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Biomedical subjects

H Inoue

Publications and source records attributed to H Inoue.

At least 1,657 records · Page 92Linked to original sources
PubMed1987

Immunohistochemical studies on synapse formation by embryonic cerebellar tissue transplanted into the cerebellum of the weaver mutant mouse.

Normal cerebellar tissue, obtained from 15-day-old CBA/JNCij mouse embryos, was transplanted into the cerebellum of 4-week-old weaver mice. At the 6th week after the transplantation, the grafted tissue was distinguishable from the host cerebellum, developing a trilaminar organization. The formation of synapses by the implanted granule cells was analyzed immunohistochemically with antiserum against synaptic vesicle protein, Synapsin I. Some areas in the host cerebellum as well as in the grafted tissue were intensely stained by anti-Synapsin I serum, suggesting that the implanted granule cells make synaptic contacts with the neuronal cells.

Animals↗
PubMed1987

Synthesis and hybridization studies on two complementary nona(2'-O-methyl)ribonucleotides.

2'-O-Methyl derivatives of the common ribonucleosides except for guanosine were synthesized via the 2'-O-methylation of appropriately-protected nucleosides with CH3I in the presence of Ag2O. The 2'-O-methylguanosine derivative was prepared by the monomethylation of a 2',3'-cis-diol system with diazomethane. These derivatives were converted to protected 2'-O-methylribonucleoside 3'-phosphates and used for oligonucleotide synthesis on polymer supports. Thus, oligo(2'-O-methyl-ribonucleotides) having the sequence identical to the consensus sequence of the 5'-splice junction CAGGUAAGU and its complement were synthesized in a stepwise manner using the phosphotriester method. Thermal stabilities (Tm's) of the duplex of these 2'-O-methyl ribo-oligomers and eight related duplexes containing ribo- or deoxyribo-oligomers were examined. It was found that the 2'-O-methyl oligoribonucleotides can be utilized as an alternative to an oligoribonucleotide probe in RNA hybridizations as the hybrid formed has a high, or a higher Tm, the probe is much easier to synthesize and it is less likely to be enzymatically degraded.

Acetylation↗
PubMed1987

Site-directed cleavage of RNA.

Using complementary chimeric oligonucleotides containing deoxyribonucleotides and 2'-O-methylribonucleotides (1), enzymatically synthesized RNA (90 mer) were cleaved at a single site with Escherichia coli RNaseH, either at a hairpin loop or at a stem region. Especially, site-specific cleavage occurred in even the target region being enclosed within a stable, base-paired stem. The method is proved to be generally applicable to RNA containing secondary structures.

Base Sequence↗
PubMed1987

Metabolism of 3-phosphoglyceroyl phosphate in phosphoenolpyruvate-enriched human erythrocytes.

The concentration of 3-phosphoglyceroyl phosphate in erythrocytes was increased by more than 100-fold when red cells were incubated with extracellular phosphoenolpyruvate at 37 degrees C. Since these elevated levels were maintained for 60 min, the metabolism of 3-phosphoglyceroyl phosphate and related compounds could be investigated in phosphoenolpyruvate-treated erythrocytes. 2,3-Bisphosphoglycerate synthesis was not affected by intracellular pH when the 3-phosphoglyceroyl phosphate level was constant but did vary with 3-phosphoglyceroyl phosphate concentration. On the other hand, the relationship between the rate of 2,3-bisphosphoglycerate synthesis and 3-phosphoglyceroyl phosphate concentration was not straightforward. At relatively low concentrations of 3-phosphoglyceroyl phosphate, the observed rate of 2,3-bisphosphoglycerate synthesis agreed with a rate calculated from a formula incorporating kinetic parameters of purified 2,3-bisphosphoglycerate synthase (Rose, Z.B. (1973) Arch. Biochem. Biophys. 158, 903-910). However, at high concentrations of 3-phosphoglyceroyl phosphate, the observed rate of 2,3-bisphosphoglycerate synthesis was lower than the calculated value. The concentration of glucose 1,6-bisphosphate did not increase even when 3-phosphoglyceroyl phosphate was elevated to 200 microM. Elevated levels of intracellular 2,3-bisphosphoglycerate did not inhibit glycolytic activity in these erythrocytes. These results suggest that incubation of erythrocytes with phosphoenolpyruvate is a useful technique to investigate the effect of metabolic perturbations at the intermediate stages of glycolysis.

