In this study, we used small interfering RNA (siRNA) directed against vascular endothelial growth factor receptor 1 (vegfr1) mRNA to investigate the role of VEGFR1 in ocular neovascularization (NV). After evaluating many siRNAs, Sirna-027 was identified; it cleaved vegfr1 mRNA at the predicted site and reduced its levels in cultured endothelial cells and in mouse models of retinal and choroidal neovascularization (CNV). Compared to injection of an inverted control sequence, quantitative reverse transcriptase-PCR demonstrated statistically significant reductions of 57 and 40% in vegfr1 mRNA after intravitreous or periocular injection of Sirna-027, respectively. Staining showed uptake of 5-bromodeoxyuridine-labeled Sirna-027 in retinal cells that lasted between 3 and 5 days after intravitreous injection and was still present 5 days after periocular injection. In a CNV model, intravitreous or periocular injections of Sirna-027 resulted in significant reductions in the area of NV ranging from 45 to 66%. In mice with ischemic retinopathy, intravitreous injection of 1.0 mg of Sirna-027 reduced retinal NV by 32% compared to fellow eyes treated with 1.0 mg of inverted control siRNA. These data suggest that VEGFR1 plays an important role in the development of retinal and CNV and that targeting vegfr1 mRNA with siRNA has therapeutic potential. Gene Therapy (2006) 13, 225-234.
Pseudomonas putida flagella were examined. Also, changes in motile behavior in response to chemoattractants were analyzed quantitatively by computer. Reversals in the rotation direction of bundles of polar flagella resulted in changes in swimming direction. Cells swimming in buffer changed direction once every 2 s on average, whereas cells exposed to the attractant benzoate changed direction an average of once every 10 s. The findings show that P. putida responds to temporal gradients of chemoattractant by suppressing changes in the direction of rotation of flagella.Aromatic compounds are a major component of plants and also constitute a substantial proportion of the toxic wastes that are released into the environment (3, 5). Many kinds of bacteria can grow on various aromatic compounds, and several groups of motile soil bacteria, including Psei(domonas, Rhizobium, and Agrobacterium spp., can also sense and respond behaviorally to the presence of aromatics in the environment (1, 8, 17, 18 P. piutida PRS2000 (wild type) (16) was cultivated in defined mineral medium as described previously (8).Cells for light-microscopy observations and videomicroscopy were harvested by centrifugation in the early to midlogarithmic phase of growth and suspended in chemotaxis buffer (50 mM potassium phosphate [pH 7.0], 10 ,uM EDTA) to a density of 4 x 106 to 6 x 106 cells per ml. Approximately 80 to 90% of the cells were motile for at least 1 h.Cells for electron microscopy were harvested in the early to mid-logarithmic phase of growth and suspended in distilled water to a concentration of 108/ml. Cell suspensions were dried onto Formvar-coated grids and shadowed with platinum-palladium (80 and 20%, respectively) at an angle of 200 before examination with a Philips 300 transmission electron microscope operating at 80 kV.High-intensity illumination dark-field microscopy observations were made by using a 450-W xenon short-arc lamp and an oil immersion dark-field condenser, as described previously (13
siRNAs confer sequence specific and robust silencing of mRNA. By virtue of these properties, siRNAs have become therapeutic candidates for disease intervention. However, their use as therapeutic agents can be hampered by unintended off-target effects by either or both strands of the siRNA duplex. We report here that unlocked nucleobase analogs (UNAs) confer desirable properties to siRNAs. Addition of a single UNA at the 5′-terminus of the passenger strand blocks participation of the passenger strand in RISC-mediated target down-regulation with a concomitant increase in guide strand activity. Placement of a UNA in the seed region of the guide strand prevents miRNA-like off-target silencing without compromising siRNA activity. Most significantly, combined substitution of UNA at the 3′-termini of both strands, the addition of a UNA at the 5′-terminus of the passenger strand, and a single UNA in the seed region of the guide strand, reduced the global off-target events by more than 10-fold compared to unmodified siRNA. The reduction in off-target events was specific to UNA placement in the siRNA, with no apparent new off-target events. Taken together, these results indicate that when strategically placed, UNA substitutions have important implications for the design of safe and effective siRNA-based therapeutics.
A computer program has been designed to study behavior in populations of Spirochaeta aurantia cells, and this program has been used to analyze changes in behavior in response to chemoattractants. Three kinds of behavior were distinguished: smooth swimming, flexing, and reversals in direction of swimming after a short pause (120 ms). Cell populations exposed to chemoattractants spent, on average, 66, 33, and 1% of the time in these modes, respectively. After the addition of a chemoattractant, behavior was modifed transientlysmooth swimming increased, flexing decreased, and reversals were suppressed. After addition of D-xylose (final concentration, 10 mM), the adaptation time (the time required for the populations to return to the unmodified behavior) for S. aurantia was 1.5 to 2.0 min. A model to explain the behavior of S. aurantia and the response of cells to chemoattractants is described. This model includes a coordinating mechanism for flagellar motor Qperation and a motor switch synchronizing device.Spirochetes are relatively long, slender, helical bacteria that have flagella contained within the outer membrane, where they wrap around the peptidoglycan layer of the cell cylinder (9, 10, 18). Spirochaeta aurantia has two flagella which are inserted through the peptidoglycan into the cytoplasmic membrane at opposite poles of the cell (6; see Fig. 1). Except for location, the S. aurantia periplasmic flagella appear analogous to other bacterial flagella; they propel the cell by rotation driven by a proton motive force (2, 13). S. aurantia cells generally swim in relatively straight lines (runs), but occasionally they reverse swimming direction, the cell anterior becoming posterior, and often they stop running and flex. The duration of a flex can vary from a fraction of a second to several seconds (16,17).Berg (2) proposed a model to explain the behavior of S. aurantia. This model requires three assumptions for which there is strong supportive evidence: that the peptidoglycanbound cell cylinder is semirigid, that the flagella rotate, and that the outer membrane is flexible and not fixed to the protoplasmic cylinder. According to this model, when the flagella rotate in concert they slip agairqt the cylinder and roll against the outer membrane, causing the two to move in opposite directions. The cell rotates about its longitudinal axis and moves along it, due to the helical configuration.Reversals occur when both flagella switch their directjn of rotation synchronously. If switching is asynchronous, i.e., if only one flagellum switches, the ends twist in oppositior to each other and a flex is generated (Fig. 1).According to the model for motility of S. aurantia, runs occur when the flagellar motor at one cell end is rotating counterclockwise (CCW) and the motor at the other cell end is rotating clockwise (CW). Synchronous switching of these two motors generates a run in the reverse orientation. When both motors rotate CW or CCW, the spirochetal cell flexes.
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