bluuebunny/crossref_metadata_2025 huggingface dataset:
Expansions, contractions, and fragility of the spinocerebellar ataxia type 10 pentanucleotide repeat in yeast
Authors: Cherng Nicole , Shishkin Alexander A. , Schlager Lucas I. , Tuck Ryan H. , Sloan Laura , Matera Robert , Sarkar Partha S. , Ashizawa Tetsuo , Freudenreich Catherine H. , Mirkin Sergei M.
Published: February 2011

Spinocerebellar ataxia 10 (SCA10) is an autosomal dominant disease caused by large-scale expansions of the (ATTCT)
n
repeat within an intron of the human
ATXN10
gene. In contrast to other expandable repeats, this pentanucleotide repeat does not form stable intra- or interstranded DNA structures, being a DNA unwinding element instead. We analyzed the instability of the (ATTCT)
n
repeat in a yeast experimental system, where its expansions led to inactivation of the
URA3
reporter gene. The inactivation was due to a dramatic decrease in the mRNA levels owing to premature transcription termination and RNA polyadenylation at the repeat. The rates of expansions strongly increased with the repeat's length, mimicking genetic anticipation in human pedigrees. A first round of genetic analysis showed that a functional
TOF1
gene precludes, whereas a functional
RAD5
gene promotes, expansions of the (ATTCT)
n
repeat. We hypothesize that repeat expansions could occur upon fortuitous template switching during DNA replication. The rate of repeat contractions was elevated in the Tof1 knockout strain, but it was not affected by the
RAD5
gene. Supporting the notion of replication irregularities, we found that (ATTCT)
n
repeats also cause length-dependent chromosomal fragility in yeast. Repeat-mediated fragility was also affected by the Tof1 and Rad5 proteins, being reduced in their absence.
Human endothelial cells generate Th17 and regulatory T cells under inflammatory conditions
Authors: Taflin Cécile , Favier Benoit , Baudhuin Jeremy , Savenay Alain , Hemon Patrice , Bensussan Armand , Charron Dominique , Glotz Denis , Mooney Nuala
Published: February 2011

Organ transplantation represents a unique therapeutic option for irreparable organ dysfunction and rejection of transplants results from a breakdown in operational tolerance. Although endothelial cells (ECs) are the first target in graft rejection following kidney transplantation, their capacity to alloactivate and generate particular T lymphocyte subsets that could intervene in this process remains unknown. By using an experimental model of microvascular endothelium, we demonstrate that, under inflammatory conditions, human ECs induced proliferation of memory CD4+CD45RA−T cells and selectively amplified proinflammatory Th17 and suppressive CD45RA−HLA-DR+FoxP3brightregulatory CD4+T lymphocytes (Tregs). Although HLA-DR expression on resting microvascular ECs was sufficient to induce proliferation of memory CD4+T cells, Treg amplification was dependent on the interaction with CD54, highly expressed only under inflammatory conditions. Moreover, expansion of Th17 cells was dependent on IL-6 and STAT-3, and inhibition of either specifically impaired Th17, without altering Treg expansion. Collectively these data reveal that the HLA-DR+ECs regulate the local inflammatory allogeneic response, promoting either an IL-6/STAT-3–dependent Th17 response or a contact-CD54–dependent regulatory response according to the cytokine environment. Finally, these data open therapeutic perspectives in human organ transplantation based on targeting the IL-6/STAT-3 pathway and/or promoting CD54 dependent Treg proliferation.
Serotonin reverts age-related capillarization and failure of regeneration in the liver through a VEGF-dependent pathway
Authors: Furrer Katarzyna , Rickenbacher Andreas , Tian Yinghua , Jochum Wolfram , Bittermann Anne Greet , Käch Andres , Humar Bostjan , Graf Rolf , Moritz Wolfgang , Clavien Pierre-Alain
Published: February 2011

The function of the liver is well-preserved during the aging process, although some evidence suggests that liver regeneration might be impaired with advanced age. We observed a decreased ability of the liver to restore normal volume after partial hepatectomy in elderly mice, and we identified a pathway that rescued regeneration and was triggered by serotonin. 2,5-dimethoxy-4-iodoamphetamine (DOI), a serotonin receptor agonist, reversed the age-related pseudocapillarization of old liver and improved hepatosinusoidal blood flow. After hepatectomy, the open fenestrae were associated with a restored attachment of platelets to endothelium and the initiation of a normal regenerative response, including the up-regulation of essential growth mediators and serotonin receptors. In turn, hepatocyte proliferation recovered along with regain of liver volume and animal survival. DOI operates through the release of VEGF, and its effects could be blocked with anti-VEGF antibodies both in vitro and in vivo. These results suggest that pseudocapillarization in the aged acts as a barrier to liver regeneration. DOI breaks this restraint through an endothelium-dependent mechanism driven by VEGF. This pathway highlights a target for reversing the age-associated decline in the capacity of the liver to regenerate.
Hundred years of genetic structure in a sediment revived diatom population
Authors: Härnström Karolina , Ellegaard Marianne , Andersen Thorbjørn J. , Godhe Anna
Published: February 2011

