形态性状、分子性状与同源性

“同源性（homology）”是生物学中最基本的概念之一。近年来,随着分子生物学、生物信息学、发育生物学以及进化发育遗传学等学科的快速发展,同源性一词在形态性状的比较、核苷酸和氨基酸序列的分析以及探讨形态性状进化的分子机制等方面都有广泛应用。然而,由于不同的研究者对同源性概念的理解有所不同,在实际应用中难免会出现不恰当使用“同源性”一词并得出错误结论的情况。本文从不同的角度介绍了如何对同源性进行判断以及影响同源性判断的因素。并指出正确理解同源性这一概念的含义,以及通过综合各方面的证据对同源性进行推断对于揭示基因型和表型的进化以及二者之间的关系非常重要。     

A novel synthetic small molecule YH‐306 suppresses colorectal tumour growth and metastasis via FAK pathway

Cell migration and invasion are key processes in the metastasis of cancer, and suppression of these steps is a promising strategy for cancer therapeutics. The aim of this study was to explore small molecules for treating colorectal cancer (CRC) and to investigate their anti‐metastatic mechanisms. In this study, six CRC cell lines were used. We showed that YH‐306 significantly inhibited the migration and invasion of CRC cells in a dose‐dependent manner. In addition, YH‐306 inhibited cell adhesion and protrusion formation of HCT116 and HT‐29 CRC cells. Moreover, YH‐306 potently suppressed uninhibited proliferation in all six CRC cell lines tested and induced cell apoptosis in four cell lines. Furthermore, YH‐306 inhibited CRC colonization in vitro and suppressed CRC growth in a xenograft mouse model, as well as hepatic/pulmonary metastasis in vivo. YH‐306 suppressed the activation of focal adhesion kinase (FAK), c‐Src, paxillin, and phosphatidylinositol 3‐kinases (PI3K), Rac1 and the expression of matrix metalloproteases (MMP) 2 and MMP9. Meanwhile, YH‐306 also inhibited actin‐related protein (Arp2/3) complex‐mediated actin polymerization. Taken together, YH‐306 is a candidate drug in preventing growth and metastasis of CRC by modulating FAK signalling pathway.     

Homology, biogeography and areas of endemism

Hypotheses of biogeographic homology constitute the basis of historical biogeography. Primary biogeographic homology refers to a conjecture on a common biogeographic history, and secondary biogeographic homology refers to the cladistic test of the formerly recognized homology. Panbiogeography deals with the former, through the recognition of generalized tracks and areas of endemism, whereas cladistic biogeography deals with the latter, through the generation of general area cladograms. A historical biogeographic analysis may include both approaches, in a two‐stage analysis.     

通过改换大肠杆菌腺苷酸代谢途径来增加胞内能荷水平

原核生物细胞能量和物质代谢的途径是一个很复杂的网络,改变代谢途径中的基因会对能量和物质流产生怎样的影响,仍然不是很清楚.以往的文献和研究已经将大肠杆菌的腺嘌呤核苷酸补救合成途径研究的很透彻.使用HPLC对删除了add基因的大肠杆菌细胞内腺苷类核苷酸分析表明,在腺嘌呤核苷酸补救途径中单一基因的途径操作不能有效改变腺嘌呤类核苷酸的代谢流向.实验中通过删除大肠杆菌JM83株中的add基因(编码腺苷脱氨酶[EC:3.5.4.4][1,2]),deoD基因(编码嘌呤核苷磷酸酶[EC:2.4.2.1][3,4]),amn基因(编码AMP核苷酶[EC:3.2.2.4][5])并引入外源ado1基因(来自酵母编码腺苷激酶[EC:2.7.1.20][6,7,8]),构建了菌株J991 (add-,deoD-,amn-,ado1 ,JM83),将其在含腺苷的LB培养基培养,使用HPLC分析其胞内腺苷类能量物质发现,ATP,ADP,AMP胞内含量都有所增加,分别都比对照JM83菌株提高一倍左右,大大加强了腺苷转化AMP的代谢流量,实现了改变物质代谢流向并使ATP积累的目的.该菌种实现了高产ATP代谢通路的构建,为下游生物工程发酵提供了较野生菌更高效的菌种,有望通过发酵工程优化培养,大幅提高ATP产量.同时,"尝试改变AMP的浓度而非直接针对ATP调节代谢途径,达到ATP积累的目"这一思路为同类研究提供参考.最后也表明在腺嘌呤核苷酸补救代谢途径中,为达到物质代谢流改变的目的,多基因联合操作较之单基因敲除更为有效.     

