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Biomedical subjects

M Hagiwara

Publications and source records attributed to M Hagiwara.

At least 91 records · Page 5Linked to original sources
PubMed1996

A novel kinase cascade mediated by mitogen-activated protein kinase kinase 6 and MKK3.

A cDNA encoding a novel member of the mitogen-activated protein kinase kinase (MAPKK) family, MAPKK6, was isolated and found to encode a protein of 334 amino acids, with a calculated molecular mass of 37 kDa that is 79% identical to MKK3. MAPKK6 was shown to phosphorylate and specifically activate the p38/MPK2 subgroup of the mitogen-activated protein kinase superfamily and could be demonstrated to be phosphorylated and activated in vitro by TAK1, a recently identified MAPKK kinase. MKK3 was also shown to be a good substrate for TAK1 in vitro. Furthermore, when co-expressed with TAK1 in cells in culture, both MAPKK6 and MKK3 were strongly activated. In addition, co-expression of TAK1 and p38/MPK2 in cells resulted in activation of p38/MPK2. These results indicate the existence of a novel kinase cascade consisting of TAK1, MAPKK6/MKK3, and p38/MPK2.

Amino Acid Sequence↗
PubMed1996

Suppression of CD44 expression decreases migration and invasion of human glioma cells.

We have reported high expression of CD44H in human glioma cells. To investigate the role of CD44H in the invasion of human glioma, we established a CD44-anti-sense-gene-expression glioma cell line named U-251A1. The expression of CD44H in the G-418-selected U-251A1 cells was reduced to 20% of that in the parental U-251SP cells, as determined by flow-cytometry analysis. We first examined the migratory responses of U-251A1 cells in vitro by time-lapse video-microscopic sparse cell-migration assay on hyaluronic acid or on chondroitin 6 sulfate. U-251A1 cells did not show significant differences in motility on any substrate, while U-251SP and other CD44H-positive cells showed dose-dependent increase of migration specifically on hyaluronic acid. To examine the physiologic function of CD44H in gliomas in vivo, U-251A1 and its control cells, U-251S1, which retain CD44-sense-expression vector, were injected stereotactically into the brains of nude mice. U-251A1 cells were localised in the region of the injection site, with relatively well demarcated borders between tumour and brain tissue, while the control cells demonstrated a cell-infiltration pattern. Our data suggest that CD44H may be required for infiltration of glioma cells through its interaction with hyaluronic acid, a major component of the brain extracellular matrix.

Animals↗
PubMed1996

Molecular cloning and characterization of a cDNA encoding a novel basic helix-loop-helix protein structurally related to Neuro-D/BHF1.

We cloned a novel basic helix-loop-helix protein, NDRF (NeuroD-related factor), cDNA. NDRF contains 383 amino acids and exhibits 53.4% and 52.2% sequence identity to NeuroD and MATH-2/NEX-1, respectively. NDRF mRNA appears in the brain of 12-day-old mouse embryos and is localized in certain regions of the adult brain, such as the hippocampus, dentate gyrus and cerebellum, Thus, the structure and expression patterns of NDRF are similar to but distinct from those of NeuroD and MATH-2/NEX-1, suggesting that NDRF may play distinct roles in neural development and plasticity as a novel member of the NeuroD family.

Amino Acid Sequence↗
PubMed1996

Effects of anandamide and arachidonic acid on specific binding of (+) -PN200-110, diltiazem and (-) -desmethoxyverapamil to L-type Ca2+ channel.

We examined the effects of anandamide (N-arachidonoylethanolamine) on the binding of three types of Ca2+ channel antagonists for L-type Ca2+ channel, i.e., 1,4-dihydropyridine, 1,5-benzothiazepine and phenylalkylamine, to rabbit skeletal muscle membranes. Anandamide inhibited the binding of all three ligands. Arachidonic acid, a putative metabolite or a precursor of anandamide, inhibited 1,4-dihydropyridine binding, whereas it augmented both 1,5-benzothiazepine and phenylalkylamine binding. The involvement of prostaglandins synthesized from arachidonic acid was considered to be minor. These findings indicate that both anandamide and arachidonic acid interact not only with 1,4-dihydropyridine but also with 1,5-benzothiazepine and phenylalkylamine binding sites as a common feature of unsaturated lipids.

