Biomedical subjects

T Imanari

Publications and source records attributed to T Imanari.

At least 37 records · Page 2Linked to original sources

PubMed1999

Structural characteristics of oversulfated chondroitin/dermatan sulfates in the fibrous lesions of the liver with cirrhosis.

The structural characteristics of oversulfated chondroitin/dermatan sulfates (C/DSs) in the fibrous lesions of the rat liver with cirrhosis were examined. Long-Evans Cinnamon rats were subjected to the present study as the model animals with cirrhosis. The serial polyester wax sections of liver with cirrhosis were processed into the fibrous lesions and the nonfibrous lesions. The oversulfated C/DSs in the tissue sections on a glass slide were degraded to unsaturated disaccharides by chondroitinase ABC and ACII digestion in the presence of bacterial collagenase. Subsequently, the resulting unsaturated disaccharides were determined by the reversed-phase ion-pair high-performance liquid chromatography with fluorometric postcolumn derivatization using 2-cyanoacetamide as a reagent. Through these in situ investigations, we found some facts as follows: (i) in the fibrous lesion, the remarkable increase of the oversulfated C/DSs content and the decrease of the oversulfation degree of the C/DSs were observed compared with those in the nonfibrous lesion, (ii) the proportion of the iduronic acid content in the C/DSs in the fibrous lesion was significantly low compared with that in the nonfibrous lesion, and (iii) in the nonfibrous lesion close to the fibrous lesion, both quantitative and qualitative alterations of C/DSs were not observed at all. These findings indicate that the oversulfated C/DSs with low iduronic acid content are possible marker for the fibrogenesis of liver with cirrhosis.

Animals↗

PubMed1999

A new sulfated beta-galactan from clams with anti-HIV activity.

A new polysaccharide composed of galactan sulfate with a beta-(1-->3)-glycosidic linkage has been isolated from the marine clam species Meretrix petechialis. The polysaccharide was homogeneous in its composition containing D-galactose. The glycosidic linkage was examined by 2D DQF-COSY and 2D NOESY spectroscopy. The coupling constant of anomeric proton was 7.8 Hz, suggesting a beta-galacto configuration. The downfield shift of H-2 of galactose residue demonstrated the presence of 2-O-sulfonate group. TQF-COSY confirmed that the C-6 position was substituted with a sulfonate group. The anti-HIV activity of the polysaccharides has been evaluated by the inhibition of syncytia formation. The fusion index and percentage fusion inhibition of sulfated galactan were 0.34 and 56% at 200 micrograms/mL.

Animals↗

PubMed1999

Quantitative alterations of hyaluronan and dermatan sulfate in the hairless mouse dorsal skin exposed to chronic UV irradiation.

The quantitative alterations of hyaluronan and dermatan sulfate in the upper dermis (fibrous tissue) and the lower dermis (adipose tissue) of the hairless mouse skin chronically exposed to the UV irradiation as solar-simulating irradiation (lambda(max) 352 nm, UV distribution: 300-310 nm, 0.9%; 310-320 nm, 2.0%; 320-420 nm, 97.1%) were evaluated. Hyaluronan and dermatan sulfate contents in each part of dermis were determined as follows: skin sections on a glass slide prepared by histological technique were processed into the upper dermis and the lower dermis with a small surgical knife, and treated with chondroitinase ABC and ACII in the presence of bacterial collagenase. The resulting unsaturated disaccharides were determined by HPLC method. By applying this method to the UV-irradiated hairless mouse skin, it was found that the chronic UV irradiation increased dermatan sulfate in the upper dermis, whereas an increase of hyaluronan content was not statistically significant. In the lower dermis, on the contrary, both hyaluronan and dermatan sulfate contents remarkably increased as compared with the control mice. Furthermore, the histological study showed the accumulation of the collagen fibers in the lower dermis of the UV-irradiated hairless mouse skin following the disappearance of adipocytes. These findings indicate that the increases of glycosaminoglycan contents in the UV-irradiated skin are related to the accumulation of the extracellular matrix components in the lower dermis.

Animals↗

PubMed1999

Quantification of hyaluronan and chondroitin/dermatan sulfates in the tissue sections on glass slides.

