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.

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.

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.

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.

Probing the interaction of dengue virus envelope protein with heparin: assessment of glycosaminoglycan-derived inhibitors.

A structure-activity relationship study was carried out to facilitate development of inhibitors of dengue virus infectivity. Previous studies demonstrated that a highly charged heparan sulfate, a heparin-like glycosaminoglycan found on the cell surface, serves as a receptor for dengue virus by binding to its envelope protein. Interventions that disrupt this binding effectively inhibit infectivity. A competitive binding assay was developed to screen polyanionic compounds for activity in preventing binding of dengue virus envelope protein to immobilized heparin; compounds tested included drugs, excipients, and larger glycosaminoglycans and their semisynthetic derivatives. Results of this competitive binding assay were used to select agents for detailed evaluation of interactions by surface plasmon resonance spectroscopy, which afforded binding on-rates, off-rates, and dissociation constants. From these data, an understanding of the structural requirements for polyanion binding to dengue virus envelope protein has been established.

Prevention of the photodamage in the hairless mouse dorsal skin by kojic acid as an iron chelator.

Kojic acid, a fungal metabolic product, has been used as a skin-depigmenting agent in skin care products marketed in Japan. Iron in the skin is known to be involved in wrinkling as a result of chronic photodamage. Kojic acid was expected to have anti-wrinkling activity, since it possesses iron-chelating activity. We now evaluated the anti-wrinkling activity of kojic acid by using hairless mice exposed to chronic solar-simulating ultraviolet (UV) irradiation as model animal. At the end of a 20-week irradiation period, topical application of kojic acid before UV irradiation was observed to dramatically prevent: (1) the wrinkling, (2) hyperplasia of the epidermis, (3) fibrosis of the lower dermis, and (4) the increase of extracellular matrix components in the upper dermis. These findings indicate that kojic acid is a typical agent preventing wrinkling of the skin due to chronic photodamage.

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.

Sensitive radiochemical esterolytic assays for urokinase.

Two radiochemical esterolytic assays for urokinase are described. One assay is based on the urokinase-dependent hydrolysis of Nalpha-acetyl-glycyl-L-lysine [3H]methyl ester and the other on the urokinase-dependent activation of plasminogen and assay of generated plasmin with Nalpha-tosyl-L-arginine [3H]methyl ester. The assays are performed in tubes placed in liquid scintillation counting vials. At the end of the experiment generated [3H]methanol is extracted into the liquid scintillation cocktail and counted. Unhydrolyzed substrate largely remains in the aqueous phase and contributes only a small fraction of the counts. This facile separation of 3H-labeled alcohol from the ester substrate allows the simple and highly sensitive assay for urokinase. The assays give results in good agreement with the classical fibrin plate assay.

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.

Quantitative changes in glycosaminoglycans in the lungs of rats exposed to diesel exhaust.

Exposure to diesel exhaust (DE) induces lesions in lung epithelium by generation of reactive oxygen species. Glycosaminoglycans (GAG), components of extracellar matrix, are thought to play important roles in cell proliferation and differentiation in the repair process of injured tissue. We investigated how GAG are related to the recovery of lung tissue from injury. Using high-performance liquid chromatography analysis, we determined the amounts of GAG, such as chondroitin sulfate (CS), dermatan sulfate (DS), and hyaluronan (HA) in the lungs of rats exposed to DE for 4 weeks at concentrations of 0.3 or 3 mg/m(3) as suspended particulate matter, or to filtered air. The contents of CS and HA in the surroundings of the bronchi were significantly increased after exposure to DE. In addition, immunohistochemical staining showed that the number of 8-hydroxydeoxyguanosine-positive cells as a marker of cell damage, and proliferating cell nuclear antigen-positive cells also increased in the same areas in which the levels of GAG were elevated in the lungs of rats exposed to 3 mg/m(3) DE. These results suggest that CS and HA in the lung contribute to cell proliferation and remodeling in the process of recovery from injury caused by exposure to DE.

High-performance liquid chromatographic analysis of glycosaminoglycan-derived oligosaccharides.

High-performance liquid chromatography of glycosaminoglycan (GAG)-derived oligosaccharides has been employed for the structural analysis and measurement of hyaluronan, chondroitin sulphate, dermatan sulphate, keratan sulphate, heparan sulphate and heparin. Recent developments in the separation and detection of unsaturated disaccharides and oligosaccharides derived from GAGs by enzymatic or chemical degradation are reviewed.

Separation of chondroitin sulfate and hyaluronic acid fragments by centrifugal precipitation chromatography.

Centrifugal precipitation chromatography (CPC) was applied for the first time to the separation of fragments of chondroitin sulfate (ChS) and hyaluronic acid (HA). The separation was performed using a gradient elution system between ethanol and water since solubility of these biopolymers highly depends on the concentration of ethanol in aqueous solution. ChS and HA were each eluted into several peaks through a flow-through UV detector at 275 nm, despite they have almost no absorbance at this wavelength in an aqueous solution. The separation was also confirmed by redissolving the dried fraction in water and measuring the absorbance at 210 nm. These results suggest that the CPC system can detect small precipitates of these biopolymers by light scattering at 275 nm. The separated fragments of biopolymers are not easily characterized because no suitable analytical method is available for identification of these compounds. However, the overall results demonstrate that CPC may be a useful separation of biopolymers such as glycosaminoglycans which quantitatively produce precipitates in an organic solvent mixture.

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.

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.

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.

Localization and characterization of acharan sulfate in the body of the giant African snail Achatina fulica.

