来源于中国南海的微生物新种乙醇速生杆菌(Celeribacter ethanolicus)多相分类研究（英文）

【目的】研究来源于南海海水的一株速生杆菌属菌株NH195T的多相分类。【方法】采用表型、基因型和化学分类方法,并综合系统发育关系结果,分析菌株NH195T的分类学地位。【结果】菌株NH195T是一株革兰氏阴性、好氧、杆状、无运动性细菌;能积累Poly-β-hydroxy butyrate(PHB);能在0.5%-10.0%(质量体积比)NaCl浓度,pH 5.0-9.0和20-40°C条件下生长,最适NaCl生长浓度为1.0%-3.0%;氧化酶、触酶和脲酶反应结果阳性。菌株NH195T主要呼吸醌为Q-10,主要脂肪酸为C18:1ω7c、C18:1ω6c和11 methyl C_(18:1)ω7c,主要极性脂为磷脂酰胆碱、磷脂酰甘油、双磷脂酰甘油、一个未知的氨基脂和两个未知脂。基因组G+C含量为61.3 mol%。基于16S rRNA基因的系统发育结果显示,菌株NH195T隶属于速生杆菌属;其与速生杆菌属标准菌株的16S rRNA基因相似性范围为94.4%-97.7%。菌株NH195T与速生杆菌属标准菌株C.halophilus ZXM137T和C.indicus P73T的平均核苷酸一致性(ANI)分别为78.6%和78.0%;基于基因组数据计算所得的DNA杂交同源率分别为26.1%和23.0%。【结论】基于表型和基因型结果,菌株NH195T代表了速生杆菌属一个新物种,命名为Celeribacter ethanolicus,标准菌株为NH195T(CGMCC 1.15406T=JCM 31095T)。     

河南省白龟山水库下游水体氨化细菌分离鉴定及其降解有机氮条件

【目的】从河南省白龟山水库下游水体中分离筛选氨化细菌,并研究其降解有机氮的条件。【方法】利用选择性培养基从微污染水源水中筛选氨化细菌,进一步比较了不同氨化细菌降解有机氮的效果;采用单因子法研究菌株N24降解有机氮的条件;通过形态、生理生化特征及16S rRNA基因序列分析对菌株N24进行鉴定。【结果】从微污染水源水中分离筛选到4株氨化细菌,其中菌株N24培养48 h后氨氮浓度较高,达到138.926 mg/L。菌株N24降解有机氮最适温度为30-35°C,最适初始pH值为6.0,500 mL摇瓶最适装液量75 mL。菌株N24被鉴定为弯曲芽孢杆菌(Bacillus flexus,GenBank登录号:JX291240.1),其16S rRNA基因序列与基因库中芽孢杆菌属菌株的16S rRNA基因序列有99%-100%的相似性,与弯曲芽孢杆菌(Bacillus flexus IFO15717T,GenBank登录号:NR024691.1)的遗传距离最近。【结论】菌株N24是一株高效降解有机氮的弯曲芽孢杆菌;本研究丰富了降解有机氮菌种资源,可为该菌在环境工程领域的实际应用提供理论基础。     

一个可降解直链烷基苯磺酸盐的新种

本文鉴定了一株可完全降解直链烷基苯磺酸盐（Linear Alkylbenzene Sulphonate,简称LAS）的菌株GZ6。革兰氏染色阴性,细胞为杆状或短杆状,大小为0.5μm～0.8 μm×1.0μm～2.0 μm,其生长pH范围为pH6.0～10.0,最适生长pH为7.0,生长温度范围为4℃～40℃,最适生长温度为30℃。生化特征测定除过氧化氢酶、尿酶、精氨酸脱羧酶反应为阳性,其它均为阴性。可利用Chloridazon、安替比林（antipyrin）以及LAS等为碳源。不能利用大多数糖醇。醌组分以泛醌Q10为主。菌体脂肪酸主要为C18∶1、C16∶0及C16∶1。DNA中G+C mol％含量为7010。16S rRNA 序列分析表明菌株GZ6 与其亲缘关系最近的菌株Phenylobacterium immobile DSM1986T序列相似值为9749％,DNADNA杂交率为40%。菌株GZ6具极生鞭毛,可运动,两者在细胞形态有很大差异。故将菌株GZ6定为苯基杆菌属的新种可动苯基杆菌（Phenylobaterium mobile） GZ6。     

