The transcriptional regulator IscR integrates host-derived nitrosative stress and iron starvation in activation of the vvhBA operon in Vibrio vulnificus.

For successful infection of their hosts, pathogenic bacteria recognize host-derived signals that induce the expression of virulence factors in a spatiotemporal manner. The fulminating food-borne pathogen Vibrio vulnificus produces a cytolysin/hemolysin protein encoded by the vvhBA operon, which is a virulence factor preferentially expressed upon exposure to murine blood and macrophages. The Fe-S cluster containing transcriptional regulator IscR activates the vvhBA operon in response to nitrosative stress and iron starvation, during which the cellular IscR protein level increases. Here, electrophoretic mobility shift and DNase I protection assays revealed that IscR directly binds downstream of the vvhBA promoter P vvhBA , which is unusual for a positive regulator. We found that in addition to IscR, the transcriptional regulator HlyU activates vvhBA transcription by directly binding upstream of P vvhBA , whereas the histone-like nucleoid-structuring protein (H-NS) represses vvhBA by extensively binding to both downstream and upstream regions of its promoter. Of note, the binding sites of IscR and HlyU overlapped with those of H-NS. We further substantiated that IscR and HlyU outcompete H-NS for binding to the P vvhBA regulatory region, resulting in the release of H-NS repression and vvhBA induction. We conclude that concurrent antirepression by IscR and HlyU at regions both downstream and upstream of P vvhBA provides V. vulnificus with the means of integrating host-derived signal(s) such as nitrosative stress and iron starvation for precise regulation of vvhBA transcription, thereby enabling successful host infection.

Identification and characterization of Vibrio vulnificus plpA encoding a phospholipase A2 essential for pathogenesis.

The marine bacterium Vibrio vulnificus causes food-borne diseases, which may lead to life-threatening septicemia in some individuals. Therefore, identifying virulence factors in V. vulnificus is of high priority. We performed a transcriptome analysis on V. vulnificus after infection of human intestinal HT29-methotrexate cells and found induction of plpA, encoding a putative phospholipase, VvPlpA. Bioinformatics, biochemical, and genetic analyses demonstrated that VvPlpA is a phospholipase A2 secreted in a type II secretion system-dependent manner. Compared with the wild type, the plpA mutant exhibited reduced mortality, systemic infection, and inflammation in mice as well as low cytotoxicity toward the human epithelial INT-407 cells. Moreover, plpA mutation attenuated the release of actin and cytosolic cyclophilin A from INT-407 cells, indicating that VvPlpA is a virulence factor essential for causing lysis and necrotic death of the epithelial cells. plpA transcription was growth phase-dependent, reaching maximum levels during the early stationary phase. Also, transcription factor HlyU and cAMP receptor protein (CRP) mediate additive activation and host-dependent induction of plpA Molecular biological analyses revealed that plpA expression is controlled via the promoter, P plpA , and that HlyU and CRP directly bind to P plpA upstream sequences. Taken together, this study demonstrated that VvPlpA is a type II secretion system-dependent secretory phospholipase A2 regulated by HlyU and CRP and is essential for the pathogenicity of V. vulnificus.

OxyR2 Functions as a Three-state Redox Switch to Tightly Regulate Production of Prx2, a Peroxiredoxin of Vibrio vulnificus.

The bacterial transcriptional regulator OxyR is known to function as a two-state redox switch. OxyR senses cellular levels of H2O2 via a "sensing cysteine" that switches from the reduced to a disulfide state upon H2O2 exposure, inducing the expression of antioxidant genes. The reduced and disulfide states of OxyR, respectively, bind to extended and compact regions of DNA, where the reduced state blocks and the oxidized state allows transcription and further induces target gene expression by interacting with RNA polymerase. Vibrio vulnificus OxyR2 senses H2O2 with high sensitivity and induces the gene encoding the antioxidant Prx2. In this study, we used mass spectrometry to identify a third redox state of OxyR2, in which the sensing cysteine was overoxidized to S-sulfonated cysteine (Cys-SO3H) by high H2O2 in vitro and in vivo, where the modification deterred the transcription of prx2 The DNA binding preferences of OxyR25CA-C206D, which mimics overoxidized OxyR2, suggested that overoxidized OxyR2 binds to the extended DNA site, masking the -35 region of the prx2 promoter. These combined results demonstrate that OxyR2 functions as a three-state redox switch to tightly regulate the expression of prx2, preventing futile production of Prx2 in cells exposed to high levels of H2O2 sufficient to inactivate Prx2. We further provide evidence that another OxyR homolog, OxyR1, displays similar three-state behavior, inviting further exploration of this phenomenon as a potentially general regulatory mechanism.

