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PubMed2005

Fluorescent proteins as proteomic probes.

Protein complexes mediate the majority of cellular processes. Knowledge of the localization and composition of such complexes provides key insights into their functions. Although green fluorescent protein (GFP) has been widely applied for in vivo visualization of proteins, it has been relatively little used as a tool for the isolation of protein complexes. Here we describe the use of the standard GFP tag to both visualize proteins in living cells and capture their interactions via a simple immunoaffinity purification procedure. We applied this method to the analysis of a variety of endogenous protein complexes from different eukaryotic cells. We show that efficient isolations can be achieved in 5-60 min. This rapid purification helps preserve protein complexes close to their original state in the cell and minimizes nonspecific interactions. Given the wide use and availability of GFP-tagged protein reagents, the present method should greatly facilitate the elucidation of many cellular processes.

Animals↗
PubMed2001

Characterization of intracellular reverse transcription complexes of human immunodeficiency virus type 1.

To examine the early events of the life cycle of human immunodeficiency virus type 1 (HIV-1), we analyzed the intracellular complexes mediating reverse transcription isolated from acutely infected cells. Partial purification of the reverse transcription complexes (RTCs) by equilibrium density fractionation and velocity sedimentation indicated that two species of RTCs are formed but only one species is able to synthesize DNA. Most of the capsid, matrix, and reverse transcriptase (RT) proteins dissociate from the complex soon after cell infection, but Vpr remains associated with the RTC. The RTCs isolated 1, 4, and 7 h after infection are competent for reverse transcription in vitro, indicating that a small proportion of RT remains associated with them. HIV RTCs isolated early after infection have a sedimentation velocity of approximately 560S. Later, different species with a sedimentation velocity ranging from 350S to 100S appear. Nuclear-associated RTCs have a sedimentation velocity of 80S. Shortly after initiation of reverse transcription, the viral strong-stop DNA within the RTC is sensitive to nuclease digestion and becomes protected when reverse transcription is almost completed.

Blotting, Western↗
PubMed1990

IgD immune complex vasculitis in a patient with hyperimmunoglobulinemia D and periodic fever.

We describe a 27-year-old Dutch woman with the hyperimmunoglobulinemia D and periodic fever syndrome. During febrile attacks she occasionally presented with skin lesions on the distal parts of her upper and lower extremities, with the histologic picture of a leukocytoclastic vasculitis. Clear perivascular deposits of IgD and C3 were presented in early lesional skin on immunofluorescence investigation. Circulating IgD immune complexes were demonstrated on several occasions, both during and in between clinical attacks. These findings are consistent with an IgD immune complex-mediated pathogenesis for the skin lesions. In 10 patients with other forms of immune complex vasculitis of the skin, minimal perivascular deposits of IgD were found in four cases. In these cases, however, IgD was never found as the solitary immunoglobulin class.

Adult↗
PubMed1986

Surfactant-anti-surfactant immune complexes in infants with respiratory distress syndrome.

The authors sought to determine whether treatment of respiratory distress syndrome (RDS) with human surfactant resulted in the formation of detectable circulating immune complexes. Preterm infants with severe RDS were divided into two groups: one group received human surfactant by intratracheal instillation and the other group did not. Both groups received ventilatory management involving intermittent mandatory ventilation. Plasma samples were drawn from these babies prior to treatment and at intervals thereafter. The authors developed an ELISA assay specific for surfactant-anti-surfactant immune complexes and analyzed the plasma samples for such immune complexes. Complement levels were also measured. They found that with time plasma from RDS infants in both groups showed evidence of surfactant-anti-surfactant immune complex formation. The concentrations of immune complexes generally peaked within the first week of life and then appeared to diminish over 1-4 weeks after birth in RDS infants. There was no evidence at any time in either group of immune-complex-mediated injury or of decreased serum complement levels. It is concluded that circulating immune complexes between surfactant and antibodies to surfactant are probably found in most neonates with respiratory distress syndrome, that they do not produce pulmonary damage detectable by clinical and serologic means, and that treatment of neonatal RDS with human surfactant similarly does not produce lung injury as determined with these techniques.

