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Biomedical subjects

N Takamatsu

Publications and source records attributed to N Takamatsu.

At least 19 recordsLinked to original sources
PubMed2001

HNF-1 regulates the liver-specific transcription of the chipmunk HP-20 gene.

The chipmunk hibernation-specific protein HP-20 is a component of the 140 kDa complex that drastically decreases in the blood during hibernation, and its gene is expressed specifically in the liver. To reveal molecular mechanisms underlying the liver-specific transcription of the HP-20 gene, we isolated chipmunk HP-20 genomic clones. The HP-20 gene spans approximately 6 kb, and consists of three exons. The transcription start site, as determined by 5' RACE-PCR analysis, was found to be 160 bp upstream of the translation initiation codon. Transient transfection studies in HepG2 cells revealed that the 57 bp 5' flanking sequence was sufficient for the liver-specific promoter activity. A database search revealed that this region contains a potential binding site for hepatocyte nuclear factor-1 (HNF-1). In a gel retardation assay, in vitro-synthesized HNF-1 bound to the 5' flanking sequence from -52 to -26. A similar shifted band was also observed with HepG2 nuclear extracts, and this complex was super-shifted by an anti-(HNF-1) Ig. When transfected into COS-7 cells, HNF-1 transactivated transcription from the HP-20 gene promoter, and this activity was abolished by a mutation of the HNF-1 binding site, indicating that HNF-1 plays an important role in HP-20 gene expression.

5' Flanking Region↗
PubMed2001

Expression of JNK cascade scaffold protein JSAP1 in the mouse nervous system.

The mitogen-activated protein kinase (MAPK) cascades consist of MAPK, MAPK kinase (MAPKK), and MAPKK kinase (MAPKKK). The specificity of activation of MAPK cascades may be determined, in part, by scaffold proteins that organize multi-enzyme complexes. We have earlier reported a scaffold protein JSAP1 (also known as JIP3) in the JNK MAPK cascade. We also showed that, of the adult mouse tissues tested, JSAP1 mRNA was predominantly expressed in brain. Here we report the localization of JSAP1 protein in mouse embryos and adult brain by immunohistochemical analysis. In embryos (E11-16), JSAP1 immunoreactivity was mainly found in the central and peripheral nervous systems, where it was localized to the cell bodies and/or axons of developing neurons, but not neural precursor cells. In the adult brain, immunoreactive JSAP1 was localized mostly to cell bodies in almost all neurons. We also showed that the expression of JSAP1 transcripts and proteins gradually increased during the neural differentiation of mouse P19 embryonal carcinoma (EC) cells. Furthermore, we showed that overexpressed JSAP1 facilitated the efficient activation of JNK by MEKK1 in P19 cells. These results suggest that JSAP1 may function as a scaffold protein for the JNK signaling module in neuronal cells.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Human jejunal permeability of two polar drugs: cimetidine and ranitidine.

PURPOSE: To determine the human jejunal permeability of cimetidine and ranitidine using a regional jejunal perfusion approach, and to integrate such determinations with previous efforts to establish a baseline correlation between permeability and fraction dose absorbed in humans for soluble drugs. METHODS: A sterile multi-channel perfusion tube, Loc-I-Gut, was inserted orally and positioned in the proximal region of the jejunum. A solution containing cimetidine or ranitidine and phenylalanine, propranolol, PEG 400, and PEG 4000 was perfused through a 10 cm jejunal segment in 6 and 8 subjects, respectively. RESULTS: The mean Peff (+/- se) of cimetidine and ranitidine averaged over both phases were 0.30 (0.045) and 0.27 (0.062) x 10(-4) cm/s, respectively, and the differences between the two were found to be statistically insignificant. The mean permeabilities for propranolol, phenylalanine, and PEG 400 averaged over both phases and studies were 3.88 (0.72), 3.36 (0.50), and 0.56 (0.08) x 10(-4) cm/s, respectively. The differences in permeability for a given marker were not significant between phases or between the two studies. CONCLUSIONS: The 10-fold lower permeabilities found for cimetidine and ranitidine in this study, compared to propranolol and phenylalanine, appear to be consistent with their less than complete absorption in humans.

Adolescent↗
PubMed2001

The tree squirrel HP-25 gene is a pseudogene.

