Membrane proteome of the green sulfur bacterium Chlorobium tepidum (syn. Chlorobaculum tepidum) analyzed by gel-based and gel-free methods

Chlorobium tepidum is a Gram-negative bacterium of the green sulfur phylum (Chlorobia). Chlorobia are obligate anaerobic photolithoautotrophs that are widely distributed in aquatic environments where anoxic layers containing reduced sulfur compounds are exposed to light. The envelope of C. tepidum is a complex organelle composed of the outer membrane, the periplasm–peptidoglycan layer, and the cytoplasmic membrane. In addition to the outer and plasma membranes, C. tepidum contains chlorosomes attached to the cytoplasmic side of the plasma membrane. Each cellular compartment has a unique set of proteins, called sub-proteome. An important aim of proteome analysis is to study the level of the expressed genes and their response to environmental changes. Membrane protein studies are of primary importance to understand how nutrients are transported inside the cell, how toxic molecules are exported, and the mechanisms of photosynthesis and energy metabolism.     

Functionality of the GAL4/UAS system in <Emphasis Type="Italic">Tribolium</Emphasis> requires the use of endogenous core promoters

Background  

The red flour beetle Tribolium castaneum has developed into an insect model system second only to Drosophila. Moreover, as a coleopteran it represents the most species-rich metazoan taxon which also includes many pest species. The genetic toolbox for Tribolium research has expanded in the past years but spatio-temporally controlled misexpression of genes has not been possible so far.     

The IGF-1 network in lung carcinoma therapeutics

Elucidation of the molecular events that underlie respiratory epithelium carcinogenesis still remains a largely unresolved issue. Various new therapeutic interventions are in advanced clinical testing or in daily clinical practice based on available preclinical findings. However, the complex molecular interplay that characterizes carcinogenesis requires further investigation to identify the pivotal factors and their interactions that might render the treatment of these malignancies more effective. Insulin-like growth factor-1 (IGF-1) network is a new important signalling cascade in lung carcinogenesis. Here, we integrate updated results that further support the significance of IGF-1 molecular circuitry in respiratory epithelium tumourigenesis, and pose future perspectives regarding its optimal use in the therapeutic field.     

A phase I trial of adoptive transfer of allogeneic natural killer cells in patients with advanced non-small cell lung cancer

HLA-mismatched natural killer (NK) cells have shown efficacy in acute myeloid leukemia, and their adoptive transfer in patients with other malignancies has been proven safe. This phase I clinical trial was designed to evaluate safety (primary endpoint) and possible clinical efficacy (secondary endpoint) of repetitive administrations of allogeneic, in vitro activated and expanded NK cells along with chemotherapy in patients with advanced non-small cell lung cancer (NSCLC). Patients with unresectable, locally advanced/metastatic NSCLC receiving 1st/2nd line chemotherapy were eligible to receive 2–4 doses of activated NK cells from two relative donors. Donor’s CD56⁺ cells were cultured for 20–23 days with interleukin-15 (IL-15) and hydrocortisone (HC) and administered intravenously between chemotherapy cycles. Premedication with corticosteroids and/or H1 inhibitors was allowed. Sixteen patients (performance status 0–1) with adenocarcinoma (n = 13) or squamous cell carcinoma (n = 3) at stage IIIb (n = 5) or IV (n = 11) receiving 1st (n = 13) or 2nd (n = 3) line treatment were enrolled. Fifteen patients received 2–4 doses of allogeneic activated NK cells (0.2–29 × 10⁶/kg/dose, median 4.15 × 10⁶/kg/dose). No side effects (local or systemic) were observed. At a median 22-month follow-up (range, 16.5–26 months) 2 patients with partial response and 6 patients with disease stabilization were recorded. Median progression free survival and overall survival were 5.5 and 15 months, respectively. A 56% 1-year survival and a 19% 2-year survival were recorded. In conclusion, repetitive infusions of allogeneic, in vitro activated and expanded with IL-15/HC NK cells, in combination with chemotherapy are safe and potentially clinically effective.     

Ecosystem services and biodiversity conservation: concepts and a glossary

The RUBICODE project draws on expertise from a range of disciplines to develop and integrate frameworks for assessing the impacts of environmental change on ecosystem service provision, and for rationalising biodiversity conservation in that light. With such diverse expertise and concepts involved, interested parties will not be familiar with all the key terminology. This paper defines the terms as used within the project and, where useful, discusses some reasoning behind the definitions. Terms are grouped by concept rather than being listed alphabetically.     

