Nutrition and its role in human evolution.

Our understanding of human evolution has improved rapidly over recent decades, facilitated by large-scale cataloguing of genomic variability amongst both modern and archaic humans. It seems clear that the evolution of the ancestors of chimpanzees and hominins separated 7-9 million years ago with some migration out of Africa by the earlier hominins; Homo sapiens slowly emerged as climate change resulted in drier, less forested African conditions. The African populations expanded and evolved in many different conditions with slow mutation and selection rates in the human genome, but with much more rapid mutation occurring in mitochondrial DNA. We now have evidence stretching back 300 000 years of humans in their current form, but there are clearly four very different large African language groups that correlate with population DNA differences. Then, about 50 000-100 000 years ago a small subset of modern humans also migrated out of Africa resulting in a persistent signature of more limited genetic diversity amongst non-African populations. Hybridization with archaic hominins occurred around this time such that all non-African modern humans possess some Neanderthal ancestry and Melanesian populations additionally possess some Denisovan ancestry. Human populations both within and outside Africa also adapted to diverse aspects of their local environment including altitude, climate, UV exposure, diet and pathogens, in some cases leaving clear signatures of patterns of genetic variation. Notable examples include haemoglobin changes conferring resistance to malaria, other immune changes and the skin adaptations favouring the synthesis of vitamin D. As humans migrated across Eurasia, further major mitochondrial changes occurred with some interbreeding with ancient hominins and the development of alcohol intolerance. More recently, an ability to retain lactase persistence into adulthood has evolved rapidly under the environmental stimulus of pastoralism with the ability to husband lactating ruminants. Increased amylase copy numbers seem to relate to the availability of starchy foods, whereas the capacity to desaturase and elongate monounsaturated fatty acids in different societies seems to be influenced by whether there is a lack of supply of readily available dietary sources of long-chain polyunsaturated fatty acids. The process of human evolution includes genetic drift and adaptation to local environments, in part through changes in mitochondrial and nuclear DNA. These genetic changes may underlie susceptibilities to some modern human pathologies including folate-responsive neural tube defects, diabetes, other age-related pathologies and mental health disorders.

Evidence for topologically protected surface states and a superconducting phase in [Tl4](Tl(1-x)Sn(x))Te3 using photoemission, specific heat, and magnetization measurements, and density functional theory.

We report the discovery of surface states in the perovskite superconductor [Tl4]TlTe3 (Tl5Te3) and its nonsuperconducting tin-doped derivative [Tl4](Tl0.4Sn0.6)Te3 as observed by angle-resolved photoemission spectroscopy. Density functional theory calculations predict that the surface states are protected by a Z2 topology of the bulk band structure. Specific heat and magnetization measurements show that Tl5Te3 has a superconducting volume fraction in excess of 95%. Thus Tl5Te3 is an ideal material in which to study the interplay of bulk band topology and superconductivity.

Maternally transmitted diabetes and deafness associated with a 10.4 kb mitochondrial DNA deletion.

Diabetes mellitus (DM) is one of the most common chronic disorders of children and adults. Several reports have suggested an increased incidence of maternal transmission in some forms of DM. Therefore, we tested a pedigree with maternally transmitted DM and deafness for mitochondrial DNA mutations and discovered a 10.4 kilobase (kb) mtDNA deletion. This deletion is unique because it is maternally inherited, removes the light strand origin (OL) of mtDNA replication, inhibits mitochondrial protein synthesis, and is not associated with the hallmarks of mtDNA deletion syndromes. This discovery demonstrates that DM can be caused by mtDNA mutations and suggests that some of the heterogeneity of this disease results from the novel features of mtDNA genetics.

Mitochondrial DNA deletions in human brain: regional variability and increase with advanced age.

We have examined the role of somatic mitochondrial DNA (mtDNA) mutations in human ageing by quantitating the accumulation of the common 4977 nucleotide pair (np) deletion (mtDNA4977) in the cortex, putamen and cerebellum. A significant increase in the mtDNA4977 deletion was seen in elderly individuals. In the cortex, the deleted to total mtDNA ratio ranged from 0.00023 to 0.012 in 67-77 year old brains and up to 0.034 in subjects over 80. In the putamen, the deletion level ranged from 0.0016 to 0.010 in 67 to 77 years old up to 0.12 in individuals over the age of 80. The cerebellum remained relatively devoid of mtDNA deletions. Similar changes were observed with a different 7436 np deletion. These changes suggest that somatic mtDNA deletions might contribute to the neurological impairment often associated with ageing.

