bluuebunny/crossref_metadata_2025 huggingface dataset:
EphrinA1 inactivates integrin-mediated vascular smooth muscle cell spreading via the Rac/PAK pathway
Authors: Deroanne Christophe , Vouret-Craviari Valérie , Wang Bingcheng , Pouysségur Jacques
Published: March 2003

Interactions between the Eph receptor tyrosine kinase and ephrin ligands transduce short-range signals regulating axon pathfinding, development of the cardiovascular system, as well as migration and spreading of neuronal and non-neuronal cells. Some of these effects are believed to be mediated by alterations in actin dynamics. The members of the small Rho GTPase family elicit various effects on actin structures and are probably involved in Eph receptor-induced actin modulation. EphrinA1 is proposed to contribute to angiogenesis as it is strongly expressed at sites of neovascularization. Moreover, angiogenic factors induce the expression of ephrinA1 in endothelial cells. In this study, using rat vascular smooth muscle cells (VSMCs), we investigated the contribution of the small Rho GTPases in ephrinA1-induced integrin inactivation. EphrinA1 did not significantly affect early adhesion of VSMCs on purified laminin or fibronectin, but strongly impaired cell spreading. The Rho kinase inhibitor Y-27632 partly reversed the ephrinA1 effect, suggesting involvement of Rho in this model. However, inhibition of RhoA synthesis with short interfering (si)RNA had a modest effect, suggesting that RhoA plays a limited role in ephrinA1-mediated inhibition of spreading in VSMCs. The ephrinA1-mediated morphological alterations correlated with inhibition of Rac1 and p21-activated kinase 1 (PAK1) activity, and were antagonized by the expression of a constitutively active Rac mutant. Moreover,repression of Rac1 synthesis with siRNA amplifies the ephrinA1-induced inhibition of spreading. Finally, sphingosine-1-phosphate (S1P), a lipid mediator known to inhibit Rac activation in VSMCs amplifies the ephrinA1 effect. In conclusion, our results emphasize the role of the Rac/PAK pathway in ephrinA1-mediated inhibition of spreading. In this way, ephrinA1, alone or in synergy with S1P, can participate in blood vessel destabilization, a prerequisite for angiogenesis.
Polo boxes form a single functional domain that mediates interactions with multiple proteins in fission yeast polo kinase
Authors: Reynolds Nicola , Ohkura Hiroyuki
Published: March 2003

Polo kinases play multiple roles in cell cycle regulation in eukaryotic cells. In addition to the kinase domain, conservation at the primary sequence level is also found in the non-catalytic region mainly in three blocks, namely the polo boxes. Although several studies have implicated the polo boxes in protein localisation, no systematic study to elucidate the roles of individual polo boxes has been carried out. Here we show, by using fission yeast, that the polo boxes form a single functional unit that is essential for both cellular function and cell-cycle-regulated localisation to the spindle pole bodies. Various polo box mutations abolish the mitotic arrest seen upon overexpression of plo1 but do not prevent the untimely septation seen under these conditions, showing that the functions of Plo1 may be separated. Plo1 interacts with multiple proteins including cell cycle regulators in a polo-box-dependent manner. Isolation of mutants that differentially disrupt these interactions revealed a role for the polo boxes in mediating protein-protein interactions.
Control of morphogenesis and actin localization by thePenicillium marneffei RAChomolog
Authors: Boyce Kylie J. , Hynes Michael J. , Andrianopoulos Alex
Published: March 2003

Rac proteins control polarized growth in many organisms but the specific function of these proteins remains undefined. In this study, we describe the cloning and functional characterization of a RAC homolog, cflB, from the dimorphic fungus Penicillium marneffei. P. marneffei produces asexual spores on complex structures (conidiophores)and switches between hyphal and yeast growth. CflB colocalizes with actin at the tips of vegetative hyphal cells and at sites of cell division. Deletion of cflB results in cell division (septation) and growth defects in both vegetative hyphal and conidiophore cell types such that cells become depolarized, exhibit inappropriate septation and the actin cytoskeleton is severely disrupted. This data suggests that Rac proteins play a crucial role in actin dependent polarized growth and division. The CDC42 ortholog in P. marneffei, cflA, controls vegetative hyphal and yeast growth polarization but does not affect asexual development. By contrast, CflB affects cellular polarization during asexual development and hyphal growth but not during yeast growth. This shows that these two GTPases have both overlapping and distinct roles during growth and development. RAC orthologs are not found in less morphologically complex eukaryotes such as Saccharomyces cerevisiae, suggesting that RAC genes might have evolved with increasing cellular complexity.
Telomere-based proliferative lifespan barriers in Werner-syndrome fibroblasts involve both p53-dependent and p53-independent mechanisms
Authors: Davis Terence , Singhrao Sim K. , Wyllie Fiona S. , Haughton Michele F. , Smith Paul J. , Wiltshire Marie , Wynford-Thomas David , Jones Christopher J. , Faragher Richard G. A. , Kipling David
Published: March 2003

