Tropomyosin in preimplantation mouse development: identification, expression, and organization during cell division and polarization

Tropomyosin is an actin-binding cytoskeletal protein which has been extensively characterized in a variety of cell types and tissues, with the exception of very early developmental stages during which cellular polarization first occurs. We have identified five polypeptides in mouse preimplantation conceptuses which show many of the characteristics of tropomyosin. They form the major portion of the heat-stable cytoskeletal protein fraction of blastomeres and have the characteristic isoelectric and SDS-PAGE migration characteristics on 1-D and 2-D gels. All five polypeptides were synthesized in late 2- and 4-cell, and all 8-cell stages, with three of the five polypeptides showing lower synthetic levels in fertilized eggs and early 2-cell conceptuses. These heat-stable proteins showed specific differences from proteins isolated from mouse 3T3 fibroblasts by the same method, namely higher Mr isoforms were not represented, also some of the isoforms can be labeled by incorporation of [14C]proline. The cellular distribution of tropomyosin in early stage conceptuses was examined using monoclonal and affinity-purified polyclonal antibodies. Tropomyosin becomes associated both with the blastomere cortex postfertilization and with the cleavage furrow during cytokinesis. The interphase cortical association is uniform until the 8-cell stage, when tropomyosin becomes associated with the developing apical pole and is excluded from the basolateral cortex. This polar localization is inherited along with the pole at the 8- to 16-cell division, but experiments in which cell division is artificially prolonged show that tropomyosin localization does not represent a permanent marking of the pole. We conclude that the early mouse conceptus contains a unique and specific set of tropomyosins which respond to polarizing signals.     

Tight junction proteins form large complexes and associate with the cytoskeleton in an ATP depletion model for reversible junction assembly

A key feature of the ischemic epithelial cell phenotype is the disruption of tight junctions (TJ). In a Manin-Darby canine kidney cell model for ischemia-reperfusion/hypoxia-reoxygenation injury which employs inhibitors of glycolysis (2-deoxy-D-glucose) and oxidative phosphorylation (antimycin A), transepithelial electrical resistance, a measure of TJ integrity, dropped rapidly, correlating well with declining ATP levels. Although immunocytochemical studies revealed only subtle changes in the distribution of the TJ proteins, zonula occludens (ZO)-1, ZO-2, and cingulin, examination of the Triton X-100 solubilities of these proteins, an indicator of cytoskeletal association, revealed a striking shift of all three TJ proteins into the insoluble pool, consistent with increased cytoskeletal interaction during ATP depletion. In addition, rate-zonal centrifugation analysis of a detergent-soluble fraction showed an increase in the amount of ZO-1 and ZO-2 in high density fractions following ATP depletion, providing further evidence for association of TJ proteins into a large complex possibly involving the cytoskeleton. Analysis of immunoprecipitation data from [35S]methionine-labeled cells revealed that ATP depletion led to the association of a 240-kDa protein with the ZO-1-containing complex. Western blots of this protein immunoprecipitated with anti-ZO-1 antibodies confirmed its identity as fodrin, a protein believed to link membrane and other proteins to the actin-based cytoskeleton. Together, our data suggest that in the absence of major immunocytochemical changes, ATP depletion leads TJ proteins to form large insoluble complexes and associate with the cytoskeleton. We propose a model in which a key, potentially regulated, step in the generation of the ischemic epithelial cell phenotype is the interaction between TJ proteins and fodrin and/or other cytoskeletal proteins.     

Generation of monoclonal antibodies against human prion proteins in PrP0/0 mice

BACKGROUND: Prion diseases belong to a group of neurodegenerative disorders affecting humans and animals. The human diseases include kuru, Creutzfeldt-Jakob disease (CJD), Gerstmann-Str?ussler-Scheinker syndrome (GSS), and fatal familial insomnia (FFI). The pathogenic mechanisms of the prion diseases are not yet understood. Monoclonal antibodies provide valuable tools in the diagnosis, as well as in the basic research, of several diseases; however, monospecific antisera or monoclonal antibodies (mAbs) against human prion proteins were, until now, not available. MATERIALS AND METHODS: We have developed an immunization protocol based on nucleic acid injection into nontolerant PrP0/0 mice. DNA or RNA coding for different human prion proteins including the mutated sequences associated with CJD, GSS, and FFI were injected into muscle tissue. Mice were primarily inoculated with DNA plasmids encoding the prion protein (PRNP) gene and boosted either with DNA, RNA, or recombinant Semliki Forest Virus particles expressing PRNP. Hybridomas were then prepared. RESULTS: Different mAbs against human prion proteins were obtained, and their binding behavior was analyzed by peptide enzyme-linked immunosorbent assay, Western blot, immunofluorescence, and immunoprecipitation. Their cross-reactivity with prion protein from other species was also determined. Our mAbs are directed against four different linear epitopes and may also recognize discontinuous regions of the native prion protein. CONCLUSIONS: These antibodies should allow us to address questions concerning the nature of the prion protein as well as the initiation and progression of prion diseases. Moreover, these mAbs can now be used for the diagnosis of prion diseases of humans and animals.     

