Phylogeography of native ploidy levels and invasive tetraploids of Solidago gigantea

Ploidy level is an important aspect of the genetic makeup of a plant, and can strongly influence ecological characteristics such as invasiveness. We used a phylogeographical approach to elucidate the history of polyploidization and colonization success of diploid and tetraploid Solidago gigantea Aiton (Asteraceae) within its native range in North America. We were also able to identify the probable source material of the haplotype lineages invasive in Europe and Asia, where only tetraploid plants occur. To do this, we sequenced 1275 bp of chloroplast intergenic spacer DNA in 268 individuals from 57 populations. In addition, we performed a crossing experiment, which supported the hypothesis that chloroplast inheritance in this species is maternal. The phylogeographical analysis showed a complex pattern of 20 haplotypes of diploid and tetraploid plants. In North America, we found significant differentiation among regions, private haplotypes, and isolation by distance. Ploidy levels were more differentiated in the northern regions than in the South. The haplotype network was shallow and included one tetraploid-only, star-shaped cluster of haplotypes that were particularly successful colonizers. Post-glacial migration of diploid S. gigantea occurred mainly northwards east of the Appalachian Mountains, and to a lesser degree also southward. Our data suggest that tetraploids have formed several times in North America. Haplotype number and diversity were lower in European populations than in the native range, and we found evidence that four haplotypes were introduced to Europe from two source areas, New England and the Southern Appalachian Mountains.     

Vector transmission of Bartonella species with emphasis on the potential for tick transmission

Bartonella species are gram-negative bacteria that infect erythrocytes, endothelial cells and macrophages, often leading to persistent blood-borne infections. Because of the ability of various Bartonella species to reside within erythrocytes of a diverse number of animal hosts, there is substantial opportunity for the potential uptake of these blood-borne bacteria by a variety of arthropod vectors that feed on animals and people. Five Bartonella species are transmitted by lice, fleas or sandflies. However, Bartonella DNA has been detected or Bartonella spp. have been cultured from numerous other arthropods. This review discusses Bartonella transmission by sandflies, lice and fleas, the potential for transmission by other vectors, and data supporting transmission by ticks. Polymerase chain reaction (PCR) or culture methods have been used to detect Bartonella in ticks, either questing or host-attached, throughout the world. Case studies and serological or molecular surveys involving humans, cats and canines provide indirect evidence supporting transmission of Bartonella species by ticks. Of potential clinical relevance, many studies have proposed co-transmission of Bartonella with other known tick-borne pathogens. Currently, critically important experimental transmission studies have not been performed for Bartonella transmission by many potential arthropod vectors, including ticks.     

Effects of streptozotocin-induced diabetes on connexin43 mRNA and protein expression in ventricular muscle

Abnormal QT prolongation with the associated arrhythmias is a significant predictor of mortality in diabetic patients. Gap junctional intercellular communication allows electrical coupling between heart muscle cells. The effects of streptozotocin (STZ)-induced diabetes mellitus on the expression and distribution of connexin 43 (Cx43) in ventricular muscle have been investigated. Cx43 mRNA expression was measured in ventricular muscle by quantitative PCR. The distribution of total Cx43, phosphorylated Cx43 (at serine 368) and non-phosphorylated Cx43 was measured in ventricular myocytes and ventricular muscle by immunocytochemistry and confocal microscopy. There was no significant difference in Cx43 mRNA between diabetic rat ventricle and controls. Total and phosphorylated Cx43 were significantly increased in ventricular myocytes and ventricular muscle and dephosphorylated Cx43 was not significantly altered in ventricular muscle from diabetic rat hearts compared to controls. Disturbances in gap junctional intercellular communication, which in turn may be attributed to alterations in balance between total, phosphorylated and dephosporylated Cx43, might partly underlie prolongation of QRS and QT intervals in diabetic heart.     

