The Interaction of Equine Lysozyme:Oleic Acid Complexes with Lipid Membranes Suggests a Cargo Off-Loading Mechanism

The normal function of equine lysozyme (EL) is the hydrolysis of peptidoglycan residues of bacterial cell walls. EL is closely related to α-lactalbumins with respect to sequence and structure and further possesses the calcium binding site of α-lactalbumins. Recently, EL multimeric complexes with oleic acids (ELOAs) were shown to possess tinctorial and morphological properties, similar to amyloidal aggregates, and to be cytotoxic. ELOA's interactions with phospholipid membranes appear to be central to its biological action, similar to human α-lactalbumin made lethal to tumor cells. Here, we describe the interaction of ELOA with phospholipid membranes. Confocal scanning laser microscopy shows that ELOA, but not native EL, accumulates on the surface of giant unilamellar vesicles, without inducing significant membrane permeability. Quartz crystal microbalance with dissipation data indicated an essentially non-disruptive binding of ELOA to supported lipid bilayers, leading to formation of highly dissipative and “soft” lipid membrane; at higher concentrations of ELOA, the lipid membrane desorbs from the surface probably as bilayer sheets of vesicles. This membrane rearrangement occurred to a similar extent when free oleic acid (OA) was added, but not when free OA was removed from ELOA by prior incubation with bovine serum albumin, emphasizing the role of OA in this process. NMR data indicated an equilibrium between free and bound OA, which shifts towards free OA as ELOA is progressively diluted, indicating that OA is relatively loosely bound. Activity measurements together with fluorescence spectroscopy and circular dichroism suggested a conversion of ELOA towards a more native-like state on interaction with lipid membranes, although complete refolding was not observed. Altogether, these results suggest that ELOA may act as an OA carrier and facilitate OA transfer to the membrane. ELOA's properties illustrate that protein folding variants may possess specific functional properties distinct from the native protein.     

The feet of the measles virus polymerase bind the viral nucleocapsid protein at a single site

Measles virus has a single‐stranded RNA genome that is organized into a helical complex by the viral N protein. The resulting structure is termed the nucleocapsid and is traversed by the viral polymerase during RNA synthesis. The P protein, the noncatalytic subunit of the polymerase, provides the “legs and feet” that allow the polymerase to walk along its protein‐RNA template. The polymerase feet are very simple three‐helix bundles, only 50 amino acids in size. Previously, we have shown that these feet grasp the viral N protein during movement by attaching to a short sequence (amino acids 487–503) within the disordered and surface‐exposed tail of N, causing it to fold into a helix. The result is a weak‐affinity complex with a short lifetime, which would allow the polymerase to take rapid steps forward. The structure of the complex was determined using X‐ray crystallography. This simple model of binding was challenged by a paper in this journal, claiming that a downstream sequence in the tail of N (amino acids 517–525) was also critical for the association. Its presence was reported to enhance the overall affinity of the polymerase feet for N by three orders of magnitude. We have, therefore, examined binding of the polymerase foot domain to amino acids 477–525 of N using quantitative biophysical techniques, and compared the results to our previous binding studies, performed using amino acids 477–505 of N. We find no evidence that the sequence downstream of amino acid 505 influences binding, validating the original single‐site binding model.     

Five Cyanophora (Cyanophorales,Glaucophyta) species delineated based on morphological and molecular data

Cyanophora is an important glaucophyte genus of unicellular biflagellates that may have retained ancestral features of photosynthetic eukaryotes. The nuclear genome of Cyanophora was recently sequenced, but taxonomic studies of more than two strains are lacking for this genus. Furthermore, no study has used molecular methods to taxonomically delineate Cyanophora species. Here, we delimited the species of Cyanophora using light and electron microscopy, combined with molecular data from several globally distributed strains, including one newly established. Using a light microscope, we identified two distinct morphological groups: one with ovoid to ellipsoidal vegetative cells and another with dorsoventrally flattened or broad, bean‐shaped vegetative cells containing duplicated plastids. Our light and scanning electron microscopy clearly distinguished three species with ovoid to ellipsoidal cells (C. paradoxa Korshikov, C. cuspidata Tos.Takah. & Nozaki sp. nov., and C. kugrensii Tos.Takah. & Nozaki sp. nov.) and two species with broad, bean‐shaped cells (C. biloba Kugrens, B.L.Clay, C.J.Mey. & R.E.Lee and C. sudae Tos.Takah. & Nozaki sp. nov.) based on differences in cell shape and surface ornamentations of the vegetative cells under the field‐emission scanning electron microscope. Molecular phylogenetic analyses of P700 chl a apoprotein A2 (psaB) genes and internal transcribed spacer (ITS) regions of nuclear ribosomal DNA (rDNA), as well as a comparison of secondary structures of nuclear rDNA ITS‐2 and genetic distances of psaB genes, supported the delineation of five morphological species of Cyanophora.     