This paper presents research on the genetic structure and diversity of populations of a common marine protist and their changes over time. The bloom-forming diatom
Skeletonema marinoi
was used as a model organism. Strains were revived from anoxic discrete layers of a
210
Pb-dated sediment core accumulated over more than 100 y, corresponding to >40,000 diatom mitotic generations. The sediment core was sampled from the highly eutrophic Mariager Fjord in Denmark. The genetic structure of
S. marinoi
was examined using microsatellite markers, enabling exploration of changes through time and of the effect of environmental fluctuations. The results showed a stable population structure among and within the examined sediment layers, and a similar genetic structure has been maintained over thousands of generations. However, established populations from inside the fjord were highly differentiated from open-sea populations. Despite constant water exchange and influx of potential colonizers into the fjord, the populations do not mix. One fjord population, accumulated in 1980, was significantly differentiated from the other groups of strains isolated from the fjord. This differentiation could have resulted from the status of Mariager Fjord, which was considered hypereutrophic, around 1980. There was no significant genetic difference between pre- and posteutrophication groups of strains. Our data show that dispersal potential and generation time do not have a large impact on the genetic structuring of the populations investigated here. Instead, the environmental conditions, such as the extreme eutrophication of the Mariager Fjord, are deemed more important.
Lethal Arg9Cys phospholamban mutation hinders Ca
2+
-ATPase regulation and phosphorylation by protein kinase A
Authors: Ha Kim N. , Masterson Larry R. , Hou Zhanjia , Verardi Raffaello , Walsh Naomi , Veglia Gianluigi , Robia Seth L.
Published: February 2011

The regulatory interaction of phospholamban (PLN) with Ca
2+
-ATPase controls the uptake of calcium into the sarcoplasmic reticulum, modulating heart muscle contractility. A missense mutation in PLN cytoplasmic domain (R9C) triggers dilated cardiomyopathy in humans, leading to premature death. Using a combination of biochemical and biophysical techniques both in vitro and in live cells, we show that the R9C mutation increases the stability of the PLN pentameric assembly via disulfide bridge formation, preventing its binding to Ca
2+
-ATPase as well as phosphorylation by protein kinase A. These effects are enhanced under oxidizing conditions, suggesting that oxidative stress may exacerbate the cardiotoxic effects of the PLN
R9C
mutant. These results reveal a regulatory role of the PLN pentamer in calcium homeostasis, going beyond the previously hypothesized role of passive storage for active monomers.
CAF-I-dependent control of degradation of the discontinuous strands during mismatch repair
Authors: Kadyrova Lyudmila Y. , Blanko Elena Rodriges , Kadyrov Farid A.
Published: February 2011

DNA mismatch repair (MMR) is a multifunctional process that promotes genetic stability and suppresses carcinogenesis. Correction of DNA replication errors is its major function. Despite the importance of MMR, its functioning in eukaryotes is not well understood. Here we report that human mismatch correction reactions in cell-free extracts occur during concomitant nick-dependent nucleosome assembly shaped by the replication histone chaperone CAF-I. Concomitant nucleosome assembly protects the discontinuous mismatch-containing strands from excessive degradation by MMR machinery. Such protection is also demonstrated in a defined purified system that supports both mismatch correction and CAF-I-dependent histone H3–H4 deposition reactions. In addition, we find that the mismatch recognition factor MutSα suppresses CAF-I-dependent histone H3–H4 deposition in a mismatch-dependent manner. We suggest that there is active crosstalk between MMR and replication-dependent nucleosome assembly during the correction of DNA replication errors and, as a result, the nascent mismatch-containing strands are degraded in a controlled manner.
Polarity proteins Bem1 and Cdc24 are components of the filamentous fungal NADPH oxidase complex
Authors: Takemoto Daigo , Kamakura Sachiko , Saikia Sanjay , Becker Yvonne , Wrenn Ruth , Tanaka Aiko , Sumimoto Hideki , Scott Barry
Published: February 2011

Regulated synthesis of reactive oxygen species (ROS) by membrane-bound fungal NADPH oxidases (Nox) plays a key role in fungal morphogenesis, growth, and development. Generation of reactive oxygen species (ROS) by the plant symbiotic fungus,
Epichloë festucae
, requires functional assembly of a multisubunit complex composed of NoxA, a regulatory component, NoxR, and the small GTPase RacA. However, the mechanism for assembly and activation of this complex at the plasma membrane is unknown. We found by yeast two-hybrid and coimmunoprecipitation assays that
E. festucae
NoxR interacts with homologs of the yeast polarity proteins, Bem1 and Cdc24, and that the Phox and Bem1 (PB1) protein domains found in these proteins are essential for these interactions. GFP fusions of BemA, Cdc24, and NoxR preferentially localized to actively growing hyphal tips and to septa. These proteins interact with each other in vivo at these same cellular sites as shown by bimolecular fluorescent complementation assays. The PB1 domain of NoxR is essential for localization to the hyphal tip. An
E. festucae
Δ
bemA
mutant was defective in hyphal morphogenesis and growth in culture and
in planta
. The changes in fungal growth
in planta
resulted in a defective symbiotic interaction phenotype. Our inability to isolate a Δ
cdc24
mutant suggests this gene is essential. These results demonstrate that BemA and Cdc24 play a critical role in localizing NoxR protein to sites of fungal hyphal morphogenesis and growth. Our findings identify a potential shared ancestral link between the protein machinery required for fungal polarity establishment and the Nox complex controlling cellular differentiation.
Signaling mechanisms in the restoration of impaired immune function due to diet-induced obesity
Authors: Zhou Qingde , Leeman Susan E. , Amar Salomon
Published: February 2011

