Search PubMed⌕ Search

Biomedical subjects

H Inokuchi

Publications and source records attributed to H Inokuchi.

At least 91 records · Page 5Linked to original sources
PubMed1993

A simplified method for generating step-wise deletions using PCR.

A simple and general method is described for the generation of ordered deletions for DNA sequencing. Nicked plasmids, the substrates for step-wise digestion by exonuclease III, are obtained after the self-ligation of PCR products with phosphorylated and non-phosphorylated primers and plasmid DNAs as template. The method is suitable for use with any plasmid vector and for generation of deletion clones with deletions in both possible directions.

Base Sequence↗
PubMed1993

Membrane properties and dendritic arborization of the intermediolateral nucleus neurons in the guinea-pig thoracic spinal cord in vitro.

The morphological and electrophysiological properties of neurons in the intermediolateral nucleus (IML) were studied in the transverse and longitudinal slice of guinea-pig thoracic spinal cord (T2-T3) using intracellular staining and recording techniques. Two morophologically different types of neurons were observed: fusiform cells with craniocaudally oriented dendrites, and multipolar cells with dendrites diffusely extending in the IML. The ratio of fusiform to multipolar cells was 4:1. The fusiform cells were identified as sympathetic preganglionic neurons (SPNs) by their antidromic responses to stimulation of the ventral root exit zone, while the multipolar cells were not antidromically activated by stimulation of this site. Both cell types showed similar resting membrane potential and input resistance. The tonic responses of these neurons to hyperpolarizing current pulses were characteristically different: the SPNs had a marked hyperpolarizing sag at the break of the pulse, caused by an A current, while the unidentified neurons showed no A current. In addition, the SPNs had much longer duration of spike and afterhyperpolarization, as well as lower frequency of spontaneous or current-evoked firing, than the unidentified neurons. These observations suggest that, in the absence of the criterion of antidromic activation by stimulation of the axon, it is still possible to differentiate SPNs from other IML neurons on the basis of morphological and electrophysiological properties of the neuron.

Animals↗
PubMed1993

Selectivity and specificity in the recognition of tRNA by E coli glutaminyl-tRNA synthetase.

The specific recognition by Escherichia coli glutaminyl-tRNA synthetase (GlnRS) of tRNA(Gln) is mediated by extensive protein:RNA contacts and changes in the conformation of tRNA(Gln) when complexed with GlnRS. In vivo accuracy of aminoacylation depends on two factors: competition between synthetases, and the context and recognition of identity elements in the tRNA. The structure of the tRNA(Gln):GlnRS complex supports studies from amber and opal suppressor tRNAs, complemented by in vitro aminoacylation of the mutated tRNA transcripts, that the glutamine identity elements are located in the anticodon and acceptor stem of tRNA(Gln). Recognition of individual functional groups in tRNA, for example the 2-amino group of guanosine, is also evident from the result with inosine-substituted tRNAs. Communication between anticodon and acceptor stem recognition is indicated by mutants in GlnRS isolated by genetic selection with opal suppressor tRNAs which are altered in interactions with the inside of the L-shaped tRNA. We have also used genetic selection to obtain mutants of GlnRS altered in acceptor stem recognition with relaxed specificity for amber suppressor tRNAs, and a more extensive mutational analysis shows the importance of the acceptor binding domain to accurate recognition of tRNA.

Amino Acyl-tRNA Synthetases↗
PubMed1993

Ionic mechanisms underlying the depolarizing and hyperpolarizing afterpotentials of single spike in guinea-pig cingulate cortical neurons.

