bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Spontaneous Bilayer Curvature on Influenza Virus–mediated Fusion Pores
Authors: Razinkov Vladimir I. , Melikyan Grigory B. , Epand Richard M. , Epand Raquel F. , Cohen Fredric S.
Published: July 2002

Cells expressing the hemagglutinin protein of influenza virus were fused to planar bilayer membranes containing the fluorescent lipid probes octadecylrhodamine (R18) or indocarbocyanine (DiI) to investigate whether spontaneous curvature of each monolayer of a target membrane affects the growth of fusion pores. R18 and DiI lowered the transition temperatures for formation of an inverted hexagonal phase, indicating that these probes facilitate the formation of negative curvature structures. The probes are known to translocate from one monolayer of a bilayer membrane to the other in a voltage-dependent manner. The spontaneous curvature of the cis monolayer (facing the cells) or the trans monolayer could therefore be made more negative through control of the polarity of voltage across the planar membrane. Electrical admittance measurements showed that the open times of flickering fusion pores were shorter when probes were in trans monolayers and longer when in cis monolayers compared with times when probe was symmetrically distributed. Open times were the same for probe symmetrically distributed as when probes were not present. Thus, open times were a function of the asymmetry of the spontaneous curvature between the trans and cis monolayers. Enriching the cis monolayer with a negative curvature probe reduced the probability that a small pore would fully enlarge, whereas enriching the trans monolayer promoted enlargement. Lysophosphatidylcholine has positive spontaneous curvature and does not translocate. When lysophosphatidylcholine was placed in trans leaflets of planar membranes, closing of fusion pores was rare. The effects of the negative and positive spontaneous curvature probes do not support the hypothesis that a flickering pore closes from an open state within a hemifusion diaphragm (essentially a “flat” structure). Rather, such effects support the hypothesis that the membrane surrounding the open pore forms a three-dimensional hourglass shape from which the pore flickers shut.
Single-Channel Characteristics of Wild-Type IKs Channels and Channels formed with Two MinK Mutants that Cause Long QT Syndrome
Authors: Sesti Federico , Goldstein Steve A.N.
Published: July 2002

IKs channels are voltage dependent and K+ selective. They influence cardiac action potential duration through their contribution to myocyte repolarization. Assembled from minK and KvLQT1 subunits, IKs channels are notable for a heteromeric ion conduction pathway in which both subunit types contribute to pore formation. This study was undertaken to assess the effects of minK on pore function. We first characterized the properties of wild-type human IKs channels and channels formed only of KvLQT1 subunits. Channels were expressed in Xenopus laevis oocytes or Chinese hamster ovary cells and currents recorded in excised membrane patches or whole-cell mode. Unitary conductance estimates were dependent on bandwidth due to rapid channel “flicker.” At 25 kHz in symmetrical 100-mM KCl, the single-channel conductance of IKs channels was ∼16 pS (corresponding to ∼0.8 pA at 50 mV) as judged by noise-variance analysis; this was fourfold greater than the estimated conductance of homomeric KvLQT1 channels. Mutant IKs channels formed with D76N and S74L minK subunits are associated with long QT syndrome. When compared with wild type, mutant channels showed lower unitary currents and diminished open probabilities with only minor changes in ion permeabilities. Apparently, the mutations altered single-channel currents at a site in the pore distinct from the ion selectivity apparatus. Patients carrying these mutant minK genes are expected to manifest decreased K+ flux through IKs channels due to lowered single-channel conductance and altered gating.
Voltage-dependent Gating of Single Wild-Type and S4 Mutant KAT1 Inward Rectifier Potassium Channels
Authors: Zei Paul C. , Aldrich Richard W.
Published: July 2002

The voltage-dependent gating mechanism of KAT1 inward rectifier potassium channels was studied using single channel current recordings from Xenopus oocytes injected with KAT1 mRNA. The inward rectification properties of KAT1 result from an intrinsic gating mechanism in the KAT1 channel protein, not from pore block by an extrinsic cation species. KAT1 channels activate with hyperpolarizing potentials from −110 through −190 mV with a slow voltage-dependent time course. Transitions before first opening are voltage dependent and account for much of the voltage dependence of activation, while transitions after first opening are only slightly voltage dependent. Using burst analysis, transitions near the open state were analyzed in detail. A kinetic model with multiple closed states before first opening, a single open state, a single closed state after first opening, and a closed-state inactivation pathway accurately describes the single channel and macroscopic data. Two mutations neutralizing charged residues in the S4 region (R177Q and R176L) were introduced, and their effects on single channel gating properties were examined. Both mutations resulted in depolarizing shifts in the steady state conductance–voltage relationship, shortened first latencies to opening, decreased probability of terminating bursts, and increased burst durations. These effects on gating were well described by changes in the rate constants in the kinetic model describing KAT1 channel gating. All transitions before the open state were affected by the mutations, while the transitions after the open state were unaffected, implying that the S4 region contributes to the early steps in gating for KAT1 channels.
Extracellular Zinc Ion Inhibits ClC-0 Chloride Channels by Facilitating Slow Gating
Authors: Chen Tsung-Yu
Published: July 2002

