Biomedical subjects

Hiroshi Sakai

Publications and source records attributed to Hiroshi Sakai.

At least 19 recordsLinked to original sources

PubMed2006

Influence of cationic molecules on the hairpin to duplex equilibria of self-complementary DNA and RNA oligonucleotides.

A self-complementary nucleotide sequence can form both a unimolecular hairpin and a bimolecular duplex. In this study, the secondary structures of the self-complementary DNA and RNA oligonucleotides with different sequences and lengths were investigated under various solution conditions by gel electrophoresis, circular dichroism (CD) and electron paramagnetic resonance (EPR) spectroscopy and a ultraviolet (UV) melting analysis. The DNA sequences tended to adopt a hairpin conformation at low cation concentrations, but a bimolecular duplex was preferentially formed at an elevated cationic strength. On the other hand, fully matched RNA sequences adopted a bimolecular duplex regardless of the cation concentration. The thermal melting experiments indicated a greater change in the melting temperature of the bimolecular duplexes (by approximately 20 degrees C) than that of the hairpin (by approximately 10 degrees C) by increasing the NaCl concentration from 10 mM to 1 M. Hairpin formations were also observed for the palindrome DNA sequences derived from Escherichia coli, but association of the complementary palindrome sequences was observed when spermine, one of the major cationic molecules in a cell, existed at the physiological concentration. The results indicate the role of cations for shifting the structural equilibrium toward a nucleotide assembly and implicate nucleotide structures in cells.

Cations↗

PubMed2006

Effects of low- or medium-pressure ultraviolet lamp irradiation on Microcystis aeruginosa and Anabaena variabilis.

A pure culture of Microcystis aeruginosa or Anabaena variabilis, the representatives of water blooming algae, was exposed to low-pressure (LP) or medium-pressure (MP) UV lamps. Irradiated pure culture suspension was subsequently incubated for 7d under white light fluorescent lamps. During incubation, profiles of the number of cells, DNA damage and photosynthetic activity were determined. When UV fluence was 600mJ/cm(2), M. aeruginosa cell numbers decreased throughout the 7-d incubation period, to produce 1.5log reduction (LP) or 1.2log reduction (MP) compared with control. The amount of DNA damage was 2.02x10(-4) ESS/base (LP) and 3.42x10(-4) ESS/base (MP) just after UV irradiation, which became 0.05x10(-4) ESS/base and 0.23x10(-4) ESS/base, respectively, after 3d incubation. However, cell number kept decreasing, even after DNA repair. Photosynthetic activity decreased by 1.5log within 1d (LP) or 3d (MP). Thus, reduction in photosynthetic activity could contribute to the reduction in M. aeruginosa cell numbers. A. variabilis cell numbers reduced by 2.3log (LP) or 2.2log (MP) during the 7-d incubation period; however, after DNA damage repair, cell number began to increase. The amount of DNA damage was 6.07x10(-4) ESS/base (LP) and 4.48x10(-4) ESS/base (MP) just after UV irradiation, which became 0.23x10(-4) ESS/base and 0.40x10(-4) ESS/base, respectively, after 3d incubation. No reduction was observed in photosynthetic activity/cell. Therefore, DNA damage is the main contributor of the reduction in cell number of A. variabilis.

Anabaena variabilis↗

PubMed2006

Total synthesis of (-)-5,6,11-trideoxytetrodotoxin and its 4-epimer.

[reaction: see text] The first total synthesis of 5,6,11-trideoxytetrodotoxin (1) and its 4-epimer were achieved. The synthesis is characterized by the stereoselective construction of the quaternary amino carbon center at C8a by an asymmetric transferring Strecker synthesis and the highly efficient conversion of cyanohydrin 4 to 1 via intramolecular cyclization reactions.

Molecular Structure↗

PubMed2006

Once-weekly epoetin-beta improves hemoglobin levels in cancer patients with chemotherapy-induced anemia: A randomized, double-blind, dose-finding study.

