bluuebunny/crossref_metadata_2025 huggingface dataset:
Systematic analysis of protein subcellular localization and interaction using high‐throughput transient transformation of Arabidopsis seedlings
Authors: Marion Jessica , Bach Lien , Bellec Yannick , Meyer Christian , Gissot Lionel , Faure Jean‐Denis
Published: July 2008

The functional genomics approach requires systematic analysis of protein subcellular distribution and interaction networks, preferably by optimizing experimental simplicity and physiological significance. Here, we present an efficient in planta transient transformation system that allows single or multiple expression of constructs containing various fluorescent protein tags in Arabidopsis cotyledons. The optimized protocol is based on vacuum infiltration of agrobacteria directly into young Arabidopsis seedlings. We demonstrate that Arabidopsis epidermal cells show a subcellular distribution of reference markers similar to that in tobacco epidermal cells, and can be used for co‐localization or bi‐molecular fluorescent complementation studies. We then used this new system to investigate the subcellular distribution of enzymes involved in sphingolipid metabolism. In contrast to transformation systems using tobacco epidermal cells or cultured Arabidopsis cells, our system provides the opportunity to take advantage of the extensive collections of mutant and transgenic lines available in Arabidopsis. The fact that this assay uses conventional binary vectors and a conventional Agrobacterium strain, and is compatible with a large variety of fluorescent tags, makes it a versatile tool for construct screening and characterization before stable transformation. Transient expression in Arabidopsis seedlings is thus a fast and simple method that requires minimum handling and potentially allows medium‐ to high‐throughput analyses of fusion proteins harboring fluorescent tags in a whole‐plant cellular context.
AtMYB12 regulates caffeoyl quinic acid and flavonol synthesis in tomato: expression in fruit results in very high levels of both types of polyphenol
Authors: Luo Jie , Butelli Eugenio , Hill Lionel , Parr Adrian , Niggeweg Ricarda , Bailey Paul , Weisshaar Bernd , Martin Cathie
Published: July 2008

Plant polyphenolics exhibit a broad spectrum of health‐promoting effects when consumed as part of the diet, and there is considerable interest in enhancing the levels of these bioactive molecules in plants used as foods. AtMYB12 was originally identified as a flavonol‐specific transcriptional activator in Arabidopsis thaliana, and this has been confirmed by ectopic expression in tobacco. AtMYB12 is able to induce the expression of additional target genes in tobacco, leading to the accumulation of very high levels of flavonols. When expressed in a tissue‐specific manner in tomato, AtMYB12 activates the caffeoyl quinic acid biosynthetic pathway, in addition to the flavonol biosynthetic pathway, an activity which probably mirrors that of the orthologous MYB12‐like protein in tomato. As a result of its broad specificity for transcriptional activation in tomato, AtMYB12 can be used to produce fruit with extremely high levels of multiple polyphenolic anti‐oxidants. Our data indicate that transcription factors may have different specificities for target genes in different plants, which is of significance when designing strategies to improve metabolite accumulation and the anti‐oxidant capacity of foods.
Proteolytic processing of a precursor protein for a growth‐promoting peptide by a subtilisin serine protease in Arabidopsis
Authors: Srivastava Renu , Liu Jian‐Xiang , Howell Stephen H.
Published: July 2008

Phytosulfokines (PSKs) are secreted, sulfated peptide hormones derived from larger prepropeptide precursors. Proteolytic processing of one of the precursors, AtPSK4, was demonstrated by cleavage of a preproAtPSK4–myc transgene product to AtPSK4–myc. Cleavage of proAtPSK4 was induced by placing root explants in tissue culture. The processing of proAtPSK4 was dependent on AtSBT1.1, a subtilisin‐like serine protease, encoded by one of 56 subtilase genes in Arabidopsis. The gene encoding AtSBT1.1 was up‐regulated following the transfer of root explants to tissue culture, suggesting that activation of the proteolytic machinery that cleaves proAtPSK4 is dependent on AtSBT1.1 expression. We also demonstrated that a fluorogenic peptide representing the putative subtilase recognition site in proAtPSK4 is cleaved in vitro by affinity‐purified AtSBT1.1. An alanine scan through the recognition site peptide indicated that AtSBT1.1 is fairly specific for the AtPSK4 precursor. Thus, this peptide growth factor, which promotes callus formation in culture, is proteolytically cleaved from its precursor by a specific plant subtilase encoded by a gene that is up‐regulated during the process of transfering root explants to tissue culture.
Overexpression of the lemon basil α‐zingiberene synthase gene increases both mono‐ and sesquiterpene contents in tomato fruit
Authors: Davidovich‐Rikanati Rachel , Lewinsohn Efraim , Bar Einat , Iijima Yoko , Pichersky Eran , Sitrit Yaron
Published: July 2008

