bluuebunny/crossref_metadata_2025 huggingface dataset:
Crystal structure analysis reveals
Pseudomonas
PilY1 as an essential calcium-dependent regulator of bacterial surface motility
Authors: Orans Jillian , Johnson Michael D. L. , Coggan Kimberly A. , Sperlazza Justin R. , Heiniger Ryan W. , Wolfgang Matthew C. , Redinbo Matthew R.
Published: December 2009

Several bacterial pathogens require the “twitching” motility produced by filamentous type IV pili (T4P) to establish and maintain human infections. Two cytoplasmic ATPases function as an oscillatory motor that powers twitching motility via cycles of pilus extension and retraction. The regulation of this motor, however, has remained a mystery. We present the 2.1 Å resolution crystal structure of the
Pseudomonas aeruginosa
pilus-biogenesis factor PilY1, and identify a single site on this protein required for bacterial translocation. The structure reveals a modified β-propeller fold and a distinct EF-hand-like calcium-binding site conserved in pathogens with retractile T4P. We show that preventing calcium binding by PilY1 using either an exogenous calcium chelator or mutation of a single residue disrupts
Pseudomonas
twitching motility by eliminating surface pili. In contrast, placing a lysine in this site to mimic the charge of a bound calcium interferes with motility in the opposite manner—by producing an abundance of nonfunctional surface pili. Our data indicate that calcium binding and release by the unique loop identified in the PilY1 crystal structure controls the opposing forces of pilus extension and retraction. Thus, PilY1 is an essential, calcium-dependent regulator of bacterial twitching motility.
c-Abl tyrosine kinase regulates cardiac growth and development
Authors: Qiu Zhaozhu , Cang Yong , Goff Stephen P.
Published: December 2009

The c-Abl protein is a ubiquitously expressed nonreceptor tyrosine kinase involved in the development and function of many mammalian organ systems, including the immune system and bone. Here we show that homozygous
Abl
mutant embryos and newborns on the C57BL/6J background, but not on other backgrounds, display dramatically enlarged hearts and die perinatally. The heart defects can be largely rescued by cardiomyocyte-specific restoration of the full-length c-Abl protein. The cardiac hyperplasia phenotype is not caused by decreased apoptosis, but rather by abnormally increased cardiomyocyte proliferation during later stages of embryogenesis. Genes involved in cardiac stress and remodeling and cell cycle regulation are also up-regulated in the mutant hearts. These findings reveal an essential role for c-Abl in mammalian heart growth and development.
Dual viewpoint gestures
Authors: Parrill Fey
Published: December 2009

This paper examines gestures that simultaneously express multiple physical perspectives, known as dual viewpoint gestures. These gestures were first discussed in McNeill’s 1992 book, Hand and mind. We examine a corpus of approximately fifteen hours of narrative data, and use these data to extend McNeill’s observations about the different possibilities for combining viewpoints. We also show that a phenomenon thought to be present only in the narrations of children is present in the narrations of adults. We discuss the significance of these gestures for theories of speech-gesture integration.
Gesture–speech integration in narrative
Authors: Alibali Martha W. , Evans Julia L. , Hostetter Autumn B. , Ryan Kristin , Mainela-Arnold Elina
Published: December 2009

Speakers sometimes express information in gestures that they do not express in speech. In this research, we developed a system that could be used to assess the redundancy of gesture and speech in a narrative task. We then applied this system to examine whether children and adults produce non-redundant gesture–speech combinations at similar rates. The coding system was developed based on a sample of 30 children. A crucial feature of the system is that gesture meanings can be assessed based on form alone; thus, the meanings speakers express in gesture and speech can be assessed independently and compared. We then collected narrative data from a new sample of 17 children (ages 5–10), as well as a sample of 20 adults, and we determined the average proportion of non-redundant gesture–speech combinations produced by individuals in each group. Children produced more non-redundant gesture–speech combinations than adults, both at the clause level and at the word level. These findings suggest that gesture–speech integration is not constant over the life span, but instead appears to change with development.
Repetition in infant-directed action depends on the goal structure of the object
Authors: Brand Rebecca J. , McGee Anna , Kominsky Jonathan F. , Briggs Kristen , Gruneisen Aline , Orbach Tessa
Published: December 2009

Adults automatically adjust their speech and actions in a way that may facilitate infants’ processing (e.g., Brand, Baldwin, & Ashburn, 2002). This research examined whether mothers’ use of repetition for infants depended on whether the object being demonstrated required a series of actions in sequence in order to reach a salient goal (called an “enabling” sequence). Mothers (n = 39) demonstrated six objects, three with an enabling sequence and three with an arbitrary sequence, to their 6- to 8- or 11- to 13-month-olds. As predicted, in demonstrations of objects with an enabling sequence, mothers were more likely to repeat series of actions, whereas for those without such structure, mothers were more likely to repeat individual units of action. This may or may not have been deliberately pedagogical on mothers’ part, but nevertheless indicates another way in which input to infants is richly patterned to support their learning.
Mitochondrial proteases strike the right balance
Authors: Short Ben
Published: December 2009

Two studies reveal how proteases combine to regulate mitochondrial fusion.
Elaine Fuchs: A love for science that's more than skin deep
Authors: Short Ben
Published: December 2009

Fuchs is too passionate about her work to rest on her recently awarded laurels.
The binding of NCAM to FGFR1 induces a specific cellular response mediated by receptor trafficking
Authors: Francavilla Chiara , Cattaneo Paola , Berezin Vladimir , Bock Elisabeth , Ami Diletta , de Marco Ario , Christofori Gerhard , Cavallaro Ugo
Published: December 2009