Acharan sulfate is a glycosaminoglycan (GAG), having the structure -->4)-2-acetamido-2-deoxy-alpha-D-glucopyranose(1-->4)-2-sulfo-alpha-L-idopyranosyluronic acid (1-->, isolated from the body of the giant African snail Achatina fulica. This GAG represents 3-5% of the dry weight of this snail's soft body tissues. Frozen sections and polyester wax sections of the snail's body were stained by Alcian blue-periodic acid-Schiff's reagent (PAS) to localize acharan sulfate. Alcian blue staining indicated that GAG was mainly secreted into the outer surface of the body from internal granules. A highly mucous material was collected and treated and the acharan sulfate was recovered by ethanol and cetyl pyridinium chloride precipitation. Crude acharan sulfate was purified by DEAE-Sephacel ion-exchange chromatography. Depolymerization of intact mucus and purified acharan sulfate fractions by heparin lyase II (heparitinase I) from Flavobacterium heparinum produced an unsaturated disaccharide as a major product, establishing the repeating unit of acharan sulfate. These results demonstrate that mucus in the granule and secreted to the outside of the body is composed entirely of acharan sulfate.

Preparation and anticoagulant activity of fully O-sulphonated glycosaminoglycans.

Glycosaminoglycans including dermatan sulphate, hyaluronan, heparan sulphate and heparin were chemically modified by O-sulphonation. By altering the reaction conditions, products having a different degree of O-sulphonation could be obtained. Glycosaminoglycan derivatives were prepared having no free hydroxyl groups, with sulphoester group/disaccharide unit ratios of 4.0 for dermatan sulphate and hyaluronan, and sulphoester and sulphamide group/disaccharide unit ratios of 4.22 and 4.88 for heparan sulphate and heparin, respectively. 1H NMR spectroscopy showed that the fully O-sulphonated hyaluronan derivative had a glucuronate residue with an altered conformation. Since glycosaminiglycans and their derivatives are often used as anticoagulant/antithrombotic agents, their anti-amidolytic activities were determined. The anti-factor IIa activity of fully O-sulphonated dermatan sulphate, hyaluronan and heparan sulphate ranged from 40 to 80 units/mg, while no anti-factor Xa activity of the fully O-sulphonated glycosaminoglycans was detected. These values are lower than those reported for low-molecular-weight heparins and are consistent with the requirement of an antithrombin III pentasaccharide binding site for anti-factor Xa activity. Interestingly, the anti-factor Xa of heparin is lost by chemical O-sulphonation.

[Metabolic study on chondroitin sulfates in rabbits].

The metabolic studies on three types of chondroitin sulfates in rabbits were performed. The analyses of chondroitin sulfates gave the following results concerning their metabolic behavior. 1) Desulfation of chondroitin sulfates on GalNAc C6 position was observed more clearly after the administration of low molecular weight chondroitin sulfate (molecular weight, 6000 and 16,000) than after that of high molecular weight one (molecular weight, 50,000). 2) The disappearance velocities of the administered low molecular weight chondroitin sulfates in the blood and those of excretion into the urine were faster than those of high molecular weight one. 3) Intact chondroitin sulfates were excreted into the urine after the administration of low molecular weight chondroitin sulfates, while chondroitin sulfates having more than 30000 molecular weight were not detected in the collected urine after the administration of high molecular weight one.

[Activation of skin cells by inorganic compounds].

It has been known that the stimulation of skin cells by physiologically active substances is accompanied by the quantitative and qualitative alterations of the glycosaminoglycans (GAGs) in skin. This phenomenon make it possible to evaluate the activity of test substances for the stimulation of skin cells. The purpose of our study is to elucidate the effectiveness of inorganic compounds for the stimulation of skin cells. (1) The cultured skin cells including keratinocytes and fibroblasts, (2) the cultured model skin constituted of collagen gel, fibroblasts and keratinocytes, and (3) the mouse damaged skin were used in the present study. The results obtained from these studies demonstrate that inorganic substances commonly have a possibility to activate cyclic AMP-dependent protein kinase (A-kinase), and the A-kinase activation results in an increase in GAGs. These findings brought the scientific basis for the effectiveness of the Japanese traditional balneotherapy for damaged skin.

[Continuous intraarterial infusion of 5-fluorouracil plus leucovorin for liver metastases from colorectal cancer].

Between September 1990 and August 1994, 11 patients (pts) with liver metastases (mets) from colorectal cancer were treated with continuous hepatic arterial infusion chemotherapy of 5-fluorouracil (FU) plus leucovorin (LV). Eight pts had non-resectable liver mets (H3: 7, H2: 1), and 3 had residual small mets after resection of major mets. Drugs were administered via an extracorporeal infusion device connected to the hepatic arterial infusion port. 5-FU and LV were given through a 5- to 7-day continuous infusion at 500-750 mg/body/day and 30 mg/body/day, respectively, with a 3- to 4-week rest period. In the recent 6 pts, cisplatin was administered as a 2-hour infusion at 25 mg/body, one or two times simultaneously. Grade 2 toxicity was noted in two pts (18%). One was stomatitis and another was uncontrolled ascites in an advanced cirrhotic pt. The response rate in the 9 evaluable pts was 67% with 6 PR and no CR. The duration of the response was 5 to 9 months. One- and two-year survival rates were 75% and 22%, respectively. These results were superior to those of the intermittent bolus injection of 5-FU plus MMC (or epirubicin) in 40 pts from 1977 to 1994. These results suggest that continuous 5-FU plus LV arterial infusion is an effective regimen in pts with liver metastases from colorectal cancer. However, the infusion with an extracorporeal device limits the pts' quality of life. Further investigation is needed for a schedule that can be practiced for a longer period.