Comparison of full-length genomics sequences between dengue virus serotype 3, parental strain,and its derivatives,and B-cell epitopes prediction from envelope region

Biological markers are normally used to evaluate the candidate of live-attenuated dengue vaccines. D3V 16562 Vero 23 and D3V 16562 Vero 33 which were derivatives of D3V 16562, parental strain, showed the similar biological data. We used molecular techniques and computational tools to evaluate these derivatives. The nucleotide and amino acid sequences of the derivatives were compared to their parent. The secondary structures of untranslated regions and B-cell epitopes were predicted. The results showed that nucleotide substitutions mostly occurred in NS5 and NS5 of V2 was unusual because of amino acid change at 3349 (tryptophan →stop codon). The nucleotide substitutions in 5''UTR, prM, E, NS1, NS2A, NS3, and 3''UTR were 4, 1, 2, 2, 1, 3, and 2, respectively. The secondary structure of 5''UTR of V2 was different from P and V1. The secondary structure of 3''UTR of V2 was similar to P and certainly distinct from V1. Furthermore, B-cell epitopes prediction revealed that there were 21 epitopes of envelope and the interesting epitope was at position 297-309 because it was in domain III in which the neutralizing antibody is induced. For this study, the attenuation of derivatives was caused by the nucleotide substitutions in 5''UTR, 3''UTR, and NS5 regions. The genotypic data and B-cell epitope make the derivatives attractive for the chimeric and peptide DENV vaccine development.     

The excision of AP sites by the 3'-5' exonuclease activity of the Klenow fragment of Escherichia coli DNA polymerase I

The 3' AP endonucleases (class I) are said to hydrolyze the phosphodiester bond 3' to AP sites yielding 3'-OH and 5'-phosphate ends; on the other hand, the resulting 3' terminal AP site is not removed by the 3'-5' exonuclease activity of the Klenow fragment [1]. We show that AP sites in DNA are easily removed by the 3'-5' exonuclease activity of the Klenow fragment and that they are excised as deoxyribose-5-phosphate. It is suggested that the 3' AP endonucleases are perhaps not the hydrolases they are supposed to be.     

Sulfolobus acidocaldarius DHH超家族核酸酶Saci0542的酶学特征研究

【目的】以嗜酸嗜热硫化叶菌Sulfolobus acidocaldarius的DHH超家族核酸酶（Saci0542）为例,研究其核酸外切酶活性特点,为阐明其在DNA代谢中的具体功能提供生化基础。【方法】将嗜酸嗜热硫化叶菌DHH超家族核酸酶Saci0542基因在大肠杆菌中重组表达,经亲和层析纯化得到电泳纯的重组蛋白;利用荧光标记的寡核苷酸作为底物,用尿素变性聚丙烯酰胺凝胶电泳技术,鉴定Saci0542的酶学特征。【结果】重组表达的DHH超家族核酸酶Saci0542具有典型的单链核酸特异性的3’-5’外切酶活性。进一步酶学特征表征结果如下:酶活性依赖于二价金属离子Mn²⁺,而Ca²⁺、Mg²⁺、Zn²⁺等二价金属离子对活性没有明显的促进作用;Saci0542在pH5.5–10的广泛范围内均表现出较高酶活性;高于200 mmol/L的NaCl强烈抑制酶活性;最适反应温度为50–55℃;末端磷酸基团抑制3’-5’外切酶活性。【结论】本研究证实,Saci0542是一种Mn²⁺依赖型3’-...     

