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PubMed2004

Prediction of alpha-turns in proteins using PSI-BLAST profiles and secondary structure information.

In this paper a systematic attempt has been made to develop a better method for predicting alpha-turns in proteins. Most of the commonly used approaches in the field of protein structure prediction have been tried in this study, which includes statistical approach "Sequence Coupled Model" and machine learning approaches; i) artificial neural network (ANN); ii) Weka (Waikato Environment for Knowledge Analysis) Classifiers and iii) Parallel Exemplar Based Learning (PEBLS). We have also used multiple sequence alignment obtained from PSIBLAST and secondary structure information predicted by PSIPRED. The training and testing of all methods has been performed on a data set of 193 non-homologous protein X-ray structures using five-fold cross-validation. It has been observed that ANN with multiple sequence alignment and predicted secondary structure information outperforms other methods. Based on our observations we have developed an ANN-based method for predicting alpha-turns in proteins. The main components of the method are two feed-forward back-propagation networks with a single hidden layer. The first sequence-structure network is trained with the multiple sequence alignment in the form of PSI-BLAST-generated position specific scoring matrices. The initial predictions obtained from the first network and PSIPRED predicted secondary structure are used as input to the second structure-structure network to refine the predictions obtained from the first net. The final network yields an overall prediction accuracy of 78.0% and MCC of 0.16. A web server AlphaPred (http://www.imtech.res.in/raghava/alphapred/) has been developed based on this approach.

Amino Acid Sequence↗

PubMed2001

An alternative solid phase peptide fragment condensation protocol with improved efficiency.

The success of solid phase peptide synthesis is often limited by the aggregation of the growing peptide chains on the resin. Working from the results of a study of model coupling reactions in solution between Z-Gly-Phe-OH and H-Phe-OBzl, we have achieved higher efficiency in the repetitive solid phase fragment condensation of VGVAPG, in a 3:1 chloroform-phenol solvent system, using diisopropylcarbodiimide (DIC) as coupling agent, and a combination of 3-hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine (HODhbt) and its tetrabutyl ammonium salt as additive, than in DMF with DIC and HODhbt alone.

Amino Acid Sequence↗

PubMed2000

Prediction of tight turns and their types in proteins.

A tight turn in protein structure is defined as a site where (i) a polypeptide chain reverses its overall direction, i.e., leads the chain to fold back on itself by nearly 180 degrees, and (ii) the amino acid residues directly involved in forming the turn are no more than six. Tight turns are generally categorized as delta-turn, gamma-turn, beta-turn, alpha-turn, and pi-turn, which are formed by two-, three-, four-, five-, and six-amino-acid residues, respectively. According to the folding mode, each of such tight turns can be further classified into several different types. Tight turns play an important role in globular proteins from both the structural and functional points of view. In view of this, various efforts have been made to predict tight turns and their types. This Review summarizes the development in this area, with an emphasis focused on the most recent work concerned that is featured by the sequence-coupled model. Meanwhile, the future challenge in this area has also been briefly addressed.

Algorithms↗

PubMed2002

Non-newtonian behavior of an insoluble monolayer: effects of inertia.

Interfacial velocity measurements were performed in an optical annular channel, consisting of stationary inner and outer cylinders, a floor rotating at a constant rate, and a flat free surface on which an insoluble monolayer was initially spread. Measurements for essentially inviscid monolayers and some viscous monolayers on water show good agreement with numerical predictions for a Newtonian interface (Boussinesq-Scriven surface model) coupled to a bulk flow described by the Navier-Stokes equations. Here, we consider in detail a viscous monolayer, namely hemicyanine, and find that above a certain concentration, the monolayer does not behave Newtonian at a Reynolds number of about 250. We show that the discrepancies between the measurements and predicted Newtonian behavior are not due to compositional effects (i.e., nonuniform monolayer distribution), Reynolds number (i.e., inertia and/or secondary flows), or surface dilatational viscosity (which does not play any role in the parameter regime investigated). We show prima facie evidence that the observed shear thinning nature of the velocity profile is associated with a phase transition at C approximately 0.9 mg/m(2) at low Reynolds numbers. At large Reynolds numbers (Re=8500), hemicyanine is found to flow like a viscous Newtonian monolayer on the air/water interface, with viscosity dependent only on the local concentration.