Intracellular recordings and hybrid single-microelectrode voltage-clamp techniques were used to study the ionic mechanisms underlying the afterdepolarization and the subsequent slow afterhyperpolarization that followed a single action potential in layers V/VI neurons of the guinea-pig anterior cingulate cortex in in vitro slices. Both the afterdepolarization and afterhyperpolarization were markedly suppressed in size by addition of Co2+ or Cd2+, reduction in extracellular Ca2+, and intracellular EGTA injection. On the other hand, elevation of extracellular Ca2+ concentration augmented the amplitudes of the afterpotentials. The afterdepolarization amplitude was selectively depressed by the stilbene derivatives, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonate, disodium 3H2O, and 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid, disodium salt. Reduction in external Cl- and intracellular Cl- injection enhanced the afterdepolarization amplitude without affecting the afterhyperpolarization. The null potentials for the afterdepolarizations recorded with K acetate- and Cs acetate-electrodes were -68 and -63 mV, respectively. The slope of the null potential obtained with K acetate electrodes or Cs acetate electrodes was 49 and 53 mV, respectively, per log unit of the external Cl- concentration. Reduction in external K+ markedly depressed the afterdepolarization and augmented the afterhyperpolarization in size, whereas rise in external K+ markedly augmented the afterdepolarization and depressed the afterhyperpolarization. The null potential for the afterhyperpolarization recorded with K acetate electrodes was -94 mV. The slope of the null potential was 57 mV per log unit of the external K+ concentration. Reduction in extracellular Na+ concentration slightly depressed both the amplitudes of the afterdepolarization and afterhyperpolarization. A hybrid voltage-clamp analysis revealed a slow decaying inward current and a subsequent outward current that followed an action potential. Both the amplitudes of the inward current corresponding to afterdepolarization and the outward current corresponding to afterhyperpolarization were suppressed by addition of Co2+. Reduction in extracellular Cl- concentration augmented the inward current amplitude without significantly affecting the outward current. These results indicate that the afterdepolarization is mainly due to an increase in a Ca(2+)-activated Cl- conductance, while the afterhyperpolarization is mainly generated by an activation of Ca(2+)-mediated K+ conductance.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1993

Pepsinogen C gene polymorphisms associated with gastric body ulcer.

This study was aimed to investigate the association of restriction fragment length polymorphisms (RFLPs) for pepsinogen genes with peptic ulcer disease. Eighty unrelated controls, 61 patients with gastric ulcer, and 57 patients with duodenal ulcer were studied. No genetic polymorphisms for pepsinogen A were detected by EcoRI digestion in Japanese subjects but a 100 base pairs insertion-deletion RFLP for the pepsinogen C gene was observed. The allele frequencies of the large (3.6 kilobase EcoRI fragment) and the small fragment (3.5 kilobase EcoRI fragment) were 80.6% and 19.4% respectively in controls, 55.4% and 44.6% in patients with gastric body ulcer, 79.4% and 20.6% in patients with gastric angular ulcer, 71.4% and 28.6% in patients with gastric antral ulcer, and 75.4% and 24.6% in patients with duodenal ulcer. The allele frequency of the small fragment was significantly higher in patients with gastric body ulcer than in controls and in patients with gastric angular or antral ulcer. The genotypes which possessed the small fragment were significantly more frequent in patients with gastric body ulcer (78.4%) than in controls (33.8%) and in patients with gastric angular or antral ulcer (37.5%). These results suggest that there is a significant association between the genetic polymorphism at the pepsinogen C gene locus and gastric body ulcer, and that the pepsinogen C RFLP is a useful marker of the genetic predisposition to this disorder. These results also indicate genetic heterogeneity of gastric ulcer disease, and suggest that the pepsinogen C RFLP may be a useful subclinical marker to explain the differences in genetic aetiologies of gastric body ulcer and gastric angular or antral ulcer.

Adult↗
PubMed1993

Heterogeneity of the afterhyperpolarization of sympathetic preganglionic neurons.

The components of the afterhyperpolarization (AHP) of the sympathetic preganglionic neuron were studied in the slice of the upper thoracic spinal cord of the cat. In this neuron, the AHP consists of a fast (fAHP) followed by a slow (sAHP) component. While the fAHP had similar time course in all neurons studied (n = 116), the sAHP had considerable variability. Two components could be identified and the variability resulted from variation in the relative magnitude of these. Fifteen percent of neurons showed only an early component of the sAHP, 16% showed only a late component, and 69% showed both components. The early component of the sAHP was selectively blocked by apamin and d-tubocurarine, while the late component was selectively blocked by ryanodine, procaine or phenylephrine.

Action Potentials↗
PubMed1993

The recognition of E. coli glutamine tRNA by glutaminyl-tRNA synthetase.

