Balancing selection, random genetic drift, and genetic variation at the major histocompatibility complex in two wild populations of guppies (Poecilia reticulata)

Our understanding of the evolution of genes of the major histocompatibility complex (MHC) is rapidly increasing, but there are still enigmatic questions remaining, particularly regarding the maintenance of high levels of MHC polymorphisms in small, isolated populations. Here, we analyze the genetic variation at eight microsatellite loci and sequence variation at exon 2 of the MHC class IIB (DAB) genes in two wild populations of the Trinidadian guppy, Poecilia reticulata. We compare the genetic variation of a small (Ne, 100) and relatively isolated upland population to that of its much larger (Ne approximately 2400) downstream counterpart. As predicted, microsatellite diversity in the upland population is significantly lower and highly differentiated from the population further downstream. Surprisingly, however, these guppy populations are not differentiated by MHC genetic variation and show very similar levels of allelic richness. Computer simulations indicate that the observed level of genetic variation can be maintained with overdominant selection acting at three DAB loci. The selection coefficients differ dramatically between the upland (s > or = 0.2) and lowland (s < or = 0.01) populations. Parasitological analysis on wild-caught fish shows that parasite load is significantly higher on upland than on lowland fish, which suggests that large differences in selection intensity may indeed exist between populations. Based on the infection intensity, a substantial proportion of the upland fish would have suffered direct or indirect fitness consequences as a result of their high parasite loads. Selection by parasites plays a particularly important role in the evolution of guppies in the upland habitat, which has resulted in high levels of MHC diversity being maintained in this population despite considerable genetic drift.     

Sphingolipid biosynthesis in pathogenic fungi: identification and characterization of the 3-ketosphinganine reductase activity of Candida albicans and Aspergillus fumigatus

An early step in sphingolipid biosynthesis, the reduction of 3-ketosphinganine, is catalyzed in the yeast Saccharomyces cerevisiae by Tsc10p (TSC10 (YBR265W)). We have identified orthologs of TSC10 in two clinically important fungal pathogens, Candida albicans and Aspergillus fumigatus. The translated sequences of the putative C. albicans ortholog, KSR1 (orf6.5112), and the putative A. fumigatus ortholog, ksrA, show significant homology to the yeast protein. All three proteins contain the signature motifs of NAD(P)H-dependent oxidoreductases in the short-chain dehydrogenase/reductase family and a conserved putative substrate-binding domain. Despite being essential in S. cerevisiae, we demonstrate that the C. albicans ortholog, KSR1, is not required for cell viability. However, ksr1 null mutants produce lower levels of inositolphosphorylceramides, are significantly more sensitive than the wildtype to an inhibitor of a subsequent step in sphingolipid biosynthesis, and are defective for the transition from yeast to filamentous growth, a key virulence determinant. Recombinant, purified Ksr1p and KsrA can carry out the reduction of 3-ketosphinganine in an NADPH-dependent manner. Molecular modeling of Ksr1p with bound substrates suggests that a significant portion of the aliphatic chain of 3-ketosphinganine protrudes from the enzyme. Guided by this molecular model, we developed shorter, water-soluble derivatives of 3-ketosphinganine that are substrates for 3-ketosphinganine reductase.     

Discovery of 4H-pyrazolo[1,5-a]pyrimidin-7-ones as potent inhibitors of hepatitis C virus polymerase

A series of 4H-pyrazolo[1,5-a]pyrimidin-7-one derivatives was synthesized and evaluated for inhibitory activity against HCV NS5B RNA-dependent RNA polymerase. A number of these compounds exhibited potent activity in enzymatic assay. The synthesis and structure–activity relationship are also described.     

Antagonistic interactions between garden yeasts and microfungal garden pathogens of leaf-cutting ants

We investigate the diversity of yeasts isolated in gardens of the leafcutter ant Atta texana. Repeated sampling of gardens from four nests over a 1-year time period showed that gardens contain a diverse assemblage of yeasts. The yeast community in gardens consisted mostly of yeasts associated with plants or soil, but community composition changed between sampling periods. In order to understand the potential disease-suppressing roles of the garden yeasts, we screened isolates for antagonistic effects against known microfungal garden contaminants. In vitro assays revealed that yeasts inhibited the mycelial growth of two strains of Escovopsis (a specialized attine garden parasite), Syncephalastrum racemosum (a fungus often growing in gardens of leafcutter lab nests), and the insect pathogen Beauveria bassiana. These garden yeasts add to the growing list of disease-suppressing microbes in attine nests that may contribute synergistically, together with actinomycetes and Burkholderia bacteria, to protect the gardens and the ants against diseases. Additionally, we suggest that garden immunity against problem fungi may therefore derive not only from the presence of disease-suppressing Pseudonocardia actinomycetes, but from an enrichment of multiple disease-suppressing microorganisms in the garden matrix.     

