Magnetic microsphere and its use in biochemical separation and analysis

Magnetic microsphere comprises a magnetically responsive metal or metal oxide core surrounded by a polymer shell with active groups. Nowadays, methods of directly coating polymer, monomer polymerazation, impregnation, extrusion and biological synthesis are generally used to prepare magnetic particles. This kind of superparamagnetic microspheres can be attached to chemical, biochemical and biological substances by their active groups, then applying a magnetic field to separate from the media. Preparation and utilization of magnetic microspheres in immunoassay, nucleic acid hybrization assay, gene sequencing, cell isolation, enzyme immoblization, receptor isolation and other Gelds are reviewed with 44 references in this paper. Also, the further development is outlooked.

Progress in electrochemiluminescent analysis

The recent progress in electrochemiluminescent (ECL) assay is reviewed. This review begins with the fundamental researches in ECL, including the discovery of new ECL-active species, such as biochemical, organic and metallorganic materials, digital modeling of ECL process, the flow cells used in ECL assay, and electrochemiluminescent sensor. The application of ECL in environmental analysis, immunoassay, nucleotide acid hybridization sensor. The applications of ECL in environmental analysis, immunooassay, nucleic acid hybridization assay, and other aspects are reviewed with the latest references in detail. Finally, the main problems in the further investigation are outlooked, so are its prospects.

Synthesis, electrochemistry, fluorescence and ECL of Ru (phen)(2) (dcbpy) (PF6)(2)

A new ECL-active species, Ru (phen)(2) (dcbpy) (PF6)(2), has been designed and synthesized. Its structure was confirmed by means of IR, ESI-MS and 2D NMR. Also, its properties of electrochemistry, fluorescence and ECL are reported, which have suggested a good hope of being used in electrochemiluminescent immunoassay and nucleic acid hybridization.

Potential-dependent adsorption/desorption of organic adsorbate at HOPG electrode and accompanying delamination of graphite surface

In situ electrochemical scanning tunneling microscopy, alternating current voltammetry, and electrochemical quartz crystal microbalance have been employed to follow the potential-dependent adsorption/desorption processes of nucleic acid bases on highly oriented pyrolytic graphite (HOPG) electrode. The results show that (i) potential-dependent adsorption/desorption of nucleic acid bases on HOPG electrode was accompanied by delamination of the HOPG surface, and the delamination initiates from steps or kinks on the electrode surface, which provide highly active sites for adsorption; (ii) the delamination usually occurred when the electrode potential was changed or when the electrode was at potentials where the phase transition of adsorbate occurred. These results suggest that the surface stress resulting from the interaction between the substrate and adsorbate, as well as the interaction due to potential-induced surface charge distribution and the hysteresis of charge equilibrium are the main factors resulting in HOPG delamination. (C) 1999 The Electrochemical Society. S0013-4651(97)12-013-4. All rights reserved.

Changes in RNA, DNA, protein contents and growth of turbot Scophthalmus maximus larvae and juveniles

The growth potential of turbot Scophthalmus maximus larvae and juveniles was studied using nucleic acid-based indices and protein variables. The experiment was carried out from 4 to 60 days post hatching (dph). A significant increase in instantaneous growth rate during metamorphosis and retarded growth rate during post-metamorphic phase were observed. Ontogenetic patterns of DNA, RNA and protein all showed developmental stage-specific traits. The RNA:DNA ratio decreased up to 12 dph, then increased rapidly till 19 dph and fluctuated until 35 dph followed by a decline to the end. The RNA:DNA ratio was positively correlated with growth rate of juveniles during the post-metamorphic phase, whereas this ratio was not a sensitive indicator of growth during the pre-metamorphic phase and metamorphosis. The protein:DNA ratio showed a similar tendency to the RNA:DNA ratio. Changes of DNA content and protein:DNA ratio revealed that growth of S. maximus performed mainly by hyperplasia from 4 to 12 dph and hypertrophy until 21 dph during the pre-metamorphic larval phase. Growth was dominantly hypertrophical from the early- to mid-metamorphosing phase and hyperplastic thereafter. The results show that the DNA content and protein:DNA ratio can evaluate growth rates of larval and juvenile S. maximus on a cellular level.

