bluuebunny/crossref_metadata_2025 huggingface dataset:
roundabout4
is essential for angiogenesis
in vivo
Authors: Bedell Victoria M. , Yeo Sang-Yeob , Park Kye Won , Chung Jeffrey , Seth Pankaj , Shivalingappa Venkatesha , Zhao Jinhua , Obara Tomoko , Sukhatme Vikas P. , Drummond Iain A. , Li Dean Y. , Ramchandran Ramani
Published: April 2005

Stereotypical patterns of vascular and neuronal networks suggest that specific genetic programs tightly control path determination and, consequently, angiogenesis and axon-guidance mechanisms. Our study focuses on one member of the roundabout family of receptors, which traditionally mediate repulsion from the midline. Here, we characterize a fourth member of this family,
roundabout4
(
robo4
), which is the predominant roundabout (robo) that is expressed in embryonic zebrafish vasculature. Gene knockdown and overexpression approaches show that
robo4
is essential for coordinated symmetric and directed sprouting of intersomitic vessels and provide mechanistic insights into this process. Also, human
robo4
gene functionally compensates for loss of
robo4
gene function, suggesting evolutionary conservation. This article reports an endothelial-specific function for a robo gene in vertebrates
in vivo
.
Localized glucose and water influx facilitatesCryptosporidium parvumcellular invasion by means of modulation of host-cell membrane protrusion
Authors: Chen Xian-Ming , O'Hara Steven P. , Huang Bing Q. , Splinter Patrick L. , Nelson Jeremy B. , LaRusso Nicholas F.
Published: April 2005

Dynamic membrane protrusions such as lamellipodia and filopodia are driven by actin polymerization and often hijacked by intracellular microbes to enter host cells. The overall rate of membrane protrusion depends on the actin polymerization rate and the increase of localized cell volume. Although the signaling pathways involving actin polymerization are well characterized, the molecular mechanisms regulating local cell volume associated with membrane protrusion are unclear.Cryptosporidium parvum, an intracellular parasite, depends on host-cell membrane protrusion to accomplish cell entry and form the parasitophorous vacuole. Here, we report thatC. parvuminfection of cholangiocytes recruits host-cell SGLT1, a Na+/glucose cotransporter, and aquaporin 1 (AQP1), a water channel, to the attachment site. SGLT1-dependent glucose uptake occurs at the attachment site. Concordantly, the region of attachment displays localized water influx that is inhibited by either suppression of AQP1 by means of AQP1-small interfering RNA (siRNA) or inhibition of SGLT1 by a specific pharmacologic inhibitor, phlorizin. Inhibition of SGLT1 does not affect actin accumulation but decreases the membrane protrusion at the attachment site. Moreover, functional inhibition of host-cell AQP1 and SGLT1 hampersC. parvuminvasion of cholangiocytes. Thus, glucose-driven, AQP-mediated localized water influx is involved in the membrane protrusion duringC. parvumcellular invasion, phenomena that may also be relevant to the mechanisms of cell membrane protrusion in general.
Quantum corrections in vibrational and electronic condensed phase spectroscopy: Line shapes and echoes
Authors: Lawrence C. P. , Skinner J. L.
Published: April 2005

Various linear and nonlinear vibrational and electronic spectroscopy experiments in liquids are usually analyzed within the second-cumulant approximation, and therefore the fundamental quantity of interest is the equilibrium time-correlation function of the fluctuating transition frequency. In the usual approach the “bath” variables responsible for the fluctuating frequency are treated classically, leading to a classical time-correlation function. Alternatively, sometimes a quantum correction appropriate for relatively high temperatures is included, which adds an imaginary part to the classical time-correlation function. This approach, although appealing, does not satisfy detailed balance. One can consider a similar correction, but where detailed balance is satisfied, by using the harmonic quantum correction factor. In this article, we compare these approaches for a model system and two realistic examples. Our conclusion is that for linear spectroscopy the classical result is usually adequate, whereas for nonlinear spectroscopy it can be more important to include quantum corrections.
A rhodopsin exhibiting binding ability to agonist
all-trans-retinal
Authors: Tsukamoto Hisao , Terakita Akihisa , Shichida Yoshinori
Published: April 2005

Rhodopsins are the members of the family of G protein-coupled receptors that have diverged from ligand-binding receptors into photoreceptive pigments. Vertebrate rhodopsins are able to bind the inverse agonist 11-
cis
-retinal but are unable to bind the agonist
all-trans
-retinal, indicating that vertebrate rhodopsin changed its binding ability during the course of molecular evolution. Here, we show that unlike vertebrate rhodopsin, amphioxus rhodopsin is still able to bind the agonist
all-trans
-retinal. The opsin of amphioxus rhodopsin can also bind 11-
cis
-retinal to form a photoreceptive pigment that can convert to a red-shifted photoproduct through
cis-trans
isomerization of the chromophore upon photon absorption. The red-shifted photoproduct is the stable G protein activating state. Incubation of the opsin with
all-trans
-retinal produces a G protein activating state that is spectroscopically and biochemically indistinguishable from the red-shifted photoproduct, indicating that the opsin possesses agonist-binding ability. The opsin exhibits an ≈50-fold higher affinity for 11-
cis
-retinal than for
all-trans
-retinal, and mutational analyses revealed that Trp-265 situated in helix VI is important for the increase in binding affinity to 11-
cis
-retinal. These properties of amphioxus rhodopsin suggest that an ancestral rhodopsin increased the affinity for 11-
cis
-retinal by rearrangement of a structure including Trp-265 to act as a photoreceptor. In addition, an additional mechanism was acquired in vertebrate rhodopsin to prevent completely the binding of exogenous
all-trans
-retinal during molecular evolution.
A point mutation in the groove of HLA-DO allows egress from the endoplasmic reticulum independent of HLA-DM
Authors: Deshaies Francis , Brunet Alexandre , Diallo Djibril A. , Denzin Lisa K. , Samaan Angela , Thibodeau Jacques
Published: April 2005

