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PubMed1995

Dehalogenation of haloalkanes by Rhodococcus erythropolis Y2. The presence of an oxygenase-type dehalogenase activity complements that of an halidohydrolase activity.

Rhodococcus erythropolis Y2 produced two types of dehalogenase: a hydrolytic enzyme, that is an halidohydrolase, which was induced by C3 to C6 1-haloalkane substrates, and at least one oxygenase-type dehalogenase induced by C7 to C16 1-haloalkanes and n-alkanes. The oxygenase-type activity dehalogenated C4 to C18 1-chloroalkanes with an optimum activity towards 1-chlorotetradecane. The halidohydrolase catalysed the dehalogenation of a wide range of 1- and alpha,omega-disubstituted haloalkanes and alpha,omega-substituted haloalcohols. In resting cell suspensions of hexadecane-grown R. erythropolis Y2 the oxygenase-type dehalogenase had a specific activity of 12.9 mU (mg protein)-1 towards 1-chlorotetradecane (3.67 mU mg-1 towards 1-chlorobutane) whereas the halidohydrolase in 1-chlorobutane-grown batch cultures had a specific activity of 44 mU (mg protein)-1 towards 1-chlorobutane. The significance of the two dehalogenase systems in a single bacterial strain is discussed in terms of their contribution to the overall catabolic potential of the organism.

Alkanes↗
PubMed1973

The membrane attack mechanism of complement. Reversible interactions among the five native components in free solution.

Reversible interactions in free solution were demonstrated to occur (a) between C5 and C8, (b) between C5, 6, 7 and C8, and (c) between C8 and C9. No interaction was observed between C8 and C6 or C7 and between C9 and C5, 6, 7. Interactions between C8 and C9 were enhanced at lowered ionic strength (0.05) and a molar excess of C8 over C9. Complex formation was independent of pH over the range of 6.5-8.5. Under optimal conditions the C8, 9 complex had a sedimentation coefficient of 10.2-10.6S, while native C8 and C9 sedimented at 8.5 and 4.8S, respectively. Specificity and reversibility of these interactions were established. In spite of the limited number of interactions observed, all five of the native proteins of the membrane attack mechanism interacted to form an association product that sedimented at 10.8-11.2S. Demonstration of this product in free solution supports the concept that C5-9 on acquisition of cytolytic activity assemble into a stable multimolecular complex.

Animals↗
PubMed1984

Correlation between covalent attachment of C3 and calcium uptake in antibody-stimulated L cells.

The nature of C3 binding to cell surfaces was examined in L cells treated with antibody and complement, immunological reagents which we have previously shown to be capable of producing stimulation of several important cellular processes. In the presence of serums containing C3 and under experimental conditions where complement activation could take place, selective binding of C3 to antibody treated L cells was observed (maximum 1.1 X 10(6) C3 molecules per cell). Under similar conditions there was a C3 dependent increased calcium uptake (3.4 pmol) by antibody treated cells. Purified C3 was able to selectively restore C3 binding to cells treated with serum depleted of C3 through C9. C7-deficient serum was almost as good a source of activated C3 as its normal serum counterpart. Strong chemical nucleophiles such as salicylhydroxamic acid, which are capable of covalently coupling to the labile internal thiolester of C3, prevented the binding of C3 to cells. We conclude that C3 is covalently bound to antibody and complement treated L cells, possibly serving as an important signal in subsequent enhancement of phospholipid metabolism, DNA synthesis, and cell growth.

Animals↗
PubMed1994

Receptor-independent activation of guanine nucleotide-binding regulatory proteins by terminal complement complexes.

