Molecular genetic studies on the biosynthesis of aldosterone in humans

Corticosterone methyl oxidase Type I (CMO I) and II (CMO II) have been postulated to be the enzymes involved in the final two steps of aldosterone biosynthesis in humans. We have isolated human cDNAs for P450c11 and P450c18 as well as the corresponding genes, CYP11B1 and CYP11B2. Both protein products of these two genes as expressed in COS-7 cells exhibit steroid 11β-hydroxylase activity, but only P450c18, a product of CYP11B2, carried steroid 18-hydroxylase activity to form aldosterone. These results indicate that CYP11B2 encodes CMO, the actual catalytic function of which is retained by P450c18, a multifunctional enzyme. This conclusion is further supported by the finding that the P450c18 gene, CYP11B2, is mutated at several different loci in patients deficient in CMO I or II.     

Steroid metabolism by constitutive cytochromes P450

In rat liver endoplasmic reticulum some 16 different cytochromes P450 have been identified as constitutive, sequenced from recombinant DNA, and shown to be distinct gene products. These forms are “multipurpose”, i.e. functional in xenobiotic metabolism as well as endogenous substrate metabolism. In the latter case, these forms metabolize steroids, fatty acids, prostaglandins and even ketone bodies, indicating an involvement in homeostasis. In steroid metabolism, in contrast to “biosynthetic” forms of P450 which generally yield one product, the multipurpose forms exhibit broad, overlapping metabolite profiles, with isomeric and epimeric specificity and different mechanisms of product formation. The nature of the substrate docking region is of much interest and attempts have been made to rationalize the manner in which multiple metabolites are produced from a single substrate. Brain, with a very low level of P450 relative to liver also catalyzes steroid metabolism. The nature of the forms involved are not yet known.     

Screening of yeast strains for transfructosylating activity

Fructooligosaccharides (FOSs) are functional food ingredients with prebiotic properties, and a recent increase in the use of oligosaccharides in the food industry has led to the search for “new” microorganisms and enzymes for the production of oligosaccharides. This paper focuses on the screening of yeasts obtained from fruits and flowers (from Brazilian tropical forests), and capable of secreting extra-cellular enzymes with high fructosyl transferase activity (FTA). The screening and isolation procedures resulted in four potentially interesting yeast strains: Candida sp. (LEB-I3), Rhodotorula sp. (LEB-U5.), Cryptococcus sp. (LEB-V2) and Rhodotorula sp. LEB-V10. All were able to produce more then 100 g l⁻¹ of FOS from a 500 g l⁻¹ sucrose solution, but only the last one, (LEB-V10), showed no hydrolytic activity with respect to the FOS produced, giving a continuous increase in FOS content up to the end of the reaction, when it was about 50% of the total carbohydrates.     

Novel biosynthetic routes to non-proteinogenic amino acids as chiral pharmaceutical intermediates

Transaminases catalyse the reversible transfer of amino and keto groups between an amino acid and keto acid substrate pair. Many bacterial transaminases accept a wide array of keto acids as amino acceptors and are useful as commercial biocatalysts in the preparation of amino acids. Since the reaction equilibrium typically lies close to unity, several approaches have been described to improve upon the 50% product yield, using additional enzymes. The present work describes an efficient means to significantly increase product yield in transamination using the aromatic transaminase of Escherichia coli encoded by the tyrB gene, with -aspartate as the amino donor. This is achieved by the introduction of the alsS gene encoding the acetolactate synthase of Bacillus subtilis, which eliminates pyruvate and alanine produced as a by-product of aspartate transamination. The biosynthesis of the non-proteinogenic amino acid -2-aminobutyrate is described using a recombinant strain of E. coli containing the cloned tyrB and alsS genes. The strain additionally carries the cloned ilvA gene of E. coli encoding threonine deaminase to produce the substrate 2-ketobutyrate from -threonine. An alternate coupled process uses lysine -aminotransferase in concert with a transaminase using -glutamate as the amino donor.     

Poly-γ-glutamate depolymerase of Bacillus subtilis: production, simple purification and substrate selectivity

The Bacillus subtilis pgdS gene, which is located at the immediate downstream of the pgs operon for poly-γ-glutamate (PGA) biosynthesis, encodes a PGA depolymerase. The pgdS gene product shows the structural feature of a membrane-associated protein. The mature form of the gene product, identified as a B. subtilis extracellular protein, was produced in Escherichia coli clone cells. Since the mature PGA depolymerase has been modified with the histidine-tag at its C-terminus, it could be simply purified by metal-chelating affinity chromatography. This purified enzyme digested PGAs from B. subtilis ( -glutamate content, 70%) and from Bacillus megaterium (30%) in an endopeptidase-like fashion. In contrast, PGA from Natrialba aegyptiaca, which consists only of -glutamate, was resistant to the enzyme, suggesting that, unlike fungal PGA endo-depolymerases, the bacterial enzyme recognizes the -glutamate unit in PGA.     