MUNIN: A new approach to multi-dimensional NMR spectra interpretation

A new method, MUNIN (Multi-dimensional NMR spectra interpretation), is introduced for the automated interpretation of three-dimensional NMR spectra. It is based on a mathematical concept referred to as three-way decomposition. An NMR spectrum is decomposed into a sum of components, with each component corresponding to one or a group of peaks. Each component is defined as the direct product of three one-dimensional shapes. A consequence is reduction in dimensionality of the spectral data used in further analysis. The decomposition may be applied to frequency-domain or time-domain data, or to a mixture of these. Features of MUNIN include good resolution in crowded regions and the absence of assumptions about line shapes. Uniform sampling of time-domain data, a prerequisite for discrete Fourier transform, is not required. This opens an avenue for the processing of NMR data that do not follow oscillating behaviour, e.g. from relaxation measurements. The application of MUNIN is illustrated for a ¹H-¹⁵N-NOESY-HSQC, where each component is defined as the set of all NOE peaks formed by a given amide group. As a result, the extraction of structural information simply consists of one-dimensional peak picking of the shape along the NOE-axis obtained for each amide group.     

Backbone dynamics of the channel-forming antibiotic zervamicin IIB studied by 15N NMR relaxation

The backbone dynamics of the channel-forming peptide antibiotic zervamicin IIB (Zrv-IIB) in methanol were studied by 15N nuclear magnetic resonance relaxation measurements at 11.7, 14.1 and 18.8 T magnetic fields. The anisotropic overall rotation of the peptide was characterized based on 15N relaxation data and by hydrodynamic calculations. 'Model-free' analysis of the relaxation data showed that the peptide is fairly rigid on a sub-nanosecond time-scale. The residues from the polar side of Zrv-IIB helix are involved in micro-millisecond time-scale conformational exchange. The conformational exchange observed might indicate intramolecular processes or specific intermolecular interactions of potential relevance to Zrv-IIB ion channel formation.     

Dynamics-modulated biological activity of transforming growth factor beta3

Transforming growth factor beta3 (TGF-beta3) is an important mediator of growth, maintenance, and repair processes in human cells. Internal dynamic properties have been derived from (15)N NMR relaxation data and mapped onto the spatial structure of TGF-beta3. The pattern of internal dynamics in the structure identifies potential "hot spots" of binding free energy and reveals the importance of conformational entropy in the interaction of TGF-beta3 with the receptors. The observed internal dynamics set TGF-beta3 apart from other TGF-beta isoforms, with which it shares the same fold. These findings may explain functional differences among the various TGF-beta isoforms and thus prove essential in the search for related therapeutic agents.     

Perforin oligomers form arcs in cellular membranes: a locus for intracellular delivery of granzymes

