Mechanism involved in enhancement of osteoblast differentiation by hyaluronic acid

Objectives

Bone morphogenetic protein-2 (BMP-2) is expected to be utilized to fill bone defects and promote healing of fractures. However, it is unable to generate an adequate clinical response for use in bone regeneration. Recently, it was reported that glycosaminoglycans, including heparin, heparan sulfate, keratan sulfate, dermatan sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, and hyaluronic acid (HA), regulate BMP-2 activity, though the mechanism by which HA regulates osteogenic activities has not been fully elucidated. The aim of this study was to investigate the effects of HA on osteoblast differentiation induced by BMP-2.

Materials and methods

Monolayer cultures of osteoblastic lineage MG63 cells were incubated with BMP-2 and HA for various time periods. To determine osteoblastic differentiation, alkaline phosphatase (ALP) activity in the cell lysates was quantified. Phosphorylation of Smad 1/5/8, p38, and ERK proteins was determined by Western blot analysis. To elucidate the nuclear translocation of phosphorylated Smad 1/5/8, stimulated cells were subjected to immunofluorescence microscopy. To further elucidate the role of HA in enhancement of BMP-2-induced Smad signaling, mRNA expressions of the BMP-2 receptor antagonists noggin and follistatin were detected using real-time RT-PCR.

Results

BMP-2-induced ALP activation, Smad 1/5/8 phosphorylation, and nuclear translocation were up-regulated when MG63 cells were cultured with both BMP-2 and HA. Western blot analysis revealed that phosphorylation of ERK protein was diminished by HA. Furthermore, the mRNA expressions of noggin and follistatin induced by BMP-2 were preferentially blocked by HA.

Conclusions

These results indicate that HA enhanced BMP-2 induces osteoblastic differentiation in MG63 cells via down-regulation of BMP-2 antagonists and ERK phosphorylation.     

Intermedilysin induces EGR-1 expression through calcineurin/NFAT pathway in human cholangiocellular carcinoma cells

RNF8 is a nuclear protein having an N-terminal forkhead-associated (FHA) domain and a C-terminal RING-finger (RF) domain. Depletion of RNF8 caused cell growth inhibition and cell cycle arrest at not only S but also G2/M phases. In addition, cell death was frequently observed in RNF8-depleted cells. Analyses of time-lapse microscopy revealed that the cells died in mitosis and interphase. To elucidate the RNF8 function in M phase, the Plk1 content in RNF8-depleted cells was examined. The amount of RNF8 decreased time-dependently, whereas Plk1 reciprocally increased by transfection of RNF8 siRNA. Protein contents of RNF8 and Plk1 among various cell lines were also compared. RNF8 in normal cell lines was much higher than that in many cancer cell lines. Conversely, Plk1 in normal cell lines was lower than in cancer cell lines. These results suggest that RNF8 is downregulated in many cancer cells and inversely correlated with Plk1.     

Regulatory role of cardiolipin in the activity of an ATP-dependent protease, Lon, from Escherichia coli

Lon is an ATP-dependent serine protease that plays a significant role in the quality control of proteins in cells, degrading misfolded proteins and certain short-lived regulatory proteins under stresses as such heat-shock and UV irradiation. It is known that some polymers containing phosphate groups regulate enzymatic activity by binding with Lon. We focused on the phospholipids of biological membrane components such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylglycerol and cardiolipin (CL), and examined whether or not liposomes containing these phospholipids regulate the enzymatic activity of Lon. CL-containing liposomes specifically inhibited both the proteolytic and ATPase activities of Lon in a dose-dependent manner. In addition, on pull-down assay, we found that CL-containing liposomes selectively bound to Lon. The interaction between CL-containing liposomes and Lon changed with the order of addition of Mg(2+)/ATP. When CL-containing liposomes were added after the addition of Mg(2+)/ATP to Lon, the binding of CL-containing liposomes to Lon was significantly decreased as compared with the reversed order. In fact, we found that CL-containing liposomes bound to Lon, resulting in inhibition of the enzymatic activity of Lon. These results suggest that Lon interacts with CL in biological membranes, which may regulate the functions of Lon as a protein-degrading centre in accordance with environmental changes inside cells.     

Enumeration of viable Enterococcus faecalis, a predominant apical periodontitis pathogen, using propidium monoazide and quantitative real-time polymerase chain reaction

To discriminate between viable and non-viable Enterococcus faecalis, the predominant pathogen in apical periodontitis, a real-time PCR method combined with propidium monoazide (PMA) was developed and evaluated. PMA had no antimicrobial effect on E. faecalis cells and permitted enumeration of both viable and non-viable cells. Therefore, E. faecalis cells from the root canals of nine patients with apical periodontitis were analyzed to evaluate the diagnostic usefulness of this approach. Viable and non-viable E. faecalis cells were successfully discriminated in these clinical specimens. A real-time PCR assay combined with PMA will contribute to the precise diagnosis of apical periodontitis.     

