This study investigated the antidiabetic effect of amaranth grain ethanol extract (AEE) in a diabetic animal model, db/db mouse. The mice were divided into 4 groups: normal control mice (C57BL/6J), diabetic mice (C57BL/6J db/db), diabetic mice fed a lower concentration of AEE (0.3 mg/kg), and diabetic mice fed a higher concentration of AEE (0.5 mg/kg). After 10 weeks of treatment, body weights, blood insulin levels and blood glucose levels of each group were compared. At the end of treatment, the results showed that both AEE supplemented groups had lower body weights than those in the diabetic groups although higher than those in the normal groups. Moreover, in both AEE supplemented groups, serum insulin levels were higher and blood glucose levels were lower than those in the diabetic groups although both values were higher than those in the normal groups. The results of this study suggest that AEE can alleviate many of the common symptoms of diabetes in diabetic mice and, therefore, has potential as a therapeutic supplement for normalization of blood glucose and insulin levels in humans.

Natural biopolymers such as collagen and fibrin have been widely used in bone regenerative applications. Despite the frequent use, their comparative biological propertiesis are largely unknown. In a previous study, we found the superiority of fibrin to collagen in the adsorption of serum proteins and the proliferation and differentiation of cultured osteoblasts. In this study, we used an in vivo model to evaluate how effectively fibrin supports bone regeneration, as compared with collagen. Collagen and fibrin were placed in critical size defects made on rat calvarial bones. Compared with collagen, fibrin supported substantially more new bone tissue formation, which was confirmed by micro-CT measurement and histological analyses. The cells in the regenerative tissues of the fibrin-filled defects were immunostained strongly for Runx2, while collagen-placed defects were stained weakly. These in vivo results demonstrate that fibrin is superior to collagen in supporting bone regeneration

Cytokines are known to function as regulatory molecules that can be produced by virtually every nucleated cell type in the body, including lymphocytes, monocytes/macrophages, epithelial cells, fibroblasts, and many others. Cytokines include lymphocyte-derived factors (lymphokines), monocyte-derived factors (monokines), hematopoietic factors (colony-stimulating factors), connective tissue/ growth factors, and chemotactic chemokines. Cytokines released in response to infection can affect tumor development in different ways. When exposed to infectious agents, cytokines are secreted by sentinel cells, such as macrophages and dendritic cells. These cytokines include interleukin 1 (IL-1) and tumor necrosis factor-α, as well as others, such as IL-6, IL-12, and IL-18. When released in sufficient quantities, these molecules can cause inflammation. Chronic inflammation is highly associated with tumor initiation, promotion, and progression. In this article, we review the roles and mechanisms of cytokines in tumor development.

Mamestra brassicae (cabbage moth) is a common European moth of the order Lepidoptera and the family Noctuidae. The larval stage is highly polyphagous and is known to feed on more than 70 species of host plants from 22 families, including Brassica species, lettuce, onion, potato, pea, tomato and apple. M. brassicae has become a significant pest also in Asia due to the damage caused to agriculturally and economically important Brassica crops. It is difficult to control M. brassicae using chemical insecticide because of its rapid development of resistance. The objective of our study, therefore, was the mass production and formulation of a local strain of M. brassicae nucleopolyhedrovirus-K1 (MabrNPV-K1) for the development of viral insecticide to control it. In production efficiency of MabrNPV-K1 using M. brassicae larvae, the mortality of the 3rd instar larvae was 100% when inoculated with 1.0 × 105 PIBs/larva and the yield of MabrNPV-K1 was maximal. Regarding the mortality, yield of polyhedra, inoculation doses and required time, the 1.0 × 104 PIBs/larva at 30°C was determined as optimal conditions producing polyhedra efficiently. To formulate MabrNPV-K1, feeding toxicities of various supplements including spreader and ultraviolet (UV) -protectant were determined. Tinopal UNPA-GX which is UV-protectants was effective for protection of polyhedra from UV and showed the increased mortality when added with 1% concentration. Other supplements did not influence significantly the mortality of MabrNPV-K1. Formulated MabrNPV-K1 with several supplements showed higher pathogencity than un-formulated MabrNPV-K1.

