Quantitative and specific detection of a trichloroethylene-degrading methanotroph, Methylocystis sp. strain M, by a most probable number-polymerase chain reaction method

We developed a rapid and specific enumeration method for a trichloroethylene-degrading methanotroph, Methylocystis sp. strain M, based on a most probable number-polymerase chain reaction method for monitoring the bacterium at bioremediation sites. The primers designed for the mmoC gene of the soluble methane monooxygenase gene cluster were specific to strain M. Recovery of the cells with a membrane filter enabled us to detect strain M in trichloroethylene-contaminated groundwater. We used the enumeration method to monitor the number of strain M cells in effluent from soil columns supplied with trichloroethylene-contaminated groundwater. The number of strain M cells in the effluent depended on the amount of the strain M inoculated and the number of cells measured by the most probable number-polymerase chain reaction method was correlated with that measured by a culture method. The detection limit for strain M in effluent detected by MPN-PCR method was 4 to 8 x 10(2) cells/ml.     

Tamarindus indica L. leaf is a source of allelopathic substance

The allelopathic potential of the Tamarindus indica L. leaf was investigated through bioassay guided studies using several weed and edible crop species. Both radicle and hypocotyl growth of all the plant species tested was strongly inhibited by the tamarind leaf using a sandwich method. The growth of weed species was reduced more than that of edible crop species. Among the weed species, barnyard grass followed by white clover, and in the edible crop species, lettuce followed by radish ranked top in terms of growth inhibition. Different concentrations of tamarind leaf crude water-soluble extract exhibited a strong inhibition in all the plant species tested and, by contrast, the magnitude of inhibition in the weed species was higher than in edible crop species and ranged from 30–75%. The 10% concentration of the tamarind leaf crude water-soluble extract was most potent against growth of seedlings. The concentrations of the nutrient components were linearly correlated with an increase in the concentration of tamarind leaf crude water-soluble extract. No significant changes in either pH or EC were found in the variations of different concentrations of tamarind leaf crude water-soluble extracts. As compared to control, growth of both radicle and hypocotyl in weed (barnyard grass and white clover) and in edible crop (lettuce and radish) species were significantly reduced when blended tamarind leaves at different concentrations were incorporated into the growth medium. The inhibitory magnitude increased with an increase in the concentration of the tamarind leaf. In terms of growth inhibition, among these tested plants, weed species particularly barnyard grass were most sensitive to the allelochemicals exuded from blended tamarind leaves. When the blended tamarind leaves were removed from the growth medium, all the seedlings grew quickly and the percentage of recovery was between 76–97% of the corresponding controls. Reduction in the fresh and dry weight of these 4 plant species was observed under the experimental conditions, and ranged between 33–42% and 40–53% in the radicle and hypocotyl, respectively. The fresh and dry weight, and total chlorophyll content declined significantly in the incorporated tamarind leaf treatments. Compared to the control, the highest drop in the chlorophyll content of 60% in barnyard grass was observed with the 10% concentration of the leaf treatment. These results clearly indicate that the tamarind leaf contains one or more strong biologically active allelochemical(s) that function as true growth regulator(s) and is involved in plant growth regulation, particularly in weed species.     

Myotube formation is affected by adipogenic lineage cells in a cell-to-cell contact-independent manner

Intramuscular adipose tissue (IMAT) formation is observed in some pathological conditions such as Duchenne muscular dystrophy (DMD) and sarcopenia. Several studies have suggested that IMAT formation is not only negatively correlated with skeletal muscle mass but also causes decreased muscle contraction in sarcopenia. In the present study, we examined w hether adipocytes affect myogenesis. For this purpose, skeletal muscle progenitor cells were transfected with siRNA of PPARγ (siPPARγ) in an attempt to inhibit adipogenesis. Myosin heavy chain (MHC)-positive myotube formation was promoted in cells transfected with siPPARγ compared to that of cells transfected with control siRNA. To determine whether direct cell-to-cell contact between adipocytes and myoblasts is a prerequisite for adipocytes to affect myogenesis, skeletal muscle progenitor cells were cocultured with pre- or mature adipocytes in a Transwell coculture system. MHC-positive myotube formation was inhibited when skeletal muscle progenitor cells were cocultured with mature adipocytes, but was promoted when they were cocultured with preadipocytes. Similar effects were observed when pre- or mature adipocyte-conditioned medium was used. These results indicate that preadipocytes play an important role in maintaining skeletal muscle mass by promoting myogenesis; once differentiated, the resulting mature adipocytes negatively affect myogenesis, leading to the muscle deterioration observed in skeletal muscle pathologies.     

