Antidiabetic effect of an acidic polysaccharide (TAP) from Tremella aurantia and its degradation product (TAP-H).

Continuous oral administration of the acidic polysaccharide (TAP) solution (0.5 g/l) and the TAP-H (degradation products of TAP) solution (1.5 g/l) instead of water for 10 weeks were found to depress plasma glucose increases in diabetes using genetically non-insulin-dependent diabetic model (KK-Ay) mice. TAP and TAP-H significantly lowered levels of insulin, total-cholesterol and triglyceride in the blood of the mice. In excretion to feces, TAP and TAP-H significantly increased the total bile acid, while the cholesterol content of both groups was less than that of the control. Furthermore, TAP and TAP-H significantly decreased the plasma lipoperoxide level. The study shows that TAP and TAP-H have an antidiabetic effect on diabetes model mice.

Structural features of an anti-diabetic polysaccharide (TAP) from Tremella aurantia.

The structure of an anti-diabetic polysaccharide (TAP) obtained from the fruiting bodies of Tremella aurantia was investigated by methylation analysis, Smith degradation, partial acid hydrolysis, 13C-NMR spectrometry, and enzymatic digestion. The results suggested that TAP was composed of (1-->3)-linked alpha-D-mannopyranosyl residues as a backbone, some of which were substituted at position 2 with (1-->3)-linked beta-D-xylopyranose side chains and with beta-D-glucopyransyluronic acid at position 4 linked to terminal alpha-D-mannopyranose.

Effect of a polysaccharide (TAP) from the fruiting bodies of Tremella aurantia on glucose metabolism in mouse liver.

An acidic polysaccharide (TAP) obtained from the fruiting bodies of Tremella aurantia significantly increased the activities of glucokinase, hexokinase, and glucose-6-phosphate dehydrogenase, and decreased the activity of glucose-6-phosphatase in normal and diabetic mouse liver after intraperitoneal administration, while the glycogen content in the liver was reduced. Furthermore, TAP lowered the plasma cholesterol level in normal and diabetic mice.

Conformational analysis and docking study of potent factor XIIIa inhibitors having a cyclopropenone ring.

A conformational analysis and docking study of potent factor XIIIa inhibitors having a cyclopropenone ring were carried out in an attempt to obtain structural insight into the inhibition mechanism. First, stable conformers of the inhibitors alone were obtained from the conformational analysis by systematic search and molecular dynamics. Next, a binding form model of factor XIIIa was built based on an X-ray crystal structure of the enzyme. Finally, the docking study of the inhibitors into the model's binding site was performed. From the resulting stable complex structures, it was found that the cyclopropenone ring fits the active site located at the base of the binding cavity with high complementarity. The carbonyl oxygen of the cyclopropenone ring formed a hydrogen bond to the indole NH group of Trp279 and the terminal carbon atom of the reactive C=C double bond was in close proximity to the sulfur atom of the catalytic residue, Cys314. This binding mode suggests a possible inhibition mechanism, whereby the cysteine residue reacts with the cyclopropenone ring of the inhibitor, forming an enzyme-ligand adduct. In addition, the higher interaction energies between factor XIIIa and the inhibitors alluded to the probable binding sites of the ligand side chain.

Structural features and hypoglycemic activity of a polysaccharide (CS-F10) from the cultured mycelium of Cordyceps sinensis.

A polysaccharide (CS-F10) purified from a hot water extract of the cultured mycelium of Cordyceps sinensis was composed of galactose, glucose and mannose in a molar ratio of 43:33:24; its molecular weight was estimated to be about 15000. The results of chemical and spectroscopic investigations suggest that CS-F10 has a comb-type structure, and has alpha-D-glucopyranosyl residues on the terminal of the side-chains and characteristic sugar residues of C. sinensis i.e., 1,5-linked beta-D-galactofuranosyl residues. CS-F10 significantly lowered the plasma glucose level in normal, streptozotocin (STZ)-induced diabetic and epinephrine-induced hyperglycemic mice after intraperitoneal administration (50 mg/kg). Administration of CS-F10 to STZ-induced diabetic mice significantly increased the activity of hepatic glucokinase. A significant reduction in the hepatic glucose output was observed following the infusion of CS-F10 using the perfused rat liver. CS-F10 also significantly decreased protein content of facilitative glucose transporter isoform 2 (GLUT2) from rat liver following i.p. administration. These effects presumably contribute to the hypoglycemic activity.

