Heat-stress response of maize mitochondria.

We have identified maize (Zea mays L. inbred B73) mitochondrial homologs of the Escherichia coli molecular chaperones DnaK (HSP70) and GroEL (cpn60) using two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblots. During heat stress (42 degrees C for 4 h), levels of HSP70 and cpn60 proteins did not change significantly. In contrast, levels of two 22-kD proteins increased dramatically (HSP22). Monoclonal antibodies were developed to maize HSP70, cpn60, and HSP22. The monoclonal antibodies were characterized with regard to their cross-reactivity to chloroplastic, cytosolic, and mitochondrial fractions, and to different plant species. Expression of mitochondrial HSP22 was evaluated with regard to induction temperature, time required for induction, and time required for degradation upon relief of stress. Maximal HSP22 expression occurred in etiolated seedling mitochondria after 5 h of a +13 degrees C heat stress. Upon relief of heat stress, the HSP22 proteins disappeared with a half-life of about 4 h and were undetectable after 21 h of recovery. Under continuous heat-stress conditions, the level of HSP22 remained high. A cDNA for maize mitochondrial HSP22 was cloned and extended to full length with sequences from an expressed sequence tag database. Sequence analysis indicated that HSP22 is a member of the plant small heat-shock protein superfamily.

Sequential acquisition of mitochondrial and plasma membrane alterations during early lymphocyte apoptosis.

When cells undergo nuclear apoptosis (chromatin condensation, DNA fragmentation), they already manifest at least three alterations that can be quantified cytofluorometrically at the single-cell level: 1) a loss of mitochondrial transmembrane potential (delta psi m), 2) an increased production of superoxide anions, and 3) the aberrant exposure of phosphatidylserine (PS) residues on the outer plasma membrane leaflet. This latter alteration allows for the phagocytic recognition/elimination of apoptotic cells. In this work, we show that cells first undergo the delta psi m disruption and that PS exposure only affects cells that already have a low delta psi m. Pharmacologic modulation of apoptosis with inhibitors of macromolecule synthesis or proteases, as well as with drugs stabilizing the delta psi m, indicates that delta psi m disruption and PS exposure are coregulated. Interventions on apoptosis-regulatory genes (p53, bcl-2) confirm the coregulation of delta-psi-m disruption, PS exposure, and nuclear signs of apoptosis. In all conditions in which apoptosis is prevented, the delta psi m remains stable and PS cannot be detected on the cell surface. Reactive oxygen species do not contribute to PS exposure, based on two lines of evidence. First, among thymocytes undergoing apoptosis in response to dexamethasone, delta psi mlow cells first expose PS and then hyperproduce superoxide anion. Second, exogenous sources of reactive oxygen species or the superoxide anion-generating drug menadione fail to cause rapid PS exposure. Instead, direct interventions on mitochondria using inhibitors of the respiratory chain or the F1 ATP synthase cause PS exposure in cells subsequent to delta psi m disruption. This effect is also obtained in anucleate cells, indicating that the nucleus does not intervene in the sequence of events coupling mitochondrial dysfunction to PS exposure. Altogether, these data underline the functional impact of mitochondrial alterations on the apoptotic process.

[Detection of antibodies to submitochondrial particles, F1-ATPase complex and liver specific lipoprotein in liver diseases using ELISA]. / Detekce protilátek proti submitochondriálnym cásticím, F1-ATP-ázovému komplexu a jaternímu specifickému lipoproteínu u jaterních onemocnení pomocí ELISA.

ELISA was elaborated for the determination of antibodies against liver antigen complexes--submitochondrial particles, F1-ATPase and liver specific lipoprotein. The parameters achieved so far allow to use the assay as an undemanding complementary laboratory technique in diagnosing and monitoring hepatopathies of autoimmune origin. Cross reactivity between individual antigen complexes was recorded in the majority of sera from positively reacting patients. The preliminary results show that individual antigen complexes have similar antigen structures bound to high-molecular membrane complexes, some of which, however, can be solubilized on maintaining antigen activity in ELISA.

