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Biomedical subjects

S Fujiwara

Publications and source records attributed to S Fujiwara.

At least 55 records · Page 3Linked to original sources
PubMed2001

Control of electron transfer in neuronal NO synthase.

The nitric oxide synthases (NOSs) are dimeric flavocytochromes consisting of an oxygenase domain with cytochrome P450-like Cys-ligated haem, coupled to a diflavin reductase domain, which is related to cytochrome P450 reductase. The NOSs catalyse the sequential mono-oxygenation of arginine to N-hydroxyarginine and then to citrulline and NO. The constitutive NOS isoforms (cNOSs) are regulated by calmodulin (CaM), which binds at elevated concentrations of free Ca(2+), whereas the inducible isoform binds CaM irreversibly. One of the main structural differences between the constitutive and inducible isoforms is an insert of 40-50 amino acids in the FMN-binding domain of the cNOSs. Deletion of the insert in rat neuronal NOS (nNOS) led to a mutant enzyme which binds CaM at lower Ca(2+) concentrations and which retains activity in the absence of CaM. In order to resolve the mechanism of action of CaM activation we determined reduction potentials for the FMN and FAD cofactors of rat nNOS in the presence and absence of CaM using a recombinant form of the reductase domain. The results indicate that CaM binding does not modulate the reduction potentials of the flavins, but appears to control electron transfer primarily via a large structural rearrangement. We also report the creation of chimaeric enzymes in which the reductase domains of nNOS and flavocytochrome P450 BM3 (Bacillus megaterium III) have been exchanged. Despite its very different flavin redox potentials, the BM3 reductase domain was able to support low levels of CaM-dependent NO synthesis, whereas the NOS reductase domain did not effectively substitute for that of cytochrome P450 BM3.

Amino Acid Substitution↗
PubMed2001

Intestinal transit of an orally administered streptomycin-rifampicin-resistant variant of Bifidobacterium longum SBT2928: its long-term survival and effect on the intestinal microflora and metabolism.

AIMS: The objectives of this study are to investigate the fate of a streptomycin-rifampicin-resistant variant of Bifidobacterium longum SBT2928 (BL2928SR) and the influence of its oral administration on the composition and metabolism of the intestinal microflora. METHODS AND RESULTS: Intestinal passage of BL2928SR was monitored by a combination of selection with antibiotics and identification by a randomly amplified polymorphic DNA (RAPD)-PCR method. Intestinal microflora was analysed by the method developed by Mitsuoka et al. (1965, 1974). Long-term survival of orally administered BL2928SR in the human intestine was confirmed. BL2928SR ingestion specifically lowered faecal populations of Enterobacteriaceae and clostridia, including lecithinase-positive Clostridium spp. CONCLUSION: BL2928SR and its parent strain, BL2928, are considered to be appropriate candidates for probiotics. SIGNIFICANCE AND IMPACT OF THE STUDY: It is clarified that BL2928SR has the ability for long-term survival in the human gastrointestinal tract, and alters the composition and metabolism of the intestinal microflora.

Actinomycetales Infections↗
PubMed2001

Establishment of orally-administered Lactobacillus gasseri SBT2055SR in the gastrointestinal tract of humans and its influence on intestinal microflora and metabolism.

AIMS: To investigate the fate of a streptomycin-rifampicin-resistant variant of Lactobacillus gasseri SBT2055 (LG2055SR) and the influence of its oral administration on the composition and metabolism of the intestinal microflora. METHODS AND RESULTS: Intestinal passage of LG2055SR was monitored by a combination of selection with antibiotics and identification by a randomly amplified polymorphic DNA (RAPD)-PCR METHOD: Composition of intestinal microflora was analysed by the method developed by Mitsuoka et al. (1965, 1974). Establishment of orally-administered LG2055SR in the human intestine was confirmed in this study. LG2055SR ingestion specifically lowered faecal populations of Staphylococcus and faecal contents of p-cresol. CONCLUSION: LG2055SR and its parent strain, LG2055, are considered to be appropriate candidates for probiotics. SIGNIFICANCE AND IMPACT OF THE STUDY: It is clarified that LG2055SR has the ability to establish in the human gastrointestinal tract and alters the composition and metabolism of the intestinal microflora and physical characteristics of faeces.