Perforin-mediated cytotoxicity is an essential host defense, in which defects contribute to tumor development and pathogenic disorders including autoimmunity and autoinflammation. How perforin (PFN) facilitates intracellular delivery of pro-apoptotic and inflammatory granzymes across the bilayer of targets remains unresolved. Here we show that cellular susceptibility to granzyme B (GzmB) correlates with rapid PFN-induced phosphatidylserine externalization, suggesting that pores are formed at a protein-lipid interface by incomplete membrane oligomers (or arcs). Supporting a role for these oligomers in protease delivery, an anti-PFN antibody (pf-80) suppresses necrosis but increases phosphatidylserine flip-flop and GzmB-induced apoptosis. As shown by atomic force microscopy on planar bilayers and deep-etch electron microscopy on mammalian cells, pf-80 increases the proportion of arcs which correlates with the presence of smaller electrical conductances, while large cylindrical pores decline. PFN appears to form arc structures on target membranes that serve as minimally disrupting conduits for GzmB translocation. The role of these arcs in PFN-mediated pathology warrants evaluation where they may serve as novel therapeutic targets.The cytotoxic cell granule-secretory pathway depends on perforin (PFN) to deliver granzyme (Gzm) proteases to the cytosol of target cells where they induce apoptosis and other biological effects, such as inflammation.¹ Ring-shaped transmembrane PFN pores hereafter called ‘cylindrical pores'', are presumed to act as the gateway for cytosolic entry, either at the plasma membrane or after endocytosis.^{2, 3, 4} In either case the highly cationic Gzms are thought to diffuse through these cylindrical pores formed by poly-PFN. Nevertheless, a mechanistic understanding of the phenomenon (how the cationic globular protein exchanges from its carrier proteoglycan, serglycin, to the pore and crosses the plasma and/or vesicular membranes) has been lacking due to limitations in imaging technology and in our detailed understanding of the molecular forms that PFN may adopt following interaction with a target cell plasma membrane.Here we show under conditions where cylindrical pore formation is minimal,⁵ that granzyme B (GzmB) translocation readily occurs. We previously demonstrated that a prelude to granzyme translocation is PFN-mediated, Ca-independent phosphatidylserine (PS) externalization (flip-flop) measured by annexin-V and lactadherin binding.⁶ This rapid PS flip-flop also occurs when mouse CD8 cells contact antigen-pulsed target cells. Inasmuch as the proteinaceous cylinders offer a formidable barrier to lipid flow, we have speculated that the observed movement of anionic phospholipids to the external leaflet is due to the formation of proteo-lipidic structures, which consists of oligomerized PFN monomers bearing an arc morphology and plasma membrane lipids.^{6, 7, 8}In the work reported here, the topology of PFN embedded into homogeneous planar bilayers and tumor cell plasma membranes was imaged by atomic force microscopy (AFM) and deep etch electron microscopy (DEEM), respectively. Further, the influence of an anti-human PFN mAb (pf-80) that rescues target cells from necrosis,⁹ was examined. The AFM data show that PFN forms arcs as well as rings in planar bilayers, while conductance measurements across equivalent membranes in parallel experiments measured functional pore sizes consistent with these varied structures. The pf-80 mAb increased the frequency of arc formation and reduced conductance values. Interestingly, PS flip-flop and granzyme delivery were both increased in target cells after PFN oligomerization was interrupted by the pf-80 mAb. A similar effect was seen in T24 bladder carcinoma cells imaged by DEEM. Treatment with PFN leads to deposition of rings (barrel stave pores) and arcs and the pf-80 mAb increased the ratio of arcs to rings on the surface of these cells. We suggest that the observed protein arcs function as toroidal pores in whole cells, explaining PS flip-flop, and act as focal points for granzyme translocation across lipid bilayer.     

Aerobic fitness and performance in elite female futsal players

Despite its growing popularity, few studies have investigated specific physiological demands for elite female futsal. The aim of this study was to determine aerobic fitness in elite female futsal players using laboratory and field testing. Fourteen female futsal players from the Venezuelan National team (age =21.2±4.0 years; body mass =58.6±5.6 kg; height =161±5.0 cm) performed a progressive maximal treadmill test under laboratory conditions. Players also performed a progressive intermittent futsal-specific field test for endurance, the Futsal Intermittent Endurance Test (FIET), until volitional fatigue. Outcome variables were exercise heart rate (HR), VO₂, post-exercise blood lactate concentrations ([La]b) and running speeds (km · h^-1). During the treadmill test, VO₂max, maximal aerobic speed (MAS), HR and peak [La]b were 45.3±5.6 ml · kg^-1 · min^-1, 12.5±1.77 km · h^-1, 197±8 beats · min^-1 and 11.3±1.4 mmol · l^-1, respectively. The FIET total distance, peak running velocity, peak HR and [La]b were 1125.0±121.0 m, 15.2±0.5 km · h^-1, 199±8 beats · min^-1 and 12.5±2.2 mmol · l^-1, respectively. The FIET distance and peak speed were strongly associated (r= 0.85-87, p < 0.0001) with VO₂max and MAS, respectively. Peak HR and [La]b were not significantly different between tests. Elite female futsal players possess moderate aerobic fitness. Furthermore, the FIET can be considered as a valid field test to determine aerobic fitness in elite level female futsal players.