The method for the determination of hyaluronan and chondroitin/dermatan sulfates in the tissue sections on a glass slide, which were prepared by histological technique, was established by applying to porcine skin. The degradation of these glycosaminoglycans to the unsaturated disaccharides in porcine skin sections on a glass slide was achieved by chondroitinase ABC and ACII in the presence of highly purified bacterial collagenase. Subsequently, the resulting unsaturated disaccharides were determined by HPLC with fluorometric postcolumn derivatization using 2-cyanoacetamide as a reagent. So far, the determination of the glycosaminoglycans in the tissues has taken up more than 5 days, whereas the determination of the glycosaminoglycans in the frozen sections by the present method was completed within a day. In addition, applications of the present method to the serial polyester wax sections processed with a small surgical knife made it possible to determine the glycosaminoglycans in a local part in the tissue section. The present method should open a way for the clinical analysis of glycosaminoglycans in the pathological tissue samples.

Animals↗

PubMed1999

Oral absorption and clearance of partially depolymerized fucosyl chondroitin sulfate from sea cucumber.

Partially depolymerized holothurian glycosaminoglycan (DHG), a fucosyl chondroitin sulfate chains isolated from sea cucumber, was administered by the intravenous and oral routes to experimental animal. After intravenous injection, clearance of DHG, as measured by the postcolumn HPLC, displayed complex kinetics that were not dose dependent. DHG was excreted unchanged in the urine. No degradation products of DHG were detected by either gel filtration or anion exchange HPLC at any time in plasma, indicating that administered DHG is not catabolyzed by mammalian. Anion exchange chromatographic behavior of DHG excreted into urine after oral administration showed that partial desulfation might occur through intestinal absorption. After oral administration of DHG (50 mg/kg), 0.1% of the dose was found in urine collected for 24 hours. More than 5% of intravenously administered DHG (1 mg/kg) was excreted into urine in 24 hours. These results are suggesting that orally administered macromolecules such as DHG are absorbed in the gastrointestinal tract.

Administration, Oral↗

PubMed1999

Alterations of hairless mouse skin exposed to chronic UV irradiation and its prevention by hydrocortisone.

Ultraviolet-induced alterations of skin were investigated in a murine animal model. Groups of hairless mice were exposed to UV (black light, lambda max 352 nm; UV distribution: 300-310 nm, 0.9%; 310-320 nm, 2.0%; 320-420 nm, 97.1%) for 20 weeks at a dose of 16.3 J/cm2 five times weekly on weekdays. At the end of 20 weeks irradiation, the dorsal skins were biochemically and histologically examined. Ultraviolet caused remarkable increases in amounts of hyaluronan, chondroitin sulfates and dermatan sulfates in skin (microgram/cm2). Interestingly, a significant change in a collagen content (hydroxyproline, microgram/g of dry powder) caused by UV irradiation was not observed, whereas the amount of collagen (hydroxyproline, microgram/cm2) increased remarkably. Histologically, no distinguishable thickening was observed in both upper dermis and lower dermis, but thickening of the epidermis was observed. Furthermore, the histological study indicated that UV irradiation caused a disappearance of crowds of adipocytes, alternative appearance of numerous fibroblasts and accumulation of collagen bundles and hyaluronan in lower dermis. Hydrocortisone, an anti-inflammatory agent, prevented both the fibrosis of lower dermis and the accumulation of the extracellular matrix components. Based on these results, it seems reasonable that UV penetrates into the lower dermis and causes fibrosis there, resulting from the inflammatory responses.

Animals↗

PubMed1998

Enzymatic method for the simultaneous determination of hyaluronan and chondroitin sulfates using high-performance liquid chromatography.

A chromatographic method for the simultaneous determination of hyaluronan and chondroitin sulfates was examined. Hyaluronan differs from chondroitin sulfates in the susceptibility to chondroitinase ABC under alkaline conditions. When hyaluronan and chondroitin sulfates were treated with chondroitinase ABC in the buffered solution (pH 9.1), chondroitin sulfates were selectively degraded to the unsaturated disaccharides, whereas hyaluronan was not. Subsequently, hyaluronan in the reaction mixture was digested to the unsaturated tetrasaccharide and hexasaccharide at pH 6.0 by Streptomyces hyaluronidase in the presence of zinc ion (inhibitor for chondroitinase ABC). The separation of the resulting unsaturated disaccharides from chondroitin sulfates and the unsaturated oligosaccharides from hyaluronan was achieved by a reversed-phase ion-pair HPLC. The structural polydispersities of hyaluronan and chondroitin sulfates in the molecular weight, the sulfation position, or the components of uronic acid did not affect their determination. The usefulness of the present method was proved by application to the porcine skin samples.