α‐Zingiberene synthase (ZIS), a sesquiterpene synthase gene that was isolated from lemon basil (Ocimum basilicum L.), encodes an enzyme that catalyzes the formation of α‐zingiberene, and other sesquiterpenes, from farnesyl diphosphate. Transgenic tomato fruits overexpressing ZIS under the control of the fruit ripening‐specific tomato polygalacturonase promoter (PG) accumulated high levels of α‐zingiberene (224–1000 ng g−1 fresh weight) and other sesquiterpenes, such as α‐bergamotene, 7‐epi‐sesquithujene, β‐bisabolene and β‐curcumene, whereas no sesquiterpenes were detected in non‐transformed control fruits. The ZIS‐transgenic fruits also produced monoterpenes, such as α‐thujene, α‐pinene, β‐phellandrene and γ‐terpinene (1–22 ng g−1 fresh weight), which were either not detected or were found only in minute concentrations in control fruits. Recombinant ZIS overexpressed in Escherichia coli catalyzed the formation of these monoterpenes from geranyl diphosphate. As the ZIS protein apparently lacks a transit peptide, and is localized in the cytosol, the production of monoterpenes in the transgenic tomatoes suggests that a pool of geranyl diphosphate is available in the cytosol. The phenotype of the ZIS‐transgenic tomatoes was the same as that for wild‐type tomatoes, with regard to plant vigor and shape, but transgenic plants exhibited a small decrease in lycopene content. This study thus showed that the synthesis of both mono‐ and sesquiterpenes can be enhanced by the ectopic expression of a single transgene in tomato fruit, and it further demonstrated the interconnection between the pools of terpenoid precursors in the plastids and the cytosol.
Dynamics of RpaB–promoter interaction during high light stress, revealed by chromatin immunoprecipitation (ChIP) analysis in Synechococcus elongatus PCC 7942
Authors: Hanaoka Mitsumasa , Tanaka Kan
Published: July 2008

In cyanobacteria, a series of genes are induced by, and cause tolerance to, high light stress conditions. Some of these genes share a short, repeated sequence motif known as a high light regulatory 1 (HLR1) element in their promoter regions. Previously, RpaB, a two‐component response regulator, was shown to interact with the HLR1 element of several high light‐responsive promoters in vitro. However, how RpaB regulates target promoters in vivo remained elusive. In this study, we analyzed the role of RpaB in transcriptional regulation of high light‐responsive genes by chromatin immunoprecipitation (ChIP) analysis, which has been recently developed and utilized to study in vivo interactions between DNA‐binding proteins and the relevant target DNA. One of the advantages of this method is the ability to detect dynamic interaction patterns in response to various growth and/or environmental conditions instantaneously at the time of the analysis. Here we examined the binding patterns of RpaB under various light conditions using ChIP assays. We found that strong interactions of RpaB with target promoters were weakened in a high light‐dependent manner, and that the lower binding level of RpaB continued as long as the high light conditions were maintained. Thus, in regulation of high light‐inducible genes, we suggest that RpaB functions as a repressor under normal light conditions, and that high light conditions result in release of the repression.
Tissue‐dependent limited pleiotropy affects gene expression in barley
Authors: Potokina Elena , Druka Arnis , Luo Zewei , Moscou Matthew , Wise Roger , Waugh Robbie , Kearsey Mike
Published: July 2008

Non‐synonymous coding mutations in a gene change the resulting protein, no matter where it is expressed, but the effects of cis‐regulatory mutations could be spatially or temporally limited – a phenomenon termed limited pleiotropy. Here, we report the genome‐wide occurrence of limited pleiotropy of cis‐regulatory mutations in barley (Hordeum vulgare L.) using Affymetrix analysis of 22 840 genes in a population of 139 doubled haploid lines derived from a cross between the cultivars Steptoe (St) and Morex (Mx). We identified robust cis‐acting expression regulators that segregate as major genes in two successive ontogenetic stages: germinating embryo tissues and seedling leaves from the embryonic axis. We show that these polymorphisms may be consistent in both tissues or may cause a dramatic change in transcript abundance in one tissue but not in another. We also show that the parental allele that increases expression can vary with the tissue, suggesting nucleotide polymorphism in enhancer sequences. Because of the limited pleiotropy of cis‐regulating mutations, the number of cis expression quantitative trait loci (cis‐eQTLs) discovered by ‘genetical genomics’ is strongly affected by the particular tissue or developmental stage studied. Given that limited pleiotropy is a common feature of cis‐regulatory mutations in barley, we predict that the phenomenon would be relevant to developmental and/or tissue‐specific interactions across wide taxonomic boundaries in both plants and animals.
Functional gene‐mining for salt‐tolerance genes with the power of Arabidopsis
Authors: Du Jin , Huang Yue‐Ping , Xi Jing , Cao Min‐Jie , Ni Wan‐Song , Chen Xi , Zhu Jian‐Kang , Oliver David J. , Xiang Cheng‐Bin
Published: July 2008

Here we report on a functional gene‐mining method developed to isolate stress tolerance genes without any prior knowledge of the genome or genetic mapping of the source germplasms. The feasibility of this approach was demonstrated by isolating novel salt stress tolerance genes from salt cress (Thellungiella halophila), an extremophile that is adapted to a harsh saline environment and a close relative of the model plant Arabidopsis thaliana. This gene‐mining method is based on the expression of salt cress cDNA libraries in Arabidopsis. A cDNA expression library of the source germplasm, salt cress, was constructed and used to transform Arabidopsis via Agrobacterium‐mediated gene transfer. A transgenic seed library consisting of >125 000 independent lines was generated and screened for salt‐tolerant lines via a high‐throughput genetic screen. A number of salt‐tolerant lines were isolated, and the salt cress cDNAs were identified by PCR amplification and sequencing. Among the genes isolated, several novel small protein‐encoding genes were discovered. The homologs of these genes in Arabidopsis have not been experimentally analyzed, and their functions remain unknown. The function of two genes isolated by this method, ST6‐66 and ST225, and their Arabidopsis homologs, were investigated in Arabidopsis using gain‐ and loss‐of‐function analyses, and their importance in salt tolerance was demonstrated. Thus, our functional gene‐mining method was validated by these results. Our method should be applicable for the functional mining of stress tolerance genes from various germplasms. Future improvements of the method are also discussed.
Overexpression of a fatty acid amide hydrolase compromises innate immunity in Arabidopsis
Authors: Kang Li , Wang Yuh‐Shuh , Uppalapati Srinivasa Rao , Wang Keri , Tang Yuhong , Vadapalli Vatsala , Venables Barney J. , Chapman Kent D. , Blancaflor Elison B. , Mysore Kirankumar S.
Published: July 2008

