bluuebunny/crossref_metadata_2025 huggingface dataset:
Assembly of nonnative floras along elevational gradients explained by directional ecological filtering
Authors: Alexander Jake M. , Kueffer Christoph , Daehler Curtis C. , Edwards Peter J. , Pauchard Aníbal , Seipel Tim ,   MIREN Consortium, Arévalo José , Cavieres Lohengrin , Dietz Hansjoerg , Jakobs Gabi , McDougall Keith , Naylor Bridgett , Otto Rüdiger , Parks Catherine G. , Rew Lisa , Walsh Neville
Published: December 2010

Nonnative species richness typically declines along environmental gradients such as elevation. It is usually assumed that this is because few invaders possess the necessary adaptations to succeed under extreme environmental conditions. Here, we show that nonnative plants reaching high elevations around the world are not highly specialized stress tolerators but species with broad climatic tolerances capable of growing across a wide elevational range. These results contrast with patterns for native species, and they can be explained by the unidirectional expansion of nonnative species from anthropogenic sources at low elevations and the progressive dropping out of species with narrow elevational amplitudes—a process that we call directional ecological filtering. Independent data confirm that climatic generalists have succeeded in colonizing the more extreme environments at higher elevations. These results suggest that invasion resistance is not conferred by extreme conditions at a particular site but determined by pathways of introduction of nonnative species. In the future, increased direct introduction of nonnative species with specialized ecophysiological adaptations to mountain environments could increase the risk of invasion. As well as providing a general explanation for gradients of nonnative species richness and the importance of traits such as phenotypic plasticity for many invasive species, the concept of directional ecological filtering is useful for understanding the initial assembly of some native floras at high elevations and latitudes.
Mechanism of tripartite RNA genome packaging in Rift Valley fever virus
Authors: Terasaki Kaori , Murakami Shin , Lokugamage Kumari G. , Makino Shinji
Published: December 2010

The
Bunyaviridae
family includes pathogens of medical and veterinary importance. Rift Valley fever virus (RVFV), a member in the
Phlebovirus
genus of the family
Bunyaviridae
, is endemic to sub-Saharan Africa and causes a mosquito-borne disease in ruminants and humans. Viruses in the family
Bunyaviridae
carry a tripartite, single-stranded, negative-sense RNA genome composed of L, M, and S RNAs. Little is known about how the three genomic RNA segments are copackaged to generate infectious bunyaviruses. We explored the mechanism that governs the copackaging of the three genomic RNAs into RVFV particles. The expression of viral structural proteins along with replicating S and M RNAs resulted in the copackaging of both RNAs into RVFV-like particles, while replacing M RNA with M1 RNA, lacking a part of the M RNA 5′ UTR, abrogated the RNA copackaging. L RNA was efficiently packaged into virus particles released from cells supporting the replication of L, M, and S RNAs, and replacing M RNA with M1 RNA abolished the packaging of L RNA. Detailed analyses using various combinations of replicating viral RNAs suggest that M RNA alone or a coordinated function of M and S RNAs exerted efficient L RNA packaging either directly or indirectly. Collectively, these data are consistent with the possibility that specific intermolecular interactions among the three viral RNAs drive the copackaging of these RNAs to produce infectious RVFV.
Krüppel-like factor 2 (KLF2) regulates B-cell reactivity, subset differentiation, and trafficking molecule expression
Authors: Hart Geoffrey T. , Wang Xiaodan , Hogquist Kristin A. , Jameson Stephen C.
Published: December 2010

The transcription factor Krüppel-like factor 2 (KLF2) is critical for normal trafficking of T lymphocytes, but its role in B cells is unclear. We report that B cell-specific KLF2 deficiency leads to decreased expression of the trafficking molecules CD62L and β7-integrin, yet expression of sphingosine-1 phosphate receptor 1 (which is a critical target of KLF2 in T cells) was, unexpectedly, minimally altered. Unexpectedly,Klf2deletion led to a drastic reduction in the B1 B-cell pool and a substantial increase in transitional and marginal zone B-cell numbers. In addition, we observed that KLF2-deficient B cells showed increased apoptosis and impaired proliferation after B-cell receptor cross-linking. Gene expression analysis indicated that KLF2-deficient follicular B cells display numerous characteristics shared by normal marginal zone B cells, including reduced expression of several signaling molecules that may contribute to defective activation of these cells. Hence, our data indicate that KLF2 plays a critical role in dictating normal subset differentiation and functional reactivity of mature B cells.
Hexagonal assembly of a restricting TRIM5α protein
Authors: Ganser-Pornillos Barbie K. , Chandrasekaran Viswanathan , Pornillos Owen , Sodroski Joseph G. , Sundquist Wesley I. , Yeager Mark
Published: December 2010