Inhibition of CYP4A reduces hepatic endoplasmic reticulum stress and features of diabetes in mice.

BACKGROUND & AIMS: Endoplasmic reticulum (ER) stress is implicated in the development of type 2 diabetes mellitus. ER stress activates the unfolded protein response pathway, which contributes to apoptosis and insulin resistance. We investigated the roles of cytochrome P450 4A (CYP4A) in the regulation of hepatic ER stress, insulin resistance, and the development of diabetes in mice. METHODS: We used mass spectrometry to compare levels of CYP450 proteins in livers from C57BL/6J and C57BL/KsJ-db/db (db/db) mice; findings were confirmed by immunoblot and real-time PCR analyses. To create a model of diet-induced diabetes, C57BL/6J mice were placed on high-fat diets. Mice were given intraperitoneal injections of an inhibitor (HET0016) or an inducer (clofibrate) of CYP4A, or tail injections of small hairpin RNAs against CYP4A messenger RNA; liver tissues were collected and analyzed for ER stress, insulin resistance, and apoptosis. The effect of HET0016 and CYP4A knockdown also were analyzed in HepG2 cells. RESULTS: Levels of the CYP4A isoforms were highly up-regulated in livers of db/db mice compared with C57BL/6J mice. Inhibition of CYP4A in db/db and mice on high-fat diets reduced features of diabetes such as insulin hypersecretion, hepatic steatosis, and increased glucose tolerance. CYP4A inhibition reduced levels of ER stress, insulin resistance, and apoptosis in the livers of diabetic mice; it also restored hepatic functions. Inversely, induction of CYP4A accelerated ER stress, insulin resistance, and apoptosis in livers of db/db mice. CONCLUSIONS: CYP4A proteins are up-regulated in livers of mice with genetically induced and diet-induced diabetes. Inhibition of CYP4A in mice reduces hepatic ER stress, apoptosis, insulin resistance, and steatosis. Strategies to reduce levels or activity of CYP4A proteins in liver might be developed for treatment of patients with type 2 diabetes.

Proteomic and bioinformatic analysis of membrane proteome in type 2 diabetic mouse liver.

Type 2 diabetes mellitus (T2DM) is the most prevalent and serious metabolic disease affecting people worldwide. T2DM results from insulin resistance of the liver, muscle, and adipose tissue. In this study, we used proteomic and bioinformatic methodologies to identify novel hepatic membrane proteins that are related to the development of hepatic insulin resistance, steatosis, and T2DM. Using FT-ICR MS, we identified 95 significantly differentially expressed proteins in the membrane fraction of normal and T2DM db/db mouse liver. These proteins are primarily involved in energy metabolism pathways, molecular transport, and cellular signaling, and many of them have not previously been reported in diabetic studies. Bioinformatic analysis revealed that 16 proteins may be related to the regulation of insulin signaling in the liver. In addition, six proteins are associated with energy stress-induced, nine proteins with inflammatory stress-induced, and 14 proteins with endoplasmic reticulum stress-induced hepatic insulin resistance. Moreover, we identified 19 proteins that may regulate hepatic insulin resistance in a c-Jun amino-terminal kinase-dependent manner. In addition, three proteins, 14-3-3 protein beta (YWHAB), Slc2a4 (GLUT4), and Dlg4 (PSD-95), are discovered by comprehensive bioinformatic analysis, which have correlations with several proteins identified by proteomics approach. The newly identified proteins in T2DM should provide additional insight into the development and pathophysiology of hepatic steatosis and insulin resistance, and they may serve as useful diagnostic markers and/or therapeutic targets for these diseases.

Roles of peroxiredoxin II in the regulation of proinflammatory responses to LPS and protection against endotoxin-induced lethal shock.

