Heteroplasmic ratio of the A3243G mitochondrial DNA mutation in single pancreatic beta cells.

AIMS/HYPOTHESIS: To examine whether there is a high content of mutated mitochondrial DNA in individual pancreatic beta cells from a patient with the A3243G mitochondrial DNA mutation. METHODS: Tissues were available from a patient with diabetes and the A3243G mutation including pancreatic tissue. We quantified the amount of mutated mitochondrial DNA in tissue homogenates and single pancreatic beta cells using hot last cycle PCR. RESULTS: The percentage ratio of mutated to wild-type mtDNA was high in tissues such as muscle and brain (>60%), but surprisingly low in both pancreatic islets and in individual beta cells from these islets. The islets were smaller in the patient than in control subjects in keeping with a decreased beta-cell mass. CONCLUSIONS/INTERPRETATION: These observations suggest that either the beta cells show increased sensitivity to the effects mtDNA mutations on respiratory chain function, and/or cells with a high mutant load are preferentially removed leading to a progressive decrease in the islet beta-cell mass.

The role of cytochrome c oxidase deficient hippocampal neurones in Alzheimer's disease.

Defects of mitochondrial function have been proposed as a potential mechanism in the development and pathogenesis of Alzheimer's disease (AD) and neuronal apoptosis. Mitochondrial enzyme-deficient pyramidal neurones are found in greater quantities in the hippocampus of AD patients than in age-matched controls. The presence of these neurones indicates that high levels of mutant mtDNA (mitochondrial DNA), sufficient to cause a biochemical deficiency within individual neurones, occur more frequently in AD than in normal ageing. This study analyses the relationship of cytochrome c oxidase (COX)-deficient neurones with the neuropathological markers of AD, neurofibrillary tangles (NFTs) and amyloid plaques, as well as markers of neuronal apoptosis known to occur in AD brains. Frozen sections of hippocampi from three AD patients were used to directly colocalize in situ the presence of histochemically COX-deficient neurones with immunohistology for the classical neuropathological markers of AD, tau and beta-amyloid. In addition, we also directly colocalized these mitochondrial-enzyme deficient neurones using terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling and cleaved caspase-3. The distribution of amyloid plaques is anatomically distinct from the COX-deficient hippocampal pyramidal neurones and the neurones that contained NFTs or apoptotic labelling were always COX-positive. COX-deficient, succinate dehydrogenase-positive hippocampal neurones indicative of high mtDNA mutation load do not appear to be prone to apoptosis or to directly participate in the over production of tau or beta-amyloid. Biochemically significant mitochondrial defects do occur in AD and are likely to contribute to the overall central nervous system dysfunction in impairing neuronal function and possibly causing neurodegeneration via mechanisms other than apoptosis.

Mitochondrial DNA mutations in disease and ageing.

The chronological accumulation of mitochondrial DNA mutations has been proposed as a potential mechanism in the physiological processes of ageing and age-related disease. We discuss the evidence behind this theory and relate some of the ageing mitochondrial changes to mitochondrial DNA disorders. In particular, we describe the aggregation of cytochrome c oxidase-deficient cells in both skeletal muscle and the CNS in normal ageing as seen in the mitochondrial DNA disorders. These mitochondrial enzyme-deficient cells have been shown to occur in significant quantities in both muscle and CNS in patients with mitochondrial DNA disorders. In both ageing and mtDNA disorder muscle these cytochrome c-deficient fibres contain high levels of a single mutant strain of mitochondrial DNA. Whether these mutations are a primary or secondary event in the physiology of ageing remains to be determined.

Cytochrome c oxidase deficient cells accumulate in the hippocampus and choroid plexus with age.

The chronological accumulation of mitochondrial dysfunction has been proposed as a potential mechanism in the physiological processes of aging. Cytochrome c oxidase deficient, succinate dehydrogenase positive muscle fibers containing high copy numbers of a mitochondrial DNA mutation are a pathological hallmark of mitochondrial DNA disorders. We show that there is an age-related increase in cytochrome c oxidase-deficient cells in both hippocampal pyramidal neurons and choroid plexus epithelial cells. We suggest that these cells contribute to the cell death and dysfunction in CNS aging.

Role of mitochondrial DNA mutations in disease and aging.

Since Harman in 1972 first proposed a role in the process of aging for the mitochondrial genome, a wealth of evidence has been accumulated to support this theory. We discuss the hereditary mitochondrial DNA disorders, which we believe may give insight into both normal aging and neurodegenerative conditions. We then review the evidence for the role of mitochondrial DNA mutations in both aging and age-related disorders and also discuss new approaches for investigating the mitochondrial genome at a single cell level, by observing the activity of the mitochondrial enzyme cytochrome c oxidase.

