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Umami is one of the 5 basic taste qualities. The umami taste of L-glutamate can be drastically enhanced by 5' ribonucleotides and the synergy is a hallmark of this taste quality. The umami taste receptor is a heteromeric complex of 2 class C G-protein-coupled receptors, T1R1 and T1R3. Here we elucidate the molecular mechanism of the synergy using chimeric T1R receptors, site-directed mutagenesis, and molecular modeling. We propose a cooperative ligand-binding model involving the Venus flytrap domain of T1R1, where L-glutamate binds close to the hinge region, and 5' ribonucleotides bind to an adjacent site close to the opening of the flytrap to further stabilize the closed conformation. This unique mechanism may apply to other class C G-protein-coupled receptors.

Mechanical stress and hypoxia during episodes of ocular hypertension (OHT) trigger glial activation and neuroinflammation in the retina. Glial activation and release of pro-inflammatory cytokines TNFα and IL-1β, complement, and other danger factors was shown to facilitate injury and loss of retinal ganglion cells (RGCs) that send visual information to the brain. However, cellular events linking neuroinflammation and neurotoxicity remain poorly characterized. Several pro-inflammatory and danger signaling pathways, including P2X7 receptors and Pannexin1 (Panx1) channels, are known to activate inflammasome caspases that proteolytically activate gasdermin D channel-formation to export IL-1 cytokines and/or induce pyroptosis. In this work, we used molecular and genetic approaches to map and characterize inflammasome complexes and detect pyroptosis in the OHT-injured retina. Acute activation of distinct inflammasome complexes containing NLRP1, NLRP3 and Aim2 sensor proteins was detected in RGCs, retinal astrocytes and Muller glia of the OHT-challenged retina. Inflammasome-mediated activation of caspases-1 and release of mature IL-1β were detected within 6 h and peaked at 12-24 h after OHT injury. These coincided with the induction of pyroptotic pore protein gasdermin D in neurons and glia in the ganglion cell layer (GCL) and inner nuclear layer (INL). The OHT-induced release of cytokines and RGC death were significantly decreased in the retinas of Casp1 Casp4(11) , Panx1 and in Wild-type (WT) mice treated with the Panx1 inhibitor probenecid. Our results showed a complex spatio-temporal pattern of innate immune responses in the retina. Furthermore, they indicate an active contribution of neuronal NLRP1/NLRP3 inflammasomes and the pro-pyroptotic gasdermin D pathway to pathophysiology of the OHT injury. These results support the feasibility of inflammasome modulation for neuroprotection in OHT-injured retinas.

This article presents the results of an experimental study of the coolant flow in a fuel rod bundle of a nuclear reactor fuel assembly of a small modular reactor for a small ground-based nuclear power plant. The aim of the work is to experimentally determine the hydrodynamic characteristics of the coolant flow in a fuel rod bundle of a fuel assembly. For this purpose, experimental studies were conducted in an aerodynamic model that included simulators of fuel elements, burnable absorber rods, spacer grids, a central displacer, and stiffening corners. During the experiments, the water coolant flow was modeled using airflow based on the theory of hydrodynamic similarity. The studies were conducted using the pneumometric method and the contrast agent injection method. The flow structure was visualized by contour plots of axial and tangential velocity, as well as the distribution of the contrast agent. During the experiments, the features of the axial flow were identified, and the structure of the cross-flows of the coolant was determined. The database obtained during the experiments can be used to validate CFD programs, refine the methods of thermal-hydraulic calculation of nuclear reactor cores, and also to justify the design of fuel assemblies.

