Effect of estradiol‐17β during in vitro growth culture on the growth,maturation, cumulus expansion and development of porcine oocytes from early antral follicles

Growing porcine oocytes from early antral follicles can acquire meiotic and developmental competence under suitable culture conditions, but at lower rates compared to full‐grown oocytes. We postulated that estradiol‐17β (E₂) supported the acquisition of meiotic and developmental competence as well as cumulus‐expansion ability during growth culture. Growing oocytes from early antral follicles (1.2 to 1.5 mm in diameter) were grown in vitro for 5 days in a medium containing 0, 10^?7, 10^?6, 10^?5 or 10^?4 mol/L E₂; after in vitro maturation, 35, 58, 47, 74 and 49% of oocytes matured to metaphase II, 25, 79, 77, 90 and 97% acquired cumulus‐expansion ability, and 23, 54, 63, 89 and 64% were fully surrounded by cumulus cells, respectively. Following maturation, electro‐stimulation was applied to the oocytes grown with 10^?5 mol/L E₂. After 6 days of culture, in vitro‐grown oocytes developed to the blastocyst stage at a rate similar to that for full‐grown oocytes (31% and 40%, respectively). Therefore, we suggest that the use of E₂ during growth culture improves the meiotic and developmental competence of oocytes, cumulus‐expansion ability, and cumulus cell attachment to the oocytes.     

Antioxidant activity of prune (Prunus domestica L.) constituents and a new synergist

Ethanol extract of prune was separated into hexane-soluble and H(2)O-soluble fractions, and the H(2)O-soluble fraction was further separated into a methanol (MeOH) eluate and an H(2)O eluate by Diaion HP-20 column chromatography. The MeOH eluate exhibited the strongest antioxidant activity among the separated fractions evaluated by oxygen radical absorbance capacity (ORAC). Further purification of the MeOH eluate led to isolation of a novel compound, 4-amino-4-carboxychroman-2-one, together with four known compounds (p-coumaric acid, vanillic acid beta-glucoside, protocatechuic acid, and caffeic acid), structures of which were identified by NMR and MS analyses. The ORAC values of these isolated compounds showed 0.15-1.43 units (micromol of Trolox equiv)/micromol, and the new compound showed a remarkable synergistic effect on caffeoylquinic acid isomers. The antioxidant activity of the MeOH eluate was highly dependent on the major prune components, caffeoylquinic acid isomers, with a contribution from the new synergist.     

Cyanobacterial communities of rice straw left on the soil surface of a paddy field

To estimate diversity, seasonal variation, and phylogeny of the cyanobacterial communities in rice straw placed in nylon mesh bags and left on the soil surface of a paddy field, total DNA was extracted from straw, amplified by polymerase chain reaction targeting 16S rRNA genes of cyanobacteria, and the amplicons were separated by denaturing gradient gel electrophoresis (DGGE). These DGGE bands were sequenced. The paddy field was under flooded condition after transplanting of rice (Experiment 1) and under drained conditions after harvest (Experiment 2). The residual samples on the soil surface under upland conditions were collected just before spring plowing and were placed again on the soil surface after transplanting under flooded conditions. DGGE band patterns of cyanobacterial communities of rice straw were different under drained conditions, under flooded conditions when fresh rice straw samples were placed (Experiment 1), and under flooded conditions when residual rice straw samples were replaced (Experiment 2), indicating that the communities were influenced by both water regime of the paddy field and the degree of the rice straw decomposition. Sequence analysis of DGGE bands indicated that most of the cyanobacteria in rice straw on the soil surface in the paddy field were filamentous members belonging to Subsections III and IV. Filamentous cyanobacterial cells were observed in rice straw under flooded conditions by epifluorescence microscopy.     

