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Biomedical subjects

Y Ushio

Publications and source records attributed to Y Ushio.

At least 19 recordsLinked to original sources
PubMed1997

Cloning and characterization of NE-dlg: a novel human homolog of the Drosophila discs large (dlg) tumor suppressor protein interacts with the APC protein.

We have cloned a cDNA for a novel human homolog of the Drosophila discs large (dig) tumor suppressor protein, termed NE-dlg (neuronal and endocrine dig). Northern blot analysis revealed that the gene is highly expressed in neuronal and endocrine tissues. Fluorescence in situ hybridization (FISH) and radiation hybrid mapping studies localized the NE-dlg gene to chromosome Xq13. We also found that the NE-dlg gene encoded a 100 kDa protein. Immunolocalization studies using an NE-dlg antibody showed that the protein tended to be expressed in non-proliferating cells, such as neurons, cells in Langerhans islets of the pancreas, myocytes of the heart muscles, and the prickle and functional layer cells of the esophageal epithelium. Proliferative cells, including various cultured cancer cell lines and basal cells in the esophageal epithelium, showed little expression of the NE-dlg protein. In addition, yeast two-hybrid screening and in vitro binding assays revealed that the NE-dlg interacted with the carboxyl-terminal region of the APC tumor suppressor protein. These data suggest that NE-dlg negatively regulates cell proliferation through its interaction with the APC protein.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Postnatal ontogeny of striatal-enriched protein tyrosine phosphatase (STEP) in rat striatum.

The present study was undertaken to examine developmental change in expression of striatal-enriched protein tyrosine phosphatase (STEP) in the postnatal striatum of rats. For this purpose, immunohistochemical staining and transimmunoblotting analyses were carried out using a cDNA-generated polyclonal antibody to the STEP with a molecular weight of 46 kDa. Immunostaining showed that in neonatal striatum STEP-immunoreactivity was found in discrete patches composed of many immature cells, which corresponded to the tyrosine hydroxylase-immunopositive "dopamine islands." With development there was an increase in staining intensity and in the number of positively reacting cells. By 4 weeks postnatally, STEP-immunoreactivity was almost homogeneously distributed throughout the striatum, as was seen at the adult stage. Immunoblotting analysis showed that STEP protein expression abruptly increased from 2 to 4 weeks postnatally when it reached the adult level. These findings suggest that STEP is involved in development and maturation of the striatal neurons.

Animals↗
PubMed1997

Calcineurin in the adult rat hippocampus: different distribution in CA1 and CA3 subfields.

We examined the immunohistochemical regional distribution of calcineurin (Ca2+/calmodulin-dependent protein phosphatase) in the adult rat hippocampus, following various regional destruction. In the normal adult rat hippocampus, the calcineurin immunoreactivity showed a characteristic pattern. This protein phosphatase was detected in all layers of the CA1 subfield, including the cytoplasm of the pyramidal cells, whereas it was strongly evident in the stratum lucidum and moderately so in the cytoplasm of pyramidal cells in the CA3 subfield. Seven days after transient forebrain ischemia, which induced destruction of CA1 pyramidal cells, the calcineurin immunoreactivity decreased in all layers of the CA1 subfield, while the immunoreactivity for synapsin I, a marker of the presynaptic site, was preserved. Seven days after the intraventricular injection of kainate, which induced destruction of CA3 pyramidal cells, the calcineurin immunoreactivity in the stratum lucidum was preserved, although the immunostaining pattern of the stratum lucidum changed when CA3 pyramidal cells were destroyed. Seven days after mechanical destruction of the dentate gyrus and CA4 subfield, which induced destruction of mossy fibers, the calcineurin immunoreactivity in the stratum lucidum was lost, except in the far site of the stratum lucidum. In the CA1 subfield, calcineurin was mainly located in postsynaptic sites, while it was mainly located in the presynaptic sites in the mossy fibers of the CA3 subfield. The immunohistochemistry of adjacent sections with antibodies of microtubule-associated protein 2 and synapsin I, which are markers of postsynaptic and presynaptic sites respectively, supports these results. Thus, calcineurin has a different synaptical distribution in the rat hippocampus.

Animals↗
PubMed1997

Ruptured distal accessory anterior cerebral artery aneurysm: case report.

