Structural features of the glycogen branching enzyme encoding genes from aspergilli

A maltose binding protein, p78, was purified to homogeneity from Aspergillus nidulans by a single column chromatography step on cross-linked amylose. The partial amino acid sequence was highly homologous to the glycogen branching enzymes (GBEs) of human and yeast, and p78 did show branching enzyme activity. The genomic gene and its cDNA encoding GBE (p78) were isolated from the A. nidulans genomic and cDNA libraries. Furthermore, a cDNA encoding A. oryzae GBE was entirely sequenced. A. nidulans GBE shared overall and significant amino acid sequence identity with GBEs from A. oryzae (83.9%), Saccharomyces cerevisiae (61.1%) and human (63.0%), and with starch branching enzymes from green plants (55–56%).     

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.     

Characterization of a mutant of Anacystis nidulans r2 resistant to the natural herbicide, cyanobacterin

Cyanobacterin, a secondary metabolite produced by the cysnobacterium, Scytonema hofmanni, inhibits electron transport at a site in photosystem II. It was previously shown that a DCMU-resistant mutant of A. nidulans R2 was still susceptible to cyanobacterin (Gleason et al., Plant Science, 46 (1986) 5–10). Apparently, cyanobacterin acts at a site different from that of DCMU and similar PS II inhibitors. To confirm this conclusion, a cyanobacterin-resistant strain of A. nidulans R2 was produced by nitrosoguanidine mutagenesis and selected by growth in the presence of 4.7 μM cyanobacterin. Hill activity in mutant thylakoids was compared to that of the wild type membranes in the presence of ferricyanide and silicomolybdate as electron acceptors. Photosynthetic electron transport in the mutant membranes shows a high degree of resistance to cyanobacterin in both reactions. In contrast, the mutant exhibits the same susceptibility to DCMU inhibition as the wild type R2. Cyanobacterin acts at a unique site, inhibiting electron flow from quinone-A to quinone-B.     

Binding of the C6-zinc cluster protein, AFLR, to the promoters of aflatoxin pathway biosynthesis genes in Aspergillus parasiticus

AFLR is a Zn₂Cys₆-type sequence-specific DNA-binding protein that is thought to be necessary for expression of most of the genes in the aflatoxin pathway gene cluster in Aspergillus parasiticus and A. flavus, and the sterigmatocystin gene cluster in A. nidulans. However, it was not known whether AFLR bound to the promoter regions of each of the genes in the cluster. Recently, A. nidulans AFLR was shown to bind to the motif 5′-TCGN₅CGA-3′. In the present study, we examined the binding of AFLR to promoter regions of 11 genes in the A. parasiticus cluster. Based on electrophoretic mobility shift assays, the genes nor1, pksA, adhA, norA, ver1, omtA, ordA, and, vbs, had at least one 5′-TCGN₅CGA-3′ binding site within 200 bp of the translation start site, and pksA and ver1 had an additional binding site further upstream. Although the promoter region of avnA lacked this motif, AFLR bound weakly to the sequence 5′-TCGCAGCCCGG-3′ at −110 bp. One region in the promoter of the divergently transcribed genes aflR/aflJ bound weakly to AFLR even though it contained a site with at most only 7 bp of the 5′-TCGN₅CGA-3′ motif. This partial site may be recognized by a monomeric form of AFLR. Based on a comparison of 16 possible sites, the preferred binding sequence was 5′-TCGSWNNSCGR-3′.     

