Activation of adenylate cyclase by forskolin increases the protein stability of RCAN1 (DSCR1 or Adapt78)

Overexpression of Regulator of Calcineurin 1 (RCAN1/DSCR1/Adapt78) is known to inhibit the calcineurin-NFAT dependent signaling pathway. In this report, we find that activation of adenylate cyclase by forskolin increases the expression of RCAN1 through the increase of the protein’s half-life. The ability of forskolin to increase the accumulation of RCAN1 protein is significantly inhibited with protein kinase A inhibitors such as KT5720 and H-89. Furthermore, forskolin targets the central and C-terminal region of RCAN1 and enhances the inhibitory effect of RCAN1 on the calcineurin-mediated activation of NFAT. Our findings provide the first evidence that the accumulation of the RCAN1 protein by cAMP acts as an important regulatory mechanism in the control of the calcineurin-dependent cellular pathway.

Structured summary

MINT-7262390: PKA (uniprotkb:P22694) phosphorylates (MI:0217) RCAN1 (uniprotkb:P53805) by protein kinase assay (MI:0424)     

New discovery rarely runs smooth: an update on progranulin/TNFR interactions

Progranulin (PGRN) is a growth factor implicated in various pathophysiological processes, including wound healing, inflammation, tumorigenesis, and neurodegeneration. It was previously reported that PGRN binds to tumor necrosis factor receptors (TNFR) and has therapeutic effects in inflammatory arthritis (Tang et. al, in Science 332:478−484, 2011); however, Chen et al. reported their inability to demonstrate the PGRN-TNFR interactions under their own conditions (Chen et. al, in J Neurosci 33:9202−9213, 2013). A letter-to-editor was then published by the original group in response to the Chen et al. paper that discussed the reasons for the latter’s inability to recapitulate the interactions. In addition, the group published follow-up studies that further reinforced and dissected the interactions of PGRNTNFR. Recently, the dispute about the legitimacy of PGRN-TNFR interactions appears to be finally settled with independent confirmations of these interactions in various conditions by numerous laboratories. This review presents a chronological update on the story of PGRN-TNFR interactions, highlighting the independent confirmations of these interactions in various diseases and conditions.     

Long Noncoding RNA Expression Profiling Reveals Upregulation of Uroplakin 1A and Uroplakin 1A Antisense RNA 1 under Hypoxic Conditions in Lung Cancer Cells

Hypoxia plays important roles in cancer progression by inducing angiogenesis, metastasis, and drug resistance. However, the effects of hypoxia on long noncoding RNA (lncRNA) expression have not been clarified. Herein, we evaluated alterations in lncRNA expression in lung cancer cells under hypoxic conditions using lncRNA microarray analyses. Among 40,173 lncRNAs, 211 and 113 lncRNAs were up- and downregulated, respectively, in both A549 and NCI-H460 cells. Uroplakin 1A (UPK1A) and UPK1A-antisense RNA 1 (AS1), which showed the highest upregulation under hypoxic conditions, were selected to investigate the effects of UPK1A-AS1 on the expression of UPK1A and the mechanisms of hypoxia-inducible expression. Following transfection of cells with small interfering RNA (siRNA) targeting hypoxia-inducible factor 1α (HIF-1α), the hypoxia-induced expression of UPK1A and UPK1A-AS1 was significantly reduced, indicating that HIF-1α played important roles in the hypoxia-induced expression of these targets. After transfection of cells with UPK1A siRNA, UPK1A and UPK1A-AS1 levels were reduced. Moreover, transfection of cells with UPK1A-AS1 siRNA downregulated both UPK1A-AS1 and UPK1A. RNase protection assays demonstrated that UPK1A and UPK1A-AS1 formed a duplex; thus, transfection with UPK1A-AS1 siRNA decreased the RNA stability of UPK1A. Overall, these results indicated that UPK1A and UPK1A-AS1 expression increased under hypoxic conditions in a HIF-1α-dependent manner and that formation of a UPK1A/UPK1A-AS1 duplex affected RNA stability, enabling each molecule to regulate the expression of the other.     

