Biomedical subjects

R Fujii

Publications and source records attributed to R Fujii.

At least 37 records · Page 2Linked to original sources

PubMed2000

Identification of neuromedin U as the cognate ligand of the orphan G protein-coupled receptor FM-3.

Neuromedin U is a bioactive peptide first isolated from porcine spinal cord. In this paper, we demonstrate that neuromedin U is the cognate ligand for the orphan G protein-coupled receptor, FM-3, isolated originally as a homologue of neurotensin and growth hormone secretogogue receptors. Neuromedin U induced specific and evident elevation of extracellular acidification rates, arachidonic acid metabolite release, and intracellular Ca(2+) mobilization in Chinese hamster ovary cells expressing human FM-3. In addition, radiolabeled neuromedin U specifically bound to membrane fractions prepared from these cells with high affinity. We subsequently analyzed the tissue distribution of neuromedin U and FM-3 mRNAs in rats using quantitative reverse transcription-polymerase chain reaction. Neuromedin U mRNA was highly expressed in the gastrointestinal tract, and the highest expression was detected in the pituitary gland. On the other hand, FM-3 mRNA was highly expressed in the small intestine and lung, suggesting that neuromedin U plays important roles in these tissues. The identification of a specific and functional receptor for neuromedin U will facilitate studies on their physiological roles and the search for receptor agonists and antagonists.

Amino Acid Sequence↗

PubMed2000

Molecular and functional characteristics of APJ. Tissue distribution of mRNA and interaction with the endogenous ligand apelin.

We have recently identified apelin as the endogenous ligand for human APJ. In rats, the highest expression of APJ mRNA was detected in the lung, suggesting that APJ and its ligand play an important role in the pulmonary system. When apelin-36 and its pyroglutamylated C-terminal peptide, [<Glu(65)]apelin-13, were compared in microphysiometric analyses, the elevation of extracellular acidification induced in cells expressing APJ by [<Glu(65)]apelin-13 was transient, whereas that by apelin-36 was sustained. These responses were almost completely inhibited by a specific inhibitor for G(i) or that for Na(+)/H(+) exchanger. (125)I -Labeled [<Glu(65)]apelin-13 analogue specifically bound to APJ with a high affinity, and [<Glu(65)]apelin-13 was more potent than apelin-36 in competitive inhibition assays. Because pretreatment with apelin-36 but not [<Glu(65)]apelin-13 drastically reduced the binding of the labeled apelin to APJ, the different patterns of acidification induced by these two peptides appeared to reflect their dissociation rather than association with APJ. Apelin elicited the migration of APJ-expressing cells, and [<Glu(65)]apelin-13 was more potent than apelin-36 in this activity. Heterogeneous molecular forms of apelin corresponding to apelin-36 and [<Glu(65)]apelin-13 were produced in bovine colostrum. Apelin-36 and [<Glu(65)]apelin-13 might have different functions in vivo and in vitro.

Amino Acid Sequence↗

PubMed2000

Cell type-dependent transactivation or repression of mesoderm-restricted basic helix-loop-helix protein, POD-1/Capsulin.

A family of basic-helix-loop-helix (bHLH) nuclear factors play important roles in controlling cell growth and differentiation as critical regulatory components in transcription. Here we describe molecular characterization of mesoderm-specific bHLH protein, POD-1/Capsulin. Transactivation property of POD-1/Capsulin was analyzed by the Gal4 fusion system in six mammalian cell lines. The results indicated that an activation property was shown in HT1080 and HeLa cells, but a repression activity in HepG2 cells. Mapping analysis for the transactivation and repression activities revealed that the C-terminal domain of POD-1/Capsulin is essential for the transactivation and both the N-terminal and C-terminal domains are contributed to the repression activities. Furthermore, in order to identify possible interactants of the POD-1/Capsulin, we performed yeast two-hybrid screen in a human kidney cDNA library, and identified a class A bHLH protein, ITF-2 as potential heterodimeric partner of the bHLH protein.

