Basis for Phospholipid Incorporation into Peripheral Nerve Myelin

Abstract: To characterize the mechanism(s) for targeting of phospholipids to peripheral nerve myelin, we examined the kinetics of incorporation of tritiated choline-, glycerol-, and ethanolamine-labeled phospholipids into four subfractions: microsomes, mitochondria, myelin-like material, and purified myelin at 1, 6, and 24 h after precursors were injected into sciatic nerves of 23–24-day-old rats. As validation of the fractionation scheme, a lag (> 1 h) in the accumulation of labeled phospholipids in the myelin-containing subfractions was found. This lag signifies the time between synthesis on organelles in Schwann cell cytoplasm and transport to myelin. In the present study, we find that sphingomyelin (choline-labeled) accumulated in myelin-rich subfractions only at 6 and 24 h, whereas phosphatidylserine (glycerol-labeled) and plasmalogen (ethanolamine-labeled) accumulated in the myelin-rich fractions by 1 h. The later phospholipids accumulate preferentially in the myelin-like fraction. These results are consistent with the notion that the targeting of sphingomyelin, a lipid present in the outer myelin leaflet, is different from the targeting of phosphatidylserine and ethanolamine plasmalogen, lipids in the inner leaflet. These findings are discussed in light of the possibility that sphingomyelin targeting is Golgi apparatus based, whereas phosphatidylserine and ethanolamine plasmalogen use a more direct transport system. Furthermore, the routes of phospholipid targeting mimic routes taken by myelin proteins P₀ (Golgi) and myelin basic proteins (more direct).     

Laminin A, B1, and B2 Chain Gene Expression in Transected and Regenerating Nerves: Regulation by Axonal Signals

Abstract: Laminin A, B1, and B2 chain mRNA levels in degenerating and regenerating mouse sciatic nerves were examined using northern blot analysis. In normal intact nerves, B1 and B2 mRNA steady-state levels were high, but when the nerves were crushed, the steady-state levels of B1 and B2 mRNA per milligram wet tissue weight of the distal segments of the nerves increased five- to eightfold over that of control levels as the total RNA and β-actin mRNA levels increased, suggesting that these increases were the consequence of Schwann cell proliferation after axotomy. When the steady-state levels of B1 and B2 mRNA were normalized as the ratio to total RNA or β-actin mRNA levels, however, they drastically decreased to about 20% of the normal nerve levels in the nerve segments distal to both the crush and transaction sites 1 day after injury. In the crushed nerves, B1 and B2 mRNA levels gradually increased as the regenerating nerves arrived at the distal segments and reestablished normal axon–Schwann cell contact, and then returned to normal levels on the 21 st day. In the transected nerves, where Schwann cells continued to be disconnected from axons, both B1 and B2 mRNA levels remained low. Cultured Schwann cells expressed detectable levels of B1 and B2 chain mRNA which significantly increased when the cells were cocultured with sensory neurons. However, mRNA for A chain was not detectable in the normal, axotomized nerves or in cultured Schwann cells. These data indicate that Schwann cells express laminin B1 and B2 chain mRNA that are up-regulated by axonal or neuronal contact, but they do not express A chain mRNA.     

K+ channels of stomatal guard cells: Abscisic-acid-evoked control of the outward rectifier mediated by cytoplasmic pH

