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Biomedical subjects

S Fujiwara

Publications and source records attributed to S Fujiwara.

At least 91 records · Page 5Linked to original sources
PubMed2000

[Na+]i, [K+]i and [Cl-]i regulation of exocytosis in guinea-pig antral mucous cells.

Effects of intracellular Na+, K+ and Cl- on Ca(2+)-regulated exocytosis activated by 10 microM acetylcholine (ACh) were studied in guinea-pig antral mucous cells which are permeabilized by nystatin treatment. Ca(2+)-regulated exocytotic events were modulated by [Na+]i, [K+]i and [Cl-]i via mediation of PTX-sensitive G proteins. Increases in [Na+]i and PTX inhibit G protein (G(Na)), which suppressed the exocytosis. Increases in [K+]i caused the exchange of G proteins (from G(Na) to G(K)) to increase, and GK evoked activation of the exocytosis and was inhibited by PTX. Increases in [Cl-]i and PTX inhibit G protein (G(Cl)), which stimulates exocytotic events. Based on these observations, the exocytosis in antral mucous cells were modulated by intracellular ions, concentration of which were increased or decreased by cell volume changes caused by Ach.

Acetylcholine↗
PubMed2000

Quantitation of macrophage migration inhibitory factor (MIF) using the one-step sandwich enzyme immunosorbent assay: elevated serum MIF concentrations in patients with autoimmune diseases and identification of MIF in erythrocytes.

We raised monoclonal antibodies against human macrophage migration inhibitory factor (MIF), and developed a one-step sandwich enzyme-linked immunosorbent assay (ELISA) method highly specific for human MIF. The ELISA system utilizes a solid phase monoclonal antibody as a capture antibody and a horseradish peroxidase-conjugated monoclonal antibody as a detector antibody. We used this ELISA method to evaluate the serum level of MIF in 240 healthy volunteers (140 males and 100 females). We found no significant difference in MIF concentration with respect to age. A significant difference was found with respect to sex, with the mean value (+/- SD) for male subjects of 5.3+/-2.3, and that for female subjects of 4.6+/-2.3 ng/ml (p<0.05). We next measured the serum MIF contents of patients with autoimmune diseases, and found that MIF levels were significantly elevated in patients with systemic lupus erythematosus and rheumatoid arthritis, 20.0+/-11.0 ng/ml and 21. 7+/-11.2 ng/ml, respectively. Using anti-MIF antibody-immobilized sepharose column chromatography, we discovered for the first time that MIF was present in erythrocytes. Taken together these results suggest that MIF plays a major role in autoimmune diseases and, moreover, potentially induces various patho-logical outcomes in cases of hemolytic disorders.

Adult↗
PubMed2000

[A case report of pulmonary embolectomy for acute pulmonary thromboembolism without extra-corporeal circulation].

A 69-year-old man complaining of abrupt dyspnea was admitted by ambulance. Acute massive pulmonary thromboembolism was diagnosed by pulmonary arteriography but after PAG cardiac standstill developed. Infusing of heparin and tPA immediately, cardiopulmonary resuscitation was successful after 5 minutes. Repeated PAG showed that thrombus in the right intermediate pulmonary artery was not detected, but was still detected in the left main pulmonary artery. The emergency embolectomy of left main pulmonary artery was performed without extracorporeal circulation and massive thrombi were removed. Mechanical respiratory support was required and we suffered from the frequent bleeding of the air way for one night. The patient was discharged about one month without any complaints.

Aged↗
PubMed2000

Ethanol patch test: a simple method for identifying the effectiveness of cyanamide in alcoholics.