B lymphocytes express the nonclassical class II molecule HLA-DO, which modulates the peptide loading activity of HLA-DM in the endocytic pathway. Binding to HLA-DM is required for HLA-DO to egress from the endoplasmic reticulum (ER). To gain insights into the mode of action of DO and on the role of DM in ER release, we sought to identify DM-binding residues on DO. Our results show that DOα encompasses the binding site for HLA-DM. More specifically, mutation of residue DOα41 on an exposed lateral loop of the α1 domain affects the binding to DM, ER egress, and activity of DO. Using a series of chimeric DR/DO molecules, we confirmed the role of the α chain and established that a second DM-binding region is located C-terminal to the DOα80 residue, most probably in the α2 domain. Interestingly, after mutation of a buried proline (α11) on the floor of the putative peptide-binding groove, HLA-DO remained functional but became independent of HLA-DM for ER egress and intracellular trafficking. Collectively, these results suggest that the binding of HLA-DM to DOα allows the complex to egress from the ER by stabilizing intramolecular contacts between the N-terminal antiparallel β-strands of the DOαβ heterodimer.
Invasion success of vertebrates in Europe and North America
Authors: Jeschke Jonathan M. , Strayer David L.
Published: April 2005

Species become invasive if they (
i
) are introduced to a new range, (
ii
) establish themselves, and (
iii
) spread. To address the global problems caused by invasive species, several studies investigated steps
ii
and
iii
of this invasion process. However, only one previous study looked at step
i
and examined the proportion of species that have been introduced beyond their native range. We extend this research by investigating all three steps for all freshwater fish, mammals, and birds native to Europe or North America. A higher proportion of European species entered North America than vice versa. However, the introduction rate from Europe to North America peaked in the late 19th century, whereas it is still rising in the other direction. There is no clear difference in invasion success between the two directions, so neither the imperialism dogma (that Eurasian species are exceptionally successful invaders) is supported, nor is the contradictory hypothesis that North America offers more biotic resistance to invaders than Europe because of its less disturbed and richer biota. Our results do not support the tens rule either: that ≈10% of all introduced species establish themselves and that ≈10% of established species spread. We find a success of ≈50% at each step. In comparison, only ≈5% of native vertebrates were introduced in either direction. These figures show that, once a vertebrate is introduced, it has a high potential to become invasive. Thus, it is crucial to minimize the number of species introductions to effectively control invasive vertebrates.
E1A and a nuclear receptor corepressor splice variant (N-CoR
I
) are thyroid hormone receptor coactivators that bind in the corepressor mode
Authors: Meng Xianwang , Webb Paul , Yang Yong-Fan , Shuen Michael , Yousef Ahmed F. , Baxter John D. , Mymryk Joe S. , Walfish Paul G.
Published: April 2005

Unliganded thyroid hormone (TH) receptors (TRs) and other nuclear receptors (NRs) repress transcription of hormone-activated genes by recruiting corepressors (CoRs), such as NR CoR (N-CoR) and SMRT. Unliganded TRs also activate transcription of TH-repressed genes. Some evidence suggests that these effects also involve TR/CoR contacts; however, the precise reasons that CoRs activate transcription in these contexts are obscure. Unraveling these mechanisms is complicated by the fact that it is difficult to decipher direct vs. indirect effects of TR-coregulator contacts in mammalian cells. In this study, we used yeast,
Saccharomyces cerevisiae
, which lack endogenous NRs and NR coregulators, to determine how unliganded TRs can activate transcription. We previously showed that adenovirus 5 early-region 1A coactivates unliganded TRs in yeast, and that these effects are blocked by TH. We show here that human adenovirus type 5 early region 1A (E1A) contains a short peptide (LDQLIEEVL amino acids 20-28) that resembles CoR-NR interaction motifs (CoRNR boxes), and that this motif is required for TR binding and coactivation. Although full-length N-CoR does not coactivate TR in yeast, a naturally occurring N-CoR variant (N-CoR
I
) and an artificial N-CoR truncation (N-CoR
C
) that retain CoRNR boxes but lack N-terminal repressor domains behave as potent and direct TH-repressed coactivators for unliganded TRs. We conclude that E1A and N-CoR
I
are naturally occurring TR coactivators that bind in the typical CoR mode and suggest that similar factors could mediate transcriptional activation by unliganded TRs in mammals.
Speciation in birds: Genes, geography, and sexual selection
Authors: Edwards Scott V. , Kingan Sarah B. , Calkins Jennifer D. , Balakrishnan Christopher N. , Jennings W. Bryan , Swanson Willie J. , Sorenson Michael D.
Published: April 2005

