Expression of recombinant CD59 with an N-terminal peptide epitope facilitates analysis of residues contributing to its complement-inhibitory function.

PubMed

Zhou, Q; Zhao, J; Hüsler, T; Sims, P J

1996-10-01

CD59 is a plasma membrane-anchored glycoprotein that serves to protect human cells from lysis by the C5b-9 complex of complement. The immunodominant epitopes of CD59 are known to be sensitive to disruption of native tertiary structure, complicating immunological measurement of expressed mutant constructs for structure function analysis. In order to quantify cell-surface expression of wild-type and mutant forms of this complement inhibitor, independent of CD59 antigen, an 11-residue peptide (TAG) recognized by monoclonal antibody (mAb) 9E10 was inserted before the N-terminal codon (L1) of mature CD59, in a pcDNA3 expression plasmid. SV-T2 cells were transfected with this plasmid, yielding cell lines expressing 0 to > 10(5) CD59/cell. The TAG-CD59 fusion protein was confirmed to be GPI-anchored, N-glycosylated and showed identical complement-inhibitory function to wild-type CD59, lacking the TAG peptide sequence. Using this construct, the contribution of each of four surface-localized aromatic residues (4Y, 47F, 61Y, and 62Y) to CD59's complement-inhibitory function was examined. These assays revealed normal surface expression with complete loss of complement-inhibitory function in the 4Y --> S, 47F --> G and 61Y --> S mutants. By contrast, 62Y --> S mutants retained approximately 40% of function of wild-type CD59. These studies confirmed the utility of the TAG-CD59 construct for quantifying CD59 surface expression and activity, and implicate surface aromatic residues 4Y, 47F, 61Y and 62Y as essential to maintenance of CD59's normal complement-regulatory function.

Analysis of the interaction between respiratory syncytial virus and lipid-rafts in Hep2 cells during infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brown, Gaie; Jeffree, Chris E.; McDonald, Terence

2004-10-01

The assembly of respiratory syncytial virus (RSV) in lipid-rafts was examined in Hep2 cells. Confocal and electron microscopy showed that during RSV assembly, the cellular distribution of the complement regulatory proteins, decay accelerating factor (CD55) and CD59, changes and high levels of these cellular proteins are incorporated into mature virus filaments. The detergent-solubility properties of CD55, CD59, and the RSV fusion (F) protein were found to be consistent with each protein being located predominantly within lipid-raft structures. The levels of these proteins in cell-released virus were examined by immunoelectronmicroscopy and found to account for between 5% and 15% of themore » virus attachment (G) glycoprotein levels. Collectively, our findings suggest that an intimate association exists between RSV and lipid-raft membranes and that significant levels of these host-derived raft proteins, such as those regulating complement activation, are subsequently incorporated into the envelope of mature virus particles.« less

Smallpox Inhibitor of Complement Enzymes (SPICE): Dissecting Functional Sites and Abrogating Activity1

PubMed Central

Liszewski, M. Kathryn; Leung, Marilyn K.; Hauhart, Richard; Fang, Celia J.; Bertram, Paula; Atkinson, John P.

2010-01-01

Although smallpox was eradicated as a global illness more than 30 years ago, variola virus and other related pathogenic poxviruses, such as monkeypox, remain potential bioterrorist weapons or could re-emerge as natural infections. Poxviruses express virulence factors that down-modulate the host’s immune system. We previously compared functional profiles of the poxviral complement inhibitors of smallpox, vaccinia, and monkeypox known as SPICE, VCP (or VICE), and MOPICE, respectively. SPICE was the most potent regulator of human complement and attached to cells via glycosaminoglycans. The major goals of the present study were to further characterize the complement regulatory and heparin binding sites of SPICE and to evaluate a mAb that abrogates its function. Using substitution mutagenesis, we established that (1) elimination of the three heparin binding sites severely decreases but does not eliminate glycosaminoglycan binding, (2) there is a hierarchy of activity for heparin binding among the three sites, and (3) complement regulatory sites overlap with each of the three heparin binding motifs. By creating chimeras with interchanges of SPICE and VCP residues, a combination of two SPICE amino acids (H77 plus K120) enhances VCP activity ~200-fold. Also, SPICE residue L131 is critical for both complement regulatory function and accounts for the electrophoretic differences between SPICE and VCP. An evolutionary history for these structure-function adaptations of SPICE is proposed. Finally, we identified and characterized a mAb that inhibits the complement regulatory activity of SPICE, MOPICE, and VCP and thus could be used as a therapeutic agent. PMID:19667083

Complement Evasion Strategies of Viruses: An Overview

PubMed Central

Agrawal, Palak; Nawadkar, Renuka; Ojha, Hina; Kumar, Jitendra; Sahu, Arvind

2017-01-01

Being a major first line of immune defense, the complement system keeps a constant vigil against viruses. Its ability to recognize large panoply of viruses and virus-infected cells, and trigger the effector pathways, results in neutralization of viruses and killing of the infected cells. This selection pressure exerted by complement on viruses has made them evolve a multitude of countermeasures. These include targeting the recognition molecules for the avoidance of detection, targeting key enzymes and complexes of the complement pathways like C3 convertases and C5b-9 formation – either by encoding complement regulators or by recruiting membrane-bound and soluble host complement regulators, cleaving complement proteins by encoding protease, and inhibiting the synthesis of complement proteins. Additionally, viruses also exploit the complement system for their own benefit. For example, they use complement receptors as well as membrane regulators for cellular entry as well as their spread. Here, we provide an overview on the complement subversion mechanisms adopted by the members of various viral families including Poxviridae, Herpesviridae, Adenoviridae, Flaviviridae, Retroviridae, Picornaviridae, Astroviridae, Togaviridae, Orthomyxoviridae and Paramyxoviridae. PMID:28670306

Clinical roundtable monograph: Paroxysmal nocturnal hemoglobinuria: a case-based discussion.

PubMed

Szer, Jeff; Hill, Anita; Weitz, Ilene Ceil

2012-11-01

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, acquired disorder characterized by chronic intravascular hemolysis as the primary clinical manifestation and morbidities that include anemia, thrombosis, renal impairment, pulmonary hypertension, and bone marrow failure. The prevalence of the PNH clone (from <1-100% PNH granulocytes) is approximately 16 per million, and careful monitoring is required. The average age of onset of the clinical disease is the early 30s, although it can present at all ages. PNH is caused by the acquisition of a somatic mutation of the gene phosphatidylinositol glycan anchor (PIG-A) in a multipotent hematopoietic stem cell (HSC), with clonal expansion of the mutated HSC. The mutation causes a deficiency in the synthesis of glycosylphosphatidylinositol (GPI). In cells derived from normal HSCs, the complement regulatory proteins CD55 and CD59 are anchored to the hematopoietic cell membrane surface via GPI, protecting the cells from complement-mediated lysis. However, in patients with PNH, these 2 proteins, along with numerous other GPI-linked proteins, are absent from the cell surface of red cells, granulocytes, monocytes, and platelets, resulting in complement-mediated intravascular hemolysis and other complications. Lysis of red blood cells is the most obvious manifestation, but as other cell lineages are also affected, this complement-mediated attack contributes to additional complications, such as thrombosis. Eculizumab, a humanized monoclonal antibody against the C5 complement protein, is the only effective drug therapy for PNH patients. The antibody prevents cleavage of the C5 protein by C5 convertase, in turn preventing generation of C5b-9 and release of C5a, thereby protecting from hemolysis of cells lacking the CD59 surface protein and other complications associated with complement activation. Drs. Ilene C. Weitz, Anita Hill, and Jeff Szer discuss 3 recent cases of patients with PNH.

Dialysis in rats with acute renal failure: evaluation of three different dialyzer membranes.

PubMed

Kränzlin, B; Gretz, N; Kirschfink, M; Mujais, S K

1996-11-01

Exposure to complement-activating cellulosic dialysis membranes has been claimed to adversely affect the course of acute renal failure (ARF). To test this hypothesis, male Sprague-Dawley rats were allocated to 2 groups: in Group 1, ARF was induced by bilateral renal artery clamping whereas in Group 2, animals underwent a sham procedure. In each group, rats were further allocated to undergo hemodialysis with either a Cuprophan, a Hemophan, or a polyacrylonitrile minidialyzer on Days 4 and 8 after surgery, or no dialysis. Renal function was measured by inulin clearance on the days after dialysis. Additionally, total complement activity (CH50) was estimated on Days 1, 2, 4, and 8, and complement factor C3 was detected immunohistochemically. The degree of renal failure and the rate of recovery of renal function were similar in all the ARF groups irrespective of whether they had undergone dialysis or not, or of the type of the dialysis membrane. Furthermore, there were no significant differences in the course of CH50 or in the amount and distribution of complement factor C3 in the kidney tissue between the rats of Groups 1 and 2. Our findings refute the hypothesis that in ischemic ARF exposure to complement-activating cellulosic dialysis membranes impairs the recovery of renal function in rats.

Pull-down Assay to Characterize Ca2+/Calmodulin Binding to Plant Receptor Kinases.

PubMed

Kaufmann, Christine; Sauter, Margret

2017-01-01

Plant receptor-like kinases (RLKs) are regulated by posttranscriptional modification and by interaction with regulatory proteins. A common modification of RLKs is (auto)phosphorylation, and a common regulatory protein is the calcium sensor calmodulin (CaM). We have developed protocols to detect the interaction of an RLK with CaM. The interaction with CaM was shown by bimolecular fluorescence complementation (BiFC) (see Chapter 14) and pull-down assay (this chapter). Both methods offer unique advantages. BiFC is useful in showing interaction of soluble as well as of membrane-bound proteins in planta. Pull-down assays are restricted to soluble proteins and provide in vitro data. The pull-down assay provides the advantage that proteins can be modified prior to binding and that experimental conditions such as the concentration of Ca 2+ or other divalent cations can be controlled. This chapter provides a pull-down protocol to study RLK-CaM interaction with optional steps to investigate the impact of RLK phosphorylation or of Ca 2+ .

Structure and dynamics of thylakoids in land plants.

PubMed

Pribil, Mathias; Labs, Mathias; Leister, Dario

2014-05-01

Thylakoids of land plants have a bipartite structure, consisting of cylindrical grana stacks, made of membranous discs piled one on top of the other, and stroma lamellae which are helically wound around the cylinders. Protein complexes predominantly located in the stroma lamellae and grana end membranes are either bulky [photosystem I (PSI) and the chloroplast ATP synthase (cpATPase)] or are involved in cyclic electron flow [the NAD(P)H dehydrogenase (NDH) and PGRL1-PGR5 heterodimers], whereas photosystem II (PSII) and its light-harvesting complex (LHCII) are found in the appressed membranes of the granum. Stacking of grana is thought to be due to adhesion between Lhcb proteins (LHCII or CP26) located in opposed thylakoid membranes. The grana margins contain oligomers of CURT1 proteins, which appear to control the size and number of grana discs in a dosage- and phosphorylation-dependent manner. Depending on light conditions, thylakoid membranes undergo dynamic structural changes that involve alterations in granum diameter and height, vertical unstacking of grana, and swelling of the thylakoid lumen. This plasticity is realized predominantly by reorganization of the supramolecular structure of protein complexes within grana stacks and by changes in multiprotein complex composition between appressed and non-appressed membrane domains. Reversible phosphorylation of LHC proteins (LHCPs) and PSII components appears to initiate most of the underlying regulatory mechanisms. An update on the roles of lipids, proteins, and protein complexes, as well as possible trafficking mechanisms, during thylakoid biogenesis and the de-etiolation process complements this review.

Protective responses to sublytic complement in the retinal pigment epithelium

PubMed Central

Tan, Li Xuan; Toops, Kimberly A.; Lakkaraju, Aparna

2016-01-01

The retinal pigment epithelium (RPE) is a key site of injury in inherited and age-related macular degenerations. Abnormal activation of the complement system is a feature of these blinding diseases, yet how the RPE combats complement attack is poorly understood. The complement cascade terminates in the cell-surface assembly of membrane attack complexes (MACs), which promote inflammation by causing aberrant signal transduction. Here, we investigated mechanisms crucial for limiting MAC assembly and preserving cellular integrity in the RPE and asked how these are compromised in models of macular degeneration. Using polarized primary RPE and the pigmented Abca4−/− Stargardt disease mouse model, we provide evidence for two protective responses occurring within minutes of complement attack, which are essential for maintaining mitochondrial health in the RPE. First, accelerated recycling of the membrane-bound complement regulator CD59 to the RPE cell surface inhibits MAC formation. Second, fusion of lysosomes with the RPE plasma membrane immediately after complement attack limits sustained elevations in intracellular calcium and prevents mitochondrial injury. Cholesterol accumulation in the RPE, induced by vitamin A dimers or oxidized LDL, inhibits these defense mechanisms by activating acid sphingomyelinase (ASMase), which increases tubulin acetylation and derails organelle traffic. Defective CD59 recycling and lysosome exocytosis after complement attack lead to mitochondrial fragmentation and oxidative stress in the RPE. Drugs that stimulate cholesterol efflux or inhibit ASMase restore both these critical safeguards in the RPE and avert complement-induced mitochondrial injury in vitro and in Abca4−/− mice, indicating that they could be effective therapeutic approaches for macular degenerations. PMID:27432952

Identity of the segment of human complement C8 recognized by complement regulatory protein CD59.

PubMed

Lockert, D H; Kaufman, K M; Chang, C P; Hüsler, T; Sodetz, J M; Sims, P J

1995-08-25

CD59 antigen is a membrane glycoprotein that inhibits the activity of the C5b-9 membrane attack complex (MAC), thereby protecting human cells from lysis by human complement. The inhibitory function of CD59 derives from its capacity to interact with both the C8 and C9 components of MAC, preventing assembly of membrane-inserted C9 polymer. MAC-inhibitory activity of CD59 is species-selective and is most effective when both C8 and C9 derive from human or other primate plasma. Rabbit C8 and C9, which can substitute for human C8 and C9 in MAC, mediate virtually unrestricted lysis of human cells expressing CD59. In order to identify the segment of human C8 that is recognized by CD59, recombinant peptides containing human or rabbit C8 sequence were expressed in Escherichia coli and purified. CD59 was found to specifically bind to a peptide corresponding to residues 334-385 of the human C8 alpha-subunit, and to require a disulfide bond between Cys345 and Cys369. No specific binding was observed to the corresponding sequence from rabbit C8 alpha (residues 334-386). To obtain functional evidence that this segment of human C8 alpha is selectively recognized by CD59, recombinant C8 proteins were prepared by co-transfecting COS-7 cells with human/rabbit chimeras of the C8 alpha cDNA, and cDNAs encoding the C8 beta and C8 gamma chains. Hemolytic activity of MAC formed with chimeric C8 was analyzed using target cells reconstituted with CD59. These experiments confirmed that CD59 recognizes a conformationally sensitive epitope that is within a segment of human C8 alpha internal to residues 320-415. Our data also suggest that optimal interaction of CD59 with this segment of human C8 alpha is influenced by N-terminal flanking sequence in C8 alpha and by human C8 beta, but is unaffected by C8 gamma.

Expression of complement membrane regulators membrane cofactor protein (CD46), decay accelerating factor (CD55), and protectin (CD59) in human malignant gliomas.

PubMed Central

Mäenpää, A.; Junnikkala, S.; Hakulinen, J.; Timonen, T.; Meri, S.

1996-01-01

Gliomas are malignant brain tumors, which, despite recent progress in surgical and radiological treatment, still have a poor prognosis. Since gliomas apparently resist immunological clearance mechanisms, we became interested in examining bow gliomas resist killing by the human complement system. The resistance of human cells to complement-mediated damage is, in large part, mediated by specific inhibitors of complement:membrane cofactor protein (CD46), decay-accelerating factor (CD55), and protectin (CD59). In the present study we examined the expression of complement regulators in 14 human glioma tumors and in 7 glioma cell lines (U251, U87, HS683, U373, U138, U118, and H2). Protectin was found to be strongly expressed by all glioma tumors and cell lines. Northern blotting analysis demonstrated the typical pattern of four to five protectin mRNAs in the glioma cells. Except for blood vessels, the expression of decay-accelerating factor was weak or absent in the tumors in situ, whereas in the cell lines its expression varied, ranging from negative to intermediate. Membrane cofactor protein was moderately expressed by all the cell lines but only weakly in the tumors. Cell-killing experiments demonstrated that the glioma cell lines were exceptionally resistant to C-mediated lysis. Five of the seven cell lines (U373, HS683, U118, U138, and H2) resisted complement lysis under conditions where most other cell lines were sensitive to killing. Neutralization experiments using specific monoclonal antibodies indicated that protectin was functionally the most important complement regulator in the glioma cells. The killing of the U87 and U251 cells could be significantly increased by a blocking anti-protectin monoclonal antibody, whereas for the other cell lines only moderate or no response was observed. The H2 cell line resisted killing by all antibodies and by complement. These results show that protectin is the most important complement regulator on human glioma cells. The exceptional complement resistance of some glioma cell lines suggests that they may utilize other, hitherto less well characterized, mechanisms to resist complement killing. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 6 Figure 7 PMID:8644856

Inhibition of the Membrane Attack Complex by Dengue Virus NS1 through Interaction with Vitronectin and Terminal Complement Proteins

PubMed Central

Conde, Jonas Nascimento; da Silva, Emiliana Mandarano; Allonso, Diego; Coelho, Diego Rodrigues; Andrade, Iamara da Silva; de Medeiros, Luciano Neves; Menezes, Joice Lima; Barbosa, Angela Silva

2016-01-01

ABSTRACT Dengue virus (DENV) infects millions of people worldwide and is a major public health problem. DENV nonstructural protein 1 (NS1) is a conserved glycoprotein that associates with membranes and is also secreted into the plasma in DENV-infected patients. The present study describes a novel mechanism by which NS1 inhibits the terminal complement pathway. We first identified the terminal complement regulator vitronectin (VN) as a novel DENV2 NS1 binding partner by using a yeast two-hybrid system. This interaction was further assessed by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) assay. The NS1-VN complex was also detected in plasmas from DENV-infected patients, suggesting that this interaction occurs during DENV infection. We also demonstrated that the DENV2 NS1 protein, either by itself or by interacting with VN, hinders the formation of the membrane attack complex (MAC) and C9 polymerization. Finally, we showed that DENV2, West Nile virus (WNV), and Zika virus (ZIKV) NS1 proteins produced in mammalian cells inhibited C9 polymerization. Taken together, our results points to a role for NS1 as a terminal pathway inhibitor of the complement system. IMPORTANCE Dengue is the most important arthropod-borne viral disease nowadays and is caused by dengue virus (DENV). The flavivirus NS1 glycoprotein has been characterized functionally as a complement evasion protein that can attenuate the activation of the classical, lectin, and alternative pathways. The present study describes a novel mechanism by which DENV NS1 inhibits the terminal complement pathway. We identified the terminal complement regulator vitronectin (VN) as a novel DENV NS1 binding partner, and the NS1-VN complex was detected in plasmas from DENV-infected patients, suggesting that this interaction occurs during DENV infection. We also demonstrated that the NS1-VN complex inhibited membrane attack complex (MAC) formation, thus interfering with the complement terminal pathway. Interestingly, NS1 itself also inhibited MAC activity, suggesting a direct role of this protein in the inhibition process. Our findings imply a role for NS1 as a terminal pathway inhibitor of the complement system. PMID:27512066

Release of complement regulatory proteins from ocular surface cells in infections.

PubMed

Cocuzzi, E; Guidubaldi, J; Bardenstein, D S; Chen, R; Jacobs, M R; Medof, E M

2000-11-01

The decay accelerating factor (DAF or CD55) and the membrane inhibitor of reactive lysis (MIRL or CD59), two complement regulatory proteins that protect self cells from autologous complement-mediated injury, are attached to corneal and cqonjunctival epithelial cells by glycosylphos-phatidylinositol (GPI) anchors. We sought to 1) determine the frequency with which bacteria recovered from patients with infections of the eye elaborate factors that can remove these surface proteins from ocular cells, 2) determine the spectrum of bacteria from other sites that have similar effects, and 3) establish the time interval required for reconstitution of the two regulators. Culture supernatants of 18 ocular isolates [P. aeruginosa (n = 3), S. marcescens (n = 1), S. epidermidis (n = 9), and S. aureus (n = 5)], and > 100 other clinical specimens isolated in the hospital's microbiology laboratory [P. mirabilis (n = 1), S. aureus (n = 65), S. epidermidis (n = 24), B. cereus (n = 12), H. influenzae (n = 15), and Enterobacter sp. (n = 21)] were incubated at 37 degrees C for various times with conjunctival epithelial cells, conjunctival fibroblasts or HeLa cells and the release of DAF and CD59 proteins from the surfaces of the cells analyzed by 2-site immunoradiometric assays and by Western blotting. The kinetics of recovery of DAF and CD59 expression on the cells was measured by flow cytometry. DAF and/or CD59 release from the cell monolayers varied from < 5% to > 99% at as much as a 1:81 dilution of the supernatant from some bacteria. On conjunctival epithelial cells, more than 8 hr was required for 44% recovery of DAF expression and for 50% recovery of CD59 expression. Bacteria produce phospholipases and/or other enzymes which can efficiently remove DAF and CD59 from ocular cell surfaces. This phenomenon may correlate with their in vivo pathogenicity.

Trichinella spiralis Paramyosin Binds to C8 and C9 and Protects the Tissue-Dwelling Nematode from Being Attacked by Host Complement

PubMed Central

Zhang, Zhifei; Yang, Jing; Wei, Junfei; Yang, Yaping; Chen, Xiaoqin; Zhao, Xi; Gu, Yuan; Cui, Shijuan; Zhu, Xinping

2011-01-01

Background Paramyosin is a thick myofibrillar protein found exclusively in invertebrates. Evidence suggested that paramyosin from helminths serves not only as a structural protein but also as an immunomodulatory agent. We previously reported that recombinant Trichinella spiralis paramyosin (Ts-Pmy) elicited a partial protective immunity in mice. In this study, the ability of Ts-Pmy to bind host complement components and protect against host complement attack was investigated. Methods and Findings In this study, the transcriptional and protein expression levels of Ts-Pmy were determined in T. spiralis newborn larva (NBL), muscle larva (ML) and adult worm developmental stages by RT-PCR and western blot analysis. Expression of Ts-Pmy at the outer membrane was observed in NBL and adult worms using immunogold electron microscopy and immunofluorescence staining. Functional analysis revealed that recombinant Ts-Pmy(rTs-Pmy) strongly bound to complement components C8 and C9 and inhibited the polymerization of C9 during the formation of the membrane attack complex (MAC). rTs-Pmy also inhibited the lysis of rabbit erythrocytes (ER) elicited by an alternative pathway-activated complement from guinea pig serum. Inhibition of native Ts-Pmy on the surface of NBL with a specific antiserum reduced larvae viability when under the attack of complement in vitro. In vivo passive transfer of anti-Ts-Pmy antiserum and complement-treated larvae into mice also significantly reduced the number of larvae that developed to ML. Conclusion These studies suggest that the outer membrane form of T. spiralis paramyosin plays an important role in the evasion of the host complement attack. PMID:21750743

The functional interactome of GSTP: A regulatory biomolecular network at the interface with the Nrf2 adaption response to oxidative stress.

PubMed

Bartolini, Desirée; Galli, Francesco

2016-04-15

Glutathione S-transferase P (GSTP), and possibly other members of the subfamily of cytosolic GSTs, are increasingly proposed to have roles far beyond the classical GSH-dependent enzymatic detoxification of electrophilic metabolites and xenobiotics. Emerging evidence suggests that these are essential components of the redox sensing and signaling platform of cells. GSTP monomers physically interact with cellular proteins, such as other cytosolic GSTs, signaling kinases and the membrane peroxidase peroxiredoxin 6. Other interactions reported in literature include that with regulatory proteins such as Fanconi anemia complementation group C protein, transglutaminase 2 and several members of the keratin family of genes. Transcription factors downstream of inflammatory and oxidative stress pathways, namely STAT3 and Nrf2, were recently identified to be further components of this interactome. Together these pieces of evidence suggest the existence of a regulatory biomolecular network in which GSTP represents a node of functional convergence and coordination of signaling and transcription proteins, namely the "GSTP interactome", associated with key cellular processes such as cell cycle regulation and the stress response. These aspects and the methodological approach to explore the cellular interactome(s) are discussed in this review paper. Copyright © 2016 Elsevier B.V. All rights reserved.

Homologous species restriction of the complement-mediated killing of nucleated cells.

PubMed Central

Yamamoto, H; Blaas, P; Nicholson-Weller, A; Hänsch, G M

1990-01-01

The homologous restriction of complement (C) lysis is attributed to membrane proteins: decay-accelerating factor (DAF), C8 binding protein (C8bp) and P18/CD59. Since these proteins are also expressed on peripheral blood cells, species restriction was tested for in the complement-mediated killing of antibody-coated human leucocytes by human or rabbit complement. Killing was more efficient when rabbit complement was used. Preincubation of cells with an antibody to DAF abolished the difference. When C1-7 sites were first attached to the cells and either rabbit or human C8, C9 were added, the killing of monocytes and lymphocytes was equally efficient; only in polymorphonuclear neutrophils was a higher efficiency of rabbit C8, C9 seen. Thus, in contrast to haemolysis, restriction occurred predominantly at the C3 level and the action of the terminal complement components was not inhibited. Since C8bp isolated from peripheral blood cells showed essentially similar characteristics as the erythrocyte-derived C8bp, the failure of C8bp to inhibit the action of the terminal components on nucleated cells might reflect differences of the complement membrane interactions between erythrocytes or nucleated cells, respectively. Images Figure 5 PMID:1697561

Mutants in three novel complementation groups inhibit membrane protein insertion into and soluble protein translocation across the endoplasmic reticulum membrane of Saccharomyces cerevisiae

PubMed Central

1992-01-01

We have isolated mutants that inhibit membrane protein insertion into the ER membrane of Saccharomyces cerevisiae. The mutants were contained in three complementation groups, which we have named SEC70, SEC71, and SEC72. The mutants also inhibited the translocation of soluble proteins into the lumen of the ER, indicating that they pleiotropically affect protein transport across and insertion into the ER membrane. Surprisingly, the mutants inhibited the translocation and insertion of different proteins to drastically different degrees. We have also shown that mutations in SEC61 and SEC63, which were previously isolated as mutants inhibiting the translocation of soluble proteins, also affect the insertion of membrane proteins into the ER. Taken together our data indicate that the process of protein translocation across the ER membrane involves a much larger number of gene products than previously appreciated. Moreover, different translocation substrates appear to have different requirements for components of the cellular targeting and translocation apparatus. PMID:1730771

Inhibition of the Membrane Attack Complex by Dengue Virus NS1 through Interaction with Vitronectin and Terminal Complement Proteins.

PubMed

Conde, Jonas Nascimento; da Silva, Emiliana Mandarano; Allonso, Diego; Coelho, Diego Rodrigues; Andrade, Iamara da Silva; de Medeiros, Luciano Neves; Menezes, Joice Lima; Barbosa, Angela Silva; Mohana-Borges, Ronaldo

2016-11-01

Dengue virus (DENV) infects millions of people worldwide and is a major public health problem. DENV nonstructural protein 1 (NS1) is a conserved glycoprotein that associates with membranes and is also secreted into the plasma in DENV-infected patients. The present study describes a novel mechanism by which NS1 inhibits the terminal complement pathway. We first identified the terminal complement regulator vitronectin (VN) as a novel DENV2 NS1 binding partner by using a yeast two-hybrid system. This interaction was further assessed by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) assay. The NS1-VN complex was also detected in plasmas from DENV-infected patients, suggesting that this interaction occurs during DENV infection. We also demonstrated that the DENV2 NS1 protein, either by itself or by interacting with VN, hinders the formation of the membrane attack complex (MAC) and C9 polymerization. Finally, we showed that DENV2, West Nile virus (WNV), and Zika virus (ZIKV) NS1 proteins produced in mammalian cells inhibited C9 polymerization. Taken together, our results points to a role for NS1 as a terminal pathway inhibitor of the complement system. Dengue is the most important arthropod-borne viral disease nowadays and is caused by dengue virus (DENV). The flavivirus NS1 glycoprotein has been characterized functionally as a complement evasion protein that can attenuate the activation of the classical, lectin, and alternative pathways. The present study describes a novel mechanism by which DENV NS1 inhibits the terminal complement pathway. We identified the terminal complement regulator vitronectin (VN) as a novel DENV NS1 binding partner, and the NS1-VN complex was detected in plasmas from DENV-infected patients, suggesting that this interaction occurs during DENV infection. We also demonstrated that the NS1-VN complex inhibited membrane attack complex (MAC) formation, thus interfering with the complement terminal pathway. Interestingly, NS1 itself also inhibited MAC activity, suggesting a direct role of this protein in the inhibition process. Our findings imply a role for NS1 as a terminal pathway inhibitor of the complement system. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Ectromelia virus inhibitor of complement enzymes protects intracellular mature virus and infected cells from mouse complement.

PubMed

Moulton, Elizabeth A; Bertram, Paula; Chen, Nanhai; Buller, R Mark L; Atkinson, John P

2010-09-01

Poxviruses produce complement regulatory proteins to subvert the host's immune response. Similar to the human pathogen variola virus, ectromelia virus has a limited host range and provides a mouse model where the virus and the host's immune response have coevolved. We previously demonstrated that multiple components (C3, C4, and factor B) of the classical and alternative pathways are required to survive ectromelia virus infection. Complement's role in the innate and adaptive immune responses likely drove the evolution of a virus-encoded virulence factor that regulates complement activation. In this study, we characterized the ectromelia virus inhibitor of complement enzymes (EMICE). Recombinant EMICE regulated complement activation on the surface of CHO cells, and it protected complement-sensitive intracellular mature virions (IMV) from neutralization in vitro. It accomplished this by serving as a cofactor for the inactivation of C3b and C4b and by dissociating the catalytic domain of the classical pathway C3 convertase. Infected murine cells initiated synthesis of EMICE within 4 to 6 h postinoculation. The levels were sufficient in the supernatant to protect the IMV, upon release, from complement-mediated neutralization. EMICE on the surface of infected murine cells also reduced complement activation by the alternative pathway. In contrast, classical pathway activation by high-titer antibody overwhelmed EMICE's regulatory capacity. These results suggest that EMICE's role is early during infection when it counteracts the innate immune response. In summary, ectromelia virus produced EMICE within a few hours of an infection, and EMICE in turn decreased complement activation on IMV and infected cells.

Myasthenia gravis: the role of complement at the neuromuscular junction.

PubMed

Howard, James F

2018-01-01

Generalized myasthenia gravis (gMG) is a rare autoimmune disorder characterized by skeletal muscle weakness caused by disrupted neurotransmission at the neuromuscular junction (NMJ). Approximately 74-88% of patients with gMG have acetylcholine receptor (AChR) autoantibodies. Complement plays an important role in innate and antibody-mediated immunity, and activation and amplification of complement results in the formation of membrane attack complexes (MACs), lipophilic proteins that damage cell membranes. The role of complement in gMG has been demonstrated in animal models and patients. Studies in animals lacking specific complement proteins have confirmed that MAC formation is required to induce experimental autoimmune MG (EAMG) and NMJ damage. Complement inhibition in EAMG models can prevent disease induction and reverse its progression. Patients with anti-AChR + MG have autoantibodies and MACs present at NMJs. Damaged NMJs are associated with more severe disease, fewer AChRs, and MACs in synaptic debris. Current MG therapies do not target complement directly. Eculizumab is a humanized monoclonal antibody that inhibits cleavage of complement protein C5, preventing MAC formation. Eculizumab treatment improved symptoms compared with placebo in a phase II study in patients with refractory gMG. Direct complement inhibition could preserve NMJ physiology and muscle function in patients with anti-AChR + gMG. © 2017 The Authors. Annals of the New York Academy of Sciences published by Wiley Periodicals Inc. on behalf of The New York Academy of Sciences.

Predictive value of the complement system for sepsis-induced disseminated intravascular coagulation in septic patients in emergency department.

PubMed

Zhao, Xin; Chen, Yun-Xia; Li, Chun-Sheng

2015-04-01

To investigate changes in circulating complement component C3, membrane attack complex (MAC), and mannose-binding lectin (MBL) in patients with sepsis-induced disseminated intravascular coagulation (DIC). Adult septic patients admitted to the emergency department (ED) of Beijing Chao-Yang Hospital were enrolled. A DIC score of 5 or higher was considered sepsis-induced DIC. Circulating C3, MAC, and MBL levels were detected on ED arrival and compared between patients with and without DIC. The predictive value of C3, MAC, and MBL for sepsis-induced DIC at ED arrival and development of DIC after admission were assessed by receiver operating characteristic curve and logistic regression. We enrolled 267 septic patients between February and December 2013. Complement 3, MAC, and MBL were higher in the DIC group (P < .01). Membrane attack complex was the independent predictor of sepsis-induced DIC. The area under the curve of MAC in predicting sepsis-induced DIC was 0.793. During hospitalization, 25 patients without DIC at enrollment developed DIC. Membrane attack complex and Sequential Organ Failure Assessment independently predicted progress to DIC. The area under the curve of MAC was 0.741. Complement 3, MAC, and MBL were significantly increased in septic patients with DIC. Membrane attack complex independently predicted sepsis-induced DIC and development of DIC after ED admission. Copyright © 2014 Elsevier Inc. All rights reserved.

Identification and cloning of a regulatory gene for nitrogen assimilation in the cyanobacterium Synechococcus sp. strain PCC 7942.

PubMed Central

Vega-Palas, M A; Madueño, F; Herrero, A; Flores, E

1990-01-01

Twenty-seven mutants that were unable to assimilate nitrate were isolated from Synechococcus sp. strain PCC 7942. In addition to mutants that lacked nitrate reductase or nitrite reductase, seven pleiotropic mutants impaired in both reductases, glutamine synthetase, and methylammonium transport were also isolated. One of the pleiotropic mutants was complemented by transformation with a cosmid gene bank from wild-type strain PCC 7942. Three complementing cosmids were isolated, and a 3.1-kilobase-pair DNA fragment that was still able to complement the mutant was identified. The regulatory gene that was cloned (ntcA) appeared to be required for full expression of proteins subject to ammonium repression in Synechococcus sp. PMID:1967601

BIOSENSORS FOR ENVIRONMENTAL MONITORING: A REGULATORY PERSPECTIVE

EPA Science Inventory

Biosensors show the potential to complement laboratory-based analytical methods for environmental applications. Although biosensors for potential environmental-monitoring applications have been reported for a wide range of environmental pollutants, from a regulatory perspective, ...

Protection by meningococcal outer membrane protein PorA-specific antibodies and a serogroup B capsular polysaccharide-specific antibody in complement-sufficient and C6-deficient infant rats.

PubMed

Toropainen, Maija; Saarinen, Leena; Vidarsson, Gestur; Käyhty, Helena

2006-05-01

The relative contributions of antibody-induced complement-mediated bacterial lysis and antibody/complement-mediated phagocytosis to host immunity against meningococcal infections are currently unclear. Further, the in vivo effector functions of antibodies may vary depending on their specificity and Fc heavy-chain isotype. In this study, a mouse immunoglobulin G2a (mIgG2a) monoclonal antibody (MN12H2) to meningococcal outer membrane protein PorA (P1.16), its human IgG subclass derivatives (hIgG1 to hIgG4), and an mIgG2a monoclonal antibody (Nmb735) to serogroup B capsular polysaccharide (B-PS) were evaluated for passive protection against meningococcal serogroup B strain 44/76-SL (B:15:P1.7,16) in an infant rat infection model. Complement component C6-deficient (PVG/c-) rats were used to assess the importance of complement-mediated bacterial lysis for protection. The PorA-specific parental mIgG2a and the hIgG1 to hIgG3 derivatives all induced efficient bactericidal activity in vitro in the presence of human or infant rat complement and augmented bacterial clearance in complement-sufficient HsdBrlHan:WIST rats, while the hIgG4 was unable to do so. In C6-deficient PVG/c- rats, lacking complement-mediated bacterial lysis, the augmentation of bacterial clearance by PorA-specific mIgG2a and hIgG1 antibodies was impaired compared to that in the syngeneic complement-sufficient PVG/c+ rat strain. This was in contrast to the case for B-PS-specific mIgG2a, which conferred similar protective activity in both rat strains. These data suggest that while anti-B-PS antibody can provide protection in the infant rats without membrane attack complex formation, the protection afforded by anti-PorA antibody is more dependent on the activation of the whole complement pathway and subsequent bacterial lysis.

BiFC Assay to Detect Calmodulin Binding to Plant Receptor Kinases.

PubMed

Fischer, Cornelia; Sauter, Margret; Dietrich, Petra

2017-01-01

Plant receptor-like kinases (RLKs) are regulated at various levels including posttranscriptional modification and interaction with regulatory proteins. Calmodulin (CaM) is a calcium-sensing protein that was shown to bind to some RLKs such as the PHYTOSULFOKINE RECEPTOR1 (PSKR1). The CaM-binding site is embedded in subdomain VIa of the kinase domain. It is possible that many more of RLKs interact with CaM than previously described. To unequivocally confirm CaM binding, several methods exist. Bimolecular fluorescence complementation (BiFC) and pull-down assays have been successfully used to study CaM binding to PSKR1 and are described in this chapter (BiFC) and in Chapter 15 (pull down). The two methods are complementary. BiFC is useful to show localization and interaction of soluble as well as of membrane-bound proteins in planta.

PASSIVE HEMOLYSIS BY SERUM AND COBRA VENOM FACTOR: A NEW MECHANISM INDUCING MEMBRANE DAMAGE BY COMPLEMENT*

PubMed Central

Pickering, R. J.; Wolfson, M. R.; Good, R. A.; Gewurz, H.

1969-01-01

The studies presented here indicate that activation of the complement (C′) system by a foreign protein will cause membrane injury and passive lysis of unsensitized erythrocytes present at the time of the reaction. These observations suggest that in addition to the classical antibody-C′-induced cytolysis, there are alternative pathways or mechanisms for activation and participation of the terminal C′ components in the production of cell membrane injury. We have shown that a substance derived from cobra venom and eluted from a single protein band on polyacrylamide can promote lysis of unsensitized autologous or heterologous erythrocytes in the presence of fresh guinea pig serum and that this lysis-inducing activity and C′-inhibiting activity appear to reside in the same fractions. The lytic activity is prevented by several agents known to impair classical C′3 activity, but is unaffected by certain procedures which interfere with the function of C′ components C′1 and C′2, a suggestion that this reaction involves chiefly C′3-C′9. Further, the cobra venom (CV) factor depletes C′ activity in cobra serum, and the CV factor (with its 5S serum cofactor) converts purified C′3 to its inactive form,1 indicating that the reaction of this complex with the complement system occurs without participation of antibody. Therefore, since the lysis-inducing and C′-inhibiting activity of the CV factor appear to result from similar interactions with the complement system, these observations suggest that cell membrane damage and cell lysis can be accomplished through activation of the complement system by a mechanism involving little or no participation of classical antibody or C′ components C′1, 4, or 2. Images PMID:4978744

Expression of proteins in serum, synovial fluid, synovial membrane, and articular cartilage samples obtained from dogs with stifle joint osteoarthritis secondary to cranial cruciate ligament disease and dogs without stifle joint arthritis.

PubMed

Garner, Bridget C; Kuroki, Keiichi; Stoker, Aaron M; Cook, Cristi R; Cook, James L

2013-03-01

To identify proteins with differential expression between healthy dogs and dogs with stifle joint osteoarthritis secondary to cranial cruciate ligament (CCL) disease. Serum and synovial fluid samples obtained from dogs with stifle joint osteoarthritis before (n = 10) and after (8) surgery and control dogs without osteoarthritis (9) and archived synovial membrane and articular cartilage samples obtained from dogs with stifle joint osteoarthritis (5) and dogs without arthritis (5). Serum and synovial fluid samples were analyzed via liquid chromatography-tandem mass spectrometry; results were compared against a nonredundant protein database. Expression of complement component 3 in archived tissue samples was determined via immunohistochemical methods. No proteins had significantly different expression between serum samples of control dogs versus those of dogs with stifle joint osteoarthritis. Eleven proteins (complement component 3 precursor, complement factor I precursor, apolipoprotein B-100 precursor, serum paraoxonase and arylesterase 1, zinc-alpha-2-glycoprotein precursor, serum amyloid A, transthyretin precursor, retinol-binding protein 4 precursor, alpha-2-macroglobulin precursor, angiotensinogen precursor, and fibronectin 1 isoform 1 preproprotein) had significantly different expression (> 2.0-fold) between synovial fluid samples obtained before surgery from dogs with stifle joint osteoarthritis versus those obtained from control dogs. Complement component 3 was strongly expressed in all (5/5) synovial membrane samples of dogs with stifle joint osteoarthritis and weakly expressed in 3 of 5 synovial membrane samples of dogs without stifle joint arthritis. Findings suggested that the complement system and proteins involved in lipid and cholesterol metabolism may have a role in stifle joint osteoarthritis, CCL disease, or both.

Identification of a central role for complement in osteoarthritis

PubMed Central

Wang, Qian; Rozelle, Andrew L.; Lepus, Christin M.; Scanzello, Carla R.; Song, Jason J.; Larsen, D. Meegan; Crish, James F.; Bebek, Gurkan; Ritter, Susan Y.; Lindstrom, Tamsin M.; Hwang, Inyong; Wong, Heidi H.; Punzi, Leonardo; Encarnacion, Angelo; Shamloo, Mehrdad; Goodman, Stuart B.; Wyss-Coray, Tony; Goldring, Steven R.; Banda, Nirmal K.; Thurman, Joshua M.; Gobezie, Reuben; Crow, Mary K.; Holers, V. Michael; Lee, David M.; Robinson, William H.

2011-01-01

Osteoarthritis, characterized by the breakdown of articular cartilage in synovial joints, has long been viewed as the result of “wear and tear”1. Although low-grade inflammation is detected in osteoarthritis, its role is unclear2–4. Here we identify a central role for the inflammatory complement system in the pathogenesis of osteoarthritis. Through proteomic and transcriptomic analyses of synovial fluids and membranes from individuals with osteoarthritis, we find that expression and activation of complement is abnormally high in human osteoarthritic joints. Using mice genetically deficient in C5, C6, or CD59a, we show that complement, and specifically the membrane attack complex (MAC)-mediated arm of complement, is critical to the development of arthritis in three different mouse models of osteoarthritis. Pharmacological modulation of complement in wild-type mice confirmed the results obtained with genetically deficient mice. Expression of inflammatory and degradative molecules was lower in chondrocytes from destabilized joints of C5-deficient mice than C5-sufficient mice, and MAC induced production of these molecules in cultured chondrocytes. Furthermore, MAC co-localized with matrix metalloprotease (MMP)-13 and with activated extracellular signal-regulated kinase (ERK) around chondrocytes in human osteoarthritic cartilage. Our findings indicate that dysregulation of complement in synovial joints plays a critical role in the pathogenesis of osteoarthritis. PMID:22057346

Regulation of age-related macular degeneration-like pathology by complement factor H

PubMed Central

Toomey, Christopher B.; Kelly, Una; Saban, Daniel R.; Bowes Rickman, Catherine

2015-01-01

Complement factor H (CFH) is a major susceptibility gene for age-related macular degeneration (AMD); however, its impact on AMD pathobiology is unresolved. Here, the role of CFH in the development of AMD pathology in vivo was interrogated by analyzing aged Cfh+/− and Cfh−/− mice fed a high-fat, cholesterol-enriched diet. Strikingly, decreased levels of CFH led to increased sub-retinal pigmented epithelium (sub-RPE) deposit formation, specifically basal laminar deposits, following high-fat diet. Mechanistically, our data show that deposits are due to CFH competition for lipoprotein binding sites in Bruch’s membrane. Interestingly and despite sub-RPE deposit formation occurring in both Cfh+/− and Cfh−/− mice, RPE damage accompanied by loss of vision occurred only in old Cfh+/− mice. We demonstrate that such pathology is a function of excess complement activation in Cfh+/− mice versus complement deficiency in Cfh−/− animals. Due to the CFH-dependent increase in sub-RPE deposit height, we interrogated the potential of CFH as a previously unidentified regulator of Bruch’s membrane lipoprotein binding and show, using human Bruch’s membrane explants, that CFH removes endogenous human lipoproteins in aged donors. Thus, advanced age, high-fat diet, and decreased CFH induce sub-RPE deposit formation leading to complement activation, which contributes to RPE damage and visual function impairment. This new understanding of the complicated interactions of CFH in AMD-like pathology provides an improved foundation for the development of targeted therapies for AMD. PMID:25991857

BvrR/BvrS-Controlled Outer Membrane Proteins Omp3a and Omp3b Are Not Essential for Brucella abortus Virulence▿

PubMed Central

Manterola, Lorea; Guzmán-Verri, Caterina; Chaves-Olarte, Esteban; Barquero-Calvo, Elías; de Miguel, María-Jesús; Moriyón, Ignacio; Grilló, María-Jesús; López-Goñi, Ignacio; Moreno, Edgardo

2007-01-01

The Brucella abortus two-component regulatory system BvrR/BvrS controls the expression of outer membrane proteins (Omp) Omp3a (Omp25) and Omp3b (Omp22). Disruption of bvrS or bvrR generates avirulent mutants with altered cell permeability, higher sensitivity to microbicidal peptides, and complement. Consequently, the role of Omp3a and Omp3b in virulence was examined. Similar to bvrS or bvrR mutants, omp3a and omp3b mutants displayed increased attachment to cells, indicating surface alterations. However, they showed unaltered permeability; normal expression of Omp10, Omp16, Omp19, Omp2b, and Omp1; native hapten polysaccharide; and lipopolysaccharide and were resistant to complement and polymyxin B at ranges similar to those of the wild-type (WT) counterpart. Likewise, omp3a and omp3b mutants were able to replicate in murine macrophages and in HeLa cells, were resistant to the killing action of human neutrophils, and persisted in mice, like the WT strain. Murine macrophages infected with the omp3a mutant generated slightly higher levels of tumor necrosis factor alpha than the WT, whereas the bvrS mutant induced lower levels of this cytokine. Since the absence of Omp3a or Omp3b does not result in attenuation, it can be concluded that BvrR/BvrS influences additional Brucella properties involved in virulence. Our results are discussed in the light of previous works suggesting that disruption of omp3a generates attenuated Brucella strains, and we speculate on the role of group 3 Omps. PMID:17664262

Deletion of Crry and DAF on murine platelets stimulates thrombopoiesis and increases factor H-dependent resistance of peripheral platelets to complement attack.

PubMed

Barata, Lidia; Miwa, Takashi; Sato, Sayaka; Kim, David; Mohammed, Imran; Song, Wen-Chao

2013-03-15

Complement receptor 1-related gene/protein y (Crry) and decay-accelerating factor (DAF) are two murine membrane C3 complement regulators with overlapping functions. Crry deletion is embryonically lethal whereas DAF-deficient mice are generally healthy. Crry(-/-)DAF(-/-) mice were viable on a C3(-/-) background, but platelets from such mice were rapidly destroyed when transfused into C3-sufficient mice. In this study, we used the cre-lox system to delete platelet Crry in DAF(-/-) mice and studied Crry/DAF-deficient platelet development in vivo. Rather than displaying thrombocytopenia, Pf4-Cre(+)-Crry(flox/flox) mice had normal platelet counts and their peripheral platelets were resistant to complement attack. However, chimera mice generated with Pf4-Cre(+)-Crry(flox/flox) bone marrows showed platelets from C3(-/-) but not C3(+/+) recipients to be sensitive to complement activation, suggesting that circulating platelets in Pf4-Cre(+)-Crry(flox/flox) mice were naturally selected in a complement-sufficient environment. Notably, Pf4-Cre(+)-Crry(flox/flox) mouse platelets became complement susceptible when factor H function was blocked. Examination of Pf4-Cre(+)-Crry(flox/flox) mouse bone marrows revealed exceedingly active thrombopoiesis. Thus, under in vivo conditions, Crry/DAF deficiency on platelets led to abnormal platelet turnover, but peripheral platelet count was compensated for by increased thrombopoiesis. Selective survival of Crry/DAF-deficient platelets aided by factor H protection and compensatory thrombopoiesis demonstrates the cooperation between membrane and fluid phase complement inhibitors and the body's ability to adaptively respond to complement regulator deficiencies.

Biocompatibility differences with respect to the dialyzer sterilization method.

PubMed

Müller, T F; Seitz, M; Eckle, I; Lange, H; Kolb, G

1998-01-01

The impact of the method of sterilization (steam vs. ethylene oxide, ETO) on indices of biocompatibility is investigated using polysulfone membranes. Eight patients were treated with a random choice of the high-flux membranes F60S (steam) and F60 (ETO) and the low-flux membrane F6 (ETO). Blood samples were taken prior to and 5, 15, 30, 60, and 180 min after the start of hemodialysis. White blood cell count, platelet count, and plasma concentrations of polymorphonuclear neutrophil elastase, complements C3a and C5a, and beta2-microglobulin were determined. The dialysis procedure was associated with a significant decrease in white blood cell count and beta2-microglobulin level and a significant increase in polymorphonuclear neutrophil elastase and complement C3a and C5a levels. However, the steam-sterilized F60S membrane had a significantly lower impact on the biocompatibility indices than the ETO-sterilized F60 and F6 membranes (p < 0.05 or p < 0.001 for the individual markers). We conclude that using steam instead of ETO for sterilization may improve the biocompatibility of membranes.

Effect of complement and its regulation on myasthenia gravis pathogenesis

PubMed Central

Kusner, Linda L; Kaminski, Henry J; Soltys, Jindrich

2015-01-01

Myasthenia gravis (MG) is primarily caused by antibodies directed towards the skeletal muscle acetylcholine receptor, leading to muscle weakness. Although these antibodies may induce compromise of neuromuscular transmission by blocking acetylcholine receptor function or antigenic modulation, the predominant mechanism of injury to the neuromuscular junction is complement-mediated lysis of the postsynaptic membrane. The vast majority of data to support the role of complement derives from experimentally acquired MG (EAMG). In this article, we review studies that demonstrate the central role of complement in EAMG and MG pathogenesis along with the emerging role of complement in T- and B-cell function, as well as the potential for complement inhibitor-based therapy to treat human MG. PMID:20477586

Obstetrical APS: is there a place for hydroxychloroquine to improve the pregnancy outcome?

PubMed

Mekinian, Arsene; Costedoat-Chalumeau, Nathalie; Masseau, Agathe; Tincani, Angela; De Caroli, Sara; Alijotas-Reig, Jaume; Ruffatti, Amelia; Ambrozic, Ales; Botta, Angela; Le Guern, Véronique; Fritsch-Stork, Ruth; Nicaise-Roland, Pascale; Carbonne, Bruno; Carbillon, Lionel; Fain, Olivier

2015-01-01

The use of the conventional APS treatment (the combination of low-dose aspirin and LMWH) dramatically improved the obstetrical prognosis in primary obstetrical APS (OAPS). The persistence of adverse pregnancy outcome raises the need to find other drugs to improve obstetrical outcome. Hydroxychloroquine is widely used in patients with various autoimmune diseases, particularly SLE. Antimalarials have many anti-inflammatory, anti-aggregant and immune-regulatory properties: they inhibit phospholipase activity, stabilize lysosomal membranes, block the production of several pro-inflammatory cytokines and, in addition, impair complement-dependent antigen-antibody reactions. There is ample evidence of protective effects of hydroxychloroquine in OAPS similar to the situation in SLE arising from in vitro studies of pathophysiological working mechanism of hydroxychloroquine. However, the clinical data on the use of hydroxychloroquine in primary APS are lacking and prospective studies are necessary. Copyright © 2014 Elsevier B.V. All rights reserved.

Complement activation on B lymphocytes opsonized with rituximab or ofatumumab produces substantial changes in membrane structure preceding cell lysis.

PubMed

Beum, Paul V; Lindorfer, Margaret A; Beurskens, Frank; Stukenberg, P Todd; Lokhorst, Henk M; Pawluczkowycz, Andrew W; Parren, Paul W H I; van de Winkel, Jan G J; Taylor, Ronald P

2008-07-01

Binding of the CD20 mAb rituximab (RTX) to B lymphocytes in normal human serum (NHS) activates complement (C) and promotes C3b deposition on or in close proximity to cell-bound RTX. Based on spinning disk confocal microscopy analyses, we report the first real-time visualization of C3b deposition and C-mediated killing of RTX-opsonized B cells. C activation by RTX-opsonized Daudi B cells induces rapid membrane blebbing and generation of long, thin structures protruding from cell surfaces, which we call streamers. Ofatumumab, a unique mAb that targets a distinct binding site (the small loop epitope) of the CD20 Ag, induces more rapid killing and streaming on Daudi cells than RTX. In contrast to RTX, ofatumumab promotes streamer formation and killing of ARH77 cells and primary B cells from patients with chronic lymphocytic leukemia. Generation of streamers requires C activation; no streaming occurs in media, NHS-EDTA, or in sera depleted of C5 or C9. Streamers can be visualized in bright field by phase imaging, and fluorescence-staining patterns indicate they contain membrane lipids and polymerized actin. Streaming also occurs if cells are reacted in medium with bee venom melittin, which penetrates cells and forms membrane pores in a manner similar to the membrane-attack complex of C. Structures similar to streamers are demonstrable when Ab-opsonized sheep erythrocytes (non-nucleated cells) are reacted with NHS. Taken together, our findings indicate that the membrane-attack complex is a key mediator of streaming. Streamer formation may, thus, represent a membrane structural change that can occur shortly before complement-induced cell death.

Epstein-Barr virus/complement fragment C3d receptor (CR2) reacts with p53, a cellular antioncogene-encoded membrane phosphoprotein: detection by polyclonal anti-idiotypic anti-CR2 antibodies.

PubMed Central

Barel, M; Fiandino, A; Lyamani, F; Frade, R

1989-01-01

Epstein-Barr virus and the C3d fragment of the third component of complement are specific extracellular ligands for complement receptor type 2 (CR2). However, intracellular proteins that react specifically with CR2 and are involved in post-membrane signals remain unknown. We recently prepared polyclonal anti-idiotypic anti-CR2 antibodies (Ab2) by using the highly purified CR2 molecule as original immunogen. We showed that Ab2 contained anti-idiotypic specificities that mimicked extracellular domains of CR2 and detected two distinct binding sites on CR2 for its specific extracellular ligands, Epstein-Barr virus and C3d. We postulated that Ab2 might also contain specificities that could mimic intracellular domains of CR2. Here we report that Ab2, which did not react with Raji B-lymphoma cell surface components, detected specifically, among all components solubilized from Raji cell membranes, a single intracellular membrane protein of apparent molecular mass of 53 kDa. This protein was identified as the p53 cellular antioncogene-encoded membrane phosphoprotein by analyzing its antigenic properties with Pab1801, a monoclonal anti-p53 antibody, and by comparing its biochemical properties with those of p53. Additionally, solubilized and purified CR2 bound to solubilized p53 immobilized on Pab1801-Sepharose. p53, like CR2, was localized only in purified plasma membranes and nuclei of Raji cells. These data suggest strongly that p53, a cellular antioncogene-encoded phosphoprotein, reacted specifically with CR2 in Raji membranes. This interaction may represent one of the important steps through which CR2 could be involved in human B-lymphocyte proliferation and transformation. Images PMID:2557614

Role of Complement in Red Cell Dysfunction in Trauma

DTIC Science & Technology

2013-12-01

fragmentation 2. Erythrocyte membrane has there major components: 1) membrane proteins, that are either transmembrane or attached to the plasma membrane...through GPI- or lipid-anchors (glycophorins, CD47, CR1, band 3, CD55, CD59, flotillin, stomatin etc.) 2) skeletal proteins, located below the plasma ...glycophorin C with spectrin skeleton 3. More recently, adducin and dematin have also been implicated in linking plasma membrane protein Glut-1

Role of Complement in Red Cell Dysfunction in Trauma

DTIC Science & Technology

2012-12-01

there major components: 1) membrane proteins, that are either transmembrane or attached to the plasma membrane through GPI- or lipid-anchors...glycophorins, CD47, CR1, band 3, CD55, CD59, flotillin, stomatin etc.) 2) skeletal proteins, located below the plasma membrane, conferring the erythrocyte...skeleton 3. More recently, adducin and dematin have also been implicated in linking plasma membrane protein Glut-1 (glucose transporter-1) to spectrin 4

Adaptation of Tri-molecular fluorescence complementation allows assaying of regulatory Csr RNA-protein interactions in bacteria.

PubMed

Gelderman, Grant; Sivakumar, Anusha; Lipp, Sarah; Contreras, Lydia

2015-02-01

sRNAs play a significant role in controlling and regulating cellular metabolism. One of the more interesting aspects of certain sRNAs is their ability to make global changes in the cell by interacting with regulatory proteins. In this work, we demonstrate the use of an in vivo Tri-molecular Fluorescence Complementation assay to detect and visualize the central regulatory sRNA-protein interaction of the Carbon Storage Regulatory system in E. coli. The Carbon Storage Regulator consists primarily of an RNA binding protein, CsrA, that alters the activity of mRNA targets and of an sRNA, CsrB, that modulates the activity of CsrA. We describe the construction of a fluorescence complementation system that detects the interactions between CsrB and CsrA. Additionally, we demonstrate that the intensity of the fluorescence of this system is able to detect changes in the affinity of the CsrB-CsrA interaction, as caused by mutations in the protein sequence of CsrA. While previous methods have adopted this technique to study mRNA or RNA localization, this is the first attempt to use this technique to study the sRNA-protein interaction directly in bacteria. This method presents a potentially powerful tool to study complex bacterial RNA protein interactions in vivo. © 2014 Wiley Periodicals, Inc.

Identification and functional characterisation of an allene oxide synthase from grapevine (Vitis vinifera L. Sauvignon blanc).

PubMed

Dumin, Walftor; Rostas, Michael; Winefield, Christopher

2018-06-01

Jasmonic acid (JA) is known to be an important phytohormone that orchestrates plant defence mechanisms against a range of herbivores and pathogens. Studies have suggested allene oxide synthase (AOS; E.C 4.2.1.92), the first committed step in JA biosynthesis, is essential for JA biosynthesis, yet clear evidence of its role as a biosynthetic regulatory point is lacking, in the main due to conflicting results derived from transgenic studies. However other studies lend support to a biosynthetic regulatory role for AOS. These studies have suggested that certain amino acid substitutions can increase the biosynthetic capacity of the enzyme and consequently improve pathogen tolerance in plants. To explore the role of AOS in Grapevine we isolated and functionally characterised this enzyme for the first time from Vitis vinifera L. Sauvignon blanc. The cloned AOS consisted of a single 1563 bp open reading frame. Comparative sequence analysis showed that the cloned gene (VvAOS) was highly conserved compared to those from other species. Complementation of an Arabidopsis AOS null mutant (aos) with VvAOS recovered the male sterile mutant phenotype and confirmed its function. Transcript analysis showed that VvAOS was wound responsive in leaves and was detectable in most tissues, with the highest levels of transcript in the mesocarp (pulp) of mature berries. Sub-cellular localisation of the VvAOS protein indicated that VvAOS is associated with the chloroplast membrane. Unexpectedly high levels of VvAOS transcript in complemented aos lines did not lead to predicted increases in JA. We have functionally characterised the sole AOS from Grapevine. Patterns of transcript accumulation in grapevine suggest roles in growth, development as well as an important role for JA in fruit ripening. Expression of VvAOS in Arabidopsis suggest complex epigenetic interactions between transgenic and endogenous AOS alleles, providing a possible explanation for why transgenic studies of AOS have delivered conflicting data pointing to a questionable role of AOS as a key regulatory point in JA biosynthesis.

Novel Two-Component System of Streptococcus sanguinis Affecting Functions Associated with Viability in Saliva and Biofilm Formation.

PubMed

Camargo, Tarsila M; Stipp, Rafael N; Alves, Lívia A; Harth-Chu, Erika N; Höfling, José F; Mattos-Graner, Renata O

2018-04-01

Streptococcus sanguinis is a pioneer species of teeth and a common opportunistic pathogen of infective endocarditis. In this study, we identified a two-component system, S. sanguinis SptRS (SptRS Ss ), affecting S. sanguinis survival in saliva and biofilm formation. Isogenic mutants of sptR Ss (SKsptR) and sptS Ss (SKsptS) showed reduced cell counts in ex vivo assays of viability in saliva compared to those of parent strain SK36 and complemented mutants. Reduced counts of the mutants in saliva were associated with reduced growth rates in nutrient-poor medium (RPMI) and increased susceptibility to the deposition of C3b and the membrane attach complex (MAC) of the complement system, a defense component of saliva and serum. Conversely, sptR Ss and sptS Ss mutants showed increased biofilm formation associated with higher levels of production of H 2 O 2 and extracellular DNA. Reverse transcription-quantitative PCR (RT-qPCR) comparisons of strains indicated a global role of SptRS Ss in repressing genes for H 2 O 2 production (2.5- to 15-fold upregulation of spxB , spxR , vicR , tpk , and ackA in sptR Ss and sptS Ss mutants), biofilm formation, and/or evasion of host immunity (2.1- to 11.4-fold upregulation of srtA , pcsB , cwdP , iga , and nt5e ). Compatible with the homology of SptR Ss with AraC-type regulators, duplicate to multiple conserved repeats were identified in 1,000-bp regulatory regions of downstream genes, suggesting that SptR Ss regulates transcription by DNA looping. Significant transcriptional changes in the regulatory genes vicR , spxR , comE , comX , and mecA in the sptR Ss and sptS Ss mutants further indicated that SptRS Ss is part of a regulatory network that coordinates cell wall homeostasis, H 2 O 2 production, and competence. This study reveals that SptRS Ss is involved in the regulation of crucial functions for S. sanguinis persistence in the oral cavity. Copyright © 2018 American Society for Microbiology.

Introducing multiple bio-functional groups on the poly(ether sulfone) membrane substrate to fabricate an effective antithrombotic bio-interface.

PubMed

Wang, Lingren; He, Min; Gong, Tao; Zhang, Xiang; Zhang, Lincai; Liu, Tao; Ye, Wei; Pan, Changjiang; Zhao, Changsheng

2017-11-21

It has been widely recognized that functional groups on biomaterial surfaces play important roles in blood compatibility. To construct an effective antithrombotic bio-interface onto the poly(ether sulfone) (PES) membrane surface, bio-functional groups of sodium carboxylic (-COONa), sodium sulfonic (-SO 3 Na) and amino (-NH 2 ) groups were introduced onto the PES membrane surface in three steps: the synthesis of PES with carboxylic (-COOH) groups (CPES) and water-soluble PES with sodium sulfonic (-SO 3 Na) groups and amino (-NH 2 ) groups (SNPES); the introduction of carboxylic groups onto the PES membrane by blending CPES with PES; and the grafting of SNPES onto CPES/PES membranes via the coupling of amino groups and carboxyl groups. The physical/chemical properties and bioactivities were dependent on the proportions of the additives. After introducing bio-functional groups, the excellent hemocompatibility of the modified membranes was confirmed by the inhibited platelet adhesion and activation, prolonged clotting times, suppressed blood-related complement and leukocyte-related complement receptor activations. Furthermore, cell tests indicated that the modified membranes showed better cytocompatibility in endothelial cell proliferation than the pristine PES membrane due to the synergistic promotion of the functional groups. To sum up, these results suggested that modified membranes present great potential in fields using blood-contacting materials, such as hemodialysis and surface endothelialization.

Bioincompatibility of dialyzer membranes may have a negative impact on outcome of acute renal failure, independent of the dose of dialysis delivered: a retrospective multicenter analysis.

PubMed

Schiffl, H; Lang, S M; Haider, M

1998-01-01

The mortality rate of critically ill patients with acute renal failure (ARF) has remained high. The impact of vigorous intermittent hemodialysis (IHD) on the outcome of ARF has not been validated. In this retrospective multicenter analysis, 154 patients with ARF were treated daily (intensive) or on alternate days (conventional) using complement and cell activating cuprophane (bioincompatible) or high-flux polysulfone dialyzer membranes with insignificant effects on circulating complement or cells (biocompatible). At initiation of IHD, all four groups were similar in patient characteristics and ARF factors. The use of synthetic membranes resulted in a reduced mortality rate (18% vs 45%; p < 0.001) and shorter duration of ARF (8 vs 15 sessions; p < 0.001). Daily IHD with cellulose based membranes tended to increase mortality rates compared with conventional cuprophane dialysis (37% vs 53%). Intensive IHD with polysulfone membranes resulted in a further decrease in overall mortality rates (15% vs 22%). This retrospective analysis shows that bioincompatibility of dialyzer membranes may be more important for the outcome of patients with ARF than the dose of dialysis. Its impact on outcome occurs independently of the dose of dialysis delivered.

Complement activation and choriocapillaris loss in early AMD: Implications for pathophysiology and therapy

PubMed Central

Whitmore, S.Scott; Sohn, Elliott H.; Chirco, Kathleen R.; Drack, Arlene V.; Stone, Edwin M.; Tucker, Budd A.; Mullins, Robert F.

2015-01-01

Age-related macular degeneration (AMD) is a common and devastating disease that can result in severe visual dysfunction. Over the last decade, great progress has been made in identifying genetic variants that contribute to AMD, many of which lie in genes involved in the complement cascade. In this review we discuss the significance of complement activation in AMD, particularly with respect to the formation of the membrane attack complex in the aging choriocapillaris. We review the clinical, histological and biochemical data that indicate that vascular loss in the choroid occurs very early in the pathogenesis of AMD, and discuss the potential impact of vascular dropout on the retinal pigment epithelium, Bruch's membrane and the photoreceptor cells. Finally, we present a hypothesis for the pathogenesis of early AMD and consider the implications of this model on the development of new therapies. PMID:25486088

Complement activation and choriocapillaris loss in early AMD: implications for pathophysiology and therapy.

PubMed

Whitmore, S Scott; Sohn, Elliott H; Chirco, Kathleen R; Drack, Arlene V; Stone, Edwin M; Tucker, Budd A; Mullins, Robert F

2015-03-01

Age-related macular degeneration (AMD) is a common and devastating disease that can result in severe visual dysfunction. Over the last decade, great progress has been made in identifying genetic variants that contribute to AMD, many of which lie in genes involved in the complement cascade. In this review we discuss the significance of complement activation in AMD, particularly with respect to the formation of the membrane attack complex in the aging choriocapillaris. We review the clinical, histological and biochemical data that indicate that vascular loss in the choroid occurs very early in the pathogenesis of AMD, and discuss the potential impact of vascular dropout on the retinal pigment epithelium, Bruch's membrane and the photoreceptor cells. Finally, we present a hypothesis for the pathogenesis of early AMD and consider the implications of this model on the development of new therapies. Copyright © 2014 Elsevier Ltd. All rights reserved.

Mouse genetics and proteomic analyses demonstrate a critical role for complement in a model of DHRD/ML, an inherited macular degeneration

PubMed Central

Garland, Donita L.; Fernandez-Godino, Rosario; Kaur, Inderjeet; Speicher, Kaye D.; Harnly, James M.; Lambris, John D.; Speicher, David W.; Pierce, Eric A.

2014-01-01

Macular degenerations, inherited and age related, are important causes of vision loss. Human genetic studies have suggested perturbation of the complement system is important in the pathogenesis of age-related macular degeneration. The mechanisms underlying the involvement of the complement system are not understood, although complement and inflammation have been implicated in drusen formation. Drusen are an early clinical hallmark of inherited and age-related forms of macular degeneration. We studied one of the earliest stages of macular degeneration which precedes and leads to the formation of drusen, i.e. the formation of basal deposits. The studies were done using a mouse model of the inherited macular dystrophy Doyne Honeycomb Retinal Dystrophy/Malattia Leventinese (DHRD/ML) which is caused by a p.Arg345Trp mutation in EFEMP1. The hallmark of DHRD/ML is the formation of drusen at an early age, and gene targeted Efemp1R345W/R345W mice develop extensive basal deposits. Proteomic analyses of Bruch's membrane/choroid and Bruch's membrane in the Efemp1R345W/R345W mice indicate that the basal deposits comprise normal extracellular matrix (ECM) components present in abnormal amounts. The proteomic analyses also identified significant changes in proteins with immune-related function, including complement components, in the diseased tissue samples. Genetic ablation of the complement response via generation of Efemp1R345W/R345W:C3−/− double-mutant mice inhibited the formation of basal deposits. The results demonstrate a critical role for the complement system in basal deposit formation, and suggest that complement-mediated recognition of abnormal ECM may participate in basal deposit formation in DHRD/ML and perhaps other macular degenerations. PMID:23943789

Arabidopsis CPR5 regulates ethylene signaling via molecular association with the ETR1 receptor.

PubMed

Wang, Feifei; Wang, Lijuan; Qiao, Longfei; Chen, Jiacai; Pappa, Maria Belen; Pei, Haixia; Zhang, Tao; Chang, Caren; Dong, Chun-Hai

2017-11-01

The plant hormone ethylene plays various functions in plant growth, development and response to environmental stress. Ethylene is perceived by membrane-bound ethylene receptors, and among the homologous receptors in Arabidopsis, the ETR1 ethylene receptor plays a major role. The present study provides evidence demonstrating that Arabidopsis CPR5 functions as a novel ETR1 receptor-interacting protein in regulating ethylene response and signaling. Yeast split ubiquitin assays and bi-fluorescence complementation studies in plant cells indicated that CPR5 directly interacts with the ETR1 receptor. Genetic analyses indicated that mutant alleles of cpr5 can suppress ethylene insensitivity in both etr1-1 and etr1-2, but not in other dominant ethylene receptor mutants. Overexpression of Arabidopsis CPR5 either in transgenic Arabidopsis plants, or ectopically in tobacco, significantly enhanced ethylene sensitivity. These findings indicate that CPR5 plays a critical role in regulating ethylene signaling. CPR5 is localized to endomembrane structures and the nucleus, and is involved in various regulatory pathways, including pathogenesis, leaf senescence, and spontaneous cell death. This study provides evidence for a novel regulatory function played by CPR5 in the ethylene receptor signaling pathway in Arabidopsis. © 2017 Institute of Botany, Chinese Academy of Sciences.

Proteomic analysis in peritoneal dialysis patients with different peritoneal transport characteristics.

PubMed

Wen, Qiong; Zhang, Li; Mao, Hai-Ping; Tang, Xue-Qing; Rong, Rong; Fan, Jin-Jin; Yu, Xue-Qing

2013-08-30

Peritoneal membranes can be categorized as high, high average, low average, and low transporters, based on the removal or transport rate of solutes. In this study, we used proteomic analysis to determine the differences in proteins removed by different types of peritoneal membranes. Peritoneal transport characteristics in patients who received peritoneal dialysis therapy were assessed by a peritoneal equilibration test. Two-dimensional differential gel electrophoresis technology followed by quantitative analysis was performed to study the variation in protein expression from peritoneal dialysis effluents (PDE) among different groups. Proteins were identified by MALDI-TOF-MS/MS analyses. Further validation in PDE or serum was performed utilizing ELISA analysis. Proteomics analysis revealed ten protein spots with significant differences in intensity levels among different groups, including vitamin D-binding protein, complement C3, apolipoprotein-A1, complement factor C4A, haptoglobin, alpha-1 antitrypsin, immunoglobulin kappa light chain, alpha-2-microglobulin, retinol-binding protein 4 and transthyretin. The levels of vitamin D-binding protein, complement C3, and apolipoprotein-A1 in PDE derived from different groups were greatly varied (P<0.05). However, no significant difference was found in the serum levels of these proteins among different groups (P>0.05 for all groups). This study provides a novel overview of the differences in PDE proteomes of four types of peritoneal membranes. Vitamin D-binding protein, complement C3, and apolipoprotein-A1 showed enhanced expression in PDE of patients with high transporter. Copyright © 2013 Elsevier Inc. All rights reserved.

The Complement System in Dialysis: A Forgotten Story?

PubMed Central

Poppelaars, Felix; Faria, Bernardo; Gaya da Costa, Mariana; Franssen, Casper F. M.; van Son, Willem J.; Berger, Stefan P.; Daha, Mohamed R.; Seelen, Marc A.

2018-01-01

Significant advances have lead to a greater understanding of the role of the complement system within nephrology. The success of the first clinically approved complement inhibitor has created renewed appreciation of complement-targeting therapeutics. Several clinical trials are currently underway to evaluate the therapeutic potential of complement inhibition in renal diseases and kidney transplantation. Although, complement has been known to be activated during dialysis for over four decades, this area of research has been neglected in recent years. Despite significant progress in biocompatibility of hemodialysis (HD) membranes and peritoneal dialysis (PD) fluids, complement activation remains an undesired effect and relevant issue. Short-term effects of complement activation include promoting inflammation and coagulation. In addition, long-term complications of dialysis, such as infection, fibrosis and cardiovascular events, are linked to the complement system. These results suggest that interventions targeting the complement system in dialysis could improve biocompatibility, dialysis efficacy, and long-term outcome. Combined with the clinical availability to safely target complement in patients, the question is not if we should inhibit complement in dialysis, but when and how. The purpose of this review is to summarize previous findings and provide a comprehensive overview of the role of the complement system in both HD and PD. PMID:29422906

Immunophenotypic analysis of mast cells in mastocytosis: When and how to do it. Proposals of the Spanish Network on Mastocytosis (REMA).

PubMed

Escribano, Luis; Diaz-Agustin, Beatriz; López, Antonio; Núñez López, Rosa; García-Montero, Andrés; Almeida, Julia; Prados, Aranzazu; Angulo, Miguel; Herrero, Sonia; Orfao, Alberto

2004-03-01

Mastocytosis is a term used for a heterogeneous group of disorders characterized by an abnormal proliferation and accumulation of mast cells (MCs) in one or multiple tissues including skin, bone marrow, liver, spleen, and lymph nodes, among others. In recent years, multiparameter flow cytometric studies have shown that pathologic MCs from patients with mastocytosis display unique aberrant immunophenotypic characteristics as compared with normal MCs. Among other features, pathologic MCs show aberrant expression of CD25 and CD2 antigens and abnormally high levels of the CD11c and CD35 complement receptors, the CD59 complement regulatory molecule, the CD63 lysosomal membrane antigen, and the CD69 early-activation antigen. In addition, MCs from mastocytosis express abnormally low levels of CD117 and unexpectedly high light scatter and autofluorescence characteristics. These aberrant immunophenotypic features are of great relevance for the assessment of tissue involvement in mastocytosis with consequences in the diagnosis, classification, and follow-up of the disease and in its differential diagnosis with other entities. In this paper we provide the reader with information for the objective and reproducible identification of pathologic MCs by using quantitative multiparametric flow cytometry, information for their phenotypic characterization, and the criteria currently used for a correct interpretation of the immunophenotypic results obtained. Copyright 2004 Wiley-Liss, Inc.

Update on hemolytic uremic syndrome: Diagnostic and therapeutic recommendations

PubMed Central

Salvadori, Maurizio; Bertoni, Elisabetta

2013-01-01

Hemolytic uremic syndrome (HUS) is a rare disease. In this work the authors review the recent findings on HUS, considering the different etiologic and pathogenetic classifications. New findings in genetics and, in particular, mutations of genes that encode the complement-regulatory proteins have improved our understanding of atypical HUS. Similarly, the complement proteins are clearly involved in all types of thrombotic microangiopathy: typical HUS, atypical HUS and thrombotic thrombocytopenic purpura (TTP). Furthermore, several secondary HUS appear to be related to abnormalities in complement genes in predisposed patients. The authors highlight the therapeutic aspects of this rare disease, examining both “traditional therapy” (including plasma therapy, kidney and kidney-liver transplantation) and “new therapies”. The latter include anti-Shiga-toxin antibodies and anti-C5 monoclonal antibody “eculizumab”. Eculizumab has been recently launched for the treatment of the atypical HUS, but it appears to be effective in the treatment of typical HUS and in TTP. Future therapies are in phases I and II. They include anti-C5 antibodies, which are more purified, less immunogenic and absorbed orally and, anti-C3 antibodies, which are more powerful, but potentially less safe. Additionally, infusions of recombinant complement-regulatory proteins are a potential future therapy. PMID:24255888

Altered Lipid Synthesis by Lack of Yeast Pah1 Phosphatidate Phosphatase Reduces Chronological Life Span*

PubMed Central

Park, Yeonhee; Han, Gil-Soo; Mileykovskaya, Eugenia; Garrett, Teresa A.; Carman, George M.

2015-01-01

In Saccharomyces cerevisiae, Pah1 phosphatidate phosphatase, which catalyzes the dephosphorylation of phosphatidate to yield diacylglycerol, plays a crucial role in the synthesis of the storage lipid triacylglycerol. This evolutionarily conserved enzyme also plays a negative regulatory role in controlling de novo membrane phospholipid synthesis through its consumption of phosphatidate. We found that the pah1Δ mutant was defective in the utilization of non-fermentable carbon sources but not in oxidative phosphorylation; the mutant did not exhibit major changes in oxygen consumption rate, mitochondrial membrane potential, F1F0-ATP synthase activity, or gross mitochondrial morphology. The pah1Δ mutant contained an almost normal complement of major mitochondrial phospholipids with some alterations in molecular species. Although oxidative phosphorylation was not compromised in the pah1Δ mutant, the cellular levels of ATP in quiescent cells were reduced by 2-fold, inversely correlating with a 4-fold increase in membrane phospholipids. In addition, the quiescent pah1Δ mutant cells had 3-fold higher levels of mitochondrial superoxide and cellular lipid hydroperoxides, had reduced activities of superoxide dismutase 2 and catalase, and were hypersensitive to hydrogen peroxide. Consequently, the pah1Δ mutant had a shortened chronological life span. In addition, the loss of Tsa1 thioredoxin peroxidase caused a synthetic growth defect with the pah1Δ mutation. The shortened chronological life span of the pah1Δ mutant along with its growth defect on non-fermentable carbon sources and hypersensitivity to hydrogen peroxide was suppressed by the loss of Dgk1 diacylglycerol kinase, indicating that the underpinning of pah1Δ mutant defects was the excess synthesis of membrane phospholipids. PMID:26338708

The prognostic performance of the complement system in septic patients in emergency department: a cohort study.

PubMed

Zhao, Xin; Chen, Yun-Xia; Li, Chun-Sheng

2015-01-01

To investigate the prognostic performance of complement components in septic patients, complement 3, membrane attack complex (MAC) and mannose-binding lectin were measured and compared among adult patients with sepsis, severe sepsis and septic shock, as well as between in-hospital nonsurvivors and survivors. The prognostic value of complement components was compared with mortality in emergency department sepsis (MEDS) score. Median complement 3, MAC and mannose-binding lectin increased directly with the sepsis, severe sepsis and septic shock groups, and were significantly higher in nonsurvivors than in survivors. MEDS and MAC independently predicted in-hospital mortality. The prognostic performance of MAC was superior to MEDS as analyzed by receiver operating characteristic curve and area under the curve.

Functional Characterization of LcpA, a Surface-Exposed Protein of Leptospira spp. That Binds the Human Complement Regulator C4BP▿

PubMed Central

Barbosa, Angela S.; Monaris, Denize; Silva, Ludmila B.; Morais, Zenaide M.; Vasconcellos, Sílvio A.; Cianciarullo, Aurora M.; Isaac, Lourdes; Abreu, Patricia A. E.

2010-01-01

We have previously shown that pathogenic leptospiral strains are able to bind C4b binding protein (C4BP). Surface-bound C4BP retains its cofactor activity, indicating that acquisition of this complement regulator may contribute to leptospiral serum resistance. In the present study, the abilities of seven recombinant putative leptospiral outer membrane proteins to interact with C4BP were evaluated. The protein encoded by LIC11947 interacted with this human complement regulator in a dose-dependent manner. The cofactor activity of C4BP bound to immobilized recombinant LIC11947 (rLIC11947) was confirmed by detecting factor I-mediated cleavage of C4b. rLIC11947 was therefore named LcpA (for leptospiral complement regulator-acquiring protein A). LcpA was shown to be an outer membrane protein by using immunoelectron microscopy, cell surface proteolysis, and Triton X-114 fractionation. The gene coding for LcpA is conserved among pathogenic leptospiral strains. This is the first characterization of a Leptospira surface protein that binds to the human complement regulator C4BP in a manner that allows this important regulator to control complement system activation mediated either by the classical pathway or by the lectin pathway. This newly identified protein may play a role in immune evasion by Leptospira spp. and may therefore represent a target for the development of a human vaccine against leptospirosis. PMID:20404075

Human NKCC2 cation–Cl– co-transporter complements lack of Vhc1 transporter in yeast vacuolar membranes.

PubMed

Petrezselyova, Silvia; Dominguez, Angel; Herynkova, Pavla; Macias, Juan F; Sychrova, Hana

2013-10-01

Cation–chloride co-transporters serve to transport Cl– and alkali metal cations. Whereas a large family of these exists in higher eukaryotes, yeasts only possess one cation–chloride co-transporter, Vhc1, localized to the vacuolar membrane. In this study, the human cation–chloride co-transporter NKCC2 complemented the phenotype of VHC1 deletion in Saccharomyces cerevisiae and its activity controlled the growth of salt-sensitive yeast cells in the presence of high KCl, NaCl and LiCl. A S. cerevisiae mutant lacking plasma-membrane alkali–metal cation exporters Nha1 and Ena1-5 and the vacuolar cation–chloride co-transporter Vhc1 is highly sensitive to increased concentrations of alkali–metal cations, and it proved to be a suitable model for characterizing the substrate specificity and transport activity of human wild-type and mutated cation–chloride co-transporters. Copyright © 2013 John Wiley & Sons, Ltd.

Acute symptoms during and between hemodialysis: the relative role of speed, duration, and biocompatibility of dialysis.

PubMed

Skroeder, N R; Jacobson, S H; Lins, L E; Kjellstrand, C M

1994-12-01

The relationship between hemodialysis (HD) symptoms and dialyzer membrane composition and area, blood-flow, treatment duration, urea removal, ultrafiltration volume, leukocyte activation, and complement generation (C3a) was studied in 20 patients undergoing 234 HD treatments by 12 different modes in random order using Cuprophan, hemophane, or polyamide membranes with small or large membrane areas with high Qb (400 ml/min) and short duration (2 h) or low Qb (200 ml/min) and long duration (4 h). Fewer symptoms occurred during the 2-h HD at high Qb than during the 4-h HD with low Qb (19% vs. 32%, p = 0.0351). No differences were observed between different dialyzer membranes or areas. More intradialytic symptoms occurred when urea elimination was high than it was low (p = 0.0044). Leukocyte activation (leukocyte drop) after 15 min of dialysis and complement generation did not influence symptom incidence. Blood pressure changes were mainly influenced by ultrafiltration volume (p < 0.001). Symptoms between dialyses were determined by urea removal and ultrafiltration. Membrane, area, or Qb were of no importance. Thus, duration of dialysis, urea removal, and demand for ultrafiltration, but not membrane composition, area, or biocompatability, are important for the development of HD-related symptoms.

Novel Evasion Mechanisms of the Classical Complement Pathway

PubMed Central

Garcia, Brandon L.; Zwarthoff, Seline A.; Rooijakkers, Suzan H. M.; Geisbrecht, Brian V.

2016-01-01

Complement is a network of soluble and cell surface-associated proteins which gives rise to a self-amplifying, yet tightly regulated system with fundamental roles in immune surveillance and clearance. Complement becomes activated on the surface of ‘non-self’ cells by one of three initiating mechanisms known as the classical, lectin, or alternative pathways. Evasion of complement function is a hallmark of invasive pathogens and hematophagous organisms. While many complement inhibition strategies hinge on hijacking activities of endogenous complement regulatory proteins, an increasing number of uniquely evolved evasion molecules have been discovered over the past decade. In this review we focus on several recent investigations which have revealed mechanistically distinct inhibitors of the classical pathway. Because the classical pathway is an important and specific mediator of various autoimmune and inflammatory disorders, in-depth knowledge of novel evasion mechanisms could direct future development of therapeutic anti-inflammatory molecules. PMID:27591336

Novel Evasion Mechanisms of the Classical Complement Pathway.

PubMed

Garcia, Brandon L; Zwarthoff, Seline A; Rooijakkers, Suzan H M; Geisbrecht, Brian V

2016-09-15

Complement is a network of soluble and cell surface-associated proteins that gives rise to a self-amplifying, yet tightly regulated system with fundamental roles in immune surveillance and clearance. Complement becomes activated on the surface of nonself cells by one of three initiating mechanisms known as the classical, lectin, and alternative pathways. Evasion of complement function is a hallmark of invasive pathogens and hematophagous organisms. Although many complement-inhibition strategies hinge on hijacking activities of endogenous complement regulatory proteins, an increasing number of uniquely evolved evasion molecules have been discovered over the past decade. In this review, we focus on several recent investigations that revealed mechanistically distinct inhibitors of the classical pathway. Because the classical pathway is an important and specific mediator of various autoimmune and inflammatory disorders, in-depth knowledge of novel evasion mechanisms could direct future development of therapeutic anti-inflammatory molecules. Copyright © 2016 by The American Association of Immunologists, Inc.

Changes in membrane lipid composition in ethanol- and acid-adapted Oenococcus oeni cells: characterization of the cfa gene by heterologous complementation.

