Kiminori Matsubara

Anticoagulant properties of a sulfated galactan preparation from a marine green alga, Codium cylindricum.

An anticoagulant was isolated from a marine green alga, Codium cylindricum. The anticoagulant was composed mainly of galactose with a small amount of glucose, and was highly sulfated (13.1% as SO3Na). The anticoagulant properties of the purified anticoagulant were compared with that of heparin by assays of activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) using normal human plasma. The anticoagulant showed similar activities with heparin, however, weaker than heparin. On the other hand, the anticoagulant did not affect PT even at the concentration at which APTT and TT were strongly prolonged. The anticoagulant did not potentiate antithrombin III (AT III) and heparin cofactor II (HC II), thus the anticoagulant mechanism would be different from that of other anticoagulants isolated so far from the genus Codium.

Purification and characterization of a fibrinolytic enzyme and identification of fibrinogen clotting enzyme in a marine green alga, Codium divaricatum.

A fibrinolytic enzyme was isolated from a marine green alga, Codium divaricatum, and designated C. divaricatum protease (CDP). This protease effectively hydrolyzed fibrinogen A alpha chain, while it had very low hydrolyzing efficiency for B beta and gamma chains. This property was similar to that of alpha-fibrinogenase isolated from snake venom. Protease activity peaked at pH 9, and was completely inhibited by diisopropyl fluorophosphate (DFP) and phenylmethylsulfonyl fluoride (PMSF), identifying it as a serine protease. Its molecular form was single polypeptide structure and molecular weight was estimated as 31,000 by SDS-PAGE. Fibrinogen clotting enzyme was also identified in a fraction by ion-exchange chromatography. Analysis of clots formed by the enzyme and by thrombin by SDS-PAGE showed that the fibrinogen clotting enzyme would act like thrombin and have high substrate specificity.

Vitamin B6-mediated suppression of colon tumorigenesis, cell proliferation, and angiogenesis (review)

This review describes current research on the preventive effect of dietary vitamin B(6) against colon tumorigenesis and its possible mechanisms. Studies in cell culture have demonstrated that high levels of vitamin B(6) suppress growth of some cancer cells. From these studies it has been considered that supraphysiological doses of vitamin B(6) suppress tumor growth and metastasis. However, recent rodent study has indicated that azoxymethane-induced colon tumorigenesis in mice is suppressed by moderate doses of dietary vitamin B(6.) Epidemiological studies also support an inverse relationship between vitamin B(6) intake and colon cancer risk. Potential mechanisms underlying the preventive effect of dietary vitamin B(6) have been suggested to include the suppression of cell proliferation, oxidative stress, nitric oxide (NO) synthesis, and angiogenesis.

An anticoagulant proteoglycan from the marine green alga, Codium pugniformis

An anticoagulant isolated from the marine green alga Codium pugniformis was composed mainly of glucose with minor amounts of arabinose and galactose. It was highly sulfated (326 μg mg-1 polysaccharide) and contained protein(52 μg mg-1 polysaccharide) and was thus a proteoglycan. The anticoagulant properties of the purified proteoglycan were compared with those of heparin by studying the activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time(TT) using normal human plasma. The proteoglycan showed similar activities to heparin, but was weaker than heparin. On the other hand, the proteoglycan did not affect PT even at the concentration at which APTT and TT were prolonged. The anticoagulation mechanism of this proteoglycan was due to the direct inhibition of thrombin and the potentiation of antithrombin III.

Inhibitory Effect of Carotenoids on the Degranulation of Mast Cells via Suppression of Antigen-induced Aggregation of High Affinity IgE Receptors

Carotenoids have been demonstrated to possess antioxidative and anti-inflammatory effects. However, there is no report that the effects of carotenoids on degranulation of mast cell is critical for type I allergy. In this study, we focused on the effect of carotenoids on antigen-induced degranulation of mast cells. Fucoxanthin, astaxanthin, zeaxanthin, and β-carotene significantly inhibited the antigen-induced release of β-hexosaminidase in rat basophilic leukemia 2H3 cells and mouse bone marrow-derived mast cells. Those carotenoids also inhibited antigen-induced aggregation of the high affinity IgE receptor (FcϵRI), which is the most upstream of the degranulating signals of mast cells. Furthermore, carotenoids inhibited FcϵRI-mediated intracellular signaling, such as phosphorylation of Lyn kinase and Fyn kinase. It suggests that the inhibitory effect of carotenoids on the degranulation of mast cells were mainly due to suppressing the aggregation of FcϵRI followed by intracellular signaling. In addition, those carotenoids inhibited antigen-induced translocation of FcϵRI to lipid rafts, which are known as platforms of the aggregation of FcϵRI. We assume that carotenoids may modulate the function of lipid rafts and inhibit the translocation of FcϵRI to lipid rafts. This is the first report that focused on the aggregation of FcϵRI to investigate the mechanism of the inhibitory effects on the degranulation of mast cells and evaluated the functional activity of carotenoids associated with lipid rafts.