Neural cell adhesion molecule (NCAM) associates with fibroblast growth factor (FGF) receptor-1 (FGFR1). However, the biological significance of this interaction remains largely elusive. In this study, we show that NCAM induces a specific, FGFR1-mediated cellular response that is remarkably different from that elicited by FGF-2. In contrast to FGF-induced degradation of endocytic FGFR1, NCAM promotes the stabilization of the receptor, which is recycled to the cell surface in a Rab11- and Src-dependent manner. In turn, FGFR1 recycling is required for NCAM-induced sustained activation of various effectors. Furthermore, NCAM, but not FGF-2, promotes cell migration, and this response depends on FGFR1 recycling and sustained Src activation. Our results implicate NCAM as a nonconventional ligand for FGFR1 that exerts a peculiar control on the intracellular trafficking of the receptor, resulting in a specific cellular response. Besides introducing a further level of complexity in the regulation of FGFR1 function, our findings highlight the link of FGFR recycling with sustained signaling and cell migration and the critical role of these events in dictating the cellular response evoked by receptor activation.
Role of A-type lamins in signaling, transcription, and chromatin organization
Authors: Andrés Vicente , González José M.
Published: December 2009

A-type lamins (lamins A and C), encoded by the LMNA gene, are major protein constituents of the mammalian nuclear lamina, a complex structure that acts as a scaffold for protein complexes that regulate nuclear structure and functions. Interest in these proteins has increased in recent years with the discovery that LMNA mutations cause a variety of human diseases termed laminopathies, including progeroid syndromes and disorders that primarily affect striated muscle, adipose, bone, and neuronal tissues. In this review, we discuss recent research supporting the concept that lamin A/C and associated nuclear envelope proteins regulate gene expression in health and disease through interplay with signal transduction pathways, transcription factors, and chromatin-associated proteins.
Cellular IAPs inhibit a cryptic CD95-induced cell death by limiting RIP1 kinase recruitment
Authors: Geserick Peter , Hupe Mike , Moulin Maryline , Wong W. Wei-Lynn , Feoktistova Maria , Kellert Beate , Gollnick Harald , Silke John , Leverkus Martin
Published: December 2009

A role for cellular inhibitors of apoptosis (IAPs [cIAPs]) in preventing CD95 death has been suspected but not previously explained mechanistically. In this study, we find that the loss of cIAPs leads to a dramatic sensitization to CD95 ligand (CD95L) killing. Surprisingly, this form of cell death can only be blocked by a combination of RIP1 (receptor-interacting protein 1) kinase and caspase inhibitors. Consistently, we detect a large increase in RIP1 levels in the CD95 death-inducing signaling complex (DISC) and in a secondary cytoplasmic complex (complex II) in the presence of IAP antagonists and loss of RIP1-protected cells from CD95L/IAP antagonist–induced death. Cells resistant to CD95L/IAP antagonist treatment could be sensitized by short hairpin RNA–mediated knockdown of cellular FLICE-inhibitory protein (cFLIP). However, only cFLIPL and not cFLIPS interfered with RIP1 recruitment to the DISC and complex II and protected cells from death. These results demonstrate a fundamental role for RIP1 in CD95 signaling and provide support for a physiological role of caspase-independent death receptor–mediated cell death.
Inducible proteolytic inactivation of OPA1 mediated by the OMA1 protease in mammalian cells
Authors: Head Brian , Griparic Lorena , Amiri Mandana , Gandre-Babbe Shilpa , van der Bliek Alexander M.
Published: December 2009

The mammalian mitochondrial inner membrane fusion protein OPA1 is controlled by complex patterns of alternative splicing and proteolysis. A subset of OPA1 isoforms is constitutively cleaved by YME1L. Other isoforms are not cleaved by YME1L, but they are cleaved when mitochondria lose membrane potential or adenosine triphosphate. In this study, we show that this inducible cleavage is mediated by a zinc metalloprotease called OMA1. We find that OMA1 small interfering RNA inhibits inducible cleavage, helps retain fusion competence, and slows the onset of apoptosis, showing that OMA1 controls OPA1 cleavage and function. We also find that OMA1 is normally cleaved from 60 to 40 kD by another as of yet unidentified protease. Loss of membrane potential causes 60-kD protein to accumulate, suggesting that OMA1 is attenuated by proteolytic degradation. We conclude that a proteolytic cascade controls OPA1. Inducible cleavage provides a mechanism for quality control because proteolytic inactivation of OPA1 promotes selective removal of defective mitochondrial fragments by preventing their fusion with the mitochondrial network.
Regulation of OPA1 processing and mitochondrial fusion by m-AAA protease isoenzymes and OMA1
Authors: Ehses Sarah , Raschke Ines , Mancuso Giuseppe , Bernacchia Andrea , Geimer Stefan , Tondera Daniel , Martinou Jean-Claude , Westermann Benedikt , Rugarli Elena I. , Langer Thomas
Published: December 2009

