Search PubMed⌕ Search

Biomedical subjects

Y Ninomiya

Publications and source records attributed to Y Ninomiya.

At least 127 records · Page 7Linked to original sources
PubMed1997

Kinetic diversity in the fusion of exocytotic vesicles.

The speed at which secretory vesicles fuse with the plasma membrane is a key parameter for neuronal and endocrine functions. We determined the precise time courses for fusion of small clear and large dense-core vesicles in PC12 and chromaffin cells by simultaneously measuring both plasma membrane areas and release of vesicular contents. We found that instantaneous increases in cytosolic Ca2+ concentration evoked vesicle fusion, but with time constants that varied over four orders of magnitude among different types of vesicles and cells. This indicates that the molecular machinery for the final Ca2+-dependent fusion steps of exocytosis is highly variable and is as critical as Ca2+ signalling processes in determining the speed and amount of secretion of neurotransmitters and hormones. Our results suggest a new possibility that the molecules responsible for the final fusion reaction that leads to vesicle fusion are key determinants for neuronal plasticity and hormonal disorders.

Acetylcholine↗
PubMed1997

Transcriptional regulation by competition between ELP isoforms and nuclear receptors.

ELP is a transcription factor belonging to the nuclear receptor superfamily. The consensus binding sequence for ELP contains a half site of the nuclear receptor recognition element. We demonstrated previously that ELP1, the repressor type isoform of ELP, competes for binding with the retinoic acid receptor and represses retinoic acid-induced transactivation. In this study, competitive repression by ELP1 was investigated for several other nuclear receptors. As in the case of the retinoic acid receptor, binding of vitamin D receptor, thyroid hormone receptor, and estrogen receptor could be competed by ELP1, resulting in repression of their ligand-dependent transactivation. Interestingly, the activator-type ELP isoforms were capable of repressing retinoic acid-induced transactivation through binding to the retinoic acid receptor binding element. These data suggest that competition for target DNA binding is a general mechanism of transcriptional repression by ELP isoforms.

3T3 Cells↗
PubMed1997

Purification and characterization of human nephritogenic antigen that induces anti-GBM nephritis in rats.

Human nephritogenic antigen induces anti-glomerular basement membrane antibody glomerulonephritis in rats. This antigen was purified from collagenase-solubilized renal basement membrane by means of gel filtration and affinity chromatography using a rabbit antibody. Western blots of the purified nephritogenic antigen using epitope-defined monoclonal antibodies showed that it contains the NC1 domains of the a1 to a6 chains of type IV collagen. Nephritogenicity was thought to be a feature of the NC1 domains of the a3 to a5 chains, because the a6 chain is not located in the glomerular basement membrane, and because an NC1 fraction consisting of the NC1 domains of the a1 and a2 chains was poorly nephritogenic. Autoantibodies in the sera of patients with Goodpasture's syndrome were detected by ELISA using the purified nephritogenic antigen. These results indicate that the nephritogenic antigen contains the Goodpasture antigen, defined as the antigen reactive with sera from patients with Goodpasture's syndrome.

Animals↗
PubMed1997

Apoptotic cell death in neuronal differentiation of P19 EC cells: cell death follows reentry into S phase.

Apoptotic cell death was observed during aggregate culture of the mouse embryonal carcinoma cell line P19 exposed to all-trans retinoic acid (tRA). This finding was confirmed by genomic DNA agarose gel electrophoresis and transmission electron microscopy. Apoptosis was associated with P19 cell neuronal differentiation; alternative causes of cell death, i.e., cavitation-related, cytotoxicity of tRA, or spontaneous cell death were excluded. Analysis by flow cytometry revealed that the apoptosis was likely to occur in multiplying cells that underwent to reentering into S phase. We therefore examined 5-bromo-2'-deoxyuridine (BrdU) incorporation and proliferating cell nuclear antigen (PCNA) expression and localization in the aggregates by immunofluorescent staining. Although the P19 cells in the aggregates exposed to tRA incorporated BrdU at an equivalent level to those not exposed to tRA, the cells showed diminished PCNA expression and nuclear accumulation. We propose that P19 apoptosis during neuronal differentiation is a model system in which programmed cell death occurs simultaneously with cell division leading to differentiation.