N‐acylethanolamines are a group of lipid mediators that accumulate under a variety of neurological and pathological conditions in mammals. N‐acylethanolamine signaling is terminated by the action of diverse hydrolases, among which fatty acid amide hydrolase (FAAH) has been well characterized. Here, we show that transgenic Arabidopsis lines overexpressing an AtFAAH are more susceptible to the bacterial pathogens Pseudomonas syringae pv. tomato and P. syringae pv. maculicola. AtFAAH overexpressors also were highly susceptible to non‐host pathogens P. syringae pv. syringae and P. syringae pv. tabaci. AtFAAH overexpressors had lower amounts of jasmonic acid, abscisic acid and both free and conjugated salicylic acid (SA), compared with the wild‐type. Gene expression studies revealed that transcripts of a number of plant defense genes, as well as genes involved in SA biosynthesis and signaling, were lower in AtFAAH overexpressors than wild‐type plants. Our data suggest that FAAH overexpression alters phytohormone accumulation and signaling which in turn compromises innate immunity to bacterial pathogens.
HAHB4, a sunflower HD‐Zip protein, integrates signals from the jasmonic acid and ethylene pathways during wounding and biotic stress responses
Authors: Manavella Pablo A. , Dezar Carlos A. , Bonaventure Gustavo , Baldwin Ian T. , Chan Raquel L.
Published: July 2008

The Helianthus annuus (sunflower) HAHB4 transcription factor belongs to the HD‐Zip family and its transcript levels are strongly induced when sunflower plants are attacked by herbivores, mechanically damaged or treated with methyl‐jasmonic acid (MeJA) or ethylene (ET). Promoter fusion analysis, in Arabidopsis and in sunflower, demonstrated that induction of HAHB4 expression by these treatments is regulated at the transcriptional level. In transiently transformed sunflower plants HAHB4 expression upregulates the transcript levels of several genes involved in JA biosynthesis and defense‐related processes such as the production of green leaf volatiles and trypsin protease inhibitors (TPI). In HAHB4 sunflower overexpressing tissue, increased activities of lipoxygenase, hydroperoxide lyase and TPI are detected whereas in HAHB4‐silenced tissue these activities are reduced. Transgenic Arabidopsis thaliana and Zea mays plants ecotopically expressing HAHB4 also exhibit higher transcript levels of defense‐related genes and when Spodoptera littoralis or Spodoptera frugiperda larvae are placed on each species, respectively, larvae consumed less and gain less mass compared with larvae feeding on control plants. Arabidopsis plants ectopically expressing HAHB4 had higher amounts of JA, JA‐isoleucine and ET compared with control plants both before and after wounding, but reduced levels of salicylic acid (SA) after wounding and bacterial infection. We conclude that HAHB4 coordinates the production of phytohormones during biotic stress responses and mechanical damage, specifically by positively regulating JA and ET production and negatively regulating ET sensitivity and SA accumulation.
Molecular basis of the functional specificities of phototropin 1 and 2
Authors: Aihara Yusuke , Tabata Ryohei , Suzuki Tomomi , Shimazaki Ken‐ichiro , Nagatani Akira
Published: July 2008

A blue‐light photoreceptor in plants, phototropin, mediates phototropism, chloroplast relocation, stomatal opening, and leaf‐flattening responses. Phototropin is divided into two functional moieties, the N‐terminal photosensory and the C‐terminal signaling moieties. Phototropin perceives light stimuli by the light, oxygen or voltage (LOV) domain in the N‐terminus; the signal is then transduced intramolecularly to the C‐terminal kinase domain. Two phototropins, phot1 and phot2, which have overlapping and distinct functions, exist in Arabidopsis thaliana. Phot1 mediates responses with higher sensitivity than phot2. Phot2 mediates specific responses, such as the chloroplast avoidance response and chloroplast dark positioning. To elucidate the molecular basis for the functional specificities of phot1 and phot2, we exchanged the N‐ and C‐terminal moieties of phot1 and phot2, fused them to GFP and expressed them under the PHOT2 promoter in the phot1 phot2 mutant background. With respect to phototropism and other responses, the chimeric phototropin consisting of phot1 N‐terminal and phot2 C‐terminal moieties (P1n/2cG) was almost as sensitive as phot1; whereas the reverse combination (P2n/1cG) functioned with lower sensitivity. Hence, the N‐terminal moiety mainly determined the sensitivity of the phototropins. Unexpectedly, both P1n/2cG and P2n/1cG mediated the chloroplast avoidance response, which is specific to phot2. Hence, chloroplast avoidance activity appeared to be suppressed specifically in the combination of N‐ and C‐terminal moieties of phot1. Unlike the chloroplast avoidance response, chloroplast dark positioning was observed for P2G and P2n/1cG but not for P1G or P1n/2cG, suggesting that a specific structure in the N‐terminal moiety of phot2 is required for this activity.
Genome‐scale Arabidopsis promoter array identifies targets of the histone acetyltransferase GCN5
Authors: Benhamed Moussa , Martin‐Magniette Marie‐Laure , Taconnat Ludivine , Bitton Frédérique , Servet Caroline , De Clercq Rebecca , De Meyer Björn , Buysschaert Caroline , Rombauts Stéphane , Villarroel Raimundo , Aubourg Sébastien , Beynon Jim , Bhalerao Rishikesh P. , Coupland George , Gruissem Wilhelm , Menke Frank L.H. , Weisshaar Bernd , Renou Jean‐Pierre , Zhou Dao‐Xiu , Hilson Pierre
Published: July 2008

