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PubMed2002

The follicular dendritic cell restricted epitope, FDC-M2, is complement C4; localization of immune complexes in mouse tissues.

We have identified the murine follicular dendritic cell (FDC) marker, FDC-M2, recognized by monoclonal antibody mAb209, as complement component C4. Consistent with this, FDC-M2 was detectable at sites of immune complex-mediated inflammatory disease. Analysis of FDC-M2 distribution in complement-deficient mice highlighted the differences in immune complex clearance between these mice, andshowed that a population of uncharacterized FDC-M2+ reticular and perivascular cells in the spleen, distinct from FDC, are also involved in immune complex capture and possibly retention. These results demonstrate that mAb209, in addition to its role as an FDC marker, is a valuable reagent for the analysis of complement deposition in vivo.

Animals↗
PubMed2002

Assays for the functional activity of the mannan-binding lectin pathway of complement activation.

Mannan-binding lectin (MBL) activates complement independently of the adaptive, clonal immune system and thus presents an innate anti-microbial defence mechanism. Events in the MBL pathway of complement activation involve the binding of MBL to patterns of carbohydrate structures presented by the surface of micro-organisms. For the activation of complement to occur MBL must be associated with serine proteases (MBL-associated serine proteases, MASPs) in an MBL/MASP complex. When bound to micro-organisms, the MBL complex mediates the activation of C4 and C2, generating the C3 convertase, C4bC2b. The C4/C2 cleaving activity of the MBL complex is shared with the C1 complex of the classical pathway of complement activation. Different assays that allow for determination of the activity of the MBL complex in serum samples have been developed and are discussed in this report. We present data from one such assay (MBL/MASP activity test), which we have found useful for the routine evaluation of clinical samples. In this assay any influence of the classical pathway has been eliminated by using a hypertonic buffer, which inhibits the binding of C1q to immuncomplexes and disrupt the C1 complex, while leaving the function of the MBL complex intact. In parallel we determine the MBL concentration in the sample. As predicted a very high correlation is observed between the results of the two assays.

Complement Activation↗
PubMed1995

Glutathione and free amino acids form stable complexes with DNA following exposure of intact mammalian cells to chromate.

Exposure of cells to carcinogenic Cr(VI) compounds results in the formation of several types of DNA lesions such as strand breaks, DNA-protein crosslinks and uncharacterized DNA-Cr adducts. Hexavalent chromium compounds are positive in most bacterial and eukaryotic mutagenic systems, although the nature of DNA modifications underlying the chromium-induced mutagenesis is not known. Hexavalent chromate(VI) is very active in cellular systems because it is actively transported into cells, but intracellularly it is ultimately reduced to Cr(III). Here we show that exposure of Chinese hamster ovary (CHO) cells to potassium chromate(VI) leads to the formation of stable complexes between DNA and amino acids or glutathione. Cysteine, glutamic acid and histidine were the major amino acids crosslinked to DNA in chromate-treated cells. Incubation of purified DNA in the presence of EDTA dissociated SDS stable amino acid-DNA complexes, which indicates that these DNA adducts are most likely to represent ternary coordination complexes mediated by Cr(III) rather than covalent linkage between amino acids/glutathione and DNA. The amino acids that were found complexed with DNA purified from chromate-exposed cells did not orginate from previously crosslinked proteins during DNA isolation, but represented authentic reactions of free amino acids and glutathione with chromium and DNA in cells. Ternary complexes of glutathione or amino acids with Cr(III) and DNA were estimated to account for as much as 50% of DNA-bound chromium following exposure to < or = 25 microM chromate.

Amino Acids↗
PubMed1991

Lack of binding between cryoimmunoglobulins, immunoglobulins and fibronectin: implications for immune complex vasculitis.

