Isolation of Oospores of Sunflower Downy Mildew, Plasmopara halstedii, and Microscopical Studies on Oospore Germination

A method was elaborated to isolate oospores of Plasmopara halstedii from tissue of its host, Helianthus annuus . Isolated oospores were studied microscopically and germination was documented with respect to the time course and the mode of germination. Formation of primary sporangia was similarly observed in oospores, harvested from 4- to 6-week-old systemically infected sunflower plants, grown under constant conditions at 16°C, as well as from field plants, harvested late in the season. Pretreatment of oospores with cold temperatures, previously assumed to stimulate the rate and to accelerate the speed of oospore germination, did not result in such effects. Germination usually occurred within 10–30 days of incubation at a highly variable rate of about 1 to 17% (average 6.7%) in deionized water.     

Enhanced exon skipping and prolonged dystrophin restoration achieved by TfR1-targeted delivery of antisense oligonucleotide using FORCE conjugation in mdx mice

Current therapies for Duchenne muscular dystrophy (DMD) use phosphorodiamidate morpholino oligomers (PMO) to induce exon skipping in the dystrophin pre-mRNA, enabling the translation of a shortened but functional dystrophin protein. This strategy has been hampered by insufficient delivery of PMO to cardiac and skeletal muscle. To overcome these limitations, we developed the FORCE^TM platform consisting of an antigen-binding fragment, which binds the transferrin receptor 1, conjugated to an oligonucleotide. We demonstrate that a single dose of the mouse-specific FORCE–M23D conjugate enhances muscle delivery of exon skipping PMO (M23D) in mdx mice, achieving dose-dependent and robust exon skipping and durable dystrophin restoration. FORCE–M23D-induced dystrophin expression reached peaks of 51%, 72%, 62%, 90% and 77%, of wild-type levels in quadriceps, tibialis anterior, gastrocnemius, diaphragm, and heart, respectively, with a single 30 mg/kg PMO-equivalent dose. The shortened dystrophin localized to the sarcolemma, indicating expression of a functional protein. Conversely, a single 30 mg/kg dose of unconjugated M23D displayed poor muscle delivery resulting in marginal levels of exon skipping and dystrophin expression. Importantly, FORCE–M23D treatment resulted in improved functional outcomes compared with administration of unconjugated M23D. Our results suggest that FORCE conjugates are a potentially effective approach for the treatment of DMD.     

Enzymatic stereospecific preparation of fluorescent S-adenosyl-l-methionine analogs

S-Adenosyl-l-methionine (SAM) is the preferred cofactor for biological methyl group transfers to various substrates such as nucleic acids, proteins, and lipids. Here we present stereospecific (>95% of the desired enantiomer) and high-yield preparation of four fluorescent and biologically active SAM analogs and demonstrate their usefulness in binding studies. Using a fluorescence titration experiment, we obtained a K_d of 0.38 μM for the S-2,6-diaminopurinylmethionine-SAM-III riboswitch complex.     

Influence of Microbially Reducible Fe(III) on the Bacterial Community Structure of Estuarine Surface Sediments

We analyzed PCR-amplified 16S rRNA genes from native and Fe(III)-enriched surface sediments of a major tidal channel in the Tijuana River Estuary, California, USA. Clones from native sediments were most closely affiliated with photosynthetic taxa (Cyanobacteria, Chloroflexi, and Halochromatium) and microorganisms known to reduce (Desulfatibacillus, Desulfobacterium, and Desulfuromusa) or oxidize (Microcoleus, Phormidium, and Halochromatium) various sulfur species, reflective of the fluctuating redox conditions in the tidal zone. Fe(III) was rapidly reduced in anaerobic microcosms amended with 2-line ferrihydrite, with or without the sulfate reduction inhibitor sodium molybdate. The addition of ferrihydrite without molybdate caused a major shift in community structure to a dominance of the Fe(III)-reducing genus Shewanella, while at the same time the sulfate-reducing and sulfide-oxidizing populations were replaced by taxa known to cycle elemental sulfur. Sediments amended with both ferrihydrite and molybdate were again populated by Shewanella clones, but also numerically important were clones most similar to Marinobacterium, Pseudomonas, and Bacillus, suggesting a role for these taxa in Fe(III) reduction in marine habitats.     

Ion transport by mitochondria-rich cells in toad skin

Summary The optical sectioning video imaging technique was used for measurements of the volume of mitochondria-rich (m.r.) cells of the isolated epithelium of toad skin. Under short-circuit conditions, cell volume decreased by about 14% in response to bilateral exposure to Cl-free (gluconate substitution) solutions, apical exposure to ouabain resulted in a large increase in volume, which could be prevented either by the simultaneous application of amiloride in the apical solution or by the exposure of the epithelium to bilateral Cl-free solutions. Unilateral exposure to a Cl-free solution did not prevent ouabain-induced cell swelling. It is concluded that m.r. cells have an amiloride-blockable Na conductance in the apical membrane, a ouabain-sensitive Na pump in the basolateral membrane, and a passive Cl permeability in both membranes. From the initial rate of ouabain-induced cell volume increase the active Na current carried by a single m.r. cell was estimated to be 9.9±1.3 pA. Voltage clamping of the preparation in the physiological range of potentials (0 to –100 mV, serosa grounded) resulted in a cell volume increase with a time course similar to that of the stimulation of the voltage-dependent activation were prevented by exposure of the tissue to a Cl-free apical solution. The steady-state volume of the m.r. cells increased with the clamping voltage, and at –100 mV the volume was about 1.15 times that under short-circuit conditions. The rate of volume increase during current passage was significantly decreased by lowering the serosal K concentration (K _i ) to 0.5mm, but was independent of whether K _i was 2.4, 5, or 10mm. This indicates that the K conductance of the serosal membrane becomes rate limiting for the uptake of KCl when K _i is significantly lower than its physiological value. It is concluded that the voltage-activated Cl currents flow through the m.r. cells and that swelling is caused by an uptake of Cl ions from the apical bath and K ions from the serosal bath. Bilateral exposure of the tissue to hypo- or hypertonic bathing solutions changed cell volume without detectable changes in the Cl conductance. The volume response to external osmotic perturbations followed that of an osmometer with an osmotically inactive volume of 21%. Using this value and the change in cell volume in response to bilateral Cl-free solutions, we calculated an intracellular steady-state Cl concentration of 19.8±1.7mm (n=6) of the short-circuited cell.     

Liposomes of controllable size in the range of 40 to 180 nm by defined dialysis of lipid/detergent mixed micelles

Liposomes, in the size range of 40–180 nm, are formed when lipid and additives are solubilized with detergent, yielding defined mixed micelles, and the detergent is subsequently removed by controlled dialysis. Their most important properties are that they are indeed unilamellar with usefully large encapsulated volumes and are homogeneous in size. Liposomes have been formed from both natural and synthetic phospholipids with cholesterol and charged molecules added. This relatively simple technique may be particularly useful for encapsulating drugs, enzymes and other macromolecules and in studies of reconstitution of membrane proteins.