腺苷对缺氧时海马神经元内游离Ca^2＋的影响

实验用Ｆｌｕｏ－３负载细胞,在激光扫描共聚焦显微镜下直接监测缺氧后分散培养的大鼠海马ＣＡ１区神经元内游离Ｃａ＾２＋浓度（［Ｃａ＾２＋］ｉ）的变化,观察腺苷对这种变化的影响并初步探讨其作用机制。结果发现,急性缺氧使海马神经元［Ｃａ＾２＋］ｉ显著升高;腺苷（１００μｍｏｌ／Ｌ）明显抑制缺氧引起的［Ｃａ＾２＋］ｉ增高,腺苷Ａ１受体拮抗剂ＣＰＴ以及Ｋ＾＋通道阻断剂４－ＡＰ和ＡＴＰ敏感性Ｋ＾＋通道阻断剂ｇｌ     

培养乳鼠心肌细胞自发性〔Ca~（2＋）〕_i瞬间变化的实验模型

使用Ｃａ￣（２＋）敏感的荧光指示剂Ｆｕｒａ－２／ＡＭ,对培养的心肌细胞测定细胞内Ｃａ￣（２＋）的瞬间变化。结果为,在一个心搏周期中,经过大约１８０ｍｓ的时间,［Ｃａ￣（２＋）］＿ｉ瞬间变化达到最大值,然后恢复到基线水平（最小值）,经历了２６０ｍｓ。测得平均［Ｃａ￣（２＋）］＿ｉ瞬间变化的最大值及最小值分别为３４６±３８ｎｍｏｌ／Ｌ和１０２±１８ｎｍｏｌ／Ｌ。血管紧张素Ⅱ１００ｎｍｏｌ／Ｌ引起［Ｃａ￣（２＋）］＿ｉ瞬间变化最大值明显增加,但对［Ｃａ￣（２＋）］＿ｉ瞬间变化的基线水平没有明显的影响。ｒｙａｎｏｄｉｎｅ３μｍｏｌ／Ｌ使［Ｃａ￣（２＋）］＿ｉ瞬间变化的幅度明显降低。ＫＣｌ５０ｍｍｏｌ／Ｌ使［Ｃａ￣（２＋）］＿ｉ瞬间变化完全停止,并明显增加［ｃａ￣（２＋）］＿ｉ的基线水平。结果提示,本实验模型可用来观察［Ｃａ￣（２＋）］＿ｉ的瞬间变化。     

SNP抑制5-HT诱导的胞内游离钙浓度升高和内钙释放

用Ｆｕｒａ － ２／ＡＭ 荧光测量技术研究了５ － 羟色胺（５－ ＨＴ） 诱导的大鼠尾动脉平滑肌细胞胞内钙升高和一氧化氮（ＮＯ） 的抑制效应。实验表明, 胞外０ｍ ｍｏｌ／ Ｌ Ｃａ２ ＋ 时胞内静息［Ｃａ２ ＋ ］ ｉ 为２０ ．２±８ ．６ｎｍｏｌ／Ｌ（ｎ ＝ ８） 。１０μｍｏｌ／Ｌ ５－ ＨＴ 可诱导出胞内钙库释放引起的瞬态［Ｃａ２ ＋］ｉ 升高,其峰值达２４５ ．７ ±７１．６ｎｍｏｌ／ Ｌ（ｎ ＝ ６） 。１０ － ７ ｍｏｌ／Ｌ 硝普钠（ＳＮＰ） 可抑制５－ ＨＴ 诱导的［Ｃａ２ ＋］ｉ 升高,其峰值浓度降为７５．１±３５ ．９ｎｍｏｌ／Ｌ（ｎ ＝ ５） 。当细胞浴液含２．５ｍ ｍｏｌ／Ｌ Ｃａ２ ＋ 时,静息［Ｃａ２ ＋］ｉ为１１２ ．８ ±１０ ．３ｎｍｏｌ／ Ｌ（ｎ ＝ ５） , 这时１０μｍｏｌ／ Ｌ ５ － ＨＴ 可诱导［Ｃａ２ ＋ ］ ｉ 的峰值为２５２ ．３ ±８０ ．６ｎｍｏｌ／Ｌ（ｎ ＝ ４） ,以及其后平台浓度为１４３ ．０ ±３７ ．６ｎｍｏｌ／Ｌ（ｎ ＝ ４） ,略大于［Ｃａ２ ＋］ｉ 为１１２．８ ±１０ ．３ｎｍｏｌ／Ｌ 的静息浓度,为外钙内流引起。１０ － ７ ｍｏｌ／Ｌ ＳＮＰ 也可抑制５－ ＨＴ 诱导［Ｃａ２ ＋ ］ｉ 平台相浓度。平台浓度由１４３ ±４７     