Immune-complex-mediated vasculitis is a frequent complication of rheumatoid arthritis and systemic lupus erythematosus. The mechanism of deposition of immune complexes within the vessel wall in these diseases remains unknown, but probably involves other proteins. Fibronectin is a likely candidate since it possesses the ability to bind to collagen, endothelial cells, and possibly immunoglobulins and immune complexes. In this study, the binding of fibronectin to IgG and IgM cryoglobulins, cold soluble IgM, IgG, IgG subclasses and IgG fragments was investigated in the solution phase. Static light scattering, fluorescence anisotropy, fluorescence intensity, and PEG precipitation studies were used to investigate binding under different conditions of temperature and ionic strength. These studies failed to demonstrate significant binding between fibronectin and IgM, IgG, IgG subclasses and IgG fragments under the conditions studied. These findings argue against solution phase binding of fibronectin and immunoglobulins contributing to immune complex vasculitis. The possibility of important surface interactions between these proteins has not been ruled out.

Cryoglobulins↗
PubMed1992

Cyclic AMP in prokaryotes.

Cyclic AMP (cAMP) is found in a variety of prokaryotes including both eubacteria and archaebacteria. cAMP plays a role in regulating gene expression, not only for the classic inducible catabolic operons, but also for other categories. In the enteric coliforms, the effects of cAMP on gene expression are mediated through its interaction with and allosteric modification of a cAMP-binding protein (CRP). The CRP-cAMP complex subsequently binds specific DNA sequences and either activates or inhibits transcription depending upon the positioning of the complex relative to the promoter. Enteric coliforms have provided a model to explore the mechanisms involved in controlling adenylate cyclase activity, in regulating adenylate cyclase synthesis, and in performing detailed examinations of CRP-cAMP complex-regulated gene expression. This review summarizes recent work focused on elucidating the molecular mechanisms of CRP-cAMP complex-mediated processes. For other bacteria, less detail is known. cAMP has been implicated in regulating antibiotic production, phototrophic growth, and pathogenesis. A role for cAMP has been suggested in nitrogen fixation. Often the only data that support cAMP involvement in these processes includes cAMP measurement, detection of the enzymes involved in cAMP metabolism, or observed effects of high concentrations of the nucleotide on cell growth.

Adenylyl Cyclases↗
PubMed1992

GABA- and glutamate-gated ion channels as molecular sites of alcohol and anesthetic action.

The evidence presented above indicates that GABA- and glutamate-activated ion channels are molecular sites of alcohol and anesthetic action. In view of the important role that these channels play in CNS excitability, it seems likely that the actions of alcohol and anesthetics on these channels contribute significantly to the behavioral effects of these agents. Although the behavioral effects of alcohol and anesthetics may well result from a combination of actions on different ion channels and other molecular sites in the CNS, it is of interest to consider whether the actions of these agents on particular types of ion channels may contribute to particular behavioral effects. In this regard, it should be noted that benzodiazepines potentiate GABAA responses, but do not produce intoxication or general anesthesia in their clinical dose range. Benzodiazepines are widely used clinically, primarily for their anxiolytic actions (26), suggesting that the potentiation of GABAA responses by ethanol and barbiturates may contribute to the anxiolytic effects of these agents. Since kainate and quisqualate channels mediate fast excitatory transmission in the CNS, inhibition of kainate and quisqualate receptor-activated responses would be expected to result in general CNS depression. This suggests that inhibition of kainate and quisqualate receptor-mediated responses may contribute to the general anesthetic effects of ethanol, trichloroethanol and barbiturates. NMDA channels are thought to mediate complex excitatory neural phenomena and cognitive function. In view of this, the observation that ethanol inhibits NMDA receptor-mediated responses over the concentration range that produces intoxication and the correlation between the potency of different alcohols for inhibiting NMDA-activated current and their potency for producing intoxication suggest that ethanol-induced inhibition of NMDA receptor-mediated responses may contribute to the intoxicating effects of ethanol. Although these speculations are no doubt oversimplifications, the recognition that GABA- and glutamate-gated ion channels are molecular sites of alcohol and anesthetic action provides a basis for investigating the molecular mechanisms involved in the action of these agents and the behavioral significance of those actions.

Alcohols↗
PubMed1993

Identification of residues 413-433 of plasma protein S as essential for binding to C4b-binding protein.