Carbohydrate Sequence↗

PubMed1998

Formation of a complex containing ATP, Mg2+, and spermine. Structural evidence and biological significance.

The conformation of ATP in the presence of Mg2+ and/or spermine was studied by 31P and 1H NMR, to clarify how polyamines interact with ATP. Spermine predominantly interacted with the beta- and gamma-phosphates of ATP in the presence of Mg2+. A conformational change of the beta- and gamma-phosphate of ATP with spermine could not be observed in the absence of Mg2+ by 31P NMR. It was found by 1H NMR that the conformation of adenosine moiety of ATP was not influenced significantly by spermine. The binding of Mg2+ to ATP was slightly inhibited by spermine and vice versa. The results indicate that the binding sites of Mg2+ and spermine on ATP only partially overlap. The PotA protein, an ATP-dependent enzyme, was used as a model system to study the biological role of the ATP-Mg2+-spermine complex. The ATPase activity of PotA was greatly enhanced by spermine. Double reciprocal plots at several concentrations of spermine as an activator indicate that spermine interacts with ATP, but not with PotA. The activity of protein kinase A was also stimulated about 2-fold by spermine. The results suggest that a ternary complex of ATP-Mg2+-spermine may play an important role in some ATP-dependent reactions in vivo and in the physiological effects of endogenous polyamines.

ATP-Binding Cassette Transporters↗

PubMed1998

Degradation of dehydroascorbate to 2,3-diketogulonate in blood circulation.

In our previous paper (Biochim. Biophys. Acta 1379 (1998) 257-263), we demonstrated that bicarbonate promotes a cleavage of lactone ring of dehydroascorbate (DHA) on the basis of in vitro experiments. In the present study, we examined the degradation of DHA in blood circulation in vivo by using a high-performance liquid chromatographic method for the determination of ascorbate (AsA), DHA and 2,3-diketogulonate (2,3-DKG), which required no pretreatment of biological fluids. When DHA was intravenously administered to rats, a rapid disappearance of DHA (t1/2 < 1 min) and a concomitant appearance of 2,3-DKG in blood circulation were observed. Approximately 90% of the administered DHA were excreted into urine as resulting 2,3-DKG (55%) and AsA (31%), respectively. Furthermore, we elucidated that rat plasma lacks an enzyme having an aldonolactonase-like activity. The result of the present study suggests that this DHA disappearance is a function of both a chemical degradation to 2,3-DKG and a reduction to AsA.

2,3-Diketogulonic Acid↗

PubMed1998

Downregulation of the Klotho gene in the kidney under sustained circulatory stress in rats.

We recently reported the isolation of the klotho gene, which in predominantly expressed in the kidney and involved in human aging phenotypes. In our previous studies, we demonstrated that the Klotho protein or its metabolites may possibly function as humoral factor(s) and protect against endothelial dysfunction because acetylcholine-mediated NO production in arteries was impaired in heterozygous klotho deficient mice (kl/+). However, the pathophysiological significance of the Klotho protein has not been clarified yet. In the present study, we examined expression of the klotho gene in the kidney of the following rat models for human diseases: (1) spontaneously hypertensive rat, (2) deoxycorticosterone acetate-salt hypertensive rat, (3) 5/6 nephrectomized rat, (4) non-insulin-dependent diabetes mellitus rat (the Otsuka Long-Evans Tokushima Fatty rat), and (5) rat with acute myocardial infarction. The expression levels of klotho mRNA in the kidney in these models were significantly lower than controls except for MI rats. This is the first report showing the expression of the klotho gene in the kidney is regulated under sustained circulatory stress such as long-term hypertension, diabetes mellitus, and chronic renal failure.

Aging↗

PubMed1998

Quantification of carbamylated dehydroascorbate derivative produced from cyanate and dehydroascorbate.

