溶剂极性及盐酸胍对碳酸酐酶构象的影响

以紫外荧光光谱为检测手段,比较研究了溶剂极性及盐酸胍对碳酸酐酶构象的影响,结果表明,当盐酸胍浓度小于１ｍｏｌ／Ｌ时酶呈天然构象,１．５ｍｏｌ／Ｌ－２．０ｍｏｌ／Ｌ的盐酸胍导致该酶呈伸展构象;而乙醇不以导致该酶产生稳定的中间态构象,１ｍｏｌ／Ｌ和３ｍｏｌ／Ｌ的甲醇以及３ｍｏｌ／Ｌ的乙醇对盐酸胍致碳酸酐酶中间态的影响不大,甲醇略使致中间态的盐酸胍浓度下降,乙醇略使之上升。     

Thiophosphorylation of (Na + K+)-ATPase yields an ADP-sensitive phosphointermediate

(1) Treatment of $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ from rabbit kidney outer medulla with the $\text{γ-}{}^{35}\text{S}$ labeled thio-analogue of ATP in the presence of $\text{Na}{}^{\mathrm{+}}\text{+ Mg}{}^{\mathrm{2+}}$ and the absence of K⁺ leads to thiophosphorylation of the enzyme. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value for [γ-S]ATP is 2.2 μM and for Na⁺ 4.2 mM at 22°C. Thiophosphorylation is a sigmoidal function of the Na⁺ concentration, yielding a Hill coefficient $\text{n}\text{H}\text{= 2.6}$. (2) The thio-analogue ($\text{K}{}_{\mathrm{<mtext>m</mtext><mtext>\; =\; 35\; \mu M</mtext>}}$) can also support overall $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ activity, but $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ at 37°C is only $\text{1.3 γ}\text{mol}\text{· (mg protein)}{}^{\mathrm{?}}\text{·}$ h^?1 or 0.09% of the specific activity for ATP ($\text{K}{}_{\mathrm{<mtext>m</mtext><mtext>\; =\; 0.43\; mM</mtext>}}$). (3) The thiophosphoenzyme intermediate, like the natural phosphoenzyme, is sensitive to hydroxylamine, indicating that it also is an acylphosphate. However, the thiophosphoenzyme, unlike the phosphoenzyme, is acid labile at temperatures as low as 0°C. The acid-denatured thiophosphoenzyme has optimal stability at pH 5–6. (4) The thiophosphorylation capacity of the enzyme is equal to its phosphorylation capacity, indicating the same number of sites. Phosphorylation by ATP excludes thiophosphorylation, suggesting that the two substrates compete for the same phosphorylation site. (5) The (apparent) rate constants of thiophosphorylation (0.4 s^?1 vs. 180 s^?1), spontaneous dethiophosphorylation (0.04 s^?1 vs. 0.5 s^?1) and K⁺-stimulated dethiophosphorylation (0.54 s^?1 vs. 230 s^?1) are much lower than those for the corresponding reactions based on ATP. (6) In contrast to the phosphoenzyme, the thiophosphoenzyme is ADP-sensitive (with an apparent rate constant in ADP-induced dethiophosphorylation of 0.35 s^?1, $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$$\text{ADP = 48 μM at 0.1 mM ATP}$) and is relatively K⁺-insensitve. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for K⁺ in dethiophosphorylation is 0.9 mM and in dephosphorylation 0.09 mM. The thiophosphoenzyme appears to be for 75–90% in the ADP-sensitive E₁-conformation.     

