bluuebunny/crossref_metadata_2025 huggingface dataset:
Directed evolution of a monomeric, bright and photostable version of Clavularia cyan fluorescent protein: structural characterization and applications in fluorescence imaging
Authors: Ai Hui-wang , Henderson J. Nathan , Remington S. James , Campbell Robert E.
Published: November 2006

The arsenal of engineered variants of the GFP [green FP (fluorescent protein)] from Aequorea jellyfish provides researchers with a powerful set of tools for use in biochemical and cell biology research. The recent discovery of diverse FPs in Anthozoa coral species has provided protein engineers with an abundance of alternative progenitor FPs from which improved variants that complement or supersede existing Aequorea GFP variants could be derived. Here, we report the engineering of the first monomeric version of the tetrameric CFP (cyan FP) cFP484 from Clavularia coral. Starting from a designed synthetic gene library with mammalian codon preferences, we identified dimeric cFP484 variants with fluorescent brightness significantly greater than the wild-type protein. Following incorporation of dimer-breaking mutations and extensive directed evolution with selection for blue-shifted emission, high fluorescent brightness and photostability, we arrived at an optimized variant that we have named mTFP1 [monomeric TFP1 (teal FP 1)]. The new mTFP1 is one of the brightest and most photostable FPs reported to date. In addition, the fluorescence is insensitive to physiologically relevant pH changes and the fluorescence lifetime decay is best fitted as a single exponential. The 1.19 Å crystal structure (1 Å=0.1 nm) of mTFP1 confirms the monomeric structure and reveals an unusually distorted chromophore conformation. As we experimentally demonstrate, the high quantum yield of mTFP1 (0.85) makes it particularly suitable as a replacement for ECFP (enhanced CFP) or Cerulean as a FRET (fluorescence resonance energy transfer) donor to either a yellow or orange FP acceptor.
Late 20th century landscape-wide expansion of Allosyncarpia ternata (Myrtaceae) forests in Kakadu National Park, northern Australia
Authors: Bowman David M. J. S. , Dingle Joanne K.
Published: November 2006

Allosyncarpia ternata S.T.Blake is a large tree endemic to the rugged western edge of the Arnhem Land Plateau, northern Australia, with most of the species conserved in Kakadu National Park (KNP). A. ternata stems suffer substantial mortality following wildfire but the species resprouts prolifically from root stocks. Nonetheless, there is concern about the persistence of A. ternata rainforest patches following breakdown of traditional Aboriginal landscape burning that generated a mosaic of burnt and unburnt areas. Generalised linear modelling was used to identify the landscape features associated with the fragmentary distribution of A. ternata rainforest. We randomly sampled 12 areas that together made up 12.6% of the total coverage of A. ternata in KNP (12 191 ha) that spanned the geographic range of this vegetation type within the Park. The modelling of these data showed that A. ternata forests were most likely to occur at sites with fire protection, as inferred from the small number of fire scars apparent on sequences of satellite imagery, steep slope angles and proximity to drainage lines. Analysis of historical aerial photography revealed that, despite considerable negative and positive variation, there has been a 21% expansion of A. ternata forests over the last 50 years. Expansion occurred by incremental growth from existing forest boundaries and not by nucleation, reflecting the poor seed dispersal of the tree. The forest expansion was negatively correlated with fire activity. A regionally wetter climate since the mid-20th century may be an important cause of the expansion despite currently unfavourable fire regimes.
Prediction of spatial ET-fluxes using remote sensing and field data of selected areas in the Eastern Part of Abu Dhabi, United Arab Emirates
Authors: Howari Fares M. , Murad Ahmed , Garamoon Hassan
Published: November 2006

Evapotranspiration (ET) is a major source of water depletion in arid and semi-arid environments; and it is a poorly quantified variable in the hydrological cycle. Remote sensing has the potential application to quantify this variable especially at large scale. The present study reports methodology useful to determine whether derived variables from remotely sensed data, such as vegetation and soil brightness indices, could be used to predict ET. To achieve this goal, various regression analyses were conducted between data derived from satellites, field meteorological stations, and ET values. Selected sub-scenes of Landsat Enhanced Thematic Mapper images free of cloud were used to derive Normalized Difference Vegetation Index (NDVI) and Soil Brightness Index using ER-Mapper and JMP software packages. From the obtained relationship between NDVI and ET, it was observed that ET increases sharply with increase in NDVI. The predicted ET results obtained from the multiple regression functions of field ET, NDVI, solar radiation, wind velocity, and/or temperature are comparable with the ET values obtained by Penman-Monteith method. The results showed that a remotely sensed vegetation index could be used, indirectly, to determine ET values. However, there is still considerable work to be done before simple and full automated extraction of ET from the reported methods can be achieved for large-scale applications.
Empirical Bayes hierarchical models for regularizing maximum likelihood estimation in the matrix Gaussian Procrustes problem
Authors: Theobald Douglas L. , Wuttke Deborah S.
Published: November 2006

Procrustes analysis involves finding the optimal superposition of two or more “forms” via rotations, translations, and scalings. Procrustes problems arise in a wide range of scientific disciplines, especially when the geometrical shapes of objects are compared, contrasted, and analyzed. Classically, the optimal transformations are found by minimizing the sum of the squared distances between corresponding points in the forms. Despite its widespread use, the ordinary unweighted least-squares (LS) criterion can give erroneous solutions when the errors have heterogeneous variances (heteroscedasticity) or the errors are correlated, both common occurrences with real data. In contrast, maximum likelihood (ML) estimation can provide accurate and consistent statistical estimates in the presence of both heteroscedasticity and correlation. Here we provide a complete solution to the nonisotropic ML Procrustes problem assuming a matrix Gaussian distribution with factored covariances. Our analysis generalizes, simplifies, and extends results from previous discussions of the ML Procrustes problem. An iterative algorithm is presented for the simultaneous, numerical determination of the ML solutions.
Evidence for structural constraint on ovulin, a rapidly evolving
Drosophila melanogaster
seminal protein
Authors: Wong Alex , Albright Shannon N. , Wolfner Mariana F.
Published: November 2006

