bluuebunny/crossref_metadata_2025 huggingface dataset:
Respiratory syncytial virus uses a Vps4-independent budding mechanism controlled by Rab11-FIP2
Authors: Utley Thomas J. , Ducharme Nicole A. , Varthakavi Vasundhara , Shepherd Bryan E. , Santangelo Philip J. , Lindquist Michael E. , Goldenring James R. , Crowe James E.
Published: July 2008

Respiratory syncytial virus (RSV) infects polarized epithelia, which have tightly regulated trafficking because of the separation and maintenance of the apical and basolateral membranes. Previously we established a link between the apical recycling endosome (ARE) and the assembly of RSV. The current studies tested the role of a major ARE-associated protein, Rab11 family interacting protein 2 (FIP2) in the virus life cycle. A dominant-negative form of FIP2 lacking its N-terminal C2 domain reduced the supernatant-associated RSV titer 1,000-fold and also caused the cell-associated virus titer to increase. These data suggested that the FIP2 C2 mutant caused a failure at the final budding step in the virus life cycle. Additionally, truncation of the Rab-binding domain from FIP2 caused its accumulation into mature filamentous virions. RSV budding was independent of the ESCRT machinery, the only well-defined budding mechanism for enveloped RNA viruses. Therefore, RSV uses a virus budding mechanism that is controlled by FIP2.
Structure of the α
2
ε
2
Ni-dependent CO dehydrogenase component of the
Methanosarcina barkeri
acetyl-CoA decarbonylase/synthase complex
Authors: Gong Weimin , Hao Bing , Wei Zhiyi , Ferguson Donald J. , Tallant Thomas , Krzycki Joseph A. , Chan Michael K.
Published: July 2008

Ni-dependent carbon monoxide dehydrogenases (Ni-CODHs) are a diverse family of enzymes that catalyze reversible CO:CO
2
oxidoreductase activity in acetogens, methanogens, and some CO-using bacteria. Crystallography of Ni-CODHs from CO-using bacteria and acetogens has revealed the overall fold of the Ni-CODH core and has suggested structures for the C cluster that mediates CO:CO
2
interconversion. Despite these advances, the mechanism of CO oxidation has remained elusive. Herein, we report the structure of a distinct class of Ni-CODH from methanogenic archaea: the α
2
ε
2
component from the α
8
β
8
γ
8
δ
8
ε
8
CODH/acetyl-CoA decarbonylase/synthase complex, an enzyme responsible for the majority of biogenic methane production on Earth. The structure of this Ni-CODH component provides support for a hitherto unobserved state in which both CO and H
2
O/OH
-
bind to the Ni and the exogenous FCII iron of the C cluster, respectively, and offers insight into the structures and functional roles of the ε-subunit and FeS domain not present in nonmethanogenic Ni-CODHs.
Uncoupling of behavioral and autonomic responses after lesions of the primate orbitofrontal cortex
Authors: Reekie Y. L. , Braesicke K. , Man M. S. , Roberts A. C.
Published: July 2008

Successful adaptation to changes in an animal's emotional and motivational environment depends on behavioral flexibility accompanied by changes in bodily responses, e.g., autonomic and endocrine, which support the change in behavior. Here, we identify the orbitofrontal cortex (OFC) as pivotal in the flexible regulation and coordination of behavioral and autonomic responses during adaptation. Using an appetitive Pavlovian task, we demonstrate that OFC lesions in the marmoset (
i
) impair an animal's ability to rapidly suppress its appetitive cardiovascular arousal upon termination of a conditioned stimulus and (
ii
) cause an uncoupling of the behavioral and autonomic components of the adaptive response after reversal of the reward contingencies. These findings highlight the role of the OFC in emotional regulation and are highly relevant to our understanding of disorders such as schizophrenia and autism in which uncoupling of emotional responses may contribute to the experiential distress and disadvantageous behavior associated with these disorders.
Coordinated evolution of the hepatitis C virus
Authors: Campo D. S. , Dimitrova Z. , Mitchell R. J. , Lara J. , Khudyakov Y.
Published: July 2008

Hepatitis C virus is a genetically heterogeneous RNA virus that is a major cause of liver disease worldwide. Here, we show that, despite its extensive heterogeneity, the evolution of hepatitis C virus is primarily shaped by negative selection and that numerous coordinated substitutions in the polyprotein can be organized into a scale-free network whose degree of connections between sites follows a power-law distribution. This network shares all major properties with many complex biological and technological networks. The topological structure and hierarchical organization of this network suggest that a small number of amino acid sites exert extensive impact on hepatitis C virus evolution. Nonstructural proteins are enriched for negatively selected sites of high centrality, whereas structural proteins are enriched for positively selected sites located in the periphery of the network. The complex network of coordinated substitutions is an emergent property of genetic systems with implications for evolution, vaccine research, and drug development. In addition to such properties as polymorphism or strength of selection, the epistatic connectivity mapped in the network is important for typing individual sites, proteins, or entire genetic systems. The network topology may help devise molecular intervention strategies for disrupting viral functions or impeding compensatory changes for vaccine escape or drug resistance mutations. Also, it may be used to find new therapeutic targets, as suggested in this study for the NS4A protein, which plays an important role in the network.
Cyanobacteriochrome CcaS is the green light receptor that induces the expression of phycobilisome linker protein
Authors: Hirose Yuu , Shimada Takashi , Narikawa Rei , Katayama Mitsunori , Ikeuchi Masahiko
Published: July 2008

