Biomedical subjects

M Sakaguchi

Publications and source records attributed to M Sakaguchi.

At least 73 records · Page 4Linked to original sources

PubMed2001

Microsomal p450s use specific proline-rich sequences for efficient folding, but not for maintenance of the folded structure.

The amino-terminal region of microsomal P450s contains three distinct sequence motifs, the signal-anchor sequence (SA), the basic sequence (BS), and the proline-rich sequence (PR). Studies with two P450s of the CYP2C subfamily, P4502C11 (CYP2C11) and P4502C2 (CYP2C2), have indicated that upon expression in eukaryotic cells (yeast, COS cells, and insect cells), specific proline residues in PR are important for proper folding. In the present study, we have established that the PR region in a very different CYP gene family, P450c17 (CYP17), is also important for efficient folding. These studies have been carried out using expression in Escherichia coli. Using P4502C11, we have established that the folding requirements for P450s in bacteria are very similar to those in eukaryotic cells. Interestingly, when the PR from P450c17 is swapped for that of P4502C11 and visa versa, complete misfolding is observed. However, both the BS and SA can be swapped between these P450s without affecting folding. After proper folding of P450c17, removal of the PR by factor Xa protease has no effect on the maintenance of the P450 structure. Inspection of the sequences of many different CYP gene families indicates that the PR sequence is conserved within a gene family but varies considerably between families. We conclude that PR is important for directing the folding pathway leading to the functional P450, but not for maintaining the functional form.

Cytochrome P-450 Enzyme System↗

PubMed2001

Importance of a proline-rich sequence in the amino-terminal region for correct folding of mitochondrial and soluble microbial p450s.

All microsomal P450s have a proline-rich sequence (PR) in the amino-terminal region that is needed for proper folding [Kusano, K., Sakaguchi, M., Kagawa, N., Waterman, M.R. and Omura, T. (2001) J. Biochem., 129, 259-269]. There are also multiple proline residues near the amino-termini of the mature forms of all mitochondrial P450s and the amino-termini of soluble microbial P450s. To examine the functional significance of the PR in mitochondrial P450s, we expressed human P450c27 (CYP27) and bovine P450scc (CYP11A1) in an Escherichia coli heterologous expression system, and found that in each one specific proline residue is important for correct folding. Deletions from the amino-terminus further indicated the importance of the PR for the expression of a spectrally normal P450c27. Essentially the same results were obtained with two soluble microbial P450s, P450cam (CYP101) and P450nor, in each of which a PR is important for proper folding. We conclude that in all P450s (mitochondrial, microbial and microsomal P450s), a proline-rich sequence located in the amino-terminal region is important for proper folding. Furthermore, we predict that the importance of the PR in P450 folding is to reduce the tendency of the polypeptide to misfold prior to heme binding.

Amino Acid Sequence↗

PubMed2001

Preoperative small-dose ketamine prevented tourniquet-induced arterial pressure increase in orthopedic patients under general anesthesia.

UNLABELLED: The mechanism of tourniquet-induced arterial pressure increase is not known. We investigated the effect of preoperative ketamine on tourniquet-induced arterial pressure and heart rate changes in 85 patients undergoing knee surgery with a tourniquet under general anesthesia. Patients were randomly assigned into three groups; Large Ketamine (n = 28; ketamine 1.0 mg/kg), Small Ketamine (n = 28; ketamine 0.25 mg/kg), and Control (n = 29; normal saline) groups. Anesthesia was maintained with 1.5%-2.5% sevoflurane and 66% N(2)O in oxygen with endotracheal intubation. Ketamine or normal saline was given in a double-blinded fashion before skin incision and tourniquet inflation. Arterial pressure and heart rate were recorded every 10 min until 60 min after the start of tourniquet inflation and again after deflation. Arterial pressure and heart rate were compared among the three groups by using repeated-measures analysis of variance. In the Large and Small Ketamine groups, arterial pressure was not significantly changed, but in the Control group arterial pressure was significantly increased 40, 50, and 60 min after the start of tourniquet inflation (P < 0.05). Development of more than a 30% increase in systolic arterial pressure during tourniquet inflation was more frequent in the Control group than the other groups. The results show that preoperative IV ketamine, 0.25 mg/kg or more, significantly prevented tourniquet-induced systemic arterial pressure increase in patients under general anesthesia. IMPLICATIONS: Preoperative small-dose ketamine, IV, significantly prevented a systemic arterial pressure increase during prolonged tourniquet inflation in patients under general anesthesia.

