Biomedical subjects

Y Sugimoto

Publications and source records attributed to Y Sugimoto.

At least 145 records · Page 8Linked to original sources

PubMed1999

Identification of substrate sequences for membrane type-1 matrix metalloproteinase using bacteriophage peptide display library.

Membrane type-1 matrix metalloproteinase (MT1-MMP) has been reported to mediate the activation of progelatinase A (proMMP-2) which is associated with tumor invasion and metastasis, and also known to have an ability to digest extracellular matrix components. To clarify substrate specificity of MT1-MMP, we have searched for amino acid sequences cleaved by this protease using the hexamer substrate phage library consisting of a large number of randomized amino acids sequences. The consensus substrate sequences for MT1-MMP were deduced from the selected clones and appeared to be P-X-G/P-L at the P3-P1' sites. Peptide cleavage assay revealed that MT1-MMP preferentially digested a synthetic substrate containing Pro of the P1 position compared to that being substituted with Gly. Our results may have an important implication to identifying new target proteins for MT1-MMP and leading to the design of its selective inhibitors suitable for cancer chemotherapy.

Bacteriophages↗

PubMed1999

Cell migration from the ganglionic eminence to the neocortex investigated by labeling nuclei with UV irradiation via a fiber-optic cable.

Recent studies have shown that the ganglionic eminence is one of the sources of tangentially migrating cells in the developing neocortex. Since the migration of the DiI-labeled cells from the ganglionic eminence to the neocortex was not monitored by videomicroscopy in these reports, we devised a novel method to study cell migration in vitro and in vivo. The new method involves ultraviolet (UV) irradiation of the cells through a fiber-optic cable and subsequent identification of the irradiated cells on the basis of the formation of thymine dimers in the nuclei. First, we tested the new method (UV-thymine dimer-labeling method) by applying it to monitor the cell migration of neuronal precursor cells in the rostral migratory stream in the neonatal rat telencephalon. In vitro, UV irradiation for 1 s through the fiber-optic cable resulted in the formation of sufficient thymine dimers as to allow immunohistochemical detection after 6 h of incubation; a significant proportion of the irradiated cells continued to migrate in the same direction and at the same speed as those before irradiation. There was no significant difference in the cell migration distance over 6 h between cells exposed and not exposed to the UV irradiation in vitro. In vivo, this method revealed that three times as many cells in the subventricular zone of the olfactory bulb migrated rostrally as caudally. The new method also allowed us to measure the speed of cell migration, which was estimated to be about 70 microm/h at the maximum in the rostral direction. After these examinations of reliability of the method, we applied it to the rat embryo brain. One day after UV irradiation of the ganglionic eminence, labeled migrating cells were found in the striatum, in the internal capsule, and in the intermediate zone of the neocortex. The observation period of cell migration to the neocortex was extended by the use of a xeroderma pigmentosum group A gene mutant mouse, which lacked an ability to remove thymine dimer from the UV-irradiated nuclei. Two days after the UV irradiation, labeled migrating cells from the ganglionic eminence of the mutant mouse embryos were found both in the cortical plate and in the intermediate zone of the neocortex.

Animals↗

PubMed1999

The serotonin precursor 5-hydroxytryptophan elevates serum leptin levels in mice.

The effects of a serotonin (5-HT) precursor 5-hydroxytryptophan (5-HTP) on serum leptin levels were investigated in mice. 5-HTP dose dependently increased serum leptin levels in mice. Pretreatment of the peripheral aromatic amino acid decarboxylase inhibitor carbidopa suppressed 5-HTP-induced hyperleptinemia. These results suggest that the secretion of leptin may be modified by serotonergic mechanisms.

5-Hydroxytryptophan↗

PubMed1999

Induction of prostaglandin I(2) receptor by tumor necrosis factor alpha in osteoblastic MC3T3-E1 cells.

Mouse osteoblastic cells MC3T3-E1 produced prostaglandin E(2) via the reaction of cyclooxygenase-2 enzyme induced by tumor necrosis factor alpha (TNFalpha). Originally, the mRNA level for prostaglandin I(2) receptor (IP) was low in the cells. However, the addition of TNFalpha brought about a marked increase in the IP mRNA with a lag of about 3 h up to an about 8-fold higher level for 24 h. In addition, the induction of IP was supported by a binding experiment of [(3)H]iloprost (a stable analogue of prostaglandin I(2)). The amount of iloprost bound to the TNFalpha-stimulated cell membranes increased to a saturation level around 30 nM. Dexamethasone, cycloheximide and cyclooxygenase inhibitor suppressed the IP mRNA induction. The finding with the latter two compounds suggested a TNFalpha-dependent de novo synthesis of a protein, which is involved in the IP mRNA induction and may be attributed partially to the induced cyclooxygenase-2.

