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membrane protein - Top 30 Publications

Genetic profiling and surface proteome analysis of human atrial stromal cells and rat ventricular epicardium-derived cells reveals novel insights into their cardiogenic potential.

Epicardium-derived cells (EPDC) and atrial stromal cells (ASC) display cardio-regenerative potential, but the molecular details are still unexplored. Signals which induce activation, migration and differentiation of these cells are largely unknown. Here we have isolated rat ventricular EPDC and rat/human ASC and performed genetic and proteomic profiling. EPDC and ASC expressed epicardial/mesenchymal markers (WT-1, Tbx18, CD73, CD90, CD44, CD105), cardiac markers (Gata4, Tbx5, troponin T) and also contained phosphocreatine. We used cell surface biotinylation to isolate plasma membrane proteins of rEPDC and hASC, Nano-liquid chromatography with subsequent mass spectrometry and bioinformatics analysis identified 396 rat and 239 human plasma membrane proteins with 149 overlapping proteins. Functional GO-term analysis revealed several significantly enriched categories related to extracellular matrix (ECM), cell migration/differentiation, immunology or angiogenesis. We identified receptors for ephrin and growth factors (IGF, PDGF, EGF, anthrax toxin) known to be involved in cardiac repair and regeneration. Functional category enrichment identified clusters around integrins, PI3K/Akt-signaling and various cardiomyopathies. Our study indicates that EPDC and ASC have a similar molecular phenotype related to cardiac healing/regeneration. The cell surface proteome repository will help to further unravel the molecular details of their cardio-regenerative potential and their role in cardiac diseases.

HOCl and the control of oncogenesis.

Sustained generation of extracellular superoxide anions by membrane-associated NADPH oxidase-1 is a hallmark of malignant transformation. The resulting H2O2 drives the proliferation of malignant cells and is converted to HOCl by a Dual oxidase-related peroxidase domain that acts analogously to myeloperoxidase. Whereas H2O2 induces apoptosis nonselectively in nontransformed and transformed cells, HOCl selectively affects malignant cells, as the interaction between HOCl and extracellular superoxide anions allows for site-specific generation of apoptosis-inducing hydroxyl radicals. Transformed cells (early stages of tumor progression) and bona fide tumor cells (representing late stages of tumor progression) respond to exogenous HOCl or HOCl generated by professional phagocytes with induction of apoptosis. In contrast, only transformed cells have the potential to synthesize HOCl through interaction between their superoxide anions/H2O2 and Dual oxidase-related peroxidase released by themselves or neighbouring nontransformed or transformed effector cells. Tumor cells prevent HOCl synthesis through membrane-associated catalase that decomposes H2O2, the substrate for peroxidase, and thus prevents HOCl synthesis. Elimination of malignant cells through HOCl signaling is prevented by Helicobacter pylori-associated catalase and superoxide dismutase, whereas it is enhanced by low dose irradiation and by H2O2-producing lactobacilli in the presence of myeloperoxidase. Peroxidase and catalase that are involved in the control of HOCl signaling are also affecting apoptosis-inducing pathways based on reactive nitrogen species. Modification of tumor cell proteins by HOCl enhances the establishment of a T cell response and thus might be involved in immunogenic modulation. Therefore, targeting the control of HOCl signaling system should allow one to establish novel rational therapeutic approaches.

Validating Coarse Grained Voltage Activation Model by Comparing the Performance to the Results of MC Simulations.

Simulating the nature of voltage activated systems is a problem of major current interest, covering the action of voltage gated ion channels to energy storage batteries. However, fully microscopic converging molecular simulations of external voltage effects present a major challenge and macroscopic models are associated with major uncertainties about the dielectric treatment and the underlying physical basis. Recently we developed a coarse grained (CG) model that represents explicit the electrodes, the electrolytes and the membrane / protein system. The CG model provides a semi-macroscopic way for capturing the microscopic physics of voltage activated systems. Our method was originally validated by reproducing macroscopic and analytical results for key test cases and then used in modeling voltage activated ion channels and related problems. In this work, we further establish the reliability of the CG voltage model by comparing it to the results of Monte Carlo (MC) simulations with a microscopic electrolyte model. The comparison explores different aspects of membrane, electrolytes, electrodes systems ranging from the Gouy-Chapman model to the determination of the electrolyte charge distribution in the solution between two electrodes (without and with separating membrane), as well as the evaluation of gating charges. Overall the agreement is very impressive. This provides a confidence in the CG model and, also shows that the MC model can be used in realistic simulation of voltage activation of proteins with a sufficient computer time.

Novel chloroacetamido compound CWR-J02 is an anti-inflammatory glutaredoxin-1 inhibitor.