Werner-syndrome fibroblasts have a reduced in vitro life span before entering replicative senescence. Although this has been thought to be causal in the accelerated ageing of this disease, controversy remains as to whether Werner syndrome is showing the acceleration of a normal cellular ageing mechanism or the occurrence of a novel Werner-syndrome-specific process. Here,we analyse the signalling pathways responsible for senescence in Werner-syndrome fibroblasts. Cultured Werner-syndrome (AG05229) fibroblasts senesced after ∼20 population doublings with most of the cells having a 2N content of DNA. This was associated with hypophosphorylated pRb and high levels of p16Ink4a and p21Waf1. Senescent AG05229 cells re-entered the cell cycle following microinjection of a p53-neutralizing antibody. Similarly, production of the human papilloma virus 16 E6 oncoprotein in presenescent AG05229 cells resulted in senescence being bypassed and extended cellular life span. Werner-syndrome fibroblasts expressing E6 did not proliferate indefinitely but reached a second proliferative lifespan barrier, termed Mint, that could be bypassed by forced production of telomerase in post-M1 E6-producing cells. The conclusions from these studies are that: (1) replicative senescence in Werner-syndrome fibroblasts is a telomere-induced p53-dependent event; and(2) the intermediate lifespan barrier Mint is also a telomere-induced event, although it appears to be independent of p53. Werner-syndrome fibroblasts resemble normal human fibroblasts for both these proliferative lifespan barriers, with the strong similarity between the signalling pathway linking telomeres to cell-cycle arrest in Werner-syndrome and normal fibroblasts providing further support for the defect in Werner syndrome causing the acceleration of a normal ageing mechanism.
Microtubule-dependent redistribution of the type-1 inositol 1,4,5-trisphosphate receptor in A7r5 smooth muscle cells
Authors: Vermassen Elke , Van Acker Kristel , Annaert Wim G. , Himpens Bernard , Callewaert Geert , Missiaen Ludwig , De Smedt Humbert , Parys Jan B.
Published: March 2003

In A7r5 vascular smooth muscle cells, the two expressed inositol 1,4,5-trisphosphate receptor (IP3R) isoforms were differentially localized. IP3R1 was predominantly localized in the perinuclear region, whereas IP3R3 was homogeneously distributed over the cytoplasm. Prolonged stimulation (1-5 hours) of cells with 3 μM argininevasopressin induced a redistribution of IP3R1 from the perinuclear region to the entire cytoplasm, whereas the localization of IP3R3 appeared to be unaffected. The redistribution process occurred independently of IP3R downregulation. No structural changes of the endoplasmic reticulum were observed, but SERCA-type Ca2+ pumps redistributed similarly to IP3R1. The change in IP3R1 localization induced by arginine-vasopressin could be blocked by the simultaneous addition of nocodazole or taxol and depended on Ca2+ release from intracellular stores since Ca2+-mobilizing agents such as thapsigargin and cyclopiazonic acid could induce the redistribution. Furthermore, various protein kinase C inhibitors could inhibit the redistribution of IP3R1, whereas the protein kinase C activator 1-oleoyl-2-acetyl-sn-glycerol induced the redistribution. Activation of protein kinase C also induced an outgrowth of the microtubules from the perinuclear region into the cytoplasm, similar to what was seen for the redistribution of IP3R1. Finally, blocking vesicular transport at the level of the intermediate compartment inhibited the redistribution. Taken together, these findings suggest a role for protein kinase C and microtubuli in the redistribution of IP3R1, which probably occurs via a mechanism of vesicular trafficking.
Preparation, Characterisation and Application of Metal-Doped Carbons for Hydrogen Cyanide Removal
Authors: Barnes P.A. , Chinn M.J. , Dawson E.A. , Norman P.R.
Published: March 2003

A novel method for the production of metal-doped activated carbons was developed. Evaluation of the materials produced showed that the metals were well dispersed throughout the carbon pore structure and had a high degree of accessibility. The filtration performance of these materials was assessed against HCN using inverse gas chromatography. The performance was affected both by the selection of metals and the conditions used in the production of the carbon. In-depth studies varying the synthesis parameters were performed with the aim of optimising the manufacturing process. Significant HCN adsorption capacity was developed which exceeded that of the current best available ASC-type carbons.
Combination of a Dielectric Continuum Model with Inverse Gas Chromatography for the Characterization of Solid Surfaces
Authors: Mehler C. , Thielmann F. , Peukert W.
Published: March 2003

The use of a dielectric continuum model for the characterization of solid surfaces was combined for the first time with inverse gas chromatography. Extension of dielectric continuum models to adsorption from the gaseous phase allowed the distributed surface properties of solid surfaces to be determined. An inverse gas chromatograph was used for the measurement of adsorption equilibria as a quick alternative to time-consuming measurements by gravimetric or volumetric set-ups. Combination of the two techniques allowed the rapid determination of the distributed properties of solid surfaces to be effected and the results were interpreted in a fundamental physical sense. This led to a novel and promising way for the rapid and exact characterization of solid surfaces.
Physicochemical and Catalytic Properties of Vanadia/Titania Catalysts. IV. Catalytic Oxidation of o-Xylene
Authors: El-Nabarawy Th. , Alaya M.N. , Ahmed S.A. Sayed , Youssef A.M.
Published: March 2003

Titania samples were prepared by precipitation at pH 3.0 (to provide ‘Ta’ samples) or at pH 9.0 (to provide ‘Tb’ samples). These materials were then impregnated with NH4VO3 to obtain vanadia/titania catalysts of different V2O5 content. These V2O5/TiO2 samples were subsequently calcined at 400°C or 600°C to obtain two series. The oxidation of o-xylene was undertaken at 250°C, 300°C or 350°C using a conventional flow apparatus at atmospheric pressure. The reaction products were detected and separated via a gas chromatograph fitted with a flame ionization detector. The chemical composition of the catalyst, the calcination temperature, the reaction mixture employed and the reaction temperature were among the variables considered.

