Ⅰ型人免疫缺陷病毒（HIV—1）逆转录酶在大杆菌中的表达及其初步纯化

Ⅰ型人免疫缺陷病毒逆转录酶在抗感染及ＡＩＤＳ治疗药物的设计中是一个重要的靶分子,并且可作为工具酶应用于逆转录ＰＣＲ等分子生物学研究中。本研究将ＨＩＶ－１ＲＴ基因经ＰＣＲ扩增并修饰后克隆入大杆菌表达载体ｐＢＶ２２０,所获重组子所表达的ＨＩＶ－１ＲＴ蛋白占菌体总蛋白的８％左右,且经〔＾３Ｈ〕ｄＴＴＰ掺入法证实该重组ＨＩＶ－１ＲＴ具有ＲＴ聚合酶活性。用Ｑ－Ｓｅｐｈａｒｏｓｅ层析柱对重组ＨＩＶ－１ＲＴ蛋白     

斑点杂交检测肾综合征出血热病毒核酸的研究

本研究用非放射性标记物—地高辛碱性磷酸酶标记肾综合征出血热病毒（ＨＦＲＳＶ）ｃＤＮＡ制备探针,检测恙螨、鼠肺中ＨＦＲＳＶＲＮＡ的ＲＴＰＣＲ扩增产物中目的片段。结果表明：ＨＦＲＳＶ抗原阳性鼠体螨５０只组、１０只组,游离螨５０只组、１０只组,ＨＦＲＳＶ抗原阳性鼠肺１０００ｍｇ、５００ｍｇ,抗原阴性鼠肺１０００ｍｇ检测结果为阳性,呈现明显紫褐色斑点,而ＲＴＰＣＲ检测时未能扩增出ＨＦＲＳＶ抗原阴性鼠肺１０００ｍｇ和游离螨１０只组中的目的条带。实验结果说明,斑点杂交检测方法比ＲＴＰＣＲ敏感。     

日本血吸虫(中国大陆株)谷胱甘肽转移酶编码区基因的体外扩增、克隆及在原核细胞的高效表达

为表达、获取日本血吸早谷胱甘肽转移酶（ＳｊＧＳＴ）基因工程重组蛋白,以日本血吸虫（中国大陆株）ｃＤＮＡ为模板,设计、合成特定寡核苷酸引物,ＲＴ－ＰＣＲ法扩增ＧＳＴ编码基因序列,将扩增产物连接ｐＧＥＭ－Ｔ克隆载体,再亚克隆到真、原核表达质粒ｐＢＫ－ＣＭＶ中,转染大肠杆菌ＸＬ１－ｂｌｕｅ,经ＩＰＴＧ诱导后用ＳＤＳ－ＰＡＧＥ分析表达效果。结果 ＲＴ－ＰＣＲ法特异性扩增出日本血吸虫ＧＳＴ编码区基因片段,其     

应用RT—PCR检测流产胎儿组织中猪繁殖与呼吸综合征病毒

根据猪繁殖与呼吸综合征病毒（ＰＲＲＳＶ）美洲型膜蛋白和核衣壳蛋白基因序列,设计了一对含有ＥｃｏＲＩ和ＢａｍＨＩ酶切位点的引物,用ＲＴ－ＰＣＲ对四个流产猪场的病料进行了检测,扩增出约９１８ｂｐ的基因片段。通过病毒分离、酶切鉴定和序列分析证实为ＰＲＲＳＶ感染。结果说明应用所设计的引物进行ＲＴ－ＰＣＲ快速检测ＰＲＲＳ是可行的,为我国快速特异诊为ＰＲＲＳ和ＰＲＲＳＶ强毒株的深入研究奠定了基础。     