The activation by abscisic acid (ABA) of current through outward-rectifying K⁺ channels and its dependence on cytoplasmic pH (pH_i) was examined in stomatal guard cells of Vicia faba L. Intact guard cells were impaled with multibarrelled and H⁺-selective microelectrodes to record membrane potentials and pH_i during exposures to ABA and the weak acid butyrate. Potassium channel currents were monitored under voltage clamp and, in some experiments, guard cells were loaded with pH buffers by iontophoresis to suppress changes in pH_i. Following impalements, stable pH_i values ranged between 7.53 and 7.81 (7.67±0.04, n = 17). On adding 20 M ABA, pH_i rose over periods of 5–8 min to values 0.27±0.03 pH units above the pH_i before ABA addition, and declined slowly thereafter. Concurrent voltage-clamp measurements showed a parallel rise in the outward-rectifying K⁺ channel current (I_{K, out}) and, once evoked, both pH_i and I_{K, out} responses were unaffected by ABA washout. Acid loads, imposed with external butyrate, abolished the ABA-evoked rise in I_{K, out}. Butyrate concentrations of 10 and 30 mM (pH₀ 6.1) caused pH_i to fall to values near 7.0 and below, both before and after adding ABA, consistent with a cytoplasmic buffer capacity of 128±12 mM per pH unit (n = 10) near neutrality. Butyrate washout was characterised by an appreciable alkaline overshoot in pH_i and concomitant swell in the steady-state conductance of I_{K, out}. The rise in pH_i and i_{K, out} in ABA were also virtually eliminated when guard cells were first loaded with pH buffers to raise the cytoplasmic buffer capacity four- to sixfold; however, buffer loading was without appreciable effect on the ABA-evoked inactivation of a second, inward-rectifying class of K⁺ channels (I_{K, in}). The pH_i dependence of I_{K, out} was consistent with a cooperative binding of at least 2H⁺ (apparent pK_a = 8.3) to achieve a voltage-independent block of the channel. These results establish a causal link previously implicated between cytoplasmic alkalinisation and the activation of I_{K, out} in ABA and, thus, affirm a role for H⁺ in signalling and transport control in plants distinct from its function as a substrate in H⁺-coupled transport. Additional evidence implicates a coordinate control of I_{K, in} by cytoplasmic-free [Ca²⁺] and pH_i.Abbreviations ABA abscisic acid - [Ca²⁺]_i cytoplasmic free [Ca²⁺]_i - E_K K⁺ equilibrium potential - I_{K, out}, I_{K, in} outward-, inward-rectifying K⁺ channel (current) - I-V current-voltage (relation) - Mes 2-(N-morpholino)ethanesulfonic acid - pH_i cytoplasmic pH - Tes 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]-amino}ethanesulfonic acid - V_m membrane potential We are grateful to G. Thiel (Pflanzenphysiologisches Institut, Universität Göttingen, Germany) for helpful discussions. This work was possible with equipment grants-in-aid from the Gatsby Charitable Foundation, the Royal Society and the University of London Central Research Fund. F.A. holds a Sainsbury Studentship.     

N-Terminal arginylation of proteins in explants of injured sciatic nerves and embryonic brains of rats

Posttranslational modification of proteins by arginine and lysine has been demonstrated in crude extracts of vertebrate nerves and brain but not in intact cells. In the present experiments we have exploited the fact that Arg is added posttranslationally only at the N-terminus of target proteins, to demonstrate these reactions in intact cells of sciatic nerves and embryonic brains of rats. Sciatic nerves were crushed in anaesthesized rats and 2 hrs later segments of nerve, including the site of the crush, were removed and incubated in media containing [³H]Arg. Incorporation of [³H]Arg into total proteins was analyzed by acid precipitation and the presence of label at the N-terminus was determined by a modification of the Edman degradation procedure. Approximately 25% of protein bound [³H]Arg was released from the N-terminus by the Edman reaction indicating that it was added posttranslationally rather than through protein synthesis. N-terminal labeling was not detectable in nerves not crushed prior to explant and incubation. Slices of embryonic day 20 visual cortex, when incubated under similar conditions as injured sciatic nerves, also showed approximately 25% of the protein incorporated [³H]Arg at the N-terminus, while arginylation was not detectable in adult rat brain slices. Since Lys is not added posttranslationally to the N-terminus, we have attempted to observe lysylation of proteins in intact cells by using cycloheximide (Cx) to block protein synthesis without interfering with protein modification. The posttranslational incorporation of Arg/Lys into proteins was found to be insensitive to up to 2.0 mM Cx in tissue extracts (in vitro). However, in intact cells, doses as low as 10 uM Cx completely inhibited the incorporation of [³H]Arg/Lys into proteins. One uM Cx allowed for some incorporation of [³H]Arg/Lys into protein and approximately 40% of the Cx insensitive Arg was incorporated into the N-terminal. These results show that in vivo but not in vitro, Cx can block protein modification, suggesting that either in intact cells protein modification requires protein synthesis, or that Cx has effects other than as an inhibitor of protein synthesis on cells in culture, effects that it does not have on the partially purified components of the reaction.     

Gonadotropin-releasing hormone (GnRH) pathways and reproductive control in elasmobranchs

Synopsis Immunoreactive (ir) gonadotropin-releasing hormone (GnRH) is localized in many neurons of the terminal nerve (TN) and midbrain tegmentum, while few ir-cells are observed in the preoptic area and ventral hypothalamus. The paucity of preoptic ir-cells may relate to an unusual feature of the elasmobranch pituitary, i.e. a lack of portal control of gonadotropin-producing cells. TN and midbrain GnRH-ir neurons may be major sources of GnRH used to modulate or otherwise control both pituitary and brain cells via delivery through the systemic circulation. These ir-nuclei also appear to directly innervate CNS regions (the preoptic area, habenula and clasper control area of the spinal cord) involved in sexual functions. Important regulatory mechanisms, represented by interactions between GnRH pathways and sex-steroid concentrating neurons, are likely to occur in the preoptic area, habenula and midbrain tegmentum.     