TRIM5α proteins are restriction factors that protect mammalian cells from retroviral infections by binding incoming viral capsids, accelerating their dissociation, and preventing reverse transcription of the viral genome. Individual TRIM5 isoforms can often protect cells against a broad range of retroviruses, as exemplified by rhesus monkey TRIM5α and its variant, TRIM5-21R, which recognize HIV-1 as well as several distantly related retroviruses. Although capsid recognition is not yet fully understood, previous work has shown that the C-terminal SPRY/B30.2 domain of dimeric TRIM5α binds directly to viral capsids, and that higher-order TRIM5α oligomerization appears to contribute to the efficiency of capsid recognition. Here, we report that recombinant TRIM5-21R spontaneously assembled into two-dimensional paracrystalline hexagonal lattices comprising open, six-sided rings. TRIM5-21R assembly did not require the C-terminal SPRY domain, but did require both protein dimerization and a B-box 2 residue (Arg121) previously implicated in TRIM5α restriction and higher-order assembly. Furthermore, TRIM5-21R assembly was promoted by binding to hexagonal arrays of the HIV-1 CA protein that mimic the surface of the viral capsid. We therefore propose that TRIM5α proteins have evolved to restrict a range of different retroviruses by assembling a deformable hexagonal scaffold that positions the capsid-binding domains to match the symmetry and spacing of the capsid surface lattice. Capsid recognition therefore involves a synergistic combination of direct binding interactions, avidity effects, templated assembly, and lattice complementarity.
Tuning the ion selectivity of tetrameric cation channels by changing the number of ion binding sites
Authors: Derebe Mehabaw G. , Sauer David B. , Zeng Weizhong , Alam Amer , Shi Ning , Jiang Youxing
Published: December 2010

Selective ion conduction across ion channel pores is central to cellular physiology. To understand the underlying principles of ion selectivity in tetrameric cation channels, we engineered a set of cation channel pores based on the nonselective NaK channel and determined their structures to high resolution. These structures showcase an ensemble of selectivity filters with a various number of contiguous ion binding sites ranging from 2 to 4, with each individual site maintaining a geometry and ligand environment virtually identical to that of equivalent sites in K
+
channel selectivity filters. Combined with single channel electrophysiology, we show that only the channel with four ion binding sites is K
+
selective, whereas those with two or three are nonselective and permeate Na
+
and K
+
equally well. These observations strongly suggest that the number of contiguous ion binding sites in a single file is the key determinant of the channel’s selectivity properties and the presence of four sites in K
+
channels is essential for highly selective and efficient permeation of K
+
ions.
Identification of a gain-of-function mutation in a Golgi P-type ATPase that enhances Mn
2+
efflux and protects against toxicity
Authors: Mukhopadhyay Somshuvra , Linstedt Adam D.
Published: December 2010

P-type ATPases transport a wide array of ions, regulate diverse cellular processes, and are implicated in a number of human diseases. However, mechanisms that increase ion transport by these ubiquitous proteins are not known. SPCA1 is a P-type pump that transports Mn
2+
from the cytosol into the Golgi. We developed an intra-Golgi Mn
2+
sensor and used it to screen for mutations introduced in SPCA1, on the basis of its predicted structure, which could increase its Mn
2+
pumping activity. Remarkably, a point mutation (Q747A) predicted to increase the size of its ion permeation cavity enhanced the sensor response and a compensatory mutation restoring the cavity to its original size abolished this effect. In vivo and in vitro Mn
2+
transport assays confirmed the hyperactivity of SPCA1-Q747A. Furthermore, increasing Golgi Mn
2+
transport by expression of SPCA1-Q747A increased cell viability upon Mn
2+
exposure, supporting the therapeutic potential of increased Mn
2+
uptake by the Golgi in the management of Mn
2+
-induced neurotoxicity.
Heterodimer of two bHLH-PAS proteins mediates juvenile hormone-induced gene expression
Authors: Li Meng , Mead Edward A. , Zhu Jinsong
Published: December 2010

Juvenile hormone (JH) plays crucial roles in many aspects of insect life. TheMethoprene-tolerant(Met) gene product, a member of the bHLH-PAS family of transcriptional regulators, has been demonstrated to be a key component of the JH signaling pathway. However, the molecular function of Met in JH-induced signal transduction and gene regulation remains to be fully elucidated. Here we show that a transcriptional coactivator of the ecdysteroid receptor complex, FISC, acts as a functional partner of Met in mediating JH-induced gene expression. Met and FISC appear to use their PAS domains to form a dimer only in the presence of JH or JH analogs. In newly emerged adult female mosquitoes, expression of some JH responsive genes is considerably dampened when Met or FISC is depleted by RNAi. Met and FISC are found to be associated with the promoter of the early trypsin gene (AaET) when transcription of this gene is activated by JH. A juvenile hormone response element (JHRE) has been identified in theAaETupstream regulatory region and is bound in vitro by the Met-FISC complex present in the nuclear protein extracts of previtellogenic adult female mosquitoes. In addition, theDrosophilahomologs of Met and FISC can also use this mosquito JHRE to activate gene transcription in response to JH in a cell transfection assay. Together, the evidence indicates that Met and FISC form a functional complex on the JHRE in the presence of JH and directly activate transcription of JH target genes.
Structure-guided reprogramming of serine recombinase DNA sequence specificity
Authors: Gaj Thomas , Mercer Andrew C. , Gersbach Charles A. , Gordley Russell M. , Barbas Carlos F.
Published: December 2010

