Induction of endoplasmic reticulum-endosome fusion for antigen cross-presentation induced by poly (γ-glutamic acid) nanoparticles

We previously reported that poly (γ-glutamic acid)-based nanoparticles (γ-PGA NPs) are excellent vaccine carriers for inducing efficient cross-presentation in dendritic cells, thereby producing strong antitumor immunity in vivo. Analyzing the mechanism of cross-presentation induced by γ-PGA NPs will be useful toward designing novel vaccine carriers. In this study, we show an intracellular mechanism of efficient cross-presentation induced by OVA-loaded γ-PGA NPs. Cross-presentation induced by γ-PGA NPs depended on cytoplasmic proteasomes and TAP, similar to the classical MHC class I presentation pathway for endogenous Ags. Intracellular behavior analyzed by confocal laser scanning microscopy revealed that encapsulated OVA and γ-PGA accumulated in both the endoplasmic reticulum (ER) and endosome compartments within 2 h. At the same time, electron microscopy analysis clearly showed that intracellular γ-PGA NPs and encapsulated Au NPs were enveloped in endosome-like vesicles, not in the ER. These findings strongly suggest that γ-PGA NPs enhance ER-endosome fusion for cross-presentation. Moreover, inhibition of ER translocon sec61 significantly decreased the γ-PGA NP/OVA-mediated cross-presentation efficiency, indicating that sec61 is important for transporting Ags from the fused ER-endosome to the cytoplasm. These findings imply that the ER-endosome complex is key for the efficient cross-presentation of Ags encapsulated in γ-PGA NPs.     

Myosin XI-dependent formation of tubular structures from endoplasmic reticulum isolated from tobacco cultured BY-2 cells

The reticular network of the endoplasmic reticulum (ER) consists of tubular and lamellar elements and is arranged in the cortical region of plant cells. This network constantly shows shape change and remodeling motion. Tubular ER structures were formed when GTP was added to the ER vesicles isolated from tobacco (Nicotiana tabacum) cultured BY-2 cells expressing ER-localized green fluorescent protein. The hydrolysis of GTP during ER tubule formation was higher than that under conditions in which ER tubule formation was not induced. Furthermore, a shearing force, such as the flow of liquid, was needed for the elongation/extension of the ER tubule. The shearing force was assumed to correspond to the force generated by the actomyosin system in vivo. To confirm this hypothesis, the S12 fraction was prepared, which contained both cytosol and microsome fractions, including two classes of myosins, XI (175-kD myosin) and VIII (BY-2 myosin VIII-1), and ER-localized green fluorescent protein vesicles. The ER tubules and their mesh-like structures were arranged in the S12 fraction efficiently by the addition of ATP, GTP, and exogenous filamentous actin. The tubule formation was significantly inhibited by the depletion of 175-kD myosin from the S12 fraction but not BY-2 myosin VIII-1. Furthermore, a recombinant carboxyl-terminal tail region of 175-kD myosin also suppressed ER tubule formation. The tips of tubules moved along filamentous actin during tubule elongation. These results indicated that the motive force generated by the actomyosin system contributes to the formation of ER tubules, suggesting that myosin XI is responsible not only for the transport of ER in cytoplasm but also for the reticular organization of cortical ER.     

Catalase overexpression fails to attenuate allergic airways disease in the mouse

Oxidative stress is a hallmark of asthma, and increased levels of oxidants are considered markers of the inflammatory process. Most studies to date addressing the role of oxidants in the etiology of asthma were based on the therapeutic administration of low m.w. antioxidants or antioxidant mimetic compounds. To directly address the function of endogenous hydrogen peroxide in the pathophysiology of allergic airway disease, we comparatively evaluated mice systemically overexpressing catalase, a major antioxidant enzyme that detoxifies hydrogen peroxide, and C57BL/6 strain matched controls in the OVA model of allergic airways disease. Catalase transgenic mice had 8-fold increases in catalase activity in lung tissue, and had lowered DCF oxidation in tracheal epithelial cells, compared with C57BL/6 controls. Despite these differences, both strains showed similar increases in OVA-specific IgE, IgG1, and IgG2a levels, comparable airway and tissue inflammation, and identical increases in procollagen 1 mRNA expression, following sensitization and challenge with OVA. Unexpectedly, mRNA expression of MUC5AC and CLCA3 genes were enhanced in catalase transgenic mice, compared with C57BL/6 mice subjected to Ag. Furthermore, when compared with control mice, catalase overexpression increased airway hyperresponsiveness to methacholine both in naive mice as well as in response to Ag. In contrast to the prevailing notion that hydrogen peroxide is positively associated with the etiology of allergic airways disease, the current findings suggest that endogenous hydrogen peroxide serves a role in suppressing both mucus production and airway hyperresponsiveness.     

Identification of radicals formed in the reaction mixtures of rat liver microsomes with ADP, Fe3+ and NADPH using HPLC EPR and HPLC EPR MS

The reaction of rat liver microsomes with Fe(3+), ADP and NADPH was examined using EPR, HPLC-EPR and HPLC-EPR-MS combined use of spin trapping technique. A prominent EPR spectrum (alpha(N) = 1.58 mT and alpha(H)beta = 0.26 mT) was observed in the complete reaction mixture. The EPR spectrum was hardly observed for the complete reaction mixture without rat liver microsomes. The radicals appear to be derived from microsomal components. The EPR spectrum was also hardly observed in the absence of Fe(3+). Addition of some iron chelators such as EDTA, citrate and ADP resulted in the dramatic change in the EPR intensity. Iron ions seem to be essential for this reaction. For the complete reaction mixture with boiled microsomes, a weak EPR spectrum was observed, suggesting that enzymes participate in the reaction. Five peaks were separated on the HPLC-EPR elution profile of the complete reaction mixture of rat liver microsomes with ADP, Fe(3+) and NADPH. The retention times of the peaks 1 to 5 were 19.4, 22.5, 27.3, 29.8 and 31.4 min, respectively. To identify the radical adducts, HPLC-EPR-MS analyses were performed for the three prominent peaks. The HPLC-EPR-MS analyses showed that a new radical adduct, 4-POBN/1-hydroxypentyl radical, in addition to 4-POBN/ethyl radical adducts, forms in a reaction mixture of rat liver microsomes with ADP, Fe(3+) and NADPH.     