Analysis of mitochondrial DNA mutations : deletions.

Although the precise mechanisms of the aging process remain poorly understood, a plausible theory for cellular dysfunction and deterioration during aging involves mitochondria (1, 2). The major function of mitochondria is to generate energy for cellular processes in the form of ATP by oxidative phosphorylation. Mitochondria contain their own DNA (mtDNA), a small 16.5 kb circular molecule that encodes 13 essential polypeptides of the mitochondrial respiratory chain, as well as 2 rRNAs and 22 tRNAs required for intramitochondrial protein synthesis (3). The mitochondrial respiratory chain is a series of five, multisubunit protein complexes located within the inner mitochondrial membrane. The first four of these (complexes I-IV) reoxidize reduced cofactors (NADH and FADH(2)) generated by the oxidation of foodstuffs, thereby generating an electrochemical gradient across the inner mitochondrial membrane which is harnessed by the fifth complex, the ATP synthetase, to drive the formation of ATP.

Mitochondrial enzyme activity in amyotrophic lateral sclerosis: implications for the role of mitochondria in neuronal cell death.

The mechanism of selective loss of motor neurons in amyotrophic lateral sclerosis (ALS) has not been clarified. Mitochondrial pathology is present in central nervous system tissue from ALS cases and occurs as an early event in a mouse model of ALS. We demonstrate that, in sporadic ALS, there is a selective decrease in the activity of the mitochondrial DNA-encoded enzyme cytochrome c oxidase in human spinal cord motor neurons. We propose that this may not only be important in neuronal cell death but could well be caused by oxidative damage to mitochondrial DNA leading to the accumulation of mitochondrial DNA mutations.

Immunocytochemical study of the distribution of the free radical scavenging enzymes Cu/Zn superoxide dismutase (SOD1); MN superoxide dismutase (MN SOD) and catalase in the normal human spinal cord and in motor neuron disease.

Interest in free radical-mediated mechanisms of motor neuron injury has arisen following the discovery of point mutations in the Cu/Zn superoxide dismutase (SOD1) gene in a proportion of cases of familial motor neuron disease (MND). Evidence is emerging which indicates that oxidative stress may contribute to the pathophysiology of sporadic MND. Free radical scavenging enzymes form a major component of the anti-oxidant defense system. The aim of this study was to compare the distribution and density of immunoreactivity to Cu/Zn SOD. Mn SOD and catalase in the spinal cord of sporadic MND cases (n = 10) compared to normal controls (n = 8). There was abundant expression of Cu/Zn SOD, Mn SOD and catalase in spinal motor neurons, suggesting important roles for these enzymes in neuroprotective pathways in this cell group. In MND cases, there was no evidence in surviving motor neurons of a consistent alteration in the protein expression of any of these enzymes. There was evidence of increased expression of these enzymes in glial cells present in the ventral and intermediate grey matter and degenerating descending motor pathways of the spinal cord in MND cases. The changes observed were more marked in the cervical compared to lumbar spinal segments. Further investigation is required to determine whether these findings represent a compensatory response to a pathophysiological process involving oxidative stress.

Inhibin and relaxin concentrations in early singleton, multiple, and failing pregnancy: relationship to gonadotropin and steroid profiles.

OBJECTIVE: To examine the inter-relationships between inhibin, relaxin, steroid concentrations, estradiol (E2), progesterone (P), and gonadotropins in early pregnancy. DESIGN: Hormone concentrations in plasma were measured during the luteal phase of subjects who became pregnant (n = 58) or failed to become pregnant (n = 47) after ovarian hyperstimulation for in vitro fertilization-gamete intrafallopian transfer (IVF-GIFT) (group 1). A further group of subjects became pregnant (n = 7) or failed to become pregnant (n = 8) during endocrinology tracking of a natural cycle (group 2). Blood was obtained every 3 days in the luteal phase from day 5 in group I (day 0 was oocyte recovery) and from day 0 (first increase in luteinizing hormone [LH]) in group II. RESULTS: Progesterone and E2 were increased over nonpregnant values by day 11 (P) and day 16 (E2) in group I and by day 11 (E2 and P) in group II. Inhibin and relaxin concentrations were significantly increased by day 16 in group I (often by day 11) and by day 14 in group II pregnancy subjects. A direct relationship existed between inhibin, P, relaxin, and human chorionic gonadotropin (hCG). Subjects who had twin pregnancies demonstrated higher concentrations of all hormones and often exhibited increases earlier (by day 11 in group I) than singleton pregnancy subjects. Pregnancies that ended in miscarriages tended to have lower concentrations of P and inhibin. None of the hormones reliably discriminated between the clinical conditions of blighted ovum and of spontaneous abortion, and the predictive value of any hormone measured for miscarriage was not high. CONCLUSIONS: The trend of inhibin and relaxin concentrations closely parallels rises in P during early pregnancy. Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels are suppressed very early in pregnancy. The suppression of LH and FSH in hyperstimulated cycles is more governed by E2 than inhibin in stimulated cycles. Some subjects destined to miscarry exhibit abnormal endocrine changes very early in the luteal phase.

