Monitoring the Activity of Single Translocons

Recent studies introduced a novel view that the SecYEG translocon functions as a monomer and interacts with the dimeric SecA ATPase, which fuels the preprotein translocation reaction. Here, we used nanodisc-reconstituted SecYEG to characterize the functional properties of single copies of the translocon. Using a method based on intermolecular Förster resonance energy transfer, we show for the first time that isolated nanodisc-reconstituted SecYEG monomers support preprotein translocation. When several copies of SecYEG were co-reconstituted within a nanodisc, no change in translocation kinetics was observed, suggesting that SecYEG oligomers do not facilitate enhanced translocation. In contrast, nanodisc-reconstituted monomers of the PrlA4 variant of SecYEG showed increased translocation rates. Experiments based on intramolecular Förster resonance energy transfer within the nanodisc-isolated monomeric SecYEG demonstrated a nucleotide-dependent opening of the channel upon interaction with SecA. In conclusion, the nanodisc-reconstituted SecYEG monomers are functional for preprotein translocation and provide a new prospect for single-molecule analysis of dynamic aspects of protein translocation.     

Elucidating the Native Architecture of the YidC: Ribosome Complex

Membrane protein biogenesis in bacteria occurs via dedicated molecular systems SecYEG and YidC that function independently and in cooperation. YidC belongs to the universally conserved Oxa1/Alb3/YidC family of membrane insertases and is believed to associate with translating ribosomes at the membrane surface. Here, we have examined the architecture of the YidC:ribosome complex formed upon YidC-mediated membrane protein insertion. Fluorescence correlation spectroscopy was employed to investigate the complex assembly under physiological conditions. A slightly acidic environment stimulates binding of detergent-solubilized YidC to ribosomes due to electrostatic interactions, while YidC acquires specificity for translating ribosomes at pH-neutral conditions. The nanodisc reconstitution of the YidC to embed it into a native phospholipid membrane environment strongly enhances the YidC:ribosome complex formation. A single copy of YidC suffices for the binding of translating ribosome both in detergent and at the lipid membrane interface, thus being the minimal functional unit. Data reveal molecular details on the insertase functioning and interactions and suggest a new structural model for the YidC:ribosome complex.     

Inhibition of proinflammatory macrophage responses and lymphocyte proliferation in vitro by ethyl acetate leaf extract from Daphne gnidium

Medicinal plants are considered immunomodulatory as they display various biological activities. There is no report addressing the anti-inflammatory effects of Daphne gnidium. In this study, we investigated the effects of D. gnidium ethyl acetate (EA) leaf extract on mice immune cell function in vitro. Production of pro-inflammatory cytokines (IL-1β and TNF-α), cyclooxygenase-2-derived prostaglandinE2 (PGE2) and iNOS-II-synthesised nitric oxide (NO) were examined. EA extract effect on mitogen-induced lymphocyte proliferation was also investigated. We reported for the first time that D. gnidium EA leaf extract dose-dependently inhibits macrophage proinflammatory function by reducing LPS-induced production of IL-1β, TNF-α, COX-2-derived PGE2 and iNOS-II-synthesised NO. Mitogen-induced lymphocyte proliferation was also dose-dependently inhibited by the extract. Lectin-induced response appears to be more sensitive to the suppressive effects of the extract than LPS-stimulated response. Collectively, these results demonstrate that D. gnidium EA leaf extract acts as an in vitro anti-inflammatory factor by inhibiting mice macrophage and lymphocyte activities.     

1,25-dihydroxyvitamin D3 induces biphasic NF-kappaB responses during HL-60 leukemia cells differentiation through protein induction and PI3K/Akt-dependent phosphorylation/degradation of IkappaB

