Clathrin-mediated Pathway Research Articles

Haemolysin Sph2 of Leptospira interrogans induces cell apoptosis via intracellular reactive oxygen species elevation and mitochondrial membrane injury.

Leptospira interrogans causes widespread leptospirosis in humans and animals, with major symptoms of jaundice and haemorrhage. Sph2, a member of the sphingomyelinase haemolysins, is an important virulence factor for leptospire. In this study, the function and mechanism of Sph2 in the pathogenesis of leptospirosis were investigated to further understand the pathogenesis of leptospire. Real-time PCR analysis of expression levels during cell invasion showed that sph2 gene expression was transiently induced in human umbilical vein endothelial cells (HUVECs), human embryo liver cells (L02), and human epithelial lung cells (L132), with expression levels reaching a peak after 45min of infection. Further functional analysis of recombinant Sph2 (rSph2) by LDH assays and confocal microscopy showed that rSph2 can be internalised by cells both by causing cell membrane damage and by a damage-independent clathrin-mediated endocytosis pathway. Subsequently, rSph2 is able to translocate to mitochondria, which led to an increase in the levels of reactive oxygen species (ROS) and a decrease of the mitochondrial membrane potential (ΔΨm ). Further flowcytometry analyses after rSph2 exposure showed that 28.7%, 31%, and 27.3% of the HUVEC, L02, and L132 cells, respectively, became apoptotic. Because apoptosis could be decreased with the ROS inhibitor N-acetyl cysteine, these experiments suggested that rSph2 triggers apoptosis through mitochondrial membrane damage and ROS elevation. The ability of leptospiral haemolysin rSph2 to cause apoptosis likely contributes to the pathogenesis of leptospirosis.

Functionalized selenium nanoparticles for targeted delivery of doxorubicin to improve non-small-cell lung cancer therapy.

BackgroundSelenium nanoparticles (SeNPs) loaded with chemotherapeutic drugs provided a novel perspective for cancer therapy.Materials and methodsHere, SeNPs were modified with cyclic peptide (Arg–Gly–Asp–d-Phe–Cys [RGDfC]) to fabricate tumor-targeting delivery carrier RGDfC-SeNPs and, then, doxorubicin (DOX) was loaded to the surface of RGDfC-SeNPs for improving the antitumor efficacy of DOX in non-small-cell lung carcinoma therapy.ResultsThe chemical structure characterization of RGDfC-Se@DOX showed that DOX was successfully loaded to the surface of RGDfC-SeNPs to prepare functionalized antitumor drug delivery system RGDfC-Se@DOX. RGDfC-Se@DOX exhibited effective cellular uptake in A549 cells and entered A549 cells mainly by clathrin-mediated endocytosis pathway. Compared to free DOX or Se@DOX at the equivalent dose of DOX, RGDfC-Se@DOX showed greater activity to inhibit A549 cells’ proliferation and migration/invasion and induce A549 cells’ apoptosis. More importantly, compared with passive targeting delivery system Se@DOX, active targeting delivery system RGDfC-Se@DOX exhibited more significant antitumor efficacy in vivo.ConclusionTaken together, RGDfC-Se@DOX may be a novel promising drug candidate for the lung carcinoma therapy.

Iron Oxide Magnetie-Polyethylene Glycol-Layered Double Hydroxide Core Shell Nanoparticles for Drug Delivery

Recently, micro- and nanoparticles with magnetic core and functional shell structures have received much attention because of their potential applications in drug delivery, biosensors catalysis, selective separation and chromatography etc. Layered double hydroxides (LDHs) nanoparticles (NPs) are an efficient cellular delivery vehicle for many negatively charged chemical species. Recently, Mg and Zn/Al-layered double hydroxide (Mg/Al-LDH) and (Zn/Al-LDH) has emerged as a promising nanocarrier for drug delivery system due to its unique properties such as low cytotoxicity, pH-controlled release, good biocompatibility, tunable particle size, and protection of drugs in the interlayer. Various anionic biofunctional molecules, including DNA, SiRNA, drugs and vitamins have been successfully intercalated into LDH interlayers, protected from enzyme degradation and delivered into the cytoplasm in mammalian cells through the clathrin-mediated endocytosis pathway. In this study, a magnetite core shell nanodrug delivery formulation was designed based on Fe3O4-PEG-Zn/Al-LHDs or Mg/Al-LDHs loaded with anticancer drug 5-Fluoro-2,4(1H,3H)-pyrimidinedione commonly called 5-fluorouracil. Designed core shell magnetite nanoparticles formulation was characterized using XRD, zeta sizer and high performance liquid chromatography (HPLC).

