Analyzing Responses of Mouse Olfactory Sensory Neurons Using the Air-phase Electroolfactogram Recording

Animals depend on olfaction for many critical behaviors, such as finding food sources, avoiding predators, and identifying conspecifics for mating and other social interactions. The electroolfactogram (EOG) recording is an informative, easy to conduct, and reliable method to assay olfactory function at the level of the olfactory epithelium. Since the 1956 description of the EOG by Ottoson in frogs1, the EOG recording has been applied in many vertebrates including salamanders, rabbits, rats, mice, and humans (reviewed by Scott and Scott-Johnson, 2002, ref. 2). The recent advances in genetic modification in mice have rekindled interest in recording the EOG for physiological characterization of olfactory function in knock-out and knock-in mice. EOG recordings have been successfully applied to demonstrate the central role of olfactory signal transduction components3-8, and more recently to characterize the contribution of certain regulatory mechanisms to OSN responses9-12

Olfactory CNG Channel Desensitization by Ca2+/CaM via the B1b Subunit Affects Response Termination but Not Sensitivity to Recurring Stimulation

SummaryCa2+/calmodulin-mediated negative feedback is a prototypical regulatory mechanism for Ca2+-permeable ion channels. In olfactory sensory neurons (OSNs), such regulation on the cyclic nucleotide-gated (CNG) channel is considered a major mechanism of OSN adaptation. To determine the role of Ca2+/calmodulin desensitization of the olfactory CNG channel, we introduced a mutation in the channel subunit CNGB1b in mice that rendered the channel resistant to fast desensitization by Ca2+/calmodulin. Contrary to expectations, mutant OSNs showed normal receptor current adaptation to repeated stimulation. Rather, they displayed slower response termination and, consequently, reduced ability to transmit olfactory information to the olfactory bulb. They also displayed reduced response decline during sustained odorant exposure. These results suggest that Ca2+/calmodulin-mediated CNG channel fast desensitization is less important in regulating the sensitivity to recurring stimulation than previously thought and instead functions primarily to terminate OSN responses

Numerical analysis of the engine with spark ignition and compression ignition

U radu se prikazuju istraživanja simulacije izgaranja u testiranom motoru s četiri cilindra, na osnovu motora Toyote Yaris 1,3 dm3 s paljenjem iskrom i opremljenim s dva sustava napajanja energijom: osnovnim i paljenjem. Primijenjene su dvije metode pokretanja postupka izgaranja: paljenje iskrom i ponovo, poslije isključenja, ubrizgavanje zapaljive doze. Cilj je istraživanja bilo utvrđivanje razlika u postupku izgaranja i radu motora, prije svega promjena tlaka do kojih je došlo u cilindru te vrste motora s dva sustava napajanja energijom. Zapaljiva doza uštrcana u cilindar bila je jednaka otprilike 5 ÷ 8 % ukupne mase goriva u motoru. Gorivo je direktno ubrizgavano tijekom nekoliko desetaka stupnjeva rotacije koljenaste osovine prije dostizanja gornje mrtve točke. Pripremljena je prostorna mreža modela motora (predprocesor), a napravljena je i modifikacija izvornog KIVA 3V programa kako bi se uzela u obzir dva sustava ubrizgavanja. Treba spomenuti da standardni KIVA 3V program ima, po defaultu, samo jedan izvor ubrizgavanja goriva.The article presents simulation researches on the combustion process in the four-cylinder testing engine, based on the spark ignition engine Toyota Yaris 1,3 dm3 which was equipped with two systems of power supply i.e.: fundamental and ignition. Two methods of initiating the combustion process were applied: by spark ignition and, after it was turned off, by means of the ignition dose injection. The researches aimed at revealing the differences in the combustion process and engine performance, above all the changes in pressure that occurred in the cylinder for that kind of two-fuel power supply of the engine. The ignition dose injected into the cylinder was equal to about 5 ÷ 8 % of the general mass of the fuel supplied to the engine. The mass of the fuel injected into the approach collector determined the remaining part, and the direct injection of the fuel took place over several dozen of rotation degrees of the crankshaft before TDC (Top Dead Centre). A spatial grid of the engine’s model (pre-processor) was prepared, and also a modification to the source KIVA 3V program was made consisting in taking into account of two systems of injection. It is necessary to mention that the standard KIVA 3V program contains, as a default, only one source of fuel injection

Modelling of the injection and combustion processes in the gasoline direct injection engine

Rad prikazuje modeliranje procesa ubrizgavanja pomoću KIVA programa. Složeni matematički modeli koji se koriste u izračunima i koji opisuju procese koji se odvijaju unutar cilindra motora omogućuju generiranje kompliciranih virtualnih modela koji odražavaju stvarne uvjete na zadovoljavajući način. Točnost rezultata proračuna određena je kvalitetom ulaznih podataka, koji se mogu dobiti na temelju ispitivanja s modelom. Na temelju modeliranja procesa ubrizgavanja i izgaranja u GDI motoru, predstavljenih u radu, dobivena je osnova za širu analizu plinsko-dinamičkih pojava.The paper presents modelling of the injection process by means of KIVA program. The elaborate mathematical models used in calculations describing the processes occurring inside the engine cylinder make it possible to create complicated virtual models reflecting real conditions in a satisfactory way. The accuracy of calculation results is determined by the quality of input data, which can be obtained based on model investigations. Based on the modeling of injection and combustion processes in the GDI engine, presented in the work, the basis for a broader analysis of the gas-dynamic phenomena was obtained

The Na(+)/Ca(2+) exchanger NCKX4 governs termination and adaptation of the mammalian olfactory response

