Effectors of the mammalian plasma membrane NADH-oxidoreductase system. Short-chain ubiquinone analogues as potent stimulators.

In the presence of effectors variations in the two recognized activities of the plasma membrane NADH-oxidoreductase system were studied in separate, specific in vitro assays. We report here that ubiquinone analogues that contain a short, less hydrophobic side chain than coenzyme Q-10 dramatically stimulate the NADH-oxidase activity of isolated rat liver plasma membranes whereas they show no effect on the reductase activity of isolated membranes. If measured in assays of the NADH:ferricyanide reductase of living cultured cells these compounds have only a limited effect; the oxidase activity of whole cells is not measurable in our hands. We have furthermore identified selective inhibitors of both enzyme activities. In particular, the NADH-oxidase activity can be significantly inhibited by structural analogues of ubiquinone, such as capsaicin and resiniferatoxin. The NADH:ferricyanide reductase, on the other hand, is particularly sensitive to pCMBS, indicating the presence of a sulfhydryl group of groups at its active site. The identification of these specific effectors of the different enzyme activities of the PMOR yields further insights into the function of this system.

The specificity of mitochondrial complex I for ubiquinones.

We report the first detailed study on the ubiquinone (coenzyme Q; abbreviated to Q) analogue specificity of mitochondrial complex I, NADH:Q reductase, in intact submitochondrial particles. The enzymic function of complex I has been investigated using a series of analogues of Q as electron acceptor substrates for both electron transport activity and the associated generation of membrane potential. Q analogues with a saturated substituent of one to three carbons at position 6 of the 2,3-dimethoxy-5-methyl-1,4-benzoquinone ring have the fastest rates of electron transport activity, and analogues with a substituent of seven to nine carbon atoms have the highest values of association constant derived from NADH:Q reductase activity. The rate of NADH:Q reductase activity is potently but incompletely inhibited by rotenone, and the residual rotenone-insensitive rate is stimulated by Q analogues in different ways depending on the hydrophobicity of their substituent. Membrane potential measurements have been undertaken to evaluate the energetic efficiency of complex I with various Q analogues. Only hydrophobic analogues such as nonyl-Q or undecyl-Q show an efficiency of membrane potential generation equivalent to that of endogenous Q. The less hydrophobic analogues as well as the isoprenoid analogue Q-2 are more efficient as substrates for the redox activity of complex I than for membrane potential generation. Thus the hydrophilic Q analogues act also as electron sinks and interact incompletely with the physiological Q site in complex I that pumps protons and generates membrane potential.

Immunolocalisation of interferon-alpha in hepatitis C patients and its correlation with response to interferon-alpha therapy.

Localised interferon-alpha production was investigated in hepatitis C patients entered into a trial of interferon-alpha-2a therapy. Antibodies capable of reacting specifically with interferon-alpha-2, interferon-alpha-4 or with all interferon-alpha subtypes were used as immunohistochemical and immunofluorescence probes to study interferon-alpha production in liver biopsy tissue, and peripheral blood mononuclear cells prior to and after stimulation with Sendai virus. Measurement of cytoplasmic interferon-alpha, specifically interferon-alpha-2 and interferon-alpha-4, in peripheral blood mononuclear cells isolated from the hepatitis C patients and of total interferon-alpha secreted into culture supernatants by these cells showed interferon-alpha production similar to that of peripheral blood mononuclear cells isolated from normal individuals. Interferon-alpha-positive cells were observed in the infiltrating mononuclear cells of the liver biopsy tissue obtained from 8 of the 14 patients. Lymphocytes, fibroblasts, Kupffer cells, polymorphonuclear cells and monocytes stained positive for interferon-alpha, and specifically interferon-alpha-4, in all of the eight patients. The cytoplasm of hepatocytes also stained weakly positive in three of these patients. Interferon-alpha positive cells showed a good correlation with the degree of histological damage observed in the liver biopsies but not with presence of antibodies towards hepatitis C virus or levels of serum alanine aminotransferase measured prior to interferon-alpha-2a therapy. Interestingly, response to therapy seemed linked to local interferon-alpha production status. Those patients who responded best to therapy displayed no or only low levels of interferon-alpha positive cells in liver biopsy tissue. Thus patients with a lower activation of their endogenous interferon-alpha system may benefit from administration of exogenous interferon-alpha.

Detection of cells producing murine interferon-alpha using antipeptide antibodies.

