hierfstat,a package for r to compute and test hierarchical F‐statistics

The package hierfstat for the statistical software r , created by the R Development Core Team, allows the estimate of hierarchical F‐statistics from a hierarchy with any numbers of levels. In addition, it allows testing the statistical significance of population differentiation for these different levels, using a generalized likelihood‐ratio test. The package hierfstat is available at http://www.unil.ch/popgen/softwares/hierfstat.htm .     

A Brassica S-locus gene promoter targets toxic gene expression and cell death to the pistil and pollen of transgenic Nicotiana

The S-locus glycoprotein gene of Brassica is derived from the genetic locus that controls the self-incompatibility response and the specific recognition between pollen and stigma. The promoter of this gene was used to direct expression of the diphtheria toxin A chain gene and the Escherichia coli beta-glucuronidase gene in transgenic Nicotiana tabacum. Expression of the promoter in cells of the pistil and in pollen suggests that a single gene may direct the self-incompatibility response in the two interacting cell types. Additionally, the fusion genes were expressed gametophytically in the heterologous host species, Nicotiana, rather than sporophytically as expected for Brassica. Thus, although the genes involved in self-incompatibility in Brassica and Nicotiana are not homologous in their coding regions, signals for expression of these genes are apparently conserved between the two genera. Our analysis of toxic gene fusion transformants shows that genetic ablation is useful for probing developmental processes and for studying temporal and spatial patterns of gene expression in plants.     

Surface analysis of nitrogen plasma-treated C<Subscript>60</Subscript>/PS nanocomposite films for antibacterial activity

The influence of N₂ plasma on the antibacterial properties of polystyrene/fullerene (C₆₀/PS) nanocomposite films with two concentrations is investigated. A comparison is made between the surface characteristics of the films before and after plasma irradiation for different time intervals. The alterations induced on the surface of the films after treatment are analyzed by contact angle and surface energy measurements, FTIR spectroscopy, and atomic force microscopy. The antibacterial properties, growth, biofilm formation, and adhesion of the nanocomposite films against two multidrug-resistant bacterial strains, Staphylococcus aureus KT337489 and Pseudomonas aeruginosa KT337488, are investigated before and after plasma irradiation. The results indicate that P. aeruginosa is more sensitive to treatment than S. aureus as well as an enhancement of the anti-adhesion of both strains to treated surfaces through exposure.     

A role for dendritic translation of CaMKIIα mRNA in olfactory plasticity

Local protein synthesis in dendrites contributes to the synaptic modifications underlying learning and memory. The mRNA encoding the α subunit of the calcium/calmodulin dependent Kinase II (CaMKIIα) is dendritically localized and locally translated. A role for CaMKIIα local translation in hippocampus-dependent memory has been demonstrated in mice with disrupted CaMKIIα dendritic translation, through deletion of CaMKIIα 3'UTR. We studied the dendritic localization and local translation of CaMKIIα in the mouse olfactory bulb (OB), the first relay of the olfactory pathway, which exhibits a high level of plasticity in response to olfactory experience. CaMKIIα is expressed by granule cells (GCs) of the OB. Through in situ hybridization and synaptosome preparation, we show that CaMKIIα mRNA is transported in GC dendrites, synaptically localized and might be locally translated at GC synapses. Increases in the synaptic localization of CaMKIIα mRNA and protein in response to brief exposure to new odors demonstrate that they are activity-dependent processes. The activity-induced dendritic transport of CaMKIIα mRNA can be inhibited by an NMDA receptor antagonist and mimicked by an NMDA receptor agonist. Finally, in mice devoid of CaMKIIα 3'UTR, the dendritic localization of CaMKIIα mRNA is disrupted in the OB and olfactory associative learning is severely impaired. Our studies thus reveal a new functional modality for CaMKIIα local translation, as an essential determinant of olfactory plasticity.     

