DNA-drug interactions. The crystal structures of d(TGTACA) and d(TGATCA) complexed with daunomycin

The anticancer drug daunomycin has been co-crystallized with the hexanucleotide duplex sequences d(TGTACA) and d(TGATCA) and single crystal X-ray diffraction studies of these two complexes have been carried out. Structure solution of the d(TGTACA) and d(TGATCA) complexes to 1.6 and 1.7 Angstrom resolution, respectively, shows two daunomycin molecules bound to the DNA hexamer. Binding occurs via intercalation of the drug chromophore at the d(TpG) step, and hydrogen bonding interactions involving the drug, DNA and solvent molecules. The daunomycin sugar is located in the minor groove of the DNA hexamer and is stabilized by hydrogen bonds between the amino group of the sugar and functional groups on the floor of the groove. The amino sugar of the d(TGATCA) duplex interacts directly with the DNA sequence, while in the d(TGTACA) duplex, the interaction is via solvent molecules. Two other complexes d(CGTACG)-daunomycin and d(CGATCG)-daunomycin have previously been structurally characterized. Comparison of the four structures with daunomycin bound to the triplet sequences 5'TGT, 5'TGA, 5'CGT and 5'CGA reveals changes in the conformation of both the DNA hexamer and the daunomycin upon complexation, as well as the hydrogen bonding and van der Waals' interactions.     

Emerging infectious diseases and animal social systems

Emerging infectious diseases threaten a wide diversity of animals, and important questions remain concerning disease emergence in socially structured populations. We developed a spatially explicit simulation model to investigate whether—and under what conditions—disease-related mortality can impact rates of pathogen spread in populations of polygynous groups. Specifically, we investigated whether pathogen-mediated dispersal (PMD) can occur when females disperse after the resident male dies from disease, thus carrying infections to new groups. We also examined the effects of incubation period and virulence, host mortality and rates of background dispersal, and we used the model to investigate the spread of the virus responsible for Ebola hemorrhagic fever, which currently is devastating African ape populations. Output was analyzed using regression trees, which enable exploration of hierarchical and non-linear relationships. Analyses revealed that the incidence of disease in single-male (polygynous) groups was significantly greater for those groups containing an average of more than six females, while the total number of infected hosts in the population was most sensitive to the number of females per group. Thus, as expected, PMD occurs in polygynous groups and its effects increase as harem size (the number of females) increases. Simulation output further indicated that population-level effects of Ebola are likely to differ among multi-male–multi-female chimpanzees and polygynous gorillas, with larger overall numbers of chimpanzees infected, but more gorilla groups becoming infected due to increased dispersal when the resident male dies. Collectively, our results highlight the importance of social system on the spread of disease in wild mammals.     

Molecular cloning of a malyl coenzyme A lyase gene from Pseudomonas sp. strain AM1, a facultative methylotroph

A genomic library containing HindIII partial digests of Pseudomonas sp. strain AM1 DNA was constructed in the broad-host-range cosmid pVK100. PCT57, a Pseudomonas sp. strain AM1 methanol mutant deficient in malyl coenzyme A lyase activity, was complemented to a methanol-positive phenotype by mobilization of the pVK100 library into PCT57 recipients with the ColE1/RK2 mobilizing plasmid pRK2013. Six different complemented isolates all contained a recombinant plasmid carrying the same 19.6-kilobase-pair Pseudomonas sp. strain AM1 DNA insert. Subcloning and complementation analysis demonstrated that the gene deficient in PCT57 (mcl-1) was located in a 1.6-kilobase-pair region within a 7.4-kilobase-pair EcoRI-HindIII fragment.     

Fc receptors on mouse effector cells mediating natural cytotoxicity against tumor cells.

Mouse effector cells mediating natural cytotoxicity against tumor cells have been previously thought to be lymphocytes that lack any detectable cell surface markers. The present study presents evidence for receptors for the Fc portion of IgG on these cells. By adsorption of cytotoxic spleen cells on monolayers of sheep erythrocytes (E) plus IgG antibodies to sheep erythrocytes (EA), 50 to 96% of the total cytotoxic reactivity could be removed. Parallel adsorption of cells on E monolayers or on EA monolayers coated with protein A, to block the Fc portion of IgG, resulted in little or no depletion of cytotoxic activity. The presence of Fc receptors on the NK cells was confirmed by combining EA rosette formation with velocity sedimentation at unit gravity. Peak cytotoxicity occurred at the same sedimentation velocity as the peak of Fc-positive cells. After EA rosette formation, there was a shift to a higher sedimentation velocity in the Fc-positive cells and in the natural cytotoxic activity. The increase in sedimentation velocity of NK activity that was observed in these experiments indicated that most of the cells had only bound a small number (three or four) of antibody-coated erythrocytes. Together, these data indicate that cells with Fc receptors account for most of the total lytic activity of normal mouse spleen cells.     

