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M Kotani

Publications and source records attributed to M Kotani.

At least 55 records · Page 3Linked to original sources
PubMed1997

An immunohistochemical technique with a series of monoclonal antibodies to gangliosides: their differential distribution in the rat cerebellum.

Gangliosides, sialic acid-containing glycosphingolipids, are normal membrane constituents and are highly expressed in the vertebral central nervous system. Owing to their topological localization on the outer surface of neural plasma membranes and their unique chemical structure, gangliosides have been implicated in a variety of phenomena. It was, however, difficult to study the localization of gangliosides in the central nervous system because of the lack of useful probes for gangliosides. We recently established an improved method for the generation of mouse MAbs to gangliosides by immunizing C3H/HeN mice with purified gangliosides. Using this method, we succeeded in generating a large number of the MAbs specific for gangliosides. These MAbs enabled us to determine the localization of gangliosides in the rat brain. We previously described the differential distribution patterns of gangliosides in the brain regions by an immunohistochemical technique with MAbs. In the present paper, we describe an immunofluorescence technique for the detection of ganglioside distribution in the postnatal rat cerebellar cortex in detail. The principles of immunohistochemistry have been described in several review articles.

Animals↗
PubMed1997

Tissue factor expression and fibrin deposition in the lungs of patients with idiopathic pulmonary fibrosis and systemic sclerosis.

Although abnormalities of alveolar fibrin turnover have been reported to play a role in the development of idiopathic pulmonary fibrosis (IPF), the pathophysiological relevance remains unclear. We therefore investigated the localization of tissue factor (TF) and fibrin deposition in patients with IPF using immunohistochemistry and compared the results with those from patients who had interstitial pneumonia associated with systemic sclerosis (IP-SSc) and idiopathic bronchiolitis obliterans with organizing pneumonia (BOOP). Expression of TF-mRNA was also assessed, using in situ hybridization with a digoxigenin-labeled cRNA probe. In patients with IPF, IP-SSc, and idiopathic BOOP, the TF antigen was positively stained in type II pneumocytes and in some alveolar macrophages. The fibrin antigen was stained in the type II pneumocytes and the adjacent area. Tissue factor-mRNA was expressed in the type II pneumocytes and in some alveolar macrophages. Neither TF antigens nor TF-mRNA were detected in the normal lung. These results indicate that type II pneumocytes are a major source of TF, suggesting that TF production in these cells is closely related to fibrin deposition in the lungs of people with these diseases.

Base Sequence↗
PubMed1997

Experimental study of the discrimination of two signal sources in the brain by three-dimensional vector magnetic field measurement of a magnetoencephalogram.

To determine the possibility of discriminating multi-sources in the brain by 3D vector magnetic field measurement of a magnetoencephalogram (MEG), measurements were made of magnetic fields produced by two current dipoles implanted in a spherical head model. The 3D vector magnetic field measurements were made by using a 3D second-order gradiometer connected to three rf-SQUIDs, which can detect magnetic field components perpendicular to and tangential to the scalp. The MEG distribution measuring the magnetic field perpendicular to the scalp was not helpful in estimating the location and number of sources because of the lack of a dipole pattern. By referring to the MEG distribution measuring the magnetic field distribution tangential to the scalp, however, two current sources could be clearly discriminated in a spherical head model. It was found that this MEG distribution measuring tangential to the scalp could provide information on new constraint conditions for the calculation of inverse problems with multi-sources. These results were also confirmed by measurement of the mixed somatosensory evoked fields elicited by simultaneous electric stimulation to the median nerve and the thumb.

Adult↗
PubMed1997

Measurements of the quantities of particles accumulated in the lungs of workers.

The human body is enveloped in weak (10-9 T) magnetic fields generated by magnetic particles accumulated in the lungs and other organs. We studied magnetic fields from the lungs (magnetopneumogram) in order to determine how they may be utilized in the early diagnosis of pneumonoconiosis and other diseases. The effects of these diseases have been positively correlated with the size of the magnetic particle deposits in the lungs. When we measured the relaxation effect and the secondary magnetization for smokers and non-smokers, we found there was a large difference in these characteristics between these groups.