We have assembled approximately 20 000 Arabidopsis thaliana promoter regions, compatible with functional studies that require cloning and with microarray applications. The promoter fragments can be captured as modular entry clones (MultiSite Gateway format) via site‐specific recombinational cloning, and transferred into vectors of choice to investigate transcriptional networks. The fragments can also be amplified by PCR and printed on glass arrays. In combination with immunoprecipitation of protein–DNA complexes (ChIP–chip), these arrays enable characterization of binding sites for chromatin‐associated proteins or the extent of chromatin modifications at genome scale. The Arabidopsis histone acetyltransferase GCN5 associated with 40% of the tested promoters. At most sites, binding did not depend on the integrity of the GCN5 bromodomain. However, the presence of the bromodomain was necessary for binding to 11% of the promoter regions, and correlated with acetylation of lysine 14 of histone H3 in these promoters. Combined analysis of ChIP–chip and transcriptomic data indicated that binding of GCN5 does not strictly correlate with gene activation. GCN5 has previously been shown to be required for light‐regulated gene expression and growth, and we found that GCN5 targets were enriched in early light‐responsive genes. Thus, in addition to its transcriptional activation function, GCN5 may play an important role in priming activation of inducible genes under non‐induced conditions.
Arabinogalactan proteins 6 and 11 are required for stamen and pollen function in Arabidopsis
Authors: Levitin Bella , Richter Dganit , Markovich Inbal , Zik Moriyah
Published: July 2008

Successful male reproductive function in plants is dependent on the correct development and functioning of stamens and pollen. AGP6 and AGP11 are two homologous Arabidopsis genes encoding cell wall‐associated arabinogalactan glycoproteins (AGPs). Both genes were found to be specifically expressed in stamens, pollen grains and pollen tubes, suggesting that these genes may play a role in male organ development and function. RNAi lines with reduced AGP6 and AGP11 expression were generated. These, together with lines harboring point mutations in the coding region of AGP6, were used to show that loss of function in AGP6 and AGP11 led to reduced fertility, at least partly as a result of inhibition of pollen tube growth. Our results also suggest that AGP6 and AGP11 play an additional role in the release of pollen grains from the mature anther. Thus, our study demonstrates the involvement of specific AGPs in pollen tube growth and stamen function.
Getting the most out of publicly available T‐DNA insertion lines
Authors: Ülker Bekir , Peiter Edgar , Dixon David P. , Moffat Caroline , Capper Richard , Bouché Nicolas , Edwards Robert , Sanders Dale , Knight Heather , Knight Marc R.
Published: July 2008

In the course of several different projects, we came to realize that there is a significant amount of untapped potential in the publicly available T‐DNA insertion lines. In addition to the GABI‐Kat lines, which were designed specifically for activation tagging, lines from the SAIL and FLAGdb collections are also useful for this purpose. As well as the 35S promoter chosen for activation tagging in GABI‐Kat lines, we found that the 1′2′ bidirectional promoter is capable of activating expression of flanking genomic sequences in both GABI‐Kat and SAIL lines. Thus these lines have added potential for activation tagging. We also show that these lines are capable of generating antisense transcripts and so have the potential to be used for suppression (loss/reduction of function) studies. By virtue of weak terminator sequences in some T‐DNA constructs, transcript read‐through from selectable markers is also possible, which again has the potential to be exploited in activation/suppression studies. Finally, we show that, by selecting and characterizing lines in which the T‐DNA insertions are present specifically within introns of a target gene, an allelic series of mutants with varying levels of reduced expression can be generated, due to differences in efficiency of intron splicing. Taken together, our analyses demonstrate that there is a wealth of untapped potential within existing insertion lines for studies on gene function, and the effective exploitation of these resources is discussed.
Quantitative analysis of heterogeneous spatial distribution of Arabidopsis leaf trichomes using micro X‐ray computed tomography
Authors: Kaminuma Eli , Yoshizumi Takeshi , Wada Takuji , Matsui Minami , Toyoda Tetsuro
Published: July 2008