Expression of a Vibrio parahaemolyticus toxin in Escherichia coli results in chromosomal DNA degradation

A toxin-antitoxin system, vp1842/vp1843, locates within a superintegron on the Vibrio parahaemolyticus genome chromosome I whose toxin gene vp1843 encodes a DNA nicking endonuclease. We found that the vp1843 expression in Escherichia coli cells strongly induced chromosomal DNA degradation. On the basis of these observations, we discuss a possible physiological role of vp1842/vp1843 in V. parahaemolyticus.     

Polyphosphates and enzymes of polyphosphate metabolism in Escherichia coli

This review summarizes the results of our study of polyphosphate and enzymes of polyphosphate metabolism in E. coli and their regulation by exogenous orthophosphate and other physiological and genetic factors.     

Screening of the small molecule library of Meinox enables the identification of anticancer compounds in pathologically distinct cancers

Small molecules are widely used for the modulation of the molecular basis of diseases. This makes them the perfect tool for discovering and developing new therapeutics. In this work, we have established a library of small molecules in house and characterized its molecular and druglike properties. We have shown that most small molecules have molecular weights less than 450. They have pharmaceutically relevant cLogP, cLogS, and druglikeness value distributions. In addition, Meinox’s small molecule library contained small molecules with polar surface areas that are less than 60 square angstroms, suggesting their potent ability to cross the blood-brain barrier. Meinox’s small molecule library was also tested in vitro for pathologically distinct forms of cancer, including pancreatic adenocarcinoma PANC1, breast carcinoma MCF7, and lymphoblastic carcinoma RS4-11 cell lines. Analysis of this library at a dose of 1 μM allowed the discovery of potent, specific or broadly active anticancer compounds against pathologically distinct cancers. This study shows that in vitro analysis of different cancers or other phenotypic assays with Meinox small molecule library may generate novel and potent bioassay-specific compounds.     

Membrane localization of small proteins in Escherichia coli

Escherichia coli synthesize over 60 poorly understood small proteins of less than 50 amino acids. A striking feature of these proteins is that 65% contain a predicted α-helical transmembrane (TM) domain. This prompted us to examine the localization, topology, and membrane insertion of the small proteins. Biochemical fractionation showed that, consistent with the predicted TM helix, the small proteins generally are most abundant in the inner membrane fraction. Examples of both N(in)-C(out) and N(out)-C(in) orientations were found in assays of topology-reporter fusions to representative small TM proteins. Interestingly, however, three of nine tested proteins display dual topology. Positive residues close to the transmembrane domains are conserved, and mutational analysis of one small protein, YohP, showed that the positive inside rule applies for single transmembrane domain proteins as has been observed for larger proteins. Finally, fractionation analysis of small protein localization in strains depleted of the Sec or YidC membrane insertion pathways uncovered differential requirements. Some small proteins appear to be affected by both Sec and YidC depletion, others showed more dependence on one or the other insertion pathway, whereas one protein was not affected by depletion of either Sec or YidC. Thus, despite their diminutive size, small proteins display considerable diversity in topology, biochemical features, and insertion pathways.     