3T3 Cells↗

PubMed2000

The regulation of motile activity in fish chromatophores.

Chromatophores, including melanophores, xanthophores, erythrophores, leucophores and iridophores, are responsible for the revelation of integumentary coloration in fish. Recently, blue chromatophores, also called cyanophores, were added to the list of chromatophores. Many of them are also known to possess cellular motility, by which fish are able to change their integumentary hues and patterns, thus enabling them to execute remarkable or subtle chromatic adaptation to environmental hues and patterns, and to cope with various ethological encounters. Such physiological color changes are indeed crucial for them to survive, either by protecting themselves from predators or by increasing their chances of feeding. Sometimes, they are also useful in courtship and mutual communications among individuals of the same species, leading to an increased rate of species survival. Such strategies are realized by complex mechanisms existing in the endocrine and/or nervous systems. Current studies further indicate that some paracrine factors such as endothelins (ETs) are involved in these processes. In this review, the elaborate mechanisms regulating chromatophores in these lovely aquatic animals are described.

Amino Acid Sequence↗

PubMed2000

New neuropeptides containing carboxy-terminal RFamide and their receptor in mammals.

Only a few RFamide peptides have been identified in mammals, although they have been abundantly found in invertebrates. Here we report the identification of a human gene that encodes at least three RFamide-related peptides, hRFRP-1-3. Cells transfected with a seven-transmembrane-domain receptor, OT7T022, specifically respond to synthetic hRFRP-1 and hRFRP-3 but not to hRFRP-2. RFRP and OT7T022 mRNAs are expressed in particular regions of the rat hypothalamus, and intracerebroventricular administration of hRFRP-1 increases prolactin secretion in rats. Our results indicate that a variety of RFamide-related peptides may exist and function in mammals.

Amino Acid Sequence↗

PubMed2000

Mutations in the hepatocyte nuclear factor-4alpha gene in Japanese with non-insulin-dependent diabetes: a nucleotide substitution in the polypyrimidine tract of intron 1b.

Mutations of the hepatocyte nuclear factor 4 alpha (HNF-4alpha) gene have been demonstrated in maturity-onset diabetes of the young (MODY) 1 families. To investigate the possibility that the HNF-4alpha gene contributes to the onset of non-insulin-dependent diabetes mellitus (NIDDM) in Japanese patients, we screened all exons and flanking introns of this gene for mutations in 100 patients with NIDDM diagnosed after 25 years of age. We identified two missense mutations: M49V in exon 1c and T1301 in exon 4; and two nucleotide substitutions in introns: cytosine to thymidine at -5 nt in intron 1b and adenine to thymidine at -21 nt in intron 5. We screened an additional 220 diabetic subjects for the polymorphism in intron 1b. The c/t substitution in intron 1b was associated with NIDDM. This substitution in the polypyrimidine tract, an important cis-acting element directing intron removal, is likely to influence pre-mRNA splicing of this gene. T1301 in exon 4 was observed in only two diabetic subjects. This mutation could influence the conformation of this peptide, resulting in changes in ligand binding domain function. M49V in exon 1c was found in both diabetic and non-diabetic subjects; isoforms HNF-4alpha 4, 5, and 6 with this mutation may impair glucose metabolism in tissue. In contrast to the primary cause of nonsense and missense mutations of the HNF-4alpha gene in MODY1, the nucleotide substitution in intron 1b may partially contribute to development of NIDDM in combination with other genetic and environmental factors.

Aged↗

PubMed2000

[Clinical evaluation of commercial serological test for Bartonella infection].