Mitochondrial fusion depends on the dynamin-like guanosine triphosphatase OPA1, whose activity is controlled by proteolytic cleavage. Dysfunction of mitochondria induces OPA1 processing and results in mitochondrial fragmentation, allowing the selective removal of damaged mitochondria. In this study, we demonstrate that two classes of metallopeptidases regulate OPA1 cleavage in the mitochondrial inner membrane: isoenzymes of the adenosine triphosphate (ATP)–dependent matrix AAA (ATPase associated with diverse cellular activities [m-AAA]) protease, variable assemblies of the conserved subunits paraplegin, AFG3L1 and -2, and the ATP-independent peptidase OMA1. Functionally redundant isoenzymes of the m-AAA protease ensure the balanced accumulation of long and short isoforms of OPA1 required for mitochondrial fusion. The loss of AFG3L2 in mouse tissues, down-regulation of AFG3L1 and -2 in mouse embryonic fibroblasts, or the expression of a dominant-negative AFG3L2 variant in human cells decreases the stability of long OPA1 isoforms and induces OPA1 processing by OMA1. Moreover, cleavage by OMA1 causes the accumulation of short OPA1 variants if mitochondrial DNA is depleted or mitochondrial activities are impaired. Our findings link distinct peptidases to constitutive and induced OPA1 processing and shed new light on the pathogenesis of neurodegenerative disorders associated with mutations in m-AAA protease subunits.
Opposite-polarity motors activate one another to trigger cargo transport in live cells
Authors: Ally Shabeen , Larson Adam G. , Barlan Kari , Rice Sarah E. , Gelfand Vladimir I.
Published: December 2009

Intracellular transport is typically bidirectional, consisting of a series of back and forth movements. Kinesin-1 and cytoplasmic dynein require each other for bidirectional transport of intracellular cargo along microtubules; i.e., inhibition or depletion of kinesin-1 abolishes dynein-driven cargo transport and vice versa. Using Drosophila melanogaster S2 cells, we demonstrate that replacement of endogenous kinesin-1 or dynein with an unrelated, peroxisome-targeted motor of the same directionality activates peroxisome transport in the opposite direction. However, motility-deficient versions of motors, which retain the ability to bind microtubules and hydrolyze adenosine triphosphate, do not activate peroxisome motility. Thus, any pair of opposite-polarity motors, provided they move along microtubules, can activate one another. These results demonstrate that mechanical interactions between opposite-polarity motors are necessary and sufficient for bidirectional organelle transport in live cells.
The Chlamydomonas reinhardtii BBSome is an IFT cargo required for export of specific signaling proteins from flagella
Authors: Lechtreck Karl-Ferdinand , Johnson Eric C. , Sakai Tsuyoshi , Cochran Deborah , Ballif Bryan A. , Rush John , Pazour Gregory J. , Ikebe Mitsuo , Witman George B.
Published: December 2009

In humans, seven evolutionarily conserved genes that cause the cilia-related disorder Bardet-Biedl syndrome (BBS) encode proteins that form a complex termed the BBSome. The function of the BBSome in the cilium is not well understood. We purified a BBSome-like complex from Chlamydomonas reinhardtii flagella and found that it contains at least BBS1, -4, -5, -7, and -8 and undergoes intraflagellar transport (IFT) in association with a subset of IFT particles. C. reinhardtii insertional mutants defective in BBS1, -4, and -7 assemble motile, full-length flagella but lack the ability to phototax. In the bbs4 mutant, the assembly and transport of IFT particles are unaffected, but the flagella abnormally accumulate several signaling proteins that may disrupt phototaxis. We conclude that the BBSome is carried by IFT but is an adapter rather than an integral component of the IFT machinery. C. reinhardtii BBS4 may be required for the export of signaling proteins from the flagellum via IFT.
The nonpolymorphic MHC Qa-1b mediates CD8+ T cell surveillance of antigen-processing defects
Authors: Oliveira Cláudia C. , van Veelen Peter A. , Querido Bianca , de Ru Arnoud , Sluijter Marjolein , Laban Sandra , Drijfhout Jan W. , van der Burg Sjoerd H. , Offringa Rienk , van Hall Thorbald
Published: December 2009

The nonclassical major histocompatibility complex (MHC) Qa-1b accommodates monomorphic leader peptides and functions as a ligand for germ line receptors CD94/NKG2, which are expressed by natural killer cells and CD8+ T cells. We here describe that the conserved peptides are replaced by a novel peptide repertoire of surprising diversity as a result of impairments in the antigen-processing pathway. This novel peptide repertoire represents immunogenic neoantigens for CD8+ T cells, as we found that these Qa-1b–restricted T cells dominantly participated in the response to tumors with processing deficiencies. A surprisingly wide spectrum of target cells, irrespective of transformation status, MHC background, or type of processing deficiency, was recognized by this T cell subset, complying with the conserved nature of Qa-1b. Target cell recognition depended on T cell receptor and Qa-1b interaction, and immunization with identified peptide epitopes demonstrated in vivo priming of CD8+ T cells. Our data reveal that Qa-1b, and most likely its human homologue human leukocyte antigen-E, is important for the defense against processing-deficient cells by displacing the monomorphic leader peptides, which relieves the inhibition through CD94/NKG2A on lymphocytes, and by presenting a novel repertoire of immunogenic peptides, which recruits a subset of cytotoxic CD8+ T cells.
Thymic development beyond β-selection requires phosphatidylinositol 3-kinase activation by CXCR4
Authors: Janas Michelle L. , Varano Gabriele , Gudmundsson Kristjan , Noda Mamiko , Nagasawa Takashi , Turner Martin
Published: December 2009