The purpose of this study was to produce antibodies which could be used to investigate the expression of murine (Mu)IFN-alpha. Rabbits were immunized with a peptide, corresponding to the 15 COOH-terminal amino acids of MuIFN-alpha-1, conjugated to keyhole limpet haemocyanin (KLH), and the resulting antipeptide antibodies were identified by indirect ELISA. Antipeptide antibodies were purified from rabbit immune sera by immunoadsorption to peptide immobilized on nitrocellulose and any remaining antibodies to KLH removed by immunoadsorption to KLH-Sepharose. The characterization of the antipeptide antibodies by ELISA, immunoprecipitation, affinity chromatography and immunofluorescence demonstrated that the antibodies recognize the peptide immunogen and the native IFN-alpha molecule. Using these antibodies for immunofluorescence staining and flow cytometric analyses of stained cells, we have shown that unstimulated murine spleen cells produce IFN-alpha. This finding is in agreement with the recent demonstration of constitutive IFN-alpha production by unstimulated human leucocytes and has important implications for the functions of interferons. The production, characterization and use of antipeptide antibodies as described herein may also have broader application for studies of the expression of other cytokines.

Differing epitope selection of experimentally-induced and natural antibodies to a disease-specific autoantigen, the E2 subunit of pyruvate dehydrogenase complex (PDC-E2).

Naturally-occurring autoantibodies to a family of mitochondrial enzymes, the 2-oxoacid dehydrogenase complexes (2-OADC), characterize the human liver disease primary biliary cirrhosis. The immunodominant epitope for these autoantibodies is associated with the lipoyl-binding domain of the E2 subunit of the enzymes. The reactivity of these disease-associated autoantibodies was compared with that of antibodies raised in rats and rabbits, by immunization with various preparations derived from the 2-OADC enzymes, using immunization protocols that have successfully induced various organ-specific autoimmune diseases in animals. The immunogens included the intact pyruvate dehydrogenase complex (PDC) from bovine heart, human recombinant PDC-E2, and short synthetic peptides representing the immunodominant lipoic acid binding sequences of the 2-OADC enzymes. The techniques for antibody analysis included immunofluorescence, immunoblotting on mitochondrial extracts, ELISAs using entire PDC, PDC-E2, or synthetic peptides, epitope mapping by peptide scanning on overlapping octameric peptides representing the human PDC-E2 sequence, affinity purification on PDC-E2, and inhibition in vitro by sera of the catalytic function of PDC. Experimental immunization did not elicit any evidence of autoimmune disease. Moreover, the experimentally-induced antibodies in striking contrast to the natural autoantibodies showed preferential reactivity with PDC-E2 rather than with intact PDC, failed to inhibit in vitro the catalytic function of PDC, and, on peptide scanning, reacted with discrete epitopes, but at sites other than the lipoyl-binding region of PDC-E2. Our data indicate that 'multisystem' autoimmune diseases including primary biliary cirrhosis may not be elicitable experimentally because a critical disease-relevant autoepitope is not engaged by the immune system.

Improved production and purification of interferon-alpha-4a using a T7 RNA polymerase expression system.

An improved method for the synthesis and purification of human interferon-alpha-4a is presented. Interferon-alpha-4a was prepared using a T7 RNA polymerase expression system, where it was expressed from the vector pET-3a. Biologically active interferon-alpha-4a was isolated from the E. coli cells and purified using protamine sulphate precipitation, anion-exchange chromatography and affinity chromatography utilising a monoclonal antibody specific for human interferons-alpha.

Selective production of interferon-alpha subtypes by cultured peripheral blood mononuclear cells and lymphoblastoid cell lines.

The biological significance of the existence of multiple interferon-alpha (IFN-alpha) subtypes is unknown but may represent a finely tuned mechanism whereby different subtypes are produced in response to different stimuli. To investigate the expression of individual IFN-alpha subtypes, polyclonal antipeptide antisera designed to react with all IFN-alpha subtypes, or with a particular subtype, IFN-alpha 2 or IFN-alpha 4, have been produced. In this study we demonstrate the utility of these antisera for the detection, using indirect immunofluorescence staining, of intracellular IFN-alpha produced by human peripheral blood mononuclear cells (PBMC) and lymphoblastoid cells. Secreted IFN-alpha was also investigated by bioassay and a sandwich radioimmunoassay (RIA), using two monoclonal antibodies (mAb) and specific for IFN-alpha 4. The PBMC were shown to produce IFN reactive with all three polyclonal antisera, after stimulation with Sendai virus. The lymphoblastoid cells also produced IFN, including IFN-alpha 2, but IFN-alpha 4 was not detected either intracellularly, by immunofluorescence, or in the medium, by sandwich RIA. The immunofluorescence studies also demonstrate that in the absence of viral stimulation IFN-alpha is found in the cytoplasm of PBMC and lymphoblastoid cells but not secreted in detectable levels. The finding that two lymphoblastoid cell lines do not produce the subtype IFN-alpha 4 raises important questions as to whether other cell lines and cell types produce IFN-alpha subtypes selectively, and whether individual IFN-alpha subtypes have different roles in human physiology and pathology.