Primary structures of two hemagglutinins from the marine red alga, Hypnea japonica

As the first examples among marine algal hemagglutinins, the primary structures of two hemagglutinins, named hypnin A-1 and A-2, from the red alga Hypnea japonica, were determined by Edman degradation. Both hemagglutinins were single-chain polypeptides composed of 90 amino acid residues including four half-cystines, all of which were involved in two intrachain disulfide bonds, Cys(5)-Cys(62) and Cys(12)-Cys(89). Hypnin A-1 and A-2 had calculated molecular masses of 9146.7 and 9109.7 Da which coincided with determined values, 9148 and 9109 Da, by electrospray ionization-mass spectrometry, respectively. Both hemagglutinins only differed from each other at three positions; Pro(19), Arg(31) and Phe(52) of hypnin A-1 as compared with Leu(19), Ser(31), and Tyr(52) of hypnin A-2. Approximately 43% of total residual numbers consisted of three kinds of amino acids: serine, glycine and proline. The hemagglutination activities were lost by reduction and alkylation of the disulfide bonds. The nature of the small-sized polypeptides, including disulfide bonds, may contribute to the extreme thermostability of the hemagglutinins. Sequence having overall similarity to hypnin A-1 or A-2 was not detected in databases. Unexpectedly, however, hypnins contained a motif similar to the alignment of the C-terminal conserved amino acids within carbohydrate-recognition domains of C-type animal lectins. Furthermore, interestingly, the hemagglutination activities were inhibited by a protein, phospholipase A-2 besides some glycoproteins, suggesting that hypnins may possess both a protein-recognition site(s) and a carbohydrate-recognition site(s).

Anti-angiogenic effect of siphonaxanthin from green alga, Codium fragile.

Since anti-angiogenic therapy has becoming a promising approach in the prevention of cancer and related diseases, the present study was aimed to examine the anti-angiogenic effect of siphonaxanthin from green alga (Codium fragile) in cell culture model systems and ex vivo approaches using human umbilical vein endothelial cells (HUVECs) and rat aortic ring, respectively. Siphonaxanthin significantly suppressed HUVEC proliferation (p<0.05) at the concentration of 2.5 μM (50% as compared with control) and above, while the effect on chemotaxis was not significant. Siphonaxanthin exhibited strong inhibitory effect on HUVEC tube formation. It suppressed the formation of tube length by 44% at the concentration of 10 μM, while no tube formation was observed at 25 μM, suggesting that it could be due to the suppression of angiogenic mediators. The ex vivo angiogenesis assay exhibited reduced microvessel outgrowth in a dose dependent manner and the reduction was significant at more than 2.5 μM. Our results imply a new insight on the novel function of siphonaxanthin in preventing angiogenesis related diseases.

1,4-Naphthoquinone is a potent inhibitor of human cancer cell growth and angiogenesis

Angiogenesis inhibitors are beneficial for the prevention and treatment of angiogenesis-dependent diseases including cancer. Vitamin K2 and K3, which are naphthoquinone derivatives, inhibit angiogenesis. We examined the anti-cancer and anti-angiogenic effects of naphthoquinones and its structurally related compounds. Among these 13 compounds, 1,4-naphthoquinone strongly inhibited both human colon cancer cell (HCT116) growth and angiogenesis. To clarify the anti-angiogenic mechanism, the effects of 1,4-naphthoquinone on human umbilical vein endothelial cell (HUVEC) tube formation, proliferation and chemotaxis were examined. Consequently, 1,4-naphthoquinone inhibited HUVEC functions. These results suggest that 1,4-naphthoquinone may be useful to cancer therapy.

PURIFICATION AND CHARACTERIZATION OF TWO FIBRINOLYTIC ENZYMES FROM A MARINE GREEN ALGA, CODIUM INTRICATUM

Abstract Buffer extracts from five species of marine green algae (Codium sp.) were examined for fibrinolytic and protease activities using a fibrin plate method and chromogenic assay, respectively. Extracts of Codium fragile, C. divaricatum, C. pugniformis, and C. intricatum, contained both activities. From the extract of C. intricatum, which showed the highest activity in both assays, two fibrinolytic enzymes, named CIP-I and CIP-II, were purified to homogeneity by gel filtration followed by ion-exhange chromatography. The two enzymes (Mr, c. 20 k) hydrolyzed fibrinogen with preference to the Aα chain over Bβ or γ chains. Protease activities peaked between pH 8 and 9, and were completely inhibited by diisopropyl fluorophosphate (DFP), identifying them as serine proteases.

A fibrinolytic enzyme from a marine green alga, Codium latum

A fibrinolytic enzyme was isolated from a marine green alga, Codium latum, and designated C. latum protease (CLP). It also had fibrinogenolytic activity, hydrolyzing A alpha, B beta and gamma chains with preference in this order. As CLP hydrolyzed oxidized insulin B chain at position Arg22-Gly23, and the peptide map of lysozyme digested with CLP was similar to that with trypsin, CLP would be expected to have a high substrate specificity, similar to that of trypsin. Protease activity peaked at pH 10, and was completely inhibited by diisopropyl fluorophosphate (DFP). Therefore, we conclude that CLP is a trypsin-like serine protease.