Synergy between methylerythritol phosphate pathway and mevalonate pathway for isoprene production in Escherichia coli

Isoprene, a key building block of synthetic rubber, is currently produced entirely from petrochemical sources. In this work, we engineered both the methylerythritol phosphate (MEP) pathway and the mevalonate (MVA) pathway for isoprene production in E. coli. The synergy between the MEP pathway and the MVA pathway was demonstrated by the production experiment, in which overexpression of both pathways improved the isoprene yield about 20-fold and 3-fold, respectively, compared to overexpression of the MEP pathway or the MVA pathway alone. The ¹³C metabolic flux analysis revealed that simultaneous utilization of the two pathways resulted in a 4.8-fold increase in the MEP pathway flux and a 1.5-fold increase in the MVA pathway flux. The synergy of the dual pathway was further verified by quantifying intracellular flux responses of the MEP pathway and the MVA pathway to fosmidomycin treatment and mevalonate supplementation. Our results strongly suggest that coupling of the complementary reducing equivalent demand and ATP requirement plays an important role in the synergy of the dual pathway. Fed-batch cultivation of the engineered strain overexpressing the dual pathway resulted in production of 24.0 g/L isoprene with a yield of 0.267 g/g of glucose. The synergy of the MEP pathway and the MVA pathway also successfully increased the lycopene productivity in E. coli, which demonstrates that it can be used to improve the production of a broad range of terpenoids in microorganisms.     

D-arabinose metabolism in Escherichia coli B: induction and cotransductional mapping of the L-fucose-D-arabinose pathway enzymes.

D-Arabinose is degraded by Escherichia coli B via some of the L-fucose pathway enzymes and a D-ribulokinase which is distinct from the L-fuculokinase of the L-fucose pathway. We found that L-fucose and D-arabinose acted as the apparent inducers of the enzymes needed for their degradation. These enzymes, including D-ribulokinase, appeared to be coordinately regulated, and mutants which constitutively synthesized the L-fucose enzymes also constitutively synthesized D-ribulokinase. In contrast to D-arabinose-positive mutants of E. coli K-12, in which L-fuculose-1-phosphate and D-ribulose-1-phosphate act as inducers of the L-fucose pathway, we found that these intermediates did not act as inducers in E. coli B. To further characterize the E. coli B system, some of the L-fucose-D-arabinose genes were mapped by using bacteriophage P1 transduction. A transposon Tn10 insertion near the E. coli B L-fucose regulon was used in two- and three-factor reciprocal crosses. The gene encoding D-ribulokinase, designated darK, was found to map within the L-fucose regulon, and the partial gene order was found to be Tn10-fucA-darK-fucI-fucK-thyA.     

Biosynthesis of geranate via isopentenol utilization pathway in Escherichia coli

Isoprenoids are a large family of natural products with diverse structures, which allow them to play diverse and important roles in the physiology of plants and animals. They also have important commercial uses as pharmaceuticals, flavoring agents, fragrances, and nutritional supplements. Recently, metabolic engineering has been intensively investigated and emerged as the technology of choice for the production of isoprenoids through microbial fermentation. Isoprenoid biosynthesis typically originates in plants from acetyl-coA in central carbon metabolism, however, a recent study reported an alternative pathway, the isopentenol utilization pathway (IUP), that can provide the building blocks of isoprenoid biosynthesis from affordable C5 substrates. In this study, we expressed the IUP in Escherichia coli to efficiently convert isopentenols into geranate, a valuable isoprenoid compound. We first established a geraniol-producing strain in E. coli that uses the IUP. Then, we extended the geraniol synthesis pathway to produce geranate through two oxidation reactions catalyzed by two alcohol/aldehyde dehydrogenases from Castellaniella defragrans. The geranate titer was further increased by optimizing the expression of the two dehydrogenases and also parameters of the fermentation process. The best strain produced 764 mg/L geranate in 24 h from 2 g/L isopentenols (a mixture of isoprenol and prenol). We also investigated if the dehydrogenases could accept other isoprenoid alcohols as substrates.     

Interrelationships between the enzymes of ethanolamine metabolism in Escherichia coli

The activities of the enzymes ethanolamine ammonia-lyase, CoA-dependent and CoA-independent aldehyde dehydrogenases, and isocitrate lyase were assayed in Escherichia coli which had been grown on various sources of carbon and nitrogen. Induction of ethanolamine ammonia-lyase and of maximal levels of both aldehyde dehydrogenases required the concerted effects of ethanolamine and vitamin (or coenzyme) B12. Molecular exclusion chromatography revealed that, in the absence of one or both co-inducers, two repressible isoenzymes of CoA-dependent aldehyde dehydrogenase (mol. wts 900000 and 120000) were produced, these being replaced by two inducible isoenzymes (mol. wts 520000 and 370000) in the presence of both co-inducers. A similar inducible repressible series of isoenzymes was also observed for CoA-independent aldehyde dehydrogenase. No evidence was found for structural relationships between ethanolamine ammonia-lyase, CoA-dependent aldehyde dehydrogenase and CoA-independent aldehyde dehydrogenase, but mutant and physiological studies demonstrated that the induction of the first two enzymes is under common control. Evidence is presented for the operation of a previously unreported pathway of ethanolamine metabolism in E. coli.     