PubMed

Grandvalet, Cosette; Assad-García, Juan Simón; Chu-Ky, Son; Tollot, Marie; Guzzo, Jean; Gresti, Joseph; Tourdot-Maréchal, Raphaëlle

2008-09-01

Cyclopropane fatty acid (CFA) synthesis was investigated in Oenococcus oeni. The data obtained demonstrated that acid-grown cells or cells harvested in the stationary growth phase showed changes in fatty acid composition similar to those of ethanol-grown cells. An increase of the CFA content and a decrease of the oleic acid content were observed. The biosynthesis of CFAs from unsaturated fatty acid phospholipids is catalysed by CFA synthases. Quantitative real-time-PCR experiments were performed on the cfa gene of O. oeni, which encodes a putative CFA synthase. The level of cfa transcripts increased when cells were harvested in stationary phase and when cells were grown in the presence of ethanol or at low pH, suggesting transcriptional regulation of the cfa gene under different stress conditions. In contrast to Escherichia coli, only one functional promoter was identified upstream of the cfa gene of O. oeni. The function of the cfa gene was confirmed by complementation of a cfa-deficient E. coli strain. Nevertheless, the complementation remained partial because the conversion percentage of unsaturated fatty acids into CFA of the complemented strain was much lower than that of the wild-type strain. Moreover, a prevalence of cycC19 : 0 was observed in the membrane of the complemented strain. This could be due to a specific affinity of the CFA synthase from O. oeni. In spite of this partial complementation, the complemented strain of E. coli totally recovered its viability after ethanol shock (10 %, v/v) whereas its viability was only partly recovered after an acid shock at pH 3.0.

High-Mobility Group Box 1-Induced Complement Activation Causes Sterile Inflammation.

PubMed

Kim, Sook Young; Son, Myoungsun; Lee, Sang Eun; Park, In Ho; Kwak, Man Sup; Han, Myeonggil; Lee, Hyun Sook; Kim, Eun Sook; Kim, Jae-Young; Lee, Jong Eun; Choi, Ji Eun; Diamond, Betty; Shin, Jeon-Soo

2018-01-01

High-mobility group box 1 (HMGB1), a well-known danger-associated molecular pattern molecule, acts as a pro-inflammatory molecule when secreted by activated immune cells or released after necrotic cell damage. HMGB1 binds to immunogenic bacterial components and augments septic inflammation. In this study, we show how HMGB1 mediates complement activation, promoting sterile inflammation. We show that HMGB1 activates the classical pathway of complement system in an antibody-independent manner after binding to C1q. The C3a complement activation product in human plasma and C5b-9 membrane attack complexes on cell membrane surface are detected after the addition of HMGB1. In an acetaminophen (APAP)-induced hepatotoxicity model, APAP injection reduced HMGB1 levels and elevated C3 levels in C1q-deficient mouse serum samples, compared to that in wild-type (WT) mice. APAP-induced C3 consumption was inhibited by sRAGE treatment in WT mice. Moreover, in a mouse model of brain ischemia-reperfusion injury based on middle cerebral arterial occlusion, C5b-9 complexes were deposited on vessels where HMGB1 was accumulated, an effect that was suppressed upon HMGB1 neutralization. We propose that the HMGB1 released after cell necrosis and in ischemic condition can trigger the classical pathway of complement activation to exacerbate sterile inflammation.

High-Mobility Group Box 1-Induced Complement Activation Causes Sterile Inflammation

PubMed Central

Kim, Sook Young; Son, Myoungsun; Lee, Sang Eun; Park, In Ho; Kwak, Man Sup; Han, Myeonggil; Lee, Hyun Sook; Kim, Eun Sook; Kim, Jae-Young; Lee, Jong Eun; Choi, Ji Eun; Diamond, Betty; Shin, Jeon-Soo

2018-01-01

High-mobility group box 1 (HMGB1), a well-known danger-associated molecular pattern molecule, acts as a pro-inflammatory molecule when secreted by activated immune cells or released after necrotic cell damage. HMGB1 binds to immunogenic bacterial components and augments septic inflammation. In this study, we show how HMGB1 mediates complement activation, promoting sterile inflammation. We show that HMGB1 activates the classical pathway of complement system in an antibody-independent manner after binding to C1q. The C3a complement activation product in human plasma and C5b-9 membrane attack complexes on cell membrane surface are detected after the addition of HMGB1. In an acetaminophen (APAP)-induced hepatotoxicity model, APAP injection reduced HMGB1 levels and elevated C3 levels in C1q-deficient mouse serum samples, compared to that in wild-type (WT) mice. APAP-induced C3 consumption was inhibited by sRAGE treatment in WT mice. Moreover, in a mouse model of brain ischemia–reperfusion injury based on middle cerebral arterial occlusion, C5b-9 complexes were deposited on vessels where HMGB1 was accumulated, an effect that was suppressed upon HMGB1 neutralization. We propose that the HMGB1 released after cell necrosis and in ischemic condition can trigger the classical pathway of complement activation to exacerbate sterile inflammation. PMID:29696019

Complement in Non-Antibody-Mediated Kidney Diseases

PubMed Central

Angeletti, Andrea; Reyes-Bahamonde, Joselyn; Cravedi, Paolo; Campbell, Kirk N.

2017-01-01

The complement system is part of the innate immune response that plays important roles in protecting the host from foreign pathogens. The complement components and relative fragment deposition have long been recognized to be strongly involved also in the pathogenesis of autoantibody-related kidney glomerulopathies, leading to direct glomerular injury and recruitment of infiltrating inflammation pathways. More recently, unregulated complement activation has been shown to be associated with progression of non-antibody-mediated kidney diseases, including focal segmental glomerulosclerosis, C3 glomerular disease, thrombotic microangiopathies, or general fibrosis generation in progressive chronic kidney diseases. Some of the specific mechanisms associated with complement activation in these diseases were recently clarified, showing a dominant role of alternative activation pathway. Over the last decade, a growing number of anticomplement agents have been developed, and some of them are being approved for clinical use or already in use. Therefore, anticomplement therapies represent a realistic choice of therapeutic approaches for complement-related diseases. Herein, we review the complement system activation, regulatory mechanisms, their involvement in non-antibody-mediated glomerular diseases, and the recent advances in complement-targeting agents as potential therapeutic strategies. PMID:28748184

Factor H-related proteins.

PubMed

Józsi, Mihály; Meri, Seppo

2014-01-01

Factor H-related proteins (CFHRs) are plasma glycoproteins related in structure and antigenicity to each other and to the complement inhibitory protein factor H. Such proteins are found in most mammals but their number and domain composition vary. This chapter summarizes our current knowledge on the human factor H-related proteins. In contrast to factor H, they have no strong complement inhibitory activity, although for some of them regulatory or complement modulatory activity has been reported. A common feature of CFHRs is that they bind to the C3b component of complement. Novel links between CFHRs and various diseases (C3 glomerulopathies, atypical hemolytic uremic syndrome and age-related macular degeneration) have been revealed in recent years, but we are still far from understanding their biological function.

Neutrophilic leukocyte membrane proteins. I. Isolation.

PubMed

Hawkins, D; Sauvé, M

1978-03-01

Rabbit exudate-derived PMN were homogenized and the cell membranes isolated on a two-phase aqueous system. Glycoproteins were extracted from cell membranes with lithium diiodosalicylate. SDS polyacrylamide gel electrophoretic analysis showed a consistent pattern of three major glycoprotein entities. Cells radioiodinated supravitally showed most of the radioactivity associated with larger glycoprotein entities whereas PMN membranes radiolabeled after isolation yielded a single major peak of radioactivity associated with a much smaller protein entity. Heterologous antisera against rabbit PMN, PMN membranes, and membrane glycoproteins were all cytotoxic for PMN in the presence of complement, and all bound to the PMN surface as demonstrated with immunocolloidal gold on electron microscopy. The data suggest that one or more glycoprotein entities are membrane-associated ectoglycoproteins which can be radiolabeled supravitally.

THE PATHOPHYSIOLOGY OF GEOGRAPHIC ATROPHY SECONDARY TO AGE-RELATED MACULAR DEGENERATION AND THE COMPLEMENT PATHWAY AS A THERAPEUTIC TARGET

PubMed Central

Schmidt-Erfurth, Ursula; van Lookeren Campagne, Menno; Henry, Erin C.; Brittain, Christopher

2017-01-01

Purpose: Geographic atrophy (GA) is an advanced, vision-threatening form of age-related macular degeneration (AMD) affecting approximately five million individuals worldwide. To date, there are no approved therapeutics for GA treatment; however, several are in clinical trials. This review focuses on the pathophysiology of GA, particularly the role of complement cascade dysregulation and emerging therapies targeting the complement cascade. Methods: Primary literature search on PubMed for GA, complement cascade in age-related macular degeneration. ClinicalTrials.gov was searched for natural history studies in GA and clinical trials of drugs targeting the complement cascade for GA. Results: Cumulative damage to the retina by aging, environmental stress, and other factors triggers inflammation via multiple pathways, including the complement cascade. When regulatory components in these pathways are compromised, as with several GA-linked genetic risk factors in the complement cascade, chronic inflammation can ultimately lead to the retinal cell death characteristic of GA. Complement inhibition has been identified as a key candidate for therapeutic intervention, and drugs targeting the complement pathway are currently in clinical trials. Conclusion: The complement cascade is a strategic target for GA therapy. Further research, including on natural history and genetics, is crucial to expand the understanding of GA pathophysiology and identify effective therapeutic targets. PMID:27902638

Functional Evolution of a cis-Regulatory Module

PubMed Central

Palsson, Arnar; Alekseeva, Elena; Bergman, Casey M; Nathan, Janaki; Kreitman, Martin

2005-01-01

Lack of knowledge about how regulatory regions evolve in relation to their structure–function may limit the utility of comparative sequence analysis in deciphering cis-regulatory sequences. To address this we applied reverse genetics to carry out a functional genetic complementation analysis of a eukaryotic cis-regulatory module—the even-skipped stripe 2 enhancer—from four Drosophila species. The evolution of this enhancer is non-clock-like, with important functional differences between closely related species and functional convergence between distantly related species. Functional divergence is attributable to differences in activation levels rather than spatiotemporal control of gene expression. Our findings have implications for understanding enhancer structure–function, mechanisms of speciation and computational identification of regulatory modules. PMID:15757364

Induction of passive Heymann nephritis in complement component 6-deficient PVG rats.

PubMed

Spicer, S Timothy; Tran, Giang T; Killingsworth, Murray C; Carter, Nicole; Power, David A; Paizis, Kathy; Boyd, Rochelle; Hodgkinson, Suzanne J; Hall, Bruce M

2007-07-01

Passive Heymann nephritis (PHN), a model of human membranous nephritis, is induced in susceptible rat strains by injection of heterologous antisera to rat renal tubular Ag extract. PHN is currently considered the archetypal complement-dependent form of nephritis, with the proteinuria resulting from sublytic glomerular epithelial cell injury induced by the complement membrane attack complex (MAC) of C5b-9. This study examined whether C6 and MAC are essential to the development of proteinuria in PHN by comparing the effect of injection of anti-Fx1A antisera into PVG rats deficient in C6 (PVG/C6(-)) and normal PVG rats (PVG/c). PVG/c and PVG/C6(-) rats developed similar levels of proteinuria at 3, 7, 14, and 28 days following injection of antisera. Isolated whole glomeruli showed similar deposition of rat Ig and C3 staining in PVG/c and PVG/C6(-) rats. C9 deposition was abundant in PVG/c but was not detected in PVG/C6(-) glomeruli, indicating C5b-9/MAC had not formed in PVG/C6(-) rats. There was also no difference in the glomerular cellular infiltrate of T cells and macrophages nor the size of glomerular basement membrane deposits measured on electron micrographs. To examine whether T cells effect injury, rats were depleted of CD8+ T cells which did not affect proteinuria in the early heterologous phase but prevented the increase in proteinuria associated with the later autologous phase. These studies showed proteinuria in PHN occurs without MAC and that other mechanisms, such as immune complex size, early complement components, CD4+ and CD8+ T cells, disrupt glomerular integrity and lead to proteinuria.

Dynamic nuclear polarization methods in solids and solutions to explore membrane proteins and membrane systems.

PubMed

Cheng, Chi-Yuan; Han, Songi

2013-01-01

Membrane proteins regulate vital cellular processes, including signaling, ion transport, and vesicular trafficking. Obtaining experimental access to their structures, conformational fluctuations, orientations, locations, and hydration in membrane environments, as well as the lipid membrane properties, is critical to understanding their functions. Dynamic nuclear polarization (DNP) of frozen solids can dramatically boost the sensitivity of current solid-state nuclear magnetic resonance tools to enhance access to membrane protein structures in native membrane environments. Overhauser DNP in the solution state can map out the local and site-specific hydration dynamics landscape of membrane proteins and lipid membranes, critically complementing the structural and dynamics information obtained by electron paramagnetic resonance spectroscopy. Here, we provide an overview of how DNP methods in solids and solutions can significantly increase our understanding of membrane protein structures, dynamics, functions, and hydration in complex biological membrane environments.

Therapeutic inhibition of the complement system. Y2K update.

PubMed

Asghar, S S; Pasch, M C

2000-09-01

Activation of complement is an essential part of the mechanism of pathogenesis of a large number of human diseases; its inhibition by pharmacological means is likely to suppress disease processes in complement mediated diseases. From this point of view low molecular weight synthetic inhibitors of complement are being developed and high molecular weight natural inhibitors of human origin present in plasma or embedded in cell membrane are being purified or produced in their recombinant forms. This review is concerned with high molecular weight inhibitors, some of which are already in clinical use but may be efficacious in many other diseases in which they have not yet been tried. C1-esterase inhibitor (C1-INH) concentrate prepared from human plasma is being successfully used for the treatment of hereditary angioneurotic edema. Recently, C1-INH has been found to be consumed in severe inflammation and has been shown to exert beneficial effects in several inflammatory conditions such as human sepsis, post-operative myocardial dysfunction due to reperfusion injury, severe capillary leakage syndrome after bone marrow transplantation, reperfusion injury after lung transplantation, burn, and cytotoxicity caused by IL-2 therapy in cancer. Factor I has been used for the treatment of factor I deficiency. Recombinant soluble forms of membrane cofactor protein (MCP), and decay accelerating factor (DAF) have not yet been tried in humans but have been shown to be effective in immune complex mediate inflammation in animals. Organs of pigs transgenic for one or more of human membrane regulators of complement namely membrane cofactor protein (MCP), decay accelerating factor (DAF) or CD59, are being produced for transplantation into humans. They have been shown to be resistant to hyperacute rejection in non-human primates; acute vascular rejection is still a problem in their clinical use. It is hoped that these observations together with future developments will make xeno-transplantation in clinical practice a reality. Several recombinant variants of complement receptor 1 (CR1) have been produced. The most effective of these appears to be sCR1-SLe x, sCR1 part of which inhibits complement and carbohydrate Sle x moiety inhibits selectin mediated interactions of neutrophils and lymphocytes with endothelium. Although clinical trials of sCR1 in humans is eagerly awaited, several of the recombinant versions of sCR1 have been shown to suppress ischemia/reperfusion injury, thermal trauma, and immune complex mediated inflammation. They have also been shown to be effective in experimental models of systemic sclerosis, arthritis, myasthenia gravis, Guillain Barré syndrome and glomerulonephritis. Intravenous immunoglobulin, three of the most prominent properties of which are neutralization of autoantibody activity, suppression of autoantibody production and inhibition of complement activity, is being used in several diseases. These include autoimmune thrombocyopenic purpura, Kawasaki disease and several neurological diseases such as myasthenia gravis and Guillain Barre syndrome. In many uncontrolled small scale studies intravenous immunoglobulin has been shown to be effective in many immunological including dermatological diseases; controlled clinical trials in a large number of patients with these diseases is needed to establish the efficacy. It is hoped that in future therapeutic inhibition of complement will be one of the major approaches to combat many human diseases.

Anchoring tick salivary anti-complement proteins IRAC I and IRAC II to membrane increases their immunogenicity

PubMed Central

Gillet, Laurent; Schroeder, Hélène; Mast, Jan; Thirion, Muriel; Renauld, Jean-Christophe; Dewals, Benjamin; Vanderplasschen, Alain

2009-01-01

Tick salivary proteins are promising targets for the development of anti-tick vaccines. Recently, we described two paralogous anti-complement proteins, called Ixodes ricinus anti-complement (IRAC) proteins I and II, that are co-expressed in tick I. ricinus salivary glands. However, our previous attempts to immunize rabbits against IRAC via infection with recombinant Bovine herpesvirus 4 (BoHV-4) vectors invariably failed although both recombinants expressed high levels of functional IRAC proteins in vitro. As IRAC are soluble monovalent antigens, one of the possible explanations is that monovalent ligation of the B-cell receptor induces receptor activation but fails to promote antigen presentation, a phenomenon that is thought to induce a state of B-cell tolerance. In the present study, we tried to increase IRAC immunogenicity by expressing them as oligovalent antigens. To this end, IRAC were fused to membrane anchors and BoHV-4 vectors expressing these recombinant forms were produced. The immunization potentials of recombinant viruses expressing either secreted or transmembrane IRAC proteins were then compared. While the former did not induce a detectable immune response against IRAC, the latter led to high titres of anti-IRAC antibodies that only marginally affected tick blood feeding. All together, the data presented in this study demonstrate that the immunogenicity of a soluble antigen can be greatly improved by anchoring it in membrane. PMID:19531344

Anchoring tick salivary anti-complement proteins IRAC I and IRAC II to membrane increases their immunogenicity.

PubMed

Gillet, Laurent; Schroeder, Hélène; Mast, Jan; Thirion, Muriel; Renauld, Jean-Christophe; Dewals, Benjamin; Vanderplasschen, Alain

2009-01-01

Tick salivary proteins are promising targets for the development of anti-tick vaccines. Recently, we described two paralogous anti-complement proteins, called Ixodes ricinus anti-complement (IRAC) proteins I and II, that are co-expressed in tick I. ricinus salivary glands. However, our previous attempts to immunize rabbits against IRAC via infection with recombinant Bovine herpesvirus 4 (BoHV-4) vectors invariably failed although both recombinants expressed high levels of functional IRAC proteins in vitro. As IRAC are soluble monovalent antigens, one of the possible explanations is that monovalent ligation of the B-cell receptor induces receptor activation but fails to promote antigen presentation, a phenomenon that is thought to induce a state of B-cell tolerance. In the present study, we tried to increase IRAC immunogenicity by expressing them as oligovalent antigens. To this end, IRAC were fused to membrane anchors and BoHV-4 vectors expressing these recombinant forms were produced. The immunization potentials of recombinant viruses expressing either secreted or transmembrane IRAC proteins were then compared. While the former did not induce a detectable immune response against IRAC, the latter led to high titres of anti-IRAC antibodies that only marginally affected tick blood feeding. All together, the data presented in this study demonstrate that the immunogenicity of a soluble antigen can be greatly improved by anchoring it in membrane.

Complement component C3 plays a critical role in protecting the aging retina in a murine model of age-related macular degeneration.

PubMed

Hoh Kam, Jaimie; Lenassi, Eva; Malik, Talat H; Pickering, Matthew C; Jeffery, Glen

2013-08-01

Complement component C3 is the central complement component and a key inflammatory protein activated in age-related macular degeneration (AMD). AMD is associated with genetic variation in complement proteins that results in enhanced activation of C3 through the complement alternative pathway. These include complement factor H (CFH), a negative regulator of C3 activation. Both C3 inhibition and/or CFH augmentation are potential therapeutic strategies in AMD. Herein, we examined retinal integrity in aged (12 months) mice deficient in both factors H and C3 (CFH(-/-).C3(-/-)), CFH alone (CFH(-/-)), or C3 alone (C3(-/-)), and wild-type mice (C57BL/6). Retinal function was assessed by electroretinography, and retinal morphological features were analyzed at light and electron microscope levels. Retinas were also stained for amyloid β (Aβ) deposition, inflammation, and macrophage accumulation. Contrary to expectation, electroretinograms of CFH(-/-).C3(-/-) mice displayed more severely reduced responses than those of other mice. All mutant strains showed significant photoreceptor loss and thickening of Bruch's membrane compared with wild-type C57BL/6, but these changes were greater in CFH(-/-).C3(-/-) mice. CFH(-/-).C3(-/-) mice had significantly more Aβ on Bruch's membrane, fewer macrophages, and high levels of retinal inflammation than the other groups. Our data show that both uncontrolled C3 activation (CFH(-/-)) and complete absence of C3 (CFH(-/-).C3(-/-) and C3(-/-)) negatively affect aged retinas. These findings suggest that strategies that inhibit C3 in AMD may be deleterious. Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Low-pressure membrane integrity tests for drinking water treatment: A review.

PubMed

Guo, H; Wyart, Y; Perot, J; Nauleau, F; Moulin, P

2010-01-01

Low-pressure membrane systems, including microfiltration (MF) and ultrafiltration (UF) membranes, are being increasingly used in drinking water treatments due to their high level of pathogen removal. However, the pathogen will pass through the membrane and contaminate the product if the membrane integrity is compromised. Therefore, an effective on-line integrity monitoring method for MF and UF membrane systems is essential to guarantee the regulatory requirements for pathogen removal. A lot of works on low-pressure membrane integrity tests have been conducted by many researchers. This paper provides a literature review about different low-pressure membrane integrity monitoring methods for the drinking water treatment, including direct methods (pressure-based tests, acoustic sensor test, liquid porosimetry, etc.) and indirect methods (particle counting, particle monitoring, turbidity monitoring, surrogate challenge tests). Additionally, some information about the operation of membrane integrity tests is presented here. It can be realized from this review that it remains urgent to develop an alternative on-line detection technique for a quick, accurate, simple, continuous and relatively inexpensive evaluation of low-pressure membrane integrity. To better satisfy regulatory requirements for drinking water treatments, the characteristic of this ideal membrane integrity test is proposed at the end of this paper.

Relative importance of complement-mediated bactericidal and opsonic activity for protection against meningococcal disease.

PubMed

Granoff, Dan M

2009-06-24

Killing of Neisseria meningitidis can result from complement-mediated serum bactericidal activity (SBA) or opsonophagocytosis (OPA), or a combination of the two mechanisms. While SBA titers > or =1:4 confer protection, recent evidence suggests that this threshold titer may not be required. For example, the incidence of meningococcal disease declines between ages 1 and 4 years without evidence of acquisition of SBA titers > or =1:4. Meningococcal polysaccharide vaccination also elicited OPA and lowered the risk of disease in patients with late complement component deficiencies whose sera did not support SBA. Sera from healthy adults immunized with an outer membrane vesicle vaccine showed OPA killing of N. meningitidis with C6-depleted complement, and whole blood from complement-sufficient non-immunized adults with SBA titers <1:4 also frequently had killing activity. Collectively the data indicate that SBA titers <1:4 and/or vaccine-induced OPA can confer protection against meningococcal disease.

Relative importance of complement-mediated bactericidal and opsonic activity for protection against meningococcal disease

PubMed Central

Granoff, Dan M.

2009-01-01

Killing of Neisseria meningitidis can result from complement-mediated bactericidal activity (SBA) or opsonophagocytosis (OPA), or a combination of the two mechanisms. While SBA titers ≥1:4 confer protection, recent evidence suggests that this threshold titer may not be required. For example, the incidence of meningococcal disease declines between ages 1 and 4 years without evidence of acquisition of SBA titers ≥1:4. Meningococcal polysaccharide vaccination also elicited OPA and lowered the risk of disease in patients with late complement component deficiencies whose sera did not support SBA. Sera from healthy adults immunized with an outer membrane vesicle vaccine showed OPA killing of N. meningitidis with C6-depleted complement, and whole blood from complement-sufficient non-immunized adults with SBA titers <1:4 also frequently had killing activity. Collectively the data indicate that SBA titers <1:4 and/or vaccine-induced OPA can confer protection against meningococcal disease. PMID:19477054

Resolution of Dialyzer Membrane-Associated Thrombocytopenia with Use of Cellulose Triacetate Membrane: A Case Report

PubMed Central

Olafiranye, Feyisayo; Kyaw, Win; Olafiranye, Oladipupo

2011-01-01

Blood and dialyzer membrane interaction can cause significant thrombocytopenia through the activation of complement system. The extent of this interaction determines the biocompatibility of the membrane. Although the newer synthetic membranes have been shown to have better biocompatibility profile than the cellulose-based membranes, little is known about the difference in biocompatibility between synthetic membrane and modified cellulose membrane. Herein, we report a case of a patient on hemodialysis who developed dialyzer-membrane-related thrombocytopenia with use of synthetic membrane (F200NR polysulfone). The diagnosis of dialyzer membrane-associated thrombocytopenia was suspected by the trend of platelet count before and after dialysis, and the absence of other possible causes of thrombocytopenia. We observed significant improvement in platelet count when the membrane was changed to modified cellulose membrane (cellulose triacetate). In patients at high risk for thrombocytopenia, the modified cellulose membrane could be a better alternative to the standard synthetic membranes during hemodialysis. PMID:21547252

Structural basis for the stabilization of the complement alternative pathway C3 convertase by properdin

PubMed Central

Alcorlo, Martín; Tortajada, Agustín; Rodríguez de Córdoba, Santiago; Llorca, Oscar

2013-01-01

Complement is an essential component of innate immunity. Its activation results in the assembly of unstable protease complexes, denominated C3/C5 convertases, leading to inflammation and lysis. Regulatory proteins inactivate C3/C5 convertases on host surfaces to avoid collateral tissue damage. On pathogen surfaces, properdin stabilizes C3/C5 convertases to efficiently fight infection. How properdin performs this function is, however, unclear. Using electron microscopy we show that the N- and C-terminal ends of adjacent monomers in properdin oligomers conform a curly vertex that holds together the AP convertase, interacting with both the C345C and vWA domains of C3b and Bb, respectively. Properdin also promotes a large displacement of the TED (thioester-containing domain) and CUB (complement protein subcomponents C1r/C1s, urchin embryonic growth factor and bone morphogenetic protein 1) domains of C3b, which likely impairs C3-convertase inactivation by regulatory proteins. The combined effect of molecular cross-linking and structural reorganization increases stability of the C3 convertase and facilitates recruitment of fluid-phase C3 convertase to the cell surfaces. Our model explains how properdin mediates the assembly of stabilized C3/C5-convertase clusters, which helps to localize complement amplification to pathogen surfaces. PMID:23901101

The plant cytoskeleton controls regulatory volume increase.

PubMed

Liu, Qiong; Qiao, Fei; Ismail, Ahmed; Chang, Xiaoli; Nick, Peter

2013-09-01

The ability to adjust cell volume is required for the adaptation to osmotic stress. Plant protoplasts can swell within seconds in response to hypoosmotic shock suggesting that membrane material is released from internal stores. Since the stability of plant membranes depends on submembraneous actin, we asked, whether this regulatory volume control depends on the cytoskeleton. As system we used two cell lines from grapevine which differ in their osmotic tolerance and observed that the cytoskeleton responded differently in these two cell lines. To quantify the ability for regulatory volume control, we used hydraulic conductivity (Lp) as readout and demonstrated a role of the cytoskeleton in protoplast swelling. Chelation of calcium, inhibition of calcium channels, or manipulation of membrane fluidity, did not significantly alter Lp, whereas direct manipulation of the cytoskeleton via specific chemical reagents, or indirectly, through the bacterial elicitor Harpin or activation of phospholipase D, was effective. By optochemical engineering of actin using a caged form of the phytohormone auxin we can break the symmetry of actin organisation resulting in a localised deformation of cell shape indicative of a locally increased Lp. We interpret our findings in terms of a model, where the submembraneous cytoskeleton controls the release of intracellular membrane stores during regulatory volume change. Copyright © 2013 Elsevier B.V. All rights reserved.

Advancing Sensor Technology to Monitor Wildfires

EPA Pesticide Factsheets

EPA and partners are looking at ways to use miniature sensors to monitor air quality near wildfires. Data from these small sensors can complement measurements obtained from more complex regulatory-grade monitors that are stationary.

Control of photosynthetic membrane assembly in Rhodobacter sphaeroides mediated by puhA and flanking sequences.

PubMed Central

Sockett, R E; Donohue, T J; Varga, A R; Kaplan, S

1989-01-01

A reaction center H- strain (RCH-) of Rhodobacter sphaeroides, PUHA1, was made by in vitro deletion of an XhoI restriction endonuclease fragment from the puhA gene coupled with insertion of a kanamycin resistance gene cartridge. The resulting construct was delivered to R. sphaeroides wild-type 2.4.1, with the defective puhA gene replacing the wild-type copy by recombination, followed by selection for kanamycin resistance. When grown under conditions known to induce intracytoplasmic membrane development, PUHA1 synthesized a pigmented intracytoplasmic membrane. Spectral analysis of this membrane showed that it was deficient in B875 spectral complexes as well as functional reaction centers and that the level of B800-850 spectral complexes was greater than in the wild type. The RCH- strain was photosythetically incompetent, but photosynthetic growth was restored by complementation with a 1.45-kilobase (kb) BamHI restriction endonuclease fragment containing the puhA gene carried in trans on plasmid pRK404. B875 spectral complexes were not restored by complementation with the 1.45-kb BamHI restriction endonuclease fragment containing the puhA gene but were restored along with photosynthetic competence by complementation with DNA from a cosmid carrying the puhA gene, as well as a flanking DNA sequence. Interestingly, B875 spectral complexes, but not photosynthetic competence, were restored to PUHA1 by introduction in trans of a 13-kb BamHI restriction endonuclease fragment carrying genes encoding the puf operon region of the DNA. The effect of the puhA deletion was further investigated by an examination of the levels of specific mRNA species derived from the puf and puc operons, as well as by determinations of the relative abundances of polypeptides associated with various spectral complexes by immunological methods. The roles of puhA and other genetic components in photosynthetic gene expression and membrane assembly are discussed. Images PMID:2644200

Effects of a polyelectrolyte additive on the selective dialysis membrane permeability for low-molecular-weight proteins.

PubMed

Krieter, Detlef H; Morgenroth, Andreas; Barasinski, Artur; Lemke, Horst-Dieter; Schuster, Oliver; von Harten, Bodo; Wanner, Christoph

2007-02-01

Improving the sieving characteristics of dialysis membranes enhances the clearance of low-molecular-weight (LMW) proteins and may have an impact on outcome in patients receiving haemodialysis. To approach this goal, a novel polyelectrolyte additive process was applied to a polyethersulphone (PES) membrane. Polyelectrolyte-modified PES was characterized in vitro by measuring complement activation and sieving coefficients of cytochrome c and serum albumin. In a prospective, randomized, cross-over study, instantaneous plasma water clearances and reduction rates of LMW proteins [beta(2)-microglobulin (b2m), cystatin c, myoglobin, retinol binding protein] were determined in eight patients receiving dialysis treatment with PES in comparison with polysulphone (PSU). Biocompatibility was assessed by determination of transient leucopenia, plasma levels of complement C5a, thrombin-antithrombin III (TAT), myeloperoxidase (MPO) and elastase (ELT). PES showed a steeper sieving profile and lower complement activation in vitro compared with PSU. Instantaneous clearance (69 +/- 8 vs. 58 +/- 3 ml/min; P < 0.001) and reduction rate (72.3 +/- 1 5% vs 66.2 +/- 6.1%; P < 0.001) of b2m were significantly higher with PES as compared with PSU. With higher molecular weight of proteins, differences in the solute removal between PES and PSU further increased, whereas albumin loss remained low (PES, 0.53 +/- 0.17 vs PSU, <0.22 g/dialysis). MPO, ELT and TAT did not differ between the two membranes. In contrast, leucopenia was less pronounced and C5a generation was significantly lower during dialysis with PES. Polyelectrolyte modification of PES results in a higher LMW protein removal and in optimized biocompatibility. Whether these findings translate into better outcome of patients receiving haemodialysis requires further studies.

Statistical Inference and Reverse Engineering of Gene Regulatory Networks from Observational Expression Data

PubMed Central

Emmert-Streib, Frank; Glazko, Galina V.; Altay, Gökmen; de Matos Simoes, Ricardo

2012-01-01

In this paper, we present a systematic and conceptual overview of methods for inferring gene regulatory networks from observational gene expression data. Further, we discuss two classic approaches to infer causal structures and compare them with contemporary methods by providing a conceptual categorization thereof. We complement the above by surveying global and local evaluation measures for assessing the performance of inference algorithms. PMID:22408642

Molecular and expression analysis of complement component C5 in the nurse shark (Ginglymostoma cirratum) and its predicted functional role.

PubMed

Graham, Matthew; Shin, Dong-Ho; Smith, Sylvia L

2009-07-01

We present the complete cDNA sequence of shark (Ginglymostoma cirratum) pro-C5 and its molecular characterization with a descriptive analysis of the structural elements necessary for its potential functional role as a potent mediator of inflammation (fragment C5a) and initiator molecule (fragment C5b) for the assembly of the membrane attack complex (MAC) upon activation by C5 convertase. In mammals the three complement activation cascades, the classical, alternative and lectin pathways, converge at the activation of C3, a pivotal complement protein. It is, however, the subsequent activation of the next complement component, C5, which is the focal point at which the initiation of the terminal lytic pathway takes place and involves the stepwise assembly of the MAC. The effector cytolytic function of complement occurs with the insertion of MAC into target membranes causing dough-nut like holes and cell leakage. The lytic activity of shark complement results in structurally similar holes in target membranes suggesting the assembly of a shark MAC that likely involves a functional analogue of C5. The composition of shark MAC remains unresolved and to date conclusive evidence has been lacking for shark C5. The gene has not been cloned nor has the serum protein been characterized for any elasmobranch species. This report is the first to confirm the presence of C5 homologue in the shark. GcC5 is remarkably similar to human C5 in overall structure and domain arrangement. The GcC5 cDNA measured 5160-bp with 5' and 3' UTRs of 35 bp and 79 bp, respectively. Structural analysis of the derived protein sequence predicts a molecule that is a two-chain structure which lacks a thiolester bond and contains a C5 convertase cleavage site indicating that activation will generate two peptides, akin to C5b and C5a. The putative GcC5 molecule also contains the C-terminal C345C/Netrin module that characterizes C3, C4 and C5. Multiple alignment of deduced amino acid sequences shows that GcC5 shares more amino acid identities/similarities with mammals than that with bony fish. We conclude that at the time of emergence of sharks the elaborate mosaic structure of C5 had already evolved.

Molecular and expression analysis of complement component C5 in the nurse shark (Ginglymostoma cirratum) and its predicted functional role

PubMed Central

Graham, Matthew; Shin, Dong-Ho; Smith, Sylvia L.

2009-01-01

We present the complete cDNA sequence of shark (Ginglymostoma cirratum) pro-C5 and its molecular characterization with a descriptive analysis of the structural elements necessary for its potential functional role as a potent mediator of inflammation (fragment C5a) and initiator molecule (fragment C5b) for the assembly of the membrane attack complex (MAC) upon activation by C5 convertase. In mammals the three complement activation cascades, the classical, alternative and lectin pathways, converge at the activation of C3, a pivotal complement protein. It is, however, the subsequent activation of the next complement component, C5, which is the focal point at which the initiation of the terminal lytic pathway takes place and involves the stepwise assembly of the MAC. The effector cytolytic function of complement occurs with the insertion of MAC into target membranes causing dough-nut like holes and cell leakage. The lytic activity of shark complement results in structurally similar holes in target membranes suggesting the assembly of a shark MAC that likely involves a functional analogue of C5. The composition of shark MAC remains unresolved and to date conclusive evidence has been lacking for shark C5. The gene has not been cloned nor has the serum protein been characterized for any elasmobranch species. This report is the first to confirm the presence of C5 homologue in the shark. GcC5 is remarkably similar to human C5 in overall structure and domain arrangement. The GcC5 cDNA measured 5160-bp with 5′ and 3′ UTRs of 35bp and 79bp, respectively. Structural analysis of the derived protein sequence predicts a molecule that is a two-chain structure which lacks a thiolester bond and contains a C5 convertase cleavage site indicating that activation will generate two peptides, akin to C5b and C5a. The putative GcC5 molecule also contains the C-terminal C345C/Netrin module that characterizes C3, C4 and C5. Multiple alignment of deduced amino acid sequences show that GcC5 shares more amino acid identities/similarities with mammals than that with bony fish. We conclude that at the time of emergence of sharks the elaborate mosaic structure of C5 had already evolved. PMID:19410004

A compact, in vivo screen of all 6-mers reveals drivers of tissue-specific expression and guides synthetic regulatory element design.

PubMed

Smith, Robin P; Riesenfeld, Samantha J; Holloway, Alisha K; Li, Qiang; Murphy, Karl K; Feliciano, Natalie M; Orecchia, Lorenzo; Oksenberg, Nir; Pollard, Katherine S; Ahituv, Nadav

2013-07-18

Large-scale annotation efforts have improved our ability to coarsely predict regulatory elements throughout vertebrate genomes. However, it is unclear how complex spatiotemporal patterns of gene expression driven by these elements emerge from the activity of short, transcription factor binding sequences. We describe a comprehensive promoter extension assay in which the regulatory potential of all 6 base-pair (bp) sequences was tested in the context of a minimal promoter. To enable this large-scale screen, we developed algorithms that use a reverse-complement aware decomposition of the de Bruijn graph to design a library of DNA oligomers incorporating every 6-bp sequence exactly once. Our library multiplexes all 4,096 unique 6-mers into 184 double-stranded 15-bp oligomers, which is sufficiently compact for in vivo testing. We injected each multiplexed construct into zebrafish embryos and scored GFP expression in 15 tissues at two developmental time points. Twenty-seven constructs produced consistent expression patterns, with the majority doing so in only one tissue. Functional sequences are enriched near biologically relevant genes, match motifs for developmental transcription factors, and are required for enhancer activity. By concatenating tissue-specific functional sequences, we generated completely synthetic enhancers for the notochord, epidermis, spinal cord, forebrain and otic lateral line, and show that short regulatory sequences do not always function modularly. This work introduces a unique in vivo catalog of short, functional regulatory sequences and demonstrates several important principles of regulatory element organization. Furthermore, we provide resources for designing compact, reverse-complement aware k-mer libraries.

Transient Receptor Potential Channel 6 (TRPC6) Protects Podocytes during Complement-mediated Glomerular Disease*

PubMed Central

Kistler, Andreas D.; Singh, Geetika; Altintas, Mehmet M.; Yu, Hao; Fernandez, Isabel C.; Gu, Changkyu; Wilson, Cory; Srivastava, Sandeep Kumar; Dietrich, Alexander; Walz, Katherina; Kerjaschki, Dontscho; Ruiz, Phillip; Dryer, Stuart; Sever, Sanja; Dinda, Amit K.; Faul, Christian; Reiser, Jochen

2013-01-01

Gain-of-function mutations in the calcium channel TRPC6 lead to autosomal dominant focal segmental glomerulosclerosis and podocyte expression of TRPC6 is increased in some acquired human glomerular diseases, particularly in membranous nephropathy. These observations led to the hypothesis that TRPC6 overactivation is deleterious to podocytes through pathological calcium signaling, both in genetic and acquired diseases. Here, we show that the effects of TRPC6 on podocyte function are context-dependent. Overexpression of TRPC6 alone did not directly affect podocyte morphology and cytoskeletal structure. Unexpectedly, however, overexpression of TRPC6 protected podocytes from complement-mediated injury, whereas genetic or pharmacological TRPC6 inactivation increased podocyte susceptibility to complement. Mechanistically, this effect was mediated by Ca2+/calmodulin-dependent protein kinase II (CaMKII) activation. Podocyte-specific TRPC6 transgenic mice showed stronger CaMKII activation, reduced podocyte foot process effacement and reduced levels of proteinuria during nephrotoxic serum nephritis, whereas TRPC6 null mice exhibited reduced CaMKII activation and higher levels of proteinuria compared with wild type littermates. Human membranous nephropathy biopsy samples showed podocyte staining for active CaMKII, which correlated with the degree of TRPC6 expression. Together, these data suggest a dual and context dependent role of TRPC6 in podocytes where acute activation protects from complement-mediated damage, but chronic overactivation leads to focal segmental glomerulosclerosis. PMID:24194522

Evasion Mechanisms Used by Pathogens to Escape the Lectin Complement Pathway.

PubMed

Rosbjerg, Anne; Genster, Ninette; Pilely, Katrine; Garred, Peter

2017-01-01

The complement system is a crucial defensive network that protects the host against invading pathogens. It is part of the innate immune system and can be initiated via three pathways: the lectin, classical and alternative activation pathway. Overall the network compiles a group of recognition molecules that bind specific patterns on microbial surfaces, a group of associated proteases that initiates the complement cascade, and a group of proteins that interact in proteolytic complexes or the terminal pore-forming complex. In addition, various regulatory proteins are important for controlling the level of activity. The result is a pro-inflammatory response meant to combat foreign microbes. Microbial elimination is, however, not a straight forward procedure; pathogens have adapted to their environment by evolving a collection of evasion mechanisms that circumvent the human complement system. Complement evasion strategies features different ways of exploiting human complement proteins and moreover features different pathogen-derived proteins that interfere with the normal processes. Accumulated, these mechanisms target all three complement activation pathways as well as the final common part of the cascade. This review will cover the currently known lectin pathway evasion mechanisms and give examples of pathogens that operate these to increase their chance of invasion, survival and dissemination.

Relative Contribution of Cellular Complement Inhibitors CD59, CD46, and CD55 to Parainfluenza Virus 5 Inhibition of Complement-Mediated Neutralization

PubMed Central

Li, Yujia; Parks, Griffith D.

2018-01-01

The complement system is a part of the innate immune system that viruses need to face during infections. Many viruses incorporate cellular regulators of complement activation (RCA) to block complement pathways and our prior work has shown that Parainfluenza virus 5 (PIV5) incorporates CD55 and CD46 to delay complement-mediated neutralization. In this paper, we tested the role of a third individual RCA inhibitor CD59 in PIV5 interactions with complement pathways. Using a cell line engineered to express CD59, we show that small levels of functional CD59 are associated with progeny PIV5, which is capable of blocking assembly of the C5b-C9 membrane attack complex (MAC). PIV5 containing CD59 (PIV5-CD59) showed increased resistance to complement-mediated neutralization in vitro comparing to PIV5 lacking regulators. Infection of A549 cells with PIV5 and RSV upregulated CD59 expression. TGF-beta treatment of PIV5-infected cells also increased cell surface CD59 expression and progeny virions were more resistant to complement-mediated neutralization. A comparison of individual viruses containing only CD55, CD46, or CD59 showed a potency of inhibiting complement-mediated neutralization, which followed a pattern of CD55 > CD46 > CD59. PMID:29693588

Complement System in Dermatological Diseases – Fire Under the Skin

PubMed Central

Panelius, Jaana; Meri, Seppo

2015-01-01

The complement system plays a key role in several dermatological diseases. Overactivation, deficiency, or abnormality of the control proteins are often related to a skin disease. Autoimmune mechanisms with autoantibodies and a cytotoxic effect of the complement membrane attack complex on epidermal or vascular cells can cause direct tissue damage and inflammation, e.g., in systemic lupus erythematosus (SLE), phospholipid antibody syndrome, and bullous skin diseases like pemphigoid. By evading complement attack, some microbes like Borrelia spirochetes and staphylococci can persist in the skin and cause prolonged symptoms. In this review, we present the most important skin diseases connected to abnormalities in the function of the complement system. Drugs having an effect on the complement system are also briefly described. On one hand, drugs with free hydroxyl on amino groups (e.g., hydralazine, procainamide) could interact with C4A, C4B, or C3 and cause an SLE-like disease. On the other hand, progress in studies on complement has led to novel anti-complement drugs (recombinant C1-inhibitor and anti-C5 antibody, eculizumab) that could alleviate symptoms in diseases associated with excessive complement activation. The main theme of the manuscript is to show how relevant the complement system is as an immune effector system in contributing to tissue injury and inflammation in a broad range of skin disorders. PMID:25688346

Peptidoglycan Association of Murein Lipoprotein Is Required for KpsD-Dependent Group 2 Capsular Polysaccharide Expression and Serum Resistance in a Uropathogenic Escherichia coli Isolate.

PubMed

Diao, Jingyu; Bouwman, Catrien; Yan, Donghong; Kang, Jing; Katakam, Anand K; Liu, Peter; Pantua, Homer; Abbas, Alexander R; Nickerson, Nicholas N; Austin, Cary; Reichelt, Mike; Sandoval, Wendy; Xu, Min; Whitfield, Chris; Kapadia, Sharookh B

2017-05-23

Murein lipoprotein (Lpp) and peptidoglycan-associated lipoprotein (Pal) are major outer membrane lipoproteins in Escherichia coli Their roles in cell-envelope integrity have been documented in E. coli laboratory strains, and while Lpp has been linked to serum resistance in vitro , the underlying mechanism has not been established. Here, lpp and pal mutants of uropathogenic E. coli strain CFT073 showed reduced survival in a mouse bacteremia model, but only the lpp mutant was sensitive to serum killing in vitro The peptidoglycan-bound Lpp form was specifically required for preventing complement-mediated bacterial lysis in vitro and complement-mediated clearance in vivo Compared to the wild-type strain, the lpp mutant had impaired K2 capsular polysaccharide production and was unable to respond to exposure to serum by elevating capsular polysaccharide amounts. These properties correlated with altered cellular distribution of KpsD, the predicted outer membrane translocon for "group 2" capsular polysaccharides. We identified a novel Lpp-dependent association between functional KpsD and peptidoglycan, highlighting important interplay between cell envelope components required for resistance to complement-mediated lysis in uropathogenic E. coli isolates. IMPORTANCE Uropathogenic E. coli (UPEC) isolates represent a significant cause of nosocomial urinary tract and bloodstream infections. Many UPEC isolates are resistant to serum killing. Here, we show that a major cell-envelope lipoprotein (murein lipoprotein) is required for serum resistance in vitro and for complement-mediated bacterial clearance in vivo This is mediated, in part, through a novel mechanism by which murein lipoprotein affects the proper assembly of a key component of the machinery involved in production of "group 2" capsules. The absence of murein lipoprotein results in impaired production of the capsule layer, a known participant in complement resistance. These results demonstrate an important role for murein lipoprotein in complex interactions between different outer membrane biogenesis pathways and further highlight the importance of lipoprotein assembly and transport in bacterial pathogenesis. Copyright © 2017 Diao et al.

Effect of dialyzer geometry on granulocyte and complement activation.

PubMed

Schaefer, R M; Heidland, A; Hörl, W H

1987-01-01

During hemodialysis with cuprophan membranes, the complement system as well as leukocytes become activated. In order to clarify the role of dialyzer geometry, the effect of hollow-fiber versus flat-sheet dialyzers and of different surface areas on C3a generation and leukocyte degranulation was investigated. Plasma levels of leukocyte elastase in complex with alpha 1-proteinase inhibitor were significantly increased after 1 h (+55%) and 3 h (+62%) of hemodialysis with flat-sheet dialyzers as compared to hollow-fiber devices. In addition, plasma levels of lactoferrin, released from the specific granules of leukocytes during activation, were significantly higher (+42%) 3 h after the onset of dialysis treatment with flat-sheet than with hollow-fiber dialyzers. With respect to surface area, larger dialyzers tended to cause more release of leukocyte elastase as compared to dialyzers with smaller surface areas, irrespectively of the configuration of the dialyzer used. On the other hand, activation of the complement system, as measured by the generation of C3a-desarg, did not differ with both types of configurations. The same held true for leukopenia, which was almost identical for hollow-fiber and flat-sheet dialyzers. From these findings two lines of evidence emerge: First, not only the type of membrane material used in a dialyzer may influence its biocompatibility, but the geometry of the extracorporeal device also determines the degree of compatibility. Hence, the extent of leukocyte activation correlated with both configuration of the dialyzer and surface area of the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Deletion of the membrane complement inhibitor CD59a drives age and gender-dependent alterations to bone phenotype in mice.

PubMed

Bloom, Anja C; Collins, Fraser L; Van't Hof, Rob J; Ryan, Elizabeth S; Jones, Emma; Hughes, Timothy R; Morgan, B Paul; Erlandsson, Malin; Bokarewa, Maria; Aeschlimann, Daniel; Evans, Bronwen A J; Williams, Anwen S

2016-03-01

Degenerative joint diseases such as osteoarthritis are characterised by aberrant region-specific bone formation and abnormal bone mineral content. A recent study suggested a role for the complement membrane attack complex in experimental models of osteoarthritis. Since CD59a is the principal regulator of the membrane attack complex in mice, we evaluated the impact of CD59a gene deletion upon maintenance of bone architecture. In vivo bone morphology analysis revealed that male CD59a-deficient mice have increased femur length and cortical bone volume, albeit with reduced bone mineral density. However, this phenomenon was not observed in female mice. Histomorphometric analysis of the trabecular bone showed increased rates of bone homeostasis, with both increased bone resorption and mineral apposition rate in CD59a-deficient male mice. When bone cells were studied in isolation, in vitro osteoclastogenesis was significantly increased in male CD59a-deficient mice, although osteoblast formation was not altered. Our data reveal, for the first time, that CD59a is a regulator of bone growth and homeostasis. CD59a ablation in male mice results in longer and wider bones, but with less density, which is likely a major contributing factor for their susceptibility to osteoarthritis. These findings increase our understanding of the role of complement regulation in degenerative arthritis. Copyright © 2016 Amgen Inc. Published by Elsevier Inc. All rights reserved.

Regulatory roles of mast cells in immune responses.

PubMed

Morita, Hideaki; Saito, Hirohisa; Matsumoto, Kenji; Nakae, Susumu

2016-09-01

Mast cells are important immune cells for host defense through activation of innate immunity (via toll-like receptors or complement receptors) and acquired immunity (via FcεRI). Conversely, mast cells also act as effector cells that exacerbate development of allergic or autoimmune disorders. Yet, several lines of evidence show that mast cells act as regulatory cells to suppress certain inflammatory diseases. Here, we review the mechanisms by which mast cells suppress diseases.

Future perspectives in target-specific immunotherapies of myasthenia gravis

PubMed Central

Dalakas, Marinos C.

2015-01-01

Myasthenia gravis (MG) is an autoimmune disease caused by complement-fixing antibodies against acetylcholine receptors (AChR); antigen-specific CD4+ T cells, regulatory T cells (Tregs) and T helper (Th) 17+ cells are essential in antibody production. Target-specific therapeutic interventions should therefore be directed against antibodies, B cells, complement and molecules associated with T cell signaling. Even though the progress in the immunopathogenesis of the disease probably exceeds any other autoimmune disorder, MG is still treated with traditional drugs or procedures that exert a non-antigen specific immunosuppression or immunomodulation. Novel biological agents currently on the market, directed against the following molecular pathways, are relevant and specific therapeutic targets that can be tested in MG: (a) T cell intracellular signaling molecules, such as anti-CD52, anti-interleukin (IL) 2 receptors, anti- costimulatory molecules, and anti-Janus tyrosine kinases (JAK1, JAK3) that block the intracellular cascade associated with T-cell activation; (b) B cells and their trophic factors, directed against key B-cell molecules; (c) complement C3 or C5, intercepting the destructive effect of complement-fixing antibodies; (d) cytokines and cytokine receptors, such as those targeting IL-6 which promotes antibody production and IL-17, or the p40 subunit of IL-12/1L-23 that affect regulatory T cells; and (e) T and B cell transmigration molecules associated with lymphocyte egress from the lymphoid organs. All drugs against these molecular pathways require testing in controlled trials, although some have already been tried in small case series. Construction of recombinant AChR antibodies that block binding of the pathogenic antibodies, thereby eliminating complement and antibody-depended-cell-mediated cytotoxicity, are additional novel molecular tools that require exploration in experimental MG. PMID:26600875

Glomeruli of Dense Deposit Disease contain components of the alternative and terminal complement pathway

PubMed Central

Sethi, Sanjeev; Gamez, Jeffrey D.; Vrana, Julie A.; Theis, Jason D.; Bergen, H. Robert; Zipfel, Peter F.; Dogan, Ahmet; Smith, Richard J. H.

2009-01-01

Dense Deposit Disease (DDD), or membranoproliferative glomerulonephritis type II, is a rare renal disease characterized by dense deposits in the mesangium and along the glomerular basement membranes that can be seen by electron microscopy. Although these deposits contain complement factor C3, as determined by immunofluorescence microscopy, their precise composition remains unknown. To address this question, we used mass spectrometry to identify the proteins in laser microdissected glomeruli isolated from paraffin-embedded tissue of eight confirmed cases of DDD. Compared to glomeruli from five control patients, we found that all of the glomeruli from patients with DDD contain components of the alternative pathway and terminal complement complex. Factor C9 was uniformly present as well as the two fluid-phase regulators of terminal complement complex clusterin and vitronectin. In contrast, in nine patients with immune complex–mediated membranoproliferative glomerulonephritis, glomerular samples contained mainly immunoglobulins and complement factors C3 and C4. Our study shows that in addition to fluid-phase dysregulation of the alternative pathway, soluble components of the terminal complement complex contribute to glomerular lesions found in DDD. PMID:19177158

Complement System Part II: Role in Immunity

PubMed Central

Merle, Nicolas S.; Noe, Remi; Halbwachs-Mecarelli, Lise; Fremeaux-Bacchi, Veronique; Roumenina, Lubka T.

2015-01-01

The complement system has been considered for a long time as a simple lytic cascade, aimed to kill bacteria infecting the host organism. Nowadays, this vision has changed and it is well accepted that complement is a complex innate immune surveillance system, playing a key role in host homeostasis, inflammation, and in the defense against pathogens. This review discusses recent advances in the understanding of the role of complement in physiology and pathology. It starts with a description of complement contribution to the normal physiology (homeostasis) of a healthy organism, including the silent clearance of apoptotic cells and maintenance of cell survival. In pathology, complement can be a friend or a foe. It acts as a friend in the defense against pathogens, by inducing opsonization and a direct killing by C5b–9 membrane attack complex and by triggering inflammatory responses with the anaphylatoxins C3a and C5a. Opsonization plays also a major role in the mounting of an adaptive immune response, involving antigen presenting cells, T-, and B-lymphocytes. Nevertheless, it can be also an enemy, when pathogens hijack complement regulators to protect themselves from the immune system. Inadequate complement activation becomes a disease cause, as in atypical hemolytic uremic syndrome, C3 glomerulopathies, and systemic lupus erythematosus. Age-related macular degeneration and cancer will be described as examples showing that complement contributes to a large variety of conditions, far exceeding the classical examples of diseases associated with complement deficiencies. Finally, we discuss complement as a therapeutic target. PMID:26074922

Anti-GM2 gangliosides IgM paraprotein induces neuromuscular block without neuromuscular damage.

PubMed

Santafé, Manel M; Sabaté, M Mar; Garcia, Neus; Ortiz, Nico; Lanuza, M Angel; Tomàs, Josep

2008-11-15

We analyzed the effect on the mouse neuromuscular synapses of a human monoclonal IgM, which binds specifically to gangliosides with the common epitope [GalNAc beta 1-4Gal(3-2 alpha NeuAc)beta 1-]. We focused on the role of the complement. Evoked neurotransmission was partially blocked by IgM both acutely (1 h) and chronically (10 days). Transmission electron microscopy shows important nerve terminal growth and retraction remodelling though axonal injury can be ruled out. Synapses did not show mouse C5b-9 immunofluorescence and were only immunolabelled when human complement was added. Therefore, the IgM-induced synaptic changes occur without complement-mediated membrane attack.

Reciprocal expression of integration host factor and HU in the developmental cycle and infectivity of Legionella pneumophila.

PubMed

Morash, Michael G; Brassinga, Ann Karen C; Warthan, Michelle; Gourabathini, Poornima; Garduño, Rafael A; Goodman, Steven D; Hoffman, Paul S

2009-04-01

Legionella pneumophila is an intracellular parasite of protozoa that differentiates late in infection into metabolically dormant cysts that are highly infectious. Regulation of this process is poorly understood. Here we report that the small DNA binding regulatory proteins integration host factor (IHF) and HU are reciprocally expressed over the developmental cycle, with HU expressed during exponential phase and IHF expressed postexponentially. To assess the role of these regulatory proteins in development, chromosomal deletions were constructed. Single (ihfA or ihfB) and double deletion (Deltaihf) IHF mutants failed to grow in Acanthamoeba castellanii unless complemented in trans when expressed temporally from the ihfA promoter but not under P(tac) (isopropyl-beta-d-thiogalactopyranoside). In contrast, IHF mutants were infectious for HeLa cells, though electron microscopic examination revealed defects in late-stage cyst morphogenesis (thickened cell wall, intracytoplasmic membranes, and inclusions of poly-beta-hydroxybutyrate), and were depressed for the developmental marker MagA. Green fluorescent protein promoter fusion assays indicated that IHF and the stationary-phase sigma factor RpoS were required for full postexponential expression of magA. Finally, defects in cyst morphogenesis noted for Deltaihf mutants in HeLa cells correlated with a loss of both detergent resistance and hyperinfectivity compared with results for wild-type cysts. These studies establish IHF and HU as markers of developmental stages and show that IHF function is required for both differentiation and full virulence of L. pneumophila in natural amoebic hosts.

Targeting the Human Complement Membrane Attack Complex to Selectively Kill Prostate Cancer Cells

DTIC Science & Technology

2013-10-01

prostate cancer cells in vitro . Evaluate CD59 expression in human prostate cancer microarrays. Aim 4: Evaluate toxicity and efficacy of the lead PAC5...fragment in vitro . Since PSA is the major chymotrypsin-like serine protease in the seminal plasma and prostatic fluid, we hypothesized that PSA was...that the evolution -related complement protein C5, but not C4, is a substrate of PSA as well. *Department of Pharmacology and Molecular Sciences, The

Virulence Associated Gene 8 of Bordetella pertussis Enhances Contact System Activity by Inhibiting the Regulatory Function of Complement Regulator C1 Inhibitor

PubMed Central

Hovingh, Elise S.; de Maat, Steven; Cloherty, Alexandra P. M.; Johnson, Steven; Pinelli, Elena; Maas, Coen; Jongerius, Ilse

2018-01-01

Bordetella pertussis is a Gram-negative bacterium and the causative agent of whooping cough. Whooping cough is currently re-emerging worldwide and, therefore, still poses a continuous global health threat. B. pertussis expresses several virulence factors that play a role in evading the human immune response. One of these virulence factors is virulence associated gene 8 (Vag8). Vag8 is a complement evasion molecule that mediates its effects by binding to the complement regulator C1 inhibitor (C1-INH). This regulatory protein is a fluid phase serine protease that controls proenzyme activation and enzyme activity of not only the complement system but also the contact system. Activation of the contact system results in the generation of bradykinin, a pro-inflammatory peptide. Here, the activation of the contact system by B. pertussis was explored. We demonstrate that recombinant as well as endogenous Vag8 enhanced contact system activity by binding C1-INH and attenuating its inhibitory function. Moreover, we show that B. pertussis itself is able to activate the contact system. This activation was dependent on Vag8 production as a Vag8 knockout B. pertussis strain was unable to activate the contact system. These findings show a previously overlooked interaction between the contact system and the respiratory pathogen B. pertussis. Activation of the contact system by B. pertussis may contribute to its pathogenicity and virulence. PMID:29915576

Variola virus immune evasion proteins.

PubMed

Dunlop, Lance R; Oehlberg, Katherine A; Reid, Jeremy J; Avci, Dilek; Rosengard, Ariella M

2003-09-01

Variola virus, the causative agent of smallpox, encodes approximately 200 proteins. Over 80 of these proteins are located in the terminal regions of the genome, where proteins associated with host immune evasion are encoded. To date, only two variola proteins have been characterized. Both are located in the terminal regions and demonstrate immunoregulatory functions. One protein, the smallpox inhibitor of complement enzymes (SPICE), is homologous to a vaccinia virus virulence factor, the vaccinia virus complement-control protein (VCP), which has been found experimentally to be expressed early in the course of vaccinia infection. Both SPICE and VCP are similar in structure and function to the family of mammalian complement regulatory proteins, which function to prevent inadvertent injury to adjacent cells and tissues during complement activation. The second variola protein is the variola virus high-affinity secreted chemokine-binding protein type II (CKBP-II, CBP-II, vCCI), which binds CC-chemokine receptors. The vaccinia homologue of CKBP-II is secreted both early and late in infection. CKBP-II proteins are highly conserved among orthopoxviruses, sharing approximately 85% homology, but are absent in eukaryotes. This characteristic sets it apart from other known virulence factors in orthopoxviruses, which share sequence homology with known mammalian immune regulatory gene products. Future studies of additional variola proteins may help illuminate factors associated with its virulence, pathogenesis and strict human tropism. In addition, these studies may also assist in the development of targeted therapies for the treatment of both smallpox and human immune-related diseases.

Virulence Associated Gene 8 of Bordetella pertussis Enhances Contact System Activity by Inhibiting the Regulatory Function of Complement Regulator C1 Inhibitor.

PubMed

Hovingh, Elise S; de Maat, Steven; Cloherty, Alexandra P M; Johnson, Steven; Pinelli, Elena; Maas, Coen; Jongerius, Ilse

2018-01-01

Bordetella pertussis is a Gram-negative bacterium and the causative agent of whooping cough. Whooping cough is currently re-emerging worldwide and, therefore, still poses a continuous global health threat. B. pertussis expresses several virulence factors that play a role in evading the human immune response. One of these virulence factors is virulence associated gene 8 (Vag8). Vag8 is a complement evasion molecule that mediates its effects by binding to the complement regulator C1 inhibitor (C1-INH). This regulatory protein is a fluid phase serine protease that controls proenzyme activation and enzyme activity of not only the complement system but also the contact system. Activation of the contact system results in the generation of bradykinin, a pro-inflammatory peptide. Here, the activation of the contact system by B. pertussis was explored. We demonstrate that recombinant as well as endogenous Vag8 enhanced contact system activity by binding C1-INH and attenuating its inhibitory function. Moreover, we show that B. pertussis itself is able to activate the contact system. This activation was dependent on Vag8 production as a Vag8 knockout B. pertussis strain was unable to activate the contact system. These findings show a previously overlooked interaction between the contact system and the respiratory pathogen B. pertussis . Activation of the contact system by B. pertussis may contribute to its pathogenicity and virulence.

COMPLEMENT FIXATION IN DISEASED TISSUES

PubMed Central

Burkholder, Peter M.

1961-01-01

An immunohistologic complement fixation test has been used in an effort to detect immune complexes in sections of kidney from rats injected with rabbit anti-rat kidney serum and in sections of biopsied kidneys from four humans with membranous glomerulonephritis. Sections of the rat and human kidneys were treated with fluorescein-conjugated anti-rabbit globulin or antihuman globulin respectively. Adjacent sections in each case were incubated first with fresh guinea pig serum and then in a second step were treated with fluorescein-conjugated antibodies against fixed guinea pig complement to detect sites of fixation of the complement. It was demonstrated that the sites of rabbit globulin in glomerular capillary walls of the rat kidneys and the sites of localized human globulin in thickened glomerular capillary walls and swollen glomerular endothelial cells of the human kidneys were the same sites in which guinea pig complement was fixed in vitro. It was concluded from these studies that rabbit nephrotoxic antibodies localize in rat glomeruli in complement-fixing antigen-antibody complexes. Furthermore, it was concluded that the deposits of human globulin in the glomeruli of the human kidneys behaved like antibody globulin in complement-fixing antigen-antibody complexes. The significance of demonstrating complement-fixing immune complexes in certain diseased tissues is discussed in regard to determination of the causative role of allergic reactions in disease. PMID:19867205

T cells and cytokines in the pathogenesis of acquired myasthenia gravis.

PubMed

Milani, Monica; Ostlie, Norma; Wang, Wei; Conti-Fine, Bianca M

2003-09-01

Although the symptoms of myasthenia gravis (MG) and experimental MG (EAMG) are caused by autoantibodies, CD4(+) T cells specific for the target antigen, the nicotinic acetylcholine receptor, and the cytokines they secrete, have an important role in these diseases. CD4(+) T cells have a pathogenic role, by permitting and facilitating the synthesis of high-affinity anti-AChR antibodies. Th1 CD4(+) cells are especially important because they drive the synthesis of anti-AChR complement-fixing IgG subclasses. Binding of those antibodies to the muscle AChR at the neuromuscular junction will trigger the complement-mediated destruction of the postsynaptic membrane. Thus, IL-12, a crucial cytokine for differentiation of Th1 cells, is necessary for development of EAMG. Th2 cells secrete different cytokines, with different effects on the pathogenesis of EAMG. Among them, IL-10, which is a potent growth and differentiation factor for B cells, facilitates the development of EAMG. In contrast, IL-4 appears to be involved in the differentiation of AChR-specific regulatory CD4(+) T cells, which can prevent the development of EAMG and its progression to a self-maintaining, chronic autoimmune disease. Studies on the AChR-specific CD4(+) cells commonly present in the blood of MG patients support a crucial role of CD4(+) T cells in the development of MG. Circumstantial evidence supports a pathogenic role of IL-10 also in human MG. On the other hand, there is no direct or circumstantial evidence yet indicating a role of IL-4 in the modulatory or immunosuppressive circuits in MG.

Biological effects of contaminated silicon carbide particles from a workstation in a plant producing abrasives.

PubMed

Governa, M; Valentino, M; Amati, M; Visonà, I; Botta, G C; Marcer, G; Gemignani, C

1997-06-01

A sample of silicon carbide dust taken in the field from a plant producing abrasives was studied in vitro. The SiC particles (part unmilled and part milled) were able to disturb the structure of erythrocyte membranes and to lead to blood red-cell lysis; they also either interfered with complement and activated the alternate pathway, or interacted with biological media and polymorphonuclear leucocyte membranes, thus eliciting reactive oxygen species production. These in vitro properties were detected both in original large particles and unmilled particles, over 40% of which were of respirable size. The ability of these SiC particles to produce complement activation in vitro lends support to the previous hypothesis, that the radiographic opacities found in two workers employed in the same area of the plant from which the dust tested was taken are due to a reaction by pulmonary interstitial structures to SiC particle inhalation. It is speculated that SiC particles could act like asbestos, the ability of which to activate complement through the alternate pathway is considered to be one of the mechanisms by which the initial asbestotic lesions and subsequent fibrotic inflammatory infiltrates are generated in the lung.

Molecular characterization of the alpha subunit of complement component C8 (GcC8α) in the nurse shark (Ginglymostoma cirratum)

PubMed Central

Aybar, Lydia; Shin, Dong-Ho; Smith, Sylvia L.

2009-01-01

Target cell lysis by complement is achieved by the assembly and insertion of the membrane attack complex (MAC) composed of glycoproteins C5b through C9. The lytic activity of shark complement involves functional analogues of mammalian C8 and C9. Mammalian C8 is composed of α, β, and γ subunits. The subunit structure of shark C8 is not known. This report describes a 2341 nucleotide sequence that translates into a polypeptide of 589 amino acid residues, orthologue to mammalian C8α and has the same modular architecture with conserved cysteines forming the peptide bond backbone. The C8γ-binding cysteine is conserved in the perforin-like domain. Hydrophobicity profile indicates the presence of hydrophobic residues essential for membrane insertion. It shares 41.1% and 47.4 % identity with human and Xenopus C8α respectively. Southern blot analysis showed GcC8α exists as a single copy gene expressed in most tissues except the spleen with the liver being the main site of synthesis. Phylogenetic analysis places it in a clade with C8α orthologs and as a sister taxa to the Xenopus. PMID:19524681

Evasion of Complement-Mediated Lysis and Complement C3 Deposition Are Regulated by Francisella tularensis Lipopolysaccharide O Antigen1

PubMed Central

Clay, Corey D.; Soni, Shilpa; Gunn, John S.; Schlesinger, Larry S.

2009-01-01

The bacterium Francisella tularensis (Ft) is a potential weapon of bioterrorism when aerosolized. Macrophage infection is necessary for disease progression and efficient phagocytosis by human macrophages requires serum opsonization by complement. Microbial complement activation leads to surface deposition of a highly regulated protein complex resulting in opsonization or membrane lysis. The nature of complement component C3 deposition, i.e., C3b (opsonization and lysis) or C3bi (opsonization only) fragment deposition, is central to the outcome of activation. In this study, we examine the mechanisms of Ft resistance to complement-mediated lysis, C3 component deposition on the Ft surface, and complement activation. Upon incubation in fresh nonimmune human serum, Schu S4 (Ft subsp. tularensis), Fn (Ft subsp. novicida), and LVS (Ft subsp. holarctica live vaccine strain) were resistant to complement-mediated lysis, but LVSG and LVSR (LVS strains altered in surface carbohydrate structures) were susceptible. C3 deposition, however, occurred on all strains. Complement-susceptible strains had markedly increased C3 fragment deposition, including the persistent presence of C3b compared with C3bi, which indicates that C3b inactivation results in survival of complement-resistant strains. C1q, an essential component of the classical activation pathway, was necessary for lysis of complement-susceptible strains and optimal C3 deposition on all strains. Finally, use of Francisella LPS mutants confirmed O Ag as a major regulator of complement resistance. These data provide evidence that pathogenic Francisella activate complement, but are resistant to complement-mediated lysis in part due to limited C3 deposition, rapid conversion of surface-bound C3b to C3bi, and the presence of LPS O Ag. PMID:18832715

Characterization of noncoding regulatory DNA in the human genome.

PubMed

Elkon, Ran; Agami, Reuven

2017-08-08

Genetic variants associated with common diseases are usually located in noncoding parts of the human genome. Delineation of the full repertoire of functional noncoding elements, together with efficient methods for probing their biological roles, is therefore of crucial importance. Over the past decade, DNA accessibility and various epigenetic modifications have been associated with regulatory functions. Mapping these features across the genome has enabled researchers to begin to document the full complement of putative regulatory elements. High-throughput reporter assays to probe the functions of regulatory regions have also been developed but these methods separate putative regulatory elements from the chromosome so that any effects of chromatin context and long-range regulatory interactions are lost. Definitive assignment of function(s) to putative cis-regulatory elements requires perturbation of these elements. Genome-editing technologies are now transforming our ability to perturb regulatory elements across entire genomes. Interpretation of high-throughput genetic screens that incorporate genome editors might enable the construction of an unbiased map of functional noncoding elements in the human genome.

Mesophilic Aeromonas sp. serogroup O:11 resistance to complement-mediated killing.

PubMed Central

Merino, S; Rubires, X; Aguilar, A; Albertí, S; Hernandez-Allés, S; Benedí, V J; Tomas, J M

1996-01-01

The complement activation by and resistance to complement-mediated killing of Aeromonas sp. strains from serogroup O:11 were investigated by using different wild-type strains (with an S-layer characteristic of this serogroup) and their isogenic mutants characterized for their surface components (S-layer and lipopolysaccharide [LPS]). All of the Aeromonas sp. serogroup O:11 wild-type strains are unable to activate complement, which suggested that the S-layer completely covered the LPS molecules. We found that the classical complement pathway is involved in serum killing of susceptible Aeromonas sp. mutant strains of serogroup O11, while the alternative complement pathway seems not to be involved, and that the complement activation seems to be independent of antibody. The smooth mutant strains devoid of the S-layer (S-layer isogenic mutants) or isogenic LPS mutant strains with a complete or rather complete LPS core (also without the S-layer) are able to activate complement but are resistant to complement-mediated killing. The reasons for this resistance are that C3b is rapidly degraded, and therefore the lytic membrane attack complex (C5b-9) is not formed. Isogenic LPS rough mutants with an incomplete LPS core are serum sensitive because they bind more C3b than the resistant strains, the C3b is not completely degraded, and therefore the lytic complex (C5b-9) is formed. PMID:8945581

Complement in autoimmune diseases.

PubMed

Vignesh, Pandiarajan; Rawat, Amit; Sharma, Madhubala; Singh, Surjit

2017-02-01

The complement system is an ancient and evolutionary conserved element of the innate immune mechanism. It comprises of more than 20 serum proteins most of which are synthesized in the liver. These proteins are synthesized as inactive precursor proteins which are activated by appropriate stimuli. The activated forms of these proteins act as proteases and cleave other components successively in amplification pathways leading to exponential generation of final effectors. Three major pathways of complement pathways have been described, namely the classical, alternative and lectin pathways which are activated by different stimuli. However, all the 3 pathways converge on Complement C3. Cleavage of C3 and C5 successively leads to the production of the membrane attack complex which is final common effector. Excessive and uncontrolled activation of the complement has been implicated in the host of autoimmune diseases. But the complement has also been bemusedly described as the proverbial "double edged sword". On one hand, complement is the final effector of tissue injury in autoimmune diseases and on the other, deficiencies of some components of the complement can result in autoimmune diseases. Currently available tools such as enzyme based immunoassays for functional assessment of complement pathways, flow cytometry, next generation sequencing and proteomics-based approaches provide an exciting opportunity to study this ancient yet mysterious element of innate immunity. Copyright © 2017 Elsevier B.V. All rights reserved.

Cysteine proteinase from Streptococcus pyogenes enables evasion of innate immunity via degradation of complement factors.

PubMed

Honda-Ogawa, Mariko; Ogawa, Taiji; Terao, Yutaka; Sumitomo, Tomoko; Nakata, Masanobu; Ikebe, Kazunori; Maeda, Yoshinobu; Kawabata, Shigetada

2013-05-31

Streptococcus pyogenes is an important human pathogen that causes invasive diseases such as necrotizing fasciitis, sepsis, and streptococcal toxic shock syndrome. We investigated the function of a major cysteine protease from S. pyogenes that affects the amount of C1-esterase inhibitor (C1-INH) and other complement factors and aimed to elucidate the mechanism involved in occurrence of streptococcal toxic shock syndrome from the aspect of the complement system. First, we revealed that culture supernatant of a given S. pyogenes strain and recombinant SpeB degraded the C1-INH. Then, we determined the N-terminal sequence of the C1-INH fragment degraded by recombinant SpeB. Interestingly, the region containing one of the identified cleavage sites is not present in patients with C1-INH deficiency. Scanning electron microscopy of the speB mutant incubated in human serum showed the abnormal superficial architecture and irregular oval structure. Furthermore, unlike the wild-type strain, that mutant strain showed lower survival capacity than normal as compared with heat-inactivated serum, whereas it had a significantly higher survival rate in serum without the C1-INH than in normal serum. Also, SpeB degraded multiple complement factors and the membrane attack complex. Flow cytometric analyses revealed deposition of C9, one of the components of membrane the attack complex, in greater amounts on the surface of the speB mutant, whereas lower amounts of C9 were bound to the wild-type strain surface. These results suggest that SpeB can interrupt the human complement system via degrading the C1-INH, thus enabling S. pyogenes to evade eradication in a hostile environment.

Pseudomonas aeruginosa Regulated Intramembrane Proteolysis (RIP): Protease MucP can Overcome Mutations in the AlgO Periplasmic Protease to Restore Alginate Production in Nonmucoid Revertants.

PubMed

Delgado, Camila; Florez, Laura; Lollett, Ivonne; Lopez, Christine; Kangeyan, Shiva; Kumari, Hansi; Stylianou, Marios; Smiddy, Robert J; Schneper, Lisa; Sautter, Robert T; Szatmari, George; Mathee, Kalai

2018-05-21

The progression of cystic fibrosis (CF) from an acute to a chronic disease is often associated with the conversion of the opportunistic pathogen Pseudomonas aeruginosa from a nonmucoid form to a mucoid form in the lung. This conversion involves the overproduction of the exopolysaccharide alginate, whose production is under control of the AlgT/U sigma factor. This factor is regulated posttranslationally by an extremely unstable process and has been commonly attributed to mutations in the algT/U gene. By exploiting this unstable phenotype, we isolated 34 spontaneous nonmucoid variants arising from the mucoid strain PDO300, a PAO1 derivative containing the mucA22 allele commonly found in mucoid CF isolates. Complementation analysis using a minimal tiling path cosmid library revealed that most of these mutants mapped to two protease-encoding genes, algO also known as prc or PA3257 , and mucP. Interestingly, our algO mutations were complemented by both mucP and algO , leading us to delete, clone and overexpress mucP , algO , mucE and mucD in both wild-type PAO1 and in PDO300 backgrounds to better understand the regulation of this complex regulatory mechanism. Our findings suggest the regulatory proteases follow two pathways for regulated intramembrane proteolysis (RIP), where both the AlgO/MucP pathway and MucE/AlgW pathway are required in the wild type strain, but where the AlgO/MucP pathway can bypass the MucE/AlgW pathway in mucoid strains with membrane-associated forms of MucA with shortened C-termini, such as the MucA22 variant. This work gives us a better understanding of how alginate production is regulated in the clinically important mucoid variants of Pseudomonas aeruginosa. IMPORTANCE: Infection by the opportunistic pathogen Pseudomonas aeruginosa is the leading cause of morbidity and mortality seen in cystic fibrosis (CF) patients. Poor patient prognosis correlates with the genotypic and phenotypic change of the bacteria from a typical nonmucoid to a mucoid form in the CF lung, characterized by the overproduction of alginate. The expression of this exopolysaccharide is under the control an alternate sigma factor, AlgT/U, that is regulated post translationally by a series of proteases. A better understanding of this regulatory phenomenon will help in the development of therapies targeting alginate production, ultimately leading to an increase in the length and quality of life for those suffering from CF. Copyright © 2018 American Society for Microbiology.

The C8-binding protein of human erythrocytes: interaction with the components of the complement-attack phase.

PubMed Central

Schönermark, S; Filsinger, S; Berger, B; Hänsch, G M

1988-01-01

C8-binding protein is an intrinsic membrane protein of the human erythrocyte. It inhibits the complement (C5b-9)-mediated lysis in a species-restricting manner. In the present study we incorporated C8bp, isolated from human erythrocytes, into sheep erythrocytes (SRBC). SRBC, normally sensitive to lysis by human C5b-9, became insensitive to lysis. Furthermore, we found that C8bp is incorporated into the membrane-attack complex C5b-9, most probably by interacting with C8, since C8bp has an affinity for C8, particularly for the C8 alpha-gamma-subunit. Antibodies to C8bp react with the C8 alpha-subunits and with C9, pointing to the possibility of a partial homology between these proteins. Images Figure 4 Figure 6 Figure 7 PMID:3366469

Complement in Lupus Nephritis: New Perspectives.

PubMed

Bao, Lihua; Cunningham, Patrick N; Quigg, Richard J

2015-09-01

Systemic lupus erythematosus (SLE) is an autoimmune disorder caused by loss of tolerance to self-antigens, the production of autoantibodies and deposition of complement-fixing immune complexes (ICs) in injured tissues. SLE is characterized by a wide range of clinical manifestations and targeted organs, with lupus nephritis being one of the most serious complications. The complement system consists of three pathways and is tightly controlled by a set of regulatory proteins to prevent injudicious complement activation on host tissue. The involvement of the complement system in the pathogenesis of SLE is well accepted; yet, its exact role is still not clear. Complement plays dual roles in the pathogenesis of SLE. On the one hand, the complement system appears to have protective features in that hereditary homozygous deficiencies of classical pathway components, such as C1q and C4, are associated with an increased risk for SLE. On the other hand, IC-mediated activation of complement in affected tissues is clearly evident in both experimental and human SLE along with pathological features that are logical consequences of complement activation. Studies in genetically altered mice have shown that lack of complement inhibitors, such as complement factor H (CFH) or decay-accelerating factor (DAF) accelerates the development of experimental lupus nephritis, while treatment with recombinant protein inhibitors, such as Crry-Ig, CR2-Crry, CR2-DAF and CR2-CFH, ameliorates the disease development. Complement-targeted drugs, including soluble complement receptor 1 (TP10), C1 esterase inhibitor and a monoclonal anti-C5 antibody (eculizumab), have been shown to inhibit complement safely, and are now being investigated in a variety of clinical conditions. SLE is an autoimmune disorder which targets multiple systems. Complement is centrally involved and plays dual roles in the pathogenesis of SLE. Studies from experimental lupus models and clinical trials support the use of complement-targeted therapy in the treatment of SLE.

Modelling and analysis of gene regulatory network using feedback control theory

NASA Astrophysics Data System (ADS)

El-Samad, H.; Khammash, M.

2010-01-01

Molecular pathways are a part of a remarkable hierarchy of regulatory networks that operate at all levels of organisation. These regulatory networks are responsible for much of the biological complexity within the cell. The dynamic character of these pathways and the prevalence of feedback regulation strategies in their operation make them amenable to systematic mathematical analysis using the same tools that have been used with success in analysing and designing engineering control systems. In this article, we aim at establishing this strong connection through various examples where the behaviour exhibited by gene networks is explained in terms of their underlying control strategies. We complement our analysis by a survey of mathematical techniques commonly used to model gene regulatory networks and analyse their dynamic behaviour.

Targeted complement inhibition as a promising strategy for preventing inflammatory complications in hemodialysis

PubMed Central

DeAngelis, Robert A.; Reis, Edimara S.; Ricklin, Daniel; Lambris, John D.

2012-01-01

Hemodialysis is the most common method used to remove waste and hazardous products of metabolism in patients suffering from renal failure. Hundreds of thousands of people with end-stage renal disease undergo hemodialysis treatment in the United States each year. Strikingly, the 5-year survival rate for all dialysis patients is only 35%. Most of the patients succumb to cardiovascular disease that is exacerbated by the chronic induction of inflammation caused by contact of the blood with the dialysis membrane. The complement system, a strong mediator of pro-inflammatory networks, is a key contributor to such biomaterial-induced inflammation. Though only evaluated in experimental ex vivo settings, specific targeting of complement activation during hemodialysis has uncovered valuable information that points towards the therapeutic use of complement inhibitors as means to control the unwelcomed inflammatory responses and consequent pathologies in hemodialysis patients. PMID:22964235

CFHR1-Modified Neural Stem Cells Ameliorated Brain Injury in a Mouse Model of Neuromyelitis Optica Spectrum Disorders.

PubMed

Shi, Kaibin; Wang, Zhen; Liu, Yuanchu; Gong, Ye; Fu, Ying; Li, Shaowu; Wood, Kristofer; Hao, Junwei; Zhang, Guang-Xian; Shi, Fu-Dong; Yan, Yaping

2016-11-01

A major hurdle for effective stem cell therapy is ongoing inflammation in the target organ. Reconditioning the lesion microenvironment may be an effective way to promote stem cell therapy. In this study, we showed that engineered neural stem cells (NSCs) with complement factor H-related protein 1, a complement inhibitor protein, can attenuate inflammatory infiltration and immune-mediated damage of astrocytes, an important pathogenic progress in patients with neuromyelitis optica spectrum disorders. Furthermore, we demonstrated that transplantation of the complement factor H-related protein 1-modified NSCs effectively blocked the complement activation cascade and inhibited formation of the membrane attack complex, thus contributing to the protection of endogenous and transplanted NSC-differentiated astrocytes. Therefore, manipulation of the lesion microenvironment contributes to a more effective cell replacement therapeutic strategy for autoimmune diseases of the CNS. Copyright © 2016 by The American Association of Immunologists, Inc.

Evasion Mechanisms Used by Pathogens to Escape the Lectin Complement Pathway

PubMed Central

Rosbjerg, Anne; Genster, Ninette; Pilely, Katrine; Garred, Peter

2017-01-01

The complement system is a crucial defensive network that protects the host against invading pathogens. It is part of the innate immune system and can be initiated via three pathways: the lectin, classical and alternative activation pathway. Overall the network compiles a group of recognition molecules that bind specific patterns on microbial surfaces, a group of associated proteases that initiates the complement cascade, and a group of proteins that interact in proteolytic complexes or the terminal pore-forming complex. In addition, various regulatory proteins are important for controlling the level of activity. The result is a pro-inflammatory response meant to combat foreign microbes. Microbial elimination is, however, not a straight forward procedure; pathogens have adapted to their environment by evolving a collection of evasion mechanisms that circumvent the human complement system. Complement evasion strategies features different ways of exploiting human complement proteins and moreover features different pathogen-derived proteins that interfere with the normal processes. Accumulated, these mechanisms target all three complement activation pathways as well as the final common part of the cascade. This review will cover the currently known lectin pathway evasion mechanisms and give examples of pathogens that operate these to increase their chance of invasion, survival and dissemination. PMID:28553281

Complement Membrane Attack and Tumorigenesis: A SYSTEMS BIOLOGY APPROACH.

