Chronic ethanol consumption increases myocardial mitochondrial DNA mutations: a potential contribution by mitochondrial topoisomerases.

AIMS: Alcoholic cardiomyopathy (ACM) presents as decreased myocardial contractility, arrhythmias and secondary non-ischemic dilated cardiomyopathy leading to heart failure. Mitochondrial dysfunction is known to have a significant role in the development and complications of ACM. This study investigated if chronic ethanol feeding promoted myocardial mitochondrial topoisomerase dysfunction as one underlying cause of mitochondrial DNA (mtDNA) damage and mitochondrial dysfunction in ACM. METHODS: The impact of chronic ethanol exposure on the myocardial mitochondria was examined in both neonatal cardiomyocytes using 50 mM ethanol for 6 days and in rats assigned to control or ethanol feeding groups for 4 months. RESULTS: Chronic ethanol feeding led to significant (P < 0.05) decreases in M-mode Fractional Shortening, ejection fraction, and the cardiac output index as well as increases in Tau. Ethanol feeding promoted mitochondrial dysfunction as evidenced by significantly decreased left ventricle cytochrome oxidase activity and decreases in mitochondrial protein content. Both in rats and in cultured cardiomyocytes, chronic ethanol presentation significantly increased mtDNA damage. Using isolated myocardial mitochondria, both mitochondrial topoisomerase-dependent DNA cleavage and DNA relaxation were significantly altered by ethanol feeding. CONCLUSION: Chronic ethanol feeding compromised cardiovascular and mitochondrial function as a result of a decline in mtDNA integrity that was in part the consequence of mitochondrial topoisomerase dysfunction. Understanding the regulation of the mitochondrial topoisomerases is critical for protection of mtDNA, not only for the management of alcoholic cardiomyopathy, but also for the many other clinical treatments that targets the topoisomerases in the alcoholic patient.

Membrane fluidity and lipid content of human spermatozoa selected by swim-up method.

In this work, we examined whether spermatozoa (spz) from normospermic fertile patients and selected by a swim-up (S-U) procedure had a particular membrane fluidity related to their maturity and their lipid content as compared with the sperm cells from the whole ejaculate (total sperm). Swim-up selected sperm had a reduced cytoplasmic space as revealed by a lower creatine kinase (CK) activity compared with total sperm (2 +/- 1 vs. 12 +/- 5 mUI/10(7) spz, p < 0.05). The cholesterol (Chol) and total phospholipid (PL) contents were significantly lower in S-U selected sperm than in total sperm (0.72 +/- 0.08 vs. 1.20 +/- 0.30 nmol/10(6) spz for Chol and 1.77 +/- 0.17 vs. 2.78 +/- 0.50 nmol/10(6) spz for PL, p < 0.05) and such a decrease was observed for the three major membrane PL: phosphatidylethanolamine (PE), phosphatidylcholine (PC) and sphingomyelin (SM). However, these decreases were not associated with a change in either Chol/PL or PC/(PC + PE) molar ratios. Membrane fluidity estimated by fluorescence polarization remained comparable between the S-U sperm fraction and total sperm (fluorescence polarization anisotropy, r, which is inversely proportional to the fluidity: 0.235 +/- 0.006 vs. 0.230 +/- 0.005). The sperm membrane fluidity obtained in normospermic patients was compared with abnormospermic ones (oligoasthenoteratospermia). In abnormospermic patients, the membrane fluidity was decreased in migrated spermatozoa compared with total sperm (anisotropy: 0.210 +/- 0.010 vs. 0.250 +/- 0.013, p < 0.01). Our data suggest that the S-U method selected a subpopulation of mature spermatozoa characterised by a low content of Chol and PL, likely related to a reduced membrane area. The fact that Chol/PL and PC/(PC + PE) molar ratios were unchanged shows a maintenance of the membrane quality. This was confirmed by the fluorescence anisotropy measurement showing no difference in plasma membrane fluidity between S-U selected sperm and total sperm. In abnormal semen the migrated spermatozoa had a lower fluidity compared with total sperm suggesting a defective sperm function. These results bring new elements characterizing the S-U selected spermatozoa.

The probability of preservation of a newly arisen gene duplicate.

