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PubMed1994

Is primary biliary cirrhosis an autoimmune disease?

This review summarizes the experimental and clinical support for an autoimmune origin of primary biliary cirrhosis (PBC). Direct proof is lacking, but indications in favour of an immunologic destructive mechanism include the demonstration of antibodies and T cell clones with specificity for mitochondrial autoantigens, and the lymphocytic infiltration/destruction of small bile ducts similar to that of graft-vs-host disease and rejection. There is a weak association with other autoimmune diseases, but no clear HLA linkage. Spontaneous animal models for PBC are lacking, and immunization of animals with purified autoantigen does not result in typical disease. Anti-mitochondrial antibodies (AMAs) of M2 type are diagnostic of PBC, and are mainly directed against a functional, restricted epitope on the E2 subunit of the pyruvate dehydrogenase complex (PDC). PDC-E2 shows several similarities to other classical autoantigens. The pathogenic role of AMA remains elusive. Recent studies have shown that AMAs detect an antigenic epitope expressed on the luminal surface of biliary epithelium in PBC liver. The initial triggering event might represent a microbial infection (molecular mimicry), or an aberrant surface expression of a true autoepitope.

Animals↗
PubMed1993

Potentiation of decreased pyruvate dehydrogenase activity by inflammatory stimuli in sepsis.

The effect of inflammation and chronic sepsis on the activity of pyruvate dehydrogenase complex (PDH) in skeletal muscle was investigated in rats. Inflammation was induced by the placement of a catheter in the carotid artery. Sepsis was induced by repeated (every 48 hr) injections of an inoculum composed of Staphylococcus aureus, Escherichia coli, and Bacteroides fragilis organisms into a preformed subcutaneous abscess. Hindlimb muscle was sampled 7 or 14 days following the initial injection of the inoculum into the abscess. Total PHD activity was not altered by any of the conditions examined. There were no differences in the proportion of active PDH complex after 7 days in any of the conditions examined. In contrast, 14 days after the initial bacterial injection, the concentration of active PDH complex in skeletal muscle was reduced by 50% in the septic rats. The combination of intravascular catheterization and infection resulted in a further decrease in the concentration of active PDH complex. The decreased concentration of active PDH complex was associated with increased plasma lactate concentrations in septic rats. Catheterization exacerbated the rise in plasma lactate in sepsis. In this model of chronic sepsis, the magnitude of the hyperlactatemia and the inhibition of the PDH complex in skeletal muscle appear dependent upon the length of time of the septic insult and are potentiated by addition of an intravascular focus of inflammation.

Animals↗
PubMed1991

Phosphorylation of pyruvate dehydrogenase in the hippocampal slice: time course of response to cellular depolarization.

In the rat hippocampal slice, we evaluated the effects of potassium depolarization on phosphorylation of the alpha subunit of the pyruvate dehydrogenase complex (E1 alpha, mol. wt. approximately 41 kDa) using an in vitro back-titration assay. The in vitro back-titration phosphorylation of El alpha was maximal following 30 s of depolarization and remained elevated for up to 3 min following termination of the depolarizing pulse. Possible significance of and mechanisms for the depolarization-induced changes in E1 alpha phosphorylation are discussed.

Animals↗
PubMed2000

Thermally induced disintegration of the Bacillus stearothermophilus dihydrolipoamide dehydrogenase.

Upon heat treatment of the pyruvate dehydrogenase complex from Bacillus stearothermophilus, the most thermostable component is a dihydrolipoamide dehydrogenase (E3c). To understand this stability, the thermal disintegration of E3 dissociated from the complex (E3d) was examined, comparing with that of E3c. Judging from residual activity and inactivation rate, E3d was less thermostable than E3c; E3d and E3c lost half of their original activities upon incubations for 30 min at 79 degrees C and 90 degrees C, respectively. Heat treatment of E3d raised the fluorescence intensities of Trp residue, intrinsic FAD, and extrinsic 8-anilinonaphthalene-1-sulfonate. E3d lost FAD, and inactive E3d polypeptides were aggregated. The sulfonate bound to the aggregate became notably fluorescent. The thermal disintegration of E3d was speculated to be a consecutive reaction that was different from the concurrent disintegration reaction of the complex. Some interactions with other component polypeptides was suggested to improve the thermostability of E3c.

