The 3F3A monoclonal antibody to autocrine motility factor receptor (AMFR) labels mitochondria-associated smooth endoplasmic reticulum (ER) tubules. siRNA down-regulation of AMFR expression reduces mitochondria-associated 3F3A labelling. The 3F3A-labelled ER domain does not overlap with reticulon-labelled ER tubules, the nuclear membrane or perinuclear ER markers and only partially overlaps with the translocon component Sec61α. Upon overexpression of FLAG-tagged AMFR, 3F3A labelling is mitochondria associated, excluded from the perinuclear ER and co-distributes with reticulon. 3F3A labelling therefore defines a distinct mitochondria-associated ER domain. Elevation of free cytosolic Ca2+ levels with ionomycin promotes dissociation of 3F3A-labelled tubules from mitochondria and, judged by electron microscopy, disrupts close contacts (<50 nm) between smooth ER tubules and mitochondria. The ER tubule-mitochondria association is similarly disrupted upon thapsigargin-induced release of ER Ca2+ stores or purinergic receptor stimulation by ATP. The inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3] receptor (IP3R) colocalises to 3F3A-labelled mitochondria-associated ER tubules, and conditions that induce ER tubule-mitochondria dissociation disrupt continuity between 3F3A- and IP3R-labelled ER domains. RAS-transformed NIH-3T3 cells have increased basal cytosolic Ca2+ levels and show dissociation of the 3F3A-labelled, but not IP3R-labelled, ER from mitochondria. Our data indicate that regulation of the ER-mitochondria association by free cytosolic Ca2+ is a characteristic of smooth ER domains and that multiple mechanisms regulate the interaction between these organelles.

Parkinson’s disease (PD) is the second most common neurodegenerative disorder worldwide. Many factors have been shown to contribute to its pathogenesis including genetic and environmental factors. Ubiquitin C-terminal hydrolase L1 (UCHL1) is also known to be involved in the pathogenesis of PD. We herein modeled the study of UCHL1 in Drosophila melanogaster and investigated its functions in PD. The specific knockdown of the Drosophila ortholog of UCHL1 (dUCH) in dopaminergic neurons (DA neurons) led to the underdevelopment and/or degeneration of these neurons, specifically in DL1 DA neuron cluster in the larval brain lobe and PPM2, PPM3, PPL2ab, and VUM DA neuron clusters in the adult brain. These defects were followed by a shortage of dopamine in the brain, which subsequently resulted in locomotor dysfunction. The degeneration of DA neurons in dUCH knockdown adult brain, which occurred progressively and severely during the course of aging, mimics the epidemiology of PD. DA neuron and locomotor defects were rescued when dUCH knockdown flies were treated with vitamin C, a well-known antioxidant. These results suggest that dUCH knockdown fly is a promising model for studying the pathogenesis and epidemiology of PD as well as the screening of potential antioxidants for PD therapeutics.

SummaryGp78 (also known as AMFR and RNF45) is an E3 ubiquitin ligase that targets proteins for proteasomal degradation through endoplasmic reticulum (ER)-associated degradation (ERAD). In this study, we showed that gp78-mediated ubiquitylation is initiated in the peripheral ER. Substrate monoubiquitylation and gp78 CUE domain integrity restricted substrate to the peripheral ER, where CUE domain interactions and polyubiquitylation reduced gp78 mobility. Derlin-1 and derlin-2, which are involved in the retrotranslocation of ERAD substrates, localized to a central, juxtanuclear ER domain, where polyubiquitylated proteins accumulated upon proteasome inhibition. Transfer of polyubiquitylated substrate to the central ER was dependent on ubiquitin chain elongation and recruitment of the AAA ATPase p97 (also known as VCP). HT-1080 fibrosarcoma cells expressed elevated levels of endogenous gp78, which was associated with segregation of ubiquitylated substrate to the peripheral ER and its polyubiquitin-dependent redistribution to the central ER upon proteasome inhibition. Therefore, the peripheral ER is the site of gp78 ubiquitin ligase activity. Delivery of ubiquitylated substrate to the central ER was regulated by ubiquitin chain elongation and opposing actions of gp78 CUE domain interactions and p97 recruitment.