Diagnostic Imaging↗
PubMed1996

Gene expression of the human prostaglandin E receptor EP4 subtype: differential regulation in monocytoid and lymphoid lineage cells by phorbol ester.

We isolated a cDNA clone encoding the human prostaglandin (PG) E receptor EP4 subtype and examined the gene expression in human blood cells. Northern blot analysis revealed that the EP4 gene is expressed at a high level in peripheral blood mononuclear cells, and at lower levels in cultured human blood cell lines, THP-1 and U937 (monocytoid cell lines), MOLT-4 and Jurkat (T-cell lines), and Raji (B-cell line). To examine regulation of the EP4 gene expression in the immune system, we studied the effects of phorbol 12-myristate 13-acetate (PMA) on these cell lines. Gene expression was upregulated in THP-1, U937, and Raji cells by PMA, and was downregulated in MOLT-4 and Jurkat cells. In THP-1 cells the effects of PMA were further analyzed, and the upregulation of the EP4 gene was shown to be followed by an increase in PGE2 binding sites and in PGE2-induced cAMP accumulation. In the striking contrast, other PGE receptor subtypes (EP1, EP2 and EP3) and other prostanoid receptors (IP and DP) were shown not to be upregulated by PMA. Therefore, this is the first demonstration of a highly specific upregulation of the EP4 subtype in THP-1 cells treated with PMA, suggesting the importance of the EP4 subtype in the immune system. In the present study we also clarified that EP4 gene expression is regulated differently among human monocytoid and lymphoid lineage cells, thus leading to the better understanding of the regulatory mechanisms for the human EP4 gene expression in the immune system.

Burkitt Lymphoma↗
PubMed1995

Developmental changes of ganglioside expressions in postnatal rat cerebellar cortex.

We previously described the differential distribution of gangliosides in adult rat brain as detected by specific antibodies. We report here the distribution of gangliosides during the development of postnatal rat cerebellum by an immunofluorescence technique with mouse monoclonal antibodies (mAbs). Eleven mAbs that specifically recognize each ganglioside were used. Our study revealed that the expression of each ganglioside changed dramatically during the development. GD3 and O-Ac-GD3 were expressed intensely in the external granular layer at 1, 5, and 10 days, whereas GD2 was firstly detected in the internal granular layer at 5 days and GD1b WAS diffusely detected throughout all layers of the cerebellar cortex at early postnatal days. GD2 and GD1b were more intensely expressed in the granular layer at 20, 30, and 80 days, suggesting that premature granule cells expressed GD3 and its derivative, O-Ac-GD3, whereas mature granule cells express GD2 and GD1B intensely. On the other hand, GM1 was exclusively detected in the external granular layer and the molecular layer at 1 and 5 days. The staining sites spread gradually from these outer layers into the internal granular layer and the white matter after 10 days. The positive cells in the external granular layer and the molecular layer appeared to be Bergmann glial cells and their radially ascending cytoplasmic processes. The intensity of the staining in these specialized astroglial cells decreased gradually during postnatal days. In contrast, the expression of GQ1b was very faint at birth, but gradually increased during the development and was detected intensely in the internal granular layer, particularly in the cerebellar glomeruli in adulthood, suggesting that GQ1b expression may be associated with synapse-related structures. The developmental changes of the expression of other gangliosides were also recognized in the postnatal rat cerebellum. These results suggest that specific gangliosides may play an important role in regulating the early events responsible for the orderly formation of the cerebellar cortex.

Animals↗
PubMed1995

Promotion by estriol of the development of grafted fetal livers in mice.