OBJECTIVE: To determine a recommended dose of once-weekly epoetin-beta administration for anemic cancer patients receiving myelosuppressive chemotherapy, we conducted a multicenter, randomized, double-blind trial. METHODS: A total of 86 patients with malignant lymphoma or lung cancer who received chemotherapy containing platinum, taxanes or anthracyclines were enrolled in the study. Patients were randomly assigned into groups that received three dose levels of epoetin-beta (9000, 18,000 or 36,000 IU) administered subcutaneously once a week for 12 weeks. The primary endpoint was change in hemoglobin, while the secondary endpoints were quality of life (QOL) assessed by Functional Assessment of Cancer Therapy-Anemia (FACT-An) questionnaire and transfusion requirements. RESULTS: Among the 69 patients (per protocol set population) assessable for efficacy, hemoglobin level change in the 36,000 IU group was significantly greater than that in the 9000 IU group (1.75 +/- 2.15 versus 0.04 +/- 1.98 g/dl; P = 0.009), and a significant dose-response relationship was observed for the change in hemoglobin level (P = 0.003). Although changes in FACT-An Total Fatigue subscale (Fatigue subscale) scores were similar for the three dosage groups, there was a statistically significant correlation (r = 0.435, P < 0.001) between the change in hemoglobin levels and the change in Fatigue subscale scores. The proportion of transfused patients was significantly smaller in the 36 000 IU group compared with that in the 9000 IU group (P = 0.022, not adjusted for pre-study transfusions). The incidence of adverse events was similar in the three dosage groups. CONCLUSIONS: Once-weekly epoetin-beta 36,000 IU for 12 weeks was well tolerated and significantly increased hemoglobin levels in anemic cancer patients receiving chemotherapy.

Adult↗

PubMed2006

Effect of disulfide-bond introduction on the activity and stability of the extended-spectrum class A beta-lactamase Toho-1.

The production of class A beta-lactamases is a major cause of clinical resistance to beta-lactam antibiotics. Some of class A beta-lactamases are known to have a disulfide bridge. Both narrow spectrum and extended spectrum beta-lactamases of TEM and the SHV enzymes possess a disulfide bond between Cys77 and Cys123, and the enzymes with carbapenem-hydrolyzing activity have a well-conserved disulfide bridge between Cys69 and Cys238. We produced A77C/G123C mutant of the extended-spectrum beta-lactamase Toho-1 in order to introduce a disulfide bond between the cysteine residues at positions 77 and 123. The result of 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) titrations confirmed formation of a new disulfide bridge in the mutant. The results of irreversible heat inactivation and circular dichroism (CD) melting experiments indicated that the disulfide bridge stabilized the enzyme significantly. Though kinetic analysis indicated that the catalytic properties of the mutant were quite similar to those of the wild-type enzyme, E. coli producing this mutant showed drug resistance significantly higher than E. coli producing the wild-type enzyme. We speculate that the stability of the enzymes provided by the disulfide bond may explain the wide distribution of TEM and SHV derivatives and explain how various mutations can cause broadened substrate specificity without loss of stability.

Amino Acid Sequence↗

PubMed2006

The influence of proprioceptive impairment on hand function in patients with cervical myelopathy.

STUDY DESIGN: A prospective clinical study. OBJECTIVES: To quantitatively evaluate hand function in patients with cervical myelopathy and to assess the influence of proprioceptive impairment on hand function in patients with cervical myelopathy. SUMMARY OF BACKGROUND DATA: Clumsiness of the hands is one of the most common complaints in patients with cervical myelopathy. However, few reports have been published that evaluate hand dysfunction, and no reports have assessed the association between hand function and proprioceptive impairment. PATIENTS AND METHODS: A total of 129 patients (36 women and 93 men) with cervical myelopathy were examined. Hand function was evaluated as scores of ten subtests designed on the basis of various hand activities. The degree of proprioceptive impairment of the hand as well as that of light touch sensation loss is classified into four grades. Scores of the hand function test are compared according to the grade of proprioceptive impairment of the hands. RESULTS: Significant relation between the impairment of hand function and the extent of cervical myelopathy was observed. There was good correlation between the hand function scores and the grade of impairment of proprioception and light touch sensation. Furthermore, patients with impairment of proprioception obtained significantly lower scores in the hand function test than those with loss of light touch sensation. CONCLUSIONS: The hand function test is a useful battery to objectively and quantitatively evaluate impairment of cervical myelopathy, although longitudinal studies are needed to determine its practical usability. There was some relation between scores of hand function test and the extent of proprioceptive impairment. Thus, hand function test may be partly influenced by proprioceptive loss.