The complete mitochondrial genomes of two common shrimps (Litopenaeus vannamei and Fenneropenaeus chinensis) and their phylogenomic considerations

Complete mitochondrial genomes have proven extremely valuable in helping to understand the evolutionary relationships among metazoans. However, uneven taxon sampling may lead to unclear or even erroneous phylogenetic topologies. The decapod crustaceans are relatively well-sampled, but sampling is still uneven within this group. We have sequenced the mitochondrial genomes of two shrimps Litopenaeus vannamei and Fenneropenaeus chinensis. As seen in other metazoans, the genomes contain a standard set of 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes and an AT-rich non-coding region. The gene arrangements are consistent with the pancrustacean ground pattern. Both the pattern of gene rearrangements and phylogenomic analyses using concatenated nucleic acid and amino acid sequences of the 13 mitochondrial protein-coding genes strengthened the support that Caridea and Palinura are primitive members of Pleocyemata. These sequences, in combination with two previously published penaeid mitochondrial genomes, suggest that genera within the family Penaeidae have the following relationship: (((Penaeits + Fenneropenaett.) + Litopeiiaelts) + Marsupenaeus). The analyses of nucleic acid and amino acid sequences of the mitochondrial genomes also strongly support the monophyly of Penaeidae, Brachyura and Pleocyemata. In addition, the analyses of the average Ka/Ks in the 13 mitochondrial protein-coding genes of penaeid shrimps indicated a strong purifying selection within this group.

Correction to the pathogenic alternative splicing, caused by the common GNB3 c.825C>T allele, using a novel, antisense morpholino

The very common GNB3 c.825C>T polymorphism (rs5443), is present in approximately half of all human chromosomes. Significantly the presence of the GNB3 825T allele has been strongly associated, with predisposition to essential hypertension. Paradoxically the presence of the GNB3 825T allele, in exon 10, introduces a pathogenic alternative RNA splice site into the middle of exon 9. To attempt to correct this pathogenic aberrant splicing, we therefore bioinformatically designed, using a Gene Tools® algorithm, a GNB3 specific, antisense morpholino. It was hoped that this morpholino would behave in vitro as either a potential “ splice blocker and/or exon skipper, to both bind and inhibit/reduce the aberrant splicing of the GNB3, 825T allele. On transfecting a human lymphoblast cell line homozygous for the 825T allele, with this antisense morpholino, we encouragingly observed both a significant reduction (from ~58% to ~5%) in the production of the aberrant smaller GNB3 transcript, and a subsequent increase in the normal GNB3 transcript (from ~42% to ~95%). Our results demonstrate the potential use of a GNB3 specific antisense morpholino, as a pharmacogenetic therapy for essential hypertension.

Controlo do tratamento endodontico não-cirúrgico de dentes com diagnóstico de necrose pulpar associada ou não a lesão apical

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Medicina Dentária

Vírus e retrovírus

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

3'-UTR SIRF: A database for identifying clusters of short interspersed repeats in 3' untranslated regions

BACKGROUND:Short (~5 nucleotides) interspersed repeats regulate several aspects of post-transcriptional gene expression. Previously we developed an algorithm (REPFIND) that assigns P-values to all repeated motifs in a given nucleic acid sequence and reliably identifies clusters of short CAC-containing motifs required for mRNA localization in Xenopus oocytes.DESCRIPTION:In order to facilitate the identification of genes possessing clusters of repeats that regulate post-transcriptional aspects of gene expression in mammalian genes, we used REPFIND to create a database of all repeated motifs in the 3' untranslated regions (UTR) of genes from the Mammalian Gene Collection (MGC). The MGC database includes seven vertebrate species: human, cow, rat, mouse and three non-mammalian vertebrate species. A web-based application was developed to search this database of repeated motifs to generate species-specific lists of genes containing specific classes of repeats in their 3'-UTRs. This computational tool is called 3'-UTR SIRF (Short Interspersed Repeat Finder), and it reveals that hundreds of human genes contain an abundance of short CAC-rich and CAG-rich repeats in their 3'-UTRs that are similar to those found in mRNAs localized to the neurites of neurons. We tested four candidate mRNAs for localization in rat hippocampal neurons by in situ hybridization. Our results show that two candidate CAC-rich (Syntaxin 1B and Tubulin beta4) and two candidate CAG-rich (Sec61alpha and Syntaxin 1A) mRNAs are localized to distal neurites, whereas two control mRNAs lacking repeated motifs in their 3'-UTR remain primarily in the cell body.CONCLUSION:Computational data generated with 3'-UTR SIRF indicate that hundreds of mammalian genes have an abundance of short CA-containing motifs that may direct mRNA localization in neurons. In situ hybridization shows that four candidate mRNAs are localized to distal neurites of cultured hippocampal neurons. These data suggest that short CA-containing motifs may be part of a widely utilized genetic code that regulates mRNA localization in vertebrate cells. The use of 3'-UTR SIRF to search for new classes of motifs that regulate other aspects of gene expression should yield important information in future studies addressing cis-regulatory information located in 3'-UTRs.

