A tree-based method for the rapid screening of chemical fingerprints

Background  

The fingerprint of a molecule is a bitstring based on its structure, constructed such that structurally similar molecules will have similar fingerprints. Molecular fingerprints can be used in an initial phase of drug development for identifying novel drug candidates by screening large databases for molecules with fingerprints similar to a query fingerprint.     

Recrafting the neighbor-joining method

Background  

The neighbor-joining method by Saitou and Nei is a widely used method for constructing phylogenetic trees. The formulation of the method gives rise to a canonical Θ(n ³) algorithm upon which all existing implementations are based.     

Dysfunction of store-operated calcium channel in muscle cells lacking mg29

The store-operated calcium channel (SOC) located in the plasma membrane (PM) mediates capacitative entry of extracellular calcium after depletion of intracellular calcium stores in the endoplasmic or sarcoplasmic reticulum (ER/SR). An intimate interaction between the PM and the ER/SR is essential for the operation of this calcium signalling pathway. Mitsugumin 29 (MG29) is a synaptophysin-family-related protein located in the junction between the PM and SR of skeletal muscle. Here, we identify SOC in skeletal muscle and characterise its regulation by MG29 and the ryanodine receptor (RyR) located in the SR. Targeted deletion of mg29 alters the junctional membrane structure, causes severe dysfunction of SOC and SR calcium homeostasis and increases the susceptibility of muscle to fatigue stimulation. Severe dysfunction of SOC is also identified in muscle cells lacking both type 1 and type 3 RyRs, indicating that SOC activation requires an intact interaction between the PM and the SR, and is linked to conformational changes of RyRs. Whereas defective SOC seems to be inconsequential to short-term excitation-contraction coupling, the slow cumulative calcium entry through SOC is crucial for long-term calcium homeostasis, such that reduced SOC activity exaggerates muscle fatigue under conditions of intensive exercise.     

δ-Toxin,unlike melittin,has only hemolytic activity and no antimicrobial activity: Rationalization of this specific biological activity

The antimicrobial activity of a synthetic peptide corresponding to -hemolysin had been examined. The peptide didnot exhibit antimicrobial activity against gram negative and gram positive micro-organisms unlike other hemolytic peptides like melittin. This lack of antibacterial activity arises due to the inability of -hemolysin to perturb the negatively charged bacterial cell surface and permeabilize the bacterial plasma membrane. However, the red blood cell surface has a structure considerably different from bacteria, and does not act as a barrier to molecules reaching the lipid membrane. Hence -toxin can lyse erythrocytes. Thus, the specificity in biological activity has been rationalized in terms of differences, in the interaction of the toxin with the bacterial and red blood cell surfaces.     

Ionophore-mediated transmembrane movement of divalent cations in small unilamellar liposomes: An evaluation of the chlortetracycline fluorescence technique and correlations with black lipid membrane studies

Summary Conceptual advances in the field of membrane transport have, in the main, utilized artificial membranes, both planar and vesicular. Systems of biological interest,viz., cells and organelles, resemble vesicles in size and geometry. Methods are, therefore, required to extend the results obtained with planar membranes to liposome systems. In this report we present an analysis of a fluorescence technique, using the divalent cation probe chlortetracycline, in small, unilamellar vesicles, for the study of divalent cation fluxes. An ion carrier (X537 A) and a pore former (alamethicin) have been studied. The rate of rise of fluorescence signal and the transmembrane ion gradient have been related to transmembrane current and potential, respectively. A second power dependence of ion conduction-including the electrically silent portion thereof — on X537 A concentration, has been observed. An exponential dependence of current on transmembrane potential in the case of alamethicin is also confirmed. Possible errors in the technique are discussed.     

Choline Transporter regulates olfactory habituation via a neuronal triad of excitatory,inhibitory and mushroom body neurons

Choline is an essential component of Acetylcholine (ACh) biosynthesis pathway which requires high-affinity Choline transporter (ChT) for its uptake into the presynaptic terminals of cholinergic neurons. Previously, we had reported a predominant expression of ChT in memory processing and storing region of the Drosophila brain called mushroom bodies (MBs). It is unknown how ChT contributes to the functional principles of MB operation. Here, we demonstrate the role of ChT in Habituation, a non-associative form of learning. Odour driven habituation traces are laid down in ChT dependent manner in antennal lobes (AL), projection neurons (PNs), and MBs. We observed that reduced habituation due to knock-down of ChT in MBs causes hypersensitivity towards odour, suggesting that ChT also regulates incoming stimulus suppression. Importantly, we show for the first time that ChT is not unique to cholinergic neurons but is also required in inhibitory GABAergic neurons to drive habituation behaviour. Our results support a model in which ChT regulates both habituation and incoming stimuli through multiple circuit loci via an interplay between excitatory and inhibitory neurons. Strikingly, the lack of ChT in MBs shows characteristics similar to the major reported features of Autism spectrum disorders (ASD), including attenuated habituation, sensory hypersensitivity as well as defective GABAergic signalling. Our data establish the role of ChT in habituation and suggest that its dysfunction may contribute to neuropsychiatric disorders like ASD.     

