基因组水平蝙蝠嗅觉受体基因的分子进化

翼手目动物（俗称蝙蝠）的食性分化显著,不同食性的蝙蝠具有显著不同的嗅球大小。为了研究嗅觉是否影响了蝙蝠食性的进化,我们利用网上已公布的10种蝙蝠基因组的数据,通过同源比对的方法鉴定出所有的嗅觉受体基因,并进行嗅觉受体基因亚家族的分类,进而比较嗅觉受体基因亚家族的数目差异。结果显示,蝙蝠的嗅觉受体基因与其它哺乳动物一样,都可以分为13个单系起源的亚家族;在Yinpterochiroptera亚目中,OR1/3/7、OR2/13、OR5/8/9等3个嗅觉受体亚家族在食果蝙蝠中均发生了不同程度的扩张,基因数目显著地多于食虫蝙蝠,提示嗅觉在食果蝙蝠取食过程中具有重要的作用。因此,本研究在基因组水平上重现了蝙蝠嗅觉受体基因的进化历史,揭示了3个嗅觉受体基因亚家族的功能可能与食果蝙蝠的食性相关,突出了嗅觉对动物食性的重要作用.     

Representation of odorants by receptor neuron input to the mouse olfactory bulb.

To visualize odorant representations by receptor neuron input to the mouse olfactory bulb, we loaded receptor neurons with calcium-sensitive dye and imaged odorant-evoked responses from their axon terminals. Fluorescence increases reflected activation of receptor neuron populations converging onto individual glomeruli. We report several findings. First, five glomeruli were identifiable across animals based on their location and odorant responsiveness; all five showed complex response specificities. Second, maps of input were chemotopically organized at near-threshold concentrations but, at moderate concentrations, involved many widely distributed glomeruli. Third, the dynamic range of input to a glomerulus was greater than that reported for individual receptor neurons. Finally, odorant activation slopes could differ across glomeruli, and for different odorants activating the same glomerulus. These results imply a high degree of complexity in odorant representations at the level of olfactory bulb input.     

Odour discrimination by olfactory bulb neurons: statistical analysis of electrophysiological responses and comparison with odour discrimination by receptor cells

Electrophysiological responses of olfactory bulb neurons todifferent odorants have been presented and discussed with referenceto homologous properties of olfactory receptor cells. This paperdeals with further mathematical processings of part (experimentA) of these original data and proposes a comparison with a similarstudy performed in receptor cells, using the same odorants presentedat the same concentrations. In the olfactory bulb the patternof similarities and differences among odorants was found tobe almost the same whether the mathematical processings wereapplied to the odor-evoked discharge frequencies or to excitatoryresponses considered separately. By contrast, separate processingof inhibitory responses led to a different organization of odorantsimilarities, indicating that inhibitory responses were lessdiscriminating than excitatory responses. This was discussedin relation to the synaptic organization of the olfactory bulb.The comparison of these findings with those previously obtainedin receptor cells showed indisputable resemblances between thepatterns of discrimination among odorants at both levels ofthe olfactory pathways, especially in the grouping of some odorantsin pairs and in the overall organization of the olfactory spaceas determined by factor analysis. The findings also suggestedthat odorant discrimination was slightly improved in the olfactorybulb but no sign of a novel, specific odour categorization couldbe found.     

Oscillatory current responses of olfactory receptor neurons to odorants and computer simulation based on a cyclic AMP transduction model