Quantitative morphological traits may be defined based on the 3D anatomy reconstructed from micro X‐ray computed tomography (μCT) images. In this study, the heterogeneous spatial distribution of trichomes (hairs) on the adaxial leaf blade surface in Arabidopsis was evaluated in terms of 3D quantitative traits, including trichome number, average nearest‐neighbour distance between trichomes, and proportion of large trichomes. The data reflect spatial heterogeneity in the radial direction, in that a greater number of trichomes were observed on the leaf blade margins relative to the non‐margins, a distribution effect caused by the CAPRICE (CPC) and GLABRA3 (GL3) genes, which have previously been shown to affect trichome density. We further determined that the proportion of large trichomes on the blade mid‐rib increases from the proximal end to the distal leaf tip in both wild‐type plants and GL3 mutants. Our results indicate that the GL3 gene affects trichome distribution, rather than trichome growth, causing trichome initiation at the proximal base rather than the distal tip. On the other hand, CPC does affect trichome growth and developmental progression. Hence, quantitative phenotyping based on μCT enables precise phenotypic description for elucidation of gene control in morphological mutants.
Exploring the nuclear proteome ofMedicago truncatulaat the switch towards seed filling
Authors: Repetto Ombretta , Rogniaux Hélène , Firnhaber Christian , Zuber Hélène , Küster Helge , Larré Colette , Thompson Richard , Gallardo Karine
Published: July 2008

Despite its importance in determining seed composition, and hence quality, regulation of the development of legume seeds is incompletely understood. Because of the cardinal role played by the nucleus in gene expression and regulation, we have characterized the nuclear proteome ofMedicago truncatulaat the 12 days after pollination (dap) stage that marks the switch towards seed filling. Nano‐liquid chromatography–tandem mass spectrometry analysis of nuclear protein bands excised from one‐dimensional SDS‐PAGE identified 179 polypeptides (143 different proteins), providing an insight into the complexity and distinctive feature of the seed nuclear proteome and highlighting new plant nuclear proteins with possible roles in the biogenesis of ribosomal subunits (PESCADILLO‐like) or nucleocytoplasmic trafficking (dynamin‐like GTPase). The results revealed that nuclei of 12‐dap seeds store a pool of ribosomal proteins in preparation for intense protein synthesis activity, occurring subsequently during seed filling. Diverse proteins of the molecular machinery leading to the synthesis of ribosomal subunits were identified along with proteins involved in transcriptional regulation, RNA processing or transport. Some had already been shown to play a role during the early stages of seed formation whereas for others the findings are novel (e.g. the DIP2 and ES43 transcriptional regulators or the RNA silencing‐related ARGONAUTE proteins). This study also revealed the presence of chromatin‐modifying enzymes and RNA interference proteins that have roles in RNA‐directed DNA methylation and may be involved in modifying genome architecture and accessibility during seed filling and maturation.
A new method for isolating large quantities of Arabidopsis trichomes for transcriptome, cell wall and other types of analyses
Authors: Marks M. David , Betancur Lissete , Gilding Edward , Chen Fang , Bauer Stefan , Wenger Jonathan P. , Dixon Richard A. , Haigler Candace H.
Published: July 2008

A new procedure has been developed for the isolation of wild‐type and mutant Arabidopsis trichomes. The isolated trichomes maintained enzymatic activity and were used for DNA, protein, and RNA isolation. The RNA was used to generate probes suitable for Affymetrix analysis. The validity of the Affymetrix results was confirmed by quantitative PCR analysis on a subset of genes that are preferentially expressed in trichomes or leaves. Sufficient quantities of trichomes were isolated to probe the biochemical nature of trichome cell walls. These analyses provide evidence for the presence of lignin in Arabidopsis trichome cell walls. The monosaccharide analysis and positive staining with ruthenium red indicates that the walls also contain a large portion of pectin. The 2.23‐fold ratio of pectin‐related sugars compared with potential cellulosic glucose suggests that the polysaccharides of the trichome cell walls are more like those of typical primary walls even though the wall becomes quite thick. Overall, these analyses open the door to using the Arabidopsis trichome cell wall as an excellent model to probe various questions concerning plant cell wall biosynthesis.
Multicolor bimolecular fluorescence complementation reveals simultaneous formation of alternative CBL/CIPK complexes in planta
Authors: Waadt Rainer , Schmidt Lena K. , Lohse Marc , Hashimoto Kenji , Bock Ralph , Kudla Jörg
Published: July 2008

The specificity of intracellular signaling and developmental patterning in biological systems relies on selective interactions between different proteins in specific cellular compartments. The identification of such protein–protein interactions is essential for unraveling complex signaling and regulatory networks. Recently, bimolecular fluorescence complementation (BiFC) has emerged as a powerful technique for the efficient detection of protein interactions in their native subcellular localization. Here we report significant technical advances in the methodology of plant BiFC. We describe a series of versatile BiFC vector sets that are fully compatible with previously generated vectors. The new vectors enable the generation of both C‐terminal and N‐terminal fusion proteins and carry optimized fluorescent protein genes that considerably improve the sensitivity of BiFC. Using these vectors, we describe a multicolor BiFC (mcBiFC) approach for the simultaneous visualization of multiple protein interactions in the same cell. Application to a protein interaction network acting in calcium‐mediated signal transduction revealed the concurrent interaction of the protein kinase CIPK24 with the calcium sensors CBL1 and CBL10 at the plasma membrane and tonoplast, respectively. We have also visualized by mcBiFC the simultaneous formation of CBL1/CIPK1 and CBL9/CIPK1 protein complexes at the plasma membrane. Thus, mcBiFC provides a useful new tool for exploring complex regulatory networks in plants.
New visions for basic research and primary prevention of pediatric allergy: An iPAC summary and future trends
Authors: Hamelmann Eckard , Herz Udo , Holt Pat , Host Arne , Lauener Roger P. , Matricardi Paolo M. , Wahn Ulrich , Wickman Magnus
Published: July 2008