Antibody Specificity↗
PubMed1981

Immune complex-induced human monocyte procoagulant activity. I. a rapid unidirectional lymphocyte-instructed pathway.

It has previously been described that soluble antigen:antibody complexes in antigen excess can induce an increase in the procoagulant activity of human peripheral blood mononuclear cells. It has been proposed that this response may explain the presence of fibrin in immune complex-mediated tissue lesions. In the present study we define cellular participants and their roles in the procoagulant response to soluble immune complexes. Monocytes were shown by cell fractionation and by a direct cytologic assay to be the cell of origin of the procoagulant activity; and virtually all monocytes were able to participate in the response. Monocytes, however, required the presence of lymphocytes to respond. The procoagulant response required cell cooperation, and this collaborative interaction between lymphocytes and monocytes appeared to be unidirectional. Lymphocytes once triggered by immune complexes induced monocytes to synthesize the procoagulant product. Intact viable lymphocytes were required to present instructions to monocytes; no soluble mediator could be found to subserve this function. Indeed, all that appeared necessary to induce monocytes to produce procoagulant activity was an encounter with lymphocytes that had previously been in contact with soluble immune complexes. The optimum cellular ratio for this interaction was four lymphocytes per monocyte, about half the ratio in peripheral blood. The procoagulant response was rapid, reaching a maximum within 6 h after exposure to antigen:antibody complexes. The procoagulant activity was consistent with tissue factor because Factors VII and X and prothrombin were required for clotting of fibrinogen. WE propose that this pathway differs from a number of others involving cells of the immune system. Elucidation of the pathway may clarify the role of this lymphocyte-instructed monocyte response in the Shwartzman phenomenon and other thrombohemorrhagic events associated with immune cell function and the formation of immune complexes.

Animals↗
PubMed2004

The interaction between levofloxacine hydrochloride and DNA mediated by Cu2+.

Levofloxacin (LEV) is a fluoroquinolone antimicrobial agent. LEV also inhibits DNA synthesis and is bactericidal. The mechanisms of the interaction among LEV, DNA and Cu2+ are studied by fluorescence method. In the paper, the results show that LEV and Cu2+ can form binary complex, and also LEV and DNA can form complex mediated by Cu2+. The composition of the complex is determined. The affects to the reaction are also found.

Animals↗
PubMed2003

Capillary electrophoresis determination of the binding affinity of bioactive sulfated polysaccharides to proteins: study of the binding properties of fucoidan to antithrombin.

The interaction of proteins with polysaccharides represents a major and challenging topic in glycobiology, since such complexes mediate fundamental biological mechanisms. An affinity capillary electrophoresis method has been developed to evidence the complex formation and to determine the binding properties between an anticoagulant polysaccharide of marine origin, fucoidan, and a potential target protein, antithrombin. This method is a variant of zonal electrophoresis in the mobility shift format. A fixed amount of protein was injected into a capillary filled with a background electrolyte containing the polysaccharide in varying concentrations. The effective mobility data of the protein were processed according to classical linearization treatments to obtain the binding constant for the polysaccharide/antithrombin complex. The results indicate that fucoidan binds to antithrombin in a 1:1 stoichiometry and with an affinity depending on the molecular weight of the polysaccharide. For heparin, the binding constant obtained similarly is in accordance with the literature. This is the first report showing the implementation of a capillary electrophoresis method contributing to the mechanistic understanding of the biological activities of fucoidan and providing evidence for the complex formation between fucoidan and the protein inhibitor of the coagulation antithrombin.

Antithrombins↗
PubMed2002

Neural Wiskott-Aldrich syndrome protein (N-WASP) is the specific ligand for Shigella VirG among the WASP family and determines the host cell type allowing actin-based spreading.