Catalysts calcined at 400°C were more active than those calcined at 600°C, thereby indicating that anatase was the proper titania phase for supporting oxidation catalysts. Titania samples precipitated at pH 3.0 were more effective as a support for vanadia than those precipitated at pH 9.0 despite the fact that they possessed lower surface areas. This demonstrated that surface area was not a determining factor in catalyst performance.

The conversion of o-xylene to phthalic anhydride increased with increasing vanadia content in the catalysts employed and with increasing reaction temperature. Surface acidity was one factor amongst others contributing to the catalytic activity. It is thought that acid sites enhance the reduction of V5+ to lower oxidation states to provide the active oxidation sites. Mechanisms for the partial oxidation of o-xylene were advanced and explained.
Surface Flocculation as a New Tool for Controlling Adsorption Processes
Authors: Milichovský Miloslav , Češek Bretislav
Published: March 2003

The dyeing of modified pulp, particularly with regard to the mechanism involved, was studied. The reaction mechanism for the modification of cellulose fibres in aqueous solution by cationic agents was also described. The results obtained were expressed in the form of adsorption isotherms. The capture of molecules, colloidal particles, etc. during the adsorption processes involving modified pulp could be explained by two possible mechanisms: (i) aggregation (coagulation or flocculation) in solution followed by adsorption and (ii) surface aggregation (coagulation or flocculation).

Surface aggregation appeared to be the most likely for pulp with a low degree of modification. The typical Langmuir adsorption isotherm correlated the data obtained for unmodified pulp very well.
The adjustment of stock prices to Wall Street journal corrections
Authors: Helmuth John A. , Robin Ashok J. , Zdanowicz John S.
Published: March 2003

This paper employs standard event study methodology to study the response of stock prices to relatively unanticipated events in the ‘Corrections and Amplifications’ section of the Wall Street Journal. Our results indicate that the market reaction to these corrections is statistically significant. We also find that the market fully adjusts to all new information on the day of the unanticipated Wall Street Journal correction. We find no discernible pattern of abnormal returns after the event day. The main implication of these findings is that researchers conducting event studies and using the Wall Street Journal Index for event dates, should screen their data for corrections.
Evidence for a large surface ablation zone in central East Antarctica during the last Ice Age
Authors: Siegert Martin J. , Hindmarsh Richard C. A. , Hamilton Gordon S.
Published: March 2003

Internal isochronous ice sheet layers, recorded by airborne ice-penetrating radar, were measured along an ice flowline across a large (>1 km high) subglacial hill in the foreground of the Transantarctic Mountains. The layers, dated through an existing stratigraphic link with the Vostok ice core, converge with the ice surface as ice flows over the hill without noticeable change to their separation with each other or the ice base. A two-dimensional ice flow model that calculates isochrons and particle flowpaths and accounts for ice flow over the hill under steady-state conditions requires net ablation (via sublimation) over the stoss face for the predicted isochrons to match the measured internal layers. Satellite remote sensing data show no sign of exposed ancient ice at this site, however. Given the lack of exposed glacial ice, surface balance conditions must have changed recently from the net ablation that is predicted at this site for the last 85,000 years to accumulation.
The postglacial history of threePiceaspecies in New England, USA
Authors: Lindbladh Matts , Jacobson George L. , Schauffler Molly
Published: March 2003

Given the difficulty of separating the threePiceaspecies—P. glauca, P. mariana,andP. rubens(white, black, and red spruce)—in the pollen record, little is known about their unique histories in eastern North America following deglaciation. Here we report the first use of a classification tree analysis (CART) to distinguish pollen grains of these species. It was successfully applied to fossil pollen from eight sites in Maine and one in Massachusetts. We focused on the late glacial/early Holocene (14,000 to 8000 cal yr B.P.) and the late Holocene (1400 cal yr B.P. to present)—the two key periods since deglaciation whenPiceahas been abundant in the region. The result shows a shift from aPiceaforest ofP. glaucaandP. marianain the late glacial to a forest ofP. rubensandP. marianain the late Holocene. The small number ofP. rubensgrains identified from the late glacial/early Holocene samples (<5%) suggests that that species was either absent or rare at most of the sites. The occurrence and distribution of the three species do not reveal any geographic or temporal trend during late glacial time, but the data suggest that they were distributed in local patches on the landscape. The results of this study indicate that the recent population expansion ofPicea(1000 to 500 cal yr B.P.) was likely the first time since deglaciation thatP. rubenswas abundant in the region.
Homogenization of the Dirichlet problem for a system of quasilinear elliptic equations in a domain with fine-grained boundary
Authors: Sango Mamadou
Published: March 2003

We study the Dirichlet problem for a system of nonlinear elliptic equations of Leray–Lions type in a sequence of domains

Ω(s)

,

s=1,2,…,

with fine-grained boundaries. Under appropriate structure conditions on the system and the geometry of

Ω(s)

, we prove that the sequence of solutions of the problem converges in suitable topologies to the solution of a limit problem which contains an additional term of capacity type. We construct the limit problem.

