bluuebunny/crossref_metadata_2025 huggingface dataset:
Structure-based conformational preferences of amino acids
Authors: Koehl Patrice , Levitt Michael
Published: July 2002

Proteins can be very tolerant to amino acid substitution, even within their core. Understanding the factors responsible for this behavior is of critical importance for protein engineering and design. Mutations in proteins have been quantified in terms of the changes in stability they induce. For example, guest residues in specific secondary structures have been used as probes of conformational preferences of amino acids, yielding propensity scales. Predicting these amino acid propensities would be a good test of any new potential energy functions used to mimic protein stability. We have recently developed a protein design procedure that optimizes whole sequences for a given target conformation based on the knowledge of the template backbone and on a semiempirical potential energy function. This energy function is purely physical, including steric interactions based on a Lennard-Jones potential, electrostatics based on a Coulomb potential, and hydrophobicity in the form of an environment free energy based on accessible surface area and interatomic contact areas. Sequences designed by this procedure for 10 different proteins were analyzed to extract conformational preferences for amino acids. The resulting structure-based propensity scales show significant agreements with experimental propensity scale values, both for α-helices and β-sheets. These results indicate that amino acid conformational preferences are a natural consequence of the potential energy we use. This confirms the accuracy of our potential and indicates that such preferences should not be added as a design criterion.
Energy-based
de novo
protein folding by conformational space annealing and an off-lattice united-residue force field: Application to the 10-55 fragment of staphylococcal protein A and to apo calbindin D9K
Authors: Lee Jooyoung , Liwo Adam , Scheraga Harold A.
Published: July 2002

The conformational space annealing (CSA) method for global optimization has been applied to the 10-55 fragment of the B-domain of staphylococcal protein A (protein A) and to a 75-residue protein, apo calbindin D9K (PDB ID code
1CLB
), by using the UNRES off-lattice united-residue force field. Although the potential was not calibrated with these two proteins, the native-like structures were found among the low-energy conformations, without the use of threading or secondary-structure predictions. This is because the CSA method can find many distinct families of low-energy conformations. Starting from random conformations, the CSA method found that there are two families of low-energy conformations for each of the two proteins, the native-like fold and its mirror image. The CSA method converged to the same low-energy folds in all cases studied, as opposed to other optimization methods. It appears that the CSA method with the UNRES force field, which is based on the thermodynamic hypothesis, can be used in prediction of protein structures in real time.
DRC1,
DNA replication and checkpoint protein 1, functions with
DPB11
to control DNA replication and the S-phase checkpoint in
Saccharomyces cerevisiae
Authors: Wang Hong , Elledge Stephen J.
Published: July 2002

In addition to DNA polymerase complexes, DNA replication requires the coordinate action of a series of proteins, including regulators Cdc28/Clb and Dbf4/Cdc7 kinases, Orcs, Mcms, Cdc6, Cdc45, and Dpb11. Of these, Dpb11, an essential BRCT repeat protein, has remained particularly enigmatic. The
Schizosaccharomyces pombe
homolog of
DPB11
,
cut5
, has been implicated in the DNA replication checkpoint as has the
POL2
gene with which
DPB11
genetically interacts. Here we describe a gene,
DRC1
, isolated as a dosage suppressor of
dpb11–1
.
DRC1
is an essential cell cycle-regulated gene required for DNA replication. We show that both Dpb11 and Drc1 are required for the S-phase checkpoint, including the proper activation of the Rad53 kinase in response to DNA damage and replication blocks. Dpb11 is the second BRCT-repeat protein shown to control Rad53 function, possibly indicating a general function for this class of proteins.
DRC1
and
DPB11
show synthetic lethality and reciprocal dosage suppression. The Drc1 and Dpb11 proteins physically associate and function together to coordinate DNA replication and the cell cycle.
Effects of salicylates and aminoglycosides on spontaneous otoacoustic emissions in the Tokay gecko
Authors: Stewart Charles E. , Hudspeth A. James
Published: July 2002

The high sensitivity and sharp frequency discrimination of hearing depend on mechanical amplification in the cochlea. To explore the basis of this active process, we examined the pharmacological sensitivity of spontaneous otoacoustic emissions (SOAEs) in a lizard, the Tokay gecko. In a quiet environment, each ear produced a complex but stable pattern of emissions. These SOAEs were reversibly modulated by drugs that affect mammalian otoacoustic emissions, the salicylates and the aminoglycoside antibiotics. The effect of a single i.p. injection of sodium salicylate depended on the initial power of the emissions: ears with strong control SOAEs displayed suppression at all frequencies, whereas those with weak control emissions showed enhancement. Repeated oral administration of acetylsalicylic acid reduced all emissions. Single i.p. doses of gentamicin or kanamycin suppressed SOAEs below 2.6 kHz, while modulating those above 2.6 kHz in either of two ways. For ears whose emission power at 2.6–5.2 kHz encompassed more than half of the total, individual emissions displayed facilitation as great as 35-fold. For the remaining ears, emissions dropped to as little as one-sixth of their initial values. The similarity of the responses of reptilian and mammalian cochleas to pharmacological intervention provides further evidence for a common mechanism of cochlear amplification.
Atomic force microscopy captures quantized plastic deformation in gold nanowires
Authors: Marszalek Piotr E. , Greenleaf William J. , Li Hongbin , Oberhauser Andres F. , Fernandez Julio M.
Published: July 2002

