bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural changes at microtubule ends accompanying GTP hydrolysis: Information from a slowly hydrolyzable analogue of GTP, guanylyl (α,β)methylenediphosphonate
Authors: Müller-Reichert Thomas , Chrétien Denis , Severin Fedor , Hyman Anthony A.
Published: July 2002

Microtubules are dynamic polymers that interconvert between periods of slow growth and fast shrinkage. The energy driving this nonequilibrium behavior comes from the hydrolysis of GTP, which is required to destabilize the microtubule lattice. To understand the mechanism of this destabilization, cryo-electron microscopy was used to compare the structure of the ends of shrinking microtubules assembled in the presence of either GTP or the slowly hydrolyzable analogue guanylyl (α,β)methylenediphosphonate (GMPCPP). Depolymerization was induced by cold or addition of calcium. With either nucleotide, we have observed curled oligomers at the ends of shrinking microtubules. However, GDP oligomers were consistently more curved than GMPCPP oligomers. This difference in curvature between depolymerizing GDP and GMPCPP protofilaments suggests that GTP hydrolysis is accompanied by an increase in curvature of the protofilaments, thereby destabilizing the lateral interactions between tubulin subunits in the microtubule lattice.
The BCR-ABL oncoprotein potentially interacts with the xeroderma pigmentosum group B protein
Authors: Takeda Norifumi , Shibuya Masabumi , Maru Yoshiro
Published: July 2002

The previously uncharacterized CDC24 homology domain of BCR, which is missing in the P185 BCR-ABL oncogene of Philadelphia chromosome (Ph1)-positive acute lymphocytic leukemia but is retained in P210 BCR-ABL of chronic myelogeneous leukemia, was found to bind to the xeroderma pigmentosum group B protein (XPB). The binding appeared to be required for XPB to be tyrosine-phosphorylated by BCR-ABL. The interaction not only reduced both the ATPase and the helicase activities of XPB purified in the baculovirus system but also impaired XPB-mediated cross-complementation of the repair deficiency in rodent UV-sensitive mutants of group 3. The persistent dysfunction of XPB may in part underlie genomic instability in blastic crisis.
Calcium signaling induces acquisition of dendritic cell characteristics in chronic myelogenous leukemia myeloid progenitor cells
Authors: Engels Friederike H. C. , Koski Gary K. , Bedrosian Isabelle , Xu Shuwen , Luger Selina , Nowell Peter C. , Cohen Peter A. , Czerniecki Brian J.
Published: July 2002

Effective host T lymphocyte sensitization to malignant cells depends on successful antigen presentation. In this study, we examined the capacity of malignant myeloid progenitor cells of patients in the chronic phase of chronic myelogenous leukemia (CML) to acquire characteristics of activated dendritic cells (DCs) after intracellular calcium mobilization, thereby bypassing a need for third-party antigen-presenting cells. Treatment of purified CD33+CML cells from 15 patients with calcium ionophore (CI) consistently resulted inde novoexpression of the costimulatory molecules CD80 (B7.1) and CD86 (B7.2), CD40 and the DC-specific activation marker CD83, as well as marked up-regulation of MHC class I and II molecules and the adhesion molecule CD54. Most of these changes occurred within 24 hr of treatment. Morphologically, CI-treated CML cells developed long dendritic projections similar to those seen in mature DCs. Functionally, CI-treated CML cells provided stimulation of allogeneic T lymphocytes 10- to 20-fold that of untreated CML cells or untreated monocytes. Fluorescentin situhybridization of CI-activated CML cells confirmed their leukemic origin by displaying the typicalbcr/ablfusion signal. No difference inbcr/abltranslocation percentages between untreated and CI-treated CML nuclei was observed. These observations indicate that calcium mobilization may constitute a valuable approach for rapidly and reliably generating CML-derived DCs for immunotherapy of CML.
Extended random sequential adsorption model of irreversible deposition processes: From simulations to experiments
Authors: Lavalle P. , Schaaf P. , Ostafin M. , Voegel J.-C. , Senger B.
Published: July 2002

An experimental study of the irreversible deposition of colloidal particles of various radii
R
on a solid surface is presented over a wide range of the Péclet number,
Pe
, or reduced radius
R
* (
Pe
=
R
*
4
). The experimental data are analyzed by means of a new generalized random sequential adsorption model that takes explicitly the diffusion of the particles during the deposition into account. It allows description of the continuous transition from a random sequential adsorption-like to a ballistic-like deposition behavior. It depends on three parameters:
d
s
, related to the diffusion of the particles before adhesion;
n
s
, related to the number of allowed adhesion trials of a particle; and
R
e
, representing the effective particle radius. The model allows accounting for all of the experimental observations relative to the radial distribution functions and the number density fluctuations over the whole coverage range and all investigated values of
R
*. In addition, it is found that
d
s
/
R
is proportional to
R
*
−2
as expected for a diffusional process. Moreover, the parameters
d
s
and
n
s
appear to be connected through the empirical relation (
d
s
/
R
)
n
s
2/3
=
C
, where
C
is found to be of the order of 50. This unique statistical model allows an accurate description of the irreversible deposition process, whatever the influence of gravity with respect to diffusion.
Multinuclear solid-state three-dimensional MRI of bone and synthetic calcium phosphates
Authors: Wu Yaotang , Chesler David A. , Glimcher Melvin J. , Garrido Leoncio , Wang Jinxi , Jiang Hong J. , Ackerman Jerome L.
Published: July 2002