Our previous data have linked obesity with immune dysfunction. It is known that physical exercise with dietary control has beneficial effects on immune function and the comorbidities of obesity. However, the mechanisms underlying the improvement of immune function in obesity after physical exercise with dietary control remain unknown. Here we show that moderate daily exercise with dietary control restores the impaired cytokine responses in diet-induced obese (DIO) mice and improves the resolution of
Porphyromonas gingivalis
-induced periodontitis. This restoration of immune responses is related to the reduction of circulating free fatty acids (FFAs) and TNF. Both FFAs and TNF induce an Akt inhibitor, carboxyl-terminal modulator protein (CTMP). The expression of CTMP is also observed increased in bone marrow-derived macrophages (BMMΦ) from DIO mice and restored after moderate daily exercise with dietary control. Toll-like receptor 2 (TLR2), which increases CTMP induction by FFAs, is inhibited in BMMΦ from DIO mice or after either FFA or TNF treatment, but unexpectedly is not restored by moderate daily exercise with dietary control. Furthermore, BMMΦ from DIO mice display reduced histone H3 (Lys-9) acetylation and NF-κB recruitment to TNF, IL-10, and TLR2 promoters after
P. gingivalis
infection. However, moderate daily exercise with dietary control restores these defects at promoters for TNF and IL-10, but not for TLR2. Thus, metabolizing FFAs and TNF by moderate daily exercise with dietary control improves innate immune responses to infection in DIO mice via restoration of CTMP and chromatin modification.
Hypoxia-inducible factor 2α (HIF-2α) heterozygous-null mice exhibit exaggerated carotid body sensitivity to hypoxia, breathing instability, and hypertension
Authors: Peng Ying-Jie , Nanduri Jayasri , Khan Shakil A. , Yuan Guoxiang , Wang Ning , Kinsman Brian , Vaddi Damodara R. , Kumar Ganesh K. , Garcia Joseph A. , Semenza Gregg L. , Prabhakar Nanduri R.
Published: February 2011

Cardiorespiratory functions in mammals are exquisitely sensitive to changes in arterial O
2
levels. Hypoxia-inducible factors (e.g., HIF-1 and HIF-2) mediate transcriptional responses to reduced oxygen availability. We demonstrate that haploinsufficiency for the O
2
-regulated HIF-2α subunit results in augmented carotid body sensitivity to hypoxia, irregular breathing, apneas, hypertension, and elevated plasma norepinephrine levels in adult

Hif-2α
+/−

mice. These dysregulated autonomic responses were associated with increased oxidative stress and decreased mitochondrial electron transport chain complex I activity in adrenal medullae as a result of decreased expression of major cytosolic and mitochondrial antioxidant enzymes. Systemic administration of a membrane-permeable antioxidant prevented oxidative stress, normalized hypoxic sensitivity of the carotid body, and restored autonomic functions in

Hif-2α
+/−

mice. Thus, HIF-2α–dependent redox regulation is required for maintenance of carotid body function and cardiorespiratory homeostasis.
BRCA1 on the move
Authors: Short Ben
Published: February 2011

The tumor suppressor functions outside the nucleus to regulate cell adhesion and migration.
Kolmogorov complexity and strong approximation of Brownian motion
Authors: Kjos-Hanssen Bjørn , Szabados Tamás
Published: February 2011

Brownian motion and scaled and interpolated simple random walk can be jointly embedded in a probability space in such a way that almost surely the

n
n

-step walk is within a uniform distance

O
(

n

−
1

/

2

log
⁡
n
)

O(n^{-1/2}\log n)

of the Brownian path for all but finitely many positive integers

n
n

. Almost surely this

n
n

-step walk will be incompressible in the sense of Kolmogorov complexity, and all Martin-Löf random paths of Brownian motion have such an incompressible close approximant. This strengthens a result of Asarin, who obtained instead the bound

O
(

n

−
1

/

6

log
⁡
n
)

O(n^{-1/6} \log n)

. The result cannot be improved to

o
(

n

−
1

/

2

log
⁡
n

)

o(n^{-1/2}{\sqrt {\log n}})

.
The incidence correspondence and its associated maps in homotopy
Authors: Lopez Luis
Published: February 2011

The incidence correspondence in the grassmannian which determines the tautological bundle defines a map between cycle spaces on grassmannians. These cycle spaces decompose canonically into a product of Eilenberg-MacLane spaces. These decompositions and the associated maps are calculated up to homotopy.
On local Khintchine inequalities for spaces of exponential integrability
Authors: Carrillo-Alanís Javier
Published: February 2011

We prove a local version of the Khintchine inequality for the spaces

E
x
p

L
p

(
[
0
,
1
]
)

{\mathrm {Exp} \,} L^p([0,1])

of functions having

p
p

-th exponential integrability, for

1
≤
p
≤
2

1 \leq p \leq 2

. The result also holds for the lacunary Walsh series.
First Report of the β-Tubulin E198A Mutation Conferring Resistance to Methyl Benzimidazole Carbamates in European Isolates of Monilinia fructicola
Authors: Weger J. , Schanze M. , Hilber-Bodmer M. , Smits T. H. M. , Patocchi A.
Published: February 2011

