Modification of acetylcholinesterase with the fluorescent thiol reagent S-mercuric-N-dansylcysteine

11s acetylcholinesterase (EC 3.1.1.7) from Torpedo californica electroplax, purified by a combination of affinity and gel chromatography was found to react stoichiometrically with S-mercuric-N-dansylcysteine. Approximately four mols of reagent per mol of enzyme were incorporated when the modification was carried out in 1.0 mM Tris-C1, pH 7.5, either in the presence or absence of 0.1 M NaCl. Prior incubation of the enzyme with 1.0 x 10(-4) M Zn2+ allowed the incorporation of about six mols of reagent per mol of enzyme. Binding of the reagent produced shifts in the emission and excitation wavelength maxima which were similar for all reaction conditions; however the enhancement of fluorescence intensity which accompanied binding of reagent was dependent on the ionic composition of the reaction medium. The modified enzyme remained active towards the active site titrant 7-(dimethylcarbamoyloxy)-N-methylquinolinium and retained its sensitivity towards inactivation by Zn2+. The results suggest that acetylcholinesterase as prepared contains several accessible thiol groups, and that the bound reagent may prove to be a useful probe of ligand-induced conformational changes in acetylcholinesterase.     

昆虫嗅觉受体及其介导的信号转导机制研究进展

高度灵敏的嗅觉系统,能够帮助昆虫准确识别环境中不同来源的挥发性化合物,在昆虫觅食、交配和产卵等生命活动过程中起着至关重要的作用.通过感觉神经元膜上数量巨大且种类繁多的嗅觉受体,昆虫可以识别不同的气味物质,进而调控其行为.已知的昆虫嗅觉受体主要有三种,离子型受体、气味受体和响应二氧化碳及信息素的味觉受体.目前嗅觉受体的分子结构及其介导的信号转导机制仍然没有得到完整的阐释,嗅觉受体配体的鉴定工作也还任重道远.本综述就昆虫嗅觉受体的结构、进化、功能表征方法以及气味受体介导信号转导的机制等方面的研究进展进行了综述,以期对研究昆虫嗅觉编码和调控,以及昆虫与植物间互作提供一定的理论参考.     

Determination of total tiopronin in human plasma by LC-ESI-MS using tris (2-carboxy-ethyl) phosphine as reducing reagent and methyl acrylate as derivatization reagent for the thiol group

A quantitative method for the determination of total tiopronin (TP) in human plasma was developed by liquid chromatography with electrospray ionisation (ESI) mass spectrometric detection. After reduction with tris (2-carboxy-ethyl) phosphine (TCEP) and derivatization with methyl acrylate (MA) for the thiol group of TP, plasma samples were processed successively by deproteinization and solid phase extraction. N-acetyl-l-cysteine (NAC) was selected as internal standard undergoing the same treatment as TP. The method was validated that it could meet the need of biological analysis. The lower limit of quantitation (LLOQ) of TP in plasma was 0.02microg/mL. Finally, the method was successfully applied to a pharmacokinetic study in 20 healthy Chinese male volunteers after an oral dose of 200mg TP tablets.     

Cell dynamics in the olfactory mucosa

By means of ultrastructural and autoradiographic observations from the olfactory mucosa of frog, it has been shown that olfactory receptor neurons as well as supporting cells are continuously replaced during the adult life of the animal. The severing of the olfactory nerve in adult frogs results in rapid degeneration of all mature olfactory neurons. An increased mitotic activity of the basal cells accompanies the degeneration of the mature neurons and precedes the regeneration of new neurons. The capability of these newly formed neurons to re-establish their connections in the olfactory bulb has been ascertained and the modalities of the process will be dealt with in a further report.     

Gas chromatographic—mass spectrometric determination of tiopronin in human blood using acrylic acid esters as a derivatization reagent for the thiol group

A gas chromatographic—mass spectrometric method for determining tiopronin, which has a thiol group, in human blood has been described. To prevent the oxidative degradation of tiopronin in the blood, its thiol group was immediately protected by treatment with isobutyl acrylate, which reacted readily with tiopronin in a 0.1 M Na₂HPO₄ solution (pH 9.1). The reaction was quantitative within 30 min. The blood sample was deproteinized and purified by a combination of liquid—liquid extraction and solid-phase extraction. Finally, the carboxyl moiety of the ester adduct was derivatized to the pentafluorobenzyl ester. The derivatives of tiopronin and the internal standard were analysed with gas chromatography—mass spectrometry. The precision of the method was satisfactory, and the calibration curve had good linearity in the concentration range investigated. The limit of determination of tiopronin in blood was estimated to be ca. 1 ng/ml.     

