Biotransformation of enones with biocatalysts — two enone reductases from Astasia longa

The stereochemistry and mechanism in the reduction of the C–C double bond of carvone by the cultured cells of Astasia longa, a nonchlorophyllous cell line classified in Euglenales, was studied. The reduction of the C–C double bond of carvone with the cultured cells involved the anti-addition of hydrogen atom from the si face at the -position and the re face at the β-position of carbonyl group. Two different enone reductases were isolated from the cultured cells of A. longa. Both reductases catalyzed stereospecifically the anti-addition of hydrogen atoms from the si face at C-1 and the re face at C-6. However, one of the reductases participated in a hydrogen transfer of the pro-4R hydrogen of NADH to C-6 position of carvone and the other used the pro-4S hydrogen of NADH.     

The Aspergillus niger carbon catabolite repressor encoding gene, creA

In order to undertake a comparative analysis of carbon catabolite repression in two Aspergillus species, the creA gene has been isolated from A. niger by cross hybridization, using the cloned A. nidulans gene. The A. niger gene has been shown to be functional in A. nidulans by heterologous complementation of the creA204 mutation of A. nidulans. Overall, the genes show 90% sequence similarity (82% identity) at the amino acid (aa) level. There were some striking similarities between the aa sequences encoded by the two fungal creA genes and two genes involved in carbon catabolite repression in Saccharomyces cerevisiae. The zinc-finger regions showed 96% similarity (84% identity) with the zinc-finger region of the MIG1 gene of S. cerevisiae. The CREA protein contains a stretch of 42 aa that is identical in A. niger and A. nidulans, and these show 81% similarity (33% identity) with a region of the S. cerevisiae RGR1 gene.     

Phylogenetic and biosystematic relationships in four highly disjunct polyploid complexes in the subgenera Ceterach and Phyllitis in Asplenium (Aspleniaceae)

Phylogenetic studies using DNA sequences of two chloroplast regions, rbcL and trnL-F, demonstrate that the proposed genus Ceterach is a small clade within the large genus Asplenium, and sister to the Phyllitis clade. The Ceterach clade is characterised by irregular anastomosing veins and often densely scaled leaf blades. Its taxonomic status as a group nested within Asplenium is confirmed, and it is accepted here as a subgenus with seven species. The Ceterach clade comprises four lineages that correspond to disjunct polyploid complexes: the A. aureum clade forming a polyploid complex (4×, 6×, 8×) in Macaronesia, the A. ceterach clade forming a polyploid complex (2×, 4×, 6×) in the Mediterranean Basin, the A. paucivenosum clade (4×, 6×) in central Asia, and the A. dalhousiae clade (2×) with a disjunct distribution in the Himalaya, Yemen and Eritrea, and southwestern North America. Asplenium paucivenosum is sister to all other members of the Ceterach clade, whereas A. dalhousiae is sister to the A. aureum clade that includes tetraploid A. aureum, hexaploid A. lolegnamense, and octoploid A. parvifolium. Asplenium ceterach and its variations – including the hexaploid A. ceterach subsp. mediterraneum subsp. nov. first described below – form a monophyletic unit, sister to a clade consisting of A. aureum and A. dalhousiae. Asplenium cordatum from Africa and A. haugthonii from the isolated atlantic island of St. Helena are not members of the Ceterach clade, which suggests that leaf blades with dense indumenta have evolved at least twice within asplenioid ferns. The allotetraploid species A. hybridum has the chloroplast DNA from A. ceterach, and therefore the latter species is the maternal ancestor of the former. The other parent of this hybrid species is A. sagittatum that is nested within the sister clade of Ceterach, the Phyllitis clade comprising A. sagittatum and A. scolopendrium. The findings suggest that the current distribution of Ceterach is either the result of long-distance dispersal or represents fragmented relicts of a previously more widely distributed species.     

Antechiniscus in Australia: Description of Antechiniscus moscali sp. n. and Redescription of Antechiniscus parvisentus (Horning & Schuster, 1983) (Heterotardigrada: Echiniscidae)

Two species of the genus Antechiniscus have been found in Nothofagus forests in Australia. The new species A. moscali sp. n. is described. The New Zealand species A. parvisentus (Horning & Schuster, 1983) is redescribed from type material and from the new material from Australia. The taxonomic status of the two species within the genus is discussed.     