Adolescent↗

PubMed2001

Phase Ia study of a hypoxic cell sensitizer doranidazole (PR-350) in combination with conventional radiotherapy.

A phase Ia study of a 2-nitroimidazole nucleoside analog radiosensitizer doranidazole was conducted to evaluate its toxicity and pharmacokinetics in patients undergoing conventional external beam radiotherapy. Twenty-nine patients, aged 40-74 years, with a WHO performance status of 0-2 and with adequate organ functions, were entered in the study. Single administration of doranidazole was investigated first with 13 patients and then a course of five consecutive daily administrations was tested in 16 patients. Doranidazole was given i.v. 25 min before irradiation. Doranidazole doses of 400, 800, 1300 and 2000 mg/m2 were evaluated in the former study, and daily doses of 800, 1300 and 2000 mg/m2 were investigated in the latter study. All patients tolerated doranidazole administration. Although a transient decrease in the 24-h creatinine clearance rate was observed in five patients (one in the single administration study and four in the repeat administration study), this was not considered to be the dose-limiting toxicity. Other toxicities (hematological and gastrointestinal), which may not be related to doranidazole administration, were also mild and were not dose limiting. No neurotoxicity was observed. The average maximum concentration, area under the time-concentration curve and half-life of doranidazole in serum were 172-194 microg/ml, 502-582 microg x h/l and 4.2-4.6 h, respectively, at 2000 mg/m2. At the tested doses, administration of doranidazole was tolerable and achieved serum concentrations at which reasonable radiosensitization could be expected. A phase Ib/II study to evaluate the feasibility and efficacy of up to 30 repeat administrations seems to be warranted.

Adult↗

PubMed2001

Molecular characterization of Lactobacillus plantarum genes for beta-ketoacyl-acyl carrier protein synthase III (fabH) and acetyl coenzyme A carboxylase (accBCDA), which are essential for fatty acid biosynthesis.

Genes for subunits of acetyl coenzyme A carboxylase (ACC), which is the enzyme that catalyzes the first step in the synthesis of fatty acids in Lactobacillus plantarum L137, were cloned and characterized. We identified six potential open reading frames, namely, manB, fabH, accB, accC, accD, and accA, in that order. Nucleotide sequence analysis suggested that fabH encoded beta-ketoacyl-acyl carrier protein synthase III, that the accB, accC, accD, and accA genes encoded biotin carboxyl carrier protein, biotin carboxylase, and the beta and alpha subunits of carboxyltransferase, respectively, and that these genes were clustered. The organization of acc genes was different from that reported for Escherichia coli, for Bacillus subtilis, and for Pseudomonas aeruginosa. E. coli accB and accD mutations were complemented by the L. plantarum accB and accD genes, respectively. The predicted products of all five genes were confirmed by using the T7 expression system in E. coli. The gene product of accB was biotinylated in E. coli. Northern and primer extension analyses demonstrated that the five genes in L. plantarum were regulated polycistronically in an acc operon.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗

PubMed2001

Significance of earlier carotid atherosclerosis for stroke subtypes.

BACKGROUND AND PURPOSE: In addition to advanced stenosis, earlier stages of carotid atherosclerosis are associated with the risk for stroke. However, the significance has not been established for specific stroke subtypes. This study examines the association of earlier carotid atherosclerosis with stroke subtypes. METHODS: The subjects comprised 1059 patients (mean+/-SD age, 62+/-11 years) with <60% carotid stenosis. With the use of ultrasound, carotid atherosclerosis was evaluated by the plaque score, as defined by the sum of all plaque heights in bilateral carotid arteries. On the basis of neurological signs and symptoms, medical history, and brain MRI, we diagnosed stroke and its subtypes as follows: no stroke (n=738), atherothrombotic infarction (AI) (n=56), lacunar infarction (LI) (n=117), cardioembolic infarction (n=65), cerebral hemorrhage (n=26), and other or unclassified stroke (n=57). RESULTS: The plaque score was higher in AI (10.5+/-5.9) and LI (6.0+/-5.1) groups than in the no-stroke group (4.3+/-4.9) (both P<0.05), although it was similar between other stroke groups and the no-stroke group. Each 1 SD greater plaque score was associated with 2.5-fold (95% CI, 2.0 to 3.2) higher risk for AI and 1.4-fold (95% CI, 1.2 to 1.7) higher risk for LI compared with the no-stroke group. When we adjusted for cardiovascular risk factors, plaque score remained significantly associated with AI but not with LI. By receiver operating characteristic curve analyses, the receiver operating characteristic area for AI (0.81 to 0.86) was greater than that for LI (0.62 to 0.67) when we used plaque score either alone or in combination with cardiovascular risk factors. CONCLUSIONS: Although evaluation of carotid atherosclerosis may aid in the risk assessment for AI and LI, the benefit appears to be greater for AI.