Cyanobacteriochromes are a newly recognized group of photoreceptors that are distinct relatives of phytochromes but are found only in cyanobacteria. A putative cyanobacteriochrome, CcaS, is known to chromatically regulate the expression of the phycobilisome linker gene (
cpcG2
) in
Synechocystis
sp. PCC 6803. In this study, we isolated the chromophore-binding domain of CcaS from
Synechocystis
as well as from phycocyanobilin-producing
Escherichia coli
. Both preparations showed the same reversible photoconversion between a green-absorbing form (Pg, λ
max
= 535 nm) and a red-absorbing form (Pr, λ
max
= 672 nm). Mass spectrometry and denaturation analyses suggested that Pg and Pr bind phycocyanobilin in a double-bond configuration of C15-
Z
and C15-
E
, respectively. Autophosphorylation activity of the histidine kinase domain in nearly full-length CcaS was up-regulated by preirradiation with green light. Similarly, phosphotransfer to the cognate response regulator, CcaR, was higher in Pr than in Pg. From these results, we conclude that CcaS phosphorylates CcaR under green light and induces expression of
cpcG2
, leading to accumulation of CpcG2-phycobilisome as a chromatic acclimation system. CcaS is the first recognized green light receptor in the expanded phytochrome superfamily, which includes phytochromes and cyanobacteriochromes.
Functional architecture of inositol 1,4,5-trisphosphate signaling in restricted spaces of myoendothelial projections
Authors: Ledoux Jonathan , Taylor Mark S. , Bonev Adrian D. , Hannah Rachael M. , Solodushko Viktoriya , Shui Bo , Tallini Yvonne , Kotlikoff Michael I. , Nelson Mark T.
Published: July 2008

Calcium (Ca
2+
) release through inositol 1,4,5-trisphosphate receptors (IP
3
Rs) regulates the function of virtually every mammalian cell. Unlike ryanodine receptors, which generate local Ca
2+
events (“sparks”) that transmit signals to the juxtaposed cell membrane, a similar functional architecture has not been reported for IP
3
Rs. Here, we have identified spatially fixed, local Ca
2+
release events (“pulsars”) in vascular endothelial membrane domains that project through the internal elastic lamina to adjacent smooth muscle membranes. Ca
2+
pulsars are mediated by IP
3
Rs in the endothelial endoplasmic reticulum of these membrane projections. Elevation of IP
3
by the endothelium-dependent vasodilator, acetylcholine, increased the frequency of Ca
2+
pulsars, whereas blunting IP
3
production, blocking IP
3
Rs, or depleting endoplasmic reticulum Ca
2+
inhibited these events. The elementary properties of Ca
2+
pulsars were distinct from ryanodine-receptor-mediated Ca
2+
sparks in smooth muscle and from IP
3
-mediated Ca
2+
puffs in
Xenopus
oocytes. The intermediate conductance, Ca
2+
-sensitive potassium (K
Ca
3.1) channel also colocalized to the endothelial projections, and blockage of this channel caused an 8-mV depolarization. Inhibition of Ca
2+
pulsars also depolarized to a similar extent, and blocking K
Ca
3.1 channels was without effect in the absence of pulsars. Our results support a mechanism of IP
3
signaling in which Ca
2+
release is spatially restricted to transmit intercellular signals.
Autoreactive IgG memory antibodies in patients with systemic lupus erythematosus arise from nonreactive and polyreactive precursors
Authors: Mietzner Brun , Tsuiji Makoto , Scheid Johannes , Velinzon Klara , Tiller Thomas , Abraham Klaus , Gonzalez Jose B. , Pascual Virginia , Stichweh Dorothee , Wardemann Hedda , Nussenzweig Michel C.
Published: July 2008

Persistent autoantibody production in patients with systemic lupus erythematosus (SLE) suggests the existence of autoreactive humoral memory, but the frequency of self-reactive memory B cells in SLE has not been determined. Here, we report on the reactivity of 200 monoclonal antibodies from single IgG+ memory B cells of four SLE patients. The overall frequency of polyreactive and HEp-2 self-reactive antibodies in this compartment was similar to controls. We found 15% of IgG memory B cell antibodies highly reactive and specific for SLE-associated extractable nuclear antigens (ENA) Ro52 and La in one patient with serum autoantibody titers of the same specificity but not in the other three patients or healthy individuals. The germ-line forms of the ENA antibodies were non-self-reactive or polyreactive with low binding to Ro52, supporting the idea that somatic mutations contributed to autoantibody specificity and reactivity. Heterogeneity in the frequency of memory B cells expressing SLE-associated autoantibodies suggests that this variable may be important in the outcome of therapies that ablate this compartment.
Tools for neuroanatomy and neurogenetics in
Drosophila
Authors: Pfeiffer Barret D. , Jenett Arnim , Hammonds Ann S. , Ngo Teri-T B. , Misra Sima , Murphy Christine , Scully Audra , Carlson Joseph W. , Wan Kenneth H. , Laverty Todd R. , Mungall Chris , Svirskas Rob , Kadonaga James T. , Doe Chris Q. , Eisen Michael B. , Celniker Susan E. , Rubin Gerald M.
Published: July 2008

We demonstrate the feasibility of generating thousands of transgenic
Drosophila melanogaster
lines in which the expression of an exogenous gene is reproducibly directed to distinct small subsets of cells in the adult brain. We expect the expression patterns produced by the collection of 5,000 lines that we are currently generating to encompass all neurons in the brain in a variety of intersecting patterns. Overlapping 3-kb DNA fragments from the flanking noncoding and intronic regions of genes thought to have patterned expression in the adult brain were inserted into a defined genomic location by site-specific recombination. These fragments were then assayed for their ability to function as transcriptional enhancers in conjunction with a synthetic core promoter designed to work with a wide variety of enhancer types. An analysis of 44 fragments from four genes found that >80% drive expression patterns in the brain; the observed patterns were, on average, comprised of <100 cells. Our results suggest that the
D. melanogaster
genome contains >50,000 enhancers and that multiple enhancers drive distinct subsets of expression of a gene in each tissue and developmental stage. We expect that these lines will be valuable tools for neuroanatomy as well as for the elucidation of neuronal circuits and information flow in the fly brain.
Isolation of aDrosophilaamplification origin developmentally activated by transcription
Authors: Xie Fang , Orr-Weaver Terry L.
Published: July 2008