腺苷A2a受体在博莱霉素诱导的小鼠肺纤维化中的作用

目的：观察腺苷A_2a受体（A_2aR）在小鼠肺纤维化形成中的调控作用。方法：30只雄性SPF级野生型BALB/C小鼠和20只A_2aR基因敲除BALB/C小鼠,随机分为以下5组：野生型小鼠对照组（A组）、野生型小鼠纤维化组（B组）、A_2aR基因敲除小鼠对照组（C组）、A_2aR基因敲除小鼠纤维化组（D组）、野生型小鼠纤维化+A_2aR激动剂（CGS21680）组（E组）,每组各10只。纤维化组小鼠气管内注入博莱霉素（bleomycin,BLM）溶液50μl （5 mg/kg体重）,对照组在气管内注入等体积生理盐水,注射后立即将小鼠直立旋转3~5 min,使其均匀分布于两肺。A、B、C、D各组每天腹腔注射生理盐水0.5 ml,E组每天腹腔注射A_2aR激动剂（CGS21680）0.5 ml （0.25 mg/kg体重）,连续28 d。第29天取血检测血清转化生长因子β1（TGF-β1）含量;检测肺组织中羟脯氨酸（hydroxyproline,Hyp）、TGF-β1和A_2aR蛋白质含量,TGF-β1 mRNA、A_2aR mRNA的表达,并观察肺组织光镜和超微结构。结果：①光镜和超微结构提示,B组肺泡壁增厚破坏,肺泡腔狭窄或部分陷闭,纤维增生,炎细胞浸润,I型、Ⅱ型肺泡上皮细胞明显空泡化,D组较B组明显,E组表现较B组减轻。Masson染色提示B组、D组小鼠肺组织纤维增生明显,E组明显减少,提示基因缺失加重了小鼠肺纤维化的形成。②与A组比较,B组血清TGF-β1含量,肺组织Hyp含量,TGF-β1、A_2aR蛋白质含量和TGF-β1 mRNA、A_2aR mRNA的表达均明显增高（P<0.01,P<0.05）。与C组比较,D组血清TGF-β1含量、肺组织Hyp、TGF-β1含量和TGF-β1 mRNA表达明显增高（P<0.01,P<0.05）,提示肺纤维化小鼠肺组织Hyp含量增高,TGF-β1表达上调。③与B组相比,D组血清TGF-β1、肺组织Hyp、TGF-β1含量和TGF-β1 mRNA表达明显增高（P<0.01）。与B组比较,E组A_2aR蛋白质含量和A_2aR mRNA的表达均增高（P<0.01）,而血清TGF-β1、肺组织Hyp和TGF-β1蛋白含量及TGF-β1 mRNA表达较B组明显下降（P<0.01,P<0.05）,提示A_2aR基因敲除小鼠肺组织Hyp含量增高、TGF-β1表达上调;A_2aR激动剂可使A_2aR表达上调、TGF-β1表达下降。结论：A_2aR基因敲除小鼠的肺纤维化明显增加。A_2aR在肺纤维化形成中反应性增高,可通过降低TGF-β1的表达而抑制肺纤维化形成。     

Logic regression analysis of association of gene polymorphisms with low HDL: Tehran Lipid and Glucose Study

Introduction

Logic regression is a generalized regression method that can recognize complex Boolean interactions of binary variables. It has been successfully applied to single-nucleotide polymorphism (SNP) data because of the importance of interactions in SNP association studies. The objective of this study is to assess the association between high-density lipoprotein (HDL) function and some related gene polymorphisms after adjusting for potential confounders using logic regression.

Methods

Subjects in this cross-sectional study were randomly selected from among participants of the Tehran Lipid and Glucose Study (TLGS). A total of 436 subjects (172 men and 264 women), aged ≥ 20 years, were selected to be included in the current study. Logic regression analysis was used in order to recognize the combination of genetic main effects and possible interactions associated with HDL-C level. Cross validation and randomization test were carried out to avoid over fitting of the models.

Results

The cross validation test suggested three Boolean combinations with four predictors for a fully-adjusted logic model. The fully adjusted model showed that those who carry an Apo E gene E3 allele or have high TG level have an odds ratio of 2.35 (95% CI:1.3–4.25) for having low HDL compared to other subjects. In addition, subjects with high TG level have an odds ratio of 2.73 (95% CI: 1.65, 4.53) for having low HDL.