Pea Ferritin Stability under Gastric pH Conditions Determines the Mechanism of Iron Uptake in Caco-2 Cells

BackgroundIron deficiency is an enduring global health problem that requires new remedial approaches. Iron absorption from soybean-derived ferritin, an ∼550-kDa iron storage protein, is comparable to bioavailable ferrous sulfate (FeSO4). However, the absorption of ferritin is reported to involve an endocytic mechanism, independent of divalent metal ion transporter 1 (DMT-1), the transporter for nonheme iron.ObjectiveOur overall aim was to examine the potential of purified ferritin from peas (Pisum sativum) as a food supplement by measuring its stability under gastric pH treatment and the mechanisms of iron uptake into Caco-2 cells.MethodsCaco-2 cells were treated with native or gastric pH–treated pea ferritin in combination with dietary modulators of nonheme iron uptake, small interfering RNA targeting DMT-1, or chemical inhibitors of endocytosis. Cellular ferritin formation, a surrogate measure of iron uptake, and internalization of pea ferritin with the use of specific antibodies were measured. The production of reactive oxygen species (ROS) in response to equimolar concentrations of native pea ferritin and FeSO4 was also compared.ResultsPea ferritin exposed to gastric pH treatment was degraded, and the released iron was transported into Caco-2 cells by DMT-1. Inhibitors of DMT-1 and nonheme iron absorption reduced iron uptake by 26–40%. Conversely, in the absence of gastric pH treatment, the iron uptake of native pea ferritin was unaffected by inhibitors of nonheme iron absorption, and the protein was observed to be internalized in Caco-2 cells. Chlorpromazine (clathrin-mediated endocytosis inhibitor) reduced the native pea ferritin content within cells by ∼30%, which confirmed that the native pea ferritin was transported into cells via a clathrin-mediated endocytic pathway. In addition, 60% less ROS production resulted from native pea ferritin in comparison to FeSO4.ConclusionWith consideration that nonheme dietary inhibitors display no effect on iron uptake and the low oxidative potential relative to FeSO4, intact pea ferritin appears to be a promising iron supplement.

Antilung cancer effect of ergosterol and cisplatin-loaded liposomes modified with cyclic arginine-glycine-aspartic acid and octa-arginine peptides.

Lung cancer is one of the most important diseases threatening human health, and targeted therapy has become the main research direction. This work, therefore, aimed to develop cyclic arginine-glycine-aspartic (RGD) and octa-arginine (R8) peptide-modified ergosterol (ERG)-combined cisplatin (diamminedichloridoplatinum(II) [DDP]) liposomes (LIP) as a drug delivery system. Soybean phospholipids (SPC) and cholesterol (Chol) were selected to prepare different LIPs: ERG-loaded LIP (ERG-LIP), DDP and ERG-LIP (DDP/ERG-LIP), R8 peptide-modified DDP and ERG-LIP (R8-DDP/ERG-LIP), and cyclic RGD and R8-DDP/ERG-LIP (RGD/R8-DDP/ERG-LIP). The quality, tumor sphere penetrating ability, in vitro cellular uptake, mechanism of cellular uptake, and in vitro cytotoxicity of RGD/R8-DDP/ERG-LIP were evaluated. The LIP quality evaluation revealed that RGD/R8-DDP/ERG-LIP is round with a double-layer structure. The average particle size, dispersion coefficient of the polydispersity index (PDI), and zeta potential of RGD/R8-DDP/ERG-LIP were 155.2 ± 8.7 nm, 0.102, and 4.74 ± 0.7 mV, respectively. Furthermore, the LIPs were stable in the serum, and obviously inhibited the growth of A549 lung cancer cells with RGD/R8-DDP/ERG-LIP exhibiting the strongest inhibitory effect. The highest cellular uptake rate, which was at 4 hours, was exhibited by RGD/R8-DDP/ERG-LIP in a concentration-dependent manner. The results showed that LIP uptake by A549 cells was mainly by the clathrin-mediated endocytosis pathway (chlorpromazine). The results also suggest that RGD/R8-DDP/ERG-LIP might be a promising drug delivery system to improve antilung cancer drug effect and tumor-targeting in vitro.