Activation of macrophages and neutrophils by an endothelium growth suppressing factor.

An endothelial cell growth-suppressing factor (EGSF) was purified from the serum-free conditioned medium of the mouse P388D1 culture in the presence of carboxymethylated curdlan. The purified EGSF showed two bands corresponding to the molecular masses of 55 and 63 kDa by silver staining on a SDS-polyacrylamide gel under reducing conditions. This factor strongly suppressed the proliferation of endothelial cells from bovine artery, human umbilical vein, and human dermal vas capillare and this suppression was observed to be reversible. We found that EGSF was a potent chemoattractant for macrophages and neutrophils. EGSF mediated the adhesion of neutrophils to BAEs and transendothelial migration of neutrophils. Macrophages stimulated by EGSF produced nitrite in a dose-dependent manner. EGSF did not affect the proliferation of T lymphocytes. These findings suggest that EGSF acts not only as a potent inhibitor for the growth of endothelial cells but also an activator for macrophages and neutrophils. Thus EGSF plays a role in an inflammatory response in the endothelium.

Growth suppressing factor for endothelial cells exhibits tumor regressing activity.

Endothelium growth suppressing and tumor-regressing activities were copurified from the conditioned medium of P388D1 culture in the presence of 100 microg/ml carboxymethylated curdlan by a procedure including ammonium sulfate fractionation and six column chromatographies of Ceramic hydroxyapatite, Q-Sepharose, Sephacryl S-300 HR, Matrex PBA-30, PBE94, and anti-bovine serum albumin (anti-BSA) agarose. The intravenous administration of the purified growth suppressing factor for endothelial cells to sarcoma 180-bearing mouse caused a rapid decrease in the number of viable tumor cells in tumor lumps within 16 h. Immunohistochemical study showed that the intravenous injection of the purified factor to sarcoma 180-bearing mouse resulted in hemorrhagic disorder all over the tissue in the tumor lamp. Thus, the purified factor exhibited not only growth suppressing activity for endothelial cells but also tumor regressing activity at a concentration as low as about 15 ng/mouse. The purified factor significantly inhibited in vitro tubulogenesis of bovine artery, human umbilical vein, and adult human darmal microvascular endothelial cells on collagen gel at a concentration of about 5 ng/ml. After the tube formation of endothelial cells was completed on a collagen gel, the purified factor disrupted the tubes at a concentration of about 5 ng/ml within 48 h. These findings demonstrate that endothelium growth suppressing factor is a potent inhibitor of angiogenesis as well as the growth of endothelial cells, and may bring about the regression of a solid tumor by inhibiting angiogenesis.

Purification of a growth-suppressing factor for bovine artery endothelial cells from mouse lymphoma P388D1 cells.

A growth-suppressing factor for bovine artery endothelial cells (BAEGSF) was purified from the conditioned medium of a mouse lymphoma P388D1 cell culture in the presence of 100 microg/ml carboxymethylated curdlan. The purification steps included, in order, ammonium sulfate fractionation and eight stages of column chromatography on Macro-Prep Ceramic Hydroxyapatite, Q-Sepharose, Sephacryl S-300 HR, Matrex PBA-30, CHT II, Resource-Q, anti-bovine serum albumin (BSA) agarose, and Superdex 200HR columns. The purified BAEGSF showed two bands with silver staining on a sodium dodecyl sulfate polyacrylamide gel under reducing conditions (SDS-PAGE) and their molecular weights were estimated as approximately 55 and 63 kDa, while the molecular weight of the purified BAEGSF was estimated as about 65 kDa by gel filtration using Superdex 200HR. This result shows that BAEGSF obtained from Superdex 200HR chromatography is a partially purified preparation and suggests that one of the two bands on SDS-PAGE corresponds to BAEGSF. BAEGSF was shown not to have a lethal effect on endothelial cells, but had an inhibitory action on the proliferation of these cells. Furthermore, the growth-suppressing activity of BAEGSF for bovine artery endothelial cells (BAE) was not inhibited by anti-transforming growth factor-beta (TGF-beta), anti-tumor necrosis factor-alpha (TNF-alpha), and anti-interleukin-1 (IL-1) antibodies. These results suggest that BAEGSF is different from TGF-beta, TNF-alpha, and IL-1 which have been reported to inhibit BAE growth.