OBJECTIVE AND IMPORTANCE: Accessory anterior cerebral artery (ACA), which is a type of median artery of anomalous triplicate ACA, is not rare, but aneurysmal formation is extremely rare. We report a rare case with ruptured aneurysm arising from a distal accessory ACA. We discuss the characteristics and causes of this type of aneurysm and classification of this anomaly. CLINICAL PRESENTATION: A 63-year-old man suddenly developed severe headache and then loss of consciousness and paraplegia. Computed tomography disclosed thick and diffuse subarachnoid hemorrhage and interhemispheric hematoma. Subsequent bleeding occurred 2 hours after the first hemorrhage. Cerebral angiography disclosed a saccular aneurysm arising from the distal accessory ACA. INTERVENTION: Neck clipping of the aneurysm was performed 22 hours after the second episode, using an interhemispheric approach. CONCLUSION: Although transient paraplegia occurred 8 days after onset, the patient recovered well after surgery without neurological deficit. The characteristics of the aneurysm arising from distal accessory ACA are considered similar to those of distal ACA aneurysm. There is, however, some confusion regarding the terminology of the anterior communicating artery complex anomalies, which we discuss.

Aneurysm, Ruptured↗
PubMed1997

Two distinct populations of primary cytotoxic cells infiltrating into allografted tumor rejection sites: infiltration of macrophages cytotoxic against allografted tumor precedes that of multiple sets of cytotoxic T lymphocytes with distinct specificity to alloantigens.

It has been reported that the rejection of tumor allografts is mainly mediated by cytotoxic T lymphocytes (CTLs). Here, we characterized the cytotoxic effector cells of C57BL/6 (B6; H-2b) mice infiltrating into the rejection site of the i.p. allografted Meth A fibrosarcoma (or P815 mastocytoma) cells of H-2d origin. Two types of cytotoxic cells (i.e., CD8+ CTLs and macrophages (M phi s)) were identified by flow cytometric fractionation of the infiltrates or by specific in vitro elimination of cells either with antibody (Ab)-coated beads or with an Ab-plus complement. Of particular interest, these effector cells showed distinct and unique target specificities. First, the CTLs were inactive against transplanted tumor (e.g., Meth A) cells, whereas they were cytotoxic against donor-related concanavalin A (Con A) blasts as well as CTLL-2 (H-2b) cells transfected with a class I gene of H-2d origin. A cold target competition assay suggested that the CTLs were composed of multiple sets of T cells, each of which specifically recognized different allo-antigens. Second, the M phi s lysed the allografted tumor cells but were inert toward the Con A blasts and the CTLL-2 transfectants. Unexpectedly, the infiltration of M phi s preceded the infiltration of CTLs by several days during the course of rejection. These results indicate that two distinct populations of unique cytotoxic cells (i.e., CTLs and M phi s) are induced in the allografted tumor rejection site, and that the infiltration of cytotoxic M phi s responsible for rejection precedes that of the CTLs cytotoxic against cells expressing donor-related allo-antigens.

Animals↗
PubMed1997

Epidemiological study of primary intracranial tumours in elderly people.

The incidence of primary intracranial tumours in a well defined population of persons older than 70 years (elderly) who resided in Kumamoto prefecture was examined. During the period from 1989 to 1995, primary intracranial tumours were diagnosed in 271 elderly people; of these, 155 (57.2%) tumours were confirmed microscopically. In a mean population of 216,000 people over the age of 70 years, this yields an average annual incidence rate of 18.1 cases/100,000 population/year. The incidence was lower in men (15.2/100,000 population) than women (20.3/100,000 population). The age specific incidence/100,000/year was 23.2 for the 70-74 year age group, 18.1 for the 75-79 year age group, 15.1 for the 80-84 year age group, and 7.6 for persons older than 85 years. The most common tumours were meningiomas (50.6%), followed by malignant gliomas (13.3%), pituitary adenomas (12.9%), schwannomas (6.6%), malignant lymphomas (3.7%), and benign astrocytomas (3.7%).

Age Distribution↗
PubMed1997

Effectiveness of retrograde perfusion of the cerebral vein for attenuating neuronal injury after transient ischemia following reperfusion in the rat brain.

The effect of retrograde perfusion of the cerebral vein (RPCV) with antioxidant LY231617 on neuronal injury after transient ischemia in rat brain was examined. Transient ischemia was caused by left middle cerebral artery (MCA) occlusion and reperfusion. Rats were assigned to three groups: Group A (n = 6), MCA occlusion only; Group B (n = 8), RPCV with saline (flow rate 4.95 ml/hr) into the left inferior cerebral vein; and Group C (n = 6), RPCV with LY231617 (20 mg/kg/hr, flow rate 4.95 ml/hr). RPCV in Groups B and C was performed simultaneously with occlusion. Both occlusion and RPCV were performed for 30 minutes in awake animals. Seven days later, all rats were investigated for rotational behavior elicited by apomorphine (1.0 mg/kg), and then immunohistochemical analysis of brain specimens was carried out using calcineurin as a neuronal marker in the striatum to detect the ischemic damaged area. The number of turns to the left (lesioned side) in both Groups B (42 +/- 12) and C (46 +/- 14) was significantly lower (p < 0.01) than in Group A (222 +/- 45), but there was no significant difference between Groups B and C. The percentage ischemic damaged area in both Groups B (17.9 +/- 6.2%) and C (1.6 +/- 1.0%) was significantly less (p < 0.01) in Group A (51.1 +/- 2.1%). RPCV with and without LY231617 during occlusion was effective for attenuating reperfusion injury.

