Biomedical subjects

M Yamada

Publications and source records attributed to M Yamada.

At least 775 records · Page 43Linked to original sources

PubMed1997

Expression and clustered distribution of an inwardly rectifying potassium channel, KAB-2/Kir4.1, on mammalian retinal Müller cell membrane: their regulation by insulin and laminin signals.

Inwardly rectifying potassium (K+) channels (Kir) in Müller cells, the dominant glial cells in the retina, are supposed to be responsible for the spatial buffering action of K+ ions. The molecular properties and subcellular localization of Müller cell Kir channels in rat and rabbit retinas were examined by using electrophysiological, molecular biological, and immunostaining techniques. Only a single population of Kir channel activity, the properties of which were identical to those of KAB-2/Kir4.1 expressed in HEK293T cells, could be recorded from endfoot to the distal portion of Müller cells. Consistently, Northern blot, in situ hybridization, and RT-PCR analyses indicated expression of Kir4. 1 in Müller cells per se. The Kir4.1 immunoreactivity was distributed in clusters throughout Müller cell membrane. The Kir4.1 expression in Müller cells disappeared promptly after culturing. When the dissociated Müller cells were cultured on laminin-coated dishes in the presence of insulin, Kir4.1 immunoreactivity was detected in a clustered manner on the cell membrane. Because insulin and laminin exist in the surrounding of Müller cells in the retina, these substances possibly may be physiological regulators of expression and distribution of Kir4.1 in Müller cells in vivo.

Animals↗

PubMed1997

Identification and characterization of an anti-glial fibrillary acidic protein antibody with a unique specificity in a demented patient with an autoimmune disorder.

We detected an antibody to a 48 kd antigen of the central nervous system in the serum from a demented patient with an autoimmune disorder. To identify and characterize the antigen, we screened a human cerebral cDNA library and performed immunoblot analysis following two-dimensional gel electrophoresis (2-D blotting). The sequences of the isolated cDNA fragments were homologous to human glial fibrillary acidic protein (GFAP). Two-D blotting using patient serum revealed that the antibody reacted with a restricted subset of GFAP molecules which exhibited relatively high isoelectric points. Furthermore, to elucidate the importance of the anti-GFAP antibody in dementia, we screened for the presence of an anti-GFAP antibody in the sera of 46 demented patients: 26 with Alzheimer's disease and 20 with vascular dementia (VD). We found an anti-GFAP antibody in the serum of only one patient with VD. Two-D blotting revealed that the anti-GFAP antibody in the serum from the VD patient reacted with a more acidic subset of GFAP molecules compared with the anti-GFAP antibody from our patient. In conjunction with the fact that the GFAP molecule with high isoelectric point was insoluble and less degraded, these results suggested that the anti-GFAP antibody in the serum of our patient was not generated due to a secondary response to soluble and degraded GFAP which leaked through the damaged blood-brain barrier as found in the VD patient, but was generated actively on the basis of dysregulation of the immune system. Possible effects of the autoantibody on astrocytic function and the pathogenesis in dementia are discussed.

Alzheimer Disease↗

PubMed1997

Tertiary hypothyroidism and hyperglycemia in mice with targeted disruption of the thyrotropin-releasing hormone gene.

Thyrotropin-releasing hormone (TRH) is a brain hypothalamic hormone that regulates thyrotropin (TSH) secretion from the anterior pituitary and is ubiquitously distributed throughout the brain and other tissues including pancreas. To facilitate studies into the role of endogenous TRH, we have used homologous recombination to generate mice that lack TRH. These TRH-/- mice are viable, fertile, and exhibit normal development. However, they showed obvious hypothyroidism with characteristic elevation of serum TSH level and diminished TSH biological activity. Their anterior pituitaries exhibited an apparent decrease in TSH immunopositive cells that was not due to hypothyroidism. Furthermore, this decrease could be reversed by TRH, but not thyroid hormone replacement, suggesting a direct involvement of TRH in the regulation of thyrotrophs. The TRH-/- mice also exhibited hyperglycemia, which was accompanied by impaired insulin secretion in response to glucose. These findings indicate that TRH-/- mice provide a model of exploiting tertiary hypothyroidism, and that TRH gene abnormalities cause disturbance of insulin secretion resulting in marked hyperglycemia.