Topological and functional characterization of the F0I subunit of the membrane moiety of the mitochondrial H+-ATP synthase.

Using isolated polypeptides of the F0 sector of bovine heart mitochondrial H+-ATPase, antisera were developed detecting specifically two components of F0. These two components were identified as F0I and oligomycin-sensitivity-conferring protein (OSCP) respectively. Both F0I and OSCP were digested by mild trypsin treatment of submitochondrial particles depleted of the catalytic part of H+-ATPase (USMP). Proteolysis was largely prevented by binding of F1 to F0. Proteolysis of F0I resulted in the formation of three immunoreactive, membrane-bound fragments of apparently 26 kDa, 25.5 kDa and 18 kDa, respectively, indicating that F0I contains trypsin-accessible Arg or Lys residues located close to the end and the middle part of the protein, respectively, which are in intimate contact with F1. Digestion of USMP with trypsin resulted in depression of passive H+ conduction through F0 which could be ascribed to proteolysis of F0I.

Discrimination between M2 and M4 antimitochondrial antibodies in primary biliary cirrhosis.

10 sera were studied from patients with primary biliary cirrhosis (PBC), that were anomalous in their reactivity against mitochondrial antigens as detected by Western blotting. They had low reactivity against the major, M2 reactive antigen (Mr for beef heart mitochondria, 74 Kd) but reacted against an antigen of Mr 52 Kd (species independent) which was apparently inaccessible in submitochondrial particles (SMP) on ELISA and which was not present in chloroform-released ATPase preparations. In all respects this differed from the characteristics of the M2 antigens and it is concluded that these sera are detecting predominantly the M4-reactive antigen.

Mitochondrial antibodies in primary biliary cirrhosis: species and nonspecies specific determinants of M2 antigen.

Sera from patients with primary biliary cirrhosis reacted with four major bands in beef heart mitochondria and ATPase extract when analyzed by immunoblot after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These four immunologically reactive bands corresponded to protein bands with molecular weights of about (a) 80,000; (b) 63,000; (c) 56,000; and (d) 43,000 to 46,000. An additional immunoreactive band was found with some high-titered primary biliary cirrhosis sera at 36,000. No association with any ATPase subunits was found, except for band c which migrated between the alpha- and beta-subunit of ATPase. Most ATPase fractions did not contain this band c, indicating that M2 determinants, as defined by immunoblot, are not identical with any ATPase subunit. Species and nonspecies-specific determinants of M2 were identified using mitochondria from rat liver and human heart and liver. Antigenic bands a, c and d were nonspecies-specific. Band b and e occurred only in beef heart. An additional determinant at about 38,000 was detected using human heart and liver mitochondria. Primary biliary cirrhosis sera showed a typical reaction with two protein bands of Escherichia coli, one at about 85,000 to 90,000 and the other at 60,000. Antibodies against both determinants could be absorbed with submitochondrial particles of beef heart showing that E. coli shares cross-reacting determinants with mitochondria. Sera from 56 primary biliary cirrhosis patients were tested using beef heart mitochondria.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of two monoclonal antibodies obtained after immunization with human liver mitochondrial membrane preparations.

Two monoclonal antibodies have been generated by fusion of mouse myeloma cells with spleen cells from mice immunized with human liver mitochondrial membranes. One antibody, 1H6/C12, an immunoglobulin G2a (IgG2a), binds to the inner membrane of rat hepatocyte mitochondria, and immunoperoxidase staining demonstrates that its epitope has an intracellular particulate distribution within rat and human hepatocytes and human brain neurons. The epitope reactive with 1H6/C12 is partially sensitive to proteinase digestion. The second antibody, 3F12/F2, an IgG1, binds to a contaminating cell type, namely the granulocyte, but it does not bind to monocytes, lymphocytes and red cells in human blood. This antibody reacts with cells in the portal tract and sinusoids of rat and human liver, as shown by immunoperoxidase staining. The epitope for 3F12/F2 is extremely sensitive to proteinase digestion and is only exposed when granulocytes are fixed in acetone, indicating an internal localization.

