Biomedical subjects

M Takagi

Publications and source records attributed to M Takagi.

At least 91 records · Page 5Linked to original sources

PubMed2001

Polymorphism of tumor necrosis factor-beta and alcohol dehydrogenase genes and alcoholic brain atrophy in Japanese patients.

BACKGROUND: Alcohol abuse can induce brain atrophy, but it only occurs in some alcoholics. Many inflammatory cytokines such as tumor necrosis factor (TNF) are produced rapidly in the brain by experimental or clinical injury. METHOD: To investigate whether genetic polymorphism of TNF was related to alcoholic brain atrophy, we determined restriction fragment-length polymorphisms of the TNF-beta genes in 72 male alcoholics. Computed tomography was used to determine the severity of brain atrophy. RESULTS: Digestion with NcoI and MspI after polymerase chain reaction amplification showed that the TNFB1 allele frequency was significantly higher in patients with brain atrophy than in those without brain atrophy (chi2 = 10.20, p = 0.0034). A multivariate analysis that included age, total alcohol intake, ADH2 genotype, and TNF-beta genotype showed that the ADH21/21 genotype and TNFB1/B1 genotype are independently associated with alcoholic brain atrophy. These findings suggest that the TNFB1 allele may be associated with alcoholic brain atrophy.

Adult↗

PubMed2001

Ultrastructural analysis of MHC class II molecule-expressing cells in experimentally induced periapical lesions in the rat.

Periapical lesions were induced by making 28 days of unsealed pulp exposures in the lower first molars of Wistar rats. Major histocompatibility complex class II molecule-expressing cells were then demonstrated by means of immunoperoxidase staining using a monoclonal antibody OX6, and the ultrastructure of these cells was analyzed under electron microscopy. OX6+ cells were classified into two major populations, (i.e. macrophages and dendritic cell (DC)-like cells. DC-like cells had elongated cytoplasmic processes, contained a few lysosomal structures, lacked distinct phagosomes, and were the most predominant cell type in the established lesion. Some of lymphocytes and plasma cells also showed a positive immunoreactivity. Both OX6+ macrophages and DC-like cells often showed a cell-to-cell attachment with lymphocytes. These findings suggested that major histocompatibility complex class 11 molecule-expressing macrophages and DC-like cells may play a crucial role in periapical lesion development by acting as antigen-presenting cells to memory T lymphocytes.

Animals↗

PubMed2001

Active subtilisin-like protease from a hyperthermophilic archaeon in a form with a putative prosequence.

The gene encoding subtilisin-like protease T. kodakaraensis subtilisin was cloned from a hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. T. kodakaraensis subtilisin is a member of the subtilisin family and composed of 422 amino acid residues with a molecular weight of 43,783. It consists of a putative presequence, prosequence, and catalytic domain. Like bacterial subtilisins, T. kodakaraensis subtilisin was overproduced in Escherichia coli in a form with a putative prosequence in inclusion bodies, solubilized in the presence of 8 M urea, and refolded and converted to an active molecule. However, unlike bacterial subtilisins, in which the prosequence was removed from the catalytic domain by autoprocessing upon refolding, T. kodakaraensis subtilisin was refolded in a form with a putative prosequence. This refolded protein of recombinant T. kodakaraensis subtilisin which is composed of 398 amino acid residues (Gly(-82) to Gly(316)), was purified to give a single band on a sodium dodecyl sulfate (SDS)-polyacrylamide gel and characterized for biochemical and enzymatic properties. The good agreement of the molecular weights estimated by SDS-polyacrylamide gel electrophoresis (44,000) and gel filtration (40,000) suggests that T. kodakaraensis subtilisin exists in a monomeric form. T. kodakaraensis subtilisin hydrolyzed the synthetic substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide only in the presence of the Ca(2+) ion with an optimal pH and temperature of pH 9.5 and 80 degrees C. Like bacterial subtilisins, it showed a broad substrate specificity, with a preference for aromatic or large nonpolar P1 substrate residues. However, it was much more stable than bacterial subtilisins against heat inactivation and lost activity with half-lives of >60 min at 80 degrees C, 20 min at 90 degrees C, and 7 min at 100 degrees C.