Host specificity dynamics: observations on gyrodactylid monogeneans

The directly transmitted viviparous gyrodactylids have high species richness but low morphological and biological diversity, and many species are recorded from only a single host. They therefore constitute a guild of species ideal for studies of the evolutionary significance of host specificity. The group has the widest host range of any monogenean family, being found on 19 orders of bony fish. However, individual species range from narrowly specific (71% of 402 described species recorded from a single host) to extremely catholic (Gyrodactylus alviga recorded from 16 hosts). Gyrodactylid-host interactions extend from 60 mya (G. lotae, G. lucii) down to 150 years (G. derjavini on Oncorhynchus mykiss). Co-evolution with the host is comparatively rare within the gyrodactylids, but host switching or ecological transfer is common, and has been facilitated by the mixing of fish strains that followed glaciation. In this review, we consider the factors responsible for gyrodactylid specificity patterns, using examples from our work on salmonid gyrodactylids including G. salaris, responsible for major epidemics on wild Atlantic salmon (Salmo salar) in Norway since 1975, and G. thymalli from grayling and G. derjavini from trout.G. salaris has a wide host range with highest population growth rates on Norwegian salmon strains. However, growth rates are variable on both host strains and species, because of the multitude of micro- and macro-environmental factors influencing parasite mortality and fecundity. A better predictor of performance is the proportion of fishes of a strain which are innately resistant to the parasite, a measure which is negatively correlated with the time to peak infection in a host strain. Population growth rate is also negatively correlated with age of infection; the initial rate, therefore, predicts best the suitability of a fish as host for G. salaris. The host response to gyrodactylids appears to be the same mechanism in all salmonids with innate resistance as one end of a spectrum, but influenced by stress and probably under polygenic control. Hybrid experiments show that performance of G. salaris on a host is heritable, and usually intermediate between that of the parents. This host response mechanism, coupled with the initial parasite population growth on a fish, determines the host specificity, i.e. whether the fish will be susceptible, a responder or innately resistant. The use of population growth rate parameters allows comparison of different hosts as a resource for a gyrodactylid. In the case of G. salaris, East Atlantic and Baltic strains of Atlantic salmon are core hosts, but other salmonids can physiologically sustain infections for considerable periods, and may be important in parasite dispersal and transmission. A further group of non-salmonid fishes are unable to sustain G. salaris reproduction, but can act as transport hosts.Population growth parameters are very labile to stressors and environmental factors, particularly temperature and salinity, and also other aspects of host ecology and water quality. These factors may also influence the spectrum of hosts that can be infected under particular conditions, and probably favoured ecological transfer of gyrodactylids between host species in periglacial conditions. G. salaris may still be undergoing post-glacial range expansion (aided by anthropogenic spread) as shown by the increase in the species range over the last 25 years. The origin of G. salaris, G. teuchis and G. thymalli is discussed in relation to glacial refugiums during the last ice age.     

Time course and differential responses of the major heat shock protein families in human skeletal muscle following acute nondamaging treadmill exercise.

The exercise-induced expression of heat shock proteins (HSPs) in rodent models is relatively well defined. In contrast, comparable data from human studies are limited and the exercise-induced stress response of human skeletal muscle is far from understood. This study has characterized the time course and magnitude of the HSP response in the skeletal muscles of a healthy active, but untrained, young male population following a running exercise protocol. Eight subjects performed 45 min of treadmill running at a speed corresponding to their lactate threshold (11.7 +/- 0.5 km/h; 69.8 +/- 4.8% maximum O2 uptake). Muscle biopsies were obtained from the vastus lateralis muscle immediately before and at 24 h, 48 h, 72 h, and 7 days postexercise. Exercise induced a significant (P < 0.05) but variable increase in HSP70, heat shock cognate (HSC) 70, and HSP60 expression with peak increases (typically occurring at 48 h postexercise) to 210, 170, and 139% of preexercise levels, respectively. In contrast, exercise did not induce a significant increase in either HSP27, alphaB-crystallin, SOD 2 (MnSOD) protein content, or the activity of SOD and catalase. When examining baseline protein levels, HSC70, HSP27, and alphaB-crystallin appeared consistently expressed between subjects, whereas HSP70 and MnSOD displayed marked individual variation of up to 3- and 1.5-fold, respectively. These data are the first to define the time course and extent of HSP production in human skeletal muscle following a moderately demanding and nondamaging running exercise protocol. Data demonstrate a differential effect of aerobic exercise on specific HSPs.     