A mouse model for mitochondrial myopathy and cardiomyopathy resulting from a deficiency in the heart/muscle isoform of the adenine nucleotide translocator.

In an attempt to create an animal model of tissue-specific mitochondrial disease, we generated 'knockout' mice deficient in the heart/muscle isoform of the adenine nucleotide translocator (Ant1). Histological and ultrastructural examination of skeletal muscle from Ant1 null mutants revealed ragged-red muscle fibers and a dramatic proliferation of mitochondria, while examination of the heart revealed cardiac hypertrophy with mitochondrial proliferation. Mitochondria isolated from mutant skeletal muscle exhibited a severe defect in coupled respiration. Ant1 mutant adults also had a resting serum lactate level fourfold higher than that of controls, indicative of metabolic acidosis. Significantly, mutant adults manifested severe exercise intolerance. Therefore, Ant1 mutant mice have the biochemical, histological, metabolic and physiological characteristics of mitochondrial myopathy and cardiomyopathy.

A novel neurological phenotype in mice lacking mitochondrial manganese superoxide dismutase.

Reactive oxygen species (ROS) have been implicated in a wide range of degenerative processes including amyotrophic lateral sclerosis, ischemic heart disease, Alzheimer disease, Parkinson disease and aging. ROS are generated by mitochondria as the toxic by-products of oxidative phosphorylation, their energy generating pathway. Genetic inactivation of the mitochondrial form of superoxide dismutase in mice results in dilated cardiomyopathy, hepatic lipid accumulation and early neonatal death. We report that treatment with the superoxide dismutase (SOD) mimetic Manganese 5, 10, 15, 20-tetrakis (4-benzoic acid) porphyrin (MnTBAP) rescues these Sod2tm1Cje(-/-) mutant mice from this systemic pathology and dramatically prolongs their survival. The animals instead develop a pronounced movement disorder progressing to total debilitation by three weeks of age. Neuropathologic evaluation reveals a striking spongiform degeneration of the cortex and specific brain stem nuclei associated with gliosis and intramyelinic vacuolization similar to that observed in cytotoxic edema and disorders associated with mitochondrial abnormalities such as Leighs disease and Canavans disease. We believe that due to the failure of MnTBAP to cross the blood brain barrier progressive neuropathology is caused by excessive mitochondrial production of ROS. Consequently, MnTBAP-treated Sod2tm1Cje(-/-) mice may provide an excellent model for examining the relationship between free radicals and neurodegenerative diseases and for screening new drugs to treat these disorders.

Dilated cardiomyopathy and neonatal lethality in mutant mice lacking manganese superoxide dismutase.

The Sod2 gene for Mn-superoxide dismutase (MnSOD), an intramitochondrial free radical scavenging enzyme that is the first line of defense against superoxide produced as a byproduct of oxidative phosphorylation, was inactivated by homologous recombination. Homozygous mutant mice die within the first 10 days of life with a dilated cardiomyopathy, accumulation of lipid in liver and skeletal muscle, and metabolic acidosis. Cytochemical analysis revealed a severe reduction in succinate dehydrogenase (complex II) and aconitase (a TCA cycle enzyme) activities in the heart and, to a lesser extent, in other organs. These findings indicate that MnSOD is required for normal biological function of tissues by maintaining the integrity of mitochondrial enzymes susceptible to direct inactivation by superoxide.

Cytoplasmic transfer of chloramphenicol resistance in human tissue culture cells.

The cytoplasmic inheritance of human chloramphenicol (cap) resistance has been demonstrated by removing the nuclei of cells of the CAP-resistant HeLa strain 296-1 (enucleation) and fusing them to a CAP-sensitive HeLa strain lacking nuclear thymidine kinase. Plating the fusion products in bromodeoxyuridine and CAP resulted in the growth of about 150 colonies/10(6) parent cells plated. Permanent cell lines (cybrids) grown from such fusions have been designated HEB. A recloned HEB cybrid (HEB7A) has also been enucleated and fused to hypoxanthine phosphoribosyl transferase (HPRT)-deficient HeLa cells (S3AG1) and HPRT-deficient lymphocytes (WAL-2A). Cybrids were selected in thioguanine and CAP. In the fusion of enucleated (en) HEB7A to S3AG1, 1,200 colonies/10(6) parents were observed. Fusion of enHEB7A to WAL-2A was done in mass culture and cybrids were obtained on three separate occasions. In every case the parental controls were negative. All isolates tested from the above fusions have the CAP-resistant characteristics, in vivo and in vitro, of the enucleated parent and the nuclear characteristics of the CAP-sensitive parent, such as chromosome number, morphology, and specific isozyme and chromosome markers. Therefore, it can be concluded that CAP resistance is coded in the cytoplasm and not in the nucleus of 296-1 cells. Furthermore, this resistance can be transferred to cells of widely different origin and differentiated state. These studies represent the first genetic evidence of cytoplasmic inheritance in human cells.