Molecular studies of speciation in birds over the last three decades have been dominated by a focus on the geography, ecology, and timing of speciation, a tradition traceable to Mayr'sSystematics and the Origin of Species. However, in the recent years, interest in the behavioral and molecular mechanisms of speciation in birds has increased, building in part on the older traditions and observations from domesticated species. The result is that many of the same mechanisms proffered for model lineages such asDrosophila—mechanisms such as genetic incompatibilities, reinforcement, and sexual selection—are now being seriously entertained for birds, albeit with much lower resolution. The recent completion of a draft sequence of the chicken genome, and an abundance of single-nucleotide polymorphisms on the autosomes and sex chromosomes, will dramatically accelerate research on the molecular mechanisms of avian speciation over the next few years. The challenge for ornithologists is now to inform well studied examples of speciation in nature with increased molecular resolution—to clone speciation genes if they exist—and thereby evaluate the relative roles of extrinsic, intrinsic, deterministic, and stochastic causes for avian diversification.
Capturing protein interactions in the secretory pathway of living cells
Authors: Nyfeler Beat , Michnick Stephen W. , Hauri Hans-Peter
Published: April 2005

The secretory pathway is composed of membrane compartments specialized in protein folding, modification, transport, and sorting. Numerous transient protein-protein interactions guide the transport-competent proteins through the secretory pathway. Here we have adapted the yellow fluorescent protein (YFP)-based protein fragment complementation assay (PCA) to detect protein-protein interactions in the secretory pathway of living cells. Fragments of YFP were fused to the homooligomeric cargo-receptor lectin endoplasmic reticulum Golgi intermediate compartment (ERGIC)-53, to the ERGIC-53-interacting multicoagulation factor deficiency protein MCFD2, and to ERGIC-53's cargo glycoprotein cathepsin Z. YFP PCA analysis revealed the oligomerization of ERGIC-53 and its interaction with MCFD2, as well as its lectin-mediated interaction with cathepsin Z. Mutation of the lectin domain of ERGIC-53 selectively decreased YFP complementation with cathepsin Z. Using YFP PCA, we discovered a carbohydrate-mediated interaction between ERGIC-53 and cathepsin C. We conclude that YFP PCA can detect weak and transient protein interactions in the secretory pathway and hence is a powerful approach to study luminal processes involved in protein secretion. The study extends the application of PCA to carbohydrate-mediated protein-protein interactions of low affinity.
Genetics and genomics of
Drosophila
mating behavior
Authors: Mackay Trudy F. C. , Heinsohn Stefanie L. , Lyman Richard F. , Moehring Amanda J. , Morgan Theodore J. , Rollmann Stephanie M.
Published: April 2005

The first steps of animal speciation are thought to be the development of sexual isolating mechanisms. In contrast to recent progress in understanding the genetic basis of postzygotic isolating mechanisms, little is known about the genetic architecture of sexual isolation. Here, we have subjected
Drosophila melanogaster
to 29 generations of replicated divergent artificial selection for mating speed. The phenotypic response to selection was highly asymmetrical in the direction of reduced mating speed, with estimates of realized heritability averaging 7%. The selection response was largely attributable to a reduction in female receptivity. We assessed the whole genome transcriptional response to selection for mating speed using Affymetrix GeneChips and a rigorous statistical analysis. Remarkably, >3,700 probe sets (21% of the array elements) exhibited a divergence in message levels between the Fast and Slow replicate lines. Genes with altered transcriptional abundance in response to selection fell into many different biological process and molecular function Gene Ontology categories, indicating substantial pleiotropy for this complex behavior. Future functional studies are necessary to test the extent to which transcript profiling of divergent selection lines accurately predicts genes that directly affect the selected trait.
Prospects for identifying functional variation across the genome
Authors: Macdonald Stuart J. , Long Anthony D.
Published: April 2005

The genetic factors contributing to complex trait variation may reside in regulatory, rather than protein-coding portions of the genome. Within noncoding regions, SNPs in regulatory elements are more likely to contribute to phenotypic variation than those in nonregulatory regions. Thus, it is important to be able to identify and annotate noncoding regulatory elements. DNA conservation among diverged species successfully identifies noncoding regulatory regions. However, because rapidly evolving regulatory regions will not generally be conserved across species, these will not detected by using purely conservation-based methods. Here we describe additional approaches that can be used to identify putative regulatory elements via signatures of nonneutral evolution. An examination of the pattern of polymorphism both within and between populations of
Drosophila melanogaster
, as well as divergence with its sibling species
Drosophila simulans
, across 24.2 kb of noncoding DNA identifies several nonneutrally evolving regions not identified by conservation. Because different methods tag different regions, it appears that the methods are complementary. Patterns of variation at different elements are consistent with the action of selective sweeps, balancing selection, or population differentiation. Together with regions conserved between
D. melanogaster
and
Drosophila pseudoobscura
, we tag 5.3 kb of noncoding DNA as potentially regulatory. Ninety-seven of the 408 common noncoding SNPs surveyed are within putatively regulatory regions. If these methods collectively identify the majority of functional noncoding polymorphisms, genotyping only these SNPs in an association mapping framework would reduce genotyping effort for noncoding regions 4-fold.
Induction of CD4+CD25+regulatory T cells by copolymer-I through activation of transcription factor Foxp3
Authors: Hong Jian , Li Ningli , Zhang Xuejun , Zheng Biao , Zhang Jingwu Z.
Published: April 2005

