Biomedical subjects
Publications and source records attributed to R Ishizaki.
In a woman aged 73 years who recovered from hypoglycemic coma without neurologic deficit after glucose infusion, admission diffusion-weighted MRI showed the presence of hyperintensive lesions. The lesions regressed after glucose infusion.
p21/WAF1 blocks cell cycle progression through inhibition of cyclin/cyclin-dependent kinases (cdks) complexes and, simultaneously, has been associated with cell cycle exit and the process of differentiation. In this series, the expression of p21/WAF1 was assessed immunohistochemically in 47 cases of pituitary adenomas in relation to endocrine activity and cell proliferation. To evaluate cell proliferation, a monoclonal antibody, MIB-1, against Ki-67 antigen was used in all of the available cases. The study revealed positive p21/WAF1 staining in 24 cases of 26 functioning parenchymas, whereas 14 cases of 21 non-functioning parenchymas stained negative. The MIB-1 index ranged from less than 1% to 5.1% (mean: less than 1.7%) in functioning adenomas, and from less than 1% to 3.6% (mean: less than 1.6%) in non-functioning adenomas. Regardless of endocrine activity, p21/WAF1 positivity did not correlate with the MIB-1 index. Double staining techniques revealed the co-expression of p21/WAF1/GH or p21/WAF1/PRL in functioning adenomas. In 22 cases of p21/WAF1-positive functioning adenomas, p21/WAF1 immunoreactivity was seen in the cytoplasma as well as nuclei. These results indicate that in pituitary adenomas, p21/WAF1 expression is associated with endocrine activity, but not with cell proliferation. Taken together with recent findings demonstrating that cytoplasmic p21/WAF1 acts as an inhibitor of apoptosis, it is possible that pituitary adenomas expressing cytoplasmic p21/WAF1 have resistance against DNA-damaging agents such as ionizing radiation.
OBJECTIVE: To investigate the immunology of host-tumour interaction, critical for the development of immunotherapy against cancers, by assessing the major histocompatibility complex (MHC) class I expression in both benign and malignant prostate disease, and the relationship between their expression and degree of tumour-infiltrating lymphocytes. MATERIALS AND METHODS: Direct serial analysis of gene expression in tumours is an extremely sensitive and powerful tool for monitoring immunological changes in the immunotherapy of solid tumours. Most previous monitoring protocols rely mainly on the analysis of patient's peripheral blood but in the present study the direct molecular analysis of small tissue samples was used, and its accuracy compared with that of conventional immunohistochemical analysis. Twenty-four formalin-fixed, paraffin-embedded prostate samples (11 benign and 13 carcinoma) were used for the immunohistochemical analysis of CD8+ T lymphocytes and MHC class I expression. CD8+ T lymphocytes were counted using an ocular grid and MHC class I measured using digital image-analysis software. Twenty-seven frozen prostate tissue samples (12 benign and 15 carcinoma) were used for direct gene measurements of CD8 and interferon-gamma using a quantitative real-time polymerase chain reaction. RESULTS: There were significantly fewer CD8+ T lymphocytes in prostate carcinoma nests than in benign prostate. There was a significant correlation between the number of CD8+ T lymphocytes and MHC class I expression in the prostate. There was a strong correlation between the immunohistochemical estimates of CD8+ T lymphocytes and CD8 gene by polymerase chain reaction, but no significant difference between benign prostate and prostate carcinoma tissue in gene measurements. CONCLUSION: Down-regulation of MHC class I expression by prostate cancer cells is associated with fewer CD8+ T lymphocytes and hence might be important in cancer growth. In addition, the measurement of gene expression in small tissue samples might be useful for monitoring the efficacy of treatment throughout cancer therapy.
The aim of this study was to establish a cellular system to investigate the requirement for cell surface and diffusible molecules in the differentiation of fetal mesencephalic cells toward the dopamine lineage. Toward this end, we immortalized rat embryonic day 14 (E14) mesencephalon with a regulatable retroviral vector encoding v-myc. The stably transduced cells were pooled and designated as VME14 cells. VME14 cells proliferated rapidly, stopped proliferating, extended processes, and expressed GFAP after suppression of the v-myc expression with tetracycline, suggesting that VME14 cells differentiated into glial cells. Dissociated cells derived from the E11 rat mesencephalon gave rise to only a small number of tyrosine hydroxylase (TH)-positive neurons. However, when grown on a monolayer of the differentiated VME14 cells, a significantly higher number of cells differentiated into TH-positive neurons. VME14 cells were transduced with the secreted N-terminal cleavage product of the Sonic hedgehog gene (SHH-N), an inducer of mesencephalic dopaminergic neurons. This monoclonal, SHH-N-overexpressing cell line further enhanced dopaminergic differentiation of E11 rat mesencephalon cells. Thus, SHH-N and signals derived from fetal mesencephalic glia act cooperatively to facilitate dopaminergic differentiation. These fetal mesencephalon-derived cell lines will provide tools for the study of signals involved in dopaminergic differentiation.