Adult↗

PubMed2006

Crystallization and molecular-replacement studies of the monoclonal antibody mAbR310 specific for the (R)-HNE-modified protein.

4-hydroxy-2-nonenal (HNE), a major racemic product of lipid peroxidation, reacts with histidine to form a stable HNE-histidine Michael addition-type adduct possessing three chiral centres in the cyclic hemiacetal structure. Monoclonal antibodies against HNE-modified protein have been widely used for assessing oxidative stress in vitro and in vivo. Here, the purification, crystallization and preliminary crystallographic analysis of a Fab fragment of novel monoclonal antibody R310 (mAbR310), which recognizes (R)-HNE-modified protein, are reported. The Fab fragment of mAbR310 was obtained by digestion with papain, purified and crystallized. Using hanging-drop vapour-diffusion crystallization techniques, crystals of mAbR310 Fab were obtained. The crystal belongs to the monoclinic space group C2 (unit-cell parameters a = 127.04, b = 65.31, c = 64.29 A, beta = 118.88 degrees ) and diffracted X-rays to a resolution of 1.84 A. The asymmetric unit contains one molecule of mAbR310, with a corresponding crystal volume per protein weight of 2.51 A(3) Da(-1) and a solvent content of 51.0%.

Aldehydes↗

PubMed2006

Src homology 2 domain of overexpressed Lyn kinase is responsible for the acceleration of granulocyte colony-stimulating factor-induced neutrophilic nuclear lobulation.

We have previously shown that the overexpression of a Src family kinase, Lyn, and its kinase-negative form, LynKN, in a granulocyte progenitor cell line, GM-I62M, accelerates neutrophilic nuclear lobulation when the cells are cultured in the presence of granulocyte colony-stimulating factor. In this study, we investigated the role of the Src homology 2 (SH2) and SH3 domains of Lyn in the accelerated induction of nuclear lobulation. In contrast to wild-type Lyn, the overexpression of its SH2 domain mutant did not induce the accelerated nuclear morphological changes, but the overexpressed SH3 domain mutant had the same effects as wild-type Lyn. Therefore, the SH2 domain of Lyn is responsible for the accelerated induction of neutrophilic nuclear lobulation upon G-CSF stimulation.

Animals↗

PubMed2006

Relationship between renal function and plasma brain natriuretic peptide in patients with heart failure.

OBJECTIVES: This study sought to evaluate the relationship between brain natriuretic peptide (BNP), renal function, and the severity of congestive heart failure (CHF). BACKGROUND: Both BNP and renal function are prognostic predictors in CHF patients. METHODS: We measured the plasma BNP level in the aortic root and coronary sinus in 366 consecutive patients with CHF. Estimated glomerular filtration rate (eGFR) by the Cockcroft-Gault equation was used as an indicator of renal function. RESULTS: By stepwise multivariate analyses, hemodynamic parameters such as left ventricular ejection fraction (LVEF) and left ventricular end-diastolic pressure (LVEDP) but not eGFR were independent predictors of a transcardiac increase (coronary sinus-aortic root) in BNP. Regarding the plasma level of BNP in the aortic root, not only LVEF (p < 0.0001) and LVEDP (p < 0.0001) but also eGFR (p < 0.0001) were independent predictors. Patients were divided into two groups, patients with an eGFR > or =60 ml/min (group 1, n = 229) and patients with an eGFR <60 ml/min (group 2, n = 137). There was no difference in LVEF, LVEDP, or the transcardiac gradient of BNP between the two groups, but the plasma level of BNP in the aortic root was approximately two-fold greater in group 2 than in the group 1. CONCLUSIONS: These findings suggest that decreased clearance from the kidney contributes to the elevated BNP in CHF patients with renal dysfunction, especially in patients with an eGFR <60 ml/min.

