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.     

Expression of glucose-regulated proteins (GRP78 and GRP94) in hearts and fore-limb buds of mouse embryos exposed to hypoglycemia in vitro

Hypoglycemia, the classic inducer of glucose-related protein (GRP) synthesis, is dysmorphogenic in rodent embryos and detrimentally affects the heart. This study compares GRP induction in a target vs non-target tissue by evaluating GRP expression in hearts and fore-limb buds of mouse embryos following exposure to hypoglycemia in vitro. Gestational day 9.5 embryos were exposed to 2, 6, and 24 h of either mild (80 mg/dl glucose) or severe (40 mg/dl glucose) hypoglycemia using the method of whole-embryo culture. GRP78 increased in a dose- and time-dependent fashion in embryonic hearts exposed to either 40 mg/dl or 80 mg/dl glucose, whereas GRP94 levels increased in hearts only after 24 h of hypoglycemia. In contrast to the heart, GRP induction in fore-limb buds occurred only with GRP78 following the most severe level and duration of hypoglycemia. RT-PCR analysis demonstrated an elevation in GRP78 and GRP94 message levels in embryonic hearts following severe hypoglycemia. However, mRNA levels did not increase in response to mild hypoglycemia. Overall, these data demonstrate the preferential induction of GRPs in the heart as compared to fore-limb buds in mouse embryos exposed to hypoglycemia. Increases in GRP protein levels may be a more reliable biomarker of stress than message levels. However, both tissues and methods should be examined for enhanced biomarker sensitivity.     

Interactions among unit-price, fixed-ratio value, and dosing regimen in determining effects of repeated cocaine administration

Previous research has shown tolerance to cocaine that was dependent on fixed-ratio (FR) parameter size in the context of a multiple FR schedule of food reinforcement. Completion of the FR requirement in these studies resulted in the same magnitude of reinforcement, regardless of ratio size. The cost-to-benefits ratio (unit-price) was therefore not equated across the different FR components. The current study examined the effects of repeated administration of cocaine to pigeons when unit-price under FR schedules was either the same or different. Additionally, the role of a chronic variable-dosing versus chronic fixed-dosing procedure was examined when unit-price was equated across different ratio values. Pigeons were trained to key peck in daily sessions under a three-component multiple FR schedule of food presentation, according to which either 10, 30, or 100 pecks were required for each delivery of food. In Experiment 1, completion of the FR 10, FR 30, and FR 100 resulted in 1.5, 4.5, and 15.0 s access to food, respectively. That is, the response requirement was correlated with access to food time so that unit-price (pecks per second of access to food) was equated across components. After assessing acute effects of a range of doses of cocaine, drug administration occurred daily before each session, with the dose varying from day to day. Tolerance, the magnitude of which was unrelated to the peck requirement, developed under the repeated-dosing regimen. In order to assess whether having equal unit-price was responsible for producing similar levels of tolerance across components, daily drug administration continued in Experiment 2 using the variable-dose regimen, but the amount of food presented each time was fixed at 4.5 s access to food, yielding different unit-prices under the three pecking requirements. Subsequently, the conditions of Experiment 1 were reinstated, i.e., unit-price was equated. Making unit-price different or the same had little influence on effects of cocaine. In Experiment 3, a fixed dose of cocaine was administered before each session while programmed unit-price remained the same across components. Under these conditions, tolerance became peck-requirement related. Specifically, tolerance was most prevalent under the smaller requirements and less robust or absent when the largest requirement was in effect. Differences in unit-price, therefore, were not related to degree of tolerance, but work requirement was. Differences in effects of cocaine across responses requirements, however, were observed only when each session was preceded by the same dose, not when dose varied from session to session.     

Local and systemic inhibition of lung tumor growth after nanoparticle-mediated mda-7/IL-24 gene delivery

The human melanoma differentiation associated gene-7 (mda-7), also known as interleukin-24 (IL-24), is a novel gene with tumor suppressor, antiangiogenic, and cytokine properties. In vitro adenovirus-mediated gene transfer of the human mda-7/IL-24 gene (Ad-mda-7) results in ubiquitous growth suppression of human cancer cells with minimal toxicity to normal cells. Intratumoral administration of Ad-mda-7 to lung tumor xenografts results in growth suppression via induction of apoptosis and antiangiogenic mechanisms. Although these results are encouraging, one limitation of this approach is that its locoregional clinical application-systemic delivery of adenoviruses for treatment of disseminated cancer is not feasible at the present time. An alternative approach that is suitable for systemic application is non-viral gene delivery. We recently demonstrated that DOTAP:cholesterol (DOTAP:Chol) nanoparticles effectively deliver tumor suppressor genes to primary and disseminated lung tumors. In the present study, therefore, we evaluated nanoparticle-mediated delivery of the human mda-7/IL-24 gene to primary and disseminated lung tumors in vivo. We demonstrate that DOTAP:Chol efficiently delivers the mda-7/IL-24 gene to human lung tumor xenografts, resulting in suppression of tumor growth. Growth-inhibitory effects were observed in both primary (P=0.001) and metastatic lung tumors (P=0.02). Furthermore, tumor vascularization was reduced in mda-7/IL-24-treated tumors. Finally, growth was also inhibited in murine syngenic tumors treated with DOTAP:Chol-mda-7 nanoparticles (P=0.01). This is the first report demonstrating (1) systemic therapeutic effects of mda-7/IL-24 in lung cancer, and (2) antitumor effects of human mda-7 in syngeneic cancer models. Our findings are important for the development of mda-7/IL-24 treatments for primary and disseminated cancers.     