1,25-dihydroxyvitamin D(3) (VD(3)) induces differentiation in a number of leukemia cell lines and under various conditions is able to either stimulate or inhibit nuclear factor kappa B (NF-kappaB) activity. Here we report a time-dependent biphasic regulation of NF-kappaB in VD(3)-treated HL-60 leukemia cells. After VD(3) treatment there was an early approximately 4 h suppression and a late 8-72 h prolonged reactivation of NF-kappaB. The reactivation of NF-kappaB was concomitant with increased IKK activities, IKK-mediated IkappaBalpha phosphorylation, p65 phosphorylation at residues S276 and S536, p65 nuclear translocation and p65 recruitment to the NF-kappaB/vitamin D responsive element promoters. In parallel with NF-kappaB stimulation, there was an up-regulation of NF-kappaB controlled inflammatory and anti-apoptotic genes such as TNFalpha, IL-1beta and Bcl-xL. VD(3)-triggered reactivation of NF-kappaB was associated with PI3K/Akt phosphorylation. PI3K/Akt antagonists suppressed VD(3)-stimulated IkappaBalpha phosphorylation as well as NF-kappaB-controlled gene expression. The early approximately 4 h VD(3)-mediated NF-kappaB suppression coincided with a prolonged increase of IkappaBalpha protein which require de novo protein synthesis, lasted for as least 72 h and was insensitive to MAPK, IKK or PI3K/Akt inhibitors. Our data suggest a novel biphasic regulation of NF-kappaB in VD(3)-treated leukemia cells and our results may have provided the first molecular explanation for the contradictory observations reported on VD(3)-mediated immune-regulation.     

Probing receptor-translocator interactions in the oligopeptide ABC transporter by fluorescence correlation spectroscopy

The oligopeptide transporter Opp is a five-component ABC uptake system. The extracytoplasmic lipid-anchored substrate-binding protein (or receptor) OppA delivers peptides to an integral membrane complex OppBCDF (or translocator), where, on ATP binding and hydrolysis, translocation across the membrane takes place. OppA and OppBCDF were labeled with fluorescent probes, reconstituted into giant unilamellar vesicles, and the receptor-translocator interactions were investigated by fluorescence correlation spectroscopy. Lateral mobility of OppA was reduced on incorporation of OppBCDF into giant unilamellar vesicles, and decreased even further on the addition of peptide. Fluorescence cross-correlation measurements revealed that OppBCDF distinguished liganded from unliganded OppA, binding only the former. Addition of ATP or its nonhydrolyzable analog AMP-PNP resulted in release of OppA from OppBCDF. In vanadate-trapped “transition state” conditions, OppA also was not bound by OppBCDF. A model is presented in which ATP-binding to OppDF results in donation of peptide to OppBC and simultaneous release of OppA. ATP-hydrolysis would complete the peptide translocation and reset the transporter for another catalytic cycle. Implications in terms of a general transport mechanism for ABC importers and exporters are discussed.     

Dysfunction of regulatory volume increase is a key component of apoptosis

Sustained cell shrinkage is a major hallmark of apoptotic cell death. In apoptotic cells, whole cell volume reduction, called apoptotic volume decrease (AVD), proceeds until fragmentation of cells. Under non-apoptotic conditions, human epithelial HeLa cells exhibited a slow regulatory volume increase (RVI) after osmotic shrinkage induced by exposure to hypertonic solution. When AVD was induced by treatment with a Fas ligand, TNF-alpha or staurosporine, however, it was found that HeLa cells failed to undergo RVI. When RVI was inhibited by combined application of Na+/H+ exchanger (NHE) and anion exchanger blockers, hypertonic stress induced prolonged shrinkage followed by caspase-3 activation in HeLa cells. Hypertonicity also induced apoptosis in NHE1-deficient PS120 fibroblasts, which lack the RVI response. When RVI was restored by transfection of these cells with NHE1, hypertonicity-induced apoptosis was completely prevented. Thus, it is concluded that RVI dysfunction is indispensable for the persistence of AVD and induction of apoptosis.     

Involvement of proton-sensing receptor TDAG8 in the anti-inflammatory actions of dexamethasone in peritoneal macrophages

Dexamethasone (DEX), a potent glucocorticoid, increased the expression of T-cell death associated gene 8 (TDAG8), a proton-sensing G protein-coupled receptor, which is associated with the enhancement of acidic pH-induced cAMP accumulation, in peritoneal macrophages. We explored the role of increased TDAG8 expression in the anti-inflammatory actions of DEX. The treatment of macrophages with either DEX or acidic pH induced the cell death of macrophages; however, the cell death was not affected by TDAG8 deficiency. While DEX inhibited lipopolysaccharide-induced production of tumor necrosis factor-α, an inflammatory cytokine, which was independent of TDAG8, at neutral pH, the glucocorticoid enhanced the acidic pH-induced inhibition of tumor necrosis factor-α production in a manner dependent on TDAG8. In conclusion, the DEX-induced increase in TDAG8 expression is in part involved in the glucocorticoid-induced anti-inflammatory actions through the inhibition of inflammatory cytokine production under the acidic pH environment. On the other hand, the role of TDAG8 in the DEX-induced cell death is questionable.     

