TGF-β and TNF-α stimulate MMP-9 production in murine epidermal keratinocytes

Matrix metalloproteinases 2 and 9 (MMP‐2 and ‐9) are zinc‐dependent metalloenzymes and have gelatin‐degrading activity. Both MMP are known to be secreted by many types of cells and play important roles in several biological changes including tissue remodeling and wound healing. In the present study, a primary culture of murine epidermal keratinocytes was prepared and effects of transforming growth factor‐β (TGF‐β), tumor necrosis factor‐α (TNF‐α) and interferon‐γ (IFN‐γ) on expression of MMP‐2 and MMP‐9 by the keratinocytes was examined. Gelatin zymography revealed that murine epidermal keratinocytes secreted proenzyme forms of MMP‐2 and MMP‐9, but the active forms of both MMP were hardly detectable, indicating that in vitro autoactivation of these proenzymes did not occur. Both TGF‐β and TNF‐α stimulated MMP‐9 production in a dose‐dependent manner, but the MMP‐2 level was not changed. Interferon‐γ hardly affected production of MMP‐2 or MMP‐9. Ribonuclease protection assay demonstrated that TNF‐α increased the level of MMP‐9 mRNA 6‐fold compared to the control, whereas TGF‐β slightly up‐regulated it. These results suggest that expression of MMP‐9 could be regulated by several cytokines in murine epidermal keratinocytes.     

Isolation and identification of indigestible pyroglutamyl peptides in an enzymatic hydrolysate of wheat gluten prepared on an industrial scale

An enzymatic hydrolysate of wheat gluten was further digested in vitro with porcine pepsin and pancreatin to obtain an indigestible peptide. Indigestible pyroglutamyl peptide was isolated from the digest by strong cation-exchange, size-exclusion, and reversed-phase chromatographies. The pyroglutamyl peptide was digested with pyroglutamate aminopeptidase, and the digest was reacted with phenyl isothiocyanate. The resultant phenylthiocarbamyl (PTC) peptides were purified by reversed-phase HPLC by using binary gradient elution with ammonium acetate buffer, pH 6.0, and acetonitrile. The PTC peptides were analyzed with an automatic peptide sequencer on the basis of the Edman degradation method with a modified program. Some pyroglutamyl peptides were also analyzed by fast-atom bombardment ionization mass spectrometry without the pyroglutamate amino peptidase digestion. Consequently, pyroGlu-Asn-Pro-Gln, pyroGlu-Gln-Gln-Pro-Gln, pyroGlu-Gln-Pro-Gln, pyroGlu-Gln-Pro-Gly-Gln-Gly-Gln, pyroGlu-Gln, pyroGlu-Gln-Pro, pyroGlu-Ile-Pro-Gln, pyroGlu-Ile-Pro, pyroGlu-Gln-Pro-Leu, pyroGlu-Gln-Phe-Pro-Gln, pyroGlu-Ser-Phe-Pro-Gln, pyroGlu-Phe-Pro-Gln, and pyroGlu-Gln-Pro-Pro-Phe-Ser were identified.     

Growth and yield of potato plants grown from microtubers in fields

Despite many reports of thein vitro production of microtubers, little is known about plant growth and yield from microtubers planted in the field. This study clarified differences in growth and yields between potato plants grown in the field from microtubers and from conventional seed tubers. The experiments were performed at Hokkaido University, Japan, over four years. Conventional seed tubers of about 50 g and microtubers of two sizes (0.5–1.0 g and 1.0–3.0 g) of the latematurity cultivar Norin 1 were planted, and plant growth and tuber yields were analyzed. The microtuber plants had a lower initial increase in root and leaf area index than conventional seed tuber plants, but had the same leaf area index after about 40 days from emergence. The first tuber formation in microtuber plants was about 7 days later than in conventional seed tuber plants, while tuber bulking occurred about 14 days later in microtuber plants. Consequently, the onset of tuber weight increase was later in microtuber plants, but the rate of increase thereafter was similar between conventional seed tuber and microtuber plants. At harvest the tuber fresh weight of microtuber plants was 82% that of conventional tuber plants, suggesting a potential for using microtubers for field planting.     

