Prostaglandin E₂ increases both osteoblastic and osteoclastic activity in the scales and participates in calcium metabolism in goldfish

Using our original in vitro assay system with goldfish scales, we examined the direct effect of prostaglandin E? (PGE?) on osteoclasts and osteoblasts in teleosts. In this assay system, we measured the activity of alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) as respective indicators of each activity in osteoblasts and osteoclasts. ALP activity in scales significantly increased following treatment at high concentration of PGE?(10?? and 10?? M) over 6 hrs of incubation. At 18 hrs of incubation, ALP activity also significantly increased in the PGE? (10?? to 10?? M)-treated scale. In the case of osteoclasts, TRAP activity tended to increase at 6 hrs of incubation, and then significantly increased at 18 hrs of incubation by PGE? (10(-7) to 10?? M) treatment. At 18 hrs of incubation, the mRNA expression of osteoclastic markers (TRAP and cathepsin K) and receptor activator of the NF-κB ligand (RANKL), an activating factor of osteoclasts expressed in osteoblasts, increased in PGE? treated-scales. Thus, PGE? acts on osteoblasts, and then increases the osteoclastic activity in the scales of goldfish as it does in the bone of mammals. In an in vivo experiment, plasma calcium levels and scale TRAP and ALP activities in the PGE?-injencted goldfish increased significantly. We conclude that, in teleosts, PGE? activates both osteoblasts and osteoclasts and participates in calcium metabolism.     

Thermal denaturation of a recombinant mouse amelogenin: circular dichroism and differential scanning calorimetric studies

Conformational analyses of a recombinant mouse tooth enamel amelogenin (rM179) were performed using circular dichroism (CD), fluorescence, differential scanning calorimetry, and sedimentation equilibrium studies. The results show that the far-UV CD spectra of rM179 at acidic pH and 10 degrees C are different from the spectra of random coil in 6 M GdnHCl. A near-UV CD spectrum of rM179 at 10 degrees C is similar to that of rM179 in 6 M GdnHCl, which indicates that aromatic residues of native structure are exposed to solvent and rotate freely. Far-UV CD values of rM179 at 80 degrees C are different from that of random-coil structure in 6 M GdnHCl, which suggests that rM179 at 80 degrees C has specific secondary structures. A gradual thermal transition was observed by far-UV CD, which is interpreted as a weak cooperative transition from specific secondary structures to other specific secondary structures. The fluorescence emission maximum for the spectrum due to Trp residues in rM179 at 10 degrees C shows the same fluorescence emission maximum as rM179 in 6 M GdnHCl and amino acid Trp, which indicates that the three Trp in rM179 are exposed to solvent. Deconvolution of differential scanning calorimetry curve gives the population of three states (A, I, and C states). These results indicate that three states (A, I, and C) have specific secondary structures, in which hydrophobic and Trp residues are exposed to the solvent. The thermodynamic characteristics of rM179 are unique and different from a typical globular protein, proline-rich peptides, and a molten globule state.     

Wireless electrodeless piezomagnetic biosensor with an isolated nickel oscillator

This study presents a fundamental concept of piezomagnetic biochemical sensor driven in a wireless-electrodeless manner. A stepped cylindrical rod of nickel is used as the oscillator, which traps the vibrational energy of axially-polarized surface-shear waves in the central part, where the diameter is slightly larger. A meander-line coil surrounding the oscillator with an air gap can cause and detect the resonant vibrations of the surface-shear waves via the piezomagnetic effect. The resonant frequency of the trapped-mode resonance is continuously measured to detect human immunoglobulin G (IgG). It decreased by 0.08% when a solution containing IgG was injected into the glass cell where the oscillator was placed alone. This oscillator is useful for fundamental studies of various biochemical reactions in a closed system in different environmental gases and different pressures.     

Proteomic analysis of responses to osmotic stress in laboratory and sake-brewing strains of Saccharomyces cerevisiae

The difference in responses to osmotic stress between the laboratory and sake-brewing strains of Saccharomyces cerevisiae at the translational level was compared by two-dimensional polyacrylamide gel electrophoresis. Proteins, whose production was significantly changed by the osmotic stress, were identified by peptide mass fingerprinting. In the laboratory strain, translation of Hor2p, the protein responsible for glycerol biosynthesis, and Ald6p, related to acetate biosynthesis, was induced under high osmotic pressure conditions. In addition, production of proteins related to translation and stress response was also changed under this condition. On the other hand, in the sake-brewing strain, translation of Hor2p, Hsp26p, and some stress-related proteins was upregulated. The change in the production of enzymes related to glycolysis and ethanol formation was small; however, the production of enzymes related to glycerol formation increased in both strains. These results suggest that enhancement of glycerol formation due to enhancement of the translation of proteins, such as Hor2p, is required for growth of S. cerevisiae under high osmotic pressure condition. This is the first report on the analysis of responses of a sake-brewing strain to high osmotic pressure stress based on proteomics.     

The nucleosome binding protein HMGN3 is expressed in pancreatic α‐cells and affects plasma glucagon levels in mice

Glucose homeostasis requires the coordinated actions of various organs and is critically dependent on the proper functioning of the various cell types present in the pancreatic Langerhans islets. Here we report that chromatin architectural protein HMGN3 is highly expressed in all pancreatic endocrine islet cells, and that Hmgn3?/? mice which have a mild diabetic phenotype, have reduced glucagon levels in their blood. To elucidate the mechanism leading to altered glucagon secretion of Hmgn3?/? mice, we tested whether HMGN3 affect glucagon synthesis and secretion in αTC1‐9 cells, a glucagon secreting cell line that is used to study pancreatic α‐cell function. We find that in these cells deletion of either HMGN3 or other HMGN variants, does not significantly affect glucagon gene expression or glucagon secretion. Our studies demonstrate a link between HMGN3 and glucagon blood levels that is not directly dependent of the function of pancreatic α‐cells. J. Cell. Biochem. 109: 49–57, 2010. Published 2009 Wiley‐Liss, Inc.     

Preparation of enzymatically active human Myc-tagged-NCre recombinase exhibiting immunoreactivity with anti-Myc antibody

The Cre-loxP system has been recognized as a tool for conditional gene targeting in mice. However, most anti-Cre antibodies fail to react with Cre expressed in vivo. In an attempt to directly detect Cre by antibodies in vivo, we constructed the tagged-NCre (NCreMH) gene by connecting the human Myc and His tag sequences to the 3' end of the NCre gene carrying a nuclear localizing signal (NLS) sequence. The production of NCre protein and the recombinase activity were detected after co-transfection with pCMV-NCreMH and pCETZ-17 carrying the loxP-flanked lacZ gene into NIH3T3 cells. This activity was also confirmed in vivo after gene transfer of pCMV-NCreMH and pCRTEIL-6 carrying loxP-flanked HcRed1 and EGFP cDNAs, into oviductal epithelium by electroporation. Immunohistochemical staining using anti-Myc antibody demonstrated that the area positive for enhanced green fluorescent protein (EGFP) fluorescence was immunostained with the antibody. These findings indicate that NCreMH is useful as an alternative to NCre for gene targeting.