The causal agent of brown rot on stone and pome fruits, Monilinia fructicola (G. Wint.), is a quarantine pathogen in Europe. It has been detected in Austria (later eradicated), Spain, the Czech Republic, Italy, Germany, and Switzerland (1). In the United States and other countries, M. fructicola isolates were reported to show resistance to different classes of fungicides, including methyl benzimidazole carbamates (MBC) (2). Lichou et al. (2) reported the presence of isolates resistant to the MBC carbendazim in France, but the mechanisms inducing MBC resistance in these isolates were not studied. Ma et al. (3) in California, and more recently, Zhu et al. (4) in South Carolina, demonstrated that the molecular mechanisms accounting for low and high levels of resistance to MBC fungicides in M. fructicola isolates were the mutations H6Y and E198A, respectively, in the β-tubulin gene. Four M. fructicola isolates each from Italy, France, Spain, and Switzerland (16 isolates total), all having an unknown level of MBC resistance, were selected. In each isolate, the section of the β-tubulin gene containing the two potentially mutant codons was PCR-amplified with the primers TubA and TubR1 (3) and the amplicons were sequenced directly. Sequence analysis revealed the amino acid histidine (H) at codon 6 in all the isolates, which would not predict MBC resistance, while alanine (A) at codon 198 (the mutation predictive of a high level of MBC resistance) was found in all isolates from Spain and Switzerland and in three isolates each from France and Italy. A representative sequence of the four identical partial β-tubulin gene sequences from the Swiss isolates was submitted to GenBank under the Accession No. HQ709265. All isolates were tested in a potato dextrose agar (PDA) petri dish assay for resistance to the MBC fungicide thiophanate-methyl (Nippon Soda Co., Ltd., Tokyo, Japan) at the discriminatory dose of 50 μg/ml (4). All isolates with the E198A mutation were able to grow on the media, while the two isolates without the E198A mutation were not able to grow. The result indicated that most isolates had a high level of resistance to the MBC fungicide. To our knowledge, this is the first report of the presence of the E198A mutation conferring resistance to MBC fungicides in European isolates of M. fructicola. As the mutation appears to be widely distributed, we anticipate that MBC fungicides may be ineffective at controlling brown rot in countries with occurrence of M. fructicola.

References: (1) M. Hilber-Bodmer et al. Plant Dis. 94:643, 2010. (2) J. Lichou et al. Phytoma 547:22, 2002. (3) Z. H. Ma et al. Appl. Environ. Microbiol. 69:7145, 2003. (4) F. X. Zhu et al. Plant Dis. 94:1511, 2010.
Ploidy and the evolution of parasitism
Authors: M'Gonigle Leithen K. , Otto Sarah P.
Published: February 2011

Levels of parasitism are continuously distributed in nature. Models of host–parasite coevolution, however, typically assume that species can be easily characterized as either parasitic or non-parasitic. Consequently, it is poorly understood which factors influence the evolution of parasitism itself. We investigate how ploidy level and the genetic mechanisms underlying infection influence evolution along the continuum of parasitism levels. In order for parasitism to evolve, selective benefits to the successful invasion of hosts must outweigh the losses when encountering resistant hosts. However, we find that exactly where this threshold occurs depends not only on the strength of selection, but also on the genetic model of interaction, the ploidy level in each species, and the nature of the costs to virulence and resistance. With computer simulations, we are able to incorporate more realistic dynamics at the loci underlying species interactions and to extend our analyses in a number of directions, including finite population sizes, multiple alleles and different generation times.
Elimination of a specific histone H3K14 acetyltransferase complex bypasses the RNAi pathway to regulate pericentric heterochromatin functions
Authors: Reddy Bharat D. , Wang Yu , Niu Lifang , Higuchi Emily C. , Marguerat Samuel B. , Bähler Jürg , Smith Gerald R. , Jia Songtao
Published: February 2011

In Schizosaccharomyces pombe, the RNAi pathway is required for the formation of pericentric heterochromatin, proper chromosome segregation, and repression of pericentric meiotic recombination. Here we demonstrate that, when the activity of the histone H3 Lys 14 (H3K14) acetyltransferase Mst2 is eliminated, the RNAi machinery is no longer required for pericentric heterochromatin functions. We further reveal that reducing RNA polymerase II recruitment to pericentric regions is essential for maintaining heterochromatin in the absence of RNAi.
Strange as it may seem: the many links between Wnt signaling, planar cell polarity, and cilia: Figure 1.
Authors: Wallingford John B. , Mitchell Brian
Published: February 2011

Cilia are important cellular structures that have been implicated in a variety of signaling cascades. In this review, we discuss the current evidence for and against a link between cilia and both the canonical Wnt/β-catenin pathway and the noncanonical Wnt/planar cell polarity (PCP) pathway. Furthermore, we address the evidence implicating a role for PCP components in ciliogenesis. Given the lack of consensus in the field, we use new data on the control of ciliary protein localization as a basis for proposing new models by which cell type-specific regulation of ciliary components via differential transport, regulated entry and exit, or diffusion barriers might generate context-dependent functions for cilia.
DOT1L regulates dystrophin expression and is critical for cardiac function
Authors: Nguyen Anh T. , Xiao Bin , Neppl Ronald L. , Kallin Eric M. , Li Juan , Chen Taiping , Wang Da-Zhi , Xiao Xiao , Zhang Yi
Published: February 2011

Histone methylation plays an important role in regulating gene expression. One such methylation occurs at Lys 79 of histone H3 (H3K79) and is catalyzed by the yeast DOT1 (disruptor of telomeric silencing) and its mammalian homolog, DOT1L. Previous studies have demonstrated that germline disruption of Dot1L in mice resulted in embryonic lethality. Here we report that cardiac-specific knockout of Dot1L results in increased mortality rate with chamber dilation, increased cardiomyocyte cell death, systolic dysfunction, and conduction abnormalities. These phenotypes mimic those exhibited in patients with dilated cardiomyopathy (DCM). Mechanistic studies reveal that DOT1L performs its function in cardiomyocytes through regulating Dystrophin (Dmd) transcription and, consequently, stability of the Dystrophin–glycoprotein complex important for cardiomyocyte viability. Importantly, expression of a miniDmd can largely rescue the DCM phenotypes, indicating that Dmd is a major target mediating DOT1L function in cardiomyocytes. Interestingly, analysis of available gene expression data sets indicates that DOT1L is down-regulated in idiopathic DCM patient samples compared with normal controls. Therefore, our study not only establishes a critical role for DOT1L-mediated H3K79 methylation in cardiomyocyte function, but also reveals the mechanism underlying the role of DOT1L in DCM. In addition, our study may open new avenues for the diagnosis and treatment of human heart disease.
Essential role for the interaction between hnRNP H/F and a G quadruplex in maintaining p53 pre-mRNA 3′-end processing and function during DNA damage
Authors: Decorsière Adrien , Cayrel Anne , Vagner Stéphan , Millevoi Stefania
Published: February 2011

