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Biomedical subjects

M Nishiyama

Publications and source records attributed to M Nishiyama.

At least 19 recordsLinked to original sources
PubMed1995

Ornithine decarboxylase induction during liver regeneration in IRS-1-deficient mice.

We investigated the induction of ornithine decarboxylase during liver regeneration after partial hepatectomy in IRS-1-deficient mice. There were no significant differences in ODC activity or the time course of changes in ODC activity between IRS-1-deficient mice and wild-type mice. PI 3'-kinase activity showed similar increases in both groups of mice. Furthermore, ODC induction in IRS-1 transfected CHO cells was studied after stimulation by addition of FCS. The maximal ODC activity was 2.5-fold greater in IRS-1-transfected CHO cells than in control CHO cells. Our results suggest that the IRS-1 pathway may be involved in ODC induction. The absence of a difference in ODC and PI 3'-kinase activity in the regenerating liver between IRS-1-deficient mice and wild-type mice may have been related to the compensatory effects of IRS-2/pp190 [Araki et al. Nature (1994) 372, 186-190; Tobe et al. J.Biol.Chem. (1995) 270, 5698-5701].

Animals↗
PubMed1995

Structure of Alcaligenes faecalis nitrite reductase and a copper site mutant, M150E, that contains zinc.

The structures at 2.0 and 2.25 A resolution of native and recombinant nitrite reductase from Alcaligenes faecalis show that they are identical to each other and very similar to nitrite reductase from Achromobacter cycloclastes. The crystallographic structure of a mutant, M150E, which unlike the wild-type protein cannot be reduced by pseudoazurin, shows that the glutamate replacement for methionine binds to a metal at the type I Cu site via only one oxygen. Anomalous scattering data collected at wavelengths of 1.040 and 1.377 A reveal that the metal at the type I site is a Zn. No significant differences from the native structure other than local perturbations at the type I site are seen. A local pseudo 2-fold axis relates the two domains of different monomers which form the active site. The two residues, Asp98 and His255, believed to be involved in catalysis are related by this 2-fold. An unusual (+)-(+) charge interaction between Lys269, Glu279, and His100 helps to orient the active site Cu ligand, His100. A number of negatively charged surface residues create an electrostatic field whose shape suggests that it may serve to direct incoming negatively charged nitrite as well as to dock the electron donor partner, pseudoazurin.

Alcaligenes↗
PubMed1995

Characterization of a novel brain neutral glycosphingolipid composition in house musk shrew (Suncus murinus).

Glycosphingolipids were extracted from the brain of house musk shrew (Suncus murinus). Neutral glycosphingolipids were purified by QAE-Sephadex column chromatography followed by high-performance liquid chromatography using an Iatrobeads column. Purified glycosphingolipids were identified by high-performance thin-layer chromatography, carbohydrate analysis, fast-atom bombardment mass spectrometry and TLC immunostaining. The ganglioside pattern was almost the same as the pattern obtained for rat brain gangliosides. The brain of S. murinus, however, was unique in its neutral glycosphingolipid composition; it contained gangliotriaosylceramide and gangliotetraosylceramide as the major neutral glycosphingolipids in addition to monohexosylceramides. Mass spectroscopy analysis showed that C18:1 sphingosine and C24:0 normal fatty acids were the major ceramide constituents in the glycosphingolipis, except in the case where a slower-migrating monohexosylceramide that contained C24 h:0 hydroxy fatty acids was observed. A day after birth, monohexosylceramide contained only normal fatty acids. The amounts of hydroxy-fatty-acid-containing monohexosylceramide increased rapidly as the age of the animals increased, and the ratio of these two kinds of monohexosylceramides was reversed within five weeks. Monohexosylceramide, ganglioside, and sulfatide contents in bulbus olfactorius were almost equal in amount in contrast to the glycolipids in the cerebrum and cerebellum that contained monohexosylceramide as the major constituent. The amount of monohexosylceramide in the bulbus olfactorius was 0.3-0.4 times the values obtained for the cerebrum and cerebellum.

