Mg~(2+)和SeO_3~(2-)对鸡胚软骨细胞受模拟微重力不良影响的拮抗

在回转模拟微重力条件下 ,研究了鸡胚负重软骨细胞骨架的微管系统和碱性磷酸酶活性两项指标的变化 ,以及 1mg/L亚硒酸钠和 5mmol/LMg2 + 对这些指标的影响 .流式细胞仪对微管含量的测定显示回转后微管蛋白含量的减少 ,说明微管系统受到不良影响 .碱性磷酸酶活性比对照组明显降低 ,表明模拟微重力能降低软骨细胞的钙化能力 .如果在回转前加入SeO2 -3 和Mg2 + ,发现SeO2 -3 可以在一定程度上拮抗模拟微重力引起的微管蛋白及碱性磷酸酶活性改变 ,而Mg2 + 基本上可以完全拮抗模拟微重力对这两项指标的不良影响     

Mg2+和SeO2-3对鸡胚软骨细胞受模拟微重力不良影响的拮抗

在回转模拟微重力条件下,研究了鸡胚负重软骨细胞骨架的微管系统和碱性磷酸酶活性两项指标的变化,以及1 mg/L亚硒酸钠和5 mmol/L Mg²⁺对这些指标的影响.流式细胞仪对微管含量的测定显示回转后微管蛋白含量的减少,说明微管系统受到不良影响.碱性磷酸酶活性比对照组明显降低,表明模拟微重力能降低软骨细胞的钙化能力.如果在回转前加入SeO^2-₃和Mg²⁺,发现SeO^2-₃可以在一定程度上拮抗模拟微重力引起的微管蛋白及碱性磷酸酶活性改变,而Mg²⁺基本上可以完全拮抗模拟微重力对这两项指标的不良影响.     

野黄芩苷对内毒素抑制人牙周膜细胞碱性磷酸酶活性的影响

目的观察野黄芩苷对内毒素(LPS)抑制人牙周膜细胞的碱性磷酸酶活性的影响。方法原代培养人牙周膜细胞,采用酶动力学方法观察野黄芩苷对LPS抑制人牙周-膜细胞碱性磷酸酶活性的影响。结果100μg/mL LPS可显著抑制体外培养的人牙周膜细胞碱性磷酸酶活性。加入0.001-10μg/ml野黄芩苷干预后,对LPS抑制碱性磷酸酶活性有一定的拮抗作用,在1μg/ml时达到高峰。结果 野黄芩苷可能通过拮抗LPS抑制牙周膜细胞碱性磷酸酶的活性,促使牙周膜细胞向成骨细胞分化而利于牙周组织再生修复。     

丹皮酚对过氧亚硝基阴离子致大鼠成骨细胞分化抑制的影响

探讨丹皮酚（Pae）拮抗过氧亚硝基阴离子（ONOO^-）对体外培养大鼠成骨细胞分化的影响。用改良的组织块法分离培养新生大鼠颅骨成骨细胞,采用淬灭流动反应方法体外制备ONOO^-,以不同终浓度加入成骨细胞培养体系,在作用不同时间后,用对硝基苯二钠动力学（PNPP）法检测细胞内碱性磷酸酶（ALP）的活性,用Lowry法测定蛋白含量,并以不同终浓度Pae消除ONOO^-（1000μmoL/L）对成骨细胞分化的影响。结果显示,不同浓度的ONOO^-（50-1000μmoL/L）均能抑制碱性磷酸酶的活性,影响分化;高浓度的丹皮酚（10^-3-10^-6mol/L）能消除ONOO^-（1000μmoL/L）对碱性磷酸酶活性的抑制,拮抗ONOO^-抑制成骨细胞分化的作用。     

肌球蛋白轻链激酶CaM结合位点突变体对肌球蛋白ATP酶活性的影响

目的:探讨肌球蛋白轻链激酶(MLCK)钙调蛋白(CaM)结合位点突变体对肌球蛋白ATP酶活性的影响.方法:构建牛胃重组全长野生型MLCK CaM结合位点突变型蛋白(△CaM/MLCK);孔雀绿方法检测△CaM/MLCK对肌球蛋白的Mg2+-ATP酶活性的影响.结果:在无Ca2+/CaM存在时,随着△△CaM/MLCK浓度的增加,非磷酸化肌球蛋白的Mg2+-ATP酶活性明显增加;而磷酸化肌球蛋白的Mg2+-ATP酶活性明显降低.结论:△CaM/MLCK对肌球蛋白Mg2+-ATP酶活性的影响表明MLCK具有非激酶活性.     