Scanning probe microscopy has become a powerful tool to detect structural changes in small clusters of atoms. Herein, we use an atomic force microscope to measure the length of gold nanowire structures during extension and compression cycles. We have found that nanowires elongate under force in quantized steps of up to three integer multiples of 1.76 Å and that they shorten spontaneously in steps of 1.52 Å. Our results can be explained by the sliding of crystal planes within the gold nanowires creating stacking faults that change the local structure from face-centered cubic to hexagonal close packed. Our data also show that there can be up to three simultaneous slip events, in good agreement with the tetrahedral arrangement of slip planes in a gold crystal. These experiments provide direct evidence for the mechanism underlying the plastic deformation of a nanowire. A similar approach can be used to examine the atomic events underlying the plastic failure of other metals and their alloys.
Mechanisms and kinetics of β-hairpin formation
Authors: Klimov D. K. , Thirumalai D.
Published: July 2002

Thermodynamics and kinetics of off-lattice models with side chains for the β-hairpin fragment of immunoglobulin-binding protein and its variants are reported. For all properties (except refolding time τ
F
) there are no qualitative differences between the full model and the Gō version. The validity of the models is established by comparison of the calculated native structure with the Protein Data Bank coordinates and by reproducing the experimental results for the degree of cooperativity and τ
F
. For the full model τ
F
≈ 2 μs at the folding temperature (experimental value is 6 μs); the Gō model folds 50 times faster. Upon refolding, structural changes take place over three time scales. On the collapse time scale compact structures with intact hydrophobic cluster form. Subsequently, hydrogen bonds form, predominantly originating from the turn by a kinetic zipping mechanism. The assembly of the hairpin is complete when most of the interstrand contacts (the rate-limiting step) is formed. The dominant transition state structure (located by using cluster analysis) is compact and structured. We predict that when hydrophobic cluster is moved to the loop τ
F
marginally increases, whereas moving the hydrophobic cluster closer to the termini results in significant decrease in τ
F
relative to wild type. The mechanism of hairpin formation is predicted to depend on turn stiffness.
IMP and AMP deaminase in reperfusion injury down-regulates neutrophil recruitment
Authors: Qiu Fei-Hua , Wada Koichiro , Stahl Gregory L. , Serhan Charles N.
Published: July 2002

We examined gene regulation in murine lungs after hind-limb vessel occlusion and reperfusion. A rapid increase of transcript for the AMP deaminase 3 gene (AMPD3) and its enzymatic activity (EC3.5.4.6) generating inosine monophosphate (IMP) were identified with transcripts located in bronchial and alveolar epithelium. AMP deaminase inhibitor decreased IMP levels and significantly enhanced neutrophil recruitment within lung tissue during reperfusion. In addition, IMP inhibited cytokine-initiated neutrophil infiltrationin vivoand selectively attenuated neutrophil rolling by 90% in microvessels. We prepared labeled IMP and demonstrated that IMP specifically binds to neutrophils. IMP also stimulated binding of γ-[35S]thio-GTP, suggesting that IMP is a potent regulator of neutrophils. Taken together, these results elucidate a previously unrecognized mechanism that protects tissues from the potentially deleterious consequences of aberrant neutrophil accumulation. Moreover, they are relevant for new therapeutic approaches to regulate neutrophil responses in inflammation and vascular disease.
Sufism and Politics in the North Caucasus
Authors: Yemelianova Galina M.
Published: July 2002

After the collapse of communism in Russia, which is the home of more than 14 million Muslims, there has been an Islamic revival that has been part of the process of political and intellectual liberalization of society. The major Islamic enclaves of the Russian Federation are located in the Volga-Urals, the North Caucasus, and central Russia. Russian Muslims are concentrated in the eight autonomous republics of Tatarstan, Bashkortostan, Adyghea, Kabardino-Balkaria, Karachay-Cherkessia, Ingushetia, Dagestan, and Chechnya. Most Muslims belong to theHanafi madhhab(the juridical school) of Sunni Islam, although Dagestani and Chechen Muslims adhere to theShafii madhhabof Sunni Islam. There is also a small Shia community in southern Dagestan. A large number of Dagestanis, as well as Chechens and Ingushes, profess Sufism—a mystical form of Islam, which is also known as parallel Islam.
Attrition in group therapy with alcoholics: a survival analysis
Authors: ARNAU MIQUEL MONRAS , GUAL ANTONI
Published: July 2002

Three hundred and twenty‐nine alcohol dependent patients, diagnosed according to DSM. III‐R criteria who had participated in one of five different psychotherapy groups over a 5‐year period, were studied using survival analysis statistics. Patients were followed‐up until their discharge or withdrawal from the group to determine their retention in therapy, and to examine the effect of gender, age and family situation on compliance. Retention rates at 6, 12 and 24 months were 61%, 49% and 37%, respectively. Median retention time was 46 weeks. Women remained longer in treatment than men (median period 98 vs. 34 weeks), and this was due mainly to lower dropout rates in the first 3 months. Dropout rates were also higher in patients aged 35 years or younger. The first 3 months seem to be crucial in the process of deciding whether or not to remain in therapy.
The acute cardiovascular effects of 3,4‐methylenedioxymethamphetamine (MDMA) and p‐methoxyamphetamine (PMA)
Authors: Irvine Rodney J. , Toop Natasha P. , Phillis Benjamin D. , Lewanowitsch Tanya
Published: July 2002