Probing the relationship between RNA-stimulated ATPase and helicase activities of HCV NS3 using 2'-O-methyl RNA substrates

The hepatitis C virus (HCV) NS3 protein contains an amino terminal protease (NS3 aa. 1-180) and a carboxyl terminal RNA helicase (NS3 aa. 181-631). NS3 functions as a heterodimer of NS3 and NS4A (NS3/4A). NS3 helicase, a nucleic acid stimulated ATPase, can unwind RNA, DNA, and RNA:DNA duplexes, provided that at least one strand of the duplex contains a single-stranded 3' overhang (this strand of the duplex is referred to as the 3' strand). We have used 2'-O-methyl RNA (MeRNA) substrates to study the mechanism of NS3 helicase activity and to probe the relationship between its helicase and RNA-stimulated ATPase activities. NS3/4A did not unwind double-stranded (ds) MeRNA. NS3/4A unwinds hybrid RNA:MeRNA duplex containing MeRNA as the 5' strand but not hybrid duplex containing MeRNA as the 3' strand. The helicase activity of NS3/4A was 50% inhibited by 40 nM single-stranded (ss) RNA but only 35% inhibited by 320 nM ss MeRNA. Double-stranded RNA was 17 times as effective as double-stranded MeRNA in inhibiting NS3/4A helicase activity, while the apparent affinity of NS3/4A for ds MeRNA differed from ds RNA by only 2.4-fold. However ss MeRNA stimulated NS3/4A ATPase activity similar to ss RNA. These results indicate that the helicase mechanism involves 3' to 5' procession of the NS3 helicase along the 3' strand and only weak association of the enzyme with the displaced 5' strand. Further, our findings show that maximum stimulation of NS3 ATPase activity by ss nucleic acid is not directly related to procession of the helicase along the 3' strand.     

Aminothiazole inhibitors of HCV RNA polymerase

Aminothiazole-based inhibitors designed for HCV polymerase display low micromolar potencies in biochemical assays. These compounds show a stringent preference for a cyclohexyl hydrophobe at the 2-amino position. The composition of these compounds suggests that they may be interacting at a recently discovered allosteric site on the polymerase.     

Isolation of a functional copy of the human BRCA1 gene by transformation-associated recombination in yeast

The BRCA1 gene, mutations of which contribute significantly to hereditary breast cancer, was not identified in the existing YAC and BAC libraries. The gene is now available only as a set of overlapping fragments that form a contig. In this work we describe direct isolation of a genomic copy of BRCA1 from human DNA by transformation-associated recombination (TAR) cloning. Despite the presence of multiple repeats, most of the primary BRCA1 YAC isolates did not contain detectable deletions and could be stably propagated in a host strain with conditional RAD52. Similar to other circular YACs, 90 kb BRCA1 YACs were efficiently and accurately retrofitted into bacterial artificial chromosomes (BACs) with the Neo^R mammalian selectable marker and transferred as circular BAC/YACs in E. coli cells. The BRCA1 BAC/YAC DNAs were isolated from bacterial cells and were used to transfect mouse cells using the Neo^R gene as selectable marker. Western blot analysis of transfectants showed that BRCA1 YACs isolated by a TAR cloning contained a functional gene. The advantage of this expression vector is that the expression of BRCA1 is generated from its own regulatory elements and does not require additional promoter elements that may result in overexpression of the protein. In contrast to the results with cDNA expression vectors, the level of BRCA1 expression from this TAR vector is stable, does not induce cell death, maintains serum regulation, and approximates the level of endogenously expressed BRCA1 in human cells. The entire isolation procedure of BRCA1 described in this paper can be accomplished in approximately 10 days and can be applied to isolation of gene from clinical material. We propose that the opportunity to directly isolate normal and mutant forms of BRCA1 will greatly facilitate analysis of the gene and its contribution to breast cancer.