2001—2017年我国森林火灾时空分布特征

基于遥感的过火面积产品可提供连续、时空特征明确的火斑信息,是研究区域尺度森林火灾分布特征的重要数据来源,但精度仍有待改进。本研究结合250 m全球过火面积产品(CCI_Fire)和30 m全球森林变化(GFC)产品,通过数据整合获取改进后的森林火斑产品(CCI_GFC),并运用已有火斑数据对CCI_GFC产品进行精度验证;结合我国宏观生态区划与网格化(0.05°×0.05°)分析,利用改进后产品对2001—2017年间全国林火特征进行分析。结果表明: CCI_GFC产品的火斑识别率(RR)、变异解释量(R²)、均方根误差(RMSE)、均方绝对百分比误差(MAPE)等精度指标(分别为83%、0.91、0.28、8.5%)均优于原始CCI_Fire产品(分别为74%、0.86、0.36、11.8%)和MCD64A1产品(分别为35%、0.78、0.48、17.3%)。2001—2017年,全国总森林过火面积约为1211万hm²,年过火面积呈显著下降趋势;低(0<森林过火面积比例BFR≤40%)火烧频率区为主,占总过火区域的79%,中(40%相似文献   

Spontaneous hypomorphic mutations in antioxidant enzymes of mice

An antioxidant enzymatic system is pivotal for aerobic animals to minimize the damage induced by reactive oxygen species. Spontaneous mutant animals with altered antioxidant enzyme activity should be useful for the study of the function of these enzymes in vivo. We examined the nucleotide sequences of the genes for the major antioxidant enzymes, including catalase (Cat), superoxide dismutase (Sod1, Sod2, Sod3), glutathione peroxidase (Gpx1, Gpx2, Gpx3, Gpx4, Gpx5), and glutathione reductase (Gsr) in 10 inbred mouse strains. Nonsynonymous nucleotide polymorphisms were identified in all genes, except for Gpx1, Gpx3, and Gpx4. Notably, the SJL/J mouse strain possessed unique nucleotide substitutions in the Gsr and Sod2 genes, which led to Asp39Ala and Val138Met amino acid substitutions in GSR and SOD2, respectively. The specific activity of GSR of SJL/J mice was reduced to 65% of that of NZB/N mice. In vivo activity, however, was higher in SJL/J, due to upregulated expression of the enzyme. The SOD2 activity in SJL/J mice was reduced to half that of other mouse strains. Consistent with this reduction, oxidative damage in the mitochondria was increased as demonstrated by a decrease of total glutathione and an increase in the levels of protein oxidation. These spontaneous hypomorphic alleles would be valuable in the study of free radical biology.     

金针菇细胞色素c过氧化物酶基因（ffccp）及其在菌柄伸长的差异表达初步分析

细胞色素c过氧化物酶（cytochrome C peroxidase,CcP）是细胞内H₂O₂的主要降解酶,参与真菌的氧化应答过程。本研究基于基因组数据获得一个金针菇细胞色素c过氧化物酶编码基因,命名为ffccp。该基因全长1 913bp,包含一个1 098bp的完整开放阅读框,编码365个氨基酸。生物信息学分析结果显示,该基因编码的蛋白质（FfCcP）无跨膜结构和信号肽,不形成二硫键结构,亚细胞定位于线粒体上,具备血红素结合蛋白的保守位点。序列比对和进化分析结果显示,金针菇与糙皮侧耳Pleurotus ostreatus等大型真菌的CcP序列高度相似（相似度均超过70%）,属于CcP家族蛋白。RT-qPCR的检测结果显示,氧化胁迫和损伤胁迫均能诱导ffccp基因上调表达,但两种胁迫对ffccp表达的调控机制可能并不相同。进一步检测ffccp在子实体发育过程中的差异表达情况,发现ffccp在伸长期菌柄出现显著上调表达,且表达量与菌柄伸长速度的相关性达到0.998,为极强正相关。推测ffccp的上调表达可能有利于菌柄的伸长。     

Recovery of protein and chlorophyll from alfalfa by simultaneous lactic acid fermentation and enzyme hydrolysis (ENLAC)