PubMed

Towner, Laurence D; Wheat, Richard A; Hughes, Timothy R; Morgan, B Paul

2016-07-15

Tumor development driven by inflammation is now an established phenomenon, but the role that complement plays remains uncertain. Recent evidence has suggested that various components of the complement (C) cascade may influence tumor development in disparate ways; however, little attention has been paid to that of the membrane attack complex (MAC). This is despite abundant evidence documenting the effects of this complex on cell behavior, including cell activation, protection from/induction of apoptosis, release of inflammatory cytokines, growth factors, and ECM components and regulators, and the triggering of the NLRP3 inflammasome. Here we present a novel approach to this issue by using global gene expression studies in conjunction with a systems biology analysis. Using network analysis of MAC-responsive expression changes, we demonstrate a cluster of co-regulated genes known to have impact in the extracellular space and on the supporting stroma and with well characterized tumor-promoting roles. Network analysis highlighted the central role for EGF receptor activation in mediating the observed responses to MAC exposure. Overall, the study sheds light on the mechanisms by which sublytic MAC causes tumor cell responses and exposes a gene expression signature that implicates MAC as a driver of tumor progression. These findings have implications for understanding of the roles of complement and the MAC in tumor development and progression, which in turn will inform future therapeutic strategies in cancer. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Cyclosporine Induces Endothelial Cell Release of Complement-Activating Microparticles

PubMed Central

Renner, Brandon; Klawitter, Jelena; Goldberg, Ryan; McCullough, James W.; Ferreira, Viviana P.; Cooper, James E.; Christians, Uwe

2013-01-01

Defective control of the alternative pathway of complement is an important risk factor for several renal diseases, including atypical hemolytic uremic syndrome. Infections, drugs, pregnancy, and hemodynamic insults can trigger episodes of atypical hemolytic uremic syndrome in susceptible patients. Although the mechanisms linking these clinical events with disease flares are unknown, recent work has revealed that each of these clinical conditions causes cells to release microparticles. We hypothesized that microparticles released from injured endothelial cells promote intrarenal complement activation. Calcineurin inhibitors cause vascular and renal injury and can trigger hemolytic uremic syndrome. Here, we show that endothelial cells exposed to cyclosporine in vitro and in vivo release microparticles that activate the alternative pathway of complement. Cyclosporine-induced microparticles caused injury to bystander endothelial cells and are associated with complement-mediated injury of the kidneys and vasculature in cyclosporine-treated mice. Cyclosporine-induced microparticles did not bind factor H, an alternative pathway regulatory protein present in plasma, explaining their complement-activating phenotype. Finally, we found that in renal transplant patients, the number of endothelial microparticles in plasma increases 2 weeks after starting tacrolimus, and treatment with tacrolimus associated with increased C3 deposition on endothelial microparticles in the plasma of some patients. These results suggest that injury-associated release of endothelial microparticles is an important mechanism by which systemic insults trigger intravascular complement activation and complement-dependent renal diseases. PMID:24092930

[Construction of enterohemorrhagic Escherichia coli O157:H7 strains with espF gene deletion and complementation].

PubMed

Hua, Ying; Sun, Qi; Wang, Xiangyu; DU, Yanli; Shao, Na; Zhang, Qiwei; Zhao, Wei; Wan, Chengsong

2015-11-01

To construct enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains with delection espF gene and its nucleotide fragment and with espF gene complementation. A pair of homologous arm primers was designed to amplify the gene fragment of kanamycin resistance, which was transformed into EHEC O157:H7 EDL933w strain via the PKD46 plasmid by electroporation. The replacement of the espF gene by kanamycin resistance gene through the PKD46-mediated red recombination system was confirmed by PCR and sequencing. The entire coding region of espF along with its nucleotide fragment was amplified by PCR and cloned into pBAD33 plasmid, which was transformed into a mutant strain to construct the strain with espF complementation. RT-PCR was used to verify the transcription of espF and its nucleotide fragment in the complemented mutant strain. We established EHEC O157:H7 EDL933w strains with espF gene deletion and with espF gene complementation. Both espF and its nucleotide fragment were transcribed in the complemented mutant strain. The two strains provide a basis for further study of the regulatory mechanism of espF.

Complement activation by carbon nanotubes and its influence on the phagocytosis and cytokine response by macrophages.

PubMed

Pondman, Kirsten M; Sobik, Martin; Nayak, Annapurna; Tsolaki, Anthony G; Jäkel, Anne; Flahaut, Emmanuel; Hampel, Silke; Ten Haken, Bennie; Sim, Robert B; Kishore, Uday

2014-08-01

Carbon nanotubes (CNTs) have promised a range of applications in biomedicine. Although influenced by the dispersants used, CNTs are recognized by the innate immune system, predominantly by the classical pathway of the complement system. Here, we confirm that complement activation by the CNT used continues up to C3 and C5, indicating that the entire complement system is activated including the formation of membrane-attack complexes. Using recombinant forms of the globular regions of human C1q (gC1q) as inhibitors of CNT-mediated classical pathway activation, we show that C1q, the first recognition subcomponent of the classical pathway, binds CNTs via the gC1q domain. Complement opsonisation of CNTs significantly enhances their uptake by U937 cells, with concomitant downregulation of pro-inflammatory cytokines and up-regulation of anti-inflammatory cytokines in both U937 cells and human monocytes. We propose that CNT-mediated complement activation may cause recruitment of cellular infiltration, followed by phagocytosis without inducing a pro-inflammatory immune response. This study highlights the importance of the complement system in response to carbon nanontube administration, suggesting that the ensuing complement activation may cause recruitment of cellular infiltration, followed by phagocytosis without inducing a pro-inflammatory immune response. Copyright © 2014 Elsevier Inc. All rights reserved.

Trypanosoma cruzi H+-ATPase 1 (TcHA1) and 2 (TcHA2) genes complement yeast mutants defective in H+ pumps and encode plasma membrane P-type H+-ATPases with different enzymatic properties.

PubMed

Luo, Shuhong; Scott, David A; Docampo, Roberto

2002-11-15

Previous studies in Trypanosoma cruzi have shown that intracellular pH homeostasis requires ATP and is affected by H(+)-ATPase inhibitors, indicating a major role for ATP-driven proton pumps in intracellular pH control. In the present study, we report the cloning and sequencing of a pair of genes linked in tandem (TcHA1 and TcHA2) in T. cruzi which encode proteins with homology to fungal and plant P-type proton-pumping ATPases. The genes are expressed at the mRNA level in different developmental stages of T. cruzi: TcHA1 is expressed maximally in epimastigotes, whereas TcHA2 is expressed predominantly in trypomastigotes. The proteins predicted from the nucleotide sequence of the genes have 875 and 917 amino acids and molecular masses of 96.3 and 101.2 kDa, respectively. Full-length TcHA1 and an N-terminal truncated version of TcHA2 complemented a Saccharomyces cerevisiae strain deficient in P-type H(+)-ATPase activity, the proteins localized to the yeast plasma membrane, and ATP-driven proton pumping could be detected in proteoliposomes reconstituted from plasma membrane purified from transfected yeast. The reconstituted proton transport activity was reduced by inhibitors of P-type H(+)-ATPases. C-terminal truncation did not affect complementation of mutant yeast, suggesting the lack of C-terminal autoinhibitory domains in these proteins. ATPase activity in plasma membrane from TcHA1- and (N-terminal truncated) TcHA2-transfected yeast was inhibited to different extents by vanadate, whereas the latter yeast strain was more resistant to extremes of pH, suggesting that the native proteins may serve different functions at different stages in the T. cruzi life cycle.

Systemic Lupus Erythematosus and Deficiencies of Early Components of the Complement Classical Pathway

PubMed Central

Macedo, Ana Catarina Lunz; Isaac, Lourdes

2016-01-01

The complement system plays an important role in the innate and acquired immune response against pathogens. It consists of more than 30 proteins found in soluble form or attached to cell membranes. Most complement proteins circulate in inactive forms and can be sequentially activated by the classical, alternative, or lectin pathways. Biological functions, such as opsonization, removal of apoptotic cells, adjuvant function, activation of B lymphocytes, degranulation of mast cells and basophils, and solubilization and clearance of immune complex and cell lysis, are dependent on complement activation. Although the activation of the complement system is important to avoid infections, it also can contribute to the inflammatory response triggered by immune complex deposition in tissues in autoimmune diseases. Paradoxically, the deficiency of early complement proteins from the classical pathway (CP) is strongly associated with development of systemic lupus erythematous (SLE) – mainly C1q deficiency (93%) and C4 deficiency (75%). The aim of this review is to focus on the deficiencies of early components of the CP (C1q, C1r, C1s, C4, and C2) proteins in SLE patients. PMID:26941740

Autoantibody-induced internalization of CNS AQP4 water channel and EAAT2 glutamate transporter requires astrocytic Fc receptor.

PubMed

Hinson, Shannon R; Clift, Ian C; Luo, Ningling; Kryzer, Thomas J; Lennon, Vanda A

2017-05-23

Aquaporin-4 (AQP4) water channel-specific IgG distinguishes neuromyelitis optica (NMO) from multiple sclerosis and causes characteristic immunopathology in which central nervous system (CNS) demyelination is secondary. Early events initiating the pathophysiological outcomes of IgG binding to astrocytic AQP4 are poorly understood. CNS lesions reflect events documented in vitro following IgG interaction with AQP4: AQP4 internalization, attenuated glutamate uptake, intramyelinic edema, interleukin-6 release, complement activation, inflammatory cell recruitment, and demyelination. Here, we demonstrate that AQP4 internalization requires AQP4-bound IgG to engage an astrocytic Fcγ receptor (FcγR). IgG-lacking Fc redistributes AQP4 within the plasma membrane and induces interleukin-6 release. However, AQP4 endocytosis requires an activating FcγR's gamma subunit and involves astrocytic membrane loss of an inhibitory FcγR, CD32B. Interaction of the IgG-AQP4 complex with FcγRs triggers coendocytosis of the excitatory amino acid transporter 2 (EAAT2). Requirement of FcγR engagement for internalization of two astrocytic membrane proteins critical to CNS homeostasis identifies a complement-independent, upstream target for potential early therapeutic intervention in NMO.

Complement Component 3C3 and C3a Receptor Are Required in Chitin-Dependent Allergic Sensitization to Aspergillus fumigatus but Dispensable in Chitin-Induced Innate Allergic Inflammation

PubMed Central

Roy, René M.; Paes, Hugo C.; Nanjappa, Som G.; Sorkness, Ron; Gasper, David; Sterkel, Alana; Wüthrich, Marcel; Klein, Bruce S.

2013-01-01

ABSTRACT Levels of the anaphylatoxin C3a are increased in patients with asthma compared with those in nonasthmatics and increase further still during asthma exacerbations. However, the role of C3a during sensitization to allergen is poorly understood. Sensitization to fungal allergens, such as Aspergillus fumigatus, is a strong risk factor for the development of asthma. Exposure to chitin, a structural polysaccharide of the fungal cell wall, induces innate allergic inflammation and may promote sensitization to fungal allergens. Here, we found that coincubation of chitin with serum or intratracheal administration of chitin in mice resulted in the generation of C3a. We established a model of chitin-dependent sensitization to soluble Aspergillus antigens to test the contribution of complement to these events. C3−/− and C3aR−/− mice were protected from chitin-dependent sensitization to Aspergillus and had reduced lung eosinophilia and type 2 cytokines and serum IgE. In contrast, complement-deficient mice were not protected against chitin-induced innate allergic inflammation. In sensitized mice, plasmacytoid dendritic cells from complement-deficient animals acquired a tolerogenic profile associated with enhanced regulatory T cell responses and suppressed Th2 and Th17 responses specific for Aspergillus. Thus, chitin induces the generation of C3a in the lung, and chitin-dependent allergic sensitization to Aspergillus requires C3aR signaling, which suppresses regulatory dendritic cells and T cells and induces allergy-promoting T cells. PMID:23549917

Bacterial Reaction Centers Purified with Styrene Maleic Acid Copolymer Retain Native Membrane Functional Properties and Display Enhanced Stability**

PubMed Central

Swainsbury, David J K; Scheidelaar, Stefan; van Grondelle, Rienk; Killian, J Antoinette; Jones, Michael R

2014-01-01

Integral membrane proteins often present daunting challenges for biophysical characterization, a fundamental issue being how to select a surfactant that will optimally preserve the individual structure and functional properties of a given membrane protein. Bacterial reaction centers offer a rare opportunity to compare the properties of an integral membrane protein in different artificial lipid/surfactant environments with those in the native bilayer. Here, we demonstrate that reaction centers purified using a styrene maleic acid copolymer remain associated with a complement of native lipids and do not display the modified functional properties that typically result from detergent solubilization. Direct comparisons show that reaction centers are more stable in this copolymer/lipid environment than in a detergent micelle or even in the native membrane, suggesting a promising new route to exploitation of such photovoltaic integral membrane proteins in device applications. PMID:25212490

Sequential Actions of the AAA-ATPase Valosin-containing Protein (VCP)/p97 and the Proteasome 19 S Regulatory Particle in Sterol-accelerated, Endoplasmic Reticulum (ER)-associated Degradation of 3-Hydroxy-3-methylglutaryl-coenzyme A Reductase*

PubMed Central

Morris, Lindsey L.; Hartman, Isamu Z.; Jun, Dong-Jae; Seemann, Joachim; DeBose-Boyd, Russell A.

2014-01-01

Accelerated endoplasmic reticulum (ER)-associated degradation (ERAD) of the cholesterol biosynthetic enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase results from its sterol-induced binding to ER membrane proteins called Insig-1 and Insig-2. This binding allows for subsequent ubiquitination of reductase by Insig-associated ubiquitin ligases. Once ubiquitinated, reductase becomes dislocated from ER membranes into the cytosol for degradation by 26 S proteasomes through poorly defined reactions mediated by the AAA-ATPase valosin-containing protein (VCP)/p97 and augmented by the nonsterol isoprenoid geranylgeraniol. Here, we report that the oxysterol 25-hydroxycholesterol and geranylgeraniol combine to trigger extraction of reductase across ER membranes prior to its cytosolic release. This conclusion was drawn from studies utilizing a novel assay that measures membrane extraction of reductase by determining susceptibility of a lumenal epitope in the enzyme to in vitro protease digestion. Susceptibility of the lumenal epitope to protease digestion and thus membrane extraction of reductase were tightly regulated by 25-hydroxycholesterol and geranylgeraniol. The reaction was inhibited by RNA interference-mediated knockdown of either Insigs or VCP/p97. In contrast, reductase continued to become membrane-extracted, but not cytosolically dislocated, in cells deficient for AAA-ATPases of the proteasome 19 S regulatory particle. These findings establish sequential roles for VCP/p97 and the 19 S regulatory particle in the sterol-accelerated ERAD of reductase that may be applicable to the ERAD of other substrates. PMID:24860107

Molecular characterization of the alpha subunit of complement component C8 (GcC8alpha) in the nurse shark (Ginglymostoma cirratum).

PubMed

Aybar, Lydia; Shin, Dong-Ho; Smith, Sylvia L

2009-09-01

Target cell lysis by complement is achieved by the assembly and insertion of the membrane attack complex (MAC) composed of glycoproteins C5b through C9. The lytic activity of shark complement involves functional analogues of mammalian C8 and C9. Mammalian C8 is composed of alpha, beta, and gamma subunits. The subunit structure of shark C8 is not known. This report describes a 2341 nucleotide sequence that translates into a polypeptide of 589 amino acid residues, orthologue to mammalian C8alpha and has the same modular architecture with conserved cysteines forming the peptide bond backbone. The C8gamma-binding cysteine is conserved in the perforin-like domain. Hydrophobicity profile indicates the presence of hydrophobic residues essential for membrane insertion. It shares 41.1% and 47.4% identity with human and Xenopus C8alpha respectively. Southern blot analysis showed GcC8alpha exists as a single copy gene expressed in most tissues except the spleen with the liver being the main site of synthesis. Phylogenetic analysis places it in a clade with C8alpha orthologs and as a sister taxa to the Xenopus. 2009 Elsevier Ltd.

Peptidoglycan Association of Murein Lipoprotein Is Required for KpsD-Dependent Group 2 Capsular Polysaccharide Expression and Serum Resistance in a Uropathogenic Escherichia coli Isolate

PubMed Central

Diao, Jingyu; Bouwman, Catrien; Yan, Donghong; Kang, Jing; Katakam, Anand K.; Liu, Peter; Pantua, Homer; Abbas, Alexander R.; Nickerson, Nicholas N.; Austin, Cary; Reichelt, Mike; Sandoval, Wendy; Xu, Min

2017-01-01

ABSTRACT Murein lipoprotein (Lpp) and peptidoglycan-associated lipoprotein (Pal) are major outer membrane lipoproteins in Escherichia coli. Their roles in cell-envelope integrity have been documented in E. coli laboratory strains, and while Lpp has been linked to serum resistance in vitro, the underlying mechanism has not been established. Here, lpp and pal mutants of uropathogenic E. coli strain CFT073 showed reduced survival in a mouse bacteremia model, but only the lpp mutant was sensitive to serum killing in vitro. The peptidoglycan-bound Lpp form was specifically required for preventing complement-mediated bacterial lysis in vitro and complement-mediated clearance in vivo. Compared to the wild-type strain, the lpp mutant had impaired K2 capsular polysaccharide production and was unable to respond to exposure to serum by elevating capsular polysaccharide amounts. These properties correlated with altered cellular distribution of KpsD, the predicted outer membrane translocon for “group 2” capsular polysaccharides. We identified a novel Lpp-dependent association between functional KpsD and peptidoglycan, highlighting important interplay between cell envelope components required for resistance to complement-mediated lysis in uropathogenic E. coli isolates. PMID:28536290

SEDIMENT TOXICITY AS AN INDICATOR OF CONTAMINANT STRESS IN EMAP-ESTUARIES

EPA Science Inventory

Toxicity of sediments is widely used in EPA, ACOE, and NOAA monitoring and regulatory programs as a complement to measuring of chemical concentrations as it provides an indication of the bioavailability of sediment contaminants. Sediment toxicity was included as an abiotic condit...

Lipid Interaction Sites on Channels, Transporters and Receptors: Recent Insights from Molecular Dynamics Simulations

PubMed Central

Hedger, George; Sansom, Mark S. P.

2017-01-01

Lipid molecules are able to selectively interact with specific sites on integral membrane proteins, and modulate their structure and function. Identification and characterisation of these sites is of importance for our understanding of the molecular basis of membrane protein function and stability, and may facilitate the design of lipid-like drug molecules. Molecular dynamics simulations provide a powerful tool for the identification of these sites, complementing advances in membrane protein structural biology and biophysics. We describe recent notable biomolecular simulation studies which have identified lipid interaction sites on a range of different membrane proteins. The sites identified in these simulation studies agree well with those identified by complementary experimental techniques. This demonstrates the power of the molecular dynamics approach in the prediction and characterization of lipid interaction sites on integral membrane proteins. PMID:26946244

Smoke Exposure Causes Endoplasmic Reticulum Stress and Lipid Accumulation in Retinal Pigment Epithelium through Oxidative Stress and Complement Activation*

PubMed Central

Kunchithapautham, Kannan; Atkinson, Carl; Rohrer, Bärbel

2014-01-01

Age-related macular degeneration (AMD) is a complex disease caused by genetic and environmental factors, including genetic variants in complement components and smoking. Smoke exposure leads to oxidative stress, complement activation, endoplasmic reticulum (ER) stress, and lipid dysregulation, which have all been proposed to be associated with AMD pathogenesis. Here we examine the effects of smoke exposure on the retinal pigment epithelium (RPE). Mice were exposed to cigarette smoke or filtered air for 6 months. RPE cells grown as stable monolayers were exposed to 5% cigarette smoke extract (CSE). Effects of smoke were determined by biochemical, molecular, and histological measures. Effects of the alternative pathway (AP) of complement and complement C3a anaphylatoxin receptor signaling were analyzed using knock-out mice or specific inhibitors. ER stress markers were elevated after smoke exposure in RPE of intact mice, which was eliminated in AP-deficient mice. To examine this relationship further, RPE monolayers were exposed to CSE. Short term smoke exposure resulted in production and release of complement C3, the generation of C3a, oxidative stress, complement activation on the cell membrane, and ER stress. Long term exposure to CSE resulted in lipid accumulation, and secretion. All measures were reversed by blocking C3a complement receptor (C3aR), alternative complement pathway signaling, and antioxidant therapy. Taken together, our results provide clear evidence that smoke exposure results in oxidative stress and complement activation via the AP, resulting in ER stress-mediated lipid accumulation, and further suggesting that oxidative stress and complement act synergistically in the pathogenesis of AMD. PMID:24711457

SynechoNET: integrated protein-protein interaction database of a model cyanobacterium Synechocystis sp. PCC 6803.

PubMed

Kim, Woo-Yeon; Kang, Sungsoo; Kim, Byoung-Chul; Oh, Jeehyun; Cho, Seongwoong; Bhak, Jong; Choi, Jong-Soon

2008-01-01

Cyanobacteria are model organisms for studying photosynthesis, carbon and nitrogen assimilation, evolution of plant plastids, and adaptability to environmental stresses. Despite many studies on cyanobacteria, there is no web-based database of their regulatory and signaling protein-protein interaction networks to date. We report a database and website SynechoNET that provides predicted protein-protein interactions. SynechoNET shows cyanobacterial domain-domain interactions as well as their protein-level interactions using the model cyanobacterium, Synechocystis sp. PCC 6803. It predicts the protein-protein interactions using public interaction databases that contain mutually complementary and redundant data. Furthermore, SynechoNET provides information on transmembrane topology, signal peptide, and domain structure in order to support the analysis of regulatory membrane proteins. Such biological information can be queried and visualized in user-friendly web interfaces that include the interactive network viewer and search pages by keyword and functional category. SynechoNET is an integrated protein-protein interaction database designed to analyze regulatory membrane proteins in cyanobacteria. It provides a platform for biologists to extend the genomic data of cyanobacteria by predicting interaction partners, membrane association, and membrane topology of Synechocystis proteins. SynechoNET is freely available at http://synechocystis.org/ or directly at http://bioportal.kobic.kr/SynechoNET/.

Complement factor H protects mice from ischemic acute kidney injury but is not critical for controlling complement activation by glomerular IgM.

PubMed

Goetz, Lindsey; Laskowski, Jennifer; Renner, Brandon; Pickering, Matthew C; Kulik, Liudmila; Klawitter, Jelena; Stites, Erik; Christians, Uwe; van der Vlag, Johan; Ravichandran, Kameswaran; Holers, V Michael; Thurman, Joshua M

2018-05-01

Natural IgM binds to glomerular epitopes in several progressive kidney diseases. Previous work has shown that IgM also binds within the glomerulus after ischemia/reperfusion (I/R) but does not fully activate the complement system. Factor H is a circulating complement regulatory protein, and congenital or acquired deficiency of factor H is a strong risk factor for several types of kidney disease. We hypothesized that factor H controls complement activation by IgM in the kidney after I/R, and that heterozygous factor H deficiency would permit IgM-mediated complement activation and injury at this location. We found that mice with targeted heterozygous deletion of the gene for factor H developed more severe kidney injury after I/R than wild-type controls, as expected, but that complement activation within the glomeruli remained well controlled. Furthermore, mice that are unable to generate soluble IgM were not protected from renal I/R, even in the setting of heterozygous factor H deficiency. These results demonstrate that factor H is important for limiting injury in the kidney after I/R, but it is not critical for controlling complement activation by immunoglobulin within the glomerulus in this setting. IgM binds to glomerular epitopes after I/R, but it is not a significant source of injury. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Heterotetramerization of Plant PIP1 and PIP2 Aquaporins Is an Evolutionary Ancient Feature to Guide PIP1 Plasma Membrane Localization and Function

PubMed Central

Bienert, Manuela D.; Diehn, Till A.; Richet, Nicolas; Chaumont, François; Bienert, Gerd P.

2018-01-01

Aquaporins (AQPs) are tetrameric channel proteins regulating the transmembrane flux of small uncharged solutes and in particular water in living organisms. In plants, members of the plasma membrane intrinsic protein (PIP) AQP subfamily are important for the maintenance of the plant water status through the control of cell and tissue hydraulics. The PIP subfamily is subdivided into two groups: PIP1 and PIP2 that exhibit different water-channel activities when expressed in Xenopus oocytes or yeast cells. Most PIP1 and PIP2 isoforms physically interact and assemble in heterotetramers to modulate their subcellular localization and channel activity when they are co-expressed in oocytes, yeasts, and plants. Whether the interaction between different PIPs is stochastic or controlled by cell regulatory processes is still unknown. Here, we analyzed the water transport activity and the subcellular localization behavior of the complete PIP subfamily (SmPIP1;1, SmPIP2;1, and SmPIP2;2) of the lycophyte Selaginella moellendorffii upon (co-)expression in yeast and Xenopus oocytes. As observed for most of the PIP1 and PIP2 isoforms in other species, SmPIP1;1 was retained in the ER while SmPIP2;1 was found in the plasma membrane but, upon co-expression, both isoforms were found in the plasma membrane, leading to a synergistic effect on the water membrane permeability. SmPIP2;2 behaves as a PIP1, being retained in the endoplasmic reticulum when expressed alone in oocytes or in yeasts. Interestingly, in contrast to the oocyte system, in yeasts no synergistic effect on the membrane permeability was observed upon SmPIP1;1/SmPIP2;1 co-expression. We also demonstrated that SmPIP2;1 is permeable to water and the signaling molecule hydrogen peroxide. Moreover, growth- and complementation assays in the yeast system showed that heteromerization in all possible SmPIP combinations did not modify the substrate specificity of the channels. These results suggest that the characteristics known for angiosperm PIP1 and PIP2 isoforms in terms of their water transport activity, trafficking, and interaction emerged already as early as in non-seed vascular plants. The existence and conservation of these characteristics may argue for the fact that PIP2s are indeed involved in the delivery of PIP1s to the plasma membrane and that the formation of functional heterotetramers is of biological relevance. PMID:29632543

Heterotetramerization of Plant PIP1 and PIP2 Aquaporins Is an Evolutionary Ancient Feature to Guide PIP1 Plasma Membrane Localization and Function.

PubMed

Bienert, Manuela D; Diehn, Till A; Richet, Nicolas; Chaumont, François; Bienert, Gerd P

2018-01-01

Aquaporins (AQPs) are tetrameric channel proteins regulating the transmembrane flux of small uncharged solutes and in particular water in living organisms. In plants, members of the plasma membrane intrinsic protein (PIP) AQP subfamily are important for the maintenance of the plant water status through the control of cell and tissue hydraulics. The PIP subfamily is subdivided into two groups: PIP1 and PIP2 that exhibit different water-channel activities when expressed in Xenopus oocytes or yeast cells. Most PIP1 and PIP2 isoforms physically interact and assemble in heterotetramers to modulate their subcellular localization and channel activity when they are co-expressed in oocytes, yeasts, and plants. Whether the interaction between different PIPs is stochastic or controlled by cell regulatory processes is still unknown. Here, we analyzed the water transport activity and the subcellular localization behavior of the complete PIP subfamily (SmPIP1;1, SmPIP2;1, and SmPIP2;2) of the lycophyte Selaginella moellendorffii upon (co-)expression in yeast and Xenopus oocytes. As observed for most of the PIP1 and PIP2 isoforms in other species, SmPIP1;1 was retained in the ER while SmPIP2;1 was found in the plasma membrane but, upon co-expression, both isoforms were found in the plasma membrane, leading to a synergistic effect on the water membrane permeability. SmPIP2;2 behaves as a PIP1, being retained in the endoplasmic reticulum when expressed alone in oocytes or in yeasts. Interestingly, in contrast to the oocyte system, in yeasts no synergistic effect on the membrane permeability was observed upon SmPIP1;1/SmPIP2;1 co-expression. We also demonstrated that SmPIP2;1 is permeable to water and the signaling molecule hydrogen peroxide. Moreover, growth- and complementation assays in the yeast system showed that heteromerization in all possible SmPIP combinations did not modify the substrate specificity of the channels. These results suggest that the characteristics known for angiosperm PIP1 and PIP2 isoforms in terms of their water transport activity, trafficking, and interaction emerged already as early as in non-seed vascular plants. The existence and conservation of these characteristics may argue for the fact that PIP2s are indeed involved in the delivery of PIP1s to the plasma membrane and that the formation of functional heterotetramers is of biological relevance.

Anopheles Midgut Epithelium Evades Human Complement Activity by Capturing Factor H from the Blood Meal

PubMed Central

Khattab, Ayman; Barroso, Marta; Miettinen, Tiera; Meri, Seppo

2015-01-01

Hematophagous vectors strictly require ingesting blood from their hosts to complete their life cycles. Exposure of the alimentary canal of these vectors to the host immune effectors necessitates efficient counteractive measures by hematophagous vectors. The Anopheles mosquito transmitting the malaria parasite is an example of hematophagous vectors that within seconds can ingest human blood double its weight. The innate immune defense mechanisms, like the complement system, in the human blood should thereby immediately react against foreign cells in the mosquito midgut. A prerequisite for complement activation is that the target cells lack complement regulators on their surfaces. In this work, we analyzed whether human complement is active in the mosquito midgut, and how the mosquito midgut cells protect themselves against complement attack. We found that complement remained active for a considerable time and was able to kill microbes within the mosquito midgut. However, the Anopheles mosquito midgut cells were not injured. These cells were found to protect themselves by capturing factor H, the main soluble inhibitor of the alternative complement pathway. Factor H inhibited complement on the midgut cells by promoting inactivation of C3b to iC3b and preventing the activity of the alternative pathway amplification C3 convertase enzyme. An interference of the FH regulatory activity by monoclonal antibodies, carried to the midgut via blood, resulted in increased mosquito mortality and reduced fecundity. By using a ligand blotting assay, a putative mosquito midgut FH receptor could be detected. Thereby, we have identified a novel mechanism whereby mosquitoes can tolerate human blood. PMID:25679788

Methods to Assess the Direct Interaction of C. jejuni with Mucins.

PubMed

Clyne, Marguerite; Duggan, Gina; Naughton, Julie; Bourke, Billy

2017-01-01

Studies of the interaction of bacteria with mucus-secreting cells can be complemented at a more mechanistic level by exploring the interaction of bacteria with purified mucins. Here we describe a far Western blotting approach to show how C. jejuni proteins separated by SDS PAGE and transferred to a membrane or slot blotted directly onto a membrane can be probed using biotinylated mucin. In addition we describe the use of novel mucin microarrays to assess bacterial interactions with mucins in a high-throughput manner.

Chemical studies on damages of Escherichea coli by the immune bactericidal reaction. II. Release of phosphatidylethanolamine from a phospholipase A-deficient mutant of E. coli during the immune bactericidal reaction.

PubMed

Inoue, K; Yano, K; Amano, T

1974-12-01

When an antibody-sensitized, phospholipase A-deficient mutant of Escherichia coli B/SM was treated with complement in the absence of lysozyme, bacterial phosphatidylethanolamine (PE) was liberated into the lipid fraction of the surrounding medium, but only traces of its degradation products were found in this fraction. Therefore, most of the degradation of bacterial PE to FFA and LPE observed in the usual immune bactericidal reaction (Inoue et al., 1974) must be the result of the action of bacterial phospholipase A which is activated or becomes accessible to its substrate on formation of lesions by complement. The mechanism of complement-mediated formation of membrane lesions is discussed on the basis of these results.

The structural requirements for immunoglobulin aggregates to localize in germinal centres.

PubMed Central

Embling, P H; Evans, H; Guttierez, C; Holborow, E J; Johns, P; Johnson, P M; Papamichail, M; Stanworth, D R

1978-01-01

The capacity of non-heat-aggregated monoclonal human immunoglobulins of different classes, to localize in murine splenic germinal centres within 24 h of intravenous injection has been investigated. It has been shown that at least trimerization of polyclonal IgG must occur before any germinal centre trapping is manifest. Studies of complement fixation by these IgG preparations in vivo, together with studies of the germinal centre trapping of various monoclonal immunoglobulins, have indicated that the sole structural requirement for germinal centre localization of immunoglobulin aggregates is the ability to fix complement. Results suggest that immunoglobulin aggregates are transported to germinal centres via membrane C3 receptors of mobile cells, and then are released with loss of complement to become fixed to dendritic macrophages by a separate mechanism. PMID:363602

The role of cell membranes in the regulation of lignification in pine cells

NASA Technical Reports Server (NTRS)

Hendrix, D. L.

1978-01-01

The identity of pine cell membranes bearing PAL enzyme activity, the isolation of a plasma membrane preparation from pine cells for testing as a regulatory barrier in lignification, and the measurement of the geopotential effect in pine stems are presented. A model to describe and predict the interaction of gravity and lignification of higher plants was developed.

Functional diversification of grapevine MYB5a and MYB5b in the control of flavonoid biosynthesis in a petunia anthocyanin regulatory mutant.

PubMed

Cavallini, Erika; Zenoni, Sara; Finezzo, Laura; Guzzo, Flavia; Zamboni, Anita; Avesani, Linda; Tornielli, Giovanni Battista

2014-03-01

Flavonoids play a key role in grapevine physiology and also contribute substantially to the quality of berries and wines. VvMYB5a and VvMYB5b are R2R3-MYB transcription factors previously proposed to control the spatiotemporal expression of flavonoid structural genes during berry development. We investigated the functions of these two proteins in detail by heterologous expression in a petunia an2 mutant, which has negligible anthocyanin levels in the petals because it lacks the MYB protein PhAN2. We also expressed VvMYBA1, the grapevine ortholog of petunia PhAN2, in the same genetic background. The anthocyanin profiles induced by expressing these transgenes in the petals revealed that VvMYBA1 is the functional ortholog of PhAN2 and that, unlike VvMYB5a, VvMYB5b can partially complement the an2 mutation. Transcriptomic analysis of petals by microarray hybridization and quantitative PCR confirmed that VvMYB5b up-regulates a subset of anthocyanin structural genes, whereas VvMYB5a has a more limited impact on the expression of genes related to anthocyanin biosynthesis. Furthermore, we identified additional specific and common targets of these two regulators, related to vacuolar acidification and membrane remodeling. Taken together, these data provide insight into the role of VvMYB5a and VvMYB5b in flavonoid biosynthesis and provide evidence for additional regulatory roles in distinct pathways.

Functional Analysis of Vaccinia Virus B5R Protein: Essential Role in Virus Envelopment Is Independent of a Large Portion of the Extracellular Domain

PubMed Central

Herrera, Elizabeth; del Mar Lorenzo, María; Blasco, Rafael; Isaacs, Stuart N.

1998-01-01

Vaccinia virus has two forms of infectious virions: the intracellular mature virus and the extracellular enveloped virus (EEV). EEV is critical for cell-to-cell and long-range spread of the virus. The B5R open reading frame (ORF) encodes a membrane protein that is essential for EEV formation. Deletion of the B5R ORF results in a dramatic reduction of EEV, and as a consequence, the virus produces small plaques in vitro and is highly attenuated in vivo. The extracellular portion of B5R is composed mainly of four domains that are similar to the short consensus repeats (SCRs) present in complement regulatory proteins. To determine the contribution of these putative SCR domains to EEV formation, we constructed recombinant vaccinia viruses that replaced the wild-type B5R gene with a mutated gene encoding a B5R protein lacking the SCRs. The resulting recombinant viruses produced large plaques, indicating efficient cell-to-cell spread in vitro, and gradient centrifugation of supernatants from infected cells confirmed that EEV was formed. In contrast, phalloidin staining of infected cells showed that the virus lacking the SCR domains was deficient in the induction of thick actin bundles. Thus, the highly conserved SCR domains present in the extracellular portion of the B5R protein are dispensable for EEV formation. This indicates that the B5R protein is a key viral protein with multiple functions in the process of virus envelopment and release. In addition, given the similarity of the extracellular domain to complement control proteins, the B5R protein may be involved in viral evasion from host immune responses. PMID:9420227

Complement Evasion by Pathogenic Leptospira.

PubMed

Fraga, Tatiana Rodrigues; Isaac, Lourdes; Barbosa, Angela Silva

2016-01-01

Leptospirosis is a neglected infectious disease caused by spirochetes from the genus Leptospira . Pathogenic microorganisms, notably those which reach the blood circulation such as Leptospira , have evolved multiple strategies to escape the host complement system, which is important for innate and acquired immunity. Leptospira avoid complement-mediated killing through: (i) recruitment of host complement regulators; (ii) acquisition of host proteases that cleave complement proteins on the bacterial surface; and, (iii) secretion of proteases that inactivate complement proteins in the Leptospira surroundings. The recruitment of host soluble complement regulatory proteins includes the acquisition of Factor H (FH) and FH-like-1 (alternative pathway), C4b-binding protein (C4BP) (classical and lectin pathways), and vitronectin (Vn) (terminal pathway). Once bound to the leptospiral surface, FH and C4BP retain cofactor activity of Factor I in the cleavage of C3b and C4b, respectively. Vn acquisition by leptospires may result in terminal pathway inhibition by blocking C9 polymerization. The second evasion mechanism lies in plasminogen (PLG) binding to the leptospiral surface. In the presence of host activators, PLG is converted to enzymatically active plasmin, which is able to degrade C3b, C4b, and C5 at the surface of the pathogen. A third strategy used by leptospires to escape from complement system is the active secretion of proteases. Pathogenic, but not saprophytic leptospires, are able to secrete metalloproteases that cleave C3 (central complement molecule), Factor B (alternative pathway), and C4 and C2 (classical and lectin pathways). The purpose of this review is to fully explore these complement evasion mechanisms, which act together to favor Leptospira survival and multiplication in the host.

Complement Evasion by Pathogenic Leptospira

PubMed Central

Fraga, Tatiana Rodrigues; Isaac, Lourdes; Barbosa, Angela Silva

2016-01-01

Leptospirosis is a neglected infectious disease caused by spirochetes from the genus Leptospira. Pathogenic microorganisms, notably those which reach the blood circulation such as Leptospira, have evolved multiple strategies to escape the host complement system, which is important for innate and acquired immunity. Leptospira avoid complement-mediated killing through: (i) recruitment of host complement regulators; (ii) acquisition of host proteases that cleave complement proteins on the bacterial surface; and, (iii) secretion of proteases that inactivate complement proteins in the Leptospira surroundings. The recruitment of host soluble complement regulatory proteins includes the acquisition of Factor H (FH) and FH-like-1 (alternative pathway), C4b-binding protein (C4BP) (classical and lectin pathways), and vitronectin (Vn) (terminal pathway). Once bound to the leptospiral surface, FH and C4BP retain cofactor activity of Factor I in the cleavage of C3b and C4b, respectively. Vn acquisition by leptospires may result in terminal pathway inhibition by blocking C9 polymerization. The second evasion mechanism lies in plasminogen (PLG) binding to the leptospiral surface. In the presence of host activators, PLG is converted to enzymatically active plasmin, which is able to degrade C3b, C4b, and C5 at the surface of the pathogen. A third strategy used by leptospires to escape from complement system is the active secretion of proteases. Pathogenic, but not saprophytic leptospires, are able to secrete metalloproteases that cleave C3 (central complement molecule), Factor B (alternative pathway), and C4 and C2 (classical and lectin pathways). The purpose of this review is to fully explore these complement evasion mechanisms, which act together to favor Leptospira survival and multiplication in the host. PMID:28066433

The hemodialysis membranes: a historical perspective, current state and future prospect.

PubMed

Cheung, A K; Leypoldt, J K

1997-05-01

Transport and biocompatibility characteristics are two important considerations when choosing hemodialysis membranes. Dialyzer performance depends on clearances of small solutes, middle molecules, and oncotically active proteins. Although complement and neutrophil activation have become the gold standards for biocompatibility testing of dialysis membranes, alterations of other cellular and noncellular blood elements as a result of blood-membrane interactions are also important. Because of concerns about middle molecule transport and biocompatibility, the original cellophane membrane has been gradually replaced by modified cellulosic membranes and synthetic membranes for clinical use. Recent studies suggest that the choice of dialysis membrane influences the clinical outcome of patients in several areas, including intradialytic acute anaphylactoid reactions, beta 2-microglobulin associated amyloidosis, recovery from acute renal failure, and mortality of chronic hemodialysis patients. However, the relative contributions of middle molecule transport, biocompatibility, and other factors in determining these differences in outcome are unclear. Future development of hemodialysis membranes should focus on improving biocompatibility and enhancing clearances of small solutes and middle molecules, while minimizing the loss of larger plasma proteins.

Novel Functional Complexity of Polycystin-1 by GPS Cleavage In Vivo: Role in Polycystic Kidney Disease

PubMed Central

Kurbegovic, Almira; Kim, Hyunho; Xu, Hangxue; Yu, Shengqiang; Cruanès, Julie; Maser, Robin L.; Boletta, Alessandra; Trudel, Marie

2014-01-01

Polycystin-1 (Pc1) cleavage at the G protein-coupled receptor (GPCR) proteolytic site (GPS) is required for normal kidney morphology in humans and mice. We found a complex pattern of endogenous Pc1 forms by GPS cleavage. GPS cleavage generates not only the heterodimeric cleaved full-length Pc1 (Pc1cFL) in which the N-terminal fragment (NTF) remains noncovalently associated with the C-terminal fragment (CTF) but also a novel (Pc1) form (Pc1deN) in which NTF becomes detached from CTF. Uncleaved Pc1 (Pc1U) resides primarily in the endoplasmic reticulum (ER), whereas both Pc1cFL and Pc1deN traffic through the secretory pathway in vivo. GPS cleavage is not a prerequisite, however, for Pc1 trafficking in vivo. Importantly, Pc1deN is predominantly found at the plasma membrane of renal epithelial cells. By functional genetic complementation with five Pkd1 mouse models, we discovered that CTF plays a crucial role in Pc1deN trafficking. Our studies support GPS cleavage as a critical regulatory mechanism of Pc1 biogenesis and trafficking for proper kidney development and homeostasis. PMID:24958103

Molecular Cloning and Characterization of cgs, the Brucella abortus Cyclic β(1-2) Glucan Synthetase Gene: Genetic Complementation of Rhizobium meliloti ndvB and Agrobacterium tumefaciens chvB Mutants

PubMed Central

Iñón de Iannino, Nora; Briones, Gabriel; Tolmasky, Marcelo; Ugalde, Rodolfo A.

1998-01-01

The animal pathogen Brucella abortus contains a gene, cgs, that complemented a Rhizobium meliloti nodule development (ndvB) mutant and an Agrobacterium tumefaciens chromosomal virulence (chvB) mutant. The complemented strains recovered the synthesis of cyclic β(1-2) glucan, motility, virulence in A. tumefaciens, and nitrogen fixation in R. meliloti; all traits were strictly associated with the presence of an active cyclic β(1-2) glucan synthetase protein in the membranes. Nucleotide sequencing revealed the presence in B. abortus of an 8.49-kb open reading frame coding for a predicted membrane protein of 2,831 amino acids (316.2 kDa) and with 51% identity to R. meliloti NdvB. Four regions of the B. abortus protein spanning amino acids 520 to 800, 1025 to 1124, 1284 to 1526, and 2400 to 2660 displayed similarities of higher than 80% with R. meliloti NdvB. Tn3-HoHo1 mutagenesis showed that the C-terminal 825 amino acids of the Brucella protein, although highly conserved in Rhizobium, are not necessary for cyclic β(1-2) glucan synthesis. Confirmation of the identity of this protein as B. abortus cyclic β(1-2) glucan synthetase was done by the construction of a B. abortus Tn3-HoHo1 insertion mutant that does not form cyclic β(1-2) glucan and lacks the 316.2-kDa membrane protein. The recovery of this mutant from the spleens of inoculated mice was decreased by 3 orders of magnitude compared with that of the parental strain; this result suggests that cyclic β(1-2) glucan may be a virulence factor in Brucella infection. PMID:9721274

Phospholipid Binding Protein C Inhibitor (PCI) Is Present on Microparticles Generated In Vitro and In Vivo

PubMed Central

Einfinger, Katrin; Badrnya, Sigrun; Furtmüller, Margareta; Handschuh, Daniela; Lindner, Herbert; Geiger, Margarethe

2015-01-01

Protein C inhibitor is a secreted, non-specific serine protease inhibitor with broad protease reactivity. It binds glycosaminoglycans and anionic phospholipids, which can modulate its activity. Anionic phospholipids, such as phosphatidylserine are normally localized to the inner leaflet of the plasma membrane, but are exposed on activated and apoptotic cells and on plasma membrane-derived microparticles. In this report we show by flow cytometry that microparticles derived from cultured cells and activated platelets incorporated protein C inhibitor during membrane blebbing. Moreover, protein C inhibitor is present in/on microparticles circulating in normal human plasma as judged from Western blots, ELISAs, flow cytometry, and mass spectrometry. These plasma microparticles are mainly derived from megakaryocytes. They seem to be saturated with protein C inhibitor, since they do not bind added fluorescence-labeled protein C inhibitor. Heparin partially removed microparticle-bound protein C inhibitor, supporting our assumption that protein C inhibitor is bound via phospholipids. To assess the biological role of microparticle-bound protein C inhibitor we performed protease inhibition assays and co-precipitated putative binding partners on microparticles with anti-protein C inhibitor IgG. As judged from amidolytic assays microparticle-bound protein C inhibitor did not inhibit activated protein C or thrombin, nor did microparticles modulate the activity of exogenous protein C inhibitor. Among the proteins co-precipitating with protein C inhibitor, complement factors, especially complement factor 3, were most striking. Taken together, our data do not support a major role of microparticle-associated protein C inhibitor in coagulation, but rather suggest an interaction with proteins of the complement system present on these phospholipid vesicles. PMID:26580551

Chitin-induced and CHITIN ELICITOR RECEPTOR KINASE1 (CERK1) phosphorylation-dependent endocytosis of Arabidopsis thaliana LYSIN MOTIF-CONTAINING RECEPTOR-LIKE KINASE5 (LYK5).

PubMed

Erwig, Jan; Ghareeb, Hassan; Kopischke, Michaela; Hacke, Ronja; Matei, Alexandra; Petutschnig, Elena; Lipka, Volker

2017-07-01

To detect potential pathogens, plants perceive the fungal polysaccharide chitin through receptor complexes containing lysin motif receptor-like kinases (LysM-RLKs). To investigate the ligand-induced spatial dynamics of chitin receptor components, we studied the subcellular behaviour of two Arabidopsis thaliana LysM-RLKs involved in chitin signalling, CHITIN ELICITOR RECEPTOR KINASE1 (CERK1) and LYSIN MOTIF-CONTAINING RECEPTOR-LIKE KINASE5. We performed standard and quantitative confocal laser scanning microscopy on stably transformed A. thaliana plants expressing fluorescently tagged CERK1 and LYK5 from their native promoters. Microscopy approaches were complemented by biochemical analyses in plants and in vitro. Both CERK1 and LYK5 localized to the plasma membrane and showed constitutive endomembrane trafficking. After chitin treatment, however, CERK1 remained at the plasma membrane while LYK5 relocalized into mobile intracellular vesicles. Detailed analyses revealed that chitin perception transiently induced the internalization of LYK5 into late endocytic compartments. Plants that lacked CERK1 or expressed an enzymatically inactive CERK1 variant did not exhibit chitin-induced endocytosis of LYK5. CERK1 could phosphorylate LYK5 in vitro and chitin treatment induced CERK1-dependent phosphorylation of LYK5 in planta. Our results suggest that chitin-induced phosphorylation by CERK1 triggers LYK5 internalization. Thus, our work identifies phosphorylation as a key regulatory step in endocytosis of plant RLKs and also provides evidence for receptor complex dissociation after ligand perception. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

A second-generation blood substitute (perfluorodichlorooctane emulsion) does not activate complement during an ex vivo circulation model of bypass.

PubMed

Rosoff, J D; Soltow, L O; Vocelka, C R; Schmer, G; Chandler, W L; Cochran, R P; Kunzelman, K S; Spiess, B D

1998-08-01

To examine whether a second-generation perfluorocarbon (PFC) blood substitute added to the cardiopulmonary bypass (CPB) prime influences complement production. A prospective, randomized, single-blinded, ex vivo model. A university hospital, laboratory, and clinics. Ten healthy adult consented volunteer blood donors (five men, five women). Ex vivo closed-loop extracorporeal circuit including membrane oxygenator, tubing, and filter primed with crystalloid or crystalloid plus PFC was circulated for 1 hour with the addition of 500 mL of heparinized fresh human whole blood. Laboratory specimens were drawn from the circuit at 10-minute intervals for 1 hour and measured for complement (C3a, Bb fragment) concentrations, blood gases, fibrinogen concentration, platelet count, and hematocrit. In the PFC group, C3a and Bb fragments were equal to or less than those in the group that received crystalloid alone. The second-generation PFC added to the prime of a CPB circuit does not independently increase complement production.

Susceptibility of pathogenic and nonpathogenic Naegleria ssp

DOE Office of Scientific and Technical Information (OSTI.GOV)

Whiteman, L.Y.

1988-01-01

The susceptibility of four species of Naegleria amoebae to complement-mediated lysis was determined. The amoebicidal activity of normal human serum (NHS) and normal guinea pig serum (NGPS) for Naegleria amoebae was measured by an in vitro cytotoxicity assay. Release of radioactivity from amoebae labeled with {sup 3}H-uridine and visual observation with a compound microscope were used as indices of lysis. Susceptibility or resistance to complement-mediated lysis in vitro correlated with the in vivo pathogenic potential. Nonpathogenic Naegleria amoebae were lysed at a faster rate and at higher cell concentrations than were pathogenic amoebae. Electrophoretic analysis of NHS incubated with pathogenicmore » or nonpathogenic Naegleria spp. demonstrated that amoebae activate the complement cascade resulting in the production of C3 and C5 complement cleavage products. Treatment with papain or trypsin for 1 h, but not with sialidase, increase the susceptibility of highly pathogenic, mouse-passaged N. fowleri to lysis. Treatment with actinomycin D, cycloheximide or various protease inhibitors for 4 h did not increase susceptibility to lysis. Neither a repair process involving de novo protein synthesis nor a complement-inactivating protease appear to account for the increase resistance of N. fowleri amoebae to complement-mediated lysis. A binding study with {sup 125}I radiolabeled C9 indicated that the terminal complement component does not remain stably bound to the membrane of pathogenic amoebae.« less

Identification of C3b-Binding Small-Molecule Complement Inhibitors Using Cheminformatics.

PubMed

Garcia, Brandon L; Skaff, D Andrew; Chatterjee, Arindam; Hanning, Anders; Walker, John K; Wyckoff, Gerald J; Geisbrecht, Brian V

2017-05-01

The complement system is an elegantly regulated biochemical cascade formed by the collective molecular recognition properties and proteolytic activities of more than two dozen membrane-bound or serum proteins. Complement plays diverse roles in human physiology, such as acting as a sentry against invading microorganisms, priming of the adaptive immune response, and removal of immune complexes. However, dysregulation of complement can serve as a trigger for a wide range of human diseases, which include autoimmune, inflammatory, and degenerative conditions. Despite several potential advantages of modulating complement with small-molecule inhibitors, small-molecule drugs are highly underrepresented in the current complement-directed therapeutics pipeline. In this study, we have employed a cheminformatics drug discovery approach based on the extensive structural and functional knowledge available for the central proteolytic fragment of the cascade, C3b. Using parallel in silico screening methodologies, we identified 45 small molecules that putatively bind C3b near ligand-guided functional hot spots. Surface plasmon resonance experiments resulted in the validation of seven dose-dependent C3b-binding compounds. Competition-based biochemical assays demonstrated the ability of several C3b-binding compounds to interfere with binding of the original C3b ligand that guided their discovery. In vitro assays of complement function identified a single complement inhibitory compound, termed cmp-5, and mechanistic studies of the cmp-5 inhibitory mode revealed it acts at the level of C5 activation. This study has led to the identification of a promising new class of C3b-binding small-molecule complement inhibitors and, to our knowledge, provides the first demonstration of cheminformatics-based, complement-directed drug discovery. Copyright © 2017 by The American Association of Immunologists, Inc.

Identification of C3b-binding Small Molecule Complement Inhibitors Using Cheminformatics

PubMed Central

Garcia, Brandon L.; Skaff, D. Andrew; Chatterjee, Arindam; Hanning, Anders; Walker, John K.; Wyckoff, Gerald J.; Geisbrecht, Brian V.

2017-01-01

The complement system is an elegantly regulated biochemical cascade formed by the collective molecular recognition properties and proteolytic activities of over two dozen membrane-bound or serum proteins. Complement plays diverse roles in human physiology which include acting as a sentry against invading microorganisms, priming of the adaptive immune response, and removal of immune complexes. However, dysregulation of complement can serve as a trigger for a wide range of human diseases which include autoimmune, inflammatory, and degenerative conditions. Despite several potential advantages of modulating complement with small molecule inhibitors, small molecule drugs are highly underrepresented in the current complement-directed therapeutics pipeline. In this study we have employed a cheminformatics drug discovery approach based on the extensive structural and functional knowledge available for the central proteolytic fragment of the cascade, C3b. Using parallel in silico screening methodologies we identified 45 small molecules which putatively bind C3b near ligand-guided functional hot-spots. Surface plasmon resonance experiments resulted in the validation of seven dose-dependent C3b-binding compounds. Competition-based biochemical assays demonstrated the ability of several C3b-binding compounds to interfere with binding of the original C3b ligand which guided their discovery. In vitro assays of complement function identified a single complement inhibitory compound, termed cmp-5, and mechanistic studies of the cmp-5 inhibitory mode revealed it acts at the level of C5 activation. This study has led to the identification of a promising new class of C3b-binding small molecule complement inhibitors, and to our knowledge, provides the first demonstration of cheminformatics-based complement-directed drug discovery. PMID:28298523

Complement is activated in progressive multiple sclerosis cortical grey matter lesions.

PubMed

Watkins, Lewis M; Neal, James W; Loveless, Sam; Michailidou, Iliana; Ramaglia, Valeria; Rees, Mark I; Reynolds, Richard; Robertson, Neil P; Morgan, B Paul; Howell, Owain W

2016-06-22

The symptoms of multiple sclerosis (MS) are caused by damage to myelin and nerve cells in the brain and spinal cord. Inflammation is tightly linked with neurodegeneration, and it is the accumulation of neurodegeneration that underlies increasing neurological disability in progressive MS. Determining pathological mechanisms at play in MS grey matter is therefore a key to our understanding of disease progression. We analysed complement expression and activation by immunocytochemistry and in situ hybridisation in frozen or formalin-fixed paraffin-embedded post-mortem tissue blocks from 22 progressive MS cases and made comparisons to inflammatory central nervous system disease and non-neurological disease controls. Expression of the transcript for C1qA was noted in neurons and the activation fragment and opsonin C3b-labelled neurons and glia in the MS cortical and deep grey matter. The density of immunostained cells positive for the classical complement pathway protein C1q and the alternative complement pathway activation fragment Bb was significantly increased in cortical grey matter lesions in comparison to control grey matter. The number of cells immunostained for the membrane attack complex was elevated in cortical lesions, indicating complement activation to completion. The numbers of classical (C1-inhibitor) and alternative (factor H) pathway regulator-positive cells were unchanged between MS and controls, whilst complement anaphylatoxin receptor-bearing microglia in the MS cortex were found closely apposed to cortical neurons. Complement immunopositive neurons displayed an altered nuclear morphology, indicative of cell stress/damage, supporting our finding of significant neurodegeneration in cortical grey matter lesions. Complement is activated in the MS cortical grey matter lesions in areas of elevated numbers of complement receptor-positive microglia and suggests that complement over-activation may contribute to the worsening pathology that underlies the irreversible progression of MS.

Temperature-sensitive Mutants of Sindbis Virus: Biochemical Correlates of Complementation

PubMed Central

Burge, Boyce W.; Pfefferkorn, E. R.

1967-01-01

Temperature-sensitive mutants of Sindbis virus fail to grow at a temperature that permits growth of the wild type, but when certain pairs of these mutants, mixed together, infect cells at that temperature, viral growth (i.e., complementation) occurs. The yield from this complementation, however, is of the same order of magnitude as the infectivity in the inoculum. Since in animal virus infections the protein components of the virion probably enter the cell with the viral nucleic acid, it was necessary to demonstrate that the observed complementation required synthesis of new viral protein and nucleic acid rather than some sort of rearrangement of the structural components of the inoculum. To demonstrate that complementation does require new biosynthesis, three biochemical events of normal virus growth have been observed during complementation and correlated with the efficiency of viral growth seen in complementation. These events include: (i) entrance of parental viral ribonucleic acid (RNA) into a double-stranded form; (ii) subsequent synthesis of viral RNA; and (iii) synthesis and subsequent incorporation of viral protein(s) into cell membranes where they were detected by hemadsorption. Although the infecting single-stranded RNA genome of the wild type was converted to a ribonuclease-resistant form, the genome of a mutant (ts-11) incapable of RNA synthesis at a nonpermissive temperature was not so converted. However, during complementation with another mutant also defective in viral RNA synthesis, some of the RNA of mutant ts-11 was converted to a ribonuclease-resistant form, and total synthesis of virus-specific RNA was markedly enhanced. The virus-specific alteration of the cell surface, detected by hemadsorption, was also extensively increased during complementation. These observations support the view that complementation between temperature-sensitive mutants and replication of wild-type virus are similar processes. PMID:5630228

Water Activated Graphene Oxide Transfer Using Wax Printed Membranes for Fast Patterning of a Touch Sensitive Device.

PubMed

Baptista-Pires, Luis; Mayorga-Martínez, Carmen C; Medina-Sánchez, Mariana; Montón, Helena; Merkoçi, Arben

2016-01-26

We demonstrate a graphene oxide printing technology using wax printed membranes for the fast patterning and water activation transfer using pressure based mechanisms. The wax printed membranes have 50 μm resolution, longtime stability and infinite shaping capability. The use of these membranes complemented with the vacuum filtration of graphene oxide provides the control over the thickness. Our demonstration provides a solvent free methodology for printing graphene oxide devices in all shapes and all substrates using the roll-to-roll automatized mechanism present in the wax printing machine. Graphene oxide was transferred over a wide variety of substrates as textile or PET in between others. Finally, we developed a touch switch sensing device integrated in a LED electronic circuit.

Transcriptomic insights into citrus segment membrane's cell wall components relating to fruit sensory texture.

PubMed

Wang, Xun; Lin, Lijin; Tang, Yi; Xia, Hui; Zhang, Xiancong; Yue, Maolan; Qiu, Xia; Xu, Ke; Wang, Zhihui

2018-04-23

During fresh fruit consumption, sensory texture is one factor that affects the organoleptic qualities. Chemical components of plant cell walls, including pectin, cellulose, hemicellulose and lignin, play central roles in determining the textural qualities. To explore the genes and regulatory pathways involved in fresh citrus' perceived sensory texture, we performed mRNA-seq analyses of the segment membranes of two citrus cultivars, Shiranui and Kiyomi, with different organoleptic textures. Segment membranes were sampled at two developmental stages of citrus fruit, the beginning and end of the expansion period. More than 3000 differentially expressed genes were identified. The gene ontology analysis revealed that more categories were significantly enriched in 'Shiranui' than in 'Kiyomi' at both developmental stages. In total, 108 significantly enriched pathways were obtained, with most belonging to metabolism. A detailed transcriptomic analysis revealed potential critical genes involved in the metabolism of cell wall structures, for example, GAUT4 in pectin synthesis, CESA1, 3 and 6, and SUS4 in cellulose synthesis, CSLC5, XXT1 and XXT2 in hemicellulose synthesis, and CSE in lignin synthesis. Low levels, or no expression, of genes involved in cellulose and hemicellulose, such as CESA4, CESA7, CESA8, IRX9 and IRX14, confirmed that secondary cell walls were negligible or absent in citrus segment membranes. A chemical component analysis of the segment membranes from mature fruit revealed that the pectin, cellulose and lignin contents, and the segment membrane's weight (% of segment) were greater in 'Kiyomi'. Organoleptic quality of citrus is easily overlooked. It is mainly determined by sensory texture perceived in citrus segment membrane properties. We performed mRNA-seq analyses of citrus segment membranes to explore the genes and regulatory pathways involved in fresh citrus' perceived sensory texture. Transcriptomic data showed high repeatability between two independent biological replicates. The expression levels of genes involved in cell wall structure metabolism, including pectin, cellulose, hemicellulose and lignin, were investigated. Meanwhile, chemical component contents of the segment membranes from mature fruit were analyzed. This study provided detailed transcriptional regulatory profiles of different organoleptic citrus qualities and integrated insights into the mechanisms affecting citrus' sensory texture.

Life and Death of Glucose-6-Phosphate Dehydrogenase (G6PD) Deficient Erythrocytes - Role of Redox Stress and Band 3 Modifications.

PubMed

Arese, Paolo; Gallo, Valentina; Pantaleo, Antonella; Turrini, Franco

2012-10-01

G6PD catalyzes the first, pace-making reaction of pentosephosphate cycle (PPC) which produces NADPH. NADPH maintains glutathione and thiol groups of proteins and enzymes in the reduced state which is essential for protection against oxidative stress. Individuals affected by G6PD deficiency are unable to regenerate reduced glutathione (GSH) and are undefended against oxidative stress. G6PD deficiency accelerates normal senescence and enhances the precocious removal of chronologically young, yet biologically old cells. The term hemolytic anemia is misleading because RBCs do not lyse but are removed by phagocytosis. Acute hemolysis by fava bean ingestion in G6PD deficient individuals (favism) is described being the best-studied natural model of oxidant damage. It bears strong analogies to hemolysis by oxidant drugs or chemicals. Membrane alterations observed in vivo during favism are superimposable to changes in senescent RBCs. In summary, RBC membranes isolated from favic patients contained elevated amounts of complexes between IgG and the complement fragment C3b/C3c and were prone to vesiculation. Anti-band 3 IgG reacted to aggregated band 3-complement complexes. In favism extensive clustering of band 3 and membrane deposition of hemichromes were also observed. Severely damaged RBCs isolated from early crises had extensive membrane cross-bonding and very low GSH levels and were phagocytosed 10-fold more intensely compared to normal RBCs.

75 FR 54374 - BOEMRE Information Collection Activity: 1010-0112, Performance Measures Data, Revision of a...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-09-07

... industry as a complement to current regulatory efforts to protect people and the environment during OCS oil... ______ ENVIRONMENT G. No. of EPA NPDES Noncompliances _________ H. For Oil Spills ... oil, gas, and sulphur resources; make such resources available to meet the Nation's energy needs as...

Induction, transcription, synthesis, and adsorption of interleukin-1 by dialyzer membranes.

PubMed

Vaziri, N D; Wang, J; Cesario, T; Yousefi, S; Valenzuela, R; Carandang, G

1994-05-01

This study was designed to dissect the direct effect of dialyzer membrane on interleukin-1 (IL-1) induction from those of complement activation, mechanical stimulation, acetate/bicarbonate and endotoxin diffusion, and cell type interactions. To this end, a suspension of P388D1 murine macrophages in a complement-free culture medium containing 10% heat-inactivated serum, a closed-loop system consisting of tubing alone or with a hollow-fiber cuprammonium cellulose (CU) or polyacrylonitrile (PAN) dialyzer, and a roller pump were used. The dialysate compartment was filled with the same medium and capped. Cell suspension was recirculated at 300 mL/min for 3 h. Cells and supernates were separated, and adhering proteins were eluted. All samples tested negative for endotoxin. IL-1 mRNA was greatest with CU, followed by PAN and tubing alone. IL-1 in the supernate was greater with CU than with either tubing alone or PAN (P < 0.005; analysis of variance), which showed comparable values. IL-1 eluted from loops was greatest with PAN dialyzers, followed by sets with CU dialyzers and tubing alone (P < 0.001; analysis of variance). Thus, both CU and PAN membranes directly induce IL-1. However, avid adsorption by PAN attenuates the rise in circulating IL-1.

Autoantibody-induced internalization of CNS AQP4 water channel and EAAT2 glutamate transporter requires astrocytic Fc receptor

PubMed Central

Hinson, Shannon R.; Clift, Ian C.; Luo, Ningling; Kryzer, Thomas J.; Lennon, Vanda A.

2017-01-01

Aquaporin-4 (AQP4) water channel-specific IgG distinguishes neuromyelitis optica (NMO) from multiple sclerosis and causes characteristic immunopathology in which central nervous system (CNS) demyelination is secondary. Early events initiating the pathophysiological outcomes of IgG binding to astrocytic AQP4 are poorly understood. CNS lesions reflect events documented in vitro following IgG interaction with AQP4: AQP4 internalization, attenuated glutamate uptake, intramyelinic edema, interleukin-6 release, complement activation, inflammatory cell recruitment, and demyelination. Here, we demonstrate that AQP4 internalization requires AQP4-bound IgG to engage an astrocytic Fcγ receptor (FcγR). IgG-lacking Fc redistributes AQP4 within the plasma membrane and induces interleukin-6 release. However, AQP4 endocytosis requires an activating FcγR’s gamma subunit and involves astrocytic membrane loss of an inhibitory FcγR, CD32B. Interaction of the IgG–AQP4 complex with FcγRs triggers coendocytosis of the excitatory amino acid transporter 2 (EAAT2). Requirement of FcγR engagement for internalization of two astrocytic membrane proteins critical to CNS homeostasis identifies a complement-independent, upstream target for potential early therapeutic intervention in NMO. PMID:28461494

Biocompatibility of nanoporous alumina membranes for immunoisolation

PubMed Central

La Flamme, Kristen E.; Popat, Ketul C.; Leoni, Lara; Markiewicz, Erica; LaTempa, Thomas J.; Roman, Brian B.; Grimes, Craig A.; Desai, Tejal A.

2011-01-01

Cellular immunoisolation using semi-permeable barriers has been investigated over the past several decades as a promising treatment approach for diseases such as Parkinson’s, Alzheimer’s, and Type 1 diabetes. Typically, polymeric membranes are used for immunoisolation applications; however, recent advances in technology have led to the development of more robust membranes that are able to more completely meet the requirements for a successful immunoisolation device, including well controlled pore size, chemical and mechanical stability, non-biodegradability, and biocompatibility with both the graft tissue as well as the host. It has been shown previously that nanoporous alumina biocapsules can act effectively as immunoisolation devices, and support the viability and functionality of encapsulated β cells. The aim of this investigation was to assess the biocompatibility of the material with host tissue. The cytotoxicity of the capsule, as well as its ability to activate complement and inflammation was studied. Further, the effects of PEG-modification on the tissue response to implanted capsules were studied. Our results have shown that the device is non-toxic and does not induce significant complement activation. Further, in vivo work has demonstrated that implantation of these capsules into the peritoneal cavity of rats induces a transient inflammatory response, and that PEG is useful in minimizing the host response to the material. PMID:17335895

Isolation and properties of mesosomal membrane fractions from Micrococcus lysodeikticus

PubMed Central

Owen, Peter; Freer, John H.

1972-01-01

1. A method is described for the isolation of pure mesosomal membrane fractions from Micrococcus lysodeikticus. 2. Plasmolysis of cells, before wall digestion, was necessary for effective mesosome release. 3. The effect of mild shearing forces, temperature and time upon the release of mesosomal membrane from protoplasts was investigated. 4. The optimum yield of mesosomal membranes from stable protoplasts was achieved at 10mm-Mg2+. 5. Mesosomal membrane vesicle fractions prepared at differing Mg2+ concentrations above 10mm were similar in chemical composition. 6. Comparison of the properties of peripheral and mesosomal membrane fractions revealed major differences in the distribution of protein components, membrane phosphorus, mannose and dehydrogenase activities between the two fractions. 7. Only cytochrome b556 was detected in mesosomal membranes, whereas peripheral membranes contained a full complement of cytochromes. 8. Preliminary investigations suggested the localization of an autolytic enzyme(s) in the mesosomal vesicles. 9. The anatomy of mesosomal and peripheral membrane have been compared by the negative-staining and freeze-fracture technique. 10. The results are discussed in relation to a plausible role for the mesosome. ImagesPLATE 1PLATE 2PLATE 3PLATE 4 PMID:4655825

Detection and characterisation of Complement protein activity in bovine milk by bactericidal sequestration assay.

PubMed

Maye, Susan; Stanton, Catherine; Fitzgerald, Gerald F; Kelly, Philip M

2015-08-01

While the Complement protein system in human milk is well characterised, there is little information on its presence and activity in bovine milk. Complement forms part of the innate immune system, hence the importance of its contribution during milk ingestion to the overall defences of the neonate. A bactericidal sequestration assay, featuring a Complement sensitive strain, Escherichia coli 0111, originally used to characterise Complement activity in human milk was successfully applied to freshly drawn bovine milk samples, thus, providing an opportunity to compare Complement activities in both human and bovine milks. Although not identical in response, the levels of Complement activity in bovine milk were found to be closely comparable with that of human milk. Differential counts of Esch. coli 0111 after 2 h incubation were 6.20 and 6.06 log CFU/ml, for raw bovine and human milks, respectively - the lower value representing a stronger Complement response. Exposing bovine milk to a range of thermal treatments e.g. 42, 45, 65, 72, 85 or 95 °C for 10 min, progressively inhibited Complement activity by increasing temperature, thus confirming the heat labile nature of this immune protein system. Low level Complement activity was found, however, in 65 and 72 °C heat treated samples and in retailed pasteurised milk which highlights the outer limit to which high temperature, short time (HTST) industrial thermal processes should be applied if retention of activity is a priority. Concentration of Complement in the fat phase was evident following cream separation, and this was also reflected in the further loss of activity recorded in low fat variants of retailed pasteurised milk. Laboratory-based churning of the cream during simulated buttermaking generated an aqueous (buttermilk) phase with higher levels of Complement activity than the fat phase, thus pointing to a likely association with the milk fat globule membrane (MFGM) layer.

Molecular characterization of the rhesus rhadinovirus (RRV) ORF4 gene and the RRV complement control protein it encodes.

PubMed

Mark, Linda; Spiller, O Brad; Okroj, Marcin; Chanas, Simon; Aitken, Jim A; Wong, Scott W; Damania, Blossom; Blom, Anna M; Blackbourn, David J

2007-04-01

The diversity of viral strategies to modulate complement activation indicates that this component of the immune system has significant antiviral potential. One example is the Kaposi's sarcoma-associated herpesvirus (KSHV) complement control protein (KCP), which inhibits progression of the complement cascade. Rhesus rhadinovirus (RRV), like KSHV, is a member of the subfamily Gammaherpesvirinae and currently provides the only in vivo model of KSHV pathobiology in primates. In the present study, we characterized the KCP homologue encoded by RRV, RRV complement control protein (RCP). Two strains of RRV have been sequenced to date (H26-95 and 17577), and the RCPs they encode differ substantially in structure: RCP from strain H26-95 has four complement control protein (CCP) domains, whereas RCP from strain 17577 has eight CCP domains. Transcriptional analyses of the RCP gene (ORF4, referred to herein as RCP) in infected rhesus macaque fibroblasts mapped the ends of the transcripts of both strains. They revealed that H26-95 encodes a full-length, unspliced RCP transcript, while 17577 RCP generates a full-length unspliced mRNA and two alternatively spliced transcripts. Western blotting confirmed that infected cells express RCP, and immune electron microscopy disclosed this protein on the surface of RRV virions. Functional studies of RCP encoded by both RRV strains revealed their ability to suppress complement activation by the classical (antibody-mediated) pathway. These data provide the foundation for studies into the biological significance of gammaherpesvirus complement regulatory proteins in a tractable, non-human primate model.

Molecular Characterization of the Rhesus Rhadinovirus (RRV) ORF4 Gene and the RRV Complement Control Protein It Encodes▿

PubMed Central

Mark, Linda; Spiller, O. Brad; Okroj, Marcin; Chanas, Simon; Aitken, Jim A.; Wong, Scott W.; Damania, Blossom; Blom, Anna M.; Blackbourn, David J.

2007-01-01

The diversity of viral strategies to modulate complement activation indicates that this component of the immune system has significant antiviral potential. One example is the Kaposi's sarcoma-associated herpesvirus (KSHV) complement control protein (KCP), which inhibits progression of the complement cascade. Rhesus rhadinovirus (RRV), like KSHV, is a member of the subfamily Gammaherpesvirinae and currently provides the only in vivo model of KSHV pathobiology in primates. In the present study, we characterized the KCP homologue encoded by RRV, RRV complement control protein (RCP). Two strains of RRV have been sequenced to date (H26-95 and 17577), and the RCPs they encode differ substantially in structure: RCP from strain H26-95 has four complement control protein (CCP) domains, whereas RCP from strain 17577 has eight CCP domains. Transcriptional analyses of the RCP gene (ORF4, referred to herein as RCP) in infected rhesus macaque fibroblasts mapped the ends of the transcripts of both strains. They revealed that H26-95 encodes a full-length, unspliced RCP transcript, while 17577 RCP generates a full-length unspliced mRNA and two alternatively spliced transcripts. Western blotting confirmed that infected cells express RCP, and immune electron microscopy disclosed this protein on the surface of RRV virions. Functional studies of RCP encoded by both RRV strains revealed their ability to suppress complement activation by the classical (antibody-mediated) pathway. These data provide the foundation for studies into the biological significance of gammaherpesvirus complement regulatory proteins in a tractable, non-human primate model. PMID:17287274

The Arabidopsis R-SNARE VAMP721 Interacts with KAT1 and KC1 K+ Channels to Moderate K+ Current at the Plasma Membrane.

PubMed

Zhang, Ben; Karnik, Rucha; Wang, Yizhou; Wallmeroth, Niklas; Blatt, Michael R; Grefen, Christopher

2015-06-01

SNARE (soluble N-ethylmaleimide-sensitive factor protein attachment protein receptor) proteins drive vesicle traffic, delivering membrane and cargo to target sites within the cell and at its surface. They contribute to cell homeostasis, morphogenesis, and pathogen defense. A subset of SNAREs, including the Arabidopsis thaliana SNARE SYP121, are known also to coordinate solute uptake via physical interactions with K(+) channels and to moderate their gating at the plasma membrane. Here, we identify a second subset of SNAREs that interact to control these K(+) channels, but with opposing actions on gating. We show that VAMPs (vesicle-associated membrane proteins), which target vesicles to the plasma membrane, also interact with and suppress the activities of the inward-rectifying K(+) channels KAT1 and KC1. Interactions were evident in yeast split-ubiquitin assays, they were recovered in vivo by ratiometric bimolecular fluorescence complementation, and they were sensitive to mutation of a single residue, Tyr-57, within the longin domain of VAMP721. Interaction was also recovered on exchange of the residue at this site in the homolog VAMP723, which normally localizes to the endoplasmic reticulum and otherwise did not interact. Functional analysis showed reduced channel activity and alterations in voltage sensitivity that are best explained by a physical interaction with the channel gates. These actions complement those of SYP121, a cognate SNARE partner of VAMP721, and lead us to propose that the channel interactions reflect a "hand-off" in channel control between the two SNARE proteins that is woven together with vesicle fusion. © 2015 American Society of Plant Biologists. All rights reserved.

Functional complementation of yeast cytosolic pyrophosphatase by bacterial and plant H+-translocating pyrophosphatases.

PubMed

Perez-Castineira, Jose R; Lopez-Marques, Rosa L; Villalba, Jose M; Losada, Manuel; Serrano, Aurelio

2002-12-10

Two types of proteins that hydrolyze inorganic pyrophosphate (PPi), very different in both amino acid sequence and structure, have been characterized to date: soluble and membrane-bound proton-pumping pyrophosphatases (sPPases and H(+)-PPases, respectively). sPPases are ubiquitous proteins that hydrolyze PPi releasing heat, whereas H+-PPases, so far unidentified in animal and fungal cells, couple the energy of PPi hydrolysis to proton movement across biological membranes. The budding yeast Saccharomyces cerevisiae has two sPPases that are located in the cytosol and in the mitochondria. Previous attempts to knock out the gene coding for a cytosolic sPPase (IPP1) have been unsuccessful, thus suggesting that this protein is essential for growth. Here, we describe the generation of a conditional S. cerevisiae mutant (named YPC-1) whose functional IPP1 gene is under the control of a galactose-dependent promoter. Thus, YPC-1 cells become growth arrested in glucose but they regain the ability to grow on this carbon source when transformed with autonomous plasmids bearing diverse foreign H+-PPase genes under the control of a yeast constitutive promoter. The heterologously expressed H+-PPases are distributed among different yeast membranes, including the plasma membrane, functional complementation by these integral membrane proteins being consistently sensitive to external pH. These results demonstrate that hydrolysis of cytosolic PPi is essential for yeast growth and that this function is not substantially affected by the intrinsic characteristics of the PPase protein that accomplishes it. Moreover, this is, to our knowledge, the first direct evidence that H+-PPases can mediate net hydrolysis of PPi in vivo. YPC-1 mutant strain constitutes a convenient expression system to perform studies aimed at the elucidation of the structure-function relationships of this type of proton pumps.

Loss of endocytic clathrin-coated pits upon acute depletion of phosphatidylinositol 4,5-bisphosphate.

PubMed

Zoncu, Roberto; Perera, Rushika M; Sebastian, Rafael; Nakatsu, Fubito; Chen, Hong; Balla, Tamas; Ayala, Guillermo; Toomre, Derek; De Camilli, Pietro V

2007-03-06

Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)], a phosphoinositide concentrated predominantly in the plasma membrane, binds endocytic clathrin adaptors, many of their accessory factors, and a variety of actin-regulatory proteins. Here we have used fluorescent fusion proteins and total internal reflection fluorescence microscopy to investigate the effect of acute PI(4,5)P(2) breakdown on the dynamics of endocytic clathrin-coated pit components and of the actin regulatory complex, Arp2/3. PI(4,5)P(2) breakdown was achieved by the inducible recruitment to the plasma membrane of an inositol 5-phosphatase module through the rapamycin/FRB/FKBP system or by treatment with ionomycin. PI(4,5)P(2) depletion resulted in a dramatic loss of clathrin puncta, which correlated with a massive dissociation of endocytic adaptors from the plasma membrane. Remaining clathrin spots at the cell surface had only weak fluorescence and were static over time. Dynamin and the p20 subunit of the Arp2/3 actin regulatory complex, which were concentrated at late-stage clathrin-coated pits and in lamellipodia, also dissociated from the plasma membrane, and these changes correlated with an arrest of motility at the cell edge. These findings demonstrate the critical importance of PI(4,5)P(2) in clathrin coat dynamics and Arp2/3-dependent actin regulation.

Heparin free coating on PLA membranes for enhanced hemocompatibility via iCVD

NASA Astrophysics Data System (ADS)

Wang, Hui; Shi, Xiao; Gao, Ailin; Lin, Haibo; Chen, Yongliang; Ye, Yumin; He, Jidong; Liu, Fu; Deng, Gang

2018-03-01

In the present work, we report one-step immobilization of nano-heparin coating on PLA membranes via initiated chemical vapor deposition (iCVD) for enhanced hemocompatibility. The nano-coating introduced onto the membrane surface via the crosslinking of P(MAA-EGDA) was confirmed by the FTIR, SEM and weight measurement respectively. The negative carboxyl groups could form the hydration interaction with the protein and platelets and electrostatic interaction with amide groups of thrombin by the mediation of antithrombin, which is similar but different with heparin. The P(MAA-EGDA) coated membranes showed suppressed platelet adhesion and prolonged clotting time (APTTs increased to 59 s, PTs increased to 20.4 s, TTs increased to 17.5 s, and the FIBs declined by 30 mg/dL). Moreover, the complement activation tests demonstrated the formation of C3a and C5a was inhibited. All results demonstrated that the nano-coating of P(MAA-EGDA) via iCVD significantly enhanced the hemocompatibility of PLA membranes, which is also applicable for various membranes.

Cpa, the outer membrane protease of Cronobacter sakazakii, activates plasminogen and mediates resistance to serum bactericidal activity.

PubMed

Franco, A A; Kothary, M H; Gopinath, G; Jarvis, K G; Grim, C J; Hu, L; Datta, A R; McCardell, B A; Tall, B D

2011-04-01

Cronobacter spp. are emerging neonatal pathogens in humans, associated with outbreaks of meningitis and sepsis. To cause disease, they must survive in blood and invade the central nervous system by penetrating the blood-brain barrier. C. sakazakii BAA-894 possesses an ~131-kb plasmid (pESA3) that encodes an outer membrane protease (Cpa) that has significant identity to proteins that belong to the Pla subfamily of omptins. Members of this subfamily of proteins degrade a number of serum proteins, including circulating complement, providing protection from the complement-dependent serum killing. Moreover, proteins of the Pla subfamily can cause uncontrolled plasmin activity by converting plasminogen to plasmin and inactivating the plasmin inhibitor α2-antiplasmin (α2-AP). These reactions enhance the spread and invasion of bacteria in the host. In this study, we found that an isogenic cpa mutant showed reduced resistance to serum in comparison to its parent C. sakazakii BAA-894 strain. Overexpression of Cpa in C. sakazakii or Escherichia coli DH5α showed that Cpa proteolytically cleaved complement components C3, C3a, and C4b. Furthermore, a strain of C. sakazakii overexpressing Cpa caused a rapid activation of plasminogen and inactivation of α2-AP. These results strongly suggest that Cpa may be an important virulence factor involved in serum resistance, as well as in the spread and invasion of C. sakazakii.

Cholesterol transfer at endosomal-organelle membrane contact sites.

PubMed

Ridgway, Neale D; Zhao, Kexin

2018-06-01

Cholesterol is delivered to the limiting membrane of late endosomes by Niemann-Pick Type C1 and C2 proteins. This review summarizes recent evidence that cholesterol transfer from endosomes to the endoplasmic reticulum and other organelles is mediated by lipid-binding proteins that localize to membrane contact sites (MCS). LDL-cholesterol in the late endosomal/lysosomes is exported to the plasma membrane, where most cholesterol resides, and the endoplasmic reticulum, which harbors the regulatory complexes and enzymes that control the synthesis and esterification of cholesterol. A major advance in dissecting these cholesterol transport pathways was identification of frequent and dynamic MCS between endosomes and the endoplasmic reticulum, peroxisomes and plasma membrane. Positioned at these MCS are members of the oxysterol-binding protein (OSBP) and steroidogenic acute regulatory protein-related lipid-transfer family of lipid transfer proteins that bridge the opposing membranes and directly or indirectly mediate cholesterol transfer. OSBP-related protein 1L (ORP1L), ORP5 and ORP6 mediate cholesterol transfer to the endoplasmic reticulum that regulates cholesterol homeostasis. ORP1L and STARD3 also move cholesterol from the endoplasmic reticulum-to-late endosomal/lysosomes under low-cholesterol conditions to facilitate intraluminal vesicle formation. Cholesterol transport also occurs at MCS with peroxisomes and possibly the plasma membrane. Frequent contacts between organelles and the endo-lysosomal vesicles are sites for bidirectional transfer of cholesterol.

78 FR 1158 - Anesthesiology Devices; Reclassification of Membrane Lung for Long-Term Pulmonary Support...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-01-08

... controls) for conditions where imminent death is threatened by cardiopulmonary failure in neonates and... to the same regulatory controls, all of the device components used in an ECMO procedure are being... regulatory controls needed to provide reasonable assurance of their safety and effectiveness. The three...

Logarithmic phase Escherichia coli K1 efficiently avoids serum killing by promoting C4bp-mediated C3b and C4b degradation

PubMed Central

Wooster, David G; Maruvada, Ravi; Blom, Anna M; Prasadarao, Nemani V

2006-01-01

Meningitis caused by Escherichia coli K1 is a serious illness in neonates with neurological sequelae in up to 50% of survivors. A high degree of bacteremia is required for E. coli K1 to cross the blood–brain barrier, which suggests that the bacterium must evade the host defence mechanisms and survive in the bloodstream. We previously showed that outer membrane protein A (OmpA) of E. coli binds C4b-binding protein (C4bp), an inhibitor of complement activation via the classical pathway. Nevertheless, the exact mechanism by which E. coli K1 survives in serum remains elusive. Here, we demonstrate that log phase (LP) OmpA+E. coli K1 avoids serum bactericidal activity more effectively than postexponential phase bacteria. OmpA–E. coli cannot survive in serum grown to either phase. The increased serum resistance of LP OmpA+E. coli is the result of increased binding of C4bp, with a concomitant decrease in the deposition of C3b and the downstream complement proteins responsible for the formation of the membrane attack complex. C4bp bound to E. coli K1 acts as a cofactor to factor I in the cleavage of both C3b and C4b, which shuts down the ensuing complement cascade. Accordingly, a peptide corresponding to the complement control protein domain 3 of C4bp sequence, was able to compete with C4bp binding to OmpA and cause increased deposition of C3b. Thus, binding of C4bp appears to be responsible for survival of E. coli K1 in human serum. PMID:16556262

The membrane attack complex of complement contributes to plasmin-induced synthesis of platelet-activating factor by endothelial cells and neutrophils

PubMed Central

Lupia, Enrico; Del Sorbo, Lorenzo; Bergerone, Serena; Emanuelli, Giorgio; Camussi, Giovanni; Montrucchio, Giuseppe

2003-01-01

Thrombolytic agents, used to restore blood flow to ischaemic tissues, activate several enzymatic systems with pro-inflammatory effects, thus potentially contributing to the pathogenesis of ischaemia–reperfusion injury. Platelet-activating factor (PAF), a phospholipid mediator of inflammation, has been implicated in the pathogenesis of this process. We previously showed that the infusion of streptokinase (SK) induces the intravascular release of PAF in patients with acute myocardial infarction (AMI), and that cultured human endothelial cells (EC) synthesized PAF in response to SK and plasmin (PLN). In the present study, we investigated the role of the membrane attack complex (MAC) of complement in the PLN-induced synthesis of PAF. In vivo, we showed a correlation between the levels of soluble terminal complement components (sC5b-9) and the concentrations of PAF detected in blood of patients with AMI infused with SK. In vitro both EC and polymorphonuclear neutrophils (PMN), incubated in the presence of PLN and normal human serum, showed an intense staining for the MAC neoepitope, while no staining was detected when they were incubated with PLN in the presence of heat-inactivated normal human serum. Moreover, the insertion of MAC on EC and PMN plasmamembrane elicited the synthesis of PAF. In conclusion, our results elucidate the mechanisms involved in PAF production during the activation of the fibrinolytic system, showing a role for complement products in this setting. The release of PAF may increase the inflammatory response, thus limiting the beneficial effects of thrombolytic therapy. Moreover, it may have a pathogenic role in other pathological conditions, such as transplant rejection, tumoral angiogenesis, and septic shock, where fibrinolysis is activated. PMID:12871223

The membrane attack complex of complement contributes to plasmin-induced synthesis of platelet-activating factor by endothelial cells and neutrophils.

