Analysis of anti-rotavirus activity of extract from Stevia rebaudiana.

Anti-human rotavirus (HRV) activity of hot water extracts from Stevia rebaudiana (SE) was examined. SE inhibited the replication of all four serotypes of HRV in vitro. This inhibitory effect of SE was not reduced on the prior exposure of SE to HCl for 30 min at pH 2. Binding assay with radiolabeled purified viruses indicated that the inhibitory mechanism of SE is the blockade of virus binding. The SE inhibited the binding of anti-VP7 monoclonal antibody to HRV-infected MA104 cells. The inhibitory components of SE were found to be heterogeneous anionic polysaccharides with different ion charges. The component analyses suggested that the purified fraction named as Stevian with the highest inhibitory activity consists of the anionic polysaccharide with molecular weight of 9800, and contains Ser and Ala as amino acids. Analyses of sugar residues suggest uronic acid(s) as sugar components. It did not contain amino and neutral sugars and sulfate residues. These findings suggest that SE may bind to 37 kD VP7 and interfere with the binding of VP7 to the cellular receptors by steric hindrance, which results in the blockade of the virus attachment to cells.

Induction of apoptosis and inhibition of DNA topoisomerase-I in K-562 cells by a marine microalgal polysaccharide.

We have previously purified an extracellular polysaccharide, D-galactan sulfate associated with L(+)-lactic acid, produced from a marine microalga Dinoflagellate Gymnodinium sp. A3 (GA3). The GA3 polysaccharide, irrespective of presence or absence of lactic acid, exhibited significant cytotoxicity, which is based on an induction of apoptotic cell death, toward human myeloid leukemia K562 cells. Furthermore, we found that the GA3 polysaccharide with or without lactic acid possesses an inhibitory effect on topoisomerase-I (topo-I). The potent cytotoxic effect of GA3 polysaccharide may result from its inhibitory effect on topo-I, because the topo-I inhibition is known to trigger apoptotic cell death.

Antiviral Activities of Marine Pseudomonas Polysaccharides and Their Oversulfated Derivatives.

: A marine Pseudomonas species WAK-1 strain simultaneously produces extracellular glycosaminoglycan and sulfated polysaccharide. Among the antiviral activities tested for these polysaccharides, the latter showed anti-HSV-1 activity in RPMI 8226 cells (50% effective concentration is 1.4 µg/ml). Oversulfated derivatives of these polysaccharides prepared by dicyclohexylcarbodiimide-mediated reaction for both polysaccharides showed antiviral activities against influenza virus type A (for glycosaminoglycan, 50% effective concentration is 11.0 µg/ml; for another, 2.9 µg/ml). Glycosaminoglycan, sulfated polysaccharide, and their chemically synthesized oversulfated derivatives did not show antiviral activities against influenza virus type B and human immunodeficiency virus type 1. No cytotoxicity of these products was noted against host cells at the 50% cytotoxic concentration of 100 µg/ml, except that naturally occurring sulfated polysaccharide had 50% cytotoxicity against MT-4 cells at 8-21 µg/ml.

Revelation of Antiviral Activities by Artificial Sulfation of a Glycosaminoglycan from a Marine Pseudomonas.

: Sulfated derivatives of a glycosaminoglycan containing l-glutamic acid produced by a marine Pseudomonas species, No. 42 strain, were prepared by the method of dicyclohexyl-carbodiimide-mediated reaction. Both low and high degrees of sulfation of the polysaccharides (products A1 and A2, respectively) were investigated for their antiviral activities against influenza virus type A (FluV-A) and B (FluV-B) in MDCK cells. Both preparations showed antiviral activity against FluV-A at the 50% antiviral effective concentration of 17.3 and 5.2 µg/ml, respectively, whereas they had no antiviral activity against FluV-B. No cytotoxicity of either product was noted against MDCK cells at the 50% cytotoxic concentration of 100 µg/ml.

Decrease of nuclear protein phosphatase 1 activity and induction of mitotic arrest and apoptosis by a marine microalgal polysaccharide in human myeloid leukemia U937 cells.

An extracellular polysaccharide, which we designate GA3P, produced from a marine microalga dinoflagellate Gymnodinium sp. A3, has been previously reported to induce apoptosis in lymphoid and myeloid cell lines. We found that the GA3P accumulates cells into the mitotic phase of the cell cycle and decreases nuclear protein phosphatase 1 (PP1) activity in a dose-dependent manner in myeloid leukemia U937 cells. Dose-dependent patterns in the decrease of nuclear PP1 activity and in the accumulation of cells into mitotic phase or apoptotic status by the GA3P were concordant with each other, indicating that the decrease of nuclear PP1 activity at least mediates some of the etiological steps in development of mitotic arrest and apoptosis induced by the GA3P. In addition, the GA3P repressed the expression of protein levels of the PP1 catalytic subunit isoform PP1 gamma 1 gamma 1. We thus suggest that the decrease of nuclear PP1 activity is due to down-regulation of the protein levels of the PP1 gamma 1.