We report experimental results on hydrodynamic perturbation transfer from the rear to the front of laser-irradiated targets. Flat polystyrene foils with rear-surface perturbations were irradiated by partially coherent light. We observed phase inversion of the rear surface after the shock breakout at the rear surface. Perturbations on the laser-irradiated surface arose due to the rippled rarefaction wave. Experimental results were well reproduced by a simple model with unperturbed hydrodynamic quantities calculated from the one-dimensional simulation.
OBJECT: The evolution and severity of hydrocephalus in animal models varies in the species and mode of induction. This makes comparisons of the physiological system under investigation difficult between models. We noted that injection of kaolin into neonatal rats results in a dichotomous outcome into either an acute or subacute form. We investigated the clinical and functional transmitter system changes to compare these two types of hydrocephalus evolution. METHODS: Hydrocephalus was induced in Wistar neonatal rats (within a week after their birth) by intracisternal injection of 0.02 ml volume of 25% kaolin solution under microscopic guidance. The same volume of sterile saline was injected into 12 neonatal rats as control group. The animals were assigned to either the acute or subacute group according to their head size, and sacrificed at 2, 4 and 8 weeks after injection. Biparietal diameter, ventricular size, cholinergic interneurons in the neostriatum and dopaminergic projection neurons in the substantia nigra were analyzed at each stage. RESULTS: Animals affected with the acute type of hydrocephalus had obvious head enlargement, rapid ventricular enlargement, and all died at about 4 weeks. Animals with subacute type had slowly progressive ventricular enlargement, and all survived until 8 weeks. There appeared to be more kaolin ventral to the brainstem in the acute type. The number of cholinergic neostriatal neurons was significantly reduced at 2 and 4 weeks in the acute type, but only at 8 weeks in the subacute type. The number of dopaminergic nigral neurons was decreased at 4 weeks in the acute type, but unaffected in the subacute type. CONCLUSIONS: The severity of onset of hydrocephalus in this animal model also correlates with the clinical outcome and changes in functional transmitter systems.
Southern blot hybridization of DNA samples from 9 primary tumors of avian lymphoid leukosis (LL) rapidly induced by ALV infection 27-74 days post inoculation was carried out to search for rearrangement of the c-myc gene with human c-myc gene exon III as a probe. Rearrangement of the c-myc gene was detected by appearance of new EcoRI fragments in 7 out of 9 tumors examined. The size of the fragments ranged from 3.1 to 4.0 kilobases (kb). In addition to these fragments, two fragments (9.0 kb and 13 kb) were observed in one tumor, and a faint fragment (3.5 kb) was observed in another tumor. Rearrangement of the c-myc gene was not detected in the remaining two tumors kept in unsuitable condition. These results suggest that rearrangement of c-myc gene was induced even in rapidly induced LL as well as that induced after long incubation period. This is the first report of involvement of c-myc gene in rapidly induced B-cell lymphoma (LL).
OBJECT: Prominent features of moyamoya disease are intimal thickening of the cerebral arterial trunks and abundant angiogenesis for collateral blood supplies, but its pathogenesis is still unknown. The aim of this study was to test the possibility that transforming growth factor-beta1 (TGFbeta1) may play a role in the pathogenesis of moyamoya disease. METHODS: The authors used reverse transcription-polymerase chain reaction to analyze the expression level of TGFbeta1 in smooth-muscle cells cultured from the superficial temporal arteries (STAs) and measured the serum level of TGFbeta1 by using enzyme-linked immunosorbent assay. Although the STA is not predominantly involved with moyamoya disease, it has been used in studies of the pathogenesis of this disease. In this report, the STAs from six patients with moyamoya disease and four with arteriosclerotic cerebrovascular disease, along with sera from 14 patients with moyamoya disease and 10 normal healthy volunteers, were studied. The expression of TGFbeta1 was significantly higher in cultured smooth-muscle cells derived from the STAs of patients with moyamoya disease than in those derived from the STAs of patients with arteriosclerotic cerebrovascular disease (p < 0.05). The serum level of TGFbeta1 was also significantly higher in patients with moyamoya disease than in controls (p < 0.0005). CONCLUSIONS: Taking into account the functional roles of TGFbeta1 in the expression of connective tissue genes and angiogenesis, these investigators suggest that TGFbeta1 is associated with the pathogenesis of moyamoya disease, including abundant neovascularization, although their findings do not necessarily mean that TGFbeta1 is a causative factor in this disease.