We evaluated the usefulness of a serological diagnostic kit (Bartonella IFA IgG, IgM; MRL Diagnostics) for Bartonella henselae infection. Of the 110 healthy individuals, 107 (97.3%) were with titers being less than 1:64 for IgG antibody to B. henselae, 2 were with titers being 1:64 and 1 with 1:128, IgM antibody to B. henselae was negative in all individuals. Serological diagnosis of cat scratch disease (CSD) using indirect fluorescence antibody (IFA) methods (in-house and diagnostic kit) was made in either elevated titers of IgM (> or = 1:20) or IgG (> or = 1:256) antibodies, or a four-fold rise in IgG titer between acute and convalescent sera. Of the 18 individuals with serological diagnosis of CSD by in-house IFA method in 26 CSD clinical diagnosed patients, 15 (83%) were compatible with the results of the diagnostic kit, whereas 3 (17%) were not compatible. Of the 8 without serological diagnosis, 1 (13%) was serologically diagnosed as CSD, and the others were negative. Overall, the serological diagnosis was made in 16 of 26 (62%). The specificity and sensitivity of this kit were 100% and 62%, respectively. The cross-reaction between B. henselae and Bartonella quintana was observed in sera from controls and patients. Our results show that the diagnostic kit as well as in-house method is an useful tool for the serological diagnosis of cat scratch disease.

Adolescent↗

PubMed2000

Neutralization assay for human group C rotaviruses using a reverse passive hemagglutination test for endpoint determination.

A novel neutralization assay for human group C rotavirus (CHRV) was developed by using a reverse passive hemagglutination (RPHA) test for endpoint determination. In this assay, the neutralization (N)-RPHA test, serial twofold dilutions of sera were mixed with a solution of CHRV that yielded an RPHA test titer of 8 at 3 days after infection. The mixtures were incubated at 37 degrees C for 1 h and were inoculated onto CaCo-2 cell monolayers in a 96-well microplate. Maintenance medium containing 100 microgram of pancreatin per ml was placed in each well. The plate was sealed with sticky plastic film and was incubated at 37 degrees C for 3 days under continuous rotation. Then, the RPHA test titer of each well was determined. The neutralization titer was expressed as the reciprocal of the maximum dilution of the serum that exhibited a fourfold (75%) or greater reduction in the RPHA test titer (8 to 2 or less). Seroconversion of neutralizing antibody was demonstrated by this method in four sets of paired serum specimens from patients with diarrheal disease caused by CHRV. The seroprevalence of CHRV in the general population in Okayama Prefecture was 26.8% by immunofluorescence and 25.5% by the N-RPHA test. The N-RPHA test described here is the first system used to assay for a neutralization antibody against CHRV and is applicable in both clinical and epidemiological settings.

Adolescent↗

PubMed2000

Analyses for susceptibility of rat anterior pituitary cells to prolactin-releasing peptide.

We validated the effect of prolactin-releasing peptide (PrRP) on prolactin (PRL) secretion from rat anterior pituitary cells in in vitro culture. We found that culture conditions considerably influenced the response of the anterior pituitary cells to PrRP. Longer culture term (4 d) was required to obtain better responses of the anterior pituitary cells to PrRP in comparison to thyrotropin-releasing hormone (TRH). Under the culture conditions employed here, PrRP was comparable to TRH in the potency promoting PRL secretion, and the action of PrRP was very specific for PRL secretion. The susceptibility of the anterior pituitary cells to PrRP varied in female rats depending on the process of reproduction: the cells prepared from lactating rats were the most sensitive to PrRP compared with those from random-cycle and pregnant rats. Because the expression levels of PrRP receptor mRNA in the pituitary varied during the reproductive process, we speculated that the susceptibility of the anterior pituitary cells would reflect cellular changes including the expression level of PrRP receptors. In addition, treatment with estrogen in vivo enhanced the susceptibility of the cultured anterior pituitary cells in male rats. Our results indicate that the susceptibility of the rat anterior pituitary cells to PrRP is regulated by physiological mechanisms.

Animals↗

PubMed2000

Proliferation of immature myeloma cells by interleukin-6 is associated with CD45 expression in human multiple myeloma.