Animals↗

PubMed1997

Molecular cloning and expression of fatty acid alpha-hydroxylase from Sphingomonas paucimobilis.

Fatty acid alpha-hydroxylase (FAAH) catalyzes the initial reaction in alpha-oxidation of fatty acid to produce 2-hydroxy fatty acid. FAAH activity has been detected in a wide range of organisms from prokaryotes to eukaryotes. Here, we describe cloning of the FAAH gene from Sphingomonas paucimobilis, a sphingolipid- and 2-hydroxymyristic acid-rich bacterium. The isolated gene encoded 415 amino acids. A homology search revealed that amino acid sequences highly conserved in cytochrome P450 (P450) were present in FAAH. Although the heme-binding cysteine was recognizable at position 361, the consensus in the heme-binding region was modified by an insertion. Overall, FAAH has no significant identity to the known P450s. CO difference spectrum of recombinant FAAH showed the characteristic one of P450, except this peak was at 445 nm. These results suggest bacterial FAAH is a novel member of the P450 superfamily.

Amino Acid Sequence↗

PubMed1997

Transcription initiation sites and promoter structure of the mouse type 2 inositol 1,4,5-trisphosphate receptor gene.

Transcription initiation sites and the promoter sequence of the ubiquitously expressed mouse type 2 inositol 1,4,5-trisphosphate receptor (IP3R2) gene were determined. In contrast to the nervous system-enriched IP3R1, the IP3R2 gene had multiple (seven major) transcription initiation sites located 334 to 269 bp upstream from the first ATG codon. Transient luciferase assay revealed promoter activity of the IP3R2 sequence upstream from the transcription initiation sites. The IP3R2 promoter was GC-rich and had no conventional TATA box, but had a GC box in the proximal promoter. Multiple transcription start sites were flanked by CpG islands, and various cis elements were located in the promoter. These structural features are considered to be responsible for a profile of IP3R2 gene expression.

Animals↗

PubMed1997

Chicken Nkx-2.8: a novel homeobox gene expressed in early heart progenitor cells and pharyngeal pouch-2 and -3 endoderm.

Members of the NK family of homeobox transcription factors regulate critical steps of organogenesis during vertebrate development. In the studies described in this report, we have isolated and functionally characterized the chicken Nkx-2.8 (cNkx-2.8) cDNA and protein and defined the temporal and spatial pattern of cNkx-2.8 gene expression during chicken development. cNkx-2.8 transcripts are first detectable at HH stage 7 in the splanchnopleura. At stage 10(+), the cNkx-2.8 gene is expressed in the linear heart tube and the dorsal half of the vitelline vein. However, after looping, HH stage 13, cNkx-2.8 is no longer expressed in the looped heart tube, but is expressed in the ventral pharyngeal endoderm. At stage 15, in addition to the pharyngeal expression pattern, cNkx-2.8 is expressed in the ectoderm of the pharyngeal arches and the aortic sac. By HH Stage 17, cNkx-2.8 expression is detectable in lateral endoderm of the second and third pharyngeal pouches, the posterior portion of the aortic sac, and the sinus venosus. cNkx-2.8 binds to previously characterized Nkx2-1 and Nkx2-5 DNA-binding sites and overexpression of cNkx-2.8 transactivates a minimal promoter which contains multimerized Nkx-2 DNA-binding sites. In addition, cNkx-2.8 and serum response factor can coactivate a minimal cardiac alpha-actin promoter. These data are consistent with a model in which cNkx-2.8 performs a unique temporally and spatially restricted function in the developing embryonic heart and pharyngeal region. Moreover, the coexpression of cNkx-2.5 and -2.8 raises the possibility that cNkx-2. 8 may have a redundant role with cNkx-2.5 in the coalescing heart tube and may play an important role in the transcriptional program(s) that underlies thymus formation. The existence of multiple NK-2 family members and their partially overlapping patterns of expression are discussed within the framework of a "Nkx code."