The mechanism of Mycobacterium tuberculosis alkylhydroperoxidase AhpD as defined by mutagenesis,crystallography, and kinetics

AhpD, a protein with two cysteine residues, is required for physiological reduction of the Mycobacterium tuberculosis alkylhydroperoxidase AhpC. AhpD also has an alkylhydroperoxidase activity of its own. The AhpC/AhpD system provides critical antioxidant protection, particularly in the absence of the catalase-peroxidase KatG, which is suppressed in most isoniazid-resistant strains. Based on the crystal structure, we proposed recently a catalytic mechanism for AhpD involving a proton relay in which the Glu118 carboxylate group, via His137 and a water molecule, deprotonates the catalytic residue Cys133 (Nunn, C. M., Djordjevic, S., Hillas, P. J., Nishida, C., and Ortiz de Montellano, P. R. (2002) J. Biol. Chem. 277, 20033-20040). A possible role for His132 in subsequent formation of the Cys133-Cys130 disulfide bond was also noted. To test this proposed mechanism, we have expressed the H137F, H137Q, H132F, H132Q, E118F, E118Q, C133S, and C130S mutants of AhpD, determined the crystal structures of the H137F and H132Q mutants, estimated the pKa values of the cysteine residues, and defined the kinetic properties of the mutant proteins. The collective results strongly support the proposed catalytic mechanism for AhpD.     

The diets and parasites of larval and 0+ juvenile twaite shad in the lower reaches and estuaries of the rivers Wye,Usk and Towy,UK

Aquatic predators and habitat permanence can jointly affect benthic invertebrate biomass and community composition. In 2006 I sampled fish and invertebrates in ten ponds embedded in a seasonal wetland before and after a natural drought. Drought reduced fish biomass and density leaving some ponds in a fishless condition when rains returned in July. In July, large aquatic insects and crayfish colonized and reproduced in the ponds, but did not colonize all of the ponds equally. Using measurements of fish abundance and other environmental parameters of the ponds, I conducted linear regression analyses to explore potential drivers of variable invertebrate biomass in July. Fish biomass had a negative effect on invertebrate biomass and it explained more of the variation in total invertebrate biomass and total non-shrimp biomass than fish abundance (number of fish caught). Dissolved oxygen and pond depth were both correlated with fish biomass, but were poorer predictors of invertebrate biomass. Ponds with few or no fish had 20× greater total biomass and 200× more non-shrimp biomass than ponds with high fish biomass. Shrimp dominated the invertebrate composition, and were only found in the two deepest ponds with the highest fish biomass; predatory insects and crayfish dominated the other eight ponds. When taxa were analyzed separately, fish biomass explained a large portion of the variation for predatory insects (Coleoptera, Hemiptera, and Odonata) and crayfish (Procambarus alleni), but dissolved oxygen was the best predictor of larval stratiomyid (order Diptera) biomass. These results are generally consistent with studies demonstrating negative effects of fish on large predatory invertebrates, but also suggest that more severe local droughts can seasonally enhance insect and crayfish populations by generating fishless or nearly fishless conditions. Handling editor: J. Trexler     

Parasite-mediated evolution of the functional part of the MHC in primates

The major histocompatibility complex (MHC) is a key model of genetic polymorphism, but the mechanisms underlying its extreme variability are debated. Most hypotheses for MHC diversity focus on pathogen-driven selection and predict that MHC polymorphism evolves under the pressure of a diverse parasite fauna. Several studies reported that certain alleles offer protection against certain parasites, yet it remains unclear whether variation in parasite pressure more generally covaries with allelic diversity and rates of molecular evolution of MHC across species. We tested this prediction in a comparative study of 41 primate species. We characterized polymorphism of the exon 2 of DRB region of the MHC class II. Our phylogenetic analyses controlled for the potential effects of neutral mutation rate, population size, geographic origin and body mass and revealed that nematode species richness associates positively with nonsynonymous nucleotide substitution rate at the functional part of the molecule. We failed to find evidence for allelic diversity being strongly related to parasite species richness. Continental distribution was a strong predictor of both allelic diversity and substitution rate, with higher values in Malagasy and Neotropical primates. These results indicate that parasite pressure can influence the different estimates of MHC polymorphism, whereas geography plays an independent role in the natural history of MHC.     

Behavioural defences against sexually transmitted diseases in primates

Sexually transmitted diseases (STDs) are known to exist in wild and domesticated animals, but little is know about behavioural defences that animals use to reduce the risk of acquiring STDs. Using comparative data and a phylogeny of primates, I investigated whether behaviours hypothesized to reduce STD transmission are correlated with measures of STD risk involving mating promiscuity and life-history traits. Comparative tests revealed no support for genital inspection as a means to identify and avoid infected individuals with genital inspection was performed more commonly by males than females and uncorrelated with mating promiscuity. Primate species characterized by increased promiscuity were not more likely to display genital self-grooming following mating. Similarly, males and females of these species were not more likely to urinate immediately after mating, counter to suggestions that urination flushes microorganisms from the urethra and surrounding genital areas. Finally, monogamy was not correlated with a slow life history, which differs from predictions that monogamy is a response to increased STD risk in long-lived animals. Tests involving monogamy remained unsupported after controlling for potentially confounding variables, and all tests yielded similar results in phylogenetic and nonphylogenetic tests. Few results were significant even before controlling statistically for multiple comparisons, but nonsignificance was unlikely due to low statistical power or poor data quality in all tests. Instead, the comparative patterns were consistent with theoretical models showing that precopulatory behavioural defences to STDs, such as mate choice, are unlikely to be fully effective. In addition, many putative behavioural defences to STDs in primates entail substantial fitness costs in terms of reproductive output, offspring quality and infanticide avoidance. Copyright 2003 Published by Elsevier Ltd on behalf of The Association for the Study of Animal Behaviour.