Degradation of N-acyl homoserine lactone quorum sensing signal molecules by forest root-associated fungi

A collection of mycorrhizal and nonmycorrhizal root-associated fungi coming from forest environments was screened for their ability to degrade N-acyl homoserine lactones (AHL) or to prevent AHL recognition by producing quorum sensing inhibitors (QSI). No production of QS-inhibitors or -activators was detected using the two biosensors Chromobacterium violaceum CV026 and Agrobacterium tumefaciens in the culture supernatant of these fungi. However, the ability to degrade C6- and 3O,C6-HSL was detected for three fungal isolates. Acidification assay revealed that the AHL were degraded by a lactonase activity for two of these isolates. These results demonstrated for the first time that the forest root-associated fungi are capable of degrading the AHL signal molecules.     

塔里木河下游不同退化区地表植被和土壤种子库特征

对塔里木河下游不同退化程度的4个典型断面进行了植被和土壤种子库的取样调查,采用种子萌发试验研究了不同退化区植被和土壤种子库的特征,结果表明:(1)塔里木河下游地表植被表现为严重的逆行演替,具体体现为胡杨林都为过熟林,几乎没有胸径在10cm以下的幼林;植被盖度、密度和多样性指数均维持在一个较低的水平上;随退化程度不断加重,地表植被中草本植物的相对密度、相对盖度和相对频度逐渐降低,而灌木和乔木的相对密度、相对盖度和相对频度逐渐增加;(2)研究区土壤种子库的基本特征是:土壤种子库种类贫乏、密度低、多样性指数和相似性系数不高;(3) 随退化程度的加重,土壤种子库物种数不断减少、密度明显下降、优势种组成趋于单一、表层种子库比例升高、1年生草本植物占优势逐渐向多年生草本植物和灌木植物转变及土壤种子库物种组成与地上植被物种组成上差异显著.     

BYC, an atypical aspartic endopeptidase from Rhipicephalus (Boophilus) microplus eggs

An aspartic endopeptidase was purified in our laboratory from Rhipicephalus (Boophilus) microplus eggs [Logullo, C., Vaz, I.S., Sorgine, M.H., Paiva-Silva, G.O., Faria, F.S., Zingali, R.B., De Lima, M.F., Abreu, L., Oliveira, E.F., Alves, E.W., Masuda, H., Gonzales, J.C., Masuda, A., and Oliveira, P.L., 1998. Isolation of an aspartic proteinase precursor from the egg of a hard tick, Rhipicephalus (Boophilus) microplus. Parasitology 116, 525–532]. Boophilus yolk cathepsin (BYC) was tested as component of a protective vaccine against the tick, inducing a significant immune response in cattle [da Silva, V.I., Jr., Logullo, C., Sorgine, M., Velloso, F.F., Rosa de Lima, M.F., Gonzales, J.C., Masuda, H., Oliveira, P.L., and Masuda, A., 1998. Immunization of bovines with an aspartic proteinase precursor isolated from Rhipicephalus (Boophilus) microplus eggs. Vet. Immunol. Immunopathol. 66, 331–341]. In this work, BYC was cloned and its primary sequence showed high similarity with other aspartic endopeptidases. In spite of this similarity, BYC sequence shows many important differences in relation to other aspartic peptidases, the most important being the lack of the second catalytic Asp residue, considered to be essential for the catalysis of this class of endopeptidases. When we determined BYC cleavage specificity by LC-MS, we found out that it presents a preference for hydrophobic residues in P1 and P1' in accordance to most aspartic endopeptidases. Also, when analyzed by circular dicroism, BYC presented high β sheet content, also a characteristic of aspartic endopeptidases. On the other hand, although both native and recombinant BYC are catalytically active, they present a very low specific activity, what seems to indicate that this peptidase will digest its natural substrate, vitellin, very slowly. We speculate that such a slow Vn degradative process might constitute an important strategy to preserve egg protein content to the hatching larvae.     