Shigella, the causative agent of bacillary dysentery, is capable of directing its movement within host cells by forming an actin comet tail. The VirG (IcsA) pro-tein expressed at one pole of the bacterium recruits neural Wiskott-Aldrich syndrome protein (N-WASP), a member of the WASP family, which in turn stimulates actin-related protein (Arp) 2/3 complex-mediated actin polymerization. As all the WASP family proteins induce actin polymerization by recruiting Arp2/3 complex, we investigated their involvement in Shigella motility. Here, we show that VirG binds to N-WASP but not to the other WASP family proteins. Using a series of chimeras obtained by swapping N-WASP and WASP domains, we demonstrated that the specificity of VirG to interact with N-WASP lies in the N-terminal region containing the pleckstrin homology (PH) domain and calmodulin-binding IQ motif of N-WASP. A conformational change in N-WASP was important for the VirG-N-WASP interaction, as elimination of the C-terminal acidic region, which is responsible for the intramolecular interaction with the central basic region of N-WASP, affected the specific binding to VirG. We observed that, in haematopoietic cells such as macrophages, polymorphonuclear leucocytes (PMNs) and platelets, WASP was predominantly expressed, whereas the expression of N-WASP was greatly suppressed. Indeed, unlike Listeria, Shigella was unable to move in macrophages at all, although the movement was restored as N-WASP was expressed ectopically. Thus, our findings demonstrate that N-WASP is a specific ligand of VirG, which determines the host cell type allowing actin-based spreading of Shigella.

Actins↗
PubMed1993

Clustering of neutrophil leucocytes in serum: possible role of C1q-containing immune complexes.

Clustering activity for neutrophil granulocytes was generated in pooled normal human serum (NHS) by incubation of the serum with preformed IgG aggregates, but not in heat-treated NHS (56 degrees C, 30 min), indicating that the function was complement-dependent. Judging from results of experiments with complement-deficient sera, and serum depleted of C1q, factor D and properdin, recruitment of the complement system beyond C1 was not required for induction of the activity. Zymosan treatment of NHS resulted in some neutrophil clustering activity, but recombinant C5a had a limited effect. C1q added to heat-treated NHS in conjunction with performed IgG aggregates supported neutrophil clustering in a dose-dependent manner. The serum C1q inhibitor, a chondroitin 4-sulphate proteoglycan known to interact with the collagenous part of C1q, clearly reduced neutrophil clustering in heat-treated NHS supplemented with C1q and IgG aggregates. The C1q inhibitor also reduced the inherent neutrophil clustering activity of some sera from patients with systemic lupus erythematosus (SLE). Neutrophil clustering activity in SLE serum was earlier shown to be inversely related to the number of circulating neutrophils in vivo. Although the precise mechanisms remain unclear, we propose that C1q-containing immunoglobulin complexes mediate neutrophil clustering through C1q receptors, and that this might contribute to pathogenesis of immune complex diseases such as SLE.

Antigen-Antibody Complex↗
PubMed2005

In vitro gamma-secretase cleavage of the Alzheimer's amyloid precursor protein correlates to a subset of presenilin complexes and is inhibited by zinc.

The gamma-secretase complex mediates the final proteolytic event in Alzheimer's disease amyloid-beta biogenesis. This membrane complex of presenilin, anterior pharynx defective, nicastrin, and presenilin enhancer-2 cleaves the C-terminal 99-amino acid fragment of the amyloid precursor protein intramembranously at gamma-sites to form C-terminally heterogeneous amyloid-beta and cleaves at an epsilon-site to release the intracellular domain or epsilon-C-terminal fragment. In this work, two novel in vitro gamma-secretase assays are developed to further explore the biochemical characteristics of gamma-secretase activity. During development of a bacterial expression system for a substrate based on the amyloid precursor protein C-terminal 99-amino acid sequence, fragments similar to amyloid-beta and an epsilon-C-terminal fragment were observed. Upon purification this substrate was used in parallel with a transfected source of substrate to measure gamma-secretase activity from detergent extracted membranes. With these systems, it was determined that recovery of size-fractionated cellular and tissue-derived gamma-secretase activity is dependent upon detergent concentration and that activity correlates to a subset of high molecular mass presenilin complexes. We also show that by changing the solvent environment with dimethyl sulfoxide, detection of epsilon-C-terminal fragments can be elevated. Lastly, we show that zinc causes an increase in the apparent molecular mass of an amyloid precursor protein gamma-secretase substrate and inhibits its cleavage. These studies further refine our knowledge of the complexes and biochemical factors needed for gamma-secretase activity and suggest a mechanism by which zinc dysregulation may contribute to Alzheimer's disease pathogenesis.

