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Masato Shimizu

Publications and source records attributed to Masato Shimizu.

At least 19 recordsLinked to original sources
PubMed2007

Alanine scanning mutational analysis of the ligand binding pocket of the human Vitamin D receptor.

We achieved exhaustive alanine scanning mutational analysis of the amino acid residues lining the ligand binding pocket of the Vitamin D receptor to investigate the mechanism of the ligand recognition by the receptor. This is the first exhaustive analysis in the nuclear receptor superfamily. Our results demonstrated the role and importance of all the residues lining the ligand binding pocket. In addition, this analysis was found to indicate ligand-specific ligand-protein interactions, which have key importance in determining the transactivation potency of the individual ligands. Thus, the analysis using 1beta-methyl-1alpha,25-dihydroxyvitamin D(3) revealed the specific van der Waals interactions of 1beta-methyl group with the receptor.

Alanine↗
PubMed2007

Tautomerism of histidine 64 associated with proton transfer in catalysis of carbonic anhydrase.

The imidazole (15)N signals of histidine 64 (His(64)), involved in the catalytic function of human carbonic anhydrase II (hCAII), were assigned unambiguously. This was accomplished by incorporating the labeled histidine as probes for solution NMR analysis, with (15)N at ring-N(delta1) and N(epsilon2), (13)Cat ring-Cepsilon1, (13)C and (15)N at all carbon and nitrogen, or (15)N at the amide nitrogen and the labeled glycine with (13)C at the carbonyl carbon. Using the pH dependence of ring-(15)N signals and a comparison between experimental and simulated curves, we determined that the tautomeric equilibrium constant (K(T)) of His(64) is 1.0, which differs from that of other histidine residues. This unique value characterizes the imidazole nitrogen atoms of His(64) as both a general acid (a) and base (b): its epsilon2-nitrogen as (a) releases one proton into the bulk, whereas its delta1-nitrogen as (b) extracts another proton from a water molecule within the water bridge coupling to the zinc-bound water inside the cave. This accelerates the generation of zinc-bound hydroxide to react with the carbon dioxide. Releasing the productive bicarbonate ion from the inside separates the water bridge pathway, in which the next water molecules move into beside zinc ion. A new water molecule is supplied from the bulk to near the delta1-nitrogen of His(64). These reconstitute the water bridge. Based on these features, we suggest here a catalytic mechanism for hCAII: the tautomerization of His(64) can mediate the transfers of both protons and water molecules at a neutral pH with high efficiency, requiring no time- or energy-consuming processes.

Binding Sites↗
PubMed2006

Identification of a highly potent vitamin D receptor antagonist: (25S)-26-adamantyl-25-hydroxy-2-methylene-22,23-didehydro-19,27-dinor-20-epi-vitamin D3 (ADMI3).

We synthesized four new vitamin D derivatives, diastereomers at C20 and C25 of 26-adamantyl-1,25-dihydroxy-2-methylene-22,23-didehydro-19,27-dinorvitamin D3 (ADMI1-4), which have the bulky and rigid adamantane ring system at the side chain terminus. These compounds had significant VDR affinity (1/6-1/30 that of the natural hormone) but their efficacies of transactivation in transient transcription assay was low (approximately 1/10). All ADMI compounds antagonized the action of 1,25(OH)2D3 in transient transcription assay in COS-7 cells with ADMI3 (20S,25S-isomer) was the most potent (IC50, 3 nM). ADMI3 (1 microM) suppressed the endogenous CYP24A1 gene expression induced by 1,25(OH)2D3 (10 nM) in HEK293 cells to nearly control level. Thus we have identified 26-adamantyl vitamin D compound as a novel highly potent VDR antagonist/partial agonist. A docking model of ADMI3 reveals that a terminal part of the large adamantane ring crowds the H12 residues (Val318 and Phe422) and this would prevent the H12 adopting the active conformation.

Adamantane↗
PubMed2006

Structure-activity relationships of 19-norvitamin D analogs having a fluoroethylidene group at the C-2 position.