We exploited theDrosophilaAmplicon in Follicle Cells,DAFC-62D, to identify a new metazoan amplification origin,ori62. In addition to the origin,DAFC-62Dcontains two other developmental stage-specific binding regions for the Origin Recognition Complex (ORC) and the replicative helicase MCM2–7. All three of these regions are required for proper amplification. There are two rounds of amplification initiation atori62, and the second round is preceded by transcription acrossori62. We show by α-amanitin inhibition that RNA polymerase II (RNAPII) transcription is required to localize MCM2–7 (but not ORC) to permit the second round of origin firing. This role for transcription appears unique toDAFC-62D, because neither otherDAFCs nor ectopic transposons with theDAFC-62Dreplication elements bounded by functional chromatin insulators are affected by α-amanitin. By sequential chromatin immunoprecipitation, we show that the MCM complex and RNAPII are bound to the same 100–500 bp pieces of chromatin during late origin firing. These results raise the possibility that RNAPII may recruit MCM2–7 at some metazoan replication origins.
The MCK mouse heart model of Friedreich's ataxia: Alterations in iron-regulated proteins and cardiac hypertrophy are limited by iron chelation
Authors: Whitnall Megan , Rahmanto Yohan Suryo , Sutak Robert , Xu Xiangcong , Becker Erika M. , Mikhael Marc R. , Ponka Prem , Richardson Des R.
Published: July 2008

There is no effective treatment for the cardiomyopathy of the most common autosomal recessive ataxia, Friedreich's ataxia (FA). The identification of potentially toxic mitochondrial (MIT) iron (Fe) deposits in FA suggests that Fe plays a role in its pathogenesis. This study used the muscle creatine kinase conditional frataxin (Fxn) knockout (mutant) mouse model that reproduces the classical traits associated with cardiomyopathy in FA. We examined the mechanisms responsible for the increased cardiac MIT Fe loading in mutants. Moreover, we explored the effect of Fe chelation on the pathogenesis of the cardiomyopathy. Our investigation showed that increased MIT Fe in the myocardium of mutants was due to marked transferrin Fe uptake, which was the result of enhanced transferrin receptor 1 expression. In contrast to the mitochondrion, cytosolic ferritin expression and the proportion of cytosolic Fe were decreased in mutant mice, indicating cytosolic Fe deprivation and markedly increased MIT Fe targeting. These studies demonstrated that loss of Fxn alters cardiac Fe metabolism due to pronounced changes in Fe trafficking away from the cytosol to the mitochondrion. Further work showed that combining the MIT-permeable ligand pyridoxal isonicotinoyl hydrazone with the hydrophilic chelator desferrioxamine prevented cardiac Fe loading and limited cardiac hypertrophy in mutants but did not lead to overt cardiac Fe depletion or toxicity. Fe chelation did not prevent decreased succinate dehydrogenase expression in the mutants or loss of cardiac function. In summary, we show that loss of Fxn markedly alters cellular Fe trafficking and that Fe chelation limits myocardial hypertrophy in the mutant.
Plasma membranes are poised for activation of raft phase coalescence at physiological temperature
Authors: Lingwood Daniel , Ries Jonas , Schwille Petra , Simons Kai
Published: July 2008

Cell membranes are not randomly organized, but rather are populated by fluctuating nanoassemblies of increased translational order termed lipid rafts. This lateral heterogeneity can be biophysically extended because cooling formaldehyde-isolated plasma membrane preparations results in separation into phases similar to the liquid-ordered (Lo) and liquid-disordered (Ld) states seen in model membrane systems [Baumgart T,
et al.
(2007)
Proc Natl Acad Sci USA
104:3165–3170]. In this work we demonstrate that raft clustering, i.e., amplifying underlying raft-based connectivity to a larger scale, makes an analogous capacity accessible at 37°C. In plasma membranes at this temperature, cholera toxin-mediated cross-linking of the raft ganglioside GM1 induced the sterol-dependent emergence of a slower diffusing micrometer-scale phase that was enriched in cholesterol and selectively reorganized the lateral distribution of membrane proteins. Although parallels can be drawn, we argue that this raft coalescence in a complex biological matrix cannot be explained by only those interactions that define Lo formation in model membranes. Under this light, our induction of raft-phase separation suggests that plasma membrane composition is poised for selective and functional raft clustering at physiologically relevant temperature.
Ecosystem services: From theory to implementation
Authors: Daily Gretchen C. , Matson Pamela A.
Published: July 2008

Around the world, leaders are increasingly recognizing ecosystems as natural capital assets that supply life-support services of tremendous value. The challenge is to turn this recognition into incentives and institutions that will guide wise investments in natural capital, on a large scale. Advances are required on three key fronts, each featured here: the science of ecosystem production functions and service mapping; the design of appropriate finance, policy, and governance systems; and the art of implementing these in diverse biophysical and social contexts. Scientific understanding of ecosystem production functions is improving rapidly but remains a limiting factor in incorporating natural capital into decisions, via systems of national accounting and other mechanisms. Novel institutional structures are being established for a broad array of services and places, creating a need and opportunity for systematic assessment of their scope and limitations. Finally, it is clear that formal sharing of experience, and defining of priorities for future work, could greatly accelerate the rate of innovation and uptake of new approaches.
Host plants and associated trophobionts of the weaver ants
Oecophylla
spp. (Hymenoptera: Formicidae).
Authors: Lim G. T. , Kirton L. G. , Salom S. M. , Kok L. T. , Fell R. D. , Pfeiffer D. G.
Published: July 2008