Glutaredoxin (Grx1) is a ubiquitously expressed thiol-disulfide oxidoreductase that specifically catalyzes reduction of S-glutathionylated substrates. Grx1 is known to be a key regulator of pro-inflammatory signaling, and Grx1 silencing inhibits inflammation in inflammatory disease models. Therefore, we anticipate that inhibition of Grx1 could be an anti-inflammatory therapeutic strategy. We used a rapid screening approach to test 504 novel electrophilic compounds for inhibition of Grx1, which has a highly reactive active-site cysteine residue (pKa 3.5). From this chemical library a chloroacetamido compound, CWR-J02, was identified as a potential lead compound to be characterized. CWR-J02 inhibited isolated Grx1 with an IC50 value of 32 μM in the presence of 1 mM glutathione. Mass spectrometric analysis documented preferential adduction of CWR-J02 to the active site Cys-22 of Grx1, and molecular dynamics simulation identified a potential non-covalent binding site. Treatment of the BV2 microglial cell line with CWR-J02 led to inhibition of intracellular Grx1 activity with an IC50 value (37 μM). CWR-J02 treatment decreased lipopolysaccharide-induced inflammatory gene transcription in the microglial cells in a parallel concentration-dependent manner, documenting the anti-inflammatory potential of CWR-J02. Exploiting the alkyne moiety of CWR-J02, we used click chemistry to link biotin azide to CWR-J02-adducted proteins, isolating them with streptavidin beads. Tandem mass spectrometric analysis identified many CWR-J02-reactive proteins, including Grx1 and several mediators of inflammatory activation. Taken together, these data identify CWR-J02 as an intracellularly effective Grx1 inhibitor that may elicit its anti-inflammatory action in a synergistic manner by also disabling other pro-inflammatory mediators. The CWR-J02 molecule provides a starting point for developing more selective Grx1 inhibitors and anti-inflammatory agents for therapeutic development.

Soluble CD40 ligand directly alters glomerular permeability and may act as a circulating permeability factor in FSGS.

CD40/CD40 ligand (CD40L) dyad, a co-stimulatory bi-molecular complex involved in the adaptive immune response, has also potent pro-inflammatory actions in haematopoietic and non-haematopoietic cells. We describe here a novel role for soluble CD40L (sCD40L) as modifier of glomerular permselectivity directly acting on glomerular epithelial cells (GECs). We found that stimulation of CD40, constitutively expressed on GEC cell membrane, by the sCD40L rapidly induced redistribution and loss of nephrin in GECs, and increased albumin permeability in isolated rat glomeruli. Pre-treatment with inhibitors of CD40-CD40L interaction completely prevented these effects. Furthermore, in vivo injection of sCD40L induced a significant reduction of nephrin and podocin expression in mouse glomeruli, although no significant increase of urine protein/creatinine ratio was observed after in vivo injection. The same effects were induced by plasma factors partially purified from post-transplant plasma exchange eluates of patients with focal segmental glomerulosclerosis (FSGS), and were blocked by CD40-CD40L inhibitors. Moreover, 17 and 34 kDa sCD40L isoforms were detected in the same plasmapheresis eluates by Western blotting. Finally, the levels of sCD40Lwere significantly increased in serum of children both with steroid-sensitive and steroid-resistant nephrotic syndrome (NS), and in adult patients with biopsy-proven FSGS, compared to healthy subjects, but neither in children with congenital NS nor in patients with membranous nephropathy. Our results demonstrate that sCD40L directly modifies nephrin and podocin distribution in GECs. Moreover, they suggest that sCD40L contained in plasmapheresis eluates from FSGS patients with post-transplant recurrence may contribute, presumably cooperating with other mediators, to FSGS pathogenesis by modulating glomerular permeability.

Synthesis of Core-shell Lanthanide-doped Upconversion Nanocrystals for Cellular Applications.

Lanthanide-doped upconversion nanocrystals (UCNs) have attracted much attention in recent years based on their promising and controllable optical properties, which allow for the absorption of near-infrared (NIR) light and can subsequently convert it into multiplexed emissions that span over a broad range of regions from the UV to the visible to the NIR. This article presents detailed experimental procedures for high-temperature co-precipitation synthesis of core-shell UCNs that incorporate different lanthanide ions into nanocrystals for efficiently converting deep-tissue penetrable NIR excitation (808 nm) into a strong blue emission at 480 nm. By controlling the surface modification with biocompatible polymer (polyacrylic acid, PAA), the as-prepared UCNs acquires great solubility in buffer solutions. The hydrophilic nanocrystals are further functionalized with specific ligands (dibenzyl cyclooctyne, DBCO) for localization on the cell membrane. Upon NIR light (808 nm) irradiation, the upconverted blue emission can effectively activate the light-gated channel protein on the cell membrane and specifically regulate the cation (e.g., Ca(2+)) influx in the cytoplasm. This protocol provides a feasible methodology for the synthesis of core-shell lanthanide-doped UCNs and subsequent biocompatible surface modification for further cellular applications.

