Infection, autoimmune diseases, atherosclerosis, infarct, stroke, cancerogenesis: what is in common

The problem of the transformation of specific antibodies into nonspecific polyreactive immunoglobulins (PRIG) was considered. On the basis of the obtained data the conclusion has been made that this process can occur not only in vitro, but also in vivo. The biological consequences of such a transformation of circulated specific antibodies into PRIG or increase of PRIG reactivity because of their unblocking are discussed. It was supposed that PRIG was able to play positive role in the defence of organisms against some infections. Meanwhile, disregulation of the PRIG production in vivo can, probably, lead to their participation in induction and/or in aggravation of some danger diseases, such as autoimmune diseases, atherosclerosis, infarct, stroke, and cancer. If so, farther investigation of PRIG involvement in these pathological processes could open new ways for their curing or even prophylaxis.     

Lipid-protein interactions and conformation of proteinaceous macromolecules

Analysis of the data as to the mechanism of specific antibody transformation into polyreactive immunoglobulins (PRIG) shows that for this transformation it is necessary and sufficiently to deprive antibodies of lipids, which are in norm tightly bound to the antibodies. Removal of these lipids by any methods (by treatment of antibodies with chaotropic ions, low/high pH, reactive oxygen species and lipases) leads to the loose by antibodies of their specificity and acquiring the ability to react with various non-related antigens, i.e. to their conversion into PRIG. Mathematical modeling of the PRIG--antigen interaction and values of thermodynamic characteristics of this process shows that antigen-binding domains of PRIG are in semi-melted state, thanks to what they can fit their structure to be complementary to structurally different antigens. Thus, we conclude that lipids bound to the so-called "hydrophobic pockets" of immunoglobulins (Ig) can stabilize the conformation of Ig and increase their rigidity, and removal of these lipids induce flexibility of Ig domains, responsible for interaction with antigens. It was presumed that lipids could exert the similar function of conformation stabilization not only in the case of antibodies, but also combining with some other proteins, for example, enzymes. Their removal could lead to the changing of protein conformation and loosing its biological activity. In this case the function of lipid removing and protein inactivation could exert cellular reactive oxygen species and cellular lipases and lipoxygenases.     

Transformation of specific antibodies to polyreactive immunoglobulins and their binding to blood vessel walls

It was shown that 30-50% ethanol or 40-70% dimetilsulfoxide could efficiently induce in vitro transformation of specific monoclonal antibodies (mAbs) into non-specific polyreactive immunoglobulins (PRIG). Intravenous injection 0.4 ml of FeSO4-EDTA mixture (60 and 30 mkM respectively) could induce increase of PRIG reactivity in the blood-stream. Intramuscle injection of either 0.1 ml of 40% ethanol, or 0.1 ml of FeSO4-EDTA mixture into muscle of hind limb of C57B1 mice leads to the substantial binding of circulated immunoglobulins to the blood vessels of the muscle. The similar effect could also be induced by ischemia/reperfusion of mice hind limb. In the case of intravenous injection of specific to ovalbumin biotinilated mAbs, the subsequent intramuscle injection of 0.1 ml of 40% ethanol induces apparent transformation of these mAbs into PRIG and their binding to the blood vessels. Intramuscle injection of 0.1 ml of FeSO4-EDTA mixture induces less than ethanol though noticeable effect. The obtained data have shown that cord-blood circulating specific antibodies could be transformed into PRIG at some conditions in vivo. If so, this process might play an important role in the organism defence against infections but could, probably, facilitate the development of atherosclerosis, cardiac infarct, cerebral stroke or tumors.     

Complement-binding and immuno-modulating properties of polyreactive immunoglobulins

New data concerning biological properties of polyreactive immunoglobulins (PRIG) were obtained as a result of treatment of mouse serum immunoglobulins by 4 M KSCN and are presented in the paper. In particular, the capacity of PRIG to bind C1q, the subunit of the first component of complement was studied. It was shown that PRIG's binding capacity to C1q is similar to that of intact immunoglobulins. Intravenous administration of PRIG into mice together with either sheep red blood cells or heat-inactivated staphylococcal bacteria did not affect the immune response to these antigens. Meanwhile, the same administration of PRIG together with the purified protein derivate of tuberculin resulted in 10-fold increase of mouse antibody response to PPD. These results demonstrate that PRIG can have some immuno-modulating properties concerning low-immunogenic antigens.     