Mouse fetal livers of 12 days gestation were transplanted beneath the kidney capsule of syngeneic castrated male hosts. Recipients received a single intraperitoneal injection of 5 mg estriol (E3) immediately after transplantation and were sacrificed 4, 7 and 14 days later. Control mice received injections of solvent only. The fetal liver grafts of the E3 groups showed remarkable growth compared with the control grafts at each corresponding time. In the grafted fetal livers of the E3 groups, many more basophilic hepatocytes appeared for much longer periods, and the early formation of wide sinusoids was noted. Although hemopoietic activity had almost ceased a short time after transplantation in the control grafts, many prominent extrasinusoidal erythroid, granulocytic and megakaryocytic hemopoietic foci developed and persisted to the end of the experiments in the grafted fetal livers of the E3 groups. In the next experiment, the recipients received three injections of 0.5-5 mg E3 at 5-day intervals, and were sacrificed 21 days after transplantation. Control animals received solvent only in the same manner. The fetal liver grafts of the E3 groups also showed remarkable, dose dependent growth. The growth was particularly striking at doses of 3-5 mg E3. In the grafts of these groups, basophilic hepatocytes were predominant, and hepatic cords and sinusoids were well formed. Moreover, sinusoidal erythropoiesis was intense. These results provide evidence for the possible role of E3, secreted from the placenta into the fetal circulation, in the development of the fetal liver.

Animals↗
PubMed1995

Molecular cloning and expression of multiple isoforms of human prostaglandin E receptor EP3 subtype generated by alternative messenger RNA splicing: multiple second messenger systems and tissue-specific distributions.

Five distinct cDNA clones encoding four different isoforms of human prostaglandin (PG) E receptor EP3 subtype were isolated from a human kidney cDNA library. Two cDNA clones differed only in their 3'-untranslated regions. The four isoforms, tentatively named EP3-I, EP3-II, EP3-III, and EP3-IV, which were generated by alternative mRNA splicing, had identical amino acid sequences except for their different carboxyl-terminal tails. Transfection experiments revealed that all the four isoforms show high binding affinities to PGE2, PGE1, and M&B28767, an EP3-specific agonist, whereas their downstream signaling pathways are divergent. M&B28767 increased cAMP concentrations in cells expressing EP3-II and EP3-IV, whereas it inhibited forskolin-induced cAMP accumulations in cells expressing all EP3 isoforms. M&B28767 also stimulated phosphoinositide turnover in cells expressing EP3-I and EP3-II. Northern blot analysis revealed that the EP3 gene is expressed in a wide variety of human tissues. The human EP3 mRNA was present most abundantly in the kidney, pancreas, and uterus. A substantial expression was also detected in the heart, liver, skeletal muscle, small intestine, colon, prostate, ovary, and testis. Furthermore, reverse transcription-polymerase chain reaction analysis demonstrated tissue-specific expressions of the five different EP3 mRNA species. The present study suggests the presence of the multiple systems of PGE2/EP3 isoforms and leads to the better understanding of its physiological and pathophysiological implications in humans.

Alternative Splicing↗
PubMed1995

Localization of sulfatides in the epithelial lining of gastric mucosa: studies with a monoclonal antibody to sulfatides.

A specific monoclonal antibody against sulfatides was used to examine the cellular localization of sulfatides, the most predominant acidic glycosphingolipids in both rabbit and human gastric mucosa. The monoclonal antibody selectively recognized sulfatides among the acidic glycolipids extracted from rabbit gastric mucosa. Immunofluorescence staining revealed that specific staining was localized in the epithelial cells of the stomach. Both epithelial and glandular cells were stained in the fundic mucosa. In human stomach, the staining pattern was essentially identical to that in rabbit stomach. The specific localization of sulfatides in the epithelial lining supports our hypothesis that they may be a component of mucosal defensive substances and an adhesion receptor for Helicobacter pylori.

Animals↗
PubMed1994

Generation of monoclonal antibodies specific for ganglioside lactones: evidence of the expression of lactone on human melanoma cells.