Following DNA damage, mRNA 3′-end formation is inhibited, contributing to repression of mRNA synthesis. Here we investigated how DNA-damaged cells accomplish p53 mRNA 3′-end formation when normal mechanisms of pre-mRNA 3′-end processing regulation are inhibited. The underlying mechanism involves the interaction between a G-quadruplex structure located downstream from the p53 cleavage site and hnRNP H/F. Importantly, this interaction is critical for p53 expression and contributes to p53-mediated apoptosis. Our results uncover the existence of a specific rescue mechanism of 3′-end processing regulation allowing stress-induced p53 accumulation and function in apoptosis.
Clcn4-2 genomic structure differs between the X locus in Mus spretus and the autosomal locus in Mus musculus: AT motif enrichment on the X
Authors: Nguyen Di Kim , Yang Fan , Kaul Rajinder , Alkan Can , Antonellis Anthony , Friery Karen F. , Zhu Baoli , de Jong Pieter J. , Disteche Christine M.
Published: February 2011

In Mus spretus, the chloride channel 4 gene Clcn4-2 is X-linked and dosage compensated by X up-regulation and X inactivation, while in the closely related mouse species Mus musculus, Clcn4-2 has been translocated to chromosome 7. We sequenced Clcn4-2 in M. spretus and identified the breakpoints of the evolutionary translocation in the Mus lineage. Genetic and epigenetic differences were observed between the 5′ends of the autosomal and X-linked loci. Remarkably, Clcn4-2 introns have been truncated on chromosome 7 in M. musculus as compared with the X-linked loci from seven other eutherian mammals. Intron sequences specifically preserved in the X-linked loci were significantly enriched in AT-rich oligomers. Genome-wide analyses showed an overall enrichment in AT motifs unique to the eutherian X (except for genes that escape X inactivation), suggesting a role for these motifs in regulation of the X chromosome.
Estimation of DNA Concentration in Forensic Samples Prior to Multiplex PCR Amplification
Authors: Butler John M.
Published: February 2011

INTRODUCTION

This protocol describes the use of quantitative polymerase chain reaction (qPCR) for quantitation of DNA in forensic samples prior to PCR amplification. It is adapted from the Armed Forces DNA Identification Laboratory (Rockville, MD) Standard Operating Procedure.
In Situ Lymph Node Imaging: FIGURE 1.
Authors: Matheu Melanie P. , Cahalan Michael D. , Parker Ian
Published: February 2011

INTRODUCTION

Imaging the single-cell dynamics of the immune system within an intact environment requires the ability to look deep inside tissues and organisms with spatial and temporal resolutions adequate to track cell morphology, motility, and signaling processes, all while minimizing perturbation of the system under study. Fluorescence techniques are highly suited for this purpose, permitting both labeling of specific cells, organelles, or proteins and functional readout of physiological events, and two-photon microscopy allows these processes to be visualized within native tissue environments. Immunoimaging preparations using explanted tissues and organs maintained by superfusion with warmed oxygenated medium are more physiological than in vitro cell systems and are easy to work with. They can be cleaned of surrounding tissue, oriented as desired with respect to the microscope objective, are readily accessible for administration of pharmaceutical agents, provide a robust and relatively drift-free preparation for long-term imaging, and minimize animal welfare issues. Explant imaging can yield results indistinguishable from intravital imaging, although intravital preparations are essential if intact blood and lymphatic flow are essential for the experimental question. This protocol describes in situ imaging of lymph nodes (LNs).
Labeling Pinocytotic Vesicles and Cytoplasm with Fluorescently Labeled Ficoll or Dextran
Authors: Chazotte Brad
Published: February 2011

INTRODUCTION

Dextran, a high molecular weight poly-D-glucose, and ficoll, a synthetic polymer of epichlorhydrin and sucrose, are electroneutral hydrophilic polysaccharides whose size can be varied. This variability in size coupled with their membrane impermeability can make them useful for studying fluid phase pinocytosis, the size of membrane pores such as nuclear membrane pores, or the environmental conditions and the size of a cell compartment. In this protocol, two fluorophores based on fluorescein and rhodamine are used to label either dextran or ficoll. The resulting probes can then be used to label cells.
Assay for Courtship Suppression in Drosophila
Authors: Ejima Aki , Griffith Leslie C.
Published: February 2011

INTRODUCTION

Unsuccessful courtship reduces subsequent courtship behavior of male Drosophila. This experience-dependent behavior modification is called courtship conditioning and has been one of the major paradigms used to study learning and memory in Drosophila. This article describes a basic behavioral assay for quantifying short-term memory of courtship suppression induced by training with a mated female.
Immunoimaging: Studying Immune System Dynamics Using Two-Photon Microscopy
Authors: Matheu Melanie P. , Cahalan Michael D. , Parker Ian
Published: February 2011

