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Biomedical subjects

M Funakoshi

Publications and source records attributed to M Funakoshi.

At least 19 recordsLinked to original sources
PubMed2001

A MEK inhibitor, PD98059 enhances IL-1-induced NF-kappaB activation by the enhanced and sustained degradation of IkappaBalpha.

Interleukin-1 (IL-1) mediates numerous host responses through rapid activation of nuclear factor-kappaB (NF-kappaB), but signal pathways leading to the NF-kappaB activation appear to be complicated and multiplex. We propose a novel regulatory system for NF-kappaB activation by the extracellular signal-related kinase (ERK) pathway. In a human glioblastoma cell line, T98G, IL-1-induced NF-kappaB activation was significantly augmented by the pretreatment of a specific MEK inhibitor, PD98059. In contrast, ectopic expression of a constitutive activated form of Raf (v-Raf) reduced IL-1-induced NF-kappaB activation, and this inhibition was completely reversed by PD98059. Interestingly, PD98059 sustained IL-1-induced NF-kappaB DNA binding activity by an electrophoretic mobility shift assay and also IkappaBalpha degradation, presumably by augmenting and sustaining the proteasome activation. Concomitantly, two NF-kappaB dependent genes, A20 and IkappaBalpha expression were prolonged with PD98059. These data suggested that MEK-ERK pathway exerts a regulatory effect on NF-kappaB activation, providing a novel insight on the role of MEK-ERK pathway.

Binding, Competitive↗
PubMed2001

The N-terminal helix of Xenopus cyclins A and B contributes to binding specificity of the cyclin-CDK complex.

Mitotic cyclins A and B contain a conserved N-terminal helix upstream of the cyclin box fold that contributes to a significant interface between cyclin and cyclin-dependent kinase (CDK). To address its contribution on cyclin-CDK interaction, we have constructed mutants in conserved residues of the N-terminal helix of Xenopus cyclins B2 and A1. The mutants showed altered binding affinities to Cdc2 and/or Cdk2. We also screened for mutations in the C-terminal lobe of CDK that exhibited different binding affinities for the cyclin-CDK complex. These mutations were at residues that interact with the cyclin N-terminal helix motif. The cyclin N-terminal helix mutations have a significant effect on the interaction between the cyclin-CDK complex and specific substrates, Xenopus Cdc6 and Cdc25C. These results suggest that the N-terminal helix of mitotic cyclins is required for specific interactions with CDKs and that to interact with CDK, specific substrates Cdc6 and Cdc25C require the CDK to be associated with a cyclin. The interaction between the cyclin N-terminal helix and the CDK C-terminal lobe may contribute to binding specificity of the cyclin-CDK complex.

Amino Acid Sequence↗
PubMed2001

Differential involvement of p38 mitogen-activated protein kinase and phosphatidyl inositol 3-kinase in the IL-1-mediated NF-kappa B and AP-1 activation.

Interleukin-1 (IL-1) is a central regulator of the immune and inflammatory responses by which various inflammatory genes are induced. Although IL-1 signaling is known to involve PI3-kinase, p38 mitogen-activated protein (MAP) kinase and extracellular signal-regulated kinase (ERK), the crosstalk of these kinases on the IL-1-mediated signal transduction is not clear. We used two specific inhibitors, SB203580 which selectively inhibits p38 MAP kinase and LY294002 which inhibits PI3-kinase, respectively, to explore the involvement of these kinases in the IL-1-induced NF-kappa B activation, using a human glioblastoma cell line, T98G. Two kinase inhibitors decreased IL-1-induced IL-8 mRNA and protein levels markedly. IL-1 caused phosphorylation of p38 MAP kinase with concomitant recruitment of PI3-kinase to IL-1 receptor I (IL-1RI) and its activation. In this context, pretreatment of LY294002, but not SB203580, inhibited IL-1-induced NF-kappa B activation significantly. While IL-1 induced-AP-1 activation was moderate, both LY294002 and SB203580 suppressed IL-1-induced AP-1 activation. These observations were prominent particularly in the TRAF6 transfection system, in which overexpression of wild type TRAF6 augmented the IL-1 mediated NF-kappa B and AP-1 activation, while dominant negative TRAF6 construct (delta TRAF6) suppressed these activation. Namely, LY294002 inhibited TRAF6-mediated IL-1-induced NF-kappa B and AP-1 activation markedly, while SB203580 inhibited TRAF6-induced AP-1 activation but not NF-kappa B activation. Above results indicated that both PI3-kinase and p38 MAP kinase are differentially involved in IL-1-induced NF-kappa B and AP-1 activation.