Visualization and quantification of endoplasmic reticulum Ca in renal cells using confocal microscopy and Fluo5F

Sarcoplasmic/endoplasmic reticulum (ER) Ca²⁺ is the most abundant store of intracellular Ca²⁺, and its release is an important trigger of physiological and cell death pathways. Previous work in our laboratory revealed the importance of ER Ca²⁺ in toxicant-induced renal proximal tubular cell (RPTC) death. The purpose of this study was to evaluate the use of confocal microscopy and Fluo5F, a low affinity Ca²⁺ indicator, to directly monitor changes in RPTC ER Ca²⁺. Fluo5F staining reflected ER Ca²⁺, resolved ER structure, and showed no colocalization with tetramethyl rhodamine methyl ester (TMRM), a marker of mitochondrial membrane potential. Thapsigargin, an ER Ca²⁺ pump inhibitor, decreased ER fluorescence by 30% and 55% at 5 and 15 min, respectively, whereas A23187, a Ca²⁺ ionophore caused more rapid ER Ca²⁺ release (55% and 75% decrease in fluorescence at 5 and 15 min).Carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP), a mitochondrial uncoupler, added at the end of the experiment, further decreased ER fluorescence after thapsigargin treatment, revealing that thapsigargin did not release all ER Ca²⁺. In contrast, FCCP did not decrease ER fluorescence after A23187 treatment, suggesting complete ER Ca²⁺ release. ER Ca²⁺ release in response to A23187 or thapsigargin resulted in a modest but significant decrease in mitochondrial membrane potential. These data provide evidence that confocal microscopy and Fluo5F are useful and effective tools for directly monitoring ER Ca²⁺ in live cells.     

New effects in polynucleotide release from cationic lipid carriers revealed by confocal imaging, fluorescence cross-correlation spectroscopy and single particle tracking

We report on new insights into the mechanisms of short single and double stranded oligonucleotide release from cationic lipid complexes (lipoplexes), used in gene therapy. Specifically, we modeled endosomal membranes using giant unilamellar vesicles and investigated the roles of various individual cellular phospholipids in interaction with lipoplexes. Our approach uses a combination of confocal imaging, fluorescence cross-correlation spectroscopy and single particle tracking, revealing several new aspects of the release: (a) phosphatidylserine and phosphatidylethanolamine are equally active in disassembling lipoplexes, while phosphatidylcholine and sphingomyelin are inert; (b) in contrast to earlier findings, phosphatidylethanolamine alone, in the absence of anionic phosphatidylserine triggers extensive release; (c) a double-stranded DNA structure remains well preserved after release; (d) lipoplexes exhibited preferential binding to transient lipid domains, which appear at the onset of lipoplex attachment to originally uniform membranes and vanish after initiation of polynucleotide release. The latter effect is likely related to phosphatidyleserine redistribution in membranes due to lipoplex binding. Real time tracking of single DOTAP/DOPE and DOTAP/DOPC lipoplexes showed that both particles remained compact and associated with membranes up to 1-2 min before fusion, indicating that a more complex mechanism, different from suggested earlier rapid fusion, promotes more efficient transfection by DOTAP/DOPE complexes.     

Connexin32 protects against vascular inflammation by modulating inflammatory cytokine expression by endothelial cells

Gap junctions (GJs) play an important role in vascular function, stability, and homeostasis in endothelial cells (ECs), and GJs are comprised of members of the connexin (Cx) family. GJs of vascular ECs are assembled from Cx37, Cx40, and Cx43, and we showed that ECs also express Cx32. In this study, we investigated a potential role for Cx32 during vascular inflammation. Expression of Cx32 mRNA and protein by human umbilical venous ECs (HUVECs) decreased following treatment with tumor necrosis factor (TNF)-α, but lipopolysaccharide (LPS) and interleukin (IL)-1β did not affect Cx32 expression. Intracellular transfer of an inhibitory anti-Cx32 monoclonal antibody significantly enhanced TNF-α-induced monocyte chemotactic protein (MCP)-1 and IL-6 expression, but overexpression of Cx32 abrogated TNF-α-induced MCP-1 and IL-6 expression. LPS treatment of Cx32 knock-out mice significantly increased the serum concentrations of TNF-α, interferon-γ, IL-6 and MCP-1, compared to wild-type littermate mice. These data suggest that Cx32 protects ECs from inflammation by regulating cytokine expression and plays an important role in the maintenance of vascular function.     