Polyreactive immunoglobulins recognize hydrophobic parts of proteins

It was shown that polyreactive immunoglobulins (PRIG), which are capable to interact non-specifically with various antigens, could differ from so called natural antibodies, which have also the capacity to react non-specifically with various antigens, including self-antigens. The main differences are that in contrast to the natural antibodies, which mainly interact, probably, with hydrophilic epitopes, PRIG recognise and bond preferentially to hydrophobic epitopes. We can consider this binding as a new type of interaction between immunoglobulins and antigens.     

Polyreactive immunoglobulins and natural antibodies are different substances

It was shown that the polyreactive immunoglobulins of intact animal or human sera and the natural antibodies of these sera have different properties. Polyreactive immunoglobulins interact non-specifically with various antigens and this interaction is strongly dependent on an exposure of hydrophobic sites by antigens and, probably, by polyreactive immunoglobulins. Tween 20 and low temperature can substantially suppress this reaction. Various non-related soluble antigens can inhibit the binding of PRIG to any immobilized denatured antigen with similar efficiency. In contrast, natural antibodies interact specifically with appropriate antigens and this interaction can be suppressed only by the same or serologically similar competing antigens. Intact sera contain appreciable amount of polyreactive immunoglobulins, apparently much higher concentration than the concentration of natural antibodies. Biological functions of polyreactive immunoglobulins still remain unknown.     

Dynamics of the interaction of polyreactive immunoglobulins with immobilized antigens]

The kinetic of polyreactive immunoglobulins (PRIG) and immobilized antigen interaction was examined at different temperatures. It was shown that this process can be described by the so-called "competitive" model, and the relatively simple method for the rate constant determination for this process was developed. According to the "competitive" model PRIG molecule could be either in "active" or in "inactive" state and dynamic equilibrium exist between "active" and "inactive" molecules which strongly depend on incubation temperature. Only "active" PRIG can interact with antigens, and this is the reason of strong temperature dependence of PRIG-antigen interaction. The data also show that the mechanism of PRIG-antigen interaction differ from that of antibody-antigen interaction.     

Mechanisms of interaction of polyreactive immunoglobulins and protein antigens

New features of interaction between polyreactive immunoglobulins (PRIG) and protein antigens were considered. It was shown that unlike specific antibodies, recognizing mainly hydrophilic epitops of proteins and interacting against them with high affinity according to the mechanisms "lock-and-key" and/or "induced fit", PRIG recognized and nonspecifically bound to hydrophobic patches of protein antigens. On this reason it is possible to prevent or markedly diminish PRIG-antigen interaction using the reagents that have high affinity to hydrophobic regions of proteins and therefore are capable to block these regions. The obtained data are in a good agreement with the former data concerning the kinetic and thermodynamics characteristics of PRIG-antigen interaction described by us earlier.     

Antibodies induced by liposomal protein exhibit dual binding to protein and lipid epitopes

Natural polyreactive antibodies can accommodate chemically unrelated epitopes, such as lipids and proteins, in a single antigen binding site. Because liposomes containing lipid A as an adjuvant can induce antibodies directed against specific lipids, we immunized mice with liposomes containing lipid A together with a protein or peptide antigen to determine whether monoclonal antibodies generated after immunization would be specifically directed both to the liposomal lipid (either cholesterol or galactosylceramide) and also to the accompanying liposomal protein or peptide. Monoclonal antibodies were obtained that bound, by ELISA, to cholesterol and to recombinant gp140 envelope protein from HIV-1, or to galactosylceramide and to an HIV-1 envelope peptide. Surface plasmon resonance studies with the former antibody showed that the liposomal cholesterol and liposomal gp140 each contributed to the overall binding energy of the antibody to liposomes containing cholesterol and protein.     