Chromones↗
PubMed2001

Secretion of inhibin A and inhibin B throughout pregnancy and the early postpartum period in chimpanzees.

Plasma concentrations of inhibin A and inhibin B during pregnancy and early lactation in chimpanzees were determined by enzyme-linked immunosorbent assay (ELISA). Plasma samples were taken from five pregnant chimpanzees at 6-9, 10, 20 and 25 weeks of pregnancy, and following parturition. Throughout pregnancy and the early postpartum period, circulating inhibin A and inhibin B concentrations remained low, at similar levels to those during the normal menstrual cycle in chimpanzees. Concentrations of inhibin A in the placental homogenate were high enough to be measured by the ELISA and by bioassay, whereas circulating inhibin bioactivities in late pregnancy were too low to be measured. Plasma concentrations of FSH remained low with no significant changes throughout pregnancy and the postpartum period. Plasma concentrations of oestradiol-17beta and progesterone at 25 weeks of pregnancy were much higher than normal menstrual cycle levels. It was concluded that in chimpanzees the levels of circulating inhibin A and inhibin B remained low throughout pregnancy and the early postpartum period, and that the concentrations of bioactive dimeric inhibin did not increase towards the end of pregnancy. The suppression of circulating FSH levels during pregnancy is suggested to be controlled by steroid hormones that increased significantly in late pregnancy, and the present findings further suggest that the secretory pattern and role of inhibin during pregnancy in chimpanzees may be different from that in human and other primates.

Animals↗
PubMed2000

Anti-apoptotic role of focal adhesion kinase (FAK). Induction of inhibitor-of-apoptosis proteins and apoptosis suppression by the overexpression of FAK in a human leukemic cell line, HL-60.

Focal adhesion kinase (FAK) has an anti-apoptotic role in anchorage-dependent cells via an unknown mechanism. To elucidate the role of FAK in anti-apoptosis, we have established several FAK cDNA-transfected HL-60 cell lines and examined whether FAK-transfected cells have resistance to apoptotic stimuli. FAK-transfected HL-60 (HL-60/FAK) cells were highly resistant to apoptosis induced with hydrogen peroxide (1 mm) and etoposide (50 microg/ml) compared with the parental HL-60 cells or the vector-transfected cells, when determined using viability assay, DNA fragmentation, and flow cytometry analysis. Because no proteolytic cleavage of pro-caspase 3 to mature caspase 3 fragment was observed in HL-60/FAK cells, FAK was presumed to inhibit an upstream signal pathway leading to the activation of caspase 3. HL-60/FAK activated the phosphatidylinositide 3'-OH-kinase-Akt survival pathway and exhibited significant activation of NF-kappaB with marked induction of inhibitor-of-apoptosis proteins (IAPs: cIAP-1, cIAP-2, XIAP), regardless of the hydrogen peroxide-treated or untreated conditions, whereas no significant IAPs were detected in the parental or vector-transfected HL-60 cells. Apoptotic agents induced higher NF-kappaB activation in HL-60/FAK cells than in HL-60/Vect cells, and it appeared that sustained NF-kappaB activation is critical to the anti-apoptotic states in HL-60/FAK cells. Mutagenesis of FAK cDNA revealed that Y397 and Y925, which are involved in the tyrosine-phosphorylation sites, were prerequisite for the anti-apoptotic activity as well as induction of IAPs, and that K454, which is involved in the kinase activity, was also required for the full anti-apoptotic activity of FAK. Taken together, we have demonstrated definitively that FAK-transfected HL-60 cells, otherwise sensitive to apoptosis, become resistant to the apoptotic stimuli. We conclude that FAK activates the phosphatidylinositide 3'-OH-kinase-Akt survival pathway with the concomitant activation of NF-kB and induction of IAPs, which ultimately inhibit apoptosis by inhibiting caspase-3 cascade.