Activation of heterotrimeric guanine nucleotide-binding proteins (G proteins) by terminal complement complexes (TCC) was investigated on human lymphoblastoid B-cell line JY25 and its mutant JY5 deficient in glycosylphosphatidylinositol-anchored proteins. TCC assembly achieved by antibody-dependent activation of C7-deficient serum reconstituted with C7 increased specific guanosine-5'-(gamma-thio)triphosphate (GTP gamma S) binding, 4- and 8-fold, in JY25 and JY5 membranes, respectively, between 2 and 10 min, over the level without C7. TCC also increased GTPase activity 5- and 4-fold in JY25 and JY5, respectively, between 5 and 10 min. Increased GTPase activity was noted first with C5b-7 assembly, which increased further with C5b-8 and C5b-9. The presence of G proteins in anti-TCC immunoprecipitates of cell lysates was investigated by demonstration of G alpha subunit that can be ADP-ribosylated by pertussis toxin (PTX). Immunoprecipitated TCC complexes contained a PTX-sensitive 41-kDa Gi alpha/Go alpha subunit, as shown by SDS-PAGE and Western blotting. These complexes were functionally active as determined by GTP gamma S binding. We have further shown that enhanced TCC elimination from the plasma membrane induced by TCC-generated signals was inhibited by PTX. In conclusion the biological activities induced by TCC in nucleated cells may be mediated in part by activation of PTX-sensitive G proteins.

B-Lymphocytes↗
PubMed1988

Complement inhibitor S protein is associated with membranes of red blood cells from patients with paroxysmal nocturnal haemoglobinuria.

S protein is a plasma glycoprotein (Mr = 78,000) which binds to nascent C5b-7 complexes upon complement activation in the fluid phase in whole serum. It thereby protects innocent bystander cells from complement mediated lysis. It is unknown whether S protein also functions as complement inhibitor on cell surfaces. We here report that S protein is recognized on red blood cells (RBC) from patients with paroxysmal nocturnal haemoglobinuria (PNH), but not on normal RBC. RBC from eight PNH patients showed 12-48% haemolysis subsequent to complement activation in the fluid phase, while normal RBC did not respond. Preincubation of the PNH cells with affinity-purified antibodies against human S protein resulted in a three- to five-fold increase of haemolysis, while preincubation of these cells with S protein decreased haemolysis by 40%. In contrast, haemolysis remained unaffected by other unrelated antibodies, i.e. IgG anti-Rh(D) and anti-A. If PNH RBC, normal RBC pretreated with 2-amino-ethylisouronium bromide (AET), or untreated normal RBC, respectively, were incubated with purified S protein in vitro, the uptake of antibodies against S protein was significantly enhanced with PNH and with AET-treated, but not with untreated normal RBC. Additionally, while normal RBC did not respond to reactive lysis initiated by purified C5b-6 and C7, PNH as well as AET-RBC showed significant haemolysis that could be inhibited by S protein in a dose-dependent fashion. These findings strengthen the assumption that the increased sensitivity of PNH cells towards reactive complement lysis is either due to the lack of an inhibitor of the terminal complement sequence and/or enhanced insertion of the membrane attack complex. These defects of PNH RBC may partly be overcome by the fluid phase complement inhibitor S protein which binds to PNH RBC and may thereby suppress homologous cytolysis.

Complement Inactivator Proteins↗
PubMedDate not supplied

Domain structure, functional activity, and polymerization of trout complement protein C9.

The 3' region of trout C9 has been resequenced and found to differ from the previously published sequence (Stanley and Herz, EMBO J. 6:1951; 1987). In contrast to other sequenced C9 molecules, but in common with the other terminal complement components, trout C9 was found to contain an additional carboxy terminal thrombospondin domain. This domain does not restrict polymerization, as has been previously suggested (Stanley and Luzio, Nature 334:475; 1988), since alternative pathway activation of trout complement by rabbit erythrocytes lead to the formation of circular membrane attack complement lesions on the erythrocyte membrane. Although the trout C9 molecule is larger than human C9, the diameters of circular trout membrane attack complexes were approximately 30% smaller than their human counterparts. No lysis of erythrocytes bearing human C5b-7 or C5b-8 complexes was detected following incubation with trout serum containing EDTA, which suggests that trout C8 and C9 are unable to bind to human C7 and C8, respectively. Finally, trout and human serum were equally effective at killing the human serum-sensitive strain Salmonella minnesota Re595.

Amino Acid Sequence↗
PubMed1990

Patterns of complement activation in idiopathic membranoproliferative glomerulonephritis, types I, II, and III.