Neural oscillatory activities triggered by odorant stimulation have been often reported at various levels of olfactory nervous systems in vertebrates. To elucidate the origin of neural oscillations, we studied first the oscillatory properties of current responses of isolated olfactory receptor neurons (ORNs) of the rainbow trout to amino acid odorants, using a whole-cell voltage-clamp technique and found that the damped current oscillations were intrinsic in both ciliated and microvillous ORNs and occurred when ORNs were stimulated by odorants at high intensities. Continuous wavelet analysis using the Gabor function revealed that the dominant frequency of oscillations was 1.89 +/- 0.50 Hz (mean +/- SD, n = 92). There was no significant difference in oscillation frequency between the two types of ORNs and between different perfusion conditions with standard and Na(+)-free (choline) Ringer's solutions, but there was a slight difference in oscillation frequency between different holding potential conditions of negative and positive potentials. We then performed a computer simulation of the current responses with a cAMP olfactory transduction model. The model was based on the assumption that the current responses of ORNs were linearly related to the sum of concentrations of active cyclic-nucleotide-gated channels and Ca(2+)-activated Cl(-) channels, and was expressed by 12 differential equations with 44 different parameters. The simulation revealed that the oscillations of current responses of ORNs were mainly due to the oscillatory properties of intracellular cAMP and Ca(2+) concentrations. The necessary reaction component for the oscillations in the transduction model was direct inhibition of adenylate cyclase activity by Ca(2+). High Ca(2+) efflux by the Na(+)-Ca(2+) exchanger and cAMP-phosphodiesterase activity were most influential on the oscillations. The simulation completely represented the characteristics of current responses of ORNs: odorant-intensity-dependent response, intensity-dependent latency and adaptation. Thus, the simulation is generally applicable to current and voltage responses of ORNs equipped with cAMP olfactory transduction pathway in other vertebrate species. The simulation programs for Macintosh (cAMP 9.2.7 and 9.2.8 for MacOS 8.1 or later) and cAMP JAVA applet versions based on cAMP 9.2.8 have been published on the world wide web (http://bio2.sci.hokudai.ac.jp/bio/chinou1/noriyo_home.html).     

Pharmacological study of inhibited frog olfactory bulb glomerular input produced by a puff of air on the olfactory mucosa

The effects of applying 4-aminopyridine (10^–2 M), aminooxyacetic acid (AOAA — 10^–4–10^–3 M), -alanine (10^–3–10^–2 M), and bicuculline (10^–5, 10^–4 M) to the intact frog olfactory bulb were investigated. Having measured inhibition of orthodromic potential postsynaptic components produced either by a puff of air on the olfactory mucosa (OB input inhibition) or by single electrical stimulation of the olfactory nerve (postsynaptic inhibition) or by single electrical stimulation of the olfactory nerve (postsynaptic inhibition), it was found that 4-aminopyridine greatly intensified postsynaptic inhibition but strongly reduced that of OB input; inhibition of the latter was raised by AOAA or bicuculline and decreased by -alanine. These substances failed to exert any consistent, clear-cut effects on postsynaptic inhibition. Findings would support the hypothesis that OB input inhibition produced by a puff of air on the olfactory mucosa could occur as a result of GABA release from glial cells and subsequent binding of GABA to presynaptic GABA_B-receptors in glomeruli.M. V. Lomonosov Moscow State University. Translated from Neirofiziologiya, Vol. 19, No. 1, pp. 12–20, January–February, 1987.     

New studies on odour discrimination in the frog's olfactory receptor cells. II. Mathematical analysis of electrophysiological responses

Revial et al. (1982) reported the experimental findings obtainedin cell units stimulated with two new sets of odorants. Thepresent paper deals with mathematical processing of these electrophysiologicaldata. The multidimensionality of the odour space was confirmed.Camphor, isoborneol and cineole appeared to represent a ‘camphorgroup’ markedly discriminated from other odorants. A‘terpenegroup’ including terpinene, dimethylstyrene, limoneneand cymene and related to menthane and menthene was observed.A series of 8 cycloketones was found to have a linear patternin the factorial planes and to be subdivided into three subsets.Cyclooctadecanone and cyclotetradecanone displayed correlationsand spatial relationships with musk ketone while cycloundecanone,cyclodecanone and especially cyclononanone were more relatedto the‘camphor group’. Separate processing of theunitary responses of 12 receptor cells recorded from a singlepreparation led to the building of a factorial space resemblingthat constructed with the full collection of data. The incidenceof odorant concentration on similarity evaluation is discussedon the basis of receptor responses to two different concentrationsof some of the odorants.     

