稳定表达人源GABAAR-CHO细胞株的建立

目的: 构建α₁亚基诱导表达、β₂和γ_2L亚基稳定表达的人源α₁β₂γ_2L-GABA_AR-CHO(Chinese hamster ovary)细胞株。方法: 从人cDNA文库中扩增α₁、β₂、γ_2L亚基编码基因,分别构建亚基表达载体;将三个亚基表达载体共转染CHO-K1细胞,通过抗性筛选、膜电位检测法进行稳定表达克隆筛选;通过qPCR、Western blot对亚基表达进行鉴定;以激动剂GABA、阳性变构调节剂地西泮(diazepam,Dia)、拮抗剂荷包牡丹碱(bicuculine)为工具药,采用全细胞膜片钳方法及膜电位检测法对稳定表达细胞的药理学功能进行鉴定。结果: 经克隆筛选获得表达量较高的α₁β₂γ_2L-GABA_AR-CHO并对其亚基表达鉴定,结果显示该细胞稳定表达α₁、β₂、γ_2L亚基,构建的α₁β₂γ_2L-GABA_AR-CHO细胞仅在加入四环素(tetracyclin)诱导的情况下表达α₁亚基并与β₂、γ_2L组装成具有功能活性的α₁β₂γ_2L-GABA_AR;对其进行全细胞膜片钳检测研究发现,GABA可对其产生激动效应,引起α₁β₂γ_2L-GABA_AR-CHO细胞产生氯离子通道特征性电流变化,Dia可剂量依赖性地增强GABA对α₁β₂γ_2L-GABA_AR的激动效应;在膜电位检测研究中,获得GABA激动效应EC₅₀为(177.72 ± 15.92)nmol/L,Dia变构效应EC₅₀为(3.63±0.52)μmol/L,拮抗剂Bicuculine拮抗效应IC₅₀为(538.83±29.55)nmol/L。结论: 通过采用诱导表达策略,成功构建了α₁β₂γ_2L-GABA_AR-CHO稳定表达细胞株,该细胞株具有对激动剂、阳性变构剂、拮抗剂特异性检测的药理学功能。     

60Co-γ辐照对越南油茶种子发芽及幼苗形态指标的影响

为了研究⁶⁰Co-γ辐照对越南油茶Camellia drupifera种子发芽和表观形态指标的影响,选取越南油茶璠龙3号‘Fanglong 3’和璠龙5号‘Fanglong 5’种子分别进行0、40 Gy、50 Gy、60 Gy、70 Gy ⁶⁰Co-γ辐照并测定种子发芽率和出苗率以及幼苗生长表观形态指标(苗高、地径、叶长、叶宽、叶面积)。结果表明,⁶⁰Co-γ辐照对越南油茶种子发芽率影响小,对其出苗率、苗高、地径以及叶片生长有明显抑制作用,尤其是高剂量(60 Gy、70 Gy)辐照对越南油茶种子损伤较大,严重影响其出苗及幼苗生长。高剂量辐照明显延迟越南油茶的出苗时间,抑制幼苗生长,导致苗木瘦弱、生长缓慢,植株呈现矮化现象。高剂量亦抑制越南油茶叶片生长,导致叶片瘦小,生长缓慢。璠龙3号种子⁶⁰Co-γ辐照半致死剂量为64 Gy,璠龙5号为57 Gy。     