The egg-laying hormone ovulin (Acp26Aa) is among the most rapidly evolving proteins in the
Drosophila
genome. Against the background of ovulin's high sequence variability within and between species, we have identified highly conserved motifs that may play an important structural role. Using yeast two-hybrid and GST-pull-down assays, we show that ovulin interacts with itself. The C terminus of ovulin is necessary and sufficient for self-interaction, with its C-terminal 45 aa playing a major role. Under nonreducing conditions, ovulin participates in a high-molecular-mass complex, suggesting that it occurs in an oligomeric form. One or more of three predicted coiled-coil domains in the C terminus of ovulin may be involved in its self-interaction. These structural elements are conserved between species despite an overall rapid pace of evolution in ovulin's primary sequence. We therefore suggest that domains involved in ovulin's self-interaction form a conserved structural backbone for the protein, resulting in greater evolutionary flexibility at other sites.
Klf4 and corticosteroids activate an overlapping set of transcriptional targets to acceleratein uteroepidermal barrier acquisition
Authors: Patel Satyakam , Xi Zong Fang , Seo Eun Young , McGaughey David , Segre Julia A.
Published: November 2006

Premature infants are at an increased risk for infections and dehydration because of incomplete development of the epidermis, which attains its essential function as a barrier only during the last stages ofin uterodevelopment. When a premature birth is anticipated, antenatal corticosteroids are administered to accelerate lung epithelium differentiation. One pleiotropic, but beneficial, effect of antenatal corticosteroids is acceleration of skin barrier establishment by an unknown mechanism. In mice, the transcription factor Klf4 is both necessary and sufficient, within a developmental field of competence, to establish this skin barrier, as demonstrated by targeted ablation and transgenic expression of Klf4, respectively. Here, we report that Klf4 and corticosteroid treatment coordinately accelerate barrier acquisitionin vivo. Transcriptional profiling reveals that the genes regulated by corticosteroids and Klf4 during the critical window of epidermal development significantly overlap. KLF4 activates the proximal promoters of a significant subset of these genes. Dissecting the intersection of the genetic and pharmacological pathways, regulated by KLF4 and corticosteroids, respectively, leads to a mechanistic understanding of the normal process of epidermal developmentin utero.
Sharing of T cell receptors in antigen-specific responses is driven by convergent recombination
Authors: Venturi Vanessa , Kedzierska Katherine , Price David A. , Doherty Peter C. , Douek Daniel C. , Turner Stephen J. , Davenport Miles P.
Published: November 2006

Public responses where identical T cell receptors (TCRs) are clonally dominant and shared between different individuals are a common characteristic of CD8+T cell-mediated immunity. Focusing on TCR sharing, we analyzed ≈3,400 TCR β chains (TCRβs) from mouse CD8+T cells responding to the influenza A virus DbNP366and DbPA224epitopes. Both the “public” DbNP366-specific and “private” DbPA224-specific TCR repertoires contain a high proportion (≈36%) of shared TCRβs, although the numbers of mice sharing TCRβs in each repertoire varies greatly. Sharing of both the TCRβ amino acid and TCRβ nucleotide sequence was negatively correlated with the prevalence of random nucleotide additions in the sequence. However, the extent of TCRβ amino acid sequence sharing among mice was strongly correlated with the level of diversity in the encoding nucleotide sequences, suggesting that a key feature of public TCRs is that they can be made in a variety of ways. Using a computer simulation of random V(D)J recombination, we estimated the relative production frequencies and variety of production mechanisms for TCRβ sequences and found strong correlations with the sharing of both TCRβ amino acid sequences and TCRβ nucleotide sequences. The overall conclusion is that “convergent recombination,” rather than a bias in recombination or subsequent selection, provides the mechanistic basis for TCR sharing between individuals responding to identical peptide plus MHC class I glycoprotein complexes.
A driving and coupling “Pac-Man” mechanism for chromosome poleward translocation in anaphase A
Authors: Liu Jian , Onuchic José N.
Published: November 2006

During mitosis, chromatid harnesses its kinetochore translocation at the depolymerizing microtubule ends for its poleward movement in anaphase A. The force generation mechanism for such movement remains unknown. Analysis of the current experimental results shows that the bending energy release from the bound tubulin subunits alone cannot provide sufficient driving force. Additional contribution from effective electrostatic attractions between the kinetochore and the microtubule is needed for kinetochore translocation. Interestingly, as the kinetochore moves to inside the microtubule, the microtubule tip is free to bend outward so that the instantaneous distance between the kinetochore and the microtubule tip is much closer than the rest of the microtubule. This close contact yields much larger electrostatic attraction than that from the rest of the microtubule under physiological ionic conditions. As a result, the effective electrostatic interaction hinders the further kinetochore poleward translocation until the microtubule tip dissociates. Thus, the kinetochore translocation is strongly coupled at the depolymerizing microtubule end. This driving-coupling mechanism indicates that the kinetochore velocity is largely controlled by the microtubule dissociation rate, which explains the insensitivity of kinetochore velocity to its viscous drag and the large redundancy in its stalling force.
Unliganded and hormone-bound glucocorticoid receptors interact with distinct hydrophobic sites in the Hsp90 C-terminal domain
Authors: Fang L. , Ricketson D. , Getubig L. , Darimont B.
Published: November 2006

