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Objective:To construct an in vitro reconstitution system for inverse autotransporters in order to further investigate their translocation mechanism. Methods:Intimin from Escherichi coli was used as a model substrate. Spheroplasts were prepared from Escherichi coli strains overexpressing Intimin to induce the expression of Intimin. Recombinant β-barrel assembly machinery (BAM) complex was obtained and purified, and then proteoliposomes containing BAM were prepared. Following the digestion with proteinase K, the translocation was detected by SDS-PAGE. Results:Spheroplasts were induced to express Intimin, and then BAM-containing proteoliposomes were added to the system. Compared with control and liposomes groups, the experimental group showed that Intimin was resistant to proteinase K treatment, indicating that Intimin was successfully translocated.Conclusions:The translocation of Intimin required the participation of BAM complex. An in vitro reconstitution system for inverse autotransporters was constructed in this study, providing a method to study the translocation mechanism of inverse autotransporters.
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@#By regulating gene expression, nucleic acid drugs functioning in the cytoplasm or nucleus are of great significance in the treatment of acquired or inherited diseases and vaccine development.A variety of nucleic acid delivery vectors currently developed are suffering from low transfection efficiency due to endosome/lysosome entrapment.This paper introduces and summarizes the nucleic acid delivery strategies that bypass the endosomal/lysosomal pathway, including membrane translocation, membrane fusion, receptor/transporter-mediated non-endocytic uptake and caveolae-mediated endocytosis, and discusses the problems and challenges facing such strategies, aiming to facilitate the development of intracellular delivery of nucleic acid drugs bypassing lysosomal pathway.
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Objective To explore the role of protein kinase(PKC)in the nosogenesis of hypoxic-ischemic encephalopathy(HIE).Methods Twenty-four seven-day-old SD rats were divided randomly into normal group (N),model control group(C)and HIE group(HIE).The biochemistry techniques of SDS-PAGE and western bolt were used to examine the level of PKC membrane translocation of cortex and hippocampi.Results The level of PKC membrane translocation of cortex and hippocampi in N group was 0.28±0.02,0.29±0.02 respectively,and 0.29±0.02,0.30±0.02 in C group,and 0.38±0.04,0.40±0.06 in group HIE.The level of PKC membrane translocation of cortex and hippocampi was higher in HIE group as compared with that in N,C group(P＜0.01),but there was no different between N group and C group(P＞0.05).Conclusion PKC are activated in HIE,which indicates that PKC may involve in the pathophysiologic mechanism of HIE.
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Objective To explore the role of conventional protein kinase C(cPKCs) in delayed hypoxic preconditioning.Methods The biochemistry techniques of SDS-PAGE,Western bolt and Gel Doc imagine were applied to analyze the effect of repetitive hypoxic exposure(H5,H6) on the level of cPKC?,? membrane translocation and protein expression in murine brain.Results We found that cPKC? protein expression was significantly decreased(P
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Objective To identify certain isoforms involved in the onset of retinal ischemic preconditioning (IPC), the effects of ischemic pretreatment (IP) were observed on level of conventional protein kinase C (cPKC) ?,?Ⅰ、?Ⅱand ? isoform-specific membrane translocation and protein expression in retina of rats. Methods Retinal IP was produced by intra-ocular pressure (IOP) elevation for 5 minutes in anesthetized Wistar rats. Sham operation was similar to IP except the pressure elevation. 10, 20, or 40 minutes and 1, 12, 24, 72 or 168 hours after the procedure, cPKC isoform-specific membrane translocation and protein expression were analyzed using Western-blot. Results cPKC? protein expression significantly increased from 12 h to 168 h. A peak reached at 72 h after IP. cPKC? membrane translocation enhanced during 20 min to 1 hours with a peak at 40 min. cPKC? protein expressionlevels significantly increased from 12 hours to 72 hours. A peak was found at 24 hours after IP. However, there were no significant changes in both membrane translocation of cPKC?, ?Ⅰ、?Ⅱand protein expression of cPKC?Ⅰ, ?Ⅱ in retina of rats following IP.Conclusion The enhanced cPKC? membrane translocation, the increased cPKC? and ? protein expressions might be involved in the onset and sustain of retinal IPC in rats respectively.
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Despite the small size of its genome (3.2 kb) and having only four genes that are encoded within it, the hepatitis B virus (HBV) is one of the most successful viral pathogens in human history. It is estimated that there are about 350-400 million people worldwide who are chronically infected with HBV, and even with the extensive efforts that are being done with preventive vaccination, this malady still remains a clear and present danger to the public health. How is it possible that this small double-stranded DNA virus can escape and outfox the surveillance of the complex human immune system? One explanation is that HBV gene products play multiple roles in infections and throughout the viral life cycle so that the virus can effectively survive under various hostile circumstances. Indeed, the HBV DNA polymerase, for example, exerts several functions such as reverse transcription and RNA degradation, and the HBV X protein not only acts as a transcriptional activator, but it also interferes with the host cells' DNA repair mechanism as well as inducing apoptosis and controlling signal transduction. The HBV surface protein, which is encoded in the env gene, is another intriguing example of such multifunctionality. Thus, our present article overviews and summarizes the multifaceted role of this membrane protein as shown in 1) its role as a structural protein of the virus envelope; 2) its function as the viral ligand for interacting with the viral receptors on host cells; 3) its characteristics as an energy-independent transporter molecule that can mediate the nuclear accumulation of itself and other tagged molecules; 4) its role as a viral transactivator protein that can cause hepatocellular carcinoma; 5) its hypothetical function in viral apoptotic mimicry that results in host anti-inflammatory responses; and last 6) its immunostimulatory property by providing for strong and well-defined B- and T-cell epitopes. Understanding these various functions and the versatility of this single protein will help us decipher and understand the viral- and immuno-pathogenesis of HBV itself.