一株新的耐辐射菌Deinococcus guangxiensis sp.nov.的分离鉴定及耐辐射特性分析

摘要：【目的】从广西大学辐射中心辐射源附近被常年辐射的水样中分离并鉴定出新的耐辐射菌株,并对其耐辐射特性进行研究。【方法】通过GBM培养基分离培养得到一株新的耐辐射菌株,命名为WGR700T。应用生理生化试验,脂肪酸含量,(G+C) mol％含量测定以及16S rRNA序列同源性分析等方法对菌株进行鉴定,同时对WGR700T的耐辐射特性进行分析。【结果】菌株WGR700T为革兰氏阴性,杆状,没有鞭毛,不能运动,厌氧并能产生红色素。最佳生长温度和PH分别为37℃和pH7.0,主要的呼吸醌是MK-8,细胞壁内还有鸟氨酸,主要脂肪酸为16:1ω7c, 16:0, 15:1ω6c, iso-15:0和 iso-17:0。G+C含量为64.7mol%。菌株WGR700T具有很强的UV(>728 J/m2)和电离辐射抗性(D10 = 9.8 kGy)。菌株WGR700T和奇异球菌属（Deinococcus）内其它菌种16S rRNA有很高的相似性（87.1-95.6%）。【结论】根据16S rRNA及生理生化特征区别,菌株WGR700T应是奇异球菌属的一个新种,命名为Deinococcus guangxiensis sp. nov. 模式菌株为WGR700T(= CGMCC1.7045T =CICC 10360T = JCM 15082T)。     

一株羽毛降解菌的分离与鉴定

【目的】从土壤中分离并鉴定羽毛降解菌,测定其生长最适温度及起始pH,并观察酶活动态。【方法】采用系列稀释法和选择培养基法筛选目的菌株,基于16S rRNA基因序列及Biolog方法鉴定其分类地位,利用全自动生长曲线分析仪监测菌株的最适生长条件,并通过测定蛋白水解活性观察其酶活动态。【结果】从混合羽毛的土壤样品中筛选到一株羽毛降解菌,命名为菌株GIMN1.015,初步判定该菌株属于芽孢八叠球菌属(Sporosarcina)。最适生长pH为9.0,温度为30°C。蛋白水解活性最高值出现在培养后96 h。【结论】菌株GIMN1.015在利用羽毛角蛋白资源中具有潜在的应用价值。这是芽孢八叠球菌在羽毛降解方面的首次报道。     

近海柴油降解菌群的构建及其对柴油的降解特性

【目的】实施近海柴油污染的生物治理。【方法】以柴油为唯一碳源,从深圳港口海域富集筛选柴油降解菌;采用复配、正交试验等方式构建混合菌群;通过单因素试验研究环境因素对菌群降解柴油的影响;使用气相色谱-氢火焰检测器(GC-FID)分析降解前后柴油各组分的变化;通过生理生化试验和16S rRNA基因序列分析对菌株进行鉴定。【结果】获得了16株柴油降解菌,7 d内对柴油的降解率最高达40.8%;选择菌株C1-8、C2-10、C3-13构建了混合菌群CQ1,投加量分别为0.5%、2.0%和1.0%,CQ1对柴油去除率比单菌提高了10%以上;CQ1的最适环境条件为：温度30 °C、pH 7.6、摇床转速220 r/min、柴油浓度20 g/L,优化后9 d内对柴油去除率达60%以上;GC-FID结果显示,菌群CQ1可降解大部分C11?C27的正构烷烃,对C21?C27的烷烃降解可达100%。经鉴定,菌株C1-8、C2-10和C3-13分别为微杆菌(Microbacterium sp.)、剑菌(Ensifer sp.)和变异棒杆菌(Corynebacterium variabile)。【结论】CQ1在近海柴油污染的生物修复中具有良好的应用前景。     