Application of Aorta-gonad-mesonephros Explant Culture System in Developmental Hematopoiesis.

The limitation of using mouse embryos for hematopoiesis studies is the added inconvenience in operations, which is largely due to the intrauterine development of the embryo. Although genetic data from knockout (KO) mice are convincing, it is not realistic to generate KO mice for all genes as needed. In addition, performing in vivo rescue experiments to consolidate the data obtained from KO mice is not convenient. To overcome these limitations, the Aorta-Gonad-Mesonephros (AGM) explant culture was developed as an appropriate system to study hematopoietic stem cell (HSC) development. Especially for rescue experiments, it can be used to recover the impaired hematopoiesis in KO mice. By adding the appropriate chemicals into the medium, the impaired signaling can be reactivated or up-regulated pathways can be inhibited. With the use of this method, many experiments can be performed to identify the critical regulators of HSC development, including HSC related gene expression at mRNA and protein levels, colony formation ability, and reconstitution capacity. This series of experiments would be helpful in defining the underlying mechanisms essential for HSC development in mammals.

Ground State Depletion Super-resolution Imaging in Mammalian Cells.

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to dramatic leaps in our understanding of how cells function. The development and availability of super-resolution microscopy has considerably extended the limits of optical resolution from ~250-20 nm. Biologists are no longer limited to describing molecular interactions in terms of colocalization within a diffraction limited area, rather it is now possible to visualize the dynamic interactions of individual molecules. Here, we outline a protocol for the visualization and quantification of cellular proteins by ground-state depletion microscopy for fixed cell imaging. We provide examples from two different membrane proteins, an element of the endoplasmic reticulum translocon, sec61β, and a plasma membrane-localized voltage-gated L-type Ca(2+) channel (CaV1.2). Discussed are the specific microscope parameters, fixation methods, photo-switching buffer formulation, and pitfalls and challenges of image processing.

Optimized LC-MS/MS Method for the High-throughput Analysis of Clinical Samples of Ivacaftor, Its Major Metabolites, and Lumacaftor in Biological Fluids of Cystic Fibrosis Patients.

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening pulmonary manifestations. Ivacaftor (IVA) and ivacaftor-lumacaftor (LUMA) combination are two new breakthrough CF drugs that directly modulate the activity and trafficking of the defective CFTR-protein. However, there is still a dearth of understanding on pharmacokinetic/pharmacodynamic parameters and the pharmacology of ivacaftor and lumacaftor. The HPLC-MS technique for the simultaneous analysis of the concentrations of ivacaftor, hydroxymethyl-ivacaftor, ivacaftor-carboxylate, and lumacaftor in biological fluids in patients receiving standard ivacaftor or ivacaftor-lumacaftor combination therapy has previously been developed by our group and partially validated to FDA standards. However, to allow the high-throughput analysis of a larger number of patient samples, our group has optimized the reported method through the use of a smaller pore size reverse-phase chromatography column (2.6 µm, C8 100 Å; 50 x 2.1 mm) and a gradient solvent system (0-1 min: 40% B; 1-2 min: 40-70% B; 2-2.7 min: held at 70% B; 2.7-2.8 min: 70-90% B; 2.8-4.0 min: 90% B washing; 4.0-4.1 min: 90-40% B; 4.1-6.0 min: held at 40% B) instead of an isocratic elution. The goal of this study was to reduce the HPLC-MS analysis time per sample dramatically from ~15 min to only 6 min per sample, which is essential for the analysis of a large amount of patient samples. This expedient method will be of considerable utility for studies into the exposure-response relationships of these breakthrough CF drugs.

Determination of Hydrophobic Lengths of Membrane Proteins with the HDGB Implicit Membrane Model.

A protocol for predicting the hydrophobic length of membrane proteins using the heterogeneous dielectric generalized Born (HDGB) implicit membrane model is presented. The method involves optimal positioning in the membrane, identification of lipid-facing and inward-facing residues, followed by energy optimization of the implicit membrane model to obtain the hydrophobic length from the optimal membrane width. The latest HDGB version 3 (HDGBv3) and HDGB van der Waals (HDGBvdW) models were applied to a test set containing 15 proteins (seven β-barrel and eight α-helical proteins), for which matching membrane widths are available from experiment, and an additional set contains ten α-helical and ten β-barrel proteins without any experimental data. The results with the HDGB model compare favorably with predictions from methods used in the Orientations of Proteins in Membranes (OPM) and Protein Data Bank of Transmembrane Proteins (PDB-TM) databases.

Expression of CD40/CD40L in colon cancer, and its effect on proliferation and apoptosis of SW48 colon cancer cells.

