Biomedical subjects

S Miyazawa

Publications and source records attributed to S Miyazawa.

At least 19 recordsLinked to original sources

PubMed2002

Alterations of insulin sensitivity by the implantation of 3T3-L1 cells in nude mice. A role for TNF-alpha?

AIMS/HYPOTHESIS: Visceral adipocytes have different functions than those from the subcutaneous fat area. These differences could contribute to the pathological significance of excessive visceral fat accumulation for accompanying insulin resistance and hyperinsulinaemia. This study addresses this hypothesis and describes a unique method to clarify whether the functional differences between visceral and subcutaneous adipocytes depend on their anatomical location. METHODS: 3T3-L1 cells or TNF-alpha overexpressing CHO cells were implanted into subcutaneous fat area or mesenteric area as visceral fat area in athymic mice of BALB/C strain. Then, serum insulin, glucose, TNF-alpha, and several markers of lipid metabolism were measured in the fasting condition. OGTT was also analysed. RESULTS: During the course of glucose loading, the mice which had 3T3-L1 cells implanted into mesenteric area but not into subcutaneous fat area showed remarkably increased serum insulin and TMF-alpha concentrations, compared to the control mice. Moreover, serum insulin concentrations of the mice, implanted with TNF-alpha overexpressing cells into subcutaneous fat area, were apparently higher than that of control mice. CONCLUSION/INTERPRETATION: This method of implanting adipose cells into subcutaneous or visceral fat area showed high TNF-alpha concentration and insulin resistance by the adipose cells in visceral area of nude mice. Furthermore, we found that the functional significance of visceral fat accumulation for TNF-alpha-induced insulin resistance is partly caused by the interaction of adipocytes with surrounding conditions in mesenteric area.

3T3 Cells↗

PubMed2001

The Sos1-Rac1 signaling. Possible involvement of a vacuolar H(+)-ATPase E subunit.

We have purified and identified a 32-kDa protein interacting with the Dbl oncogene homology domain of mSos1(Sos-DH) from rat brains by glutathione S-transferase-Sos-DH affinity chromatography. Peptide sequencing revealed that the protein is identical to a positive regulatory E subunit (V-ATPase E) of a vacuolar H(+)-ATPase, which is responsible for acidification of endosome and alkalinization of intracellular pH. The interaction between V-ATPase E and Sos-DH was confirmed by yeast two-hybrid assay. A coimmunoprecipitation assay demonstrated that a V-ATPase E protein physiologically bound to mSos1, and the protein was colocalized with mSos1 in the cytoplasm, as determined by immunohistochemistry. mSos1 was found in the early endosome fraction together with V-ATPase E and Rac1, suggesting the functional involvement of mSos1/V-ATPase E complexes in the Rac1 activity at endosomes. Overexpression of V-ATPase E in COS cells enhanced the ability of mSos1 to promote the guanine nucleotide exchange activity for Rac1 and stimulated the kinase activity of Jun kinase, a downstream target of Rac1. Thus, the data indicate that V-ATPase E may participate in the regulation of the mSos1-dependent Rac1 signaling pathway involved in growth factor receptor-mediated cell growth control.

3T3 Cells↗

PubMed2001

Cloning and characterization of integrin alpha subunits from the solitary ascidian, Halocynthia roretzi.