一株分离自胡杨的赖氨酸芽孢杆菌(Lysinibacillus)ML-64的微生物学特性

摘要:【目的】从胡杨茎秆液中分离得到的一株菌株ML-64,对其进行微生物学特性的分析。【方法】通过细菌培养和染色的方法进行了形态和培养特征的检测,使用多相分类学方法测定菌株的各项生理生化指标,脂肪酸组分、醌组分、极性脂类型、16S rRNA基因系统发育分析、(G+C)mol%含量的测定和DNA-DNA杂交分析。【结果】菌株ML-64为革兰氏阳性菌,杆状,产生芽孢。菌落为圆形,淡黄色,表面光滑。菌株生长温度范围为10－45℃(最适37℃),pH范围为7.0－9.0(最适pH7.0),NaCl浓度范围为0－6%(W/V) (最适0－2%)。菌株ML-64 的类脂酯酶,精氨酸双水解酶,脲酶活性和VP-实验为阳性。在API 50CH酶活性检测实验中菌株不能发酵任何糖类。可利用的碳源有: L-丝氨酸,丙酮酸甲酯,α-酮-丁酸,乙酰乙酸。对多粘菌素B(30 μg)、新生霉素(30 μg)、青霉素G(10 U)不敏感。16S rRNA基因序列分析结果表明,菌株ML-64T与ysinibacillus chungkukjangi 2RL3-2T,Lysinibacillus sinduriensis BLB-1T有密切亲缘关系。相似率分别为100%和99.1%。菌株ML-64与Lysinibacillus chungkukjangi 2RL3-2T和Lysinibacillus massiliensis CIP108446T之间的杂交率分别为82%和50.9%。基因组DNA(G+C)mol%含量为36.8%。菌株ML-64优势脂肪酸类型为iso-C15:0(55.05%)和anteiso-C15:0(20.70%),醌组分类型是MK-7。【结论】基于表型特征、遗传型特征和系统发育分析,将菌株定为Lysinibacillus chungkukjangi的新变种,获得序列注册号为KC609752。胡杨内生菌ML-64的基因组结构已与最近源菌株L．chungkukjangi产生了较大的分化,选择性地适应了胡杨内生环境。     

铜绿假单胞菌SJTD-2降解长链烷烃与原油的特性

摘要:【目的】石油污染严重威胁生态系统和生物圈,微生物修复被认为是一种安全有效可代替物化方法来治理石油污染的办法。本文对我们从石油污染土壤中分离获得的一株可分解正烷烃和原油的革兰氏阴性菌SJTD-2的理化性质和降解效能进行了研究。【方法】利用菌株表型和生理性质、16S rRNA序列比较分析与进化树绘制,确定新分离菌株SJTD-2的种属;测定菌株SJTD-2的生长曲线,确定其利用不同长度烷烃和原油为单一碳源的效能;利用GC-MS检测烷烃类物质的残留量,确定菌株SJTD-2降解烷烃和原油SJTD-2的降解效率和降解周期。【结果】菌株表型与16S rRNA序列比较及进化树比对分析结果显示,菌株SJTD-2与假单胞菌属的亲缘关系十分接近,为铜绿假单胞菌。菌株SJTD-2 可有效分解C10到C26的中链和长链烷烃及原油,利用它们作为其单一碳源生长;该菌株对长链烷烃(C18－C22)的利用效果较中链烷烃好,其中正二十二烷被认为是其最佳碳源。48 h内,该菌株可完全降解500 mg/L正二十二烷;72h 后,2 g/L的正二十二烷可几乎被菌株全部分解利用。此外,菌株SJTD-2在7 d内可将2 g/L的原油分解88%以上。【结论】与现有其它烷烃降解菌相比,铜绿假单胞菌SJTD-2具有突出的长链烷烃与原油降解效能及耐受能力,该菌株的发现与研究将有助于烷烃降解机制的研究和环境修复的进程。     