Copolymer-I (COP-I) has unique immune regulatory properties and is a treatment option for multiple sclerosis (MS). This study revealed that COP-I induced the conversion of peripheral CD4+CD25-to CD4+CD25+regulatory T cells through the activation of transcription factor Foxp3. COP-I treatment led to a significant increase in Foxp3 expression in CD4+T cells in MS patients whose Foxp3 expression was reduced at baseline. CD4+CD25+T cell lines generated by COP-I expressed high levels of Foxp3 that correlated with an increased regulatory potential. Furthermore, we demonstrated that the induction of Foxp3 in CD4+T cells by COP-I was mediated through its ability to produce IFN-γ and, to a lesser degree, TGF-β1, as shown by antibody blocking and direct cytokine induction of Foxp3 expression in T cells. It was evident thatin vitrotreatment and administration with COP-I significantly raised the level of Foxp3 expression in CD4+T cells and promoted conversion of CD4+CD25+regulatory T cells in wild-type B6 mice but not in IFN-γ knockout mice. This study provides evidence for the role and mechanism of action of COP-I in the induction of CD4+CD25+regulatory T cells in general and its relevance to the treatment of MS.
Mutagenesis by reversible promoter insertion to study the activation of NF-κB
Authors: Kandel Eugene S. , Lu Tao , Wan Youzhong , Agarwal Mukesh K. , Jackson Mark W. , Stark George R.
Published: April 2005

Genetic dissection of signaling pathways in mammalian cells involves screening or selecting phenotypic mutants obtained by a variety of techniques. Limitations in current methods include inadequate genome coverage and difficulty in validating the link between mutation and phenotype. We describe an improved method for insertional mutagenesis with retroviral vectors and show that the ability to induce mutations increases greatly if a randomly inserted promoter directs transcription into the host DNA. The mutant phenotype is due to the expression of a hybrid transcript derived from the vector and the insertion site. Because other alleles of the affected gene remain intact, the phenotype is dominant, but is reversible by inactivating the promoter, for example, by site-specific recombination. Importantly, in mutant clones with multiple inserts, limited excision yields progeny with different patterns of inserts remaining. Characterizing these progeny allows the mutant phenotype to be associated with a specific target gene. Relative simplicity and robust target validation make the method suitable for a broad range of applications. We have used this technique to search for proteins that regulate NF-κB-dependent signaling in human cells. Two validated targets are the
relA
gene, which codes for the NF-κB p65 subunit, and the NF-κB regulator
act1
. Overexpression of the corresponding proteins, caused by insertion of a promoter into the first intron of each gene, leads to NF-κB-dependent secretion of factors that activate NF-κB through cell-surface receptors, establishing an autocrine loop.
Explanation, Emergence, and Quantum Entanglement
Authors: Hüttemann Andreas
Published: April 2005

This paper tries to get a grip on two seemingly conflicting intuitions about reductionism in quantum mechanics. On one hand it is received wisdom that quantum mechanics puts an end to ‘reductionism’. Quantum entanglement is responsible for such features of quantum mechanics as holism, the failure of supervenience, and emergence. While I agree with these claims, I will argue that it is only part of the story. Quantum mechanics provides us with thoroughgoing reductionist explanations. I will distinguish two kinds of microexplanation (or micro-‘reduction’). I will argue that even though quantum entanglement provides an example of the failure of one kind of microexplanation, it does not affect the other. Contrary to a recent paper by Kronz and Tiehen, I claim that the explanation of the dynamics of quantum mechanical systems is just as reductionist as it used to be in classical mechanics.
Heloise and the Paraclete
Authors: Mews Constant J.
Published: April 2005

Heloise and the Paraclete. This chapter considers Abelard’s decision to entrust his foundation of the Paraclete to Heloise and his writing of the Historia calamitatum in 1132–33, as well as Heloise’s reaction to this autobiographical narrative. It examines the impact of Heloise’s arguments about their past relationship and about the religious life for women on Abelard’s writings for the Paraclete. It also considers Abelard’s poetic laments in the light of Heloise’s interest in the human side of the Bible.
Morality
Authors: Gert Bernard
Published: April 2005

Morality: Its Nature and Justification provides an analysis of the concepts of morality, rationality and irrationality, reasons, goods and evils, moral rules, and impartiality. It uses these analyses to describe a justified moral system that includes moral rules that prohibit doing the kinds of actions that need justification so as not to be immoral; moral ideals that encourage doing the kinds of actions that, if they do not involve violations of moral rules, are universally considered to be morally good actions; and a two-step procedure for determining when the violations of a moral rule are strongly justified, weakly justified, and unjustified. The first step of this two-step procedure uses the morally relevant features to provide a morally relevant description of the action. The second step involves estimating whether more harm would be caused by publicly allowing such a violation or by not allowing it. This justified moral system is commonly used, although usually only implicitly, by people when they are making thoughtful moral decisions and judgments. Morality is a public system that is known by all normal adults, and that is not irrational for any of them to use to govern their own behavior and to judge the behavior of others. These analyses and justifications are used to provide an account of virtues and vices and of moral judgments, to answer the question of why one should be moral, and show that the same morality applies to governments as to individuals.
Towards a Labour Market in China
Authors: Knight John , Song Lina
Published: April 2005