Influence of stimulus intensity on odour discrimination by olfactory bulb neurons as compared with receptor cells

Previously reported electrophysiological responses recordedfrom individual neurons in the olfactory bulb of frogs stimulatedwith odorous compounds were further analyzed using statisticalmethods. Five of the odorants were delivered at two concentrations.The pattern of discrimination among these odorants was investigatedwith the aid of the Pearson's correlation test and Benzecri's‘analyse des correspondances’. Special attentionwas paid to the incidence of odour concentration on this discriminationpattern. The results were compared with those of a similar studyperformed on receptor cells in the same experimental conditions.The comparison indicated that the information processing inthe olfactory bulb seems to improve discrimination between chemicallydifferent stimuli, especially those poorly discriminated byreceptor cell responses, whereas it protects this discriminationagainst a massive influence of the intensity of the stimuli.     

Observations on the cytolemma of the olfactory receptor cell in the newt

Summary The fine structure of the cytolemma of olfactory receptor cells in the newt was studied by the freeze-fracture replica method. Two kinds of receptor cells were recognized, namely ciliated cells (ciliary type) and non-ciliated cells (microvilli type). The cytolemma of olfactory knobs as well as their processes from both types of receptor cells showed an abundance of large membrane particles 80110Å in diameter. The large square aggregation of membrane particles, 0.1×0.1 m to 0.2×0.3 m in size, consisting of 50100 cuboidal subunits, were found in the cytolemma of the dendrite. A structural model of aggregation is presented. The soma of the receptor cell revealed large pitted membrane particles about 140Å in diameter. These particles are possibly the morphologic counterpart to ionophores which have been proposed by electrophysiological studies.     

Quantitative analysis of the role of receptor recycling in T cell polarization

Activation of T cells of the immune system involves recognition of the antigen by the T cell receptor and subsequent internalization and recycling of this receptor. We present a numerical model for this process that accounts for the polarity of the intracellular traffic determined by the polarization of the microtubule-organizing center to the immunological synapse. Unexpectedly, the model explains the observed accumulation of receptors at the immunological synapse mainly as dynamic maintenance of the receptor density there, while the surface receptors everywhere else are depleted, even though the internalization occurs primarily at the synapse. In the case of an unsuccessful polarization of the microtubule-organizing center, which alters the polarity of the receptor trafficking, the model explains the absence of receptor accumulation as a dynamic downregulation at the synapse. The experiment shows that in this case the interaction of the T cell with its target is aborted. Disruption of recycling leads in the experiment to accumulation of the incompletely polarized cells. We propose that receptor recycling is a mechanism whereby the cell can sense its internal structure and detect polarity errors, analogous to checkpoint signaling mechanisms that ensure fidelity of cell division.     

The effect of molecular structure on olfactory discrimination by the parasitoid Microplitis croceipes

Flight chamber experiments were conducted to examine the capacity of the larval parasitoid Microplitis croceipes (Hymenoptera: Braconidae) to learn to distinguish between structurally related aliphatic alcohols differing in the carbon chain-length and the position of the functional group, and between an alcohol and the respective aldehyde. The parasitoid's ability to discriminate between the components depended on the chain-length of the alcohol to which they had been conditioned. Discrimination improved with increasing difference in carbon chain-length, e.g. the parasitoids made clear distinction between 1-hexanol and 1-octanol. Microplitis croceipes could also distinguish different isomers of six-carbon alcohols on the basis of the position of the alcoholic group as well as between 1-hexanol and 1-hexanal. The learning abilities of M. croceipes correspond to the specificity of antennal odour receptors towards aliphatic alcohols and aldehydes in previous electrophysiological studies of M. croceipes and other insects. Differences in perception or processing of single compounds might reflect differences of their ecological relevance.     