一株产蛋白酶嗜碱菌株的分离、鉴定及酶学特性

【目的】筛选产蛋白酶嗜碱菌并对其进行鉴定和特性分析。【方法】利用碱性脱脂牛奶培养基分离纯化产蛋白酶嗜碱菌,通过形态特征、生理生化、16S rRNA基因序列分析以及DNA-DNA杂交实验确定菌株的分类地位,利用酪蛋白水解法分析所产蛋白酶的pH和温度作用范围、稳定性和耐氧化剂能力。【结果】从我国西藏盐碱湖样品中分离到一株产碱性蛋白酶的菌株ZL223,该菌株为革兰氏阳性菌,最适生长温度为37℃,最适生长pH9.0,16S rRNA基因序列分析显示,菌株ZL223与假强芽孢杆菌Bacillus pseudo firmus OF4亲缘关系最近,16S rRNA基因序列相似性为98.6%,DNA-DNA杂交结果显示与B.pseudofirmus OF4同源性为86%。菌株ZL223产生的蛋白酶作用的最适pH为12.0,最适温度为40℃。【结论】结合生理生化指标测定的结果,鉴定菌株为假强芽孢杆菌ZL223(B.pseudofirmus ZL223)。该菌株产生的碱性蛋白酶具有较高的pH适应性,值得进一步研究。     

一株中度嗜盐芽孢杆菌(Bacillus sp. BZ-SZ-XJ39)的微生物学特性

【目的】了解一株来自新疆盐碱湖的中度嗜盐菌(Bacillus sp.BZ-SZ-XJ39)的微生物学特性,为进一步阐明该菌独特的盐适应机制奠定基础。【方法】通过16S rRNA基因序列分析、G+C mol%含量测定、细胞形态和菌落观察、培养条件确定、营养与生理生化指标测定以及细胞化学组分分析等描述该菌的生物特性。【结果】基于16S rRNA基因序列的同源性和系统发育树分析,菌株BZ-SZ-XJ39与Bacillus saliphilus 6AGT(序列相似性为97.5%),Bacillus daliensis DLS13T(96.5%),Bacillus luteus JC167T(96.2%),Bacillus chagannorensis CG-15T(95.5%)和Bacillus agaradhaerens DSM8721T(95.3%)有密切的亲缘关系。基因组DNA G+C含量为44.4 mol%±1.2 mol%(Tm)。菌株BZ-SZ-XJ39为好氧、短杆状、革兰氏阳性菌。菌落呈黄色、圆形凸起、表面光滑且边缘整齐。菌株生长盐度范围为0.5%–28%(最适8%),pH范围为5.5–9.5(最适pH 8.0),温度范围为4–41oC(最适33oC)。菌株BZ-SZ-XJ39合成的主要脂肪酸包括anteiso-C15:0(50.2%)和anteiso-C17:0(16.3%)。合成极性脂为磷脂(PL)、磷脂酰乙醇胺(PE)、磷脂酰甘油(PG)、二磷脂酰甘油(DPG)和磺酸基异鼠李糖基二脂酰基甘油(SQDG)。呼吸醌类型为甲基萘醌(MK-7)。菌株BZ-SZ-XJ39已经在中国普通微生物菌种保藏管理中心(CGMCC1.12936)和日本微生物菌种保藏中心(JCM30194)冻干保藏,在Gen Bank中的序列注册号为KP456019。【结论】基于BZ-SZ-XJ39菌株的遗传型、表型、生理生化以及化学组成特征,初步确定该菌为Bacillus属中的一个新成员。研究将为探索生命在高盐环境中存在的本质提供新素材,也可为生物技术的潜在应用提供新视角。     