PubMed

Lupia, Enrico; Del Sorbo, Lorenzo; Bergerone, Serena; Emanuelli, Giorgio; Camussi, Giovanni; Montrucchio, Giuseppe

2003-08-01

Thrombolytic agents, used to restore blood flow to ischaemic tissues, activate several enzymatic systems with pro-inflammatory effects, thus potentially contributing to the pathogenesis of ischaemia-reperfusion injury. Platelet-activating factor (PAF), a phospholipid mediator of inflammation, has been implicated in the pathogenesis of this process. We previously showed that the infusion of streptokinase (SK) induces the intravascular release of PAF in patients with acute myocardial infarction (AMI), and that cultured human endothelial cells (EC) synthesized PAF in response to SK and plasmin (PLN). In the present study, we investigated the role of the membrane attack complex (MAC) of complement in the PLN-induced synthesis of PAF. In vivo, we showed a correlation between the levels of soluble terminal complement components (sC5b-9) and the concentrations of PAF detected in blood of patients with AMI infused with SK. In vitro both EC and polymorphonuclear neutrophils (PMN), incubated in the presence of PLN and normal human serum, showed an intense staining for the MAC neoepitope, while no staining was detected when they were incubated with PLN in the presence of heat-inactivated normal human serum. Moreover, the insertion of MAC on EC and PMN plasmamembrane elicited the synthesis of PAF. In conclusion, our results elucidate the mechanisms involved in PAF production during the activation of the fibrinolytic system, showing a role for complement products in this setting. The release of PAF may increase the inflammatory response, thus limiting the beneficial effects of thrombolytic therapy. Moreover, it may have a pathogenic role in other pathological conditions, such as transplant rejection, tumoral angiogenesis, and septic shock, where fibrinolysis is activated.

Study of the plant COPII vesicle coat subunits by functional complementation of yeast Saccharomyces cerevisiae mutants.

PubMed

De Craene, Johan-Owen; Courte, Fanny; Rinaldi, Bruno; Fitterer, Chantal; Herranz, Mari Carmen; Schmitt-Keichinger, Corinne; Ritzenthaler, Christophe; Friant, Sylvie

2014-01-01

The formation and budding of endoplasmic reticulum ER-derived vesicles depends on the COPII coat protein complex that was first identified in yeast Saccharomyces cerevisiae. The ER-associated Sec12 and the Sar1 GTPase initiate the COPII coat formation by recruiting the Sec23-Sec24 heterodimer following the subsequent recruitment of the Sec13-Sec31 heterotetramer. In yeast, there is usually one gene encoding each COPII protein and these proteins are essential for yeast viability, whereas the plant genome encodes multiple isoforms of all COPII subunits. Here, we used a systematic yeast complementation assay to assess the functionality of Arabidopsis thaliana COPII proteins. In this study, the different plant COPII subunits were expressed in their corresponding temperature-sensitive yeast mutant strain to complement their thermosensitivity and secretion phenotypes. Secretion was assessed using two different yeast cargos: the soluble α-factor pheromone and the membranous v-SNARE (vesicle-soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor) Snc1 involved in the fusion of the secretory vesicles with the plasma membrane. This complementation study allowed the identification of functional A. thaliana COPII proteins for the Sec12, Sar1, Sec24 and Sec13 subunits that could represent an active COPII complex in plant cells. Moreover, we found that AtSec12 and AtSec23 were co-immunoprecipitated with AtSar1 in total cell extract of 15 day-old seedlings of A. thaliana. This demonstrates that AtSar1, AtSec12 and AtSec23 can form a protein complex that might represent an active COPII complex in plant cells.

Characterization of complementary patterned metallic membranes produced simultaneously by a dual fabrication process

NASA Astrophysics Data System (ADS)

Hao, Qingzhen; Zeng, Yong; Wang, Xiande; Zhao, Yanhui; Wang, Bei; Chiang, I.-Kao; Werner, Douglas H.; Crespi, Vincent; Huang, Tony Jun

2010-11-01

An efficient technique is developed to fabricate optically thin metallic films with subwavelength patterns and their complements simultaneously. By comparing the spectra of the complementary films, we show that Babinet's principle nearly holds for these structures in the optical domain. Rigorous full-wave simulations are employed to verify the experimental observations. It is further demonstrated that a discrete-dipole approximation can qualitatively describe the spectral dependence of the metallic membranes on the geometry of the constituent particles as well as the illuminating polarization.

Bimolecular fluorescence complementation (BiFC) analysis as a probe of protein interactions in living cells.

PubMed

Kerppola, Tom K

2008-01-01

Protein interactions are a fundamental mechanism for the generation of biological regulatory specificity. The study of protein interactions in living cells is of particular significance because the interactions that occur in a particular cell depend on the full complement of proteins present in the cell and the external stimuli that influence the cell. Bimolecular fluorescence complementation (BiFC) analysis enables direct visualization of protein interactions in living cells. The BiFC assay is based on the association between two nonfluorescent fragments of a fluorescent protein when they are brought in proximity to each other by an interaction between proteins fused to the fragments. Numerous protein interactions have been visualized using the BiFC assay in many different cell types and organisms. The BiFC assay is technically straightforward and can be performed using standard molecular biology and cell culture reagents and a regular fluorescence microscope or flow cytometer.

When the model fits the frame: the impact of regulatory fit on efficacy appraisal and persuasion in health communication.

PubMed

Bosone, Lucia; Martinez, Frédéric; Kalampalikis, Nikos

2015-04-01

In health-promotional campaigns, positive and negative role models can be deployed to illustrate the benefits or costs of certain behaviors. The main purpose of this article is to investigate why, how, and when exposure to role models strengthens the persuasiveness of a message, according to regulatory fit theory. We argue that exposure to a positive versus a negative model activates individuals' goals toward promotion rather than prevention. By means of two experiments, we demonstrate that high levels of persuasion occur when a message advertising healthy dietary habits offers a regulatory fit between its framing and the described role model. Our data also establish that the effects of such internal regulatory fit by vicarious experience depend on individuals' perceptions of response-efficacy and self-efficacy. Our findings constitute a significant theoretical complement to previous research on regulatory fit and contain valuable practical implications for health-promotional campaigns. © 2015 by the Society for Personality and Social Psychology, Inc.

Non-random distribution and co-localization of purine/pyrimidine-encoded information and transcriptional regulatory domains.

PubMed

Povinelli, C M

1992-01-01

In order to detect sequence-based information predictive for the location of eukaryotic transcriptional regulatory domains, the frequencies and distributions of the 36 possible purine/pyrimidine reverse complement hexamer pairs was determined for test sets of real and random sequences. The distribution of one of the hexamer pairs (RRYYRR/YYRRYY, referred to as M1) was further examined in a larger set of sequences (> 32 genes, 230 kb). Predominant clusters of M1 and the locations of eukaryotic transcriptional regulatory domains were found to be associated and non-randomly distributed along the DNA consistent with a periodicity of approximately 1.2 kb. In the context of higher ordered chromatin this would align promoters, enhancers and the predominant clusters of M1 longitudinally along one face of a 30 nm fiber. Using only information about the distribution of the M1 motif, 50-70% of a sequence could be eliminated as being unlikely to contain transcriptional regulatory domains with an 87% recovery of the regulatory domains present.

Computing Curvature Sensitivity of Biomolecules in Membranes by Simulated Buckling.

PubMed

Elías-Wolff, Federico; Lindén, Martin; Lyubartsev, Alexander P; Brandt, Erik G

2018-03-13

Membrane curvature sensing, where the binding free energies of membrane-associated molecules depend on the local membrane curvature, is a key factor to modulate and maintain the shape and organization of cell membranes. However, the microscopic mechanisms are not well understood, partly due to absence of efficient simulation methods. Here, we describe a method to compute the curvature dependence of the binding free energy of a membrane-associated probe molecule that interacts with a buckled membrane, which has been created by lateral compression of a flat bilayer patch. This buckling approach samples a wide range of curvatures in a single simulation, and anisotropic effects can be extracted from the orientation statistics. We develop an efficient and robust algorithm to extract the motion of the probe along the buckled membrane surface, and evaluate its numerical properties by extensive sampling of three coarse-grained model systems: local lipid density in a curved environment for single-component bilayers, curvature preferences of individual lipids in two-component membranes, and curvature sensing by a homotrimeric transmembrane protein. The method can be used to complement experimental data from curvature partition assays and provides additional insight into mesoscopic theories and molecular mechanisms for curvature sensing.

The Surface-Exposed Protein SntA Contributes to Complement Evasion in Zoonotic Streptococcus suis.

PubMed

Deng, Simin; Xu, Tong; Fang, Qiong; Yu, Lei; Zhu, Jiaqi; Chen, Long; Liu, Jiahui; Zhou, Rui

2018-01-01

Streptococcus suis is an emerging zoonotic pathogen causing streptococcal toxic shock like syndrome (STSLS), meningitis, septicemia, and even sudden death in human and pigs. Serious septicemia indicates this bacterium can evade the host complement surveillance. In our previous study, a functionally unknown protein SntA of S. suis has been identified as a heme-binding protein, and contributes to virulence in pigs. SntA can interact with the host antioxidant protein AOP2 and consequently inhibit its antioxidant activity. In the present study, SntA is identified as a cell wall anchored protein that functions as an important player in S. suis complement evasion. The C3 deposition and membrane attack complex (MAC) formation on the surface of sntA -deleted mutant strain Δ sntA are demonstrated to be significantly higher than the parental strain SC-19 and the complementary strain CΔ sntA . The abilities of anti-phagocytosis, survival in blood, and in vivo colonization of Δ sntA are obviously reduced. SntA can interact with C1q and inhibit hemolytic activity via the classical pathway. Complement activation assays reveal that SntA can also directly activate classical and lectin pathways, resulting in complement consumption. These two complement evasion strategies may be crucial for the pathogenesis of this zoonotic pathogen. Concerning that SntA is a bifunctional 2',3'-cyclic nucleotide 2'-phosphodiesterase/3'-nucleotidase in many species of Gram-positive bacteria, these complement evasion strategies may have common biological significance.

TANGO1 recruits ERGIC membranes to the endoplasmic reticulum for procollagen export

PubMed Central

Santos, António JM; Raote, Ishier; Scarpa, Margherita; Brouwers, Nathalie; Malhotra, Vivek

2015-01-01

Previously we showed that membrane fusion is required for TANGO1-dependent export of procollagen VII from the endoplasmic reticulum (ER) (Nogueira, et al., 2014). Along with the t-SNARE Syntaxin 18, we now reveal the complete complement of SNAREs required in this process, t-SNAREs BNIP1 and USE1, and v-SNARE YKT6. TANGO1 recruits YKT6-containing ER Golgi Intermediate Compartment (ERGIC) membranes to procollagen VII-enriched patches on the ER. Moreover residues 1214-1396, that include the first coiled coil of TANGO1, specifically recruit ERGIC membranes even when targeted to mitochondria. TANGO1 is thus pivotal in concentrating procollagen VII in the lumen and recruiting ERGIC membranes on the cytoplasmic surface of the ER. Our data reveal that growth of a mega transport carrier for collagen export from the ER is not by acquisition of a larger patch of ER membrane, but instead by addition of ERGIC membranes to procollagen-enriched domains of the ER by a TANGO1-mediated process. DOI: http://dx.doi.org/10.7554/eLife.10982.001 PMID:26568311

Automatic reconstruction of a bacterial regulatory network using Natural Language Processing

PubMed Central

Rodríguez-Penagos, Carlos; Salgado, Heladia; Martínez-Flores, Irma; Collado-Vides, Julio

2007-01-01

Background Manual curation of biological databases, an expensive and labor-intensive process, is essential for high quality integrated data. In this paper we report the implementation of a state-of-the-art Natural Language Processing system that creates computer-readable networks of regulatory interactions directly from different collections of abstracts and full-text papers. Our major aim is to understand how automatic annotation using Text-Mining techniques can complement manual curation of biological databases. We implemented a rule-based system to generate networks from different sets of documents dealing with regulation in Escherichia coli K-12. Results Performance evaluation is based on the most comprehensive transcriptional regulation database for any organism, the manually-curated RegulonDB, 45% of which we were able to recreate automatically. From our automated analysis we were also able to find some new interactions from papers not already curated, or that were missed in the manual filtering and review of the literature. We also put forward a novel Regulatory Interaction Markup Language better suited than SBML for simultaneously representing data of interest for biologists and text miners. Conclusion Manual curation of the output of automatic processing of text is a good way to complement a more detailed review of the literature, either for validating the results of what has been already annotated, or for discovering facts and information that might have been overlooked at the triage or curation stages. PMID:17683642

Solving the problems we face: the United States Environmental Protection Agency, sustainability, and the challenges of the twenty-first century

EPA Science Inventory

Addressing the problems of the twenty-first century will require new initiatives that complement traditional regulatory activities. Existing regulations, such as the Clean Air Act and Clean Water Act are important safety nets in the United States for protecting human health and t...

A Model Formative Assessment Strategy to Promote Student-Centered Self-Regulated Learning in Higher Education

ERIC Educational Resources Information Center

Bose, Jayakumar; Rengel, Zed

2009-01-01

Adult learners are already involved in the process of self-regulation; hence, higher education institutions should focus on strengthening students' self-regulatory skills. Self-regulation can be facilitated through formative assessment. This paper proposes a model formative assessment strategy that would complement existing university teaching,…

Membrane Contact Sites: Complex Zones for Membrane Association and Lipid Exchange

PubMed Central

Quon, Evan; Beh, Christopher T.

2015-01-01

Lipid transport between membranes within cells involves vesicle and protein carriers, but as agents of nonvesicular lipid transfer, the role of membrane contact sites has received increasing attention. As zones for lipid metabolism and exchange, various membrane contact sites mediate direct associations between different organelles. In particular, membrane contact sites linking the plasma membrane (PM) and the endoplasmic reticulum (ER) represent important regulators of lipid and ion transfer. In yeast, cortical ER is stapled to the PM through membrane-tethering proteins, which establish a direct connection between the membranes. In this review, we consider passive and facilitated models for lipid transfer at PM–ER contact sites. Besides the tethering proteins, we examine the roles of an additional repertoire of lipid and protein regulators that prime and propagate PM–ER membrane association. We conclude that instead of being simple mediators of membrane association, regulatory components of membrane contact sites have complex and multilayered functions. PMID:26949334

Regulatory elements of the floral homeotic gene AGAMOUS identified by phylogenetic footprinting and shadowing.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hong, R. L., Hamaguchi, L., Busch, M. A., and Weigel, D.

2003-06-01

OAK-B135 In Arabidopsis thaliana, cis-regulatory sequences of the floral homeotic gene AGAMOUS (AG) are located in the second intron. This 3 kb intron contains binding sites for two direct activators of AG, LEAFY (LFY) and WUSCHEL (WUS), along with other putative regulatory elements. We have used phylogenetic footprinting and the related technique of phylogenetic shadowing to identify putative cis-regulatory elements in this intron. Among 29 Brassicaceae, several other motifs, but not the LFY and WUS binding sites previously identified, are largely invariant. Using reporter gene analyses, we tested six of these motifs and found that they are all functionally importantmore » for activity of AG regulatory sequences in A. thaliana. Although there is little obvious sequence similarity outside the Brassicaceae, the intron from cucumber AG has at least partial activity in A. thaliana. Our studies underscore the value of the comparative approach as a tool that complements gene-by-gene promoter dissection, but also highlight that sequence-based studies alone are insufficient for a complete identification of cis-regulatory sites.« less

Audio visual summary: Implementing PURPA in Mid-America

DOE Office of Scientific and Technical Information (OSTI.GOV)

Not Available

The audio-visual presentation, Implementing PURPA in Mid-America, is a slide presentation designed to complement deliverable W-101-2, a booklet entitled Implementing PURPA in Mid-America: A Guide to the Public Utility Regulatory Policies Act. The presentation lasts 10 to 12 min and explains the major sections of PURPA, the rules promulgated by the Federal Energy Regulatory Commission to implement PURPA, and the implications of PURPA and its rules. It delineates the rights and responsibilities of citizens who want to sell electricity to utilities, explains the certification process, and discusses the rights and responsibilities of the utilities.

Distinct contributions of complement factors to platelet activation and fibrin formation in venous thrombus development

PubMed Central

Jäckel, Sven; Saffarzadeh, Mona; Langer, Florian

2017-01-01

Expanding evidence indicates multiple interactions between the hemostatic system and innate immunity, and the coagulation and complement cascades. Here we show in a tissue factor (TF)–dependent model of flow restriction-induced venous thrombosis that complement factors make distinct contributions to platelet activation and fibrin deposition. Complement factor 3 (C3) deficiency causes prolonged bleeding, reduced thrombus incidence, thrombus size, fibrin and platelet deposition in the ligated inferior vena cava, and diminished platelet activation in vitro. Initial fibrin deposition at the vessel wall over 6 hours in this model was dependent on protein disulfide isomerase (PDI) and TF expression by myeloid cells, but did not require neutrophil extracellular trap formation involving peptidyl arginine deiminase 4. In contrast to C3−/− mice, C5-deficient mice had no apparent defect in platelet activation in vitro, and vessel wall platelet deposition and initial hemostasis in vivo. However, fibrin formation, the exposure of negatively charged phosphatidylserine (PS) on adherent leukocytes, and clot burden after 48 hours were significantly reduced in C5−/− mice compared with wild-type controls. These results delineate that C3 plays specific roles in platelet activation independent of formation of the terminal complement complex and provide in vivo evidence for contributions of complement-dependent membrane perturbations to prothrombotic TF activation on myeloid cells. PMID:28223279

Fusobacterium nucleatum binding to complement regulatory protein CD46 modulates the expression and secretion of cytokines and matrix metalloproteinases by oral epithelial cells.

PubMed

Mahtout, Hayette; Chandad, Fatiha; Rojo, Jose M; Grenier, Daniel

2011-02-01

Periodontitis is a chronic inflammatory disease that results in the destruction of the supporting tissues of the teeth. Gingival epithelial cells are an important mechanical barrier and participate in the host inflammatory response to periodontopathogens. The aim of the present study is to investigate the capacity of Fusobacterium nucleatum to bind to the complement regulatory protein CD46 expressed by oral epithelial cells and to determine the impact of the binding on the gene expression and protein secretion of interleukin (IL)-6, IL-8, and matrix metalloproteinase (MMP)-9 by oral epithelial cells. Binding of recombinant human CD46 to the surface of F. nucleatum was demonstrated by immunologic assays. After stimulation of oral epithelial cells with F. nucleatum, gene expression was determined by real-time polymerase chain reaction analysis while protein secretion was monitored by enzyme-linked immunosorbent assays. Heat and protease treatments of bacterial cells reduced CD46 binding. F. nucleatum-bound CD46 mediated the cleavage of C3b in the presence of factor I. Stimulating oral epithelial cells with F. nucleatum at a multiplicity of infection of 50 resulted in a significant upregulation of the gene expression and protein secretion of IL-6, IL-8, and MMP-9 by oral epithelial cells. However, pretreating the epithelial cells with an anti-CD46 polyclonal antibody attenuated the production of IL-6, IL-8, and MMP-9 in response to F. nucleatum. Such an inhibitory effect was not observed with non-specific antibodies. The present study demonstrates that F. nucleatum can bind the complement regulatory protein CD46. The interaction of F. nucleatum with epithelial cell surface CD46 may contribute to increasing the levels of proinflammatory mediators and MMPs in periodontal sites and consequently modulate tissue destruction.

Spontaneous abortion is associated with elevated systemic C5a and reduced mRNA of complement inhibitory proteins in placenta

PubMed Central

Banadakoppa, M; Chauhan, M S; Havemann, D; Balakrishnan, M; Dominic, J S; Yallampalli, C

2014-01-01

Spontaneous abortion in early pregnancy due to unknown reasons is a common problem. The excess complement activation and consequent placental inflammation and anti-angiogenic milieu is emerging as an important associated factor in many pregnancy-related complications. In the present study we sought to examine the expression of complement inhibitory proteins at the feto–maternal interface and levels of complement split products in the circulation to understand their role in spontaneous abortion. Consenting pregnant women who either underwent elective abortion due to non-clinical reasons (n = 13) or suffered miscarriage (n = 14) were recruited for the study. Systemic levels of complement factors C3a and C5a were measured by enzyme-linked immunosorbent assay (ELISA). Plasma C5 and C3 protein levels were examined by Western blot. Expressions of complement regulatory proteins such as CD46 and CD55 in the decidua were investigated by quantitative polymerase chain reaction (PCR) and Western blot. The median of plasma C3a level was 82·83 ng/ml and 66·17 ng/ml in elective and spontaneous abortion patients, respectively. Medians of plasma C5a levels in elective and spontaneous abortion patients were 0·96 ng/ml and 1·14 ng/ml, respectively. Only plasma C5a levels but not C3a levels showed significant elevation in spontaneous abortion patients compared to elective abortion patients. Further, there was a threefold decrease in the mRNA expressions of complement inhibitory proteins CD46 and CD55 in the decidua obtained from spontaneous abortion patients compared to that of elective abortion patients. These data suggested that dysregulated complement cascade may be associated with spontaneous abortion. PMID:24802103

Eculizumab for dense deposit disease and C3 glomerulonephritis.

PubMed

Bomback, Andrew S; Smith, Richard J; Barile, Gaetano R; Zhang, Yuzhou; Heher, Eliot C; Herlitz, Leal; Stokes, M Barry; Markowitz, Glen S; D'Agati, Vivette D; Canetta, Pietro A; Radhakrishnan, Jai; Appel, Gerald B

2012-05-01

The principle defect in dense deposit disease and C3 glomerulonephritis is hyperactivity of the alternative complement pathway. Eculizumab, a monoclonal antibody that binds to C5 to prevent formation of the membrane attack complex, may prove beneficial. In this open-label, proof of concept efficacy and safety study, six subjects with dense deposit disease or C3 glomerulonephritis were treated with eculizumab every other week for 1 year. All had proteinuria >1 g/d and/or AKI at enrollment. Subjects underwent biopsy before enrollment and repeat biopsy at the 1-year mark. The subjects included three patients with dense deposit disease (including one patient with recurrent dense deposit disease in allograft) and three patients with C3 glomerulonephritis (including two patients with recurrent C3 glomerulonephritis in allograft). Genetic and complement function testing revealed a mutation in CFH and MCP in one subject each, C3 nephritic factor in three subjects, and elevated levels of serum membrane attack complex in three subjects. After 12 months, two subjects showed significantly reduced serum creatinine, one subject achieved marked reduction in proteinuria, and one subject had stable laboratory parameters but histopathologic improvements. Elevated serum membrane attack complex levels normalized on therapy and paralleled improvements in creatinine and proteinuria. Clinical and histopathologic data suggest a response to eculizumab in some but not all subjects with dense deposit disease and C3 glomerulonephritis. Elevation of serum membrane attack complex before treatment may predict response. Additional research is needed to define the subgroup of dense deposit disease/C3 glomerulonephritis patients in whom eculizumab therapy can be considered.

Cell micro-irradiation with MeV protons counted by an ultra-thin diamond membrane

NASA Astrophysics Data System (ADS)

Barberet, Philippe; Pomorski, Michal; Muggiolu, Giovanna; Torfeh, Eva; Claverie, Gérard; Huss, Cédric; Saada, Samuel; Devès, Guillaume; Simon, Marina; Seznec, Hervé

2017-12-01

We report the development of thin single crystal diamond membranes suitable for dose control in targeted cell irradiation experiments with a proton microbeam. A specific design was achieved to deliver single protons with a hit detection efficiency approaching 100%. The membranes have thicknesses between 1.8 and 3 μm and are used as vacuum windows on the microbeam line. The impact of these transmission detectors on the microbeam spot size is estimated by Monte-Carlo simulations, indicating that a beam lateral resolution below 2 μm is achieved. This is confirmed by experiments showing the accumulation online of X-ray Repair Cross-Complementing protein 1 (XRCC1)-Green Fluorescent Protein (GFP) at DNA damaged sites in living cells.

Demonstration and Validation of a Regenerated Cellulose Dialysis Membrane Diffusion Sampler for Monitoring Ground Water Quality and Remediation Progress at DoD Sites

DTIC Science & Technology

2007-08-30

ITRC Interstate Technology Regulatory Council LRL Laboratory reporting level LDPE Low-density polyethylene MDL Minimum detection limit MNA...diameter of the well. Another diffusion membrane sampler design consists of a tubular-shaped bag made of flexible low-density polyethylene ( LDPE ...

A 3' UTR-Derived Small RNA Provides the Regulatory Noncoding Arm of the Inner Membrane Stress Response.

PubMed

Chao, Yanjie; Vogel, Jörg

2016-02-04

Small RNAs (sRNAs) from conserved noncoding genes are crucial regulators in bacterial signaling pathways but have remained elusive in the Cpx response to inner membrane stress. Here we report that an alternative biogenesis pathway releasing the conserved mRNA 3' UTR of stress chaperone CpxP as an ∼60-nt sRNA provides the noncoding arm of the Cpx response. This so-called CpxQ sRNA, generated by general mRNA decay through RNase E, acts as an Hfq-dependent repressor of multiple mRNAs encoding extracytoplasmic proteins. Both CpxQ and the Cpx pathway are required for cell survival under conditions of dissipation of membrane potential. Our discovery of CpxQ illustrates how the conversion of a transcribed 3' UTR into an sRNA doubles the output of a single mRNA to produce two factors with spatially segregated functions during inner membrane stress: a chaperone that targets problematic proteins in the periplasm and a regulatory RNA that dampens their synthesis in the cytosol. Copyright © 2016 Elsevier Inc. All rights reserved.

A comparison of the endotoxin biosynthesis and protein oxidation pathways in the biogenesis of the outer membrane of Escherichia coli and Neisseria meningitidis

PubMed Central

Piek, Susannah; Kahler, Charlene M.

2012-01-01

The Gram-negative bacterial cell envelope consists of an inner membrane (IM) that surrounds the cytoplasm and an asymmetrical outer-membrane (OM) that forms a protective barrier to the external environment. The OM consists of lipopolysaccahride (LPS), phospholipids, outer membrane proteins (OMPs), and lipoproteins. Oxidative protein folding mediated by periplasmic oxidoreductases is required for the biogenesis of the protein components, mainly constituents of virulence determinants such as pili, flagella, and toxins, of the Gram-negative OM. Recently, periplasmic oxidoreductases have been implicated in LPS biogenesis of Escherichia coli and Neisseria meningitidis. Differences in OM biogenesis, in particular the transport pathways for endotoxin to the OM, the composition and role of the protein oxidation, and isomerization pathways and the regulatory networks that control them have been found in these two Gram-negative species suggesting that although form and function of the OM is conserved, the pathways required for the biosynthesis of the OM and the regulatory circuits that control them have evolved to suit the lifestyle of each organism. PMID:23267440

Membrane lipid-protein interactions modify the regulatory role of adenosine-deaminase complexing protein: a phase fluorometry study of a malignancy marker

NASA Astrophysics Data System (ADS)

Parola, Abraham H.; Porat, Nurith; Caiolfa, Valeria R.; Gill, David; Kiesow, Lutz A.; Weisman, Mathew; Nemschitz, S.; Yaron, Dahlia; Singer, Karen; Solomon, Ethel

1990-05-01

The role of membrane lipid-protein interactions in malignant cell transformation was examined with adenosine deaminase (ADA) as a representative membrane protein. ADA's activity changes dramatically in transformed cells and accordingly it is a malignancy marker. Yet, the mechanisms controlling its variable activity are unknown. We undertook the spectroscopic deciphering of its interactions with its lipidic environment in normal and malignant cells. ADA exists in two interconvertible forms, small (45 KD) and large (21OKD). The large form consists of two small catalytic subunits (55-ADA) and a dimeric complexing protein ADCP. The physiological role of ADCP was not known either. Our studies were carried out at three levels.: 1. Solution enzyme kinetics, 2. The interaction of 55-ADA with ADCP reconstituted in liposomes: Effect of cholesterol and 3. Multifrequency phase modulation spectrofluorometry of pyrene-labeled 55-ADA bound to ADCP on the membranes of normal and RSV or RSV Ts68 transformed chick embryo fibroblasts. We found: 1. ADCP has an allosteric regulatory role on 55-ADA, which may be of physiological relevance: It inhibits 55-ADA activity at low physiological adenosine concentrations but accelerates deamination at high substrate concentration. 2. When reconstituted in DMPC liposomes, it retains 55-ADA activity (in its absence the activity is lost) and upon rigidification with cholesterol, a three fold increase in 55-ADA activity is attained, contrary to ADCP's regulatory activity when free of lipids. 3. The reduced ADA activity in transformed chick embryo fibroblasts is associated with increased membrane lipid fluidity (reduced order parameter), reduced accessibility of ADCP and increase rotational dynamics of the complex. We thus obtained spectroscopic deciphering of the vertical motion of ADCP, controlled by lipid-protein interaction, resulting in variable activity of this malignancy marker.

Variola virus immune evasion design: expression of a highly efficient inhibitor of human complement.

PubMed

Rosengard, Ariella M; Liu, Yu; Nie, Zhiping; Jimenez, Robert

2002-06-25

Variola virus, the most virulent member of the genus Orthopoxvirus, specifically infects humans and has no other animal reservoir. Variola causes the contagious disease smallpox, which has a 30-40% mortality rate. Conversely, the prototype orthopoxvirus, vaccinia, causes no disease in immunocompetent humans and was used in the global eradication of smallpox, which ended in 1977. However, the threat of smallpox persists because clandestine stockpiles of variola still exist. Although variola and vaccinia share remarkable DNA homology, the strict human tropism of variola suggests that its proteins are better suited than those of vaccinia to overcome the human immune response. Here, we demonstrate the functional advantage of a variola complement regulatory protein over that of its vaccinia homologue. Because authentic variola proteins are not available for study, we molecularly engineered and characterized the smallpox inhibitor of complement enzymes (SPICE), a homologue of a vaccinia virulence factor, vaccinia virus complement control protein (VCP). SPICE is nearly 100-fold more potent than VCP at inactivating human C3b and 6-fold more potent at inactivating C4b. SPICE is also more human complement-specific than is VCP. By inactivating complement components, SPICE serves to inhibit the formation of the C3/C5 convertases necessary for complement-mediated viral clearance. SPICE provides the first evidence that variola proteins are particularly adept at overcoming human immunity, and the decreased function of VCP suggests one reason why the vaccinia virus vaccine was associated with relatively low mortality. Disabling SPICE may be therapeutically useful if smallpox reemerges.

Variola virus immune evasion design: Expression of a highly efficient inhibitor of human complement

PubMed Central

Rosengard, Ariella M.; Liu, Yu; Nie, Zhiping; Jimenez, Robert

2002-01-01

Variola virus, the most virulent member of the genus Orthopoxvirus, specifically infects humans and has no other animal reservoir. Variola causes the contagious disease smallpox, which has a 30–40% mortality rate. Conversely, the prototype orthopoxvirus, vaccinia, causes no disease in immunocompetent humans and was used in the global eradication of smallpox, which ended in 1977. However, the threat of smallpox persists because clandestine stockpiles of variola still exist. Although variola and vaccinia share remarkable DNA homology, the strict human tropism of variola suggests that its proteins are better suited than those of vaccinia to overcome the human immune response. Here, we demonstrate the functional advantage of a variola complement regulatory protein over that of its vaccinia homologue. Because authentic variola proteins are not available for study, we molecularly engineered and characterized the smallpox inhibitor of complement enzymes (SPICE), a homologue of a vaccinia virulence factor, vaccinia virus complement control protein (VCP). SPICE is nearly 100-fold more potent than VCP at inactivating human C3b and 6-fold more potent at inactivating C4b. SPICE is also more human complement-specific than is VCP. By inactivating complement components, SPICE serves to inhibit the formation of the C3/C5 convertases necessary for complement-mediated viral clearance. SPICE provides the first evidence that variola proteins are particularly adept at overcoming human immunity, and the decreased function of VCP suggests one reason why the vaccinia virus vaccine was associated with relatively low mortality. Disabling SPICE may be therapeutically useful if smallpox reemerges. PMID:12034872

Multiple N-glycans cooperate in the subcellular targeting and functioning of Arabidopsis KORRIGAN1.

PubMed

Rips, Stephan; Bentley, Nolan; Jeong, In Sil; Welch, Justin L; von Schaewen, Antje; Koiwa, Hisashi

2014-09-01

Arabidopsis thaliana KORRIGAN1 (KOR1) is an integral membrane endo-β1,4-glucanase in the trans-Golgi network and plasma membrane that is essential for cellulose biosynthesis. The extracellular domain of KOR1 contains eight N-glycosylation sites, N1 to N8, of which only N3 to N7 are highly conserved. Genetic evidence indicated that cellular defects in attachment and maturation of these N-glycans affect KOR1 function in vivo, whereas the manner by which N-glycans modulate KOR1 function remained obscure. Site-directed mutagenesis analysis of green fluorescent protein (GFP)-KOR1 expressed from its native regulatory sequences established that all eight N-glycosylation sites (N1 to N8) are used in the wild type, whereas stt3a-2 cells could only inefficiently add N-glycans to less conserved sites. GFP-KOR1 variants with a single N-glycan at nonconserved sites were less effective than those with one at a highly conserved site in rescuing the root growth phenotype of rsw2-1 (kor1 allele). When functionally compromised, GFP-KOR1 tended to accumulate at the tonoplast. GFP-KOR1Δall (without any N-glycan) exhibited partial complementation of rsw2-1; however, root growth of this line was still negatively affected by the absence of complex-type N-glycan modifications in the host plants. These results suggest that one or several additional factor(s) carrying complex N-glycans cooperate(s) with KOR1 in trans to grant proper targeting/functioning in plant cells. © 2014 American Society of Plant Biologists. All rights reserved.

N-Acetylcysteine Amide Protects Against Oxidative Stress–Induced Microparticle Release From Human Retinal Pigment Epithelial Cells

PubMed Central

Carver, Kyle A.; Yang, Dongli

2016-01-01

Purpose Oxidative stress is a major factor involved in retinal pigment epithelium (RPE) apoptosis that underlies AMD. Drusen, extracellular lipid- and protein-containing deposits, are strongly associated with the development of AMD. Cell-derived microparticles (MPs) are small membrane-bound vesicles shed from cells. The purpose of this study was to determine if oxidative stress drives MP release from RPE cells, to assess whether these MPs carry membrane complement regulatory proteins (mCRPs: CD46, CD55, and CD59), and to evaluate the effects of a thiol antioxidant on oxidative stress–induced MP release. Methods Retinal pigment epithelium cells isolated from human donor eyes were cultured and treated with hydrogen peroxide (H2O2) to induce oxidative stress. Isolated MPs were fixed for transmission electron microscopy or processed for component analysis by flow cytometry, Western blot analysis, and confocal microscopy. Results Transmission electron microscopy showed that MPs ranged in diameter from 100 to 1000 nm. H2O2 treatment led to time- and dose-dependent elevations in MPs with externalized phosphatidylserine and phosphatidylethanolamine, known markers of MPs. These increases were strongly correlated to RPE apoptosis. Oxidative stress significantly increased the release of mCRP-positive MPs, which were prevented by a thiol antioxidant, N-acetylcysteine amide (NACA). Conclusions This is the first evidence that oxidative stress induces cultured human RPE cells to release MPs that carry mCRPs on their surface. The levels of released MPs are strongly correlated with RPE apoptosis. N-acetylcysteine amide prevents oxidative stress–induced effects. Our findings indicate that oxidative stress reduces mCRPs on the RPE surface through releasing MPs. PMID:26842754

Comparative studies of gene expression and the evolution of gene regulation

PubMed Central

Romero, Irene Gallego; Ruvinsky, Ilya; Gilad, Yoav

2014-01-01

The hypothesis that differences in gene regulation play an important role in speciation and adaptation is more than 40 years old. With the advent of new sequencing technologies, we are able to characterize and study gene expression levels and associated regulatory mechanisms in a large number of individuals and species at unprecedented resolution and scale. We have thus gained new insights into the evolutionary pressures that shape gene expression levels, as well as developed an appreciation for the relative importance of evolutionary changes in different regulatory genetic and epigenetic mechanisms. The current challenge is to link gene regulatory changes to adaptive evolution of complex phenotypes. Here we mainly focus on comparative studies in primates, and how they are complemented by studies in model organisms. PMID:22705669

BINDING OF SOLUBLE IMMUNE COMPLEXES TO HUMAN LYMPHOBLASTOID CELLS

PubMed Central

Theofilopoulos, Argyrios N.; Dixon, Frank J.; Bokisch, Viktor A.

1974-01-01

In the present work we studied the expression of membrane-bound Ig (MBIg) as well as receptors for IgG Fc and complement on nine human lymphoblastoid cell lines. When MBIg and receptors for IgG Fc were compared, four categories of cell lines could be distinguished: (a) cell lines having both MBIg and receptors for IgG Fc, (b) cell lines having MBIg but lacking receptors for IgG Fc, (c) cell lines lacking MBIg but having receptors for IgG Fc, and (d) cell lines lacking both MBIg and receptors for IgG Fc. Two types of receptors for complement could be detected on the cell lines studied, one for C3-C3b and one for C3d. When sensitized red cells carrying C3b or C3d were used for rosette tests, three categories of cell lines could be distinguished: (a) cell lines having receptors for C3b and C3d, (b) cell lines having receptors only for C3d and (c) cell lines lacking both receptors. However, when a more sensitive immunofluorescent method was used instead of the rosette technique, it was found that cell lines unable to form rosettes with EAC1423bhu were able to bind soluble C3 or C3b which indicated the presence of these receptors on the cell surface. Inhibition experiments showed that receptors for C3-C3b and receptors for C3d are distinct and that receptors for C3-C3b and C3d are different from receptors for IgG Fc. A cell line (Raji) without MBIg but with receptors for IgG Fc, C3-C3b, and C3d was selected for use in studying the binding mechanism of soluble immune complexes to cell surface membrane. Aggregated human gamma globulin was used in place of immune complexes. Immune complexes containing complement bind to Raji cells only via receptors for complement, namely receptors for C3-C3b and C3d. Binding of immune complexes containing complement to cells is much greater than that of complexes without complement. Immune complexes bound to cells via receptors for complement can be partially released from the cell surface by addition of normal human serum as well as isolated human C3 or C3b. We postulate that such release is due to competition of immune complex bound C3b and free C3 or C3b for the receptors on Raji cells. PMID:4139225

Interactions between muscle and the immune system during modified musculoskeletal loading

NASA Technical Reports Server (NTRS)

Tidball, James G.

2002-01-01

Interactions between the immune system and skeletal muscle may play a significant role in modulating the course of muscle injury and repair after modified musculoskeletal loading. Current evidence indicates that activation of the complement system is an early event during modified loading, which then leads to inflammatory cell invasion. However, the functions of those inflammatory cells are complex and they seem to be capable of promoting additional injury and repair. Recent findings implicate an early invading neutrophil population in increasing muscle damage that is detected by the presence of muscle membrane lesions. Macrophages that invade subsequently serve to remove cellular debris, and seem to promote repair. However, macrophages also have the ability to increase damage in muscle in which there is an impaired capacity to generate nitric oxide. In vivo and in vitro evidence indicates that muscle-derived nitric oxide can serve an important role in protecting muscle from membrane damage by invading inflammatory cells. Collectively, these findings indicate that the dynamic balance between inflammatory cells, the complement system, and muscle-derived free radicals can play important roles in the secondary damage of muscle during modified musculoskeletal loading.

A chromosomally encoded virulence factor protects the Lyme disease pathogen against host-adaptive immunity.

PubMed

Yang, Xiuli; Coleman, Adam S; Anguita, Juan; Pal, Utpal

2009-03-01

Borrelia burgdorferi, the bacterial pathogen of Lyme borreliosis, differentially expresses select genes in vivo, likely contributing to microbial persistence and disease. Expression analysis of spirochete genes encoding potential membrane proteins showed that surface-located membrane protein 1 (lmp1) transcripts were expressed at high levels in the infected murine heart, especially during early stages of infection. Mice and humans with diagnosed Lyme borreliosis also developed antibodies against Lmp1. Deletion of lmp1 severely impaired the pathogen's ability to persist in diverse murine tissues including the heart, and to induce disease, which was restored upon chromosomal complementation of the mutant with the lmp1 gene. Lmp1 performs an immune-related rather than a metabolic function, as its deletion did not affect microbial persistence in immunodeficient mice, but significantly decreased spirochete resistance to the borreliacidal effects of anti-B. burgdorferi sera in a complement-independent manner. These data demonstrate the existence of a virulence factor that helps the pathogen evade host-acquired immune defense and establish persistent infection in mammals.

Molecular cloning of a murine homologue of membrane cofactor protein (CD46): preferential expression in testicular germ cells.

PubMed Central

Tsujimura, A; Shida, K; Kitamura, M; Nomura, M; Takeda, J; Tanaka, H; Matsumoto, M; Matsumiya, K; Okuyama, A; Nishimune, Y; Okabe, M; Seya, T

1998-01-01

Human membrane cofactor protein (MCP, CD46) has been suggested, although no convincing evidence has been proposed, to be a fertilization-associated protein, in addition to its primary functions as a complement regulator and a measles virus receptor. We have cloned a cDNA encoding the murine homologue of MCP. This cDNA showed 45% identity in deduced protein sequence and 62% identity in nucleotide sequence with human MCP. Its ectodomains were four short consensus repeats and a serine/threonine-rich domain, and it appeared to be a type 1 membrane protein with a 23-amino acid transmembrane domain and a short cytoplasmic tail. The protein expressed on Chinese hamster ovary cell transfectants was 47 kDa on SDS/PAGE immunoblotting, approximately 6 kDa larger than the murine testis MCP. It served as a cofactor for factor I-mediated inactivation of the complement protein C3b in a homologous system and, to a lesser extent, in a human system. Strikingly, the major message of murine MCP was 1.5 kb and was expressed predominantly in the testis. It was not detected in mice defective in spermatogenesis or with immature germ cells (until 23 days old). Thus, murine MCP may be a sperm-dominant protein the message of which is expressed selectively in spermatids during germ-cell differentiation. PMID:9461505

The complex nature of calcium cation interactions with phospholipid bilayers

PubMed Central

Melcrová, Adéla; Pokorna, Sarka; Pullanchery, Saranya; Kohagen, Miriam; Jurkiewicz, Piotr; Hof, Martin; Jungwirth, Pavel; Cremer, Paul S.; Cwiklik, Lukasz

2016-01-01

Understanding interactions of calcium with lipid membranes at the molecular level is of great importance in light of their involvement in calcium signaling, association of proteins with cellular membranes, and membrane fusion. We quantify these interactions in detail by employing a combination of spectroscopic methods with atomistic molecular dynamics simulations. Namely, time-resolved fluorescent spectroscopy of lipid vesicles and vibrational sum frequency spectroscopy of lipid monolayers are used to characterize local binding sites of calcium in zwitterionic and anionic model lipid assemblies, while dynamic light scattering and zeta potential measurements are employed for macroscopic characterization of lipid vesicles in calcium-containing environments. To gain additional atomic-level information, the experiments are complemented by molecular simulations that utilize an accurate force field for calcium ions with scaled charges effectively accounting for electronic polarization effects. We demonstrate that lipid membranes have substantial calcium-binding capacity, with several types of binding sites present. Significantly, the binding mode depends on calcium concentration with important implications for calcium buffering, synaptic plasticity, and protein-membrane association. PMID:27905555

Msp1 Is a Membrane Protein Dislocase for Tail-Anchored Proteins.

PubMed

Wohlever, Matthew L; Mateja, Agnieszka; McGilvray, Philip T; Day, Kasey J; Keenan, Robert J

2017-07-20

Mislocalized tail-anchored (TA) proteins of the outer mitochondrial membrane are cleared by a newly identified quality control pathway involving the conserved eukaryotic protein Msp1 (ATAD1 in humans). Msp1 is a transmembrane AAA-ATPase, but its role in TA protein clearance is not known. Here, using purified components reconstituted into proteoliposomes, we show that Msp1 is both necessary and sufficient to drive the ATP-dependent extraction of TA proteins from the membrane. A crystal structure of the Msp1 cytosolic region modeled into a ring hexamer suggests that active Msp1 contains a conserved membrane-facing surface adjacent to a central pore. Structure-guided mutagenesis of the pore residues shows that they are critical for TA protein extraction in vitro and for functional complementation of an msp1 deletion in yeast. Together, these data provide a molecular framework for Msp1-dependent extraction of mislocalized TA proteins from the outer mitochondrial membrane. Copyright © 2017 Elsevier Inc. All rights reserved.

Membrane cholesterol plays an important role in enteropathogen adhesion and the activation of innate immunity via flagellin-TLR5 signaling.

PubMed

Zhou, Mingxu; Duan, Qiangde; Li, Yinchau; Yang, Yang; Hardwidge, Philip R; Zhu, Guoqiang

2015-08-01

Lipid rafts are cholesterol- and sphingolipid-rich ordered microdomains distributed in the plasma membrane that participates in mammalian signal transduction pathways. To determine the role of lipid rafts in mediating interactions between enteropathogens and intestinal epithelial cells, membrane cholesterol was depleted from Caco-2 and IPEC-J2 cells using methyl-β-cyclodextrin. Cholesterol depletion significantly reduced Escherichia coli and Salmonella enteritidis adhesion and invasion into intestinal epithelial cells. Complementation with exogenous cholesterol restored bacterial adhesion to basal levels. We also evaluated the role of lipid rafts in the activation of Toll-like receptor 5 signaling by bacterial flagellin. Depleting membrane cholesterol reduced the ability of purified recombinant E. coli flagellin to activate TLR5 signaling in intestinal cells. These data suggest that both membrane cholesterol and lipid rafts play important roles in enteropathogen adhesion and contribute to the activation of innate immunity via flagellin-TLR5 signaling.

The complex nature of calcium cation interactions with phospholipid bilayers

NASA Astrophysics Data System (ADS)

Melcrová, Adéla; Pokorna, Sarka; Pullanchery, Saranya; Kohagen, Miriam; Jurkiewicz, Piotr; Hof, Martin; Jungwirth, Pavel; Cremer, Paul S.; Cwiklik, Lukasz

2016-12-01

Understanding interactions of calcium with lipid membranes at the molecular level is of great importance in light of their involvement in calcium signaling, association of proteins with cellular membranes, and membrane fusion. We quantify these interactions in detail by employing a combination of spectroscopic methods with atomistic molecular dynamics simulations. Namely, time-resolved fluorescent spectroscopy of lipid vesicles and vibrational sum frequency spectroscopy of lipid monolayers are used to characterize local binding sites of calcium in zwitterionic and anionic model lipid assemblies, while dynamic light scattering and zeta potential measurements are employed for macroscopic characterization of lipid vesicles in calcium-containing environments. To gain additional atomic-level information, the experiments are complemented by molecular simulations that utilize an accurate force field for calcium ions with scaled charges effectively accounting for electronic polarization effects. We demonstrate that lipid membranes have substantial calcium-binding capacity, with several types of binding sites present. Significantly, the binding mode depends on calcium concentration with important implications for calcium buffering, synaptic plasticity, and protein-membrane association.

Innate Immune Mechanisms in Transplant Allograft Vasculopathy

PubMed Central

Jane-wit, D; Fang, C; Goldstein, DR

2016-01-01

Purpose of Review Allograft vasculopathy (AV) is the leading cause of late allograft loss following solid organ transplantation. Ischemia reperfusion injury (IRI) and donor specific antibody (DSA)-induced complement activation confer heightened risk for AV via numerous innate immune mechanisms including MyD88, HMGB1, and complement induced non-canonical NF-kB signaling. Recent Findings The role of MyD88, a signal adaptor downstream of the toll-like receptors (TLR), has been defined in an experimental heart transplant model, which demonstrated that recipient MyD88 enhanced AV. Importantly, triggering receptor on myeloid receptor 1(Trem1), a MyD88 amplifying signal, was present in rejecting human cardiac transplant biopsies and enhanced the development of AV in mice. HMGB1, a nuclear protein that activates TLRs, also enhanced the development of AV. Complement activation elicits assembly of membrane attack complexes (MAC) on endothelial cells which activate non-canonical NF-kB signaling, a novel complement effector pathway that induces pro-inflammatory genes and potentiates endothelial cell mediated alloimmune T cell activation, processes which enhance AV. Summary Innate immune mediators including HMGB1, MyD88, and non-canonical NFκB signaling via complement activation contribute to AV. These pathways represent potential therapeutic targets to reduce AV after solid organ transplantation. PMID:27077602

Phosphoinositide signaling in sperm development.

PubMed

Brill, Julie A; Yildirim, Sukriye; Fabian, Lacramioara

2016-11-01

Phosphatidylinositol phosphates (PIPs) 1 are membrane lipids with crucial roles during cell morphogenesis, including the establishment of cytoskeletal organization, membrane trafficking, cell polarity, cell-cycle control and signaling. Recent studies in mice (Mus musculus), fruit flies (Drosophila melanogaster) and other organisms have defined germ cell intrinsic requirements for these lipids and their regulatory enzymes in multiple aspects of sperm development. In particular, PIP levels are crucial in germline stem cell maintenance, spermatogonial proliferation and survival, spermatocyte cytokinesis, spermatid polarization, sperm tail formation, nuclear shaping, and production of mature, motile sperm. Here, we briefly review the stages of spermatogenesis and discuss the roles of PIPs and their regulatory enzymes in male germ cell development. Copyright © 2016 Elsevier Ltd. All rights reserved.

Cpa, the Outer Membrane Protease of Cronobacter sakazakii, Activates Plasminogen and Mediates Resistance to Serum Bactericidal Activity▿

PubMed Central

Franco, A. A.; Kothary, M. H.; Gopinath, G.; Jarvis, K. G.; Grim, C. J.; Hu, L.; Datta, A. R.; McCardell, B. A.; Tall, B. D.

2011-01-01

Cronobacter spp. are emerging neonatal pathogens in humans, associated with outbreaks of meningitis and sepsis. To cause disease, they must survive in blood and invade the central nervous system by penetrating the blood-brain barrier. C. sakazakii BAA-894 possesses an ∼131-kb plasmid (pESA3) that encodes an outer membrane protease (Cpa) that has significant identity to proteins that belong to the Pla subfamily of omptins. Members of this subfamily of proteins degrade a number of serum proteins, including circulating complement, providing protection from the complement-dependent serum killing. Moreover, proteins of the Pla subfamily can cause uncontrolled plasmin activity by converting plasminogen to plasmin and inactivating the plasmin inhibitor α2-antiplasmin (α2-AP). These reactions enhance the spread and invasion of bacteria in the host. In this study, we found that an isogenic cpa mutant showed reduced resistance to serum in comparison to its parent C. sakazakii BAA-894 strain. Overexpression of Cpa in C. sakazakii or Escherichia coli DH5α showed that Cpa proteolytically cleaved complement components C3, C3a, and C4b. Furthermore, a strain of C. sakazakii overexpressing Cpa caused a rapid activation of plasminogen and inactivation of α2-AP. These results strongly suggest that Cpa may be an important virulence factor involved in serum resistance, as well as in the spread and invasion of C. sakazakii. PMID:21245266

Cyclopropanation of membrane unsaturated fatty acids is not essential to the acid stress response of Lactococcus lactis subsp. cremoris.

PubMed

To, Thi Mai Huong; Grandvalet, Cosette; Tourdot-Maréchal, Raphaëlle

2011-05-01

Cyclopropane fatty acids (CFAs) are synthetized in situ by the transfer of a methylene group from S-adenosyl-L-methionine to a double bond of unsaturated fatty acid chains of membrane phospholipids. This conversion, catalyzed by the Cfa synthase enzyme, occurs in many bacteria and is recognized to play a key role in the adaptation of bacteria in response to a drastic perturbation of the environment. The role of CFAs in the acid tolerance response was investigated in the lactic acid bacterium Lactococcus lactis MG1363. A mutant of the cfa gene was constructed by allelic exchange. The cfa gene encoding the Cfa synthase was cloned and introduced into the mutant to obtain the complemented strain for homologous system studies. Data obtained by gas chromatography (GC) and GC-mass spectrometry (GC-MS) validated that the mutant could not produce CFA. The CFA levels in both the wild-type and complemented strains increased upon their entry to stationary phase, especially with acid-adapted cells or, more surprisingly, with ethanol-adapted cells. The results obtained by performing quantitative reverse transcription-PCR (qRT-PCR) experiments showed that transcription of the cfa gene was highly induced by acidity (by 10-fold with cells grown at pH 5.0) and by ethanol (by 9-fold with cells grown with 6% ethanol) in comparison with that in stationary phase. Cell viability experiments were performed after an acidic shock on the mutant strain, the wild-type strain, and the complemented strain, as a control. The higher viability level of the acid-adapted cells of the three strains after 3 h of shock proved that the cyclopropanation of unsaturated fatty acids is not essential for L. lactis subsp. cremoris survival under acidic conditions. Moreover, fluorescence anisotropy data showed that CFA itself could not maintain the membrane fluidity level, particularly with ethanol-grown cells.

Cell lines that support replication of a novel herpes simplex virus 1 U{sub L}31 deletion mutant can properly target U{sub L}34 protein to the nuclear rim in the absence of U{sub L}31

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liang Li; Tanaka, Michiko; Kawaguchi, Yasushi

2004-11-10

Previous results indicated that the herpes simplex virus 1 (HSV-1) U{sub L}31 gene is necessary and sufficient for localization of the U{sub L}34 protein exclusively to the nuclear membrane of infected Hep2 cells. In the current studies, a bacterial artificial chromosome containing the entire HSV-1 strain F genome was used to construct a recombinant viral genome in which a gene encoding kanamycin resistance was inserted in place of 262 codons of the 306 codon U{sub L}31 open reading frame. The deletion virus produced virus titers approximately 10- to 50-fold lower in rabbit skin cells, more than 2000-fold lower in Veromore » cells, and more than 1500-fold lower in CV1 cells, compared to a virus bearing a restored U{sub L}31 gene. The replication of the U{sub L}31 deletion virus was restored on U{sub L}31-complementing cell lines derived either from rabbit skin cells or CV1 cells. Confocal microscopy indicated that the majority of U{sub L}34 protein localized aberrantly in the cytoplasm and nucleoplasm of Vero cells and CV1 cells, whereas U{sub L}34 protein localized at the nuclear membrane in rabbit skin cells, and U{sub L}31 complementing CV1 cells infected with the U{sub L}31 deletion virus. We conclude that rabbit skin cells encode a function that allows proper localization of U{sub L}34 protein to the nuclear membrane. We speculate that this function partially complements that of U{sub L}31 and may explain why U{sub L}31 is less critical for replication in rabbit skin cells as opposed to Vero and CV1 cells.« less

Cyclopropanation of Membrane Unsaturated Fatty Acids Is Not Essential to the Acid Stress Response of Lactococcus lactis subsp. cremoris ▿

PubMed Central

To, Thi Mai Huong; Grandvalet, Cosette; Tourdot-Maréchal, Raphaëlle

2011-01-01

Cyclopropane fatty acids (CFAs) are synthetized in situ by the transfer of a methylene group from S-adenosyl-l-methionine to a double bond of unsaturated fatty acid chains of membrane phospholipids. This conversion, catalyzed by the Cfa synthase enzyme, occurs in many bacteria and is recognized to play a key role in the adaptation of bacteria in response to a drastic perturbation of the environment. The role of CFAs in the acid tolerance response was investigated in the lactic acid bacterium Lactococcus lactis MG1363. A mutant of the cfa gene was constructed by allelic exchange. The cfa gene encoding the Cfa synthase was cloned and introduced into the mutant to obtain the complemented strain for homologous system studies. Data obtained by gas chromatography (GC) and GC-mass spectrometry (GC-MS) validated that the mutant could not produce CFA. The CFA levels in both the wild-type and complemented strains increased upon their entry to stationary phase, especially with acid-adapted cells or, more surprisingly, with ethanol-adapted cells. The results obtained by performing quantitative reverse transcription-PCR (qRT-PCR) experiments showed that transcription of the cfa gene was highly induced by acidity (by 10-fold with cells grown at pH 5.0) and by ethanol (by 9-fold with cells grown with 6% ethanol) in comparison with that in stationary phase. Cell viability experiments were performed after an acidic shock on the mutant strain, the wild-type strain, and the complemented strain, as a control. The higher viability level of the acid-adapted cells of the three strains after 3 h of shock proved that the cyclopropanation of unsaturated fatty acids is not essential for L. lactis subsp. cremoris survival under acidic conditions. Moreover, fluorescence anisotropy data showed that CFA itself could not maintain the membrane fluidity level, particularly with ethanol-grown cells. PMID:21421775

Sequestration of host-CD59 as potential immune evasion strategy of Trichomonas vaginalis.

PubMed

Ibáñez-Escribano, Alexandra; Nogal-Ruiz, Juan José; Pérez-Serrano, Jorge; Gómez-Barrio, Alicia; Escario, J Antonio; Alderete, J F

2015-09-01

Trichomonas vaginalis is known to evade complement-mediated lysis. Because the genome of T. vaginalis does not possess DNA sequence with homology to human protectin (CD59), a complement lysis restricting factor, we tested the hypothesis that host CD59 acquisition by T. vaginalis organisms mediates resistance to complement killing. This hypothesis was based on the fact that trichomonads are known to associate with host proteins. No CD59 was detected on the surface of T. vaginalis grown in serum-based medium using as probe anti-CD59 monoclonal antibody (MAb). We, therefore, infected mice intraperitoneally with live T. vaginalis, and trichomonads harvested from ascites were tested for binding of CD59. Immunofluorescence showed that parasites had surface CD59. Furthermore, as mouse erythrocytes (RBCs) possess membrane-associated CD59, and trichomonads use RBCs as a nutrient source, organisms were co-cultured with murine RBCs for one week. Parasites were shown to have detectable surface CD59. Importantly, live T. vaginalis with bound CD59 were compared with batch-grown parasites without surface-associated CD59 for sensitivity to complement in human serum. Trichomonads without surface-bound CD59 had a higher level of killing by complement than did parasites with surface CD59. These data show that host CD59 acquired onto the surface by live T. vaginalis may be an alternative mechanism for complement evasion. We describe a novel strategy by T. vaginalis consistent with host protein procurement by this parasite to evade the lytic action of complement. Copyright © 2015 Elsevier B.V. All rights reserved.

M. leprae components induce nerve damage by complement activation: identification of lipoarabinomannan as the dominant complement activator.

PubMed

Bahia El Idrissi, Nawal; Das, Pranab K; Fluiter, Kees; Rosa, Patricia S; Vreijling, Jeroen; Troost, Dirk; Morgan, B Paul; Baas, Frank; Ramaglia, Valeria

2015-05-01

Peripheral nerve damage is the hallmark of leprosy pathology but its etiology is unclear. We previously identified the membrane attack complex (MAC) of the complement system as a key determinant of post-traumatic nerve damage and demonstrated that its inhibition is neuroprotective. Here, we determined the contribution of the MAC to nerve damage caused by Mycobacterium leprae and its components in mouse. Furthermore, we studied the association between MAC and the key M. leprae component lipoarabinomannan (LAM) in nerve biopsies of leprosy patients. Intraneural injections of M. leprae sonicate induced MAC deposition and pathological changes in the mouse nerve, whereas MAC inhibition preserved myelin and axons. Complement activation occurred mainly via the lectin pathway and the principal activator was LAM. In leprosy nerves, the extent of LAM and MAC immunoreactivity was robust and significantly higher in multibacillary compared to paucibacillary donors (p = 0.01 and p = 0.001, respectively), with a highly significant association between LAM and MAC in the diseased samples (r = 0.9601, p = 0.0001). Further, MAC co-localized with LAM on axons, pointing to a role for this M. leprae antigen in complement activation and nerve damage in leprosy. Our findings demonstrate that MAC contributes to nerve damage in a model of M. leprae-induced nerve injury and its inhibition is neuroprotective. In addition, our data identified LAM as the key pathogen associated molecule that activates complement and causes nerve damage. Taken together our data imply an important role of complement in nerve damage in leprosy and may inform the development of novel therapeutics for patients.

Genetic evidence for involvement of classical complement pathway in induction of experimental autoimmune myasthenia gravis.

PubMed

Tüzün, Erdem; Scott, Benjamin G; Goluszko, Elzbieta; Higgs, Stephen; Christadoss, Premkumar

2003-10-01

Abs to acetylcholine receptor (AChR) and complement are the major constituents of pathogenic events causing neuromuscular junction destruction in both myasthenia gravis (MG) and experimental autoimmune MG (EAMG). To analyze the differential roles of the classical vs alternative complement pathways in EAMG induction, we immunized C3(-/-), C4(-/-), C3(+/-), and C4(+/-) mice and their control littermates (C3(+/+) and C4(+/+) mice) with AChR in CFA. C3(-/-) and C4(-/-) mice were resistant to disease, whereas mice heterozygous for C3 or C4 displayed intermediate susceptibility. Although C3(-/-) and C4(-/-) mice had anti-AChR Abs in their sera, anti-AChR IgG production by C3(-/-) mice was significantly suppressed. Both C3(-/-) and C4(-/-) mice had reduced levels of B cells and increased expression of apoptotis inducers (Fas ligand, CD69) and apoptotic cells in lymph nodes. Immunofluorescence studies showed that the neuromuscular junction of C3(-/-) and C4(-/-) mice lacked C3 or membrane attack complex deposits, despite having IgG deposits, thus providing in vivo evidence for the incapacity of anti-AChR IgGs to induce full-blown EAMG without the aid of complements. The data provide the first direct genetic evidence for the classical complement pathway in the induction of EAMG induced by AChR immunization. Accordingly, severe MG and other Ab- and complement-mediated diseases could be effectively treated by inhibiting C4, thus leaving the alternative complement pathway intact.

Inhibition of RAS activation due to a homozygous ezrin variant in patients with profound intellectual disability.

PubMed

Riecken, Lars Björn; Tawamie, Hasan; Dornblut, Carsten; Buchert, Rebecca; Ismayel, Amina; Schulz, Alexander; Schumacher, Johannes; Sticht, Heinrich; Pohl, Katja J; Cui, Yan; Reis, André; Morrison, Helen; Abou Jamra, Rami

2015-02-01

Gain-of-function alterations in several components and modulators of the Ras-MAPK pathway lead to dysregulation of the pathway and cause a broad spectrum of autosomal dominant developmental disorders, collectively known as RASopathies. These findings demonstrate the importance of tight multilevel Ras regulation to safeguard signaling output and prevent aberrant activity. We have recently identified ezrin as a novel regulatory element required for Ras activation. Homozygosity mapping and exome sequencing have now revealed the first presumably disease-causing variant in the coding gene EZR in two siblings with a profound intellectual disability. Localization and membrane targeting of the altered ezrin protein appeared normal but molecular modeling suggested protein interaction surfaces to be disturbed. Functional analysis revealed that the altered ezrin protein is no longer able to bind Ras and facilitate its activation. Furthermore, expression of the altered ezrin protein in different cell lines resulted in abnormal cellular processes, including reduced proliferation and neuritogenesis, thus revealing a possible mechanism for its phenotype in humans. To our knowledge, this is the first report of an autosomal recessively inherited loss-of-function mutation causing reduced Ras activity and thus extends and complements the pathogenicity spectrum of known Ras-MAPK pathway disturbances. © 2014 WILEY PERIODICALS, INC.

Prevention of PVDF ultrafiltration membrane fouling by coating MnO2 nanoparticles with ozonation

PubMed Central

Yu, Wenzheng; Brown, Matthew; Graham, Nigel. J. D.

2016-01-01

Pre-treatment is normally required to reduce or control the fouling of ultrafiltration (UF) membranes in drinking water treatment process. Current pre-treatment methods, such as coagulation, are only partially effective to prevent long-term fouling. Since biological activities are a major contributor to accumulated fouling, the application of an oxidation/disinfection step can be an effective complement to coagulation. In this study, a novel pre-treatment method has been evaluated at laboratory scale consisting of the addition of low dose ozone into the UF membrane tank after coagulation and the use of a hollow-fibre membrane coated with/without MnO2 nanoparticles over a test period of 70 days. The results showed that there was minimal fouling of the MnO2 coated membrane (0.5 kPa for 70 days), while the uncoated membrane experienced both reversible and irreversible fouling. The difference was attributed to the greatly reduced presence of bacteria and organic matter because of the catalytic decomposition of ozone to hydroxyl radicals and increase of the hydrophilicity of the membrane surface. In particular, the MnO2 coated membrane had a much thinner cake layer, with significantly less polysaccharides and proteins, and much less accumulated organic matter within the membrane pores. PMID:27436142

Prevention of PVDF ultrafiltration membrane fouling by coating MnO2 nanoparticles with ozonation

NASA Astrophysics Data System (ADS)

Yu, Wenzheng; Brown, Matthew; Graham, Nigel. J. D.

2016-07-01

Pre-treatment is normally required to reduce or control the fouling of ultrafiltration (UF) membranes in drinking water treatment process. Current pre-treatment methods, such as coagulation, are only partially effective to prevent long-term fouling. Since biological activities are a major contributor to accumulated fouling, the application of an oxidation/disinfection step can be an effective complement to coagulation. In this study, a novel pre-treatment method has been evaluated at laboratory scale consisting of the addition of low dose ozone into the UF membrane tank after coagulation and the use of a hollow-fibre membrane coated with/without MnO2 nanoparticles over a test period of 70 days. The results showed that there was minimal fouling of the MnO2 coated membrane (0.5 kPa for 70 days), while the uncoated membrane experienced both reversible and irreversible fouling. The difference was attributed to the greatly reduced presence of bacteria and organic matter because of the catalytic decomposition of ozone to hydroxyl radicals and increase of the hydrophilicity of the membrane surface. In particular, the MnO2 coated membrane had a much thinner cake layer, with significantly less polysaccharides and proteins, and much less accumulated organic matter within the membrane pores.

Effects of PKA phosphorylation on the conformation of the Na,K-ATPase regulatory protein FXYD1

PubMed Central

Teriete, Peter; Thai, Khang; Choi, Jungyuen; Marassi, Francesca M.

2009-01-01

FXYD1 (phospholemman) is a member of an evolutionarily conserved family of membrane proteins that regulate the function of the Na,K-ATPase enzyme complex in specific tissues and specific physiological states. In heart and skeletal muscle sarcolemma, FXYD1 is also the principal substrate of hormone-regulated phosphorylation by c-AMP dependent protein kinase A and by protein kinase C, which phosphorylate the protein at conserved Ser residues in its cytoplasmic domain, altering its Na,K-ATPase regulatory activity. FXYD1 adopts an L-shaped α-helical structure with the transmembrane helix loosely connected to a cytoplasmic amphipathic helix that rests on the membrane surface. In this paper we describe NMR experiments showing that neither PKA phosphorylation at Ser68 nor the physiologically relevant phosphorylation mimicking mutation Ser68Asp induces major changes in the protein conformation. The results, viewed in light of a model of FXYD1 associated with the Na,K-ATPase α and β subunits, indicate that the effects of phosphorylation on the Na,K-ATPase regulatory activity of FXYD1 could be due primarily to changes in electrostatic potential near the membrane surface and near the Na+/K+ ion binding site of the Na,K-ATPase α subunit. PMID:19761758

High-resolution Structures of Protein-Membrane Complexes by Neutron Reflection and MD Simulation: Membrane Association of the PTEN Tumor Suppressor

NASA Astrophysics Data System (ADS)

Lösche, Matthias

2012-02-01

The lipid matrix of biomembranes is an in-plane fluid, thermally and compositionally disordered leaflet of 5 nm thickness and notoriously difficult to characterize in structural terms. Yet, biomembranes are ubiquitous in the cell, and membrane-bound proteins are implicated in a variety of signaling pathways and intra-cellular transport. We developed methodology to study proteins associated with model membranes using neutron reflection measurements and showed recently that this approach can resolve the penetration depth and orientation of membrane proteins with ångstrom resolution if their crystal or NMR structure is known. Here we apply this technology to determine the membrane bindung and unravel functional details of the PTEN phosphatase, a key player in the PI3K apoptosis pathway. PTEN is an important regulatory protein and tumor suppressor that performs its phosphatase activity as an interfacial enzyme at the plasma membrane-cytoplasm boundary. Acting as an antagonist to phosphoinositide-3-kinase (PI3K) in cell signaling, it is deleted in many human cancers. Despite its importance in regulating the levels of the phosphoinositoltriphosphate PI(3,4,5)P3, there is little understanding of how PTEN binds to membranes, is activated and then acts as a phosphatase. We investigated the structure and function of PTEN by studying its membrane affinity and localization on in-plane fluid, thermally disordered synthetic membrane models. The membrane association of the protein depends strongly on membrane composition, where phosphatidylserine (PS) and phosphatidylinositol diphosphate (PI(4,5)P2) act synergetically in attracting the enzyme to the membrane surface. Membrane affinities depend strongly on membrane fluidity, which suggests multiple binding sites on the protein for PI(4,5)P2. Neutron reflection measurements show that the PTEN phosphatase ``scoots'' along the membrane surface (penetration < 5 å) but binds the membrane tightly with its two major domains, the C2 and phosphatase domains. In the bound state, PTEN's regulatory C-terminal tail is displaced from the membrane and organized on the far side of the protein, ˜ 60 å away from the bilayer surface, in a rather compact structure. The combination of binding studies and neutron reflection allows us to distinguish between PTEN mutant proteins and ultimately may identify the structural features required for membrane binding and activation of PTEN. Molecular dynamics simulations, currently in progress, refine this structural picture further.

Layer-by-layer structured polysaccharides-based multilayers on cellulose acetate membrane: Towards better hemocompatibility, antibacterial and antioxidant activities

NASA Astrophysics Data System (ADS)

Peng, Lincai; Li, Hui; Meng, Yahong

2017-04-01

The development of multifunctional cellulose acetate (CA) membranes with enhanced hemocompatibility and antibacterial and antioxidant activities is extremely important for biomedical applications. In this work, significant improvements in hemocompatibility and antibacterial and antioxidant activities of cellulose acetate (CA) membranes were achieved via layer-by-layer (LBL) deposition of chitosan (CS) and water-soluble heparin-mimicking polysaccharides (i.e., sulfated Cantharellus cibarius polysaccharides, SCP) onto their surface. The surface chemical compositions, growth manner, surface morphologies, and wetting ability of CS/SCP multilayer-modified CA membranes were characterized, respectively. The systematical evaluation of hemocompatibility revealed that CS/SCP multilayer-modified CA membranes significantly improved blood compatibility including resistance to non-specific protein adsorption, suppression of platelet adhesion and activation, prolongation of coagulation times, inhibition of complement activation, as well as reduction in blood hemolysis. Meanwhile, CS/SCP multilayer-modified CA membranes exhibited strong growth inhibition against Escherichia coli and Staphylococcus aureus, as well as high scavenging abilities against superoxide and hydroxyl radicals. In summary, the CS/SCP multilayers could confer CA membranes with integrated hemocompatibility and antibacterial and antioxidant activities, which might have great potential application in the biomedical field.

Transepithelial glucose transport and Na+/K+ homeostasis in enterocytes: an integrative model

PubMed Central

Drengstig, Tormod; Ruoff, Peter

2014-01-01

The uptake of glucose and the nutrient coupled transcellular sodium traffic across epithelial cells in the small intestine has been an ongoing topic in physiological research for over half a century. Driving the uptake of nutrients like glucose, enterocytes must have regulatory mechanisms that respond to the considerable changes in the inflow of sodium during absorption. The Na-K-ATPase membrane protein plays a major role in this regulation. We propose the hypothesis that the amount of active Na-K-ATPase in enterocytes is directly regulated by the concentration of intracellular Na+ and that this regulation together with a regulation of basolateral K permeability by intracellular ATP gives the enterocyte the ability to maintain ionic Na+/K+ homeostasis. To explore these regulatory mechanisms, we present a mathematical model of the sodium coupled uptake of glucose in epithelial enterocytes. Our model integrates knowledge about individual transporter proteins including apical SGLT1, basolateral Na-K-ATPase, and GLUT2, together with diffusion and membrane potentials. The intracellular concentrations of glucose, sodium, potassium, and chloride are modeled by nonlinear differential equations, and molecular flows are calculated based on experimental kinetic data from the literature, including substrate saturation, product inhibition, and modulation by membrane potential. Simulation results of the model without the addition of regulatory mechanisms fit well with published short-term observations, including cell depolarization and increased concentration of intracellular glucose and sodium during increased concentration of luminal glucose/sodium. Adding regulatory mechanisms for regulation of Na-K-ATPase and K permeability to the model show that our hypothesis predicts observed long-term ionic homeostasis. PMID:24898586

The Iron-Responsive Fur/RyhB Regulatory Cascade Modulates the Shigella Outer Membrane Protease IcsP ▿ †

PubMed Central

Africa, Lia A. A.; Murphy, Erin R.; Egan, Nicholas R.; Wigley, Amanda F.; Wing, Helen J.

2011-01-01

Actin-based motility is central to the pathogenicity of the intracellular bacterial pathogen Shigella. Two Shigella outer membrane proteins, IcsA and IcsP, are required for efficient actin-based motility in the host cell cytoplasm, and the genes encoding both proteins are carried on the large virulence plasmid. IcsA triggers actin polymerization on the surface of the bacterium, leading to the formation of an actin tail that allows both intra- and intercellular spread. IcsP, an outer membrane protease, modulates the amount and distribution of the IcsA protein on the bacterial surface through proteolytic cleavage of IcsA. Transcription of icsP is increased in the presence of VirB, a DNA-binding protein that positively regulates many genes carried on the large virulence plasmid. In Shigella dysenteriae, the small regulatory RNA RyhB, which is a member of the iron-responsive Fur regulon, suppresses several virulence-associated phenotypes by downregulating levels of virB in response to iron limitation. Here we show that the Fur/RyhB regulatory pathway downregulates IcsP levels in response to low iron concentrations in Shigella flexneri and that this occurs at the level of transcription through the RyhB-dependent regulation of VirB. These observations demonstrate that in Shigella species the Fur/RyhB regulatory pathway provides a mechanism to finely tune the expression of icsP in response to the low concentrations of free iron predicted to be encountered within colonic epithelial cells. PMID:21859852

Mammalian synthetic biology for studying the cell

PubMed Central

Mathur, Melina; Xiang, Joy S.

2017-01-01

Synthetic biology is advancing the design of genetic devices that enable the study of cellular and molecular biology in mammalian cells. These genetic devices use diverse regulatory mechanisms to both examine cellular processes and achieve precise and dynamic control of cellular phenotype. Synthetic biology tools provide novel functionality to complement the examination of natural cell systems, including engineered molecules with specific activities and model systems that mimic complex regulatory processes. Continued development of quantitative standards and computational tools will expand capacities to probe cellular mechanisms with genetic devices to achieve a more comprehensive understanding of the cell. In this study, we review synthetic biology tools that are being applied to effectively investigate diverse cellular processes, regulatory networks, and multicellular interactions. We also discuss current challenges and future developments in the field that may transform the types of investigation possible in cell biology. PMID:27932576

Variants in Complement Factor H and Complement Factor H-Related Protein Genes, CFHR3 and CFHR1, Affect Complement Activation in IgA Nephropathy.

PubMed

Zhu, Li; Zhai, Ya-Ling; Wang, Feng-Mei; Hou, Ping; Lv, Ji-Cheng; Xu, Da-Min; Shi, Su-Fang; Liu, Li-Jun; Yu, Feng; Zhao, Ming-Hui; Novak, Jan; Gharavi, Ali G; Zhang, Hong

2015-05-01

Complement activation is common in patients with IgA nephropathy (IgAN) and associated with disease severity. Our recent genome-wide association study of IgAN identified susceptibility loci on 1q32 containing the complement regulatory protein-encoding genes CFH and CFHR1-5, with rs6677604 in CFH as the top single-nucleotide polymorphism and CFHR3-1 deletion (CFHR3-1∆) as the top signal for copy number variation. In this study, to explore the clinical effects of variation in CFH, CFHR3, and CFHR1 on IgAN susceptibility and progression, we enrolled two populations. Group 1 included 1178 subjects with IgAN and available genome-wide association study data. Group 2 included 365 subjects with IgAN and available clinical follow-up data. In group 1, rs6677604 was associated with mesangial C3 deposition by genotype-phenotype correlation analysis. In group 2, we detected a linkage between the rs6677604-A allele and CFHR3-1∆ and found that the rs6677604-A allele was associated with higher serum levels of CFH and lower levels of the complement activation split product C3a. Furthermore, CFH levels were positively associated with circulating C3 levels and negatively associated with mesangial C3 deposition. Moreover, serum levels of the pathogenic galactose-deficient glycoform of IgA1 were also associated with the degree of mesangial C3 deposition in patients with IgAN. Our findings suggest that genetic variants in CFH, CFHR3, and CFHR1 affect complement activation and thereby, predispose patients to develop IgAN. Copyright © 2015 by the American Society of Nephrology.

The purification and characterization of ATP synthase complexes from the mitochondria of four fungal species.

PubMed

Liu, Sidong; Charlesworth, Thomas J; Bason, John V; Montgomery, Martin G; Harbour, Michael E; Fearnley, Ian M; Walker, John E

2015-05-15

The ATP synthases have been isolated by affinity chromatography from the mitochondria of the fungal species Yarrowia lipolytica, Pichia pastoris, Pichia angusta and Saccharomyces cerevisiae. The subunit compositions of the purified enzyme complexes depended on the detergent used to solubilize and purify the complex, and the presence or absence of exogenous phospholipids. All four enzymes purified in the presence of n-dodecyl-β-D-maltoside had a complete complement of core subunits involved directly in the synthesis of ATP, but they were deficient to different extents in their supernumerary membrane subunits. In contrast, the enzymes from P. angusta and S. cerevisiae purified in the presence of n-decyl-β-maltose neopentyl glycol and the phospholipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, cardiolipin (diphosphatidylglycerol) and 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] had a complete complement of core subunits and also contained all of the known supernumerary membrane subunits, e, f, g, j, k and ATP8 (or Aap1), plus an additional new membrane component named subunit l, related in sequence to subunit k. The catalytic domain of the enzyme from P. angusta was more resistant to thermal denaturation than the enzyme from S. cerevisiae, but less stable than the catalytic domain of the bovine enzyme, but the stator and the integrity of the transmembrane proton pathway were most stable in the enzyme from P. angusta. The P. angusta enzyme provides a suitable source of enzyme for studying the structure of the membrane domain and properties associated with that sector of the enzyme complex.

The purification and characterization of ATP synthase complexes from the mitochondria of four fungal species

PubMed Central

Liu, Sidong; Charlesworth, Thomas J.; Bason, John V.; Montgomery, Martin G.; Harbour, Michael E.; Fearnley, Ian M.; Walker, John E.

2015-01-01

The ATP synthases have been isolated by affinity chromatography from the mitochondria of the fungal species Yarrowia lipolytica, Pichia pastoris, Pichia angusta and Saccharomyces cerevisiae. The subunit compositions of the purified enzyme complexes depended on the detergent used to solubilize and purify the complex, and the presence or absence of exogenous phospholipids. All four enzymes purified in the presence of n-dodecyl-β-D-maltoside had a complete complement of core subunits involved directly in the synthesis of ATP, but they were deficient to different extents in their supernumerary membrane subunits. In contrast, the enzymes from P. angusta and S. cerevisiae purified in the presence of n-decyl-β-maltose neopentyl glycol and the phospholipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, cardiolipin (diphosphatidylglycerol) and 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] had a complete complement of core subunits and also contained all of the known supernumerary membrane subunits, e, f, g, j, k and ATP8 (or Aap1), plus an additional new membrane component named subunit l, related in sequence to subunit k. The catalytic domain of the enzyme from P. angusta was more resistant to thermal denaturation than the enzyme from S. cerevisiae, but less stable than the catalytic domain of the bovine enzyme, but the stator and the integrity of the transmembrane proton pathway were most stable in the enzyme from P. angusta. The P. angusta enzyme provides a suitable source of enzyme for studying the structure of the membrane domain and properties associated with that sector of the enzyme complex. PMID:25759169

Eculizumab for Dense Deposit Disease and C3 Glomerulonephritis

PubMed Central

Smith, Richard J.; Barile, Gaetano R.; Zhang, Yuzhou; Heher, Eliot C.; Herlitz, Leal; Stokes, M. Barry; Markowitz, Glen S.; D’Agati, Vivette D.; Canetta, Pietro A.; Radhakrishnan, Jai; Appel, Gerald B.

2012-01-01

Summary Background and objectives The principle defect in dense deposit disease and C3 glomerulonephritis is hyperactivity of the alternative complement pathway. Eculizumab, a monoclonal antibody that binds to C5 to prevent formation of the membrane attack complex, may prove beneficial. Design, setting, participants, & measurements In this open-label, proof of concept efficacy and safety study, six subjects with dense deposit disease or C3 glomerulonephritis were treated with eculizumab every other week for 1 year. All had proteinuria >1 g/d and/or AKI at enrollment. Subjects underwent biopsy before enrollment and repeat biopsy at the 1-year mark. Results The subjects included three patients with dense deposit disease (including one patient with recurrent dense deposit disease in allograft) and three patients with C3 glomerulonephritis (including two patients with recurrent C3 glomerulonephritis in allograft). Genetic and complement function testing revealed a mutation in CFH and MCP in one subject each, C3 nephritic factor in three subjects, and elevated levels of serum membrane attack complex in three subjects. After 12 months, two subjects showed significantly reduced serum creatinine, one subject achieved marked reduction in proteinuria, and one subject had stable laboratory parameters but histopathologic improvements. Elevated serum membrane attack complex levels normalized on therapy and paralleled improvements in creatinine and proteinuria. Conclusions Clinical and histopathologic data suggest a response to eculizumab in some but not all subjects with dense deposit disease and C3 glomerulonephritis. Elevation of serum membrane attack complex before treatment may predict response. Additional research is needed to define the subgroup of dense deposit disease/C3 glomerulonephritis patients in whom eculizumab therapy can be considered. PMID:22403278

STriatal-Enriched protein tyrosine Phosphatase (STEP) Regulates the PTPα/Fyn Signaling Pathway

PubMed Central

Xu, Jian; Kurup, Pradeep; Foscue, Ethan; Lombroso, Paul J.

2015-01-01

The tyrosine kinase Fyn has two regulatory tyrosine residues that when phosphorylated either activate (Tyr420) or inhibit (Tyr531) Fyn activity. Within the central nervous system, two protein tyrosine phosphatases (PTPs) target these regulatory tyrosines in Fyn. PTPα dephosphorylates Tyr531 and activates Fyn, while STEP (STriatal-Enriched protein tyrosine Phosphatase) dephosphorylates Tyr420 and inactivates Fyn. Thus, PTPα and STEP have opposing functions in the regulation of Fyn; however, whether there is cross talk between these two PTPs remains unclear. Here, we used molecular techniques in primary neuronal cultures and in vivo to demonstrate that STEP negatively regulates PTPα by directly dephosphorylating PTPα at its regulatory Tyr789. Dephosphorylation of Tyr789 prevents the translocation of PTPα to synaptic membranes, blocking its ability to interact with and activate Fyn. Genetic or pharmacologic reduction of STEP61 activity increased the phosphorylation of PTPα at Tyr789, as well as increased translocation of PTPα to synaptic membranes. Activation of PTPα and Fyn and trafficking of GluN2B to synaptic membranes are necessary for ethanol intake behaviors in rodents. We tested the functional significance of STEP61 in this signaling pathway by ethanol administration to primary cultures as well as in vivo, and demonstrated that the inactivation of STEP61 by ethanol leads to the activation of PTPα, its translocation to synaptic membranes, and the activation of Fyn. These findings indicate a novel mechanism by which STEP61 regulates PTPα and suggest that STEP and PTPα coordinate the regulation of Fyn. PMID:25951993

Drug Transporters and Na+/H+ Exchange Regulatory Factor PSD-95/Drosophila Discs Large/ZO-1 Proteins

PubMed Central

Walsh, Dustin R.; Nolin, Thomas D.

2015-01-01

Drug transporters govern the absorption, distribution, and elimination of pharmacologically active compounds. Members of the solute carrier and ATP binding-cassette drug transporter family mediate cellular drug uptake and efflux processes, thereby coordinating the vectorial movement of drugs across epithelial barriers. To exert their physiologic and pharmacological function in polarized epithelia, drug transporters must be targeted and stabilized to appropriate regions of the cell membrane (i.e., apical versus basolateral). Despite the critical importance of drug transporter membrane targeting, the mechanisms that underlie these processes are largely unknown. Several clinically significant drug transporters possess a recognition sequence that binds to PSD-95/Drosophila discs large/ZO-1 (PDZ) proteins. PDZ proteins, such as the Na+/H+ exchanger regulatory factor (NHERF) family, act to stabilize and organize membrane targeting of multiple transmembrane proteins, including many clinically relevant drug transporters. These PDZ proteins are normally abundant at apical membranes, where they tether membrane-delimited transporters. NHERF expression is particularly high at the apical membrane in polarized tissue such as intestinal, hepatic, and renal epithelia, tissues important to drug disposition. Several recent studies have highlighted NHERF proteins as determinants of drug transporter function secondary to their role in controlling membrane abundance and localization. Mounting evidence strongly suggests that NHERF proteins may have clinically significant roles in pharmacokinetics and pharmacodynamics of several pharmacologically active compounds and may affect drug action in cancer and chronic kidney disease. For these reasons, NHERF proteins represent a novel class of post-translational mediators of drug transport and novel targets for new drug development. PMID:26092975

Membrane Recruitment of the Non-receptor Protein GIV/Girdin (Gα-interacting, Vesicle-associated Protein/Girdin) Is Sufficient for Activating Heterotrimeric G Protein Signaling.