Paradoxical protective effect of central obesity in patients with suspected stable coronary artery disease

Objective:

Increased body mass index (BMI) has been paradoxically inversely associated with the presence of angiographic coronary artery disease (CAD). Central obesity measures, considered to be more appropriate for assessing obesity‐related cardiovascular risk, have been little studied in relation to the presence of CAD. The aim was to investigate the association of central obesity with the presence of angiographic CAD as well as the prognostic significance of obesity measures in CAD prediction when added to other cardiovascular risk factors.

Design and Methods:

Patients with suspected stable CAD (n = 403, age 61 ± 10 years, 302 males) referred for diagnostic coronary angiography with documented anthropometric data were enrolled.

Results:

Significant angiographic CAD was found in 51% of patients. Both BMI (OR = 0.64 per 1 SD increase, P = 0.001) and waist circumference (WC) (OR = 0.54 per 1 SD increase, P < 0.001) were inversely associated with the presence of CAD even after adjustment for cardiovascular risk factors. In subgroup analysis, BMI and WC were significantly inversely associated with the presence of CAD in males, non diabetics, patients >60 years old and patients with Framingham risk score (FRS) >20% (P < 0.01 for all). The addition of BMI or WC in FRS‐based regression models improved prediction of CAD (P = 0.03 and P < 0.001 for BMI and WC respectively) without a significant difference between the two models (P = 0.08).

Conclusions:

Central and overall obesity were independently associated with a reduced prevalence of angiographic CAD, lending further credence to the existence of the ‘obesity paradox’. Obesity measures may further improve risk discrimination for the presence of CAD when added in an established risk score such as FRS.     