Animals↗

PubMed1999

Selective serotonin reuptake inhibitors fluoxetine and fluvoxamine induce hyperglycemia by different mechanisms.

The effects of the selective serotonin reuptake inhibitors, fluoxetine and fluvoxamine, on plasma glucose levels were investigated in mice. Both fluoxetine and fluvoxamine elicited significant hyperglycemia, while a selective noradrenaline reuptake inhibitor maprotiline had no effect. Fluoxetine and fluvoxamine did not change serum insulin levels, although they elicited hyperglycemia. Pretreatment with the serotonin (5-hydroxytryptamine, 5-HT) depleter, p-chlorophenylalanine (pCPA), abolished fluvoxamine-induced hyperglycemia, although pCPA did not affect the fluoxetine-induced glycemic effects. These results suggest that the selective serotonin reuptake inhibitors fluoxetine and fluvoxamine induce hyperglycemia by inhibition of insulin release. Moreover, our findings indicate that the glycemic effects of these drugs are differentially associated with serotonergic mechanisms.

Animals↗

PubMed1999

Abortive expansion of the cumulus and impaired fertility in mice lacking the prostaglandin E receptor subtype EP(2).

Female mice lacking the gene encoding the prostaglandin (PG) E(2) receptor subtype EP(2) (EP(2)(-/-)) become pregnant and deliver their pups at term, but with a much reduced litter size. A decrease in ovulation number and a much reduced fertilization rate were observed in EP(2)(-/-) females without difference of the uterus to support implantation of wild-type embryos. Treatment with gonadotropins induced EP(2) mRNA expression in the cumulus cells of ovarian follicles of wild-type mice. The immature cumuli oophori from wild-type mice expanded in vitro in response to both follicle-stimulating hormone and PGE(2), but the response to PGE(2) was absent in those from EP(2)(-/-) mice. Cumulus expansion proceeded normally in preovulatory follicles but became abortive in a number of ovulated complexes in EP(2)(-/-) mice, indicating that EP(2) is involved in cumulus expansion in the oviduct in vivo. No difference in the fertilization rate between wild-type and EP(2)(-/-) mice was found in in vitro studies using cumulus-free oocytes. These results indicate that PGE(2) cooperates with gonadotropin to complete cumulus expansion for successful fertilization.

Abortion, Spontaneous↗

PubMed1999

Effects of histamine on MK-801-induced memory deficits in radial maze performance in rats.

The effects of histamine on the spatial memory deficits induced by MK-801 were investigated using the eight-arm radial maze paradigm in rats. Intracerebroventricular (i.c.v.) injection of histamine or thioperamide, and intraperitoneal (i.p.) injection of histidine improved the spatial memory deficits induced by MK-801. Similar results were obtained with 2-thiazolylethylamine. In contrast, 4-methylhistamine showed no significant effect. Based on these observations, it seems likely that the protective effect of histamine on MK-801-induced spatial memory deficit is mediated by H(1)-receptors.

Analysis of Variance↗

PubMed1999

Neuron-specific expression of cationic amino acid transporter 3 in the adult rat brain.

CAT3 (cationic amino acid transporter 3) is a member of the murine CAT family which bears a system y(+) transport activity. On the Northern blot of adult rat tissues, the expression of CAT3 is restricted to the brain. In the present study, cellular localization of CAT3 mRNA and protein in the adult rat brain sections was examined by in situ hybridization with cRNA and immunostaining with a CAT3-specific antiserum, respectively. CAT3 mRNA was present both in the cerebral and cerebellar gray matter but most prominently in the nuclei located in the ventromedial part of the brain. These included preoptic nucleus, hypothalamic nucleus, reticular nucleus of thalamus, substantia nigra, central gray around the third ventricle and amygdala. CAT3 protein was also detected both in the cerebral and cerebellar gray matter and strong immunostaining was obtained in the olfactory cortex, hippocampus and cerebellar granular and Purkinje cell layers. Observations at higher magnifications revealed that both mRNA and protein were expressed by neurons but neither by glial nor endothelial cells. These results confirm the neuron-specificity of CAT3 in the adult rat brain and indicate that CAT3 is responsible for the neuronal system y(+) activity. The discrepancy between the distribution of mRNA and its translation product suggests a regional difference in the translation rate of the CAT3 transcript.

