A chemical,genetic, and structural analysis of the nuclear bile acid receptor FXR

The farnesoid X receptor (FXR) functions as a bile acid (BA) sensor coordinating cholesterol metabolism, lipid homeostasis, and absorption of dietary fats and vitamins. However, BAs are poor reagents for characterizing FXR functions due to multiple receptor independent properties. Accordingly, using combinatorial chemistry we evolved a small molecule agonist termed fexaramine with 100-fold increased affinity relative to natural compounds. Gene-profiling experiments conducted in hepatocytes with FXR-specific fexaramine versus the primary BA chenodeoxycholic acid (CDCA) produced remarkably distinct genomic targets. Highly diffracting cocrystals (1.78 A) of fexaramine bound to the ligand binding domain of FXR revealed the agonist sequestered in a 726 A(3) hydrophobic cavity and suggest a mechanistic basis for the initial step in the BA signaling pathway. The discovery of fexaramine will allow us to unravel the FXR genetic network from the BA network and selectively manipulate components of the cholesterol pathway that may be useful in treating cholesterol-related human diseases.     

Na+-dependent transport of large neutral amino acids occurs at the abluminal membrane of the blood-brain barrier

Several Na+-dependent carriers of amino acids exist on the abluminal membrane of the blood-brain barrier (BBB). These Na+-dependent carriers are in a position to transfer amino acids from the extracellular fluid of brain to the endothelial cells and thence to the circulation. To date, carriers have been found that may remove nonessential, nitrogen-rich, or acidic (excitatory) amino acids, all of which may be detrimental to brain function. We describe here Na+-dependent transport of large neutral amino acids across the abluminal membrane of the BBB that cannot be ascribed to currently known systems. Fresh brains, from cows killed for food, were used. Microvessels were isolated, and contaminating fragments of basement membranes, astrocyte fragments, and pericytes were removed. Abluminal-enriched membrane fractions from these microvessels were prepared. Transport was Na+ dependent, voltage sensitive, and inhibited by 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid, a particular inhibitor of the facilitative large neutral amino acid transporter 1 (LAT1) system. The carrier has a high affinity for leucine (Km 21 +/- 7 microM) and is inhibited by other neutral amino acids, including glutamine, histidine, methionine, phenylalanine, serine, threonine, tryptophan, and tyrosine. Other established neutral amino acids may enter the brain by way of LAT1-type facilitative transport. The presence of a Na+-dependent carrier on the abluminal membrane capable of removing large neutral amino acids, most of which are essential, from brain indicates a more complex situation that has implications for the control of essential amino acid content of brain.     

Human skeletal muscle creatine transporter mRNA and protein expression in healthy,young males and females

The present study investigated whether there were any differences between males and females in respect to creatine transporter (CreaT) gene expression and/or total creatine (TCr) content in human vastus lateralis muscle. Skeletal muscle obtained from young healthy male (n = 13, age: 23.2 ± 5.0 years) and female subjects (n = 12, age: 21.7 ± 4.3 years) was analyzed for CreaT mRNA, CreaT protein and TCr content. Total CreaT protein content in the muscle was similar (p > 0.05) between the sexes. Two bands (~ 55 and 73 kDa) of the CreaT protein were detected in all muscle samples. Both the 55 and the 73 kDa bands were present in similar (p > 0.05) amounts in males compared with females. The 73 kDa band was in greater abundance (p < 0.05) than the 55 kDa band, irrespective of gender. In addition, CreaT mRNA expression relative to -actin mRNA and the TCr content (males: 117.8 ± 2.2, females: 125.3 ± 4.3 mmol.kg^–1 dry mass) were also unaffected (p > 0.05) by gender. These data demonstrate that gender does not influence skeletal muscle TCr content and CreaT gene expression in young human subjects.     