Routine manipulation of cellular genomes is contingent upon the development of proteins and enzymes with programmable DNA sequence specificity. Here we describe the structure-guided reprogramming of the DNA sequence specificity of the invertase Gin from bacteriophage Mu and Tn3 resolvase from
Escherichia coli
. Structure-guided and comparative sequence analyses were used to predict a network of amino acid residues that mediate resolvase and invertase DNA sequence specificity. Using saturation mutagenesis and iterative rounds of positive antibiotic selection, we identified extensively redesigned and highly convergent resolvase and invertase populations in the context of engineered zinc-finger recombinase (ZFR) fusion proteins. Reprogrammed variants selectively catalyzed recombination of nonnative DNA sequences > 10,000-fold more effectively than their parental enzymes. Alanine-scanning mutagenesis revealed the molecular basis of resolvase and invertase DNA sequence specificity. When used as rationally designed ZFR heterodimers, the reprogrammed enzyme variants site-specifically modified unnatural and asymmetric DNA sequences. Early studies on the directed evolution of serine recombinase DNA sequence specificity produced enzymes with relaxed substrate specificity as a result of randomly incorporated mutations. In the current study, we focused our mutagenesis exclusively on DNA determinants, leading to redesigned enzymes that remained highly specific and directed transgene integration into the human genome with > 80% accuracy. These results demonstrate that unique resolvase and invertase derivatives can be developed to site-specifically modify the human genome in the context of zinc-finger recombinase fusion proteins.
Flotillins play an essential role in Niemann-Pick C1-like 1-mediated cholesterol uptake
Authors: Ge Liang , Qi Wei , Wang Li-Juan , Miao Hong-Hua , Qu Yu-Xiu , Li Bo-Liang , Song Bao-Liang
Published: December 2010

Dietary absorption is a major way for mammals to obtain cholesterol, which is mediated by Niemann-Pick C1-like 1 (NPC1L1) via vesicular endocytosis. One fundamental question in this process is how free cholesterol is efficiently taken up through the internalization of NPC1L1. Using exogenously expressed NPC1L1-EGFP, we show that the lipid raft proteins flotillins associate with NPC1L1 and their localization is regulated by NPC1L1 during intracellular trafficking. Furthermore, flotillins are essential for NPC1L1-mediated cellular cholesterol uptake, biliary cholesterol reabsorption, and the regulation of lipid levels in mice. Together with NPC1L1, they form cholesterol-enriched membrane microdomains, which function as carriers for bulk of cholesterol. The hypocholesterolemic drug ezetimibe disrupts the association between NPC1L1 and flotillins, which blocks the formation of the cholesterol-enriched microdomains. Our findings reveal a functional role of flotillins in NPC1L1-mediated cholesterol uptake and elucidate the formation of NPC1L1–flotillins-postive cholesterol-enriched membrane microdomains as a mechanism for efficient cholesterol absorption.
Vitamin A-dependent transcriptional activation of the nuclear factor of activated T cells c1 (NFATc1) is critical for the development and survival of B1 cells
Authors: Maruya Mikako , Suzuki Keiichiro , Fujimoto Hanae , Miyajima Michio , Kanagawa Osami , Wakayama Teruhiko , Fagarasan Sidonia
Published: December 2010

B1 cells represent a distinct subset of B cells that produce most of the natural serum IgM and much of the gut IgA and function as an important component of early immune responses to pathogens. The development of B1 cells depends on the nuclear factor of activated T cells c1 (NFATc1), a transcription factor abundantly expressed by B1 cells but not by conventional B2 cells. However, the factors that regulate the expression of NFATc1 in B1 cells remain unknown. Here we show that a vitamin A-deficient diet results in reduction of NFATc1 expression in B1 cells and almost complete loss of the B1 cell compartment. As a consequence, vitamin A-deficient mice have reduced serum IgM and are unable to mount T cell-independent antibody responses against bacterial antigens. We demonstrate that injection of
all-trans
retinoic acid induces the expression of NFATc1, particularly from the constitutive P2 promoter, and leads to the increase of the B1 cells. Thus, the retinoic acid-dependent pathway is critical for regulating NFATc1 expression and for maintenance of the natural memory B cell compartment.
Distortions of mind perception in psychopathology
Authors: Gray Kurt , Jenkins Adrianna C. , Heberlein Andrea S. , Wegner Daniel M.
Published: December 2010

It has long been known that psychopathology can influence social perception, but a 2D framework of mind perception provides the opportunity for an integrative understanding of some disorders. We examined the covariation of mind perception with three subclinical syndromes—autism-spectrum disorder, schizotypy, and psychopathy—and found that each presents a unique mind-perception profile. Autism-spectrum disorder involves reduced perception of agency in adult humans. Schizotypy involves increased perception of both agency and experience in entities generally thought to lack minds. Psychopathy involves reduced perception of experience in adult humans, children, and animals. Disorders are differentially linked with the over- or underperception of agency and experience in a way that helps explain their real-world consequences.
High-precision radiocarbon dating shows recent and rapid initial human colonization of East Polynesia
Authors: Wilmshurst Janet M. , Hunt Terry L. , Lipo Carl P. , Anderson Atholl J.
Published: December 2010

The 15 archipelagos of East Polynesia, including New Zealand, Hawaii, and Rapa Nui, were the last habitable places on earth colonized by prehistoric humans. The timing and pattern of this colonization event has been poorly resolved, with chronologies varying by >1000 y, precluding understanding of cultural change and ecological impacts on these pristine ecosystems. In a meta-analysis of 1,434 radiocarbon dates from the region, reliable short-lived samples reveal that the colonization of East Polynesia occurred in two distinct phases: earliest in the Society Islands A.D. ∼1025–1120, four centuries later than previously assumed; then after 70–265 y, dispersal continued in one major pulse to all remaining islands A.D. ∼1190–1290. We show that previously supported longer chronologies have relied upon radiocarbon-dated materials with large sources of error, making them unsuitable for precise dating of recent events. Our empirically based and dramatically shortened chronology for the colonization of East Polynesia resolves longstanding paradoxes and offers a robust explanation for the remarkable uniformity of East Polynesian culture, human biology, and language. Models of human colonization, ecological change and historical linguistics for the region now require substantial revision.
Targeting membrane heat-shock protein 70 (Hsp70) on tumors by cmHsp70.1 antibody
Authors: Stangl Stefan , Gehrmann Mathias , Riegger Julia , Kuhs Kristin , Riederer Isabelle , Sievert Wolfgang , Hube Kathrin , Mocikat Ralph , Dressel Ralf , Kremmer Elisabeth , Pockley Alan G. , Friedrich Lars , Vigh Laszlo , Skerra Arne , Multhoff Gabriele
Published: December 2010

