Serum IL-17F does not predict poor response to IM IFNß-1a in relapsing-remitting MS.

OBJECTIVE: There is a significant unmet need for serum biomarkers in relapsing-remitting multiple sclerosis (RRMS) that are predictive of therapeutic response to disease-modifying therapies. Following a recent Stanford study which reported that pretreatment levels of serum interleukin (IL)-17F could predict poor response to interferon-ß (IFNß) therapy, we sought to validate the finding using samples from a large clinical trial. METHODS: The validation cohort included 54 good responders (GR) and 64 poor responders (PR) selected from 762 subjects with RRMS from the IM IFNß-1a dose comparison study (Avonex study C94-805). Subjects were classified as GR and PR based on the number of relapses, Expanded Disability Status Scale score, and new and enlarging T2 lesions on MRI. Serum samples were assayed for IL-17F using a multiplexed Luminex assay and for IL-17F/F using an ELISA. Replicate aliquots from the Stanford study were also assayed to assure reproducibility of methods. RESULTS: Median pretreatment and post-treatment serum IL-17F levels were not statistically significantly different between GR and PR, and serum IL-7/IL-17F ratios were also not predictive of response status. Replicate aliquots from the Stanford study showed good correlation to their original cohort (r = 0.77). CONCLUSIONS: We were unable to validate the finding that serum IL-17F is a predictor of PR in a large independent cohort of subjects with RRMS. Differences in patient populations and methodology might explain the failure to validate the results from the Stanford study.

Long-acting risperidone improves negative symptoms in stable psychotic patients.

The aim of this paper was to evaluate the efficacy of risperidone long-acting injectable (RLAI) for reducing negative symptoms of schizophrenia in patients with predominantly negative symptoms at baseline. A subanalysis was performed on data from the 6-month, open-label Switch to Risperidone Microspheres trial. Patients with Positive and Negative Syndrome Scale (PANSS) negative subscale score > or = 21, which was higher than their PANSS positive subscale score, were included in this subanalysis. Improvement in negative symptoms was measured by assessing change in the PANSS negative subscale and a negative factor score. Additional outcome variables included measures in general functioning, quality of life and patient satisfaction. A total of 842 patients were eligible for inclusion in this subanalysis. Six months of treatment was completed by 631 (74.9%) patients. Forty-three (5.1%) patients discontinued treatment due to an adverse event. Negative symptoms were significantly reduced by 6.1 +/- 6.3 points for the PANSS negative score and 6.1 +/- 6.4 points for the negative factor score (P < 0.0001 for both). Significant improvements were also noted for total PANSS and other PANSS subscale scores, general functioning, quality of life and patient satisfaction (P < 0.0001). The most common treatment-emergent adverse events (>5%) were: anxiety (6.8% of patients), exacerbation of disease (6.2%) and insomnia (5.7%). Overall, RLAI was well tolerated and associated with significant reductions in movement disorder severity. The treatment resulted in a significant improvement in negative symptom severity and was well tolerated in patients with predominantly negative symptoms, who switched from a stable antipsychotic regimen

Long-term remission in schizophrenia and related psychoses with long-acting risperidone: results obtained in an open-label study with an observation period of 18 months.

OBJECTIVE: To monitor long-term symptomatic tolerability and remission in patients with stable but suboptimally treated psychoses after switching to risperidone long-acting injectable (RLAI). METHOD: This subgroup analysis of the Switch to Risperidone Microspheres (StoRMi) open-label trial followed up patients with psychoses who were converted to RLAI for a period of 18 months or until RLAI became commercially available in their country of residence. It included patients from seven European countries. Dosage adjustments were performed as clinically necessary. The efficacy endpoint was achieving and maintaining remission, defined as absent to mild core schizophrenia symptoms for > or = 6 months. A schizophrenia assessment was also completed and patients were monitored for the development of adverse events (AEs). Discontinuation rates were calculated based on Kaplan-Meier estimates where patients switching to commercial RLAI were used as censored observations. RESULTS: A total of 529 patients were followed for up to 18 months. At 18 months, the discontinuation rate was 55.7% based on Kaplan-Meier estimates. The median time to discontinuation was 15.7 months (95% CI (14.0; 17.5)). RLAI was generally well tolerated with most AEs mild-to-moderate in severity. 13% of patients discontinued treatment because of an AE. Body weight of patients increased by a mean A+/- SD of 1.0 A+/- 6.1 kg from treatment initiation to endpoint (p = 0.0001). Glucose-related AEs occurred in four patients (0.8%). Among those patients not meeting severity remission criteria at baseline, 44.8% were in remission at endpoint. Among those patients meeting severity criteria for remission at baseline, 84.2% were in remission at endpoint. A total of 93.7% of the patients who achieved or maintained remission at 6 months were in remission at endpoint. CONCLUSIONS: RLAI is safe during long-term treatment up to 18 months in adults requiring antipsychotics. Conversion to RLAI resulted in improved symptom control. Most patients achieved and maintained a sustained remission (> or = 6 months) after conversion to RLAI.