Screening of chemical composition, antimicrobial and antioxidant activities of Artemisia essential oils

The chemical composition of essential oils isolated from aerial parts of seven wild sages from Western Canada – Artemisia absinthium L., Artemisia biennis Willd., Artemisia cana Pursh, Artemisia dracunculus L., Artemisia frigida Willd., Artemisia longifolia Nutt. and Artemisia ludoviciana Nutt., was investigated by GC–MS. A total of 110 components were identified accounting for 71.0–98.8% of the oil composition. High contents of 1,8-cineole (21.5–27.6%) and camphor (15.9–37.3%) were found in Artemisia cana, A. frigida, A. longifolia and A. ludoviciana oils. The oil of A. ludoviciana was also characterized by a high content of oxygenated sesquiterpenes with a 5-ethenyltetrahydro-5-methyl-2-furanyl moiety, of which davanone (11.5%) was the main component identified. A. absinthium oil was characterized by high amounts of myrcene (10.8%), trans-thujone (10.1%) and trans-sabinyl acetate (26.4%). A. biennis yielded an oil rich in (Z)-beta-ocimene (34.7%), (E)-beta-farnesene (40.0%) and the acetylenes (11.0%) (Z)- and (E)-en-yn-dicycloethers. A. dracunculus oil contained predominantly phenylpropanoids such as methyl chavicol (16.2%) and methyl eugenol (35.8%). Artemisia oils had inhibitory effects on the growth of bacteria (Escherichia coli, Staphylococcus aureus, and Staphylococcus epidermidis), yeasts (Candida albicans, Cryptococcus neoformans), dermatophytes (Trichophyton rubrum, Microsporum canis, and Microsporum gypseum), Fonsecaea pedrosoi and Aspergillus niger. A. biennis oil was the most active against dermatophytes, Cryptococcus neoformans, Fonsecaea pedrosoi and Aspergillus niger, and A. absinthium oil the most active against Staphylococcus strains. In addition, antioxidant (beta-carotene/linoleate model) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activities were determined, and weak activities were found for these oils.     

Asymmetric synthesis of arylselenoalcohols by means of the reduction of organoseleno acetophenones by whole fungal cells

A series of novel organoseleno acetophenones (3a–f) have been synthesized. The microbial reduction of the seleno ketones (3) has been evaluated using whole cells of Rhizopus oryzae CCT 4964, Aspergillus terreus CCT 3320, A. terreus CCT 4083 and Emericella nidulans CCT 3119. These microorganisms showed Prelog and anti-Prelog stereoselectivity, leading to the arylselenoalcohols in moderate to high enantiomeric excesses. The organoselenium compounds were compatible with the biocatalytic conditions employed.     

Structure and properties of regenerated Antheraea pernyi silk fibroin in aqueous solution

Antheraea pernyi silk fibroin fibers were dissolved by aqueous lithium thiocyanate to obtain regenerated A. pernyi silk fibroin solution. By means of circular dichroism, ¹³C NMR and Raman spectroscopy, the molecular conformation of regenerated A. pernyi silk fibroin in aqueous solution was investigated. The relationship of environmental factors and sol–gel transformation behavior of regenerated A. pernyi silk fibroin was also studied. The molecular conformations of regenerated A. pernyi silk fibroin mainly were -helix and random coil in solution. There also existed a little β-sheet conformation. It was obviously different with Bombyx mori silk fibroin, whose molecular conformation in solution was only random coil but no -helix existence. With the increase of temperature and solution concentration and with the decrease of solution pH value, the gelation velocity of regenerated A. pernyi silk fibroin solution increased. Especially, it showed that A. pernyi silk fibroin was more sensitive to temperature than B. mori silk fibroin during the sol–gel transformation. The velocity increased obviously when the temperature was above 30 °C. During the sol–gel transformation, the molecular conformation of regenerated A. pernyi silk fibroin changed from random coil to β-sheet structure. The results of these studies provided important insight into the preparation of new biomaterials by silk fibroin protein.     

Growth-mediated metabolic activation of promutagens in Aspergillus nidulans

7 procarcinogens belonging to different chemical classes (nitrosamines, hydrazoalkanes, oxazaphosphorines and aromatic amines) were tested in A. nidulans for the induction of point mutations with two genetic systems (8-AG resistance and induction of methionine suppressors).