贵州荔波玉屏洞穴陆生动物食物来源及营养级

洞穴是“极端地下生物多样性”的聚集地, 为了揭示洞穴陆生动物的食物来源以及营养级关系, 本文于2016年10月对贵州荔波玉屏镇的3个代表性洞穴内的陆生动物进行调查, 应用¹³C和¹⁵N稳定同位素方法, 分析了洞内陆生动物和基础碳源的δ¹³C、δ¹⁵N值变化特征, 探究了各动物类群的营养级位置以及不同的基础碳源对动物的食物源的贡献率。结果表明: 洞穴植物的δ¹³C值范围为-38.25‰ ± 0.95‰至-29.44‰ ± 0.49‰, 土壤有机质δ¹³C值范围为-32.73‰ ± 0.03‰至-24.68‰ ± 0.41‰, 大部分不在洞穴植物的δ¹³C变化范围内。洞穴动物δ¹³C、δ¹⁵N值范围分别为-34.22‰ ± 0.39‰至-14.83‰ ± 0.78‰、1.86‰ ± 0.20‰至16.43‰ ± 0.05‰, 表现出较大的种(或类群)间差异。IsoSource软件估算结果表明, 洞穴土壤有机质对78.3%动物类群的食物源的贡献率高于植物。荔波玉屏洞穴消费者的营养级范围为1.01-5.32, 其中, 巨蚓科、螺类、潮虫科、马陆、大蚊科、夜蛾科主要处于第2营养层次; 蚰蜒科、蚁蛉科(幼虫)、蝙蝠处于第3营养层次; 蜚蠊科处于第2-3营养层次; 驼螽科、蠼螋科处于第2-4营养层次; 蜘蛛处于第2-5营养层次, 跨3个营养级。综上认为, 洞穴土壤有机质是洞穴消费者的主要食物基础。大部分同一类群的动物在洞穴中所处的营养级位置较稳定, 少部分同属于一个类群的动物在不同洞穴或同一洞穴不同光带所处的营养级位置有差异。     

副干酪乳杆菌β-葡糖苷酶的表达、纯化及酶学性质研究 *

为了实现糖苷类物质的高效转化,将来源于副干酪乳杆菌(Lactobacillus paracasei)TK1501 β-葡糖苷酶基因连接于表达载体pET28a(+)上,在E. coli BL21中表达,重组酶经镍离子亲和层析分离得到纯酶,其分子质量和比酶活分别为86.63kDa和675.56U/mg。最适作用温度和pH分别为30℃和6.5。 Mg ²⁺和Ca ²⁺对β-葡糖苷酶酶活抑制作用最小,Cu ²⁺几乎使其丧失催化活性。其底物特异性较宽泛,对大豆异黄酮、栀子苷、水杨苷、七叶苷、虎杖苷、熊果苷均有降解作用。以β-pNPG为底物时,该酶的K_m和V_max分别为1.44mmol/L和58.32mmol/(L·s),催化系数k_cat为3 982/s。结果与分析表明,来源于副干酪乳杆菌TK1501 β-葡糖苷酶对水解大豆异黄酮和合成糖苷将会发挥重要作用。     

人β-半乳糖苷酶R299L突变体的获得与活性研究*

目的: GM1神经节苷脂贮积症是一种由半乳糖苷酶beta 1(galactosidase beta 1, GLB1)基因突变引起的β-半乳糖苷酶(β-galactosidase,β-gal)活性降低导致的严重的溶酶体贮积病。该病以进行性、致命性神经退行性病变为特征,目前尚无有效的治疗手段,AAV载体介导的基因治疗被认为是最有希望的治疗方法。通过基因定点突变获得具有较高β-gal活性的GLB1突变体,以期用于后续AAV介导的基因治疗。方法: 对人类和其他6种脊椎动物GLB1基因进行多序列比对分析,筛选出部分氨基酸位点进行定点突变,采用携带突变位点的重组质粒和AAV9载体转染或感染HEK-293细胞,比较突变体与未突变体的活性差异。对GM1模型鼠注射携带coGLB1-R299L的rAAV9病毒,探究该突变体的体内活性表达。结果: 从15个突变体中筛选出coGLB1-R299L突变体,经质粒转染导入细胞后,其β-gal活性比具有野生型氨基酸序列的coGLB1增加了30%~40%。AAV体外感染实验中,rAAV9-coGLB1-R299L组的β-gal活性较未感染的细胞对照组提升了约2.2倍。体内结果显示,rAAV9-coGLB1-R299L在模型鼠体内广泛表达,心脏、肝脏、脾脏、肺、脑组织中β-gal活性显著提升。结论: 获得了具有更高β-gal活性的突变体coGLB1-R299L,初步探究了rAAV9-coGLB1-R299L的体外表达效果和模型鼠体内β-半乳糖苷酶的表达与分布,为该突变体应用于AAV介导的GM1神经节苷脂病治疗奠定基础。     