A number of reports of severe acute toxicity as a result of recreational ingestion of what was believed to be 3,4‐methylenedioxymethamphetamine (MDMA) (ecstasy) have revealed, on chemical analysis of the victim's blood, to be due to p‐methoxyamphetamine (PMA). Increased blood pressure and heart rate have been associated with this toxicity, which is exacerbated by high ambient temperature. We compared MDMA and PMA effects on blood pressure and heart rate in conscious freely moving rats at ambient temperatures of 20°C and 30°C. Drugs were administered at 5, 10, 15 and 20 mg/kg s.c. and cardiovascular recordings continued 2–4 hours post‐drug. At 20°C MDMA had a slight lowering effect on blood pressure or heart rate at all doses tested. In contrast, PMA administered at the same ambient temperature caused a dose‐dependent long‐lasting bradycardia and lowering of blood pressure. At 30°C MDMA caused a slight increase in heart rate and a moderate rise in blood pressure. PMA did not significantly change heart rate or blood pressure at any of the doses tested. ECG was only influenced at the highest dose (20 mg/kg) of PMA and MDMA administered at 30°C where ST elongation was observed. These data indicate that although these drugs are considered similar in their psychotrophic effects they have different actions on the cardiovascular system of the rat. These findings may have implications in the diagnosis and emergency treatment of victims of MDMA/PMA toxicity.
Physiological concentrations of nicotine do not affect prostacyclin, thromboxane or PGE production from perfused human umbilical veins
Authors: Cook Jocelynn L. , Keith Brad A. , White Nancy M. , Randall Carrie L.
Published: July 2002

Smoking during pregnancy is associated with a number of maternal and fetal complications. The mechanisms underlying the effects of nicotine on fetal growth and development are not known, but may involve decreases in umbilical–placental blood flow. Prostaglandins are vasoactive mediators that regulate umbilical–placental blood flow and there is evidence that nicotine may alter prostaglandin production. Thus, alterations in vasoactive prostaglandins from the human umbilical vein may explain nicotine‐induced decreases in vessel blood flow and may underlie some of the negative effects of nicotine on the fetus. This study was designed to determine the relationship between nicotine and human umbilical vein prostaglandin production. Prostacyclin, thromboxane and prostaglandin E were measured from human umbilical veins collected from women at the time of cesarean section. Vessels were perfused with either vehicle (Dulbecco's modified Eagle's media), 5 mM, 10 mM or 20 mM nicotine for 60 minutes. Samples were collected from the perfusate after 5, 15, 30 and 60 minutes of perfusion, and prostaglandins were measured by radioimmunoassay. Data were analyzed by ANOVA. Perfusion with 10 mM or 20 mm significantly reduced prostacyclin production by the human umbilical veins, but nicotine did not affect prostaglandin E. The effects of nicotine on thromboxane were less clear. However, nicotine concentrations required to illicit an effect on prostacyclin production were much larger than circulating levels associated with smoking, so it is only pharmacological concentrations that have a significant effect. Thus, nicotine‐induced alterations in umbilical vein prostaglandin production does not appear to be a mechanism by which nicotine adversely affects fetal growth and development.
Book and Media Reviews
Authors: BALL DAVID
Published: July 2002

Books reviewed in this article:

Book reviews in this column will primarily be of titles focusing completely, or in part, on biological aspects of addiction. However, significant titles of general relevance to the addictions field will also be included, even if they are not “biological”, as will titles of general methodological and clinical relevance, even if they are not on “addictions”. Similar considerations will apply to other media (software, audio tapes and CDs, videos, etc). However, specific “addictions” software applications seem to be relatively uncommon and, as these items are rarely reviewed elsewhere, we will endeavour to include reviews of some of the older programmes that are still useful, as well as new titles that appear. I would appreciate suggestions of any items suitable for reviews, but especially software and other media of specific relevance to the addictions. Please contact: Dr David Ball, National Addiction Centre, 4 Windsor Walk, London SE5 8AF, UK. Email: media@addictionbiology.com

Essential Psychophar macology, 2nd edition STEPHEN M. STAHL

Drug and Alcohol Abuse: A Clinical Guide to Diagnosis and Treatment, 5th edition MARC A SCHUCKIT

Drugs: Dilemmas and Choices Working Party of the Royal College of Psychiatrists and the Royal College of Physicians

Alcohol and Cardiovascular Diseases: Novartis Foundation Symposium TIMOTHY PETERS (Ed.)

Directory of Biomedical and Health Care Grants 1999, 13th edition
Differential Effect of PKC Isoform on Insulin‐ and Phorbol Ester‐Stimulated Glucose Uptake Mechanism in Rat Adipocytes
Authors: Ishizuka Tatsuo , Miura Atsushi , Kajita Kazuo , Ishizawa Masayoshi , Kimura Mika , Huang Yannan , Kawai Yasunori , Morita Hiroyuki , Uno Yoshihiro , Yasuda Keigo
Published: July 2002