金属框架结构材料MOF-199对漆酶的固定化及其性质

以金属框架结构材料MOF-199为载体对漆酶进行固定化,并对固定化酶的性质进行初步研究。首先,以3-氨基丙基三乙氧基硅烷对载体MOF-199进行表面氨基化修饰,再用戊二醛对载体进行活化,最后对漆酶进行固定化。固定化条件优化结果表明:在漆酶质量浓度0.3 g/L,戊二醛用量1%(体积分数),pH 4.8下固定7 h,制得固定化酶活性最高。对固定化酶的研究发现:最适反应温度为40℃,最适pH为5.2,在连续操作7次后,固定化酶的活力仍能保持在51%。固定化漆酶热稳定性,pH耐受性,贮存稳定性均明显高于游离漆酶。     

Expression of Distinct Self-Incompatibility Specificities in Arabidopsis thaliana

The interplay of balancing selection within a species and rapid gene evolution between species can confound our ability to determine the functional equivalence of interspecific and intergeneric pairs of alleles underlying reproduction. In crucifer plants, mating specificity in the barrier to self-fertilization called self-incompatibility (SI) is controlled by allele-specific interactions between two highly polymorphic and co-evolving proteins, the S-locus receptor kinase (SRK) and its S-locus cysteine rich (SCR) ligand. These proteins have diversified both within and between species such that it is often difficult to determine from sequence information alone if they encode the same or different SI specificity. The self-fertile Arabidopsis thaliana was derived from an obligate outbreeding ancestor by loss of self-incompatibility, often in conjunction with inactivation of SRK or SCR. Nevertheless, some accessions of A. thaliana can express self-incompatibility upon transformation with an SRK–SCR gene pair isolated from its self-incompatible close relative A. lyrata. Here we show that several additional and highly diverged SRK/SCR genes from A. lyrata and another crucifer plant, Capsella grandiflora, confer self-incompatibility in A. thaliana, either as intact genes isolated from genomic libraries or after manipulation to generate chimeric fusions. We describe how the use of this newly developed chimeric protein strategy has allowed us to test the functional equivalence of SRK/SCR gene pairs from different taxa and to assay the functionality of endogenous A. thaliana SRK and SCR sequences.MATING reactions in plants, fungi, and animals are strongly influenced by molecular recognition machineries that act as gauges of genetic relatedness (Brown and Casselton 2001; Nasrallah 2005; Yamazaki and Beauchamp 2007). Many plants with hermaphroditic flowers have evolved inbreeding avoidance mechanisms, known as self-incompatibility (SI) systems. These systems are based on the ability of the female reproductive apparatus (the pistil) to discriminate among genetically distinct pollen grains, resulting in the failure of self-pollination despite functional female and male reproductive structures. In the Brassicaceae (crucifers), specific recognition of pollen by the epidermal cells of the stigma (a structure located at the tip of the pistil) is controlled by haplotypes of the S locus, and activation of the SI response leading to inhibition of pollen tube growth occurs if pollen and stigma are derived from plants that express the same S-locus haplotype (S haplotype). Within self-incompatible crucifer species, the number of S haplotypes and corresponding SI specificities is usually high, with >50 reported in some species (Watanabe et al. 2000), and SI dictates that self-incompatible plants are typically heterozygous and carry two S haplotypes. Each S haplotype is composed of two highly polymorphic genes that are the determinants of SI specificity in stigma and pollen (Stein et al. 1991; Schopfer et al. 1999). The S-locus receptor kinase (SRK) gene encodes a single-pass transmembrane serine/threonine kinase localized on the surface of stigma epidermal cells, and the S-locus cysteine-rich protein (SCR) gene encodes a small peptide localized in the pollen coat. SCR is the ligand for SRK and will bind to the extracellular domain of SRK (hereafter eSRK) only if both proteins are encoded by the same S-locus haplotype (Kachroo et al. 2001; Takayama et al. 2001; Chookajorn et al. 2004). The binding of SCR to its cognate eSRK triggers an intracellular phosphorylation cascade that results in pollen rejection by a poorly understood mechanism.A mechanistic understanding of the recognition phase of SI requires detailed structure–function analyses of SRK and SCR aimed at identifying the amino acid residues that determine their allele-specific interaction and explaining the puzzling dominance/recessive interactions exhibited by different SRK alleles in the heterozygous stigmas of self-incompatible plants (Hatakeyama et al. 2001; Mable et al. 2003; Prigoda et al. 2005). Such structure–function studies require an experimental system that allows efficient in vivo functional analysis