Recent molecular and biochemical analysis has revealed the presence of an opsonic complement system in the solitary ascidian, Halocynthia roretzi, composed of at least C3, two mannan binding protein-associated serine proteases, and factor B. To elucidate further the structure and function of this apparently primitive complement system in the urochordates, we looked for the ascidian complement receptor type 3 (CR3), or type 4 (CR4), which are members of the leukocyte integrin family in mammals. Using degenerate primers, we isolated two integrin alpha subunits (alpha(Hr1) and alpha(Hr2)) from the hemocyte mRNA of H. roretzi, by RT-PCR, and the entire coding sequence of alpha(Hr1) was determined from cDNA clones. alpha(Hr1) contains an I domain, the inserted domain characteristic of a subset of mammalian alpha subunits, including the leukocyte integrin family. A phylogenetic tree constructed for the alpha subunits also supports the ancestral position of alpha(Hr1) in the monophyletic cluster of I domain-containing alpha integrins. The alpha(Hr1) gene shows hemocyte-specific expression on Northern blot analysis. Western blot analysis and immunocytochemical staining of the hemocytes of H. roretzi using anti-alpha(Hr1) Ab showed that alpha(Hr1) subunits exist on the surface of a subpopulation of phagocytic hemocytes. Furthermore, anti-alpha(Hr1) Ab inhibited C3-dependent phagocytosis, but not basic phagocytosis, of yeast cells by ascidian hemocytes. These observations strongly suggest that alpha(Hr1) constitutes an integrin molecule on the hemocytes of H. roretzi that functions as an ancestral form of CR3 and CR4 and mediates phagocytosis in the primitive complement system of the ascidian.

Amino Acid Sequence↗

PubMed2000

Augmentative effects of L-cysteine and methylmethionine sulfonium chloride on mucin secretion in rabbit gastric mucous cells.

BACKGROUND: Our previous study showed that L-cysteine (Cys) and methylmethionine sulfonium chloride (MMSC) inhibited ethanol-induced gastric mucosal damage and increased the amount of surface mucin in rats. This study examined whether Cys and MMSC augmented mucin secretion and changed distribution of mucin vesicles ultrastructurally in mucous cells by using primary cultured mucous cells from rabbit glandular stomach. Changes in intracellular cyclic adenosine 3',5'-monophosphate (cAMP) and in levels of cytosolic free Ca2+ were investigated by treatment with Cys and MMSC. METHODS: Mucin content was measured by an enzyme-linked lectin assay. Transmission electron micrography was used to examine ultrastructural distribution of mucin granules. The amount of cAMP or levels of free Ca2+ were measured by enzyme immunoassay or by fura-2. 16,16-Dimethyl prostaglandin E2 (dmPGE2) or ATP was used as the positive control. RESULTS: L-Cysteine and MMSC increased mucin secretion and decreased cellular mucin content. The same was noted for dmPGE2. Accelerated mucin granule movements toward the plasma membrane were shown by these agents. Intracellular cAMP increased with exposure to dmPGE2 for 20 min, while neither Cys nor MMSC increased cAMP. No increase in cytosolic free Ca2+ levels occurred after treatment with Cys or MMSC, but an increase was induced 10 s after the addition of ATP. CONCLUSIONS: The present findings indicate that the increase in mucin secretion by Cys and MMSC was not mediated through the cAMP or Ca2+ signal transduction pathway, but might occur through non-receptor-mediated mechanisms.

Animals↗

PubMed2000

Characterization of recombinant human adipocyte-derived leucine aminopeptidase expressed in Chinese hamster ovary cells.

Adipocyte-derived leucine aminopeptidase (A-LAP) is a recently identified novel member of the M1 family of zinc-metallopeptidases. Transfection of the A-LAP cDNA into COS-7 cells resulted in the secretion of the enzyme. In this study, recombinant A-LAP was expressed in Chinese hamster ovary cells, purified to homogeneity and its enzymatic properties were characterized. The purified enzyme was active towards a synthetic substrate, L-leucyl-p-nitroanilide, yielding a V(max) of 3.55 micromol/min/mg and a K(m) of 1.28 mM, and was shown to be a monomeric protein with molecular mass of 120 kDa in solution. By monitoring the sequential N-terminal amino acid liberation, it was found that the enzyme hydrolyzes a variety of bioactive peptides, including angiotensin II and kallidin. Immunohistochemical analysis indicated that the enzyme is expressed in the cortex of the human kidney, where tissue kallikrein is localized. Taken together, these results indicate that A-LAP possesses a broad substrate specificity towards naturally occurring peptide hormones and suggest that it plays a role in the regulation of blood pressure through the inactivation of angiotensin II and/or the generation of bradykinin in the kidney.