缺氧时大鼠红细胞变形性损伤的机制研究

本实验通过测定平原和模拟高原减压缺氧３０天大鼠红细胞滤过指数（ＩＦ）、红细胞内［ｐＨ］ｉ、［Ｋ＋］ｉ／［Ｎａ＋］ｉ比值、［Ｃａ２＋］ｉ、［Ｍｇ２＋］ｉ、平均红细胞体积（ＭＣＶ）及平均红细胞血红蛋白浓度（ＭＣＨＣ）,从而探讨缺氧条件下大鼠红细胞变形性损害的机制。结果发现：１．缺氧组大鼠红细胞［Ｃａ２＋］ｉ明显升高,且与ＩＦ呈显著正相关,但［Ｍｇ２＋］ｉ无明显差异;２．缺氧组［Ｋ＋］ｉ／［Ｎａ＋］ｉ值较平原组明显降低,且与ＩＦ呈显著负相关;３．缺氧组ＭＣＨＣ与平原组无明显差异,但ＭＣＶ显著升高;４．缺氧组红细胞内［ｐＨ］ｉ较平原组明显升高。提示：缺氧时红细胞［Ｃａ２＋］ｉ升高,［Ｋ＋］ｉ／［Ｎａ＋］ｉ值降低,ＭＣＶ增大以及红细胞［ｐＨ］ｉ值的改变在其变形性损伤中起重要作用。     

氨对脑细胞胞浆游离钙含量的影响

目的与方法：采用Ｆｕｒａ－２／ＡＭ探针技术观察ＮＨ４Ｃｌ对离体急性分离之Ｗｉｓｔａｒ乳鼠大脑细胞胞头游离钙「Ｃａ＾２＋」ｉ含量的影响。结果：ＮＨ４＾＋浓度为２．５ｍｍｏｌ／Ｌ时脑细胞内「Ｃａ＾２＋」ｉ含量升高。在一定范围内,随着ＮＨ４＾＋浓度的加大,细胞内Ｃａ＾２＋持续升高。ＮＨ４＾＋的升钙作用主要被Ｎｉｃａｒｄｉｐｉｎｅ所阴断,其变化特征类以ＫＣｌ。结论：ＮＨ４＾＋主要通过影响电压依赖性钙离子通     

糖皮质激素对肝细胞内游离钙的作用

应用钙离子荧光指示剂ｆｕｒａ－２,对糖皮质激素（Ｇｌｕｃｏｃｏｒｔｉｃｏｉｄ,ＧＣ）是否影响肝细胞内游离钙（Ｉｎｔｒａｃｅｌｌｕａｌａｒ ｆｒｅｅ ｃａｌｃｉｕｍ,［Ｃａ＾２＋］ｉ作了初步探讨,结果发现,ＧＣ在短期内能升高肝细胞［Ｃａ＾２＋］水入１．０μｍｏｌ／Ｌ的Ｃｏｒｔｉｓｏ０．２５ｍｉｎ即可引起肝细胞［Ｃａ＾２＋］的明显升高,到１０分钟时效应达高峰。此时与静息状态的肝细胞水平相比胞浆内游离钙     

