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N Howell

Publications and source records attributed to N Howell.

At least 73 records · Page 4Linked to original sources
PubMed1993

Abnormal RNA processing associated with a novel tRNA mutation in mitochondrial DNA. A potential disease mechanism.

A patient with a mitochondrial myopathy and biochemically proven profound complex I deficiency has a new mutation in mtDNA. This A-to-G transition at position 3302, involving the aminoacyl stem of tRNA(Leu(UUR)), is associated with abnormal mitochondrial RNA processing. Northern analysis demonstrates marked accumulation of a polycistronic RNA precursor containing sequence for 16 S rRNA, tRNA(Leu(UUR)), and ND1. Comparison of skeletal muscle and skin fibroblasts suggests that the processing error may be quantitatively less severe in this tissue, and biochemical analysis shows that fibroblasts do not express a biochemical defect despite containing the mutation. Important qualitative differences in the processing of this RNA precursor were found when comparing muscle and skin fibroblasts. In muscle, processing appears to occur first at the 5'-end of the tRNA, generating 16 S rRNA plus a tRNA + ND1 intermediate. In fibroblasts, processing occurs at the 3'-end of the tRNA, generating a 16 S rRNA + tRNA intermediate. We suggest that the mutation at position 3302 induces abnormal mitochondrial RNA processing that is linked to the biochemical defect (profound loss of complex I activity), either by qualitative or quantitative abnormalities in the ND1 message. The restriction to skeletal muscle of both the processing error and the biochemical defect suggests that the observed tissue differences in RNA processing play a protective role in skin fibroblasts.

Adult↗
PubMed1993

Sequence analysis of mitochondrial chloramphenicol resistance mutations in Chinese hamster cells.

A series of mitochondrially inherited chloramphenicol-resistant (CAP-R) mutants were isolated in Chinese hamster cells. To determine whether the Chinese hamster CAP-R mutations were homologous to those isolated in mouse and human cell culture systems, we determined the nucleotide sequence of the region of the mitochondrial 16S rRNA gene spanning the peptidyl transferase-encoding region for eight CAP-R mutant lines in addition to the parental wild-type line. Three main conclusions are drawn from these studies. (1) Although the region of the gene encoding the peptidyl transferase domain is highly conserved relative to that of mice and rats, the contiguous sequences show less conservation. This sequence divergence not only includes the accumulation of single base pair replacements, but also the presence of small insertions or deletions. (2) For six of the CAP-R mutants, heteroplasmic single base pair changes were detected. These mapped to the same sites within the peptidyl transferase domain as the mutations found previously in mouse and human CAP-R mutants. (3) Two Chinese hamster CAP-R mutants, both with an unusual drug resistance phenotype, did not carry any mutations within the CAP-R peptidyl transferase domain. However, both carried a heteroplasmic mutation at the position corresponding to nucleotide 2505 of the mouse 16S rRNA gene, a site predicted to map within a stem/loop structure attached to this key domain of the ribosome. This is the first evidence for mitochondrial CAP-R mutations that map outside the peptidyl transferase region.

Animals↗
PubMed1993

Pulse intravenous calcitriol therapy of secondary hyperparathyroidism in peritoneal dialysis patients.

This study was undertaken to evaluate high-dose intravenous (IV) calcitriol therapy in chronic peritoneal dialysis (PD) patients. Nine stable chronic PD patients (7 male, 2 female, mean age 54 years) with secondary hyperparathyroidism and a mean duration of PD of 33 months, were studied for 6 months. Pulse calcitriol was administered IV in dosages of 4-8 micrograms every 2 weeks. Eight patients received oral calcitriol prior to IV pulse. Five patients were on continuous ambulatory peritoneal dialysis, and 4 patients were on continuous cycling peritoneal dialysis. Seven patients were on low-calcium dialysate (2.5 mEq/L) and 2 on high-calcium dialysate (3.5 mEq/L). All patients received calcium acetate or calcium carbonate to control hyperphosphatemia during the study. Mean serum parathyroid hormone N-terminal (PTHN) levels (35.4 +/- 9.5 pg/mL) decreased significantly from premean serum PTHN levels (79.7 +/- 10.4 pg/mL). No significant changes were observed in premean serum total calcium, ionized calcium, and phosphorus levels and posttherapy levels. No correlation was observed between serum PTHN and serum calcium, 1,25(OH)2D3, serum aluminum, and duration of dialysis. No significant difference in total body calcium and total body bone mineral density (BMD) was observed between pre and post study periods. In conclusion, biweekly IV calcitriol therapy is effective in suppressing secondary hyperparathyroidism in PD patients.

Adult↗
PubMed1992

Low calcium (2.5 mEq/l) and high calcium (3.5 mEq/l) dialysate in peritoneal dialysis patients.

