The influence of the thymine C5 methyl group on spontaneous base pair breathing in DNA

Sequences of four or more AT base pairs without a 5'-TA-3' step, so-called A-tracts, influence the global properties of DNA by causing curvature of the helix axis if phased with the helical repeat and also influence nucleosome packaging. Hence it is interesting to understand this phenomenon on the molecular level, and numerous studies have been devoted to investigations of dynamical and structural features of A-tract DNA. It was early observed that anomalously slow base pair-opening kinetics were a striking physical property unique to DNA A-tracts (Leroy, J. L., Charretier, E., Kochoyan, M., and Gueron, M. (1988) Biochemistry 27, 8894-8898). Furthermore, a strong correlation between DNA curvature and anomalously slow base pair-opening dynamics was found. In the present work it is shown, using imino proton exchange measurements by NMR spectroscopy that the main contribution to the dampening of the base pair-opening fluctuations in A-tracts comes from the C5 methylation of the thymine base. Because the methyl group has been shown to have a very limited effect on the DNA curvature as well as the structure of the DNA helix, the thymine C5 methyl group stabilizes the helix directly. Empirical potential energy calculations show that methylation of the tract improves the stacking energy of a base pair with its neighbors in the tract by 3-4 kcal/mol.     

Stable variants of sperm aneuploidy among healthy men show associations between germinal and somatic aneuploidy

Repeated semen specimens from healthy men were analyzed by sperm fluorescence in situ hybridization (FISH), to identify men who consistently produced elevated frequencies of aneuploid sperm and to determine whether men who were identified as stable variants of sperm aneuploidy also exhibited higher frequencies of aneuploidy in their peripheral blood lymphocytes. Seven semen specimens were provided by each of 15 men over a 2-year period and were evaluated by the X-Y-8 multicolor sperm FISH method (i.e., approximately 1,050,000 sperm were analyzed from 105 specimens). Three men were identified as stable aneuploidy variants producing significantly higher frequencies of XY, disomy X, disomy Y, disomy 8, and/or diploid sperm over time. In addition, one man and three men were identified as sperm-morphology and sperm-motility variants, respectively. Strong correlations were found between the frequencies of sperm with autosomal and sex-chromosome aneuploidies and between the two types of meiosis II diploidy; but not between sperm aneuploidy and semen quality. A significant association was found between the frequencies of sex-chromosome aneuploidies in sperm and lymphocytes in a subset of 10 men (r2=0.67, P=.004), especially between XY sperm and sex-chromosome aneuploidy in lymphocytes (r2=0.70, P=.003). These findings suggest that certain apparently healthy men can produce significantly higher frequencies of both aneuploid sperm and lymphocytes. Serious long-term somatic and reproductive health consequences may include increased risks of aneuploidy-related somatic diseases and of having children with paternally transmitted aneuploidies, such as Klinefelter, Turner, triple-X, and XYY syndromes.     

Diverse requirements for Notch signalling in mammals

The Notch signalling pathway has a central role in a wide variety of developmental processes and it is not therefore surprising that mutations in components of this pathway can cause dramatic human genetic disorders. One developmental process in which the Notch pathway is involved at multiple levels is somitogenesis, the mechanism by which the embryo is divided into segments that ultimately form structures such as the axial skeleton and skeletal muscle of the trunk. We are investigating the human genetic disorder spondylocostal dysplasia (SCD), which is a group of malsegmentation syndromes that occur when this process is disrupted. Mutations in the Notch ligand DELTA-LIKE 3 (DLL3) are responsible for cases of autosomal recessive SCD type I (SCDO1), and we are using information derived from these mutations to study the structure of the DLL3 protein. To aid in elucidation of the underlying developmental defect in SCDO1, we have generated a mouse model by targeted deletion of the Dll3 gene (Dunwoodie et al., 2002). These mice show segmentation defects similar to those seen in SCDO1. In addition, these mice have a distinct set of neural defects that may be useful in future neurological assessment of affected individuals. Finally, since not all cases of SCD are due to mutation of DLL3, we are investigating various genes to find other candidates involved in this genetic disease.     