Amino Acid Sequence↗

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

Urinary prostaglandin D synthase (beta-trace) excretion increases in the early stage of diabetes mellitus.

OBJECTIVE: Circulating levels of lipocalin-type prostaglandin D synthase (L-PGDS)/beta-trace reportedly increase in renal failure as well as in cardiovascular injuries. We investigated the alterations of L-PGDS in urine and plasma in the early stage of type-2 diabetic patients. METHOD: Thirty-six type-2 diabetic patients and 29 normal subjects were studied. Overnight spot urine and plasma samples were obtained in the morning. L-PGDS was measured by ELISA method using anti-L-PGDS antibody. Variables indicating renal function were determined. RESULTS: Plasma L-PGDS concentration was slightly higher in the patients with diabetes mellitus than in the control subjects, whereas the urinary L-PGDS excretion almost doubled in the diabetic patients as compared with that in the control subjects. Plasma L-PGDS was determined by plasma creatinine (Cr) concentration while urinary L-PGDS excretion was correlated solely with urinary protein excretion. There was no relationship between plasma L-PGDS concentration and urinary L-PGDS excretion. The averaged plasma concentration of L-PGDS in the diabetics with a normal Cr level in plasma, corresponding to that in the controls, was determined by the plasma Cr concentration. On the other hand, the urinary L-PGDS excretion was determined by the amount of proteinuria and greater in the diabetics with a normal Cr level in plasma than in the controls even when the patients exhibited urinary protein excretion equal to that in the control subjects. CONCLUSIONS: Urinary L-PGDS excretion increased in the early stage of kidney injury in patients with type-2 diabetes mellitus. The urinary excretion was correlated independently with urinary protein excretion even when there was no difference in urinary protein or albumin excretions, thereby suggesting that urinary L-PGDS excretion is possibly a more sensitive indicator of renal injuries than proteinuria. Urinary L-PGDS may thus predict the progression of renal injuries in diabetic patients.

Analysis of Variance↗

PubMed2001

Inhibitory effects of TA-993 and its metabolite MB3 on platelet activation induced by collagen and U-46619 in human platelets.

In the present study, we investigated the effects of TA-993 and its metabolite MB3 on platelet activation in vitro. TA-993 and MB3 concentration-dependently inhibited platelet aggregation and ATP release induced by collagen in human platelets. Thromboxane (Tx) A2 formation, as determined by the production of TxB2, and the increase in intracellular Ca2+ concentration ([Ca2+]i) were also suppressed by TA-993 and MB3. TA-993 and MB3 did not inhibit TxA2 formation caused by arachidonic acid. These results suggest that the inhibition of platelet activation by TA-993 and MB3 is partly mediated by an inhibition of TxA2 formation at a step prior to cyclooxygenase. Furthermore, TA-993 and MB3 inhibited U-46619-induced platelet aggregation without blockade of the increase in [Ca2+]i, suggesting that they are likely to exert some additional effects on the intracellular events induced by Ca2+.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed2001

Four novel withanolide-type steroids from the leaves of Solanum cilistum.

Four novel withanolide-type steroids named cilistols p, pm, p1 and u (1-4, respectively), were isolated from the leaves of Solanum cilistum. The respective structures were characterized by spectroscopic means as follows: cilistol p (1) was (22R,24R,25R,26S)-1-oxo-22,26-epoxy-3alpha,5alpha-cycloergostane-6beta,17alpha, 24,25,26-pentaol 26-O-beta-D-glucopyranoside, cilistol pm (2) corresponded to the 6-O-methyl ether derivative of 1; cilistol p1 (3) was represented as the 24-O-methyl ether of 1, and cilistol u (4) was shown to be the epoxide between C-24 and -25, presumably bearing cilistols p, pm and p1 by ring-opening.