Adipocytes↗

PubMed2000

Identifying sequence-structure pairs undetected by sequence alignments.

We examine how effectively simple potential functions previously developed can identify compatibilities between sequences and structures of proteins for database searches. The potential function consists of pairwise contact energies, repulsive packing potentials of residues for overly dense arrangement and short-range potentials for secondary structures, all of which were estimated from statistical preferences observed in known protein structures. Each potential energy term was modified to represent compatibilities between sequences and structures for globular proteins. Pairwise contact interactions in a sequence-structure alignment are evaluated in a mean field approximation on the basis of probabilities of site pairs to be aligned. Gap penalties are assumed to be proportional to the number of contacts at each residue position, and as a result gaps will be more frequently placed on protein surfaces than in cores. In addition to minimum energy alignments, we use probability alignments made by successively aligning site pairs in order by pairwise alignment probabilities. The results show that the present energy function and alignment method can detect well both folds compatible with a given sequence and, inversely, sequences compatible with a given fold, and yield mostly similar alignments for these two types of sequence and structure pairs. Probability alignments consisting of most reliable site pairs only can yield extremely small root mean square deviations, and including less reliable pairs increases the deviations. Also, it is observed that secondary structure potentials are usefully complementary to yield improved alignments with this method. Remarkably, by this method some individual sequence-structure pairs are detected having only 5-20% sequence identity.

Algorithms↗

PubMed2000

Protein sequence-structure alignment based on site-alignment probabilities.

A protein sequence-structure alignment method for database searches is examined on how effectively this method together with a simple scoring function previously developed can identify compatibilities between sequences and structures of proteins. The scoring function consists of pairwise contact energies, repulsive packing potentials of residues for overly dense arrangement and short-range potentials for secondary structures. Pairwise contact interactions in a sequence-structure alignment are evaluated in a mean field approximation on the basis of probabilities of site pairs to be aligned. Gap penalties are assumed to be proportional to the number of contacts at each residue position, and as a result gaps will be more frequently placed on protein surfaces than in cores. In addition to minimum energy alignments, we use probability alignments made by successively aligning site pairs in order by pairwise alignment probabilities. Results show that the present energy function and alignment method can detect well both folds compatible with a given sequence and, inversely, sequences compatible with a given fold. Probability alignments consisting of most reliable site pairs only can yield small root mean square deviations, and including less reliable pairs increases the deviations. Remarkably, by this method some individual sequence-structure pairs are detected having only 5-20% sequence identity.

17-Hydroxysteroid Dehydrogenases↗

PubMed1999

Evaluation of short-range interactions as secondary structure energies for protein fold and sequence recognition.

Short-range interactions for secondary structures of proteins are evaluated as potentials of mean force from the observed frequencies of secondary structures in known protein structures which are assumed to have an equilibrium distribution with the Boltzmann factor of secondary structure energies. A secondary conformation at each residue position in a protein is described by a tripeptide, including one nearest neighbor on each side. The secondary structure potentials are approximated as additive contributions from neighboring residues along the sequence. These are part of an empirical potential to provide a crude estimate of protein conformational energy at a residue level. Unlike previous works, interactions are decoupled into intrinsic potentials of residues, potentials of backbone-backbone interactions, and of side chain-backbone interactions. Also interactions are decoupled into one-body, two-body, and higher order interactions between peptide backbone and side chain and between backbones. These decouplings are essential to correctly evaluate the total secondary structure energy of a protein structure without overcounting interactions. Each interaction potential is evaluated separately by taking account of the correlation in the amino acid order of protein sequences. Interactions among side chains are neglected, because of the relatively limited number of protein structures. Proteins 1999;36:347-356. Published 1999 Wiley-Liss, Inc.

Mathematics↗

PubMed1999

An empirical energy potential with a reference state for protein fold and sequence recognition.