Adult↗
PubMed2001

Effects of pressure on the activity and spectroscopic properties of carboxyl proteinases. Apparent correlation of pepstatin-insensitivity and pressure response.

The pressure dependence of the activity and spectroscopic properties of four carboxyl proteinases were investigated. Two were pepstatin-sensitive carboxyl proteinases (porcine pepsin and proteinase A from baker's yeast) and two were pepstatin-insensitive carboxyl proteinases (from Pseudomonas sp. 101 (pseudomonapepsin; PCP) and Xanthomonas sp. T-22 (xanthomonapepsin; XCP)). The specificity constant [k(cat)/K(m(app))] of PCP and XCP for a synthetic peptide substrate showed only a slight decrease with increasing pressure, whereas pepsin and proteinase A showed substantial disactivation at higher pressures. The calculated apparent activation volume (Delta V((k(cat)/(K(m)) was about 1, 3, 13, and 14 mL.mol(-1) for PCP, XCP, pepsin, and proteinase A, respectively. The hydrolysis of acid-denatured myoglobin by the four carboxyl proteinases was only slightly affected by high pressure (except for proteinase A at 400 MPa), in contrast to the results for the peptide hydrolysis. In fact, PCP, XCP, and proteinase A actually showed slightly higher degradations of acid-denatured myoglobin at higher pressures. The residual activities of these enzymes after the incubation at high pressures implied a pressure-induced stabilization towards autolysis. The changes in the fourth derivative near-UV absorbance spectrum of the four enzymes in aqueous solution were measured at various pressures from 0.1 to 400 MPa. Upon an increase in pressure, the peaks from PCP and XCP red-shifted slightly, whereas pepsin and proteinase A blue-shifted substantially, thus indicating a more polar environment. The intrinsic fluorescence also decreased upon increasing pressure. However, the change for XCP was rather small, but the change for the other three was very large. The changes in the peak wavelength for pepsin and proteinase A were characteristic, and also indicated a more polar environment under high pressure. An analysis by the center of spectra mass (CSM) gave the Delta G and Delta V of transition as 9.8 kJ x mol(-1) and -24 mL x mol(-1) (pepsin) and 11.7 kJ x mol(-1) and -43 mL x mol(-1) (proteinase A), respectively, by assuming a simple two-state transition. The circular dichroism (CD) showed relatively small changes after 1-h incubations at 400 MPa, indicating that the secondary structures were largely maintained.

Animals↗
PubMed2001

Comparative analyses of the conformational stability of a hyperthermophilic protein and its mesophilic counterpart.

Comparison of the conformational stability of an O(6)-methylguanine-DNA methyltransferase (MGMT) from the hyperthermophilic archaeon Thermococcus kodakaraensis strain KOD1 (Tk-MGMT), and its mesophilic counterpart C-terminal Ada protein from Escherichia coli (Ec-AdaC) was performed in order to obtain information about the relationship between thermal stability and other factors, such as thermodynamic parameters, thermodynamic stability and other unfolding conditions. Tk-MGMT unfolded at Tm = 98.6 degrees C, which was 54.8 degrees C higher than the unfolding temperature of Ec-AdaC. The maximum free energy (DeltaG(max)) of the proteins were different; the value of Tk-MGMT (42.9 kJ.mol-1 at 29.5 degrees C) was 2.6 times higher than that of Ec-AdaC (16.6 kJ.mol-1 at 7.4 degrees C). The high conformational stability of Tk-MGMT was attributed to a 1.6-fold higher enthalpy value than that of Ec-AdaC. In addition, the DeltaG(max) temperature of Tk-MGMT was considerably higher (by 22.1 degrees C). The apparent heat capacity of denaturation (DeltaC(p)) of Tk-MGMT was 0.7-fold lower than that of Ec-AdaC. These three synergistic effects, increasing DeltaGmax, shifted DeltaG vs. temperature curve, and low DeltaC(p), give Tk-MGMT its thermal stability. Unfolding profiles of the two proteins, tested with four alcohols and three denaturants, showed that Tk-MGMT possessed higher stability than Ec-AdaC in all conditions studied. These results indicate that the high stability of Tk-MGMT gives resistance to chemical unfolding, in addition to thermal unfolding.