Newly emerging data from genome sequencing projects suggest that gene duplication, often accompanied by genetic map changes, is a common and ongoing feature of all genomes. This raises the possibility that differential expansion/contraction of various genomic sequences may be just as important a mechanism of phenotypic evolution as changes at the nucleotide level. However, the population-genetic mechanisms responsible for the success vs. failure of newly arisen gene duplicates are poorly understood. We examine the influence of various aspects of gene structure, mutation rates, degree of linkage, and population size (N) on the joint fate of a newly arisen duplicate gene and its ancestral locus. Unless there is active selection against duplicate genes, the probability of permanent establishment of such genes is usually no less than 1/(4N) (half of the neutral expectation), and it can be orders of magnitude greater if neofunctionalizing mutations are common. The probability of a map change (reassignment of a key function of an ancestral locus to a new chromosomal location) induced by a newly arisen duplicate is also generally >1/(4N) for unlinked duplicates, suggesting that recurrent gene duplication and alternative silencing may be a common mechanism for generating microchromosomal rearrangements responsible for postreproductive isolating barriers among species. Relative to subfunctionalization, neofunctionalization is expected to become a progressively more important mechanism of duplicate-gene preservation in populations with increasing size. However, even in large populations, the probability of neofunctionalization scales only with the square of the selective advantage. Tight linkage also influences the probability of duplicate-gene preservation, increasing the probability of subfunctionalization but decreasing the probability of neofunctionalization.

Zebrafish comparative genomics and the origins of vertebrate chromosomes.

To help understand mechanisms of vertebrate genome evolution, we have compared zebrafish and tetrapod gene maps. It has been suggested that translocations are fixed more frequently than inversions in mammals. Gene maps showed that blocks of conserved syntenies between zebrafish and humans were large, but gene orders were frequently inverted and transposed. This shows that intrachromosomal rearrangements have been fixed more frequently than translocations. Duplicated chromosome segments suggest that a genome duplication occurred in ray-fin phylogeny, and comparative studies suggest that this event happened deep in the ancestry of teleost fish. Consideration of duplicate chromosome segments shows that at least 20% of duplicated gene pairs may be retained from this event. Despite genome duplication, zebrafish and humans have about the same number of chromosomes, and zebrafish chromosomes are mosaically orthologous to several human chromosomes. Is this because of an excess of chromosome fissions in the human lineage or an excess of chromosome fusions in the zebrafish lineage? Comparative analysis suggests that an excess of chromosome fissions in the tetrapod lineage may account for chromosome numbers and provides histories for several human chromosomes.

Extended longevity in Drosophila is consistently associated with a decrease in developmental viability.

It has proven relatively easy to select normal-lived strains of Drosophila for extended longevity in the laboratory. Long-lived strains have not been observed in the wild as yet. Of the various life-history traits that have been investigated for their role in modulating the evolution of extended longevity, none have yet shown a consistent or convincing relationship. Other than developmental time, the traits usually investigated in this regard are those associated with the adult phase of the life cycle. We assayed developmental timing and viability in six pairs of normal- and long-lived strains, four pairs of which are from previously described strains and two pairs of which are new strains that have been independently and recently selected. We find that the life-history trait most obviously associated with all our long-lived strains is a significantly reduced developmental viability, with the long-lived strains' having as much as twice the developmental lethality as do any of the normal-lived strains. The long-lived strains also pupate closer to the food, a behavior known to decrease fitness. Thus the reduced fitness of the long-lived strains appears to be due to both physiological and behavioral factors and may well explain why long lived strains are not usually found in the wild. The extension of longevity involves costs as well as benefits that, in this case, are borne by different individuals.

The probability of duplicate gene preservation by subfunctionalization.