Subtype-specificity of antipeptide antibodies raised against unique sequences of human interferons-alpha.

A strategy for the production of human interferon-alpha (IFN-alpha) subtype-specific antibodies, based on immunizing rabbits with short unique synthetic peptides coupled to protein carriers, has been validated. These peptides correspond to amino acid residues 99-111 of IFN-alpha 1, 50-57 and 103-116 of IFN-alpha 2, and 37-50 of IFN-alpha 4. The antipeptide antibodies [anti-IFN alpha 1(99-111), anti-IFN alpha 2(50-57C), anti-IFN alpha 2(103-116) and anti-IFN alpha 4(C37-50)] were tested by ELISA and Western blotting for their reactivity with immunoaffinity-purified recombinant human IFN-alpha 1, -alpha 2b and -alpha 4a. The anti-IFN alpha 1(99-111) and anti-IFN alpha 2(50-57C) reacted with their corresponding IFN-alpha and did not crossreact with the other IFN subtypes. The anti-IFN alpha 2(103-116) reacted with IFN-alpha 2b and also crossreacted slightly with the other subtypes. The anti-IFN alpha 4(C37-50) reacted well with IFN-alpha 4a, crossreacted with significantly lower affinity with IFN-alpha 1 and did not bind IFN-alpha 2b. Residues 104-107 and 108-111 are the major components of the epitopes recognized by anti-IFN alpha 1(99-111) and anti-IFN alpha 2(103-116), respectively, as determined by ELISA against overlapping octapeptides.

Universal antibodies to human interferon-alpha subtypes--the production of antipeptide antibodies to conserved regions of interferon-alpha.

Antibodies to three conserved regions of all human interferon (IFN)-alpha 1 subtypes were raised by immunizing rabbits with short synthetic peptides coupled to a carrier. These peptides correspond to amino acid residues 29-36, 31-36, 126-131, 139-151, and 142-151 of the consensus sequence of IFN-alpha 1. The antibodies were tested for reactivity with IFN-alpha 1, -alpha 2a, -alpha 2b, -alpha 4a, and -alpha 14 and IFN-beta. Most antipeptide antibodies react only weakly with IFN-alpha subtypes; only the antibodies raised against the peptide corresponding to residues 142-151 conjugated to keyhole limpet hemocyanin using glutaraldehyde react appreciably with all IFN-alpha subtypes tested. These antipeptide antibodies are potentially universal antibodies to the human IFN-alpha 1 subtypes, since they exhibit similar binding affinity to all the IFN-alpha subtypes tested and do not react with IFN-beta.

Antipeptide antibodies against conserved regions of human interferon-alpha: evidence for conformational variations between IFN-alpha subtypes.

Antibodies to two conserved regions (residues 29-36 and 139-151) of human interferon-alpha were raised by immunizing rabbits with four short synthetic peptides coupled to carriers. The antibodies were tested for reactivity with recombinant interferon-alpha by ELISA. Despite the amino acid conservation of the two regions, there are significant variations in the reactivity of the antibodies with the IFN-alpha subtypes. The reactivity is enhanced significantly when the disulfide bonds of the interferon molecule are reduced. The results indicate that there are subtype-specific differences in the presentation of the epitopes in these conserved regions of human interferon-alpha.

Functional domains of human interferon gamma probed with antipeptide antibodies.

Functional domains of biologically active polypeptide molecules can be sought by raising antibodies to synthetic peptides. Human interferon gamma (HuIFN gamma) was thus studied, using two peptides based on candidate regions representing amino acids 7-16 and 121-130 of the HuIFN gamma molecule. These were conjugated to bovine serum albumin prior to immunization of rabbits. High titres of antipeptide antibodies which recognized the synthetic peptides were elicited in all of the four rabbits injected. The antipeptide antibodies from one of the rabbits immunized with the C-terminal (121-130) peptide detected native HuIFN gamma at a concn as low as 300 IU/ml, but the antipeptide antibodies from the rabbit immunized with the N-terminal (7-16) peptide did not detect HuIFN gamma. The IFN gamma-reactive antipeptide antibodies (anti-121-130) did not neutralize the antiviral activity of HuIFN gamma in a cytoprotection assay. These data and other studies establish that the C-terminus of HuIFN gamma is not essential for full antiviral activity and indicate an application of antipeptide antibodies in the analysis of structure-function relationships of cytokine molecules.

Production of subtype-specific antipeptide antibodies to human interferon-alpha 1 and -alpha 4.

