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DNA Technology (dna + technology)
Kinds of DNA Technology Selected AbstractsRapid Polymerase Chain Reaction-based Screening Assay for Bacterial Biothreat AgentsACADEMIC EMERGENCY MEDICINE, Issue 4 2008Samuel Yang MD Abstract Objectives:, To design and evaluate a rapid polymerase chain reaction (PCR)-based assay for detecting Eubacteria and performing early screening for selected Class A biothreat bacterial pathogens. Methods:, The authors designed a two-step PCR-based algorithm consisting of an initial broad-based universal detection step, followed by specific pathogen identification targeted for identification of the Class A bacterial biothreat agents. A region in the bacterial 16S rRNA gene containing a highly variable sequence flanked by clusters of conserved sequences was chosen as the target for the PCR assay design. A previously described highly conserved region located within the 16S rRNA amplicon was selected as the universal probe (UniProbe, Integrated DNA Technology, Coralville, IA). Pathogen-specific TaqMan probes were designed for Bacillus anthracis, Yersinia pestis, and Francisella tularensis. Performance of the assay was assessed using genomic DNA extracted from the aforementioned biothreat-related organisms (inactivated or surrogate) and other common bacteria. Results:, The UniProbe detected the presence of all tested Eubacteria (31/31) with high analytical sensitivity. The biothreat-specific probes accurately identified organisms down to the closely related species and genus level, but were unable to discriminate between very close surrogates, such as Yersinia philomiragia and Bacillus cereus. Conclusions:, A simple, two-step PCR-based assay proved capable of both universal bacterial detection and identification of select Class A bacterial biothreat and biothreat-related pathogens. Although this assay requires confirmatory testing for definitive species identification, the method has great potential for use in ED-based settings for rapid diagnosis in cases of suspected Category A bacterial biothreat agents. [source] Applications of mass spectrometry for the structural characterization of recombinant protein pharmaceuticalsMASS SPECTROMETRY REVIEWS, Issue 3 2007Catherine A. Srebalus Barnes Abstract Therapeutic proteins produced using recombinant DNA technologies are generally complex, heterogeneous, and subject to a variety of enzymatic or chemical modifications during expression, purification, and long-term storage. The use of mass spectrometry (MS) for the evaluation of recombinant protein sequence and structure provides detailed information regarding amino acid modifications and sequence alterations that have the potential to affect the safety and activity of therapeutic protein products. General MS approaches for the characterization of recombinant therapeutic protein products will be reviewed with particular attention given to the standard MS tools available in most biotechnology laboratories. A number of recent examples will be used to illustrate the utility of MS strategies for evaluation of recombinant protein heterogeneity resulting from post-translational modifications (PTMs), sequence variations generated from proteolysis or transcriptional/translational errors, and degradation products which are formed during processing or final product storage. Specific attention will be given to the MS characterization of monoclonal antibodies as a model system for large, glycosylated, recombinant proteins. Detailed examples highlighting the use of MS for the analysis of monoclonal antibody glycosylation, deamidation, and disulfide mapping will be used to illustrate the application of these techniques to a wide variety of heterogeneous therapeutic protein products. The potential use of MS to support the selection of cell line/clone selection and formulation development for therapeutic antibody products will also be discussed. © 2007 Wiley Periodicals, Inc., Mass Spec Rev [source] Detection of resistance to acetolactate synthase inhibitors in weeds with emphasis on DNA-based techniques: a reviewPEST MANAGEMENT SCIENCE (FORMERLY: PESTICIDE SCIENCE), Issue 7 2006Cheryl-Ann L Corbett Abstract Resistance to herbicides inhibiting acetolactate synthase (ALS) has been increasing at a faster rate than in any other herbicide group. The great majority of these cases are due to various single-nucleotide polymorphisms in the ALS gene endowing target site resistance. Many diagnostic techniques have been devised in order to confirm resistance and help producers to adopt the best management strategies. Recent advances in DNA technologies coupled with the knowledge of sequence information have allowed the development of accurate and rapid diagnostic tests. While whole plant-based diagnostic techniques such as seedling bioassays or enzyme-based in vitro bioassays provide accurate results, they tend to be labour- and/or space-intensive and will only respond to the particular herbicides tested, making resolution of cross-resistance patterns more difficult. Successful DNA-based diagnosis of ALS inhibitor resistance has been achieved with three main techniques, (1) restriction fragment