Ca2+independent cytotoxicity of Vibrio parahaemolyticus thermostable direct hemolysin (TDH) on Intestine 407, a cell line derived from human embryonic intestine

Abstract The effects of Vibrio parahaemolyticus thermostable direct hemolysin on Intestine 407, a cell line derived from the intestine of human embryo, were investigated. The hemolysin was shown to be cytotoxic to Intestine 407. This cytotoxicity is accompanied by the damage of plasma membrane and lysosomes, as well as cellular degeneration in the form of large transparent blebs. Although an increase in cytosolic free Ca²⁺ due to the influx of extracellular Ca²⁺ was observed in cells treated with thermostable direct hemolysin, it was found to be irrelevant to any of the above effects. These results suggest that the effects of thermostable direct hemolysin observed in this study on Intestine 407 are not mediated by Ca²⁺-dependent pathways.     

Plant regeneration on cultured root segments of Cephaelis ipecacuanha A. Richard

Summary Simple one step micropropagation system for Cephaelis ipecacuanha A. Richard was developed using root cultures grown in vitro. Adventitious shoots were directly formed on the cut end of root segments without callus formation, on phytohormone-free B5 solid medium in the dark. When the shoots attached with root segments were further cultured under 16 h light / 8 h dark, they developed into plantlets, which could be transplanted to soil. The regenerated plants grew well in a greenhouse with showing normal appearance and accumulated alkaloids. The influence of auxin on adventitious shoot formation was also investigated.Abbreviations MS Murashige-Skoog (Murashige and Skoog 1962) - 1/2 MS half strength MS - B5 Gamborg B5(Gamborg et al. 1968) - WP woody plant (Lloyd and McCowm 1980) - RC root culture (Thomas and Davey 1982) - HF phytohormone free - IAA indole-3-acetic acid - NAA 1-naphtaleneacetic acid - TIBA 2,3,5-triiodobenzoic acid - 2,4,6-T 2,4,6-trichlorophenoxyacetic acid - SEM scanning electron microscopy - C.V. coefficient of variation     

Recognition by restriction endodeoxyribonuclease EcoRI of octadeoxyribonucleotides containing modified sugar and base moieties

The modified base and sugar moieties, 8-oxo-7,8-dihydroadenine (HO8A),8-methoxyadenine (MeO8A),8-methoxyguanine (MeO8G) and 9-[(2-hydroxy-1-(hydroxymethyl)-ethoxy)-methyl]-adenine (acA), were chemically introduced in place of the adenine or guanine in the octa-deoxyribonucleotides [d(GGAATTCC)] containing recognition sequence of Eco RI. to the regular DNA-fragment.     

Clinical pathology data from cynomolgus monkeys from China in which diarrhea was observed during quarantine.

Shin Nippon Biomedical Laboratories, Ltd. (SNBL) imported and quarantined 3,148 cynomolgus monkeys (aged 2.5 to 6.5 years) from China in 2002. The hematology and blood biochemistry data obtained from these monkeys on Day 32 of quarantine were analyzed separately by sex [2,890 animals in which no abnormalities were observed during the 35-day quarantine period (normal group), and 258 animals which exhibited diarrhea 1 to 12 times (diarrhea group)]. The values obtained for all parameters were within the normal range (mean +/- SD), and no significant abnormalities were noted in either sex. The clinical pathology data from 11 animals (6 males and 5 females) exhibiting diarrhea repeatedly (10 to 12 times) were statistically analyzed, and significant differences were noted in PLT and ALP in both sexes. The PLT values of these animals were within the normal group mean +/- 2 SD, and were considered within the normal range. A significant difference was noted in some individual ALP values (males: Nos. 2 and 3, females: Nos. 1, 3, and 4). The clinical pathology data obtained from the normal group in this study basically correspond to the widely reported results already obtained from healthy cynomolgus monkeys, from which it can be concluded that the cynomolgus monkeys from China were generally healthy and presenting no particular abnormality. The clinical pathology data from the normal group will serve as valuable baseline data for experimenters using cynomolgus monkeys.     

A novel low-density lipoprotein with large amounts of phospholipid found in the egg yolk of crustacea sand crayfish Ibacus ciliatus: its function as vitellogenin-degrading proteinase.

Low-density lipoprotein (LDL) with large amounts of phospholipid but not triacylglycerol was isolated from the egg yolk of crustacea sand crayfish Ibacus ciliatus as well as lipovitellin. LDL possessed vitellogenin-degrading proteinase activity. Hemolymph vitellogenin was degraded by incubating with LDL at pH 4.5 for 72 hr at 35 degrees C and apolipoprotein profiles of vitellogenin degraded by LDL were very similar to those of lipovitellin in the egg.     