Bone-targeted delivery of simvastatin-loaded PEG-PLGA micelles conjugated with tetracycline for osteoporosis treatment.

This study aimed to investigate the improved therapeutic efficacy and pharmacokinetic profiles of simvastatin (SIM) with imparted bone targeting potential using tetracycline-mediated PEG-PLGA (TC-PEG-PLGA) micelles in osteoporotic rats. The SIM-loaded TC-PEG-PLGA (TC-PEG-PLGA/SIM) micelles were evaluated for particle size, morphology, stability, loading efficiency, cell viability, bone mineral binding ability in vitro, mineralization, pharmacokinetics, and pharmacodynamics. TC-PEG-PLGA conjugates were successfully and could self-assembly form micelles in aqueous medium with a 19.4μg/mL critical micelle concentration. Then, TC-PEG-PLGA/SIM micelles were prepared with solvent diffusion method, and the obtained micelles (56.21 ± 7.39nm average size; 81.8 ± 3.1% encapsulation efficiency; and 7.56 ± 0.27% drug loading) led to the prolonged release of SIM from micelles. Cellular uptake test indicated that TC had no effects on micellar internalization and micellar internalization was mainly involved with clathrin-mediated endocytic pathway. In vivo pharmacokinetic results indicated that TC-PEG-PLGA/SIM micelles exhibited a significantly prolonged time in systemic circulation and were preferentially accumulated in bone tissue. TC-PEG-PLGA/SIM micelles showed better therapeutic effects, as reflected by the improved bone mineral density, bone mineral content, and bone mechanical strength. Overall, these results suggested that TC-PEG-PLGA/SIM micelles provide several advantages, including prolonged systemic circulation, enhanced bone tissue distribution, and improved therapeutic outcomes in osteoporotic rats.

Oxidized phospholipids are ligands for LRP6

Low-density lipoprotein receptor–related protein 6 (LRP6) is a co-receptor for Wnt signaling and can be recruited by multiple growth factors/hormones to their receptors facilitating intracellular signaling activation. The ligands that bind directly to LRP6 have not been identified. Here, we report that bioactive oxidized phospholipids (oxPLs) are native ligands of LRP6, but not the closely related LRP5. oxPLs are products of lipid oxidation involving in pathological conditions such as hyperlipidemia, atherosclerosis, and inflammation. We found that cell surface LRP6 in bone marrow mesenchymal stromal cells (MSCs) decreased rapidly in response to increased oxPLs in marrow microenvironment. LRP6 directly bound and mediated the uptake of oxPLs by MSCs. oxPL-LRP6 binding induced LRP6 endocytosis through a clathrin-mediated pathway, decreasing responses of MSCs to osteogenic factors and diminishing osteoblast differentiation ability. Thus, LRP6 functions as a receptor and molecular target of oxPLs for their adverse effect on MSCs, revealing a potential mechanism underlying atherosclerosis-associated bone loss.