INTRODUCTION

Cells of the immune system explore a wider territory than any other cells in the body. Responses to a pathogen typically require long-range migration of cells, short-range communication by local chemical signaling, and direct cell-cell contact. Two-photon microscopy allows these processes to be visualized within native tissue environments. Immunoimaging is rapidly developing from a merely descriptive technique into a set of methods and analytical tools that can be used to quantify and to characterize an immune response at the cellular level. This article outlines the hardware required for immunoimaging and discusses methods for quantitative analysis of multidimensional image stacks.
The PeaGIGASGene Is aFLOWERING LOCUS THomolog Necessary for Graft-Transmissible Specification of Flowering but Not for Responsiveness to Photoperiod
Authors: Hecht Valérie , Laurie Rebecca E. , Vander Schoor Jacqueline K. , Ridge Stephen , Knowles Claire L. , Liew Lim Chee , Sussmilch Frances C. , Murfet Ian C. , Macknight Richard C. , Weller James L.
Published: February 2011

Garden pea (Pisum sativum) was prominent in early studies investigating the genetic control of flowering and the role of mobile flowering signals. In view of recent evidence that genes in the FLOWERING LOCUS T (FT) family play an important role in generating mobile flowering signals, we isolated the FT gene family in pea and examined the regulation and function of its members. Comparison with Medicago truncatula and soybean (Glycine max) provides evidence of three ancient subclades (FTa, FTb, and FTc) likely to be common to most crop and model legumes. Pea FT genes show distinctly different expression patterns with respect to developmental timing, tissue specificity, and response to photoperiod and differ in their activity in transgenic Arabidopsis thaliana, suggesting they may have different functions. We show that the pea FTa1 gene corresponds to the GIGAS locus, which is essential for flowering under long-day conditions and promotes flowering under short-day conditions but is not required for photoperiod responsiveness. Grafting, expression, and double mutant analyses show that GIGAS/FTa1 regulates a mobile flowering stimulus but also provide clear evidence for a second mobile flowering stimulus that is correlated with expression of FTb2 in leaf tissue. These results suggest that induction of flowering by photoperiod in pea results from interactions among several members of a diversified FT family.
Chilling of Dormant Buds HyperinducesFLOWERING LOCUS Tand Recruits GA-Inducible 1,3-β-Glucanases to Reopen Signal Conduits and Release Dormancy inPopulus
Authors: Rinne Päivi L.H. , Welling Annikki , Vahala Jorma , Ripel Linda , Ruonala Raili , Kangasjärvi Jaakko , van der Schoot Christiaan
Published: February 2011

In trees, production of intercellular signals and accessibility of signal conduits jointly govern dormancy cycling at the shoot apex. We identified 10 putative cell wall 1,3-β-glucanase genes (glucan hydrolase family 17 [GH17]) in Populus that could turn over 1,3-β-glucan (callose) at pores and plasmodesmata (PD) and investigated their regulation in relation to FT and CENL1 expression. The 10 genes encode orthologs of Arabidopsis thaliana BG_ppap, a PD-associated glycosylphosphatidylinositol (GPI) lipid-anchored protein, the Arabidopsis PD callose binding protein PDCB, and a birch (Betula pendula) putative lipid body (LB) protein. We found that these genes were differentially regulated by photoperiod, by chilling (5°C), and by feeding of gibberellins GA3 and GA4. GA3 feeding upregulated all LB-associated GH17s, whereas GA4 upregulated most GH17s with a GPI anchor and/or callose binding motif, but only GA4 induced true bud burst. Chilling upregulated a number of GA biosynthesis and signaling genes as well as FT, but not CENL1, while the reverse was true for both GA3 and GA4. Collectively, the results suggest a model for dormancy release in which chilling induces FT and both GPI lipid-anchored and GA3-inducible GH17s to reopen signaling conduits in the embryonic shoot. When temperatures rise, the reopened conduits enable movement of FT and CENL1 to their targets, where they drive bud burst, shoot elongation, and morphogenesis.
Foraging, bioenergetic and predation constraints on diel vertical migration: field observations and modelling of reverse migration by young‐of‐the‐year herring Clupea harengus
Authors: Jensen O. P. , Hansson S. , Didrikas T. , Stockwell J. D. , Hrabik T. R. , Axenrot T. , Kitchell J. F.
Published: February 2011

Diel vertical migration (DVM) of young‐of‐the‐year (YOY) herring Clupea harengus and one of their major predators, pikeperch Sander lucioperca, was examined using bottom‐mounted hydroacoustics in Himmerfjärden, a brackish bay of the Baltic Sea, in summer. In contrast to previous studies on DVM of C. harengus aggregated across size and age classes, YOY C. harengus showed a reverse DVM trajectory, deeper at night and, on average, shallower during the day. This pattern was observed consistently on five acoustic sampling occasions in 3 years and was corroborated by two out of three trawl surveys. Large acoustic targets (target strength >−33 dB, probably piscivorous S. lucioperca >45 cm) showed a classic DVM trajectory, shallow at night and deeper during the day. Variability in YOY C. harengus vertical distribution peaked at dawn and dusk, and their vertical distribution at midday was distinctly bimodal. This reverse DVM pattern was consistent with bioenergetic model predictions for YOY C. harengus which have rapid gut evacuation rates and do not feed at night. Reverse DVM also resulted in low spatial overlap with predators.
Oncogenic phosphatase Wip1 is a novel prognostic marker for lung adenocarcinoma patient survival
Authors: Satoh Naoyuki , Maniwa Yoshimasa , Bermudez Vladimir P. , Nishimura Kunihiro , Nishio Wataru , Yoshimura Masahiro , Okita Yutaka , Ohbayashi Chiho , Hurwitz Jerard , Hayashi Yoshitake
Published: February 2011