Journal Article↗

PubMed1998

Contribution to the Analysis of the 3A2 <-- &Xtilde;1A1 "Wulf" Transition of Ozone by High-Resolution Fourier Transform Spectrometry.

The analysis of the rotational structure of the high-resolution Fourier transform 0(0)0 absorption spectrum of the 3A2 <-- &Xtilde;1A1 band system of the "Wulf" transition of the isotopomer 16O3 of ozone is reported for the first time. With a near pure case (b) coupling model for the upper triplet state, we have assigned a significant portion of the spectrum, mainly the F1 (J = N + 1) and F2 (J = N) spin components, primarily in the lower frequency region of the band. The lines corresponding to the F3 (J = N - 1) component are weak at lower frequencies and heavily congested in the central and higher frequency regions of the spectrum. Perturbations and predissociation phenomena have reduced the effective lifetime of the metastable 3A2 state and have also limited the number of transitions included in the least-squares fit of the band. Approximately 100 lines have been assigned in the range from 9100-9550 cm-1. Three rotational, three centrifugal distortion, three spin-rotation, and one spin-spin constant were varied. The geometry of the molecule in the 3A2 state, as determined from these constants, is r = 1.345 Å and theta = 98.9 degrees, in good agreement with ab initio results. Copyright 1998 Academic Press.

Journal Article↗

PubMed1999

Modulation of myofibroblast and smooth-muscle phenotypes in the lung.

Considerable progress has been made in defining the phenotype of contractile cells of the lung during development, in the adult and during the remodeling process. The high degree of phenotypic heterogeneity of subgroups of these cells, such as SMCs, is appreciated. Recent studies also have explored the relationship between phenotype and cell function, though this remains an important area for research in the coming years. Similarly, though our understanding of the regulation of cell phenotype is expanding rapidly, much remains to be done, particularly at the level of gene regulation. New transgenic models, coupled with gene-promotor analyses in transgenic animals and in cultured cells should allow rapid progress. Studies of the regulation of specific contractile and cytoskeletal proteins at the gene level by specific cytokines and extracellular-matrix elements will be particularly important.

Actins↗

PubMed1995

Two types of A-channels in Lymnaea neurons.

The gating mechanism of A-channels of Lymnaea neurons and the effect of tetraethylammonium (TEA) on these channels were studied using macroscopic recording techniques. Along with the fast-inactivating A-current (Iaf) described earlier we found a slow-inactivating A-current (Ias) in some neurons of the visceral ganglion. Both currents have revealed similar activation kinetics, but differ in the inactivation kinetics and mechanisms. The inactivation kinetics of Ias were satisfactorily described by a sum of two exponentials with rate constants (tau -1) of 28 s-1 and 4.5 s-1 at V = -20 mV. Intracellular TEA reduced the peak amplitudes of Iaf and Ias and slowed the rate of the fast phase of inactivation of Iaf. This resulted in a crossover of the current traces in the presence and absence of TEA, as though it competes with the binding of the inactivating particle. The mechanism of the fast phase of inactivation of Iaf is similar to that of fast inactivation of the Shaker K+ channels which appears to be due to a ball-and-chain mechanism. The slow phases of inactivation of Iaf and Ias reveal properties characteristic of C-type inactivation shown in Shaker K+ channels. A partially coupled model including three pathways for transition of a channel from the closed to open states accurately reproduces all of the experimental data. It has voltage-independent transitions to the inactivation states indicating that inactivation of A-current is not associated with charge movement through the membrane. The results suggest that Lymnaea A-channels seem to be heteromultimeric.

Action Potentials↗

PubMed1989

Membrane bending energy and shape determination of phospholipid vesicles and red blood cells.

A procedure is developed to calculate red blood cell and phospholipid vesicle shapes within the bilayer couple model of the membrane. The membrane is assumed to consist of two laterally incompressible leaflets which are in close contact but unconnected. Shapes are determined by minimizing the membrane bending energy at a given volume of a cell (V), given average membrane area (A) and given difference of the areas of two leaflets (delta A). Different classes of shapes exist in parts of the v/delta a phase diagram, where v and delta a are the volume and the leaflet area difference relative to the sphere with area A. The limiting shapes are composed of sections of spheres with only two values allowed for their radii. Two low energy axisymmetrical classes, which include discocyte and stomatocyte shapes are studied and their phase diagrams are analyzed. For v= 0.6, the discocyte is the lowest energy shape, which transforms by decreasing delta a continuously into a stomatocyte. The spontaneous membrane curvature (C0) and compressibility of membrane leaflets can be incorporated into the model. A model, where delta A is free and C0 determines the shapes at given V and A, is also studied. In this case, by decreasing C0, a discocyte transforms discontinuously into an almost closed stomatocyte.

