Mutation of alpha-tubulin genes in trifluralin-resistant water foxtail (Alopecurus aequalis)

BACKGROUND: Trifluralin‐resistant biotypes of water foxtail (Alopecurus aequalis) have been identified in wheat fields from northern Kyushu, Japan. Water foxtail is a winter‐annual grassy weed, causing substantial crop losses. This study reports on mutation in α‐tubulin (TUA) genes from water foxtail, the site of action of trifluralin. RESULTS: Two trifluralin‐sensitive (S) Chikugo and Ukiha biotypes and four trifluralin‐resistant (R) Asakura‐1, Asakura‐2, Tamana and Tosu biotypes of water foxtail were used for herbicide resistance analysis. R biotypes showed 5.7–30.7‐fold trifluralin resistance compared with the S biotypes. No differences in the uptake and translocation of ¹⁴C‐trifluralin were observed between Chikugo (S) biotype and Asakura‐1 (R) biotype. Most of the ¹⁴C detected in the plant material was in the root tissue, and no substantial increases were noted in shoot tissues. Comparative TUA sequence analysis revealed two independent single amino acid changes: change of Val into Phe at position 202 in TUA1 and change of Leu into Met at position 125 in TUA3 in Asakura‐1 biotype. In the Tamana (R) biotype, two amino acid changes of Leu to Phe at position 136 and Val to Phe at position 202 were observed in the predicted amino acid sequence of TUA1, compared with Chikugo (S) biotype. CONCLUSION: The results provide preliminary molecular explanation for the resistance of water foxtail to trifluralin, a phenomenon that has arisen as a result of repeated exposure to this class of herbicide. This is the first report of α‐tubulin mutation in water foxtail and for any Alopecurus species reported in the literature. Copyright © 2011 Society of Chemical Industry     

Urinary free metanephrines measurement in dogs with adrenal gland diseases using a new simple liquid chromatography tandem mass spectrometry method

Measurement of urinary metanephrines in spot samples is used for the diagnosis of canine pheochromocytoma (PC). We describe a simple analytical method based on liquid chromatography tandem mass spectrometry (LC-MS/MS) for measuring free metanephrine (MN) and normetanephrine (NMN) in spot urine samples. Using the developed method, we evaluated the stability of urinary free-MN and free-NMN at various storing conditions. In addition, we assessed the feasibility of urinary free-MN and -NMN measurement for diagnosing PC. Urine samples were mixed with stable isotope internal standards and thereafter purified by ultrafiltration. The purified samples were analyzed by LC-MS/MS in the multiple reaction monitoring mode after separation on a multimode octa decyl silyl column. The coefficient of variation of free-MN and -NMN measurement was 7.6% and 5.5%, respectively. The linearity range was 0.5–10 µg/l for both analytes. Degradation was less than 10% for both analytes under any of the storage conditions. The median free-NMN ratio to creatinine of 9 PC dogs (595, range 144–47,961) was significantly higher (P<0.05) than that of 13 dogs with hypercortisolism (125, range 52–224) or 15 healthy dogs (85, range 50–117). The developed method is simple and may not require acidification of spot urine. The results of this preliminary retrospective study suggest that the measurement of urinary free metanephrines is a promising tool for diagnosing canine PC.     

Relationship between diarrhea and peripheral leukocyte population in neonatal Japanese black calves

Neonatal Japanese Black (JB) calves show a high incidence of diarrhea. The objective of this study was to analyze the immune cell populations of neonatal JB calves in detail and examine its correlation with the incidence of diarrhea immediately after birth. Understanding the immune cell populations is helpful in clinics in order to determine the condition of the immune system for prevention of diseases. Blood samples were obtained from JB calves on the day of birth. The peripheral leukocyte populations were analyzed separately for calves that had diarrhea within 2 weeks after birth (diarrhea group; n = 26) and for calves without diarrhea (control group; n = 74). The numbers of the peripheral blood CD3(+)TcR1-N12(+) and CD8(+) T cells were significantly lower in the diarrhea group compared with the control group. These findings suggest that the congenital lower peripheral γδ and CD8(+) T cells results in a high risk of diarrhea in neonatal JB calves.     

A rapid and simple method to obtain canine peripheral blood-derived macrophages

Macrophages play an important role in a variety of situations, including pathogen elimination, inflammation, and tissue repair. However, these cells are not fully studied in dogs, in part, due to the difficulty of efficiently isolating and culturing them in vitro. In this study, we cultured canine peripheral blood mononuclear cells (PBMCs) with 10 ng/ml of phorbol 12-myristate-13-acetate (PMA) for 5 days to obtain macrophages. A high number of round-adherent cells were obtained without the addition of any cytokine. These cells showed active phagocytic activity and a cell surface antigen profile different from dendritic cells. Our method facilitates a high yield of macrophages in a short cultivation period compared with previous studies. This method might be a powerful tool to study macrophage functions in dogs.     