The nature of gene action in a Nicotiana rustica cross revealed by the recombinant inbred and second-cycle hybrid analysis

 A unique set of data recorded on 60 randomly extracted single-seed-descent (F_∞) lines of a highly heterotic cross between two varieties of Nicotiana rustica and their 870 reciprocally produced pairwise crosses, the second-cycle hybrids (SCH), are analysed to investigate the true nature of genetical control in the cross and the results are compared with those in earlier publications. The analysis revealed that epistasis, genotype-by-micro-environmental interaction, maternal effects and linkage are significant for several characters and the additive and non-additive components of variation take large values for all of the traits. Epistasis is predominantly duplicate and not complementary. Dominance is high but partial, all estimates of dominance ratio lying between 0.5 and 0.9. Dominance is predominantly unidirectional for leaf length, leaf width and final height, while for the remaining traits, some genes show ambidirectional dominance, although the incidence of unidirectional dominance is much higher throughout. The direction of dominance is predominantly for the increased score, except for flowering time where alleles conferring earliness are up to five times more frequently dominant. The present study has also confirmed that the F₂ and SCH_i distributions are very similar and that the former can be used to predict the transgression in the latter with confidence. The reduced range of the SCH _i families compared to the recombinant inbreds, further indicated that heterosis among many of the SCH_i is due to gene dispersion and there is little evidence for the presence of over-dominance. Received: 8 July 1996 / Accepted: 8 November 1996     

Time course of miniature end-plate currents at different sites on the frog neuromuscular junction

Miniature end-plate currents (MEPC) were recorded from proximal and distal sections of the frog sartorius and cutaneo-pectoral synapses by means of glass microelectrodes using extracellular techniques. Higher MEPC amplitudes and half-decay times were found in the proximal than the distal sections. These differences disappeared under the effects of tubocurarine and augmented under the action of armine. A significant positive correlation was noted between amplitude and duration of MEPC half decay time in approximately 80% of experiments — an indication of repeated binding between acetylcholine molecules and cholinoreceptors. This correlation was observed in practically all the proximal sites investigated, but only in half of distal sites tested. Findings obtained using electronmicroscopy showed that synaptic contact is about twice as extensive at proximal as at distal sites, while postsynaptic folds are poor in arborization. It is deduced that the high amplitude and longer time course of MEPC at proximal synaptic sites are due to more pronounced repeated binding between acetylcholine molecules and cholinergic receptors of the postsynaptic membrane, which could be put down to the density of the receptor population and geometrical aspects of the synaptic cleft.S. V. Kurashov Medical Institute, Ministry of Public Health of the RSFSR, Kazan'. A. A. Zhdanov State University, Leningrad. Institute of Biophysics, Academy of Sciences of the USSR, Puschino-on-Oka. Translated from Neirofiziologiya, Vol. 19, No. 6, pp. 779–788, November–December, 1987.     

Rapid discrimination of MHC class I and killer cell lectin-like receptor allele variants by high-resolution melt analysis

Ly49G and H-2 class I D(k) molecules are critical to natural killer cell-mediated viral control. To examine their contributions in greater depth, we established NK gene complex (NKC)/Ly49 congenic strains and a novel genetic model defined by MHC class I D(k) disparity in congenic and transgenic mouse strains. Generation and maintenance of Ly49 and H-2 class I select strains require efficient and reproducible genotyping assays for highly polygenic and polymorphic sequences. Thus, we coupled gene- and allele-specific PCR with high-resolution melt (HRM) analysis to discriminate Ly49g and H-2 class I D and K alleles in select strains and in the F(2) and backcross hybrid offspring of different genetic crosses. We show that HRM typing for these critical immune response genes is fast, accurate, and dependable. We further demonstrate that H-2 class I D HRM typing is competent to detect and quantify transgene copy numbers in different mice with distinct genetic backgrounds. Our findings substantiate the utility and practicality of HRM genotyping for highly related genes and alleles, even those belonging to clustered multigene families. Based on these findings, we envision that HRM is capable to interrogate and quantify gene- and allele-specific variations due to differential regulation of gene expression.     