We have synthesized four new geometric isomers of 1alpha,25-dihydroxy-2-(2'-fluoroethylidene)-19-norvitamin D analogs 1 and 2 having a 20R- and 20S-configuration, whose structures are correlated with 2MD possessing high potencies in stimulating bone formation in vitro and in vivo. The E-isomers of (20R)- and (20S)-2-fluoroethylidene analogs 1a and 1b were comparable with the natural hormone 1alpha,25-(OH)(2)D(3) in binding to the vitamin D receptor (VDR), while two Z-isomers 2a and 2b were about 15-20 times less active than the hormone. In inducing expression of the vitamin D responsive element-based luciferase reporter gene, the E-isomers 1a and 1b were 1.2- and 8.6-fold more potent than the hormone, respectively, while the Z-isomers 2a and 2b had 27-55% of the potency. On the basis of the biological activities and a docking simulation based on X-ray crystallographic analysis of the VDR ligand-binding pocket, the structure-activity relationships of the fluorinated 19-norvitamin D analogs are discussed.

Animals↗
PubMed2006

New 19-nor-(20S)-1alpha,25-dihydroxyvitamin D3 analogs strongly stimulate osteoclast formation both in vivo and in vitro.

2-Methylene-19-nor-(20S)-1alpha,25-dihydroxyvitamin D3 (2MD), an analog of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3], has been shown to strongly induce bone formation both in vitro and in vivo. We have synthesized four substituents at carbon 2 of 2MD (2MD analogs), four stereoisomers at carbon 20 of the respective 2MD analogs (2MD analog-C20 isomers) and four 2MD analogs with an oxygen atom at carbon 22 (2MD-22-oxa analogs) and examined their ability to stimulate osteoclastogenesis and induce hypercalcemia. 2MD analogs were 100 times as potent as 1alpha,25(OH)2D3 in stimulating the formation of osteoclasts in vitro and in inducing the expression of receptor activator of NF-kappaB ligand (RANKL) and 25-hydroxyvitamin D3-24 hydroxylase mRNAs in osteoblasts. The osteoclast-inducing activities of 2MD analog-C20 isomers and 2MD 22-oxa analogs were much weaker than those of 2MD analogs. In addition, the activity of a 2MD analog in inducing dentine resorption was much stronger than that of 1alpha,25(OH)2D3 in the pit formation assay. Affinities to the vitamin D receptor and transcriptional activities of these compounds did not always correlate with their osteoclastogenic activities. Osteoprotegerin-deficient (OPG-/-) mice provide a suitable model for investigating in vivo effects of 2MD analogs because they exhibit extremely high concentrations of serum RANKL. The same amounts of 2MD analogs and 1alpha,25(OH)2D3 were administered daily to OPG-/- mice for 2 days. The elevation in serum concentrations of RANKL and calcium was much greater in 2MD analog-treated OPG-/- mice than in 1alpha,25(OH)2D3-treated ones. A 2MD analog was much more potent than 1alpha,25(OH)2D3 in causing hypercalcemia and in increasing soluble RANKL with enhanced osteoclastogenesis even in wild-type mice. In contrast, the administration of the 2MD analog to c-fos-deficient mice failed to induce osteoclastogenesis and hypercalcemia. These results suggest that new substituents at carbon 2 of 2MD strongly stimulate osteoclast formation in vitro and in vivo, and that osteoclastic bone resorption is indispensable for their hypercalcemic action of 2MD analogs in vivo.

Animals↗
PubMed2006

A novel way to express proline-selectively labeled proteins with a wheat germ cell-free protein synthesis system.

For high-throughput protein structural analyses, it is essential to develop a reliable protein overexpression system. Although many protein overexpression systems, such as ones involving Escherichia coli cells, have been developed, the number of overexpressed proteins exhibiting the same biological activities as those of the native ones is limited. A novel wheat germ cell-free protein synthesis system was developed recently, and most of the synthesized proteins that should function in solution were found to be in soluble forms. This suggests the applicability of this protein synthesis method to determination of the functional structures of soluble proteins. In our previous work, we developed a selective labeling technique for amino acids having amide functional groups (other than proline residues) involving the use of several inhibitors for transaminases. This paper in turn describes a proline-selective labeling technique. Based on our results, we have succeeded in constructing a complete amino acid selective labeling technique for the wheat germ cell-free protein synthesis system.

Cell-Free System↗
PubMed2006

Isolation and identification of 2alpha,25-dihydroxyvitamin D3, a new metabolite from Pseudonocardia autotrophica 100U-19 cells incubated with Vitamin D3.