We established a high-performance liquid chromatographic method for separating and quantifying carbamylated dehydroascorbate derivative (CDA), a reaction product of cyanate with dehydroascorbate. The separation of CDA from interfering substances was achieved by anion-exchange HPLC using a TSK gel SAX (250x4.6 mm I.D.) column and 0.12 M NaCl eluent. The detection of CDA was achieved through two steps: (1) degradation of CDA to cyanate and amino compounds in alkaline solution, and (2) detection of these products by an indophenol reaction. For the processing of plasma and urine samples, anion-exchange solid-phase extraction was used. The detection limit for quantitative determination was 0.1 microM CDA (S/N=3). The linear range found applying the optimized conditions was 0.2 to 200 microM. The intra- and inter-day assay precision (R.S.D.) of CDA (10 microM) were 4.8 and 7.2% for rat plasma, and 4.0 and 4.9% for rat urine, respectively. The usefulness of the present method was proved by the application to plasma and urine samples. The study of the biokinetics of CDA in rats revealed that the elimination of CDA is due to urinary excretion.

Animals↗

PubMed1998

Bicarbonate promotes a cleavage of lactone ring of dehydroascorbate.

The half-life of dehydroascorbate (DHA) in human plasma is only a few minutes. This DHA disappearance is caused by a cleavage of lactone ring. Similarly, when DHA was incubated in Dulbecco's modified Eagle's medium (D-MEM), which stood in atmosphere of 5% CO2-95% air, the rapid transformation of DHA into 2,3-diketogulonate (2,3-DKG) is also observed. These observations suggest that both human plasma and D-MEM contain a common component, which promotes the hydrolysis of DHA. In the present study, this component was identified to be bicarbonate which acts as a general base catalyst. Direct evidence for this mechanism was obtained as follows: (1) significant hydrolysis of DHA in the bicarbonate-free D-MEM (pH 7.40) was not observed; (2) hydrolysis of DHA in Tris-HCl buffer at constant pH (7.4) increases with increasing bicarbonate concentration; and (3) significant hydrolysis of DHA in the decarbonated ultrafiltrate of plasma was not observed. These results suggest that DHA hydrolysis may be controlled by the variation of CO2 pressure in circulating blood.

Bicarbonates↗

PubMed1998

Conformational changes and anticoagulant activity of chondroitin sulfate following its O-sulfonation.

Chondroitin sulfate from bovine tracheal cartilage, with the basic structure (4-O-sulfo-D-GalpNAc beta-1-->4-D-GlcpA)n, was chemically modified by O-sulfonation. Depending on the reaction conditions, the products showed a different degree of O-sulfonation. A fully O-sulfonated chondroitin sulfate, having no free hydroxyl groups, and a sulfo ester group:disaccharide unit ratio of 4.0 was prepared. This chondroitin sulfate derivative was shown by 1H NMR spectroscopy to have a uronate residue with an altered conformation. Usually, the uronate residue in chondroitin sulfate resides in the 4C1 form. Fully O-sulfonated chondroitin sulfate had an uronate residue in the 1C4 form at 30 degrees C, similar to the preferred conformation of the 2-O-sulfo-iduronate residue most commonly found in heparin. The 2S0 form of the uronate residue was also found in fully O-sulfonated chondroitin sulfate at 60 degrees C. The anti-factor IIa activity of fully O-sulfonated chondroitin sulfate was 40 units/mg. This value is similar to the activities reported for various low-molecular-weight heparins, and substantially higher than those previously reported for partially O-sulfonated chondroitin sulfates having an average sulfate group/disaccharide unit of 2.5 to 3.3. The anti-factor Xa activity of the fully O-sulfonated chondroitin sulfate was 12 units/mg. This value is considerably lower than the activities reported for various low-molecular-weight heparins, consistent with the critical importance of an antithrombin III pentasaccharide binding site for anti-factor Xa activity. These findings suggest that the conformational change of glucuronic acid residue in chondroitin sulfate resulting from its full O-sulfonation can result in enhanced anticoagulant activity, particularly as measured by anti-factor IIa assay.

Animals↗

PubMed1998

Characterization and determination of human urinary keratan sulfate.

