Cyclophilin 40: An Hsp90-cochaperone associated with apo-steroid receptors

Cyclophilin 40, a divergent loop cyclophilin first identified in association with the estrogen receptor α, contains a C-terminal tetratricopeptide repeat domain through which it shares structural identity with FK506-binding protein 52 (FKBP52) and other partner cochaperones in steroid receptor-heat shock protein 90 (Hsp90) complexes. By dynamically competing for Hsp90 interaction, the cochaperones allow the receptors to establish distinct Hsp90-chaperone complexes, with the potential to exert tissue-specific control over receptor activity. Cyclophilin 40 regulates Hsp90 ATPase activity during receptor-Hsp90 assembly. Functional deletion of the cyclophilin 40 yeast homologue, Cpr7, adversely affected estrogen receptor α and glucocorticoid receptor activity that could be fully restored, either with wild type Cpr7 or Cpr7 with a cyclophilin domain lacking isomerase activity. We draw parallels with the mechanism already established for FKBP52 and propose that the cyclophilin 40 divergent loop interfaces with a contact surface on the steroid receptor ligand-binding domain to achieve an optimal orientation for receptor activity.     

A solid-phase screen for protein kinase substrate selectivity.

Cassette mutagenesis was used to synthesize an Escherichia coli expression library of unique phosphorylation sites. The cassette encodes a central serine residue surrounded by every combination of Ala, Arg, Gln, Glu, Gly, and Pro residues over a 7-residue segment (a total of 6(7) approximately 2.8 x 10(5) sequences). The cassette was inserted into the gene of a suitable carrier protein and expressed in E. coli with the T7 expression system, and the resultant library was subjected to solid-phase protein phosphorylation assays on nitrocellulose filters. When the library was screened with TPK1 delta, the modified catalytic subunit of the Saccharomyces cerevisiae cAMP-dependent protein kinase, individual colonies that expressed substrates for this kinase were identified. By DNA sequencing through the cassette region of positive clones, the consensus recognition sequence for TPK1 delta was deduced and found to conform with the well-established substrate selectivity of its mammalian homolog (Arg-Arg-Xaa-Ser). Because a large number of clones can be sequenced rapidly, and the positions of invariant residues composing a recognition site identified, this approach may be useful as a general screen of protein kinase substrate selectivity.     

An inquiry into the evolutionary status and apparent identity of San hunter-gatherers

An attempt is made to reconcile a popular view of the Bushmen or San of southern Africa with reality. Following an analysis of the assumed relationships of living hunter-gatherers with their Pleistocene forebears and modem neighbors, the identity of the San is explored using archaeological and historical evidence. Finally an alternative view of modern San, consistent with this evidence, is proposed.     

White adipose tissue reference network: a knowledge resource for exploring health-relevant relations

Optimal health is maintained by interaction of multiple intrinsic and environmental factors at different levels of complexity—from molecular, to physiological, to social. Understanding and quantification of these interactions will aid design of successful health interventions. We introduce the reference network concept as a platform for multi-level exploration of biological relations relevant for metabolic health, by integration and mining of biological interactions derived from public resources and context-specific experimental data. A White Adipose Tissue Health Reference Network (WATRefNet) was constructed as a resource for discovery and prioritization of mechanism-based biomarkers for white adipose tissue (WAT) health status and the effect of food and drug compounds on WAT health status. The WATRefNet (6,797 nodes and 32,171 edges) is based on (1) experimental data obtained from 10 studies addressing different adiposity states, (2) seven public knowledge bases of molecular interactions, (3) expert’s definitions of five physiologically relevant processes key to WAT health, namely WAT expandability, Oxidative capacity, Metabolic state, Oxidative stress and Tissue inflammation, and (4) a collection of relevant biomarkers of these processes identified by BIOCLAIMS (http://bioclaims.uib.es). The WATRefNet comprehends multiple layers of biological complexity as it contains various types of nodes and edges that represent different biological levels and interactions. We have validated the reference network by showing overrepresentation with anti-obesity drug targets, pathology-associated genes and differentially expressed genes from an external disease model dataset. The resulting network has been used to extract subnetworks specific to the above-mentioned expert-defined physiological processes. Each of these process-specific signatures represents a mechanistically supported composite biomarker for assessing and quantifying the effect of interventions on a physiological aspect that determines WAT health status. Following this principle, five anti-diabetic drug interventions and one diet intervention were scored for the match of their expression signature to the five biomarker signatures derived from the WATRefNet. This confirmed previous observations of successful intervention by dietary lifestyle and revealed WAT-specific effects of drug interventions. The WATRefNet represents a sustainable knowledge resource for extraction of relevant relationships such as mechanisms of action, nutrient intervention targets and biomarkers and for assessment of health effects for support of health claims made on food products.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-014-0439-x) contains supplementary material, which is available to authorized users.     