Conclusion

The results showed that logic regression is a powerful method to find the interaction between high TG level and Apo E polymorphism associated with low HDL.     

Does cytochrome P450 1A1 MspI polymorphism increase acute lymphoblastic leukemia risk? Evidence from 2013 cases and 2903 controls

Previous studies indicated that cytochrome P450 1A1 (CYP1A1) MspI polymorphism might be a possible risk factor for several malignancies. Increasing investigations have been conducted on the association of CYP1A1 MspI polymorphisms with acute lymphoblastic leukemia (ALL). However, the results were controversial. The goal of the present study was to address this controversy by pooling and analyzing the published data. Therefore, quantitative meta-analyses evaluating the association of CYP1A1 MspI variation with ALL were performed and subgroup analyses on ethnicity, age groups and source of controls were further carried out. After a rigorous search in the Medline, EMBASE, OVID, ScienceDirect, and CNKI databases, all eligible studies for the period up to May 2012 were identified and screened according to the inclusion and exclusion criteria. Consequently, a total of fourteen case–control studies including 2013 cases and 2903 controls were selected for analysis. The overall data indicated a significant association of CYP1A1 MspI polymorphism with ALL risk (CC + TC vs TT: OR = 1.33; 95%CI = 1.05–1.69). In a subgroup analysis according to ethnicity, no associations were shown among Asians, Caucasians and Mixed ethnicity subgroups. In the subgroup analysis regarding age groups, increased risk was observed in the childhood ALL subgroup (C vs T: OR = 1.23; 95%CI = 1.04–1.45; CC + TC vs TT: OR = 1.31; 95%CI = 1.08–1.59). In the subgroup analysis stratified by source of controls, significant associations were observed in the population-based subgroup (CC + TC vs TT: OR = 1.33; 95%CI = 1.03–1.71). In conclusion, the results of the present study suggest that CYP1A1 MspI polymorphism might be a risk factor for ALL, particularly childhood ALL. Future well-designed high quality investigations with large sample sizes are required to elucidate the gene polymorphism–ALL relationship and gene–environment interactions.     

DNA family shuffling within the chicken avidin protein family - A shortcut to more powerful protein tools

Avidins represent an interesting group of proteins showing high structural similarity and ligand-binding properties but low similarity in primary structure. In this study, we show that it is possible to create functional chimeric proteins from the avidin protein family when applying DNA family shuffling to the genes of the avidin protein family: avidin, avidin related gene 2 and biotin-binding protein A. The novel chimeric proteins were selected by phage display biopanning against biotin, and the selected enriched proteins were characterized, displaying diverse features distinct from the parental genes, including binding to cysteine.     

安徽上新五褶兔两新种及淮南上新五褶兔化石(英文)

近年,作者在安徽淮南大居山陆续发现了数量很多的上新五褶兔化石,其中包括保存较完整的数十个头骨及上百个下颌骨,均产自新洞、无名洞及铁四局洞穴、裂隙堆积,分别记述为大居山上新五褶兔Pliopentalagus dajushanensis sp.nov.和安徽上新五褶兔Pl.anhuiensis sp.nov.两新种。淮南地区的晚新生代洞穴和裂隙堆积十分发育,其中常含有丰富的脊椎动物化石,而且在时、空分布上有一定的规律。区内洞穴和裂隙堆积垂直分布至少有6个水平层,最高的第6层海拔高度为160 m(如大居山老洞),第5层(如大居山新洞、无名洞)及第4层(如大居山铁四局洞穴)的海拔高度分别为130 m和90 m,属于新近纪,常含有丰富的上新五褶兔化石;第3层为第四纪早期(如大居山西裂隙),未见上新五褶兔,代之出现丝绸兔(Sericolagus sp.),第2层为中更新世(如大顶山西裂隙),出现野兔(Lepus sp.)。迄今为止,安徽淮南地区共发现了3种上新五褶兔化石,即淮南上新五褶兔Pl.huainanensis (金昌柱,2004)、大居山上新五褶兔Pl.dajushanensis sp.nov.及安徽上新五褶兔Pl. anhuiensis sp.nov.,至少涉及了3个不同地质时期的动物群:一为Pl.huainanensis,Kowalskia neimengensis,Adcrocuta eximia等代表的老洞晚中新世动物群,二为Pl.dajushanensis sp.nov.和Promimomys asiaticus等代表的新洞早上新世动物群,三为Pl.anhuiensis sp.nov.和Kowalskia yinanensis所代表的晚上新世动物群。淮南地区发现的3种上新五褶兔地史分布较连续,演化特征明显,它们构成上新五褶兔连续的进化系列。从晚中新世至晚上新世,淮南大居山上新五褶兔体型从小变大;p3较原始的釉岛状后内褶沟逐渐向舌侧开放(晚中新世种Pl. huainanensis的后内褶沟均为釉岛状,早上新世种Pl.dajushanensis为83.9%,晚上新世种Pl. anhuiensis为33.3%);p4-m2的前外褶沟逐渐退化,其下跟座舌侧的釉质层越来越变细。依性状分析,安徽发现的3种上新五褶兔化石与日本琉球奄美黑兔(Pentalagus furnessi)具有密切的亲缘关系,但从日本奄美黑兔的p4-m2完全缺失前外褶沟、颊齿褶沟釉质层的褶曲异常复杂等衍生性状看,上新五褶兔和奄美黑兔的系统演化关系较为复杂,尚有待发现更多早更新世的材料进一步探讨。     