Amino Acid Sequence↗

PubMed1997

Biologic activity of proteoglycan macrophage colony-stimulating factor.

We compared the biologic activities of 85-kDa macrophage-CSF (85-kDa M-CSF), which is fully active, and a proteoglycan M-CSF (PG-M-CSF). Both originate from the same precursor, but the latter retains the carboxyl-terminal portion, which must be proteolytically removed from the precursor to generate 85-kDa M-CSF and which is uniquely modified by a chondroitin sulfate glycosaminoglycan chain. PG-M-CSF supported the formation of murine macrophage colonies such as 85-kDa M-CSF. Furthermore, PG-M-CSF stimulated the proliferation of murine bone marrow macrophages, an M-CSF-dependent murine cell line, and an M-CSF-responsive human cell line established by transfer of the human M-CSF receptor gene. PG-M-CSF and 85-kDa M-CSF had equivalent specific biologic activities on a molar basis in all bioassays. The activity of PG-M-CSF was not affected by enzymatically removing the glycosaminoglycan chain when assayed by the formation of macrophage colonies and proliferation of the bone marrow macrophages. We analyzed the phosphorylation on tyrosine residue(s) of the M-CSF receptor in response to these M-CSFs that trigger mitogenic responses. PG-M-CSF rapidly (within 10 min) induced receptor phosphorylation in human cells with the same potency as 85-kDa M-CSF. These results indicate that PG-M-CSF is not a latent form or precursor of 85-kDa M-CSF but a fully biologically active cytokine.

Animals↗

PubMed1997

cDNA cloning of a heterogeneous two-chain anticoagulant protein IX-bp.

IX-bp and IX/X-bp are heterogeneous two-chain anticoagulant proteins isolated from the venom of habu snake, Trimeresurus flavoviridis. The amino acid sequence of one (B chain) of their two chains is identical. We recently reported the cloning of cDNA encoding the B chain and that of the A chain of IX/X-bp. Here, we report the isolation and characterization of cDNA clones encoding the A chain of IX-bp. The 697-bp sequence showed a putative ORF capable of encoding a 152-amino-acid protein containing a 23-amino-acid signal peptide. The overall amino acid sequence identity between the pre-A chain of IX-bp and that of IX/X-bp is 84%, whereas the cDNA sequence identity of the two ORFs is 91% indicating that habu snake acquired the variety of venom proteins by efficient mutations of antonymous sites.

Amino Acid Sequence↗

PubMed1997

Different element ratios of red cosmetics excavated from ancient burials of Japan.

Marker elements of red cosmetics, collected from ancient burials of Matsuyama, Tokushima and Nara Japan, were determined by emission spectrometry (ICP/AES). The mass ratios of Hg, Fe, Cu, and Zn were different between samples. Element levels were compared with reference to relative amounts of sulfur. Of the possible contaminants from the bone and sand of burials, the relative amounts of Hg and Fe to S were most commonly available to evaluate the difference between the cosmetics. The cosmetics were divided into four groups; type I (high Hg with less Fe), type II (both moderate Hg and Fe), type III (moderate Hg with high Fe) and type IV (less Hg with high Fe). The main constituents of cosmetics are mercury sulfide (cinnabar) or ferric oxide mixed with trace metals. Zinc contents differ between the Fe and Hg amounts for the three areas. Cosmetic compositions varied with each burial site, suggesting that they were derived from different mines of ancient Japan.

Anthropology↗

PubMed1997

Neovascularization induced by anulus and its inhibition by cartilage endplate. Its role in disc absorption.