Bone morphogenetic protein (BMP) 2 is a potent osteogenic factor. Although both Smad1/5 and mitogenactivated protein kinases (MAPKs) are activated by BMP2, the hierarchical relationship between them is unclear. In this study, we examined if BMP2-stimulated MAPK activation is regulated by Smad1/5 or vice versa. When C2C12 cells were treated with BMP2, the activation of extracellular signal-regulated kinase (ERK), p38 MAPK and c-Jun-N-terminal kinase was evident within 5 min. The knockdown of both Smad1 and Smad5 by small interfering RNA did not affect the activation of these MAPKs. In addition, neither the overexpression of Smad1 nor Smad5 induced ERK activation. When ERK activation was induced by constitutively active MEK1 expression, the protein level and activation of Smad1 increased. Furthermore, the inhibition of constitutively active BMP receptor type IB-induced ERK activation significantly suppressed Smad1 activation. These results indicate that Smad1/5 activation is not necessary for BMP2-induced MAPK activation and also that ERK positively regulates Smad1 activation.

Mutations in DLX3 are associated with both autosomal dominant hypoplastic hypomaturation amelogenesis imperfecta (ADHHAI) and tricho-dento-osseous (TDO) syndrome. ADHHAI is caused by a c.561_562delCT (2bp-del DLX3) mutation whereas TDO syndrome is associated with a c.571_574delGGGG (4bp-del DLX3) mutation. However, although the causal relationships between DLX3 and an enamel phenotype have been established, the pathophysiological role of DLX3 mutations in enamel development has not yet been clarified. In our current study, we prepared expression vectors for wild type and deletion mutant DLX3 products (4bp-del DLX3, 2bp-del DLX3) and examined the effects of their overexpression on the expression of the enamel matrix proteins and proteases. Wild type DLX3 enhanced the expression of matrix metalloprotease 20 (MMP20) mRNA and protein in murine ameloblast-like cells. However, neither a 4bp-del nor 2bp-del DLX3 increased MMP20 expression. Wild type DLX3, but not the above DLX3 mutants, also increased the activity of reporters containing 1.5 kb or 0.5 kb of the MMP20 promoter. An examination of protein stability showed that the half-life of wild type DLX3 protein was less than 12 h whilst that of both deletion mutants was longer than 24 h. Endogenous Dlx3 was also found to be continuously expressed during ameloblast differentiation. Since inactivating mutations in the gene encoding MMP20 are associated with amelogenesis imperfecta, the inability of 4bp-del or 2bp-del DLX3 to induce MMP20 expression suggests a possible involvement of such mutations in the enamel phenotype associated with TDO syndrome or ADHHAI.

To determine the characteristics of the Korean porcine reproductive and respiratory syndrome virus (PRRSV), CA, which was isolated from the serum of an infected pig in 2006, we investigated the nucleotide sequence and expression of the structural ORFs (ORFs 2 to 7) using the bApGOZA system. We found that the structural ORFs 2 to 7 of CA consisted of 3188 nucleotides that were the same as those formed from VR-2332. Comparison of the CA with the other strains revealed nucleotide sequence identity ranging from 89.8 to 99.5%. To better understand the genetic relationships between other strains, phylogenetic analyses were performed. The CA strain was closely related to the other North American genotype strains but formed a distinct branch with high bootstrap support. Additionally, expression levels of the PRRSV proteins in Sf21 cells were strong or partially weak. The results of this study have implications for both the taxonomy of PRRSV and vaccine development.

The porcine reproductive and respiratory syndrome virus (PRRSV) has six structural proteins which encoded by ORFs 2 to 7 are designated as GP2, 3, 4, 5, M and N, repectively. In this study, we determined the expression of each protein using novel transfer vector, pBmKSK4 which has the polyhedrin promoter of BmNPV and 6xHis tag. The recombinant transfer vector was co-transfected into Bm5 cells along with bBpGOZA DNA. Recombinant virus was purified by plaque assay and amplified in Bm5 cells. Expression of each protein was identified by SDS-PAGE and Western blot analysis using anti-6xHis monoclonal antibody. The expression levels of the structural proteins in Bm5 cells were stronger than the expression system using pBacPAK9 transfer vector in Sf21 cells. As expected, GP5 was expressed at low levels from its structural properties and its toxicity for cells. In addition, each recombinant protein was purified using Ni-NTA spin columns. The ability to produce each protein in the baculovirus system indicates that these could be major candidates for the development of a vaccine against PRRSV.