Unlike most chaperones, heat-shock protein 90 (Hsp90) interacts with a select group of “client proteins” that regulate essential biological processes. Little is known about how Hsp90 recognizes and binds these proteins. The glucocorticoid receptor (GR) is a well characterized Hsp90 client protein, whose hormone binding, nuclear-cytoplasmic trafficking, and transcriptional activity are regulated by Hsp90. Here, we provide evidence that unliganded and hormone-bound GR interact with two distinct, solvent-exposed hydrophobic sites in the Hsp90 C-terminal domain that contain the sequences “MxxIM” (HM10) and “L/MxxIL” (HM9). Our results indicate that binding of Hsp90 HM10 to unliganded GR stabilizes the unliganded ligand-binding pocket of GR indirectly by promoting an intramolecular interaction between the C-terminal α-helix (H12) and a solvent-exposed hydrophobic groove in the GR ligand binding domain. In the presence of hormone, Hsp90 appears to bind the hydrophobic groove of GR directly by mimicking the interactions of GR with transcriptional coactivators. The identified interactions provide insights into the mechanisms that enable Hsp90 to regulate the activity of both unliganded and hormone-bound GR and to sharpen the cellular response to hormone.
β2 Adrenoceptor (ADRB2) Pharmacogenetics and Cardiovascular Phenotypes during Laryngoscopy and Tracheal Intubation
Authors: Kaymak Çetin , Kocabaş Neslihan Aygün , Durmaz Emre , Öztuna Derya
Published: November 2006

Pharmacogenetics is the study of genetic variations that cause a variable drug response characterized by alteration in drug metabolism or in pharmacodynamics. The polymorphisms in genes encoding receptors relevant to treatment cause variation in sensitivity to many drugs. β2 Adrenoceptor genetic variation contributes to regulation of blood pressure and hemodynamic changes by mediating peripheral vasodilatation. Laryngoscopy and tracheal intubation associated with hemodynamic changes. Although there are four nonsynomic single-nucleotide polymorphisms (SNPs) of β2 adrenoceptor gene, codon 16 (Arg16Gly) and codon 27 (Gln27Glu) SNPs are both common and functionally important. In this paper, the authors investigated the β2 adrenoceptor Gly16 and Glu27 SNPs in response to drugs relevant to anesthesia and how these SNPs impacted upon the cardiovascular phenotypes. The authors measured arterial systolic and diastolic blood pressure, heart rate, and rate-pressure product before induction of anesthesia and 1 min following laryngoscopy and tracheal intubation. Genomic DNA was amplified and genotyped using allele-specific polymerase chain reaction (ASPCR) and restriction fragment length polymorphism (RFLP) assays, respectively. When the authors compared hemodynamic results according to genotypes, the patients with Gln homozygote allele at codon 27 exhibited significant increase of heart rate than patients with Glu allele after laryngoscope and tracheal intubation.
Acute and Subchronic Toxicity Studies on Sel-Plex®, a Standardized, Registered High-selenium Yeast
Authors: Griffiths James C. , Matulka Ray A. , Power Ronan
Published: November 2006

Selenium has been recognized as an essential nutrient for human health; however, its bioavailability is primarily dependent upon the type of selenium, elemental versus organic. In geographic areas low in selenium, there is the potential for animals (including humans) to become selenium deficient and this potential deficiency can be remedied by consumption of exogenous selenium, including selenium-enriched yeast ( Saccharomyces cerevisiae) that contains high levels of organic selenium (e.g., selenized yeast). The present studies were conducted to investigate potential oral toxicity of a unique selenized yeast preparation (Sel-Plex®) when administered to (1) adult female CHS Swiss mice ICo:OFI (IOPS Caw); (2) adult female CHS Sprague-Dawley rats; and (3) adult male and female Sprague-Dawley CD rats. For the 28- and 90-day toxicity studies, (1) adult male and female Sprague-Dawley CRL:CD® (SD) IGS BR strain rats and (2) adult male and female 6- to 7-month-old Beagle dogs were used. The LD50 for mice was ® ≥2000 mg Sel-Plex® /kg (≥4.06 mg Se/kg) and for rats, was greater ® than ≥2000 mg Sel-Plex® /kg (≥4.06 mg Se/kg). In the two 28-day studies, for rats, the no observed adverse effects level (NOAEL) was 50 mg Sel-Plex®/kg/day (0.1 mg Se/kg/day), and for the dogs, the NOAEL was 22.5 mg Sel-Plex®/kg/day (0.045 mg Se/kg/day). For the two 90-day studies, for rats the NOAEL for Sel-Plex® was 114 mg/kg/day (0.23 mg Se/kg/day), and for dogs, the NOAEL was 30 mg Sel-Plex®/kg/day (0.06 mg Se/kg/day): the latter being the NOAEL in the most sensitive species.
Genotoxicity Studies on Sel-Plex®, a Standardized, Registered High-Selenium Yeast
Authors: Griffiths James C. , Matulka Ray A. , Power Ronan
Published: November 2006