Simultaneous lactic acid fermentation and enzyme hydrolysis by cell wall degrading enzymes (ENLAC for short) was tested for improving the recovery of cell content, such as protein and chlorophyll, from alfalfa. Alfalfa was ensiled with the addition of 1.0% (wet weight) of a variety of commercial enzymes or enzyme cocktails. The best result was achieved by the addition of a 1:1 mixture of Novo Viscozyme (containing mainly hemicellulases, cellulases, and pectinases) and Novo Celluclast 1.5 L or Genencore Cellulase 150 L (containing T. reesei cellulases). ENLAC improved the recovery of protein by 160%, chlorophyll by 240%. The same enzyme treatment in a 24-h reaction without ensiling resulted in only a 14% increase in protein recovery. ENLAC provided optimal acidic conditions for enzyme action, and due to the preservation of the plant material during ensiling, it made possible the efficient use of a low concentration of enzymes during a longer reaction time, compared to conventional 24-h enzyme treatments.     

模拟酸雨对毛竹叶片抗氧化酶活性及释放绿叶挥发物的影响

为了探讨酸雨胁迫与毛竹(Phyllostachys pubescens)绿叶挥发物(green leaf volatiles, GLVs)释放规律以及抗氧化酶活性的关系, 通过盆栽试验, 采用不同pH值(5.6、4.0、2.5)的模拟酸雨对毛竹三年生实生苗进行处理, 研究酸雨对毛竹叶片可溶性蛋白质含量、丙二醛(MDA)含量和抗氧化酶活性的影响, 并采用热脱附/气相色谱/质谱联用技术对毛竹释放的GLVs成分和含量进行分析。结果表明: 酸雨胁迫下毛竹叶片MDA含量明显增加, pH 2.5模拟酸雨胁迫处理45天毛竹叶片MDA含量与对照相比增加了43.0% (p < 0.01); pH 4.0处理MDA含量增加缓慢, 处理75天时MDA含量比对照增加了0.36倍(p < 0.01)。pH 4.0和pH 2.5模拟酸雨胁迫处理45天时, 毛竹叶片可溶性蛋白质含量极显著增加, 与对照相比分别增加了32.0%和65.0% (p < 0.01)。在酸雨胁迫下, 毛竹叶片超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和过氧化物酶(POD)的响应时间存在一定差异, 表现为互相协调, pH 2.5模拟酸雨胁迫处理SOD活性和POD活性分别在45天和60天时达到最大值, 分别为对照的1.67倍和1.31倍(p < 0.01), 随后降低。pH 4.0和pH 2.5模拟酸雨胁迫处理, 毛竹叶片GLVs含量比对照分别增加26.4%和132.9% (p < 0.01), 新增GLVs为 (E)-2-辛烯醛、2-乙基己醛、(E)-2-己烯醛和(E)-2-壬烯醛。研究表明: 酸雨胁迫条件下, 毛竹可以通过调节保护酶活性、可溶性蛋白质含量和释放GLVs来提高适应环境的能力。     

Biohydroxylation of N,N-dialkylarylamines by the isolated topsoil bacterium Bacillus megaterium

Topsoil microorganisms were screened for their acceptability of the standard substrate N,N-dimethylaniline in bacterial ‘whole-cell’ incubations. One bacterium converted N,N-dimethylaniline and was identified as Bacillus megaterium by 16S rDNA analysis and DNA/DNA-hybridization. In contrast to the well-known C-hydroxylation by liver microsomes, leading to p-hydroxylation, B. megaterium formed o- and p-monohydroxylated products, i.e. N,N-dimethyl-2-aminophenol and N,N-dimethyl-4-aminophenol, both identified by gas chromatography–mass spectrometry (GC–MS) using synthesized reference compounds. The observed hydroxylation showed slight regioselectivity in favour of the o-hydroxylated product. Two further substrates, N,N-diethylaniline and N-ethyl-N-methylaniline, were also successfully biohydroxylated by B. megaterium with corresponding regioselectivity. Interestingly, aniline, known to be transformed easily by cytochrome P-450meg into p-aminophenol, was not accepted as substrate.     