Antineoplastic Agents, Phytogenic↗

PubMed2001

Six new withanolide-type steroids from the leaves of Solanum cilistum.

Six new withanolide-type steroids, designated cilistols v, t, i, j, y and w (1-6, respectively), were obtained from the leaves of Solanum cilistum. Their respective structures were characterized by spectroscopic means as follows: Cilistol v (1) was (22R,24Z)-1alpha,3beta,22,26-tetrahydroxyergost-5,24-diene 26-O-beta-D-glucopyranoside, which is regarded as the precursor of other withanolide-type steroids included in this title plant. Cilistol t (2) was (22R,24S,25R,26S)-24,25;22,26-diepoxy-1alpha,3beta,26-trihydroxyergost-5-ene 26-O-beta-D-glucopyranoside, and cilistols j (3) and i (4) corresponded to the substances probably formed by the cleavage of the epoxy ring at C-24 and 25 of 2. Cilistol y (5) was 3-O-sulphonyl (22R,24S,25R,26R)-1-oxo-24,25; 22,26-diepoxy-3beta,17alpha,26-trihydroxyergost-5-ene 26-O-beta-D-glucopyranoside, and cilistol w (6) corresponded to the substance obtained by the fission of the epoxy ring at C-24 and 25 of 5. The occurrence of these withanolide type steroids from Solanum genera is rare and worthy of note.

Ergosterol↗

PubMed2001

Enhanced signal transduction by a directly fused protein of interleukin-6 and its receptor.

In order to develop a new type of agonist for the interleukin 6 (IL-6) signal, the gene encoding a directly fused protein (DFP) was constructed by joining the N-terminal portion of IL-6 and the C-terminal (soluble) portion of IL-6R (sIL-6R) without using a flexible polypeptide linker. The biological activity if DFP from a recombinant Pichia pastoris was examined by growth stimulation of IL-6-dependent BAF130 cells expressing human gp130, a membrane receptor. The recombinant DFP exhibited a much stronger growth stimulation (10 times) than the independent IL-6 and sIL-6R (IL-6/sIL-6R), mainly because association of the IL-6 and IL-6R could be maintained even at lower concentrations of DFP. Surface plasmon resonance (SPR) analysis showed that DFP bound to the extracellular portion of gp130 in the biphasic mode, and the dissociation constants of DFP for two phases were the same as those of IL-6/sIL-6R. In cells treated with DFP, stimulation of Stat3 phosphorylation was maintained for a longer period (150 min) than in cells treated with IL6/Il-6R, suggesting that the signal mediated by the DFP was more durable than that mediated by IL-6/sIL-6R, although the signal transduction mechanisms are almost the same for both DFP and IL-6/IL-6R. Therefore, the stronger activity of DFP was attributed to the maintained association of its subunits and/or prolonged phosphorylation of Stat3.

Journal Article↗

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

Effects of shifts up and down in osmotic pressure on production of tissue plasminogen activator by Chinese hamster ovary cells in suspension.

The effects of changes in osmotic pressure on the production of tissue plasminogen activator (tPA) by Chinese hamster ovary (CHO) cells in suspension were investigated. The osmotic pressure was shifted up from 300 mOsm/kg and down from 400 or 500 mOsm/kg to 400, 350, or 300 mOsm/kg during the cell growth and tPA production phases. In both phases, the specific cell growth rate was higher at a lower osmotic pressure after the shift, whatever it was up or down. After the osmotic pressure was shifted up during the production phase, the specific tPA production rate was high at a high osmotic pressure; however, shifting the osmotic pressure down also resulted in a high specific rate of tPA production. Additionally, specific production rate increased for 2 d after upward or downward shifts during the growth phase. Shifting the osmotic pressure cyclically between 300 and 500 mOsm/kg during the production phase improved the tPA production 1.13-fold compared with the amount produced at a constant osmotic pressure of 300 mOsm/kg.

