Osteoprotegerin reduces the serum level of receptor activator of NF-kappaB ligand derived from osteoblasts

Osteoprotegerin (OPG) is a decoy receptor for receptor activator of NF-kappaB ligand (RANKL). We previously reported that OPG deficiency elevated the circulating level of RANKL in mice. Using OPG(-/-) mice, we investigated whether OPG is involved in the shedding of RANKL by cells expressing RANKL. Osteoblasts and activated T cells in culture released a large amount of RANKL in the absence of OPG. OPG or a soluble form of receptor activator of NF-kappaB (the receptor of RANKL) suppressed the release of RANKL from those cells. OPG- and T cell-double-deficient mice showed an elevated serum RANKL level equivalent to that of OPG(-/-) mice, indicating that circulating RANKL is mainly derived from bone. The serum level of RANKL in OPG(-/-) mice was increased by ovariectomy or administration of 1alpha,25-dihydroxyvitamin D(3). Expression of RANKL mRNA in bone, but not thymus or spleen, was increased in wild-type and OPG(-/-) mice by 1alpha,25-dihydroxyvitamin D(3). These results suggest that OPG suppresses the shedding of RANKL from osteoblasts and that the serum RANKL in OPG(-/-) mice exactly reflects the state of bone resorption.     

Four new crystal structures of Tk-subtilisin in unautoprocessed, autoprocessed and mature forms: insight into structural changes during maturation

Subtilisin from the hyperthermophilic archaeon Thermococcus kodakaraensis (Tk-subtilisin) is matured from Pro-Tk-subtilisin upon autoprocessing and degradation of the propeptide. The crystal structures of the autoprocessed and mature forms of Tk-subtilisin were determined at 1.89 A and 1.70 A resolution, respectively. Comparison of these structures with that of unautoprocessed Pro-Tk-subtilisin indicates that the structure of Tk-subtilisin is not seriously changed during maturation. However, one unique Ca(2+)-binding site (Ca-7) is identified in these structures. In addition, the N-terminal region of the mature domain (Gly70-Pro82), which binds tightly to the main body in the unautoprocessed form, is disordered and mostly truncated in the autoprocessed and mature forms, respectively. Interestingly, this site is formed also in the unautoprocessed form when its crystals are soaked with 10 mM CaCl(2), as revealed by the 1.87 A structure. Along with the formation of this site, the N-terminal region (Leu75-Thr80) is disordered, with the scissile peptide bond contacting with the active site. These results indicate that the calcium ion binds weakly to the Ca-7 site in the unautoprocessed form, but is trapped upon autoprocessing. We propose that the Ca-7 site is required to promote the autoprocessing reaction by stabilizing the autoprocessed form, in which the new N terminus of the mature domain is structurally disordered. Furthermore, the crystal structure of the Tk-propeptide:S324A-subtilisin complex, which was formed by the addition of separately expressed proteins, was determined at 1.65 A resolution. This structure is virtually identical with that of the autoprocessed form, indicating that the interaction between the two domains is highly intensive and specific.     

Regulation of the phosphatidylinositol 3-kinase-Akt and the mitogen-activated protein kinase pathways by ursolic acid in human endometrial cancer cells

The phosphatidylinositol 3-kinase-Akt (PI3K-Akt) pathway and the mitogen activated protein kinase (MAPK) pathway are important in the development and proliferation of various human cancers. It has been found recently that ursolic acid treatment affects growth and apoptosis in cancer cells. We sought to determine whether prominent signaling pathways, including the PI3K-Akt pathway and the MAPK (JNK, P38, and P44/42) pathway mediate these effects. Endometrial cancer cells often have high levels of phosphorylated Akt seen in conjunction with a PTEN mutation or deletion. Elevation in Akt protects the cancer cell from apoptosis. Ursolic acid treatment moderately decreased PI3K levels in SNG-II cells. Treatment also decreased phospho-Akt and phospho-P44/42 in a dose- and time-dependent fashion, dramatically in SNG-II cells and moderately in HEC108 cells. This effect was most pronounced following treatment with 50 mum ursolic acid for 72 h. Our study found inhibition of both the PI3K-Akt pathway and the MAPK pathway in two endometrial cancer cell lines, SNG-II and the poorly differentiated HEC108 cell line.     

5-hydroxytryptamine synthesis in HL-1 cells and neonatal rat cardiocytes

Some reports showed that serotonergic system might have existed and that 5-hydroxytryptamine (5-HT) was detected in the hamster heart. The source of 5-HT in the heart, however, remains to be fully elucidated. So the present study was designed to define serotonergic system and to clarify which cell could produce 5-HT in the heart. As a result, 5-HT was detected in homogenates of HL-1 cardiomyocytes by high performance liquid chromatography with fluorescence detection, but not in those of neonatal rat non-cardiomyocytes (NMCs). And TPH and AADC mRNAs were expressed in HL-1 cardiomyocytes and neonatal rat cardiomyocytes (MCs), not in NMCs. mRNAs of 5-HT(2A) receptor were detected in both MCs and NMCs, and those of 5-HT(2B) receptor in NMCs. These findings definitively demonstrate that 5-HT is secreted from the myocytes of the heart and strongly implied that 5-HT might play a certain role in cardiac physiology.     