Complement profiles on 22 hypocomplementemic patients with membranoproliferative glomerulonephritis (MPGN) type I, on 11 with MPGN II, and on 16 with MPGN III, gave evidence that the nephritic factor of the amplification loop (NFa) is responsible for the hypocomplementemia in MPGN II and the nephritic factor of the terminal pathway (NFt) for the hypocomplementemia in MPGN III. In contrast, in MPGN I, there was evidence for three complement-activating modalities, NFa, NFt, and immune complexes. As a result, four different patterns of complement activation were seen. NFa, found in MPGN II, produces a complement profile characterized mainly by C3 depression. In addition, four of seven (57%) severely hypocomplementemic MPGN II patients (C3 less than 30 mg/dL) had slightly depressed levels of factor B, and one of seven (14%) of properdin, but in all the C5 concentration was normal. In contrast, all eight severely hypocomplementemic patients with MPGN II had depressed C5 and properdin levels, and six of eight (75%) depressed levels of C6, C7, and/or C9. Of eight MPGN III patients with moderate hypocomplementemia, 50% had depressed C5 and properdin levels and the remainder, depressed C3 only. This spectrum of profiles is most likely produced by varying concentrations of NFt. In MPGN I, nine of 23 (39%) had a profile indicating only classical pathway activation; seven of 23 (39%), a pattern compatible with NFt alone; four of 23 (9%), evidence for both classical pathway activation and NFt; and three of 23 (13%), a pattern compatible with NFa. The unique multifactorial origin of the hypocomplementemia in MPGN I, often giving evidence of classical pathway activation, together with previously reported differences in glomerular morphology and clinical features at onset, makes it distinct from MPGN III. Depressed C8 levels were found to some extent in all hypocomplementemic states. The levels were uncommonly depressed in patients with NFa, most markedly depressed with NFt, and moderately reduced with classical pathway activation. The cause is not known. Diagnostically, profiles showing classical pathway activation and low levels of C6, C7, and/or C9 are specific for MPGN I. Those showing only classical activation are likewise diagnostic of MPGN I if systemic lupus erythematosus (SLE) and chronic bacteremia are ruled out.

Complement Activation↗
PubMed1985

The killer molecule of complement.

Cell injury by complement occurs as a consequence of activation of either the classical or the alternative pathway on the surface of a cell. It is accomplished by the membrane attack complex (MAC). Its precursor proteins, C5, C6, C7, C8, and C9, are hydrophilic glycoproteins with Mr ranging from 70,000-180,000. When C5 is cleaved by the serine protease C5 convertase which covalently attaches to target cells, nascent C5b is produced and forms together with C6 a soluble and stable bimolecular complex (C5b,6). Upon binding of C5b,6 to C7 a trimolecular complex (C5b-7) is formed which expresses a metastable membrane-binding site. Membrane-bound C5b-7 constitutes the receptor for C8 and the tetramolecular C5b-8 complex binds and polymerizes C9. During the assembly process the proteins undergo hydrophilic-amphiphilic transition and the end product consists of C5b-8 (Mr approximately 550,000) and of tubular poly C9 (Mr approximately 1,100,000). The functional channel size varies but its maximal diameter is approximately 100 A. C9 polymerization appears to involve initial reversible association of several C9 molecules which is followed by temperature-dependent, constrained unfolding. Unfolded C9 monomers then associate laterally with each other and polymerization terminates with closure of the circular structure which consists of 12-18 C9 monomers. Amino acid composition and sequence indicate that the N-terminal half of the single chain C9 molecule is hydrophilic and the C-terminal half rather hydrophobic. Phospholipid-binding and insertion into membranes are functions of the C-terminal portion of the molecule. Control of the MAC is exerted by the S-protein (Mr 80,000) which binds to the forming complex and prevents its attachment to the cell membrane. Control is also exerted by certain species-specific membrane proteins which interfere with C5 convertase and C9 function.

Binding Sites↗
PubMed1990

Incorporation of SP-40,40 into the soluble membrane attack complex (SMAC, SC5b-9) of complement.

When SC5b-7 was prepared from the C8-depleted serum activated with inulin, it contained SP-40,40 as well as S-protein. From the densitometry of each component in SC5b-9 after SDS-PAGE, it was estimated that SC5b-9 was constituted of one molecule each of C5b, C6, C7, C8, S-protein, and SP-40,40 and two molecules of C9. SP-40,40 was depleted from normal serum with an affinity column using mouse monoclonal anti-SP-40,40 antibody. When the resulting SP-40,40-depleted serum was activated with inulin, SC5b-9 lacking SP-40,40 could be formed. S-Protein-depleted serum was also prepared with an affinity column using mouse monoclonal anti-S-protein antibody. Similarly, SC5b-9 lacking S-protein could be formed by the inulin activation of the S-protein-depleted serum. These results indicate that either SP-40,40 or S-protein should be able to form a soluble C5b-9 complex.

