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Biomedical subjects

Hiroshi Fujii

Publications and source records attributed to Hiroshi Fujii.

At least 19 recordsLinked to original sources
PubMed2006

LXR alpha transactivates mouse organic solute transporter alpha and beta via IR-1 elements shared with FXR.

PURPOSE: Recently identified organic solute transporter (Ost) alpha and beta are located on the basolateral membrane of enterocytes and may be responsible for the intestinal absorption of many substrates including bile acids. In the present study, the mechanism governing the transcriptional regulation of their expression was investigated. METHODS AND RESULTS: To clarify the transcriptional regulation of Osts, reporter gene assays were performed using mouse Ostalpha/beta promoter-luciferase reporter constructs. Co-transfection of the constructs with farnesoid X receptor (FXR) and retinoid X receptor alpha (RXRalpha) or liver X receptor alpha (LXRalpha) and RXRalpha into Caco-2 cells induced the transcriptional activities of both Ost alpha and beta and further increases were observed following treatment with each agonist. Sequence analyses indicated the presence of IR-1 regions in Ostalpha and Ostbeta promoters, which was confirmed by the finding that the deletion of IR-1 sequences abolished the response to FXR and LXRalpha. Furthermore, mutations in IR-1 reduced the FXR- and LXRalpha-dependent transactivation of Ostalpha/beta. Together with the detection of direct binding of FXR/RXRalpha and LXRalpha/RXRalpha to the IR-1 elements, the presence of functional FXRE/LXRE was revealed in the promoter region of both Ostalpha and Ostbeta. In addition, the stimulatory effect of FXR/RXRalpha and LXRalpha/RXRalpha on Ostalpha, but not on Ostbeta, was further enhanced by HNF-4alpha. CONCLUSIONS: It was concluded that LXRalpha/RXRalpha transcriptionally regulate mouse Ostalpha/beta via IR-1 elements shared with FXR/RXRalpha. Exposure to FXR/LXRalpha modulators may affect the disposition of Ostalpha/beta substrates.

5' Flanking Region↗
PubMed2006

Ethylene-regulation of fruit softening and softening-related genes in peach.

To investigate the role of ethylene in peach fruit softening during ripening, stony hard peach fruit, in which ethylene production is suppressed during ripening, were treated with various concentrations of ethylene. There was no noticeable decrease in flesh firmness without ethylene treatment, while applied ethylene, in the range 0.1-100 microl l(-1), resulted in fruit softening. Furthermore, the fruit softened more rapidly when the applied ethylene concentration was higher. When ethylene treatment was interrupted, the degree of softening was greatly reduced. These results indicated that continuous ethylene treatment was required for the initiation and progression of fruit softening and that ethylene concentration is also an important factor in regulating the rate of softening. Eight genes, which putatively encode cell wall metabolism-related proteins, were investigated for mRNA accumulation patterns in the two different softening phenotypes of melting and stony hard peaches. All of the mRNAs investigated accumulated in fruit of the melting-flesh "Akatsuki" during ripening. By contrast, in the stony hard-flesh "Manami", the mRNAs for a putative endopolygalacturonase (PpPG), an alpha-L-arabinofuranosidase/beta-xylosidase (PpARF/XYL), and an expansin (PpExp3) showed either much lower levels or did not accumulate, and were identified as softening-related genes. Interruption of ethylene treatment indicated that these genes were regulated at the transcriptional level, and quickly responded to the presence or absence of ethylene before the softening response occurred, suggesting that ethylene directly regulates the transcription of these softening-related genes. These results suggested that cell wall metabolism, causing a rapid loss of firmness in peach fruit, may be controlled by ethylene at the transcriptional level.

Cell Wall↗
PubMed2006

Genes encoding small heat shock proteins of the silkworm, Bombyx mori.