Switching to long-acting injectable risperidone is beneficial with regard to clinical outcomes, regardless of previous conventional medication in patients with schizophrenia.

INTRODUCTION: Using an atypical long-acting antipsychotic may improve patient outcome by offering the good efficacy and tolerability of an atypical antipsychotic with improved compliance through depot administration. METHODS: This subanalysis of an international, 6-month, open-label trial of risperidone long-acting injectable (RLAI) focused on non-acute schizophrenic adult patients switching from oral or depot conventional antipsychotic. Efficacy assessments included Positive and Negative Syndrome Scale (PANSS), Global Assessment of Functioning (GAF), quality of life, treatment satisfaction, hospitalization rates, and treatment-emergent adverse events (TEAEs). RESULTS: Over 70% of patients switching from oral (n=100) or depot (n=565) conventional medication completed treatment. Improvements were observed for PANSS total and subscale scores, GAF, quality of life, treatment satisfaction and hospitalization. Overall RLAI was well tolerated. TEAEs occurring in >5% were: anxiety (11.0%), insomnia (9.0%), weight increase (6.0%) for patients switching from oral, and weight increase (6.0%) and disease exacerbation (5.3%) for patients switching from depot medication. CONCLUSION: Patients with schizophrenia, unsatisfactorily treated with oral or depot conventional antipsychotics, showed improvement in symptom control, tolerability, and patient satisfaction after switching to RLAI.

Symptom control in children and adolescents with attention-deficit/hyperactivity disorder on switching from immediate-release MPH to OROS MPH Results of a 3-week open-label study.

AIM: The aim of this study was to assess the impact of switching from immediate-release (IR) methylphenidate (MPH) to OROS MPH (CONCERTA, a once-daily long-acting MPH formulation, in children and adolescents with attention-deficit/hyperactivity disorder (ADHD). METHODS: Subjects with ADHD aged 6-16 (n=105),who were stably maintained on their current IR MPH regimen (10-60 mg/day), were switched to 18, 36 or 54 mg OROS MPH once daily for 21 days, depending on pre-study daily MPH dose. ADHD symptoms were assessed by parents, teachers and investigators. RESULTS: By Day 21, parent/caregiver IOWA Conners ratings had decreased from baseline by 2.7 points to 5.2 (I/O), and by 1.8 points to 5.0 (O/D). Teacher IOWA Conners ratings were maintained. Decreases in IOWA Conners ratings are indicative of ADHD symptom improvement. Approximately 75% of parents and investigators rated therapy as good or excellent. OROS MPH therapy was well tolerated. CONCLUSIONS: Switching from IR MPH to OROS MPH maintained and may have improved symptom control in children and adolescents with ADHD, during the course of this study. The changes in parent/caregiver IOWA Conners ratings suggest that OROS MPH improves symptom control in the after-school period. This is consistent with the 12-h duration of action previously demonstrated for OROS MPH.

12-month efficacy and safety of OROS MPH in children and adolescents with attention-deficit/hyperactivity disorder switched from MPH.

PURPOSE: The aim of this study was to evaluate long-term clinical treatment with OROS methylphenidate (MPH) (Concerta) in children and adolescents with attention-deficit/hyperactivity disorder (ADHD) who had been previously treated with immediate release (IR) MPH. METHODS: Subjects aged 6-16 years (n=105) who were stable on IR MPH (10-60 mg/day) were switched to 18, 36 or 54 mg OROS MPH once daily for 21 days, depending on prestudy MPH dose. Subjects who benefited from OROS MPH could continue in a 12-month extension period. ADHD symptoms and treatment response were assessed by parents/caregivers and investigators. RESULTS: Out of 105 enrolled children, 101 completed the 21-day treatment phase. In all, 89 parents/caregivers (88.1%) wanted their child to continue with the study treatment into the extension phase, and 56 children (63 %) completed the 1-year trial. The parent/caregiver global assessment of satisfaction ranged from 49 to 69% during the extension phase, and 49 to 71% of investigators rated the treatment as adequate. Efficacy and satisfaction were found more commonly in patients in the older age group (10-16 years), those on a higher dose (36 mg or 54 mg) and with the predominantly inattentive ADHD subtype. OROS MPH was well tolerated. CONCLUSIONS: Children and adolescents can effectively and safely be switched from IR MPH to OROS MPH with improved symptom control and compliance.

