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Biomedical subjects

M Hagiwara

Publications and source records attributed to M Hagiwara.

At least 19 recordsLinked to original sources
PubMed2000

Cellular immunologic parameters related to age, gender, and stage in lung cancer patients.

Several immunologic parameters have been reported to correlate with the clinicopathologic status of lung cancer patients. However, these studies were based on relatively small numbers of patients and often yielded conflicting results. We prospectively studied cellular immunologic parameters related to age, gender, and stage in lung cancer patients. We obtained pretreatment peripheral blood samples from 287 lung cancer patients. Lymphocyte subsets (percentage of lymphocytes positive for CD3, CD4, CD8, HLA-DR, or representing FcgammaR IIIa-positive T cells), natural killer (NK) cell activity, and lymphoblastogenesis (LB) after stimulation by phytohemagglutinin (PHA) were evaluated. Significant decline was seen in older patients in percentages of cells positive for CD3 or CD4, in the CD4/CD8 ratio and in LB. The percentage of FcgR IIIa-positive T cells increased with age. LB as well as CD4 positivity were significantly greater in women than in men. NK cell activity showed the greatest cytotoxic responses in stage IIIA, with significantly less response in stage IV than in IIIA. Node-negative patients showed higher reactivities for LB and lower positivity for HLA-DR than node-positive patients. Patients with no distant metastases had a higher level of NK cell activity than patients with distant metastases. Immune parameters are variously related to age, gender, and the stage in lung cancer patients, some may prove to be useful predictors of survival.

Adult↗
PubMed2000

A fluorescent indicator for visualizing cAMP-induced phosphorylation in vivo.

We have developed a method for visualizing phosphorylation of proteins in living cells using a novel fluorescent indicator composed of two green fluorescent protein (GFP) variants joined by the kinase-inducible domain (KID) of the transcription factor cyclic adenosine monophosphate (cAMP)-responsive element binding protein (CREB). Phosphorylation of KID by the cAMP-dependent protein kinase A (PKA) decreased the fluorescence resonance energy transfer (FRET) among the flanking GFPs. By transfecting COS-7 cells with an expression vector encoding this indicator protein (termed ART for cAMP-responsive tracer), we were able to visualize activation dynamics of PKA in living cells.

Amino Acid Sequence↗
PubMed2000

Tetracycline-induced expression of an anti-c-Myb single-chain antibody and its inhibitory effect on proliferation of the human leukemia cell line K562.

Ablation of c-Myb function might be an effective approach for the therapy of chronic myelogenous leukemia or other c-myb-dependent malignancies. To this end, we have previously used an intracellular anti-c-Myb single-chain antibody (sFv) to achieve the functional knockout of the c-Myb oncoprotein. In this study, we have employed a tetracycline-inducible system to control the expression of the sFv. A nuclear-localizing form of an anti-c-Myb sFv was cloned into a tet-regulated plasmid vector. Using a transient expression system in COS-1 cells, we observed that doxycycline (Dox) induced expression of the sFv in a dose-dependent manner, and that the sFv was localized mainly in the nucleus. The Dox-induced anti-c-Myb sFv also inhibited the transactivating activity of c-Myb in a dose-dependent manner. We subsequently confirmed the Dox-induced expression of the sFv in the leukemia cell line K562. Proliferation of the target leukemia cells was also inhibited. These results suggest that the anti-c-Myb sFv may represent a viable method for gene therapy of c-myb-dependent hematopoietic malignancies.

Animals↗
PubMed2000

Induction of mammalian cell transformation and genotoxicity by 2-methoxyestradiol, an endogenous metabolite of estrogen.