Hydrolyzed formula feeding, delayed introduction of solid food, indoor allergen avoidance, smoke and pollutants avoidance have been applied for several decades as primary preventive measures for allergic diseases. Unfortunately, some of these strategies have had no or modest success. Therefore, resources need to be focused on better understanding of the early allergic events and on interventional studies to investigate new strategies of primary and secondary prevention. Accordingly, this review summarizes the state‐of‐the‐art of genetic, immunological and clinical aspects of primary prevention of allergic diseases. Studies investigating gene‐by‐gene and gene‐by‐environment interactions suggest that prevention of allergic diseases must be tailored to the individual genetic susceptibilities (‘gene profiling’) and environmental exposures. The expanding knowledge on new T cell populations (Th17, TSLP (thymic stromal derived lymphopoietin)‐dependent ‘inflammatory Th2 cells’) is also inspiring new concepts on the origins of allergic diseases. The old concept of ‘blocking immunoglobulin G antibodies’ has been re‐appraised and it is likely to generate novel preventive and therapeutic strategies. The major task for future clinical research is to clearly define the timing of optimal exposure to potential allergens. In addition, the role of microbial products such as certain bacteria, or their components, and of helminths or their larvae at different times in early life, alone or with potential allergens, definitely need to be further investigated. The benefit of efficient allergy prevention, based on focusing resources on novel and promising research lines, will be of prime importance to both affluent countries and other parts of the world where allergy is only currently emerging.
Differences in incidence of suicide attempts during phases of bipolar I and II disorders
Authors: Valtonen Hanna M , Suominen Kirsi , Haukka Jari , Mantere Outi , Leppämäki Sami , Arvilommi Petri , Isometsä Erkki T
Published: July 2008

Background:  Differences in the incidence of suicide attempts during various phases of bipolar disorder (BD), or the relative importance of static versus time‐varying risk factors for overall risk for suicide attempts, are unknown.

Methods:  We investigated the incidence of suicide attempts in different phases of BD as a part of the Jorvi Bipolar Study (JoBS), a naturalistic, prospective, 18‐month study representing psychiatric in‐ and outpatients with DSM‐IV BD in three Finnish cities. Life charts were used to classify time spent in follow‐up in the different phases of illness among the 81 BD I and 95 BD II patients.

Results:  Compared to the other phases of the illness, the incidence of suicide attempts was 37‐fold higher [95% confidence interval (CI) for relative risk (RR): 11.8–120.3] during combined mixed and depressive mixed states, and 18‐fold higher (95% CI: 6.5–50.8) during major depressive phases. In Cox’s proportional hazards regression models, combined mixed (mixed or depressive mixed) or major depressive phases and prior suicide attempts independently predicted suicide attempts. No other factor significantly modified the risks related to these time‐varying risk factors; their population‐attributable fraction was 86%.

Conclusions:  The incidence of suicide attempts varies remarkably between illness phases, with mixed and depressive phases involving the highest risk by time. Time spent in high‐risk illness phases is likely the major determinant of overall risk for suicide attempts among BD patients. Studies of suicidal behavior should investigate the role of both static and time‐varying risk factors in overall risk; clinically, management of mixed and depressive phases may be crucial in reducing risk.
Quality of life and its association with insight, adverse effects of medication and use of atypical antipsychotics in patients with bipolar disorder and schizophrenia in remission
Authors: Yen Cheng‐Fang , Cheng Chung‐Ping , Huang Chi‐Fen , Yen Ju‐Yu , Ko Chih‐Hung , Chen Cheng‐Sheng
Published: July 2008

Objective:  The present study aimed: (i) to compare the level of quality of life (QOL) among subjects with bipolar disorder (BD) and schizophrenia who were in remission and healthy control subjects and (ii) to examine the association of QOL with insight, adverse effects of medication and use of atypical antipsychotics among subjects with BD and schizophrenia who were in remission by controlling other confounding factors.

Methods:  The QOL on the four domains of the World Health Organization Questionnaire on Quality of Life: Short Form – Taiwan version (WHOQOL‐BREF) were compared between 96 subjects with BD in remission, 96 subjects with schizophrenia in remission and 106 healthy control subjects. The association between the four QOL domains and subjects’ insight, adverse effects of medication and use of atypical antipsychotics were examined using multiple regression analyses in the subjects with BD and schizophrenia in remission.

Results:  The results demonstrated that the subjects with BD in remission had similarly poor levels of QOL in all four domains as those subjects with schizophrenia in remission, and both subjects with BD and schizophrenia had poorer QOL than those in the control group. For both subjects with BD and schizophrenia in remission, insight was negatively associated with QOL on the physical domain, and adverse effects of medication were negatively associated with QOL on the physical and environment domains. Use of atypical antipsychotics was not associated with QOL, but subjects with BD receiving olanzapine perceived better psychological QOL than those receiving risperidone and better psychological and social relationship QOL than those receiving no atypical antipsychotic.

Conclusions:  The results of the present study indicate that subjects with BD are dissatisfied with their QOL, even when they are in a remitted state. Clinicians must consider the negative influences of insight and adverse effects of medication on QOL of patients with BD and schizophrenia in remission.
Gender influences the detection of spatial working memory deficits in bipolar disorder
Authors: Barrett Suzanne Lucia , Kelly Christopher , Bell Robert , King David John
Published: July 2008

Objective:  Despite evidence that gender may influence neurocognitive functioning, few studies have examined its effects in bipolar disorder (BD) a priori. The aim of this study was to examine how gender influences executive‐type functions, which are potentially useful as endophenotypes for BD.

