Nuclear integrations of mitochondrial DNA in primates: inference of associated mutational events

To infer the possible mutational events taking place along the interorganellar transfer of genetic material from mitochondria to the nucleus, four integrations of mitochondrial DNA (mtDNA) in the human genome were characterized together with their flanking nuclear sequences. By determining their presence/absence status in different primate species, these integrations were inferred to have occurred on the lineages leading to catarrhines (Old World monkeys and hominoids), to hominoids and to humans, respectively. In case of a polymorphic state, with respect to its presence in a certain species, each preintegration sequence was either cloned in the same species or in a primate taxon that branched off before the transfer of the mtDNA to the nucleus took place. For the four mtDNA integrations presented here, random mobilization of the mtDNA and differing mechanisms for generating free ends in the nuclear target sequences can be inferred. Additionally, no common sequence features at the preintegration sites could be observed for these integrations. Moreover, the comparisons of the sites before and after integration suggest different ways of integration. Thus, mtDNA integrations represent unique molecular recombinations in the evolutionary history and can, according to their presence/absence status in different species, help to determine the branching order in phylogenetic trees.     

Phantom mutation hotspots in human mitochondrial DNA

Phantom mutations are systematic artifacts generated in the course of the sequencing process. Contra common belief these artificial mutations are nearly ubiquitous in sequencing results, albeit at frequencies that may vary dramatically. The amount of artifacts depends not only on the sort of automated sequencer and sequencing chemistry employed, but also on other lab-specific factors. An experimental study executed on four samples under various combinations of sequencing conditions revealed a number of phantom mutations occurring at the same sites of mitochondrial DNA (mtDNA) repeatedly. To confirm these and identify further hotspots for artifacts, > 5000 mtDNA electropherograms were screened for artificial patterns. Further, > 30 000 published hypervariable segment I sequences were compared at potential hotspots for phantom mutations, especially for variation at positions 16085 and 16197. Resequencing of several samples confirmed the artificial nature of these and other polymorphisms in the original publications. Single-strand sequencing, as typically executed in medical and anthropological studies, is thus highly vulnerable to this kind of artifacts. In particular, phantom mutation hotspots could easily lead to misidentification of somatic mutations and to misinterpretations in all kinds of clinical mtDNA studies.     

Automated amplification and sequencing of human mitochondrial DNA

Part of the human mitochondrial D-loop region was amplified by two successive rounds of polymerase chain reaction (PCR) amplification. In the second PCR reaction, nested primers were used, of which one contained the M13-21 universal primer sequence. By using nonequal concentrations of primers in the second amplification, single-stranded DNA was generated. This was then sequenced directly by the diodeoxy chain termination method using dye-labelled universal sequencing primers in conjunction with a fluorescence-based DNA sequencer. This enabled a 403-base-pair hypervariable segment of the D-loop region to be readily sequenced in a single reaction. This paper describes a protocol which enables mitochondrial sequence information to be generated rapidly and automatically. It is likely to be of importance in forensic analysis where the DNA is too degraded or of insufficient quantity to be analysed by other techniques.     

Electrophoretic analysis of sequence variability in three mitochondrial DNA regions for ascaridoid parasites of human and animal health significance

Sequence variability in three mitochondrial DNA (mtDNA) regions, namely cytochrome c oxidase subunit 1 (cox1), NADH dehydrogenase subunits 1 and 4 (nad1 and nad4), among and within Toxocara canis, T. cati, T. malaysiensis, T. vitulorum and Toxascaris leonina from different geographical origins was examined by a mutation-scanning approach. A portion of the cox1 gene (pcox1), a portion of the nad1 and nad4 genes (pnad1 and pnad4) were amplified separately from individual ascaridoid nematodes by polymerase chain reaction and the amplicons analyzed by single-strand conformation polymorphism (SSCP). Representative samples displaying sequence variation in SSCP profiles were subjected to sequencing in order to define genetic markers for their specific identification and differentiation. While the intra-specific sequence variations within each of the five ascaridoid species were 0.2-3.7% for pcox1, 0-2.8% for pnad1 and 0-2.3% for pnad4, the inter-specific sequence differences were significantly higher, being 7.9-12.9% for pcox1, 10.7-21.1% for pnad1 and 12.9-21.7% for pnad4, respectively. Phylogenetic analyses based on the combined sequences of pcox1, pnad1 and pnad4 revealed that the recently described species T. malaysiensis was more closely related to T. cati than to T. canis. These findings provided mtDNA evidence for the validity of T. malaysiensis and also demonstrated clearly the usefulness and attributes of the mutation-scanning sequencing approach for studying the population genetic structures of these and other nematodes of socio-economic importance.     