To study was to study the expression of CD40/CD40L in colon cancer and investigate the effects of CD40/CD40L overexpression on the proliferation and apoptosis of SW48 colon cancer cell line.

PHISTc protein family members localize to different sub-cellular organelles and bind Plasmodium falciparum major virulence factor 'PfEMP-1'.

Plasmodium falciparum (Pf) encodes a novel repertoire of exported protein family called as Plasmodium helical interspersed sub-telomeric (PHIST) that plays diverse roles in infected red blood cells (iRBCs), contributing to malaria pathogenesis. PHIST proteins are central to parasite biology, and modify human erythrocytes by executing interactions with parasite and host proteins. Here, we have attempted to understand the localization and function of two unexplored proteins of PHISTc sub-family 'PFD1140w and PF11_0503', and compared our findings with a well characterized member 'PFI1780w'. We demonstrate that Phist domains assume different oligomeric states owing to a distinct array of subunit interface residues. Co-localization with a Maurer's cleft (MC) signature protein Plasmodium falciparum skeleton binding protein-1 (PfSBP-1), and Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP-1) revealed different sub-cellular destinations for these PHIST members. We further depict the binding of recombinant PHIST proteins to cytoplasmic tail of PfEMP-1, and a novel interaction with PfSBP-1. Interestingly, PFD1140w interacts with PfEMP-1 and PfSBP-1 simultaneously in vitro leading to formation of a complex. These two distant PHISTc members also bind PfEMP-1 on distinct sites, despite sharing the Phist domain. Our data re-emphasize a supportive role for PHIST proteins in cytoadhesion, and identify a new binding partner 'PfSBP-1' for members of this family. This information therefore adds another chapter to the understanding of Pf biology, and highlights the significance of the unexplored PHIST family. This article is protected by copyright. All rights reserved.

Localization of surfactant protein-D in the rheumatoid synovial membrane.

Surfactant protein-D (SP-D) is a collectin, which plays an important role in airway protection and inflammation. The molecule has both pro- and anti-inflammatory capacities depending on its molecular size. Its involvement in joint diseases is largely unknown and the aim of this investigation was to study SP-D occurrence and distribution in the synovial membrane of patients with long-standing rheumatoid arthritis (RA) and osteoarthritis (OA). Six RA patients and six OA patients, who underwent total hip arthroplasty, were included in the study. Synovial tissue biopsies were obtained during surgery and subsequently prepared for immunohistochemistry. In this first, small-scale comparative study on the occurrence of SP-D in the synovial membrane of RA and OA, we report that SP-D was only present in the microvascular endothelium in subsynovial and pannus tissue and that the immunostaining was much stronger than in OA. This distribution pattern suggests that SP-D modulates RA inflammatory activities.

The PMCA Pumps in genetically determined neuronal pathologies.

Ca(2+) signals regulate most aspects of animal cell life. They are of particular importance to the nervous system, in which they regulate specific functions, from neuronal development to synaptic plasticity. The homeostasis of cell Ca(2+) must thus be very precisely regulated: in all cells Ca(2+) pumps transport it from the cytosol to the extracellular medium (the Plasma Membrane Ca(2+) ATPases, hereafter referred to as PMCA pumps) or to the lumen of intracellular organelles (the Sarco/Endoplasmatic Reticulum Ca(2+) ATPase and the Secretory Pathway Ca(2+) ATPase, hereafter referred to as SERCA and SPCA pumps, respectively). In neurons and other excitable cells a powerful plasma membrane Na(+)/Ca(2+) exchanger (NCX) also exports Ca(2+) from cells. Quantitatively, the PMCA pumps are of minor importance to the bulk regulation of neuronal Ca(2+). However, they are important in the regulation of Ca(2+) in specific sub-plasma membrane microdomains which contain a number of enzymes that are relevant to neuronal function. The PMCA pumps (of which 4 basic isoforms are expressed in animal cells) are P-type ATPases that are characterized by a long C-terminal cytosolic tail which is the site of interaction with most of the regulatory factors of the pump, the most important being calmodulin. In resting neurons, at low intracellular Ca(2+)the C-terminal tail of the PMCA interacts with the main body of the protein keeping it in an autoinhibited state. Local Ca(2+) increase activates calmodulin that removes the C-terminal tail from the inhibitory sites. Dysregulation of the Ca(2+) signals are incompatible with healthy neuronal life. A number of genetic mutations of PMCA pumps are associated with pathological phenotypes, those of the neuron-specific PMCA 2 and PMCA 3 being the best characterized. PMCA 2 mutations are associated with deafness and PMCA 3 mutations are linked to cerebellar ataxias. Biochemical analysis of the mutated pumps overexpressed in model cells have revealed their decreased ability to export Ca(2+). The defect in the bulk cytosolic Ca(2+) homeostasis is minor, in keeping with the role of the PMCA pumps in the local control of Ca(2+) in specialized plasma membrane microdomains.