Expression of class I and II major histocompatibility complex antigens in Wilms' tumour and normal developing human kidney.

The Wilms' tumour is a solid childhood tumour of the kidney, consisting of blastema, tubules and mesenchyme. Embryonic tumours, such as Wilms', may arise as a result of a developmental disturbance in differentiation. The expression of class I and II major histocompatibility complex (MHC) antigens was investigated on 6 Wilms' tumours and related to that in the developing human kidney in this immunohistological study, using a panel of monoclonal antibodies. The Wilms' tumour blastemal cells were class I MHC antigen negative, but differentiated structures were positive. Class II MHC antigens were not observed in Wilms' tumours. In the developing human kidney class I MHC antigen expression was observed on glomeruli from 8 weeks and on tubules from 13 weeks gestational age. Class II MHC antigen expression was observed on glomeruli from 11 weeks and on tubules from 13 weeks gestation. These results suggest that the blastemal cells within the Wilms' tumour may reflect an early stage of development with respect to the expression of MHC antigens.

Abnormal expression of class II MHC antigens in placentae from patients with pemphigoid gestationis: analysis of class II MHC subregion product expression.

Abnormal expression of class II MHC antigens was consistently observed in an immunohistological study on placentae from patients with pemphigoid gestationis. The area affected in all the placentae was the chorionic villi adjacent to the maternal decidua. The villous stroma, and in some cases the chorionic fetal endothelium, had abnormal expression of class II MHC antigens. Monoclonal antibodies specific for the class II MHC subregion products (DR, DP and DQ) were used to analyse the class II MHC antigen expression. Differential expression of the class II MHC subregion products was observed on the villous stroma and chorionic fetal endothelium; DR and DP were always expressed but DQ in some cases was heterogeneous. This abnormal expression of class II MHC antigens may reflect an immunological attack on the placenta in pemphigoid gestationis.

Ultrastructural pathology of S-antigen uveoretinitis.

The morphology of S-antigen-induced uveoretinitis in guinea pigs has been studied using transmission electron microscopy. Purified bovine retinal S-antigen was shown to produce a focal chorioretinitis, characterized by selective damage to the outer retina and, almost exclusively, a mononuclear cell infiltration of the choroid and retina. Even at high doses, extensive rod outer segment damage was associated predominantly with lymphocytic and mononuclear cell infiltration. A single immunizing injection of S-antigen was sufficient to produce a chronic ocular inflammation lasting many months. Focal lesions evolved rapidly and reached an end-stage within days to weeks. Accordingly, eyes examined at any time during the disease contained areas of normal retina coexistent with fibrotic lesions. With time, the number of advanced or end stage lesions became more frequent, thereby involving a more widespread area of the retina. Examination of early stage lesions suggest that the rod outer segment is the target for immune damage in this disease, but the mechanism of damage remains to be elucidated.

Abnormal expression of HLA-DR antigen in the placenta of a patient with pemphigoid gestationis.

The villous stroma and fetal endothelium in chorionic villi adjacent to maternal decidua in a placenta of a woman suffering from pemphigoid gestationis were found to have abnormal expression of HLA-DR antigen. This aberrant DR expression may be a reflection of an immune attack on the placenta.

Purification of retinal S-antigen by ion-exchange chromatography and chromatofocusing.

A simple method for the isolation of bovine retinal S-antigen by ion-exchange chromatography on DEAE Sephadex, alone or in combination with a chromatafocusing step is described. High yields of highly purified S-antigen were obtained. Analytical studies indicated that the isolated protein was a single chain, 55 000 Mr glycoprotein with little tendency to self-association and a pI of 5.5. S-antigen prepared in the absence of protease inhibitors migrated on SDS-PAGE as a doublet but close similarity between both protein bands was observed by analysis of papain digests, suggesting that the protein was readily susceptible to proteolysis. S-antigen prepared by this method induces a selectively posterior focal uveoretinitis in a dose-dependent manner.