High-throughput technique for comprehensive analysis of Japanese green tea quality assessment using ultra-performance liquid chromatography with time-of-flight mass spectrometry (UPLC/TOF MS)

Applications of metabolomics techniques along with chemometrics provide an understanding in the relationship between metabolome of green tea and its quality. A coupled of ultra-performance liquid chromatography with time-of-flight mass spectrometry (UPLC/TOF MS) allowed a high-throughput and comprehensive analysis with minimal sample preparation. Using this technique, a wide range of metabolites were investigated. Data analysis was rapid, considering that the fingerprinting technique was performed. A set of green tea samples from 2006 tea contest of the Kansai area was analyzed to prove usefulness of the developed technique. Green tea with different qualities were discriminated through principal component analysis (PCA). Consequently, projection to latent structure by means of partial least-squares (PLS) was performed to create a constructive quality-predictive model by means of metabolic fingerprinting. Beside epigallocatechin, other predominant catechins, including epigallocatechin gallate and epicatechin gallate, detected in green tea were found to be significant biomarkers to the high quality of Japanese green tea (Sencha).     

Influence of foliar phosphorus and nitrogen contents on chemical properties of water extractable organic matter derived from fresh and decomposed sugar maple leaves

The effects of watershed-scale experimental acidification on the macronutrient content and decomposition of sugar maple (Acer saccharum Marsh) leaves were investigated. Bear Brook Watershed in Maine (BBWM) is a paired forest watershed study where the West Bear (WB) watershed has been treated bi-monthly with 1800 eq ha^?1 yr^?1 of (NH₄)₂SO₄ since 1989, and the adjacent East Bear (EB) watershed has acted as a reference. Leaf samples collected from the treated WB watershed had significantly higher concentrations of N and P than leaves from the reference EB watershed. Leaves from both watersheds were decomposed for a 10-day laboratory incubation. Extractable total soluble carbon (C_TS) content of the leaves decreased following decomposition to a greater extent in WB leaves than in EB leaves. Spectroscopic and chromatographic chemical analyses indicated similar chemical properties for the fresh WB and EB WEOM. However, after decomposition, the WB WEOM was more humified as compared to EB WEOM indicating that the watershed treatment resulted in leaves which were more biodegradable than those in the reference watershed. Multi-dimensional fluorescence spectroscopy with parallel factor analysis (PARAFAC) modeled five components: tyrosine-like, three humic substance-like, and terrestrial/anthropogenic associated-like fluorophores. Following decomposition, the relative concentrations of two of the humic-associated components increased to a significantly greater extent for WB than for EB WEOM. These observations were consistent with greater decomposition-related changes to the WEOM from WB samples relative to EB samples. Pearson correlation analysis showed that foliar N and P concentrations were positively correlated with indices of humification. Adsorption of WEOM to goethite and gibbsite was significantly greater for decomposed WB WEOM than EB WEOM. These results demonstrate that greater leaf N and P contents can increase short-term decomposition, accelerate production of more humic-like WEOM, and thereby potentially influence the distribution of organic matter within the soil carbon pool.     

Kisspeptin‐10 stimulates the release of luteinizing hormone and testosterone in pre‐ and post‐pubertal male goats

The aims of the present study were to clarify the effect of kisspeptin‐10 (Kp10) on the secretion of luteinizing hormone (LH) and testosterone (T) in pre‐pubertal and post‐pubertal male ruminants. Four male goats (Shiba goats) were given an intravenous (i.v.) injection of Kp10 (5 µg/kg body weight (b.w.)), gonadotoropin‐releasing hormone (GnRH, 1 µg/kg b.w.), or 2 mL of saline as a control at the ages of 3 (pre‐pubertal) and 6 (post‐pubertal) months. A single i.v. injection of Kp10 significantly stimulated the release of LH and T in both groups. The area under the response curve (AUC) of LH for a 60‐min period after the i.v. injection of Kp10 was significantly greater in the pre‐pubertal goats (P < 0.05). The AUC of T for a 120 min period post‐injection did not differ between the two age groups. A single i.v. injection of GnRH also significantly stimulated the release of LH and T in both groups (P < 0.05). The secretory pattern of LH and T in response to GnRH resembled that in response to Kp10. These results show that the LH‐releasing response to Kp10 is greater in pre‐pubertal than post‐pubertal male goats. They also show that Kp10, as well as GnRH, is able to stimulate the release of T in male goats.     