2-methoxyestradiol (2-MeOE(2)) is an endogenous metabolite of 17beta-estradiol and a proposed inhibitor of tumor growth and angiogenesis. However, 2-MeOE(2) is also an inhibitor of microtubule assembly and other microtubule inhibitors, e.g. colcemid and diethylstilbestrol, induce aneuploidy and cell transformation in cultured mammalian cells. To assess the in vitro carcinogenicity and related activity of 2-MeOE(2), the abilities of this metabolite to induce cell transformation and genetic effects were studied simultaneously using Syrian hamster embryo (SHE) fibroblasts. Growth of these cells was reduced by treatment with 2-MeOE(2) at 0.1-1.0 microg/ml in a concentration-dependent manner. Treatment of SHE cells with 2-MeOE(2) at 0.3 or 1.0 microg/ml for 2-48 h also resulted in a concentration- and treatment time-related increase in the mitotic index and the percentage of multinucleated cells. Treatment with 2-MeOE(2) at 0.1-1.0 microg/ml for 48 h induced a statistically significant increase in the frequencies of morphological transformation of SHE cells in a concentration-dependent manner. A statistically significant increase in the frequencies of somatic mutations at the Na(+)/K(+) ATPase or hprt locus was also observed in cells treated with 2-MeOE(2) for 48 h at 0.1 or 0.3 microg/ml, respectively. Treatment of SHE cells with 2-MeOE(2) at 0.3 or 1.0 microg/ml for 24 h induced chromosome aberrations, mainly breaks, exchanges and chromosome pulverization. The incidence of chromosome aberrations was not affected by co-treatment with alpha-naphthoflavone, an inhibitor of 2-hydroxylase that inhibits oxidative conversion of 2-MeOE(2) to 2-hydroxyestradiol, but the incidence was slightly increased by co-treatment with L-ascorbic acid. Numerical chromosomal changes in the near diploid range and in the tetraploid and near tetraploid ranges were also detected in 2-MeOE(2)-treated cells. These findings indicate that 2-MeOE(2) has cell transforming and genotoxic activities in cultured mammalian cells and potential carcinogenic activity.

2-Methoxyestradiol↗
PubMed2000

Delayed onset of pulmonary hypertension associated with an appetite suppressant, mazindol: a case report.

The use of the appetite suppressant agents aminorex and fenfluramine derivatives has been reported as a risk factor for the development of pulmonary hypertension. A 29-year-old female developed pulmonary hypertension suspected to be due to an amphetamine-like appetite suppressant agent, mazindol ((+/-)-5-(p-chlorophenyl)-2,5-dihydro-3H-imidazo [2,1-a] isoindol-5-ol). She was admitted to Sapporo Medical University Hospital with dyspnea due to severe pulmonary hypertension. Twelve months prior to admission, she had taken mazindol continuously for a period of 10 weeks. As yet, her pulmonary hypertension has not completely improved. This is the first reported case of mazindol-associated pulmonary hypertension, which developed after a long latent interval, and it suggests that mazindol is also a risk factor for the development of pulmonary hypertension, making long-term follow-up necessary for patients taking this anorectic agent.

Adult↗
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

Song-induced phosphorylation of cAMP response element-binding protein in the songbird brain.

We have investigated the participation of cAMP response element-binding protein (CREB) in the response of the songbird brain to a natural auditory stimulus, a conspecific song. The cells in the two song control nuclei, the higher vocal center (HVC) and area X of zebra finches (Taeniopygia guttata), were intensely stained with an anti-CREB monoclonal antibody. Double-labeling studies showed that CREB immunoreactivity was detected only in area X-projecting neurons in the HVC. The cloned CREB cDNA from zebra finches (zCREB) is highly homologous to mammalian delta CREB. Phosphorylation of zCREB at Ser119 in area X-projecting HVC neurons was induced by hearing tape-recorded conspecific songs of zebra finches, but not by birdsongs of another species or white noise. These results raise the possibility that zCREB plays a crucial role in the sensory process of song learning.

Amino Acid Sequence↗
PubMed1999

The subcellular localization of SF2/ASF is regulated by direct interaction with SR protein kinases (SRPKs).