Insulin stimulates glucose uptake in association with phosphatidylinositol (PI) 3‐kinase activation mechanisms in rat adipocytes. Insulin stimulated glucose uptake to 6.5‐fold, and 12‐ o ‐tetradecanoyl phorbol 13‐acetate (TPA) also stimulated glucose uptake to 4.5‐fold in rat adipocytes. We examined these differences in glucose uptake, PKC ∂activation, and PI 3‐kinase activation after stimulation with insulin and TPA. TPA stimulated PI 3‐kinase activity and increased the p85 subunit of PI 3‐kinase immunoreactivity in anti‐phosphotyrosine antibody‐immunoprecipitated protein. Insulin and TPA provoked increases in membrane PKC ∂immunoreactivity.The PI 3‐kinase inhibitor,wortmannin, suppressed insulininduced increases in glucose uptake, PI 3‐kinase activity, and PKC ∂activation. Wortmannin also suppressed TPA‐induced PI 3‐kinase activity and PKC ∂activation but suppressed TPA‐induced glucose uptake to only a small extent. The PKC inhibitor, Go6976, which only inhibits conventional PKC αand _, suppressed TPA‐induced glucose uptake, but suppressed insulin‐induced glucose uptake to only a small extent. On the other hand, the PKC inhibitor, RO32‐0432, which inhibits conventional, novel, and atypical PKCs, markedly suppressed both insulin‐ and TPA‐induced glucose uptake. These results suggest that insulininduced glucose uptake is mainly mediated by PI 3‐kinase‐PKC ∂signaling, whereas phorbol ester‐induced glucose uptake is mainly mediated by conventional PKC despite PI 3‐kinase and PKC ∂activations.
Genomic Organization of BmTXKβ and BmTXKS2, Two Scorpion Venom Peptides From Buthus martensii Karsch
Authors: Cao Zhijian , Zhu Shunyi , Li Wenxin , Jiang Dahe , He Xiaohua , Liu Hui
Published: July 2002

Toelucidate genomic organization of BmTXK βand BmTXKS2, two scorpion venom peptides from Chinese scorpion Buthus martensii Karsch(BmK) were first isolated and their genomic regions characterized using the PCR method. Analysis of nucleotide sequence shows that there exists different intron location in the venom genes. The region encoding mature peptide of BmTXK βis disrupted by an intron with 886 bp, whereas the intron of BmTXKS2 is located within its propeptide coding region, which is different from other scorpion toxin genes with their introns within the signal peptide coding region.
Structural Features of Membrane Fusion between Influenza Virus and Liposome as Revealed by Quick-Freezing Electron Microscopy
Authors: Kanaseki Toku , Kawasaki Kazunori , Murata Masayuki , Ikeuchi Yoko , Ohnishi Shun-ichi
Published: July 2002

The structure of membrane fusion intermediates between the A/PR/8(H1N1) strain of influenza virus and a liposome composed of egg phosphatidylcholine, cholesterol, and glycophorin was studied using quick-freezing electron microscopy. Fusion by viral hemagglutinin protein was induced at pH 5.0 and 23°C. After a 19-s incubation under these conditions, small protrusions with a diameter of 10–20 nm were found on the fractured convex faces of the liposomal membranes, and small pits complementary to the protrusions were found on the concave faces. The protrusions and pits corresponded to fractured parts of outward bendings of the lipid bilayer or “microprotrusions of the lipid bilayer.” At the loci of the protrusions and pits, liposomal membranes had local contacts with viral membranes. In many cases both the protrusions and the pits were aligned in regular polygonal arrangements, which were thought to reflect the array of hemagglutinin spikes on the viral surface. These structures were induced only when the medium was acidic with the virus present. Based on these observations, it was concluded that the microprotrusions of the lipid bilayer are induced by hemagglutinin protein. Furthermore, morphological evidence for the formation of the “initial fusion pore” at the microprotrusion was obtained. The protrusion on the convex face sometimes had a tiny hole with a diameter of <4 nm in the center. The pits transformed into narrow membrane connections <10 nm in width, bridging viruses and liposomes. The structures of the fusion pore and fusion neck with larger sizes were also observed, indicating growth of the protrusions and pits to distinct fusion sites. We propose that the microprotrusion of the lipid bilayer is a fusion intermediate induced by hemagglutinin protein, and suggest that the extraordinarily high curvature of this membrane structure is a clue to the onset of fusion. The possible architecture of the fusion intermediate is discussed with regard to the localization of intramembrane particles at the microprotrusion.
Role of Ryanodine Receptors in the Assembly of Calcium Release Units in Skeletal Muscle
Authors: Protasi Feliciano , Franzini-Armstrong Clara , Allen Paul D.
Published: July 2002

Abstract. In muscle cells, excitation–contraction (e–c) coupling is mediated by “calcium release units,” junctions between the sarcoplasmic reticulum (SR) and exterior membranes. Two proteins, which face each other, are known to functionally interact in those structures: the ryanodine receptors (RyRs), or SR calcium release channels, and the dihydropyridine receptors (DHPRs), or L-type calcium channels of exterior membranes. In skeletal muscle, DHPRs form tetrads, groups of four receptors, and tetrads are organized in arrays that face arrays of feet (or RyRs). Triadin is a protein of the SR located at the SR–exterior membrane junctions, whose role is not known. We have structurally characterized calcium release units in a skeletal muscle cell line (1B5) lacking Ry1R. Using immunohistochemistry and freeze-fracture electron microscopy, we find that DHPR and triadin are clustered in foci in differentiating 1B5 cells. Thin section electron microscopy reveals numerous SR–exterior membrane junctions lacking foot structures (dyspedic). These results suggest that components other than Ry1Rs are responsible for targeting DHPRs and triadin to junctional regions. However, DHPRs in 1B5 cells are not grouped into tetrads as in normal skeletal muscle cells suggesting that anchoring to Ry1Rs is necessary for positioning DHPRs into ordered arrays of tetrads. This hypothesis is confirmed by finding a “restoration of tetrads” in junctional domains of surface membranes after transfection of 1B5 cells with cDNA encoding for Ry1R.
Dynamics of Axonal Microtubules Regulate the Topology of New Membrane Insertion into the Growing Neurites
Authors: Zakharenko Stanislav , Popov Sergey
Published: July 2002