Use of ATPase-associated antigen (M2) for detection of antimitochondrial antibodies in primary biliary cirrhosis by fluorometric immunoassay.

An indirect binding assay, the fluorometric immunoassay (FIAX), was established for the detection of anti-M2 antibodies which are specific markers for primary biliary cirrhosis (PBC). Submitochondrial particles (SMP) from beef heart and rat liver and the ATPase-associated antigen (M2) were used. The antigens were fixed to a cellulose acetate surface, SMP at a concentration of 2 mg/ml, ATPase at a concentration of 0.2 mg/ml. Sera were used at 1:60 and 1:120 and bound antimitochondrial antibodies (AMA) were demonstrated by fluorescent isothiocyanate labelled monospecific anti-human IgG, IgM and IgA antibodies. The fluorescent signals were proportional to the AMA titre in the serum samples and were measured in a fluorometer (FIAX 100). Of 94 patients with PBC, 92 had AMA against SMP from beef heart compared with 76 in the complement fixation test (CFT) and 84 in the immunofluorescence test (IFL). Ninety reacted with the ATPase-associated M2 antigen. Sera from patients known to have AMA of different specificities (anti-M1, anti-M3, anti-M5, anti-M6) reacted with SMP from beef heart and/or rat liver but not with M2.

Antimitochondrial antibodies (AMA) in primary biliary cirrhosis. I. Separation of the PBC antigen activity from mitochondrial ATPase activity.

Antimitochondrial antibodies are found in a variety of autoimmune liver diseases, particularly primary biliary cirrhosis. The antigen against which these antibodies are directed is localized on the inner mitochondrial membrane. Earlier work suggested that this antigen was associated with the mitochondrial ATPase. However, we have succeeded in separating the enzyme activity from the antigenic activity using gel filtration and ion-exchange chromatography. Furthermore, the antigenic activity is not affected by modulators of ATPase enzymatic activity like aurovertin or oligomycin. The antigenic activity is, however, very susceptible to reagents which block thiol groups. The mitochondrial antigen, in contrast to the ATPase enzyme, is found in high amounts in brown fat mitochondria. Identification of this antigen may help to explain why specific antimitochondrial antibodies arise in the sera of patients with primary biliary cirrhosis.

Mitochondrial antibodies in chronic liver diseases and connective tissue disorders: further characterization of the autoantigens.

The heterogeneity of mitochrondrial autoantibodies in a variety of diseases states has been critically re-examined by a combination of immunofluorescence staining (IFL) and complement fixation tests (CFT). The different mitochondrial IFL patterns described by other workers were confirmed and extra criteria using new substrates are presented for their differential recognition. Biochemically defined mitochondrial subfractions were used in the CFT to confirm and extend the IFL classifications. The 'M1' cardiolipin antibodies of syphilis did not react with the ATPase fraction but the antigen was present in all membrane preparations and found to be chemically resistant. The major antibody specificity of the 'M3' pattern associated with drug-induced pseudolupus syndrome is a firmly bound, outer membrane component; and a second, minor reactivity is apparently to a mercurial-insensitive antigen present in the chloroform-released ATPase preparation. The 'M5' antibody pattern correlates with a digitonin-sensitive outer membrane component. Although it was not possible to differentiate within the group of liver diseases between the 'M2' antibodies of primary biliary cirrhosis and the previously described 'M4' antibodies of other chronic liver diseases, several antibody specificities were demonstrated. All sera from liver disease patients contain the antibody directed against a mercurial-sensitive protein found in the chloroform-released ATPase preparation, and, in addition, varying titres of antibodies against two or more mercurial-resistant membrane components, of which at least one is on the inner membrane and one on the outer membrane.