The cytotoxicity of Bacillus thuringiensis subsp. coreanensis A1519 strain against the human leukemic T cell

A novel cytotoxic protein was isolated from the crystal produced by Bacillus thuringiensis subsp. coreanensis A1519 strain. Upon treatment of the crystal proteins by proteinase K, the significant cytotoxicity toward the leukemic T cell, MOLT-4, was exhibited. The microscopic observation indicated that the cell death was accompanied by no extensive rupture of the cell membrane. It was, therefore, suggested that the cell death of MOLT-4 was induced through a mechanism other than the colloid-osmotic swelling and cell lysis as caused by hitherto known B. thuringiensis crystal proteins. The 29-kDa polypeptide proved to be an active component of the proteinase K-digested A1519 crystal proteins. EC(50) of the purified 29-kDa polypeptide was 0.078 microg/ml. The N-terminal amino acid sequence of the 29-kDa polypeptide shared no significant homology with all the known proteins, suggesting that this polypeptide belong to a new family of B. thuringiensis crystal proteins. In the ligand blotting analysis, specific binding proteins for the 29-kDa polypeptide were detected from the cell membrane of MOLT-4.     

In vitro RNA editing in pea mitochondria requires NTP or dNTP,suggesting involvement of an RNA helicase

To analyze the biochemical parameters of RNA editing in plant mitochondria and to eventually characterize the enzymes involved we developed a novel in vitro system. The high sensitivity of the mismatch-specific thymine glycosylase is exploited to facilitate reliable quantitative evaluation of the in vitro RNA editing products. A pea mitochondrial lysate correctly processes a C to U editing site in the cognate atp9 template. Reaction conditions were determined for a number of parameters, which allow first conclusions on the proteins involved. The apparent tolerance against specific Zn2+ chelators argues against the involvement of a cytidine deaminase enzyme, the theoretically most straightforward catalysator of the deamination reaction. Participation of a transaminase was investigated by testing potential amino group receptors, but none of these increased the RNA editing reaction. Most notable is the requirement of the RNA editing activity for NTPs. Any NTP or dNTP can substitute for ATP to the optimal concentration of 15 mm. This observation suggests the participation of an RNA helicase in the predicted RNA editing protein complex of plant mitochondria.     

Ovarian primary tissue culture of the kuruma shrimp Marsupenaeus japonicus

Cell growth in ovarian primary culture of the kuruma shrimp, Marsupenaeus japonicus, was examined under various culture conditions. The best growth of ovarian cells was obtained in a culture system consisting of double strength of Leibovitz-15 medium supplemented with 10% fetal bovine serum, glucose (1 g/L), proline (0.1 g/L), TC-Yeastolate (1 g/L), and lactalbumin hydrolysate (1 g/L). The cells survived in this medium at 25 degrees C for 45 d. The epithelial-like cells predominated in 10-d-old cultures, covering >80% of the surface area on the bottom of flask. Cells in mitosis were often observed. Cell proliferation was monitored by incorporation of 5-bromo-2'-deoxyuridine (BrdU), an analog of thymidine. 5-Bromo-2'-deoxyuridine-associated cells accounted for 11.5 and 35.0% of cell populations at 2 and 24 h, respectively, after BrdU treatment. Our results provide an improved culture technique for ovarian tissue of the kuruma shrimp.     

Simple and sensitive method for the determination of chlorpheniramine maleate in human plasma using liquid chromatography-mass spectrometry

A convenient liquid chromatographic-single quadrupole mass spectrometric (LC-MS) method was developed and validated for the determination of chlorpheniramine maleate (INN name: chlorphenamine) in human plasma. The method had advantages of a single liquid-liquid extraction with diethylether and high sensitivity. The linearity was also excellent over the concentration range of 0.52-20.8 ng/ml of chlorpheniramine maleate. The intra- and inter-day precision and accuracy ranged between 0.0 and 13.9%, showing a good reproducibility. This developed method was successfully applied to analysis of chlorpheniramine maleate in clinical studies.     

A Novel Fission Yeast Gene, tht1+, Is Required for the Fusion of Nuclear Envelopes during Karyogamy

We have isolated a fission yeast karyogamy mutant, tht1, in which nuclear congression and the association of two spindle pole bodies occurs but the subsequent fusion of nuclear envelopes is blocked. The tht1 mutation does not prevent meiosis, so cells execute meiosis with two unfused nuclei, leading to the production of aberrant asci. The tht1⁺ gene was cloned and sequenced. Predicted amino acid sequence has no significant homology to previously known proteins but strongly suggests that it is a type I membrane protein. The tht1⁺ gene is dispensable for vegetative growth and expressed only in conjugating cells. Tht1p is a glycoprotein susceptible to endoglycosilase H digestion. Site- directed mutagenesis showed that the N-glycosylation site, as well as the COOH-terminal region of Tht1p, is essential for its function. A protease protection assay indicated that the COOH terminus is cytoplasmic. Immunocytological analysis using a HA-tagged Tht1p suggested that the protein is localized in nuclear envelopes and in the ER during karyogamy and that its levels are reduced in cells containing fused nuclei.     

Cultivation of microalgae in the solution from the desalting process of soy sauce waste treatment and utilization of the algal biomass for ethanol fermentation

The growth of four marine microalgae was examined in a medium prepared from the effluent from the desalting process of soy sauce waste. A strain of Dunaliella that showed abundant growth on soy sauce waste extract was selected, and optimum cultivation conditions were determined. The algal cells produced were disrupted, and saccharified with glucoamylase. The saccharified solution was fermented using Saccharomyces cerevisiae IAM 4140. Stoichiometric study revealed that 11mg of ethanol was produced from 1g (dry cell weight) of Dunaliella cells. This work indicates a new method for removing waste products of the soy sauce production industry.     

Distribution of the tracheal mite,Acarapis woodi,among the mesothoracic tracheal trunks of the honey bee,Apis mellifera

The selection of honey-bee tracheal trunks by host-seeking tracheal mites, Acarapis woodi, was examined in 307 samples of worker honey bees. The number of bees with 0, 1, and 2 infested tracheal trunks was determined for each sample and compared to an expected distribution based on the assumption that the probability that a tracheal trunk is infested is the same for all tracheal trunks. A Monte-Carlo simulation technique was employed to evaluate the significance of the test statistic. Observed distributions of bees with 0, 1, and 2 infested tracheal trunks were found to differ significantly from the expected distributions in 74 of the 307 samples. There were more uninfested bees and bilaterally infested bees than expected in all 74 cases. Further analysis revealed that the left and right tracheal trunks were equally likely to be infested.