操纵茶树类黄酮3′-羟基化酶生物合成B环-3′,4′-二羟基黄酮类化合物

【目的】操纵茶树类黄酮3′-羟基化酶,生物合成B环-3′,4′-二羟基黄酮类化合物圣草酚、二氢槲皮素和槲皮素。【方法】构建了4个茶树类黄酮3′-羟基化酶基因(CsF3′H)和拟南芥的P450还原酶基因(ATR)融合表达质粒:SUMO-CsF3'H[7-517]::ATR1[49-688]3 AA、SUMO-CsF3'H[28-517]::ATR1[49-688]3 AA、SUMO-CsF3'H[7-517]::ATR2[75-711]3 AA和SUMO-CsF3'H[28-517]::ATR2[75-711]3 AA,分别转化大肠杆菌菌株TOP10、DH5α和BL21,获得12个转化菌株S1–S12;构建了茶树类黄酮3′-羟基化酶基因CsF3′H表达质粒p YES-Dest52-CsF3′H,转化酵母菌株WAT11,得到转化菌株S13;构建了茶树类黄酮3′-羟基化酶基因CsF3′H表达质粒pES-URA-CsF3′H,及茶树黄烷酮3-羟基化酶基因CsF3H与拟南芥黄酮醇合成酶基因At FLS的融合表达质粒pES-HIS-CsF3H::At FLS 9AA,二者共转化酵母菌株WAT11,获得转化菌株S14。【结果】转化SUMO-CsF3'H[28-517]::ATR1[49-688]3 AA质粒的TOP10菌株S6在25°C条件下发酵,转化效率最高,能将1000μmol/L柚皮素、二氢山奈酚和山奈酚,分别转化生成287.93μmol/L圣草酚、131.76μmol/L二氢槲皮素和188.62μmol/L槲皮素。发酵菌株S13能分别将1000μmol/L柚皮素、二氢山奈酚和山奈酚,最多能转化生成734.32μmol/L圣草酚、446.07μmol/L二氢槲皮素和594.64μmol/L槲皮素。喂食S14发酵菌株5 mmol/L的底物柚皮素,在发酵36–48 h中,最多能生成1412.16μmol/L圣草酚、490.25μmol/L山奈酚、445.75μmol/L槲皮素、66.75μmol/L二氢槲皮素和73.50μmol/L二氢山奈酚。【结论】本研究首次将茶树类黄酮3′-羟基化酶基因应用于B环-3′,4′-二羟基黄酮类化合物圣草酚、二氢槲皮素和槲皮素的生物合成。     

Discovery of new VEGFR-2 inhibitors based on bis([1, 2, 4]triazolo)[4,3-a:3',4'-c]quinoxaline derivatives as anticancer agents and apoptosis inducers

Herein, a new wave of bis([1, 2, 4]triazolo)[4,3-a:3'',4''-c]quinoxaline derivatives have been successfully designed and synthesised. The synthesised derivatives were biologically investigated for their cytotoxic activities against HepG2 and MCF-7. Also, the tested compounds were further examined in vitro for their VEGFR-2 inhibitory activity. The most promising derivative 23j was further investigated for its apoptotic behaviour in HepG2 cell lines using flow cytometric and western-plot analyses. Additional in-silico studies were performed to predict how the synthesised compounds can bind to VEGFR-2 and to determine the drug-likeness profiling of these derivatives. The results revealed that compounds 23a, 23i, 23j, 23l, and 23n displayed the highest antiproliferative activities against the two cell lines with IC₅₀ values ranging from 6.4 to 19.4 µM. Furthermore, compounds 23a, 23d, 23h, 23i, 23j, 23l, 23 m, and 23n showed the highest VEGFR-2 inhibitory activities with IC₅₀ values ranging from 3.7 to 11.8 nM, comparing to sorafenib (IC₅₀ = 3.12 nM). Moreover, compound 23j arrested the HepG2 cell growth at the G2/M phase and induced apoptosis by 40.12% compared to the control cells (7.07%). As well, such compound showed a significant increase in the level of caspase-3 (1.36-fold), caspase-9 (2.80-fold), and BAX (1.65-fold), and exhibited a significant decrease in Bcl-2 level (2.63-fold).     

miR-200c suppression increases tau hyperphosphorylation by targeting 14-3-3γ in early stage of 5xFAD mouse model of Alzheimer's disease

Background and Purpose: Recently, several abnormally regulated microRNAs (miRNAs) have been identified in patients with Alzheimer''s disease (AD). The purpose of this study was to identify abnormally expressed miRNAs and to investigate whether they affect pathological changes in AD in the 5xFAD AD mouse model.Experimental Approach: Using microarray analysis and RT-qPCR, miRNA expression in the hippocampus of a 4-month-old 5xFAD mouse model of AD was investigated. A dual-luciferase assay was performed to determine whether the altered miR-200c regulates the translation of the target mRNA, Ywhag. Whether miR-200c modulates AD pathology was determined in primary hippocampal neurons and C57BL/6J mice transfected with miR-200c inhibitor. In addition, total miRNAs were extracted from the serums of 28 healthy age-matched controls and 22 individual participants with cognitive impairment, and RT-qPCR was performed.Key results: miR-200c expression was reduced in the hippocampus of 5xFAD mice. In primary hippocampal neurons, miR-200c regulated the translation of 14-3-3γ and increased tau phosphorylation (p-tau) by increasing p-GSK-3β (GSK-3β phosphorylation). It was also confirmed that miR-200c inhibition in the hippocampus of C57BL/6J mice induces cognitive impairment and increases tau phosphorylation through 14-3-3γ activation. Finally, aberrant expression of miR-200c was confirmed in the blood serum of human AD patients.Conclusion and Implications: Our results strongly suggest that dysregulation of miR-200c expression contributes to the pathogenesis of AD, including cognitive impairment through hyperphosphorylated tau.     