Aged↗

PubMed2006

Evaluation of impairment of hand function in patients with cervical myelopathy.

Clumsiness of the hands is one of the most common complaints in patients with cervical myelopathy. However, few reports have been published that evaluate hand dysfunction. One hundred and twenty-nine patients (36 women and 93 men) with cervical myelopathy were examined and results compared with those of age- and sex-matched controls. Hand function was evaluated as scores of 10 subtests designed on the basis of various hand activities. Patients with cervical myelopathy obtained significantly lower scores on the hand function test in each age-group than controls. There was good correlation between hand function scores and the extent of cervical myelopathy. Therefore, the hand function test is a useful battery to objectively and quantitatively evaluate impairment of cervical myelopathy, although longitudinal studies are needed to determine its practical usability.

Adult↗

PubMed2006

Kruppel-like factor 6 (KLF6) affects the promoter activity of the alpha1-proteinase inhibitor gene.

PURPOSE: Keratoconus is a progressive disease that thins and scars the cornea. In keratoconus corneas, levels of degradative enzymes, including lysosomal acid phosphatase (LAP) and cathepsin B, are elevated, and those of inhibitors alpha1-proteinase inhibitor (alpha1-PI) and alpha2-macroglobulin (alpha2-M) are reduced. The present study explored the possible involvement in keratoconus of Krüppel-like factor 6 (KLF6), a transcription factor previously described to be essential for the integrity of the corneal epithelium. The transcript and proteins level of KLF6 and its action in regulating the genes affected in keratoconus were examined in this study. METHODS: Semiquantitative RT-PCR, Western blot analysis, immunofluorescence and in situ hybridization were used to investigate the expression of KLF6 mRNA and protein in normal and keratoconus corneas. Modulation by KLF6 of the promoter activity of alpha1-PI, LAP, cathepsin B, and alpha2-M genes was studied after transient transfection of KLF6 expression plasmid into corneal epithelial cells using promoter-reporter gene assays. Chromatin immunoprecipitation (ChIP) assays were performed to confirm the interactions between KLF6 and promoters of the genes affected in keratoconus. RESULTS: A global increased expression of the transcription factor KLF6 in terms of mRNAs and proteins was observed in total cornea and/or the epithelium in a substantial number of the keratoconus specimens. The promoter activity of the human alpha1-PI gene was suppressed by expression of KLF6 in corneal epithelial cells. The ChIP assay confirmed a physical interaction between KLF6 and the alpha1-PI promoter. CONCLUSIONS: Transcription factor KLF6 downregulates the alpha1-PI gene in corneal epithelial cells and may thereby be involved in keratoconus.

Adolescent↗

PubMed2006

Transduction of TAT fusion proteins into the human and bovine trabecular meshwork.

