Cellular Localization of the Molecular Forms of Acetylcholinesterase in Primary Cultures of Rat Sympathetic Neurons and Analysis of the Secreted Enzyme

The secretion and cellular localization of the molecular forms of acetylcholinesterase (AChE) were studied in primary cultures of rat sympathetic neurons. When cultured under conditions favoring a noradrenergic phenotype, these neurons synthesized and secreted large quantities of the tetrameric G4, and the dodecameric A12 forms, and minor amounts of the G1 and G2 forms. When these neurons adopted the cholinergic phenotype, i.e., in the presence of muscle-conditioned medium, the development of the cellular A12 form was completely inhibited. These neurons secreted only globular, mainly G4, AChE. Both cellular and secreted A12 AChE in adrenergic cultures aggregated at an ionic strength similar to that of the culture medium, raising the hypothesis that this form was associated with a polyanionic component of basal lamina. In noradrenergic neurons, 60-80% of the catalytic sites were exposed at the cell surface. In particular, 80% of G4 form, but only 60% of the A12 form, was external, demonstrating for the A12 form a sizeable intracellular pool. The hydrophobic character of the molecular forms was studied in relation to their cellular localization. As in muscle cells, most of the G4 form was membrane-bound. Whereas 76% of the cell surface A12 form was solubilized in the aqueous phase by high salt concentrations, only 50% of the intracellular A12 form was solubilized under these conditions. The rest of intracellular A12 could be solubilized by detergents and was thus either membrane-bound or entrapped in vesicles originating from, e.g., the Golgi apparatus.     

Brain Regional Distribution of Glutamic Acid Decarboxylase, Choline Acetyltransferase, and Acetylcholinesterase in the Rat: Effects of Chronic Manganese Chloride Administration after Two Years

Abstract: Rats were treated chronically with manganese chloride from conception onward for a period of over 2 years in order to study the effects of manganese and aging on the activities of glutamic acid decarboxylase (GAD), choline acetyltransferase (ChAT), and acetylcholinesterase (AChE) in hypothalamus, cerebellum, pons and medulla, striatum, midbrain, and cerebral cortex (which included the hippocampus). Manganese-treated 2-month-old and 24- to 28-month-old rats and age-matched controls were studied. In control rats during aging the activities of GAD decreased in hypothalamus (19%), pons and medulla (28%), and midbrain (22%) whereas the activities of AChE decreased in all regions (20–48%), particularly in the striatum (44–48%). Changes in ChAT activities in aging were observed only in one region—a decrease (23%) in the striatum. Life-long treatment with manganese appeared to abolish partially the decreases in aging in AChE activities in hypothalamus, cerebellum and striatum, and striatal ChAT activity. Manganese treatment also seemed to abolish the age-related decreases in GAD activities, since GAD activities in various brain regions of manganese-treated senescent rats were not significantly different from those of control young rats. These results are discussed in relation to other metabolic changes associated with aging and manganese toxicity.     

2''-Deoxycoformycin Inhibition of Adenosine Deaminase in Rat Brain: In Vivo and In Vitro Analysis of Specificity, Potency, and Enzyme Recovery

2'-Deoxycoformycin (DCF), a potent inhibitor of adenosine deaminase (ADA), is increasingly used as a tool to investigate adenosine metabolism and neuromodulation. To advance further the usefulness of DCF for studies of purines in the CNS, we determined the inhibitory potency of this compound against ADA and adenylate deaminase (AMPDA) in brain, the rate of ADA recovery in various brain regions after single or repeated intraperitoneal DCF administrations, and the effect of DCF on several neurotransmitter synthetic enzymes. In vitro, the Ki values for inhibition of ADA and AMPDA were found to be 23 pM and 233 microM, respectively. In vivo, DCF inhibited ADA with ED50 values ranging from 155 to 280 micrograms/kg at 2 h posttreatment, and 98% inhibition was achieved with 1 mg/kg. AMPDA activity was not affected by doses up to 5.0 mg/kg. In contrast to the greater than 95% inhibition of ADA seen 1 day after DCF at 5 mg/kg, the effectiveness of a second similar DCF treatment on the activity that had recovered by 14 days was dramatically reduced. Eight days after DCF treatment with doses of 5-50 mg/kg, the degree of ADA activity recovery in 10 brain regions examined was similar; it averaged 35% of control values at the low dose but showed some heterogeneity, ranging from 15 to 54% of control values, at the higher doses. Forty days after treatment with a single dose of 5 mg/kg, ADA activity recovered by 68-78% of control values in brain regions with normally high levels of activity and by 44-59% of control values in other regions. The activities of choline acetyltransferase, glutamic acid decarboxylase, and histidine decarboxylase (an enzyme colocalized with ADA in hypothalamic neurons) were unaffected by DCF treatment, a result suggesting the lack of a generalized neurotoxic effect. The very low doses of DCF required for ADA inhibition in vivo are consistent with the high potency of this drug against ADA in vitro, and any physiological effects observed at low doses might therefore be ascribed to inhibition of ADA.     