Immunization of mice with a 14-mer peptide TKDNNLLGRFELSG, termed “TKD,” comprising amino acids 450–461 (aa450–461) in the C terminus of inducible Hsp70, resulted in the generation of an IgG1 mouse mAb cmHsp70.1. The epitope recognized by cmHsp70.1 mAb, which has been confirmed to be located in the TKD sequence by SPOT analysis, is frequently detectable on the cell surface of human and mouse tumors, but not on isogenic cells and normal tissues, and membrane Hsp70 might thus serve as a tumor-specific target structure. As shown for human tumors, Hsp70 is associated with cholesterol-rich microdomains in the plasma membrane of mouse tumors. Herein, we show that the cmHsp70.1 mAb can selectively induce antibody-dependent cellular cytotoxicity (ADCC) of membrane Hsp70+mouse tumor cells by unstimulated mouse spleen cells. Tumor killing could be further enhanced by activating the effector cells with TKD and IL-2. Three consecutive injections of the cmHsp70.1 mAb into mice bearing CT26 tumors significantly inhibited tumor growth and enhanced the overall survival. These effects were associated with infiltrations of NK cells, macrophages, and granulocytes. The Hsp70 specificity of the ADCC response was confirmed by preventing the antitumor response in tumor-bearing mice by coinjecting the cognate TKD peptide with the cmHsp70.1 mAb, and by blocking the binding of cmHsp70.1 mAb to CT26 tumor cells using either TKD peptide or the C-terminal substrate-binding domain of Hsp70.
Nanoparticle-mediated delivery of siRNA targeting Parp1 extends survival of mice bearing tumors derived from Brca1-deficient ovarian cancer cells
Authors: Goldberg Michael S. , Xing Deyin , Ren Yin , Orsulic Sandra , Bhatia Sangeeta N. , Sharp Phillip A.
Published: December 2010

Inhibition of the DNA repair enzyme poly(ADP-ribose) polymerase 1 (PARP1) with small molecules has been shown to be an effective treatment for ovarian cancer with BRCA mutations. Here, we report the in vivo administration of siRNA to Parp1 in mouse models of ovarian cancer. A unique member of the lipid-like materials known as lipidoids is shown to deliver siRNA to disseminated murine ovarian carcinoma allograft tumors following intraperitoneal (i.p.) injection. siParp1 inhibits cell growth, primarily by induction of apoptosis, in Brca1-deficient cells both in vitro and in vivo. Additionally, the treatment extends the survival of mice bearing tumors derived from Brca1-deficient ovarian cancer cells but not from Brca1 wild-type cells, confirming the proposed mechanism of synthetic lethality. Because there are 17 members of the Parp family, the inherent complementarity of RNA affords a high level of specificity for therapeutically addressing Parp1 in the context of impaired homologous recombination.
Na,K-ATPase α4 isoform is essential for sperm fertility
Authors: Jimenez Tamara , McDermott Jeffrey P. , Sánchez Gladis , Blanco Gustavo
Published: December 2010

Regulation of ion balance in spermatozoa has been shown to be essential for sperm motility and fertility. Control of intracellular ion levels requires the function of distinct ion-transport mechanisms at the cell plasma membrane. Active Na+and K+exchange in sperm is under the control of the Na,K-ATPase. Two molecular variants of the catalytic subunit of the Na,K-ATPase, α1 and α4, coexist in sperm. These isoforms exhibit different biochemical properties; however, their function in sperm fertility is unknown. In this work, we show that Na,K-ATPase α4 is essential for sperm fertility. Knockout male mice lacking α4 are completely sterile and spermatozoa from these mice are unable of fertilizing eggs in vitro. Furthermore, α4 deletion results in severe reduction in sperm motility and hyperactivation typical of sperm capacitation. In addition, absence of α4 causes a characteristic bend in the sperm flagellum, indicative of abnormal sperm ion regulation. Accordingly, α4-null sperm present increased intracellular Na+and cell plasma membrane depolarization. These results are unique in demonstrating the absolute requirement of α4 for sperm fertility. Moreover, the inability of α1 to compensate for α4 suggests that α4 is the Na,K-ATPase-α isoform directly involved in sperm fertility. Our findings show α4 as an attractive target for male contraception and open the possibility for the potential use of this Na,K-ATPase isoform as a biomarker for male fertility.
High-quality draft assemblies of mammalian genomes from massively parallel sequence data
Authors: Gnerre Sante , MacCallum Iain , Przybylski Dariusz , Ribeiro Filipe J. , Burton Joshua N. , Walker Bruce J. , Sharpe Ted , Hall Giles , Shea Terrance P. , Sykes Sean , Berlin Aaron M. , Aird Daniel , Costello Maura , Daza Riza , Williams Louise , Nicol Robert , Gnirke Andreas , Nusbaum Chad , Lander Eric S. , Jaffe David B.
Published: December 2010