Patients in the early phases of schizophrenia and schizoaffective disorders effectively treated with risperidone long-acting injectable.

The efficacy and safety of risperidone long-acting injectable (RLAI) was investigated in patients in the early phases of schizophrenia and schizoaffective disorders (< or = 3 years). Patients who required a treatment change received RLAI (2-weekly gluteal injections of 25, 37.5 or 50 mg, per clinical judgement), without an oral risperidone run-in phase.A total of 382 patients were included in this 6-month open-label study; 73% of patients completed the study. A total of 84% had schizophrenia with a median duration of 1.0 year since diagnosis. Previous medications were mainly atypical antipsychotics (70%) and depot neuroleptics (24%). The main reasons for treatment change were non-compliance (42%) and insufficient efficacy (31%) of previous medication. The total Positive and Negative Syndrome Scale (PANSS) and all its subscale scores improved significantly (p < or = 0.0001), with 40% of patients showing a 20% improvement on total PANSS. Global Assessment of Functioning, quality of life, patient satisfaction and movement disorders also improved significantly. Tolerability of RLAI was generally good and no unexpected adverse events were reported. The ensured delivery of medication with RLAI resulted in significant symptom improvement in this patient population. Direct initiation of RLAI is well accepted by patients. RLAI might represent a novel option for patients in the early phases of psychosis.

Direct transition to long-acting risperidone--analysis of long-term efficacy.

This report presents data from the extension phase of a 6-month trial that evaluated the efficacy of risperidone long-acting injectable (RLAI) in stable psychotic patients requiring a treatment change. Patients continued to receive RLAI every 2 weeks for a maximum of 12 months from study entry. Symptoms were assessed using the PANSS after 1, 3, 6, 9 and 12 months of treatment (or treatment endpoint). Remission of severity criteria were defined as < or =3 points in all PANSS items suggested by the Remission in Schizophrenia Working Group.715 patients (63% male) entered the extension phase and 508 completed the 12-month study. The mean PANSS total score at Day 0 was 74.9+/-22.7. This was significantly reduced after 1 month (67.7 +/-22.3, p< or =0.001), with continued improvements over the 12 months of the study until treatment endpoint (59.7+/-21.9). Significant improvements from Day 0 to endpoint were also seen in the scores for all PANSS subscales and symptom factors. The proportion of patients who met the PANSS severity criteria for remission increased from 29% at Day 0 to 60% at endpoint, and the proportion of patients who met these criteria for < or = 6 months increased from 24% at Month 6 to 45% at endpoint. Treatment with RLAI for up to 12 months provided significant and sustained improvements in symptom control in patients with schizophrenia. These improvements may help patients to achieve and remain in remission.

Long-acting risperidone in stable patients with schizoaffective disorder.

Oral and long-acting risperidone has been shown to be effective for acute and maintenance treatment of patients with schizoaffective disorders. The present analysis investigated the efficacy and tolerability of direct transition from other antipsychotics to risperidone long-acting injectable in patients with schizoaffective disorder. Patients aged > or = 18 years with schizoaffective disorder (DSM-IV), who required a change of medication, received risperidone long-acting injectable 25 mg (increased to 37.5 or 50 mg, if necessary) every 2 weeks for 6 months. The analysis included 249 patients (47% male; mean age 43 years), of whom 74% completed the 6-month study. Mean scores for the total Positive and Negative Syndrome Scale (PANSS) and all three subscales were significantly reduced from baseline to week 4 (p < 0.001), with further improvements until treatment endpoint. Significant improvements from baseline to endpoint were seen in the mood symptom domains of anxiety/depression (10.4+/-4.1 vs 8.7+/-3.9) and uncontrolled hostility/excitement (7.6+/-3.6 vs 6.9+/-3.8). Mean Global Assessment of Function (GAF) score improved significantly from 59.4+/-15.6 at baseline to 66.4+/-17.7 (p < 0.001) at endpoint. Of 87 patients hospitalized at baseline, 67% were discharged at endpoint. Both quality of life (SF-36) and satisfaction with treatment were improved significantly at endpoint. Total ESRS scores fell progressively throughout the study, and the reduction was already statistically significant (p < 0.001) at 4 weeks. Small but statistically significant (p < 0.001) mean shifts of 1.8% were seen in body weight and Body Mass Index (BMI). Patients with schizoaffective disorder derived several benefits from a change to risperidone long-acting injectable, including reductions in psychiatric symptoms (particularly the mood symptom domains) and a reduction in the severity of drug-induced neurological movement disorders.