Identification and relative quantification of specific glycation sites in human serum albumin

Glycation (or non-enzymatic glycosylation) is a common non-enzymatic covalent modification of human proteins. Glucose, the highest concentrated monosaccharide in blood, can reversibly react with amino groups of proteins to form Schiff bases that can rearrange to form relatively stable Amadori products. These can be further oxidized to advanced glycation end products (AGEs). Here, we analyzed the glycation patterns of human serum albumin (HSA) in plasma samples obtained from five patients with type 2 diabetes mellitus. Therefore, glycated peptides from a tryptic digest of plasma were enriched with m-aminophenylboronic acid (mAPBA) affinity chromatography. The glycated peptides were then further separated in the second dimension by RP-HPLC coupled on-line to an electrospray ionization (ESI) tandem mass spectrometer (MS/MS). Altogether, 18 Amadori peptides, encompassing 40% of the HSA sequence, were identified. The majority of the peptides were detected and relatively quantified in all five samples with a high reproducibility among the replicas. Eleven Lys-residues were glycated at similar quantities in all samples, with glycation site Lys₅₄₉ (K_Am(Glc)QTALVELVK) being the most abundant. In conclusion, the established mAPBA/nanoRP-HPLC-ESI-MS/MS approach could reproducibly identify and quantify glycation sites in plasma samples, potentially useful in diagnosis and therapeutic control.     

Identification of the active sites and mechanism for partial methane oxidation to methanol over copper-exchanged CHA zeolites

To make methane a suitable energy carrier and transport less costly, it is an urgent and challenging task for us to convert methane to liquid under mild conditions efficiently. In this study, we explored partial methane oxidation to methanol by density functional theory(DFT) calculations using a hybrid functional(HSE06) with van der Waals(vdW) interactions. The stabilities of different active sites over SSZ-13 and SAPO-34, two CHA type zeolites, are thoroughly investigated by ab initio molecular dynamics(AIMD) simulations and ab initio thermodynamics analyses. Four possible active sites, namely [CuOHCu]~(2+), [Cu(OH)_2Cu]~(2+),[CuOCu]~(2+) and [CuOH]~+, are identified stable. Methane-to-methanol reaction mechanisms are further studied upon these most stable active sites, among which [CuOCu]~(2+) and [CuOH]~+ are proved to be reactive. The migration of species among zeolite pores are also discussed, which accounts for the activity on [CuOH]~+ sites. This concept may represent a more complete picture of catalytic reactions over zeolites in general.     

Identification of N-linked glycosylation sites in human nephrin using mass spectrometry

Nephrin is a type-1 transmembrane glycoprotein and the first identified principal component of the glomerular filtration barrier. Ten potential asparagine (N)-linked glycosylation sites have been predicted within the ectodomain of nephrin. However, it is not known which of these potential sites are indeed glycosylated and what type of glycans are involved. In this work, we have identified the terminal sugar residues on the ectodomain of human nephrin and utilized a straightforward and reliable mass spectrometry-based approach to selectively identify which of the ten predicted sites are glycosylated. Purified recombinant nephrin was subjected to peptide-N-glycosidase F (PNGase F) to enzymatically remove all the N-linked glycans. Since PNGase F is an amidase, the asparagine residues from which the glycans have been removed are deaminated to aspartic acid residues, resulting in an increase in the peptide mass with 1 mass unit. Following trypsin digestion, deglycosylated tryptic peptides were selectively identified by MALDI-TOF MS and their sequence was confirmed by tandem TOF/TOF. The 1 Da increase in peptide mass for each asparagine-to-aspartic acid conversion, along with preferential cleavage of the amide bond carboxyl-terminal to aspartic acid residues in peptides where the charge is immobilized by an arginine residue, was used as a diagnostic signature to identify the glycosylated peptides. Thus, nine of ten potential glycosylation sites in nephrin were experimentally proven to be modified by N-linked glycosylation.     

