Inhibitory effect of ribbon-type NF-kappaB decoy oligodeoxynucleotides on osteoclast induction and activity in vitro and in vivo

In this study we examined the effect of ribbon-type (circular-type) NF-kappaB decoy oligodeoxynucleotides (RNODN) on osteoclast induction and activity. We extracted bone marrow cells from the femurs of rats and incubated non-adherent cells with receptor activator of nuclear factor kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). First, transfer efficiency into osteoclasts and their precursors, resistance to exonuclease, and binding activity of decoy to NF-kappaB were examined. Next, to examine the effect of RNODN on osteoclast induction and activity, osteoclast differentiation and pit formation assays were performed. RNODN were injected into the ankle joints of rats with collagen-induced arthritis. Joint destruction and osteoclast activity were examined by histological study. The resistance of RNODN to exonuclease and their binding activity on NF-kappaB were both greater than those of phosphorothionated NF-kappaB decoy oligodeoxynucleotides. The absolute number of multinucleate cells scoring positive for tartrate-resistant acid phosphatase was significantly decreased in the RNODN-treated group. The average calcified matrix resorbed area was significantly decreased in the RNODN-treated group. Histological study showed marked suppression of joint destruction and osteoclast activity by intra-articular injection of RNODN. These results suggest the inhibitory effect of RNODN on the induction and activity of osteoclasts. Direct intra-articular injection of RNODN into the joints may be an effective strategy for the treatment of arthritis.     

Human autologous serum obtained using a completely closed bag system as a substitute for foetal calf serum in human mesenchymal stem cell cultures

The major problem in cell therapy is the possibility of viral or bacterial infection and immune reactions. Therefore, it is expected of culture cells which are intended to be re-implanted with autologous serum rather than conventional bovine serum. Cell therapy with human mesenchymal stem cells (hMSC), differentiating to various cells, is thought to be curative. To culture hMSC with human autologous serum (HAS) and re-implant them for cell therapy, we developed a completely closed bag system separating serum, comparing proliferation and multipotency of hMSC cultured in HAS with those in foetal calf serum (FCS). HAS was simply, safely and efficiently obtained with the developed closed bag system. Cell proliferation of hMSC cultured in HAS was greater than that in FCS. hMSC, exposed to the defined induction medium containing HAS as well as FCS, differentiated into osteoblasts and adipocytes. These findings suggest that HAS obtained with the developed closed bag system is advantageous in a point of decrease in risk of virus or bacterial infection and foreign protein contamination and enhancement of proliferation of hMSC.     

New filarial nematode from Japanese serows (Naemorhedus crispus: Bovidae) close to parasites from elephants

A new onchocercid species, Loxodontofilaria caprini n. sp. (Filarioidea: Nematoda), found in subcutaneous tissues of 37 (33%) of 112 serows (Noemorhedus crispus) examined in Japan, is described. The female worm had the characteristics of Loxodontofilaria, e.g., the large body size, well-developed esophagus with a shallow buccal cavity, and the long tail with three caudal lappets. The male worm of the new species, which was first described in the genus, had unequal length of spicules, 10 pairs of pre- and post-caudal papillae, and three terminal caudal lappets. Deirids were present in both sexes. Among four species of the genus loxodontofiloria: one from the hippopotamus and three from the Elepantidae, L. caprini n. sp. appears close to L. asiatica Bain, Baker & Chabaud, 1982, a subcutaneous parasite of Elephas indicus in Myanmar (Burma). However, L. caprini n. sp. is distinct from L. asiatica in that the Japanese female worm has an esophagus half as long and the microfilariae also half as long with a coiled posterior. The microfilariae were found in the skin of serows. The new parasite appears to clearly illustrate a major event in the evolution of onchocercids: the host-switching. This might have occurred on the Eurasian continent, where elephantids and the lineage of rupicaprines diversified during the Pliocene-Pleistocene, or in Japan, into which some of these hosts migrated.     

Site-2 proteases in prokaryotes: regulated intramembrane proteolysis expands to microbial pathogenesis

Regulated intramembrane proteolysis (RIP) is a widely distributed mechanism of signal transduction in which membrane-bound proteases cleave transmembrane domains of substrate proteins. The site-2 protease (S2P) class of RIP metalloproteases is present in most bacterial genomes but is generally of unknown function except for the well-characterized proteases RseP and SpoIVFB. In this review we will discuss the biochemical functions and physiologic roles of S2P proteases in bacteria and highlight recent data implicating S2P family members in host-pathogen interactions.     

Genome sequence of the cat pathogen, Chlamydophila felis.

Chlamydophila felis (Chlamydia psittaci feline pneumonitis agent) is a worldwide spread pathogen for pneumonia and conjunctivitis in cats. Herein, we determined the entire genomic DNA sequence of the Japanese C. felis strain Fe/C-56 to understand the mechanism of diseases caused by this pathogen. The C. felis genome is composed of a circular 1,166,239 bp chromosome encoding 1005 protein-coding genes and a 7552 bp circular plasmid. Comparison of C. felis gene contents with other Chlamydia species shows that 795 genes are common in the family Chlamydiaceae species and 47 genes are specific to C. felis. Phylogenetic analysis of the common genes reveals that most of the orthologue sets exhibit a similar divergent pattern but 14 C. felis genes accumulate more mutations, implicating that these genes may be involved in the evolutional adaptation to the C. felis-specific niche. Gene distribution and orthologue analyses reveal that two distinctive regions, i.e. the plasticity zone and frequently gene-translocated regions (FGRs), may play important but different roles for chlamydial genome evolution. The genomic DNA sequence of C. felis provides information for comprehension of diseases and elucidation of the chlamydial evolution.     

Overexpression of heat shock protein 70 restores the structural stability and functional defects of temperature-sensitive mutant of large T antigen at nonpermissive temperature

The effects of heat shock protein 70 (Hsp70), a molecular chaperone, on the degradation and functional alterations of a mutant large T antigen induced by a nonpermissive temperature were examined. In this study, mouse tracheal epithelial TM02-3 cells harboring temperature-sensitive simian virus 40 large T antigen and stable TM02-3 cells overexpressing human Hsp70 and/or Hsp40 were used. Although the temperature shift from 33 degrees C (permissive temperature) to 39 degrees C (nonpermissive temperature) induced increases in the endogenous chaperones including Hsp70 and Hsp40, degradation of the T antigen, activation of the p53-p21(waf1) pathway, and an arrest of cell growth were observed in the mock cells. In contrast, these changes induced by the temperature shift were partially but significantly prevented in stable cells overexpressing human Hsp70 and/or Hsp40. A combination of Hsp70 and Hsp40 was the most effective, suggesting that Hsp40 may cooperate with Hsp70. Moreover, immunocytochemical observation indicated that human Hsp70 was expressed in the cytoplasm at 33 degrees C, but it colocalized with T antigen in the nucleus at 39 degrees C. These results suggest that overexpressed Hsp70 translocates from the cytoplasm to nucleus, and significantly restores the structural stability and functional defects of mutant large T antigen in the cells.