Expression of the 78 kD glucose-regulated protein is induced by endoplasmic reticulum stress in the development of hepatopulmonary syndrome

Objective

This study is to explore the role of 78 kD glucose-regulated protein (GRP78) in the development of hepatopulmonary syndrome (HPS) in rats.

Methods

The rat model of liver cirrhosis and HPS were induced with multiple pathogenic factors. Hematoxylin and eosin (H & E) staining was performed to detect the pathological changes of the lung and liver tissues. The levels of alanine transferase (ALT), endotoxin, and tumor necrosis factor-α (TNF-α) in plasma and TNF-α and malondialdehyde (MDA) in lung tissues were detected. RT-PCR and Western blotting were conducted to detect the mRNA and protein expression levels of GRP78 in lungs.

Results

The plasma endotoxin level was gradually increased as HPS developed, and the mRNA and protein expression levels of GRP78 in lungs were also increased as the disease progressed. The levels of ALT and TNF-α in plasma and the contents of TNF-α and MDA in lung tissues were gradually increased along with the disease progression, with a strong positive correlation. Compared with controls, the plasma TNF-α level and the mRNA and protein expression levels of GRP78 in lung tissues were significantly higher in rats with HPS. The levels of endotoxin and ALT in plasma and the level of MDA in lungs were significantly higher in rats with HPS than controls.

Conclusions

The increased GRP78 expression is indicative of endoplasmic reticulum stress response during HPS, which may play an important role in the disease pathogenesis.     

Role of heat shock protein 47 in intestinal fibrosis of experimental colitis

Background and aims

Intestinal fibrosis is a clinically important issue of inflammatory bowel disease (IBD). It is unclear whether or not heat shock protein 47 (HSP47), a collagen-specific molecular chaperone, plays a critical role in intestinal fibrosis. The aim of this study is to investigate the role of HSP47 in intestinal fibrosis of murine colitis.

Methods

HSP47 expression and localization were evaluated in interleukin-10 knockout (IL-10KO) and wild-type (WT, C57BL/6) mice by immunohistochemistry. Expression of HSP47 and transforming growth factor-β1 (TGF-β1) in colonic tissue was measured. In vitro studies were conducted in NIH/3T3 cells and primary culture of myofibroblasts separated from colonic tissue of IL-10KO (PMF KO) and WT mice (PMF WT) with stimulation of several cytokines. We evaluated the inhibitory effect of administration of small interfering RNA (siRNA) targeting HSP47 on intestinal fibrosis in IL-10KO mice in vivo.

Results

Immunohistochemistry revealed HSP47 positive cells were observed in the mesenchymal and submucosal area of both WT and IL-10 KO mice. Gene expressions of HSP47 and TGF-β1 were significantly higher in IL-10KO mice than in WT mice and correlated with the severity of inflammation. In vitro experiments with NIH3T3 cells, TGF-β1 only induced HSP47 gene expression. There was a significant difference of HSP47 gene expression between PMF KO and PMF WT. Administration of siRNA targeting HSP47 remarkably reduced collagen deposition in colonic tissue of IL-10KO mice.

Conclusions

Our results indicate that HSP47 plays an essential role in intestinal fibrosis of IL-10KO mice, and may be a potential target for intestinal fibrosis associated with IBD.     

Oxidative stress plays a role in high glucose-induced activation of pancreatic stellate cells

The activation of pancreatic stellate cells (PSCs) is thought to be a potential mechanism underlying islet fibrosis, which may contribute to progressive β-cell failure in type 2 diabetes. Recently, we demonstrated that antioxidants reduced islet fibrosis in an animal model of type 2 diabetes. However, there is no in vitro study demonstrating that high glucose itself can induce oxidative stress in PSCs. Thus, PSCs were isolated and cultured from Sprague Dawley rats, and treated with high glucose for 72 h. High glucose increased the production of reactive oxygen species. When treated with high glucose, freshly isolated PSCs exhibited myofibroblastic transformation. During early culture (passage 1), PSCs treated with high glucose contained an increased number of α-smooth muscle actin-positive cells. During late culture (passages 2–5), PSCs treated with high glucose exhibited increases in cell proliferation, the expression of fibronectin and connective tissue growth factor, release of interleukin-6, transforming growth factor-β and collagen, and cell migration. Finally, the treatment of PSCs with high glucose and antioxidants attenuated these changes. In conclusion, we demonstrated that high glucose increased oxidative stress in primary rat PSCs, thereby facilitating the activation of these cells, while antioxidant treatment attenuated high glucose-induced PSC activation.     