Extracellular Zn2+ was found to reversibly inhibit the ClC-0 Cl− channel. The apparent on and off rates of the inhibition were highly temperature sensitive, suggesting an effect of Zn2+ on the slow gating (or inactivation) of ClC-0. In the absence of Zn2+, the rate of the slow-gating relaxation increased with temperature, with a Q10 of ∼37. Extracellular Zn2+ facilitated the slow-gating process at all temperatures, but the Q10 did not change. Further analysis of the rate constants of the slow-gating process indicates that the effect of Zn2+ is mostly on the forward rate (the rate of inactivation) rather than the backward rate (the rate of recovery from inactivation) of the slow gating. When ClC-0 is bound with Zn2+, the equilibrium constant of the slow-gating process is increased by ∼30-fold, reflecting a 30-fold higher Zn2+ affinity in the inactivated channel than in the open-state channel. As examined through a wide range of membrane potentials, Zn2+ inhibits the opening of the slow gate with equal potency at all voltages, suggesting that a two-state model is inadequate to describe the slow-gating transition. Following a model originally proposed by Pusch and co-workers (Pusch, M., U. Ludewig, and T.J. Jentsch. 1997. J. Gen. Physiol. 109:105–116), the effect of Zn2+ on the activation curve of the slow gate can be well described by adding two constraints: (a) the dissociation constant for Zn2+ binding to the open channel is 30 μM, and (b) the difference in entropy between the open state and the transition state of the slow-gating process is increased by 27 J/ mol/°K for the Zn2+-bound channel. These results together indicate that extracellular Zn2+ inhibits ClC-0 by facilitating the slow-gating process.
The Position of the Fast-Inactivation Gate during Lidocaine Block of Voltage-gated Na+ Channels
Authors: Vedantham Vasanth , Cannon Stephen C.
Published: July 2002

Lidocaine produces voltage- and use-dependent inhibition of voltage-gated Na+ channels through preferential binding to channel conformations that are normally populated at depolarized potentials and by slowing the rate of Na+ channel repriming after depolarizations. It has been proposed that the fast-inactivation mechanism plays a crucial role in these processes. However, the precise role of fast inactivation in lidocaine action has been difficult to probe because gating of drug-bound channels does not involve changes in ionic current. For that reason, we employed a conformational marker for the fast-inactivation gate, the reactivity of a cysteine substituted at phenylalanine 1304 in the rat adult skeletal muscle sodium channel α subunit (rSkM1) with [2-(trimethylammonium)ethyl]methanethiosulfonate (MTS-ET), to determine the position of the fast-inactivation gate during lidocaine block. We found that lidocaine does not compete with fast-inactivation. Rather, it favors closure of the fast-inactivation gate in a voltage-dependent manner, causing a hyperpolarizing shift in the voltage dependence of site 1304 accessibility that parallels a shift in the steady state availability curve measured for ionic currents. More significantly, we found that the lidocaine-induced slowing of sodium channel repriming does not result from a slowing of recovery of the fast-inactivation gate, and thus that use-dependent block does not involve an accumulation of fast-inactivated channels. Based on these data, we propose a model in which transitions along the activation pathway, rather than transitions to inactivated states, play a crucial role in the mechanism of lidocaine action.
Occupancy of the Chromophore Binding Site of Opsin Activates Visual Transduction in Rod Photoreceptors
Authors: Kefalov Vladimir J. , Carter Cornwall M. , Crouch Rosalie K.
Published: July 2002

The retinal analogue β-ionone was used to investigate possible physiological effects of the noncovalent interaction between rod opsin and its chromophore 11-cis retinal. Isolated salamander rod photoreceptors were exposed to bright light that bleached a significant fraction of their pigment, were allowed to recover to a steady state, and then were exposed to β-ionone. Our experiments show that in bleach-adapted rods β-ionone causes a decrease in light sensitivity and dark current and an acceleration of the dim flash photoresponse and the rate constants of guanylyl cyclase and cGMP phosphodiesterase. Together, these observations indicate that in bleach-adapted rods β-ionone activates phototransduction in the dark. Control experiments showed no effect of β-ionone in either fully dark-adapted or background light-adapted cells, indicating direct interaction of β-ionone with the free opsin produced by bleaching. We speculate that β-ionone binds specifically in the chromophore pocket of opsin to produce a complex that is more catalytically potent than free opsin alone. We hypothesize that a similar reaction may occur in the intact retina during pigment regeneration. We propose a model of rod pigment regeneration in which binding of 11-cis retinal to opsin leads to activation of the complex accompanied by a decrease in light sensitivity. The subsequent covalent attachment of retinal to opsin completely inactivates opsin and leads to the recovery of sensitivity. Our findings resolve the conflict between biochemical and physiological data concerning the effect of the occupancy of the chromophore binding site on the catalytic potency of opsin. We show that binding of β-ionone to rod opsin produces effects opposite to its previously described effects on cone opsin. We propose that this distinction is due to a fundamental difference in the interaction of rod and cone opsins with retinal, which may have implications for the different physiology of the two types of photoreceptors.
Sequence of Events Underlying the Allosteric Transition of Rod Cyclic Nucleotide–gated Channels
Authors: Sunderman Elizabeth R. , Zagotta William N.
Published: July 2002