Animals↗
PubMed1997

Treatment of cancer using pulsed electric field in combination with chemotherapeutic agents or genes.

Electroporation is a standard laboratory technique originally developed for in vitro transfer of molecules into cells. It involves application of electrical pulses ranging from micro- to milliseconds that create transient pores in the cell membrane allowing intracellular access of exogenous molecules. This technique has been successfully applied to regress tumors in animal models by combining electroporation with chemotherapeutic agents--a process known as electrochemotherapy (ECT) which substantially enhance cytotoxicity of some antineoplastic agents. Recently ECT has moved into clinical arena and patients with cutaneous tumors and head and neck cancers have been treated very effectively with ECT. Parallel to ECT, a technique has also been developed which makes it possible to inject plasmid DNA and combine it with in vivo electroporation--electro--genetherapy (EGT)--to deliver in a highly efficient manner both marker and functional genes into target tissue and achieve gene expression. Thus, in vivo electroporation is contributing to the development of a new strategy for cancer treatment with both drugs and genes.

Animals↗
PubMed1996

Involvement of N-methyl-D-aspartate receptor in the delayed transneuronal regression of substantia nigra neurons in rats.

The substantia nigra pars reticulata (SNr) receives both inhibitory GABAergic and excitatory glutamatergic afferents from diverse origins. Ischemic injury to the striatum and/or the globus pallidus causes delayed transneuronal death of the SNr neurons, in the course of which neuronal disinhibition induced by loss of GABAergic inputs is supposed to trigger a lethal hypermetabolic process. In the in vivo experiment presented herein, we clarified the role of glutamatergic action via the N-methyl-D-aspartate receptor in this cell death process. Continuous intraventricular infusion (0.5 microliter/h) of the N-methyl-D-aspartate receptor antagonist MK-801 (1000 micrograms/ml), or of saline (control group) was initiated 24 h after 2 h of transient middle cerebral artery (MCA) occlusion in rats, by which massive ischemic injury was produced in the striatopallidal regions. The measured rectal temperature was not significantly altered in the MK-801-infused and in the control rats throughout the time period examined. The rats were killed at 15 days after MCA occlusion. The volume of the focal ischemic infarction of the MK-801-infused group did not significantly differ from that of controls. Also, MK-801-infusion did not significantly ameliorate the nigral atrophy subsequent to MCA occlusion. In association with a marked depletion of GABAergic afferent fibers, neuronal cell number in the ipsilateral SNr was significantly decreased in the control group. In contrast, the neuronal cell loss in the nucleus was completely prevented in the MK-801-infusion group. The data suggested that withdrawal of GABAergic inputs may cause a severe imbalance between excitation and inhibition of the SNr neurons and may eventually result in neurotoxicity mediated by the N-methyl-D-aspartate receptor. Suppression of glutamatergic excitatory effects by suitable drugs may be a reasonable therapy for the transneuronal death of the SNr neurons.

Animals↗
PubMed1996

Regulation of CCAAT/enhancer-binding protein family members by stimulation of glutamate receptors in cultured rat cortical astrocytes.

Regulation of mRNA levels, DNA binding activities, and phosphorylation of CCAAT/enhancer-binding protein (C/EBP) family members by stimulation of glutamate receptors were studied in cultured rat cortical astrocytes. Indirect immunofluorescence and immunoblot analyses with specific antibodies to C/EBP family members revealed that both C/EBPbeta and C/EBPdelta but not C/EBPalpha are expressed in the nuclei of astrocytes. After exposure to glutamate, C/EBPbeta mRNA levels increased within 10 min, reached the maximal level at about 1 h, and returned to the basal level within 6 h. In contrast, C/EBPdelta mRNA levels decreased by 6 h and were recovered within 12 h. These changes in mRNA levels were accompanied by an increase and a decrease in proteins for C/EBPbeta and C/EBPdelta, respectively. Elevation of C/EBPbeta mRNA levels by glutamate treatment required an increase in intracellular Ca2+ concentration and depended on activations of protein kinase C and calmodulin-dependent protein kinases. Gel mobility shift analysis using nuclear extracts from the glutamate-treated cells showed increases in C/EBP site binding activities 2 h after the exposure to glutamate. Moreover, glutamate stimulated phosphorylation of C/EBPbeta in 32P-labeled astrocytes in a Ca2+-dependent manner. These results suggest that glutamate regulates functions of C/EBP family members in brain astrocytes through changes in mRNA levels of C/EBPbeta and C/EBPdelta as well as through phosphorylation of C/EBPbeta.