A variety of genetic, biochemical and structural studies have been used to determine factors ensuring the accuracy of recognition by aminoacyl-tRNA synthetases for tRNA. The identity elements of Escherichia coli tRNA(Gln) are located mainly in the anticodon and acceptor stem, and ensure the accurate recognition of the tRNA by glutaminyl-tRNA synthetase. We summarize a number of experimental techniques to define the accuracy of aminoacylation in vivo and in vitro.

Amino Acyl-tRNA Synthetases↗
PubMed1992

Accumulation of protoporphyrin IX in light-sensitive mutants of Escherichia coli.

The accumulation of protoporphyrin IX (Proto IX) in light-sensitive mutants of Escherichia coli was detected by spectrofluorimetry. Fluorescence emission and excitation spectra were recorded from extracts of bacterial cells. Proto IX clearly accumulated in cells with mutations in the visA (hemH) gene but not in the wild-type strain CA274 or in visA mutants that had been rendered light-resistant by introduction of the wild-type visA+ gene. Accumulation of Proto IX was also not observed in cells with a mutation in the visB gene. These results confirm the hypothesis that the sensitivity of the visA mutants to light is caused by the abnormal accumulation of Proto IX, a substrate of ferrochelatase, as the result of a genetic defect in the gene for ferrochelatase.

Escherichia coli↗
PubMed1992

Switching tRNA(Gln) identity from glutamine to tryptophan.

The middle base (U35) of the anticodon of tRNA(Gln) is a major element ensuring the accuracy of aminoacylation by Escherichia coli glutaminyl-tRNA synthetase (GlnRS). An opal suppressor of tRNA(Gln) (su+2UGA) containing C35 (anticodon UCA) was isolated by genetic selection and mutagenesis. Suppression of a UGA mutation in the E. coli fol gene followed by N-terminal sequence analysis of purified dihydrofolate reductase showed that this tRNA was an efficient suppressor that inserted predominantly tryptophan. Mutations of the 3-70 base pair (U70 and A3U70) were made. These mutants of su+2UGA are less efficient suppressors and inserted predominantly tryptophan in vivo; alanine insertion was not observed. Mutations of the discriminator nucleotide (A73, U73, C73) result in very weak opal suppressors. Aminoacylation in vitro by E. coli TrpRS of tRNA(Gln) transcripts mutated in the anticodon demonstrate that TrpRS recognizes all three nucleotides of the anticodon. The results show the interchangeability of the glutamine and tryptophan identities by base substitutions in their respective tRNAs. The amber suppressor (anticodon CUA) tRNA(Trp) was known previously to insert predominantly glutamine. We show that the opal suppressor (anticodon UCA) tRNA(Gln) inserts mainly tryptophan. Discrimination by these synthetases for tRNA includes position 35, with recognition of C35 by TrpRS and U35 by GlnRS. As the use of the UGA codon as tryptophan in mycoplasma and in yeast mitochondria is conserved, recognition of the UCA anticodon by TrpRS may also be maintained in evolution.

Amino Acyl-tRNA Synthetases↗
PubMed1992

Pancreatic arteriovenous malformation with pancreatitis involving a pancreatico-venous fistula.

An arteriovenous malformation of the pancreas is a very rare disease, but its presentation is distinct and unique. In this report, we describe a patient who presented with this malformation which was localized in the tail of pancreas and demonstrated by abnormal angiography findings. The patient was a 60-year-old male with severe left hypochondralgia. Angiography revealed an increased blood volume in the tail of the pancreas with arteriovenous shunting. Secondary pancreatitis caused by the arteriovenous malformation was suspected by abnormal laboratory data, and confirmed by histology from the resected tail of the pancreas. This is a very rare report in which pancreatic arteriovenous malformation involving a pancreatico-venous fistula was confirmed by endoscopic retrograde cholangiopancreatography (ERCP).

Arteriovenous Fistula↗
PubMed1992

Fast inhibitory postsynaptic potentials and responses to inhibitory amino acids of sympathetic preganglionic neurons in the adult cat.