T cell development requires phosphatidylinositol 3-kinase (PI3K) signaling with contributions from both the class IA, p110δ, and class IB, p110γ catalytic subunits. However, the receptors on immature T cells by which each of these PI3Ks are activated have not been identified, nor has the mechanism behind their functional redundancy in the thymus. Here, we show that PI3K signaling from the preTCR requires p110δ, but not p110γ. Mice deficient for the class IB regulatory subunit p101 demonstrated the requirement for p101 in T cell development, implicating G protein–coupled receptor signaling in β-selection. We found evidence of a role for CXCR4 using small molecule antagonists in an in vitro model of β-selection and demonstrated a requirement for CXCR4 during thymic development in CXCR4-deficient embryos. Finally, we demonstrate that CXCL12, the ligand for CXCR4, allows for Notch-dependent differentiation of DN3 thymocytes in the absence of supporting stromal cells. These findings establish a role for CXCR4-mediated PI3K signaling that, together with signals from Notch and the preTCR, contributes to continued T cell development beyond β-selection.
Generation of PLZF+ CD4+ T cells via MHC class II–dependent thymocyte–thymocyte interaction is a physiological process in humans
Authors: Lee You Jeong , Jeon Yoon Kyung , Kang Byung Hyun , Chung Doo Hyun , Park Chung-Gyu , Shin Hee Young , Jung Kyeong Cheon , Park Seong Hoe
Published: December 2009

Human thymocytes, unlike mouse thymocytes, express major histocompatibility complex (MHC) class II molecules on their surface, especially during the fetal and perinatal stages. Based on this observation, we previously identified a novel developmental pathway for the generation of CD4+ T cells via interactions between MHC class II–expressing thymocytes (thymocyte–thymocyte [T–T] interactions) with a transgenic mouse system. However, the developmental dissection of this T–T interaction in humans has not been possible because of the lack of known cellular molecules specific for T–T CD4+ T cells. We show that promyelocytic leukemia zinc finger protein (PLZF) is a useful marker for the identification of T–T CD4+ T cells. With this analysis, we determined that a substantial number of fetal thymocytes and splenocytes express PLZF and acquire innate characteristics during their development in humans. Although these characteristics are quite similar to invariant NKT (iNKT) cells, they clearly differ from iNKT cells in that they have a diverse T cell receptor repertoire and are restricted by MHC class II molecules. These findings define a novel human CD4+ T cell subset that develops via an MHC class II–dependent T–T interaction.
Decoration of T-independent antigen with ligands for CD22 and Siglec-G can suppress immunity and induce B cell tolerance in vivo
Authors: Duong Bao Hoa , Tian Hua , Ota Takayuki , Completo Gladys , Han Shoufa , Vela José Luis , Ota Miyo , Kubitz Michael , Bovin Nicolai , Paulson James C. , Nemazee David
Published: December 2009

Autoreactive B lymphocytes first encountering self-antigens in peripheral tissues are normally regulated by induction of anergy or apoptosis. According to the “two-signal” model, antigen recognition alone should render B cells tolerant unless T cell help or inflammatory signals such as lipopolysaccharide are provided. However, no such signals seem necessary for responses to T-independent type 2 (TI-2) antigens, which are multimeric antigens lacking T cell epitopes and Toll-like receptor ligands. How then do mature B cells avoid making a TI-2–like response to multimeric self-antigens? We present evidence that TI-2 antigens decorated with ligands of inhibitory sialic acid–binding Ig-like lectins (siglecs) are poorly immunogenic and can induce tolerance to subsequent challenge with immunogenic antigen. Two siglecs, CD22 and Siglec-G, contributed to tolerance induction, preventing plasma cell differentiation or survival. Although mutations in CD22 and its signaling machinery have been associated with dysregulated B cell development and autoantibody production, previous analyses failed to identify a tolerance defect in antigen-specific mutant B cells. Our results support a role for siglecs in B cell self-/nonself-discrimination, namely suppressing responses to self-associated antigens while permitting rapid “missing self”–responses to unsialylated multimeric antigens. The results suggest use of siglec ligand antigen constructs as an approach for inducing tolerance.
Calcium-activated chloride current amplifies the response to urine in mouse vomeronasal sensory neurons
Authors: Yang Chun , Delay Rona J.
Published: December 2009

The vomeronasal organ (VNO) is an odor detection system that mediates many pheromone-sensitive behaviors. Vomeronasal sensory neurons (VSNs), located in the VNO, are the initial site of interaction with odors/pheromones. However, how an individual VSN transduces chemical signals into electrical signals is still unresolved. Here, we show that a Ca2+-activated Cl− current contributes ∼80% of the response to urine in mouse VSNs. Using perforated patch clamp recordings with gramicidin, which leaves intracellular chloride undisrupted, we found that the urine-induced inward current (Vhold = −80 mV) was decreased in the presence of chloride channel blockers. This was confirmed using whole cell recordings and altering extracellular chloride to shift the reversal potential. Further, the urine-induced currents were eliminated when both extracellular Ca2+ and Na+ were removed. Using inside-out patches from dendritic tips, we recorded Ca2+-activated Cl− channel activity. Several candidates for this Ca2+-activated Cl− channel were detected in VNO by reverse transcription–polymerase chain reaction. In addition, a chloride cotransporter, Na+-K+-2Cl− isoform 1, was detected and found to mediate much of the chloride accumulation in VSNs. Collectively, our data demonstrate that chloride acts as a major amplifier for signal transduction in mouse VSNs. This amplification would increase the responsiveness to pheromones or odorants.
Inactivation of the KcsA potassium channel explored with heterotetramers
Authors: Rotem Dvir , Mason Amy , Bayley Hagan
Published: December 2009

The tetrameric prokaryotic potassium channel KcsA is activated by protons acting on the intracellular aspect of the protein and inactivated through conformational changes in the selectivity filter. Inactivation is modulated by a network of interactions within each protomer between the pore helix and residues at the external entrance of the channel. Inactivation is suppressed by the E71A mutation, which perturbs the stability of this network. Here, cell-free protein synthesis followed by protein purification by sodium dodecyl sulfate–polyacrylamide gel electrophoresis was used to produce heterotetramers of KcsA that contain different combinations of wild-type and E71A subunits. Single-channel recordings from these heterotetramers reveal how the network of interactions in individual protomers affects ionic conduction and channel inactivation, suggesting that the latter is a cooperative process.
The activation mechanism of α1β2γ2S and α3β3γ2S GABAA receptors
Authors: Keramidas Angelo , Harrison Neil L.
Published: December 2009