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Viscous material secreted from NMSG 10 cells cultured from malignant fibrous histiocytoma of human bone was demonstrated using two-dimensional electrophoresis to be glycosaminoglycans (GAGs) consisting of mainly hyaluronic acid (HA), along with small amounts of heparan sulfate (HS) and chondroitin sulfate (ChS). The biosynthesis of HA in a cell-free system was located at the plasma membrane fraction by colloidal-iron-reactive ultrastructural staining. The molecular weight of the HA synthesized by the plasma membrane fraction was estimated to be about 90,000 D by gel filtration. The material secreted by the original tumor tissue of NMSG 10 cells was demonstrated to be GAGs consisting of HA, HS, ChS and dermatan sulfate using two-dimensional electrophoresis. These findings suggest that transformed tumor cells could synthesize HA at the plasma membrane and release it into the stroma of tumor tissue.
A fibrous histiocytoma cell line, NMSG 10, was derived from a malignant fibrous histiocytoma (MFH), of human tibia, and its characteristics were examined. MFH was a pleomorphic subtype associated with myxoid and storiform areas. Primary culture revealed a mixture of histiocyte-like cells, fibroblast-like cells and giant cells, but fibroblast-like cells became the major population after several passages in vitro. In addition, NMSG 10 produced a large amount of viscous material which stained with alcian blue and was digested by hyaluronidase. Thus, this viscous material was a single component of glycosaminoglycans: hyaluronic acid (HA). The cells were spindle-shaped with well-developed cytoplasmic organelles and collagenous filaments, and a colloid iron-positive substance was observed in intercellular spaces. In scid mice, the mixed populations of neoplastic cells appeared similar in histology to that of the original tumor. These findings indicated that NMSG 10 expresses the unique properties of MFH, and should therefore be useful in studies on the biological behavior, and especially the presence of HA in MFH.
Previously, we have reported the molecular cloning of ara genes encoding a small GTP-binding protein from Arabidopsis thaliana. The criterion based on amino acid sequences suggest that such an ara gene family can be classified to be of the YPT/rab type. To examine the biochemical properties of ARA proteins, several deletions and point mutations were introduced into ara cDNAs. Mutant proteins were expressed in E. coli as GST-chimeric molecules and analyzed in terms of their GTP-binding or GTP-hydrolysing ability in vitro. The results indicate that four conserved amino acid sequence regions of ARA proteins are necessary for GTP-binding. A point mutation of Asn at position 72 for ARA-2, or 71 for ARA-4, to Ile decreased GTP-binding and a point mutation of Gln at position 126 for ARA-2, or 125 for ARA-4, to Leu suppressed GTP-hydrolysis activity. Furthermore, certain factors associated with the membrane fraction accelerated GTPase activities of ARA proteins, suggesting the presence of GTPase activating protein(s) (GAP(s)) in the vesicular transport system of higher plant cells.
Neuroblastic tumor cases in our departments were evaluated in terms of the stage of the tumor, N-myc amplification, urinary vanillylamndelic acid (VMA)/homovanillic acid (HVA) and survival rate. Two asymptomatic cases, diagnosed when under a year old by mass screening, had no amplifications of N-myc but showed more than one value of urinary VMA/HVA ratio. The patients are now doing well eight years after complete excision of the neuroblastoma which had originated in the sympathetic ganglion. On the other hand, two other symptomatic cases, operated on at the ages of 3 and 5 years, showed remarkable amplifications with less than one value of urinary VMA/HVA, and died from the tumor soon after partial resection of the neuroblastoma and ganglioneuroblastoma which had originated in the adrenal gland and the sympathetic ganglion, respectively. The present monograph reports our cases and discusses prognostic factors.