PubMed

Parag-Sharma, Kshitij; Leyme, Anthony; DiGiacomo, Vincent; Marivin, Arthur; Broselid, Stefan; Garcia-Marcos, Mikel

2016-12-30

GIV (aka Girdin) is a guanine nucleotide exchange factor that activates heterotrimeric G protein signaling downstream of RTKs and integrins, thereby serving as a platform for signaling cascade cross-talk. GIV is recruited to the cytoplasmic tail of receptors upon stimulation, but the mechanism of activation of its G protein regulatory function is not well understood. Here we used assays in humanized yeast models and G protein activity biosensors in mammalian cells to investigate the role of GIV subcellular compartmentalization in regulating its ability to promote G protein signaling. We found that in unstimulated cells GIV does not co-fractionate with its substrate G protein Gα i3 on cell membranes and that constitutive membrane anchoring of GIV in yeast cells or rapid membrane translocation in mammalian cells via chemically induced dimerization leads to robust G protein activation. We show that membrane recruitment of the GIV "Gα binding and activating" motif alone is sufficient for G protein activation and that it does not require phosphomodification. Furthermore, we engineered a synthetic protein to show that recruitment of the GIV "Gα binding and activating" motif to membranes via association with active RTKs, instead of via chemically induced dimerization, is also sufficient for G protein activation. These results reveal that recruitment of GIV to membranes in close proximity to its substrate G protein is a major mechanism responsible for the activation of its G protein regulatory function. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The regulation of MADS-box gene expression during ripening of banana and their regulatory interation with ethylene

USDA-ARS?s Scientific Manuscript database

MADS-box genes (MaMADS1-6), potential components of the developmental control of ripening have been cloned from Grand Nain banana cultivar. Similarity of these genes to tomato LeRIN is very low and neither MaMADS2 nor MaMADS1 complement the tomato rin mutation. Nevertheless, the expression patterns...

Design principles and operating principles: the yin and yang of optimal functioning.

PubMed

Voit, Eberhard O

2003-03-01

Metabolic engineering has as a goal the improvement of yield of desired products from microorganisms and cell lines. This goal has traditionally been approached with experimental biotechnological methods, but it is becoming increasingly popular to precede the experimental phase by a mathematical modeling step that allows objective pre-screening of possible improvement strategies. The models are either linear and represent the stoichiometry and flux distribution in pathways or they are non-linear and account for the full kinetic behavior of the pathway, which is often significantly effected by regulatory signals. Linear flux analysis is simpler and requires less input information than a full kinetic analysis, and the question arises whether the consideration of non-linearities is really necessary for devising optimal strategies for yield improvements. The article analyzes this question with a generic, representative pathway. It shows that flux split ratios, which are the key criterion for linear flux analysis, are essentially sufficient for unregulated, but not for regulated branch points. The interrelationships between regulatory design on one hand and optimal patterns of operation on the other suggest the investigation of operating principles that complement design principles, like a user's manual complements the hardwiring of electronic equipment.

Gal knockout and beyond.

PubMed

Zhong, R

2007-01-01

Recently, Galalpha1-3Galbeta1-4GlcNAc (Gal) knockout (k/o) pigs have been developed using genetic cloning technologies. This remarkable achievement has generated great enthusiasm in xenotransplantation studies. This review summarizes the current status of nonhuman primate experiments using Gal k/o pig organs. Briefly, when Gal k/o pig organs are transplanted into primates, hyperacute rejection does not occur. Although graft survival has been prolonged up to a few months in some cases, the overall results were not better than those using Gal-positive pig organs with human complement regulatory protein transgenes. Gal k/o pig kidneys rapidly developed rejection which was associated with increased anti-non-Gal antibodies. Although the precise mechanisms of Gal k/o pig organ rejection are not clear, it could result from incomplete deletion of Gal, up-regulation of new antigen (non-Gal antigen) and/or production of non-Gal antibodies. Future work in xenotransplantation should place emphasis on further modification of donors, such as combining human complement regulatory genes with Gal k/o, deleting non-Gal antigens and adding protective/surviving genes or a gene that inhibits coagulation. Induction of donor-specific T- and B-cell tolerance and promotion of accommodation are also warranted.

Insulin stimulates syntaxin4 SNARE complex assembly via a novel regulatory mechanism.

PubMed

Kioumourtzoglou, Dimitrios; Gould, Gwyn W; Bryant, Nia J

2014-04-01

Insulin stimulates glucose transport into fat and muscle cells by increasing the exocytic trafficking rate of the GLUT4 facilitative glucose transporter from intracellular stores to the plasma membrane. Delivery of GLUT4 to the plasma membrane is mediated by formation of functional SNARE complexes containing syntaxin4, SNAP23, and VAMP2. Here we have used an in situ proximity ligation assay to integrate these two observations by demonstrating for the first time that insulin stimulation causes an increase in syntaxin4-containing SNARE complex formation in adipocytes. Furthermore, we demonstrate that insulin brings about this increase in SNARE complex formation by mobilizing a pool of syntaxin4 held in an inactive state under basal conditions. Finally, we have identified phosphorylation of the regulatory protein Munc18c, a direct target of the insulin receptor, as a molecular switch to coordinate this process. Hence, this report provides molecular detail of how the cell alters membrane traffic in response to an external stimulus, in this case, insulin.

Sphingolipid topology and the dynamic organization and function of membrane proteins.

PubMed

van Meer, Gerrit; Hoetzl, Sandra

2010-05-03

When acquiring internal membranes and vesicular transport, eukaryotic cells started to synthesize sphingolipids and sterols. The physical differences between these and the glycerophospholipids must have enabled the cells to segregate lipids in the membrane plane. Localizing this event to the Golgi then allowed them to create membranes of different lipid composition, notably a thin, flexible ER membrane, consisting of glycerolipids, and a sturdy plasma membrane containing at least 50% sphingolipids and sterols. Besides sorting membrane proteins, in the course of evolution the simple sphingolipids obtained key positions in cellular physiology by developing specific interactions with (membrane) proteins involved in the execution and control of signaling. The few signaling sphingolipids in mammals must provide basic transmission principles that evolution has built upon for organizing the specific regulatory pathways tuned to the needs of the different cell types in the body. Copyright 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Binding of human and rat CD59 to the terminal complement complexes.

PubMed Central

Lehto, T; Morgan, B P; Meri, S

1997-01-01

CD59-antigen (protectin) is a widely distributed glycolipid-anchored inhibitor of complement lysis. CD59 interacts with complement components C8 and C9 during assembly of the membrane attack complex (MAC). To evaluate species specificity of these interactions we have in the present study examined cross-species binding of isolated human and rat CD59 to the terminal complement components C8 and C9. By using primarily soluble CD59 isolated from urine (CD59U) potentially non-specific binding interactions of the phospholipid portion of the membrane forms of CD59 could be avoided. Sucrose density gradient ultracentrifugation analysis showed that human CD59U bound to both human and rat C8 in the SC5b-8 complexes. Similar binding occurred when rat CD59U was used. The degree of binding did not significantly differ between the heterologous and homologous CD59-C8 combinations. C9 from both species inhibited the binding of CD59 to soluble SC5b-8. In ligand blotting analysis human and rat CD59U bound to human and rat C8 alpha gamma-subunit and C9. Binding of human and rat CD59U was stronger to human than rat C9. In plate binding assays the erythrocyte form of CD59 (CD59E) bound to both human and rat C8. Binding of CD59E to heterologous C9 was considerably weaker than to homologous C9. Our results imply that the reciprocal binding sites between C8 and CD59 and to a lesser degree between CD59 and C9 are conserved between human and rat. Interactions of CD59 with the terminal C components are thus species selective but not 'homologously restricted'. Images Figure 4 Figure 5 PMID:9038722

Role of a disulfide-bonded peptide loop within human complement C9 in the species-selectivity of complement inhibitor CD59.

PubMed

Husler, T; Lockert, D H; Sims, P J

1996-03-12

CD59 antigen is a membrane glycoprotein that inhibits the activity of the C9 component of the C5b-9 membrane attack complex (MAC), thereby protecting human cells from lysis by human complement. The complement-inhibitory activity of CD59 is species-selective, and is most effective toward C9 derived from human or other primate plasma. The species-selective activity of CD59 was recently used to map the segment of human C9 that is recognized by this MAC inhibitor, using recombinant rabbit/human C9 chimeras that retain lytic function within the MAC [Husler, T., Lockert, D. H., Kaufman, K. M., Sodetz, J. M., & Sims, P. J. (1995) J. Biol. Chem. 270,3483-3486]. These experiments suggested that the CD59 recognition domain was contained between residues 334 and 415 in human C9. By analyzing the species-selective lytic activity of recombinant C9 with chimeric substitutions internal to this segment, we now demonstrate that the site in human C9 uniquely recognized by CD59 is centered on those residues contained between C9 Cys359/Cys384, with an additional contribution by residues C-terminal to this segment. Consistent with its role as a CD59 recognition domain, CD59 specifically bound a human C9-derived peptide corresponding to residues 359-384, and antibody (Fab) raised against this C9-derived peptide inhibited the lytic activity of human MAC. Mutant human C9 in which Ala was substituted for Cys359/384 was found to express normal lytic activity and to be fully inhibited by CD59. This suggests that the intrachain Cys359/Cys384 disulfide bond within C9 is not required to maintain the conformation of this segment of C9 for interaction with CD59.

Phylogenetic aspects of the complement system.

PubMed

Zarkadis, I K; Mastellos, D; Lambris, J D

2001-01-01

During evolution two general systems of immunity have emerged: innate or, natural immunity and adaptive (acquired), or specific immunity. The innate system is phylogenetically older and is found in some form in all multicellular organisms, whereas the adaptive system appeared about 450 million years ago and is found in all vertebrates except jawless fish. The complement system in higher vertebrates plays an important role as an effector of both the innate and the acquired immune response, and also participates in various immunoregulatory processes. In lower vertebrates complement is activated by the alternative and lectin pathways and is primarily involved in the opsonization of foreign material. The Agnatha (the most primitive vertebrate species) possess the alternative and lectin pathways while cartilaginous fish are the first species in which the classical pathway appears following the emergence of immunoglobulins. The rest of the poikilothermic species, ranging from teleosts to reptilians, appear to contain a well-developed complement system resembling that of the homeothermic vertebrates. It seems that most of the complement components have appeared after the duplication of primordial genes encoding C3/C4/C5, fB/C2, C1s/C1r/MASP-1/MASP-2, and C6/C7/C8/C9 molecules, in a process that led to the formation of distinct activation pathways. However, unlike homeotherms, several species of poikilotherms (e.g. trout) have recently been shown to possess multiple forms of complement components (C3, factor B) that are structurally and functionally more diverse than those of higher vertebrates. We hypothesize that this remarkable diversity has allowed these animals to expand their innate capacity for immune recognition and response. Recent studies have also indicated the possible presence of complement receptors in protochordates and lower vertebrates. In conclusion, there is considerable evidence suggesting that the complement system is present in the entire lineage of deuterostomes, and regulatory complement components have been identified in all species beyond the protochordates, indicating that the mechanisms of complement activation and regulation have developed in parallel.

Systemic human CR2-targeted complement alternative pathway inhibitor ameliorates mouse laser-induced choroidal neovascularization.

PubMed

Rohrer, Bärbel; Coughlin, Beth; Bandyopadhyay, Mausumi; Holers, V Michael

2012-08-01

Genetic associations and the presence of complement components within pathological structures of age-related macular degeneration (AMD) have generated the hypothesis that AMD is caused by chronic local complement activation. Since the majority of activity in the common terminal pathway results from engagement of the amplification loop, the alternative pathway has been proposed as a logical therapeutic target. We recently generated a factor H (fH)-based complement inhibitor (CR2-fH) with the capacity to be "targeted" to sites of complement C3 activation. We asked whether the human therapeutic (TT30) is effective in a mouse model of AMD. Choroidal neovascularization (CNV) was induced by argon laser photocoagulation of Bruch's membrane. Every other day, mice received intravenous injections of TT30 or vehicles, and after 6 days, the presence or absence of CNV and CNV-related changes were evaluated. Area of CNV, photoreceptor cell function, gene expression for complement components and cytokines, vascular endothelial growth factor (VEGF) protein levels, and TT30 bioavailability were determined. CNV development, which has previously been shown to require local complement activation, could be reduced by intravenous TT30 delivery. Specific inhibition of the alternative pathway not only reduced angiogenesis in CNV, but also ameliorated changes in several associated disease-related biomarkers, including diminished retinal function and molecular events known to be involved in AMD such as VEGF production. After intravenous injection, TT30 localized to CNV lesion sites in the retinal pigmented epithelium-choroid. Systemic administration of TT30 was found to reduce CNV pathology. These data may open new avenues for novel systemic AMD treatment strategies.

Microinjection of cytoplasm as a test of complementation in Paramecium

PubMed Central

1982-01-01

Mutants in Paramecium tetraurelia, unable to generate action potentials, have been isolated as cells which show no backward swimming in response to ionic stimulation. These "pawn" mutants belong to at least three complementation groups designated pwA, pwB, and pwC. We have found that microinjection of cytoplasm from a wild-type donor into a pawn recipient of any of the three complementation groups restores the ability of the pawn to generate action potentials and hence swim backward. In addition, the cytoplasm from a pawn cannot restore a recipient of the same complementation group, but that from a pawn of a different group can. Electrophysiological analysis had demonstrated that the restoration of backward swimming is not due to a simple addition of ions but represents a profound change in the excitable membrane of the recipient pawn cells. Using known pawn mutants and those which had previously been unclassified, we have been able to establish a perfect concordance of genetic complementation and complementation by cytoplasmic transfer through microinjection. This method has been used to classify pawn mutants that are sterile or hard- to-mate and to examine the ability of cytoplasms from different species of ciliated protozoa to restore the ability to swim backward in the pawn mutants of P. tetraurelia. A cell homogenate has also been fractionated by centrifugation to further purify the active components. These results demonstrate that transfer of cytoplasm between cells by microinjection can be a valid and systematic method to classify mutants. This test is simpler to perform than the genetic complementation test and can be used under favorable conditions in mutants that are sterile and in cells of different species. PMID:7061597

Polyreactive Antibodies Plus Complement Enhance the Phagocytosis of Cells Made Apoptotic by UV-Light or HIV

PubMed Central

Zhou, Zhao-hua; Wild, Teresa; Xiong, Ying; Sylvers, Peter; Zhang, Yahong; Zhang, Luxia; Wahl, Larry; Wahl, Sharon M.; Kozlowski, Steven; Notkins, Abner L.

2013-01-01

Polyreactive antibodies are a major component of the natural antibody repertoire and are capable of binding a variety of structurally unrelated antigens. Many of the properties attributed to natural antibodies, in fact, are turning out to be due to polyreactive antibodies. In humans, each day, billions of cells undergo apoptosis. In the present experiments, we show by ImageStream technology that although polyreactive antibodies do not bind to live T cells they bind to both the plasma membrane and cytoplasm of late apoptotic cells, fix complement, generate the anaphylatoxin C5a and increase by as much as 5 fold complement-mediated phagocytosis by macrophages. Of particular importance, T cells undergoing apoptosis following infection with HIV also bind polyreactive antibodies and are phagocytosed. We conclude that the polyreactive antibodies in the natural antibody repertoire contribute in a major way to the clearance of cells made apoptotic by a variety of natural and infectious processes. PMID:23881356

Thrombopoietic status of patients on haemodialysis

PubMed Central

Bat, Taha; Bat, Betul Emine; El-Moghraby, Ahmed; Patel, Samir; Feng, Xingmin; Dunbar, Cynthia E.; Sarac, Erdal

2015-01-01

Thrombocytopenia is a potential dialysis-related treatment complication. Developments in bio-compatible dialyser membranes have decreased the occurrence of thrombocytopenia. We investigated whether thrombopoiesis is impaired in haemodialysis patients by measuring the thrombopoietin level and absolute immature platelet number (AIPN) in the blood of patients undergoing haemodialysis. Samples were collected from the dialysis tubing pre- and post- haemodialysis in a cohort of 45 well-characterized haemodialysis patients. Thrombopoietin levels and AIPN increased following haemodialysis, despite no change in platelet count. Observed increase in release of immature platelets from the bone marrow following haemodialysis indicates possible complement activation secondary to interaction between blood constituents and the dialysis membrane. PMID:26887628

Differences in bio-incompatibility among four biocompatible dialyzer membranes using in maintenance hemodialysis patients.

PubMed

Zhang, Dong-Liang; Liu, Jing; Cui, Wen-Ying; Ji, Dan-Ying; Zhang, Yue; Liu, Wen-Hu

2011-01-01

Following the introduction of modified cellulosic and then synthetic membrane dialyzers, it was realized that the dialyzer bio-incompatibility depends on the membrane composition. We designed a prospective, randomized, cohort study of 6 months to determine several parameters of biocompatibility in maintenance hemodialysis (MHD) patients treated with four different membrane dialyzers. There were 60 MHD patients enrolled in the study. In baseline, synthetic low-flux dialyzer, polysulfone (PS) membrane was used in all patients for at least 3 months. Then the patients were randomly divided into three groups according to different dialyzer membranes. Synthetic high-flux dialyzer group, polyethersulfone membrane, cellulose triacetate (CTA) high-flux membrane, and synthetic low-flux dialyzer, polymethylmethacrylate (PMMA) membrane were used in 6 months. A new dialyzer was used for each study treatment, and there was no dialyzer reuse. The biocompatibility markers and solutes removal markers were detected repeatedly at different time points. The blood levels of highly sensitive C reactive protein, interleukin (IL)-1β, and interleukin (IL)-13 showed no difference among different groups at al time points. However, the blood complement levels and white blood cell counts were significantly different among three groups. When the dialyzers changed from PS to PMMA membrane, C3a levels and white blood cell counts changed significantly (p < 0.05). Moreover, the changes of C5a levels were significantly different between group CTA and group PMMA in month 3 (p < 0.05). There were much more differences on bio-incompatibility among different dialyzer membranes.

Spontaneous abortion is associated with elevated systemic C5a and reduced mRNA of complement inhibitory proteins in placenta.

PubMed

Banadakoppa, M; Chauhan, M S; Havemann, D; Balakrishnan, M; Dominic, J S; Yallampalli, C

2014-09-01

Spontaneous abortion in early pregnancy due to unknown reasons is a common problem. The excess complement activation and consequent placental inflammation and anti-angiogenic milieu is emerging as an important associated factor in many pregnancy-related complications. In the present study we sought to examine the expression of complement inhibitory proteins at the feto-maternal interface and levels of complement split products in the circulation to understand their role in spontaneous abortion. Consenting pregnant women who either underwent elective abortion due to non-clinical reasons (n = 13) or suffered miscarriage (n = 14) were recruited for the study. Systemic levels of complement factors C3a and C5a were measured by enzyme-linked immunosorbent assay (ELISA). Plasma C5 and C3 protein levels were examined by Western blot. Expressions of complement regulatory proteins such as CD46 and CD55 in the decidua were investigated by quantitative polymerase chain reaction (PCR) and Western blot. The median of plasma C3a level was 82·83 ng/ml and 66·17 ng/ml in elective and spontaneous abortion patients, respectively. Medians of plasma C5a levels in elective and spontaneous abortion patients were 0·96 ng/ml and 1·14 ng/ml, respectively. Only plasma C5a levels but not C3a levels showed significant elevation in spontaneous abortion patients compared to elective abortion patients. Further, there was a threefold decrease in the mRNA expressions of complement inhibitory proteins CD46 and CD55 in the decidua obtained from spontaneous abortion patients compared to that of elective abortion patients. These data suggested that dysregulated complement cascade may be associated with spontaneous abortion. © 2014 British Society for Immunology.

Immune response and histology of humoral rejection in kidney transplantation.

PubMed

González-Molina, Miguel; Ruiz-Esteban, Pedro; Caballero, Abelardo; Burgos, Dolores; Cabello, Mercedes; Leon, Miriam; Fuentes, Laura; Hernandez, Domingo

2016-01-01

The adaptive immune response forms the basis of allograft rejection. Its weapons are direct cellular cytotoxicity, identified from the beginning of organ transplantation, and/or antibodies, limited to hyperacute rejection by preformed antibodies and not as an allogenic response. This resulted in allogenic response being thought for decades to have just a cellular origin. But the experimental studies by Gorer demonstrating tissue damage in allografts due to antibodies secreted by B lymphocytes activated against polymorphic molecules were disregarded. The special coexistence of binding and unbinding between antibodies and antigens of the endothelial cell membranes has been the cause of the delay in demonstrating the humoral allogenic response. The endothelium, the target tissue of antibodies, has a high turnover, and antigen-antibody binding is non-covalent. If endothelial cells are attacked by the humoral response, immunoglobulins are rapidly removed from their surface by shedding and/or internalization, as well as degrading the components of the complement system by the action of MCP, DAF and CD59. Thus, the presence of complement proteins in the membrane of endothelial cells is transient. In fact, the acute form of antibody-mediated rejection was not demonstrated until C4d complement fragment deposition was identified, which is the only component that binds covalently to endothelial cells. This review examines the relationship between humoral immune response and the types of acute and chronic histological lesion shown on biopsy of the transplanted organ. Copyright © 2016 Sociedad Española de Nefrología. Published by Elsevier España, S.L.U. All rights reserved.

The shape of things to come: regulation of shape changes in endoplasmic reticulum.

PubMed

Paiement, J; Bergeron, J

2001-01-01

Shape changes in the endoplasmic reticulum control fundamental cell processes including nuclear envelope assembly in mitotic cells, calcium homeostasis in cytoplasmic domains of secreting and motile cells, and membrane traffic in the early secretion apparatus between the endoplasmic reticulum and Golgi. Opposing forces of assembly (membrane fusion) and disassembly (membrane fragmentation) ultimately determine the size and shape of this organelle. This review examines some of the regulatory mechanisms involved in these processes and how they occur at specific sites or subcompartments of the endoplasmic reticulum.

Membranous glomerulonephritis in a child asymptomatic for hepatitis B virus. Concomitant seropositivity for HBsAG and anti-HBs.

PubMed

Hirsch, H Z; Ainsworth, S K; DeBeukelaer, M; Brissie, R M; Hennigar, G R

1981-04-01

The presence of hepatitis B surface antigen (HBsAg) in association with immunoglobulins and complement components within the glomerular basement membranes of adults having chronic active hepatitis has been well documented. In addition, investigators in Poland have demonstrated HBsAg immune complexes in glomeruli of children who did not have clinical evidence of hepatitis. More recently, a single case of childhood membranous glomerulonephritis in an asymptomatic carrier of hepatitis B virus was cited by observers in Canada. Reported here is the deposition of HBsAg immune complexes in the glomerular basement membranes of a 13-year-old black boy who had membranous glomerulopathy but not clinical evidence of hepatitis. This may be the first reported case in the United States of HbsAg-associated membranous glomerulonephritis in a child asymptomatic for hepatitis B virus, and only the second such case in North America. However, unlike previous studies of childhood glomerulopathy in association with hepatitis B virus, this patient is seropositive for both HBsAg and anti-HBs (antibody for hepatitis B surface antigen). Similar "rare" serologic findings were found for the patient's eldest male sib.

Post-Fusion Membrane Reorganization.

DTIC Science & Technology

1993-01-27

diphosphoglycerate , and NEM (a crosslinking agent), and ethanol treatments all had reproducible and very specific effects on the kinetic phases and the fusion product...actually, at the ultrastructure level , a double membrane multiply perforated with fusion sites (or pores). Also, because the heat treatment was within...relationships. Moreover. 2.3- Diphosphoglycerate (2-3-DPG). a naturally occuring metabolite which is known to have a regulatory role in spectrin-cytoskeletal

Reciprocal Regulation of Endocytosis and Metabolism

PubMed Central

Antonescu, Costin N.; McGraw, Timothy E.; Klip, Amira

2014-01-01

The cellular uptake of many nutrients and micronutrients governs both their cellular availability and their systemic homeostasis. The cellular rate of nutrient or ion uptake (e.g., glucose, Fe3+, K+) or efflux (e.g., Na+) is governed by a complement of membrane transporters and receptors that show dynamic localization at both the plasma membrane and defined intracellular membrane compartments. Regulation of the rate and mechanism of endocytosis controls the amounts of these proteins on the cell surface, which in many cases determines nutrient uptake or secretion. Moreover, the metabolic action of diverse hormones is initiated upon binding to surface receptors that then undergo regulated endocytosis and show distinct signaling patterns once internalized. Here, we examine how the endocytosis of nutrient transporters and carriers as well as signaling receptors governs cellular metabolism and thereby systemic (whole-body) metabolite homeostasis. PMID:24984778

Oligomeric state regulated trafficking of human platelet-activating factor acetylhydrolase type-II.

PubMed

Monillas, Elizabeth S; Caplan, Jeffrey L; Thévenin, Anastasia F; Bahnson, Brian J

2015-05-01

The intracellular enzyme platelet-activating factor acetylhydrolase type-II (PAFAH-II) hydrolyzes platelet-activating factor and oxidatively fragmented phospholipids. PAFAH-II in its resting state is mainly cytoplasmic, and it responds to oxidative stress by becoming increasingly bound to endoplasmic reticulum and Golgi membranes. Numerous studies have indicated that this enzyme is essential for protecting cells from oxidative stress induced apoptosis. However, the regulatory mechanism of the oxidative stress response by PAFAH-II has not been fully resolved. Here, changes to the oligomeric state of human PAFAH-II were investigated as a potential regulatory mechanism toward enzyme trafficking. Native PAGE analysis in vitro and photon counting histogram within live cells showed that PAFAH-II is both monomeric and dimeric. A Gly-2-Ala site-directed mutation of PAFAH-II demonstrated that the N-terminal myristoyl group is required for homodimerization. Additionally, the distribution of oligomeric PAFAH-II is distinct within the cell; homodimers of PAFAH-II were localized to the cytoplasm while monomers were associated to the membranes of the endoplasmic reticulum and Golgi. We propose that the oligomeric state of PAFAH-II drives functional protein trafficking. PAFAH-II localization to the membrane is critical for substrate acquisition and effective oxidative stress protection. It is hypothesized that the balance between monomer and dimer serves as a regulatory mechanism of a PAFAH-II oxidative stress response. Copyright © 2015 Elsevier B.V. All rights reserved.

The Lectin Pathway of Complement and Rheumatic Heart Disease

PubMed Central

Beltrame, Marcia Holsbach; Catarino, Sandra Jeremias; Goeldner, Isabela; Boldt, Angelica Beate Winter; de Messias-Reason, Iara José

2014-01-01

The innate immune system is the first line of host defense against infection and is comprised of humoral and cellular mechanisms that recognize potential pathogens within minutes or hours of entry. The effector components of innate immunity include epithelial barriers, phagocytes, and natural killer cells, as well as cytokines and the complement system. Complement plays an important role in the immediate response against microorganisms, including Streptococcus sp. The lectin pathway is one of three pathways by which the complement system can be activated. This pathway is initiated by the binding of mannose-binding lectin (MBL), collectin 11 (CL-K1), and ficolins (Ficolin-1, Ficolin-2, and Ficolin-3) to microbial surface oligosaccharides and acetylated residues, respectively. Upon binding to target molecules, MBL, CL-K1, and ficolins form complexes with MBL-associated serine proteases 1 and 2 (MASP-1 and MASP-2), which cleave C4 and C2 forming the C3 convertase (C4b2a). Subsequent activation of complement cascade leads to opsonization, phagocytosis, and lysis of target microorganisms through the formation of the membrane-attack complex. In addition, activation of complement may induce several inflammatory effects, such as expression of adhesion molecules, chemotaxis and activation of leukocytes, release of reactive oxygen species, and secretion of cytokines and chemokines. In this chapter, we review the general aspects of the structure, function, and genetic polymorphism of lectin-pathway components and discuss most recent understanding on the role of the lectin pathway in the predisposition and clinical progression of Rheumatic Fever. PMID:25654073

Prolactin Regulatory Element Binding Protein Is Involved in Hepatitis C Virus Replication by Interaction with NS4B

PubMed Central

Kong, Lingbao; Fujimoto, Akira; Nakamura, Mariko; Aoyagi, Haruyo; Matsuda, Mami; Watashi, Koichi; Suzuki, Ryosuke; Arita, Minetaro; Yamagoe, Satoshi; Dohmae, Naoshi; Suzuki, Takehiro; Sakamaki, Yuriko; Ichinose, Shizuko; Suzuki, Tetsuro; Wakita, Takaji

2016-01-01

ABSTRACT It has been proposed that the hepatitis C virus (HCV) NS4B protein triggers the membranous HCV replication compartment, but the underlying molecular mechanism is not fully understood. Here, we screened for NS4B-associated membrane proteins by tandem affinity purification and proteome analysis and identified 202 host proteins. Subsequent screening of replicon cells with small interfering RNA identified prolactin regulatory element binding (PREB) to be a novel HCV host cofactor. The interaction between PREB and NS4B was confirmed by immunoprecipitation, immunofluorescence, and proximity ligation assays. PREB colocalized with double-stranded RNA and the newly synthesized HCV RNA labeled with bromouridine triphosphate in HCV replicon cells. Furthermore, PREB shifted to detergent-resistant membranes (DRMs), where HCV replication complexes reside, in the presence of NS4B expression in Huh7 cells. However, a PREB mutant lacking the NS4B-binding region (PREBd3) could not colocalize with double-stranded RNA and did not shift to the DRM in the presence of NS4B. These results indicate that PREB locates at the HCV replication complex by interacting with NS4B. PREB silencing inhibited the formation of the membranous HCV replication compartment and increased the protease and nuclease sensitivity of HCV replicase proteins and RNA in DRMs, respectively. Collectively, these data indicate that PREB promotes HCV RNA replication by participating in the formation of the membranous replication compartment and by maintaining its proper structure by interacting with NS4B. Furthermore, PREB was induced by HCV infection in vitro and in vivo. Our findings provide new insights into HCV host cofactors. IMPORTANCE The hepatitis C virus (HCV) protein NS4B can induce alteration of the endoplasmic reticulum and the formation of a membranous web structure, which provides a platform for the HCV replication complex. The molecular mechanism by which NS4B induces the membranous HCV replication compartment is not understood. We screened for NS4B-associated membrane proteins by tandem affinity purification and proteome analysis, followed by screening with small interfering RNA. We identified prolactin regulatory element binding (PREB) to be a novel HCV host cofactor. PREB is induced by HCV infection and recruited into the replication complex by interaction with NS4B. Recruited PREB promotes HCV RNA replication by participating in the formation of the membranous HCV replication compartment. To our knowledge, the effect of NS4B-binding protein on the formation of the membranous HCV replication compartment is newly described in this report. Our findings are expected to provide new insights into HCV host cofactors. PMID:26739056

Mammalian synthetic biology for studying the cell.

PubMed

Mathur, Melina; Xiang, Joy S; Smolke, Christina D

2017-01-02

Synthetic biology is advancing the design of genetic devices that enable the study of cellular and molecular biology in mammalian cells. These genetic devices use diverse regulatory mechanisms to both examine cellular processes and achieve precise and dynamic control of cellular phenotype. Synthetic biology tools provide novel functionality to complement the examination of natural cell systems, including engineered molecules with specific activities and model systems that mimic complex regulatory processes. Continued development of quantitative standards and computational tools will expand capacities to probe cellular mechanisms with genetic devices to achieve a more comprehensive understanding of the cell. In this study, we review synthetic biology tools that are being applied to effectively investigate diverse cellular processes, regulatory networks, and multicellular interactions. We also discuss current challenges and future developments in the field that may transform the types of investigation possible in cell biology. © 2017 Mathur et al.

Golgi Glycosylation

PubMed Central

Stanley, Pamela

2011-01-01

Glycosylation is a very common modification of protein and lipid, and most glycosylation reactions occur in the Golgi. Although the transfer of initial sugar(s) to glycoproteins or glycolipids occurs in the ER or on the ER membrane, the subsequent addition of the many different sugars that make up a mature glycan is accomplished in the Golgi. Golgi membranes are studded with glycosyltransferases, glycosidases, and nucleotide sugar transporters arrayed in a generally ordered manner from the cis-Golgi to the trans-Golgi network (TGN), such that each activity is able to act on specific substrate(s) generated earlier in the pathway. The spectrum of glycosyltransferases and other activities that effect glycosylation may vary with cell type, and thus the final complement of glycans on glycoconjugates is variable. In addition, glycan synthesis is affected by Golgi pH, the integrity of Golgi peripheral membrane proteins, growth factor signaling, Golgi membrane dynamics, and cellular stress. Knowledge of Golgi glycosylation has fostered the development of assays to identify mechanisms of intracellular vesicular trafficking and facilitated glycosylation engineering of recombinant glycoproteins. PMID:21441588

Atox1 Contains Positive Residues That Mediate Membrane Association and Aid Subsequent Copper Loading

PubMed Central

Flores, Adrian G.; Unger, Vinzenz M.

2013-01-01

Copper chaperones bind intracellular copper and ensure proper trafficking to downstream targets via protein-protein interactions. In contrast to the mechanisms of copper binding and transfer to downstream targets, the mechanisms of initial copper loading of the chaperones are largely unknown. Here we demonstrate that antioxidant protein 1 (Atox1 in human cells), the principal cellular copper chaperone responsible for delivery of copper to the secretory pathway, possesses the ability to interact with negatively charged lipid headgroups via distinct surface lysine residues. Moreover, loss of these residues lowers the efficiency of copper loading of Atox1 in vivo, suggesting that the membrane may play a scaffolding role in copper distribution to Atox1. These findings complement the recent discovery that the membrane also facilitates copper loading of the copper chaperone for superoxide dismutase 1 and provide further support for the emerging paradigm that the membrane bilayer plays a central role in cellular copper acquisition and distribution. PMID:24036897

Atox1 contains positive residues that mediate membrane association and aid subsequent copper loading.

PubMed

Flores, Adrian G; Unger, Vinzenz M

2013-12-01

Copper chaperones bind intracellular copper and ensure proper trafficking to downstream targets via protein-protein interactions. In contrast to the mechanisms of copper binding and transfer to downstream targets, the mechanisms of initial copper loading of the chaperones are largely unknown. Here, we demonstrate that antioxidant protein 1 (Atox1 in human cells), the principal cellular copper chaperone responsible for delivery of copper to the secretory pathway, possesses the ability to interact with negatively charged lipid headgroups via distinct surface lysine residues. Moreover, loss of these residues lowers the efficiency of copper loading of Atox1 in vivo, suggesting that the membrane may play a scaffolding role in copper distribution to Atox1. These findings complement the recent discovery that the membrane also facilitates copper loading of the copper chaperone for superoxide dismutase 1 and provide further support for the emerging paradigm that the membrane bilayer plays a central role in cellular copper acquisition and distribution.

Characterization of a monoclonal antibody against P57, the C3/C3b-cleaving proteinase expressed in human erythrocyte membranes.

PubMed

Fiandino-Tirel, A; Barel, M; Lyamani, F; Gauffre, A; Hermann, J; Frade, R

1991-08-01

A monoclonal antibody was raised against p57, a serine proteinase, characterized by an apparent molecular weight of 57 kDa, and purified from human erythrocyte membranes. P57 proteinase cleaves the human third component of complement, C3. The antibody selected, MP1, of IgG2a isotype, precipitated specifically the p57 antigen which carried the C3/C3b-cleaving activity present in membrane crude extract of human erythrocytes. P57 proteinase eluted from MP1-sepharose was inhibited by 5 x 10(-4) M PMSF, enhanced by 0.5% SDS and generated C3 fragments identical to those generated by membrane crude extract of human erythrocytes. All these properties were identical to those of the p57 previously purified by biochemical procedures. In addition, 5000 binding sites were detected on cell surface. This MP1 monoclonal antibody will be helpful to analyse the role of p57 in human erythrocytes.

δ-COP contains a helix C-terminal to its longin domain key to COPI dynamics and function

PubMed Central

Arakel, Eric C.; Richter, Kora P.; Clancy, Anne; Schwappach, Blanche

2016-01-01

Membrane recruitment of coatomer and formation of coat protein I (COPI)-coated vesicles is crucial to homeostasis in the early secretory pathway. The conformational dynamics of COPI during cargo capture and vesicle formation is incompletely understood. By scanning the length of δ-COP via functional complementation in yeast, we dissect the domains of the δ-COP subunit. We show that the μ-homology domain is dispensable for COPI function in the early secretory pathway, whereas the N-terminal longin domain is essential. We map a previously uncharacterized helix, C-terminal to the longin domain, that is specifically required for the retrieval of HDEL-bearing endoplasmic reticulum-luminal residents. It is positionally analogous to an unstructured linker that becomes helical and membrane-facing in the open form of the AP2 clathrin adaptor complex. Based on the amphipathic nature of the critical helix it may probe the membrane for lipid packing defects or mediate interaction with cargo and thus contribute to stabilizing membrane-associated coatomer. PMID:27298352

Complementation of a red-light-indifferent cyanobacterial mutant.

PubMed Central

Chiang, G G; Schaefer, M R; Grossman, A R

1992-01-01

Many cyanobacteria alter their phycobilisome composition in response to changes in light wavelength in a process termed complementary chromatic adaptation. Mutant strains FdR1 and FdR2 of the filamentous cyanobacterium Fremyella diplosiphon are characterized by aberrant chromatic adaptation. Instead of adjusting to different wavelengths of light, FdR1 and FdR2 behave as if they are always in green light; they do not respond to red light. We have previously reported complementation of FdR1 by conjugal transfer of a wild-type genomic library. The complementing DNA has now been localized by genetic analysis to a region on the rescued genomic subclone that contains a gene designated rcaC. This region of DNA is also able to complement FdR2. Southern blot analysis of genomic DNA from FdR1 and FdR2 indicates that these strains harbor DNA insertions within the rcaC sequence that may have resulted from the activity of transposable genetic elements. The predicted amino acid sequence of RcaC shares strong identity to response regulators of bacterial two-component regulatory systems. This relationship is discussed in the context of the signal-transduction pathway mediating regulation of genes encoding phycobilisome polypeptides during chromatic adaptation. Images PMID:1409650

Phenotypic characterization of ten methanol oxidation (Mox) mutant classes in methylobacterium AM1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nunn, D.N.; Lidstrom, M.E.

Twenty-five methanol oxidation mutants of the facultative methylotroph Methylobacterium strain AM1 have been characterized by complementation analysis and assigned to ten complementation groups, Mox A1,A2,A3 and B-H. We have characterized each of the mutants belonging to the ten Mox complementation groups by PMS-DCPIP dye linked methanol dehydrogenase activity, by methanol-dependent whole cell oxygen consumption, by the presence or absence of methanol dehydrogenase protein by SDS-polyacrylamide gels and Western blotting, by the absorption spectra of purified mutant methanol dehydrogenase proteins and by the presence or absence of the soluble cytochrome c proteins of Methylobacterium AM1. We propose functions for each ofmore » the genes deficient in the mutants of the ten Mox complementation groups. These functions include two linked genes that encode the methanol dehydrogenase structural protein and the soluble cytochrome c/sub L/, a gene encoding a secretion function essential for the synthesis and export of methanol dehydrogenase and cytochrome c/sub L/, three gene functions responsible for the proper association of the PQQ prosthetic group with the methanol dehydrogenase apoprotein and four positive regulatory gene functions controlling the expression of the ability to oxidize methanol. 24 refs., 5 figs., 2 tabs.« less

Evolutionary conservation of regulatory elements in vertebrate HOX gene clusters

DOE Office of Scientific and Technical Information (OSTI.GOV)

Santini, Simona; Boore, Jeffrey L.; Meyer, Axel

2003-12-31

Due to their high degree of conservation, comparisons of DNA sequences among evolutionarily distantly-related genomes permit to identify functional regions in noncoding DNA. Hox genes are optimal candidate sequences for comparative genome analyses, because they are extremely conserved in vertebrates and occur in clusters. We aligned (Pipmaker) the nucleotide sequences of HoxA clusters of tilapia, pufferfish, striped bass, zebrafish, horn shark, human and mouse (over 500 million years of evolutionary distance). We identified several highly conserved intergenic sequences, likely to be important in gene regulation. Only a few of these putative regulatory elements have been previously described as being involvedmore » in the regulation of Hox genes, while several others are new elements that might have regulatory functions. The majority of these newly identified putative regulatory elements contain short fragments that are almost completely conserved and are identical to known binding sites for regulatory proteins (Transfac). The conserved intergenic regions located between the most rostrally expressed genes in the developing embryo are longer and better retained through evolution. We document that presumed regulatory sequences are retained differentially in either A or A clusters resulting from a genome duplication in the fish lineage. This observation supports both the hypothesis that the conserved elements are involved in gene regulation and the Duplication-Deletion-Complementation model.« less

IFN-α and CD46 stimulation are associated with active lupus and skew natural T regulatory cell differentiation to type 1 regulatory T (Tr1) cells

PubMed Central

Le Buanec, Hélène; Gougeon, Marie-Lise; Mathian, Alexis; Lebon, Pierre; Dupont, Jean-Michel; Peltre, Gabriel; Hemon, Patrice; Schmid, Michel; Bizzini, Bernard; Künding, Thomas; Burny, Arsène; Bensussan, Armand; Amoura, Zahir; Gallo, Robert C.; Zagury, Daniel

2011-01-01

Immune suppressive activities exerted by regulatory T-cell subsets have several specific functions, including self-tolerance and regulation of adaptive immune reactions, and their dysfunction can lead to autoimmune diseases and contribute to AIDS and cancer. Two functionally distinct regulatory T-cell subsets are currently identified in peripheral tissues: thymus-developed natural T regulatory cells (nTregs) controlling self-tolerance and antiinflammatory IL-10–secreting type 1 regulatory T cells (Tr1) derived from Ag-stimulated T cells, which regulate inflammation-dependent adaptive immunity and minimize immunopathology. We establish herein that cell contact-mediated nTreg regulatory function is inhibited by inflammation, especially in the presence of the complement C3b receptor (CD46). Instead, as with other T-cell subsets, the latter inflammatory conditions of stimulation skew nTreg differentiation to Tr1 cells secreting IL-10, an effect potentiated by IFN-α. The clinical relevance of these findings was verified in a study of 152 lupus patients, in which we showed that lupus nTreg dysfunction is not due to intrinsic defects but is rather induced by C3b stimulation of CD46 and IFN-α and that these immune components of inflammation are directly associated with active lupus. These results provide a rationale for using anti–IFN-α Ab immunotherapy in lupus patients. PMID:22065791

A small diffusible signal molecule is responsible for the global control of virulence and exoenzyme production in the plant pathogen Erwinia carotovora.

PubMed Central

Pirhonen, M; Flego, D; Heikinheimo, R; Palva, E T

1993-01-01

Virulence of the plant pathogen Erwinia carotovora subsp. carotovora is dependent on the production and secretion of a complex arsenal of plant cell wall-degrading enzymes. Production of these exoenzymes is controlled by a global regulatory mechanism. A virulent mutants in one of the regulatory loci, expI, show a pleiotropic defect in the growth phase-dependent transcriptional activation of exoenzyme gene expression. The expI gene encodes a 26 kDa polypeptide that is structurally and functionally related to the luxI gene product of Vibrio fischeri. Functional similarity of expI and luxI has been demonstrated by reciprocal genetic complementation experiments. LuxI controls bioluminescence in V.fischeri in a growth phase-dependent manner by directing the synthesis of the diffusible autoinducer, N-(3-oxohexanoyl) homoserine lactone. E.c. subsp. carotovora expI+ strains or Escherichia coli harboring the cloned expI gene excrete a small diffusible signal molecule that complements the expI mutation of Erwinia as well as a luxI mutation of V.fischeri. This extracellular complementation can also be achieved by E.coli harboring the luxI gene from V.fischeri or by adding the synthetic V.fischeri autoinducer. Both the production of the plant tissue-macerating exoenzymes and the ability of the bacteria to propagate in planta are restored in expI mutants by autoinducer addition. These data suggest that the same signal molecule is employed in control of such diverse processes as virulence in a plant pathogen and bioluminescence in a marine bacterium, and may represent a general mechanism by which bacteria modulate gene expression in response to changing environmental conditions. Images PMID:8508772

A small diffusible signal molecule is responsible for the global control of virulence and exoenzyme production in the plant pathogen Erwinia carotovora.

PubMed

Pirhonen, M; Flego, D; Heikinheimo, R; Palva, E T

1993-06-01

Virulence of the plant pathogen Erwinia carotovora subsp. carotovora is dependent on the production and secretion of a complex arsenal of plant cell wall-degrading enzymes. Production of these exoenzymes is controlled by a global regulatory mechanism. A virulent mutants in one of the regulatory loci, expI, show a pleiotropic defect in the growth phase-dependent transcriptional activation of exoenzyme gene expression. The expI gene encodes a 26 kDa polypeptide that is structurally and functionally related to the luxI gene product of Vibrio fischeri. Functional similarity of expI and luxI has been demonstrated by reciprocal genetic complementation experiments. LuxI controls bioluminescence in V.fischeri in a growth phase-dependent manner by directing the synthesis of the diffusible autoinducer, N-(3-oxohexanoyl) homoserine lactone. E.c. subsp. carotovora expI+ strains or Escherichia coli harboring the cloned expI gene excrete a small diffusible signal molecule that complements the expI mutation of Erwinia as well as a luxI mutation of V.fischeri. This extracellular complementation can also be achieved by E.coli harboring the luxI gene from V.fischeri or by adding the synthetic V.fischeri autoinducer. Both the production of the plant tissue-macerating exoenzymes and the ability of the bacteria to propagate in planta are restored in expI mutants by autoinducer addition. These data suggest that the same signal molecule is employed in control of such diverse processes as virulence in a plant pathogen and bioluminescence in a marine bacterium, and may represent a general mechanism by which bacteria modulate gene expression in response to changing environmental conditions.

Upregulation of CD59

PubMed Central

Griesemer, Adam D.; Okumi, Masayoshi; Shimizu, Akira; Moran, Shannon; Ishikawa, Yoshinori; Iorio, Justin; Arn, J. Scott; Yamada, Kazuhiko

2009-01-01

Background Survival of ABO-mismatched kidneys with stable renal function despite the persistence of anti-ABO antibodies is called accommodation. The mechanism of accommodation is unclear, but may involve complement regulatory proteins such as CD59. The development of alpha-1,3-Galactosyltransferase knock-out (GalT-KO) swine that produce anti-Gal antibodies provides a large animal model capable of determining the role of complement regulatory proteins in accommodation. Methods ELISA and antibody FACS were used to examine the rate of anti-Gal antibody expression as a function of age. MHC-matched kidneys were transplanted from Gal-positive MGH miniature swine to MGH GalT-KO swine with systemic immunosuppression. One recipient underwent adsorbtion of anti-Gal antibodies prior to transplantation. Graft survival, antibody and complement deposition patterns and CD59 expression were determined. Results Three animals rejected Gal-positive kidneys via humoral mechanisms. One animal with low titers of anti-Gal Ab displayed spontaneous accommodation and the animal that was treated with Ab adsorbtion also displayed accommodation. Rejected grafts had deposition of IgM, IgG, C3 and C5b-9 with low expression of CD59, while accommodated grafts had low deposition of C5b-9 and high expression of CD59. Re-transplantation of one accommodated graft to a naïve GalT-KO animal confirmed that changes in the graft were responsible for the lack of C5b-9 deposition. Conclusion GalT-KO miniature swine produce anti-Gal antibodies and titers increase with age. These anti-Gal antibodies can cause rejection of MHC matched kidneys unless accommodation occurs. CD59 upregulation appears to be involved in the mechanism of accommodation by preventing the formation of the MAC on the accommodated graft. PMID:19424030

[In vitro function of outer membrane protease T of Escherichia coli K1 pathogenic strain].

PubMed

Hui, Changye; Guo, Yan; Wu, Shuchi; Peng, Liang; Cao, Hong; Huang, Shenghe

2010-01-01

Plasminogen activation and antimicrobial peptide hydrolysis contribute to pathogens invasion and survival in vivo. To demonstrate the expression of outer membrane protease T in E. coli K1 pathogenic strain E44, its activity of plasminogen activator and protamine hydrolysis. After Benzamidine Sepharose Fast Flow and SOURCE 30Q chromatography, we got E44 outer membrane mixed fraction, and examined its activity of plasminogen activation with chromogenic substrate S-2251 method. An ompT deletion mutant of E44 was constructed by using the suicide vector pCVD442, termed as E44ompT. We examined 0.1 mg/mL cationic antimicrobial peptide protamine susceptibility of E44, ompT mutant strain E44ompT and E44ompT harboring pUCT, which was constructed by inserting complete ompT open reading frame into pUC13. We got about 37 kDa E44 membrane extract, which could activate plasminogen, and activation was membrane extract dose dependent. This confirmed the expression of outer membrane protease T in the outer membrane of E44. E44ompT was, more susceptible to 0.1 mg/mL protamine than E44, and E440mpT was partially complemented by pUCT. Outer membrane protease T is expressed in E. coli K1 pathogenic strain E44, and can activate plasminogen and hydrolyze protamine.

Ammonium-sensitive protein kinase activity in plasma membranes of the cyanobacterium Anacystis nidulans.

PubMed

Rodríguez, R; García-González, M; Guerrero, M G; Lara, C

1994-08-15

Cytoplasmic membranes prepared from nitrate-grown Anacystis nidulans cells exhibit a Mg(2+)-dependent protein kinase activity able to phosphorylate in vitro plasma membrane polypeptides with molecular masses of 98, 93, 83, 47, 44 and 31 kDa. The protein kinase activity was inhibited in cytoplasmic membrane preparations from nitrate-grown cells which had been exposed to ammonium for 5 min. Parallely, ammonium exposure also resulted in a more than two-fold activation of an alkaline phosphatase activity present in the soluble fraction. These results are discussed in relation to the well-known inhibition by ammonium of nitrate transport activity, and a hypothesis for the regulatory mechanism involved is presented.

Opposing Effects of cAMP and T259 Phosphorylation on Plasma Membrane Diffusion of the Water Channel Aquaporin-5 in Madin-Darby Canine Kidney Cells

PubMed Central

Koffman, Jennifer S.; Arnspang, Eva C.; Marlar, Saw; Nejsum, Lene N.

2015-01-01

Aquaporin-5 (AQP5) facilitates passive water transport in glandular epithelia in response to secretory stimuli via intracellular pathways involving calcium release, cAMP and protein kinase A (PKA). In epithelial plasma membranes, AQP5 may be acutely regulated to facilitate water transport in response to physiological stimuli by changes in protein modifications, interactions with proteins and lipids, nanoscale membrane domain organization, and turnover rates. Such regulatory mechanisms could potentially be associated with alteration of diffusion behavior, possibly resulting in a change in the plasma membrane diffusion coefficient of AQP5. We aimed to test the short-term regulatory effects of the above pathways, by measuring lateral diffusion of AQP5 and an AQP5 phospho-mutant, T259A, using k-space Image Correlation Spectroscopy of quantum dot- and EGFP-labeled AQP5. Elevated cAMP and PKA inhibition significantly decreased lateral diffusion of AQP5, whereas T259A mutation showed opposing effects; slowing diffusion without stimulation and increasing diffusion to basal levels after cAMP elevation. Thus, lateral diffusion of AQP5 is significantly regulated by cAMP, PKA, and T259 phosphorylation, which could be important for regulating water flow in glandular secretions. PMID:26218429

GARP (LRRC32) is essential for the surface expression of latent TGF-beta on platelets and activated FOXP3+ regulatory T cells.

PubMed

Tran, Dat Q; Andersson, John; Wang, Rui; Ramsey, Heather; Unutmaz, Derya; Shevach, Ethan M

2009-08-11

TGF-beta family members are highly pleiotropic cytokines with diverse regulatory functions. TGF-beta is normally found in the latent form associated with latency-associated peptide (LAP). This latent complex can associate with latent TGFbeta-binding protein (LTBP) to produce a large latent form. Latent TGF-beta is also found on the surface of activated FOXP3(+) regulatory T cells (Tregs), but it is unclear how it is anchored to the cell membrane. We show that GARP or LRRC32, a leucine-rich repeat molecule of unknown function, is critical for tethering TGF-beta to the cell surface. We demonstrate that platelets and activated Tregs co-express latent TGF-beta and GARP on their membranes. The knockdown of GARP mRNA with siRNA prevented surface latent TGF-beta expression on activated Tregs and recombinant latent TGF-beta1 is able to bind directly with GARP. Confocal microscopy and immunoprecipitation strongly support their interactions. The role of TGF-beta on Tregs appears to have dual functions, both for Treg-mediated suppression and infectious tolerance mechanism.

GARP (LRRC32) is essential for the surface expression of latent TGF-β on platelets and activated FOXP3+ regulatory T cells

PubMed Central

Tran, Dat Q.; Andersson, John; Wang, Rui; Ramsey, Heather; Unutmaz, Derya; Shevach, Ethan M.

2009-01-01

TGF-β family members are highly pleiotropic cytokines with diverse regulatory functions. TGF-β is normally found in the latent form associated with latency-associated peptide (LAP). This latent complex can associate with latent TGFβ-binding protein (LTBP) to produce a large latent form. Latent TGF-β is also found on the surface of activated FOXP3+ regulatory T cells (Tregs), but it is unclear how it is anchored to the cell membrane. We show that GARP or LRRC32, a leucine-rich repeat molecule of unknown function, is critical for tethering TGF-β to the cell surface. We demonstrate that platelets and activated Tregs co-express latent TGF-β and GARP on their membranes. The knockdown of GARP mRNA with siRNA prevented surface latent TGF-β expression on activated Tregs and recombinant latent TGF-β1 is able to bind directly with GARP. Confocal microscopy and immunoprecipitation strongly support their interactions. The role of TGF-β on Tregs appears to have dual functions, both for Treg-mediated suppression and infectious tolerance mechanism. PMID:19651619

Staphylococcus aureus SdrE captures complement factor H's C-terminus via a novel ‘close, dock, lock and latch' mechanism for complement evasion

PubMed Central

Zhang, Yingjie; Wu, Minhao; Hang, Tianrong; Wang, Chengliang; Yang, Ye; Pan, Weimin; Zang, Jianye

2017-01-01

Complement factor H (CFH) is a soluble complement regulatory protein essential for the down-regulation of the alternative pathway on interaction with specific markers on the host cell surface. It recognizes the complement component 3b (C3b) and 3d (C3d) fragments in addition to self cell markers (i.e. glycosaminoglycans, sialic acid) to distinguish host cells that deserve protection from pathogens that should be eliminated. The Staphylococcus aureus surface protein serine–aspartate repeat protein E (SdrE) was previously reported to bind human CFH as an immune-evasion tactic. However, the molecular mechanism underlying SdrE–CFH-mediated immune evasion remains unknown. In the present study, we identified a novel region at CFH's C-terminus (CFH1206–1226), which binds SdrE N2 and N3 domains (SdrEN2N3) with high affinity, and determined the crystal structures of apo-SdrEN2N3 and the SdrEN2N3–CFH1206–1226 complex. Comparison of the structure of the CFH–SdrE complex with other CFH structures reveals that CFH's C-terminal tail flips from the main body to insert into the ligand-binding groove of SdrE. In addition, SdrEN2N3 adopts a ‘close’ state in the absence of CFH, which undergoes a large conformational change on CFH binding, suggesting a novel ‘close, dock, lock and latch' (CDLL) mechanism for SdrE to recognize its ligand. Our findings imply that SdrE functions as a ‘clamp' to capture CFH's C-terminal tail via a unique CDLL mechanism and sequesters CFH on the surface of S. aureus for complement evasion. PMID:28258151

Staphylococcus aureus SdrE captures complement factor H's C-terminus via a novel 'close, dock, lock and latch' mechanism for complement evasion.

PubMed

Zhang, Yingjie; Wu, Minhao; Hang, Tianrong; Wang, Chengliang; Yang, Ye; Pan, Weimin; Zang, Jianye; Zhang, Min; Zhang, Xuan

2017-05-04

Complement factor H (CFH) is a soluble complement regulatory protein essential for the down-regulation of the alternative pathway on interaction with specific markers on the host cell surface. It recognizes the complement component 3b (C3b) and 3d (C3d) fragments in addition to self cell markers (i.e. glycosaminoglycans, sialic acid) to distinguish host cells that deserve protection from pathogens that should be eliminated. The Staphylococcus aureus surface protein serine-aspartate repeat protein E (SdrE) was previously reported to bind human CFH as an immune-evasion tactic. However, the molecular mechanism underlying SdrE-CFH-mediated immune evasion remains unknown. In the present study, we identified a novel region at CFH's C-terminus (CFH 1206-1226 ), which binds SdrE N2 and N3 domains (SdrE N2N3 ) with high affinity, and determined the crystal structures of apo-SdrE N2N3 and the SdrE N2N3 -CFH 1206-1226 complex. Comparison of the structure of the CFH-SdrE complex with other CFH structures reveals that CFH's C-terminal tail flips from the main body to insert into the ligand-binding groove of SdrE. In addition, SdrE N2N3 adopts a 'close' state in the absence of CFH, which undergoes a large conformational change on CFH binding, suggesting a novel 'close, dock, lock and latch' (CDLL) mechanism for SdrE to recognize its ligand. Our findings imply that SdrE functions as a 'clamp' to capture CFH's C-terminal tail via a unique CDLL mechanism and sequesters CFH on the surface of S. aureus for complement evasion. © 2017 The Author(s).

Structural analysis of autoinhibition in the Ras-specific exchange factor RasGRP1

PubMed Central

Iwig, Jeffrey S; Vercoulen, Yvonne; Das, Rahul; Barros, Tiago; Limnander, Andre; Che, Yan; Pelton, Jeffrey G; Wemmer, David E; Roose, Jeroen P; Kuriyan, John

2013-01-01

RasGRP1 and SOS are Ras-specific nucleotide exchange factors that have distinct roles in lymphocyte development. RasGRP1 is important in some cancers and autoimmune diseases but, in contrast to SOS, its regulatory mechanisms are poorly understood. Activating signals lead to the membrane recruitment of RasGRP1 and Ras engagement, but it is unclear how interactions between RasGRP1 and Ras are suppressed in the absence of such signals. We present a crystal structure of a fragment of RasGRP1 in which the Ras-binding site is blocked by an interdomain linker and the membrane-interaction surface of RasGRP1 is hidden within a dimerization interface that may be stabilized by the C-terminal oligomerization domain. NMR data demonstrate that calcium binding to the regulatory module generates substantial conformational changes that are incompatible with the inactive assembly. These features allow RasGRP1 to be maintained in an inactive state that is poised for activation by calcium and membrane-localization signals. DOI: http://dx.doi.org/10.7554/eLife.00813.001 PMID:23908768

Complement mutations in diacylglycerol kinase-ε-associated atypical hemolytic uremic syndrome.

PubMed

Sánchez Chinchilla, Daniel; Pinto, Sheila; Hoppe, Bernd; Adragna, Marta; Lopez, Laura; Justa Roldan, Maria Luisa; Peña, Antonia; Lopez Trascasa, Margarita; Sánchez-Corral, Pilar; Rodríguez de Córdoba, Santiago

2014-09-05

Atypical hemolytic uremic syndrome is characterized by vascular endothelial damage caused by complement dysregulation. Consistently, complement inhibition therapies are highly effective in most patients with atypical hemolytic uremic syndrome. Recently, it was shown that a significant percentage of patients with early-onset atypical hemolytic uremic syndrome carry mutations in diacylglycerol kinase-ε, an intracellular protein with no obvious role in complement. These data support an alternative, complement-independent mechanism leading to thrombotic microangiopathy that has implications for treatment of early-onset atypical hemolytic uremic syndrome. To get additional insights into this new form of atypical hemolytic uremic syndrome, the diacylglycerol kinase-ε gene in a cohort with atypical hemolytic uremic syndrome was analyzed. Eighty-three patients with early-onset atypical hemolytic uremic syndrome (<2 years) enrolled in the Spanish atypical hemolytic uremic syndrome registry between 1999 and 2013 were screened for mutations in diacylglycerol kinase-ε. These patients were also fully characterized for mutations in the genes encoding factor H, membrane cofactor protein, factor I, C3, factor B, and thrombomodulin CFHRs copy number variations and rearrangements, and antifactor H antibodies. Four patients carried mutations in diacylglycerol kinase-ε, one p.H536Qfs*16 homozygote and three compound heterozygotes (p.W322*/p.P498R, two patients; p.Q248H/p.G484Gfs*10, one patient). Three patients also carried heterozygous mutations in thrombomodulin or C3. Extensive plasma infusions controlled atypical hemolytic uremic syndrome recurrences and prevented renal failure in the two patients with diacylglycerol kinase-ε and thrombomodulin mutations. A positive response to plasma infusions and complement inhibition treatment was also observed in the patient with concurrent diacylglycerol kinase-ε and C3 mutations. Data suggest that complement dysregulation influences the onset and disease severity in carriers of diacylglycerol kinase-ε mutations and that treatments on the basis of plasma infusions and complement inhibition are potentially useful in patients with combined diacylglycerol kinase-ε and complement mutations. A comprehensive understanding of the genetic component predisposing to atypical hemolytic uremic syndrome is, therefore, critical to guide an effective treatment. Copyright © 2014 by the American Society of Nephrology.

Complement Mutations in Diacylglycerol Kinase-ε–Associated Atypical Hemolytic Uremic Syndrome

PubMed Central

Sánchez Chinchilla, Daniel; Pinto, Sheila; Hoppe, Bernd; Adragna, Marta; Lopez, Laura; Justa Roldan, Maria Luisa; Peña, Antonia; Lopez Trascasa, Margarita; Sánchez-Corral, Pilar; Rodríguez de Córdoba, Santiago

2014-01-01

Background and objectives Atypical hemolytic uremic syndrome is characterized by vascular endothelial damage caused by complement dysregulation. Consistently, complement inhibition therapies are highly effective in most patients with atypical hemolytic uremic syndrome. Recently, it was shown that a significant percentage of patients with early-onset atypical hemolytic uremic syndrome carry mutations in diacylglycerol kinase-ε, an intracellular protein with no obvious role in complement. These data support an alternative, complement-independent mechanism leading to thrombotic microangiopathy that has implications for treatment of early-onset atypical hemolytic uremic syndrome. To get additional insights into this new form of atypical hemolytic uremic syndrome, the diacylglycerol kinase-ε gene in a cohort with atypical hemolytic uremic syndrome was analyzed. Design, setting, participants, & measurements Eighty-three patients with early-onset atypical hemolytic uremic syndrome (<2 years) enrolled in the Spanish atypical hemolytic uremic syndrome registry between 1999 and 2013 were screened for mutations in diacylglycerol kinase-ε. These patients were also fully characterized for mutations in the genes encoding factor H, membrane cofactor protein, factor I, C3, factor B, and thrombomodulin CFHRs copy number variations and rearrangements, and antifactor H antibodies. Results Four patients carried mutations in diacylglycerol kinase-ε, one p.H536Qfs*16 homozygote and three compound heterozygotes (p.W322*/p.P498R, two patients; p.Q248H/p.G484Gfs*10, one patient). Three patients also carried heterozygous mutations in thrombomodulin or C3. Extensive plasma infusions controlled atypical hemolytic uremic syndrome recurrences and prevented renal failure in the two patients with diacylglycerol kinase-ε and thrombomodulin mutations. A positive response to plasma infusions and complement inhibition treatment was also observed in the patient with concurrent diacylglycerol kinase-ε and C3 mutations. Conclusions Data suggest that complement dysregulation influences the onset and disease severity in carriers of diacylglycerol kinase-ε mutations and that treatments on the basis of plasma infusions and complement inhibition are potentially useful in patients with combined diacylglycerol kinase-ε and complement mutations. A comprehensive understanding of the genetic component predisposing to atypical hemolytic uremic syndrome is, therefore, critical to guide an effective treatment. PMID:25135762

Impact of dialyzer membrane on apoptosis and function of polymorphonuclear cells and cytokine synthesis by peripheral blood mononuclear cells in hemodialysis patients.

PubMed

Andreoli, Maria C C; Dalboni, Maria A; Watanabe, Renato; Manfredi, Silvia R; Canziani, Maria E F; Kallás, Esper G; Sesso, Ricardo C; Draibe, Sergio A; Balakrishnan, Vaidyanathapuram S; Jaber, Bertrand L; Liangos, Orfeas; Cendoroglo, Miguel

2007-12-01

In an in vivo crossover trial, we compared a cellulosic with a synthetic dialyzer with respect to polymorphonuclear cells (PMN) function and apoptosis, cytokine serum levels and synthesis by peripheral blood mononuclear cells (PBMC), and complement activation. Twenty hemodialysis (HD) patients were assigned in alternate order to HD with cellulose acetate (CA) or polysulfone (PS) dialyzer. After 2 weeks, patients were crossed over to the second dialyzer and treated for another 2 weeks. Apoptosis was assessed by flow cytometry in freshly isolated PMN. Phagocytosis and production of peroxide by PMN were studied by flow cytometry in whole blood. PBMC were isolated from blood samples and incubated for 24 h with or without lipopolysaccharide (LPS). There was no impact of dialyzer biocompatibility on PMN apoptosis and function, cytokine synthesis by PBMC or on their serum levels, serum levels of C3a, and terminal complement complex (TCC). Nevertheless, after HD, serum levels of complement correlated negatively with PMN phagocytosis and peroxide production, and positively with PMN apoptosis and cytokine production by PBMC. Although the results did not show a dialyzer advantage on the immunologic parameters, complement activation may have modulated cell function and apoptosis after HD.

Detection of antisperm antibodies: their localization to human sperm antigens that are transferred to the surface of zona-free hamster oocytes during the sperm penetration assay.

PubMed

Wiley, L M; Obasaju, M F; Overstreet, J W; Cross, N L; Hanson, F W; Chang, R J

1987-08-01

The authors have developed an extension of the sperm penetration assay for detecting serum immunoglobulins to sperm antigens that are transferred to the plasma membrane of a sperm-penetrated hamster oocyte. After the hamster oocytes have been scored for sperm penetration by observing for the presence of swollen sperm heads, they are incubated in serum followed by either a 20-minute treatment with rhodamine-conjugated protein A (which binds to most subclasses of IgA, IgG, and IgM) or a 2-hour incubation in guinea pig serum (complement). Positive fluorescence indicates that the serum contains antibodies to sperm antigens that were transferred to the surface of an oocyte during gamete fusion. Complement-mediated lysis indicates that the immunoglobulin that is bound can also fix complement. The advantages of this assay for detection of serum antisperm antibodies are that it is an extension of a widely used assay, is rapid and requires readily available reagents and equipment, can detect most subclasses of IgA, IgG, and IgM, detects antibodies to those sperm antigens that may be transferred to the oocyte during fertilization, and indicates whether the detected antisperm antibodies can mediate complement-dependent lysis of the fertilized oocyte.

Molecular and functional characterization of CD59 from Nile tilapia (Oreochromis niloticus) involved in the immune response to Streptococcus agalactiae.

PubMed

Gan, Zhen; Wang, Bei; Zhou, Wei; Lu, Yishan; Zhu, Weiwei; Tang, Jufen; Jian, JiChang; Wu, Zaohe

2015-05-01

CD59, the major inhibitor of membrane attack complex, plays a crucial role in regulation of complement activation. In this paper, a CD59 gene of Nile tilapia, Oreochromis niloticus (designated as On-CD59) was cloned and its expression pattern under the stimulation of Streptococcus agalactiae was investigated. Sequence analysis showed main structural features required for complement-inhibitory activity were detected in the deduced amino acid sequence of On-CD59. In healthy Nile tilapia, the On-CD59 transcripts could be detected in all the examined tissues, with the most abundant expression in the brain. When immunized with inactivated S. agalactiae, there was a clear time-dependent expression pattern of On-CD59 in the skin, brain, head kidney, thymus and spleen, with quite different kinetic expressions. The assays for the complement-inhibitory activity suggested that recombinant On-CD59 protein had a species-selective inhibition of complement. Moreover, our works showed that recombinant On-CD59 protein may possess both binding activities to PGN and LTA and inhibiting activity of S. agalactiae. These findings indicated that On-CD59 may play important roles in the immune response to S. agalactiae in Nile tilapia. Copyright © 2015 Elsevier Ltd. All rights reserved.

Mutants in the Candida glabrata Glycerol Channels Are Sensitized to Cell Wall Stress

PubMed Central

Beese-Sims, Sara E.; Pan, Shih-Jung; Lee, Jongmin; Hwang-Wong, Elizabeth; Cormack, Brendan P.

2012-01-01

Many fungal species use glycerol as a compatible solute with which to maintain osmotic homeostasis in response to changes in external osmolarity. In Saccharomyces cerevisiae, intracellular glycerol concentrations are regulated largely by the high osmolarity glycerol (HOG) response pathway, both through induction of glycerol biosynthesis and control of its flux through the plasma membrane Fps1 glycerol channel. The channel activity of Fps1 is also controlled by a pair of positive regulators, Rgc1 and Rgc2. In this study, we demonstrate that Candida glabrata, a fungal pathogen that possesses two Fps1 orthologs and two Rgc1/-2 orthologs, accumulates glycerol in response to hyperosmotic stress. We present an initial characterization of mutants with deletions in the C. glabrata FPS1 (CAGL0C03267 [www.candidagenome.org]) and FPS2 (CAGL0E03894) genes and find that a double mutant accumulates glycerol, experiences constitutive cell wall stress, and is hypersensitive to treatment by caspofungin, an antifungal agent that targets the cell wall. This mutant is cleared more efficiently in mouse infections than is wild-type C. glabrata by caspofungin treatment. Finally, we demonstrate that one of the C. glabrata RGC orthologs complements an S. cerevisiae rgc1 rgc2 null mutant, supporting the conclusion that this regulatory assembly is conserved between these species. PMID:23087370

Disruption of the nitrogen regulatory gene AcareA in Acremonium chrysogenum leads to reduction of cephalosporin production and repression of nitrogen metabolism.

PubMed

Li, Jinyang; Pan, Yuanyuan; Liu, Gang

2013-12-01

AcareA, encoding a homologue of the fungal nitrogen regulatory GATA zinc-finger proteins, was cloned from Acremonium chrysogenum. Gene disruption and genetic complementation revealed that AcareA was required for nitrogen metabolism and cephalosporin production. Disruption of AcareA resulted in growth defect in the medium using nitrate, uric acid and low concentration of ammonium, glutamine or urea as sole nitrogen source. Transcriptional analysis showed that the transcription of niaD/niiA was increased drastically when induced with nitrate in the wild-type and AcareA complemented strains but not in AcareA disruption mutant. Consistent with the reduction of cephalosporin production, the transcription of pcbAB, cefD2, cefEF and cefG encoding the enzymes for cephalosporin production was reduced in AcareA disruption mutant. Band shift assays showed that AcAREA bound to the promoter regions of niaD, niiA and the bidirectional promoter region of pcbAB-pcbC. Sequence analysis showed that all the AcAREA binding sites contain the consensus GATA elements. These results indicated that AcAREA plays an important role both in the regulation of nitrogen metabolism and cephalosporin production in A. chrysogenum. Copyright © 2013 Elsevier Inc. All rights reserved.

Sequence- and structure-based computational analyses of Gram-negative tripartite efflux pumps in the context of bacterial membranes

DOE PAGES

Travers, Timothy; Wang, Katherine J.; Lopez, Cesar A.; ...

2018-02-09

Gram-negative multidrug resistance currently presents a serious threat to public health with infections effectively rendered untreatable. Multiple molecular mechanisms exist that cause antibiotic resistance and in addition, the last three decades have seen slowing rates of new drug development. In this paper, we summarize the use of various computational techniques for investigating the mechanisms of multidrug resistance mediated by Gram-negative tripartite efflux pumps and membranes. Recent work in our lab combines data-driven sequence and structure analyses to study the interactions and dynamics of these bacterial components. Computational studies can complement experimental methodologies for gaining crucial insights into combatting multidrug resistance.

Sequence- and structure-based computational analyses of Gram-negative tripartite efflux pumps in the context of bacterial membranes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Travers, Timothy; Wang, Katherine J.; Lopez, Cesar A.

Gram-negative multidrug resistance currently presents a serious threat to public health with infections effectively rendered untreatable. Multiple molecular mechanisms exist that cause antibiotic resistance and in addition, the last three decades have seen slowing rates of new drug development. In this paper, we summarize the use of various computational techniques for investigating the mechanisms of multidrug resistance mediated by Gram-negative tripartite efflux pumps and membranes. Recent work in our lab combines data-driven sequence and structure analyses to study the interactions and dynamics of these bacterial components. Computational studies can complement experimental methodologies for gaining crucial insights into combatting multidrug resistance.

Electrochemical insights into the mechanism of NiFe membrane-bound hydrogenases

PubMed Central

Flanagan, Lindsey A.; Parkin, Alison

2016-01-01

Hydrogenases are enzymes of great biotechnological relevance because they catalyse the interconversion of H2, water (protons) and electricity using non-precious metal catalytic active sites. Electrochemical studies into the reactivity of NiFe membrane-bound hydrogenases (MBH) have provided a particularly detailed insight into the reactivity and mechanism of this group of enzymes. Significantly, the control centre for enabling O2 tolerance has been revealed as the electron-transfer relay of FeS clusters, rather than the NiFe bimetallic active site. The present review paper will discuss how electrochemistry results have complemented those obtained from structural and spectroscopic studies, to present a complete picture of our current understanding of NiFe MBH. PMID:26862221

Clinical characterization of a new polymeric membrane for use in renal replacement therapy.

PubMed

Hoenich, Nicholas A; Katopodis, Kostas P

2002-09-01

Renal replacement therapy makes extensive use of semi-permeable membranes, ideal requirements for such membranes are good solute transport characteristics and a low reactivity with blood. Membranes manufactured from synthetic polymers fulfil these requirements. Such membranes have asymmetric and anisotropic structures characterized by a dense layer with which the blood is in contact supported by a thicker solid structure with containing interlinked voids, providing support. The nature of the structures are critically dependent upon the polymer blend and the control of parameters during manufacture such as the temperature or additive concentrations. In this prospective study, we have evaluated the clinical performance of a new membrane manufactured from a blend of polyamide, polyarylethersulfone and polyvinylpyrrolidone (Polyflux, Gambro GmbH, Hechingen, Germany), and compared it with that of polysulfone blended with polyvinylpyrrolidone (Fresenius Polysulfone, Fresenius Medical Care, Bad Homburg, Germany), a material widely acknowledged as providing an optimal biocompatibility in terms of solute removal and complement activation. The clearance of small molecules (urea, creatinine, phosphate) for both membranes was comparable. Both membranes removed beta2 microglobulin during treatment (50.2% reduction with Polyflux and 54.5% reduction with polysulfone. This removal due to the non-selectivity of the membranes was associated with protein loss during therapy which was similar for both the membranes (7.7 g). The biocompatibility profiles of the membranes indicated slight neutropenia and platelet adhesion and minimal C3a, C5a and SC5b-9 generation which were independent of the membrane material. These findings indicate that despite the differences in microstructure of the membranes, their functional performance in the clinical setting is comparable.

Loss of the interferon-γ-inducible regulatory immunity-related GTPase (IRG), Irgm1, causes activation of effector IRG proteins on lysosomes, damaging lysosomal function and predicting the dramatic susceptibility of Irgm1-deficient mice to infection.

PubMed

Maric-Biresev, Jelena; Hunn, Julia P; Krut, Oleg; Helms, J Bernd; Martens, Sascha; Howard, Jonathan C

2016-04-20

The interferon-γ (IFN-γ)-inducible immunity-related GTPase (IRG), Irgm1, plays an essential role in restraining activation of the IRG pathogen resistance system. However, the loss of Irgm1 in mice also causes a dramatic but unexplained susceptibility phenotype upon infection with a variety of pathogens, including many not normally controlled by the IRG system. This phenotype is associated with lymphopenia, hemopoietic collapse, and death of the mouse. We show that the three regulatory IRG proteins (GMS sub-family), including Irgm1, each of which localizes to distinct sets of endocellular membranes, play an important role during the cellular response to IFN-γ, each protecting specific membranes from off-target activation of effector IRG proteins (GKS sub-family). In the absence of Irgm1, which is localized mainly at lysosomal and Golgi membranes, activated GKS proteins load onto lysosomes, and are associated with reduced lysosomal acidity and failure to process autophagosomes. Another GMS protein, Irgm3, is localized to endoplasmic reticulum (ER) membranes; in the Irgm3-deficient mouse, activated GKS proteins are found at the ER. The Irgm3-deficient mouse does not show the drastic phenotype of the Irgm1 mouse. In the Irgm1/Irgm3 double knock-out mouse, activated GKS proteins associate with lipid droplets, but not with lysosomes, and the Irgm1/Irgm3(-/-) does not have the generalized immunodeficiency phenotype expected from its Irgm1 deficiency. The membrane targeting properties of the three GMS proteins to specific endocellular membranes prevent accumulation of activated GKS protein effectors on the corresponding membranes and thus enable GKS proteins to distinguish organellar cellular membranes from the membranes of pathogen vacuoles. Our data suggest that the generalized lymphomyeloid collapse that occurs in Irgm1(-/-) mice upon infection with a variety of pathogens may be due to lysosomal damage caused by off-target activation of GKS proteins on lysosomal membranes and consequent failure of autophagosomal processing.

Unmasking of complements using proteinase-K in formalin fixed paraffin embedded renal biopsies.

PubMed

Nada, R; Kumar, A; Kumar, V G; Gupta, K L; Joshi, K

2016-01-01

Renal biopsy interpretation requires histopathology, direct immunofluorescence (DIF) and electron microscopy. Formalin-fixed, paraffin-embedded tissue (FFPE) sent for light microscopy can be used for DIF after antigen retrieval. However, complement staining has not been satisfactory. We standardized DIF using proteinase-K for antigen retrieval in FFPE renal biopsies. A pilot study was conducted on known cases of membranous glomerulonephritis (MGN), membranoproliferative type-1 (MPGN-1), immunoglobulin A nephropathy (IgAN), and anti-glomerular basement disease (anti-GBM). Immunofluorescence panel included fluorescein isothiocyanate (FITC) conjugated IgG, IgA, IgM, complements (C3 and C1q), light chains (kappa, lambda) and fibrinogen antibodies. After standardization of the technique, 75 renal biopsies and 43 autopsies cases were stained. Out of 43 autopsy cases, immune-complex mediated glomerulonephritis (GN) was confirmed in 18 cases (Lupus nephritis-11, IgAN-6, MGN-1), complement-mediated dense deposit disease (DDD-1) and monoclonal diseases in 4 cases (amyloidosis-3, cast nephropathy-1). Immune-mediated injury was excluded in 17 cases (focal segmental glomerulosclerosis -3, crescentic GN-6 [pauci-immune-3, anti-GBM-3], thrombotic microangiopathy-5, atherosclerosis-3). Renal biopsies (n-75) where inadequate or no frozen sample was available; this technique classified 52 mesangiocapillary pattern as MPGN type-1-46, DDD-2 and (C3GN-4). Others were diagnosed as IgAN-3, lupus nephritis-2, MGN-4, diffuse proliferative glomerulonephritis (DPGN)-1, Non-IC crescentic GN-1, monoclonal diseases-3. In nine cases, DIF on FFPE tissue could not help in making diagnosis. Proteinase-K enzymatic digestion of FFPE renal biopsies can unmask complements (both C3 and C1q) in immune-complexes mediated and complement-mediated diseases. This method showed good results on autopsy tissues archived for as long as 15 years.

Marked central nervous system pathology in CD59 knockout rats following passive transfer of Neuromyelitis optica immunoglobulin G.

PubMed

Yao, Xiaoming; Verkman, Alan S

2017-02-17

Neuromyelitis optica spectrum disorders (herein called NMO) is an inflammatory demyelinating disease of the central nervous system in which pathogenesis involves complement-dependent cytotoxicity (CDC) produced by immunoglobulin G autoantibodies targeting aquaporin-4 (AQP4-IgG) on astrocytes. We reported evidence previously, using CD59 -/- mice, that the membrane-associated complement inhibitor CD59 modulates CDC in NMO (Zhang and Verkman, J. Autoimmun. 53:67-77, 2014). Motivated by the observation that rats, unlike mice, have human-like complement activity, here we generated CD59 -/- rats to investigate the role of CD59 in NMO and to create NMO pathology by passive transfer of AQP4-IgG under conditions in which minimal pathology is produced in normal rats. CD59 -/- rats generated by CRISPR/Cas9 technology showed no overt phenotype at baseline except for mild hemolysis. CDC assays in astrocyte cultures and cerebellar slices from CD59 -/- rats showed much greater sensitivity to AQP4-IgG and complement than those from CD59 +/+ rats. Intracerebral administration of AQP4-IgG in CD59 -/- rats produced marked NMO pathology, with astrocytopathy, inflammation, deposition of activated complement, and demyelination, whereas identically treated CD59 +/+ rats showed minimal pathology. A single, intracisternal injection of AQP4-IgG in CD59 -/- rats produced hindlimb paralysis by 3 days, with inflammation and deposition of activated complement in spinal cord, optic nerves and brain periventricular and surface matter, with most marked astrocyte injury in cervical spinal cord. These results implicate an important role of CD59 in modulating NMO pathology in rats and demonstrate amplification of AQP4-IgG-induced NMO disease with CD59 knockout.

Functional complementation between a novel mammalian polygenic transport complex and an evolutionarily ancient organic solute transporter, OSTalpha-OSTbeta.

PubMed

Seward, David J; Koh, Albert S; Boyer, James L; Ballatori, Nazzareno

2003-07-25

These studies identify an organic solute transporter (OST) that is generated when two novel gene products are co-expressed, namely human OSTalpha and OSTbeta or mouse OSTalpha and OSTbeta. The results also demonstrate that the mammalian proteins are functionally complemented by evolutionarily divergent Ostalpha-Ostbeta proteins recently identified in the little skate, Raja erinacea, even though the latter exhibit only 25-41% predicted amino acid identity with the mammalian proteins. Human, mouse, and skate OSTalpha proteins are predicted to contain seven transmembrane helices, whereas the OSTbeta sequences are predicted to have a single transmembrane helix. Human OSTalpha-OSTbeta and mouse Ostalpha-Ostbeta cDNAs were cloned from liver mRNA, sequenced, expressed in Xenopus laevis oocytes, and tested for their ability to functionally complement the corresponding skate proteins by measuring transport of [3H]estrone 3-sulfate. None of the proteins elicited a transport signal when expressed individually in oocytes; however, all nine OSTalpha-OSTbeta combinations (i.e. OSTalpha-OSTbeta pairs from human, mouse, or skate) generated robust estrone 3-sulfate transport activity. Transport was sodium-independent, saturable, and inhibited by other steroids and anionic drugs. Human and mouse OSTalpha-OSTbeta also were able to mediate transport of taurocholate, digoxin, and prostaglandin E2 but not of estradiol 17beta-d-glucuronide or p-aminohippurate. OSTalpha and OSTbeta were able to reach the oocyte plasma membrane when expressed either individually or in pairs, indicating that co-expression is not required for proper membrane targeting. Interestingly, OSTalpha and OSTbeta mRNAs were highly expressed and widely distributed in human tissues, with the highest levels occurring in the testis, colon, liver, small intestine, kidney, ovary, and adrenal gland.

The Hepatitis C Virus NS4B Protein Can trans-Complement Viral RNA Replication and Modulates Production of Infectious Virus▿

PubMed Central

Jones, Daniel M.; Patel, Arvind H.; Targett-Adams, Paul; McLauchlan, John

2009-01-01

Studies of the hepatitis C virus (HCV) life cycle have been aided by development of in vitro systems that enable replication of viral RNA and production of infectious virus. However, the functions of the individual proteins, especially those engaged in RNA replication, remain poorly understood. It is considered that NS4B, one of the replicase components, creates sites for genome synthesis, which appear as punctate foci at the endoplasmic reticulum (ER) membrane. In this study, a panel of mutations in NS4B was generated to gain deeper insight into its functions. Our analysis identified five mutants that were incapable of supporting RNA replication, three of which had defects in production of foci at the ER membrane. These mutants also influenced posttranslational modification and intracellular mobility of another replicase protein, NS5A, suggesting that such characteristics are linked to focus formation by NS4B. From previous studies, NS4B could not be trans-complemented in replication assays. Using the mutants that blocked RNA synthesis, defective NS4B expressed from two mutants could be rescued in trans-complementation replication assays by wild-type protein produced by a functional HCV replicon. Moreover, active replication could be reconstituted by combining replicons that were defective in NS4B and NS5A. The ability to restore replication from inactive replicons has implications for our understanding of the mechanisms that direct viral RNA synthesis. Finally, one of the NS4B mutations increased the yield of infectious virus by five- to sixfold. Hence, NS4B not only functions in RNA replication but also contributes to the processes engaged in virus assembly and release. PMID:19073716

Membrane attack complex of complement is not essential for immune mediated demyelination in experimental autoimmune neuritis.

