Recombinant Expression and Characterization of α-Conotoxin LvIA in Escherichia coli

α-Conotoxin LvIA is derived from Conus lividus, native to Hainan, and is the most selective inhibitor of α3β2 nicotinic acetylcholine receptors (nAChRs) known to date. In this study, an efficient approach for the production of recombinant α-Conotoxin LvIA is described. Tandem repeats of a LvIA gene fragment were constructed and fused with a KSI gene and a His₆ tag in a Escherichia coli (E. coli) expression vector pET-31b(+). The recombinant plasmids were transformed into E. coli and were found to express well. The KSI-(LvIA)_n-His₆ fusion protein was purified by metal affinity chromatography and then cleaved with CNBr to release recombinant LvIA (rLvIA). High yields of fusion protein ranging from 100 to 500 mg/L culture were obtained. The pharmacological profile of rLvIA was determined by two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes expressing rat nAChR subtypes. The rLvIA antagonized the α3β2 nAChR subtype selectively with a nano-molar IC₅₀. The rLvIA was analgesic in a mouse hot-plate test model of pain. Overall, this study provides an effective method to synthesize α-conotoxin LvIA in an E. coli recombinant expression system, and this approach could be useful to obtain active conopeptides in large quantity and at low cost.     

Marine Natural Products Acting on the Acetylcholine-Binding Protein and Nicotinic Receptors: From Computer Modeling to Binding Studies and Electrophysiology

For a small library of natural products from marine sponges and ascidians, in silico docking to the Lymnaea stagnalis acetylcholine-binding protein (AChBP), a model for the ligand-binding domains of nicotinic acetylcholine receptors (nAChRs), was carried out and the possibility of complex formation was revealed. It was further experimentally confirmed via competition with radioiodinated α-bungarotoxin ([¹²⁵I]-αBgt) for binding to AChBP of the majority of analyzed compounds. Alkaloids pibocin, varacin and makaluvamines С and G had relatively high affinities (K_i 0.5–1.3 μM). With the muscle-type nAChR from Torpedo californica ray and human neuronal α7 nAChR, heterologously expressed in the GH₄C₁ cell line, no competition with [¹²⁵I]-αBgt was detected in four compounds, while the rest showed an inhibition. Makaluvamines (K_i ~ 1.5 μM) were the most active compounds, but only makaluvamine G and crambescidine 359 revealed a weak selectivity towards muscle-type nAChR. Rhizochalin, aglycone of rhizochalin, pibocin, makaluvamine G, monanchocidin, crambescidine 359 and aaptamine showed inhibitory activities in electrophysiology experiments on the mouse muscle and human α7 nAChRs, expressed in Xenopus laevis oocytes. Thus, our results confirm the utility of the modeling studies on AChBPs in a search for natural compounds with cholinergic activity and demonstrate the presence of the latter in the analyzed marine biological sources.     

棉铃虫烟碱类乙酰胆碱受体α7亚基基因的鉴定及功能分析

为探究和挖掘更多杀虫剂靶标受体,采用PCR方法结合RACE技术克隆了棉铃虫烟碱类乙酰胆碱受体（nicotinic acetylcholine receptor,nAChR）的α7亚基（nAChR-α7）基因,通过荧光定量PCR技术进行时空表达分析,并采用电生理检测以及RNAi技术研究该基因对棉铃虫生长发育的调控作用。结果表明,棉铃虫nAChR-α7基因（GenBank登录号：KM884875）全长3 632 bp,包含编码496个氨基酸的1 491 bp开放阅读框。该基因具有nAChR典型结构,与其他鳞翅目昆虫nAChR-α7基因同源性较高,且包括胞外N端配体结合区、半胱氨酸环、Loop A~Loop F、跨膜区（TM1~TM4）和1个短的C端胞外尾。nAChR-α7基因在棉铃虫幼虫头部及成虫期高表达;通过将nAChR-α7基因在非洲爪蟾Xenopus oocytes卵母细胞中表达,并检测电生理信号,确定该基因是nAChR受体基因,但不能持续响应ACh信号。干扰试验结果显示nAChR-α7基因对棉铃虫幼虫生长发育无显著影响。推测该基因可能不是主要的nAChR基因,而是与其他亚基基因共同起作用。     

电生理技术在昆虫毒理学上的应用

对电生理技术在杀虫剂作用机制，新杀虫剂筛选及害虫防治等昆虫毒理学领域上的应用进行了综述，并讨论了电生理技术在昆虫毒理研究上的发展前景。     

Intracardiac electrophysiological conduction parameters in adult dogs

Background: Invasive electrophysiology study (EPS) is one of the most important adjunct tests for the evaluation of arrhythmias in human medicine. However, EPS is neither well known nor widely used in veterinary medicine.

Objective: To define the values for intracardiac conduction parameters determined during invasive EPS in dogs.

Animals and methods: The study included 16 admitted dogs of various breeds, sex and ages and 6 control Beagles. In the Beagles, EPS was performed twice at 6-month intervals in order to verify the reproducibility of the results.

Results: No significant differences were found between the results of the baseline and repeated EPS performed in the Beagles. We found retrograde conduction in 13 (59%) out of 22 dogs and including 4 (31%) animals with concomitant ‘jumps’ in the atrioventricular node conduction curve, pointing to the presence of dual conduction in this node. The mean values of the AV and retrograde VA Wenckebach points equaled to 220 and 360 ms, respectively, suggesting that the capability of the descending route is higher than that of the ascending route.

