Background:The involvement of autophagy in heart disease has been reported. Transgenic mice expressing GFP-LC3 have been a useful tool in detecting autophagosomes systemically. It is difficult to differentiate increased formation of autophagosomes from decreased clearance of autophagosomes in the heart using GFP-LC3 mice. Methods and Results:We generated transgenic mice expressing mCherry-LC3 under αMyHC promoter and crossed the mice with transgenic mice expressing GFP-LC3. The deference of resistance to acidic conditions between GFP and mCherry overcame the limitation. Conclusions:This method is an innovative approach to examine the role of autophagy and to analyze autophagosome maturation in cardiomyocytes. (Circ J 2010; 74: 203 - 206)

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We investigated the in situ localization of the 50 kDa protein encoded by ORF2 of Apple chlorotic leaf spot virus (ACLSV) genome which is thought to be a movement protein. In immunogold electron microscopy of ACLSV-infected Chenopodium quinoa leaves, the 50 kDa protein was localized on plasmodesmata and nearby cytoplasm. Observation of transgenic Nicotiana occidentalis leaves expressing the 50 kDa protein fused to enhanced green fluorescent protein (EGFP) by fluorescence and confocal laser scanning microscopes revealed that green fluorescence was observed as spots on the cell wall or strands passing through the cell wall of several cell types, i.e., epidermal, palisade and spongy mesophyll and collenchyma cells. In transverse and longitudinal sections of leaf veins of transgenic plants showed that the 50K-EGFP fusion accumulated in sieve elements and formed an extensive interconnecting network of threadlike structure. These results indicated that ACLSV 50 kDa protein can target plasmodesmata and traffic into sieve elements.

A 60-year-old woman with unoperated single ventricle (SV) had been uneventful until infective endocarditis occurred. Only 9 cases of unoperated SV in patients older than 60 years have been reported. Previous examinations had demonstrated a prolonged PQ interval, double-inlet left ventricle, levo-transposition of the great arteries, and bicuspid pulmonary valve (PV) with severe stenosis with a 104 mm Hg pressure gradient (A to E, Online Video 1). The pulmonary stenosis had resulted in a delicate balance between the systemic and pulmonary circulation and was thought to have allowed her long-term survival. On admission, echocardiography revealed mobile vegetation on the PV, suggesting infective endocarditis. Despite antibiotic therapy, severe pulmonary insufficiency developed (F and G, Online Video 2), and broke the hemodynamic balance, resulting in death. Autopsy confirmed the SV and a bicuspid PV with infective endocarditis (H to J).

To understand why transgenic Nicotiana occidentalis plants expressing a functional movement protein (MP) of Apple chlorotic leaf spot virus (ACLSV) show specific resistance to Grapevine berry inner necrosis virus (GINV), the MPs of ACLSV (50KP) and GINV (39KP) were fused to green, yellow, or cyan fluorescent proteins (GFP, YFP, or CFP). These fusion proteins were transiently expressed in leaf cells of both transgenic (50KP) and nontransgenic (NT) plants, and the intracellular and intercellular trafficking and tubule-inducing activity of these proteins were compared. The results indicate that in epidermal cells and protoplasts from 50KP plant leaves, the trafficking and tubule-inducing activities of GINV-39KP were specifically blocked while those of ACLSV-50KP and Apple stem grooving virus MP (36KP) were not affected. Additionally, when 39KP-YFP and 50KP-CFP were coexpressed in the leaf epidermis of NT plants, the fluorescence of both proteins was confined to single cells, indicating that 50KP-CFP interferes with the cell-to-cell trafficking of 39KP-YFP and vice versa. Mutational analyses of 50KP showed that the deletion mutants that retained the activities described above still blocked cell-to-cell trafficking of 39KP, but the dysfunctional 50KP mutants could no longer impede cell-to-cell movement of 39KP. Transgenic plants expressing the functional 50KP deletion mutants showed specific resistance against GINV. In contrast, transgenic plants expressing the dysfunctional 50KP mutants did not show any resistance to the virus. From these results, we conclude that the specific resistance of 50KP plants to GINV is due to the ability of the 50KP to block intracellular and intercellular trafficking of GINV 39KP.