Stereoselective metabolism of 7-methylbenz[a]anthracene: absolute configuration of five dihydrodiol metabolites and the effect of dihydrodiol conformation on circular dichroism spectra

7-Methylbenz[a]anthracene (7-MBA) was metabolized stereoselectively by rat liver microsomes to form five optically active dihydrodiols as the predominant metabolites. The dihydrodiols were purified by a combination of reversed-phase and normal-phase high performance liquid chromatography (HPLC). By comparison of their circular dichroism (CD) spectra with the corresponding benz[a]anthracene (BA) dihydrodiols of known absolute stereochemistry, the major dihydrodiol enantiomers of 7-MBA have been determined to have 1R,2R-, 3R,4R- and 10R , 11R - absolute configurations, respectively. Due to their quasi- diaxial conformations, the absolute configuration of trans-5,6- and trans-8,9-dihydrodiols, the two most abundant metabolites of 7-MBA, could not be determined by simple comparisons of their circular dichroism spectra with those of the quasidi -equatorial BA 5R, 6R - and 8R , 9R -dihydrodiols. The major enantiomers of the quasi- diaxial trans-5,6- and trans-8,9-dihydrodiol metabolites of 7-MBA were determined by comparison to the CD spectrum of 7-bromo-BA 5R, 6R -dihydrodiol and by the exciton chirality method to have R,R absolute stereochemistry. This study also revealed that the circular dichroism Cotton effects of an enantiomeric dihydrodiol of polycyclic aromatic hydrocarbons can be drastically altered if the conformation (quasi- diaxial vs. quasi di-equatorial ) of the dihydrodiol is changed.     

Marijuana exposure in vivo and human lymphocyte chromosomes.

Sequential chromosome examinations of peripheral lymphocte cultures were carried out on 21 adult male volunteers who smoked natural blend marijuana cigarettes containing about 1%, 2%, or no delta9-THC. For a limited number of subjects, blood samples from a single venipuncture were cultured independently in two cytogenetic laboratories, and later the slides were exchaged for re-analysis. There were significant differences between laboratories in the absolute break frequencies recorded. These inter-laboratory differences were demonstrated for both techniques of cell culture and metaphase analysis. Neither laboratory found a statistically significant increase in break frequencies asssociated with marijuana smoking. The present study, therefore, failed to detect a measurable effect of marijuana smoking on chromosomal aberrations in subjects experienced in the use of the drug.     

Application of CNDO2 theoretical calculations to interpretation of the chemical reactivity and biological activity of the syn and anti diolepoxides of benzo[a]pyrene

$\text{CNDO}\text{2}$ molecular orbital theoretical calculations performed on the anti and syn diolepoxides (1 and 2) of the potent carcinogen benzo[a]pyrene provide insight into the molecular structure and reactivity of these mutagenic and carcinogenic hydrocarbon metabolites. Hydrogen-bonded interaction between the 7-HO proton and the epoxide oxygen atom of 2 is shown to be absent in the normal semichair conformation of the tetrahydro ring, (H…O bond distance = 2.7 Å), but is energetically favored in a somewhat distorted puckered structure (H…O bond distance = 1.7 Å). Unexpectedly, internal H-bonding alters the relative electron density at C₉ and C₁₀, leading to prediction of the former as the more electrophilic center. Since all reactions of 2 take place exclusively at C₁₀, transannular H-bonding is concluded not to contribute significantly to the structure of 2. Diolepoxide reactions with both weak and strong nucleophiles and with DNA are discussed and the mechanisms interpreted in terms of molecular structure as determined by the theoretical calculations.     

Release of heat pretreatment-induced dormancy in lettuce seeds by ethylene or cytokinin in relation to the production of ethylene and the synthesis of 1-aminocyclopropane-1-carboxylic acid during germination

The germination of lettuce (Lactuca sativa L.) seeds was greatly reduced when the seeds were heated at 97°C for 30 h prior to imbibition. This dormancy was effectively released when ethylene (1–100 ppm) or benzyladenine (BA) (0.005–0.05 mM) was applied during the imbibition period. Ethylene was not required during the early part of imbibition, but was essential during the period immediately prior to radicle protrusion. Treatment with 1-aminocyclopropane-1-carboxylic acid (ACC) (0.1–10 mM) stimulated germination, but was not as effective as ethylene or cytokinin treatment. During the germination of nondormant lettuce seeds, ethylene production increased rapidly and reached a peak at 24 h, which coincided with the emergence of the radicle, and then declined; the level of ACC increased as ethylene production rate increased, but remained at a high level after radicle protrusion. In heat-pretreated dormant lettuce seeds, the increases in percent germination, ethylene production, and ACC levels were all delayed and lower than those of nondormant seeds, and these increases were accelerated by treatment with ethylene or cytokinin.     