Lack of Drosophila cytoskeletal tropomyosin affects head morphogenesis and the accumulation of oskar mRNA required for germ cell formation

Drosophila encodes five muscle and one cytoskeletal isoform of the actin-binding protein tropomyosin. We have identified a lack-of-function mutation in the cytoskeletal isoform (cTmII). Zygotic mutant embryos show a defect in head morphogenesis, while embryos lacking maternal cTmII are defective in germ cell formation but otherwise give rise to viable adults. oskar mRNA, which is required for both germ cell formation and abdominal segmentation, fails to accumulate at the posterior pole in these embryos. nanos mRNA, however, which is required exclusively for abdominal segmentation, is localized at wild-type levels. These results indicate that head morphogenesis and the accumulation of high levels of oskar mRNA necessary for germ cell formation require tropomyosin-dependent cytoskeleton.     

Cytoskeleton and tissue origin in the anterior cynomolgus monkey eye

We studied cytoskeletal proteins and other markers for embryologic origin in the outflow pathways of the aqueous humor, cornea, sclera, and ciliary muscle of the cynomolgus monkey. The corneal endothelium and trabecular cells stained with markers for vimentin, smooth muscle cell alpha-actin, F-actin, spectrin, vinculin, and talin. The endothelium of Schlemm's canal stained with markers for vimentin, spectrin, and F-actin. These results suggest that trabecular cells are a kind of myofibroblast and support the belief that the endothelial cells of Schlemm's canal are vascular in origin. Fibrillary staining with antibodies to vimentin, spectrin, neurofilament protein, and glial acid fibrillary protein was observed along and between the ciliary muscle cells. Cells in the deep sclera adjacent to the supraciliary space stained with antibodies to smooth muscle alpha-actin, alpha-vinculin, talin, and desmin. These cells may anchor ciliary muscle cells into the sclera or may be developmental remnants of ciliary muscle cells. Leu 19 immunoreactivity was found in the corneal endothelium, in all trabecular cells, in ciliary muscle cells, and in keratocytes and fibroblasts in the superficial part of the cornea and sclera. All of these cells are therefore likely to express neural cell adhesion molecules indicating neuroectodermal origin.     

An improved method to create nitrocellulose particles suitable for the immobilization of antigen and antibody

A method is described for producing 1-3 microns sized particles of nitrocellulose (NC) which are able to absorb protein. Protein is absorbed onto preformed particles made by first dissolving a sheet of nitrocellulose paper in DMSO, and then precipitating it with sodium carbonate/bicarbonate buffer. The efficiency of binding is the same as that of an equivalent sheet of non-processed NC filter paper. Antibodies absorbed onto preformed particles are not exposed to DMSO and carbonate buffer and therefore retain a high antigen binding capacity. Antigen and antibody-absorbed NC preformed particles were used to capture antibody and antigen, respectively. Using lysis buffer, the captured antibodies and antigens were readily released from the NC particles. This makes the latter an appropriate matrix for immunoprecipitation assays either for an antigen or for specific antibody. Antigen-coated NC particles were specifically aggregated ('agglutinated') by specific antibodies and thus can be used in semi-quantitative tests.     

