Proteomic analysis and identification of cell surface-associated proteins of Clostridium chauvoei.

Blackleg is a highly fatal disease of cattle and sheep, caused by Clostridium chauvoei, a Gram positive, anaerobic, spore forming bacteria. Cell surface-associated proteins play a major role in inducing the protective immunity. However, the identity of a majority of cell surface-associated proteins of C. chauvoei is not known. In the present investigation, we have used SDS-PAGE, 2D-gel electrophoresis and Western blotting followed by mass spectrometry to identify cell surface-associated proteins of C. chauvoei. Among the identified proteins, which have shown to offer protective antigencity in other bacteria, Enolase, Chaperonin, Ribosomal protein L10, Glycosyl Hydrolase and Flavoprotein were characterized by sequencing and their overexpression in Escherichia coli. In conclusion, cell surface-associated proteins were identified using proteomic approach and the genes for the immunoreactive proteins were expressed, which may prove to be potential diagnostic or vaccine candidates.

IL4I1 Is a Novel Regulator of M2 Macrophage Polarization That Can Inhibit T Cell Activation via L-Tryptophan and Arginine Depletion and IL-10 Production.

Interleukin 4-induced gene-1 (IL4I1) was initially described as an early IL-4-inducible gene in B cells. IL4I1 protein can inhibit T cell proliferation by releasing its enzymatic catabolite, H2O2, and this effect is associated with transient down-regulation of T cell CD3 receptor-zeta (TCRζ) expression. Herein, we show that IL4I1 contributes to the regulation of macrophage programming. We found that expression of IL4I1 increased during bone marrow-derived macrophage (BMDM) differentiation, expression of IL4I1 is much higher in primary macrophages than monocytes, and IL4I1 expression in BMDMs could be induced by Th1 and Th2 cytokines in two different patterns. Gene expression analysis revealed that overexpression of IL4I1 drove the expression of M2 markers (Fizz1, Arg1, YM-1, MR) and inhibited the expression of M1-associated cytokines. Conversely, knockdown of IL4I1 by siRNA resulted in opposite effects, and also attenuated STAT-3 and STAT-6 phosphorylation. Furthermore, IL4I1 produced by macrophages catalyzed L-tryptophan degradation, while levo-1-methyl-tryptophan (L-1-MT), but not dextro-1-methyl-tryptophan, partially rescued IL4I1-dependent inhibition of T cell activation. Other inhibitors, such as diphenylene iodonium (DPI), an anti-IL-10Rα blocking antibody, and a nitric oxide synthase inhibitor, NG-monomethyl-L-arginine, also had this effect. Overall, our findings indicate that IL4I1 promotes an enhanced M2 functional phenotype, which is most likely associated with the phosphorylation of STAT-6 and STAT-3. Moreover, DPI, L-1-MT, NG-monomethyl-L-arginine, and anti-IL-10Rα blocking antibody were all found to be effective IL4I1 inhibitors in vitro.

Dichotomy between factors inducing the immunosuppressive enzyme IL-4-induced gene 1 (IL4I1) in B lymphocytes and mononuclear phagocytes.

MPhi and DC are key elements in the control of tissue homeostasis and response to insult. In this work, we demonstrate that MPhi and DC are the major producers of the phenylalanine catabolizing enzyme IL-4-induced gene 1 (IL4I1) under inflammatory conditions. IL4I1 was first described in B cells, which indeed can produce IL4I1 in vitro, although at much lower levels. In vivo, IL4I1 is highly expressed by MPhi and DC of Th1 granulomas (sarcoidosis, tuberculosis) but poorly detected in Th2 granulomas (schistosomiasis). In vitro, expression of the enzyme is induced in mononuclear phagocytes by various pro-inflammatory stimuli through the activation of the transcription factors NF-kappaB and/or STAT1. B cells also express IL4I1 in response to NF-kappaB-activating stimuli such as CD40L; however, in contrast to myeloid cells, B cells are insensitive to IFN-gamma but respond to stimulation of the IL-4/STAT6 axis. As we show that the expression of IL4I1 by a monocytic cell line inhibits T-cell proliferation and production of IFN-gamma and inflammatory cytokines, we propose that IL4I1 participates in the downregulation of Th1 inflammation in vivo.

Peripheral blood T lymphocyte sensitisation against calsequestrin and flavoprotein in patients with Graves' ophthalmopathy.