Aged↗

PubMed2001

Direct targeting of cis-Golgi matrix proteins to the Golgi apparatus.

The targeting route of newly synthesized GM130 and GRASP65 to the Golgi apparatus was investigated by three different approaches. First, localization of pulse labeled GM130 and GRASP65 in normal rat kidney (NRK) cells was traced by subcellular fractionation followed by immunoprecipitation. Immediately after the pulse labeling, GM130 and GRASP65 were found in the Golgi but not in the endoplasmic reticulum (ER) membrane fractions, whereas a control Golgi membrane protein was still found in the ER membrane fractions. Second, epitope tagged GM130 and GRASP65 were expressed in NRK cells by plasmid microinjection into the nuclei and their localization was analyzed by immunofluorescence. When ER to Golgi transport was inhibited by prior microinjection of a GTP-restricted mutant of Sar1 protein into the cytosol, the expressed GM130 and GRASP65 showed clear Golgi localization. Last, binding of GM130 and GRASP65 to the membranes was analyzed in vitro. In vitro synthesized GM130 and GRASP65 specifically bound to purified Golgi membranes but not to microsomal membranes. The bound GM130 and GRASP65 were found to form a complex with pre-existing counterparts on the Golgi membrane. These results strongly suggested that GM130 and GRASP65 are directly targeted to the Golgi membrane without initial assembly on the ER and subsequent vesicular transport to the Golgi apparatus.

Animals↗

PubMed2001

An orally active chymase inhibitor, BCEAB, suppresses heart chymase activity in the hamster.

We investigated the effects of a novel chymase inhibitor, BCEAB (4-[1-[[bis-(4-methyl-phenyl)-methyl]-carbamoyl]-3-(2-ethoxy-benzyl)-4-oxo-azetidine-2-yloxy]-benzoic acid). The IC50 value of BCEAB for purified human chymase was 5.4 nM, whereas BCEAB did not inhibit the angiotensin-converting enzyme, elastase and tryptase. In isolated dog arteries, the IC50 value of BCEAB for the angiotensin I-induced contraction in the presence of 1 microM lisinopril was 2.8 microM. In the hamster, the heart chymase activities were significantly suppressed to 42.0% and 26.9% 3 h after oral administration of 100 and 300 mg of BCEAB/kg of body weight, respectively. In conclusion, BCEAB is a useful chymase inhibitor for studying the role of chymase in vivo.

Angiotensin II↗

PubMed2001

Possible roles of cardiac chymase after myocardial infarction in hamster hearts.

The significance of cardiac chymase after myocardial infarction (MI) was evaluated using a hamster model of MI. At 1, 3, 7, 14, 28 and 56 days after MI, tissues were removed for measurements of angiotensin-converting enzyme (ACE) and chymase activities. The mean infarct size 3 days after left coronary artery ligation was 47.3 +/- 5.9% of the left ventricle circumference. The ratio of left ventricle weight to body weight was significantly increased from 3 days after MI. The level of plasma renin activity in the MI hamsters was significantly increased at the early phase of MI (1-3 days), while no significant changes in plasma ACE activity were observed. The ACE activity in the infarcted left ventricle was significantly increased starting from 3 days after MI and this increase was sustained up to 28 days. The chymase activity in the infarcted left ventricle was significantly increased starting from 1 day after MI and this increase was sustained up to 56 days. The number of chymase-positive mast cells in the infarcted left ventricle was significantly higher than in the sham group 3 and 7 days after operation. Treatment with an angiotensin (Ang) II type 1 receptor antagonist (candesartan cilexetil, 10 mg/kg per day) starting 3 days before the induction of MI significantly reduced the mortality rate during 14 days of observation following MI, whereas treatment with an ACE inhibitor (lisinopril, 20 mg/kg per day) did not. A significant improvement in hemodynamics (maximal negative and positive rates of pressure development, left ventricular systolic pressure and end-diastolic pressure, mean arterial blood pressure) was observed by the treatment with candesartan cilexetil, but not with lisinopril, 3 and 14 days after MI. These results suggested that Ang II produced by chymase may participate in the pathophysiologic state after MI in hamsters.

Angiotensin Receptor Antagonists↗

PubMed2001

Effects of beta-casomorphins on neuronal survival in culture of embryonic chick dorsal root ganglion neurons.