Mitochondrial genetics: a paradigm for aging and degenerative diseases?

Studies of diseases caused by mitochondrial DNA mutations suggest that a variety of degenerative processes may be associated with defects in oxidative phosphorylation (OXPHOS). Application of this hypothesis has provided new insights into such diverse clinical problems as ischemic heart disease, late-onset diabetes, Parkinson's disease, Alzheimer's disease, and aging.

Mitochondrial diseases in man and mouse.

Over the past 10 years, mitochondrial defects have been implicated in a wide variety of degenerative diseases, aging, and cancer. Studies on patients with these diseases have revealed much about the complexities of mitochondrial genetics, which involves an interplay between mutations in the mitochondrial and nuclear genomes. However, the pathophysiology of mitochondrial diseases has remained perplexing. The essential role of mitochondrial oxidative phosphorylation in cellular energy production, the generation of reactive oxygen species, and the initiation of apoptosis has suggested a number of novel mechanisms for mitochondrial pathology. The importance and interrelationship of these functions are now being studied in mouse models of mitochondrial disease.

Extension of life-span with superoxide dismutase/catalase mimetics.

We tested the theory that reactive oxygen species cause aging. We augmented the natural antioxidant systems of Caenorhabditis elegans with small synthetic superoxide dismutase/catalase mimetics. Treatment of wild-type worms increased their mean life-span by a mean of 44 percent, and treatment of prematurely aging worms resulted in normalization of their life-span (a 67 percent increase). It appears that oxidative stress is a major determinant of life-span and that it can be counteracted by pharmacological intervention.

Mitochondrial DNA mutation associated with Leber's hereditary optic neuropathy.

Leber's hereditary optic neuropathy is a maternally inherited disease resulting in optic nerve degeneration and cardiac dysrhythmia. A mitochondrial DNA replacement mutation was identified that correlated with this disease in multiple families. This mutation converted a highly conserved arginine to a histidine at codon 340 in the NADH dehydrogenase subunit 4 gene and eliminated an Sfa NI site, thus providing a simple diagnostic test. This finding demonstrated that a nucleotide change in a mitochondrial DNA energy production gene can result in a neurological disease.

Rapid identification of mitochondrial DNA (mtDNA) mutations in neuromuscular disorders by using surveyor strategy.

Mutations of mitochondrial genome are responsible for respiratory chain defects in numerous patients. We have used a strategy, based on the use of a mismatch-specific DNA endonuclease named " Surveyor Nuclease", for screening the entire mtDNA in a group of 50 patients with neuromuscular features, suggesting a respiratory chain dysfunction. We identified mtDNA mutations in 20% of patients (10/50). Among the identified mutations, four are not found in any mitochondrial database and have not been reported previously. We also confirm that mtDNA polymorphisms are frequently found in a heteroplasmic state (15 different polymorphisms were identified among which five were novel).

Mitochondrial mutations in cancer.