The gene for the hibernation-specific protein HP-25 is expressed in the liver in hibernating species of the squirrel family (chipmunk and ground squirrel), but not in a nonhibernating species (tree squirrel). To investigate why the HP-25 gene is not expressed in the tree squirrel, we isolated the tree squirrel HP-25 gene and compared its gene structure and promoter activity with that of the chipmunk. The tree squirrel HP-25 gene is composed of three exons, and the gene structures are conserved between the tree squirrel and chipmunk. However, the tree squirrel HP-25 gene has an insertional mutation of 13 nucleotides in exon 2 that disrupts the ORF. In the chipmunk HP-25 gene, the 80-bp 5' flanking sequence is sufficient for the liver-specific promoter activity, and HNF-4, which binds to the sequence from nucleotides -67 to -51, is involved in its transcriptional regulation. In contrast, the corresponding tree squirrel 5' flanking sequence had almost no promoter activity in HepG2 cells, and HNF-4 did not bind to the corresponding region of the tree squirrel HP-25 gene. Furthermore, a tree squirrel-type G to A mutation at -57 in the chipmunk HP-25 gene promoter context abolished its binding to and transactivation by HNF-4. Thus, the point mutation in the HNF-4-binding site is likely to be involved in the lack of HP-25 gene expression in the tree squirrel.

Amino Acid Sequence↗
PubMed2001

Decreased absorption and retention rates of magnesium in the rats fed on spinach-supplemented diets: possible explanations.

We studied the bioavailability of magnesium (Mg) in spinach after boiling with distilled water, using Mg-deficient growing male rats. The rats were fed a semipurified diet (Mg:0.063 per cent (w/w)) for 3 days. then a Mg-deficient diet (Mg:0.001 per cent (w/w)) for 5 days. They were then divided randomly into 7 groups of 6 rats each, and fed the semipurified diet (Mg: 0.063, 0.045 or 0.027 per cent (w/w)), or the spinach-supplemented diet (10 per cent (w/w) dried and powdered spinach after boiling with distilled water for 3 min at 100 degrees C). The Mg content of the diets supplemented with spinach grown on chemical nutrients, and on manure from pigs, cattle and fowl, was 0.069, 0.051, 0.043 and 0.036 per cent (w/w), respectively. Water intake and volumes of urine and faeces were significantly greater in the rats fed the spinach-supplemented diets than in those fed the semipurified diets. Apparent absorption of Mg, and urinary and faecal excretions of Mg were directly related to Mg intake: no significant difference was observed amongst the groups. Both the ratios of Mg absorption and retention were significantly lower in the rats fed diets supplemented with spinach than in those fed semipurified diets. The plasma Mg level was directly related to Mg intake in the rats fed the semipurified diets and the spinach-supplemented diets. However, the plasma Mg level in the rats fed spinach grown organically on manure from fowl tended to be higher than in the other groups. From these results, it was concluded that bound Mg in spinach was effectively utilized by Mg-deficient rats, however, the absorption and retention rates of Mg in rats fed diets supplemented with spinach were decreased. Possible explanations were discussed.

Animals↗
PubMed2001

Nitric oxide-mediated antitumor activity induced by the extract from Grifola frondosa (Maitake mushroom) in a macrophage cell line, RAW264.7.

We have investigated D-fraction (MDF) extracted from Grifola frondosa (Maitake mushroom) on the inducible nitric oxide synthase (iNOS)-mediated nitric oxide (NO) production in RAW264.7 (RAW) cells, a murine monocyte/macrophage cell line, with special reference to antitumor activity of MDF against human hepatoma-derived huH-1 cells. MDF could induce iNOS mRNA expression in RAW cells in a dose range of more than 30 microg/ml, but the effect of 10 microg/ml of MDF was negligible. The iNOS mRNA expression induced by 100 microg/ml of MDF was 6 hrs later, but lasted for a longer time than that of lipopolysaccharide (LPS), a representative iNOS inducer. Although iNOS mRNA levels in MDF-stimulated cells were almost equal to LPS-stimulated cells at the peak time, the cumulative amount of nitrite was only about 50% compared with that of LPS-treated cells. When huH-I cells were cultured in MDF containing media in a 24-well plate with inserted porous bottom in the presence or absence of RAW cells, the viability of huH-1 cells decreased significantly only in the presence of RAW cells in MDF dose-dependent manner. This antitumor activity of RAW cells in the presence of MDF was abolished or attenuated by the addition of L-NAME, a NOS inhibitor, confirming that this phenomenon is due to iNOS-mediated NO production by RAW cells, but not direct cytotoxic activity of MDF against huH-1 cells. These data suggest that MDF is a novel inducer for iNOS which contributes at least in part to antitumor activity of MDF.