Compartmentalized structure of the plasma membrane for receptor movements as revealed by a nanometer-level motion analysis

Movements of transferrin and alpha 2-macroglobulin receptor molecules in the plasma membrane of cultured normal rat kidney (NRK) fibroblastic cells were investigated by video-enhanced contrast optical microscopy with 1.8 nm spatial precision and 33 ms temporal resolution by labeling the receptors with the ligand-coated nanometer-sized colloidal gold particles. For both receptor species, most of the movement trajectories are of the confined diffusion type, within domains of approximately 0.25 microns2 (500-700 nm in diagonal length). Movement within the domains is random with a diffusion coefficient approximately 10(-9) cm2/s, which is consistent with that expected for free Brownian diffusion of proteins in the plasma membrane. The receptor molecules move from one domain to one of the adjacent domains at an average frequency of 0.034 s-1 (the residence time within a domain approximately 29 s), indicating that the plasma membrane is compartmentalized for diffusion of membrane receptors and that long- range diffusion is the result of successive intercompartmental jumps. The macroscopic diffusion coefficients for these two receptor molecules calculated on the basis of the compartment size and the intercompartmental jump rate are approximately 2.4 x 10(-11) cm2/s, which is consistent with those determined by averaging the long-term movements of many particles. Partial destruction of the cytoskeleton decreased the confined diffusion mode, increased the simple diffusion mode, and induced the directed diffusion (transport) mode. These results suggest that the boundaries between compartments are made of dynamically fluctuating membrane skeletons (membrane-skeleton fence model).     

The adaptation of the frog tongue to various taste solutions: the effect on gustatory neural responses to bitter stimuli

1. After the frog tongue was adapted for 10 sec to various salts and sugars, the initial phasic component of gustatory neural responses to almost all of quinine hydrochloride (Q-HCl), quinine sulfate (Q-H2SO4). Brucine, caffeine and picric acid was suppressed. 2. Following 10 sec adaptation to acetic acid, the phasic responses to Q-HCl and Q-H2SO4 were unchanged, those to brucine and caffeine were enhanced, and that to picric acid was depressed slightly. 3. The response to any one of Q-HCl, Q-H2SO4. brucine and caffeine was suppressed after adaptation to the other three, while those to picric acid and nicotine were unchanged or enhanced after adaptation to another bitter solution.     

The influence of olfactory concept on the probability of detecting sub- and peri-threshold components in a mixture of odorants.

The headspace of apple juice was analysed to obtain an ecologically relevant stimulus model mixture of apple volatiles. Two sets of volatiles were made up: a set of eight supra-threshold volatiles (MIX) and a set of three sub-threshold volatiles. These sets were used to test the hypothesis that sub-threshold components can change the quality of a familiar smelling mixture of odorants when added to this mixture. In order to test this hypothesis, three successive dilutions of the sub-threshold volatiles were prepared in such a way that the strongest was at the threshold concentration and the two lower concentrations were below the threshold. The detection probabilities of the sub-threshold components in a blank stimulus were compared with the detectabilities in MIX. The sub- and peri-threshold volatiles were detected no better in MIX than in a blank. On the contrary, sub- and peri-threshold volatiles were better detected alone than when added to MIX. However, when the group of subjects was split into two sub-groups, employing either a rough or a detailed concept definition of the target stimulus, respectively, the subjects with highly refined concepts were better able to detect the presence of sub-threshold volatiles in MIX than those with poorly refined stimulus concepts. The effect of stimulus concept definition occurred independently of the proportions of correct detections of sub-threshold volatiles in a blank.     

Activation of the frog sartorius acetylcholine receptor by a covalently attached group

Summary The frog sartorius motor endplate was treated with the specific disulfide bond reducing agent dithiothreitol and subsequently exposed to a covalently reacting compound (the nitrophenyl ester ofp-carboxyphenyltrimethylammonium iodide, NPTMB) known to activate the dithiothreitol-reduced acetylcholine receptor inElectrophorus electroplax. NPTMB causes a maximum depolarization of about 35 mV when applied to the dithiothreitol-treated sartorius motor endplate. It is ineffective on postjunctional membrane prior to disulfide bond reduction and on extrajunctional regions, reduced or unreduced. High concentrations of a competitive antagonist such as (+)-tubocurarine prevent reaction between NPTMB and the reduced receptor and cause a repolarization of the membrane when applied to the already-depolarized preparation. We conclude that in frog muscle, as in electroplax, the attached activator bridges the acetylcholine binding site of the reduced receptor between a sulfhydryl group, to which it is covalently bound, and a negative subsite, with which it forms a reversible ionic bond.