Amyloid Precursor Protein Secretases↗
PubMed2005

Effects of thalidomide on intracellular Mycobacterium leprae in normal and activated macrophages.

Thalidomide is an effective drug for the treatment of erythema nodosum leprosum (ENL). ENL is an inflammatory reaction that may occur in multibacillary leprosy patients. Its cause(s) as well as the mechanism of thalidomide in arresting this condition are not fully understood. It has been suggested that ENL is an immune complex-mediated hypersensitivity precipitated by the release of Mycobacterium leprae from macrophages. The released antigen may complex with precipitating antibodies, initiating complement fixation and the production of inflammatory cytokines like tumor necrosis factor alpha (TNF-alpha). Thalidomide has been shown in vitro to reduce antigen- or mitogen-activated macrophage production of TNF-alpha. We investigated if thalidomide could also influence the viability of intracellular M. leprae. Mouse peritoneal macrophages were infected with M. leprae, activated with gamma interferon and endotoxin, or nonactivated, and treated with thalidomide. Intracellular bacilli were recovered, and metabolic activity was assessed by a radiorespirometric procedure. Thalidomide did not possess antimicrobial action against M. leprae in normal and activated host macrophages. This suggests that thalidomide does not retard the release of mycobacterial antigens, a possible prelude or precipitating factor for ENL. A distinct sequence of events explaining the mechanism of action for thalidomide's successful treatment of ENL has yet to be established.

Animals↗
PubMed1985

New developments in immunopathology with special reference to complement and immune complexes.

The last few years have seen rapid advances in our understanding of immune phenomena, and these are now being translated into a new understanding of immunopathology. This paper reviews a number of areas where important new knowledge has been gained in the area of complement and immune complexes as a cause of disease. An understanding of the role of the metabolism of complement proteins has led to an understanding of how these proteins interact with receptors to cause phagocytosis. New understanding of the lysis-producing steps has led to new diagnostic tests for complement-induced damage as well as new knowledge of how complement functions to kill microorganisms. New methods for purifying anaphylatoxins have made these proteins available for study in man. A model of serum sickness in man has allowed for the more detailed understanding of how immune complexes cause disease and has led to the appreciation of new clinical signs of immune-complex-mediated disease in man.

Antigen-Antibody Complex↗
PubMed1987

Accelerated glomerulosclerosis in diabetic rats with immune complex injury.

Immune complex-mediated injury has been postulated to contribute to diabetic microangiopathy. To test this hypothesis, immune complex disease was induced in both insulin-deficient (I-) and insulin-treated (I+) rats with streptozocin-induced diabetes mellitus (DM), and the rats were compared with their respective controls. Heymann nephritis (HN), an animal model of membranous nephropathy, was induced in rats by immunization with proximal renal tubular brush border antigen. In addition to the homogeneous mesangial deposits of IgG that developed in diabetic rats, diabetic rats with immune injury also developed immune deposits of IgG and tubular antigen. Diabetic animals with Heymann nephritis developed more intense granular mesangial and capillary wall immune deposits, detected by immunofluorescence (ranked-sums test, P = .002) and electron microscopy. Mesangial immune deposits were associated with mesangial hypercellularity, determined by counting nuclei per glomerular cross section. Diabetic animals with immune injury had an increased number of nuclei (DM, I-, HN: 70 +/- 4; DM, I+, HN: 65 +/- 3) compared with animals with only Heymann nephritis (55 +/- 4) or only diabetes [DM, I-: 52 +/- 4; DM, I+: 54 +/- 3 (mean +/- SE); P less than .05, ANOVA]. An increase in the accumulation of mesangial matrix in diabetic animals with Heymann nephritis was also apparent by light microscopy and immunofluorescence staining of the mesangium for fibronectin. Insulin treatment and control of hyperglycemia did not prevent the development of these changes. Animals with only Heymann nephritis had lesser amounts of immune deposits, which were limited to the subepithelial space and not associated with structural alterations of the mesangium.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2002

Rho-kinase and myosin-II control phagocytic cup formation during CR, but not FcgammaR, phagocytosis.