Oncogene amplification and chromosomal abnormalities in small cell lung cancer

Twelve cell lines isolated from patients with small cell lung cancer have been studied for amplification of the three characterised members of the myc proto-oncogene family (c-myc, N-myc, and L-myc) and for abnormalities of chromosome 3. Ten of these lines were being studied for the first time. Ten of the 12 small cell lung cancer cell lines had amplification of one member of the myc proto-oncogene family. Amplification of c-myc was observed in only one small cell lung line--a "morphological variant". One "classic" small cell lung cancer line expressed c-myc but had no obvious amplification of the gene. N-myc and L-myc were more commonly amplified than c-myc. Chromosomal abnormalities (mainly deletions) in chromosome 3 were observed in all small cell lung carcinoma cell lines examined. When the small cell lung carcinoma lines were grouped according to "classic" or "variant" characteristics, it was found that the "classics" had deletions of the short arm of chromosome 3, whereas the "biochemical variants" had deletions of the long arm of chromosome 3. The extent of the deletions varied between cell lines. For the deletion in the short arm of chromosome 3 the minimum common region of overlap was assigned to bands 3p23-3p24.     

过表达羧化途径及失活苹果酸酶基因对大肠杆菌好氧发酵产苹果酸的影响

苹果酸是一种重要的C4二羧酸,在食品、医药、化工等领域有广泛的应用。本文主要研究羧化途径强化及苹果酸酶失活对大肠杆菌好氧发酵生产苹果酸的影响。首先在大肠杆菌E2中过表达了磷酸烯醇式丙酮酸羧化酶基因ppc,得到菌株E21,苹果酸积累量从0.57 g/L提高到3.83 g/L。随后,分别过表达来自谷氨酸棒杆菌的丙酮酸羧化酶基因pyc和来自琥珀酸放线杆菌的磷酸烯醇式丙酮酸激酶pck基因,相应的工程菌株E21(pTrcpyc)和E21(pTrc-A-pck)分别产6.04和5.01 g/L苹果酸,得率分别达到0.79和0.65 mol/mol葡萄糖。敲除E21中的苹果酸酶基因mae A和mae B,苹果酸产量也显著提高了36%,达到5.21 g/L,得率为0.62 mol/mol。然而,在过表达pyc的基础上敲除苹果酸酶基因并不能进一步提高苹果酸的产量。经过摇瓶发酵条件的初步优化,菌株E21(pTrcpyc)生产12.45 g/L苹果酸,得率为0.84 mol/mol,达到理论得率的63.2%。     

Combinational biosynthesis of isoprene by engineering the MEP pathway in Escherichia coli

As an important feedstock in petrochemistry, isoprene is used in a wide range of industrial applications. It is produced almost entirely from petrochemical sources; however, these sources are being progressively depleted. A reliable biological process for isoprene production utilizing renewable feedstocks would be an industry-redefining development. There are two biosynthetic pathways producing isoprene: the mevalonate (MVA) pathway and the methyl erythritol 1-phosphate (MEP) pathway. In this study, the MEP pathway was modified in Escherichia coli BL21 (DE3) to produce isoprene. The isoprene synthase (IspS) gene chemically synthesized from Populus alba after codon optimization for expression in E. coli was heterologously expressed. The endogenous genes of 1-deoxy-d-xylulose-5-phosphate synthase (DXS) and 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) were over-expressed. The isopentenyl pyrophosphate isomerase (Idi) gene from Streptococcus pneumoniae was exogenously over-expressed, and farnesyl diphosphate synthase (ispA) was weakened to enhance the yield. The control strain harboring empty plasmids did not emit any isoprene. The over-expression of the DXR gene only had little impact on the yield of isoprene. Idi from S. pneumoniae played a significant role in the improvement of isoprene production. The highest yield was achieved by an ispA-weakened DXS-IDI-IspS recombinant with 19.9 mg/l isoprene, which resulted in a 33-fold enhancement of the isoprene yield from the IspS recombinant.     