The α1β2γ2 and α3β3γ2 are two isoforms of γ-aminobutyric acid type A (GABAA) receptor that are widely distributed in the brain. Both are found at synapses, for example in the thalamus, where they mediate distinctly different inhibitory postsynaptic current profiles, particularly with respect to decay time. The two isoforms were expressed in HEK293 cells, and single-channel activity was recorded from outside-out patches. The kinetic characteristics of both isoforms were investigated by analyzing single-channel currents over a wide range of GABA concentrations. α1β2γ2 channels exhibited briefer active periods than α3β3γ2 channels over the entire range of agonist concentrations and had lower intraburst open probabilities at subsaturating concentrations. Activation mechanisms were constructed by fitting postulated schemes to data recorded at saturating and subsaturating GABA concentrations simultaneously. Reaction mechanisms were ranked according to log-likelihood values and how accurately they simulated ensemble currents. The highest ranked mechanism for both channels consisted of two sequential binding steps, followed by three conducting and three nonconducting configurations. The equilibrium dissociation constant for GABA at α3β3γ2 channels was ∼2.6 µM compared with ∼19 µM for α1β2γ2 channels, suggesting that GABA binds to the α3β3γ2 channels with higher affinity. A notable feature of the mechanism was that two consecutive doubly liganded shut states preceded all three open configurations. The lifetime of the third shut state was briefer for the α3β3γ2 channels. The longer active periods, higher affinity, and preference for conducting states are consistent with the slower decay of inhibitory currents at synapses that contain α3β3γ2 channels. The reaction mechanism we describe here may also be appropriate for the analysis of other types of GABAA receptors and provides a framework for rational investigation of the kinetic effects of a variety of therapeutic agents that activate or modulate GABAA receptors and hence influence synaptic and extrasynaptic inhibition in the central nervous system.
Epac1 mediates protein kinase A–independent mechanism of forskolin-activated intestinal chloride secretion
Authors: Hoque Kazi Mirajul , Woodward Owen M. , van Rossum Damian B. , Zachos Nicholas C. , Chen Linxi , Leung George P.H. , Guggino William B. , Guggino Sandra E. , Tse Chung-Ming
Published: December 2009

Intestinal Cl− secretion is stimulated by cyclic AMP (cAMP) and intracellular calcium ([Ca2+]i). Recent studies show that protein kinase A (PKA) and the exchange protein directly activated by cAMP (Epac) are downstream targets of cAMP. Therefore, we tested whether both PKA and Epac are involved in forskolin (FSK)/cAMP-stimulated Cl− secretion. Human intestinal T84 cells and mouse small intestine were used for short circuit current (Isc) measurement in response to agonist-stimulated Cl− secretion. FSK-stimulated Cl− secretion was completely inhibited by the additive effects of the PKA inhibitor, H89 (1 µM), and the [Ca2+]i chelator, 1,2-bis-(o-aminophenoxy)-ethane-N,N,N’,N’-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM; 25 µM). Both FSK and the Epac activator 8-pCPT-2’-O-Me-cAMP (50 µM) elevated [Ca2+]i, activated Ras-related protein 2, and induced Cl− secretion in intact or basolateral membrane–permeabilized T84 cells and mouse ileal sheets. The effects of 8-pCPT-2’-O-Me-cAMP were completely abolished by BAPTA-AM, but not by H89. In contrast, T84 cells with silenced Epac1 had a reduced Isc response to FSK, and this response was completely inhibited by H89, but not by the phospholipase C inhibitor U73122 or BAPTA-AM. The stimulatory effect of 8-pCPT-2’-O-Me-cAMP on Cl− secretion was not abolished by cystic fibrosis transmembrane conductance (CFTR) inhibitor 172 or glibenclamide, suggesting that CFTR channels are not involved. This was confirmed by lack of effect of 8-pCPT-2’-O-Me-cAMP on whole cell patch clamp recordings of CFTR currents in Chinese hamster ovary cells transiently expressing the human CFTR channel. Furthermore, biophysical characterization of the Epac1-dependent Cl− conductance of T84 cells mounted in Ussing chambers suggested that this conductance was hyperpolarization activated, inwardly rectifying, and displayed a Cl−>Br−>I− permeability sequence. These results led us to conclude that the Epac-Rap-PLC-[Ca2+]i signaling pathway is involved in cAMP-stimulated Cl− secretion, which is carried by a novel, previously undescribed Cl− channel.
Barriers to Hepatitis B Vaccine Coverage among Healthcare Workers in the Republic of Georgia: An International Perspective
Authors: Topuridze M. , Butsashvili M. , Kamkamidze G. , Kajaia M. , Morse D. , McNutt L. A.
Published: December 2009

Background.

While the Republic of Georgia has a high prevalence of hepatitis B virus (HBV) infection (3.4% of blood donors tested positive for HBV surface antigen [HBsAg]), relatively few healthcare workers (HCWs) are thought to be immunized.

Objective.

To measure rates of HBV vaccination coverage and identify predictors of vaccine acceptance among HCWs.

Design.

Cross-sectional survey.

Methods.

A study was conducted among full-time physicians and nurses at 2 large hospitals. Self-administered questionnaires included questions about demographic characteristics, HBV vaccine status, willingness to recommend vaccination to other HCWs, and barriers to vaccination. Laboratory tests were conducted for identification of HBsAg and antibody to hepatitis B core antigen.