The metabolism of solid tumors is associated with high lactate production while growing in oxygen (aerobic glycolysis) suggesting that tumors may have defects in mitochondrial function. The mitochondria produce cellular energy by oxidative phosphorylation (OXPHOS), generate reactive oxygen species (ROS) as a by-product, and regulate apoptosis via the mitochondrial permeability transition pore (mtPTP). The mitochondria are assembled from both nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) genes. The mtDNA codes for 37 genes essential of OXPHOS, is present in thousands of copies per cell, and has a very high mutations rate. In humans, severe mtDNA mutations result in multisystem disease, while some functional population-specific polymorphisms appear to have permitted humans to adapt to new environments. Mutations in the nDNA-encoded mitochondrial genes for fumarate hydratase and succinate dehydrogenase have been linked to uterine leiomyomas and paragangliomas, and cancer cells have been shown to induce hexokinase II which harnesses OXPHOS adenosine triphosphate (ATP) production to drive glycolysis. Germline mtDNA mutations at nucleotides 10398 and 16189 have been associated with breast cancer and endometrial cancer. Tumor mtDNA somatic mutations range from severe insertion-deletion and chain termination mutations to mild missense mutations. Surprisingly, of the 190 tumor-specific somatic mtDNA mutations reported, 72% are also mtDNA sequence variants found in the general population. These include 52% of the tumor somatic mRNA missense mutations, 83% of the tRNA mutations, 38% of the rRNA mutations, and 85% of the control region mutations. Some associations might reflect mtDNA sequencing errors, but analysis of several of the tumor-specific somatic missense mutations with population counterparts appear legitimate. Therefore, mtDNA mutations in tumors may fall into two main classes: (1) severe mutations that inhibit OXPHOS, increase ROS production and promote tumor cell proliferation and (2) milder mutations that may permit tumors to adapt to new environments. The former may be lost during subsequent tumor oxygenation while the latter may become fixed. Hence, mitochondrial dysfunction does appear to be a factor in cancer etiology, an insight that may suggest new approaches for diagnosis and treatment.

Mitochondrial diseases: genotype versus phenotype.

Recently, a variety of degenerative diseases have been attributed to mutations in mitochondrial DNA. Even though these mutations are inherited and present throughout the body, they frequently cause late-onset, tissue-specific disease. This may be explained by a combination of the tissue-specific accumulation of somatic mtDNA mutations with age and the variation between tissues in the expression of nuclear genes that encode mitochondrial functions.

Mitochondrial DNA mutations and neuromuscular disease.

Mitochondrial DNA mutations have been identified in patients with certain neuromuscular diseases. Point mutations have been associated with maternally inherited diseases, while deletions have been identified in some 'spontaneous' cases.

Assignment of the chloramphenicol resistance gene to mitochondrial deoxyribonucleic acid and analysis of its expression in cultured human cells.

The mitochondrial deoxyribonucleic acids (mtDNA's) from human HeLa and HT1080 cells differed in their restriction endonuclease cleavage patterns for HaeII, HaeIII, and HhaI. HaeII digestion yielded a 9-kilobase fragment in HT1080, which was replaced by 4.5-, 2.4-, and 2.1-kilobase fragments in HeLa. HaeIII and HhaI yielded distinctive 1.35- and 0.68-kilobase HeLa fragments. These restriction endonuclease polymorphisms were used as mtDNA markers in HeLa-HT1080 cybrid and hybrid crosses involving the cytoplasmic chloramphenicol resistance mutation was used. mtDNA's were purified and digested with the restriction endonucleases, the fragments were separated on agarose gels, and the bands were detected by ethidium bromide staining and Southern transfer analysis. Three cybrids and four hybrids (four expressing HeLa and three expressing HT1080 chloramphenicol resistance) contained 2- to 10-fold excesses of the mtDNA of the chloramphenicol-resistant parent. One cybrid, which was permitted to segregate chloramphenicol resistance and was then rechallenged with chloramphenicol, had approximately equal proportions of the two mtDNA's. Only one hybrid was discordant. These results indicated that chloramphenicol resistance is encoded in mtDNA and that expression of chloramphenicol resistance is related to the ratio of chloramphenicol-resistant and -sensitive genomes within cells.

Assignment of two mitochondrially synthesized polypeptides to human mitochondrial DNA and their use in the study of intracellular mitochondrial interaction.

Two mitochondrially synthesized marker polypeptides, MV-1 and MV-2, were found in human HeLa and HT1080 cells. These were assigned to the mitochondrial DNA in HeLa-HT1080 cybrids and hybrids by demonstrating their linkage to cytoplasmic genetic markers. These markers include mitochondrial DNA restriction site polymorphisms and resistance to chloramphenicol, an inhibitor of mitochondrial protein synthesis. In the absence of chloramphenicol, the expression of MV-1 and MV-2 in cybrids and hybrids was found to be directly proportional to the ratio of the parental mitochondrial DNAs. In the presence of chloramphenicol, the marker polypeptide linked to the chloramphenicol-sensitive mitochondrial DNA continued to be expressed. This demonstrated that resistant and sensitive mitochondrial DNAs can cooperate within a cell for gene expression and that the CAP-resistant allele was dominant or codominant to sensitive. Such cooperation suggests that mitochondrial DNAs can be exchanged between mitochondria.