Clinical relevance of autoantibodies in systemic rheumatic diseases

Autoantibodies directed to intracellular antigens are serological hallmarks of systemic rheumatic diseases. Identification of circulating autoantibodies is helpful in establishing the correct diagnosis, indicating the prognosis and providing a guide to treatment and follow-up. Some autoantibodies are included in diagnostic and classification criteria for diseases such as anti-Sm antigen and anti-double-stranded DNA antibodies in systemic lupus erythematosus, anti-U1 nuclear ribonucleoprotein antibodies in mixed connective tissue disease, and anti-SS-A/Ro and anti-SS-B/La antibodies in Sjögren's syndrome. Over the past 30 years, the identification of new autoantibody systems was advanced by the initiation or adaptation of novel techniques such as double immunodiffusion to detect antibodies to saline-soluble nuclear antigens, extraction-reconstitution and ELISA techniques to detect histone and chromatin antibodies, immunoblotting and immunoprecipitation to detect a wide range of antibodies directed against naturally occurring and recombinant proteins. These techniques have been made possible by advances in cellular and molecular biology and in turn, the sera from index patients have been important reagents to identify novel intracellular macromolecules. This paper will focus on the clinical relevance of several autoantibody systems described by Tan and his colleagues over the past 30 years.Abbreviations ANA antinuclear antibody - CENPs centromere proteins - CTD connective tissue disease - DIA drug-induced autoimmunity - DIL drug-induced lupus - HIV human immunodeficiency virus - IIF indirect immunofluorescence - JCA juvenile chronic arthritis - MCTD mixed connective tissue disease - MSA mitotic spindle apparatus - NOR nucleolar organizer - NuMA nuclear mitosis antigen - PBC primary biliary cirrhosis - PCNA proliferating cell nuclear antigen - PM polymyositis - RA rheumatoid arthritis - RNP ribonucleoprotein - SLE systemic lupus erythematosus - SS Sjögren's syndrome - SSc systemic sclerosis - UCTD undifferentiated connective tissue disease     

Antinuclear antibodies defining autoimmunity pathways

Immunofluorescent imaging has been a powerful technique in helping to identify intracellular nuclear and cytoplasmic molecules which are target antigens of autoantibodies in systemic autoimmune disorders. Patterns of staining can be correlated with molecules engaged in specific cellular functions and distributed in distinct cellular domains. Different autoimmune disorders have different profiles of autoantibodies, and immunodiagnostics has become an important adjunct in differential diagnosis. An important finding that has eluded explanation is the presence of autoantibodies to many different antigens, manifested strikingly in systemic lupus erythematosus. In cancer, the occurrence of autoantibodies to tumor-associated antigens is not uncommon and a characteristic feature is also the presence of multiple autoantibodies. The targeted tumor-associated antigens are either oncogene or tumor suppressor gene products or their coactivators, which are altered or mutated and driving the autoimmune response. Most cancer cells have between two and eight mutated genes before oncogenic transformation occurs, initiating a process called synthetic lethality in tumorigenesis pathways. These observations beg the question of whether there are similar mechanisms in systemic lupus erythematosus and other disorders driving autoimmunity pathways. Targeting molecules that are synthetic lethal to each other is in the forefront of the search for anticancer therapy, and this could also be an objective in systemic autoimmune disorders.     

The DNA segregation mechanism of Epstein-Barr virus nuclear antigen 1

Latent Epstein–Barr virus (EBV) genomes are maintained in human cells as low copy number episomes that are thought to be partitioned by attachment to the cellular mitotic chromosomes through the viral EBNA1 protein. We have identified a human protein, EBP2, which interacts with the EBNA1 sequences that govern EBV partitioning. Here we show that, in mitosis, EBP2 localizes to the condensed cellular chromosomes producing a staining pattern that is indistinguishable from that of EBNA1. The localization of EBNA1 proteins with mutations in the EBP2 binding region was also examined. An EBNA1 mutant (Δ325–376) disrupted for EBP2 binding and segregation function was nuclear but failed to attach to the cellular chromosomes in mitosis. Our results indicate that amino acids 325–376 mediate the binding of EBNA1 to mitotic chromosomes and strongly suggest that EBNA1 mediates EBV segregation by attaching to EBP2 on the cellular mitotic chromosomes.     