Motor Axon Guidance in Drosophila.

The Drosophila motor system starts to assemble during embryonic development. It is composed of 30 muscles per abdominal hemisegment and 36 motor neurons assembling into nerve branches to exit the CNS, navigate within the muscle field and finally establish specific connections with their target muscles. Several families of guidance molecules that play a role controlling this process as well as transcriptional regulators that program the behavior of specific motoneurons have been identified. In this review we summarize the role of both groups of molecules in the motor system as well as their relationship where known. It is apparent that partially redundant guidance protein families and membrane molecules with different functional output direct guidance decisions cooperatively. Some distinct transcriptional regulators seem to control guidance of specific nerve branches globally directing the expression of groups of pathfinding molecules in all motoneurons within the same motor branch.

Cell membrane dynamics induction using optogenetic tools.

Structures arising from actin-based cell membrane movements, including ruffles, lamellipodia, and filopodia, play important roles in a broad spectrum of cellular functions, such as cell motility, axon guidance in neurons, wound healing, and micropinocytosis. Previous studies investigating these cell membrane dynamics often relied on pharmacological inhibition, RNA interference, and constitutive active/dominant negative protein expression systems. However, such studies did not allow the modulation of protein activity at specific regions of cells, tissues, and organs in animals with high spatial and temporal precision. Recently, optogenetic tools for inducing cell membrane dynamics have been developed which address several of the disadvantages of previous techniques. In a recent study, we developed a powerful optogenetic tool, called the Magnet system, to change cell membrane dynamics through Tiam1 and PIP3 signal transductions with high spatial and temporal resolution. In this review, we summarize recent advances in optogenetic tools that allow us to induce actin-regulated cell membrane dynamics and unique membrane ruffles that we discovered using our Magnet system.

Dibromoacetic acid induced Cl.Ly1+2/-9 T-cell apoptosis and activation of MAPKs signaling cascades.

Dibromoacetic acid (DBA), a haloacetic acid by-product of disinfection of drinking water, can cause many adverse effects in test animals, including immunotoxicity. However, the underlying molecular mechanism for the immunomodulatory effects remains unclear. The present study was undertaken to help in defining some potential mechanisms for this type of toxicity. Here, Cl.Ly1+2/-9 T-cells were exposed to varying levels of DBA and then several parameters, including cell survival, apoptosis, changes in mitochondrial potentials, and effects on select kinases (i.e., p38, ERK1/2, JNK1/2) were examined. The data showed that DBA significantly decreased Cl.Ly1+2/-9 cell viability in a dose-related manner. DBA also induced apoptosis, a decrease in mitochondrial trans-membrane potential, and up-regulated the protein expression of cleaved caspase-3. Moreover, DBA increased the phosphorylation of all three mitogen-activated protein kinases (MAPKs) evaluated. Pre-treatment with specific p38, ERK1/2, and JNK1/2 inhibitors (SB203580, U0126, SP600125, respectively) attenuated the inducible phosphorylation events. DBA also induced up-regulation of mRNA levels of the MAPKs downstream transcription factors ATF-2 and Elk-1. When taken together, the results suggest that DBA could induce murine Cl.Ly1+2/-9 T-cells apoptosis through mitochondria-dependent way, and activate the MAPKs pathways and downstream transcription factors ATF-2 and Elk-1.

MDR1 gene C3435T polymorphism in chronic hepatitis C patients.

MDR1 is a highly polymorphic gene that encodes P-glycoprotein (P-gp). This protein anchor to the cell membrane and transports toxins, xenobiotic, chemicals, and drugs from the intracellular to extracellular and thus protect cells. Polymorphism of the MDR1 gene seems to be effective in gene expression and response to treatment. Since one of the main mechanisms of drug resistance is the removal of the drug from the cell by ATP-dependent efflux proteins, thus MDR1, single nucleotide polymorphism (SNP) C3435T can be used as a predictor for treatment outcomes.

Asymmetric distribution of CRUMBS polarity complex proteins from compacted 8-cell to blastocyst stage during mouse preimplantation development.

During mouse preimplantation development, blastomeres are equipotent until polarity establishment at compacted 8-cell stage. The intrinsic nature of polarity is the asymmetric distribution of polarity proteins between inside and outside blastomeres along the direction of apical-basal axis. This study investigated the early developmental temporal and spatial expression of the main CRUMBS polarity complex proteins in the mouse preimplantation embryo. We observed that Crb3, Pals1, Patj and Mpdz are transcribed in the mouse preimplantation embryo. However, the asymmetric distribution of these polarity proteins is not established until the compacted 8-cell stage. From compaction and thereafter, CRB3 and PALS1 are progressively enriched in the apical membrane, while PATJ and MPDZ are discretely localized at both tight junctions and the apical membrane adjacent to tight junctions. These temporal and spatial distribution patterns suggest that CRUMBS polarity complex might be involved in the cell polarity establishment in the early mouse embryo and reinforce the viewpoint that developmentally spatial asymmetries are first set up at the compaction stage. The present study provides a foundation for further investigation on the functions of CRUMBS polarity complex in trophectoderm specification and blastocyst morphogenesis.