Both ganglioside GM2 and cholesterol in the cell membrane are essential for Bombyx mori cypovirus cell entry

Bombyx mori cypovirus (BmCPV) enters permissive cells via clathrin-mediated endocytosis pathway. However, the distinct entry mechanism for BmCPV is still ambiguous. The aim of this study is to investigate the role of gangliosides and cholesterol in BmCPV cell entry. The number of BmCPV virions attached to the cell surface and the expression level of BmCPV vp1 gene was significantly decreased by digestion of terminal sialic acids in gangliosides with neuraminidase (NA). Preincubation of different concentration of ganglioside GM1, GM2 or GM3 with BmCPV prior to infection, the reduction of BmCPV infectivity was found by GM2-treated in a dose-depend manner. BmCPV virions were found to colocalize with GM2 in the cell surface. The infectivity of BmCPV was reduced by anti-GM2 antibody treatment cells. Moreover, BmCPV infection was impaired by depletion of membrane cholesterol with MβCD, but the inhibitory effect of MβCD was restored by supplementing with cholesterol. The number of viral particles attached on the BmN cells was significantly decreased by pretreated with MβCD, and BmCPV infection was inhibited by silencing the expression of 3-hydroxy-3-methylglutaryl-CoA reductase gene (Hmg-r) in cholesterol biosynthesis pathway. These results indicate that ganglioside GM2 and cholesterol in membrane lipid rafts are essential for BmCPV attachment to cell surface for its cell entry.

Polysaccharide-modified nanoparticles with intelligent CD44 receptor targeting ability for gene delivery.

BackgroundHyaluronic acid (HA) and chondroitin sulfate (CD) are endogenous polysaccharides. In recent years, they have aroused the interest of scientists because of specific binding to CD44 receptors, which are overexpressed in several types of tumors.MethodsIn this study, HA- and CD-modified poly(D,L-lactide-co-glycolide)-poly(ethylene glycol) (PLGA-PEG) copolymers were synthesized and applied to encapsulate 1,2-Dioleoyl-3-trimethylammonium-propane (DOTAP)/pDNA (D/P) lipoplex as CD44 receptor targeting gene delivery nanoparticles (NPs).ResultsThe particle size of CD-PEG-PLGA-D/P (186.8 ± 21.7 nm) was smaller than that of HA-PEG-PLGA-D/P (270.2 ± 13.8 nm), with narrow size distribution, and both HA-PEG-PLGA-D/P NPs and CD-PEG-PLGA NPs possessed negative zeta potentials (−39.63 ± 5.44 mV and −38.9 ± 2.0 mV, respectively), which prevent erythrocytes from agglutination. Both NPs exhibited pH-dependent release and had faster release in pH 4.0 than in pH 7.4. Generally, the CD-PEG-PLGA-D/P NPs possessed less cytotoxicity than HA-PEG-PLGA-D/P NPs. The D/P-loaded HA-PEG-PLGA and CD-PEG-PLGA NPs expressed significantly higher transfection in CD44 high-expressed U87 (30.1% ± 2.1% and 40.7% ± 4.3%, respectively) than in CD44-negative HepG2 (3.3% ± 1.5% and 1.4% ± 1.0%, respectively) (p < 0.001). It was revealed that the endocytosis of HA-PEG-PLGA-D/P NPs was majorly dominated by macropinocytosis and the endocytosis of CD-PEG-PLGA-D/P NPs was dominated by clathrin-mediated endocytosis pathway (p < 0.001).ConclusionThe high selectivity to CD44-positive U87 cancer cells and low cytotoxicity in L929 normal cells assured the promising potential of CD-PEG-PLGA NPs as gene delivery nano-carriers.

Epidermal growth factor receptor is a co-factor for transmissible gastroenteritis virus entry

Transmissible gastroenteritis virus (TGEV) causes severe diarrhea and high mortality in newborn piglets. It is well established that porcine intestinal epithelium is the target of the TGEV infection, however the mechanism that TGEV invades the host epithelium remains largely unknown. Aminopeptidase N (APN) is a known receptor of TGEV. This study discovered that the extracellular receptor binding domain 1 pertaining to epidermal growth receptor (EGFR) interact with TGEV spike protein. APN and EGFR synergistically promote TGEV invasion. TGEV promotes APN and EGFR clustering early in infection. Furthermore APN and EGFR synergistically stimulate PI3K/AKT as well as MEK/ERK1/2 endocytosis signaling pathways. TGEV entry is via clathrin and caveolin mediated endocytosis in IPEC-J2 cells. TGEV binds with EGFR, and subsequently promotes EGFR internalization by a clathrin-mediated endocytosis pathway. These results show that EGFR is a co-factor of TGEV, and that it plays a synergistic role with APN early in TGEV infection.