Extremely high levels of human mitochondrial DNA heteroplasmy in single hair roots

For many years it has been assumed that the vast majority of mitochondrial genomes of a single individual are identical, both in the same tissue and within different tissues. Incidences of heteroplasmy (i.e., the occurrence of two or more codominating types of molecules within the mitochondrial DNA population of the same individual) were thought to be extremely rare. This study strongly supports the thesis that heteroplasmy is a principle, rather than an exception, in mitochondrial DNA genetics. During direct sequencing of the first hypervariable segment of the human mitochondrial control region (HV1) in 100 single hair roots obtained from 35 individuals, 24 different heteroplasmic positions were identified. Unusually high levels of heteroplasmy (up to six positions in the HV1 region) were encountered in two individuals. Two individuals related in maternal lineage shared the same heteroplasmic positions. Moreover, highly variable levels of heteroplasmy were observed even among roots from the same individual. The most probable mechanisms involved in generating so many mismatches are mutations occurring presumably in the female germline, followed by differential segregation of mitotypes during the development of individual hairs. Generally, heteroplasmy complicates sequence comparisons in mitochondrial DNA testing performed for forensic purposes, but in some cases it can substantially increase the discriminating power of the analysis.     

Identification of specific protein carbonylation sites in model oxidations of human serum albumin

Human serum albumin (HSA) was subjected to oxidative stress and the locations of the resulting protein carbonyls were determined using mass spectrometry in conjunction with a hydrazide labeling scheme. To model oxidative stress, HSA samples were subjected to metal-catalyzed oxidation (MCO) conditions or treated with hypochlorous acid (HOCl). Oxidation led to the conversion of lysine residues to 2-aminoadipic semi-aldehyde residues, which were subsequently labeled with biotin hydrazide. Analysis of the tryptic peptides from the samples indicates that the oxidations are highly selective. Under MCO conditions, only two of the 59 lysine residues appeared to be modified (Lys-97 and Lys-186). With HOCl, five different lysine modification sites were identified (Lys-130, Lys-257, Lys-438, Lys-499, and Lys-598). These results strongly suggest that the preferred site of modification is dependent on the nature of the oxidant and that the process relies on specific structural motifs in the protein to direct the oxidation. The high selectivity seen here provides insights into the factors that in vivo drive the selective carbonylation of specific proteins in systems under oxidative stress.     

Identification of the tyrosine nitration sites in human endothelial nitric oxide synthase by liquid chromatography-mass spectrometry

The formation of nitric oxide (NO) in biological systems has led to the discovery of a number of post- translational protein modifications that can affect biological conditions such as vasodilation. Studies both from our laboratory and others have shown that beside its effect on cGMP generation from soluble guanylate cylcase, NO can produce protein modifications through both S-nitrosylation of cysteine residues. Previously, we have identified the potential S-nitrosylation sites on endothelial NO synthase (eNOS). Thus, the goal of this study was to further increase our understanding of reactive nitrogen protein modifications of eNOS by identifing tyrosine residues within eNOS that are susceptible to nitration in vitro. To accomplish this, nitration was carried out using tetranitromethane followed by tryptic digest of the protein. The resulting tryptic peptides were analyzed by liquid chromatography/mass spectrometry (LC/MS) and the position of nitrated tyrosines in eNOS were identified. The eNOS sequence contains 30 tyrosine residues and our data indicate that multiple tyrosine residues are capable of being nitrated. We could identify 25 of the 30 residues in our tryptic digests and 19 of these were susceptible to nitration. Interstingly, our data identified four tyrosine residues that can be modified by nitration that are located in the region of eNOS responsible for the binding to heat shock protein 90 (Hsp90), which is responsible for ensuring efficient coupling of eNOS.     

Primary photodamage sites and mitochondrial events after Foscan photosensitization of MCF-7 human breast cancer cells

To determine the initial photodamage sites of Foscan-mediated photodynamic treatment, we evaluated the enzymatic activities in selected organelles immediately after light exposure of MCF-7 cells. The measurements indicated that the enzymes located in the Golgi apparatus (uridine 5'-diphosphate galactosyl transferase) and in the endoplasmic reticulum (ER) (nicotinamide adenine dinucleotide [reduced] [NADH] cytochrome c [cyt c] reductase) are inactivated by the treatment, whereas mitochondrial marker enzymes (cyt c oxidase and dehydrogenases) were unaffected. This indicates that the ER and the Golgi apparatus are the primary intracellular sites damaged by Foscan-mediated PDT in MCF-7 cells. We further investigated whether the specific mitochondria events could be associated with Foscan photoinduced cell death. The dose response profiles of mitochondrial depolarization and cytochrome c release immediately after Foscan-based PDT were very different from that of overall cell death. By 24 h post-PDT the fluence dependency was strikingly similar for both mitochondrial alterations and cell death. Therefore, although mitochondria are not directly affected by the treatment, they can be strongly implicated in Foscan-mediated MCF-7 cell death by late and indirect mechanism.     