Muramyl dipeptide enhances osteoclast formation induced by lipopolysaccharide, IL-1 alpha, and TNF-alpha through nucleotide-binding oligomerization domain 2-mediated signaling in osteoblasts

Muramyl dipeptide (MDP) is the minimal essential structural unit responsible for the immunoadjuvant activity of peptidoglycan. As well as bone-resorbing factors such as 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3) and PGE2, LPS and IL-1alpha stimulate osteoclast formation in mouse cocultures of primary osteoblasts and hemopoietic cells. MDP alone could not induce osteoclast formation in the coculture, but enhanced osteoclast formation induced by LPS, IL-1alpha, or TNF-alpha but not 1alpha,25(OH)2D3 or PGE2. MDP failed to enhance osteoclast formation from osteoclast progenitors induced by receptor activator of NF-kappaB ligand (RANKL) or TNF-alpha. MDP up-regulated RANKL expression in osteoblasts treated with LPS or TNF-alpha but not 1alpha,25(OH)2D3. Osteoblasts expressed mRNA of nucleotide-binding oligomerization domain 2 (Nod2), an intracellular sensor of MDP, in response to LPS, IL-1alpha, or TNF-alpha but not 1alpha,25(OH)2D3. Induction of Nod2 mRNA expression by LPS but not by TNF-alpha in osteoblasts was dependent on TLR4 and MyD88. MDP also enhanced TNF-alpha-induced osteoclast formation in cocultures prepared from Toll/IL-1R domain-containing adapter protein (TIRAP)-deficient mice through the up-regulation of RANKL mRNA expression in osteoblasts, suggesting that TLR2 is not involved in the MDP-induced osteoclast formation. The depletion of intracellular Nod2 by small interfering RNA blocked MDP-induced up-regulation of RANKL mRNA in osteoblasts. LPS and RANKL stimulated the survival of osteoclasts, and this effect was not enhanced by MDP. These results suggest that MDP synergistically enhances osteoclast formation induced by LPS, IL-1alpha, and TNF-alpha through RANKL expression in osteoblasts, and that Nod2-mediated signals are involved in the MDP-induced RANKL expression in osteoblasts.     

Exploration of genes that encode a carbocycle-forming enzyme involved in biosynthesis of aminoglycoside antibiotics from the environmental DNA

2-Deoxy-scyllo-inosose (DOI) synthase is the enzyme participating in biosynthesis of 2-deoxystreptamine (DOS)-containing aminoglycoside antibiotics. The gene which encodes the enzyme can be a marker for screening of DOS-containing aminoglycoside-producer and exploration of its biosynthetic gene. Further, this enzyme is expected to be of use in industry, because it converts sugar into 6-membered carbocycle. In the present study, we identified 21 clones encoding DOI synthase from environmental DNA by degenerate PCR. They were clearly divided into two groups. One appeared to derive from actinomycetes, and the other from non-actinomycetes. The latter group was larger (17 clones) than the former (four clones) despite the fact that only one strain of non-actinomycete was identified for DOS-containing aminoglycoside production. This result indicates that there are still many unidentified non-actinomycetes for DOS-containing aminoglycoside biosynthesis. We showed the possibility of identification of novel aminoglycoside-producing non-actinomycete from soil, and for development of more efficient enzymes for industrial use.     

Gastric inhibitory polypeptide as an endogenous factor promoting new bone formation after food ingestion

Calcium plays a fundamental role as second messenger in intracellular signaling and bone serves as the body's calcium reserve to tightly maintain blood calcium levels. Calcium in ingested meal is the main supply and inadequate calcium intake causes osteoporosis and bone fracture. Here, we describe a novel mechanism of how ingested calcium is deposited on bone. Meal ingestion elicits secretion of the gut hormone gastric inhibitory polypeptide (GIP) from endocrine K cells in the duodenum. Bone histomorphometrical analyses revealed that bone formation parameters in the mice lacking GIP receptor (GIPR(-/-)) were significantly lower than those of wild-type (GIPR(+/+)) mice, and that the number of osteoclasts, especially multinuclear osteoclasts, was significantly increased in GIPR(-/-) mice, indicating that GIPR(-/-) mice have high-turnover osteoporosis. In vitro examination showed the percentage of osteoblastic cells undergoing apoptosis to be significantly decreased in the presence of GIP. Because GIPR(-/-) mice exhibited an increased plasma calcium concentration after meal ingestion, GIP directly links calcium contained in meal to calcium deposition on bone.     

Effects of chloromethanes on growth of and deletion of the pce gene cluster in dehalorespiring Desulfitobacterium hafniense strain Y51

The dehalorespiring Desulfitobacterium hafniense strain Y51 efficiently dechlorinates tetrachloroethene (PCE) to cis-1,2-dichloroethene (cis-DCE) via trichloroethene by PceA reductive dehalogenase encoded by the pceA gene. In a previous study, we found that the significant growth inhibition of strain Y51 occurred in the presence of commercial cis-DCE. In this study, it turned out that the growth inhibition was caused by chloroform (CF) contamination of cis-DCE. Interestingly, CF did not affect the growth of PCE-nondechlorinating SD (small deletion) and LD (large deletion) variants, where the former fails to transcribe the pceABC genes caused by a deletion of the promoter and the latter lost the entire pceABCT gene cluster. Therefore, PCE-nondechlorinating variants, mostly LD variant, became predominant, and dechlorination activity was significantly reduced in the presence of CF. Moreover, such a growth inhibitory effect was also observed in the presence of carbon tetrachloride at 1 microM, but not carbon dichloride even at 1 mM.