Inhibition of topoisomerase I by heparin

DNA topoisomerase I isolated from mouse mammary cacinoma cells was shown to be inhibited by heparin, the dose giving 50% inhibition (IC₅₀) being 0.20 μg/ml. Other chemically related acid mucopolysaccharides including heparan sulfate, dermatan sulfate etc. were more than 500 times less active than heparin. When the amount of enzyme was doubled relative to the substrate the inhibition was reversed. Addition of heparin to assay mixtures after the initiation of the reaction immediately inhibited the enzyme reaction.     

Interactions of sera from patients with autoimmune diseases with cDNA expressed fragment of topoisomerase I and monoclonal antibodies against enzyme

The interaction of sera from 34 patients with different autoimmune diseases with the expressed fusion protein cloned in lambda gt11 vector (topoisomerase I--beta galactosidase) and monoclonal antibodies against enzyme was studied. It was demonstrated that 100% of Scl cases possessed positive activity against fusion protein. It was shown that this test is not absolutely specific for Scl, i. e. 57.1% of Sle and 84.6% of RA demonstrated positive activity against "topoisomerase test". Autoimmune sera had the positive activity against monoclonal antibodies for topoisomerase I. This activity was shown to be due to the presence of antiidiotypic antibodies against topoisomerase in the autoimmune sera.     

Scl 70 autoantibodies from scleroderma patients recognize a 95 kDa protein identified as DNA topoisomerase I

Sera of patients suffering from the autoimmune disease progressive systemic sclerosis (PSS) are known to contain autoantibodies which have been reported to recognize a 70 kDa antigenic protein, designated the Scl 70 antigen. By immunoblotting of nuclear extracts from HeLa cells with sera from scleroderma patients we observed that the size of the antigen present in such cells depends on the conditions of antigen isolation. When protease inhibitors were included in the extraction buffer, a 95 kDa protein was identified instead of a 70 kDa protein. When protease inhibitors were omitted, a number of polypeptides in the size range 66 to 95 kDa was found. Furthermore, antibodies which had been affinity purified on the 95 kDa antigen, crossreacted with the 66 to 95 kDa polypeptides. These results suggest that the smaller proteins were degradation products of the 95 kDa antigen. Immunofluorescence studies on PtK-2 cells with the antibody specific for the 95 kDa protein gave staining of nuclei, nucleoli and of chromosomes and the nucleolar organizer region in mitotic cells. Since this distribution of antigens within the nucleus was reminiscent of the intranuclear distribution of DNA topoisomerase I found by others we probed purified DNA topoisomerase I from calf thymus directly with the autoantibodies from PSS patients, and also the 95 kDa antigens of HeLa cell nuclei with antibodies raised against the bovine DNA topoisomerase I. From the crossreaction pattern observed with the different antigens and antibodies we conclude that DNA topoisomerase I is one of the antigenic components against which autoantibodies are formed in scleroderma patients.     

Inhibition of Mycobacterium smegmatis topoisomerase I by specific oligonucleotides

DNA topoisomerase I from Mycobacterium smegmatis unlike many other type I topoisomerases is a site specific DNA binding protein. We have investigated the sequence specific DNA binding characteristics of the enzyme using specific oligonucleotides of varied length. DNA binding, oligonucleotide competition and covalent complex assays show that the substrate length requirement for interaction is much longer ( approximately 20 nucleotides) in contrast to short length substrates (eight nucleotides) reported for Escherichia coli topoisomerase I and III. P1 nuclease and KMnO(4) footprinting experiments indicate a large protected region spanning about 20 nucleotides upstream and 2-3 nucleotides downstream of the cleavage site. Binding characteristics indicate that the enzyme interacts efficiently with both single-stranded and double-stranded substrates containing strong topoisomerase I sites (STS), a unique property not shared by any other type I topoisomerase. The oligonucleotides containing STS effectively inhibit the M. smegmatis topoisomerase I DNA relaxation activity.     

Anti-interferon-gamma antibodies in sera from HIV infected patients

High serum levels of antibodies to interferon-gamma (IFN-gamma) have been found in patients infected with human immunodeficiency virus (HIV). A radioimmunoassay (RIA) with a recombinant IFN-gamma protein or an affinity purified IFN-gamma preparation as antigens, was developed to detect the specific anti-IFN-gamma antibodies. Reactivity of sera to IFN-gamma was confirmed by Western blot analysis. These antibodies, however, do not seem to recognize the active site of the molecule, since they do not neutralize the antiviral IFN-gamma activity in a biological assay. These results enforce the hypothesis of the role of autoimmunization during the course of the disease.     