Animals↗
PubMed1996

Multiple expression of Ly-6C and accumulation of a Ly-6C pre-mRNA in activated macrophages involved in rejection of an allografted tumor.

We established a monoclonal antibody against a surface antigen of activated macrophages (Møs) involved in the rejection of an allografted mouse tumor, Meth A. Bacteria-elicited Møs also expressed the antigen but resident or inflammatory Møs, or other leukocytes, did not. The antigen was identified as Ly-6C by isolation of cDNA clones encoding it. Two-dimensional immunoblotting revealed that Ly-6C of the Møs exists in multiple forms with a similar size but with different isoelectric points. A Ly-6C pre-mRNA also accumulated abundantly in the Møs. Thus, the multiple expression of Ly-6C and the accumulation of the pre-mRNA are the features of allograft- and bacteria-elicited Møs.

Amino Acid Sequence↗
PubMed1996

Intrathecal chemotherapy with 1,3-bis(2-chloroethyl)-1-nitrosourea encapsulated into hybrid liposomes for meningeal gliomatosis: an experimental study.

1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), one of the chloroethyl nitrosoureas, is effective against malignant glioma. To develop its use in intrathecal chemotherapy, we encapsulated BCNU in hybrid liposomes composed of dimyristoylphosphatidylcholine and micellar surfactants (Tween 20) and dissolved it in artificial cerebrospinal fluid (lipo-BCNU). We then studied the toxicity of hybrid liposomes and cellular proliferation inhibition of lipo-BCNU in vitro. We found that 3 mM hybrid liposomes did not affect the viability of human endothelial cells and that lipo-BCNU inhibited the proliferation of human glioma cell lines U-105MG, U-251MG, and U-373MG, and rat glioma cell lines C6 and 9L in a concentration-dependent fashion. Wistar rats that were administered lipo-BCNU intracisternally showed no weight loss, neurological symptoms, or histological changes of the brain and spinal cord. A Wistar rat model of meningeal gliomatosis was established by intracisternal inoculation of 0.1 ml cell suspension containing 1 x 10(6) or 5 x 10(6) viable C6 glioma cells. Two days after inoculation, lipo-BCNU (BCNU, 2.5 mg/kg) was administered intracisternally. When 1 x 10(6) glioma cells were inoculated (experiments 1 and 2), the median survival times were 24.5 and 26 days in the control groups and 32 and 45 days in the lipo-BCNU-treated groups. respectively. When 5 x 10(6) glioma cells were inoculated (experiments 3-6), the median survival times were 17-29.5 days in the control groups and 23-44 days in the treated groups, respectively. Significantly prolonged survival was obtained in three of six experimental groups. After the administration of 1 ml lipo-BCNU (BCNU, 4.67 mM) or 1 ml BCNU solubilized with 5% dextrose/water (BCNU, 4.67 mM) into the cisterna magna of dogs, the cisterna magna cerebrospinal fluid was sampled, and the BCNU concentrations were assayed by high-performance liquid chromatography. The half-life of the lipo-BCNU was longer than that of BCNU solubilized with 5% dextrose/water. These results suggest that the intrathecal administration of lipo-BCNU may be possible for the treatment of meningeal gliomatosis.

Animals↗
PubMed1996

Inducing the loss of immunoglobulin lambda light chain production and the rearrangement of a previously excluded allele in human plasma B cell lines with concanavalin A.