It has often been argued that gene-duplication events are most commonly followed by a mutational event that silences one member of the pair, while on rare occasions both members of the pair are preserved as one acquires a mutation with a beneficial function and the other retains the original function. However, empirical evidence from genome duplication events suggests that gene duplicates are preserved in genomes far more commonly and for periods far in excess of the expectations under this model, and whereas some gene duplicates clearly evolve new functions, there is little evidence that this is the most common mechanism of duplicate-gene preservation. An alternative hypothesis is that gene duplicates are frequently preserved by subfunctionalization, whereby both members of a pair experience degenerative mutations that reduce their joint levels and patterns of activity to that of the single ancestral gene. We consider the ways in which the probability of duplicate-gene preservation by such complementary mutations is modified by aspects of gene structure, degree of linkage, mutation rates and effects, and population size. Even if most mutations cause complete loss-of-subfunction, the probability of duplicate-gene preservation can be appreciable if the long-term effective population size is on the order of 10(5) or smaller, especially if there are more than two independently mutable subfunctions per locus. Even a moderate incidence of partial loss-of-function mutations greatly elevates the probability of preservation. The model proposed herein leads to quantitative predictions that are consistent with observations on the frequency of long-term duplicate gene preservation and with observations that indicate that a common fate of the members of duplicate-gene pairs is the partitioning of tissue-specific patterns of expression of the ancestral gene.

Preservation of duplicate genes by complementary, degenerative mutations.

The origin of organismal complexity is generally thought to be tightly coupled to the evolution of new gene functions arising subsequent to gene duplication. Under the classical model for the evolution of duplicate genes, one member of the duplicated pair usually degenerates within a few million years by accumulating deleterious mutations, while the other duplicate retains the original function. This model further predicts that on rare occasions, one duplicate may acquire a new adaptive function, resulting in the preservation of both members of the pair, one with the new function and the other retaining the old. However, empirical data suggest that a much greater proportion of gene duplicates is preserved than predicted by the classical model. Here we present a new conceptual framework for understanding the evolution of duplicate genes that may help explain this conundrum. Focusing on the regulatory complexity of eukaryotic genes, we show how complementary degenerative mutations in different regulatory elements of duplicated genes can facilitate the preservation of both duplicates, thereby increasing long-term opportunities for the evolution of new gene functions. The duplication-degeneration-complementation (DDC) model predicts that (1) degenerative mutations in regulatory elements can increase rather than reduce the probability of duplicate gene preservation and (2) the usual mechanism of duplicate gene preservation is the partitioning of ancestral functions rather than the evolution of new functions. We present several examples (including analysis of a new engrailed gene in zebrafish) that appear to be consistent with the DDC model, and we suggest several analytical and experimental approaches for determining whether the complementary loss of gene subfunctions or the acquisition of novel functions are likely to be the primary mechanisms for the preservation of gene duplicates. For a newly duplicated paralog, survival depends on the outcome of the race between entropic decay and chance acquisition of an advantageous regulatory mutation. Sidow 1996(p. 717) On one hand, it may fix an advantageous allele giving it a slightly different, and selectable, function from its original copy. This initial fixation provides substantial protection against future fixation of null mutations, allowing additional mutations to accumulate that refine functional differentiation. Alternatively, a duplicate locus can instead first fix a null allele, becoming a pseudogene. Walsh 1995 (p. 426) Duplicated genes persist only if mutations create new and essential protein functions, an event that is predicted to occur rarely. Nadeau and Sankoff 1997 (p. 1259) Thus overall, with complex metazoans, the major mechanism for retention of ancient gene duplicates would appear to have been the acquisition of novel expression sites for developmental genes, with its accompanying opportunity for new gene roles underlying the progressive extension of development itself. Cooke et al. 1997 (p. 362)

Zebrafish hox clusters and vertebrate genome evolution.

HOX genes specify cell fate in the anterior-posterior axis of animal embryos. Invertebrate chordates have one HOX cluster, but mammals have four, suggesting that cluster duplication facilitated the evolution of vertebrate body plans. This report shows that zebrafish have seven hox clusters. Phylogenetic analysis and genetic mapping suggest a chromosome doubling event, probably by whole genome duplication, after the divergence of ray-finned and lobe-finned fishes but before the teleost radiation. Thus, teleosts, the most species-rich group of vertebrates, appear to have more copies of these developmental regulatory genes than do mammals, despite less complexity in the anterior-posterior axis.

Vertebrate genome evolution and the zebrafish gene map.