Agaricales↗
PubMed2000

A scaffold protein in the c-Jun NH2-terminal kinase signaling pathways suppresses the extracellular signal-regulated kinase signaling pathways.

We previously reported that c-Jun NH(2)-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1) functions as a putative scaffold factor in the JNK mitogen-activated protein kinase (MAPK) cascades. In that study we also found MEK1 and Raf-1, which are involved in the extracellular signal-regulated kinase (ERK) MAPK cascades, bind to JSAP1. Here we have defined the regions of JSAP1 responsible for the interactions with MEK1 and Raf-1. Both of the binding regions were mapped to the COOH-terminal region (residues 1054-1305) of JSAP1. We next examined the effect of overexpressing JSAP1 on the activation of ERK by phorbol 12-myristate 13-acetate in transfected COS-7 cells and found that JSAP1 inhibits ERK's activation and that the COOH-terminal region of JSAP1 was required for the inhibition. Finally, we investigated the molecular mechanism of JSAP1's inhibitory function and showed that JSAP1 prevents MEK1 phosphorylation and activation by Raf-1, resulting in the suppression of the activation of ERK. Taken together, these results suggest that JSAP1 is involved both in the JNK cascades, as a scaffolding factor, and the ERK cascades, as a suppressor.

Animals↗
PubMed2000

Immunoglobulin treatment ameliorates murine myocarditis associated with reduction of neurohumoral activity and improvement of extracellular matrix change.

OBJECTIVES: We examined effects of immunoglobulin on murine myocarditis induced by encephalomyocarditis virus, not pathogenic to humans, and analyzed the plasma cytokine and catecholamine levels and the changes of the extracellular matrix with or without the treatment. BACKGROUND: We have previously shown that immunoglobulin therapy suppressed murine coxsackievirus B3 myocarditis by an antiviral effect. However, it is not yet determined whether beneficial effects of immunoglobulin for myocarditis are due to antiviral effects or to other unknown effects. METHODS: Antiviral activity of human immunoglobulin (Polyglobin-N) against encephalomyocarditis virus was determined in vitro. Immunoglobulin (1 g/kg/day) was administered intraperitoneally to the virus-infected mice daily for two weeks, beginning simultaneously with virus inoculation in experiment I and on day 14 after virus inoculation in experiment II. RESULTS: Antiviral activity of immunoglobulin could not be detected in the assay of a plaque-reduction method in vitro. The in vivo study showed that immunoglobulin administration ameliorated both myocardial necrosis with interstitial fibrin deposition in experiment I and interstitial fibrosis with the improvement of ventricular remodeling in experiment II by the reduction of plasma catecholamines, interferon-alpha, and soluble intercellular adhesion molecule-1. CONCLUSIONS: Immunoglobulin therapy could suppress myocarditis associated with the improvement of extracellular matrix changes by the reduction of neurohumoral activity.

Animals↗
PubMed2000

Isoforms of JSAP1 scaffold protein generated through alternative splicing.

We have identified four isoforms of c-Jun NH(2)-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1), a scaffold protein that participates in JNK mitogen-activated protein kinase cascades, termed JSAP1a, JSAP1b, JSAP1c, and JSAP1d. The previously identified JSAP1 was renamed JSAP1a to avoid confusion. Analyses of the exon-intron structure of the jsap1 gene indicated that the isoforms are generated through alternative splicing involving exons 5 and 6. The mRNA expression levels of the JSAP1 isoforms differed among the mouse tissues examined. We also investigated the region of JSAP1 responsible for its interaction with JNK, and found that the JNK-binding domain is located between aa residues 201 and 217 in JSAP1a, which is encoded by part of exon 6. As all the JSAP1 isoforms contain this binding domain, we examined the binding affinity of the JSAP1 isoforms for JNK1, JNK2, and JNK3. JSAP1c and JSAP1d, which contain a 31-aa sequence not present in JSAP1a or JSAP1b, had a lower binding affinity for the JNKs, especially JNK3. These results suggest that JSAP1c and JSAP1d may attenuate the scaffolding activity of JSAP1a and/or JSAP1b in JNK cascades, especially the JNK3 cascades.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Characterization of Solt, a novel SoxLZ/Sox6 binding protein expressed in adult mouse testis.