Apoptosis↗
PubMed2000

Differential induction of JE/MCP-1 in subclones from a murine macrophage cell line, RAW 264.7: role of kappaB-3 binding protein.

The JE/MCP-1 gene is an immediate-early gene, and its product is a CC chemokine that attracts monocytes, basophils and T lymphocytes. JE/MCP-1 gene expression is induced by various inflammatory stimuli, but its transcriptional mechanism is not fully understood. To address this question, we obtained two subclones from a parental RAW264.7 cell line, one subline with low JE/MCP-1-producing capacity (named RAW.c11) and the other with high JE/MCP-1-producing capacity (named RAW.c25), in response to lipopolysaccharide (LPS). These subclones have no significant differences in CD14 expression, nitric oxide production, or production of other cytokines, including TNF-alpha or IL-1alpha/beta. In electrophoretic mobility shift assays (EMSA), there were no significant differences in DNA binding to the NF-kappaB-consensus sequence and interferon regulatory factor (IRF)-1,2 binding sequences. However, significantly higher binding activity to the NF-kappaB-like sequence (kappaB-3), which is located in the promoter region of the JE/MCP-1 gene, was shown by a high producer subclone than by a low producer subclone. Transient transfection analysis using deletion mutants of a 0.5-kb region from -467 to +59 identified an LPS-responsive region in a kappaB-3 site (from -169 to -132) in the high producer subclone. Mutation of this site markedly reduced sensitivity to LPS in the high producer subclone. These data suggest that a yet undefined nuclear factor may be involved in differential JE/MCP-1 gene transcription.

Animals↗
PubMed2000

Isolation and characterisation of a mutation in the PMR1 gene encoding a Golgi membrane ATPase, which causes hypersensitivity to over-expression of Clb3 in Saccharomyces cerevisiae.

We screened for mutant strains of Saccharomyces cerevisiae that are sensitive to overexpression of specific cyclins, and identified mutations in two genes that caused growth inhibition in response to mild overexpression of Clb3. One was the ANP1 gene, which encodes a glycosyltransferase previously identified by a similar strategy using Clb2 instead of Clb3. This paper describes the second strain of S. cerevisiae that is hypersensitive to Clb3 expression. The gene mutated in this strain was identified as PMR1, which encodes a Ca2+-ATPase located in the Golgi membrane. The protein product of pmr1-1 was truncated at residue 409 and thus lacked the C-terminal ATPase domain. The pmr1-1 strain was hypersensitive to over-expression of Clb3, but not Cln2, Clb5 or Clb2. The lethality due to Clb3 expression in pmr1-1 could be suppressed by adding Ca2+ ions to the medium. The pmr1-1 strain proved to be defective in glycosylation, and the defects in glycosylation were exacerbated by high levels of Clb3. On induction of Clb3 expression in the pmr1-1 strain, the cells arrested at anaphase with an elongated daughter bud. We discuss possible interpretations of this synthetic lethal phenotype.

Calcium↗
PubMed2000

Changes in plasma concentrations of inhibin A and inhibin B throughout sexual maturation in the male chimpanzee.

Profiles of circulating plasma inhibin A and inhibin B during sexual maturation in male chimpanzees were investigated by using two-site enzyme-linked immunoassay (ELISA). Plasma concentrations of testosterone and pituitary gonadotropins were also measured. Concentrations of inhibin B, testosterone, luteinizing hormone (LH) and prolactin increased with age throughout prepuberty to adulthood, whereas inhibin A level was low and there were no age-related changes in concentrations of either inhibin A and follicle-stimulating hormone (FSH). Inhibin B showed an inverse correlation with FSH in adult (7 years or order) but not in immature (6 years or younger) male chimpanzees. There was no correlation between plasma levels of FSH and testosterone throughout the period of sexual maturation. However, testosterone levels were positively correlated with inhibin B levels. These results suggest that circulating inhibin B is involved in the regulation of FSH secretion after puberty in adult male chimpanzees, and also that circulating inhibin B is an important form of inhibin as a marker of Sertoli cell function in adult male chimpanzees.