Weaver ants (
Oecophylla
spp.) are often found on plants with insect symbionts (trophobionts), but the extent of such associations is not known. Examination of literature records of weaver ant host plants from 1900 to 2006 revealed that
Oecophylla smaragdina
, native to Asia, was recorded on 175 plant species in 46 families, with 28 associated trophobiont species in 7 families.
Oecophylla longinoda
, native to Africa, was recorded on 66 plant species in 34 families with 17 associated trophobiont species in 6 families. Both
Oecophylla
spp. shared host records on 17 economically important plant species. Such host plants could be used to augment establishment of weaver ants, facilitating their role as deterrents of phytophagous insect pests of economically important plants.
O. smaragdina
-tended trophobionts were recorded associating with the ants on several crops but rarely considered to be pests. Thus, the risk of trophobionts associated with
Oecophylla
ants being pests is considered minimal.
Large-core needle biopsy versus fine-needle aspiration biopsy in solid breast lesions: comparison of costs and diagnostic value
Authors: Vimpeli S.-M. , Saarenmaa I. , Huhtala H. , Soimakallio S.
Published: July 2008

Background: In the current climate of budget constraints and personnel shortages, hospitals are required to demonstrate ever-greater cost effectiveness. In the diagnosis of breast lesions, it is useful to compare the costs and benefits of fine-needle aspiration biopsy (FNAB) and core-needle biopsy (CNB).

Purpose: To determine the total costs of FNAB and CNB of breast lesions, and to compare the costs and diagnostic value of these methods.

Material and Methods: The material consisted of 688 breast lesion cases from a Finnish breast-imaging unit. All cases underwent FNAB and/or CNB. Based on the primary biopsy method used, the cases were allocated to the FNAB ( n=590) or CNB ( n=98) group. Data on costs, sensitivity, and specificity were used to compare the cost effectiveness of the two methods.

Results: 590 FNABs were performed in the FNAB group and 98 CNBs in the CNB group. In addition, we needed 78 CNBs in the FNAB group and 11 FNABs in the CNB group for further assessment. The false-negative rate for FNAB was 19% and for CNB 11%; the false-positive rates were 9% and 1%, respectively. The average cost of biopsies per case in the FNAB group was €66 and in the CNB group €221. FNAB was most expensive when performed under ultrasound guidance (€44.65) and CNB when performed under stereotactic guidance (€246.37).

Conclusion: FNAB is a less reliable and less informative diagnostic method than CNB. Although a negative or indeterminate FNAB result requires follow-up or a re-biopsy with core needle, it is still a cost-effective procedure. Stereotactic guidance considerably increases the costs of CNB, and therefore US guidance should be used whenever possible.
On conformally Kähler, Einstein manifolds
Authors: Chen Xiuxiong , LeBrun Claude , Weber Brian
Published: July 2008

We prove that any compact complex surface with

c
1

>
0

c_1>0

admits an Einstein metric which is conformally related to a Kähler metric. The key new ingredient is the existence of such a metric on the blow-up

C
P

2

#
2

C
P

2

¯

\mathbb {CP}_2\# 2\overline {\mathbb {CP}_2}

of the complex projective plane at two distinct points.
Disk enumeration on the quintic 3-fold
Authors: Pandharipande R. , Solomon J. , Walcher J.
Published: July 2008

Holomorphic disk invariants with boundary in the real Lagrangian of a quintic 3-fold are calculated by localization and proven mirror transforms. A careful discussion of the underlying virtual intersection theory is included. The generating function for the disk invariants is shown to satisfy an extension of the Picard-Fuchs differential equations associated to the mirror quintic. The Ooguri-Vafa multiple cover formula is used to define virtually enumerative disk invariants. The results may also be viewed as providing a virtual enumeration of real rational curves on the quintic.
Correction to “Formal degrees and adjoint 𝛾-factors”
Authors: Hiraga Kaoru , Ichino Atsushi , Ikeda Tamotsu
Published: July 2008

This is a correction to the paper Formal degrees and adjoint

γ
\gamma

-factors, J. Amer. Math. Soc. 21 (2008), 283–304.
Phosphatidylinositol (4,5)-bisphosphate Modulates Nox5 Localization via an N-Terminal Polybasic Region
Authors: Kawahara Tsukasa , Lambeth J. David
Published: July 2008

Nox5, an EF-hand–containing reactive oxygen species (ROS)-generating NADPH oxidase, contains two conserved polybasic regions: one N-terminal (PBR-N), located between the fourth EF-hand and the first transmembrane region, and one C-terminal (PBR-C), between the first and second NADPH-binding subregions. Here, we show that phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P2], a major phosphoinositide in plasma membrane, binds to human Nox5 causing Nox5 to localize from internal membranes to the plasma membrane. Enzymatic modulation of PtdIns(4,5)P2levels in intact cells altered cell surface localization of Nox5 in parallel with extracellular ROS generation. Mutations in PBR-N prevented PtdIns(4,5)P2-dependent localization of Nox5 to the plasma membrane and decreased extracellular ROS production. A synthetic peptide corresponding to PBR-N bound to PtdIns(4,5)P2, but not to PtdIns, whereas mutations in the PBR-N peptide abrogated the binding to PtdIns(4,5)P2. Arginine-197 in PBR-N was a key residue to regulate subcellular localization of Nox5 and its interaction with PtdIns(4,5)P2. In contrast, mutation in PBR-C did not affect localization. Thus, extracellular ROS production by Nox5 is modulated by PtdIns(4,5)P2by localizing Nox5 to the plasma membrane.
Drosophila Sec16 Mediates the Biogenesis of tER Sites Upstream of Sar1 through an Arginine-Rich Motif
Authors: Ivan Viorica , de Voer Gert , Xanthakis Despina , Spoorendonk Kirsten M. , Kondylis Vangelis , Rabouille Catherine
Published: July 2008