Multinuclear three-dimensional solid-state MRI of bone, tooth, and synthetic calcium phosphates is demonstratedin vitroandin vivowith a projection reconstruction technique based on acquisition of free induction decays in the presence of fixed amplitude magnetic field gradients. Phosphorus-31 solid-state MRI provides direct images of the calcium phosphate constituents of bone substance and is a quantitative measurement of the true volumetric bone mineral density of the bone. Proton solid-state MRI shows the density of bone matrix including its organic constituents, which consist principally of collagen. These solid-state MRI methods promise to yield a biological picture of bone richer in information concerning the bone composition and short range-crystalline order than the fluid-state images provided by conventional proton MRI or the density images produced by radiologic imaging techniques. Three-dimensional solid-state projection reconstruction MRI should be readily adaptable to both human clinical use and nonmedical applications for a variety of solids in materials science.
A model for studying megakaryocyte development and biology
Authors: Murphy George J. , Leavitt Andrew D.
Published: July 2002

The limited current understanding of megakaryocyte-lineage development and megakaryocyte biology is in large part because of a paucity of useful systems in which to conduct experiments. To overcome this problem, we have developed a transgenic mouse that uses the
GP-Ib
α regulatory sequences to achieve megakaryocyte-lineage restricted expression of an avian retroviral receptor. Through the transgenic avian receptor, avian retroviruses can efficiently and selectively infect megakaryocyte-lineage cells
in vitro
and
in vivo
. Serial infections can be performed to introduce and express multiple genes in the same cell. We have used this system to generate and characterize a pure population of primary CD41-positive megakaryocyte progenitors.
Millimeter-scale positioning of a nerve-growth-factor source and biological activity in the brain
Authors: Mahoney Melissa J. , Saltzman W. Mark
Published: July 2002

Toxicity prevents the systemic administration of many therapeutic proteins, and attempts at protein targeting via the circulatory system (i.e., “magic bullets”) have failed in all but a few special cases. Direct administration at the target site is a logical alternative, particularly in the central nervous system, but the limits of direct administration have not been defined clearly. Nerve growth factor (NGF) enhances survival of cholinergic neurons and, therefore, has generated considerable interest for the treatment of Alzheimer’s disease. We tested the effectiveness of local delivery by implanting small polymer pellets that slowly released NGF into the central nervous system of adult rats at controlled distances from a target site containing transplanted fetal cholinergic cells. NGF-releasing implants placed within 1–2 mm of the treatment site enhanced the biological function of cellular targets, whereas identical implants placed ≈3 mm from the target site of treatment produced no beneficial effect. Effective NGF therapy required millimeter-scale positioning of the NGF source, and efficacy correlated with the spatial distribution of NGF concentration in the tissue; this result suggests that NGF must be delivered within several millimeters of the target to be effective in treating Alzheimer’s disease. Because the human brain is divided into functional regions that are typically several centimeters in diameter and often irregular in shape, new methods for sculpting larger-scale drug fields are needed. We illustrate a concept, called pharmacotectonics, in which drug-delivery systems are arranged spatially in tissues to shape concentration fields for potent agents.
In vivo
selection of functional ribosomes with variations in the rRNA-binding site of
Escherichia coli
ribosomal protein S8: Evolutionary implications
Authors: Moine Hervé , Squires Catherine L. , Ehresmann Bernard , Ehresmann Chantal
Published: July 2002

The highly conserved nature of rRNA sequences throughout evolution allows these molecules to be used to build philogenic trees of different species. It is unknown whether the stability of specific interactions and structural features of rRNA reflects an optimal adaptation to a functional task or an evolutionary trap. In the work reported here, we have applied an
in vivo
selection strategy to demonstrate that unnatural sequences do work as a functional replacement of the highly conserved binding site of ribosomal protein S8. However, growth competition experiments performed between
Escherichia coli
isolates containing natural and unnatural S8-binding sites showed that the fate of each isolate depended on the growth condition. In exponentially growing cells, one unnatural variant was found to be equivalent to wild type in competition experiments performed in rich media. In culture conditions leading to slow growth, however, cells containing the wild-type sequence were the ultimate winner of the competition, emphasizing that the wild-type sequence is, in fact, the most fit solution for the S8-binding site.
Landscape approaches for determining the ensemble of folding transition states: Success and failure hinge on the degree of frustration
Authors: Nymeyer Hugh , Socci Nicholas D. , Onuchic José Nelson
Published: July 2002

We present a method for determining structural properties of the ensemble of folding transition states from protein simulations. This method relies on thermodynamic quantities (free energies as a function of global reaction coordinates, such as the percentage of native contacts) and not on “kinetic” measurements (rates, transmission coefficients, complete trajectories); consequently, it requires fewer computational resources compared with other approaches, making it more suited to large and complex models. We explain the theoretical framework that underlies this method and use it to clarify the connection between the experimentally determined Φ value, a quantity determined by the ratio of rate and stability changes due to point mutations, and the average structure of the transition state ensemble. To determine the accuracy of this thermodynamic approach, we apply it to minimalist protein models and compare these results with the ones obtained by using the standard experimental procedure for determining Φ values. We show that the accuracy of both methods depends sensitively on the amount of frustration. In particular, the results are similar when applied to models with minimal amounts of frustration, characteristic of rapid-folding, single-domain globular proteins.
Anti-*that- trace Effects in Norwegian and Optimality Theory
Authors: Keer Edward
Published: July 2002