应用循环逆转录PCR技术检测丙型肝炎病毒RNA

循环逆转录（ｃｉｒｃｕｌａｔｏｒｙ ｒｅｖｅｒｓｅ ｔｒａｎｓｃｒｉｐｔｉｏｎ,ＣＲＴ）是线性增长逆转录ｃＤＮＡ产量的一种新技术。为了将该技术用于检测ＨＣＶ ＲＮＡ,通过改变ＣＲＴ的循环次数,结合竞争ＰＣＲ,作出标准曲线。采用１６次ＣＲＴ加３４次循环ＰＣＲ检测了１３６例ＨＣＶ ＥＬＩＳＡ阳性、５４例ＨＣＶ ＥＬＩＳＡ阴性和１０８例临床可疑病人全血标本,并与逆转录ＰＣＲ（ＲＴ－ＰＣＲ）和巢式ＰＣＲ（     

猪水泡病病毒强致病性毒株主要保护性抗原蛋白基因cDNA的序列测定 …

从猪水泡病病毒细胞培养物的ＰＥＧ浓缩毒中提取病毒ＲＮＡ,经ＲＴ－ＰＣＲ和套式ＰＣＲ扩增病毒主要保护性抗原蛋白基因,将扩增产生１．６ｋｂ插入ｐＵＣ１８载体中,经亚克隆后用双脱氧链终止法测定其序列,与已发表的ＳＶＤＶ分离物该区序列作比较。     

水稻草矮病毒血清学和分子检测方法的比较

将间接ＥＬＩＳＡ、非放射性分子杂交和ＲＴ－ＰＣＲ三种方法应用于水稻草矮病毒（ＲＧＳＶ）的检测。结果表明,利用自制的融合蛋白ＧＳＴ－ＮＣ的抗血清检测ＲＧＳＶ的灵敏度为１ｍｇ鲜重的病株叶片或８４ｎｇ提纯病毒,利用地高辛（ＤＩＧ）标记的ＤＮＡ探针ＮＣ的点杂交方法检测ＲＧＳＶ的灵敏度为５０μｇ病叶或６ｎｇ病毒,而ＲＴ－ＰＣＲ的检测灵敏度则为１０μｇ病叶或２ｎｇ的病毒,对上述三种方法的灵敏度和可操作性也进行     

直接逆转录原位PCR检测实验性汉坦病毒感染乳鼠脑组织中病毒RNA及其与原位分子杂交的比较

出生后２～３ｄ的昆明种乳鼠,经腹腔接种１００个半数致死量的陈株汉坦病毒,每只０．０５ｍｌ,于接种后１、２、４、６、８、１０、１２、１４ｄ处死动物,每个时间点３～６只不等,取其脑组织固定于４％的多聚甲醛中,石蜡包埋制备５μｍ的连续组织切片,每时间点取１～２例组织切片,用逆转录原位ＰＣＲ（ＲＴ－ＩＳＰＣＲ）方法检测组织中病毒Ｓ片段ＲＮＡ,组织脱蜡后,经ＤＮａｓｅ、蛋白酶Ｋ、消化等予处理,用汉坦病毒ＲＮＡＳ片段特异的一对引物,在组织切片上进行病毒ＲＮＡ的逆转录和ＰＣＲ过程,直接将ｄｉｇｏｘｉｇｅｎｉｎ－１１－ｄＵＴＰ掺入到扩增产物中,经过３０个ＰＣＲ循环后,用碱性磷酸酶标记的抗ｄｉｇｏｘｉｇｅｎｉｎ抗体免疫组化检测扩增产物,连续组织切片用ｄｉｇｏｘｉｇｅｎｉｎ标记的汉坦病毒Ｍ片段Ｇ２编码区ＲＴ－ＰＣＲ扩增产物的和Ｓ片段特异性探针进行原位分子杂交并与ＲＴ－ＩＳＰＣＲ结果进行比较,另外应用免疫组化检测该基因表达产物病毒核抗原（ＮＰ）,结果,ＲＴ－ＩＳＰＣＲ在病毒感染１ｄ的乳鼠脑组织中检测到病毒ＲＮＡ扩增产物,扩增产物定位于神经细胞胞浆内,而原位分子杂交和免疫组化检测阴性。在病毒感染２ｄ及２ｄ以后的乳鼠脑组织中ＲＴ－Ｉ     