PubMed

Tran, Giang T; Hodgkinson, Suzanne J; Carter, Nicole M; Killingsworth, Murray; Nomura, Masaru; Verma, Nirupama D; Plain, Karren M; Boyd, Rochelle; Hall, Bruce M

2010-12-15

Antibody deposition and complement activation, especially membrane attack complex (MAC) formation are considered central for immune mediated demyelination. To examine the role of MAC in immune mediated demyelination, we studied experimental allergic neuritis (EAN) in Lewis rats deficient in complement component 6 (C6) that cannot form MAC. A C6 deficient Lewis (Lewis/C6-) strain of rats was bred by backcrossing the defective C6 gene, from PVG/C6- rats, onto the Lewis background. Lewis/C6- rats had the same C6 gene deletion as PVG/C6- rats and their sera did not support immune mediated haemolysis unless C6 was added. Active EAN was induced in Lewis and Lewis/C6- rats by immunization with bovine peripheral nerve myelin in complete Freund's adjuvant (CFA), and Lewis/C6- rats had delayed clinical EAN compared to the Lewis rats. Peripheral nerve demyelination in Lewis/C6- was also delayed but was similar in extent at the peak of disease. Compared to Lewis, Lewis/C6- nerves had no MAC deposition, reduced macrophage infiltrate and IL-17A, but similar T cell infiltrate and Th1 cytokine mRNA expression. ICAM-1 and P-selectin mRNA expression and immunostaining on vascular endothelium were delayed in Lewis C6- compared to Lewis rats' nerves. This study found that MAC was not required for immune mediated demyelination; but that MAC enhanced early symptoms and early demyelination in EAN, either by direct lysis or by sub-lytic induction of vascular endothelial expression of ICAM-1 and P-selectin. Copyright © 2010 Elsevier B.V. All rights reserved.

Early Elevations of the Complement Activation Fragment C3a and Adverse Pregnancy Outcomes

PubMed Central

Lynch, Anne M.; Gibbs, Ronald S.; Murphy, James R.; Giclas, Patricia C.; Salmon, Jane E.; Holers, V. Michael

2016-01-01

OBJECTIVE To estimate whether elevations of complement C3a early in pregnancy are predictive of the subsequent development of adverse pregnancy outcomes. METHODS A plasma sample was obtained from each enrolled pregnant woman before 20 weeks of gestation. The cohort (n=1,002) was evaluated for the development of adverse pregnancy outcomes defined as hypertensive diseases of pregnancy (gestational hypertension or preeclampsia), preterm birth (before 37 weeks of gestation), premature rupture of the membranes, pregnancy loss (during the embryonic and fetal period), intrauterine growth restriction, and the composite outcome of any adverse outcome. RESULTS One or more adverse pregnancy outcomes occurred in 211 (21%) of the cohort. The mean levels (ng/mL) of C3a in early pregnancy were significantly (P=<.001) higher among women with one or more adverse outcomes (858±435) compared with women with an uncomplicated pregnancy (741±407). Adjusted for parity and prepregnancy body mass index, women with levels of C3a in the upper quartile in early pregnancy were three times more likely to have an adverse outcome later in pregnancy compared with women in the lowest quartile (95% confidence interval, 1.8–4.8; P<.001). The link between early elevated C3a levels and adverse pregnancy outcomes was driven primarily by individual significant (P<.05) associations of C3a with hypertensive diseases of pregnancy, preterm birth, and premature rupture of the membranes. CONCLUSION Elevated C3a as early as the first trimester of pregnancy is an independent predictive factor for adverse pregnancy outcomes, suggesting that complement-related inflammatory events in pregnancy contribute to the subsequent development of poor outcomes at later stages of pregnancy. PMID:21173647

Covalent binding of C3b to tetanus toxin: influence on uptake/internalization of antigen by antigen-specific and non-specific B cells.

PubMed Central

Villiers, M B; Villiers, C L; Jacquier-Sarlin, M R; Gabert, F M; Journet, A M; Colomb, M G

1996-01-01

Antigen opsonization by the C3b fragment of complement is a significant event in the modulation of cell-mediated immune response, but its mechanism is still largely unknown. The structural characteristics of C3b allow it to act as a bifunctional ligand between antigen and cells via their membrane C3b receptors. It was thus of interest to study the influence of the covalent link between C3b and antigen on the fixation and internalization of this antigen by antigen-presenting cells. Tetanus toxin (TT) was used as antigen, either free or covalently linked to C3b (TT-C3b). The antigen-presenting cells were TT-specific (4.2) or non-specific (BL15) Epstein-Barr virus (EBV)-transformed B cells. C3b was found to play an important role in antigen fixation and internalization by both antigen-specific and antigen non-specific cells. Covalent binding of C3b on TT (1) permitted fixation and internalization of this antigen by non-specific cells via their complement receptors; (2) enhanced antigen fixation and resulted in cross-linking between membrane immunoglobulins and complement receptors on antigen-specific cells. The consequences of covalent C3b binding to TT were analysed using antigen-specific and antigen-nonspecific cells. In both cases, a net increase in antigen fixation was observed. At the intracellular level, covalent C3b binding to TT resulted in a large TT incorporation in endosomes of nonspecific cells, similar to that observed in antigen-specific cells. Thus, C3b covalently linked to antigen enlarges the array of B-cell types capable of presenting antigen, including non-specific cells. Images Figure 2 PMID:8958046

Biochemical Reconstitution of the WAVE Regulatory Complex

PubMed Central

Chen, Baoyu; Padrick, Shae B.; Henry, Lisa; Rosen, Michael K.

2014-01-01

The WAVE regulatory complex (WRC) is a 400-KDa heteropentameric protein assembly that plays a central role in controlling actin cytoskeletal dynamics in many cellular processes. The WRC acts by integrating diverse cellular cues and stimulating the actin nucleating activity of the Arp2/3 complex at membranes. Biochemical and biophysical studies of the underlying mechanisms of these processes require large amounts of purified WRC. Recent success in recombinant expression, reconstitution, purification and crystallization of the WRC has greatly advanced our understanding of the inhibition, activation and membrane recruitment mechanisms of this complex. But many important questions remain to be answered. Here we summarize and update the methods developed in our laboratory, which allow reliable and flexible production of tens of milligrams of recombinant WRC of crystallographic quality, sufficient for many biochemical and structural studies. PMID:24630101

Quantifying lipid changes in various membrane compartments using lipid binding protein domains.

PubMed

Várnai, Péter; Gulyás, Gergő; Tóth, Dániel J; Sohn, Mira; Sengupta, Nivedita; Balla, Tamas

2017-06-01

One of the largest challenges in cell biology is to map the lipid composition of the membranes of various organelles and define the exact location of processes that control the synthesis and distribution of lipids between cellular compartments. The critical role of phosphoinositides, low-abundant lipids with rapid metabolism and exceptional regulatory importance in the control of almost all aspects of cellular functions created the need for tools to visualize their localizations and dynamics at the single cell level. However, there is also an increasing need for methods to determine the cellular distribution of other lipids regulatory or structural, such as diacylglycerol, phosphatidic acid, or other phospholipids and cholesterol. This review will summarize recent advances in this research field focusing on the means by which changes can be described in more quantitative terms. Published by Elsevier Ltd.

Mitochondrial metabolic regulation by GRP78

PubMed Central

Prasad, Manoj; Pawlak, Kevin J.; Burak, William E.; Perry, Elizabeth E.; Marshall, Brendan; Whittal, Randy M.; Bose, Himangshu S.

2017-01-01

Steroids, essential for mammalian survival, are initiated by cholesterol transport by steroidogenic acute regulatory protein (StAR). Appropriate protein folding is an essential requirement of activity. Endoplasmic reticulum (ER) chaperones assist in folding of cytoplasmic proteins, whereas mitochondrial chaperones fold only mitochondrial proteins. We show that glucose regulatory protein 78 (GRP78), a master ER chaperone, is also present at the mitochondria-associated ER membrane (MAM), where it folds StAR for delivery to the outer mitochondrial membrane. StAR expression and activity are drastically reduced following GRP78 knockdown. StAR folding starts at the MAM region; thus, its cholesterol fostering capacity is regulated by GRP78 long before StAR reaches the mitochondria. In summary, GRP78 is an acute regulator of steroidogenesis at the MAM, regulating the intermediate folding of StAR that is crucial for its activity. PMID:28275724

Regulation of extracellular matrix vesicles via rapid responses to steroid hormones during endochondral bone formation.

PubMed

Asmussen, Niels; Lin, Zhao; McClure, Michael J; Schwartz, Zvi; Boyan, Barbara D

2017-12-09

Endochondral bone formation is a precise and highly ordered process whose exact regulatory framework is still being elucidated. Multiple regulatory pathways are known to be involved. In some cases, regulation impacts gene expression, resulting in changes in chondrocyte phenotypic expression and extracellular matrix synthesis. Rapid regulatory mechanisms are also involved, resulting in release of enzymes, factors and micro RNAs stored in extracellular matrisomes called matrix vesicles. Vitamin D metabolites modulate endochondral development via both genomic and rapid membrane-associated signaling pathways. 1α,25-dihydroxyvitamin D3 [1α,25(OH) 2 D 3 ] acts through the vitamin D receptor (VDR) and a membrane associated receptor, protein disulfide isomerase A3 (PDIA3). 24R,25-dihydroxyvitamin D3 [24R,25(OH) 2 D 3 ] affects primarily chondrocytes in the resting zone (RC) of the growth plate, whereas 1α,25(OH) 2 D 3 affects cells in the prehypertrophic and upper hypertrophic cell zones (GC). This includes genomically directing the cells to produce matrix vesicles with zone specific characteristics. In addition, vitamin D metabolites produced by the cells interact directly with the matrix vesicle membrane via rapid signal transduction pathways, modulating their activity in the matrix. The matrix vesicle payload is able to rapidly impact the extracellular matrix via matrix processing enzymes as well as providing a feedback mechanism to the cells themselves via the contained micro RNAs. Copyright © 2017. Published by Elsevier Inc.

Blood coagulation reactions on nanoscale membrane surfaces

NASA Astrophysics Data System (ADS)

Pureza, Vincent S.

Blood coagulation requires the assembly of several membrane-bound protein complexes composed of regulatory and catalytic subunits. The biomembranes involved in these reactions not only provide a platform for these procoagulant proteins, but can also affect their function. Increased exposure of acidic phospholipids on the outer leaflet of the plasma membrane can dramatically modulate the catalytic efficiencies of such membrane-bound enzymes. Under physiologic conditions, however, these phospholipids spontaneously cluster into a patchwork of membrane microdomains upon which membrane binding proteins may preferentially assemble. As a result, the membrane composition surrounding these proteins is largely unknown. Through the development and use of a nanometer-scale bilayer system that provides rigorous control of the phospholipid membrane environment, I investigated the role of phosphatidylserine, an acidic phospholipid, in the direct vicinity (within nanometers) of two critical membrane-bound procoagulant protein complexes and their respective natural substrates. Here, I present how the assembly and function of the tissue factor˙factor VIIa and factor Va˙factor Xa complexes, the first and final cofactor˙enzyme complexes of the blood clotting cascade, respectively, are mediated by changes in their immediate phospholipid environments.

The presence of OMP inclusion bodies in a Escherichia coli K-12 mutated strain is not related to lipopolysaccharide structure.

PubMed

Corsaro, M Michela; Parrilli, Ermenegilda; Lanzetta, Rosa; Naldi, Teresa; Pieretti, Giuseppina; Lindner, Buko; Carpentieri, Andrea; Parrilli, Michelangelo; Tutino, M Luisa

2009-08-01

The role of lipopolysaccharides (LPSs) in the biogenesis of outer membrane proteins have been investigated in several studies. Some of these analyses showed that LPS is required for correct and efficient folding of outer membrane proteins; other studies support the idea of independence of outer membrane proteins biogenesis from LPS structure. In this article, we investigated the involvement of LPS structure in the anomalous aggregation of outer membrane proteins in a E. coli mutant strain (S17-1(lambdapir)). To achieve this aim, the LPS structure of the mutant strain was carefully determined and compared with the E. coli K-12 one. It turned out that LPS of these two strains differs in the inner core for the absence of a heptose residue (HepIII). We demonstrated that this difference is due to a mutation in waaQ, a gene encoding the transferase for the branch heptose HepIII residue. The mutation was complemented to find out if the restoration of LPS structure influenced the observed outer membrane proteins aggregation. Data reported in this work demonstrated that, in E. coli S17-1(lambdapir) there is no influence of LPS structure on the outer membrane proteins inclusion bodies formation.

Lateral Membrane Diffusion Modulated by a Minimal Actin Cortex

PubMed Central

Heinemann, Fabian; Vogel, Sven K.; Schwille, Petra

2013-01-01

Diffusion of lipids and proteins within the cell membrane is essential for numerous membrane-dependent processes including signaling and molecular interactions. It is assumed that the membrane-associated cytoskeleton modulates lateral diffusion. Here, we use a minimal actin cortex to directly study proposed effects of an actin meshwork on the diffusion in a well-defined system. The lateral diffusion of a lipid and a protein probe at varying densities of membrane-bound actin was characterized by fluorescence correlation spectroscopy (FCS). A clear correlation of actin density and reduction in mobility was observed for both the lipid and the protein probe. At high actin densities, the effect on the protein probe was ∼3.5-fold stronger compared to the lipid. Moreover, addition of myosin filaments, which contract the actin mesh, allowed switching between fast and slow diffusion in the minimal system. Spot variation FCS was in accordance with a model of fast microscopic diffusion and slower macroscopic diffusion. Complementing Monte Carlo simulations support the analysis of the experimental FCS data. Our results suggest a stronger interaction of the actin mesh with the larger protein probe compared to the lipid. This might point toward a mechanism where cortical actin controls membrane diffusion in a strong size-dependent manner. PMID:23561523

Persistent complement activation on tumor cells in breast cancer.

PubMed Central

Niculescu, F.; Rus, H. G.; Retegan, M.; Vlaicu, R.

1992-01-01

The neoantigens of the C5b-9 complement complex, IgG, C3, C4, S-protein/vitronectin, fibronectin, and macrophages were localized on 17 samples of breast cancer and on 6 samples of benign breast tumors using polyclonal or monoclonal antibodies and the streptavidin-biotin-peroxidase technique. All the tissue samples with carcinoma in each the TNM stages presented C5b-9 deposits on the membranes of tumor cells, thin granules on cell remnants, and diffuse deposits in the necrotic areas. When chemotherapy and radiation therapy preceded surgery, C5b-9 deposits were more intense and extended. The C5b-9 deposits were absent in all the samples with benign lesions. S-protein/vitronectin was present as fibrillar deposits in the connective tissue matrix and as diffuse deposits around the tumor cells, less intense and extended than fibronectin. IgG, C3, and C4 deposits were present only in carcinoma samples. The presence of C5b-9 deposits is indicative of complement activation and its subsequent pathogenetic effects in breast cancer. Images Figure 1 PMID:1374587

Detailed Investigation of Separation Performance of a MMM for Removal of Higher Hydrocarbons under Varying Operating Conditions

PubMed Central

Mushardt, Heike; Müller, Marcus; Shishatskiy, Sergey; Wind, Jan; Brinkmann, Torsten

2016-01-01

Mixed-matrix membranes (MMMs) are promising candidates to improve the competitiveness of membrane technology against energy-intensive conventional technologies. In this work, MMM composed of poly(octylmethylsiloxane) (POMS) and activated carbon (AC) were investigated with respect to separation of higher hydrocarbons (C3+) from permanent gas streams. Membranes were prepared as thin film composite membranes on a technical scale and characterized via scanning electron microscopy (SEM) and permeation measurements with binary mixtures of n-C4H10/CH4 under varying operating conditions (feed and permeate pressure, temperature, feed gas composition) to study the influence on separation performance. SEM showed good contact and absence of defects. Lower permeances but higher selectivities were found for MMM compared to pure POMS membrane. Best results were obtained at high average fugacity and activity of n-C4H10 with the highest selectivity estimated to be 36.4 at n-C4H10 permeance of 12 mN3/(m2·h·bar). Results were complemented by permeation of a multi-component mixture resembling a natural gas application, demonstrating the superior performance of MMM. PMID:26927194

An anaerobic membrane bioreactor - membrane distillation hybrid system for energy recovery and water reuse: Removal performance of organic carbon, nutrients, and trace organic contaminants.

PubMed

Song, Xiaoye; Luo, Wenhai; McDonald, James; Khan, Stuart J; Hai, Faisal I; Price, William E; Nghiem, Long D

2018-07-01

In this study, a direct contact membrane distillation (MD) unit was integrated with an anaerobic membrane bioreactor (AnMBR) to simultaneously recover energy and produce high quality water for reuse from wastewater. Results show that AnMBR could produce 0.3-0.5L/g COD added biogas with a stable methane content of approximately 65%. By integrating MD with AnMBR, bulk organic matter and phosphate were almost completely removed. The removal of the 26 selected trace organic contaminants by AnMBR was compound specific, but the MD process could complement AnMBR removal, leading to an overall efficiency from 76% to complete removal by the integrated system. The results also show that, due to complete retention, organic matter (such as humic-like and protein-like substances) and inorganic salts accumulated in the MD feed solution and therefore resulted in significant fouling of the MD unit. As a result, the water flux of the MD process decreased continuously. Nevertheless, membrane pore wetting was not observed throughout the operation. Crown Copyright © 2018. Published by Elsevier B.V. All rights reserved.

Outer membrane proteins of pathogenic spirochetes

PubMed Central

Cullen, Paul A.; Haake, David A.; Adler, Ben

2009-01-01

Pathogenic spirochetes are the causative agents of several important diseases including syphilis, Lyme disease, leptospirosis, swine dysentery, periodontal disease and some forms of relapsing fever. Spirochetal bacteria possess two membranes and the proteins present in the outer membrane are at the site of interaction with host tissue and the immune system. This review describes the current knowledge in the field of spirochetal outer membrane protein (OMP) biology. What is known concerning biogenesis and structure of OMPs, with particular regard to the atypical signal peptide cleavage sites observed amongst the spirochetes, is discussed. We examine the functions that have been determined for several spirochetal OMPs including those that have been demonstrated to function as adhesins, porins or to have roles in complement resistance. A detailed description of the role of spirochetal OMPs in immunity, including those that stimulate protective immunity or that are involved in antigenic variation, is given. A final section is included which covers experimental considerations in spirochetal outer membrane biology. This section covers contentious issues concerning cellular localization of putative OMPs, including determination of surface exposure. A more detailed knowledge of spirochetal OMP biology will hopefully lead to the design of new vaccines and a better understanding of spirochetal pathogenesis. PMID:15449605

Altered Subcellular Localization of a Tobacco Membrane Raft-Associated Remorin Protein by Tobamovirus Infection and Transient Expression of Viral Replication and Movement Proteins

PubMed Central

Sasaki, Nobumitsu; Takashima, Eita; Nyunoya, Hiroshi

2018-01-01

Remorins are plant specific proteins found in plasma membrane microdomains (termed lipid or membrane rafts) and plasmodesmata. A potato remorin is reported to be involved in negatively regulating potexvirus movement and plasmodesmal permeability. In this study, we isolated cDNAs of tobacco remorins (NtREMs) and examined roles of an NtREM in infection by tomato mosaic virus (ToMV). Subcellular localization analysis using fluorescently tagged NtREM, ToMV, and viral replication and movement proteins (MPs) indicated that virus infection and transient expression of the viral proteins promoted the formation of NtREM aggregates by altering the subcellular distribution of NtREM, which was localized uniformly on the plasma membrane under normal conditions. NtREM aggregates were often observed associated closely with endoplasmic reticulum networks and bodies of the 126K replication and MPs. The bimolecular fluorescence complementation assay indicated that NtREM might interact directly with the MP on the plasma membrane and around plasmodesmata. In addition, transient overexpression of NtREM facilitated ToMV cell-to-cell movement. Based on these results, we discuss possible roles of the tobacco remorin in tobamovirus movement. PMID:29868075

Haemodialysis-membrane biocompatibility and mortality of patients with dialysis-dependent acute renal failure: a prospective randomised multicentre trial. International Multicentre Study Group.

PubMed

Jörres, A; Gahl, G M; Dobis, C; Polenakovic, M H; Cakalaroski, K; Rutkowski, B; Kisielnicka, E; Krieter, D H; Rumpf, K W; Guenther, C; Gaus, W; Hoegel, J

1999-10-16

There is controversy as to whether haemodialysis-membrane biocompatibility (ie, the potential to activate complement and neutrophils) influences mortality of patients with acute renal failure. We did a prospective randomised multicentre trial in patients with dialysis-dependent acute renal failure treated with two different types of low-flux membrane. 180 patients with acute renal failure were randomly assigned bioincompatible Cuprophan (n=90) or polymethyl-methacrylate (n=90) membranes. The main outcome was survival 14 days after the end of therapy (treatment success). Odds ratios for survival were calculated and the two groups were compared by Fisher's exact test. Analyses were based on patients treated according to protocol (76 Cuprophan, 84 polymethyl methacrylate). At the start of dialysis, the groups did not differ significantly in age, sex, severity of illness (as calculated by APACHE II scores), prevalence of oliguria, or biochemical measures of acute renal failure. 44 patients (58% [95% CI 46-69]) assigned Cuprophan membranes and 50 patients (60% [48-70]) assigned polymethyl-methacrylate membranes survived. The odds ratio for treatment failure on Cuprophan compared with polymethyl-methacrylate membranes was 1.07 (0.54-2.11; p=0.87). No difference between Cuprophan and polymethyl-methacrylate membranes was detected when the analysis was adjusted for age and APACHE II score. 18 patients in the Cuprophan group and 20 in the polymethyl-methacrylate group had clinical complications of therapy (mainly hypotension). There were no differences in outcome for patients with dialysis-dependent acute renal failure between those treated with Cuprophan membranes and those treated with polymethyl-methacrylate membranes.

Neurotensin effect on Na+, K+-ATPase is CNS area- and membrane-dependent and involves high affinity NT1 receptor.

PubMed

López Ordieres, María Graciela; Rodríguez de Lores Arnaiz, Georgina

2002-11-01

We have previously shown that peptide neurotensin inhibits cerebral cortex synaptosomal membrane Na+, K+-ATPase, an effect fully prevented by blockade of neurotensin NT1 receptor by antagonist SR 48692. The work was extended to analyze neurotensin effect on Na+, K+-ATPase activity present in other synaptosomal membranes and in CNS myelin and mitochondrial fractions. Results indicated that, besides inhibiting cerebral cortex synaptosomal membrane Na+, K+-ATPase, neurotensin likewise decreased enzyme activity in homologous striatal membranes as well as in a commercial preparation obtained from porcine cerebral cortex. However, the peptide failed to alter either Na+, K+-ATPase activity in cerebellar synaptosomal and myelin membranes or ATPase activity in mitochondrial preparations. Whenever an effect was recorded with the peptide, it was blocked by antagonist SR 48692, indicating the involvement of the high affinity neurotensin receptor (NT1), as well as supporting the contention that, through inhibition of ion transport at synaptic membrane level, neurotensin plays a regulatory role in neurotransmission.

Hemocompatibility evaluation of poly(1,8-octanediol citrate) blend polyethersulfone membranes.

PubMed

Zailani, Muhamad Zulhilmi; Ismail, Ahmad Fauzi; Sheikh Abdul Kadir, Siti Hamimah; Othman, Mohd Hafiz Dzarfan; Goh, Pei Sean; Hasbullah, Hasrinah; Abdullah, Mohd Sohaimi; Ng, Be Cheer; Kamal, Fatmawati

2017-05-01

In this study, poly (1,8-octanediol citrate) (POC) was used to modify polyethersulfone (PES)-based membrane to enhance its hemocompatibility. Different compositions of POC (0-3%) were added into the polyethersulfone (PES) dope solutions and polyvinylpyrrolidone (PVP) was used as pore forming agent. The hemocompatible POC modified PES membranes were fabricated through phase-inversion technique. The prepared membranes were characterized using attenuated total reflectance-Fourier transform infrared (ATR-FTIR), thermogravimetric analysis (TGA), scanning electron microscopy (SEM), Atomic-force microscopy (AFM), contact angle, Zeta-potential, membrane porosity and pore size and pure water flux (PWF) and BSA rejection. The hemocompatibility of the modified PES membranes was evaluated by human serum fibrinogen (FBG) protein adsorption, platelet adhesion, activated partial thromboplastin time (APTT) and prothrombin time (PT), and thrombin-antithrombin III (TAT), complement (C3a and C5a) activation and Ca 2+ absorption on membrane. Results showed that by increasing POC concentration, FBG adsorption was reduced, less platelets adhesion, prolonged APTT and PT, lower TAT, C5a and C3a activation and absorb more Ca 2+ ion. These results indicated that modification of PES with POC has rendered improved hemocompatibility properties for potential application in the field of blood purification, especially in hemodialysis. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1510-1520, 2017. © 2017 Wiley Periodicals, Inc.

A critical review of the application of polymer of low concern and regulatory criteria to fluoropolymers.

PubMed

Henry, Barbara J; Carlin, Joseph P; Hammerschmidt, Jon A; Buck, Robert C; Buxton, L William; Fiedler, Heidelore; Seed, Jennifer; Hernandez, Oscar

2018-05-01

Per- and polyfluoroalkyl substances (PFAS) are a group of fluorinated substances that are in the focus of researchers and regulators due to widespread presence in the environment and biota, including humans, of perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA). Fluoropolymers, high molecular weight polymers, have unique properties that constitute a distinct class within the PFAS group. Fluoropolymers have thermal, chemical, photochemical, hydrolytic, oxidative, and biological stability. They have negligible residual monomer and oligomer content and low to no leachables. Fluoropolymers are practically insoluble in water and not subject to long-range transport. With a molecular weight well over 100 000 Da, fluoropolymers cannot cross the cell membrane. Fluoropolymers are not bioavailable or bioaccumulative, as evidenced by toxicology studies on polytetrafluoroethylene (PTFE): acute and subchronic systemic toxicity, irritation, sensitization, local toxicity on implantation, cytotoxicity, in vitro and in vivo genotoxicity, hemolysis, complement activation, and thrombogenicity. Clinical studies of patients receiving permanently implanted PTFE cardiovascular medical devices demonstrate no chronic toxicity or carcinogenicity and no reproductive, developmental, or endocrine toxicity. This paper brings together fluoropolymer toxicity data, human clinical data, and physical, chemical, thermal, and biological data for review and assessment to show that fluoropolymers satisfy widely accepted assessment criteria to be considered as "polymers of low concern" (PLC). This review concludes that fluoropolymers are distinctly different from other polymeric and nonpolymeric PFAS and should be separated from them for hazard assessment or regulatory purposes. Grouping fluoropolymers with all classes of PFAS for "read across" or structure-activity relationship assessment is not scientifically appropriate. Integr Environ Assess Manag 2018;14:316-334. © 2018 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals, Inc. on behalf of Society of Environmental Toxicology & Chemistry (SETAC). © 2018 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals, Inc. on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Study of the Electrical Impedance Scanning

DTIC Science & Technology

2001-10-25

extremely promising technique to complement other conventional image examinations. Areas presenting abnormalities and/or malignant neoplasia evidence...presenting abnormalities and/or malignant neoplasia evidence exhibit conductive changes (in the structure of cell’s membrane, in its permeability, in the...Calas , R. R. de Cas and V. R Manoel , “Scanning de Impedância Transpectral Elétrica da Mama ”, CEPEM – Centro de Estudos e Pesquisas da Mulher

Rituximab for Treatment of Membranoproliferative Glomerulonephritis and C3 Glomerulopathies

PubMed Central

2017-01-01

Membranoproliferative glomerulonephritis (MPGN) is a histological pattern of injury resulting from predominantly subendothelial and mesangial deposition of immunoglobulins or complement factors with subsequent inflammation and proliferation particularly of the glomerular basement membrane. Recent classification of MPGN is based on pathogenesis dividing MPGN into immunoglobulin-associated MPGN and complement-mediated C3 glomerulonephritis (C3GN) and dense deposit disease (DDD). Current guidelines suggest treatment with steroids, cytotoxic agents with or without plasmapheresis only for subjects with progressive disease, that is, nephrotic range proteinuria and decline of renal function. Rituximab, a chimeric B-cell depleting anti-CD20 antibody, has emerged in the last decade as a treatment option for patients with primary glomerular diseases such as minimal change disease, focal-segmental glomerulosclerosis, or idiopathic membranous nephropathy. However, data on the use of rituximab in MPGN, C3GN, and DDD are limited to case reports and retrospective case series. Patients with immunoglobulin-associated and idiopathic MPGN who were treated with rituximab showed partial and complete responses in the majorities of cases. However, rituximab was not effective in few cases of C3GN and DDD. Despite promising results in immunoglobulin-associated and idiopathic MPGN, current evidence on this treatment remains weak, and controlled and prospective data are urgently needed. PMID:28573137

A monogalactosyldiacylglycerol synthase found in the green sulfur bacterium Chlorobaculum tepidum reveals important roles for galactolipids in photosynthesis.

PubMed

Masuda, Shinji; Harada, Jiro; Yokono, Makio; Yuzawa, Yuichi; Shimojima, Mie; Murofushi, Kazuhiro; Tanaka, Hironori; Masuda, Hanako; Murakawa, Masato; Haraguchi, Tsuyoshi; Kondo, Maki; Nishimura, Mikio; Yuasa, Hideya; Noguchi, Masato; Oh-Oka, Hirozo; Tanaka, Ayumi; Tamiaki, Hitoshi; Ohta, Hiroyuki

2011-07-01

Monogalactosyldiacylglycerol (MGDG), which is conserved in almost all photosynthetic organisms, is the most abundant natural polar lipid on Earth. In plants, MGDG is highly accumulated in the chloroplast membranes and is an important bulk constituent of thylakoid membranes. However, precise functions of MGDG in photosynthesis have not been well understood. Here, we report a novel MGDG synthase from the green sulfur bacterium Chlorobaculum tepidum. This enzyme, MgdA, catalyzes MGDG synthesis using UDP-Gal as a substrate. The gene encoding MgdA was essential for this bacterium; only heterozygous mgdA mutants could be isolated. An mgdA knockdown mutation affected in vivo assembly of bacteriochlorophyll c aggregates, suggesting the involvement of MGDG in the construction of the light-harvesting complex called chlorosome. These results indicate that MGDG biosynthesis has been independently established in each photosynthetic organism to perform photosynthesis under different environmental conditions. We complemented an Arabidopsis thaliana MGDG synthase mutant by heterologous expression of MgdA. The complemented plants showed almost normal levels of MGDG, although they also had abnormal morphological phenotypes, including reduced chlorophyll content, no apical dominance in shoot growth, atypical flower development, and infertility. These observations provide new insights regarding the importance of regulated MGDG synthesis in the physiology of higher plants.

Potential influences of complement factor H in autoimmune inflammatory and thrombotic disorders.

PubMed

Ferluga, Janez; Kouser, Lubna; Murugaiah, Valarmathy; Sim, Robert B; Kishore, Uday

2017-04-01

Complement system homeostasis is important for host self-protection and anti-microbial immune surveillance, and recent research indicates roles in tissue development and remodelling. Complement also appears to have several points of interaction with the blood coagulation system. Deficiency and altered function due to gene mutations and polymorphisms in complement effectors and regulators, including Factor H, have been associated with familial and sporadic autoimmune inflammatory - thrombotic disorders, in which autoantibodies play a part. These include systemic lupus erythematosus, rheumatoid arthritis, atypical haemolytic uremic syndrome, anti-phospholipid syndrome and age-related macular degeneration. Such diseases are generally complex - multigenic and heterogeneous in their symptoms and predisposition/susceptibility. They usually need to be triggered by vascular trauma, drugs or infection and non-complement genetic factors also play a part. Underlying events seem to include decline in peripheral regulatory T cells, dendritic cell, and B cell tolerance, associated with alterations in lymphoid organ microenvironment. Factor H is an abundant protein, synthesised in many cell types, and its reported binding to many different ligands, even if not of high affinity, may influence a large number of molecular interactions, together with the accepted role of Factor H within the complement system. Factor H is involved in mesenchymal stem cell mediated tolerance and also contributes to self-tolerance by augmenting iC3b production and opsonisation of apoptotic cells for their silent dendritic cell engulfment via complement receptor CR3, which mediates anti-inflammatory-tolerogenic effects in the apoptotic cell context. There may be co-operation with other phagocytic receptors, such as complement C1q receptors, and the Tim glycoprotein family, which specifically bind phosphatidylserine expressed on the apoptotic cell surface. Factor H is able to discriminate between self and nonself surfaces for self-protection and anti-microbe defence. Factor H, particularly as an abundant platelet protein, may also modulate blood coagulation, having an anti-thrombotic role. Here, we review a number of interaction pathways in coagulation and in immunity, together with associated diseases, and indicate where Factor H may be expected to exert an influence, based on reports of the diversity of ligands for Factor H. Copyright © 2017 Elsevier Ltd. All rights reserved.

Complement factor H is expressed in adipose tissue in association with insulin resistance.

PubMed

Moreno-Navarrete, José María; Martínez-Barricarte, Rubén; Catalán, Victoria; Sabater, Mònica; Gómez-Ambrosi, Javier; Ortega, Francisco José; Ricart, Wifredo; Blüher, Mathias; Frühbeck, Gema; Rodríguez de Cordoba, Santiago; Fernández-Real, José Manuel

2010-01-01

Activation of the alternative pathway of the complement system, in which factor H (fH; complement fH [CFH]) is a key regulatory component, has been suggested as a link between obesity and metabolic disorders. Our objective was to study the associations between circulating and adipose tissue gene expressions of CFH and complement factor B (fB; CFB) with obesity and insulin resistance. Circulating fH and fB were determined by enzyme-linked immunosorbent assay in 398 subjects. CFH and CFB gene expressions were evaluated in 76 adipose tissue samples, in isolated adipocytes, and in stromovascular cells (SVC) (n = 13). The effects of weight loss and rosiglitazone were investigated in independent cohorts. Both circulating fH and fB were associated positively with BMI, waist circumference, triglycerides, and inflammatory parameters and negatively with insulin sensitivity and HDL cholesterol. For the first time, CFH gene expression was detected in human adipose tissue (significantly increased in subcutaneous compared with omental fat). CFH gene expression in omental fat was significantly associated with insulin resistance. In contrast, CFB gene expression was significantly increased in omental fat but also in association with fasting glucose and triglycerides. The SVC fraction was responsible for these differences, although isolated adipocytes also expressed fB and fH at low levels. Both weight loss and rosiglitazone led to significantly decreased circulating fB and fH levels. Increased circulating fH and fB concentrations in subjects with altered glucose tolerance could reflect increased SVC-induced activation of the alternative pathway of complement in omental adipose tissue linked to insulin resistance and metabolic disturbances.

The Scl1 protein of M6-type group A Streptococcus binds the human complement regulatory protein, factor H, and inhibits the alternative pathway of complement.

PubMed

Caswell, Clayton C; Han, Runlin; Hovis, Kelley M; Ciborowski, Pawel; Keene, Douglas R; Marconi, Richard T; Lukomski, Slawomir

2008-02-01

Non-specific activation of the complement system is regulated by the plasma glycoprotein factor H (FH). Bacteria can avoid complement-mediated opsonization and phagocytosis through acquiring FH to the cell surface. Here, we characterize an interaction between the streptococcal collagen-like protein Scl1.6 of M6-type group A Streptococcus (GAS) and FH. Using affinity chromatography with immobilized recombinant Scl1.6 protein, we co-eluted human plasma proteins with molecular weight of 155 kDa, 43 kDa and 38 kDa. Mass spectrometry identified the 155 kDa band as FH and two other bands as isoforms of the FH-related protein-1. The identities of all three bands were confirmed by Western immunoblotting with specific antibodies. Structure-function relation studies determined that the globular domain of the Scl1.6 variant specifically binds FH while fused to collagenous tails of various lengths. This binding is not restricted to Scl1.6 as the phylogenetically linked Scl1.55 variant also binds FH. Functional analyses demonstrated the cofactor activity of the rScl1.6-bound FH for factor I-mediated cleavage of C3b. Finally, purified FH bound to the Scl1.6 protein present in the cell wall material obtained from M6-type GAS. In conclusion, we have identified a functional interaction between Scl1 and plasma FH, which may contribute to GAS evasion of complement-mediated opsonization and phagocytosis.

N-acetylcysteine (NAC) ameliorates Epstein-Barr virus latent membrane protein 1 induced chronic inflammation.

PubMed

Gao, Xiao; Lampraki, Eirini-Maria; Al-Khalidi, Sarwah; Qureshi, Muhammad Asif; Desai, Rhea; Wilson, Joanna Beatrice

2017-01-01

Chronic inflammation results when the immune system responds to trauma, injury or infection and the response is not resolved. It can lead to tissue damage and dysfunction and in some cases predispose to cancer. Some viruses (including Epstein-Barr virus (EBV)) can induce inflammation, which may persist even after the infection has been controlled or cleared. The damage caused by inflammation, can itself act to perpetuate the inflammatory response. The latent membrane protein 1 (LMP1) of EBV is a pro-inflammatory factor and in the skin of transgenic mice causes a phenotype of hyperplasia with chronic inflammation of increasing severity, which can progress to pre-malignant and malignant lesions. LMP1 signalling leads to persistent deregulated expression of multiple proteins throughout the mouse life span, including TGFα S100A9 and chitinase-like proteins. Additionally, as the inflammation increases, numerous chemokines and cytokines are produced which promulgate the inflammation. Deposition of IgM, IgG, IgA and IgE and complement activation form part of this process and through genetic deletion of CD40, we show that this contributes to the more tissue-destructive aspects of the phenotype. Treatment of the mice with N-acetylcysteine (NAC), an antioxidant which feeds into the body's natural redox regulatory system through glutathione synthesis, resulted in a significantly reduced leukocyte infiltrate in the inflamed tissue, amelioration of the pathological features and delay in the inflammatory signature measured by in vivo imaging. Reducing the degree of inflammation achieved through NAC treatment, had the knock on effect of reducing leukocyte recruitment to the inflamed site, thereby slowing the progression of the pathology. These data support the idea that NAC could be considered as a treatment to alleviate chronic inflammatory pathologies, including post-viral disease. Additionally, the model described can be used to effectively monitor and accurately measure therapies for chronic inflammation.

Self-regulatory processes mediate the intention-behavior relation for adherence and exercise behaviors.

PubMed

de Bruin, Marijn; Sheeran, Paschal; Kok, Gerjo; Hiemstra, Anneke; Prins, Jan M; Hospers, Harm J; van Breukelen, Gerard J P

2012-11-01

Understanding the gap between people's intentions and actual health behavior is an important issue in health psychology. Our aim in this study was to investigate whether self-regulatory processes (monitoring goal progress and responding to discrepancies) mediate the intention-behavior relation in relation to HIV medication adherence (Study 1) and intensive exercise behavior (Study 2). In Study 1, questionnaire and electronically monitored adherence data were collected at baseline and 3 months later from patients in the control arm of an HIV-adherence intervention study. In Study 2, questionnaire data was collected at 3 time points 6-weeks apart in a cohort study of physical activity. Complete data at all time points were obtained from 51 HIV-infected patients and 499 intensive exercise participants. Intentions were good predictors of behavior and explained 25 to 30% of the variance. Self-regulatory processes explained an additional 11% (Study 1) and 6% (Study 2) of variance in behavior on top of intentions. Regression and bootstrap analyses revealed at least partial, and possibly full, mediation of the intention-behavior relation by self-regulatory processes. The present studies indicate that self-regulatory processes may explain how intentions drive behavior. Future tests, using different health behaviors and experimental designs, could firmly establish whether self-regulatory processes complement current health behavior theories and should become routine targets for intervention. (PsycINFO Database Record (c) 2012 APA, all rights reserved).

Relationship between platelet count and hemodialysis membranes

PubMed Central

Nasr, Rabih; Saifan, Chadi; Barakat, Iskandar; Azzi, Yorg Al; Naboush, Ali; Saad, Marc; Sayegh, Suzanne El

2013-01-01

Background One factor associated with poor outcomes in hemodialysis patients is exposure to a foreign membrane. Older membranes are very bioincompatible and increase complement activation, cause leukocytosis by activating circulating factors, which sequesters leukocytes in the lungs, and activates platelets. Recently, newer membranes have been developed that were designed to be more biocompatible. We tested if the different “optiflux” hemodialysis membranes had different effects on platelet levels. Methods Ninety-nine maintenance hemodialysis patients with no known systemic or hematologic diseases affecting their platelets had blood drawn immediately prior to, 90 minutes into, and immediately following their first hemodialysis session of the week. All patients were dialyzed using a Fresenius Medical Care Optiflux polysulfone membrane F160, F180, or F200 (polysulfone synthetic dialyzer membranes, 1.6 m2, 1.8 m2, and 2.0 m2 surface area, respectively, electron beam sterilized). Platelet counts were measured from each sample by analysis using a CBC analyzer. Results The average age of the patients was 62.7 years; 36 were female and 63 were male. The mean platelet count pre, mid, and post dialysis was 193 (standard deviation ±74.86), 191 (standard deviation ±74.67), and 197 (standard deviation ±79.34) thousand/mm3, respectively, with no statistical differences. Conclusion Newer membranes have no significant effect on platelet count. This suggests that they are, in fact, more biocompatible than their predecessors and may explain their association with increased survival. PMID:23983482

The single-subunit RING-type E3 ubiquitin ligase RSL1 targets PYL4 and PYR1 ABA receptors in plasma membrane to modulate abscisic acid signaling.

PubMed

Bueso, Eduardo; Rodriguez, Lesia; Lorenzo-Orts, Laura; Gonzalez-Guzman, Miguel; Sayas, Enric; Muñoz-Bertomeu, Jesús; Ibañez, Carla; Serrano, Ramón; Rodriguez, Pedro L

2014-12-01

Membrane-delimited events play a crucial role for ABA signaling and PYR/PYL/RCAR ABA receptors, clade A PP2Cs and SnRK2/CPK kinases modulate the activity of different plasma membrane components involved in ABA action. Therefore, the turnover of PYR/PYL/RCARs in the proximity of plasma membrane might be a step that affects receptor function and downstream signaling. In this study we describe a single-subunit RING-type E3 ubiquitin ligase RSL1 that interacts with the PYL4 and PYR1 ABA receptors at the plasma membrane. Overexpression of RSL1 reduces ABA sensitivity and rsl1 RNAi lines that impair expression of several members of the RSL1/RFA gene family show enhanced sensitivity to ABA. RSL1 bears a C-terminal transmembrane domain that targets the E3 ligase to plasma membrane. Accordingly, bimolecular fluorescent complementation (BiFC) studies showed the RSL1-PYL4 and RSL1-PYR1 interaction is localized to plasma membrane. RSL1 promoted PYL4 and PYR1 degradation in vivo and mediated in vitro ubiquitylation of the receptors. Taken together, these results suggest ubiquitylation of ABA receptors at plasma membrane is a process that might affect their function via effect on their half-life, protein interactions or trafficking. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

Estradiol's interesting life at the cell's plasma membrane.

PubMed

Caldwell, J D; Gebhart, V M; Jirikowski, G F

2016-07-01

Clearly, we have presented here evidence of a very complex set of mechanisms and proteins involved with various and intricate actions of steroids at the plasma membrane. Steroids do MUCH more at the plasma membrane than simply passing passively through it. They may sit in the membrane; they are bound by numerous proteins in the membrane, including ERs, SHBG, steroid-binding globulin receptors, and perhaps elements of cellular architecture such as tubulin. It also seems likely that the membrane itself responds graphically to the presence of steroids by actually changing its shape as well, perhaps, as accumulating steroids. Clara Szego suggested in the 1980s that actions of E2 at one level would act synergistically with its actions at another level (e.g. membrane actions would complement nuclear actions). Given the sheer number of proteins involved in steroid actions, just at the membrane level, it seems unlikely that every action of a steroid on every potential protein effector will act to the same end. It seems more likely that these multiple effects and sites of effect of steroids contribute to the confusion that exists as to what actions steroids always have. For example, there is confusion with regard to synthetic agents (SERMs etc.) that have different and often opposite actions depending on which organ they act upon. A better understanding of the basic actions of steroids should aid in understanding the variability of their clinical effects. Copyright © 2016 Elsevier Inc. All rights reserved.

Plasma membrane associated membranes (PAM) from Jurkat cells contain STIM1 protein is PAM involved in the capacitative calcium entry?

PubMed

Kozieł, Katarzyna; Lebiedzinska, Magdalena; Szabadkai, Gyorgy; Onopiuk, Marta; Brutkowski, Wojciech; Wierzbicka, Katarzyna; Wilczyński, Grzegorz; Pinton, Paolo; Duszyński, Jerzy; Zabłocki, Krzysztof; Wieckowski, Mariusz R

2009-12-01

A proper cooperation between the plasma membrane, the endoplasmic reticulum and the mitochondria seems to be essential for numerous cellular processes involved in Ca(2+) signalling and maintenance of Ca(2+) homeostasis. A presence of microsomal and mitochondrial proteins together with those characteristic for the plasma membrane in the fraction of the plasma membrane associated membranes (PAM) indicates a formation of stabile interactions between these three structures. We isolated the plasma membrane associated membranes from Jurkat cells and found its significant enrichment in the plasma membrane markers including plasma membrane Ca(2+)-ATPase, Na(+), K(+)-ATPase and CD3 as well as sarco/endoplasmic reticulum Ca(2+) ATPase as a marker of the endoplasmic reticulum membranes. In addition, two proteins involved in the store-operated Ca(2+) entry, Orai1 located in the plasma membrane and an endoplasmic reticulum protein STIM1 were found in this fraction. Furthermore, we observed a rearrangement of STIM1-containing protein complexes isolated from Jurkat cells undergoing stimulation by thapsigargin. We suggest that the inter-membrane compartment composed of the plasma membrane and the endoplasmic reticulum, and isolated as a stabile plasma membrane associated membranes fraction, might be involved in the store-operated Ca(2+) entry, and their formation and rebuilding have an important regulatory role in cellular Ca(2+) homeostasis.

In vitro mitochondrial test to assess haemodialyser biocompatibility.

PubMed

Tabouy, L J; Chauvet-Monges, A M; Brunet, P J; Braguer, D L; García, P A; Berland, Y F; Crevat, A D

1997-08-01

This paper describes an in vitro mitochondrial test to assess the biocompatibility of haemodialysers. We tested on isolated liver mitochondria the effect of solutions obtained by an aqueous rinse of different haemodialysers (cuprophane, cellulose acetate, Hemophan, polyacrylonitrile, polymethylmethacrylate, polysulphone, polyamide). Moreover, to determine the penetration into the cell and the cytotoxicity of these solutions from haemodialysers, we examined the effect of rinse solutions on HT29-D4 cells. Our results showed that rinse solutions from haemodialysers decrease the mitochondrial ATP synthesis. Cuprophane has the most marked effect, and the synthetic membranes exhibited only mild effects. Rinse solutions penetrated the cell and were cytotoxic by acting on mitochondria in the cell. In this respect, cellulosic membranes were the most toxic. Taken together our findings lead to a classification of haemodialyser membranes which is identical to one based on criteria such as activation of complement (cuprophane > other cellulosics > synthetics). Moreover isolated mitochondria make it possible to differentiate among the synthetic membranes. Isolated mitochondria thus appear to be a good in vitro test to assess the biocompatibility of haemodialysers.

Endocytosis and membrane receptor internalization: implication of F-BAR protein Carom.

PubMed

Xu, Yanjie; Xia, Jixiang; Liu, Suxuan; Stein, Sam; Ramon, Cueto; Xi, Hang; Wang, Luqiao; Xiong, Xinyu; Zhang, Lixiao; He, Dingwen; Yang, William; Zhao, Xianxian; Cheng, Xiaoshu; Yang, Xiaofeng; Wang, Hong

2017-03-01

Endocytosis is a cellular process mostly responsible for membrane receptor internalization. Cell membrane receptors bind to their ligands and form a complex which can be internalized. We previously proposed that F-BAR protein initiates membrane curvature and mediates endocytosis via its binding partners. However, F-BAR protein partners involved in membrane receptor endocytosis and the regulatory mechanism remain unknown. In this study, we established database mining strategies to explore mechanisms underlying receptor-related endocytosis. We identified 34 endocytic membrane receptors and 10 regulating proteins in clathrin-dependent endocytosis (CDE), a major process of membrane receptor internalization. We found that F-BAR protein FCHSD2 (Carom) may facilitate endocytosis via 9 endocytic partners. Carom is highly expressed, along with highly expressed endocytic membrane receptors and partners, in endothelial cells and macrophages. We established 3 models of Carom-receptor complexes and their intracellular trafficking based on protein interaction and subcellular localization. We conclude that Carom may mediate receptor endocytosis and transport endocytic receptors to the cytoplasm for receptor signaling and lysosome/proteasome degradation, or to the nucleus for RNA processing, gene transcription and DNA repair.

Changes in cis-regulatory elements of a key floral regulator are associated with divergence of inflorescence architectures.

PubMed

Kusters, Elske; Della Pina, Serena; Castel, Rob; Souer, Erik; Koes, Ronald

2015-08-15

Higher plant species diverged extensively with regard to the moment (flowering time) and position (inflorescence architecture) at which flowers are formed. This seems largely caused by variation in the expression patterns of conserved genes that specify floral meristem identity (FMI), rather than changes in the encoded proteins. Here, we report a functional comparison of the promoters of homologous FMI genes from Arabidopsis, petunia, tomato and Antirrhinum. Analysis of promoter-reporter constructs in petunia and Arabidopsis, as well as complementation experiments, showed that the divergent expression of leafy (LFY) and the petunia homolog aberrant leaf and flower (ALF) results from alterations in the upstream regulatory network rather than cis-regulatory changes. The divergent expression of unusual floral organs (UFO) from Arabidopsis, and the petunia homolog double top (DOT), however, is caused by the loss or gain of cis-regulatory promoter elements, which respond to trans-acting factors that are expressed in similar patterns in both species. Introduction of pUFO:UFO causes no obvious defects in Arabidopsis, but in petunia it causes the precocious and ectopic formation of flowers. This provides an example of how a change in a cis-regulatory region can account for a change in the plant body plan. © 2015. Published by The Company of Biologists Ltd.

The Segregated Expression of Voltage-Gated Potassium and Sodium Channels in Neuronal Membranes: Functional Implications and Regulatory Mechanisms

PubMed Central

Duménieu, Maël; Oulé, Marie; Kreutz, Michael R.; Lopez-Rojas, Jeffrey

2017-01-01

Neurons are highly polarized cells with apparent functional and morphological differences between dendrites and axon. A critical determinant for the molecular and functional identity of axonal and dendritic segments is the restricted expression of voltage-gated ion channels (VGCs). Several studies show an uneven distribution of ion channels and their differential regulation within dendrites and axons, which is a prerequisite for an appropriate integration of synaptic inputs and the generation of adequate action potential (AP) firing patterns. This review article will focus on the signaling pathways leading to segmented expression of voltage-gated potassium and sodium ion channels at the neuronal plasma membrane and the regulatory mechanisms ensuring segregated functions. We will also discuss the relevance of proper ion channel targeting for neuronal physiology and how alterations in polarized distribution contribute to neuronal pathology. PMID:28484374

Mitochondrial antibodies in primary biliary cirrhosis

PubMed Central

Berg, P. A.; Roitt, I. M.; Doniach, D.; Cooper, H. M.

1969-01-01

The effect on the mitochondrial antigen of different agents known to influence the integrity and structure of membranes has been studied using quantitative complement fixation with autoantibodies from the serum of a patient with primary biliary cirrhosis. The susceptibility to proteolytic enzymes suggests that the antigen is a protein. Activity depends upon an association with phospholipids. Addition of phospholipids prevents loss of antigen during artificial ageing of mitochondria at 37°. Activity is lost after treatment with phospholipases or solvents which extract phospholipids. Antigen is also destroyed by surface active agents which dissociate the link with phospholipid but those which weaken bonds between phospholipids and hydrophobic molecules yield fragments of antigen-containing membrane structures which, nonetheless, still react with the mitochondrial autoantibody. ImagesFIG. 2FIG. 4 PMID:5804537

GFP-complementation assay to detect functional CPP and protein delivery into living cells

PubMed Central

Milech, Nadia; Longville, Brooke AC; Cunningham, Paula T; Scobie, Marie N; Bogdawa, Heique M; Winslow, Scott; Anastasas, Mark; Connor, Theresa; Ong, Ferrer; Stone, Shane R; Kerfoot, Maria; Heinrich, Tatjana; Kroeger, Karen M; Tan, Yew-Foon; Hoffmann, Katrin; Thomas, Wayne R; Watt, Paul M; Hopkins, Richard M

2015-01-01

Efficient cargo uptake is essential for cell-penetrating peptide (CPP) therapeutics, which deliver widely diverse cargoes by exploiting natural cell processes to penetrate the cell’s membranes. Yet most current CPP activity assays are hampered by limitations in assessing uptake, including confounding effects of conjugated fluorophores or ligands, indirect read-outs requiring secondary processing, and difficulty in discriminating internalization from endosomally trapped cargo. Split-complementation Endosomal Escape (SEE) provides the first direct assay visualizing true cytoplasmic-delivery of proteins at biologically relevant concentrations. The SEE assay has minimal background, is amenable to high-throughput processes, and adaptable to different transient and stable cell lines. This split-GFP-based platform can be useful to study transduction mechanisms, cellular imaging, and characterizing novel CPPs as pharmaceutical delivery agents in the treatment of disease. PMID:26671759

Intramolecular Dynamics within the N-Cap-SH3-SH2 Regulatory Unit of the c-Abl Tyrosine Kinase Reveal Targeting to the Cellular Membrane*♦

PubMed Central

de Oliveira, Guilherme A. P.; Pereira, Elen G.; Ferretti, Giulia D. S.; Valente, Ana Paula; Cordeiro, Yraima; Silva, Jerson L.

2013-01-01

c-Abl is a key regulator of cell signaling and is under strict control via intramolecular interactions. In this study, we address changes in the intramolecular dynamics coupling within the c-Abl regulatory unit by presenting its N-terminal segment (N-Cap) with an alternative function in the cell as c-Abl becomes activated. Using small angle x-ray scattering, nuclear magnetic resonance, and confocal microscopy, we demonstrate that the N-Cap and the Src homology (SH) 3 domain acquire μs-ms motions upon N-Cap association with the SH2-L domain, revealing a stabilizing synergy between these segments. The N-Cap-myristoyl tether likely triggers the protein to anchor to the membrane because of these flip-flop dynamics, which occur in the μs-ms time range. This segment not only presents the myristate during c-Abl inhibition but may also trigger protein localization inside the cell in a functional and stability-dependent mechanism that is lost in Bcr-Abl+ cells, which underlie chronic myeloid leukemia. This loss of intramolecular dynamics and binding to the cellular membrane is a potential therapeutic target. PMID:23928308

Intramolecular dynamics within the N-Cap-SH3-SH2 regulatory unit of the c-Abl tyrosine kinase reveal targeting to the cellular membrane.

PubMed

de Oliveira, Guilherme A P; Pereira, Elen G; Ferretti, Giulia D S; Valente, Ana Paula; Cordeiro, Yraima; Silva, Jerson L

2013-09-27

c-Abl is a key regulator of cell signaling and is under strict control via intramolecular interactions. In this study, we address changes in the intramolecular dynamics coupling within the c-Abl regulatory unit by presenting its N-terminal segment (N-Cap) with an alternative function in the cell as c-Abl becomes activated. Using small angle x-ray scattering, nuclear magnetic resonance, and confocal microscopy, we demonstrate that the N-Cap and the Src homology (SH) 3 domain acquire μs-ms motions upon N-Cap association with the SH2-L domain, revealing a stabilizing synergy between these segments. The N-Cap-myristoyl tether likely triggers the protein to anchor to the membrane because of these flip-flop dynamics, which occur in the μs-ms time range. This segment not only presents the myristate during c-Abl inhibition but may also trigger protein localization inside the cell in a functional and stability-dependent mechanism that is lost in Bcr-Abl(+) cells, which underlie chronic myeloid leukemia. This loss of intramolecular dynamics and binding to the cellular membrane is a potential therapeutic target.

Spatio-mechanical EphA2/ephrin-A1 Signaling in Cancer Cells

NASA Astrophysics Data System (ADS)

Xu, Qian

2011-12-01

Communication strategies in nature are an integral part to the survival of multi-cellular organisms. Cell membranes provide the chemical environment in which intercellular signaling begins. The vast complexity of this signaling requires that a relatively conserved set of chemical constituents be able to generate enormous signal diversity. Spatial sorting of signaling molecules within the membrane allows for this diversity. My research uses synthetic lipid membranes, solid-state nanostructures, and high-resolution imaging to study a potentially novel spatio-mechanical regulatory mechanism in the EphA2 signaling pathway. My hypothesis is that the multi-scale organization of the EphA2 receptor in the cell membrane regulates its biochemical function. This hypothesis is motivated by the idea that extracellular mechanical inputs have an important role in intracellular signaling cascades.

What makes a natural clay antibacterial?

USGS Publications Warehouse

Williams, Lynda B.; Metge, David W.; Eberl, Dennis D.; Harvey, Ronald W.; Turner, Amanda G.; Prapaipong, Panjai; Port-Peterson, Amisha T.

2011-01-01

Chemical analyses of E. coli killed by aqueous leachates of an antibacterial clay show that intracellular concentrations of Fe and P are elevated relative to controls. Phosphorus uptake by the cells supports a regulatory role of polyphosphate or phospholipids in controlling Fe2+. Fenton reaction products can degrade critical cell components, but we deduce that extracellular processes do not cause cell death. Rather, Fe2+ overwhelms outer membrane regulatory proteins and is oxidized when it enters the cell, precipitating Fe3+ and producing lethal hydroxyl radicals.

Glomerular clusterin is associated with PKC-alpha/beta regulation and good outcome of membranous glomerulonephritis in humans.

PubMed

Rastaldi, M P; Candiano, G; Musante, L; Bruschi, M; Armelloni, S; Rimoldi, L; Tardanico, R; Sanna-Cherchi, S; Cherchi, S Sanna; Ferrario, F; Montinaro, V; Haupt, R; Parodi, S; Carnevali, M L; Allegri, L; Camussi, G; Gesualdo, L; Scolari, F; Ghiggeri, G M

2006-08-01

Mechanisms for human membranous glomerulonephritis (MGN) remain elusive. Most up-to-date concepts still rely on the rat model of Passive Heymann Nephritis that derives from an autoimmune response to glomerular megalin, with complement activation and membrane attack complex assembly. Clusterin has been reported as a megalin ligand in immunodeposits, although its role has not been clarified. We studied renal biopsies of 60 MGN patients by immunohistochemistry utilizing antibodies against clusterin, C5b-9, and phosphorylated-protien kinase C (PKC) isoforms (pPKC). In vitro experiments were performed to investigate the role of clusterin during podocyte damage by MGN serum and define clusterin binding to human podocytes, where megalin is known to be absent. Clusterin, C5b-9, and pPKC-alpha/beta showed highly variable glomerular staining, where high clusterin profiles were inversely correlated to C5b-9 and PKC-alpha/beta expression (P=0.029), and co-localized with the low-density lipoprotein receptor (LDL-R). Glomerular clusterin emerged as the single factor influencing proteinuria at multivariate analysis and was associated with a reduction of proteinuria after a follow-up of 1.5 years (-88.1%, P=0.027). Incubation of podocytes with MGN sera determined strong upregulation of pPKC-alpha/beta that was reverted by pre-incubation with clusterin, serum de-complementation, or protein-A treatment. Preliminary in vitro experiments showed podocyte binding of biotinilated clusterin, co-localization with LDL-R and specific binding inhibition with anti-LDL-R antibodies and with specific ligands. These data suggest a central role for glomerular clusterin in MGN as a modulator of inflammation that potentially influences the clinical outcome. Binding of clusterin to the LDL-R might offer an interpretative key for the pathogenesis of MGN in humans.

Untangling the evolution of Rab G proteins: implications of a comprehensive genomic analysis

PubMed Central

2012-01-01

Background Membrane-bound organelles are a defining feature of eukaryotic cells, and play a central role in most of their fundamental processes. The Rab G proteins are the single largest family of proteins that participate in the traffic between organelles, with 66 Rabs encoded in the human genome. Rabs direct the organelle-specific recruitment of vesicle tethering factors, motor proteins, and regulators of membrane traffic. Each organelle or vesicle class is typically associated with one or more Rab, with the Rabs present in a particular cell reflecting that cell's complement of organelles and trafficking routes. Results Through iterative use of hidden Markov models and tree building, we classified Rabs across the eukaryotic kingdom to provide the most comprehensive view of Rab evolution obtained to date. A strikingly large repertoire of at least 20 Rabs appears to have been present in the last eukaryotic common ancestor (LECA), consistent with the 'complexity early' view of eukaryotic evolution. We were able to place these Rabs into six supergroups, giving a deep view into eukaryotic prehistory. Conclusions Tracing the fate of the LECA Rabs revealed extensive losses with many extant eukaryotes having fewer Rabs, and none having the full complement. We found that other Rabs have expanded and diversified, including a large expansion at the dawn of metazoans, which could be followed to provide an account of the evolutionary history of all human Rabs. Some Rab changes could be correlated with differences in cellular organization, and the relative lack of variation in other families of membrane-traffic proteins suggests that it is the changes in Rabs that primarily underlies the variation in organelles between species and cell types. PMID:22873208

A Conserved Circular Network of Coregulated Lipids Modulates Innate Immune Responses

PubMed Central

Köberlin, Marielle S.; Snijder, Berend; Heinz, Leonhard X.; Baumann, Christoph L.; Fauster, Astrid; Vladimer, Gregory I.; Gavin, Anne-Claude; Superti-Furga, Giulio

2015-01-01

Summary Lipid composition affects the biophysical properties of membranes that provide a platform for receptor-mediated cellular signaling. To study the regulatory role of membrane lipid composition, we combined genetic perturbations of sphingolipid metabolism with the quantification of diverse steps in Toll-like receptor (TLR) signaling and mass spectrometry-based lipidomics. Membrane lipid composition was broadly affected by these perturbations, revealing a circular network of coregulated sphingolipids and glycerophospholipids. This evolutionarily conserved network architecture simultaneously reflected membrane lipid metabolism, subcellular localization, and adaptation mechanisms. Integration of the diverse TLR-induced inflammatory phenotypes with changes in lipid abundance assigned distinct functional roles to individual lipid species organized across the network. This functional annotation accurately predicted the inflammatory response of cells derived from patients suffering from lipid storage disorders, based solely on their altered membrane lipid composition. The analytical strategy described here empowers the understanding of higher-level organization of membrane lipid function in diverse biological systems. PMID:26095250

A cellular backline: specialization of host membranes for defence.

PubMed

Faulkner, Christine

2015-03-01

In plant-pathogen interactions, the host plasma membrane serves as a defence front for pathogens that invade from the extracellular environment. As such, the lipid bilayer acts as a scaffold that targets and delivers defence responses to the site of attack. During pathogen infection, numerous changes in plasma membrane composition, organization, and structure occur. There is increasing evidence that this facilitates the execution of a variety of responses, highlighting the regulatory role membranes play in cellular responses. Membrane microdomains such as lipid rafts are hypothesized to create signalling platforms for receptor signalling in response to pathogen perception and for callose synthesis. Further, the genesis of pathogen-associated structures such as papillae and the extra-haustorial membrane necessitates polarization of membranes and membrane trafficking pathways. Unlocking the mechanisms by which this occurs will enable greater understanding of how targeted defences, some of which result in resistance, are executed. This review will survey some of the changes that occur in host membranes during pathogen attack and how these are associated with the generation of defence responses. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Incorporating comparative genomics into the design-test-learn cycle of microbial strain engineering.

PubMed

Sardi, Maria; Gasch, Audrey P

2017-08-01

Engineering microbes with new properties is an important goal in industrial engineering, to establish biological factories for production of biofuels, commodity chemicals and pharmaceutics. But engineering microbes to produce new compounds with high yield remains a major challenge toward economically viable production. Incorporating several modern approaches, including synthetic and systems biology, metabolic modeling and regulatory rewiring, has proven to significantly advance industrial strain engineering. This review highlights how comparative genomics can also facilitate strain engineering, by identifying novel genes and pathways, regulatory mechanisms and genetic background effects for engineering. We discuss how incorporating comparative genomics into the design-test-learn cycle of strain engineering can provide novel information that complements other engineering strategies. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Arabidopsis PPP family of serine/threonine protein phosphatases: many targets but few engines.

PubMed

Uhrig, R Glen; Labandera, Anne-Marie; Moorhead, Greg B

2013-09-01

The major plant serine/threonine protein phosphatases belong to the phosphoprotein phosphatase (PPP) family. Over the past few years the complement of Arabidopsis thaliana PPP family of catalytic subunits has been cataloged and many regulatory subunits identified. Specific roles for PPPs have been characterized, including roles in auxin and brassinosteroid signaling, in phototropism, in regulating the target of rapamycin pathway, and in cell stress responses. In this review, we provide a framework for understanding the PPP family by exploring the fundamental role of the phosphatase regulatory subunits that drive catalytic engine specificity. Although there are fewer plant protein phosphatases compared with their protein kinase partners, their function is now recognized to be as dynamic and as regulated as that of protein kinases. Copyright © 2013 Elsevier Ltd. All rights reserved.

Reconstructing the regulatory network controlling commitment and sporulation in Physarum polycephalum based on hierarchical Petri Net modelling and simulation.

PubMed

Marwan, Wolfgang; Sujatha, Arumugam; Starostzik, Christine

2005-10-21

We reconstruct the regulatory network controlling commitment and sporulation of Physarum polycephalum from experimental results using a hierarchical Petri Net-based modelling and simulation framework. The stochastic Petri Net consistently describes the structure and simulates the dynamics of the molecular network as analysed by genetic, biochemical and physiological experiments within a single coherent model. The Petri Net then is extended to simulate time-resolved somatic complementation experiments performed by mixing the cytoplasms of mutants altered in the sporulation response, to systematically explore the network structure and to probe its dynamics. This reverse engineering approach presumably can be employed to explore other molecular or genetic signalling systems where the activity of genes or their products can be experimentally controlled in a time-resolved manner.

Morphological and biochemical characterization of the membranous hepatitis C virus replication compartment.

PubMed

Paul, David; Hoppe, Simone; Saher, Gesine; Krijnse-Locker, Jacomine; Bartenschlager, Ralf

2013-10-01

Like all other positive-strand RNA viruses, hepatitis C virus (HCV) induces rearrangements of intracellular membranes that are thought to serve as a scaffold for the assembly of the viral replicase machinery. The most prominent membranous structures present in HCV-infected cells are double-membrane vesicles (DMVs). However, their composition and role in the HCV replication cycle are poorly understood. To gain further insights into the biochemcial properties of HCV-induced membrane alterations, we generated a functional replicon containing a hemagglutinin (HA) affinity tag in nonstructural protein 4B (NS4B), the supposed scaffold protein of the viral replication complex. By using HA-specific affinity purification we isolated NS4B-containing membranes from stable replicon cells. Complementing biochemical and electron microscopy analyses of purified membranes revealed predominantly DMVs, which contained viral proteins NS3 and NS5A as well as enzymatically active viral replicase capable of de novo synthesis of HCV RNA. In addition to viral factors, co-opted cellular proteins, such as vesicle-associated membrane protein-associated protein A (VAP-A) and VAP-B, that are crucial for viral RNA replication, as well as cholesterol, a major structural lipid of detergent-resistant membranes, are highly enriched in DMVs. Here we describe the first isolation and biochemical characterization of HCV-induced DMVs. The results obtained underline their central role in the HCV replication cycle and suggest that DMVs are sites of viral RNA replication. The experimental approach described here is a powerful tool to more precisely define the molecular composition of membranous replication factories induced by other positive-strand RNA viruses, such as picorna-, arteri- and coronaviruses.

Morphological and Biochemical Characterization of the Membranous Hepatitis C Virus Replication Compartment

PubMed Central

Hoppe, Simone; Saher, Gesine; Krijnse-Locker, Jacomine

2013-01-01

Like all other positive-strand RNA viruses, hepatitis C virus (HCV) induces rearrangements of intracellular membranes that are thought to serve as a scaffold for the assembly of the viral replicase machinery. The most prominent membranous structures present in HCV-infected cells are double-membrane vesicles (DMVs). However, their composition and role in the HCV replication cycle are poorly understood. To gain further insights into the biochemcial properties of HCV-induced membrane alterations, we generated a functional replicon containing a hemagglutinin (HA) affinity tag in nonstructural protein 4B (NS4B), the supposed scaffold protein of the viral replication complex. By using HA-specific affinity purification we isolated NS4B-containing membranes from stable replicon cells. Complementing biochemical and electron microscopy analyses of purified membranes revealed predominantly DMVs, which contained viral proteins NS3 and NS5A as well as enzymatically active viral replicase capable of de novo synthesis of HCV RNA. In addition to viral factors, co-opted cellular proteins, such as vesicle-associated membrane protein-associated protein A (VAP-A) and VAP-B, that are crucial for viral RNA replication, as well as cholesterol, a major structural lipid of detergent-resistant membranes, are highly enriched in DMVs. Here we describe the first isolation and biochemical characterization of HCV-induced DMVs. The results obtained underline their central role in the HCV replication cycle and suggest that DMVs are sites of viral RNA replication. The experimental approach described here is a powerful tool to more precisely define the molecular composition of membranous replication factories induced by other positive-strand RNA viruses, such as picorna-, arteri- and coronaviruses. PMID:23885072

The effectiveness of styrene-maleic acid (SMA) copolymers for solubilisation of integral membrane proteins from SMA-accessible and SMA-resistant membranes.

PubMed

Swainsbury, David J K; Scheidelaar, Stefan; Foster, Nicholas; van Grondelle, Rienk; Killian, J Antoinette; Jones, Michael R

2017-10-01

Solubilisation of biological lipid bilayer membranes for analysis of their protein complement has traditionally been carried out using detergents, but there is increasing interest in the use of amphiphilic copolymers such as styrene maleic acid (SMA) for the solubilisation, purification and characterisation of integral membrane proteins in the form of protein/lipid nanodiscs. Here we survey the effectiveness of various commercially-available formulations of the SMA copolymer in solubilising Rhodobacter sphaeroides reaction centres (RCs) from photosynthetic membranes. We find that formulations of SMA with a 2:1 or 3:1 ratio of styrene to maleic acid are almost as effective as detergent in solubilising RCs, with the best solubilisation by short chain variants (<30kDa weight average molecular weight). The effectiveness of 10kDa 2:1 and 3:1 formulations of SMA to solubilise RCs gradually declined when genetically-encoded coiled-coil bundles were used to artificially tether normally monomeric RCs into dimeric, trimeric and tetrameric multimers. The ability of SMA to solubilise reaction centre-light harvesting 1 (RC-LH1) complexes from densely packed and highly ordered photosynthetic membranes was uniformly low, but could be increased through a variety of treatments to increase the lipid:protein ratio. However, proteins isolated from such membranes comprised clusters of complexes in small membrane patches rather than individual proteins. We conclude that short-chain 2:1 and 3:1 formulations of SMA are the most effective in solubilising integral membrane proteins, but that solubilisation efficiencies are strongly influenced by the size of the target protein and the density of packing of proteins in the membrane. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Suppression of complement regulatory protein C1 inhibitor in vascular endothelial activation by inhibiting vascular cell adhesion molecule-1 action

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Haimou; Qin, Gangjian; Liang, Gang

Increased expression of adhesion molecules by activated endothelium is a critical feature of vascular inflammation associated with the several diseases such as endotoxin shock and sepsis/septic shock. Our data demonstrated complement regulatory protein C1 inhibitor (C1INH) prevents endothelial cell injury. We hypothesized that C1INH has the ability of an anti-endothelial activation associated with suppression of expression of adhesion molecule(s). C1INH blocked leukocyte adhesion to endothelial cell monolayer in both static assay and flow conditions. In inflammatory condition, C1INH reduced vascular cell adhesion molecule (VCAM-1) expression associated with its cytoplasmic mRNA destabilization and nuclear transcription level. Studies exploring the underlying mechanismmore » of C1INH-mediated suppression in VCAM-1 expression were related to reduction of NF-{kappa}B activation and nuclear translocation in an I{kappa}B{alpha}-dependent manner. The inhibitory effects were associated with reduction of inhibitor I{kappa}B kinase activity and stabilization of the NF-{kappa}B inhibitor I{kappa}B. These findings indicate a novel role for C1INH in inhibition of vascular endothelial activation. These observations could provide the basis for new therapeutic application of C1INH to target inflammatory processes in different pathologic situations.« less

Reducing abrupt climate change risk using the Montreal Protocol and other regulatory actions to complement cuts in CO2 emissions.

PubMed

Molina, Mario; Zaelke, Durwood; Sarma, K Madhava; Andersen, Stephen O; Ramanathan, Veerabhadran; Kaniaru, Donald

2009-12-08

Current emissions of anthropogenic greenhouse gases (GHGs) have already committed the planet to an increase in average surface temperature by the end of the century that may be above the critical threshold for tipping elements of the climate system into abrupt change with potentially irreversible and unmanageable consequences. This would mean that the climate system is close to entering if not already within the zone of "dangerous anthropogenic interference" (DAI). Scientific and policy literature refers to the need for "early," "urgent," "rapid," and "fast-action" mitigation to help avoid DAI and abrupt climate changes. We define "fast-action" to include regulatory measures that can begin within 2-3 years, be substantially implemented in 5-10 years, and produce a climate response within decades. We discuss strategies for short-lived non-CO(2) GHGs and particles, where existing agreements can be used to accomplish mitigation objectives. Policy makers can amend the Montreal Protocol to phase down the production and consumption of hydrofluorocarbons (HFCs) with high global warming potential. Other fast-action strategies can reduce emissions of black carbon particles and precursor gases that lead to ozone formation in the lower atmosphere, and increase biosequestration, including through biochar. These and other fast-action strategies may reduce the risk of abrupt climate change in the next few decades by complementing cuts in CO(2) emissions.

Genetic analysis of indefinite division in human cells: Evidence for a cell senescence-related gene(s) on human chromosome 4

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yi Ning; Ledbetter, D.H.; Smith, J.R.

1991-07-01

Earlier studies had demonstrated that fusion of normal with immortal human cells yielded hybrids having limited division potential. This indicated that the phenotype of limited proliferation (cellular senescence) is dominant and that immortal cells result from recessive changes in normal growth-regulatory genes. In additional studies, the authors exploited the fact that the immortal phenotype is recessive and, by fusing various immortal human cell lines with each other, identified four complementation groups for indefinite division. Assignment of cell lines to specific groups allowed us to take a focused approach to identify the chromosomes and genes involved in growth regulation that havemore » been modified in immortal cells. They report here that introduction of a normal human chromosome 4 into three immortal cell lines (HeLa, J82, T98G) assigned to complementation group B resulted in loss of proliferation and reversal of the immortal phenotype. No effect on the proliferation potential of cell lines representative of the other complementation groups was observed. This result suggests that a gene(s) involved in cellular senescence and normal growth regulation resides on chromosome 4.« less

MEMBRANE TECHNOLOGIES FOR REMEDIATING CONTAMINATED SOILS: A CRITICAL REVIEW

EPA Science Inventory

Regulatory compliance requires the cleanup of soils contaminated with toxic organic and metallic compounds. Several chemical and thermal detoxification technologies have been tested on soils excavated from contaminated sites. Soil washing with aqueous solutions transfers the cont...

Demonstration and Validation of a Regenerated Cellulose Dialysis Membrane Diffusion Sampler for Monitoring Ground-Water Quality and Remediation Progress at DoD Site: Perchlorate and Ordnance Compounds

DTIC Science & Technology

2011-10-01

Regulatory Council LDPE low-density polyethylene MDL minimum detection limit NAVFAC ESC Naval Facilities Engineering Command Engineering Service...membrane sampler design consists of a tubular-shaped bag made of flexible low-density polyethylene ( LDPE ) (Vroblesky, 2001a, 2001b). The LDPE tube is...requirements, and can be constructed from small-diameter LDPE tubing that fits into small- 4 diameter wells. These polyethylene diffusion bag

The MARVEL transmembrane motif of occludin mediates oligomerization and targeting to the basolateral surface in epithelia.

PubMed

Yaffe, Yakey; Shepshelovitch, Jeanne; Nevo-Yassaf, Inbar; Yeheskel, Adva; Shmerling, Hedva; Kwiatek, Joanna M; Gaus, Katharina; Pasmanik-Chor, Metsada; Hirschberg, Koret

2012-08-01

Occludin (Ocln), a MARVEL-motif-containing protein, is found in all tight junctions. MARVEL motifs are comprised of four transmembrane helices associated with the localization to or formation of diverse membrane subdomains by interacting with the proximal lipid environment. The functions of the Ocln MARVEL motif are unknown. Bioinformatics sequence- and structure-based analyses demonstrated that the MARVEL domain of Ocln family proteins has distinct evolutionarily conserved sequence features that are consistent with its basolateral membrane localization. Live-cell microscopy, fluorescence resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC) were used to analyze the intracellular distribution and self-association of fluorescent-protein-tagged full-length human Ocln or the Ocln MARVEL motif excluding the cytosolic C- and N-termini (amino acids 60-269, FP-MARVEL-Ocln). FP-MARVEL-Ocln efficiently arrived at the plasma membrane (PM) and was sorted to the basolateral PM in filter-grown polarized MDCK cells. A series of conserved aromatic amino acids within the MARVEL domain were found to be associated with Ocln dimerization using BiFC. FP-MARVEL-Ocln inhibited membrane pore growth during Triton-X-100-induced solubilization and was shown to increase the membrane-ordered state using Laurdan, a lipid dye. These data demonstrate that the Ocln MARVEL domain mediates self-association and correct sorting to the basolateral membrane.

Selective Regulation of Maize Plasma Membrane Aquaporin Trafficking and Activity by the SNARE SYP121[W

PubMed Central

Besserer, Arnaud; Burnotte, Emeline; Bienert, Gerd Patrick; Chevalier, Adrien S.; Errachid, Abdelmounaim; Grefen, Christopher; Blatt, Michael R.; Chaumont, François

2012-01-01

Plasma membrane intrinsic proteins (PIPs) are aquaporins facilitating the diffusion of water through the cell membrane. We previously showed that the traffic of the maize (Zea mays) PIP2;5 to the plasma membrane is dependent on the endoplasmic reticulum diacidic export motif. Here, we report that the post-Golgi traffic and water channel activity of PIP2;5 are regulated by the SNARE (for soluble N-ethylmaleimide-sensitive factor protein attachment protein receptor) SYP121, a plasma membrane resident syntaxin involved in vesicle traffic, signaling, and regulation of K+ channels. We demonstrate that the expression of the dominant-negative SYP121-Sp2 fragment in maize mesophyll protoplasts or epidermal cells leads to a decrease in the delivery of PIP2;5 to the plasma membrane. Protoplast and oocyte swelling assays showed that PIP2;5 water channel activity is negatively affected by SYP121-Sp2. A combination of in vitro (copurification assays) and in vivo (bimolecular fluorescence complementation, Förster resonance energy transfer, and yeast split-ubiquitin) approaches allowed us to demonstrate that SYP121 and PIP2;5 physically interact. Together with previous data demonstrating the role of SYP121 in regulating K+ channel trafficking and activity, these results suggest that SYP121 SNARE contributes to the regulation of the cell osmotic homeostasis. PMID:22942383

Selective regulation of maize plasma membrane aquaporin trafficking and activity by the SNARE SYP121.

PubMed

Besserer, Arnaud; Burnotte, Emeline; Bienert, Gerd Patrick; Chevalier, Adrien S; Errachid, Abdelmounaim; Grefen, Christopher; Blatt, Michael R; Chaumont, François

2012-08-01

Plasma membrane intrinsic proteins (PIPs) are aquaporins facilitating the diffusion of water through the cell membrane. We previously showed that the traffic of the maize (Zea mays) PIP2;5 to the plasma membrane is dependent on the endoplasmic reticulum diacidic export motif. Here, we report that the post-Golgi traffic and water channel activity of PIP2;5 are regulated by the SNARE (for soluble N-ethylmaleimide-sensitive factor protein attachment protein receptor) SYP121, a plasma membrane resident syntaxin involved in vesicle traffic, signaling, and regulation of K(+) channels. We demonstrate that the expression of the dominant-negative SYP121-Sp2 fragment in maize mesophyll protoplasts or epidermal cells leads to a decrease in the delivery of PIP2;5 to the plasma membrane. Protoplast and oocyte swelling assays showed that PIP2;5 water channel activity is negatively affected by SYP121-Sp2. A combination of in vitro (copurification assays) and in vivo (bimolecular fluorescence complementation, Förster resonance energy transfer, and yeast split-ubiquitin) approaches allowed us to demonstrate that SYP121 and PIP2;5 physically interact. Together with previous data demonstrating the role of SYP121 in regulating K(+) channel trafficking and activity, these results suggest that SYP121 SNARE contributes to the regulation of the cell osmotic homeostasis.

Membrane remodeling by amyloidogenic and non-amyloidogenic proteins studied by EPR

NASA Astrophysics Data System (ADS)

Varkey, Jobin; Langen, Ralf

2017-07-01

The advancement in site-directed spin labeling of proteins has enabled EPR studies to expand into newer research areas within the umbrella of protein-membrane interactions. Recently, membrane remodeling by amyloidogenic and non-amyloidogenic proteins has gained a substantial interest in relation to driving and controlling vital cellular processes such as endocytosis, exocytosis, shaping of organelles like endoplasmic reticulum, Golgi and mitochondria, intracellular vesicular trafficking, formation of filopedia and multivesicular bodies, mitochondrial fusion and fission, and synaptic vesicle fusion and recycling in neurotransmission. Misregulation in any of these processes due to an aberrant protein (mutation or misfolding) or alteration of lipid metabolism can be detrimental to the cell and cause disease. Dissection of the structural basis of membrane remodeling by proteins is thus quite necessary for an understanding of the underlying mechanisms, but it remains a formidable task due to the difficulties of various common biophysical tools in monitoring the dynamic process of membrane binding and bending by proteins. This is largely since membranes generally complicate protein structure analysis and this problem is amplified for structural analysis in the presence of different types of membrane curvatures. Recent EPR studies on membrane remodeling by proteins show that a significant structural information can be generated to delineate the role of different protein modules, domains and individual amino acids in the generation of membrane curvature. These studies also show how EPR can complement the data obtained by high resolution techniques such as X-ray and NMR. This perspective covers the application of EPR in recent studies for understanding membrane remodeling by amyloidogenic and non-amyloidogenic proteins that is useful for researchers interested in using or complimenting EPR to gain better understanding of membrane remodeling. We also discuss how a single protein can generate different type of membrane curvatures using specific conformations for specific membrane structures and how EPR is a versatile tool well-suited to analyze subtle alterations in structures under such modifying conditions which otherwise would have been difficult using other biophysical tools.

Complement Factor H Is Expressed in Adipose Tissue in Association With Insulin Resistance

PubMed Central

Moreno-Navarrete, José María; Martínez-Barricarte, Rubén; Catalán, Victoria; Sabater, Mònica; Gómez-Ambrosi, Javier; Ortega, Francisco José; Ricart, Wifredo; Blüher, Mathias; Frühbeck, Gema; Rodríguez de Cordoba, Santiago; Fernández-Real, José Manuel

2010-01-01

OBJECTIVE Activation of the alternative pathway of the complement system, in which factor H (fH; complement fH [CFH]) is a key regulatory component, has been suggested as a link between obesity and metabolic disorders. Our objective was to study the associations between circulating and adipose tissue gene expressions of CFH and complement factor B (fB; CFB) with obesity and insulin resistance. RESEARCH DESIGN AND METHODS Circulating fH and fB were determined by enzyme-linked immunosorbent assay in 398 subjects. CFH and CFB gene expressions were evaluated in 76 adipose tissue samples, in isolated adipocytes, and in stromovascular cells (SVC) (n = 13). The effects of weight loss and rosiglitazone were investigated in independent cohorts. RESULTS Both circulating fH and fB were associated positively with BMI, waist circumference, triglycerides, and inflammatory parameters and negatively with insulin sensitivity and HDL cholesterol. For the first time, CFH gene expression was detected in human adipose tissue (significantly increased in subcutaneous compared with omental fat). CFH gene expression in omental fat was significantly associated with insulin resistance. In contrast, CFB gene expression was significantly increased in omental fat but also in association with fasting glucose and triglycerides. The SVC fraction was responsible for these differences, although isolated adipocytes also expressed fB and fH at low levels. Both weight loss and rosiglitazone led to significantly decreased circulating fB and fH levels. CONCLUSIONS Increased circulating fH and fB concentrations in subjects with altered glucose tolerance could reflect increased SVC-induced activation of the alternative pathway of complement in omental adipose tissue linked to insulin resistance and metabolic disturbances. PMID:19833879

Mapping the local organization of cell membranes using excitation-polarization-resolved confocal fluorescence microscopy.

PubMed

Kress, Alla; Wang, Xiao; Ranchon, Hubert; Savatier, Julien; Rigneault, Hervé; Ferrand, Patrick; Brasselet, Sophie

2013-07-02

Fluorescence anisotropy and linear dichroism imaging have been widely used for imaging biomolecular orientational distributions in protein aggregates, fibrillar structures of cells, and cell membranes. However, these techniques do not give access to complete orientational order information in a whole image, because their use is limited to parts of the sample where the average orientation of molecules is known a priori. Fluorescence anisotropy is also highly sensitive to depolarization mechanisms such as those induced by fluorescence energy transfer. A fully excitation-polarization-resolved fluorescence microscopy imaging that relies on the use of a tunable incident polarization and a nonpolarized detection is able to circumvent these limitations. We have developed such a technique in confocal epifluorescence microscopy, giving access to new regions of study in the complex and heterogeneous molecular organization of cell membranes. Using this technique, we demonstrate morphological changes at the subdiffraction scale in labeled COS-7 cell membranes whose cytoskeleton is perturbed. Molecular orientational order is also seen to be affected by cholesterol depletion, reflecting the strong interplay between lipid-packing regions and their nearby cytoskeleton. This noninvasive optical technique can reveal local organization in cell membranes when used as a complement to existing methods such as generalized polarization. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

A role for CSLD3 during cell-wall synthesis in apical plasma membranes of tip-growing root-hair cells.