Thyroid-associated ophthalmopathy (TAO) is an orbital autoimmune disorder of the extraocular and eyelid muscles and surrounding connective and adipose tissue. Although mononuclear cell infiltration of orbital tissue is a characteristic feature of TAO the likely role of T lymphocyte reactivity against eye muscle antigens in the initiation of eye muscle damage in TAO has not been explored in detail. Therefore, we tested for T lymphocyte sensitisation to three eye muscle antigens namely, calsequestrin, G2s and flavoprotein (Fp), in patients with Graves' ophthalmopathy (GO), Graves' hyperthyroidism (GH) without ophthalmopathy and age and sex matched normal subjects. T lymphocyte reactivity was determined in a proliferation assay, results being expressed as stimulation index (SI). Mean ( +/- SE) SI for patients with GO, but not GH without ophthalmopathy, were significantly greater than that for normal subjects for calsequestrin and Fp, but not G2s. Mean ( +/- SE) SI was also significantly increased in patients with active ophthalmopathy, but not chronic ophthalmopathy, compared to normal subjects, for calsequestrin and Fp, but not G2s. Overall, positive lymphocyte proliferation to calsequestrin was demonstrated in 59% of patients with GO and 33% of patients with GH, which was significantly greater than in normals for both groups. In conclusion, we have demonstrated significant T lymphocyte reactivity to calsequestrin and, to a lesser extent, Fp in patients with GO. Because calsequestrin is located in the cell membranes of the eye muscle cell during the myotubular stage of the cell cycle, its targeting might be the primary reaction which leads to extraocular muscle inflammation in patients with GH.

[Current understanding of pathogenesis of Graves ophthalmopathy].

Graves ophthalmopathy most frequently develops in a patient suffering from Graves disease in which autoantibodies to a target antigen, thyrotropin receptor, activate the autoantigen leading to hyperthyroidism. It is well known that in Graves ophthalmopathy inflammatory cells infiltrate and hydrophobic glycosaminoglycan accumulates in the retro -occular tissues. However, in contrast to Graves disease, little has been known as to how this eye disease develops. Here we review recent advance in understanding of pathogenesis of Graves ophthalmopathy.

Study of serum antibodies against three eye muscle antigens and the connective tissue antigen collagen XIII in patients with Graves' disease with and without ophthalmopathy: correlation with clinical features.

OBJECTIVE: The extraocular muscles are one of the primary tissues implicated in the autoimmune-mediated inflammation of thyroid-associated ophthalmopathy (TAO). Our aim was to determine the prevalence and level of antibodies against three candidate eye muscle antigens and the orbital fibroblast membrane antigen collagen XIII, in well-characterized patient groups. STUDY DESIGN AND PATIENTS: The study cohort consisted of patients with Graves' hyperthyroidism with and without ophthalmopathy, controls patients with other thyroid or other autoimmune disorders and healthy subjects. The presence of eye muscle antibodies was determined using an optimized and standardized enzyme-linked immunosorbent assay. We measured antibodies against (i) the 67-kDa flavoprotein (Fp) subunit of the mitochondrial enzyme succinate dehydrogenase; (ii) G2s, a 141 amino acid fragment of the winged-helix transcription factor FOXP1; (iii) calsequestrin, a 63-kDa calcium-binding protein; and (iv) collagen XIII, a connective tissue protein that is closely linked to the congestive ophthalmopathy subtype of TAO. Eye muscle antibody levels were correlated with clinical diagnosis and presence or not of ophthalmopathy. RESULTS: Prevalences of positive antibody tests to calsequestrin (75.0%) and collagen XIII (43.8%) were significantly greater in Graves' disease (GD) patients with ophthalmopathy than in healthy subjects, whereas modest significance was demonstrated with antibodies against Fp, but not G2s. Significantly greater serum levels of antibodies against calsequestrin, G2s, and collagen XIII, but not Fp, were found in GD patients with ophthalmopathy compared to control patients without eye disease and healthy subjects. CONCLUSIONS: Calsequestrin and collagen XIII antibodies are the most specific to TAO, whereas antibodies against G2s, and to a lesser extent Fp, are also markers of ophthalmopathy, but less reliable. These results are unique in that it is the first time the significance of a panel of three candidate eye muscle antibodies and a connective tissue antibody have been evaluated in the same patients with ophthalmopathy.

