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Biomedical subjects

M Takagi

Publications and source records attributed to M Takagi.

At least 127 records · Page 7Linked to original sources
PubMed2000

Multifocal Electroretinogram in Central Serous Chorioretinopathy.

Purpose: An assessment of retinal function topographically in the affected eyes with central serous chorioretinopathy (CSC).Participants and Method: We recorded multifocal electroretimogram (mERG) in 5 patients with unilateral CSC, and analyzed the topographical properties in the central visual field (rings #1 + 2).Results: mERG amplitudes in the CSC eyes were significantly reduced compared with the fellow eyes, for P 1-N 1 (58.5 +/- 23.5%, p < 0.05) and for P 1-N 2 (47.5 +/- 15.0%, p < 0.05). mERG latencies in the CSC eyes were significantly increased compared with those in the fellow eyes, for N 1 (by 9.0 +/- 8.1%, p < 0.05) and for P 1 (by 8.4 +/- 7.0%, p < 0.05). mERG in the CSC eyes gradually recovered in the follow-up period. However, the responses did not recover to normal value during follow-up, even when the subretinal fluid disappeared ophthalmoscopically.Conclusions: These results show that a topographical analysis of the mERG is useful for clinical observation of CSC.

Journal Article↗
PubMed2000

Antiplatelet mechanisms of TA-993 and its metabolite MB3 in ADP-induced platelet aggregation.

We investigated the antiplatelet mechanisms of TA-993 [(-)-cis-3-acetoxy-5-(2-(dimethylamino)ethyl)-2, 3-dihydro-8-methyl-2-(4-methylphenyl)-1,5-benzothiazepin-4(5H)-one maleate] and its metabolite MB3 (deacetyl and N-monomethyl TA-993) in human platelets stimulated by ADP in vitro. TA-993 and MB3 concentration-dependently inhibited fibrinogen binding to the ADP-stimulated platelets as well as inhibiting platelet aggregation. The antiplatelet effect of MB3 was about 300 times more potent than those of TA-993 and a glycoprotein IIb/IIIa receptor antagonist, Arg-Gly-Asp-Ser (RGDS). Aggregation of ADP-treated fixed platelets caused by the addition of fibrinogen was inhibited by RGDS but not by TA-993 and MB3. TA-993 and MB3 inhibited ADP-induced polymerization of actin filaments. Neither TA-993 nor MB3 affected cyclic AMP and cyclic GMP levels in resting platelets, and nor suppressed the increase in intracellular Ca(2+) concentration induced by ADP. These results suggest that the antiplatelet mechanisms of TA-993 and MB3 may involve inactivation of glycoprotein IIb/IIIa receptors via inhibition of the polymerization of actin.

Actins↗
PubMed2000

Inhibition of Fas-mediated apoptosis by Trypanosoma cruzi infection.

Trypanosoma cruzi-infected and normal control mammalian cells were subjected to analysis of Fas-mediated apoptosis stimulated by an agonistic anti-Fas monoclonal antibody. The infected cells showed markedly hampered apoptotic changes in nuclear morphology, phosphatidylethanolamine translocation from the inside to the outside of the plasma membrane, and DNA fragmentation into multiples of 180 bp, relative to normal control cells. Upstream of these morphological and biochemical consequences, the caspase-3 activity was elevated by the Fas stimulation in a significantly greater proportion of intact control cells, but at a highly reduced rate of infected cells. The rapid elevation of caspase-8 activity in control, apoptotic cells was completely inhibited in infected cells. In an examination of the specificity of other stimulants, X-ray radiation or chemicals such as hydrogen peroxide, colchicine or etoposide did not cause significant differences in apoptotic rates between control and infected cells; tumor necrosis factor-alpha, however, induced a high rate of apoptosis in control cells, with an extremely lowered rate in infected cells. This study demonstrates, for the first time, that T. cruzi infection inhibits one of the earliest steps of death receptor-mediated apoptosis, an effect that most probably involves the inhibition of caspase-8. Differential apoptotic responses in cells infected with T. cruzi and other intracellular parasites are discussed.

Animals↗
PubMed2000

Characterization of 1-deoxy-D-xylulose 5-phosphate reductoisomerase, an enzyme involved in isopentenyl diphosphate biosynthesis, and identification of its catalytic amino acid residues.