Effect of oxygen free radicals on ubiquinone in aqueous solution and phospholipid vesicles

The purpose of this study was to evaluate the direct effect of oxygen free radicals produced by ultrasonic irradiation on ubiquinone and to compare the efficiency with which the antioxidant can compete with these radicals when it is both in aqueous solution and within the lipid bilayer. The main product obtained after insonation of aqueous solutions of ubiquinone-0 was ubiquinol, moreover some degradation occurred. The direct electron donor responsible for most of the ubiquinol generated by ultrasonic irradiation appeared to be superoxide radical. Addition reactions of hydroxyl radicals with aromatic ring structure led probably to degradation products of ubiquinone, which were not identified. Experiments were also performed to evaluate the efficiency with which ubiquinone-3 could react with oxygen radicals when it was within the lipid bilayer. The effect of presence or absence of a net surface charge was studied selecting a suitable bilayer including dimyristylphosphatidic acid or stearylamine in uncharged dimyristylphosphatidylcholine vesicles. In these systems hydroxyl radicals did not represent a potential danger for the antioxidant, the reaction between superoxide and ubiquinone-3 instead was significant only in positively charged membranes and gave rise to ubiquinol. It is suggested that ubiquinone acts as an antioxidant by stopping the propagation reaction.     

Susceptibility of glutathione peroxidase to proteolysis after oxidative alteration by peroxides and hydroxyl radicals.

Glutathione peroxidase is a key enzyme in the antioxidant system of the cells. This enzyme has been shown to be irreversibly inactivated by H2O2, tert-butyl hydroperoxide (tert-BHP) and hydroxyl radicals when incubated without GSH. We observed that in our experimental conditions glutathione peroxidase was not degraded by trypsin or chymotrypsin while degraded by pronase, papa?n, pepsin, and lysosomal proteases. Hydroxyl radicals and superoxide anions but not H2O2 or tert-BHP could also fragment the enzyme on their own. A former incubation with H2O2, tert-BHP, or hydroxyl radicals also increased the proteolytic susceptibility of glutathione peroxidase. Like superoxide dismutase (SOD) and other oxidatively denatured proteins, glutathione peroxidase inactivated by peroxides or free radicals seems to be degraded preferentially by proteases. As hydroxyl radicals can fragment the enzyme by themselves, the increased proteolytic susceptibility afterwards is easily understood while the increased susceptibility induced by H2O2 and tert-BHP seems to be more specific.     

Peroxule extension over ER-defined paths constitutes a rapid subcellular response to hydroxyl stress

Plants survive against myriad environmental odds while remaining rooted to a single spot. The time scale over which plant cells can respond to environmental cues is seldom appreciated. Fluorescent protein-assisted live imaging of peroxisomes reveals that they respond within seconds of exposure to hydrogen peroxide and hydroxyl radicals by producing dynamic extensions called peroxules. Observations of the Arabidopsis flu mutant and treatments with xenobiotics eliciting singlet oxygen and superoxide reactive oxygen species suggest that the observed responses are specific for hydroxyl radicals. Prolonged exposure to hydroxyl radicals inhibits peroxule extension, and instead causes motile and spherical peroxisomes in a cell to become immotile and elongate several-fold. Expression of photo-convertible EosFP–PTS1 demonstrates that vermiform peroxisomes result from rapid stretching of individual peroxisomes, while the subsequent 'beads-on-a-string' morphology results from differential protein distribution within an elongated tubule. Over time, the beads in elongated peroxisomes also extend peroxules randomly before undergoing asynchronous, asymmetrical fission. Peroxule extension does not appear to involve cytoskeletal elements directly, but is closely aligned with and reflects the dynamics of ER tubules. Peroxisomal responses reveal a rapidly invoked subcellular machinery that is involved in recognition of hydroxyl stress thresholds, and its possible remediation locally through extension of peroxules or globally by increasing peroxisome numbers. A matrix protein retro-flow mechanism that supports peroxisome–ER connectivity in plant cells is suggested.     

The broad antibacterial activity of the natural antibody repertoire is due to polyreactive antibodies

Polyreactive antibodies bind to a variety of structurally unrelated antigens. The function of these antibodies, however, has remained an enigma, and because of their low binding affinity their biological relevance has been questioned. Using a panel of monoclonal polyreactive antibodies, we showed that these antibodies can bind to both Gram-negative and Gram-positive bacteria and acting through the classical complement pathway can inhibit bacterial growth by lysis, generate anaphylatoxin C5a, enhance phagocytosis, and neutralize the functional activity of endotoxin. Polyreactive antibody-enriched, but not polyreactive antibody-reduced, IgM prepared from normal human serum displays antibacterial activity similar to that of monoclonal polyreactive IgM. We conclude that polyreactive antibodies are a major contributor to the broad antibacterial activity of the natural antibody repertoire.     