Chromatography, Gel↗
PubMed1993

Identification of the essential cysteine residue in the active site of bovine pyruvate dehydrogenase.

Pyruvate dehydrogenase (E1), the first catalytic component of the bovine pyruvate dehydrogenase complex, is composed of two nonidentical subunits in a tetrameric alpha 2 beta 2 form. The sulfhydryl-specific reagent N-ethylmaleimide (NEM) was used to identify the reactivities and function of cysteinyl residues and subsequent identification of these residues in the active site of bovine E1. Treatment of E1 with 0.2 mM NEM resulted in loss (90%) of enzymatic activity; the inactivation followed bimolecular reaction kinetics. The inactivation was almost entirely prevented by thiamin pyrophosphate (TPP) and pyruvate; protection is probably due to formation of the hydroxyethylidene-TPP intermediate. To identify the reactive cysteinyl residues in the active site region, the nonessential SH groups in E1 were first modified with NEM in the presence of TPP and pyruvate. After quenching with dithiothreitol and removal of the substrate and cofactor by dialysis, the modified E1 was treated with [14C]NEM to label the exposed cysteinyl residue(s) in or near the active site region. The data indicate that NEM reacted in the active site region of the E1 component with a stoichiometry of 2 mol of [14C]NEM bound per mol of E1 tetramer. The initial rapid labeling of E1 with [14C]NEM established that incorporation was predominantly into the alpha subunit. A single radiolabeled peptide was isolated following V8 protease digestion of radiolabeled E1 by [14C]NEM. Sequence analysis of the labeled peptide derived from bovine E1 demonstrated that the labeled cysteinyl residue was equivalent to Cys-62 in the alpha subunit (mature form) of human E1.

Amino Acid Sequence↗
PubMed2004

New recombinant vaccines based on the use of prokaryotic antigen-display systems.

A major challenge in vaccine design has been to identify antigen presentation systems that elicit strong T- and B-cell responses. In the authors' laboratory, two new delivery vehicles derived from nonpathogenic prokaryotic organisms were recently designed and investigated. Conserved antigenic determinants were inserted into the N-terminal region of the major pVIII coat protein of bacteriophage fd virions or on the surface of an icosahedral scaffold formed by the acyltransferase component (E2 protein) of the pyruvate dehydrogenase complex of Bacillus stearothermophilus. The data indicate that the antigenic determinant displayed by either fd virions or on the surface of the E2 lattice are accessible to the immune system, and are able to trigger a humoral response as well as a potent helper and cytolytic response in vitro and in vivo. These systems offer the potential for safe and inexpensive vaccines to elicit full-spectrum immune responses.

Animals↗
PubMed2005

Gibberellin regulates mitochondrial pyruvate dehydrogenase activity in rice.

Pyruvate dehydrogenase kinase (PDK) is a negative regulator of the mitochondrial pyruvate dehydrogenase complex (mtPDC) that plays a key role in intermediary metabolism. OsPDK1 was identified as a gibberellin-up-regulated gene using a cDNA microarray. The full-length cDNA for OsPDK1 was 1498 bp and encoded a predicted polypeptide of 363 amino acids. Genomic DNA analysis showed the presence of another isoform of PDK, OsPDK2, in rice. Reverse transcriptase-PCR analysis revealed differential expression of the two isoforms. OsPDK1 was expressed in leaf blade and leaf sheath but not in callus and root, while OsPDK2 was expressed constitutively in all tissues examined. Maximum expression of OsPDK1 in leaf sheath was detected by Northern blot analysis when seedlings were treated with 5 microM GA3 for 24 h. OsPDK1 expression was up-regulated by GA3, and there was little effect of other plant hormones. Mitochondrial pyruvate dehydrogenase (PDH) activity was reduced compared with control plants in 2-week-old seedlings treated with GA3. The beta-glucuronidase (GUS) reporter gene, driven by a 2,067 bp OsPDK1 promoter region fragment, was mainly expressed in the aleurone layer of germinating seed and leaf sheath. Transgenic rice expressing PDK1 RNAi had altered vegetative growth with reduced accumulation of vegetative tissues. These results suggest that gibberellin modulates the activity of mtPDC by regulating OsPDK1 expression and subsequently controlling plant growth and development.