Massively parallel DNA sequencing technologies are revolutionizing genomics by making it possible to generate billions of relatively short (~100-base) sequence reads at very low cost. Whereas such data can be readily used for a wide range of biomedical applications, it has proven difficult to use them to generate high-quality de novo genome assemblies of large, repeat-rich vertebrate genomes. To date, the genome assemblies generated from such data have fallen far short of those obtained with the older (but much more expensive) capillary-based sequencing approach. Here, we report the development of an algorithm for genome assembly, ALLPATHS-LG, and its application to massively parallel DNA sequence data from the human and mouse genomes, generated on the Illumina platform. The resulting draft genome assemblies have good accuracy, short-range contiguity, long-range connectivity, and coverage of the genome. In particular, the base accuracy is high (≥99.95%) and the scaffold sizes (N50 size = 11.5 Mb for human and 7.2 Mb for mouse) approach those obtained with capillary-based sequencing. The combination of improved sequencing technology and improved computational methods should now make it possible to increase dramatically the de novo sequencing of large genomes. The ALLPATHS-LG program is available at
http://www.broadinstitute.org/science/programs/genome-biology/crd
.
Pre- and postexposure protection against virulent anthrax infection in mice by humanized monoclonal antibodies to
Bacillus anthracis
capsule
Authors: Chen Zhaochun , Schneerson Rachel , Lovchik Julie , Lyons C. Rick , Zhao Huaying , Dai Zhongdong , Kubler-Kielb Joanna , Leppla Stephen H. , Purcell Robert H.
Published: December 2010

One of the two essential virulence factors of
Bacillus anthracis
is the poly-γ-
d
-glutamic acid (γDPGA) capsule. Five γDPGA-specific antibody antigen-binding fragments (Fabs) were generated from immunized chimpanzees. The two selected for further study, Fabs 11D and 4C, were both converted into full-length IgG1 and IgG3 mAbs having human IgG1 or IgG3 constant regions. These two mAbs had similar binding affinities, in vitro opsonophagocytic activities, and in vivo efficacies, with the IgG1 and IgG3 subclasses reacting similarly. The mAbs bound to γDPGA specifically with estimated binding affinities (
K
d
) of 35–70 nM and effective affinities (effective
K
d
) of 0.1–0.3 nM. The LD
50
in an opsonophagocytic bactericidal assay was ≈10 ng/mL of 11D or 4C. A single 30-μg dose of either mAb given to BALB/c mice 18 h before challenge conferred about 50% protection against a lethal intratracheal spore challenge by the virulent
B. anthracis
Ames strain. More importantly, either mAb given 8 h or 20 h after challenge provided significant protection against lethal infection. Thus, these anti-γDPGA mAbs should be useful, alone or in combination with antitoxin mAbs, for achieving a safe and efficacious postexposure therapy for anthrax.
Folding of the Pit1 homeodomain near the speed limit
Authors: Banachewicz Wiktor , Johnson Christopher M. , Fersht Alan R.
Published: December 2010

Current questions in protein folding mechanisms include how fast can a protein fold and are there energy barriers for the folding and unfolding of ultrafast folding proteins? The small 3-helical engrailed homeodomain protein folds in 1.7 μs to form a well-characterized intermediate, which rearranges in 17 μs to native structure. We found that the homologous pituitary-specific transcription factor homeodomain (Pit1) folded in a similar manner, but in two better separated kinetic phases of 2.3 and 46 μs. The greater separation and better fluorescence changes facilitated a detailed kinetic analysis for the ultrafast phase for formation of the intermediate. Its folding rate constant changed little with denaturant concentration or mutation but unfolding was very sensitive to denaturant and energy changes on mutation. The folding rate constant of 3 × 10
5
 s
-1
in water decreased with increasing viscosity, and was extrapolated to 4.4 × 10
5
 s
-1
at zero viscosity. Thus, the formation of the intermediate was partly rate limited by chain diffusion and partly by an energy barrier to give a very diffuse transition state, which was followed by the formation of structure. Conversely, the unfolding reaction required the near complete disruption of the tertiary structure of the intermediate in a highly cooperative manner, being exquisitely sensitive to individual mutations. The folding is approaching, but has not reached, the downhill-folding scenario of energy landscape theory. Under folding conditions, there is a small energy barrier between the denatured and transition states but a larger barrier between native and transition states.
Adaptin-like protein TPLATE and clathrin recruitment during plant somatic cytokinesis occurs via two distinct pathways
Authors: Van Damme Daniël , Gadeyne Astrid , Vanstraelen Marleen , Inzé Dirk , Van Montagu Marc C. E. , De Jaeger Geert , Russinova Eugenia , Geelen Danny
Published: December 2010

Plant cytokinesis deploys a transport system that centers cell plate-forming vesicles and fuses them to form a cell plate. Here we show that the adaptin-like protein TPLATE and clathrin light chain 2 (CLC2) are targeted to the expanding cell plate and to the equatorial subregion of the plasma membrane referred to as the cortical division zone (CDZ). Bimolecular fluorescence complementation and immunodetection indicates that TPLATE interacts with clathrin. Pharmacological tools as well as analysis of protein targeting in a mutant background affecting cell plate formation allowed to discriminate two recruitment pathways for TPLATE and CLC2. The cell plate recruitment pathway is dependent on phragmoplast microtubule organization and the formation and transport of secretory vesicles. The CDZ recruitment pathway, on the other hand, is activated at the end of cytokinesis and independent of trans-Golgi–derived vesicle trafficking. TPLATE and CLC2 do not accumulate at a narrow zone central of the CDZ. We have dubbed this subdomain the cortical division site and show that it corresponds precisely with the position where the cell plate merges with the parental wall. These data provide evidence that the plasma membrane is subject to localized endocytosis or membrane remodeling processes that are required for the fusion of the cell plate with a predefined region of the plasma membrane.
Arc-dependent synapse-specific homeostatic plasticity
Authors: Béïque Jean-Claude , Na Youn , Kuhl Dietmar , Worley Paul F. , Huganir Richard L.
Published: December 2010