Animals↗
PubMed1997

Sucrose consumption in mice: major influence of two genetic loci affecting peripheral sensory responses.

Individual variability in sucrose consumption is prominent in humans and other species. To investigate the genetic contribution to this complex behavior, we conducted behavioral, electrophysiological, and genetic studies, using male progeny of two inbred mouse strains (C57BL/6ByJ [B6] and 129/J [129]) and their F2 hybrids. Two loci on Chromosome (Chr) 4 were responsible for over 50% of the genetic variability in sucrose intake. These loci apparently modulated intake by altering peripheral neural responses to sucrose. One locus affected the response threshold, whereas the other affected the response magnitude. These findings suggest that the majority of difference in sucrose intake between male B6 and 129 mice is due to polymorphisms of two genes that influence receptor or peripheral nervous system activity.

Animals↗
PubMed1997

Taste in chimpanzees II: single chorda tympani fibers.

Data are presented from 48 taste fibers in chorda tympani nerves of 10 chimpanzees during taste stimulation with 29 stimuli. The results demonstrated a higher taste fiber specificity than in any other mammalian species reported; breadth of tuning equals 0.3. Hierarchical cluster analysis separated an S-cluster (50% of all fibers), an N-cluster (31%), and a Q-cluster (19%). The S-cluster showed the highest specificity. Its fibers responded, with few exceptions, to every sweetener tested, including the sweet proteins brazzein and monellin. The response grew with increasing sweetener concentration. A large response to one sweetener was generally accompanied by a large response to all other sweeteners, and vice versa. Except for one broadly tuned fiber, the fibers of the S-cluster never responded to the bitter compounds. The fibers of the Q-cluster were more broadly tuned than any other fibers. Quinine hydrochloride was their best stimulus, but most fibers were also stimulated by KCl and NaCl with amiloride. Acids stimulated some of these fibers. The N-cluster could be divided into 3 subclusters: an Na-subcluster (3 fibers), Na-K subcluster (10 fibers), and M-subcluster (3 fibers). The Na-fibers responded strongly to, and were quite specific to, NaCl and LiCl stimulation but not to KCl, and fibers of the Na-K subcluster responded equally well to NaCl and KCl. The response to NaCl was suppressed by amiloride in the fibers of the Na-subcluster, but not in the fibers of the Na-K subcluster. Umami compounds elicited the strongest responses in the M-subcluster.

Animals↗
PubMed1997

p53 gene mutation analysis in porokeratosis and porokeratosis-associated squamous cell carcinoma.

In this and previous studies, we have shown p53 overexpression immunohistochemically in 14 of 17 porokeratotic specimens obtained from 14 lesions of nine cases, and in all six specimens of squamous cell carcinoma (SCC) arising on porokeratotic lesions of two cases. We screened mutations in exons 5 to 10 of the p53 gene in all these specimens by polymerase chain reaction-single strand conformation polymorphism analysis. Mutations of the p53 gene were detected in two of the six SCCs but not in any of the 17 porokeratotic specimens. These two mutations were C to T transitions at codons 146 and 175 in exon 5, which were a nonsense mutation at a dipyrimidine site and a missense mutation at a CG site, respectively. To our knowledge, neither of these mutations has been identified in skin cancers before. Our observations indicate that mutations of the p53 gene are not the major molecular etiology for porokeratosis, but are related to its skin carcinogenesis, and that p53 overexpression in porokeratosis is not due to p53 gene mutations.

Adult↗
PubMed1997

Differential expression of alpha 1(IV), alpha 2(IV), alpha 5(IV) and alpha 6(IV) collagen chains in the basement membrane of basal cell carcinoma.