Blood Proteins↗
PubMed2002

Identification of a DtxR-regulated operon that is essential for siderophore-dependent iron uptake in Corynebacterium diphtheriae.

The diphtheria toxin repressor (DtxR) uses Fe(2+) as a corepressor and inhibits transcription from iron-regulated promoters (IRPs) in Corynebacterium diphtheriae. A new IRP, designated IRP6, was cloned from C. diphtheriae by a SELEX-like procedure. DtxR bound to IRP6 in vitro only in the presence of appropriate divalent metal ions, and repression of IRP6 by DtxR in an Escherichia coli system was iron dependent. The open reading frames (ORFs) downstream from IRP6 and previously described promoter IRP1 were found to encode proteins homologous to components of ATP-binding cassette (ABC) transport systems involved in high-affinity iron uptake in other bacteria. IRP1 and IRP6 were repressed under high-iron conditions in wild-type C. diphtheriae C7(beta), but they were expressed constitutively in C7(beta) mutant strains HC1, HC3, HC4, and HC5, which were shown previously to be defective in corynebactin-dependent iron uptake. A clone of the wild-type irp6 operon (pCM6ABC) complemented the constitutive corynebactin production phenotype of HC1, HC4, and HC5 but not of HC3, whereas a clone of the wild-type irp1 operon failed to complement any of these strains. Complementation by subclones of pCM6ABC demonstrated that mutant alleles of irp6A, irp6C, and irp6B were responsible for the phenotypes of HC1, HC4, and HC5, respectively. The irp6A allele in HC1 and the irp6B allele in HC5 encoded single amino acid substitutions in their predicted protein products, and the irp6C allele in HC4 caused premature chain termination of its predicted protein product. Strain HC3 was found to have a chain-terminating mutation in dtxR in addition to a missense mutation in its irp6B allele. These findings demonstrated that the irp6 operon in C. diphtheriae encodes a putative ABC transporter, that specific mutant alleles of irp6A, irp6B, and irp6C are associated with defects in corynebactin-dependent iron uptake, and that complementation of these mutant alleles restores repression of corynebactin production under high-iron growth conditions, most likely as a consequence of restoring siderophore-dependent iron uptake mediated by the irp6 operon.

Bacterial Proteins↗
PubMed1983

The fifth component of complement (C5): purification without activation.

In the course of our studies on the structural change of C5 by acidification (U. Rother et al., 1978), we found that the C5 preparations purified according to published methods contained more or less activated C56. When added to sensitive target cells (guinea pig or chicken erythrocytes), C5 mediated lysis by C7-C9 without the addition of C6 or any activation procedure. Generation of C56 was probably due to drastic changes in the physicochemical environment during purification. Such changes like high or low pH or high ionic strength were shown to cause activation. A method for purification of C5 is described in which polyethyleneglycol (PEG) or (NH4)2SO4 precipitation, as well as low or high pH, was avoided. As a last step, traces of C6 were removed by affinity chromatography. The resulting preparation was free of C56. Activation by acidification was not possible without the addition of C6. The total recovery of C5 was 12% with almost no loss of specific activity.

Animals↗
PubMed1988

Hydrolysis of myelin basic protein in human myelin by terminal complement complexes.

The participation of terminal complement complexes (TCC) in demyelination has been shown in rodent cerebellar cultures. Since TCC modulates activities of various membrane-associated enzymes and increases the level of cellular Ca2+ we investigated whether TCC could activate Ca2+-dependent neutral proteases in myelin that would lead to hydrolysis of myelin basic protein (BP). Addition of antibody and C7-deficient serum plus C7 to sealed myelin vesicles of two to six bilayers caused significant BP hydrolysis compared to the hydrolysis caused by antibody and C7-deficient serum. Significant hydrolysis occurred at the stage of C5b6,7 assembly, which increased in magnitude at the C5b6-8 stage. C5b6-9 formation did not enhance the effect of C5b6-8. BP hydrolysis by C5b6,7 did not require Ca2+ whereas the effect of C5b6-8/C5b6-9 was, in part, Ca2+-dependent. We postulated that TCC formation in myelin membranes causes activation of myelin-associated neutral proteases with subsequent hydrolysis of BP as a consequence of complement peptide insertion and channel formation. Such processes may alter the structure of myelin and augment the action of other inflammatory cells and their products in demyelinating diseases that could ultimately lead to the loss of myelin.