Rodent islet cell antigens recognized by antibodies in sera from diabetic patients

Rat islets, rat insulinoma cells and islets from three different mouse strains were labelled with 35S-cysteine and/or 35S-methionine. Detergent lysates of the cells were subjected to immunoprecipitation with sera from 5 newly diagnosed diabetic children and 5 control sera. The immunoprecipitates were analysed by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis followed by autoradiography. One of the sera immunoprecipitated a protein of Mr 64K from lysates of rat islets, rat insulinoma cells, A. TH and NMRI but not CBA/H mouse islets. This protein was not consistently immunoprecipitated by the other diabetic sera, however, it was never found with control sera nor was it detected in rodent lymphocytes. Some proteins of lower molecular weight (59K, 57K, 40K, 29K) were specifically immunoprecipitated by one or more diabetic sera from some of the rodent islet cell preparations. It is concluded that rodent islet cells contain a protein of Mr 64K which may be antigenically related to a 64K protein previously detected in immunoprecipitates of human islet cells with the same diabetic sera. The variable results with rat and mouse islet cell material suggest that the level of cross-reactivity is low. Further studies are needed to clarify whether the lower molecular components detected in some immunoprecipitates represent other antigenic determinants or degradation products of the 64K protein.     

HIV-1 integrase-hydrolyzing antibodies from sera of HIV-infected patients

Autoantibodies with enzymic activities (abzymes) are a distinctive feature of autoimmune diseases. It was interesting whether Abs from patients with viral diseases can hydrolyze viral proteins. Electrophoretically and immunologically homogeneous IgGs were isolated from sera of AIDS patients by chromatography on several affinity sorbents. We present evidence showing that 89.5% IgGs purified from the sera of HIV-infected patients using several affinity resins including Sepharose with immobilized integrase specifically hydrolyze only HIV integrase (IN) but not many other tested proteins. Several rigid criteria have been applied to show that the IN-hydrolyzing activity is an intrinsic property of AIDS IgGs but not from healthy donors. Similar to autoimmune proteolytic abzymes, IN-hydrolyzing IgGs from some patients were inhibited by specific inhibitors of serine and metal-dependent proteases but a significant inhibition of the activity by specific inhibitors of acidic- and thiol-like proteases was observed for the first time. Although HIV infection leads to formation of Abs to many viral and human antigens, no possible biological role for most of them is known. Since anti-IN IgG can efficiently hydrolyze IN, a positive role of abzymes in counteracting the infection cannot be excluded. In addition, detection of IN-hydrolyzing activity can be useful for diagnostic purposes and for estimation of the immune status in AIDS patients.     

Inhibition of Mycobacterium tuberculosis topoisomerase I by m-AMSA,a eukaryotic type II topoisomerase poison

m-AMSA, an established inhibitor of eukaryotic type II topoisomerases, exerts its cidal effect by binding to the enzyme–DNA complex thus inhibiting the DNA religation step. The molecule and its analogues have been successfully used as chemotherapeutic agents against different forms of cancer. After virtual screening using a homology model of the Mycobacterium tuberculosis topoisomerase I, we identified m-AMSA as a high scoring hit. We demonstrate that m-AMSA can inhibit the DNA relaxation activity of topoisomerase I from M. tuberculosis and Mycobacterium smegmatis. In a whole cell assay, m-AMSA inhibited the growth of both the mycobacteria.     

Inhibition of HeLa cell DNA topoisomerase I by ATP and phosphate.

The relaxation activity of DNA topoisomerase I from HeLa cell nuclei is strongly inhibited by a variety of purine nucleotides in the presence but not absence of 1 mM potassium phosphate. For ATP, 3-4 mM causes nearly complete inhibition. The 2'-and 3'-AMP isomer are active as well in the presence of 1 mM phosphate, but the 5'-AMP isomer and adenosine are inert. At 3 mM ATP, the titration curve for phosphate is sigmoidal with inhibition beginning abruptly at about 0.5 mM. The negatively-supercoiled DNA isolated from an "inhibited" reaction is relaxed as well as the standard DNA template in the absence of ATP and phosphate suggesting that inhibition does not result from an alteration of the template which protects against its relaxation. Relaxation of positively-supercoiled DNA is also inhibited. Catalysis by E. coli DNA topoisomerase I and HeLa DNA topoisomerase II is not inhibited at concentrations of ATP and phosphate sufficient to cause 80-90% inhibition of HeLa type 1 enzyme.     

Monoclonal antibodies neutralizing mammalian DNA topoisomerase I activity

We have isolated three different monoclonal antibodies specific for mammalian type-I DNA topoisomerase. The antibodies react with three closely adjacent epitopes located in a central section of the enzyme (between amino acid residues 344 and 483). Two of the antibodies inhibit an early step of the nicking/closing pathway. We provide evidence showing that the antibodies do not block the association of the enzyme with DNA. The antibodies are useful for immunocytochemical investigation and for further exploration of the biochemical function of mammalian type-I DNA topoisomerase.     