Type IV collagen, the major component of basement membrane, consists primarily of alpha 1(IV) and alpha 2(IV) chains. Recently, other types of collagen IV chains, i.e. alpha 3(IV), alpha 4(IV), alpha 5(IV) and alpha 6(IV) chains, have been identified by protein chemistry and molecular cloning. We have examined the diversity of the assembly of alpha (IV) chains of the basement membrane surrounding tumour nests of basal cell carcinomas, in tissues from 11 patients, by immunohistochemical analysis using specific monoclonal antibodies to six alpha (IV) chain. The immunostaining profile of each chain differed with respect to the histological subtypes of basal cell carcinoma. In the morphea-like subtype, which was more invasive, alpha 1(IV) and alpha 2(IV) chains were discontinuously stained, and alpha 5(IV) and alpha 6(IV) chains were entirely absent. However, in the superficial subtype, which was non-aggressive, alpha 1(IV), alpha 2(IV), alpha 5(IV) and alpha 6(IV) chains were well stained compared with the other subtypes of basal cell carcinoma. In addition, in the solid subtype, which showed slow growth and ulceration, alpha 1(IV) and alpha 2(IV) chains were continuously stained, and alpha 5(IV) and alpha 6(IV) chains were discontinuous or absent. The assembly of alpha 5(IV) and alpha 6(IV) chains into the basement membrane was inhibited in the solid and morphea subtypes of BCC. This differential expression of type IV collagen chains seems to be associated with the invasive potential of basal cell carcinoma.

Adult↗
PubMed1997

Cranial anomaly of homozygous rSey rat is associated with a defect in the migration pathway of midbrain crest cells.

Craniofacial development of vertebrates depends largely on neural crest contribution and each subdomain of the crest-derived ectomesenchyme follows its specific genetic control. The rat small eye (rSey) involves a mutation in the Pax-6 gene and the external feature of rSey homozygous embryos exhibits craniofacial defects in ocular and frontonasal regions. In order to identify the mechanism of craniofacial development, we examined the cranial morphology and migration of cephalic crest cells in rSey embryos. The chondrocranial defects of homozygous rSey embryos primarily consisted of spheno-orbital and ethmoidal anomalies. The former defects appeared to be brought about by the lack of the eye. In the ethmoid region, the nasal septum and the derivative of the medial nasal prominence were present, while the rest of the nasal capsule, as well as the nasal and lachrymal bones, were totally absent except for a pair of cartilaginous rods in place of the nasal capsule. This suggests that the primary cranial defect is restricted to the lateral nasal prominence derivatives. Dil labeling revealed the abnormal migration of crest cells specifically from the anterior midbrain to the lateral nasal prominence in homozygous rSey embryos. Pax-6 was not expressed in the crest cells but was strongly expressed in the frontonasal ectoderm. To determine whether or not this migratory defect actually resides in environmental cues, normal midbrain crest cells from wild-type embryos were labeled with Dil and were orthotopically injected into host rSey embryos. Migration of the donor crest cells into the lateral nasal prominence was abnormal in homozygous host embryos, while they migrated normally in wild-type or heterozygous embryos. Therefore, the cranial defects in rSey homozygous embryos are due to inappropriate substrate for crest cell migration towards the lateral nasal prominence, which consistently explains the cranial morphology of homozygous rSey embryos.

Animals↗
PubMed1997

Epitope-defined monoclonal antibodies against type-IV collagen for diagnosis of Alport's syndrome.

BACKGROUND: Alport's syndrome can be diagnosed by staining the alpha 5 chain of type IV collagen in kidney biopsy specimens with a monoclonal antibody. Because antibodies already established against the alpha 5 chain require denaturation treatment of cryostat sections to expose their epitopes. To save time and effort for staining, a new epitope-defined monoclonal antibody whose epitope is initially exposed on the surface of the molecule was established. METHODS: Two monoclonal antibodies against the triple-helical domains of the type IV collagen alpha 2 and alpha 5 chains were established with synthetic peptides as immunogens by the rat lymph node method. Their epitope were EAIQP at the positions of 675-679 of the alpha 2 chain, and IDVEF at the positions of 251-255 of the alpha 5 chain, respectively. They were purified with synthetic peptide-coupled affinity columns, and then conjugated with Texas red and FITC, respectively. RESULTS: The mixture of fluorochrome-conjugated antibodies was able to detect the distribution of the alpha 2 and alpha 5 chains in the normal and Alport kidney and skin by direct immunofluorescence staining with and without denaturation treatment of the sections. CONCLUSIONS: The direct double immunofluorescence staining of kidney and skin cryostat sections with the fluorochrome-conjugated antibodies is useful, reliable, and convenient for diagnosis of Alport's syndrome.