湖北省不同地区汉坦病毒M片段的分析研究

参考汉坦病毒（ＨＶ）的基因文库软件设计Ｍ片段Ｇ－１区型特异性引物,以ＨＶ　７６／１１８、Ｈ－１１４、Ａ－９及Ｒ－｛２２｝株ＲＮＡ为阳性模板,建立ＨＶ的分型方法──逆转录一半巢式聚合酶链反应（ＲＴ－ｈｅｍｉｎｅｓｔｅｄ　ＰＣＲ）,对湖北省不同地区分离的３０株ＨＶ进行分　型。结果显示：（１）建立的ＲＴ－ｈｅｍｉｎｅｓｔｅｄ　ＰＣＲ分型方法对ＨＶ两型的代表株７６／１１８（ＨＴＮ）、Ａ－９（ＨＴＮ）、Ｈ－１１４（ＨＴＮ）及Ｒ－｛２２｝（ＳＥＯ）ＲＮＡ进行了特异性扩增,其大小与理论值一致;此　法只能检测ＨＶ　ＲＮＡ,不能检测其它病毒ＲＮＡ。（２）分离于湖北省不同地区的３０株ＨＶ的分型结果　为汉滩型２１株、汉城型９株,其中长江以南汉滩型１２株、汉城型２株;长江以北汉滩型９株、　汉城型７株。这表明建立的ＲＴ－ｈｅｍｉｎｅｓｔｅｄ　ＰＣＲ用于ＨＶ的检测特异性好;分析湖北省不同　地区分离的３０株ＨＶ,显示湖北省ＨＦＲＳ流行为混合疫区;且在湖北地区具有一定的地理聚集性。     

利用RT—PCR,DNA序列分析及原核基因表达对我国大豆花叶病毒进行分 …

大豆花叶病毒（ＳＭＶ）在大豆（Ｇｋｌｙｃｉｎｅ ｍａｘ Ｌ．）上引起严重病害。利用ＲＴ－ＰＣＲ扩增并克隆了ＳＭＶ－ＺＫ（一个中国ＳＭＶ分离株）基因组中全部蛋白质编码区的ｄＤＮＡ,通过对ＨＣ－ＰＲＯ,ＮＩｂ和ＣＰ编码区进行序列测定与分析,发现ＳＭＶ－ＺＫ与ＭＳＶ－Ｇ２高度同源,从而在分子水平上证明在我国大豆作物中存在ＳＭＶ－Ｇ２类似株系。     

Reconstitution of vacuolar-type rotary H+-ATPase/synthase from Thermus thermophilus

Vacuolar-type rotary H(+)-ATPase/synthase (V(o)V(1)) from Thermus thermophilus, composed of nine subunits, A, B, D, F, C, E, G, I, and L, has been reconstituted from individually isolated V(1) (A(3)B(3)D(1)F(1)) and V(o) (C(1)E(2)G(2)I(1)L(12)) subcomplexes in vitro. A(3)B(3)D and A(3)B(3) also reconstituted with V(o), resulting in a holoenzyme-like complexes. However, A(3)B(3)D-V(o) and A(3)B(3)-V(o) did not show ATP synthesis and dicyclohexylcarbodiimide-sensitive ATPase activity. The reconstitution process was monitored in real time by fluorescence resonance energy transfer (FRET) between an acceptor dye attached to subunit F or D in V(1) or A(3)B(3)D and a donor dye attached to subunit C in V(o). The estimated dissociation constants K(d) for V(o)V(1) and A(3)B(3)D-V(o) were ～0.3 and ～1 nm at 25 °C, respectively. These results suggest that the A(3)B(3) domain tightly associated with the two EG peripheral stalks of V(o), even in the absence of the central shaft subunits. In addition, F subunit is essential for coupling of ATP hydrolysis and proton translocation and has a key role in the stability of whole complex. However, the contribution of the F subunit to the association of A(3)B(3) with V(o) is much lower than that of the EG peripheral stalks.     