The cytochromes of Chlorobium thiosulfatophilum

Three c-type cytochromes (c-551, c-553, c-555) have been isolated and characterized from a strain of the green photosynthetic bacterium Chlorobium thiosulfatophilum. These cytochromes are atypical when compared to horse heart cytochrome c in many properties, among them: oxidation-reduction potential at pH 7.0 (c-551, 135 mV; c-553, 98 mV; c-555, 145 mV), molecular weight (c-551, 45000–60000; c-553, 50000; c-555, 10000) and isolelectric point (c-551, 6.0; c-553, 6.7). No protoheme was detected in whole cells or cell-free extracts.     

Chemical structure and conformational features of cell-wall polysaccharides isolated from Aphanoascus mephitalus and related species

The structures of cell-wall mannans isolated from Aphamoascus mephitalus, A. Fulvescens, A. verrucosus, and A. reticulisporus have been investigated by chemical analyses and 1D and 2D ¹H and ¹³C NMR techniques. It was found that all of them consists of a relatively simple comb-like structure of the disaccharide repeating block {→ 6)-[-Man p-(1 → 2)]--Man p-(1 →}. The conformations around the -(1 → 2) and -(1 → 6) linkages in thes kinds of polymers were also studied by using molecular mechanics and dynamics calculations, together with NOE data. The results are similar to those found within the oligosaccharide chains of glycoproteins, with a well-defined conformation for the -(1 → 2) linkage and a certain restriction around the -(1 → 6) bonding imposed by the 2-substitution.     

The role of a cytochrome c-552-cytochrome c complex in the oxidation of sulfide in Chromatium vinosum

The sulfide:cytochrome c oxidoreductase activity of the flavocytochrome c-522 from the purple sulfur bacterium Chromatium vinosum has been investigated. The oxidized sulfur product of the sulfide:cytochrome c reductase activity has been shown to be elemental sulfur. Cytochrome c-552 has been found to form a stable complex with horse heart cytochrome c that appears to be held together by electrostatic interactions. The stability of this complex and the sulfide:cytochrome c reductase activity of cytochrome c-552 are both ionic strength dependent, with maximal rates of cytochrome c reduction and extent of complex formation occurring over the same ionic strength range. Trifluoroacetylated cytochrome c is not reduced in the presence of cytochrome c-552 and sulfide, nor does it form a complex with cytochrome c-552. These results suggest the possible involvement of cytochrome c lysine residues in complex formation. Cytochrome c-552 migrates with an anomalously high apparent molecular weight on gel filtration columns equilibrated with low ionic strength buffers, suggesting the possibility of conformational changes or dimerization of the protein. However, complexation of cytochrome c-552 with cytochrome c still occurs at low ionic strength.     

Construction and characterisation of a BAC library from Arabidopsis halleri: Evaluation of physical mapping based on conserved synteny with Arabidopsis thaliana

We constructed and characterized the first large-insert DNA BAC library for Arabidopsis halleri, a close relative of Arabidopsis thaliana. Double size selection of high molecular weight DNA was performed to increase the average insert size. The BAC library consists of 6128 clones of which 87% have an insert size above 125 kb. Organellar DNA contamination is estimated to 1.4%. The coverage of the library is equivalent to 4.5 times the haploid genome (250 Mb), indicating the library is suitable for almost any application. We explored the possibility of generating a physical map of A. halleri using the high conserved synteny existing between this species and A. thaliana. A set of unigenes separated by 50 kb in a 850 kb region of A. thaliana chromosome II was used to probe the library. The A. halleri BAC clones isolated with these probes were grouped into two contigs. Analysis of BAC-end sequences revealed that the two A. halleri genomic contigs were highly colinear with the A. thaliana genome. Therefore, the exploitation of the conserved synteny existing between the two species will greatly facilitate the construction of a raw full physical map of A. halleri.     

Geographic differentiation of monoterpenes from Abies procera and Abies magnifica

Cortical oleoresins from 352 Abies magnifica and A. procera trees, collected in 35 localities, were analysed for composition of their monoterpene fractions. The monoterpenes were composed primarily of -pinene, β-pinene, limonene and β-phellandrene, with camphene and 3-carene appearing only sporadically in larger amounts. Limonene, β-phellandrene, -pinene and 3-cerene were used to study the transitional populations in northern California and southern and central Oregon and the transitional-like populations in southern Sierra Nevada. The populations segregated latitudinally into three related clusters—above 44° (A.procera), between 44° and 40° (transitional), and below 40° (A.magnifica). A.magnifica from southern Sierra Nevada and A.magnifica from Mtn. Shasta differed in a number of parameters, with southern Sierra Nevada populations being chemically much closer to typical A. magnifica from central and northern Sierra Nevada. While monoterpene and morphological data did not allow a clear Interpretation of the status of the transitional and southern populations, the paleobotanical evidence favored the recent evolution over introgression. The desirability of additional studies was indicated.     

