Biotransformation of linear alkylbenzene sulfonate (LAS) by Phanerochaete chrysosporium: oxidation of alkyl side-chain

The white rot fungus Phanerochaete chrysosporium, which generally mineralizes substituted aromatics to CO₂, transformed linear alkylbenzene sulfonate (LAS) surfactants mainly at their alkyl side chain. Degradation of LAS was evidenced by a zone of clearing on LAS-containing agar plates and colorimetric analysis of liquid cultures. Disappearance of LAS was virtually complete within 10 days in low nitrogen (2.4 mM N), high nitrogen (24 mM N) and malt extract (ME) liquid media. After 5 days of incubation in ME medium, transformation of LAS was complete at concentrations4 mg l^-1, but decreased at higher concentrations. The LAS degradation was not dependent on lignin peroxidases (LiPs) and manganese-dependent peroxidases (MnPs). Mineralization of¹⁴C-ring-LAS to ¹⁴CO₂ by P. chrysosporium was <1% regardless of the culture conditions used. Thin layer chromatography and mass spectral analyses indicated that P. chrysosporium transformed LAS to sulfophenyl carboxylates (SPCs) through oxidative shortening of the alkyl side-chains. While LAS disappearance in the cultures was not dependent on LiPs and MnPs, transformation of the parent LAS moieties to SPCs was more extensive in low N medium that favors expression of these enzymes. The SPCs produced in LN cultures were shorter in chain-length than those produced in ME cultures. Also there was a notable shift in the relative abundance of odd and even chain length metabolites compared to the starting LAS particularly in the low N cultures suggesting the possible involvement of processes other than or in addition to-oxidation in the chain-shortening process.     

全氟辛烷磺酰基化合物对剑尾鱼肝脏抗氧化物酶活性的影响

目的研究全氟辛烷磺酰基化合物(PFOS)暴露对剑尾鱼(Xiphophorus helleri Heckel)抗氧化物酶活性的影响,探讨PFOS对鱼类的致毒机理。方法使用浸润法以3.5、7.0、14.0和28.0 mg/L四个PFOS浓度为剑尾鱼染毒,定量测定了96 h内肝脏组织中的超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-PX)活性及丙二醛(MDA)含量的变化。结果 PFOS暴露12 h后,除28.0 mg/L组SOD活性被显著性抑制外,其余各组与对照组均无显著性差异(P>0.05);7.0 mg/L组和14.0 mg/L组在24 h被极显著诱导(P<0.01),并且一直保持至96 h。CAT活性随PFOS浓度的升高而降低,12 h时,除3.5 mg/L组外,其余各组CAT活性被显著或极显著抑制,至24 h时,各组CAT活性有上升趋势,但48 h后,各组呈不断下降趋势持续至96 h,其CAT活性恢复到12 h水平。GSH-PX活性变化与CAT活性变化趋势相似,其中28.0 mg/L组在不同时间均被显著性抑制,并在96 h时抑制率达到最高值64.8%。MDA含量在12 h时呈小幅下降趋势,但随着暴露时间的延长,各处理组MDA含量呈连续上升趋势,并在96 h时达到最高点,诱导率分别为71.2%、70.1%和85.1%。结论结果表明,SOD的高活性是由于机体中超氧阴离子的存在,而高浓度的超氧阴离子能够灭活CAT和GSH-PX活性,因此,CAT和GSH-PX活性始终低于对照组。GSH-PX对PFOS的敏感性高于CAT。MDA含量持续升高反映出细胞组织已经遭受到氧化损伤。剑尾鱼活体的实验表明,PFOS能够诱导肝脏氧化应激反应,氧化损伤是PFOS致毒的主要途径之一。     

Specificity of Na+ binding to phosphatidylserine vesicles from A 23Na NMR relaxation rate study

²³Na NMR relaxation rate measurements show that Na⁺ binds specificially to phosphatidylserine vesicles and is displaced partially from the binding site by K⁺ and Ca²⁺ but to a considerably less extent by tetraethylammonium ion. The data indicate that tetraethylammonium ion affects the binding of Na⁺ only slightly, by affecting the surface potential through its presence in the double layer, without competing for a phosphatidylserine binding site. Values for the intrinsic binding constant for the Na⁺-phosphatidylserine complex that would be consistent with the competition experiments (and the dependence of the relaxation rate on concentration of free Na⁺) fall in the range 0.4–1.2 M^?1 with a better fit towards the higher values. We conclude that in the absence of competing cations in solution an appreciable fraction of the phosphatidylserine sites could be associated with bound Na⁺ at 0.1 M Na⁺ concentration.     