This article argues that the difference between English and Norwegian with respect to the presence of a complementizer in embedded subject questions is attributable to a larger difference between the two languages, namely that Norwegian is a verb second language while English is not. Verb second forces subject wh- phrases to move to the specifier of a higher projection in Norwegian. The movement creates the need for a complementizer. In English, there is no such pressure for subject wh- phrases to move. Therefore there is no need for a complementizer. The so called anti-*that- trace effects in Norwegian and their lack in English supports the use of ranked and violable constraints in an Optimality-Theoretic system.
Cytoplasmic Regulation of the Movement of E-Cadherin on the Free Cell Surface as Studied by Optical Tweezers and Single Particle Tracking: Corralling and Tethering by the Membrane Skeleton
Authors: Sako Yasushi , Nagafuchi Akira , Tsukita Shoichiro , Takeichi Masatoshi , Kusumi Akihiro
Published: July 2002

The translational movement of E-cadherin, a calcium-dependent cell–cell adhesion molecule in the plasma membrane in epithelial cells, and the mechanism of its regulation were studied using single particle tracking (SPT) and optical tweezers (OT). The wild type (Wild) and three types of artificial cytoplasmic mutants of E-cadherin were expressed in L-cells, and their movements were compared. Two mutants were E-cadherins that had deletions in the COOH terminus and lost the catenin-binding site(s) in the COOH terminus, with remaining 116 and 21 amino acids in the cytoplasmic domain (versus 152 amino acids for Wild); these are called Catenin-minus and Short-tailed in this paper, respectively. The third mutant, called Fusion, is a fusion protein between E-cadherin without the catenin-binding site and α-catenin without its NH2-terminal half. These cadherins were labeled with 40-nm φ colloidal gold or 210-nm φ latex particles via a monoclonal antibody to the extracellular domain of E-cadherin for SPT or OT experiments, respectively. E-cadherin on the dorsal cell surface (outside the cell–cell contact region) was investigated. Catenin-minus and Short-tailed could be dragged an average of 1.1 and 1.8 μm by OT (trapping force of 0.8 pN), and exhibited average microscopic diffusion coefficients (Dmicro) of 1.2 × 10−10 and 2.1 × 10−10 cm2/s, respectively. Approximately 40% of Wild, Catenin-minus, and Short-tailed exhibited confined-type diffusion. The confinement area was 0.13 μm2 for Wild and Catenin-minus, while that for Short-tailed was greater by a factor of four. In contrast, Fusion could be dragged an average of only 140 nm by OT. Average Dmicro for Fusion measured by SPT was small (0.2 × 10−10 cm2/s). These results suggest that Fusion was bound to the cytoskeleton. Wild consists of two populations; about half behaves like Catenin- minus, and the other half behaves like Fusion. It is concluded that the movements of the wild-type E-cadherin in the plasma membrane are regulated via the cytoplasmic domain by (a) tethering to actin filaments through catenin(s) (like Fusion) and (b) a corralling effect of the network of the membrane skeleton (like Catenin-minus). The effective spring constants of the membrane skeleton that contribute to the tethering and corralling effects as measured by the dragging experiments were 30 and 5 pN/μm, respectively, indicating a difference in the skeletal structures that produce these two effects.
Three-dimensional Structure of Acanthamoeba castellanii Myosin-IB (MIB) Determined by Cryoelectron Microscopy of Decorated Actin Filaments
Authors: Jontes James D. , Ostap E. Michael , Pollard Thomas D. , Milligan Ronald A.
Published: July 2002

The Acanthamoeba castellanii myosin-Is were the first unconventional myosins to be discovered, and the myosin-I class has since been found to be one of the more diverse and abundant classes of the myosin superfamily. We used two-dimensional (2D) crystallization on phospholipid monolayers and negative stain electron microscopy to calculate a projection map of a “classical” myosin-I, Acanthamoeba myosin-IB (MIB), at ∼18 Å resolution. Interpretation of the projection map suggests that the MIB molecules sit upright on the membrane. We also used cryoelectron microscopy and helical image analysis to determine the three-dimensional structure of actin filaments decorated with unphosphorylated (inactive) MIB. The catalytic domain is similar to that of other myosins, whereas the large carboxy-terminal tail domain differs greatly from brush border myosin-I (BBM-I), another member of the myosin-I class. These differences may be relevant to the distinct cellular functions of these two types of myosin-I. The catalytic domain of MIB also attaches to F-actin at a significantly different angle, ∼10°, than BBM-I. Finally, there is evidence that the tails of adjacent MIB molecules interact in both the 2D crystal and in the decorated actin filaments.
Analysis of C-cadherin Regulation during Tissue Morphogenesis with an Activating Antibody
Authors: Zhong Yun , Brieher William M. , Gumbiner Barry M.
Published: July 2002

The regulation of cadherin-mediated adhesion at the cell surface underlies several morphogenetic processes. To investigate the role of cadherin regulation in morphogenesis and to begin to analyze the molecular mechanisms of cadherin regulation, we have screened for monoclonal antibodies (mAbs) that allow us to manipulate the adhesive state of the cadherin molecule.