Mutagenicity of C24H14 PAH in human cells expressing CYP1A1

Relatively little is known about the mutagenicity of C24H14 PAH, a diverse group of five- and six-ring PAH, some of which are present at trace levels in the environment. To better understand the mutagenicity of this class of compounds, 11 C24H14 PAH, including benzo[a]perylene, benzo[b]perylene, dibenzo[a,e]fluoranthene, dibenzo[a,f]fluoranthene, dibenzo[j,l]fluoranthene, dibenzo[a,h]pyrene, dibenzo[a,i]pyrene, dibenzo[e,l]pyrene, naphtho[1,2-b]fluoranthene, naphtho[2,3-a]pyrene, and naphtho[2,3-e]pyrene, were tested in a mutagenicity assay based on human h1A1v2 cells. h1A1v2 cells are a line of human B-lymphoblastoid cells that have been engineered to express cytochrome P4501A1 (CYP1A1), an enzyme capable of metabolizing promutagenic PAH. Mutagenicity was measured at the thymidine kinase (tk) locus following a 72-h exposure period. Our results show that nine of the compounds were mutagenic. Benzo[a]perylene, dibenzo[a,e]fluoranthene, dibenzo[a,i]pyrene, and naphtho[2,3-a]pyrene were the most potent mutagens, having minimum mutagenic concentrations (MMC) (i.e., the dose at which the induced response was twice that of the negative controls) in the 1-5 ng/ml range. Benzo[b]perylene, dibenzo[a,h]pyrene, dibenzo[a,f]fluoranthene, and naphtho[2,3-e]pyrene were somewhat less potent mutagens, having MMC in the 10-30 ng/ml range. Dibenzo[e,l]pyrene, which had an MMC of 280 ng/ml, was the least potent mutagen. Dibenzo[j,l]fluoranthene and naphtho[1,2-b]fluoranthene were not mutagenic at the doses tested (1-3000 ng/ml). The most mutagenic compounds were also quite toxic. At the highest doses tested, benzo[a]perylene, dibenzo[a,e]fluoranthene, dibenzo[a,i]pyrene, dibenzo[a,h]pyrene, and dibenzo[a,f]fluoranthene induced > 60% killing, and naphtho[2,3-a]pyrene and naphtho[2,3-e]pyrene induced > 50% killing. Benzo[b]perylene, dibenzo[e,l]pyrene, dibenzo[j,l]fluoranthene, and naphtho[1,2-b]fluoranthene induced < 50% killing at the highest doses tested. Comparing these results to a previous study in which nine other C24H14 PAH were tested for mutagenicity in this same assay, it was found that dibenzo[a]pyrene isomers were generally more mutagenic than the other groups of C24H14 PAH tested. These observations are discussed with emphasis given to identifying C24H14 PAH that may be important environmental mutagens.     

Apo[a] size and PNR explain African American-Caucasian differences in allele-specific apo[a] levels for small but not large apo[a

Apolipoprotein [a] (apo[a]) gene size is a major predictor of lipoprotein [a] level. To determine genetic predictors of allele-specific apo[a] levels beyond gene size, we evaluated the upstream C/T and pentanucleotide repeat (PNR) polymorphisms. We determined apo[a] sizes, allele-specific apo[a] levels, and C/T and PNR in 215 Caucasians and 139 African Americans. For Caucasians, apo[a] size affected allele-specific levels substantially greater in subjects with apo[a] < 24 K4; for African Americans, the size effect was smaller than in Caucasians, <24 K4, but did not decrease at higher repeats. In both groups, the level decreased with increasing size of the other allele. Controlling for apo[a] sizes, PNR decreased allele-specific apo[a] levels in Caucasians with increasing PNR > 8. In a multiple regression model, apo[a] allele size and size and expression of the other apo[a] allele (and PNR > 8 for Caucasians) significantly predicted allele-specific apo[a] levels. For a common PNR 8 allele, predicted values were similar in the two ethnicities for small size apo[a]. Allele-specific apo[a] levels were influenced by the other allele size and expression. Observed differences between Caucasians and African Americans in allele-specific apo[a] levels were explained for small apo[a] sizes by the other allele size and PNR; the ethnicity differences remain unexplained for larger sizes.     