Blotting, Northern↗
PubMed1999

Multiple alignment and sorting of peptides derived from phage-displayed random peptide libraries with polyclonal sera allows discrimination of relevant phagotopes.

Biopanning of phage-displayed random peptide libraries is a powerful technique for identifying peptides that mimic epitopes (mimotopes) for monoclonal antibodies (mAbs). However, peptides derived using polyclonal antisera may represent epitopes for a diverse range of antibodies. Hence following screening of phage libraries with polyclonal antisera, including autoimmune disease sera, a procedure is required to distinguish relevant from irrelevant phagotopes. We therefore applied the multiple sequence alignment algorithm PILEUP together with a matrix for scoring amino acid substitutions based on physicochemical properties to generate guide trees depicting relatedness of selected peptides. A random heptapeptide library was biopanned nine times using no selecting antibodies, immunoglobulin G (IgG) from sera of subjects with autoimmune diseases (primary biliary cirrhosis (PBC) and type 1 diabetes) and three murine ascites fluids that contained mAbs to overlapping epitope(s) on the Ross River Virus envelope protein 2. Peptides randomly sampled from the library were distributed throughout the guide tree of the total set of peptides whilst many of the peptides derived in the absence of selecting antibody aligned to a single cluster. Moreover peptides selected by different sources of IgG aligned to separate clusters, each with a different amino acid motif. These alignments were validated by testing all of the 53 phagotopes derived using IgG from PBC sera for reactivity by capture ELISA with antibodies affinity purified on the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2), the major autoantigen in PBC: only those phagotopes that aligned to PBC-associated clusters were reactive. Hence the multiple sequence alignment procedure discriminates relevant from irrelevant phagotopes and thus a major difficulty with biopanning phage-displayed random peptide libraries with polyclonal antibodies is surmounted.

Algorithms↗
PubMed2002

Effect of dietary fish oil on insulin sensitivity and metabolic fate of glucose in the skeletal muscle of normal rats.

The aim of this work was to study the effect of the administration of cod liver oil on the non-oxidative and oxidative fate of glucose metabolism in the skeletal muscle of normal rats. To achieve this goal, the gastrocnemius was examined regarding glucose oxidation, glycogen synthase activity and glycogen storage both at baseline and during euglycemic hyperinsulinemic clamping. The results show that dietary fish oil decreases plasma insulin levels without alteration in glucose homeostasis (at baseline). In addition, the observed enhancement in whole body glucose utilization during clamping suggests an increased peripheral insulin sensitivity. Furthermore, under insulin-stimulated glucose disposal, an enhancement in the glycolytic pathway (increased levels of muscle glucose-6-phosphate and plasma lactate) rather than changes in the oxidation (pyruvate dehydrogenase complex) and storage components of glucose metabolism was observed in the skeletal muscle of rats fed dietary fish oil. These results coupled with the hypolipidemic effects of fish oil may have implications for the prevention and/or management of some pathological states manifested by insulin resistance with or without dyslipidemia.

Animals↗
PubMedDate not supplied

Addison's other disease: primary biliary cirrhosis as a model autoimmune disease.

