Molecular characterization and phylogenetic analysis of a penaeidin-like antimicrobial peptide, Fi-penaeidin from Fenneropenaeus indicus

Antimicrobial peptides (AMPs) play a major role in innate immunity. Penaeidins are a family of AMPs that appear to be expressed in all penaeid shrimps. Penaeidins are composed of an N-terminal proline-rich domain, followed by a C-terminal domain containing six cysteine residues organized in two doublets. This study reports the first penaeidin AMP sequence, Fi-penaeidin (GenBank accession number HM243617) from the Indian white shrimp, Fenneropenaeus indicus. The full length cDNA consists of 186 base pairs encoding 61 amino acidswith an ORF of 42 amino acids and contains a putative signal peptide of 19 amino acids. Comparison of F. indicus penaeidin (Fi-penaeidin) with other known penaeidins showed that it shared maximum similarity with penaeidins of Farfantepenaeus paulensis and Farfantepenaeus subtilis (96% each). Fi-penaeidin has a predicted molecular weight (MW) of 4.478 kDa and theoretical isoelectric point (pI) of 5.3

Molecular Characterisation and Phylogenetic Analysis of a Novel Isoform of Hepatic Antimicrobial Peptide, Hepcidin (Zc-hepc1), from the Coral Fish Moorish idol, Zanclus cornutus (Linnaeus,1758)

Hepcidin is a family of short cysteine-rich antimicrobial peptides (AMPs) participating in various physiological functions with inevitable role in host immune responses. Present study deals with identification and characterisation of a novel hepcidin isoform from coral fish Zanclus cornutus. The 81 amino acid (aa) preprohepcidin obtained from Z. cornutus consists of a hydrophobic aa rich 22 mer signal peptide, a highly variable proregion of 35 aa and a bioactive mature peptide with 8 conserved cysteine residues which contribute to the disulphide back bone. The mature hepcidin, Zc-hepc1 has a theoretical isoelectric point of 7.46, a predicted molecular weight of 2.43 kDa and a net positive charge of ?1. Phylogenetic analysis grouped Z. cornutus hepcidin with HAMP2 group hepcidins confirming the divergent evolution of hepcidin-like peptide in fishes. Zc-hepc1 can attain a b-hairpin-like structure with two antiparallel b-sheets. This is the first report of an AMP from the coral fish Z. cornutus.

Production, purification and partial characterization of a novel protease from marine Engyodontium album BTMFS10 under solid state fermentation

Engyodontium album isolated from marine sediment produced protease, which was active at pH 11. Process parameters influencing the production of alkaline protease by marine E. album was optimized. Particle size of <425 mm, 60% initial moisture content and incubation at 25 8C for 120 h were optimal for protease production under solid state fermentation (SSF) using wheat bran. The organism has two optimal pH (5 and 10) for maximal enzyme production. Sucrose as carbon source, ammonium hydrogen carbonate as additional inorganic nitrogen source and amino acid leucine enhanced enzyme production during SSF. The protease was purified and partially characterized. A 16-fold purified enzyme was obtained after ammonium sulphate precipitation and ion-exchange chromatography. Molecular weight of the purified enzyme protein was recorded approximately 38 kDa by SDS-PAGE. The enzyme showed maximum activity at pH 11 and 60 8C. Activity at high temperature and high alkaline pH suggests suitability of the enzyme for its application in detergent industry

Extracellular β-glucosidase Production by a Marine Aspergillus sydowii BTMFS 55 under Solid State Fermentation Using Statistical Experimental Design