Inhibitory effect of a marine microalgal polysaccharide on the telomerase activity in K562 cells.

An extracellular polysaccharide from marine microalga, dinoflagellate Gymnodinium sp. A3 (GA3), showed cytotoxicity to human myeloid leukemia K562 cells. We measured telomerase activity in K562 cells cultured with GA3 polysaccharide. 10.0 micrograms/ml of GA3 polysaccharide inhibited the telomerase activity in the cells completely. Also, we found a decrease in expression of the catalytic subunit of protein phosphatase (PP) type 1, PP1 gamma 1, in K562 cells cultured with GA3 polysaccharide.

Induction of apoptosis by a marine microalgal polysaccharide in a human leukemic cell line.

An extracellular polysaccharide produced by a marine microalga Gymnodinium A3 OKU-1 strain showed a strong cytotoxicity for the human leukemia K-562 cells (CC50 = 11.6 µg/ml). We revealed that this polysaccharide induced apoptosis of K-562 cells, as shown by cell morphology, nuclear fragmentation, in situ end-labeling of fragmented DNA, and internucleosomal DNA fragmentation, suggesting that the mechanism of cytotoxicity of this polymer was based on the induction of apoptosis.

Enhanced expression of PP1 gamma 1, a catalytic subunit isoform of protein phosphatase type 1, in invasive ductal carcinoma of the breast.

Breast cancer is one of the most common malignancies of women. Assessing the biological parameters of malignant tumors may facilitate predictions of clinical outcome. The expression of the three catalytic subunits of protein phosphatase (PP) type 1, PP1 alpha, PP1 gamma 1 and PP1 delta, as well as the one catalytic subunit of PP type 2, PP2AC, were examined in ten cases of mammary dysplasia, ten cases of fibroadenoma and 12 cases of invasive ductal carcinoma, using immunohistochemical analysis. Moreover, we measured the S-phase fraction of the cell cycle for use as a marker value of cell growth, using flow cytometric analysis. The percentage of proliferating cells that stained positive with antisera against PP1 gamma 1 was significantly higher in invasive ductal carcinoma than in mammary dysplasia and fibroadenoma. Furthermore, invasive ductal carcinoma showed a markedly high number of tumor cells in the S-phase of the cell cycle, as compared to mammary dysplasia and fibroadenoma. Our results indicate that PP1 gamma 1 may be involved in the accelerated growth of malignant cells in breast tumors.

In vitro antiviral activities of sulfated polysaccharides from a marine microalga (Cochlodinium polykrikoides) against human immunodeficiency virus and other enveloped viruses.

A marine microalga, Cochlodinium polykrikoides, produces extracellular sulfated polysaccharides. Isolation and purification of the polysaccharides were accomplished by precipitation with ethanol and Cetavlon, followed by DEAE-cellulose column chromatography (polysaccharides A1 and A2). These polysaccharides, which were homogeneous when analysed by both ultracentrifugal and electrophoretic methods, were composed of mannose, galactose, glucose and uronic acid, together with sulfate groups (S = 7-8% w/w). Both A1 and A2 inhibited the cytopathic effect of influenza virus types A and B in MDCK cells, that of respiratory syncytial virus types A and B in HEp-2 cells, that of human immunodeficiency virus type 1 in MT-4 cells; and, except A1 for herpes simplex virus type 1 and A2 for parainfluenza virus type 2 in HMV-2 cells, the cochlodinium polysaccharides showed no antiviral activity against parainfluenza virus types 2 and 3, measles virus, mumps virus or herpes simplex virus type 1 in HMV-2 cells. No cytotoxicity for host cells was observed with these polysaccharides at a concentration of 100 micrograms ml-1. Inhibitory effects on various viruses were achieved at concentrations that were not markedly inhibitory to the blood coagulation process.

Structural investigation of Klebsiella serotype K46 polysaccharide.

The structure of the capsular polysaccharide from Klebsiella type K46 has been investigated by using the techniques of methylation analysis, periodate oxidation, and partial hydrolysis. The anomeric linkages were determined by 1H- and 13C-n.m.r. spectroscopy of the polysaccharide and of derived poly- and oligo-saccharides obtained through degradative procedures. 1H-N.m.r. spectroscopy of the polysaccharide in D2O showed clearly a ratio of one (1-carboxyethylidene) group (CH3, delta 1.47) to six anomeric protons (delta 4.62--5.29). The polysaccharide was shown to consist of the following hexasaccharide repeating unit, which is unique in this series in having a (1-carboxyethylidene) acetal group on a lateral, but nonterminal, sugar residue. (Formula: see text).