新型染料木素磺酸酯的前药判定与大鼠体内生物利用度

为寻找染料木素(GE)新的前药,采用建立的生物样品中药物浓度测定的液相色谱法对新型大豆异黄酮染料木素磺酸酯(GB)进行前药判定以及大鼠体内药物动力学研究,以考察前药中染料木素的口服生物利用度是否改善.在大鼠体内药物代谢实验中,灌胃给予GB的大鼠血浆中能检测到明显的GE.在临床前药物动力学实验中,该前体静注给药和以40 mg/kg灌胃药后,GE在大鼠体内的动力学过程均符合一室模型.GB中GE的相对口服生物利用度为原药的110.9％.研究表明,相对于原药GE,前药中GE的相对口服生物利用度达到预期的改善,该前药有进一步研究意义.     

基因组编辑对酿酒酵母DNA的损伤作用及修复响应

【目的】为了研究基因组编辑工具CRISPR/Cas9和CRISPR/Cpf1所产生的DNA双链断裂(DNA doublestrandbreak,DSB)对酿酒酵母DNA的损伤作用及修复响应情况,对比化学物质甲基磺酸甲酯(methyl methanesulfonate,MMS)对酿酒酵母基因组DNA的损伤和修复,阐明编辑细胞在细胞水平和转录水平上的变化。【方法】起始细胞分为两种情况,包括未进行细胞周期同步化和被α-因子同步化细胞周期至G0/G1期。检测CRISPR/Cas9和CRISPR/Cpf1处理后编辑细胞的生长情况。利用流式细胞术检测编辑细胞的细胞周期延滞的情况。利用荧光定量PCR检测编辑细胞和MMS处理细胞后DNA损伤响应关键基因转录表达水平的变化情况。【结果】起始细胞无论是未同步化还是同步化,其生长均受到基因组编辑抑制,细胞存活率降低,细胞周期被滞留在G2/M期,而MMS处理导致细胞周期S期的滞留。此外,随编辑时间的延长,突变率增加,细胞存活率降低。CRISPR/Cpf1编辑细胞的突变率和存活率均低于CRISPR/Cas9,由此可见,CRISPR/Cpf1对细胞的损伤强度高于CRISPR/Cas9。两种编辑均诱导酵母DNA损伤响应关键基因RNR3及HUG1转录水平显著上调,并且CRISPR/Cpf1介导的上调幅度大于CRISPR/Cas9,但两者均低于MMS的处理。【结论】本研究解析了CRISPR/Cas9和CRISPR/Cpf1介导的基因组编辑在细胞水平和转录水平上对DNA损伤作用及修复响应,初步揭示了酿酒酵母应对不同类型的DSB损伤时响应程度的差异,为提高基因组编辑工具的编辑能力和评估基因编辑安全性提供了重要依据。     

Phosphorylation in isolated chloroplasts coupled to dichlorophenyldimethylurea-insensitive silicomolybdate reduction

1. The electron transport in isolated chloroplasts with silicomolybdate as electron acceptor has been reinvestigated. The silicomolybdate reduction has been directly measured as ΔA₇₅₀ or indirectly as O₂ evolution (in the presence or absence of ferricyanide).2. Silicomolybdate-dependent O₂ evolution is inhibited to a similar extent by 3-(3,4-dichlorophenyl) 1, 1-dimethylurea (DCMU) or dibromothymoquinone (DBMIB), indicating the existence of two different sites of silicomolybdate reduction: one before the DCMU block (i.e. at Photosystem II) and one after the DBMIB block (i.e. at Photosystem I).3. Silicomolybdate-dependent O₂ evolution is coupled to ATP synthesis with an $\text{ATP}\text{2}\text{e}{}^{\mathrm{?}}$ ratio of 1.0 to 1.1. The presence of ferricyanide inhibits this ATP synthesis ($\text{ATP}\text{2}\text{e}{}^{\mathrm{?}}$ ratio then is about 0.3).4. Silicomolybdate-dependent O₂ evolution is also coupled to ATP-synthesis in the presence of DCMU with an $\text{ATP}\text{2}\text{e}{}^{\mathrm{?}}$ ratio of 0.6–0.8 characteristic of Site II; in this case the electron transport itself is not affected by uncouplers or energy-transfer inbihitors.5. The data are interpreted as a further demonstration that the water-splitting reaction is responsible for the conservation of energy at Photosystem II.     