Nous étudions le problème de Dirichlet pour un système d’équations non linéaires élliptiques de type Leray–Lions dans une suite de domaines

Ω(s)

,

s=1,2,…,

avec des frontières finement granulées. Sous des conditions de structure appropriées que nous imposons sur le système et sur la géometrie de

Ω(s)

, nous démontrons que la suite de solutions du problème tend dans des topologies appropriées vers la solution d’un problème limite qui contient un terme additionnel de type capacitaire. Nous construisons le problème limite.
Elections in Poland 2001: electoral manipulation and party upheaval
Authors: Millard F.
Published: March 2003

The Polish parliamentary elections of 2001 took place in a context of fresh upheavals in the configuration of political parties. The architects of the new electoral law aimed to reduce the seats gained by the social democrats and increase their own. They succeeded in the first aim by a change of electoral formula, forcing the victorious social democratic electoral coalition to seek a third coalition partner. They did not achieve the second aim, as their own failures in government drastically reduced their electoral support and facilitated the breakthrough of populist formations. The result had implications for party development and the composition and workings of both parliament and government. While representation was enhanced by a parliament more accurately reflecting the voters’ choice, the impact appeared potentially harmful to Polish democracy as a whole.
An Unexpected Adverse Drug Effect
Authors: Krulewitch Cara J.
Published: March 2003

Adverse drug‐drug interactions can occur between active and/or inactive ingredients in different formulations. The occurrence of a disulfiram reaction that developed postpartum following a 7‐day course of metronidazole is presented. The case is presented, followed by a discussion of the mechanism of action and treatment. Recommendations for prevention of adverse drug effects are reviewed.
Diethylstilbestrol (DES) Update: Recommendations for the Identification and Management of DES‐Exposed Individuals
Authors: Hammes Barbara , Laitman Cynthia J.
Published: March 2003

Diethylstilbestrol (DES) was etiologically linked to clear cell adenocarcinoma of the vagina in 1971. This article reviews on‐going research and emerging information relevant to DES‐related health risks, thereby enabling women's health care providers to maintain an evidence‐based practice for their DES‐exposed patients. To accomplish these goals, the Center for Disease Control and Prevention (CDC) has initiated a national education campaign. This article describes the reasons for this new initiative, the target audiences, the DES historical framework (including major studies and findings), and populations that are affected. Clinical steps for the identification and management of the DES‐exposed individual and resultant implications for midwifery and women's health practices are reviewed.
Management of T3 N0 and T4 N0 Glottic Carcinomas: Results of a National Survey
Authors: Greene Ryan M. , Dewitt Adriane I. , Otto RANDAL A.
Published: March 2003

OBJECTIVE

A survey was undertaken to document the clinical management of T3 and T4 pure glottic primary carcinomas and the management of the N0 neck by otolaryngologists and radiation oncologists.

STUDY DESIGN AND SETTING

This study represents the results of a national survey of 250 otolaryngologists and 250 radiation oncologists regarding management of T3 N0 M0 and T4 N0 M0 glottic carcinomas.

RESULTS

Of the surveys sent, 208 completed questionnaires were received. Results of this survey suggest that 87% and 90% will treat the neck for a T3 N0 M0 and T4 N0 M0 glottic tumor, respectively, with a large number choosing to perform a radical neck dissection.

CONCLUSIONS

A significant percentage of otolaryngologists perform neck dissections in the management of T3 N0 M0 and T4 N0 M0 glottic carcinomas. Given the relatively low risk of occult metastasis, potentially high morbidity associated with overtreatment, and the lack of a well‐designed outcome study investigating treatment alternatives, a prospective randomized study is needed to address the issue.
The Effect of Capillary Ultrafiltration Probes on Skin Flap Edema
Authors: Odland Rick M. , Kizziar Ron , Rheuark Daryl , Simental Alfred
Published: March 2003

OBJECTIVE

Edema clearly has deleterious effects on the microcirculation and, consequently, cell viability. Prior work from this laboratory demonstrated that hyperosmolar microdialysis can reduce tissue edema, but this method is technically challenging. A new, simpler technique of microdialysis using capillary ultrafiltration probes (CUPs) has been studied to determine if CUP microdialysis is as effective in reducing tissue edema in the same animal model.

METHODS

Twenty‐four Sprague‐Dawley rats were studied using a modified McFarlane skin flap. Microdialysis was accomplished using a catheter constructed of four 4‐cm hollow fibers that were connected to polyethylene tubing. Catheters on the experimental side of the flap were attached to a vacuum manifold for 8 hours. The control side was treated in 2 ways. In group 1, catheters were placed but not applied to suction. In group 2, no catheters are placed on the control side. Tissue water content was determined by a biopsy‐drying technique.

RESULTS

Tissue water content was significantly reduced (by paired t test) in both groups by a mean of 3.2 mL/100 g of wet tissue.