tER sites are specialized cup-shaped ER subdomains characterized by the focused budding of COPII vesicles. Sec16 has been proposed to be involved in the biogenesis of tER sites by binding to COPII coat components and clustering nascent-coated vesicles. Here, we show that Drosophila Sec16 (dSec16) acts instead as a tER scaffold upstream of the COPII machinery, including Sar1. We show that dSec16 is required for Sar1-GTP concentration to the tER sites where it recruits in turn the components of the COPII machinery to initiate coat assembly. Last, we show that the dSec16 domain required for its localization maps to an arginine-rich motif located in a nonconserved region. We propose a model in which dSec16 binds ER cups via its arginine-rich domain, interacts with Sar1-GTP that is generated on ER membrane by Sec12 and concentrates it in the ER cups where it initiates the formation of COPII vesicles, thus acting as a tER scaffold.
The Yeast Aac2 Protein Exists in Physical Association with the Cytochromebc1-COX Supercomplex and the TIM23 Machinery
Authors: Dienhart Mary K. , Stuart Rosemary A.
Published: July 2008

The ADP/ATP carrier (AAC) proteins play a central role in cellular metabolism as they facilitate the exchange of ADP and ATP across the mitochondrial inner membrane. We present evidence here that in yeast (Saccharomyces cerevisiae) mitochondria the abundant Aac2 isoform exists in physical association with the cytochrome c reductase (cytochrome bc1)-cytochrome c oxidase (COX) supercomplex and its associated TIM23 machinery. Using a His-tagged Aac2 derivative and affinity purification studies, we also demonstrate here that the Aac2 isoform can be affinity-purified with other AAC proteins. Copurification of the Aac2 protein with the TIM23 machinery can occur independently of its association with the fully assembled cytochrome bc1-COX supercomplex. In the absence of the Aac2 protein, the assembly of the cytochrome bc1-COX supercomplex is perturbed, whereby a decrease in the III2-IV2assembly state relative to the III2-IV form is observed. We propose that the association of the Aac2 protein with the cytochrome bc1-COX supercomplex is important for the function of the OXPHOS complexes and for the assembly of the COX complex. The physiological implications of the association of AAC with the cytochrome bc1-COX-TIM23 supercomplex are also discussed.
Nicotiana benthamiana: Its History and Future as a Model for Plant–Pathogen Interactions
Authors: Goodin Michael M. , Zaitlin David , Naidu Rayapati A. , Lommel Steven A.
Published: July 2008

Nicotiana benthamiana is the most widely used experimental host in plant virology, due mainly to the large number of diverse plant viruses that can successfully infect it. Additionally, N. benthamiana is susceptible to a wide variety of other plant-pathogenic agents (such as bacteria, oomycetes, fungi, and so on), making this species a cornerstone of host–pathogen research, particularly in the context of innate immunity and defense signaling. Moreover, because it can be genetically transformed and regenerated with good efficiency and is amenable to facile methods for virus-induced gene silencing or transient protein expression, N. benthamiana is rapidly gaining popularity in plant biology, particularly in studies requiring protein localization, interaction, or plant-based systems for protein expression and purification. Paradoxically, despite being an indispensable research model, little is known about the origins, genetic variation, or ecology of the N. benthamiana accessions currently used by the research community. In addition to addressing these latter topics, the purpose of this review is to provide information regarding sources for tools and reagents that can be used to support research in N. benthamiana. Finally, we propose that N. benthamiana is well situated to become a premier plant cell biology model, particularly for the virology community, who as a group were the first to recognize the potential of this unique Australian native.
Evaluation of Constitutive Viral Promoters in Transgenic Soybean Roots and Nodules
Authors: Govindarajulu Manjula , Elmore James M. , Fester Thomas , Taylor Christopher G.
Published: July 2008

The efficiency of β-glucuronidase (GUS) expression was evaluated with five viral promoters to identify the most suitable promoter or promoters for use in soybean hairy roots, including applications to study the symbiotic interaction with Bradyrhizobium japonicum. Levels of GUS activity were fluorimetrically and histochemically assayed when the GUS (uidA) gene was driven by the Cauliflower mosaic virus (CaMV) 35S promoter and enhanced 35S (E35S) promoter, the Cassava vein mosaic virus (CsVMV) promoter, the Figwort mosaic virus (FMV) promoter, and the Strawberry vein banding virus (SVBV2) promoter. We demonstrate that GUS activity was highest when driven by the FMV promoter and that the promoter activity of 35S and SVBV2 was significantly lower than that of the CsVMV and E35S promoters when tested in soybean hairy roots. In mature soybean root nodules, strong GUS activity was evident when the FMV, 35S, and CsVMV promoters were used. These results indicate that the FMV promoter facilitates the strong expression of target genes in soybean hairy roots and root nodules.
A Single Amino Acid of NIaPro of Papaya ringspot virus Determines Host Specificity for Infection of Papaya
Authors: Chen Kuan-Chun , Chiang Chu-Hui , Raja Joseph A. J. , Liu Fang-Lin , Tai Chun-Hsi , Yeh Shyi-Dong
Published: July 2008

Most strains of Papaya ringspot virus (PRSV) belong to type W, causing severe loss on cucurbits worldwide, or type P, devastating papaya in tropical areas. While the host range of PRSV W is limited to plants of the families Chenopodiaceae and Cucuribitaceae, PRSV P, in addition, infects plants of the family Caricaceae (papaya family). To investigate one or more viral genetic determinants for papaya infection, recombinant viruses were constructed between PRSV P-YK and PRSV W-CI. Host reactions to recombinant viruses indicated that the viral genomic region covering the C-terminal region (142 residues) of NIaVPg, full NIaPro, and N-terminal region (18 residues) of NIb, is critical for papaya infection. Sequence analysis of this region revealed residue variations at position 176 of NIaVPg and positions 27 and 205 of NIaPro between type P and W viruses. Host reactions to the constructed mutants indicated that the amino acid Lys27 of NIaPro determines the host-specificity of PRSV for papaya infection. Predicted three-dimensional structures of NIaPros of parental viruses suggested that Lys27 does not affect the protease activity of NIaPro. Recovery of the infected plants from certain papaya-infecting mutants implied involvement of other viral factors for enhancing virulence and adaptation of PRSV on papaya.
Expression of the Bradyrhizobium japonicum Type III Secretion System in Legume Nodules and Analysis of the Associated tts box Promoter
Authors: Zehner Susanne , Schober Grit , Wenzel Mandy , Lang Kathrin , Göttfert Michael
Published: July 2008