SoxLZ/Sox6, a member of the Sox protein family, contains a leucine zipper motif in addition to an HMG box, which is its DNA binding domain. Here we have identified a novel SoxLZ/Sox6 binding protein, termed Solt, which we obtained independently using both a far-Western blot and a yeast two-hybrid screen. Like SoxLZ/Sox6 mRNA, Solt mRNA was exclusively expressed in the testis in mouse. Solt contains an unusual leucine zipper, which bound to the leucine zipper region of SoxLZ/Sox6 in vitro. In transient transfection assays in CHO cells with SoxLZ/Sox6 containing the transactivational region of herpes simplex virus VP16, expression of a reporter gene that carries a cis binding region for Sox proteins was significantly enhanced by the co-expression of Solt and Ca(2+)/calmodulin-dependent protein kinase IV.

Amino Acid Sequence↗
PubMed2000

Barnacle cement proteins. Importance of disulfide bonds in their insolubility.

Barnacles produce a cement that is a proteinaceous underwater adhesive for their secure attachment to the substratum. The biochemical properties of the cement have not previously been elucidated, because the insolubility of the cement proteins hampers their purification and characterization. We developed a non-hydrolytic method to render soluble most of the cement components, thereby allowing the proteins to be analyzed. Megabalanus rosa cement could be almost completely rendered soluble by its reduction with 0.5 m dithiothreitol at 60 degrees C in a 7 m guanidine hydrochloride solution, the high concentration of dithiothreitol being indispensable to achieve this. The effectiveness of this reduction treatment was confirmed by the detachment of the barnacle from the substratum. Three proteins comprising up to 94% of the whole cement were identified as the major cement components. The cDNA clone of one of these major proteins was isolated, and the site-specific expression of the gene in the basal portion of the adult barnacle, where the cement glands are located, was demonstrated. A sequence analysis revealed this cement component to be a novel protein of 993 amino acid residues, including a signal peptide. This is the first report of the major component of the barnacle cement protein complex.

Amino Acid Sequence↗
PubMed2000

Prevalence of Bacteroides forsythus, Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans in subgingival microflora of Japanese patients with adult and rapidly progressive periodontitis.

BACKGROUND, AIMS: This study investigated the prevalence of Bacteroides forsythus, Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans among various periodontitis patients and healthy individuals in Japan, and correlated it with clinical parameters. METHOD: Subgingival plaque samples were collected from 21 patients with adult periodontitis (AP), 8 with rapidly progressive periodontitis (RPP) and 15 healthy individuals. RESULTS: The frequency detected in culture was as follows: B. forsythus was found in 47.6% of AP sites and in 37.5% of RPP sites. P. gingivalis was identified in 64.3% of AP and 59.4% of RPP sites. A. actinomycetemcomitans was detected in 4.8% of AP and 3.1% of RPP sites. The 3 species were detected in only 2 of the healthy individuals. The proportion of B. forsythus in the total microflora in culture was 0.07% in the healthy group, 4.1% in AP and 2.4% in RPP. The proportions of P. gingivalis were 0% in the healthy group, 18.8% in AP and 16.2% in RPP. The proportion of A. actinomycetemcomitans was very low in all 3 groups. A DNA probe detected B. forsythus in 78.6% of AP and 65.6% of RPP sites, as well as P.gingivalis in 58.3% of AP and 59.4% of RPP sites. A. actinomycetemcomitans was detected in only 1.2% of AP sites. The 3 species were undetectable in the healthy group. CONCLUSIONS: The prevalence and the proportion of B. forsythus and P. gingivalis were significantly correlated with clinical parameters, suggesting that B. forsythus and P. gingivalis are closely related to AP and RPP in the Japanese population.

Aggregatibacter actinomycetemcomitans↗
PubMed2000

HNF-4 plays a pivotal role in the liver-specific transcription of the chipmunk HP-25 gene.

