Marine bacterial isolates were screened for phospholipase C (PLC) activity on PCY agar plates containing phosphatidylcholine (PC) as substrate. The strain that showed the highest activity on a PCY screening agar plate and a thin-layer chromatography was identified as a strain of Pseudoalteromonas and subsequently designated Pseudoalteromonas sp. J937. The extracellular PLC of the strain J937 was purified to a specific activity of 33 U mg(-1) protein by serial ion exchange and gel filtration column chromatography. It had a molecular mass of 32 kDa estimated by SDS-PAGE. The optimal pH and temperature of the enzyme were about pH 8 and 45 degrees C, respectively. The PLC hydrolyzed phosphatidylethanolamine as well as PC but not other glycerophospholipids. Its activity was enhanced by 150% with Ca2+ (200 mM) and by 180% with Na+ (500 mM), suggesting that the purified PLC is a marine-type enzyme.
ObjectivesTo determine whether a novel marine micro-organism with anticancer properties, H31, the metabolic product of Bacillus SW31, has anti-tumor effects on head and neck cancer, and potential for apoptotic-enhancing anti-cancer treatment of affected patients.MethodsThe cell viability and apoptosis assays were performed. Changes in the signal pathway related to apoptosis were investigated. Then, the therapeutic effects of H31 were explored in mouse xenograft model and drug toxicity of H31 was examined in zebrafish model.ResultsWe identified the anticancer activity of H31, a novel metabolic product of Bacillus SW31. Bacillus SW31, a new marine micro-organism, has 70% homology with Bacillus firmus and contains potent cytotoxic bioactivity in head and neck cancer cells using MTT assay. Combined with c-JUN, p53, cytochrome C, and caspase-3, H31 induced apoptosis of KB cells, a head and neck cancer cell line. In a separate in vivo model, tumor growth in C3H/HeJ syngeneic mice was suppressed by H31. In addition, in a zebrafish model used for toxicity testing, a considerable dose of H31 did not result in embryo or neurotoxicity.ConclusionGrowth inhibition and apoptosis were achieved both in vitro and in vivo in head and neck cancer cells after exposure to H31, a metabolite from the marine Bacillus species, without any significant toxicity effects even at considerable H31 dose concentrations.
A novel extracellular phospholipase C (PLC) was purified from a marine streptomycete. It had a molecular mass of 28 kDa as estimated by SDS-polyacrylamide gel electrophoresis. Its enzyme activity was optimal at pH 8.0 at 45 degrees Celsius. The PLC hydrolyzed only phosphatidylcholine. Its activity was enhanced 300% by Na(+) (200 mM), suggesting that the purified PLC is a typical marine-type enzyme.
A new bioluminescent assay method for the activity of sphingolipid ceramide N-deacylase (SCDase: EC 3.5.1.69) as well as ceramidase (CDase: EC 3.5.1.23) was developed using bioluminescent marine bacteria. Enzymatically synthesized ceramide (N-myristoyl sphigosine, C14:0-18:l) and commercial SCDase were used in this demonstration, and myristic (tetradecanoic, C14:0) acid produced by the SCDase hydrolysis was quantified using Vibrio harveyi M-17, a dark mutant of V. harveyi. The in vivo light intensity of M-17 was stimulated up to thousands fold in the presence of myristic acid, was used for this assay. SCDase activity with as little as 10 microU and 5 nM of myristic acid production were detected in less than one min. The assay worked well for the determination of Km and chromatographic fraction assay.
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