The autophagy-lysosome pathway is a major protein degradation pathway stimulated by multiple cellular stresses, including nutrient or growth factor deprivation, hypoxia, misfolded proteins, damaged organelles, and intracellular pathogens. Recent studies have revealed that transcription factor EB (TFEB) and transcription factor E3 (TFE3) play a pivotal role in the biogenesis and functions of autophagosome and lysosome. Here we report that three translation inhibitors (cycloheximide, lactimidomycin, and rocaglamide A) can facilitate the nuclear translocation of TFEB/TFE3 via dephosphorylation and 14-3-3 dissociation. In addition, the inhibitor-mediated TFEB/TFE3 nuclear translocation significantly increases the transcriptional expression of their downstream genes involved in the biogenesis and function of autophagosome and lysosome. Furthermore, we demonstrated that translation inhibition increased autophagosome biogenesis but impaired the degradative autolysosome formation because of lysosomal dysfunction. These results highlight the previously unrecognized function of the translation inhibitors as activators of TFEB/TFE3, suggesting a novel biological role of translation inhibition in autophagy regulation.

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Chronic leg ulcer is one of the diabetic complications causing continuous pain and higher mortality risk. The therapeutic treatment of chronic diabetic wounds can be studied through animal models which simulate the human pathology. Experimental diabetes in mice induced by streptozotocin (STZ) is an easy method with low cost. There have been several studies using STZ for diabetic mice model of cutaneous excisional wound. However, those procedures are different in many factors such as dose of STZ, mouse weight, blood glucose level and the method for assessing health status in mice. We have constructed the criteria to evaluate the health status of the experimental mice including skin color, fur condition, ability to move and body condition. These criteria of health status wereused together with the blood glucose level to evaluate the male mice’s sensitivity to a single high dose (100-150 mg/kg) of STZ. Approximately 40 % of 20–28 g weighted mice, which were injected by 125 mg/kg STZ, developed diabetes in a good health status in the first two weeks after injection. Mice with blood glucose level higher than 200 mg/dL had damaged islet and stably high blood glucose as well as stable health status after two weeks carrying wounds. The wound healing process of the diabetic wounded mice occured relatively slower than that of the control group (non diabetic). The model of diabetic wounded mice has been used to evaluate of the wound healing effect of the rhPDGF-BB protein which has been used in the treatment of chronic wounds caused by human diabetes.

Melicope pteleifolia (Champ. ex benth.) T.g. Hartley, a folk medicinal plant, is used by ethnic minorities in Bidoup–Nui Ba National Park, Lam Dong Province, Vietnam to treat effectively wound, inflammation and skin ulcer. To scientifically prove the claimed utilization and understand the mechanism of action of the plant, the in vitro and in vivo healing properties of the extract and fractions of the plant were investigated. The ethanol 70 % extract (50 – 400 mg/mL), aqueous (200 mg/mL), ethyl acetate (100 mg/mL) and petroleum ether (50 mg/mL) fractions were used to evaluate the antibacterial activities by using agar diffusion method. The healing properties were in vitro investigated through fibroblasts and keratinocytes proliferation and migration (7.8 g/mL to 250 g/mL in accordance with each extract and fraction). Besides, the macrophage-induced inhibition of the nitric oxide (NO) production was examined (15.6 – 62.5 g/mL). In addition, the excision wound model was used to test the wound healing activity on mice model. We found that the ethanol extract and the ethyl acetate fraction showed potent activity against Staphylococcus aureus, Enterococcus feacalis and Pseudomonas aeruginosa. The extract and fractions stimulated fibroblasts and keratinocytes proliferation in a concentration-dependent way. They also inhibited macrophage produce NO. In addition, mice treated by the extract formed scabs on wound excision of mice model faster than the control group. The wound healing efficiency seems to involve antibacterial, stimulating fibroblasts and keratinocytes proliferation, inhibition of macrophages produce NO.

Parkinson's is the second most common neurodegenerative disease in the world (after Alzheimer's), characterized by the degeneration of dopaminergic neurons and mobility impairment, which consequently severely reduces patient life quality. Up to now, Parkinson's Disease cannot be completely cured. Base on the relationship between oxidative stress and the formation, the progression of Parkinson's Disease, antioxidant compounds have been studied as potential candidates in rescue or retard disease progression. Resveratrol is a strong antioxidant, anti-inflammatory, anti-apoptosis compound that exists in many fruits, especially grapes, strawberries..., and has activity depends on the used concentration. In this study, we evaluated the potential of resveratrol for treating Parkinson's Disease at concentrations of 0.064 mg/g, 0.32 mg/g, 0.64 mg/g by utilizing the knockdown dUCH (Drosophila ubiquitin carboxyl-terminal hydrolase) Drosophila melanogaster model. This model can mimic the typical symptoms of Parkinson's Disease and has been proved for the efficiency in screening drugs to treat the disease. Our results showed that the use of resveratrol at a concentration of 0.32 mg/g was effective in preventing the degeneration of dopaminergic neurons and improving mobility in dUCH knockdown flies. These results provided scientific evidence for the development of functional products or drugs to support Parkinson's Disease treatment.