Mammalian 2-Cys peroxiredoxin II (Prx II) is a cellular peroxidase that eliminates endogenous H(2)O(2). The involvement of Prx II in the regulation of lipopolysaccharide (LPS) signaling is poorly understood. In this report, we show that LPS induces substantially enhanced inflammatory events, which include the signaling molecules nuclear factor kappaB and mitogen-activated protein kinase (MAPK), in Prx II-deficient macrophages. This effect of LPS was mediated by the robust up-regulation of the reactive oxygen species (ROS)-generating nicotinamide adenine dinucleotide phosphate (NADPH) oxidases and the phosphorylation of p47(phox). Furthermore, challenge with LPS induced greater sensitivity to LPS-induced lethal shock in Prx II-deficient mice than in wild-type mice. Intravenous injection of Prx II-deficient mice with the adenovirus-encoding Prx II gene significantly rescued mice from LPS-induced lethal shock as compared with the injection of a control virus. The administration of catalase mimicked the reversal effects of Prx II on LPS-induced inflammatory responses in Prx II-deficient cells, which suggests that intracellular H(2)O(2) is attributable, at least in part, to the enhanced sensitivity to LPS. These results indicate that Prx II is an essential negative regulator of LPS-induced inflammatory signaling through modulation of ROS synthesis via NADPH oxidase activities and, therefore, is crucial for the prevention of excessive host responses to microbial products.

ATP-binding cassette B4, an auxin-efflux transporter, stably associates with the plasma membrane and shows distinctive intracellular trafficking from that of PIN-FORMED proteins.

Intracellular trafficking of auxin transporters has been implicated in diverse developmental processes in plants. Although the dynamic trafficking pathways of PIN-FORMED auxin efflux proteins have been studied intensively, the trafficking of ATP-binding cassette protein subfamily B proteins (ABCBs; another group of auxin efflux carriers) still remains largely uncharacterized. In this study, we address the intracellular trafficking of ABCB4 in Arabidopsis (Arabidopsis thaliana) root epidermal cells. Pharmacological analysis showed that ABCB4 barely recycled between the plasma membrane and endosomes, although it slowly endocytosed via the lytic vacuolar pathway. Fluorescence recovery after photobleaching analysis revealed that ABCB4 is strongly retained in the plasma membrane, further supporting ABCB4's nonrecycling property. The endocytosis of ABCB4 was not dependent on the GNOM-LIKE1 function, and the sensitivity of ABCB4 to brefeldin A required guanine nucleotide exchange factors for adenosyl ribosylation factor other than GNOM. These characteristics of intracellular trafficking of ABCB4 are well contrasted with those of PIN-FORMED proteins, suggesting that ABCB4 may be a basic and constitutive auxin efflux transporter for cellular auxin homeostasis.

Regulation of the major vacuolar Ca²âº transporter genes, by intercellular Ca²âº concentration and abiotic stresses, in tip-burn resistant Brassica oleracea.

Calcium is an essential plant macronutrient that has unique structural and signaling roles related to tip-burn disorder in Brassica spp. crops. For two types of cabbage inbred lines, tip-burn susceptible and resistant, we measured and compared major macronutrient cations, including Ca(2+), in leaves. In both lines, Ca(2+), Mg(2+), Na(+), and K(+), accumulated more in leaf base than in leaf apex. Ca(2+) and K(+) were >2 times more abundant in the tip-burn resistant line, while Na(+) was higher in the susceptible line. Ca(2+) differences between the two lines resulted from differential accumulation of calcium into cell vacuoles. We profiled major vacuolar Ca(2+) transporters, in both cabbage lines, by growth time and intercellular Ca(2+) concentration. Expression pattern of several Ca(2+) transporter genes differed between tip-burn susceptible and resistant lines by growth time points. We also identified promoter regions of the major Ca(2+) vacuole transporter genes, CAX1, ACA4, and ACA11, which displayed hormonal, light and defense-related cis-acting regulatory elements. Finally, transporter genes in the two cabbage lines responded differently to abiotic stresses, demonstrating diversity in gene regulation among orthologous genes.

Profiling and semiquantitative analysis of the cell surface proteome in human mesenchymal stem cells.