Proteome analysis of whole and water‐soluble proteins in masseter and semitendinosus muscles of Holstein cows

To assess both quantitative and qualitative differences between the slow‐ and fast‐type muscles, masseter (slow) and semitendinosus (fast) from four Holstein cows were analyzed by two‐dimensional difference gel electrophoresis (2D DIGE) and mass spectrometry. The proteome analysis identified 27 spots as 20 proteins in the whole protein fraction extracted with 8 mol/L urea solution, and 16 spots were identified as 11 proteins in the water‐soluble protein fraction. Two slow‐type myofibrillar proteins (myosin light chain‐1 slow‐b and myosin light chain‐2 slow), and aconitase‐2 mitochondria were present at higher levels in the masseter muscle (P < 0.05). Four fast‐type myofibrillar proteins (myosin light chain‐1 fast, myosin light chain‐2 fast, myosin light chain‐3 fast and tropomyosin‐1), and three enzymes of glycolytic pathway (enolase‐3, aldolase‐A and triosephosphate isomerase), were present at higher levels in the semitendinosus muscle (P < 0.05). Our proteome analysis showed that the composition of sarcoplasmic proteins as well as myofibrillar proteins was clearly different between slow‐ and fast‐type muscles.     

Characteristics of cytochrome P450-dependent metabolism in the liver of the wild raccoon,Procyon lotor

Wildlife is exposed to a wide range of xenobiotics in the natural environment. In order to appropriately assess xenobiotic-induced toxicity in wildlife, it is necessary to understand metabolic capacities. Carnivores, in general, have low metabolic abilities, making them vulnerable to a variety of chemicals. Raccoons (Procyon lotor) in the wild have been found to have high levels of xenobiotics. However, little is known about the metabolic capacity of the cytochrome P450 (CYP) enzymes in this species. Thus, this study used liver samples to investigate the characteristics of CYP enzymes in wild raccoons. In 22 wild raccoons, CYP concentrations in hepatic microsomes were examined. To better understand the properties of CYP-dependent metabolism, in vitro metabolic activity studies were performed using ethoxyresorufin, pentoxyresorufin and testosterone as substrates. In addition, three raccoons were fed commercial dog food in the laboratory for one week, and the effects on CYP-dependent metabolism were investigated. In comparison to other mammalian species, raccoons had very low concentrations of CYP in their livers. In an in vitro enzymatic analysis, raccoons’ ethoxyresorufin O-deethylase (EROD) and pentoxyresorufin O-depentylase (PROD) metabolic capacities were less than one-fifth and one-tenth of rats’, respectively. These results indicate the possible high risk in raccoons if exposed to high levels of environmental xenobiotics because of their poor CYP activity. In this study, the features of CYP-dependent metabolism in wild raccoons are described for the first time.     

National surveillance of Salmonella enterica in food-producing animals in Japan

A total of 518 fecal samples collected from 183 apparently healthy cattle, 180 pigs and 155 broilers throughout Japan in 1999 were examined to determine the prevalence and antimicrobial susceptibility of Salmonella. The isolation rates were 36.1% in broilers, 2.8% in pigs and 0.5% in cattle. S. enterica Infantis was the most frequent isolate, found in 22.6% of broiler fecal samples. Higher resistance rates were observed against oxytetracycline (82.0%), dihydrostreptomycin (77.9%), kanamycin (41.0%) and trimethoprim (35.2%). Resistance rates to ampicillin, ceftiofur, bicozamycin, chloramphenicol and nalidixic acid were <10%. CTX-M-2 β-lactamase producing S. enterica Senftenberg was found in the isolates obtained from one broiler fecal sample. This is the first report of cephalosporin-resistant Salmonella directly isolated from food animal in Japan.     

Sooty spot caused by Cladosporium cladosporioides in postharvest Satsuma mandarin grown in heated greenhouses

In June 2006, a large number of small black spots containing a branching network of black hyphae developed on shipped Satsuma mandarin fruits grown in greenhouses, which markedly decreased their commercial value. The fungus that was frequently isolated from the damaged fruits was identified as Cladosporium cladosporioides. Inoculation of fruits with the fungus caused the same symptoms, and the fungus was reisolated from the diseased fruits. The disease caused by this fungus has not been previously documented in citrus fruits; we name this new disease “sooty spot” on the basis of its symptoms.