Inhibition of bovine adrenocortical cytochrome P-450scc by 3,3'-dimethoxybenzidine

The effect of 3,3'-dimethoxybenzidine ($\text{o}$-dianisidine) on the conversion of cholesterol to pregnenolone was investigated in a reconstituted side chain cleavage system using enzymes purified from bovine adrenal cortex; d-$\text{p}$-aminoglutethimide was also assayed under similar conditions for comparison. 3,3'-Dimethoxybenzidine was found to be a potent inhibitor of pregnenolone formation, causing 50% inhibition at a concentration of 1.5 μM when using 70 μM cholesterol — this dose is approximately one fourth that required of 3-methoxybenzidine and one twentieth that required of benzidine for equal inhibition. In the same system, d-$\text{p}$-aminoglutethimide exhibited an I₅₀ value of about 55 μM. No effects of 3,3'-dimetoxybenzidine on adrenodoxin reductase or adrenodoxin activities could be detected, and inhibition of side chain cleavage could be relieved by dilution suggesting that the inhibitor acts by reversibly binding to cytochrome P-450scc.     

Crystallographic studies of drug-nucleic acid interactions: Proflavine intercalation between the non-complementary base-pairs of cytidilyl-3′,5′-adenosine

In order to get insights into the binding of dyes and mutagens with denatured and single-stranded nucleic acids and the possible implications in frameshift mutagenesis, a 1:1 complex between the non-self-complementary dinucleoside monophosphate cytidilyl-3′,5′-adenosine (CpA) and proflavine was crystallized. The crystals belong to the tetragonal space group P4₂2₁2 with cell constants $\text{a = b = 19.38(1)}\text{A}\text{?}\text{and}\text{c = 27.10(1)}\text{A}\text{?}$. The asymmetric unit contains one CpA, one proflavine and nine water molecules by weight. The structure was determined using Patterson and direct methods and refined to an R-value of 11% using 2454 diffractometer intensities.The non-self-complementary dinucleoside monophosphate CpA forms a selfpaired parallel chain dimer with a proflavine molecule intercalated between the protonated cytosine-cytosine (C · C) pair and the neutral adenine-adenine (A · A) pair. The dimer complex exhibits a right-handed helical twist and an irregular girth. The neutral A · A pair is doubly hydrogen-bonded through the N(6) and N(7) sites (C(1′)C(1′) distance: 10.97(2) Å) and the protonated C · C pair is triply hydrogen-bonded with a proton shared between the N(3) sites (C(1′)C(1′) distance: 9.59(2) Å). To accommodate the intercalating dye, the sugars of successive nucleotide residues adopt the two fundamental conformations (5′ end: 3′-endo, 3′ end: 2′-endo), the backbone adopts torsion angle values that fluctuate within their preferred conformational domains: the PO bonds (ω, ω′) adopt the characteristic helical (gauche^?-gauche^?) conformation, the CO bonds (φ, φ′) are both in the trans domain and the C(4′)C(5′) bonds (ψ) are in the gauche⁺ region. The bases of both residues are disposed in the preferred anti domain with the glycosyl torsion angles (χ) correlated to the puckering mode of the sugar so that the cytidine residue is C(3′)-endo, low χ (12 dg), and the adenosine residue is C(2′)-endo, high χ (84 °). The intercalated proflavine stacks more extensively with the C · C pair than the A · A pair. Between 4₂-related CpA proflavine units there is a second proflavine which stacks well with both the A · A and the C · C pairs sandwiching it. Both proflavine molecules are positionally disordered. In each of its two disordered sites, the intercalated proflavine forms hydrogen-bonded interactions with only one sugar-phosphate backbone. A total of 26 water sites has been characterized of which only two are fully occupied. These hydration sites are involved in an intricate network of hydrogen bonds with both the dye and CpA and provide insights on the various modes of interactions between water molecules and between water molecules and nucleic acids.The structure of the proflavine-CpA complex shows that intercalation of planar drugs can occur between non-complementary base-pairs. This result can be relevant for understanding the strong binding of acridine dyes to denatured DNA, single-stranded RNA, and single-stranded polynucleotides. Also, the ability of proflayine to promote self-pairs of adenine and cytosine bases could provide a chemical basis for an alternative mechanism of frameshift mutagenesis.     