Small heat shock protein (sHSP) is a family of ubiquitous polypeptides involved in a variety of physiological phenomena. From the silkworm, Bombyx mori, we isolated and sequenced the following cDNAs encoding sHSPs: shsp19.9, shsp20.1, shsp20.4, shsp20.8, shsp21.4, and shsp23.7. shsp21.4 was nearly twice as large in size as other shsps. The deduced amino acid sequence of sHSP21.4 was similar to that of Drosophila melanogaster CG14207-PA. Other sHSPs were highly similar to each other and, in a phylogenetic tree, formed a cluster including Plodia interpunctella alphaCP25. It was speculated that shsp21.4 has at least one intron in genome while other shsps do not. The transcripts of all shsps were subtle, but were constitutively detected in various tissues. Heat shock triggered a substantial increase in the transcripts other than shsp21.4. The B. mori sHSPs are perhaps classified into at least two groups: sHSP21.4 and others.

Animals↗
PubMed2006

Trigonal-bipyramidal geometry induced by an external water ligand in a sterically hindered iron salen complex, related to the active site of protocatechuate 3,4-dioxygenase.

A unique distorted trigonal-bipyramidal geometry observed for the non-heme iron center in protocatechuate 3,4-dioxygenase (3,4-PCD) was carefully examined utilizing a sterically hindered iron salen complex, which well reproduces the endogenous His2Tyr2 donor set with water as an external ligand. X-ray crystal structures of a series of iron model complexes containing bis(3,5-dimesitylsalicylidene)-1,2-dimesitylethylenediamine indicate that a distorted trigonal-bipyramidal geometry is achieved upon binding of water as an external ligand. The extent of a structural change of the iron center from a preferred square-pyramidal to a distorted trigonal-bipyramidal geometry varies with the external ligand that is bound in the order Cl << EtO < H2O, which is consistent with the spectrochemical series. The distortion in the model system is not due to steric repulsions but electronic interactions between the external ligand and the iron center, as evidenced from the X-ray crystal structures of another series of iron model complexes with a less-hindered bis(3-xylylsalicylidene)-1,2-dimesitylethylenediamine ligand, as well as by density functional theory calculations. Further spectroscopic investigations indicate that a unique distorted trigonal-bipyramidal geometry is indeed maintained even in solution. The present model study provides a new viewpoint that a unique distorted trigonal-bipyramidal iron site might not be preorganized by a 3,4-PCD protein but could be electronically induced upon the binding of an external hydroxide ligand to the iron(III) center. The structural change induced by the external water ligand is also discussed in relation to the reaction mechanism of 3,4-PCD.

Binding Sites↗
PubMed2006

Rapid GC-MS analysis of methamphetamine and its metabolites in urine--application of a short narrow-bore capillary column to GC-MS.

A rapid analysis of methamphetamine and its metabolites in urine was performed by gas chromatography-mass spectrometry (GC-MS) using a short narrow-bore capillary column (NBC) (5 m x 0.1 mm I.D.). For detection, selected ion monitoring (SIM) was performed for the characteristic ions of each of the compounds. The analytes were independently detected within 2 min. Linearity was demonstrated over a range from 25-2500 ng/ml. As an application of this study, a urine sample from a drug-abuse suspect was analyzed. The analytes from the actual sample were detected with reasonable reproducibility. The results indicate the possibility of rapid analysis using a conventional GC-MS with a short NBC at a relatively low inlet pressure.

Female↗
PubMed2006

Fatty acid binding protein 6 is overexpressed in colorectal cancer.

PURPOSE: Fatty acid binding protein 6 (FABP6) is a cancer-related protein that acts as an intracellular transporter of bile acid in the ileal epithelium. Because bile acids are implicated in the carcinogenesis of colorectal cancer, we evaluated FABP6 expression in colorectal cancer. EXPERIMENTAL DESIGN: The expression of FABP6 mRNA was evaluated in 78 paired samples of cancer/normal tissue representing colorectal cancer cases, plus 16 adenomas, and 16 metastatic lymph nodes. An immunohistochemical study was conducted with paraffin sections. In vitro transfection was done to determine FABP6's biological roles. RESULTS: The expression of FABP6 mRNA was significantly higher in cancer (75 of 78, 96.2%) than in normal tissue (P<0.001). The expression of mRNA was increased in cancer compared with adenoma, but was dramatically decreased in node metastases. Tumors with high FABP6 expression were smaller in size (P<0.01), more often in the left colon (P<0.05), and had shallower invasion into the bowel wall (P<0.05) compared with those with low expression. There was no significant difference between high- and low-expression tumors regarding clinicopathologic variables such as histologic type, lymph node, or liver metastasis, Dukes' classification, and prognosis. Immunohistochemical study revealed that FABP6 expression was primarily observed in cancer cells. In vitro transfection revealed that transfectants showed weaker invasiveness (P<0.05), more dominant proliferation (P<0.001), and less apoptosis than mock cells. CONCLUSIONS: The expression of FABP6 was higher in primary colorectal cancers and adenomas than in normal epithelium, but was dramatically decreased in lymph node metastases, suggesting that FABP6 may play an important role in early carcinogenesis.

