Tk-PTP,protein tyrosine/serine phosphatase from hyperthermophilic archaeon Thermococcus kodakaraensis KOD1: enzymatic characteristics and identification of its substrate proteins

The Tk-ptp gene encoding a protein tyrosine phosphatase from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 was cloned and biochemical characteristics of the recombinant protein (Tk-PTP) were examined. A series of mutants, D63A (replacing Asp-63 with Ala), C93S, C93A, R99K, and R99M, were also constructed and analyzed. Two unique features were found. First, the Tk-PTP showed the phosphatase activity not only toward phosphotyrosine but also toward phosphoserine. Second, the conserved Asp-63, which corresponds to a critical residue among other known PTPs, was not essential for catalysis. Cys-93 and Arg-99 residues played a crucial role in substrate binding and catalysis. To know a specific substrate for Tk-PTP, C93S mutant was used to trap substrate proteins from cell extract of KOD1. Phenylalanyl-tRNA synthetase subunit beta-chain, one of the gene products of RNA terminal phosphate cyclase operon and phosphomannomutase, was identified, suggesting that they functioned for phosphate donation.     

Isolation and identification of an allelopathic substance from peel of Citrus junos

The inhibitory effect of Citrus junos peel on plant growth using lettuce (Lactuca sativa L.) as a bioassay material was investigated, since the powder of the peel had been found to inhibit growth of weeds. Basic, neutral and acidic fractions were separated from the aqueous fraction obtained from the methanol extract of C. junos peel. All fractions inhibited the growth of lettuce seedlings, but by far the greatest inhibition was observed with the neutral fraction. Thus, the latter was further purified and an allelopathically active substance was isolated. The structure of the substance was determined from high-resolution MS and 1H and 13C NMR spectral data as abscisic acid-beta-D-glucopyranosyl ester (ABA-GE). ABA-GE inhibited hypocotyl and root growth of lettuce seedlings at concentrations greater than 0.3 microM, and the concentrations for 50% inhibition of hypocotyl and root growth were 2.3 and 1.4 microM, respectively. The effectiveness of ABA-GE on inhibition of growth and the occurrence of ABA-GE in the peel itself suggested that ABA-GE may play an important role in the allelopathic potential of C. junos peel. The peel may be potentially useful for weed management in a field setting.     

Cloning of the maltose phosphorylase gene from Bacillus sp. strain RK-1 and efficient production of the cloned gene and the trehalose phosphorylase gene from Bacillus stearothermophilus SK-1 in Bacillus subtilis

The maltose phosphorylase (MPase) gene of Bacillus sp. strain RK-1 was cloned by PCR with oligonucleotide primers designed on the basis of a partial N-terminal amino acid sequence of the purified enzyme. The MPase gene consisted of 2,655 bp encoding a theoretical protein with a Mr of 88,460, and had no secretion signal sequence, although most of the MPase activity was detected in the culture supernatant of RK-1. This cloned MPase gene and the trehalose phosphorylase (TPase) gene from Bacillus stearothermophilus SK-1 were efficiently expressed intracellularly under the control of the Bacillus amyloliquefaciens alpha-amylase promoter in Bacillus subtilis. The production yields were estimated to be more than 2 g of enzyme per liter of medium, about 250 times the production of the original strains, in a simple shake flask. About 60% of maltose was converted into trehalose by the simultaneous action of both enzymes produced in B. subtilis.     

Preliminary examination on lapillus utility for otolith increment analysis in Malawian cyprinid <Emphasis Type="Italic">Engraulicypris sardella</Emphasis>

Features and increments of otoliths in Engraulicypris sardella were observed. The sagitta was arrowhead shaped with an obvious core and rostra, the latter being fragile and easily destroyed by extracting and grinding processes. Increments around the core were readable but not in rostra. The lapillus was round fan-shaped with an obvious core. Increments in the lapillus were clearly deposited from the core to the margin. The asteriscus had an ambiguous core that led to difficulty in discerning the first increment and had fewer increments than the lapilli. Thus, the lapillus is the most appropriate for reading otolith increments.     

Individuals exhibiting conspicuous nevi (lentigo simplex) are resistant to allergic rhinitis/conjunctivitis (pollinosis), but those who do not show increased susceptibility to pollinosis

We investigated 115 individuals in Tokyo from two groups of persons with conspicuous nevi on the head, neck, and arms, etc. (nevus-positive group) and persons who do not exhibit such nevi (nevi-negative group) interviewing them to determine whether they suffered from allergic rhinitis/conjunctivitis (pollinosis) and examining them for the presence and extent of nevi. There was a distinct correlation: only 5 persons among 85 individuals of the nevus-positive group suffered from pollinosis, and all 30 people in the nevus-negative group had pollinosis. Thus, individuals exhibiting conspicuous nevi are resistant to pollinosis, but those who do not show increased susceptibility to pollinosis.     