血清sPLA2-X、8-羟基-2'-脱氧鸟苷及hs-CRP水平诊断慢性阻塞性肺疾病急性加重的价值

摘要 目的：探究血清sPLA2-X、8-羟基-2''-脱氧鸟苷及hs-CRP水平诊断慢性阻塞性肺疾病急性加重的价值。方法：随机选取2021年3月~2023年11月在我院进行治疗的COPD患者128例,以患者是否出现急性加重分为稳定组和AECOPD组,其中稳定组29例,AECOPD组99例。比较两组研究对象外周血sPLA2-X、8-OHdG及hs-CRP水平水平差异;COX比例风险因素回归模型分析AECOPD的影响因素,采用ROC曲线分析相关指标单独及联合检测诊断AECOPD的价值。结果：AECOPD组FEV₁指标显著低于稳定组,差异具有统计学意义（P<0.05）。与稳定组组相比,AECOPD组患者外周血sPLA2-X、8-OHdG及hs-CRP水平均明显升高,差异具有统计学意义（P<0.01）。外周血sPLA2-X、8-OhdG、hs-CRP水平与GOLD分级成正相关（P<0.05）,外周血sPLA2-X、8-OhdG、hs-CRP水平与FEV₁/FVC（%）、FEV₁%pred成负相关（P<0.05）。采用COX比例风险因素回归模型分析,外周血sPLA2-X、8-OhdG、hs-CRP均为影响AECOPD的独立危险因素。ROC曲线分析外周血sPLA2-X、8-OhdG、hs-CRP诊断AECOPD的曲线下面积（AUC）分别为0.689、0.708、0.642、0.875,联合各指标预测价值明显高于单独检测。结论：AECOPD 患者外周血sPLA2-X、8-OhdG、hs-CRP水平高表达。外周血sPLA2-X、8-OhdG、hs-CRP水平与肺功能严重程度、肺功能指标相关,同时三者可用作诊断COPD患者急性加重的潜在预测指标。三者联合具有更高的诊断价值。     

The effect of prolonged dietary administration of the non-carcinogen, 4-acetylaminofluorene on the liver of the rat. An electron microscope study

A diet containing 0.05 % of the non-carcinogen 4-acetylaminofluorene (4-AAF) was fed to male Leeds strain rats for periods of up to 10 months. Some animals were killed after 8–12 weeks, 6 months and 8–10 months of 4-AAF feeding, while further groups were returned to a normal diet after 10 months of treatment and then killed 2, 5, 9 and 12 months later. The hepatic tissues were removed and prepared for electron microscopy. The main fine structural changes induced by 4-AAF were a prominent hypertrophy of the agranular endoplasmic reticulum, glycogen depletion and lipid accumulation. It was noted that these changes persisted following withdrawal of dietary 4-AAF, for the duration of the experiment. The effects of 4-AAF are compared with those of its carcinogenic isomer, 2-acetylaminofluorene (2-AAF) and the possibility is discussed that the clear differences revealed in this study may be directly related to the relative carcinogenicities of these two compounds.     

Inhibition of 2'-phosphodiesterase by cAMP-dependent protein kinase. Involvement of phosphorylation of protein inhibitor

2'-Phosphodiesterase from NIH 3T3 cells was purified about 530-fold. Treatment of the cell lysate with the cAMP-dependent protein kinase causing the 2'-phosphodiesterase inhibition did not result in phosphorylation of the enzyme itself. The kinase was found to phosphorylate a specific 18-kDa protein, the phosphorylated form of this protein being the inhibitor of 2'-phosphodiesterase.     