Fragmented landscapes, habitat specificity, and conservation genetics of three lizards in Florida scrub

Dry, sandy scrub habitats of the Floridapeninsula represent naturally fragmentedremnants of xeric ecosystems that werewidespread during the Pliocene and earlyPleistocene. This habitat is characterized byhigh endemism, and distribution of genetic andevolutionary diversity among scrub ``islands' isof compelling interest because Florida scrub israpidly disappearing under human development. We compare range-wide diversity inmitochondrial cytochrome b sequences forthree scrub-associated lizards with contrastinglevels of habitat specificity. All speciesshow strong geographic partitioning of geneticdiversity, supporting the hypothesis that scrubfauna is highly restricted by vicariantseparations. The mole skink (Eumecesegregius), the least habitat specific, has thelowest phylogeographic structure among thelizards (_st = 0.631). The mtDNAgeneology for E. egregius is not entirelyconcordant with the five recognized subspeciesand supports a link between populations incentral Florida (E. e. lividus) and theFlorida Keys (E.e. egregius) rather thana previously proposed affiliation betweennorthern and southern populations. The Floridascrub lizard (Sceloporus woodi) is themost habitat specific of the lizards and hasthe strongest phylogeographic structure (_st = 0.876). The sand skink (Neosepsreynoldsi) falls between the moleskink and scrub lizard in terms of habitatspecificity and phylogeographic structure (_st = 0.667). For all three species,networks of mtDNA haplotypes coalesce on twocentral ridges that contain the oldest scrub. The geographic structure and deep evolutionarylineages observed in these species have strongimplications for conservation, includingstrategies for translocation, reserve design,and management of landscape connectivity.     

Hermansky-Pudlak Syndrome Protein Complexes Associate with Phosphatidylinositol 4-Kinase Type II �� in Neuronal and Non-neuronal Cells