Results.

A total of 297 (91%) of 325 randomly selected HCWs provided information for the study (124 physicians and 173 nurses). The rate of HBV vaccination coverage was 12%, and 54% of respondents indicated that they would recommend vaccination to other HCWs. Perception of vaccine safety was identified as the most important predictor for acceptance (prevalence ratio [PR] 3.3 [95% confidence ratio {CI}, 1.2-8.9]) and for willingness to recommend HBV vaccination to other HCWs (PR, 5.5 [95% CI, 3.1-9.4]). Vaccinated HCWs were more likely to recommend vaccination to other healthcare personnel (PR, 1.7 [95% CI, 1.5-2.1]), as were those younger than 40 years of age (PR, 6.0 [95% CI, 2.8-12.6]). Multivariate analyses identified 2 additional factors associated with vaccine acceptance and willingness to recommend vaccination: the hospital at which the HCW was employed and the perception of risk of infection for HCWs.

Conclusion.

Georgia plans a major HBV vaccination campaign for HCWs in 2009. The campaign's success will depend on addressing vaccine safety concerns identified in this study and educating HCWs about risk factors for infection and benefits of immunization.
Enhancing Patient Safety by Reducing Healthcare-Associated Infections: The Role of Discovery and Dissemination
Authors:   The Research Committee of the Society of Healthcare Epidemiology of America
Published: December 2009

Healthcare-associated infections (HAIs) take a major human toll on society and reduce public confidence in the healthcare system. The current convergence of scientific, public, and legislative interest in reducing rates of HAI can provide the necessary momentum to address and answer important questions in HAI research. This position paper outlines priorities for a national approach to HAIs: scrutinizing the science base, developing a prioritized research agenda, conducting studies that address the questions that have been identified, creating and deploying guidelines that are based on the outcomes of these studies, and then initiating new studies that assess the efficacy of the interventions.
Expanding Roles of Healthcare Epidemiology and Infection Control in Spite of Limited Resources and Compensation
Authors: Wright Sharon B. , Ostrowsky Belinda , Fishman Neil , Deloney Valerie M. , Mermel Leonard , Perl Trish M.
Published: December 2009

Objective.

Data on the resources and staff compensation of hospital epidemiology and infection control (HEIC) departments are limited and do not reflect current roles and responsibilities, including the public reporting of healthcare-associated infections. This study aimed to obtain information to assist HEIC professionals in negotiating resources.

Methods.

A 28-question electronic survey was sent via e-mail to all Society for Healthcare Epidemiology of America (SHEA) members in October 2006 with the use of enterprise feedback management solution software. The survey responses were analyzed using Microsoft Excel.

Results.

Responses were received from 526 (42%) of 1,255 SHEA members. Of the respondents, 84% were doctors of medicine (MDs) or doctors of osteopathy (DOs), 6% were registered nurses, and 21% had a master of public health or master of science degree. Sixty-two percent were male (median age range, 50-59 years). Their practice locations varied across the United States and internationally. Two-thirds of respondents practiced in a hospital setting, and 63% were the primary or associate hospital epidemiologist. Although 91% provided HEIC services, only 65% were specifically compensated. In cases of antimicrobial management, patient safety, employee health, and emergency preparedness, 75%-80% of respondents provided expertise but were compensated in less than 25% of cases. Of the US-based MD and DO respondents, the median range of earnings was $151,000-$200,000, regardless of their region (respondents selected salary ranges instead of specifying their exact salaries). Staffing levels varied: the median number of physician full-time equivalents (FTEs) was 1.0 (range, 1-5); only about 25% of respondents had 3 or more infection control practitioner FTEs.

Conclusions.

Most professionals working in HEIC have had additional training and provide a wide, growing range of services. In general, only traditional HEIC work is compensated and at levels much less than the time dedicated to those services. Most HEIC departments are understaffed. These data are essential to advocate for needed funding and resources as the roles of HEIC departments expand.
Antiproliferative Activity of Cinnamomum cassia Constituents and Effects of Pifithrin‐Alpha on Their Apoptotic Signaling Pathways in Hep G2 Cells
Authors: Ng Lean-Teik , Wu Shu-Jing
Published: December 2009

Cinnamaldehyde (Cin), cinnamic acid (Ca) and cinnamyl alcohol (Cal), major constituents of Cinnamomum cassia, have been shown to possess antioxidant, anti‐inflammatory, anticancer and other activities. In this study, our aim was to evaluate the antiproliferative activity of these compounds in human hepatoma Hep G2 cells and examine the effects of pifithrin‐alpha (PFTα; a specific p53 inhibitor) on their apoptotic signaling transduction mechanism. The antiproliferative activity was measured by XTT assay. Expression of apoptosis‐related proteins was detected by western blotting. Results showed that at a concentration of 30 μM, the order of antiproliferative activity in Hep G2 cells was Cin > Ca > Cal. Cin (IC50 9.76 ± 0.67 μM) demonstrated an antiproliferative potency as good as 5‐fluorouracil (an anti‐cancer drug; IC50 9.57 ± 0.61 μM). Further studies on apoptotic mechanisms of Cin showed that it downregulated the expression of Bcl‐XL, upregulated CD95 (APO‐1), p53 and Bax proteins, as well as cleaving the poly (ADP‐ribose) polymerase (PARP) in a time‐dependent pattern. PFTα pre‐incubation significantly diminished the effect of Cin‐induced apoptosis. It markedly upregulated the anti‐apoptotic (Bcl‐XL) expression and downregulated the pro‐apoptotic (Bax) expression, as well as effectively blocking the CD95 (APO‐1) and p53 expression, and PARP cleavage in Cin‐treated cells. This study indicates that Cin was the most potent antiproliferative constituent of C. cassia, and its apoptotic mechanism in Hep G2 cells could be mediated through the p53 induction and CD95 (APO‐1) signaling pathways.
Plasma lipoproteins are important components of the immune system
Authors: Han Runlin
Published: December 2009