Breed differences in genotype and allele frequency of catechol O-methyltransferase gene polymorphic regions in dogs

Catechol O-methyltransferase (COMT) inactivates catecholamines and catechol-containing drugs such as L-DOPA. The common genetic polymorphism Val158Met in the human COMT gene is suspected to be associated with "persistence" or risk for schizophrenia. In this study, we attempted to identify the canine COMT gene fragment and to find a similar polymorphism and to reveal its genetic distribution among five representative canine breeds. We found that the amplified gene consisted of 663 bp nucleotides and was 84% homologous with the human COMT gene. The single nucleotide polymorphisms, guanine adenine substitution, were observed at the 39th, 216th and 482nd nucleotides. From the genotyping of the 216th polymorphism among 266 dogs by the polymerase chain reaction-restriction fragment length polymorphism method with restriction enzyme EagI, and that of the 482nd polymorphism with restriction enzyme SfcI, we found inter-breed variations of genotypes as well as of allelic frequencies for both of these polymorphic regions. These results suggest that the identified polymorphisms will be useful tools in elucidating the genetic background of canine behavioral traits.     

Yield losses of soybean and maize by competition with interseeded cover crops and weeds in organic-based cropping systems

Weed management is a major issue in organic farming systems. Although interseeding cover crops is one alternative to herbicides, cover crops often suppress not only weeds but also main crops. Therefore, using cover crops for weed control without adverse effects on main crop growth is important. To verify the effect of cover crops on competition between main crops, cover crops and weeds in a snowy-cold region, main crops soybean (Glycine max Merr.) in 2005 and maize (Zea mays L.) in 2006 were grown with cover crops winter rye (Secale cereale L.) and hairy vetch (Vicia villosa Roth), respectively. The cover crops were sown on three sowing dates: before main crop planting (Pre-MC), on the same date of main crop planting (Syn-MC) and after main crop planting (Post-MC). A plot without cover crops (Sole-MC) was used as a control. The dry weight (DW), vegetation cover ratio (VCR), vertical community structure and chlorophyll content were measured to estimate the competition between main crops, cover crops and weeds. Weed DW was suppressed significantly by sowing cover crops in both soybean and maize. This weed suppression was associated with the increase of VCR of main crops plus cover crops at the early growth stage of main crops. Soil seed banks of dominant weed also became lower by sowing cover crops, implying the importance of proper weed management for suppressing weeds successively. In addition, the sowing dates of cover crops had large effects on main crops DW especially in maize, i.e., it was significantly lower in Pre-MC and Syn-MC than in Post-MC. Although the cover crop height was markedly shorter than the main crop height, the chlorophyll content of the main crops was significantly lower when cover crops were sown earlier. These results suggest that the growth inhibition of main crops by cover crops was partly caused by competition for nutrients between main crops and cover crops, and this growth inhibition was alleviated when cover crops were sown after the establishment of main crops. Consequently, soybean yield was the highest in Post-MC and decreased by 29%, 18% and 7% in Sole-MC, Pre-MC and Syn-MC, respectively, and maize yield was also the highest in Post-MC and decreased by 68%, 100% and 24% in Sole-MC, Pre-MC and Syn-MC, respectively. It was concluded that weeds could be controlled effectively by sowing cover crops after planting main crops in organic farming systems in a snowy-cold region.     

Novel Wx alleles induced by chemical mutagens in rice,Oryza sativa L.

From mutant pools of two Taiwanese elite japonica cultivars, Tainung 67 and Taikeng 8, we identified 13 mutant lines possessing opaque endosperm with relatively low amylose contents (AC) ranging from 1.5% to 7.1%. Because of different AC, paste viscosities of these 13 mutant lines differed, as revealed by palatability and physicochemical properties. The mutated gene conferring opaque endosperm was isolated from the F₂ population of one mutant line, WY1× indica cv. ‘Taichung Sen 17’, by positional cloning, revealing a G3018→A3018 substitution at exon 9 of Waxy leading to a non‐synonymous mutation from alanine to valine. Two additional alleles were identified from the other 12 mutant lines, for which single‐nucleotide substitutions G2708 → A2708 and G3029 → A3029 occurred in exons 8 and 9, leading to non‐synonymous mutations from arginine to histidine and glutamic acid to lysine, respectively. The three novel wx alleles had different effects on grain quality, specifically on eating and cooking quality, and could be applied in rice breeding programmes to develop new low AC varieties by marker‐assisted selection.     