Animals↗
PubMed1995

A new type of two-dimensional thin-layer chromatography (TLC mapping) for analysis of acidic glycosphingolipid molecular species.

A new approach to the analysis of acidic glycosphingolipid molecular species has been developed by using two-dimensional thin-layer chromatography: TLC mapping. TLC mapping was accomplished by use of two types of high-performance thin-layer chromatographic (HPTLC) plates. A NH2-silica gel HPTLC plate developed with chloroform:methanol:1% diethylamine (50:47:15) was used for the first-dimension chromatography. Acidic glycosphingolipids on the plate were transferred to a normal silica gel HPTLC plate by developing the plate with chloroform:methanol:ammonia (4:10:6) after the NH2 plate was placed face to face on top of the silica gel HPTLC plate. Acidic glycophingolipids on the HPTLC plate were then developed with chloroform: methanol:0.2% CaCl2 (60:40:10) for the second-dimension chromatography. The use of the TLC-mapping method allowed us to compare detailed membrane constituents, including minor molecular species with regard to acidic glycophingolipids. The method is rapid and simple and is feasible for determining acidic glycosphingolipid from biological samples.

Animals↗
PubMed1995

Retrovirally transmitted gene therapy for gastric carcinoma using herpes simplex virus thymidine kinase gene.

BACKGROUND: Herpes simplex thymidine kinase (HTK) is known to phosphorylate ganciclovir (GCV). Phosphorylated GCV is incorporated into genomic DNA, which leads to inhibition of cell growth and cell death in the replicating cells. Recently, much attention has been drawn to the use of retrovirally mediated gene therapy using HTK as a new therapeutic approach for brain tumors. However, little is known about this phenomenon in gastrointestinal carcinomas. METHODS: The authors transfected the HTK gene packaged in retroviral vector into TMK-1 gastric carcinoma cells (TMK-HTK cells). Sensitivity of TMK-HTK cells to GCV was examined in vitro. Moreover, TMK-HTK cells were transduced into nude mice subcutaneously, and the effects of GCV therapy was examined at the concentration of 20 mg/kg daily for 14 days. RESULTS: TMK-HTK cells were sensitive to GCV at the concentration of 0.1 to 100 micrograms/ml in a dose- and time-dependent manner in vitro. Moreover, 12 of 13 TMK-HTK tumors, which were transduced into nude mice subcutaneously, shrunk from the average diameter of 6.5 mm. CONCLUSION: The results indicate that retrovirally transmitted gene therapy with GCV may provide a new therapeutic approach for treatment of gastric carcinomas.

Animals↗
PubMed1995

Identification of interaction site of pseudoazurin with its redox partner, copper-containing nitrite reductase from Alcaligenes faecalis S-6.

Pseudoazurin, a low molecular weight protein containing a single type I copper, functions as an electron donor to a copper-containing nitrite reductase (NIR) in a denitrifying bacterium Alcaligenes faecalis S-6. To elucidate the protein-protein interaction between these two copper-containing proteins, each of nine out of 13 lysine residues on the surface of pseudoazurin were independently replaced by alanine or aspartate, and the effects of the mutations on the interaction with NIR, as well as the physicochemical properties of pseudoazurin, were analyzed. All of the mutated pseudoazurins showed optical spectra and oxidation-reduction potentials almost identical to those of wild-type pseudoazurin, suggesting that none of the replacements of these lysine residues affected the environment around the type I copper site. Kinetic analysis of electron transfer between mutated pseudoazurins and NIR reveals that the lysine mutations have very little effect on the rate of electron transfer to NIR, but substitution at residues 10, 38, 57 and 77, all close to the copper site, substantially decreases the affinity of pseudoazurin for NIR. This suggests that pseudoazurin interacts with NIR through the region close to the type I copper site. The refined X-ray structures of Lys38Asp and Lys10Asp/Lys38Asp show that the molecular structure has indeed changed little. A new space group is observed for the Lys109Ala mutant crystal. Crystal packing interactions change for the Lys10Asp/Lys38Asp mutant but remain the same for Lys38Asp and Lys59Ala mutants.