Immuno-isolation of highly purified peroxisomes using magnetic beads and continuous immunomagnetic sorting

Immuno-isolation is a powerful technique for the isolation of cells as well as subcellular organelle populations based on their antigenic properties. We have established a method for immuno-isolation of peroxisomes (PO) from both rat liver and the human hepatoblastoma cell line HepG2 using magnetic beads as solid support. A polyclonal antibody raised against the cytoplasmic C-terminal 10 amino acids of the rat 70 kDa peroxisomal membrane protein was covalently bound to magnetic beads (Dynabeads M-450). The coated beads were incubated with a light mitochondrial fraction and the organelle-bead complexes formed were separated by magnetic sorting in a free-flow system without pelleting the complexes during the isolation procedure. Scanning electron microscopy revealed decoration of beads with particles measuring 150-400 nm in diameter. The particles were identified as PO by catalase cytochemistry and biochemically by marker enzyme analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as well as immunoblotting for specific detection of peroxisomal matrix, core and membrane proteins. The functional significance of PO in man is emphasized by the existence of inherited diseases such as the Zellweger syndrome in which intact PO are lacking, but peroxisomal remnants called "ghosts" are observed instead. Peroxisomal disorders are usually studied using skin fibroblast cell lines derived from afflicted patients and immuno-magnetic separation may prove particularly useful for the investigation of such cultured cells and for further elucidation of the pathogenesis of fatal peroxisomal disorders.     

Neuronal nicotinic ACh receptor antibody in subacute autonomic neuropathy and cancer-related syndromes

BACKGROUND: Autoantibodies specific for the acetylcholine receptor (AChR) of skeletal muscle (containing the alpha1 subunit) impair neuromuscular transmission in myasthenia gravis (MG). AChRs mediating fast synaptic transmission through autonomic ganglia are structurally similar to muscle AChR, but contain the alpha3 subunit. We propose that ganglionic AChR autoimmunity may cause dysautonomia. OBJECTIVE: To test serum of patients with autonomic neuropathy for autoantibodies of neuronal ganglionic AChR specificity. METHODS: We developed an immunoprecipitation radioassay by complexing epibatidine (125I-labeled high affinity agonist) to a Triton X-100-solubilized AChR antigen from peripheral neuroblastoma membranes. Monoclonal rat immunoglobulins (IgG) specific for muscle or neuronal AChRs validated the assay's specificity. We tested serum from 52 healthy subjects, 12 patients with subacute autonomic neuropathy, and 248 patients with other neurologic disorders. RESULTS: Twelve patients had antibodies that bound unequivocally to ganglionic AChR. Five had subacute autonomic neuropathy, and three (of six tested) had Isaacs' syndrome; four of these eight had a carcinoma (lung, bladder, rectum, thyroid). The remaining four seropositive patients (two Lambert-Eaton syndrome, one dementia, one sensory neuronopathy) all had Ca2+ channel antibodies and three had small cell lung carcinoma. No healthy subject had ganglionic AChR antibodies, nor did 62 patients with MG and muscle AChR antibodies. CONCLUSION: Neuronal AChR antibodies are a novel serologic marker of neurologic autoimmunity. The pathogenicity of neuronal AChR autoantibodies in autonomic neuropathy, Isaacs' syndrome, or other neurologic disorders remains to be shown, as has been demonstrated for muscle AChR antibodies in MG. An autoimmune and potentially paraneoplastic etiology is implicated in seropositive patients.     

Colony-stimulating factor-1 stimulates the formation of multimeric cytosolic complexes of signaling proteins and cytoskeletal components in macrophages

Stimulation of macrophages with colony-stimulating factor-1 (CSF-1) results in the protein tyrosine phosphorylation of the CSF-1 receptor (CSF-1R) and many other, primarily cytosolic, proteins. Stimulation by CSF-1 at 4 degreesC was used to facilitate the purification and identification of the proteins of the cytosolic anti-phosphotyrosine (PY)-reactive fraction (alphaPY-RF) involved in downstream signaling pathways. Confocal microscopy revealed that the PY proteins are in close proximity to the CSF-1R at the plasma membrane. The alphaPY-RF contained pre-existing complexes of PY proteins and non-PY proteins which generally increased in size and PY protein content following CSF-1 stimulation. PY proteins identified by microsequencing and Western blotting include Cbl, STAT3, STAT5a, STAT5b, SHP-1, Shc, and two novel proteins pp57 and pp37. Other proteins included cytoskeletal/contractile proteins (paxillin, vimentin, elongation factor-1alpha, F-actin, tropomyosin, and myosin regulatory light chain), Ras family signaling proteins (p85 (phosphoinositide 3-kinase), Vav, Ras-GTPase-activating protein SH3 domain-binding protein, and Grb2), DnaJ-like protein, and glyceraldehyde-3-phosphate dehydrogenase. CSF-1 induced the de novo recruitment of Cbl, STAT3, STAT5a, STAT5b, p85, SHP-1, Shc, vimentin, and Grb2 to complexes and caused pre-existing complexes involving Vav, elongation factor-1alpha, and F-actin to increase in size. These studies indicate that CSF-1-induced protein tyrosine phosphorylation is associated with the reorganization of complexes of cytoskeletal, signaling, and other proteins that mediate CSF-1-regulated motility and growth.     