Methods:  The performance of 26 euthymic patients (12 males, 14 females) with DSM‐IV BD (20 BD type I and six BD type II) was compared to that of 26 controls (12 males, 14 females) on tests of executive function. Controls were matched to patients on an individual basis for sex, age and premorbid IQ. Tests assessed spatial working memory (SWM), planning, attentional set‐shifting and verbal fluency.

Results:  Overall, patients showed deficits in SWM strategy (p < 0.001) and made more SWM errors relative to controls (p < 0.001). These deficits were more apparent in male‐only comparisons (both p < 0.001) than in female‐only comparisons (both p < 0.05). When examined in isolation, male controls were significantly better at performing the SWM task than female controls (both p < 0.05). This pattern was not observed in the patient cohort: male patients had poorer strategy scores than female patients (p < 0.05), but made a similar number of SWM errors.

Conclusions:  These findings provide evidence that gender can influence the detection of SWM deficits in the euthymic phase of BD, as the sex‐related disequilibrium in SWM identified in healthy controls was disrupted in BD.
Metabolic syndrome in bipolar disorder: a cross‐sectional assessment of a Health Management Organization database
Authors: Sicras Antoni , Rejas Javier , Navarro Ruth , Serrat Josep , Blanca Milagrosa
Published: July 2008

Objective:  To estimate the prevalence of metabolic syndrome (MS) in patients with bipolar disorder (BD) included in a Health Management Organization (HMO) database.

Methods:  A cross‐sectional analysis of the administrative claim database of Badalona Serveis Assistencials (BSA) was performed. All patients of either sex over 16 years of age and receiving treatment for BD for more than three weeks were included in the study group. The reference group comprised the rest of patients in the BSA database without BD. MS was defined according to the National Cholesterol Education Program (NCEP) Adult Treatment Panel (ATP) III modified criteria and required fulfillment of at least three of the following five components: body mass index (BMI) ≥28.8 kg/m2, triglycerides ≥150 mg/dL, high‐density lipoprotein (HDL) cholesterol <40 mg/dL (males)/<50 mg/dL (females), blood pressure ≥130/85 mmHg, and fasting glucose ≥110 mg/dL. Descriptive statistics, bivariate analysis, and logistic regression models were applied.

Results:  We identified 178 patients with BD out of 86,028 subjects (50.5% women; 45.5 ± 17.8 years, mean ± SD) included in the BSA database. MS prevalence was significantly higher in BD: 24.7% [95% confidence interval (CI): 18.6–31.7] versus 14.4% (CI: 14.2–14.7) with no statistically significant differences between genders; age‐adjusted odds ratio (OR) = 1.65 (1.11–2.44, p = 0.013). All MS components were higher in the BD group, particularly BMI >28.8 kg/m2 [33.1% (26.3–40.6) versus 17.9% (17.7–18.2), adjusted OR = 2.05 (1.46–2.87, p < 0.001)], high triglyceride levels [23.0% (17.1–29.9) versus 11.3% (11.1–11.5), adjusted OR = 2.09 (1.45–3.02, p < 0.001)], and low HDL cholesterol levels [54.5% (46.9–62.0) versus 29.4% (29.1–29.7), adjusted OR = 2.77 (2.02–3.80, p < 0.001)]. Furthermore, patients with BD showed a significantly higher frequency of obesity [41.4% (32.3–50.9) versus 27.1% (26.6–27.5); adjusted OR = 1.83 (1.24–2.68, p = 0.002)].

Conclusions:  Compared with the general population managed by the BSA, the prevalence of MS was significantly higher in patients with BD, mainly due to a higher prevalence of obesity, high triglyceride levels, and low HDL cholesterol levels. These findings strongly support the development of health policies addressing this problem in BD patients.
Rapid switching of mood in families with familial bipolar disorder
Authors: Nwulia Evaristus A , Zandi Peter P , McInnis Melvin G , Raymond DePaulo Jr J , MacKinnon Dean F
Published: July 2008

Objective: Rapid switching of moods in bipolar disorder has been associated with early age at onset, panic comorbidity, and suicidality. This study aims to confirm these associations and investigate other potential correlates of rapid switching of mood using families from a multisite bipolar linkage study.

Methods: The subjects were comprised of 1,143 probands and relatives with diagnosis of bipolar disorder. All subjects were interviewed directly with a standard diagnostic instrument, and all subjects who met criteria for bipolar disorder were asked if their moods had ever switched rapidly.

Results: Individuals with rapid mood switching had significantly earlier age at onset (18 versus 21 years, p < 0.00001), higher comorbid anxiety (47% versus 26%, p < 0.00001) and substance use disorders (52% versus 42%, p = 0.0006), higher rate of violent behavior (6% versus 3%, p < 0.004), suicidal behavior (46% versus 31%, p < 0.00001), and nonsuicidal self‐harm (13% versus 6%, p < 0.0002). Multiple logistic regression analysis found significant net effects on rapid mood switching for early emergence of symptoms [odds ratio (OR) = 0.62; 95% confidence interval (CI): 0.45–0.85]; anxiety comorbidity (OR = 2.31; 95% CI: 1.34–3.98); and hypersensitivity to antidepressants (OR = 2.05; 95% CI: 1.49–2.83) as the strongest predictors.