To advance our understanding how the outer eye interacts with its environment, we asked which cellular receptors are expressed in the cornea, focusing on G protein-coupled receptors. Total RNA from the mouse cornea was subjected to next-generation sequencing using the Illumina platform. The data was analyzed with TopHat and CuffLinks software packages. Expression of a representative group of genes detected by RNA-seq was further analyzed by RT-PCR and in situ hybridization using RNAscope technology and fluorescent microscopy. We generated more than 46 million pair-end reads from mouse corneal RNA. Bioinformatics analysis revealed that the mouse corneal transcriptome reconstructed from these reads represents over 10,000 gene transcripts. We identified 194 GPCR transcripts, of which 96 were putative olfactory receptors. RT-PCR analysis confirmed the presence of several olfactory receptors and related genes, including olfactory marker protein and the G protein associated with olfaction, Gαolf. In situ hybridization showed that mRNA for olfactory marker protein, Gαolf and possibly some olfactory receptors were found in the corneal epithelial cells. In addition to the corneal epithelium, Gαolf was present in the ganglionic and inner nuclear layers of the retina. One of the olfactory receptors, Olfr558, was present primarily in vessels of the eye co-stained with antibodies against alpha-smooth muscle actin, indicating expression in arterioles. Several species of mRNA encoding putative olfactory receptors and related genes are expressed in the mouse cornea and other parts of the eye indicating they may play a role in sensing chemicals in the ocular environment.

Pannexin 1 (Panx1) forms ATP-permeable membrane channels that play a key role in purinergic signaling in the nervous system in both normal and pathological conditions. In the retina, particularly high levels of Panx1 are found in retinal ganglion cells (RGCs), but the normal physiological function in these cells remains unclear. In this study, we used patch clamp recordings in the intact inner retina to show that evoked currents characteristic of Panx1 channel activity were detected only in RGCs, particularly in the OFF-type cells. The analysis of pattern electroretinogram (PERG) recordings indicated that Panx1 contributes to the electrical output of the retina. Consistently, PERG amplitudes were significantly impaired in the eyes with targeted ablation of the Panx1 gene in RGCs. Under ocular hypertension and ischemic conditions, however, high Panx1 activity permeated cell membranes and facilitated the selective loss of RGCs or stably transfected Neuro2A cells. Our results show that high expression of the Panx1 channel in RGCs is essential for visual function in the inner retina but makes these cells highly sensitive to mechanical and ischemic stresses. These findings are relevant to the pathophysiology of retinal disorders induced by increased intraocular pressure, such as glaucoma.

We studied the function of G protein-coupled receptor kinases (GRKs) in the regulation of thrombin-activated signaling in endothelial cells. GRK2, GRK5, and GRK6 isoforms were expressed predominantly in endothelial cells. The function of these isoforms was studied by expressing wild-type and dominant negative (dn) mutants in endothelial cells. We determined the responses to thrombin, which activates intracellular signaling in endothelial cells by cleaving the NH2terminus of the G protein-coupled proteinase-activated receptor-1 (PAR-1). We measured changes in phosphoinositide hydrolysis and intracellular Ca2+concentration ([Ca2+]i) in response to thrombin as well as the state of endothelial activation. In the latter studies, the transendothelial monolayer electrical resistance, a measure of the loss of endothelial barrier function, was measured in real time. Of the three isoforms, GRK5 overexpression was selective in markedly reducing the thrombin-activated phosphoinositide hydrolysis and increased [Ca2+]i. GRK5 overexpression also inhibited the thrombin-induced decrease in endothelial monolayer resistance by 75%. These effects of GRK5 overexpression occurred in association with the specific increase in the thrombin-induced phosphorylation of PAR-1. In contrast to the effects of GRK5 overexpression, the expression of the dn-GRK5 mutant produced a long-lived increase in [Ca2+]iin response to thrombin, whereas dn-GRK2 had no effect. These results indicate the crucial role of the GRK5 isoform in the mechanism of thrombin-induced desensitization of PAR-1 in endothelial cells.

