青花菜谷胱甘肽-S-转移酶基因克隆及其表达分析

为探讨青花菜在模拟酸雨胁迫下谷胱甘肽-S-转移酶的表达变化,克隆了青花菜谷胱甘肽-S-转移酶基因(glutathione-S-transferase,GST)的cDNA序列全长,并进行了生物信息学和表达分析。结果表明:青花菜GST基因cDNA全长为915bp,开放阅读框为642bp,编码213个氨基酸,推测分子式为C1091H1719N289O306S5,分子量为23 940.7,没有跨膜螺旋区域和信号肽。系统进化树分析表明,该青花菜基因GST与芥菜的GST聚类关系最近。实时荧光定量PCR结果显示,在模拟酸雨胁迫下,GST基因的表达量在胁迫初期显著增大,随时间延长开始下降,表明其参与了青花菜抗酸雨的应答反应。     

Proteomic characterization of the released/secreted proteins of Leishmania (Viannia) braziliensis promastigotes

Extracellular proteins secreted/released by protozoan parasites are key mediators of the host–parasite interaction. To characterise the profile of proteins secreted/released by Leishmania (Viannia) braziliensis promastigotes, a proteomic approach combining two-dimensional electrophoresis (2DE), tandem matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF/TOF) mass spectrometry, and data mining was carried out. The 2DE map revealed a set of 270 secreted protein spots from which 42 were confidently identified and classified into 11 categories according to Gene Ontology (GeneDB database) and KEEG Ontology annotation of biological processes. Parasite promastigotes were able to secrete/release proteins involved in immunomodulation, signal transduction, and intracellular survival, such as HSP70, acid phosphatase, activated protein kinase C receptor (LACK), elongation factor 1β, and tryparedoxin peroxidase. Data mining showed that ~ 5% of identified proteins present a classical secretion signal whereas ~ 57% were secreted following non-classical secretion mechanisms, indicating that protein export in this primitive eukaryote might proceed mainly by unconventional pathways. This study reports a suitable approach to identify secreted proteins in the culture supernatant of L. braziliensis and provides new perspectives for the study of molecules potentially involved in the early stages of infection.     

NADPH : protochlorophyllide oxidoreductases in white pine (Pines strobes) and loblolly pine (P. taeda)

Summary NADPH : protochlorophyllide oxidoreductase (pchlide reductase, EC 1.6.99.1) catalyzes the light-dependent reduction of protochlorophyllide in higher plants. Cloned cDNAs encoding two distinct pchlide reductases were isolated from a gt11 library constructed from poly(A)⁺ RNA prepared from the cotyledons of dark-grown white pine (Pines strobes) seedlings and a nuclear gene (lpcr) analogous to one of these cDNAs has been characterized from loblolly pine (P. taeda). The pine gene encodes an approximately 43 kDa precursor polypeptide consisting of a 334-amino acid mature protein and a 66-amino acid transit peptide. The deduced primary structures for the pine proteins are highly homologous to those reported from monocots and dicots. The coding portion of the pine lpcr gene is interrupted by four introns. The placement of these introns within the pine lpcr gene is identical to that observed in pea (Pisum sativum), suggesting conservation in gene organization between dicot and gymnosperm species. Western blot analysis using polyclonal antiserum against oat pchlide reductase detected in extracts of dark-grown pine cotyledons a single immunoreactive protein, which declined in abundance during a 48 h period of illumination with white light. Cotyledons of dark-grown seedlings were also found to accumulate high levels of pchlide reductase mRNA; however, little or no change in the steady-state levels of mRNA encoding pchlide reductase was observed in these tissues following illumination. Stem tissue of dark-grown seedlings did not contain significant levels of pchlide reductase mRNA, whereas stems of light-grown plants of the same age accumulated substantial amounts of the message. These results suggest that light and the developmental age of the tissue affect regulation of lpcr expression in pine.     