STUDY DESIGN: A study of neovascularization with respect to the anulus and the endplate. OBJECTIVES: To determine the ability of the anulus and the endplate to induce neovascularization and to study the effect of the endplate on the neovascularizing activity of the anulus. SUMMARY OF BACKGROUND DATA: Previous studies showed that herniations contain varying amounts of nucleus pulposus, anulus fibrosus, and endplate. Results of recent studies have shown preferential neovascularization around the herniated anulus rather than the endplate. METHODS: Human L4-L5 discs were harvested from three specimens. In the left cornea of New Zealand White rabbits, an anulus (AF group) or an endplate (EP group) was implanted 4 mm from the superior corneoscleral limbus. In a third group (AF and EP group), an endplate was implanted between the anulus and the limbus. Twelve assays were done for each group. Sham surgeries were done on a fourth group as a control. Neovascularization was monitored by stereomicroscopy and scored from 0 (no neovascularization) to +5 (capillaries growing around the implant). Histologic studies were done at weekly intervals. RESULTS: In the AF group, the anulus induced neovascularization in 11 or 12 corneas. By the third week, the anulus appeared thinner, and the newly formed vessels regressed. In the EP group, only two corneas showed neovascularization; 10 corneas showed no vascularization. In the AF and EP group, three corneas showed no neovascularization, seven had sustained neovascularization, and two had vascularization. In the corneas containing the endplates, the implants remained unchanged. Control corneas did show vascularization. Histologic sections showed that neovascularization and cell infiltration were most abundant in the AF group, followed by the AF&EP and the EP groups. CONCLUSION: The reactions to anulus fibrosus and to cartilage endplate differ. Anulus fibrosus may contain soluble substances that induce neovascularization, leading to its degradation and absorption. Cartilage endplate fails to induce neovascularization or diminishes the neovascular response, preventing its degradation. Disc herniations that contain a significant amount of endplate may not be degraded nor absorbed, leading to failure in spontaneous remission of symptoms.

Animals↗

PubMed1997

A distinct function of STAT proteins in erythropoietin signal transduction.

The Janus kinase (JAK)-signal transducers and activators of transcription (STAT) pathway is an important signaling pathway of interferons and cytokines. We examined the activation of STAT proteins induced by interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or erythropoietin (EPO) using the human leukemia cell line, UT-7, which requires these cytokines for growth. IL-3, GM-CSF, and EPO induced DNA-binding activity to the oligonucleotides corresponding to the sis-inducible elements (SIE) of c-fos, in addition to the beta-casein promoter (beta-CAP), SIE- and beta-CAP-binding proteins were identical to Stat1alpha and Stat3 complex and to Stat5 protein, respectively. This indicates that IL-3, GM-CSF, and EPO commonly activated Stat1alpha, Stat3, and Stat5 proteins in UT-7. However, EPO hardly activated Stat1alpha and Stat3 in UT-7/GM, which is a subline of UT-7 that grows slightly in response to EPO. Transfection studies revealed that UT-7/GM cells constitutively expressing Stat1alpha, but not Stat3, can grow as well in response to EPO as GM-CSF, suggesting that Stat1alpha is involved in the EPO-induced proliferation of UT-7. Thus, although Stat1alpha, Stat3, and Stat5 proteins are activated by GM-CSF, IL-3, and EPO, our data suggest that each STAT protein has a distinctive role in the actions of cytokines.

Cell Division↗

PubMed1997

An ATP-dependent inwardly rectifying potassium channel, KAB-2 (Kir4. 1), in cochlear stria vascularis of inner ear: its specific subcellular localization and correlation with the formation of endocochlear potential.

Cochlear endolymph has a highly positive potential of approximately +80 mV. This so-called endocochlear potential (EP) is essential for hearing. Although pivotal roles of K+ channels in the formation of EP have been suggested, the types and distribution of K+ channels in cochlea have not been characterized. Because EP was depressed by vascular perfusion of Ba2+, an inhibitor of inwardly rectifying K+ (Kir) channels, but not by either 4-aminopyridine or tetraethylammonium, we examined the expression of Kir channel subunits in cochlear stria vascularis, the tissue that is supposed to play the central role in the generation of positive EP. Of 11 members of the Kir channel family examined with reverse transcription-PCR, we could detect only expression of KAB-2 (Kir4.1) mRNA in stria vascularis. KAB-2 immunoreactivity was specifically localized at the basolateral membrane of marginal cells but not in either basal or intermediate cells. Developmental expression of KAB-2 in marginal cells paralleled formation of EP. Furthermore, deaf mutant mice (viable dominant spotting; WV/WV) expressed no KAB-2 in their marginal cells. These results suggest that KAB-2 in marginal cells may be critically involved in the generation of positive EP.