Amino Acid Sequence↗
PubMed1997

There is temporal and spatial expression of alpha1 (IV), alpha2 (IV), alpha5 (IV), alpha6 (IV) collagen chains and beta1 integrins during the development of the basal lamina in an "in vitro" skin model.

Temporal and spatial expression of alpha1 (IV), alpha2 (IV), alpha3 (IV), alpha4 (IV), alpha5 (IV), and alpha6 (IV) collagen chains was studied during the formation of the basal lamina in an "in vitro" skin model. A sequential study was performed at 7-d and 14-d cultures (lamina densa absent) and at 28-, 36-, and 56-d cultures (lamina densa present). Expression of beta1, beta4, alpha1, alpha2, alpha3, alpha5, alpha6 integrin subunits and co-localization with collagen IV was studied by regular and laser confocal indirect immunofluorescence microscopy. mRNA expression of alpha2 (IV) and alpha6 (IV) chains was estimated by northern blots. The earliest expression of alpha1 (IV) and alpha2 (IV) collagen chains was noted in 7-d cultures restricted to basal keratinocytes. At 14-d cultures, alpha1 (IV) and alpha2 (IV) chains were noted in basal keratinocytes and as a broad band (10 microm) in the adjacent dermis. At this stage 80% of the alpha2 (IV) mRNA was expressed in the dermis and 20% in the epidermis. At 28-, 36-, and 56-d cultures the alpha1 (IV) and alpha2 (IV) chains were present in a linear distribution at the epidermo-dermal junction and in the upper dermis. The alpha6 (IV) collagen chains were expressed much later at 36-d cultures and the alpha5 (IV) at 56 d, both mostly in a linear distribution but also in the adjacent dermis. Alpha6 (IV) mRNA was demonstrated in the dermis of 36-d cultures. There was co-localization of collagen IV and beta1 integrin subunits in 14-d cultures at the matrix site of keratinocytes. Functional perturbation studies with AIIB2 monoclonal antibody (anti-beta1 subunits) and competitive inhibition with a collagen cyanogen bromide digestion derived fragment (CB3[IV]) that contains the collagen IV ligand for alpha1beta1, alpha2beta1 integrins, altered the pattern of collagen IV deposition.

Antibodies, Monoclonal↗
PubMed1997

Lack of gurmarin sensitivity of sweet taste receptors innervated by the glossopharyngeal nerve in C57BL mice.

Effects of a sweet response inhibitor, gurmarin, on responses of the chorda tympani and glossopharyngeal nerves were studied in the C57BL/KsJ strain of mice. The lingual application of gurmarin at 3.0 microg/ml (approxiamtely 0.7 microM) or more significantly suppressed chorda tympani responses to 0.5 M sucrose, as previously reported. The magnitude of gurmarin inhibition of the chorda tympani responses reached a plateau (approximately 45% of control) at 50 microg/ml (approximately 11.9 microM). In contrast, no such gurmarin inhibition of sucrose responses was observed in the glossopharyngeal nerve even at 100 microg/ml (approximately 23.8 microM). The lingual application of a proteolytic enzyme, pronase, suppressed sucrose responses to <20% of control in both chorda tympani and glossopharyngeal nerves. These results suggest differential sensitivity to gurmarin by sweet taste receptors innervated by the chorda tympani and the glossopharyngeal nerves. The former apparently possess gurmarin sensitivity, whereas most of the latter may be lacking sensitivity.

Animals↗
PubMed1997

Primate sense of taste: behavioral and single chorda tympani and glossopharyngeal nerve fiber recordings in the rhesus monkey, Macaca mulatta.