Solution conformations of peptides representing the sequence of the toxin pardaxin and analogues in trifluoroethanol-water mixtures: Analysis of CD spectra

The cytolytic activities and conformational properties of pardaxin (GFFALIPKIISSPLFKTLLSAVGSALSSSGEQE), a 33-residue linear peptide that exhibits unusual shark repellent and cytolytic activities, and its analogues have been examined in aqueous environment and trifluoroethanol (TFE) using CD spectroscopy. A peptide corresponding to the 1–26 segment and an analogue where P7 has been changed to A show greater hemolytic activity than pardaxin. While the peptide corresponding to the N-terminal 18-residue segment does not exhibit hemolytic activity, its analogue where P7 is replaced by A is hemolytic. The secondary structural propensities of the peptides were inferred by deconvolution of the experimental spectra into pure components. Pardaxin, its variant where proline at position 7 was replaced by alanine, and shorter peptides corresponding to N-terminal segments exist in multiple conformations in aqueous medium that are comprised of β-turn, β-sheet, and distorted helical structures. With increasing proportions of TFE, while helical conformation predominates in all the peptides, both distorted and the regular α-helices appear to be populated. Analysis of CD spectra by deconvolution methods appears to be a powerful tool for delineating multiple conformations in peptides, especially membrane-active peptides that encounter media of different polarity ranging from aqueous environment to one of low dielectric constant in the hydrophobic interior of membranes. Our study provides further insights into the structural requirements for the biological activity of pardaxin and related peptides. © 1997 John Wiley & Sons, Inc. Biopoly 41: 635–645, 1997     

Detection of peptides covalently modified with multiple fatty acids by MALDI-TOF mass spectrometry.

Analysis and characterization of membrane proteins and hydrophobic peptides by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) is a considerable challenge because of their lower ionization efficiency. Detergents are used to solubilize hydrophobic peptides and proteins. However, in MALDI-MS, the presence of detergents can cause considerable loss of signal intensity. The extent of interference depends on the matrix/sample preparation method and experimental conditions. In the present study, we have analyzed the MALDI response of multiple fatty acylated peptides in the presence of the matrices alpha-cyano-4-hydroxy cinnamic acid (HCCA) and 2,5-dihydroxy benzoic acid (DHB). The effect of adding the nonionic detergent n-octylglucoside (OG) was also examined. The presence of OG facilitated detection of tetrapalmitoylated peptide, particularly when HCCA was used as the matrix. When DHB was used as the matrix, good signal intensity was observed in the absence of OG. Lower laser pulse rate in the linear mode of analysis resulted in good signal intensity for the tetrapalmitoylated peptide. Conditions for obtaining good signal intensities for dipalmitoylated and N-myristoyl peptides with both HCCA and DHB as matrices were also investigated.     

Defective maintenance of intracellular Ca^2＋ homeostasis is linked to increasedmuscle fatigability in the MG29 null mice

Mitsugumin 29 (MG29) is a transmembrane protein that is normally found in the triad junction of skeletal muscle. Our previous studies have shown that targeted deletion of rag29 from the skeletal muscle resulted in abnormality of the triad junction structure, and also increased susceptibility to muscle fatigue. To elucidate the basis of these effects, we investigated the properties of Ca^2 uptake and -release in toxin-skinned Extensor Digitorium Longus (EDL) muscle fibers from control and rag29 knockout mice. Compared with the control muscle, submaximal Ca^2 uptake into the sarcoplasmic reticulum (SR) was slower and the storage of Ca^2 inside the SR was less in the mutant muscle, due to increased leakage process of Ca^2 movement across the SR. The leakage pathway is associated with the increased sensitivity of Ca^2 /caffeine -induced Ca^2 release to myoplasmic Ca^2 . Therefore, the increased fatigability of mutant EDL muscles can result from a combination of a slowing of Ca^2 uptake, modification of Ca^2 -induced Ca^2 release (CICR), and a reduction in total SR Ca^2 content.