We consider modifications of an empirical energy potential for fold and sequence recognition to represent approximately the stabilities of proteins in various environments. A potential used here includes a secondary structure potential representing short-range interactions for secondary structures of proteins, and a tertiary structure potential consisting of a long-range, pairwise contact potential and a repulsive packing potential. This potential is devised to evaluate together the total conformational energy of a protein at the coarse grained residue level. It was previously estimated from the observed frequencies of secondary structures, from contact frequencies between residues, and from the distributions of the number of residues in contact in known protein structures by regarding those distributions as the equilibrium distributions with the Boltzmann factor of these interaction energies. The stability of native structures is assumed as a primary requirement for proteins to fold into their native structures. A collapse energy is subtracted from the contact energies to remove the protein size dependence and to represent protein stabilities for monomeric and multimeric states. The free energy of the whole ensemble of protein conformations that is subtracted from the conformational energy to represent protein stability is approximated as the average energy expected for a typical native structure with the same amino acid composition. This term may be constant in fold recognition but essentially varies in sequence recognition. A simple test of threading sequences into structures without gaps is employed to demonstrate the importance of the present modifications that permit the same potential to be utilized for both fold and sequence recognition. Proteins 1999;36:357-369. Published 1999 Wiley-Liss, Inc.

Models, Chemical↗

PubMed1999

Self-consistent estimation of inter-residue protein contact energies based on an equilibrium mixture approximation of residues.

Pairwise contact energies for 20 types of residues are estimated self-consistently from the actual observed frequencies of contacts with regression coefficients that are obtained by comparing "input" and predicted values with the Bethe approximation for the equilibrium mixtures of residues interacting. This is premised on the fact that correlations between the "input" and the predicted values are sufficiently high although the regression coefficients themselves can depend to some extent on protein structures as well as interaction strengths. Residue coordination numbers are optimized to obtain the best correlation between "input" and predicted values for the partition energies. The contact energies self-consistently estimated this way indicate that the partition energies predicted with the Bethe approximation should be reduced by a factor of about 0.3 and the intrinsic pairwise energies by a factor of about 0.6. The observed distribution of contacts can be approximated with a small relative error of only about 0.08 as an equilibrium mixture of residues, if many proteins were employed to collect more than 20,000 contacts. Including repulsive packing interactions and secondary structure interactions further reduces the relative errors. These new contact energies are demonstrated by threading to have improved their ability to discriminate native structures from other non-native folds.

Computer Simulation↗

PubMed1999

Analysis of human error in nursing care.

Analysis of reports about incidental and accidental events in nursing care were made using a reliability engineering method. Unnatural working hours, such as evening duty, night duty falling next to a holiday, two consecutive night-duty shifts, and two consecutive evening-duty shifts were major factors in the occurrence of errors. In a mixed-division ward (a ward containing patients belonging to different divisions), rule-based errors happened more frequently than in a single-division ward. Also, less experienced nursing staffs made errors more frequently than experienced nursing staffs.

Cardiology Service, Hospital↗

PubMed1999

Mice with early onset of death (EOD) due to lupus glomerulonephritis.

Both MRL-lpr/lpr (lpr) and BXSB mice fall victim to autoimmune disease as a function of age. To combine their properties, brother-sister mating of (female lpr x male BXSB)F1 mice was done. Mice for mating were selected according to indicators of early onset of glomerulonephritis and subsequent early death (i.e., EOD). This mating was continued for more than 16 generations. The EOD mice thus established had homozygous H-2k/k, lpr/lpr, and possible yaa/- (in the case of males). The average life span of males was 83 days while that of females was 126 days. After 12 weeks of age, the majority (> 80%) of male EOD mice were characterized by the abnormality of urine due to glomerulonephritis. We then characterized how glomerulonephritis was evoked, especially in terms of expanding lymphocyte subsets in various immune organs. Similar to the case of parental lpr mice, the major expanding cells were CD4-8-B220+ TCRint cells in the immune organs and kidney. In addition, myeloid cells were found to infiltrate the kidney. This massive infiltration of both TCRint cells and myeloid cells might be responsible for the onset of acute glomerulonephritis. Even after more than 50 generations, these EOD mice still carry both lpr and yaa genes. These results suggest that EOD mice might be a very useful tool for the study of acute lupus glomerulonephritis which is evoked by the genetic abnormalities.