We generated 3 murine monoclonal antibodies (MAbs) specific for ganglioside lactones by immunizing C3H/HeN mice with purified lactones adsorbed to Salmonella minnesota followed by fusion with mouse myeloma cells. The use of a wide variety of glycolipids, including ganglioside lactones, enabled the precise structures recognized by these MAbs to be elucidated through an ELISA and by immunostaining on thin-layer chromatography. MAb AMR38, which was generated with GM1 lactone, showed restricted specificity, detecting only the GM1 lactone used for immunization. None of the other ganglioside lactones, intact gangliosides (including GM1) or neutral glycolipids tested were recognized. In contrast, MAbs AMR40 and AMR19, which were generated with GD1a lactone and GD3 lactone, respectively, showed broader specificities, recognizing several ganglioside lactones. However, the precise epitopes were different. MAb AMR40 reacted intensely with ganglioside lactones having an external NeuAc alpha 2-->3Gal-sequence (GD1a, GM3, GM1b, GT1b, and IV3NeuAc alpha-nLc4Cer), but not with those having a NeuAc alpha 2-->8NeuAc alpha 2-->3Gal- sequence. On the other hand, MAb AMR19 reacted with ganglioside lactones having a NeuAc alpha 2-->8NeuAc alpha 2-->3Gal- sequence (GD3, O-Ac-GD3, GD2, GDlb, GTlb, GQlb and GTla), but not with those having a NeuAc alpha 2-->3Gal- sequence. None of the intact gangliosides or neutral glycolipids tested were recognized by the MAbs. We also determined the expression of ganglioside lactones on human melanoma cells grown in athymic nude mice by means of an immunofluorescence technique.

Animals↗
PubMed1994

Site-specific alteration of arginine 376, the unique positively charged amino acid residue in the mid-membrane-spanning regions of the proline carrier of Escherichia coli.

An alignment of 5 amino acids in the Escherichia coli proline carrier (G328-A366-L371-GR376) is common in the amino acid sequences of several Na+ symport carriers, and it has been proposed as the putative sodium binding motif (Deguchi, Y., Yamato, I., and Anraku, Y. (1990) J. Biol. Chem. 265, 21704-21708). To determine whether these amino acids are essential for Na+ symport activity as the Na+ binding site, one of the amino acids in this alignment, Arg-376, which is the only positively charged amino acid in the innermost part of the predicted membrane-spanning regions, was changed to either lysine, glutamine, or glutamic acid by oligonucleotide dependent site-specific mutagenesis. The transport and binding activities of the proline of the R376K mutant carrier were not detected at all. The activities of the other mutant carriers for uptake and binding of proline were as high as those of the wild-type carrier. These two mutant carriers were as sensitive to the proline analogue azetidine-2-carboxylate and to N-ethylmaleimide as the wild-type carrier, indicating that they have the same properties as the wild-type. The amounts of the carrier proteins expressed from these mutated putP genes were similar to that from the wild-type gene. These results imply that the Arg-376 in the proline carrier does not reside at the sodium binding site, suggesting that the similar alignment found in the amino acid sequences of several Na+ symport carriers is not essential for the transport or binding activities, although this similar alignment may have some relevance to the structure of the Na+ symporter. Furthermore, that the only Arg residue in the middle part of the predicted membrane-spanning regions is dispensable for the energy coupling activity indicates a unique difference of the coupling mechanism from the other secondary active transport systems, such as that of the lactose permease and the tetracycline/H+ antiporter.

Amino Acid Sequence↗
PubMed1994

Generation of one set of murine monoclonal antibodies specific for globo-series glycolipids: evidence for differential distribution of the glycolipids in rat small intestine.