Selenium, recognized as an essential nutrient for human health, is a component of proteins and enzymes required for various biological functions and is currently being used as a feed supplement for livestock in geographical areas that are naturally low in selenium. Selenium is structurally similar to sulfur, replacing the sulfur atom in stoichiometric amounts and thus functions through an association with proteins, termed selenoproteins. In geographic areas low in selenium, there is the potential for animals (including humans) to become selenium deficient and this potential deficiency can be remedied by consumption of exogenous selenium, including selenium-enriched yeast ( Saccharomyces cerevisiae) that contains high levels of organic selenium (e.g., selenized yeast). A unique, standardized, registered high selenium food-grade baker’s yeast ( S. cerevisiae; Sel-Plex®), was tested in the following battery of Genotoxicity assays; (1) a bacterial reverse mutation test (Ames test); (2) an in vitro mammalian chromosome aberration test; and (3) a mouse micronucleus test. Under the conditions of this assay, Sel-Plex® showed no evidence of mutagenic activity in Salmonella typhimurium, in the bacterial reverse mutation test. Sel-Plex® did not induce significant chromosomal aberrations in cultured human lymphocytes in the in vitro mammalian chromosome aberration test. Sel-Plex® did not statistically increase the frequency or proportion of micronucleated immature erythrocytes in the mouse micronucleus test. Thus, from the studies presented here, the authors conclude that Sel-Plex® is nongenotoxic.
Comparative Immunotoxicity Evaluation of Amphotericin B and ABELCET®, an Amphotericin B Lipid Complex (ABLC)
Authors: Zhang Zhihua , Lipman Jack M. , Diener Robert M. , Thomas Peter
Published: November 2006

ABELCET®, an amphotericin B lipid complex formulation (ABLC) and an aqueous, non–lipid-containing formulation with sodium deoxycholate (AmBd), were evaluated for their potential to induce immunotoxicity in B6C3F1 female mice. ABLC was administered intravenously at doses of 1, 3, and 10 mg/kg daily for 28 days, while AmBd at 1 mg/kg was administered by the same route and duration. The effect of ABLC and AmBd on clinical signs, body weight, and spleen weight was determined. Peritoneal macrophage function was measured by phagocytosis of 51Cr-labeled chicken red blood cells and generation of hydrogen peroxide during respiratory burst. The ability of natural killer cells to lyse radiolabeled tumor target cells was evaluated in a short-term chromium-release assay. The ability of splenic T and B cells to undergo blastogenesis and of splenic T cells to recognize alloantigens present on foreign cells was assessed in a splenic lymphocyte assay and the ability of mice to generate antibody-forming cells following immunization with sheep red blood cells was measured. Neither ABLC nor AmBd affected the metabolic or functional activity of murine phagocytic cells. These agents also did not cause any biologically significant or dose-related changes in B- or T-cell responses to mitogens, T-cell responses to allogeneic cells in the mixed lymphocyte culture assay, or natural killer cell function. The ability to generate a primary antibody response to a T cell–dependent antigen was also unimpaired. Based on the results of this study, it was concluded that neither ABLC at dose up to 10 mg/kg nor AmBd at dose up to 1.0 mg/kg produce biologically significant immunologic changes in B6C3F1 mice.
Chronic Contamination with 137Cesium in Rat: Effect on Liver Cholesterol Metabolism
Authors: Souidi M. , Tissandie E. , Grandcolas L. , Grison S. , Paquet F. , Voisin P. , Aigueperse J. , Gourmelon P. , Guéguen Y.
Published: November 2006

After the Chernobyl nuclear accident, epidemiological studies on human populations living in 137Cs-contaminated areas revealed the increase frequencies of thyroid cancer and evoked the apparition of cardiovascular diseases, hormonal effect, liver alteration, and lipid disorder. Actually, it raises a problem of public safety for the populations living on these territories that are exposed to low levels of 137Cs during a long period through food. Then it is necessary to study potential effect of this chronic contamination. To mimic this situation, the authors investigate the potential biological effects of chronic exposure to 137Cs at a postaccidental dose (150 Bq/rat/day) on hepatic metabolism of cholesterol in rat. Plasma lipid level, gene expression and activity were analyzed. It was observed that in 137Cs-exposed rats, gene expression of low-density lipoprotein receptor (LDLr), apolipoprotein B (apoB), and liver X receptor α (LXR α) are increased (95%, p < .05; 34%, p < .05; 20%, p < 0.05, respectively), whereas transporter adenosine triphosphate–binding cassette transporter G5 (ABCG5) is decreased (42%, p < .05). In addition, cytochrome P450 27A1 (CYP27A1) activity is increased (34%, p < .05) in contaminated rat liver. In conclusion, the results suggest that 137Cs contamination at low-level induces molecular modifications of the liver cholesterol metabolism without leading to a dysregulation of its homeostasis. These results suggest that chronic long term exposure at low-level of 137Cs may evolve to lipid disorder.
Nonclinical Vehicle Use in Studies by Multiple Routes in Multiple Species
Authors: Gad Shayne C. , Cassidy Crystal D. , Aubert Nicolas , Spainhour Bart , Robbe Heide
Published: November 2006

The laboratory toxicologist is frequently faced with the challenge of selecting appropriate vehicles or developing utilitarian formulations for use in in vivo nonclinical safety assessment studies. Although there are many vehicles available that may meet physical and chemical requirements for chemical or pharmaceutical formulation, there are wide differences in species and route of administration specific to tolerances to these vehicles. In current practice, these differences are largely approached on a basis of individual experience as there is only scattered literature on individual vehicles and no comprehensive treatment or information source. This approach leads to excessive animal use and unplanned delays in testing and development. To address this need, a consulting firm and three contract research organizations conducted a rigorous data mining operation of control (vehicle) data from studies dating from 1991 to present. The results identified 65 single component vehicles used in 368 studies across multiple species (dog, primate, rat, mouse, rabbit, guinea pig, minipig, chick embryo, and cat) by multiple routes. Reported here are the results of this effort, including maximum tolerated use levels by species, route, and duration of study, with accompanying dose limiting toxicity. Also included are basic chemical information and a review of available literature on each vehicle, as well as guidance on volume limits and pH by route and some basic guidance on nonclinical formulation development.
Lack of Trigeminal Nerve Toxicity in Rats Exposed to Trichloroethylene Vapor for 13 Weeks
Authors: Albee Ralph R. , Spencer Pamela J. , Johnson Keith A. , Bradley Greg J. , Marable Brian R. , Wilmer Jan W. , Mattsson Joel L.
Published: November 2006

