Probing an open CFTR pore with organic anion blockers

The cystic fibrosis transmembrane conductance regulator (CFTR) is an ion channel that conducts Cl- current. We explored the CFTR pore by studying voltage-dependent blockade of the channel by two organic anions: glibenclamide and isethionate. To simplify the kinetic analysis, a CFTR mutant, K1250A-CFTR, was used because this mutant channel, once opened, can remain open for minutes. Dose-response relationships of both blockers follow a simple Michaelis-Menten function with K(d) values that differ by three orders of magnitude. Glibenclamide blocks CFTR from the intracellular side of the membrane with slow kinetics. Both the on and off rates of glibenclamide block are voltage dependent. Removing external Cl- increases affinity of glibenclamide due to a decrease of the off rate and an increase of the on rate, suggesting the presence of a Cl- binding site external to the glibenclamide binding site. Isethionate blocks the channel from the cytoplasmic side with fast kinetics, but has no measurable effect when applied extracellularly. Increasing the internal Cl- concentration reduces isethionate block without affecting its voltage dependence, suggesting that Cl- and isethionate compete for a binding site in the pore. The voltage dependence and external Cl- concentration dependence of isethionate block are nearly identical to those of glibenclamide block, suggesting that these two blockers may bind to a common binding site, an idea further supported by kinetic studies of blocking with glibenclamide/isethionate mixtures. By comparing the physical and chemical natures of these two blockers, we propose that CFTR channel has an asymmetric pore with a wide internal entrance and a deeply embedded blocker binding site where local charges as well as hydrophobic components determine the affinity of the blockers.     

Block of Voltage-dependent Calcium Channel by the Green Mamba Toxin Calcicludine

A number of peptide toxins derived from marine snails and various spiders have been shown to potently inhibit voltage-dependent calcium channels. Here, we describe the effect of calcicludine, a 60 amino-acid peptide isolated from the venom of the green mamba (Dendroaspis angusticeps), on transiently expressed high voltage-activated calcium channels. Upon application of calcicludine, L-type (α_{1 C} ) calcium channels underwent a rapid, irreversible decrease in peak current amplitude with no change in current kinetics, or any apparent voltage-dependence. However, even at saturating toxin concentrations, block was always incomplete with a maximum inhibition of 58%, indicating either partial pore block, or an effect on channel gating. Block nonetheless was of high affinity with an IC₅₀ value of 88 nm. Three other types of high voltage activated channels tested (α_{1 A} , α_{1 B} , and α_{1 E} ) exhibited a diametrically different response to calcicludine. First, the maximal inhibition observed was around 10%, furthermore, the voltage-dependence of channel activation was shifted slightly towards more negative potentials. Thus, at relatively hyperpolarized test potentials, calcicludine actually upregulated current activity of (N-type) α_{1 B} channels by as much as 50%. Finally, the use of several chimeric channels combining the major transmembrane domains of α_{1 C} and α_{1 E} revealed that calcicludine block of L-type calcium channels involves interactions with multiple structural domains. Overall, calcicludine is a potent and selective inhibitor of neuronal L-type channels with a unique mode of action. Received: 22 September 1999/Revised: 1 December 1999     

Root apical meristems ofAllium porrum L. as affected by arbuscular mycorrhizae and phosphorus

Summary Arbuscular mycorrhizal (AM) fungi significantly improve plant growth in soils with low phosphorus availability and cause many changes in root morphology, similar to those produced by increased P nutrition, mainly depending on root apex size and activity. The aim of this work was to discriminate between the morphogenetic role of AM fungi and P in leek (Allium porrum L.) by feeding mycorrhizal and nonmycorrhizal plants with two nutrient solutions containing 3.2 or 96 M P and examining specific parameters related to adventitious root apices (apex size, mitotic cycle, and RNA synthesis). The results showed that AM fungi blocked meristem activity as indicated by the higher percentages of inactive apices and metaphases in the apical meristem of mycorrhizal plants, whereas the high P supply lengthened the mitotic cycle without blocking the apices, resulting in steady, slow root growth. The possible involvement of abscisic acid in the regulation of root apex activity is discussed.Abbreviations ABA abscisic acid - AM arbuscular mycorrhizae - CI and CII nonmycorrhizal control plants grown with low or high phosphorus concentration - MI and MII mycorrhizal plants grown with low or high phosphorus concentration - PGR plant growth regulator     

Time-irreversible Subconductance Gating Associated with Ba2+ Block of Large Conductance Ca2+-activated K+ Channels