东方蜜蜂入侵群体的建立机制

蜜蜂、胡蜂及蚂蚁等是具有高度入侵性的社会性昆虫.理论上,在奠基者群体数量较少时,其单一位点互补性性别决定机制会限制入侵群体的发展.在人类活动影响下,东方蜜蜂已扩散至其自然分布区之外的很多地区,给当地饲养的西方蜜蜂带来很大影响.本文简要介绍了东方蜜蜂的分布、性别决定机制以及近几年来针对澳大利亚东方蜜蜂入侵群体的系列研究结果,从蜂王的交尾行为、工蜂的繁殖行为、平衡选择作用、产雌孤雌生殖等方面分析了东方蜜蜂群体如何在奠基者效应带来的遗传负荷压力下快速发展起来,为揭示相关社会性昆虫入侵群体的建立机制提供了很好的借鉴.     

Brown tide alga, Aureococcus anophagefferens, can affect growth but not survivorship of Mercenaria mercenaria larvae

Since the collapse of populations of northern quahogs (hard clam), Mercenaria mercenaria, in Long Island bays, brown tide blooms have been proposed to pose a barrier to recovery. We tested whether the brown tide alga, Aureococcus anophagefferens, affects survivorship, development or growth in the larvae of M. mercenaria. There was no effect of A. anophagefferens (clone CCMP1708) on survivorship of hard clam larvae, even at bloom concentrations. Under most experimental conditions, larvae fed a mixed diet of Isochrysis galbana (T-Iso) and A. anophagefferens or a single species diet of A. anophagefferens, developed faster than those fed a single species diet of Isochrysis. A mixed diet of I. galbana and A. anophagefferens either had no effect on larval growth, or produced enhanced growth at moderate cell densities (8 × 10⁴ cells ml⁻¹ of A. anophagefferens). Similarly, moderate cell densities of a single food diet of A. anophagefferens (1.6 × 10⁵ cells ml⁻¹) generally had no effect on the growth of larvae. When fed bloom concentrations (10⁶ cells ml⁻¹) of A. anophagefferens, larvae developed faster, but growth was reduced, compared to those fed an equal biovolume of Isochrysis. Larvae fed slow growing or near stationary phase cultures of A. anophagefferens experienced reduced growth and slowed development. These data suggest a qualitative difference between slow or stationary phase and fast growing cultures of the brown tide alga. They also suggest that impacts of A. anophagefferens, when present, are likely to be due to the nutritional quality of this alga as a food source for hard clam larvae, which could have a lasting legacy through ontogeny. Additional studies are needed to test whether our findings apply to more recently isolated strains of A. anophagefferens.     

The cardenolide content of Asclepias linaria

The cardenolides of the aerial parts of Asclepias linaria were isolated and identified as the known calactin, calotoxin, proceroside, gomphoside, desglucouzarin and the new cardenolide 6′-β-coumaroyl desglucouzarin.     

Experimental hybridization of Angiostrongylus mackerrasae, Bhaibulaya, 1968 and Angiostrongylus cantonensis (Chen, 1935)

Angiostrongylus mackerrasae and A. cantonensis are closely related and their status as distinct species was tested by experimental hybridization. Hybridization between A. mackerrasae and A. cantonensis was observed and the morphology of the F₁ hybrids was found to be intermediate between that of the parental species which suggests strongly that A. mackerrasae and A. cantonensis are both valid species. However, male F₁ hybrids were sterile, whereas females were fertile; no evidence of parthenogenesis or other form of asexual reproduction were observed in either species. It is suggested that A. mackerrasae and A. cantonensis may cross breed in nature.     

Genes encoding multiple drug resistance-like proteins in Aspergillus fumigatus and Aspergillus flavus

Polymerase chain reaction using degenerate primers was used to identify genes encoding proteins of the ATP-binding cassette superfamily in Aspergillus fumigatus and Aspergillus flavus. In A. fumigatus, two genes (AfuMDR1 and AfuMDR2) encoding proteins of the ATP-binding cassette superfamily were identified. One gene (AflMDR1) was isolated from A. flavus and is the apparent homologue to AfuMDR1. AfuMDR1and AflMDR1 encode proteins of molecular weights 148 000 and 143 000, respectively, each containing 12 putative transmembrane regions and two ATP-binding sites. These proteins are arranged in two homologous halves, each half consisting of a hydrophobic region (encoding six putative transmembrane domains) and an ATP-binding site. The AfuMDR1 and AflMDR1-encoded proteins bear a high degree of similarity to the Schizosaccharomyces pombe leptomycin B resistance protein and to human MDR1. The second gene identified in A. fumigatus, AfuMDR2, encodes a protein of molecular weight 85 000, containing four putative transmembrane domains and an ATP binding domain. The encoded protein is similar to those encoded by MDL1 and MDL2, two MDR-like genes of Saccharomyces cerevisiae. Expression of AFUMDR1 in S. cerevisiae conferred increased resistance to the antifungal agent cilofungin (LY121019), an echinocandin B analog.     