Erythrocyte Membrane↗

PubMed1995

The quality of DNA double-strand breaks: a Monte Carlo simulation of the end-structure of strand breaks produced by protons and alpha particles.

The quality of DNA damage induced by protons and alpha-particles of various linear energy transfer (LET) was studied. The aim was to single out specific lesions in the DNA molecule that might lead to biological endpoints such as inactivation. A DNA model coupled with a track structure code (MOCA-15) were used to simulate the lesions induced on the two helixes. Four categories of DNA breaks were considered: single-strand breaks (ssb), blunt-ended double-strand breaks (dsb, with no or few overlapping bases), sticky-ended double-strand breaks (with cohesive free ends of many bases), and deletions (complex lesions which involve at least two dsb within a small number of base pairs). Calculations were carried out assuming various sets of parameters characterizing the production of these different DNA breaks. No large variations in the yields of ssb and blunt- or sticky-ended dsb were found in the LET range between 10 and 200 keV/mu m. On the other hand, the yield of deletions increases up to about 100 keV/mu m and seems to reach a plateau at higher LET values. In the LET interval from 30 to 60 keV/mu m, protons proved to be more efficient than alpha-particles in inducing deletions. The induction of these complex lesions is thus dependent not simply on LET but also on the characteristics of the track structure. Comparison with RBE values for cell killing shows that this special class of dsb might play an important role in radiation-induced cell inactivation.

Alpha Particles↗

PubMedDate not supplied

Low-conductivity calcium channels in the macrophage plasma membrane: activation by inositol-1,4,5-triphosphate.

Local voltage clamping was applied to mouse macrophage plasma membrane to study calcium channels activated by inositol-1,4,5-triphosphate (IP3) and blocked by heparin. These channels were clearly distinguished from IP3-activated channels of the endoplasmic reticulum by their low conductivity (about 1 pSm for 100 mM Ca2+), high selectivity for Ca2+ relative to K+ (P(Ca):P(K) > 1000), calcium inactivation, and activation on hyperpolarization; these properties allowed them to be assigned to the I(CRAC) family. On the other hand, the properties of the IP3 receptors of these channels (IP3R), i.e., the dose-dependent effect of IP3, the IP3 desensitization of the receptor, and the sensitivity to micromolar concentrations of heparin and arachidonic acid were close to those of the endoplasmic reticulum IP3 receptor. The most likely interpretation of these data is that IP3R are not located in the endoplasmic reticulum, but, acting via some kind of conformational change occurring on binding of IP3, transmit a signal from the endoplasmic reticulum to the highly selective Ca2+ channels. This point of view is in agreement with the published "coupling model" [1].

Animals↗

PubMed2006

Theoretical analysis of contributions of disuse, basic multicellular unit activation threshold, and osteoblastic formation threshold to changes in bone mineral density at menopause.

Although bone loss at menopause is known to be associated with disuse and estrogen deficit, their contributions and mechanisms are not completely clear. This article presents a computer simulation aimed at clarifying the contributions of various factors that may be responsible for osteoporosis in women. We utilized a mechanical-biological coupled model at the level of basic multicellular units (BMUs) to quantify disuse, changes in BMU activation threshold, and changes in osteoblastic formation threshold on a representative cross section of 100 mm(2) of cancellous bone in the lumbar spine of a postmenopausal woman. The separate and combined contributions were determined by a total of 17 parametrical analyses through comparison to clinical data presented in the literature. An increase in osteoblastic formation threshold alone cannot explain the bone loss patterns seen clinically. Bone mineral density predictions were much closer to the clinical data for the simulation of a maintained increase in BMU activation threshold during menopause or some time after menopause. Maintained disuse alone would result in high rates of bone loss long after menopause. A combination of disuse and an increase in osteoblastic formation threshold, or of an increase in BMU activation threshold and an increase in osteoblastic formation threshold, or of disuse and an increase in BMU activation threshold, or a combination of these three is capable of describing the bone loss found at menopause. It was found that disuse and an increase in the BMU activation threshold may be the major contributors to bone loss at menopause. Understanding the contributions of disuse and the changes in the BMU activation threshold and in the osteoblastic formation threshold that occur at menopause may help to gain more insight into the mechanism of osteoporosis and identify improved osteoporosis treatment and prevention.