Activation of cyclic nucleotide–gated (CNG) ion channels involves a conformational change in the channel protein referred to as the allosteric transition. The amino terminal region and the carboxyl terminal cyclic nucleotide–binding domain of CNG channels have been shown to be involved in the allosteric transition, but the sequence of molecular events occurring during the allosteric transition is unknown. We recorded single-channel currents from bovine rod CNG channels in which mutations had been introduced in the binding domain at position 604 and/or the rat olfactory CNG channel amino terminal region had been substituted for the bovine rod amino terminal region. Using a hidden Markov modeling approach, we analyzed the kinetics of these channels activated by saturating concentrations of cGMP, cIMP, and cAMP. We used thermodynamic mutant cycles to reveal an interaction during the allosteric transition between the purine ring of the cyclic nucleotides and the amino acid at position 604 in the binding site. We found that mutations at position 604 in the binding domain alter both the opening and closing rate constants for the allosteric transition, indicating that the interactions between the cyclic nucleotide and this amino acid are partially formed at the time of the transition state. In contrast, the amino terminal region affects primarily the closing rate constant for the allosteric transition, suggesting that the state-dependent stabilizing interactions between amino and carboxyl terminal regions are not formed at the time of the transition state for the allosteric transition. We propose that the sequence of events that occurs during the allosteric transition involves the formation of stabilizing interactions between the purine ring of the cyclic nucleotide and the amino acid at position 604 in the binding domain followed by the formation of stabilizing interdomain interactions.
Allosteric Voltage Gating of Potassium Channels II
Authors: Horrigan Frank T. , Aldrich Richard W.
Published: July 2002

Large-conductance Ca2+-activated K+ channels can be activated by membrane voltage in the absence of Ca2+ binding, indicating that these channels contain an intrinsic voltage sensor. The properties of this voltage sensor and its relationship to channel activation were examined by studying gating charge movement from mSlo Ca2+-activated K+ channels in the virtual absence of Ca2+ (<1 nM). Charge movement was measured in response to voltage steps or sinusoidal voltage commands. The charge–voltage relationship (Q–V) is shallower and shifted to more negative voltages than the voltage-dependent open probability (G–V). Both ON and OFF gating currents evoked by brief (0.5-ms) voltage pulses appear to decay rapidly (τON = 60 μs at +200 mV, τOFF = 16 μs at −80 mV). However, QOFF increases slowly with pulse duration, indicating that a large fraction of ON charge develops with a time course comparable to that of IK activation. The slow onset of this gating charge prevents its detection as a component of IgON, although it represents ∼40% of the total charge moved at +140 mV. The decay of IgOFF is slowed after depolarizations that open mSlo channels. Yet, the majority of open channel charge relaxation is too rapid to be limited by channel closing. These results can be understood in terms of the allosteric voltage-gating scheme developed in the preceding paper (Horrigan, F.T., J. Cui, and R.W. Aldrich. 1999. J. Gen. Physiol. 114:277–304). The model contains five open (O) and five closed (C) states arranged in parallel, and the kinetic and steady-state properties of mSlo gating currents exhibit multiple components associated with C–C, O–O, and C–O transitions.
Tuning the Voltage Dependence of Tetraethylammonium Block with Permeant Ions in an Inward-Rectifier K+ Channel
Authors: Spassova Maria , Lu Zhe
Published: July 2002

To understand the role of permeating ions in determining blocking ion–induced rectification, we examined block of the ROMK1 inward-rectifier K+ channel by intracellular tetraethylammonium in the presence of various alkali metal ions in both the extra- and intracellular solutions. We found that the channel exhibits different degrees of rectification when different alkali metal ions (all at 100 mM) are present in the extra- and intracellular solution. A quantitative analysis shows that an external ion site in the ROMK1 pore binds various alkali metal ions (Na+, K+, Rb+, and Cs+) with different affinities, which can in turn be altered by the binding of different permeating ions at an internal site through a nonelectrostatic mechanism. Consequently, the external site is saturated to a different level under the various ionic conditions. Since rectification is determined by the movement of all energetically coupled ions in the transmembrane electrical field along the pore, different degrees of rectification are observed in various combinations of extra- and intracellular permeant ions. Furthermore, the external and internal ion-binding sites in the ROMK1 pore appear to have different ion selectivity: the external site selects strongly against the smaller Na+, but only modestly among the three larger ions, whereas the internal site interacts quite differently with the larger K+ and Rb+ ions.
High-Level Expression, Functional Reconstitution, and Quaternary Structure of a Prokaryotic Clc-Type Chloride Channel
Authors: Maduke Merritt , Pheasant Deborah J. , Miller Christopher
Published: July 2002

ClC-type anion-selective channels are widespread throughout eukaryotic organisms. BLAST homology searches reveal that many microbial genomes also contain members of the ClC family. An Escherichia coli–derived ClC Cl− channel homologue, “EriC,” the product of the yadQ gene, was overexpressed in E. coli and purified in milligram quantities in a single-step procedure. Reconstitution of purified EriC into liposomes confers on these membranes permeability to anions with selectivity similar to that observed electrophysiologically in mammalian ClC channels. Cross-linking studies argue that EriC is a homodimer in both detergent micelles and reconstituted liposomes, a conclusion corroborated by gel filtration and analytical sedimentation experiments.
Modulation of the Shaker K+Channel Gating Kinetics by the S3–S4 Linker
Authors: Gonzalez Carlos , Rosenman Eduardo , Bezanilla Francisco , Alvarez Osvaldo , Latorre Ramon
Published: July 2002

