Diversity of Melissococcus plutonius from honeybee larvae in Japan and experimental reproduction of European foulbrood with cultured atypical isolates

European foulbrood (EFB) is an important infectious disease of honeybee larvae, but its pathogenic mechanisms are still poorly understood. The causative agent, Melissococcus plutonius, is a fastidious organism, and microaerophilic to anaerobic conditions and the addition of potassium phosphate to culture media are required for growth. Although M. plutonius is believed to be remarkably homologous, in addition to M. plutonius isolates with typical cultural characteristics, M. plutonius-like organisms, with characteristics seemingly different from those of typical M. plutonius, have often been isolated from diseased larvae with clinical signs of EFB in Japan. Cultural and biochemical characterization of 14 M. plutonius and 19 M. plutonius-like strain/isolates revealed that, unlike typical M. plutonius strain/isolates, M. plutonius-like isolates were not fastidious, and the addition of potassium phosphate was not required for normal growth. Moreover, only M. plutonius-like isolates, but not typical M. plutonius strain/isolates, grew anaerobically on sodium phosphate-supplemented medium and aerobically on some potassium salt-supplemented media, were positive for β-glucosidase activity, hydrolyzed esculin, and produced acid from L-arabinose, D-cellobiose, and salicin. Despite the phenotypic differences, 16S rRNA gene sequence analysis and DNA-DNA hybridization demonstrated that M. plutonius-like organisms were taxonomically identical to M. plutonius. However, by pulsed-field gel electrophoresis analysis, these typical and atypical (M. plutonius-like) isolates were separately grouped into two genetically distinct clusters. Although M. plutonius is known to lose virulence quickly when cultured artificially, experimental infection of representative isolates showed that atypical M. plutonius maintained the ability to cause EFB in honeybee larvae even after cultured in vitro in laboratory media. Because the rapid decrease of virulence in cultured M. plutonius was a major impediment to elucidation of the pathogenesis of EFB, atypical M. plutonius discovered in this study will be a breakthrough in EFB research.     

A 50-kilodalton Cry2A peptide is lethal to Bombyx mori and Lymantria dispar

The Cry2Aa3 gene was introduced into asporogenic Bacillus thuringiensis, and the synthesized protoxin killed Bombyx mori and Lymantria dispar larvae. Chymotrypsin hydrolyzed the linkages between 49Tyr/Val50 and 145Lys/Ser146 in the protoxin, and 50- and 58-kDa fragments were generated, respectively. Both peptides killed the larvae of both insects.     

Synthesis and SAR study of imidazoquinolines as a novel structural class of microsomal prostaglandin E₂ synthase-1 inhibitors

The imidazoquinoline derivative 1 was found as a novel mPGES-1 inhibitor. Optimization of 1 led to the identification of the 2-chlorophenyl group at the C(2)-position and the quinolone structure at the C(4)-position. Compound 33, the most potent synthesized compound, showed excellent mPGES-1 inhibition (IC(50)=9.1nM) with high selectivity (>1000-fold) over both COX-1 and COX-2.     

In situ expression of ribosomal protein L21 in developing tooth germ of the mouse lower first molar

We previously performed cDNA subtraction between the mouse mandibles at embryonic day 10.5 (E10.5) in the pre-initiation stage of the odontogenesis and E12.0 in the late initiation stage to investigate the key regulator genes in odontogenesis. Ribosomal protein L21 (Rpl21) is one of differentially expressed genes in the E12.0 mandible. This study examined the precise expression pattern of Rpl21 mRNA in the mouse mandibular first molar by in situ hybridization. Rpl21 mRNA was expressed in the presumptive dental epithelium and the underlying mesenchyme at E10.5, and in the thickened dental epithelium at E12.0. Strong in situ signals were observed in the epithelial bud at E14.0, and in the enamel organ at E15.0. However, either no (E14.0) or only a weak (E15.0) in situ signal was found in the primary enamel knot at these gestational days. Rpl21 was strongly expressed in the inner enamel epithelium, cervical loop and dental lamina from E16.0 to E18.0. In addition, Rpl21 mRNA was also demonstrated in various developing cranio-facial organs. These results suggest that Rpl21 participates in the synthesis of various polypeptides which might be related to the initiation and the development of such tooth germ, and also in the synthesis of enamel components in the presecretory stage of the ameloblast. Rpl21 for protein synthesis might also be related to the morphogenesis of the developing cranio-facial organs.     

Behavior of psychrotrophic lactic acid bacteria isolated from spoiling cooked meat products

Three kinds of lactic acid bacteria were isolated from spoiling cooked meat products stored below 10 degrees C. They were identified as Leuconostoc mesenteroides subsp. mesenteroides, Lactococcus lactis subsp. lactis, and Leuconostoc citreum. All three strains grew well in MRS broth at 10 degrees C. In particular, L. mesenteroides subsp. mesenteroides and L. citreum grew even at 4 degrees C, and their doubling times were 23.6 and 51.5 h, respectively. On the other hand, although the bacteria were initially below the detection limit (<10 CFU/g) in model cooked meat products, the bacterial counts increased to 10(8) CFU/g at 10 degrees C after 7 to 12 days.     