Regulation of neural ion channels by muscarinic receptors.

The excitable behaviour of neurons is determined by the activity of their endogenous membrane ion channels. Since muscarinic receptors are not themselves ion channels, the acute effects of muscarinic receptor stimulation on neuronal function are governed by the effects of the receptors on these endogenous neuronal ion channels. This review considers some principles and factors determining the interaction between subtypes and classes of muscarinic receptors with neuronal ion channels, and summarizes the effects of muscarinic receptor stimulation on a number of different channels, the mechanisms of receptor - channel transduction and their direct consequences for neuronal activity. Ion channels considered include potassium channels (voltage-gated, inward rectifier and calcium activated), voltage-gated calcium channels, cation channels and chloride channels.

The mitochondrial TMEM177 associates with COX20 during COX2 biogenesis.

The three mitochondrial-encoded proteins, COX1, COX2, and COX3, form the core of the cytochrome c oxidase. Upon synthesis, COX2 engages with COX20 in the inner mitochondrial membrane, a scaffold protein that recruits metallochaperones for copper delivery to the CuA-Site of COX2. Here we identified the human protein, TMEM177 as a constituent of the COX20 interaction network. Loss or increase in the amount of TMEM177 affects COX20 abundance leading to reduced or increased COX20 levels respectively. TMEM177 associates with newly synthesized COX2 and SCO2 in a COX20-dependent manner. Our data shows that by unbalancing the amount of TMEM177, newly synthesized COX2 accumulates in a COX20-associated state. We conclude that TMEM177 promotes assembly of COX2 at the level of CuA-site formation.

Identification and characterization of a calcium-dependent lily-type lectin from black rockfish (Sebastes schlegelii): Molecular antennas are involved in host defense via pathogen recognition.

Lily-type lectins (LTLs) are soluble pathogen recognition receptors (PRRs) that contain one or more characteristic carbohydrate recognition domains (CRDs), through which LTLs bind reversibly and specifically to cognate sugar moieties present on the invading pathogen membrane and trigger the host innate immune responses. In this study, we identified a LTL homolog (SsLTL) from black rockfish (Sebastes schlegelii) transcriptome database and its functional roles in innate immunity was investigated in vitro and in vivo. Three mannose-binding sites were found in the protein sequence of SsLTL, among which two sites are conserved with those in mannose-binding lectins of monocotyledonous plants. SsLTL were highly expressed in both the external and internal mucosal tissues of healthy rockfish. During the immune challenge, early up-regulation of SsLTL mRNA expression showed in gill and blood upon both poly I:C and S. iniae challenges. In contrast, the challenge with lipopolysaccharide significantly down-regulated SsLTL expression in both examined tissues. Recombinant SsLTL showed a hemagglutination activity toward fish erythrocytes, which could be enhanced by the addition of calcium ions. Furthermore, strong agglutination activity of SsLTL was also observed with a broad range of fish pathogenic bacteria. Our results implied the crucial role of SsLTL as a PRR molecule in the black rockfish defense mechanism against invading microbial pathogens.

Significant changes in ITIH4, AHSG, ORM1, and CD46 content in milk fat globule membrane proteins of ketotic dairy cows.

High concentrations of non-esterified fatty acid (NEFA) and β-hydroxybutyrate (BHBA) in cows' blood caused by ketosis are associated with inflammatory states. We hypothesised that ketosis in postparturient dairy cows would result in altered levels on inflammation-related proteins not only in plasma but also in the milk fat globule membranes (MFGM). Thirty cows were selected from a dairy farm in Heilongjiang, China. Inflammatory milk fat globule membrane proteins were detected using ELISA kits, and a fully automatic biochemical analyser was used to measure the concentrations of BHBA, NEFA, glucose (GLU) and triglyceride (TG) in plasma. MFGM protein from milk of ketotic cows contained significantly different concentrations of acute-phase response proteins (complement C3 (C3), prothrombin (F2), alpha-1-acid glycoprotein (ORM1), inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), alpha-2-HS-glycoprotein (AHSG), complement C9 (C9), complement regulatory protein variant 4 (CD46)) in comparison with milk from non-ketotic cows. Blood concentrations of C3, complement C9 (C9), tumour necrosis factor α (TNFα), MFGM C3, monocyte differentiation antigen CD14 (CD14) and ORM1 levels were correlated with energy balance. ITIH4 and CD46 increased, and AHSG and ORM1 decreased before the onset of ketosis. These biomarkers offer potential as predictors and monitors of ketosis in at-risk cows.