Rational design of antitumor prostaglandins with high biological stability.

microolecular design can overcome the metabolic instability of Delta7-PGA1, while maintaining its antitumor potency. Saturation of the C(13)-C(14) double bond enhances the biological stability but decreases the antiproliferative activity. Configurational inversion of the isomerase-sensitive C(12) stereocenter from the natural S to the unnatural R geometry not only enhances biological stability but also significantly suppresses the growth of the tumor cells. The 12R derivatives markedly increase the induction of p21, a Cdk inhibitor, leading to sharp cell cycle arrest at the G1 phase at a dose level so low that at this dose Delta7-PGA1 methyl ester scarcely exerts an effect. These conspicuous biological properties lead to long-term suppression of tumor cell growth. The structure-stability relationship demonstrates that the stability of prostaglandins (PGs) is crucially controlled by the C(12) configuration and is unaffected by the geometry of the hydroxy-bearing C(15). The successful design of antitumor PGs resistant to enzymatic metabolism provides a new strategy applicable to creating a useful PG for cancer chemotherapy.

Potent prostaglandin A1 analogs that suppress tumor cell growth through induction of p21 and reduction of cyclin E.

Although the cyclopentenone prostaglandin A1 (PGA1) is known to arrest the cell cycle at the G1 phase in vitro and to suppress tumor growth in vivo, its relatively weak activity limits its usefulness in cancer chemotherapy. In an attempt to develop antitumor drugs of greater potency and conspicuous biological specificity, we synthesized novel analogs based on the structure of PGA1. Of the newly synthesized analogs, 15-epi-delta7-PGA1 methyl ester (NAG-0092), 12-iso-delta7-PGA1 methyl ester (NAG-0093), and ent-delta7-PGA1 methyl ester (NAG-0022) possess a cross-conjugated dienone structure around the five-member ring with unnatural configurations at C(12) and/or C(15) and were found to be far more potent than native PGA1 in inhibiting cell growth and causing G1 arrest in A172 human glioma cells. These three analogs induced the expression of p21 at both RNA and protein levels in a time- and dose-dependent fashion. Kinase assays with A172 cells treated with these analogs revealed that both cyclin A- and E-dependent kinase activities were markedly reduced, although cyclin D1-dependent kinase activity was unaffected. Immunoprecipitation-Western blot analysis showed that the decrease in cyclin A-dependent kinase activity was due to an increased association of p21 with cyclin A-cyclin-dependent kinase 2 complexes, whereas the decrease in cyclin E-dependent activity was due to a combined mechanism involving reduction in cyclin E protein itself and increased association of p21. Thus, these newly synthesized PGA1 analogs may prove to be powerful tools in cancer chemotherapy as well as in investigations of the structural basis of the antiproliferative activity of A series prostaglandins.

Biological activities of (1-->3)-beta-D-glucans with reducing glucose side chains.

Newly synthesized (1-->3)-beta-D-glucans with reducing glucose side chains (6-O-glucopyranosylated curdlan and 3-O-glucopyranosylated curdlan, with glucose linked directly (except for anomeric carbon) had antitumor activity against mice sarcoma 180 in mice. The two glucans potentiated the reticuloendotheliai system and activated macrophages (increased their glucose consumption). The activity inducing tumor regressing factor of the glucan derivatives was stronger than a linear (1-->3)-beta-D-glucan (curdlan).

Growth suppressing activity for endothelial cells induced from macrophages by carboxymethylated curdlan.

A carboxymethylated derivative of a linear (1-->3)-beta-D-glucan (CMCD) from Alcaligenes faecalis var. myxogenes acted directly on mouse peritoneal macrophages and mouse lymphoma P388D1 cells, and induced a growth suppressing activity for bovine artery endothelial cells (BAEs) from themselves at a concentration of 100 micrograms/ml. The suppressing activity was also detected in the mouse serum administered as an i.p. injection of CMCD at a dose of 100 mg/kg, suggesting that the growth suppressing activity was induced from macrophages potentiated by CMCD in vivo.

Analysis of wool fiber by alkali-catalyzed pyrolysis gas chromatography.