Characterisation of the Nucleotide Exchange Factor ITSN1L: Evidence for a Kinetic Discrimination of GEF-Stimulated Nucleotide Release from Cdc42

Cdc42, a member of the Ras superfamily of small guanine nucleotide binding proteins, plays an important role in regulating the actin cytoskeleton, intracellular trafficking, and cell polarity. Its activation is controlled by guanine nucleotide exchange factors (GEFs), which stimulate the dissociation of bound guanosine-5′-diphosphate (GDP) to allow guanosine-5′-triphosphate (GTP) binding. Here, we investigate the exchange factor activity of the Dbl-homology domain containing constructs of the adaptor protein Intersectin1L (ITSN1L), which is a specific GEF for Cdc42. A detailed kinetic characterisation comparing ITSN1L-mediated nucleotide exchange on Cdc42 in its GTP- versus GDP-bound state reveals a kinetic discrimination for GEF-stimulated dissociation of GTP: The maximum acceleration of the intrinsic mGDP [2′/3′-O-(N-methyl-anthraniloyl)-GDP] release from Cdc42 by ITSN1L is accelerated at least 68,000-fold, whereas the exchange of mGTP [2′/3′-O-(N-methyl-anthraniloyl)-GTP] is stimulated only up to 6000-fold at the same GEF concentration. The selectivity in nucleotide exchange kinetics for GDP over GTP is even more pronounced when a Cdc42 mutant, F28L, is used, which is characterised by fast intrinsic dissociation of nucleotides. We furthermore show that both GTP and Mg²⁺ ions are required for the interaction with effectors. We suggest a novel model for selective nucleotide exchange residing on a conformational change of Cdc42 upon binding of GTP, which enables effector binding to the Cdc42 · GTP complex but, at the same time, excludes efficient modulation by the GEF. The higher exchange activity of ITSN1L towards the GDP-bound conformation of Cdc42 could represent an evolutionary adaptation of this GEF that ensures nucleotide exchange towards the formation of the signalling-active GTP-bound form of Cdc42 and avoids dissociation of the active complex.     

嗜盐菌Halobacterium species NRC-1不同盐浓度下基因表达差异分析

用RNA随机起始PCR（RAP—PCR）技术分析了盐杆菌NRC-1（Halobacterium NRC-1）不同NaCl盐度下基因表达的差异。81条引物用于比较17％、30％两种盐度下基因表达的差异,每条引物平均可以产生10条以上扩增条带,共得到15条在2种盐度下差异表达的条带,这些差异扩增条带的获得将有助从分子水平上了解Halobacterium NRC-1在高盐环境下的适应机制。     

Differential selection of baculovirus genotypes mediated by different species of host food plant

Recent studies have demonstrated high levels of genotypic and phenotypic variation in populations of parasites, even within individual hosts. Several genetic, immunological and epidemiological mechanisms have been postulated as promoters of such variation, but little empirical work has addressed the role of host ecology. A nucleopolyhedrovirus that attacks larvae of the pine beauty moth, Panolis flammea , exists as a complex mixture of genotypes within individual host larvae. We demonstrate that the food plant species eaten by the host (Scots pine vs. lodgepole pine) differentially affects the pathogenicity and productivity of two virus genotypes originally purified from a single host individual. We hypothesize that such food plant-mediated differential selection will promote genotypic variation between baculovirus populations, and that subsequent remixing of virus genotypes could maintain genotypic variation within individual hosts. Our results provide a tritrophic explanation for the genotypic and phenotypic complexity of host–parasite interactions with complex ecologies.     