Xenopus C-cadherin is regulated during convergent extension movements of gastrulation. Treatment of animal pole tissue explants (animal caps) with the mesoderm-inducing factor activin induces tissue elongation and decreases the strength of C-cadherin–mediated adhesion between blastomeres (Brieher, W.M., and B.M. Gumbiner. 1994. J. Cell Biol. 126:519–527). We have generated a mAb to C-cadherin, AA5, that restores strong adhesion to activin-treated blastomeres. This C-cadherin activating antibody strongly inhibits the elongation of animal caps in response to activin without affecting mesodermal gene expression. Thus, the activin-induced decrease in C-cadherin adhesive activity appears to be required for animal cap elongation.

Regulation of C-cadherin and its activation by mAb AA5 involve changes in the state of C-cadherin that encompass more than changes in its homophilic binding site. Although mAb AA5 elicited a small enhancement in the functional activity of the soluble C-cadherin ectodomain (CEC1-5), it was not able to restore cell adhesion activity to mutant C-cadherin lacking its cytoplasmic tail. Furthermore, activin treatment regulates the adhesion of Xenopus blastomeres to surfaces coated with two other anti–C-cadherin mAbs, even though these antibodies probably do not mediate adhesion through a normal homophilic binding mechanism. Moreover, mAb AA5 restores strong adhesion to these antibodies. mAb AA5 only activates adhesion of blastomeres to immobilized CEC1-5 when it binds to C-cadherin on the cell surface. It does not work when added to CEC1-5 on the substrate. Together these findings suggest that the regulation of C-cadherin by activin and its activation by mAb AA5 involve changes in its cellular organization or interactions with other cell components that are not intrinsic to the isolated protein.
Intracellular Movement of Green Fluorescent Protein–Tagged Phosphatidylinositol 3-Kinase in Response to Growth Factor Receptor Signaling
Authors: Gillham Helen , Golding Matthew C.H.M. , Pepperkok Rainer , Gullick William J.
Published: July 2002

Phosphatidylinositol 3-kinase (PI 3-kinase) is a lipid kinase which has been implicated in mitogenesis, protein trafficking, inhibition of apoptosis, and integrin and actin functions. Here we show using a green fluorescent protein–tagged p85 subunit that phosphatidylinositol 3-kinase is distributed throughout the cytoplasm and is localized to focal adhesion complexes in resting NIH-3T3, A431, and MCF-7 cells. Ligand stimulation of an epidermal growth factor receptor/c-erbB-3 chimera expressed in these cells results in a redistribution of p85 to the cell membrane which is independent of the catalytic activity of the enzyme and the integrity of the actin cytoskeleton. The movement is, however, dependent on the phosphorylation status of the erbB-3 chimera. Using rhodamine-labeled epidermal growth factor we show that the phosphatidylinositol 3-kinase and the receptors colocalize in discrete patches on the cell surface. Low concentrations of ligand cause patching only at the periphery of the cells, whereas at high concentrations patches were seen over the whole cell surface. Using green fluorescent protein–tagged fragments of p85 we show that binding to the receptor requires the NH2-terminal part of the protein as well as its SH2 domains.
Influence of the NH2-terminal Amino Acid of the T Cell Receptor α Chain on Major Histocompatibility Complex (MHC) Class II + Peptide Recognition
Authors: Seibel Jeffrey L. , Wilson Nancy , Kozono Haruo , Marrack Philippa , Kappler John W.
Published: July 2002

The α/β T cell receptor (TCR) recognizes peptide fragments bound in the groove of major histocompatibility complex (MHC) molecules. We modified the TCR α chain from a mouse T cell hybridoma and tested its ability to reconstitute TCR expression and function in an α chain–deficient variant of the hybridoma. The modified α chain differed from wild type only in its leader peptide and mature NH2-terminal amino acid. Reconstituted cell surface TCR complexes reacted normally with anti-TCR and anti-CD3 antibodies. Although cross-linking of this TCR with an antibody to the TCR idiotype elicited vigorous T cell hybridoma activation, stimulation with its natural MHC + peptide ligand did not. We demonstrated that this phenotype could be reproduced simply by substituting the glutamic acid (E) at the mature NH2 terminus of the wild type TCR α chain with aspartic acid (D). The substitution also dramatically reduced the affinity of soluble α/β-TCR heterodimers for soluble MHC + peptide molecules in a cell-free system, suggesting that it did not exert its effect simply by disrupting TCR interactions with accessory molecules on the hybridoma. These results demonstrate for the first time that amino acids which are not in the canonical TCR complementarity determining regions can be critical in determining how the TCR engages MHC + peptide.
Hypermutation of Immunoglobulin Genes in Memory B Cells of DNA Repair–deficient Mice
Authors: Jacobs Heinz , Fukita Yosho , van der Horst Gijsbertus T.J. , de Boer Jan , Weeda Geert , Essers Jeroen , de Wind Niels , Engelward Bevin P. , Samson Leona , Verbeek Sjef , de Murcia Josiane Ménissier , de Murcia Gilbert , e Riele Hein t , Rajewsky Klaus
Published: July 2002

