Characterization of three strains of Capnocytophaga canis isolated from patients with sepsis

Capnocytophaga canimorsus and Capnocytophaga cynodegmi, both commensal bacteria in the oral cavities of dogs and cats, are zoonotic pathogens. In particular, C. canimorsus causes sepsis and fatal septic shock. Recently, a novel Capnocytophaga species, C. canis, was isolated from the oral cavities of healthy dogs. It is reportedly oxidase‐negative and therefore considered avirulent in humans. In the present study, three strains of C. canis were isolated from Japanese patients with sepsis. All three strains, HP20001, HP33001 and HP40001, were oxidase‐positive. Nucleotide sequence identities of the 16S rRNA gene of the three strains to the C. canimorsus type strain ATCC35979, C. cynodegmi type strain ATCC49044 and C. canis type strain LMG29146 were 96.9–97.0%, 96.9–97.0% and 99.7–99.8%, respectively. Multi‐locus sequence analysis based on seven house‐keeping genes, dnaJ, fumC, glyA, gyrB, murG, trpB and tuf, revealed that the oxidase‐positive C. canis strains isolated in Japan and oxidase‐negative strains of C. canis from canine oral cavities in Switzerland were clustered in different genetic subgroups. These results indicate that the virulence of C. canis strains in humans is associated with oxidase activity.

     

Sterigmatocystin and aflatoxin B<Subscript>1</Subscript> contamination of corn,soybean meal,and formula feed in Japan

Sterigmatocystin (STC) and aflatoxin B₁ (AFB₁) were analyzed in 246 corn samples, 126 soybean meal samples, and 861 formula feed samples from the Japanese market between April 2010 and March 2015. The detection rate, the highest concentration, and the mean concentration of STC were respectively 14%, 6.4 μg/kg, and 1.2 μg/kg for corn; 14%, 1.1 μg/kg, and 0.63 μg/kg for soybean meal; and 43%, 9.1 μg/kg, and 0.97 μg/kg for formula feed. The detection rate, the highest concentration, and the mean concentration of AFB₁ were respectively 46%, 24 μg/kg, and 3.9 μg/kg for corn; 30%, 6.7 μg/kg, and 1.1 μg/kg for soybean meal; and 47%, 20 μg/kg, and 1.6 μg/kg for formula feed. A weak negative correlation between the STC and AFB₁ concentrations was observed: there was a high concentration of AFB₁ in samples that contained a lower concentration of STC and vice versa. Spearman’s rank correlation coefficient showed a weak negative correlation of ? 0.30 (p < 0.001, n = 128) for corn and ? 0.23 (p < 0.001, n = 575) for formula feed. In conclusion, no correlation was observed between the mean concentrations of STC contamination in formula feed (0.97 μg/kg) and in corn (1.2 μg/kg) and the blending rate (approximately 50%). The rate of STC contamination in the formula feed (43%) was higher than that in corn (14%). Therefore, it is likely that ingredients other than corn contribute to the contamination of formula feed with STC. In this study, regarding STC, problematic samples were not found.     

Loss of chloroplast‐localized protein phosphatase 2Cs in Arabidopsis thaliana leads to enhancement of plant immunity and resistance to Xanthomonas campestris pv. campestris infection

Protein phosphatases (PPs) counteract kinases in reversible phosphorylation events during numerous signal transduction pathways in eukaryotes. PP2Cs, one of the four major classes of the serine/threonine‐specific PP family, are greatly expanded in plants. Thus, PP2Cs are thought to play a specific role in signal transduction pathways. Some rice PP2Cs classified in subgroup K are responsive to infection by the compatible Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight. In Arabidopsis thaliana, orthologous PP2C genes (AtPP2C62 and AtPP2C26) classified to subgroup K are also responsive to Xanthomonas campestris pv. campestris (Xcc, causal agent of black rot) infection. To elucidate the function of these subgroup K PP2Cs, atpp2c62‐ and atpp2c26‐deficient A. thaliana mutants were characterized. A double mutant plant which was inoculated with a compatible Xcc showed reduced lesion development, as well as the suppression of bacterial multiplication. AtPP2C62 and AtPP2C26 localized to the chloroplast. Furthermore, the photosynthesis‐related protein, chaperonin‐60, was indicated as the potential candidate for the dephosphorylated substrate catalysed by AtPP2C62 and AtPP2C26 using two‐dimensional isoelectric focusing sodium dodecylsulfate‐polyacrylamide gel electrophoresis (2D‐IDF‐SDS‐PAGE). Taken together, AtPP2C62 and AtPP2C26 are suggested to be involved in both photosynthesis and suppression of the plant immune system. These results imply the occurrence of crosstalk between photosynthesis and the plant defence system to control productivity under pathogen infection.     

