Prokofieff\u27s Second Piano Sonata Transcribed for Sring Quartet

The primary purpose of the transcription is to contribute new and fresh material to the ever expanding body of string quartet literature. A new work by a composer of Prokofieff\u27s stature will be a creditable addition to the chamber music repertoire. The purpose of the accompanying text is threefold: (1) to give a brief history of Serge Prokofieff, (2) to give a brief history of the string quartet, and (3) to examine the methods chosen by the writer to transcribe the sonata tor string quartet

Bradykinin B2 receptor-mediated transport into intact cells : anti-receptor antibody-based cargoes

Endocytosis of the bradykinin-stimulated B2 receptors is parallel to the transport and subsequent degradation of the ligand. To implement biotechnological applications based on receptor-mediated transport, one strategy is to conjugate the agonist ligand to a cargo. Alternatively, we studied whether the B2 receptor can transport large antibody-based cargoes into intact cells and characterized the ensuing endosomal routing. Myc-tagged B2 receptors (coded by the vector myc-B2R) and a truncated construction devoid of the Ser–Thr phosphorylation domain (myc-B2Rtrunc vector) were coupled to anti-myc monoclonal antibodies that did not impair bradykinin binding or elicit calcium signaling in intact cells. Anti-myc antibodies, conjugated or not with secondary antibodies optionally coupled to Qdot nanomaterials, were transported into early endosome autoantigen 1-, and β-arrestin-positive vesicles in bradykinin-stimulated intact cells expressing receptors encoded by myc-B2R. Antibody-conjugated cargoes progressed into late-endosomes-lysosomes within 3 h without evidence of autophagy. Receptors encoded by myc-B2Rtrunc did not support the ligand-controlled endocytosis of anti-myc antibodies. Aside from small ligand-conjugated cargoes, very large antibody-based cargoes can be transported by agonist-stimulated B2 receptors into intact cells. The latter type of cargo requires a receptor competent for interaction with β-arrestins, enters the degradation pathway separately from the receptor as a function of time and has the potential to confer a qualitatively novel function to a receptor

A tagged parathyroid hormone derivative as a carrier of antibody cargoes transported by the G protein coupled PTH1 receptor

Based on the known fact that the parathyroid hormone (PTH) might be extended at its C-terminus with biotechnological protein cargoes, a vector directing the secretion of PTH1–84 C-terminally fused with the antigenic epitope myc (PTH-myc) was exploited. The functional properties and potential of this analog for imaging PTH1R-expressing cells were examined. The PTH-myc construct was recombinantly produced as a conditioned medium (CM) of transfected HEK 293a cells (typical concentrations of 187 nM estimated with ELISAs for PTH). PTH-myc CM induced cyclic AMP formations (10 min), with a minor loss of potency relative to authentic PTH1–84, and c-Fos expression (1–3 h). Treatment of recipient HEK 293a cells transiently expressing PTH1R with PTH-myc CM (supplemented with a fluorescent monoclonal anti-myc tag antibody, either 4A6 or 9E10) allowed the labeling of endosomal structures positive for Rab5 and/or for β-arrestin1 (microscopy, cytofluorometry). Authentic PTH was inactive in this respect, ruling out a non-specific form of endocytosis like pinocytosis. Using a horseradish peroxidase-conjugated secondary antibody, the endocytosis of the PTH-myc-based antibody complex by endogenous PTH1R was evidenced in MG-63 osteoblastoid cells. The secreted construct PTH-myc represents a bona fide agonist that supports the feasibility of transporting cargoes of considerable molecular weight inside cells using arrestin and Rab5-mediated PTH1R endocytosis. PTH-myc is also transported into cells that express PTH1R at a physiological level. Such tagged peptide hormones may be part of a cancer chemotherapy scheme exploiting a modular cytotoxic secondary antibody and the receptor repertoire expressed in a given tumor

Inhibitory effects of cytoskeleton disrupting drugs and GDP-locked Rab mutants on bradykinin B2 receptor cycling

The bradykinin (BK) B2 receptor (B2R) is G protein coupled and phosphorylated upon agonist stimulation; its endocytosis and recycling are documented. We assessed the effect of drugs that affect the cytoskeleton on B2R cycling. These drugs were targeted to tubulin (paclitaxel, or the novel combretastatin A-4 mimetic 3,4,5-trimethoxyphenyl-4-(2-oxoimidazolidin-1-yl)benzenesulfonate [IMZ-602]) and actin (cytochalasin D). Tubulin ligands did not alter agonist-induced receptor endocytosis, as shown using antibodies reactive with myc-tagged B2Rs (microscopy, cytofluorometry), but rather reduced the progression of the ligand–receptor–β-arrestin complex from the cell periphery to the interior. The 3 fluorescent probes of this complex (B2R-green fluorescent protein [B2R-GFP], the fluorescent agonist fluorescein-5-thiocarbamoyl-D-Arg-[Hyp3, Igl5, Oic7, Igl8]-BK and β-arrestin2–GFP) were condensed in punctuate structures that remained close to the cell surface in the presence of IMZ-602. Cytochalasin D selectively inhibited the recycling of endocytosed B2R-GFP (B2R-GFP imaging, [3H]BK binding). Dominant negative (GDP-locked)-Rab5 and -Rab11 reproduced the effects of inhibitors of tubulin and actin, respectively, on the cycling of B2R-GFP. GDP-locked-Rab4 also inhibited B2R-GFP recycling to the cell surface. Consistent with the displacement of cargo along specific cytoskeletal elements, Rab5-associated progression of the endocytosed BK B2R follows microtubules toward their (−) end, while its recycling progresses along actin fibers to the cell surface. However, tubulin ligands do not suppress the tested desensitization or resensitization mechanisms of the B2