The genetic mechanisms of warfarin resistance in Rattus rattus found in the wild in Japan

Warfarin is commonly used worldwide as a rodenticide. It inhibits blood coagulation by inhibiting vitamin K 2,3-epoxide reductase (VKOR) activity leading to hemorrhage. However, it has been reported that repeated or long-term treatment with warfarin results in resistance emerging in wild rodents. Such resistance may explain why it is difficult to control rodents in many regions in Japan. In this report, we studied mutations in the VKOR gene (including the VKOR complex subunit 1 (VKORC1)), while also analyzing VKOR and clotting factor activity in black rats (Rattus rattus) in order to understand better the mechanism of warfarin resistance in this species.We sequenced the VKORC1 gene from 275 rats living in the wild in Japan. We found several types of novel base substitutions, some of which conferred warfarin resistance.There was no difference in coagulation times between warfarin-sensitive and resistant rats measured under physiological conditions. However, after warfarin administration, no effect was noted in warfarin-resistant rats, although a prolonged coagulation time was noted in warfarin-sensitive rats.We also determined the kinetic differences in hepatic microsomal VKOR-dependent activity between warfarin-resistant and sensitive rats. Warfarin-resistant rats showed 2-3-fold lower V_max/K_m values than did sensitive rats. In addition, we report that resistant rats found in the Tokyo area had a VKOR activity which was poorly inhibited by warfarin.Finally, we conclude that reduced VKOR activity and warfarin resistance in the Japanese black rat might be due to mutations in the VKORC1 gene. However, further study is needed to clarify how such rats can maintain adequate vitamin K-dependent clotting factor levels, while simultaneously exhibiting low VKOR activity and warfarin resistance.     

Comparative study of the plasma globulin level, CD21(-) B-cell counts and FOXP3 mRNA expression level in CD4(+) T-cells for different clinical stages of feline immunodeficiency virus infected cats

Feline immunodeficiency virus (FIV) infection leads to hypergammaglobulinemia through mechanisms that remain poorly understood. We investigated changes in plasma globulin level, B cells, and T cells with progression of the clinical stage of FIV-infected cats. We classified FIV-infected cats into the stage of Asymptomatic carrier (AC) and AIDS-related complex (ARC) based on the clinical symptoms, and measured the plasma globulin level, the CD4(+) T-cell counts, and analyzed surface markers of B cells. We investigated the relationship between the plasma globulin level and regulatory T cells (Tregs) using the Forkhead box P3 (FOXP3) mRNA expression level. In FIV-infected cats, the plasma globulin level and the surface immunoglobulin (sIg)(+) CD21(-) B-cell counts were increased, whereas the CD4(+) T-cell counts were decreased compared with specific-pathogen free (SPF) cats. The mRNA expression of Blimp-1 (master gene of plasma cells) was increased in peripheral blood, and the FOXP3 mRNA expression level was decreased in CD4(+) T-cells. These immunological changes were marked in the ARC stage. These data indicate that the decrease of Tregs and the increase of plasma cells lead to hypergammaglobulinemia.     

Characteristics of growth and quality,and factors contributing to high yield in newly developed rice variety ‘Akidawara’

The newly developed rice variety ‘Akidawara’ (AKI) combines the traits of high yield and highly palatability, and its cultivation is expected to spread. We examined characteristics of growth and quality, and factors contributing to high yield in AKI by comparing with ‘Nipponbare’ (NIP). Grain yield for AKI were 703 g m^?2 (9% more than NIP) and 781 g m^?2 (14% more than NIP) under the standard and heavy fertilizer regimes. It was also suggested that increase in the sink capacity was the key contributors to the high yield in AKI resulting from a conspicuous increase in the number of spikelets, which is likely due to introgression of the high-yielding variety allele. Furthermore, AKI achieved the similar degree of sink filling in spite of its larger sink capacity. In this point, panicle dry weight increase during ripening (ΔP) was significantly higher for AKI than for NIP despite the fact that no differences in shoot dry weight increase were observed between varieties. The greater ΔP in AKI might be derived from its larger sink capacity and the difference between varieties involves the translocation of nonstructural carbohydrate. In the grain quality, the reduction in perfect grain ratio was negligible and regarded as a small trade-off for AKI’s 14% increase in yield, and grain protein content increased to a lesser degree in AKI at the same yield level. These results suggest that over 700 g m^?2 high yield can be achieved with relative high grain quality and lower protein content in AKI.     