In chordate phylogeny, changes in the nervous system, jaws, and appendages transformed meek filter feeders into fearsome predators. Gene duplication is thought to promote such innovation. Vertebrate ancestors probably had single copies of genes now found in multiple copies in vertebrates and gene maps suggest that this occurred by polyploidization. It has been suggested that one genome duplication event occurred before, and one after the divergence of ray-finned and lobe-finned fishes. Holland et al., however, have argued that because various vertebrates have several HOX clusters, two rounds of duplication occurred before the origin of jawed fishes. Such gene-number data, however, do not distinguish between tandem duplications and polyploidization events, nor whether independent duplications occurred in different lineages. To investigate these matters, we mapped 144 zebrafish genes and compared the resulting map with mammalian maps. Comparison revealed large conserved chromosome segments. Because duplicated chromosome segments in zebrafish often correspond with specific chromosome segments in mammals, it is likely that two polyploidization events occurred prior to the divergence of fish and mammal lineages. This zebrafish gene map will facilitate molecular identification of mutated zebrafish genes, which can suggest functions for human genes known only by sequence.

valentino: a zebrafish gene required for normal hindbrain segmentation.

Mutational analysis can serve both to identify new genes essential for patterning embryonic development and to determine their functions. Here we describe the identification and phenotypic characterization of alleles of valentino, which we recovered in a genetic screen that sought to identify mutations in the zebrafish that disrupt region-specific gene expression patterns in the embryonic brain. valentino is required for normal hindbrain segmentation and the hindbrain of valentino mutant embryos is shortened by the length of one rhombomere. We demonstrate that valentino is required cell-autonomously in the development of rhombomeres 5 and 6, and propose that valentino functions in the subdivision and expansion of a common precursor region in the presumptive hindbrain into the definitive rhombomeres 5 and 6. These results provide genetic evidence for a two-segment periodicity in the hindbrain and suggest that this periodicity arises sequentially, through the specification and later subdivision of a two-rhombomere unit, or 'protosegment'.

Factors contributing to the plasticity of the extended longevity phenotypes of Drosophila.

A number of laboratories have constructed independently derived long-lived strains of Drosophila, each of which have similar but not identical patterns of variability in their adult longevity. Given the observed plasticity of longevity within each of these strains, it would be useful to review the operational and environmental factors that give rise to this phenotypic plasticity and ascertain whether they are common or strain specific. Our review of the more extensively analyzed strains suggests that the allelic composition of the initial genomes and the selection/transgene strategy employed yield extended longevity strains with superficially similar phenotypes but which are probably each the result of different proximal genetic mechanisms. This then offers a plausible explanation for the differential effects of various environmental factors on each strain's particular pattern of phenotypic plasticity. It also illustrates that the species has the potential to employ any one of a number of different proximal mechanisms, each of which give rise to a similar longevity phenotype.

Comparative biochemical and stress analysis of genetically selected Drosophila strains with different longevities.

We have performed a comparative analysis of the effects of age of reproduction on the biochemical (protein, lipid, and glycogen content) and stress resistance (ability to survive starvation, desiccation, and exogenous paraquat) parameters on 10 sister lines of five different Drosophila strains. Four pairs of these sister lines were selected under different regimens for either early or delayed reproduction; the fifth pair was maintained in a nonselected state and served as the baseline strain to which all others were compared. It is generally accepted that the early regimens give rise to short-lived phenotypes, whereas the delayed regimens give rise to long-lived phenotypes. Our results suggest that a mechanism involving lipid and starvation resistance is not operative in our long-lived strains. In addition, a mechanism involving glycogen content and desiccation resistance is only weakly supported. Finally, there is strong support for a mechanism that gives rise to enhanced paraquat resistance and therefore may involve regulatory changes in the pattern of ADS gene expression. In addition, the 15-day early age of reproduction regimen (M type) shows qualitatively similar responses to that of the late age at reproduction regimen (L type). These results suggest that correlations between biochemical traits and longevity must be interpreted with caution. We discuss possible reasons for these results, including the possibility of multiple mechanisms, each leading to a different extended longevity phenotype.

Larval regulation of adult longevity in a genetically-selected long-lived strain of Drosophila.