Biodegradation of aniline, anthracene, chlornitrophen, fenitrothion and linear alkylbenzene sulphonate in pond water

Biodegradation of five chemicals (aniline, anthracene, chlornitrophen (CNP), fenitrothion (FNT) and linear alkylbenzene sulphonate (LAS)) by aquatic bacteria in three different types of ponds was determined according to the cultivation method developed by this group. The degradability toward these chemicals was varied among the ponds, except for LAS which was decomposed well in all samples. Higher degradability towards the two agrochemicals, CNT and FNT, was found in the pond surrounded by paddy fields, whereas aniline and anthracene were decomposed more rapidly in the pond located in the industrial area. Water from the pond in the botanical garden, with the least exposure to any chemicals, exhibited the lowest degradation toward all chemicals tested. There was no significant seasonal variation in the biodegradation of chemicals in these ponds. It was deduced that biodegradability toward certain chemicals could be a result of acclimatization of the microbial community by chemical contamination present and past, suggesting the possible use of biodegradation profiles as an indicator for chemical pollution in the aquatic environment.     

DYRK2 is targeted to the nucleus and controls p53 via Ser46 phosphorylation in the apoptotic response to DNA damage

Genotoxic stress exerts biological activity by activating downstream effectors, including the p53 tumor suppressor. p53 regulates cell-cycle checkpoint and induction of apoptosis in response to DNA damage; however, molecular mechanisms responsible for committing to these distinct functions remain to be elucidated. Recent studies demonstrated that phosphorylation of p53 at Ser46 is associated with induction of p53AIP1 expression, resulting in commitment to apoptotic cell death. In this regard, the role for Ser46 kinases in p53-dependent apoptosis has been established; however, the kinases responsible for Ser46 phosphorylation have yet to be identified. Here, we demonstrate that the dual-specificity tyrosine-phosphorylation-regulated kinase 2 (DYRK2) directly phosphorylates p53 at Ser46. Upon exposure to genotoxic stress, DYRK2 translocates into the nucleus for Ser46 phosphorylation. Consistent with these results, DYRK2 induces p53AIP1 expression and apoptosis in a Ser46 phosphorylation-dependent manner. These findings indicate that DYRK2 regulates p53 to induce apoptosis in response to DNA damage.     

Induction of cytotoxic T cell responses and tumor immunity against unrelated tumors using telomerase reverse transcriptase RNA transfected dendritic cells

The polypeptide component of telomerase (TERT) is an attractive candidate for a broadly expressed tumor rejection antigen because telomerase is silent in normal tissues but is reactivated in more than 85% of cancers. Here we show that immunization against TERT induces immunity against tumors of unrelated origin. Immunization of mice with TERT RNA-transfected dendritic cells (DC) stimulated cytotoxic T lymphocytes (CTL), which lysed melanoma and thymoma tumor cells and inhibited the growth of three unrelated tumors in mice of distinct genetic backgrounds. TERT RNA-transfected human DC stimulated TERT-specific CTL in vitro that lysed human tumor cells, including Epstein Barr virus (EBV)-transformed B cells as well as autologous tumor targets from patients with renal and prostate cancer. Tumor RNA-transfected DC were used as surrogate targets in the CTL assays, obviating the difficulties in obtaining tumor cells from cancer patients. In one instance, where a tumor cell line was successfully established in culture from a patient with renal cancer, the patient's tumor cells were efficiently lysed by the CTL. Immunization with tumor RNA was generally more effective than immunization with TERT RNA, suggesting that an optimal immunization protocol may have to include TERT as well as additional tumor antigens.     

Microdose clinical trial: quantitative determination of fexofenadine in human plasma using liquid chromatography/electrospray ionization tandem mass spectrometry

A sample treatment procedure and high-sensitive liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method for quantitative determination of fexofenadine in human plasma was developed for a microdose clinical trial with a cold drug, i.e., a non-radioisotope-labeled drug. Fexofenadine and terfenadine, as internal standard, were extracted from plasma samples using a 96-well solid-phase extraction plate (Oasis HLB). Quantitation was performed on an ACQUITY UPLC system and an API 5000 mass spectrometer by multiple reaction monitoring. Chromatographic separation was achieved on an XBridge C18 column (100 mm x 2.1 mm i.d., particle size 3.5 microm) using acetonitrile/2 mM ammonium acetate (91:9, v/v) as the mobile phase at a flow rate of 0.6 ml/min. The analytical method was validated in accordance with the FDA guideline for validation of bioanalytical methods. The calibration curve was linear in the range of 10-1000 pg/ml using 200 microl of plasma. Analytical method validation for the clinical dose, for which the calibration curve was linear in the range of 1-500 ng/ml using 20 microl of plasma, was also conducted. Each method was successfully applied for making determinations in plasma using LC/ESI-MS/MS after administration of a microdose (100 microg solution) and a clinical dose (60 mg dose) in eight healthy volunteers.