Methionine sulfoximine suppressed the stimulation of dark carbon fixation by ammonium nutrition in wheat roots

To evaluate the role of NH₄ ⁺ assimilates in dark carbon fixation in roots in providing carbon skeletons expended for NH₄ ⁺ assimilation, the rate of dark carbon fixation in roots was measured using NaH¹⁴CO₃. The ¹⁴C-metabolites were analyzed in wheat (Triticum aestivum L.) plants grown in NH₄ ⁺ media for various periods of time with or without methionine sulfoximine (MSX) treatment. The dark carbon fixation rate in the roots of wheat plants that had been grown with NH₄ ⁺ for 1 d was approximately 6-fold higher than the rate in control roots. The stimulation of dark carbon fixation in NH₄ ⁺-grown plants, however, was not observed in MSX-treated roots. In the roots of NH₄ ⁺-grown plants, the concentration and ¹⁴C-Iabeling of acidic metabolites such as citrate and malate considerably decreased whereas those of basic metabolites, especially asparagine, increased noticeably. With MSX treatment, the incorporation of ¹⁴C into basic metabolites was negligible. In response to NH₄ ⁺, phosphoenolpyruvate carboxylase (PEPC) activity increased, and PEPC proteins accumulated in wheat roots. Neither activity nor amounts of PEPC in roots increased in the presence of MSX. These findings suggest that primary assimilation of NH₄ ⁺ in roots is essential for the stimulation of dark carbon fixation, which coincides with the increased activity of root PEPC, to sufficiently replenish carbon skeletons necessary for NH₄ ⁺ assimilation.     

Effects of astaxanthin‐enriched yeast on mucosal IgA induction in the jejunum and ileum of weanling mice

The present study was conducted to clarify the effects of astaxanthin‐enriched yeast on the concentration of immunoglobulin A (IgA), the numbers of IgA antibody‐secreting cells (ASC) and the messenger RNA (mRNA) expression of IgA C‐region in the jejunum and ileum of weanling mice. Weanling mice were fed rodent feed or astaxanthin‐enriched yeast‐supplemented rodent feed for 7, 14 or 21 days. Supplemental astaxanthin‐enriched yeast increased the numbers of IgA ASC in the jejunum and ileum after 7, 14 and 21 days of treatment. Supplemental astaxanthin‐enriched yeast increased IgA concentrations in the jejunum after 21 days of treatment, but IgA concentrations in the ileum were not affected by the treatment. The mRNA expressions of IgA C‐region in the jejunum after 14 and 21 days of treatment and the ileum after 14 days of treatment were enhanced by supplementation of astaxanthin‐enriched yeast. These results indicate that supplementation of astaxanthin‐enriched yeast is effective to enhance the numbers of IgA ASC in the jejunum and ileum and IgA concentrations in the ileum of weanling mice.     

Effects of high potassium chloride supplementation on water intake and bodyweight gains in pregnant and lactating mice

Thirty‐one ICR pregnant mice were assigned to a control or a potassium chloride (KCl) diet group to clarify the effects of KCl supplementation on water intake, bodyweight gains and serum components in pregnant and lactating mice, and 5% KCl was supplemented in KCl diets from 6.5 days post coitus to 1 or 14 days after parturition. Feed intake was not affected by treatment, but supplemental KCl decreased bodyweight gains of lactating mice and their neonatal mice. Water intake and urine volume of KCl supplemented mice were significantly higher than those of control mice during pregnancy and supplemental KCl decreased serum urea N in pregnant mice. Supplemental KCl increased water intake drastically in lactating mice immediately after parturition and increased serum K at 14 days after parturition. Histological alteration using hematoxylin–eosin was not found in the kidney of each mouse at 1 or 14 days after parturition. These results indicate that high KCl supplementation accelerates water intake in lactating mice and prevents bodyweight gains of maternal and neonatal mice during lactation.     

Welfare of lactating Holstein cows under outdoor grazing and indoor housing in relation to temperature and humidity in summer in Japan

Aim

The aim of this study was to investigate the differences in the physiological responses of lactating Holstein cows between outdoor grazing and indoor housing in summer in Japan.

Method

The cows (n=16) were grazed outside from indoor housing and returned indoors 3 weeks later, with a 1-week transition phase to mitigate each environmental change. Grazing cows remained in pasture except for milking twice per day and concentrate feeding before milking. Indoors, the cows were tethered except for milking twice per day and about 5.5 h of exercise in an open-air paddock.

Results

When the Temperature–Humidity Index (THI) threshold was above 72, urinary cortisol levels were higher only in the grazing phase. The indoor cows took a longer time to prepare to lie down compared with grazing cows.

