Impacts of bottom and suspended cultures of mussels <Emphasis Type="Italic">Mytilus</Emphasis> spp. on the surrounding sedimentary environment and macrobenthic biodiversity

The aim of this study was to quantify the effect of bottom and suspended mussel cultures, cultured in different physical environments, on the sedimentary environmental conditions and thereby the biodiversity structure of the associated macrofaunal community. We compared two bottom cultures (Limfjorden: microtidal, wind-driven; Oosterschelde: macrotidal) and one suspended culture (Ria de Vigo in an upwelling coastal region). The sedimentary environmental conditions (mud fraction, POC, PON, phosphorus content, chl a breakdown products) were significantly elevated underneath and surrounding bottom and suspended cultures compared to culture-free sediments that were nearby and hydrodynamically similar. The relative change in environmental conditions was more pronounced in the Oosterschelde compared to Limfjorden, most likely due to differences in hydrodynamic forcing and characteristics of the mussel bed. The effect of the suspended cultures in Ria de Vigo on the surrounding sediments was influenced by local topographic and hydrodynamic conditions. The impact of mussels on the benthic community due to biodeposition was clearly seen in the community structure. The species composition changed from species which are typically present in sandy environments to more small opportunistic species, which are typically present in organically enriched sediments. The impact of bottom cultures on the benthic community due to changes in the habitat under the presence of mussels was positive, especially in the Oosterschelde where an increase in the number of epibenthic species was seen. The influence of bottom cultures on the sedimentary environment and on the macrobenthic community seems to be very local. Within the mussel site in Limfjorden, differences were detected between sites where none or almost no mussels were present with sites where mussels were very abundant.     

类泛素修饰蛋白质ISG15及其修饰酶系的功能

受干扰素诱导表达的干扰素刺激基因15编码蛋白质(ISG15)是第1个被鉴定的类泛素修饰蛋白质.目前已在病毒感染细胞和肿瘤细胞中发现了多种ISG15的作用靶蛋白,提示ISG15可能在免疫调节和肿瘤发生等方面发挥重要作用.本文介绍ISG15的结构与生化特点,探讨ISG15在相关酶系作用下修饰目标蛋白质的机制,总结ISG15及其修饰酶系的抗病毒和抗肿瘤作用及其相关机制.     

Evolution of haploid–diploid life cycles when haploid and diploid fitnesses are not equal

Many organisms spend a significant portion of their life cycle as haploids and as diploids (a haploid–diploid life cycle). However, the evolutionary processes that could maintain this sort of life cycle are unclear. Most previous models of ploidy evolution have assumed that the fitness effects of new mutations are equal in haploids and homozygous diploids, however, this equivalency is not supported by empirical data. With different mutational effects, the overall (intrinsic) fitness of a haploid would not be equal to that of a diploid after a series of substitution events. Intrinsic fitness differences between haploids and diploids can also arise directly, for example because diploids tend to have larger cell sizes than haploids. Here, we incorporate intrinsic fitness differences into genetic models for the evolution of time spent in the haploid versus diploid phases, in which ploidy affects whether new mutations are masked. Life‐cycle evolution can be affected by intrinsic fitness differences between phases, the masking of mutations, or a combination of both. We find parameter ranges where these two selective forces act and show that the balance between them can favor convergence on a haploid–diploid life cycle, which is not observed in the absence of intrinsic fitness differences.     

Mitochondrial tRNAGlu 14693G variant may modulate the phenotypic manifestation of deafness-associated 12S rRNA A1555G mutation in a Hah Chinese family

Mutations in mitochondrial 12S rRNA gene are one of the most important causes of aminoglycoside-induced and nonsyndromic hearing loss. Here we report the characterization of one Han Chinese pedigree with aminoglycoside-induced and nonsyndromic hearing loss.This Chinese family carrying the 12S rRNA A1555G mutation exhibited high penetrance and expressivity of hearing impairment. In particular, penetrances of hearing loss in this family pedigree were 43.8% and 25%, respectively, when aminoglycoside-induced heating loss was included or excluded. Mutational analysis of entire mitochondrial genomes in this family showed the homoplasmic A1555G mutation and a set of variants belonging to haplogroup Y2. Of these, the A14693G variant occurred at the extremely conserved nucleotide (conventional position 54) of the TψC-loop of tRNAGlu and was absent in 156 Chinese controls. Nucleotides at position 54 of tRNAs are often modified, thereby contributing to the structural formation and stabilization of functional tRNAs. Thus, the structural alteration of tRNA by the A14693G variant may lead to a failure in tRNA metabolism and impair mitochondrial protein synthesis, thereby worsening mitochondrial dysfunctions altered by the A1555G mutation. Therefore, the tRNAalu A14693G variant may have a potential modifier role in increasing the penetrance and expressivity of the deafness-associated AI555G mutation in this Chinese pedigree.     