Reversible association of protein S with C4b-binding protein (C4BP) in plasma down-regulates protein S activity, since free protein S but not the protein S.C4BP complex is an anticoagulant cofactor for activated protein C. To identify regions on the surface of protein S that mediate complex formation with C4BP, a number of nonoverlapping synthetic pentadecapeptides comprising protein S sequences were prepared and tested for their ability to inhibit complex formation. The most potent pentadecapeptide, residues 420-434 (PSP-420) (SGIKEIIQEKQNKHC), gave half-maximal effect at 20 microM. A peptide with the reverse sequence, 434-420, did not inhibit. A peptide containing the sequence of protein S residues 408-434 inhibited complex formation by > 95% with 50% inhibition at 5 microM peptide. Biotinylated C4BP bound specifically to plates coated with PSP-420 but not with the 434-420 peptide; and biotinylated PSP-420 bound to plates coated with C4BP. Rabbit antibodies were raised against several keyhole limpet hemocyanin-conjugated peptides, and each was tested for ability to inhibit complex formation. Anti-PSP-420 antibody potently inhibited complex formation with half-maximal effect at 25 nM IgG. A monoclonal antibody (LJ-56) made against PSP-420 showed high affinity for protein S and inhibited complex formation; this monoclonal antibody specifically recognized free protein S but not the protein S.C4BP complex. These results imply that the PSP-420 sequence is surface-exposed, capable of binding to C4BP, and essential for protein S binding to C4BP.

Amino Acid Sequence↗
PubMed2002

Combinatorial diversity of fission yeast SCF ubiquitin ligases by homo- and heterooligomeric assemblies of the F-box proteins Pop1p and Pop2p.

BACKGROUND: SCF ubiquitin ligases share the core subunits cullin 1, SKP1, and HRT1/RBX1/ROC1, which associate with different F-box proteins. F-box proteins bind substrates following their phosphorylation upon stimulation of various signaling pathways. Ubiquitin-mediated destruction of the fission yeast cyclin-dependent kinase inhibitor Rum1p depends on two heterooligomerizing F-box proteins, Pop1p and Pop2p. Both proteins interact with the cullin Pcu1p when overexpressed, but it is unknown whether this reflects their co-assembly into bona fide SCF complexes. RESULTS: We have identified Psh1p and Pip1p, the fission yeast homologues of human SKP1 and HRT1/RBX1/ROC1, and show that both associate with Pop1p, Pop2p, and Pcu1p into a ~500 kDa SCFPop1p-Pop2p complex, which supports polyubiquitylation of Rum1p. Only the F-box of Pop1p is required for SCFPop1p-Pop2p function, while Pop2p seems to be attracted into the complex through binding to Pop1p. Since all SCFPop1p-Pop2p subunits, except for Pop1p, which is exclusively nuclear, localize to both the nucleus and the cytoplasm, the F-box of Pop2p may be critical for the assembly of cytoplasmic SCFPop2p complexes. In support of this notion, we demonstrate individual SCFPop1p and SCFPop2p complexes bearing ubiquitin ligase activity. CONCLUSION: Our data suggest that distinct homo- and heterooligomeric assemblies of Pop1p and Pop2p generate combinatorial diversity of SCFPop function in fission yeast. Whereas a heterooligomeric SCFPop1p-Pop2p complex mediates polyubiquitylation of Rum1p, homooligomeric SCFPop1p and SCFPop2p complexes may target unknown nuclear and cytoplasmic substrates.

Binding Sites↗
PubMed2006

Activator-specific recruitment of Mediator in vivo.

The Mediator complex associates with eukaryotic RNA polymerase (Pol) II and is recruited to transcriptional enhancers by activator proteins. It is believed that Mediator is a general component of the Pol II machinery that is crucial to connect enhancer-bound activators to basic transcription factors. However, we show that Mediator does not detectably associate with many highly active Pol II promoters in yeast cells. Furthermore, in response to stress conditions, Mediator association is not directly related to Pol II association and in some cases is not detectable at highly activated promoters. Thus, Mediator is recruited to enhancers in an activator-specific manner, and it does not seem to be a stoichiometric component of the basic Pol II machinery in vivo. Mediator is recruited by many activators involved in stress responses, but not by the major activators that function under optimal conditions.

Copper↗
PubMed2006

Roles of cortactin, an actin polymerization mediator, in cell endocytosis.