Nerve growth depends on the delivery of cell body–synthesized material to the growing neuronal processes. The cellular mechanisms that determine the topology of new membrane addition to the axon are not known. Here we describe a technique to visualize the transport and sites of exocytosis of cell body– derived vesicles in growing axons. We found that in Xenopus embryo neurons in culture, cell body–derived vesicles were rapidly transported all the way down to the growth cone region, where they fused with the plasma membrane. Suppression of microtubule (MT) dynamic instability did not interfere with the delivery of new membrane material to the growth cone region; however, the insertion of vesicles into the plasma membrane was dramatically inhibited. Local disassembly of MTs by focal application of nocodazole to the middle axonal segment resulted in the addition of new membrane at the site of drug application. Our results suggest that the local destabilization of axonal MTs is necessary and sufficient for the delivery of membrane material to specific neuronal sites.
Attenuation of Colon Inflammation through Activators of the Retinoid X Receptor (Rxr)/Peroxisome Proliferator–Activated Receptor γ (Pparγ) Heterodimer
Authors: Desreumaux Pierre , Dubuquoy Laurent , Nutten Sophie , Peuchmaur Michel , Englaro Walter , Schoonjans Kristina , Derijard Benoit , Desvergne Beatrice , Wahli Walter , Chambon Pierre , Leibowitz Mark D. , Colombel Jean-Frédéric , Auwerx Johan
Published: July 2002

The peroxisome proliferator–activated receptor γ (PPARγ) is highly expressed in the colon mucosa and its activation has been reported to protect against colitis. We studied the involvement of PPARγ and its heterodimeric partner, the retinoid X receptor (RXR) in intestinal inflammatory responses. PPARγ1/− and RXRα1/− mice both displayed a significantly enhanced susceptibility to 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis compared with their wild-type littermates. A role for the RXR/PPARγ heterodimer in the protection against colon inflammation was explored by the use of selective RXR and PPARγ agonists. TNBS-induced colitis was significantly reduced by the administration of both PPARγ and RXR agonists. This beneficial effect was reflected by increased survival rates, an improvement of macroscopic and histologic scores, a decrease in tumor necrosis factor α and interleukin 1β mRNA levels, a diminished myeloperoxidase concentration, and reduction of nuclear factor κB DNA binding activity, c-Jun NH2-terminal kinase, and p38 activities in the colon. When coadministered, a significant synergistic effect of PPARγ and RXR ligands was observed. In combination, these data demonstrate that activation of the RXR/PPARγ heterodimer protects against colon inflammation and suggest that combination therapy with both RXR and PPARγ ligands might hold promise in the clinic due to their synergistic effects.
Quantitative Regulation of Class Switch Recombination by Switch Region Transcription
Authors: Lee Chung-Gi , Kinoshita Kazuo , Arudchandran Arulvathani , Cerritelli Susana M. , Crouch Robert J. , Honjo Tasuku
Published: July 2002

The isotype specificity of immunoglobulin (Ig) class switching is regulated by a cytokine which induces transcription of a specific switch (S) region, giving rise to so-called germline transcripts. Although previous studies have demonstrated that germline transcription of an S region is required for class switch recombination (CSR) of that particular S region, it has not been shown whether the level of S region transcription affects the efficiency of CSR. We addressed this question by using an artificial DNA construct containing a constitutively transcribed μ switch (Sμ) region and an α switch (Sα) region driven by a tetracycline-responsive promoter. The construct was introduced into a switch-inducible B lymphoma line and the quantitative correlation between Sα region transcription and class switching efficiency was evaluated. The level of Sα transcription was linearly correlated with CSR efficiency, reaching a plateau at saturation. On the other hand, we failed to obtain the evidence to support involvement of either RNA–DNA heteroduplex or trans germline transcripts in CSR. Taken together, it is likely that S region transcription and/or transcript processing in situ may be required for CSR. We propose that because of the unusual properties of S region DNA, transcription induces the DNA to transiently be single stranded, permitting secondary structure(s) to form. Such structures may be recognition targets of a putative class switch recombinase.
Proton Pump Activity of Mitochondria-rich Cells
Authors: Jensen Lars J. , Sørensen Jens N. , Larsen E. Hviid , Willumsen Niels J.
Published: July 2002

We have hypothesized that a major role of the apical H+-pump in mitochondria-rich (MR) cells of amphibian skin is to energize active uptake of Cl− via an apical Cl−/HCO3−-exchanger. The activity of the H+ pump was studied by monitoring mucosal [H+]-profiles with a pH-sensitive microelectrode. With gluconate as mucosal anion, pH adjacent to the cornified cell layer was 0.98 ± 0.07 (mean ± SEM) pH-units below that of the lightly buffered bulk solution (pH = 7.40). The average distance at which the pH-gradient is dissipated was 382 ± 18 μm, corresponding to an estimated “unstirred layer” thickness of 329 ± 29 μm. Mucosal acidification was dependent on serosal pCO2, and abolished after depression of cellular energy metabolism, confirming that mucosal acidification results from active transport of H+. The [H+] was practically similar adjacent to all cells and independent of whether the microelectrode tip was positioned near an MR-cell or a principal cell. To evaluate [H+]-profiles created by a multitude of MR-cells, a mathematical model is proposed which assumes that the H+ distribution is governed by steady diffusion from a number of point sources defining a set of particular solutions to Laplace's equation. Model calculations predicted that with a physiological density of MR cells, the [H+] profile would be governed by so many sources that their individual contributions could not be experimentally resolved. The flux equation was integrated to provide a general mathematical expression for an external standing [H+]–gradient in the unstirred layer. This case was treated as free diffusion of protons and proton-loaded buffer molecules carrying away the protons extruded by the pump into the unstirred layer; the expression derived was used for estimating stationary proton-fluxes. The external [H+]-gradient depended on the mucosal anion such as to indicate that base (HCO3−) is excreted in exchange not only for Cl −, but also for Br− and I−, indicating that the active fluxes of these anions can be attributed to mitochondria-rich cells.
Effect of Alkali Metal Cations on Slow Inactivation of Cardiac Na+ Channels
Authors: Townsend Claire , Horn Richard
Published: July 2002