Angiotensin II indirectly vasoconstricts the ovine uterine circulation

The uterine vasculature of women and sheep predominantly expresses type 2 ANG II receptors that do not mediate vasoconstriction. Although systemic ANG II infusions increase uterine vascular resistance (UVR), this could reflect indirect mechanisms. Thus we compared systemic and local intra-arterial ANG II infusions in six near-term pregnant and five ovariectomized nonpregnant ewes to determine how ANG II increases UVR. Systemic ANG II dose-dependently (P > 0.001) increased arterial pressure (MAP) and UVR and decreased uterine blood flow (UBF) in pregnant and nonpregnant ewes; however, nonpregnant responses exceeded pregnant (P < 0.001). In contrast, local ANG II infusions at rates designed to achieve concentrations in the uterine circulation comparable to those seen during systemic infusions did not significantly decrease UBF in either group, and changes in MAP and UVR were absent or markedly attenuated. When MAP rose during local ANG II, which only occurred with doses > or =2 ng/ml, increases in MAP were delayed more than twofold compared with responses during systemic ANG II infusions and always preceded decreases in UBF, resembling that observed during systemic ANG II infusions. These observations demonstrate attenuated uterine vascular responses to systemic ANG II during pregnancy and suggest that systemic ANG II may increase UVR through release of another potent vasoconstrictor(s) into the systemic circulation.     

Kinesin has three nucleotide-dependent conformations. Implications for strain-dependent release

Although crystallographic information is available on several nucleotide-induced states in myosin, little is known about the corresponding structural changes in kinesin, since a crystallographic model is only available for the kinesin:ADP complex. This makes it difficult to characterize at a molecular level the structural changes that occur in this motor through the course of its ATPase cycle. In this study, we report on the production of a series of single tryptophan mutants of a monomeric human kinesin motor domain, which demonstrate nucleotide-dependent changes in microtubule affinity that are similar to wild type. We have used these mutations to measure intramolecular distances in both strong and weak binding states, using fluorescence resonance energy transfer. This work provides direct evidence that movement of the switch II loop and helix are essential to mediate communication between the catalytic and microtubule binding sites, evidence that is supported as well by molecular modeling. Kinetic studies of fluorescent nucleotide binding to these mutants are consistent with these distance changes, and demonstrate as well that binding of ADP produces two structural transitions, neither of which are identical to that produced by the binding of ATP. This study provides a basis for understanding current structural models of the kinesin mechanochemical cycle.     

The taxonomic treatment of agamosperms in the genusLimonium mill. (Plumbaginaceae)

Agamospermous species account for a large proportion of the species ofLimonium. Agamospermy is indicated by uneven polyploidy or aneuploidy, low pollen stainability and by the presence of monomorphic self-incompatible populations. The taxonomic treatment of agamospermous taxa varies from recognition of all of them at the same specific rank or by utilising a range of ranks in the taxonomic hierarchy. The influence of evolutionary hypotheses on taxonomic systems is considered. Molecular data provide a means of measuring the genetical relationships of taxa and establishing groups in a taxonomic hierarchy.     

Stable individual differences in separation calls during early development in cats and mice

Background

The development of ethologically meaningful test paradigms in young animals is an essential step in the study of the ontogeny of animal personality. Here we explore the possibility to integrate offspring separation (distress) calls into the study of consistent individual differences in behaviour in two species of mammals, the domestic cat (Felis silvestris catus) and the mound-building mouse (Mus spicilegus). Such vocal responses in young mammals are a potentially useful test option as they represent an important element of mother-offspring communication with strong implications for offspring survival. In addition, the neural control of vocalisation is closely associated with emotional state.

Results

We found marked similarities in the pattern of individual responses of the young of both species to separation from their mother and littermates. In the domestic cat as well as in the mound-building mouse, individual differences in the frequency of calls and to a lesser extent in locomotor activity were repeatable across age, indicating the existence of personality types. Such consistencies across age were also apparent when only considering relative individual differences among litter siblings. In both species, however, individual patterns of vocalisation and locomotor activity were unrelated. This suggests that these two forms of behavioural responses to isolation represent different domains of personality, presumably based on different underlying neurophysiological mechanisms.

Conclusions

Brief separation experiments in young mammals, and particularly the measurement of separation calls, provide a promising approach to study the ontogeny of personality traits. Future long-term studies are needed to investigate the association of these traits with biologically meaningful and potentially repeatable elements of behaviour during later life.