Our previous work has shown that the major genes involved in the expression of the extended-longevity phenotype are located on the third chromosome. Furthermore, their expression is negatively and positively influenced by chromosomes 2 and 1, respectively. In this report we show that the expression of the extended-longevity phenotype is dependent on the larval environment. A controlled chromosome substitution experiment was carried out using a strain selected for long life (L) and its parent (R) strain. Twenty different combinations of the three major chromosomes were conducted and their longevities were determined under both high (HD) and low (LD) larval density conditions. The extended-longevity phenotype was only expressed under HD conditions. The chromosome interactions were not apparent under LD conditions. Density-shift experiments delineate a critical period for expression of the extended-longevity phenotype, extending from 60 h after egg laying (AEL) to 96 h AEL, during which the developing animal must be exposed to HD conditions if the extended-longevity phenotype is to be expressed. The change from HD to LD conditions is accompanied by statistically significant increases in body weight. The possible role of a dietary restriction phenomenon is examined and the implications of these findings discussed. It is now apparent, however, that the extended-longevity phenotype in Drosophila is a developmental genetic process.

In-vivo transperitoneal fertilization.

We present the technique of in-vivo transperitoneal fertilization (IVTPF) as a first approach to infertility treatment in couples with male subfertility or unexplained factors. The technique is statistically less successful but also less invasive than either gamete intra-Fallopian transfer (GIFT) or in-vitro fertilization - embryo transfer (IVFET) and offers considerable advantages over intrauterine insemination (IUI). The IVTPF technique involves transperitoneal transfer of processed spermatozoa within the pouch of Douglas after induction of ovulation. We report our 4-year experience with IVTPF which includes 136 treatment cycles in 89 couples. Eight pregnancies were achieved in 89 patients (9%) and 136 treatment cycles (7%). Fifty-one patients (57%) received IVTPF for only one treatment cycle; seven of the eight IVTPF pregnancies occurred in this group. An ectopic pregnancy resulted in one of the eight IVTPF pregnancies (13%). The functional quality of the sperm in those couples who achieved pregnancy was statistically superior to those couples who did not conceive. However, pregnancy was also obtained in case of severe oligozoospermia. Based on our experience, we feel IVTPF to be a very reasonable first approach in patients with no pelvic pathology and with infertility secondary to male factors or unexplained causes.

Production and glycosylation of sperm constitutive proteins in the lizard Lacerta vivipara. Evolution during the reproductive period.

From epididymal fluid samples taken at three different times during the reproductive period (early April, late April, mid-May), the soluble proteins were separated with one dimensional electrophoresis on polyacrylamide gel. Their evolution was studied: firstly quantitatively, after staining with Coomassie blue, or, for one protein (the "L" protein), by immunodetection; secondly, according to their glycosylation after transfer to nitrocellulose and treatment with a set of labelled lectins: from Wheat germ, Ricinus communis, Lens culinaris, Asparagus pea or Canavalia ensiformis, with or without use of their specific inhibitor sugars. At least 15 proteins underwent a quantitative and/or qualitative evolution, mainly during the month of April. Protein "L" (19 kDa), which is androgen dependent and which fixates on to spermatozoa during their epididymal transit, appears to be little or not glycosylated. By contrast its accumulation in the epididymal canal increases considerably during the month of April. Five other proteins proved to be especially interesting because of their evolution during this same period, notably the MW 94, 67, 35, 29 and 25.5 kDa proteins. With the exception of the 67 kDa all the others increased quantitatively. All were decisively enriched in mannose or in methyl-mannoside residues. The proteins of MW 29 and 25.5 kDa were also enriched in galactose or N-acetyl galactosamine residues. These findings are of physiological significance since they are set up concomitantly with the acquisition of maximum motility of spermatozoa in the distal segment of the epididymis, and they coincide with a very great increase in testosteronemia.

Laser remote sensing of atmospheric ammonia using a CO2 lidar system.

A CO2 differential-absorption lidar system has been used for the remote sensing of ammonia in the atmosphere. For CO2 lidar returns backscattered from topographic targets at ranges up to 2.7 km, the path-averaged sensitivity of the DIAL system was 5 ppb of NH3 . Concentrations of atmospheric ammonia were found to vary during the day from undetectable levels (<5 ppb) to as high as 20 ppb, depending on temperature and humidity conditions.