In Bradyrhizobium japonicum, as in some other rhizobia, symbiotic efficiency is influenced by a type III secretion system (T3SS). Most genes encoding the transport machinery and secreted proteins are preceded by a conserved 30-bp motif, the type-three secretion (tts) box. In this study, we found that regions downstream of 34 tts boxes are transcribed. For nopB, nopL, and gunA2, the transcriptional start sites were found to be 12, 11, and 10 bp downstream of their tts boxes, respectively. The deletion of this motif or modification of two or more conserved residues strongly reduced expression of nopB. This indicates that the tts box is an essential promoter element. Data obtained with lacZ reporter gene fusions of five genes preceded by a tts box (gunA2, nopB, rhcV, nopL, and blr1806) revealed that they are expressed in 4-week-old nodules of Macroptilium atropurpureum. These data suggest that the T3SS is active in mature nitrogen-fixing nodules. The two-component response regulator TtsI is required for the expression of rhcV, nopL, and blr1806 in bacteroids. Staining of inoculated roots showed that nopB is also expressed in early infection stages.
Quorum-Sensing System Affects Gall Development Incited by Pantoea agglomerans pv. gypsophilae
Authors: Chalupowicz Laura , Manulis-Sasson Shulamit , Itkin Maxim , Sacher Ayelet , Sessa Guido , Barash Isaac
Published: July 2008

The quorum-sensing (QS) regulatory system of the gall-forming Pantoea agglomerans pv. gypsophilae was identified. Mass spectral analysis, together with signal-specific biosensors, demonstrated that P. agglomerans pv. gypsophilae produced N-butanoyl-l-homoserine lactone (C4-HSL) as a major and N-hexanoyl-l-homoserine lactone (C6-HSL) as a minor QS signal. Homologs of luxI and luxR regulatory genes, pagI and pagR, were characterized in strain P. agglomerans pv. gypsophilae Pag824-1 and shown to be convergently transcribed and separated by 14 bp. The deduced PagI (23.8 kDa) and PagR (26.9 kDa) show high similarity with SmaI (41% identity) and SmaR (43% identity), respectively, of Serratia sp. American Type Culture Collection 39006. PagR possesses characteristic autoinducer binding and a helix-turn-helix DNA-binding domain. Gall formation by P. agglomerans pv. gypsophilae depends on a plasmid-borne hrp/hrc gene cluster, type III effectors, and phytohormones. Disruption of pagI, pagR, or both genes simultaneously in Pag824-1 reduced gall size in gypsophila cuttings by 50 to 55% when plants were inoculated with 106 CFU/ml. Higher reductions in gall size (70 to 90%) were achieved by overexpression of pagI or addition of exogenous C4-HSL. Expression of the hrp/hrc regulatory gene hrpL and the type III effector pthG in the pagI mutant, as measured with quantitative reverse-transcriptase polymerase chain reaction, was reduced by 5.8 and 6.6, respectively, compared with the wild type, suggesting an effect of the QS system on the Hrp regulon.
Leaf Stripe and Stem Rot Caused by Burkholderia gladioli, a New Disease of Maize in México
Authors: Gijón-Hernandez A. , Téliz-Ortiz D. , Cárdenas-Soriano E. , De León C. , Mora-Aguilera A. , Mejía-Sánchez D. , De La Torre-Almaraz R.
Published: July 2008

A new maize disease appeared in the State of Veracruz, Mexico during 2003–2004. Initial symptoms in the leaves were small, white-yellow, watery spots, which coalesced into dry necrotic stripes that were 0.3 wide and 8 cm long. Reddening sometimes developed on these leaves. Stems developed a rot in the crown. The flag leaf showed a rot and necrosis at the base, rolled inward, and dried out. Necrosis developed at the base of the corn ears and their growth was halted. A bacterium characterized by white colonies was consistently isolated from lesions on casamino acid peptone and glucose (CPG), King's medium B, and nutrient agar media. Ten isolates were chosen for further characterization. Pathogenicity was confirmed in the greenhouse (25 to 30°C) on 45-day-old ASGROW 7573 maize plants by injection of bacterial suspensions (107 CFU/ml) at the base of the stem. Control maize seedlings were injected with phosphate buffer. Symptoms similar to those observed in the field were observed after 3 days on all inoculated plants but were not observed on control plants. Koch's postulates were fulfilled with reisolation of the inoculated strain from the inoculated tissues and confirmation was fulfilled by phenotypic characters and 16S rDNA sequences. The white colonies on CPG were slightly convex, shiny, circular with entire margins, gram negative, lacked arginine dihydrolase, did not produce fluorescent pigment on Pseudomonas F medium, and grew aerobically. The strains were able to utilize l-arabinose, d-mannitol, and cellobiose, but unable to utilize d-maltose and l-rhamnose. Gel hydrolysis was positive but starch hydrolysis was not positive. Colonies grew at 40°C. These characteristics are the same as those described previously for Burkholderia gladioli (3). The identity of these isolates was confirmed by 16S analysis with two universal primers, FD1 and RD1, for Eubacteria (2), which generate a 1,600-bp fragment. Two primers specific for the genus Burhkolderia, RHG-F and RHG-R that generate a 500-bp fragment (1) were also used for amplification. Comparison of sequences using Clustal W and Megaline, DNAstar software showed 100% similarity among strains isolated from the three counties (GenBank Accession Nos. EU161873 to EU161878) and 99% similarity of field isolates with B. gladioli in the GenBank database. To our knowledge, this is the first report of B. gladioli causing leaf stripe and stem rot of maize in Mexico.