1-Deoxy-d-xylulose 5-phosphate (DXP) reductoisomerase, which simultaneously catalyzes the intramolecular rearrangement and reduction of DXP to form 2-C-methyl-d-erythritol 4-phosphate, constitutes a key enzyme of an alternative mevalonate-independent pathway for isopentenyl diphosphate biosynthesis. The dxr gene encoding this enzyme from Escherichia coli was overexpressed as a histidine-tagged protein and characterized in detail. DNA sequencing analysis of the dxr genes from 10 E. coli dxr-deficient mutants revealed base substitution mutations at four points: two nonsense mutations and two amino acid substitutions (Gly(14) to Asp(14) and Glu(231) to Lys(231)). Diethyl pyrocarbonate treatment inactivated DXP reductoisomerase, and subsequent hydroxylamine treatment restored the activity of the diethyl pyrocarbonate-treated enzyme. To characterize these defects, we overexpressed the mutant enzymes G14D, E231K, H153Q, H209Q, and H257Q. All of these mutant enzymes except for G14D were obtained as soluble proteins. Although the purified enzyme E231K had wild-type K(m) values for DXP and NADPH, the mutant enzyme had less than a 0.24% wild-type k(cat) value. K(m) values of H153Q, H209Q, and H257Q for DXP increased to 3.5-, 7.6-, and 19-fold the wild-type value, respectively. These results indicate that Glu(231) of E. coli DXP reductoisomerase plays an important role(s) in the conversion of DXP to 2-C-methyl-d-erythritol 4-phosphate, and that His(153), His(209), and His(257), in part, associate with DXP binding in the enzyme molecule.

Aldose-Ketose Isomerases↗
PubMed2000

DNA sensing on a DNA probe-modified electrode using ferrocenylnaphthalene diimide as the electrochemically active ligand.

Naphthalene diimide derivative 1 carrying ferrocenyl moieties at the termini of imide substituents binds intact calf thymus DNA 4 times more strongly than the denatured DNA, and its complex with the intact DNA dissociates 80 times more slowly than that with the denatured DNA. On the basis of these observations, ligand 1 was applied to a probe of electrochemical DNA sensing. A thiol-linked single-stranded DNA probe was immobilized through the S-Au bonding to 20-30 pmol/mm2 on a gold electrode. Following hybridization with the complementary DNA, the electrode was soaked in a solution containing 1 (intercalation step) and then washed with buffer for 5 s. The cyclic voltammogram and differential pulse voltammogram for this electrode gave an electrochemical signal due to the redox reaction of 1 that was bound to the double-stranded DNA on the electrode. Thus, dA20 and the yeast choline transport gene were quantitated at the subpicomole level. The sensitivity of DNA detection was improved to 10 zmol by reducing the amount of immobilized DNA probe and protecting the uncovered surface of the electrode with 2-mercaptoethanol.

Base Sequence↗
PubMed2000

Estrogen deficiency is a potential cause for osteopenia in adult male patients with Noonan's syndrome.

Osteopenia is frequently observed in patients with Turner's syndrome. By contrast, there is no report concerning bone metabolism in patients with Noonan's syndrome which comprises Turner's phenotypic characteristics without any sex chromosome abnormalities. In the present investigation, we determined bone mineral density (BMD) as well as serum and urine indices of bone turnover in two male patients with Noonan's syndrome. Both patients showed remarkably decreased BMD, measured at two sites on the lumbar spine (L2-L4) and the distal end of the radius using dual energy X-ray absorptiometry (DXA). Urinary pyridinoline (PYD) and deoxypyridinoline (DPD) concentrations were significantly elevated in both patients, and serum osteocalcin and carboxyterminal propeptide of type I procollagen (PICP) concentrations were elevated in one patient. Surprisingly, both patients had a low level of serum 17beta-estradiol compared with control males, whereas they had normal levels of serum testosterone and dihydrotestosterone. Conjugated estrogens (Premarin 0.625 mg/day) were continued to be administered to these patients, followed up for 12 months. Urinary PYD and DPD concentrations gradually decreased, followed by an increase in their BMD. This is the first report that male patients with Noonan's syndrome showed osteopenia associated with increased bone resorption. Our data indicate that hypoestrogenism plays a potentially significant role in the abnormal bone metabolism in these patients.

Adolescent↗
PubMed2000

Limited feeding of potassium nitrate for intracellular lipid and triglyceride accumulation of Nannochloris sp. UTEX LB1999.