Diphosphoglyceric Acids↗
PubMed1987

Interchange reaction of disulfides and denaturation of oxytocin by copper(II)/ascorbic acid/O2 system.

The interchange reaction of disulfides was caused by the copper(II)/ascorbic acid/O2 system. The incubation of two symmetric disulfides, L-cystinyl-bis-L-phenylalanine (PP) and L-cystinyl-bis-L-tyrosine (TT), with L-ascorbic acid and CuSO4 in potassium phosphate buffer (pH 7.2, 50 mM) resulted in the formation of an asymmetric disulfide, L-cystinyl-L-phenylalanine-L-tyrosine (PT), and the final ratio of PP:PT:TT was 1:2:1. As the reaction was inhibited by catalase and DMSO only at the initial time, hydroxyl radical generated by the copper(II)/ascorbic acid/O2 system seemed to be responsible for the initiation of the reaction. Oxytocin and insulin were denatured by this system, and catalase and DMSO similarly inhibited these denaturations. As the composition of amino acids was unchanged after the reaction, hydroxyl radical was thought to cause the cleavage and/or interchange reaction of disulfides to denature the peptides.

Amino Acids↗
PubMed1987

Effects of sialagogues on the syntheses of polyamines and DNA in murine parotid gland.

When rat parotid explants were cultured on siliconized lens paper floating on chemically defined 199 medium, all of sialagogues tested increased ornithine decarboxylase activity, which was roughly proportional to the amylase released into the culture medium. S-Adenosylmethionine decarboxylase and DNA synthesis were also induced by isoproterenol, methoxamine, carbachol and pilocarpine, but not by serotonin or substance P. The increases of the two decarboxylase activities and DNA synthesis were observed in vivo in mouse parotid gland after repeated injections of carbachol or pilocarpine. These results indicate that both adrenergic and cholinergic sialagogues stimulate the syntheses of polyamines and DNA in murine parotid gland.

Adenosylmethionine Decarboxylase↗
PubMed1987

Sequence-dependent hydrolysis of RNA using modified oligonucleotide splints and RNase H.

We found that, in the presence of chimeric oligonucleotides containing complementary deoxyribo- and 2'-O-methylnucleosides, a nonaribonucleotide, [5'-32P]pACUUACCUG, was cleaved specifically upon treatment with RNase H. When 3'm(UG)d(AATG)m(GAC)5' was used as a hybridization strand, pACUUACCUG was cleaved between C6 and C7 to yield pACUUAC. In the presence of 3'm(UGAA)d(TGGA)m(C)5', the nonaribonucleotide was hydrolyzed, mainly between U8 and C9, to give pACUUACCU. This method will have a variety of applications in the field of RNA engineering.

Autoradiography↗
PubMed1987

A new solid-phase synthesis of oligoribonucleotides by the phosphoro-p-anisidate method using tetrahydrofuranyl protection of 2'-hydroxyl groups.

Six nonaribonucleotides containing the 5'-splice site, one complementary nonamer and an octadecamer containing the 3'-splice site have been synthesized on a polymer support using the phosphoro-p-anisidate method. A 5'-linked 2'-O-tetrahydrofuranyl-N-protected nucleoside 3'-(o-chlorophenyl)phosphoro-p-anisidate was used as the starting nucleotide, and the chain elongated in the 3'-direction by removing the p-anisidate protecting group with isoamyl nitrite under neutral conditions. The octadecamer has been synthesized using dinucleotide blocks and a 3'-terminal trinucleotide.

Base Sequence↗
PubMed1987

Quantitative tissue isolation from Drosophila freeze-dried in acetone.

Freeze-drying procedures were developed to enable collection of tissues from Drosophila flies. The flies were frozen in acetone at -86 or -94 degrees C, and dehydrated therein. After drying, many tissues could be easily taken in entirety and free of neighbouring tissues without action of degradative enzymes. Seven polypeptide species specific to retina, and nine specific to cornea, were identified on two-dimensional electrophoretograms. Phospholipids of the dried tissues could be studied by t.l.c., and phosphatidic acid of the fly head was found to occur predominantly in the retina. Activity of three enzymes in the dried tissues could be assayed. The results of protein, phospholipid and enzyme analyses were corroborated by analyses by 'genetic dissection' using an eyeless mutant line.