Journal Article↗

PubMed2001

Effects of high concentrations of energy sources and metabolites on suspension culture of Chinese hamster ovary cells producing tissue plasminogen activator.

A high-concentration medium was developed for the high-density cultivation of Chinese hamster ovary (CHO) cells to produce tissue plasminogen activator (tPA). First, the effects of the concentrations of several medium components on the growth and metabolism of CHO cells were studied particularly in relation to the serum concentration, in order to develop a suitable concentrated medium. High concentrations of serum, glucose, lactate, and glutamine and a low ammonia concentration increased the specific growth rate and decreased the specific tPA production rate. Among these components, the effect of the serum concentration was the most significant. There was an inverse correlation between the specific rates of cell growth and tPA production. Using a medium containing glutamine at twice the concentration of the original medium (F-12) allowed the medium volume to be reduced to seven-ninths that of the original, without any reduction in tPA productivity. When the F-12 medium was supplemented five-fold with amino acids and vitamins, the total amount of tPA produced, specific rate of tPA production, and yield of tPA per unit volume of medium employed were respectively enhanced 1.7, 1.5, and 3.6 times. As a consequence, the frequency of medium exchange could be reduced. The results strongly indicate that the supplementation of nutrient amino acids and vitamins to a medium with a low concentration of serum are the most critical factors for cost-effective tPA production in high-density cell cultivation.

Journal Article↗

PubMed2001

Effect of antioxidants on the apoptosis of CHO cells and production of tissue plasminogen activator in suspension culture.

The effects of antioxidants on the apoptosis of Chinese hamster ovary (CHO) 1-15,500 cells and production of tissue plasminogen activator (tPA) in suspension culture were investigated. After cell growth to 2 x 10(5) cells/ml in Ham's F12 medium containing 10% newborn bovine serum (NBS) in a spinner bottle, CHO cells were maintained for 6 d in Ham's F12 medium containing 0 or 0.4% NBS and 10 mM antioxidants, namely, l-ascorbic acid 2-phosphate (VCP) or the reduced form of glutathione (GSH). The viable cell concentrations at day 6 in the serum-free culture with GSH and in the low-serum culture wiht VCP or GSH were 0.57, 1.04 and 1.69 x 10(5) cells/ml, respectively, while those in the serum-free and low-serum cultures without the antioxidants were only 2.33 and 1.17 x 10(3) cells/ml, respectively. The percentages of apoptotic cells in the serum-free and low-serum cultures with VCP (73.2, 44.6%) and GSH (76.9, 38.6%) measured using a flowcytometer after annexin V-FITC/propidium iodide staining were markedly lower than those in the cultures without antioxidants (96.3, 92.5%). The percentage of cells having a high mitochondrial membrane potential among the viable cells in the cultures with antioxidants determined using a flowcytometer after 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide staining was clearly higher than those in the cultures without the antioxidants. The production of tPA in the serum-free and low-serum media with VCP (0.282, 2.92 mg/l) or GSH (1.01, 1.61 mg/l) was markedly higher than that in the cultures without the antioxidants (0.275, 0.689 mg/l). Consequently, the suppression of apoptosis through the maintenance of the membrane potential of mitochondria by VCP or GSH resulted in a marked increase in tPA production by CHO cells in the serum-free and low-serum cultures.

Journal Article↗

PubMed2001

Synthesis of methyl (5Z,9Z,17R)-17-methylnonadeca-5,9-dienoate, the (R)-enantiomer of the structure proposed for a metabolite of the Philippine sponge Plakinastrella sp.

The (R)-enantiomer (1) of methyl (5Z,9Z)-17-methylnonadeca-5,9-dienoate, the structure proposed for a metabolite of the Philippine sponge, Plakinastrella sp., was synthesized. The 1H- and 13C-NMR spectra of the synthetic material were different from those reported for the natural product. The proposed structure 1 is therefore incorrect.