of large numbers of SRK and SCR sequence variants generated in vitro by site-directed mutagenesis or domain swapping between proteins that determine different SI specificities. The recent transfer of the SI trait into Arabidopsis thaliana has established this species as a model organism for mechanistic and evolutionary studies of mating systems in crucifers (Nasrallah et al. 2002, 2004). However, to date, only one SI specificity, that which is determined by the Sb haplotype of A. lyrata, has been successfully introduced into A. thaliana and shown to alter the plant''s mating reaction from strict autogamy to full SI. To exploit fully the A. thaliana transgenic SI model, additional S haplotypes must be introduced into this species. In addition to facilitating mechanistic studies of the SRK–SCR interaction and dominance relationships, the expression of multiple SI specificities in A. thaliana promises to shed light on processes underlying the diversification of SRK and SCR genes. For example, expression in A. thaliana of SI specificities derived from different crucifer species will allow direct assays of the functional equivalence or nonequivalence of the corresponding S haplotypes, an issue that is difficult to resolve on the basis of sequence information alone.Although conceptually simple, expressing different SI specificities by transformation with different SRK/SCR gene pairs is not a straightforward proposition. Difficulties stem largely from the availability of appropriate cloned SRK/SCR variants for use in transformation experiments. A large number of SRK/SCR gene pairs are available from Brassica species as a result of extensive and long-standing studies of SI. However, attempts to restore SI in transgenic A. thaliana using Brassica S-locus genes had met with failure (Bi et al. 2000; J. B. Nasrallah, unpublished data), possibly because of the inability of Brassica SRKs to interact productively with A. thaliana components of the SI signal transduction pathway. In the past few years, studies of SI were initiated in self-incompatible species more closely related to A. thaliana, such as A. lyrata, A. halleri, and Capsella grandiflora. However, with a few exceptions, these studies produced only partial SRK and SCR sequences amplified from genomic DNA (Schierup et al. 2001; Prigoda et al. 2005; Bechsgaard et al. 2006; Paetsch et al. 2006). The challenging task of cloning the very highly polymorphic SCR sequences and complete SRK and SCR genes, which requires genomic library construction and in many cases chromosome walking, has only been accomplished for two S haplotypes of A. lyrata, Sb (hereafter AlSb, which was used in previous transformation studies (Nasrallah et al. 2002, 2004), and Sa (AlSa; Kusaba et al. 2001), and for the S7 haplotype of C. grandiflora (CgS7; Nasrallah et al. 2007).In this article, we report the isolation of two new SRK/SCR gene pairs from genomic libraries of A. lyrata and expression of the corresponding SI specificities in A. thaliana. We also describe a novel strategy for rapid and efficient transfer of several distinct SI specificities into A. thaliana, which only requires knowledge of the eSRK sequence and SCR second-exon sequences that encode the mature SCR protein.     

Alanine metabolism, transport, and cycling in the brain

Brain glutamate/glutamine cycling is incomplete without return of ammonia to glial cells. Previous studies suggest that alanine is an important carrier for ammonia transfer. In this study, we investigated alanine transport and metabolism in Guinea pig brain cortical tissue slices and prisms, in primary cultures of neurons and astrocytes, and in synaptosomes. Alanine uptake into astrocytes was largely mediated by system L isoform LAT2, whereas alanine uptake into neurons was mediated by Na⁺-dependent transporters with properties similar to system B⁰ isoform B⁰AT2. To investigate the role of alanine transport in metabolism, its uptake was inhibited in cortical tissue slices under depolarizing conditions using the system L transport inhibitors 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid and cycloleucine (1-aminocyclopentanecarboxylic acid; cLeu). The results indicated that alanine cycling occurs subsequent to glutamate/glutamine cycling and that a significant proportion of cycling occurs via amino acid transport system L. Our results show that system L isoform LAT2 is critical for alanine uptake into astrocytes. However, alanine does not provide any significant carbon for energy or neurotransmitter metabolism under the conditions studied.     