Impact of heat shock protein 60KD in combination with outer membrane proteins on immune response against Brucella melitensis.

Brucellosis caused by the bacterium Brucella affects various domestic and wild species. The outer membrane proteins 25 and 31 play key roles on stimulation of cell-mediated immune response against Brucella. GroEL as one of the major Brucella antigens stimulates the immune system and increases intracellular survival of bacteria. In the present study, we assumed injection of GroEL in combination with OMP25 and OMP31 would offer higher immunity levels. So, the impact of GroEL with different concentrations of recombinant outer membrane proteins emulsified in Chitosan Nanoparticles on immune responses was evaluated in mice model. Results showed both univalent (except rGroEL) and divalent immunized groups induced higher IFN-γ, TNF-α, and IL-4 titers in comparison to negative control groups. While GroEL showed negative effect on TNF-α titer, there were positive increase trends in IFN-γ in some treatments. Analysis of humoral antibody response revealed both univalent and divalent immunized groups induced higher IgG2a titer than IgG1 titer, indicating strong bent of Th1 immune response. Also, results showed GroEL can have positive impact on lymphocyte proliferation response. Overall, mice immunization using individual OMP25 or OMP31 demonstrated more effective cell-mediated immunity, although some combinations of rGroEL and rOMP31 vaccines were more efficient than other divalent ones.

Subjective reality and brain topology: Inversion transformations on non-orientable atomic surfaces of membrane channels.

Subject-object relations reflect the relation of phenomenology and physics and are at the centre of interest in brain research and neuro-psychology. The unresolved dichotomy behind this relation is one of the most challenging questions of our time. Setting out from causal modelling I suggest a particular topology for subject-object relations and argue that we can find a physical realization in living organism that provides a continuous transform between both domains. In a geometrical metaphor this transform has the topological properties of a one-sided surface or non-orientable flat. I argue that such a surface can be found within the electronic organization of atomic linings in the filter region of ion-conducting membrane proteins. Electron transfer along these atomic surfaces makes chiral induced spin changes to a promising signature of subject-object relations and has found experimental evidence in previous studies. I finally advocate the view that there is a basic dualism between subject and object which is physical on both sides and realized by an inversion relation along one-sided surfaces. The transition between these two aspects however is non-physical and hosts the phenomenology that characterizes subjectivity.

Proliferative and metastatic roles for Phospholipase D in mouse models of cancer.

Phospholipase D (PLD) activity has been proposed to facilitate multiple steps in cancer progression including growth, metabolism, angiogenesis, and mobility. The canonical enzymes PLD1 and PLD2 enact their diverse effects through hydrolyzing the membrane lipid phosphatidylcholine to generate the second messenger and signaling lipid phosphatidic acid (PA). However, the widespread expression of PLD1 and PLD2 in normal tissues and the additional distinct enzymatic mechanisms through which PA can be generated have produced uncertainty regarding the optimal settings in which PLD inhibition might ameliorate cancer. Recent studies in mouse model systems have demonstrated that inhibition or elimination of PLD activity reduces tumor growth and metastasis. One mechanism proposed for this outcome involves proliferative signaling mediated by receptor tyrosine kinases (RTK) and G protein-coupled receptors (GPCR), which is attenuated when downstream PLD signal propagation is suppressed. The reduced proliferative signaling has been reported to be compounded by dysfunctional energetic metabolism in the tumor cells under conditions of nutrient deprivation. Moreover, cancer cells lacking PLD activity display inefficiencies across multiple steps of the metastatic cascade, limiting the tumor's lethal spread. Using PLD isoform knockout mice, recent studies have reported on the net effects of inhibition and ablation in multiple cancer models through examining the role of PLD in the non-tumor cells comprising the stroma and microenvironment. The promising results of such in vivo studies, combined with the apparent low toxicity of highly-specific and potent inhibitors, highlights PLD as an attractive target for therapeutic inhibition in cancer. We discuss here the array of anti-tumor effects produced by PLD inhibition and ablation in cancer models with a focus on animal studies.

The endometria of women with endometriosis exhibit dysfunctional expression of complement regulatory proteins during the mid secretory phase.