Aging↗
PubMed1999

Identification of XDRP1; a Xenopus protein related to yeast Dsk2p binds to the N-terminus of cyclin A and inhibits its degradation.

Using the N-terminus of cyclin A1 in a two-hybrid screen as a bait, we identified a Xenopus protein, XDRP1, that contains a ubiquitin-like domain in its N-terminus and shows significant homology in its C-terminal 50 residues to Saccharomyces cerevisiae Dsk2 and Schizosaccharomyces pombe dph1. XDRP1 is a nuclear phosphoprotein in Xenopus cells, and its phosphorylation is mediated by cyclin A-dependent kinase. XDRP1 binds to both embryonic and somatic forms of cyclin A (A1 and A2) in Xenopus cells, but not to B-type cyclins. The N-terminal ubiquitin-like domain of XDRP1, but not the C-terminal Dsk2-like domain, is required for interaction with cyclin A. XDRP1 requires residues 130-160 of cyclin A1 for efficient binding, which do not include the destruction box of cyclin A. The addition of bacterially expressed XDRP1 protein to frog egg extract inhibited the Ca(2+)-induced degradation of cyclin A, but not that of cyclin B. The injection of XDRP1 protein into fertilized Xenopus eggs blocked embryonic cell division.

Amino Acid Sequence↗
PubMed1999

Emerin and cardiomyopathy in Emery-Dreifuss muscular dystrophy.

Emery-Dreifuss muscular dystrophy (EDMD) is an inherited disorder characterized by the clinical triad of life-threatening progressive cardiomyopathy with conduction defect, early onset joint contractures and slow progressive muscle weakness in scapulo-humero-peroneal distribution. Cardiomyopathy in EDMD is usually noticed after the second to third decade of life, and becomes worse with age. Permanent auricular paralysis occurs frequently and is considered a hallmark of EDMD cardiomyopathy. Cardiac involvement may also occur in female carriers. In autopsy cases, enlargement of the atria with remarkable thinning have been observed. Identification of the gene responsible for X-linked EDMD (X-EDMD) and the protein product, emerin, provided a diagnostic clue for EDMD. Since the emerin gene is rather small, the entire sequence can easily be surveyed. Western blot and immunohistochemistry show an absence of emerin in muscle and skin tissues and oral exfoliating cells in male patients with X-EDMD, and a reduction of the protein content with a mosaic expression pattern in female carriers. Emerin anchors at the inner nuclear membrane of cardiac, skeletal and smooth muscles, and interacts with lamins and nucleoplasm, thereby possibly maintaining the mechanical stability of the nuclear membrane of muscle cells that shows rigorous contraction/relaxation. More recently, positive emerin staining at the cardiac demosomes and fasciae adherentes was noticed in addition to the specific localization at the inner nuclear membrane. This localization implies a physiological role for the protein in cardiac conduction.

Cardiomyopathies↗
PubMed1999

Maize genes specifically expressed in the outermost cells of root cap.

The cells at the periphery of the root cap are continuously sloughed off from the root into the mucilage, and are thought to be programmed to die. By using subtractive hybridization/differential screening and a transmembrane-domain trapping screening strategy involving expression screening in transfected COS-7 cells, we isolated two related maize cDNAs (ZmRCP1 and ZmRCP2) that are specifically expressed in the outermost one to three cells of the cap. ZmRCP1 and ZmRCP2 are homologous proteins of 37 kDa mature polypeptides with a region of regularly-spaced Cys residues and putative N-terminal signal peptides, and represent members of a novel protein family which is conserved among angiosperm and gymnosperm.

Amino Acid Sequence↗
PubMed1998

Epilepsy and mental retardation in a subset of early onset 4q35-facioscapulohumeral muscular dystrophy.

