Heat-shock protein 70 (Hsp70) as a biochemical stress indicator: an experimental field test in two congeneric intertidal gastropods (genus: Tegula)

Although previous studies have demonstrated that heat-shock protein 70 (Hsp70) can be induced by environmental stress, little is known about natural variation in this response over short time scales. We examined how Hsp70 levels varied over days to weeks in two intertidal snail species of the genus Tegula: Sampling was conducted both under naturally changing environmental conditions and in different vertical zones on a rocky shore. The subtidal to low-intertidal T. brunnea was transplanted into shaded and unshaded mid-intertidal cages to assess temporal variation in Hsps under conditions of increased stress. For comparison, the low to mid-intertidal T. funebralis was transplanted into mid-intertidal cages, within this species' natural zone of occurrence. Snails were sampled every 3 to 4 days for one month, and endogenous levels of two Hsp70-kDa family members (Hsp72 and Hsp74) were quantified using solid-phase immunochemistry. Following periods of midday low tides, levels of Hsps increased greatly in transplanted T. brunnea but not in T. funebralis. Levels of Hsps increased less in T. brunnea transplanted to shaded cages than to unshaded cages, suggesting that prolonged emersion and reduction in feeding time per se are factors that are only mildly stressful. Upregulated levels of Hsps returned to base levels within days. In unmanipulated snails collected from their natural zones, Hsp levels showed little change with thermal variation, indicating that these species did not experience thermally stressful conditions during this study. However, under common conditions in the mid-intertidal zone, Hsp70 levels reflected the different thermal sensitivities of the physiological systems of these two species.     

Dispersal biology of Liatris scariosa var. novae-angliae (Asteraceae), a rare New England grassland perennial

Propagule dispersal biology is a crucial avenue of research for rare plant species, especially those adapted to disturbance, such as northern blazing star (Liatris scariosa var. novae-angliae), a rare, early-successional New England grassland perennial. We examined the dispersal ability of northern blazing star propagules collected from 14 populations covering the entire latitudinal range of the taxon. Multiple regression demonstrated that dispersal ability, as measured by drop time in still air and flight distance in a low-speed wind tunnel, decreased significantly with propagule size and achene length, and increased with achene width and (for flight distance) pappus length. We used this multiple regression model to test for differences in predicted dispersal capability among maternal families, populations, and inland, coastal, and island habitats. Dispersal capability differed significantly among families and populations but not regions, and allometric relationships between morphological measurements were consistent across populations. Overall, dispersal capability was negatively correlated with germination success in a common greenhouse environment. However, germination success for a given dispersal ability, as well as achene shape, differed among populations. These results suggest specific populations to be targeted for management efforts promoting dispersal and establishment.     

A simple nonparametric test for linkage with sib-pair censored event time observations

Consider the case that individual phenotype and genotype observations were collected from a large or moderate number of pedigrees. Some of the pedigrees have multi-generation nuclear families. For each nuclear family, the phenotype trait value of each sibling is the time to onset for a specific event (e.g., disease). Often, this event time may be right censored, that is, an individual is event-free at the study examination time point. In this article, we propose a purely nonparametric test for testing if the distribution of a Haseman-Elston distance measure between two siblings' event times is independent of their mean genetic sharing identical by descent at a genetic marker based on such incomplete observations from all the nuclear families. The new test can be implemented easily and is illustrated with a data set from the Genetic Analysis Workshop 12. The validity of the new test is examined via a simulation study.     

Linkage disequilibrium and inference of ancestral recombination in 538 single-nucleotide polymorphism clusters across the human genome

The prospect of using linkage disequilibrium (LD) for fine-scale mapping in humans has attracted considerable attention, and, during the validation of a set of single-nucleotide polymorphisms (SNPs) for linkage analysis, a set of data for 4,833 SNPs in 538 clusters was produced that provides a rich picture of local attributes of LD across the genome. LD estimates may be biased depending on the means by which SNPs are first identified, and a particular problem of ascertainment bias arises when SNPs identified in small heterogeneous panels are subsequently typed in larger population samples. Understanding and correcting ascertainment bias is essential for a useful quantitative assessment of the landscape of LD across the human genome. Heterogeneity in the population recombination rate, rho=4Nr, along the genome reflects how variable the density of markers will have to be for optimal coverage. We find that ascertainment-corrected rho varies along the genome by more than two orders of magnitude, implying great differences in the recombinational history of different portions of our genome. The distribution of rho is unimodal, and we show that this is compatible with a wide range of mixtures of hotspots in a background of variable recombination rate. Although rho is significantly correlated across the three population samples, some regions of the genome exhibit population-specific spikes or troughs in rho that are too large to be explained by sampling. This result is consistent with differences in the genealogical depth of local genomic regions, a finding that has direct bearing on the design and utility of LD mapping and on the National Institutes of Health HapMap project.     