To investigate the possible involvement of DNA repair in the process of somatic hypermutation of rearranged immunoglobulin variable (V) region genes, we have analyzed the occurrence, frequency, distribution, and pattern of mutations in rearranged Vλ1 light chain genes from naive and memory B cells in DNA repair–deficient mutant mouse strains. Hypermutation was found unaffected in mice carrying mutations in either of the following DNA repair genes: xeroderma pigmentosum complementation group (XP)A and XPD, Cockayne syndrome complementation group B (CSB), mutS homologue 2 (MSH2), radiation sensitivity 54 (RAD54), poly (ADP-ribose) polymerase (PARP), and 3-alkyladenine DNA-glycosylase (AAG). These results indicate that both subpathways of nucleotide excision repair, global genome repair, and transcription-coupled repair are not required for somatic hypermutation. This appears also to be true for mismatch repair, RAD54-dependent double-strand–break repair, and AAG-mediated base excision repair.
Binding and Antigen Presentation of Ceramide-Containing Glycolipids by Soluble Mouse and Human Cd1d Molecules
Authors: Naidenko Olga V. , Maher Juli K. , Ernst William A. , Sakai Teruyuki , Modlin Robert L. , Kronenberg Mitchell
Published: July 2002

We have purified soluble mouse and human CD1d molecules to assess the structural requirements for lipid antigen presentation by CD1. Plate-bound CD1d molecules from either species can present the glycolipid α-galactosyl ceramide (α-GalCer) to mouse natural killer T cells, formally demonstrating both the in vitro formation of antigenic complexes, and the presentation of α-GalCer by these two CD1d molecules. Using surface plasmon resonance, we show that at neutral pH, mouse CD1 and human CD1d bind to immobilized α-GalCer, unlike human CD1b, which requires acidic pH for lipid antigen binding. The CD1d molecules can also bind both to the nonantigenic β-GalCer and to phosphatidylethanolamine, indicating that diverse lipids can bind to CD1d. These studies provide the first quantitative analysis of monomeric lipid antigen–CD1 interactions, and they demonstrate that the orientation of the galactose, or even the nature of the polar head group, are likely to be more important for T cell receptor contact than CD1d binding.
Anti–Interleukin 10 Receptor Monoclonal Antibody Is an Adjuvant for T Helper Cell Type 1 Responses to Soluble Antigen Only in the Presence of Lipopolysaccharide
Authors: Castro Antonio G. , Neighbors Margaret , Hurst Stephen D. , Zonin Francesca , Silva Regina A. , Murphy Erin , Liu Yong-Jun , O'Garra Anne
Published: July 2002

Soluble foreign antigen usually leads to a transient clonal expansion of antigen-specific T cells followed by the deletion and/or functional inactivation of the cells. As interleukin (IL)-10 is a key immunoregulatory cytokine, we questioned whether neutralization of IL-10 during priming with soluble antigen could prime for a subsequent T helper cell type 1 (Th1) effector recall response. By using an adoptive transfer model to track the fate of antigen-specific T cell receptor (TCR)-transgenic CD4+ T cells, we show that administration of soluble ovalbumin (OVA) protein, but not OVA323–339 peptide antigen, together with an anti–IL-10 receptor (R) mAb led to the enhancement of a Th1 response upon rechallenge. Lipopolysaccharide (LPS) present in the protein was necessary for priming for Th1 recall responses in the presence of anti–IL-10R mAb, as removal of LPS abrogated this effect. Moreover, addition of LPS to the peptide did not itself allow priming for recall Th1 effector responses unless endogenous levels of IL-10 were neutralized with an anti–IL-10R mAb. A significant increase in OVA-specific IgG1 and IgG2a isotypes was observed when the protein antigen was administered with anti–IL-10R mAb; however, this was not the case with peptide antigen administered together with anti–IL-10R and LPS. Our data, showing that LPS receptor signaling and neutralization of endogenous immunosuppressive cytokines is essential for Th1 priming, has important implications for the design of relevant vaccines for effective in vivo immunotherapy.
Functional Consequences of a Decreased Potassium Affinity in a Potassium Channel Pore
Authors: Ogielska Eva M. , Aldrich Richard W.
Published: July 2002

Ions bound near the external mouth of the potassium channel pore impede the C-type inactivation conformational change (Lopez-Barneo, J., T. Hoshi, S. Heinemann, and R. Aldrich. 1993. Receptors Channels. 1:61– 71; Baukrowitz, T., and G. Yellen. 1995. Neuron. 15:951–960). In this study, we present evidence that the occupancy of the C-type inactivation modulatory site by permeant ions is not solely dependent on its intrinsic affinity, but is also a function of the relative affinities of the neighboring sites in the potassium channel pore. The A463C mutation in the S6 region of Shaker decreases the affinity of an internal ion binding site in the pore (Ogielska, E.M., and R.W. Aldrich, 1998). However, we have found that this mutation also decreases the C-type inactivation rate of the channel. Our studies indicate that the C-type inactivation effects observed with substitutions at position A463 most likely result from changes in the pore occupancy of the channel, rather than a change in the C-type inactivation conformational change. We have found that a decrease in the potassium affinity of the internal ion binding site in the pore results in lowered (electrostatic) interactions among ions in the pore and as a result prolongs the time an ion remains bound at the external C-type inactivation site. We also present evidence that the C-type inactivation constriction is quite local and does not involve a general collapse of the selectivity filter. Our data indicate that in A463C potassium can bind within the selectivity filter without interfering with the process of C-type inactivation.
Cystic Fibrosis Transmembrane Conductance Regulator
Authors: Smith Stephen S. , Steinle Erich D. , Meyerhoff Mark E. , Dawson David C.
Published: July 2002