Expression and Purification of Human Keratinocyte Growth Factor 2 by Fusion with SUMO

Small ubiquitin-related modifier (SUMO) fusion system has been shown to be efficient for enhancing expression and preventing degradation of the target protein. We showed herein that SUMO fusion to human keratinocyte growth factor 2 (hKGF-2) gene was feasible and it significantly enhanced protein expression and its efficiency. The fusion DNA fragment composed of SUMO gene, which was fused to hexahistidine tag, and hKGF-2 gene was amplified by PCR and inserted into the expression vector pET28a to construct the recombinant plasmid, pET28a-SUMO-hKGF-2. The plasmid was then transformed into Escherichia coli Rosetta^TM2(DE3), and the recombinant fusion protein SUMO-hKGF-2 was expressed at 30°C for 6 h, with the induction of IPTG at the final concentration of 0.4 mM. The expression level of the fusion protein was up to 30% of the total cellular protein. The fusion protein was purified by Ni-NTA affinity chromatography. After desalting by Sephadex G-25 size exclusion chromatography, the hexahistidine-SUMO-hKGF-2 was digested by SUMO proteases. The recombinant hKGF-2 was purified again with Ni-NTA column and the purity was about 95% with a total yield of 13.9 mg/l culture. The result of mitogenicity assay suggests that the recombinant hKGF-2 can significantly promote the proliferation of normal rat kidney epithelial (NRK-52E) cells. Xiaoping Wu, and Changjun Nie contributed equally to the work.     

High-level expression and characterization of an anti-VEGF165 single-chain variable fragment (scFv) by small ubiquitin-related modifier fusion in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Antibodies currently constitute the most rapidly growing class of human therapeutics; however, the high-yield production of recombinant antibodies and antibody fragments is a real challenge. High expression of active single-chain antibody fragment (scFv) in Escherichia coli has not been successful, as the protein contains three intramolecular disulfide bonds that are difficult to form correctly in the bacterial intracellular environment. To solve this problem, we fused the scFv gene against VEGF165 with a small ubiquitin-related modifier gene (SUMO) by synthesizing an artificial SUMO–scFv fusion gene that was highly expressed in the BL21(DE3) strain. The optimal expression level of the soluble fusion protein, SUMO–scFv, was up to 28.5% of the total cellular protein. The fusion protein was purified by Ni nitrilotriacetic acid (NTA) affinity chromatography and cleaved by a SUMO-specific protease to obtain the native scFv, which was further purified by Ni-NTA affinity chromatography. The result of the high-performance liquid chromatography showed that the purity of the recombinant cleaved scFv was greater than 98%. The primary structure of the purified scFv was confirmed by N-terminal amino acid sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy analysis. In vitro activity assay demonstrated that the recombinant scFv could dose-dependently inhibit VEGF165-induced human umbilical vein-derived endothelial cell proliferation. The expression strategy presented in this study allows convenient high yield and easy purification of recombinant scFv with native sequences.     

Introducing desirable transgenes into insect populations using Y-linked meiotic drive - a theoretical assessment

The use of genetic drive mechanisms to replace native mosquito genotypes with individuals bearing antipathogen transgenes is a potential strategy for repressing insect transmission of human diseases such as malaria and dengue. Antipathogen transgenes have been developed and tested, but efficient gene drive mechanisms are lacking. Here we theoretically assess the feasibility of introducing antipathogen genes into wild Aedes aegypti populations by using a naturally occurring meiotic drive system. We consider the release of males having both a Y-linked meiotic drive gene and an X-linked drive-insensitive response allele to which an antipathogen gene is linked. We use mathematical models and computer simulations to determine how the post-introduction dynamics of the antipathogen gene are affected by specific genetic characteristics of the system. The results show that when the natural population is uniformly sensitive to the meiotic drive gene, the antipathogen gene may be driven close to fixation if the fitness costs of the drive gene, the insensitive response allele, and the antipathogen gene are low. However, when the natural population has a small proportion of an X-linked insensitive response allele or an autosomal gene that strongly reduces the effect of the drive gene, the antipathogen gene does not spread if it has an associated fitness cost. Our modeling results provide a theoretical foundation for further experimental tests.     

