Base composition and nucleosome density in exonic and intronic regions in genes of the filamentous ascomycetes Aspergillus nidulans and Aspergillus oryzae

We sequenced nucleosomal DNA fragments of the filamentous ascomycetes Aspergillus nidulans and Aspergillus oryzae and then mapped those sequences on their genomes. We compared the GC content and nucleosome density in the exonic and intronic regions in the genes of A. nidulans and A. oryzae. Although the GC content and nucleosome density in the exonic regions tended to be higher than those in the intronic regions, the difference in the distribution of the GC content was more notable than that of the nucleosome density. Next, we compared the GC content and nucleosome density in the exonic regions of 9616 orthologous gene pairs. In both Aspergillus species, the GC content did not correlate with the nucleosome density. In addition, the Spearman's rank correlation coefficient (ρ = 0.51) between the GC content of the exonic regions of the 9616 orthologous gene pairs was higher than that (ρ = 0.31) of the nucleosome densities of A. nidulans and A. oryzae. These results strongly suggest that the GC content in the exons of the orthologous gene pairs has been conserved during evolution but the nucleosome density has varied throughout.     

Ly6C+Ly6G− Myeloid‐derived suppressor cells play a critical role in the resolution of acute inflammation and the subsequent tissue repair process after spinal cord injury

Acute inflammation is a prominent feature of central nervous system (CNS) insult and is detrimental to the CNS tissue. Although this reaction spontaneously diminishes within a short period of time, the mechanism underlying this inflammatory resolution remains largely unknown. In this study, we demonstrated that an initial infiltration of Ly6C⁺Ly6G^? immature monocyte fraction exhibited the same characteristics as myeloid‐derived suppressor cells (MDSCs), and played a critical role in the resolution of acute inflammation and in the subsequent tissue repair by using mice spinal cord injury (SCI) model. Complete depletion of Ly6C⁺Ly6G^? fraction prior to injury by anti‐Gr‐1 antibody (clone: RB6‐8C5) treatment significantly exacerbated tissue edema, vessel permeability, and hemorrhage, causing impaired neurological outcomes. Functional recovery was barely impaired when infiltration was allowed for the initial 24 h after injury, suggesting that MDSC infiltration at an early phase is critical to improve the neurological outcome. Moreover, intraspinal transplantation of ex vivo‐generated MDSCs at sites of SCI significantly reduced inflammation and promoted tissue regeneration, resulting in better functional recovery. Our findings reveal the crucial role of an Ly6C⁺Ly6G^? fraction as MDSCs in regulating inflammation and tissue repair after SCI, and also suggests an MDSC‐based strategy that can be applied to acute inflammatory diseases.     

Inhibitory effect of novel somatostatin peptide analogues on human cancer cell growth based on the selective inhibition of DNA polymerase β

The present study was designed to investigate the anticancer activity of novel nine small peptides (compounds 1–9) derived from TT-232, a somatostatin structural analogue, by analyzing the inhibition of mammalian DNA polymerase (pol) and human cancer cell growth. Among the compounds tested, compounds 3 [tert-butyloxycarbonyl (Boc)-Tyr-Phe-1-naphthylamide], 4 (Boc-Tyr-Ile-1-naphthylamide), 5 (Boc-Tyr-Leu-1-naphthylamide) and 6 (Boc-Tyr-Val-1-naphthylamide) containing tyrosine (Tyr) but no carboxyl groups, selectively inhibited the activity of rat pol β, which is a DNA repair-related pol. Compounds 3–6 strongly inhibited the growth of human colon carcinoma HCT116 p53^+/+ cells. The influence of compounds 1–9 on HCT116 p53^?/? cell growth was similar to that observed for HCT116 p53^+/+ cells. These results suggest that the cancer cell growth suppression induced by these compounds might be related to their inhibition of pol. Compound 4 was the strongest inhibitor of pol β and cancer cell growth among the nine compounds tested. This compound specifically inhibited rat pol β activity, but had no effect on the other 10 mammalian pols investigated. Compound 4 combined with methyl methane sulfonate (MMS) treatment synergistically suppressed HCT116 p53^?/? cell growth compared with MMS alone. This compound also induced apoptosis in HCT116 cells with or without p53. From these results, the influence of compound 4, a specific pol β inhibitor, on the relationship between DNA repair and cancer cell growth is discussed.     

Protoplast-transfection of Streptomyces chartreusis SF1623 with Actinophage Φr5 DNA

To establish a procedure for high frequency transfection in streptomycetes, the conditions and factors affecting the polyethyleneglycol (PEG) mediated transfection of S. chartreusis SF1623 by actinophage Φr5 DNA were studied. Protoplasts of S. chartreusis SF1623 prepared by treatment with lysozyme and achromopeptidase were very stable. Protoplasts from 20 to 22hr culture cells were more competent for transfection. The optimal pH of the medium for transfection was pH 7.6. The presence of NaCl, thymidine, ATP, ADP or adenosine in the transfection medium enhanced the frequency of transfection. The optimal conditions determined for protoplast transfection were 12.5% PEG 4,000, 300 mm NaCl, 1 mm thymidine, final concentration, Φr5 DNA and protoplasts in P3 medium (pH 7.6). The frequency of transfection under the optimal conditions was 5 × 10⁵ per μg Φr5 DNA and was about 3 × 10^?3 per regenerated protoplasts.