This book presents a compilation of studies on China’s labour market. These explore institutional and political constraints on the operation of the market, and their changes over time. The book is divided into four parts. Part I studies the Chinese labour and wage system under the planned economy, labour market reforms, their evolution, and their consequences. Part II examines the various aspects of the labour market such as wage structure and segmentation. Part III analyses the rural labour market. Part IV discusses the imperfect labour market.
The Evangelical Conversion Narrative
Authors: Hindmarsh D. Bruce
Published: April 2005

This book is about conversion narrative, a popular genre of spiritual autobiography that proliferated during the last two-thirds of the eighteenth century within the context of the Evangelical Revival in England. The subject is set in a large chronological frame, beginning with the rise of the genre in the mid-seventeenth century and ending with the ‘fall’ of the genre among some of the non-Western converts of early nineteenth-century missionaries. This large frame allows the genre to be seen whole, and draws attention to the particular conditions under which early modern people turned to spiritual autobiography. Tracing the development of the genre across the period of the Evangelical Revival through different communities and representative writings, the book provides a comprehensive typology of conversion and evangelical self-identity as it differed among the Arminian and perfectionist followers of Wesley, the Moravians under the influence of ‘stillness’, the moderate Calvinists in the Church of England, the Particular Baptists who continued to embrace high Calvinism, and others. A chapter is also included on conversion narrative among evangelical Presbyterians involved in the Cambuslang Revival in Scotland. On the basis of extensive, untapped archival sources, The Evangelical Conversion Narrative explores the different forms of expression among the educated and uneducated, pastors and laypeople, women and men, and Western and non-Western peoples. By being on the trailing edge of Christendom and the leading edge of modernity, eighteenth-century England provided the right conditions for evangelical conversion narrative to flourish, and the concluding chapter examines afresh the significance of the appearance of the genre in this context. This book is concerned with the history of autobiography, the study of eighteenth-century religion and culture, and our understanding of the Evangelical Revival.
John Calvin's Ideas
Authors: Helm Paul
Published: April 2005

John Calvin is universally acknowledged as one of the formative Christian theologians and one of the great doctors of the Church. His work continues to be the subject of almost continuous exposition and scholarly appraisal. This book is the first at length treatment of some of his key ideas and his theological positions that have a philosophical aspect to them. Work has been done on the philosophical sources of some of Calvin's work, but little or nothing on how Calvin actually made use of philosophical ideas in his work. Calvin has frequently been thought of as anti philosophical in his bent, with particular focus being placed on his intense dislike of speculation. Emphasis has been placed on his role as a theologian of 'the Word' and on his Renaissance background. It is not denied that Calvin was first and foremost a theologian, and not a philosopher, and the influence of the Renaissance upon him, particularly on his style, must be recognized. However, careful analysis of his theology reveals both Calvin's thorough familiarity with a range of philosophical ideas, and a willingness to use these, putting them to work in elucidation of his own theological positions, and even on occasion indulging in a little speculation on his own account.

In order to emphasis Calvin's often positive relationship to philosophical ideas, the chapters of the book are arranged in philosophical rather than theological order. So there are chapters on metaphysics, epistemology, and ethics. As well as examining Calvin's theology in its late mediaeval context, attention is given to the way in which Calvin has been appealed to in contemporary philosophy by 'reformed' epistemology.

It is believed that this book should lead to a reappraisal not of Calvin's theology as such, but of his theological method, and of the way in which his work relates not only to late mediaeval theology but also to later developments in Reformed theology, in Puritanism, and Reformed Scholasticism.
Ethylene-Regulated Floral Volatile Synthesis in Petunia Corollas
Authors: Underwood Beverly A. , Tieman Denise M. , Shibuya Kenichi , Dexter Richard J. , Loucas Holly M. , Simkin Andrew J. , Sims Charles A. , Schmelz Eric A. , Klee Harry J. , Clark David G.
Published: April 2005

In many flowering plants, such as petunia (Petunia × hybrida), ethylene produced in floral organs after pollination elicits a series of physiological and biochemical events, ultimately leading to senescence of petals and successful fertilization. Here, we demonstrate, using transgenic ethylene insensitive (44568) and Mitchell Diploid petunias, that multiple components of emission of volatile organic compounds (VOCs) are regulated by ethylene. Expression of benzoic acid/salicylic acid carboxyl methyltransferase (PhBSMT1 and 2) mRNA is temporally and spatially down-regulated in floral organs in a manner consistent with current models for postpollination ethylene synthesis in petunia corollas. Emission of methylbenzoate and other VOCs after pollination and exogenous ethylene treatment parallels a reduction in PhBSMT1 and 2 mRNA levels. Under cyclic light conditions (day/night), PhBSMT mRNA levels are rhythmic and precede emission of methylbenzoate by approximately 6 h. When shifted into constant dark or light conditions, PhBSMT mRNA levels and subsequent methylbenzoate emission correspondingly decrease or increase to minimum or maximum levels observed during normal conditions, thus suggesting that light may be a more critical influence on cyclic emission of methylbenzoate than a circadian clock. Transgenic PhBSMT RNAi flowers with reduced PhBSMT mRNA levels show a 75% to 99% decrease in methylbenzoate emission, with minimal changes in other petunia VOCs. These results implicate PhBSMT1 and 2 as genes responsible for synthesis of methylbenzoate in petunia.
Spatial and Temporal Distribution of Polyamine Levels and Polyamine Anabolism in Different Organs/Tissues of the Tobacco Plant. Correlations with Age, Cell Division/Expansion, and Differentiation
Authors: Paschalidis Konstantinos A. , Roubelakis-Angelakis Kalliopi A.
Published: April 2005