Complement Membrane Attack Complex↗
PubMed1994

Blocking antibodies specific for human albumin interfere with the hemolytic activity of the membrane attack complex of complement.

Earlier, we showed that antibodies specific for human serum albumin are able to bind to erythrocyte-associated albumin and inhibit complement mediated hemolysis. In the present study we determine if inhibition is occurring at the membrane attack phase of complement activation or at an earlier step. We show that although cell-bound anti-albumin antibodies do not inhibit binding and activation of C3 or uptake of C9, they do appear to cause cells to become refractory to lysis by the membrane attack complex as they inhibit both the kinetics and the extent of hemolysis in a reactive lysis system which employs preformed C5b6 plus C7, C8, and C9. We believe that this is the first report of inhibition of the hemolytic activity of the membrane attack complex by antibodies bound to an erythrocyte surface antigen.

Animals↗
PubMed1986

Isolation of a human erythrocyte membrane protein capable of inhibiting expression of homologous complement transmembrane channels.

Erythrocytes are poorly lysed by homologous complement, whereas they are readily lysed by heterologous complement. This phenomenon had been attributed to an interference by the cell surface with the action of complement components C8 and C9. To isolate the responsible membrane constituent, detergent-solubilized human erythrocyte (EH) membranes were subjected to affinity chromatography by using human C9-Sepharose. The isolated protein had a mass of 38 kDa and, incorporated into liposomes, was highly effective in inhibiting complement-mediated channel expression, including the C5b-8, membrane attack complex, and tubular polymer of C9 channels. Antibody produced to the 38-kDa protein caused a 20-fold increase in reactive lysis of EH by isolated C5b6, C7, C8, and C9. The antibody did not enhance C5b-7 uptake, but it affected C9 binding to the target cell membrane. Antibody to human decay-accelerating factor, used as a control, had no effect on reactive lysis of EH. Anti-38-kDa protein did not enhance the action on EH of C8 and C9 from other species, indicating that the action of this regulatory protein is species specific. It was therefore termed homologous restriction factor (HRF). Blood cells other than erythrocytes, such as polymorphonuclear leukocytes, also exhibited cell-surface HRF activity. In immunoblots of freshly isolated EH membranes, anti-38-kDa HRF detected primarily a 65-kDa protein, suggesting that the 38-kDa protein constitutes an active fragment of membrane HRF. Because of the specific binding reaction observed between HRF and C8 or C9, HRF was tested with anti-human C8 and anti-human C9. A limited immunochemical relationship of HRF to C8 and C9 could be established and solid-phase anti-C9 proved an efficient tool for the isolation of HRF from solubilized EH membranes.

Blood Proteins↗
PubMed1992

Effects of complement activation on platelet-activating factor and eicosanoid synthesis in rat mesangial cells.

The effects of in vitro complement activation and of isolated complement components (C3a, C5a, C3b) on the activity of the microsomal enzyme acetyl coenzyme A: 1-0-alkyl-glycero-3-phosphocholine acetyl transferase (AcTr) in cultured mesangial cells and on the synthesis of prostaglandin E2 (PGE2) were assessed. In vitro complement activation induced by the introduction of purified cobra venom factor (CVF) in culture media containing human serum enhanced mesangial cell PGE2 synthesis and had no effect on microsomal AcTr activity. When media containing C6-deficient serum were used, the stimulatory effect of CVF on PGE2 synthesis was abolished. Introduction of CVF in these media enhanced mesangial cell AcTr activity. These effects were partially reversed when the C6-deficient serum was supplemented with C7-deficient serum to allow formation of the C5b-9 complex. Isolated C3a, C5a, and C3b had opposite effects on PGE2 synthesis and on AcTr activity. Specifically, all components enhanced mesangial cell PGE2 synthesis. In contrast, AcTr activity was enhanced by C3a alone; C5a had no effect, whereas C3b had an inhibitory effect. The observations indicate that in response to complement activation and specific anaphylatoxin agonists, mesangial cell eicosanoid synthesis is not coupled with changes in the activity of AcTr and PAF synthesis.

