The measurement of chemically-induced DNA repair synthesis in human cells by BND-cellulose chromatography.

Repair synthesis in human cells in tissue culture can be readily separated from semi-conservative DNA synthesis with the aid of a benzoylated naphthoylated DEAE cellulose (BND-cellulose) column. Cells are incubated with a radioactive DNA precursor during treatment with a repair-inducing agent. An inhibitor of semi-conservative DNA synthesis (hydroxyurea) is added to slow the progression of the DNA growing point. The cells are lysed and after treatment with ribonuclease and pronase the lysates are sheared and passed through a BND-cellulose column. Native DNA is eluted with I M NaCl. Any increase in radioactivity in the native DNA is due to repair synthesis and the specific repair activity (nucleotides inserted per mug of DNA) can be determined from radioactivity and absorbancy measurements. Repair can also be measured in the region of the DNA growing point by fractionation of the material eluted from BND-cellulose with 50% formamide. Repair was not detected in N-acetoxy-2-acetylaminofluorene (AAAF)-treated lymphoblasts derived from an individual with xeroderma pigmentosum although methyl methanesulfonate (MMS)-induced repair was observed in these cells.     

Incorporation of methionine-[methyl-2H3] and mevalonic acid-[2-14C,(4R)-4-3H1] into Phycomyces blakesleeanus sterols

Ergosterol, episterol, 4α-methyl-5α-ergosta-8,24(28)-dien-3β-ol and 24-methylene-24,25-dihydrolanosterol, isolated from Phycomyces blakesleeanus grown in the presence of methionine-[methyl-²H₃], each contained two deuterium atoms; lanosterol, however, was unlabelled. The ¹⁴C:³H atomic ratio of the following sterols isolated from P. blakesleeanus grown in the presence of mevalonic acid-[2-¹⁴C,(4R)-4-³H₁], was: ergosterol, 5:3; episterol, 5:4; ergosta-5,7,24(28)-trien-3β-ol, 5:3; 4α-methyl-5α-ergosta-8,24(28)-dien-3β-ol, 5:4; 24-methylene-24,25-dihydrolanosterol, 6:5; lanosterol, 6:5. The significance of these results in terms of ergosterol biosynthesis is discussed.     

Ultraviolet-mediated antibiotic activity of thiophene compounds of Tagetes

Two intensely mauve UV fluorescent compounds isolated from Tagetes root were found to be phototoxic to Candida albicans. By chromatography on alumina followed by gel filtration on Sephadex LH-20, the compounds were identified as 5-(3-buten-1-ynyl)-2,2′-bithienyl and α-terthienyl.     

In vivo metabolism of 3H-testosterone in adult male rats: effects of estrogen administration.

The metabolism of testosterone (T) was studied in normal adult male rats using a constant infusion of trace amounts of the 3H-steroid into a tail vein for 3 h in order to attain a state of equilibrium. Samples of plasma, liver, kidney, prostate, seminal vesicles and muscle were analysed for 3H-testosterone, 3H-5alpha-dihydrotestosterone (5alphaDHT) and 3H-5alpha-androstanediol (Adiol). When compared to the 3H-T level in plasma there were high levels of 3H-T in kidney and of 3H-5alphaDHT in prostate and seminal vesicles. Intraperitoneal estradiol valerate administration (100 mug/day) for 4 days decreased and 3H-5alphaDHT levels in the prostate and seminal vesicles. The estrogen administration increased the T metabolic clearance rate from 17.5 1/24 h/100 g body wt to 22.6 1/24 h/100 g body wt.     

Biotransformation of pregnenolone - 7alpha-3H, progesterone - 7alpha-3H and dehydroepiandrosterone - 7alpha-3H by porcine fetal and maternal adrenal homogenate preparations.