Dimethylnitrosamine, diethylnitrosamine, nitrosomorpoline, dimethyl-hydrazine, procarbazine and cyclophosphamide gave positive results with a good dose—effect relationship in the growth-mediated assay, whereas they gave negative or borderline positive results in the plate incorporation assay. 2-Aminoanthracene was completely negative with both experimental procedures.

DMN, DEN and NM were also tested for their ability to induce somatic segregation: all were positive when assayed in the growth-mediated assay.     

Dithioerythritol (DTE) prevents inhibitory effects of triphenyltin (TPT) on the key enzymes of the human sex steroid hormone metabolism

Organotins are known to induce imposex (pseudohermaphroditism) in marine neogastropods and are suggested to act as specific endocrine disruptors, inhibiting the enzyme-mediated conversion of steroid hormones. Therefore, we investigated the in vitro effects of triphenyltin (TPT) on human 5-reductase type 2 (5-Re 2), cytochrome P450 aromatase (P450arom), 17β-hydroxysteroid dehydrogenase type 3 (17β-HSD 3), 3β-HSD type 2 and 17β-HSD type 1 activity. First, the present study demonstrates that significant amounts of TPT occurred in the blood of eight human volunteers (0.17–0.67 μg organotin cation/l, i.e. 0.49–1.92 nmol cation/l). Second, TPT showed variable inhibitory effects on all the enzymes investigated. The mean IC₅₀ values were 0.95 μM for 5-Re 2 (mean of n=4 experiments), 1.5 μM for P450arom (n=5), 4.0 μM for 3β-HSD 2 (n=1), 4.2 μM for 17β-HSD 3 (n=3) and 10.5 μM for 17β-HSD 1 (n=3). To exclude the possibility that the impacts of TPT are mediated by oxidizing essential thiol residues of the enzymes, the putative compensatory effects of the reducing agent dithioerythritol (DTE) were investigated. Co-incubation with DTE (n=3) resulted in dose-response prevention of the inhibitory effects of 100 μM deleterious TPT concentrations on 17β-HSD 3 (EC₅₀ value of 12.9 mM; mean of n=3 experiments), 3β-HSD 2 (0.90 mM; n=3), P450arom (0.91 mM; n=3) and 17β-HSD 1 (0.21 mM; n=3) activity. With these enzymes, the use of 10 mM DTE resulted in an at least 80% antagonistic effect, whereas, the effect of TPT on 5-Re 2 was not compensated. In conclusion, the present study shows that TPT acts as an unspecific, but significant inhibitor of human sex steroid hormone metabolism and suggests that the inhibitory effects are mediated by the interaction of TPT with critical cysteine residues of the enzymes.     

Chromosome polymorphism in Astyanax fasciatus (Teleostei, Characidae), Part 3: Analysis of the RAPD and ISSR molecular markers

The Randomly Amplified Polymorphic DNA (RAPD) and Inter-Simple Sequence Repeats (ISSR) molecular markers were used to complement the study of chromosomal polymorphism in Astyanax fasciatus (Teleostei, Characidae) from the Mogi-Guaçu River (Southeastern Brazil), analyzed in three collection sites along the river (Ouro Fino – MG, Cachoeira de Emas – SP and Barrinha – SP). Two cytotypes (or karyotypic types), denominated standard cytotypes, were previously characterized, one including 2n = 46 chromosomes and the other 2n = 48 chromosomes, where all the chromosomes of the complement form homologous pairs. Additionally, variant karyotypic forms with 2n = 45, 46 and 47 chromosomes were also detected, although with a lower frequency in relation to the standard cytotypes. RAPD turned out little informative in the analysis of the observed situation, indicating a high value of migrants per generation among the cytotypes. On the other hand, ISSR showed a small structure, especially among the standard cytotypes from the Barrinha region where the Nm was 0.4301 with a genetic identity of 0.6862 and genetic distance of 0.3765. However, the general results obtained do not discard the possibility of interbreeding between both standard cytotypes and/or their descendants as a source of chromosome variation. The association between the cytogenetic and molecular markers viabilized putative explanatory scenery for the origin and evolution of the forms seen in A. fasciatus.     