Bone Density↗

PubMed2006

Molecular mobility in glass forming fananserine: a dielectric, NMR, and TMDSC investigation.

PURPOSE: This study was conducted to characterize the molecular mobility of supercooled fananserine and derive from this analysis the non-Arrhenius and nonexponential properties of the primary alpha-relaxation. METHODS: The use of three investigation techniques of the molecular mobility, namely, dielectric relaxation, modulated differential scanning calorimetry, and proton nuclear magnetic resonance, allowed us to describe the dynamic properties of supercooled fananserine on a wide range of frequencies and temperatures, ranging from the melting temperature T(m) = 372 K down to the glass transition temperature T(g) = 292 K. RESULTS: We emphasized the capacity of these three techniques to give a coherent set of information. We used the coupling-model theory to interpret the dielectric results. It allowed us to identify two relaxation processes (alpha and beta), corresponding to different molecular motions. The temperature evolution of the alpha-relaxation indicates that fananserine is a fragile glass former, as reflected by the steepness index value, m = 77. The temperature T(o) where the relaxation times diverge was also determined. CONCLUSIONS: The description of the dielectric relaxation data in terms of the Kohlrausch-Williams-Watt relaxation function has shown the existence of an additional low-amplitude relaxation process assigned to the so-called Johari-Goldstein process. Mainly concerned by the primary alpha-process directly involved in the glass formation, we derived from this analysis the characteristic features of this process and showed that supercooled fananserine is characterized by a strongly non-Arrhenius and nonexponential behavior.

Air Pressure↗

PubMed1985

The effect of temperature and chronic ethanol feeding on the proton electrochemical potential and phosphate potential in rat liver mitochondria.

The relationship between the proton electrochemical potential (delta mu H) and the maximal free energy of ATP hydrolysis (delta GP) in coupled respiring rat liver mitochondria was investigated as a function of temperature and chronic ethanol-feeding. The flow dialysis method was utilized to measure the temperature dependence of delta mu H from the uptake of 86Rb (in the presence of valinomycin) and [14C]DMO. delta GP in state 4 was determined by a null-point titration of the reversible, H+-coupled ATPase against the phosphate potential. delta mu H increases with temperature from 196 mV at 10 degrees C, to 217 mV at 40 degrees C. The maximal delta GP at state 4 decreases as a function of temperature from 67.8 kJ/mol at 10 degrees C, to 54.8 kJ/mol at 40 degrees C. As a result, the ratio delta GP/delta mu H decreases with temperature from 3.56 at 10 degrees C to 2.60 at 40 degrees C. Similar studies with mitochondria from rats which were chronically fed with ethanol show that, while delta GP at state 4 decreases in these rats from 61.2 to 56.0 (25 degrees C), the delta mu H is essentially unchanged at 212 mV. Thus the ratio delta GP/delta mu H in ethanol-fed rats at 25 degrees C is 2.77 as compared with 2.97 in control. Similar reduction of delta GP was observed in inverted inner membranes from ethanol-fed rats. Both the temperature dependence of delta GP/delta mu H and the effect of ethanol-feeding cannot be easily explained by the chemiosmotic hypothesis which postulates that delta mu H is the only driving force for ATP synthesis. In contrast, a parallel coupling model, which postulates that intramembrane proton transfer from redox pumps to ATPase is mediated by the formation of dynamic aggregates of the mitochondrial inner-membrane proteins, can easily accommodate these findings. Accordingly, the temperature effect is due to weakening of these fragile aggregates, while the ethanol-feeding effect is the result of reduced concentration of active pumps, which decrease the frequency of formation of functional aggregates.

Adenosine Triphosphate↗

PubMed1993

Effects of exogenous phospholipids on platelet activation.