Unilateral incompatibility as a major cause of skewed segregation in the cross between Lycopersicon esculentum and L. pennellii

Skewed segregations are frequent events in segregating populations derived from different interspecific crosses in tomato. To determine a basis for skewed segregations in the progeny of the cross between Lycopersicon esculentum and L. pennellii, monogenic segregations of 16 isozyme loci were analyzed in an F₂ and two backcross populations of this cross. In the F₂, 9 loci mapping to chromosomes 1, 2, 4, 9, 10 and 12 exhibited skewed segregations and in all cases there was an excess of L. pennellii homozygotes. The genotypic frequencies at all but one locus were at Hardy-Weinberg equilibria. In the backcross populations, all except two loci exhibited normal Mendelian segregations. No post-zygotic selection model could statistically or biologically explain the observed segregation patterns in the F₂ and backcross populations. A pre-zygotic selection model, assuming selective elimination of the male gametophytes during pollen function (i.e., from pollination to karyogamy), could adequately explain the observed segregations in all three populations. The direction of the skewed segregations in the F₂ population was consistent with that expected based on the effects of unilateral incompatibility reactions between the two species. In addition, the chromosomal locations of 5 of the 9 markers that exhibited skewed segregations coincided with the locations of several known compatibility-related genes in tomato. Multigenic unilateral incompatibility reactions between L. esculentum pollen and the stigma or style of L. pennellii (or its hybrid derivatives) are suggested to be the major cause of the skewed segregations in the F₂ progeny of this cross.     

The role of protonation in protein fibrillation

Many proteins fibrillate at low pH despite a high population of charged side chains. Therefore exchange of protons between the fibrillating peptide and its surroundings may play an important role in fibrillation. Here, we use isothermal titration calorimetry to measure exchange of protons between buffer and the peptide hormone glucagon during fibrillation. Glucagon absorbs or releases protons to an extent which allows it to attain a net charge of zero in the fibrillar state, both at acidic and basic pH. Similar results are obtained for lysozyme. This suggests that side chain pK_a values change dramatically in the fibrillar state.     

‘寒富’苹果二倍体及其同源四倍体叶片超微结构和叶绿素荧光参数特征

采用扫描电镜和石蜡切片法,以‘寒富’苹果二倍体及经秋水仙素加倍获得的同源四倍体植株为材料,比较两种倍性植株叶片超微结构、叶绿素含量及叶绿素荧光参数的日变化规律。结果显示:(1)与二倍体植株相比较,其同源四倍体叶片厚度、栅海比、气孔长、气孔宽、分别增加了15.1%、16.1%、70.5%、27.2%,而气孔密度显著减少了58.7%;其同源四倍体叶保卫细胞中叶绿体数和叶绿素含量分别比二倍体植株高出125.3%、37.7%。(2)‘寒富’苹果同源四倍体与其二倍体的叶绿素荧光参数PSⅡ的原初光能转化效率(Fv/Fm)、PSⅡ的潜在光化学效率(Fv/F0)和以吸收光能为基础的光合性能指数(PI)值的日变化趋势相似,但PI平均值比二倍体显著高出38.6%。研究表明,同源四倍体较二倍体叶片在形态上更大、更厚,气孔更大、密度更小,栅海比更大,表现出抗病的叶片结构;同时同源四倍体较二倍体含有更高的叶绿素含量,表现更优良的光合特性。     

广西桉树人工林叶面积指数的估测及其校正

叶面积指数(Leaf area index, LAI)是森林生态系统重要的结构参数,通过遥感技术可反演区域LAI,但其可靠性需要地面准确的实测数据进行验证。选取广西国有高峰林场不同林龄的桉树(Eucalyptus robusta)人工林为对象,以异速生长法(Allometry)为对照,综合利用植物冠层分析仪法(LAI-2200)、跟踪辐射和冠层结构分析仪法(TRAC)、半球摄影法(DHP)以及地基激光雷达法(TLS)等间接法估测样地的LAI,并考虑木质成分以及聚集效应影响,进行相应的校正处理,为地面快速、准确测量桉树人工林LAI提供参考。结果表明：桉树人工林的比叶面积为125.37±13.38 cm~2/g,通过Allometry获得的LAI变化范围在1.65—3.84,平均为2.73,不同林龄间的差异均显著(P<0.05),随着林龄的增加呈现先增加后减少的趋势。在未校正情况下,LAI-2200、TRAC、DHP、TLS估算的LAI存在显著差异(P<0.05)。与对照相比,LAI-2200在幼龄林和过熟林中估算误差最小,TRAC在成熟林中估算误差最小。相对于完全去除法,利用...