Deriving behavioural receptive fields for visually completed contours

The visual system is constantly faced with the problem of identifying partially occluded objects from incomplete images cast on the retinae. Phenomenologically, the visual system seems to fill in missing information by interpolating illusory and occluded contours at points of occlusion, so that we perceive complete objects. Previous behavioural [1] [2] [3] [4] [5] [6] [7] and physiological [8] [9] [10] [11] [12] studies suggest that the visual system treats illusory and occluded contours like luminance-defined contours in many respects. None of these studies has, however, directly shown that illusory and occluded contours are actually used to perform perceptual tasks. Here, we use a response-classification technique [13] [14] [15] [16] [17] [18] [19] [20] to answer this question directly. This technique provides pictorial representations - 'classification images' - that show which parts of a stimulus observers use to make perceptual decisions, effectively deriving behavioural receptive fields. Here we show that illusory and occluded contours appear in observers' classification images, providing the first direct evidence that observers use perceptually interpolated contours to recognize objects. These results offer a compelling demonstration of how visual processing acts on completed representations, and illustrate a powerful new technique for constraining models of visual completion.     

Aryl hydrocarbon receptor-mediated activity of mutagenic polycyclic aromatic hydrocarbons determined using in vitro reporter gene assay

Activation of aryl hydrocarbon receptor (AhR) by 30 polycyclic aromatic hydrocarbons (PAHs) was determined in the chemical-activated luciferase expression (CALUX) assay, using two exposure times (6 and 24h), in order to reflect the metabolization of PAHs. AhR-inducing potencies of PAHs were expressed as induction equivalency factors (IEFs) relative to benzo[a]pyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In 24h exposure assay, the highest IEFs were found for benzo[k]fluoranthene, dibenzo[a,h]anthracene and dibenzo[a,k]fluoranthene (approximately three orders of magnitude lower than TCDD) followed by dibenzo[a,j]anthracene, benzo[j]fluoranthene, indeno[1,2,3-cd]pyrene, and naphtho[2,3-a]pyrene. The 6h exposure to PAHs led to a significantly higher AhR-mediated activity than the 24h exposure (generally by two orders of magnitude), probably due to the high rate of PAH metabolism. The strongest AhR inducers showed IEFs approaching that of TCDD. Several PAHs, including some strong mutagens, such as dibenzo[a,l]pyrene, cyclopenta[cd]pyrene, and benzo[a]perylene, elicited only partial agonist activity. Calculation of IEFs based on EC25 values and/or 6h exposure data is suggested as an alternative approach to estimation of toxic potencies of PAHs with high metabolic rates and/or the weak AhR agonists. The IEFs, together with the recently reported relative mutagenic potencies of PAHs [Mutat. Res. 371 (1996) 123; Mutat. Res. 446 (1999) 1] were combined with data on concentrations of PAHs in extracts of model environmental samples (river sediments) to calculate AhR-mediated induction equivalents and mutagenic equivalents. The highest AhR-mediated induction equivalents were found for benzo[k]fluoranthene and benzo[j]fluoranthene, followed by indeno[1,2,3-cd]pyrene, dibenzo[a,h]anthracene, benzo[a]pyrene, dibenzo[a,j]anthracene, chrysene, and benzo[b]fluoranthene. High mutagenic equivalents in the river sediments were found for benzo[a]pyrene, dibenzo[a,e]pyrene, and naphtho[2,3-a]pyrene and to a lesser extent also for benzo[a]anthracene, benzo[b]fluoranthene, indeno[1,2,3-cd]pyrene, benzo[j]fluoranthene, dibenzo[a,e]fluoranthene and dibenzo[a,i]pyrene. These data illustrate that AhR-mediated activity of PAHs, including the highly mutagenic compounds, occurring in the environment but not routinely monitored, could significantly contribute to their adverse effects.     