Sustained improvement of clinical outcome with risperidone long-acting injectable in psychotic patients previously treated with olanzapine.

The efficacy and tolerability of risperidone long-acting injectable were investigated in patients with schizophrenia or other psychotic disorders who had previously been symptomatically stable on olanzapine treatment. Patients received risperidone long-acting injectable, 25 mg, by intramuscular injection every 2 weeks; the dose could be increased to 37.5 or 50 mg if necessary. Patients were transferred directly from their previous medication to risperidone long-acting injectable without a run-in period of oral risperidone treatment. Of 192 patients recruited, 134 patients (70%) completed the study. The principal reasons for discontinuation were withdrawal of consent (8%), adverse events (6%), insufficient response (5%) and non-compliance (4%). Risperidone long-acting injectable produced a significant improvement (p = 0.0001) in Positive and Negative Syndrome Scale (PANSS) total scores, from 74.2+/-21.3 at baseline to 65.8+/-21.4 at endpoint. There were also significant reductions in PANSS subscales (positive symptoms, negative symptoms, general psycho-pharmacology) and Marder factor scores. The Clinical Global Impression increased significantly from baseline to endpoint (p = 0.0001), as reflected by the increase in the proportion of patients rated as 'not ill' or 'borderline ill' from 10% at baseline to 21% at endpoint. Risperidone long-acting injectable was also associated with significant improvements in Global Assessment of Function, patient satisfaction with treatment, and quality of life, measured on the SF-36 scale. Movement disorders, measured on the Extrapyramidal Symptom Rating Scale, were significantly reduced following the change to risperidone long-acting injectable. Treatment with risperidone long-acting injectable was well tolerated, and no significant weight gain occurred during the study. This open study suggests that risperidone long-acting injectable produces symptomatic improvement in schizophrenia patients previously considered symptomatically stable with olanzapine, along with improvement in movement disorders. The combination of improved efficacy and good tolerability may have important implications for patient adherence to therapy and subsequent long-term outcomes.

A randomized controlled study of pergolide in patients with restless legs syndrome.

BACKGROUND: Open clinical trials indicate that low doses of pergolide, a long-acting D1 and D2 dopamine agonist, lead to a reduction in the symptoms of restless legs syndrome (RLS) with subjective improvement in sleep quality. OBJECTIVE: To assess the therapeutic efficacy of pergolide in improving sleep and subjective measures of well-being in patients with idiopathic RLS using polysomnography and clinical ratings. METHODS: In a randomized, double-blind, placebo-controlled crossover design we enrolled 30 patients with idiopathic RLS according to the criteria of the International RLS Study Group. All patients were free of psychoactive drugs for at least 2 weeks before the study. Patients were monitored using polysomnography, clinical ratings, and sleep diaries at baseline and at the end of a 4-week pergolide or placebo treatment period. The initial dosage of 0.05 mg pergolide was increased to the best subjective improvement paralleled by 20 mg domperidone tid. RESULTS: At a mean dosage of 0.51 mg pergolide as a single daily dose 2 hours before bedtime, there were fewer periodic leg movements per hour of time in bed (5.7 versus 54.9, p < 0.0001), and total sleep time was significantly longer (373 versus 261 minutes, p < 0.0001). Ratings of subjective sleep quality, quality of life, and severity of RLS were improved significantly without relevant adverse events. CONCLUSION: Pergolide given as a single low-to-medium bedtime dose in combination with domperidone provides a well-tolerated and effective treatment of sensorimotor symptoms and sleep disturbances in patients with primary RLS.

Dystrophin and its isoforms in a sympathetic ganglion of normal and dystrophic mdx mice: immunolocalization by electron microscopy and biochemical characterization.