Pseudonocardia autotrophica converted Vitamin D(3) to 25-hydroxyvitamin D(3) and 1alpha,25-dihydroxyvitamin D(3). The hydroxylation of Vitamin D(3) with P. autotrophica was enhanced by the addition of cyclodextrin. In this microbial hydroxylation, a new Vitamin D(3) metabolite was observed in the reaction mixture of P. autotrophica and Vitamin D(3), and was isolated in a pure form by several steps of chromatography. The structure of the new metabolite was determined to be 2alpha,25-dihydroxyvitamin D(3) by UV, NMR and mass spectroscopic analyses. Biological evaluation of the new metabolite was conducted by means of several experiments.

Animals↗
PubMed2006

Analogs of 1alpha,25-dihydroxyvitamin D3 with high potency in induction of osteoclastogenesis and prevention of dendritic cell differentiation: synthesis and biological evaluation of 2-substituted 19-norvitamin D analogs.

In our previous papers, we found that introduction of a substituent at C(2) into 1alpha,25-dihydroxy-19-norvitamin D(3) (2a) caused dramatic changes in binding affinity for the vitamin D receptor (VDR) and in transcriptional activity compared with the parent compound. To investigate the broad biological activity of 2-substituted 19-norvitamin D analogs, we synthesized two new (20S)-2-hydroxyethylidene-19-norvitamin D derivatives (3b and 4b) and a total of 16 A-ring-modified analogs including 3b and 4b were tested for the following in vitro and in vivo biological activities: (1) affinity for the VDR, (2) transcriptional activity, (3) osteoclast formation, (4) bone calcium mobilization in rats, and (5) effects on differentiation of dendritic cells (DCs). The biological effects of the analogs were compared with those of 1alpha,25-dihydroxyvitamin D(3) (1a) and 2MD, which is being developed for the treatment of osteoporosis. The efficacy of the (20S)-19-norvitamin D analogs with 2-hydroxyethylidene, 2-hydroxyethoxy, and 2-methyl moieties (3b, 5b, 6b, and 9b) was more than 10-fold stronger than that of 1a with respect to transcriptional activity, ability to induce osteoclast formation, and ability to inhibit CD86 expression, a marker of mature DCs, and was similar to that of 2MD. The (20S)-2beta-hydroxyethoxy derivative 6b was 2 orders of magnitude more active than 1a and approximately twice as potent as 2MD in preventing CD86 production. The 2-epoxy derivatives 7 and 8 were relatively poor ligands for the VDR and exhibited activity lower than that of the natural hormone 1a.

Animals↗
PubMed2006

Synthesis and biological activities of new 1alpha,25-dihydroxy-19-norvitamin D3 analogs with modifications in both the A-ring and the side chain.

In a series of studies on structure-activity relationships of 2-substituted 19-norvitamin D analogs, we found that 1alpha,25-dihydroxy-19-norvitamin D3 analogs with 2beta-hydroxyethoxy or 2E-hydroxyethylidene moieties show strong binding affinity for the vitamin D receptor (VDR) as well as marked transcriptional activity. To further examine the effects of side chain structure on the activity of 2-substituted 19-norvitamin D analogs, we have synthesized new 19-norvitamin D3 analogs with modifications in both the A-ring at the C(2) position and the side chain. The side chains of these analogs contained a double bond between C(22) and C(23) or an oxygen atom at C(22). The biological activity of the analogs was evaluated in vitro. All the side chain-modified analogs were less active than 1alpha,25-dihydroxyvitamin D31e and the parent compounds 3-6e possessing a natural 20R-configuration in binding to the VDR, but, except for the (20R)-22-oxa analogs 3-6d, were significantly more potent in transcriptional activity. Of the side-chain-modified analogs 4 and 5, the 2beta-hydroxyethoxy- and 2E-hydroxyethylidene-22,24-diene-24a,26a,27a-trihomo analogs showed markedly higher transcriptional activity (25- and 17.5-fold, respectively) compared with 1e. Elongation of the side chain at the C-24, C-26, and C-27 positions and introduction of a 22,24-diene moiety strongly increased transcriptional activity, as seen in the 20-epi analogs 3-6f.