Mutational and pseudomutational effects of 5-bromodeoxyuridine in human lymphoblasts

We have studied the effects of 5-bromodeoxyuridine (BrdUrd) at two genetic loci in diploid human lymphoblast cells. In thymidine kinase heterozygotes (tk +/-), a 2-h dose of BrdUrd induced a transient, non-heritable resistance to the thymidine analogue, trifluorothymidine (F3TdR). We have called this phenomenon pseudomutation and have shown that affected cells acquire the ability to survive in the presence of F3TdR and then, after degradation of F3TdR in the medium, return to an apparently normal wild-type state. Our data suggest that BrdUrd incorporation into DNA as a thymidine analogue is responsible for the effect, which we interpret as a temporary loss of thymidine kinase activity. This effect is not seen in tk +/+ homozygotes. In contrast, at the hypoxanthine-guanine phosphoribosyl transferase locus in tk +/- heterozygotes, BrdUrd did not induce a permanent, heritable resistance to 6-thioguanine (gene locus mutation). We detected such mutations only in the tk +/+ homozygote and only at external BrdUrd concentrations considerably higher than those which saturate the uptake of BrdUrd into DNA as a thymidine analogue. We postulate that the reduced TK enzyme levels (30%) in the heterozygote prevent the build-up of a sufficiently high intracellular BrdUrd triphosphate pool to promote the misincorporations as deoxycytidine triphosphate which may be responsible for gene locus mutation.     

丹参酮Ⅱ-A磺酸钠对异丙基肾上腺素引起的心肌线粒体积蓄钙离子的保护作用

 临床报道丹参酮Ⅱ-A磺酸钠(T)对冠心病心绞痛和胸闷症状有一定疗效,并能改善缺血性心电图。药理研究证明,具有抗钙和抗钙调蛋白(CaM)作用。本文进一步研究了T对正常的和异丙基肾上腺素(Isp)兴奋的家兔心肌线粒体摄取~(45)Ca~(2+)的影响。本文制备的心肌线粒体经透射和扫描电镜检查证明形态和内部结构均正常。线粒体蛋白在0.1mg/ml以下时其钙摄取与浓度呈线性关系。Isp10~(-6)mol/L可使线粒体钙摄取提高20±5％。T在10μg/ml时对正常的和Isp兴奋的线粒体摄取钙的抑制率分别为61.2±3.9％和54.2±5.1％。已知大剂量Isp可引起心肌线粒体钙超载和细胞坏死。戊脉安可减少以上病理性损害。本文结果表明T也具有类似戊脉安的保护心肌的作用。     

Tricyclic coumarin sulphonate derivatives with alkaline phosphatase inhibitory effects: in vitro and docking studies

Tissue-nonspecific alkaline phosphatase (TNAP) is an important isozyme of alkaline phosphatases, which plays different pivotal roles within the human body. Most importantly, it is responsible for maintaining the balanced ratio of phosphate and inorganic pyrophosphate, thus regulates the extracellular matrix calcification during bone formation and growth. The elevated level of TNAP has been linked to vascular calcification and end-stage renal diseases. Consequently, there is a need to search for highly potent and selective inhibitors of alkaline phosphatases (APs) for treatment of disorders associated with the over-expression of APs. Herein, a series of tricyclic coumarin sulphonate 1a-za with known antiproliferative activity, was evaluated for AP inhibition against human tissue nonspecific alkaline phosphatase (h-TNAP) and human intestinal alkaline phosphatase (h-IAP). The methylbenzenesulphonate derivative 1f (IC₅₀?=?0.38?±?0.01?μM) was found to be the most active h-TNAP inhibitor. Another 4-fluorobenzenesulphonate derivative 1i (IC₅₀?=?0.45?±?0.02?μM) was found as the strongest inhibitor of h-IAP. Some of the derivatives were also identified as highly selective inhibitors of APs. Detailed structure-activity relationship (SAR) was investigated to identify the functional groups responsible for the effective inhibition of AP isozymes. The study was also supported by the docking studies to rationalise the most possible binding site interactions of the identified inhibitors with the targeted enzymes.     

Corneal Dystrophy Mutations Drive Pathogenesis by Targeting TGFBIp Stability and Solubility in a Latent Amyloid-forming Domain

Numerous mutations in the corneal protein TGFBIp lead to opaque extracellular deposits and corneal dystrophies (CDs). Here we elucidate the molecular origins underlying TGFBIp's mutation-induced increase in aggregation propensity through comprehensive biophysical and bioinformatic analyses of mutations associated with every major subtype of TGFBIp-linked CDs including lattice corneal dystrophy (LCD) and three subtypes of granular corneal dystrophy (GCD 1–3). LCD mutations at buried positions in the C-terminal Fas1–4 domain lead to decreased stability. GCD variants show biophysical profiles distinct from those of LCD mutations. GCD 1 and 3 mutations reduce solubility rather than stability. Half of the 50 positions within Fas1–4 most sensitive to mutation are associated with at least one known disease-causing mutation, including 10 of the top 11 positions. Thus, TGFBIp aggregation is driven by mutations that despite their physico-chemical diversity target either the stability or solubility of Fas1–4 in predictable ways, suggesting straightforward general therapeutic strategies.