Identification of a second cytotoxic protein produced by Bacillus thuringiensis A1470

Bacillus thuringiensis A1470 produces multiple proteins with similar molecular masses (~30 kDa) with cytotoxicity against human cell lines. One that was previously identified, parasporin-4, is a β-pore-forming toxin. The N-terminal sequence of a second cytotoxic protein was identical to a partial sequence of parasporin-2 produced by B. thuringiensis A1547. PCR was performed on total plasmid DNA from A1470 by using primers for parasporin-2 to amplify a gene which was then cloned. The cloned gene differed from A1547 parasporin-2 by 8 bp and the predicted protein differed by four amino acids. The gene was expressed in Escherichia coli, and the cytotoxic activities of the recombinant protein against four human cell lines (MOLT-4, Jurkat, HeLa, and HepG2) were similar to those of A1547 parasporin-2. We then confirmed that the A1470 strain simultaneously produces parasporin-2 and parasporin-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.     

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.     

Negligible impact of deer-induced habitat degradation on the genetic diversity of extant <Emphasis Type=Italic>Bombus diversus</Emphasis> populations in comparison with museum specimens

The genetic diversity of bumblebees can be adversely affected by habitat degradation. An overabundance of deer has altered the composition and diversity of herbaceous plants in many places of the world, resulting in decreases of herbaceous flowers. Populations of Bombus diversus may be strongly affected by this degradation of habitat in the Ashiu primary beech forest in Kyoto, Japan. To estimate the effects of deer browsing on B. diversus populations, we analyzed and compared the genetic diversity of the extant population in Ashiu to museum specimens collected prior to heavy deer browsing in Ashiu (1980s) and the extant population in Hyonosen primary beech forest in Tottori, Japan, which has not been as severely degraded by deer. We successfully amplified DNA from ~20-year-old museum specimens and determined the genetic diversity of B. diversus in Ashiu populations from the 1980s. Results were analyzed for indications of a bottleneck as well as estimates of N _e, allelic richness, rare allelic richness, expected heterozygosity, and the effective number of alleles. Our findings did not reveal clear evidence of degradation in genetic diversity of the extant Ashiu population compared to the museum specimens or to the Hyonosen population. Thus, the Ashiu population of B. diversus appears to have maintained a level of genetic diversity during 20 years irrespective of habitat degradation and the levels have been similar to that of the Hyonosen population.     

Differential contributions of protein kinase C isoforms in the regulation of group IIA secreted phospholipase A2 expression in cytokine-stimulated rat fibroblasts

Protein kinase C (PKC) is a family of serine/threonine kinases involved in various signal transduction pathways. We investigated the roles of PKC in the regulation of group IIA secreted phospholipase A₂ (sPLA₂-IIA) expression in cytokine-stimulated rat fibroblastic 3Y1 cells. Here we show that the induction of sPLA₂-IIA by proinflammatory cytokines was under the control of both classical cPKCα and atypical aPKCλ/ι pathways by using PKC inhibitors, a PKC activator, and PKC knockdowns. Treatment of 3Y1 cells with PKC selective inhibitors having broad specificity, such as chelerythrine chloride and GF109203X, blocked IL-1β/TNFα-dependent induction of sPLA₂-IIA protein in a dose-dependent manner. Treatment with the PKC activator phorbol 12-myristate 13-acetate (PMA), which activates cPKC and novel nPKC isoforms, markedly attenuated the cytokine-dependent induction of sPLA₂-IIA expression. In comparison, 24-h pretreatment with PMA, which down-regulates these PKC isoforms, markedly enhanced sPLA₂-IIA expression. Results with short hairpin RNA (shRNA)-mediated knockdown of PKC isoforms revealed that the cytokine-induced sPLA₂-IIA expression was markedly enhanced in cPKCα knockdown cells compared to those in replicate control cells. In contrast, knockdown of the aPKCλ/ι isoform reduced the cytokine-induced expression of sPLA₂-IIA. These results suggest that the aPKCλ/ι pathway is required for the induction of sPLA₂-IIA expression and that the cPKCα pathway acts as a negative regulator of sPLA₂-IIA expression in cytokine-stimulated rat fibroblasts.     

Redescription of Dendrochirus zebra (Scorpaenidae: Pteroinae) with a new species of Dendrochirus from the Ogasawara Islands,Japan

Ichthyological Research - Dendrochirus zebra (Cuvier in Cuvier and Valenciennes 1829) (Scorpaenidae: Pteroinae) is redescribed on the basis of 405 specimens from the Indo-West Pacific region. The...     

Anthocyanin synthesis potential in betalain-producing Caryophyllales plants

Journal of Plant Research - Although anthocyanins are widely distributed in higher plants, betalains have replaced anthocyanins in most species of the order Caryophyllales. The accumulation of...