Animals↗
PubMed2006

Vitamin D receptor: ligand recognition and allosteric network.

To investigate the allosteric effects of ligands in the function of nuclear receptors, we performed exhaustive alanine scanning mutational analysis (ASMA) of the residues lining the ligand-binding pocket (LBP) of the human vitamin D receptor. The effects of ligands were examined in this system (termed two-dimensional (2D) ASMA) using 10 structurally and biologically characteristic ligands that included agonists, partial agonists, and a full antagonist. The results clearly revealed the role and importance of all the amino acid residues lining the LBP and the relationships between ligand binding and transcriptional potency. 2D ASMA indicated ligand-specific ligand-protein interactions, which have key importance in determining the transactivation potency of the ligand. Taking the results as a whole, we suggest a ligand-mediated allosteric network through which information from ligands is transmitted to the interfaces with protein cofactors and which was shown to be linked to part of the network found by statistical coupling analysis.

Alanine↗
PubMed2006

A case of interstitial pneumonia caused by bucillamine: a study using serological markers.

The patient was a 61-year-old man diagnosed with rheumatoid arthritis (RA) in 2001. He initially received treatment at a nearby clinic, but his condition could not be satisfactorily controlled. He subsequently consulted our hospital during the same year. Although his symptoms improved in response to treatment at our hospital, slight fever, cough, and then high fever and dyspnea subsequently developed. A diagnosis of interstitial pneumonia was made on the basis of findings of diagnostic imaging. The time course of changes in serological markers, including surfactant protein A (SP-A), surfactant protein D (SP-D), and KL-6, as well as markers of inflammatory reaction and lactate dehydrogenase was examined to determine the clinical significance of serological markers in the management of interstitial pneumonia.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed2005

New derivatives of 1alpha,25-dihydroxy-19-norvitamin D3 with two substituents at C-2: synthesis and biological activity.

To examine the effect of 2,2-disubstitution on the biological activities of 19-norvitamin D analogs, novel 2,2-disubstituted-(20R)- and (20S)-1alpha,25-dihydroxy-19-norvitamin D3 analogs were prepared and their biological activities were studied. All the synthesized analogs possessing hydrophobic 2alpha-substituents were more active than the corresponding 2beta-isomers both in binding to the vitamin D receptor and in activating gene transcription. The 2alpha-methyl-2beta-hydroxy analog 9b was found to have markedly higher transcriptional activity (32-fold) than the natural ligand 1a, although the two had the same binding affinity to the vitamin D receptor. To our knowledge, this analog is among the most potent of 19-norvitamin D analogs.

Animals↗
PubMed2005

Identification of putative metabolites of docosahexaenoic acid as potent PPARgamma agonists and antidiabetic agents.

We found that putative metabolites of docosahexaenoic acid (DHA) are strong PPARgamma activators and potential antidiabetic agents. We designed DHA derivatives based on the crystal structure of PPARgamma, synthesized them and evaluated their activities in vitro and in vivo. The efficacy of 5E-4-hydroxy-DHA 2a as a PPARgamma activator was about fourfold stronger than that of pioglitazone. Furthermore, the 4-keto derivative (10b) showed antidiabetic activity in animal models without producing undesirable effects such as obesity and hepatotoxicity.

Animals↗
PubMed2004

Perfusion flow assessment of coronary shunt during off-pump coronary artery bypass grafting.

BACKGROUND: Coronary shunts are widely used to prevent myocardial ischemia during off-pump coronary artery bypass graft (OPCAB) procedures. Although clinical effectiveness has been reported, actual perfusion flow has not been well assessed. The purpose of this study was to evaluate actual shunt flow and its pattern during passive coronary perfusion in clinical OPCAB. METHODS: In 15 OPCAB cases, the coronary perfusion flow of the external shunt (1.7 or 2.0 mm) during anastomosis and the free flow of the shunt were measured with an in-line electromagnetic or ultrasonic flow probe. The perfused coronary blood vessel was either the left anterior descending coronary artery or the right coronary artery. The inflow vessel of the external shunt was either the femoral artery (FA) or the ascending aorta (AA). RESULTS: Free flow values of a 1.7-mm FA shunt, 1.7-mm AA shunt, and 2.0-mm FA shunt were 34+/- 7, 39 +/- 3, and 44 +/- 7 mL/min. Perfusion flows were 13 +/- 4, 14 +/- 3, and 22 +/- 4 mL/min, respectively. Perfusion flow was significantly lower than free flow and correlated well with coronary resistance. Although inflow site did not influence net perfusion flow, diastolic/systolic flow fraction ratio was significantly greater when the shunt was perfused from the FA. CONCLUSIONS: External shunt from FA would provide limited but effective perfusion flow with a physiological pattern, which is passively regulated by coronary resistance.

