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Detection of metazoan species as a public health issue: simple methods for the validation of food safety and quality.

Species identification represents a critical issue in food chain safety and quality control. Several procedures are available to detect animal proteins in cattle feed or to trace transgenic foods. The most effective approach is based on the use of DNA as a marker. Amplification of DNA provides rapid, sensitive and specific protocols. Several target genes can be used, but new insights come from the mitochondrial genome, which is naturally amplified in each cell and shows a remarkable resistance to degradation. These are key points when analysing complex matrices such as foods, animal feedstuff or environmental samples. Traceability is important to prevent BSE or to monitor novel foods, such as genetically modified organisms. Amplification is commonly performed, but it requires expertise and a molecular biology laboratory to perform restriction analysis, electrophoresis or gel staining for the visualisation of results. Hereby, we consider a strategy based on multiple nested amplification and reverse hybridisation assay that virtually requires only a thermocycler and a water bath. The protocol is rapid and simple and can simultaneously detect different species in a DNA sample. This promising approach allows microarray developments, opening up to further perspectives. An international application has been published under the patent cooperation treaty. Presently, a ban on feeding ruminants on cattle-derived proteins is in force in Europe and USA. The identification of metazoan traces in a sample is not only a mere preventive measure for BSE, but represents a possible screening system for monitoring biotechnology products and procedures, as well as a quality control strategy to assure consumer's rights.

Animals↗

The development and evaluation of a homogeneous immunoassay for the isoenzymes of aspartate aminotransferase.

Purified isoenzymes of aspartate aminotransferase (AST) from human liver (mitochondrial) and erythrocytes (cytoplasmic) were used to elicit antisera in rabbits. Each antiserum was characterized for titer and specificity. Complexes formed upon addition of each isoenzyme to its specific antiserum were demonstrated to be catalytically inactive. Results obtained when either filtration or centrifugation was used for separating the complexes (heterogeneous assay) were comparable to those obtained when the complexes were not separated (homogeneous assay) from the mixture before assay. A quality control system was designed to monitor specificity in addition to the usual parameters. The precision for the inhibition of cytoplasmic (CV 4.8%) and mitochondrial (CV 3.5%) isoenzymes was within that of the enzymatic assay. Several parametric conditions of the enzyme-antibody reactions were examined, and the assay was adapted for semi-automation. The homogeneous assay was evaluated with a series of pseudo specimens containing known mixtures of pure isoenzymes to determine the extent of recovery (99.8%) for a particular isoenzyme in the presence of varying concentrations of the other isoenzyme. In addition, sera from patients having elevated AST concentrations were examined for isoenzyme contributions to total AST activity. A mean recovery of 96% was obtained for these specimens.

Antibody Specificity↗

Structure and function of Hsp78, the mitochondrial ClpB homolog.

The cellular role of Hsp100/Clp chaperones in maintaining protein stability is based on two functional aspects. Under normal growth conditions they represent components of cellular protein quality control machineries that selectively remove damaged or misfolded polypeptides in cooperation with specific proteases. After thermal stress, proteins of the ClpB subfamily have the unique ability to directly resolubilize aggregated polypeptides in concert with Hsp70-type chaperones, leading to the recovery of enzymatic activity. Hsp78, the homolog of the bacterial chaperone ClpB in mitochondria of eukaryotic organisms, participates in both protective activities. Hsp78 is involved in conferring thermotolerance to the mitochondrial compartment but also participates in protein degradation by the matrix protease Pim1. Despite the high sequence conservation between Hsp78 and ClpB, an analysis of the structural properties revealed significant differences. The identified mitochondrial Hsp78s do not contain N-terminal substrate-binding domains. In addition, formation of the oligomeric chaperone complex was more variable as anticipated from the studies with bacterial ClpB. Hsp78 predominantly formed a trimeric complex under in vivo conditions. Hence, mitochondrial Hsp78s form a distinct subgroup of the ClpB chaperone family, exhibiting specific structural and functional properties.

Amino Acid Sequence↗

Artificial recombination in forensic mtDNA population databases.

Artificial recombination of two or more mitochondrial DNA fragments from different samples would constitute a serious cause of error in forensic DNA typing, and yet one can demonstrate that such events have happened in the preparation of several published mtDNA databases. Focussed database searches, phylogenetic analysis, and network representations can highlight mosaic patterns and thus pinpoint sample mix-up. Therefore, we suggest that this approach should be applied to data prior to publication in order to uncover such errors in time.