PubMed

Park, Sungjin; Szumlanski, Amy L; Gu, Fangwei; Guo, Feng; Nielsen, Erik

2011-07-17

In plants, cell shape is defined by the cell wall, and changes in cell shape and size are dictated by modification of existing cell walls and deposition of newly synthesized cell-wall material. In root hairs, expansion occurs by a process called tip growth, which is shared by root hairs, pollen tubes and fungal hyphae. We show that cellulose-like polysaccharides are present in root-hair tips, and de novo synthesis of these polysaccharides is required for tip growth. We also find that eYFP-CSLD3 proteins, but not CESA cellulose synthases, localize to a polarized plasma-membrane domain in root hairs. Using biochemical methods and genetic complementation of a csld3 mutant with a chimaeric CSLD3 protein containing a CESA6 catalytic domain, we provide evidence that CSLD3 represents a distinct (1→4)-β-glucan synthase activity in apical plasma membranes during tip growth in root-hair cells.

Super-Anticoagulant Heparin-Mimicking Hydrogel Thin Film Attached Substrate Surfaces to Improve Hemocompatibility.

PubMed

He, Min; Cui, Xiaofei; Jiang, Huiyi; Huang, Xuelian; Zhao, Weifeng; Zhao, Changsheng

2017-02-01

In this study, heparin-mimicking hydrogel thin films are covalently attached onto poly(ether sulfone) membrane surfaces to improve anticoagulant property. The hydrogel films display honeycomb-like porous structure with well controlled thickness and show long-term stability. After immobilizing the hydrogel films, the membranes show excellent anticoagulant property confirmed by the activated partial thromboplastin time values exceeding 600 s. Meanwhile, the thrombin time values increase from 20 to 61 s as the sodium allysulfonate proportions increase from 0 to 80 mol%. In vitro investigations of protein adsorption and blood-related complement activation also confirm that the membranes exhibit super-anticoagulant property. Furthermore, gentamycin sulfate is loaded into the hydrogel films, and the released drug shows significant inhibition toward E. coli bacteria. It is believed that the surface attached heparin-mimicking hydrogel thin films may show high potential for the applications in various biological fields, such as blood contacting materials and drug loading materials. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

X-ray Reflectivity Characterization of Ion Distribution at Biomimetic Membrane Surfaces

NASA Astrophysics Data System (ADS)

Krüger, Peter; Pittler, Jens; Vaknin, David; Lösche, Mathias

2003-03-01

Ions at cell membrane surfaces may control the function and conformation of nearby biomolecules, thus playing an important role in inter- and intracellular transport as well as in biorecognition processes. Moreover, charge patterns at membrane surfaces may direct the growth of inorganic crystals in biomineralization. Langmuir monolayers are widely employed as model systems for studying charge distribution and growth processes at the organic/inorganic interface. We present a novel x-ray reflectivity technique that provides detailed information on ion distribution at biomembrane surfaces by using monochromatic x-rays at various energies at and away from the ion x-ray absorption edges. As a model, the interaction of Ba^2+ with DMPA^- (dimyristoyl phosphatidic acid) monolayers at the aqueous surface was studied. We find an unexpectedly large concentration of the cations near the interface where they form a Stern layer of bound ions. These studies have been complemented with conventional x-ray reflectivity measurements and extended to other anionic lipid species (DMPS, DMPG) and cations (Ca^2+).

Formation and organization of protein domains in the immunological synapse

NASA Astrophysics Data System (ADS)

Carlson, Andreas; Mahadevan, L.

2014-11-01

The cellular basis for the adaptive immune response during antigen recognition relies on a specialized protein interface known as the immunological synapse. Here, we propose a minimal mathematical model for the dynamics of the IS that encompass membrane mechanics, hydrodynamics and protein kinetics. Simple scaling laws describe the dynamics of protein clusters as a function of membrane stiffness, rigidity of the adhesive proteins, and fluid flow in the synaptic cleft. Numerical simulations complement the scaling laws by quantifying the nucleation, growth and stabilization of proteins domains on the size of the cell. Direct comparison with experiment suggests that passive dynamics suffices to describe the short-time formation and organization of protein clusters, while the stabilization and long time dynamics of the synapse is likely determined by active cytoskeleton processes triggered by receptor binding. Our study reveals that the fluid flow generated by the interplay between membrane deformation and protein binding kinetics can assist immune cells in regulating protein sorting.

A novel pair of inducible expression vectors for use in Methylobacterium extorquens.

PubMed

Chubiz, Lon M; Purswani, Jessica; Carroll, Sean Michael; Marx, Chistopher J

2013-05-06

Due to the ever increasing use of diverse microbial taxa in basic research and industrial settings, there is a growing need for genetic tools to alter the physiology of these organisms. In particular, there is a dearth of inducible expression systems available for bacteria outside commonly used γ-proteobacteria, such as Escherichia coli or Pseudomonas species. To this end, we have sought to develop a pair of inducible expression vectors for use in the α-proteobacterium Methylobacterium extorquens, a model methylotroph. We found that the P(R) promoter from rhizobial phage 16-3 was active in M. extorquens and engineered the promoter to be inducible by either p-isopropyl benzoate (cumate) or anhydrotetracycline. These hybrid promoters, P(R/cmtO) and P(R/tetO), were found to have high levels of expression in M. extorquens with a regulatory range of 10-fold and 30-fold, respectively. Compared to an existing cumate-inducible (10-fold range), high-level expression system for M. extorquens, P(R/cmtO) and P(R/tetO) have 33% of the maximal activity but were able to repress gene expression 3 and 8-fold greater, respectively. Both promoters were observed to exhibit homogeneous, titratable activation dynamics rather than on-off, switch-like behavior. The utility of these promoters was further demonstrated by complementing loss of function of ftfL--essential for growth on methanol--where we show P(R/tetO) is capable of not only fully complementing function but also producing a conditional null phenotype. These promoters have been incorporated into a broad-host-range backbone allowing for potential use in a variety of bacterial hosts. We have developed two novel expression systems for use in M. extorquens. The expression range of these vectors should allow for increased ability to explore cellular physiology in M. extorquens. Further, the P(R/tetO) promoter is capable of producing conditional null phenotypes, previously unattainable in M. extorquens. As both expression systems rely on the use of membrane permeable inducers, we suspect these expression vectors will be useful for ectopic gene expression in numerous proteobacteria.

Taming the Sphinx: Mechanisms of Cellular Sphingolipid Homeostasis

PubMed Central

Olson, D. K.; Fröhlich, F.; Farese, R; Walther, T. C.

2016-01-01

Sphingolipids are important structural membrane components of eukaryotic cells, and potent signaling molecules. As such, their levels must be maintained to optimize cellular functions in different cellular membranes. Here, we review the current knowledge of homeostatic sphingolipid regulation. We describe recent studies in Saccharomyces cerevisiae that have provided insights into how cells sense changes in sphingolipid levels in the plasma membrane and acutely regulate sphingolipid biosynthesis by altering signaling pathways. We also discuss how cellular trafficking has emerged as an important determinant of sphingolipid homeostasis. Finally, we highlight areas where work is still needed to elucidate the mechanisms of sphingolipid regulation and the physiological functions of such regulatory networks, especially in mammalian cells. PMID:26747648

A novel strategy for hemolytic uremic syndrome: successful treatment with thrombomodulin α.

PubMed

Honda, Takashi; Ogata, Shohei; Mineo, Eri; Nagamori, Yukako; Nakamura, Shinya; Bando, Yuki; Ishii, Masahiro

2013-03-01

Hemolytic uremic syndrome (HUS) is a life-threatening infectious disease in childhood for which there is no confirmed therapeutic strategy. Endothelial inflammation leading to microthrombosis formation via complement activation is the main pathology of HUS. Thrombomodulin is an endothelial membrane protein that has anticoagulation and anti-inflammatory effects, including the suppression of complement activity. Recombinant human soluble thrombomodulin (rTM) is a novel therapeutic medicine for disseminated intravascular coagulation. We administered rTM to 3 patients with HUS for 7 days and investigated the outcomes in view of the patients' prognoses, changes in biochemical markers, complications, and adverse effects of rTM. Symptoms and laboratory data improved after initiation of rTM in all 3 patients. Abnormal activation of complements was also dramatically suppressed in 1 patient. The patients recovered without any complications or adverse effects of rTM. They were discharged having normal neurologic status and with no renal dysfunction. To our knowledge, this is the first report of rTM being used to treat HUS. These case reports show the positive effect of rTM in patients with HUS. Randomized controlled studies should be performed to assess the efficacy and safety of rTM for children with HUS.

The expression of Fc and complement receptors in young, adult and aged mice.

PubMed Central

Vĕtvicka, V; Fornůsek, L; Zídková, J

1985-01-01

Age-dependent changes in the expression of Fc receptors (FcR) for different isotypes of immunoglobulins and receptors for C3b, C5b and C3bi fragments of complement on the membranes of peritoneal macrophages were studied with mice of different ages. An age-related increase in expression of Fc receptors for IgM, IgE, IgA, IgG2b and IgG3, and a decrease in the expression of Fc receptors for IgG1 was observed. The expression of FcR on macrophages of donors of different ages corresponded with Fc-receptor mediated phagocytosis. The highest number of C3b-binding macrophages was found in aged mice, in contrast to low numbers of C3bi-binding macrophages at this age. The percentage of C5b-binding macrophages was lowest in adult animals. We also observed effective inhibition of binding of the C3b component of complement by preincubation of macrophages with aggregated IgG and vice versa. These observations suggest that fluctuation in expression of Fc but not C receptors may be important to the generalized changes that occur in macrophage function during development and ageing. PMID:2931351

Interaction of Human Complement Factor H Variants Tyr402 and His402 with Leptospira spp.

PubMed Central

Silva, Aldacilene Souza; Valencia, Mónica Marcela Castiblanco; Cianciarullo, Aurora Marques; Vasconcellos, Sílvio Arruda; Barbosa, Angela Silva; Isaac, Lourdes

2011-01-01

Leptospirosis is a zoonosis caused by pathogenic bacteria from the genus Leptospira. The disease represents a serious public health problem in underdeveloped tropical countries. Leptospires infect hosts through small abrasions in the skin or mucous membranes and they rapidly disseminate to target organs. The capacity of some pathogenic leptospiral strains to acquire the negative complement regulators factor H (FH) and C4b binding protein correlates with their ability to survive in human serum. In this study we assessed the functional consequences of the age macular degeneration-associated polymorphism FH His402 or FH Tyr402 on FH–Leptospira interactions. In binding assays using sub-saturating amounts of FH, the FH Tyr402 variant interacted with all the strains tested more strongly than the FH His402 variant. At higher concentrations, differences tended to disappear. We then compared cofactor activities displayed by FH His402 and FH Tyr402 bound to the surface of L. interrogans. Both variants exhibit similar activity as cofactors for Factor I-mediated cleavage of C3b, thus indicating that they do not differ in their capacity to regulate the complement cascade. PMID:22566834

Hha Controls Escherichia coli O157:H7 Biofilm Formation by Differential Regulation of Global Transcriptional Regulators FlhDC and CsgD

PubMed Central

Bearson, Bradley L.

2013-01-01

Although molecular mechanisms promoting adherence of enterohemorrhagic Escherichia coli (EHEC) O157:H7 on epithelial cells are well characterized, regulatory mechanisms controlling biofilm formation are not fully understood. In this study, we demonstrate that biofilm formation in EHEC O157:H7 strain 86-24 is highly repressed compared to that in an isogenic hha mutant. The hha mutant produced large quantities of biofilm compared to the wild-type strain at 30°C and 37°C. Complementation of the hha mutant reduced the level of biofilm formation to that of the wild-type strain, indicating that Hha is a negative regulator of biofilm production. While swimming motility and expression of the flagellar gene fliC were significantly reduced, the expression of csgA (encoding curlin of curli fimbriae) and the ability to bind Congo red were significantly enhanced. The expression of both fliC and csgA and the phenotypes of motility and curli production affected by these two genes, respectively, were restored to wild-type levels in the complemented hha mutant. The csgA deletion abolished biofilm formation in the hha mutant and wild-type strain, and csgA complementation restored biofilm formation to these strains, indicating the importance of csgA and curli in biofilm formation. The regulatory effects of Hha on flagellar and curli gene expression appear to occur via the induction and repression of FlhDC and CsgD, as demonstrated by reduced flhD and increased csgD transcription in the hha mutant, respectively. In gel shift assays Hha interacted with flhDC and csgD promoters. In conclusion, Hha regulates biofilm formation in EHEC O157:H7 by differential regulation of FlhDC and CsgD, the global regulators of motility and curli production, respectively. PMID:23377937

Hha controls Escherichia coli O157:H7 biofilm formation by differential regulation of global transcriptional regulators FlhDC and CsgD.

PubMed

Sharma, Vijay K; Bearson, Bradley L

2013-04-01

Although molecular mechanisms promoting adherence of enterohemorrhagic Escherichia coli (EHEC) O157:H7 on epithelial cells are well characterized, regulatory mechanisms controlling biofilm formation are not fully understood. In this study, we demonstrate that biofilm formation in EHEC O157:H7 strain 86-24 is highly repressed compared to that in an isogenic hha mutant. The hha mutant produced large quantities of biofilm compared to the wild-type strain at 30°C and 37°C. Complementation of the hha mutant reduced the level of biofilm formation to that of the wild-type strain, indicating that Hha is a negative regulator of biofilm production. While swimming motility and expression of the flagellar gene fliC were significantly reduced, the expression of csgA (encoding curlin of curli fimbriae) and the ability to bind Congo red were significantly enhanced. The expression of both fliC and csgA and the phenotypes of motility and curli production affected by these two genes, respectively, were restored to wild-type levels in the complemented hha mutant. The csgA deletion abolished biofilm formation in the hha mutant and wild-type strain, and csgA complementation restored biofilm formation to these strains, indicating the importance of csgA and curli in biofilm formation. The regulatory effects of Hha on flagellar and curli gene expression appear to occur via the induction and repression of FlhDC and CsgD, as demonstrated by reduced flhD and increased csgD transcription in the hha mutant, respectively. In gel shift assays Hha interacted with flhDC and csgD promoters. In conclusion, Hha regulates biofilm formation in EHEC O157:H7 by differential regulation of FlhDC and CsgD, the global regulators of motility and curli production, respectively.

Dynamic Network-Based Epistasis Analysis: Boolean Examples

PubMed Central

Azpeitia, Eugenio; Benítez, Mariana; Padilla-Longoria, Pablo; Espinosa-Soto, Carlos; Alvarez-Buylla, Elena R.

2011-01-01

In this article we focus on how the hierarchical and single-path assumptions of epistasis analysis can bias the inference of gene regulatory networks. Here we emphasize the critical importance of dynamic analyses, and specifically illustrate the use of Boolean network models. Epistasis in a broad sense refers to gene interactions, however, as originally proposed by Bateson, epistasis is defined as the blocking of a particular allelic effect due to the effect of another allele at a different locus (herein, classical epistasis). Classical epistasis analysis has proven powerful and useful, allowing researchers to infer and assign directionality to gene interactions. As larger data sets are becoming available, the analysis of classical epistasis is being complemented with computer science tools and system biology approaches. We show that when the hierarchical and single-path assumptions are not met in classical epistasis analysis, the access to relevant information and the correct inference of gene interaction topologies is hindered, and it becomes necessary to consider the temporal dynamics of gene interactions. The use of dynamical networks can overcome these limitations. We particularly focus on the use of Boolean networks that, like classical epistasis analysis, relies on logical formalisms, and hence can complement classical epistasis analysis and relax its assumptions. We develop a couple of theoretical examples and analyze them from a dynamic Boolean network model perspective. Boolean networks could help to guide additional experiments and discern among alternative regulatory schemes that would be impossible or difficult to infer without the elimination of these assumption from the classical epistasis analysis. We also use examples from the literature to show how a Boolean network-based approach has resolved ambiguities and guided epistasis analysis. Our article complements previous accounts, not only by focusing on the implications of the hierarchical and single-path assumption, but also by demonstrating the importance of considering temporal dynamics, and specifically introducing the usefulness of Boolean network models and also reviewing some key properties of network approaches. PMID:22645556

Structural Characterization of a Novel Polysaccharide from Lepidium meyenii (Maca) and Analysis of Its Regulatory Function in Macrophage Polarization in Vitro.

PubMed

Zhang, Mengmeng; Wu, Wenjia; Ren, Yao; Li, Xiaofeng; Tang, Yuqian; Min, Tian; Lai, Furao; Wu, Hui

2017-02-15

In our previous study, three novel polysaccharides, named MC-1, MC-2, and MC-3, were separated from the roots of maca (Lepidium meyenii), which is a food source from the Andes region. The structural information and immunomodulatory activity of MC-1 were then investigated. The structure and activity of MC-2 are still unknown. In this study, structural characterization revealed that MC-2 has an average molecular weight of 9.83 kDa and is composed of arabinose (20.9%), mannose (4.5%), glucose (71.9%), and galactose (2.7%). The main linkage types of MC-2 were proven to be (1→5)-α-l-Ara, (1→3)-α-l-Man, (1→)-α-d-Glc, (1→4)-α-d-Glc, (1→6)-α-d-Glc, and (1→6)-β-d-Gal by methylation and NMR analyses. Congo red assay showed that MC-2 possesses a triple-helix conformation. Immunostimulating assays indicated that MC-2 could induce M1 polarization of original macrophages and convert M2 macrophages into M1 phenotype. Although MC-2 could not shift M1 macrophages into M2, it could still inhibit inflammatory reactions induced by lipopolysaccharide. Furthermore, Toll-like receptor 2, tTll-like receptor 4, complement receptor 3, and mannose receptor were confirmed as the membrane receptors for MC-2 on macrophages. These results indicate that MC-2 could potentially be used toward hypoimmunity and tumor therapies.

Regulation of Flagellum Biosynthesis in Response to Cell Envelope Stress in Salmonella enterica Serovar Typhimurium

PubMed Central

2018-01-01

ABSTRACT Flagellum-driven motility of Salmonella enterica serovar Typhimurium facilitates host colonization. However, the large extracellular flagellum is also a prime target for the immune system. As consequence, expression of flagella is bistable within a population of Salmonella, resulting in flagellated and nonflagellated subpopulations. This allows the bacteria to maximize fitness in hostile environments. The degenerate EAL domain protein RflP (formerly YdiV) is responsible for the bistable expression of flagella by directing the flagellar master regulatory complex FlhD4C2 with respect to proteolytic degradation. Information concerning the environmental cues controlling expression of rflP and thus about the bistable flagellar biosynthesis remains ambiguous. Here, we demonstrated that RflP responds to cell envelope stress and alterations of outer membrane integrity. Lipopolysaccharide (LPS) truncation mutants of Salmonella Typhimurium exhibited increasing motility defects due to downregulation of flagellar gene expression. Transposon mutagenesis and genetic profiling revealed that σ24 (RpoE) and Rcs phosphorelay-dependent cell envelope stress response systems sense modifications of the lipopolysaccaride, low pH, and activity of the complement system. This subsequently results in activation of RflP expression and degradation of FlhD4C2 via ClpXP. We speculate that the presence of diverse hostile environments inside the host might result in cell envelope damage and would thus trigger the repression of resource-costly and immunogenic flagellum biosynthesis via activation of the cell envelope stress response. PMID:29717015

The Three Streptomyces lividans HtrA-Like Proteases Involved in the Secretion Stress Response Act in a Cooperative Manner

PubMed Central

Vicente, Rebeca L.; Gullón, Sonia; Marín, Silvia; Mellado, Rafael P.

2016-01-01

Overproduction of Sec-proteins in S. lividans accumulates misfolded proteins outside of the cytoplasmic membrane where the accumulated proteins interfere with the correct functioning of the secretion machinery and with the correct cell functionality, triggering the expression in S. lividans of a CssRS two-component system which regulates the degradation of the accumulated protein, the so-called secretion stress response. Optimization of secretory protein production via the Sec route requires the identification and characterisation of quality factors involved in this process. The phosphorylated regulator (CssR) interacts with the regulatory regions of three genes encoding three different HtrA-like proteases. Individual mutations in each of these genes render degradation of the misfolded protein inoperative, and propagation in high copy number of any of the three proteases encoding genes results on indiscriminate alpha-amylase degradation. None of the proteases could complement the other two deficiencies and only propagation of each single copy protease gene can restore its own deficiency. The obtained results strongly suggest that the synthesis of the three HtrA-like proteases needs to be properly balanced to ensure the effective degradation of misfolded overproduced secretory proteins and, at the same time, avoid negative effects in the secreted proteins and the secretion machinery. This is particularly relevant when considering the optimisation of Streptomyces strains for the overproduction of homologous or heterologous secretory proteins of industrial application. PMID:27977736

A Receptor-Like Kinase Mediates Ammonium Homeostasis and Is Important for the Polar Growth of Root Hairs in Arabidopsis[W

PubMed Central

Bai, Ling; Ma, Xiaonan; Zhang, Guozeng; Song, Shufei; Zhou, Yun; Gao, Lijie; Miao, Yuchen; Song, Chun-Peng

2014-01-01

Ammonium (NH4+) is both a necessary nutrient and an important signal in plants, but can be toxic in excess. Ammonium sensing and regulatory mechanisms in plant cells have not been fully elucidated. To decipher the complex network of NH4+ signaling, we analyzed [Ca2+]cyt-associated protein kinase (CAP) genes, which encode signaling components that undergo marked changes in transcription levels in response to various stressors. We demonstrated that CAP1, a tonoplast-localized receptor-like kinase, regulates root hair tip growth by maintaining cytoplasmic Ca2+ gradients. A CAP1 knockout mutant (cap1-1) produced elevated levels of cytoplasmic NH4+. Furthermore, root hair growth of cap1-1 was inhibited on Murashige and Skoog medium, but NH4+ depletion reestablished the Ca2+ gradient necessary for normal growth. The lower net NH4+ influx across the vacuolar membrane and relatively alkaline cytosolic pH of cap1-1 root hairs implied that mutation of CAP1 increased NH4+ accumulation in the cytoplasm. Furthermore, CAP1 functionally complemented the npr1 (nitrogen permease reactivator protein) kinase yeast mutant, which is defective in high-affinity NH4+ uptake via MEP2 (methylammonium permease 2), distinguishing CAP1 as a cytosolic modulator of NH4+ levels that participates in NH4+ homeostasis-regulated root hair growth by modulating tip-focused cytoplasmic Ca2+ gradients. PMID:24769480

Autoinhibition of a calmodulin-dependent calcium pump involves a structure in the stalk that connects the transmembrane domain to the ATPase catalytic domain

NASA Technical Reports Server (NTRS)

Curran, A. C.; Hwang, I.; Corbin, J.; Martinez, S.; Rayle, D.; Sze, H.; Harper, J. F.; Evans, M. L. (Principal Investigator)

2000-01-01

The regulation of Ca(2+)-pumps is important for controlling [Ca(2+)] in the cytosol and organelles of all eukaryotes. Here, we report a genetic strategy to identify residues that function in autoinhibition of a novel calmodulin-activated Ca(2+)-pump with an N-terminal regulatory domain (isoform ACA2 from Arabidopsis). Mutant pumps with constitutive activity were identified by complementation of a yeast (K616) deficient in two Ca(2+)-pumps. Fifteen mutations were found that disrupted a segment of the N-terminal autoinhibitor located between Lys(23) and Arg(54). Three mutations (E167K, D219N, and E341K) were found associated with the stalk that connects the ATPase catalytic domain (head) and with the transmembrane domain. Enzyme assays indicated that the stalk mutations resulted in calmodulin-independent activity, with V(max), K(mATP), and K(mCa(2+)) similar to that of a pump in which the N-terminal autoinhibitor had been deleted. A highly conservative substitution at Asp(219) (D219E) still produced a deregulated pump, indicating that the autoinhibitory structure in the stalk is highly sensitive to perturbation. In plasma membrane H(+)-ATPases from yeast and plants, similarly positioned mutations resulted in hyperactive pumps. Together, these results suggest that a structural feature of the stalk is of general importance in regulating diverse P-type ATPases.

Protection of Nonself Surfaces from Complement Attack by Factor H-Binding Peptides: Implications for Therapeutic Medicine

PubMed Central

Wu, You-Qiang; Qu, Hongchang; Sfyroera, Georgia; Tzekou, Apostolia; Kay, Brian K.; Nilsson, Bo; Ekdahl, Kristina Nilsson; Ricklin, Daniel; Lambris, John D.

2011-01-01

Exposure of nonself surfaces such as those of biomaterials or transplanted cells and organs to host blood frequently triggers innate immune responses, thereby affecting both their functionality and tolerability. Activation of the alternative pathway of complement plays a decisive role in this unfavorable reaction. Whereas previous studies demonstrated that immobilization of physiological regulators of complement activation (RCA) can attenuate this foreign body-induced activation, simple and efficient approaches for coating artificial surfaces with intact RCA are still missing. The conjugation of small molecular entities that capture RCA with high affinity is an intriguing alternative, as this creates a surface with autoregulatory activity upon exposure to blood. We therefore screened two variable cysteine-constrained phage-displayed peptide libraries for factor H-binding peptides. We discovered three peptide classes that differed with respect to their main target binding areas. Peptides binding to the broad middle region of factor H (domains 5–18) were of particular interest, as they do not interfere with either regulatory or binding activities. One peptide in this group (5C6) was further characterized and showed high factor H-capturing activity while retaining its functional integrity. Most importantly, when 5C6 was coated to a model polystyrene surface and exposed to human lepirudin-anticoagulated plasma, the bound peptide captured factor H and substantially inhibited complement activation by the alternative pathway. Our study therefore provides a promising and novel approach to produce therapeutic materials with enhanced biocompatibility. PMID:21339361

Expression of Human Complement Factor H Prevents Age-Related Macular Degeneration–Like Retina Damage and Kidney Abnormalities in Aged Cfh Knockout Mice

PubMed Central

Ding, Jin-Dong; Kelly, Una; Landowski, Michael; Toomey, Christopher B.; Groelle, Marybeth; Miller, Chelsey; Smith, Stephanie G.; Klingeborn, Mikael; Singhapricha, Terry; Jiang, Haixiang; Frank, Michael M.; Bowes Rickman, Catherine

2016-01-01

Complement factor H (CFH) is an important regulatory protein in the alternative pathway of the complement system, and CFH polymorphisms increase the genetic risk of age-related macular degeneration dramatically. These same human CFH variants have also been associated with dense deposit disease. To mechanistically study the function of CFH in the pathogenesis of these diseases, we created transgenic mouse lines using human CFH bacterial artificial chromosomes expressing full-length human CFH variants and crossed these to Cfh knockout (Cfh−/−) mice. Human CFH protein inhibited cleavage of mouse complement component 3 and factor B in plasma and in retinal pigment epithelium/choroid/sclera, establishing that human CFH regulates activation of the mouse alternative pathway. One of the mouse lines, which express relatively higher levels of CFH, demonstrated functional and structural protection of the retina owing to the Cfh deletion. Impaired visual function, detected as a deficit in the scotopic electroretinographic response, was improved in this transgenic mouse line compared with Cfh−/− mice, and transgenics had a thicker outer nuclear layer and less sub–retinal pigment epithelium deposit accumulation. In addition, expression of human CFH also completely protected the mice from developing kidney abnormalities associated with loss of CFH. These humanized CFH mice present a valuable model for study of the molecular mechanisms of age-related macular degeneration and dense deposit disease and for testing therapeutic targets. PMID:25447048

Phenotypic characterization of 10 methanol oxidation mutant classes in Methylobacterium sp. strain AM1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nunn, D.N.; Lidstrom, M.E.

Twenty-five methanol oxidation mutants of the facultative methylotroph Methylobacterium sp. strain AM1 have been characterized by complementation analysis and assigned to 10 complementation groups, Mox A1, A2, A3, and B through H. In this study we have characterized each of the mutants belonging to the 10 Mox complementation groups for the following criteria: (i) phenazine methosulfate-dichlorophenolindophenol dye-linked methanol dehydrogenase activity; (ii) methanol-dependent whole-cell oxygen consumption; (iii) the presence or absence of methanol dehydrogenase protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting; (iv) the absorption spectra of purified mutant methanol dehydrogenase proteins; and (v) the presence or absence ofmore » the soluble cytochrome c proteins of Methylobacterium sp. strain AM1, as determined by reduced-oxidized difference spectra and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With this information, we have proposed functions for each of the genes deficient in the mutants of the 10 Mox complementation groups. These proposed gene functions include two linked genes that encode the methanol dehydrogenase structural protein and the soluble cytochrome c/sub L/, a gene encoding a secretion function essential for the synthesis and export of methanol dehydrogenase and cytochrome c/sub L/, three gene functions responsible for the proper association of the pyrrolo-quinoline quinone prosthetic group with the methanol dehydrogenase apoprotein, and four positive regulatory gene functions controlling the expression of the ability to oxidize methanol.« less

The Multifaceted Role of SNARE Proteins in Membrane Fusion

PubMed Central

Han, Jing; Pluhackova, Kristyna; Böckmann, Rainer A.

2017-01-01

Membrane fusion is a key process in all living organisms that contributes to a variety of biological processes including viral infection, cell fertilization, as well as intracellular transport, and neurotransmitter release. In particular, the various membrane-enclosed compartments in eukaryotic cells need to exchange their contents and communicate across membranes. Efficient and controllable fusion of biological membranes is known to be driven by cooperative action of SNARE proteins, which constitute the central components of the eukaryotic fusion machinery responsible for fusion of synaptic vesicles with the plasma membrane. During exocytosis, vesicle-associated v-SNARE (synaptobrevin) and target cell-associated t-SNAREs (syntaxin and SNAP-25) assemble into a core trans-SNARE complex. This complex plays a versatile role at various stages of exocytosis ranging from the priming to fusion pore formation and expansion, finally resulting in the release or exchange of the vesicle content. This review summarizes current knowledge on the intricate molecular mechanisms underlying exocytosis triggered and catalyzed by SNARE proteins. Particular attention is given to the function of the peptidic SNARE membrane anchors and the role of SNARE-lipid interactions in fusion. Moreover, the regulatory mechanisms by synaptic auxiliary proteins in SNARE-driven membrane fusion are briefly outlined. PMID:28163686

Endocytosis and membrane receptor internalization: implication of F-BAR protein Carom

PubMed Central

Xu, Yanjie; Liu, Suxuan; Xia, Jixiang; Stein, Sam; Ramon, Cueto; Xi, Hang; Wang, Luqiao; Xiong, Xinyu; Zhang, Lixiao; He, Dingwen; Yang, William; Zhao, Xianxian; Cheng, Xiaoshu; Yang, Xiaofeng; Wang, Hong

2016-01-01

Endocytosis is a cellular process mostly responsible for membrane receptor internalization. Cell membrane receptors bind to their ligands and form a complex which can be internalized. We previously proposed that F-BAR protein initiates membrane curvature and mediates endocytosis via their binding partners. However, F-BAR protein partners involved in membrane receptor endocytosis and the regulatory mechanism remain unknown. In this study, we established a group of database mining strategies to explore mechanisms underlying receptor-related endocytosis. We identified 34 endocytic membrane receptors and 10 regulating proteins for vesicle formation in clathrin-dependent endocytosis (CDE), a major process of membrane receptor internalization. We found that F-BAR protein FCHSD2 (Carom) may facilitate endocytosis via 9 endocytic partners. Carom is highly expressed, along with highly expressed endocytic membrane receptors and partners, in endothelial cells and macrophages. We established 3 models of Carom-receptor complex and their intracellular trafficking based on protein-protein interaction and subcellular localization. We conclude that Carom may mediate receptor endocytosis and transport endocytic receptors to the cytoplasm for receptor signaling and lysosome/proteasome degradation, or to the nucleus for RNA processing, gene transcription and DNA repair. PMID:28199211

The Multifaceted Role of SNARE Proteins in Membrane Fusion.

PubMed

Han, Jing; Pluhackova, Kristyna; Böckmann, Rainer A

2017-01-01

Membrane fusion is a key process in all living organisms that contributes to a variety of biological processes including viral infection, cell fertilization, as well as intracellular transport, and neurotransmitter release. In particular, the various membrane-enclosed compartments in eukaryotic cells need to exchange their contents and communicate across membranes. Efficient and controllable fusion of biological membranes is known to be driven by cooperative action of SNARE proteins, which constitute the central components of the eukaryotic fusion machinery responsible for fusion of synaptic vesicles with the plasma membrane. During exocytosis, vesicle-associated v-SNARE (synaptobrevin) and target cell-associated t-SNAREs (syntaxin and SNAP-25) assemble into a core trans-SNARE complex. This complex plays a versatile role at various stages of exocytosis ranging from the priming to fusion pore formation and expansion, finally resulting in the release or exchange of the vesicle content. This review summarizes current knowledge on the intricate molecular mechanisms underlying exocytosis triggered and catalyzed by SNARE proteins. Particular attention is given to the function of the peptidic SNARE membrane anchors and the role of SNARE-lipid interactions in fusion. Moreover, the regulatory mechanisms by synaptic auxiliary proteins in SNARE-driven membrane fusion are briefly outlined.

Some properties of electrolyte solutions in nanoconfinement revealed by the measurement of transient filtration potential after pressure switch off.

PubMed

Yaroshchuk, Andriy E; Boiko, Yuriy P; Makovetskiy, Alexandre L

2005-08-16

We have demonstrated that with a composite nanoporous ceramic membrane in a batch membrane cell it is technically feasible to switch off the trans-membrane hydrostatic pressure difference within tens of milliseconds. That enabled us to resolve practically the whole time evolution of transient filtration potential. Measurements of the latter have been complemented by measurements of steady-state salt rejection by the composite membrane and by measurements of the streaming potential and hydraulic permeability of membrane supports available separately. A theory has been developed in terms of network thermodynamics for the electrical response of a bilayer membrane to a pressure perturbation. In combination with the results of salt rejection measurements, from the time transients of filtration potential we could determine the ion transport numbers within the nanoporous layer. Besides that, from the dependence of steady-state salt rejection on the trans-membrane volume flow, we have determined the diffusion permeability of and the salt reflection coefficient in the nanoporous layer. This has enabled us to estimate the contributions of Donnan and non-Donnan mechanisms to the rejection of ions by the nanoporous membrane used in this study. It has been unexpectedly found that the Donnan exclusion played only a secondary role. Our hypothesis is that the non-Donnan exclusion of ions from the nanopores might be caused by changes in water properties in nanoconfinement. Proceeding from the results of steady-state filtration experiments with the membrane and the support, we also concluded that the nanoporous layer was imperfection-free and had a quite narrow pore size distribution, which made it a suitable object for fundamental studies of ion transfer mechanisms in nanopores.

Non-Native Metal Ion Reveals the Role of Electrostatics in Synaptotagmin 1-Membrane Interactions.

PubMed

Katti, Sachin; Nyenhuis, Sarah B; Her, Bin; Srivastava, Atul K; Taylor, Alexander B; Hart, P John; Cafiso, David S; Igumenova, Tatyana I

2017-06-27

C2 domains are independently folded modules that often target their host proteins to anionic membranes in a Ca 2+ -dependent manner. In these cases, membrane association is triggered by Ca 2+ binding to the negatively charged loop region of the C2 domain. Here, we used a non-native metal ion, Cd 2+ , in lieu of Ca 2+ to gain insight into the contributions made by long-range Coulombic interactions and direct metal ion-lipid bridging to membrane binding. Using X-ray crystallography, NMR, Förster resonance energy transfer, and vesicle cosedimentation assays, we demonstrate that, although Cd 2+ binds to the loop region of C2A/B domains of synaptotagmin 1 with high affinity, long-range Coulombic interactions are too weak to support membrane binding of individual domains. We attribute this behavior to two factors: the stoichiometry of Cd 2+ binding to the loop regions of the C2A and C2B domains and the impaired ability of Cd 2+ to directly coordinate the lipids. In contrast, electron paramagnetic resonance experiments revealed that Cd 2+ does support membrane binding of the C2 domains in full-length synaptotagmin 1, where the high local lipid concentrations that result from membrane tethering can partially compensate for lack of a full complement of divalent metal ions and specific lipid coordination in Cd 2+ -complexed C2A/B domains. Our data suggest that long-range Coulombic interactions alone can drive the initial association of C2A/B with anionic membranes and that Ca 2+ further augments membrane binding by the formation of metal ion-lipid coordination bonds and additional Ca 2+ ion binding to the C2 domain loop regions.

G-protein-coupled receptors for neurotransmitter amino acids: C-terminal tails, crowded signalosomes.

PubMed Central

El Far, Oussama; Betz, Heinrich

2002-01-01

G-protein-coupled receptors (GPCRs) represent a superfamily of highly diverse integral membrane proteins that transduce external signals to different subcellular compartments, including nuclei, via trimeric G-proteins. By differential activation of diffusible G(alpha) and membrane-bound G(beta)gamma subunits, GPCRs might act on both cytoplasmic/intracellular and plasma-membrane-bound effector systems. The coupling efficiency and the plasma membrane localization of GPCRs are regulated by a variety of interacting proteins. In this review, we discuss recently disclosed protein interactions found with the cytoplasmic C-terminal tail regions of two types of presynaptic neurotransmitter receptors, the group III metabotropic glutamate receptors and the gamma-aminobutyric acid type-B receptors (GABA(B)Rs). Calmodulin binding to mGluR7 and other group III mGluRs may provide a Ca(2+)-dependent switch for unidirectional (G(alpha)) versus bidirectional (G(alpha) and G(beta)gamma) signalling to downstream effector proteins. In addition, clustering of mGluR7 by PICK1 (protein interacting with C-kinase 1), a polyspecific PDZ (PSD-95/Dlg1/ZO-1) domain containing synaptic organizer protein, sheds light on how higher-order receptor complexes with regulatory enzymes (or 'signalosomes') could be formed. The interaction of GABA(B)Rs with the adaptor protein 14-3-3 and the transcription factor ATF4 (activating transcription factor 4) suggests novel regulatory pathways for G-protein signalling, cytoskeletal reorganization and nuclear gene expression: processes that may all contribute to synaptic plasticity. PMID:12006104

A Multifunctional ATP-Binding Cassette Transporter System from Vibrio cholerae Transports Vibriobactin and Enterobactin

PubMed Central

Wyckoff, Elizabeth E.; Valle, Ana-Maria; Smith, Stacey L.; Payne, Shelley M.

1999-01-01

Vibrio cholerae uses the catechol siderophore vibriobactin for iron transport under iron-limiting conditions. We have identified genes for vibriobactin transport and mapped them within the vibriobactin biosynthetic gene cluster. Within this genetic region we have identified four genes, viuP, viuD, viuG and viuC, whose protein products have homology to the periplasmic binding protein, the two integral cytoplasmic membrane proteins, and the ATPase component, respectively, of other iron transport systems. The amino-terminal region of ViuP has homology to a lipoprotein signal sequence, and ViuP could be labeled with [3H]palmitic acid. This suggests that ViuP is a membrane lipoprotein. The ViuPDGC system transports both vibriobactin and enterobactin in Escherichia coli. In the same assay, the E. coli enterobactin transport system, FepBDGC, allowed the utilization of enterobactin but not vibriobactin. Although the entire viuPDGC system could complement mutations in fepB, fepD, fepG, or fepC, only viuC was able to independently complement the corresponding fep mutation. This indicates that these proteins usually function as a complex. V. cholerae strains carrying a mutation in viuP or in viuG were constructed by marker exchange. These mutations reduced, but did not completely eliminate, vibriobactin utilization. This suggests that V. cholerae contains genes in addition to viuPDGC that function in the transport of catechol siderophores. PMID:10601218

Interplay among Membrane-Bound Lytic Transglycosylase D1, the CreBC Two-Component Regulatory System, the AmpNG-AmpDI-NagZ-AmpR Regulatory Circuit, and L1/L2 β-Lactamase Expression in Stenotrophomonas maltophilia

PubMed Central

Huang, Yi-Wei; Wu, Chao-Jung; Hu, Rouh-Mei; Lin, Yi-Tsung

2015-01-01

Lytic transglycosylases (LTs) are an important class of enzymes involved in peptidoglycan (PG) cleavage, with the concomitant formation of an intramolecular 1,6-anhydromuramoyl reaction product. There are six annotated LT genes in the Stenotrophomonas maltophilia genome, including genes for five membrane-bound LTs (mltA, mltB1, mltB2, mltD1, and mltD2) and a gene for soluble LT (slt). Six LTs of S. maltophilia KJ were systematically mutated, yielding the ΔmltA, ΔmltB1, ΔmltB2, ΔmltD1, ΔmltD2, and Δslt mutants. Inactivation of mltD1 conferred a phenotype of elevated uninduced β-lactamase activity. The underlying mechanism responsible for this phenotype was elucidated by the construction of several mutants and determination of β-lactamase activity. The expression of the genes assayed was assessed by quantitative reverse transcriptase PCR and a promoter transcription fusion assay. The results demonstrate that ΔmltD1 mutant-mediated L1/L2 β-lactamase expression involved the creBC two-component regulatory system (TCS) and the ampNG-ampDI-nagZ-ampR regulatory circuit. The inactivation of mltD1 resulted in mltB1 and mltD2 upexpression in a creBC- and ampNG-dependent manner. The overexpressed MltB1 and MltD2 activity contributed to the expression of the L1/L2 β-lactamase genes via the ampNG-ampDI-nagZ-ampR regulatory circuit. These findings reveal, for the first time, a linkage between LTs, the CreBC TCS, the ampNG-ampDI-nagZ-ampR regulatory circuit, and L1/L2 β-lactamase expression in S. maltophilia. PMID:26282431

[Free from stress by autogenic therapy. Relaxation technique yielding peace of mind and self-insight].

PubMed

Broms, C

1999-02-10

The utilisation of self-regulatory capacity is one of the purposes of autogenic therapy, a method consisting of exercises focused on the limbs, lungs, heart, diaphragm and head. The physiological response is muscle relaxation, increased peripheral blood flow, lower heart rate and blood pressure, slower and deeper breathing, and reduced oxygen consumption. Autogenic training is applicable in most pathological conditions associated with stress, and can be used preventively or as a complement to conventional treatment.

Dysregulated humoral immunity to nontyphoidal Salmonella in HIV-infected African adults

PubMed Central

MacLennan, Calman A.; Gilchrist, James J.; Gordon, Melita A.; Cunningham, Adam F.; Cobbold, Mark; Goodall, Margaret; Kingsley, Robert A.; van Oosterhout, Joep J. G.; Msefula, Chisomo L.; Mandala, Wilson L.; Leyton, Denisse L.; Marshall, Jennifer L.; Gondwe, Esther N.; Bobat, Saeeda; López-Macías, Constantino; Doffinger, Rainer; Henderson, Ian R.; Zijlstra, Eduard E.; Dougan, Gordon; Drayson, Mark T.; MacLennan, Ian C. M.; Molyneux, Malcolm E.

2013-01-01

Nontyphoidal Salmonellae are a major cause of life-threatening bacteremia among HIV-infected individuals. Although cell-mediated immunity controls intracellular infection, antibody protects against Salmonella bacteremia. We report that high titer antibodies specific for Salmonella lipopolysaccharide (LPS) associate with absent Salmonella-killing in HIV-infected African adults. Killing was restored by genetically shortening LPS from target Salmonella, or removing LPS-specific antibodies from serum. Complement-mediated killing of Salmonella by healthy serum is shown to be induced specifically by antibodies against outer membrane proteins. This killing is lost when excess antibody against Salmonella LPS is added. Thus our study indicates impaired immunity against nontyphoidal Salmonella bacteremia in HIV infection results from excess inhibitory antibodies against Salmonella LPS, whilst serum killing of Salmonella is induced by antibodies against outer membrane proteins. PMID:20413503

[The effect of substance P on functional proteins in human neutrophil].

PubMed

Yang, Lin; Fa, Xiang-guang

2002-02-01

To explore the effect of substance P (SP) on the functional proteins on plasma membrane of neutrophil (Np). The response of Np to SP was examined by measuring the level of respiratory burst, the activities of ACP and ALP, the fluoroscopy intensity of CR3, CD45 and FM-LP. It was found that SP could increase respiratory burst of Np, decrease the activity of acid phosphatase (ACP), but had no effect on alkaline phosphatase (ALP). SP could also promote the amount of CD45, complement receptor type 3 (CR3) and N-Formyl-Met-Leu-Phe (FMLP) receptors. The results showed that the effects of SP on functional proteins in human Np membrane were universality and diversity. It implied that SP could affect various inflammation responses in Np.

Polymethylmethacrylate membrane with a series of serendipity.

PubMed

Sakai, Yoshitada

2011-01-01

Forty years have passed since the polymethylmethacrylate (PMMA) membrane was first developed. This article reviews its history and explains its longevity. The membrane was developed through application of a stereocomplex phenomenon that is observed upon mixture of isotactic and syndiotactic PMMA polymers. Filtryzer(TM) B1 and B2 were approved in Japan in 1977. B1 was the pioneer high-performance membrane model and B2 was a model that simulated a low-flux cellulosic membrane. The development of B1 led to the development of the dialysis machine with an ultrafiltration rate (UFR)-controlling function because the UFR of B1 was too high to control using transmembrane pressure control. B1 was used not only as a dialyzer but also as a hemodiafilter by combination with a UFR controller. Biocompatibility of the dialysis membrane, complement activation and/or transient leukopenia was studied with B2. Cooperative studies between Niigata University and Toray resulted in Gejyo's finding regarding the harmfulness of β(2)-microglobulin (BMG). Long-term follow-up of patients dialyzed using the BK membrane revealed that plasma BMG levels were significantly low and that the occurrence ratio of carpal tunnel syndrome was suppressed. These results were obtained by the adsorptive removal of BMG onto a PMMA membrane. Several papers have discussed new aspects in succession mainly based on clinical experiences that were not aimed at a development stage, i.e. they were kinds of serendipity. For the BK-F membrane with the largest pore size, this includes anemia and removal or modification of furancarboxylic acid, homocysteine, pentosidine and soluble CD40. For the BG membrane with a slightly anionic component, this includes pruritus and removal of free immunoglobulin light chains. Even patients' prognoses may be modified by the use of PMMA membrane. The mechanisms of these findings have been clarified bit by bit and the membrane will further open new frontiers in dialysis treatment. Copyright © 2011 S. Karger AG, Basel.

Characterization of shark complement factor I gene(s): genomic analysis of a novel shark-specific sequence.

PubMed

Shin, Dong-Ho; Webb, Barbara M; Nakao, Miki; Smith, Sylvia L

2009-07-01

Complement factor I is a crucial regulator of mammalian complement activity. Very little is known of complement regulators in non-mammalian species. We isolated and sequenced four highly similar complement factor I cDNAs from the liver of the nurse shark (Ginglymostoma cirratum), designated as GcIf-1, GcIf-2, GcIf-3 and GcIf-4 (previously referred to as nsFI-a, -b, -c and -d) which encode 689, 673, 673 and 657 amino acid residues, respectively. They share 95% (

Characterization of shark complement factor I gene(s): genomic analysis of a novel shark-specific sequence

PubMed Central

Shin, Dong-Ho; Webb, Barbara M.; Nakao, Miki; Smith, Sylvia L.

2009-01-01

Complement factor I is a crucial regulator of mammalian complement activity. Very little is known of complement regulators in non-mammalian species. We isolated and sequenced four highly similar complement factor I cDNAs from the liver of the nurse shark (Ginglymostoma cirratum), designated as GcIf-1, GcIf-2, GcIf-3 and GcIf-4 (previously referred to as nsFI-a, -b, -c and –d) which encode 689, 673, 673 and 657 amino acid residues, respectively. They share 95% (≤) amino acid identities with each other, 35.4 ~ 39.6% and 62.8 ~ 65.9% with factor I of mammals and banded houndshark (Triakis scyllium), respectively. The modular structure of the GcIf is similar to that of mammals with one notable exception, the presence of a novel shark-specific sequence between the leader peptide (LP) and the factor I membrane attack complex (FIMAC) domain. The cDNA sequences differ only in the size and composition of the shark-specific region (SSR). Sequence analysis of each SSR has identified within the region two novel short sequences (SS1 and SS2) and three repeat sequences (RS1, 2 and 3). Genomic analysis has revealed the existence of three introns between the leader peptide and the FIMAC domain, tentatively designated intron 1, intron 2, and intron 3 which span 4067, 2293 and 2082 bp, respectively. Southern blot analysis suggests the presence of a single gene copy for each cDNA type. Phylogenetic analysis suggests that complement factor I of cartilaginous fish diverged prior to the emergence of mammals. All four GcIf cDNA species are expressed in four different tissues and the liver is the main tissue in which expression level of all four is high. This suggests that the expression of GcIf isotypes is tissue-dependent. PMID:19423168

Pathogenic Variants in Complement Genes and Risk of Atypical Hemolytic Uremic Syndrome Relapse after Eculizumab Discontinuation.

PubMed

Fakhouri, Fadi; Fila, Marc; Provôt, François; Delmas, Yahsou; Barbet, Christelle; Châtelet, Valérie; Rafat, Cédric; Cailliez, Mathilde; Hogan, Julien; Servais, Aude; Karras, Alexandre; Makdassi, Raifah; Louillet, Feriell; Coindre, Jean-Philippe; Rondeau, Eric; Loirat, Chantal; Frémeaux-Bacchi, Véronique

2017-01-06

The complement inhibitor eculizumab has dramatically improved the outcome of atypical hemolytic uremic syndrome. However, the optimal duration of eculizumab treatment in atypical hemolytic uremic syndrome remains debated. We report on the French atypical hemolytic uremic syndrome working group's first 2-year experience with eculizumab discontinuation in patients with atypical hemolytic uremic syndrome. Using the French atypical hemolytic uremic syndrome registry database, we retrospectively identified all dialysis-free patients with atypical hemolytic uremic syndrome who discontinued eculizumab between 2010 and 2014 and reviewed their relevant clinical and biologic data. The decision to discontinue eculizumab was made by the clinician in charge of the patient. All patients were closely monitored by regular urine dipsticks and blood tests. Eculizumab was rapidly (24-48 hours) restarted in case of relapse. Among 108 patients treated with eculizumab, 38 patients (nine children and 29 adults) discontinued eculizumab (median treatment duration of 17.5 months). Twenty-one patients (55%) carried novel or rare complement genes variants. Renal recovery under eculizumab was equally good in patients with and those without complement gene variants detected. After a median follow-up of 22 months, 12 patients (31%) experienced atypical hemolytic uremic syndrome relapse. Eight of 11 patients (72%) with complement factor H variants, four of eight patients (50%) with membrane cofactor protein variants, and zero of 16 patients with no rare variant detected relapsed. In relapsing patients, early reintroduction (≤48 hours) of eculizumab led to rapid (<7 days) hematologic remission and a return of serum creatinine to baseline level in a median time of 26 days. At last follow-up, renal function remained unchanged in nonrelapsing and relapsing patients compared with baseline values before eculizumab discontinuation. Pathogenic variants in complement genes were associated with higher risk of atypical hemolytic uremic syndrome relapse after eculizumab discontinuation. Prospective studies are needed to identify biomarkers predictive of relapse and determine the best strategy of retreatment in relapsing patients. Copyright © 2016 by the American Society of Nephrology.

The Lectin Complement Pathway Is Involved in Protection Against Enteroaggregative Escherichia coli Infection.

PubMed

Adler Sørensen, Camilla; Rosbjerg, Anne; Hebbelstrup Jensen, Betina; Krogfelt, Karen Angeliki; Garred, Peter

2018-01-01

Enteroaggregative Escherichia coli (EAEC) causes acute and persistent diarrhea worldwide. Still, the involvement of host factors in EAEC infections is unresolved. Binding of recognition molecules from the lectin pathway of complement to EAEC strains have been observed, but the importance is not known. Our aim was to uncover the involvement of these molecules in innate complement dependent immune protection toward EAEC. Binding of mannose-binding lectin, ficolin-1, -2, and -3 to four prototypic EAEC strains, and ficolin-2 binding to 56 clinical EAEC isolates were screened by a consumption-based ELISA method. Flow cytometry was used to determine deposition of C4b, C3b, and the bactericidal C5b-9 membrane attack complex (MAC) on the bacteria in combination with different complement inhibitors. In addition, the direct serum bactericidal effect was assessed. Screening of the prototypic EAEC strains revealed that ficolin-2 was the major binder among the lectin pathway recognition molecules. However, among the clinical EAEC isolates only a restricted number ( n  = 5) of the isolates bound ficolin-2. Using the ficolin-2 binding isolate C322-17 as a model, we found that incubation with normal human serum led to deposition of C4b, C3b, and to MAC formation. No inhibition of complement deposition was observed when a C1q inhibitor was added, while partial inhibition was observed when ficolin-2 or factor D inhibitors were used separately. Combining the inhibitors against ficolin-2 and factor D led to virtually complete inhibition of complement deposition and protection against direct bacterial killing. These results demonstrate that ficolin-2 may play an important role in innate immune protection against EAEC when an appropriate ligand is exposed, but many EAEC strains evade lectin pathway recognition and may, therefore, circumvent this strategy of innate host immune protection.

Granular C3 Dermatosis.

PubMed

Hashimoto, Takashi; Tsuruta, Daisuke; Yasukochi, Atsushi; Imanishi, Hisayoshi; Sekine, Hideharu; Fujita, Teizo; Wanibuchi, Hideki; Gi, Min; Kárpáti, Sarolta; Sitaru, Cassian; Zone, John J; Endo, Daisuke; Abe, Shinichi; Nishino, Tomoya; Koji, Takehiko; Ishii, Norito

2016-08-23

There has been no previous systematic study of bullous skin diseases with granular basement membrane zone deposition exclusively of C3. In this study we collected 20 such patients, none of whom showed cutaneous vasculitis histopathologically. Oral dapsone and topical steroids were effective. Various serological tests detected no autoantibodies or autoantigens. Direct immunofluorescence for various complement components revealed deposition only of C3 and C5-C9, indicating that no known complement pathways were involved. Studies of in situ hybridization and micro-dissection with quantitative RT-PCR revealed a slight reduction in expression of C3 in patient epidermis. These patients may represent a new disease entity, for which we propose the term "granular C3 dermatosis". The mechanism for granular C3 deposition in these patients is unknown, but it is possible that the condition is caused by autoantibodies to skin or aberrant C3 expression in epidermal keratinocytes.

[Atipical uremic hemolityc syndrome in pregnancy].

PubMed

Pérez-Calatayud, Ángel Augusto; Briones-Garduño, Jesús Carlos; Álvarez-Goris, Mercedes Del Pilar; Sánchez Zamora, Ricardo; Torres Aguilar, Angélica A; Mendoza-Mórales, Rosa Elba

2016-01-01

Atypical haemolytic uraemic syndrome is one of the main variants of thrombotic microangiopathy, and is characterized by excessive complement activation in the microvasculature. It is also characterised by the clinical triad; non-immune haemolytic anaemia, thrombocytopenia, and acute renal failure. In addition, 60% of patients have mutations in the genes encoding complement regulators (factor H, factor I, membrane cofactor proteins, and thrombomodulin), activators (factor B and C3), as well as autoantibodies against factor H. Multiple factors are required for the disease to manifest itself, including a trigger and gene mutations with adequate penetration. Being one of the differential diagnoses of preeclampsia- eclampsia and HELLP syndrome means that the clinician must be familiar with the disease due to its high mortality, which can be modified with early diagnosis and comprehensive treatment. Copyright © 2016 Academia Mexicana de Cirugía A.C. Published by Masson Doyma México S.A. All rights reserved.

2-DE analysis indicates that Acinetobacter baumannii displays a robust and versatile metabolism

PubMed Central

Soares, Nelson C; Cabral, Maria P; Parreira, José R; Gayoso, Carmen; Barba, Maria J; Bou, Germán

2009-01-01

Background Acinetobacter baumannii is a nosocomial pathogen that has been associated with outbreak infections in hospitals. Despite increasing awareness about this bacterium, its proteome remains poorly characterised, however recently the complete genome of A. baumannii reference strain ATCC 17978 has been sequenced. Here, we have used 2-DE and MALDI-TOF/TOF approach to characterise the proteome of this strain. Results The membrane and cytoplasmatic protein extracts were analysed separately, these analyses revealed the reproducible presence of 239 and 511 membrane and cytoplamatic protein spots, respectively. MALDI-TOF/TOF characterisation identified a total of 192 protein spots (37 membrane and 155 cytoplasmatic) and revealed that the identified membrane proteins were mainly transport-related proteins, whereas the cytoplasmatic proteins were of diverse nature, although mainly related to metabolic processes. Conclusion This work indicates that A. baumannii has a versatile and robust metabolism and also reveal a number of proteins that may play a key role in the mechanism of drug resistance and virulence. The data obtained complements earlier reports of A. baumannii proteome and provides new tools to increase our knowledge on the protein expression profile of this pathogen. PMID:19785748

Diphtheria toxin translocation across cellular membranes is regulated by sphingolipids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Spilsberg, Bjorn; Hanada, Kentaro; Sandvig, Kirsten

2005-04-08

Diphtheria toxin is translocated across cellular membranes when receptor-bound toxin is exposed to low pH. To study the role of sphingolipids for toxin translocation, both a mutant cell line lacking the first enzyme in de novo sphingolipid synthesis, serine palmitoyltransferase, and a specific inhibitor of the same enzyme, myriocin, were used. The serine palmitoyltransferase-deficient cell line (LY-B) was found to be 10-15 times more sensitive to diphtheria toxin than the genetically complemented cell line (LY-B/cLCB1) and the wild-type cell line (CHO-K1), both when toxin translocation directly across the plasma membrane was induced by exposing cells with surface-bound toxin to lowmore » pH, and when the toxin followed its normal route via acidified endosomes into the cytosol. Toxin binding was similar in these three cell lines. Furthermore, inhibition of serine palmitoyltransferase activity by addition of myriocin sensitized the two control cell lines (LY-B/cLCB1 and CHO-K1) to diphtheria toxin, whereas, as expected, no effect was observed in cells lacking serine palmitoyltransferase (LY-B). In conclusion, diphtheria toxin translocation is facilitated by depletion of membrane sphingolipids.« less

Cytolytic pore-forming protein associated with the surface membrane of Naegleria fowleri

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lowrey, D.M.

1985-01-01

Whole cell homogenates of Naegleria fowleri were examined by hemolytic and /sup 51/Cr-release assays for the presence of cytolytic molecules which may participate in the cytopathogenic action of this amoeba. Two distinct cytolytic activities were found. A surface membrane cytolysin was identified which was found to be avidly associated with membranes possessing an equilibrium density of 1.135 g/cm/sup 3/ in isopycnic sucrose gradients. The activity of the surface membrane cytolysin was not affected by heating at 75/sup 0/C for 30 min. The second cytolytic activity was found in putative lysosomes possessing an equilibrium density of 1.162 g/cm/sup 3/ and wasmore » completely inactivated by heating at 75/sup 0/C for 30 min. Cytolysis produced in the presence of both cytolysins was consistently synergistic with respect to the activity of either cytolysin alone. The lesions produced on erythrocytes by this cooperative process were characterized by electron microscopy as transmembrane pores resembling a number of other cytolytic effector molecules including the ninth component of complement, perforins of cytolytic T lymphocytes, and the alphatoxin of Staphylococcus aureus.« less

C1q-targeted inhibition of the classical complement pathway prevents injury in a novel mouse model of acute motor axonal neuropathy.

PubMed

McGonigal, Rhona; Cunningham, Madeleine E; Yao, Denggao; Barrie, Jennifer A; Sankaranarayanan, Sethu; Fewou, Simon N; Furukawa, Koichi; Yednock, Ted A; Willison, Hugh J

2016-03-02

Guillain-Barré syndrome (GBS) is an autoimmune disease that results in acute paralysis through inflammatory attack on peripheral nerves, and currently has limited, non-specific treatment options. The pathogenesis of the acute motor axonal neuropathy (AMAN) variant is mediated by complement-fixing anti-ganglioside antibodies that directly bind and injure the axon at sites of vulnerability such as nodes of Ranvier and nerve terminals. Consequently, the complement cascade is an attractive target to reduce disease severity. Recently, C5 complement component inhibitors that block the formation of the membrane attack complex and subsequent downstream injury have been shown to be efficacious in an in vivo anti-GQ1b antibody-mediated mouse model of the GBS variant Miller Fisher syndrome (MFS). However, since gangliosides are widely expressed in neurons and glial cells, injury in this model was not targeted exclusively to the axon and there are currently no pure mouse models for AMAN. Additionally, C5 inhibition does not prevent the production of early complement fragments such as C3a and C3b that can be deleterious via their known role in immune cell and macrophage recruitment to sites of neuronal damage. In this study, we first developed a new in vivo transgenic mouse model of AMAN using mice that express complex gangliosides exclusively in neurons, thereby enabling specific targeting of axons with anti-ganglioside antibodies. Secondly, we have evaluated the efficacy of a novel anti-C1q antibody (M1) that blocks initiation of the classical complement cascade, in both the newly developed anti-GM1 antibody-mediated AMAN model and our established MFS model in vivo. Anti-C1q monoclonal antibody treatment attenuated complement cascade activation and deposition, reduced immune cell recruitment and axonal injury, in both mouse models of GBS, along with improvement in respiratory function. These results demonstrate that neutralising C1q function attenuates injury with a consequent neuroprotective effect in acute GBS models and promises to be a useful new target for human therapy.

Membrane development in purple photosynthetic bacteria in response to alterations in light intensity and oxygen tension.

PubMed

Niederman, Robert A

2013-10-01

Studies on membrane development in purple bacteria during adaptation to alterations in light intensity and oxygen tension are reviewed. Anoxygenic phototrophic such as the purple α-proteobacterium Rhodobacter sphaeroides have served as simple, dynamic, and experimentally accessible model organisms for studies of the photosynthetic apparatus. A major landmark in photosynthesis research, which dramatically illustrates this point, was provided by the determination of the X-ray structure of the reaction center (RC) in Blastochloris viridis (Deisenhofer and Michel, EMBO J 8:2149-2170, 1989), once it was realized that this represented the general structure for the photosystem II RC present in all oxygenic phototrophs. This seminal advance, together with a considerable body of subsequent research on the light-harvesting (LH) and electron transfer components of the photosynthetic apparatus has provided a firm basis for the current understanding of how phototrophs acclimate to alterations in light intensity and quality. Oxygenic phototrophs adapt to these changes by extensive thylakoid membrane remodeling, which results in a dramatic supramolecular reordering to assure that an appropriate flow of quinone redox species occurs within the membrane bilayer for efficient and rapid electron transfer. Despite the high level of photosynthetic unit organization in Rba. sphaeroides as observed by atomic force microscopy (AFM), fluorescence induction/relaxation measurements have demonstrated that the addition of the peripheral LH2 antenna complex in cells adapting to low-intensity illumination results in a slowing of the rate of electron transfer turnover by the RC of up to an order of magnitude. This is ascribed to constraints in quinone redox species diffusion between the RC and cytochrome bc1 complexes arising from the increased packing density as the intracytoplasmic membrane (ICM) bilayer becomes crowded with LH2 rings. In addition to downshifts in light intensity as a paradigm for membrane development studies in Rba. sphaeroides, the lowering of oxygen tension in chemoheterotropically growing cells results in a gratuitous formation of the ICM by an extensive membrane biogenesis process. These membrane alterations in response to lowered illumination and oxygen levels in purple bacteria are under the control of a number of interrelated two-component regulatory circuits reviewed here, which act at the transcriptional level to regulate the formation of both the pigment and apoprotein components of the LH, RC, and respiratory complexes. We have performed a proteomic examination of the ICM development process in which membrane proteins have been identified that are temporally expressed both during adaptation to low light intensity and ICM formation at low aeration and are spatially localized in both growing and mature ICM regions. For these proteomic analyses, membrane growth initiation sites and mature ICM vesicles were isolated as respective upper-pigmented band (UPB) and chromatophore fractions and subjected to clear native electrophoresis for isolation of bands containing the LH2 and RC-LH1 core complexes. In chromatophores, increasing levels of LH2 polypeptides relative to those of the RC-LH1 complex were observed as ICM membrane development proceeded during light-intensity downshifts, along with a large array of other associated proteins including high spectral counts for the F1FO-ATP synthase subunits and the cytochrome bc1 complex, as well as RSP6124, a protein of unknown function, that was correlated with increasing LH2 spectral counts. In contrast, the UPB was enriched in cytoplasmic membrane (CM) markers, including electron transfer and transport proteins, as well as general membrane protein assembly factors confirming the origin of the UPB from both peripheral respiratory membrane and sites of active CM invagination that give rise to the ICM. The changes in ICM vesicles were correlated to AFM mapping results (Adams and Hunter, Biochim Biophys Acta 1817:1616-1627, 2012), in which the increasing LH2 levels were shown to form densely packed LH2-only domains, representing the light-responsive antenna complement formed under low illumination. The advances described here could never have been envisioned when the author was first introduced in the mid-1960s to the intricacies of the photosynthetic apparatus during a lecture delivered in a graduate Biochemistry course at the University of Illinois by Govindjee, to whom this volume is dedicated on the occasion of his 80th birthday.

Differential Association of the Na+/H+ Exchanger Regulatory Factor (NHERF) Family of Adaptor Proteins with the Raft-and the Non-Raft Brush Border Membrane Fractions of NHE3

PubMed Central

Sultan, Ayesha; Luo, Min; Yu, Qin; Riederer, Brigitte; Xia, Weiliang; Chen, Mingmin; Lissner, Simone; Gessner, Johannes E.; Donowitz, Mark; Yun, C. Chris; deJonge, Hugo; Lamprecht, Georg; Seidler, Ursula

2014-01-01

Background/Aims Trafficking, brush border membrane (BBM) retention, and signal-specific regulation of the Na+/H+ exchanger NHE3 is regulated by the Na+/H+ Exchanger Regulatory Factor (NHERF) family of PDZ-adaptor proteins, which enable the formation of multiprotein complexes. It is unclear, however, what determines signal specificity of these NHERFs. Thus, we studied the association of NHE3, NHERF1 (EBP50), NHERF2 (E3KARP), and NHERF3 (PDZK1) with lipid rafts in murine small intestinal BBM. Methods Detergent resistant membranes (“lipid rafts”) were isolated by floatation of Triton X-incubated small intestinal BBM from a variety of knockout mouse strains in an Optiprep step gradient. Acid-activated NHE3 activity was measured fluorometrically in BCECF-loaded microdissected villi, or by assessment of CO2/HCO3− mediated increase in fluid absorption in perfused jejunal loops of anethetized mice. Results NHE3 was found to partially associate with lipid rafts in the native BBM, and NHE3 raft association had an impact on NHE3 transport activity and regulation in vivo. NHERF1, 2 and 3 were differentially distributed to rafts and non-rafts, with NHERF2 being most raft-associated and NHERF3 entirely non-raft associated. NHERF2 expression enhanced the localization of NHE3 to membrane rafts. The use of acid sphingomyelinase-deficient mice, which have altered membrane lipid as well as lipid raft composition, allowed us to test the validity of the lipid raft concept in vivo. Conclusions The differential association of the NHERFs with the raft-associated and the non-raft fraction of NHE3 in the brush border membrane is one component of the differential and signal-specific NHE3 regulation by the different NHERFs. PMID:24297041

Differential association of the Na+/H+ Exchanger Regulatory Factor (NHERF) family of adaptor proteins with the raft- and the non-raft brush border membrane fractions of NHE3.

PubMed

Sultan, Ayesha; Luo, Min; Yu, Qin; Riederer, Brigitte; Xia, Weiliang; Chen, Mingmin; Lissner, Simone; Gessner, Johannes E; Donowitz, Mark; Yun, C Chris; deJonge, Hugo; Lamprecht, Georg; Seidler, Ursula

2013-01-01

Trafficking, brush border membrane (BBM) retention, and signal-specific regulation of the Na+/H+ exchanger NHE3 is regulated by the Na+/H+ Exchanger Regulatory Factor (NHERF) family of PDZ-adaptor proteins, which enable the formation of multiprotein complexes. It is unclear, however, what determines signal specificity of these NHERFs. Thus, we studied the association of NHE3, NHERF1 (EBP50), NHERF2 (E3KARP), and NHERF3 (PDZK1) with lipid rafts in murine small intestinal BBM. Detergent resistant membranes ("lipid rafts") were isolated by floatation of Triton X-incubated small intestinal BBM from a variety of knockout mouse strains in an Optiprep step gradient. Acid-activated NHE3 activity was measured fluorometrically in BCECF-loaded microdissected villi, or by assessment of CO2/HCO3(-) mediated increase in fluid absorption in perfused jejunal loops of anethetized mice. NHE3 was found to partially associate with lipid rafts in the native BBM, and NHE3 raft association had an impact on NHE3 transport activity and regulation in vivo. NHERF1, 2 and 3 were differentially distributed to rafts and non-rafts, with NHERF2 being most raft-associated and NHERF3 entirely non-raft associated. NHERF2 expression enhanced the localization of NHE3 to membrane rafts. The use of acid sphingomyelinase-deficient mice, which have altered membrane lipid as well as lipid raft composition, allowed us to test the validity of the lipid raft concept in vivo. The differential association of the NHERFs with the raft-associated and the non-raft fraction of NHE3 in the brush border membrane is one component of the differential and signal-specific NHE3 regulation by the different NHERFs. © 2013 S. Karger AG, Basel.

Membrane Stored Curvature Elastic Stress Modulates Recruitment of Maintenance Proteins PspA and Vipp1

PubMed Central

McDonald, Christopher; Jovanovic, Goran; Ces, Oscar

2015-01-01

ABSTRACT Phage shock protein A (PspA), which is responsible for maintaining inner membrane integrity under stress in enterobacteria, and vesicle-inducting protein in plastids 1 (Vipp1), which functions for membrane maintenance and thylakoid biogenesis in cyanobacteria and plants, are similar peripheral membrane-binding proteins. Their homologous N-terminal amphipathic helices are required for membrane binding; however, the membrane features recognized and required for expressing their functionalities have remained largely uncharacterized. Rigorously controlled, in vitro methodologies with lipid vesicles and purified proteins were used in this study and provided the first biochemical and biophysical characterizations of membrane binding by PspA and Vipp1. Both proteins are found to sense stored curvature elastic (SCE) stress and anionic lipids within the membrane. PspA has an enhanced sensitivity for SCE stress and a higher affinity for the membrane than Vipp1. These variations in binding may be crucial for some of the proteins’ differing roles in vivo. Assays probing the transcriptional regulatory function of PspA in the presence of vesicles showed that a relief of transcription inhibition occurs in an SCE stress-specific manner. This in vitro recapitulation of membrane stress-dependent transcription control suggests that the Psp response may be mounted in vivo when a cell’s inner membrane experiences increased SCE stress. PMID:26330516

[Interaction of free fatty acids with mitochondria during uncoupling of oxidative phosphorylation].

PubMed

Samartsev, V N; Rybakova, S R; Dubinin, M V

2013-01-01

The activity of free saturated fatty acids (caprylic, capric, lauric, myristic, palmitic and stearic) as inducers and regulators of uncoupling of oxidative phosphorylation with participation of ADP/ATP antiporter, aspartate/glutamate antiporter and cyclosporin A-sensitive structure was investigated in experiments on rat liver mitochondria. It is established that at equal uncoupling activity of fatty acids the regulatory effect is minimal for caprylic acid and raised with increasing the hydrophobicity of fatty acids reaching the maximum value for stearic acid. There exists the linear dependence of the regulatory effect value of fatty acids on fatty acids content in the hydrophobic region of the inner membrane. The model that describes the interaction of fatty acids with the hydrophobic region of the mitochondrial inner membrane preserving functional activity of organelles is developed. It is established that if molecules of various fatty acids being in the hydrophobic region of the membrane are equally effective as uncoupling regulators, their specific uncoupling activity is different. Caprylic acid, a short-chain fatty acid, possesses the highest uncoupling activity. As the acyl chain length increases, the specific uncoupling activity of fatty acids reduces exponentially. Under these conditions components of the uncoupling activity sensitive to glutamate and carboxyatractylate and glutamate and insensitive to these reagents (but sensitive to cyclosporin A) change approximately equally.

Membrane-bound Dickkopf-1 in Foxp3+ regulatory T cells suppresses T-cell-mediated autoimmune colitis.

PubMed

Chae, Wook-Jin; Park, Jong-Hyun; Henegariu, Octavian; Yilmaz, Saliha; Hao, Liming; Bothwell, Alfred L M

2017-10-01

Induction of tolerance is a key mechanism to maintain or to restore immunological homeostasis. Here we show that Foxp3 + regulatory T (Treg) cells use Dickkopf-1 (DKK-1) to regulate T-cell-mediated tolerance in the T-cell-mediated autoimmune colitis model. Treg cells from DKK-1 hypomorphic doubleridge mice failed to control CD4 + T-cell proliferation, resulting in CD4 T-cell-mediated autoimmune colitis. Thymus-derived Treg cells showed a robust expression of DKK-1 but not in naive or effector CD4 T cells. DKK-1 expression in Foxp3 + Treg cells was further increased upon T-cell receptor stimulation in vitro and in vivo. Interestingly, Foxp3 + Treg cells expressed DKK-1 in the cell membrane and the functional inhibition of DKK-1 using DKK-1 monoclonal antibody abrogated the suppressor function of Foxp3 + Treg cells. DKK-1 expression was dependent on de novo protein synthesis and regulated by the mitogen-activated protein kinase pathway but not by the canonical Wnt pathway. Taken together, our results highlight membrane-bound DKK-1 as a novel Treg-derived mediator to maintain immunological tolerance in T-cell-mediated autoimmune colitis. © 2017 The Authors. Immunology Published by John Wiley & Sons Ltd.

Diversity and regulation of plant Ca2+ pumps: insights from expression in yeast

NASA Technical Reports Server (NTRS)

Sze, H.; Liang, F.; Hwang, I.; Curran, A. C.; Harper, J. F.; Evans, M. L. (Principal Investigator)

2000-01-01

The spatial and temporal regulation of calcium concentration in plant cells depends on the coordinate activities of channels and active transporters located on different organelles and membranes. Several Ca2+ pumps have been identified and characterized by functional expression of plant genes in a yeast mutant (K616). This expression system has opened the way to a genetic and biochemical characterization of the regulatory and catalytic features of diverse Ca2+ pumps. Plant Ca(2+)-ATPases fall into two major types: AtECA1 represents one of four or more members of the type IIA (ER-type) Ca(2+)-ATPases in Arabidopsis, and AtACA2 is one of seven or more members of the type IIB (PM-type) Ca(2+)-ATPases that are regulated by a novel amino terminal domain. Type IIB pumps are widely distributed on membranes, including the PM (plasma membrane), vacuole, and ER (endoplasmic reticulum). The regulatory domain serves multiple functions, including autoinhibition, calmodulin binding, and sites for modification by phosphorylation. This domain, however, is considerably diverse among several type IIB ATPases, suggesting that the pumps are differentially regulated. Understanding of Ca2+ transporters at the molecular level is providing insights into their roles in signaling networks and in regulating fundamental processes of cell biology.

Demonstration and Validation of a Regenerated Cellulose Dialysis Membrane Diffusion Sampler for Monitoring Ground Water Quality and Remediation Progress at DoD Sites for Perchlorate and Explosives Compounds (ER-0313)

DTIC Science & Technology

2010-09-30

Inductively coupled plasma – mass spectrometry ITRC Interstate Technology Regulatory Council LRL Laboratory reporting level LDPE Low-density polyethylene...diameter of the well. Another diffusion membrane sampler design consists of a tubular-shaped bag made of flexible low-density polyethylene ( LDPE ...Vroblesky, 2001a, 2001b). The LDPE tube is heat-sealed on one end, filled with high-purity water, heat-sealed at the top, and then suspended in a well to

Preventing tissue fibrosis by local biomaterials interfacing of specific cryptic extracellular matrix information

PubMed Central

Horejs, Christine-Maria; St-Pierre, Jean-Philippe; Ojala, Juha R. M.; Steele, Joseph A. M.; da Silva, Patricia Barros; Rynne-Vidal, Angela; Maynard, Stephanie A.; Hansel, Catherine S.; Rodríguez-Fernández, Clara; Mazo, Manuel M.; You, Amanda Y. F.; Wang, Alex J.; von Erlach, Thomas; Tryggvason, Karl; López-Cabrera, Manuel; Stevens, Molly M.

2017-01-01

Matrix metalloproteinases (MMPs) contribute to the breakdown of tissue structures such as the basement membrane, promoting tissue fibrosis. Here we developed an electrospun membrane biofunctionalized with a fragment of the laminin β1-chain to modulate the expression of MMP2 in this context. We demonstrate that interfacing of the β1-fragment with the mesothelium of the peritoneal membrane via a biomaterial abrogates the release of active MMP2 in response to transforming growth factor β1 and rescues tissue integrity ex vivo and in vivo in a mouse model of peritoneal fibrosis. Importantly, our data demonstrate that the membrane inhibits MMP2 expression. Changes in the expression of epithelial-to-mesenchymal transition (EMT)-related molecules further point towards a contribution of the modulation of EMT. Biomaterial-based presentation of regulatory basement membrane signals directly addresses limitations of current therapeutic approaches by enabling a localized and specific method to counteract MMP2 release applicable to a broad range of therapeutic targets. PMID:28593951

Preventing tissue fibrosis by local biomaterials interfacing of specific cryptic extracellular matrix information

NASA Astrophysics Data System (ADS)

Horejs, Christine-Maria; St-Pierre, Jean-Philippe; Ojala, Juha R. M.; Steele, Joseph A. M.; da Silva, Patricia Barros; Rynne-Vidal, Angela; Maynard, Stephanie A.; Hansel, Catherine S.; Rodríguez-Fernández, Clara; Mazo, Manuel M.; You, Amanda Y. F.; Wang, Alex J.; von Erlach, Thomas; Tryggvason, Karl; López-Cabrera, Manuel; Stevens, Molly M.

2017-06-01

Matrix metalloproteinases (MMPs) contribute to the breakdown of tissue structures such as the basement membrane, promoting tissue fibrosis. Here we developed an electrospun membrane biofunctionalized with a fragment of the laminin β1-chain to modulate the expression of MMP2 in this context. We demonstrate that interfacing of the β1-fragment with the mesothelium of the peritoneal membrane via a biomaterial abrogates the release of active MMP2 in response to transforming growth factor β1 and rescues tissue integrity ex vivo and in vivo in a mouse model of peritoneal fibrosis. Importantly, our data demonstrate that the membrane inhibits MMP2 expression. Changes in the expression of epithelial-to-mesenchymal transition (EMT)-related molecules further point towards a contribution of the modulation of EMT. Biomaterial-based presentation of regulatory basement membrane signals directly addresses limitations of current therapeutic approaches by enabling a localized and specific method to counteract MMP2 release applicable to a broad range of therapeutic targets.

Electron transport and light-harvesting switches in cyanobacteria

PubMed Central

Mullineaux, Conrad W.

2014-01-01

Cyanobacteria possess multiple mechanisms for regulating the pathways of photosynthetic and respiratory electron transport. Electron transport may be regulated indirectly by controlling the transfer of excitation energy from the light-harvesting complexes, or it may be more directly regulated by controlling the stoichiometry, localization, and interactions of photosynthetic and respiratory electron transport complexes. Regulation of the extent of linear vs. cyclic electron transport is particularly important for controlling the redox balance of the cell. This review discusses what is known of the regulatory mechanisms and the timescales on which they occur, with particular regard to the structural reorganization needed and the constraints imposed by the limited mobility of membrane-integral proteins in the crowded thylakoid membrane. Switching mechanisms requiring substantial movement of integral thylakoid membrane proteins occur on slower timescales than those that require the movement only of cytoplasmic or extrinsic membrane proteins. This difference is probably due to the restricted diffusion of membrane-integral proteins. Multiple switching mechanisms may be needed to regulate electron transport on different timescales. PMID:24478787

Inconsistencies in the red blood cell membrane proteome analysis: generation of a database for research and diagnostic applications

PubMed Central

Hegedűs, Tamás; Chaubey, Pururawa Mayank; Várady, György; Szabó, Edit; Sarankó, Hajnalka; Hofstetter, Lia; Roschitzki, Bernd; Sarkadi, Balázs

2015-01-01

Based on recent results, the determination of the easily accessible red blood cell (RBC) membrane proteins may provide new diagnostic possibilities for assessing mutations, polymorphisms or regulatory alterations in diseases. However, the analysis of the current mass spectrometry-based proteomics datasets and other major databases indicates inconsistencies—the results show large scattering and only a limited overlap for the identified RBC membrane proteins. Here, we applied membrane-specific proteomics studies in human RBC, compared these results with the data in the literature, and generated a comprehensive and expandable database using all available data sources. The integrated web database now refers to proteomic, genetic and medical databases as well, and contains an unexpected large number of validated membrane proteins previously thought to be specific for other tissues and/or related to major human diseases. Since the determination of protein expression in RBC provides a method to indicate pathological alterations, our database should facilitate the development of RBC membrane biomarker platforms and provide a unique resource to aid related further research and diagnostics. Database URL: http://rbcc.hegelab.org PMID:26078478

Dual role of K ATP channel C-terminal motif in membrane targeting and metabolic regulation.

PubMed

Kline, Crystal F; Kurata, Harley T; Hund, Thomas J; Cunha, Shane R; Koval, Olha M; Wright, Patrick J; Christensen, Matthew; Anderson, Mark E; Nichols, Colin G; Mohler, Peter J

2009-09-29

The coordinated sorting of ion channels to specific plasma membrane domains is necessary for excitable cell physiology. K(ATP) channels, assembled from pore-forming (Kir6.x) and regulatory sulfonylurea receptor subunits, are critical electrical transducers of the metabolic state of excitable tissues, including skeletal and smooth muscle, heart, brain, kidney, and pancreas. Here we show that the C-terminal domain of Kir6.2 contains a motif conferring membrane targeting in primary excitable cells. Kir6.2 lacking this motif displays aberrant channel targeting due to loss of association with the membrane adapter ankyrin-B (AnkB). Moreover, we demonstrate that this Kir6.2 C-terminal AnkB-binding motif (ABM) serves a dual role in K(ATP) channel trafficking and membrane metabolic regulation and dysfunction in these pathways results in human excitable cell disease. Thus, the K(ATP) channel ABM serves as a previously unrecognized bifunctional touch-point for grading K(ATP) channel gating and membrane targeting and may play a fundamental role in controlling excitable cell metabolic regulation.

Fatty Acids in Membranes as Homeostatic, Metabolic and Nutritional Biomarkers: Recent Advancements in Analytics and Diagnostics.

PubMed

Ferreri, Carla; Masi, Annalisa; Sansone, Anna; Giacometti, Giorgia; Larocca, Anna Vita; Menounou, Georgia; Scanferlato, Roberta; Tortorella, Silvia; Rota, Domenico; Conti, Marco; Deplano, Simone; Louka, Maria; Maranini, Anna Rosaria; Salati, Arianna; Sunda, Valentina; Chatgilialoglu, Chryssostomos

2016-12-22

Fatty acids, as structural components of membranes and inflammation/anti-inflammatory mediators, have well-known protective and regulatory effects. They are studied as biomarkers of pathological conditions, as well as saturated and unsaturated hydrophobic moieties in membrane phospholipids that contribute to homeostasis and physiological functions. Lifestyle, nutrition, metabolism and stress-with an excess of radical and oxidative processes-cause fatty acid changes that are examined in the human body using blood lipids. Fatty acid-based membrane lipidomics represents a powerful diagnostic tool for assessing the quantity and quality of fatty acid constituents and also for the follow-up of the membrane fatty acid remodeling that is associated with different physiological and pathological conditions. This review focuses on fatty acid biomarkers with two examples of recent lipidomic research and health applications: (i) monounsaturated fatty acids and the analytical challenge offered by hexadecenoic fatty acids (C16:1); and (ii) the cohort of 10 fatty acids in phospholipids of red blood cell membranes and its connections to metabolic and nutritional status in healthy and diseased subjects.

Fatty Acids in Membranes as Homeostatic, Metabolic and Nutritional Biomarkers: Recent Advancements in Analytics and Diagnostics

PubMed Central

Ferreri, Carla; Masi, Annalisa; Sansone, Anna; Giacometti, Giorgia; Larocca, Anna Vita; Menounou, Georgia; Scanferlato, Roberta; Tortorella, Silvia; Rota, Domenico; Conti, Marco; Deplano, Simone; Louka, Maria; Maranini, Anna Rosaria; Salati, Arianna; Sunda, Valentina; Chatgilialoglu, Chryssostomos

2016-01-01

Fatty acids, as structural components of membranes and inflammation/anti-inflammatory mediators, have well-known protective and regulatory effects. They are studied as biomarkers of pathological conditions, as well as saturated and unsaturated hydrophobic moieties in membrane phospholipids that contribute to homeostasis and physiological functions. Lifestyle, nutrition, metabolism and stress—with an excess of radical and oxidative processes—cause fatty acid changes that are examined in the human body using blood lipids. Fatty acid-based membrane lipidomics represents a powerful diagnostic tool for assessing the quantity and quality of fatty acid constituents and also for the follow-up of the membrane fatty acid remodeling that is associated with different physiological and pathological conditions. This review focuses on fatty acid biomarkers with two examples of recent lipidomic research and health applications: (i) monounsaturated fatty acids and the analytical challenge offered by hexadecenoic fatty acids (C16:1); and (ii) the cohort of 10 fatty acids in phospholipids of red blood cell membranes and its connections to metabolic and nutritional status in healthy and diseased subjects. PMID:28025506

The identification of N-linked oligosaccharides on the human CR2/Epstein-Barr virus receptor and their function in receptor metabolism, plasma membrane expression, and ligand binding.