Intercalation of amphipaths into the plasma membrane of platelets has a marked effect on their morphology. Incubation of platelets with phosphatidylcholines (PC) results in rounding of the platelet body and speculation, while incubation with aminophospholipids such as dilauroylphosphatidylserine (DLPS) results in a biphasic shape change consistent with the bilayer couple model (Sheetz, M.P. and Singer, S.J. (1982) Proc. Natl. Acad. Sci. USA 71, 4457-4461) and with the activity of an aminophospholipid translocator facilitating transverse bilayer diffusion (Daleke, D.L. and Huestis, W.H. (1985) Biochemistry 24, 5406-5416). The present study extends this work to investigate the effects of PC and PS on platelet responses to a natural agonist, thrombin. PC incorporation produces a concentration-dependent progression of shape changes, beginning with surface ruffling and development of fine spicules, followed by sphering of the cell body, and ending with the apparent loss of spicules. PC reduces platelet responses to thrombin only under conditions that promote membrane vesiculation, seen morphologically as a loss of spicules and biochemically as a loss of 14C-PC labeled membrane. PS homologues of varying acyl chain composition induce concentration- and time-dependent platelet sphering. Incorporation of PS inhibits thrombin-induced platelet shape change, granule secretion, and protein phosphorylation. Inhibition of these responses requires transit of the exogenous PS to the cytofacial leaflet of the membrane bilayer.

Blood Platelets↗

PubMed1984

On the logic of the application of double-inhibitor titrations for the elucidation of the mechanisms of energy coupling.

In the last couple of years the chemiosmotic hypothesis has been severely criticised, such that many research groups now consider it to be a less than exact description of biological energy transduction. The most potent experimental support for this view is based upon the technique known as the double-inhibitor titration [e.g., (1982) Biochem. J. 206, 351-357]. The results of such experiments have been considered by many to exclude 'unequivocally' the chemiosmotic coupling model. It will be shown that such unequivocal statements are not possible. An argument is put forward which shows how the chemiosmotic model may explain these titrations without any further elaborations of the original hypothesis.

Bacteria↗

PubMed1991

Diffusion of water in rhyolitic glasses.

Water dehydration experiments on rhyolitic glasses have been carried out at 400-550 degrees C under a N2 atmosphere. Concentration profiles of both H2O molecules and OH groups were measured by Fourier transform infrared spectroscopy. As found in previous studies of water diffusion in rhyolitic melts, the measured total water concentration profiles do not match expectations based on a single constant diffusion coefficient for total water. The diffusion of total water is described by considering the diffusion of both H2O molecules and OH groups and the reaction between them. The concentration relationship between the two species has been obtained from direct infrared measurement on quenched experimental charges. The quench is inferred to be rapid enough to preserve concentrations of both species at experimental temperature based on experimental results designed to examine reaction kinetics. The measured species concentrations along diffusion profiles show that local equilibrium between H2O and OH is approximately reached at high temperatures and high water contents. However, at lower water content or lower temperature, local equilibrium is not reached. In treating the diffusion problem, this disequilibrium effect is partially compensated by using empirical relationships between H2O and OH concentrations based on measurements, instead of using an equilibrium relationship. It is thus possible to obtain diffusion coefficients for both species from their concentration profiles. The diffusion coefficient of OH is found to be negligible compared to that of H2O at 403-530 degrees C (DOH < 0.02 DH2O and could be much smaller); i.e., H2O is the dominant diffusing species even at total water concentration as low as 0.2 wt%. The variation of OH concentration along the diffusion profile is inferred to be due to the local interconversion between OH groups and H2O molecules; the reaction also provides the diffusing H2O species. DH20 values are found to vary by less than a factor of 2 over a total water concentration range of 0.2 to 1.7 wt%. This simple model, coupled with the assumption of local equilibrium between H2O and OH, yields a very good fit to the data from diffusion-couple experiments of LAPHAM et al. (1984) at 850 degrees C. When our data are combined with DH2O obtained from that fit, DH2O (in m2/s) is given by: ln DH2O = (-14.59 +/- 1.59) - (103000 +/- 5000) / RT; 673 K < or = T < or = 1123 K, where T is temperature in K and R is the gas constant in J K-1 mol-1. This equation also approximates well DH2O values calculated from previous measurements of concentration-dependent bulk water diffusion coefficients of KARSTEN et al. (1982). The diffusion of H2O is also compared to the diffusion of the noble gas elements. The activation energy for diffusion in rhyolitic glasses is well correlated with neutral species radii of He, Ne, H2O, and Ar. This supports the contention that the diffusing species for "water" is neutral molecular H2O. The role of speciation may also be important in understanding the diffusion of many other multi-species components, and the effect can be treated in a similar fashion as that during water diffusion.

