Exercise training changes autonomic cardiovascular balance in mice.

Experiments were performed to investigate the influence of exercise training on cardiovascular function in mice. Heart rate, arterial pressure, baroreflex sensitivity, and autonomic control of heart rate were measured in conscious, unrestrained male C57/6J sedentary (n = 8) and trained mice (n = 8). The exercise training protocol used a treadmill (1 h/day; 5 days/wk for 4 wk). Baroreflex sensitivity was evaluated by the tachycardic and bradycardic responses induced by sodium nitroprusside and phenylephrine, respectively. Autonomic control of heart rate and intrinsic heart rate were determined by use of methylatropine and propranolol. Resting bradycardia was observed in trained mice compared with sedentary animals [485 +/- 9 vs. 612 +/- 5 beats/min (bpm)], whereas mean arterial pressure was not different between the groups (106 +/- 2 vs. 108 +/- 3 mmHg). Baroreflex-mediated tachycardia was significantly enhanced in the trained group (6.97 +/- 0.97 vs. 1.6 +/- 0.21 bpm/mmHg, trained vs. sedentary), whereas baroreflex-mediated bradycardia was not altered by training. The tachycardia induced by methylatropine was significantly increased in trained animals (139 +/- 12 vs. 40 +/- 9 bpm, trained vs. sedentary), whereas the propranolol effect was significantly reduced in the trained group (49 +/- 11 vs. 97 +/- 11 bpm, trained vs. sedentary). Intrinsic heart rate was similar between groups. In conclusion, dynamic exercise training in mice induced a resting bradycardia and an improvement in baroreflex-mediated tachycardia. These changes are likely related to an increased vagal and decreased sympathetic tone, similar to the exercise response observed in humans.     

Structures of lysenin reveal a shared evolutionary origin for pore-forming proteins and its mode of sphingomyelin recognition

Pore-forming proteins insert from solution into membranes to create lesions, undergoing a structural rearrangement often accompanied by oligomerization. Lysenin, a pore-forming toxin from the earthworm Eisenia fetida, specifically interacts with sphingomyelin (SM) and may confer innate immunity against parasites by attacking their membranes to form pores. SM has important roles in cell membranes and lysenin is a popular SM-labeling reagent. The structure of lysenin suggests common ancestry with other pore-forming proteins from a diverse set of eukaryotes and prokaryotes. The complex with SM shows the mode of its recognition by a protein in which both the phosphocholine headgroup and one acyl tail are specifically bound. Lipid interaction studies and assays using viable target cells confirm the functional reliance of lysenin on this form of SM recognition.     

Role of NF-κB and p38 MAPK activation in mediating angiotensin II and endothelin-1-induced stimulation in leptin production and cardiomyocyte hypertrophy

We recently identified leptin as a downstream factor mediating the hypertrophic effects of both angiotensin II and endothelin-1 in cardiomyocytes, an effect dependent on increased leptin biosynthesis, however, the mechanism for such increased leptin production is not known. This study was designed to elucidate the mechanisms underlying angiotensin II- and endothelin-1-stimulated synthesis in cultured ventricular myocytes. The hypertrophic effects of both angiotensin II (100 nM) and endothelin-1 (10 nM) were associated with increased leptin secretion and gene expression by 40 and 50 %, and 86 and 68 %, respectively. These effects were associated with significantly increased nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) phosphorylation by 34 and 52 %, as well as enhanced translocation of NF-κB into nuclei and also the NF-κB-DNA binding activity by 35 and 31 % induced by angiotensin II and endothelin-1, respectively. On their own, 24 h treatment with either angiotensin II or endothelin-1 increased cell surface area by 30 and 40 %, protein synthesis by 30 % and the α-skeletal actin gene by 53 and 68 %, respectively, indicating a robust hypertrophic effect whereas this was completely prevented by NF-κB inhibition. In addition, NF-κB inhibition significantly attenuated angiotensin II and endothelin-1-induced p38 MAPK activation whereas inhibition of p38 MAPK blocked both angiotensin II- and endothelin-1-induced increases in leptin secretion. The ability of both angiotensin II- and endothelin-1 to increase leptin production in cardiomyocytes and the resultant hypertrophic response are mediated by NF-κB and dependent on p38 MAPK activation.     

Diversity of North American map and sawback turtles (Testudines: Emydidae: Graptemys)

Map turtles of the genus Graptemys are native to North America, where a high degree of drainage endemism is believed to have shaped current diversity. With 14 species and one additional subspecies, Graptemys represents the most diverse genus in the family Emydidae. While some Graptemys species are characterized by pronounced morphological differences, previous phylogenetic analyses have failed yet to confirm significant levels of genetic divergence for many taxa. As a consequence, it has been debated whether Graptemys is taxonomically inflated or whether the low genetic divergence observed reflects recent radiations or ancient hybridization. In this study, we analysed three mtDNA blocks (3228 bp) as well as 12 nuclear loci (7844 bp) of 89 specimens covering all species and subspecies of Graptemys. Our analyses of the concatenated mtDNA sequences reveal that the widespread G. geographica constitutes the sister taxon of all other Graptemys species. These correspond to two clades, one comprised of all broad‐headed Graptemys species and another clade containing the narrow‐headed species. Most species of the broad‐headed clade are reciprocally monophyletic, except for G. gibbonsi and G. pearlensis, which are not differentiated. By contrast, in the narrow‐headed clade, many currently recognized species are not monophyletic and divergence is significantly less pronounced. Haplotype networks of phased nuclear loci show low genetic divergence among taxa and many shared haplotypes. Principal component analyses using coded phased nuclear DNA sequences revealed eight distinct clusters within Graptemys that partially conflict with the terminal mtDNA clades. This might be explained by male‐mediated gene flow across drainage basins and female philopatry within drainage basins. Our results support that Graptemys is taxonomically oversplit and needs to be revised.     