细胞色素P450还原酶与CYP17的共表达及其功能分析*

17α-羟基黄体酮(17α-OH-PROG)是甾体激素类药物的关键中间体,其生物合成主要由细胞色素单加氧酶(CYP17)催化生成。在此过程中,细胞色素 P450还原酶(cytochrome P450 reductase,CPR)作为细胞色素P450 酶电子传递链的重要组成部分,直接影响CYP17的催化效率。为研究不同来源CPR与17α-羟化酶的适配性,首先以人源17α-羟化酶作为研究对象,构建了表达质粒pPIC3.5k-hCYP17,获得了重组毕赤酵母菌株。其次筛选获得3种不同来源CPR,构建了表达质粒 pPICZX-CPR,获得17α-羟化酶与CPR共表达菌株,并在毕赤酵母中进行转化实验,对转化产物进行薄层色谱(TLC)和高效液相色谱(HPLC)分析。结果显示,重组菌株具有17α-羟化酶活性,能够催化黄体酮生成目标产物17α-OH-PROG 以及副产物16α-羟基黄体酮(16α-OH-PROG)。不同来源的CPR与17α-羟化酶共表达与仅表达17α-羟化酶的产率相比均有所提高,其中hCPR-CYP17共表达菌株表现出最高的转化水平,17α-OH-PROG产率提高42%。上述结果表明:17α-羟化酶基因与CPR共表达能够提高其黄体酮17α-羟基化水平。为甾体黄体酮17α-羟基化的生物催化研究提供思路,对甾体药物的工业生产具有重要意义。     

毛头鬼伞子实体化学成分及抗糖尿病活性初探

从毛头鬼伞子实体中萃取得到乙醇、乙酸乙酯、石油醚3种有机提取物,采用α-葡萄糖苷酶活性抑制实验对3种有机提取物的抗糖尿病活性进行评价,结果显示,乙酸乙酯提取物对α-葡萄糖苷酶有较强的抑制活性。采用柱层析技术从乙酸乙酯提取物中分离纯化出10种化合物,经核磁等方法鉴定为：（1）顺,顺-9,12-十八(碳)二烯酸;（2）顺式-9-十八烯酸;（3）(22E,24R)-麦角甾烷-5,7,22-三烯-3β醇;（4）3β-5α-6α-22E-麦角甾-7,22-双烯-3,5,6-三醇-6-亚油酸酯;（5）3β-5α-6α-22E-麦角甾-7,22-双烯-3,5,6-三醇-6-油酸酯;（6）邻苯二甲酸二异丁酯;（7）对羟基苯乙醇;（8）4-羟基苯乙基乙酸酯;（9）3-(4-羟基-3-甲氧苯基)败脂酸;（10）N-反式-3,4亚甲二氧基肉桂酰基-3-甲氧基酪胺。对分离化合物的α-葡萄糖苷酶活性抑制实验结果显示,N-反式-3,4亚甲二氧基肉桂酰基-3-甲氧基酪胺对α-葡萄糖苷酶具有较强的抑制活性,其IC₅₀值为4.17mg/mL。     