Serine/arginine-rich (SR) proteins play an important role in constitutive and alternative pre-mRNA splicing. The C-terminal arginine-serine domain of these proteins, such as SF2/ASF, mediates protein-protein interactions and is phosphorylated in vivo. Using glutathione S-transferase (GST)-SF2/ASF-affinity chromatography, the SF2/ASF kinase activity was co-purified from HeLa cells with a 95-kDa protein, which was recognized by an anti-SR protein kinase (SRPK) 1 monoclonal antibody. Recombinant SRPK1 and SRPK2 bound to and phosphorylated GST-SF2/ASF in vitro. Phosphopeptide mapping showed that identical sites were phosphorylated in the pull-down kinase reaction with HeLa extracts and by recombinant SRPKs. Epitope-tagged SF2/ASF transiently expressed in COS7 cells co-immunoprecipitated with SRPKs. Deletion analysis mapped the phosphorylation sites to a region containing an (Arg-Ser)8 repeat beginning at residue 204, and far-Western analysis showed that the region is required for binding of SRPKs to SF2/ASF. Further binding studies showed that SRPKs bound unphosphorylated SF2/ASF but did not bind phosphorylated SF2/ASF. Expression of an SRPK2 kinase-inactive mutant caused accumulation of SF2/ASF in the cytoplasm. These results suggest that the formation of complexes between SF2/ASF and SRPKs, which is influenced by the phosphorylation state of SF2/ASF, may have regulatory roles in the assembly and localization of this splicing factor.

Glutathione Transferase↗
PubMed1999

Successive learning in hetero-associative memory using chaotic neural networks.

In this paper, we propose a successive learning method in hetero-associative memories, such as Bidirectional Associative Memories and Multidirectional Associative Memories, using chaotic neural networks. It can distinguish unknown data from the stored known data and can learn the unknown data successively. The proposed model makes use of the difference in the response to the input data in order to distinguish unknown data from the stored known data. When input data is regarded as unknown data, it is memorized. Furthermore, the proposed model can estimate and learn correct data from noisy unknown data or incomplete unknown data by considering the temporal summation of the continuous data input. In addition, similarity to the physiological facts in the olfactory bulb of a rabbit found by Freeman are observed in the behavior of the proposed model. A series of computer simulations shows the effectiveness of the proposed model.

Artificial Intelligence↗
PubMed1999

[UFT-E granule compliance in postoperative adjuvant chemotherapy].

UFT-E granules were administered as postoperative adjuvant chemotherapy to patients who had undergone surgery for gastric cancer, colorectal cancer or breast cancer. After treatment for one year, the dose conditions were investigated. The subjects were patients under 75 years of age in whom malignant tumors had been confirmed histologically, who had undergone curative resections, had no marked complications, and from whom personal informed consent had been obtained. As a rule, UFT-E granules were administered orally 450 mg (t.i.d.)/day continuously for one year beginning two weeks after surgery. The dose rate was studied from patient records and the tegafur blood concentrations on the 1st, 2nd, 6th, 8th and 12th month. The appearance of complications or clinical lab test abnormalities was also checked. A total of 19 cases were compliant among the 5 gastric cancer, 10 colorectal cancer, and 7 breast cancer patients. The mean administration period was 459 days (29-879 days), and the mean completion rate was 92.5%. The complications in 4 cases (21.1%) were relatively mild. A comparison of the prescribed dosage and patient records revealed a mean dose rate of 86.3%. From these findings, long-term administration of UFT-E granules with mild side effects is considered feasible. However, to achieve high compliance, it is considered necessary to gain a clear picture of dose conditions from patients records and other sources.

Administration, Oral↗
PubMed1998

cdc2 kinase-mediated phosphorylation of splicing factor SF2/ASF.

SR proteins are a family of splicing factors which are important components of spliceosomes. Recent studies suggested that phosphorylation of SR protein might be a key event for the regulation of pre-mRNA splicing and is prevalent in metaphase cells. To investigate the role of cdc2 kinase in cell cycle-dependent phosphorylation of SR protein, we examined its phosphorylation of SF2/ASF, a representative SR protein. SF2/ASF was phosphorylated both by recombinant cdc2 kinase, a cdc2-cyclin B complex, and by cdc2 kinase immunoprecipitated from G2/M phase HeLa cells. In vitro phosphorylation and phosphopeptide mapping of several mutant proteins revealed that cdc2 kinase specifically phosphorylates the RS domain of SF2/ASF with serines 227, 238 and presumably 199 as major phosphorylation sites. These findings suggest the possibility that cdc2 kinase takes part in the cell cycle-dependent phosphorylation of SR protein which regulates the function of spliceosomes.