Role of calcineurin in striated muscle: development, adaptation, and disease

Striated muscles, cardiac and skeletal muscles, use calcium as a second messenger to respond and adapt to environmental stimuli. Elevations in intracellular calcium activate calcineurin, a serine/threonine phosphatase, resulting in expression of a set of genes involved in remodeling striated muscle. Activation of calcineurin in hearts produces cardiac hypertrophy, and in skeletal muscle promotes cell differentiation and transforms fiber type specificity. In this review we discuss the effects of calcineurin activity on development, adaptation, and disease of striated muscle.     

Achieving selectivity between highly homologous tyrosine kinases: a novel selective erbB2 inhibitor

The discovery of small molecule kinase inhibitors for use as drugs is a promising approach for the treatment of cancer and other diseases, but the discovery of highly specific agents is challenging because over 850 kinases are expressed in mammalian cells. Systematic modification of the 4-anilino functionality of a selective quinazoline inhibitor of the epidermal growth factor receptor (EGFR) tyrosine kinase can invert selectivity to favor inhibition of the highly homologous erbB2 tyrosine kinase. The selectivity pattern was demonstrated in assays of recombinant kinases and recapitulated in measures of kinase activity in intact cells. The most potent and selective erbB2 inhibitor of the analog series has anti-proliferative activity against an erbB2-overexpressing cell line that was lacking in the original EGFR-selective compound. Subtle changes to the molecular structure of ATP-competitive small molecule inhibitors of tyrosine kinases can yield dramatic changes in potency and selectivity. These results suggest that the discovery of highly selective small molecule inhibitors of very homologous kinases is achievable.     

The KiSS-1 receptor GPR54 is essential for the development of the murine reproductive system

GPR54 is a G-protein-coupled receptor that displays a high percentage of identity in the transmembrane domains with the galanin receptors. The ligand for GPR54 has been identified as a peptide derived from the KiSS-1 gene. KiSS-1 has been shown to have anti-metastatic effects, suggesting that KiSS-1 or its receptor represents a potential therapeutic target. To further our understanding of the physiological function of this receptor, we have generated a mutant mouse line with a targeted disruption of the GPR54 receptor (GPR54 -/-). The analysis of the GPR54 mutant mice revealed developmental abnormalities of both male and female genitalia and histopathological changes in tissues which normally contain sexually dimorphic features. These data suggest a role for GPR54/KiSS-1 in normal sexual development, and indicate that study of the GPR54 mutant mice may provide valuable insights into human reproductive syndromes.     

Structure of mammalian cytochrome P450 2C5 complexed with diclofenac at 2.1 A resolution: evidence for an induced fit model of substrate binding

The structure of the anti-inflammatory drug diclofenac bound in the active site of rabbit microsomal cytochrome P450 2C5/3LVdH was determined by X-ray crystallography to 2.1 A resolution. P450 2C5/3LVdH and the related enzyme 2C5dH catalyze the 4'-hydroxylation of diclofenac with apparent K(m) values of 80 and 57 microM and k(cat) values of 13 and 16 min(-1), respectively. Spectrally determined binding constants are similar to the K(m) values. The structure indicates that the pi-electron system of the dichlorophenyl moiety faces the heme Fe with the 3'- and 4'-carbons located 4.4 and 4.7 A, respectively, from the Fe. The carboxyl moiety of the substrate is hydrogen bonded to a cluster of waters that are also hydrogen bonded to the side chains of N204, K241, S289, and D290 as well as the backbone of the protein. The proximity of the diclofenac carboxylate to the side chain of D290 together with an increased binding affinity at lower pH suggests that diclofenac is protonated when bound to the enzyme. The structure exhibits conformational changes indicative of an adaptive fit to the substrate reflecting both the hydration and size of the substrate. These results indicate how structurally diverse substrates are recognized by drug-metabolizing P450 enzymes.