DNA damage response pathways are important for maintaining genomic stability. The oncogenic phosphatase Wip1 plays a crucial role in DNA damage response by inhibiting several cell cycle proteins, including p53. Although Wip1 gene amplification has been reported in various primary tumors, including lung cancer, its biological significance for survival of primary lung tumor patients remains unclear. We investigated the expression of Wip1 in cancer epithelial cells immunohistochemically in 84 consecutive resected cases of lung adenocarcinoma. Increased Wip1 expression was observed in 54 (64.3%) of the 84 cases. Wip1 expression was found to be correlated significantly with two clinicopathological factors: γ‐H2AX expression, and invasion to the pulmonary vein. A univariate analysis and log–rank test indicated a significant association between Wip1 expression and lower overall survival rate (P = 0.019 and P = 0.0099, respectively). A multivariate analysis also indicated a statistically significant association between increased Wip1 expression and lower overall survival rate (hazard ratio, 4.3; P = 0.026). The Ki67 index level was higher in the Wip1‐positive group than in the negative group (P < 0.04, Mann–Whitney U‐test). Moreover, in a subgroup analysis of only stage I patients, increased Wip1 expression was also significantly associated with a lower overall survival rate (P = 0.023, log–rank test). These results indicate that the increased expression of Wip1 in cancer epithelial cells has significant value for tumor progression and the clinical prognosis of patients with primary lung adenocarcinoma. (Cancer Sci, 2011; 102: 1101–1106)
Inhibition of nuclear factor‐kappaB suppresses peritoneal dissemination of gastric cancer by blocking cancer cell adhesion
Authors: Mino Kazuhiro , Ozaki Michitaka , Nakanishi Kazuaki , Haga Sanae , Sato Masanori , Kina Masaya , Takahashi Masato , Takahashi Norihiko , Kataoka Akihiko , Yanagihara Kazuyoshi , Ochiya Takahiro , Kamiyama Toshiya , Umezawa Kazuo , Todo Satoru
Published: February 2011

Currently, patients with peritoneal dissemination of gastric cancer must accept a poor prognosis because there is no standard effective therapy. To inhibit peritoneal dissemination it is important to inhibit interactions between extracellular matrices (ECM) and cell surface integrins, which are important for cancer cell adhesion. Although nuclear factor‐kappa B (NF‐κB) is involved in various processes in cancer progression, its involvement in the expression of integrins has not been elucidated. We used a novel NF‐κB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), to study whether NF‐κB blocks cancer cell adhesion via integrins in a gastric cancer dissemination model in mice and found that DHMEQ is a potent suppressor of cancer cell dissemination. Dehydroxymethylepoxyquinomicin suppressed the NF‐κB activity of human gastric cancer cells NUGC‐4 and 44As3Luc and blocked the adhesion of cancer cells to ECM when compared with the control. Dehydroxymethylepoxyquinomicin also inhibited expression of integrin (α2, α3, β1) in in vitro studies. In the in vivo model, we injected 44As3Luc cells pretreated with DHMEQ into the peritoneal cavity of mice and performed peritoneal lavage after the injection of cancer cells. Viable cancer cells in the peritoneal cavities were evaluated sequentially by in vivo imaging. In mice injected with DHMEQ‐pretreated cells and lavaged, live cancer cells in the peritoneum were significantly reduced compared with the control, and these mice survived longer. These results indicate that DHMEQ could inhibit cancer cell adhesion to the peritoneum possibly by suppressing integrin expression. Nuclear factor‐kappa B inhibition may be a new therapeutic option for suppressing postoperative cancer dissemination. (Cancer Sci 2011; 102: 1052–1058)
Garlic virus X11‐kDa protein granules move within the cytoplasm and traffic a host protein normally found in the nucleolus
Authors: LU YUWEN , YAN FEI , GUO WEI , ZHENG HONGYING , LIN LIN , PENG JIEJUN , ADAMS MICHAEL J. , CHEN JIANPING
Published: February 2011

The subcellular localization of the 11‐kDa protein (p11) encoded by ORF3 ofGarlic virus X(GarVX; genusAllexivirus, familyAlphaflexiviridae) was examined by confocal microscopy. Granules with intense fluorescence were visible on the endoplasmic reticulum when p11 fused with green or red fluorescent protein (GFP or RFP) was expressed in epidermal cells ofNicotiana benthamiana. Moreover, the p11‐RFP granules moved in the cytoplasm, along the cell periphery and through the cell membranes to adjacent cells. A 17‐kDa protein (p17) of garlic interacting with p11 was identified by yeast two‐hybridization and bimolecular fluorescence complementation assay. When p17 fused to GFP was expressed in epidermal cells ofN. benthamiana, it localized to the nucleolus. However, in the presence of GarVX p11, the distribution of p17 changed to that of p11, but did not appear to affect the pattern of movement of p11.
Effects of harvesting seasons and maturity stages on internal characteristics of the mangosteen having different surface properties
Authors: Chaisrichonlathan Preedawan , Noomhorm Athapol
Published: February 2011