Multiple myeloma (MM) is a hematologic malignancy of human plasma cells, and myeloma cells can be classified into several subpopulations according to phenotypic differences, such as CD38 MPC-1- CD49e- immature, CD38 MPC-1+ CD49e- intermediate and CD38 MPC-1+ CD49e+ mature myeloma cells. The expression of the CD45 molecule on myeloma cells is quite variable, and the physiological consequence of CD45 on myeloma cells is still unknown. Recently, we have found that a few MPC-1- immature myeloma cells express CD45 antigens while most myeloma cells do not express the CD45. MPC-1- CD45+ CD49e- but not MPC-1- CD45- CD49e- immature cells contain proliferating cells in response to interleukin-6 (IL-6). IL-6 can also induce expression of CD45 on the MPC-1- CD45- subpopulation of immature myeloma cells. In addition, myeloma cell lines responding to IL-6 express CD45, whereas cell lines proliferating independent of IL-6 do not express CD45. In the U266 cell line, IL-6 leads to the induction of CD45 expression and cell proliferation, indicating that IL-6-induced effects are closely linked to CD45 expression. Thus, there is a heterogeneity in human myeloma cells, and among these subpopulations immature myeloma cells expressing the CD45 molecules appear to proliferate in response to IL-6. In this review we propose the involvement of CD45 in MM pathogenesis, and the possible implications of CD45 as both a phenotypic marker and a functional molecule is discussed.

Antigens, Neoplasm↗

PubMed2000

Simplification for measuring input function of FDG PET: investigation of 1-point blood sampling method.

UNLABELLED: The current method for quantitative FDG PET study requires application of multiple arterial blood sampling for measuring the input function, but the procedure is invasive and complicated. The purpose of this study was to establish a 1-point blood sampling technique that gives data comparable with the data of more elaborate serial arterial sampling. METHODS: We established a time point for 1-point arterial sampling that exhibited the highest correlation between plasma radioactivity at the time point and the real integrated value (IV) of the measured input function obtained by multiple arterial sampling in 120 patients and the smallest coefficient of variation of the real IV divided by plasma radioactivity at the time point in 120 patients. Scaling factors for estimation at each sampling point were determined, and a reference table was established to make the supposed input function. RESULTS: The optimal time for 1-point arterial sampling was 12 min after FDG injection. A good correlation was observed between the real IVs and those estimated from 1-point arterial blood sampling at 12 min using the supposed input function (n = 120; P < 0.001). The time point at which the difference between values of arterial and venous blood disappeared was 40 min after FDG injection. The percentage errors of IV estimation by 1-point sampling were 1.70% (n = 120) for arterial blood at 12 min and 3.64% (n = 10) for venous blood at 40 min. CONCLUSION: We conclude that the simplified 1-point sample method works in a manner that is comparable with serial arterial sampling and should be useful for clinical PET.

Adolescent↗

PubMed2000

Visualization of myocardial phosphoinositide turnover with 1-[1-(11)C]-butyryl-2-palmitoyl-rac-glycerol in rats with myocardial infarction.