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.     

云南省可培养丝孢真菌资源的调查研究

本文报道曲霉属及其相关的有性型属、即散囊菌属和裸胞壳属的分类群共15个,其中新变种1个,我国新记录3个。它们是：日本曲霉小囊变种(新变种),赭曲霉,蜂蜜曲霉,孔曲霉,埋藏曲霉(新记录),佩特曲霉(新记录)、亮白曲霉,阿姆斯特丹散囊菌,谢瓦散囊菌,腊叶散囊菌,赤散囊菌,匍匐散囊菌原变种,构巢裸胞壳,无冠裸胞壳和刺孢裸胞壳(新记录)。     

Contribution of several nitrogen sources to growth of Alexandrium catenella during blooms in Thau lagoon, southern France

A monitoring program with a weekly sampling frequency over a 15-month period indicates that urea concentrations above a certain threshold level may trigger the blooms of Alexandrium catenella in Thau lagoon. However, urea concentrations were also sometimes related to ammonium and dissolved organic nitrogen concentrations, indicating that the role of urea may not be a direct one. An original approach is used to assess the relative contribution of several nitrogen sources (nitrate, nitrite, ammonium, urea) to growth of A. catenella by comparing nitrogen uptake rates to nitrogen-based growth rates estimated from dilution experiments during four blooms over a 4-year period (2001–2004) in Thau lagoon. Nitrate and nitrite contributed 0.1–14% and 0.1–5% respectively of growth requirements. Ammonium and urea were the main N sources fueling growth of A. catenella (30–100% and 2–59%, respectively). Indirect estimates indicated that an unidentified N source could also contribute significantly to growth at specific times. Concerning ammonium and urea uptake kinetics, half-saturation constants varied between 0.2 and 20 μgat N L⁻¹ for ammonium and between 0.1 and 44 μgat N L⁻¹ over the 4-year period, indicating that A. catenella can have a competitive advantage over other members of the phytoplankton even under low concentrations of ammonium and urea. However, the observed large changes in ammonium and urea uptake kinetics on a short time scale (days) during blooms preclude more precise estimates of those contributions to growth and require further investigation.     

pH control of the chlorophyll a fluorescence in algae

The pH of the suspension medium was found to have a remarkable influence on the “slow” (min) time course of Chlorophyll a fluorescence yield in the green alga Chlorella pyrenoidosa and in the blue-green alga Anacystis nidulans. In Chlorella, the decay of fluorescence yield, in the 1- to 5-min region, is strongly retarded at alkaline pH; this decay rate shows an optimum at pH 6–7. In Anacystis, the rise of fluorescence yield, in the same time range, is decreased optimally at pH 6–7; poisoning with 3(3,4-dichlorophenyl)-1,1-dimethylurea reverses the direction of this pH effect. These observations suggest a correlation of the H⁺ status (or the processes associated with it such as photophosphorylation and resulting conformational changes) of the chloroplast to the yield of chlorophyll a fluorescence in vivo.     

果肉和埋土深度对新疆野杏种子萌发与幼苗生长的影响

为探明新疆野杏（Armeniaca vulgaris）种子萌发与幼苗生长对果肉和埋土深度的响应,以期为新疆野杏的天然更新与实生苗培育提供理论参考。通过2种果皮结构（有果肉和无果肉）的种子在不同埋土深度（地表至18.0 cm的14个梯度）对新疆野杏种子萌发和幼苗生长进行研究,旨在揭示果皮结构和埋土深度对新疆野杏种子萌发与成苗能力的影响。结果表明：果肉和埋土深度显著影响野杏种子的萌发、幼苗生长与质量（P<0.05）。埋土深度<3.0 cm不利于成苗,埋土深度>6.0 cm时,萌发能力与幼苗生长量随埋土深度的增加而降低,3.0～6.0cm为适宜埋土深度。无果肉种子萌发优于有果肉种子,萌发率、萌发指数、成苗率、活力指数分别增长了37.18%、3.88%、37.18%、26.59%,幼苗高、基径、叶片数量、根冠比、幼苗质量指数分别增长了36.99%、7.48%、68.69%、20.61%、14.29%,其萌发能力与幼苗生长量显著高于有果肉种子（P<0.05）。有无果肉种子的萌发和幼苗生长指标与埋土深度呈显著负相关（P<0.05）。研究结果表明,无果肉处理对新疆野杏种...     