Cortactin, an actin-binding protein and a substrate of Src, is encoded by the EMS1 oncogene. Cortactin is known to activate Arp2/3 complex-mediated actin polymerization and interact with dynamin, a large GTPase and proline rich domain-containing protein. Transferrin endocytosis was significantly reduced in cells by knock-down of cortactin expression as well as in vivo introduction of cortactin immunoreagents. Cortactin-dynamin interaction displayed morphologically dynamic co-distribution with a change in the endocytosis level in cells treated with an actin depolymerization reagent, cytochalasin D. In an in vitro beads assay, a branched actin network was recruited onto dynamin-coated beads in a cortactin Src homology domain 3 (SH3)-dependent manner. In addition, cortactin was found to function in the late stage of clathrin coated vesicle formation. Taken together, cortactin is required for optimal clathrin mediated endocytosis in a dynamin directed manner.

Actin-Related Protein 2↗
PubMed1978

Merocyanine 540 as a fluorescent probe of membranes: staining of electrically excitable cells.

With the exception of certain blood cells considered in the accompanying paper (Valinsky, Easton and Reich, 1978), merocyanine 540 (MC 540), a fluorescent membrane probe, selectively strains the membranes of a wide variety of electrically excitable cells, but not those of nonexcitable cells. This reaction is Ca2+-dependent when staining is performed in buffered iso-osmotic sucrose, Ca2+-independent when staining proceeds at high ionic strength, inhibited by La3+ and sodium Suramin, enhanced by controlled, low level photosensitization of cell-associated dye and essentially irreversible. These characteristics of the staining reaction depend upon the maintenance of both cell viability and a normal unperturbed membrane structure. Although the mechanisms involved in the staining specificity remain unknown, observation of MC 540 partitioning between benzene and water in model reactions indicates that dye transport into hydrophobic solvents is accompanied by the formation of stoichiometric complexes with cations and phospholipids. These results may suggest the existence of specific, possibly phospholipid-rich membrane domains that mediate complex formation with MC 540 in excitable cells; comparable domains either would not exist, or would be inaccessible at the external surfaces of nonexcitable cells.

Benzoxazoles↗
PubMed2006

Capturing protein-protein complexes at equilibrium: the holdup comparative chromatographic retention assay.

The popular pulldown chromatographic assay detects complexes mediated by fusion proteins retained on affinity resin. The main limitation of this method is that it does not analyze complexes at equilibrium but after several washing steps. Consequently, fast-dissociating complexes may remain undetected. Here, we present the holdup assay, based on the principle of comparative chromatographic retention which eliminates the use of washing steps. The assay evaluates fractions of free and bound species at equilibrium. We used human papillomavirus oncoprotein E6, an E6-binding peptide and an E6-binding PDZ domain, to test several protocols utilizing pure proteins or expression extracts. The holdup assay is faster and more informative than the pulldown assay. It detects fast-dissociating complexes and it is also suited for evaluating equilibrium constants. It is potentially adaptable for automated determination of affinity constants and high-throughput analysis of interactions between proteins and other proteins, peptides, nucleic acids, or small regulatory molecules.

Cell Line, Tumor↗
PubMed2000

Establishment of monoclonal anti-retroviral gp70 autoantibodies from MRL/lpr lupus mice and induction of glomerular gp70 deposition and pathology by transfer into non-autoimmune mice.

Several strains of mice, including MRL/MpJ mice homozygous for the Fas mutant lpr gene (MRL/lpr mice), F(1) hybrids of New Zealand Black and New Zealand White mice, and BXSB/MpJ mice carrying a Y-linked autoimmune acceleration gene, spontaneously develop immune complex-mediated glomerulonephritis. The involvement of the envelope glycoprotein gp70 of an endogenous xenotropic virus in the formation of circulating immune complexes and their deposition in the glomerular lesions have been demonstrated, as has the pathogenicity of various antinuclear, antiphospholipid, and rheumatoid factor autoantibodies. In recent genetic linkage studies as well as in a study of cytokine-induced protection against nephritis development, the strongest association of serum levels of gp70-anti-gp70 immune complexes, rather than the levels of antinuclear autoantibodies, with the development and severity of glomerulonephritis has been demonstrated, suggesting a major pathogenic role of anti-gp70 autoantibodies in the lupus-prone mice. However, the pathogenicity of anti-gp70 autoantibodies has not yet been directly tested. To examine if anti-gp70 autoantibodies induce glomerular pathology, we established from unmanipulated MRL/lpr mice hybridoma clones that secrete monoclonal antibodies reactive with endogenous xenotropic viral env gene products. Upon transplantation, a high proportion of these anti-gp70 antibody-producing hybridoma clones induced in syngeneic non-autoimmune and severe combined immunodeficiency mice proliferative or wire loop-like glomerular lesions. Furthermore, deposition of gp70 in glomeruli and pathological changes were observed after intravenous injection of representative clones of purified anti-gp70 immunoglobulin G, demonstrating pathogenicity of at least some anti-gp70 autoantibodies.