Application of protein microarrays for multiplexed detection of antibodies to tumor antigens in breast cancer

There is strong preclinical evidence that cancer, including breast cancer, undergoes immune surveillance. This continual monitoring, by both the innate and the adaptive immune systems, recognizes changes in protein expression, mutation, folding, glycosylation, and degradation. Local immune responses to tumor antigens are amplified in draining lymph nodes, and then enter the systemic circulation. The antibody response to tumor antigens, such as p53 protein, are robust, stable, and easily detected in serum; may exist in greater concentrations than their cognate antigens; and are potential highly specific biomarkers for cancer. However, antibodies have limited sensitivities as single analytes, and differences in protein purification and assay characteristics have limited their clinical application. For example, p53 autoantibodies in the sera are highly specific for cancer patients, but are only detected in the sera of 10-20% of patients with breast cancer. Detection of p53 autoantibodies is dependent on tumor burden, p53 mutation, rapidly decreases with effective therapy, but is relatively independent of breast cancer subtype. Although antibodies to hundreds of other tumor antigens have been identified in the sera of breast cancer patients, very little is known about the specificity and clinical impact of the antibody immune repertoire to breast cancer. Recent advances in proteomic technologies have the potential for rapid identification of immune response signatures for breast cancer diagnosis and monitoring. We have adapted programmable protein microarrays for the specific detection of autoantibodies in breast cancer. Here, we present the first demonstration of the application of programmable protein microarray ELISAs for the rapid identification of breast cancer autoantibodies.     

Tumor-associated autoantibodies as diagnostic and prognostic biomarkers

In the process of tumorigenesis, normal cells are remodeled to cancer cells and protein expression patterns are changed to those of tumor cells. A newly formed tumor microenvironment elicits the immune system and, as a result, a humoral immune response takes place. Although the tumor antigens are undetectable in sera at the early stage of tumorigenesis, the nature of an antibody amplification response to antigens makes tumor-associated autoantibodies as promising early biomarkers in cancer diagnosis. Moreover, the recent development of proteomic techniques that make neo-epitopes of tumor-associated autoantigens discovered concomitantly has opened a new area of ‘immuno-proteomics’, which presents tumor-associated autoantibody signatures and confers information to redefine the process of tumorigenesis. In this article, the strategies recently used to identify and validate serum autoantibodies are outlined and tumor-associated antigens suggested until now as diagnostic/prognostic biomarkers in various tumor types are reviewed. Also, the meaning of autoantibody signatures and their clinical utility in personalized medicine are discussed. [BMB Reports 2012; 45(12): 677-685]     

Antinuclear autoantibodies: probes for defining proteolytic events associated with apoptosis

Antinuclear autoantibodies (ANAs) derived from patients with systemic autoimmune diseases have proven to be powerful tools in cell and molecular biology. The availability of these autoantibodies has been instrumental in the identification and characterization of a wide range of intracellular proteins involved in essential cellular activities. Recently, these autoantibodies have been used in molecular studies of apoptosis, particularly in the identification of substrates cleaved by proteases of the ICE/CED-3 family during this cell death pathway. The identification of these substrates may help to understand the role of proteolysis in apoptosis. Examples of nuclear autoantigens whose cleavage during apoptosis have been defined using ANAs include the 70 kD protein of the U1 small nuclear ribonucleoprotein particle (U1-70 kD), the nuclear mitotic apparatus protein (NuMA), DNA topoisomerase I, the RNA polymerase I upstream binding factor (UBF), and the catalytic subunit of DNA-dependent protein kinase (DNA-PK_cs). The use of ANAs as probes for defining proteolytic events associated with apoptosis promises to yield important insights into the mechanisms driving this cell death pathway.Abbreviations ANAs Antinuclear autoantibodies - DNA-PK_cs DNA-dependent protein kinase - ICE interleukin-1 (IL-1)-converting enzyme - MARs or SARs matrix or scaffold attachment regions - NuMA nuclear mitotic apparatus protein - PARP poly (ADP) ribose polymerase - snRNPs small nuclear ribonucleoproteins - SLE systemic lupus erythematosus - UBF upstream binding factor     