UNLABELLED: Phosphoinositide turnover mediates the signaling of angiotensin II, which plays a pivotal role in ventricular remodeling after myocardial infarction (MI). We tested the hypothesis that phosphoinositide turnover can be visualized by 1-[1 -(11)C]butyryl-2-palmitoyl-rac-glycerol (11C-DAG) in both infarcted and noninfarcted myocardium after MI in rats. METHODS: Rats received an injection of 11C-DAG 7 d after left coronary artery ligation, and myocardial lipids were extracted from both infarcted and noninfarcted areas of myocardium (n = 3). Metabolites of 11C-DAG were determined by thin-layer chromatography. Quantitative autoradiography of hearts was performed to visualize myocardial phosphoinositide turnover in rats that received an injection of 11C-DAG 1 d (n = 3) and 7 d (n = 5) after MI and 7 d after a sham operation (n = 3). Quantitative autoradiography with 201TlCl was also performed to evaluate myocardial blood flow in rats 7 d after MI (n = 3). Cells occupying the infarcted myocardium were identified by immunohistochemistry. RESULTS: The radioactivity incorporated into the intermediates of phosphoinositide turnover was predominant in both the infarcted (67.1% +/- 5.2% of the total activity) and the noninfarcted (57.4% +/- 3.2%) myocardium. 11C-DAG radioactivity in the infarcted region normalized to that in the noninfarcted region was 1.09 +/- 0.04 in rats 7 d after MI, which was significantly higher than that in rats 1 d after MI (0.38 +/- 0.03, P < 0.001). 201Tl radioactivity in the infarcted region normalized to that in the noninfarcted region was only 0.19 +/- 0.01 7 d after MI. 11C-DAG radioactivity in the noninfarcted region normalized to that in the right ventricular free wall tended to be increased in rats 1 and 7 d after MI compared with the sham-operated rats; the differences, however, were not statistically significant (1.30 +/- 0.15, 1.20 +/- 0.07, and 1.13 +/- 0.02, respectively). Immunohistochemistry revealed that abundant fibroblasts, myofibroblasts, and macrophages occupied the infarcted myocardium 7 d after MI, but the cellularity was low during the first day after MI. CONCLUSION: These data suggest that 11C-DAG may be useful for visualizing regions with activated phosphoinositide turnover after MI. Because wound healing and fibrogenic processes are important factors of ventricular remodeling, 11C-DAG and PET may offer new information benefiting patient management after MI.

Animals↗

PubMed1999

Hypernuclear acetylation in atherosclerotic lesions and activated vascular smooth muscle cells.

Recent studies have implicated acetylation of several nuclear proteins such as histones and p53 on their epsilon-portion of lysine residues in eukaryotic transcription. Here we raised a specific polyclonal antibody against epsilon-acetylated lysine. Using the antibody, we detected hypernuclear acetylation (HNA) in atherosclerotic vascular smooth muscle cells (VSMCs). Thrombin, a humoral factor known to cause activation and proliferation of VSMCs, strongly potentiated HNA in cultured VSMCs. MAP kinase pathway and a signal coactivator CREB binding protein (CBP) were involved in thrombin-induced HNA of VSMCs. Our results suggest that coactivators cooperating with signal-dependent transcription activators play an important role in atherosclerogenesis via HNA in VSMCs.

Acetylation↗

PubMed1999

Enforced CD19 expression leads to growth inhibition and reduced tumorigenicity.

In multiple myeloma (MM), the cell surface protein, CD19, is specifically lost while it continues to be expressed on normal plasma cells. To examine the biological significance of loss of CD19 in human myeloma, we have generated CD19 transfectants of a tumorigenic human myeloma cell line (KMS-5). The CD19 transfectants showed slower growth rate in vitro than that of control transfectants. They also showed a lower capability for colony formation as evaluated by anchorage-independent growth in soft agar assay. The CD19 transfectants also had reduced tumorigenicity in vivo when subcutaneously implanted into severe combined immunodeficiency (SCID)-human interleukin-6 (hIL-6) transgenic mice. The growth-inhibitory effect was CD19-specific and probably due to CD19 signaling because this effect was not observed in cells transfected with a truncated form of CD19 that lacks the cytoplasmic signaling domain. The in vitro growth-inhibitory effect was confirmed in a nontumorigenic human myeloma cell line (U-266). However, introduction of the CD19 gene into a human erythroleukemia cell line (K-562) also induced growth inhibition, suggesting that this effect is CD19-specific, but not restricted to myeloma cells. These data suggest that the specific and generalized loss of CD19 in human myeloma cells could be an important factor contributing to the proliferation of the malignant plasma cell clones in this disease.

Antigens, CD19↗

PubMed1999

Apelin, the natural ligand of the orphan receptor APJ, is abundantly secreted in the colostrum.