胡萝卜及愈伤组织细胞质Ca^2＋水平分析的研究

为测定植物细胞质内［Ｃａ＾２＋］ｉ,对胡萝卜（Ｄａｕｃｕｓｃａｒｔａｖａｒ．ｓａｔｉｖａＤＣ．）原生质体制备介质做了改进,并在正常生理条件,用温和的、非损伤性的方法将Ｃａ＾２＋荧光指示剂ｉｎｄｏ－１Ｋ＾＋和ｆｕｒａ－２Ｋ＾＋地入该原生体,能很好地标记细胞质的游离Ｃａ＾２＋。     

大鼠嗜铬细胞分泌的膜电容和微碳纤电极检测

在原代培养的大鼠肾上腺嗜铬细胞上,综合运用细胞内钙测定法和全细胞膜片钳法,以检测膜电容变化为手段测定单一肾上腺嗜铬细胞的胞吐过程。－７０ｍＶ到＋２０ｍＶ去极化引起的钙电流和细胞膜电容的变化以及吹加６０ｍｍｏｌ／ＬＫＣｌ时,细胞内游离钙离子浓度［Ｃａ２＋］ｉ和细胞膜电容变化的同时检测,表明了Ｃａ２＋对细胞胞吐的控制作用。而用微碳纤电极则能检测到吹加６０ｍｍｏｌ／ＬＫＣｌ导致嗜铬细胞胞吐时儿茶酚胺的量子化释放。细胞膜电容检测和微碳纤电极检测从不同侧面动态的反映了细胞胞吐过程与Ｃａ２＋的相关性     

糖皮质激素对肝细胞内游离钙的作用

应用钙离子荧光指示剂ｆｕｒａ－２,对糖皮质激素（Ｇｌｕｃｏｃｏｒｔｉｃｏｉｄ,ＧＣ）是否影响肝细胞内游离钙（Ｉｎｔｒａｃｅｌｌｕｌａｒ　ｆｒｅｅ　ｃａｌｃｉｕｍ,［Ｃａ２＋］ｉ作了初步探讨。结果发现,ＧＣ在短期内能升高肝细胞［Ｃａ２＋］ｉ,水平,并具有明显的量效关系。以１．０μｍｏｌ／Ｌ的Ｃｏｒｔｉｓｏｌ和１０．０μｍｏｌ／Ｌ的Ｄｅｘａｍｅｔｈａｓｏｎｅ效果最好。加入１．０μｍｏｌ／Ｌ的Ｃｏｒｔｉｓｏｌ０．２５ｍｉｎ即可引起肝细胞［Ｃａ２＋］ｉ的明显升高,到１０分钟时效应达高峰。此时与静息状态的肝细胞［Ｃａ２＋］ｉ水平相比,胞浆内游离钙升高了近３倍;与相应对照组比较,胞浆内游离钙升高具有明显的统计学意义,Ｐ＜０．０１。ＲＵ４８６为一种人工合成的糖皮质激素受体（Ｇｌｕｃｏｃｏｒｔｉｃｏｉｄ　ｒｅｃｅｐｔｏｒ,ＧＲ）的拮抗剂,它可以取消ＧＣ升高肝细胞［Ｃａ２＋］ｉ的效应,提示ＧＣ升高肝细胞内［Ｃａ２＋］ｉ可能与ＧＲ介导有一定关系。鉴于ＧＣ升高［Ｃａ２＋］ｉ时间较短,推测与肝细胞膜ＧＲ的非基因快速调节作用影响钙离子通道有关。     

降钙素基因相关肽对缺氧时海马细胞内游离Ca^2＋的影响

本实验用Ｆｕｒａ－２荧光测定技术直接监测了缺氧时大鼠海马细胞内游离Ｃａ２＋浓度（［Ｃａ２＋］ｉ）的变化,并观察了降钙素基因相关肽（ＣＧＲＰ）对这种变化的影响。结果发现,缺氧可使海马细胞［Ｃａ２＋］ｉ显著增高;４或８ｎｍｏｌ／ＬＣＧＲＰ能明显地降低缺氧引起的［Ｃａ２＋］ｉ增高,但在无胞外Ｃａ２＋的情况下,ＣＧＲＰ的作用消失。结果表明,ＣＧＲＰ的降钙作用是通过抑制缺氧时胞外Ｃａ２＋的内流来实现的。     