length polymorphism, (2) polymerase chain reaction amplification of specific alleles and (3) denaturing high-performance liquid chromatography. All DNA-based techniques are relatively rapid and provide clear identification of the mutations causing resistance. Resistance based on non-target mechanisms is not identified by these DNA-based methods; however, given the prevalence of target site-based ALS inhibitor resistance, this is a minor inconvenience. Copyright © 2006 Society of Chemical Industry [source] A novel T cell cytokine, secreted osteoclastogenic factor of activated T cells, induces osteoclast formation in a RANKL-independent mannerARTHRITIS & RHEUMATISM, Issue 11 2009Leonard Rifas Objective Chronic T cell activation is central to the etiology of rheumatoid arthritis (RA), an inflammatory autoimmune disease that leads to severe focal bone erosions and generalized systemic osteoporosis. Previous studies have shown novel cytokine-like activities in medium containing activated T cells, characterized by potent induction of the osteoblastic production of interleukin-6 (IL-6), an inflammatory cytokine and stimulator of osteoclastogenesis, as well as induction of an activity that directly stimulates osteoclast formation in a manner independent of the key osteoclastogenic cytokine RANKL. This study was undertaken to identify the factors secreted by T cells that are responsible for these activities. Methods Human T cells were activated using anti-human CD3 and anti-human CD28 antibodies for 72 hours in AIM V serum-free medium to obtain T cell,conditioned medium, followed by concentration and fractionation of the medium by fast-protein liquid chromatography. Biologically active fractions were resolved using sodium dodecyl sulfate,polyacrylamide gel electrophoresis. Major bands were analyzed by mass spectrometry, and a major candidate protein was identified. This novel cytokine was cloned, and its expression was analyzed using recombinant DNA technologies. Results A single novel cytokine that could induce both osteoblastic IL-6 production and functional osteoclast formation in the absence of osteoblasts or RANKL and that was insensitive to the effects of the RANKL inhibitor osteoprotegerin was identified in the activated T cell,conditioned medium; this cytokine was designated secreted osteoclastogenic factor of activated T cells (SOFAT). Further analysis of SOFAT revealed that it was derived from an unusual messenger RNA splice variant coded by the threonine synthase,like 2 gene homolog, which is a conserved gene remnant coding for threonine synthase, an enzyme that functions only in microorganisms and plants. Conclusion SOFAT may act to exacerbate inflammation and/or bone turnover under inflammatory conditions such as RA or periodontitis and in conditions of estrogen deficiency. [source] Application of genomics to grapevine improvementAUSTRALIAN JOURNAL OF GRAPE AND WINE RESEARCH, Issue 2010G. DI GASPERO Abstract Imagine a breeder browsing a grape chromosome nucleotide-by-nucleotide around a trait locus, scrolling down the list of catalogued genes along a genetic interval, resequencing for a few thousand dollars a potential parent or a selected breeding line. In the past couple of years, this vision has become a reality. The availability of the reference genome sequence has provided significant assistance in the saturation of loci with targeted genetic markers. Grape breeders are now offered unprecedented possibilities for selecting plants using deoxyribonucleic acid (DNA) sequences within or near the gene that controls a desirable trait rather than handling their phenotypes. Genomics-assisted selection offers unique advantages in the correct choice of elite genotypes, in order to improve traits for which limitations of phenotyping technologies or low hereditability adversely affect the efficiency of phenotypic selection. DNA technologies enable the application of marker-assisted selection to thousands of grape seedlings every year, which was previously feasible only for cereals and annuals, enhancing the possibilities of finding an ideal recombinant in populations bred from highly heterozygous parents. The expected outcome is a renewal of the varietal choices available to viticulturists, with novel genotypes that meet the demand for disease-free vines and flavourful grapes. The depth of exploration and characterisation of the existing germplasm is crucial for translating natural diversity into new varieties that could perform beyond the fence of the experimental vineyards and gain substantial market share. We review here how current achievements in genomics and genome sequencing are expected to increase the efficiency of grapevine breeding programs. [source] Mouse × pig chimeric antibodies expressed in Baculovirus retain the same properties of their parent antibodiesBIOTECHNOLOGY PROGRESS, Issue 2 2009Ana M. Jar Abstract The development of hybridoma and recombinant DNA technologies has made it possible to use antibodies against cancer, autoimmune disorders, and infectious diseases in humans. These advances in therapy, as well as immunoprophylaxis, could also make it possible to use these technologies in agricultural species of economic