Acetone↗
PubMed1987

A sensitive colorimetric detection of virus DNA and oncogene.

Advantage of cloning probe DNA fragment in phage M13 DNA was taken to provide a larger single stranded DNA as a hybridization probe. High level of direct enzyme labels was introduced via the M13 DNA moiety as well as probe DNA. A highly sensitive colorimetric detection of virus DNA and oncogene was developed.

Coliphages↗
PubMed1987

Four synaptic vesicle-specific proteins: identification by monoclonal antibodies and distribution in the nervous tissue and the adrenal medulla.

Synaptic vesicles from the guinea-pig cerebrum were isolated and administered to mice for the production of monoclonal antibodies (MAb). Four vesicle-associated proteins in the guinea-pig nervous tissue were specifically and differentially recognized by MAbs thus obtained. These proteins had molecular weights of 30,000, 36,000, 38,000 and 65,000 Da and were named SVPs (synaptic vesicle proteins) 30, 36, 38 and 65, respectively. Immunohistochemistry demonstrated that all SVPs were localized in the synaptic regions throughout the central nervous system and in the adrenal medulla. Nerve terminals in skeletal muscle, smooth muscle and sympathetic ganglion contained SVPs 36 and 38. Immunoelectron microscopy of the cerebellar cortex confirmed the localization of SVPs in the synaptic vesicles and the adjacent membranes of the presynaptic nerve terminals. Fractionation of the cerebral tissue and treatment with various agents showed that SVPs were localized in the synaptic vesicles and the synaptic plasma membrane and that SVPs were integrated within the membrane and liberated only after solubilization of the membrane.

Adrenal Medulla↗
PubMed1987

The enhanced 32P labeling of CDP-diacylglycerol in c-myc gene expressed human kidney cancer cells.

Correlation between c-myc gene expression and phosphatidylinositol (PI) metabolism was studied using seven human kidney cancer cell lines. We found that the exceptional incorporation of 32P into CDP-diacylglycerol (CDP-DG) was observed in only two cell lines, in which c-myc expression was increased among seven cell lines tested. In these two cell lines, PI was also labeled. The other five cell lines indicated the accumulation of radioactive PI, whereas CDP-DG was unlabeled.

Cell Division↗
PubMed1987

Effect of 5'-difluoromethylthioadenosine, an inhibitor of methylthioadenosine phosphorylase, on proliferation of cultured cells.

5'-Methylthioadenosine (MTA) is formed from decarboxylated S-adenosylmethionine during biosynthesis of polyamines. This nucleoside is cleaved by methylthioadenosine phosphorylase (MTA Pase) to adenine and 5-methylthioribose-I-phosphate in mammalian cells. 5'-Difluoromethylthioadenosine (DFMTA), a synthetic analog of MTA, was not a substrate for MTA Pase, but was a strong competitive inhibitor of the enzyme (Ki = 0.48 microM). DFMTA caused marked accumulation of labeled MTA formed from [35S]methionine in Raji cells, which contain MTA Pase, but not in CCRF-CEM cells, which do not contain this enzyme, suggesting that it also inhibits the enzyme in intact cells. DFMTA inhibited the growth of a variety of cultured cells and its cytostatic effect was roughly proportional to the MTA Pase activity of the cells. MTA also depressed the growth of cultured cells but, in contrast with DFMTA, its inhibitory effect was greater in MTA Pase-deficient cells (CCRF-CEM) than MTA Pase-containing cells (Raji). Inhibition of growth of Raji cells by DFMTA was partially reversed by exogenous adenine, a reaction product of MTA Pase. These results suggest that the utilization of adenine formed from MTA was important for proliferation of cells containing MTA Pase under the culture conditions employed, and that DFMTA inhibited cell growth by inhibiting MTA Pase activity.

Adenosine↗
PubMed1987

Combined effects of somatomedin-like growth factors with fibroblast growth factor or epidermal growth factor in DNA synthesis in rabbit chondrocytes.