Cation trapping by cellular acidic compartments: beyond the concept of lysosomotropic drugs

“Lysosomotropic” cationic drugs are known to concentrate in acidic cell compartments due to low retro-diffusion of the protonated molecule (ion trapping); they draw water by an osmotic mechanism, leading to a vacuolar response. Several aspects of this phenomenon were recently reexamined. (1) The proton pump vacuolar (V)-ATPase is the driving force of cationic drug uptake and ensuing vacuolization. In quantitative transport experiments, V-ATPase inhibitors, such as bafilomycin A1, greatly reduced the uptake of cationic drugs and released them in preloaded cells. (2) Pigmented or fluorescent amines are effectively present in a concentrated form in the large vacuoles. (3) Consistent with V-ATPase expression in trans-Golgi, lysosomes and endosomes, a fraction of the vacuoles is consistently labeled with trans-Golgi markers and protein secretion and endocytosis are often inhibited in vacuolar cells. (4) Macroautophagic signaling (accumulation of lipidated and membrane-bound LC3 II) and labeling of the large vacuoles by the autophagy effector LC3 were consistently observed in cells, precisely at incubation periods and amine concentrations that cause vacuolization. Vacuoles also exhibit late endosome/lysosome markers, because they may originate from such organelles or because macroautophagosomes fuse with lysosomes. Autophagosome persistence is likely due to the lack of resolution of autophagy, rather than to nutritional deprivation. (5) Increased lipophilicity decreases the threshold concentration for the vacuolar and autophagic cytopathology, because simple diffusion into cells is limiting. (6) A still unexplained mitotic arrest is consistently observed in cells loaded with amines. An extended recognition of relevant clinical situations is proposed for local or systemic drug administration

The effect of grazing management on the hydrological balance of natural pastures on the North-West Slopes of New South Wales

Natural pastures, which are dominated by native plant species, occupy an extensive proportion of Australia (432 M ha, or 56% of the continental landmass). Traditional grazing methods (continuous set-stocking) can lead to low levels of herbage mass, litter mass and ground cover, which in turn leads to high surface runoff, high soil evaporation, and poor pasture growth. A key component of designing a sustainable grazing system for these pastures includes a sound knowledge of the impact of that system on the hydrological balance. A grazing management experiment was established at Springmount near Barraba on the North-West Slopes to study the effect of five grazing treatments on pasture characteristics while monitoring the associated impact on selected components of the hydrological balance. The grazing treatments included: continuous grazing (4 and 6 sheep/11a), continuous grazing with subterranean clover and fertiliser applied (8 sheep/ha), and rotational grazing (4 sheep/ha) with pastures grazed for four weeks and rested for four weeks (two paddock rotation) or rested for 12 weeks (four paddock rotation). The continuous grazing treatments had significantly lower mean levels of herbage mass (1500-1800 kg DM/ha), litter mass (1)0-110 kg DM/ha) and ground cover (70-73%) compared with either rotational grazing or over-sowing with subterranean clover (3000-3500 kg DM/ha, 210-260 kg DM/ha, and 83-90% for herbage mass, litter mass and ground cover, respectively)

Rapid and accurate analysis of stem cell-derived extracellular vesicles with super resolution microscopy and live imaging

Extracellular vesicles (EVs) have prevalent roles in cancer biology and regenerative medicine. Conventional techniques for characterising EVs including electron microscopy (EM), nanoparticle tracking analysis (NTA) and tuneable resistive pulse sensing (TRPS), have been reported to produce high variability in particle count (EM) and poor sensitivity in detecting EVs below 50 nm in size (NTA and TRPS), making accurate and unbiased EV analysis technically challenging. This study introduces direct stochastic optical reconstruction microscopy (d-STORM) as an efficient and reliable characterisation approach for stem cell-derived EVs. Using a photo-switchable lipid dye, d-STORM imaging enabled rapid detection of EVs down to 20–30 nm in size with higher sensitivity and lower variability compared to EM, NTA and TRPS techniques. Imaging of EV uptake by live stem cells in culture further confirmed the potential of this approach for downstream cell biology applications and for the analysis of vesicle-based cell-cell communication

Quantitative and Rapid DNA Detection by Laser Transmission Spectroscopy

Laser transmission spectroscopy (LTS) is a quantitative and rapid in vitro technique for measuring the size, shape, and number of nanoparticles in suspension. Here we report on the application of LTS as a novel detection method for species-specific DNA where the presence of one invasive species was differentiated from a closely related invasive sister species. The method employs carboxylated polystyrene nanoparticles functionalized with short DNA fragments that are complimentary to a specific target DNA sequence. In solution, the DNA strands containing targets bind to the tags resulting in a sizable increase in the nanoparticle diameter, which is rapidly and quantitatively measured using LTS. DNA strands that do not contain the target sequence do not bind and produce no size change of the carboxylated beads. The results show that LTS has the potential to become a quantitative and rapid DNA detection method suitable for many real-world applications