4-Aminopyridine↗

PubMed1997

In vitro development of erythroid and megakaryocytic cells from a UT-7 subline, UT-7/GM.

UT-7 is a human megakaryoblastic leukemia cell line with absolute dependence on interleukin-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), or erythropoietin (EPO) for growth and survival. We isolated a novel subline, UT-7/GM after long-term culture of UT-7 with GM-CSF. The hemoglobin concentration and gamma-globin and EPO-receptor mRNA levels were significantly higher in EPO-treated UT-7/GM cells than in untreated cells. In contrast, the platelet factor 4 and glycoprotein IIb mRNA levels were much higher in thrombopoietin (TPO)-treated UT-7/GM cells than in untreated cells. Some TPO-treated cells had morphologically mature megakaryocytic characteristics such as a developed demarcation membrane in the cytoplasm and multilobular nuclei. These findings indicate that UT-7/GM is a bipotential cell line that can be induced to differentiate into erythroid and megakaryocytic lineages by EPO and TPO, respectively. Moreover, a minority of UT-7/GM cells acquired a high hemoglobin concentration by treatment with TPO, suggesting that TPO in part induced the erythroid differentiation of the UT-7/GM cells. Interestingly, GM-CSF inhibited the EPO- or TPO-induced erythroid differentiation and the TPO-induced megakaryocytic differentiation of UT-7/GM cells. These results support the hypothesis that cytokines influence the programming of gene expression required for lineage commitment or differentiation.

Cell Differentiation↗

PubMed1997

Clustering and enhanced activity of an inwardly rectifying potassium channel, Kir4.1, by an anchoring protein, PSD-95/SAP90.

An inwardly rectifying potassium channel predominantly expressed in glial cells, Kir4.1/KAB-2, has a sequence of Ser-Asn-Val in its carboxyl-terminal end, suggesting a possible interaction with an anchoring protein of the PSD-95 family. We examined the effects of PSD-95 on the distribution and function of Kir4.1 in a mammalian cell line. When Kir4.1 was expressed alone, the channel immunoreactivity was distributed homogeneously. In contrast, when co-expressed with PSD-95, prominent clustering of Kir4.1 in the cell membrane occurred. Kir4.1 was co-immunoprecipitated with PSD-95 in the co-expressed cells. Glutathione S-transferase-fusion protein of COOH terminus of Kir4.1 bound to PSD-95. These interactions disappeared when the Ser-Asn-Val motif was deleted. The magnitude of whole-cell Kir4.1 current was increased by 2-fold in cells co-expressing Kir4.1 and PSD-95 compared with cells expressing Kir4. 1 alone. SAP97, another member of the PSD-95 family, showed similar effects on Kir4.1. Furthermore, we found that Kir4.1 as well as SAP97 distributed not diffusely but clustered in retinal glial cells. Therefore, PSD-95 family proteins may be a physiological regulator of the distribution and function of Kir4.1 in glial cells.

Adaptor Proteins, Signal Transducing↗

PubMed1997

Ultrastructural localization of CD38 immunoreactivity in rat brain.

The subcellular localization of CD38 in the rat cerebral and cerebellar cortices was studied using immunoelectron microscopy. In the cerebral cortex, immunoreactivity was present in a subset of pyramidal neurons, and was distributed predominantly in the perikarya and dendrites. It was found in association with rough endoplasmic reticulum, ribosomes, small vesicles, mitochondria and the plasma membrane including the postsynaptic densities. In the cerebellum, labeling was observed in several types of neuron such as granule, Golgi, basket and Purkinje cells. In contrast to the cerebrum, immunoreactivity was accentuated in the perikarya or axon terminals, and the synaptic vesicles represented another organelle that was immunopositive for CD38. In both of these CNS regions, the nuclear envelope, particularly the outer membrane, showed constant labeling. Diffuse immunoreactivity was also present in the astrocytes from the perikarya to the processes including the perivascular glia limitans. Oligodendrocytes and microglia were immunonegative for CD38. The pattern of distribution of CD38 in the CNS is suggestive of multiple roles for this molecule at various functional sites in both neurons and astrocytes.