In Shaker K+ channels depolarization displaces outwardly the positively charged residues of the S4 segment. The amount of this displacement is unknown, but large movements of the S4 segment should be constrained by the length and flexibility of the S3–S4 linker. To investigate the role of the S3–S4 linker in the ShakerH4Δ(6–46) (ShakerΔ) K+ channel activation, we constructed S3–S4 linker deletion mutants. Using macropatches of Xenopus oocytes, we tested three constructs: a deletion mutant with no linker (0 aa linker), a mutant containing a linker 5 amino acids in length, and a 10 amino acid linker mutant. Each of the three mutants tested yielded robust K+ currents. The half-activation voltage was shifted to the right along the voltage axis, and the shift was +45 mV in the case of the 0 aa linker channel. In the 0 aa linker, mutant deactivation kinetics were sixfold slower than in ShakerΔ. The apparent number of gating charges was 12.6 ± 0.6 eo in ShakerΔ, 12.7 ± 0.5 in 10 aa linker, and 12.3 ± 0.9 in 5 aa linker channels, but it was only 5.6 ± 0.3 eo in the 0 aa linker mutant channel. The maximum probability of opening (Pomax) as measured using noise analysis was not altered by the linker deletions. Activation kinetics were most affected by linker deletions; at 0 mV, the 5 and 0 aa linker channels' activation time constants were 89× and 45× slower than that of the ShakerΔ K+ channel, respectively. The initial lag of ionic currents when the prepulse was varied from −130 to −60 mV was 0.5, 14, and 2 ms for the 10, 5, and 0 aa linker mutant channels, respectively. These results suggest that: (a) the S4 segment moves only a short distance during activation since an S3–S4 linker consisting of only 5 amino acid residues allows for the total charge displacement to occur, and (b) the length of the S3–S4 linker plays an important role in setting ShakerΔ channel activation and deactivation kinetics.
Membrane Cholesterol Content Modulates Activation of Volume-Regulated Anion Current in Bovine Endothelial Cells
Authors: Levitan Irena , Christian Aimee E. , Tulenko Thomas N. , Rothblat George H.
Published: July 2002

Activation of volume-regulated anion current (VRAC) plays a key role in the maintenance of cellular volume homeostasis. The mechanisms, however, that regulate VRAC activity are not fully understood. We have examined whether VRAC activation is modulated by the cholesterol content of the membrane bilayer. The cholesterol content of bovine aortic endothelial cells was increased by two independent methods: (a) exposure to a methyl-β-cyclodextrin saturated with cholesterol, or (b) exposure to cholesterol-enriched lipid dispersions. Enrichment of bovine aortic endothelial cells with cholesterol resulted in a suppression of VRAC activation in response to a mild osmotic gradient, but not to a strong osmotic gradient. Depletion of membrane cholesterol by exposing the cells to methyl-β-cyclodextrin not complexed with cholesterol resulted in an enhancement of VRAC activation when the cells were challenged with a mild osmotic gradient. VRAC activity in cells challenged with a strong osmotic gradient were unaffected by depletion of membrane cholesterol. These observations show that changes in membrane cholesterol content shift VRAC sensitivity to osmotic gradients. Changes in VRAC activation were not accompanied by changes in anion permeability ratios, indicating that channel selectivity was not affected by the changes in membrane cholesterol. This suggests that membrane cholesterol content affects the equilibrium between the closed and open states of VRAC channel rather than the basic pore properties of the channel. We hypothesize that changes in membrane cholesterol modulate VRAC activity by affecting the membrane deformation energy associated with channel opening.
Histidine Scanning Mutagenesis of Basic Residues of the S4 Segment of the Shaker K+ Channel
Authors: Starace Dorine M. , Bezanilla Francisco
Published: July 2002

The voltage sensor of the Shaker potassium channel is comprised mostly of positively charged residues in the putative fourth transmembrane segment, S4 (Aggarwal, S.K., and R. MacKinnon. 1996. Neuron. 16:1169–1177; Seoh, S.-A., D. Sigg, D.M. Papazian, and F. Bezanilla. 1996. Neuron. 16:1159–1167). Movement of the voltage sensor in response to a change in the membrane potential was examined indirectly by measuring how the accessibilities of residues in and around the sensor change with voltage. Each basic residue in the S4 segment was individually replaced with a histidine. If the histidine tag is part of the voltage sensor, then the gating charge displaced by the voltage sensor will include the histidine charge. Accessibility of the histidine to the bulk solution was therefore monitored as pH-dependent changes in the gating currents evoked by membrane potential pulses. Histidine scanning mutagenesis has several advantages over other similar techniques. Since histidine accessibility is detected by labeling with solution protons, very confined local environments can be resolved and labeling introduces minimal interference of voltage sensor motion. After histidine replacement of either residue K374 or R377, there was no titration of the gating currents with internal or external pH, indicating that these residues do not move in the transmembrane electric field or that they are always inaccessible. Histidine replacement of residues R365, R368, and R371, on the other hand, showed that each of these residues traverses entirely from internal exposure at hyperpolarized potentials to external exposure at depolarized potentials. This translocation enables the histidine to transport protons across the membrane in the presence of a pH gradient. In the case of 371H, depolarization drives the histidine to a position that forms a proton pore. Kinetic models of titrateable voltage sensors that account for proton transport and conduction are presented. Finally, the results presented here are incorporated into existing information to propose a model of voltage sensor movement and structure.
Interdomain Interactions within Ryanodine Receptors Regulate Ca2+ Spark Frequency in Skeletal Muscle
Authors: Shtifman Alexander , Ward Christopher W. , Yamamoto Takeshi , Wang Jianli , Olbinski Beth , Valdivia Hector H. , Ikemoto Noriaki , Schneider Martin F.
Published: July 2002