Serological responses of FldA and small-molecular-weight proteins of Helicobacter pylori: correlation with the presence of the gastric MALT tissue.

PURPOSE: We tested whether the serological response to Flavodoxin-A (FldA) protein and anti-Helicobacter pylori immunoblots correlated to the degree of mucosaassociated lymphoid tissue (MALT) in the stomach. METHODS: Eighty H. pyloni-infected patients with different degrees of MALT in the stomach were investigated with serum sampling and endoscopy on enrolment, the 2nd and the 12th months after anti-H. pylori therapy. All sera were tested for the anti-FldA protein and anti-H. pylon immunoblots, including 19.5, 26.5, 30, 35, 89, and 116 KDa (CagA). Regression of follicular gastritis was assessed by histology. RESULTS: Patients with dense lymphoid follicles had higher prevalence rates of anti-FldA protein, 19.5, 26.5, and 30 KDa antibodies of H. pylori (p < .05). Histologic downgrade of MALT was observed in 25% (10/40) of patients in the 2nd month and in 60% (23/38) in the 12th month after H. pylori therapy. After H. pylori eradication, the patients with MALT and dense lymphoid follicles had significantly negative seroconversions of 19.5, 26.5, 30, and 35 KDa antibodies (p < .05), but not of CagA and FldA. CONCLUSION: Patients with gastric MALT and dense lymphoid follicles had different anti-H. pylori serological responses to those with scanty or an absence of lymphoid follicles. The negative seroconversion of the smaller-molecular-weight proteins, but not CagA and FldA, may correlate with the regression of MALT by H. pylori eradication.

Trichomonas vaginalis degrades nitric oxide and expresses a flavorubredoxin-like protein: a new pathogenic mechanism?

Besides possessing many physiological roles, nitric oxide (NO) produced by the immune system in infectious diseases has antimicrobial effects. Trichomoniasis, the most widespread non-viral sexually transmitted disease caused by the microaerophilic protist Trichomonas vaginalis, often evolves into a chronic infection, with the parasite able to survive in the microaerobic, NO-enriched vaginal environment. We relate this property to the finding that T. vaginalis degrades NO under anaerobic conditions, as assessed amperometrically. This activity, which is maximal (133 +/- 41 nmol NO/10(8) cells per minute at 20 degrees C) at low NO concentrations (< or = 1.2 microM), was found to be: (i) NADH dependent, (ii) cyanide insensitive and (iii) inhibited by O(2). These features are consistent with those of the Escherichia coli A-type flavoprotein (ATF), recently discovered to be endowed with NO reductase activity. Using antibodies against the ATF from E. coli, a protein band was immunodetected in the parasite grown in a standard medium. If confirmed, the expression of an ATF in eukaryotes suggests that the genes coding for ATFs were transferred during evolution from anaerobic Prokarya to pathogenic protists, to increase their fitness for the microaerobic, parasitic life style. Thus the demonstration of an ATF in T. vaginalis would appear relevant to both pathology and evolutionary biology. Interestingly, genomic analysis has recently demonstrated that Giardia intestinalis and other pathogenic protists have genes coding for ATFs.

Second class minors: molecular identification of the autosomal H46 histocompatibility locus as a peptide presented by major histocompatibility complex class II molecules.

CD4 T cells regulate immune responses that cause chronic graft rejection and graft versus host disease but their target antigens remain virtually unknown. We developed a new method to identify CD4 T cell-stimulating antigens. LacZ-inducible CD4 T cells were used as a probe to detect their cognate peptide/MHC II ligand generated in dendritic cells fed with Escherichia coli expressing a library of target cell genes. The murine H46 locus on chromosome 7 was thus found to encode the interleukin 4-induced IL4i1 gene. The IL4i1 precursor contains the HAFVEAIPELQGHV peptide which is presented by A(b) major histocompatibility complex class II molecule via an endogenous pathway in professional antigen presenting cells. Both allelic peptides bind A(b) and a single alanine to methionine substitution at p2 defines nonself. These results reveal novel features of H loci that regulate CD4 T cell responses as well as provide a general strategy for identifying elusive antigens that elicit CD4 T cell responses to tumors or self-tissues in autoimmunity.

Mediation of poly(ADP-ribose) polymerase-1-dependent cell death by apoptosis-inducing factor.