Sensory perception requires accurate encoding of stimulus information by sensory receptor cells. We identified NCKX4, a potassium-dependent Na(+)/Ca(2+) exchanger, as being necessary for rapid response termination and proper adaptation of vertebrate olfactory sensory neurons (OSNs). Nckx4(-/-) (also known as Slc24a4) mouse OSNs displayed substantially prolonged responses and stronger adaptation. Single-cell electrophysiological analyses revealed that the majority of Na(+)-dependent Ca(2+) exchange in OSNs relevant to sensory transduction is a result of NCKX4 and that Nckx4(-/-) mouse OSNs are deficient in encoding action potentials on repeated stimulation. Olfactory-specific Nckx4(-/-) mice had lower body weights and a reduced ability to locate an odorous source. These results establish the role of NCKX4 in shaping olfactory responses and suggest that rapid response termination and proper adaptation of peripheral sensory receptor cells tune the sensory system for optimal perception

Origin of basal activity in mammalian olfactory receptor neurons

Mammalian odorant receptors form a large, diverse group of G protein–coupled receptors that determine the sensitivity and response profile of olfactory receptor neurons. But little is known if odorant receptors control basal and also stimulus-induced cellular properties of olfactory receptor neurons other than ligand specificity. This study demonstrates that different odorant receptors have varying degrees of basal activity, which drives concomitant receptor current fluctuations and basal action potential firing. This basal activity can be suppressed by odorants functioning as inverse agonists. Furthermore, odorant-stimulated olfactory receptor neurons expressing different odorant receptors can have strikingly different response patterns in the later phases of prolonged stimulation. Thus, the influence of odorant receptor choice on response characteristics is much more complex than previously thought, which has important consequences on odor coding and odor information transfer to the brain

Essential Roles of the Tap42-Regulated Protein Phosphatase 2A (PP2A) Family in Wing Imaginal Disc Development of Drosophila melanogaster

Protein ser/thr phosphatase 2A family members (PP2A, PP4, and PP6) are implicated in the control of numerous biological processes, but our understanding of the in vivo function and regulation of these enzymes is limited. In this study, we investigated the role of Tap42, a common regulatory subunit for all three PP2A family members, in the development of Drosophila melanogaster wing imaginal discs. RNAi-mediated silencing of Tap42 using the binary Gal4/UAS system and two disc drivers, pnr- and ap-Gal4, not only decreased survival rates but also hampered the development of wing discs, resulting in a remarkable thorax cleft and defective wings in adults. Silencing of Tap42 also altered multiple signaling pathways (HH, JNK and DPP) and triggered apoptosis in wing imaginal discs. The Tap42RNAi-induced defects were the direct result of loss of regulation of Drosophila PP2A family members (MTS, PP4, and PPV), as enforced expression of wild type Tap42, but not a phosphatase binding defective Tap42 mutant, rescued fly survivorship and defects. The experimental platform described herein identifies crucial roles for Tap42•phosphatase complexes in governing imaginal disc and fly development

NMR Studies of the C-Terminus of alpha4 Reveal Possible Mechanism of Its Interaction with MID1 and Protein Phosphatase 2A

Alpha4 is a regulatory subunit of the protein phosphatase family of enzymes and plays an essential role in regulating the catalytic subunit of PP2A (PP2Ac) within the rapamycin-sensitive signaling pathway. Alpha4 also interacts with MID1, a microtubule-associated ubiquitin E3 ligase that appears to regulate the function of PP2A. The C-terminal region of alpha4 plays a key role in the binding interaction of PP2Ac and MID1. Here we report on the solution structure of a 45-amino acid region derived from the C-terminus of alpha4 (alpha45) that binds tightly to MID1. In aqueous solution, alpha45 has properties of an intrinsically unstructured peptide although chemical shift index and dihedral angle estimation based on chemical shifts of backbone atoms indicate the presence of a transient α-helix. Alpha45 adopts a helix-turn-helix HEAT-like structure in 1% SDS micelles, which may mimic a negatively charged surface for which alpha45 could bind. Alpha45 binds tightly to the Bbox1 domain of MID1 in aqueous solution and adopts a structure consistent with the helix-turn-helix structure observed in 1% SDS. The structure of alpha45 reveals two distinct surfaces, one that can interact with a negatively charged surface, which is present on PP2A, and one that interacts with the Bbox1 domain of MID1

Functional Evidence of Multidrug Resistance Transporters (MDR) in Rodent Olfactory Epithelium

Background: P-glycoprotein (Pgp) and multidrug resistance-associated protein (MRP1) are membrane transporter proteins which function as efflux pumps at cell membranes and are considered to exert a protective function against the entry of xenobiotics. While evidence for Pgp and MRP transporter activity is reported for olfactory tissue, their possible interaction and participation in the olfactory response has not been investigated. Principal Findings: Functional activity of putative MDR transporters was assessed by means of the fluorometric calcein acetoxymethyl ester (calcein-AM) accumulation assay on acute rat and mouse olfactory tissue slices. Calcein-AM uptake was measured as fluorescence intensity changes in the presence of Pgp or MRP specific inhibitors. Epifluorescence microscopy measured time course analysis in the olfactory epithelium revealed significant inhibitor-dependent calcein uptake in the presence of each of the selected inhibitors. Furthermore, intracellular calcein accumulation in olfactory receptor neurons was also significantly increased in the presence of either one of the Pgp or MRP inhibitors. The presence of Pgp or MRP1 encoding genes in the olfactory mucosa of rat and mouse was confirmed by RT-PCR with appropriate pairs of speciesspecific primers. Both transporters were expressed in both newborn and adult olfactory mucosa of both species. To assess a possible involvement of MDR transporters in the olfactory response, we examined the electrophysiological response to odorants in the presence of the selected MDR inhibitors by recording electroolfactograms (EOG). In both animal species