In normal mouse superior cervical ganglion, dystrophin immunoreactivity is present in ganglionic neurons, satellite cells and Schwann cells. It is associated with several cytoplasmic organelles and specialized plasma membrane domains, including two types of structurally and functionally different intercellular junctions: synapses, where it is located at postsynaptic densities, and adherens junctions. Dystrophin immunostaining can be ascribed to the 427,000 mol. wt full-length dystrophin, as well as to the several dystrophin isoforms present in superior cervical ganglion, as revealed by western immunoblots. In mdx mouse superior cervical ganglion, which lacks the 427,000 mol. wt dystrophin, the unchanged pattern of dystrophin immunolabelling observed at several subcellular structures indicates the presence of dystrophin isoforms at these sites. Moreover, the absence of labelled adherens junctions indicates the presence of full-length dystrophin at this type of junction in the normal mouse superior cervical ganglion. The lower number of immunopositive postsynaptic densities in mdx mouse superior cervical ganglion than in normal mouse ganglion suggests the presence, in the latter, of postsynaptic densities with differently organized dystrophin cytoskeleton: some containing dystrophin isoforms alone or together with 427,000 mol. wt dystrophin, and others containing 427,000 mol. wt dystrophin alone.

Lipoic (thioctic) acid increases brain energy availability and skeletal muscle performance as shown by in vivo 31P-MRS in a patient with mitochondrial cytopathy.

A woman affected by chronic progressive external ophthalmoplegia and muscle mitochondrial DNA deletion was studied by phosphorus magnetic resonance spectroscopy (31P-MRS) prior to and after 1 and 7 months of treatment with oral lipoic acid. Before treatment a decreased phosphocreatine (PCr) content was found in the occipital lobes, accompanied by normal inorganic phosphate (Pi) level and cytosolic pH. Based on these findings, we found a high cytosolic adenosine diphosphate concentration [ADP] and high relative rate of energy metabolism together with a low phosphorylation potential. Muscle MRS showed an abnormal work-energy cost transfer function and a low rate of PCr recovery during the post-exercise period. All of these findings indicated a deficit of mitochondrial function in both brain and muscle. Treatment with 600 mg lipoic acid daily for 1 month resulted in a 55% increase of brain [PCr], 72% increase of phosphorylation potential, and a decrease of calculated [ADP] and rate of energy metabolism. After 7 months of treatment MRS data and mitochondrial function had improved further. Treatment with lipoate also led to a 64% increase in the initial slope of the work-energy cost transfer function in the working calf muscle and worsened the rate of PCr resynthesis during recovery. The patient reported subjective improvement of general conditions and muscle performance after therapy. Our results indicate that treatment with lipoate caused a relevant increase in levels of energy available in brain and skeletal muscle during exercise.

Oxidative stress and mitochondrial dysfunction in neurodegeneration.

In the last 4 years much progress has been made in the understanding of mitochondrial disorders. Point-mutations, deletions and depletion of the mitochondrial genome are associated with disorders like Leber's disease, MERRF (Myoclonus Epilepsia with Ragged Red Fibers), MELAS (mitochondrial Myopathy, Encephalopathy, Lactic acidosis and Stroke-like episodes) and several others. Recently, mitochondrial dysfunctions have been also related to neurodegenerative disorders like Parkinson's disease and to aging. Since the brain depends mostly on mitochondrial energy supply, mitochondrial dysfunctions may affect the nervous system more severely than other tissues causing or worsening diseases and playing a role in the biological deterioration of aging. Furthermore, the mitochondrial energy supply is associated with the production of highly reactive oxygen species. Ninety-five percent of the molecular oxygen is metabolized within the mitochondria by the electron-transport chain so that mitochondria are highly exposed to oxidative stress which may damage selected neuronal populations. Oxygen radicals created during respiration induce mitochondrial dysfunction which accelerates the production of more deleterious species of oxygen. The latter step further increases mitochondrial malfunction, thus intensifying and perpetuating the cycle. These two mechanisms combined may lead to cell death in brain and other tissues with high metabolic rate. Therefore, in neurodegenerative disorders such as Parkinson's disease mitochondrial dysfunction and oxidative stress may cause or worsen the clinical features.

Pyruvate dehydrogenase deficiency: molecular basis for intrafamilial heterogeneity.