Limited feeding of nitrate during culture of Nannochloris sp. UTEX LB1999 for intracellular lipid and triglyceride accumulation was investigated with the aim of obtaining cells superior for liquefaction into a fuel oil. The intracellular lipid contents and the percentage of triglycerides in the lipids of cells grown in a nitrogen-limited medium (0.9 mM KNO3) were 1.3 times as high as those grown in a modified NORO medium containing 2.0-9.9 mM KNO3. However, the cell concentration was too low for the practical production of fuel oil by high-pressure liquefaction of the cell mass. A single feeding of 0.9 mM nitrate after nitrate depletion during cultivation in a nitrate-limited medium increased the cell concentration to twice that obtained without such feeding, and the lipid content was maintained at a high level. The timing of nitrate feeding, i.e., whether it was given during the log phase (before nitrate depletion), the constant growth phase (just after the depletion), or the stationary phase (after the depletion), had negligible effect on the intracellular lipid content and percentage of triglycerides in the lipids. When 0.9 mM nitrate was intermittently fed ten times during the log phase in addition to the initial nitrate feed (0.9 mM), the cell concentration reached almost the same (2.16 g/l) and the intracellular lipid content and the percentage of triglycerides in the lipids increased from 31.0 to 50.9% and 26.0 to 47.6%, respectively, compared with those of cells cultured in a modified NORO medium containing 9.9 mM KNO3 without additional nitrate feeding.

Chlorophyta↗
PubMed2000

Multiple occurrence of osteochondromas in dysplasia epiphysealis hemimelica.

Dysplasia epiphysealis hemimelica was defined by Trevor (1950) as a rare congenital growth disorder of the tarsus and of the epiphysis of the long bone. In this report, a rare case of dysplasia epiphysealis hemimelica associated with multiple extraskeletal osteochondromas is presented. Although different modes of expression of the same pathologic process have been suggested for dysplasia epiphysealis hemimelica and osteochondroma, the biological feature of cartilaginous overgrowth in the skeletal system still seems unclear.

Child↗
PubMed2000

The role of beta-tricalcium phosphate in vascularized periosteum.

To investigate the osteogenic capacity of vascularized periosteum in bore grafting, we prepared experimental groups by wrapping beta-tricalcium phosphate (beta-TCP) with vascularized periosteum of the femur of 12-week-old Japanese white rabbits, and evaluated osteogenesis histologically and biochemically. Bone formation was observed in the group with vascularized periosteum and in the group with bone marrow fluid added to the vascularized periosteum. In particular, woven bone was observed in the group that had added bone marrow. Osteogenesis appeared earlier in the group with bone marrow fluid added to the vascularized periosteum, but histologically, there was no significant difference between this group and the group with vascularized periosteum without bone marrow fluid added at week 24 after the operation. In the group with non-vascularized periosteum, slight osteogenesis was found at week 6 after the operation, but in the control group, with beta-TCP implanted in soft tissue, osteogenesis did not occur at all. Alkaline phosphatase (ALP) activity reached a peak at week 2 after the operation in the group with vascularized periosteum, but only half the peak value was then maintained until week 8. In the group with bone marrow fluid added to the vascularized periosteum, similar values were found from immediately after the operation until week 8. ALP activity did not show any significant difference between these two groups at week 8 postoperatively. In the group with beta-TCP implanted in the soft tissue, ALP activity was low at all times measured. These results suggested that the periosteum had osteoinduction capacity and beta-TCP had osteoconduction capacity; that better osteogenesis occurred with vascularized periosteum; and that bone marrow fluid was involved in the promotion of osteoblastic activity, but not in calcification.

Alkaline Phosphatase↗
PubMed2000

Mizuo phenomenon observed by scanning laser ophthalmoscopy in a patient with Oguchi disease.

PURPOSE: To elucidate the origin of the abnormal fundus reflex in Oguchi disease. METHODS: The ocular fundus of a 63-year-old woman who showed a homozygous arrestin 1147delA mutation was observed by scanning laser ophthalmoscopy with the use of an argon blue laser (wavelength, 488 nm), a helium-neon laser (633 nm), and an infrared laser (780 nm). RESULTS: Diffuse, fine, white particles, which do not exist in normal subjects, were clearly demonstrated only with the helium-neon laser. After 4-hour dark adaptation, the abnormal particles disappeared, but then they reappeared gradually during 30 minutes of light adaptation, in accordance with the golden metallic reflex. CONCLUSION: The white particles found by helium-neon laser could be the origin of the abnormal fundus reflex in Oguchi disease.

Arrestin↗
PubMed2000

Impairment of insulin-induced vasodilation is associated with muscle insulin resistance in type 2 diabetes.