DNA Fingerprinting↗

Real-time DNA quantification of nuclear and mitochondrial DNA in forensic analysis.

The rapid development of molecular genetic analysis tools has made it possible to analyze most biological materialfound at the scene of a crime. Evidence materials containing DNA quantities too low to be analyzed using nuclear markers can be analyzed using the highly abundant mtDNA. However, there is a shortage of sensitive nDNA and mtDNA quantification assays. In this study, an assay for the quantification of very small amounts of DNA, based on the real-time Taq-Man assay, has been developed. This analysis will provide an estimate of the total number of nDNA copies and the total number of mtDNA molecules in a particular evidence material. The quantification is easy to perform, fast, and requires a minimum of the valuable DNA extracted from the evidence materiaL The results will aid in the evaluation of whether the specific sample is suitable for nDNA or mtDNA analysis. Furthermore, the optimal amount of DNA to be used in further analysis can be estimated ensuring that the analysis is successful and that the DNA is retained for future independent analysis. This assay has significant advantages over existing techniques because of its high sensitivity, accuracy, and the combined analysis of nDNA and mtDNA. Moreover, it has the potential to provide additional information about the presence of inhibitors in forensic samples. Subsequent mitochondrial and nuclear analysis of quantified samples illustrated the potential to predict the number of DNA copies required for a successful analysis in a certain typing assay.

Cell Nucleus↗

Short-term aerobic training response in chronic myopathies.

We have previously demonstrated that patients with mitochondrial myopathies can benefit from short-term aerobic exercise training. In this study, we compared the responses to short-term aerobic training of patients with mitochondrial myopathies, patients with nonmetabolic myopathies, and sedentary normal subjects. Training consisted of 8 weeks of treadmill exercise at 70% to 85% of estimated maximum heart rate reserve. All groups showed significant improvements in estimated aerobic capacity as well as heart rate and blood lactate at submaximal exercise intensities. The increase in estimated aerobic capacity was greater in the mitochondrial myopathy patients than in the other two groups. Phosphorus magnetic resonance spectroscopy demonstrated increased oxidative capacity of muscle in patients with mitochondrial myopathies in response to this training but not in patients with other, nonmetabolic myopathies or sedentary control subjects. A self-assessed measurement of functional status (SF-36) suggested improved quality of life associated with the training. This study demonstrates that short-term aerobic training at low intensity can benefit patients with nonmetabolic myopathies but to a lesser extent than patients with mitochondrial myopathies.

Adolescent↗

Polymerase chain reaction inhibition assay documenting the amotosalen-based photochemical pathogen inactivation process of platelet concentrates.

BACKGROUND: The INTERCEPT Blood System (Baxter Healthcare Corp.) for platelets (PLTs) uses amotosalen-HCl (S-59) in conjunction with ultraviolet A (UVA) light to inactivate contaminating pathogens by modifying the nucleic acids of pathogens. The success of this photochemical treatment (PCT) process can be documented indirectly with a high-performance liquid chromatography assay measuring the photodegradation of amotosalen and measurement of the UVA light dose delivered by the illumination system. STUDY DESIGN AND METHODS: To develop an assay that documents the success of PCT directly on the effector molecule DNA, the effect of PCT on PLT-derived mitochondrial DNA (mtDNA) was examined. mtDNA-specific polymerase chain reaction (PCR) assays were tested with regard to their susceptibility for PCT, their reliability in terms of PCR performance, and the absence of polymorphic sites in primer hybridization loci. RESULTS: Suitable PCR amplification targets were found in the regions of 16S rDNA, cytochrome c oxidase I, and cytochrome c oxidase III of mitochondria. Amplicon sizes between 868 and 1248 bp gave consistent signals before PCT and complete inhibition of the PCR signal after PCT. Amplicons of less than 300 bp were found to be transparent to PCT. CONCLUSION: Based on PCT-mediated mtDNA modifications in PLTs, a PCR inhibition assay was established with a large amplicon documenting the success of PCT and a small amplicon serving as an internal control.

Adenine↗

The EDNAP mitochondrial DNA population database (EMPOP) collaborative exercises: organisation, results and perspectives.