Neurogenesis drives stimulus decorrelation in a model of the olfactory bulb

The reshaping and decorrelation of similar activity patterns by neuronal networks can enhance their discriminability, storage, and retrieval. How can such networks learn to decorrelate new complex patterns, as they arise in the olfactory system? Using a computational network model for the dominant neural populations of the olfactory bulb we show that fundamental aspects of the adult neurogenesis observed in the olfactory bulb – the persistent addition of new inhibitory granule cells to the network, their activity-dependent survival, and the reciprocal character of their synapses with the principal mitral cells – are sufficient to restructure the network and to alter its encoding of odor stimuli adaptively so as to reduce the correlations between the bulbar representations of similar stimuli. The decorrelation is quite robust with respect to various types of perturbations of the reciprocity. The model parsimoniously captures the experimentally observed role of neurogenesis in perceptual learning and the enhanced response of young granule cells to novel stimuli. Moreover, it makes specific predictions for the type of odor enrichment that should be effective in enhancing the ability of animals to discriminate similar odor mixtures.     

Influence of a detergent on the catfish olfactory mucosa

The olfactory mucosa of the catfish (Ictulurus punctatus) has been briefly exposed to various concentrations of the non-ionic detergent Triton X-100. At high concentrations (1–4%) the upper layer of cells constituting the sensory and non-sensory areas of the lamellae is extensively damaged and new receptor cells do not appear in significant number before 2 months after treatment. Respiratory cells regenerate first followed by sustentacular and olfactory receptors. The regenerative process is very similar to that described previously after prolonged contact between the mucosa and ZnSO₄. Low detergent concentrations 0.03 – 0.1% affect only the sensory area. Olfactory and sustentacular microvilli and cilia, are immediately severed by the chemical. Regeneration occurs within the next 4 days. The cellular membranes appear also to be affected. From anatomical, electrophysiological and biochemical studies both in vivo and in vitro, it can be hypothesized that receptors involved in the transduction process are solubilized by the detergent but reappear at a level corresponding to 50–60% of their original activity within 2 h.Proteins, having an amino acid binding effectiveness correlated to the amino acid electrophysiological activities measured in vivo, can be isolated from the solubilized material. Further studies will be necessary to confirm that some of these molecules are involved in the olfactory transduction mechanism.     

Study of orexins signal transduction pathways in rat olfactory mucosa and in olfactory sensory neurons-derived cell line Odora: multiple orexin signalling pathways

Orexins A and B (OxA and OxB) are multifunctional neuropeptides implicated in the regulation of energy metabolism, wakefulness but also in a broad range of motivated behaviours. They signal through two G-protein-coupled receptors: orexin receptor 1 and 2 (Ox1R and Ox2R). The orexins and their receptors are present at all levels of the rat olfactory system: epithelium, bulb, piriform cortex but their signalling mechanisms remain unknown. We have studied orexins signal transduction pathways in the rat olfactory mucosa (OM) and in the Odora cell line derived from olfactory sensory neurons and heterologously expressing Ox1R or Ox2R. We have demonstrated by western blot and RT-PCR that multiple components of adenylyl cyclase (AC) and phospholipase C (PLC) signalling pathways were identical in OM and Odora cells. OxA and OxB induced a weak increase in IP3 in OM; they induced a significant rise in cAMP and IP3 in Odora transfected cells, suggesting the activation of AC and PLC pathways. Both OxA and OxB induced intracellular calcium elevation and transient activation of MAP kinases (ERK42/44) in Odora/Ox1R and Odora/Ox2R cells. These results suggest the existence of multiple orexins signalling pathways in Odora cells and probably in OM, corresponding to different possible roles of these peptides.     

Regulatory factors in the vertebrate olfactory mucosa

Access to and clearance of ligands from binding sites on olfactorycilia are regulated by a complex interplay of molecular, physicaland cellular factors. Nasal/olfactory glands secrete mucus thatcontains many proteins, among them odorant-binding proteins(OBP) that may solubilize lipophilic odorants in the aqueousmucous phase and subsequently transport them to receptor sites.The rate of transport of the ligand–OBP complex or unboundodorant is a function of the diffusion coefficient that, underphysiological conditions, is determined largely by the molecularsize of the complex or unbound odorant, the viscosity of mucusand the tortuosity factor. The binding constants must favorassociation of the ligand with the binding protein, dissociationof the complex and possible reassociation of the ligand withthe odorant receptor. Neural regulation of secretion determinesthe properties of the olfactory mucus that affect ligand accessand clearance, including viscosity, water content and depth.Extrinsic autonomic (adrenergic, cholinergic) and peptidergic(substance P/CGRP, VIP) neurons innervate olfactory glands andregulate both secretory granule release and electrolyte/waterbalance. Extrinsic peptidergic (substance (P/CGRP, VIP) neuronsterminate near the epithelial surface in close apposition tosustentacular cells and olfactory receptor neurons. The substanceP/CGRP fibers, in addition to functioning as sensory fibers,appear to regulate secretion from sustentacular cells througha secretomotor reflex and to neuromodulate the sensitivity ofolfactory receptor neurons to odorant stimulation. The actionof regulatory factors in the olfactory mucosa is an emergingtopic of research focused on molecular, physical and cellularfactors that affect sensory transduction.     