STUDY OBJECTIVE: To compare the effects of low-calcium and high-calcium dialysate in stable ESRD patients on peritoneal dialysis (PD). DESIGN: Dialysate containing 2.5 mEq/l and 3.5 mEq/l calcium in combination with oral calcium salts as phosphate binders were evaluated. SETTING: Tertiary-referral university hospital. PATIENTS AND METHODS: Fifteen patients (6 male, 9 female) on low-calcium (2.5 mEq/l) and 15 patients (6 male, 9 female) on high-calcium (3.5 mEq/l) dialysate were studied for 6 months. All patients received calcium acetate or calcium carbonate to control hyperphosphatemia before the study. RESULTS: Serum calcium, phosphorus and albumin did not differ before and after between the two groups. Three patients in low-calcium and five in high-calcium group developed hypercalcemia. Three in low-calcium group and four in high-calcium group required sucralfate to control hyperphosphatemia and hypercalcemia. Mean dose of elemental calcium was 1152 mg/day in low-calcium group and 790 mg/day in high-calcium group. A negative correlation (r = -0.82, p < 0.005) was observed between serum calcium and PTH at the end of study period in the low-calcium group. No such relationship was observed in the high-calcium group. CONCLUSIONS: Degree and frequency of hypercalcemia appeared similar with low-calcium and high-calcium dialysate in peritoneal dialysis patients.

Calcium↗
PubMed1992

A variant of Leber hereditary optic neuropathy characterized by recovery of vision and by an unusual mitochondrial genetic etiology.

The Tas2 and Vic2 Australian families are affected with a variant of Leber hereditary optic neuropathy (LHON). The risk of developing the optic neuropathy shows strict maternal inheritance, and the ophthalmological changes in affected family members are characteristic of LHON. However, in contrast to the common form of the disease, members of these two families show a high frequency of vision recovery. To ascertain the mitochondrial genetic etiology of the LHON in these families, both (a) the the nucleotide sequences of the seven mitochondrial genes encoding subunits of respiratory-chain complex I and (b) the mitochondrial cytochrome b gene were determined for representatives of both families. Neither family carries any of the previously identified primary mitochondrial LHON mutations: ND4/11778, ND1/3460, or ND1/4160. Instead, both LHON families carry multiple nucleotide changes in the mitochondrial complex I genes, which produce conservative amino acid changes. From the available sequence data, it is inferred that the Vic2 and Tas2 LHON families are phylogenetically related to each other and to a cluster of LHON families in which mutations in the mitochondrial cytochrome b gene have been hypothesized to play a primary etiological role. However, sequencing analysis establishes that the Vic2 and Tas2 LHON families do not carry these cytochrome b mutations. There are two hypotheses to account for the unusual mitochondrial genetic etiology of the LHON in the Tas2 and Vic2 LHON families. One possibility is that there is a primary LHON mutation within the mitochondrial genome but that it is at a site that was not included in the sequencing analyses. Alternatively, the disease in these families may result from the cumulative effects of multiple secondary LHON mutations that have less severe phenotypic consequences.

Adolescent↗
PubMed1991

Multiple defects of the mitochondrial respiratory chain in a mitochondrial encephalopathy (MERRF): a clinical, biochemical and molecular study.

We describe a young man with a progressive neurological disorder including myoclonus, mental retardation, muscle weakness and a mitochondrial myopathy (myoclonus epilepsy and ragged red fibres--MERRF). Multiple abnormalities of the mitochondrial respiratory chain in skeletal muscle are shown by direct measurement of the flux through the individual complexes, low-temperature redox spectroscopy and decreased immunodetectable subunits of complexes I and IV by immunoblotting. No abnormality of mitochondrial DNA was found. This is the first report of combined defects of complexes I, III and IV as a cause of this clinical syndrome. However, we propose that the occurrence of multiple respiratory chain defects may be more common than previously recognised and that this particular combination of defects, involving complexes I, III and IV, may be the predominant biochemical abnormality in MERRF.

Adult↗
PubMed1991

Leber hereditary optic neuropathy: identification of the same mitochondrial ND1 mutation in six pedigrees.