Cr-Polluted Soil Studied by High Gradient Magnetic Separation and Electron Probe

An Fe-rich soil from the site of a former leather tannery, heavily polluted with Cr, was studied using a combination of wet chemical analysis, high gradient magnetic separation (HGMS), and electron probe microanalysis (EPMA). It is demonstrated that such a combination is a powerful tool for the characterization of polluted soils, especially in cases where the pollution is present as discrete particles. Both EPMA and magnetic separation data indicated that the Cr pollution was present as a hydrous Cr-oxide phase. The Cr does not correlate with the Fe minerals, most likely as a result of the initial high Cr concentrations in the soil, which lead to precipitation of separate hydrous Cr-oxide minerals and Fe minerals. The Cr-containing material is present as (layered) aggregates, which are formed around larger quartz grains or around very small other particles that served as precipitation nuclei. Magnetic separation tests show that the Cr pollution can largely be removed by HGMS.     

Influence of antigen organization on the development of lupus autoantibodies

OBJECTIVE: To investigate the reason for grouping of antibodies against small nuclear RNP (snRNP) particles, which are major autoantigens in systemic lupus erythematosus (SLE). METHODS: Mice were immunized with biochemically purified native snRNP particles or recombinant proteins, followed by assessment of antibody and T cell responses. Since mouse (self) snRNPs are not immunogenic in mice, a eukaryotic expression vector was constructed to induce high-level expression of the human U1 snRNP-associated A protein in murine cells. Native chimeric (mouse/human) snRNP particles were used to immunize normal mice of both H-2k and H-2b backgrounds. We also disrupted the native snRNPs by digestion with ribonuclease and used this mixture of proteins to immunize mice. RESULTS: Immunization with native chimeric snRNPs resulted in the development of antibodies against a set of snRNP-associated proteins, a response which was accompanied by breakdown in T cell tolerance to mouse snRNPs in mice immunized with chimeric snRNPs. We also demonstrated that the ordered production of these antibodies was due to the fact that snRNP-associated proteins are grouped together in snRNP particles, since disruption of the particles resulted in development of antibodies in a random order, distinct from antibodies seen with intact particles. CONCLUSION: Our findings directly demonstrate that the pattern of development of antibodies to native snRNPs is similar to that which is commonly observed in SLE, and that disruption of the particles results in disappearance of this ordered pattern. These results suggest that the autoimmune response to snRNPs, and possibly to other autoantigens, in lupus is a specific reaction similar to that seen in a typical immune response to foreign immunogens.     

Paxillin phosphorylation and association with Lck and Pyk2 in anti-CD3- or anti-CD45-stimulated T cells

Antibodies to either CD3 or CD45 have been shown to induce dramatic changes in cell morphology, increased tyrosine phosphorylation of cellular proteins, and the association of a subset of these proteins with the tyrosine kinase Lck. The current study was initiated to determine the identity of the tyrosine-phosphorylated 70-80 kDa protein that becomes Lck-associated after stimulation with anti-CD45 or anti-CD3. We demonstrate that the cytoskeletal protein paxillin becomes tyrosine-phosphorylated when cells are plated on immobilized antibodies specific for CD45 or CD3. Only tyrosine-phosphorylated paxillin is associated with Lck, suggesting that the association is through the SH2 domain of Lck. Consistent with this we demonstrate that the SH2 domain of Lck binds tyrosine-phosphorylated paxillin. In contrast, the association of paxillin with the FAK-related kinase Pyk2 was found to be constitutive and not altered by the phosphorylation of either protein. Finally, we establish that the phosphorylation of paxillin is dependent on the expression of Lck. Taken together, these results demonstrate that paxillin is physically associated with kinases from two different families in T cells and suggest that paxillin may function as an adaptor protein linking cellular signals with cytoskeletal changes during T cell activation.     