Expression of pheA gene using PR-PL tandem promoter of bacteriophage lambda

Summary The genepheA ⁺ coding chorismate niutase P-prephenate dehydratase, one of the regulatory enzymes of phenylalanine biosynthesis, was cloned into the down-stream of P_R-P_L tandem promoter. In this construction, both the native promoter-operator region and the attenuator region ofpheA ⁺ operon were eliminated so as to avoid the repression and attenuation ofpheA ⁺ gene expression. The expression ofpheA ⁺ gene was directed by P_R-P_L tandem promoter of bacteriophage lambda and controlled by a temperature sensitive repressor, cI₈₅₇.It was shown that the expression as well as phenylalanine production was regulated by temperature. Maximum production of phenylalanine, 170 mg/l, was obtained at 40°C. The host strain, MC1065, produced a trace (4 mg/l) of phenylalanine at the same temperature.     

Two morphological groups derived fromClonostachys cylindrospora and their relationship totrans-4-hydroxy-(l)-proline productivity

Thetrans-4-hydroxy-(l)-proline productivity and morphological characteristics of strains isolated from two different conidium-producing structures inClonostachys cylindrospora were compared.trans-4-Hydroxy-(l)-proline was found to be produced only by strains originating from conidia on penicillate conidiophores, not by strains originating from conidia on verticillate conidiophores. Strains from each conidium-producing structure were also segregated by morphological properties; i.e., the amount of aerial hypha produced on modified Weitzman's agar, production of water-soluble yellow pigment in potato-dextrose agar, the primarily formed conidium-producing structures, and sclerotium formation. These phenomena indicate that morphological characteristics and metabolite productivity are correlated and destined at the stage of conidial production inC. cylindrospora.     

The TraB protein, which mediates the intermycelial transfer of the Streptomyces plasmid pSN22, has functional NTP-binding motifs and is localized to the cytoplasmic membrane

The traB gene on the Streptomyces conjugative plasmid pSN22 is required for intermycelial plasmid transfer and the mobilization of chromosomal markers (Cma). The predicted amino acid sequence of TraB contains one Walker type-A and two type-B NTP-binding motifs. Site-directed mutagenesis revealed that the type-A motif and one of the type-B motifs, 109 amino acid residues downstream of the type-A motif, were essential for both plasmid transfer and Cma. The second type-B sequence could be changed without any phenotypic effect. A modified traB gene was constructed, resulting in the production of a functional protein with an amino-terminal c-Myc epitope tag for immunological analysis. This protein was associated with the cytoplasmic membrane, suggesting that TraB is a membrane protein that uses energy from ATP hydrolysis to transport DNA between mycelia. The c-Myc tagging of TraB decreased the efficiency of intramycelial plasmid spread, suggesting that TraB is involved in both inter- and intramycelial transfer processes.     

Molecular structure of the replication origin of a Bacillus amyloliquefaciens plasmid pFTB14

Summary The structure of a 1.5-kb DNA sequence that is necessary and sufficient for the replication of an 8.2-kb cryptic plasmid, pFTB14, isolated from a strain of Bacillus amyloliquefaciens has been characterized. The 1.5-kb DNA sequence contains an open reading frame, rep, stretching for 1017 bp, a promoter region for rep expression, and a possible replication origin for the plasmid upstream of the promoter. The rep product is trans-active and essential for plasmid replication. The predicted rep protein is a basic protein, as are the RepC protein of pT181, RepB of pUB110 and protein A of pC194 (all these found in staphylococci) and the protein of the R6K plasmid of Escherichia coli. The predicted rep protein has highly homologous amino acid sequences with protein A of pC194 and RepC of pUB110 throughout the protein molecule, but not with RepC of pT181, of R6K or protein RepH encoded by and iniating the replication of pC194.     

Growth-inhibitory activity of the d-mannan of saccharomyces cerevisiae X2180-1A-5 mutant strain against mouse-implanted sarcoma 180 and ehrlich-carcinoma solid tumor

The d-mannan of Saccharomyces cerevisiae X2180-1A-5 mutant strain, which possesses a main chain composed of α-(1→6) linked d-mannopyranosyl residues and a small proportion of branches composed of α-(1→2)- and α-(1→3)-linked d-mannopyranosyl residues, showed strong growth-inhibitory activity against mouse-implanted Sarcoma 180 and Ehrlich-carcinoma solid tumor. The observation that the level of this activity was nearly identical with that of the d-mannan of a wild-type strain of bakers' yeast, which possesses a high proportion of branches composed of α-(1→2)- and α-(1→3)-linked d-mannopyranosyl residues, suggests that the branches are not essential for antitumor activity. The partial acid-degradation products of both d-mannans, the molecular weight of which was one-third of that of each parent d-mannan, had only one half of the antitumor activity of the parent d-mannans. This suggests that molecular size is the most important factor for the differences in activity of the polysaccharides of wild and mutant strains.