PURPOSE: To examine the applicability of TAT (the protein transduction domain of transactivating transcription polypeptide)-mediated protein-transduction technology, in introducing proteins of interest into trabecular meshwork (TM) cells in various culture systems. METHODS: Normal human TM cell cultures, human tissues in organ cultures, and bovine eyes in perfusion organ cultures were incubated or perfused for various lengths of time with TAT- and hemagglutinin (HA)-tagged fusion proteins, TAT-HA-beta-galactosidase (TAT-HA-beta-gal), TAT-HA-myocilin, and TAT-HA-myocilin-EGFP. Transduction of TAT-HA-beta-gal was detected by X-gal staining. Transduction of myocilin or myocilin-EGFP was evaluated by immunostaining or fluorescence. beta-Gal and EGFP proteins were used as the negative control. RESULTS: Blue X-gal staining, signifying beta-gal activity resulting from transduction, was observed in cultured TM cells in a concentration- and time-dependent manner. TAT-HA-beta-gal was also transduced into cells in all regions of TM tissues in organ cultures. TM cell cultures, after TAT-HA-myocilin incubation, showed an enhanced myocilin staining compared with the control cultures. Stronger myocilin or HA staining was also noted in TM tissues of TAT-HA-myocilin-incubated or -perfused eyes. Myocilin transduction resulted in a loss of actin stress fibers and focal adhesions in TM cells in culture. The level of phosphorylated myosin light chain was reduced. Human and bovine TM tissues after TAT-myocilin transduction also exhibited a diminished actin and paxillin-vinculin staining. CONCLUSIONS: TAT fusion proteins can be efficiently transduced into TM cells and tissues. The TAT-mediated protein transduction technology may be valuable in studies of proteins such as myocilin in the TM.

Actins↗

PubMed2006

Serum level of uric acid, partly secreted from the failing heart, is a prognostic marker in patients with congestive heart failure.

BACKGROUND: A recent study suggested that xanthine oxidase is activated in congestive heart failure (CHF). However, whether uric acid (UA) is secreted from the failing heart remains unknown, so it is currently unclear whether serum UA can provide prognostic information independent of brain natriuretic peptide (BNP). METHODS AND RESULTS: Serum UA was measured in the aortic root (AO) and the coronary sinus (CS) of 74 patients with CHF. The serum UA level was significantly higher in the CS than in the AO. The transcardiac gradient of UA (CS-AO) increased with the severity of CHF, inversely correlated with left ventricular ejection fraction (LVEF) and positively correlated with left ventricular end-diastolic volume index. The plasma levels of norepinephrine, BNP, UA, and LVEF were monitored prospectively in 150 CHF patients for a mean follow-up of 3 years. High plasma levels of UA (p<0.001) and BNP (p<0.001) were shown by multivariate stepwise analysis to be independent predictors of mortality. CONCLUSIONS: High plasma UA level, partly secreted from the failing heart, is a prognostic predictor independent of BNP in patients with CHF. Monitoring a combination of BNP and UA may be useful for the management of patients with CHF.

Aged↗

PubMed2006

Prediction of mortality by high-sensitivity C-reactive protein and brain natriuretic peptide in patients with dilated cardiomyopathy.

BACKGROUND: The prognostic role of serum C-reactive protein (CRP) in chronic heart failure (CHF) patients, especially those with nonischemic dilated cardiomyopathy (DCM), remains unknown. In the present study, whether CRP provides prognostic information in DCM patients was evaluated. METHODS AND RESULTS: Neurohumoral factors and hemodynamics in 84 consecutive DCM patients were measured and these patients were followed up for a mean period of 42 months. During the follow-up period, 23 patients developed cardiac events and 18 patients died of cardiac causes. Using stepwise multivariate Cox proportional hazards regression analyses, log brain natriuretic peptide (BNP) (p = 0.007) and high-sensitivity CRP (hsCRP) > 1 mg/L (p = 0.008) were significant independent predictors of cardiac events. The patients were stratified into 4 groups based on the normal serum concentration of hsCRP (1 mg/L) and median plasma concentration of BNP (110 pg/ml). Survival rates were significantly higher in patients with hsCRP < 1 mg/L and BNP < 110 pg/ml. The hazard ratio of patients with BNP > 110 pg/ml and hsCRP > 1 mg/L was 15.8 (95% confidence interval, 1.9-127.2) compared with those with BNP < 110 pg/ml and hsCRP < 1 mg/L for cardiac death. CONCLUSIONS: Serum hsCRP level is an independent prognostic predictor in patients with DCM and the combination of hsCRP and BNP may be useful for the management of CHF patients with DCM.

C-Reactive Protein↗

PubMed2006

Development of a new pulsatile ventricular assist device.