Adenoma↗
PubMed2006

13C and 15N NMR studies of iron-bound cyanides of heme proteins and related model complexes: sensitive probe for detecting hydrogen-bonding interactions at the proximal and distal sides.

Studies of the 13C and 15N NMR paramagnetic shifts of the iron-bound cyanides in the ferric cyanide forms of various heme proteins containing the proximal histidine and related model complexes are reported. The paramagnetic shifts of the 13C and 15N NMR signals of the iron-bound cyanide are not significantly affected by the substitution of the porphyrin side chains. On the other hand, the paramagnetic shifts of both the 13C and 15N NMR signals decrease with an increase in the donor effect of the proximal ligand, and the 13C NMR signal is more sensitive to a modification of the donor effect of the proximal ligand than the 15N NMR signal. With the tilt of the iron-imidazole bond, the paramagnetic shift of the 13C NMR signal increases, whereas that of the 15N NMR signal decreases. The hydrogen-bonding interaction of the iron-bound cyanide with a solvent decreases the paramagnetic shift of both 13C and 15N NMR signals, and the effect is more pronounced for the 15N NMR signal. Data on the 13C and 15N NMR signals of iron-bound cyanide for various heme proteins are also reported and analyzed in detail. Substantial differences in the 13C and 15N NMR shifts for the heme proteins can be explained on the basis of the results for the model complexes and structures around the heme in the heme proteins. The findings herein show that the paramagnetic shift of the 13C NMR signal of the iron-bound cyanide is a good probe to estimate the donor effect of the proximal imidazole and that the ratio of 15N/13C NMR shifts allows the hydrogen-bonding interaction on the distal side to be estimated.

Carbon Isotopes↗
PubMed2006

Construction of a single nucleotide polymorphism linkage map for the silkworm, Bombyx mori, based on bacterial artificial chromosome end sequences.

We have developed a linkage map for the silkworm Bombyx mori based on single nucleotide polymorphisms (SNPs) between strains p50T and C108T initially found on regions corresponding to the end sequences of bacterial artificial chromosome (BAC) clones. Using 190 segregants from a backcross of a p50T female x an F1 (p50T x C108T) male, we analyzed segregation patterns of 534 SNPs between p50T and C108T, detected among 3840 PCR amplicons, each associated with a p50T BAC end sequence. This enabled us to construct a linkage map composed of 534 SNP markers spanning 1305 cM in total length distributed over the expected 28 linkage groups. Of the 534 BACs whose ends harbored the SNPs used to construct the linkage map, 89 were associated with 107 different ESTs. Since each of the SNP markers is directly linked to a specific genomic BAC clone and to whole-genome sequence data, and some of them are also linked to EST data, the SNP linkage map will be a powerful tool for investigating silkworm genome properties, mutation mapping, and map-based cloning of genes of industrial and agricultural interest.

Animals↗
PubMed2006

17O NMR study of oxo metalloporphyrin complexes: correlation with electronic structure of M=O moiety.