Onconeural antibodies in sera from patients with various types of tumours

Purpose  We assessed the frequency and levels of onconeural antibodies in 974 patients with various types of tumours, but without apparent paraneoplastic neurological syndromes (PNS). Patients and methods  We included patients with the following tumours: 200 small-cell lung cancer (SCLC) patients, 253 breast cancer patients, 182 ovarian cancer patients, 266 uterine cancer patients and 73 thymoma patients, as well as 52 patients with PNS and cancer and 300 healthy blood donors. Sera were screened for amphiphysin, CRMP5, Hu, Ma2, Ri and Yo antibodies using a multi-well immunoprecipitation technique. Results  The most frequently detected antibodies were Hu followed by CRMP5. Ma2, Yo, amphiphysin and Ri antibodies were less common, but each was found at similar frequencies. Onconeural antibodies were present at similar levels in sera from the PNS control group and from cancer patients. Hu antibodies were rare in cancers other than SCLC. CRMP5 was the only antibody found in patients with thymoma and this antibody was more common among patients with thymoma than in other tumour patients. With one exception, coexisting antibodies were only found in patients with SCLC. The presence of onconeural antibodies in SCLC patients was not associated with prolonged survival. Conclusion  Onconeural antibodies are associated with various types of tumours suggesting that all antibodies should be included in the serological screening for possible PNS. The levels of onconeural antibody are not sufficiently sensitive to discriminate between cancer patients with PNS and those without.     

Plant DNA topoisomerase I is recognized and inhibited by human Scl-70 sera autoantibodies

Type I topoisomerases (EC 5.99.1.2) are those enzymes capable of relaxing negatively supercoiled DNA without the need for ATP. The central role played by these enzymes in cell function suggests that the structure of type I topoisomerases may be highly conserved in eukaryotic cells. However, the extent of the conservation among eukaryotes is unknown. Human DNA topoisomerase I is an autoimmune antigen (Scl-70) of scleroderma patients. We have found that the autoimmune antibodies in human Scl-70 sera recognize protein from various plants, and these proteins display DNA relaxation function. In addition, Scl-70 antibodies were able to inhibit enzymatic activity of plant topoisomerase I. Therefore, the immunological cross-reactivity of the plant topoisomerase with human antibodies demonstrates that, despite divergence of eukaryotic organisms, these plant and animal enzymes retain structurally similar enzymatic features.     

Specific antibodies and mycobacterial antigens in pulmonary tuberculosis patients sera. Note I

206 sera collected from different groups of subjects were analyzed by immunoenzymatic methods regarding the content of Mycobacterium tuberculosis specific antibodies and mycobacterial antigens. The results underlined that two assays offered improved serologic diagnosis of tuberculosis over a single antibody test. BCG vaccination interferes with serologic tests for tuberculosis when polyspecific antibodies or mixture of common and specific antigens are used as immunologic reagents. A mycobacterial antigens circadian variation in correlation with vesperal fever in tuberculous patients was not revealed. The mycobacterial antigens seem to become undetectable, in sera, after 6 months of efficient treatment.     

Plant DMA topoisomerase I is recognized and inhibited by human Scl-70 sera autoantibodies

Type I topoisomerases (EC 5.99.1.2) are those enzymes capable of relaxing negatively supercoiled DNA without the need for ATP. The central role played by these enzymes in cell function suggests that the structure of type I topoisomerases may be highly conserved in eukaryotic cells. However, the extent of the conservation among eukaryotes is unknown. Human DNA topoisomerase I is an autoimmune antigen (Scl-70) of scleroderma patients. We have found that the autoimmune antibodies in human Scl-70 sera recognize protein from various plants, and these proteins display DNA relaxation function. In addition, Scl-70 antibodies were able to inhibit enzymatic activity of plant topoisomerase I. Therefore, the immunological cross-reactivity of the plant topoisomerase with human antibodies demonstrates that, despite divergence of eukaryotic organisms, these plant and animal enzymes retain structurally similar enzymatic features.     

Inhibition of adenovirus DNA synthesis in vitro by sera from patients with systemic lupus erythematosus

Sera containing antinuclear antibodies from patients with systemic lupus erythematosus (SLE) and related disorders were tested for their effect on the synthesis of adenovirus (Ad) DNA in an in vitro replication system. After being heated at 60 degrees C for 1 h, some sera from patients with SLE inhibited Ad DNA synthesis by 60 to 100%. Antibodies to double-stranded DNA were present in 15 of the 16 inhibitory sera, and inhibitory activity copurified with anti-double-stranded DNA in the immunoglobulin G fraction. These SLE sera did not inhibit the DNA polymerases alpha, beta, gamma and had no antibody to the 72,000-dalton DNA-binding protein necessary for Ad DNA synthesis. The presence of antibodies to single-stranded DNA and a variety of saline-extractable antigens (Sm, Ha, nRNP, and rRNP) did not correlate with SLE serum inhibitory activity. Methods previously developed for studying the individual steps in Ad DNA replication were used to determine the site of inhibition by the SLE sera that contained antibody to double-stranded DNA. Concentrations of the SLE inhibitor that decreased the elongation of Ad DNA by greater than 85% had no effect on either the initiation of Ad DNA synthesis or the polymerization of the first 26 deoxyribonucleotides.     

