How does exercise intensity and type affect equine distal tarsal subchondral bone thickness?

Adaptation of osteochondral tissues is based on the strains experienced during exercise at each location within the joint. Different exercise intensities and types may induce particular site-specific strains, influencing osteochondral adaptation and potentially predisposing to injury. Our hypotheses were that patterns of equine distal tarsal subchondral bone (SCB) thickness relate to the type and intensity of exercise, and that high-intensity exercise leads to site-specific increases in thickness. SCB thickness was measured at defined dorsal and plantar locations on magnetic resonance images of cadaver tarsi collected from horses with a history of low [general purpose (n=20) and horse walker (n=6)] or high [elite competition (n=12), race training (n=15), and treadmill training (n=4)] exercise intensity. SCB thickness was compared between sites within each exercise group and between exercise groups. SCB thickness in elite competition and race training, but not treadmill training, was greater than low-intensity exercise. For general purpose horses, lateral SCB thickness was greater than medial throughout. Horse walker exercise led to relatively thicker lateral and medial SCB compared with the midline. Elite competition was associated with increased SCB thickness of the proximal small tarsal bones medially and the distal bones laterally. For race training and treadmill training, there were minimal differences between sites overall, although the lateral aspect was greater than medial, and medial greater than midline at a few sites for race training. In conclusion, different types of high-intensity exercise were associated with different patterns of SCB thickness across the joints from medial to lateral and proximal to distal, indicating that both exercise intensity and type of exercise affect the SCB response at any particular site within the equine distal tarsal joints.     

Structure and function of a novel purine specific nucleoside hydrolase from Trypanosoma vivax

The purine salvage pathway of parasitic protozoa is currently considered as a target for drug development because these organisms cannot synthesize purines de novo. Insight into the structure and mechanism of the involved enzymes can aid in the development of potent inhibitors, leading to new curative drugs. Nucleoside hydrolases are key enzymes in the purine salvage pathway of Trypanosomatidae, and they are especially attractive because they have no equivalent in mammalian cells. We cloned, expressed and purified a nucleoside hydrolase from Trypanosoma vivax. The substrate activity profile establishes the enzyme to be a member of the inosine-adenosine-guanosine-preferring nucleoside hydrolases (IAG-NH). We solved the crystal structure of the enzyme at 1.6 A resolution using MAD techniques. The complex of the enzyme with the substrate analogue 3-deaza-adenosine is presented. These are the first structures of an IAG-NH reported in the literature. The T. vivax IAG-NH is a homodimer, with each subunit consisting of ten beta-strands, 12 alpha-helices and three small 3(10)-helices. Six of the eight strands of the central beta-sheet form a motif resembling the Rossmann fold. Superposition of the active sites of this IAG-NH and the inosine-uridine-preferring nucleoside hydrolase (IU-NH) of Crithidia fasciculata shows the molecular basis of the different substrate specificity distinguishing these two classes of nucleoside hydrolases. An "aromatic stacking network" in the active site of the IAG-NH, absent from the IU-NH, imposes the purine specificity. Asp10 is the proposed general base in the reaction mechanism, abstracting a proton from a nucleophilic water molecule. Asp40 (replaced by Asn39 in the IU-NH) is positioned appropriately to act as a general acid and to protonate the purine leaving group. The second general acid, needed for full enzymatic activity, is probably part of a flexible loop located in the vicinity of the active site.     

Decreased density of [3H]TCP binding following antipsychotic drug withdrawal in rats

Antipsychotic drugs have been reported to increase the expression of subunits of the NMDA receptor at the level of mRNA but it is not clear whether such effects are apparent at the level of the radioligand binding or receptor protein. Therefore, we examined the effect of treatment of, and withdrawal from, haloperidol, chlorpromazine, olanzapine or clozapine on the binding of [3H]N-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP ) to the open ion channel of the NMDA receptor in rat caudate-putamen, hippocampus and frontal cortex. [3H]TCP binding was not significantly different in the caudate-putamen, hippocampus and cortex after three months of treatment with any antipsychotic drug. There were significant decreases in [3H]TCP binding in rat caudate-putamen and cortex, but not hippocampus, one month after ceasing treatment. Decreases in the caudate-putamen were detected in rats previously treated with chlorpromazine (0.1 mg/kg/day) and clozapine (0.1 and 1.0 mg/kg/day). In the cortex, decreases in [3H]TCP binding were also detected in rats previously treated with olanzapine (0.1 mg/kg/day) for three months. These data suggest that changes in the NMDA receptor associated ion channels occur following antipsychotic drug withdrawal.     