Clathrin-mediated endocytosis is a candidate entry sorting mechanism for Bombyx mori cypovirus

Bombyx mori cypovirus (BmCPV), a member of the Reoviridae, specifically infects silkworms and causes extensive economic losses to the sericulture industry. To date, the entry mechanism of BmCPV into cells is unclear. Here we used electron microscopy to study the route of entry of BmCPV into cells, and the results demonstrated that the entry of BmCPV into BmN cells was mediated by endocytosis. Blocking the entry pathway with four endocytosis inhibitors, including dansylcadaverine, chlorpromazine, genistein, and PP2, significantly decreased the infectivity of BmCPV. This indicates that BmCPV enters BmN cells via endocytosis, and that clathrin-mediated sorting is the predominant entry method. After the relative expression levels of clathrin heavy chain (clathrin, GenBank accession No. NM_001142971.1) and the adaptor protein complex-1 gamma subunit AP-1 (AP-1, GenBank accession No. JQ824201.1), which are involved in clathrin-mediated endocytosis, were inhibited by RNA interference or abolishing the functions of clathrin and AP-1 with their corresponding antibodies, the infectivity of BmCPV was reduced significantly, which suggests that clathrin-mediated endocytosis contributed to the entry of BmCPV into cells. Our findings suggest that the clathrin-mediated endocytosis pathway is a candidate for the development of therapeutics for silkworm cytoplasmic polyhedrosis.

Single arginine modified chitosan as gene carriers

Objective To explore a new method for synthesizing arginine modified chitosan (AC) with mono-arginine substitution and high degree of substitution, and to evaluate the biological effect of AC as gene carriers. Methods The single arginine modified chitosan (sAC) was synthesized by means of protecting and de-protecting the arginine amino group before and after chitosan modified arginine reaction. Liu's arginine-modified chitosan (LAC) was prepared according to the methods reported in the literature. The conjunction of arginine to chitosan was detected by infrared spectroscopy and nuclear magnetic resonance spectroscopy. Three kinds of chitosan gene nanoparticles were respectively prepared by complex coacervation and characterized, including sAC gene nanoparticles (sACGNs), the LAC gene nanoparticles (LACGNs) and the chitosan gene nanoparticles (CGNs). A10 rat vascular smooth cells transfected with sACGNs were used to estimate the in vitro cellular uptake, internalization mechanisms and transfection efficiency. Thiazolyl blue tetrazolium blue (MTT) assay was used to measure the cytotoxicity. Results The infrared spectrum analysis confirmed that sAC was obtained via the conjunction of arginine to chitosan. Nuclear magnetic resonance spectrum analysis showed that the degree of substitution of arginine in sAC and LAC was 21.3% and 6.4%, respectively. When the ratio of nitrogen to phosphorus (N/P ratio) was 2:1, the particle sizes of CGN, LACGN, and sACGN were (94.81±2.93) nm, (124.53±2.55) nm, and (128.53±2.04) nm, respectively, and the Zeta potentials were (3.50±1.61) mV, (5.74±0.41) mV, and (6.04±1.39) mV, respectively. For the cellular uptake, CGNs were mainly through the clathrin-mediated endocytic pathway, and LACGNs and sACGN were mainly through the caveolin-mediated endocytic pathway. Compared with CGNs, LACGNs and sACGNs showed higher cellular uptake and transfection efficiency, and the differences were statistically significant (all P<0.05). sACGNs achieved the highest transfection efficiency in the near-neutral pH environment. MTT results showed that when the mass concentration of sACGNs reached 100 μg/ml, the survival rate of A10 cells was still higher than 90%, indicating the non-cytotoxicity of sACGNs. Conclusion The new method successfully synthesized single arginine-modified chitosan. As a gene carrier, sACGNs show higher gene transfection efficiency and lower cytotoxicity than CGNs and LACGNs in near neutral pH environment. Key words: Chitosan; Single arginine modified chitosan; Gene carrier; Nanoparticles