Conclusions: This confirms earlier reports associating rapid switching with a more complex clinical course, in particular early emergence of bipolar symptomatology, antidepressant activation, and anxiety comorbidity. These results support a clinical differentiation of bipolar disorder into subtypes based on symptom stability.
Association between brain‐derived neurotrophic factor gene and a severe form of bipolar disorder, but no interaction with the serotonin transporter gene
Authors: Vincze Ilona , Perroud Nader , Buresi Catherine , Baud Patrick , Bellivier Frank , Etain Bruno , Fournier Claire , Karege Felicien , Matthey Marie‐Louise , Preisig Martin , Leboyer Marion , Malafosse Alain
Published: July 2008

Background:  Recent data suggest that brain‐derived neurotrophic factor (BDNF) and the serotonergic system are involved and interact in major depressive disorder and suicidal behavior (SB). Several family and population‐based studies have reported associations between the BDNF gene and serotonin‐related genes, specifically the serotonin transporter (5HTT) gene, with bipolar disorder (BD) and SB. However, despite the fact that gene‐by‐gene interaction between BDNF and 5HTT has been demonstrated in monoamine deficiencies in animals, this kind of interaction has never been tested in humans. Our hypothesis is that some BDNF and 5HTT polymorphisms might confer increased risk for BD and SB and that both genes may interact with each other.

Methods:  To test this hypothesis, we genotyped the most common BDNF polymorphisms, G196A (Val66Met), A‐633T and BDNF‐LCPR, as well as 5HTT (5HTT‐LPR), in 447 BD patients and 370 controls.

Results:  We replicated the association previously reported between BDNF G196A (Val66Met) polymorphism and BD. We also observed a correlation between the number of G196 alleles and short alleles of 5HTT‐LPR and the severity of SB in BD. However, we found no significant interaction between these two markers.

Conclusions:  These results suggest that BDNF G196A as well as 5HTT‐LPR polymorphisms confer risk for SB in BD, but we did not observe any evidence for an interaction between them.
Quality of life in bipolar disorder patients: a comparison with a general population sample
Authors: Gutiérrez‐Rojas Luis , Gurpegui Manuel , Ayuso‐Mateos José L , Gutiérrez‐Ariza José A , Ruiz‐Veguilla Miguel , Jurado Dolores
Published: July 2008

Objectives:  To compare the Quality of Life (QoL) of bipolar disorder (BD) patients with that of the general population; and, within the BD patients, to find the demographic and clinical variables associated with low QoL, controlling for the effects of potential confounders.

Methods:  Based on the 25th percentile of the physical (PCS) and the mental (MCS) component scores (PCS <53 and MCS < 50, respectively) of the Medical Outcomes Survey 36‐item Short‐Form Health‐Survey (SF‐36) of a general population representative sample (n = 1,210), we compared by logistic regression the QoL of 48 euthymic and 60 non‐euthymic BD outpatients and the general population. Within BD patients, we analyzed the clinical and course‐of‐illness variables associated with low physical and mental QoL, including manic and depressive symptoms and consumption of addictive substances; in addition, we calculated the partial correlation of the different variables with the dimensional PCS and MCS through multiple linear regression.

Results:  Low physical QoL was significantly more frequent among both euthymic [odds ratio (OR) = 3.5; 95% confidence interval (CI): 1.9–6.5] and non‐euthymic (OR = 4.0; 95% CI: 2.3–7.0) BD patients than in the general population; the respective values for low mental QoL were OR = 2.2; 95% CI: 1.2–4.0 and OR = 8.5; 95% CI: 4.6–15.7. Low mental QoL was more frequent among non‐euthymic than euthymic BD patients (OR = 3.9; 95% CI: 1.6–9.1). Within BD patients, low mental QoL was associated with the length of illness (or early onset), the presence of depressive symptoms, nicotine dependence and the lack of social support.

Conclusions:  Among the BD patients, who experience lower physical and mental QoL even in a euthymic period, the optimal control of depressive symptoms as well as the availability of social support may enhance their well‐being.
Appreciation of research information in patients with bipolar disorder
Authors: Misra Sahana , Socherman Robert , Hauser Peter , Ganzini Linda
Published: July 2008

Objective:  Ethicists have debated whether patients with serious mental illness can appreciate the risks of research participation and make autonomous decisions. We compared the abilities of euthymic and manic bipolar patients to appreciate and make voluntary decisions regarding research participation.

Methods:  Twenty‐six subjects with mania and 25 euthymic subjects reviewed hypothetical consent forms for three research studies of varying risk. We assessed subjects’ appreciation of: their diagnosis and need for treatment; the researcher’s role; the risks of participation; and the degree of influence of family, the treating clinician, and payment on decisions to participate.

Results:  Most subjects (92%) agreed they had bipolar disorder requiring medication treatment. Subjects were less likely to participate in riskier studies. About half of subjects erroneously believed that researchers would make decisions based solely on what would be the best care for them (therapeutic misconception); and in randomized medication trials, they mistakenly believed they had improved chances of receiving one treatment over another. There were no differences between mood groups on these measures. Over half of subjects (59%) indicated that their mental health provider might influence them to participate in a study even when they did not want to, but most rejected a role for family in decision making. Payment was rated as having little impact on decisions to participate in research.

Conclusion:  Mania does not substantially influence appreciation of research participation. Subjects with bipolar disorder, regardless of mood state, are at risk for therapeutic misconception and optimistic bias. Special protections may be needed when mental health professionals approach their own patients to participate in research.