Acaricidal activity of the essential oil from Tetradenia riparia (Lamiaceae) on the cattle tick Rhipicephalus (Boophilus) microplus (Acari; Ixodidae)

Tetradenia riparia (Lamiaceae) is a well-known herbal medicine with a variety of useful properties, including its acaricidal effect. This experiment was carried out to study the bioacaricidal activity of T. riparia essential oil (EO) against engorged females of Rhipicephalus (Boophilus) microplus (Acari; Ixodidae). For this purpose, nine serial concentrations (12.50%, 6.25%, 3.75%, 1.80%, 0.90%, 0.45%, 0.22%, 0.11%, and 0.056% w/v) of T. riparia were used for the adult immersion test (AIT). For the larval packet test (LPT), we used 14 serial concentrations (100.00%, 50.00%, 25.00%, 12.50%, 6.25%, 3.65%, 1.82%, 0.91%, 0.45%, 0.228%, 0.114%, 0.057%, 0.028%, and 0.014% w/v). The results for AIT showed 100.00% and 2.05% mortality, 19.00 and 90.20% for the total number of eggs, egg-laying inhibition of 0.00% and 90.20%, hatchability inhibition of 0.00% and 70.23%, and product effectiveness of 100.00% and 2.89%, respectively. The AIT indicated that the LC₅₀ and LC_99.9, calculated using the Probit test, were for mortality (%) 0.534 g/mL (0.436–0.632) and 1.552 g/mL (1.183–1.92); for total number of eggs were 0.449 g/mL (0.339–0.558) and 1.76 g/mL (1.27–2.248); and for hatchability inhibition were 0.114 g/mL (0.0–0.31) and 2.462 g/mL (1.501–3.422), respectively. Larvae between 14 and 21 days old were fasted and placed in each envelope. Bioassays were performed at 27° ± 1 °C, RH ? 80%. Larval mortality was observed 24 h after treatment and showed 10.60–100% mortality in the LPT bioassay. The LPT showed that the LC₅₀ and LC_99.9 were 1.222 g/mL (0.655–1.788) and 11.382 g/mL (7.84–14.91), respectively. A positive correlation between T. riparia EO concentration and tick control, was observed by the strong acaricidal effects against R. (B.) microplus, and the mortality rate of ticks was dose-dependent. Our results showed that T. riparia is a promising candidate as an acaricide against resistant strains of R. (B.) microplus.     

Flavonoids and other constituents of Hymenostegia afzelii (Caesalpiniaceae)

Phytochemical investigation of the stem bark and leaves of Hymenostegia afzelii resulted in the isolation of three new flavonoids, named afzelin A-C (1–3), together with five known compounds: 7,6-(2″,2″-dimethylpyrano)-3,5,4′-trihydroxyflavone, apigenin, 3-O-α-l-rhamnopyranosylcincholic acid, 3-O-β-d-glucopyranosylcincholic acid, and dodecanoic acid 1,1′-[(1S)-1-(hydroxymethyl)-1,2-ethanediyl] ester. The structures of 1–3 were determined by means of spectroscopic methods. Compounds 1–3 were tested in vitro for their preliminary cytotoxicity using the Artemia salina assay.     

Nucleotide sequence of genes hrdA, hrdC, and hrdD from Streptomyces coelicolor A3(2) having similarity to rpoD genes

Summary The complete nucleotide sequences were determined of hrdA, hrdC, and hrdD from Streptomyces coelicolor A3(2). They indicate the presence of a single open reading frame in each gene coding for polypeptides of 396 (43747 daltons), 339 (38173 daltons), and 332 amino acid residues (37190 daltons), respectively. These amino acid sequences revealed extensive similarities with the principal sigma factors of Bacillus subtilis, Escherichia coli, Mxyococcus xanthus, Pseudomonas aeruginosa, and also the katF gene product of E. coli. Besides the highly conserved amino acid residues in the rpoD box region, alignment of hrd gene products and the known principal sigma factors and sigma-related factors allowed us to postulate a common basic structure for the principal sigma type factors as distinct from the alternative sigma factors.     