Ba²⁺ block of large conductance Ca²⁺-activated K⁺ channels was studied in patches of membrane excised from cultures of rat skeletal muscle using the patch clamp technique. Under conditions in which a blocking Ba²⁺ ion would dissociate to the external solution (150 mM N-methyl-d-glucamine⁺ _o, 500 mM K⁺ _i, 10 μM Ba²⁺ _i, +30 mV, and 100 μM Ca²⁺ _i to fully activate the channel), Ba²⁺ blocks with a mean duration of ∼2 s occurred, on average, once every ∼100 ms of channel open time. Of these Ba²⁺ blocks, 78% terminated with a single step in the current to the fully open level and 22% terminated with a transition to a subconductance level at ∼0.26 of the fully open level (preopening) before stepping to the fully open level. Only one apparent preclosing was observed in ∼10,000 Ba²⁺ blocks. Thus, the preopenings represent Ba²⁺-induced time-irreversible subconductance gating. The fraction of Ba²⁺ blocks terminating with a preopening and the duration of preopenings (exponentially distributed, mean = 0.75 ms) appeared independent of changes in [Ba²⁺]_i or membrane potential. The fractional conductance of the preopenings increased from 0.24 at +10 mV to 0.39 at +90 mV. In contrast, the average subconductance level during normal gating in the absence of Ba²⁺ was independent of membrane potential, suggesting different mechanisms for preopenings and normal subconductance levels. Preopenings were also observed with 10 mM Ba²⁺ _o and no added Ba²⁺ _i. Adding K⁺, Rb⁺, or Na⁺ to the external solution decreased the fraction of Ba²⁺ blocks with preopenings, with K⁺ and Rb⁺ being more effective than Na⁺. These results are consistent with models in which the blocking Ba²⁺ ion either induces a preopening gate, and then dissociates to the external solution, or moves to a site located on the external side of the Ba²⁺ blocking site and acts directly as the preopening gate.     

Strategies and counterstrategies to infanticide in mammals

I analyse and summarize the empirical evidence in mammals supporting alternative benefits that individuals may accrue when committing nonparental infanticide. Nonparental infanticide may provide the perpetrator with nutritional benefits, increased access to limited resources, increased reproductive opportunities, or it may prevent misdirecting parental care to unrelated offspring. The possibility that infanticide is either a neutral or maladaptive behaviour also is considered. I devote the second half of this article to reviewing potential mechanisms that individuals may use to prevent infanticide. These counterstrategies include the early termination of pregnancy, direct aggression by the mother against intruders, the formation of coalitions for group defence, the avoidance of infanticidal conspecifics, female promiscuity, and territoriality. I evaluate the support for each benefit and counterstrategy across different groups of mammals and make suggestions for future research.     

地塞米松复合罗哌卡因臂丛神经阻滞对儿童肱骨髁上骨折患儿术后镇痛的影响

摘要 目的：探讨地塞米松复合罗哌卡因臂丛神经阻滞（BPB）对儿童肱骨髁上骨折患儿术后镇痛效果的影响。方法：择期行肱骨髁上骨折手术的患儿140例，随机分组为对照组70例与试验组70例。麻醉后两组均于超声引导下实施BPB，其中对照组予以0.25%罗哌卡因药液，试验组予以0.25%罗哌卡因、0.1 mg/kg地塞米松所组成的混合药液。记录两组患儿痛觉阻滞时间；于患儿苏醒后10 min、术后2 h、术后6 h、术后12 h及术后24 h，采用FLACC评分对患儿疼痛程度进行评估；记录两组患儿术后24 h内镇痛药物使用情况；记录两组患儿术后首次下床活动时间和术后住院时间；记录两组术后24 h内不良反应发生情况。结果：与对照组相比，试验组痛觉阻滞时间显著延长（P<0.05）。与对照组相比，试验组术后2~24 h的疼痛评分均显著降低（P<0.05）。试验组术后24 h布洛芬混悬液使用次数显著少于对照组（P<0.05），曲马多使用率显著低于对照组（P<0.05）。与对照组相比，试验组下床活动时间提前（P<0.05），术后住院时间缩短（P<0.05）。两组不良反应发生率无统计学差异（P>0.05）。结论：地塞米松复合罗哌卡因行BPB能够为肱骨髁上骨折患儿提供良好术后镇痛效果，利于患儿术后恢复。     

Molecular mechanisms of congenital heart block

Autoantibody-associated congenital heart block (CHB) is a passively acquired autoimmune condition associated with maternal anti-Ro/SSA antibodies and primarily affecting electric signal conduction at the atrioventricular node in the fetal heart. CHB occurs in 1–2% of anti-Ro/SSA antibody-positive pregancies and has a recurrence rate of 12–20% in a subsequent pregnancy. Despite the long-recognized association between maternal anti-Ro/SSA autoantibodies and CHB, the molecular mechanisms underlying CHB pathogenesis are not fully understood, but several targets for the maternal autoantibodies in the fetal heart have been suggested. Recent studies also indicate that fetal susceptibility genes determine whether an autoantibody-exposed fetus will develop CHB or not, and begin to identify such genes. In this article, we review the different lines of investigation undertaken to elucidate the molecular pathways involved in CHB development and reflect on the hypotheses put forward to explain CHB pathogenesis as well as on the questions left unanswered and that should guide future studies.     

Genome Re-duplication and Irregular Segregation Occur During the Cell Cycle of Entamoeba histolytica

Heterogeneity of genome content is commonly observed in axenic cultures of Entamoeba histolytica. Cells with multiple nuclei and nuclei with heterogenous genome contents suggest that regulatory mechanisms that ensure alternation of DNA synthesis and mitosis are absent in this organism. Therefore, several endo-reduplicative cycles may occur without mitosis. The data also shows that unlike other endo-reduplicating organisms, E.histolytica does not undergo a precise number of endo-reduplicative cycles. We propose that irregular endo-reduplication and genome partitioning lead to heterogeneity in the genome content of E.histolytica trophozoites in their proliferative phase. The goal of future studies should be aimed at understanding the mechanisms that are involved in (a) accumulation of multiple genome contents in a single nucleus; (b) genome segregation in nuclei that contain multiple genome contents and (c) maintenance of genome fidelity in E. histolytica.