Animals↗

PubMed2001

Antimicrobial susceptibility of Staphylococcus intermedius isolated from healthy and diseased dogs.

A total of 90 strains of Staphylococcus intermedius isolated from dogs were examined for antimicrobial susceptibility. There were no significant differences in the distribution patterns of MICs between strains from 1982 to 1985 and those from 1999, and between strains from healthy dogs and those from diseased dogs. All of the strains were susceptible to ABPC, DMPPC, CEX, TDM, ERFX, BFLX, and FF at concentrations of 0.05 to 6.25 microg/ml. The MICs of OTC, KM, EM, AIV-TS, and LCM were distributed in a broad range of 0.1 to >100 microg/ml, indicating the existence of resistant as well as susceptible populations of S. intermedius. Thirty-three strains (36.7%) were resistant to one or more anitmicrobial agents such as OTC (n=32), KM (n=9), EM (n=7), AIV-TS (n=7), and LCM (n=7).

Animals↗

PubMed2001

High-turnover periprosthetic bone remodeling and immature bone formation around loose cemented total hip joints.

Aseptic loosening and periprosthetic osteolysis are the major problems awaiting solution in total hip surgery. The clinical investigation focused on the analysis of periprosthetic bone remodeling to clarify one important key event in the cascade of periprosthetic connective tissue weakening and osteolysis around loose artificial hip joints. Twelve acetabular bone samples adjacent to granulomatous synovial-like membrane of loose hip prosthesis were retrieved at revision surgery and processed for Villanueva bone staining for morphological observation and bone histomorphometric analysis. Eight well-fixed bony samples were used as control. Although osteoclastic surface and eroded surface by osteoclasts were evident in the periprosthetic bone from loose hip joints (p = 0.003 and p = 0.027), increased osteoid/low-mineralized bone matrix (p < 0.001) and osteoid width (p < 0.001) also were significant findings in structural analysis. In addition, not only elevated mineral apposition rate (MAR; p = 0.044) but also increased mineralizing surface (p = 0.044) and bone formation rate (BFR; p = 0.002) in loose periprosthetic bones were shown in dynamic data analysis. These results were confirmed by precise morphological observation by confocal laser scanning microscopy. Active coupling of bone formation and resorption and increased osteocytes with abundant bone canalicular projections were found in combined with the presence of immature bone matrices (osteoid and low-mineralized bone areas) in periprosthetic bones from loose hip joints. These results indicated that active osteoclastic bone resorption and/or defective bone formation are coupled with monocyte/macrophage-mediated foreign body-type granuloma in the synovial-like interface membrane of loose hip joints. Thus, this unique high-turnover periprosthetic bone remodeling with bad bone quality probably is caused by the result of cellular host response combined with inappropriate cyclic mechanical loading. The fragile periprosthetic bone may contribute to hip prosthesis loosening.

Aged↗

PubMed2001

Acid attack and cathepsin K in bone resorption around total hip replacement prosthesis.

Normal bone remodeling and pathological bone destruction have been considered to be osteoclast-driven. Osteoclasts are able to attach to bare bone surface and produce an acidic subcellular space. This leads to acid dissolution of hydroxyapatite, allowing cathepsin K to degrade the organic type I collagen-rich osteoid matrix under the acidic condition prevailing in Howship lacunae. Using a sting pH electrode, the interface membrane around a loosened total hip replacement prosthesis was found to be acidic. Confocal laser scanning disclosed irregular demineralization of the bone surface in contact with the acidic interface. Cathepsin K, an acidic collagenolytic enzyme, was found in interface tissue macrophages/giant cells and pseudosynovial fluid. Tissue extracts contained high levels of cathepsin K messenger RNA (mRNA) and protein. These observations suggest the presence of an acid- and cathepsin K-driven pathological mechanism of bone resorption, mediated not by osteoclasts in subosteoclastic space, but rather by the uncontrolled activity of macrophages in extracellular space.

Acids↗