基于稳定同位素技术的塔里木河下游不同林龄胡杨的水分利用来源

水分是制约很多陆地生态系统植物生长和繁殖的重要因素, 在干旱地区尤为明显。利用稳定同位素技术探究塔里木河下游不同林龄胡杨(Populus euphratica)的水分来源情况, 了解生态输水背景下荒漠河岸林的水分利用循环与利用策略, 可为生态输水提供科学依据, 同时也可对同类地区的生态恢复提供借鉴。本研究通过测定塔里木河下游胡杨茎干水和各潜在水源(土壤水、地下水)的稳定氢氧同位素值(δD、δ¹⁸O), 应用多源线性混合模型(IsoSource)分析了各潜在水源对不同林龄胡杨的贡献比例, 并结合3种林龄胡杨不同土壤深度含水量的变化, 分析了胡杨的主要吸水层位。结果表明: (1)不同林龄胡杨样地的不同深度区间上的土壤水δ¹⁸O值存在显著差异(P < 0.05): 胡杨幼龄木、成熟木、过熟木木质部δ¹⁸O分别为-7.83 ± 0.07‰、-8.53 ± 0.11‰、-9.36 ± 0.21‰; 而δD值不存在显著差异(P > 0.05)。可据此来推断胡杨的主要吸水层位。(2)总体上, 三种林龄胡杨土壤水δ¹⁸O值随土壤深度增加而减小, 并趋于接近地下水的δ¹⁸O值。其中, 0-60 cm土壤水受蒸发影响比较大, 其同位素组成经历了强烈的蒸发分馏过程, 土壤含水量极少, 土壤水δ¹⁸O值偏正。(3)不同林龄胡杨所利用的水分来源不同: 胡杨幼龄木对于地表80 cm以下的土壤水以及地下水均有一定程度的利用, 对80-140 cm、140-220 cm和220-340 cm的土壤水平均利用比率依次为16.2%、21.4%和24.6%, 对地下水平均利用比率为24.5%; 成熟木主要利用220-340 cm的土壤水及地下水, 平均利用比率分别为36.9%和42.3%; 过熟木主要利用140-340 cm的土壤水及地下水, 平均利用比率分别为32.8%和49.3%。     

黑河中游春玉米叶片水δD和δ18O的富集过程和影响因素

植物水的稳定同位素分馏过程是水在土壤-植物-大气连续体中循环的重要环节。以往研究由于叶片水¹⁸O同位素比值(δ¹⁸O _l,b)和氘(D)同位素比值(δD_l,b)(合称δ_l,b)实测数量少只能作为模型验证数据, 导致δ_l,b富集机制研究多集中于模型研究, 缺乏基于野外试验条件的δ_l,b富集的控制机制研究。叶片水δD_l,b和δ¹⁸O _l,b的富集程度(ΔD_l,b和Δ¹⁸O _l,b, 合称Δ_l,b)通常表示为δ_l,b与茎秆水D同位素比值(δD_x)和¹⁸O同位素比值(δ¹⁸O_x) (合称δ_x)之差, 即Δ_l,b = δ_l,b - δ_x。该研究以黑河中游沙漠绿洲春玉米(Zea mays)生态系统为研究对象, 重点采集和分析了季节和日尺度δ_l,b和δ_x数据, 配套开展了大气水汽δ¹⁸O和δD (合称δ_v)等辅助变量的原位连续观测, 探讨了季节和日尺度上的δ_l,b富集特征及其影响因素。结果表明: 叶片水δ_l,b和Δ_l,b的季节变化趋势不明显, 而受蒸腾作用影响表现出白天富集夜间贫化的单峰日变化特征。对于D来说, 无论季节尺度上还是日尺度上, 大气水汽δ_v和相对湿度是δD_l,b和ΔD_l,b的主要环境控制因素; 而对于¹⁸O来说, 无论季节尺度上还是日尺度上, 相对湿度是δ¹⁸O _l,b和Δ¹⁸O _l,b的主要环境控制因素。由于D和¹⁸O在热力学平衡分馏上有约8倍差异, 直接分析叶片水ΔD_l,b和Δ¹⁸O_l,b与影响因素的差异性, 有助于理解叶片水δD和δ¹⁸O富集过程以及对模型发展有一定的指导意义。     

鼎湖山森林演替序列植物-土壤碳氮同位素特征

植物群落对水分利用和养分利用的优化策略, 土壤碳周转和氮循环过程对演替变化如何响应, 森林土壤有机碳积累机制等都是森林生态学需要解决的关键问题。然而, 这些生态学过程的变化在短时间内通过传统的研究手段难以被精确观测, 碳氮同位素(¹³C、¹⁵N)技术的应用或许能提供更多有价值的信息。该文通过对鼎湖山森林演替序列代表性群落——马尾松(Pinus massoniana)针叶林(PF)、针阔叶混交林(MF)和季风常绿阔叶林(BF)植物-土壤碳氮同位素自然丰度的测定, 分析了叶片稳定碳同位素比率(δ¹³C)和稳定氮同位素比率(δ¹⁵N)与其叶片元素含量的关系, 以及叶片-凋落物-土壤δ¹³C、δ¹⁵N在演替水平和垂直方向上的变化特征。结果显示: 1)主要优势树种叶片δ¹³C与其C:N极显著正相关(p < 0.01), 凋落物和各层土壤δ¹³C均表现为PF > MF > BF, 沿演替方向逐渐降低; 2)叶片δ¹⁵N与叶片N含量正相关(p = 0.05), 凋落物和表层土壤(0-10 cm) δ¹⁵N沿演替方向逐渐增大; 3)不同演替阶段土壤δ¹³C、δ¹⁵N均沿垂直剖面呈现增大的趋势。结果表明: 南亚热带地区植物群落的发展并不一定受水分利用和氮素利用的补偿制约; δ¹³C自然丰度法的应用有助于森林土壤有机碳积累机制, 尤其有助于成熟森林土壤“碳汇”机制的阐释; 植物-土壤δ¹⁵N值可作为评估土壤氮素有效性和生态系统“氮饱和”状态的潜在指标。     