Acute Disease↗

PubMed1998

Serum and antral gastrin levels in fed and fasted rats: relation to aging.

The effect of short fasting for up to 24 h on serum and antral gastrin concentrations and G cell ultrastructure has been examined in the young rat compared with the middle-aged rat. The serum gastrin levels at 24-h fasting were markedly reduced in both young and middle-aged rats. There was also a significant decrease in antral gastrin concentrations after 24 h of fasting in the middle-aged rats. By contrast, the antral gastrin concentrations in the young rats increased progressively and significantly with fasting for up to 24 h. These responses were associated with a significant increase in the content of secretory granules of G cell, which was at its greatest by 24 h. The antral gastrin level in the 48-h fasting rats was markedly reduced to a level below that for the prestarvation group. By 48 h of starvation, the amount of secretory granules in G cell was significantly reduced (P < 0.05) compared with the 24-h fasting group. These results indicate that the effects of fasting on the antral gastrin levels in young rats differ from those of the middle-aged and that starvation for up to 24 h caused a significant increase in the antral gastrin content in the young rats' stomach.

Aging↗

PubMed1998

Patients' understanding and opinion about informed consent for coronary angiography in a rural Japanese hospital.

Based on an anonymous questionnaire obtained from 102 Japanese in-patients at a rural Japanese hospital who underwent coronary angiography, the patients understanding of information about the procedure, their perception of consent and their attitude toward it were investigated. The patients were able to recall 63.8% of the contents of the information. There was a statistically significant correlation between the patients' educational status and the recall test score of the information. Older (65 years old and above) patients had a tendency to entrust decision making to their physicians. Only 19.6% of the patients made the decision to undergo a coronary angiography by themselves. After being informed of the risks 40.2% of the patients felt more anxious. In conclusion, our patients could not recall the contents of the information sufficiently. There still existed a tendency to entrust important decisions to the physician especially in older patients. The patients had a tendency not to face difficult problems solely by themselves but with the support of family members.

Aged↗

PubMed1997

Cloning and expression of cDNA for a newly identified isozyme of bovine liver 3-hydroxyacyl-CoA dehydrogenase and its import into mitochondria.

cDNA for a heretofore undescribed mitochondrial 3-hydroxyacyl-CoA dehydrogenase, designated as the type II enzyme with different molecular and catalytic properties, compared to those of the classical mitochondrial beta-oxidation enzyme (type I enzyme), was cloned from a bovine liver cDNA library. Nucleotide sequence of the cDNA encoded 261 amino acids with a subunit molecular weight of 27,140. The deduced primary structure of the type II enzyme showed no significant homology to the reported amino acid sequence of the classical 3-hydroxyacyl-CoA dehydrogenases. On SDS-PAGE, no differences in subunit molecular weights were observed among the in vitro translation products, the recombinant type II enzyme produced in Escherichia coli and the purified enzyme. NH2-terminal and COOH-terminal amino acid sequence analysis of the purified type II enzyme revealed that the mature enzyme had not been proteolytically processed. The in vitro translation products of the type II enzyme were efficiently incorporated into isolated rat liver mitochondria, without changes in size, thereby suggesting that unlike other mitochondrial enzymes of fatty acid beta-oxidation, the type II enzyme had no cleavable signal peptide upon import into mitochondria.

3-Hydroxyacyl CoA Dehydrogenases↗

PubMed1997

D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein deficiency: a newly identified peroxisomal disorder.