Features of autoantigens

The major cellular antigens recognized by autoantibodies in SLE and other systemic autoimmune diseases have been identified and characterized over the past 25 years. The pioneering studies of Eng Tan demonstrate the importance of autoantibodies as diagnostic markers. However, why certain autoantibodies, such as anti-Sm, are pathognomonic of SLE, while others are markers of othe autoimmune disease subsets, remains unanswered. This central question continues to drive much current research into the pathogenesis of SLE. Features of the autoantigens recognized by autoantibodies may provide important clues to the causes of lupus. Most autoantigens in systemic autoimmunity are multicomponent nucleoprotein complexes. These particles are encountered by the immune system as units, resulting in the tandem production of autoantibodies recognizing several components of the same complex. However, the intermolecular-intrastructural spreading of autoimmunity is regulated by mechanisms that at present are defined poorly. Also unexplained is the observation that the antigenic determinants recognized by autoantibodies are restricted and frequently correspond to active sites or functional domains. Analysis of experimental models of autoimmunity suggests that altering the structure of autoantigens, due to abnormal protein-protein interactions, hapten binding, altered degradation, or other mechanisms, could help to explain both the restricted patterns of autoantibody spreading and the selective targeting of antigenic sites. This may be a worthwhile area for further investigation of the pathogenesis of systemic autoimmune diseases.Abbreviations MCTD mixed connective tissue disease - PM/DM polymyositis / dermatomyositis - SLE Systemic lupus erythematosus - SSc systemic sclerosis - SVT simian virus 40 large T antigen     

Ultrastructural basis of mitosis in the fungusNectria haematococca (sexual stage ofFusarium solani)

Summary Microtubules (MTs) in the mitotic asters of the fungusNectria haematococca (teleomorph ofFusarium solani f. sp.pisi) pull on the spindle pole bodies (SPBs) during anaphase. To elucidate the structural basis of astral forces, we conducted an ultrastructural study using primarily freeze-substitution, three-dimensional reconstruction, and computerized numerical data acquisition and analysis. The asters were composed of numerous (68–171), mostly short (<0.5 m) MTs and varied widely in total MT length (34–83 m). Both the number and total length of MTs varied up to twofold or more among asters, even between the two asters of the same mitotic apparatus (MA). Surprisingly, less than one half (38%) of the MTs in each aster were attached to the SPB. Both the number and total length of these polar MTs varied up to twofold between the two asters of the same MA. Some asters included MTs oriented back toward the opposite SPB, whereas others did not, and the number and total length of such MTs varied among asters. These results are best interpreted by assuming that astral MTs inN. haematococca have a rapid rate of turnover and exhibit dynamic instability. Any of these parameters of astral architecture could vary during mitosis and thereby give rise to the oscillations of the mitotic apparatus that occur during anaphase B by generating unequal and fluctuating forces in the two sister asters. Astral MTs were arranged asymmetrically around the astral axis, and this asymmetry could produce the lateral movements of the SPB that occur during anaphase B. An apparently extensive system of 10nm filaments occurred in these cells, and some astral MTs were associated either terminally (at the plasma membrane) or laterally with these filaments. Such associations could be involved in the development and maintenance of astral forces.Abbreviations fMT free microtubule - MA mitotic apparatus - MT microtubule - pMT polar microtubule - SPB spindle pole body     