References: (1) J. J. Lipuma et al. J. Clin. Microbiol. 37:3167, 1999. (2) L. M. Rodrigues et al. Appl. Environ. Microbiol. 69:4249, 2003. 3) N. W. Shaad et al., eds. 3rd ed. Laboratory Guide for Identification of Plant Pathogenic Bacteria. The American Phytopathological Society, St. Paul, MN, 2001.
GR24, a Synthetic Analog of Strigolactones, Stimulates the Mitosis and Growth of the Arbuscular Mycorrhizal FungusGigaspora roseaby Boosting Its Energy Metabolism
Authors: Besserer Arnaud , Bécard Guillaume , Jauneau Alain , Roux Christophe , Séjalon-Delmas Nathalie
Published: July 2008

Arbuscular mycorrhizal (AM) fungi are obligate biotrophs that participate in a highly beneficial root symbiosis with 80% of land plants. Strigolactones are trace molecules in plant root exudates that are perceived by AM fungi at subnanomolar concentrations. Within just a few hours, they were shown to stimulate fungal mitochondria, spore germination, and branching of germinating hyphae. In this study we show that treatment of Gigaspora rosea with a strigolactone analog (GR24) causes a rapid increase in the NADH concentration, the NADH dehydrogenase activity, and the ATP content of the fungal cell. This fully and rapidly (within minutes) activated oxidative metabolism does not require new gene expression. Up-regulation of the genes involved in mitochondrial metabolism and hyphal growth, and stimulation of the fungal mitotic activity, take place several days after this initial boost to the cellular energy of the fungus. Such a rapid and powerful action of GR24 on G. rosea cells suggests that strigolactones are important plant signals involved in switching AM fungi toward full germination and a presymbiotic state.
Mobilization of Rubisco and Stroma-Localized Fluorescent Proteins of Chloroplasts to the Vacuole by anATGGene-Dependent Autophagic Process
Authors: Ishida Hiroyuki , Yoshimoto Kohki , Izumi Masanori , Reisen Daniel , Yano Yuichi , Makino Amane , Ohsumi Yoshinori , Hanson Maureen R. , Mae Tadahiko
Published: July 2008

During senescence and at times of stress, plants can mobilize needed nitrogen from chloroplasts in leaves to other organs. Much of the total leaf nitrogen is allocated to the most abundant plant protein, Rubisco. While bulk degradation of the cytosol and organelles in plants occurs by autophagy, the role of autophagy in the degradation of chloroplast proteins is still unclear. We have visualized the fate of Rubisco, stroma-targeted green fluorescent protein (GFP) and DsRed, and GFP-labeled Rubisco in order to investigate the involvement of autophagy in the mobilization of stromal proteins to the vacuole. Using immunoelectron microscopy, we previously demonstrated that Rubisco is released from the chloroplast into Rubisco-containing bodies (RCBs) in naturally senescent leaves. When leaves of transgenic Arabidopsis (Arabidopsis thaliana) plants expressing stroma-targeted fluorescent proteins were incubated with concanamycin A to inhibit vacuolar H+-ATPase activity, spherical bodies exhibiting GFP or DsRed fluorescence without chlorophyll fluorescence were observed in the vacuolar lumen. Double-labeled immunoelectron microscopy with anti-Rubisco and anti-GFP antibodies confirmed that the fluorescent bodies correspond to RCBs. RCBs could also be visualized using GFP-labeled Rubisco directly. RCBs were not observed in leaves of a T-DNA insertion mutant in ATG5, one of the essential genes for autophagy. Stroma-targeted DsRed and GFP-ATG8 fusion proteins were observed together in autophagic bodies in the vacuole. We conclude that Rubisco and stroma-targeted fluorescent proteins can be mobilized to the vacuole through an ATG gene-dependent autophagic process without prior chloroplast destruction.
SENSITIVE TO FREEZING6 Integrates Cellular and Environmental Inputs to the Plant Circadian Clock
Authors: Knight Heather , Thomson Adrian J.W. , McWatters Harriet G.
Published: July 2008

The sensitive to freezing6 (sfr6) mutant of Arabidopsis (Arabidopsis thaliana) is late flowering in long days due to reduced expression of components in the photoperiodic flowering pathway in long-day photoperiods. Microarray analysis of gene expression showed that a circadian clock-associated motif, the evening element, was overrepresented in promoters of genes down-regulated in sfr6 plants. Analysis of leaf movement rhythms found sfr6 plants showed a sucrose (Suc)-dependent long period phenotype; unlike wild-type Arabidopsis, the clock in sfr6 plants did not have a shorter rhythm in the presence of Suc. Other developmental responses to Suc were unaltered in sfr6 plants, suggesting insensitivity to Suc is restricted to the clock. We investigated the effect of sfr6 and Suc upon clock gene expression over 24 h. The sfr6 mutation resulted in reduced expression of the clock components CIRCADIAN CLOCK ASSOCIATED1, GIGANTEA, and TIMING OF CAB1. These changes occurred independently of Suc supplementation. Wild-type plants showed small increases in clock gene expression in the presence of Suc; this response to Suc was reduced in sfr6 plants. This study shows that large changes in level and timing of clock gene expression may have little effect upon clock outputs. Moreover, although Suc influences the period and accuracy of the Arabidopsis clock, it results in relatively minor changes in clock gene expression.
The Medium‐Chain Dehydrogenase/Reductase Engineering Database: A systematic analysis of a diverse protein family to understand sequence–structure–function relationship
Authors: Knoll Michael , Pleiss Jürgen
Published: July 2008