Mulitpotent mesenchymal stem cells (MSCs) derived from human bone marrow are promising candidates for the development of cell therapeutic strategies. MSC surface protein profiles provide novel biological knowledge concerning the proliferation and differentiation of these cells, including the potential for identifying therapeutic targets. Basic fibroblast growth factor (bFGF) affects cell surface proteins, which are associated with increased growth rate, differentiation potential, as well as morphological changes of MSCs in vitro. Cell surface proteins were isolated using a biotinylation-mediated method and identified using a combination of one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry. The resulting gel lines were cut into 20 bands and digested with trypsin. Each tryptic fragment was analyzed by liquid chromatography-electrospray ionization tandem mass spectrometry. Proteins were identified using the Mascot search program and the International Protein Index human database. Noble MSC surface proteins (n = 1,001) were identified from cells cultured either with (n = 857) or without (n = 667) bFGF-containing medium in three independent experiments. The proteins were classified using FatiGO to elucidate their function. We also confirmed the proteomics results using Western blotting and immunofluorescence microscopic analysis. The nature of the proteins identified makes it clear that MSCs express a wide variety of signaling molecules, including those related to cell differentiation. Among the latter proteins, four Ras-related Rab proteins, laminin-R, and three 14-3-3 proteins that were fractionated from MSCs cultured on bFGF-containing medium are implicated in bFGF-induced signal transduction of MSCs. Consequently, these finding provide insight into the understanding of the surface proteome of human MSCs.

Mycobacterium tuberculosis eis regulates autophagy, inflammation, and cell death through redox-dependent signaling.

The "enhanced intracellular survival" (eis) gene of Mycobacterium tuberculosis (Mtb) is involved in the intracellular survival of M. smegmatis. However, its exact effects on host cell function remain elusive. We herein report that Mtb Eis plays essential roles in modulating macrophage autophagy, inflammatory responses, and cell death via a reactive oxygen species (ROS)-dependent pathway. Macrophages infected with an Mtb eis-deletion mutant H37Rv (Mtb-Δeis) displayed markedly increased accumulation of massive autophagic vacuoles and formation of autophagosomes in vitro and in vivo. Infection of macrophages with Mtb-Δeis increased the production of tumor necrosis factor-α and interleukin-6 over the levels produced by infection with wild-type or complemented strains. Elevated ROS generation in macrophages infected with Mtb-Δeis (for which NADPH oxidase and mitochondria were largely responsible) rendered the cells highly sensitive to autophagy activation and cytokine production. Despite considerable activation of autophagy and proinflammatory responses, macrophages infected with Mtb-Δeis underwent caspase-independent cell death. This cell death was significantly inhibited by blockade of autophagy and c-Jun N-terminal kinase-ROS signaling, suggesting that excessive autophagy and oxidative stress are detrimental to cell survival. Finally, artificial over-expression of Eis or pretreatment with recombinant Eis abrogated production of both ROS and proinflammatory cytokines, which depends on the N-acetyltransferase domain of the Eis protein. Collectively, these data indicate that Mtb Eis suppresses host innate immune defenses by modulating autophagy, inflammation, and cell death in a redox-dependent manner.

Dexamethasone Induces FcγRIIb Expression in RBL-2H3 Cells.

Mast cells are involved in allergic responses, protection against pathogens and autoimmune diseases. Dexamethasone (Dex) and other glucocorticoids suppress FcεRI-mediated release of inflammatory mediators from mast cells. The inhibition mechanisms were mainly investigated on the downstream signaling of Fc receptor activations. Here, we addressed the effects of Dex on Fc receptor expressions in rat mast cell line RBL-2H3. We measured mRNA levels of Fc receptors by real-time PCR. As expected, Dex decreased the mRNA levels of activating Fc receptor for IgE (FcεR) I and increased the mRNA levels of the inhibitory Fc receptor for IgG FcγRIIb. Interestingly, Dex stimulated transcriptions of other activating receptors such as Fc receptors for IgG (FcγR) I and FcγRIII. To investigate the mechanisms underlying transcriptional regulation, we employed a transcription inhibitor actinomycin D and a translation inhibitor cycloheximide. The inhibition of protein synthesis without Dex treatment enhanced FcγRI and FcγRIII mRNA levels potently, while FcεRI and FcγRIIb were minimally affected. Next, we examined expressions of the Fc receptors on cell surfaces by the flow cytometric method. Only FcγRIIb protein expression was significantly enhanced by Dex treatment, while FcγRI, FcγRIII and FcεRI expression levels were marginally changed. Our data showed, for the first time, that Dex regulates Fc receptor expressions resulting in augmentation of the inhibitory receptor FcγRIIb.

1,3-Dibenzyl-5-Fluorouracil Prevents Ovariectomy-Induced Bone Loss by Suppressing Osteoclast Differentiation.