This paper presents an overview of the organisation and the results of the collaborative exercises (CE) of the European DNA Profiling (EDNAP) Group's mitochondrial DNA population database project (EMPOP). The aim of the collaborative exercises was to determine whether uniformity of mtDNA sequencing results could be achieved among different laboratories. These were asked to sequence either the complete mtDNA control region or the two hypervariable regions HVI (16024-16365) and HVII (73-340) from DNA extracts, buccal swabs or bloodstains, proceeding in accordance with the protocol and strategies used in each individual laboratory. The results of the collaborative exercises were employed to identify possible sources of errors that could arise during the analysis and interpretation of mtDNA profiles. These findings were taken as a basis to tentatively make suitable arrangements for the construction of a high quality mtDNA database. One hundred fifty mtDNA profiles were submitted to the evaluating laboratory, and disaccording profiles were classified into four groups corresponding to the source of error: clerical errors, sample mix-ups, contaminations and discrepancies with respect to the mtDNA nomenclature. Overall, 14 disaccording haplotypes (16 individual errors) were observed. The errors included 10 clerical errors, 3 interpretation problems, 2 cases of sample mix-up and 1 case of point heteroplasmic mixture, where the 2 sequencing reactions brought inconsistent base calls. This corresponds to an error rate of 10.7% in a virtual mtDNA database consisting of the collaborative exercise results. However, this estimate is still conservative compared to conclusions drawn by authors of meanwhile numerous publications critically reviewing published mtDNA population databases. Our results and earlier published concerns strongly emphasize the need for appropriate safety regulations when mtDNA profiles are compiled for database purposes in order to accomplish the high standard required for mtDNA databases that are used in the forensic context.

Clinical Laboratory Techniques↗

Fertilization and elimination of the paternal mitochondrial genome.

With rare exceptions, mammalian mitochondria are inherited through the female. This probably serves to minimize lethal cytoplasmic gene competition and to prevent the inheritance of sperm mitochondrial DNA that has been subject to degradation by free radicals. In general, organisms are intolerant of mitochondrial heteroplasmy and, when this occurs in humans, it frequently presents as progressive and lethal bioenergetic or neurological disease. The mitochondria of spermatozoa are specifically destroyed by proteolysis in early embryonic development, in mice at the 4- to 8-cell transition. While there are concerns in human assisted reproduction that microinjection of abnormal or immature sperm cells could lead to lasting harm in the offspring through transmission of abnormal mitochondria, there is no clinical evidence to support this. There is more potential for harm through attempts to 'rescue' poor quality oocytes by cytoplasmic or nuclear transfer, as it is not currently possible to control the final fate of the donated mitochondria in relation to nuclear-mitochondrial interactions or the embryonic axes. Moreover, the balance between nuclear and mitochondrial genes and the role of cytoplasmic factors in epigenesis are still poorly understood. The future challenge for biologists is to comprehend the nature of the selective destruction of paternal mitochondria, as it appears to be a species-specific recognition phenomenon.

Animals↗

Search for mitochondrial A3243G tRNA(Leu) mutation in Polish patients with type 2 diabetes mellitus.

BACKGROUND: The influences of genetic and environmental factors form a clinical picture of type 2 diabetes mellitus. Genetic studies of type 2 diabetes mellitus become increasingly important. The knowledge of the molecular background of type 2 diabetes has been growing rapidly over recent years. One of the forms of the disease defined on the molecular level is maternally inherited type 2 diabetes mellitus. This diabetes, which is frequently accompanied by hearing impairment of deafness (maternally inherited diabetes with deafness-MIDD), was linked with sequence differences in mitochondrial DNA. The most frequent cause of MIDD is A3243G substitution in a mitochondrial tRNA(Leu) gene. While this mutation was identified in different races in several populations, it is still important and valuable to evaluate its prevalence in various ethnic groups. The aim of the project was to determine the prevalence of A3243G substitution in a mitochondrial tRNA(Leu) gene among Polish diabetic subjects. MATERIAL AND METHODS: In total 129 individuals, with type 2 diabetes and 12 with gestational diabetes were selected for this study. Two techniques based on restriction fragment length polymorphism (RFLP) method were used to screen for A3243G mutation. In the first approach, non-radioactive PCR reactions of mitochondrial DNA region of interest were performed using DNA of the study participants. This was followed by Apa I restriction enzyme digestion of the PCR product. Subsequently an electrophoretic separation was done on 2% agarose gel with ethidium bromide staining. In the second, more sensitive, modification of RFLP, [alpha 32P]dCTP was used for internal primer labeling and the electrophoresis was done on acrylamide gel. A positive sample was used to control the quality of the genotyping. RESULTS: In both approaches none of the samples, except for the positive control, showed the evidence of the G variant. CONCLUSIONS: In summary, the A3243G mutation in mitochondrial tRNA(Leu) gene is not a frequent cause of diabetes in the Polish population. Further screening of enlarging study group is necessary to fully determine the prevalence of this mutation in our population. This, together with the search for other mitochondrial mutations, should allow to fully determine the prevalence of MIDD and its specific molecular background in the Polish population.