Pine wilt is the most important disease of pine trees in Korea, Japan and China. The pathogen causing this disease, the pinewood nematode (Bursaphelenchus xylophylus), is transmitted vectored by adults of some cerambycid beetle species and the Japanese pine sawyer, Monochamus alternatus, is the major vector species in Korea. Although chemical insecticides have been used to kill vector insect and thus prevent transmission of the pathogen, the efficacy is not good. In Japan, to control this insect, an entomopathogenic fungus was studied and developed as an insecticide. This is thought to be the convenient and effective method to control M. alternatus. Recently, there are several reports about the pinewood nematode is vectored by also the pine sawyer, M. saltuarius, in Korea. The objective of this study, therefore, was to isolate and identify entomopathogenic fungi from M. saltuarius cadaver to control it. We collected the cadaver of M. saltuarius and then screened several fungi colonies. The pathogenicity of each fungus was tested using oak longicorn beetle, Moechotypa diphysis, as substitutive insect. M. diphysis is also serious pest to various trees in forest. As the result, only one of them showed high pathogenicity against M. diphysis. Selected fungus was identified by microscopic examination and DNA analysis. Pathogenicity was also evaluated to M. saltuarius.

In the process of bone remodeling, mineral phase of bone is dissolved by osteoclasts, resulting in elevation of calcium concentration in micro-environment. This study was performed to explore the effect of high extracellular calcium (Cα²+e) on mineralized nodule formation and on the expression of progressive ankylosis (Ank), plasma cell membrane glycoprotein-1 (PC-1) and osteopontin by primary cultured mouse calvarial cells. Osteoblastic differentiation and mineralized nodule formation was induced by culture of mouse calvarial cells in osteoblast differentiation medium containing ascorbic acid and β-glycerophosphate. Although Ank, PC-1 and osteopontin are well known inhibitors of mineralization, expression of these genes were induced at the later stage of osteoblast differentiation during when expression of osteocalcin, a late marker gene of osteoblast differentiation, was induced and mineralization was actively progressing. High Cα²+e(10 mM) treatment highly enhanced mRNA expression of Ank, PC-1 and osteopontin in the late stage of osteoblast differentiation but not in the early stage. Inhibition of p44/42 MAPK activation but not that of protein kinase C suppressed high Cα²+e- induced expression of Ank, PC-1 and osteopontin. When high Cα²+e (5 mM or 10 mM) was present in culture medium during when mineral deposition was actively progressing, matrix calcifiation was significantly increased by high Cα²+e. This stimulatory effect was abolished by pyrophosphate (5 mM) or levamisole (0.1-0.5 mM), an alkaline phosphatase inhibitor. In addition, probenecid (2mM), an inhibitor of Ank, suppressed matrix calcification in both control and high Cα²+e- treated group, suggesting the possible role of Ank in matrix calcification by osteoblasts. Taken together, these results showed that high Cα²+e stimulates expression of Ank, PC-1 and osteopontin as well as matrix calcification in late differentiation stage of osteoblasts and that p44/42 MAPK activation is involved in high Cα²+e- induced expression of Ank, PC-1 and osteopontin.