A review of the systematics of the sleeper shark genus Somniosus with redescriptions of Somniosus ( Somniosus) antarcticus and Somniosus ( Rhinoscymnus) longus (Squaliformes: Somniosidae)

Past treatments of the sleeper shark genus Somniosus generally recognize three species: S. microcephalus, S. pacificus, and S. rostratus. Based on morphometrics and meristics, we conclude that this genus includes two subgenera (Somniosus and Rhinoscymnus) and five species. Subgenus Somniosus differs from Rhinoscymnus by being much larger when adult and in having more numerous tooth rows in the lower jaw, hooklike rather than leaf-shaped dermal denticles, more numerous spiral valve and vertebral counts, and a poorly calcified vertebral column. Subgenus Somniosus includes S. (Somniosus) microcephalus and S. (S.) pacificus of the Northern Hemisphere and S. (S.) antarcticus of the Southern Hemisphere. Although Somniosus antarcticus has been synonymized with S. microcephalus and identified as S. pacificus in past literature, it differs from S. microcephalus in having a shorter interdorsal space, a more posterior first dorsal fin, lower dorsal fins, more numerous tooth rows in the lower jaw, more numerous spiral valve counts, and fewer precaudal vertebrae. Somniosus antarcticus also differs from S. pacificus by having a shorter prebranchial length, lower dorsal fins, more numerous spiral valve counts, and slightly more precaudal vertebrae. Subgenus Rhinoscymnus includes S. (Rhinoscymnus) rostratus from the eastern North Atlantic and Mediterranean Sea (senior synonym of S. bauchotae) and S. (R.) longus from the western Pacific Ocean. Somniosus longus has been synonymized with S. rostratus, but differs in having a relatively longer second dorsal fin, a slightly larger eye, more lower tooth rows, and slightly higher spiral valve counts. Both Somniosus (Somniosus) antarcticus and S. (Rhinoscymnus) longus from the Pacific Ocean were redescribed. A key to the species and the geographical distribution of all species are provided.     

大黄属(蓼科) 植物ｎｄｈＦ 基因的适应性进化

大黄属(Rheum L.)是蓼科(Polygonaceae)中一个高度分化的大属,广泛分布在亚洲和欧洲的高山和沙漠地区,全世界约60种,其中在青藏高原及其邻近地区发现了约40种。该属种的高度分化曾被推测是第三纪末青藏高原的快速隆升以及第四纪气候的反复变化所引发的适应性辐射导致。为进一步了解大黄属植物辐射式物种分化的分子适应机制,该研究选取34个形态上多样化的大黄属物种,利用系统发育分析软件,在时间框架下采用位点模型和分支模型对大黄属的叶绿体ndhF基因进行了适应性进化分析。结果表明：大黄属植物的分子进化系统树呈现短而平行的辐射式分支式样,显示出典型的物种快速辐射多样化特征;用位点模型检验ndhF基因是否存在经受正向选择(ω>1)时,在氨基酸水平上共鉴定出3个NDHF亚基的正选择位点(188H,465H,551L),对NDHF亚基的二级结构进行分析后发现编码的188H氨基酸位于α螺旋上。大黄属植物可能通过这些结构域的适应性进化,适应青藏高原的快速隆升以及第四纪气候的反复变化而引发的陆地生态系统改变。该研究结果可为今后对该属植物的实验分析提供首选位点。     

Induction of a non-specific permeability transition in mitochondria from Yarrowia lipolytica and Dipodascus (Endomyces) magnusii yeasts