Multisite promiscuity in the processing of endogenous substrates by human carboxylesterase 1

Human carboxylesterase 1 (hCE1) is a drug and endobiotic-processing serine hydrolase that exhibits relatively broad substrate specificity. It has been implicated in a variety of endogenous cholesterol metabolism pathways including the following apparently disparate reactions: cholesterol ester hydrolysis (CEH), fatty acyl Coenzyme A hydrolysis (FACoAH), acyl-Coenzyme A:cholesterol acyltransfer (ACAT), and fatty acyl ethyl ester synthesis (FAEES). The structural basis for the ability of hCE1 to perform these catalytic actions involving large substrates and products has remained unclear. Here we present four crystal structures of the hCE1 glycoprotein in complexes with the following endogenous substrates or substrate analogues: Coenzyme A, the fatty acid palmitate, and the bile acids cholate and taurocholate. While the active site of hCE1 was known to be promiscuous and capable of interacting with a variety of chemically distinct ligands, these structures reveal that the enzyme contains two additional ligand-binding sites and that each site also exhibits relatively non-specific ligand-binding properties. Using this multisite promiscuity, hCE1 appears structurally capable of assembling several catalytic events depending, apparently, on the physiological state of the cellular environment. These results expand our understanding of enzyme promiscuity and indicate that, in the case of hCE1, multiple non-specific sites are employed to perform distinct catalytic actions.     

The Cytochrome P4501A1 gene polymorphisms and idiopathic male infertility risk: A meta-analysis

Studies of the relationship between male infertility and CYP1A1 polymorphisms are inconclusive. To drive a more precise estimation, we performed a meta-analysis based on 1060cases and 1225 controls from 7 published case–control studies. PubMed and CNKI literature search were conducted to identify all eligible studies investigating such a relationship. Crude odds ratios (ORs) with 95% confidence intervals (CIs) were used to assess the strength of association in the additive model, dominant model, recessive model, and allele-frequency genetic model. In the overall analysis, the frequency of CYP1A1*2A genotype was significantly associated with susceptibility to idiopathic male infertility. Further stratified analysis by ethnicity showed notable association between the polymorphism and the risk of idiopathic male infertility in Asians. In conclusion, these results support that the CYP1A1*2A genotype polymorphism mainly contributes to idiopathic male infertility susceptibility in Asians but not in Caucasians.     