Incorporation of DUF/FACT into chromatin enhances the accessibility of nucleosomal DNA

DNA unwinding factor (DUF) was discovered as an essential DNA replication factor in Xenopus egg extracts. DUF consists of an HMG protein and a homolog of Cdc68p/Spt16p, and has the capability of unwinding dsDNA. Here we have examined the interaction of DUF with chromatin. DUF was incorporated into chromatin assembled from sperm heads and from plasmid DNA in egg extracts. It was revealed that the chromatin assembled in egg extracts immunodepleted of DUF is less sensitive to micrococcal nuclease (NNase) digestion than that assembled in control extracts, indicating that chromatin containing DUF has more decompact structure than that without DUF. Also we found that DUF has a high affinity for core histones in vitro. We suggest that the function of DUF may be to make the chromatin structure accessible to replication factors.     

Src homology region 2 domain-containing phosphatase 1 positively regulates B cell receptor-induced apoptosis by modulating association of B cell linker protein with Nck and activation of c-Jun NH2-terminal kinase

Src homology region 2 domain-containing phosphatase 1 (SHP-1) is a key mediator in lymphocyte differentiation, proliferation, and activation. We previously showed that B cell linker protein (BLNK) is a physiological substrate of SHP-1 and that B cell receptor (BCR)-induced activation of c-Jun NH(2)-terminal kinase (JNK) is significantly enhanced in cells expressing a form of SHP-1 lacking phosphatase activity (SHP-1-C/S). In this study, we confirmed that SHP-1 also exerts negative regulatory effects on JNK activation in splenic B cells. To further clarify the role of SHP-1 in B cells, we examined how dephosphorylation of BLNK by SHP-1 affects downstream signaling events. When a BLNK mutant (BLNK Delta N) lacking the NH(2)-terminal region, which contains four tyrosine residues, was introduced in SHP-1-C/S-expressing WEHI-231 cells, the enhanced JNK activation was inhibited. Among candidate proteins likely to regulate JNK activation through BLNK, Nck adaptor protein was found to associate with tyrosine-phosphorylated BLNK and this association was more pronounced in SHP-1-C/S-expressing cells. Furthermore, expression of dominant-negative forms of Nck inhibited BCR-induced JNK activation. Finally, BCR-induced apoptosis was suppressed in SHP-1-C/S-expressing cells and coexpression of Nck SH2 mutants or a dominant-negative form of SEK1 reversed this phenotype. Collectively, these results suggest that SHP-1 acts on BLNK, modulating its association with Nck, which in turn negatively regulates JNK activation but exerts a positive effect on apoptosis.     

Evaluation of the ESPLINE INFLUENZA A&B-N Kit for the diagnosis of avian and swine influenza

The ESPLINE INFLUENZA A&B-N kit was evaluated for its applicability to the rapid diagnosis of influenza in chickens and pigs. The kit specifically detected viral antigens in tracheal swabs and tissue homogenates of the trachea, liver, spleen, and colon of chickens inoculated with a highly pathogenic avian influenza virus strain, A/chicken/Yamaguchi/7/04 (H5N1), at 48 hr post-inoculation (p.i.) as well as in the tracheal and cloacal swabs and tissue homogenates of dead chickens. For those infected with a low pathogenic strain, A/chicken/aq-Y-55/01 (H9N2), antigens were detected only in the samples from tracheal swabs and organs 1-4 days p.i. The kit also detected viral antigens in the nasal swabs of miniature pigs infected with swine and avian influenza viruses. The kit was found to be sensitive and specific enough for the rapid diagnosis of infections of influenza A virus in chickens and pigs.     

Lipid peroxidation of a human hepatoma cell line (HepG2) after incorporation of linoleic acid, arachidonic acid, and docosahexaenoic acid

Lipid peroxidation of human heptoma cell line, HepG2, after incorporation of linoleic acid (LA), arachidonic acid (AA), and docosahexaenoic acid (DHA) was measured with a fluorescent probe and gas chromatography-mass spectrometry (GC-MS) analysis. The analysis with a fluorescent probe showed that incorporation of each polyunsaturated fatty acid (PUFA) enhanced the cellular lipid peroxidation level, but there was little difference in the effect of LA, AA, or DHA on the enhancement of cellular lipid peroxidation. The fluorescent analysis also showed that the addition of H(2)O(2) (0.5 mM) enhanced the cellular lipid peroxidation levels in LA and AA supplemented cells as compared with those without H(2)O(2). However, the enhancement of lipid peroxidation by H(2)O(2) was not observed in DHA-supplemented cells. The same result was obtained in the GC-MS analysis of total amounts of monohydroperoxides (MHP) formed in the cellular phospholipid oxidation. In this case, the main source for MHP was LA in LA-, AA-, and DHA-supplemented cells. A significant amount of AA-MHP and a small amount of DHA-MHP were observed in AA- and DHA-supplemented cells respectively. GC-MS analysis also indicated the specific positional distribution of DHA-MHP isomers. The isomers were formed only by hydrogen abstraction at the C-18 (16-MHP + 20-MHP; 46.5%), C-6 (4-MHP + 8-MHP; 38.5%), and C-12 (10-MHP + 14-MHP; 15.1%) positions, but not at the C-9 or C-15 positions.