Polyamine (PA) titers and biosynthesis follow a basipetal decrease along the tobacco (Nicotiana tabacum) plant axis, and they also correlate negatively with cell size. On the contrary, the titers of arginine (Arg), ornithine (Orn), and arginase activity increase with age. The free (soluble)/total-PA ratios gradually increase basipetally, but the soluble conjugated decrease, with spermidine (Spd) mainly to determine these changes. The shoot apical meristems are the main site of Spd and spermine biosynthesis, and the hypogeous tissues synthesize mostly putrescine (Put). High and low Spd syntheses are correlated with cell division and expansion, respectively. Put biosynthetic pathways are differently regulated in hyper- and hypogeous tobacco tissues: Only Arg decarboxylase is responsible for Put synthesis in old hypergeous vascular tissues, whereas, in hypogeous tissues, arginase-catalyzed Orn produces Put via Orn decarboxylase. Furthermore, Orn decarboxylase expression coincides with early cell divisions in marginal sectors of the lamina, and Spd synthase strongly correlates with later cell divisions in the vascular regions. This detailed spatial and temporal profile of the free, soluble-conjugated, and insoluble-conjugated fractions of Put, Spd, and spermine in nearly all tobacco plant organs and the profile of enzymes of PA biosynthesis at the transcript, protein, and specific activity levels, along with the endogenous concentrations of the precursor amino acids Arg and Orn, offer new insight for further understanding the physiological role(s) of PAs. The results are discussed in the light of age dependence, cell division/expansion, differentiation, phytohormone gradients, senescence, and sink-source relationships.
Genome-Based Examination of Chlorophyll and Carotenoid Biosynthesis in Chlamydomonas reinhardtii
Authors: Lohr Martin , Im Chung-Soon , Grossman Arthur R.
Published: April 2005

The unicellular green alga Chlamydomonas reinhardtii is a particularly important model organism for the study of photosynthesis since this alga can grow heterotrophically, and mutants in photosynthesis are therefore conditional rather than lethal. The recently developed tools for genomic analyses of this organism have allowed us to identify most of the genes required for chlorophyll and carotenoid biosynthesis and to examine their phylogenetic relationships with homologous genes from vascular plants, other algae, and cyanobacteria. Comparative genome analyses revealed some intriguing features associated with pigment biosynthesis in C. reinhardtii; in some cases, there are additional conserved domains in the algal and plant but not the cyanobacterial proteins that may directly influence their activity, assembly, or regulation. For some steps in the chlorophyll biosynthetic pathway, we found multiple gene copies encoding putative isozymes. Phylogenetic studies, theoretical evaluation of gene expression through analysis of expressed sequence tag data and codon bias of each gene, enabled us to generate hypotheses concerning the function and regulation of the individual genes, and to propose targets for future research. We have also used quantitative polymerase chain reaction to examine the effect of low fluence light on the level of mRNA accumulation encoding key proteins of the biosynthetic pathways and examined differential expression of those genes encoding isozymes that function in the pathways. This work is directing us toward the exploration of the role of specific photoreceptors in the biosynthesis of pigments and the coordination of pigment biosynthesis with the synthesis of proteins of the photosynthetic apparatus.
Characterization and Expression Patterns of UDP-d-Glucuronate Decarboxylase Genes in Barley
Authors: Zhang Qisen , Shirley Neil , Lahnstein Jelle , Fincher Geoffrey B.
Published: April 2005

UDP-d-glucuronate decarboxylase (EC 4.1.1.35) catalyzes the synthesis of UDP-d-xylose from UDP-d-glucuronate in an essentially irreversible reaction that is believed to commit glycosyl residues to heteroxylan and xyloglucan biosynthesis. Four members of the barley (Hordeum vulgare) UDP-d-glucuronate decarboxylase gene family, designated HvUXS1 to HvUXS4, have been cloned and characterized. Barley HvUXS1 appears to be a cytosolic enzyme, while the others are predicted to be membrane-bound proteins with single transmembrane helices. Heterologous expression of a barley HvUXS1 cDNA in Escherichia coli yields a soluble enzyme that converts UDP-d-glucuronate to UDP-d-xylose, is associated with a single molecule of bound NAD+, and is subject to feedback inhibition by UDP-d-xylose. Quantitative PCR shows that the HvUXS1 mRNA is most abundant among the 4 HvUXS genes, accounting for more than 80% of total HvUXS transcripts in most of the tissues examined. The abundance of HvUXS1 mRNA is 10-fold higher in mature roots and stems than in leaves, developing grains, or floral tissues. Transcriptional activities of HvUXS2 and HvUXS4 genes are relatively high in mature roots, coleoptiles, and stems compared with root tips, leaves, and floral tissues, while HvUXS3 mRNA is low in all tissues. In barley leaf sections, levels of the most abundant mRNA, encoding HvUXS1, reflect the amount of soluble enzymic protein and activity. In selected tissues where HvUXS1 transcript levels are high, cell walls have higher arabinoxylan contents.
A Novel Short-Root Gene Encodes a Glucosamine-6-Phosphate Acetyltransferase Required for Maintaining Normal Root Cell Shape in Rice
Authors: Jiang Huawu , Wang Shaomin , Dang Lei , Wang Shoufeng , Chen Hanmin , Wu Yunrong , Jiang Xinhang , Wu Ping
Published: April 2005