Thomas Addison described three of the classical autoimmune diseases, so can justifiably be regarded as one of the fathers of the study of autoimmunity. The least well recognised of these conditions, the autoimmune liver disease primary biliary cirrhosis (PBC), is in some ways the most interesting. As a result of the relatively advanced state of knowledge of its immunopathogenesis, it represents a good model for the study of autoimmune disease. The classical pathological lesion of PBC is apoptotic damage to the biliary epithelial cells lining the small intrahepatic bile ducts. The disease is typified by two symptom sets: fatigue and pruritus, which can occur at any stage of the disease process, and the features of advanced liver disease, which occur when secondary liver damage results from bile retention. Although autoantibodies directed at, in particular, pyruvate dehydrogenase complex (PDC) are almost universally present in PBC (and represent an important diagnostic tool), it appears likely that CD8+ cytotoxic T cells reactive with self-PDC derived epitopes are directly responsible for target cell damage. Recent studies in humans and a novel murine disease model have shed light on the mechanism of breakdown of immune tolerance to self-PDC; they provide important insights into the pathogenesis of PBC in particular and of autoimmunity in general.

Animals↗
PubMed2000

Autoantigens in primary biliary cirrhosis.

The automimmune liver disease primary biliary cirrhosis (PBC) is characterised by serum autoantibodies directed at mitochondrial and nuclear antigens (seen in most patients and a subset of patients, respectively). The antimitochondrial antibodies (AMA) characteristic of PBC are directed at members of the 2-oxoacid dehydrogenase components of multienzyme complexes; in particular, the E2 and E3 binding protein (E3BP) components of the pyruvate dehydrogenase complex (PDC). The presence of autoantibodies reactive with PDC-E2 and/or E3BP is strongly predictive of the presence of PBC. Therefore, the detection of these antibodies plays a very important role in the diagnosis of PBC. Originally demonstrated using immunofluorescence approaches, AMA can now be detected by the use of commercially available enzyme linked immunosorbent assays (ELISAs). Although the ELISA based approaches have advantages in terms of laboratory practicality, they are slightly less sensitive for the diagnosis of PBC than immunofluorescence (occasional patients with PBC show reactivity with PDC related antigens not present in the antigen preparations available for use with ELISA). Therefore, immunofluorescence should continue to be available as a complementary diagnostic test for use in occasional patients. In a subset of patients with PBC, autoantibodies are directed at increasingly well characterised nuclear antigens. Antinuclear antibody (ANA) positive patients are typically AMA negative. There are no significant differences in disease phenotype between AMA positive and AMA negative groups. At present, the clinical detection of ANA is mostly by Hep2 immunofluorescence, although ELISA kits for individual nuclear antigens are increasingly becoming available.

Antigens, Nuclear↗
PubMed1979

Pyruvate carboxylase deficiency and lactic acidosis in a retarded child without Leigh's disease.

A child with lactic acidosis, severe mental and developmental retardation, and proximal renal tubular acidosis is presented. Biopsy and autopsy studies show severe hepatic, renal cortical, and cerebral deficiencies in pyruvate carboxylase (EC 6.4.1.1) activity. The patient had 1.81 +/- 0.20 units/g fresh weight at biopsy and 0.75 +/- 0.07 units/g fresh weight hepatic pyruvate carboxylase activity at autopsy compared with 10.9, 11.3, and 9.5 units/g fresh weight in two autopsy and one biopsy controls, respectively. The patient's renal cortical pyruvate carboxylase activity at autopsy was 0.008 +/- 0.004 units/g fresh weight compared with 5.05 units/g in the autopsy control. The patient had no detectable (less than 0.018 units/g fresh weight) cerebral pyruvate carboxylase activity at autopsy compared with 0.44, 0.53, and 0.695 units/g in the autopsy cerebrum of one human and two rhesus monkeys, respectively. Pyruvate dehydrogenase complex, phosphoenolpyruvate carboxykinase (PEPCK, EC 4.1.1.32), and fructose-1,6-bisphosphatase (EC 3.1.3.11) activities were in the normal range. The patient's urine pH was above 7.9 when the total serum CO2 was greater than 7.8 mM. However, the patient was able to acidify the urine to pH 5.1 when the total serum CO2 was 1.6 mM. The neuropathologic examination of the brain at autopsy revealed no sign of Leigh's disease, although developmental and degenerative lesions were observed. This is the first reported patient with a primary deficiency in hepatic, renal, and cerebral pyruvate carboxylase deficiency in whom the neuropathologic lesions, distinct from those of Leigh's disease, and proximal renal tubular acidosis have both been documented.

