Evaluation of the drug sensitivity and expression of 16 drug resistance-related genes in canine histiocytic sarcoma cell lines

Canine histiocytic sarcoma (HS) is an aggressive tumor type originating from histiocytic cell lineages. This disease is characterized by poor response to chemotherapy and short survival time. Therefore, it is of critical importance to identify and develop effective antitumor drugs against HS. The objectives of this study were to examine the drug sensitivities of 10 antitumor drugs. Using a real-time RT-PCR system, the mRNA expression levels of 16 genes related to drug resistance in 4 canine HS cell lines established from dogs with disseminated HS were determined and compared to 2 canine lymphoma cell lines (B-cell and T-cell). These 4 canine HS cell lines showed sensitivities toward microtubule inhibitors (vincristine, vinblastine and paclitaxel), comparable to those in the canine B-cell lymphoma cell line. Moreover, it was shown that P-gp in the HS cell lines used in this study did not have enough function to efflux its substrate. Sensitivities to melphalan, nimustine, methotrexate, cytarabine, doxorubicin and etoposide were lower in the 4 HS cell lines than in the 2 canine lymphoma cell lines. The data obtained in this study using cultured cell lines could prove helpful in the developing of advanced and effective chemotherapies for treating dogs that are suffering from HS.     

Effects of utilization of local food by‐products as total mixed ration silage materials on fermentation quality and intake,digestibility, rumen condition and nitrogen availability in sheep

Four wethers were used in a 4 × 4 Latin square design experiment to evaluate in vivo digestibility of total mixed ration (TMR) silage with food by‐products for dairy cows, and the ruminal condition and nitrogen (N) balance were examined. Five by‐products (i.e. potato waste, noodle waste, soybean curd residue, soy sauce cake and green tea waste) were obtained. Four types of TMR silage were used: control (C) containing roughage and commercial concentrate, T1:20% and T1:40% containing the five by‐products replacing 20% and 40% of the commercial concentrate on a dry matter (DM) basis, respectively, and T2:40% containing three by‐products (potato waste, noodle waste and soybean curd residue) replacing 40% of the commercial concentrate on a DM basis. The ingredients were mixed and preserved in oil drum silos for 4 months. The TMR silages showed 4.02–4.44% and 1.75–2.19% for pH and lactic acid contents, respectively. The digestibility of DM and neutral detergent fiber, and total digestible nutrient content were higher (P < 0.05) for T2:40% feeding than for C feeding. Urinary nitrogen excretion tended to be lower (P = 0.07) for T2:40% than for C. The results suggested 40% replacing of commercial concentrate by using the three food by‐products can be most suitable for TMR silage.     

Intramammary infusion of an Enterococcus faecium SF68 preparation promoted the involution of drying off Holstein cows partly related to neutrophil‐associated matrix metalloproteinase 9

A problem for dairy cows following milk stasis is to cope with a high risk of intramammary infection and there is a need to initiate an extensive renewal of secretory modules in mammary glands so that milk production in next lactation may be optimized. We recently reported that ultrasonicated Enterococcus faecium SF68 (SF68) is compatible with cow mammary glands and an enhancer of innate immunity during the immediate post‐milk stasis period. The current study further examines the concomitant effect of ultrasonicated SF68 on mammary tissue remodeling. Four Holstein cows each received intramammary infusions of regular antibiotic dry‐cow formula (positive control) and two different doses of SF68 in different quarters. Analyses of individual quarter secretion samples showed faster neutrophil infiltration, earlier modifications in protein composition, including caseins and lactoferrins, as well as more prompt elevation of the specific unit of 92‐kDa matrix metalloproteinase 9 (MMP9) in SF68‐infused quarters compared to the positive controls. Intramammary infusion of ultrasonicated SF68 seems able to accelerate the regression of mammary synthetic capacity and potentiate the breakdown of glandular extracellular matrix, indicating a more efficient mammary gland involution. Correlation analyses imply that the ability of ultrasonicated SF68 to induce faster neutrophil chemotaxis and the associated MMP9 release is partly responsible.     