By using a strategy that we have developed to search for the ligands of orphan seven-transmembrane-domain receptors [S. Hinuma et al., Nature 393 (1998) 272-276], we have recently identified a natural ligand, apelin, for the orphan 7TMR, APJ [K. Tatemoto et al., Biochem. Biophys. Res. Commun. 251 (1998) 471-476]. In this paper, we isolated rat and mouse apelin cDNAs, and analyzed the tissue distribution of apelin mRNA in rats. Although apelin mRNA was widely detected in a variety of tissues, the highest expression of apelin mRNA was detected in the mammary gland of pregnant rats. In the mammary gland, biologically active apelin and its mRNA considerably increased during pregnancy and lactation, and reached a maximal level around parturition. Moreover, a large amount of apelin (14-93 pmol/ml) was found to be secreted in the bovine colostrum, and it was still detectable even in commercial bovine milk. Since apelin partially suppressed cytokine production by mouse spleen cells in response to T cell receptor/CD3 cross-linking, the oral intake of apelin in the colostrum and milk might modulate immune responses in neonates.

Adipokines↗

PubMed1999

Visualization of mRNA expression in CNS using 11C-labeled phosphorothioate oligodeoxynucleotide.

Antisense phosphorothioate oligodeoxynucleotide for mRNA of glial fibrillary acidic protein (GFAP) was labeled with the positron emitter 11C and administered i.v. to rats bearing glioma, which were expected to exhibit active expression of GFAP. Antisense oligodeoxynucleotide was retained in tumor cells, yielding clear images of tumors, while the control 20% mismatch oligodeoxynucleotide and sense-strand oligodeoxynucleotide were not retained in tumor cells. Findings revealed sequence-specific binding of the antisense oligodeoxynucleotide to the GFAP mRNA. Our methods can be used directly for non-invasive imaging of human gene expression using PET, a frequently used method of clinical examination.

Animals↗

PubMed1999

Tissue distribution of prolactin-releasing peptide (PrRP) and its receptor.

Prolactin-releasing peptide (PrRP) is a novel bioactive peptide, originally isolated from bovine hypothalamus by utilizing an orphan seven-transmembrane-domain receptor expressed in the human pituitary gland. In this paper, we analyzed the tissue distribution of rat and human PrRP and their receptor mRNAs by quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Northern blotting. In RT-PCR analysis, rat PrRP receptor mRNA was detected in the central nervous system, and the highest expression was detected in the pituitary gland. In addition, in situ hybridization revealed that rat PrRP receptor mRNA was highly expressed in the anterior lobe of the pituitary. On the other hand, rat PrRP mRNA was most abundantly expressed in the medulla oblongata, while significant levels of expression were widely detected in other tissues. In Northern blot analyses, human PrRP receptor mRNA was detected only in the pituitary gland among tissues examined. Human PrRP mRNA was detected in the medulla oblongata and in the pancreas. In contrast to the pattern of mRNA expression, the highest content of bioactive PrRP was found in the hypothalamus rather than the medulla oblongata in the rat brain, indicating that PrRP mRNA does not always parallel with mature PrRP in tissue distribution. The wide distribution of PrRP and its receptor suggests that they have various functions not only in the pituitary gland but also in the other tissues.

Amino Acid Sequence↗

PubMed1999

Aberrant plasticity in Alzheimer's disease.

Alzheimer's disease (AD) is a region-specific degenerative disease that mainly impairs the temporal and parietal lobes, with preservation of the frontal lobes until advanced stages of disease. Since [11C]diacylglycerol PET facilitates examination of loci exhibiting plasticity, it was performed in eight patients with AD and six age-matched normal control subjects to evaluate frontal lobe function. [11C]Diacylglycerol tomograms obtained from patients with AD demonstrated strong spotty incorporation of [11C]diacylglycerol mainly in the frontal association areas. However, [18F]fluorodeoxyglucose tomograms exhibited region-specific findings such as decreased CMRGIc in the parietotemporal association areas, which are involved in impairment of cognitive function. The strong spotty incorporation of [11C]diacylglycerol suggested a compensatory plastic process in the frontal lobes in response to involvement by Alzheimer's disease of the posterior association areas.

Adult↗