Chaperone protein 14-3-3 and protein kinase A increase the relative abundance of low agonist sensitivity human alpha 4 beta 2 nicotinic acetylcholine receptors in Xenopus oocytes

Alpha4 and beta2 nicotinic acetylcholine (nACh) receptor subunits expressed heterologously in Xenopus oocytes assemble into a mixture of receptors with high and low agonist sensitivity whose relative abundance is influenced by the heteropentamer subunit ratio. We have found that inhibition of protein kinase A by KT5720 decreased maximal [3H]cytisine binding and acetylcholine (ACh)-induced current responses, and increased the relative proportion of alpha4beta2 receptors with high agonist sensitivity. Mutation of serine 467, a putative protein kinase A substrate in a chaperone protein binding motif within the large cytoplasmic domain of the alpha4 subunit, to alanine or asparate decreased or increased, respectively, maximal [3H]cytisine binding and ACh response amplitude. Expression of alpha4S467A mutant subunits decreased steady levels of alpha4 and the relative proportion of alpha4beta2 receptors with low agonist sensitivity, whilst expression of alpha4S467D increased steady levels of alpha4 and alpha4beta2 receptors with low agonist sensitivity. Difopein, an inhibitor of chaperone 14-3-3 proteins, decreased [3H]cytisine binding and ACh responses and increased the proportion of alpha4beta2 with high sensitivity to activation by ACh. Thus, post-translational modification affecting steady-state levels of alpha4 subunits provides a possible means for physiologically relevant, chaperone-mediated variation in the relative proportion of high and low agonist sensitivity alpha4beta2 nACh receptors.     

Bakuchalcone,a dihydrofuranochalcone from the seeds of Psoralea corylifolia

A new dihydrofuranochalcone has been identified in seeds of Psoralea corylifolia and its structure confirmed by synthesis.     

喜盐鸢尾花色形成关键基因的克隆及表达分析

喜盐鸢尾(Iris halophila Pall.)及其变种蓝花喜盐鸢尾(I.halophila Pall.var.sogdiana(Bung)Grubov)因耐盐碱及其多种花色而具有盐碱地园艺开发价值。本文根据喜盐鸢尾内轮花被转录组测序结果,利用基因特异性引物从这2种植物中分别克隆了编码查尔酮合成酶(CHS)、查尔酮异构酶(CHI)、类黄酮-3',5'-羟基化酶类(F3'5'H-like)等基因的部分片段,并对它们在内轮花被中的表达水平进行实时定量PCR分析。序列分析结果确认在喜盐鸢尾中所克隆的CHS(311 bp)、CHI(457 bp)、F3'5'H-like(496 bp)3个基因(部分)未见文献报道与NCBI等数据库记录。其中F3'5'H-like基因与经典的属于细胞色素P450CYP75A亚家族的F3'5'H不同,而与万带兰的F3'5'H-like同属于CYP76AB亚家族,为一类新的蓝花相关基因。实时定量PCR表达分析结果表明,与黄花的喜盐鸢尾相比,蓝花喜盐鸢尾中CHS与F3'5'H-like显著上调表达,可能是其花色不同于喜盐鸢尾的主要原因。     

The effects of the continuous administration of N,N-dimethyl-4-phenylazoaniline (DAB) on the activities and the inducibilities of some drug-metabolizing enzymes in rat liver.

(1) The effect of feeding a relatively low-protein diet containing 0.06% DAB for 29 weeks on the activity of DAB-azoreductase, nitroreductase (p-nitrobenzoic acid), N-oxidase (N,N-dimethylaniline), N-demethylase (DAB), cytochrome P-450, NADPH-cytochrome c reductase, beta-glucuronidase and arylsulphatase A were studied. Rapid decreases occurred in the activities of the first six enzymes, reaching minimal values at between 4 and 8 weeks. Activities then increased in all cases to control or nearly control levels. This rate of increase was least for cytochrome P-450. At 4 weeks azoreductase activity with the chemotherapeutic agent CB10-252 (I) as substrate was significantly higher than in control rats. Early increases occurred in the activities of beta-glucuronidase and arylsulphatase A and the activity of the latter never dropped below the control level. (2) An investigation was made of the differential effects of dye feeding on some of the enzyme activities in the two major liver lobes and differences were found. (3) The effect of phenobarbital (PB) pretreatment on the DAB-fed rats was studied at 4-week intervals. The activities of DAB-azoreductase and of nitroreductase increased throughout the whole period, while the activities of the lysosomal enzymes were decreased. (4) After feeding DAB for 4 weeks the effect of PB and 3-methylcholanthrene (MC) on the activities of DAB-azoreductase, CB10-252-azoreductase and components of the azoreductases-cytochrome P-450, NADPH-cytochrome c reductase, the CO-CB10-252-azoreductase was not induced by PB or MC, and CO did not inhibit its reduction. Its reduction depended only slightly on NADH. CO caused a greater relative decrease in the activity of DAB-azoreductase in dye-fed animals and also in animals following PB and MC pretreatment, implying a greater role of cytochrome P-450 in dye-fed animals.