The Medium‐Chain Dehydrogenase/Reductase Engineering Database (MDRED, http://www.mdred.uni‐stuttgart.de) has been established to serve as an analysis tool for a systematic investigation of sequence–structure–function relationships. It includes sequence and structure information of 2684 and 42 medium‐chain dehydrogenases/reductases (MDRs), respectively. Although MDRs are very diverse in sequence, they have a conserved tertiary structure. MDRs are assigned to 199 homologous families and 29 superfamilies. For each family, annotated multiple sequence alignments are provided, and functionally relevant residues are annotated. Twenty‐five superfamilies were classified as zinc‐containing MDRs, four as non‐zinc‐containing MDRs. For the zinc‐containing MDRs, three subclasses were identified by systematic analysis of a variable loop region, the quaternary structure determining loop (QSDL): the class of short, medium, and long QSDL, which include 11, 3, and 5 superfamilies, respectively. The length of the QSDL is predictive for tetramer (short QSDL) and dimer (long QSDL) formation. The class of medium QSDL includes both tetrameric and dimeric MDRs. The shape of the substrate‐binding site is highly conserved in all zinc‐containing MDRs with the exception of two variable regions, the substrate recognition sites (SRS): two residues located on the QSDL (SRS1) and, for the class of long QSDL, one residue located in the catalytic domain (SRS2). The MDRED is the first online‐accessible resource of MDRs that integrates information on sequence, structure, and function. Annotation of functionally relevant residues assist the understanding of sequence–structure–function relationships. Thus, the MDRED serves as a valuable tool to identify potential hotspots for engineering properties such as substrate specificity.
Bullous pemphigoid with a prolonged prodrome
Authors: NAKATANI Tomomi , INAOKI Makoto , TAKEHARA Kazuhiko
Published: July 2008

Bullous pemphigoid is an autoimmune blistering skin disease of the elderly that may be preceded by a pruritic, urticarial or eczematous eruption. We report a case of bullous pemphigoid preceded by prodromal eczematous eruptions that lasted an unusually long time of 11 years. Elderly patients with persistent pruritic or eczematous eruption of unknown etiology should be carefully followed, as bullous pemphigoid may be a potential diagnosis.
Palisading granuloma presenting in a patient with systemic lupus erythematosus
Authors: OHTSUKA Tsutomu
Published: July 2008

A patient with systemic lupus erythematosus who developed a distinctive nodular eruption demonstrating typical features of palisading granuloma is presented herein. A 60‐year‐old woman was admitted complaining of an elastic‐hard, indurated nodule on the right dorsal aspect of the metacarpophalangeal joint of her third finger with a history of several years. She was successfully treated for her lupus nephritis with oral predonisolone. She had experienced erythema on her cheeks after sun exposure and polyarthralgia for a decade. Laboratory examination revealed positive results for antinuclear antibody and rheumatoid factor. Peripheral blood cell counts showed leukocytopenia and lymphocytopenia. Results of hand X‐ray were unremarkable. Histological examination of the skin biopsy specimen from the nodule revealed a structure composed of a central area of fibrinoid necrosis, surrounded by a middle zone of palisading cells and an outer zone of chronic lymphocyte infiltrate. These findings led us to the histological diagnosis of palisading granuloma. She was diagnosed as having systemic lupus erythematosus complicated with a rheumatoid nodule. She is currently under treatment with cyclophosphamide (50 mg/day) without exaggeration of her systemic lupus erythematosus and nodule.
Cutaneous extramedullary hematopoiesis in a patient with idiopathic myelofibrosis
Authors: MIYATA Toshiko , MASUZAWA Mikio , KATSUOKA Kensei , HIGASHIHARA Masaaki
Published: July 2008

Idiopathic myelofibrosis (IM) is a chronic myeloproliferative disorder and some cases of IM have extramedullary hematopoiesis. Extramedullary hematopoiesis is commonly seen in the liver, spleen and lymph nodes, but cutaneous extramedullary hematopoiesis (CEH) is very rare in cases of IM. We report a case of CEH in a 65‐year‐old Japanese woman with IM. This patient had many hard brownish nodules on her chest, abdomen and scalp. Histopathological examination of the nodule on her chest showed the existence of various stages of immature erythrocytes, leukocytes and megakaryocytes indicating that these hard nodules showed extramedullary hematopoiesis in the dermis. The proliferation of these immature cells in the dermis plays an important role in the pathogenesis of CEH. CEH is a very rare manifestation of IM and progresses slowly.
Adult cutaneous alveolar rhabdomyosarcoma on the face diagnosed by the expression of PAX3‐FKHR gene fusion transcripts
Authors: NAKAGAWA Noboru , TSUDA Tatsuya , YAMAMOTO Mika , ITO Takaaki , FUTANI Hiroshi , YAMANISHI Kiyofumi
Published: July 2008

A 26‐year‐old woman presented with an indurated subcutaneous tumor on her left cheek. The histology was compatible with alveolar rhabdomyosarcoma, but immunohistochemistry showed that the tumor cells were negative for desmin, α‐smooth muscle actin and α‐Sr‐1, but were positive for CD56, vimentin and myogenin. The diagnosis of alveolar rhabdomyosarcoma was confirmed by the detection of PAX3‐FKHR fusion gene transcripts in the paraffin‐embedded tumor tissue. The tumor was unresponsive to chemotherapy with pirarubicin, carboplatin and ifosfamide, and the patient died 9 months after the diagnosis. This adult case of an alveolar rhabdomyosarcoma primarily occurring on the face is very rare, and the detection of PAX3‐FKHR fusion gene transcripts was useful for diagnosis of the disease.