ABSTRACT
Objective@#To study the intracellular location and autophagosome production of rhinovirus 16 2B protein using miniSOG labeling technique.@*Methods@#2B was fused with miniSOG and flag tags to construct pcDNA3.1-2B-miniSOG-flag plasmid, which was used to transfect HEK293 cells, LC3 protein was detected by western blot. The transfected cells were fixed, stained with DAB through the photooxidation activity of miniSOG, and used to prepare ultrathin sections. Localization of 2B-miniSOG protein in cells and ultrastructural changes of cells were observed under electron microscope.@*Results@#2B-miniSOG protein glows green under a fluorescence microscopy. Green flourescence coold be observed in the cells expressing 2B-miniSOG protein.LC-II protein increased in the cells transfected with pcDNA3.1-2B-miniSOG-flag. Under electron microscopy it was observed that 2B-miniSOG protein was located in the mitochondria, and a large number of vesicular structures appeared in the cytoplasm. Both autophagosomes and autophagic lysosomes can be observed.@*Conclusions@#Non-structural protein 2B of HRV16 can induce autophagy.
ABSTRACT
The JMJD family is an important group of histone demethylase. JMJD2B belongs to one of its family members,and is a JMJD containing a JmjC domain. It mainly regulates chromatin structure,transcription and cell appearance. Recent studies from domestic and foreign have demonstrated that JMJD2B protein is high expressed in human malignant tumors,such as liver cancer,gas-tric cancer,breast cancer,kidney cancer,and skin cancer. Moreover,it also shows that JMJD2B is involved in the occurrence,develop-ment,migration,infiltration,and proliferation of malignant tumors.
ABSTRACT
BACKGROUND: The developing brain has a distinctive set of characteristics that make it have different susceptibility to excitotoxins. Using primary tissue cultures of rat hippocampus, we investigated the developmental susceptibility to N-methyl-D-aspartic acid (NMDA)-induced cell death at various days in vitro in relation to the appearance of Bcl-2 protein and NMDA receptor 2B subunit. METHODS: Six, 12, and 18 days-in-vitro (DIV) hippocampal tissue cultures derived from 7-day-old Sprague-Dawley rat pups were used. Each group was treated with 100 micrometer NMDA in 5% CO2 incubator at 36 degrees C for 30 min. A western blot was then performed for the NeuN, Bcl-2 and NMDA receptor 2B subunit and propidium Iodide (PI) staining. RESULTS: The NeuN and Bcl-2 were most highly expressed in 12 DIV tissues. The reductions of the NeuN and Bcl-2 protein expressions by NMDA were significant at the 12 and the 18 DIV tissues, but less at 6 DIV tissues (p<0.05). The PI staining showed that the area of fluorescence of the 7 DIV tissues after NMDA exposure was less than the DIV 13 and 19 tissues. Without NMDA treatment, the NMDA receptor 2B subunit protein expressions at the 6 DIV tissues were highest and decreased with maturation. CONCLUSIONS: These results suggest that the immature tissues were more resistant to NMDA toxicity than the mature tissues, and further studies are needed to establish its relationship with the Bcl-2 protein and NMDA receptor 2B subunit.
Subject(s)
Animals , Rats , Blotting, Western , Brain , Cell Death , Fluorescence , Hippocampus , Incubators , N-Methylaspartate , Neurotoxins , Propidium , Rats, Sprague-DawleyABSTRACT
The 2b protein encoded by Cucumber mosaic virus(CMV)plays an important pathogenicity role in many solanaceous hosts,but mechanism of inducing disease is still unknown.In order to investigate virulence of the 2b protein on Nicotiana glutinosa plants,in terms of chloroplast structure and photosynthesis,a mutant Fny-CMV?2bpro,which cannot express the 2b protein,was achieved by introducing mutant sites in the 2b gene of Fny-CMV.N.glutinosa seedlings were inoculated with wild-type Fny-CMV and the mutant Fny-CMV?2bpro,and were analyzed for symptom expression,chlorophyll content,photosynthetic rate,and ultra-structural alteration of chloroplast.Up to 30 days post inoculation,wild-type Fny-CMV caused symptoms of severe mosaic,leaf deformation,and stunting,reduced photosynthetic rate and chlorophyll content,and altered the ultra-structure and morphological characters of the chloroplasts.However,host seedlings inoculated with the mutant Fny-CMV?2bpro expressed only slight mosaic symptom.Their photosynthetic rates and chlorophyll contents were not significantly different from those of the mock-inoculated plants,and the ultra-structure and morphological characters of their chloroplasts appeared to be normal.The observed low photosynthetic rates and chlorophyll contents were related to the breakage of the chloroplast morphology and ultra-structure.Results of Northern blotting showed that the virulence of 2b protein was associated with high accumulation level of CMV progeny RNAs in systemic leaves.Non-expression of the 2b protein reduced the accumulation levels of its genomic RNAs 1 and 2.The level of subgenomic RNA4,encoding CP protein,was found to be significantly decreased.