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Animals , Humans , English Abstract , Hepatitis B Surface Antigens/physiologyABSTRACT
BACKGROUND: This study was aimed to differentiate two forms of CTLA-4 (CD152) in activated peripheral blood lymphocyte and clarify the mechanism how cytoplasmic form of this molecule is targeted to cell surface. METHODS: For this purpose we generated 2 different anti-human CD152 peptide antibodies and 5 different N'-terminal deletion mutant CTLA4Ig fusion proteins and carried out a series of Western blot and ELISA analyses. Antipeptide antibodies made in this study were anti-CTLA4pB and anti- CTLA4pN. The former recognized a region on extracellular single V-like domain and the latter recognized N'-terminal sequence of leader domain of human CD152. RESULTS: In Western blot, the former antibody recognized recombinant human CTLA4Ig fusion protein as an antigen. And this recognition was completely blocked by preincubating antipeptide antibody with the peptide used for the antibody generation at the peptide concentration of 200 ug/ml. These antibodies were recognized human CD152 as a cytoplasmic sequestered- and a membrane bound- forms in phytohemagglutinin (PHA)- stimulated peripheral blood lymphocyte (PBL). These two forms of CD152 were further differentiated by using anti-CTLA4pN and anti-CTLA4pB antibodies such that former recognized cytosolic form only while latter recognized both cytoplasmic- and membrane- forms of this molecule. Furthermore, in a transfection expression study of 5 different N'-terminal deletion mutant CTLA4Ig, mutated proteins were secreted out from transfected cell surface only when more than 6 amino acids from N'-terminal were deleted. CONCLUSION: Our results implies that cytosolic form of CTLA-4 has leader sequence while membrane form of this molecule does not. And also suggested is that at least N'-terminal 6 amino acid residues of human CTLA-4 are required for regulation of targeting this molecule from cytosolic- to membrane- area of activated human peripheral blood T lymphocyte.
Subject(s)
Humans , Amino Acids , Antibodies , Blotting, Western , Cytoplasm , Cytosol , Enzyme-Linked Immunosorbent Assay , Lymphocytes , Membranes , Staphylococcal Protein A , TransfectionABSTRACT
AIM: To explore the role of cPKCs in the development of cerebral hypoxic preconditioning, the effect of repetitive hypoxia on the level of protein kinases C ?I and ?II (cPKC ?I and ?II ) membrane translocation in the brain of mice was observed. METHODS: The hypoxic preconditioned mouse model was adapted with minor modification from our previous report. The biochemistry techniques of SDS-PAGE and Western blotting were applied to determine the level of cPKC ?I and ?II membrane translocation in cortex and hippocampus of mice. RESULTS: cPKC ?II translocated from the cytosolic fraction to the particulate fraction in response to the repetitive hypoxic exposure (H1-H4) both in hippocampus and cortex of mice, which was regarded as membrane translocation. The significant membrane translocation of cPKC ?II was found in hippocampus of H4 group (173.3%?21.3% vs H0: 100% or H1: 79.5%?10.7%, P0.05, n=8). CONCLUSION: These results suggest that cPKC ?II may play an important role in the development of cerebral hypoxic preconditioning, but the changes of novel and typical PKC isoenzymes are still under investigation.
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Objective To construct the vector that expresses the fusion protein of HIV-Tat protein and red fluorescent protein(mCherry) in mammalian cells,and observe by fluorescence microscopy the intracellular transduction and localization of recombinant protein in cells,in order to obtain a useful tool for the study of the uptake mechanism and intracellular localization of HIV-TAT.Methods With the designed primer coding mCherry sequence,the mCherry gene was amplified by PCR with the vector pmCherry-C2 as template,and inserted into vector pET14b-His-TAT to construct the expression vector pET14b-His-TAT-mCherry.The constructed vector was then transformed into E.coli BL21(DE3),which had been identified by PCR and double digested with restriction endonuclease,followed by sequencing.After IPTG induction,the recombinant protein of His-TAT-mCherry was lyzed and analyzed with SDS-PAGE.Purified His-TAT-mCherry recombinant protein was added to Hela cells and the fluorescence was observed to evaluate the transduction efficiency.Results The results of identification by PCR,digestion with restriction endonuclease and sequencing indicated that the vector His-TAT-mCherry was correctly constructed.His-TAT-mCherry fusion protein was expressed in mammalian Hela cell line and purified successfully,and the fusion protein showed cellular transduction activity.It was found by fluorescence microscopy that the red fluorescence protein located mainly over the cytoplasm,and also the membrane to some extent.Conclusion The expression vector is successfully constructed for HIV-TAT labeled with mCherry sequence.Effective expression and purification of this fusion protein is achieved.It has been observed that the constructed vector may be expressed in mammalian Hela cell under active condition.Thus,it might be useful in the study of uptake mechanism and intracellular localization of HIV-TAT.