Poly(ADP-ribose) polymerase-1 (PARP-1) protects the genome by functioning in the DNA damage surveillance network. PARP-1 is also a mediator of cell death after ischemia-reperfusion injury, glutamate excitotoxicity, and various inflammatory processes. We show that PARP-1 activation is required for translocation of apoptosis-inducing factor (AIF) from the mitochondria to the nucleus and that AIF is necessary for PARP-1-dependent cell death. N-methyl-N'-nitro-N-nitrosoguanidine, H2O2, and N-methyl-d-aspartate induce AIF translocation and cell death, which is prevented by PARP inhibitors or genetic knockout of PARP-1, but is caspase independent. Microinjection of an antibody to AIF protects against PARP-1-dependent cytotoxicity. These data support a model in which PARP-1 activation signals AIF release from mitochondria, resulting in a caspase-independent pathway of programmed cell death.

Structural characterization of the 60-kDa bermuda grass pollen isoallergens, a covalent flavoprotein.

Our studies suggest a tripartite structure for the 60-kDa allergen of Bermuda grass pollen (BG60) including a short N-terminal segment, a FAD-binding domain, and a C-terminal domain. The lower molecular weight isoallergens lack the N-terminal segment. The higher protease susceptibility and the lower melting temperature of approximately 20 degrees C of the lower molecular weight isoforms suggest that the N-terminal segment is essential for a compact structure. Database screening reveals that the protease-digested peptide sequences (approximately 180 residues in total) share 40% identity with the plant berberine bridge enzymes. In particular, a 24-residue peptide sequence displays high similarity to a conserved FAD-binding motif. The spectroscopic and SDS-PAGE analyses suggest that the cofactor FAD is covalently linked to the central domain. Therefore, we conclude that BG60 is identified as the first flavinylated allergen.

Coxsackievirus B3 infection induces anti-flavoprotein antibodies in mice.

Enteroviruses, the most common cause of acute myocarditis, are also supposed aetiological agents of dilated cardiomyopathy. Autoantibodies (anti-M7; Klein & Berg, Clin Exp Immunol 1990; 58:283-92) directed against flavoproteins with covalently bound flavin (alphaFp-Ab; Otto et al., Clin Exp Immunol 1998; 111:541-2) are detected in up to 30% of sera of patients with myocarditis and idiopathic dilated cardiomyopathy (IDCM). Mice inoculated with a myocarditic variant of coxsackievirus B3 (CVB3) were employed to study the occurrence of serum alphaFp-Ab following viral infection. The presence of alphaFp-Ab was analysed by Western blotting with the flavoprotein antigens 6-hydroxy-D-nicotine oxidase (6HDNO) and sarcosine oxidase (SaO). Of 10 sera from CVB3-infected mice, five showed a strong reaction with both antigens. The sera were reactive also to the mitochondrial covalently flavinylated proteins dimethylglycine dehydrogenase and sarcosine dehydrogenase. Sera of non-infected mice did not react with these antigens. A 6HDNO mutant protein with non-covalently bound FAD no longer reacted on Western blots with sera of CVB3-infected mice. Preincubation with FAD abolished or reduced the reaction of the sera with the 6HDNO antigen. At 2 weeks p.i. the alphaFp-Ab were of the IgM and IgG isotypes, at 7 and 9 weeks p.i. of the IgG isotype. The sera of CVB3-infected mice reproduced closely the antigenic specificity of the anti-M7 sera of patients, lending further support to the role of coxsackieviruses in the pathogenesis of IDCM.

Autoantibodies to the flavoprotein subunit of succinate dehydrogenase: analysis of specificity in autoimmune thyroid disease.

