Role of lysosomal acid lipase in the intracellular metabolism of LDL-transported dehydroepiandrosterone-fatty acyl esters

Dehydroepiandrosterone-fatty acyl esters (DHEA-FAE) belong to a unique family of naturally occurring hydrophobic steroid hormone derivatives that are transported in circulating lipoproteins and may act as a source of dehydroepiendrosterone (DHEA) and other biologically active steroid hormones in cells. Here, we studied the metabolic fate of low-density lipoprotein-associated [(3)H]DHEA-FAE ([(3)H]DHEA-FAE-LDL) and the possible role of lysosomal acid lipase (LAL) in the hydrolysis of DHEA-FAE in cultured human cells. When HeLa cells were incubated with [(3)H]DHEA-FAE-LDL, the accumulation of label in the cellular fraction increased with incubation time and could be inhibited by excess unlabeled LDL, suggesting LDL receptor or LDL receptor-related receptor-dependent uptake. During 48 h of chase, decreasing amounts of [(3)H]DHEA-FAE were found in the cellular fraction, while in the medium increasing amounts of unesterified [(3)H]DHEA and its two metabolites, [(3)H]-5alpha-androstanedione (5alpha-adione) and [(3)H]androstenedione (4-adione), appeared. As LDL-cholesteryl ester hydrolysis is dependent on LAL activity, we depleted LAL from HeLa cells using small interfering RNAs and compared the hydrolysis of [(3)H]DHEA-FAE-LDL and [(3)H]cholesteryl-FAE-LDL. The results demonstrated a more modest but significant reducing effect on the hydrolysis of [(3)H]DHEA-FAE compared with [(3)H]cholesteryl-FAE. Moreover, experiments in LAL-deficient human fibroblasts (Wolman disease patient cells) showed that [(3)H]DHEA-FAE hydrolysis was not completely dependent on LAL activity. In summary, LDL-transported [(3)H]DHEA-FAE entered cells via LDL receptor or LDL receptor-related receptor-mediated uptake, followed by intracellular hydrolysis and further metabolism into 5alpha-adione and 4-adione that were excreted from cells. Although LAL contributed to the deesterification of DHEA-FAE, it was not solely responsible for the hydrolysis.     

MGMT Promoter Methylation Correlates with an Overall Survival Benefit in Chinese High-Grade Glioblastoma Patients Treated with Radiotherapy and Alkylating Agent-Based Chemotherapy: A Single-Institution Study

Promoter methylation of the O⁶-methylguanine-DNA-methyltransferase (MGMT) gene has been considered a prognostic marker and has become more important in the treatment of glioblastoma. However, reports on the correlation between MGMT and clinical outcomes in Chinese glioblastoma patients are very scarce. In this study, quantitative methylation data were obtained by the pyrosequencing of tumor tissues from 128 GBM patients. The median overall survival (OS) was 13.1 months, with a 1-year survival of 45.3%. The pyrosequencing data were reproducible based on archived samples yielding data for all glioblastomas. MGMT promoter methylation was detected in 75/128 cases (58.6%), whereas 53/128 (41.4%) cases were unmethylated. Further survival analysis also revealed that methylation was an independent prognostic factor associated with prolonged OS but not with progression-free survival (PFS) (p = 0.029 and p = 0.112, respectively); the hazard radios were 0.63 (95% CI: 0.42–0.96) and 0.72 (95% CI: 0.48–1.09), respectively. These data indicated that MGMT methylation has prognostic significance in patients with newly diagnosed high-grade glioblastoma undergoing alkylating agent-based chemotherapy after surgical resection.     

Corrigendum to ‘Nanopore-based Fourth-generation DNA Sequencing Technology' [GPB 144(2015)–GPB 13/1(4–16)]

<正>The authors regret that there were typos in the online version of Figure 4 published in Issue 1,2015.In the figure legend boxes of panels F and G,‘‘Ni’’should be corrected to‘‘N’’for the labeling of the green curves.The figure in the printed version has been corrected.The correct Figure 4 is shown below.The authors would like to apologize for any inconvenience caused.     