Acidosis↗
PubMed1984

Effects of dichloroacetate on pyruvate metabolism in rat brain in vivo.

The effects of dichloroacetate (DCA) on the activity of the pyruvate dehydrogenase (PDH) complex and associated changes in the lactate and glucose levels in rat brain were investigated in vivo. The average activities of the active form of the PDH complex in the brain, liver and muscle of starved rats were respectively 0.40 +/- 0.04, 0.07 +/- 0.04, and 0.17 +/- 0.11 mumol/min/g tissue, and amounted to 21, 11, and 16% of the total activity of the complex. Intraperitoneal injection of DCA (125 mg/kg) increased the percentage of the active form of the PDH complex in the brain, liver, and muscle to 107, 40, and 84%, respectively. DCA significantly lowered the lactate and glucose concentrations of the brain and blood. A lower dose of DCA (12.5 mg/kg) also caused significant increase in activity of the PDH complex in the brain, but did not significantly change the lactate or glucose concentration of the brain. These results suggest that DCA crosses the blood-brain barrier reasonably well.

Acetates↗
PubMed2003

3D electron microscopy reveals the variable deposition and protein dynamics of the peripheral pyruvate dehydrogenase component about the core.

Cryo-electron microscopy was exploited to reveal and study the influence of pyruvate dehydrogenase (E1) occupancy on the conformational states of the Saccharomyces cerevisiae pyruvate dehydrogenase complex (PDC). Structures representative of PDC preparations with approximately 40% and full E1 occupancy were determined after the electron microscopy images from each preparation were classified according to their sizes. The reconstructions derived from two size groups showed that the deposition of the E1 molecules associated with the larger complex is, unexpectedly, not icosahedrally arranged, whereas in the smaller complex the E1 molecules have an arrangement and architecture similar to their more ordered deposition in the WT bovine kidney PDC. This study also shows that the linker of dihydrolipamide acetyltransferase (E2) that tethers E1 to the E2 core increases in length from approximately 50 to 75 A, accounting largely for the size difference of the smaller and larger structures, respectively. Extensive E1 occupancy of its 60 E2 binding sites favors the extended conformation of the linker associated with the larger complex and appears to be related to the loss of icosahedral symmetry of the E1 molecules. However, the presence of a significant fraction of larger molecules also in the WT PDC preparation with low E1 occupancy indicates that the conformational variability of the linker contributes to the overall protein dynamics of the PDC and the variable deposition of E1. The flexibility of the complex may enhance the catalytic proficiency of this macromolecular machine by promoting the channeling of the intermediates of catalysis between the active sites.

Animals↗
PubMed1996

The treatment of congenital lactic acidoses.

Congenital lactic acidoses form a heterogeneous group of disorders: this paper considers primarily defects of the pyruvate dehydrogenase complex and the respiratory chain. Attempts to treat these disorders are hampered by uncertainty concerning the pathophysiology and by the central role of the enzymes in cellular metabolism. Few strategies are of proven efficacy, though many have been tried, including dietary manipulation, enhancement of residual enzyme activity, artificial electron acceptors and free-radical scavengers. Evaluation of treatment is complicated by the rarity, heterogeneity and unpredictable course of the diseases. Double-blind placebo-controlled trials are needed.

Acidosis, Lactic↗
PubMed1990

Preischemic hyperglycemia and postischemic alteration of rat brain pyruvate dehydrogenase activity.