水稻两性生殖细胞的N-甲基-N-亚硝基脲诱变方法

N-甲基-N-亚硝基脲(MNU)被用于水稻(Oryza sativa)受精卵的诱变。通过水稻辽盐6号成熟生殖器官的MNU体内同步处理及后代群体筛查, 确立了水稻两性生殖细胞的MNU诱变方法。与辽盐6号受精卵的MNU处理相比, 各组条件下两性生殖细胞的MNU处理明显使M₁群体生长发育的指标降低及M₁-M₂群体中突变性状的发生率升高。两性生殖细胞在含有1.5 mmol?L ^-1 MNU和10 mmol?L ^-1 PO₄ ^3-的缓冲液(pH4.8)中处理60分钟, 突变性状发生率是基于受精卵MNU处理的3倍。进一步筛查M₃群体, 获得了包含新型植株和籽粒突变体的纯合突变体系列。研究结果表明, 水稻两性生殖细胞的MNU诱变可显著提高广谱诱变效率。该技术的应用可为水稻的未知功能基因鉴定和育种所需的各种突变体规模化开发提供高效的技术支撑。     

Diphenidol-related diamines as novel muscarinic M4 receptor antagonists

A series of hydrochloride derivatives 2a–9a and quaternary ammonium derivatives 3b–9b of diphenidol have been synthesized and characterized in receptor binding and cellular functional assays versus human muscarinic M₁–M₅ receptors expressed in CHO cells. Compound 8b, a methiodide derivative with a bipiperidinyl moiety and a second diphenidol framework, showed a potent and selective M₄ activity as competitive antagonist. Moreover 8b, acting as an allosteric modulator, was able to retard the dissociation rate of [³H]-N-methylscopolamine from CHO-M₄ cell membranes exposed to atropine. Taken together, these data suggest that 8b might open new avenues to the discovery of novel multivalent antagonists for the muscarinic receptors.     

水稻两性生殖细胞的N-甲基-N-亚硝基脲诱变方法

N-甲基-N-亚硝基脲(MNU)被用于水稻(Oryza sativa)受精卵的诱变。通过水稻辽盐6号成熟生殖器官的MNU体内同步处理及后代群体筛查, 确立了水稻两性生殖细胞的MNU诱变方法。与辽盐6号受精卵的MNU处理相比, 各组条件下两性生殖细胞的MNU处理明显使M₁群体生长发育的指标降低及M₁-M₂群体中突变性状的发生率升高。两性生殖细胞在含有1.5 mmol∙L ^-1 MNU和10 mmol∙L ^-1 PO₄ ^3-的缓冲液(pH4.8)中处理60分钟, 突变性状发生率是基于受精卵MNU处理的3倍。进一步筛查M₃群体, 获得了包含新型植株和籽粒突变体的纯合突变体系列。研究结果表明, 水稻两性生殖细胞的MNU诱变可显著提高广谱诱变效率。该技术的应用可为水稻的未知功能基因鉴定和育种所需的各种突变体规模化开发提供高效的技术支撑。     

Synthesis and properties of the dichloro [tetramethyl (trifluoromethyl)cyclopentadienyl]ruthenium dimer: X-ray structure of [M2(η-C5Me4CF3)2Cl2(μ-Cl)2] (M=Ru, Rh)