Both theoretical and experimental research has indicated that the synaptic strength between neurons in a network needs to be properly fine-tuned and controlled by homeostatic mechanisms to ensure proper network function. One such mechanism that has been extensively characterized is synaptic homeostatic plasticity or global synaptic scaling. This mechanism refers to the bidirectional ability of all synapses impinging on a neuron to actively compensate for changes in the neuron's overall excitability. Here, using a combination of electrophysiological, two-photon glutamate uncaging and imaging methods, we show that mature individual synapses, independent of neighboring synapses, have the ability to autonomously sense their level of activity and actively compensate for it in a homeostatic-like fashion. This synapse-specific homeostatic plasticity, similar to global synaptic plasticity, requires the immediate early gene Arc. Together, our results document an extra level of regulation of synaptic function that bears important computational consequences on information storage in the brain.
Genetic susceptibility to systemic lupus erythematosus protects against cerebral malaria in mice
Authors: Waisberg Michael , Tarasenko Tatyana , Vickers Brandi K. , Scott Bethany L. , Willcocks Lisa C. , Molina-Cruz Alvaro , Pierce Matthew A. , Huang Chiung-yu , Torres-Velez Fernando J. , Smith Kenneth G. C. , Barillas-Mury Carolina , Miller Louis H. , Pierce Susan K. , Bolland Silvia
Published: December 2010

Plasmodium falciparum
has exerted tremendous selective pressure on genes that improve survival in severe malarial infections. Systemic lupus erythematosus (SLE) is an autoimmune disease that is six to eight times more prevalent in women of African descent than in women of European descent. Here we provide evidence that a genetic susceptibility to SLE protects against cerebral malaria. Mice that are prone to SLE because of a deficiency in FcγRIIB or overexpression of Toll-like receptor 7 are protected from death caused by cerebral malaria. Protection appears to be by immune mechanisms that allow SLE-prone mice better to control their overall inflammatory responses to parasite infections. These findings suggest that the high prevalence of SLE in women of African descent living outside of Africa may result from the inheritance of genes that are beneficial in the immune control of cerebral malaria but that, in the absence of malaria, contribute to autoimmune disease.
β Ca
2+
/CaM-dependent kinase type II triggers upregulation of GluA1 to coordinate adaptation to synaptic inactivity in hippocampal neurons
Authors: Groth Rachel D. , Lindskog Maria , Thiagarajan Tara C. , Li Li , Tsien Richard W.
Published: December 2010

Prolonged AMPA-receptor blockade in hippocampal neuron cultures leads to both an increased expression of GluA1 postsynaptically and an increase in vesicle pool size and turnover rate presynaptically, adaptive changes that extend beyond simple synaptic scaling. As a molecular correlate, expression of the β Ca
2+
/CaM-dependent kinase type II (βCaMKII) is increased in response to synaptic inactivity. Here we set out to clarify the role of βCaMKII in the various manifestations of adaptation. Knockdown of βCaMKII by lentiviral-mediated expression of shRNA prevented the synaptic inactivity-induced increase in GluA1, as did treatment with the CaM kinase inhibitor KN-93, but not the inactive analog KN-92. These results demonstrate that, spurred by AMPA-receptor blockade, up-regulation of βCaMKII promotes increased GluA1 expression. Indeed, transfection of βCaMKII, but not a kinase-dead mutant, increased GluA1 expression on dendrites and elevated vesicle turnover (Syt-Ab uptake), mimicking the effect of synaptic inactivity on both sides of the synapse. In cells with elevated βCaMKII, relief of synaptic-activity blockade uncovered an increase in the frequency of miniature excitatory postsynaptic currents that could be rapidly and fully suppressed by PhTx blockade of GluA1 receptors. This increased mini frequency involved a genuine presynaptic enhancement, not merely an increased abundance of synapses. This finding suggests that Ca
2+
flux through GluA1 receptors may trigger the acute release of a retrograde messenger. Taken together, our results indicate that synaptic inactivity-induced increases in βCaMKII expression set in motion a series of events that culminate in coordinated pre- and postsynaptic adaptations in synaptic transmission.
Leptin-dependent serotonin control of appetite: temporal specificity, transcriptional regulation, and therapeutic implications
Authors: Yadav Vijay K. , Oury Franck , Tanaka Kenji F. , Thomas Tiffany , Wang Ying , Cremers Serge , Hen Rene , Krust Andree , Chambon Pierre , Karsenty Gerard
Published: December 2010

Recent evidence indicates that leptin regulates appetite and energy expenditure, at least in part by inhibiting serotonin synthesis and release from brainstem neurons. To demonstrate that this pathway works postnatally, we used a conditional, brainstem-specific mouse CreERT2 driver to show that leptin signals in brainstem neurons after birth to decrease appetite by inhibiting serotonin synthesis. Cell-specific gene deletion experiments and intracerebroventricular leptin infusions reveal that serotonin signals in arcuate nuclei of the hypothalamus through the Htr1a receptor to favor food intake and that this serotonin function requires the expression of Creb, which regulates the expression of several genes affecting appetite. Accordingly, a specific antagonist of the Htr1a receptor decreases food intake in leptin-deficient but not in Htr1a−/− mice. Collectively, these results establish that leptin inhibition of serotonin is necessary to inhibit appetite postnatally and provide a proof of principle that selective inhibition of this pathway may be a viable option to treat appetite disorders.
Cellular cohesiveness in benign and malignant thyroid follicular tumours varies significantly, but the difference is not useful in diagnosis of individual cases
Authors: Rozkos T. , Ryska A. , Cap J. , Sobande F. , Laco J.
Published: December 2010

T. Rozkos, A. Ryska, J. Cap, F. Sobande and J. Laco  Cellular cohesiveness in benign and malignant thyroid follicular tumours varies significantly, but the difference is not useful in diagnosis of individual cases

Introduction:  The aim of our study was to search for new, readily available and statistically reliable cytological markers for differentiating benign and malignant follicular thyroid neoplasms pre‐operatively.