Photosynthetic pathway influences xylem structure and function in Flaveria (Asteraceae)

Higher water use efficiency (WUE) in C(4) plants may allow for greater xylem safety because transpiration rates are reduced. To evaluate this hypothesis, stem hydraulics and anatomy were compared in 16 C(3), C(3)-C(4) intermediate, C(4)-like and C(4) species in the genus Flaveria. The C(3) species had the highest leaf-specific conductivity (K(L)) compared with intermediate and C(4) species, with the perennial C(4) and C(4)-like species having the lowest K(L) values. Xylem-specific conductivity (K(S)) was generally highest in the C(3) species and lower in intermediate and C(4) species. Xylem vessels were shorter, narrower and more frequent in C(3)-C(4) intermediate, C(4)-like and C(4) species compared with C(3) species. WUE values were approximately double in the C(4)-like and C(4) species relative to the C(3)-C(4) and C(3) species. C(4)-like photosynthesis arose independently at least twice in Flaveria, and the trends in WUE and K(L) were consistent in both lineages. These correlated changes in WUE and K(L) indicate WUE increase promoted K(L) decline during C(4) evolution; however, any involvement of WUE comes late in the evolutionary sequence. C(3)-C(4) species exhibited reduced K(L) but little change in WUE compared to C(3) species, indicating that some reduction in hydraulic efficiency preceded increases in WUE.     

δ(13) C of leaf-respired CO(2) reflects intrinsic water-use efficiency in barley

Leaf intrinsic water-use efficiency (WUE), the ratio of photosynthetic rate to stomatal conductance (A/g(s) ), is a key plant trait linking terrestrial carbon and water cycles. A rapid, integrative proxy for A/g(s) is of benefit to crop breeding programmes aiming to improve WUE, but also for ecologists interested in plant carbon-water balance in natural systems. We hypothesize that the carbon isotope composition of leaf-respired CO(2) (δ(13) C(Rl) ), two hours after leaves are transferred to the dark, records photosynthetic carbon isotope discrimination and so provides a proxy for A/g(s) . To test this hypothesis, δ(13) C(Rl) was measured in four barley cultivars grown in the field at two levels of water availability and compared to leaf-level gas exchange (the ratio of leaf intercellular to ambient CO(2) partial pressure, C(i) /C(a) , and A/g(s) ). Leaf-respired CO(2) was more (13) C-depleted in plants grown at higher water availability, varied between days as environmental conditions changed, and was significantly different between cultivars. A strong relationship between δ(13) C(Rl) and δ(13) C of sucrose was observed. δ(13) C(Rl) was converted into apparent photosynthetic discrimination (Δ(13) C(Rl) ) revealing strong relationships between Δ(13) C(Rl) and C(i) /C(a) and A/g(s) during the vegetative stage of growth. We therefore conclude that δ(13) C(Rl) may provide a rapid, integrative proxy for A/g(s) in barley.     

3-Amidoquinuclidine derivatives: synthesis of compounds and inhibition of butyrylcholinesterase

The synthesis of racemic and enantiomerically pure 3-butanamidoquinuclidines ((+/-)-Bu, (R)-Bu and (S)-Bu), (1-3) and 3-benzamidoquinuclidines ((+/-)-Bz, (R)-Bz, and (S)-Bz), (4-6) is described. The N-quaternary derivatives, N-benzyl-3-butanamidoquinuclidinium bromides ((+/-)-BnlBu, (R)-BnlBu and (S)-BnlBu), (7-9) and N-benzyl-3-benzamidoquinuclidinium bromides ((+/-)-BnlBz, (R)-BnlBz and (S)-BnlBz), (10-12) were subsequently synthesized. The interaction of the four enantiomerically pure quaternary derivatives with horse serum butyrylcholinesterase (BChE) was tested. All tested compounds inhibited the enzyme. The best inhibitior of the enzyme was (S)-BnlBz with a K(i) = 3.7 microM. The inhibitor potency decreases in order (S)-BnlBz > (R)-BnlBz > (R)-BnlBu > (S)-BnlBu.     