The significance of persistent newly developed autoantibodies in JIA patients under long-term anti-TNF treatment

Objective: To study the significance of persistent (12 months) new autoantibodies, in Juvenile Idiopathic Arthritis (JIA) patients treated with either Infliximab (INFL) or Etanercept (ET) for 2 years. Patients-methods: 26 children under INFL (n = 12) or ET (n = 14) were prospectively studied. A large panel of autoantibodies was tested using indirect immunofluorescence (ANA, anti-dsDNA, anti-ENA, SMA, LKM, AMA, PCA, anti-R1, ATA), ELISA (ANA, anti-ENA, anti-cardiolipin, ANCA), immunoblotting assay (anti-ENA: anti-Ro, anti-La, anti-Sm, anti-URNP, anti-Jo, anti-Scl70, anti-centromere, anti-ribosomal and anti-histone) and rate nephelometry (RF). Results: Apart from the positive patients for ANA (13/26) and RF (2/26) prior to anti-TNF treatment, 6/26 patients (23%) developed new autoantibodies (SMA, anti-R1, ATA) which persisted for 12–50 months. None developed antibodies to nuclear antigens. In only one case, ATA was associated with the development of Hashimoto’s thyroiditis. Conclusions: These findings indicate that in JIA patients in contrast to adult RA patients, development of new autoantibodies to various nuclear antigens is rare. Other non relevant to rheumatic diseases autoantibodies, may appear and persist for >12 months, but very rarely they may be related to clinical entities, especially in the presence of a positive family history of autoimmunity.     

The effects of maleic anhydride on the ionic permeability of red cells

Summary Maleic anhydride (MA) has been shown to react specifically and rapidly with amino groups of proteins; the maleyl amino groups are negatively charged and completely stable at neutral pH. Treatment of human red cells with this reagent results in a significant increase in K⁺ permeability which is associated with a much smaller increase in Na⁺ permeability. Opposite effects are observed on anion permeability, the SO ₄ ^–– and Cl^– permeability being decreased to an approximately similar extent upon treatment with MA.Studies on the distribution of MA between membrane lipids and proteins shows that most of the membrane-bound MA is associated with membrane proteins. These results suggest that the observed effects of MA on the ion permeability of the red cell are caused by its combination with amino groups of cell membrane proteins.Fellow of the Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina (CONICET).Established Investigators of CONICET.     

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.     

Mitotic segregation in hybrid of methylotrophic yeastCandida pelliculosa

Protoplasts from two autoxotrophic mutants of methylotrophic yeastCandida pelliculosa were fused by the PEG technique, and the genetic structure of the hybrid obtained was studied by means of mitotic segregation. Spontaneous mitotic segregation depended on the composition of the medium and the type of carbon source used. Mechanisms by which segregants appeared were studied by means of UV- and benomyl-induced mitotic segregation. Our data suggest that one large percentage of segregants (Leu ^–) occurred as a result of mitotic chromosome loss. The other large portion of segregants (Ade ^– andLys ^–) are a consequence of mitotic recombination.     

High-performance liquid chromatographic determination of 1,3-diethyl-2-thiobarbituric acid–malonaldehyde adduct in fish meat

The reaction conditions of 1,3-diethyl-2-thiobarbituric acid (DETBA)–malonaldehyde (MA) adduct formation were examined in order to analyze MA in fish tissue by high-performance liquid chromatography. A reaction mixture containing 4 mM butyl hydroxytoluene was heated at 100°C for 150 min and the DETBA–MA adduct formed was separated by a Inertsil ODS column for 20 min. The detection limit was 5 pmol.     