The gene for facioscapulohumeral muscular dystrophy (FSHD) has been mapped to chromosome 4q35. In most patients with FSHD, a deletion of 3.3 kb tandemly repeated units within the EcoRI fragment that can be detected by probe p13E-11 is associated with the disease. To elucidate the relation between the phenotype and the genotype in FSHD, we examined 91 Japanese unrelated families with a clinical diagnosis of FSHD (140 patients, 205 healthy individuals). Of these, 78 families (86%) had 4q35-FSHD (127 patients), in which 20 patients (20/127, 16%) were classified as early onset FSHD. We found that all nine patients with the smallest EcoRI fragments (10 to 11 kb) were classified among the early onset group (9/20, 45%), and these patients showed a high frequency of both epilepsy (4/9, 44%) and mental retardation (8/9, 89%). In contrast, none of the remaining patients with 4q35-FSHD had epilepsy or mental retardation. We conclude that FSHD patients with a large gene deletion in the FSHD gene region tend to have a higher chance of showing severe clinical phenotypes with CNS abnormalities. This finding may have practical implications for genetic counseling, although the molecular genetic background remains unclear.

Adolescent↗
PubMed1997

FMLP actions and its binding sites in isolated human coronary arteries.

The chemoattractant f-Met-Leu-Phe (FMLP) can modulate human coronary arterial tone without the involvement of peripheral leukocytes. We investigated the actions of FMLP and its cellular mechanism in human coronary arteries isolated 2-3 h after death. A single dose of FMLP (0.01-10 microM) produced transient contraction (or, followed by relaxation) responses in most human coronary rings examined. These responses to FMLP were in large part mediated by the generation of cyclooxygenase products, mainly thromboxane A2 (TXA2) and prostaglandin I2 (PGI2). Radiolabeled N-formyl hexapeptide. 125I-f-Nle-Leu-Phe-Nle-Tyr-Lys bound densely to intimal and adventitial sites that accumulated macrophages (CD68-positive) with a Kd of 14-29 nM and, further, weakly to the media with a Kd of 2.4-3.6 microM. Several cell types including macrophages, endothelial cells and smooth muscle cells were positively immunostained for both TXA2 synthase and PGI2 synthase. However, there was no significant relation between the magnitude of the responses to FMLP and dense macrophage accumulation in the intimal plaques or the adventitia. A reverse transcription-polymerase chain reaction showed predominant expression of FMLP receptor homologues, FPRH1 and FPRH2 mRNA, in human coronary medial tissues relative to that in leukocytes. In conclusion. FMLP produced transient tension changes in human coronary arteries, mainly via the generation of TXA2 and PGI2. This effect of FMLP did not appear to be mediated by the activation of densely accumulated intimal and/or adventitial macrophages, but by the activation of unidentified medial tissue cells which might have functional FMLP receptor homologues.

Adolescent↗
PubMed1997

Effect of mastication on regional cerebral blood flow in humans examined by positron-emission tomography with ¹⁵O-labelled water and magnetic resonance imaging.

The interaction between mastication and cerebral blood flow was studied in 12 healthy volunteers (five males and seven females) aged 18-40 years. Positron-emission tomography (PET) autoradiography was carried out after bolus injection of 1.5 GBq H₂¹⁵O (¹⁵O-labelled water) with a half life of 2 min. The PET images were superimposed on magnetic resonance images of each participant. The regional cerebral blood flow images were normalized by the global cerebral blood flow value, and subtraction images (those during gum-chewing minus those during resting) were created and recut at the magnetic resonance image slice positions. Gum specially designed for chewing training was used. Mastication increased regional cerebral blood flow in the primary sensorimotor areas by 25-28%, in the supplementary motor areas and insulae by 9-17%, and in the cerebellum and striatum by 8-11%. These increases demonstrate that chewing activates widespread regions of the brain.

Adolescent↗
PubMed1997

Xenopus cyclin A1 can associate with Cdc28 in budding yeast, causing cell-cycle arrest with an abnormal distribution of nuclear DNA.