Alanine↗
PubMed1995

Laparoscopic omental patch repair for perforated peptic ulcer.

OBJECTIVE: The authors' initial experience with laparoscopic omental patch repair for perforated peptic ulcer is documented. Its results are compared with those of other procedures and follow-up study is reviewed. SUMMARY BACKGROUND DATA: Since the advent of H2-antagonists, the usefulness of simple closure of a perforated peptic ulcer is increasing, and improvements in laparoscopic surgery have made possible minimally invasive surgery for perforated ulcer. METHODS: From December 1992 to February 1994, laparoscopic omental patch repair followed by use of H2-antagonists was performed successfully in 11 patients. Fifty-five patients underwent other surgical procedures for perforated peptic ulcers (conventional open omental patch: 4, selective vagotomy in combination with antrectomy: 24, distal gastrectomy: 27). RESULTS: The average operation time was 135 minutes. Administration of postoperative pain medication was reduced remarkably (0.9 times per patient), and all patients recovered rapidly. No serious postoperative complications were recorded. After a mean period of 11 months, the postoperative evaluation was satisfactory for all patients, and no ulcer recurrence was found. CONCLUSIONS: In perforated peptic ulcer disease, laparoscopic omental patch repair offers a number of advantages. Because no upper abdominal incision is made, there is decreased postoperative pain, and the patient rapidly recovers with fewer and less severe complications. Although the procedure requires a surgeon with particular expertise in endoscopic suturing technique, surgeons familiar with laparoscopic cholecystectomy can readily perform it after some practice. The authors' preliminary experience suggests that this is a minimally invasive procedure for perforated peptic ulcer that offers an attractive alternative to open surgery.

Adult↗
PubMed1995

Does bacitracin have an absorption-enhancing effect in the intestine?

We studied the absorption enhancement effects of three types of protease inhibitors, aprotinin, bacitracin and soybean trypsin inhibitor, in the rat intestine. Of these protease inhibitors, bacitracin enhanced the absorption of FD-4 and phenol red from the rat small and large intestine without mucosal toxicity. Thus, it was suggested that bacitracin has not only a protease-inhibitory but also an absorption-enhancing capability.

Animals↗
PubMed1995

Two different endothelin B receptor subtypes mediate contraction of the rabbit saphenous vein.

To study endothelin receptor subtypes that mediate venous smooth muscle contraction, effects of some endothelin receptor agonists and antagonists on the rabbit lateral saphenous vein were examined and compared with those on the saphenous artery. In the artery, endothelin (ET)-1 elicited concentration-dependent contractions, while selective ETB-receptor agonists, IRL1620 (Suc-[Glu9,Ala11,15]ET-1(8-21)) and sarafotoxin 6c (S6c) had almost no effect. The ET-1-induced responses shifted in parallel to the right by BQ-123 (cyclo (-D-Trp-D-Asp-Pro-D-Val-Leu-)), an ETA-receptor antagonist, or PD142893 (Ac-D-Dip-Leu-Asp-Ile-Ile-Trp), an ETA/ETB-receptor antagonist, indicating the involvement of the ETA receptor in this response. In the saphenous vein, not only ET-1 and ET-3, but also ETB-receptor agonists, IRL1620, S6c and [Glu9]sarafotoxin 6b ([Glu9]S6b), produced concentration-dependent, BQ-123-insensitive contractions. PD142893 did not affect the ET-1-induced contraction, but it shifted greatly the IRL1620-induced concentration-response curve in parallel to the right. The major components of ET-3-, S6c- and [Glu9]S6b-induced contractions were resistant to PD142893. These results indicate that two different vasoconstrictive ETB-receptor subtypes, ETB1 (sensitive to IRL1620 and PD142893) and ETB2 (insensitive to IRL1620 and PD142893), are located on the smooth muscle of the saphenous vein.