Biochemical and molecular genetic evidence is presented that in six independent pedigrees the development of Leber hereditary optic neuropathy (LHON) is due to the same primary mutation in the mitochondrial ND1 gene. A LHON family from the Newcastle area of Great Britain was analyzed in depth to determine the mitochondrial genetic etiology of their disease. Biochemical assays of mitochondrial electron transport in organelles isolated from the platelet/white-blood-cell fraction have established that the members of this family have a substantial and specific lowering of flux through complex I (NADH-ubiquinone oxidoreductase). To determine the site of the primary mitochondrial gene mutation in this pedigree, all seven mitochondrial complex I genes were sequenced, in their entirety, from two family members. The primary mutation was identified as a homoplasmic transition at nucleotide 3460, which results in the substitution of threonine for alanine at position 52 of the ND1 protein. This residue occurs within a very highly conserved hydrophilic loop, is invariantly alanine or glycine in all ND1 proteins, and is adjacent to an invariant aspartic acid residue. This is only the second instance in which both a biochemical abnormality and a mitochondrial gene mutation have been identified in an LHON pedigree. The sequence analysis of the ND81 gene was extended to a further 11, unrelated LHON pedigrees that had been screened previously and found not to carry the mitochondrial ND4/R340H mutation. The ND1/A52T mutation at nucleotide 3460 was found in five of these 11 pedigrees. In contrast, this sequence change was not found in any of the 47 non-LHON controls. The possible role of secondary complex I mutations in the etiology of LHON is also addressed in these studies.

Amino Acid Sequence↗
PubMed1991

Leber hereditary optic neuropathy: involvement of the mitochondrial ND1 gene and evidence for an intragenic suppressor mutation.

A large Queensland family has an extreme form of Leber hereditary optic neuropathy (LHON) in which several neurological abnormalities and an infantile encephalopathy are present in addition to the characteristic ophthalmological changes. Sequence analysis of the seven mitochondrial genes encoding subunits of respiratory chain complex I (NADH-ubiquinone oxidoreductase) reveals two novel features of the etiology of this mitochondrial genetic disease. The first conclusion from these studies is that the ophthalmological and neurological deficits in this family are produced by a mutation at nucleotide 4160 of the ND1 gene. This nucleotide alteration results in the substitution of proline for the highly conserved leucine residue at position 285 of the ND1 protein. Secondary-structure analysis predicts that the proline replacement disrupts a small alpha helix in a hydrophilic loop. All nine family members analyzed were homoplasmic for this mutation. The second major result from these studies is that the members of one branch of this family carry, at nucleotide 4136 of the same gene, a second mutation, also homoplasmic, which produces a cysteine-for-tyrosine replacement at position 277. The clinical and biochemical phenotypes of the family members indicate that this second nucleotide substitution may function as an intragenic suppressor mutation which ameliorates the neurological abnormalities and complex I deficiency.

Amino Acid Sequence↗
PubMed1990

Glycine-231 residue of the mouse mitochondrial protonmotive cytochrome b: mutation to aspartic acid deranges electron transport.

The mouse LA9 HQN-R11 cytochrome b mutant, in which the glycine residue at position 231 is replaced by aspartic acid, has increased resistance to all inhibitors of the Qn redox center. It is shown here that this single amino acid alteration has multiple and unexpectedly diverse effects upon the mitochondrial protonmotive bc1 complex. (1) The specific activities of both succinate- and ubiquinol-cytochrome c oxidoreductases in isolated mitochondria are reduced by approximately 65% in the mutant. The parallel reductions in both oxidoreductase activities are not compatible with simple Q pool kinetics for mitochondrial electron transport. (2) There is also a reduction in the relative concentration of cytochrome b in the mutant when calculated on the basis of mitochondrial protein; this decrease does not account for more than a small portion of the reduced catalytic fluxes. (3) The increased antimycin resistance of the mutant is lost upon solubilization by the detergent dodecyl maltoside of the bc1 complex from mitochondria. (4) In pre-steady-state assays of cytochrome b reduction by quinol, the mutant shows a reduced extent of reduction. It was observed in other experiments that there was less oxidant-induced extrareduction of cytochrome b in the mutant. These results could arise from a lowering of the midpoint potentials of both the cytochrome b-562 and cytochrome b-566 heme groups. Alternatively, these effects may reflect changes at the Qp and Qn quinone/quinol binding sites. (5) An unexplained observation for the mutant is the increased rate of cytochrome c1 reduction in the presence of myxothiazol. (6) These functional alterations in the LA9 HQN-R11 mutant are not accompanied by detectable changes in the spectral properties of the cytochrome b or c1 heme groups.

Antimycin A↗
PubMed1990

An example of Leber hereditary optic neuropathy not involving a mutation in the mitochondrial ND4 gene.

A large Australian family afflicted with Leber's Hereditary Optic Neuropathy (LHON) is analyzed at the nucleotide sequence level in this report. Biochemical assays of platelet mitochondria isolated from members of this family have demonstrated a significant decrease in the specific activity of Complex I (NADH-ubiquinol oxidoreductase) of the electron transport chain. It is shown here, however, that neither this biochemical lesion nor the optic neuropathy are due to the mutation at nucleotide position 11,778 of the mitochondrial ND4 gene first identified by Wallace et al. in several LHON pedigrees. Furthermore, extensive DNA sequencing studies reveal no candidate mutations within the mitochondrial ND3 gene, the ND4L/ND4 genes, or the contiguous tRNA genes. These studies provide the first direct evidence that not all LHON lineages--even those associated with a biochemical defect in mitochondrial respiratory chain Complex I--carry a mutation in the ND4 gene. Members of the Australian LHON family exhibit neurological abnormalities in addition to the well-characterized ophthalmological changes. It is hypothesized that LHON may be a syndrome or set of related diseases in which the clinical abnormalities are a function, at least in part, of the mitochondrial Complex I gene in which the proximate mutation occurs.