DP4 is a 36-residue synthetic peptide that corresponds to the Leu2442-Pro2477 region of RyR1 that contains the reported malignant hyperthermia (MH) mutation site. It has been proposed that DP4 disrupts the normal interdomain interactions that stabilize the closed state of the Ca2+ release channel (Yamamoto, T., R. El-Hayek, and N. Ikemoto. 2000. J. Biol. Chem. 275:11618–11625). We have investigated the effects of DP4 on local SR Ca2+ release events (Ca2+ sparks) in saponin-permeabilized frog skeletal muscle fibers using laser scanning confocal microscopy (line-scan mode, 2 ms/line), as well as the effects of DP4 on frog SR vesicles and frog single RyR Ca2+ release channels reconstituted in planar lipid bilayers. DP4 caused a significant increase in Ca2+ spark frequency in muscle fibers. However, the mean values of the amplitude, rise time, spatial half width, and temporal half duration of the Ca2+ sparks, as well as the distribution of these parameters, remained essentially unchanged in the presence of DP4. Thus, DP4 increased the opening rate, but not the open time of the RyR Ca2+ release channel(s) generating the sparks. DP4 also increased [3H]ryanodine binding to SR vesicles isolated from frog and mammalian skeletal muscle, and increased the open probability of frog RyR Ca2+ release channels reconstituted in bilayers, without changing the amplitude of the current through those channels. However, unlike in Ca2+ spark experiments, DP4 produced a pronounced increase in the open time of channels in bilayers. The same peptide with an Arg17 to Cys17 replacement (DP4mut), which corresponds to the Arg2458-to-Cys2458 mutation in MH, did not produce a significant effect on RyR activation in muscle fibers, bilayers, or SR vesicles. Mg2+ dependence experiments conducted with permeabilized muscle fibers indicate that DP4 preferentially binds to partially Mg2+-free RyR(s), thus promoting channel opening and production of Ca2+ sparks.
Constructive Consumer Choice Processes
Authors: Bettman James R. , Luce Mary Frances , Payne John W.
Published: July 2002

Consumer decision making has been a focal interest in consumer research, and consideration of current marketplace trends (e.g., technological change, an information explosion) indicates that this topic will continue to be critically important. We argue that consumer choice is inherently constructive. Due to limited processing capacity, consumers often do not have well-defined existing preferences, but construct them using a variety of strategies contingent on task demands. After describing constructive choice, consumer decision tasks, and decision strategies, we provide an integrative framework for understanding constructive choice, review evidence for constructive consumer choice in light of that framework, and identify knowledge gaps that suggest opportunities for additional research.
Sampling in a Hilbert space
Authors: Zayed Ahmed
Published: July 2002

An analog of the Whittaker-Shannon-Kotel′nikov sampling theorem is derived for functions with values in a separable Hilbert space. The proof uses the concept of frames and frame operators in a Hilbert space. One of the consequences of this theorem is that it allows us to derive sampling theorems associated with boundary-value problems and some homogeneous integral equations, which in turn gives us a generalization of another sampling theorem by Kramer.
Newton’s formula for 𝔤𝔩_{𝔫}
Authors: Umeda Tôru
Published: July 2002

This paper presents an explicit relation between the two sets which are well-known generators of the center of the universal enveloping algebra

U
(

g
l

n

)

U(\mathfrak {gl}_{n})

of the Lie algebra

g
l

n

\mathfrak {gl}_{n}

: one by Capelli (1890) and the other by Gelfand (1950). Our formula is motivated to give an exact analogy for the classical Newton’s formula connecting the elementary symmetric functions and the power sum symmetric functions. The formula itself can be deduced from a more general result on Yangians obtained by Nazarov. Our proof is elementary and has an advantage in its direct accessibility.
Residually finite, congruence meet-semidistributive varieties of finite type have a finite residual bound
Authors: Kearnes Keith , Willard Ross
Published: July 2002

We show that a residually finite, congruence meet-semidistributive variety of finite type is residually

>
N

> N

for some finite

N
N

. This solves Pixley’s problem and a special case of the restricted Quackenbush problem.
Functional Nonequivalency of Actin Isovariants inArabidopsis
Authors: Kandasamy Muthugapatti K. , McKinney Elizabeth C. , Meagher Richard B.
Published: July 2002

