Akihiko Shiba

Identification and Characterization of the Precursors Committed to Osteoclasts Induced by TNF-Related Activation-Induced Cytokine/Receptor Activator of NF-κB Ligand

Osteoclasts are terminally differentiated from cells of monocyte/macrophage lineage by stimulation with TNF-related activation-induced cytokine (TRANCE) (receptor activator of NF-κB ligand/osteoprotegerin ligand/osteoclast differentiation factor/TNFSF11/CD254). In the present study, we attempted to determine when and how the cell fate of precursors becomes committed to osteoclasts following TRANCE stimulation. Although mouse bone marrow-derived macrophages (BMMs) were able to differentiate into either osteoclasts or dendritic cells, the cells no longer differentiated into dendritic cells after treatment with TRANCE for 24 h, indicating that their cell fate was committed to osteoclasts. Committed cells as well as BMMs were still quite weak in tartrate-resistant acid phosphatase activity, an osteoclast marker, and incorporated zymosan particles by phagocytosis. Interestingly, committed cells, but not BMMs, could still differentiate into osteoclasts even after incorporation of the zymosan particles. Furthermore, IL-4 and IFN-γ, potent inhibitors of osteoclast differentiation, failed to inhibit osteoclast differentiation from committed cells, and blocking of TRANCE stimulation by osteoprotegerin resulted in cell death. Adhesion to culture plates was believed to be essential for osteoclast differentiation; however, committed cells, but not BMMs, differentiated into multinucleated osteoclasts without adhesion to culture plates. Although LPS activated the NF-κB-mediated pathway in BMMs as well as in committed cells, the mRNA expression level of TNF-α in the committed cells was significantly lower than that in BMMs. These results suggest that characteristics of the committed cells induced by TRANCE are distinctively different from that of BMMs and osteoclasts.

Electrophoretic analysis of the protein in palatine saliva

The protein in palatine saliva was investigated and compared with that in parotid saliva by means of SDS electrophoretic method. It was found that palatine saliva contains a large amount of high molecular weight protein (higher than 300,000) which is identified as mucin-rich glycoprotein and sialoglycoprotein, while no such protein is present in parotid saliva. The protein composition of palatine saliva has qualitatively smaller individual difference when compared to that of parotid saliva. We suggest that the high molecular weight protein which is specific to palatine saliva probably contributes to the rentention of complete maxillary dentures.

Automatic assay of urinary protein using Coomassie Brilliant Blue G-250

Abstract An automatic method for the protein assay using Coomassie Brilliant Blue G-250 was developed and applied to the assay of urinary proteins. In developing the automatic system, the adhesion of protein-bound dye to the walls of the flow cell and tubes was found to be the most troublesome problem, by which the baseline was shifted upwardly to give positive errors. For the purpose of preventing such adhesion, the concentration of CBB was reduced to half of that used in the manual method, glass tubes and glass coils were changed to those made of Kel-F material, and the flow cell was coated with fluorine resin. As a result, the staining with protein-bound dye was nearly completely eliminated. The final system showed satisfactory ability in performance, namely, the value of a coefficient variation for the reproducibility within run was 1.3%, that for the carry over was 0–1.1%, and the recovery was 98.8%. The calibration curve was linear in a range of 0–1000 μg/ml, and 80 samples could be processed in 1 h. Thus, the present method may serve as an efficient automatic protein analyzer for routine clinical tests of urine samples.

A new method of collecting saliva from human palatine glands for electrophoretic study.

Abstract After stimulating the surface of palatine mucosa by applying 0.2 per cent ZnCl 2 solution, 20–30 μl of saliva secreted from the openings of the palatine gland was directly collected using a newly-developed sampler. Saliva collected with the sampler gave 12 well-defined protein bands on SDS electrophoresis, whereas saliva obtained by a conventional filter-paper disc technique gave fewer and less dense protein bands.

Re-evaluation of normal human urinary proteins fractionated by one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis

Proteins in normal human urine were clearly fractionated into 26 bands with molecular weights from 14,000 to 230,000 by means of one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) coupled with silver staining. The main band contained uromucoid, and the second main band had albumin. However, when urine samples from healthy persons were electrophoresed in the absence of SDS using polyacrylamide gel or agarose gel, or a cellulose acetate membrane, albumin but not uromucoid, frequently formed the main protein band. It is suggested that this is due to the complexing of uromucoid subunits to form a large molecule which cannot penetrate into the gel. In order to correctly fractionate all the proteins contained in normal human urine, it was concluded that it was best to treat a urine sample with SDS with pre-condensation, fractionate it by SDS-PAGE and stain fractionated proteins by a highly sensitive method such as silver staining.