Bacterial Proteins↗
PubMed2001

Prolactinoma with preferential infrasellar extension: a report of two cases.

The authors describe two male patients with prolactinomas which were incidentally found either during the course of a complete work up for headache or after minor head trauma. Since these tumours were located mainly in the sphenoid bone and did not show any upward growth to the suprasellar region, they were initially thought to be a primary skull base tumour; however, the serum prolactin levels of these patients were 1,179 ng/ml and 3,260 ng/ml, respectively. The authors thus emphasise the need to recognise this peculiar infrasellar growth pattern of prolactinoma.

Adult↗
PubMed2001

Decreased expression of signaling lymphocytic-activation molecule-associated protein (SAP) transcripts in T cells from patients with rheumatoid arthritis.

The function of Epstein-Barr virus (EBV)-specific cytotoxic T cells is disturbed in rheumatoid arthritis (RA) patients but the mechanism for this disturbance has remained unknown. In a recent study searching for the causative gene of X-linked lymphoproliferative syndrome, the gene possibly linked to EBV-specific cytotoxic T cells or NK cell-mediated cytotoxic activity to EBV-infected cells was discovered, and its product is now referred to as signaling lymphocytic-activation molecule-associated protein (SAP) or Src homology 2 domain-containing protein (SH2D1A). In the present study, we attempted to investigate the involvement of the SAP gene in RA using a quantitative real-time PCR; the expression level of SAP transcripts in peripheral leukocytes or T cells was examined for patients with RA. The expression level of SAP transcripts in peripheral leukocytes of 21 RA patients was significantly lower than that of 13 normal individuals (P = 0.0007), four patients with palindromic RA, 11 with inactive systemic lupus erythematosus (SLE) or 17 with chronic renal diseases. The decreased expression of SAP transcripts in RA patients was also observed in peripheral CD2(+) T cells compared with normal individuals. There was no mutation in the coding region of SAP cDNAs derived from peripheral leukocytes of five RA patients. The decreased expression of SAP transcripts in peripheral leukocytes or T cells of RA patients might lead to the failure of the immune system to eliminate the EBV-infected synovial lining cells in joints of RA patients. Our findings have suggested that decreased expression of the SAP gene might be involved in the onset or progress of RA.

Antigens, CD↗
PubMed2001

Characterization of cytochrome b(5) in the ascidian Polyandrocarpa misakiensis and budding-specific expression.

A cDNA for cytochrome b(5) was cloned from a cDNA library of buds of the ascidian, Polyandrocarpa misakiensis, by a hybridization method involving a digoxigenin-labeled cDNA probe of human soluble cytochrome b(5). The nucleotide sequence of the cDNA for the ascidian cytochrome b(5) (Pmb5) consisted of about 1,800 base pairs including 5'- and 3'-noncoding regions, and a coding sequence of 405 base pairs. The amino acid sequence of 135 residues deduced from the coding nucleotide sequence exhibited 54% identity and 76% similarity to chicken cytochrome b(5). A highly conserved amino acid sequence was observed in the amino-terminal domain of 96 residues containing two heme-binding histidine residues. The putative soluble form of the recombinant Pmb5 expressed in Escherichia coli was purified to homogeneity by column chromatographies on an anion-exchanger and gel filtration. The purified Pmb5 showed the typical absorption spectrum of cytochrome b(5) with an asymmetric peak at 556 nm and a shoulder at 560 nm upon reduction with NADH and NADH-cytochrome b(5) reductase. The low temperature spectrum of the dithionite-reduced form of the protein contained the split peaks at 551 and 555 nm, this spectrum being very similar to that of mammalian liver cytochrome b(5). Expression of Pmb5 in the ascidian was examined immunohistochemically with a monoclonal antibody against the Pmb5. Apparently high level expression of Pmb5 was found in the developing buds, but the levels of cytochrome b(5) in the parents and juvenile adults were very low. This is the first report on the characterization of Pmb5, and the increased expression of Pmb5 in the ascidian.