Animals↗
PubMed1989

Glomerular basement membrane-containing immune complexes stimulate tumor necrosis factor and interleukin-1 production by human monocytes.

The ability of human peripheral blood monocytes to produce tumor necrosis factor (TNF) and interleukin-1 (IL-1) in an in vitro model of immune complex-mediated glomerulonephritis was investigated. When isolated monocytes were incubated with human glomerular basement membrane (GBM) containing anti-GBM immune complexes, both TNF and IL-1 were produced and secreted into the medium. The time course of secretion differed, with IL-1 production being maximal after approximately 8 hours, whereas TNF levels continued to rise for 30 hours. The activities of the monocyte-derived TNF and IL-1 were inhibitable by specific antibodies. No effect was seen when monocytes were incubated separately with either GBM alone or anti-GBM IgG. The levels of TNF and IL-1 released were comparable with those induced by high concentrations of LPS, indicating that production was close to the maximal levels reported for these cells. High levels of TNF and IL-1 also were produced in response to soluble immune complexes. The results show that monocytes can produce significant levels of TNF and IL-1 in response to both surface-bound and soluble immune complexes and provide support for the participation of these monokines in glomerulonephritis.

Antigen-Antibody Complex↗
PubMed1997

Cross-linking of Fc gamma receptor IIa and Fc gamma receptor IIIb induces different proadhesive phenotypes on human neutrophils.

Activation of polymorphonuclear leukocytes (PMN) plays an important role in vascular injury associated with systemic vasculitis and in models of autoantibody- and immune complex-mediated disease. The potential role of intravascular activation of PMN, however, is confounded by the observation that some stimuli injected i.v. (e.g., IL-8 and C5a) lead to L-selectin shedding by PMN, which inhibits attachment to endothelium and may be functionally anti-inflammatory. To explore the impact of Fc gamma receptor (Fc gamma R)-mediated activation on the PMN adhesive phenotype, Fc gamma RIIa (CD32) and Fc gamma RIIIb (Cd16) were targeted with receptor-specific reagents, and the expression of adhesion molecules-mediating rolling (L-selectin) and firm adhesion (CD11b/CD18) was measured. Engagement of either Fc gamma RIIa or Fc gamma RIIIb leads to activation, demonstrated by degranulation (upregulation of CD66b), and to increased expression of total CD11b/CD18 and functional CD11b/CD18 (I-domain). In contrast, L-selectin shedding induced by PMN Fc gamma R was divergent. Despite the 5- to 10-fold greater expression and engagement at saturation, activation via Fc gamma RIIIb led to little or no change in L-selectin expression. Stimulation of PMN with intact murine anti-receptor IgG1 showed a contribution of Fc gamma RIIa receptor polymorphisms, underscoring the direct influences of Fc gamma R allotypes on receptor function. These observations suggest that Fc gamma RIIIb-mediated activation of circulating PMN may lead to a proadhesive phenotype likely to promote systemic vascular damage. This Fc gamma R-mediated adhesive phenotype will vary with the receptors engaged and their allotypes, which, in turn, reflect properties of the immune complex and the genetics of the host.

Alleles↗
PubMed2001

Caspases mediate nucleoporin cleavage, but not early redistribution of nuclear transport factors and modulation of nuclear permeability in apoptosis.