Regeneration of multiple shoots from transgenic potato events facilitates the recovery of phenotypically normal lines: assessing a cry9Aa2 gene conferring insect resistance

Background

Tools for authenticating cell lines are critical for quality control in cell-based biological experiments. Currently there are methods to authenticate human cell lines using short tandem repeat (STR) markers based on the technology and procedures successfully used in the forensic community for human identification, but there are no STR based methods for authenticating nonhuman cell lines to date. There is significant homology between the human and vervet monkey genome and we utilized these similarities to design the first multiplex assay based on human STR markers for vervet cell line identification.

Results

The following STR markers were incorporated into the vervet multiplex PCR assay: D17S1304, D5S1467, D19S245, D1S518, D8S1106, D4S2408, D6S1017, and DYS389. The eight markers were successful in uniquely identifying sixty-two vervet monkey DNA samples and confirmed that Vero76 cells and COS-7 cells were derived from Vero and CV-1 cells, respectively. The multiplex assay shows specificity for vervet DNA within the determined allele range for vervet monkeys; however, the primers will also amplify human DNA for each marker resulting in amplicons outside the vervet allele range in several of the loci. The STR markers showed genetic stability in over sixty-nine passages of Vero cells, suggesting low mutation rates in the targeted STR sequences in the Vero cell line.

Conclusions

A functional vervet multiplex assay consisting of eight human STR markers with heterozygosity values ranging from 0.53-0.79 was successful in uniquely identifying sixty-two vervet monkey samples. The probability of a random match using these eight markers between any two vervet samples is approximately 1 in 1.9 million. While authenticating a vervet cell line, the multiplex assay may also be a useful indicator for human cell line contamination since the assay is based on human STR markers.     

The plant ant Tetraponera aethiops (Pseudomyrmecinae) protects its host myrmecophyte Barteria fistulosa (Passifloraceae) through aggressiveness and predation

Plant ants generally provide their host myrmecophytes (i.e. plants that shelter a limited number of ant species in hollow structures) protection from defoliating insects, but the exact nature of this protection is poorly known. It was with this in mind that we studied the association between Tetraponera aethiops F. Smith (Pseudomyrmecinae) and its specific host myrmecophyte Barteria fistulosa Mast. (Passifloraceae). Workers bore entrances into the horizontal hollow branches (domatia) of their host B. fistulosa , near the base of the petiole of the alternate horizontal leaves. They then ambush intruders from the domatia, close to these entrances. After perceiving the vibrations caused when an insect lands on a leaf, they rush to it and sting and generally spreadeagle the insect (only small caterpillars are mastered by single workers). Among the insects likely to defoliate B. fistulosa , adult leaf beetles and large katydids were taken as prey and cut up; single workers then retrieved some pieces, whereas other workers imbibed the prey's haemolymph. Other insects known to defoliate this plant, if unable to escape, were killed and discarded. Small Acrea zetes L. caterpillars were stung and then discarded by single workers; whereas locusts of different sizes were mastered by groups of workers that stung and spreadeagled them before discarding them (although a part of their haemolymph was imbibed). More workers were involved and more time was necessary to master insects taken as prey than those attacked and discarded. Consequently, the protection T. aethiops workers provide to their host B. fistulosa from defoliating insects results from predation, but more often from a type of aggressiveness wherein insects are killed and then discarded.  © 2008 The Linnean Society of London, Biological Journal of the Linnean Society , 2008, 93 , 63–69.