Epipolarization Microscopic Immunogold Assay: a Combination of Immunogold Silver Staining, Enzyme-Linked-Immunosorbent Assay and Epipolarization Microscopy

We describe a new immunoassay which combines an immunosorbent assay, Immunogold silver staining and epipolarization microscopy. Our new assay procedure features multiple samples on a single microscope slide, and high sensitivity of epipolarization microscope for detection of silver-enhanced colloidal gold as a final immunoassay product. We call the new immunoassay “on slide immunogold assay” (OSIGA). This new method uses biotinylated antibody and streptavidin-gold reaction with silver enhancement technique. With OSIGA it is possible to investigate 30 samples on a single microscopic slide. Our preliminary studies used 10-20 μ1 samples and detected nanogram quantities of a standardized protein solution. Unlike enzyme linked immunosorbent assay (ELISA), which has a limited time for reading the final color products, the OSIGA specimens can be dried or resin mounted for longer storage and future reference.     

Unlocking the barley genome by chromosomal and comparative genomics

We used a novel approach that incorporated chromosome sorting, next-generation sequencing, array hybridization, and systematic exploitation of conserved synteny with model grasses to assign ~86% of the estimated ~32,000 barley (Hordeum vulgare) genes to individual chromosome arms. Using a series of bioinformatically constructed genome zippers that integrate gene indices of rice (Oryza sativa), sorghum (Sorghum bicolor), and Brachypodium distachyon in a conserved synteny model, we were able to assemble 21,766 barley genes in a putative linear order. We show that the barley (H) genome displays a mosaic of structural similarity to hexaploid bread wheat (Triticum aestivum) A, B, and D subgenomes and that orthologous genes in different grasses exhibit signatures of positive selection in different lineages. We present an ordered, information-rich scaffold of the barley genome that provides a valuable and robust framework for the development of novel strategies in cereal breeding.     

Chronic PKC-beta activation in HT-29 Cl.19a colonocytes prevents cAMP-mediated ion secretion by inhibiting apical membrane current generation

We investigated the effects of PKC-stimulating 12-deoxyphorbol 13-phenylacetate 20-acetate (DOPPA) and phorbol 12-myristate 13-acetate (PMA) phorbol esters on cAMP-dependent, forskolin (FSK)-stimulated, short-circuit Cl- current (ISC-cAMP) generation by colonocyte monolayers. These agonists elicited different actions depending on their dose and incubation time; PMA effects at the onset (<5 min) were independent of cAMP agonist and were characterized by transient anion-dependent transcellular and apical membrane ISC generation. DOPPA failed to elicit similar responses. Whereas chronic (24 h) exposure to both agents inhibited FSK-stimulated transcellular and apical membrane ISC-cAMP, the effects of DOPPA were more complex: this conventional PKC-beta-specific agonist also stimulated Ba2+-sensitive basolateral membrane-dependent facilitation of transcellular ISC-cAMP. PMA did not elicit a similar phenomenon. Prolonged exposure to high-dose PMA but not DOPPA led to apical membrane ISC-cAMP recovery. Changes in PKC alpha-, beta1-, gamma-, and epsilon-isoform membrane partitioning and expression correlated with these findings. PMA-induced transcellular ISC correlated with PKC-alpha membrane association, whereas low doses of both agents inhibited transcellular and apical membrane ISC-cAMP, increased PKC-beta1, decreased PKC-beta2 membrane association, and caused reciprocal changes in isoform mass. During the apical membrane ISC-cAMP recovery after prolonged high-dose PMA exposure, an almost-complete depletion of cellular PKC-beta1 and a significant reduction in PKC-epsilon mass occurred. Thus activated PKC-beta1 and/or PKC-epsilon prevented, whereas activated PKC-alpha facilitated, apical membrane ISC-cAMP. PKC-beta-dependent augmentation of transcellular ISC-cAMP at the level of the basolateral membrane demonstrated that transport events with geographically distinct subcellular membranes can be independently regulated by the PKC beta-isoform.     

Localisation of [3H] clonidine binding in rat neurohypophysis by means of electron-microscopic autoradiography

Summary Electron-microscopic autoradiography of rat neurohypophyses treated with [³H] clonidine, an ₂-agonist, showed that binding apparently occurred preferentially at the neurosecretory endings and blood vessels rather than on the pituicytes. Since it is known that clonidine has a high affinity for plasma proteins, the distribution over the neurosecretory nerve endings would suggest the existence of presynaptic ₂-binding sites on neurosecretory neurones, which could indicate a regulatory function for catecholamines in neurohypophysial hormone release.     

Observation of the helical structure of the bacterial polysaccharide acetan by atomic force microscopy.

A method has been developed that has been found to give reproducible images of uncoated polysaccharides by Atomic Force Microscopy (AFM). Aqueous solutions of the polysaccharide are deposited as drops onto freshly cleaved mica surfaces, air dried, and then imaged under butanol. The method has been used to obtain images of the bacterial polysaccharide acetan. In regions within the deposited sample, where the molecules are aligned side-by-side, it has been possible to observe a periodic structure along the polysaccharide chain, attributable to the helical structure of acetan.