The Escherichia coli Peripheral Inner Membrane Proteome

Biological membranes are essential for cell viability. Their functional characteristics strongly depend on their protein content, which consists of transmembrane (integral) and peripherally associated membrane proteins. Both integral and peripheral inner membrane proteins mediate a plethora of biological processes. Whereas transmembrane proteins have characteristic hydrophobic stretches and can be predicted using bioinformatics approaches, peripheral inner membrane proteins are hydrophilic, exist in equilibria with soluble pools, and carry no discernible membrane targeting signals. We experimentally determined the cytoplasmic peripheral inner membrane proteome of the model organism Escherichia coli using a multidisciplinary approach. Initially, we extensively re-annotated the theoretical proteome regarding subcellular localization using literature searches, manual curation, and multi-combinatorial bioinformatics searches of the available databases. Next we used sequential biochemical fractionations coupled to direct identification of individual proteins and protein complexes using high resolution mass spectrometry. We determined that the proposed cytoplasmic peripheral inner membrane proteome occupies a previously unsuspected ∼19% of the basic E. coli BL21(DE3) proteome, and the detected peripheral inner membrane proteome occupies ∼25% of the estimated expressed proteome of this cell grown in LB medium to mid-log phase. This value might increase when fleeting interactions, not studied here, are taken into account. Several proteins previously regarded as exclusively cytoplasmic bind membranes avidly. Many of these proteins are organized in functional or/and structural oligomeric complexes that bind to the membrane with multiple interactions. Identified proteins cover the full spectrum of biological activities, and more than half of them are essential. Our data suggest that the cytoplasmic proteome displays remarkably dynamic and extensive communication with biological membrane surfaces that we are only beginning to decipher.An in-depth understanding of cellular proteomes requires knowledge of protein subcellular topology, assembly in macromolecular complexes, and modification and degradation of poplypeptides. Escherichia coli, a model organism for many such studies, is by far the best studied. The genomes of strain K-12 derivatives MG1655 and W3110 have been sequenced (1, 2), and >75% of their genes have been functionally assigned (3). Almost 90% of the K-12 proteome has been identified experimentally, and >73% of its proteins have known structures (4, 5). Moreover, the genomes of another 38 E. coli strains have been determined (see EcoliWiki for details).In E. coli, like in all Gram-negative bacteria, the bacterial cell envelope comprises the plasma or inner membrane and the outer membrane, which are separated by the periplasmic space. The inner membrane encloses the cytoplasm and is a dynamic substructure. It harbors a wide variety of proteins that function in vital cell processes such as the trafficking of ions, molecules, and macromolecules; cell division; environmental sensing; lipid, polysaccharide, and peptidoglycan biosynthesis; and metabolism. Inner membrane proteins either fully span the lipid bilayer using one or more hydrophobic transmembrane helices (integral) or are bound either directly to phospholipid components or via protein–protein interactions to the surface of the membrane (peripheral) (6) (Fig. 1A). Peripheral inner membrane proteins exist on either side of the membrane and may be recruited in membrane-associated complexes on demand (7). Peripheral inner membrane proteins on the cytoplasmic side constitute a sub-proteome of central importance because of their interaction with the cytoplasmic proteome, the nucleoid, and most of the cell''s metabolism. Thanks to their soluble character and the nature of their interactions with the membrane (mostly electrostatic and moderate hydrophobic interactions (7)), peripheral inner membrane proteins can be extracted using high salt concentrations, extreme pH levels, or chaotropes without disrupting the lipid bilayer (8–11). In contrast, the solubilization of integral proteins requires amphiphilic detergents in order to displace the membrane phospholipids and maintain them as soluble in aqueous solutions (12).Open in a separate windowFig. 1.Bioinformatics and experimental workflow for characterizing peripheral inner membrane proteins. A, schematic representation of the subcellular localization of the E. coli inner membrane peripherome. Protein topology assignment is based on the cellular compartment: A, cytoplasmic; B, integral inner membrane proteins; F1, peripheral inner membrane proteome; r, ribosome. B, schematic diagram for PIM protein annotation. 130 cytoplasmic and PIM E. coli K-12 proteins were downloaded from Uniprot (November 2010) (81) and EchoLOCATION (23). A set of bioinformatics tools was used to predict topologies and features of the unassigned and differently assigned proteins and to further validate existing protein annotations (see supplemental text). For the annotation of additional peripheral membrane proteins, the literature was extensively searched. Additional, other E. coli K-12 databases containing gene ontology annotations (84, 85) and protein homologies through BLAST (44) were employed. Homologues of curated E. coli K-12 proteins were identified in E. coli BL21(DE3) (supplemental Table S1A). C, preparation strategy for detecting the E. coli inner membrane peripherome via nanoLC-MS/MS. Inverted membrane vesicles (IMVs) were isolated and washed extensively with the indicated chemical agents to extract cytoplasmic and PIM proteins (“IMVs washed”), and then their surface was trypsinized (gray arrow). Following digestion, soluble peptides were analyzed via nanoLC-MS/MS. D, protein enrichment at different sample preparation conditions. Top: Relative percentage of proteins detected with the proteolysis approach. Proteins are classified here in three major categories: cytoplasmic (A), ribosomal (r), and peripheral (F1). The bar graphs indicate the percentage of proteins in each category relative to the proteins in other categories at a given sample preparation condition. Bottom: Heat maps showing relative quantities of individual proteins at different sample preparation conditions. Perseus (version 1.2.0.16), a part of the MaxQuant bioinformatics platform, was used for the construction of the heat map (86). A top-three label-free quantitative method was employed (27). Individual protein values across the various treatments are given in supplemental Table S3B.Unlike the cytoplasmic proteome of E. coli, which has been extensively characterized (13), its membrane sub-proteome is still poorly defined. Of 1133 predicted integral inner membrane proteins, only half were experimentally identified through proteomics approaches (14). These figures are constantly being re-evaluated,² but most protein identifications appear robust. In contrast to integral inner membrane proteins, bioinformatics prediction of peripheral inner membrane proteins is currently not possible because they are not known to possess any specific features. Despite the occasional designation of partner proteins identified as peripheral in studies that target inner membrane complexes (15–21), no systematic effort has been undertaken to analyze the peripheral inner membrane proteome.Here we have used a multi-pronged strategy employing bioinformatics, biochemistry, proteomics, and complexomics to systematically determine the peripheral inner membrane proteome of E. coli. We focus exclusively on the peripheral inner membrane proteome that faces the cytoplasm, referred to hereinafter as PIM,¹ and do not analyze peripheral inner membrane proteins residing on the periplasm. Manually curated and re-evaluated topology of the E. coli K-12 proteome was extrapolated to the non-K-12 strain BL21(DE3) (95% proteome homology to K-12) (22). By combining various biochemical treatments, we determined experimentally that several cytoplasmic proteins are also novel PIM proteins, and many of them participate in protein complexes associated with the membrane. Collectively, we demonstrate that a significant, previously unsuspected percentage of the expressed polypeptides constitute the PIM proteome.     

