The crystal structure of ribonuclease A in complex with thymidine‐3′‐monophosphate provides further insight into ligand binding

Thymidine‐3′‐monophosphate (3′‐TMP) is a competitive inhibitor analogue of the 3′‐CMP and 3′‐UMP natural product inhibitors of bovine pancreatic ribonuclease A (RNase A). Isothermal titration calorimetry experiments show that 3′‐TMP binds the enzyme with a dissociation constant (K_d) of 15 μM making it one of the strongest binding members of the five natural bases found in nucleic acids (A, C, G, T, and U). To further investigate the molecular properties of this potent natural affinity, we have determined the crystal structure of bovine pancreatic RNase A in complex with 3′‐TMP at 1.55 Å resolution and we have performed NMR binding experiments with 3′‐CMP and 3′‐TMP. Our results show that binding of 3′‐TMP is very similar to other natural and non‐natural pyrimidine ligands, demonstrating that single nucleotide affinity is independent of the presence or absence of a 2′‐hydroxyl on the ribose moiety of pyrimidines and suggesting that the pyrimidine binding subsite of RNase A is not a significant contributor of inhibitor discrimination. Accumulating evidence suggests that very subtle structural, chemical, and potentially motional variations contribute to ligand discrimination in this enzyme. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Population history and gene dispersal inferred from spatial genetic structure of a Central African timber tree,Distemonanthus benthamianus (Caesalpinioideae)

African rainforests have undergone major distribution range shifts during the Quaternary, but few studies have investigated their impact on the genetic diversity of plant species and we lack knowledge on the extent of gene flow to predict how plant species can cope with such environmental changes. Analysis of the spatial genetic structure (SGS) of a species is an effective method to determine major directions of the demographic history of its populations and to estimate the extent of gene dispersal. This study characterises the SGS of an African tropical timber tree species, Distemonanthus benthamianus, at various spatial scales in Cameroon and Gabon. Displaying a large continuous distribution in the Lower Guinea domain, this is a model species to detect signs of past population fragmentation and recolonization, and to estimate the extent of gene dispersal. Ten microsatellite loci were used to genotype 295 adult trees sampled from eight populations. Three clearly differentiated gene pools were resolved at this regional scale and could be linked to the biogeographical history of the region, rather than to physical barriers to gene flow. A comparison with the distribution of gene pools observed for two other tree species living in the same region invalidates the basic assumption that all species share the same Quaternary refuges and recolonization pathways. In four populations, significant and similar patterns of SGS were detected. Indirect estimates of gene dispersal distances (sigma) obtained for three populations ranged from 400 to 1200 m, whereas neighbourhood size estimates ranged from 50 to 110.     

Developmental expression of alcohol dehydrogenase (ADH3) in zebrafish (Danio rerio)

Alcohol dehydrogenase (ADH) is the primary enzyme responsible for metabolism of ethanol to acetaldehyde. One class of ADH has been described in fish, and has been found to be structurally similar to mammalian class III ADH (glutathione-dependent formaldehyde dehydrogenase) but functionally similar to class I ADH (primarily responsible for ethanol metabolism). We have cloned a cDNA by RT-PCR from zebrafish (Danio rerio) liver representing the zebrafish ADH3 gene product, with a coding region of 1131 nucleotides. The deduced amino acid sequences share 90% identity to ADH3 from the marine fish Sparus aurata, and 82 and 81% identity to the mouse and human sequences, respectively. Using a quantitative competitive RT-PCR assay, ADH3 mRNA was detected at all timepoints analyzed and was lowest between 8 and 24 h postfertilization. Thus, differential ADH3 expression may be at least partly responsible for temporal variations in the sensitivity of zebrafish embryos to developmental alcohol exposure.     

