Analytical method for lipoperoxidation relevant reactive aldehydes in human sera by high-performance liquid chromatography–fluorescence detection

A validated, simple and sensitive HPLC method was developed for the simultaneous determination of lipoperoxidation relevant reactive aldehydes: glyoxal (GO), acrolein (ACR), malondialdehyde (MDA), and 4-hydroxy-2-nonenal (HNE) in human serum. The studied aldehydes were reacted with 2,2′-furil to form fluorescent difurylimidazole derivatives that were separated on a C₁₈ column using gradient elution and fluorescence detection at excitation and emission wavelengths of 250 and 355 nm, respectively. The method showed good linearity over the concentration ranges of 0.100–5.00, 0.200–10.0, 0.200–40.0, and 0.400–10.0 nmol/mL for GO, ACR, HNE, and MDA, respectively, with detection limits ranging from 0.030 to 0.11 nmol/mL. The percentage RSD of intraday and interday precision did not exceed 5.0 and 6.2%, respectively, and the accuracy (%found) ranged from 95.5 to 103%. The proposed method was applied for monitoring the four aldehydes in sera of healthy, diabetic, and rheumatic human subjects with simple pretreatment steps and without interference from endogenous components. By virtue of its high sensitivity and accuracy, our method enabled detection of differences between analytes concentrations in sera of human subjects under different clinical conditions.     

Development of a highly sensitive fluorescence probe for peptidyl arginine deiminase (PAD) activity

Peptidyl arginine deiminases (PADs) catalyze the post-translational deimination of arginine residues to citrulline residues. Aberrant levels of PAD activity are associated with various diseases, such as rheumatoid arthritis, Alzheimer’s disease, and multiple sclerosis, so there is a need for simple and convenient high-throughput screening systems to discover PAD inhibitors as candidate therapeutic agents. Here, we report a highly sensitive off/on-type fluorescence probe for PAD activity based on the donor-excited photoinduced electron transfer (d-PeT) mechanism, utilizing the specific cycloaddition reaction between the benzil group of the probe and the ureido group of the PAD product, citrulline, under acidic conditions. We synthesized and functionally evaluated a series of probes bearing substituents on the benzil phenyl group, and found that 4MEBz-FluME could successfully detect citrulline with higher sensitivity and broader dynamic range than our previously reported fluorescence probe, FGME. Moreover, we succeeded in establishing multiple assay systems for PAD subtypes activities, including PAD2 and PAD4, with 4MeBz-FluME thanks to its high sensitivity. We expect that our fluorescence probes will become a powerful tool for discovering PAD inhibitors of several subtypes. Thus, it should be suitable for high-throughput screening of chemical libraries for inhibitors of PADs.     

Habitat generalization of a predatory ladybird,Harmonia yedoensis (Coleoptera: Coccinellidae), in an allopatric area with respect to its sibling species Harmonia axyridis

In central Japan, Harmonia yedoensis is a specialist ladybird that is confined to pine tree habitats, whereas its sibling species Harmonia axyridis is a generalist that feeds on a wide range of aphid species in nature. Interestingly, H. axyridis is not distributed in the Ryukyu Archipelago, southern Japan. We hypothesized that the ecological niche of H. yedoensis should be wider in the Ryukyu Archipelago, where its competitor species in central Japan, H. axyridis, is absent. We undertook fieldwork and a survey of published works to examine habitat utilization by H. yedoensis in the Ryukyu Archipelago. We found that H. yedoensis adults in the Ryukyu Archipelago visited several kinds of deciduous trees, including wild tamarind, Chinese hibiscus, Taiwan cherry and Malayan banyan, as well as pine trees. These observations suggest that habitat generalization has occurred in H. yedoensis in the Ryukyu Archipelago, where it does not compete with H. axyridis.     