Animals↗
PubMed2001

A phycocyanin-deficient mutant of synechocystis PCC 6714 with a single-base substitution upstream of the cpc operon.

The structure and expression of the cpc operon encoding phycocyanin subunits and linker polypeptides in a phycocyanin-deficient mutant (PD-1) and the wild-type of Synechocystis PCC 6714 were analyzed. The results of sequence and Northern blot analyses of the wild type indicate that the cpc operon consists of cpcB, cpcA, cpcC1, cpcC2 and cpcD, in that order. The levels of the transcripts in PD-1 were one-tenth to one-sixth as high as those in the wild type. In the PD-1 genome, a single-base substitution of C for T has occurred at base 259 upstream of the translational initiation codon of cpcB (at three bases downstream of the putative -10 region). To evaluate the in vivo transcription activities of these promoters in a cyanobacterium, we constructed vectors for the transformation of Synechococcus PCC7942, pANY1 and pANY2, which contain the upstream region of cpcB of the wild type (pANY1) or PD-1 (pANY2) and the promoter-less luxAB fusion. The bioluminescence of the transformants with pANY2 was one-tenth to one-sixth as high as that with pANY1. The coincidence of the results of Northern analysis and the promoter assay shows that the phycocyanin deficiency of PD-1 is due to the single-base substitution in the upstream region of the cpc operon.

Base Sequence↗
PubMed2001

Reverse redistribution of 99m Tc-sestamibi after direct percutaneous transluminal coronary angioplasty in acute myocardial infarction: relationship with wall motion and functional response to dobutamine stimulation.

Reverse redistribution (RR) of 99mTc-sestamibi is observed after direct percutaneous transluminal coronary angioplasty (PTCA) in acute myocardial infarction (AMI). The purpose of this study was to clarify the functional characteristics of myocardial segments with RR after direct PTCA in AMI. Thirty patients with AMI who had undergone direct PTCA were examined. Myocardial perfusion tomography with 99mTc-sestamibi and low dose dobutamine echocardiography were performed within 2 weeks of the onset. The 99mTc-sestamibi images were obtained 1 and 3 h after tracer administration. The left ventricle was divided into nine segments, and regional 99mTc-sestamibi uptake and clearance were quantitatively evaluated in each segment. RR was defined as a decrease in 99mTc-sestamibi uptake of >10% on 3 h delayed images compared with the 1 h early images. The left ventricle in the echocardiographic images was also divided into nine segments corresponding to the scintigraphic images, and regional wall motion was assessed in the resting condition as the baseline and during dobutamine administration (5-10 microg x kg(-1) x min(-1)). Out of a total of 270 myocardial segments, 111 segments were perfused by the culprit coronary artery and were defined as ischaemic segments. There were 25 segments with RR and 86 segments without RR in the ischaemic myocardium. Enhanced clearance of 99mTc-sestamibi was observed in ischaemic segments with RR (P<0.001). Echocardiography demonstrated that 24 out of 25 segments with RR and 61 out of 86 segments without RR had wall motion abnormalities. Dobutamine infusion improved wall motion in 20 (83%) of the 24 dysfunctional segments with RR and 33 (54%) of the 61 dysfunctional segments without RR (P<0.02). These findings suggest that RR indicates reversible functional abnormalities associated with preserved contractile reserve in response to dobutamine. The early and delayed imaging of 99mTc-sestamibi provides useful information regarding the residual viability of the dysfunctional myocardium in AMI patients.

Acute Disease↗
PubMed2001

Extracellular synthesis, specific recognition, and intracellular degradation of cyclomaltodextrins by the hyperthermophilic archaeon Thermococcus sp. strain B1001.