Acetyltransferases↗
PubMed1976

The genetics of dopa decarboxylase in Drosophila melanogaster. II. Isolation and characterization of dopa-decarboxylase-deficient mutants and their relationship to the alpha-methyl-dopa-hypersensitive mutants.

Of 84 lethals isolated over the dopa decarboxylase (DDC) deficiency Df(2L)50, 8 have been identified as DDC-deficient alleles on the basis of their effect on DDC activity when heterozygous over the Cgamma-O balancer chromosome with activities ranging from 28% to 53% of controls. Some of the Ddc-deficient alleles exhibit intracistronic complementation. Most of the complementing pairs of alleles are much reduced in viability, e.g. less than 5% of expected, and express a common syndrome of mutant phenes which can reasonably be inferred to derive from inadequately sclerotinized cuticle. Individuals heterozygous for the noncomplementing allele, Ddcn7, over the 12-band DDC deficiency, Df (2L)130, die at the end of embryogenesis as unhatched larvae with unpigmented mouth parts. The Ddc alleles and the l(2) amd alpha-methyl dopa (alphaMD) hypersensitive alleles are both located within the 11 band region 37B10-C7. The l(2) and locus is immediately to the right of hk(2-53.6). Ddc has been mapped within 0.004 Map Units to the right of l(2) and with a maximum estimated recombination frequency of 0.01%. None of the Ddc/CgammaOstrains are sensitive to the dietary administration of alpha-methyl dopa (alphaMD), and complementation occurs between the Ddc deficient alleles and the l(2) amd alleles both on the basis of viability and DDC activity. No effect on DDC by the amd alleles has been found to date. Even in the complementing heterozygote, amdH1/amdH89, the level of activity, thermostability, and in vitro alphaMD inhibition of DDC remains unaffected. Although no biochemical phene has yet been established for the alphaMD hypersensitive amd alleles, it seems likely that the two groups of mutants are functionally related.

Alleles↗
PubMed1993

Use of a pool of monoclonal antibodies in diagnosing cells from serous cavities.

AIMS AND BACKGROUND: The usefulness of monoclonal antibodies that recognize markers of neoplastic lesions in complementing conventional cytology was evaluated by the avidin-biotin-peroxidase complex, indirect immunoperoxidase technique. METHODS: In order to enhance the sensitivity of the traditional method, a pool of seven combined monoclonal antibodies (Pool C7), which reacts specifically with cells of epithelial origin and is able to distinguish between mesothelial and malignant cells, was tested on cytologic smears of 262 serous effusions. The effusions were benign or neoplastic, mainly from breast, ovary and lung cancers. RESULTS: Immunocytochemical method showed an 100% specificity and 100% of predictivity whereas the sensitivity was 98%, 96% and 95% for breast ovarian and lung carcinomas, respectively. CONCLUSIONS: The results demonstrated that the pool when used together with conventional methods, is useful in analysis of serous effusions in diagnostic investigations.

Antibodies, Monoclonal↗
PubMed1989

Serum terminal complement component levels in hypocomplementemic glomerulonephritides.

Measurements of serum C3 through C9 are reported for patients with acute poststreptococcal glomerulonephritis (AGN), membranoproliferative glomerulonephritis type I (MPGN I), MPGN II, and MPGN III. Except in MPGN II, depressed C5 levels correlated with depressed C3 levels. In MPGN II, levels of C5 and of other terminal components were normal. In MPGN III, markedly depressed levels of C7 through C9 correlated strongly with depressed levels of C3 and C5. C6 was less severely depressed. In MPGN I, terminal component levels were less often depressed than in MPGN III and in AGN, depression of terminal components was seen only when levels of C3 and C5 were extremely low. The data indicate that late terminal components are activated in MPGN III to a greater extent than in the other nephritides despite C5 activation approximately equal in extent to that in AGN and MPGN I.

Acute Disease↗