模拟微重力效应通过微丝骨架调控成骨细胞BMP2-Smad信号

细胞微丝骨架在力信号传导和基因表达调控中起重要作用。为了研究微丝骨架在模拟微重力效应调控成骨细胞BMP2-Smad信号中的作用,作者通过构建反映Smad活性的报告基因载体转染MC3T3-E1细胞,并通过报告基因活性分析、Western blot等方法检测了微丝骨架解聚剂和回转模拟微重力效应对BMP2诱导Samd磷酸化、核质分布和转录活性的作用。结果显示,构建的报告基因载体在成骨细胞中正确表达并响应BMP2;破坏微丝骨架会抑制BMP2诱导的Smad1/5/8蛋白磷酸化、入核及转录活性;回转抑制Smad1/5/8磷酸化、入核及其转录活性,而微丝骨架稳定剂可对抗回转的抑制作用。因此,认为回转模拟微重力效应可通过解聚微丝骨架抑制BMP2-Smad信号传导。     

金属离子对牛小肠碱性磷酸酶的影响

经Tris-HCl缓冲液抽提,硫酸铵分级分离沉淀,2次DEAE-32柱层析和Sephadex G-150凝胶过滤,从牛小肠中得到碱性磷酸酶(ALP),提纯倍数为50.69倍,比活为48.87 U/mg,酶液经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)检测呈现单一条带.该酶催化底物对硝基苯磷酸二钠(pNPP)水解反应的最适pH值为9.7,最适温度为45℃.研究了多种金属离子对牛小肠碱性磷酸酶活性的影响,结果表明:一价金属离子Na+、K+对酶活力没有影响;不同的二价金属离子对酶的影响不同,过渡态金属离子中,Ni2+、Co2+对酶的活力影响不大,Mg2+、Ca2+、 Mn2+对酶有不同程度的激活作用,Zn2+、Cu2+对酶有抑制作用;重金属离子Pb2+、Cd2+对酶的活力起抑制作用.     

在不同营养条件下铜绿微囊藻对模拟微重力胁迫的响应

通过测定在不同重力水平和营养条件下培养的铜绿微囊藻(Microcystis aeruginosa)的各项生理生化指标,研究了培养基的营养物质浓度对微囊藻细胞响应模拟微重力胁迫的影响。结果表明,在正常浓度的BG-11(富营养)和营养盐浓度减为1/10的BG-11(贫营养)培养基中培养的微囊藻对模拟微重力胁迫都很敏感,培养2d后多项生理生化指标显著改变;但是在富营养和贫营养条件下,模拟微重力的作用效果是截然不同的。对培养在BG-11中的微囊藻细胞来说,模拟微重力抑制其生长和光合活性,导致细胞内色素(叶绿素a和类胡萝卜素)、蛋白(藻蓝蛋白和可溶性蛋白)和毒素含量显著升高,向外分泌的毒素含量降低;而对培养在1/10BG-11中的藻细胞来说,模拟微重力促进其生长和光合活性,导致细胞内色素、蛋白和毒素含量降低,并使得毒素分泌增强。模拟微重力或营养限制单独作用所造成的影响相似,且后者的作用效果强于前者。当二者同时存在时,模拟微重力可以部分抵消营养限制对微囊藻生长和代谢的影响,这可能是由于模拟微重力下藻细胞的生长受到抑制而导致营养需求降低,也可能是由于模拟微重力提高了藻细胞利用营养物质的效率。总之,微囊藻对模拟微重力胁迫的响应与培养基的营养条件有关。     

玉米果糖-6-磷酸,2-激酶/果糖-2,6-二磷酸酶基因的结构与表达分析

通过RT-PCR,结合RACE技术,得到了玉米(Zea mays L.)果糖-6-磷酸,2-激酶/果糖-2,6-二磷酸酶的全长cDNA克隆,命名为mF2KP.氨基酸序列同源性比较发现,mF2KP蛋白可以分为两个部分:C端包含高度保守的催化功能区,N端为植物中特有的多肽.将mF2KP基因中一段包含完整催化功能区的片段在大肠杆菌(Escherichia coli)中表达,融合蛋白具有果糖-6-磷酸,2-激酶/果糖-2,6-二磷酸酶活性.Northern杂交证明在种子活力不同的幼苗中,mF2KP的转录水平存在明显差异.种子活力越高,幼苗中mF2KP的转录水平越低.     

