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S Marzuki

Publications and source records attributed to S Marzuki.

At least 37 records · Page 2Linked to original sources
PubMed1993

Significance of mitochondrial DNA deletions in myotonic dystrophy.

Mitochondrial DNA (mtDNA) deletions have been noted in small quantities in a handful of atypical cases of myotonic dystrophy and there are clinical and pathological parallels between this autosomal dominant disease and certain mitochondrial myopathies where such deletions are well recognised. We studied 20 individuals from typical pedigrees of myotonic dystrophy (of whom 19 were clinically affected) with Southern blot analysis, and 2 of the affected individuals with PCR analysis of mtDNA, but were unable to demonstrate the previously noted deletions in any quantity by either method. We conclude that especially in view of known naturally occurring large scale and minor length variants in mtDNA, these previous findings are of dubious relevance to the disease.

Adolescent↗
PubMed1992

Antipeptide antibodies to the carboxy terminal and the DCCD binding region of the human mitochondrial ATP synthase beta-subunit.

Antibodies to defined epitopes on the human ATP synthase would provide a powerful tool in the definition of the subunit composition of the enzyme complex and in the characterization of any defect in its assembly in diseases associated with mitochondrial disorders. Antibodies have been thus raised against synthetic peptides, corresponding to two regions on the human ATP synthase beta-subunit: the C-terminal region, and a region which includes the two dicyclohexylcarbodiimide (DCCD)-reactive glutamic acid residues suggested to be involved in the enzyme catalytic activity. The antibodies to the C-terminal peptide reacted with the ATP synthase beta-subunit in ELISA, in Western immunoblotting and in immunohistochemical experiments, and had the ability to immunoprecipitate the enzyme complex. The antibodies to the DCCD-binding region peptide did not react to the ATP synthase beta-subunit in its native configuration, although reacted well under Western immunoblotting conditions.

Amino Acid Sequence↗
PubMed1992

Tissue segregation of a heteroplasmic mtDNA mutation in MERRF (myoclonic epilepsy with ragged red fibers) encephalomyopathy.

The distribution of the causal 8344A-->G mtDNA mutation has been examined in six tissues of a patient with myoclonic epilepsy with ragged red fibers (MERRF), to study the developmental genetics of this type of mitochondrial disorder, and to determine the pathophysiological importance of the mtDNA heteroplasmy generally observed in such patients. Heteroplasmy of the mtDNA was observed in all six tissues (cerebellum, cerebrum, pancreas, liver, muscle, and heart) suggesting that, whereas the mtDNA mutation is relatively new, the mutated population must have existed before the formation of the three primary embryonic layers. The tissue distribution reveals significant variations in the ratio between the mutated and the normal mtDNA species, indicating the randomness of mtDNA segregation during developmental cell division and differentiation events. The result suggests the existence of tissue-specific nuclear factor(s) that determines the expression of the 8344A-->G mutation in various tissues; in MERRF syndrome, expression is mainly in the central nervous system.

Adult↗
PubMed1992

Molecular genetics of Leber's hereditary optic neuropathy: study of a six-generation family from Western Australia.

Molecular genetic studies were carried out on a 6-generation family from Western Australia with Leber's hereditary optic neuropathy. Pedigree analysis confirms the maternal inheritance of the genetic lesion underlying the disorder in this family. The presence of a recently reported disease-associated mutation at nucleotide 11778 of the mtDNA was established in one clinically affected family member by the sequencing of an appropriate 1.6 kb PCR-amplified fragment of the mtDNA; this mutation leads to an Arg340----His amino acid replacement in the ND4 subunit of respiratory complex I. The 11778 G to A base substitution is associated with the loss of an SfaNI restriction site. Examination of the representative members for this site revealed that while only mtDNA carrying this substitution could be detected in the leukocytes of 4 family members of the sixth generation, the mutated mtDNA was found to co-exist with the normal mtDNA population (heteroplasmy) in a clinically unaffected member from the fifth generation. This observation suggests that the nt 11778 mutation observed in this LHON family is relatively new; the observation of both heteroplasmy and apparent homoplasmy of the mtDNA in different family members might reflect the normal progression in the establishment of a mitochondrially inherited mutation.

Australia↗
PubMed1992

A new disease-related mutation for mitochondrial encephalopathy lactic acidosis and strokelike episodes (MELAS) syndrome affects the ND4 subunit of the respiratory complex I.