Integrated genetic analysis systems

The overall objective of this thesis is to integrate a number of micro/nanotechnologies into integrated cartridge type systems to implement such biochemical protocols. Instrumentation and systems were developed to interface such cartridge systems: (i) implementing microfluidic handling, (ii) executing thermal control during biochemical protocols and (iii) detection of biomolecules associated with inherited or infectious disease. This system implements biochemical protocols for DNA extraction, amplification and detection. A digital microfluidic chip (ElectroWetting on Dielectric) manipulated droplets of sample and reagent implementing sample preparation protocols. The cartridge system also integrated a planar magnetic microcoil device to generate local magnetic field gradients, manipulating magnetic beads. For hybridisation detection a fluorescence microarray, screening for mutations associated with CFTR gene is printed on a waveguide surface and integrated within the cartridge. A second cartridge system was developed to implement amplification and detection screening for DNA associated with disease-causing pathogens e.g. Escherichia coli. This system incorporates (i) elastomeric pinch valves isolating liquids during biochemical protocols and (ii) a silver nanoparticle microarray for fluorescent signal enhancement, using localized surface plasmon resonance. The microfluidic structures facilitated the sample and reagent to be loaded and moved between chambers with external heaters implementing thermal steps for nucleic acid amplification and detection. In a technique allowing probe DNA to be immobilised within a microfluidic system using (3D) hydrogel structures a prepolymer solution containing probe DNA was formulated and introduced into the microfluidic channel. Photo-polymerisation was undertaken forming 3D hydrogel structures attached to the microfluidic channel surface. The prepolymer material, poly-ethyleneglycol (PEG), was used to form hydrogel structures containing probe DNA. This hydrogel formulation process was fast compared to conventional biomolecule immobilization techniques and was also biocompatible with the immobilised biomolecules, as verified by on-chip hybridisation assays. This process allowed control over hydrogel height growth at the micron scale.

A binding site for the cyclic adenosine 3',5'-monophosphate-response element-binding protein as a regulatory element in the grp78 promoter.

The 78-kDa glucose-regulated protein (GRP78) is ubiquitously expressed in many cell types. Its promoter contains multiple protein-binding sites and functional elements. In this study we examined a high affinity protein-binding site spanning bp -198 to -180 of the rat grp78 promoter, using nuclear extracts from both B-lymphoid and HeLa cells. This region contains a sequence TGACGTGA which, with the exception of one base, is identical to the cAMP-response element (CRE). Site-directed mutagenesis reveals that this sequence functions as a major basal level regulatory element in hamster fibroblast cells and is also necessary to maintain high promoter activity under stress-induced conditions. By gel mobility shift analysis, we detect two specific protein complexes. The major specific complex I, while immunologically distinct from the 42-kDa CRE-binding protein (CREB), binds most strongly to the grp site, but also exhibits affinity for the CRE consensus sequence. As such, complex I may consist of other members of the CREB/activating transcription factor protein family. The minor specific complex II consists of CREB or a protein antigenically related to it. A nonspecific complex III consists of the Ku autoantigen, an abundant 70- to 80-kDa protein complex in HeLa nuclear extracts. By cotransfection experiments, we demonstrate that in F9 teratocarcinoma cells, the grp78 promoter can be transactivated by the phosphorylated CREB or when the CREB-transfected cells are treated with the calcium ionophore A23187. The differential regulation of the grp78 gene by cAMP in specific cell types and tissues is discussed.