(17)O NMR spectroscopy of oxo ligand of oxo metalloporphyrin can be considered as an excellent means to derive information about structure, electronic state, and reactivity of the metal bound oxo ligand. To show the utility of (17)O NMR spectroscopy of oxo ligand of oxo metalloporphyrin, (17)O NMR spectra of oxo ligands of dioxo ruthenium(VI), oxo chromium(IV), and oxo titanium(IV) porphyrins are measured. For all oxo metalloporphyrins, well-resolved (17)O NMR signals are detected in far high frequency region. The (17)O NMR signal of the metal bound oxo ligand shifts high frequency in order of Ru(VI)<Ti(IV)<Cr(IV), thus the (17)O NMR chemical shift does not directly correlate with the oxo-transfer reactivity, Ti(IV)<Cr(IV)<Ru(VI). On the other hand, the (17)O NMR shift of oxo ligand correlates with the bond strength of metal-oxo bond. This suggests that the (17)O NMR signal of metal bound oxo ligand is a sensitive probe to study the nature of metal-oxo bond in oxo metalloporphyrin. The effect of the electron-withdrawing meso-substituent on the (17)O NMR shift of the oxo ligand is also investigated. With increase in the electron-withdrawing effect of the meso-substituent, the (17)O NMR signal of the oxo ligand of oxo chromium(IV) porphyrin shifts high frequency while that of dioxo ruthenium(VI) porphyrin hardly change resonance position. The changes in metal-oxo bonds induced by the electron-withdrawing meso-substituent are discussed on the basis of the (17)O NMR shifts, the strengths of the metal-oxo bonds, and the oxo-transfer reaction rates.

Chromium Compounds↗
PubMed2006

Roles of the heme distal residues of FixL in O2 sensing: a single convergent structure of the heme moiety is relevant to the downregulation of kinase activity.

FixL is a heme-based O(2) sensor, in which the autophosphorylation is regulated by the binding of exogenous ligands such as O(2) and CN(-). In this study, mutants of the heme distal Arg200, Arg208, Ile209, Ile210, and Arg214 residues of SmFixL were characterized biochemically and physicochemically, because it has been suggested that they are significant residues in ligand-linked kinase regulation. Measurements of the autoxidation rate, affinities, and kinetics of ligand binding revealed that all of the above residues are involved in stabilization of the O(2)-heme complex of FixL. However, Arg214 was found to be the only residue that is directly relevant to the ligand-dependent regulation of kinase activity. Although the wild type and R214K and R214Q mutants exhibited normal kinase regulation, R214A, R214M, R214H, and R214Y did not. (13)C and (15)N NMR analyses for (13)C(15)N(-) bound to the truncated heme domains of the Arg214 mutants indicated that, in the wild type and the foregoing two mutants, the heme moiety is present in a single conformation, but in the latter four, the conformations fluctuate possibly because of the lack of an interaction between the iron-bound ligand and residue 214. It is likely that such a rigid conformation of the ligand-bound form is important for the downregulation of histidine kinase activity. Furthermore, a comparison of the NMR data between the wild type and R214K and R214Q mutants suggests that a strong electrostatic interaction between residue 214 and the iron-bound ligand is not necessarily required for the single convergent structure and eventually for the downregulation of FixL.

Arginine↗
PubMed2006

Telomeres, immune aging and autoimmunity.

Telomere length is important in constraining the replicative potential of cells; cellular systems that are dependent on cell replenishment for renewal or on cell proliferation for functionality are highly sensitive to telomeric erosion. Cell replication invariably leads to telomere loss, which, in some cellular systems, is partially compensated for by telomerase activity. In addition to this typical telomere loss, several mechanisms of sporadic telomere loss exist. Heterogeneity in age-dependent telomere loss can be a consequence of increased cellular turnover during a lifetime, accelerated telomeric DNA damage, or defects in telomere repair. The immune system is a prime example of a highly dynamic cellular system, for which telomere maintenance is pivotal. Immune competence is strictly dependent on rapid expansions of clonal T- and B-cell populations, and telomere loss may contribute to defective immune responses in the elderly. Equally interestingly, accelerated T-cell aging combined with telomeric shortening may predispose for autoimmune responses and thereby explain the increased susceptibility for chronic inflammatory diseases in the elderly.

Aging↗
PubMed2006

Proteome analysis of silk gland proteins from the silkworm, Bombyx mori.