The somatomedin-like growth factors cartilage-derived factor (CDF) and multiplication-stimulating activity (MSA) stimulate DNA synthesis and proliferation of rabbit costal chondrocytes under serum-free conditions. Previously, we suggested that CDF and MSA act on chondrocytes in an early G1 phase to stimulate DNA synthesis. CDF and MSA have synergistic effects with epidermal growth factor (EGF) or fibroblast growth factor (FGF) in stimulating DNA synthesis of the cells. The mode of combined action of CDF or MSA with EGF or FGF in chondrocytes was studied by sequential treatments with these agents. EGF or FGF had synergistic effects with CDF or MSA in stimulating DNA synthesis, even when added 10 h after the latter. Synergism was also observed in cells pretreated with CDF or MSA; That is, the cultures were treated for 5 h with CDF or MSA and then washed, and treated with FGF or EGF. However, when CDF or MSA was added more than 5 h after EGF or FGF, no synergism of effects was observed. These findings suggest that the cultured chondrocytes become activated to interact with FGF or EGF for commitment to DNA synthesis when they are exposed to somatomedin-like growth factors at an early stage in the G1 phase. Thus chondrocytes are under a different mechanism of growth control from fibroblastic cells.

Animals↗
PubMed1987

Increased afferent vagal responses produced by epicardial application of nicotine on the canine posterior left ventricle.

Vagally mediated vasodepressor reflexes are observed in dogs and patients with acute myocardial ischemia or infarction and may be related to the Bezold-Jarisch reflex. To test whether nicotine and bradykinin produced site-specific vagal vasodepressor and sympathetic vasopressor responses, respectively, we compared blood pressure and heart rate responses to these drugs applied topically at six epicardial sites of the canine left ventricle: midanterior, apical anterior, midlateral, apical lateral, midposterior, and apical posterior left ventricle. In alpha-chloralose-anesthetized dogs with open chests a felt pad soaked in bradykinin (25 micrograms) or nicotine (50 micrograms) was applied to the test sites. Previous studies indicated that epicardial application of these drugs elicited a sympathetically mediated pressor response and a vagally mediated depressor response, respectively. Vasopressor response to bradykinin did not differ among six test sites in 10 dogs. However, vasodepressor response to nicotine was greater in the midposterior left ventricular epicardium than at other test sites in 16 dogs. A hypertensive response after the initial hypotensive response to nicotine was observed in the midanterior left ventricle and not in any other site. These data indicate that vagal vasodepressor responses to epicardial nicotine are greater in the canine midposterior left ventricle and are compatible with the higher incidence of bradycardia or hypotension during acute inferoposterior myocardial infarction in patients. Sympathetic vasopressor responses to epicardial bradykinin are not site specific. Half of the dogs showed no response to bradykinin or nicotine.

Afferent Pathways↗
PubMed1987

A new method of inhalation challenge with propranolol: comparison with methacholine-induced bronchoconstriction and role of vagal nerve activity.

To establish a safe procedure for examining propranolol-induced bronchoconstriction, we have developed a new method for performing inhalation challenge with propranolol. Monitoring respiratory resistance during tidal breathing with continuous inhalation of propranolol in 1.5-fold increasing concentrations from 0.78 to 30 mg/ml for 1 minute at each concentration, we tested 43 subjects with stable asthma and 10 normal subjects. We also compared bronchial responsiveness with responsiveness to inhaled methacholine on separate days. In addition, to determine the role of vagal nerve activity in propranolol-induced bronchoconstriction, we studied the effect of atropine. Inhaled propranolol caused dose-related bronchoconstriction in all subjects with asthma but not in normal subjects. None of the subjects suffered severe asthmatic attack during the test, which was performed in 15 minutes or less. The minimum cumulative dose of methacholine and of propranolol, at the point where respiratory conductance began to decrease, was not significantly correlated. Increased respiratory resistance was reversed by atropine in 70% of the subjects with asthma with marked individual differences. These data suggest that, although in most subjects with asthma, vagal nerve activity contributes in varying degree to bronchoconstriction, other constricting factors may contribute in the remaining subjects. It is also suggested that the mechanism of bronchial response to propranolol differs from that of the nonspecific airway reactivity estimated by methacholine challenge.

Adolescent↗