Glycosylation is a posttranslational modification occurring in many secreted and membrane-associated proteins in eukaryotes. It plays important roles in both physiological and pathological processes. Most of these protein modifications depend on UDP-N-acetylglucosamine. In this study, a T-DNA insertional rice (Oryza sativa) mutant exhibiting a temperature-sensitive defect in root elongation was isolated. Genetic and molecular analysis indicated that the mutated phenotype was caused by loss of function of a gene encoding a glucosamine-6-P acetyltransferase (designated OsGNA1), which is involved in de novo UDP-N-acetylglucosamine biosynthesis. The aberrant root morphology of the gna1 mutant includes shortening of roots, disruption of microtubules, and shrinkage of cells in the root elongation zone. Our observations support the idea that protein glycosylation plays a key role in cell metabolism, microtubule stabilization, and cell shape in rice roots.
Nematode Infection Triggers the de Novo Formation of Unloading Phloem That Allows Macromolecular Trafficking of Green Fluorescent Protein into Syncytia
Authors: Hoth Stefan , Schneidereit Alexander , Lauterbach Christian , Scholz-Starke Joachim , Sauer Norbert
Published: April 2005

Syncytial feeding complexes induced by the cyst nematode Heterodera schachtii represent strong metabolic sinks for photoassimilates. These newly formed structures were described to be symplastically isolated from the surrounding root tissue and their mechanism of carbohydrate import has repeatedly been under investigation. Here, we present analyses of the symplastic connectivity between the root phloem and these syncytia in nematode-infected Arabidopsis (Arabidopsis thaliana) plants expressing the gene of the green fluorescent protein (GFP) or of different GFP fusions under the control of the companion cell (CC)-specific AtSUC2 promoter. In the same plants, phloem differentiation during syncytium formation was monitored using cell-specific antibodies for CCs or sieve elements (SEs). Our results demonstrate that free, CC-derived GFP moved freely from the phloem into the syncytial domain. No or only marginal cell-to-cell passage of GFP was observed into other root cells adjacent to these syncytia. In contrast, membrane-anchored GFP variants as well as soluble GFP fusions with increased molecular masses were restricted to the SE-CC complex. The presented data also show that nematode infection triggers the de novo formation of phloem containing an approximately 3-fold excess of SEs over CCs. This newly formed phloem exhibits typical properties of unloading phloem previously described in other sink tissues. Our results reveal the existence of a symplastic pathway between phloem CCs and nematode-induced syncytia. The plasmodesmata responsible for this symplastic connectivity allow the cell-to-cell movement of macromolecules up to 30 kD and are likely to represent the major or exclusive path for the supply of assimilates from the phloem into the syncytial complex.
Localization of Soluble β-Carbonic Anhydrase in the Marine Diatom Phaeodactylum tricornutum. Sorting to the Chloroplast and Cluster Formation on the Girdle Lamellae
Authors: Tanaka Yuji , Nakatsuma Daisuke , Harada Hisashi , Ishida Maki , Matsuda Yusuke
Published: April 2005

A β-carbonic anhydrase (CA) in the marine diatom Phaeodactylum tricornutum (PtCA1) is encoded by the nuclear genome. This enzyme was previously found to be important for the operation of photosynthesis with a high affinity for dissolved inorganic carbon. A cDNA sequence that encodes PtCA1 (ptca1) was shown to possess a presequence of 138 bp (pre138), which encodes an N-terminal sequence of 46 amino acids (Pre46AA) that does not exist in the mature PtCA1. In this study, pre138 was ligated with the enhanced green fluorescent protein (GFP) gene (egfp), and introduced into P. tricornutum by microprojectile bombardment. Subsequently, the expressed Pre46AA-GFP fusion was shown to be localized in the chloroplast stroma, whereas the expressed GFP without Pre46AA was localized in the cytoplasm. Insertion of the DNA sequence, encoding a mature region of ptca1 (mptca1) between pre138 and egfp, resulted in the formation of particles with concentrated GFP fluorescence in the stroma of P. tricornutum. These particles, 0.3 to 3.0 μm in size, were shown to be distinct from the mitochondria and localized on the surface of the putative girdle lamella. The attachment of the initial one-half of the pre138 to the mptca1-egfp fusion caused the expressed GFP fusion to accumulate in areas surrounding the chloroplast, presumably due to the presence of the endoplasmic reticulum signal encoded by the initial half-sequence and to the absence of the chloroplast transit sequence. These results indicate that PtCA1 is targeted to the stroma by the bipartite sequences of Pre46AA and that the observed GFP particles are formed specifically in the stroma due to the function of the mptca1.
Sanguinarine Biosynthesis Is Associated with the Endoplasmic Reticulum in Cultured Opium Poppy Cells after Elicitor Treatment
Authors: Alcantara Joenel , Bird David A. , Franceschi Vincent R. , Facchini Peter J.
Published: April 2005