Peroxisomal beta-oxidation proceeds from enoyl-CoA through D-3-hydroxyacyl-CoA to 3-ketoacyl-CoA by the D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxy-acyl-CoA dehydrogenase bifunctional protein (d-bifunctional protein), and the oxidation of bile-acid precursors also has been suggested as being catalyzed by the d-bifunctional protein. Because of the important roles of this protein, we reinvestigated two Japanese patients previously diagnosed as having enoyl-CoA hydratase/L-3-hydroxyacyl-CoA dehydrogenase bifunctional protein (L-bifunctional protein) deficiency, in complementation studies. We found that both the protein and the enzyme activity of the d-bifunctional protein were hardly detectable in these patients but that the active L-bifunctional protein was present. The mRNA level in patient 1 was very low, and, for patient 2, mRNA was of a smaller size. Sequencing analysis of the cDNA revealed a 52-bp deletion in patient 1 and a 237-bp deletion in patient 2. This seems to be the first report of D-bifunctional protein deficiency. Patients previously diagnosed as cases of L-bifunctional protein deficiency probably should be reexamined for a possible d-bifunctional protein deficiency.

17-Hydroxysteroid Dehydrogenases↗

PubMed1997

Structure of D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein.

When D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein was purified from human liver, two preparations were obtained. One contained a 77-kDa polypeptides as the main and minor smaller polypeptides including a 46-kDa polypeptide, and this preparation showed both the dehydratase and dehydrogenase activities. The other preparation was a homodimer of the 46-kDa polypeptide and showed only the dehydratase activity. Further analysis indicated that the native enzyme is a homodimer of 77-kDa polypeptide, which was proteolytically modified during purification. The cDNA for the human 77-kDa polypeptide was cloned. The amino acid sequences of the peptides derived from the components of the enzyme preparations were located in the deduced amino acid sequence of the cDNA. The preparation containing the 77-kDa polypeptide was treated with a protease, and two monofunctional fragments were separated. The dehydrogenase and dehydratase fragments were located on the amino- and carboxyl-terminal sides, respectively, of the deduced amino acid sequence of the cDNA. The protein expressed by the cDNA with the entire coding region exhibited both the dehydratase and dehydrogenase activities, and that expressed by a truncated version covering the carboxyl-terminal side exhibited only the dehydratase activity. The cloned cDNA was identical to the human 17 beta-hydroxysteroid dehydrogenase IV cDNA.

17-Hydroxysteroid Dehydrogenases↗

PubMed1996

Residue-residue potentials with a favorable contact pair term and an unfavorable high packing density term, for simulation and threading.

Attractive inter-residue contact energies for proteins have been re-evaluated with the same assumptions and approximations used originally by us in 1985, but with a significantly larger set of protein crystal structures. An additional repulsive packing energy term, operative at higher densities to prevent overpacking, has also been estimated for all 20 amino acids as a function of the number of contacting residues, based on their observed distributions. The two terms of opposite sign are intended to be used together to provide an estimate of the overall energies of inter-residue interactions in simplified proteins without atomic details. To overcome the problem of how to utilize the many homologous proteins in the Protein Data Bank, a new scheme has been devised to assign different weights to each protein, based on similarities among amino acid sequences. A total of 1168 protein structures containing 1661 subunit sequences are actually used here. After the sequence weights have been applied, these correspond to an effective number of residue-residue contacts of 113,914, or about six times more than were used in the old analysis. Remarkably, the new attractive contact energies are nearly identical to the old ones, except for those with Leu and the rarer amino acids Trp and Met. The largest change found for Leu is surprising. The estimates of hydrophobicity from the contact energies for non-polar side-chains agree well with the experimental values. In an application of these contact energies, the sequences of 88 structurally distinct proteins in the Protein Data Bank are threaded at all possible positions without gaps into 189 different folds of proteins whose sequences differ from each other by at least 35% sequence identity. The native structures for 73 of 88 proteins, excluding 15 exceptional proteins such as membrane proteins, are all demonstrated to have the lowest alignment energies.

Amino Acids↗