Male and female Fischer-344 rats were exposed to 1,1,2-trichloroethylene (TCE) at 250, 800, or 2500 ppm for 6 h/day, 5 days/week, for 13 weeks. Weekly body weights and daily clinical observations were recorded and a functional observational battery (FOB) was performed monthly. Postexposure neurotoxicological evaluations included an electrodiagnostic evaluation of auditory function, the trigeminal nerve, and a comprehensive neuropathological examination. After 8 weeks of exposure, female, but not male, rats exposed to 2500 ppm were slightly more reactive to handling than the controls but not after 13 weeks of exposure. After 13 weeks, female rats exposed to 2500 ppm TCE were slightly more active during the 1-min observation period than the controls. There were no treatment-related differences in grip performance, landing foot splay, or on the trigeminal nerve–evoked potential at any dose. At 2500 ppm TCE, mild frequency-specific hearing deficits were observed, including elevated tone-pip auditory brainstem response thresholds. Focal loss of hair cells in the upper basal turn of the cochlea was observed in 2500 ppm–exposed rats. Except for the cochleas of 2500 ppm–exposed rats, no treatment-related lesions were noted during the neuro-histopathologic examination. The no-observable-adverse-effect level for this study was 800 ppm based on ototoxicity at 2500 ppm.
Human SHPRH suppresses genomic instability through proliferating cell nuclear antigen polyubiquitination
Authors: Motegi Akira , Sood Raman , Moinova Helen , Markowitz Sanford D. , Liu Pu Paul , Myung Kyungjae
Published: November 2006

Differential modifications of proliferating cell nuclear antigen (PCNA) determine DNA repair pathways at stalled replication forks. In yeast, PCNA monoubiquitination by the ubiquitin ligase (E3) yRad18 promotes translesion synthesis (TLS), whereas the lysine-63–linked polyubiquitination of PCNA by yRad5 (E3) promotes the error-free mode of bypass. The yRad5-dependent pathway is important to prevent genomic instability during replication, although its exact molecular mechanism is poorly understood. This mechanism has remained totally elusive in mammals because of the lack of apparent RAD5 homologues. We report that a putative tumor suppressor gene, SHPRH, is a human orthologue of yeast RAD5. SHPRH associates with PCNA, RAD18, and the ubiquitin-conjugating enzyme UBC13 (E2) and promotes methyl methanesulfonate (MMS)–induced PCNA polyubiquitination. The reduction of SHPRH by stable short hairpin RNA increases sensitivity to MMS and enhances genomic instability. Therefore, the yRad5/SHPRH-dependent pathway is a conserved and fundamental DNA repair mechanism that protects the genome from genotoxic stress.
Requirement of biphasic calcium release from the endoplasmic reticulum for Fas-mediated apoptosis
Authors: Wozniak Ann L. , Wang Xinmin , Stieren Emily S. , Scarbrough Shelby G. , Elferink Cornelis J. , Boehning Darren
Published: November 2006

Fas receptor is a member of the tumor necrosis factor-α family of death receptors that mediate physiologic apoptotic signaling. To investigate the molecular mechanisms regulating calcium mobilization during Fas-mediated apoptosis, we have analyzed the sequential steps leading to altered calcium homeostasis and cell death in response to activation of the Fas receptor. We show that Fas-mediated apoptosis requires endoplasmic reticulum–mediated calcium release in a mechanism dependent on phospholipase C-γ1 (PLC-γ1) activation and Ca2+ release from inositol 1,4,5-trisphosphate receptor (IP3R) channels. The kinetics of Ca2+ release were biphasic, demonstrating a rapid elevation caused by PLC-γ1 activation and a delayed and sustained increase caused by cytochrome c binding to IP3R. Blocking either phase of Ca2+ mobilization was cytoprotective, highlighting PLC-γ1 and IP3R as possible therapeutic targets for disorders associated with Fas signaling.
Deregulated Syk inhibits differentiation and induces growth factor–independent proliferation of pre–B cells
Authors: Wossning Thomas , Herzog Sebastian , Köhler Fabian , Meixlsperger Sonja , Kulathu Yogesh , Mittler Gerhard , Abe Akihiro , Fuchs Uta , Borkhardt Arndt , Jumaa Hassan
Published: November 2006

The nonreceptor protein spleen tyrosine kinase (Syk) is a key mediator of signal transduction in a variety of cell types, including B lymphocytes. We show that deregulated Syk activity allows growth factor–independent proliferation and transforms bone marrow–derived pre–B cells that are then able to induce leukemia in mice. Syk-transformed pre–B cells show a characteristic pattern of tyrosine phosphorylation, increased c-Myc expression, and defective differentiation. Treatment of Syk-transformed pre–B cells with a novel Syk-specific inhibitor (R406) reduces tyrosine phosphorylation and c-Myc expression. In addition, R406 treatment removes the developmental block and allows the differentiation of the Syk-transformed pre–B cells into immature B cells. Because R406 treatment also prevents the proliferation of c-Myc–transformed pre–B cells, our data indicate that endogenous Syk kinase activity may be required for the survival of pre–B cells transformed by other oncogenes. Collectively, our data suggest that Syk is a protooncogene involved in the transformation of lymphocytes, thus making Syk a potential target for the treatment of leukemia.
Conformational Dynamics of hSGLT1 during Na+/Glucose Cotransport
Authors: Loo Donald D.F. , Hirayama Bruce A. , Karakossian Movses H. , Meinild Anne-Kristine , Wright Ernest M.
Published: November 2006