We studied the effects of beta-casomorphins (beta-CMs, mu-acting opioid peptides from milk protein beta-casein) on survival of primary-cultured chick dorsal root ganglion neurons in the presence of nerve growth factor. Beta-CM-5 and beta-CM-7 had potent neuronal survival-promoting activities. Beta-CM-4 amide (morphiceptin) and des-Tyr'-beta-CM-7 also exhibited the similar promoting effects, although their effects were very weak. The promoting effect of beta-CM-5 was prevented by co-administration of naloxone, or pretreatment with pertussis toxin. These results suggest that the neuronal survival-promoting effects of beta-CMs might be mediated through opioid receptors coupled to G proteins.

Amino Acid Sequence↗

PubMed2001

Clinical usefulness of duplex ultrasonography for the assessment of renal arteriosclerosis in essential hypertensive patients.

The present study was carried out to investigate whether the renal resistive index (RRI), obtained by ultrasonic duplex scanning, is useful for the evaluation of renal arteriosclerosis in essential hypertensive patients. We also studied the relationships between RRI and other kinds of hypertensive target-organ damage, including carotid atherosclerosis. One hundred and two patients (56.4+/-9.4 years) with untreated mild or moderate essential hypertension were examined. The normal range of RRI was determined for 12 normal age-matched volunteers (55.0+/-6.6 years). Hypertensive organ damage was evaluated by funduscopy, electrocardiograms, and carotid B-mode imaging. Based on the mean and distribution of RRI in normal volunteers (0.60+/-0.05), the normal upper limit of RRI was found to be 0.7. RRI was correlated with creatinine clearance (CCr) (r=-0.61, p<0.05), and blood urea nitrogen (r=0.46, p<0.05), but not with serum creatinine. In addition, the incidence of abnormal RRI (>0.7) was higher in patients with left ventricular hypertrophy and in those with advanced carotid atherosclerosis (p<0.01, respectively). Thus, RRI appears to be more strongly associated with CCr than with serum creatinine, and it increases in patients with hypertensive end-organ damage. The assessment of RRI may be useful for the evaluation of early renal damage in essential hypertension.

Adult↗

PubMed2001

Seasonal rhinitis in a cat sensitized to Japanese cedar (Cryptomeria japonica) pollen.

A cat showing seasonal allergic symptoms of rhinitis was examined for reactivities to Japanese cedar (Cryptomeria japonica, CJ) pollen allergen by intradermal skin test (IDST), Prausnitz-Kustner (P-K) test, and lymphocyte blastogenic response. In IDST for 26 common allergens. the cat showed a positive reaction to CJ pollen allergen. P-K test using CJ pollen allergen also showed a positive reaction, indicating the presence of serum IgE specific to CJ pollen. In the lymphocyte blastogenic response, the stimulation index in the presence of CJ pollen allergen was 2.4. These data suggested that the seasonal rhinitis observed in the cat was caused by the sensitization to CJ pollen allergen.

Animals↗

PubMed2001

Loss of nuclear localization of the S100C protein in immortalized human fibroblasts.

It is well known that cancer develops through a multistep process. In vitro transformation studies of normal human cells have shown that the immortalization step is critical for neoplastic transformation of cells. Furthermore, studies of cell fusion between normal and immortalized cells have indicated that the normal phenotype is dominant and the immortal phenotype is recessive. Thus we looked for cellular proteins that were down-regulated in immortalized human cells by two-dimensional gel electrophoresis to elucidate the mechanisms of immortalization of human cells. We found that the S100C protein was down-regulated in immortalized cells. This protein was localized in the cytoplasm of cells at the semiconfluent stage, while at the confluent stage it moved into the nuclei of normal cells but not into those of immortalized cells. Microinjection of an S100C antibody into normal confluent cells diminished the level of nuclear S100C protein, resulting in DNA synthesis. Taken together, loss of nuclear localization of the S100C protein, which may be related to DNA synthesis, is thought to be one of the mechanisms of immortalization.

Amino Acid Sequence↗

PubMed2001

Mycobacterium kansasii arthritis of the foot in a patient with systemic lupus erythematosus.

Mycobacterium kansasii, an atypical Mycobacterium, may cause serious illness in humans. We describe a M. kansasii infection of the foot joint, which was diagnosed in a 46-year-old woman with systemic lupus erythematosus. The diagnosis was based on a positive culture from degenerative tissue and histological diagnosis of a synovium. We reviewed the literature regarding M. kansasii infection of the joint, bone, and periarticular structures focusing on the complication of rheumatic diseases.

Arthritis, Infectious↗

PubMed2001

Duodenal somatostatinoma and erythrocytosis in a patient with von Hippel-Lindau disease type 2A.