Proteases in autophagy

Autophagy is a process involved in the proteolytic degradation of cellular macromolecules in lysosomes, which requires the activity of proteases, enzymes that hydrolyse peptide bonds and play a critical role in the initiation and execution of autophagy. Importantly, proteases also inhibit autophagy in certain cases. The initial steps of macroautophagy depend on the proteolytic processing of a particular protein, Atg8, by a cysteine protease, Atg4. This processing step is essential for conjugation of Atg8 with phosphatidylethanolamine and, subsequently, autophagosome formation. Lysosomal hydrolases, known as cathepsins, can be divided into several groups based on the catalitic residue in the active site, namely, cysteine, serine and aspartic cathepsins, which catalyse the cleavage of peptide bonds of autophagy substrates and, together with other factors, dispose of the autophagic flux. Whilst most cathepsins degrade autophagosomal content, some, such as cathepsin L, also degrade lysosomal membrane components, GABARAP-II and LC3-II. In contrast, cathepsin A, a serine protease, is involved in inhibition of chaperon-mediated autophagy through proteolytic processing of LAMP-2A. In addition, other families of calcium-dependent non-lysosomal cysteine proteases, such as calpains, and cysteine aspartate-specific proteases, such as caspases, may cleave autophagy-related proteins, negatively influencing the execution of autophagic processes. Here we discuss the current state of knowledge concerning protein degradation by autophagy and outline the role of proteases in autophagic processes. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.     

Structural determinants of glutathione transferases with azathioprine activity identified by DNA shuffling of alpha class members

A library of alpha class glutathione transferases (GSTs), composed of chimeric enzymes derived from human (A1-1, A2-2 and A3-3), bovine (A1-1) and rat (A2-2 and A3-3) cDNA sequences was constructed by the method of DNA shuffling. The GST variants were screened in bacterial lysates for activity with the immunosuppressive agent azathioprine, a prodrug that is transformed into its active form, 6-mercaptopurine, by reaction with the tripeptide glutathione catalyzed by GSTs. Important structural determinants for activity with azathioprine were recognized by means of primary structure analysis and activities of purified enzymes chosen from the screening. The amino acid sequences could be divided into 23 exchangeable segments on the basis of the primary structures of 45 chosen clones. Segments 2, 20, 21, and 22 were identified as primary determinants of the azathioprine activity representing two of the regions forming the substrate-binding H-site. Segments 21 and 22 are situated in the C-terminal helix characterizing alpha class GSTs, which is instrumental in their catalytic function. The study demonstrates the power of DNA shuffling in identifying segments of primary structure that are important for catalytic activity with a targeted substrate. GSTs in combination with azathioprine have potential as selectable markers for use in gene therapy. Knowledge of activity-determining segments in the structure is valuable in the protein engineering of glutathione transferase for enhanced or suppressed activity.     

Impact of the loss of caveolin-1 on lung mass and cholesterol metabolism in mice with and without the lysosomal cholesterol transporter,Niemann–Pick type C1

Caveolin-1 (Cav-1) is a major structural protein in caveolae in the plasma membranes of many cell types, particularly endothelial cells and adipocytes. Loss of Cav-1 function has been implicated in multiple diseases affecting the cardiopulmonary and central nervous systems, as well as in specific aspects of sterol and lipid metabolism in the liver and intestine. Lungs contain an exceptionally high level of Cav-1. Parameters of cholesterol metabolism in the lung were measured, initially in Cav-1-deficient mice (Cav-1^−/−), and subsequently in Cav-1^−/− mice that also lacked the lysosomal cholesterol transporter Niemann–Pick C1 (Npc1) (Cav-1^−/−:Npc1^−/−). In 50-day-old Cav-1^−/− mice fed a low- or high-cholesterol chow diet, the total cholesterol concentration (mg/g) in the lungs was marginally lower than in the Cav-1^+/+ controls, but due to an expansion in their lung mass exceeding 30%, whole-lung cholesterol content (mg/organ) was moderately elevated. Lung mass (g) in the Cav-1^−/−:Npc1^−/− mice (0.356 ± 0.022) markedly exceeded that in their Cav-1^+/+:Npc1^+/+ controls (0.137 ± 0.009), as well as in their Cav-1^−/−:Npc1^+/+ (0.191 ± 0.013) and Cav-1^+/+:Npc1^−/− (0.213 ± 0.022) littermates. The corresponding lung total cholesterol contents (mg/organ) in mice of these genotypes were 6.74 ± 0.17, 0.71 ± 0.05, 0.96 ± 0.05 and 3.12 ± 0.43, respectively, with the extra cholesterol in the Cav-1^−/−:Npc1^−/− and Cav-1^+/+:Npc1^−/− mice being nearly all unesterified (UC). The exacerbation of the Npc1 lung phenotype and increase in the UC level in the Cav-1^−/−:Npc1^−/− mice imply a regulatory role of Cav-1 in pulmonary cholesterol metabolism when lysosomal sterol transport is disrupted.     