BCL2 inhibitor ABT-199 and JNK inhibitor SP600125 exhibit synergistic cytotoxicity against imatinib-resistant Ph+ ALL cells

Imatinib (IMT), a specific tyrosine kinase inhibitor (TKI), has drastically changed the treatment strategy for Ph+ ALL (Philadelphia chromosome-positive acute lymphoblastic leukemia). However, TKI resistance remains a serious problem for patient prognosis. Here, a Ph+ ALL cell line NphA2 and the IMT-resistant subline NphA2/STIR were analyzed to identify a potential novel treatment strategy. We also examined other Ph+ ALL cells, MR87 and its IMT-resistant subline, MR87/STIR. IMT induced apoptosis of NphA2 and MR87 but had no effect on resistant sublines. Increased phosphorylated ERK and BCL2, but not BCL-XL, were observed in NphA2/STIR compared with NphA2. NphA2/STIR but not NphA2 was moderately sensitive to U0126, an ERK inhibitor. Interestingly, SP600125, a JNK inhibitor, was potent in cell growth inhibition and apoptosis induction of both parental and IMT-resistant NphA2 and MR87 cells. Moreover, NphA2 and MR87 and their IMT-resistant sublines were sensitive to ABT-199, a specific BCL2 inhibitor. The combination of SP600125 and ABT-199 synergistically suppressed both parental and IMT-resistant cells, including one with T315I mutation, suggesting that Ph+ ALL exhibits high sensitivity to ABT-199 and SP600125 regardless of TKI resistance. This combination might be a possible therapeutic strategy for Ph+ ALL in the future.     

Lupin alkaloids from flowers of Echinosophora koreensis

(?)-Methyl 12-cytisineacetate (2) was isolated from methanol extracts of fresh flowers of Echinosophora koreensis together with seven known lupin alkaloids. Ethyl 12-cytisineacetate (3) was also isolated from ethanol extracts of the same flowers. Compounds 2 and 3 were artifacts and (?)-12-cytisineacetic acid (4) is assumed to be the principal source of 2 and 3. The variations in alkaloid content during growth of the flowers and the seedlings were also examined.     

Quantitative analysis of anticytokinin activity of 4-substituted-2-methylthiopyrido[2,3-d]pyrimidines

Ten 4-substituted-2-methylthiopyrido[2,3-d]pyrimidines were synthesized and tested for their cytokinin-antagonistic activity by the tobacco callus bioassay. This series of compounds constitutes the first example of anti-cytokinins which possess a fused 6-6 membered ring system. The treatment of Lineweaver and Burk, the method of classical enzyme kinetics, revealed competitive inhibition of cytokinin-induced tobacco callus growth. The variation of activity with the systematic transformation of 4-substituents was analysed quantitatively with physicochemical substituent parameters and regression analysis. The results indicated the predominant importance of substituent width for binding of the antagonists at the receptor site of cytokinins.     

Efficient flower induction from cultured buckwheat (Fagopyrum esculentum L.) node segments in vitro

Flower induction from shoot segments of buckwheat seedlings was examinedin vitro. Cytokinin, (especially kinetin at 0.1M), short day conditions and a high concentration ofsolidifying agent improved the flower induction from node segments invitro, in up to about 50% of node segments. The use of anaeration membrane on bottle caps and a high content of sucrose in the mediumimproved flower induction in vitro considerably. In theimproved conditions, flowers were induced from 100% cultures and 10bloomed flowers per explant were induced in vitro in 8weeks. Both long and short types of stigmas, and normal set of flowers wereobserved under the microscope. When pollen produced invitro was cultured on an artificial medium, 70% of the pollengrains germinated, indicating normal viability of in vitropollen, and indicating the potential for artificial pollination invitro. All the varieties examined flowered at a similar percentage,suggesting that the process was independent of variety and that flowers couldbeproduced in vitro. Flower induction from buckwheat plantsin vitro and possible cross breeding invitro are also discussed.     

Sterol 14-demethylase P450 (CYP51) provides a breakthrough for the discussion on the evolution of cytochrome P450 gene superfamily

Biodiversity is the most characteristic feature of cytochrome P450. Finding of CYP51 distributing widely in biological kingdoms provided breakthroughs for the discussion on the evolution and diversification of P450. Molecular phylogenetic analysis demonstrated that CYP51 appeared in the prokaryotic era and distributed into most kingdoms concomitant with phylogenetic divergence. This is the first evolutionary evidence indicating the prokaryotic origin of P450. Modification of substrate specificity of eukaryotic CYP51s occurred independently to adapt to the different sterol precursors existing in each kingdom. Formation of CYP51 variants through the mutation of active site and the selection of the advantageous ones from them were demonstrated by the emergence of azole-resistant CYP51s in Candida albicans under the environments rich in azole antifungal agents. These findings illustrate the most probable core process of P450 diversification consisting of modification of active site and selection of the resulting variants through interaction with endogenous and exogenous chemicals.     

Soybean-milk-coagulating activity of Bacillus pumilus derives from a serine proteinase

A proteolytic enzyme from Bacillus pumilus strain TYO-67, which was able to coagulate the protein in soybean milk, was characterized enzymologically. The optimum pH and temperature for its activities were 9.0 and 50 °C, respectively. The enzyme was strongly believed to be a serine proteinase because it was completely inhibited by the addition of diisopropyl fluorophosphate or phenylmethanesulfonyl fluoride. Hammerstein milk casein, cytochrome c and soybean protein were good substrates for the enzyme. Seven cleavages were detected using the oxidized insulin B-chain as peptide substrate for the proteolytic specificity test of the serine proteinase from B. pumilus. The bonds most susceptible to the action of the serine proteinase from B. pumilus were Leu-15–Tyr-16. The mode of action on soybean milk protein by the enzyme from B. pumilus was also investigated. The acidic subunit in glycinin and the α′-, α- and β-subunits in β-conglycinin were degraded during the enzyme reaction. However, the basic subunit in glycinin could not be degraded by the enzyme. The formation of coagula in soybean milk caused by the serine proteinase from B. pumilus was mainly due to the hydrophobic interaction. Received: 9 July 1999 / Received last revision: 22 October 1999 / Accepted: 5 November 1999