Progenitively mature phages appeared 4hr after incubation in the regeneration solution and their number continued to increase for about 11 hr. The burst size was estimated to be about 400.     

Synthesis of Prostaglandin-F1 Related Compounds

Synthesis of several prostaglandin-F₁ related compounds utilizing bicyclo(2,2,1) heptene derivatives as key intermediates were investigated.     

Phospholipids in Castor Seeds

Sites of restriction endonucleases were mapped on pOAD2, a plasmid harbored in Flavobacterium sp. KI72. The plasmid codes 6-aminohexanoic acid cyclic dimer hydrolase and 6-aminohexanoic acid linear oligomer hydrolase. pOAD2 (molecular weight: 28.8 megadaltons [Mdal]) had 6 HindIII and 5 EcoRI sites, which were located at 0, 8.4, 8.9, 11.1, 19.0 and 25.0 Mdal (for HindIII) and 3.3, 5.4, 20.4, 20.8, 22.6 Mdal (for EcoRI). A mutant which could not grow on 6-aminohexanoic acid cyclic dimer but grew on the linear dimer as the sole carbon and nitrogen source harbored a deletion plasmid pOAD21 derived from pOAD2. By comparing the restriction sites of these two plasmids, the deleted region was localized on which the 6-aminohexanoic acid cyclic dimer hydrolase was coded.     

Phylogeographic insights into the invasion history and secondary spread of the signal crayfish in Japan

Successful invasion by nonindigenous species is often attributed to high propagule pressure, yet some foreign species become widespread despite showing reduced genetic variation due to founder effects. The signal crayfish (Pacifastacus leniusculus) is one such example, where rapid spread across Japan in recent decades is believed to be the result of only three founding populations. To infer the history and explore the success of this remarkable crayfish invasion, we combined detailed phylogeographical and morphological analyses conducted in both the introduced and native ranges. We sequenced 16S mitochondrial DNA of signal crayfish from across the introduced range in Japan (537 samples, 20 sites) and the native range in western North America (700 samples, 50 sites). Because chela size is often related to aggressive behavior in crayfish, and hence, their invasion success, we also measured chela size of a subset of specimens in both introduced and native ranges. Genetic diversity of introduced signal crayfish populations was as high as that of the dominant phylogeographic group in the native range, suggesting high propagule pressure during invasion. More recently established crayfish populations in Japan that originated through secondary spread from one of the founding populations exhibit reduced genetic diversity relative to older populations, probably as a result of founder effects. However, these newer populations also show larger chela size, consistent with expectations of rapid adaptations or phenotypic responses during the invasion process. Introduced signal crayfish populations in Japan originate from multiple source populations from a wide geographic range in the native range of western North America. A combination of high genetic diversity, especially for older populations in the invasive range, and rapid adaptation to colonization, manifested as larger chela in recent invasions, likely contribute to invasion success of signal crayfish in Japan.     

uPA Attenuated LPS-induced Inflammatory Osteoclastogenesis through the Plasmin/PAR-1/Ca2+/CaMKK/AMPK Axis

Chronic inflammatory diseases, such as rheumatoid arthritis and periodontitis-caused bone destruction, results from an increase of bone-resorbing osteoclasts (OCs) induced by inflammation. However, the detailed mechanisms underlying this disorder remain unclear. We herein investigated that the effect of urokinase-type plasminogen activator (uPA) on inflammatory osteoclastogenesis induced by lipopolysaccharide (LPS), which is a potent stimulator of bone resorption in inflammatory diseases. We found that the uPA deficiency promoted inflammatory osteoclastogenesis and bone loss induced by LPS. We also showed that LPS induced the expression of uPA, and the uPA treatment attenuated the LPS-induced inflammatory osteoclastogenesis of RAW264.7 mouse monocyte/macrophage lineage cells. Additionally, we showed that the uPA-attenuated inflammatory osteoclastgenesis is associated with the activation of plasmin/protease-activated receptor (PAR)-1 axis by uPA. Moreover, we examined the mechanism underlying the effect of uPA on inflammatory osteoclastogenesis, and found that uPA/plasmin/PAR-1 activated the adenosine monophosphate-activated protein kinase (AMPK) pathway through Ca2+/calmodulin dependent protein kinase kinase (CaMKK) activation, and attenuated inflammatory osteoclastogenesis by inactivation of NF-κB in RAW264.7 cells. These data suggest that uPA attenuated inflammatory osteoclastogenesis through the plasmin/PAR-1/Ca2+/CaMKK/AMPK axis. Our findings may provide a novel therapeutic approach to bone loss caused by inflammatory diseases.