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl channel exhibits lyotropic anion selectivity. Anions that are more readily dehydrated than Cl exhibit permeability ratios (PS/PCl) greater than unity and also bind more tightly in the channel. We compared the selectivity of CFTR to that of a synthetic anion-selective membrane [poly(vinyl chloride)–tridodecylmethylammonium chloride; PVC-TDMAC] for which the nature of the physical process that governs the anion-selective response is more readily apparent. The permeability and binding selectivity patterns of CFTR differed only by a multiplicative constant from that of the PVC-TDMAC membrane; and a continuum electrostatic model suggested that both patterns could be understood in terms of the differences in the relative stabilization of anions by water and the polarizable interior of the channel or synthetic membrane. The calculated energies of anion–channel interaction, derived from measurements of either permeability or binding, varied as a linear function of inverse ionic radius (1/r), as expected from a Born-type model of ion charging in a medium characterized by an effective dielectric constant of 19. The model predicts that large anions, like SCN, although they experience weaker interactions (relative to Cl) with water and also with the channel, are more permeant than Cl because anion–water energy is a steeper function of 1/r than is the anion–channel energy. These large anions also bind more tightly for the same reason: the reduced energy of hydration allows the net transfer energy (the well depth) to be more negative. This simple selectivity mechanism that governs permeability and binding acts to optimize the function of CFTR as a Cl filter. Anions that are smaller (more difficult to dehydrate) than Cl are energetically retarded from entering the channel, while the larger (more readily dehydrated) anions are retarded in their passage by “sticking” within the channel.
The BioCatalog.
Authors: Rodriguez-Tom√© P
Published: July 2002

MOTIVATION: The BioCatalog is a database of information on software of interest in molecular biology and genetics. The programs are grouped by domain of interest. AVAILABILITY: The BioCatalog is freely distributed as ASCII files. It can be searched through its Web interface at http://www.ebi.ac.uk/biocat. CONTACT: biocat@ebi. ac.uk
The bio-objects project. Part I: the object data model core elements.
Authors: Maltchenko S Z
Published: July 2002

MOTIVATION: Scientific data reside in a number of different data sources, both public and commercial, and are accessible through various software applications. Data and software interoperability is becoming a critical issue in the research community. In our opinion, the need to develop a unified object model for biotechnology must be recognized. Herein, we present a prototype and core elements for the Object Data Model, a central part of our future commercial product development. RESULTS: We have designed a first layer of the Object Data Model that will be used for further commercial application development. The CBioBase object was designed as a citable, stable and core data element for the Object Data Model. General relationships between objects and application components were established. AVAILABILITY: Software tools will be provided with a future DNASIS product to access various elements of the Object Model. We are planning to assemble C++ libraries and Java binary files into BioObjects SDK (software development kit) for those interested in software implementation using the described object model. CONTACT: smaltch@msn.com or smaltch@hitachi-soft.com
VIRTLAB: a virtual molecular biology laboratory.
Authors: Iazzetti G , Santini G , Rau M , Bucci E , Calogero R A
Published: July 2002

SUMMARY: VIRTLAB is a self-training program based on PBL (Problem-Based-Learning Pathway) built to simulate a molecular biology laboratory. It has been designed to stimulate students in the biological sciences to analyse and solve molecular biology problems using standard laboratory techniques (e.g. restriction enzyme digestions, analytical and preparative agarose gels, DNA cloning and sequencing, etc.) and can thus be viewed as a teaching aid. AVAILABILITY: The VIRTLAB package is distributed free of charge to non-profit organisations by the authors (virtlab@biol.dgbm. unina.it). On-line help and tutorials, available now in English, French, Italian, and shortly in German, are provided with the software or at http://biol.dgbm.unina.it:8080/virtlab.html++ +
The domain-server: direct prediction of protein domain-homologies from BLAST search.
Authors: Murvai J , Vlahovicek K , Barta E , Parthasarathy S , Hegyi H , Pfeiffer F , Pongor S
Published: July 2002

RESULTS: A WWW server for protein domain homology prediction, based on BLAST search and a simple data-mining algorithm (Hegyi,H. and Pongor,S. (1993) Comput. Appl. Biosci., 9, 371-372), was constructed providing a tabulated list and a graphic plot of similarities. AVAILABILITY: http://www.icgeb.trieste.it/domain. Mirror site is available at http://sbase.abc.hu/domain. A standalone programme will be available on request. SUPPLEMENTARY INFORMATION: A series of help files is available at the above addresses.
Wavelet change-point prediction of transmembrane proteins
Authors: Lio Pietro , Vannucci Marina
Published: July 2002

Motivation: A non-parametric method, based on a wavelet data-dependent threshold technique for change-point analysis, is applied to predict location and topology of helices in transmembrane proteins. A new propensity scale generated from a transmembrane helix database is proposed.