Animals↗
PubMed1994

Involvement of the COOH-terminal pro-sequence of Serratia marcescens serine protease in the folding of the mature enzyme.

The serine protease (SSP) from a Gram-negative organism, Serratia marcescens, is excreted even from Escherichia coli. The protease is synthesized as a 1045-amino-acid precursor (preproSSP) composed of three functional portions, a typical NH2-terminal signal peptide of 27 amino acids, the mature protease part of 618 amino acids, and a large COOH-terminal part of 400 amino acids. After the mature part (Ala28 to Asp645) has been liberated into the medium, most of the COOH-terminal part (Phe717 to Phe1045) remains stably in the outer membrane. When a mutated gene encoding the prepro-SSP with deletion of the junction region (Ser646 to Gly716) between the mature protease and the processed COOH-terminal protein was expressed in E. coli, the SSP protein was not detected in any fractions of the cells nor in the medium, whereas a processed COOH-terminal protein was found in the outer membrane. However, when the outer membrane fraction prepared from the transformant exhibiting the junction region (Ser646 to Gly716) probably on the cell surface was co-cultured with the transformant, SSP was found in the medium. Furthermore, a significant portion of the SSP protein denatured with guanidine hydrochloride was correctly refolded in vitro into the active protease, only in the presence of the outer membrane preparation from the transformant exhibiting the junction region. These results suggest that the junction region is exposed outside the cells, and it plays a role for guiding the folding of SSP in the correct conformation.

Amino Acid Sequence↗
PubMed1994

Mutation of a fungal aspartic proteinase, Mucor pusillus rennin, to decrease thermostability for use as a milk coagulant.

Mutagenesis of a fungus Mucor pusillus, a producer of an aspartic proteinase named Mucor pusillus rennin (MPR), was performed to obtain the mutated enzymes with decreased thermostability, which is desirable for practical use of the enzyme as a milk coagulant for cheese manufacturing. A fungal mutant strain which produced the mutant enzyme with distinctly reduced thermostability was isolated. Two different mutant alleles of the mpr gene, one with a single amino acid exchange of Ala101 for Thr and the other of Gly186 for Asp, were cloned out of this mutant strain. The mutated mpr genes were expressed in Saccharomyces cerevisiae under the control of the yeast GAL7 promoter to produce the active enzymes in extracellular medium. Both of the mutations, especially Gly186Asp, were confirmed to cause a marked decrease in thermostability of the enzyme. All mutants possessing exchanges of Gly186 for various amino acids by site-directed mutagenesis showed a decrease in thermostability, indicating involvement of this residue to maintain a conformation of the enzyme. A double mutant having the both exchanges, Ala101Thr and Gly186Asp, in a single molecule showed the lowest thermostability without decrease in the enzymatic activity as well as the relative ratio of clotting to proteolytic activity.

Amino Acid Sequence↗
PubMed1994

Substrate-dependent change in the pH-activity profile of alkaline endo-1,4-beta-glucanase from an alkaline Bacillus sp.