Conclusions

This study suggests that forcing cows outside during hot weather can induce certain physiological stress responses in lactating Holstein cows. Cows exhibit more fluid lying-down movements on pasture, suggesting that the comfort of lying conditions of indoor housing was not ideal.     

No Mutations of Lysophosphatidic Acid Receptor Genes in Lung Adenocarcinomas Induced by N-Nitrosobis(2-hydroxypropyl)amine in Rats

Lysophosphatidic acid (LPA) is a bioactive phospholipid that stimulates cell proliferation and migration, and protects cells from apoptosis. It interacts with specific G protein-coupled transmembrane receptors. Recently, frequent mutations of the LPA receptor-1 (LPA1) gene were detected in rat lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl)amine (BHP). In this study, to evaluate the involvement of other LPA receptor gene alterations during lung carcinogenesis, we investigated mutations of the LPA2, LPA3, LPA4 and LPA5 genes in lung adenocarcinomas induced by BHP in rats. Fifteen male Wistar rats, 6 weeks of age, were given 2000 ppm BHP in their drinking water for 12 weeks and then maintained without further treatment until sacrifice at 25 weeks, and 15 adenocarcinomas were obtained. Genomic DNAs were extracted from frozen tissues, and the LPA2, LPA3, LPA4 and LPA5 genes were examined for mutations, using polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) analysis. No mutations of LPA2, LPA3, LPA4 and LPA5 were detected in the 15 adenocarcinomas. These results suggest that alterations due to LPA2, LPA3, LPA4 and LPA5 gene mutations might not be involved in the development of lung adenocarcinomas induced by BHP in rats.     

Ultrastructural analysis of responses of host and fungal cells during plant infection

Cellular responses in fungi and in susceptible or resistant hosts during fungus–plant interactions have been studied ultrastructurally to examine their role in pathogenicity. Pathogenicity is determined in some saprophytic fungi by various factors: the production of disease determinants such as the production of host-specific toxins (HSTs) or the extracellular matrix (ECM) by fungal infection structures and H₂O₂ generation from penetration pegs. Three different target sites for HSTs have been identified in host cells in many ultrastructural studies: plasma membranes, chloroplasts, and mitochondria. The mode of action of HSTs is characterized by the partial destruction of the target structures only in susceptible genotypes of host plants, with the result that the fungus can colonize the host. The infection structures of most fungal pathogen secrete ECM on plant surfaces during fungal differentiation, while the penetration pegs of some pathogens produce reactive oxygen species (ROS) in the cell walls and plasma membranes. The pathological roles of ECM and H₂O₂ generation are discussed here in light of ultrastructural evidence. Host and fungal characteristics in the incompatible interactions include the rapid formation of lignin in host epidermal cell walls, failure of penetration pegs to invade lignin-fortified pectin layers, the inhibition of subcuticular hyphal proliferation and the collapse of hyphae that have degraded cell walls within pectin layers of the host. Apoptosis-like host resistant mechanism is also discussed.     

Molecular cloning and biochemical characterization of medaka (Oryzias latipes) lysosomal neu4 sialidase

Glycoconjugates are known to be involved in many physiological events in vertebrates. Sialidase is one of the glycosidases, which removes sialic acid from glycoconjugates. In mammals, the properties and physiological functions of sialidases have been investigated, while there is little understanding of fish sialidase. Here, to investigate the significance of fish neu4 sialidase, neu4 gene was cloned from medaka brain mRNA and identified. Sialidase-specific motifs (GPG, YRVP and Asp-Box) were well conserved in the medaka neu4 polypeptide. Optimal pH of medaka neu4 sialidase was 4.6, but its activity was sustained even at neutral and weak alkaline pH. The neu4 considerably cleaved sialic acid from 4-methylumbelliferyl-N-acetyl-α-d-neuraminic acid and sialyllactose, but not from ganglioside and fetuin, which are good substrates for human NEU4. neu4 activity was mostly detected in mitochondria/lysosome fraction after biochemical fractionation, and indirect immunofluorescence assays revealed neu4 localization in lysosome in neu4 overexpressed cells. Next, developmental change in medaka neu4 and other sialidase mRNA levels were estimated by real-time PCR. Each sialidases showed different expression patterns in embryonic development: neu4 was up-regulated at late developmental stage in embryo, and neu3a mRNA level was quite high in 0.5 dpf. On the other hand, neu3b expression was drastically increased after hatching, suggesting that each sialidase may play a different role in embryonic development.     

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.