Keratan sulfate was isolated from normal human urine and was characterized by sugar compositional analysis and H-NMR spectroscopy. It was found that KS from human urine is classified as skeletal type (KS-II type chain) with an O-glycosidic linkage between galactosamine and serine (or threonine). H-NMR studies revealed that urinary KS is not a proteoglycan but a polysaccharide (molecular weight is about 5 kDa). The quantitation of human urinary KS by HPLC showed that urinary KS is excreted at constant levels (0.07 +/- 0.015 microgram/mg creatinine).

Carbohydrates↗

PubMed1998

Evaluation of an acidic deproteinization for the measurement of ascorbate and dehydroascorbate in plasma samples.

The most popular pretreatment method of plasma samples for the measurement of ascorbate (AsA) and dehydroascorbate (DHA) has been an acidic deproteinization via metaphosphoric acid or trichloroacetic acid. In general, DHA is absent in plasma samples prepared from human blood in a conventional manner. However, when these plasma samples were subjected to acidic deproteinization, DHA was detected in the acidified sample solutions. In the present study, we demonstrate that the oxidation of AsA to DHA in the solutions was promoted by at least two mechanisms, one involving catalysis by ferric ion released from transferrin, and the other involving catalysis by plasma hemoglobin. In the acidified transferrin solution by trichloroacetic acid, an oxidation of AsA to DHA proceeded with standing time, whereas the oxidation was not observed in that by metaphosphoric acid. This oxidation appeared to be catalyzed by ferric ion released from transferrin. In contrast, plasma hemoglobin functioned as a catalyst for AsA oxidation in both metaphosphoric acid and trichloroacetic acid solutions. Therefore, DHA content in the trichloroacetic acid-treated plasma sample was markedly higher than that in the metaphosphoric acid-treated one. These results suggest that DHA detected in acidified plasma samples is an artifact resulting from AsA oxidation.

Adult↗

PubMed1997

Sensitive high-performance liquid chromatographic method with fluorometric detection for the determination of heparin and heparan sulfate in biological samples: application to human urinary heparan sulfate.

A sensitive high-performance liquid chromatographic method for the determination of unsaturated disaccharides produced from heparin and heparan sulfate is described. Heparan sulfate was depolymerized using a combination of heparin lyase I (EC 4.2.2.7), heparin lyase II and heparin lyase III (EC 4.2.2.8). Seven unsaturated disaccharides were separated under isocratic conditions within 25 min using acetonitrile-H2O-0.2 M sodium phosphate buffer (pH 7.0)-3.0 M ammonium chloride (32:10:1:1) and were monitored by fluorescence detection using 2-cyanoacetamide as a post-column derivatizing reagent. As little as 2 pmol of a disaccharide could be detected with excitation at 346 nm and emission at 410 nm. This method was applied to the analysis of normal human urine. It was revealed that the concentration of normal human urinary heparan sulfate is 1.53+/-0.36 mg/mg creatinine (n=4).

Adult↗

PubMed1997

Detection of glycosaminoglycans as a copper(II) complex in high-performance liquid chromatography.

Glycosaminoglycans including heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate and hyaluronan were analyzed by high-performance gel filtration chromatography. Detection was achieved at 240 nm based on the formation of copper(II) complex in copper sulfate solution at low pH. Detection of the copper(II)-heparin complex is sensitive, permitting the analysis of as little as 10(-7) g. This method was also successfully applied to the analysis of the chemically derivatized glycosaminoglycans (de-N-acetylated and/or oversulfated chondroitin sulfate) that cannot be depolymerized by enzymes such as heparin and chondroitin lyases.

Chelating Agents↗

PubMed1997

Cyanate causes depletion of ascorbate in organisms.

Ascorbate-dehydroascorbate redox cycle plays a key role in protecting organisms from an excess of oxidants. Recently, we found a novel reaction of dehydroascorbate with cyanate under the conditions of neutral pH and ordinary temperature. In this report, we demonstrated that through this irreversible reaction, cyanate causes the depletion of ascorbate in the matrix, where the ascorbate-dehydroascorbate redox cycle revolves. When the leaves of weed (Erigeron canadensis) were soaked in sodium cyanate solution generally used as a herbicide, the depletion of ascorbate as well as dehydroascorbate in them was observed, followed by the change in color from green to brown. These results suggest that a possible way of cyanate toxicity is to inflict oxidative stress on organisms.

Animals↗