Animals↗
PubMed1996

Anticoagulant peptides; synthesis, stability and antithrombin activity of hirudin C-terminal-related peptides and their disulfated analog.

We designed a unique anticoagulant decapeptide, which possesses two O-sulfated tyrosine residues, based on the structure of hirudin's C-terminal functional domain. We first prepared a series of octa-, nona- and decapeptides with no sulfation, Suc-Phe-Glu-Pro-Ile-Pro-Glu-Tyr-Tyr-X-OH [X = bond, Leu or Leu-Gln], by a solution phase method and measured their thrombin times (TT) using human thrombin and rabbit plasma. The shortest octapeptide (3a) showed full antithrombin activity comparable to that of the lead compound hirudin (54-65), and the longest decapeptide (3c) prolonged TT most potently with an IC50 value of 5.8 microM. We consequently converted 3c to a disulfated decapeptide (NF-22) with SO3.pyridine complex and compared its antithrombin activity with that of known hirudin-related peptides: hirugen, MDL28050 and hirulog-1. NF-22 showed potent antithrombin activity with an IC50 value of 0.3 microM, being more potent than hirugen and MDL28050 (IC50 values of 4.0 microM and 1.1 microM, respectively). NF-22 was as potent as hirulog-1. NF-22 showed no change in activity in aqueous solution for 10 d at 60 degrees C, and remained about 90% unchanged in rat plasma on incubation for 24h at 37 degrees C, whereas the corresponding unsulfated peptide (3c) was completely digested under the same condition. NF-22 appears to be one of the most potent and stable peptide anticoagulants among the hirudin analogs.

Amino Acid Sequence↗
PubMed1996

[Transcriptional regulation by cAMP and calcium].

Transcription of a number of eukaryotic genes is activated in response to an increase in the intracellular cAMP concentration. These genes stimulated by cAMP have a common promoter element, cAMP response element (CRE). The CRE is recognized by a CRE binding protein, CREB. The binding of CREB to CRE does not induce transcription. Activation of transcription requires the phosphorylation or CREB at Ser-133. In the case of the cAMP pathway, the activated catalytic subunit of cAMP-dependent protein kinase (PKA) translocates to the nucleus and phosphorylates Ser-133 of CREB. In the nervous system, signals transmitted across synapses are known to regulate gene expression in the post-synaptic cell. This process often involves membrane depolarization and subsequent amplification of intracellular Ca2+. The transcriptional activation induced by membrane depolarization and Ca2+ influx is mediated by a promoter element, called the Ca(2+)-responsive element (CaRE). Recent studies of c-fos and proenkephalin gene expression have shown that the CaRE is indistinguishable from a CRE. In this paper, we focus on the possible interactions between Ca2+ and the cAMP signaling pathways into the nucleus.

Animals↗
PubMed1996

Differential distribution of CaM kinases and induction of c-fos expression by flashing and sustained light in rat retinal cells.

PURPOSE: To examine the expression of c-fos proto-oncogene and phosphorylation of cAMP responsive element binding (CREB) protein in the rat retina after changes in the light-dark condition. METHODS: Rats were exposed to both steady light and flashing light and were killed at the end of light exposure. The retinas were analyzed by in situ hybridization using single-stranded RNA probes for c-fos transcripts and by immunocytochemistry using phosphoSer-133 specific CREB antiserum, anti-calcium calmodulin dependent protein (CaM) kinase II, and anti-CaM kinase IV. RESULTS: c-fos mRNA was expressed in the outer half of the inner nuclear layer (INL) and in the ganglion cell layer (GCL) after 30 minutes of sustained light. After 30 minutes of flashing light, c-fos expression also was detected in the inner border of the INL. Phosphorylated CREB immunoreactive nuclei had similar distribution after steady and flashing light. Both CaM kinase II and CaM kinase IV, which phosphorylate CREB at Ser 133 in vitro, were expressed in the GCL and in the INL. CaM kinase II, however, was localized in the inner border of the INL, whereas CaM kinase IV was distributed in the outer half of the INL. CONCLUSIONS: These results suggest that the differential expression of c-fos mRNA induced by flashing and sustained light may reflect the CREB phosphorylation by CaM kinases in a different subpopulation of retinal cells.