Generation and characterization of an IGFBP-7 antibody: identification of 31kD IGFBP-7 in human biological fluids and Hs578T human breast cancer conditioned media

The cDNA encoding mac25 (IGFBP-7) was firsr derived from mRNA isolated from leptomeningial and senescent human mammary epithelial cells (1,2). The open reading frame was shown to predict a protein with homology to the amino terminus of the IGF binding proteins, (IGFBP)1-6. Studies in our laboratory have shown that baculovirus generated mac25 binds IGF-I and-II in a specific manner, leading to the renaming of mac25 as IGFBP-7 (3). Further studies at the cellular level, to identify the involvement of IGFBP-7 in IGF regulation and cell growth, require a specific antibody against the protein, which has yet to be identified in either cultured cells or in vivo. We have now generated three polyclonal antibodies against the purified baculovirus peptide and, by western immunoblots and immunoprecipitation, demonstrated the existence of a specific 31,000 dalton protein. It is a secreted protein, and can be identified in the conditioned media of Hs578T breast cancer cells, as well as in normal human urine, cerebrospinal fluid and amniotic fluid. Subsequent studies with these antibodies should help elucidate the physiological role(s) of this protein.     

The active state of the thin filament is destabilized by an internal deletion in tropomyosin

The function of three of tropomyosin's sequential quasiequivalent regions was studied by deletion from skeletal muscle alpha-tropomyosin of internal residues 49-167. This deletion mutant tropomyosin spans four instead of the normal seven actins, and most of the tropomyosin region believed to interact with troponin is retained and uninterrupted in the mutant. The mutant tropomyosin was compared with a full-length control molecule that was modified to functionally resemble muscle tropomyosin (Monteiro, P. B., Lataro, R. C., Ferro, J. A., and Reinach, F. C. (1994) J. Biol. Chem. 269, 10461-10466). The tropomyosin deletion suppressed the actin-myosin subfragment 1 MgATPase rate and the in vitro sliding of thin filaments over a heavy meromyosin-coated surface. This inhibition was not reversed by troponin plus Ca2+. Comparable tropomyosin affinities for actin, regardless of the deletion, suggest that the deleted region has little interaction with actin in the absence of other proteins. Similarly, the deletion did not weaken binding of the troponin-tropomyosin complex to actin. Furthermore, Ca2+ had a 2-fold effect on troponin-tropomyosin's affinity for actin, regardless of the deletion. Notably, the deletion greatly weakened tropomyosin binding to myosin subfragment 1-decorated actin, with the full-length tropomyosin having a 100-fold greater affinity. The inhibitory properties resulting from the deletion are attributed to defective stabilization of the myosin-induced active state of the thin filament.     

Clinical significance of antiheart antibodies after myocardial infarction

We used one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis of myocardial proteins followed by Western blotting to study the formation of antiheart antibodies during three months after myocardial infarction and the relationship between the appearance of antibodies and clinical and laboratory findings. Fifty-four percent of the 66 patients with infarction had different types of antiheart antibodies. The autoantibodies detected most frequently were against 35 and 42 kDa cardiac proteins. Immunoblottings with purified proteins showed that these autoantibodies reacted against myocardial tropomyosin and actin, which have been detected after acute myocardial infarction and can have immunogenetic activity through a humoral immune response. However, only the presence of autoantibody against myocardial tropomyosin correlated significantly with the presence of clinical and laboratory findings. Our results suggest that autoantibody against myocardial tropomyosin may play an immunopathogenic role in the development of symptoms in these patients.     

Defective expression of plectin/HD1 in epidermolysis bullosa simplex with muscular dystrophy

Epidermolysis bullosa simplex with muscular dystrophy (MD-EBS) is a disease characterized by generalized blistering of the skin associated with muscular involvement. We report that the skin of three MD-EBS patients is not reactive with antibodies 6C6, 10F6, or 5B3 raised against the intermediate filament-associated protein plectin. Immunofluorescence and Western analysis of explanted MD-EBS keratinocytes confirmed a deficient expression of plectin, which, in involved skin, correlated with an impaired interaction of the keratin cytoskeleton with the hemidesmosomes. Consistent with lack of reactivity of MD-EBS skin to plectin antibodies, plectin was not detected in skeletal muscles of these patients. Impaired expression of plectin in muscle correlated with an altered labeling pattern of the muscle intermediate filament protein desmin. A deficient immunoreactivity was also observed with the monoclonal antibody HD121 raised against the hemidesmosomal protein HD1. Furthermore, immunofluorescence analysis showed that HD1 is expressed in Z-lines in normal skeletal muscle; whereas this expression is deficient in patient muscle. Colocalization of HD1 and plectin in normal skin and muscle, together with their impaired expression in MD-EBS tissues, strongly suggests that plectin and HD1 are closely related proteins. Our results therefore provide strong evidence that, in MD-EBS patients, the defective expression of plectin results in an aberrant anchorage of cytoskeletal structures in keratinocytes and muscular fibers leading to cell fragility.     