Cytokine effects on cell survival and death of A549 lung carcinoma cells

PurposeIL-13, TNF-α and IL-1β have various effects on lung cancer growth and death, but the signaling pathways mediating these effects have not been extensively analyzed. Therefore, the effects of IL-13, TNF-α and IL-1β alone, and in combination with Fas, on cell viability and death as well as major signaling pathways involved in these effects were investigated in A549 lung carcinoma cells.ResultsUsing MTT and flow cytometry, IL-13, TNF-α and IL-1β pretreatment decreased Fas-induced cell death. These anti-cell death effects were attenuated by pretreatment with inhibitors of Nuclear factor-κB [NF-κB], Phoshatidylinositole-3 kinase [PI3-K], JNK, p38 and ERK1/2 pathways.Using Western blot, IL-13, TNF-α and IL-1β treated cells showed time-dependent expression of p-ERK1/2, p-p38, p-JNK, p-Akt and p-IκBα proteins, decreased IκBα protein expression, no cleavage of Caspase-3 and PARP1 proteins and no notable alterations of Fas protein. IL-13 and TNF-α treated cells showed time-dependent increase of FLIP_L expression.ConclusionIL-13, TNF-α and IL-1β attenuate the pro-cell death effects of Fas on A549 cells, at least partially, by pathways involving the NF-κB, PI3-K and MAP kinases, but not by alterations of Fas protein expression. The IL-13 and TNF-α cell survival effects may also be due to increased expression of FLIP_L protein.     

A versatile strategy for gene trapping and trap conversion in emerging model organisms

Genetic model organisms such as Drosophila, C. elegans and the mouse provide formidable tools for studying mechanisms of development, physiology and behaviour. Established models alone, however, allow us to survey only a tiny fraction of the morphological and functional diversity present in the animal kingdom. Here, we present iTRAC, a versatile gene-trapping approach that combines the implementation of unbiased genetic screens with the generation of sophisticated genetic tools both in established and emerging model organisms. The approach utilises an exon-trapping transposon vector that carries an integrase docking site, allowing the targeted integration of new constructs into trapped loci. We provide proof of principle for iTRAC in the emerging model crustacean Parhyale hawaiensis: we generate traps that allow specific developmental and physiological processes to be visualised in unparalleled detail, we show that trapped genes can be easily cloned from an unsequenced genome, and we demonstrate targeting of new constructs into a trapped locus. Using this approach, gene traps can serve as platforms for generating diverse reporters, drivers for tissue-specific expression, gene knockdown and other genetic tools not yet imagined.     

The Complementary Effects of Atorvastatin and Exercise Treatment on the Composition and Stability of the Atherosclerotic Plaques in ApoE Knockout Mice

Aim

This study aimed to investigate the effects of combined atorvastatin and exercise treatment on the composition and stability of the atherosclerotic plaques in apolipoproteinE (apoE) knockout mice.

Methods

Forty male, apoE^−/− mice were fed a high-fat diet for 16 weeks. Thereafter, while maintained on high-fat diet, they were randomized into four (n = 10) groups for 8 additional weeks: Group CO: Control. Group AT: Atorvastatin treatment (10 mg/Kg/day). Group EX: Exercise-training on treadmill. Group AT+EX: Atorvastatin and simultaneous exercise training. At the study’s end, plasma cholesterol levels, lipids and triglycerides were measured, along with the circulating concentrations of matrix-metalloproteinases (MMP-2,3,8,9) and their inhibitors (TIMP-1,2,3). Plaque area and the relative concentrations of collagen, elastin, macrophages, smooth muscle cells, MMP-2,3,8,9 and TIMP-1,2,3 within plaques were determined. Lastly, MMP activity was assessed in the aortic arch.

Results

All intervention groups showed a lower degree of lumen stenosis, with atheromatous plaques containing more collagen and elastin. AT+EX group had less stenosis and more elastin compared to single intervention groups. MMP-3,-8 -9 and macrophage intra-plaque levels were reduced in all intervention groups. EX group had increased TIMP-1 levels within the lesions, while TIMP-2 was decreased in all intervention groups. The blood levels of the above molecules increased during atherosclerosis development, but they did not change after the therapeutic interventions in accordance to their intra-plaque levels.

Conclusion

The two therapeutic strategies act with synergy regarding the extent of the lesions and lumen stenosis. They stabilize the plaque, increasing its content in elastin and collagen, by influencing the MMP/TIMP equilibrium, which is mainly associated with the macrophage amount. While the increased MMP-2,-3,-8 -9, as well as TIMP-1 and TIMP-2 circulating levels are markers of atherosclerosis, they are not correlated with their corresponding concentrations within the lesions after the therapeutic interventions, and cannot serve as markers for the disease development/amelioration.