Phagocytosis through Fcgamma receptor (FcgammaR) or complement receptor 3 (CR) requires Arp2/3 complex-mediated actin polymerization, although each receptor uses a distinct signaling pathway. Rac and Cdc42 are required for actin and Arp2/3 complex recruitment during FcgammaR phagocytosis, while Rho controls actin assembly at CR phagosomes. To better understand the role of Rho in CR phagocytosis, we tested the idea that a known target of Rho, Rho-kinase (ROK), might control phagocytic cup formation and/or engulfment of particles. Inhibitors of ROK (dominant-negative ROK and Y-27632) and of the downstream target of ROK, myosin-II (ML7, BDM, and dominant-negative myosin-II), were used to test this idea. We found that inhibition of the Rho --> ROK --> myosin-II pathway caused a decreased accumulation of Arp2/3 complex and F-actin around bound particles, which led to a reduction in CR-mediated phagocytic engulfment. FcgammaR-mediated phagocytosis, in contrast, was independent of Rho or ROK activity and was only dependent on myosin-II for particle internalization, not for actin cup formation. While myosins have been previously implicated in FcgammaR phagocytosis, to our knowledge, this is the first demonstration of a role for myosin-II in CR phagocytosis.

Actins↗
PubMed2005

LAT-mediated signaling in CD4+CD25+ regulatory T cell development.

Engagement of the T cell receptor for antigen (TCR) induces formation of signaling complexes mediated through the transmembrane adaptor protein, the linker for activation of T cells (LAT). LAT plays an important role in T cell development, activation, and homeostasis. A knock-in mutation at Tyr136, which is the phospholipase C (PLC)-gamma1-binding site in LAT, leads to a severe autoimmune disease in mice. In this study, we show that CD4+CD25+ T reg cells that expressed Foxp3 transcription factor were nearly absent in both thymus and peripheral lymphoid organs of LAT(Y136F) mice. This defect was not a result of the autoimmune environment as LAT(Y136F) T reg cells also failed to develop in healthy LAT-/- mice that received mixed wild-type and LAT(Y136F) bone marrow cells. Moreover, adoptive transfer of normal CD4+CD25+ T reg cells protected neonatal LAT(Y136F) mice from developing this disease. These T reg cells effectively controlled expansion of CD4+ T cells in LAT(Y136F) mice likely via granzymes and/or TGF-beta-mediated suppression. Furthermore, ectopic expression of Foxp3 conferred a suppressive function in LAT(Y136F) T cells. Our data indicate that the LAT-PLC-gamma1 interaction plays a critical role in Foxp3 expression and the development of CD4+CD25+ T reg cells.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Focal adhesion kinase is activated and mediates the early hypertrophic response to stretch in cardiac myocytes.

Previously we reported that the rapid activation of the Fak/Src multicomponent signaling complex mediates load-induced activation of growth and survival signaling pathways in adult rat heart. In this study, we report that 5% to 20% (10-minute) cyclic stretch (1 Hz) of neonatal rat ventricular myocytes (NRVMs) was paralleled by increases of Fak phosphorylation at Tyr-397 (from 1.5- to 2.8-fold), as detected by anti-Fak-pY397 phosphospecific antibody. Moreover, 15% cyclic stretch lasting from 10 to 120 minutes increased Fak phosphorylation at Tyr-397 by 2.5- to 3.5-fold. This activation was accompanied by a dramatic change in Fak localization in NRVMs from densely concentrated in the perinuclear regions in nonstretched cells to aggregates regularly distributed along the myofilaments in stretched cells. Furthermore, a 4-hour cyclic stretch enhanced the activity of an atrial natriuretic factor (ANF) promoter-luciferase reporter gene by 2.7-fold. Disrupting endogenous Fak/Src signaling either by expression of a dominant-negative Fak mutant with phenylalanine substituted for Tyr-397 or by treatment with a c-Src pharmacological inhibitor (PP-2) markedly attenuated stretch-induced Fak activation and clustering at myofilaments and inhibited stretch-induced ANF gene activation. On the other hand, overexpression of wild-type Fak potentiated the stretch-induced Fak phosphorylation but did not enhance either baseline or stretch-induced ANF promoter-luciferase reporter gene activity compared with the responses of nontransfected NRVMs. These findings identify Fak as an important element in the early responses induced by stretch in cardiac myocytes, indicating that it may coordinate the cellular signaling machinery that controls gene expression program associated with load-induced cardiac myocyte hypertrophy.