Purpose To advance our understanding how the outer eye interacts with its environment, we asked which cellular receptors are expressed in the cornea, focusing on G protein-coupled receptors. Methods Total RNA from the mouse cornea was subjected to next-generation sequencing using the Illumina platform. The data was analyzed with TopHat and CuffLinks software packages. Expression of a representative group of genes detected by RNA-seq was further analyzed by RT-PCR and in situ hybridization using RNAscope technology and fluorescent microscopy. Results We generated more than 46 million pair-end reads from mouse corneal RNA. Bioinformatics analysis revealed that the mouse corneal transcriptome reconstructed from these reads represents over 10,000 gene transcripts. We identified 194 GPCR transcripts, of which 96 were putative olfactory receptors. RT-PCR analysis confirmed the presence of several olfactory receptors and related genes, including olfactory marker protein and the G protein associated with olfaction, G alpha olf. In situ hybridization showed that mRNA for olfactory marker protein, G alpha olf and possibly some olfactory receptors were found in the corneal epithelial cells. In addition to the corneal epithelium, G alpha olf was present in the ganglionic and inner nuclear layers of the retina. One of the olfactory receptors, Olfr558, was present primarily in vessels of the eye co-stained with antibodies against alpha-smooth muscle actin, indicating expression in arterioles. Conclusions Several species of mRNA encoding putative olfactory receptors and related genes are expressed in the mouse cornea and other parts of the eye indicating they may play a role in sensing chemicals in the ocular environment.

Signal-transducing guanine nucleotide-binding proteins (G proteins) are made up of three subunits, α, β, and γ. Each of these subunits comprises a family of proteins. The rules for association between members of one family with members of another to form a multimer are not known; it is not clear whether associations are specific or nonspecific. Other than transducin (Gt), the G protein in rod photoreceptors, most purified G proteins contain more than one subtype of β or γ subunits. The Gtα subunit is associated only with β1and γ1. It is not known whether this specificity is due to the differential expression of these subunit types in a cell type or due to intrinsically different affinities between different β and γ subunit types. We have used a transfected cell assay system to examine the association of the β1, β2, and β3proteins with the γ1and γ2proteins. Results show that γ1does not associate with β2and that β3does not associate with γ1or γ2. Differences in affinities between types of G protein subunits will impose restrictions on the formation of certain heterotrimers and determine which G protein will be active in a cell. A chimeric molecule of β1and β2was used to broadly map the regions on these subunits that determine specificity of association.

In this thesis we consider a number of past, present, and future neutrino experiments designed to test physics beyond the Standard Model. First, we analyze potential new physics explanations of the NuTeV anomaly and check their compatibility with the most recent experimental data. The models we consider are: gauged Lµ – Lτ, gauged B – 3L µ, and S1, [special characters omitted], V1, [special characters omitted] leptoquarks. We find that only the triplet leptoquark models can explain NuTeV and be compatible with the data from other experiments at the same time, and only if the components of the triplet have different masses. Then, we analyze the prospects of discovery of heavy Majorana neutrinos (neutrissimos ) suggested by the Okamura model at the LHC. We find that these particles, if produced, will live short enough to decay inside of the detector, while long enough to lead to a narrow peak in the invariant mass spectrum of the decay products. We estimate the typical masses of the neutrissimos to be in the TeV range. However, studies exist that have shown that if their masses are larger than about 150 GeV then the production cross-section is too small to lead to an observable event rate. Thus, we conclude that it will not be possible to detect the neutrissimo at the LHC unless its mass is smaller that about 150 GeV which corresponds to a very small region close to the edge of the parameter space of the Okamura model. Nevertheless, we argue that the signature of the neutrissimo may be detectable in other neutrino experiments which may be carried out in the future. As examples, we consider the NuSOnG experiment, which is a fixed target neutrino scattering experiment proposed at Fermilab, and a hypothetical long-baseline neutrino oscillation experiment in which the Fermilab NUMI beam is aimed at the Hyper-Kamiokande detector in Japan. In addition to the sensitivity to neutrissimos, we analyze the capabilities of these experiments to constraint the coupling constants and masses of new particles in various models of new physics suggested in the literature. The models we consider are: neutrissimo models, models with generation distinguishing Z's such as topcolor assisted technicolor, models containing various types of leptoquarks, R-parity violating SUSY, and extended Higgs sector models. In several cases, we find that the limits thus obtained could be competitive with those expected from direct searches at the LHC. In the event that any of the particles discussed here are discovered at the LHC, then the observation, or non-observation, of these particles in the NuSOnG and Fermilab→Hyper-Kamiokande experiments could help in identifying what type of particle had been observed.

A study examined the membrane and cytosolic fractions from transfected cells to infer whether a particular beta protein subunit was associated with a particular gamma protein subunit.