This study examines the conformations of the Na+/glucose cotransporter (SGLT1) during sugar transport using charge and fluorescence measurements on the human SGLT1 mutant G507C expressed in Xenopus oocytes. The mutant exhibited similar steady-state and presteady-state kinetics as wild-type SGLT1, and labeling of Cys507 by tetramethylrhodamine-6-maleimide had no effect on kinetics. Our strategy was to record changes in charge and fluorescence in response to rapid jumps in membrane potential in the presence and absence of sugar or the competitive inhibitor phlorizin. In Na+ buffer, step jumps in membrane voltage elicited presteady-state currents (charge movements) that decay to the steady state with time constants τmed (3–20 ms, medium) and τslow (15–70 ms, slow). Concurrently, SGLT1 rhodamine fluorescence intensity increased with depolarizing and decreased with hyperpolarizing voltages (ΔF). The charge vs. voltage (Q-V) and fluorescence vs. voltage (ΔF-V) relations (for medium and slow components) obeyed Boltzmann relations with similar parameters: zδ (apparent valence of voltage sensor) ≈ 1; and V0.5 (midpoint voltage) between −15 and −40 mV. Sugar induced an inward current (Na+/glucose cotransport), and reduced maximal charge (Qmax) and fluorescence (ΔFmax) with half-maximal concentrations (K0.5) of 1 mM. Increasing [αMDG]o also shifted the V0.5 for Q and ΔF to more positive values, with K0.5's ≈ 1 mM. The major difference between Q and ΔF was that at saturating [αMDG]o, the presteady-state current (and Qmax) was totally abolished, whereas ΔFmax was only reduced 50%. Phlorizin reduced both Qmax and ΔFmax (Ki ≈ 0.4 μM), with no changes in V0.5's or relaxation time constants. Simulations using an eight-state kinetic model indicate that external sugar increases the occupancy probability of inward-facing conformations at the expense of outward-facing conformations. The simulations predict, and we have observed experimentally, that presteady-state currents are blocked by saturating sugar, but not the changes in fluorescence. Thus we have isolated an electroneutral conformational change that has not been previously described. This rate-limiting step at maximal inward Na+/sugar cotransport (saturating voltage and external Na+ and sugar concentrations) is the slow release of Na+ from the internal surface of SGLT1. The high affinity blocker phlorizin locks the cotransporter in an inactive conformation.
A Cation–π Interaction between Extracellular TEA and an Aromatic Residue in Potassium Channels
Authors: Ahern Christopher A. , Eastwood Amy L. , Lester Henry A. , Dougherty Dennis A. , Horn Richard
Published: November 2006

Open-channel blockers such as tetraethylammonium (TEA) have a long history as probes of the permeation pathway of ion channels. High affinity blockade by extracellular TEA requires the presence of an aromatic amino acid at a position that sits at the external entrance of the permeation pathway (residue 449 in the eukaryotic voltage-gated potassium channel Shaker). We investigated whether a cation–π interaction between TEA and such an aromatic residue contributes to TEA block using the in vivo nonsense suppression method to incorporate a series of increasingly fluorinated Phe side chains at position 449. Fluorination, which is known to decrease the cation–π binding ability of an aromatic ring, progressively increased the inhibitory constant Ki for the TEA block of Shaker. A larger increase in Ki was observed when the benzene ring of Phe449 was substituted by nonaromatic cyclohexane. These results support a strong cation–π component to the TEA block. The data provide an empirical basis for choosing between Shaker models that are based on two classes of reported crystal structures for the bacterial channel KcsA, showing residue Tyr82 in orientations either compatible or incompatible with a cation–π mechanism. We propose that the aromatic residue at this position in Shaker is favorably oriented for a cation–π interaction with the permeation pathway. This choice is supported by high level ab initio calculations of the predicted effects of Phe modifications on TEA binding energy.
Improved breast cancer prognosis through the combination of clinical and genetic markers
Authors: Sun Yijun , Goodison Steve , Li Jian , Liu Li , Farmerie William
Published: November 2006

Motivation: Accurate prognosis of breast cancer can spare a significant number of breast cancer patients from receiving unnecessary adjuvant systemic treatment and its related expensive medical costs. Recent studies have demonstrated the potential value of gene expression signatures in assessing the risk of post-surgical disease recurrence. However, these studies all attempt to develop genetic marker-based prognostic systems to replace the existing clinical criteria, while ignoring the rich information contained in established clinical markers. Given the complexity of breast cancer prognosis, a more practical strategy would be to utilize both clinical and genetic marker information that may be complementary.

Methods: A computational study is performed on publicly available microarray data, which has spawned a 70-gene prognostic signature. The recently proposed I-RELIEF algorithm is used to identify a hybrid signature through the combination of both genetic and clinical markers. A rigorous experimental protocol is used to estimate the prognostic performance of the hybrid signature and other prognostic approaches. Survival data analyses is performed to compare different prognostic approaches.

Results: The hybrid signature performs significantly better than other methods, including the 70-gene signature, clinical makers alone and the St. Gallen consensus criterion. At the 90% sensitivity level, the hybrid signature achieves 67% specificity, as compared to 47% for the 70-gene signature and 48% for the clinical makers. The odds ratio of the hybrid signature for developing distant metastases within five years between the patients with a good prognosis signature and the patients with a bad prognosis is 21.0 (95% CI:6.5–68.3), far higher than either genetic or clinical markers alone.

