47 research outputs found
Vasopressin V2R-Targeting Peptide Carrier Mediates siRNA Delivery into Collecting Duct Cells
Internalization of receptor proteins after interacting with specific ligands has been proposed to facilitate siRNA delivery into the target cells via receptor-mediated siRNA transduction. In this study, we demonstrated a novel method of vasopressin V2 receptor (V2R)-mediated siRNA delivery against AQP2 in primary cultured inner medullary collecting duct (IMCD) cells of rat kidney. We synthesized the dDAVP conjugated with nine D-arginines (dDAVP-9r) as a peptide carrier for siRNA delivery. The structure of synthetic peptide carrier showed two regions (i.e., ligand domain to V2R (dDAVP) and siRNA carrying domain (nine D-arginine)) bisected with a spacer of four glycines. The results revealed that 1) synthesized dDAVP-9r peptides formed a stable polyplex with siRNA; 2) siRNA/dDAVP-9r polyplex could bind to the V2R of IMCD cells and induced AQP2 phosphorylation (Ser 256); 3) siRNA/dDAVP-9r polyplex was stable in response to the wide range of different osmolalities, pH levels, or to the RNases; 4) fluorescein-labeled siRNA was delivered into V2R-expressing MDCK and LLC-PK1 cells by siRNA/dDAVP-9r polyplex, but not into the V2R-negative Cos-7 cells; and 5) AQP2-siRNA/dDAVP-9r polyplex effectively delivered siRNA into the IMCD cells, resulting in the significant decrease of protein abundance of AQP2, but not AQP4. Therefore, for the first time to our knowledge, we demonstrated that V2R-mediated siRNA delivery could be exploited to deliver specific siRNA to regulate abnormal expression of target proteins in V2R-expressing kidney cells. The methods could be potentially used in vivo to regulate abnormal expression of proteins associated with disease conditions in the V2R-expressing kidney cells
Source genotype influence on cross species transmission of transmissible spongiform encephalopathies evaluated by RT-QuIC.
Scrapie is a naturally occurring transmissible spongiform encephalopathy of sheep and goats. This fatal neurodegenerative disease is caused by misfolding of the cellular prion protein to pathogenic β-rich conformers (PrPSc) that accumulate in higher order structures of the brain and other tissues. This conversion has been used for in vitro assays including serial protein misfolding amplification and real-time quaking induced conversion (RT-QuIC). RT-QuIC can be used for the detection of prions and for strain discrimination in a variety of biological tissues from humans and animals. In this study, we evaluated how PrPSc isolated from sheep of different genotypes after inoculation with the scrapie agent influence the fibril formation in vitro using RT-QuIC. We found that reaction mixtures seeded with PrPSc from genotype VRQ/VRQ sheep brains have better conversion efficiency with 132M elk substrate compared to reactions seeded with PrPSc from the brains of sheep with the ARQ/ARQ genotype no matter which strain of scrapie was used to seed the reactions. We also inoculated transgenic mice expressing 132M elk PRNP (Tg12) with the scrapie agent from different genotypes of sheep to compare with our RT-QuIC results. The bioassays support the data showing a significantly shorter incubation period for inoculum from VRQ/VRQ sheep when compared to inoculum from ARQ/ARQ sheep. Thus, we conclude that the genotype of both source and recipient can strongly influence transmission
Use of bovine recombinant prion protein and real-time quaking-induced conversion to detect cattle transmissible mink encephalopathy prions and discriminate classical and atypical L- and H-Type bovine spongiform encephalopathy - Fig 6
<p><b>RT-QuIC detection of C-, L-, and H-BSE prion seeding activity using bovine prion proteins (25–241) wild type (A) and its disease associated form E211K protein (B).</b> RT-QuIC reaction mixtures were seeded with 10<sup>−4</sup> dilutions of brain tissues from uninfected, C-BSE-affected (cyan and grey lines), L-BSE-affected (red and black lines) and H-BSE-affected (pink, blue, yellow, green lines) cattle. A final SDS concentration of 0.001% in combination with 300 mM NaCl was used with the substrates. Data are presented as mean ThT fluorescence of 8 reactions conducted as 2 repeats of 4 reactions. The positive threshold was calculate as ~10,000 relative fluorescence units of normal cattle brain homogenates.</p
Animal donor information.