Identification of a family of human centromere proteins using autoimmune sera from patients with scleroderma

We have examined preimmune serum samples from a patient who progressively developed the symptoms of scleroderma CREST over a period of several years. During this period, anti-centromere antibodies (recognized by indirect immunofluorescence) appeared in the serum. Concomitant with the appearance of the anti-centromere antibodies, antibody species recognizing three chromosomal antigens in immunoblots of SDS polyacrylamide gels appeared in the patient's serum. These antigens migrate with electrophoretic mobilities corresponding to M_r=17, 80, and 140 kilodaltons (kd). Affinity-eluted antibody fractions recognizing the antigens have been prepared from sera of three other patients. Indirect immunofluorescence labeling of mitotic cells using these antibody fractions demonstrates that the antigens are centromere components. We designate them CENP (CENtromere Protein) — A (17kd), CENP-B (80kd), and CENP-C (140kd). The three CENP antigens share antigenic determinants. Immunoblotting experiments show that these patients make antibody species recognizing at least three distinct epitopes on CENP-B and two on CENP-C. Sera from different patients contain different mixtures of the antibody species.     

Inhibition of DNA topoisomerase I by natural and synthetic mono- and dimeric protoberberine alkaloids

A series of natural (i.e., 1-7) and synthetic (i.e., 8-23) protoberberine alkaloids were evaluated for their inhibitory activities towards DNA topoisomerase I. Both the natural, monomeric protoberberine alkaloids and their mono-modified congeners showed only minor activities. In contrast, most of the dimeric protoberberine alkaloids, especially compounds 12-22, were highly active, with a similar cleavage efficiency as camptothecin (CPT), a well-known, potent topoisomerase-I inhibitor. Thus, these dimeric compounds are promising candidates to be further elaborated as anticancer leads. The mechanism of topoisomerase-I inhibition seems to be dependent on drug concentration for the dimeric protoberberines. At low concentration, they exhibit similar characteristics as CPT. At high concentration, this ability is mostly lost, and the dimers inhibit the relaxation activity of topoisomerase I. Thus, we suppose that the active, dimeric protoberberines strongly bind to plasmid DNA at elevated drug concentration. This most likely results in blocking the enzyme's access to plasmid DNA, thus inhibiting its relaxation.     

Decreased catalytic function with altered sumoylation of DNA topoisomerase I in the nuclei of scleroderma fibroblasts

Introduction  

Sumoylation is involved in nucleolus-nucleoplasm transport of DNA topoisomerase I (topo I), which may associate with changes of cellular and topo I functions. Skin fibroblasts of patients with systemic sclerosis (SSc) exhibit profibrotic cellular changes. The aims of this study were to examine the catalytic function and sumoylation of topo I in the nuclei of SSc fibroblasts, a major cell type involved in the fibrotic process.     

DNA-hydrolysing activity of IgG antibodies from the sera of patients with schizophrenia

It is believed that damage to the membranes of brain cells of schizophrenia (SCZ) patients induces the formation of autoantigens and autoantibodies. Nevertheless, the importance of immunological changes leading to the loss of tolerance to self-antigens in the genesis of SCZ has not been established. The MALDI mass spectra of the IgG light chains of 20 healthy donors were relatively homogeneous and characterized by one peak with only one maximum. In contrast to the healthy donors, the MALDI mass spectra of IgG light chains corresponding to 20 SCZ patients demonstrated, similarly to 20 autoimmune systemic lupus erythematosus (SLE) patients, two maxima of a comparable intensity. In addition, the MALDI spectra of the IgG light chains of five SLE and four SCZ patients contained a small additional brightly pronounced peak with remarkably lower molecular mass compared with the main one. DNase autoantibodies (abzymes) can be found in the blood of patients with several autoimmune diseases, while the blood of healthy donors or patients with diseases without a significant disturbance of the immune status does not contain DNase abzymes. Here, we present the first analysis of anti-DNA antibodies and DNase abzymes in the sera of SCZ patients. Several strict criteria have been applied to show that the DNase activity is an intrinsic property of IgGs from the sera of SCZ patients. The sera of approximately 30% of SCZ patients displayed a higher content of antibodies (compared with 37% of SLE) interacting with single- and double-stranded DNA compared with healthy donors. Antibodies with DNase activity were revealed in 80% of the patients. These data indicate that some SCZ patients may show signs of typical autoimmune processes to a certain extent.