The responses of 51 chorda tympani proper (CT) and 33 glossopharyngeal (NG) neural taste units from the rhesus monkey (Macaca mulatta) were recorded during stimulation of either the anterior (CT) or posterior (NG) part of the tongue with 26 stimuli that taste salty, umami, sour, bitter, and sweet to humans. In the CT, hierarchical cluster analysis separated four major clusters. The N and S clusters were most populous, followed by the H cluster and a small Q cluster. NaCl, monosodium glutamate (MSG), and MSG with guanosine 5'-monophosphate were the best stimuli in the N cluster. Amiloride suppressed responses to NaCl. KCl did not stimulate fibers from this cluster. S cluster fibers were characterized by strong responses to all sweeteners. The H cluster responded best to acids but also to some of the sweeteners such as xylitol, fructose, and sucrose. Q fibers responded well to quinine hydrochloride (QHCl) and caffeine, but not to denatonium benzoate. In the NG, hierarchical cluster analysis separated three major clusters. Q fibers formed the largest cluster. QHCl, caffeine, and sucrose octa-acetate but not denatonium benzoate elicited very strong responses in these fibers. S fibers formed a second cluster. Although most of the sweeteners stimulated the S fibers, their responses were not so pronounced as in CT S fibers. The small M cluster was formed by fibers that responded best to MSG. They also responded to NaCl and acids. Two bottle preference tests showed a positive relationship between a sweetener's ability to stimulate the taste fibers and the animals' consumption. Thus the most-liked sweeteners stimulated the S cluster fibers of CT best, whereas less-liked sweeteners such as D-phenylalanine elicited a response in Q fibers and sodium cyclamate stimulated N fibers. The results show that both CT and NG taste fibers of M. mulatta group according to the human concepts of taste qualities.

Animals↗
PubMed1997

Three-dimensional analysis of vasospastic major cerebral arteries in rats with the corrosion cast technique.

BACKGROUND AND PURPOSE: Although mice, rats, and other small animals are commonly used for molecular biology research, their use in the evaluation of cerebral vasospasm after subarachnoid hemorrhage is somewhat problematic because of the correspondingly small size of their cerebral vessels. We have already reported that the corrosion cast technique was useful for evaluating newly formed cerebral vessels in neural grafts in these small animals. In the present study we applied the corrosion cast technique to the evaluation of hemolysate-induced cerebral vasospasm in rats and performed three-dimensional analysis for comparison. The casting was done 10 minutes after the hemolysate injection, so that only acute "vasospasm" was assessed. METHODS: After withdrawal of 0.1 mL cerebrospinal fluid, 0.2 mL hemolysate (n = 9) or saline (n = 10) was injected into the cisterna magna of male Sprague-Dawley rats weighing between 300 and 350 g. Ten minutes later, perfusion of a semipolymerized casting medium was performed at an injection pressure of 100 to 120 mm Hg. The brains were immersed and corroded in 10% NaOH solution. After these procedures, the basilar artery as well as peripheral vessels was analyzed morphologically with scanning electron microscopy. Conventional histological analysis with the use of paraffin-embedded section with hematoxylin-eosin staining was also performed, and the results were compared with those for the corrosion cast methods. RESULTS: In the saline-injected group, SEM showed that the inner surface of the basilar artery was smooth and the form of the endothelial cell was printed on the surface of the cast. In the hemolysate-injected group, the basilar artery showed an apparent vasospasm over its entire length, and corrugation was observed on the inner surface of the basilar artery in a three-dimensional fashion. Higher magnification revealed that the nuclei of the endothelial cells were distorted. Local narrowing of the basilar artery and vasospasm in the arteries of the anterior circulation and in peripheral arteries were also observed. Measurement of the inner diameter of the basilar artery showed 37.8% contraction in the hemolysate-injected group compared with the saline-injected group by the corrosion cast method. This degree of vasospasm was similar to that observed by the conventional histological method. CONCLUSIONS: In this report we show that detailed three-dimensional observation in the rat can be performed qualitatively and quantitatively with the corrosion cast technique. We conclude that this method derives an accurate measurement of the diameter of rat major cerebral arteries and is more reliable for analyzing vasospasm in rats than angiography and other conventional procedures.

Animals↗
PubMed1997

Laminin alpha 1, alpha 2, alpha 4 and beta 1 chain mRNA expression in mouse embryonic, neonatal, and adult hearts.