杨树受机械损伤与光肩星天牛危害的防御性反应

杨树是我国“三北”地区防护林建设的主栽树种,自20世纪70年代以来长期受到光肩星天牛的严重危害。北抗杨对光肩星天牛有一定的抗性,但产生抗性的生化机制尚不清楚。本研究采用试剂盒法与高效液相色谱法以未受害、机械损伤、虫害北抗杨为研究材料,对其树皮和木质部中的次生代谢产物和防御酶含量进行检测,以探索其抗性机制。结果表明,北抗杨受到机械损伤和光肩星天牛危害后其反应不同:1)次生代谢产物,北抗杨受到机械损伤后,树皮中的水杨苷和白杨甙含量显著上升,槲皮苷含量降低;而受光肩星天牛危害后,树皮中的水杨苷和槲皮苷含量显著上升,白杨甙含量无显著变化。机械损伤的北抗杨木质部总酚含量高于虫害与未受害木质部,后两者间无显著差异;光肩星天牛危害的北抗杨木质部白杨甙和亚麻木酚素含量高于机械损伤木质部与未受害木质部。遭受机械损伤与虫害后北抗杨木质部的总酚苷含量显著高于未受害木质部。2)防御酶活性分析表明,与未受害北抗杨树皮相比,受到机械损伤与虫害后的树皮苯丙氨酸解氨酶(PAL)活性显著升高,但两者间无差异;受到机械损伤与虫害后的树皮超氧化物歧化酶(SOD)活性高于未受害树皮,且机械损伤树皮高于虫害树皮;北抗杨受机械损伤与虫害后木质部中的过氧化物酶(POD)活性高于未受害木质部,但两者间无差异。3)与未受害北抗杨相比,北抗杨受机械损伤、虫害后部分次生代谢物和防御酶都有不同程度的增加,推测这些物质可能与北抗杨抗逆性反应有关。     

A two-step bioconversion process for vanillin production from ferulic acid combining Aspergillus niger and Pycnoporus cinnabarinus

A two-step bioconversion process of ferulic acid to vanillin was elaborated combining two filamentous fungi, Aspergillus niger and Pycnoporus cinnabarinus. In the first step, A. niger transformed ferulic acid to vanillic acid and in the second step vanillic acid was reduced to vanillin by P. cinnabarinus. Ferulic acid metabolism by A. niger occurred essentially via the propenoic chain degradation to lead to vanillic acid, which was subsequently decarboxylated to methoxyhydroquinone. In 3-day-old cultures of P. cinnabarinus supplied with vanillic-acid-enriched culture medium from A. niger as precursor source, vanillin was successfully produced. In order to improve the yields of the process, sequential additions of precursors were performed. Vanillic acid production by A. niger from ferulic acid reached 920 mg l⁻¹ with a molar yield of 88% and vanillin production by P. cinnabarinus from vanillic acid attained 237 mg l ⁻¹ with a molar yield of 22%. However, the vanillic acid oxidative system producing methoxyhydroquinone was predominant in P. cinnabarinus cultures, which explained the relatively low level in vanillin.     

Chemical composition and in vitro fermentation of tannin-rich tree fruits

Dry and mature tree fruits are a potential source of protein for goats in the semi-arid areas of southern Africa, but their chemical composition and feeding value is largely unknown. This study presents the chemical composition and in vitro fermentation of indehiscent whole fruits and separated seed and hull fractions from Acacia nilotica, Acacia erubescens, Acacia sieberiana, Acacia erioloba, Piliostigma thonningii and Dichrostachys cinerea trees. Results indicate that the N contents of whole fruits ranged between 13.5 g/kg DM (A. nilotica) and 27.1 g/kg DM (A. erubescens). Seeds had a higher N content than hulls for all tree species. A. nilotica, D. cinerea and P. thonningii fruits had high levels of extractable phenolics (758, 458 and 299 g/kg DM, respectively). Soluble phenolics (SPh) and ytterbium precipitable phenolics (YbPh) levels were negatively correlated to in vitro gas production but positively correlated to in vitro organic matter degradability (iOMD). Partition factors for whole fruits at 48 h ranged between 3.6 mg/ml for A. erioloba and 7.8 mg/ml for A. nilotica. Seeds of A. erioloba, A. erubescens and P. thonningii were consistently fermented more efficiently throughout the incubation period compared to their whole fruits or hulls. Estimating in vitro degradability of phenolic-rich substrates through filtration procedures can give erroneous results due to the loss of soluble phenolics, which are not necessarily degradable. The feeding value of fruits from D. cinerea and A. nilotica tree species may be reduced due to the presence of high levels of phenolics.     