Osteoclasts (OCs) are clinically important cells that resorb bone matrix. Accelerated bone destruction by OCs is closely linked to the development of metabolic bone diseases. In this study, we screened novel chemical inhibitors targeting OC differentiation to identify drug candidates for metabolic bone diseases. We identified that 1,3-dibenzyl-5-fluorouracil, also named OCI-101, is a novel inhibitor of osteoclastogenesis. The formation of multinucleated OCs is reduced by treatment with OCI-101 in a dose-dependent manner. OCI-101 inhibited the expression of OC markers via downregulation of receptor activator of NF-κB ligand and M-CSF signaling pathways. Finally, we showed that OCI-101 prevents ovariectomy-induced bone loss by suppressing OC differentiation in mice. Hence, these results demonstrated that OCI-101 is a good drug candidate for treating metabolic bone diseases.

NADPH oxidase 2 interaction with TLR2 is required for efficient innate immune responses to mycobacteria via cathelicidin expression.

Gp91(phox)/NADPH oxidase (NOX) 2 is the main catalytic component of NOX, which mediates the phagocytic killing of ingested pathogens via the production of reactive oxygen species (ROS). However, Mycobacterium tuberculosis (Mtb) is relatively resistant to the microbicidal effects of ROS. Thus, the exact roles of NOX2 in the innate immune control against Mtb infection are not fully resolved. In this study, we show that NOX2 is essential for TLR2-dependent inflammatory responses and 1,25-dihydroxyvitamin D(3) (1,25D(3))-mediated antimicrobial activity against Mtb via cathelicidin expression. NOX2-null macrophages prominently abrogated Mtb-induced ROS production and inflammatory signaling activation in a TLR2-dependent manner. Mtb triggered a physical association between NOX2 and TLR2. In addition, the knockdown of NOX2 inhibited 1,25D(3)-triggered antimicrobial activity against viable Mtb through the modulation of cathelicidin expression in human macrophages. Treatment of NOX2 knocked down cells with cathelicidin restored the 1,25D(3)-induced antimicrobial effect, suggesting that the NOX2-dependent induction of cathelicidin in macrophages is part of a defensive strategy against Mtb. Furthermore, cathelicidin expression was required for the Mtb-induced release of ROS and the production of proinflammatory cytokines/chemokines, indicating a positive circuit of inflammation in response to Mtb. Our data collectively demonstrate a novel regulatory mechanism for TLR2-dependent innate responses to Mtb involving crosstalk between NOX2 and TLR2 and the expression of cathelicidin.

Innate immune responses to Mycobacterium ulcerans via toll-like receptors and dectin-1 in human keratinocytes.

Mycobacterium ulcerans (MU), an environmental pathogen, causes Buruli ulcer, a severe skin disease. We hypothesized that epidermal keratinocytes might not be a simple barrier, but play a role during MU infection through pattern-recognition receptors expressed in keratinocytes. We found that keratinocyte Toll-like receptors (TLRs) 2 and 4 and Dectin-1 actively participate in the innate immune response to MU, which includes the internalization of bacteria, the production of reactive oxygen species (ROS), and the expression of chemokines and LL-37. Human keratinocytes constitutively expressed TLRs 2 and 4 and induced Dectin-1 in response to MU. Exposing keratinocytes to MU resulted in rapid ROS production, which in turn contributed to the mRNA and protein expression of LL-37. In addition, TLR2, Dectin-1 and, to an extent, TLR4 are essential for the MU-mediated expression of CXCL8, CCL2 and LL-37 in keratinocytes. Furthermore, confocal analysis showed that the Dectin-1 is necessary for keratinocytes to internalize bacilli. Importantly, blockade of ROS and LL-37 significantly increased the intracellular MU growth in keratinocytes, suggesting an important role of these mediators for cutaneous innate immune responses. Our results demonstrate that TLR2, TLR4 and Dectin-1 actively sense, internalize and respond in an innate way to MU in human epidermal keratinocytes.

The presence of monoglucosylated N196-glycan is important for the structural stability of storage protein, arylphorin.