Results: We show that wavelet change-point performs well for smoothing hydropathy and transmembrane profiles generated using different scales. We investigate which wavelet bases and threshold functions are overall most appropriate to detect transmembrane segments. Prediction accuracy is based on the analysis of two data sets used as standard benchmarks for transmembrane prediction algorithms. The analysis of a test set of 83 proteins results in accuracy per segment equal to 98.2%; the analysis of a 48 proteins blind-test set, i.e. containing proteins not used to generate the propensity scales, results in accuracy per segment equal to 97.4%. We believe that this method can also be applied to the detection of boundaries of other patterns such as G + Cisochores and dot-plots.

Availability: The transmembrane database, TMALN and source code are available upon request from the authors.

Contact: plio@hgmp.mrc.ac.uk
Polylink: to support two-point linkage analysis in
autotetraploids
Authors: He Yang , Xu Xiangming , Tobutt Kenneth R. , Ridout Martin S.
Published: July 2002

Summary: Polylink runs under Microsoft Windows (95 or later). It performs various calculations that are useful for investigating two-point linkage analysis for autopolyploids, based on the random chromosome pairing model. These include calculation of offspring phenotypic probabilities as functions of the recombination fraction, \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(r\) \end{document}calculation of theoretical standard errors for the maximum likelihood estimator of \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(r\) \end{document}and numerical computation of maximum likelihood estimates. It also includes simulation facilities.

Availability: Polylink is free and available from Xiangming Xu via email

Contact: xiangming.xu@hri.ac.uk

* To whom correspondence should be addressed.
Role of the single cysteine residue, Cys 3, of human and bovine cystatin B (stefin B) in the inhibition of cysteine proteinases
Authors: Pol Ewa , Björk Ingemar
Published: July 2002

Cystatin B is unique among cysteine proteinase inhibitors of the cystatin superfamily in having a free Cys in the N‐terminal segment of the proteinase binding region. The importance of this residue for inhibition of target proteinases was assessed by studies of the affinity and kinetics of interaction of human and bovine wild‐type cystatin B and the Cys 3‐to‐Ser mutants of the inhibitors with papain and cathepsins L, H, and B. The wild‐type forms from the two species had about the same affinity for each proteinase, binding tightly to papain and cathepsin L and more weakly to cathepsins H and B. In general, these affinities were appreciably higher than those reported earlier, perhaps because of irreversible oxidation of Cys 3 in previous work. The Cys‐to‐Ser mutation resulted in weaker binding of cystatin B to all four proteinases examined, the effect varying with both the proteinase and the species variant of the inhibitor. The affinities of the human inhibitor for papain and cathepsin H were decreased by threefold to fourfold and that for cathepsin B by ∼20‐fold, whereas the reductions in the affinities of the bovine inhibitor for papain and cathepsins H and B were ∼14‐fold, ∼10‐fold and ∼300‐fold, respectively. The decreases in affinity for cathepsin L could not be properly quantified but were greater than threefold. Increased dissociation rate constants were responsible for the weaker binding of both mutants to papain. By contrast, the reduced affinities for cathepsins H and B were due to decreased association rate constants. Cys 3 of both human and bovine cystatin B is thus of appreciable importance for inhibition of cysteine proteinases, in particular cathepsin B.
NMR of hydrogen bonding in cold‐shock protein A and an analysis of the influence of crystallographic resolution on comparisons of hydrogen bond lengths
Authors: Alexandrescu Andrei T. , Snyder Doug R. , Abildgaard Frits
Published: July 2002

Hydrogen bonding in cold‐shock protein A of Escherichia coli has been investigated using long‐range HNCO spectroscopy. Nearly half of the amide protons involved in hydrogen bonds in solution show no measurable protection from exchange in water, cautioning against a direct correspondence between hydrogen bonding and hydrogen exchange protection. The N to O atom distance across a hydrogen bond, RNO, is related to the size of the 3hJNC' trans hydrogen bond coupling constant and the amide proton chemical shift. Both NMR parameters show poorer agreement with the 2.0‐Å resolution X‐ray structure of the cold‐shock protein studied by NMR than with a 1.2‐Å resolution X‐ray structure of a homologous cold‐shock protein from the thermophile B. caldolyticus. The influence of crystallographic resolution on comparisons of hydrogen bond lengths was further investigated using a database of 33 X‐ray structures of ribonuclease A. For highly similar structures, both hydrogen bond RNO distance and Cα coordinate root mean square deviations (RMSD) show systematic increases as the resolution of the X‐ray structure used for comparison decreases. As structures diverge, the effects of coordinate errors on RNO distance and Cα coordinate root mean square deviations become progressively smaller. The results of this study are discussed with regard to the influence of data precision on establishing structure similarity relationships between proteins.
Additive noise properties of active matrix flat‐panel imagers
Authors: Maolinbay M. , El‐Mohri Y. , Antonuk L. E. , Jee K.‐W. , Nassif S. , Rong X. , Zhao Q.
Published: July 2002