Simultaneous measurements of quantum yield and CO2 uptake for the assessment of non-assimilative electron flow in tree leaves

Using attached and detached leaves ofAcer palmatum Thunb. andRhaphiolepsis umbellata Makino, pulse-modulated chlorophyll fluorescence and CO₂ exchange were measured. Quantum yield of photosynthesis was determined from the fluorescence parameter(Fm′−Fs)/Fm′, where (Fm′−Fs) was defined as the difference between steady state chlorophyll fluorescence (Fs) and maximum fluorescence (Fm′) elicited by a saturating light pulse. The rate of electron transport through photosystem II (total electron flow) was calculated from the product of quantum yield andA (PFD), whereA is the rate of absorbed photons as given by leaf absorptance, and PFD is the photon flux density at the leaf surface. The rate of electron transport dependant on CO₂ uptake (assimilative electron flow) was calculated from the gross photosynthetic rate in a leaf. The difference between the rates of total and assimilative electron transport was denoted as the rate of non-assimilative electron transport which depends on photorespiration and oxygen reduction. Available data provided quantitative information on the rate of non-assimilative electron flow in intact leaves. When leaf photosynthesis ofA. palmatum was measured under sunlight, the rates of total and assimilative electron transport were determined to be approximately 900 and 150 μmol equiv. e/mg Chl·h, respectively. The difference (750 μmol equiv. e/mg Chl·h) was attributed to the activity of non-assimilative electron flow. The ratio of total to assimilative electron flow was found to increase gradually with rising in irradiance. The results suggest that non-assimilative electron flow occurred at much higher rate than assimilative electron flow at high irradiance. Implications of the results are briefly discussed in relation to photosynthesis limitation in tree leaves.     

Pollen dispersal in a hinoki (Chamaecyparis obtusa) seed orchard detected using a chloroplast DNA marker

Pollen dispersal was estimated in two test plots in a hinoki (Chamaecyparis obtusa) seed orchard using a chloroplast DNA marker, the spacer region between thetrnD andtrnY genes, and SSCP (single strand conformation polymorphism). In Plot 1, 2,020 seeds from 40 trees within 30 m of the marker tree were analyzed using the PCR-SSCP method. In Plot 2, 1,850 seeds from 37 trees were analyzed in the same manner. The results revealed that the maximum pollen dispersal distance in the two plots exceeded 25 m. Pollen dispersal appeared to be inversely proportional to the distance from the marker tree. The effective pollen dispersal was suggested to be less than about 20 m in a mature hinoki seed orchard. Adjacent trees had an excessive influence when the pollen density was increased by artificial flower stimulation. Therefore, it was suggested that seed production better resembles ideal random mating when carried out as naturally as possible. In conclusion, the SSCP chloroplast DNA marker was a useful tool for amassing basic information on pollen management in seed orchards of coniferous species.     

Improvement of functional properties of egg white protein through phosphorylation by dry-heating in the presence of pyrophosphate

Egg white protein (EWP) was phosphorylated by dry-heating in the presence of pyrophosphate at pH 3.0-7.0 and 85 degrees C for 1 and 5 days, and the functional properties of the phosphorylated EWP (PP-EWP) were investigated. The phosphorylation was accelerated with a decrease of pH from 7.0 to 3.0 and for heating times from 1 to 5 days. The phosphorus content of EWP increased approximately 1.05% by dry-heating at pH 4.0 and 85 degrees C for 5 days in the presence of pyrophosphate, which was higher than that of casein. The electrophoretic mobility of EWP increased with an increase in the phosphorylation level. The surface hydrophobicity of EWP increased by phosphorylation. The heat stability, emulsifying properties, and digestibility of EWP were improved by phosphorylation. The calcium phosphate-solubilizing ability of EWP was enhanced by phosphorylation. A firmer and transparent heat-induced gel of PP-EWP was obtained, and the water-holding capacity of heat-induced PP-EWP gel was higher that that of the control. These results suggest that phosphorylation by dry-heating in the presence of pyrophosphate is a useful method for improving the functional properties of EWP.     