Alpha-tocopheryl succinate epitomizes a compound with a shift in biological activity due to pro-vitamin-to-vitamin conversion

With the advent of the third millennium, a number of pathologies have been eradicated or taken under control. However, the incidences, of cancer and atherosclerosis, the two most common causes of death in developed countries, have increased or, in some instances, only stagnated. Therefore there has been an intensive search for agents effective against such life-threatening conditions. Accordingly, the potential anti-atherogenic activity of vitamin E analogs has been studied extensively. Interestingly, recent reports strongly suggest that certain vitamin E analogs, represented in particular by alpha-tocopheryl succinate (alpha-TOS), also possess anti-neoplastic activity. In this communication, we review our current understanding of the molecular basis for these double effects of alpha-TOS and propose a testable hypothesis, according to which this semi-synthetic analog exerts both anti-atherogenic and anti-neoplastic activities. We propose that the prevalence of each activity depends on the actual form of the vitamin E analog. That is, the conversion of the pro-vitamin E form, alpha-TOS, to the corresponding vitamin form, alpha-tocopherol, makes this anti-neoplastic agent active against inflammatory diseases like atherosclerosis.     

Lipophilic phosphoramidates as antiviral pronucleotides

In order to overcome restrictions imposed by activation (phosphorylation) mechanism of antiviral and antitumor nucleoside analogues several prodrug approaches have been designed. Lipophilic pronucleotides are capable of intracellular delivery of monophosphates of nucleoside analogues, thus circumventing the limitations of enzymic phosphorylation. One of the successful approaches employs lipophilic amino acid ester (alanine) phenyl phosphoramidates as pronucleotides. This approach was applied to AIDS drugs such as AZT, d4T and related analogues but also to nonclassical nucleoside analogues based on allenic and methylenecyclopropane structure. Antiviral effects of the parent analogues were in many cases increased by conversion to phenyl phosphoralaninate (PPA) pronucleotides. Although cytotoxicity increase frequently accompanies antiviral effects of these pronucleotides, a favorable selectivity index can be obtained by manipulation of the parent structure as shown, e.g., for 2,6-diaminopurine methylenecyclopropane pronucleotide 15c. A lack of in vivo toxicity was demonstrated for 2-amino-6-methoxypurine methylenecyclopropane pronucleotide 15e in mice. The PPA pronucleotides can overcome deficiency of phosphorylating enzymes and offer favorable cross-resistance patterns when compared with other antiviral drugs.     

Dispersal patterns of endemic alpine butterflies with contrasting population structures:<Emphasis Type="Italic"> Erebia epiphron</Emphasis> and<Emphasis Type="Italic"> E. sudetica</Emphasis>

We studied population sizes and mobility of Erebia epiphron and Erebia sudetica, two high mountain butterflies forming endemic subspecies in the Hrubý Jeseník Mountains, Czech Republic. E. epiphron formed two continuous populations containing 100,000 and 4,500 individuals on alpine grasslands. The butterflies moved freely within their habitats, but movements between the two populations were highly unlikely. E. sudetica formed a system of colonies at timberline sites on valley headwalls and in forest clearings. Two such colonies studied in detail contained 4,500 and 450 adults and were interconnected by limited dispersal. The negative exponential function and the sigmoid function (this assumes flat decrease of movements over short distances) were superior to the inverse power function in fitting mobility data for both species. For E. sudetica, the functions describing movements within a habitat differed significantly from total movements, suggesting different behaviours of dispersing individuals. The habitats of E. epiphron are uniform and highly isolated, favouring free within-habitat mobility but prohibiting leaving their boundaries. The habitats of E. sudetica are diverse and disturbance-dependent; leaving such habitats is less risky, and a source-sink model may explain the persistence of the species in the mountains.     

Overexpression of plant uncoupling mitochondrial protein in transgenic tobacco increases tolerance to oxidative stress

An Arabidopsis thaliana cDNA clone encoding a plant uncoupling mitochondrial protein (AtPUMP1) was overexpressed in transgenic tobacco plants. Analysis of the AtPUMP1 mRNA content in the transgenic lines, determined by Northernblot, revealed variable levels of transgene expression. Antibody probing ofWestern blots of mitochondrial proteins from three independent transgenic lines showed significant accumulation of AtPUMP1 in this organelle. Overproduction of AtPUMP1 in transgenic tobacco plants led to a significantincrease in tolerance to oxidative stress promoted by exogenous hydrogen peroxide as compared to wild-type control plants. These results provide thefirst biological evidence for a role of PUMP in protection of plant cells against oxidative stress damage.