Although N-glycosylation has been known to increase the stability of glycoproteins, it is difficult to assess the structural importance of glycans in the stabilization of glycoproteins. APA (Antheraea pernyi arylphorin) is an insect hexamerin that has two N-glycosylations at Asn196 and Asn344 respectively. The glycosylation of Asn344 is critical for the folding process; however, glycosylation of Asn196 is not. Interestingly, the N196-glycan (glycosylation of Asn196) remains in an immature form (Glc1Man9GlcNAc2). The mutation of Asn196 to glutamine does not change the ecdysone-binding activity relative to that of the wild-type. In the present study, we determined the crystal structure of APA, and all sugar moieties of the N196-glycan were clearly observed in the electron-density map. Although the sugar moieties of the glycan generally have high structural flexibility, most sugar moieties of the N196-glycan were well organized in the deep cleft of the subunit interface and mediated many inter- and intrasubunit hydrogen bonds. Analytical ultracentrifugation and GdmCl (guanidinium chloride) unfolding experiments revealed that the presence of the N196-glycan was important for stabilizing the hexameric state and overall stability of APA respectively. Our results could provide a structural basis for studying not only other glycoproteins that carry an immature N-glycan, but also the structural role of N-glycans that are located in the deep cleft of a protein.

A MARTX Toxin rtxA Gene Is Controlled by Host Environmental Signals through a CRP-Coordinated Regulatory Network in Vibrio vulnificus.

A multifunctional autoprocessing repeats-in-toxin (MARTX) toxin plays an essential role in the virulence of many pathogens, including a fulminating human pathogen Vibrio vulnificus H-NS and HlyU repress and derepress expression of the MARTX toxin gene rtxA in V. vulnificus, respectively. However, little is known about other regulatory proteins and environmental signals involved in rtxA regulation. In this study, we found that a leucine-responsive regulatory protein (Lrp) activates rtxA by binding directly and specifically to the rtxA promoter, P rtxA Phased hypersensitivity resulting from DNase I cleavage of the P rtxA regulatory region suggests that Lrp probably induces DNA bending in P rtxA Lrp activates P rtxA independently of H-NS and HlyU, and leucine inhibits Lrp binding to P rtxA and reduces the Lrp-mediated activation. Furthermore, a cyclic AMP receptor protein (CRP) represses P rtxA , and exogenous glucose relieves the CRP-mediated repression. Biochemical and mutational analyses demonstrated that CRP binds directly and specifically to the upstream region of P rtxA , which presumably alters the DNA conformation in P rtxA and thus represses rtxA Moreover, CRP represses expression of lrp and hlyU by binding directly to their upstream regions, forming coherent feed-forward loops with Lrp and HlyU. In conclusion, expression of rtxA is controlled by a regulatory network comprising CRP, Lrp, H-NS, and HlyU in response to changes in host environmental signals such as leucine and glucose. This collaborative regulation enables the elaborate expression of rtxA, thereby enhancing the fitness and pathogenesis of V. vulnificus during the course of infection.IMPORTANCE A MARTX toxin, RtxA, is an essential virulence factor of many pathogens, including Vibrio species. H-NS and HlyU repress and derepress, respectively, rtxA expression of a life-threatening pathogen, Vibrio vulnificus We found that Lrp directly activates rtxA independently of H-NS and HlyU, and leucine inhibits the Lrp-mediated activation of rtxA Furthermore, we demonstrated that CRP represses rtxA but derepresses in the presence of exogenous glucose. CRP represses rtxA not only directly by binding to upstream of rtxA but also indirectly by repressing lrp and hlyU This is the first report of a regulatory network comprising CRP, Lrp, H-NS, and HlyU, which coordinates the rtxA expression in response to environmental signals such as leucine and glucose during infection. This elaborate regulatory network will enhance the fitness of V. vulnificus and contribute to its successful infection within the host.

Differential expression of cell surface proteins in human bone marrow mesenchymal stem cells cultured with or without basic fibroblast growth factor containing medium.

Mesenchymal stem cells (MSCs) are multipotent cells, which have the capability to differentiate into various mesenchymal tissues such as bone, cartilage, fat, tendon, muscle, and marrow stroma. However, they lose the capability of multi-lineage differentiation after several passages. It is known that basic fibroblast growth factor (bFGF) increases growth rate, differentiation potential, and morphological changes of MSCs in vitro. In this report, we have used 2-DE coupled to MS to identify differentially expressed proteins at the cell membrane level in MSCs growing in bFGF containing medium. The cell surface proteins isolated by the biotin-avidin affinity column were separated by 2-DE in triplicate experiments. A total of 15 differentially expressed proteins were identified by quadrupole-time of flight tandem MS. Nine of the proteins were upregulated and six proteins were downregulated in the MSCs cultured with bFGF containing medium. The expression level of three actin-related proteins, F-actin-capping protein subunit alpha-1, actin-related protein 2/3 complex subunit 2, and myosin regulatory light chain 2, was confirmed by Western blot analysis. The results indicate that the expression levels of F-actin-capping protein subunit alpha-1, actin-related protein 2/3 complex subunit 2, and myosin regulatory light chain 2 are important in bFGF-induced morphological change of MSCs.