BACKGROUND: To identify the pharmacological effectiveness of cyanamide, 144 alcoholics treated with cyanamide were subjected to a test that used an acetaldehyde dehydrogenase (ALDH) inhibitor, the ethanol patch test, which is considered to be a good indicator of ALDH2 phenotype. METHODS: We placed 100 microl of 70% ethanol on a lint pad and, as a control, placed the same volume of distilled water on a second pad. The ethanol patch test was performed on 144 alcoholics more than 2 weeks after abstinence from alcohol before and after treatment with cyanamide for 1 week. The dose of cyanamide was increased up to 150 mg until the patch test yielded a positive result. RESULTS: In the ethanol patch test, 36 alcoholics (25.0%) gave a positive result before treatment with cyanamide and might have been ALDH2(1)/2(2) heterozygotes. Among 108 alcoholics who were not positive, the distribution of the cyanamide dose that yielded a positive ethanol patch test result was 30 mg in 42 cases (38.9%), 50 mg in 33 cases (30.6%), 70 mg in 5 cases (4.6%), 100 mg in 6 cases (5.6%), and 150 mg in 2 cases (1.9%). Prevalence of liver cirrhosis was significantly higher in alcoholics who showed a positive ethanol patch test result at doses of less than 50 mg cyanamide than those at doses more than 70 mg (p = 0.029). The prevalence of adverse effects was significantly higher in alcoholics who showed a positive ethanol patch test result at doses of more than 70 mg than at doses of less than 50 mg cyanamide (p = 0.002). CONCLUSIONS: The ethanol patch test is a useful method for identifying pharmacological effectiveness of cyanamide and may reduce the prevalence of side effects in cyanamide-treated alcoholics.

Adult↗
PubMed2000

[mRNA detection of beta-myosin heavy chain gene in the autopsy cases of hypertrophic cardiomyopathy].

A known trigger of sudden cardiac death, hypertrophic cardiomyopathy (HCM) is associated with a point mutation in or overexpression of MYH7, which encodes the sarcomere protein beta-myosin heavy chain. We used nested RT-PCR to detect MYH7 mRNA in left ventricular myocardial tissue. We extracted total RNA from tissue samples that had been obtained after autopsy of 8 people who had died from HCM-related sudden death and that had been fixed in 10% formalin solution for as long as 4 years. The abundance of total RNA extracted from the 100 mg samples of cardiac muscle ranged from 10 to 24 mg/ml. The products of the nested RT-PCR were electrophoresed on a denaturing 8% polyacrylamide gel, and the MYH7 mRNA product was detected as a 424 bp band. MYH7 mRNA was easier to detect in tissue that had been fixed for shorter rather than longer periods. In addition, the greater the cardiac weight, the higher was the yield of the MYH7 mRNA product. Although mRNA had been detected by using RT-PCR on formalin-fixed paraffin-embedded tissue, no one previously had identified by using RT-PCR or nested RT-PCR on formalin-fixed tissue. By using nested RT-PCR, we were able to detect MYH7 mRNA in myocardial tissue that had been fixed in formalin solution for 4 years. Our results are applicable to retrospective examination into the cause of death in cases of sudden cardiac failure.

Adult↗
PubMed1999

IgE sensitivity and cross-reactivity to crude and purified mite allergens (Der f 1, Der f 2, Der p 1, Der p 2) in atopic dogs sensitive to Dermatophagoides mite allergens.

The present study investigates IgE-reactivity to crude and purified mite allergens by intradermal skin test (IDST), Immunodot method, and ELISA in atopic dogs sensitive to mite allergens, as well as the allergenic cross-reactivity between Dermatophgoides (D) farinae (DF) and D. pteronyssinus (DP) in dogs by IgE-ELISA inhibition. IDST and Immunodot method for crude mite allergens were performed for atopic dogs and 16 atopic dogs showed sensitivity to mite allergens. Of the 16 dogs, all dogs had anti-DF IgE and 11 had anti-DP IgE. We measured specific IgE to purified major allergens (Der f 1, Der f 2, Der p 1, Der p 2). Of the 16 atopic dogs, six had anti-Der f 1 IgE and seven had anti-Der f 2 IgE. Similarly, of the 16 dogs, six had anti-Der p 1 IgE and seven had anti-Der p 2 IgE. However, eight dogs had no specific IgE to these mite allergens. These dogs may be sensitive to other major mite allergens except Der 1 and Der 2. In the dogs that had both anti-DF and DP IgE, IgE binding to DF was greatly inhibited by DP, and reciprocal inhibition was observed. Based on these data, it appears that there is a strong cross-reactivity between DF and DP in dogs. Similarly, a cross-reactivity between DF and DP in purified allergens was also observed. IDST and Immunodot method are useful methods for the diagnosis of atopic diseases in dogs, and ELISA is a useful method for further investigation of IgE-reactivity for the allergens.

Allergens↗
PubMed1999

Single-point amino acid substitutions at the 119th residue of thermolysin and their pressure-induced activation.