Translucent flesh disorder is a major internal physical disorder of mangosteen. The internal characteristics of smooth‐ and coarse‐surface fruits in relation to their respective specific gravity values and sensory characteristics were evaluated for suitable grading process. Study on percentage of the internal characteristics was performed in different external characteristics, different harvesting seasons and different maturity stages of mangosteen. Results showed that internal physical disorders of smooth‐surface fruits were higher than coarse‐surface ones at the level of 63% and 19%, respectively. Average specific gravity of normal‐flesh fruits, mild translucent‐flesh fruits and moderate‐to‐severe translucent‐flesh fruits were observed as 0.97, 1.00 and 1.01, respectively. The sensory characteristics: sweetness, flavour and overall acceptability were significantly (P < 0.05) higher for coarse‐surface fruits compared with smooth‐surface fruits. Both results were inconsistent with market price in which smooth fruit was 50% higher than coarse‐surface fruit.
Optimisation of conditions for glucose syrup production from banana (Musa paradisiaca L.) pulp using response surface methodology
Authors: Rendón‐Villalobos J. Rodolfo , Solorza‐Feria Javier , Aguilar‐Sandoval Alondra
Published: February 2011

A central composite design was applied to assess some basic factors that affect profoundly the production of dextrose equivalent from banana pulp. Optimal conditions for the conversion of starch banana pulp to glucose were found to be a banana pulp concentration of 8.5%, reaction time of 90 min, and α‐amylase concentration of 0.04% for liquefaction and reaction time of 24 h with a glucoamylase concentration of 0.05% for saccharification. Only 16 and 12 experiments, respectively, were needed to assess these conditions. The adequacy of model was highly satisfactory, because the coefficients of determination (R2) were 0.93 and 0.91, respectively. The glucose syrup (GS) obtained was slightly yellowish with a hint of brown and slightly sweet. The syrup had a protein of 0.63% and an ash content of 0.60%, with a pH of 4.8. The characteristics of the GS produced met all the recommended standards, except for that of colour.
How and why do student teachers use ICT?
Authors: Hammond M. , Reynolds L. , Ingram J.
Published: February 2011

This paper examines how and why student teachers made use of information and communication technology (ICT) during a 1‐year initial teacher education programme from 2008 to 2009. This is a mixed methods study involving a survey (N = 340) of the entire cohort and a series of semi‐structured interviews with a sample of student teachers within the cohort (N = 21). The study explored several themes, including the nature of student teachers' use of ICT; variation in the use of ICT; support for, and constraints on, using ICT; attitudes to ICT and to teaching and learning more generally. It was found that nearly all teachers were receptive to using ICT – more so than their in‐service counterparts – and made frequent use of it during their placement (internship) experience. The Interactive Whiteboard (IWB) was central to nearly all student teachers' use of ICT, in good part, because it was already used by their mentors and was widely accessible. Student teachers' use of ICT was categorized in three levels. Routine users focused mostly on the use of the IWB for whole class teaching; extended users gave greater opportunities for pupils to use ICT for themselves; innovative student teachers used ICT in a greater range of contexts and made more effort to overcome barriers such as access. ICT use was seen as emerging from a mix of factors: chiefly student teachers' access to ICT; their feeling of ‘self‐efficacy’ when using ICT; and their belief that ICT had a positive impact on learning – in particular, the impact on pupils' behavioural and affective engagement. Factors which influenced ICT use included mentoring, training and support. Limitations on student teachers' use of ICT are explored and it is suggested that new teachers need to be supported in developing a more discerning use as they begin their teaching careers.
A shared mechanism of SoxR activation by redox‐cycling compounds
Authors: Dietrich Lars E. P. , Kiley Patricia J.
Published: February 2011

In this issue of Molecular Microbiology, Gu and Imlay show that a class of compounds known as redox‐cycling agents directly activate the transcription factor SoxR of Escherichia coli and cause cellular toxicity independent of the production of the reactive oxygen species superoxide. Despite the fact that redox‐cycling agents increase formation of superoxide in E. coli, the results described in this new publication revise the long‐held assumption that superoxide is responsible for the activation of SoxR and for all of the major toxic effects of redox‐cycling drugs. This study also suggests that the critical function of the SoxRS regulon in E. coli is in protection against redox‐cycling agents and not exclusively the defence against superoxide.
NFKBIL1 Confers Resistance to Experimental Autoimmune Arthritis Through the Regulation of Dendritic Cell Functions
Authors: Chiba T. , Matsuzaka Y. , Warita T. , Sugoh T. , Miyashita K. , Tajima A. , Nakamura M. , Inoko H. , Sato T. , Kimura M.
Published: February 2011

We and others have reported that human NF‐κB inhibitor‐like‐1 (NFKBIL1) was a putative susceptible gene for autoimmune diseases such as rheumatoid arthritis (RA). However, its precise role in the pathogenesis of RA is still largely unknown. In this study, we generated transgenic mice expressing human NFKBIL1 (NFKBIL1‐Tg) and examined whether NFKBIL1 plays some role(s) in the development of autoimmune arthritis. In both a collagen‐induced arthritis model and a collagen antibody‐induced arthritis model, NFKBIL1‐Tg mice showed resistance to arthritis compared to control mice, indicating that the gene product of NFKBIL1 was involved in the control of thusly induced arthritis. Total spleen cells of NFKBIL1‐Tg mouse showed decreased proliferation to mitogenic stimuli, consistent with its resistance to arthritis. Unexpectedly, purified T cells of NFKBIL1‐Tg mouse showed increased proliferation and cytokine production. This apparent discrepancy was accounted for by the impaired functions of antigen‐presenting cells of NFKBIL1‐Tg mouse; both T/B cell‐depleted spleen cells and bone marrow‐derived dendritic cells of the Tg mouse induced less prominent proliferation and IL‐2 production of T cells. Furthermore, dendritic cells (DCs) derived from NFKBIL1‐Tg mouse showed lower expression of co‐stimulatory molecules and decreased production of inflammatory cytokines when they were activated by lipopolysaccharide. Taken together, these results indicated that NFKBIL1 affected the pathogenesis of RA at least in part through the regulation of DC functions.