Conformational analysis of a potent SSTR3-selective somatostatin analogue by NMR in water solution.

The three-dimensional structure of a potent SSTR3-selective analogue of somatostatin, cyclo(3-14)H-Cys(3)-Phe(6)-Tyr(7)-D-Agl(8)(N(beta) Me, 2-naphthoyl)-Lys(9)-Thr(10)-Phe(11)-Cys(14)-OH (des-AA(1, 2, 4, 5, 12, 13)[Tyr(7), D-Agl(8)(N(beta) Me, 2-naphthoyl)]-SRIF) (peptide 1) has been determined by (1)H NMR in water and molecular dynamics (MD) simulations. The peptide exists in two conformational isomers differing mainly by the cis/trans isomerization of the side chain in residue 8. The structure of 1 is compared with the consensus structural motifs of other somatostatin analogues that bind predominantly to SSTR1, SSTR2/SSTR5 and SSTR4 receptors, and to the 3D structure of a non-selective SRIF analogue, cyclo(3-14)H-Cys(3)-Phe(6)-Tyr(7)-D-2Nal(8)-Lys(9)-Thr(10)-Phe(11)-Cys(14)-OH (des-AA(1, 2, 4, 5, 12, 13)[Tyr(7), D-2Nal(8)]-SRIF) (peptide 2). The structural determinant factors that could explain selectivity of peptide 1 for SSTR3 receptors are discussed.     

Cyclopeptides from Sagina japonica (Caryophyllaceae)

Two new cyclic peptides, named sajaponicin C (1) and sajaponicin D (2), were isolated from the whole plants of Sagina japonica (Caryophyllaceae). Their structures were determined as cyclo(Pro(2)-Leu(2)-Tyr-Leu(1)-Phe(1)-Pro(3)-Phe(2)-Pro(1)) (1) and cyclo(Pro(1)-Pro(2)-Pro(3)-Pro(4)-Phe(1)-Gly-Thr-Ser-Phe(2)-Ile-Tyr) (2) on the basis of spectroscopic data, especially by two-dimensional (2D) NMR techniques.     

Technical note: interpreting stable carbon isotopes in human tooth enamel: an examination of tissue spacings from South Africa

Stable isotope analysis of skeletal tissues is widely used in archeology and paleoanthropology to reconstruct diet. In material that is poorly preserved or very old, the tissue of choice is frequently tooth enamel, since this is less susceptible to diagenesis. The relationships between carbon isotope ratios in tooth enamel (δ(13) C(enamel) ), bone collagen (δ(13) C(collagen) ), and bone apatite (δ(13) C(bone apatite) ) are, however, not well understood. To elucidate these, we have measured all three indicators in archeological humans from the western and southern Cape coastal regions of South Africa. The correlation between δ(13) C(enamel) and δ(13) C(collagen) is good (R(2) = 0.71 if two outliers are excluded, n = 79). The correlation between δ(13) C(enamel) and δ(13) C(bone apatite) is weaker (R(2) = 0.37, n = 33) possibly due to bone diagenesis. No systematic offset between δ(13) C(bone apatite) and δ(13) C(enamel) was observed in this sample of archeological humans. Intertooth comparisons of δ(13) C(enamel) in three individuals showed little variation, despite the different ages of crown formation. Carbon isotope ratios in both enamel and bone collagen are good proxies for δ(13) C(diet) .     