A neutral cellulase (BSC) from Bacillus subtilis and an alkaline cellulase (NK1) from alkalophilic Bacillus sp. N-4 show significant amino acid sequence homology. Despite the high homology, the pH-activity profiles of the two enzymes for carboxymethyl cellulose (CMC) hydrolysis are quite different; BSC shows a sharp optimum pH at 6, whereas NK1 shows its full activity in a broad range, from pH 6 to 10.5. For elucidation of the reasons for the difference in their pH-activity profiles, their activities were examined at various pHs using a series of cellooligosaccharides and their derivatives, cellotetraose (G4), cellopentaose (G5), cellohexaose (G6), cellopentaitol (G5OH), and cellohexaitol (G6OH), as substrates. The optimum pH of BSC was around 6 for all the cellooligosaccharides examined. On the other hand, the optimum pH of NK1 varied depending on the substrate, i.e., a sharp optimum at pH 6 with G4 and G5OH, and a broad optimum of pH 6 to 10.5 with G5, G6, and G6OH. Comparison of the kinetic parameters of the two cellulases at pH 7 and 9 using G6OH as a substrate revealed that NK1 showed similar values at both pHs, while BSC showed a greatly increased Km value for this substrate at pH 9. In addition, NK1 showed a greatly increased Km value for G5OH hydrolysis at pH 9. Both enzymes cleaved these substrates at the same position, which suggests the same productive binding mode of these substrates with both enzymes. All these observations suggest that the reduced enzyme activity of BSC in the alkaline pH range can be attributed to a decrease in the affinity of a subsite for the third glucose moiety from the scissile site of these substrates.

Alkalies↗
PubMed1994

Nitrile hydratase gene from Rhodococcus sp. N-774 requirement for its downstream region for efficient expression.

For improvement of the production of nitrile hydratase (NHase) from Rhodococcus sp. N-774 by recombinant DNA techniques, several plasmids, each of which had a deletion of the upstream or downstream region of the genes encoding the alpha and beta subunits of NHase, were constructed. Enzyme assays of recombinant R. rhodochrous and Escherichia coli cells showed that a downstream region of the NHase genes was indispensable for the production of active NHase in both cells, but for the production of the active amidase, no genes other than the amidase structural gene were required. The nucleotide sequence of the downstream region contained a single open reading frame (Orf1188) with 396 amino acids. Orf1188 showed similarity in amino acid sequence to P47K, an open reading frame found downstream of the NHase genes from Pseudomonas chlororaphis B23, and also to the cobW gene product, which may be involved in cobalamin biosynthesis in Pseudomonas denitrificans. Because the distance between the TGA stop codon for the NHase beta-subunit and the ATG codon for Orf1188 is only 98 bp, and because production of both Orf1188 and NHase is dependent on a promoter upstream of the amidase gene, these genes appear to be co-transcribed in a polycistronic manner, forming an operon. By optimization of the culture conditions of R. rhodochrous carrying pKRNH2, which contained the amidase, NHase, and Orf1188 genes, the transformant showed the NHase activity 6-fold higher than that of the original strain, Rhodococcus sp. N-774.

Amino Acid Sequence↗
PubMed1994

[Prolongation and recovery under sevoflurane anesthesia of neuromuscular blocking action of vecuronium].

Thirty-seven patients (ASA I or II) undergoing elective abdominal surgery were randomly allocated to four groups, which were anesthetized with nitrous oxide 67%, oxygen 33% and sevoflurane 0%, 1%, 2% and 3% respectively. We administered clinically used doses of vecuronium for abdominal surgery and studied the duration (the time from 100% block after injection to 20% recovery) and the recovery time (time from the last injection of vecuronium to 20% recovery). No prolongation of neuromuscular blocking effect was demonstrated with the initial dose of vecuronium. Significant prolongations of the duration and the recovery time were demonstrated with the maintenance dose of vecuronium only with sevoflurane over 2% and the effect was concentration-dependent.

Abdomen↗
PubMed1993

Amidase coupled with low-molecular-mass nitrile hydratase from Rhodococcus rhodochrous J1. Sequencing and expression of the gene and purification and characterization of the gene product.