Dlx3 is a homeodomain protein and is known to play a role in development and differentiation of many tissues. Deletion of four base pairs in DLX3 (NT3198) is causally related to tricho-dento-osseous (TDO) syndrome (OMIM #190320), a genetic disorder manifested by taurodontism, hair abnormalities, and increased bone density in the cranium. The molecular mechanisms that explain the phenotypic characteristics of TDO syndrome have not been clearly determined. In this study, we examined phenotypic characteristics of wild type DLX3(wtDlx3) and 4-BP DEL DLX3 (TDO mtDlx3) in C2C12 cells. To investigate how wtDlx3 and TDO mtDlx3 differentially regulate osteoblastic differentiation, reporter assays were performed by using luciferase reporters containing the promoters of alkaline phosphatase, bone sialoprotein or osteocalcin. Both wtDlx3 and TDO mtDlx3 enhanced significantly all the reporter activities but the effect of mtDlx3 was much weaker than that of wtDlx3. In spite of these differences in reporter activity, electrophoretic mobility shift assay showed that both wtDlx3 and TDO mtDlx3 formed similar amounts of DNA binding complexes with Dlx3 binding consensus sequence or with ALP promoter oligonucleotide bearing the Dlx3 binding core sequence. TDO mtDlx3 exhibits a longer half-life than wtDlx3 and it corresponds to PESTfind analysis result showing that potential PEST sequence was missed in carboxy terminal of TDO mtDlx3. In addition, co-immunoprecipitation demonstrated that TDO mtDlx3 binds to Msx2 more strongly than wtDlx3. Taken together, though TDO mtDlx3 acted as a weaker transcriptional activator than wtDlx3 in osteoblastic cells, there is possibility that during in vivo osteoblast differentiation TDO mtDlx3 may antagonize transcriptional repressor activity of Msx2 more effectively and for longer period than wtDlx3, resulting in enhancement of osteoblast differentiation.

We hypothesized that plaque-associated bacteria may have a role in maintenance of alveolar bone. To test it, immortalized gingival epithelial HOK-16B cells were co-cultured with live or lysed eight plaque bacterial species and the expression levels of bone morphogenetic protein (BMP)-2 and -4 were examined by real time reverse transcription-polymerase chain reaction. Un-stimulated HOK-16B cells expressed both BMP-2 and -4. Co-culture with plaque bacterial lysates had significant effects on the level of BMP-2 but not on that of BMP-4. Five species including Streptococcus sanguinis, S. gordonii, Veillonella atypica, Porphyromonas gingivalis, and Treponema denticola substantially up-regulated the level of BMP-2. In contrary to the upregulatory effect of lysate, live T. denticola suppressed the expression of BMP-2. In addition, in vitro osteoblastic differentiation assay using C2C12 cells and the conditioned medium of HOK-16B cells confirmed the production of BMPs by gingival epithelial cells and the modulation of BMP expression by the lysates of S. sanguinis and T. denticola. In conclusion, we have shown that plaque bacteria can regulate the expression of BMP-2 by gingival epithelial cells, the physiologic meaning of which needs further investigation.

Dlx3 is a homeodomain protein and is known to playa role in development and differentiation of many tissues. Deletion of four base pairs in DLX3 (NT3198) is causally related to tricho-dento-osseous (TDO) syndrome (OMIM # 190320), a genetic disorder manifested by taurodontism, hair abnormalities, and increased bone density in the cranium. Although the observed defects of TDO syndrome involves bone, little is known about the role of Dlx3 in bone remodeling process. In this study, we examined the effect of wild type DLX3 (wtDlx3) expression on osteoclast differentiation and compared it with that of 4-BP DEL DLX3 (TDO mtDlx3). To examine whether Dlx3 is expressed during RANKL-induced osteoclast differentiation, RAW264.7 cells were cultured in the presence of receptor activator of nuclear factor-B ligand (RANKL). Dlx3 protein level increased slightly after RANKL treatment for 1 day and peaked when the fusion of prefusion osteoclasts actively progressed. When wtDlx3 and TDO mtDlx3 were overexpressed in RAW264.7 cells, they enhanced RANKL-induced osteoclastogenesis and the expression of osteoclast differentiation marker genes such as calcitonin receptor, vitronectin receptor and cathepsin K. Since osteoclast differentiation is critically regulated by the balance between RANKL and osteoprotegerin (OPG), we examined the effect of Dlx3 overexpression on expression of RANKL and OPG in C2C12 cells in the presence of bone morphogenetic protein 2. Overexpression of wtDlx3 enhanced RANKL mRNA expression while slightly suppressed OPG expression. However, TDO mtDlx3 did not exert significant effects. This result suggests that inability of TDO mtDlx3 to regulate expression of RANKL and OPG may contribute to increased bone density in TDO syndrome patients. Taken together, it is suggested that Dlx3 playa role as a positive regulator of osteoclast differentiation via up-regulation of osteoclast differentiation-associated genes in osteoclasts, as well as via increasing the ratio of RANKL to OPG in osteoblastic cells.