Effect of particulates on metabolism and mutagenicity of benzo[a]pyrene

Mechanisms of co-carcinogenicity of particulates, such as iron oxide and asbestos, and benzo[a]pyrene (B[a]P) are not completely understood. Particulates dramatically alter rates of uptake of B[a]P into membranes, a factor which could account for co-carcinogenicity. However, B[a]P must be activated to reactive forms to be carcinogenic and mutagenic so alterations in metabolism of B[a]P by particulates also could result in co-carcinogenesis. To elucidate mechanisms of particulate-B[a]P co-carcinogenesis, we have correlated rates of uptake of B[a]P into microsomes with metabolism of B[a]P and with mutagenicity of B[a]P in the Ames test. In general, aryl hydrocarbon hydroxylase (AHH) activity paralleled rates of uptake of B[a]P, though some inhibition of AHH activity by particulates which was not attributable to availability of B[a]P was evident. This inhibition was studied further by assaying separately mixed function oxidase and epoxide hydrase activities in the presence of particulates. Both chrysotile and iron oxide inhibited O-deethylation of 7-ethoxyresorufin and hydration of B[a]P-4,5-oxide. To determine effects of this inhibition on activation of B[a]P to reactive forms, we studied profiles of metabolites of B[a]P and mutagenicity of B[a]P. The only alteration in profiles of B[a]P metabolites produced by particulates was that due to effects on rates of uptake. Similarly, mutagenicity of B[a]P was positively correlated with rates of uptake into microsomes. We conclude that the predominant effects of chrysotile and iron oxide are in altering rates of uptake of particle-adsorbed B[a]P. Changes in uptake rates then result in alterations of B[a]P metabolism and mutagenicity.     

Dual-label high-performance liquid chromatographic assay for femtomole levels of benzo[a]pyrene metabolites

A dual-label HPLC assay to measure femtomole quantities of ethyl acetate-extractable [3H]benzo[a]pyrene metabolites was developed. 14C-labeled metabolites of benzo[a]pyrene formed by rat liver 9000g supernatant were used as both internal standards and chromatographic markers. The percentage deviation between assays was determined to be between 11 and 13% for 9,10-dihydro-9,10-dihydroxybenzo[a]pyrene, 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene, benzo[a]pyrene-3,6-quinone, benzo[a]pyrene-1,6-quinone, and 9-hydroxybenzo[a]pyrene, 22% for 4,5-dihydro-4,5-dihydroxybenzo[a]pyrene, and less than 5% for 3-hydroxybenzo[a]pyrene. The detection limit of this assay was between 3 and 10 fmol per metabolite. The application of this technique to the metabolism of [3H]benzo[a]pyrene by microsomes of hamster and human oral cavity tissue is described.     

Characterization of the basis of lipoprotein [a] lysine-binding heterogeneity

Although elevated plasma concentrations of lipoprotein [a] (Lp[a]) are considered to be a risk factor for atherosclerosis, the mechanisms by which Lp[a] mediates its pathogenic effects have not been conclusively determined. The apolipoprotein [a] (apo[a]) component of Lp[a] confers unique structural properties to this lipoprotein, including the ability to bind to lysine residues in biological substrates. It has been shown, however, that only a fraction of plasma Lp[a] (Lp[a]-Lys(+)) binds to lysine-Sepharose in vitro. The nature of the non-lysine-binding Lp[a] fraction in plasma (Lp[a]-Lys(-)) is currently unknown. In the present study, the Lp[a]-Lys(+) fraction was determined in the plasma of six unrelated individuals; the Lp[a]-Lys(+) fraction in these plasma samples ranged from approximately 37 to approximately 48%. Interestingly, purification of the Lp[a] by density gradient ultracentrifugation followed by gel filtration and ion-exchange chromatography resulted in progressive increases in the Lp[a]-Lys(+) fraction. Addition of either purified low density lipoprotein (LDL) or fibronectin to the purified Lp[a] at a 1:1 molar ratio reduced the Lp[a]-Lys(+) fraction (maximal decrease of 34 and 20%, respectively) whereas addition of both fibronectin and LDL to the purified Lp[a] resulted in a further decrease (45% maximally) in this fraction. Similar results were obtained by using a recombinant expression system for apo[a]: addition of a 4-fold molar excess of either LDL or fibronectin to conditioned medium containing metabolically labeled recombinant apo[a] reduced the Lys(+) fraction by 49 and 23%, respectively.Taken together, our data suggest that the lysine-binding heterogeneity of plasma Lp[a] is not primarily an intrinsic property of the lipoprotein, but rather results in large part from its ability to noncovalently associate with abundant plasma components such as LDL and fibronectin. These interactions appear to mask the lysine-binding site in apo[a] kringle IV type 10, which mediates the interaction of Lp[a] with lysine-Sepharose. The contribution of these interactions to the function of Lp[a] in vivo remains to be investigated.     