In this study we used tightly-coupled mitochondria from Yarrowia lipolytica and Dipodascus (Endomyces) magnusii yeasts, possessing a respiratory chain with the usual three points of energy conservation. High-amplitude swelling and collapse of the membrane potential were used as parameters for demonstrating induction of the mitochondrial permeability transition due to opening of a pore (mPTP). Mitochondria from Y. lipolytica, lacking a natural mitochondrial Ca²⁺ uptake pathway, and from D. magnusii, harboring a high-capacitive, regulated mitochondrial Ca²⁺ transport system (Bazhenova et al. J Biol Chem 273:4372–4377, 1998a; Bazhenova et al. Biochim Biophys Acta 1371:96–100, 1998b; Deryabina and Zvyagilskaya Biochemistry (Moscow) 65:1352–1356, 2000; Deryabina et al. J Biol Chem 276:47801–47806, 2001) were very resistant to Ca²⁺ overload. However, exposure of yeast mitochondria to 50–100 μM Ca²⁺ in the presence of the Ca²⁺ ionophore ETH129 induced collapse of the membrane potential, possibly due to activation of the fatty acid-dependent Ca²⁺/nH⁺-antiporter, with no classical mPTP induction. The absence of response in yeast mitochondria was not simply due to structural limitations, since large-amplitude swelling occurred in the presence of alamethicin, a hydrophobic, helical peptide, forming voltage-sensitive ion channels in lipid membranes. Ca²⁺- ETH129-induced activation of the Ca²⁺/H⁺-antiport system was inhibited and prevented by bovine serum albumin, and partially by inorganic phosphate and ATP. We subjected yeast mitochondria to other conditions known to induce the permeability transition in animal mitochondria, i.e., Ca²⁺ overload (in the presence of ETH129) combined with palmitic acid (Mironova et al. J Bioenerg Biomembr 33:319–331, 2001; Sultan and Sokolove Arch Biochem Biophys 386:37–51, 2001), SH-reagents, carboxyatractyloside (an inhibitor of the ADP/ATP translocator), depletion of intramitochondrial adenine nucleotide pools, deenergization of mitochondria, and shifting to acidic pH values in the presence of high phosphate concentrations. None of the above-mentioned substances or conditions induced a mPTP-like pore. It is thus evident that the permeability transition in yeast mitochondria is not coupled with Ca²⁺ uptake and is differently regulated compared to the mPTP of animal mitochondria.     

Phylogenetic relationships of Ceratitis fruit flies inferred from nuclear CAD and tango/ARNT gene fragments: Testing monophyly of the subgenera Ceratitis (Ceratitis) and C. (Pterandrus)

Systematic studies of Ceratitis (Tephritidae) fruit flies using molecular (i.e., COI, ND6, and period genes) and morphological (plus host-use characters) data have recently challenged the monophyly of the subgenera Ceratitis (Ceratitis) and Ceratitis (Pterandrus). In this paper, we report on the phylogenetic utility of three single-copy nuclear gene regions (two non-overlapping fragments of the carbamoylphosphate synthetase, CPS, locus of CAD, and a fragment of tango) within these taxa and investigate evolutionary relationships based on a concatenated ca. 3.4 kb data set that includes the six protein encoding gene regions. Results indicate that the CAD and tango genes provide useful phylogenetic signal within the taxa and are compatible with the previously studied genes. The two subgenera, as currently classified, are not monophyletic. Our molecular phylogenetic analyses support a revised classification in which (1) the subgenus C. (Pterandrus) comprises two lineages called A and B, (2) the C. (Pterandrus) B species should be included in C. (Ceratitis), and (3) the newly defined subgenera C. (Pterandrus) (=Pterandrus section A) and C. (Ceratitis) [=C. (Ceratitis) + C. (Pterandrus) section B] are reciprocally monophyletic.     

Cumanensic acid, a new chromene isolated from Piper cf. cumanense Kunth. (Piperaceae)

A new chromene, cumanensic acid (1), together with eight known compounds have been isolated from the aerial part of Piper cf. cumanense Kunth (Piperaceae). Chromene structure was identified on the basis of spectroscopic analysis and comparison with literature data. The compound showed antifungal activity against Fusarium oxysporum f. sp. dianthi and Botrytis cinerea.     