BACKGROUND: Cyclins play a regulatory role in cell cycle progression, associated with cyclin-dependent kinases. We have investigated the structure-function relationships of cyclin A, mainly using Xenopus egg extracts in vitro. To further analyse the function and structure of cyclin A in vivo, we expressed Xenopus cyclin A1 in the budding yeast Saccharomyces cerevisiae. RESULTS: We herein show that vertebrate cyclin A1 can associate with endogenous Cdc28 to form histone H1 kinase. The growth of the yeast was inhibited by the expression of indestructible cyclin A1, but not by a non-Cdk binding cyclin A1 mutant. The induction of cyclin A1 expression in yeast caused cell cycle arrest with an abnormal distribution of nuclear DNA to the daughter bud. Suppressors of the cyclin A1-mediated growth arrest were identified as new alleles of the cdc28 mutation that reduced the binding of cyclin A1 and possessed different affinities for the cyclin-Cdc28 complexes. The temperature-sensitivity of the cdc28 mutation was thus preferentially suppressed by the endogenous cyclins CLN2 and CLB2. CONCLUSIONS: These results suggest that the Cdc28 protein kinase activity mediated by vertebrate cyclin A1 may be involved in the process of nuclear movement in the yeast, and thereby affect the dependence of the M phase on the completion of the S phase through a preferential binding affinity of the cyclin-Cdc28 complex.

Amino Acid Sequence↗
PubMed1997

Role of cytosolic Ca2+ and protein kinase C in developing myogenic contraction in isolated rat small arteries.

Cytosolic Ca2+ and protein kinase C (PKC) may regulate the myogenic contraction of arterial myocytes. The role of these second messengers is examined in skeletal muscle small arteries, which have strong myogenic activity, and mesenteric small arteries, which have weak myogenic activity. The vessels were isolated and cannulated. The inner diameter was measured with a video-digitizing system. Cytosolic Ca2+ concentration was assessed by fura 2. Skeletal muscle small arteries dilated from 122 +/- 6 to 153 +/- 6 microm immediately after the transmural pressure change from 40 to 100 mmHg and constricted to 121 +/- 5 microm (myogenic contraction) with an increase in the 340/380 fluorescence ratio (by approximately 33%) in control vessels. Nifedipine abolished myogenic contraction and the increase in the fluorescence ratio. PKC inhibitors (H7 and staurosporine) abolished myogenic contraction but did not depress the increase in the fluorescence ratio. In mesenteric small arteries, myogenic contraction was insignificant in control vessels. A relatively low dose of PKC activator (4.4 +/- 1.4 nmol/l) elicited myogenic contraction, but a higher dose (21 +/- 6 nmol/l) depressed it. Thus the cytosolic Ca2+ increase and PKC activity may cooperatively act on the myogenic contraction of skeletal muscle small arteries. The activity of PKC should play an important role in myogenic contraction of rat small arteries.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1997

An altered nuclear migration into the daughter bud is induced by the cyclin A1-mediated Cdc28 kinase through an aberrant spindle movement in Saccharomyces cerevisiae.

A strain of Saccharomyces cerevisiae that contains an integrated copy of a Xenopus cyclin A1 gene under the control of the GAL1 promoter has been constructed. On inducing expression of cyclin A1, the nuclear migration that occurs prior to division becomes aberrant. Instead of migrating to the neck between the mother cell and daughter bud, the nucleus, the short mitotic spindle and its associated two spindle pole bodies entered the daughter bud. This phenotype was induced by expression of an indestructible cyclin mutant, but not by a mutated cyclin A1 unable to activate Cdc28 kinase. The nuclear abnormality induced by cyclin A1 was overcome by cdc28 mutations that abolish its ability to bind cyclin A1. Both yeast cyclin Clb3 and Xenopus mitotic cyclin B produced the same phenotype, whereas G1 cyclin Cln2 did not. The results suggest that the proper movement of the nucleus through the spindle function during mitosis requires the appropriate activity of Cdc28 kinase mediated by specific cyclins.

Animals↗