A female with von Hippel-Lindau (VHL) disease type 2A first presented with erythrocytosis at the age of 9 years. This patient revealed multiple paragangliomas at age 22. After the removal of tumors, a retinal hemangioblastoma developed. Our diagnosis of VHL disease type 2A was confirmed. Moreover, systemic examination showed a duodenal somatostatinoma. Frequent and long-term monitoring is important for patients with pheochromocytomas or paragangliomas, and a screening for VHL disease and other hereditary cancer syndromes is recommended. Recognition of neuroendocrine tumors as a manifestation of VHL disease permits earlier diagnosis and improves prognosis.

Adult↗

PubMed2001

Reactivation of liver-specific gene expression in an immortalized human hepatocyte cell line by introduction of the human HNF4alpha2 gene.

An immortalized human hepatocyte cell line (OUMS-29) was established from fetal liver by transfection with the SV-40 large T antigen gene that has certain liver-specific functions such as albumin production and enzyme activities of CYP1A1, 1A2, and 2E1. To make OUMS-29 cells express other liver-specific functions, the human hepatocyte nuclear factor 4alpha2 (HNF4alpha2) gene was introduced into the cells, because this gene was found to be markedly down-regulated. The transduced HNF4alpha2 was overexpressed in the nuclei of the transfected cells, and its DNA-binding activity was also detected. The liver-specific genes such as apolipoprotein AI, CII, CIII, blood coagulation factor X, alpha1-antitrypsin, and HNF1alpha were up-regulated. Thus, this cell line is expected to be a useful tool for studying the differentiated human hepatocyte functions.

Apolipoprotein A-I↗

PubMed2001

Overexpression of platelet-derived growth factor B and downregulation of PDGF-receptor alpha in human immortalized fibroblasts.

Since immortalization of cells is critical in multistep carcinogenesis, efforts should be made to elucidate the mechanisms of the immortalization. To determine whether platelet-derived growth factor (PDGF) signal pathways play a role in immortalization of cells, we compared mRNA expressions of PDGFs and their receptors in three immortalized human fibroblast cell lines (SUSM-1, OUMS-24F, and KMST-6) with their normal parent cells. As a result, mRNA expression of PDGF-B (oncogene: c-sis) was upregulated in these immortalized cells. Unexpectedly, the expression of alpha- and beta-PDGF receptor genes was downregulated. PDGFR-alpha mRNA was remarkably decreased. When exogenous PDGFR-alpha was expressed transiently in the KMST-6 cells, the morphology of the cells resembled that of normal cells. These results suggest that the overexpression of PDGF-B (c-sis) and downregulation of PDGFR-alpha are related to the phenotypic characteristics of immortalized human cells.

Blotting, Northern↗

PubMed2001

Effects of oxygen concentrations on human fibroblasts treated with Fe(3+)-NTA.

Free radicals derived from the reaction of iron and oxygen are thought to be one of the causes of tissue injury. In order to identify whether oxygen concentrations are an important factor in iron-mediated damage to cells, cytotoxic effects of Fe(3+)-NTA on human fibroblasts (KMST-6 line) were studied under the conditions of 1% and 20% oxygen concentrations in an incubator. A comparison of the effects of Fe(3+)-NTA on cells cultured in 1% and 20% oxygen environments showed that the following features were more prominent under the usual culture concentrations of 20% oxygen: i) cytotoxicity, ii) increase in intracellular reactive oxygen species, iii) increase in H(2)O(2) production in the cells, and iv) formation of 8-hydroxydeoxyguanosine. To elucidate the roles of endogenous antioxidants, the levels of manganese superoxide dismutase (MnSOD) and catalase were measured by Western blotting. The increase in MnSOD in the presence of Fe(3+)-NTA was greater under the condition of 20% O(2) than under the condition of 1% O(2). The expression of catalase was significantly up-regulated at 20% O(2). However, when the cells were treated with Fe(3+)-NTA, the expression of catalase was markedly down-regulated under the condition of 20% O(2). Hydroxyl radical scavengers such as vitamin E, dimethyl-sulfoxide (DMSO) and mannitol reduced endogenous ROS generation and alleviated the cytotoxic effects of iron. On the other hand, superoxide dismutase (SOD), vitamin C and catalase did not show any protective effects against Fe(3+)-NTA. These findings suggest that enhanced cytotoxic effects of Fe(3+)-NTA at 20% O(2 )are due to endogenously produced hydroxyl radicals.

8-Hydroxy-2'-Deoxyguanosine↗