Adult↗

Ram sperm selection by a dextran/swim-up procedure increases fertilization rates following intrauterine insemination in superovulated ewes.

The objective of this study was to compare the efficacy of 2 dextran/swim-up media to increase the sperm quality parameters and the maintenance of these parameters at 15 degrees C and 30 degrees C over 6 hours. Additionally, this study examined whether differences in sperm quality reflect different reproductive efficiencies following intrauterine insemination in superovulated ewes. The study involved 2 selected samples (SS) obtained by dextran/swim-up, performed either with (SS+) or without (SS-) capacitating compounds, and a control sample consisting of raw semen diluted in the same medium. The efficacies of the swim-up sperm selection procedures were similar in both media, and no significant differences were found among the evaluated parameters. Conversely, we found important differences between selected and control samples. Sperm motility, viability (as assessed by carboxifluorescein diacetate/propidium iodide [PI] staining), and mitochondrial activity (as assessed by rhodamine 123/PI) were significantly higher in the selected samples than in the control. Additionally, following incubation at 15 degrees C, the preservation of sperm quality was significantly better in the selected samples than in the control samples. After 6 hours of incubation at 15 degrees C, selected samples had a motility value of 46%, which was significantly (P < .001) higher than the value observed in control samples (27%). The percentage of viable cells observed after 6 hours of incubation at 15 degrees C was significantly (P < .0001) higher in selected samples than in the control samples. Furthermore, after 2 hours of incubation at 30 degrees C, swim-up samples had viability values that were significantly (P < .0001) higher than those of the control samples. SS+ and SS- samples did not differ significantly in spermatozoa yield, sperm quality, or survival. Differences between selected samples and controls were reflected in the fertilization rate obtained following intrauterine insemination in superovulated ewes that experienced a 52-hour interval between progestagen removal and artificial insemination. A restricted criterion for fertilization rate evaluation was established, and only the percentage of embryos recovered from the uterine horns 6 days after insemination was considered with respect to the total number of corpora lutea counted in the ovaries. The fertilization rate of SS- samples (50%) was significantly higher (P > .001) than those of the SS+ (2%) and control samples (5%).

Animals↗

Quality control of translation through the kinetic discrimination of tRNAs in the network of aminoacyl-tRNA synthetases.

It is known that each aminoacyl-tRNA synthetase (aaRS) specifically recognizes its cognate tRNAs to ensure the correct translation of the genetic information. However, we had previously demonstrated that mammalian mitochondrial seryl-tRNA synthetase (mt SerRS) can markedly misacylate mitochondrial (mt) tRNA(Gln). To investigate extensively misacylation reactions in mammalian mitochondrion, we purified overall twenty-two mt tRNAs from bovine liver, and determined their misacylation activities using mt SerRS. In addition to tRNA(Gln), tRNA(Ala) and tRNA(Asn) showed weak but significant serylation activities, which raises the possibility that each mammalian mt aaRS can misacylate several non-cognate mt tRNAs in varying degrees, but translational fidelity might be maintained by kinetic discrimination of tRNAs in the network of aaRSs.

Amino Acyl-tRNA Synthetases↗

Crystallization of the bovine ADP/ATP carrier is critically dependent upon the detergent-to-protein ratio.

The ADP/ATP carrier is an integral membrane protein located in the mitochondrial inner membrane. It mediates the exchange of cytosolic ADP for ATP generated in the mitochondrial matrix. Here, the purification and crystallization of the bovine ADP/ATP carrier in complex with the inhibitor carboxyatractyloside in the presence of LAPAO, an aminoxide detergent, is reported. High-quality crystals were only obtained when excess detergent was removed to reach a controlled detergent-to-protein ratio.

Animals↗

Quantitative and Kinetic Proteomics Reveal ApoE Isoform-dependent Proteostasis Adaptations in Mouse Brain.