<p>Brain tissues collected from cattle that were normal or clinically ill with BSE were used to prepare homogenates.</p
RT-QuIC sodium chloride titration for TME-infected and uninfected cattle brain samples.
<p>RT-QuIC reactions were seeded with 10<sup>−4</sup> (A, B) and 10<sup>−6</sup> dilution (C, D) of TME-infected and uninfected cattle brain homogenates using a range of NaCl concentrations (100–500 mM) with the full-length bPrP E211K protein (aa 25–241) as substrate. Data are presented as mean ThT fluorescence of 8 reactions conducted as 2 repeats of 4 reactions. The positive threshold was calculate as ~10,000 relative fluorescence units of normal cattle brain homogenates.</p
Western blot of a representative brain sample of each BSE strain and TME in cattle.
<p>Brainstem samples were characterized by western blotting (mAb 6H4). Lanes from left: 1. French L-type (#6895, 2 mg);; 2. U.S. H-type (#80, 2 mg); 3. Classical (#6836, 1 mg); 4.Negative Control (#6969, 2 mg); 5. Biotinylated protein marker; 6. Bovine TME (Animal #52AA, 2 mg); 7. Negative Control (#6969, 2mg); 8. Biotinylated protein marker.</p
RT-QuIC sodium chloride titration for TME-infected and uninfected cattle brain samples.
<p>RT-QuIC reactions were seeded with 10<sup>−4</sup> (A, B) and 10<sup>−6</sup> dilution (C, D) of TME-infected and uninfected cattle brain homogenates using a range of NaCl concentrations (100–500 mM) with the full-length bPrP wild type (aa 25–241) as substrate. Data are presented as mean ThT fluorescence of 8 reactions conducted as 2 repeats of 4 reactions. The positive threshold was calculate as ~10,000 relative fluorescence units of normal cattle brain homogenates.</p
RT-QuIC sodium chloride titration for different types of BSE-infected cattle brain samples.
<p>RT-QuIC reactions were seeded with 10<sup>−4</sup> and 10<sup>−6</sup> dilution of C-, L, and H-BSE-infected cattle brain homogenates using a range of NaCl concentrations (100–500 mM) with the full-length bPrP wild type (aa 25–241) as substrate. Data are presented as mean ThT fluorescence of 8 reactions conducted as 2 repeats of 4 reactions. The positive threshold was calculate as ~10,000 relative fluorescence units of normal cattle brain homogenates.</p
Changing Roles for Temporal Representation of Odorant During the Oscillatory Response of the Olfactory Bulb
An Excess-Calcium-Binding-Site Model Predicts Neurotransmitter Release at the Neuromuscular Junction
AbstractDespite decades of intense experimental studies, we still lack a detailed understanding of synaptic function. Fortunately, using computational approaches, we can obtain important new insights into the inner workings of these important neural systems. Here, we report the development of a spatially realistic computational model of an entire frog active zone in which we constrained model parameters with experimental data, and then used Monte Carlo simulation methods to predict the Ca2+-binding stoichiometry and dynamics that underlie neurotransmitter release. Our model reveals that 20–40 independent Ca2+-binding sites on synaptic vesicles, only a fraction of which need to bind Ca2+ to trigger fusion, are sufficient to predict physiological release. Our excess-Ca2+-binding-site model has many functional advantages, agrees with recent data on synaptotagmin copy number, and is the first (to our knowledge) to link detailed physiological observations with the molecular machinery of Ca2+-triggered exocytosis. In addition, our model provides detailed microscopic insight into the underlying Ca2+ dynamics during synapse activation