Human heart Na+ channels were expressed transiently in both mammalian cells and Xenopus oocytes, and Na+ currents measured using 150 mM intracellular Na+. The kinetics of decaying outward Na+ current in response to 1-s depolarizations in the F1485Q mutant depends on the predominant cation in the extracellular solution, suggesting an effect on slow inactivation. The decay rate is lower for the alkali metal cations Li+, Na+, K+, Rb+, and Cs+ than for the organic cations Tris, tetramethylammonium, N-methylglucamine, and choline. In whole cell recordings, raising [Na+]o from 10 to 150 mM increases the rate of recovery from slow inactivation at −140 mV, decreases the rate of slow inactivation at relatively depolarized voltages, and shifts steady-state slow inactivation in a depolarized direction. Single channel recordings of F1485Q show a decrease in the number of blank (i.e., null) records when [Na+]o is increased. Significant clustering of blank records when depolarizing at a frequency of 0.5 Hz suggests that periods of inactivity represent the sojourn of a channel in a slow-inactivated state. Examination of the single channel kinetics at +60 mV during 90-ms depolarizations shows that neither open time, closed time, nor first latency is significantly affected by [Na+]o. However raising [Na+]o decreases the duration of the last closed interval terminated by the end of the depolarization, leading to an increased number of openings at the depolarized voltage. Analysis of single channel data indicates that at a depolarized voltage a single rate constant for entry into a slow-inactivated state is reduced in high [Na+]o, suggesting that the binding of an alkali metal cation, perhaps in the ion-conducting pore, inhibits the closing of the slow inactivation gate.
Permeation and Gating of an Inwardly Rectifying Potassium Channel
Authors: Choe Han , Sackin Henry , Palmer Lawrence G.
Published: July 2002

Permeation, gating, and their interrelationship in an inwardly rectifying potassium (K+) channel, ROMK2, were studied using heterologous expression in Xenopus oocytes. Patch-clamp recordings of single channels were obtained in the cell-attached mode. The gating kinetics of ROMK2 were well described by a model having one open and two closed states. One closed state was short lived (∼1 ms) and the other was longer lived (∼40 ms) and less frequent (∼1%). The long closed state was abolished by EDTA, suggesting that it was due to block by divalent cations. These closures exhibit a biphasic voltage dependence, implying that the divalent blockers can permeate the channel. The short closures had a similar biphasic voltage dependence, suggesting that they could be due to block by monovalent, permeating cations. The rate of entering the short closed state varied with the K+ concentration and was proportional to current amplitude, suggesting that permeating K+ ions may be related to the short closures. To explain the results, we propose a variable intrapore energy well model in which a shallow well may change into a deep one, resulting in a normally permeant K+ ion becoming a blocker of its own channel.
Distinct Structural Requirements for Clustering and Immobilization of K+ Channels by PSD-95
Authors: Burke Nancy A. , Takimoto Koichi , Li Danqing , Han Weiping , Watkins Simon C. , Levitan Edwin S.
Published: July 2002

PDZ-domain–containing proteins such as PSD-95 have been implicated in the targeting and clustering of membrane proteins. Biochemical and immunohistochemical studies indicate that PSD-95 recognizes COOH-terminal S/TXV sequences present in Kv1 K+ channels. However, the effect of binding a PDZ domain on a target protein has not been studied in live cells. In the present study, a green fluorescent protein–Kv1.4 fusion protein is used to study the effect of PSD-95 on channel movement. Fluorescence recovery after photobleaching showed that PSD-95 can immobilize K+ channels in the plasma membrane in an all-or-none manner. Furthermore, time lapse imaging showed that channel clusters formed in the presence of PSD-95 are stable in size, shape, and position. As expected from previous reports, two green fluorescent protein–tagged COOH-terminal variants of Kv1.4, Δ15 and V655A, are not clustered by PSD-95. However, coexpression of PSD-95 with V655A, but not Δ15, leads to the appearance of PSD-95 immunoreactivity in the plasma membrane. Furthermore, fluorescence recovery after photobleaching studies show that V655A channels are immobilized by PSD-95. Thus, V655A channels can interact with PSD-95 in a manner that leads to channel immobilization, but not clustering. These experiments document for the first time that PSD-95 immobilizes target proteins. Additionally, the data presented here demonstrate that the structural requirements for protein clustering and immobilization by PSD-95 are distinct.
Regulation of Type 1 Inositol 1,4,5-Trisphosphate–gated Calcium Channels by InsP3 and Calcium
Authors: Moraru I.I. , Kaftan E.J. , Ehrlich B.E. , Watras J.
Published: July 2002