Transient cerebral ischemia in normoglycemic animals is followed by a decrease in glucose utilization, reflecting a postischemic cerebral metabolic depression and a reduction in the activity of the pyruvate dehydrogenase complex (PDHC). Preischemic hyperglycemia, which aggravates ischemic brain damage and invariably causes seizure, is known to further reduce cerebral metabolic rate. To investigate whether these effects are accompanied by changes in PDHC activity, the postischemic cerebral cortical activity of this enzyme was investigated in rats with preischemic hyperglycemia (plasma glucose 20-25 mM). The results were compared with those obtained in normoglycemic animals (plasma glucose 5-10 mM). The activated portion of PDHC and total PDHC activity were measured in neocortical samples as the rate of decarboxylation of [14C]pyruvate in crude brain mitochondrial homogenates after 5 min, 15 min, 1 h, 6 h, and 18 h of recirculation following 15 min of incomplete cerebral ischemia. In normoglycemic animals the fraction of activated PDHC, which rises abruptly during ischemia, was reduced to 19-25% during recirculation compared with 30% in sham-operated controls. In hyperglycemic rats the fraction of activated PDHC was higher during the first 15 min of recirculation. However, after 1 and 6 h of recirculation, the fraction was reduced to values similar to those measured in normoglycemic animals. Fifteen of 26 rats experienced early (1-4 h post ischemia) seizures in the recovery period. The PDHC activity appeared unchanged prior to these early postischemic seizures. We conclude that the accentuated depression of postischemic metabolic rate observed in hyperglycemic animals is not coupled to a corresponding postischemic depression of PDHC.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2003

Immunization with a xenobiotic 6-bromohexanoate bovine serum albumin conjugate induces antimitochondrial antibodies.

The E2 subunit of pyruvate dehydrogenase complex (PDC-E2) is the major autoantigen recognized by antimitochondrial Abs (AMA) in primary biliary cirrhosis (PBC). Recently, we replaced the lipoic acid moiety of PDC-E2 with a battery of synthetic structures designed to mimic a xenobiotically modified lipoyl hapten on a 12-aa peptide that was found within the immunodominant autoepitope of PDC-E2 and demonstrated that AMA in PBC reacted against several organic modified mimotopes as well as, or sometimes significantly better than, the native lipoyl domain. Based on this data, we immunized rabbits with one such xenobiotic organic compound, 6-bromohexanoate, coupled to BSA. One hundred percent of immunized rabbits developed AMA that have each and every characteristic of human AMAs with reactivity against PDC-E2, E2 subunit of branched chain 2-oxo-acid dehydrogenase, and E2 subunit of 2-oxoglutarate dehydrogenase complex. The rabbit AMA also inhibited enzymatic function of PDC-E2 and, importantly, binds to peptide sequences not present in the xenobiotic carrier immunogen. In contrast, BSA-immunized controls did not produce such activity. Our observation that animals immunized with a xenobiotic BSA complex produce autoantibodies that react not only with the xenobiotic, but also with mitochondrial autoantigens recognized by autoimmune PBC sera, suggests that environmental xenobiotic agents can be a risk factor for the induction of PBC.

Animals↗
PubMed1990

Pyruvate dehydrogenase activation by insulin in human circulating lymphocytes and the possible pathway involved.

1. The incubation of human fresh circulating lymphocytes with insulin leads to modifications in the behaviour of the pyruvate dehydrogenase complex (PDH) when the contact medium is supplemented with 50 microM Ca2+ and Mg2+. 2. To investigate the mechanism involved in the PDH responsiveness to insulin in circulating lymphocytes and the role of Ca2+ and Mg2+ in this process, the PDH activity was assayed in lymphocytes combined with insulin and/or a number of substances whose mechanism of action is partially known. 3. Of these some have been seen to mimick insulin effects on PDH, whereas other were tested for the first time in this study.

Enzyme Activation↗