Aged↗
PubMed2004

Efficient production of isotopically labeled proteins by cell-free synthesis: a practical protocol.

We provide detailed descriptions of our refined protocols for the cell-free production of labeled protein samples for NMR spectroscopy. These methods are efficient and overcome two critical problems associated with the use of conventional Escherichia coli extract systems. Endogenous amino acids normally present in E. coli S30 extracts dilute the added labeled amino acids and degrade the quality of NMR spectra of the target protein. This problem was solved by altering the protocol used in preparing the S30 extract so as to minimize the content of endogenous amino acids. The second problem encountered in conventional E. coli cell-free protein production is non-uniformity in the N-terminus of the target protein, which can complicate the NMR spectra. This problem was solved by adding a DNA sequence to the construct that codes for a cleavable N-terminal peptide tag. Addition of the tag serves to increase the yield of the protein as well as to ensure a homogeneous protein product following tag cleavage. We illustrate the method by describing its stepwise application to the production of calmodulin samples with different stable isotope labeling patterns for NMR analysis.

Animals↗
PubMed2004

Two-dimensional alanine scanning mutational analysis of the interaction between the vitamin D receptor and its ligands: studies of A-ring modified 19-norvitamin D analogs.

To clarify the structure-function relationship (SFR) of vitamin D analogs in terms of their interaction with the vitamin D receptor (VDR), we have proposed a new approach, two-dimensional alanine scanning mutational analysis (2D-ASMA). In this paper, attention was focused on the interactions around the A-ring of vitamin D. For this purpose, we synthesized four new 2-substituted 19-norvitamin D derivatives (3-6). The VDR affinity (3-6: 1, 5, 2 and 1/140, respectively) and transcriptional activity (3-6: 10, 30, 2 and 0.3, respectively) of the four compounds were evaluated relative to 1,25-(OH)(2)D(3) (5) (normalized to 1). Then, the transcriptional activities of wild-type and 18 mutant VDRs induced by the four compounds (3-6) were investigated. The results of this 18 x 4 2D-ASMA were presented as a patch table, and the effects of the mutations were analyzed in comparison with the natural hormone (1) and 2-methylene-19-nor-20-epi-1,25-(OH)(2)D(3) (2MD, 2). Of the four A-ring analogs, the 2alpha-hydroxyethoxy derivative (3) showed striking differences in the pattern on the patch table. From the results, we suggest a docking mode of this compound (3) in which the A-ring adopts the alpha conformation.

Alanine↗
PubMed2004

A novel way of amino acid-specific assignment in (1)H-(15)N HSQC spectra with a wheat germ cell-free protein synthesis system.

For high-throughput protein structural analyses, it is indispensable to develop a reliable protein overexpression system. Although many protein overexpression systems, such as ones utilizing E. coli cells, have been developed, a lot of proteins functioning in solution still were synthesized as insoluble forms. Recently, a novel wheat germ cell-free protein synthesis system was developed, and many of such proteins were synthesized as soluble forms. This means that the applicability of this protein synthesis method to determination of the functional structures of soluble proteins. In our previous work, we synthesized (15)N-labeled proteins with this wheat germ cell-free system, and confirmed this applicability on the basis of the strong similarity between the (1)H-(15)N HSQC spectra for native proteins and the corresponding ones for synthesized ones. In this study, we developed a convenient and reliable method for amino acid selective assignment in (1)H-(15)N HSQC spectra of proteins, using several inhibitors for transaminases and glutamine synthase in the process of protein synthesis. Amino acid selective assignment in (1)H-(15)N HSQC spectra is a powerful means to monitor the features of proteins, such as folding, intermolecular interactions and so on. This is also the first direct experimental evidence of the presence of active transaminases and glutamine synthase in wheat germ extracts.

Amino Acid Sequence↗