The gene for chipmunk hibernation-specific protein HP-25 is expressed specifically in the liver. To understand the transcriptional regulation of HP-25 gene expression, we isolated its genomic clones, and characterized its structural organization and 5' flanking region. The gene spans approximately 7 kb and consists of three exons. The transcription start site, as determined by primer extension analysis, is located at 113 bp upstream of the translation initiation codon. Transient transfection studies in HepG2 cells revealed that the 80 bp 5' flanking sequence was sufficient for the liver-specific promoter activity. In a gel retardation assay using HepG2 nuclear extracts, the 5' flanking sequence from -74 to -46 showed a shifted band. All cDNA clones isolated by a yeast one-hybrid system for a protein capable of binding to this 5' flanking sequence encoded HNF-4. HNF-4 synthesized in vitro bound to this sequence in a gel retardation assay. Furthermore, supershift assays with anti-(HNF-4) Ig confirmed that the protein in HepG2 or chipmunk liver nuclear extracts that bound to this sequence was HNF-4. When transfected into HeLa cells, HNF-4 transactivated transcription from the HP-25 gene promoter, and mutation of the HNF-4 binding site abolished transactivation by HNF-4, indicating that HNF-4 plays an important role in HP-25 gene expression.

Amino Acid Sequence↗
PubMed1999

A novel Jun N-terminal kinase (JNK)-binding protein that enhances the activation of JNK by MEK kinase 1 and TGF-beta-activated kinase 1.

We have identified a novel Jun N-terminal kinase (JNK)-binding protein, termed JNKBP1, and examined its binding affinity for JNK1, JNK2, JNK3, and extracellular signal-regulated kinase 2 (ERK2) in COS-7 cells. JNKBP1 preferentially interacted with the JNKs, but not with ERK2. Furthermore, we investigated the effect of overexpressing JNKBP1 on the JNK and ERK signaling pathways in COS-7 cells. JNKBP1 alone had only a marginal effect on JNK activity. However, the activation of JNK by MEK kinase 1 and TGF-beta-activated kinase 1 was significantly enhanced in the presence of JNKBP1. In contrast, JNKBP1 had no or very little effect on the ERK signaling pathway. These results suggest that JNKBP1 functions to facilitate the specific and efficient activation of the JNK signaling pathways.

Amino Acid Sequence↗
PubMed1999

JSAP1, a novel jun N-terminal protein kinase (JNK)-binding protein that functions as a Scaffold factor in the JNK signaling pathway.

The major components of the mitogen-activated protein kinase (MAPK) cascades are MAPK, MAPK kinase (MAPKK), and MAPKK kinase (MAPKKK). Recent rapid progress in identifying members of MAPK cascades suggests that a number of such signaling pathways exist in cells. To date, however, how the specificity and efficiency of the MAPK cascades is maintained is poorly understood. Here, we have identified a novel mouse protein, termed Jun N-terminal protein kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1), by a yeast two-hybrid screen, using JNK3 MAPK as the bait. Of the mammalian MAPKs tested (JNK1, JNK2, JNK3, ERK2, and p38alpha), JSAP1 preferentially coprecipitated with the JNKs in cotransfected COS-7 cells. JNK3 showed a higher binding affinity for JSAP1, compared with JNK1 and JNK2. In similar cotransfection studies, JSAP1 also interacted with SEK1 MAPKK and MEKK1 MAPKKK, which are involved in the JNK cascades. The regions of JSAP1 that bound JNK, SEK1, and MEKK1 were distinct from one another. JNK and MEKK1 also bound JSAP1 in vitro, suggesting that these interactions are direct. In contrast, only the activated form of SEK1 associated with JSAP1 in cotransfected COS-7 cells. The unstimulated SEK1 bound to MEKK1; thus, SEK1 might indirectly associate with JSAP1 through MEKK1. Although JSAP1 coprecipitated with MEK1 MAPKK and Raf-1 MAPKKK, and not MKK6 or MKK7 MAPKK, in cotransfected COS-7 cells, MEK1 and Raf-1 do not interfere with the binding of SEK1 and MEKK1 to JSAP1, respectively. Overexpression of full-length JSAP1 in COS-7 cells led to a considerable enhancement of JNK3 activation, and modest enhancement of JNK1 and JNK2 activation, by the MEKK1-SEK1 pathway. Deletion of the JNK- or MEKK1-binding regions resulted in a significant reduction in the enhancement of the JNK3 activation in COS-7 cells. These results suggest that JSAP1 functions as a scaffold protein in the JNK3 cascade. We also discuss a scaffolding role for JSAP1 in the JNK1 and JNK2 cascades.

Adaptor Proteins, Signal Transducing↗