Genome-wide analysis of substrate specificities of the Escherichia coli haloacid dehalogenase-like phosphatase family

Haloacid dehalogenase (HAD)-like hydrolases are a vast superfamily of largely uncharacterized enzymes, with a few members shown to possess phosphatase, beta-phosphoglucomutase, phosphonatase, and dehalogenase activities. Using a representative set of 80 phosphorylated substrates, we characterized the substrate specificities of 23 soluble HADs encoded in the Escherichia coli genome. We identified small molecule phosphatase activity in 21 HADs and beta-phosphoglucomutase activity in one protein. The E. coli HAD phosphatases show high catalytic efficiency and affinity to a wide range of phosphorylated metabolites that are intermediates of various metabolic reactions. Rather than following the classical "one enzyme-one substrate" model, most of the E. coli HADs show remarkably broad and overlapping substrate spectra. At least 12 reactions catalyzed by HADs currently have no EC numbers assigned in Enzyme Nomenclature. Surprisingly, most HADs hydrolyzed small phosphodonors (acetyl phosphate, carbamoyl phosphate, and phosphoramidate), which also serve as substrates for autophosphorylation of the receiver domains of the two-component signal transduction systems. The physiological relevance of the phosphatase activity with the preferred substrate was validated in vivo for one of the HADs, YniC. Many of the secondary activities of HADs might have no immediate physiological function but could comprise a reservoir for evolution of novel phosphatases.     

Effect of culture conditions on the obtention of boar epididymal epithelial cell monolayers

The goal of this study was to investigate the effect of the collagenase digestion time, the initial density of fragments and the culture temperature on the obtention of a boar epididymal epithelial cell culture, which is a useful methodology for the study of epididymal functions. A confluent monolayer of caput, corpus and cauda epididymal epithelial cells was only obtained when an adequate enzymatic digestion of the connective tissue surrounding the epididymal tubule was performed. For the correct digestion of caput and corpus fragments two collagenase digestions of 2 and 1h, respectively, were enough. Cauda fragments, however, needed two collagenase digestions of 3h each. A confluent monolayer of caput, corpus and cauda epididymal epithelial cells was obtained regardless of the initial density tested (15, 30, 60 and 90fragments/well). However, cultures originated from 15 and 30fragments/well showed higher cell concentration during the first 2 weeks of culture than cultures originated from 60 and 90fragments/well. A confluent monolayer of caput, corpus and cauda epididymal epithelial cells was obtained at both 32 and 37 degrees Celsius, but at 32 degrees Celsius cells grew very slowly and confluence was not reached until a week later than it was with cells growing at 37 degrees Celsius. In conclusion, we have observed that the time of digestion with collagenase is an important factor for the successful establishment of boar epididymal cell monolayers, and that the initial density of fragments and the culture temperature should be taken into account.     

A diet supplemented with l-carnitine improves the sperm quality of Piétrain but not of Duroc and Large White boars when photoperiod and temperature increase

It has been reported that a diet supplemented with l-carnitine can improve sperm quality in some mammalian species. Against this background, the current study seeks to determine the effects of feeding l-carnitine (625 mg·day⁻¹) on boar semen characteristics (ejaculate volume, sperm concentration, sperm viability, acrosome and mitochondrial sheath integrity, sperm motility, sperm morphology, and osmotic resistance of spermatozoa) in three different porcine breeds (Sus domesticus) (Piétrain, Duroc, and Large White) exposed to natural environmental changes in temperature and photoperiod over a 20-wk period (February to July 2007). One hundred twenty boars (40 per breed) were randomly separated into two groups (60 boars each): the first (20 boars per breed) was fed a control diet and the second (also 20 males per breed) the same diet supplemented with l-carnitine (625 mg·day⁻¹). Whereas the l-carnitine supplement did not affect ejaculate volume, concentration, motility, viability, or the osmotic resistance of spermatozoa, it did improve sperm morphology in Piétrain boars by reducing the percentage of immature spermatozoa when the temperature and the photoperiod increased. Conversely, no effect on sperm morphology from supplementing feed with l-carnitine was observed in both Duroc and Large White breeds. We can therefore conclude that the addition of l-carnitine to the diet of males may maintain the level of normal sperm morphology in Piétrain boars when a drop in sperm quality occurs (due to increases in photoperiod and temperature), without affecting the other sperm quality parameters.