Apolipoprotein E (ApoE) polymorphisms modify the risk of Alzheimer's disease with ApoE4 strongly increasing and ApoE2 modestly decreasing risk relative to the control ApoE3. To investigate how ApoE isoforms alter risk, we measured changes in proteome homeostasis in transgenic mice expressing a human ApoE gene (isoform 2, 3, or 4). The regulation of each protein's homeostasis is observed by measuring turnover rate and abundance for that protein. We identified 4849 proteins and tested for ApoE isoform-dependent changes in the homeostatic regulation of ~2700 ontologies. In the brain, we found that ApoE4 and ApoE2 both lead to modified regulation of mitochondrial membrane proteins relative to the wild-type control ApoE3. In ApoE4 mice, lack of cohesion between mitochondrial membrane and matrix proteins suggests that dysregulation of proteasome and autophagy is reducing protein quality. In ApoE2, proteins of the mitochondrial matrix and the membrane, including oxidative phosphorylation complexes, had a similar increase in degradation which suggests coordinated replacement of the entire organelle. In the liver we did not observe these changes suggesting that the ApoE-effect on proteostasis is amplified in the brain relative to other tissues. Our findings underscore the utility of combining protein abundance and turnover rates to decipher proteome regulatory mechanisms and their potential role in biology.

Animals↗

Low oocyte mitochondrial DNA content in ovarian insufficiency.

BACKGROUND: Mitochondrial biogenesis and bioenergetics play an important role in oocyte maturation and embryo development. We have investigated the relationship between defective mitochondrial biogenesis and the lack of oocyte maturity observed during IVF procedures with patients suffering from ovarian dystrophy and ovarian insufficiency. METHODS: We used real-time quantitative PCR to quantify mitochondrial DNA (mtDNA) in 116 oocytes obtained from 47 women undergoing the ICSI procedure. We compared the mtDNA content of oocytes from women with a normal ovarian profile with that of oocytes from women with ovarian dystrophy and ovarian insufficiency. RESULTS: We found an average of 256,000 +/- 213,000 mitochondrial genomes per cell. The mean mtDNA copy number was not significantly different in ovarian dystrophy compared with controls, but it was significantly lower in oocytes from women with ovarian insufficiency (100,000 +/- 99,000, P < 0.0001). CONCLUSIONS: Our results suggest that low mtDNA content is associated with the impaired oocyte quality observed in ovarian insufficiency.

Case-Control Studies↗

Controlled clinical trial of dichloroacetate for treatment of congenital lactic acidosis in children.

OBJECTIVE: Open-label studies indicate that oral dichloroacetate (DCA) may be effective in treating patients with congenital lactic acidosis. We tested this hypothesis by conducting the first double-blind, randomized, control trial of DCA in this disease. METHODS: Forty-three patients who ranged in age from 0.9 to 19 years were enrolled. All patients had persistent or intermittent hyperlactatemia, and most had severe psychomotor delay. Eleven patients had pyruvate dehydrogenase deficiency, 25 patients had 1 or more defects in enzymes of the respiratory chain, and 7 patients had a mutation in mitochondrial DNA. Patients were preconditioned on placebo for 6 months and then were randomly assigned to receive an additional 6 months of placebo or DCA, at a dose of 12.5 mg/kg every 12 hours. The primary outcome results were (1) a Global Assessment of Treatment Efficacy, which incorporated tests of neuromuscular and behavioral function and quality of life; (2) linear growth; (3) blood lactate concentration in the fasted state and after a carbohydrate meal; (4) frequency and severity of intercurrent illnesses and hospitalizations; and (5) safety, including tests of liver and peripheral nerve function. OUTCOME: There were no significant differences in Global Assessment of Treatment Efficacy scores, linear growth, or the frequency or severity of intercurrent illnesses. DCA significantly decreased the rise in blood lactate caused by carbohydrate feeding. Chronic DCA administration was associated with a fall in plasma clearance of the drug and with a rise in the urinary excretion of the tyrosine catabolite maleylacetone and the heme precursor delta-aminolevulinate. CONCLUSIONS: In this highly heterogeneous population of children with congenital lactic acidosis, oral DCA for 6 months was well tolerated and blunted the postprandial increase in circulating lactate. However, it did not improve neurologic or other measures of clinical outcome.

Acidosis, Lactic↗