Subcomplexes of mitochondrial complex V reveal mutations in mitochondrial DNA

Complex V, site of the final step in oxidative phosphorylation, uses the proton gradient across the inner mitochondrial membrane for the production of ATP. It is a multi‐subunit complex composed of a catalytic domain (F₁) and a membrane domain (F₀) linked by two stalks. Subcomplexes of complex V containing the F₁ domain have previously been reported in small series of patients. We report the results in tissue samples and/or cultured skin fibroblasts studied by blue native PAGE followed by activity staining in the gel. Catalytically active subcomplexes of complex V were detected in 66 tissues originating from 53 patients. In 29 of the latter (55%), a mitochondrial DNA (mtDNA) defect was identified. Twelve patients had a pathogenic point mutation in a mitochondrial tRNA, one a large mtDNA deletion, 12 showed mtDNA depletion and four had a mutation in the MT‐ATP6 gene. We conclude that the presence of subcomplexes of complex V is a valuable indicator in the detection of mtDNA defects.     

Profiling of length heteroplasmies in the human mitochondrial DNA control regions from blood cells in the Korean population

The length heteroplasmies in the hypervariable (HV) regions of mitochondrial DNA (mtDNA) from blood cells were examined in 57 healthy Korean donors. Interestingly, all the healthy Korean subjects displayed length heteroplasmies in both the HV1 and HV2 regions. Closer examination of the HV2 length heteroplasmies indicated that most of these donors (84%) exhibited a minimal 303-315 homopolymeric C (poly-C) tract frameshift of 1 bp (mixture of one major and minor mtDNA type). Sixteen percent of the donors however had poly-C tract frameshifts of 2 bp or more. The donor group with major length variants (two or more frameshifts) had about a two-fold decrease in mtDNA copy number compared with the group exhibiting only a 1 bp frameshift. This result supports the possibility that a severe frameshift in the 303-315 poly-C tract may also cause the impairment of mtDNA replication in hematopoietic tissue.     

Identification and partial characterization of an unusual distribution of the photosensitizer meta-tetrahydroxyphenyl chlorin (temoporfin) in human plasma

Temoporfin (m-THPC) is an extremely powerful photosensitizing drug, more than 100-fold more photocytotoxic than Photofrin and many other drugs. The reasons for this are not yet known but are likely to be associated with the mechanism of uptake of the drug and its intratumoral and intracellular localization. Uptake itself is likely to be dependent upon the plasma binding of the drug following administration. In the current work, we have shown that the addition of m-THPC to human plasma in vitro at clinically relevant doses of sensitizer and administration solvent (diluant) gives rise to a protein-binding pattern quite different to that of Photofrin and other hydrophobic drugs as judged by density-gradient ultracentrifugation. Analysis of the binding immediately after addition to human plasma has shown that lipoprotein binding accounts for only a minor proportion of the sensitizer, which is mainly associated with a high-density protein fraction that is not coincident with serum albumin. The m-THPC protein complex does not fluoresce significantly even on dilution. This binding pattern is highly dependent on administration conditions and storage. Over a period of 6-8 h at 37 degrees C the m-THPC that is associated with this unidentified fraction redistributes to the plasma lipoproteins. Plasma collected from rats after intravenous administration of m-THPC also contains this low fluorescent complex, showing that this phenomenon is not limited to human plasma and also occurs in vivo. It is postulated that the m-THPC bound to the unknown protein fraction is highly aggregated and that it is likely to be taken up into tissues in this form. This unusual uptake may possibly be associated with the very high activity of m-THPC and also to the recent finding of a second peak in the plasma pharmacokinetics of the drug.     

Dideoxy fingerprinting of low-level nucleotide variation in mitochondrial DNA of the human blood fluke, Schistosoma japonicum.

Dideoxy fingerprinting (ddF) is an efficient method for the detection of sequence changes in polymerase chain reaction (PCR)-amplified DNA fragments. It is a hybrid between single-strand conformation polymorphism (SSCP) and dideoxy sequencing, employing only one dideoxynucleotide in the reaction. We report the application of ddF for the display of low-level nucleotide variation in the mitochondrial (mt) NADH dehydrogenase subunit 1 (ND1) (404 bp) in the human blood fluke, Schistosoma japonicum. Variant samples differing by 1-6 nucleotides could be readily differentiated from one another by their characteristic and reproducible ddF profiles. The findings indicate the potential of this method to screen for point mutation in any parasite genes.     