Availability: The breast cancer dataset is available at and Matlab codes are available upon request.

Contact:  sun@dsp.ufl.edu

Supplementary information: Supplementary data are available at Bioinformatics online.
Bowen's disease with invasive adnexal carcinoma: The pluripotential nature of Bowen's disease cells
Authors: KAMIYA Hideki , KITAJIMA Yasuo , BAN Masanori
Published: November 2006

Bowen's disease rarely exhibits multiple combinations of premalignant and/or malignant skin lesions. Bowen's disease with invasive adnexal carcinoma was originally described by Kao, but is not well recognized by clinicians due to its rarity and lack of specific clinical features of this condition. Herein, we describe three unusual cases of Bowen's disease with invasive adnexal carcinoma. The two distinct neoplastic areas exhibited continuity both clinically and histologically. The plaque lesions possessed clinical features typical of Bowen's disease. In cases 1 and 3, we confirmed the adnexal tumor within tumors of Bowen's disease, the diagnosis of which is eccrine porocarcinoma. The tumor in case 2 was characteristic to trichilemmal carcinoma. Immunohistochemically, the tumor cells of Bowen's disease and the adnexal carcinoma differed in antigenicities. The present cases support a notion that Bowen's disease maintains a pluripotential nature.
Eruptive pseudoangiomatosis
Authors: YANG Jeong Hoon , KIM Jee Woong , PARK Hyun Su , JANG Sang Jai , CHOI Jung Chul
Published: November 2006

Eruptive pseudoangiomatosis (EPA) is a rare, self‐limiting exanthem, which is known to occur primarily in children. It is characterized by an eruption of distinctive erythematous angioma‐like papules often surrounded by a pale halo, with histological findings distinct from that of true angiomas. We describe three women with angioma‐like papules. The biopsy specimens from them showed a unique histological appearance consisting of dilated dermal blood vessels with plump endothelial cell and perivascular lymphocytes. On the basis of the clinical and histological findings, we present the adult cases of eruptive pseudoangiomatosis and their etiology.
Photosensitive psoriasis in a vitiligo patient
Authors: TORCHIA Daniele , TERRANOVA Margherita , FABBRI Paolo
Published: November 2006

We report a case of a 15‐year‐old Caucasian female, previously affected by non‐segmental vitiligo and psoriasis vulgaris, who developed a psoriatic eruption on sun‐exposed skin during the summer. Oral therapy with cyclosporine A achieved a rapid improvement of the clinical picture. The main features of photosensitive psoriasis and the association between psoriasis and vitiligo are discussed herein.
Effects of varying the monetary value of voucher‐based incentives on abstinence achieved during and following treatment among cocaine‐dependent outpatients
Authors: Higgins Stephen T. , Heil Sarah H. , Dantona Robert , Donham Robert , Matthews Martha , Badger Gary J.
Published: November 2006

Aims  This study examined whether increasing the amount of abstinence achieved during outpatient treatment for cocaine dependence is an effective method for increasing longer‐term cocaine abstinence.

Design  A two‐condition, parallel groups, randomized controlled trial was conducted.

Setting  The trial was conducted in a university‐based research clinic.

Participants  A total of 100 cocaine‐dependent outpatients participated in the trial.

Intervention  Participants were assigned randomly to receive treatment based on the community reinforcement approach (CRA) plus voucher‐based incentives set at a relatively high monetary value (maximal value = $1995/12 weeks) or CRA with vouchers set at a relatively low monetary value (maximal value = $499/12 weeks). Vouchers were earned contingent on cocaine‐negative urinalysis results during the initial 12 weeks of the 24‐week outpatient treatment.

Measurements  Outcomes were evaluated using urine‐toxicology testing, questionnaires and other self‐report instruments.

Findings  Increasing voucher value increased the duration of continuous cocaine abstinence achieved during the 24‐week treatment period. Point‐prevalence cocaine abstinence assessed every 3 months throughout an 18‐month follow‐up period was greater in the high‐ than low‐value voucher conditions. The duration of abstinence achieved during treatment predicted abstinence during follow‐up, although that relationship weakened over time.

Conclusions  Increasing the value of abstinence‐contingent incentives during the initial weeks of treatment appears to represent an effective method for increasing during‐treatment and longer‐term cocaine abstinence, but the positive association of during‐treatment abstinence with longer‐term outcome dissipates with time.
Review article: tolevamer, a novel toxin‐binding polymer: overview of preclinical pharmacology and physicochemical properties
Authors: BARKER R. H. , DAGHER R. , DAVIDSON D. M. , MARQUIS J. K.
Published: November 2006

Background  Tolevamer is a novel toxin‐binding polymer that is currently being investigated in clinical trials for the treatment of patients who have Clostridium difficile‐associated diarrhoea.

Aims  To summarize the results of in vitro and in vivo preclinical studies of tolevamer. In contrast to antibiotics, tolevamer binds C. difficile toxins to interrupt toxin‐mediated intestinal inflammation and tissue damage, and does not demonstrate direct antimicrobial activity.

Methods  Pharmacokinetics/pharmacodynamics were studied in rats and dogs; efficacy was studied in a hamster model.

Results  Studies in rats and dogs indicate that tolevamer is essentially non‐absorbed from the gastrointestinal tract and show that drug interactions with commonly used therapies are unlikely. Pharmacologic studies indicate that tolevamer reduces disease severity and recurrence rates in the hamster model of C. difficile‐associated diarrhoea and blocks the enterotoxic effects of toxin A in rat ileum. The binding parameters calculated for the interaction of tolevamer with toxins A and B provide a reasonable physicochemical model that supports the potential clinical utility of tolevamer.