The effect of amino acid substitution at the 119th site of thermolysin (TLN) on the pressure activation behavior of this enzyme was studied for four mutants at pressures < 300 MPa. For Q119Q, Q119N and Q119R, the highest activation was observed to be over 30 times that at atmospheric pressure and the activation volumes (deltaV++) were about -75 ml/mol. However, we obtained only 10 times higher activation for Q119E and Q119D (deltaV++ approximately -60 ml/mol). The intrinsic fluorescence of TLN changed at pressures > 300 MPa, and the latter two mutants showed a smaller deltaGapp and deltaVapp of transition than the wild type. These results are discussed with respect to the hydration change in the enzyme protein around the substituted region.

Amino Acid Substitution↗
PubMed1999

A retinoic acid-inducible modular protease in budding ascidians.

Retinoic acid-treated mesenchyme cells of the budding ascidian Polyandrocarpa misakiensis acquire an organizer activity to induce a secondary body axis when implanted into developing buds. We identified several different mRNAs that were upregulated in the mesenchyme cells after retinoic acid treatment. We isolated a cDNA clone corresponding to one of these mRNAs. The C-terminal region of the predicted protein product is homologous to the catalytic domain of serine proteases that belong to the trypsin family. The N-terminal region contains several types of protein-protein interaction domains. We therefore named this protein tunicate retinoic acid-inducible modular protease (TRAMP). Expression of the TRAMP mRNA in mesenchyme cells during budding and its upregulation by retinoic acid were demonstrated by reverse transcription-PCR and in situ hybridization. A glutathione S-transferase-TRAMP fusion protein showed a protease activity with trypsin-like substrate specificity and stimulated proliferation of the cell line established in this species.

Amino Acid Sequence↗
PubMed1999

Hyperthermostable protein structure maintained by intra and inter-helix ion-pairs in archaeal O6-methylguanine-DNA methyltransferase.

The crystal structure of O6-methylguanine-DNA methyltransferase (EC 2.1.1.63) of hyperthermophilic archaeon Pyrococcuskodakaraensis strain KOD1 (Pk -MGMT) was determined by single isomorphous replacement method with anomalous scattering (SIRAS) at 1.8 A resolution. The archaeal protein is extremely thermostable and repairs alkylated DNA by suicidal alkyl transfer from guanine O6 to its own cysteine residue. Archaea constitute the third primary kingdom of living organisms, sharing characteristics with procaryotic and eucaryotic cells. They live in various extreme environments and are thought to include the most ancient organisms on the earth. Structural studies on hyperthermophilic archaeal proteins reveal the structural features essential for stability under the extreme environments in which these organisms live, and will provide the structural basis required for stabilizing various mesophilic proteins for industrial applications. Here, we report the crystal structure of Pk-MGMT and structural comparison of Pk-MGMT and methyltransferase homologue from Escherichia coli (AdaC, C-terminal fragment of Ada protein). Analyses of solvent-accessible surface area (SASA) reveals a large discrepancy between Pk-MGMT and AdaC with respect to the property of the ASA. In the Pk-MGMT structure, the intra-helix ion-pairs contribute to reinforce stability of alpha-helices. The inter-helix ion-pairs exist in the interior of Pk-MGMT and stabilize internal packing of tertiary structure. Furthermore, structural features of helix cappings, intra and inter-helix ion-pairs are found around the active-site structure in Pk-MGMT.

Amino Acid Sequence↗
PubMed1999

Sequence and transcriptional studies of five clustered flagellin genes from hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1.

Five clustered genes (flaB1, flaB2, flaB3, flaB4 and flaB5) for multiple subunits of flagellar filaments from hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 were cloned and sequenced. Deduced amino acid sequences were aligned and it was revealed that five flagellin genes are homologous especially in the N-terminal hydrophobic region which might be important for interaction among flagellin subunits. Phylogenetic analysis was performed among archaeal flagellins from methanogens, an extreme halophile and our hyperthermophile, indicating that KOD1 flagellins were grouped together with methanogenic counterparts and were distinguishable from halophilic flagellins. Northern analysis of transcripts from flagellin genes from P. kodakaraensis KOD1 revealed that four major transcripts (0.98, 3.7, 5.4 and 9.2 kb) initiating from immediately upstream of flaB1 encode different combinations of five flagellins.

Amino Acid Sequence↗
PubMed1999

Analysis of DNA compaction profile and intracellular contents of archaeal histones from Pyrococcus kodakaraensis KOD1.