Australia↗
PubMed1989

Sequence analysis of mouse mitochondrial chloramphenicol-resistant mutants.

The nucleotide sequences of the 3' halves of the mitochondrial 16S rRNA genes from four independent mouse chloramphenicol-resistant (CAP-R) mutants were determined. Each contained a different, single base change that encodes the mutational phenotype. The mitochondrial rRNA gene from the SVA31 CAP-R mutant contains a G-to-A transition at nucleotide 2161 of the noncoding strand; the SVIS CAP-R mutant, a G-to-A transition at position 2375; the LA9 CAP-R mutant, an A-to-T transversion at position 2379; and the SVT2 CAP-R mutant, a T-to-C transition at position 2433. Three of these CAP-R mutants appear to be heteroplasmic as the mtDNA populations contain both wild-type and mutant copies of the rRNA gene. The SVIS CAP-R mutation has not been observed in other mammalian CAP-R mutants, although it occurs at a site homologous to one of the yeast mitochondrial CAP-R mutations. Based upon the locations of the mutated sites within the 16S rRNA, and their proximity to previously analyzed sites of mutations conferring increased inhibitor resistance, all these mutations occur within the ribosomal RNA peptidyltransferase domain. These results provide an explanation for the pleiotropic nature of mitochondrial CAP-R mutations in mammalian cells, particularly the observations that some of the mutant lines are partially respiration deficient.

Animals↗
PubMed1989

Evolutionary conservation of protein regions in the protonmotive cytochrome b and their possible roles in redox catalysis.

The amino acid sequences of the protonmotive cytochrome b from seven representative and phylogenetically diverse species have been compared to identify protein regions or segments that are conserved during evolution. The sequences analyzed included both prokaryotic and eukaryotic examples as well as mitochondrial cytochrome b and chloroplast b6 proteins. The principal conclusion from these analyses is that there are five protein regions--each comprising about 20 amino acid residues--that are consistently conserved during evolution. These domains are evident despite the low density of invariant residues. The two most highly conserved regions, spanning approximately consensus residues 130-150 and 270-290, are located in extramembrane loops and are hypothesized to constitute part of the Qo reaction center. The intramembrane, hydrophobic protein regions containing the heme-ligating histidines are also conserved during evolution. It was found, however, that the conservation of the protein segments extramembrane to the histidine residues ligating the low potential b566 heme group showed a higher degree of sequence conservation. The location of these conserved regions suggests that these extramembrane segments are also involved in forming the Qo reaction center. A protein segment putatively constituting a portion of the Qi reaction center, located approximately in the region spanned by consensus residues 20-40, is conserved in species as divergent as mouse and Rhodobacter. This region of the protein shows substantially less sequence conservation in the chloroplast cytochrome b6. The catalytic role of these conserved regions is strongly supported by locations of residues that are altered in mutants resistant to inhibitors of cytochrome b electron transport.

Amino Acid Sequence↗
PubMed1989

Annual tuberculin skin testing of employees at a university hospital: a cost-benefit analysis.

The usefulness of routine annual tuberculin skin testing (purified protein derivative [PPD]) of hospital employees has been questioned. Between 1984 and the end of 1987 the PPD conversion rates of hospital employees at a university and psychiatric hospital in North Florida were compared. The number of employees in both hospitals were almost equal and compliance with the annual testing was more than 95%. In the psychiatric hospital tuberculosis screening of patients was practiced on admission and annually thereafter. Although no unsuspected smear-positive tuberculosis patients were diagnosed in the psychiatric hospital as compared to four in the university hospital, the annual conversion rates of employees were 0.42% and 0.13%, respectively (p greater than 0.001). However, the ratios of these conversion rates to the incidence of tuberculosis in the counties where these hospitals are located respectively were similar (20.0 vs 24.3, p = 0.7). The community seems be the major source of the PPD conversion. At the university hospital more than +70,000 was spent on the annual PPD testing to discover 15 converters; nine had no or minimal contact with patients and only two complied with isoniazid (INH) prophylaxis. Annual PPD testing is not cost effective in hospitals with a low annual conversion rate among its employees and with low tuberculosis case rates in the hospital and the surrounding community.

Age Factors↗