Focus on Weed Control: A Novel Rice Cytochrome P450 Gene,CYP72A31, Confers Tolerance to Acetolactate Synthase-Inhibiting Herbicides in Rice and Arabidopsis

Target-site and non-target-site herbicide tolerance are caused by the prevention of herbicide binding to the target enzyme and the reduction to a nonlethal dose of herbicide reaching the target enzyme, respectively. There is little information on the molecular mechanisms involved in non-target-site herbicide tolerance, although it poses the greater threat in the evolution of herbicide-resistant weeds and could potentially be useful for the production of herbicide-tolerant crops because it is often involved in tolerance to multiherbicides. Bispyribac sodium (BS) is an herbicide that inhibits the activity of acetolactate synthase. Rice (Oryza sativa) of the indica variety show BS tolerance, while japonica rice varieties are BS sensitive. Map-based cloning and complementation tests revealed that a novel cytochrome P450 monooxygenase, CYP72A31, is involved in BS tolerance. Interestingly, BS tolerance was correlated with CYP72A31 messenger RNA levels in transgenic plants of rice and Arabidopsis (Arabidopsis thaliana). Moreover, Arabidopsis overexpressing CYP72A31 showed tolerance to bensulfuron-methyl (BSM), which belongs to a different class of acetolactate synthase-inhibiting herbicides, suggesting that CYP72A31 can metabolize BS and BSM to a compound with reduced phytotoxicity. On the other hand, we showed that the cytochrome P450 monooxygenase CYP81A6, which has been reported to confer BSM tolerance, is barely involved, if at all, in BS tolerance, suggesting that the CYP72A31 enzyme has different herbicide specificities compared with CYP81A6. Thus, the CYP72A31 gene is a potentially useful genetic resource in the fields of weed control, herbicide development, and molecular breeding in a broad range of crop species.The mechanism of herbicide tolerance can be classified roughly into two groups: target-site and non-target-site herbicide tolerance (Powles and Yu, 2010). Target-site herbicide tolerance is caused by the prevention of herbicide binding to the target enzyme, caused by point mutations occurring in the latter. It is relatively easy to elucidate the molecular mechanisms of target-site herbicide tolerance, because it is regulated mostly by a single gene encoding a target enzyme harboring point mutations. On the other hand, non-target-site herbicide tolerance is caused by reduction to a nonlethal dose of herbicide reaching the target enzyme, caused by mechanisms such as activation of herbicide detoxification, decrease of herbicide penetration, and herbicide compartmentation in plant cells (Yuan et al., 2007). Among these mechanisms, the oxidization of herbicides by endogenous cytochrome P450 monooxygenase is thought to be a major pathway in plants (Werck-Reichhart et al., 2000; Siminszky, 2006; Powles and Yu, 2010). From the point of view of weed control, non-target-site herbicide tolerance is a greater threat to crop production and in the evolution of herbicide-resistant weeds, because it is often involved in resistance to multiherbicides that inhibit different target proteins, including never-used and potential plant growth regulators (Yuan et al., 2007; Powles and Yu, 2010). Conversely, it is expected that multiherbicide-tolerant crops could be produced easily by the application of non-target-site herbicide tolerance. Moreover, information gained from study of the molecular mechanisms of non-target-site herbicide tolerance can be applied to the research and development of novel herbicides and plant growth regulators.Acetolactate synthase (ALS; also known as acetohydroxy acid synthase) plays a key role in the biosynthesis of branched-chain amino acids such as Val, Leu, and Ile in many organisms. ALS is the primary target site for at least four classes of herbicides: sulfonylurea, imidazolinone, pyrimidinyl carboxylates, and triazolopyrimidine herbicides (Shimizu et al., 2002, 2005). These herbicides can inhibit ALS activity, resulting in plant death caused by a deficiency of branched-chain amino acids. ALS-inhibiting herbicides control many weed species in addition to exhibiting high selectivity in major crops and low toxicity to mammals, which lack the branched-chain amino acid biosynthetic pathway. However, various mutations in ALS that confer ALS-inhibiting herbicide tolerance have been found in many weeds (Shimizu et al., 2005; Powles and Yu, 2010). Similar mutations in ALS have also been reported in crops (Shimizu et al., 2005). To date, crops that show tolerance to ALS-inhibiting herbicides have been produced by various approaches, such as conventional mutation breeding, conventional transformation, and pinpoint mutagenesis via gene targeting based on information obtained from analyses of ALS mutants (Shimizu et al., 2005; Endo and Toki, 2013). On the other hand, weeds that show tolerance to ALS-inhibiting herbicides by cytochrome P450-mediated detoxification have also been reported (Powles and Yu, 2010). However, compared with target-site herbicide tolerance, little is known of the molecular mechanism of herbicide metabolism mediated by cytochrome P450. In rice (Oryza sativa), an herbicide-sensitive mutant has been produced by γ-ray irradiation (Zhang et al., 2002). This mutant showed 60-fold higher sensitivity to bensulfuron-methyl (BSM), a sulfonylurea herbicide, compared with wild-type rice (Pan et al., 2006). Genetic mapping and complementation tests revealed that a cytochrome P450, CYP81A6, is involved in BSM tolerance (Pan et al., 2006). As far as we know, this is the only example of the isolation and characterization of a cytochrome P450 gene involved in nontarget herbicide tolerance in rice.Bispyribac sodium (BS), a pyrimidinyl carboxylate herbicide, is effective in controlling many annual and perennial weeds, with excellent selectivity on direct-seeded rice (Shimizu et al., 2002). Recently, it was reported that japonica rice varieties show higher sensitivity to BS compared with indica rice varieties at the early stages of plant growth (Ohno et al., 2008; Taniguchi et al., 2010). A mutated ALS gene confers BS tolerance in plants including rice (Shimizu et al., 2005; Endo and Toki, 2013). However, the deduced amino acid sequences were shown to be highly conserved among japonica and indica rice varieties, and ALS levels of sensitivity to BS were similar in japonica and indica rice varieties (Taniguchi et al., 2010). These results suggest the possibility that indica rice varieties might show higher tolerance to BS due to the acquisition of nontarget herbicide tolerance.In this study, we isolated and characterized a novel cytochrome P450 gene, CYP72A31, involved in BS tolerance in rice. We also demonstrated that overexpression of CYP72A31 confers tolerance to ALS-inhibiting herbicides, including BS and BSM, in Arabidopsis (Arabidopsis thaliana).     

