Artepillin C isoprenomics: design and synthesis of artepillin C isoprene analogues as lipid peroxidation inhibitor having low mitochondrial toxicity

We designed and synthesized isoprene analogues of artepillin C, a major component of Brazilian propolis, and investigated the inhibitory activity on lipid peroxidation of rat liver mitochondria (RLM) and RLM toxicity based on isoprenomics. We succeeded in the synthesis of artepillin C isoprene analogues using regioselective prenylation within the range from 22% to 53% total yield. Reactivity of artepillin C and its isoprene analogues with ABTS (2,2'-Azinobis(3-ethylbenzothiazoline-6-sulfonate)) radical cations showed only a slight difference among the molecules. The isoprene side-chain elongation analogues of artepillin C showed almost the same inhibitory activity against RLM lipid peroxidation as artepillin C. Artepillin C and its isoprene analogues had very weak RLM uncoupling activity. Moreover, artepillin C and its isoprene analogues exhibited a lower inhibitory activity against adenosine 5'-triphosphate (ATP) synthesis by about two orders of magnitude than the effective inhibitory activity against RLM lipid peroxidation. From these results we conclude that artepillin C isoprene analogues could be potent lipid peroxidation inhibitors having low mitochondrial toxicity. We also conclude that elongation of the isoprene side chain of artepillin C to increase lipophilicity had little influence on the inhibitory activity toward RLM lipid peroxidation.     

Role of the fly in the transport of Yersinia enterocolitica

Yersinia enterocolitica were isolated from flies collected from a piggery and a kitchen of farm and from ham hung in a piggery. The cultures were identified as Y. enterocolitica biovar 4 and serovar 3 by biochemical and serological characteristics. From these results it is suggested that flies may play an important role in food contamination by Y. enterocolitica. In this study, the probable donors of Y. enterocolitica to the flies were swine.     

Genotypic characterization of human and environmental isolates of Salmonella choleraesuis subspecies choleraesuis serovar infantis by pulsed-field gel electrophoresis.

To determine the extent of genetic diversity of Salmonella choleraesuis subspecies choleraesuis serovar Infantis and whether environmental isolates were similar or identical to human isolates, a total of 110 isolates from humans, broiler samples, egg production facilities, riverwater, sewage, and chicken meat were analyzed epidemiologically by pulsed-field gel electrophoresis. While the isolates showed 35 distinct pulsed-field profiles, none had the genotype of the human isolates. One pulsed-field profile was shared by 43 (39%) of the 110 isolates. These results indicate that relatively fewer clonal lines of S. serovar Infantis had spread widely while multiple clonal lines, including the strain involved in the outbreak, exist in Western Japan.     

Isolation of an Hswl Nav4 Influenza Virus from a Tufted Duck (Aythya fuligula) in Japan

A hemagglutinating agent was isolated from a tufted duck captured in Lake Shinji, Shimane Prefecture, Japan, and identified as influenza A by double immunodiffusion tests with the antiserum to influenza A virus ribonucleoprotein. The hemagglutinin of the isolate was antigenically related to that of A/New Jersey/8/76 but was not identical with it. The neuraminidase antigen of this Hswl subtype was closely related to that of A/turkey/Ontario/6118/68 and was shown to be Nav4 subtype. After experimental infection of 5-week-old SPF-chickens with the isolate, virus was recovered from various organs including the brain, despite the absence of signs of disease.     

Rat liver phosphoribosyl pyrophosphate synthetase: existence of the purified enzyme as heterogeneous aggregates and identification of the catalytic subunit

Mammalian phosphoribosyl pyrophosphate (PRPP) synthetase has been extensively investigated. However, considerable ambiguity remains concerning its physical and regulatory properties. We purified PRPP synthetase from rat liver and studied some of the physical properties, in parallel with cloning experiments (Taira, M. et. al. [1987] J. Biol. Chem. 262, 14867-14870). 1) The enzyme was purified to a specific activity of 7,280 milliunits/mg, the highest value in the literature for a mammalian PRPP synthetase. The apparent molecular mass was over 1,000 kDa. 2) The final preparation contained 34-kDa, 38-kDa, and 40-kDa protein species, as analyzed by SDS gel electrophoresis. 3) Further attempts at separation using conventional procedures only led to a co-purification of the components. Thus, the purified enzyme appears to exist as complex aggregates composed of heterogeneous components. 4) Gel filtration of the enzyme in the presence of 1 M MgCl2 isolated part of the 34-kDa component, free of other species. The preparation was catalytically active, indicating that this component is the catalytic subunit. 5) The amino acid composition of the 34-kDa subunit and the amino acid sequences of its N-terminal region and of two tryptic peptides were determined. The results are in accord with the results of cDNA analyses.     

Purification and functional characterization of insecticidal sphingomyelinase C produced by Bacillus cereus.

Bacillus cereus isolated from the larvae of Myrmeleon bore was found to secrete proteins that paralyze and kill German cockroaches, Blattela germanica, when injected. One of these active proteins was purified from the culture broth of B. cereus using anion-exchange and gel-filtration chromatography. The purified toxin, with a molecular mass of 34 kDa, was identified as sphingomyelinase C (EC 3.1.4.12) on the basis of its N-terminal and internal amino-acid sequences. A recombinant sphingomyelinase C expressed in Escherichia coli was as potent as the native protein in killing the cockroaches. Site-directed mutagenesis (His151Ala) that inactivated the sphingomyelinase activity also abolished the insecticidal activity, suggesting that the rapid insect toxicity of sphingomyelinase C results from its phospholipid-degrading activity.