Two histone genes, hpkA and hpkB, from hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 strain were cloned, sequenced, and expressed in Escherichia coli cells. Both hpkA and hpkB genes encoded a protein of 67 amino acids, however they possessed the different molecular weight (HpkA, 7,378:HpkB, 7,167). Deduced amino acid sequences of HpkA and HpkB were homologous to other archaeal histones and eucaryal core histones (H2A, H4). Gel mobility shift assays by purified proteins demonstrated that HpkB possessed higher affinity to DNA and more extensive ability to compact DNA than HpkA. HpkB prevented double stranded DNA from thermal denaturation in less amount than HpkA in vitro. In order to investigate intracellular contents of HpkA and HpkB in KOD1 cells, immunoblot analysis was performed by using anti-HpkA antisera obtained from immunized BALB/c mice, showing that HpkA was less abundantly expressed than HpkB in KOD1 cells. These results suggest that HpkB plays a major role to protect double stranded DNA from thermal denaturation in vivo.

Amino Acid Sequence↗
PubMed1999

Isosmotic modulation of Ca2+-regulated exocytosis in guinea-pig antral mucous cells: role of cell volume.

1. Exocytotic events and changes of cell volume in mucous cells from guinea-pig antrum were examined by video-enhanced optical microscopy. 2. Acetylcholine (ACh) evoked exocytotic events following cell shrinkage, the frequency and extent of which depended on the ACh concentration. ACh actions were mimicked by ionomycin and thapsigargin, and inhibited by Ca2+-free solution and Ca2+ channel blockers (Ni2+, Cd2+ and nifedipine). Application of 100 microM W-7, a calmodulin inhibitor, also inhibited the ACh-induced exocytotic events. These results indicate that ACh actions are mediated by intracellular Ca2+ concentration ([Ca2+]i) in antral mucous cells. 3. The effects of ion channel blockers on exocytotic events and cell shrinkage evoked by ACh were examined. Inhibition of KCl release (quinine, Ba2+, NPPB or KCl solution) suppressed both the exocytotic events and cell shrinkage evoked by ACh. 4. Bumetanide (inhibition of NaCl entry) or Cl--free solution (increasing Cl- release and inhibition of NaCl entry) evoked exocytotic events following cell shrinkage in unstimulated antral mucous cells and caused further cell shrinkage and increases in the frequency of exocytotic events in ACh-stimulated cells. However, Cl--free solution did not evoke exocytotic events in unstimulated cells in the absence of extracellular Ca2+, although cell shrinkage occurred. 5. To examine the effects of cell volume on ACh-evoked exocytosis, the cell volume was altered by increasing the extracellular K+ concentration. The results showed that cell shrinkage increases the frequency of ACh-evoked exocytotic events and cell swelling decreases them. 6. Osmotic shrinkage or swelling caused the frequency of ACh-evoked exocytotic events to increase. This suggests that the effects of cell volume on ACh-evoked exocytosis under anisosmotic conditions may not be the same as those under isosmotic conditions. 7. In antral mucous cells, Ca2+-regulated exocytosis is modulated by cell shrinkage under isosmotic conditions.

Acetylcholine↗
PubMed1999

Dermatopontin interacts with transforming growth factor beta and enhances its biological activity.

Dermatopontin, a recently found low-molecular-mass component of the extracellular matrix, was studied for its interaction with decorin and transforming growth factor beta (TGF-beta) and its influence on TGF-beta bioactivity. Dermatopontin reacted with decorin with an apparent Kd of 100 nM in a solid-phase assay. Dermatopontin inhibited the formation of the decorin-TGF-beta1 complex. Decorin also competed with dermatopontin for the binding of this cytokine. The dermatopontin-decorin complex bound 3-fold more TGF-beta1 than did each component individually, and binding was inhibited more strongly by decorin preincubated with dermatopontin than by dermatopontin or decorin alone. Dermatopontin augmented the biological activity of TGF-beta1, as analysed by the expression of luciferase in mink lung epithelial cells transfected with a plasminogen activator inhibitor-promoter-luciferase construct, although dermatopontin itself did not show apparent induction of luciferase. Dermatopontin showed weak inhibitory activity on the proliferation of mink lung epithelial cells, and it enhanced the growth-inhibitory activity of TGF-beta on these cells. Thus dermatopontin increases the cellular response to TGF-beta. These findings strongly suggest that dermatopontin modifies the behaviour of TGF-beta through interaction with decorin in the microenvironment of the extracellular matrix in vivo.