Analysis and evaluation of trans,trans-muconic acid as a biomarker for benzene exposure

Benzene is an important industrial chemical and, due to its occurrence in mineral oil and its formation in many combustion processes, a widespread environmental pollutant. Since benzene is hematoxic and has been classified as a human carcinogen, monitoring and control of benzene exposure is of importance. Although trans,trans-muconic acid (ttMA) was identified as a urinary metabolite of benzene at the beginning of this century, only recently has its application as a biomarker for occupational and environmental benzene exposure been investigated. The range of metabolic conversion of benzene to ttMA is about 2–25% and dependent on the benzene exposure level, simultaneous exposure to toluene, and probably also to genetic factors. For the quantitation of ttMA in urine, HPLC methods using UV and diode array detection as well as GC methods combined with MS or FID detection have been described. Sample pretreatment for both HPLC and GC analysis comprises centrifugation and enrichment by solid-phase extraction on anion-exchange sorbents. Described derivatization procedures prior to GC analysis include reaction with N,O-bis(trimethysilyl)acetamide, N,O-bis(trimethylsilyl)trifluoroacetamide, pentafluorobenzyl bromide and borontrifluoride–methanol. Reported limits of detection for HPLC methods range from 0.1 to 0.003 mg l⁻¹, whereas those reported for GC methods are 0.03–0.01 mg l⁻¹. Due to its higher specificity, GC methods appear to be more suitable for determination of low urinary ttMA levels caused by environmental exposure to benzene. In studies with occupational exposure to benzene (>0.1 ppm), good correlations between urinary ttMA excretion and benzene levels in breathing air are observed. From the reported regressions for these variables, mean excretion rates of ttMA of 1.9 mg g⁻¹ creatinine or 2.5 mg l⁻¹ at an exposure dose of 1 ppm over 8 h can be calculated. The smoking-related increase in urinary ttMA excretion reported in twelve studies ranged from 0.022 to 0.2 mg g⁻¹ creatinine. Only a few studies have investigated the effect of exposure to environmental levels of benzene (<0.01 ppm) on urinary ttMA excretion. A trend for slightly increased ttMA levels in subjects living in areas with high automobile traffic density was observed, whereas exposure to environmental tobacco smoke did not significantly increase the urinary ttMA excretion. It is concluded that urinary ttMA is a suitable biomarker for benzene exposure at occupational levels as low as 0.1 ppm. Biomonitoring of exposure to environmental benzene levels (<0.01 ppm) using urinary ttMA appears to be possible only if the ingestion of dietary sorbic acid, another precursor to urinary ttMA, is taken into account.     

Use of synthetic peptides for the detection and quantification of autoantibodies

Summary and conclusions The rapid progress made over the last 10 years in the identification of individual autoantigens and in the localization of the epitopes involved, has resulted in a parallel reduction in the complexity of the antigen required for the detection of autoantibodies. The ability to use synthetic peptides as antigens is a remarkable culmination of this process considering that many antigenic particles contain multiple proteins (eg. Sm consist of 8 or more individual proteins).Despite the fact that patients with SLE have a polyclonal hypergammaglobulinemia, excellent correlations between ELISAs utilizing the P2 or SmB/B synthetic peptides, ELISAs utilizing r proteins and immunoblotting were obtained [28, 38, 50]. However, false positive/non-specific binding to a P2-BSA-glutaraldehyde conjugate has been observed with serum from old MRL/lpr mice (unpublished observations). In addition, some of the results obtained in human autoimmune diseases suggest that non-specific binding may be problematic in some instances. It is difficult, at present, to know whether the higher frequencies of detection of autoantibodies to certain synthetic peptide antigens reflect increased sensitivity or decreased specificity.Synthetic peptide antigens have beeen used to detect autoantibodies in both organ specific and multisystem autoimmune diseases. In only a small number of cases have these reagents been rigorously tested for sensitivity and specificity. Despite this, synthetic peptides have been shown to be valuable for detection and quantification of autoantibodies in certain clinical situations. Undoubtedly, further progress in epitope mapping of autoantigens coupled with technological advances in protein synthesis and improved prediction of protein structure will lead to a large number of synthetic peptide antigens for research and clinical applications. It is unlikely that short synthetic peptides will substitute for native proteins in all instances since some autoantibodies show a striking preference for conformational epitopes.Abbreviations r recombinant - SLE systemic lupus erythematosus     