Tropomyosin implicated in host protective responses to microfilariae in onchocerciasis

A cDNA from adult female Onchocerca volvulus encoding the C-terminal portion of a tropomyosin isoform (termed MOv-14) has been shown previously to confer protective immunity in rodent models of onchocerciasis. The full-length sequence (designated Ov-tmy-1) obtained by PCR amplification, codes for a protein of 33 kDa and shares 91% identity with tropomyosins from other nematodes, falling to 57% identity with human alpha-tropomyosin. Ov-TMY-1 migrates with an apparent molecular mass of 42 kDa on SDS/PAGE and is present in all life-cycle stages, as determined by immunoblotting. Immunogold electron microscopy identified antigenic sites within muscle blocks and the cuticle of microfilariae and infective larvae. Anti-MOv14 antibodies were abundant in mice exhibiting serum-transferable protection against microfilariae conferred by vaccination with a PBS-soluble parasite extract. In contrast, little or no MOv14-specific antibody was present in mice inoculated with live microfilariae, in which resistance is mediated by antibody-independent mechanisms. In human infections, there was an inverse correlation between anti-tropomyosin IgG levels and densities of microfilariae in the skin. Seropositivity varied with the relative endemicity of infection. An immunodominant B cell epitope within Ov-TMY-1 (AQLLAEEADRKYD) was mapped to the N terminus of the MOv14 protein by using sera from protectively vaccinated mice. Intriguingly, the sequence coincides with an IgE-binding epitope within shrimp tropomyosin, believed to be responsible for hypersensitivity in individuals exhibiting allergy to shellfish. IgG and IgE antibodies reacting with the O. volvulus epitope were detected in human infections. It is concluded that antibody responses to tropomyosin may be important in limiting microfilarial densities in a proportion of individuals with onchocerciasis and have the potential to mediate hypersensitivity reactions to dead microfilariae, raising the possibility of a link with the immunopathology of infection.     

大冶铁矿强磁选精矿磁化焙烧热力学研究

磁化焙烧-磁选分离的技术路线,是解决菱铁矿含量较高的大冶铁矿尾矿中低品位难选红铁矿(w(TFe)=34%左右)的有效工艺.其焙烧工艺参数对工艺效率影响较大.对其热力学反应条件进行了分析,依靠碳酸铁的自身分解产物CO和还原气氛,大冶低品位含菱铁矿弱磁性铁矿能在弱还原气氛条件下,在10～0 s内完成整个磁化焙烧过程.磁化焙烧后,弱磁选精矿铁品位大大提高(w(TFe)》60%).     

Cytochrome P4502E1 hydroxyethyl radical adducts as the major antigen in autoantibody formation among alcoholics

BACKGROUND & AIMS: We have previously reported that alcoholics have increased titers of immunoglobulins reacting with protein adducts of hydroxyethyl free radicals. Because hydroxyethyl radicals are produced during ethanol metabolism by liver microsomes, the aim of this study was to determine whether such antibodies recognize microsomal proteins complexed with hydroxyethyl radicals. METHODS: Liver microsomal proteins reacting with the anti-hydroxyethyl radical antibodies were characterized by an enzyme-linked immunosorbent assay and Western blotting. RESULTS: Alcoholic cirrhotics, but not patients with nonalcoholic cirrhosis or healthy subjects, had increased serum levels of immunoglobulin G and A directed against antigens produced in microsomes incubated with reduced nicotinamide adenine dinucleotide phosphate (NADPH) and ethanol. Such immunoreactivity was completely blocked when microsomes were incubated with ethanol in the presence of the spin-trapping agent 4-pyridyl-1-oxide-t-butyl nitrone or by preincubating the sera with hydroxyethyl radical-bound human albumin. Immunoblotting of proteins from human liver microsomes incubated with NADPH and ethanol showed that 86% of the sera from alcoholic cirrhotics reacted with a 52-kilodalton protein, whereas variable reactivity was observed with proteins of 78, 60, and 40 kilodaltons, respectively, The 52-kilodalton protein was identified by immunoblotting and immunoprecipitation as ethanol-inducible cytochrome P4502E1. CONCLUSIONS: Antibodies from alcoholic cirrhotics specifically recognized hydroxyethyl radical-cytochrome P4502E1 adducts, suggesting the possible implication of these antigens in the development of autoimmune reactions in alcoholic liver disease.