Discovery of a novel small molecule agonist scaffold for the APJ receptor

The apelinergic system includes a series of endogenous peptides apelin, ELABELA/TODDLER and their 7-transmembrane G-protein coupled apelin receptor (APJ, AGTRL-1, APLNR). The APJ receptor is an attractive therapeutic target because of its involvement in cardiovascular diseases and potentially other disorders including liver fibrosis, obesity, diabetes, and neuroprotection. To date, pharmacological characterization of the APJ receptor has been limited due to the lack of small molecule functional agonists or antagonists. Through focused screening we identified a drug-like small molecule agonist hit 1 with a functional EC₅₀ value of 21.5 ± 5 μM and binding affinity (K_i) of 5.2 ± 0.5 μM. Initial structure–activity studies afforded compound 22 having a 27-fold enhancement in potency and the first sub-micromolar full agonist with an EC₅₀ value of 800 ± 0.1 nM and K_i of 1.3 ± 0.3 μM. Preliminary SAR, synthetic methodology, and in vitro pharmacological characterization indicate this scaffold will serve as a favorable starting point for further refinement of APJ potency and selectivity.

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大肠杆菌角鲨烯合成途径的构建与调控

角鲨烯因其具有良好的抗氧化功能而被广泛应用于食品、医药、化妆品、工业应用等领域。本实验在大肠杆菌中构建角鲨烯合成途径,通过对其合成途径中关键限速酶(1-脱氧-D-木酮糖-5-磷酸合酶和异戊烯基二磷酸异构酶)过表达的方法进行初步调控,使角鲨烯的产量提升了近三倍。之后采用单因素试验对其发酵培养基和培养条件进行优化,以此来提高角鲨烯的产量。优化发酵条件后,使用最优发酵培养基——TB培养基,在最佳发酵条件:37℃,220r/min培养至OD600约为1.2时加入终浓度为0.1mmol/L的IPTG诱导剂,25℃条件下诱导48h,角鲨烯产量可达73.88mg/L。     

Studies on the Robertsonian chromosomal variation of Mus musculus domesticus (Rodentia, Muridae) in Greece

The karyotype of the house mouse, Mus musculus domesticus , was examined in 282 specimens from 44 localities, in an effort to gain better understanding of the Robertsonian (Rb) variation known to exist in Greece. We consider that an Rb system exists in Peloponnisos, southern Greece, distributed in an area that is substantially larger than previously known. It consists of at least three Rb races with 2 n  = 30, 2 n  = 24 and 2 n  = 28, respectively, the last being reported for the first time in this paper and carrying Rb(3.6), Rb(8.12), Rb(10.14), Rb(13.15), Rb(9.16) and Rb(11.17) in a homozygous state. Additional instances of variation in this Rb system include individuals with 2 n  = 31 and 32 of variable Rb constitution and hybrids between the Rb races with 2 n  = 30 and 2 n  = 24. In southern Peloponnisos, Rb(10.14) was found in either a homozygous or a heterozygous state (2 n  = 38 or 39). The relationships among the Rb populations of Peloponnisos are discussed and hypotheses for their evolution are proposed. Rb variation was also recorded in two new locations of eastern Sterea Ellas (2 n  = 28 and 29) and one in Ipiros, north-west Greece (2 n  = 38). These findings corroborate the existence of two separate Rb systems in those two areas. Moreover, among a number of islands surveyed, Rb variation was only found in Kythira island, with Rb(10.14) in a heterozygous state (2 n  = 39). Finally, the typical all-acrocentric karyotype (2 n  = 40) was found in 51 of the animals studied from 13 localities. © 2005 The Linnean Society of London, Biological Journal of the Linnean Society , 2005, 84 , 503–513.