The reaction of RuCl₃(H₂O), with C₅Me₄CF₃J in refluxing EtOH gives [Ru₂(η⁵-C₅Me₁CF₂)₂ (μ-Cl₂] (20 in 44% yield. Dimer 2 antiferromagnetic (−2J=200 cm¹). The crystal structures of 2 (rhombohedral system, R3 space group, Z=9, R=0.0589) and [Rh₂(η⁵-C₅Me₄CF₃(₂Cl₂(μ-Cl)₂] (3) (rhombohedral system. space group, Z = 9, R = 0.0641) were solved; both complexes have dimeric structures with a trans arrangement of the η⁵-C₅Me₄CF₄ rings. Comparison of the geometry of 2 and 3 with those of the corresponding η⁵-C₅Me₅ complexes shows that lowering the ring symmetry causes significant distortion of the M₂(μ-Cl)₂ moiety. The analysis of the MCl₃ fragment conformations in 2 and 3 and in the η⁵-C₅ME₅ analogues shows that they are correlated with the M---M distances. The Cl atoms are displaced by Br on reaction of 2 with KBr in MeOH to give the diamagnetic dimer [Ru₂(η⁵-C₅Me₄CF₃)₂Br₂ (μ-Br₂] (4). Complex 2 reacts with O₂ in CH₂Cl₂ solution at ambient temperature to form a mixture of isomeric η⁶-fulvene dimers [Ru₂(η⁶-C₅Me₃CF₃ = CH₂)₂Cl₂(μ-Cl)₂] (5). Reactions of 5 with CO and allyl chloride give Ru(η⁵-C₅Me₃CF₃CH₂Cl)(CO)₂Cl (6) and Ru(η⁵-C₅Me₃CF₃CF₃CH₂Cl)(η³-C₃H₅)Cl₂ (7) respectively.     

Simulation of the molecular and electronic structure of heterofullerenes C55Y5 (Y=Si, Ge, Sn, B, Al, N, P, SiH, GeH, SnH) and their η-π-complexes with Li by the MNDO method

Qualitative estimates of the relative stability of hypothetical heterofullerenes C₅₅Y₅ (Y=Si, Ge, Sn, B, Al, N, P, SiH, GeH, SnH) and some η⁵-π-complexes LiC₅₅Y₅ were carried out by the MNDO method. Atoms Y (or groups XH) are assumed to substitute those C atoms in fullerene C₆₀ which are located at the -positions of a separated pentagonal face (pent^*) of this polyhedral molecule. It is shown that the spin densities in radicals C₅₅Y₅ (Y=SiH, GeH, SnH, B, Al, N, P) are localized on the separated pentagon atoms and the Li-pentagonal face (Li-pent^*) bonds in η⁵-π-complexes of these radicals with the Li atom are considerably stronger than Li-pent^* bonds in complexes [η⁵-π-LiC₆₀]⁺ and [η⁵-π-LiC₆₀] of unsubstituted C₆₀. In addition, it is established that the Li-pent^* bond energies in η⁵-π-complexes LiC₅₅B₅ and LiC₅₅Al₅ exceed the energy of the Li-pent^* bond in the η⁵-π-complex LiC₆₀H₅ studied earlier. In contrast, the energies of similar bonds for Y=N, P are close to the energy of the Li-pent^* bond in the η⁵-π-complex LiC₆₀H₅.     

Synthesis, NMR spectroscopy study, and antimuscarinic activity of a series of 2-(Acyloxymethyl)-1,3-dioxolanes

A series of 1,3-dioxolane-based ligands, bearing hydroxymethyl or ester functionalities, was synthesized and tested as potential muscarinic antagonists. The compounds display moderate to low affinity for the three receptor subtypes M₁-M₃, with some of them showing a significant selectivity for the M₃ subtype. The configurational and conformational properties were studied using NOE experiments and vicinal coupling constants. The ¹H and ¹³C NMR chemical shifts show stereochemically dependent trends. Quantitative analysis of conformer populations showed that the exocyclic CH₂N⁺(CH₃)₃ group is prevalently in a pseudo-axial orientation in the cis isomers and in a pseudo-equatorial orientation in the trans isomers.     

