Genetic isolation among mountains but not between stream types in a tropical high‐altitude mayfly

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Artificial and natural hybrids in pycnanthemum (Labiatae)

Further chromosome counts, principally from the southeastern United States, are reported for 38 accessions of 11 species, together with new or corrected counts forP. nudum (2n = 40),P. montanum (2n = 40),P. curvipes (2n = 40), andP. torrei (2n = 80). Four species have now been shown to have intraspecific polyploidy, five are known only as diploids, and nine occur only at the tetraploid level. Artificially produced interspecific hybrids range from vigorous to dwarfed or nonflowering, and from semifertile to sterile as measured by anther development and stainable pollen. Naturally occurring hybrids are described for the following species combinations:P. clinopodioides ×P. incanum,P. pilosum ×P. virginianum, P. flexuosum ×P. nudum, andP. albescens ×P. loomisii.

     

Fluorescently labeled adrenomedullin allows real‐time monitoring of adrenomedullin receptor trafficking in living cells

The human adrenomedullin (ADM) is a 52 amino acid peptide hormone belonging to the calcitonin family of peptides, which plays a major role in the development and regulation of cardiovascular and lymphatic systems. For potential use in clinical applications, we aimed to investigate the fate of the peptide ligand after binding and activation of the adrenomedullin receptor (AM₁), a heterodimer consisting of the calcitonin receptor‐like receptor (CLR), a G protein‐coupled receptor, associated with the receptor activity‐modifying protein 2 (RAMP2). Full length and N‐terminally shortened ADM peptides were synthesized using Fmoc/tBu solid phase peptide synthesis and site‐specifically labeled with the fluorophore carboxytetramethylrhodamine (Tam) either by amide bond formation or copper(I)‐catalyzed azide alkyne cycloaddition. For the first time, Tam‐labeled ligands allowed the observation of co‐internalization of the whole ligand‐receptor complex in living cells co‐transfected with fluorescent fusion proteins of CLR and RAMP2. Application of a fluorescent probe to track lysosomal compartments revealed that ADM together with the CLR/RAMP2‐complex is routed to the degradative pathway. Moreover, we found that the N‐terminus of ADM is not a crucial component of the peptide sequence in terms of AM₁ internalization behavior. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Calcium efflux of plasma membrane vesicles exposed to ELF magnetic fields-test of a nuclear magnetic resonance interaction model

The question whether very weak, low frequency magnetic fields can affect biological matter is still under debate. The theoretical possibility of such an interaction is often questioned and the site of interaction in the cell is unknown. In the present study, the influence of extremely weak 60 Hz magnetic fields on the transport of Ca²⁺ was studied in a biological system consisting of highly purified plasma membrane vesicles. We tested a newly proposed quantum mechanical model postulates that polarization of hydrogen nuclei can elicit a biological effect. Vesicles were exposed for half an hour at 32 °C and the calcium efflux was studied using radioactive ⁴⁵Ca²⁺ as a tracer. A static magnetic field of 26 µT and time‐varying magnetic fields with a frequency of 60 Hz and amplitudes between 0.6 and 6.3 µT were used. The predictions of the model, proposed by Lednev, that at a frequency of 60 Hz the biological effect under investigation would significantly be altered at the amplitudes of 1.3 and 3.9 µT could not be confirmed. Bioelectromagnetics 33:535–542, 2012. © 2012 Wiley Periodicals, Inc.     

AZD2184: a radioligand for sensitive detection of β-amyloid deposits

The presence of β‐amyloid plaques in brain is a hallmark of Alzheimer’s disease (AD) and serves as a biomarker for confirmation of diagnosis postmortem. Positron emission tomography (PET) radioligands such as Pittsburgh compound B ([¹¹C]‐2‐(3‐fluoro‐4‐methylamino‐phenyl)‐benzothiazol‐6‐ol) (PIB) binds selectively to β‐amyloid and are promising new tools supporting the clinical diagnoses of AD. In addition, such methodology may be useful for evaluation of new drugs aiming at reduction of amyloid plaque load. The objective of this study is to develop a new amyloid selective PET radioligand with higher signal‐to‐background ratio when compared with existing amyloid PET ligands. The lead compound, AZD2184, (2‐[6‐(methylamino)pyridin‐3‐yl]‐1,3‐benzothiazol‐6‐ol) was found to have high affinity for amyloid fibrils in vitro (K_d: 8.4 ± 1.0 nM). Two minutes after i.v. administration in rats, about 1% of the dose was in brain. In vitro autoradiography on cortical brain sections from amyloid‐beta precursor protein/presenilin 1 (APP/PS1) mice and AD patients showed that while [³H]AZD2184 and [³H]PIB are mutually displaceable, [³H]AZD2184 displays a higher signal‐to‐background ratio primarily by virtue of lower background binding levels. The ratio of binding ability in prefrontal cortex (high plaque load) to subcortical white matter (background) was 4.5 for [³H]AZD2184 and 0.8 for [³H]PIB at 1 nM. In adjacent cortical sections from APP/PS1 mouse as well as from AD cortical tissue, [³H]AZD2184 and antibodies to human β‐amyloid labeled identical structures. In vivo administration of [³H]AZD2184 to APP/PS1 mice further showed that [³H]AZD2184 labels amyloid deposits with low non‐specific background binding. Taken together, the pre‐clinical profile of AZD2184 in relation to the reference ligand PIB, suggests that ¹¹C‐labeled AZD2184 is a potential radioligand for PET‐visualization of β‐amyloid deposits in the living human brain.     