importance such as pigs. Porcine reproductive and respiratory syndrome virus (PRRSV) is an arterivirus causing very important economic losses to the industry. Passive transfer of antibodies obtained by biotechnology could be used in the future to complement or replace vaccination against this and other pig pathogens. To this end, we constructed and studied the properties of chimeric mouse × pig anti-PRRSV antibodies. We cloned the constant regions of gamma-1 and gamma-2 heavy chains and the lambda light chain of pig antibodies in frame with the variable regions of heavy and light chains of mouse monoclonal antibody ISU25C1, which has neutralizing activity against PRRSV. The coding regions for chimeric IgG1 and IgG2 were expressed in a baculovirus expression system. Both chimeric antibodies recognized PRRSV in ELISA as well as in a Western-blot format and, more importantly, were able to neutralize PRRSV in the same fashion as the parent mouse monoclonal antibody ISU25C1. In addition, we show that both pig IgG1 and IgG2 antibodies could bind complement component C1q, with IgG2 being more efficient than IgG1 in binding C1q. Expressing chimeric pig antibodies with protective capabilities offers a new alternative strategy for infectious disease control in domestic pigs. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] Evaluating wastewater-induced plant genotoxicity using randomly amplified polymorphic DNAENVIRONMENTAL TOXICOLOGY, Issue 1 2008K. M. Swaileh Abstract Wastewater often contains genotoxic substances that can resist different stages of the treatment process. In the present study, randomly amplified polymorphic DNA technology was applied to evaluate the genotoxic effects of wastewater (treated and raw) irrigation on oat plants (Avena sativa). RAPD profiles obtained showed that both treated and raw wastewater (RWW) were having genotoxic effects on oat plants. This was apparent by the appearance/disappearance of bands in the treatments compared with the control plants. From the 15 primers used, 186 bands were obtained with an average of 12.4 bands per primer. Irrigating plants with RWW caused 51 new bands to appear and 19 to disappear. Treated wastewater (TWW) caused only 16 new bands and the loss of 17 bands. This makes TWW less genotoxic than RWW. The Euclidean distances shown on the dendrogram, revealed the presence of two clusters according to dissimilarity values. One cluster contained the control plants and those irrigated with TWW, whereas the second contained the plants irrigated with RWW. Similarity indices calculated between the treatments and the control plants showed that the control and the plants irrigated with TWW had a similarity index of 0.87, the control and plants irrigated with RWW 0.73 and between the treatments 0.75. © 2008 Wiley Periodicals, Inc. Environ Toxicol, 2008. [source] Hb Woodville, a rare , -globin variant, caused by codon 6 mutation of the ,1 geneEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 1 2006Vip Viprakasit Abstract:, Since 1995, the national programme for the prevention and control of severe thalassaemia has been implemented in Thailand. This programme is composed of the population screening in pregnant women and couples by osmotic fragility, HbE screening and the confirmation test using haemoglobin analyses by electrophoresis or chromatography. Thereafter, several hitherto unidentified haemoglobins (Hbs) with structural defects are increasingly described and these variants are now easily studied using DNA technology. In this study, the authors describe the haematology and molecular analyses in a 28-yr-old healthy female who was identified as having an exceptionally ,high HbA2' from haemoglobin analysis. Subsequent analyses demonstrated that observed atypical ,HbA2' was, in fact, a rare innocuous , -globin variant, called Hb Woodville [alpha 2 6(A4); Asp , Tyr]. For the first time, this abnormal Hb species is characterised at the molecular level. [source] Genetic improvement of processes yielding microbial productsFEMS MICROBIOLOGY REVIEWS, Issue 2 2006Jose L. Adrio Abstract Although microorganisms are extremely good in presenting us with an amazing array of valuable products, they usually produce them only in amounts that they need for their own benefit; thus, they tend not to overproduce their metabolites. In strain improvement programs, a strain producing a high titer is usually the desired goal. Genetics has had a long history of contributing to the production of microbial products. The tremendous increases in fermentation productivity and the resulting decreases in costs have come about mainly by mutagenesis and screening/selection for higher producing microbial strains and the application of recombinant DNA technology. [source] The long and winding road from the research laboratory to industrial applications of lactic acid bacteriaFEMS MICROBIOLOGY REVIEWS, Issue 3 2005Martin Bastian Pedersen Abstract Research innovations are constantly occurring in universities, research institutions and industrial research laboratories. These are reported in the scientific literature and presented to the scientific community in various congresses and symposia as well as through