Plant ubiquitin-proteasome pathway and its role in gibberellin signaling

The ubiquitin-proteasome system (UPS) in plants, like in other eukaryotes, targets numerous intracellular regulators and thus modulates almost every aspect of growth and development. The well-known and best-characterized outcome of ubiquitination is mediating target protein degradation via the 26S proteasome, which represents the major selective protein degradation pathway conserved among eukaryotes. In this review, we will discuss the molecular composition, regulation and function of plant UPS, with a major focus on how DELLA protein degradation acts as a key in gibberellin signal transduction and its implication in the regulation of plant growth.     

Incorporation of hygromycin resistance in Brassica nigra and its transfer to B. napus through asymmetric protoplast fusion

Summary With the idea to develop a selection system for asymmetric somatic hybrids between oilseed rape (Brassica napus) and black mustard (B. nigra), the marker gene hygromycin resistance was introduced in this last species by protoplast transformation with the disarmed Agrobacterium tumefaciens strain C58 pGV 3850 HPT. The B. nigra lines used for transformation had been previously selected for resistance to two important rape pathogens (Phoma lingam, Plasmodiophora brassicae). Asymmetric somatic hybrids were obtained through fusion of X-ray irradiated (mitotically inactivated) B. nigra protoplasts from transformed lines as donor with intact protoplasts of B. napus, using the hygromycin resistance as selection marker for fusion products. The somatic hybrids hitherto obtained expressed both hygromycin phosphotransferase and nopaline synthase genes. Previous experience with other plant species had demonstrated that besides the T-DNA, other genes of the donor genome can be co-transferred. In this way, the produced hybrids constitute a valuable material for studying the possibility to transfer agronomically relevant characters — in our case, diseases resistances — through asymmetric protoplast fusion.     

Binding,internalization, and degradation of antiproliferative heparan sulfate by human embryonic lung fibroblasts

Binding, internalization, and degradation of ¹²⁵I-labeled, antiproliferative, or nonantiproliferative heparan sulfate by human embryonic lung fibroblasts was investigated. Both L-iduronate-rich, antiproliferative heparan sulfate species as well as L-iduronate-poor, inactive ones were bound to trypsin-releasable, cell-surface sites. Both heparan sulfate types were bound with approximately the same affinity to one high-affinity site (K_d approximately 10⁻⁸ M) and to one (K_d approximately 10⁻⁶ M), respectively. Results of Hill-plot analysis suggested that the two sites are independent. Competition experiments with unlabeled glycosaminoglycans indicated that the binding sites had a selective specificity for sulfated, L-iduronate-rich heparan sulfate. Dermatan sulfate, which is also antiproliferative, was weakly bound to the cells. The antiproliferative effects of heparan and dermatan sulfate appeared to be additive. Hence, the two glycosaminoglycans probably exert their effect through different mechanisms. At concentrations above 5 μg/ml (approximately 10⁻⁷ M), heparan sulfate was taken up by human embryonic lung fibroblasts, suggesting that the low-affinity site represents an endocytosis receptor. The antiproliferative effect of L-iduronate-rich heparan sulfate species was also exerted at the same concentrations. The antiproliferative species was taken up to a greater degree than the inactive one, suggesting a requirement for internalization. However, competition experiments with dextran sulfate suggested that both the high-affinity and the low-affinity sites are involved in mediating the antiproliferative effect. Structural analysis of the inactive and active heparan sulphate preparations indicated that although sulphated L-iduronate appears essential for antiproliferative activity, it is not absolutely required for binding to the cells. Degradation of internalized heparan sulfate was analyzed by polyacrylamide gel electrophoresis using a sensitive detection technique. The inactive species was partially degraded, whereas the antiproliferative one was only marginally affected. J. Cell. Biochem. 64:595–604. © 1997 Wiley-Liss, Inc.     