Structural and Genetic Basis for the Serological Differentiation of Pasteurella multocida Heddleston Serotypes 2 and 5

Pasteurella multocida is classified into 16 serotypes according to the Heddleston typing scheme. As part of a comprehensive study to define the structural and genetic basis of this scheme, we have determined the structure of the lipopolysaccharide (LPS) produced by P. multocida strains M1404 (B:2) and P1702 (E:5), the type strains for serotypes 2 and 5, respectively. The only difference between the LPS structures made by these two strains was the absence of a phosphoethanolamine (PEtn) moiety at the 3 position of the second heptose (Hep II) in M1404. Analysis of the lpt-3 gene, required for the addition of this PEtn residue, revealed that the gene was intact in P1702 but contained a nonsense mutation in M1404. Expression of an intact copy of lpt-3 in M1404 resulted in the attachment of a PEtn residue to the 3 position of the Hep II residue, generating an LPS structure identical to that produced by P1702. We identified and characterized each of the glycosyltransferase genes required for assembly of the serotype 2 and 5 LPS outer core. Monoclonal antibodies raised against serotype 2 LPS recognized the serotype 2/5-specific outer core LPS structure, but recognition of this structure was inhibited by the PEtn residue on Hep II. These data indicate that the serological classification of strains into Heddleston serotypes 2 and 5 is dependent on the presence or absence of PEtn on Hep II.Pasteurella multocida is a gram-negative pathogen that causes serious diseases in animals and humans. It is the causative agent of fowl cholera (7), hemorrhagic septicemia in cattle (9), atrophic rhinitis in pigs (6), and dog and cat bite infections in humans (28).P. multocida isolates may be grouped serologically based on capsular antigens into five serogroups—A, B, D, E, and F—using a passive hemagglutination test with erythrocytes sensitized with capsular antigen. Structural information is available for the capsular polysaccharides synthesized by serogroups A (hyaluronic acid) (22), D (heparin) (10), and F (chondroitin) (10). The genes involved in biosynthesis of the capsules have been identified for all five serogroups (27), and capsule is a critical virulence factor for serogroups A (8) and B (3).Lipopolysaccharide (LPS) is also an important virulence factor in P. multocida (13) and can be used for the identification of strains, with two main somatic typing systems reported (14, 17). The Namioka system is based on a tube agglutination test and is able to recognize 11 serotypes (17), whereas the Heddleston system uses a gel diffusion precipitation test and can recognize 16 serotypes; the Heddleston system is currently the preferred method (14). Current classification of P. multocida strains combines capsular typing with Heddleston somatic typing. Strains are given a designation in which the first letter indicates the capsular group and the number designates the Heddleston LPS serotype (e.g., A:1 indicates a strain that is capsular group A and LPS serotype 1). LPS produced by each of the 16 Heddleston serotype strains has been examined previously for sugar content and reactivity with LPS antisera (21). The LPS isolated from serotype 2 and 5 strains was virtually identical in sodium dodecyl sulfate-polyacrylamide gel electrophoresis migration profile (19), sugar composition, and serological reactivity with anti-LPS antibodies (21). Interestingly, serotypes 2 and 5 were the only serotypes found to elaborate two isomers of heptose in their LPS, namely l-glycero-d-manno-heptose (ld-Hep) and d-glycero-d-manno-heptose (dd-Hep) (21). The aims of this study were to determine whether the LPS molecules made by these two serotypes were structurally distinct and to compare the LPS structures with those previously determined for P. multocida serotypes 1 and 3 (24-26). Furthermore, we identified the transferase genes responsible for the assembly of the outer core LPS structure in each of these strains and characterized the function of each glycosyltransferase.     

Discovery of tetrahydro-cyclopenta[b]indole as selective LXRs modulator

A series of tetrahydro-cyclopenta[b]indoles modulating the activity of the liver-X-receptor (LXR) were derived from a high throughput screening hit. The potency and selectivity for LXRβ versus LXRα was improved. One compound, administered to wild-type mice modestly increased plasma HDL-cholesterol with no change in plasma triglycerides (TG) and reduced effects on liver TG content compared to T0901317. This novel series of LXR agonists shows promise to improve therapeutic efficacy with reduced potential to increase TG.     

C-Terminal Fragments of Amyloid-β Peptide Cause Cholinergic Axonal Degeneration by a Toxic Effect Rather Than by Physical Injury in the Nondemented Human Brain

Previous experimental studies have indicated that amyloid-b peptide (A) may cause axonal degeneration in the brain of individuals with Alzheimer's disease (AD) by physical injury, mass lesion, or membrane perturbation. In this study, acetylcholinesterase histochemical, and A and tau immunohistochemical double-staining were performed in nondemented elderly human hippocampal and entorhinal brain samples, to demonstrate the presence of dystrophic neurites caused by the C-terminal or N-terminal fragments of A. The early interactions between the A-stained senile plaques (SPs) and the enzyme-positive axons were investigated. The double-stained samples revealed that A deposition occurs first, followed by the development of cholinergic axonal damage. Most of the dystrophic axonal processes are incorporated in the peripheral area of the SPs and are positive for phosphorylated tau [pS²⁰²] and tau-5. The result suggests that C-terminal fragments are more harmful than N-terminal fragments of A and may induce the development of dystrophic neurites by a toxic effect rather than by physical injury.