The Metabolism of Dopamine by Both Forms of Monoamine Oxidase in the Rat Brain and Its Inhibition by Cimoxatone

In the rat brain, dopamine is metabolised by both A and B forms of monoamine oxidase (MAO), although the A form of the enzyme is the major component. The Km of MAO-A toward dopamine (120 microM) is lower than the Km of MAO-B toward this substrate (340 microM). The activity of MAO-A was lower in old rats than in young rats, and the same degree of decrease was found for 5-hydroxytryptamine as for dopamine as substrates for this enzyme form. The activity of MAO-B was higher in the old rats, the degree of increase being the same for dopamine as for beta-phenethylamine as substrates for this enzyme form. The Ki values of the inhibition of MAO-A by cimoxatone and MD770222 (the principal plasma metabolite of cimoxatone) were independent of the substrate used to assay for activity, but were lower than the Ki values for the inhibition of MAO-B by these compounds.     

改良血管套接法与血管断端吻合法对断指再植患者断指血运、断指成活以及运动功能的影响

目的:探讨改良血管套接法与血管断端吻合法对断指再植患者断指血运、断指成活以及运动功能的影响。方法:回顾性分析我院于2017年1月到2018年12月期间治疗的107例断指再植患者的临床资料。根据手术方法的不同将患者分为A组(n=55,共断指93指)和B组(n=52,共断指86指),A组给予血管断端吻合法,B组给予改良血管套接法,比较两组患者断指血运、断指成活、运动功能、生活质量及并发症。结果:B组毛细血管充盈时间、患指微循环检测值低于A组,氧饱和度、经皮氧分压则高于A组(P<0.05)。B组断指成活率、断指张力正常率及断指颜色正常率均高于A组(P<0.05)。B组术后1年社会关系领域、生理领域、心理领域以及环境领域的维度评分均高于A组(P<0.05)。B组的肌力、活动度、灵巧度、Carroll量表评分均高于A组(P<0.05)。两组并发症发生率对比未见统计学差异(P>0.05)。结论:与血管断端吻合法相比,断指再植患者采用改良血管套接法,在断指血运、断指成活、运动功能、生活质量方面的改善效果更佳,且不会增加并发症发生率。     

The Predominant Molecular State of Bound Enzyme Determines the Strength and Type of Product Inhibition in the Hydrolysis of Recalcitrant Polysaccharides by Processive Enzymes

Processive enzymes are major components of the efficient enzyme systems that are responsible for the degradation of the recalcitrant polysaccharides cellulose and chitin. Despite intensive research, there is no consensus on which step is rate-limiting for these enzymes. Here, we performed a comparative study of two well characterized enzymes, the cellobiohydrolase Cel7A from Hypocrea jecorina and the chitinase ChiA from Serratia marcescens. Both enzymes were inhibited by their disaccharide product, namely chitobiose for ChiA and cellobiose for Cel7A. The products behaved as noncompetitive inhibitors according to studies using the ¹⁴C-labeled crystalline polymeric substrates ¹⁴C chitin nanowhiskers and ¹⁴C-labeled bacterial microcrystalline cellulose for ChiA and Cel7A, respectively. The resulting observed K_i(obs) values were 0.45 ± 0.08 mm for ChiA and 0.17 ± 0.02 mm for Cel7A. However, in contrast to ChiA, the K_i(obs) of Cel7A was an order of magnitude higher than the true K_i value governed by the thermodynamic stability of the enzyme-inhibitor complex. Theoretical analysis of product inhibition suggested that the inhibition strength and pattern can be accounted for by assuming different rate-limiting steps for ChiA and Cel7A. Measuring the population of enzymes whose active site was occupied by a polymer chain revealed that Cel7A was bound predominantly via its active site. Conversely, the active-site-mediated binding of ChiA was slow, and most ChiA exhibited a free active site, even when the substrate concentration was saturating for the activity. Collectively, our data suggest that complexation with the polymer chain is rate-limiting for ChiA, whereas Cel7A is limited by dissociation.