The molecular lesions in two patients exhibiting classical clinical manifestations of MELAS (mitochondrial encephalopathy, lactic acidosis, and strokelike episodes) syndrome have been investigated. A recently reported disease-related A----G base substitution at nt 3243 of the mtDNA, in the DHU loop of tRNA(Leu), was detected by restriction-enzyme analysis of the relevant PCR-amplified segment of the mtDNA of one patient but was not observed, by either restriction-enzyme analysis or nucleotide sequencing, in the other. To define the molecular lesion in the patient who does not have the A----G base substitution at nt 3243, the total mitochondrial genome of the patient has been sequenced. An A----G base substitution at nt 11084, leading to a Thr-to-Ala amino acid replacement in the ND4 subunit of the respiratory complex I, is suggested to be a disease-related mutation.

Acidosis, Lactic↗
PubMed1992

The molecular genetics of mitochondrial cytopathies: the Melbourne experience.

Mitochondrial DNA is a unique, maternally inherited molecule encoding several subunits of the respiratory enzyme chain. In several mitochondrial cytopathies mutations have been described in this genome viz. large-scale heteroplasmic deletions in syndromes with progressive external ophthalmoplegia and point mutations in MELAS and MERRF encephalomyopathies. We here report Southern blot analyses in the cases of CPEO we have seen and describe the search for point mutations in MELAS and MERRF. Mitochondrial genetic sequencing in normal and disease controls as well as in patients has confirmed the pathogenic nature of a tRNA Lys point mutation in MERRF. We propose a novel mitochondrial structural gene mutation in a MELAS--like encephalomyopathy: an A-->G substitution at position 11084 leading to a Thr to Ala replacement in the ND4 subunit of complex I.

Base Sequence↗
PubMed1992

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.

Biological Assay↗
PubMed1991

Mitochondrial genome distribution in histochemically cytochrome c oxidase-negative muscle fibres in patients with a mixture of deleted and wild type mitochondrial DNA.

In situ hybridization studies were performed on a series of chronic progressive external ophthalmoplegia patients harbouring large mitochondrial DNA deletions, using intra- and extra-deletional probes. Clear differences in the distribution of wild type and deleted mitochondrial genomes were seen in both ragged-red and non-ragged red, cytochrome c oxidase-negative fibres, with an accumulation of deleted genomes in the subsarcolemmal zone. Wild type genome content was normal or decreased in the cytochrome c oxidase-negative regions of one case, but in two patients, wild type mtDNA content in cytochrome c oxidase-negative regions was either normal (most fibres) or increased (occasional fibres). The latter observation suggests there may be a stage in the natural history of ragged-red fibre evolution where wild type genomes are transiently increased. The significance of this finding is discussed.

Base Sequence↗
PubMed1991

The human pyruvate dehydrogenase complex: a polymorphic region of the lipoate acetyl transferase (E2) subunit gene.

A major issue in the study of the pathogenesis of primary biliary cirrhosis is whether the E2 subunit of the pyruvate dehydrogenase complex (PDH-E2), the major autoantigen in the disease, exists as a tissue-specific isoform. cDNA clones spanning a segment of the 3'-catalytic region of PDH-E2 (nt 1158-1361) have been isolated from human kidney, placenta and bile epithelium cells. Nucleotide sequence analysis of the clones showed differences consistent with the presence of normal variants of PDH-E2 in the human population. However, the existence of tissue-specific isoforms of PDH-E2 cannot yet be discounted.

Acetyltransferases↗
PubMed1991

Mitochondrial DNA sequence analysis in congenital myotonic dystrophy.

Mitochondrial genetic modifying factors have been suspected in several autosomally inherited diseases. The congenital variant of myotonic dystrophy, in which there is striking maternal inheritance pattern, is a likely candidate disease. To investigate this possibility, we sequenced completely the mitochondrial genome in 2 patients with congenital myotonic dystrophy. Comparison of the two sequences with control data failed to reveal a specific nucleotide variant or length variant in this disease. We conclude that a mitochondrial genetic modifying factor is not present in congenital myotonic dystrophy.

Adult↗
PubMed1991

Normal variants of human mitochondrial DNA and translation products: the building of a reference data base.

A good standard reference for the highly polymorphic human mitochondrial DNA (mtDNA) sequence is essential for studies of normal and disease-related nucleotide variants in the mitochondrial genome. A consensus sequence for the human mitochondrial genome has been derived from thirteen unrelated mtDNA sequences. We report 128 nucleotide variants of the human mtDNA sequence, and 62 amino acid variants of the human mitochondrial translation products, observed in the coding region of these mtDNA sequences.

Amino Acid Sequence↗
PubMed1991

Chronic progressive external ophthalmoplegia in patients with large heteroplasmic mitochondrial DNA deletions: an immunocytochemical study.

Immunocytochemical studies with a holocomplex antibody battery in patients with chronic progressive external ophthalmoplegia, with and without large mitochondrial DNA deletions, revealed positive (and often increased) immunoreactivity for all complexes studied in histochemically cytochrome oxidase (COX)-negative areas, suggesting a compensatory up-regulation of these components. Similar findings were observed with subunit-specific probes directed against both nuclear- and mitochondrially encoded gene products. Comparison of staining intensities between the different complexes revealed significantly more variability in COX-negative than COX-positive fibres, suggesting disordered stoichiometric control during up-regulation. These differences were confirmed using statistical models. This data challenges the view that COX-negative fibre segments have little or no mitochondrially coded protein translation.