Amino Acid Sequence↗
PubMed1998

CREB-induced transcriptional activation depends on mGluR6 in rod bipolar cells.

To investigate the molecular mechanisms of stimuli-induced transcriptional activation in neuronal cells, we have investigated the light-induced gene expression in the neural retina of rats. The immunoreactivity for phosphorylated cAMP responsive element binding protein (PCREB-IR) was expressed in the outer half of the inner nuclear layer (INL) and the ganglion cell layer (GCL) after 5 min exposure to steady light also in mice. In addition to these cells, PCREB-IR was also detected in the inner border of the INL after 5 min exposure to flashing light. Both steady and flashing lights induced c-fos mRNA in the same types of cells as the PCREB-IR-positive cells. Majority of PCREB immunoreactive nuclei in the outer half of the INL were also immunopositive for anti-protein kinase C alpha (PKC alpha), a marker of rod bipolar cells, while CaM kinase IV immunoreactivity was not detected in these cells. PCREB-IR and c-fos gene expression in the PKC alpha positive rod bipolar cells were lost in mice lacking metabotropic glutamate receptor 6 (mGluR6). Thus, we propose that the transcriptional response of CREB to light stimulation in rod bipolar cells is regulated via mGluR6.

Animals↗
PubMed1998

Novel SR-protein-specific kinase, SRPK2, disassembles nuclear speckles.

SR-protein-specific kinase 1 (SRPK1) is first identified as a specific kinase for SR splicing factors. By RT-PCR of a conserved kinase domain, novel SR-protein-specific kinase clones were isolated from mouse brain. The cloned cDNAs encode a 106 kDa protein (648 amino acids, 92% identical to human SRPK1) and a 120 kDa protein (681 amino acids, 58% identical to human SRPK1). Therefore, they were designated mSRPK1 and mSRPK2, respectively. Northern blotting revealed the ubiquitous expression of mSRPK1 in all tissues examined and the tissue-specific expression of mSRPK2 in testis, lung, and brain. Both kinases phosphorylated SF2/ASF, a member of SR proteins in vitro and the phosphopeptide mappings were identical, indicating that these kinases phosphorylate the same site of SF2/ASF. Overexpression of mSRPK2 caused disassembly of cotransfected SF2/ASF and endogenous SC35. Our results indicate that SRPK family members may regulate the disassembly of the SR proteins in a tissue-specific manner.

Amino Acid Sequence↗
PubMed1998

Autoantibody to thioredoxin reductase in an ovarian cancer patient.

In a patient with ovarian cancer, anti-nuclear antibodies were found by indirect immunofluorescence analysis in cultured cells. The patient's serum was used for immunoscreening of a cDNA library. Three overlapping positive clones were partial cDNA clones of thioredoxin reductase (TR), which is a member of the pyridine nucleotide-disulfide oxidoreductase family of flavoenzymes. Human autoantibodies affinity-purified with bacterial recombinant TR reacted to approximately 60-kDa polypeptide in HeLa extract in immunoblotting analysis and strongly stained around chromosome of metaphase of HeLa cells in immunofluorescence studies. Autoantibodies against recombinant TR were not found in our 100 patients with systemic autoimmune diseases. Immunoblotting analysis with bovine purified TR confirmed that the human serum had reactivities to TR. This is the first report of autoantibodies against a redox member in a cancer patient. TR has diverse functions in cell growth, death, and transformation through redox modulation with thioredoxin. Since dysregulation of TR in cancer cells is considered to be involved in some redox states of tumorigenesis, the observed antibody response in this patient could have arisen due to immunoreaction to the abnormally regulated protein.