Cytosolic calcium acts as both a coagonist and an inhibitor of the type 1 inositol 1,4,5-trisphosphate (InsP3)–gated Ca channel, resulting in a bell-shaped Ca dependence of channel activity (Bezprozvanny, I., J. Watras, and B.E. Ehrlich. 1991. Nature. 351:751–754; Finch, E.A., T.J. Turner, and S.M. Goldin. 1991. Science. 252: 443–446; Iino, M. 1990. J. Gen. Physiol. 95:1103–1122). The ability of Ca to inhibit channel activity, however, varies dramatically depending on InsP3 concentration (Combettes, L., Z. Hannaert-Merah, J.F. Coquil, C. Rousseau, M. Claret, S. Swillens, and P. Champeil. 1994. J. Biol. Chem. 269:17561–17571; Kaftan, E.J., B.E. Ehrlich, and J. Watras. 1997. J. Gen. Physiol. 110:529–538). In the present report, we have extended the characterization of the effect of cytosolic Ca on both InsP3 binding and InsP3-gated channel kinetics, and incorporated these data into a mathematical model capable of simulating channel kinetics. We found that cytosolic Ca increased the Kd of InsP3 binding ∼3.5-fold, but did not influence the maximal number of binding sites. The ability of Ca to decrease InsP3 binding is consistent with the rightward shift in the bell-shaped Ca dependence of InsP3-gated Ca channel activity. High InsP3 concentrations are able to overcome the Ca-dependent inhibition of channel activity, apparently due to a low affinity InsP3 binding site (Kaftan, E.J., B.E. Ehrlich, and J. Watras. 1997. J. Gen. Physiol. 110:529–538). Constants from binding analyses and channel activity determinations were used to develop a mathematical model that fits the complex Ca-dependent regulation of the type 1 InsP3-gated Ca channel. This model accurately simulated both steady state data (channel open probability and InsP3 binding) and kinetic data (channel activity and open time distributions), and yielded testable predictions with regard to the regulation of this intracellular Ca channel. Information gained from these analyses, and our current molecular model of this Ca channel, will be important for understanding the basis and regulation of intracellular Ca waves and oscillations in intact cells.
The Block of Shaker K+ Channels by κ-Conotoxin Pviia Is State Dependent
Authors: Terlau Heinrich , Boccaccio Anna , Olivera Baldomero M. , Conti Franco
Published: July 2002

κ-conotoxin PVIIA is the first conotoxin known to interact with voltage-gated potassium channels by inhibiting Shaker-mediated currents. We studied the mechanism of inhibition and concluded that PVIIA blocks the ion pore with a 1:1 stoichiometry and that binding to open or closed channels is very different. Open-channel properties are revealed by relaxations of partial block during step depolarizations, whereas double-pulse protocols characterize the slower reequilibration of closed-channel binding. In 2.5 mM-[K+]o, the IC50 rises from a tonic value of ∼50 to ∼200 nM during openings at 0 mV, and it increases e-fold for about every 40-mV increase in voltage. The change involves mainly the voltage dependence and a 20-fold increase at 0 mV of the rate of PVIIA dissociation, but also a fivefold increase of the association rate. PVIIA binding to Shaker Δ6-46 channels lacking N-type inactivation or to wild phenotypes appears similar, but inactivation partially protects the latter from open-channel unblock. Raising [K+]o to 115 mM has little effect on open-channel binding, but increases almost 10-fold the tonic IC50 of PVIIA due to a decrease by the same factor of the toxin rate of association to closed channels. In analogy with charybdotoxin block, we attribute the acceleration of PVIIA dissociation from open channels to the voltage-dependent occupancy by K+ ions of a site at the outer end of the conducting pore. We also argue that the occupancy of this site by external cations antagonizes on binding to closed channels, whereas the apparent competition disappears in open channels if the competing cation can move along the pore. It is concluded that PVIIA can also be a valuable tool for probing the state of ion permeation inside the pore.
The Spark and Its Ember
Authors: González A. , Kirsch W.G. , Shirokova N. , Pizarro G. , Stern M.D. , Ríos E.
Published: July 2002

Amplitude, spatial width, and rise time of Ca2+ sparks were compared in frog fast-twitch muscle, in three conditions that alter activation of release channels by [Ca2+]. A total of ∼17,000 sparks from 30 cells were evaluated. In cells under voltage clamp, caffeine (0.5 or 1 mM) increased average spark width by 28%, rise time by 18%, and amplitude by 7%. Increases in width were significant even among events of the same rise time. Spontaneous events recorded in permeabilized fibers with low internal [Mg2+] (0.4 mM), had width and rise times greater than in reference, and not significantly different than those in caffeine. The spark average in reference rides on a continuous fluorescence “ridge” and is continued by an “ember,” a prolongation of width ∼1 μm and amplitude <0.2, vanishing in ∼100 ms. Ridge and ember were absent in caffeine and in permeabilized cells. Exposure of voltage-clamped cells to high internal [Mg2+] (7 mM) had effects opposite to caffeine, reducing spark width by 26% and amplitude by 27%. In high [Mg2+], the ember was visible in individual sparks as a prolongation of variable duration and amplitude up to 1.2. Based on simulations and calculation of Ca2+ release flux from averaged sparks, the increase in spark width caused by caffeine was interpreted as evidence of an increase in radius of the release source—presumably by recruitment of additional channels. Conversely, spark narrowing suggests loss of contributing channels in high Mg2+. Therefore, these changes in spark width at constant rise times are evidence of a multichannel origin of sparks. Because ridge and ember were reduced by promoters of Ca2+-dependent activation (caffeine, low [Mg2+]) and became more visible in the presence of its inhibitors, they are probably manifestations of Ca2+ release directly operated by voltage sensors.
Ion Interactions in the High-Affinity Binding Locus of a Voltage-Gated Ca2+ Channel
Authors: Cloues Robin K. , Cibulsky Susan M. , Sather William A.
Published: July 2002