Adult↗
PubMed1991

Functional respiratory chain studies in mitochondrial cytopathies. Support for mitochondrial DNA heteroplasmy in myoclonus epilepsy and ragged red fibers (MERRF) syndrome.

Mitochondrial respiratory chain function was investigated with polarographic and enzymatic studies, and correlated with immunoblot studies using a battery of probes against respiratory chain holocomplexes in a series of patients with myoclonus epilepsy and ragged red fibers (MERRF) syndrome. State III respiration rates in intact skeletal muscle mitochondria were normal in two cases, suggested site I deficiency in one case and a mid-respiratory defect in another. Immunological studies of complex I showed reduced levels of several subunits with the apparent absence of two bands (which at 45 and 42 kDa, coincide with the predicted electrophoretic mobility of the ND5 gene product) in one case. Complex I, III and IV composition was normal in the other three cases indicating no major disruption of complex assembly. A differing severity of skeletal muscle respiratory chain impairment in a group of unrelated patients with severe cerebral clinical involvement is best explained by uneven tissue distribution between brain and muscle of a heteroplasmic mtDNA mutation. The relationship between MERRF and mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) encephalopathies is reappraised by extension of this hypothesis.

Adult↗
PubMed1991

Mitochondrial encephalomyopathies: a correlation between neuropathological findings and defects in mitochondrial DNA.

Neuropathological studies were carried out in two patients with mitochondrial encephalomyopathies in whom the underlying lesions in muscle mitochondrial DNA (mtDNA) and respiratory enzyme complexes have been investigated. The first, a man with Kearns-Sayre syndrome, died at the age of 49 years. Autopsy showed an old parietal lobe infarct, diffuse spongiform leukoencephalopathy of cerebral and cerebellar white matter and mild spongiform change in deep grey matter and brainstem nuclei. Heteroplasmy of skeletal muscle mitochondrial DNA with a 3.5 kb mtDNA deletion in one of two mtDNA populations was found. The second case, a woman, suffering from myoclonic epilepsy, cerebellar ataxia, bilateral sensorineural deafness, several 'stroke-like' episodes died at age 52. At autopsy, an old infarct was seen in the L internal capsule. Severe loss of neurons and gliosis were found in the dentate nuclei, moderate changes in the red nuclei and inferior olivary nuclei and mild changes in the substantial nigra and locus coeruleus. In both patients, skeletal muscle biopsy showed numbers of ragged-red fibres and intramitochondrial paracrystalline inclusions at electron microscopy. A defect in the synthesis of the ND5 subunit of the respiratory complex I was suggested in the second patient in whom a diagnosis of MELAS was made.

Adult↗
PubMed1991

Functional respiratory chain studies in subjects with chronic progressive external ophthalmoplegia and large heteroplasmic mitochondrial DNA deletions.

The functional consequences of large heteroplasmic mtDNA deletions were investigated in a group of 6 patients with chronic progressive external ophthalmoplegia (CPEO) syndromes. State III respiration rates corrected for age were low with site I and II substrates in all cases and cytochrome oxidase activity was depressed. The severity of impairment varied and is consistent with inclusion of a variable percentage of non-functioning mitochondria (with deleted mtDNA) in the pellet. Western blot studies with a holocomplex antibody battery revealed no abnormalities in subunit content of complexes III and IV. A deficiency of several complex I subunits in 3 cases suggests that abnormal nuclear-mitochondrial regulation of complex I assembly may follow large mtDNA deletions.

Adult↗
PubMed1991

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.

Amino Acid Sequence↗
PubMed1991

Affinity chromatography isolation of human cytochrome oxidase and small-scale Western immunoblot probing of the enzyme complex in mitochondrial cytopathy patients.

Isolation of human cytochrome oxidase by a one-step affinity chromatography procedure on a Sepharose 4B-ferrocytochrome c matrix following solubilization with the nonionic detergent laurylmaltoside yields an enzyme isolate of adequate purity for producing polyclonal antisera. Such an antiserum produced a distinctive immunoreactive profile in Western immunoblot studies to that reported using the enzyme isolated with ionic detergents. A sensitive and highly reproducible Western immunoblotting method is described for probing mitochondrial fractions prepared from small frozen skeletal muscle biopsies with an antiserum against the human placenta cytochrome oxidase. Application of this method to mitochondrial cytopathy patients with partial cytochrome oxidase deficiency shows that the detected subunits are synthesized in these patients.

Blotting, Western↗