皂角发酵物对红托竹荪病害防治及土壤主要微生物群落影响

为了研究皂角发酵物对贵州当地特色产业红托竹荪的病害防治及促生长作用,本研究在室内分离病原菌并进行平板对峙实验,田间试验设计4个处理：不施用药剂的常规处理、解淀粉芽孢杆菌HN11菌液、皂角粉末、皂角发酵物,调查防治效果,测量菌蛋大小及个数,检测土壤微生物群落变化。从发病组织中分离出一株病原菌,鉴定为阴沟肠杆菌Enterobacter cloacae。皂角发酵物对竹荪病害田间防治效果达77.86%,生长面积提高61.22%。土壤微生物群落中细菌和真菌分析结果显示,相较于其他处理组,皂角发酵物处理组中竹荪相对丰度占比最大,达25.83%。皂角发酵物能有效防治红托竹荪病害,促进红托竹荪菌蛋生长,减少化学农药的用量,促进生态循环,保障农产品食用安全,提升皂角和竹荪产业综合效益。     

Oligosaccharide and alkyl β-galactopyranoside synthesis from lactose with Caldocellum saccharolyticum β-glycosidase

The synthetic utility of the thermostable β-glycosidase from Caldocellum saccharolyticum was investigated. The ability of the enzyme to catalyze oligosaccharide and β-galactopyranoside synthesis from lactose was compared with that of the readily commercially available, moderately thermostable β-galactosidase (β- -galactoside galactohydrolase, EC 3.2.1.23) from Aspergillus oryzae. Generally, the C. saccharolyticum enzyme showed significantly greater resistance to inactivation by heat and organic solvent and better yields of product. Although the A. oryzae enzyme gave better oligosaccharide yields at lower lactose concentrations, at higher concentrations (above 50% w/w) the C. saccharolyticum enzyme was significantly better, yielding a sugar mixture containing 42% by weight of tri- plus tetra-saccharides, from a 70% w/w lactose solution, compared with 31% by weight of oligosaccharides with the A. oryzae enzyme. In ethyl galactoside synthesis from ethanol and lactose, neither enzyme appeared to hydrolyze the product to any great extent. Under all conditions tested, the product yield did not peak, even at long reaction times, when most of the lactose had been consumed. The C. saccharolyticum enzyme, however, gave slightly higher product yields and could be used at higher ethanol concentrations without serious loss of activity.     

Formation of 2-nitro-3-methylimidazo[4,5-f]quinoline, a directly mutagenic product, by near-ultraviolet irradiation of a mixture of 2-amino-3-methylimidazo[4,5-f]quinoline and N-nitrosodimethylamine

Direct-acting mutagens to Salmonella typhimurium TA98 were found to be formed from heterocyclic amines on exposure to near-ultraviolet light in the presence of N-nitrosodialkylamines. We have isolated the mutagenic photoproduct formed from 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and N-nitrosodimethylamine, and the product was identified as 3-methyl-2-nitromidazo[4,5-f]quinoline (IQ(NO₂)). The yield of IQ(NO₂) from IQ was estimated to be 17%. Similar light-dependent activation of IQ was noted with 4 different nitrosodialkylamines other than nitrosodimethylamine. Furthermore, MeIQ and MeIQx were also activated with nitrosamine and light. These reactions represent an example of interaction between 2 different classes of mutagens.     

Electrophoretic studies on the Anisakis simplex complex (Ascaridida:Anisakidae) from the Mediterranean and North-East Atlantic

Full-size image

and 1986. Electrophoretic studies on the Anisakis simplex complex (Ascaridida: Anisakidae) from the Mediterranean and North-East Atlantic. International Journal for Parasitology 16: 633–640. The genetic variation of the sibling species Anisakis simplex A and A. simplex B was investigated by electrophoretic analysis of 22 gene-enzyme systems. The two species are reproductively isolated and no gene flow takes place between them. Three loci, Sod, Adk-2 and Lap-1, show distinct alleles in A. simplex A and A. simplex B, allowing their reliable identification both at the larval and adult stages. A fourth locus, Got, appears to be diagnostic at the 95% level. The value of Nei's genetic distance found between A. simplex A and A. simplex B is 0.28. Parameters of genetic variability (He, P, A) are given for both species. The geographic distribution of A. simplex A and A. simplex B appears to be mainly Mediterranean for the former, and mainly North Atlantic for the latter. Several paratenic hosts (fish and squid) and one cetacean definitive host are identified for each of the two species. The names A. pegreffii and A. simplex are tentatively proposed for A. simplex A and A. simplex B respectively.     

植物蛋白的体外泛素化检测方法

泛素激活酶(E1)、泛素耦联酶(E2)和泛素连接酶(E3)是蛋白质泛素化修饰的关键酶。在真核基因组上有大量基因编码这些泛素化相关的酶类或蛋白。检测这些泛素化修饰酶及其底物蛋白的生化特性和特异性是分析其生物学功能的重要内容。该文提供了一种简便快速检测体外泛素化反应的方法, 不仅可通过检测对DTT敏感的硫酯键的形成来判断E2的活性、检测E3的体外泛素化活性, 而且可以检测E2-E3和E3-底物的特异性。所用蛋白主要来源于拟南芥(Arabidopsis thaliana), 包括分属于绝大多数E2亚家族的成员, 可用于不同RING类型E3的活性检测。该方法不仅可以采用多种E2进行E3活性分析, 而且可以分析不同组合的E2-RING E3、RING E3-底物的泛素化活性等, 亦可应用于真核生物蛋白质尤其是植物蛋白的体外泛素化活性分析。     