We examined the relative expression of laminin alpha 1, alpha 2, alpha 4 and beta 1 chain genes in the heart of embryonic, neonatal and adult BALb/c mice. The reverse transcriptase-polymerase chain reaction was employed to determine the mRNA expression of these chains because of the relatively small amount of RNA extracted from rat embryonic hearts. Glyceraldehyde 3-phosphate dehydrogenase and beta-actin were used as internal controls. Among the hearts examined, a relatively high expression of laminin alpha 1 chain was observed in the embryonic hearts, while its expression was very weak in the neonatal and negligible in the adult hearts. Conversely, expression of laminin alpha 2 chain was virtually not observed in the embryonic hearts, but this chain was expressed weakly in the neonatal and substantially in the adult hearts. Similar to laminin alpha 1, laminin alpha 4 was expressed in the embryonic hearts, while its expression was relatively weak in the neonatal and adult hearts. Laminin beta 1 was expressed in the hearts of mice at all stages examined. These results demonstrate that the laminin chain gene expression changes in the different developmental stages of the hearts of BALb/c mice.

Animals↗
PubMed1997

Effect of arginine vasopressin on breathing movements of chronically instrumented fetal lambs.

BACKGROUND: In hypoxic conditions, fetal breathing movements (FBM) are reportedly inhibited with simultaneous elevation of the plasma level of catecholamines and arginine vasopressin (AVP). Although some kinds of catecholamines are reported to inhibit FBM, the effects of AVP on FBM are still unknown. The present study was undertaken to elucidate whether or not AVP has an inhibitory effect on FBM and electrocortical activities in chronically instrumented fetal lambs. METHODS: Arginine vasopressin was injected slowly into the fetal external jugular vein via a catheter over a 30 minute period at an infusion rate of 4.9 approximately 10.0 mIU/minute/body. In 15 fetuses (36 estimations) the mean fetal age, mean estimated fetal body weight, and mean infusion dose were 132.4 +/- 4.5 days, 3.4 +/- 0.3 kg and 1.7 +/- 0.3 mIU/kg/min. RESULTS: The infusion caused a persistent decrease in the fetal heart rate and an increase in the fetal arterial pressure during the 30 minute period. The infusion of AVP resulted in a significant decrease in the incidence of FBM from 36.1 +/- 14.8% to 12.4 +/- 14.9% (p <0.01). However, there were no significant differences in the incidence of the low voltage fast (LVF) activity before and during infusion of AVP. CONCLUSION: AVP has an inhibitory effect on FBM. The mechanism of this inhibition was not a direct suppression of the CNS as there was a dissociation between FBM and the electrocortical activity during infusion of AVP.

Animals↗
PubMed1997

Diffuse leiomyomatosis associated with X-linked Alport syndrome: extracellular matrix study using immunohistochemistry and in situ hybridization.

Inherited diffuse esophageal leiomyomatosis a benign tumor involving smooth muscle cells of the whole esophagus, is frequently associated with X-linked Alport syndrome, a hereditary disease of type IV collagen. Families with this condition are consistently found to have deletions encompassing the 5' ends of both the alpha 5 chain of type IV collagen (COL4A5) and the alpha 6 chain of type IV collagen (COL4A6) genes, always limited in COL4A6 to exons 1', 1, and 2. On the contrary, patients with COL4A5/COL4A6 deletions extending further into COL4A6 display no such tumors. Despite the deletion, a COL4A6 transcript including exon 4, but not exon 3, was found in a tumor sample, raising the possibility of the involvement of a truncated alpha 6(IV) chain in the tumorous process. Using immunohistochemistry and in situ hybridization methods, we analyzed the expression and distribution of the alpha 6 chain of type IV collagen in tumors in comparison with that of normal, fetal, and mature esophagus. We also studied associated changes in tumor basement membrane composition and in tumor-cell integrin subunit distribution. No labeling with alpha 6(IV) antibodies was detected in tumors, ruling out the hypothesis of a stably integrated truncated alpha 6(IV) chain in tumor basement membranes. In contrast, despite the deletions of the first two exons of the gene and its 5' end, a COL4A6 transcript is clearly expressed by tumor cells. This finding raises the question of a potential role for this RNA in the tumor process. The absence of the alpha 6(IV) chain is associated with the absence of the alpha 5(IV) chain, as was suggested by the COL4A5 deletion. An additional striking feature is the absence of the beta 1 chain of laminin in tumor basement membranes and the lack of or uneven expression of the alpha 5 integrin subunit. These findings show that dramatic changes in the composition of the matrix and the expression of integrin receptors also occur in this benign tumorous process.

Basement Membrane↗