Isolation and characterization of aminopeptidase (Jc-peptidase) from Japanese cedar pollen (Cryptomeria japonica)

An aminopeptidase, Jc-peptidase, was purified from Japanese cedar pollen by seven steps, including precipitation with ammonium sulfate, ion-exchange chromatography, gel filtration, hydrophobic interaction chromatography on phenyl-agarose, and high-performance liquid chromatography. Purified Jc-peptidease has a molecular weight of 42 kDa and hydrolyzes the synthetic substrates of L-phenylalanyl-4-methylcoumaryl-7-amide (Phe-MCA) with Km = 5 x 10(-5) M, Tyr-MCA with Km = 7 x 10(-4) M, Leu-MCA with Km = 1 x 10(-3) M, and Met-MCA with Km = 1 x 10(-3) M. Other MCA analogues such as Arg-MCA or Glu-MCA failed to serve as its substrates. The activity was inhibited in the presence of phebestin, [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-L-valyl]-L-phenylalanine, with Ki = 4.7 x 10(-5) M, or bestatin, [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl]-L-leucine, with Ki = 1.1 x 10(-4) M. According to amino acid sequence analysis, the N-terminal amino group seems to be blocked. The physiological function of the aminopeptidase (Jc-peptidase) has not been clarified in vivo.     

Anticancer activities of thelephantin O and vialinin A isolated from Thelephora aurantiotincta

Thelephora aurantiotincta is an edible mushroom belonging to the genus Thelephora; it grows in symbiosis with pine trees. Recently, phytochemical investigations have revealed that the genus Thelephora is an abundant source of p-terphenyl derivatives. However, their bioactivity has not yet been well characterized. In screening for natural materials with anticancer activity, a T. aurantiotincta ethanol extract (TAE) was found to decrease cell viability in human hepatocellular carcinoma cells (HepG2). In this study, a new p-terphenyl derivative, thelephantin O, and a known compound, vialinin A, were isolated as the principal bioactive components of TAE. These compounds decreased cell viability in HepG2 and human colonic carcinoma cells (Caco2), but not in noncancerous human hepatocytes. This is the first report of the isolation from T. aurantiotincta of selective cytotoxic agents against cancer cells.     

Ability of heifers to discriminate between familiar herdmates and members of an unfamiliar group: preference test and operant conditioning test

Using a preference test and operant conditioning in a Y-maze, this experiment examined the ability of heifers to discriminate between their own familiar herdmates and member(s) of an unfamiliar group. Sixteen Danish Friesian heifers, eight older animals (360.6 ± 24.2 days of age) and eight younger ones (190.1 ± 14.1 days of age) were used. Each age group was further divided into two experimental groups. Members of each of these groups were housed together in small pens before the experiments began. In experiment 1, each of the 16 animals was allowed to approach either a familiar or an unfamiliar individual in the Y-maze. The test was repeated 12 times, with a different unfamiliar subject for each test. In experiment 2, eight heifers were individually tested in a conditioning experiment to examine whether they could learn to discriminate between a group of their three herdmates and a group of three unfamiliar heifers. Test animals were rewarded when they chose their own group. In experiment 1, heifers did not show a preference between familiar and unfamiliar individuals. Interestingly the younger stimulus heifers but not the test animals showed an ability to discriminate between unfamiliar animals by vocalizing. In experiment 2, four of the eight test animals achieved the criterion for successful discrimination between the familiar and unfamiliar group ( P  < 0.003: binomial law). There was no age group difference in the ability to discriminate between familiar and unfamiliar animals. In conclusion, heifers did not show a preference toward familiar or unfamiliar individuals; but after conditioning, some heifers could learn to discriminate between familiar and unfamiliar groups.