CONCLUSION

CUP microdialysis reduced tissue water content as effectively as did hyperosmolar microdialysis, but in a simpler and therefore more cost‐effective method. The technique could be easily adapted for clinical application.
Mexican hats and pinwheels in visual cortex
Authors: Kang Kukjin , Shelley Michael , Sompolinsky Haim
Published: March 2003

Many models of cortical function assume that local lateral connections are specific with respect to the preferred features of the interacting cells and that they are organized in a Mexican-hat pattern with strong “center” excitation flanked by strong “surround” inhibition. However, anatomical data on primary visual cortex indicate that the local connections are isotropic and that inhibition has a shorter range than excitation. We address this issue in an analytical study of a neuronal network model of the local cortical circuit in primary visual cortex. In the model, the orientation columns specified by the convergent lateral geniculate nucleus inputs are arranged in a pinwheel architecture, whereas cortical connections are isotropic. We obtain a trade-off between the spatial range of inhibition and its time constant. If inhibition is fast, the network can operate in a Mexican-hat pattern with isotropic connections even with a spatially narrow inhibition. If inhibition is not fast, Mexican-hat operation requires a spatially broad inhibition. The Mexican-hat operation can generate a sharp orientation tuning, which is largely independent of the distance of the cell from the pinwheel center.
Proteins with H-bond packing defects are highly interactive with lipid bilayers: Implications for amyloidogenesis
Authors: Fernández Ariel , Berry R. Stephen
Published: March 2003

We noticed that disease-related amyloidogenic proteins and especially cellular prion proteins have the highest proportion of incompletely desolvated backbone H bonds among soluble proteins. Such bonds are vulnerable to water attack and thus represent structural weaknesses. We have measured the adsorption of proteins onto phospholipid bilayers and found a strong correlation between the extent of underwrapping of backbone H bonds in the native structure of a protein and its extent of deposition on the bilayer: the less the H bond wrapping, the higher the propensity for protein–bilayer binding. These observations support the proposition that soluble proteins with amyloidogenic propensity and membrane proteins share a pervasive building motif: the underwrapped H bonds. Whereas in membrane proteins, this motif does not signal a structural vulnerability, in soluble proteins, it is responsible for their reactivity.
The
Tre2
(
USP6
) oncogene is a hominoid-specific gene
Authors: Paulding Charles A. , Ruvolo Maryellen , Haber Daniel A.
Published: March 2003

Gene duplication and domain accretion are thought to be the major mechanisms for the emergence of novel genes during evolution. Such events are thought to have occurred at early stages in the vertebrate lineage, but genomic sequencing has recently revealed extensive amplification events during the evolution of higher primates. We report here that the
Tre2
(
USP6
) oncogene is derived from the chimeric fusion of two genes,
USP32
(
NY-REN-60
), and
TBC1D3. USP32
is an ancient, highly conserved gene, whereas
TBC1D3
is derived from a recent segmental duplication, which is absent in most other mammals and shows rapid amplification and dispersal through the primate lineage. Remarkably, the chimeric gene
Tre2
exists only in the hominoid lineage of primates. This hominoid-specific oncogene arose as recently as 21–33 million years ago, after proliferation of the
TBC1D3
segmental duplication in the primate lineage. In contrast to the broad expression pattern of
USP32
and
TBC1D3
, expression of
Tre2
is testis-specific, a pattern proposed for novel genes implicated in the emergence of reproductive barriers. The sudden emergence of chimeric proteins, such as that encoded by
Tre2
, may have contributed to hominoid speciation.
Elucidation of crystal form diversity of the HIV protease inhibitor ritonavir by high-throughput crystallization
Authors: Morissette Sherry L. , Soukasene Stephen , Levinson Douglas , Cima Michael J. , Almarsson Örn
Published: March 2003

Pharmaceutical compounds are molecular solids that frequently exhibit polymorphism of crystal form. One high profile case of polymorphism was ritonavir, a peptidomimetic drug used to treat HIV-1 infection and introduced in 1996. In 1998, a lower energy, more stable polymorph (form II) appeared, causing slowed dissolution of the marketed dosage form and compromising the oral bioavailability of the drug. This event forced the removal of the oral capsule formulation from the market. We have carried out high-throughput crystallization experiments to comprehensively explore ritonavir form diversity. A total of five forms were found: both known forms and three previously unknown forms. The novel forms include a metastable polymorph, a hydrate phase, and a formamide solvate. The solvate was converted to form I via the hydrate phase by using a simple washing procedure, providing an unusual route to prepare the form I “disappearing polymorph” [Dunitz, J. D. & Bernstein, J. (1995)
Acc. Chem. Res.
28, 193–200]. Crystals of form I prepared by using this method retained the small needle morphology of the solvate and thus offer a potential strategy for particle size and morphology control.
Structural origins of adenine-tract bending
Authors: Barbič Andrej , Zimmer Daniel P. , Crothers Donald M.
Published: March 2003

DNA sequences containing short adenine tracts are intrinsically curved and play a role in transcriptional regulation. Despite many high-resolution NMR and x-ray studies, the origins of curvature remain disputed. Long-range restraints provided by 85 residual dipolar couplings were measured for a DNA decamer containing an adenine (A)4-tract and used to refine the structure. The overall bend in the molecule is a result of in-phase negative roll in the A-tract and positive roll at its 5′ junction, as well as positive and negative tilt inside the A-tract and near its junctions. The bend magnitude and direction obtained from NMR structures is 9.0° into the minor groove in a coordinate frame located at the third AT base pair. We evaluated long-range and wedge models for DNA curvature and concluded that our data for A-tract curvature are best explained by a “delocalized bend” model. The global bend magnitude and direction of the NMR structure are in excellent agreement with the junction model parameters used to rationalize gel electrophoretic data and with preliminary results of a cyclization kinetics assay from our laboratory.
Community disassembly by an invasive species
Authors: Sanders Nathan J. , Gotelli Nicholas J. , Heller Nicole E. , Gordon Deborah M.
Published: March 2003