Analysis of human palatine salivary proteins by isoelectric focusing in agarose

An electrophoretic method capable of analysing a small volume (5-10 microliter) of human palatine saliva without prior concentration was developed by combining the high resolution of isoelectric focusing in agarose with a highly sensitive silver protein staining method. Human palatine saliva exhibited 23 protein bands, those at pI 4.3-4.4, 4.7, 5.3 and 5.8 being major bands.

The Effect of Ozone on Collagen Type-1 and Inflammatory Cytokine Production in Human Gingival Fibroblasts

Ozone is currently being considered as a possible oral antiseptic agent because it is strongly antimicrobial and does not induce microbial resistance. In the article, we examined the effects of ozone exposure on the production of collagen type-1 and inflammatory cytokines in primary human gingival fibroblasts (HGFs) in vitro using enzyme-linked immunosorbent assays. The addition of 0.5 ppm ozone significantly enhanced collagen type-1 production by HGFs within 24 h. Secretion of the pro-inflammatory cytokines interleukin-6 (IL-6) and IL-8 by HGFs treated with lipopolysaccharide decreased when ozone was present in the medium. Together, these results suggest that clinical use of ozone would facilitate the positive balance between HGF-mediated periodontal tissue maintenance and repair and the stimulation of inflammation and tissue degeneration following exposure to microbial pathogens.

Study on Porcelain-Fused Alloy for Conical Double-Crown Telescopics Prosthesis. Part 1. Bond Strength of Multi-Purpose Gold Alloy.

Multi-purpose gold alloys (Degunorm, Degudent LTG) were investigated as casting metal for conical double-crown telescopic prosthesis. Bond strength between porcelain and metal was compared to conventional ceramo-metal system. In addition, bond strength with some four treatments was also examined. The results were as follows:1. Bond strength of multi-purpose gold alloy with its original low-fused porcelain was similar to those of conventional dental porcelain-fused metal.2. Bond strength of Degunorm painted bonding agent after sandblasting showed the highest value under all test conditions in this study.3. According to SEM observation, body-opaque interface after firing was definite. However, bondingopaque interface was not distinguishable.4. The values of Vickers hardness (Hv) for body, opaque and bonding porcelain were 712, 325 and 468respectively.5. Particle size of opaque porcelain was approximately 50 pm, and was coarser than that of bonding porcelain.6. Leucite was detected in all of porcelain powders before firing. However, the leucite peak of body powder drawn in the X-ray diffraction chart was not obviously steep because it was amorphous.

ポーセレン前装コーヌスクローネ用合金に関する研究 : 第4報 チタン含有コバルトクロム合金へのZr添加による物理的性質, 焼付強度について

To develop porcelain-facing alloys for conical double-crown telescopic prosthesis, several alloys were manufactured by adding 0.5%, 1.0%, 1.5% Zr to a Co-Cr alloy containing 2% titanium.Physical and mechanical properties and the bond strength of these alloys were examined to investigate the optimal amount.The following results were obtained:1. Oxidation at the melting point of the alloy were reduced by the addition of Zr.2. The value of Vickers hardness were reduced by the addition of Zr.3. The value of contraverse strength and elastic constant were reduced by the addition of Zr.4. The bond strength was increased by the addition of 0.5% Zr, and the optical value was obtained at 1, 050° under reduced pressure.5. The optimal additional amount was 0.5% for porcelain-facing alloys for conical double-crown telescopic prosthesis.6. The bond strength was increased by the addition of Zr, because the form of Cr2Co3and CoO at the oxide layer were restrained.

Studies on Porcelain-Facing Alloys for Conical Double-Crown Telescopic Prothesis. Part 2. Change of Bond Strength, Marginal Fitness and Casting State with Addition of Titanium in Cobalt-Chrome Alloys.

We made a Co-Cr alloy containing 0-10% titanium for Konus-Telescope Denture. The results of physical properties were published in part 1.The purpose of this study was to investigate the porcelain bonding strength, the adaptability, and the castability of these alloys.The results were as follows: The porcelain bonding strength became higher as the contained titanium percentage was increased.The alloy containing 6% titanium showed maximum porcelain bonding strength, the alloy containing 2% showed the second highest porcelain bonding strength. But there was no significant difference between alloys containing 2% titanium and 6% titanium concerning the porcelain bonding strength.Vertical displacement enlarged as the contained titanium percentage was increased after the work process.Castability was excellent as the contained titanium percentage was increased.Considering these results, the Co-Cr alloy containing 2% titanium alloy was suitable for porcelain bonding alloy.