Plasma lipoproteins (VLDL, LDL, Lp[a] and HDL) function primarily in lipid transport among tissues and organs. However, cumulative evidence suggests that lipoproteins may also prevent bacterial, viral and parasitic infections and are therefore a component of innate immunity. Lipoproteins can also detoxify lipopolysaccharide and lipoteichoic acid. Infections can induce oxidation of LDL, and oxLDL in turn plays important anti‐infective roles and protects against endotoxin‐induced tissue damage. There is also evidence that apo(a) is protective against pathogens. Taken together, the evidence suggests that it might be valuable to introduce the concept that plasma lipoproteins belong in the realm of host immune response.
Influence of the prodrugs 5‐fluorocytosine and CPT‐11 on ovarian cancer cells using genetically engineered stem cells: tumor‐tropic potential and inhibition of ovarian cancer cell growth
Authors: Kim Ki‐Yon , Kim Seung U. , Leung Peter C. K. , Jeung Eui‐Bae , Choi Kyung‐Chul
Published: December 2009

Recent studies have shown that genetically engineered stem cells (GESTECs) to produce suicide enzymes that convert non‐toxic prodrugs to toxic metabolites selectively migrate toward tumor sites and reduce tumor growth. In the present study, we evaluated whether these GESTECs were capable of migrating to human ovarian cancer cells and examined the potential therapeutic efficacy of the gene‐directed enzyme prodrug therapy against ovarian cancer cellsin vitro. The expression of cytosine deaminase (CD) or carboxyl esterase (CE) mRNA of GESTECs was confirmed by RT‐PCR. A modified transwell migration assay was performed to determine the migratory capacity of GESTECs to ovarian cancer cells. GESTECs (HB1.F3.CD or HB1.F3.CE cells) engineered to express a suicide gene (CD or CE) selectively migrated toward ovarian cancer cells. A [3H] thymidine incorporation assay was conducted to measure the proliferative index. Treatment of human epithelial ovarian cancer cell line (SKOV‐3, an ovarian adenocarcinoma derived from the ascites of an ovarian cancer patient) with the prodrugs 5‐fluorocytosine (5‐FC) or camptothecin‐11 (CPT‐11) in the presence of HB1.F3.CD or HB1.F3.CE cells resulted in the inhibition of ovarian cancer cell growth. Based on the data presented herein, we suggest that GESTECs expressing CD/CE may have a potent advantage to selectively treat ovarian cancers.

(Cancer Sci2010; 101: 955–962)
Frequent transcriptional inactivation of Kallikrein 10 gene by CpG island hypermethylation in non‐small cell lung cancer
Authors: Zhang Youwei , Song Haizhu , Miao Yufeng , Wang Rui , Chen Longbang
Published: December 2009

The role of Kallikrein 10 gene (KLK10) in non‐small cell lung cancer (NSCLC) remains largely unknown. We determined the frequency and functional significance of KLK10 hypermethylation in NSCLC. The mRNA expression and methylation status of KLK10 in 78 pairs NSCLC specimens was explored. The biological effects of KLK10 were analyzed by transfection. The results showed that, KLK10 was significantly downregulated in NSCLC (57.7%, 45/78) as compared to non‐cancer samples (P = 0.010). CpG island hypermethylation of KLK10 was detected in 46.2% (36/78) NSCLC tissues and was closely correlated with loss of transcript (P < 0.001). KLK10 methylation was associated with advanced stage (P = 0.013) and lymph metastasis (P = 0.015). Furthermore, demethylation treatment restored the expression of KLK10 in two lung adencarcinoma cell lines (A549, SPC‐A1). Forced expression of KLK10 in A549 and SPC‐A1 remarkably suppressed cells proliferation, migration in vitro and oncogenicity in vivo. Additionally, methylated KLK10 was detected in 38.7% (30/78) of plasma samples from cancer patients but rare in cancer‐free controls (P < 0.001). In conclusion, KLK10 acts as a functional tumor suppressor gene in NSCLC, epigenetic inactivation of KLK10 is a common event contributing to NSCLC pathogenesis and may be used as a potential biomarker.

(Cancer Sci 2010; 101: 934–940)
Girding for migratory cues: roles of the Akt substrate Girdin in cancer progression and angiogenesis
Authors: Weng Liang , Enomoto Atsushi , Ishida‐Takagishi Maki , Asai Naoya , Takahashi Masahide
Published: December 2009

Cell migration is a fundamental aspect of a multitude of physiological and pathological processes, including embryonic development, inflammation, angiogenesis, and cancer progression. A variety of proteins are essential for cell migration, but context‐specific signaling pathways and promigratory proteins must now be identified for our understanding of cancer biology to continue to advance. In this review, we focus on the emerging roles of Girdin (also designated KIAA1212, APE, GIV, and HkRP1), a novel component of the phosphatidylinositol 3‐kinase (PI3‐K)/Akt signaling pathway that is a core‐signaling transduction pathway in cancer progression. Girdin is expressed in some types of cancer cells and immature endothelial cells, and is therefore at the crossroads of multiple intracellular processes, including reorganization of the actin cytoskeleton, endocytosis, and modulation of Akt activity, which ultimately lead to cancer invasion and angiogenesis. It also acts as a nonreceptor guanine nucleotide exchange factor (GEF) for Gαi proteins. A significant observation is that Girdin, although vital for cancer progression and postnatal vascular remodelling, is dispensable for cell migratory events during embryonic development. These findings suggest that Girdin and its interacting proteins are potential pharmaceutical targets for cancer therapies and pathological anigiogenesis, including tumor angiogenesis.