The silk gland of Bombyx mori is an organ specialized for the synthesis and secretion of silk proteins. We report here the resolution of silk gland proteins by 2-DE and the identification of many of those proteins. This was accomplished by dissecting the glands into several sections, with each exhibiting more than 400 protein spots by 2-DE, of which 100 spots were excised and characterized by in-gel digestion followed by PMF. Ninety-three proteins were tentatively identified. These were then categorized into groups involved in silk protein secretion, transport, lipid metabolism, defense, etc. Western blotting of a 2-DE gel using an antibody of the carotenoid binding protein confirmed the presence of this protein in the silk gland. Proteins including fibroin L-chain and P25 were found as multiple isoforms, some of which contained differential amounts of phosphate residues as analyzed by on-probe dephosphorylation. The current analysis contributes to our understanding of proteins expressed by the silk gland not only of the model lepidopteran B. mori, but also to proteins from other silk-producing insects such as Philosamia cynthia ricini.

Animals↗
PubMed2006

Silkworm midgut proteins interacting with a hemolymph protease inhibitor, CI-8.

CI-8 is the chymotrypsin inhibitor in hemolymph from the silkworm, Bombyx mori. It occurs in the midgut at the spinning stage of larva, but little information on the mechanism of its uptake in the midgut is available. We found that two polypeptides interacting with CI-8 are in the midgut membrane, and we purified them using a biotinylated CI-8, viz., p29 and p60, having molecular sizes of 29 kDa and 60 kDa respectively. The structures of p29 and p60 were examined by N-terminal amino acid sequencing and peptide mass mapping, including tryptic digestion. p29 was highly similar to the matured 19G1-30K lipoprotein from hemolymph, but p60 was novel. Purified p29 was recognized by anti-19G1-30K antibody, and was confirmed to be similar to 19G1-30K. The antibody also neutralized the CI-8 binding ability of p29 in the midgut membrane. p29 and p60 are perhaps proteinaceous factors involved in the uptake of CI-8 into the midgut through the membrane.

Amino Acid Sequence↗
PubMed2005

Oxidizing intermediates from the sterically hindered iron salen complexes related to the oxygen activation by nonheme iron enzymes.

Oxidizing intermediates are generated from nonheme iron(III) complexes to investigate the electronic structure and the reactivity, in comparison with the oxoiron(IV) porphyrin pi-cation radical (compound I) as a heme enzyme model. Sterically hindered iron salen complexes, bearing a fifth ligand Cl (1), OH(2) (2), OEt (3), and OH (4), are oxidized both electrochemically and chemically. Stepwise one-electron oxidation of 1 and 2 generates iron(III)-mono- and diphenoxyl radicals, as revealed by detailed spectroscopic investigations, including UV-vis, EPR, Mössbauer, resonance Raman, and ESIMS spectroscopies. In contrast to the oxoiron(IV) formation from the hydroxoiron(III) porphyrin upon one-electron oxidation, the hydroxo complex 4 does not generate oxoiron(IV) species. Reaction of 2 with mCPBA also results in the formation of the iron(III)-phenoxyl radical. One-electron oxidation of 3 leads to oxidative degradation of the fifth EtO ligand to liberate acetaldehyde even at 203 K. The iron(III)-phenoxyl radical shows high reactivity for alcoxide on iron(III) but exhibits virtually no reactivity for alcohols including even benzyl alcohol without a base to remove an alcohol proton. This study explains unique properties of mononuclear nonheme enzymes with Tyr residues and also the poor epoxidation activity of Fe salen compared to Mn and Cr salen compounds.

Ethylenediamines↗
PubMed2005

Tissue microarray of head and neck squamous carcinoma: validation of the methodology for the study of cutaneous fatty acid-binding protein, vascular endothelial growth factor, involucrin and Ki-67.