Effects of JO 1784, A selective sigma ligand, on the autoradiographic localization of M2 and M2 muscarinic receptor subtypes in trimethyltin treated rats

The distribution patterns of M₁ and M₂ muscarinic receptor subtypes following TMT and JO 1784 administration in the male Sprague-Dawley rat were investigated. In the present study, JO 1784 was injected in doses of 1, 4 and 16 mg/kg i.p. for one week prior to the single injection of TMT (8 mg/kg i.p.) and subsequently for 33 days. The effects of JO 1784 on the density of muscarinic receptor sub-types (M₁ and M₂) in the control and trimethyltin (TMT) treated rats were then evaluated. The topographic distribution and changes in muscarinic (M₁ and M₂) receptor densities were determined by means of autoradiography using [³H]quinuclidinylbenzilate (QNB). Both sub-types of muscarinic receptors contributed to the observed decrease in total muscarinic receptor binding in TMT-treated rats. In control rats, JO 1784 alone decreased M₁ receptor density in the amygdaloid nuclei, basal ganglia, cortex and hippocampus and decreased M₂ receptor density in the amygdaloid nuclei, basal ganglia, cortex, hippocampus, hypothalamus and septal regions. In TMT treated rats, chronic JO 1784 administration has a “neuroprotective effect” on both M₁ and M₂ receptors subtypes. Thus, following chronic administration of JO 1784 to TMT treated rats, both increases and decreases in M₁ receptor density were observed relative to TMT animals. A significant increase in M₁ receptor density was found in the cortex, olfactory regions, septum, thalamus and basal forebrain nuclei. In the hippocampus (CA₂ and CA₃), a significant decrease in M₁ receptor density was observed. In TMT-treated rats, JO 1784 produced a significant increase in M₂ receptor density in several brain regions with the most marked effects occurring in the amygdaloid nuclei, basal ganglia, cortex, hippocampus and hypothalamus. The ability of the selective sigma ligand, JO 1784, to attenuate the loss of muscarinic receptors in TMT treated rats could be of importance in the development of novel neuroprotective drugs.     

The neuroprotective effect of tacrine on trimethyltin induced memory and muscarinic receptor dysfunction in the rat

In this study chronic (39 days) tacrine (3 mg/kg i.p.) treatment significantly improved trimethyltin (8 mg/kg i.p.) induced deficits in spatial navigation. Tacrine also reduced trimethyltin induced hyperactivity and passive avoidance deficits but these effects did not reach statistical significance. The effect of trimethyltin on muscarinic (M₁ and M₂) receptor sites was determined by means of quantitative autoradiography using [³H]quinuclidinyl benzilate. A selective pattern of M₁ and M₂ receptor loss was observed mainly affecting the hippocampus and other limbic structures while leaving other brain regions intact. Tacrine successfully prevented the M₁ and M₂ receptor loss in the CA₁ and CA₁ hippocampal subfields. The improvement in trimethyltin behavioural toxicity following tacrine treatment may be related to the protective effect of this compound on muscarinic receptor density in the hippocampal formation and lends support to the hypothesis that cholinergic system dysfunction may be primarily responsible for trimethyltin induced deficits in cognitive function.     

Modulation of immobilized enzyme activity by altering the hydrophobicity of nylon-grafted membranes: Part 2: Non-isothermal conditions

Lactose hydrolysis by β-galactosidase immobilized on two nylon membranes, differently grafted, has been studied in a bioreactor operating under isothermal and non-isothermal conditions. One membrane (M₁) was obtained by chemical grafting of methylmethacrylate (MAA); the other one (M₂) by a double chemical grafting: styrene (Sty) and MAA. Hexamethylenediamine was used as a spacer between the grafted membranes and the enzyme. Both membranes have been physically characterized studying their permeabilities in presence of pressure or temperature gradients. Under non-isothermal conditions, the increase in activity of membrane M₂ was higher than that of membrane M₁. The and β coefficients, giving the percentage of activity increase when a temperature difference of 1°C is applied across the catalytic membranes, have been calculated. Results have been discussed with reference to the greater hydrophobicity of membrane M₂ with respect to membrane M₁, the hydrophobicity being a prerequisite for the occurrence of the process of thermodialysis.