A unique extracellular and thermostable cyclomaltodextrin glucanotransferase (CGTase) from the hyperthermophilic archaeon Thermococcus sp. strain B1001 produces predominantly (>85%) alpha-cyclomaltodextrin (alpha-CD) from starch (Y. Tachibana, et al., Appl. Environ. Microbiol. 65:1991--1997, 1999). Nucleotide sequencing of the CGTase gene (cgtA) and its flanking region was performed, and a cluster of five genes was found, including a gene homolog encoding a cyclomaltodextrinase (CDase) involved in the degradation of CDs (cgtB), the gene encoding CGTase (cgtA), a gene homolog for a CD-binding protein (CBP) (cgtC), and a putative CBP-dependent ABC transporter involved in uptake of CDs (cgtDE). The CDase was expressed in Escherichia coli and purified. The optimum pH and temperature for CD hydrolysis were 5.5 and 95 degrees C, respectively. The molecular weight of the recombinant enzyme was estimated to be 79,000. The CDase hydrolyzed beta-CD most efficiently among other CDs. Maltose and pullulan were not utilized as substrates. Linear maltodextrins with a small glucose unit were very slowly hydrolyzed, and starch was hydrolyzed more slowly. Analysis by thin-layer chromatography revealed that glucose and maltose were produced as end products. The purified recombinant CBP bound to maltose as well as to alpha-CD. However, the CBP exhibited higher thermostability in the presence of alpha-CD. These results suggested that strain B1001 possesses a unique metabolic pathway that includes extracellular synthesis, transmembrane uptake, and intracellular degradation of CDs in starch utilization. Potential advantages of this starch metabolic pathway via CDs are discussed.

Amino Acid Sequence↗
PubMed2001

A novel C-type lectin regulating cell growth, cell adhesion and cell differentiation of the multipotent epithelium in budding tunicates.

We have isolated two Ca(2+)-dependent, galactose-binding polypeptides from the budding tunicate, Polyandrocarpa misakiensis. Based on their partial amino acid sequences, full-length cDNAs were cloned. One of them was identical with a tunicate C-type lectin (TC14-2) reported previously. The other was a novel C-type lectin, referred to as TC14-3. In living animals, they appeared to be coupled. This complex of lectins, when applied in vitro to tunicate multipotent cells of epithelial origin, blocked cell proliferation and induced cell aggregation. The aggregates expressed a homolog of the integrin alpha-chain and other differentiation markers specific for epithelial cells. Recombinant TC14-3 could reproduce all the activities of native lectins by itself, which was accelerated by recombinant TC14-2. The inhibitory activity of TC14-3 on cell growth was completely abolished by the addition of 50 microM D-galactose. Anti-TC14-3 monoclonal antibody showed that the antigen was expressed constitutively by the multipotent epithelial and mesenchymal cells. These results provide evidence that in P. misakiensis a C-type lectin plays a novel, cytostatic role in regulating cell growth, cell adhesion and cell differentiation during asexual reproduction.

Amino Acid Sequence↗
PubMed2001

Utilization of immobilized archaeal chaperonin for enzyme stabilization.

CpkA and CpkB are thermostable chaperonins from hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. CpkA or CpkB was immobilized onto agarose beads by the carbodiimide coupling method. In order to investigate whether immobilized CpkA or CpkB stabilizes a foreign enzyme, their ability to stabilize enzyme was examined using beta-D-galactosidase from Escherichia coli as a model target. In the absence of chaperonin beads, 38.1% of the total soluble protein was precipitated by heat treatment at 53 degrees C for 30 min. The protein structure of the residual soluble fraction was examined by circular dichroism, indicating that beta-D-galactosidase exists as a mixture of the active folded form and the inactive unfolded form. The specific activity of the residual soluble fraction decreased to 85.1% that of the unheated level (from 149.0 U/mg to 127.0 U/mg), indicating that the active folded form in the heated soluble fraction was 85.2%. In the presence of CpkA- or CpkB-beads, 28.6% or 34.3% of beta-d-galactosidase was precipitated by the same heat treatment. However, the specific activity in the soluble fraction was almost maintained (CpkA, from 151.0 U/mg to 140.3 U/mg; CpkB, from 149.0 U/mg to 140.3 U/mg). These results indicated that CpkA- or CpkB-beads are useful for keeping the enzyme active.

Journal Article↗
PubMed2001

Unique nucleoid structure during cell division of Thermococcus kodakaraensis KOD1.