Calcium concentration in the bone resorption lacunae is high and is in the mM concentration range. Both osteoblast and osteoclast have calcium sensing receptor in the cell surface, suggesting the regulatory role of high extracellular calcium in bone metabolism. In vitro, high extracellular calcium stimulated osteoclastogenesis in coculture of mouse osteoblasts and bone marrow cells. Therefore we examined the genes that were commonly regulated by both high extracellular calcium and 1.25(OH)₂vitaminD₃(VD3) by using mouse oligo 11 K gene chip. In the presence of 10 mM [Ca²+]e or 10 nM VD3, mouse calvarial osteoblasts and bone marrow cells were co-cultured for 4 days when tartrate resistant acid phosphatase-positive multinucleated cells start to appear. Of 11,000 genes examined, the genes commonly regulated both by high extracellular calcium and by VD3 were as follows; 1) the expression of genes which were osteoclast differentiation markers or were associated with osteoclastogenesis were up-regulated both by high extracellular calcium and by VD3; trap, mmp9, car2, ctsk, ckb, atp6b2, tm7sf4, rab7, 2) several chemokine and chemokine receptor genes such as sdf1, scya2, scyb5, scya6, scya8, scya9, and ccr1 were up-regulated both by high extracellular calcium and by VD3, 3) the genes such as mmp1b, mmp3 and c3 which possibly stimulate bone resorption by osteoclast, were commonly up-regulated, 4) the gene such as c1q and msr2 which were related with macrophage function, were commonly down-regulated, 5) the genes which possibly stimulate osteoblast differentiation and/or mineralization of extracellular matrix, were commonly down-regulated; slc8a1, admr, plod2, lox, fosb, 6) the genes which possibly suppress osteoblast differentiation and/or mineralization of extracellular matrix, were commonly up-regulated; s100a4, npr3, mme, 7) the genes such as calponin 1 and tgfbi which possibly suppress osteoblast differentiation and/or mineralization of extracellular matrix, were up-regulated by high extracellular calcium but were down-regulated by VD3. These results suggest that in coculture condition, both high extracellular calcium and VD3 commonly induce osteoclastogenesis but suppress osteoblast differentiation/mineralization by regulating the expression of related genes.

The elderly suffer from an impaired immune function being obvious in a higher susceptibility to infections. Although the inflammatory cells are the major immunomodulatory cells, fibroblasts also secrete a variety of inflammatory cytokines and chemokines. Therefore periodontal tissue aging might playa role in development and progress of periodontitis. In this study, we investigated the effect of in vitro periodontal ligament cellular aging on the inflammatory cytokines, chemokines, and matrix metalloprotease(MMP)-2 expression induced by lipopolysaccharide(LPS) treatment. Three different cell populations were used; passages 4-5, 14-15, and 24-25 (at passage 27, more than 90% cells were replicative senescent). LPS increased the expression of interleukin(IL)-1β, IL-6, and tumor necrosis factor-α, IL-8, RANTES, and MMP-2. However, the order of induction folds were passages 14-15 > 4-5 > 24-25. While the expression level of Toll-like receptor(TLR) 4 decreased according to the increase in passage number, the level of TLR2 was highest at passages 14-15 and then decreased at passages 24-25. While the spontaneous expression of IL-8 decreased according to the increase in passage number, that of RANTES and proMMP-2 increased according to the increase in passage number. These results suggest that the aging of periodontal ligament fibroblasts differentially affect the role as immunomodulatory cells in response to periodontopathic bacteria and therefore might be another risk factor of periodontitis progression.