Dose-rate and oxygen effects in models of lipid membranes: linoleic acid.

Cellular membranes have been suggested as possible loci for the development of the oxygen effect in radiobiology. Unsaturated lipids from membranes are subject to very efficient radiation-induced peroxidation, and the deleterious effects generally associated with lipid autoxidation could be initiated by ionizing radiation. Oxidative damage in lipids is characterized not only by high yields but also by a profound dose-rate effect. At dose-rates of X-irradiation below 100 rad/min, a very sharp rise occurs in oxidative damage. This damage has been quantified spectrophotometrically in terms of diene conjugation (O.D. 234 mm) and chromatographically in terms of specific 9- and 13-hydroperoxide formation in linoleic acid micelles. Radical scavenging experiments indicate that hydroxyl radical attack initiates the oxidative damage. Dimethyl sulphoxide is exceptional in that it does not protect, but sensitizes, linoleic acid to radiation induced peroxidation. The yields of hydroperoxides are substantial (G=10--40) and can be related to biological changes known to be effected by autoxidizing lipids.     

Photogeneration of free radicals (*OH and HB*-) and singlet oxygen (1O2) by hypocrellin B in TX-100 micelles microsurroundings

To solve the problems faced in clinical use of hypocrellins, a water-soluble preparation of Hypocrellin B (HB), HB-Triton X-100 (TX-100) micelles, was prepared. To evaluate the photodynamic activity, the free radicals (^•OH and HB^•¯) and singlet oxygen (

1

O

2

) generated via photosensitization of the preparation in aqueous solution were detected by using electron paramagnetic resonance (EPR) and spectrophotometric methods. It was observed that

1

O

2

was formed with a quantum yield of 0.72, similar to that for HB in organic solvents, further, hydroxyl radicals (

•

OH) could also be efficiently produced by the new preparation, which have never before been detected following HB photoactivities. In addition, the semiquinone anion radicals (HB^•-) could also be generated via the self-electron transfer between an excited triplet state and a ground state molecule. The accumulation of HB^•- would replace that of

•

OH or

1

O

2

with the depletion of oxygen in the system. All these findings suggested that the HB-TX-100 micelles could play the photodynamic action through not only the type I mechanism by free radicals (^•OH, O₂^•- and HB^•-) but also the type II mechanism by singlet oxygen (

1

O

2

). It can be concluded further that the new preparation basically maintains the inherent photodynamic activity of HB, or even higher.     

Analysis of titration curves of serum antibodies obtained by ELISA

Different forms of titration curves obtained by ELISA for serum antibodies, monoclonal antibodies and polyreactive immunoglobulins were considered. A new interpretation of dose-dependent titration curves was suggested. It was shown that our interpretation of dose-dependent titration curves for antibodies which are presented in some biological liquids allow obtaining additional information about properties of the samples. This information was not obtained earlier because of the wrong understanding of the considered problem.     

Antioxidative activity of the blue pigment formed in a D-xylose-glycine reaction system

A blue compound was prepared from 1 M D-xylose and 0.1 M glycine, and designated Blue-M1, an intermediate color product of melanoidins. As melanoidins are well known to have antioxidative activity as well as high scavenging activity against active oxygen species, the antioxidative activity of Blue-M1 against the peroxidation of linoleic acid was investigated, in addition to the scavenging activity of Blue-M1 toward hydroxyl and DPPH radicals. Blue-M1 suppressed the peroxidation of linoleic acid as effectively as melanoidins did. The scavenging activity of Blue-M1 toward hydroxyl and DPPH radicals was also as strong as that of melanoidins. Blue-M1 showed higher activity with increasing concentration. The pyrrolopyrrole ring and a methine bridge between two pyrrolopyrrole rings in Blue-M1 could be related to the ability for radical scavenging activity, but not four carboxyl groups.     

The mechanism of liver microsomal lipid peroxidation.