A detailed theoretical and empirical investigation of additive noise for indirect detection, active matrix flat‐panel imagers (AMFPIs) has been performed. Such imagers comprise a pixelated array, incorporating photodiodes and thin‐film transistors (TFTs), and an associated electronic acquisition system. A theoretical model of additive noise, defined as the noise of an imaging system in the absence of radiation, has been developed. This model is based upon an equivalent‐noise‐circuit representation of an AMFPI. The model contains a number of uncorrelated noise components which have been designated as pixel noise, data line thermal noise, externally coupled noise, preamplifier noise and digitization noise. Pixel noise is further divided into the following components: TFT thermal noise, shot and noise associated with the TFT and photodiode leakage currents, and TFT transient noise. Measurements of various additive noise components were carried out on a prototype imaging system based on a 508 μm pitch, array. Other measurements were performed in the absence of the array, involving discrete components connected to the preamplifier input. Overall, model predictions of total additive noise as well as of pixel, preamplifier, and data line thermal noise components were in agreement with results of their measured counterparts. For the imaging system examined, the model predicts that pixel noise is dominated by shot and noise components of the photodiode and TFT at frame times above ∼1 s. As frame time decreases, pixel noise is increasingly dominated by TFT thermal noise. Under these conditions, the reasonable degree of agreement observed between measurements and model predictions provides strong evidence that the role of TFT thermal noise has been properly incorporated into the model. Finally, the role of the resistance and capacitance of array data lines in the model was investigated using discrete component circuits at the preamplifier input. Measurements of preamplifier noise and data line thermal noise components as a function of input capacitance and resistance were found to be in reasonable agreement with model predictions.
A Monte Carlo calculation of dosimetric parameters of and SS sources for intravascular brachytherapy
Authors: Wang Ruqing , Li X. Allen
Published: July 2002

The AAPM TG‐60 report has proposed various dose calculation parameters for intravascular brachytherapy (IVBT). These parameters include the dose rate constant (or the dose rate at a reference position for a beta‐particle emitting source), the radial dose function, and the anisotropy function. In this work, we have used a modified EGS4 Monte Carlo system to calculate these parameters for the two most commonly used IVBT sources (the beta‐particle emitting source and the photon emitting SS source). To ensure the calculation accuracy, the present calculation was compared with several measurements and calculations reported by other authors. Excellent agreement was found for the results with the photon source. For beta‐particle source calculation, the present results for a variety of point sources agree very well with a previous work. The presently calculated radial dose functions for the source are consistent with those of a published work for intermediate radial distances. The dose uniformity in the axial direction was also studied. The contributions of bremsstrahlung photons to total doses for the beta source, and the influence of ignoring electron transport on calculated doses for the SS photon source are discussed.
Performance gain in computer‐assisted detection schemes by averaging scores generated from artificial neural networks with adaptive filtering
Authors: Zheng Bin , Chang Yuan‐Hsiang , Good Walter F. , Gur David
Published: July 2002

The authors investigated a new method to optimize artificial neural networks (ANNs) with adaptive filtering used in computer‐assisted detection schemes in digitized mammograms and to assess performance changes when averaging classification scores from three sets of optimized schemes. Two independent training and testing image databases involving 978 and 830 digitized mammograms, respectively, were used in this study. In the training data set, initial filtering and subtraction resulted in the identification of 592 mass regions and 3790 suspicious, but actually negative regions. These regions (including both true‐positive and negative regions) were segmented into three subsets three times based on the calculation of the values of three features as segmentation indices. The indices were “mass” size multiplied by their digital value contrast, conspicuity, and circularity. Nine ANN‐based classifiers were separately optimized using a genetic algorithm for each subset of regions. Each region was assigned three classification scores after applying the three adaptive ANNs. The performance gain of the CAD scheme after averaging the three scores for each suspicious region was tested using an independent data set and a ROC methodology. The experimental results showed that the areas under ROC curves for the testing database using three sets of optimized ANNs individually were and respectively. The between‐index correlations of three values were 0.013, −0.007, and 0.086. Similar to averaging diagnostic ratings from independent observers, by averaging three ANN‐generated scores for each testing region, the performance of the CAD scheme was significantly improved with value of
An oblique arc capable patient positioning system for sequential tomotherapy
Authors: Salter Bill J. , Hevezi James M. , Sadeghi Amir , Fuss Martin , Herman Terence S.
Published: July 2002

A new patient positioning system has been designed and manufactured, allowing for the accurate delivery of obliquely oriented intensity modulated treatment arcs via a commercially available IMRT system. The ability to deliver such obliquely oriented intensity modulated arcs allows the commercial system to more closely approach a 4π pencil beam delivery geometry which, in turn, allows for significant improvements in conformality for many tumor geometries. While the IMRT system delivered to this institution in the fall of 1996 was capable of planning for nonparallel plane delivery schemes, it proved incapable of delivering such treatments with acceptable accuracy. Because our early clinical experience revealed that certain patients could benefit significantly from such a delivery scheme we endeavored to design and manufacture an alternative treatment couch/patient positioning system (Xlator) which could overcome the limitations of the vendor supplied system. We present our initial evidence for the benefits of obliquely oriented intensity modulated treatment arcs, along with data demonstrating the inability of the original vendor supplied system to deliver such treatments with acceptable accuracy. The design of our new system is presented, as well as data demonstrating its ability to accurately deliver obliquely oriented intensity‐modulated arcs. A detailed comparison of the performance of the Xlator and the vendor‐supplied system is presented with regard to match line repeatability and hysteresis. Finally, the ability of the Xlator to deliver multiple couch angle sequential tomotherapy with spatial accuracy necessary to radiosurgical applications is demonstrated via a AAPM Report 54,TG‐42 hidden target test. Readers note: The Xlator patient positioning system designed and patented here has recently come to be commercially available, and is currently marketed by the vendor under the name Crane II.