Malat-dehydrogenase isoenzymbanden als potentielles chemotaxonomisches kriterium für cyanophyceen-species

Malate dehydrogenase isoenzymes from eight cyanophycean species were investigated with polyacrylamide disc gel electrophoresis. Using 7% acrylamide the pherograms from each species showed 5–8 zones with malate dehydrogenase activity. It was demonstrated that in Anabaena flos-aquae there are 8 isoenzyme bands which include 3 forms of equal molecular weight, two of which consist of several isomers differing in their net charge. The MDH zymograms of the blue-green algae investigated can be used as “fingerprints”. The isoenzyme pattern of the MDHs of Anacystis nidulans makes its position in the order Chroococcales uncertain.

Résumé

Wäßrige Extrakte aus acht Cyanophyceenarten wurden einer Polyacrylamid-Discelektrophorese unterzogen und die erhaltenen Elektropherogramme auf Malat-Dehydrogenase (MDH)-Aktivität geprüft. Dabei ergab sich, daß unter unseren Versuchsbedingungen die MDH der getesteten Cyanophyceen in 5–8 Isoenzymbanden aufspaltet. Für Anabaena flos-aquae konnte gezeigt warden, daß sich die 8 Isoenzymbanden auf wenigstens 3 Molekulargewichts-Isomere zurückführen lassen, von denen zwei noch mehrere Ladungsisomere bilden. Die erhaltenen Zymogramme zeigen “fingerprint”-Charakter. was ihre mögliche Verwendbarkeit für die Chemotaxonomie der Cyanophyta nahelegt. Die Stellung von Anacystis nidulans innerhalb der CyanophyceenOrdnungen wird diskutiert.     

Evolution in Carex L. sect. Spirostachyae (Cyperaceae): A molecular and cytogenetic approach

Carex sect. Spirostachyae comprises 25 species displaying the centre of diversity in Eurasia. Phylogenetic analysis of the ITS nrDNA region of 20 species of sect. Spirostachyae, six species of sect. Ceratocystis, five species of sect. Elatae, and eight outgroup species reveals that neither sect. Spirostachyae nor sect. Elatae is monophyletic. With the exclusion of Carex cretica, the 19 species of sect. Spirostachyae studied form a clade with the five species of tropical-subtropical sect. Elatae. Taxonomy of the core Spirostachyae is not only mostly in agreement with our phylogenetic hypothesis, but also with ecological and new cytogenetic results. Two main groups with different chromosome numbers and edaphic preferences are identified in the core Spirostachyae. One includes primarily acidophilous species with high chromosome numbers (2n=(64)68–84), whereas the other one includes mainly basophilous species with lower chromosome numbers (2n=60–74(75)). Chromosome-number variation is extremely different in the core Spirostachyae, showing great stability in some widespread species (e.g. Carex extensa) but an active chromosome evolution – faster chromosomal rearrangements, fusion and fission events than ITS nucleotide substitutions – in more restricted species (e.g. Carex troodi). Biogeography of the two amphiatlantic pairs of species reveals two independent colonizations of South America from the European continent. The geographical barrier of the Strait of Gibraltar has played different roles in the course of evolution of this section, acting as an effective barrier to gene flow in one case (Carex helodes) but as a limited barrier or recent separation in two others (Carex distans, Carex punctata).