Animals↗
PubMed1996

In vitro antiplatelet activity of the carbinol analogue of ketoprofen, 3-(1-methyl-2-hydroxyethyl)-benzophenone.

3-(1-Methyl-2-hydroxyethyl)-benzophenone (F025) is a close structural analogue of ketoprofen, in which the carboxylic acid of ketoprofen is reduced to a carbinol moiety. The in vitro effects of F025 on platelet aggregation, cyclooxygenase and hypotonic NaCl-induced erythrocyte hemolysis were examined in comparison with those of ketoprofen. Although the inhibitory activity of F025 on collagen, arachidonic acid and ADP-induced platelet aggregation was between 2 and 20 times that of ketoprofen in healthy volunteers, guinea pigs, rabbits and rats, F025 had only one-tenth of the inhibitory action of ketoprofen on sheep seminal vesicle cyclooxygenase F025, but not ketoprofen, inhibited hypotonic NaCl-induced erythrocyte hemolysis in guinea pigs. The substantial antiplatelet activity of F025 may be due to membrane stabilization as well as to cyclooxygenase inhibition.

Animals↗
PubMed1996

Gene therapy against an experimental glioma using adeno-associated virus vectors.

The efficacy of gene therapy for glioma was examined using adeno-associated virus (AAV)-based vectors to deliver genes to experimental tumors in mice. Stereotactic injection of 2 x 10(5) U-251SP human glioma cells into the brains of nude mice produced tumors of 19.06 +/- 1.79 mm2 17 days after injection. Employing a high titer preparation of AAV vector containing the gene for beta-galactosidase (AAV-lacZ), dose-dependent transduction of U-251SP cells was seen in vitro. When 1.6 x 10(10) AAV-lacZ particles were directly injected into tumors in vivo, 30-40% of the cells along the needle track expressed beta-galactosidase. Transduction of U-251SP cells in vitro with an AAV vector containing a bicistronic gene encoding both herpes simplex thymidine kinase and human interleukin-2 (AAV-tk-IRES-IL2) rendered them sensitive to the cytocidal effects of ganciclovir (GCV) and IL-2 was produced in a dose-dependent manner. Cocultures of AAV-tk-IRES-IL2 transduced cells and nontransduced cells proved highly sensitive to GCV indicating the contribution of the bystander effect. Stereotactic delivery of 6 x 10(10) AAV-tk-IRES-IL2 particles into day 7 tumors in nude mice followed by administration of GCV for 6 days, resulted in a 35-fold reduction in the mean volume of tumors compared with controls. Normal brains did not suffer from any toxic effect of the administration of AAV-tk-IRES-IL2 and GCV. These results indicate that high titer AAV vector treatment may be safe and effective for in vivo gene therapy of human brain tumors.

Animals↗
PubMed1995

Cocaine-induced CREB phosphorylation and c-Fos expression are suppressed in Parkinsonism model mice.

Cocaine exerts multiple neurochemical effects in the central nervous system through inhibition of the dopamine transporter at the synapse. Here we report that systemic administration of the drug induces rapid phosphorylation of CREB in the mouse striatum where expression of the nuclear proto-oncogene c-fos is observed. In MPTP-treated mice, in which dopaminergic neurones are degenerated and which show Parkinsonism-like behaviour, however, CREB phosphorylation is not induced by cocaine exposure and c-fos expression is significantly depressed in comparison with controls. These data suggest that CREB may play a major role in the dopaminergic activation of c-fos in the striatum and that the lack of a CREB-induced transcription cascade may have a critical relevance for long-lasting psychomotor disorders in Parkinsonism.