小麦TaMlo基因的原核表达、抗体制备及细胞化学分析(简报)

白粉病菌(Blumeria graminis)是一类高度专化性的寄生真菌,可侵染650多种单子叶植物和 9000多种双子叶植物,能够引起多种麦类作物的白粉病,给农业生产带来巨大的损失。由于白粉病菌生理小种多、变异快,所以利用专化性抗病基因难以解决植物的持久抗病性问题。人们在研究大麦白粉病时．发现大麦Mlo基因的隐性突变可导致大麦对绝大多数白粉病菌生理小种的高效持久的广谱抗病性。Schulze-Lefert等多家实验室合作于1997年成功克隆了野生的 Mlo基因。进一步研究表明．该基因编码一种植物特有的具有7个跨膜区和羧基端长尾的膜蛋白(Mlo),它可能对植物细胞的坏死起负调控作用。但Mlo基因如何表达及其在白粉病菌发育中的作用机制尚不清楚。     

Aspergillus niger DLFCC-90 rhamnoside hydrolase, a new type of flavonoid glycoside hydrolase

A novel rutin-α-L-rhamnosidase hydrolyzing α-L-rhamnoside of rutin, naringin, and hesperidin was purified and characterized from Aspergillus niger DLFCC-90, and the gene encoding this enzyme, which is highly homologous to the α-amylase gene, was cloned and expressed in Pichia pastoris GS115. The novel enzyme was classified in glycoside-hydrolase (GH) family 13.     

食管鳞癌组织中CDC2、CLDN5蛋白表达及其临床病理意义的研究

目的探讨CDC2及CLDN5在食管鳞癌中表达及其临床病理特征的关系。方法应用免疫组化Elivision法检测90例食管鳞癌组织、28例正常食管黏膜组织及16例重度不典型增生组织中CDC2和CLDN5的蛋白表达情况。结果在食管鳞癌和正常食管黏膜组织中CDC2和CI。DN5的阳性表达率分别为88．89％（80／90）、85．56％（77／90）和48．86％（12／28）、25．00％（7／28）,两者差异有统计学意义（P〈O．05）。CDC2蛋白表达在低分化食管鳞癌中明显高于高分化食管鳞癌;临床分期Ⅲ＋Ⅳ期组的CDC2蛋白的表达显著高于I期、Ⅱ期组（P〈O．05）。CDC2和CLDN5在食管鳞癌中表达呈正相关（r=0．537,P〈o．05）。结论CDC2和CLDN5在食管鳞癌的发生、发展过程中可能发挥重要作用,可能作为食管癌临床早期诊断的重要指标。     

Asymmetric reduction of ketopantolactone using a strictly (<Emphasis Type="Italic">R</Emphasis>)-stereoselective carbonyl reductase through efficient NADPH regeneration and the substrate constant-feeding strategy

Objectives

To characterize a recombinant carbonyl reductase from Saccharomyces cerevisiae (SceCPR1) and explore its use in asymmetric synthesis of (R)-pantolactone [(R)-PL].

Results

The NADPH-dependent SceCPR1 exhibited strict (R)-enantioselectivity and high activity in the asymmetric reduction of ketopantolactone (KPL) to (R)-PL. Escherichia coli, coexpressing SceCPR1 and glucose dehydrogenase from Exiguobacterium sibiricum (EsGDH), was constructed to fulfill efficient NADPH regeneration. During the whole-cell catalyzed asymmetric reduction of KPL, the spontaneous hydrolysis of KPL significantly affected the yield of (R)-PL, which was effectively alleviated by the employment of the substrate constant-feeding strategy. The established whole-cell bioreduction for 6 h afforded 458 mM (R)-PL with the enantiomeric excess value of >99.9% and the yield of 91.6%.

Conclusions

Escherichia coli coexpressing SceCPR1 and EsGDH efficiently catalyzed the asymmetric synthesis of (R)-PL through the substrate constant-feeding strategy.