Nanoparticles up-regulate tumor necrosis factor-alpha and CXCL8 via reactive oxygen species and mitogen-activated protein kinase activation.

Evaluating the toxicity of nanoparticles is an integral aspect of basic and applied sciences, because imaging applications using traditional organic fluorophores are limited by properties such as photobleaching, spectral overlaps, and operational difficulties. This study investigated the toxicity of nanoparticles and their biological mechanisms. We found that nanoparticles, quantum dots (QDs), considerably activated the production of tumor necrosis factor (TNF)-alpha and CXC-chemokine ligand (CXCL) 8 through reactive oxygen species (ROS)- and mitogen-activated protein kinases (MAPKs)-dependent mechanisms in human primary monocytes. Nanoparticles elicited a robust activation of intracellular ROS, phosphorylation of p47phox, and nicotinamide adenine dinucleotide phosphate oxidase activities. Blockade of ROS generation with antioxidants significantly abrogated the QD-mediated TNF-alpha and CXCL8 expression in monocytes. The induced ROS generation subsequently led to the activation of MAPKs, which were crucial for mRNA and protein expression of TNF-alpha and CXCL8. Furthermore, confocal and electron microscopy analyses showed that internalized QDs were trapped in cytoplasmic vesicles and compartmentalized inside lysosomes. Finally, several repeated intravenous injections of QDs caused an increase in neutrophil infiltration in the lung tissues in vivo. These results provide novel insights into the QD-mediated chemokine induction and inflammatory toxic responses in vitro and in vivo.

Crystal Structure of the Regulatory Domain of MexT, a Transcriptional Activator of the MexEFOprN Efflux Pump in Pseudomonas aeruginosa.

The Gram-negative opportunistic pathogen, Pseudomonas aeruginosa , has multiple multidrug efflux pumps. MexT, a LysR-type transcriptional regulator, functions as a transcriptional activator of the MexEF-OprN efflux system. MexT consists of an N-terminal DNA-binding domain and a C-terminal regulatory domain (RD). Little is known regarding MexT ligands and its mechanism of activation. We elucidated the crystal structure of the MexT RD at 2.0 Å resolution. The structure comprised two protomer chains in a dimeric arrangement. MexT possessed an arginine-rich region and a hydrophobic patch lined by a variable loop, both of which are putative ligand-binding sites. The three-dimensional structure of MexT provided clues to the interacting ligand structure. A DNase I footprinting assay of full-length MexT identified two MexT-binding sequence in the mexEF oprN promoter. Our findings enhance the understanding of the regulation of MexT-dependent activation of efflux pumps.

Small-molecule inhibitor of HlyU attenuates virulence of Vibrio species.

Increasing antibiotic resistance has led to the development of new strategies to combat bacterial infection. Anti-virulence strategies that impair virulence of bacterial pathogens are one of the novel approaches with less selective pressure for developing resistance than traditional strategies that impede viability. In this study, a small molecule CM14 [N-(4-oxo-4H-thieno[3,4-c]chromen-3-yl)-3-phenylprop-2-ynamide] that inhibits the activity of HlyU, a transcriptional regulator essential for the virulence of the fulminating human pathogen Vibrio vulnificus, has been identified. Without affecting bacterial growth or triggering the host cell death, CM14 reduces HlyU-dependent expression of virulence genes in V. vulnificus. In addition to the decreased hemolysis of human erythrocytes, CM14 impedes host cell rounding and lysis caused by V. vulnificus. Notably, CM14 significantly enhances survival of mice infected with V. vulnificus by alleviating hepatic and renal dysfunction and systemic inflammation. Biochemical, mass spectrometric, and mutational analyses revealed that CM14 inhibits HlyU from binding to target DNA by covalently modifying Cys30. Remarkably, CM14 decreases the expression of various virulence genes of other Vibrio species and thus attenuates their virulence phenotypes. Together, this molecule could be an anti-virulence agent against HlyU-harboring Vibrio species with a low selective pressure for the emergence of resistance.