The control of complement activation within embryo-endometrium environment is critical for embryo survival. Cell evasion from complement attack requires interaction of complement regulatory proteins (CRPs) with cell adhesion αvβ3 integrin. We aim to compare the expression of CRPs in endometria of women with and without endometriosis and to examine the molecular interaction of decay accelerating factor (DAF) with αvβ3 integrin. Endometrial expression of Membrane cofactor protein (CD46), Decay accelerating factor (DAF), Membrane attack complex inhibitory factor (CD59) and β3 integrin subunit were determined through menstrual cycle by immunohistochemistry. DAF protein quantity was determined by Western blot and mRNA levels measured in epithelial cells isolated by laser capture microdissection (LCM). Using in vitro assay, we examined DAF and β3 integrin expression through paracrine regulation between endometrial compartments. To determine whether β3 integrin and DAF interacts in vivo, endometrial samples were subjected to immunoprecipitation and colocalization using dual immunofluorescence technique. DAF and β3 integrin expression were significantly low in samples from women with endometriosis during mid secretory phase. This observation was supported by decreased DAF protein quantity; faint DAF and β3 integrin interaction and reduced mRNA levels in cells dissected by LCM. Moreover epithelial DAF and β3 integrin expression through paracrine regulation by progesterone from stromal compartment was disrupted in endometriosis. Endometria from women with endometriosis exhibits aberrant expression of complement proteins. The abnormal DAF expression potentially compromises embryo survival, contributing to understand the implantation failure in women with endometriosis.

Establishing and transducing cell polarity: common themes and variations.

All cells in vivo have a primary axis of polarity that controls many aspects of their behaviour, such as the direction of protein secretion and signalling, the orientation of cell division and directed cell movement and morphogenesis. Cell polarise in response to extracellular cues or intracellular landmarks that initiate a signal transduction process that establishes complementary cortical domains of conserved polarity factors. These cortical domains then transmit this polarity to the rest of the cell by regulating the organisation of the cytoskeleton and membrane trafficking systems. Here I review work over the past couple of years that has elucidated many key features of how polarity is established and transduced in different systems, but has also revealed unexpected variations in polarity mechanisms depending on context.

A 100-Year Review: Progress on the chemistry of milk and its components.

Understanding the chemistry of milk and its components is critical to the production of consistent, high-quality dairy products as well as the development of new dairy ingredients. Over the past 100 yr we have gone from believing that milk has only 3 protein fractions to identifying all the major and minor types of milk proteins as well as discovering that they have genetic variants. The structure and physical properties of most of the milk proteins have been extensively studied. The structure of the casein micelle has been the subject of many studies, and the initial views on submicelles have given way to the current model of the micelle as being assembled as a result of the concerted action of several types of interactions (including hydrophobic and the formation of calcium phosphate nanoclusters). The benefits of this improved knowledge of the type and nature of casein interactions include better control of the cheesemaking process, more functional milk powders, development of new products such as cream liqueurs, and expanded food applications. Increasing knowledge of proteins and minerals was paralleled by developments in the analysis of milk fat and its synthesis together with greater knowledge of its packaging in the milk fat globule membrane. Advances in analytical techniques have been essential to the isolation and characterization of milk components. Milk testing has progressed from gross compositional analyses of the fat and total solids content to the rapid analysis of milk for a wide range of components for various purposes, such as diagnostic issues related to animal health. Up to the 1950s, research on dairy chemistry was mostly focused on topics such as protein fractionation, heat stability, acid-base buffering, freezing point, and the nature of the calcium phosphate present in milk. Between the 1950s and 1970s, there was a major focus on identifying all the main protein types, their sequences, variants, association behavior, and other physical properties. During the 1970s and 1980s, one of the major emphases in dairy research was on protein functionality and fractionation processes. The negative cloud over dairy fat has lifted recently due to multiple reviews and meta-analyses showing no association with chronic issues such as cardiovascular disease, but changing consumer misconceptions will take time. More recently, there has been a great deal of interest in the biological and nutritional components in milk and how these materials were uniquely designed by the cow to achieve this type of purpose.

Association of IMMP2L deletions with autism spectrum disorder: A trio family study and meta-analysis.

IMMP2L, the gene encoding the inner mitochondrial membrane peptidase subunit 2-like protein, has been reported as a candidate gene for Tourette syndrome, autism spectrum disorder (ASD) and additional neurodevelopmental disorders. Here we genotyped 100 trio families with an index proband with autism spectrum disorder in Han Chinese population and found three cases with rare exonic IMMP2L deletions. We have conducted a comprehensive meta-analysis to quantify the association of IMMP2L deletions with ASD using 5,568 cases and 10,279 controls. While the IMMP2L deletions carried non-recurrent breakpoints, in contrast to previous reports, our meta-analysis found no evidence of association (P > 0.05) between IMMP2L deletions and ASD. We also observed common exonic deletions impacting IMMP2L in a separate control (5,971 samples) cohort where subjects were screened for psychiatric conditions. This is the first systematic review and meta-analysis regarding the effect of IMMP2L deletions on ASD, but further investigations in different populations, especially Chinese population may be still needed to confirm our results.