In eukaryotic cells, both soluble transport factors and components of the nuclear pore complex mediate protein and RNA trafficking between the nucleus and the cytoplasm. Here, we investigated whether caspases, the major execution system in apoptosis, target the nuclear pore or components of the nuclear transport machinery. Four nucleoporins, Nup153, RanBP2, Nup214 and Tpr are cleaved by caspases during apoptosis. In contrast, the nuclear transport factors, Ran, importin alpha and importin beta are not proteolytically processed, but redistribute across the nuclear envelope independently and prior to caspase activation. Also, mRNA accumulates into the nucleus before caspases become active. Microinjection experiments further revealed that early in apoptosis, the nucleus becomes permeable to dextran molecules of 70 kD molecular weight. Redistribution of import factors and mRNA, as well as nuclear permeabilisation, occur prior to caspase-mediated nucleoporin cleavage. Our findings suggest that the apoptotic programme includes modifications in the machinery responsible for nucleocytoplasmic transport, which are independent from caspase-mediated degradation of nuclear proteins.

Active Transport, Cell Nucleus↗
PubMedDate not supplied

[Desensitization to human recombinant DNA insulin in an adolescent with insulin-dependent diabetes mellitus].

BACKGROUND: Adverse reactions to drugs have increased in the last years, about 15% of all side effects are thought to be immune mediated according to the Coombs and Gell classification they can be type I (immediate) hypersensitivity, type II (cytotoxic) type III (immune complex mediated) or type IV (delay). Allergy to insulin is defined as an immunological response type I, and type II or III to exogenous insulin solutions occurring the 0.1% and 0.2% of the patients. PATIENTS: A 13 year old female with a 4-year history of insulin-dependent diabetes mellitus who presented hypersensitivity against recombinant DNA (rDNA) insulin manifested with urticaria and itching. We used a premedication therapy without good response and impossibility to use alternative therapy for her metabolic control, so she needed desensitization with insulin. METHODS: Skin prick testing with rapid insulin preparations 1:10 W/V dilution were positive. IgE antibodies to insulin weren't presented. IgE serum values were normal. We began the desensitization with a rapid 1:1000 UI insulin solution by intradermal route, than by subcutaneous route until reaching the accumulated doses necessary per day. During the process it appeared a papular rash and itching which were treated with an intravenous antihistaminic without troubles. RESULTS: The patient tolerated the desensitization procedure very well. For the past 14 months she has been treated uneventfully by subcutaneous administration of rDNA insulin. DISCUSSION: The desensitization against drugs is not a frequently process it only has to be used when it is impossible to substitute the treatment. Our patient showed probably hypersensitivity type 1 to insulin. However, we have to take into account the cytotoxic reaction caused by IgG or IgM antibodies or by immune complex. The desensitization finally was tolerated, 14 months after our patient accepts correctly her daily dose of human recombinant insulin.

Adolescent↗
PubMedDate not supplied

Immune-mediated neuropathy and myopathy in post-streptococcal disease: electron-microscopical, morphometrical and immunohistochemical studies.

A 22-year-old man suffered from a complete flaccid tetraparesis and an immune complex-mediated rapid progressive glomerulonephritis after group A streptococcal infection. Serum creatine kinase was excessively elevated and myoglobinuria occurred. Nerve conduction studies revealed evidence of axonal neuropathy. Recovery was satisfactory within 18 months. Sural nerve and peroneus muscle biopsies were performed in the 4th and 14th week of the disease. Light microscopy of the sural nerve showed an incipient axonal type of neuropathy in the first biopsy. Ultrastructurally, Wallerian degeneration and endoneurial inflammatory cells were present. In the muscle biopsy, few atrophic fibers and altered blood vessels without further anomalies were found. In the second sural nerve biopsy, macrophages were numerous, some of which were immunoreactive for HLA-DR, and only a few myelinated and some unmyelinated nerve fibers remained. Muscle fibers in the second biopsy showed high-grade atrophy and myofibrillar abnormalities. Immunohistochemistry revealed diffuse endoneurial immunoglobulin deposition in the first sample, while in the later biopsy specimen, deposits of IgG, and kappa and lambda light chains were visible in circumscribed endoneurial areas. Immune-mediated neuropathy and myopathy are not well-known complications of streptococcal disease. This is, to our knowledge, the first detailed report on morphological findings in muscle and nerve in such a disorder.

Adult↗