A potential fungal strain producing extracellular β-glucosidase enzyme was isolated from sea water and identified as ^ëéÉêJ Öáääìë=ëóÇçïáá BTMFS 55 by a molecular approach based on 28S rDNA sequence homology which showed 93% identity with already reported sequences of ^ëéÉêÖáääìë=ëóÇçïáá in the GenBank. A sequential optimization strategy was used to enhance the production of β-glucosidase under solid state fermentation (SSF) with wheat bran (WB) as the growth medium. The two-level Plackett-Burman (PB) design was implemented to screen medium components that influence β-glucosidase production and among the 11 variables, moisture content, inoculums, and peptone were identified as the most significant factors for β-glucosidase production. The enzyme was purified by (NH4)2SO4 precipitation followed by ion exchange chromatography on DEAE sepharose. The enzyme was a monomeric protein with a molecular weight of ~95 kDa as determined by SDS-PAGE. It was optimally active at pH 5.0 and 50°C. It showed high affinity towards éNPG and enzyme has a hã and sã~ñ of 0.67 mM and 83.3 U/mL, respectively. The enzyme was tolerant to glucose inhibition with a há of 17 mM. Low concentration of alcohols (10%), especially ethanol, could activate the enzyme. A considerable level of ethanol could produce from wheat bran and rice straw after 48 and 24 h, respectively, with the help of p~ÅÅÜ~êçãóÅÉë=ÅÉêÉîáëá~É in presence of cellulase and the purified β-glucosidase of ^ëéÉêÖáääìë=ëóÇçïáá BTMFS 55.

Characterization of the molecular clockwork in the cockroach Rhyparobia maderae

Der Wechsel von Tag und Nacht erzeugt einen regelmäßigen Rhythmus von verschiedenen Umweltreizen, allen voran Licht und Temperatur. Fast jedes bis zum heutigen Tage untersuchte Lebewesen besitzt einen endogenen Mechanismus zur Zeitwahrnehmung, und diese "innere Uhr" befähigt Lebewesen dazu, sich vorausschauend an rhythmische Umwelt-Änderungen anzupassen. Circadiane Rhythmen bestehen auch ohne jegliche äußere Reize und basieren auf einem molekularen Rückkopplungs-Mechanismus, der Rhythmen in Genexpression und Proteinkonzentration von etwa 24 Stunden erzeugt. Obwohl sich die grundsätzlichen Mechanismen und Komponenten dieses molekularen Uhrwerks in allen Insekten ähneln, zeigte sich jedoch immer mehr, dass es im Detail doch wesentliche Unterschiede zwischen verschiedenen Insektengruppen gibt. Während das molekulare Uhrwerk der Fruchtfliege Drosophila melanogaster inzwischen sehr gut untersucht ist, fehlen bei den meisten Insektengruppen immernoch eingehende Untersuchungen. Fast nichts ist über die molekulare Basis von circadianen Rhythmen bei der Schabe Rhyparobia maderae bekannt, obwohl diese Art bereits seit Langem als Modellorganismus in der Chronobiologie dient. Um mit der Forschung am molekularen, circadianen System von R. maderae zu beginnen, wurde die Struktur und das Expressionsprofil der core feedback loop Gene per, tim1 und cry2 analysiert. Mittels degenerierten Primern und RACE konnte das vollständige offene Leseraster (OLR) von rmPer und rmCry2, und ein Teil des rmTim1 OLR kloniert werden. Eine phylogenetische Analyse gruppierte rmPER und rmCRY2 gemeinsam mit den Orthologa hemimetaboler Insekten. Viele bei D. melanogaster funktionell charakterisierte Domänen sind bei diesen Proteinen konserviert, was auf eine ähnliche Funktion in der inneren Uhr von R. maderae hinweist. Mittels quantitativer PCR konnte gezeigt werden, dass die mRNA von rmPer, rmTim1 und rmCry2 in verschiedenen Lichtregimen in der gleichen Phasenlage Tageszeit-abhängig schwankt. Die Phasenlage stellte sich bei unterschiedlichen Photoperioden jeweils relativ zum Beginn der Skotophase ein, mit Maxima in der ersten Hälfte der Nacht. Auch im Dauerdunkel zeigen sich Rhythmen in der rmTim1 und rmCry2 Expression. Die Amplitude der rmPer Expressionsrhythmen war jedoch so gering, dass keine signifikanten Unterschiede zwischen den einzelnen Zeitgeberzeiten (ZT) festgestellt werden konnten. Mittels Laufrad-Assays wurde untersucht wie Kurz- und Langtag Lichtregime die Verhaltensrhythmen beeinflussen. Es konnten nur Unterschiede in der Periodenlänge unter freilaufenden Bedingungen festgestellt werden, wenn höhere Lichtintensitäten (1000lx) zur Synchronisation (entrainment) genutzt wurden. Die Periode des freilaufenden Rhythmus war bei Tieren aus dem Kurztag länger. Die photoperiodische Plastizität zeigte sich also auch auf Verhaltensebene, obwohl höhere Lichtintensitäten notwendig waren um einen Effekt zu beobachten. Basierend auf den Sequenzen der zuvor klonierten OLR wurden gegen rmPER, rmTIM1 und rmCRY2 gerichtete Antikörper hergestellt. Die Antikörper gegen rmPER und rmTIM1 erkannten in western blots sehr wahrscheinlich spezifisch das jeweilige Protein. Zeitreihen von Gehirngewebe-Homogenisaten zeigten keinen offensichtlichen circadianen Rhythmus in der Proteinkonzentration, wahrscheinlich auf Grund einer Oszillation mit niedriger Amplitude. In Immunhistochemischen Färbungen konnte nur mit dem gegen rmPER gerichteten Antikörper aus Kaninchen ein Signal beobachtet werden. Beinahe jede Zelle des Zentralnervensystems war rmPER-immunreaktiv im Zellkern. Es konnten keine Unterschiede zwischen den untersuchten ZTs festgestellt werden, ähnlich wie bei den western blot Zeitreihen. In dieser Studie konnten erstmals molekulare Daten der circadianen Uhr von R. maderae erfasst und dargestellt werden. Die Uhrgene per, tim1 und cry2 werden in dieser Schabenart exprimiert und ihre Domänenstruktur sowie das circadiane Expressionsmuster ähneln dem hypothetischen ursprünglichen Insektenuhrwerk, welches der circadianen Uhr von Vertebraten nahesteht. Das molekulare Uhrwerk von R. maderae kann sich an unterschiedliche Photoperioden anpassen, und diese Anpassungen manifestieren sich im Expressionsprofil der untersuchten Uhrgene ebenso wie im Verhalten.