We generated four murine monoclonal antibodies (MAbs) specific for globo-series glycolipids by immunizing C3H/HeN mice with these purified glycolipids adsorbed to Salmonella minnesota followed by fusion with mouse myeloma cells. By use of a wide variety of glycolipids, the precise structures recognized by these MAbs were elucidated through an enzyme-linked immunosorbent assay and an immunostaining on thin-layer chromatography. These four MAbs, designated as BGR23, BGR47, BMR26, and BGR27, exhibited highly restricted binding specificities, reacting only with the globo-series glycolipids Gb3Cer, III3Gal alpha-Gb3Cer, Gb4Cer, and IV3GalNAc alpha-Gb4Cer, respectively, which were used for immunization. None of the other various glycolipids or gangliosides were recognized. We determined the localization of these globo-series glycolipids in adult rat small intestine by means of an immunofluorescence technique with these MAbs. Our study revealed that there is a differential distribution of these glycolipids in the rat tissue. III3Gal alpha-Gb3Cer was demonstrated on the cryptic cells and circular muscle, whereas Gb4Cer was localized on both the circular and longitudinal muscles. The expression of Gb3Cer was associated with the epithelium and the capillary endothelial cells in the lamina propria mucosae as well as with the tunica submucosa, whereas IV3GalNAc alpha-Gb4Cer was detected on the epithelium, capillary endothelial cells in the lamina propria mucosae, and both the muscle layers.

Animals↗
PubMed1994

Immunohistochemical localization of minor gangliosides in the rat central nervous system.

We previously described the differential distribution of major gangliosides (GM1, GD1a, GD1b, GT1b and GQ1b) in adult rat brain detected by specific antibodies (Kotani, M., Kawashima, I., Ozawa, I., Terashima, T. and Tai, T. Glycobiology, 3, 137-146, 1993). We report here the distribution of minor gangliosides in the adult rat brain by an immunofluorescence technique with mouse monoclonal antibodies (MAbs). Ten MAbs (GMR6, GMB28, GMR11, GMR19, GMR2, GMR7, GGR51, AMR10, NGR54 and NGR53) that specifically recognize GM3, GM2, GT1a, GD3, O-Acdisialoganglioside, GD2, GM1b, GM4, IV3NeuAc alpha-nLc4Cer and IV6NeuAc alpha-nLc4Cer, respectively, were used. Our study revealed that there is a cell type-specific expression of minor gangliosides, as well as major gangliosides, in the rat brain. In the cerebellar cortex, GM3 was expressed intensely in the white matter and slightly in the granular layer. GD3 was present in both the granular layer and the white matter, but not in the Purkinje cell layer or in the molecular layer. An O-Ac-disialoganglioside, which was suggested to be O-Ac-LD1, was detected exclusively in both the molecular layer and Purkinje cell layer. The presence of GD2 was restricted to the granular layer. GM4 was associated with some astrocytes, but not with myelin or oligodendrocytes. GM2, GT1a, GM1b, IV3NeuAc alpha-nLc4Cer and IV6NeuAc alpha-nLc4Cer gangliosides were not clearly detected in the cerebellar cortex. In other regions, such as cerebral cortex, hippocampal formation and spinal cord, the expression of the gangliosides was also highly localized to a specific cell type and layer.

Animals↗
PubMed1994

Evidence for direct binding of intracellularly distributed ganglioside GM2 to isolated vimentin intermediate filaments in normal and Tay-Sachs disease human fibroblasts.

Although some intracellularly distributed glycosphingolipids are reported to be associated with vimentin intermediate filaments or colchicine sensitive cytoskeleton, no direct evidence for such an association has yet been shown. In this report we demonstrated that the intracellularly distributed ganglioside GM2 directly binds to isolated vimentin intermediate filaments in normal and Tay-Sachs disease human fibroblasts. Indirect immunofluorescence microscopy using a GM2-specific monoclonal antibody demonstrated filamentously distributed GM2 in the cytoplasm. A double staining of Tay-Sachs fibroblasts with anti-GM2 and anti-vimentin monoclonal antibodies strongly suggested that the GM2 positive filaments are vimentin intermediate filaments. We then isolated vimentin, in the presence of a detergent and urea, from the normal human skin fibroblasts and murine mastocytoma cells. In a solid phase enzyme-linked immunosorbent assay, the isolated vimentin dose-dependently reacted with both anti-vimentin and anti-GM2 monoclonal antibodies but not with anti-GM3 or anti-GM1 monoclonal antibody. The molar ratio of GM2 to vimentin was approximately 20:1. The lipid fraction extracted from the purified vimentin preparation was immunostained with anti-GM2 on a thin-layer chromatography plate. Furthermore, only one band was detected at the molecular weight of 57 kDa, after electroblotting and simultaneous immunostaining with anti-GM2 and anti-vimentin monoclonal antibodies. These results clearly indicated that ganglioside GM2 directly binds to vimentin.