ADP-ribosyl Cyclase↗

PubMed1997

Expression and nocturnal increase of type II iodothyronine deiodinase mRNA in rat pineal gland.

It has been demonstrated that thyroxine deiodinating activity is present in rat pineal gland, and its activity increases significantly during the night time. We have studied whether mRNA for type II iodothyronine deiodinase is expressed in rat pineal gland and whether the nocturnal rise of pineal T4 deiodinating activity is due to the change in type II iodothyronine deiodinase mRNA level. Reverse transcription-polymerase chain reaction amplification and Northern blot analyses have demonstrated that type II iodothyronine deiodinase mRNA is expressed in rat pineal gland and its mRNA level increases markedly at midnight. These results suggest that the nocturnal rise in pineal T4 deiodinating activity is due to the change in type II iodothyronine deiodinase mRNA level.

Animals↗

PubMed1997

Dual effects of auranofin on prostaglandin E2 production by rat peritoneal macrophages.

Incubation of rat peritoneal macrophages in medium containing various concentrations of auranofin (1, 3 and 10 microM) increased prostaglandin E2 production at 4 h in a concentration-dependent manner, in accordance with the increase in the release of [3H]arachidonic acid from membrane phospholipids. However, at 20 h, no stimulation of prostaglandin E2 production by auranofin was observed. When the peritoneal macrophages were incubated in the presence of 12-O-tetradecanoylphorbol 13-acetate (TPA), thapsigargin or A23187, prostaglandin E2 production at 4 and 20 h was enhanced. The stimulator-induced prostaglandin E2 production at 20 h was suppressed by 10 microM of auranofin. Western blot analysis demonstrated that auranofin inhibited the induction of cyclooxygenase 2 by TPA, thapsigargin or A23187 at 4 and 20 h. The level of cyclooxygenase 1 did not change by treatment with these stimulators in the presence or absence of auranofin. These findings suggest that auranofin has dual effects on prostaglandin E2 production: without stimulation, auranofin increases prostaglandin E2 production at 4 h due to the increased release of arachidonic acid which is converted to prostaglandin E2 mainly by cyclooxygenase 1, but inhibits the stimulator-induced late-phase prostaglandin E2 production by inhibiting the induction of cyclooxygenase 2.

Animals↗

PubMed1997

Molecular cloning and nucleotide sequence of an endo-1,5-alpha-L-arabinase gene from Bacillus subtilis.

The nucleotide sequence of the gene encoding an endo-1,5-alpha-L-arabinase (protopectinase C) of Bacillus subtilis was determined by sequencing fragments amplified by the cassette-ligation-mediated PCR (CLM-PCR). The gene covering the start and stop codon was amplified by PCR with two specific primers, which were designed from the sequence data determined by CLM-PCR. An approximately 1.5-kb amplification product was cloned into the vector pUC119, forming a plasmid termed pPPC. An ORF that encodes the arabinase composed of 324 amino acids including a 33-amino-acid signal peptide was assigned. Comparison of the deduced amino acid sequence of the enzyme with that of an Aspergillus niger endoarabinase showed 37% identity in a 207-amino-acid overlap. The optimal nucleotide sequence for catabolite repression of B. subtilis was found upstream of the structural gene. In a culture of Escherichia coli DH5alpha cells harboring pPPC, no arabinase activity was detected, either intracellularly or extracellularly, suggesting that the B. subtilis promotor is not functional in this transformant. In B. subtilis IFO 3134 strain, production of protopectinase C was repressed by readily metabolizable carbohydrates. In contrast, productivity (total enzyme activity/bacterial growth) of the enzyme was increased about fourfold in the presence of 0.75 M potassium phosphate in the culture medium. The phosphate anion seemed to be involved in the stimulation of protopectinase C production in this stain.

Amino Acid Sequence↗