Animals↗
PubMed1999

In vitro heat effect on functional and conformational changes of cyclodextrin glucanotransferase from hyperthermophilic archaea.

The in vitro heat effect on protein characteristics of thermostable enzyme was examined using a cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) from the hyperthermophilic archaeon Thermococcus sp. B1001 as a model protein. The recombinant form of CGTase was obtained as an inclusion body from Escherichia coli cells harboring a plasmid which carried the B1001 CGTase gene (cgtA). CGTase was solubilized by 6 M urea, refolded, purified to homogeneity, and heat treated at 80 degrees C for 20 min. Enzyme characteristics were examined compared with those of unheated CGTase. Cyclization activity was increased by in vitro heat treatment, while hydrolysis activity was decreased. The heated and unheated CGTases were analyzed for structures by circular dichroism (CD). The near- and far-UV CD spectra indicated that the structure of unheated CGTase with low cyclization activity was different from that of heated CGTase with high activity. Differential scanning calorimetry of unheated CGTase showed two absorption peaks at 87 and 106 degrees C with increasing temperature. After heat treatment, the minor peak at 87 degrees C disappeared, suggesting that heat-dependent structural conversion occurred in CGTase. These results indicate that the thermal environment plays an important role for the protein folding process of thermostable CGTase.

Calorimetry, Differential Scanning↗
PubMed1999

Pk-cdcA encodes a CDC48/VCP homolog in the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1: transcriptional and enzymatic characterization.

A gene encoding a cell division control protein from the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1, Pk-cdcA, was cloned and sequenced. The Pk-cdcA gene is composed of 2508 nucleotides, encoding a protein of 835 amino acids with a molecular mass of 93,666 Da. Pk-CdcA has a typical Walker-type ATPase motif and was classified as a new member of the CDC48/VCP subfamily of so-called AAA proteins. In addition, Pk-CdcA possesses a unique region composed of charged amino acids, which is not observed in other homologs from Archaea. Transcription of the gene was analyzed by primer extension and Northern analyses, revealing that Pk-cdcA is transcribed from a site 77 bases upstream of the initiation codon. Pk-CdcA and its deletion mutant Pk-CdcAdelta63, which lacks the unique inserted region, were expressed in Escherichia coli cells as His-tagged fusion proteins and purified. Both Pk-CdcA and Pk-CdcAdelta63 possess an ATPase activity, as do other CDC48/VCP proteins. However, Pk-CdcAdelta63 showed a higher level of ATPase activity and greater thermostability than Pk-CdcA. Furthermore, Pk-CdcAdelta63 has a higher Vmax value than wild type, even though the Km was unchanged. These observations indicated that the inserted region affects enzyme stability and activity. In order to investigate intracellular expression levels of Pk-CdcA, Western analysis was performed using anti-Pk-CdcA antisera obtained from immunized BALB/C mice. Equal levels of Pk-CdcA expression were observed during exponential and stationary phases. Growth phase-specific fragmentation of Pk-CdcA was found in stationary-phase cells.

Adenosine Triphosphatases↗
PubMed1999

The tryptophan biosynthesis gene cluster trpCDEGFBA from Pyrococcus kodakaraensis KOD1 is regulated at the transcriptional level and expressed as a single mRNA.

The entire gene cluster encoding enzymes involved in biosynthesis of L-tryptophan in the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 has been cloned and sequenced. Seven ORFs, which encode indole-3-glycerol phosphate synthase (trpC), anthranilate phosphoribosyltransferase (trpD), the two subunits of anthranilate synthase (trpEG), phosphoribosyl anthranilate isomerase (trpF) and the two subunits of tryptophan synthase (trpAB), were identified. The gene order is trpCDEGFBA, covering a region of 6045 bp. In order to confirm the function of the gene products, we expressed the first gene, Pk-trpC, in Escherichia coli. The protein product was purified, and was found to show the expected indole-3-glycerol phosphate synthase activity, with a temperature optimum of 85 degrees C. We could clearly identify a single mRNA transcript by Northern analysis using probes in the central and 3'-regions of the gene cluster, indicating that the gene cluster is transcribed as an operon. A significant increase in trp mRNA level was observed in cells grown in medium depleted of L-tryptophan, compared to cells grown in medium supplemented with L-tryptophan, indicating that expression of the gene cluster is regulated at the transcriptional level.

Base Sequence↗