Salmonella virulence effector SopE and Host GEF ARNO cooperate to recruit and activate WAVE to trigger bacterial invasion

Salmonella virulence effectors elicit host cell membrane ruffling to facilitate pathogen invasion. The WAVE regulatory complex (WRC) governs the underlying membrane-localized actin polymerization, but how Salmonella manipulates WRC is unknown. We show that Rho GTPase activation by the Salmonella guanine nucleotide exchange factor (GEF) SopE efficiently triggered WRC recruitment but not its activation, which required host Arf GTPase activity. Invading Salmonella recruited and activated Arf1 to facilitate ruffling and uptake. Arf3 and Arf6 could also enhance invasion. RNAi screening of host Arf-family GEFs revealed a key role for ARNO in pathogen invasion and generation of pathogen-containing macropinosomes enriched in Arf1 and WRC. Salmonella recruited ARNO via Arf6 and the phosphoinositide phosphatase effector SopB-induced PIP3 generation. ARNO in turn triggered WRC recruitment and activation, which was dramatically enhanced when SopE and ARNO cooperated. Thus, we uncover a mechanism by which pathogen and host GEFs synergize to regulate WRC and trigger Salmonella invasion.     

Bacterial delivery of large intact genomic-DNA-containing BACs into mammalian cells

Efficient delivery of large intact vectors into mammalian cells remains problematical. Here we evaluate delivery by bacterial invasion of two large BACs of more than 150 kb in size into various cells. First, we determined the effect of several drugs on bacterial delivery of a small plasmid into different cell lines. Most drugs tested resulted in a marginal increase of the overall efficiency of delivery in only some cell lines, except the lysosomotropic drug chloroquine, which was found to increase the efficiency of delivery by 6-fold in B16F10 cells. Bacterial invasion was found to be significantly advantageous compared with lipofection in delivering large intact BACs into mouse cells, resulting in 100% of clones containing intact DNA. Furthermore, evaluation of expression of the human hypoxanthine phosphoribosyltransferase (HPRT) gene from its genomic locus, which was present in one of the BACs, showed that single copy integrations of the HPRT-containing BAC had occurred in mouse B16F10 cells and that expression of HPRT from each human copy was 0.33 times as much as from each endogenous mouse copy. These data provide new evidence that bacterial delivery is a convenient and efficient method to transfer large intact therapeutic genes into mammalian cells.     

Regulation of signaling at regions of cell-cell contact by endoplasmic reticulum-bound protein-tyrosine phosphatase 1B

Protein-tyrosine phosphatase 1B (PTP1B) is a ubiquitously expressed PTP that is anchored to the endoplasmic reticulum (ER). PTP1B dephosphorylates activated receptor tyrosine kinases after endocytosis, as they transit past the ER. However, PTP1B also can access some plasma membrane (PM)-bound substrates at points of cell-cell contact. To explore how PTP1B interacts with such substrates, we utilized quantitative cellular imaging approaches and mathematical modeling of protein mobility. We find that the ER network comes in close proximity to the PM at apparently specialized regions of cell-cell contact, enabling PTP1B to engage substrate(s) at these sites. Studies using PTP1B mutants show that the ER anchor plays an important role in restricting its interactions with PM substrates mainly to regions of cell-cell contact. In addition, treatment with PTP1B inhibitor leads to increased tyrosine phosphorylation of EphA2, a PTP1B substrate, specifically at regions of cell-cell contact. Collectively, our results identify PM-proximal sub-regions of the ER as important sites of cellular signaling regulation by PTP1B.     

Biological evaluation of cobalt(II) complexes with non-steroidal anti-inflammatory drug naproxen

Cobalt(II) complexes with the non-steroidal anti-inflammatory drug naproxen in the presence or absence of nitrogen-donor heterocyclic ligands (pyridine, 2,2′-bipyridine or 1,10-phenanthroline) have been synthesized and characterized with physicochemical and spectroscopic techniques. The deprotonated naproxen acts as monodentate ligand coordinated to Co(II) ion through a carboxylato oxygen. The crystal structure of [bis(aqua)bis(naproxenato)bis(pyridine)cobalt(II)], 2 has been determined by X-ray crystallography. The EPR spectrum of complex 2 in frozen solution reveals that it retains its structure. UV study of the interaction of the complexes with calf-thymus DNA (CT DNA) has shown that the complexes can bind to CT DNA and [(2,2′-bipyridine)bis(methanol)bis(naproxenato)cobalt(II)] exhibits the highest binding constant to CT DNA. The cyclic voltammograms of the complexes recorded in DMSO solution and in the presence of CT DNA in 1/2 DMSO/buffer (containing 150 mM NaCl and 15 mM trisodium citrate at pH 7.0) solution have shown that they can bind to CT DNA by the intercalative binding mode which has also been verified by DNA solution viscosity measurements. Competitive study with ethidium bromide (EB) has shown that the complexes can displace the DNA-bound EB indicating that they bind to DNA in strong competition with EB. Naproxen and its cobalt(II) complexes exhibit good binding propensity to human or bovine serum albumin proteins having relatively high binding constant values. The antioxidant activity of the compounds has been evaluated indicating their high scavenging activity against hydroxyl free radicals and superoxide radicals.     