The cloned 9.4-kb insert of plasmid pNHJ20L containing low-molecular-mass nitrile hydratase (L-NHase) gene from Rhodococcus rhodochrous J1 [Kobayashi, M. et al. (1991) Biochim. Biophys. Acta 1129, 23-33] was digested with various restriction enzymes, and the trimmed fragments were inserted into pUC18 or pUC19. A 1.96-kb EcoRI-SphI region located 1.9-kb downstream of the L-NHase gene was found to be essential for the expression of amidase activity in Escherichia coli; the gene arrangement of the amidase and the NHase in R. rhodochrous J1 differed from those in Rhodococcus species including N-774 and Pseudomonas chlororaphis B23. The nucleotide-determined sequence indicated that the amidase consists of 515 amino acids (54626 Da) and the deduced amino acid sequence of the amidase had high similarity to those of amidases from Rhodococcus species including N-774 and P. chlororaphis B23 and to indole-3-acetamide hydrolase from Pseudomonas savastanoi. The amidase gene modified in the nucleotide sequence upstream from its start codon expressed 8% of the total soluble protein in E. coli under the control of lac promoter. The level of amidase activity in cell-free extracts of E. coli was 0.468 unit/mg using benzamide as a substrate. This amidase was purified to homogeneity from extracts of the E. coli transformant with 30.4% overall recovery. The molecular mass of the enzyme estimated by HPLC was about 110 kDa and the enzyme consists of two subunits identical in molecular mass (55 kDa). The enzyme acted upon aliphatic amides such as propionamide and also upon aromatic amides such as benzamide. The apparent Km values for propionamide and benzamide were 0.48 mM and 0.15 mM, respectively. This amidase was highly specific for the S-enantiomer of 2-phenylpropionamide, but could not recognize the configuration of 2-chloropropionamide. It also catalyzed the transfer of an acyl group from an amide to hydroxylamine to produce the corresponding hydroxamate.

Amidohydrolases↗
PubMed1993

Role of the amino-terminal amino acid sequences determining the in vitro refolding process of prochymosin polypeptide.

Prochymosin, a zymogen of an aspartic proteinase chymosin, is produced as inclusion bodies in the recombinant Escherichia coli cells. Solubilization of the inclusion bodies with 8 M urea followed by dialysis at pH 10.5 achieves correctly refolded prochymosin to some extent, which is then activated by self-processing at acidic pHs. Analyses of the alkaline dialysates by anion exchange chromatography revealed broad distribution of prochymosin polypeptides with different conformations. Stepwise dialysis with a slower decreasing rate of urea resulted in marked improvement of the yield of correctly refolded molecules. A hybrid prochymosin (CR601) possessing a short NH2-terminal replacement with the trp-leader peptide was not refolded into the correct conformation by one-step dialysis, but it was by stepwise dialysis. Replacement of Lys at the NH2-terminal second position of CR601 with Asp or Glu caused marked enhancement of correct refolding. These findings suggest that the amino acid sequence in the NH2-terminal region of prochymosin plays a crucial role in determining the whole refolding process of the polypeptide.

Amino Acid Sequence↗
PubMed1993

Endothelin-2-converting enzyme from human renal adenocarcinoma cells is a phosphoramidon-sensitive, membrane-bound metalloprotease.

From the membrane fraction of cultured human renal adenocarcinoma (ACHN) cells, two endothelin-2-converting enzymes (ECE-2A and ECE-2B) were solubilized with detergent Lubrol PX and separated by hydrophobic butyl fast-performance liquid chromatography. The pH range of the converting activity of ECE-2B for big endothelin-1 (big ET-1), big ET-2, or big ET-3, was very narrow, and the optimal pH for each substrate was significantly different; the pH optimum for big ET-1 was 6.8 and that for big ET-2 or big ET-3 was 6.4. The ET-converting activity was abolished by phosphoramidon, 1,10-phenanthroline and EDTA but was not inhibited by thiorphan, E-64, leupeptin, PCMS, p-APMSF, or pepstatin A. The conversion efficiency for big ET-2 or big ET-3 by ECE-2B was approximately one-eighth of that for big ET-1. The molecular weight of ECE-2B was estimated to be 400 kDa by gel filtration. Because these characteristics of ECE-2B are very similar to those of ET-1-converting enzyme (ECE-1) in endothelial cells, these results raise the possibility that ECE-2B is identical to ECE-1 and that a single ECE physiologically converts all big ETs to the corresponding ETs in ET-producing cells.

Adenocarcinoma↗