Invasive species pose serious threats to community structure and ecosystem function worldwide. The impacts of invasive species can be more pervasive than simple reduction of species numbers. By using 7 years of data in a biological preserve in northern California, we documented the disassembly of native ant communities during an invasion by the Argentine ant. In sites without the Argentine ant, native ant communities exhibit significant species segregation, consistent with competitive dynamics. In sites with the Argentine ant, native ant communities appear random or weakly aggregated in species co-occurrence. Comparisons of the same sites before and after invasion indicate that the shift from a structured to a random community is rapid and occurs within a year of invasion. Our results show that invasive species not only reduce biodiversity but rapidly disassemble communities and, as a result, alter community organization among the species that persist.
A triple-mutated allele of granzyme B incapable of inducing apoptosis
Authors: McIlroy Dorian , Cartron Pierre-François , Tuffery Pierre , Dudoit Yasmine , Samri Assia , Autran Brigitte , Vallette François M. , Debré Patrice , Theodorou Ioannis
Published: March 2003

Granzyme B (GzmB) is a serine protease involved in many pathologies, including viral infections, autoimmunity, transplant rejection, and antitumor immunity. To measure the extent of genetic variation in GzmB, we screened the GzmB gene for polymorphisms and defined a frequently represented triple-mutated GzmB allele. In this variant, three amino acids of the mature protein Q48P88Y245are mutated to R48A88H245. In CD8+cytotoxic T lymphocytes, GzmB was expressed at similar levels in QPY homozygous, QPY/RAH heterozygous, and RAH homozygous individuals, demonstrating that RAH GzmB is a stable protein. Active RAH GzmB expressed in glioblastoma cell lines displayed proteolytic activity, but in contrast to QPY GzmB, it did not accumulate in the nucleus and was unable to induce Bid cleavage, cytochromecrelease, or apoptosis. Molecular modeling showed that the three amino acid substitutions clustered near the C-terminal α-helix of the protein, indicating that this region of the protein may be involved in the intracellular targeting of GzmB. The triple-mutated GzmB allele that we describe appears to be incapable of inducing apoptosis in tumor cell lines, and its presence could, therefore, influence both the prognosis of cancer patients and the success rates of antitumor cellular immunotherapy.
Evidence for defective retinoid transport and function in late onset Alzheimer's disease
Authors: Goodman Ann B. , Pardee Arthur B.
Published: March 2003

The hypothesis of this article is that late onset Alzheimer's disease (AD) is influenced by the availability in brain of retinoic acid (RA), the final product of the vitamin A (retinoid) metabolic cascade. Genetic, metabolic, and environmental/dietary evidence is cited supporting this hypothesis. Significant genetic linkages to AD are demonstrated for markers close to four of the six RA receptors, RA receptor G at 12q13, retinoid X receptor B at 6p21.3, retinoid X receptor G at 1q21, and RA receptor A at 17q21. Three of the four retinol-binding proteins at 3q23 and 10q23 and the RA-degrading cytochrome P450 enzymes at 10q23 and 2p13 map to AD linkages. Synthesis of the evidence supports retinoid hypofunction and impaired transport as contributing factors. These findings suggest testable experiments to determine whether increasing the availability of retinoid in brain, possibly through pharmacologic targeting of the RA receptors and the cytochrome P450 RA-inactivating enzymes, can prevent or decrease amyloid plaque formation.
Immunomic analysis of human sarcoma
Authors: Lee Sang-Yull , Obata Yuichi , Yoshida Masahiro , Stockert Elisabeth , Williamson Barbara , Jungbluth Achim A. , Chen Yao-Tseng , Old Lloyd J. , Scanlan Matthew J.
Published: March 2003

The screening of cDNA expression libraries from human tumors with serum antibody (SEREX) has proven to be a powerful method for identifying the repertoire of tumor antigens recognized by the immune system of cancer patients, referred to as the cancer immunome. In this regard, cancer/testis (CT) antigens are of particular interest because of their immunogenicity and restricted expression patterns. Synoivial sarcomas are striking with regard to CT antigen expression, with >80% of specimens homogeneously expressing NY-ESO-1 and MAGE-A3. In the present study, 54 sarcoma patients were tested for serum antibodies to NY-ESO-1, SSX2, MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A10, CT7, and CT10. Two patients had detectable antibodies to CT antigens, and this seroreactivity was restricted to NY-ESO-1. Thus, although highly expressed in sarcoma, CT antigens do not induce frequent humoral immune responses in sarcoma patients. Sera from these two patients were used to immunoscreen cDNA libraries from two synovial sarcoma cell lines and normal testis, resulting in the identification of 113 distinct antigens. Thirty-nine antigens were previously identified by SEREX analysis of other tumor types, and 23/39 antigens (59%) had a serological profile that was not restricted to cancer patients, indicating that only a proportion of SEREX-defined antigens are cancer-related. A novel CT antigen, NY-SAR-35, mapping to chromosome Xq28 was identified among the cancer-related antigens, and encodes a putative extracellular protein. In addition to testis-restricted expression, NY-SAR-35 mRNA was expressed in sarcoma, melanoma, esophageal cancer, lung cancer and breast cancer. NY-SAR-35 is therefore a potential target for cancer vaccines and monoclonal antibody-based immunotherapies.
Aquaglyceroporin AQP9: Solute permeation and metabolic control of expression in liver
Authors: Carbrey Jennifer M. , Gorelick-Feldman Daniel A. , Kozono David , Praetorius Jeppe , Nielsen Søren , Agre Peter
Published: March 2003