Alkali-catalyzed pyrolysis gas chromatography (PyGC) has been used to identify minute samples of wool fiber. The wool sample to which aqueous sodium hydroxide was added was pyrolyzed in a Curie-point pyrolyzer attached to a gas chromatograph or a gas chromatograph-mass spectrometer. The addition of an aqueous solution of sodium hydroxide increased the production of specific volatile pyrolysis products from the constitutive amino acid residues of wool protein, i.e. acetaldehyde from alanine or proline, isobutyronitrile from valine, 2-methylbutyronitrile from isoleucine, isovaleronitrile from leucine and toluene from phenylalanine. Compared with conventional non-catalyzed PyGC, the alkali-catalyzed PyGC was found to greatly improve the detection limit of wool fiber and make it possible to analyze very minute samples. The alkali-catalyzed PyGC presented here has been shown to be applicable to minute thermally-denatured samples of wool fiber which cannot be identified successfully by morphological inspection using a microscope or by using Fourier-transform infrared microspectroscopy. Furthermore, the present PyGC method was successfully used for several protein samples and was shown to be useful for analysis of proteins other than wool fibers by using different special pyrograms reflecting different amino acid compositions.

Preparation of novel (1-->3)-beta-D-glucans having reducing glucose side chains.

Novel (1-->3)-beta-D-glucans (GPBCD, GPECD, GP6CD, and GP3CD) having reducing glucose side chains were prepared from a linear (1-->3)-beta-D-glucan (curdlan: CD) with halogeno glucose isopropylidene derivatives in dimethyl sulfoxide containing dimsyl sodium, followed by treatment with 40% trifluoroacetic acid to remove protecting isopropylidene groups. The side chain glucose moiety was linked or directly or through a spacer at various positions except for its anomeric carbon.

Polysaccharides in fungi. XXXVIII. Anti-diabetic activity and structural feature of a galactomannan elaborated by Pestalotiopsis species.

A fungus of Pestalotiopsis species produced an extracellular, water-soluble polysaccharide (PS-N). PS-N exhibited significant hypoglycemic activity in streptozotocin-induced diabetic mice following intraperitoneal administration and had an effect on oral glucose tolerance in normal mice following oral administration. PS-N ([alpha]D +34.5 degrees in water) was homogeneous on gel chromatography, it is composed of galactose and mannose in a molar ratio of 1:9, and its molecular weight was estimated by gel chromatography to be about 24,000. Its structure was investigated by a combination of chemical and spectroscopic methods. The results indicated that PS-N, a highly branched galactomannan, is composed of beta-(1-->3)-linked D-galactofuranosyl and non-reducing terminal beta-D-galactofuranosyl residues, in addition to alpha-D-mannopyranosyl residues of a yeast mannan type.

Effect of 3-deoxyglucosone on the activities of enzymes responsible for glucose metabolism in mouse liver.

Crude extracts containing the enzymes obtained from mouse liver were incubated with 3-deoxyglucosone (3-DG), and then subjected to assay of the activities of enzymes responsible for glucose metabolism. Hexokinase and glucose-6-phosphate dehydrogenase activities were decreased by 3-DG and hexokinase activity was strongly inhibited time and concentration dependently, while glucokinase, glucose-6-phosphatase, and phosphofructokinase activities were scarcely affected. These results suggest that 3-DG inhibits the intake of glucose in the liver and a connection with development of diabetes.

Polysaccharides in fungi. XXXVI. Hypoglycemic activity of a polysaccharide (CS-F30) from the cultural mycelium of Cordyceps sinensis and its effect on glucose metabolism in mouse liver.

A polysaccharide (CS-F30) obtained from the cultural mycelium of Cordyceps sinensis showed potent hypoglycemic activity in genetic diabetic mice after intraperitoneal administration, and the plasma glucose level was quickly reduced in normal and streptozotocin-induced diabetic mice after intravenous administration. Administration of CS-F-30 to normal mice significantly increased the activities of hepatic glucokinase, hexokinase and glucose-6-phosphate dehydrogenase, although the glycogen content in the liver was reduced. Furthermore, CS-F30 lowered the plasma triglyceride level and cholesterol level in mice.

Polysaccharides in fungi. XXXVII. Immunomodulating activities of carboxymethylated derivatives of linear (1-->3)-alpha-D-glucans extracted from the fruiting bodies of Agrocybe cylindracea and Amanita muscaria.