Autoantibodies↗
PubMed1998

Dymple, a novel dynamin-like high molecular weight GTPase lacking a proline-rich carboxyl-terminal domain in mammalian cells.

We have cloned human dymple, a novel dynamin family member. The full-length cDNA sequence encodes a protein composed of 736 amino acids with a molecular mass of 80 kDa. This amino acid sequence most resembles yeast DNM1P and VPS1P. Dymple lacks a proline-rich carboxyl-terminal domain through which dynamin binds to SH3 domains to be activated. Northern blot analysis revealed two transcript sizes of 2.5 and 4.2 kilobases with alternative polyadenylation at the highest levels in brain, skeletal muscle, and testis. It was further established that there are three patterns of alternative splicing producing in-frame deletions in the coding sequence of dymple in a tissue-specific manner. When overexpressed, wild-type dymple exhibited a punctate perinuclear cytoplasmic pattern, whereas an amino-terminal deletion mutant formed large aggregates bounded by a trans-Golgi network marker. Since dynamin participates in clathrin-mediated endocytosis through a well-characterized mechanism, the existence of a dynamin-like molecule in each specific vesicle transport pathway has been predicted. Our findings suggest that dymple may be the first example of such a subfamily in mammalian cells other than dynamin itself, although its precise role and membrane localization remain to be resolved.

Alternative Splicing↗
PubMed1998

[Intratumoral heterogeneity of DNA content in lung adenocarcinoma].

Quantitative microspectrophotometry was performed in 10 resected lung adenocarcinomas for the purpose of studying intratumoral heterogeneity of DNA content. Histologically, there were 3 stage I, 2 stage IIIA, 2 stage IIIB, and 3 stage IV cases, consisting of 3 well, 4 moderately and 3 poorly differentiated cases. The tumors were cut at the greatest dimension and fine needle aspirations were performed from 5 separate areas (central, cranial, caudal, inward, and outward). One of the imprint smears was submitted for cytology and the other for microspectrophotometry using Feulgen staining. Although G0G1 and G2M phase cell populations varied in each specimen, all 5 specimens showed the same DNA histogram in 9 cases. These were 4 aneuploid, 4 polyploid (euploid but not diploid) and only one diploid tumor. Two stem lines were observed in the remaining single case. One of 5 specimens showed aneuploid, the other 4 specimens showed polyploid. Namely, intratumoral heterogeneity was observed in one case. Ther was no relationship between intratumoral heterogeneity tumor size or differentiation. Most lung adenocarcinomas showed DNA stability. Fine needle aspiration specimens obtained from one area of lung adenocarcinoma represent the DNA contents of the entire tumor.

Adenocarcinoma↗
PubMed1998

[Blunt thoracic injury].

Of 161 patients with blunt thoracic injury, 135 were male (83.9%) and 26 were female. The most common cause of injury was traffic accidents (130 patients, 80.7%), followed by falls (22 patients), and crushing (7 patients). There were 46 third decade and 36 second decade patients. Thirty-two patients had single thoracic injury and the other had multiple organ injury. The most common associated injury was head injury (65 patients). Most traffic accidents involved motor cycle accident. Forty-four patients died, 32 within 24 hours, and 4 died to thoracic injury. These 4 patients were shock on arrival and died within 24 hours. The injury severity score, which was under 30 in 78.3% of patients, correlated to the mortality rate. Rib fracture was the most common thoracic injury in 96 patients followed by hemothorax in 91, pulmonary contusion in 79, and pneumothorax in 64. Most of the thoracic injuries were treated conservatively. Thoracotomy was performed in 6 patients. Other than one patient with rupture of the left pulmonary vein, 5 patients recovered. Continued bleeding at a rate of more than 200 ml/h from the chest drainage tube or no recovery from shock and large air leakage preventing re-expansion of the lung are indications for emergency thoracotomy. Thoracotomy should also be considered after conservative treatment in patients with continued air leakage or intrabronchial bleeding negatively affecting respiration. Indications for thoracotomy should be determined individually based on evaluating of vital sign.

Accidents, Traffic↗