Molecular analysis of RANKL-independent cell fusion of osteoclast-like cells induced by TNF-alpha, lipopolysaccharide, or peptidoglycan

Focusing on the final step of osteoclastogenesis, we studied cell fusion from tartrate-resistant acid phosphatase (TRAP)-positive mononuclear cells into multinuclear cells. TRAP-positive mononuclear cells before generation of multinuclear cells by cell fusion were differentiated from RAW264.7 cells by treatment with receptor activator of nuclear factor kappa B ligand (RANKL), and then the cells were treated with lipopolysaccharide (LPS), followed by culturing for further 12 h. LPS-induced cell fusion even in the absence of RANKL. Similarly, tumor necrosis factor (TNF)-alpha and peptidoglycan (PGN) induced cell fusion, but M-CSF did not. The cell fusion induced by RANKL, TNF-alpha, and LPS was specifically blocked by osteoprotegerin (OPG), anti-TNF-alpha antibody, and polymyxin B, respectively. LPS- and PGN-induced cell fusion was partly inhibited by anti-TNF-alpha antibody but not by OPG. When TRAP-positive mononuclear cells fused to yield multinuclear cells, phosphorylation of Akt, Src, extracellular signal-regulated kinase (ERK), p38MAPK (p38), and c-Jun NH2-terminal kinase (JNK) was observed. The specific chemical inhibitors LY294002 (PI3K), PP2 (Src), U0126 (MAPK-ERK kinase (MEK)/ERK), and SP600125 (JNK) effectively suppressed cell fusion, although SB203580 (p38) did not. mRNA of nuclear factor of activated T-cells c1 (NFATc1) and dendritic cell-specific transmembrane protein (DC-STAMP) during the cell fusion was quantified, however, there was no obvious difference among the TRAP-positive mononuclear cells treated with or without M-CSF, RANKL, TNF-alpha, LPS, or PGN. Collectively, RANKL, TNF-alpha, LPS, and PGN induced cell fusion of osteoclasts through their own receptors. Subsequent activation of signaling pathways involving PI3K, Src, ERK, and JNK molecules was required for the cell fusion. Although DC-STAMP is considered to be a requisite for cell fusion of osteoclasts, cell fusion-inducing factors other than DC-STAMP might be necessary for the cell fusion.     

Cloning and Characterization of Two Novel Genes, cry24B and s1orf2, from a Mosquitocidal Strain of Bacillus thuringiensis serovar sotto

Two new crystal protein genes, cry24B and s1orf2, were cloned from a mosquitocidal Bacillus thuringiensis serovar sotto strain. The cry24B and s1orf2 genes encoded a 76-kDa and 62-kDa protein, respectively. The Cry24B protein retained five conserved regions commonly found in the existing Cry proteins. The amino acid sequence of the S1ORF2 had a high homology to that of the ORF2 protein of B. thuringiensis serovar jegathesan. Southern hybridization experiments with a cry24B gene-specific probe revealed that these genes are located on two large plasmids of > 100 kb. When the two genes, cry24B and s1orf2, were expressed in an acrystalliferous B. thuringiensis host, the proteins were synthesized and accumulated as inclusions. These inclusions exhibited no larvicidal activities against three mosquito species: Aedes aegypti, Anopheles stephensi, and Culex pipiens molestus. Likewise, the inclusions contained no cytocidal activity against HeLa cells.     

Seasonality and fasting effect in raccoon dog Nyctereutes procyonoides serum leptin levels determined by canine leptin-specific enzyme-linked immunosorbent assay

Leptin is an adipocyte-derived peptide hormone that acts on the brain and regulates food intake and energy balance. Several previous reports have suggested that overwintering raccoon dogs Nyctereutes procyonoides are able to control their adiposity efficiently, but the contribution of leptin to weight regulation in these animals remains unclear. To study the seasonality of overwintering raccoon dogs as well as the effects of fasting on them, serum leptin levels were investigated using a newly established canine leptin-specific enzyme-linked immunosorbent assay (ELISA) kit. Of the nine animals studied, five were fed and four were fasted (deprived of food for 2 months in winter). Blood samples and body fat weights were monitored once a month throughout the experimental period (July 2007-March 2008). Leptin concentrations obtained by ELISA were significantly higher than and had a positive correlation with those obtained by previously used multispecies radioimmunoassay (RIA) kits. Moreover, ELISA showed a clearer correlation between the body fat weight and leptin levels compared with RIA, suggesting the efficacy of canine leptin-specific ELISA kit for leptin estimation in raccoon dogs. Autumnal fattening was observed in both groups of animals, but the wintertime loss of adipose tissue was more obvious in the fasted group. Serum leptin concentrations determined by ELISA showed seasonal changes without significant differences between the fed and fasted animals. Therefore, high levels of leptin may be responsible for the suppression of feeding behavior in raccoon dogs before winter.     