Targeted Stimuli-Responsive Dextran Conjugates for Doxorubicin Delivery to Hepatocytes

A targeted, stimuli-responsive, polymeric drug delivery vehicle is being developed in our lab to help alleviate severe side-effects caused by narrow therapeutic window drugs. Targeting specific cell types or organs via proteins, specifically, lectin-mediated targeting holds potential due to the high specificity and affinity of receptor-ligand interactions, rapid internalization, and relative ease of processing. Dextran, a commercially available, biodegradable polymer has been conjugated to doxorubicin and galactosamine to target hepatocytes in a three-step, one-pot synthesis. The loading of doxorubicin and galactose on the conjugates was determined by absorbance at 485 nm and elemental analysis, respectively. Conjugation efficiency based on the amount loaded of each reactant varies from 20% to 50% for doxorubicin and from 2% to 20% for galactosamine. Doxorubicin has also been attached to dextran through an acid-labile hydrazide bond. Doxorubicin acts by intercalating with DNA in the nuclei of cells. The fluorescence of doxorubicin is quenched when it binds to DNA. This allows a fluorescence-based cell-free assay to evaluate the efficacy of the polymer conjugates where we measure the fluorescence of doxorubicin and the conjugates in increasing concentrations of calf thymus DNA. Fluorescence quenching indicates that our conjugates can bind to DNA. The degree of binding increases with polymer molecular weight and substitution of doxorubicin. In cell culture experiments with hepatocytes, the relative uptake of polymer conjugates was evaluated using flow cytometry, and the killing efficiency was determined using the MTT cell proliferation assay. We have found that conjugate uptake is much lower than that of free doxorubicin. Lower uptake of conjugates may increase the maximum dose of drug tolerated by the body. Also, non-galactosylated conjugate uptake is lower than that of the galactosylated conjugate. Microscopy indicates that doxorubicin localizes almost exclusively at the nucleus, whereas the conjugates are present throughout the cell. Doxorubicin linked to dextran through a hydrazide bond was used to achieve improved killing efficiency. Following uptake, the doxorubicin dissociates from the polymer in an endosomal compartment and diffuses to the nucleus. The LC₅₀ of covalently linked doxorubicin is 7.4 μg/mL, whereas that of hydrazide linked doxorubicin is 4.4 μg/mL.