Methods:  Cohesiveness of tumour cells in cytology slides from a series of 58 follicular tumours diagnosed between 1998 and 2004 inclusive was studied, including 48 follicular adenomas, and eight minimally invasive and two widely invasive follicular carcinomas. Photomicrographs of the cytology slides were taken and the digital images were analysed using computer image analysis software. We evaluated the relative proportions of cells arranged in groups of various sizes. The cohesiveness of the cells in cytological smears was then correlated with the immunohistochemical expression of E‐cadherin in corresponding histological slides.

Results:  Cases from 15 men (26%) and 43 women (74%) with a mean age of 50 years (range, 19–79) were analysed. In follicular adenomas and carcinomas, respectively, isolated cells were seen in 16.8% and 24.7% (P = 0.028), groups of two to five cells in 9.7% and 11.5% (P = 0.145) and groups of more than five cells in 73.5% and 63.8% (P = 0.041). The mean cell count in groups with more than five cells was 46.5 and 27.0 in adenomas and carcinomas, respectively (P < 0.001). Cell cohesiveness, either as percentage of cells in groups of more than five (R2 = 0.026) or as mean cell count per group of more than five (R2 = 0.005), was not found to be dependent on the expression of E‐cadherin. Using a threshold of 13% isolated tumour cells in cytological smears, follicular adenomas and carcinomas could be distinguished with 90% sensitivity and 41% specificity.

Conclusions:  Although we demonstrated a statistically significant difference in cell cohesion between follicular adenomas and carcinomas, these could not be distinguished in the clinical setting by evaluation of the percentage of isolated cells in cytological smears because the specificity was too low. The absence of correlation of cellular cohesiveness with E‐cadherin expression indicates that other factors are probably responsible for the loss of cohesiveness observed in follicular thyroid malignancy.
Discretely calibrated regulatory loops controlled by ppGpp partition gene induction across the ‘feast to famine’ gradient in Escherichia coli
Authors: Traxler Matthew F. , Zacharia Vineetha M. , Marquardt Stafford , Summers Sean M. , Nguyen Huyen‐Tran , Stark S. Elizabeth , Conway Tyrrell
Published: December 2010

Bacteria comprehensively reorganize their global gene expression when faced with starvation. The alarmone ppGpp facilitates this massive response by co‐ordinating the downregulation of genes of the translation apparatus, and the induction of biosynthetic genes and the general stress response. Such a large reorientation requires the activities of multiple regulators, yet the regulatory network downstream of ppGpp remains poorly defined. Transcription profiling during isoleucine depletion, which leads to gradual starvation (over > 100 min), allowed us to identify genes that required ppGpp, Lrp and RpoS for their induction and to deduce the regulon response times. Although the Lrp and RpoS regulons required ppGpp for their activation, they were not induced simultaneously. The data suggest that metabolic genes, i.e. those of the Lrp regulon, require only a low level of ppGpp for their induction. In contrast, the RpoS regulon was induced only when high levels of ppGpp accumulated. We tested several predictions of a model that explains how bacteria allocate transcriptional resources between metabolism and stress response by discretely tuning two regulatory circuits to different levels of ppGpp. The emergent regulatory structure insures that stress survival circuits are only triggered if homeostatic metabolic networks fail to compensate for environmental deficiencies.
Spo0J regulates the oligomeric state of Soj to trigger its switch from an activator to an inhibitor of DNA replication initiation
Authors: Scholefield Graham , Whiting Rachel , Errington Jeff , Murray Heath
Published: December 2010

Control of DNA replication initiation is essential for bacterial cells to co‐ordinate the faithful replication and segregation of their genetic material. The Bacillus subtilis ATPase Soj is a dynamic protein that regulates DNA replication initiation by either inhibiting or activating the DNA replication initiator protein DnaA. Here we report that the key event which switches Soj regulatory activity is a transition in its oligomeric state from a monomer to an ATP‐dependent homodimer capable of DNA binding. We show that the DNA binding activity of the Soj dimer is required both for activation of DNA replication initiation and for interaction with Spo0J. Finally, we demonstrate that Spo0J inhibits Soj dimerization by stimulating Soj ATPase activity. The data provide a molecular explanation for the dichotomous regulatory activities of Soj, as well as assigning unique Soj conformations to distinct cellular localization patterns. We discuss how the regulation of Soj ATPase activity by Spo0J could be utilized to control the initiation of DNA replication during the cell cycle.
Cooperative amino acid changes shift the response of the σ54‐dependent regulator XylR from natural m‐xylene towards xenobiotic 2,4‐dinitrotoluene
Authors: de las Heras Aitor , de Lorenzo Víctor
Published: December 2010