Two half-brothers and their mother had symptomatic pyruvate dehydrogenase complex deficiency. The infants had severe congenital lactic acidosis, seizures, and apneic spells and died at the ages 3 and 4 months. The mother was less symptomatic with mental retardation, truncal ataxia, and dysarthria. The residual pyruvate dehydrogenase activities in cultured skin fibroblasts from the 2 infants and their mother were 7, 15, and 10% of control values. Immunoblot analysis showed negligible amounts of E1 alpha and E1 beta subunits of the complex. Northern blot analysis for the E1 alpha subunit showed normal results. In the 2 sons, complementary DNA sequence analysis revealed a cytosine to thymine mutation in exon 4, resulting in a change of arginine 127 to tryptophan in the E1 alpha subunit. Restriction enzyme analysis of the polymerase chain reaction product representing exon 4 of the E1 alpha gene revealed that the mother was a heterozygotes. Complementary DNA restriction analysis and methylation analysis of the X chromosome DXS255 loci revealed skewed activation of the mutant allele, consistent with the deficient pyruvate dehydrogenase activity in the mother's fibroblasts. The milder maternal phenotype is consistent with variable X-inactivation patterns in different organs of female heterozygotes.

Prion protein gene analysis in three kindreds with fatal familial insomnia (FFI): codon 178 mutation and codon 129 polymorphism.

Fatal familial insomnia (FFI) is a disease linked to a GAC(Asp)-->AAC(Asn) mutation in codon 178 of the prion protein (PrP) gene. FFI is characterized clinically by untreatable progressive insomnia, dysautonomia, and motor dysfunctions and is characterized pathologically by selective thalamic atrophy. We confirmed the 178Asn mutation in the PrP gene of a third FFI family of French ancestry. Three family members who are under 40 years of age and who inherited the mutation showed only reduced perfusion in the basal ganglia on single photon emission computerized tomography. Some FFI features differ from the clinical and neuropathologic findings associated with 178Asn reported elsewhere. However, additional intragenic mutations accounting for the phenotypic differences were not observed in two affected individuals. In other sporadic and familial forms of Creutzfeldt-Jakob disease and Gerstmann-Sträussler syndrome, Met or Val homozygosity at polymorphic codon 129 is associated with a more severe phenotype, younger age at onset, and faster progression. In FFI, young and old individuals at disease onset had 129Met/Val. Moreover, of five 178Asn individuals who are above age-at-onset range and who are well, two have 129Met and three have 129Met/Val, suggesting that polymorphic site 129 does not modulate FFI phenotypic expression. Genetic heterogeneity and environment may play an important role in inter- and intrafamilial variability of the 178Asn mutation.

Dystrophinopathy in two young boys with exercise-induced cramps and myoglobinuria.

Two young boys were referred for evaluation of metabolic myopathy because of elevated serum levels of creatine kinase, cramps and pigmenturia. Immunohistochemical studies of dystrophin in muscle biopsies showed reduced intensity of the stain with a patchy and discontinuous pattern in most fibers. In both patients dystrophin was undetectable by immunoblotting. DNA analysis of the dystrophin gene was not informative in one patient; in the other it revealed an in-frame deletion comprising exons 3-6. These observations suggest that the two patients are affected with an unusual phenotype of Becker muscular dystrophy. Dystrophin analysis should be included in the evaluation of patients with childhood-onset of recurrent myoglobinuria.

Mitochondrial DNA alterations as a source of human disorders.

The mitochondrial genome has an underdeveloped "DNA repair repertoire" compared with the nuclear genome, making the mitochondrial DNA more susceptible to mutations by endogenous factors such as defects of the mitochondrial polymerase itself, and by exogenous factors such as radiation and UV light. Increased sensitivity to mutagenic factors may account for the mitochondrial DNA polymorphism within ethnic groups and the mitochondrial diseases associated with all mitochondrial DNA mutations, including DNA depletion. The presence in highly developed organisms of a DNA repair repertoire less organized in the mitochondria than in the nuclei might be a source of biologic dysfunction relevant also to aging and cell death. Uncorrected mitochondrial DNA modifications may determine lethal and severe diseases or asymptomatic biochemical dysfunctions. Considering the long life span and the complex metabolism of highly developed cells, the tendency to produce and accumulate mitochondrial DNA mutations may assume a pathogenetic role with aging.

Production of single-stranded DNA for sequencing: an alternative approach.

We describe a simple procedure for the direct sequencing of single-stranded, PCR-amplified, target regions of human genomic DNA. At variance with previously reported procedures, purification of the desired double-stranded DNA was introduced. This additional step allowed the single-stranded amplification and sequencing of the target gene. This step is required for direct sequencing of some amplified regions of human genomic DNA. However, no individual technique seems suitable to generate and sequence all single-stranded DNA.