Presión arterial en preescolares de la localidad de Usaquén en Bogotá

Introducción: La hipertensión arterial es un problema de salud pública tanto en países industrializados como en vía de desarrollo. Su prevalencia en la infancia viene en aumento por lo que es relevante determinarla en niños preescolares a nivel local. Objetivo: Determinar la prevalencia de hipertensión arterial en niños de tres a cinco años de una cohorte de 14 hogares infantiles del ICBF de la localidad de Usaquén en Bogotá. Materiales y métodos: Se realizó un estudio de corte transversal analítico, utilizando la base de datos de un ensayo aleatorizado y controlado del año 200913, y se evaluaron las cifras de tensión arterial de acuerdo a sexo, edad, talla y su correlación con el IMC con un nivel de confianza del 95% y precisión del 1%. Se calcularon las medias, desviaciones estándar, percentiles y prevalencia. Resultados: Se obtuvo una muestra de 1035 casos, encontrándose una prevalencia de 4,5% de HTA sistólica, 10,4% de diastólica, ambas en estadio I; teniendo en cuenta tanto sistólica como diastólica, fue de 11,6% en estadio I. Se determinaron los valores de presión arterial sistólica y diastólica en cuartiles de acuerdo a edad, sexo y talla. El coeficiente de correlación entre el IMC y los niveles de presión arterial sistólica y diastólica fueron de 0.0992 y 0.0362 respectivamente. Conclusión: La prevalencia de HTA general fue de 11,6%, predominando la diastólica en estadio I en niños preescolares. No se encontró correlación entre el IMC y las cifras de tensión arterial sistólica y diastólica.

Bioinspired iron and manganese catalysts for the effective and selective oxidation of alkanes and alkenes

La gran eficiència, selectivitat i les condicions suaus exhibides per les reaccions que tenen lloc al centre actiu de les metal·looxigenases són la font d'inspiració per la present dissertació. Amb l'objectiu de dissenyar catalitzadors d'oxidació eficients hem fet ús de dues estratègies: la primera consisteix en el disseny de complexos amb baix pes molecular inspirats en aspectes estructurals de la primera esfera de coordinació del centre metàl·lic d'enzims de ferro i de manganès. Aquests complexos s'han estudiat com a catalitzadors en l'oxidació selectiva d'alcans i d'alquens fent servir oxidants "verds" com ara l'H2O2. La segona estratègia està basada en l'ús de la química supramolecular per tal de desenvolupar estructures moleculars auto-acoblades amb la forma i les propietats químiques desitjades. Concretament, la construcció de nanocontenidors amb un catalitzador d'oxidació incrustat a la seva estructura ens permetria dur a terme reaccions més selectives, tal com passa en les reaccions catalitzades per enzims.