Animals↗
PubMed1994

Development of a novel drug release system, time-controlled explosion system (TES). I. Concept and design.

A novel controlled drug release system. Time-Controlled Explosion System (TES) has been developed. TES has a four-layered spherical structure, which consists of core, drug, swelling agent and water insoluble polymer membrane. TES is characterized by a rapid drug release with a precisely programmed lag time; i.e. expansion of the swelling agent by water penetrating through the outer membrane, destruction of the membrane by stress due to swelling force and subsequent rapid drug release. For establishing the concept and development strategy, TES was designed using metoprolol and polystyrene balls (size: 3.2 mm in diameter) as a model drug and core particles. Among the polymers screened, low-substituted hydroxypropylcellulose (L-HPC) and ethylcellulose (EC) were selected for a swelling agent and an outer water insoluble membrane, respectively. The release profiles of metoprolol from the system were not affected by the pH of the dissolution media. Lag time was controlled by the thickness of the outer EC membrane; thus, a combination of TES particles possessing different lag times could offer any desired release profile of the model compound, metoprolol.

Chemistry, Pharmaceutical↗
PubMed1993

Generation of a monoclonal antibody specific for a new class of minor ganglioside antigens, GQ1b alpha and GT1a alpha: its binding to dorsal and lateral horn of human thoracic cord.

We have established a monoclonal antibody, GGR41, specific for a new class of minor gangliosides, such as GQ1b alpha and GT1a alpha, by immunizing mice with a GQ1b-rich ganglioside fraction extracted from bovine brain. Each of those minor gangliosides has been reported to be one of the cholinergic-specific gangliosides (Chol-1). Careful examination of binding specificity of the antibody by both an enzyme-linked immunosorbent assay and immunostaining on thin-layer chromatograms showed that the antibody recognizes three sialyl residues separately attaching to the gangliotetraosyl backbone structure. Immunohistochemical analysis revealed that GGR41 immunostained lamina I and III of dorsal horn and lateral horn of human thoracic cord but motor neurons were not immunostained. Except for negative staining of motor neurons, this distribution is similar to the distribution pattern of staining as reported in rats and humans using a polyclonal antibody against Chol-1. Thus, the antibody obtained in this study should be a useful reagent to study the function of a unique new class of the minor gangliosides.

Animals↗
PubMed1993

Generation and characterization of monoclonal antibodies against rabbit CD4, CD5 and CD11a antigens.

We describe in this report the production and characterization of monoclonal antibodies (mAb) to the rabbit homologues of CD4, CD5 and CD11a antigens, and their use for phenotypic analysis of rabbit lymphoid cell lines. All the mAbs were produced by immunizing mice with rabbit thymocytes. mAb KEN-4 apparently identified rabbit CD4, precipitated two bands of 42 and 50 kDa under reducing and non-reducing conditions and markedly inhibited allo-MLR. The distribution of antigen-positive cells were restricted to the thymus and classical T-dependent areas in peripheral lymphoid tissues. mAb KEN-5 apparently identified rabbit CD5, precipitated a single polypeptide of 67 kDa similar to other anti-CD5 mAb in the human and mouse. The use of this mAb revealed that CD5+ B cells were infrequent in this species. mAb KEN-11 apparently identified rabbit CD11a and precipitated a heterodimer of 150/95 kDa by selectively recognizing the 150 kDa moiety. It blocked cation-dependent aggregation of phorbol ester-induced rabbit Con A blasts and also allo-MLR in a similar manner to other anti-CD11a mAb in various animal species. Phenotypic examination of HTLV-1 transformed rabbit lymphoid cell lines using these mAb clearly indicated that most of them were CD4+, CD5+ and CD11a+, and hence derived from CD4+ T cells. These mAb will be useful tools for the study of the cellular immune system in the rabbit.

Animals↗