The nucleoid structure and the partition in the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 were observed by a combination of phase-contrast microscopy and fluorescence microscopy. The nucleoids occurred as rounded fluorescent foci centrally located in the cells and as differences in fluorescence intensity between exponential and stationary phases. The cellular space occupied by the nucleoid in the stationary phase was larger than that in the exponential phase. Various shapes of nucleoid in the exponential-phase cells were observed, indicating that nucleoid separation was processed under cell cycle control. The number of cells which showed distinctive division stages was counted and the proportions of dividing cells were determined. About half of the observed cells were in the replication stage. More than 40% of the counted cells possessed a fully replicated but not separated form of nucleoid. Only 8% of the total cells clearly showed visible constriction. These results suggested that the post-replication period before cell division was relatively as long as the eucaryal gap period (G2); however, the period of visible cell constriction was almost the same as that of the bacteria.

Journal Article↗
PubMed2001

A new type of allantoinase in amphibian liver.

Allantoinase and allantoicase are known to form a complex in amphibian liver. In this study, a new type of allantoinase that did not form a complex with allantoicase was found in the amphibian liver. Purified enzyme had a molecular mass of about 44 kDa both in SDS-PAGE and gel-filtrations. The enzyme cross-reacted with anti-sardine allantoinase polyclonal antibody, and it weakly cross-reacted with anti-bullfrog allantoinase polyclonal antibody.

Amidohydrolases↗
PubMed2001

Effects of antihypertensive agents on blood pressure during exercise.

The relationship between blood pressure (BP) and cardiovascular morbidity has been appreciated for many years. Casual BP may not be representative of the pressure at other times. It is recognized that BP during exercise may be a more accurate predictor than casual BP. There is, however, little information about the effects of antihypertensive drugs on the BP during exercise. This study was designed to investigate the effects of various antihypertensive agents on BP during exercise. Sixty-four patients (age, 49+/-10 years) with untreated essential hypertension (WHO I, II) were studied during a supine ergometric exercise regimen. A graded exercise test was started at a workload of 50 W, and the load was increased by 25 W every 3 min. The hemodynamic responses to exercise were evaluated by changes in systolic and diastolic BP (SBP, DBP) and heart rate (HR). Plasma norepinephrine (NE) levels were measured at rest and during submaximal exercise, and before and after 4 weeks of treatment with metoprolol (METO), doxazosin (DOXA), trichlormethiazide (TCTZ), nifedipine (NIFE), amlodipine (AMLO) and temocapril (TEMO) between left ventricular mass index (LVMI) and BP values at rest, during exercise, and during the recovery period after exercise were assessed by multiple regression analysis. The stepwise selection (forward conditional) method showed that LVMI was significantly associated with SBP during submaximal exercise and during the recovery period. All antihypertensive treatments decreased SBP and DBP (p<0.01) at rest. METO, AMLO and TEMO significantly lowered SBP (p<0.05) during exercise, whereas DOXA, TCTZ and NIFE induced no change in SBP. The exercise-induced increase of plasma NE was further enhanced by METO and NIFE but not by AMLO, DOXA, or TCTZ, and it was significantly suppressed by TEMO (p<0.01). These results suggest that BP during exercise is more highly associated with the progression of left ventricular hypertrophy (LVH) than is casual BP. Because antihypertensive agents differ in their effects on exercise hemodynamics, we recommend that hemodynamic factors during exercise be considered when selecting the optimal antihypertensive medication for highly active patients.

Adult↗
PubMed2001

Pneumocystis carinii pneumonia in a Cavalier King Charles Spaniel.

Pneumocystis carinii pneumonia was diagnosed by postmortem examination of a one-year-old Cavalier King Charles Spaniel with four-week history of dyspnea. Cytologic and histologic examination of lung tissues revealed numerous P. carinii trophozoites and cysts, and P. carinii specific DNA was detected by polymerase chain reaction. The dog showed hypogammagloblinemia and extremely low levels of serum IgG. It was considered that P. carinii pneumonia in this case was associated with an immunodeficient condition which has already been reported in Miniature Dachshunds.

Animals↗