Effective intracellular delivery and Th1 immune response induced by ovalbumin loaded in pH-responsive polyphosphazene polymersomes

A polymersome system for delivering protein antigen ovalbumin (OVA) based on amphiphilic polyphosphazene grafting with N,N-diisopropylethylenediamine (DPA) and poly(ethylene glycol) (PEG) groups (poly[(DPA)m (PEG)n phosphazene], PEDP) was designed and constructed. The 200-240 nm-size OVA-loaded polymersomes displayed high stability at physiological pH, slow internalization through clathrin-mediated endocytosis pathway, and then a pH-triggered sustained OVA release in acidic environment, leading to extensive antigen access to cytosol. Prime-boost vaccine kept high antibody titers for 8 weeks and the subcutaneous vaccine of OVA polymersomes biased the immune response towards a type 1 T helper (Th1) response. Animal experiment results showed that the antigen-specific prophylactic vaccination by PEDP polymersomes delivery was much more rapid and efficient in depressing tumor growth and progress when compared with the therapeutic vaccination. These results suggested that PEDP-based polymersomes are very promising in controlled cytosolic delivery of protein antigens, and enhanced Th1 specific immune response.

Intracellular distribution and internalization pathways of guanidinylated bioresponsive poly(amido amine)s in gene delivery

Guanidinylated bioresponsive poly(amido amine)s polymers, CAR-CBA and CHL-CBA, were synthesized by Michael-type addition reaction between guanidine hydrochloride (CAR) or chlorhexidine (CHL) and N,N′-cystaminebisacrylamide (CBA). Previous studies have shown that both polymers had high transfection efficiencies as gene delivery carriers. In this study, we investigated the nucleolus localization abilities and cellular internalization pathways of these two polymers in gene delivery. Each polymer condensed plasmid DNA (pDNA) and formed nanoparticle complexes, and then their transfection studies were performed in MCF-7 cells. Both complexes were found enriched in nucleolus after cellular transfection, and their transfection efficiencies were significantly improved when transfection was performed on MCF-7 cells arrested at M phase. The transfection efficiency of CAR-CBA-pDNA was inhibited by chlorpromazine, and cell endosomes were disrupted after being exposed to CAR-CBA-pDNA. In regards to CHL-CBA-pDNA, its transfection efficiency was not affected by three types of endocytosis inhibitors used in the study, and CHL-CBA-pDNA showed no effect on endosomes. Cellular lactate dehydrogenase release and membrane morphology were changed after cells were transfected by the two complexes. The results indicated that both CAR-CBA and CHL-CBA polymers demonstrated good nucleolus localization abilities. It was beneficial for transfection when cells were arrested at M phase. CAR-CBA-pDNA cellular internalization was involved with clathrin-mediated endocytosis pathway, and escaping from endosomal entrapment, while the cellular uptake of CHL-CBA-pDNA occurs via clathrin- and caveolae-independent mechanism.

Dileucine-like motifs in the C-terminal tail of connexin32 control its endocytosis and assembly into gap junctions.

Defects in assembly of gap junction-forming proteins, called connexins (Cxs), are observed in a variety of cancers. Connexin32 (Cx32; also known as GJB1) is expressed by the polarized cells in epithelia. We discovered two dileucine-based motifs, which govern the intracellular sorting and endocytosis of transmembrane proteins, in the C-terminal tail of Cx32 and explored their role in regulating its endocytosis and gap junction-forming abilities in pancreatic and prostate cancer cells. One motif, designated as LI, was located near the juxtamembrane domain, whereas the other, designated as LL, was located distally. We also discovered a non-canonical motif, designated as LR, in the C-terminal tail. Our results showed that rendering these motifs non-functional had no effect on the intracellular sorting of Cx32. However, rendering the LL or LR motif nonfunctional enhanced the formation of gap junctions by inhibiting Cx32 endocytosis by the clathrin-mediated pathway. Rendering the LI motif nonfunctional inhibited gap junction formation by augmenting the endocytosis of Cx32 via the LL and LR motifs. Our studies have defined distinct roles of these motifs in regulating the endocytosis of Cx32 and its gap junction-forming ability.This article has an associated First Person interview with the first author of the paper.