Actin Cytoskeleton↗
PubMed1999

Magnesium fluoride-dependent binding of small G proteins to their GTPase-activating proteins.

GTPase-activating proteins (GAPs) enhance the intrinsic GTPase activity of small G proteins, such as Ras and Rho, by contributing a catalytic arginine to the active site. An intramolecular arginine plays a similar role in heterotrimeric G proteins. Aluminum fluoride activates the GDP form of heterotrimeric G proteins, and enhances binding of the GDP form of small G proteins to their GAPs. The resultant complexes have been interpreted as analogues of the transition state of the hydrolytic reaction. Here, equilibrium binding has been measured using scintillation proximity assays to provide quantitative information on the fluoride-mediated interaction of Ras and Rho proteins with their respective GAPs, neurofibromin (NF1) and RhoGAP. High-affinity fluoride-mediated complex formation between Rho.GDP and RhoGAP occurred in the absence of aluminum; however, under these conditions, magnesium was required. Additionally, the novel observation was made of magnesium-dependent, fluoride-mediated binding of Ras.GDP to NF1 in the absence of aluminum. Aluminum was required for complex formation when the concentration of magnesium was low. Thus, either aluminum fluoride or magnesium fluoride can mediate the high-affinity binding of Rho. GDP or Ras.GDP to GAPs. It has been reported that magnesium fluoride can activate heterotrimeric G proteins. Thus, magnesium-dependent fluoride effects might be a general phenomenon with G proteins. Moreover, these data suggest that some protein.nucleotide complexes previously reported to contain aluminum fluoride may in fact contain magnesium fluoride.

Aluminum↗
PubMed1998

Differences in tumor necrosis factor-alpha soluble receptor serum concentrations between patients with Henoch-Schönlein purpura and pediatric systemic lupus erythematosus: pathogenetic implications.

OBJECTIVE: Animal models of immune complex mediated tissue injury have shown different patterns of proinflammatory cytokine production according to the subtype of immunoglobulin involved. The IgA immune complex model differs from the IgG model by the lack of involvement of tumor necrosis factor (TNF) in the pathogenesis of tissue damage. We investigated in age matched patients the possible difference in TNF involvement in a predominantly IgA mediated disease, Henoch-Schönlein purpura (HSP), in comparison with systemic lupus erythematosus (SLE), in which vascular injury is mostly associated with local deposition of IgG immune complexes. METHODS: Serum concentrations of TNF-alpha and its soluble receptors (sTNF-R) p55 and p75 were studied in 20 patients with pediatric SLE at various degrees of disease activity, in 16 patients with highly active HSP, and in 15 healthy controls by enzyme amplified sensitivity immunoassay. SLE disease activity was evaluated using 2 scores, the European Consensus Group Study for SLE Disease Activity Criteria and the SLE Disease Activity Index. RESULTS: Serum concentrations of TNF-alpha fell within the normal range in patients with both SLE and HSP irrespective of disease activity. Conversely, patients with SLE displayed increased serum levels of sTNF-R that correlated positively with the degree of disease activity (r = 0.60, p < 0.001; r = 0.71, p < 0.001, for p55 and p75, respectively). In contrast, no difference in the serum levels of sTNF-R was found between patients with highly active HSP and controls. CONCLUSION: Our study provides the first circumstantial evidence that pediatric SLE and HSP are characterized by differential involvement of TNF in the pathogenesis of tissue damage.

Adolescent↗