Selective desensitization of growth factor signaling by cell adhesion to fibronectin

Cell adhesion to the extracellular matrix is required to execute growth factor (GF)-mediated cell behaviors, such as proliferation. A major underlying mechanism is that cell adhesion enhances GF-mediated intracellular signals, such as extracellular signal-regulated kinase (Erk). However, because GFs use distinct mechanisms to activate Ras-Erk signaling, it is unclear whether adhesion-mediated enhancement of Erk signaling is universal to all GFs. We examined this issue by quantifying the dynamics of Erk signaling induced by epidermal growth factor, basic fibroblast growth factor (bFGF), and platelet-derived growth factor (PDGF) in NIH-3T3 fibroblasts. Adhesion to fibronectin-coated surfaces enhances Erk signaling elicited by epidermal growth factor but not by bFGF or PDGF. Unexpectedly, adhesion is not always a positive influence on GF-mediated signaling. At critical subsaturating doses of PDGF or bFGF, cell adhesion ablates Erk signaling; that is, adhesion desensitizes the cell to GF stimulation, rendering the signaling pathway unresponsive to GF. Interestingly, the timing of growth factor stimulation proved critical to the desensitization process. Erk activation significantly improved only when pre-exposure to adhesion was completely eliminated; thus, concurrent stimulation by GF and adhesion was able to partially rescue adhesion-mediated desensitization of PDGF- and bFGF-mediated Erk and Akt signaling. These findings suggest that adhesion-mediated desensitization occurs with rapid kinetics and targets a regulatory point upstream of Ras and proximal to GF receptor activation. Thus, adhesion-dependent Erk signaling is not universal to all GFs but, rather, is GF-specific with quantitative features that depend strongly on the dose and timing of GF exposure.     

Epigenetic Modulations Induction Using DSCR1 Ectopic Expression in Breast Cancer Cells

Today, prognosis, diagnosis and treatment of cancers are progressing with non-invasive methods, including investigation and modification of the DNA methylation profile in cancer cells. One of the effective factors in regulating gene expression in mammals is DNA methylation. Methylation alterations of genes by external factors can change the expression of genes and inhibit the cancer. In the present study, we investigated the effect of Down syndrome critical region 1 gene (DSCR1) ectopic expression on the methylation status of the BCL-XL, ITGA6, TCF3, RASSF1A, DOK7, VIM and CXCR4 genes in breast cancer cell lines. The effect of DSCR1 ectopic expression on cell viability in MCF7, MDA-MB-468, MDA-MB-231 and MCF10A cell lines was evaluated using MTT assay after the cells treated by lentivirus vectors harboring DSCR1 for 72 hours. Methylation status of BCL-XL, ITGA6, TCF3, RASSF1A, DOK7, VIM and CXCR4 genes in breast cancer cell lines was assessed by Restriction Enzyme PCR (REP) method. Also, methylation changes of these genes in breast cancer cell lines after treatment by lentivirus vectors harboring DSCR1 for 7 days were analyzed by REP method. To confirm the effect of DSCR1 on methylation of genes, Real-time PCR was performed. The MTT assay results indicated that DSCR1 ectopic expression reduced cell viability in all three human breast cancer cell lines. Our results showed that DSCR1 ectopic expression after 6 days reversed the hypomethylation status of the BCL-XL, ITGA6, TCF3, VIM and CXCR4 genes and hypermethylation of RASSF1A and DOK7 genes. The expression levels of BCL-XL, ITGA6, TCF3, VIM and CXCR4 mRNA significantly reduced (P<0.05) and the expression levels of RASSF1A and DOK7 mRNA significantly increased (P<0.05). Our findings reveal for the first time the impact of DSCR1 ectopic expression on the methylation status of breast cancer cells and identify a novel agent for epigenetic therapy.     

Loss-of-Function GAS8 Mutations Cause Primary Ciliary Dyskinesia and Disrupt the Nexin-Dynein Regulatory Complex

Multiciliated epithelial cells protect the upper and lower airways from chronic bacterial infections by moving mucus and debris outward. Congenital disorders of ciliary beating, referred to as primary ciliary dyskinesia (PCD), are characterized by deficient mucociliary clearance and severe, recurrent respiratory infections. Numerous genetic defects, most of which can be detected by transmission electron microscopy (TEM), are so far known to cause different abnormalities of the ciliary axoneme. However, some defects are not regularly discernable by TEM because the ciliary architecture of the axoneme remains preserved. This applies in particular to isolated defects of the nexin links, also known as the nexin-dynein regulatory complex (N-DRC), connecting the peripheral outer microtubular doublets. Immunofluorescence analyses of respiratory cells from PCD-affected individuals detected a N-DRC defect. Genome-wide exome sequence analyses identified recessive loss-of-function mutations in GAS8 encoding DRC4 in three independent PCD-affected families.     

Expression of the Novel Hepatitis C Virus Core+1/ARF Protein in the Context of JFH1-Based Replicons

Hepatitis C virus contains a second open reading frame within the core gene, designated core+1/ARF. Here we demonstrate for the first time expression of core+1/ARF protein in the context of a bicistronic JFH1-based replicon and report the production of two isoforms, core+1/L (long) and core+1/S (short), with different kinetics.