Evaluation of the potential biological toxicities of aqueous extracts from red tide phytoplankton cultures in in vitro and in vivo systems

The biological toxic potentials of aqueous extracts from the dinophycean flagellates Gymnodinium impudicum and Alexandrium affine and the raphidophycean flagellate Chattonella ovata were examined in both in vitro and in vivo systems. Interestingly, the extract from A. affine was the only one that showed potent cytotoxicities towards HeLa, Vero, and Neuro-2a cells in a concentration- dependent manner. Mice given intraperitoneal injections of the extracts revealed that none of the extracts exhibited serious toxicities in mice. However, temporal body weight loss was observed in the mice injected with the extract from A. affine during the early stage, and the dramatic enlargement of spleens was also observed in the mice on the 7th day after injection. Since A. affine extract showed potent hemolytic activity in vitro towards mouse erythrocytes, hemolytic anemia may be a possible mechanism responsible for the splenomegaly in the mice injected with A. affine extract. Similar marginal effects were observed in the mice injected with the extract from C. ovata; however, no significant toxic or detrimental effects were detected in the mice injected with the extract from G. impudicum. These results suggest that the extract from G. impudicum may not be contaminated with detectable levels of biologically hazardous compounds and may be relatively safe compared with the other two extracts

Heart Failure-Inducible Gene Therapy Targeting Protein Phosphatase 1 Prevents Progressive Left Ventricular Remodeling

BACKGROUND: The targeting of Ca(2+) cycling has emerged as a potential therapy for the treatment of severe heart failure. These approaches include gene therapy directed at overexpressing sarcoplasmic reticulum (SR) Ca(2+) ATPase, or ablation of phospholamban (PLN) and associated protein phosphatase 1 (PP1) protein complexes. We previously reported that PP1β, one of the PP1 catalytic subunits, predominantly suppresses Ca(2+) uptake in the SR among the three PP1 isoforms, thereby contributing to Ca(2+) downregulation in failing hearts. In the present study, we investigated whether heart-failure-inducible PP1β-inhibition by adeno-associated viral-9 (AAV9) vector mediated gene therapy is beneficial for preventing disease progression in genetic cardiomyopathic mice. METHODS: We created an adeno-associated virus 9 (AAV9) vector encoding PP1β short-hairpin RNA (shRNA) or negative control (NC) shRNA. A heart failure inducible gene expression system was employed using the B-type natriuretic protein (BNP) promoter conjugated to emerald-green fluorescence protein (EmGFP) and the shRNA sequence. AAV9 vectors (AAV9-BNP-EmGFP-PP1βshRNA and AAV9-BNP-EmGFP-NCshRNA) were injected into the tail vein (2×10(11) GC/mouse) of muscle LIM protein deficient mice (MLPKO), followed by serial analysis of echocardiography, hemodynamic measurement, biochemical and histological analysis at 3 months. RESULTS: In the MLPKO mice, BNP promoter activity was shown to be increased by detecting both EmGFP expression and the induced reduction of PP1β by 25% in the myocardium. Inducible PP1βshRNA delivery preferentially ameliorated left ventricular diastolic function and mitigated adverse ventricular remodeling. PLN phosphorylation was significantly augmented in the AAV9-BNP-EmGFP-PP1βshRNA injected hearts compared with the AAV9-BNP-EmGFP-NCshRNA group. Furthermore, BNP production was reduced, and cardiac interstitial fibrosis was abrogated at 3 months. CONCLUSION: Heart failure-inducible molecular targeting of PP1β has potential as a novel therapeutic strategy for heart failure