OBJECTIVE: Thyroid-associated ophthalmopathy is a progressive eye disorder affecting the extraocular muscle and orbital connective tissue and is considered to have an autoimmune aetiology. A recent study reported a close relationship between serum antibodies against the flavoprotein subunit of succinate dehyhdrogenase (SDHFp) and active thyroid-associated ophthalmopathy involving eye muscle damage. The aim of the present study was to develop a sensitive and quantitative radiobinding assay for the detection of antibodies to the flavoprotein subunit of succinate dehydrogenase and to use this to determine the distribution of antibodies in different patient groups. DESIGN AND PATIENTS: Serum samples from the following patient groups were analysed: 20 systemic lupus erythematosus; 20 Addison's disease; 26 autoimmune hypothyroidism; 28 Graves' hyperthyroidism; 12 pretibial myxoedema; 25 thyroid-associated ophthalmopathy. Sera from 20 healthy subjects were used as controls. [35S]-labelled succinate dehydrogenase flavoprotein was produced in an in vitro transcription-translation system and subsequently used in immunoprecipitation experiments with sera from patient and control groups to test for the presence of antibodies to the flavoprotein. RESULTS: Succinate dehydrogenase flavoprotein antibodies were detected in five of the 20 (25%) patients with Addison's disease, six of the 20 (30%) with systemic lupus erythematosus, five of the 26 (19%) with autoimmune hypothyroidsm, six of the 28 (21%) with Graves' hyperthyroidism, two of the 12 (17%) with pretibial myxoedema and three of the 25 (12%) with thyroid-associated ophthalmopathy. The frequencies of flavoprotein antibodies were significantly greater than controls (P-value < 0.05) for patients with systemic lupus erythematosus (P = 0.02), but not for patients with either Addison's disease (P = 0.05), pretibial myxoedema (P = 0.13), Graves' hyperthyroidism (P = 0.07), autoimmune hypothyroidism (P = 0.06) or thyroid-associated ophthalmopathy (P = 0.24). For the patients with thyroid-associated ophthalmopathy, the frequency of SDHFp antibodies did not appear to be related to the length of time from diagnosis: the group containing samples taken less than one year from diagnosis showed no increased frequency of SDHFp antibodies when compared to controls (P = 0.10), with three of the 18 (17%) patients being positive. With respect to seven patients with thyroid-associated ophthalmopathy diagnosed for more than a year, SDHFp antibodies were not detected in any of their serum samples. In addition, the clinical severity of the disease, as recorded by the NOSPECS classification, did not correlate with the frequency of SDHFp antibodies: P = 0.13, 0.33 and 0.38, respectively, for patients with Grade II, III and IV ophthalmopathy. Similar results were also found in the case of patients with pretibial myxoedema and eye disease: P = 0.06 for patients with Grade III ophthalmopathy and, SDHFp antibodies were not detected in any of the sera taken from patients with Grade IV ophthalmopathy. In addition, no association was found between disease duration and the frequency of antibodies to the flavoprotein in this patient group. CONCLUSIONS: Our results indicate that succinate dehydrogenase flavoprotein antibodies are not a suitable marker for thyroid-associated ophthalmopathy, at least with the assay system used, as they can be found in patients who do not have eye disease and therefore lack the disease specificity required of a diagnostic tool.

Purification and cloning of an apoptosis-inducing protein derived from fish infected with Anisakis simplex, a causative nematode of human anisakiasis.

While investigating the effect of marine products on cell growth, we found that visceral extracts of Chub mackerel, an ocean fish, had a powerful and dose-dependent apoptosis-inducing effect on a variety of mammalian tumor cells. This activity was strikingly dependent on infection of the C. mackerel with the larval nematode, Anisakis simplex. After purification of the protein responsible for the apoptosis-inducing activity, we cloned the corresponding gene and found it to be a flavoprotein. This protein, termed apoptosis-inducing protein (AIP), was also found to possess an endoplasmic reticulum retention signal (C-terminal KDEL sequence) and H2O2-producing activity, indicating that we had isolated a novel reticuloplasimin with potent apoptosis-inducing activity. AIP was induced in fish only after infection with larval nematode and was localized to capsules that formed around larvae to prevent their migration to host tissues. Our results suggest that AIP may function to impede nematode infection.

Nuclear localization of the Arabidopsis blue light receptor cryptochrome 2.

The cryptochrome blue light photoreceptor family of Arabidopsis thaliana consists of two members, CRY1 and CRY2 (PHH1). CRY2 contains a putative nuclear localization signal (NLS) within its C-terminal region. We examined whether CRY2 is localized in the nucleus and whether the C-terminal region of CRY2 is involved in nuclear targeting. Total cellular and nuclear protein extracts from Arabidopsis were subjected to immunoblot analysis with CRY2-specific antibodies. Strong CRY2 signals were obtained in the nuclear fraction. Fusion proteins consisting of the green fluorescent protein (GFP) and different fragments of CRY2 were expressed in parsley protoplasts and the localization of the fusion proteins was determined by fluorescence and confocal laser scanning microscopy. GFP-fusions containing the entire CRY2 protein or its C-terminal region were found exclusively in the nucleus. We conclude from these results that CRY2 is localized in the nucleus and that nuclear localization is mediated by the C-terminal region of CRY2.