Application of the ELISPOT method for comparative analysis of interleukin (IL)-6 and IL-10 secretion in peripheral blood of patients with astroglial tumors

Glioblastoma, (grade IV astrocytoma), is characterized by rapid growth and resistance to treatment. Identification of markers of aggressiveness in this tumor could represent new therapeutic targets. Interleukins (IL)-6 and IL-10 may be considered as possible candidates, regulating cell growth, resistance to chemotherapy and angiogenesis. ELISPOT method provides a useful tool for the determination of the exact cell number of peripheral lymphocytes secreting a specific cytokine. IL-6 and IL-10 secretion levels were determined using ELISPOT methodology in peripheral blood mononuclear cells of 18 patients with astrocytic neoplasms (3 grade II and 15 grade IV), in parallel with 18 healthy controls. Additionally, immunohistochemical expression of these two cytokines was performed in paraffin-embedded neoplastic tissue in 12 of these patients. The secretion of IL-6 from peripheral monocytes was significantly higher in glioma patients compared to controls (P = 0.0003). In addition, IL-10 secretion from peripheral mononuclear and tumor cells of glioma patients was also higher as compared to healthy controls (P = 0.0002). Based on immunohistochemical staining, IL-6 expression was localized in tumor cells and macrophages as well as in areas of large ischemic necrosis, while the major source of IL-10 expression in glioblastomas was the microglia/macrophage cells. It is suggested that IL-10 contributes to the progression of astrocytomas by suppressing the patient’s immune response, whereas IL-6 provides an additional growth advantage. This study demonstrates for the first time the usefulness of ELISPOT in estimating the secretion of IL-6 and IL-10 from peripheral blood and the correlation of their expression in neoplastic cells. Christina Piperi and Penelope Korkolopoulou have equally contributed to this work.     

Apoptosis in medfly hemocytes is regulated during pupariation through FAK, Src, ERK, PI-3K p85a, and Akt survival signaling

Focal adhesion kinase (FAK) and its downstream signaling targets are implicated in the process of apoptosis induced by external stimuli, in several mammalian systems. In this report, we demonstrate, that medfly (Ceratitis capitata) hemocytes do undergo apoptosis during larval development. In particular, we show using Western blot, ELISA and flow cytometry analysis, that FAK expression silencing in transfected by FAK double-stranded RNA (dsRNA) hemocytes, enhances twofold hemocyte apoptosis, by signaling through Src, MEK/ERK, and PI-3K/Akt signaling pathways. FAK expression silencing, in response to FAK dsRNA treatment, blocks partially the phosphorylation of its downstream targets. Pre-incubation of hemocytes, with specific inhibitors of FAK downstream signaling molecules, demonstrated that all these inhibitors reduced hemocyte viability and enhanced the magnitude of apoptosis about threefold. This data suggest that these pathways contribute to hemocyte survival and/or death during development. The expression and phosphorylation of FAK, Src, PI-3K p85a, Akt, and ERK signaling molecules appear to be dependent upon developmental stages. The expression and phosphorylation of the above signaling molecules, in annexin-positive and annexin-negative hemocytes is also distinct. The maximum expression and phosphorylation of FAK, Src, PI-3K p85a, Akt, and ERK appeared in annexin-positive hemocytes, in both early and late apoptotic hemocytes. The novel aspect of this report is based on the fact that hemocytes attempt to suppress apoptosis, by increasing the expression/phosphorylation of FAK and, hence its downstream targets signaling molecules Src, ERK, PI-3K p85a, and Akt. Evidently, the basic survival pathways among insects and mammals appear to remain unchanged, during evolution.     

A Highlights from MBoC Selection: Systematic identification of pathological lamin A interactors

Laminopathies are a collection of phenotypically diverse diseases that include muscular dystrophies, cardiomyopathies, lipodystrophies, and premature aging syndromes. Laminopathies are caused by >300 distinct mutations in the LMNA gene, which encodes the nuclear intermediate filament proteins lamin A and C, two major architectural elements of the mammalian cell nucleus. The genotype–phenotype relationship and the basis for the pronounced tissue specificity of laminopathies are poorly understood. Here we seek to identify on a global scale lamin A–binding partners whose interaction is affected by disease-relevant LMNA mutations. In a screen of a human genome–wide ORFeome library, we identified and validated 337 lamin A–binding proteins. Testing them against 89 known lamin A disease mutations identified 50 disease-associated interactors. Association of progerin, the lamin A isoform responsible for the premature aging disorder Hutchinson–Gilford progeria syndrome, with its partners was largely mediated by farnesylation. Mapping of the interaction sites on lamin A identified the immunoglobulin G (IgG)–like domain as an interaction hotspot and demonstrated that lamin A variants, which destabilize the Ig-like domain, affect protein–protein interactions more globally than mutations of surface residues. Analysis of a set of LMNA mutations in a single residue, which result in three phenotypically distinct diseases, identified disease-specific interactors. The results represent a systematic map of disease-relevant lamin A interactors and suggest loss of tissue-specific lamin A interactions as a mechanism for the tissue-specific appearance of laminopathic phenotypes.     

Exercise-induced oxidatively damaged DNA in humans: evaluation in plasma or urine?

Physical exercise can induce oxidative damage in humans. 8-Hydroxy-2′-deoxyguanosine (8-OHdG) is a widely known biomarker of DNA oxidation, which can be determined in blood and urine. The aim of the present study was to compare these two biological fluids in terms of which is more suitable for the estimation of the oxidative damage of DNA by measuring the concentration of 8-OHdG one hour after maximal exercise by enzyme immunoassay. The concentration of 8-OHdG increased with exercise only in plasma (p?<?0.001), and values differed between exercise tests in both plasma and urine (p?<?0.05). In conclusion, plasma appears to be more sensitive to exercise-induced 8-OHdG changes than urine and, hence, a more appropriate medium for assessing oxidative damage of DNA, although the poor repeatability of the measurement needs to be addressed in future studies