Antibodies that are specific to the human interferon (IFN)-alpha 1 and -alpha 4 subtypes have been produced by immunizing rabbits with two short synthetic peptides, corresponding to residues 99-111 of IFN-alpha 1 and residues 37-50 of IFN-alpha 4, respectively. The IFN-alpha 1 peptide has at least three closely clustered residues that are different from those in the other IFN-alpha subtypes, while the IFN-alpha 4 peptide has only two unique amino acid residues, separated by five common residues. The antibodies raised against the IFN-alpha 1 peptide react with recombinant human IFN-alpha 1 but do not cross-react with recombinant human IFN-alpha 4 or IFN-alpha 2. The antibodies raised against the IFN-alpha 4 peptide react with IFN-alpha 4, cross-react with IFN-alpha 1 but not with IFN-alpha 2; the affinity of the antibodies to IFN-alpha 1, however, is at least 10 times lower than their affinity to IFN-alpha 4.

Chemical synthesis of a mitochondrial gene designed for expression in the yeast nucleus.

A novel DNA sequence coding for subunit 8 of the mitochondrial ATPase of Saccharomyces cerevisiae has been constructed by chemical synthesis. The synthetic gene, termed NAP1, is designed for expression in the yeast nucleus and codes for a 48 amino acid polypeptide identical to that encoded by the mitochondrial aap1 gene of S. cerevisiae. The codons chosen for the NAP1 sequence correspond almost exclusively to those most frequently occurring in highly expressed yeast genes. The NAP1 coding region differs in 31 codons from that of aap1, and is flanked by sequences carrying restriction enzyme sites useful for cloning and for gene expression. A 170 bp double stranded DNA molecule was constructed by assembling 12 oligonucleotides (12 to 45 bases in length) in a single annealing/ligation mixture. This synthetic gene will provide a route for the systematic manipulation, through in vitro mutagenesis, of the structure of a protein normally encoded by mitochondrial DNA.

Biogenesis of mitochondria: DNA sequence analysis of mit- mutations in the mitochondrial oli1 gene coding for mitochondrial ATPase subunit 9 in Saccharomyces cerevisiae.

The nucleotide sequence of the yeast mitochondrial olil gene has been obtained in a series of mit- mutants with mutations in this gene, which codes for subunit 9 of of the mitochondrial ATPase complex. Subunit 9 is the proteolipid, 76 amino acids in length, necessary for the proton translocation function of the membrane Fo-sector. These mutants were classified on the basis of their rescue by a petite strain shown here to retain the entire wild-type olil gene. The mutation in one mit- strain removes a positively charged residue (Arg39----Met) which is likely to be located in a segment of subunit 9 that protrudes from the inner mitochondrial membrane. In a second mit- mutant, a negatively charged residue replaces a conserved glycine residue (Gly18----Asp) in a glycine-rich segment of the protein that is most likely embedded within the membrane. Other mit- mutations result in frameshifts with predicted products 7, 65 and 68 amino acid residues long. In each mit- mutant, there is the loss of one or more of the amino acid residues that are highly conserved among diverse species. The location and nature of specific changes pinpoint amino acid residues in subunit 9 essential to the activity of the mitochondrial ATPase complex.

Nucleotide sequence and expression in E. coli of a human interferon-alpha gene selected from a genomic library using synthetic oligonucleotides.

The complete nucleotide sequence of a human interferon-alpha gene is reported. The gene, designated IFN-alpha M1, was isolated from a human genomic library in phage lambda Charon 4A using synthetic oligonucleotides as hybridization probes. Based on a comparison of nucleotide sequence data obtained from this recombinant phage with published interferon-alpha gene sequences, a region of DNA capable of coding for a pre-interferon of 189 amino acids was identified. An AluI fragment containing the coding region for the mature interferon was inserted into the HincII site of the phage M13mp11, resulting in a fusion of portions of the IFN-alpha M1 and the beta-galactosidase genes. Antiviral activity was detected in extracts from E. coli infected with the recombinant M13 phage carrying the fused gene. The antiviral activity was completely neutralized by antibodies to human interferon-alpha.

Biogenesis of mitochondria: the mitochondrial gene (aap1) coding for mitochondrial ATPase subunit 8 in Saccharomyces cerevisiae.

A mitochondrial gene (denoted aap1) in Saccharomyces cerevisiae has been characterized by nucleotide sequence analysis of a region of mtDNA between the oxi3 and oli2 genes. The reading frame of the aap1 gene specifies a hydrophobic polypeptide containing 48 amino acids. The functional nature of this reading frame was established by sequence analysis of a series of mit- mutants and revertants. Evidence is presented that the aap1 gene codes for a mitochondrially synthesized polypeptide associated with the mitochondrial ATPase complex. This polypeptide (denoted subunit 8) is a proteolipid whose size has been previously assumed to be 10 kilodaltons based on its mobility on SDS-polyacrylamide gels, but the sequence of the aap1 gene predicts a molecular weight of 5,815 for this protein.