Aquaglyceroporins form the subset of the aquaporin water channel family that is permeable to glycerol and certain small, uncharged solutes. AQP9 has unusually broad solute permeability and is expressed in hepatocyte plasma membranes. Proteoliposomes reconstituted with expressed, purified rat AQP9 protein were compared with simple liposomes for solute permeability. At pH 7.5, AQP9 proteoliposomes exhibited Hg
2+
-inhibitible glycerol and urea permeabilities that were increased 63-fold and 90-fold over background. β-Hydroxybutyrate permeability was not increased above background, and osmotic water permeability was only minimally elevated. During starvation, the liver takes up glycerol for gluconeogenesis. Expression of AQP9 in liver was induced up to 20-fold in rats fasted for 24–96 h, and the AQP9 level gradually declined after refeeding. No changes in liver AQP9 levels were observed in rats fed ketogenic diets or high-protein diets, but AQP9 levels were elevated in livers of rats made diabetic by streptozotocin injection. When blood glucose levels of the diabetic rats were restored to normal by insulin treatments, the AQP9 levels returned to baseline. Confocal immunofluorescence revealed AQP9 immunostaining on the sinusoidal surfaces of hepatocyte plates throughout the livers of control rats. Denser immunostaining was observed in the same distribution in livers of fasted and streptozotocin-treated rats. We conclude that AQP9 serves as membrane channel in hepatocytes for glycerol and urea at physiological pH, but not for β-hydroxybutyrate. In addition, levels of AQP9 expression fluctuate depending on the nutritional status of the subject and the circulating insulin levels.
The FKBP-associated protein FAP48 is an antiproliferative molecule and a player in T cell activation that increases IL2 synthesis
Authors: Krummrei Ulrike , Baulieu Etienne-Emile , Chambraud Béatrice
Published: March 2003

FAP48 was identified and cloned thanks to its interaction with FK506-binding proteins (FKBPs) such as FKBP52 and FKBP12, which belong to the large family of immunophilins that bind the macrolide immunosuppressant drugs FK506 and rapamycin. We have previously shown that FAP48–FKBP complexes are dissociated by FK506 and rapamycin, suggesting that FAP48 is an endogenous ligand of FKBP. The present work describes the biochemical consequences of FAP48 overexpression, induced by the tetracycline analogue doxycycline, in an established cell line derived from Jurkat T cells. We report that overexpression of FAP48 results in the inhibition of cellular proliferation as does the exposure of Jurkat T cells to FK506. We also show that the expression levels of argininosuccinate synthetase and the Myc antagonist Mxi1 are modified by overexpression of FAP48, suggesting that these proteins could be good candidates to mediate the antiproliferative effect of FAP48. FAP48 affects neither the calcineurin-dependent nuclear factor of activated T cells (NFAT)1 nor JNK/p38-dependent pathways that mediate immunosuppression by FK506. However, contrary to FK506, which blocks IL2 synthesis, we observed that FAP48–FKBP complexes increase IL2 production, thus revealing a previously uncharacterized aspect of the immunosuppressive mechanism of FK506.
The mouse C-type transient receptor potential 2 (TRPC2) channel: Alternative splicing and calmodulin binding to its N terminus
Authors: Yildirim Eda , Dietrich Alexander , Birnbaumer Lutz
Published: March 2003

Channels of the C-type transient receptor potential (TRPC) are involved in agonist-stimulated and capacitative calcium entry. There are seven TRPCs, all of which have a Ca
2+
-dependent calmodulin (CaM)-binding domain in their C termini. We now tested binding of CaM to TRPC N termini and show that only that of TRPC2 binds CaM in a Ca
2+
-dependent manner. Four TRPC2 cDNAs have been reported: a (also clone 14), b (also clone 17), α, and β. Sequences responsible for CaM binding in TRPC2 a and b are absent from the α and β isoforms. The α and β cDNAs of TRPC2 were reported as alternative forms, when recloning of TRPC2 a and b proved impossible. Here we analyzed total RNA samples from brain and testis for presence of TRPC2 a and b and describe the splicing patterns responsible for their formation, as well as those leading to the α and β forms of TRPC2. We re-assert existence of RNA encoding the TRPC2 a and b, encoded in 21 exons with an initiator ATG in exon 2 for TRPC2a and in exon 4 for TRCP2b. The analysis of α and β TRPC2 cDNAs indicates that although the TRPC2β mRNA may exist, the TRPC2α cDNA is derived from an incompletely processed TRPC2a mRNA: It includes in its presumed 5′-untranslated sequence, 713 nt of TRPC2a cDNA fused to 291 nt of an incompletely excised intron. While encoding an active channel in the mouse, the human TRPC2 appears to be a pseudogene. We searched for the human gene in the data bank and located approximately one-half of it in a chromosomal region syntenic to that of the mouse, with similar intron–exon structure. We conclude that the human TRPC2 gene may never have been an active gene because of incomplete ancestral duplication or, if it was complete at one point, that it became inactive upon loss of chromosomal sequences.
A plant signal peptide–hepatitis B surface antigen fusion protein with enhanced stability and immunogenicity expressed in plant cells
Authors: Sojikul Punchapat , Buehner Norene , Mason Hugh S.
Published: March 2003