Three key benzylisoquinoline alkaloid biosynthetic enzymes, (S)-N-methylcoclaurine-3′-hydroxylase (CYP80B1), berberine bridge enzyme (BBE), and codeinone reductase (COR), were localized in cultured opium poppy (Papaver somniferum) cells by sucrose density gradient fractionation and immunogold labeling. CYP80B1 catalyzes the second to last step in the formation of (S)-reticuline, the last common intermediate in sanguinarine and morphine biosynthesis. BBE converts (S)-reticuline to (S)-scoulerine as the first committed step in sanguinarine biosynthesis, and COR catalyzes the penultimate step in the branch pathway leading to morphine. Sanguinarine is an antimicrobial alkaloid that accumulates in the vacuoles of cultured opium poppy cells in response to elicitor treatment, whereas the narcotic analgesic morphine, which is abundant in opium poppy plants, is not produced in cultured cells. CYP80B1 and BBE were rapidly induced to high levels in response to elicitor treatment. By contrast, COR levels were constitutive in the cell cultures, but remained low and were not induced by addition of the elicitor. Western blots performed on protein homogenates from elicitor-treated cells fractionated on a sucrose density gradient showed the cosedimentation of CYP80B1, BBE, and sanguinarine with calreticulin, and COR with glutathione S-transferase. Calreticulin and glutathione S-transferase are markers for the endoplasmic reticulum (ER) and the cytosol, respectively. In response to elicitor treatment, large dilated vesicles rapidly developed from the lamellar ER of control cells and fused with the central vacuole. Immunogold localization supported the association of CYP80B1 and BBE with ER vesicles, and COR with the cytosol in elicitor-treated cells. Our results show that benzylisoquinoline biosynthesis and transport to the vacuole are associated with the ER, which undergoes major ultrastructural modification in response to the elicitor treatment of cultured opium poppy cells.
Mutants of Arabidopsis Lacking a Chloroplastic Isoamylase Accumulate Phytoglycogen and an Abnormal Form of Amylopectin
Authors: Wattebled Fabrice , Dong Ying , Dumez Sylvain , Delvallé David , Planchot Véronique , Berbezy Pierre , Vyas Darshna , Colonna Paul , Chatterjee Manash , Ball Steven , D'Hulst Christophe
Published: April 2005

Mutant lines defective for each of the four starch debranching enzyme (DBE) genes (AtISA1, AtISA2, AtISA3, and AtPU1) detected in the nuclear genome of Arabidopsis (Arabidopsis thaliana) were produced and analyzed. Our results indicate that both AtISA1 and AtISA2 are required for the production of a functional isoamylase-type of DBE named Iso1, the major isoamylase activity found in leaves. The absence of Iso1 leads to an 80% decrease in the starch content in both lines and to the accumulation of water-soluble polysaccharides whose structure is similar to glycogen. In addition, the residual amylopectin structure in the corresponding mutant lines displays a strong modification when compared to the wild type, suggesting a direct, rather than an indirect, function of Iso1 during the synthesis of amylopectin. Mutant lines carrying a defect in AtISA3 display a strong starch-excess phenotype at the end of both the light and the dark phases accompanied by a small modification of the amylopectin structure. This result suggests that this isoamylase-type of DBE plays a major role during starch mobilization. The analysis of the Atpu1 single-mutant lines did not lead to a distinctive phenotype. However, Atisa2/Atpu1 double-mutant lines display a 92% decrease in starch content. This suggests that the function of pullulanase partly overlaps that of Iso1, although its implication remains negligible when Iso1 is present within the cell.
TheLPB1Gene Is Important for Acclimation ofChlamydomonas reinhardtiito Phosphorus and Sulfur Deprivation
Authors: Chang Chiung-Wen , Moseley Jeffrey L. , Wykoff Dennis , Grossman Arthur R.
Published: April 2005

Organisms exhibit a diverse set of responses when exposed to low-phosphate conditions. Some of these responses are specific for phosphorus limitation, including responses that enable cells to efficiently scavenge phosphate from internal and external stores via the production of high-affinity phosphate transporters and the synthesis of intracellular and extracellular phosphatases. Other responses are general and occur under a number of different environmental stresses, helping coordinate cellular metabolism and cell division with the growth potential of the cell. In this article, we describe the isolation and characterization of a mutant of Chlamydomonas reinhardtii, low-phosphate bleaching (lpb1), which dies more rapidly than wild-type cells during phosphorus limitation. The responses of this mutant to nitrogen limitation appear normal, although the strain is also somewhat more sensitive than wild-type cells to sulfur deprivation. Interestingly, depriving the cells of both nutrients simultaneously allows for sustained survival that is similar to that observed with wild-type cells. Furthermore, upon phosphorus deprivation, the lpb1 mutant, like wild-type cells, exhibits increased levels of mRNA encoding the PHOX alkaline phosphatase, the PTB2 phosphate transporter, and the regulatory element PSR1. The mutant strain is also able to synthesize the extracellular alkaline phosphatase activity upon phosphorus deprivation and the arylsulfatase upon sulfur deprivation, suggesting that the specific responses to phosphorus and sulfur deprivation are normal. The LPB1 gene was tagged by insertion of the ARG7 gene, which facilitated its isolation and characterization. This gene encodes a protein with strong similarity to expressed proteins in Arabidopsis (Arabidopsis thaliana) and predicted proteins in Oryza sativa and Parachlamydia. A domain in the protein contains some similarity to the superfamily of nucleotide-diphospho-sugar transferases, and it is likely to be localized to the chloroplast or mitochondrion based on programs that predict subcellular localization. While the precise catalytic role and physiological function of the putative protein is not known, it may function in some aspect of polysaccharide metabolism and/or influence phosphorus metabolism (either structural or regulatory) in a way that is critical for allowing the cells to acclimate to nutrient limitation conditions.