Proteome analysis of pitcher fluid of the carnivorous plant Nepenthes alata

The genus Nepenthes comprises carnivorous plants that digest insects in pitcher fluid to supplement their nitrogen uptake. In a recent study, two acid proteinases (nepenthesins I and II) were purified from the pitcher fluid. However, no other enzymes involved in prey digestion have been identified, although several enzyme activities have been reported. To identify all the proteins involved, we performed a proteomic analysis of Nepenthes pitcher fluid. The secreted proteins in pitcher fluid were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and several protein bands were detected by silver staining. The proteins were identified by in-gel tryptic digestion, de novo peptide sequencing, and homology searches against public databases. The proteins included homologues of beta-D-xylosidase, beta-1,3-glucanase, chitinase, and thaumatin-like protein, most of which are designated "pathogenesis-related proteins". These proteins presumably inhibit bacterial growth in the pitcher fluid to ensure sufficient nutrients for Nepenthes growth.     

TAK1-TAB1 fusion protein: a novel constitutively active mitogen-activated protein kinase kinase kinase that stimulates AP-1 and NF-kappaB signaling pathways

TAK1 mitogen-activated protein kinase kinase kinase (MAP3K) is activated by its specific activator, TAK1-binding protein 1 (TAB1). A constitutively active TAK1 mutant has not yet been generated due to the indispensable requirement of TAB1 for TAK1 kinase activity. In this study, we generated a novel constitutively active TAK1 by fusing its kinase domain to the minimal TAK1-activation domain of TAB1. Co-immunoprecipitation assay demonstrated that these domains interacted intra-molecularly. The TAK1-TAB1 fusion protein showed a significant MAP3K activity in vitro and activated c-Jun N-terminal kinase/p38 MAPKs and IkappaB kinase in vivo, which was followed by increased production of interleukin-6. These results indicate that the fusion protein is useful for characterizing the physiological roles of the TAK1-TAB1 complex.     

Efficient Production of 2,5-Diketo-d-Gluconate via Heterologous Expression of 2-Ketogluconate Dehydrogenase in Gluconobacter japonicus

2,5-Diketo-d-gluconate (2,5DKG) is a compound that can be the intermediate for d-tartrate and also vitamin C production. Although Gluconobacter oxydans NBRC3293 produces 2,5DKG from d-glucose via d-gluconate and 2-keto-d-gluconate (2KG), with accumulation of the product in the culture medium, the efficiency of 2,5DKG production is unsatisfactory because there is a large amount of residual d-gluconate at the end of the biotransformation process. Oxidation of 2KG to 2,5DKG is catalyzed by a membrane-bound flavoprotein-cytochrome c complex: 2-keto-gluconate dehydrogenase (2KGDH). Here, we studied the kgdSLC genes encoding 2KGDH in G. oxydans NBRC3293 to improve 2,5DKG production by Gluconobacter spp. The kgdS, kgdL, and kgdC genes correspond to the small, large, and cytochrome subunits of 2KGDH, respectively. The kgdSLC genes were cloned into a broad-host-range vector carrying a DNA fragment of the putative promoter region of the membrane-bound alcohol dehydrogenase gene of G. oxydans for expression in Gluconobacter spp. According to our results, 2KGDH that was purified from the recombinant Gluconobacter cells showed characteristics nearly the same as those reported previously. We also expressed the kgdSLC genes in a mutant strain of Gluconobacter japonicus NBRC3271 (formerly Gluconobacter dioxyacetonicus IFO3271) engineered to produce 2KG efficiently from a mixture of d-glucose and d-gluconate. This mutant strain consumed almost all of the starting materials (d-glucose and d-gluconate) to produce 2,5DKG quantitatively as a seemingly unique metabolite. To our knowledge, this is the first report of a Gluconobacter strain that produces 2,5DKG efficiently and homogeneously.