The biotransformation of pregnenolone-7alpha-3H and of progesterone-7alpha-3H by porcine fetal and maternal adrenal homogenates at 56 and 112 days of pregnancy and of dehydroepiandrosterone-7alpha-3H by fetal adrenal homogenates has been investigated in vitro. Both pregnenolone-7alpha-3H and progesterone-7alpha-3H were metabolized extensively by maternal adrenal preparations, the principal radioactive metabolites isolated being cortisol, corticosterone, 11-deoxycortisol, deoxycorticosterone, 11beta-hydroxyprogesterone and androstenedione. In addition, 17alpha-hydroxyprogesterone, 20alpha-dihydroprogesterone and cortisone were formed from both substrates and 17alpha-hydroxypregnenolone and progesterone were formed from pregnenolone. Although essentially the same radioactive metabolites were isolated after incubation of fetal adrenal glands with pregnenolone-7alpha-3H or progesterone-7alpha-3H, a greater proportion of the radioactivity was associated with corticosteroids at 112 days of pregnancy than at 56 days. 11beta-Hydroxyandrostenedione and androstenedione were isolated and identified together with an unknown polar metabolite, after incubation of fetal adrenal tissue with dehydroepiandrosterone-7alpha-3H. These results are discussed in relation to feto-placental steroid biosynthesis and metabolism and the role of the fetal adrenal in the initiation of parturition in the pig.     

Stimulation of sugar transport in rat diaphragm by 8-bromoguanosine 3′,5′-monophosphate

The cyclic GMP derivative, 8-bromo cyclic GMP, increases the uptake of D-xylose and of 2-deoxy D-glucose into intact rat diaphragm incubated in vitro. 8-Bromo cyclic GMP does not stimulate the incorporation of [¹⁴C] glucose into glycogen in the diaphragm, or the uptake of α-amino isobutyric acid into this tissue. The effect of 8-bromo cyclic GMP on the diaphragm is consistent with the hypothesis that cyclic GMP plays a role in the regulation of sugar transport in muscle.     

平滑肌22α蛋白在血管稳态和血管重构中的作用

有关血管稳态和重构的分子机制一直是近年来的研究热点,也被视为治疗血管损伤性疾病的突破点.大量研究证实,血管损伤修复及病理性重构过程与血管平滑肌细胞(vascular smooth muscle cells,VSMCs)的表型转化、异常增殖与迁移、细胞衰老关系密切.平滑肌22α(smooth muscle 22α,SM2...     

Battling for Ribosomes: Translational Control at the Forefront of the Antiviral Response

In the early stages of infection, gaining control of the cellular protein synthesis machinery including its ribosomes is the ultimate combat objective for a virus. To successfully replicate, viruses unequivocally need to usurp and redeploy this machinery for translation of their own mRNA. In response, the host triggers global shutdown of translation while paradoxically allowing swift synthesis of antiviral proteins as a strategy to limit collateral damage. This fundamental conflict at the level of translational control defines the outcome of infection. As part of this special issue on molecular mechanisms of early virus–host cell interactions, we review the current state of knowledge regarding translational control during viral infection with specific emphasis on protein kinase RNA-activated and mammalian target of rapamycin-mediated mechanisms. We also describe recent technological advances that will allow unprecedented insight into how viruses and host cells battle for ribosomes.     

N-糖基化对毕赤酵母表达的DSPAα1分泌和活性的影响(英文)

溶栓剂DSPAα1正处于治疗急性缺血性中风的III期临床研究,临床结果显示DSPAα1具有良好的药理学和安全特性。将DSPAα1基因序列按照毕赤酵母偏好密码子进行优化,并在毕赤酵母菌株GS115和KM71中进行表达,同时利用定点突变对糖基化侧链进行缺失,考察糖基侧链对毕赤酵母表达DSPAα1的影响。结果表明,野生型DSPAα1在GS115和KM71中均获得高表达,在摇瓶发酵条件下,表达量分别为70mg/L和105mg/L;利用SDS-PAGE对DSPAα1三种突变体(N117Q、N362Q和N117Q/N362Q)进行分析,与野生型蛋白质相比较,3种突变体的表达水平显著下降,同时纤溶平板测活数据显示,纯化后的突变体N117Q和N362Q比活性均低于野生型蛋白质的25%。这表明,N-型糖链(N117和N362)对毕赤酵母表达的DSPAα1分泌和酶活性具有重要作用。