BCL2-CISD2

CISD2, an ER BCL2-associated autophagy regulator also known as NAF-1, is responsible for the human degenerative disorder Wolfram Syndrome 2. In order to interrogate the physiological role of CISD2 we generated and characterized the Cisd2 gene deletion in mice. Cisd2 null mice manifest significant degeneration in skeletal muscle tissues, which is accompanied with augmented autophagy, dysregulated Ca²⁺ homeostasis and elongated mitochondria. Our findings describe a novel role for BCL2-CISD2 in the homeostatic maintenance of skeletal muscle. It remains to be elucidated how and if the antagonism of the BECN1 autophagy-initiating complex and modulation of ER Ca²⁺ homeostasis by BCL2-CISD2 are interconnected.     

Synthesis of [11-2H2], [8-2H2], [7-2H2], [6-2H2], [5-2H2], [4-2H2] and [3-2H2] cis-9-octadecenoates

Deuterated oleates have been synthesized by semihydrogenation of acetylenic intermediates. [11-²H₂]Oleate was prepared by two-carbon chain extension of the C₁₆ alcohol obtained from [1-²H₂]octyl bromide and 7-octyn-1-ol. [8-²H₂] and [7-²H₂]oleates were both prepared from dimethyl suberate, tetradeutero intermediate C₁₆ alcohols were synthesized from [1,8-²H₄] and [2,7-²H₄]octane diols by monobromination, conversion to deuterated 9-decyn-1-ols and reaction with octyl bromide. Oxidation gave [8-²H₂]-9-octadecynoate and [2,7-²H₂]-9-octadecynoate, after semihydrogenation of the latter, deuterons at C-2 were removed by exchange with aqueous alkali. [6-²H₂] and [5-²H₂]oleates were obtained from methyl 5-tetradecynoate, semihydrogenation, deuterium exchange at C-2 and two malonate extensions gave [6-²H₂]oleate; reduction with lithium aluminum deuteride, two malonate extensions and semihydrogenation gave the [5-²H₂] ester. [4-²H₂] and [3-²H₂]oleates were both obtained from methyl 7-cis-hexadecenoate, exchange of the α protons and chain extension gave the [4-²H₂] ester and reduction with lithium aluminum deuteride and chain extension gave the [3-²H₂] ester.     

Synthesis and physicochemical properties of 2'-deoxy-2',2'-difluoro-beta-D-ribofuranosyl and 2'-deoxy-2',2'-difluoro-alpha-D-ribofuranosyl oligonucleotides

We present procedures for nucleoside and oligonucleotide synthesis, binding affinity (Tm) and structural analysis (CD spectra) of 2'-deoxy-2',2'-difluoro-alpha-D-ribofuranosyl and 2'-deoxy-2',2'-difluoro-beta-D-ribofuranosyl oligothymidylates. Possible reasons for the thermal instability of duplexes formed between these compounds and RNA or DNA targets are discussed.     

Stereospecific 2'-amination and 2'-chlorination of adenosine by Actinomadura in the biosynthesis of 2'-amino-2'-deoxyadenosine and 2'-chloro-2'-deoxycoformycin