Copper-mediated cross-linking of S100A4, but not of S100A2, results in proinflammatory effects in melanoma cells

The aim of this study was to investigate the response to and the physiological consequences of copper-mediated cross-linking of S100A2 and S100A4, two members of the S100 family of EF-hand calcium-binding proteins. As demonstrated by electrophoresis and mass spectrometry techniques S100A2 and S100A4 show formation of cross-links due to copper-mediated oxidation of cysteine residues. For S100A4, but not for S100A2, this results in both increased activation of NFκB and secretion of TNF-α in human A375 and, to a higher extent, in RAGE-transfected melanoma cells. The data suggest that a prooxidative tumor microenvironment enhances proinflammatory and prometastatic action of S100A4.     

Isolation and characterization of a large soluble form of fibronectin that stimulates adhesion, spreading, and alignment of mouse erythroleukemia cells

Fibronectin (FN) is a major component of the extracellular matrix which plays important roles in a variety of cellular processes including cell adhesion, and migration. The soluble cellular form of FN has a monomer molecular weight of approximately 250 kDa, and generally exists as a dimer of 500 kDa. We have isolated a different form of soluble FN from mouse breast cancer cell line SC115 conditioned medium (CM) and purified it to homogeneity as evidenced by both native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate PAGE. It still exhibits a monomeric form of about 250 kDa while its form in the CM is stable and soluble with an apparent tetrameric molecular weight in the range of 800-1000 kDa. This form of FN is a potent cell adhesion factor (AF) that induces adhesion to polystyrene, elongation, spreading, alignment or “track” formation, and migration of mouse erythroleukemia cells. Column fractions homogeneous for AF protein were able to stimulate 10% cell adhesion at concentrations of 23 ng/ml and 1.9 ng/cm². Purified AF induced 50% cell adhesion at 94 ng/ml and 7.5 ng/cm². AF also increased the migration of human aortic smooth muscle and vascular endothelial cells. However, this form of FN differs from other forms as it does not bind tightly to either gelatin or heparin. Studies of this AF should shed light on adhesion of cells to extracellular matrix molecules and on cell migration, both of which are critical in several biological processes such as wound healing, metastasis, matrix formation and structure, and organ development.     

TRPC3-mediated Ca2+ influx contributes to Rac1-mediated production of reactive oxygen species in MLP-deficient mouse hearts

Obesity-related metabolic abnormalities, including chronic inflammation and oxidative stress, increase the risk of colorectal cancer. Dysregulation of the renin–angiotensin system (RAS) also plays a critical role in obesity-related metabolic disorders and in several types of carcinogenesis. In the present study, we examined the effects of an angiotensin-converting enzyme (ACE) inhibitor and angiotensin-II type 1 receptor blocker (ARB), both of which inhibit the RAS, on the development of azoxymethane (AOM)-initiated colonic premalignant lesions in C57BL/KsJ-db/db (db/db) obese mice. Male db/db mice were given 4 weekly subcutaneous injections of AOM (15 mg/kg body weight), and then, they received drinking water containing captopril (ACE inhibitor, 5 mg/kg/day) or telmisartan (ARB, 5 mg/kg/day) for 7 weeks. At sacrifice, administration of either captopril or telmisartan significantly reduced the total number of colonic premalignant lesions, i.e., aberrant crypt foci and β-catenin accumulated crypts, compared to that observed in the control group. The expression levels of TNF-α mRNA in the colonic mucosa of AOM-treated db/db mice were decreased by captopril and telmisartan. Captopril lowered the expression levels of TNF-α, IL-1β, IL-6, and PAI-1 mRNAs, while telmisartan lowered the expression levels of COX-2, IL-1β, IL-6, and PAI-1 mRNAs in the white adipose tissues of these mice. In addition, these agents significantly reduced the levels of urinary 8-OHdG, a surrogate marker of oxidative damage to DNA, in the experimental mice. These findings suggested that both ACE inhibitor and ARB suppress chemically-induced colon carcinogenesis by attenuating chronic inflammation and reducing oxidative stress in obese mice. Therefore, targeting dysregulation of the RAS might be an effective strategy for chemoprevention of colorectal carcinogenesis in obese individuals.