Synthetic chimeras of mouse growth factor-associated glandular kallikreins. I. Kinetic properties.

A series of six chimeric proteins, composed of fragments corresponding to either one or the other of the growth factor-associated mouse glandular kallikreins-epidermal growth factor binding protein (EGF-BP) and the gamma-subunit of nerve growth factor (gamma-NGF)--were expressed in Escherichia coli and isolated, and their kinetic properties were characterized. The assembly of these synthetic proteases involved the substitution of regions of the proteins containing four specific surface loops that have been postulated to influence both kinetic specificity and the formation of growth factor complexes. The substrates utilized in the kinetic characterization of these chimeric kallikreins were tripeptide nitroanilides representing carboxyl termini of both the EGF and beta-NGF mature hormones, putative processing sites for these kallikreins in the precursors. Characterization of these hybrid enzymes demonstrates that Km and kcat kinetic constants may be independently affected by the regions utilized in construction of these chimeric kallikreins. Specifically, loop 1, located in the amino terminal region (Bode, W., et al., J. Mol. Biol. 164, 237-282, 1983), in gamma-NGF enhanced the kcat for substrates containing threonine in the P2 position, as is the case during the processing of the carboxy terminus of the beta-NGF precursor. Also, the central regions of the kallikreins containing loop 2 and the kallikrein loop dictated the generally inverted Km and kcat kinetic constants observed between EGF-BP and gamma-NGF. Finally, in gamma-NGF the autolysis loop, found in the carboxyl terminal region, functions to lower the Km kinetic constant for a variety of substrates. The results allow previously characterized kinetic differences between EGF-BP and gamma-NGF to be interpreted in terms of specific regions of the proteins and identify a subset of amino acid positions responsible for these functional characteristics.     

超声引导下神经阻滞麻醉联合全身麻醉对股骨头置换老年患者麻醉效果及术后认知功能的影响

摘要 目的：研究超声引导神经阻滞麻醉联合全身麻醉对股骨头置换老年患者的麻醉效果及对术后认知功能的影响。方法：选取2019年1月至2021年12月期间我院收治的150例拟行股骨头置换术的老年患者,随机分为对照组和观察组,各75例。对照组患者行常规全身麻醉,观察组在对照组的基础上行超声引导下神经阻滞麻醉,比较两组患者的血流动力学指标,拔管时间、苏醒时间和复苏室停留时间,苏醒后疼痛,术后认知功能及不良反应的发生率。结果：观察组患者各时间点的心率（HR）和平均动脉压（MAP）均较对照组低（P＜0.05）。观察组拔管时间、苏醒时间和复苏室停留时间均较对照组短（P＜0.05）。观察组患者术后视觉模拟评分（VAS）均低于同一时间点的对照组（P＜0.05）。与对照组相比,观察组患者术后1 h、12 h和24 h的简易智力状态检查（MMSE）评分均较高（P＜0.05）,观察组术后1 h、12 h和24 h术后认知功能障碍（POCD）发生率较对照组低（P＜0.05）。两组患者不良反应发生率无差异（P＞0.05）。结论：超声引导神经阻滞麻醉可稳定血流动力学,缩短拔管、苏醒及复苏室停留时间,减轻术后疼痛,改善术后认知功能,减少POCD的发生,值得临床推广。     