Oxidation of benzo[a]pyrene by the filamentous fungus Cunninghamella elegans.

Cunninghamella elegans oxidized benzo[a]pyrene to several metabolic products. Compounds that were isolated and identified were: trans-9,10-dihydroxy-9,10-dihydrobenzo[a]pyrene, trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene, benzo[a]pyrene 1,6-quinone, benzo[a]pyrene 3,6-quinone, 9-hydroxybenz[a]pyrene, and 3-hydroxybenzo[a]pyrene. In addition, an unidentified dihydroxybenzo[a]pyrene metabolite was also formed. Experiments with [14C]benzo[a]pyrene showed that over a 96-h period, 18.4% of the hydrocarbon was converted to metabolic products. Most of the metabolites were sulfate conjugates as demonstrated by the formation of benzo[a]pyrene quinones and phenols after treatment with aryl sulfatase. Glucuronide and sulfate conjugates were also detected as water-soluble metabolites. The results show that benzo[a]pyrene is metabolized by a filamentous fungus in a manner that is remarkably similar to that observed in higher organisms.     

Quantification of apo[a] and apoB in human atherosclerotic lesions.

Lipoprotein[a] or Lp[a] is a cholesterol-rich plasma lipoprotein that is associated with increased risk for cardiovascular disease. To better understand this association we determined the amount of apo[a] and apoB as possible estimates for Lp[a] and low density lipoprotein (LDL) accumulation in atherosclerotic lesions and in plasma, from patients undergoing vascular surgery, using specific radioimmunoassays for apolipoprotein[a] and apolipoprotein B. Apo[a] and apoB were operationally divided into a loosely bound fraction obtained by extracting minced samples of plaque with phosphate-buffered saline (PBS), and a tightly bound fraction obtained by extracting the residual tissue with 6 M guanidine-HCl (GuHCl). We found that 83% of all apo[a] but only 32% of all apoB in lesions was in the tightly bound fraction. When normalized for corresponding plasma levels, apo[a] accumulation in plaques was more than twice that of apoB. All fractions of tissue apo[a], loosely bound, tightly bound, and total, correlated significantly with plasma apo[a]. However, no significant correlations were found between any of the tissue fractions and plasma apoB. If all apo[a] and apoB had been associated with intact Lp[a] or LDL particles, the calculated mass of tightly bound Lp[a] would actually have exceeded that of tightly bound LDL in five cases with plasma Lp[a] levels above 5 mg apo[a] protein/dl. When PBS and GuHCl extracts of lesions were subjected to one-dimensional electrophoresis, the major band stained for lipid and immunoblotted positively for apo[a] and apoB, suggesting the presence of some intact Lp[a] in these extracts. These results suggest that Lp[a] accumulates preferentially to LDL in plaques, and that plaque apo[a] is directly associated with plasma apo[a] levels and is in a form that is less easily removable than most of the apoB. This preferential accumulation of apo[a] as a tightly bound fraction in lesions, could be responsible for the independent association of Lp[a] with cardiovascular disease in humans.