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This study was performed as a part of the research on the ultrasonic measurement of bone porosity, bone density, and Young's modulus. It was found that the bone porosity estimated by Biot's theory agrees within 5% of the actual porosity. The bone's structure factor involved in the Biot's theory was determined experimentally to have a value of 2. It was also found that the use of the propagation velocity of the fast wave is more advantageous than using the slow wave in estimating bone porosity. The estimated bone density obtained by the use of Biot's theory is within ±5% of the measured value obtained by the use of Archimedes' principle. Bone density as well as Young's modulus can be estimated precisely by using the proposed ultrasonic wave method.

This study was performed as a part of the research on the ultrasonic measurement in vivo of bone porosity, density, Young modulus and hardness. It was clarified that the bone porosity estimated by Biot's theory agrees well with the actual porosity within an accuracy of 5%. In the estimation the structure factor involved in the Biot's theory was determined as a value of 2. In this evaluation, an use of the propagation velocity of the fast wave is more advantageous to estimate the bone porosity than using the one of the slow wave. The estimated bone density by Biot's theory yields only ±5% differences from the measured value by the Archimedes method. Therefore, bone density can be estimated precisely by using the proposed ultrasonic wave method.

Functional cross-linking of a single chain Fv fragment of anti-hen egg-white lysozyme antibody (scFv) and alkaline phosphatase (AP) was explored using microbial transglutaminase (MTG) from Streptomyces mobaraensis. A specific peptidyl linker for MTG was genetically fused to the N-terminus of each protein and the resultant proteins were obtained separately by bacterial expression. The recombinant peptide-tagged scFv and AP were site-specifically cross-linked by MTG through the extra peptidyl linkers in vitro, which mainly yielded the heterodimer (i.e., scFv-AP conjugate). The enzymatic cross-linking reaction had little influence on either the antigen-binding ability of the scFv moiety or the enzymatic activity of the AP moiety of the conjugate, allowing use within an enzyme-linked immunosorbent assay. The results obtained suggest that the enzymatic approach with MTG facilitates the posttranslational construction of functional fusion proteins.

Microbial transglutaminase (MTG), an enzyme that works in posttranslational modification of proteins, has been applied to in vitro preparation of a bi-functional fusion protein from separately obtained recombinant proteins. A quite simple strategy in which a specific peptidyl linker recognizable by MTG is fused to the N-terminus of proteins of interest has been verified in the preparation of a bi-functional fusion protein using an antibody variable domain (single chain Fv of anti-hen-egg white lysozyme antibody, scFv) and a fluorescent protein (enhanced yellow fluorescent protein, EYFP). The resultant peptidyl linker-fused proteins were readily cross-linked by MTG to only give the heterodimer (i.e. scFv-EYFP fusion protein), suggesting that the reaction proceeded in highly specific manner. As the fusion protein exhibited sufficient bi-functionality in fluorescence immunoassay (FIA), this work shows for the first time a successful enzymatic preparation of a bi-functional fusion protein through recombinantly incorporated specific peptidyl linkers into target proteins.

In our search for new PPARα/γ agonists, we designed and synthesized a series of benzoylazole-based carboxylic acids. Compound 9 showed potent PPARγ partial agonistic activity with modest PPARα agonistic activity. The sodium salt of 9 (9Na) demonstrated potent efficacy in lowering both blood glucose and lipids in an animal model without causing significant body weight gain, a well-known side effect associated with PPARγ full agonists.

Adiponectin plays a central role as an antidiabetic and antiatherogenic adipokine. AdipoR1 and AdipoR2 serve as receptors for adiponectin in vitro, and their reduction in obesity seems to be correlated with reduced adiponectin sensitivity. Here we show that adenovirus-mediated expression of AdipoR1 and R2 in the liver of Lepr(-/-) mice increased AMP-activated protein kinase (AMPK) activation and peroxisome proliferator-activated receptor (PPAR)-alpha signaling pathways, respectively. Activation of AMPK reduced gluconeogenesis, whereas expression of the receptors in both cases increased fatty acid oxidation and lead to an amelioration of diabetes. Alternatively, targeted disruption of AdipoR1 resulted in the abrogation of adiponectin-induced AMPK activation, whereas that of AdipoR2 resulted in decreased activity of PPAR-alpha signaling pathways. Simultaneous disruption of both AdipoR1 and R2 abolished adiponectin binding and actions, resulting in increased tissue triglyceride content, inflammation and oxidative stress, and thus leading to insulin resistance and marked glucose intolerance. Therefore, AdipoR1 and R2 serve as the predominant receptors for adiponectin in vivo and play important roles in the regulation of glucose and lipid metabolism, inflammation and oxidative stress in vivo.

We have found that ribonuclease S-peptide can work as a novel peptidyl substrate in protein cross-linking reactions catalyzed by microbial transglutaminase (MTG) from Streptomyces mobaraensis. Enhanced green fluorescent protein tethered to S-peptide at its N-terminus (S-tag-EGFP) appeared to be efficiently cross-linked by MTG. As wild-type EGFP was not susceptible to cross-linking, the S-peptide moiety is likely to be responsible for the cross-linking. A site-directed mutation study assigned Gln15 in the S-peptide sequence as the sole acyl donor. Mass spectrometric analysis showed that two Lys residues (Lys5 and Lys11) in the S-peptide sequence functioned as acyl acceptors. We also succeeded in direct monitoring of the cross-linking process by virtue of fluorescence resonance energy transfer (FRET) between S-tag-EGFP and its blue fluorescent color variant (S-tag-EBFP). The protein cross-linking was tunable by either engineering S-peptide sequence or capping the S-peptide moiety with S-protein, the partner protein of S-peptide for the formation of ribonuclease A. The latter indicates that S-protein can be used as a specific inhibitor of S-peptide-directed protein cross-linking by MTG. The controllable protein cross-linking of S-peptide as a potent substrate of MTG will shed new light on biomolecule conjugation.