不同容量胸段经椎间孔硬膜外注射阻滞范围的CT影像研究及诊断性阻滞的可行性分析

摘要 目的：探讨不同容量对胸段经椎间孔硬膜外注射（TFEI）药液扩散范围和镇痛效果的影响以及胸段TFEI用于诊断性阻滞的可行性。方法：选择2021年1月至2022年12月南京大学医学院附属鼓楼医院收治的胸段带状疱疹相关疼痛患者140例,随机分为4组,实施单次TFEI并分别注入不同容量含造影剂局麻药（A组：0.2 mL;B组：0.5 mL;C组：1.0 mL;D组：2 mL）,CT扫描并观察造影剂在硬膜外向头侧、尾侧及总扩散节段,造影剂在椎间孔、同侧椎旁间隙、同侧及对侧硬膜外间隙扩散情况,判断是否为选择性神经根阻滞。评估注射前、注射后30分钟及24小时视觉模拟评分（VAS）。结果：头侧和尾侧扩散节段以及总扩散节段数,D组最多,A组最少（P<0.05）;C组、D组造影剂扩散≥3个节段发生率明显高于B组（P<0.05）;C组、D组病例造影剂扩散至同侧椎旁间隙和对侧硬膜外间隙的发生率明显高于A组、B组（P<0.05）,仅A组37.1%的病例实现选择性神经根阻滞,其余各组均无选择性阻滞病例。注射后30分钟,C、D组VAS评分显著低于A、B组（P<0.05）;注射后24小时,D组VAS评分显著低于A、B组（P<0.05）。结论：胸段带状疱疹相关疼痛患者TFEI药液扩散范围随注射容量的增加而扩大,且在硬膜外倾向于头侧扩散,2 mL容量单次TFEI可阻滞3个以上的神经节段,获得良好的镇痛效果。胸段TFEI行诊断性阻滞的可行性较差。     

加巴喷丁联合射频脉冲、神经阻滞治疗带状疱疹后神经痛的效果

摘要 目的：探讨加巴喷丁联合脉冲射频、神经阻滞治疗带状疱疹后遗神经痛的效果。方法：本研究选取106例确诊为带状疱疹后遗神经痛的患者,采用随机数表法将其分为对照组A（36例）、试验组B（35例）和试验组C（35例）。A组患者采用口服加巴喷丁进行治疗,B组患者采用加巴喷丁联合神经阻滞治疗,C组患者采用加巴喷丁联合脉冲射频治疗,观察比较三组患者的治疗效果,分别对视觉模拟评分（VAS）、夜间睡眠评分（SRSS）、疼痛程度（NRS）、不良反应及综合疗效进行统计评估。结果：治疗3天、7天、14天和1月后,与对照组A比较,B、C组患者的VAS、SRSS和NRS评分较治疗前均有显著降低（P<0.05）;试验组B、C不良反应发生率分别为8.57%（3/35）和5.71%（2/35）,对照组A不良反应发生率为22.22%（8/36）,试验组不良反应发生率显著低于对照组,差异具有统计学意义（P<0.05）。结论：相较于使用加巴喷丁和加巴喷丁联合神经阻滞这两种治疗方案,加巴喷丁联合射频脉冲及神经阻滞治疗带状疱疹后遗神经痛能够在短期内有效缓解患者疼痛并改善睡眠状况。     

不同剂量右美托咪定联合超声引导下多点髂筋膜间隙阻滞在老年全髋关节置换术中的应用效果分析

摘要 目的：探讨超声引导下多点髂筋膜间隙阻滞联合不同剂量右美托咪定在老年全髋关节置换术（THA）中的应用效果。方法：选取2019年11月-2022年12月北京积水潭医院贵州医院择期行THA的老年患者120例,按照随机数字表法分为A组（n=40）、B组（n=40）、C组（n=40）,三组均接受超声引导下多点髂筋膜间隙阻滞治疗,在此基础上,A组、B组、C组分别给予0.5 μg/kg、1.0 μg/kg、1.5 μg/kg右美托咪定注射液。对比三组临床指标、血流动力学指标、应激反应指标、脑神经损伤情况,观察三组围术期间不良反应发生情况。结果：B组、C组的阻滞消退时间、拔管时间长于A组,首次下床时间短于A组（P<0.05）。B组、C组的阻滞消退时间、拔管时间、首次下床时间对比无差异（P>0.05）。三组麻醉诱导后即刻（T1）~手术结束时（T3）时间点心率（HR）、平均动脉压（MAP）升高后降低（P<0.05）。B组、C组的T1~T3时间点HR、MAP均低于A组,且B组低于C组（P<0.05）。三组T3时间点皮质醇（Cor）、白介素-6（IL-6）、肿瘤坏死因子-α（TNF-α）升高（P<0.05）。B组、C组的T3时间点Cor、IL-6、TNF-α均低于A组,且B组低于C组（P<0.05）。B组、C组的T3时间点中枢神经特异性蛋白（S100β）均低于A组,且B组低于C组（P<0.05）。B组、C组的T3时间点脑源性神经生长因子（BDNF）均高于A组,且B组高于C组（P<0.05）。三组不良反应发生率组间对比无差异（P>0.05）。结论：超声引导下多点髂筋膜间隙阻滞联合1.0 μg/kg右美托咪定在THA中具有更好的麻醉效果,可缩短首次下床时间,减轻机体应激反应、血流动力学波动以及脑部神经损伤。