Sterols regulate endocytic pathways during flg22-induced defense responses in Arabidopsis.

The plant transmembrane receptor kinase FLAGELLIN SENSING 2 (FLS2) is crucial for innate immunity. Although previous studies have reported FLS2-mediated signal transduction and endocytosis via the clathrin-mediated pathway, whether additional endocytic pathways affect FLS2-mediated defense responses remains unclear. Here, we show that the Arabidopsis thaliana sterol-deficient mutant steroid methyltransferase 1 displays defects in immune responses induced by the flagellin-derived peptide flg22. Variable-angle total internal reflection fluorescence microscopy (VA-TIRFM) coupled with single-particle tracking showed that the spatiotemporal dynamics of FLS2-GFP changed on a millisecond time scale and that the FLS2-GFP dwell time at the plasma membrane increased in cells treated with a sterol-extracting reagent when compared with untreated counterparts. We further demonstrate that flg22-induced FLS2 clustering and endocytosis involves the sterol-associated endocytic pathway, which is distinct from the clathrin-mediated pathway. Moreover, flg22 enhanced the colocalization of FLS2-GFP with the membrane microdomain marker Flot 1-mCherry and FLS2 endocytosis via the sterol-associated pathway. This indicates that plants may respond to pathogen attacks by regulating two different endocytic pathways. Taken together, our results suggest the key role of sterol homeostasis in flg22-induced plant defense responses.

Proteomic identification of turkey (Meleagris gallopavo) seminal plasma proteins ,

SDS-PAGE combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) and 2-dimensional electrophoresis (2DE) combined with matrix-assisted laser desorption/ionization time of flight/time of flight mass spectrometry (MALDI TOF/TOF) were applied to characterize the turkey seminal plasma proteome. LC-MS/MS led to the identification of 175 proteins, which were classified according to their function and to corresponding biochemical pathways. Using 2DE and MALDI TOF/TOF, 34 different turkey seminal plasma proteins could be identified, of which 20 were found in more than one spot, indicating different proteoforms of these proteins. For validation, antibodies against turkey albumin and ovoinhibitor as well as sperm acrosin were used in 2DE Western blots experiments. The bioinformatic analysis of the results indicates that turkey seminal plasma proteins may be involved in regulation of lipid metabolism [liver X receptor/retinoid X receptor (LXR/RXR) activation and farnesoid X receptor/retinoid X receptor (FXR/RXR) activation pathways)], endocytic entry of proteins and lipids at the plasma membrane (clathrin-mediated endocytosis pathway), and defense against pathogens (acute phase response signaling pathway) and energy production (glycolysis and gluconeogenesis). Moreover, a comparative meta-analysis of seminal plasma proteomes from other species indicated the presence of proteins specific for avian reproduction, but distinct differences between turkey and chicken seminal plasma proteomes were detected. The results of our study provide basic knowledge of the protein composition of turkey seminal plasma highlighting important physiological pathways which may play crucial roles in the sperm environment after ejaculation. This knowledge can be the basis to further develop procedures improving the reproduction of farmed turkeys.

Intracellular disposition of chitosan nanoparticles in macrophages: intracellular uptake, exocytosis, and intercellular transport.