钙、镁离子在活化HL-60细胞核酸内切酶中的作用不同

EGTA ,EDTA抑制游离HL- 6 0细胞核中核酸内切酶的活化 .EDTA对游离HL -6 0细胞核中核酸内切酶活性的抑制可被外加Ca ²⁺逆转 ,而EDTA对该酶活性的抑制却不能被外加Mg ²⁺逆转 .用CHELEX 1 0 0去除游离核孵育缓冲液中存在的Ca2 ²⁺,Mg ²⁺后 ,外界Ca2 ²⁺( 1～ 1 0mmol/L)单独可诱导游离HL -6 0细胞核中的核酸内切酶活化 ,且强度一致 ;而外加Mg ²⁺则不能诱导该酶活性 .只有在钙存在( 0 1～ 1 0mmol/L)的条件下 ,该酶活性才随Mg ²⁺浓度增高而增高 .这说明HL- 6 0细胞中核酸内切酶的活化必须有Ca ²⁺存在 ,在Ca ²⁺存在的条件下 ,其活性可被镁增强 ,Ca ²⁺和Mg ²⁺在核酸内切酶活化中的作用是不同的 .     

Synthesis and properties of 2'-O-methyl-2-thiouridine and oligoribonucleotides containing 2'-O-methyl-2-thiouridine

A new method for the synthesis of 2'-O-methyl-2-thiouridine (s2Um) found in thermophilic bacterial tRNA was developed. Structural properties of s2Um and s2Um(p)U were studied by using 1H NMR spectroscopy. A modified nonaribonucleotide (RNA*: 5'-CGUUs2UmUUGC-3') was synthesized to study the base-recognition ability of s2Um in formation of RNA-RNA and RNA DNA duplexes. The UV melting experiments revealed that RNA*-RNA and RNA*-DNA duplexes having an s2U-A base pair are more stable than those having a U-A base pair. On the contrary, the thermal stability of RNA*-RNA and RNA*-DNA duplexes having an s2U-G wobble base pair was much lower than that of the unmodified duplexes having a natural U-G base pair. It is concluded that s2Um has higher selectivity toward A over G than unmodified U.     

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.     

Stereoselective Synthesis of 2-Amino-2-deoxy-d-arabinose and 2-Deoxy-d-ribose

An efficient method for the stereoselective synthesis of 2-amino-2-deoxy-d-arabinose and 2-deoxy-d-ribose is described.

The key step in this method was accomplished by the nucleophilic addition of methyl isocyanoacetate to 2,3-O-isopropylidene-d-glyceraldehyde with high erythro-selectivity (nearly 100%).

Subsequent intermolecular cyclization predominantly gave the desired oxazoline derivative (trans-form), in which two new chiral centers were formed. The oxazoline derivative was efficiently converted to both 2-amino-2-deoxy-d-arabinose and 2-deoxy-d-ribose.     

Characterization of alpha 2 beta 2 and alpha 2 forms of kinesin

Bovine brain kinesin separates into two components on sucrose density gradient centrifugation. The predominant component is a heterotetramer of two 120 kDa alpha subunits and two 64 kDa beta subunits with an sedimentation coefficient of 9.6 S and a low Vm rate of microtubule-stimulated ATPase of 1.3 +/- 0.5 sec-1 at 25 degrees, pH 7.0. The minor element is a homodimer of two alpha subunits without beta subunits with a sedimentation coefficient of 6.9 S and a higher Vm rate of microtubule-stimulated ATPase of 7.0 +/- 1.9 sec-1. Microtubules stimulate the rate of release of ADP from the active site of the tetramer, but the rate of release is not fast enough to account for the rate of steady state ATP hydrolysis. Further complexity is indicated by biphasic release kinetics. In spite of the large difference in Vm ATPase rate for the two species, both drive the sliding of sea urchin axonemes over glass surfaces at the same velocity.     

Complexes of arabinogalactan of Pereskia aculeata and Co2+, Cu2+, Mn2+, and Ni2+

The main interest in the biopolymer arabinogalactan is that it is edible. Complementing its high protein percentage, when complexed to essential metal ions, widens the use in food and pharmacology industries and technologies. The binding constants of Co2+, Cu2+, Mn2+ and Ni2+ with arabinogalactan, extracted from the leaves of Pereskia aculeata from Brazil were determined by potentiometric titrations and also the speciation according to pH values. The complexed species proposed by potentiometric titrations and the unique complexing ability of galacturonic acid groups towards Cu2+ and Ni2+ in the tridimensional web structure of arabinogalactan were confirmed by IR and EPR spectroscopies. The thermal stability of the complexed species also varied with the metal ion employed in the complexation when compared to the biopolymer alone. These complexes are new sources of additives for the food and pharmacology industries and carriers of essential metal ions to animal and vegetal biochemistry.