We developed a small, lightweight, low-cost implantable ventricular assist device (VAD) for use in smaller Japanese subjects. The major advantage of this pump is the simplicity of its fabrication. Most parts of the pump were shaped from a transparent acrylic block by a turning process, and the diaphragm was made from a silicon sheet. Since this method of construction did not require any complex processes, we could manufacture many pumps of various shapes. We determined the -most efficient shape for the Ebacor VAD using the flow visualization technique. The pump showed an output above 6 liters/min under a driving pressure of 300/-100 mmHg. The pump performance of current VADs is superior to that of the Ebacor VAD, because these pumps are larger. Since the Ebacor VAD is small in size, it can be driven by the driving system of a normal IABP control unit, which many hospitals already have in place. During a 30-day continuous driving performance test of this pump, no problems like performance decrements or water and air leakage were observed.

Heart-Assist Devices↗

PubMed2006

[Meningeal carcinomatosis in patients with lung cancer].

Of the 1548 patients with primary lung cancer who were admitted to our hospital from January 2001 through March 2005, 37 in whom meningeal carciomatosis was diagnosed on cytologic examination of cerebrospinal fluid or magnetic resonance imaging of the brain and spinal cord were studied retrospectively. The most common histologic type was adenocarcinoma, diagnosed in 70% of those patients. The results of cytologic examination of cerebrospinal fluid were positive in 71%. The time from the date of diagnosis of lung cancer to the date of diagnosis of meningeal carcinomatosis ranged from -2 days to 8 years (median, 407 days). Survival from the date of diagnosis of meningeal carcinomatosis ranged from 10 to 392 days (median, 106 days). Treatment for meningeal carcinomatosis was decided on the basis of the patient's general condition and the contorol status of the primary lesion. Radiotherapy, systemic chemotherapy, and palliative therapy were combined. Gefitinib was most often used for chemotherapy after the onset of meningeal carcinomatosis, and 60% of the patients given gefitinib had stable disease. One of these patients (adenocarcinoma) survived for longer than 1 year. Further investigatons are needed to establish standard treatments, including the use of gefitinib, that can improve the quality of life and prolong the survival of patients with meningeal carcinomatosis.

Adenocarcinoma↗

PubMed2005

Amino acid ester salt recognition by ferrocene-based ditopic receptor bearing oligoethylene glycol with pendant bipy subunits: CV, UV-vis and ESR studies.

The novel ferrocene-based ditopic receptor 1 was synthesized. This receptor bears two oligoethylene glycol arms with pendant 2,2'-bipyridine unit at the identical cyclopentadienyl rings, Cu(I) cation binds to 1 to form the 1 : 1 complex (1.Cu(I)) with the cavity consisting of polyether, and the resulting complex acts as a receptor for amino acid ester salts to give the ditopic complex (1.Cu(I).AAOMe-HCl). The 1H NMR spectrum of 1.Cu(I).LeuOMe-HCl exhibits strong broadening at the bipyridine region, and the ESR spectrum of the same sample gives the signals assigned as Cu(II) species. With these data, the binding of 1.Cu(I) towards AAOMe.HCl leads to the conformational change, and the Cu(I) complex is simultaneously oxidized to the Cu(II) complex.

Amino Acids↗

PubMed2005

Crystallization and preliminary X-ray analysis of pyruvate kinase from Bacillus stearothermophilus.

Pyruvate kinase (PK) from a moderate thermophile, Bacillus stearothermophilus (BstPK), is an allosteric enzyme activated by AMP and ribose 5-phosphate but not by fructose 1,6-bisphosphate (FBP). However, almost all other PKs are activated by FBP. The wild-type and W416F/V435W mutant BstPKs were crystallized by the hanging-drop vapour-diffusion method. However, they were unsuitable for structural analysis because their data sets exhibited low completeness. A crystal suitable for structural analysis was obtained using C9S/C268S enzyme. The crystal belonged to space group P6(2)22, with unit-cell parameters a = b = 145.97, c = 118.03 A.

Crystallization↗