Biodegradable nanomaterials have been widely used in numerous medical fields. To further improve such efforts, this study focused on the intracellular disposition of chitosan nanoparticles (CsNPs) in macrophages, a primary cell of the mononuclear phagocyte system (MPS). Such interactions with the MPS determine the nanoparticle retention time in the body and consequently play a significant role in their own clinical safety. In this study, various dye-labeled CsNPs (about 250 nm) were prepared, and a murine macrophage cell line (RAW 264.7) was selected as a model macrophage. The results showed two mechanisms of macrophage incorporation of CsNPs, ie, a clathrin-mediated endocytosis pathway (the primary) and phagocytosis. Following internalization, the particles partly dissociated in the cells, indicating cellular digestion of the nanoparticles. It was proved that, after intracellular uptake, a large proportion of CsNPs were exocytosed within 24 h; this excretion induced a decrease in fluorescence intensity in cells by 69%, with the remaining particles possessing difficulty being cleared. Exocytosis could be inhibited by both wortmannin and vacuolin-1, indicating that CsNP uptake was mediated by lysosomal and multivesicular body pathways, and after exocytosis, the reuptake of CsNPs by neighboring cells was verified by further experiments. This study, thus, elucidated the fate of CsNPs in macrophages as well as identified cellular disposition mechanisms, providing the basis for how CsNPs are recognized by the MPS; such information is crucial to numerous medical applications of CsNPs.

Variable Heavy Chain Domain Derived from a Cell-Penetrating Anti-DNA Monoclonal Antibody for the Intracellular Delivery of Biomolecules

ABSTRACTTargeting and modification of important intracellular proteins using efficient vehicles are invaluable in diagnostic and therapeutic fields. Cell-penetrating antibodies and their fragments can be utilized as vehicles for the delivery of modifiers into cells. In this study, we explored the applicability of variable heavy chain (VH) domain as delivery vehicles for mammalian cells. The characteristics of the recombinant VH domain produced from a cell-penetrating monoclonal anti-double stranded DNA antibody 2C10 were analyzed using flow cytometry, confocal microscopy, cell proliferation assay, and cell cycle analysis in various mammalian cell lines. The VH domain penetrated into various cell lines in a time- and dose-dependent manner, although the internalization efficiency varied. The domain was localized in the nuclei as well as the cytoplasm of living cells. It was also internalized into cells mainly through the clathrin-mediated endocytosis pathway. We tested further its efficiency in delivering specific biomolecule(s) using the conjugates of the single domain molecule and small interfering RNA (siRNA) for the testicular nuclear auto-antigenic sperm protein (tNASP). It was found that the siRNA was successfully delivered by the VH domain into cancer cells, and knockdown effects from the delivered tNASP-siRNA were observed. The levels of the RNA transcript and protein of tNASP were decreased and the down-regulated tNASP inhibited cell proliferation and caused G0G1 phase arrest of the cell cycle. These results indicate that the recombinant 2C10 VH domain could be applied as an efficient vehicle capable of delivering valuable biomolecule into the cytoplasm or cell nuclei for clinical uses.

Enhanced cellular uptake and gene silencing activity of siRNA using temperature-responsive polymer-modified liposome

Short interfering RNA (siRNA) delivery systems using nanoparticle carriers have been limited by inefficient intracellular delivery. One drawback is the poor cellular uptake of siRNA/particle complexes through the plasma membrane and release of the nucleic acids into the cytosol. In this study, to develop the temperature-responsive liposome as a novel carrier for siRNA delivery, we prepared lipoplexes and assessed cellular uptake of siRNA and gene silencing activity of target genes, compared with those of a commercial transfection reagent, Lipofectamine RNAiMAX, and non-modified or PEGylated liposomes. The temperature-responsive polymer, N-isopropylacrylamide-co-N,N’-dimethylaminopropylacrylamide [P(NIPAAm-co-DMAPAAm)]-modified liposome induced faster intracellular delivery because P(NIPAAm-co-DMAPAAm) exhibits a lower critical solution temperature (LCST) changing its nature from hydrophilic to hydrophobic above the LCST. The temperature-responsive liposomes showed significantly higher gene silencing activity than other carriers with less cytotoxicity. Furthermore, we showed that the temperature-responsive lipoplexes were internalized mainly via microtubule-dependent transport and also by the clathrin-mediated endocytosis pathway. This is the first report that temperature-responsive polymer-modified liposomes thermally enhanced silencing activity of siRNA. The dehydrated polymer on the liposomes, and its aggregation caused around the LCST, can probably be attributed to effective cellular uptake of the lipoplexes for gene silencing activity by interaction with the cell membrane.