Tissue microarrays allow the simultaneous analysis of many tumours using small-diameter cores sampled from larger blocks of tissue, but may be limited by tumour heterogeneity. This study considers the validation of tissue microarray for the study of four molecules of interest as prognostic factors in head and neck squamous carcinoma, including a consideration of methods for assessing immunocytochemical scoring of microarrays. Tissue microarray blocks were constructed from 100 cases of head and neck squamous carcinoma, taking four cores from different areas of each tumour. Immunocytochemical labelling was performed for cutaneous fatty acid binding protein, involucrin, vascular endothelial growth factor and Ki-67. The extent and intensity of scoring was determined for each core and the degree of agreement determined for results from the assessment of two, three or four cores for each carcinoma. In a subset of 30 representative cases, the labelling in the tissue microarrays was compared with that in whole-tissue sections of the same carcinomas. An adequate sample of carcinoma was achieved in more than 90% of the 400 cores; unsuccessful results were attributed to uneven core alignment or to poor targeting of the tumour tissue in the donor blocks. The degree of agreement in the assessment of extent and intensity of labelling was moderate to good (weighted kappa, range 0.479-0.902) between whole-tissue sections and microarray sections depending on the antigen and the scoring system. Tissue microarray is a reliable tool to demonstrate cellular and molecular alterations in head and neck squamous carcinomas. We recommend using the mean results from four cores for biological studies, with analysis of categorical data based on quartile groups. Concordance with whole-tissue section data is reassuring, but data from microarrays need to be validated against clinical outcomes.

Adult↗
PubMed2005

Molecular and biochemical characterization of manganese-containing superoxide dismutase from the silkworm, Bombyx mori.

Superoxide dismutase (SOD) is responsible for the removal of superoxide anion from living organisms. In this study, cDNA encoding the manganese-containing SOD (MnSOD) from the silkworm, Bombyx mori, was isolated by reverse transcriptase-polymerase chain reaction and sequenced. The deduced amino acid sequence of the MnSOD revealed 62% identity to that of the Drosophila melanogaster; both were close to each other in a phylogenetic tree. The MnSOD was overproduced in Escherichia coli and purified. The internal structure of the recombinant MnSOD was confirmed by peptide mass fingerprinting method. The recombinant MnSOD facilitating the reduction reaction of superoxide anion retained 75% of its original activity after incubation at pH 4-11 for 24 h at 4 degrees C. Its activity was never affected by incubation at pH 7 for 30 min below 50 degrees C.

Amino Acid Sequence↗
PubMed2005

Ileal bile acid-binding protein, functionally associated with the farnesoid X receptor or the ileal bile acid transporter, regulates bile acid activity in the small intestine.

Bile acids secreted in the small intestine are reabsorbed in the ileum where they activate the nuclear farnesoid X receptor (FXR), which in turn stimulates expression of the ileal bile acid-binding protein (I-BABP). We first hypothesized that I-BABP may negatively regulate the FXR activity by competing for the ligands, bile acids. Reporter assays using stable HEK293 cell lines expressing I-BABP revealed that I-BABP enhances rather than attenuates FXR activity. In these cells I-BABP localizes predominantly in the cytosol and partially in the nucleus, a distribution that does not shift in response to FXR expression. In vitro binding assays reveal that recombinant I-BABP is able to bind 35S-labeled FXR and that chenodeoxycholic acid (CDCA) stimulates this interaction modestly. When FLAG-tagged FXR was expressed in stable cells, the FXR.I-BABP complex in the nuclear extracts was more efficiently immunoprecipitable with anti-FLAG antibodies in the presence of CDCA. These results indicate that I-BABP stimulates FXR activity through a mutual interaction augmented by bile acids. When stable cells were transfected with an expression plasmid of the ileal bile acid transporter 14(IBAT) essential for the reabsorption of conjugated bile acids, the C-labeled conjugated bile acid, glycocholic acid, was more efficiently imported via IBAT in the presence than absence of I-BABP, whereas no change was observed in 14C-labeled CDCA uptake, which is independent of IBAT. Immunofluorescent staining analysis revealed that these two proteins co-localize in the vicinity of the plasma membrane in stable cells. Taken together, the current data provide the first evidence that I-BABP is functionally associated with FXR and IBAT in the nucleus and on the membrane, respectively, stimulating FXR transcriptional activity and the conjugated bile acid uptake mediated by IBAT in the ileum.

Bile Acids and Salts↗