2'-Amino-2'-deoxyadenosine and 2'-chloro-2'-deoxycoformycin (2'-CldCF) are two nucleoside antibiotics produced by Actinomadura. The biosynthesis of these two nucleoside antibiotics has been studied by the addition of [U-14C]adenosine with or without unlabeled adenine to cultures of Actinomadura. By this experimental approach, it is possible to demonstrate that adenosine is the direct precursor for the biosynthesis of 2'-amino-2'-deoxyadenosine and 2'-CldCF. These conclusions are based on the observation that the percentage distribution of 14C in the aglyconic and pentofuranosyl moieties of 2'-amino-2'-deoxyadenosine and 2'-CldCF were similar to the distribution of 14C in the adenine and ribosyl moieties of the [U-14C]adenosine (i.e., 48:52) added to cultures of Actinomadura. Experimentally, the percentage distribution of 14C in the (i) adenine:2-amino-2-deoxy-beta-D-ribofuranose of 2'-amino-2'-deoxyadenosine is 51:49; (ii) 8-(R)-3,6,7,8-tetrahydroimidazo[4,5-d]-[1,3-diazepin-8-o1]:2 -chloro-2- beta-D-ribofuranose of 2'-CldCF is 45:55; and (iii) adenine:ribose of the adenosine isolated from the RNA of Actinomadura is 42:58. Further proof that adenosine is the direct precursor for the biosynthesis 2'-amino-2'-deoxyadenosine and 2'-CldCF was demonstrated by the addition of 75 mumol of unlabeled adenine together with [U-14C]adenosine to nucleoside-producing cultures of Actinomadura. The percentage distribution of 14C in the aglycon and the sugar moieties of 2'-amino-2'-deoxyadenosine and 2'-CldCF were 46:54 and 47:53, respectively; the percentage distribution of 14C in the adenine and ribose moieties of the adenosine isolated from the RNA of Actinomadura was 51:49. These data show that the hydroxyl on C-2' of the ribosyl moiety of adenosine undergoes a replacement by a 2'-amino or a 2'-chloro group to form 2'-amino-2'-deoxyadenosine or 2'-CldCF with retention of stereconfiguration at C-2'. Finally, Actinomadura can utilize inorganic chloride from the medium as demonstrated by the isolation of [36Cl]2'-CldCF following the addition of [36Cl]chloride to the culture medium. Mechanisms for the regioselective modification of the C-2' hydroxyl group and stereospecific insertion of the amino and chloro groups are discussed.     

A concise synthesis of 4-nitrophenyl 2-azido-2-deoxy- and 2-acetamido-2-deoxy-D-mannopyranosides

4-nitrophenyl 3,4,6-tri-O-acetyl-2-azido-2-deoxy-alpha- and beta-D-mannopyranosides were prepared from methyl 4,6-O-benzylidene-alpha-D-glucopyranoside and 1,3,4,6-tetra-O-acetyl-alpha-D-glucopyranose, respectively. Chemoselective reduction of both azides with hydrogen sulfide readily afforded 4-nitrophenyl 2-acetamido-4,6-di-O-acetyl-2-deoxy-alpha-D- and -beta-D-mannopyranosides in higher yields than reduction with triphenylphosphine or a polymer-supported triarylphosphine. Subsequent de-O-acetylation yielded 4-nitrophenyl 2-acetamido-2-deoxy-alpha-D-mannopyranoside and 4-nitrophenyl 2-acetamido-2-deoxy-beta-D-mannopyranoside in 20% and 44% overall yields, respectively.     

Stereospecific formation of alpha-hydroxycarboxylato oxo peroxo complexes of vanadium(V). Crystal structure of (NBu4)2[V2O2(O2)2(L-lact)2] x 2H2O and (NBu4)2[V2O2(O2)2(D-lact)(L-lact)] x 2H2O

An overview of structurally characterized alpha-hydroxycarboxylatodioxo- and alpha-hydroxycarboxylatooxoperoxovanadates(V) is presented and the geometric parameters of the V2O2 bridging core are discussed. The first case of a stereospecific formation of oxoperoxovanadates(V) is reported: The crystal structures of the isomeric compounds (NBu4)2[V2O2(O2)2(L-lact)2] x 2H2O and (NBu4)2[V2O2(O2)2(D-lact)(L-lact)] x 2H2O (lact = C3H4O3(2-), the anion of the lactic acid) differ mainly in the arrangement of the V2O2 core and in mutual orientation of the V=O bonds. The complexes with achiral ligands adopt the same structural type as the complexes formed from a racemic mixture of a chiral ligand, while the structure obtained using an enantiopure L,L-hydroxycarboxylate is different.