Interstitial insertion of varying amounts of ABL-containing genetic material into chromosome 22 in Ph-negative CML.

We studied the cells from three selected patients with Ph-chromosome-negative chronic myeloid leukemia (CML) by Southern blotting, polymerase chain reaction, and in situ hybridization of informative probes to metaphase chromosomes. All three patients had rearrangement of M-BCR sequences in the BCR gene and expression of one or other of the mRNA species characteristic of Ph-positive CML. Leukemic metaphases studied after trypsin-Giemsa banding were indistinguishable from normal. The ABL probe localized both to chromosome 9 and 22 in each case. A probe containing 3' M-BCR sequences localized only to chromosome 22, and not to chromosome 9 as would be expected in Ph-positive CML. Two new probes that recognize different polymorphic regions distal to the ABL gene on chromosome 9 in normal subjects localized exclusively to chromosome 9 in two patients and to both chromosomes 9 and 22 in one patient. These results show that Ph-negative CML with BCR rearrangement is associated with insertion of a variable quantity of chromosome 9 derived material into chromosome 22q11; there is no evidence for reciprocal translocation of material from chromosome 22 to chromosome 9.

Novel DNA sensor for guanine content.

The bifunctional complex [Ru(TAP)(2)POQ-Nmet](2+), 1, formed with a [Ru(TAP)(2)Phen](2+) metallic unit linked to a quinoline moiety, and [Ru(TAP)(2)Phen](2+), 2, as reference, have been tested as photoprobes of DNA. Interestingly, 1 exhibits an emission enhancement of a factor of 16-17 upon binding to calf thymus DNA. Moreover, this emission is modulated by the nucleic base content of the polynucleotide. It varies by almost an order of magnitude from a polynucleotide containing 100% of G-C to a guanine-free nucleic acid where the excited-state lifetime reaches about 2 micros. The origins of these interesting properties are analyzed by comparing 1 with reference 2 in the presence of different polynucleotides.

On the adsorption of hexaammineruthenium (III) at anionic self-assembled monolayers

The binding of the electroactive hexaammineruthenium (III) complex ions to anionic self-assembled monolayers (SAMs) has been investigated by means of chronocoulometry and ac voltammetry. From chronocoulometric data recorded in 10-2 M LiClO4 containing different [Ru(NH3)6]3+ concentrations, we have established the adsorption isotherm of [Ru(NH3)6]3+ on a compact monolayer of 2-mercaptobenzimidazole-5-sulfonate (MBIS) self-assembled on Au(1 1 1). The data were satisfactorily fitted to the linearized Langmuir adsorption isotherm and a binding constant of 4.0 (±0.4) × 106 M-1 has been determined. The electrostatic binding of [Ru(NH3)6]3+ to a dilute PNA-DNA monolayer formed after hybridization on a PNA-modified gold electrode by self-assembly from a mixed solution of mercaptobutan-1-ol and PNA oligonucleotides has been studied by ac voltammetry. The admittance of the PNA-modified electrode after hybridization with complementary DNA was measured in 0.01 M Tris-HCl buffer containing different [Ru(NH3)6]3+ concentrations. Based on these data, a binding constant of [Ru(NH3)6]3+ to the surface-confined PNA-DNA duplex was derived from the Langmuir isotherm and amounts to 2.9 (±0.3) × 105 M-1. As the interactions between [Ru(NH3)6]3+ and the immobilized PNA-DNA hybrids on the gold surface are essentially electrostatic, the adsorption of the highly charged cationic redox complex at low concentrations to the negatively charged PNA-DNA modified surface is in large competition with other monovalent cations present in the electrolyte at higher concentrations. The influence of competing sodium cations was thus studied by adding different NaCl concentrations in the 0.01 M Tris-HCl electrolyte. © 2008 Elsevier Ltd. All rights reserved.