The phylogenetic placement of Ostropales within Lecanoromycetes (Ascomycota) revisited

Results of molecular studies regarding the phylogenetic placement of the order Ostropales and related taxa within Lecanoromycetes were thus far inconclusive. Some analyses placed the order as sister to the rest of Lecanoromycetes, while others inferred a position nested within Lecanoromycetes. We assembled a data set of 101 species including sequences from nuLSU rDNA, mtSSU rDNA, and the nuclear protein-coding RPB1 for each species to examine the cause of incongruencies in previously published phylogenies. MP, minimum evolution, and Bayesian analyses were performed using the combined three-region data set and the single-gene data sets. The position of Ostropales nested in Lecanoromycetes is confirmed in all single-gene and concatenated analyses, and a placement as sister to the rest of Lecanoromycetes is significantly rejected using two independent methods of alternative topology testing. Acarosporales and related taxa (Acarosporaceae group) are basal in Lecanoromycetes. However, if the these basal taxa are excluded from the analyses, Ostropales appear to be sister to the rest of Lecanoromycetes, suggesting different ingroup rooting as the cause for deviating topologies in previously published phylogenies.     

Methyl palmitate: a novel product of the Medfly (Ceratitis capitata) corpus allatum

The corpus allatum (CA) of adult female Ceratitis capitata produces methyl palmitate (MP) in vitro, in addition to JHB₃ and JH III. Biosynthesized MP migrates on TLC and co-elutes from RP-18 HPLC with synthetic MP. Its identity is verified herein by GCMS. MP production is up-regulated twofold by mevastatin, an inhibitor of mevalonic acid-dependent isoprene biosynthesis. Fosmidomycin, an inhibitor of mevalonic acid-independent isoprene synthesis in graminaceous plants, up-regulates MP synthesis by about fourfold. However, it does not depress JHB₃ biosynthesis concurrently. This suggests that the initial enzyme(s) in the conversion of 1-deoxy-xylulose 5-phosphate to isoprene is presumably present in C. capitata, but is inhibited by fosmidomycin, and this inhibition diverts precursors to MP synthesis. Phytol, an acyclic diterpene, might be suppressing isoprene biosynthesis by CA, thereby resulting in a fourfold increase in the MP biosynthesis. Linolenic acid is an end-product and its presence in incubation media up-regulates MP biosynthesis by twofold, presumably due to the feedback diversion to biosynthesis of C_16:0 and its methyl ester. Biosynthesis of MP is markedly depressed after mating, while otherwise maintained at significantly higher levels in virgin females. MP biosynthesis is significantly reduced in virgin females by direct axonal control but is less consistent after mating.     

ThenadB gene ofSalmonella typhimurium complements the nicotinic acid auxotrophy ofShigella flexneri

Shigella species are characteristically nicotinic acid (NA) auxotrophs. The invasiveS. flexneri strain M90T, transformed with the multicopy plasmid pZT349 encoding thenadB gene ofSalmonella typhimurium, can grow in minimal glucose medium without exogenous NA, whereas, M90T containing the control vector, pUC18 does not, suggesting that this species lacksl-aspartic acid oxidase, the first enzyme in the de novo NAD biosynthetic pathway. The estimated growth rate of strain M90T (pZT349) in HeLa cells was identical to that of M90T (pUC18), indicating the available intracellular concentration of NA is not limiting for bacterial growth.     

荧光假单胞菌CLW17菌株pqqE和GDH基因的功能

[背景]磷是植物生长所必需的大量元素,但绝大多数不能被植物吸收利用。然而溶磷微生物能够分泌有机酸来溶解土壤中难溶性磷,提高土壤中磷的利用率,促进植物生长,提高作物的产量和品质。[目的]探究高效解磷荧光假单胞菌CLW17菌株的pqqE和GDH基因的生理学功能。[方法]利用生物在线软件对2个基因编码蛋白进行生物信息学分析。利用同源重组技术分别获得pqqE和GDH基因缺失突变株(CLW17ΔpqqE,CLW17ΔGDH),并使用接合转移的方式获得回补菌株(ΔpqqE/pqqE,ΔGDH/GDH)。分别采用NBRIP培养基、钼锑抗比色法及高压液相色谱法(HPLC)对野生型、突变株及互补株的溶磷及产有机酸能力进行检测。[结果]pqqE和GDH基因编码氨基酸数目分别为390和803,均无信号肽。pqqE无跨膜结构域,而GDH预测有5个跨膜结构域。pqqE和GDH基因是CLW17菌株的溶磷相关基因,2个基因的缺失均使该菌株的溶磷能力显著下降,而回补株可以恢复溶磷能力。CLW17野生株能分泌多种有机酸,其中葡萄糖酸(gluconic acid,GA)含量最多,其次是乙酸;但敲除株产有机酸的能力明显降低...     