Secretoglobin 1A member 1 (SCGB1A1) + 38A/G polymorphism is associated with asthma risk: A meta-analysis

Background

Epidemiological studies have evaluated the association between Secretoglobin 1A member 1 (SCGB1A1) + 38A/G polymorphism and asthma, but the results remain inconclusive. The aim of this study was to perform a meta-analysis to investigate a more authentic association between SCGB1A1 + 38A/G polymorphism and asthma.

Methods

Published literature from PubMed, Web of Science, China National Knowledge Infrastructure (CNKI), and Embase databases were searched for eligible publications. Pooled odds ratios (ORs) with 95% confidence intervals (CIs) were calculated using random or fixed-effect model according the between-study heterogeneity.

Results

A total of 19 case-control studies in 18 articles were included in the meta-analysis, including 3191 cases and 5182 controls. We found that SCGB1A1 + 38A/G polymorphism was associated with a significantly increased risk of asthma risk when all studies were pooled in a dominant model (OR = 1.29; 95% CI 1.08–1.54; P = 0.005). The cumulative meta-analysis and sensitivity analysis further strengthened the stability of the result. Furthermore, publication bias was not detected.

Conclusions

This study suggested that SCGB1A1 + 38A/G polymorphism was a risk factor for asthma. Further large and well-designed studies are needed to confirm this association.     

The WNT7A G204S mutation is associated with both Al-Awadi–Raas Rothschild syndrome and Fuhrmann syndrome phenotypes

Two syndromes are known to be associated with WNT7A mutations: Al-Awadi–Raas-Rothschild syndrome (AARRS) and Fuhrmann syndrome. Woods et al. (2006) showed that there is complete and partial loss of WNT7A function in these two syndromes respectively. Therefore, both syndromes have similar clinical features but the phenotype in Fuhrmann syndrome is less severe. The G204S mutation was previously reported to result in AARRS phenotype in three Saudi families. In the current communication, we report on a different unrelated Saudi patient with the same mutation but the patient had Fuhrmann syndrome phenotype. We believe this case is important because it questions the presence of a phenotype–genotype correlation in WNT7A mutations and because it demonstrates that the G204S mutation may be associated with both AARRS and Fuhrmann phenotypes.     

人质子感知受体G2A和OGR1在低氧性肺动脉高压患者外周血细胞中的表达

目的：检测人质子感知受体G蛋白偶联受体2A （G2A）和卵巢癌G蛋白偶联受体1（OGR1）在低氧性肺动脉高压（HPH）患者外周血细胞中的变化。方法：研究对象选取31例HPH患者为低氧性肺动脉高压组（HPH组）,男性16例,女性15例,年龄（65.19 ±5.86）岁。同时符合中华医学会呼吸病学分会慢性阻塞性肺疾病学组诊断标准和呼吸衰竭诊断标准,选取30例健康体检者为正常组（NC组）,男15例,女15例,年龄（63.47 ±6.16）岁。心脏彩超计算HPH组肺动脉压力、进行血气分析和肺功能检测,采集外周血检测G2A、OGR1基因mRNA表达水平、血清肿瘤坏死因子α（TNF-α）水平。结果：HPH组PaCO₂较NC组明显增高（P<0.05）,1 s用力呼气容积占预计值的百分比（FEV1pro%）和1 s用力呼气容积与用力肺活量比值（FEV1/FVC）明显低于NC组（P<0.05）。HPH组外周血中G2A mRNA及TNF-α含量明显高于NC组（P<0.05）。OGR1 mRNA与NC组无差别。HPH组G2A mRNA及TNF-α表达与肺动脉压力呈显著正相关。结论：肺动脉高压患者外周血细胞中质子感知受体G2A表达增加,TNF-α水平增加,G2A的表达和TNF-α水平与肺动脉压力呈明显正相关。     