The Hermansky-Pudlak syndrome is a disorder affecting endosome sorting. Disease is triggered by defects in any of 15 mouse gene products, which are part of five distinct cytosolic molecular complexes: AP-3, homotypic fusion and vacuole protein sorting, and BLOC-1, -2, and -3. To identify molecular associations of these complexes, we used in vivo cross-linking followed by purification of cross-linked AP-3 complexes and mass spectrometric identification of associated proteins. AP-3 was co-isolated with BLOC-1, BLOC-2, and homotypic fusion and vacuole protein sorting complex subunits; clathrin; and phosphatidylinositol-4-kinase type II α (PI4KIIα). We previously reported that this membrane-anchored enzyme is a regulator of AP-3 recruitment to membranes and a cargo of AP-3 (Craige, B., Salazar, G., and Faundez, V. (2008) Mol. Biol. Cell 19,1415 -1426). Using cells deficient in different Hermansky-Pudlak syndrome complexes, we identified that BLOC-1, but not BLOC-2 or BLOC-3, deficiencies affect PI4KIIα inclusion into AP-3 complexes. BLOC-1, PI4KIIα, and AP-3 belong to a tripartite complex, and down-regulation of either PI4KIIα, BLOC-1, or AP-3 complexes led to similar LAMP1 phenotypes. Our analysis indicates that BLOC-1 complex modulates the association of PI4KIIα with AP-3. These results suggest that AP-3 and BLOC-1 act, either in concert or sequentially, to specify sorting of PI4KIIα along the endocytic route.Membranous organelles along the exocytic and endocytic pathways are each defined by unique lipid and protein composition. Vesicle carriers communicate and maintain the composition of these organelles (2). Consequently defining the machineries that specify vesicle formation, composition, and delivery are central to understanding membrane protein traffic. Generally vesicle biogenesis uses multiprotein cytosolic machineries to select membrane components for inclusion in nascent vesicles (2, 3). Heterotetrameric adaptor complexes (AP-1 to AP-4) are critical to generate vesicles of specific composition from the different organelles constituting the exocytic and endocytic routes (2-4).The best understood vesicle formation machinery in mammalian cells is the one organized around the adaptor complex AP-2 (5). This complex generates vesicles from the plasma membrane using clathrin. Our present detailed understanding of AP-2 vesicle biogenesis mechanisms and interactions emerged from a combination of organellar and in vitro binding proteomics analyses together with the study of binary interactions in cell-free systems (5-9). In contrast, the vesicle biogenesis pathways controlled by AP-3 are far less understood. AP-3 functions to produce vesicles that traffic selected membrane proteins from endosomes to lysosomes, lysosome-related organelles, or synaptic vesicles (10-13). AP-3 is one of the protein complexes affected in the Hermansky-Pudlak syndrome (HPS;³ Online Mendelian Inheritance in Man (OMIM) 203300). So far, mutations in any of 15 mouse or eight human genes trigger a common syndrome. This syndrome encompasses defects that include pigment dilution, platelet dysfunction, pulmonary fibrosis, and occasionally neurological phenotypes (14, 15). All forms of HPS show defective vesicular biogenesis or trafficking that affects lysosomes, lysosome-related organelles (for example melanosomes and platelet dense granules), and, in some of them, synaptic vesicles (11-13). Most of the 15 HPS loci encode polypeptides that assemble into five distinct molecular complexes: the adaptor complex AP-3, HOPS, and the BLOC complexes 1, 2, and 3 (14). Recently binary interactions between AP-3 and BLOC-1 or BLOC-1 and BLOC-2 suggested that arrangements of these complexes could regulate membrane protein targeting (16). Despite the abundance of genetic deficiencies leading to HPS and genetic evidence that HPS complexes may act on the same pathway in defined cell types (17), we have only a partial picture of protein interactions organizing these complexes and how they might control membrane protein targeting.In this study, we took advantage of cell-permeant and reversible cross-linking of HPS complexes followed by their immunoaffinity purification to identify novel molecular interactions. Cross-linked AP-3 co-purified with BLOC-1, BLOC-2, HOPS, clathrin, and the membrane protein PI4KIIα. We previously identified PI4KIIα as a cargo and regulator of AP-3 recruitment to endosomes (1, 18). Using mutant cells deficient in either individual HPS complexes or a combination of them, we found that BLOC-1 facilitates the interaction of AP-3 and PI4KIIα. Our studies demonstrate that subunits of four of the five HPS complexes co-isolate with AP-3. Moreover BLOC-1, PI4KIIα, and AP-3 form a tripartite complex as demonstrated by sequential co-immunoprecipitations as well as by similar LAMP1 distribution phenotypes induced by down-regulation of components of this tripartite complex. Our findings indicate that BLOC-1 complex modulates the recognition of PI4KIIα by AP-3. These data suggest that AP-3, either in concert or sequentially with BLOC-1, participates in the sorting of common membrane proteins along the endocytic route.     

Tm studies of a tertiary structure from the human hepatitis delta agent which functions in vitro as a ribozyme control element.

Viroids and other circular subviral RNA pathogens, such as the hepatitis delta agent, use a rolling circle replication cycle requiring an intact circular RNA. However, many infectious RNAs have the potential to form self-cleavage structures, whose formation must be controlled in order to preserve the circular replication template. The native structure of delta RNA contains a highly conserved element of local tertiary structure which is composed of sequences partially overlapping those needed to form the self-cleavage motif. A bimolecular complex containing the tertiary structure can be made. We show that when it is part of this bimolecular complex the potential cleavage site is protected and is not cleaved by the delta ribozyme, demonstrating that the element of local tertiary structure can function as a ribozyme control element in vitro. Physical studies of the complex containing this element were carried out. The complex binds magnesium ions and is not readily dissociated by EDTA under the conditions tested; > 50% of the complexes remain following incubation in 1 mM EDTA at 60 degrees C for 81 min. The thermal stability of the complex is reduced in the presence of sodium ions. A DNA complex and a perfect RNA duplex studied in parallel showed a similar effect, but of lesser magnitude. The RNA complex melts at temperatures approximately 10 degrees C lower in buffers containing 0.5 mM MgCl2 and 100 mM NaCl than in buffers containing 0.5 mM MgCl2 with no NaCl (78.1 compared with 87.7 degrees C). The element of local tertiary structure in delta genomic RNA appears to be a molecular clamp whose stability is highly sensitive to ion concentration in the physiological range.     

Smart is the new sexy: female mountain chickadees increase reproductive investment when mated to males with better spatial cognition

Understanding the evolution of inter and intraspecific variation in cognitive abilities is one of the main goals in cognitive ecology. In scatter‐caching species, spatial memory is critical for the recovery of food caches and overwinter survival, but its effects on reproduction are less clear. Better spatial cognition may improve pre‐breeding condition allowing for earlier reproduction. Alternatively, when mated to males with better spatial memory, females may be able to invest more in reproduction which may allow increased offspring survival and hence higher fitness. Using wild food‐caching mountain chickadees, we found that when environmental conditions were favourable for breeding, females mated to males with better spatial cognition laid larger clutches and fledged larger broods than females mated to males with worse cognitive performance. Our results support the hypothesis that females may increase their reproductive investment to gain indirect, genetic benefits when mated to high‐quality males with better spatial cognitive abilities.