Conformational flexibility in the apolipoprotein E amino-terminal domain structure determined from three new crystal forms: implications for lipid binding

An amino-terminal fragment of human apolipoprotein E3 (residues 1-165) has been expressed and crystallized in three different crystal forms under similar crystallization conditions. One crystal form has nearly identical cell dimensions to the previously reported orthorhombic (P2(1)2(1)2(1)) crystal form of the amino-terminal 22 kDa fragment of apolipoprotein E (residues 1-191). A second orthorhombic crystal form (P2(1)2(1)2(1) with cell dimensions differing from the first form) and a trigonal (P3(1)21) crystal form were also characterized. The structures of the first orthorhombic and the trigonal form were determined by seleno-methionine multiwavelength anomalous dispersion, and the structure of the second orthorhombic form was determined by molecular replacement using the structure from the trigonal form as a search model. A combination of modern experimental and computational techniques provided high-quality electron-density maps, which revealed new features of the apolipoprotein E structure, including an unambiguously traced loop connecting helices 2 and 3 in the four-helix bundle and a number of multiconformation side chains. The three crystal forms contain a common intermolecular, antiparallel packing arrangement. The electrostatic complimentarity observed in this antiparallel packing resembles the interaction of apolipoprotein E with the monoclonal antibody 2E8 and the low density lipoprotein receptor. Superposition of the model structures from all three crystal forms reveals flexibility and pronounced kinks in helices near one end of the four-helix bundle. This mobility at one end of the molecule provides new insights into the structural changes in apolipoprotein E that occur with lipid association.     

Cadmium-induced crystallization of proteins: II. Crystallization of the Salmonella typhimurium histidine-binding protein in complex with L-histidine, L-arginine, or L-lysine.

To further investigate favorable effects of divalent cations on the formation of protein crystals, three complexes of Salmonella typhimurium histidine-binding protein were crystallized with varying concentrations of cadmium salts. For each of the three histidine-binding protein complexes, cadmium cations were found to promote or improve crystallization. The optimal cadmium concentration is ligand specific and falls within a narrow concentration range. In each case, crystals grown in the presence of cadmium diffract to better than 2.0 angstroms resolution and belong to the orthorhombic space group P2(1)2(1)2(1). From our results and from the analysis of cadmium sites in well-refined protein structures, we propose that cadmium addition provides a generally useful technique to modify crystal morphology and to improve diffraction quality.     

Tau,XMAP215/Msps and Eb1 co-operate interdependently to regulate microtubule polymerisation and bundle formation in axons

The formation and maintenance of microtubules requires their polymerisation, but little is known about how this polymerisation is regulated in cells. Focussing on the essential microtubule bundles in axons of Drosophila and Xenopus neurons, we show that the plus-end scaffold Eb1, the polymerase XMAP215/Msps and the lattice-binder Tau co-operate interdependently to promote microtubule polymerisation and bundle organisation during axon development and maintenance. Eb1 and XMAP215/Msps promote each other’s localisation at polymerising microtubule plus-ends. Tau outcompetes Eb1-binding along microtubule lattices, thus preventing depletion of Eb1 tip pools. The three factors genetically interact and show shared mutant phenotypes: reductions in axon growth, comet sizes, comet numbers and comet velocities, as well as prominent deterioration of parallel microtubule bundles into disorganised curled conformations. This microtubule curling is caused by Eb1 plus-end depletion which impairs spectraplakin-mediated guidance of extending microtubules into parallel bundles. Our demonstration that Eb1, XMAP215/Msps and Tau co-operate during the regulation of microtubule polymerisation and bundle organisation, offers new conceptual explanations for developmental and degenerative axon pathologies.     

Analysis of circulating microRNA biomarkers in plasma and serum using quantitative reverse transcription-PCR (qRT-PCR)

MicroRNAs (miRNAs) are small (～22 nt) RNAs that play important roles in gene regulatory networks by binding to and repressing the activity of specific target mRNAs. Recent studies have indicated that miRNAs circulate in a stable, cell-free form in the bloodstream and that the abundance of specific miRNAs in plasma or serum can serve as biomarkers of cancer and other diseases. Measurement of circulating miRNAs as biomarkers is associated with some special challenges, including those related to pre-analytic variation and data normalization. We describe here our procedure for qRT-PCR analysis of circulating miRNAs as biomarkers, and discuss relevant issues of sample preparation, experimental design and data analysis.