Significance of serum antibodies reactive with flavoprotein subunit of succinate dehydrogenase in thyroid associated orbitopathy.

AIMS: Thyroid associated orbitopathy (TAO) is an autoimmune disorder of extraocular muscles and orbital connective tissue. Identification of the principal target antigens would help the understanding of the pathogenesis of the disease and possibly lead to the development of specific therapies in the future. The purpose of this study was to measure serum antibodies against the flavoprotein subunit of succinate dehydrogenase in patients with TAO and correlate their presence with factors of TAO. METHODS: Sera of patients with active TAO of 6 months' duration or less were tested for antibodies against the flavoprotein subunit of succinate dehydrogenase. Clinical data were obtained by retrospective review of patients' charts. Enzyme linked immunosorbent assay was used to test sera for serum antibodies against purified succinate dehydrogenase. RESULTS: 38 patients with TAO and 32 healthy age and sex matched controls were included in the study. Anti-flavoprotein antibodies were detected in 24 out of 38 patients with TAO (63.16%) and in five out of 32 healthy controls (15.63%) (p<0.01). Neither age, sex, duration of thyroid disease, thyroid status, treatment of thyroid disease, smoking history, duration of orbitopathy, activity of orbitopathy, nor the presence of lid retraction were significantly associated with the presence of serum anti-flavoprotein antibodies (p>0.05). However, the total number of rectus muscles affected in both eyes of the patients was significantly correlated with the finding of a positive antibody test (p<0.05). CONCLUSIONS: Serum antibodies reactive with the flavoprotein subunit of succinate dehydrogenase are associated with extraocular muscle involvement in active TAO of recent onset.

Serum antibodies against the flavoprotein subunit of succinate dehydrogenase are sensitive markers of eye muscle autoimmunity in patients with Graves' hyperthyroidism.

Thyroid-associated ophthalmopathy is an autoimmune disorder of the extraocular muscles and orbital connective tissue, which is usually associated with Graves' hyperthyroidism. Well-studied markers of ophthalmopathy are eye muscle membrane antigens, reportedly of approximately 64-kDa molecular mass. One, originally identified only as the 64-kDa protein, has recently been shown to be the flavoprotein (Fp) subunit of mitochondrial succinate dehydrogenase, which has a correct molecular mass of 67 kDa. We have used purified beef heart Fp as antigen in an enzyme-linked immunosorbent assay for cross-reactive human autoantibodies. Sera have been screened from patients with thyroid-associated ophthalmopathy classified according to activity and presence or not of eye muscle disease, and from those with Graves' hyperthyroidism without eye involvement. Also examined were serum samples taken periodically from 20 patients with Graves' hyperthyroidism during 24 months of treatment of their hyperthyroidism with antithyroid drugs. Four of these patients had ophthalmopathy at the onset, 12 developed ophthalmopathy, and 4 did not develop any eye signs during treatment. Anti-Fp subunit antibodies were detected in 73% of patients with active ophthalmopathy and evidence of eye muscle involvement but only in 25% if there was only congestive ophthalmopathy. These values were 0% and 11% for patients with chronic ophthalmopathy, with or without eye muscle dysfunction, respectively. The antibodies were also detected in 14% of patients with Graves' hyperthyroidism without evident ophthalmopathy, 11% of patients with nonimmunologic thyroid disorders, 12% of type I diabetics, and 12% of age- and sex-matched normal subjects. Significantly, appearance of anti-Fp antibodies predicted the development of ophthalmopathy in 5 of the 6 patients with Graves' hyperthyroidism, who developed eye muscle dysfunction after treatment of the hyperthyroidism, and coincided with the onset of eye muscle signs in the other patient. Antibodies were not detected in any of 6 patients who developed congestive ophthalmopathy without evidence of eye muscle damage or in 4 patients who did not develop any eye signs. In conclusion, we have shown a close relationship between eye muscle disease and serum antibodies against the Fp subunit of succinate dehydrogenase in patients with Graves' hyperthyroidism.

Anti-mitochondrial flavoprotein autoantibodies of patients with myocarditis and dilated cardiomyopathy (anti-M7): interaction with flavin-carrying proteins, effect of vitamin B2 and epitope mapping.