The selectivity filter of voltage-gated Ca2+ channels is in part composed of four Glu residues, termed the EEEE locus. Ion selectivity in Ca2+ channels is based on interactions between permeant ions and the EEEE locus: in a mixture of ions, all of which can pass through the pore when present alone, those ions that bind weakly are impermeant, those that bind more strongly are permeant, and those that bind more strongly yet act as pore blockers as a consequence of their low rate of unbinding from the EEEE locus. Thus, competition among ion species is a determining feature of selectivity filter function in Ca2+ channels. Previous work has shown that Asp and Ala substitutions in the EEEE locus reduce ion selectivity by weakening ion binding affinity. Here we describe for wild-type and EEEE locus mutants an analysis at the single channel level of competition between Cd2+, which binds very tightly within the EEEE locus, and Ba2+ or Li+, which bind less tightly and hence exhibit high flux rates: Cd2+ binds to the EEEE locus ∼104× more tightly than does Ba2+, and ∼108× more tightly than does Li+. For wild-type channels, Cd2+ entry into the EEEE locus was 400× faster when Li+ rather than Ba2+ was the current carrier, reflecting the large difference between Ba2+ and Li+ in affinity for the EEEE locus. For the substitution mutants, analysis of Cd2+ block kinetics shows that their weakened ion binding affinity can result from either a reduction in blocker on rate or an enhancement of blocker off rate. Which of these rate effects underlay weakened binding was not specified by the nature of the mutation (Asp vs. Ala), but was instead determined by the valence and affinity of the current-carrying ion (Ba2+ vs. Li+). The dependence of Cd2+ block kinetics upon properties of the current-carrying ion can be understood by considering the number of EEEE locus oxygen atoms available to interact with the different ion pairs.
Electrostatic and Steric Contributions to Block of the Skeletal Muscle Sodium Channel by μ-Conotoxin
Authors: Hui Kwokyin , Lipkind Gregory , Fozzard Harry A. , French Robert J.
Published: July 2002

Pore-blocking toxins are valuable probes of ion channels that underlie electrical signaling. To be effective inhibitors, they must show high affinity and specificity and prevent ion conduction. The 22-residue sea snail peptide, μ-conotoxin GIIIA, blocks the skeletal muscle sodium channel completely. Partially blocking peptides, derived by making single or paired amino acid substitutions in μ-conotoxin GIIIA, allow a novel analysis of blocking mechanisms. Replacement of one critical residue (Arg-13) yielded peptides that only partially blocked single-channel current. These derivatives, and others with simultaneous substitution of a second residue, were used to elucidate the structural basis of the toxin's blocking action. The charge at residue-13 was the most striking determinant. A positive charge was necessary, though not sufficient, for complete block. Blocking efficacy increased with increasing residue-13 side chain size, regardless of charge, suggesting a steric contribution to inhibition. Charges grouped on one side of the toxin molecule at positions 2, 12, and 14 had a weaker influence, whereas residue-16, on the opposite face of the toxin, was more influential. Most directly interpreted, the data suggest that one side of the toxin is masked by close apposition to a binding surface on the pore, whereas the other side, bearing Lys-16, is exposed to an aqueous cavity accessible to entering ions. Strong charge-dependent effects emanate from this toxin surface. In the native toxin, Arg-13 probably presents a strategically placed electrostatic barrier rather than effecting a complete steric occlusion of the pore. This differs from other well-described channel inhibitors such as the charybdotoxin family of potassium channel blockers and the sodium channel-blocking guanidinium toxins (tetrodotoxin and saxitoxin), which appear to occlude the narrow part of the pore.
PASSML: combining evolutionary inference and protein secondary structure prediction.
Authors: Li√≤ P , Goldman N , Thorne J L , Jones3 D T
Published: July 2002

MOTIVATION: Evolutionary models of amino acid sequences can be adapted to incorporate structure information; protein structure biologists can use phylogenetic relationships among species to improve prediction accuracy. Results : A computer program called PASSML ('Phylogeny and Secondary Structure using Maximum Likelihood') has been developed to implement an evolutionary model that combines protein secondary structure and amino acid replacement. The model is related to that of Dayhoff and co-workers, but we distinguish eight categories of structural environment: alpha helix, beta sheet, turn and coil, each further classified according to solvent accessibility, i.e. buried or exposed. The model of sequence evolution for each of the eight categories is a Markov process with discrete states in continuous time, and the organization of structure along protein sequences is described by a hidden Markov model. This paper describes the PASSML software and illustrates how it allows both the reconstruction of phylogenies and prediction of secondary structure from aligned amino acid sequences. AVAILABILITY: PASSML 'ANSI C' source code and the example data sets described here are available at http://ng-dec1.gen.cam.ac.uk/hmm/Passml.html and 'downstream' Web pages. CONTACT: P.Lio@gen.cam.ac.uk
Disperse: a simple and efficient approach to parallel database
searching
Authors: Clifford Raphaël , Mackey Aaron J.
Published: July 2002

Summary: A general system for performing multiple independent database searches in parallel is presented. Run-time addition and removal of clients, robust failure and error trapping and near 100% efficiency with very large numbers of clients are achieved by a flexible asynchronous, client-driven approach.

Availability: Disperse software is freely available. Please visit http://www.doc.ic.ac.uk/~rc5/Disperse/for further details.

Contact: r.clifford@ic.ac.uk, amackey@virginia.edu