Conclusion: The values determined in this study may be helpful in early detection of abnormalities in the electrical conduction system of the heart.     

Clinical, electrophysiological and morphological changes in a case of hereditary retinal degeneration in the Papillon dog

An autosomal recessive retinal disease with a late onset in Swedish Papillon dogs has recently been described. A 7-year-old Papillon dog showed no obvious signs of visual impairment and only minor ophthalmoscopic changes. Cone ERG b-wave amplitudes were within normal limits, while rod responses were nonrecordable or severely abnormal. Ultrastructural examination showed a generalized retinal degenerative disease, most prominent in the peripheral areas. The inferior retina was more severely affected than the superior areas. Both rods and cones showed morphological changes. The Papillon dog is another dog breed affected by progressive rod-cone degeneration, with similarities to the canine retinal disease given the gene symbol prcd .     

Organotypic slice culture of the hypothalamic paraventricular nucleus of rat

Organotypic slice cultures have been developed as an alternative to acute brain slices because the neuronal viability and synaptic connectivity in these cultures can be preserved well for a prolonged period of time. This study evaluated a stationary organotypic slice culture developed for the hypothalamic paraventricular nucleus (PVN) of rat. The results showed that the slice cultures maintain the typical shape of the nucleus, the immunocytochemical signals for oxytocin, vasopressin, and corticotropin-releasing hormone, and the electrophysiological properties of PVN neurons for up to 3 weeks in vitro. The PVN neurons in the culture expressed the green fluorescent protein gene that had been delivered by the adenoviral vectors. The results indicate that the cultured slices preserve the properties of the PVN neurons, and can be used in longterm studies on these neurons in vitro.     

Antiarrhythmic and Electrophysiologic Effects of Flecainide on Acutely Induced Atrial Fibrillation in Healthy Horses

Background

Only few pharmacologic compounds have been validated for treatment of atrial fibrillation (AF) in horses. Studies investigating the utility and safety of flecainide to treat AF in horses have produced conflicting results, and the antiarrhythmic mechanisms of flecainide are not fully understood.

Objectives

To study the potential of flecainide to terminate acutely induced AF of short duration (≥15 minutes), to examine flecainide‐induced changes in AF duration and AF vulnerability, and to investigate the in vivo effects of flecainide on right atrial effective refractory period, AF cycle length, and ventricular depolarization and repolarization.

Animals

Nine Standardbred horses. Eight received flecainide, 3 were used as time‐matched controls, 2 of which also received flecainide.

Methods

Prospective study. The antiarrhythmic and electrophysiologic effects of flecainide were based on 5 parameters: ability to terminate acute pacing‐induced AF (≥15 minutes), and drug‐induced changes in atrial effective refractory period, AF duration, AF vulnerability, and ventricular depolarization and repolarization times. Parameters were assessed at baseline and after flecainide by programmed electrical stimulation methods.

Results

Flecainide terminated all acutely induced AF episodes (n = 7); (AF duration, 21 ± 5 minutes) and significantly decreased the AF duration, but neither altered atrial effective refractory period nor AF vulnerability significantly. Ventricular repolarization time was prolonged between 8 and 20 minutes after initiation of flecainide infusion, but no ventricular arrhythmias were detected.

Conclusions and Clinical Importance

Flecainide had clear antiarrhythmic properties in terminating acute pacing‐induced AF, but showed no protective properties against immediate reinduction of AF. Flecainide caused temporary prolongation in the ventricular repolarization, which may be a proarrhythmic effect.     

Acylated 1,3-Aminopropanols as Repellents against Bloodsucking Arthropods

Starting from a set of known repellent compounds, a general structural framework with high probability for repellent activity was developed by molecular modelling techniques. Synthesis, structure–biological activity relationships of acylated 1,3-aminopropanols on the yellow fever mosquito Aedes aegypti and the properties and activity of the new development candidate compound KBR 3023 on Ae. aegypti, the malaria mosquito Anopheles stephensi, the common house mosquito Culex quinquefasciatus and the stable fly Stomoxys calcitrans are described. The sensory effect of KBR 3023 and deet has been studied in behavioural and neurophysiological investigations on Ae. aegypti and the American cockroach Periplaneta americana. The compounds clearly reduce or even eliminate the approach behaviour towards attractants like host odours or sexual pheromones. Electrophysiological studies on the insects' olfactory receptor organs reveal that certain cell types, which are not involved in perception of the attractive odorants, respond to deet and/or KBR 3023. As soon as one of the compounds is presented together with an attractant, a new input is active in the brain, which adds to the input from other receptors activated by the attractant. This new overall pattern clearly differs from that elicited by the attractant, so that the insect is no longer able to detect the latter. The specificity and mode of action of KBR 3023 was investigated by experiments exploring second-messenger responses elicited in antennal preparations of male P. americana. KBR 3023 induced a rapid increase in the concentration of inositol triphosphate in a dose-dependent and tissue-specific manner; other second-messenger systems were not affected. These observations suggest that KBR 3023 may act via subsets of G-protein-coupled receptors in sensory neurones.