ABSTRACT
We have investigated D-fraction (MDF) extracted from Grifola frondosa (Maitake mushroom) on the inducible nitric oxide synthase (iNOS)-mediated nitric oxide (NO) production in RAW264.7 (RAW) cells, a murine monocyte/macrophage cell line, with special reference to antitumor activity of MDF against human hepatoma-derived huH-1 cells. MDF could induce iNOS mRNA expression in RAW cells in a dose range of more than 30 microg/ml, but the effect of 10 microg/ml of MDF was negligible. The iNOS mRNA expression induced by 100 microg/ml of MDF was 6 hrs later, but lasted for a longer time than that of lipopolysaccharide (LPS), a representative iNOS inducer. Although iNOS mRNA levels in MDF-stimulated cells were almost equal to LPS-stimulated cells at the peak time, the cumulative amount of nitrite was only about 50% compared with that of LPS-treated cells. When huH-I cells were cultured in MDF containing media in a 24-well plate with inserted porous bottom in the presence or absence of RAW cells, the viability of huH-1 cells decreased significantly only in the presence of RAW cells in MDF dose-dependent manner. This antitumor activity of RAW cells in the presence of MDF was abolished or attenuated by the addition of L-NAME, a NOS inhibitor, confirming that this phenomenon is due to iNOS-mediated NO production by RAW cells, but not direct cytotoxic activity of MDF against huH-1 cells. These data suggest that MDF is a novel inducer for iNOS which contributes at least in part to antitumor activity of MDF.
Subject(s)
Agaricales/chemistry , Antineoplastic Agents/pharmacology , Macrophages/drug effects , Nitric Oxide/metabolism , Plant Extracts/pharmacology , Animals , Cell Division/drug effects , Cell Line , DNA Primers/chemistry , Glucans/isolation & purification , Glucans/pharmacology , Macrophages/metabolism , Mice , Nitrates/metabolism , Nitric Oxide Synthase/genetics , Nitric Oxide Synthase/metabolism , Nitric Oxide Synthase Type II , Nitrites/metabolism , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Time FactorsABSTRACT
Blood agar plates for demonstration of the streptococcal serum opacity reaction were prepared by pouring into Petri discs four layers, one above the other, in the following sequence: 1. agar; 2. horse-sheep blood mixed with agar; 3. porcine serum in agarose; and 4. agarose. The strains to be tested were streaked with a loop on the plates and a control medium was prepared as described above, but without porcine serum. After incubation for 18 hours at 37 degrees C, the streptococci producing opacity factor showed greenish zones around the colonies or else no haemolysis at all on the plates containing porcine serum, in contrast to a clear betahaemolysis on the control plates. Opacity reaction negative steptococci showed the same clear beta-haemolysis on both plates. Concordant results were obtained by this method and a standard method for demonstration of opacity factor.