We investigated the expression of differential lambda light chains in human B cell lines secreting immunoglobulin (Ig). When these cell lines were cultured with concanavalin A for a long period of time, a subpopulation of some but not all of these cell lines was induced to express new lambda light chains replacing the original lambda chain (light chain shifting). Production of the new lambda chain, which replaces the original lambda chain, results from a VJ rearrangement at a previously excluded allele and a dramatic reduction of the original lambda chain transcript, although no difference was found in the level of heavy chain transcript. Recombination activating genes RAG-1 and RAG-2, which are normally expressed during specific early stages of lymphocyte development, were expressed in not only the light chain shifting-inducible lines but also in the non-inducible cells. Treatment of these Ig secreting cell lines with dibutyryl cAMP, which is known to enhance expression of the RAG genes, could not induce the creation of new lambda light chain-producing cells from the inducible lines, suggesting that the expression of the two RAG genes is not sufficient for inducing new lambda light chain production. Concanavalin A induced a gradual but significant production lost of the original lambda chain in a subpopulation of the light chain shifting-inducible cells but not in the non-inducible cells. Association of new lambda light chain production with loss of original lambda chain raises the possibility that, when the RAG genes are expressed, concanavalin A may act on a novel intracellular mechanism controlling lambda light chain allelic exclusion in these plasma cell lines.

Adenocarcinoma↗
PubMed1996

Expression of tissue factor correlates with grade of malignancy in human glioma.

BACKGROUND: Tissue factor (TF), a cell surface receptor of factor VII/VIIa, was initially recognized as an initiator of the extrinsic coagulation pathway. TF has recently been found to be expressed highly in certain types of malignant tumors. In addition, TF belongs to the interferon receptor family and is one of the immediate early genes, suggesting that TF may participate in the regulation of cell growth. However, the correlation between the expression of TF and cell growth is still unclear. METHODS: Expression of TF in 6 glioma cell lines and 44 glioma surgical specimens was studied by Northern blot analysis, Western blot analysis, immunohistochemistry, and in situ hybridization. RESULTS: Northern blot analysis showed that glioma cells expressed minor novel transcripts of 3.3 kb and 1.6 kb, in addition to the transcripts of 2.2 kb and 3.1 kb that were previously reported. Western blot analysis revealed that the level of TF protein did not correlate with that of TF transcripts. Although immunohistochemical analysis of surgical specimens showed that all gliomas were positive for TF, it was interesting that 1 of 10 benign gliomas (10%) was positive for TF (malignancy Grade I-II), 13 of 14 anaplastic astrocytomas (86%) (malignancy grade III) and 19 of 20 glioblastomas (95%) (malignancy grade IV) were moderately or strongly positive for TF. In situ hybridization showed the expression of TF mRNA in glioma cells. CONCLUSIONS: TF is expressed in glioma and the level of expression correlates with the histologic grade of malignancy.

Astrocytoma↗
PubMed1996

High-efficiency in vivo gene transfer using intraarterial plasmid DNA injection following in vivo electroporation.

A novel method for high-efficiency and region- controlled in vivo gene transfer was developed by combining in vivo electroporation and intraarterial plasmid DNA injection. A mammalian expression plasmid for the Escherichia coli lacZ gene (driven with a SV40 early promoter) was injected into the internal carotid artery of rats whose brain tumors (from prior inoculation) had been electroporated between two electrodes. The lacZ gene was efficiently transferred and expressed in the tumor cell 3 days after plasmid injection. However, neither any gene transfers nor any elevated lacZ activity was detected in tissues outside the electrodes. The plasmid was not transferred without electroporation. Human monocyte chemoattractant protein-1 cDNA was also transferred by this method, and its long-lasting (3 weeks) expression was confirmed by using the Epstein-Barr virus episomal replicon system. The expressed monocyte chemoattractant protein-1 protein was functional, as evident by the presence of a large number of monocytes in tumor tissue. This method, electrogene therapy, which does not require viral genes or particles, allows genes to be transferred and expressed in desired organs or tissues, and it may lead to the development of a new type of highly effective gene therapy.

Animals↗
PubMed1996

Alterations of local cerebral blood flow, phorbol 12,13-dibutyrate binding activity, and histological damage during acute focal ischaemia in rat brain. A pathophysiology of acute focal ischaemia: Part 1.

The alterations of the local cerebral blood flow (LCBF), 3H-phorbol 12,13-dibutyrate (PDBu) binding activity were measured, and histological findings were also examined during the closed time course (0, 1, 3, 5, 7 hour) after middle cerebral artery occlusion (MCAO) in rat brain to assess the complex pathophysiology of acute focal ischaemia. From 1 to 3 hours after the start of MCAO, significant (p < 0.01) hyperreactivity of the second messenger system involving PDBu binding may be present, despite low perfusion of LCBF, and severe damage in the striatum whereas sparing almost completely the cortex on histological examination. At 5 hours, the PDBu binding activity increased slightly but not significantly but is reduced markedly at 7 hours after MCAO compared with the control group. The measurement of PDBu binding activity, additionally to measuring the LCBF and observation of the histological change might be a useful indicator in determining the threshold and duration of ischaemia which cause functionally irreversible cell damage in the brain.

Animals↗