烟草赤星病菌拮抗性大豆根瘤内生菌的筛选及抑制作用

以烟草赤星病菌为供试病原菌,采用对峙法、代谢液培养法对分离自大豆根瘤的内生细菌进行抑菌筛选,对筛选菌株作用的病原菌菌丝进行显微观察,研究筛选菌株细胞培养特征及生理生化特性、16S rDNA测序序列、系统发育,以及进行温室接种防病试验.结果表明: 大豆根瘤内生菌经初筛、复筛,对烟草赤星病菌抑菌率42%以上的有7株菌,分别属于芽孢杆菌属、假单胞菌属、中华根瘤菌属和寡养单胞菌属.显微观察表明,在拮抗性内生菌作用下病原菌菌丝末端出现明显畸形,呈珊瑚状分枝、球状膨大等;代谢液试验表明,内生菌对病原菌的抑制作用主要是内生菌代谢产生的胞外物质在发挥作用;对峙试验表明,芽孢杆菌可迅速形成生物薄膜,有效阻碍病原菌丝生长和延伸.盆栽防病试验表明,拮抗性内生菌处理下烟草病情指数显著低于对照,表明筛选菌株可作为烟草赤星病的生防菌株资源.     

Reducibility of cytochromes b in aerobic beef-heart mitochondria treated with antimycin

Under anaerobic conditions cytochrome b in beef-heart mitochondria is partially reduced in the presence of NADH, whereas other cytochromes are completely reduced. Addition of antimycin together with oxygen under these conditions causes an immediate reduction of cytochromes b-558, b-562 and b-566 and oxidation of cytochrome c. During the subsequent transient aerobic steady state cytochromes b-558 and b-566 are rapidly re-oxidized without changes in redox state of cytochrome c, but cytochrome b-562 remains reduced. When oxygen is consumed by the leak through or around the antimycin-inhibition site, cytochrome b-562 becomes oxidized with concomitant reduction of cytochrome c.

The cytochromes b in lyophilized beef-heart mitochondria are more readily accessible to electrons from NADH, and in the presence of antimycin and NADH a complete and stable reduction is obtained under both aerobic and anaerobic conditions. Gradual addition of rotenone under these conditions causes re-oxidation of cytochromes b in which oxidation of cytochromes b-558 and b-566 precedes that of cytochrome b-562.

It is concluded that (1) the effect of antimycin in the presence of oxygen involves all three cytochromes b, (2) the reducibility of the cytochromes b in the aerobic steady state of antimycin-treated mitochondria is dependent upon the potential of the substrate redox couple registered on the cytochromes, and (3) the midpoint potential of cytochrome b-562 in the presence of antimycin is higher than that of cytochrome b-558 or b-566.     

Genetic diversity of an amphicarpic species, Amphicarpaea edgeworthii Benth. (Leguminosae) based on RAPD markers

Amphicarpaea edgeworthii Benth. is an amphicarpic legume widespread in China. Amphicarpy describes the phenomenon that a plant produces aerial as well as subterranean fruits. A. edgeworthii can reproduce via three kinds of flowers: aerial chasmogamous flowers, aerial cleistogamous flowers, and subterranean cleistogamous flowers. Although there are some studies on the population genetic structure of species with both chasmogamous and cleistogamous flowers, none has been done for that of an amphicarpic species so far. The present study uses random amplified polymorphic DNA (RAPD) to assess level and pattern of genetic variation in 15 natural populations of A. edgeworthii. A total of 131 stable and clearly scored RAPD bands were achieved from 13 primers. The average genetic diversity within populations estimated by Shannon's information index was 0.218 at the population level, but ranged from 0.119 to 0.302, which was significantly different (P < 0.01). Different statistical analyses revealed a high level of genetic differentiation among populations (G_ST = 0.473–0.527). Thus, the pattern of genetic structure of A. edgeworthii is consistent with that of an inbreeding species.