Effect of theanine, γ-glutamylethylamide, on bodyweight and fat accumulation in mice

To clarify the details of the anti‐obesity effect of theanine, γ‐glutamylethylamide, female ICR mice were fed diets containing theanine at the concentrations of 0.01, 0.02, 0.04, 0.08 and 0.16% for 16 weeks. Bodyweight and food intake in the mice were measured every 4 weeks, and several organs and intraperitoneal adipose tissues (IPAT) were weighed after the feeding. In addition, lipid levels in the serum and liver were analyzed. As a result, the bodyweight increase and weight of IPAT were significantly reduced in mice fed 0.04% theanine compared with controls, but not in mice fed other diets. The levels of triglycerides (TG) and non‐esterified fatty acids in the serum and TG level in the liver in mice fed the 0.04% theanine diet were remarkably decreased. These results indicated that bodyweight increase and fat accumulation were suppressed by a limited concentration of 0.04% theanine in mice.     

Relationship between fat accumulation in the liver and energy intake, milk fat yield and blood metabolites in dairy cows

In the present study, 42 multiparous Holstein cows were used to investigate the relationship between fat accumulation in the liver and dry matter intake, milk yield and blood metabolites. Based on the percentage of fat in the liver cell at 2 weeks post‐parturition, the cows were classified into three groups. These groups were: (i) less than 10% of fat (normal group, n = 29); (ii) 10–20% of fat (mild group, n = 6); and (iii) more than 20% of fat (moderate group, n = 7). The bodyweight of the moderate group was high (771 kg) before calving. The sufficiency rates of total digestible nutrients (TDN) were remarkably decreased (approximately 65%) in early lactation. The milk fat yield and milk fat composition of the moderate group were higher (P < 0.05) than the other groups at 1 and 2 weeks post‐parturition. It was suggested that non‐esterified fatty acids (NEFA) mobilized from adipose tissues was directly used by the mammary gland for synthesis of milk fat. The percentage of bromsulfalein (BSP) retention of the moderate group was high (21.1%) at 30 min, and it showed that the BSP clearance function was significantly decreased. The concentrations of NEFA, β‐hydroxybutyric acid and glucose were appropriate indicators of energy status; however, aspartate aminotransferase, γ‐glutamyl transpeptidase and total bilirubin were not sensitive indicators of a moderate fatty liver. Thus, high‐yielding cows that calve in an overweight condition are more likely to develop excessive fat accumulation in the liver because of great mobilization from adipose tissues post‐parturition. In cows with a moderately fatty liver, a decrease in TDN sufficiency rates, an increase of milk fat yield and a reduction of liver function were observed in early lactation. The increase of serum NEFA and milk fat composition resulting from mobilization of adipose tissues helped to diagnose moderate fatty liver.     

The absence of Rhodococcus equi in Mongolian horses

In native Mongolian horses, the incidence and distribution of Rhodococcus equi are poorly understood. One hundred and fourteen equine fecal samples and 71 soil samples were collected from the camp sites of 26 nomadic families located in three areas less than 100 km from Ulaanbaatar, Mongolia. Five fecal samples were also collected from foals of Przewalski's Horses introduced into the Hustai National Park, Mongolia. No R. equi was isolated from the Mongolian horses or the soil samples. However, three colonies of R. equi were isolated from two fecal samples collected from foals of Przewalski's Horses. These isolates were avirulent, with neither 15- to 17-kDa antigens (VapA) nor a 20-kDa antigen (VapB) genes being detected. We concluded that native Mongolian horses and their environment appear free from contamination with R. equi.