Co-Positivity for Anti-dsDNA, -Nucleosome and -Histone Antibodies in Lupus Nephritis Is Indicative of High Serum Levels and Severe Nephropathy

ObjectiveTo characterize the significance of correlated autoantibodies in systemic lupus erythematosus (SLE) and its complication lupus nephritis (LN) in a large cohort of patients.MethodsClinical data were statistically analyzed in 1699 SLE patients with or without nephritis who were diagnosed and treated during 2002–2013 in the northeast region of China. Reactivity to a list of 16 autoantibodies was detected by the serum test Euroline ANA profile (IgG). Serum titers of the anti-nucleosome autoantibodies were measured by ELISA assays. Kidney biopsies were examined by pathologists. Immune complex deposition was identified by immunohistochemistry stain.ResultsSimultaneous positivity of anti-dsDNA, -nucleosome and -histone antibodies (3-pos) was prevalent in SLE patients with LN compared to Non-renal SLE patients (41% vs 11%, p< 0.001). Significant correlations were found between any two of the above three anti-nucleosome antibodies in LN patients. In comparison to non-3-pos cohorts, 3-pos patients with LN had significantly higher serum levels of the three antibodies and more active disease; was associated with type IV disease; suffered from more severe renal damages; received more intensive treatment and had worse disease outcome. The serum levels of these three autoantibodies in 3-pos LN patients were significantly decreased when they underwent clinical recovery.ConclusionsSimultaneous reactivity to anti-dsDNA, -nucleosome and -histone antibodies by Euroline ANA profile (IgG) may indicate severe nephropathy in patients with SLE.     

A 68-Nucleotide Sequence within the 3′ Noncoding Region of Simian Hemorrhagic Fever Virus Negative-Strand RNA Binds to Four MA104 Cell Proteins

The 3′ noncoding region (NCR) of the negative-strand RNA [3′(−)NCR RNA] of the arterivirus simian hemorrhagic fever virus (SHFV) is 209 nucleotides (nt) in length. Since this 3′ region, designated 3′(−)209, is the site of initiation of full-length positive-strand RNA and is the template for the synthesis of the 5′ leader sequence, which is found on both full-length and subgenomic mRNAs, it is likely to contain cis-acting signals for RNA synthesis and to interact with cellular and viral proteins to form replication complexes. Gel mobility shift assays showed that cellular proteins in MA104 S100 cytoplasmic extracts formed two complexes with the SHFV 3′(−)209 RNA, and results from competition gel mobility shift assays demonstrated that these interactions were specific. Four proteins with molecular masses of 103, 86, 55, and 36 kDa were detected in UV-induced cross-linking assays, and three of these proteins (103, 55, and 36 kDa) were also detected by Northwestern blotting assays. Identical gel mobility shift and UV-induced cross-linking patterns were obtained with uninfected and SHFV-infected extracts, indicating that the four proteins detected are cellular, not viral, proteins. The binding sites for the four cellular proteins were mapped to the region between nt 117 and 184 (68-nt sequence) from the 3′ end of the SHFV negative-strand RNA. This 68-nt sequence was predicted to form two stem-loops, SL4 and SL5. The 3′(−)NCR RNA of another arterivirus, lactate dehydrogenase-elevating virus C (LDV-C), competed with the SHFV 3′(−)209 RNA in competition gel mobility shift assays. UV-induced cross-linking assays showed that four MA104 cellular proteins with the same molecular masses as those that bind to the SHFV 3′(−)209 RNA also bind to the LDV-C 3′(−)NCR RNA and equine arteritis virus 3′(−)NCR RNA. However, each of these viral RNAs also bound to an additional MA104 protein. The binding sites for the MA104 cellular proteins were shown to be located in similar positions in the LDV-C 3′(−)NCR and SHFV 3′(−)209 RNAs. These data suggest that the binding sites for a set of the cellular proteins are conserved in all arterivirus RNAs and that these cell proteins may be utilized as components of viral replication complexes.     