蛋白质修饰剂、变性剂和活性氧对菠菜叶片光系统Ⅱ光失活的影响

菠菜叶圆片以活性氧、蛋白质修饰剂和变性剂结合强光处理半小时,检测叶绿素荧光动力学并与单照强光的对照作比较。加人H2O2、·O2、OH·和O2等活性氧加剧了PSⅡ的光抑制作用,Fv／Fm,qp和PSⅡ降低,qn和1—qp相应上升。4种蛋白质修饰剂NBS、DEPC、BDE和pCMB的处理,使叶片的Ch1荧光参数发生了与活性氧作用相类似的变化,尤以精氨酸残基修饰剂BDE的影响较为显著。SDS明显地影响FV和qp但未改变qN值。Gu－HC1导致qp完全丧失,qN达最大值。低浓度的脲素和DMSO略为降低PSⅡ活性和增大qN。结果认为对叶绿体蛋白构象或结构的任何改变皆引起光下PSⅡ光失活作用的增强。     

cDNA cloning, recombinant expression and bioactivity of Père David's deer BAFF

B cell activating factor (BAFF), a member of the TNF family, is a critical cytokine for the survival, proliferation, maturation, and differentiation of B cells. In the present study, Père David's deer BAFF (miBAFF) was amplified from Elaphurus davidianus using RT-PCR. This is the first BAFF cloned from a member of Cervidae family. The open reading frame (ORF) of the miBAFF cDNA consists of 843 bases that encode a 280-amino acid protein bearing typical TNF homology domain. Sequence alignment shows that miBAFF shares 39.3%-97% sequence homology with the BAFF sequences of other mammals. Comparative protein modeling predicted that the 3D structure of the soluble mature portion of miBAFF (misBAFF) is very similar to that of human BAFF (hsBAFF). Recombinant misBAFF fused to a SUMO-tag was efficiently expressed in Escherichia coli BL21 (DE3) cells. The protein molecular weight of ~36 KDa was determined using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. In vitro, purified misBAFF was shown to promote the survival and proliferation of Père David's deer peripheral blood lymphocytes and mouse B cells. These results indicate that miBAFF plays an important role in the survival/proliferation of mouse B cells and, shows highly conserved evolutionarily, leading to functional cross-reactivity that exists between mouse and Père David's deer BAFF.     

Residue-level NMR view of the urea-driven equilibrium folding transition of SUMO-1 (1-97): native preferences do not increase monotonously

SUMO-1 (1-97) is a crucial protein in the machinery of post-translational modifications. We observed by circular dichroism and fluorescence spectroscopy that urea-induced unfolding of this protein is a complex process with the possibility of occurrence of detectable intermediates along the way. The tertiary structure is completely lost around approximately 4.5 M urea with a transition mid-point at 2.53 M urea, while the secondary structure unfolding seems to show two transitions, with mid-points at 2.42 M and 5.69 M urea. We have elucidated by systematic urea titration, the equilibrium residue level structural and dynamics changes along the entire folding/unfolding transition by multidimensional NMR. With urea dilution, the protein is seen to progressively lose most of the broad beta-domain structural preferences present at 8 M urea, acquire some helical propensities at 5 M urea, and lose some of them again on further dilution of urea. Between 3 M and 2 M urea, the protein starts afresh to acquire native structural features. These observations are contrary to the conventional notion that proteins fold with monotonously increasing native-type preferences. For folding below approximately 3 M urea, the region around the alpha1 helix appears to be a potential folding initiation site. The folding seems to start with a collapse into native-like topologies, at least in parts, and is followed by formation of secondary and tertiary structure, perhaps by cooperative rearrangements. The motional characteristics of the protein show sequence-dependent variation as the concentration of urea is progressively reduced. At the sub-nanosecond level, the features are extremely unusual for denatured states, and only certain segments corresponding to the flexible regions in the native protein display these motions at the different concentrations of urea.