Vitamin B2 and flavin cofactors are transported tightly bound to immunoglobulin in human serum. We reasoned that anti-mitochondrial flavoprotein autoantibodies (alpha Fp-AB) present in the serum of patients with myocarditis and cardiomyopathy of unknown aetiology may form immunoglobulin aggregates with these serum proteins. However, immunodiffusion and Western blot assays demonstrated that the flavin-carrying proteins were not recognized by alpha Fp-AB. Apparently the flavin moiety in the native protein conformation was inaccessible to alpha Fp-AB. This conclusion was supported by the absence of an immunoreaction between the riboflavin-binding protein from egg white and alpha FP-AB. Intravenous application of vitamin B2 to rabbits immunized with 6-hydroxy-D-nicotine oxidase, a bacterial protein carrying covalently attached FAD, did not neutralize alpha Fp-AB which had been raised in the serum of the animals. FAD-carrying peptides generated from 6-hydroxy-D-nicotine oxidase by trypsin and chymotrypsin treatment were not recognized by the alpha Fp-AB, but those generated by endopeptidase Lys were. This demonstrates that the epitope recognized by alpha Fp-AB comprises, besides the flavin moiety, protein secondary structure elements.

Thyroid-associated ophthalmopathy in black South African patients with Graves' disease: relationship to antiflavoprotein antibodies.

Thyroid-associated ophthalmopathy (TAO) is a progressive eye disorder associated with Graves' hyperthyroidism, which is generally considered to have an autoimmune etiology. Eye muscle membrane proteins of 64 kd are good markers of ophthalmopathy in patients with thyroid autoimmunity. The 64-kd protein is now shown from a partial sequence to be the flavoprotein subunit (Fp) of mitochondrial succinate dehydrogenase. Hyperthyroidism due to Graves' disease is increasing in incidence among urban black female Africans, possibly because of exposure to environmental risk factors such as increased dietary iodine ingestion and stress. Ophthalmopathy is frequently observed in this clinical context, but its association with serum autoantibodies reactive with Fp has not been examined. We studied 19 black South African patients with Graves' disease during the course of prolonged antithyroid drug administration, of whom 10 had congestive ophthalmopathy, but no clinical evidence for eye muscle damage at the onset. Anti-Fp antibodies were detected in 2 of these patients, as well as in 2 of the 9 patients who did not have overt eye disease. Additionally, the antibodies became positive in 3 patients with ophthalmopathy in whom tests were negative initially, remained positive in 1 patient throughout the study period and became negative in 1 patient with positive tests initially. Ophthalmopathy did not develop in any of the 9 patients who lacked this complication on presentation. The reasons why we failed to demonstrate a close relationship between anti-Fp antibodies and the eye muscle component of ophthalmopathy are unclear although one possibility is that ocular myopathy is an uncommon manifestation in African thyrotoxic patients compared with those of Caucasian origin. The relationship between anti-Fp antibodies and eye muscle inflammation in patients with thyroid autoimmunity of different ethnic origins and environmental settings, needs to be addressed in a large prospective study.

Characterization of photolyase/blue-light receptor homologs in mouse and human cells.

We isolated and characterized mouse photolyase-like genes, mCRY1 (mPHLL1) and mCRY2 (mPHLL2), which belong to the photolyase family including plant blue-light receptors. The mCRY1 and mCRY2 genes are located on chromosome 10C and 2E, respectively, and are expressed in all mouse organs examined. We raised antibodies specific against each gene product using its C-terminal sequence, which differs completely between the genes. Immunofluorescent staining of cultured mouse cells revealed that mCRY1 is localized in mitochondria whereas mCRY2 was found mainly in the nucleus. The subcellular distribution of CRY proteins was confirmed by immunoblot analysis of fractionated mouse liver cell extracts. Using green fluorescent protein fused peptides we showed that the C-terminal region of the mouse CRY2 protein contains a unique nuclear localization signal, which is absent in the CRY1 protein. The N-terminal region of CRY1 was shown to contain the mitochondrial transport signal. Recombinant as well as native CRY1 proteins from mouse and human cells showed a tight binding activity to DNA Sepharose, while CRY2 protein did not bind to DNA Sepharose at all under the same condition as CRY1. The different cellular localization and DNA binding properties of the mammalian photolyase homologs suggest that despite the similarity in the sequence the two proteins have distinct function(s).