Bilateral projection of the olfactory mucosa in the frog

Bulbar potentials wer bilaterally recorded in the frog following electrical stimulation of one olfactory nerve bundle. The general features of the contralateral evoked response were very similar to those of ipsilateral ones. The contralateral response was shown to be produced in situ, not being electronically transmitted from the bulb on the stimulated side. Its response disappeared after section of the olfactory interbulbar adhesion but was not affected by sectioning through either the anterior or the habenular commissure. It was concluded that messages from the neuroreceptors belonging to either the ventral or the dorsal olfactory mucosa on one side, reach both olfactory bulbs.     

Evidence for a thiol reagent inhibiting choline acetyltransferase by reacting with the thiol group of coenzyme A forming a potent ingibitor.

Evidence is presented for the thiol reagent methyl methanethiolsulfonate inhibiting choline acetyltransferase (EC 2.3.1.6), not by reaction with an enzymic thiol group, but by reaction with the thiol group of CoA. The resulting CoA methyl disulfide is a potent inhibitor of this enzyme. Its action is reversed competitively by acetyl CoA.     

Drug-induced protoporphyria in the olfactory mucosa of the hamster

Administration of 3,5-diethoxycarbonyl-4-ethyl-1,4-dihydro-2,6-dimethylpyridine (4-ethyl-DDC) to hamsters resulted in a marked loss of cytochrome P-450-dependent reactions (peroxidase, 7-ethoxycoumarin O-deethylase, and 7-ethoxyresorufin O-deethylase) in both liver and olfactory epithelium within 2 hr. This inactivation of cytochrome P-450 was accompanied by inhibition of ferrochelatase (FK), stimulation of 5-aminolevulinate synthase (ALA-S), and accumulation of protoporphyrin both in the liver and to a lesser degree, in the olfactory epithelium. These results suggest that the mechanism of induction of protoporphyria in nasal tissues is similar to that occurring in the liver, namely, suicidal metabolism of 4-ethyl DDC by cytochrome P-450 resulting in formation of N-ethylprotoporphyrin, a potent inhibitor of FK. The consequent depletion of heme leads to stimulation of ALA-S and, thus, porphyrin accumulation. Investigation of the dose-response to 4-ethyl DDC demonstrated that, in liver, maximal inhibition of FK and accumulation of protoporphyrin occurred at a dose of 50 mg/kg while ALA-S activity continued to increase up to a dose of 100 mg/kg. This is compatible with an additional effect of the drug on ALA-S involving induction of cytochrome P-450 and, thus, further depletion of heme. In the olfactory epithelium, stimulation of ALA-S was significantly less marked, suggesting that this secondary effect does not operate in nasal tissue. This is consistent with reports that olfactory cytochrome P-450s are noninducible.     

Receptor cells of the catfish olfactory mucosa

In the present study, it has been demonstrated that catfisholfactory cells, having knobs characterized by a crown of longmicrovilli, are bipolar neurons. These cells, found almost exclusivelyon the dorso-medial half of the sensory area of the olfactorylamellae, most probably function as olfactory receptor cells.They appear to be different from microvillous tufted cells describedin several other fishes as well as in the catfish.     

On the stimulus conducting structures in insect olfactory receptors

Summary In the olfactory sensilla trichodea of the silk moth, the cuticle of the sense hair is perforated by numerous pores. These pores are shown to be connected with the dendrites of the receptor cell by extracellular pore tubules. It is suggested, therefore, that odour molecules reach the stimulus transduction sites on the receptor membrane by surface diffusion alone, and need not diffuse three-dimensionally through the sensillum liquor.

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Zusammenfassung Bei den olfaktorischen Sensilla trichodea des Seidenspinners wird die Cuticula des Sinneshaares von zahlreichen Poren durchbrochen. Es wird gezeigt, daß diese Poren mit den Dendriten der Sinneszellen durch extrazelluläre Porentubuli verbunden sind. Daraus folgt, daß die Duftmoleküle den Ort der Erregungsbildung an der Rezeptormembran wahrscheinlich allein durch Oberflächendiffusion erreichen, ohne eine dreidimensionale Diffusion durch den Sensillenliquor.