Amino Acid Sequence↗

PubMed1999

Influence of the non-competitive NMDA receptor antagonist MK-801 on 2-deoxy-D-glucose-induced hyperphagia in rats.

The effects of the glutamate N-methyl-D-aspartate (NMDA) receptor antagonist (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclo-hepten-5, 10-imine (MK-801) on 2-deoxy-D-glucose-induced hyperphagia were investigated in rats. MK-801 significantly increased 2-deoxy-D-glucose-elicited eating. The facilitating effects of MK-801 on 2-deoxy-D-glucose-elicited feeding were not affected by coadministration of a nitric oxide (NO) precursor, L-arginine. Because NO synthase inhibitors inhibit 2-deoxy-D-glucose-induced hyperphagia and activation of the NMDA receptor leads to NO formation, our results suggest that blockade of the NMDA receptor increases 2-deoxy-D-glucose-induced hyperphagia, which is unrelated to inhibition of NO, and that NMDA receptors may play a role in satiety.

Animals↗

PubMed1999

[Arg8]-vasopressin-induced increase in intracellular Ca2+ concentration in cultured rat hippocampal neurons.

Changes in intracellular Ca2+ concentration ([Ca2+]i) induced by [Arg8]-vasopressin (AVP) were studied in cultured rat hippocampal neurons by fura-2 fluorometry. AVP (10-1,000 nM) caused a dose-dependent increase in [Ca2+]i. The selective V1 vasopressin receptor agonist [Phe2, Ile3, Orn8]-vasopressin also induced a significant increase in [Ca2+]i, whereas the selective V2 vasopressin receptor agonist [deamino Cys1, D-Arg8]-vasopressin showed no effect. The AVP-induced increase in [Ca2+]i was inhibited by the selective V1 vasopressin receptor antagonist d(CH2)5[Tyr2(Me), Arg8]-vasopressin and nonpeptide V1 antagonist OPC-21268. On the other hand, no antagonistic effects were observed with the V2 vasopressin antagonist desglycinamide-[d(CH2)5, D-Ile2, Ile4, Arg8]-vasopressin and nonpeptide V2 antagonist OPC-31260. The increase in [Ca2+]i induced by AVP was abolished after removal of extracellular Ca2+. In addition, AVP-induced [Ca2+]i elevation was not affected by treatment with verapamil, which blocked the [Ca2+]i increase induced by an isotonic high K(+)-medium (50 mM). However, omega-conotoxin GVIA completely inhibited the effect of AVP. These results suggested that the AVP-induced [Ca2+]i increase in cultured rat hippocampal neurons is due to influx of Ca2+ through V1 VP receptors coupled with N-type calcium channels.

Animals↗

PubMed1999

A neuronal nitric oxide synthase inhibitor 7-nitroindazole reduces the 5-HT1A receptor against 8-OH-DPAT-elicited hyperphagia in rats.

The effects of the neuronal nitric oxide (NO) synthase inhibitor 7-nitroindazole on 8-hydroxy-2-di-n-(propylamino)tetralin (8-OH-DPAT)-induced hyperphagia, which is mediated by the 5-HT1A autoreceptor, were investigated in rats. 7-Nitroindazole suppressed 8-OH-DPAT-elicited increases in food intake. The inhibitory effects of 7-nitroindazole on 8-OH-DPAT-induced feeding were prevented by the NO precursor L-arginine. Although 8-OH-DPAT decreases 5-hydroxytryptamine (5-HT) synthesis, 7-nitroindazole did not reverse the 8-OH-DPAT-elicited decrease in 5-HT synthesis. Therefore, these results indicate that NO formed in the brain is involved in 8-OH-DPAT-induced hyperphagia and that the hypophagic effects of 7-nitroindazole are not dependent on 5-HT synthesis.

8-Hydroxy-2-(di-n-propylamino)tetralin↗

PubMed1999

Parathyroid hormone-related protein is a positive regulator of keratinocyte growth factor expression by normal dermal fibroblasts.