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A possible mechanism for the nicotine-induced relaxation of circular muscle strips of the guinea-pig gastric fundus was investigated. In the presence of atropine (0.2 microM), nicotine produced concentration-dependent relaxation with a maximum effect at 100 microM (mean pEC50 value, 4.60). The maximum relaxation due to nicotine was greatly reduced by pretreatment with tetrodotoxin (0.3 microM) or hexamethonium (10 microM), but not with metitepine (0.3 microM). Combined pretreatment with timolol (0.3 microM) and phentolamine (0.3 microM) or chemical sympathectomy by 6-hydroxydopamine pretreatment partially inhibited the nicotine-induced relaxation. alpha-Chymotrypsin (2 u/ml) which abolished the equivalent relaxation induced by vasoactive intestinal polypeptide (VIP) had no effect on nicotine-induced relaxation. NG-Nitro-L-arginine (L-NNA) and NG-Nitro-L-arginine methyl ester (L-NAME) caused a concentration-dependent inhibition of the nicotine-induced relaxation (98% inhibition at 10 microM of L-NNA), but had no effect on sodium nitroprusside- or noradrenaline-induced relaxation. The inhibitory effect of L-NNA or L-NAME was reversed completely by L-arginine (3 mM), but not by D-arginine (3 mM). From these results, we concluded that nicotine-induced relaxation of the guinea-pig gastric fundus is mediated largely by the release of nitric oxide or a related substance and partially by the release of noradrenaline. Possible contributions of 5-hydroxytryptamine or VIP to the nicotine-induced relaxation appear to be negligible.
DNA fingerprinting method was applied to identify inbred strains of laboratory rats. By Southern blot hybridization with core sequence of minisatellite DNA as a probe, typical hypervariable patterns of DNA fingerprint were obtained in inbred rat strains. The patterns were completely different among 15 rat strains examined, and the patterns of the DNA fingerprint of samples obtained from the same strain were completely identical. The patterns of the DNA fingerprint of two substrains derived from the same strain were identical, indicating relative stability of the patterns over a large number of generations. Therefore, we concluded that the DNA fingerprinting method was useful for the identification of inbred strains in genetic monitoring of laboratory rats.
The 5-HT receptor that mediates relaxation of circular muscle strips of the guinea-pig stomach fundus under resting tone was investigated. Concentration-dependent relaxation was obtained in the presence of atropine (0.2 microM) with 5-hydroxytryptamine (5-HT) (apparent mean pEC50 value, 5.27), 5-carboxamidotryptamine (7.35), 5-methoxytryptamine (4.98) and 5-methyltryptamine (4.58). 1-(m-Trifluoromethyl-phenyl)piperazine and 8-hydroxy-2-(di-n- propylamino)tetralin acted as partial agonists while 2-methyl-5-hydroxytryptamine, alpha-methyl-5-hydroxytryptamine, sumatriptan, metoclopramide and cisapride had little or no effect on the guinea-pig stomach fundus. The concentration-response curve for 5-HT was not affected by tetrodotoxin (0.3 microM), guanethidine (5 microM) or indomethacin (2 microM), suggesting that the relaxation is non-neuronal in origin and is independent of the release of catecholamines or prostanoids. The non-selective 5-HT receptor antagonist, metitepine (0.03-0.1 microM), the 5-HT1C/5-HT2 receptor antagonists, mianserin (0.3-1 microM), pizotifen (0.3-1 microM), ketanserin (3-10 microM), and the 5-HT1A/5-HT2 receptor antagonist, spiperone (3 microM), shifted the concentration-response curves for 5-HT to the right. A 5-HT3 receptor antagonist, ICS205-930 (1 microM), propranolol (1 microM) and phentolamine (1 microM) failed to block the 5-HT-induced relaxation. In conclusion, the results found with agonists and antagonists are compatible with the view that a 5-HT1-like receptor is involved in 5-HT-induced direct relaxation of circular muscle of guinea-pig stomach fundus.
We previously isolated a DNA fragment from Arabidopsis thaliana homologous to the mammalian ras gene and named it ara [Matsui et al., Gene 76 (1989) 313-319]. Screening of cDNA clones homologous to ara in A. thaliana resulted in the isolation of four homologous genes. The products of these genes, ARA-2, ARA-3, ARA-4 and ARA-5, showed conservation of amino acids (aa) in four regions, all of which are present in small GTP-binding proteins, and are important for GTPase/GTP-binding activities. These products were highly homologous to those of the YPT genes of Saccharomyces cerevisiae and the ypt gene of Schizosaccharomyces pombe in the regions around aa 45, which is thought to be the site interacting with effector molecules. The products of these four genes showed characteristic aa sequence at their C termini, Cys-Cys-Xaa-Xaa. Another characteristic of this family is presence of Ser in place of Gly in the first conserved region (Gly12 of mammalian GTP-binding Ras protein).