Plants encode at least two ancient and divergent classes of actin, reproductive and vegetative, and each class produces several subclasses of actin isovariants. To gain insight into the functional significance of the actin isovariants, we generated transgenicArabidopsis lines that expressed a reproductive actin, ACT1, under the control of the regulatory sequences of a vegetative actin gene, ACT2. In the wild-type plants, ACT1 is predominantly expressed in the mature pollen, growing pollen tubes, and ovules, whereas ACT2 is constitutively and strongly expressed in all vegetative tissues and organs, but not in pollen. Misexpression of ACT1 in vegetative tissues causes dwarfing of plants and altered morphology of most organs, and the effects are in direct proportion to protein expression levels. Similar overexpression of ACT2 has little effect. Immunolocalization of actin in leaf cells from transgenic plants with highest levels of ACT1 protein revealed massive polymerization, bundling, and reorganization of actin filaments. This phenomenon suggests that misexpression of ACT1 isovariant in vegetative tissues affects the dynamics of actin and actin-associated proteins, in turn disrupting the organization of actin cytoskeleton and normal development of plants.
Genetic Ablation of Phosphatidylinositol Transfer Protein Function in Murine Embryonic Stem Cells
Authors: Alb James G. , Phillips Scott E. , Rostand Kathleen , Cui Xiaoxia , Pinxteren Jef , Cotlin Laura , Manning Timothy , Guo Shuling , York John D. , Sontheimer Harald , Collawn James F. , Bankaitis Vytas A.
Published: July 2002

Phosphatidylinositol transfer proteins (PITPs) regulate the interface between signal transduction, membrane-trafficking, and lipid metabolic pathways in eukaryotic cells. The best characterized mammalian PITPs are PITPα and PITPβ, two highly homologous proteins that are encoded by distinct genes. Insights into PITPα and PITPβ function in mammalian systems have been gleaned exclusively from cell-free or permeabilized cell reconstitution and resolution studies. Herein, we report for the first time the use of genetic approaches to directly address the physiological functions of PITPα and PITPβ in murine cells. Contrary to expectations, we find that ablation of PITPα function in murine cells fails to compromise growth and has no significant consequence for bulk phospholipid metabolism. Moreover, the data show that PITPα does not play an obvious role in any of the cellular activities where it has been reconstituted as an essential stimulatory factor. These activities include protein trafficking through the constitutive secretory pathway, endocytic pathway function, biogenesis of mast cell dense core secretory granules, and the agonist-induced fusion of dense core secretory granules to the mast cell plasma membrane. Finally, the data demonstrate that PITPα-deficient cells not only retain their responsiveness to bulk growth factor stimulation but also retain their pluripotency. In contrast, we were unable to evict both PITPβ alleles from murine cells and show that PITPβ deficiency results in catastrophic failure early in murine embryonic development. We suggest that PITPβ is an essential housekeeping PITP in murine cells, whereas PITPα plays a far more specialized function in mammals than that indicated by in vitro systems that show PITP dependence.
GeneCards: a novel functional genomics compendium with automated data mining and query reformulation support.
Authors: Rebhan M , Chalifa-Caspi V , Prilusky J , Lancet D
Published: July 2002

MOTIVATION: Modern biology is shifting from the 'one gene one postdoc' approach to genomic analyses that include the simultaneous monitoring of thousands of genes. The importance of efficient access to concise and integrated biomedical information to support data analysis and decision making is therefore increasing rapidly, in both academic and industrial research. However, knowledge discovery in the widely scattered resources relevant for biomedical research is often a cumbersome and non-trivial task, one that requires a significant amount of training and effort. RESULTS: To develop a model for a new type of topic-specific overview resource that provides efficient access to distributed information, we designed a database called 'GeneCards'. It is a freely accessible Web resource that offers one hypertext 'card' for each of the more than 7000 human genes that currently have an approved gene symbol published by the HUGO/GDB nomenclature committee. The presented information aims at giving immediate insight into current knowledge about the respective gene, including a focus on its functions in health and disease. It is compiled by Perl scripts that automatically extract relevant information from several databases, including SWISS-PROT, OMIM, Genatlas and GDB. Analyses of the interactions of users with the Web interface of GeneCards triggered development of easy-to-scan displays optimized for human browsing. Also, we developed algorithms that offer 'ready-to-click' query reformulation support, to facilitate information retrieval and exploration. Many of the long-term users turn to GeneCards to quickly access information about the function of very large sets of genes, for example in the realm of large-scale expression studies using 'DNA chip' technology or two-dimensional protein electrophoresis. AVAILABILITY: Freely available at http://bioinformatics.weizmann.ac.il/cards/ Contact: cards@bioinformatics.weizmann.ac.il
Thermodynamic database for protein–nucleic acid
interactions (ProNIT)
Authors: Prabakaran Ponraj , An Jianghong , Gromiha M. Michael , Selvaraj Samuel , Uedaira Hatsuho , Kono Hidetoshi , Sarai Akinori
Published: July 2002

Motivation: Protein–nucleic acid interactions are fundamental to the regulation of gene expression. In order to elucidate the molecular mechanism of protein–nucleic acid recognition and analyze the gene regulation network, not only structural data but also quantitative binding data are necessary. Although there are structural databases for proteins and nucleic acids, there exists no database for their experimental binding data. Thus, we have developed a Thermodynamic Database for Protein–Nucleic Acid Interactions (ProNIT).