非小细胞肺癌R A SsF IA 基因甲基化及下游基因表达

目的：研究非小细胞肺癌RASSF1A基因启动子甲基化及下游基因表达情况。方法：留取58例非小细胞肺癌患者手术标本及正常肺组织,甲基化特异性PCR分析RASSF1A基因启动子甲基化情况,同时Northern blot分析SM22、SPARC、SDHB和CC-ND3等4种RASSFIA下游基因的表达情况。结果：58例非小细胞肺癌中RASSFIA基因启动子甲基化阳性率为34．5％,甲基化与各临床参数之间无显著相关性,SM22和SPARC在RASSF1A甲基化组表迭明显下调。结论：原发性非小细胞肺癌中存在着较高比例的RASSF1A启动子过度甲基化,并与下游基因SM22和SPARC的表达下调密切相关。提示RASSF1A在非小细胞肺癌的发生中起着多种作用。     

Cholesterol 25-hydroxylation activity of CYP3A

To date, many studies have been conducted using 25-hydroxycholesterol, which is a potent regulator of lipid metabolism. However, the origins of this oxysterol have not been entirely elucidated. Cholesterol 25-hydroxylase is one of the enzymes responsible for the metabolism of 25-hydroxycholesterol, but the expression of this enzyme is very low in humans. This oxysterol is also synthesized by sterol 27-hydroxylase (CYP27A1) and cholesterol 24-hydroxylase(CYP46A1), but it is only a minor product of these enzymes. We now report that CYP3A synthesizes a significant amount of 25-hydroxycholesterol and may participate in the regulation of lipid metabolism. Induction of CYP3A by pregnenolone-16α-carbonitrile caused the accumulation of 25-hydroxycholesterol in a cell line derived from mouse liver. Furthermore, treatment of the cells with troleandomycin, a specific inhibitor of CYP3A, significantly reduced cellular 25-hydroxycholesterol concentrations. In cells that overexpressed human recombinant CYP3A4, the activity of cholesterol 25-hydroxylation was found to be higher than that of cholesterol 4β-hydroxylation, a known marker activity of CYP3A4. In addition, 25-hydroxycholesterol concentrations in normal human sera correlated positively with the levels of 4β-hydroxycholesterol (r = 0.650, P < 0.0001, n = 78), but did not significantly correlate with the levels of 27-hydroxycholesterol or 24S-hydroxycholesterol. These results demonstrate the significance of CYP3A on the production of 25-hydroxycholesterol.     

Quantitative evaluation of bromodichloromethane metabolism by recombinant rat and human cytochrome P450s

We report quantitative estimates of the parameters for metabolism of bromodichloromethane (BDCM) by recombinant preparations of hepatic cytochrome P450s (CYPs) from rat and human. Earlier work identified CYP2E1, CYP2B1/2 and CYP1A2 as activating enzymes necessary for hepatotoxicity in rat. In order to extend an existing PBPK model for rat to include a capability for extrapolation to humans, it is necessary to evaluate quantitatively the principal metabolic pathways in both species. We have conducted in vitro experiments using recombinant preparations of the three rat CYP isoenzymes mentioned above and for CYP2C11 and CYP3A1 as well. Similar experiments have been performed with human recombinant isoenzymes for CYP2E1, CYP1A2, CYP2A6, CYP2B6, CYP2D6 and CYP3A4. Results indicate that the principal metabolizing enzymes in rat are those identified previously, CYP2E1, CYP2B1/2 and CYP1A2. CYP3A1 may also have some activity. In human, CYP2E1, CYP1A2 and CYP3A4 show substantial activity, and CYP2A6 also measurably metabolizes BDCM. In both species, CYP2E1 is the low K(m) isoenzyme, with K(m) approximately 27-fold lower than those for the isoenzymes with the next lowest K(m). In addition, the metabolic parameters, K(m) and k(cat), for rat and human CYP2E1 were nearly identical. The metabolic parameters for CYP1A2, the only other isoenzyme active in both species, were not similar across species. In addition, calculations based on the kinetic constants obtained are compared to results from two in vivo experiments to show that the in vitro kinetic data is relevant to in vivo exposures. We conclude that although several CYPs metabolize BDCM, at low concentration/exposure, BDCM metabolism is dominated by CYP2E1 in both rat and human, but that other isoenzymes can be important at higher concentrations. We further conclude that the kinetic data are consistent with existing in vivo results.