To clarify the association between the actions of insulin on the vascular wall and on the muscles in diabetes, we evaluated insulin-mediated vasodilation and muscle glucose uptake simultaneously using the euglycemic hyperinsulinemic glucose clamp technique and the calculation of total peripheral vascular resistance (TPR) from arterial pulse wave analysis in 19 Japanese patients with type 2 diabetes who had no signs of atherosclerosis. During the clamp study, the plasma norepinephrine (NE) level and plasma renin activity (PRA) increased without showing any significant correlation to the glucose infusion rate (GIR); a marker of muscle insulin sensitivity, and no changes of other plasma vasoactive hormone levels were observed. TPR decreased over time during the clamp study. The decrease of TPR from baseline was 0.88 +/- 0.02 at 1 h (mean +/- S.E.M., P < 0.01) and 0.79 +/- 0.03 at 2 h (P < 0.01), and the relative change in TPR from baseline was negatively correlated with GIR (r = -0.48 at 1 and 2 h; both P < 0.05). Our results suggest that there is also insulin resistance in the vascular wall, and this phenomenon may be associated with muscle insulin resistance in type 2 diabetes.

Adult↗
PubMed2000

Effect of polyamines on histone-induced DNA compaction of hyperthermophilic archaea.

The effect of polyamines on histone-mediated DNA compaction was examined in vitro with archaeal histone HpkA from Pyrococcus kodakaraensis KOD1. An agarose gel mobility-shift experiment indicated that histone-bound DNA (compacted DNA) was further compacted by addition of a polyamine (putrescine, spermidine, or spermine) or its acetylated form (N-acetylputrescine, N1-acetylspermidine, N8-acetylspermidine, or N1-acetylspermine) when the mixture was incubated at above 75 degrees C. Spermine was most effective in compaction enhancement among all the polyamines tested. A high concentration of potassium ion (1.0 M) did not stabilize the compacted form of DNA even though double-stranded DNA was stably maintained against thermal denaturation at elevated temperatures under this condition. It appears likely that multivalent polyamines have a nucleosome maintenance function in hyperthermophilic archaea in high-temperature environments.

Journal Article↗
PubMed2000

Characterization of FtsZ homolog from hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1.

The gene of bacterial type ftsZ homolog in hyperthermophilic archaeon, Pyrococcus kodakaraensis KOD1 (Pk-ftsZ), was identified. The gene product of the Pk-ftsZ gene is composed of 380 amino acids with a molecular mass of 41,354 Da. In the deduced amino acid sequence of the Pk-ftsZ gene, a glycine-rich sequence (Gly-Gly-Gly-Thr-Gly-Ala-Gly) implicated in GTP binding was well conserved. The Pk-ftsZ gene was overexpressed using Escherichia coli as a host and the recombinant protein was purified. The purified Pk-FtsZ protein exhibited GTPase activity with optimum temperatures higher than 80 degrees C. However, the protein showed little GTPase activity at 40 degrees C, indicating that a high reaction temperature is required for the GTPase activity in accordance with the thermophilic nature of P. kodakaraensis KOD1. The GTP-binding ability of Pk-FtsZ protein could also be detected by UV-induced cross-linking of a protein to [alpha-32P] GTP. The Pk-ftsZ gene was expressed in E. coli cells with a temperature-sensitive ftsZ mutation, E. coli ftsZ84 (ts), but its mutant phenotype of elongated cell form at a nonpermissive temperature (42 degrees C) could not be compensated, possibly because of the thermophilic nature of the Pk-FtsZ. Pk-FtsZ could form protofilaments in a GTP-dependent manner at 90 degrees C. Results of phylogenetic analysis suggest that there might be additional factors required for formation of the Z ring in P. kodakaraensis KOD1.

Journal Article↗
PubMed2000

Alteration of product specificity of cyclodextrin glucanotransferase from Thermococcus sp. B1001 by site-directed mutagenesis.

Cyclodextrin glucanotransferase (CGTase) from the hyperthermophilic archaeon Thermococcus sp. B1001 catalyzed the production predominantly of alpha-cyclodextrin (CD) from starch (Tachibana, Y. et al., Appl. Environ. Microbiol., 65, 1991-1997, 1999). The CGTase gene (cgtA) from this strain was cloned and sequenced. It was composed of 2217 nucleotides, and encoded a protein (739 amino acids) with a molecular mass of 83,240 Da. Recombinant CgtA expressed in Escherichia coli also catalyzed the production predominantly of alpha-CD from starch, as did native CgtA from strain B1001. Based on a substrate binding model of Bacillus circulans no. 8 CGTase, Tyr100, Trp191 and Tyr267 were specified to locate the spiral amylose and to minimize the size of the CD by saccharide aromatics interaction. In order to determine the critical residue for catalyzing production predominantly of alpha-CD, site-directed mutations were introduced in CgtA (Y100W, Tyr100-->Trp; W191Y, Trp191-->Tyr; W191F, Trp191-->Phe; Y267W, Tyr267-->Trp; Y267F, Tyr267-->Phe). Analysis of the reaction products by HPLC revealed that the mutant enzyme Y267W produced more beta- and gamma-CD than the wild-type enzyme. However, the other mutants still produced high levels of alpha-CD, suggesting that Tyr267 plays a critical role in alpha-CD production catalyzed by B1001 CGTase.