Linear viscoelasticity from molecular dynamics simulation of entangled polymers

The linear viscoelastic (LVE) spectrum is one of the primary fingerprints of polymer solutions and melts, carrying information about most relaxation processes in the system. Many single chain theories and models start with predicting the LVE spectrum to validate their assumptions. However, until now, no reliable linear stress relaxation data were available from simulations of multichain systems. In this work, we propose a new efficient way to calculate a wide variety of correlation functions and mean-square displacements during simulations without significant additional CPU cost. Using this method, we calculate stress−stress autocorrelation functions for a simple bead−spring model of polymer melt for a wide range of chain lengths, densities, temperatures, and chain stiffnesses. The obtained stress−stress autocorrelation functions were compared with the single chain slip−spring model in order to obtain entanglement related parameters, such as the plateau modulus or the molecular weight between entanglements. Then, the dependence of the plateau modulus on the packing length is discussed. We have also identified three different contributions to the stress relaxation:  bond length relaxation, colloidal and polymeric. Their dependence on the density and the temperature is demonstrated for short unentangled systems without inertia.

Enhanced phytoextraction: In search of EDTA alternatives

Enhanced phytoextraction proposes the use of soil amendments to increase the heavy-metal content of above-ground harvestable plant tissues. This study compares the effect of synthetic aminopolycarboxylic acids [ethylenediamine tetraacetatic acid (EDTA), nitriloacetic acid (NTA), and diethylenetriamine pentaacetic acid (DTPA)] with a number of biodegradable, low-molecular weight, organic acids (citric acid, ascorbic acid, oxalic acid, salicylic acid, and NH4 acetate) as potential soil amendments for enhancing phytoextraction of heavy metals (Cu, Zn, Cd, Pb, and Ni) by Zea mays. The treatments in this study were applied at a dose of 2 mmol/kg(-1) 1 d before sowing. To compare possible effects between presow and postgermination treatments, a second smaller experiment was conducted in which EDTA, citric acid, and NH4 acetate were added 10 d after germination as opposed to 1 d before sowing. The soil used in this screening was a moderately contaminated topsoil derived from a dredged sediment disposal site. This site has been in an oxidized state for more than 8 years before being used in this research. The high carbonate, high organic matter, and high clay content characteristic to this type of sediment are thought to suppress heavy-metal phytoavailability. Both EDTA and DTPA resulted in increased levels of heavy metals in the above-ground biomass. However, the observed increases in uptake were not as large as reported in the literature. Neither the NTA nor organic acid treatments had any significant effect on uptake when applied prior to sowing. This was attributed to the rapid mineralization of these substances and the relatively low doses applied. The generally low extraction observed in this experiment restricts the use of phytoextraction as an effective remediation alternative under the current conditions, with regard to amendments used, applied dose (2 mmol/kg(-1) soil), application time (presow), plant species (Zea mays), and sediment (calcareous clayey soil) under study.

Selective and highly efficient dye scavenging by a pH-responsive molecular hydrogelator

A structurally simple low molecular weight hydrogelator derived from isophthalic acid forms robust pH-responsive hydrogels capable of highly efficient and selective dye adsorption.

Rapid parallel expression in E-coli and insect cells: Analysis of five lef gene products of the Autographa californica multiple nuclear polyhedrosis virus (AcMNPV)

A number of strategies are emerging for the high throughput (HTP) expression of recombinant proteins to enable structural and functional study. Here we describe a workable HTP strategy based on parallel protein expression in E. coli and insect cells. Using this system we provide comparative expression data for five proteins derived from the Autographa californica polyhedrosis virus genome that vary in amino acid composition and in molecular weight. Although the proteins are part of a set of factors known to be required for viral late gene expression, the precise function of three of the five, late expression factors (lefs) 6, 7 and 10, is unknown. Rapid expression and characterisation has allowed the determination of their ability to bind DNA and shown a cellular location consistent with their properties. Our data point to the utility of a parallel expression strategy to rapidly obtain workable protein expression levels from many open reading frames (ORFs).