A fusion gene coding for two different δ-endotoxinsof Bacillus thuringiensis toxic to Plutella xylostella and usefulfor resistance management

Two truncated Bacillus thuringiensis -endotoxin genes, belonging to the classes cry1Ab and cry1B, and both coding for N-terminal toxic fragments of the corresponding crystal proteins, were translationally fused. Expression of the fusion gene driven by the cry1C promoter in Escherichia coli at a very high level resulted in a protein with enhanced toxicity to the diamondback moth (Plutella xylostella).     

Degradation of agarophytic red algal cell wall components by new crude enzyme preparations

Several new crude enzyme preparations were isolated from a marine association of the agarolytic bacterium Cytophaga diffluens and the infusorium Uronema marinum, an axenic culture of Cytophaga diffluens, some species of land micro- and macromycetes adapted to assimilate red algal biomass and from the marine mollusc Littorina littorea. Fungal and mollusc enzyme preparations were shown to have cellulase, xylanase, protease and agarase activities. Fungal agarase activity was revealed only after 3–4 passages of the culture on the medium containing algal biomass. Enzyme preparations from the association and the pure bacterial culture growing on the medium with bactoagar as the sole carbon source contained only agarase activity. The maximum specific agarase activity was found in a preparation from the marine association. The preparations obtained can be used for isolating protoplasts and single cells from red seaweed thalli. This revised version was published online in June 2006 with corrections to the Cover Date.     

Phosphorylation-dependent cleavage of p130cas in apoptotic rat-1 cells

We previously demonstrated caspase-mediated cleavage of p130cas during apoptosis and identified two caspase-3 cleavage sites [1]. In this study, we investigated the phosphorylation-dependent cleavage of p130cas in apoptotic Rat-1 fibroblast cells. Lysophosphatidic acid and fibronectin induced p130cas phosphorylation, which in turn resulted in resistance to caspase-mediated cleavage. Alternatively, dephosphorylation by calf intestinal alkaline phosphatase, PP1, and LAR stimulated cleavage of p130cas by caspase-3, generating a 31-kDa fragment. During apoptosis, p130cas dephosphorylation seems to precede its cleavage. The phosphorylation of tyrosine and serine residues immediately adjacent to the two cleavage sites (DVPD(416) and DSPD(748)) strongly affected p130cas cleavage by caspase-3, both in vitro and in vivo. Furthermore, the generation of the 31-kDa cleavage fragment was strongly regulated by phosphorylation of a tyrosine residue at position 751 (DSPD(748) and GQY(751)). Our results collectively suggest that degradation of p130cas during apoptosis is modulated in a phosphorylation-dependent manner.     

Impact of microorganisms,humidity, and temperature on the enantioselective degradation of imazethapyr in two soils

Imazethapyr (IM) is a chiral herbicide composed of an (−)‐R‐enantiomer and an (+)‐S‐enantiomer with differential herbicidal activity. In this study, the effects of microbial organisms, humidity, and temperature on the selective degradation of the (−)‐R‐ and (+)‐S‐enantiomers of IM were determined in silty loam (SL) and clay loam (CL) soil with different pH values. The (−)‐R‐enantiomer of IM was preferentially degraded in two soils under different microorganism, humidity, and temperature conditions. The average half‐lives of R‐IM ranged from 43 to 66.1 days and were significantly shorter (P <  0.05) than those of S‐IM, which ranged from 51.4 to 79.8 days. The enantiomer fraction (EF = (+)‐S‐enantiomer/((−)‐R‐enantiomer + (+)‐S‐enantiomer)) values were used to describe the enantioselectivity of degradation of IM were >0.5 (P <  0.05) in two unsterilized soils under different humidity and temperature conditions. The highest EF values were observed at unsterilized CL soil samples under 50% maximum water‐holding capacity (MWHC) and 25 °C environmental conditions. The EF values of the IM enantiomers were significantly higher (P <  0.05) in CL soils (higher pH = 5.81) and were 0.581 (unsterilized) and 0.575 (50% MWHC; 25 °C) compared with those recorded in SL soil (lower pH = 4.85). In addition, this study revealed that microbial organisms preferentially utilized the more herbicidal active IM enantiomer.