Noncompetitive Activation of the Peroxidase-Catalyzed Oxidation of o-Phenylenediamine by Melamine

Peroxidase-catalyzed oxidation of o-phenylenediamine (PDA) is greatly activated with melamine (MA) in 15 mM phosphate–citrate buffer at pH 6.0–7.4 in a noncompetitive manner: k _cat and K _m increase in direct proportion to the MA concentration. An extent of the activation is quantitatively characterized with a coefficient (in M^–1), which essentially increases along with the rise in pH from 6.0 to 7.4. MA acts as a nucleophilic catalyst in the oxidation process: it most likely affects the peroxidase active site from the distal position of heme. MA noncompetitively inhibits the peroxidase oxidation of PDA at pH 4.3, since it completely loses its nucleophilic properties in acidic medium. A rapid, highly accurate, and simple analytical test system based on the kinetics of melamine-activated oxidation of PDA is proposed for the quantitative determination of melamine within the concentration range of 10^–4–10^–3 M. This test system uses the spectrophotometric determination of the PDA oxidation product at 455 nm.     

Activation of Peroxidase-Catalyzed Oxidation of Aromatic Amines with 2-Aminothiazole and Melamine

The peroxidase-catalyzed oxidation of 3,3",5,5"-tetramethylbenzidine (TMB), ortho-phenylenediamine (PDA), and 5-aminosalicylic acid (5-ASA) is significantly accelerated in the presence of 2-aminothiazole (AT) and melamine (MA), and an increase in their concentrations is associated with a parallel increase in the k _cat and K _m values for TMB and PDA. The activation of the peroxidase-catalyzed oxidation of TMB and PDA is quantitatively characterized by a coefficient (degree) (M^–1) which significantly depends on pH in the range 6.2-6.4, 6.4-7.4, and 6.0-7.4 for the TMB–AT, TMB–MA, and PDA–MA pairs, respectively. An increase in the coefficient with increase in pH confirms nucleophilicity of activation of the peroxidase-catalyzed oxidation of the aromatic amines in the presence of AT and MA. Under optimal conditions the coefficients for the TMB–AT, PDA–AT, TMB–MA, and PDA–MA pairs vary in the limits of (1.90-3.53)·10³ M^–1.     

Localization of autoepitopes on the PCM-1 autoantigen using scleroderma sera with autoantibodies against the centrosome

Characterization of epitope domains of autoantigens is important for deducing the cellular functions of autoantigens and may be important for understanding the autoimmune response. In the reported studies, epitope analysis of the centrosome autoantigen PCM-1 was performed. For these investigations, portions of the PCM-1 cDNA were subcloned into the pMAL expression plasmid, fusion proteins were induced, and aliquots of the extracts were probed by immunoblot analysis using two human autoimmune anticentrosome autoantisera. Immunoblotting identified three individual autoepitopes of 26-40 amino acid residues, amino acids 506-545, 1434–1465, and 1661–1686, within the PCM-1 protein. ELISA assays using non-denatured proteins did not identity any additional autoepitopes in the remainder of the PCM-1 molecule. To analyze the identified autoepitopes further, synthetic peptides were generated that covered each of the three autoepitopes and the synthetic peptides then were probed using the scleroderma sera. Peptides that covered the antigenic regions from amino acids 506-545 and 1434–1465 failed to react with the anticentrosome autoantisera suggesting that overall protein conformation may be important for the formation of those two autoepitopes. Peptides derived from the sequence of the third autoepitope were recognized by autoantibodies present in the anticentrosome autoantisera allowing the identification of the tripeptide KDC as the autoepitope in this region of the PCM-1 molecule. These studies lay the foundation for future investigations of the autoimmune response in scleroderma patients that are producing anticentrosome autoantibodies and should allow an investigation of the cellular role of the PCM-1 protein.