Catalytic oxidation of alcohols using molecular oxygen mediated by poly(ethylene glycol)-supported nitroxyl radicals

The selective catalytic oxidation of alcohols over a mixture of copper(l) chloride and a number of linear 'linker-less' or 'branched' poly(ethylene glycol)-supported nitroxyl radicals of the 2,2,6,6-tetramethyl-piperidine-1-oxyl (TEMPO) family as a catalyst system has been investigated in the presence of molecular oxygen in a batch reactor. It is found that the activity profile of the polymer-supported nitroxyl radicals is in good agreement with that of low-molecular weight nitroxyl catalysts, for example, allylic and benzylic alcohols are oxidised faster than aliphatic alcohols. The oxidations can be tuned to be highly selective such that aldehydes are the only oxidation products observed in the oxidation of primary alcohols and the oxidations of secondary alcohols yield the corresponding ketones. A strong structural effect of the polymeric nitroxyl species on catalytic activity that is dependent upon their spatial orientation of the nitroxyl radicals is particularly noted. The new soluble macromolecular catalysts can be recovered readily from the reaction mixture by solvent precipitation and filtration. In addition, the recycled catalysts demonstrate a similar selectivity with only a small decrease in activity compared to the fresh catalyst even after five repetitive cycles. (c) 2005 Elsevier B.V. All rights reserved.

Purification and molecular cloning of antimicrobial peptides from Scots pine seedlings

A novel protocol for rapid and efficient purification of antimicrobial peptides from plant seedlings has been developed. Two peptides with antimicrobial activity, designated p1 and p2, were purified nearly to homogeneity from Scots pine seedlings by a combination of sulfuric acid extraction, ammonium sulfate precipitation, heat-inactivation and ion-exchange chromatography on phosphocellulose. Purified proteins had molecular masses of 11 kDa (p1) and 5.8 kDa (p2) and were identified by mass spectrometry as defensin and lipid-transfer protein, respectively. We demonstrated their growth inhibitory effects against a group of phytopathogenic fungi. Furthermore, we report for the first time molecular cloning and characterization of defensin I cDNA from Scots pine. A cDNA expression library from 7 days Scots pine seedlings was generated and used to isolate a cDNA clone corresponding to Scots pine defensin, termed PsDef1. The full-length coding sequence of PsDef1 is 252 bp in length and has an open reading frame capable to encode a protein of 83 amino residues. The deduced sequence has the typical features of plant defensins, including an endoplasmic reticulum signal sequence of 33 aa, followed by a characteristic defensin domain of 50 amino acids representing its active form. The calculated molecular weight of the mature form of PsDef1 is 5601.6 Da, which correlates well with the results of SDS-PAGE analysis. Finally, the antimicrobial properties of PsDef1 against a panel of fungi and bacteria define it as a member of the morphogenic group of plant defensins. (C) 2009 Elsevier Inc. All rights reserved.

The impact of flavonoids on memory: physiological and molecular considerations

Emerging evidence suggests that a group of dietary-derived phytochemicals known as flavonoids are able to induce improvements in memory acquisition, consolidation, storage and retrieval. These low molecular weight polyphenols are widespread in the human diet, are absorbed to only a limited degree and localise in the brain at low concentration. However, they have been found to be highly effective in reversing age-related declines in memory via their ability to interact with the cellular and molecular architecture of the brain responsible for memory. These interactions include an ability to activate signalling pathways, critical in controlling synaptic plasticity, and a potential to induce vascular effects capable of causing new nerve cell growth in the hippocampus. Their ability to activate the extracellular signal-regulated kinase (ERK1/2) and the protein kinase B (PKB/Akt) signalling pathways, leading to the activation of the cAMP response element-binding protein (CREB), a transcription factor responsible for increasing the expression of a number of neurotrophins important in de. ning memory, will be discussed. How these effects lead to improvements in memory through induction of synapse growth and connectivity, increases in dendritic spine density and the functional integration of old and new neurons will be illustrated. The overall goal of this critical review is to emphasize future areas of investigation as well as to highlight these dietary agents as promising candidates for the design of memory-enhancing drugs with relevance to normal and pathological brain ageing (161 references).