Initial Studies on the Regulation of Toluene Degradation by Pseudomonas Putida F1

Pseudomonas putida Fl oxidizes toluene through cis-toluene dihydrodiol to 3-methylcatechol. The latter compound is the substrate for “meta” fission of the aromatic nucleus. Kinetic and induction experiments indicate that the genes encoding enzymes for these reactions are part of an operon, designated the tod operon, that is coordinately induced and regulated. Strains unable to utilize toluene as a growth substrate were isolated at high frequencies by using screening procedures that utilize the redox dye, 2,3,5-triphenyl-2H-tetrazolium chloride. Biochemical characterization of strains with mutations in the structural genes of the tod operon showed that toluene induces the first four enzymes in toluene degradation by P. putida Fl. The isolation and characterization of pleiotropicnegative mutants together with mutants altered in terms of their expression of tod genes suggests that the tod operon may be under the control of a positive regulatory element.     

植物蛋白的体外泛素化检测方法

泛素激活酶(E1)、泛素耦联酶(E2)和泛素连接酶(E3)是蛋白质泛素化修饰的关键酶。在真核基因组上有大量基因编码这些泛素化相关的酶类或蛋白。检测这些泛素化修饰酶及其底物蛋白的生化特性和特异性是分析其生物学功能的重要内容。该文提供了一种简便快速检测体外泛素化反应的方法, 不仅可通过检测对DTT敏感的硫酯键的形成来判断E2的活性、检测E3的体外泛素化活性, 而且可以检测E2-E3和E3-底物的特异性。所用蛋白主要来源于拟南芥(Arabidopsis thaliana), 包括分属于绝大多数E2亚家族的成员, 可用于不同RING类型E3的活性检测。该方法不仅可以采用多种E2进行E3活性分析, 而且可以分析不同组合的E2-RING E3、RING E3-底物的泛素化活性等, 亦可应用于真核生物蛋白质尤其是植物蛋白的体外泛素化活性分析。     

Ideal phenotypes and mismatching haplotypes – errors of mtDNA treeing in ants (Hymenoptera: Formicidae) detected by standardized morphometry

A total of 401 nest samples of Formica lugubris Zetterstedt, F. pratensis Retzius, F. aquilonia Yarrow, F. rufa Linnaeus, and F. polyctena Förster, covering the entire Palaearctic range of these species and including 2100 individual workers, was phenotypically investigated by a system of standardized morphometry, pairwise removal of allometric variance, and canonical discriminant functions. A mitochondrial DNA fragment including the cytochrome b gene was sequenced in 148 samples from basically the same range. In the more difficult F. pratensis vs. F. lugubris case, the phenotypic system correctly determined 99.6% of all nest samples, and 95.1% with p<0.05. In all other pairwise species discriminations any nest sample was correctly determined with p<0.01, and three samples with hybrids F. rufa×lugubris were identified. At four localities in the Pyrenees and the Urals, 9 samples with F. pratensis phenotypes (7 of them ideal) but F. lugubris mtDNA haplotypes could be identified, resulting in 14.5% of phenotype/haplotype mismatches. A local dominance of this mismatch combination was observed at one Pyrenean and one Ural locality. There was no indication of an F. pratensis haplotype associated with an F. lugubris phenotype. One ideal F. polyctena phenotype was associated with an F. aquilonia haplotype in a sample from the Urals, and one ideal F. aquilonia phenotype was combined with an F. lugubris haplotype in a sample from central Siberia, resulting in overall phenotype/haplotype mismatch frequencies of 12.5% and 11.1%, respectively. We conclude that all these samples cannot represent actual F₁ hybrids but are the result of hybridizations in the past followed by unidirectional purging of the nuclear genome. Whether this process of purging worked very fast or over longer periods of population history, and whether or not it was complete or incomplete, cannot be assessed from the available information. These facts of hybridizing in two thirds of the W Palaearctic wood ant species, of extreme regional hybrid frequencies (up to 26%), of unidirectional purging of nDNA associated with mismatching mtDNA haplotypes, and of occasional achievement of local dominance of these mismatch combinations, may serve as urgent warning not to perform isolated mtDNA phylogenetic studies without a geographically and locally wide sampling basis and without control by nDNA information or reliable phenotypic determination. The latter two systems definitely have superior significance when conflicts with mtDNA indications arise.