Results: We have collected experimentally observed binding data from the literature. ProNIT contains several important thermodynamic data for protein–nucleic acid binding, such as dissociation constant (Kd), association constant (Ka), Gibbs free energy change (ΔG), enthalpy change (ΔH), heat capacity change (ΔCp), experimental conditions, structural information of proteins, nucleic acids and the complex, and literature information. These data are integrated into a relational database system together with structural and functional information to provide flexible searching facilities by using combinations of various terms and parameters. A www interface allows users to search for data based on various conditions, with different display and sorting options, and to visualize molecular structures and their interactions.

Availability: ProNIT is freely accessible at the URL http://www.rtc.riken.go.jp/jouhou/pronit/pronit.html.

Contact: sarai@rtc.riken.go.jp

* To whom correspondence should be addressed.
PCSB—a program collection for structural biology and
biophysical chemistry
Authors: Hofmann A. , Wlodawer A.
Published: July 2002

We present the first package of Java classes specifically aimed at the handling of structural and biophysical problems. To enable object-oriented programming a basis of fundamental Java classes is required which deals with basic operations of vectors, matrices, amino acid sequences, crystal symmetries and PDB files. Five classes, which carry out these basic operations, were constructed and bundled together with several utility functions in the PCSB package. Furthermore, to demonstrate their applicability and to obtain programs handling common tasks in structural laboratories, we present the first six applications of PCSB. All applications are portable to different platforms and require only the Java Runtime Environment to be installed on the system.

Availability: http://www24.brinkster.com/hofmanna/pcsb/

Contact: hofmanna@ncifcrf.gov

Supplementary information: A manual for PCSB describing the Java classes as well as the applications is available as PDF file.

* To whom correspondence should be addressed.
STOCKS: STOChastic Kinetic Simulations of biochemical systems
with Gillespie algorithm
Authors: Kierzek Andrzej M.
Published: July 2002

Motivation: The availability of a huge amount of molecular data concerning various biochemical reactions provoked numerous attempts to study the dynamics of cellular processes by means of kinetic models and computer simulations. Biochemical processes frequently involve small numbers of molecules (e.g. a few molecules of a transcriptional regulator binding to one ‘molecule’ of a DNA regulatory region). Such reactions are subject to significant stochastic fluctuations. Monte Carlo methods must be employed to study the functional consequences of the fluctuations and simulate processes that cannot be modelled by continuous fluxes of matter. This provides the motivation to develop software dedicated to Monte Carlo simulations of cellular processes with the rigorously proven Gillespie algorithm.

Results: STOCKS, software for the stochastic kinetic simulation of biochemical processes is presented. The program uses a rigorously derived Gillespie algorithm that has been shown to be applicable to the study of prokaryotic gene expression. Features dedicated to the study of cellular processes are implemented, such as the possibility to study a process in the range of several cell generations with the application of a simple cell division model. Taking expression of Escherichia coli beta-galactosidase as an example, it is shown that the program is able to simulate systems composed of reactions varying in several orders of magnitude by means of reaction rates and the numbers of molecules involved.

Availability: The software is available at ftp://ibbrain.ibb.waw.pl/stocksand http://www.ibb.waw.pl/stocks.

Supplementary information: Parameters of the model of prokaryotic gene expression are available in example files of software distribution.

Contact: andrzejk@ibb.waw.pl
ProbeMatchDB—a web database for finding equivalent probes
across microarray platforms and species
Authors: Wang Pinglang , Ding Fei , Chiang Hsienyuan , Thompson Robert C. , Watson Stanley J. , Meng Fan
Published: July 2002

Summary: ProbeMatchDB is a web-based database designed to facilitate the search of EST/cDNA sequences or STS markers that can be used to represent the same gene across different microarray platforms and species. It can be used for finding equivalent EST clones in the Research Genetics sequence verified clone set based on results from Affymetirx GeneChip®s. It will also help to identify probes representing orthologous genes across human, mouse and rat on different microarray platforms.

Availability: The database is accessible at http://brainarray.mhri.med.umich.edu/MARRAY/BC_ASP/brainarray.htm by clicking the ‘Query ProbeMatchDB’ link.

Contact: mengf@umich.edu
Free Agency, Competitive Balance, and Diminishing Returns to Pennant Contention
Authors: Eckard E. Woodrow
Published: July 2002

This article argues that the 1976 introduction of free agency increased competitive balance in Major League Baseball. The evidence is based on a new empirical measure that captures the key dynamic element of balance: year‐to‐year fluctuations in team performance. My hypothesis is that diminishing returns to each additional year's “production” of a pennant‐contending team reduces the incentive to bid continually for top players. Free agency allows talent to be reallocated more readily to potential new contenders, given that player sales had been restricted prior to 1976. The hypothesis is supported by evidence of declining attendance during contending “streaks.”
Cell‐selective intracellular delivery of a foreign enzyme to endothelium in vivo using vascular immunotargeting
Authors: SCHERPEREEL ARNAUD , WIEWRODT RAINER , CHRISTOFIDOU‐SOLOMIDOU MELPO , GERVAIS RADJ , MURCIANO JUAN‐CARLOS , ALBELDA STEVEN M. , MUZYKANTOV VLADIMIR R.
Published: July 2002

