Induction of cytochrome P-450 by glucocorticoids in rat liver. I. Evidence that glucocorticoids and pregnenolone 16 alpha-carbonitrile regulate de novo synthesis of a common form of cytochrome P-450 in cultures of adult rat hepatocytes and in the liver in vivo

We administered a series of steroid hormones to primary nonproliferating cultures of adult rat hepatocytes and found that dexamethasone and other glucocorticoids but not sex steroid hormones, mineralocorticoids, or derivatives of pregnenolone other than pregnenolone 16 alpha-carbonitrile (PCN) stimulated de novo synthesis of an immunoreactive protein, indistinguishable from the form of cytochrome P-450 (P450PCN) induced by PCN in rat liver. No difference were discerned among purified liver cytochromes from rats treated with dexamethasone, PCN or dexamethasone plus PCN, among proteolytic digests of these proteins, or among the immunoprecipitated cytochromes prepared from cultured hepatocytes treated with these steroids as judged by electrophoresis on polyacrylamide gels containing sodium dodecyl sulfate followed by immunoblot analysis. Of the steroids tested, dexamethasone proved to be the most efficacious inducer increasing the rate of synthesis of P450PCN from 0.05% of total cellular protein synthesis in incubated control cultures (measured as incorporation of [3H]leucine into immunoprecipitable P450PCN) to as much as 9.4% in cultures incubated for 5 days in medium containing dexamethasone (10(-5) M). As with traditional glucocorticoid-responsive liver functions, induction of immunoreactive P450PCN was dependent on the concentration of dexamethasone (10(-8) to 10(-5) M) and was promptly reversed upon withdrawal of the steroid. However, during the 24-h interval between 24 to 48 h of culture age the hepatocytes were refractory to either induction or de-induction of immunoreactive P450PCN even though continuous exposure of the cells to dexamethasone (including this interval) was mandatory for maximal induction of P450PCN at 120 h in culture. Unlike cultured rat hepatocytes, HTC hepatoma cultures failed to exhibit dexamethasone-responsive expression of immunoreactive P450PCN. We conclude that glucocorticoids and PCN constitute a specific "class" of synthetic and endogenous inducers of a single form of cytochrome P-450.     

Anonymous nuclear DNA markers in the American oyster and their implications for the heterozygote deficiency phenomenon in marine bivalves

A puzzling population-genetic phenomenon widely reported in allozyme surveys of marine bivalves is the occurrence of heterozygote deficits relative to Hardy-Weinberg expectations. Possible explanations for this pattern are categorized with respect to whether the effects should be confined to protein-level assays or are genomically pervasive and expected to be registered in both protein- and DNA-level assays. Anonymous nuclear DNA markers from the American oyster were employed to reexamine the phenomenon. In assays based on the polymerase chain reaction (PCR), two DNA-level processes were encountered that can lead to artifactual genotypic scorings: (a) differential amplification of alleles at a target locus and (b) amplification from multiple paralogous loci. We describe symptoms of these complications and prescribe methods that should generally help to ameliorate them. When artifactual scorings at two anonymous DNA loci in the American oyster were corrected, Hardy-Weinberg deviations registered in preliminary population assays decreased to nonsignificant values. Implications of these findings for the heterozygote-deficit phenomenon in marine bivalves, and for the general development and use of PCR-based assays, are discussed.      

Characteristics of microsomal reduction of benzo[a]pyrene 4,5-oxide

NADPH-reduction of benzo[a]pyrene 4,5-oxide (BP-4,5-oxide) to BP required four components from rat liver: cytochrome P-450, NADPH cytochrome P-450 reductase, phosphatidylcholine and a soluble, heat-sensitive factor which was present in 105 000 × g supernatant and was also released from microsomes by sonication. The requirement for this factor contrasts with recently reported results from Sugiura et al. (Cancer Res., 40 (1980) 2910). Oxide-reduction was 40 times faster under anaerobic conditions, but oxygen did not affect the stimulation factor. This stimulation was highest (× 15) at low concentrations of microsomal protein (<0.1 mg/ml) and was almost absent at high concentrations of microsomal protein (>1 mg/ml). Oxide-reduction activity was proportional to microsomal protein concentration in the presence of added 105 000 × g supernatant, but for microsomes alone (>0.1 mg/ml) exhibited a parallel plot with an intercept at 0.08 mg/ml microsomal protein. Stimulation was highest at high concentrations of BP-4,5-oxide and a linear plot of V⁻¹ vs. [BP-4,5-oxide]⁻¹ was only obtained in the presence of 105 000 × g supernatant (K_m = 3 μM, V_max = 3.3 nmol/mg/min). Microsomal hydration of BP-4,5-oxide (inhibited in reductase assays) was unaffected by 105 000 × g supernatant, suggesting that stimulation of oxide-reduction did not derive from solubilization of BP-4,5-oxide. Stimulation was observed in the initial rate of reaction and was independent of incubation time. Inhibition of lipid peroxidation, removal of peroxides and deoxygenation were all excluded as explanations of the stimulatory effect.     