Animals↗
PubMed1995

Nitric oxide induces c-fos gene expression via cyclic AMP response element binding protein (CREB) phosphorylation in rat retinal pigment epithelium.

We have examined whether nitric oxide (NO) induces the expression of c-fos proto-oncogene and the phosphorylation of cyclic AMP response element binding protein (CREB) in the rat retina. NO donor sodium nitroprusside (SNP) was injected into the vitreal cavity of the eye, and the retina and retinal pigment epithelium (RPE) was analysed by in situ hybridization using single-stranded RNA probes for c-fos transcripts and by immunocytochemistry using an anti-phospho Ser-133 antibody 20 min after the injection of SNP, phosphorylated-CREB immunoreactivity was found in RPE cells and weakly in some cells of the INL. Forty-five min after the SNP injection, the expression of c-fos mRNA was detected in the RPE. These results suggested that NO induced the c-fos expression via the phosphorylation of CREB in RPE cells, as it has been demonstrated in PC12 cells that the transcription of c-fos gene was activated by the CREB phosphorylation.

Activating Transcription Factor 2↗
PubMed1995

The monoclonal antibody specific for the 4-hydroxy-2-nonenal histidine adduct.

Monoclonal antibodies directed against proteins modified with the major membrane lipid peroxidation product, 4-hydroxy-2-nonenal, have been established and characterized. The monoclonal antibodies specific for HNE-modified proteins were raised by immunizing mice with a HNE-keyhole limpet hemocyanin conjugate. The resulting five monoclonal antibodies (mAbs HNEJ-1-5) recognized HNE-modified bovine serum albumin (BSA), but not native BSA in Western blot studies. Of the five mAbs, HNEJ-2 exhibited the highest affinity for HNE-modified proteins and a much higher affinity for the HNE-histidine adduct than the HNE-lysine or HNE-cysteine adducts. mAb HNEJ-2 did not cross-react with proteins that had been treated with other aldehydes, such as 1-hexenal, 2-hexenal, 4-hydroxy-2-hexenal, 2-nonenal, formaldehyde, or glutaraldehyde. These results suggest that the major epitope recognized by mAb HNEJ-2 is the Michael addition-type HNE-histidine adduct.

Aldehydes↗
PubMed1995

Functional movements of putative jaw muscle insertions.

BACKGROUND: The craniomandibular muscles control jaw position and forces at the teeth and temporomandibular joints, but little is known regarding their biomechanical behaviour during dynamic function. The objective of this study was to determine how jaw muscle insertions alter position during different jaw movements in living subjects. METHODS: Computer 3D reconstruction of MR images and jaw-tracking were combined to permit the examination of movement with six degrees of freedom. Maximum mandibular opening, protrusive and laterotrusive positions were recorded in four subjects, and the translation and rotation of the putative insertions of masseter, temporal, medial, and lateral pterygoid muscles were measured. RESULTS: The sizes and shapes of regional attachments varied markedly among subjects, and their displacement patterns were different in specific muscles. For instance, when the jaw closed to the dental intercuspal position from maximum gape, the region near the superior insertion site of the masseter moved backward and upward, whereas the region near the inferior insertion site displaced mainly forward. In three subjects, the jaw's rotational center during this act was approximately 26-34 mm below the mandibular condyles. CONCLUSIONS: Since the movements of each muscle part differ according to variations in the size and shape of insertion areas, individual musculoskeletal form, and patterns of jaw motion during function, the prediction of motion-related muscle mechanics in any one subject is unlikely to be possible without direct measurement of the motion of visualized muscle parts. The present study shows that this information can be obtained.

Adult↗