Immunomodulating activities of three carboxymethylated derivatives (AG-AL-CMS, AG-AL-CMI, and AM-APP-CM) of linear (1-->3)-alpha-D-glucans from Agrocybe cylindracea and Amanita muscaria were evaluated with murine peritoneal macrophages playing an important role in tumor immunity. The ratio of macrophages in peritoneal exudate cells increased more than 50% after the administration of three carboxymethylated (1-->3)-alpha-D-glucans. These carboxymethylated (1-->3)-alpha-D-glucans exhibited higher potentiating activities for macrophages than carboxymethylated linear (1-->3)-beta-D-glucan (CMPS) in the potency of reduction of nitro blue tetrazolium, products of nitric oxide and the soluble cytotoxic factor, the amount of glucose consumption, and the activation of acid phosphatase. AG-AL-CMS, AG-AL-CMI, and AM-APP-CM were found to induce the tumor regressing factor in mouse serum, although the ability of the induction of this factor was weaker than that of CMPS. The reticuloendothelial system-potentiating activation of three carboxymethylated alpha-D-glucans was similar to that of the carboxymethylated beta-D-glucan. AG-AL-CMS and AG-AL-CMI, but not AM-APP-CM, were suggested to possess a higher-order structure, resulting from the formation of a fluorescent complex with aniline blue.

[Gas chromatographic analysis of reduction products of paraquat, diquat and the related compounds: reductive cleavage in the pyridine ring on N-alkylpyridinium derivatives with NaBH4-NiCl2 reduction system, and inhibition of the cleavage].

When N-alkylpyridinium derivatives were reduced with sodium borohydride-nickel (II) chloride reduction system, reductive cleavage occurred at the C-N bond in the pyridine ring of N-alkylpyridinium derivatives to give a small amount of reductive cleavage product along with the major perhydrogenated product. It was presumed in the previous report that this reductive cleavage in the pyridine ring proceeded through a complex of nickel ion and 1,2,3,6-tetrahydropyridine derivatives produced with NaBH4 alone reduction. The abundances of these reductive cleavage products arising from N-alkylpyridinium derivatives, i.e., paraquat, diquat and so on, are capable of giving a bad effect on the accuracy of gas chromatographic analysis. For the purpose of inhibition of the reductive cleavage in this reduction system, a suitable catalyst was examined. In addition, we pursued whether borane-1,2,3,6-tetrahydropyridine derivative complexes arose from N-alkylpyridinium derivatives by NaBH4 alone reduction or not, and whether these borane-amine complexes were the precursors of reductive cleavage products or not. N-Alkyl-1,2,3,6-tetrahydropyridine derivatives (III-I, IV-I, VI-I, VII-I and VIII-I) and the corresponding borane-amine complexes (III-II, IV-II, VI-II, VII-II and VIII-II) were synthesized by NaBH4 reduction in aqueous solution of N-alkylpyridinium salts, i.e. I, II, 1,4-dimethylpyridinium iodide (III), 1-dodecylpyridinium chloride (IV), 1,1'-diethyl-4,4'-dipyridinium dichloride (V), 1-methyl-4-phenylpyridinium iodide (VI), 1-n-propylpyridinium iodide (VII) and 1-n-butylpyridinium iodide (VIII). The structure of the borane-amine complexes were proved by the Mass spectrometry and 1H- and 13C-NMR analysis. The NiCl2-NaBH4 reduction of the borane-amine complexes gave the perhydrogenated products alone, but not reductive cleavage products. In conclusion, it was recognized that the precursors of reductive cleavage products were not borane-amine complexes, but 1,2,3,6-tetrahydropyridine. Furthermore, it was found the reductive cleavage at the C-N bond in the pyridine ring of these 1,2,3,6-tetrahydropyridine derivatives was hindered by applying Amberlite-Ni2B, NaBH4 reduction system.

Polysaccharides in fungi. XXXV. Anti diabetic activity of an acidic polysaccharide from the fruiting bodies of Tremella aurantia.

An acidic polysaccharide (TAP) was isolated from a hot-water extract of the fruiting bodies of Tremella aurantia. It showed remarkable hypoglycemic activity in normal mice and two diabetic mouse models, streptozotocin-induced diabetes and genetic diabetes, following intraperitoneal administration. Continuous oral administration of TAP solution (0.5g/l) for a long period was found to be also effective in hyperglycemia in glucose-loaded mice and no harmful physical effects were found. TAP had an [alpha]D -7 degrees in water, and its molecular weight was estimated to be about 1500000. TAP is composed of mannose, xylose, glucuronic acid and glucose (molar ratio, 4:2:1:0.3), and it contains 2.2% of O-acetyl groups.