The use of transgenic plants to express orally immunogenic protein antigens is an emerging strategy for vaccine biomanufacturing and delivery. This concept has particular suitability for developing countries. One factor that has limited the development of this technology is the relatively modest levels of accumulation of some antigenic proteins in plant tissues. We used fusion protein design to improve expression of the hepatitis B surface antigen (HBsAg) by attempting to mimic the process of HBsAg targeting to the endoplasmic reticulum of human liver cells during hepatitis B virus infection. We created a gene encoding a recombinant HBsAg modified to contain a plant signal peptide fused to its amino terminus. The signal peptide from soybean vegetative storage proteinvspA(VSPαS) directed endoplasmic reticulum targeting of HBsAg in plant cells, but was not cleaved and resulted in enhanced VSPαS-HBsAg fusion accumulation. This product was more stable and presented the protective “a” antigenic determinant to significantly higher levels than unmodified native HBsAg expressed in plant cells. It also showed a greater extent of intermolecular disulfide bond formation and formation of virus-like particles. Moreover, VSPαS-HBsAg stimulated higher levels of serum IgG than native HBsAg when injected into mice. We conclude that HBsAg tolerates a polypeptide fusion at the amino terminus and that VSPαS-HBsAg is an improved antigen for plant-based expression of a subunit vaccine for hepatitis B virus.
IL-15 availability conditions homeostasis of peripheral natural killer T cells
Authors: Ranson Thomas , Vosshenrich Christian A. J. , Corcuff Erwan , Richard Odile , Laloux Veronique , Lehuen Agnès , Di Santo James P.
Published: March 2003

Steady-state numbers of peripheral lymphocyte are tightly controlled. For conventional T cells, signals delivered through the interaction of the T cell receptor (TCR) with antigen-loaded MHC molecules are required for the peripheral survival of naive T cells and for their homeostatic expansion in lymphopenic hosts. Cytokines, including IL-7, are also essential for survival of peripheral naive T cells. CD1d-restricted, Vα14+natural killer (NK)-T cells are a specialized autoreactive T subset with immunoregulatory activity. The relative roles of TCR engagement and cytokine signaling in the peripheral homeostasis of Vα14+NK-T cells were investigated. After adoptive transfer, the survival and expansion of peripheral Vα14+NK-T cells was independent of CD1d expression in the host. In contrast, IL-15 (but not IL-7) was required for maintenance of peripheral CD1d-reactive Vα14+T cells. Comparison of Vα14+T cell transfers into NK-proficient vs. deficient hosts suggests that NK-T cells and NK cells compete for peripheral resources. Our results indicate that IL-15 maintains the homeostasis of peripheral Vα14+NK-T cells. In contrast, TCR “tickling” of NK-T cells, if it occurs under steady-state conditions, does not by itself provide a sufficient signal for their peripheral survival.
DNA molecule provides a computing machine with both data and fuel
Authors: Benenson Yaakov , Adar Rivka , Paz-Elizur Tamar , Livneh Zvi , Shapiro Ehud
Published: March 2003

The unique properties of DNA make it a fundamental building block in the fields of supramolecular chemistry, nanotechnology, nano-circuits, molecular switches, molecular devices, and molecular computing. In our recently introduced autonomous molecular automaton, DNA molecules serve as input, output, and software, and the hardware consists of DNA restriction and ligation enzymes using ATP as fuel. In addition to information, DNA stores energy, available on hybridization of complementary strands or hydrolysis of its phosphodiester backbone. Here we show that a single DNA molecule can provide both the input data and all of the necessary fuel for a molecular automaton. Each computational step of the automaton consists of a reversible software molecule/input molecule hybridization followed by an irreversible software-directed cleavage of the input molecule, which drives the computation forward by increasing entropy and releasing heat. The cleavage uses a hitherto unknown capability of the restriction enzyme
Fok
I, which serves as the hardware, to operate on a noncovalent software/input hybrid. In the previous automaton, software/input ligation consumed one software molecule and two ATP molecules per step. As ligation is not performed in this automaton, a fixed amount of software and hardware molecules can, in principle, process any input molecule of any length without external energy supply. Our experiments demonstrate 3 × 10
12
automata per μl performing 6.6 × 10
10
transitions per second per μl with transition fidelity of 99.9%, dissipating about 5 × 10
−9
W/μl as heat at ambient temperature.
Lineage-restricted retention of a primitive immunoglobulin heavy chain isotype within the Dipnoi reveals an evolutionary paradox
Authors: Ota Tatsuya , Rast Jonathan P. , Litman Gary W. , Amemiya Chris T.
Published: March 2003

The lineage leading to lungfishes is one of the few major jawed vertebrate groups in which Ig heavy chain isotype structure has not been investigated at the genetic level. In this study, we have characterized three different Ig heavy chain isotypes of the African lungfish,Protopterus aethiopicus, including an IgM-type heavy chain and short and long forms of non-IgM heavy chains. Northern blot analysis as well as patterns of VHutilization suggest that the IgM and non-IgM isotypes are likely encoded in separate loci. The two non-IgM isotypes identified inProtopterusshare structural features with the short and long forms of IgX/W/NARC (referred to hereafter as IgW), which were previously considered to be restricted to the cartilaginous fish. It seems that the IgW isotype has a far broader phylogenetic distribution than considered originally and raises questions with regard to the origin and evolutionary divergence of IgM and IgW. Moreover, its absence in other gnathostome lineages implies paradoxically that the IgW-type genes were lost from teleost and tetrapod lineages.