(Cancer Sci 2010; 101: 836–842)
The cell death machinery governed by the p53 tumor suppressor in response to DNA damage
Authors: Yoshida Kiyotsugu , Miki Yoshio
Published: December 2009

The cellular response to genotoxic stress that damages DNA includes cell cycle arrest, activation of DNA repair, and in the event of irreparable damage, induction of apoptosis. However, the signals that determine cell fate, that is, survival or apoptosis, are largely unclear. The tumor suppressor p53 has been implicated in many important cellular processes, including regulation of apoptotic cell death. When cells encounter genotoxic stress, certain sensors for DNA lesions eventually stabilize and activate p53. Subsequently, p53 exerts its tumor suppressor function by transactivating numerous target genes. Active p53 is subjected to a complex and diverse array of covalent post‐translational modifications, which selectively influence the expression of p53 target genes. In this regard, the molecular basis for how p53 induces apoptosis has been extensively studied; however, the relative contribution of each downstream effecter is still to be explored. Moreover, little is known about precise mechanisms by which modified p53 is capable of apoptosis induction. A thorough understanding for the whole picture of p53 modification in apoptosis will be extremely valuable in the development of highly effective and specific therapies for caner patients. This review is focused on the current views regarding the regulation of cell fate by p53 in the apoptotic response to DNA damage.

(Cancer Sci 2010; 101: 831–835)
Clinical and economic outcomes in a population‐based European cohort of 948 ulcerative colitis and Crohn’s disease patients by Markov analysis
Authors: ODES S. , VARDI H. , FRIGER M. , ESSER D. , WOLTERS F. , MOUM B. , WATERS H. , ELKJAER M. , BERNKLEV T. , TSIANOS E. , O’MORAIN C. , STOCKBRÜGGER R. , MUNKHOLM P. , LANGHOLZ E.
Published: December 2009

Aliment Pharmacol Ther 31, 735–744

Background Forecasting clinical and economic outcomes in ulcerative colitis (UC) and Crohn’s disease (CD) patients is complex, but necessary.

Aims To determine: the frequency of treatment‐classified clinical states; the probability of transition between states; and the economic outcomes.

Methods Newly diagnosed UC and CD patients, allocated into seven clinical states by medical and surgical treatments recorded in serial 3‐month cycles, underwent Markov analysis.

Results Over 10 years, 630 UC and 318 CD patients had 22,823 and 11 871 cycles. The most frequent clinical outcomes were medical/surgical remission (medication‐free) and mild disease (on 5‐aminosalicylates, antibiotics, topical corticosteroids), comprising 28% and 62% of UC cycles and 24% and 51% of CD cycles respectively. The probability of drug‐response in patients receiving systemic corticosteroids/immunomodulators was 0.74 in UC, 0.66 in CD. Both diseases had similar likelihood of persistent drug‐dependency or drug‐refractoriness. Surgery was more probable in CD, 0.20, than UC, 0.08. In terms of economic outcomes, surgery was costlier in UC per cycle, but the outlay over 10 years was greater in CD. Drug‐refractory UC and CD cases engendered high costs in the cohort.

Conclusions Most patients on 5‐aminosalicylates, corticosteroids and immunomodulators had favourable clinical and economic outcomes over 10 years. Drug‐refractory and surgical patients exhibited greater long‐term expenses.
Blunting effect of hypoxia on the proliferation and differentiation of human primary and rat L6 myoblasts is not counteracted by Epo
Authors: Launay T. , Hagström L. , Lottin‐Divoux S. , Marchant D. , Quidu P. , Favret F. , Duvallet A. , Darribère T. , Richalet J. P. , Beaudry M.
Published: December 2009

Objectives:  The aim of this study was to evaluate whether hypoxia and/or erythropoietin would be able to modulate proliferation/differentiation processes of rat and human myoblasts.

Materials and methods:  Rat L6 and primary human myoblasts were grown in 21% or 1% O2 in the presence or absence of recombinant human erythropoietin (RhEpo). Presence of erythropoietin receptors (EpoR) was assayed using RT‐PCR and Western blotting techniques. Cell proliferation was evaluated by determining the doubling time and kinetics of cultures by counting cells. Cell differentiation was analysed by determining myogenic fusion index using antibodies against the myosin heavy chain. Expression of myogenin and myosin heavy chain (MHC) proteins were evaluated using the Western blotting technique.

Results:  After 96 h culture in growth medium for 2.5 and 9 h, doubling time of L6 and human primary myoblasts respectively, had increased in 1% O2 conditions (P < 0.01). Kinetics of culture showed alteration in proliferation at 72 h in L6 myoblast cultures and at 4 days in human primary myoblasts. The myogenic fusion index had reduced by 30% in L6 myoblasts and by 20% in human myoblasts (P < 0.01). Expression of myogenin and MHC had reduced by around 50%. Despite presence of EpoR mRNA and protein, RhEpo did not counteract the effects of hypoxia either in L6 cells or in human myoblasts.

Conclusions:  The data show that exposure to hypoxic conditions (1% O2) of rat and human myoblasts altered their proliferation and differentiation processes. They also show that Epo is not an efficient growth factor to counteract this deleterious effect.