Parathyroid hormone-related protein (PTHrP), an important factor in the pathogenesis of humoral hypercalcemia of malignancy, is produced by many normal tissues, including the skin, where it regulates keratinocyte growth and differentiation and dermal fibroblast function. Keratinocyte growth factor (KGF), a member of the fibroblast growth factor (FGF) family, is a secretory product of stromal cells and functions as a mediator of epithelial cell growth and differentiation. Phenotypes of the skin in several transgenic mouse models, in which the KGF and PTHrP genes have been overexpressed or disrupted, suggest that these two factors interact in vivo to regulate homeostasis of the skin. In this study, we investigated the effects of KGF on PTHrP secretion and expression by normal human foreskin keratinocytes (NHFK) and the effects of PTHrP on KGF secretion and expression by normal human dermal fibroblasts (NHDF) in vitro. N-terminal PTHrP(1-36) increased KGF secretion, protein expression and mRNA expression by NHDF in a dose-dependent manner, however, KGF did not regulate PTHrP expression and secretion by NHFK. By flow cytometry, PTHrP also increased the percentage of NHDF producing KGF. Our results indicate that PTHrP produced by keratinocytes is a potential paracrine regulator of KGF expression by dermal fibroblasts in vivo. This paracrine regulation may explain, in part, the epidermal atrophy seen in the PTHrP null mice and epidermal hyperplasia seen in transgenic mice overexpressing PTHrP in their basal keratinocytes. Our results also suggest that PTHrP is an important mediator for the healing of skin wounds and growth of neoplasms of squamous origin.

Animals↗

PubMed1999

The common marmoset as a target preclinical primate model for cytokine and gene therapy studies.

Nonhuman primate models are useful to evaluate the safety and efficacy of new therapeutic modalities, including gene therapy, before the inititation of clinical trials in humans. With the aim of establishing safe and effective approaches to therapeutic gene transfer, we have been focusing on a small New World monkey, the common marmoset, as a target preclinical model. This animal is relatively inexpensive and easy to breed in limited space. First, we characterized marmoset blood and bone marrow progenitor cells (BMPCs) and showed that human cytokines were effective to maintain and stimulate in culture. We then examined their susceptibility to transduction by retroviral vectors. In a mixed culture system containing both marmoset stromal cells and retroviral producer cells, the transduction efficiency into BMPCs and peripheral blood progenitor cells (PBPCs) was 12% to 24%. A series of marmosets then underwent transplantation with autologous PBPCs transduced with a retroviral vector carrying the multidrug resistance 1 gene (MDR1) and were followed for the persistence of these cells in vivo. Proviral DNA was detectable by polymerase chain reaction (PCR) in peripheral blood granulocytes and lymphocytes in the recipients of gene transduced progenitors up to 400 days posttransplantation. To examine the function of the MDR1 gene in vivo, recipient maromsets were challenged with docetaxel, an MDR effluxed drug, yet the overall level of gene transfer attained in vivo (<1% in peripheral blood granulocytes) was not sufficient to prevent the neutropenia induced by docetaxel treatment. Using this model, we safely and easily performed a series of in vivo studies in our small animal center. Our results show that this small nonhuman primate, the common marmoset, is a useful model for the evaluation of gene transfer methods targeting hematopoietic stem cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1999

Ovalbumin in developing chicken eggs migrates from egg white to embryonic organs while changing its conformation and thermal stability.

Ovalbumin was detected in developing chicken eggs. The large majority of these ovalbumin molecules was found to be in a heat-stable form reminiscent of S-ovalbumin. About 83 and 90% of the ovalbumin population was in a heat-stable form in day 14 or stage 40 amniotic fluid and day 18 or stage 44 egg yolk, respectively, whereas ovalbumin in newly deposited eggs was in the heat-unstable, native form. Purified preparations of stable ovalbumin from egg white and amniotic fluid showed a less ordered configuration than native ovalbumin, as analyzed by circular dichroism and differential scanning calorimetry. In addition, mass spectrometric analysis exhibited distinct size microheterogeneity between the stable and native forms of ovalbumin. Immunohisotochemical study revealed that ovalbumin was present in the central nervous system and other embryonic organs. These results indicated that egg white ovalbumin migrates into the developing embryo while changing its higher order structure.

Amino Acid Sequence↗

PubMed1999

Retroviral expression of a mutant (Gly-533) human DNA topoisomerase I cDNA confers a dominant form of camptothecin resistance.