Smooth muscle and NMR review: An overview of smooth muscle metabolism

Nuclear magnetic resonance (NMR) is a non-invasive technique which allows us to examine the biochemical, physiological and metabolic events occurring inside living tissue; such as vascular and other smooth muscles.It has been found that the smooth muscle metabolism is compartmented such that mitochondrial function fuels contraction and that much glycolytic ATP production is used for membrane pumps. Using NMR we have been able to observe the ATP and phosphocreatine (PCr) concentrations and estimate the ADP concentration, as well as flux through the creatine kinase (CK) system. It has also been found that the smooth muscle metabolism is able to maintain ATP concentration in the absence of mitochondrial function (cyanide inhibition). Therefore, the vessels are able to adapt to metabolic demands as necessary.NMR is versatile in the information it can provide because it has also yielded important contributions with regard to the intracellular pH and ionic status. For example, the intracellular free Mg²⁺ ([Mg²⁺]_i) can be measured with NMR simultaneously with ATP concentrations and NMR has shown us that the [Mg²⁺]_i is highly protected in the muscle (within confined range), but also responds to the environment around it.In this review we conclude that NMR measurements of smooth muscle research is a useful technique for assessing chronic and acute changes that occur in the tissue and during diseases.     

Host range expansion by Rhopalomyia yomogicola (Diptera: Cecidomyiidae) from a native to an alien species of Artemisia (Asteraceae) in Japan

In 2001, subconical galls were found on the leaves of an alien Artemisia species (Asteraceae) in Ibusuki City, Kagoshima Prefecture, Japan. These galls were quite similar to those induced by Rhopalomyia yomogicola (Diptera: Cecidomyiidae) on Artemisia princeps, Artemisia montana, and Artemisia japonica in Japan. The morphological features of the pupal head and molecular sequencing data indicated that the gall midge from the alien Artemisia was identical to R. yomogicola. Usually, galling insects do not expand readily their host range to alien plants, but R. yomogicola is considered to have expanded its host range to the alien Artemisia by its multivoltine life history trait and oligophagous habit across two different botanical sections of the genus Artemisia. Adult abdominal tergites and sternites and immature stages of R. yomogicola are described for the first time and detailed biological information is presented.     

Contrasting Effects of the Cytotoxic Anticancer Drug Gemcitabine and the EGFR Tyrosine Kinase Inhibitor Gefitinib on NK Cell-Mediated Cytotoxicity via Regulation of NKG2D Ligand in Non-Small-Cell Lung Cancer Cells

IntroductionSeveral cytotoxic anticancer drugs inhibit DNA replication and/or mitosis, while EGFR tyrosine kinase inhibitors inactivate EGFR signalling in cancer cell. Both types of anticancer drugs improve the overall survival of the patients with non-small-cell lung cancer (NSCLC), although tumors often become refractory to this treatment. Despite several mechanisms by which the tumors become resistant having been described the effect of these compounds on anti-tumor immunity remains largely unknown.MethodsThis study examines the effect of the cytotoxic drug Gemcitabine and the EGFR tyrosine kinase inhibitor Gefitinib on the expression of NK group 2 member D (NKG2D) ligands as well as the sensitivity of NSCLC cells to the NK-mediated lysis.ResultsWe demonstrate that Gemcitabine treatment leads to an enhanced expression, while Gefitinib downregulated the expression of molecules that act as key ligands for the activating receptor NKG2D and promote NK cell-mediated recognition and cytolysis. Gemcitabine activated ATM and ATM- and Rad-3-related protein kinase (ATR) pathways. The Gemcitabine-induced phosphorylation of ATM as well as the upregulation of the NKG2D ligand expression could be blocked by an ATM-ATR inhibitor. In contrast, Gefitinib attenuated NKG2D ligand expression. Silencing EGFR using siRNA or addition of the PI3K inhibitor resulted in downregulation of NKG2D ligands. The observations suggest that the EGFR/PI3K pathway also regulates the expression of NKG2D ligands. Additionally, we showed that both ATM-ATR and EGFR regulate MICA/B via miR20a.ConclusionIn keeping with the effect on NKG2D expression, Gemcitabine enhanced NK cell-mediated cytotoxicity while Gefitinib attenuated NK cell killing in NSCLC cells.