In the presence of Fe-3+ and complexing anions, the peroxidation of unsaturated liver microsomal lipid in both intact microsomes and in a model system containing extracted microsomal lipid can be promoted by either NADPH and NADPH : cytochrome c reductase or by xanthine and xanthine oxidase. Erythrocuprein effectively inhibits the activity promoted by xanthine and xanthine oxidase but produces much less inhibition of NADPH-dependent peroxidation. The singlet-oxygen trapping agent, 1, 3-diphenylisobenzofuran, had no effect on NADPH-dependent peroxidation but strongly inhibited the peroxidation promoted by xanthine and xanthine oxidase. NADPH-dependent lipid peroxidation was also shown to be unaffected by hydroxyl radical scavengers.. The addition of catalase had no effect on NADPH-dependent lipid peroxidation, but it significantly increased the rate of malondialdehyde formation in the reaction promoted by xanthine and xanthine oxidase. The results demonstrate that NADPH-dependent lipid peroxidation is promoted by a reaction mechanism which does not involve either superoxide, singlet oxygen, HOOH, or the hydroxyl radical. It is concluded that NADPH-dependent lipid peroxidation is initiated by the reduction of Fe-3+ followed by the decomposition of hydroperoxides to generate alkoxyl radicals. The initiation reaction may involve some form of the perferryl ion or other metal ion species generated during oxidation of Fe-2+ by oxygen.     

Carbonate anions; effects on the oxidation of luminol, oxidative hemolysis, gamma-irradiation and the reaction of activated oxygen species with enzymes containing various active centres

Presence of carbonate anions increases the oxidation of luminol in different chemical systems. Lysis of human erythrocytes due to the action of dihydroxyfumaric acid or of perborate is also stimulated by carbonate ions. These anions also change considerably the loss of activity of different enzymes treated with superoxide, hydroxyl or formate radicals and can increase or decrease the effect as a function of the nature of the active centre of the enzyme. The relative effects of superoxide, hydroxyl, formate and carbonate radicals for the inactivation of various enzymes (superoxide dismutases, catalase, ribonuclease, glucose oxidase and glutathione peroxidase) have been examined. Three systems were used: gamma-irradiation under different conditions, photoproduction of radicals and sonication. Inactivation of the enzymes is a function not only of the radical used but also of the nature of the active site. Thus glutathione peroxidase is remarkably resistant to hydroxyl radicals while the superoxide dismutases are rapidly inactivated by carbonate radicals. All of the results combine to show that the presence or absence of carbonate anions must be considered in all studies of oxygen containing free radicals whether chemical, biochemical or biological or high energy irradiation.     

Regulation of CTP: phosphocholine cytidylyltransferase activity in type II pneumonocytes.

Phosphatidylcholine synthesis by rat type II pneumonocytes was altered either by depleting the cells of choline or by exposing the cells to extracellular lung surfactant. Effects of these experimental treatments on the activity of a regulatory enzyme, CTP:phosphocholine cytidylyltransferase, were investigated. Although choline depletion of type II pneumonocytes resulted in inhibition of phosphatidylcholine synthesis, cytidylyltransferase activity (measured in cell homogenates in either the absence or presence of added lipids) was greatly increased. Activation of cytidylyltransferase in choline-depleted cells was rapid and specific, and was quickly and completely reversed when choline-depleted cells were exposed to choline (but not ethanolamine). Choline-dependent changes in enzymic activity were apparently not a result of direct actions of choline on cytidylyltransferase and they were largely unaffected by cyclic AMP analogues, oleic acid, linoleic acid or cycloheximide. The Km value of cytidylyltransferase for CTP (but not phosphocholine) was lower in choline-depleted cells than in choline-repleted cells. Subcellular redistribution of cytidylyltransferase also was associated with activation of the enzyme in choline-depleted cells. When measured in the presence of added lipids, 66.5 +/- 5.0% of recovered cytidylyltransferase activity was particulate in choline-depleted cells but only 34.1 +/- 4.5% was particulate in choline-repleted cells. An increase in particulate cytidylyltransferase also occurred in type II pneumonocytes that were exposed to extracellular surfactant. This latter subcellular redistribution, however, was not accompanied by a change in cytidylyltransferase activity even though incorporation of [3H]choline into phosphatidylcholine was inhibited by approx. 50%. Subcellular redistribution of cytidylyltransferase, therefore, is associated with changes in enzymic activity under some conditions, but can also occur without a resultant alteration in enzymic activity.