In previous studies, we isolated a mutant DNA topoisomerase I cDNA from a camptothecin (CPT)-resistant human T-lymphoblastic leukemia cell line, CPT-K5, and demonstrated that an amino acid change from Asp to Gly at residue 533 is responsible for the CPT resistance of the enzyme. In the present study, we have constructed a bicistronic retroviral vector, Ha-TM1-IRES-neo, that carries the mutant (Gly-533) TOP1 cDNA (TM1) and a neomycin-resistance gene to examine the effect of mutant DNA topoisomerase I (topo I) expression on CPT resistance of cells. HeLa S3 cells were transduced with Ha-TM1-IRES-neo, and the transduced cells were selected with G418. Two independently isolated populations of the G418-resistant cells and 2 clones showed 1.7- to 1.8-fold higher resistance to CPT than the control cells. Integration and expression of the exogenous TOP1 were confirmed by genomic and RT-PCR analyses. The topo I enzyme (mixture of mutant and wild-type) expressed in the transduced cells showed 3-fold resistance to CPT in cleavable-complex-formation assay and DNA-relaxation assay. Mutant topo I activity in the transduced cells was as much as 10% that of the endogenous enzyme. Our results clearly show that expression of Gly-533 topo I confers a dominant form of CPT resistance in cells expressing wild-type topo I. The mutant TOP1 could be used for the protection of normal bone marrow cells of cancer patients from the severe hematotoxicity of CPT-derivative anti-tumor agents.

Anti-Bacterial Agents↗

PubMed1999

Modification of expression of stem cell factor by various cytokines.

The local production of stem cell factor (SCF) may be an important mechanism for regulating proliferation, differentiation, and migration of various cells bearing c-kit receptors, and might be susceptible to the cytokines that serve in inflammation and tissue repair. We have demonstrated that in three murine cell lines, Balb/3T3A31, MC3T3-E1, and C3H-2K, which constitutively produced SCF with different quantity, the SCF mRNA expression was greatly enhanced in response to basic fibroblast growth factor (bFGF) or transforming growth factor beta1 (TGF-beta1). The study was carried out by in situ hybridization utilizing nonradioactive oligonucleotide probes and quantitative image analysis. Leukemia inhibitory factor (LIF) or interleukin-4 (IL-4) moderately increased SCF mRNA in all cell lines, but IL-3 did not. The dot-blot enzyme-linked immunosorbent assay (ELISA) further confirmed that SCF protein production in these cell lines and bone marrow stromal cells was markedly enhanced by TGF-beta1, although TGF-beta1 suppressed the proliferation of all these cells. bFGF also enhanced the SCF production in these cell lines, but did not in bone marrow stromal cells, suggesting a difference in their susceptibility to the cytokine. Our results suggest that TGF-beta1 and bFGF potentially modulate the biological function of cells bearing c-kit receptors through the modulation of SCF production in fibroblasts.

3T3 Cells↗

PubMed1999

Involvement of nitric oxide in the 5-HT1A autoreceptor-mediated hyperphagia in rats.

Effects of nitric oxide synthase(NOS) inhibitors on 8-hydroxy-2-di-n-(propylamino)tetralin (8-OH-DPAT)-induced hyperphagia which is mediated by the 5-HT autoreceptor were investigated. The non-selective NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) and neuronal NOS (nNOS) inhibitor 7-nitroindazole (7-NI) clearly suppressed increases in food intake by 8-OH-DPAT. Both hypophagic effects of L-NAME and 7-NI were reversed by the nitric oxide precursor, L-arginine. The findings suggest that nitric oxide formed in the brain is involved in 8-OH-DPAT-induced hyperphagia.

8-Hydroxy-2-(di-n-propylamino)tetralin↗

PubMed1999

High-resolution, human-bovine comparative mapping based on a closed YAC contig spanning the bovine mh locus.

A closed YAC contig spanning the mh locus was assembled by STS content mapping with seven microsatellite markers, eight genes or EST, and nine STS corresponding to YAC ends. The contig comprises 27 YACs, has an average depth of 4.3 YACs, and spans an estimated 1.2 Mb. A linkage map was constructed based on five of the microsatellite markers anchored to the contig and shown to span 7 cM, yielding a ratio of 160 kb/1 cM for the corresponding chromosome region. Comparative mapping data indicate that the constructed contig spans an evolutionary breakpoint connecting two chromosome segments that are syntenic but not adjacent in the human. Consolidation of human gene order by means of whole genome radiation hybrids and its comparison with the bovine order as inferred from the contig confirm conservation of gene order within segments.

Animals↗