A hydro-environmental watershed model improved in canal-aquifer water exchange process

Long-term simulation using the distributed hydro-environmental watershed model is efficacious for assessing irrigation impacts on hydrological cycle in detail and for implementing watershed management successfully. In this article, the previously developed hydro-environmental watershed model (HEWM-1) is improved in the water exchange process caused by surface water-groundwater interaction via drainage canals and/or underdrains. The time-varying stream flow in canals is described by the complete one-dimensional shallow water equations in a newly introduced submodel, the open channel flow submodel. This submodel coordinates with the other submodels: the tank, soil moisture and groundwater flow submodels which are interlinked in a cascade manner. The improved model (HEWM-2) is applied to an agricultural watershed covering an area from an alluvial fan onto a nearly level alluvial plain, to be validated. The simulation by HEWM-2 is informative for identifying whether any drainage canal is gaining or losing water in relation to groundwater level. It could thus provide useful information for conserving a complex network of drainage canals which also functions as a passage for aquatic animals like fishes.     

Effects of the aromatase inhibitor Fadrozole on plasma sex steroid secretion and oocyte maturation in female coho salmon (Oncorhynchus kisutch) during vitellogenesis

17-estradiol, 17-20-dihydroxy-4-pregnen-3-one (17-20-P), and testosterone levels were measured in plasma samples obtained from vitellogenic coho salmon (Oncorhynchus kisutch) before and 32 days after injection of the aromatase inhibitor Fadrozole (AI). Plasma 17-estradiol levels decreased significantly 6 h after injection in all AI treated fish. The higher the dose the longer the maintenance of low plasma 17-estradiol levels. Inversely, plasma 17-20-P increased significantly 6 h after injection in all AI treated fish, and the higher the dose the longer the maintenance of high plasma 17-20-P levels. At 48 h after injection plasma testosterone levels were significantly higher in the AI treated groups. The oocyte maturation index showed that multiple injections with AI retarded oocyte development. Besides, oocyte diameter and GSI were lower in the same group, which presented high incidence of atresia of vitellogenic oocytes. The ovarian follicles and brain of the fish which received multiple injections secreted less 17-estradiol, in vitro. These findings suggest that aromatase inhibitors such as Fadrozole may have a potential as a tool to regulate sexual development in salmon.     

A retrospective study and gene analysis of canine sterile panniculitis

In this study, a retrospective analysis was conducted to assess the current aspects and predisposing factors of canine sterile panniculitis. Miniature dachshund, neutered, and younger dogs appeared to be predisposed. In addition, histories of previous surgery and injection were associated in 46.5% of the cases, with several types of surgical suture materials used. About 88% of the dogs had multifocal lesions, frequently with signs of systemic illnesses. Whereas systemic immunosuppressive therapy was effective in most dogs, surgical excision of lesions was rarely curative. In order to prevent recurrences, over 65% of the cases required prolonged immunosuppressive therapy. Polymorphism of canine alpha(1)AT gene was investigated as a candidate gene for sterile panniculitis. Eight polymorphisms were discovered in seven Miniature dachshunds by direct nucleotide sequencing, which included a 12-bp deletion, three non-synonymous single nucleotide polymorphisms, and four silent substitutions. Genotyping of the two polymorphisms, c.109_120del12 and c.483A>C, which identified at high incidence in the dachshunds, was conducted in 83 dogs of 6 popular breeds. The frequencies of neither polymorphism differed between Miniature dachshunds and other breeds, suggesting that neither is responsible for developing panniculitis.     

Evaluation of the thermal property of bovine intramuscular adipose tissue using differential scanning calorimetry

The thermal property of bovine intramuscular adipose tissue (IAT) was evaluated using differential scanning calorimetry (DSC) and compared with the melting point temperature (MP) of the fat extract of IAT, which was measured using the slip point method. The beef samples were classified according to the beef marbling score (BMS). Beef with a high BMS contained less protein than that with middle or low BMS. Beef with a high BMS contained significantly more fat than that with a low BMS (P < 0.05). The endothermic point temperature (EP) of IAT, measured by DSC, was significantly higher than the MP of IAT fat (P < 0.05). The EP showed no significant difference among the three marbling grade groups. Although the MP was correlated with the monounsaturated fatty acids (MUFA) content of IAT (R² = 0.505), there was no correlation between the EP and the MUFA (R² = 0.040). However, the EP of IAT treated with collagenase was relatively highly correlated with the MP (R² = 0.655). Thus, these results suggested that DSC analysis would give us the practical thermal information regarding the melt‐in the‐mouth of beef such as the gelatinization of collagen, along with the melting of fat in IAT.