Central somatostatin-somatostatin receptor 2 signaling mediates lactational suppression of luteinizing hormone release via the inhibition of glutamatergic interneurons during late lactation in rats

Reproductive function is suppressed during lactation owing to the suckling-induced suppression of the kisspeptin gene (Kiss1) expression in the arcuate nucleus (ARC) and subsequent suppression of luteinizing hormone (LH) release. Our previous study revealed that somatostatin (SST) neurons mediate suckling-induced suppression of LH release via SST receptor 2 (SSTR2) in ovariectomized lactating rats during early lactation. This study examined whether central SST-SSTR2 signaling mediates the inhibition of ARC Kiss1 expression and LH release in lactating rats during late lactation and whether the inhibition of glutamatergic neurons, stimulators of LH release, is involved in the suppression of LH release mediated by central SST-SSTR2 signaling in lactating rats. A central injection of the SSTR2 antagonist CYN154806 (CYN) significantly increased ARC Kiss1 expression in lactating rats on day 16 of lactation. Dual in situ hybridization revealed that few ARC Kiss1-positive cells co-expressed Sstr2, and some of the ARC Slc17a6 (a glutamatergic neuronal marker)-positive cells co-expressed Sstr2. Furthermore, almost all ARC Kiss1-positive cells co-expressed Grin1, a subunit of N-methyl-D-aspartate (NMDA) receptors. The numbers of Slc17a6/Sstr2 double-labeled and Slc17a6 single-labeled cells were significantly lower in lactating dams than in non-lactating rats whose pups had been removed after parturition. A central injection of an NMDA antagonist reversed the CYN-induced increase in LH release in lactating rats. Overall, these results suggest that central SST-SSTR2 signaling, at least partly, mediates the suppression of ARC Kiss1 expression and LH release by inhibiting ARC glutamatergic interneurons in lactating rats.     

Studies of certain factors affecting the microenvironment and microflora of the external ear of the dog in health and disease

A total of 187 dogs, 110 with clinical signs of otitis externa (OE), and 77 without history or clinical signs of OE, were examined microenvironment and microbiological analysis of their ear exudates made. The aural temperature and humidity of 160 dogs were measured. There were no significant difference between healthy dogs and OE dogs. German shepherd showed relatively lower temperature (p<0.01) and higher humidity (p<0.01). The mean log(10) number of microbial organisms of ears of OE dogs (4.16 +/- 0.31 cfu/g) was significantly increased, compared to that from the ears of non-OE group (2.55 +/- 0.24 cfu/g). Pseudomonas spp. and Proteus spp. were detected only from OE dogs. In addition, three enterotoxigenic Staphylococcus aureus were isolated from ear specimens.     

A comparative study of zinc protoporphyrin IX‐forming properties of animal by‐products as sources for improving the color of meat products

The objective of this study was to obtain fundamental data for improving the color of meat products by using animal by‐products. We investigated zinc protoporphyrin IX (ZnPP)‐forming properties of various internal organs from pigs and chickens. ZnPP was formed in the liver, heart and kidney, whereas the porcine spleen and bile, which are involved in the metabolism of heme, did not have ZnPP‐forming properties. The optimum pH values were different among the internal organs and the ZnPP‐forming properties of porcine organs were better than those of chicken organs. The porcine liver showed the greatest ZnPP‐forming properties among all of the internal organs investigated in this study. The optimum pH value for ZnPP formation in the liver was lower than that of skeletal muscle. Oxygen did not inhibit the formation of ZnPP in the liver, unlike in skeletal muscle. Animal by‐products such as the liver have good ability for the formation of ZnPP and might be useful for improving the color of meat products.     

Inhibition of post-mortem muscle softening following in situ perfusion of protease inhibitors in tilapia

ABSTRACT:     A method of introducing protease inhibitors into fish muscle through the bulbus arteriosus was developed using an in situ perfusion technique. Perfusion efficiency was initially tested using eosin and [³⁵S]-methionine. Visible fluorescence was observed in the gill, liver, intestine and dorsal muscle of the eosin-treated tilapia, and the occurrence of eosin in the blood vessels of the dorsal muscle was confirmed under a fluorescence stereoscopic microscope with ultraviolet light. The radioactivity of [³⁵S]-methionine was taken into the dorsal muscle and liver at a concentration of 7.8 Bq/g and 70.2 Bq/g, respectively, after perfusion with 1000 Bq/mL solution. Using the perfusion technique with four kinds of protease inhibitors dissolved in physiological saline, the type of proteases implicated in the post-mortem muscle softening in tilapia (867 ± 195 g, n  = 10/protease inhibitor) was investigated. After the perfusion of leupeptin (serine and cysteine protease inhibitor), benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk; caspase inhibitor), chymostatin (serine protease inhibitor) and ο -phenanthroline (metalloprotease inhibitor), the breaking strength of the perfused muscle was measured as a parameter of the meat toughness and compared with that of the control fish, which were perfused with physiological saline only. The reduction of breaking strength during storage was inhibited by the perfusion of leupeptin and Z-VAD-fmk.     