What makes wild chimpanzees wake up at night?

I examined the possible cause of night awakening among wild chimpanzees (Pan troglodytes) in Mahale Mountains National Park, Tanzania. Chimpanzee vocalizations and activity-related sounds (CVSs) were used to indicate awakening because I was unable to visually observe them. Over a 5-night observation period, CVSs (n = 128) were heard every night, and most (n = 91) were observed within 5 min of previous CVSs. Chimpanzees use CVSs as social communication to maintain spatial contact with other chimpanzees who occasionally travel at night. The first sound in a sequence of CVSs (CVS bout) was heard immediately following the vocalization or sound of another animal (n = 11), defecation or urination by a chimpanzee (n = 7), or unknown (n = 19). CVS bouts were longer when preceded by defecation or urination than when preceded by the vocalization or sound of other animals or an unknown factor. This suggests that the degree of wakefulness varies according to the possible cause of the disturbance. CVSs at night may be provoked by various factors, and awakening during the night is probably common among diurnal primates.     

Elevated mitochondrial biogenesis in skeletal muscle is associated with testosterone-induced body weight loss in male mice

Androgen reduces fat mass, although the underlying mechanisms are unknown. Here, we examined the effect of testosterone on heat production and mitochondrial biogenesis. Testosterone-treated mice exhibited elevated heat production. Treatment with testosterone increased the expression level of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α), ATP5B and Cox4 in skeletal muscle, but not that in brown adipose tissue and liver. mRNA levels of genes involved in mitochondrial biogenesis were elevated in skeletal muscle isolated from testosterone-treated male mice, but were down-regulated in androgen receptor deficient mice. These results demonstrated that the testosterone-induced increase in energy expenditure is derived from elevated mitochondrial biogenesis in skeletal muscle.     

Adipose-derived stromal cells inhibit prostate cancer cell proliferation inducing apoptosis

Mesenchymal stem cells (MSCs) have generated a great deal of interest in the field of regenerative medicine. Adipose-derived stromal cells (AdSCs) are known to exhibit extensive proliferation potential and can undergo multilineage differentiation, sharing similar characteristics to bone marrow-derived MSCs. However, as the effect of AdSCs on tumor growth has not been studied sufficiently, we assessed the degree to which AdSCs affect the proliferation of prostate cancer (PCa) cell. Human AdSCs exerted an inhibitory effect on the proliferation of androgen-responsive (LNCaP) and androgen-nonresponsive (PC3) human PCa cells, while normal human dermal fibroblasts (NHDFs) did not, and in fact promoted PCa cell proliferation to a degree. Moreover, AdSCs induced apoptosis of LNCaP cells and PC3 cells, activating the caspase3/7 signaling pathway. cDNA microarray analysis suggested that AdSC-induced apoptosis in both LNCaP and PC3 cells was related to the TGF-β signaling pathway. Consistent with our in vitro observations, local transplantation of AdSCs delayed the growth of tumors derived from both LNCaP- and PC3-xenografts in immunodeficient mice. This is the first preclinical study to have directly demonstrated that AdSC-induced PCa cell apoptosis may occur via the TGF-β signaling pathway, irrespective of androgen-responsiveness. Since autologous AdSCs can be easily isolated from adipose tissue without any ethical concerns, we suggest that therapy with these cells could be a novel approach for patients with PCa.