verified to be required for AM-toxin biosynthesis and hence pathogenicity. The fact that the two-copy mutants have a remaining copy of AMT2 suggests that multiple copies of AMT2 are prerequisite for the pathogen to produce enough AM-toxin for full pathogenicity.     

Atmospheric moisture influences on conidia development in Podosphaera xanthii through host-plant morphological responses

This study investigated the effects of atmospheric moisture on conidia development in cucurbit powdery mildew fungus (Podosphaera xanthii) through host-plant responses. Cucumber (Cucumis sativus L.) seedlings were grown under a high vapour pressure deficit (VPD) (3.8 kPa) or a low VPD (0.4 kPa). When the cotyledons had expanded, spores of P. xanthii were inoculated onto the adaxial surface. Inoculated seedlings for both treatments were then placed in a growth chamber maintained at a VPD of 2.1 kPa. The density of visible P. xanthii colonies on the high-VPD-acclimated cotyledons was 0.46 to 0.85 times that of the low-VPD-acclimated cotyledons 7 days after inoculation. It is likely the post-germination behaviour of P. xanthii such as the infection and consequent hyphal development was affected because spore germination did not differ between the treatments. The percentage of adaxial epidermal leaf cells with haustoria was also lower in the seedlings which had acclimated to a higher VPD. The high-VPD-acclimated cotyledons were thicker and had greater dry mass per area. The water potential of cotyledons did not differ between the treatments, although the stomatal conductance of high-VPD-acclimated cotyledons was lower than that of low-VPD-acclimated cotyledons. From these results, we conclude that the inhibition of P. xanthii conidia development on high-VPD-acclimated cotyledons was mainly caused by changes in leaf morphological properties.     

CT and MRI imaging diagnosis of epidural idiopathic sterile pyogranulomatous inflammation in a dog spinal canal

A 12-year-old neutered male shih tzu developed progressive pelvic limb paraparesis. Computed tomography showed a radiolucent mass lesion in the spinal canal at the left side of the 11th thoracic vertebra. The mass was not enhanced by intravenous contrast medium injection. It was hyperintense on both T1- and T2-weighted magnetic resonance images. The signal intensity of the mass was decreased with a fat suppression technique, indicating a fatty origin. After removal of the mass via T11-T12 hemilaminectomy, chronic panniculitis was confirmed by histopathological examination. This case demonstrates the utility of computed tomography and magnetic resonance imaging for the diagnosis of spinal canal pyogranulomatous inflammation.     

Association between energy status early postpartum and subsequent embryonic mortality in high‐yielding recipient cows

High‐yielding Holstein‐Friesian recipients (n = 43) were used in order to investigate the relation between energy balance status during the early postpartum period and subsequent embryonic mortality after transferring good‐quality frozen embryos. Blood samples were collected during the second, third, fourth, fifth, sixth and seventh weeks postpartum in order to measure energy status indicators. These indicators include β hydroxyl butyric acid (BHBA), non‐esterified fatty acid (NEFA), total cholesterol (T‐chol), glucose and blood urea nitrogen (BUN). Moreover, body condition scores (BCS) were assessed during the same period. Pregnancy diagnosis by ultrasonography at the 28th day postestrus and embryo viability was ascertained until 45 days postestrus in order to detect late embryonic mortality (LEM). The pregnancy rate on day 28 was 44.2% (19/43); however, five cows (11.6%) experienced LEM by day 45. Based on the non‐return rate at day 24, non‐pregnant animals, as diagnosed by ultrasonography, were allocated into animals with longer estrus intervals than 24 days (32.5%; mid‐embryonic mortality (MEM) group) and animals returning to estrus by day 24 postestrus (23.5%; early embryonic mortality (EEM) group). At week 5 postpartum, BCS was significantly (P < 0.05) lower in the LEM group than that of pregnant (PREG), EEM and MEM groups. NEFA was significantly higher in animals that experienced LEM (LEM group) at week 7 postpartum (289.6 ± 47.0 µEq/L; P < 0.01) than that of PREG (196.8 ± 16.0 µEq/L), EEM (157.2 ± 18.6 µEq/L) and MEM groups (191.5 ± 14.4 µEq/L). In conclusion, lower BCS at week 5 postpartum and higher NEFAs at week 7 postpartum may be associated with subsequent LEM in high‐yielding recipient cows.     

Molecular mechanism of the suppression of larval skeleton by polycyclic aromatic hydrocarbons in early development of sea urchin <Emphasis Type="Italic">Hemicentrotus pulcherrimus</Emphasis>

Polycyclic aromatic hydrocarbons including benz[a]anthracene (BaA) are priority pollutants in the aquatic environment. Our previous study revealed that BaA and its metabolite, 4-monohydroxylated BaA (4-OHBaA) inhibit larval skeletogenesis in the sea urchin Hemicentrotus pulcherrimus. Here we report studies to elucidate the target of skeletogenesis inhibition elicited by BaA and 4-OHBaA. First, we performed an in vitro experiment using isolated micromeres which give rise to the larval skeletogenic mesenchyme. However, skeletogenesis was not repressed by BaA and 4-OHBaA, implying that these chemicals indirectly influence on the formation of larval skeleton. Next, we analyzed their influence in vivo using embryos. Vascular endothelial growth factor (VEGF) that is expressed in the ectoderm and induces spicule formation was inhibited by BaA and 4-OHBaA treatment. These chemicals also suppressed the expression of the heparan sulfate 6-O endosulfatase (Sulf) known as a VEGF signaling modulator. We, therefore, propose that BaA and 4-OHBaA effects on larval skeletogenesis via VEGF signaling. Furthermore, we showed that the expression of Endo16 mRNA, an endodermal marker, decreased after BaA and 4-OHBaA exposure, suggesting that these chemicals affect endodermal function together with skeletogenesis. This study demonstrates that BaA and 4-OHBaA exert multiple detrimental effects on the development of H. pulcherrimus.