Vascular immunotargeting, the administration of drugs conjugated with antibodies to endothelial surface antigens, has the potential for drug delivery to the endothelium. Our previous cell culture studies showed that biotinylated antibodies to PECAM‐1 (a highly expressed endothelial surface antigen) coupled with streptavidin (SA, a cross‐linking protein that facilitates anti‐PECAM internalization and targeting) may provide a carrier for the intracellular delivery of therapeutic enzymes. This paper describes the PECAM‐directed vascular immunotargeting of a reporter enzyme (β‐galactosidase, β‐Gal) in intact animals. Intravenous injection of [125I]SA‐β‐Gal conjugated with either anti‐PECAM or IgG led to a high 125I uptake in liver and spleen, yet β‐Gal activity in these organs rapidly declined to the background levels, suggesting rapid degradation of the conjugates. In contrast, anti‐PECAM/[125I]SA‐β‐Gal, but not IgG/[125I]SA‐β‐Gal, accumulated in the lungs (36.0±1.3 vs. 3.9±0.6% injected dose/g) and induced a marked elevation of β‐Gal activity in the lung tissue persisting for up to 8 h after injection (10‐fold elevation 4 h postinjection). Using histochemical detection, the β‐Gal activity in the lungs was detected in the endothelial cells of capillaries and large vessels. The anti‐PECAM carrier also provided 125I uptake and β‐Gal activity in the renal glomeruli. Predominant intracellular localization of anti‐PECAM/SA‐β‐Gal was documented in the PECAM‐expressing cells in culture by confocal microscopy and in the pulmonary endothelium by electron microscopy. Therefore, vascular immunotargeting is a feasible strategy for cell‐selective, intracellular delivery of an active foreign enzyme to endothelial cells in vivo, and thus may be potentially useful for the treatment of acute pulmonary or vascular diseases.—Scherpereel, A., Wiewrodt, R., Christofidou‐Solomidou, M., Gervais, R., Murciano, J.‐C., Albelda, S. M., Muzykantov, V. R. Cell‐selective intracellular delivery of a foreign enzyme to endothelium in vivo using vascular immunotargeting. FASEB J. 15, 416‐426 (2001).
Theories of rotary motors
Authors: Berry Richard M.
Published: July 2002

The bacterial flagellar motor and the ATP–hydrolysing F
1
portion of the F
1
F
o
–ATPase are known to be rotary motors, and it seems highly probable that the H
+
–translocating F
o
portion rotates too. The energy source in the case of F
o
and the flagellar motor is the flow of ions, either H
+
(protons) or Na
+
, down an electrochemical gradient across a membrane. The fact that ions flow in a particular direction through a well–defined structure in these motors invites the possibility of a type of mechanism based on geometric constraints between the rotor position and the paths of ions flowing through the motor. The two beststudied examples of such a mechanism are the ‘turnstile’ model of Khan and Berg and the ‘proton turbine’ model of Lauger or Berry. Models such as these are typically represented by a small number of kinetic states and certain allowed transitions between them. This allows the calculation of predictions of motor behaviour and establishes a dialogue between models and experimental results. In the near future structural data and observations of single–molecule events should help to determine the nature of the mechanism of rotary motors, while motor models must be developed that can adequately explain the measured relationships between torque and speed in the flagellar motor.
Balancing the two photosystems: photosynthetic electron transfer governs transcription of reaction centre genes in chloroplasts
Authors: Allen John F. , Pfannschmidt Thomas
Published: July 2002

Chloroplasts are cytoplasmic organelles whose primary function is photosynthesis, but which also contain small, specialized and quasi–autonomous genetic systems. In photosynthesis, two energy converting photosystems are connected, electrochemically, in series. The connecting electron carriers are oxidized by photosystem I (PS I) and reduced by photosystem II (PS II). It has recently been shown that the oxidation–reduction state of one connecting electron carrier, plastoquinone, controls transcription of chloroplast genes for reaction centre proteins of the two photosystems. The control counteracts the imbalance in electron transport that causes it: oxidized plastoquinone induces PS II and represses PS I; reduced plastoquinone induces PS I and represses PS II. This complementarity is observed both
in vivo
, using light favouring one or other photosystem, and
in vitro
, when site–specific electron transport inhibitors are added to transcriptionally and photosynthetically active chloroplasts. There is thus a transcriptional level of control that has a regulatory function similar to that of purely post–translational ‘state transitions’ in which the redistribution of absorbed excitation energy between photosystems is mediated by thylakoid membrane protein phosphorylation. The changes in rates of transcription that are induced by spectral changes
in vivo
can be detected even before the corresponding state transitions are complete, suggesting the operation of a branched pathway of redox signal transduction. These findings suggest a mechanism for adjustment of photosystem stoichiometry in which initial events involve a sensor of the redox state of plastoquinone, and may thus be the same as the initial events of state transitions. Redox control of chloroplast transcription is also consistent with the proposal that a direct regulatory coupling between electron transport and gene expression determines the function and composition of the chloroplast's extra–nuclear genetic system.