Intracellular recordings were obtained from sympathetic preganglionic neurons (SPNs) of the intermediolateral nucleus (IML) in slices of upper thoracic spinal cord of the anesthetized cat. A total of 44 neurons was studied. Single shock stimulation of an area of white matter dorsolateral to the IML, close to the recording electrode (< 0.5 mm), evoked fast IPSPs with rise time of 3.8 ms and 1/2 decay time of 14.7 ms (n = 12). In 17 other cells only fast EPSPs were recorded but, after suppression of the EPSPs by the excitatory amino acid receptor antagonists CNQX (20 microM) and APV (100-250 microM), fast IPSPs were unmasked. The IPSP reversed polarity at -63 mV (-67 mV in the presence of CNQX and APV). The reversal potential shifted to a less negative value when the extracellular chloride concentration was reduced. The IPSP was reversibly abolished by the GABAA receptor antagonist bicuculline in 32% of the cells, by the glycine receptor antagonist strychnine in 47% of the cells and by the combination of the two in 21% of the cells. The IPSP was abolished by TTX (0.5 microM), had constant latency and showed no failures during high frequency stimulation. The IPSP presumably resulted from the excitation of inhibitory axons and/or inhibitory neuron somata with monosynaptic connections to the SPN. Glycine and GABA (1-3 mM) produced hyperpolarization associated with decreased membrane resistance. Sixty-nine percent of cells responded to both agonists, 19% to glycine only and 12% to GABA only. The GABAB agonist baclofen (5 microM) had no effect.(ABSTRACT TRUNCATED AT 250 WORDS)

2-Amino-5-phosphonovalerate↗
PubMed1992

Fast excitatory postsynaptic potentials and the responses to excitant amino acids of sympathetic preganglionic neurons in the slice of the cat spinal cord.

The properties of the excitatory postsynaptic potential evoked by focal stimulation and of the responses to excitatory amino acids were examined by intracellular recording from sympathetic preganglionic neurons in upper thoracic spinal cord slices of the adult cat. Single stimuli to the region dorsal to the intermedio-lateral nucleus evoked short-latency, presumably monosynaptic, excitatory postsynaptic potentials. The reversal potential of this response was -2.2 mV and became more negative when external Na+ or K+ concentration was decreased. The excitatory postsynaptic potential was depressed by the non-selective excitatory amino acid receptor antagonist cis-2,3-piperidine dicarboxylic acid and enhanced by a glutamate uptake inhibitor. The non-N-methyl-D-aspartate receptor antagonist 6-cyano-7-nitroquinoxaline-2.3-dione abolished the excitatory postsynaptic potential in 72% of neurons. In the remaining neurons, this antagonist only depressed the potential and unmasked a slower component which was abolished by the N-methyl-D-aspartate receptor antagonist D,L-2-amino-5-phosphonovaleric acid. In the presence of tetrodotoxin all neurons tested were depolarized by glutamate or aspartate, as well as by the selective agonists quisqualate, alpha-amino-3-hydroxy-5-methylisoxazole propionic acid, kainate and N-methyl-D-aspartate. The glutamate-evoked depolarization reversed at a membrane potential of -2.0 mV and at a more negative value when external Na+ or K+ concentration was decreased. The response to alpha-amino-3-hydroxy-5-methylisoxazole propionic acid was abolished by 6-cyano-7-nitroquinoxaline-2,3-dione in all neurons tested and that to kainate in only one-third of the cells. In the remainder the response to kainate was only slightly depressed by this antagonist. The responses to glutamate and aspartate were only slightly depressed by the combined action of the various amino acid receptor antagonists used. The responses to N-methyl-D-aspartate were abolished by D,L-2-amino-5-phosphonovaleric acid. The punched-out region of the intermedio-lateral nucleus, maintained in vitro, released glutamate and aspartate in the absence of stimulation. Field stimulation (20 Hz) enhanced release by between 40 and 100%. The increase was prevented by superfusion with calcium-free Krebs. It is concluded that excitatory amino acids, acting on both N-methyl-D-aspartate and non-N-methyl-D-aspartate receptors, but mainly on the latter, are likely mediators of the monosynaptic excitatory postsynaptic potential evoked in sympathetic preganglionic neurons by the stimulated region. The efflux data suggest that glutamate and aspartate are among the mediators.

6-Cyano-7-nitroquinoxaline-2,3-dione↗