Brain neurotransmitter receptor-binding characteristics in rats after oral administration of haloperidol, risperidone and olanzapine

The present study was conducted to characterize the binding of neurotransmitter receptors (dopamine D(2), serotonin 5-HT(2), histamine H(1), adrenaline alpha(1) and muscarine M(l) receptors) in the rat's brain after the oral administration of haloperidol, risperidone, and olanzapine. Haloperidol at 1 and 3 mg/kg displayed significant activity to bind the D(2) receptor (increase in the Kd value for [(3)H]raclopride binding) in the corpus striatum with little change in the activity toward the 5-HT(2) receptor (binding parameters for [(3)H]ketanserin). In contrast, risperidone (0.1-3 mg/kg) showed roughly 30 times more affinity for the 5-HT(2) receptor than D(2) receptor. Also, olanzapine (1-10 mg/kg) was most active toward the H(1) receptor in the cerebral cortex, corpus striatum, and hippocampus, was less active in binding 5-HT(2) and D(2) receptors, and showed the least affinity for alpha(1) and M(1) receptors. In conclusion, haloperidol and risperidone administered orally selectively bind D(2) and 5-HT(2) receptors, respectively, in the rat brain, while olanzapine binds H(1), 5-HT(2), and D(2) receptors more than alpha(1) and M(1) receptors.     

Crystal structure and aqueous solubility of ammonium D-glucarate

Ammonium D-glucarate, NH(4)(C(6)H(9)O(8)) [ammonium D-saccharate, NH(4)-SAC], has been synthesized, and its crystal structure solved by single-crystal X-ray diffraction methods. NH(4)-SAC crystallizes in the monoclinic space group P2(1) (#4) with cell parameters a = 4.8350(4) Angstroms, b = 11.0477(8) Angstroms, c = 16.7268(12) Angstroms, beta = 90.973(1) degrees, V = 894.34(12) Angstroms(3), Z = 3. The structure was refined by full-matrix least-squares on F(2) yielding final R-values (all data) R1 = 0.0353 and R(w)2 = 0.0870. The structure consists of alternating (NH(4))(+) and (C(6)H(11)O(6))(-) layers parallel to the bc plane. An extended network of N-H...O(SAC) and O(SAC)-H...O(SAC) hydrogen bonds provide the 3-D connectivity. The aqueous solubility (S(w)) has been shown to be pH independent at ambient conditions within the range 4.5 < pH < 10 with S(w) = 2.19 M/L, whose value is about a factor of two lower than that of the ammonium isosaccharate analogue.     

Sulfide oxidation at halo-alkaline conditions in a fed-batch bioreactor

A biotechnological process is described to remove hydrogen sulfide (H(2)S) from high-pressure natural gas and sour gases produced in the petrochemical industry. The process operates at halo-alkaline conditions and combines an aerobic sulfide-oxidizing reactor with an anaerobic sulfate (SO(4) (2-)) and thiosulfate (S(2)O(3) (2-)) reducing reactor. The feasibility of biological H(2)S oxidation at pH around 10 and total sodium concentration of 2 mol L(-1) was studied in gas-lift bioreactors, using halo-alkaliphilic sulfur-oxidizing bacteria (HA-SOB). Reactor operation at different oxygen to sulfide (O(2):H(2)S) supply ratios resulted in a stable low redox potential that was directly related with the polysulfide (S(x) (2-)) and total sulfide concentration in the bioreactor. Selectivity for SO(4) (2-) formation decreased with increasing S(x) (2-) and total sulfide concentrations. At total sulfide concentrations above 0.25 mmol L(-1), selectivity for SO(4) (2-) formation approached zero and the end products of H(2)S oxidation were elemental sulfur (S(0)) and S(2)O(3) (2-). Maximum selectivity for S(0) formation (83.3+/-0.7%) during stable reactor operation was obtained at a molar O(2):H(2)S supply ratio of 0.65. Under these conditions, intermediary S(x) (2-) plays a major role in the process. Instead of dissolved sulfide (HS(-)), S(x) (2-) seemed to be the most important electron donor for HA-SOB under S(0) producing conditions. In addition, abiotic oxidation of S(x) (2-) was the main cause of undesirable formation of S(2)O(3) (2-). The observed biomass growth yield under SO(4) (2-) producing conditions was 0.86 g N mol(-1) H(2)S. When selectivity for SO(4) (2-) formation was below 5%, almost no biomass growth was observed.