Effect of a peri fluoro substituent on the conformation of dihydrodiol derivatives of polycyclic aromatic hydrocarbons

$\text{Trans}$-3,4-, 5,6-, 8,9-, and 10,11-dihydrodiols formed from the metabolism of 7-fluorobenz[a]anthracene by rat liver microsomes were isolated by reversed-phase high performance liquid chromatography. Ultraviolet absorption, mass, and NMR spectral analyses indicated that the 5,6- and 8,9-dihydrodiols were preferentially in quasi-diaxial conformations, whereas the 3,4- and 10,11-dihydrodiols were preferentially in quasi-diequatorial conformations. CPK space-filling models suggest that the quasi-diaxial conformation is primarily the result of electronic repulsion between the fluorine and the $\text{peri}$ hydroxyl oxygen. These findings provide a structural basis in the interpretation of the carcinogenic potencies of some fluorinated polycyclic aromatic hydrocarbons.     

龙牙百合3个地方品种的形态特征及核型分析

采用经典测量和染色体常规压片法,对龙牙百合（Lilium brownii var.viridulum Baker）3个地方品种的形态特征及核型进行研究。植株形态分析结果显示：‘江西’龙牙的株高、开花口径、种球重量和周长、中外层鳞片重量和长度以及鳞片扦插产生小鳞茎数等指标均显著大于‘大叶’龙牙和‘平头’龙牙;‘大叶’龙牙的叶片最长,均值为14.54 cm。花粉、叶表皮气孔及鳞片淀粉粒的微形态特征分析结果显示：‘江西’龙牙的花粉粒径最大,均值达111.76 μm;‘平头’龙牙的叶表皮气孔最长,气孔密度也最大（约47.6个/mm²）;‘大叶’龙牙的淀粉粒径最大,均值为47.61 μm;‘江西’龙牙的淀粉粒大小分布更集中,差异性小。染色体核型分析结果显示：龙牙百合3个品种的染色体数目均为2n=2x=24,为二倍体,其中‘江西’龙牙核型公式为2n=2x=24=2m（2SAT）+6sm（2SAT）+12st（4SAT）+4t;‘平头’龙牙核型公式为2n=2x=24=4m+8sm+10st（4SAT）+2t;‘大叶’龙牙核型公式为2n=2x=24=2m（2SAT）+6sm+14st（4SAT）+2t,三者核型均为3B型。     

发根农杆菌介导的菠菜毛状根遗传转化体系的建立

发根农杆菌(Agrobacterium rhizogenes)侵染植物后可诱导植物产生毛状根。菠菜(Spinacia oleracea)是常见的食用蔬菜, 目前尚未见菠菜毛状根的研究报道。经筛选得到适合诱导菠菜毛状根的发根农杆菌菌株LBA9402, LBA9402侵染菠菜外植体茎后, 毛状根的诱导率最高可达16%。菠菜毛状根呈白色, 具有丰富的根毛, 能在无外源激素的固体培养基上快速增殖生长。通过诱导菠菜毛状根产生愈伤组织并进行分化, 获得了菠菜毛状根的再生植株, 再生率为8%。此外, LBA9402可将含有Ri质粒的T-DNA和携带外源GFP基因的Ti质粒T-DNA共同导入外植体中。PCR检测和荧光显微观察结果显示, rolB及GFP基因在菠菜毛状根基因组中稳定表达, 共转化频率为50%。