Journal Article↗
PubMed2000

Selective retension of active cells employing low centrifugal force at the medium change during suspension culture of Chinese hamster ovary cells producing tPA.

The effect of centrifugal force applied for cell separation at the medium change on the growth, metabolism and tissue plasminogen activator (tPA) productivity of Chinese hamster ovary (CHO) cells suspension culture was investigated. The viability of the precipitated cells increased exponentially as the centrifugal force decreased. However, the cell recovery was lower than 91% when centrifugal forces applied for 5 min was less than 67 x g. In cultures incubated for 474 h with 7 medium changes employing centrifugal forces ranging from 67 to 364 x g, a centrifugal force lower than 119 x g resulted in higher specific rates of growth, glucose consumption, and lactate and tPA production during the whole culture period. On the other hand, daily centrifugation at 67 to 537 x g without discarding the supernatant had no effect on the specific rates. The cultures inoculated with cells precipitated at a centrifugal force of 67 x g showed apparently higher specific rates of metabolism compared to those inoculated with cells in the supernatant. The cells in the supernatant and the precipitate obtained following centrifugation at 67 x g have average diameters of 15.5 and 17.4 microm, respectively. The intracellular contents of amino acids, especially nonessential amino acids, of the precipitated cells were markedly higher than those of the cells in the supernatant. These results indicate that large cells with high amino acid content and metabolic activity were selectively retained in the culture by means of centrifugation at low forces such as 67 x g. Consequently, application of a low centrifugal force is recommended for medium change in order to maintain higher specific productivity of suspended mammalian cells in perfusion culture.

Journal Article↗
PubMed2000

Acceptor specificity of 4-alpha-glucanotransferase from Pyrococcus kodakaraensis KOD1, and synthesis of cycloamylose.

4-Alpha-glucanotransferase from a hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 showed a broad acceptor specificity to various saccharides in an intermolecular transglycosylation reaction. In particular, the enzyme produced large amounts of transfer products of various acceptors such as D-glucose, methyl-alpha-D-glucoside, phenyl-alpha-D-glucoside, and D-xylose. It is suggested that the requirement for an effective acceptor in the intermolecular transglycosylation reaction catalyzed by this enzyme is the pyranose structure with the same configurations of the free C2-, C3-, and C4-hydroxyl groups as d-glucopyranose, like cyclomaltodextrin glucanotransferase (CGTase). However, the enzyme showed some acceptor specificities unlike those of CGTase. Analysis of the action of 4-alpha-glucanotransferase indicated that the enzyme catalyzes an intramolecular trans-glycosylation (cyclization) reaction of amylose to produce cyclic alpha-1,4-glucan (cycloamylose). The yield of cycloamylose reached 67%, and the degree of polymerization was found to range from 16 to above 55.

Journal Article↗
PubMed2000

Gene expression and immunohistochemical localization of biglycan in association with mineralization in the matrix of epiphyseal cartilage.

This study has used in situ hybridization, Northern blot analysis, and immunohistochemistry at the light and electron microscope levels to localize mRNAs and core proteins of biglycan in developing tibial epiphyseal cartilage of 10-day old Wistar rats. The expression of mRNAs and core proteins of biglycan appeared prominent in hypertrophic and degenerative chondrocytes associated with the epiphyseal ossification centre and the growth plate cartilage, but was not seen in the rest of epiphyseal cartilage. Northern blot analysis confirmed biglycan mRNA expression in the epiphyseal cartilage. Ultrastructural immunogold cytochemistry of the growth plate revealed that prominent immunolabelling was confined to the Golgi apparatus and cisternae of rough-surfaced endoplasmic reticulum of the hypertrophic and the degenerating chondrocytes, the early mineralized cartilage matrices of the longitudinal septum of the lower hypertrophic and the calcifying zones, and fully mineralized cartilage mitrices, which were present in the metaphyseal bone trabeculae. Furthermore, Western blot analysis of biglycan in extracts of fresh epiphyseal cartilage revealed that an EDTA extract, after chondroitinase ABC digestion, contains core proteins of biglycan, indicating the presence of biglycan in mineralized cartilage matrices. These results indicate that the distribution of biglycan is associated with cartilage matrix mineralization.

Animals↗