PubMed

Weis, J J; Fearon, D T

1985-11-05

Human complement receptor type 2 (CR2) was biosynthetically labeled by pulsing SB B lymphoblastoid cells for 25 min with [35S]methionine followed by chase in the presence of excess unlabeled methionine. An Mr 134,000 polypeptide represented the major form of the receptor at the end of the pulse period, and within 1 h of chase this disappeared coincident with the appearance of the Mr 145,000 mature form of CR2. Precursor, but not mature, CR2 was sensitive to endoglycosidase H, indicating that maturation of CR2 represented processing of N-linked high mannose oligosaccharides to the complex type. The processing of precursor CR2 was impaired by monensin. In the presence of tunicamycin an Mr 111,000 form of CR2 was synthesized by SB cells, and this did not chase into either precursor or mature CR2. This Mr 111,000 form of CR2 did not incorporate [3H]glucosamine, indicating that it lacked both N- and O-linked oligosaccharide. The half-lives of mature CR2 and nonglycosylated CR2 pulse-labeled in the presence of tunicamycin were 13.8 and 2.8 h, respectively; the turnover rate of B1, a membrane protein normally lacking carbohydrate, was unaffected by the presence of the antibiotic. The percentage of pulse-labeled, nonglycosylated CR2 that was expressed at the cell surface after 1 h of chase in the presence of tunicamycin was 30%, identical to that of mature CR2 in cells chased in the absence of the antibiotic. However, after 6 h of chase there was no additional net accumulation of nonglycosylated CR2 at the plasma membrane, while the proportion of pulse-labeled mature CR2 at this site had risen to 81%. Therefore, N-linked oligosaccharides are essential for the stability of CR2 and have some role in its plasma membrane expression. In contrast, the observation that all three forms of CR2 bound to Sepharose C3 indicates that oligosaccharides are not necessary for the interaction between CR2 and its complement ligand.

Developmental control of transcriptional and proliferative potency during the evolutionary emergence of animals

PubMed Central

Arenas-Mena, Cesar; Coffman, James A.

2016-01-01

Summary It is proposed that the evolution of complex animals required repressive genetic mechanisms for controlling the transcriptional and proliferative potency of cells. Unicellular organisms are transcriptionally potent, able to express their full genetic complement as the need arises through their life cycle, whereas differentiated cells of multicellular organisms can only express a fraction of their genomic potential. Likewise, whereas cell proliferation in unicellular organisms is primarily limited by nutrient availability, cell proliferation in multicellular organisms is developmentally regulated. Repressive genetic controls limiting the potency of cells at the end of ontogeny would have stabilized the gene expression states of differentiated cells and prevented disruptive proliferation, allowing the emergence of diverse cell types and functional shapes. We propose that distal cis-regulatory elements represent the primary innovations that set the stage for the evolution of developmental gene regulatory networks and the repressive control of key multipotency and cell-cycle control genes. The testable prediction of this model is that the genomes of extant animals, unlike those of our unicellular relatives, encode gene regulatory circuits dedicated to the developmental control of transcriptional and proliferative potency. PMID:26173445

The Plasma Membrane Calcium Pump: New Ways to Look at an Old Enzyme

PubMed Central

Lopreiato, Raffaele; Giacomello, Marta; Carafoli, Ernesto

2014-01-01

The three-dimensional structure of the PMCA pump has not been solved, but its basic mechanistic properties are known to repeat those of the other Ca2+ pumps. However, the pump also has unique properties. They concern essentially its numerous regulatory mechanisms, the most important of which is the autoinhibition by its C-terminal tail. Other regulatory mechanisms involve protein kinases and the phospholipids of the membrane in which the pump is embedded. Permanent activation of the pump, e.g. by calmodulin, is physiologically as harmful to cells as its absence. The concept is now emerging that the global control of cell Ca2+ may not be the main function of the pump; in some cell types, it could even be irrelevant. The main pump role would be the regulation of Ca2+ in cell microdomains in which the pump co-segregates with partners that modulate the Ca2+ message and transduce it to important cell functions. PMID:24570005

Meiotic chromatin diminution in a vertebrate, the holocephalan fish Hydrolagus collie (Chondrichthyes, Holocephali).

PubMed

Stanley, H P; Kasinsky, H E; Bols, N C

1984-01-01

A histochemical, microdensitometric, and electron microscopic study of testes of the ratfish Hydrolagus colliei shows that an instance of the rare phenomenon of germ line chromatin diminution occurs in this vertebrate species. In primary spermatocytes at metaphase I a spherical mass of heterochromatin accumulates at one side of the metaphase plate. At anaphase I the heterochromatic mass is left in the equatorial cytoplasm and is passed into one of the two secondary spermatocytes formed during cytokinesis. As nuclear membranes are being restored, a double membrane envelope is also formed around the heterochromatic mass, which is then termed the 'chromatin diminution body' (CDB). At second meiotic division the CDB is included in the cytoplasm of one of the four spermatids and retained there, apparently unchanged, until mid-spermiogenesis. At that time the CDB becomes adherent to the spermatid plasma membrane and is pinched off from the spermatid by a process of apocrine exocytosis, taking a layer of spermatid plasma membrane along with it. Simultaneously this tri-membrane CDB is taken into the adjacent Sertoli cell by endocytosis, thereby acquiring a fourth membrane layer, a part of the Sertoli cell plasma membrane. The CDBs are subsequently phagocytized, possibly first fusing with dense, multilaminate bodies in the Sertoli cell cytoplasm. The CDB chromatin mass is strongly positive with the Feulgen method for DNA and the alkaline fast green method for histones. Microdensitometric analysis shows that the discarded chromatin amounts to about 10% of the diploid nuclear content and that it appears to be part of the normal diploid complement rather than DNA amplified during meiosis.

Asymptotic approximations for pure bending of thin cylindrical shells

NASA Astrophysics Data System (ADS)

Coman, Ciprian D.

2017-08-01

A simplified partial wrinkling scenario for in-plane bending of thin cylindrical shells is explored by using several asymptotic strategies. The eighth-order boundary eigenvalue problem investigated here originates in the Donnel-Mushtari-Vlasov shallow shell theory coupled with a linear membrane pre-bifurcation state. It is shown that the corresponding neutral stability curve is amenable to a detailed asymptotic analysis based on the method of multiple scales. This is further complemented by an alternative WKB approximation that provides comparable information with significantly less effort.

Targeting the Human Complement Membrane Attack Complex to Selectively Kill Prostate Cancer Cells

DTIC Science & Technology

2012-10-01

prostate cancer cells in vitro . Evaluate CD59 expression in human prostate cancer microarrays. Aim 4: Evaluate toxicity and efficacy of the lead...findings suggest PSA may also have immunoregulatory activity in the seminal plasma to aid in normal fertility that may have been co-opted by prostate...cleavage fragments have not been described. PSA can cleave C3 and generate the 37 kDa fragment in vitro . PSA is the major chymotrypsin-like serine

Targeting the Human Complement Membrane Attack Complex to Selectively Kill Prostate Cancer Cells

DTIC Science & Technology

2014-12-01

These mutants will be tested for their specificity and potency against PSA positive/negative cells in conjunction with the PSMA binding urea...targeting studies. Second, to achieve cell binding and uptake, we propose to link a PSMA binding urea to the C-terminus of recombinant GZMB. This will be...will be linked to the free amine of the PSMA urea in order to covalently link the compound to the C- terminus of GZMB. The C-terminus was chosen

Outer membrane lipoprotein VacJ is required for the membrane integrity, serum resistance and biofilm formation of Actinobacillus pleuropneumoniae.

PubMed

Xie, Fang; Li, Gang; Zhang, Wanjiang; Zhang, Yanhe; Zhou, Long; Liu, Shuanghong; Liu, Siguo; Wang, Chunlai

2016-02-01

The outer membrane proteins of Actinobacillus pleuropneumoniae are mediators of infection, acting as targets for the host's defense system. The outer membrane lipoprotein VacJ is involved in serum resistance and intercellular spreading in several pathogenic bacteria. To investigate the role of VacJ in the pathogenicity of Actinobacillus pleuropneumoniae, the vacJ gene-deletion mutant MD12 ΔvacJ was constructed. The increased susceptibility to KCl, SDS plus EDTA, and several antibiotics in the MD12ΔvacJ mutant suggested that the stability of the outer membrane was impaired as a result of the mutation in the vacJ gene. The increased NPN fluorescence and significant cellular morphological variation in the MD12ΔvacJ mutant further demonstrated the crucial role of the VacJ lipoprotein in maintaining the outer membrane integrity of A. pleuropneumoniae. In addition, the MD12ΔvacJ mutant exhibited decreased survival from the serum and complement killing compared to the wild-type strain. Interestingly, the MD12ΔvacJ mutant showed reduced biofilm formation compared to the wild-type strain. To our knowledge, this is the first description of the VacJ lipoprotein contributing to bacterial biofilm formation. The data presented in this study illustrate the important role of the VacJ lipoprotein in the maintenance of cellular integrity, serum resistance, and biofilm formation in A. pleuropneumoniae. Copyright © 2015 Elsevier B.V. All rights reserved.

Visualizing the dynamic structure of the plant photosynthetic membrane.

PubMed

Ruban, Alexander V; Johnson, Matthew P

2015-11-03

The chloroplast thylakoid membrane is the site for the initial steps of photosynthesis that convert solar energy into chemical energy, ultimately powering almost all life on earth. The heterogeneous distribution of protein complexes within the membrane gives rise to an intricate three-dimensional structure that is nonetheless extremely dynamic on a timescale of seconds to minutes. These dynamics form the basis for the regulation of photosynthesis, and therefore the adaptability of plants to different environments. High-resolution microscopy has in recent years begun to provide new insights into the structural dynamics underlying a number of regulatory processes such as membrane stacking, photosystem II repair, photoprotective energy dissipation, state transitions and alternative electron transfer. Here we provide an overview of the essentials of thylakoid membrane structure in plants, and consider how recent advances, using a range of microscopies, have substantially increased our knowledge of the thylakoid dynamic structure. We discuss both the successes and limitations of the currently available techniques and highlight newly emerging microscopic methods that promise to move the field beyond the current 'static' view of membrane organization based on frozen snapshots to a 'live' view of functional membranes imaged under native aqueous conditions at ambient temperature and responding dynamically to external stimuli.

Reducing abrupt climate change risk using the Montreal Protocol and other regulatory actions to complement cuts in CO2 emissions

PubMed Central

Molina, Mario; Zaelke, Durwood; Sarma, K. Madhava; Andersen, Stephen O.; Ramanathan, Veerabhadran; Kaniaru, Donald

2009-01-01

Current emissions of anthropogenic greenhouse gases (GHGs) have already committed the planet to an increase in average surface temperature by the end of the century that may be above the critical threshold for tipping elements of the climate system into abrupt change with potentially irreversible and unmanageable consequences. This would mean that the climate system is close to entering if not already within the zone of “dangerous anthropogenic interference” (DAI). Scientific and policy literature refers to the need for “early,” “urgent,” “rapid,” and “fast-action” mitigation to help avoid DAI and abrupt climate changes. We define “fast-action” to include regulatory measures that can begin within 2–3 years, be substantially implemented in 5–10 years, and produce a climate response within decades. We discuss strategies for short-lived non-CO2 GHGs and particles, where existing agreements can be used to accomplish mitigation objectives. Policy makers can amend the Montreal Protocol to phase down the production and consumption of hydrofluorocarbons (HFCs) with high global warming potential. Other fast-action strategies can reduce emissions of black carbon particles and precursor gases that lead to ozone formation in the lower atmosphere, and increase biosequestration, including through biochar. These and other fast-action strategies may reduce the risk of abrupt climate change in the next few decades by complementing cuts in CO2 emissions. PMID:19822751

Cell surface dynamics - how Rho GTPases orchestrate the interplay between the plasma membrane and the cortical cytoskeleton.

PubMed

de Curtis, Ivan; Meldolesi, Jacopo

2012-10-01

Small GTPases are known to regulate hundreds of cell functions. In particular, Rho family GTPases are master regulators of the cytoskeleton. By regulating actin nucleation complexes, Rho GTPases control changes in cell shape, including the extension and/or retraction of surface protrusions and invaginations. Protrusion and invagination of the plasma membrane also involves the interaction between the plasma membrane and the cortical cytoskeleton. This interplay between membranes and the cytoskeleton can lead to an increase or decrease in the plasma membrane surface area and its tension as a result of the fusion (exocytosis) or internalization (endocytosis) of membranous compartments, respectively. For a long time, the cytoskeleton and plasma membrane dynamics were investigated separately. However, studies from many laboratories have now revealed that Rho GTPases, their modulation of the cytoskeleton, and membrane traffic are closely connected during the dynamic remodeling of the cell surface. Arf- and Rab-dependent exocytosis of specific vesicles contributes to the targeting of Rho GTPases and their regulatory factors to discrete sites of the plasma membrane. Rho GTPases regulate the tethering of exocytic vesicles and modulate their subsequent fusion. They also have crucial roles in the different forms of endocytosis, where they participate in the sorting of membrane domains as well as the sculpting and sealing of membrane flasks and cups. Here, we discuss how cell surface dynamics depend on the orchestration of the cytoskeleton and the plasma membrane by Rho GTPases.

Concerted regulation of retinal pigment epithelium basement membrane and barrier function by angiocrine factors.

PubMed

Benedicto, Ignacio; Lehmann, Guillermo L; Ginsberg, Michael; Nolan, Daniel J; Bareja, Rohan; Elemento, Olivier; Salfati, Zelda; Alam, Nazia M; Prusky, Glen T; Llanos, Pierre; Rabbany, Sina Y; Maminishkis, Arvydas; Miller, Sheldon S; Rafii, Shahin; Rodriguez-Boulan, Enrique

2017-05-19

The outer blood-retina barrier is established through the coordinated terminal maturation of the retinal pigment epithelium (RPE), fenestrated choroid endothelial cells (ECs) and Bruch's membrane, a highly organized basement membrane that lies between both cell types. Here we study the contribution of choroid ECs to this process by comparing their gene expression profile before (P5) and after (P30) the critical postnatal period when mice acquire mature visual function. Transcriptome analyses show that expression of extracellular matrix-related genes changes dramatically over this period. Co-culture experiments support the existence of a novel regulatory pathway: ECs secrete factors that remodel RPE basement membrane, and integrin receptors sense these changes triggering Rho GTPase signals that modulate RPE tight junctions and enhance RPE barrier function. We anticipate our results will spawn a search for additional roles of choroid ECs in RPE physiology and disease.

Concerted regulation of retinal pigment epithelium basement membrane and barrier function by angiocrine factors

PubMed Central

Benedicto, Ignacio; Lehmann, Guillermo L.; Ginsberg, Michael; Nolan, Daniel J.; Bareja, Rohan; Elemento, Olivier; Salfati, Zelda; Alam, Nazia M.; Prusky, Glen T.; Llanos, Pierre; Rabbany, Sina Y.; Maminishkis, Arvydas; Miller, Sheldon S.; Rafii, Shahin; Rodriguez-Boulan, Enrique

2017-01-01

The outer blood-retina barrier is established through the coordinated terminal maturation of the retinal pigment epithelium (RPE), fenestrated choroid endothelial cells (ECs) and Bruch's membrane, a highly organized basement membrane that lies between both cell types. Here we study the contribution of choroid ECs to this process by comparing their gene expression profile before (P5) and after (P30) the critical postnatal period when mice acquire mature visual function. Transcriptome analyses show that expression of extracellular matrix-related genes changes dramatically over this period. Co-culture experiments support the existence of a novel regulatory pathway: ECs secrete factors that remodel RPE basement membrane, and integrin receptors sense these changes triggering Rho GTPase signals that modulate RPE tight junctions and enhance RPE barrier function. We anticipate our results will spawn a search for additional roles of choroid ECs in RPE physiology and disease. PMID:28524846

Myotonic dystrophy protein kinase (DMPK) induces actin cytoskeletal reorganization and apoptotic-like blebbing in lens cells

NASA Technical Reports Server (NTRS)

Jin, S.; Shimizu, M.; Balasubramanyam, A.; Epstein, H. F.

2000-01-01

DMPK, the product of the DM locus, is a member of the same family of serine-threonine protein kinases as the Rho-associated enzymes. In DM, membrane inclusions accumulate in lens fiber cells producing cataracts. Overexpression of DMPK in cultured lens epithelial cells led to apoptotic-like blebbing of the plasma membrane and reorganization of the actin cytoskeleton. Enzymatically active DMPK was necessary for both effects; inactive mutant DMPK protein did not produce either effect. Active RhoA but not constitutive GDP-state mutant protein produced similar effects as DMPK. The similar actions of DMPK and RhoA suggest that they may function in the same regulatory network. The observed effects of DMPK may be relevant to the removal of membrane organelles during normal lens differentiation and the retention of intracellular membranes in DM lenses. Copyright 2000 Wiley-Liss, Inc.

The effect of membrane filtration on dissolved trace element concentrations

USGS Publications Warehouse

Horowitz, A.J.; Lum, K.R.; Garbarino, J.R.; Hall, G.E.M.; Lemieux, C.; Demas, C.R.

1996-01-01

The almost universally accepted operational definition for dissolved constituents is based on processing whole-water samples through a 0.45-??m membrane filter. Results from field and laboratory experiments indicate that a number of factors associated with filtration, other than just pore size (e.g., diameter, manufacturer, volume of sample processed, amount of suspended sediment in the sample), can produce substantial variations in the 'dissolved' concentrations of such elements as Fe, Al, Cu, Zn, Pb, Co, and Ni. These variations result from the inclusion/exclusion of colloidally- associated trace elements. Thus, 'dissolved' concentrations quantitated by analyzing filtrates generated by processing whole-water through similar pore- sized membrane filters may not be equal/comparable. As such, simple filtration through a 0.45-??m membrane filter may no longer represent an acceptable operational definition for dissolved chemical constituents. This conclusion may have important implications for environmental studies and regulatory agencies.

Continuous in vivo infusion of interferon-gamma (IFN-γ) enhances engraftment of syngeneic wild-type cells in Fanca–/– and Fancg–/– mice

PubMed Central

Si, Yue; Ciccone, Samantha; Yang, Feng-Chun; Yuan, Jin; Zeng, Daisy; Chen, Shi; van de Vrugt, Henri J.; Critser, John; Arwert, Fre; Haneline, Laura S.; Clapp, D. Wade

2006-01-01

Fanconi anemia (FA) is a heterogeneous genetic disorder characterized by bone marrow (BM) failure and cancer susceptibility. Identification of the cDNAs of FA complementation types allows the potential of using gene transfer technology to introduce functional cDNAs as transgenes into autologous stem cells and provide a cure for the BM failure in FA patients. However, strategies to enhance the mobilization, transduction, and engraftment of exogenous stem cells are required to optimize efficacy prior to widespread clinical use. Hypersensitivity of Fancc–/– cells to interferon-gamma (IFN-γ), a nongenotoxic immune-regulatory cytokine, enhances engraftment of syngeneic wild-type (WT) cells in Fancc–/– mice. However, whether this phenotype is of broad relevance in other FA complementation groups is unresolved. Here we show that primitive and mature myeloid progenitors in Fanca–/– and Fancg–/– mice are hypersensitive to IFN-γ and that in vivo infusion of IFN-γ at clinically relevant concentrations was sufficient to allow consistent long-term engraftment of isogenic WT repopulating stem cells. Given that FANCA, FANCC, and FANCG complementation groups account for more than 90% of all FA patients, these data provide evidence that IFN-γ conditioning may be a useful nongenotoxic strategy for myelopreparation in FA patients. PMID:16946306

Continuous in vivo infusion of interferon-gamma (IFN-gamma) enhances engraftment of syngeneic wild-type cells in Fanca-/- and Fancg-/- mice.

PubMed

Si, Yue; Ciccone, Samantha; Yang, Feng-Chun; Yuan, Jin; Zeng, Daisy; Chen, Shi; van de Vrugt, Henri J; Critser, John; Arwert, Fre; Haneline, Laura S; Clapp, D Wade

2006-12-15

Fanconi anemia (FA) is a heterogeneous genetic disorder characterized by bone marrow (BM) failure and cancer susceptibility. Identification of the cDNAs of FA complementation types allows the potential of using gene transfer technology to introduce functional cDNAs as transgenes into autologous stem cells and provide a cure for the BM failure in FA patients. However, strategies to enhance the mobilization, transduction, and engraftment of exogenous stem cells are required to optimize efficacy prior to widespread clinical use. Hypersensitivity of Fancc-/- cells to interferon-gamma (IFN-gamma), a nongenotoxic immune-regulatory cytokine, enhances engraftment of syngeneic wild-type (WT) cells in Fancc-/- mice. However, whether this phenotype is of broad relevance in other FA complementation groups is unresolved. Here we show that primitive and mature myeloid progenitors in Fanca-/- and Fancg-/- mice are hypersensitive to IFN-gamma and that in vivo infusion of IFN-gamma at clinically relevant concentrations was sufficient to allow consistent long-term engraftment of isogenic WT repopulating stem cells. Given that FANCA, FANCC, and FANCG complementation groups account for more than 90% of all FA patients, these data provide evidence that IFN-gamma conditioning may be a useful nongenotoxic strategy for myelopreparation in FA patients.

REMOVING ORGANIC CONTAMINANTS OF REGULATORY INTEREST WITH MEMBRANE PROCESSES: USEPA'S SCREENING STUDIES

EPA Science Inventory

The 1996 amendments to the Safe Drinking Water Act require the US Environmental Protection Agency (USEPA) to establish a list of unregulated microbiological and chemical contaminants to aid in priority-setting for the Agency's drinking water program. This list, known as the Cont...

ISOTONIC DOSE-RESPONSE MODELING OF THE TRANSCRIPTIONAL RESPONSE OF RAT CEREBROCORTICAL TISSUE AFTER ACUTE PYRETHROID EXPOSURE IN VIVO.

EPA Science Inventory

Pyrethroid insecticides produce neurotoxicity in mammals by disrupting ion channel function in excitable nerve membranes. Pyrethroid use has increased as regulatory guidelines have restricted the use of other pesticide classes. Currently, a sensitive, specific, and dose-responsiv...

Plasma membrane proteome analysis identifies a role of barley membrane steroid binding protein in root architecture response to salinity.

PubMed

Witzel, Katja; Matros, Andrea; Møller, Anders L B; Ramireddy, Eswarayya; Finnie, Christine; Peukert, Manuela; Rutten, Twan; Herzog, Andreas; Kunze, Gotthard; Melzer, Michael; Kaspar-Schoenefeld, Stephanie; Schmülling, Thomas; Svensson, Birte; Mock, Hans-Peter

2018-06-01

Although the physiological consequences of plant growth under saline conditions have been well described, understanding the core mechanisms conferring plant salt adaptation has only started. We target the root plasma membrane proteomes of two barley varieties, cvs. Steptoe and Morex, with contrasting salinity tolerance. In total, 588 plasma membrane proteins were identified by mass spectrometry, of which 182 were either cultivar or salinity stress responsive. Three candidate proteins with increased abundance in the tolerant cv. Morex were involved either in sterol binding (a GTPase-activating protein for the adenosine diphosphate ribosylation factor [ZIGA2], and a membrane steroid binding protein [MSBP]) or in phospholipid synthesis (phosphoethanolamine methyltransferase [PEAMT]). Overexpression of barley MSBP conferred salinity tolerance to yeast cells, whereas the knock-out of the heterologous AtMSBP1 increased salt sensitivity in Arabidopsis. Atmsbp1 plants showed a reduced number of lateral roots under salinity, and root-tip-specific expression of barley MSBP in Atmsbp1 complemented this phenotype. In barley, an increased abundance of MSBP correlates with reduced root length and lateral root formation as well as increased levels of auxin under salinity being stronger in the tolerant cv. Morex. Hence, we concluded the involvement of MSBP in phytohormone-directed adaptation of root architecture in response to salinity. © 2018 John Wiley & Sons Ltd.

A novel calmodulin-regulated Ca2+-ATPase (ACA2) from Arabidopsis with an N-terminal autoinhibitory domain

NASA Technical Reports Server (NTRS)

Harper, J. F.; Hong, B.; Hwang, I.; Guo, H. Q.; Stoddard, R.; Huang, J. F.; Palmgren, M. G.; Sze, H.; Evans, M. L. (Principal Investigator)

1998-01-01

To study transporters involved in regulating intracellular Ca2+, we isolated a full-length cDNA encoding a Ca2+-ATPase from a model plant, Arabidopsis, and named it ACA2 (Arabidopsis Ca2+-ATPase, isoform 2). ACA2p is most similar to a "plasma membrane-type" Ca2+-ATPase, but is smaller (110 kDa), contains a unique N-terminal domain, and is missing a long C-terminal calmodulin-binding regulatory domain. In addition, ACA2p is localized to an endomembrane system and not the plasma membrane, as shown by aqueous-two phase fractionation of microsomal membranes. ACA2p was expressed in yeast as both a full-length protein (ACA2-1p) and an N-terminal truncation mutant (ACA2-2p; Delta residues 2-80). Only the truncation mutant restored the growth on Ca2+-depleted medium of a yeast mutant defective in both endogenous Ca2+ pumps, PMR1 and PMC1. Although basal Ca2+-ATPase activity of the full-length protein was low, it was stimulated 5-fold by calmodulin (50% activation around 30 nM). In contrast, the truncated pump was fully active and insensitive to calmodulin. A calmodulin-binding sequence was identified within the first 36 residues of the N-terminal domain, as shown by calmodulin gel overlays on fusion proteins. Thus, ACA2 encodes a novel calmodulin-regulated Ca2+-ATPase distinguished by a unique N-terminal regulatory domain and a non-plasma membrane localization.

A Common Fold Mediates Vertebrate Defense and Bacterial Attack

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rosado, Carlos J.; Buckle, Ashley M.; Law, Ruby H.P.

2008-10-02

Proteins containing membrane attack complex/perforin (MACPF) domains play important roles in vertebrate immunity, embryonic development, and neural-cell migration. In vertebrates, the ninth component of complement and perforin form oligomeric pores that lyse bacteria and kill virus-infected cells, respectively. However, the mechanism of MACPF function is unknown. We determined the crystal structure of a bacterial MACPF protein, Plu-MACPF from Photorhabdus luminescens, to 2.0 angstrom resolution. The MACPF domain reveals structural similarity with poreforming cholesterol-dependent cytolysins (CDCs) from Gram-positive bacteria. This suggests that lytic MACPF proteins may use a CDC-like mechanism to form pores and disrupt cell membranes. Sequence similarity between bacterialmore » and vertebrate MACPF domains suggests that the fold of the CDCs, a family of proteins important for bacterial pathogenesis, is probably used by vertebrates for defense against infection.« less

Establishment and validation of new complementing cells for production of E1-deleted adenovirus vectors in serum-free suspension culture.

PubMed

Gilbert, Rénald; Guilbault, Claire; Gagnon, David; Bernier, Alice; Bourget, Lucie; Elahi, Seyyed Mehdy; Kamen, Amine; Massie, Bernard

2014-11-01

E1-deleted adenovirus vectors (AdV) are important gene transfer vehicles for gene therapy and vaccination. Amplification of AdV must take place in cells that express the adenovirus E1A and E1B genes. Sequence homology between AdV and the E1 genes integrated within the complementing cells should be minimal to reduce the odds of generating replication-competent adenovirus (RCA). The present study describes the establishment of AdV complementing cells constructed by stable transfection of the minimal E1A and E1B genes into human lung carcinoma (A549). Because some transgene products can be cytotoxic, the cells were engineered to stably express the repressor of the cumate-switch (CymR) to silence transgene transcription during vector growth. For regulatory compliance and to facilitate the scale-up, the resulting complementing cells (SF-BMAdR) were adapted to serum-free suspension culture. The best clone of SF-BMAdR produced AdV carrying an innocuous transgene to the same level as 293 cells, but titers were better for AdV carrying transgene for a cytotoxic product. Elevated titers were maintained for at least two months in suspension culture in the absence of selective agent and the cells did not produce RCA. Because of their advantageous properties, SF-BMAdR cells should become an important tool for developing large-scale production processes of AdV for research and clinical applications. Copyright © 2014. Published by Elsevier B.V.

Skipping of exon 27 in C3 gene compromises TED domain and results in complete human C3 deficiency.

PubMed

da Silva, Karina Ribeiro; Fraga, Tatiana Rodrigues; Lucatelli, Juliana Faggion; Grumach, Anete Sevciovic; Isaac, Lourdes

2016-05-01

Primary deficiency of complement C3 is rare and usually associated with increased susceptibility to bacterial infections. In this work, we investigated the molecular basis of complete C3 deficiency in a Brazilian 9-year old female patient with a family history of consanguinity. Hemolytic assays revealed complete lack of complement-mediated hemolytic activity in the patient's serum. While levels of the complement regulatory proteins Factor I, Factor H and Factor B were normal in the patient's and family members' sera, complement C3 levels were undetectable in the patient's serum and were reduced by at least 50% in the sera of the patient's parents and brother. Additionally, no C3 could be observed in the patient's plasma and cell culture supernatants by Western blot. We also observed that patient's skin fibroblasts stimulated with Escherichia coli LPS were unable to secrete C3, which might be accumulated within the cells before being intracellularly degraded. Sequencing analysis of the patient's C3 cDNA revealed a genetic mutation responsible for the complete skipping of exon 27, resulting in the loss of 99 nucleotides (3450-3549) located in the TED domain. Sequencing of the intronic region between the exons 26 and 27 of the C3 gene (nucleotides 6690313-6690961) showed a nucleotide exchange (T→C) at position 6690626 located in a splicing donor site, resulting in the complete skipping of exon 27 in the C3 mRNA. Copyright © 2016. Published by Elsevier GmbH.

Membrane remodeling by amyloidogenic and non-amyloidogenic proteins studied by EPR.

PubMed

Varkey, Jobin; Langen, Ralf

2017-07-01

The advancement in site-directed spin labeling of proteins has enabled EPR studies to expand into newer research areas within the umbrella of protein-membrane interactions. Recently, membrane remodeling by amyloidogenic and non-amyloidogenic proteins has gained a substantial interest in relation to driving and controlling vital cellular processes such as endocytosis, exocytosis, shaping of organelles like endoplasmic reticulum, Golgi and mitochondria, intracellular vesicular trafficking, formation of filopedia and multivesicular bodies, mitochondrial fusion and fission, and synaptic vesicle fusion and recycling in neurotransmission. Misregulation in any of these processes due to an aberrant protein (mutation or misfolding) or alteration of lipid metabolism can be detrimental to the cell and cause disease. Dissection of the structural basis of membrane remodeling by proteins is thus quite necessary for an understanding of the underlying mechanisms, but it remains a formidable task due to the difficulties of various common biophysical tools in monitoring the dynamic process of membrane binding and bending by proteins. This is largely since membranes generally complicate protein structure analysis and this problem is amplified for structural analysis in the presence of different types of membrane curvatures. Recent EPR studies on membrane remodeling by proteins show that a significant structural information can be generated to delineate the role of different protein modules, domains and individual amino acids in the generation of membrane curvature. These studies also show how EPR can complement the data obtained by high resolution techniques such as X-ray and NMR. This perspective covers the application of EPR in recent studies for understanding membrane remodeling by amyloidogenic and non-amyloidogenic proteins that is useful for researchers interested in using or complimenting EPR to gain better understanding of membrane remodeling. We also discuss how a single protein can generate different type of membrane curvatures using specific conformations for specific membrane structures and how EPR is a versatile tool well-suited to analyze subtle alterations in structures under such modifying conditions which otherwise would have been difficult using other biophysical tools. Copyright © 2017 Elsevier Inc. All rights reserved.

Differential effects of human and plant N-acetylglucosaminyltransferase I (GnTI) in plants.

PubMed

Henquet, Maurice; Heinhuis, Bas; Borst, Jan Willem; Eigenhuijsen, Jochem; Schreuder, Mariëlle; Bosch, Dirk; van der Krol, Alexander

2010-08-01

In plants and animals, the first step in complex type N-glycan formation on glycoproteins is catalyzed by N-acetylglucosaminyltransferase I (GnTI). We show that the cgl1-1 mutant of Arabidopsis, which lacks GnTI activity, is fully complemented by YFP-labeled plant AtGnTI, but only partially complemented by YFP-labeled human HuGnTI and that this is due to post-transcriptional events. In contrast to AtGnTI-YFP, only low levels of HuGnTI-YFP protein was detected in transgenic plants. In protoplast co-transfection experiments all GnTI-YFP fusion proteins co-localized with a Golgi marker protein, but only limited co-localization of AtGnTI and HuGnTI in the same plant protoplast. The partial alternative targeting of HuGnTI in plant protoplasts was alleviated by exchanging the membrane-anchor domain with that of AtGnTI, but in stably transformed cgl1-1 plants this chimeric GnTI still did not lead to full complementation of the cgl1-1 phenotype. Combined, the results indicate that activity of HuGnTI in plants is limited by a combination of reduced protein stability, alternative protein targeting and possibly to some extend to lower enzymatic performance of the catalytic domain in the plant biochemical environment.

Neural Correlates of the False Consensus Effect: Evidence for Motivated Projection and Regulatory Restraint.

PubMed

Welborn, B Locke; Gunter, Benjamin C; Vezich, I Stephanie; Lieberman, Matthew D

2017-04-01

The false consensus effect (FCE), the tendency to project our attitudes and opinions on to others, is a pervasive bias in social reasoning with a range of ramifications for individuals and society. Research in social psychology has suggested that numerous factors (anchoring and adjustment, accessibility, motivated projection, etc.) may contribute to the FCE. In this study, we examine the neural correlates of the FCE and provide evidence that motivated projection plays a significant role. Activity in reward regions (ventromedial pFC and bilateral nucleus accumbens) during consensus estimation was positively associated with bias, whereas activity in right ventrolateral pFC (implicated in emotion regulation) was inversely associated with bias. Activity in reward and regulatory regions accounted for half of the total variation in consensus bias across participants (R 2 = .503). This research complements models of the FCE in social psychology, providing a glimpse into the neural mechanisms underlying this important phenomenon.

An Arabidopsis Gene Regulatory Network for Secondary Cell Wall Synthesis

PubMed Central

Taylor-Teeples, M; Lin, L; de Lucas, M; Turco, G; Toal, TW; Gaudinier, A; Young, NF; Trabucco, GM; Veling, MT; Lamothe, R; Handakumbura, PP; Xiong, G; Wang, C; Corwin, J; Tsoukalas, A; Zhang, L; Ware, D; Pauly, M; Kliebenstein, DJ; Dehesh, K; Tagkopoulos, I; Breton, G; Pruneda-Paz, JL; Ahnert, SE; Kay, SA; Hazen, SP; Brady, SM

2014-01-01

Summary The plant cell wall is an important factor for determining cell shape, function and response to the environment. Secondary cell walls, such as those found in xylem, are composed of cellulose, hemicelluloses and lignin and account for the bulk of plant biomass. The coordination between transcriptional regulation of synthesis for each polymer is complex and vital to cell function. A regulatory hierarchy of developmental switches has been proposed, although the full complement of regulators remains unknown. Here, we present a protein-DNA network between Arabidopsis transcription factors and secondary cell wall metabolic genes with gene expression regulated by a series of feed-forward loops. This model allowed us to develop and validate new hypotheses about secondary wall gene regulation under abiotic stress. Distinct stresses are able to perturb targeted genes to potentially promote functional adaptation. These interactions will serve as a foundation for understanding the regulation of a complex, integral plant component. PMID:25533953

Nucleotide sequencing analysis of a LEU gene of Candida maltosa which complements leuB mutation of Escherichia coli and leu2 mutation of Saccharomyces cerevisiae.

PubMed

Takagi, M; Kobayashi, N; Sugimoto, M; Fujii, T; Watari, J; Yano, K

1987-01-01

The expression of a LEU gene from Candida maltosa (designated as C-LEU2) isolated previously (Kawamura et al. 1983) was shown to be regulated, when transferred into Saccharomyces cerevisiae, by leucine and threonine in the medium, as in the case of LEU2 gene of S. cerevisiae. The coding region together with the regulatory region was subcloned and the nucleotide sequence was determined. When the sequence of the coding region was compared with that of LEU2, the homology was 72% for base pairs and 76% for deduced amino acids. Comparison of the regulatory region of C-LEU2 with those of LEU1 and LEU2 suggested a few short consensus sequences which are involved in regulation of gene expression by leucine and threonine in the medium.

Hybrid Storage Market Assessment: A JISEA White Paper

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ericson, Sean J.; Rose, Eric; Jayaswal, Harshit

This white paper evaluates which markets are best suited for battery storage and storage hybrids and reviews regulations and incentives that support or impede the implementation of standalone storage and battery hybrids. The costs of battery storage technologies have dropped in recent years, resulting in a seven-fold increase in installed capacity over the last decade (1). These technologies offer an attractive rate of return in some locations; however, cost and regulatory barriers still limit the market for storage. Hybridizing a battery (combining the battery with a generator) can in some instances reduce total system costs and increase value compared tomore » separate installations. The fast ramping and dispatchability of a battery can complement the generator to provide services that neither battery nor generator could provide alone. Battery hybrids also benefit from some policy incentives and may be better able to meet market and regulatory requirements.« less

Two regulatory RNA elements affect TisB-dependent depolarization and persister formation.

PubMed

Berghoff, Bork A; Hoekzema, Mirthe; Aulbach, Lena; Wagner, E Gerhart H

2017-03-01

Bacterial survival strategies involve phenotypic diversity which is generated by regulatory factors and noisy expression of effector proteins. The question of how bacteria exploit regulatory RNAs to make decisions between phenotypes is central to a general understanding of these universal regulators. We investigated the TisB/IstR-1 toxin-antitoxin system of Escherichia coli to appreciate the role of the RNA antitoxin IstR-1 in TisB-dependent depolarization of the inner membrane and persister formation. Persisters are phenotypic variants that have become transiently drug-tolerant by arresting growth. The RNA antitoxin IstR-1 sets a threshold for TisB-dependent depolarization under DNA-damaging conditions, resulting in two sub-populations: polarized and depolarized cells. Furthermore, our data indicate that an inhibitory 5' UTR structure in the tisB mRNA serves as a regulatory RNA element that delays TisB translation to avoid inappropriate depolarization when DNA damage is low. Investigation of the persister sub-population further revealed that both regulatory RNA elements affect persister levels as well as persistence time. This work provides an intriguing example of how bacteria exploit regulatory RNAs to control phenotypic heterogeneity. © 2016 John Wiley & Sons Ltd.

Membrane-associated proteomics of chickpea identifies Sad1/UNC-84 protein (CaSUN1), a novel component of dehydration signaling

NASA Astrophysics Data System (ADS)

Jaiswal, Dinesh Kumar; Mishra, Poonam; Subba, Pratigya; Rathi, Divya; Chakraborty, Subhra; Chakraborty, Niranjan

2014-02-01

Dehydration affects almost all the physiological processes including those that result in the accumulation of misfolded proteins in the endoplasmic reticulum (ER), which in turn elicits a highly conserved signaling, the unfolded protein response (UPR). We investigated the dehydration-responsive membrane-associated proteome of a legume, chickpea, by 2-DE coupled with mass spectrometry. A total of 184 protein spots were significantly altered over a dehydration treatment of 120 h. Among the differentially expressed proteins, a non-canonical SUN domain protein, designated CaSUN1 (Cicer arietinum Sad1/UNC-84), was identified. CaSUN1 localized to the nuclear membrane and ER, besides small vacuolar vesicles. The transcripts were downregulated by both abiotic and biotic stresses, but not by abscisic acid treatment. Overexpression of CaSUN1 conferred stress tolerance in transgenic Arabidopsis. Furthermore, functional complementation of the yeast mutant, slp1, could rescue its growth defects. We propose that the function of CaSUN1 in stress response might be regulated via UPR signaling.

Measles Virus Fusion Protein: Structure, Function and Inhibition

PubMed Central

Plattet, Philippe; Alves, Lisa; Herren, Michael; Aguilar, Hector C.

2016-01-01

Measles virus (MeV), a highly contagious member of the Paramyxoviridae family, causes measles in humans. The Paramyxoviridae family of negative single-stranded enveloped viruses includes several important human and animal pathogens, with MeV causing approximately 120,000 deaths annually. MeV and canine distemper virus (CDV)-mediated diseases can be prevented by vaccination. However, sub-optimal vaccine delivery continues to foster MeV outbreaks. Post-exposure prophylaxis with antivirals has been proposed as a novel strategy to complement vaccination programs by filling herd immunity gaps. Recent research has shown that membrane fusion induced by the morbillivirus glycoproteins is the first critical step for viral entry and infection, and determines cell pathology and disease outcome. Our molecular understanding of morbillivirus-associated membrane fusion has greatly progressed towards the feasibility to control this process by treating the fusion glycoprotein with inhibitory molecules. Current approaches to develop anti-membrane fusion drugs and our knowledge on drug resistance mechanisms strongly suggest that combined therapies will be a prerequisite. Thus, discovery of additional anti-fusion and/or anti-attachment protein small-molecule compounds may eventually translate into realistic therapeutic options. PMID:27110811

A large-sized bubbling appearance of the glomerular basement membrane in a patient with pulmonary limited AL amyloidosis and a past history of lupus nephritis.

PubMed

Suga, Norihiro; Miura, Naoto; Uemura, Yuko; Nakamura, Toshinobu; Morita, Hiroyuki; Banno, Shogo; Imai, Hirokazu

2011-12-01

We report an unusual pathological finding, a large-sized bubbling appearance of the glomerular basement membrane (GBM), in a patient with pulmonary limited AL amyloidosis and a past history of lupus nephritis. The first renal biopsy specimen from 10 years ago, when systemic lupus erythematosus was diagnosed, demonstrated mild mesangial proliferation and subepithelial deposits (WHO classification: III + V). Light microscopy of the current biopsy using periodic acid methenamine silver (PAMS) stain demonstrated a large-sized bubbling appearance of the GBM; however, very weak immunoglobulin and complement deposition was observed in immunofluorescence studies. Routine electron microscopy demonstrated partial subendothelial expansion with electron-lucent materials, but no electron-dense deposits or amyloid fibrils. Electron microscopy with PAMS stain revealed electron-lucent endothelial scalloping, including some cellular components and microspheres in the GBM; however, it is not clear if these materials are derived from endothelial cells. One possibility is that these unique findings represent a recovery phase of lupus membranous nephritis; another is that these findings correspond to a new disease entity.

Oil-in-water monitoring using membrane inlet mass spectrometry.

PubMed

Brkić, Boris; France, Neil; Taylor, Stephen

2011-08-15

A membrane inlet mass spectrometry (MIMS) system has been used for detection and analysis of two types of North Sea crude oil. The system was installed on-field on the Flotta Oil Terminal (Orkney, UK). It consisted of a quadrupole mass spectrometer (QMS) connected to the capillary probe with a silicone-based membrane. The produced mass spectra and calibration plots from the MIMS instrument showed the capability to measure levels of individual hydrocarbons within crude oil in seawater. The generated mass spectra from the field tests also showed the ability to distinguish between different types of oil and to determine concentrations of toxic hydrocarbons in oil (e.g., benzene, toluene, and xylene (BTX)). The performance of the instrument at different temperatures of seawater and oil droplet sizes was also investigated. The results showed that the QMS-based MIMS system has a potential to complement existing oil-in-water (OiW) monitors by being able to detect different oil types and specific hydrocarbon concentrations with high accuracy, which are currently not supported in commercially available OiW monitors.

The Listeriolysin O PEST-like Sequence Co-opts AP-2-Mediated Endocytosis to Prevent Plasma Membrane Damage during Listeria Infection.

PubMed

Chen, Chen; Nguyen, Brittney N; Mitchell, Gabriel; Margolis, Shally R; Ma, Darren; Portnoy, Daniel A

2018-06-13

Listeriolysin O (LLO) is a cholesterol-dependent cytolysin that mediates escape of Listeria monocytogenes from a phagosome, enabling growth of the bacteria in the host cell cytosol. LLO contains a PEST-like sequence that prevents it from killing infected cells, but the mechanism involved is unknown. We found that the LLO PEST-like sequence was necessary to mediate removal of LLO from the interior face of the plasma membrane, where it coalesces into discrete puncta. LLO interacts with Ap2a2, an adaptor protein involved in endocytosis, via its PEST-like sequence, and Ap2a2-dependent endocytosis is required to prevent LLO-induced cytotoxicity. An unrelated PEST-like sequence from a human G protein-coupled receptor (GPCR), which also interacts with Ap2a2, could functionally complement the PEST-like sequence in L. monocytogenes LLO. These data revealed that LLO co-opts the host endocytosis machinery to protect the integrity of the host plasma membrane during L. monocytogenes infection. Copyright © 2018 Elsevier Inc. All rights reserved.

Exploiting algal NADPH oxidase for biophotovoltaic energy

DOE PAGES

Anderson, Alexander; Laohavisit, Anuphon; Blaby, Ian K.; ...

2015-01-29

Photosynthetic microbes exhibit light-dependent electron export across the cell membrane, which can generate electricity in biological photovoltaic (BPV) devices. How electrons are exported remains to be determined; the identification of mechanisms would help selection or generation of photosynthetic microbes capable of enhanced electrical output. We show that plasma membrane NADPH oxidase activity is a significant component of light-dependent generation of electricity by the unicellular green alga Chlamydomonas reinhardtii. NADPH oxidases export electrons across the plasma membrane to form superoxide anion from oxygen. The C. reinhardtii mutant lacking the NADPH oxidase encoded by RBO1 is impaired in both extracellular superoxide anionmore » production and current generation in a BPV device. Complementation with the wild-type gene restores both capacities, demonstrating the role of the enzyme in electron export. Monitoring light-dependent extracellular superoxide production with a colorimetric assay is shown to be an effective way of screening for electrogenic potential of candidate algal strains. Furthermore, the results show that algal NADPH oxidases are important for superoxide anion production and open avenues for optimizing the biological component of these devices.« less

Measles Virus Fusion Protein: Structure, Function and Inhibition.

PubMed

Plattet, Philippe; Alves, Lisa; Herren, Michael; Aguilar, Hector C

2016-04-21

Measles virus (MeV), a highly contagious member of the Paramyxoviridae family, causes measles in humans. The Paramyxoviridae family of negative single-stranded enveloped viruses includes several important human and animal pathogens, with MeV causing approximately 120,000 deaths annually. MeV and canine distemper virus (CDV)-mediated diseases can be prevented by vaccination. However, sub-optimal vaccine delivery continues to foster MeV outbreaks. Post-exposure prophylaxis with antivirals has been proposed as a novel strategy to complement vaccination programs by filling herd immunity gaps. Recent research has shown that membrane fusion induced by the morbillivirus glycoproteins is the first critical step for viral entry and infection, and determines cell pathology and disease outcome. Our molecular understanding of morbillivirus-associated membrane fusion has greatly progressed towards the feasibility to control this process by treating the fusion glycoprotein with inhibitory molecules. Current approaches to develop anti-membrane fusion drugs and our knowledge on drug resistance mechanisms strongly suggest that combined therapies will be a prerequisite. Thus, discovery of additional anti-fusion and/or anti-attachment protein small-molecule compounds may eventually translate into realistic therapeutic options.

Measurement of soluble CD59 in CSF in demyelinating disease: Evidence for an intrathecal source of soluble CD59.

PubMed

Zelek, Wioleta M; Watkins, Lewis M; Howell, Owain W; Evans, Rhian; Loveless, Sam; Robertson, Neil P; Beenes, Marijke; Willems, Loek; Brandwijk, Ricardo; Morgan, B Paul

2018-02-01

CD59, a broadly expressed glycosylphosphatidylinositol-anchored protein, is the principal cell inhibitor of complement membrane attack on cells. In the demyelinating disorders, multiple sclerosis (MS) and neuromyelitis optica spectrum disorder (NMOSD), elevated complement protein levels, including soluble CD59 (sCD59), were reported in cerebrospinal fluid (CSF). We compared sCD59 levels in CSF and matched plasma in controls and patients with MS, NMOSD and clinically isolated syndrome (CIS) and investigated the source of CSF sCD59 and whether it was microparticle associated. sCD59 was quantified using enzyme-linked immunosorbent assay (ELISA; Hycult; HK374-02). Patient and control CSF was subjected to western blotting to characterise anti-CD59-reactive materials. CD59 was localised by immunostaining and in situ hybridisation. CSF sCD59 levels were double those in plasma (CSF, 30.2 ng/mL; plasma, 16.3 ng/mL). Plasma but not CSF sCD59 levels differentiated MS from NMOSD, MS from CIS and NMOSD/CIS from controls. Elimination of microparticles confirmed that CSF sCD59 was not membrane anchored. CSF levels of sCD59 are not a biomarker of demyelinating diseases. High levels of sCD59 in CSF relative to plasma suggest an intrathecal source; CD59 expression in brain parenchyma was low, but expression was strong on choroid plexus (CP) epithelium, immediately adjacent the CSF, suggesting that this is the likely source.

The organization of the fuc regulon specifying L-fucose dissimilation in Escherichia coli K12 as determined by gene cloning.

PubMed

Chen, Y M; Zhu, Y; Lin, E C

1987-12-01

In Escherichia coli the six known genes specifying the utilization of L-fucose as carbon and energy source cluster at 60.2 min and constitute a regulon. These genes include fucP (encoding L-fucose permease), fucI (encoding L-fucose isomerase), fucK (encoding L-fuculose kinase), fucA (encoding L-fuculose 1-phosphate aldolase), fucO (encoding L-1,2-propanediol oxidoreductase), and fucR (encoding the regulatory protein). In this study the fuc genes were cloned and their positions on the chromosome were established by restriction endonuclease and complementation analyses. Clockwise, the gene order is: fucO-fucA-fucP-fucI-fucK-fucR. The operons comprising the structural genes and the direction of transcription were determined by complementation analysis and Southern blot hybridization. The fucPIK and fucA operons are transcribed clockwise. The fucO operon is transcribed counterclockwise. The fucR gene product activates the three structural operons in trans.

The chromosomes of the Didelphidae (Marsupialia) and their evolutionary significance

USGS Publications Warehouse

Reig, O.; Gardner, A.L.; Bianchi, N.O.; Patton, J.L.

1977-01-01

One hundred and seventy-seven specimens of American didelphids, representing 9 genera and 22 species have been studied for their chromosomal constitution. Didelphids are very conservative in chromosomal complements. All of the studied species can be sorted into one of three kinds of karyotypes: 2n= 14 (three species of Didelphis, one of Lutreolina, two of Philander, and one of Chironectes), 2n = 14 (eight species of Marmosa, one of Metachirus, three of Caluromys, and one of Dromiciops), and 2n= 18 (three species of Monodelphis). These karyotypes are stable, showing only minor variations within each basic pattern. It is concluded that chromosomals evolution in the Didelphidae proceededs from low numbers to higher numbers by a process of centromeric fissioning complemented by some pericentric inversions and/or translocations. The pattern of karyotypic stability is consistent with bradytely at the organismic level of evolution. This is explained by a low rate of regulatory genetic evolution promoted by epistatic selection favouring the retention of chromosomal arrangements highly advantageous for overall adaptation.

[Medical science and the human image--on the theory of medical tasks].

PubMed

Jenny, S

1993-06-22

Our science-determined medicine interprets the non-individual partial aspects of disease that are approachable by causal analytical thought; therefore, it becomes only then an art of healing when it is complemented by medical craftsmanship and philosophic reflection. Naturopathy perceives the individual as an open self-regulatory system whose innumerable mechanisms and strategies for maintenance of integrity have to be supported. In medical practise both concepts have always to be available concomitantly and and in equal rights, not only for repair of lesions but also for real healing.

Novel Mutations Causing C5 Deficiency in Three North-African Families.

PubMed

Colobran, Roger; Franco-Jarava, Clara; Martín-Nalda, Andrea; Baena, Neus; Gabau, Elisabeth; Padilla, Natàlia; de la Cruz, Xavier; Pujol-Borrell, Ricardo; Comas, David; Soler-Palacín, Pere; Hernández-González, Manuel

2016-05-01

The complement system plays a central role in defense to encapsulated bacteria through opsonization and membrane attack complex (MAC) dependent lysis. The three activation pathways (classical, lectin, and alternative) converge in the cleavage of C5, which initiates MAC formation and target lysis. C5 deficiency is associated to recurrent infections by Neisseria spp. In the present study, complement deficiency was suspected in three families of North-African origin after one episode of invasive meningitis due to a non-groupable and two uncommon Meningococcal serotypes (E29, Y). Activity of alternative and classical pathways of complement were markedly reduced and the measurement of terminal complement components revealed total C5 absence. C5 gene analysis revealed two novel mutations as causative of the deficiency: Family A propositus carried a homozygous deletion of two adenines in the exon 21 of C5 gene, resulting in a frameshift and a truncated protein (c.2607_2608del/p.Ser870ProfsX3 mutation). Families B and C probands carried the same homozygous deletion of three consecutive nucleotides (CAA) in exon 9 of the C5 gene, leading to the deletion of asparagine 320 (c.960_962del/p.Asn320del mutation). Family studies confirmed an autosomal recessive inheritance pattern. Although sharing the same geographical origin, families B and C were unrelated. This prompted us to investigate this mutation prevalence in a cohort of 768 North-African healthy individuals. We identified one heterozygous carrier of the p.Asn320del mutation (allelic frequency = 0.065 %), indicating that this mutation is present at low frequency in North-African population.

Characterization of protein phosphatase 2A acting on phosphorylated plasma membrane aquaporin of tulip petals.

PubMed

Azad, Abul Kalam; Sawa, Yoshihiro; Ishikawa, Takahiro; Shibata, Hitoshi

2004-05-01

A protein phosphatase holo-type enzyme (38, 65, and 75 kDa) preparation and a free catalytic subunit (38 kDa) purified from tulip petals were characterized as protein phosphatase 2A (PP2A) by immunological and biochemical approaches. The plasma membrane containing the putative plasma membrane aquaporin (PM-AQP) was prepared from tulip petals, phosphorylated in vitro, and used as the substrate for both of the purified PP2A preparations. Although both preparations dephosphorylated the phosphorylated PM-AQP at 20 degrees C, only the holo-type enzyme preparation acted at 5 degrees C on the phosphorylated PM-AQP with higher substrate specificity, suggesting that regulatory subunits are required for low temperature-dependent dephosphorylation of PM-AQP in tulip petals.

Reuse and Biocompatibility of Hemodialysis Membranes: Clinically Relevant?

PubMed

Upadhyay, Ashish; Jaber, Bertrand L

2017-03-01

The practice of reprocessing dialyzers for reuse, once predominant in the United States, has been steadily declining over the last 20 years. The professed roles of reuse in improving dialyzer membrane biocompatibility and lowering the risk of first-use syndrome have lost relevance with the advent of biocompatible dialyzer membranes and favorable sterilization techniques. The potential for cost-savings from reuse is also called into question by the easy availability of comparatively cheaper dialyzers and rising regulatory demands and operational cost of reprocessing systems. While the environmental concerns from additional dialyzer-related solid waste from rising single-use practice remains pertinent and requires development of safer dialyzer disposable system technologies, there is no meaningful medical rationale for the continued practice of dialyzer reuse in the twenty-first century. © 2017 Wiley Periodicals, Inc.

CD4(+) T-cell responses to Epstein-Barr virus (EBV) latent membrane protein 1 in infectious mononucleosis and EBV-associated non-Hodgkin lymphoma: Th1 in active disease but Tr1 in remission.

PubMed

Marshall, Neil A; Culligan, Dominic J; Johnston, Peter W; Millar, Colin; Barker, Robert N; Vickers, Mark A

2007-10-01

Primary infection with Epstein-Barr virus (EBV) in childhood is usually asymptomatic, whereas infection in adolescence may result in infectious mononucleosis (IM) often followed by a fatigue syndrome. EBV latent membrane protein 1 (LMP1) is expressed in latency and in many EBV-associated tumours, including non-Hodgkin lymphoma (NHL). Given the regulatory nature of the CD4(+) T-cell response against LMP1 previously reported in healthy donors, we investigated whether patients with active EBV-driven disease can nevertheless mount effector [T-helper cell, type 1 (Th1)] anti-LMP1 responses. We therefore performed a longitudinal study of the nature of CD4(+) T-cell responses to LMP1 in four patients with IM, and five patients with NHL. In both groups, responses changed with time. During symptomatic infection or active tumour growth, responses were dominated by a Th1 effector phenotype, but switched to a regulatory interleukin-10 response upon recovery. In addition, the fine specificities of the T cells driving these responses evolved. This study showed the dynamic nature of CD4(+) T-cell responses to LMP1, and demonstrated that, although patients can mount Th1 effector responses, recovery from IM and NHL is associated with regulatory responses.

Coagulation and complement activation.

PubMed

Christensen, K; Larsson, R; Emanuelsson, H; Elgue, G; Larsson, A

2001-02-01

The purpose of this investigation was to assess the effect of heparin coating of a new stent construction (Stent Graft, Jomed Implantate GmbH, Germany) on platelet and coagulation activity. Stent grafts with an ePTFE membrane interfoliated between two stents were deployed in tubings to form Chandler loops. Fresh human blood with a low concentration of heparin was rotated for 1 h, then collected and used for measurements of platelet number, thrombin-antithrombin complex (TAT), CD11b, C3a and C5b-9. There were five study groups: Group 1, conventional unmodified stents (n = 8); Group 2, untreated stent grafts (n = 8); Group 3, heparin-coated stents and untreated membrane (n = 7); Group 4, heparin-coated stents and membrane (n = 8); Group 5, heparin-coated PVC tubings with no stents (n = 8). There was a significant drop in platelet count, increase in TAT-values and CD11b expression in Groups 1-3 but not in Group 4 compared to Group 5. Examination by scanning electron microscopy revealed extensive activation on non-modified stents but almost no deposition of thrombotic material on heparin-modified stent grafts. With unmodified stents and membrane there were signs of significant activation of platelets and coagulation. In contrast, the heparin-coated stent graft induced much less alterations, indicating improved blood compatibility.

Antibacterial Free Fatty Acids and Monoglycerides: Biological Activities, Experimental Testing, and Therapeutic Applications

PubMed Central

Yoon, Bo Kyeong; Jackman, Joshua A.; Valle-González, Elba R.

2018-01-01

Antimicrobial lipids such as fatty acids and monoglycerides are promising antibacterial agents that destabilize bacterial cell membranes, causing a wide range of direct and indirect inhibitory effects. The goal of this review is to introduce the latest experimental approaches for characterizing how antimicrobial lipids destabilize phospholipid membranes within the broader scope of introducing current knowledge about the biological activities of antimicrobial lipids, testing strategies, and applications for treating bacterial infections. To this end, a general background on antimicrobial lipids, including structural classification, is provided along with a detailed description of their targeting spectrum and currently understood antibacterial mechanisms. Building on this knowledge, different experimental approaches to characterize antimicrobial lipids are presented, including cell-based biological and model membrane-based biophysical measurement techniques. Particular emphasis is placed on drawing out how biological and biophysical approaches complement one another and can yield mechanistic insights into how the physicochemical properties of antimicrobial lipids influence molecular self-assembly and concentration-dependent interactions with model phospholipid and bacterial cell membranes. Examples of possible therapeutic applications are briefly introduced to highlight the potential significance of antimicrobial lipids for human health and medicine, and to motivate the importance of employing orthogonal measurement strategies to characterize the activity profile of antimicrobial lipids. PMID:29642500

Nephrin redistribution on podocytes is a potential mechanism for proteinuria in patients with primary acquired nephrotic syndrome.

PubMed

Doublier, S; Ruotsalainen, V; Salvidio, G; Lupia, E; Biancone, L; Conaldi, P G; Reponen, P; Tryggvason, K; Camussi, G

2001-05-01

We investigated the distribution of nephrin by immunofluorescence microscopy in renal biopsies of patients with nephrotic syndrome: 13 with membranous glomerulonephritis (GN), 10 with minimal change GN, and seven with focal segmental glomerulosclerosis. As control, six patients with IgA GN without nephrotic syndrome and 10 normal controls were studied. We found an extensive loss of staining for nephrin and a shift from a podocyte-staining pattern to a granular pattern in patients with nephrotic syndrome, irrespective of the primary disease. In membranous GN, nephrin was co-localized with IgG immune deposits. In the attempt to explain these results, we investigated in vitro whether stimuli acting on the cell cytoskeleton, known to be involved in the pathogenesis of GN, may induce redistribution of nephrin on the surface of human cultured podocytes. Aggregated but not disaggregated human IgG(4), plasmalemmal insertion of membrane attack complex of complement, tumor necrosis factor-alpha, and puromycin, induced the shedding of nephrin with a loss of surface expression. This phenomenon was abrogated by cytochalasin and sodium azide. These results suggest that the activation of cell cytoskeleton may modify surface expression of nephrin allowing a dislocation from plasma membrane to an extracellular site.

Nephrin Redistribution on Podocytes Is a Potential Mechanism for Proteinuria in Patients with Primary Acquired Nephrotic Syndrome

PubMed Central

Doublier, Sophie; Ruotsalainen, Vesa; Salvidio, Gennaro; Lupia, Enrico; Biancone, Luigi; Conaldi, Pier Giulio; Reponen, Paula; Tryggvason, Karl; Camussi, Giovanni

2001-01-01

We investigated the distribution of nephrin by immunofluorescence microscopy in renal biopsies of patients with nephrotic syndrome: 13 with membranous glomerulonephritis (GN), 10 with minimal change GN, and seven with focal segmental glomerulosclerosis. As control, six patients with IgA GN without nephrotic syndrome and 10 normal controls were studied. We found an extensive loss of staining for nephrin and a shift from a podocyte-staining pattern to a granular pattern in patients with nephrotic syndrome, irrespective of the primary disease. In membranous GN, nephrin was co-localized with IgG immune deposits. In the attempt to explain these results, we investigated in vitro whether stimuli acting on the cell cytoskeleton, known to be involved in the pathogenesis of GN, may induce redistribution of nephrin on the surface of human cultured podocytes. Aggregated but not disaggregated human IgG4, plasmalemmal insertion of membrane attack complex of complement, tumor necrosis factor-α, and puromycin, induced the shedding of nephrin with a loss of surface expression. This phenomenon was abrogated by cytochalasin and sodium azide. These results suggest that the activation of cell cytoskeleton may modify surface expression of nephrin allowing a dislocation from plasma membrane to an extracellular site. PMID:11337370

Automated sample preparation using membrane microtiter extraction for bioanalytical mass spectrometry.

PubMed

Janiszewski, J; Schneider, P; Hoffmaster, K; Swyden, M; Wells, D; Fouda, H

1997-01-01

The development and application of membrane solid phase extraction (SPE) in 96-well microtiter plate format is described for the automated analysis of drugs in biological fluids. The small bed volume of the membrane allows elution of the analyte in a very small solvent volume, permitting direct HPLC injection and negating the need for the time consuming solvent evaporation step. A programmable liquid handling station (Quadra 96) was modified to automate all SPE steps. To avoid drying of the SPE bed and to enhance the analytical precision a novel protocol for performing the condition, load and wash steps in rapid succession was utilized. A block of 96 samples can now be extracted in 10 min., about 30 times faster than manual solvent extraction or single cartridge SPE methods. This processing speed complements the high-throughput speed of contemporary high performance liquid chromatography mass spectrometry (HPLC/MS) analysis. The quantitative analysis of a test analyte (Ziprasidone) in plasma demonstrates the utility and throughput of membrane SPE in combination with HPLC/MS. The results obtained with the current automated procedure compare favorably with those obtained using solvent and traditional solid phase extraction methods. The method has been used for the analysis of numerous drug prototypes in biological fluids to support drug discovery efforts.

Short-term hyperthyroidism modulates adenosine receptors and catalytic activity of adenylate cyclase in adipocytes.

PubMed Central

Rapiejko, P J; Malbon, C C

1987-01-01

The effects of short-term hyperthyroidism in vivo on the status of the components of the fat-cell hormone-sensitive adenylate cyclase were investigated. The number of beta-adrenergic receptors was elevated by about 25% in membranes of fat-cells isolated from hyperthyroid rats as compared with euthyroid rats, but their affinity for radioligand was unchanged. Membranes of hyperthyroid-rat fat-cells displayed less than 65% of the normal complement of receptors for [3H]cyclohexyladenosine. The affinity of the receptors for this ligand was normal. In contrast with the marked increase in the amounts of the alpha-subunits of the guanine nucleotide-binding proteins Gi (Mr 41,000) and Go (Mr 39,000) observed in the hypothyroid state [Malbon, Rapiejko & Mangano (1985) J. Biol. Chem. 260, 2558-2564], the amounts of alpha-Gi, alpha-Go as well as alpha-Gs subunits [Mr 42,000 (major) and 46,000/48,000 (minor)] were not changed by hyperthyroidism. Adenylate cyclase activity in response to forskolin, guanosine 5'-[gamma-thio]triphosphate or isoprenaline, in contrast, was decreased by 30-50% in fat-cell membranes from hyperthyroid rats. Fat-cells isolated from hyperthyroid rats accumulated cyclic AMP to less than 50% of the extent in their euthyroid counterparts in the presence of adenosine deaminase and either adrenaline or forskolin, suggesting a decrease in the amount or activity of the catalytic subunit of adenylate cyclase. In the absence of exogenous adenosine deaminase, cyclic AMP accumulation in response to adrenaline was elevated rather than decreased in fat-cells from hyperthyroid rats. The inhibitory influence of adenosine is apparently limited in the hyperthyroid state by the decreased complement of inhibitory R-site purinergic receptors in these fat-cells. Short-term hyperthyroidism modulates the fat-cell adenylate cyclase system at the receptor level (beta-receptor number increased, R-site purinergic-receptor number decreased) and the catalytic subunit of adenylate cyclase. Images Fig. 2. PMID:3036073

Anionic lipids and the cytoskeletal proteins MreB and RodZ define the spatio-temporal distribution and function of membrane stress controller PspA in Escherichia coli.

PubMed

Jovanovic, Goran; Mehta, Parul; Ying, Liming; Buck, Martin

2014-11-01

All cell types must maintain the integrity of their membranes. The conserved bacterial membrane-associated protein PspA is a major effector acting upon extracytoplasmic stress and is implicated in protection of the inner membrane of pathogens, formation of biofilms and multi-drug-resistant persister cells. PspA and its homologues in Gram-positive bacteria and archaea protect the cell envelope whilst also supporting thylakoid biogenesis in cyanobacteria and higher plants. In enterobacteria, PspA is a dual function protein negatively regulating the Psp system in the absence of stress and acting as an effector of membrane integrity upon stress. We show that in Escherichia coli the low-order oligomeric PspA regulatory complex associates with cardiolipin-rich, curved polar inner membrane regions. There, cardiolipin and the flotillin 1 homologue YqiK support the PspBC sensors in transducing a membrane stress signal to the PspA-PspF inhibitory complex. After stress perception, PspA high-order oligomeric effector complexes initially assemble in polar membrane regions. Subsequently, the discrete spatial distribution and dynamics of PspA effector(s) in lateral membrane regions depend on the actin homologue MreB and the peptidoglycan machinery protein RodZ. The consequences of loss of cytoplasmic membrane anionic lipids, MreB, RodZ and/or YqiK suggest that the mode of action of the PspA effector is closely associated with cell envelope organization. © 2014 The Authors.

The Borrelia afzelii outer membrane protein BAPKO_0422 binds human factor-H and is predicted to form a membrane-spanning β-barrel

PubMed Central

Dyer, Adam; Brown, Gemma; Stejskal, Lenka; Laity, Peter R.; Bingham, Richard J.

2015-01-01

The deep evolutionary history of the Spirochetes places their branch point early in the evolution of the diderms, before the divergence of the present day Proteobacteria. As a spirochete, the morphology of the Borrelia cell envelope shares characteristics of both Gram-positive and Gram-negative bacteria. A thin layer of peptidoglycan, tightly associated with the cytoplasmic membrane, is surrounded by a more labile outer membrane (OM). This OM is rich in lipoproteins but with few known integral membrane proteins. The outer membrane protein A (OmpA) domain is an eight-stranded membrane-spanning β-barrel, highly conserved among the Proteobacteria but so far unknown in the Spirochetes. In the present work, we describe the identification of four novel OmpA-like β-barrels from Borrelia afzelii, the most common cause of erythema migrans (EM) rash in Europe. Structural characterization of one these proteins (BAPKO_0422) by SAXS and CD indicate a compact globular structure rich in β-strand consistent with a monomeric β-barrel. Ab initio molecular envelopes calculated from the scattering profile are consistent with homology models and demonstrate that BAPKO_0422 adopts a peanut shape with dimensions 25×45 Å (1 Å=0.1 nm). Deviations from the standard C-terminal signature sequence are apparent; in particular the C-terminal phenylalanine residue commonly found in Proteobacterial OM proteins is replaced by isoleucine/leucine or asparagine. BAPKO_0422 is demonstrated to bind human factor H (fH) and therefore may contribute to immune evasion by inhibition of the complement response. Encoded by chromosomal genes, these proteins are highly conserved between Borrelia subspecies and may be of diagnostic or therapeutic value. PMID:26181365

The Role of Synaptopodin in Membrane Protein Diffusion in the Dendritic Spine Neck.

PubMed

Wang, Lili; Dumoulin, Andréa; Renner, Marianne; Triller, Antoine; Specht, Christian G

2016-01-01

The dynamic exchange of neurotransmitter receptors at synapses relies on their lateral diffusion in the plasma membrane. At synapses located on dendritic spines this process is limited by the geometry of the spine neck that restricts the passage of membrane proteins. Biochemical compartmentalisation of the spine is believed to underlie the input-specificity of excitatory synapses and to set the scale on which functional changes can occur. Synaptopodin is located predominantly in the neck of dendritic spines, and is thus ideally placed to regulate the exchange of synaptic membrane proteins. The central aim of our study was to assess whether the presence of synaptopodin influences the mobility of membrane proteins in the spine neck and to characterise whether this was due to direct molecular interactions or to spatial constraints that are related to the structural organisation of the neck. Using single particle tracking we have identified a specific effect of synaptopodin on the diffusion of metabotropic mGluR5 receptors in the spine neck. However, super-resolution STORM/PALM imaging showed that this was not due to direct interactions between the two proteins, but that the presence of synaptopodin is associated with an altered local organisation of the F-actin cytoskeleton, that in turn could restrict the diffusion of membrane proteins with large intracellular domains through the spine neck. This study contributes new data on the way in which the spine neck compartmentalises excitatory synapses. Our data complement models that consider the impact of the spine neck as a function of its shape, by showing that the internal organisation of the neck imposes additional physical barriers to membrane protein diffusion.

The Role of Synaptopodin in Membrane Protein Diffusion in the Dendritic Spine Neck

PubMed Central

Wang, Lili; Dumoulin, Andréa; Renner, Marianne; Triller, Antoine; Specht, Christian G.

2016-01-01

The dynamic exchange of neurotransmitter receptors at synapses relies on their lateral diffusion in the plasma membrane. At synapses located on dendritic spines this process is limited by the geometry of the spine neck that restricts the passage of membrane proteins. Biochemical compartmentalisation of the spine is believed to underlie the input-specificity of excitatory synapses and to set the scale on which functional changes can occur. Synaptopodin is located predominantly in the neck of dendritic spines, and is thus ideally placed to regulate the exchange of synaptic membrane proteins. The central aim of our study was to assess whether the presence of synaptopodin influences the mobility of membrane proteins in the spine neck and to characterise whether this was due to direct molecular interactions or to spatial constraints that are related to the structural organisation of the neck. Using single particle tracking we have identified a specific effect of synaptopodin on the diffusion of metabotropic mGluR5 receptors in the spine neck. However, super-resolution STORM/PALM imaging showed that this was not due to direct interactions between the two proteins, but that the presence of synaptopodin is associated with an altered local organisation of the F-actin cytoskeleton, that in turn could restrict the diffusion of membrane proteins with large intracellular domains through the spine neck. This study contributes new data on the way in which the spine neck compartmentalises excitatory synapses. Our data complement models that consider the impact of the spine neck as a function of its shape, by showing that the internal organisation of the neck imposes additional physical barriers to membrane protein diffusion. PMID:26840625

Detection of lipid-induced structural changes of the Marburg virus matrix protein VP40 using hydrogen/deuterium exchange-mass spectrometry

PubMed Central

Wijesinghe, Kaveesha J.; Urata, Sarah; Bhattarai, Nisha; Kooijman, Edgar E.; Gerstman, Bernard S.; Chapagain, Prem P.; Li, Sheng; Stahelin, Robert V.

2017-01-01

Marburg virus (MARV) is a lipid-enveloped virus from the Filoviridae family containing a negative sense RNA genome. One of the seven MARV genes encodes the matrix protein VP40, which forms a matrix layer beneath the plasma membrane inner leaflet to facilitate budding from the host cell. MARV VP40 (mVP40) has been shown to be a dimeric peripheral protein with a broad and flat basic surface that can associate with anionic phospholipids such as phosphatidylserine. Although a number of mVP40 cationic residues have been shown to facilitate binding to membranes containing anionic lipids, much less is known on how mVP40 assembles to form the matrix layer following membrane binding. Here we have used hydrogen/deuterium exchange (HDX) mass spectrometry to determine the solvent accessibility of mVP40 residues in the absence and presence of phosphatidylserine and phosphatidylinositol 4,5-bisphosphate. HDX analysis demonstrates that two basic loops in the mVP40 C-terminal domain make important contributions to anionic membrane binding and also reveals a potential oligomerization interface in the C-terminal domain as well as a conserved oligomerization interface in the mVP40 N-terminal domain. Lipid binding assays confirm the role of the two basic patches elucidated with HD/X measurements, whereas molecular dynamics simulations and membrane insertion measurements complement these studies to demonstrate that mVP40 does not appreciably insert into the hydrocarbon region of anionic membranes in contrast to the matrix protein from Ebola virus. Taken together, we propose a model by which association of the mVP40 dimer with the anionic plasma membrane facilitates assembly of mVP40 oligomers. PMID:28167534

Detection of lipid-induced structural changes of the Marburg virus matrix protein VP40 using hydrogen/deuterium exchange-mass spectrometry.

PubMed

Wijesinghe, Kaveesha J; Urata, Sarah; Bhattarai, Nisha; Kooijman, Edgar E; Gerstman, Bernard S; Chapagain, Prem P; Li, Sheng; Stahelin, Robert V

2017-04-14

Marburg virus (MARV) is a lipid-enveloped virus from the Filoviridae family containing a negative sense RNA genome. One of the seven MARV genes encodes the matrix protein VP40, which forms a matrix layer beneath the plasma membrane inner leaflet to facilitate budding from the host cell. MARV VP40 (mVP40) has been shown to be a dimeric peripheral protein with a broad and flat basic surface that can associate with anionic phospholipids such as phosphatidylserine. Although a number of mVP40 cationic residues have been shown to facilitate binding to membranes containing anionic lipids, much less is known on how mVP40 assembles to form the matrix layer following membrane binding. Here we have used hydrogen/deuterium exchange (HDX) mass spectrometry to determine the solvent accessibility of mVP40 residues in the absence and presence of phosphatidylserine and phosphatidylinositol 4,5-bisphosphate. HDX analysis demonstrates that two basic loops in the mVP40 C-terminal domain make important contributions to anionic membrane binding and also reveals a potential oligomerization interface in the C-terminal domain as well as a conserved oligomerization interface in the mVP40 N-terminal domain. Lipid binding assays confirm the role of the two basic patches elucidated with HD/X measurements, whereas molecular dynamics simulations and membrane insertion measurements complement these studies to demonstrate that mVP40 does not appreciably insert into the hydrocarbon region of anionic membranes in contrast to the matrix protein from Ebola virus. Taken together, we propose a model by which association of the mVP40 dimer with the anionic plasma membrane facilitates assembly of mVP40 oligomers. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Molecular characterization of the Na+/H+-antiporter NhaA from Salmonella Typhimurium.

PubMed

Lentes, Christopher J; Mir, Syed H; Boehm, Marc; Ganea, Constanta; Fendler, Klaus; Hunte, Carola

2014-01-01

Na+/H+ antiporters are integral membrane proteins that are present in almost every cell and in every kingdom of life. They are essential for the regulation of intracellular pH-value, Na+-concentration and cell volume. These secondary active transporters exchange sodium ions against protons via an alternating access mechanism, which is not understood in full detail. Na+/H+ antiporters show distinct species-specific transport characteristics and regulatory properties that correlate with respective physiological functions. Here we present the characterization of the Na+/H+ antiporter NhaA from Salmonella enterica serovar Thyphimurium LT2, the causing agent of food-born human gastroenteritis and typhoid like infections. The recombinant antiporter was functional in vivo and in vitro. Expression of its gene complemented the Na+-sensitive phenotype of an E. coli strain that lacks the main Na+/H+ antiporters. Purified to homogeneity, the antiporter was a dimer in solution as accurately determined by size-exclusion chromatography combined with multi-angle laser-light scattering and refractive index monitoring. The purified antiporter was fully capable of electrogenic Na+(Li+)/H+-antiport when reconstituted in proteoliposomes and assayed by solid-supported membrane-based electrophysiological measurements. Transport activity was inhibited by 2-aminoperimidine. The recorded negative currents were in agreement with a 1Na+(Li+)/2H+ stoichiometry. Transport activity was low at pH 7 and up-regulation above this pH value was accompanied by a nearly 10-fold decrease of KmNa (16 mM at pH 8.5) supporting a competitive substrate binding mechanism. K+ does not affect Na+ affinity or transport of substrate cations, indicating that selectivity of the antiport arises from the substrate binding step. In contrast to homologous E. coli NhaA, transport activity remains high at pH values above 8.5. The antiporter from S. Typhimurium is a promising candidate for combined structural and functional studies to contribute to the elucidation of the mechanism of pH-dependent Na+/H+ antiporters and to provide insights in the molecular basis of species-specific growth and survival strategies.

Molecular Characterization of the Na+/H+-Antiporter NhaA from Salmonella Typhimurium

PubMed Central

Lentes, Christopher J.; Mir, Syed H.; Boehm, Marc; Ganea, Constanta; Fendler, Klaus; Hunte, Carola

2014-01-01

Na+/H+ antiporters are integral membrane proteins that are present in almost every cell and in every kingdom of life. They are essential for the regulation of intracellular pH-value, Na+-concentration and cell volume. These secondary active transporters exchange sodium ions against protons via an alternating access mechanism, which is not understood in full detail. Na+/H+ antiporters show distinct species-specific transport characteristics and regulatory properties that correlate with respective physiological functions. Here we present the characterization of the Na+/H+ antiporter NhaA from Salmonella enterica serovar Thyphimurium LT2, the causing agent of food-born human gastroenteritis and typhoid like infections. The recombinant antiporter was functional in vivo and in vitro. Expression of its gene complemented the Na+-sensitive phenotype of an E. coli strain that lacks the main Na+/H+ antiporters. Purified to homogeneity, the antiporter was a dimer in solution as accurately determined by size-exclusion chromatography combined with multi-angle laser-light scattering and refractive index monitoring. The purified antiporter was fully capable of electrogenic Na+(Li+)/H+-antiport when reconstituted in proteoliposomes and assayed by solid-supported membrane-based electrophysiological measurements. Transport activity was inhibited by 2-aminoperimidine. The recorded negative currents were in agreement with a 1Na+(Li+)/2H+ stoichiometry. Transport activity was low at pH 7 and up-regulation above this pH value was accompanied by a nearly 10-fold decrease of Km Na (16 mM at pH 8.5) supporting a competitive substrate binding mechanism. K+ does not affect Na+ affinity or transport of substrate cations, indicating that selectivity of the antiport arises from the substrate binding step. In contrast to homologous E. coli NhaA, transport activity remains high at pH values above 8.5. The antiporter from S. Typhimurium is a promising candidate for combined structural and functional studies to contribute to the elucidation of the mechanism of pH-dependent Na+/H+ antiporters and to provide insights in the molecular basis of species-specific growth and survival strategies. PMID:25010413

The Caenorhabditis elegans Iodotyrosine Deiodinase Ortholog SUP-18 Functions through a Conserved Channel SC-Box to Regulate the Muscle Two-Pore Domain Potassium Channel SUP-9

PubMed Central

de la Cruz, Ignacio Perez; Ma, Long; Horvitz, H. Robert

2014-01-01

Loss-of-function mutations in the Caenorhabditis elegans gene sup-18 suppress the defects in muscle contraction conferred by a gain-of-function mutation in SUP-10, a presumptive regulatory subunit of the SUP-9 two-pore domain K+ channel associated with muscle membranes. We cloned sup-18 and found that it encodes the C. elegans ortholog of mammalian iodotyrosine deiodinase (IYD), an NADH oxidase/flavin reductase that functions in iodine recycling and is important for the biosynthesis of thyroid hormones that regulate metabolism. The FMN-binding site of mammalian IYD is conserved in SUP-18, which appears to require catalytic activity to function. Genetic analyses suggest that SUP-10 can function with SUP-18 to activate SUP-9 through a pathway that is independent of the presumptive SUP-9 regulatory subunit UNC-93. We identified a novel evolutionarily conserved serine-cysteine-rich region in the C-terminal cytoplasmic domain of SUP-9 required for its specific activation by SUP-10 and SUP-18 but not by UNC-93. Since two-pore domain K+ channels regulate the resting membrane potentials of numerous cell types, we suggest that the SUP-18 IYD regulates the activity of the SUP-9 channel using NADH as a coenzyme and thus couples the metabolic state of muscle cells to muscle membrane excitability. PMID:24586202

Biofield Physiology: A Framework for an Emerging Discipline

PubMed Central

Levin, Michael; McCraty, Rollin; Bat, Namuun; Ives, John A.; Lutgendorf, Susan K.; Oschman, James L.

2015-01-01

Biofield physiology is proposed as an overarching descriptor for the electromagnetic, biophotonic, and other types of spatially-distributed fields that living systems generate and respond to as integral aspects of cellular, tissue, and whole organism self-regulation and organization. Medical physiology, cell biology, and biophysics provide the framework within which evidence for biofields, their proposed receptors, and functions is presented. As such, biofields can be viewed as affecting physiological regulatory systems in a manner that complements the more familiar molecular-based mechanisms. Examples of clinically relevant biofields are the electrical and magnetic fields generated by arrays of heart cells and neurons that are detected, respectively, as electrocardiograms (ECGs) or magnetocardiograms (MCGs) and electroencephalograms (EEGs) or magnetoencephalograms (MEGs). At a basic physiology level, electromagnetic activity of neural assemblies appears to modulate neuronal synchronization and circadian rhythmicity. Numerous nonneural electrical fields have been detected and analyzed, including those arising from patterns of resting membrane potentials that guide development and regeneration, and from slowly-varying transepithelial direct current fields that initiate cellular responses to tissue damage. Another biofield phenomenon is the coherent, ultraweak photon emissions (UPE), detected from cell cultures and from the body surface. A physiological role for biophotons is consistent with observations that fluctuations in UPE correlate with cerebral blood flow, cerebral energy metabolism, and EEG activity. Biofield receptors are reviewed in 3 categories: molecular-level receptors, charge flux sites, and endogenously generated electric or electromagnetic fields. In summary, sufficient evidence has accrued to consider biofield physiology as a viable scientific discipline. Directions for future research are proposed. PMID:26665040

Spa47 is an oligomerization-activated type three secretion system (T3SS) ATPase from Shigella flexneri.

PubMed

Burgess, Jamie L; Jones, Heather B; Kumar, Prashant; Toth, Ronald T; Middaugh, C Russell; Antony, Edwin; Dickenson, Nicholas E

2016-05-01

Gram-negative pathogens often use conserved type three secretion systems (T3SS) for virulence. The Shigella type three secretion apparatus (T3SA) penetrates the host cell membrane and provides a unidirectional conduit for injection of effectors into host cells. The protein Spa47 localizes to the base of the apparatus and is speculated to be an ATPase that provides the energy for T3SA formation and secretion. Here, we developed an expression and purification protocol, producing active Spa47 and providing the first direct evidence that Spa47 is a bona fide ATPase. Additionally, size exclusion chromatography and analytical ultracentrifugation identified multiple oligomeric species of Spa47 with the largest greater than 8 fold more active for ATP hydrolysis than the monomer. An ATPase inactive Spa47 point mutant was then engineered by targeting a conserved Lysine within the predicted Walker A motif of Spa47. Interestingly, the mutant maintained a similar oligomerization pattern as active Spa47, but was unable to restore invasion phenotype when used to complement a spa47 null S. flexneri strain. Together, these results identify Spa47 as a Shigella T3SS ATPase and suggest that its activity is linked to oligomerization, perhaps as a regulatory mechanism as seen in some related pathogens. Additionally, Spa47 catalyzed ATP hydrolysis appears to be essential for host cell invasion, providing a strong platform for additional studies dissecting its role in virulence and providing an attractive target for anti-infective agents. © 2016 The Protein Society.