     

Multi-scale characterization of swine femoral cortical bone

Multi-scale experimental work was carried out to characterize cortical bone as a heterogeneous material with hierarchical structure, which spans from nanoscale (mineralized collagen fibril), sub-microscale (single lamella), microscale (lamellar structures), to mesoscale (cortical bone) levels. Sections from femoral cortical bone from 6, 12, and 42 months old swine were studied to quantify the age-related changes in bone structure, chemical composition, and mechanical properties. The structural changes with age from sub-microscale to mesoscale levels were investigated with scanning electron microscopy and micro-computed tomography. The chemical compositions at mesoscale were studied by ash content method and dual energy X-ray absorptiometry, and at microscale by Fourier transform infrared microspectroscopy. The mechanical properties at mesoscale were measured by tensile testing, and elastic modulus and hardness at sub-microscale were obtained using nanoindentation. The experimental results showed age-related changes in the structure and chemical composition of cortical bone. Lamellar bone was a prevalent structure in 6 months and 12 months old animals, resorption sites were most pronounced in 6 months old animals, while secondary osteons were the dominant features in 42 months old animals. Mineral content and mineral-to-organic ratio increased with age. The structural and chemical changes with age corresponded to an increase in local elastic modulus, and overall elastic modulus and ultimate tensile strength as bone matured.     

莳萝蒿适应盐渍环境的Na~+区域化方式和生理特征

莳萝蒿是广泛分布在我国北方的一种特殊类型的菊科盐生植物,阐明莳萝蒿特殊的耐盐机制和生理特征有助于丰富植物抗盐性研究的内容。用0、100、200、300、400 mmol/L Na Cl处理莳萝蒿7 d后,比较莳萝蒿盐处理植株与对照植株在生长和生理方面的差异,并详细分析了Na+在莳萝蒿体内的积累水平和区域化方式。结果显示:莳萝蒿虽然能够耐受400 mmol/L Na Cl,但盐处理显著抑制了莳萝蒿的生长,整株鲜重随着盐处理浓度的升高逐渐减小。在水分生理方面,随着盐处理浓度的升高,莳萝蒿叶片细胞的渗透调节能力逐渐增强,其叶片肉质化程度却呈逐渐降低的趋势。分析盐处理对光合作用的影响发现,盐处理后莳萝蒿叶片光合速率与气孔导度显著下降,而其PSⅡ光化学活性并未受到抑制,叶绿素含量甚至逐渐增大,说明盐处理后莳萝蒿叶片光合速率的降低主要是由于气孔因素造成的,而不是由于光合结构被破坏。莳萝蒿体内的Na+含量随着盐处理浓度的升高显著增加,400 mmol/L Na Cl条件下叶、茎、根中的Na+含量分别高达321.4、242.1和182.3μmol/g鲜重;莳萝蒿体内的Na+70%以上积累在叶片内,而叶片内98%左右的Na+积累在叶片原生质体中,叶片原生质体中的Na+平均浓度是质外体1.2—1.8倍,推测其叶片细胞内存在着有效的Na+区域化机制。盐处理后莳萝蒿叶片液泡膜V-H+-ATPase的质子泵活性比对照增加了30%—50%,液泡膜Na+/H+逆向转运活性则增加至对照的4—7倍,进一步证实莳萝蒿叶片具有较强的液泡Na+区域化能力。随着盐处理浓度的升高,Na+在叶片中的分布比例相对减少,V-H+-ATPase的质子泵活性和Na+/H+逆向转运活性增幅也减缓。这种Na+区域化能力使莳萝蒿获得了较强的耐盐性,有效保护了其光系统,降低了细胞汁液渗透势。但是盐处理后这种耐盐方式并不能阻止莳萝蒿叶片肉质化程度和光合活性下降,莳萝蒿生长仍然受盐抑制,说明Na+区域化是莳萝蒿适应盐渍环境的必要条件而非充分条件。