XylR is a σ54‐dependent transcriptional factor of Pseudomonas putida that activates the Pu promoter of the TOL plasmid upon binding its natural effector, m‐xylene. The search for mutants of the signal‐sensing module of XylR that respond to the xenobiotic compound 2,4‐dinitrotoluene recurrently yields protein variants with a broad effector range. These mutants had amino acid changes not only in the effector recognition moiety (A module), but also in the inter‐domain B linker of the protein. A random mutagenesis and selection/counterselection setup was adopted to optimize the 2,4‐DNT reaction of XylRv17, one of the best 2,4‐DNT responders and thus recreate how this regulator can adjust its specificity to novel effectors by individual changes on the evolving protein. Site‐specific mutagenesis was then used to decipher the contribution of individual mutations in XylRv17 and in one of the mutants evolved from it (XylR28) to the 2,4‐DNT response. This approach allowed us to capture a new XylR version with novel mutations that fixed the protein in an intermediate stage of the progress from an effector‐promiscuous, pluri‐potent protein type to a more specific form where the natural response to m‐xylene was decreased and the non‐native acquired response to 2,4‐DNT was increased.
Two‐component regulatory systems in Pseudomonas aeruginosa: an intricate network mediating fimbrial and efflux pump gene expression
Authors: Sivaneson Melissa , Mikkelsen Helga , Ventre Isabelle , Bordi Christophe , Filloux Alain
Published: December 2010

Pseudomonas aeruginosa is responsible for chronic and acute infections in humans. Chronic infections are associated with production of fimbriae and the formation of a biofilm. The two‐component system Roc1 is named after its role in the regulation of cup genes, which encode components of a machinery allowing assembly of fimbriae. A non‐characterized gene cluster, roc2, encodes components homologous to the Roc1 system. We show that cross‐regulation occurs between the Roc1 and Roc2 signalling pathways. We demonstrate that the sensors RocS2 and RocS1 converge on the response regulator RocA1 to control cupC gene expression. This control is independent of the response regulator RocA2. Instead, we show that these sensors act via the RocA2 response regulator to repress the mexAB‐oprM genes. These genes encode a multidrug efflux pump and are upregulated in the rocA2 mutant, which is less susceptible to antibiotics. It has been reported that in cystic fibrosis lungs, in which P. aeruginosa adopts the biofilm lifestyle, most isolates have an inactive MexAB‐OprM pump. The concomitant RocS2‐dependent upregulation of cupC genes (biofilm formation) and downregulation of mexAB‐oprM genes (antibiotic resistance) is in agreement with this observation. It suggests that the Roc systems may sense the environment in the cystic fibrosis lung.
Gene expression profiling of yeasts overexpressing wild type or misfolded Pma1 variants reveals activation of the Hog1 MAPK pathway
Authors: Eraso Pilar , Mazón María J. , Posas Francesc , Portillo Francisco
Published: December 2010

Dominant negative PMA1 mutants render misfolded proteins that are retained in the endoplasmic reticulum (ER) and slowly degraded by ER‐associated degradation. Accumulation of misfolded proteins in the ER activates an ER‐to‐nucleus signalling pathway termed the unfolded protein response (UPR). We have used a PMA1‐D378T dominant negative mutant to analyse its impact on UPR induction. Our results show that overexpression of the misfolded mutant Pma1 does not lead to activation of the UPR. In addition, in mutants with a constitutively activated UPR the turnover rate of the mutant ATPase is not altered. To determine if the expression of the misfolded mutant protein induces some other kind of response we performed global gene expression analysis experiments in yeasts overexpressing either wild type or dominant lethal PMA1 alleles. The results suggest that the high osmolarity glycerol (Hog1) mitogen‐activated protein kinase (MAPK) pathway is activated by both wild type and mutant ATPases. We show that expression of the PMA1 alleles induces phosphorylation of Hog1 and activation of the Hog1 MAPK cascade. This activation is mediated by the Sln1 branch of the stress‐dependent Hog1 MAPK network. Finally, we show that at least two other plasma membrane proteins are also able to activate the Hog1 MAPK system.
Amelioration of intestinal colitis by macrophage migration inhibitory factor isolated from intestinal parasites through Toll‐like receptor 2
Authors: CHO M. K. , LEE C. H. , YU H. S.
Published: December 2010

In a previous study, we cloned type II MIFs (As‐MIF) from Anisakis simplex 3rd stage larva and expressed a recombinant protein that suppressed allergic airway inflammation via regulatory T (CD4+CD25+Foxp3+ T; Treg)‐cell recruitment. In this study, in an effort to evaluate the function of rAs‐MIF on another immune disease, we induced intestinal inflammation in mice using dextran sodium sulphate (DSS) with or without the application of rAs‐MIF treatment to the mice. As a consequence, weight losses were recovered, and the value of disease activity index (DAI) was reduced by rAs‐MIF treatment during the experimental period. The levels of TGF‐β and IL‐10 in the spleens and mesenteric lymph nodes (MLN) from the rAs‐MIF‐treated mice were higher, but the levels of IFN‐γ, IL‐6 and IL‐13 were lower than those of the mice treated with DSS but not with rAs‐MIF. Additionally, the Treg cells observed were greatly increased in the MLNs of the rAs‐MIF‐treated mice than those of mice not treated with rAs‐MIF. The results of our in vitro experiments showed that the elevated IL‐10 production induced by rAs‐MIF was generated via toll‐like receptor 2. In conclusion, rAs‐MIF appears to ameliorate DSS‐induced colitis and may prove useful as a therapeutic agent for the treatment of intestinal inflammatory disease.