蒙古高原岩黄芪属植物的分支分类学研究

以蒙古高原岩黄芪属植物为对象, 应用徐克学的最大同步法, 探讨了蒙古高原岩黄芪属(豆科)植物的系统演化, 并根据分支分类结果对蒙古高原岩黄芪属进行了系统学处理。作者首次将蒙古高原岩黄芪属分为岩黄芪亚属、半灌木岩黄芪亚属(新拟)和无刺岩黄芪组、丛枝岩黄芪组、无茎岩黄芪组、半灌木岩黄芪组等4 个组。本文对蒙古高原岩黄芪组的划分符合苏联植物志(1945)中的观点。     

长爪沙鼠肝细胞体外分离培养体系的建立

目的建立长爪沙鼠原代肝细胞分离培养体系。方法以雄性长爪沙鼠为供体,采用组织消化法和Seglen两步灌流法分离肝细胞,以台盼蓝染色检测细胞得率和活率,过碘酸-希夫氏反应(PAS)鉴定肝细胞,倒置显微镜观察肝细胞形态变化,并使用含有多种细胞因子的培养基维持培养。结果组织消化法和Seglen两步灌流法平均每只长爪沙鼠可分别获得肝细胞(1.33±0.34)×107个、(3.97±1.15)×107个,细胞活率分别为(29.4±6.05)%、(80.3±4.56)%,这两种方法在细胞得率及活率方面存在显著差异。肝细胞内因有大量的糖原颗粒,经PAS染色后被染成红色。结果表明肝细胞在贴壁后72 h内,肝细胞形态发生显著变化。结论采用胶原酶经肝门静脉灌流分离肝细胞是一种高效获得肝细胞的方法。各种细胞因子有利于维持肝细胞在体外的生长分化,长爪沙鼠原代肝细胞分离培养体系的建立将为肝脏相关疾病研究和防治药物的开发提供技术支持。     

构建基于CRISPR/Cas9技术敲除ALOX5的质粒

旨在利用CRISPR/Cas9技术构建敲除花生四烯5-脂氧合酶基因(Arachidonate 5-lipoxygenase gene,ALOX5)的重组质粒。设计合成3对靶向敲除ALOX5第六外显子的sgRNA,将其分别插入到CRISPR/Cas9质粒骨架pX458载体中,转化感受态大肠杆菌DH5α后挑取克隆,通过测序评估重组质粒是否构建成功。将构建好的重组质粒转染293T细胞,在荧光显微镜下观察转染效果,挑取转染成功的细胞,用试剂盒提取转染细胞基因组DNA,PCR扩增含敲除位点的DNA片段,用测序技术获得核苷酸序列,用DNAStar软件分析转染细胞中ALOX5基因敲除情况。测序结果表明2对双链sgRNA寡核苷酸已插入质粒,且序列正确,靶向ALOX5基因的重组质粒pX458-sgRNAs-ALOX5构建成功。其在293T细胞中的转染效率约为50%,用一代测序法未检测到sgRNAs的切割效果。初步表明利用CRISPR/Cas9技术成功构建靶向ALOX5基因的重组质粒pX458-sgRNAs-ALOX5。     

Fluctuations in Species-Level Protein Expression Occur during Element and Nutrient Cycling in the Subsurface

While microbial activities in environmental systems play a key role in the utilization and cycling of essential elements and compounds, microbial activity and growth frequently fluctuates in response to environmental stimuli and perturbations. To investigate these fluctuations within a saturated aquifer system, we monitored a carbon-stimulated in situ Geobacter population while iron reduction was occurring, using 16S rRNA abundances and high-resolution tandem mass spectrometry proteome measurements. Following carbon amendment, 16S rRNA analysis of temporally separated samples revealed the rapid enrichment of Geobacter-like environmental strains with strong similarity to G. bemidjiensis. Tandem mass spectrometry proteomics measurements suggest high carbon flux through Geobacter respiratory pathways, and the synthesis of anapleurotic four carbon compounds from acetyl-CoA via pyruvate ferredoxin oxidoreductase activity. Across a 40-day period where Fe(III) reduction was occurring, fluctuations in protein expression reflected changes in anabolic versus catabolic reactions, with increased levels of biosynthesis occurring soon after acetate arrival in the aquifer. In addition, localized shifts in nutrient limitation were inferred based on expression of nitrogenase enzymes and phosphate uptake proteins. These temporal data offer the first example of differing microbial protein expression associated with changing geochemical conditions in a subsurface environment.     

The early life history of two sympatric New Zealand octopuses: eggs and paralarvae of Octopus huttoni and Pinnoctopus cordiformis

This study combined morphological and morphometric information on egg clutches, egg capsules and paralarvae of two sympatric coastal octopuses from New Zealand waters, Octopus huttoni and Pinnoctopus cordiformis, to provide species-specific traits to identify their early life stages obtained from field surveys. Eggs of O. huttoni (2.5 mm length; 1 mm width) were entwined with one another forming strings that ranged from 11 to 25.8 mm in length. Eggs of P. cordiformis (6.4 mm length; 1.5 mm width) were significantly bigger than those of O. huttoni and were grouped in small clusters of about seven eggs. Paralarvae O. huttoni and P. cordiformis differed in hatching size (1.4 mm versus 3.1 mm mantle length), number of suckers per arm (four versus eight), number of lamellae per outer demibranch (five versus ten) and arrangements of chromatophores in the body surface (29 to 59 versus 91 to 179), respectively. The morphological traits described in hatchlings from the laboratory allowed comparisons with field-collected paralarvae, suggesting that such characters were reliable species-specific patterns to enable a consistent differentiation between the early life stages of these two sympatric species, even in the absence of the brooding female.