Micron-scale assessment of molecular lipid organization in human stratum corneum using microprobe X-ray diffraction

Lipid and protein components of the stratum corneum (SC) are organized in complex supramolecular arrangements. Exploring spatial relations between various possible substructures is important for understanding the barrier function of this uppermost layer of epidermis. Here, we report the first study where micro-focus X-ray scattering was used for assessing fine structural variations of the human skin barrier with micrometer resolution. We found that the scattering profiles were unchanged when scanning in the direction parallel to the SC surface. Furthermore, small-angle scattering profiles did not change as a function of depth in the SC, confirming that the lipid lamellar spacings remained the same throughout the SC. However, the wide-angle scattering data showed that the orthorhombic phase was more abundant in the middle layers of the SC, whereas the hexagonal phase dominated in the surface layers both at the external and the lowest part of the SC; i.e., the lipids were most tightly packed in the middle region of the SC. Taken together, our results demonstrate that microprobe X-ray diffraction provides abundant information about spatial variations of the SC lipid structure and thus may be a promising tool for assessing the effects of topical formulations on the barrier function of skin.     

Seed dispersal effectiveness of the western lowland gorilla (Gorilla gorilla gorilla) in Gabon

The quantitative and qualitative aspects of seed dispersal by the western lowland gorilla (Gorilla gorilla gorilla) were investigated in Gabon. Fresh faeces were collected and washed to identify and count the seeds. Seed germinability after gut passage was estimated with trials in a nursery at the study site. To assess the impact of gut passage on germination success and delay, comparative trials were run with four treatments: (i) gut passed seeds cleaned of faeces, (ii) gut passed seeds within a faecal matrix, (iii) seeds from fresh fruits surrounded by pulp, and (iv) seeds from fresh fruits cleaned of pulp. The analysis of 180 faecal units resulted in the identification of 58 species of seed. Germination trials were realized for 55 species and the mean germination success reached 46%. The impact of gut passage was investigated for Santiria trimera and Chrysophyllum lacourtianum; both species displayed higher germination success after ingestion. This study shows that gorillas effectively disperse seeds of numerous plant species, many of which provide timber or nontimber forest products or are typical of Gabonese forests. Considering the high‐quality of gorilla deposition sites, gorillas is thought to play a unique role in the dynamics of Central African forest.     

Dynamic sexual dichromatism in an explosively breeding Neotropical toad

Sexual selection often promotes the evolution of elaborate colour signals in males, but the importance of sexually selected colour signals remains poorly studied in amphibians. We used reflectance spectrometry to document pronounced sexual dichromatism and dramatic colour change in Bufo luetkenii, a toad that breeds in large aggregations at the onset of the rainy season in Costa Rica. Our observations suggest that males fade rapidly from a vibrant lemon yellow to a dull brown once they have paired with a female. We demonstrate this by showing that males are much brighter than females and that unpaired males are more colourful than males in amplexus. We also show that coloration fades rapidly when males are briefly held captive. This is, to our knowledge, the first study to document such dynamic change in male coloration and sexual dichromatism in anurans.     

Structure-function relationships in spruce budworm antifreeze protein revealed by isoform diversity.

The spruce budworm, Choristoneura fumiferana, produces antifreeze protein (AFP) to assist in the protection of the overwintering larval stage. AFPs are thought to lower the freezing point of the hemolymph, noncolligatively, by interaction with the surface of ice crystals. Previously, we had identified a cDNA encoding a 9-kDa AFP with 10-30 times the thermal hysteresis activity, on a molar basis, than that shown by fish AFPs. To identify important residues for ice interaction and to investigate the basis for the hyperactivity of the insect AFPs, six new spruce budworm AFP cDNA isoforms were isolated and sequenced. They differ in amino-acid identity as much as 36% from the originally characterized AFP and can be divided into three classes according to the length of their 3' untranslated regions (UTRs). The new isoforms have at least five putative 'Thr-X-Thr' ice-binding motifs and three of the new isoforms encode larger, 12-kDa proteins. These appear to be a result of a 30 amino-acid insertion bearing two additional ice-binding motifs spaced 15 residues apart. Molecular modeling, based on the NMR structure of a short isoform, suggests that the insertion folds into two additional beta-helix loops with their Thr-X-Thr motifs in perfect alignment with the others. The first Thr of the motifs are often substituted by Val, Ile or Arg and a recombinantly expressed isoform with both Val and Arg substitutions, showed wild-type thermal hysteresis activity. The analysis of these AFP isoforms suggests therefore that specific substitutions at the first Thr in the ice binding motif can be tolerated, and have no discernible effect on activity, but the second Thr appears to be conserved. The second Thr is thus likely important for the dynamics of initial ice contact and interaction by these hyperactive antifreezes.