Notch信号途径中的Su(H)和E(spl)基因对果蝇天然免疫的影响

天然免疫系统是多细胞生物抵抗各种入侵微生物的第一道防线.Notch途径介导相邻细胞之间的相互作用,调节细胞、组织、器官的分化和发育.为了进一步探索Notch信号途径在果蝇天然免疫中的功能,利用Notch途径下游基因Su(H)和E(spl)的低表达突变体果蝇,通过体外注射病原体分析了生存率、血细胞的噬菌功能和抗菌肽的表达量以及突变体的血细胞数量.结果表明,革兰氏阴性细菌和真菌感染后果蝇E(spl)突变体的生存率、噬菌能力及抗菌肽的表达量明显降低,而且幼虫期血细胞出现异常增殖;Su(H)突变体只对真菌表现出敏感性,抗菌肽的表达量降低,但是对真菌的噬菌能力正常.此结果表明,Notch途径不仅影响个体的生长发育,而且在果蝇天然免疫中也起重要的调节作用.     

Iridoids and phenylethanoids in Lagotis integrifolia and Wulfeniopsis amherstiana (Plantaginaceae)

In a chemosystematic investigation of Wulfeniopsis amherstiana we have isolated four common iridoid glucosides as well as the esters 3″- and 4″-Cinnamoyl 6-O-rhamnopyranosylcatalpol. Furthermore the plant contained mannitol, arbutin, and four caffeoyl phenylethanoid glucosides, namely plantamajoside, aragoside and two new acetyl derivatives of the latter, named amherstianoside A and B. From Lagotis integrifolia we have obtained mannitol and three common iridoid glucosides together with the 8,9-unsaturated iridoids arborescosidic acid and anagalloside. The results show that the genus Wulfeniopsis is chemically different from Wulfenia and that it is closer related to Veronicastrum.     

Characterization of the Escherichia coli gene for 1-acyl-sn-glycerol-3-phosphate acyltransferase (pIsC)

Summary An Escherichia coli strain deficient in 1-acyl-sn-glycerol-3-phosphate acyltransferase activity has previously been isolated, and the gene (plsC) has been shown to map near min 65 on the chromosome. I precisely mapped the location of plsC on the chromosome, and determined its DNA sequence. plsC is located between parC and sufI, and is separated from sufI by 74 bp. Upstream of plsC is parC, separated by 233 bp, which includes an active promoter. parC, plsC, and sufI are all transcribed in the counterclockwise direction on the chromosome, possibly in an operon with multiple promoters. The amino-terminal sequence of the partially purified protein, combined with the DNA sequence, reveal 1-acyl-sn-glycerol-3-phosphate acyltransferase to be a 27.5 kDa highly basic protein. The plsC gene product, 1-acyl-sn-glycerol-3-phosphate acyltransferase, is localized to the cytoplasmic membrane of the cell. The amino-terminal sequence of the purified protein reveals the first amino acid to be a blocked methionine residue, most probably a formyl-methionine. The amino acid sequence of 1-acyl-sn-glycerol-3-phosphate acyltransferase has a short region of homology to two other E. coli acyltransferases that utilize acyl-acyl carrier protein as the acyl donor, sn-glycerol-3-phosphate acyltransferase and UDP-N-acetyl-glucosamine acyltransferase (involved in lipid A biosynthesis).