Carotenoid accumulations and carotenogenic gene expressions in the petals of Eustoma grandiflorum

Eustoma grandiflorum is one of the leading cut‐flowers in Japan. There are market demands for cultivars with deep‐yellow flowers, but they have never been bred successfully. By investigating the carotenoid accumulation and carotenogenic gene expressions, this study attempted to explore the reasons that block the formation of deep‐yellow colour in Eustoma. High performance liquid chromatography analysis showed that the carotenoid compositions in petals were similar to those in leaves, accumulating mainly lutein, violaxanthin and β‐carotene. The total carotenoid contents decreased as the petals matured in all the cultivars tested. Quantitative real‐time PCR analysis showed that the expression levels of PSY, LCYB, ZDS and LCYE showed significant differences between white and pale‐yellow petals or between petals and leaves, indicating that these enzymes may play a key role in the carotenoid biosynthesis in E. grandiflorum. The expression levels of CCD4 were high in both pale‐yellow and white petals during development, suggesting that carotenoid degradation activity is high in the petals. We then conclude that the total carotenoid accumulation level could be determined by the balance between carotenoid biosynthesis and degradation activities.     

Structure and possible function of <Emphasis Type="Italic">N</Emphasis>-glycans of an invertebrate C-type lectin from the acorn barnacle <Emphasis Type="Italic">megabalanus rosa</Emphasis>

ABSTRACT:   A C-type lectin (BRA-2) isolated from the acorn barnacle Megabalanus rosa , which was a glycoprotein having an N -linked sugar chain, was deglycosylated by N -glycopeptidase F. The structure of the released sugar chains was determined by a 2-D mapping method after derivatization with a fluorescent reagent, 2-aminopyridine, to be Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4(Fucα1-6)GlcNAc and Manα1-6(GlcNAcβ1-2Manα1-3)Manβ1-4GlcNAcβ1-4(Fucα1-6)GlcNAc. The structures were confirmed by matrix-assisted laser desorption ionization mass spectrometry and a comparison with authentic sugar chains by high-pressure liquid chromatography. Various properties of BRA-2 were examined before and after deglycosylation. The susceptibility of BRA-2 to protease digestion was increased by deglycosylation. However, the inhibitory activity toward calcium carbonate crystallization as well as the hemagglutinating activity of deglycosylated BRA-2 was significantly decreased. These results suggest that the sugar chains of BRA-2 are important to both its structural stability and its function.     

Application of α-glucosidase activity and drug susceptibility tests to epidemiological studies on the fish pathogen <Emphasis Type="Italic">Nocardia seriolae</Emphasis>

This study determined the minimum inhibitory concentrations (MICs) for oxytetracycline hydrochloride (OTC) and erythromycin (Em), along with the α-glucosidase (α-glu) activities in 110 Nocardia seriolae strains isolated in Miyazaki and Kagoshima prefectures in 2008–2009. The strains were examined for the presence of the tet(K), tet(L), tet(M), tet(O), tet(S), erm(A), erm(B), mph(A), mef(A), and msr(D) genes. All the α-glu-positive strains (n = 15) were OTC resistant and Em sensitive, with MICs of 32–64 and <0.125 μg/ml, respectively. All the α-glu-negative strains (n = 95) were OTC sensitive, with MICs of 2–4 μg/ml, and most of them (93 of 95) were Em resistant, with MICs of >128 μg/ml. The MICs for Em in the remaining 2 α-glu-negative strains were <0.125 μg/ml. The 15 OTC-resistant strains possessed the tet(K) and/or tet(L) gene(s), and all of the 93 Em-resistant strains possessed both the mef(A) and msr(D) genes. The relationship between α-glu activity and drug sensitivity of the N. seriolae strains may explain the difference in prevalence of each phenotype. Nevertheless, the relationship should be further explored using N. seriolae isolates collected from more prefectures and farms.