Conclusions  These preclinical results are consistent with the effectiveness and safety profile of tolevamer observed in clinical studies in patients with C. difficile‐associated diarrhoea.
Use of antisecretory drugs among consumers of non‐steroidal anti‐inflammatory drugs in the general population
Authors: REY E. , ELOLA‐OLASO C. MORENA , RODRÍGUEZ‐ARTALEJO F. , LOCKE G. R. , DIAZ‐RUBIO M.
Published: November 2006

Background  Overall success of prophylactic strategies against non‐steroidal anti‐inflammatory drug (NSAID) complications depends on the use of gastroprotective drugs.

Aim  We examined the use of antisecretory drugs in NSAID users in the general population of Spain.

Participants and Methods  In 2002, a phone interview was conducted with 2500 persons representative of the general population of Spain. Using a validated questionnaire, we asked about the use of NSAID, aspirin and antisecretory drugs, and history of digestive diseases. We estimated the use of antisecretory drugs in NSAID users, according to risk factors for gastrointestinal (GI) lesions associated with NSAID.

Results  In total, 425 persons [17.0% (95% CI 15.5–18.5%)] were NSAID users. Of them, 69 persons (16.2%; 95% CI 12.7–19.7%) used antisecretory drugs [proton pump inhibitor (PPI) 11.8% and H2‐blocker 4.9%]. Forty‐four of the 224 NSAID users (19.6%) with one risk factor for GI lesions were antisecretory drug users (PPI 16%; H2‐blocker 4%), compared with 24 of the 197 NSAID users (12.7%) without risk factors (PPI 6.6%; H2‐blocker 6.1%). NSAID users with risk factors for GI lesions but without upper GI (UGI) symptoms did not consume more antisecretory drugs than equivalent non‐NSAID users (12.9% vs. 10.7%).

Conclusion  Current strategies to prevent GI lesions in NSAID users are not effective from a population perspective, especially in subjects without UGI symptoms.
Oesophageal mucosal involvement in patients with systemic sclerosis receiving proton pump inhibitor therapy
Authors: MARIE I. , DUCROTTE P. , DENIS P. , HELLOT M. F. , LEVESQUE H.
Published: November 2006

Aim  To assess the prevalence of oesophagitis, Barrett's oesophagus (BE) and other oesophageal mucosal abnormalities in patients with systemic sclerosis (SSc) without prior selection on digestive clinical presentation. We also investigated the association between oesophageal endoscopic and manometric data with clinical manifestations of SSc.

Methods  Oesophageal endoscopy and manometry were performed in 133 consecutive patients with SSc, receiving proton pump inhibitor (PPI) therapy since SSc diagnosis.

Results  Endoscopy revealed oesophagitis in 43 patients (32.3%), BE in 9 patients (6.8%), candidiasis in 7 patients (5.3%) and hyperplastic polyp arising in ectopic gastric mucosa in 1 patient. Patients with severe oesophageal motor impairment further exhibited a higher prevalence of interstitial lung disease (ILD) when compared with those without.

Conclusion  Our study underlines the high frequency of oesophageal mucosal abnormalities in unselected SSc patients receiving long‐term PPI therapy. A relationship between oesophagitis/BE and severe manometric motor disturbances was established; these patients may require a higher regimen of PPI. Finally, our series indicates a correlation between severe oesophageal motor disturbances and evidence for ILD in patients, suggesting that gastro‐oesophageal reflux may be one of the contributing factors of ILD in SSc; this subgroup of patients may require close monitoring of lung parameters.
Inhibitory effects of suplatast tosilate on the differentiation and function of monocyte‐derived dendritic cells from patients with asthma
Authors: Tanaka A. , Minoguchi K. , Samson K. T. R. , Oda N. , Yokoe T. , Tazaki T. , Yamamoto Y. , Yamamoto M. , Ohta S. , Adachi M.
Published: November 2006

Background
Dendritic cells (DCs) are antigen‐presenting cells that efficiently activate T cells.

Objective
We examined the effects of suplatast tosilate, which prevents T‐helper type 2 responses, on the differentiation and function of monocyte‐derived DCs (moDCs).

Methods
DCs were differentiated in vitro from peripheral monocytes from patients with asthma by the addition of granulocyte macrophage colony‐stimulating factor and IL‐4 in the presence or absence of suplatast tosilate. Cell surface molecules (CD1a, CD14, CD80, CD83, CD86, HLA‐DR) on immature and mature DCs were analysed with flow cytometry, and the secretion of CC chemokine ligand (CCL)17 (thymus and activation‐regulated chemokine), IL‐12p70, IL‐12p40, and IL‐10 was measured with an ELISA. We also studied the proliferative responses of allogeneic CD4+ T cells from healthy subjects to DCs differentiated in the presence of suplatast tosilate. In addition, the production of IFN‐γ and IL‐5 by CD4+ T cells after coculture with untreated DCs or suplatast tosilate‐treated DCs was measured with ELISA.

Results
Suplatast tosilate significantly inhibited the expression of CD1a, CD80, and CD86 on immature DCs and of CD1a, CD80, CD83, and CD86 on mature DCs. Suplatast tosilate also significantly inhibited the secretion of CCL17, IL‐12p70, and IL‐12p40; however, the secretion of IL‐10 was not affected. The proliferative responses of allogeneic CD4+ T cells to suplatast tosilate‐treated DCs were suppressed. Moreover, suplatast tosilate‐treated DCs had an impaired capacity to stimulate CD4+ T cells to produce IFN‐γ and IL‐5.

Conclusion
Suplatast tosilate inhibits the differentiation, maturation, and function of moDCs.