Diffusion↗

PubMed1993

The signal transduction between beta-receptors and adenylyl cyclase.

The beta-adrenergic receptor-dependent adenylyl cyclase system is the most extensively studied G-protein-coupled system. Studies of the coupling between the receptor and effector can provide an insight into the nature of all these systems in general. In the activation of adenylyl cyclase by the receptor, the binding of an agonist to the stimulatory receptor (Rs) and the binding of GTP to the G-protein (Gs) are both required to activate the catalytic moiety (C). The active state decays as GTP is hydrolysed to GDP and inorganic phosphate (Pi), but reactivation occurs as GTP is replenished. The receptor acts as a catalyst, i.e. one agonist-bound receptor can activate numerous adenylyl cyclase molecules. Kinetic studies led to the formulation of the 'collision coupling' model of receptor activation and show that Gs protein does not shuttle between the receptor and cyclase. The Gs protein appears to undergo conformational changes between an 'open' state in which it can bind with GTP, and a 'closed' state unable to achieve this binding. This mechanism of activation does not involve the dissociation of Gs or of Gi. A model which fits the experimental data suggests that Gi*GTP affects cyclase only in its Gs-activated state via the G alpha 1 subunit, but that the oligomeric state of Gi is required for inhibition. The site on C which interacts with Gi is formed only when C is activated by Gs.

Adenylyl Cyclases↗

PubMed1994

Differential effects of carbachol on calcium entry and release in CHO cells expressing the m3 muscarinic receptor.

Calcium signalling was examined in CHO-k1 cells that stably express the m3 subtype of the muscarinic receptor. The calcium indicator Fura-2 was retained in these cells only in the presence of probenecid (1 mM), suggesting that Fura-2 efflux was mediated by an organic anion transporter. The addition of carbachol (CCh) to Fura-2 loaded cells in suspension caused a rapid transient increase in intracellular calcium [Ca]i followed by a smaller sustained plateau phase. The transient rise in [Ca]i was dose-dependent with a threshold response of 89 +/- 18 nM above baseline with 10 nM CCh and a maximum stimulation of 734 +/- 46 nM with 10 microM CCh. This phase was accompanied by a similar dose-dependent stimulation of total inositol phosphate production and was assumed to be generated by release from intracellular stores of the endoplasmic reticulum (ER). The sustained increase in [Ca]i was generated by entry from the extracellular bath since it was blocked by pretreatment with La3+ (1 microM) and was absent when bath calcium was chelated with EGTA. This phase was not dependent on CCh dose, and a stimulation of [Ca]i of approximately 90 nM above baseline was observed with CCh concentrations between 50 nM and 10 microM. With this dose range, the rate of Mn2+ quenching of Fura-2 at the Ca-insensitive excitation wavelength of 360 nm was likewise maximally stimulated. At lower CCh concentrations (10-50 nM), it was clear that the activation of Ca entry could not be dissociated from a threshold release of Ca from intracellular stores. The phorbol ester PMA, which uncouples the muscarinic receptor from phospholipase C, reduced the transient rise in [Ca]i by approximately 50% with little or no effect on Ca entry at higher CCh levels (> or = 1 microM). At lower CCh concentrations (< or = 100 nM) however, pretreatment with PMA completely blocked all Ca mobilization and supports the contention that Ca entry is coupled to Ca release from stores or to store depletion. The emptying of inositol trisphosphate-sensitive stores with thapsigargin (10 nM) stimulated Ca entry and also the rate of Mn2+ quenching. Store depletion by incubation in Ca-free media likewise stimulated Mn2+ uptake without a rise in [Ca]i. Our data are therefore consistent with a 'capacitative' coupling model, whereby the activation of the plasma membrane receptor leads to an InsP3-induced change in the degree of filling of the ER Ca pool.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