Usefulness of microchip electrophoresis for the analysis of mitochondrial DNA in forensic and ancient DNA studies

We evaluate the usefulness of a commercially available microchip CE (MCE) device in different genetic identification studies performed with mitochondrial DNA (mtDNA) targets, including the haplotype analysis of HVR1 and HVR2 and the study of interspecies diversity of cytochrome b (Cyt b) and 16S ribosomal RNA (16S rRNA) mitochondrial genes in forensic and ancient DNA samples. The MCE commercial system tested in this study proved to be a fast and sensitive detection method of length heteroplasmy in cytosine stretches produced by 16 189T>C transitions in HVR1 and by 309.1 and 309.2 C-insertions in HVR2. Moreover, the quantitative analysis of PCR amplicons performed by LIF allowed normalizing the amplicon input in the sequencing reactions, improving the overall quality of sequence data. These quantitative data in combination with the quantification of genomic mtDNA by real-time PCR has been successfully used to evaluate the PCR efficiency and detection limit of full sequencing methods of different mtDNA targets. The quantification of amplicons also provided a method for the rapid evaluation of PCR efficiency of multiplex-PCR versus singleplex-PCR to amplify short HV1 amplicons (around 100 bp) from severely degraded ancient DNA samples. The combination of human-specific (Cyt b) and universal (16S rRNA) mtDNA primer sets in a single PCR reaction followed by MCE detection offers a very rapid and simple screening test to differentiate between human and nonhuman hair forensic samples. This method was also very efficient with degraded DNA templates from forensic hair and bone samples, because of its applicability to detect small amplicon sizes. Future possibilities of MCE in forensic DNA typing, including nuclear STRs and SNP profiling are suggested.     

Identification of (2-aminopropyl)benzofuran and its metabolites in human urine

An HPLC?HRMS method for the detection of the benzofuran analog of amphetamine and its metabolites in human urine using both library search and target search of the most probable metabolites based on their calculated accurate weights and fragmentation regularities is proposed. The proposed method is confirmatory. Gas chromatography–electron impact mass spectrometry with library search is proposed as the main screening method.     

Identification of chromium and nickel sites in zinc phosphate glasses

Optical and EPR investigations of chromium and nickel doped zinc phosphate glasses are carried out at room temperature to evaluate crystal field, spin-Hamiltonian and bonding parameters. For Cr³⁺ ion, the site symmetry is ascribed to a distorted octahedron, whereas the Ni²⁺ ion is located in a near octahedral site. In addition, the bonding parameters suggest covalent nature for Cr³⁺ and a moderate covalent nature for Ni²⁺ in the zinc phosphate glasses.     

Mapping the sites for selective oxidation of guanines in DNA

The effective energy of a positive charge when it is localized at a specific guanine nucleobase in DNA was calculated using density functional theory. The results demonstrate that the efficiency of a guanine to act as a hole-trap in DNA strongly depends on the nature of the flanking nucleobases. The presence of a pyrimidine base at the 3' position adjacent to a guanine significantly increases the localization energy of the positive charge. The calculated distributions of a positive charge in sequences of two or three adjacent guanines, flanked by other nucleobases, provide an explanation for experimental literature data on the site-selective oxidation of DNA.     

Multiplex amplified product-length polymorphism analysis for rapid detection of human mitochondrial DNA variations.

A number of mutations in coding and noncoding regions of mitochondrial DNA (mtDNA) have previously been studied. In the present study, we simultaneously typed six mutation sites in the coding region by use of amplified product-length polymorphism (APLP) analysis. The mtDNA variations of 2471 individuals from 20 populations of Japanese, Korean, Chinese, and German were examined and classified into 18 haplotypes. Two of these haplotypes, B1 (estimated ancestral haplotype) and C1, were distributed among all populations tested. However, the haplotypes A1, A2, B2, B3, and C2 were mostly restricted to the Mongoloid populations, whereas haplotypes B5 and C5 appeared almost exclusively in the German population. Phylogenetic analysis by the neighbor-joining method revealed that the Japanese populations were more closely related to each other than to the other East Asian populations surveyed. The multiplex APLP method is suitable for large-scale screening studies of mtDNA variability because it is both rapid and economical.