direct contacts and collaborations. Conversion of these research results to industrially useful innovations is, however, considerably more complex than generally appreciated. The long and winding road from the research laboratory to industrial applications will be illustrated with two recent examples from Chr. Hansen A/S: the implementation in industrial scale of a new production technology based on respiration by Lactococcus lactis and the introduction to the market of L. lactis strains constructed using recombinant DNA technology. [source] The hinge region fragment of immunoglobulin G improves immunogenicity of recombinant gonadotrophin-releasing hormone conjugated to the T-helper epitope in designing peptide vaccinesIMMUNOLOGY, Issue 1pt2 2009Jinshu Xu Summary In our previous study, the hinge fragment (225,232/225,,232,) of human immunoglobulin G1 (IgG1) was used as a space peptide linker for synthesizing the GnRH3,hinge,MVP chimeric peptide, whereby three repeated gonadotrophin-releasing hormone (GnRH) units and a T-cell epitope from measles virus fusion protein (MVP) were amide-bond-linked at the N and C terminus, respectively, to the hinge peptide for producing anti-GnRH antibody responses. To investigate whether or not the hinge region fragment can improve the immunogenicity of GnRH, we further synthesized and purified GnRH3,hinge,MVP, GnRH3,hinge and GnRH3,MVP using recombinant DNA technology. Under high pH conditions, GnRH3,hinge,MVP was capable of forming double-chain structures. Immunization of male mice with the immunogens of GnRH3,hinge,MVP resulted in the generation of high-titre antibodies specific for GnRH. The synthetic GnRH3,hinge and GnRH3,MVP induced a lower titre of anti-GnRH antibody than GnRH3,hinge,MVP. This was followed by a decrease in serum testosterone levels, which resulted in a low level of expression of the relaxin-like factor gene in the testis. Our data suggest that peptide and T-cell epitopes oriented at the N-terminus or C-terminus of hinge peptides simplify the antigenic peptide conjugates and may be considered as potential synthetic immunogens. [source] Interpreting DNA Evidence: A ReviewINTERNATIONAL STATISTICAL REVIEW, Issue 3 2003L.A. Foreman Summary The paper provides a review of current issues relating to the use of DNA profiling in forensic science. A short historical section gives the main statistical milestones that occurred during a rapid development of DNA technology and operational uses. Greater detail is then provided for interpretation issues involving STR DNA profiles, including: , methods that take account of population substructure in DNA calculations; , parallel work carried out by the US National Research Council; , the move away from multiple independence testing in favour of experiments that demonstrate the robustness of casework procedures; , the questionable practice of source attribution ,with reasonable scientific certainty'; , the effect on the interpretation of profiles obtained under increasingly sensitive techniques, the LCN technique in particular; , the use of DNA profiles as an intelligence tool; , the interpretation of DNA mixtures. Experience of presenting DNA evidence within UK courts is also discussed. The paper then summarises a generic interpretation framework based on the concept of likelihood ratio within a hierarchy of propositions. Finally the use of Bayesian networks to interpret DNA evidence is reviewed. Résumé Cet article présente un inventaire des questions relativesá l'utilisation du profilage ADN dans la science légale. Une courte section historique décrit les principales étapes statistiques qui ont eu lieu pendant le rapide développement de la technologie ADN et ses utilisations opérationnelles. De plus grands détails sont ensuite donnés pour l'interprétation de questions sur les profils AND STR, ce qui inclut: ,les méthodes qui tiennent compte des sous-structures de population dans les calculs ADN; ,le travail conduit en paralléle par le Conseil de Recherche Nationale des Etats-Unis (NRC); ,l'évolution depuis les tests d'indépendance multiple vers des expériences qui démontrent la robustesse des procédures; ,la pratique contestable de l'attribution de source avec "certitude scientifique raisonnable"; ,l'effet de l'interprétation des profils obtenus sous techniques de plus en plus sensibles, la technique LCN en particulier ,l'utilisation de profils ADN comme outil d'intelligence; ,l'interprétation de mélanges ADN. L'expérience de ce type de preuve dans les tribunaux britanniques sera également présentée et commentée. L'article présentera un cavenas d'interprétation centré sur le concept de rapport de vraisemblance, inscrit dans une hérarchie de propositions. Finalement, l'utilisation de réseaux Bayesien pour interpréter la preuve par ADN sera abordée. [source] Molecular mechanisms of intercellular communication in the hormonal and neural systemsIUBMB LIFE, Issue 5-6 2006Shigetada Nakanishi Abstract This paper reviews our studies that have addressed the molecular mechanisms underlying the biosynthesis and reception of extracellular signaling molecules and integrative mechanisms of extracellular-intracellular signaling transmission in biological systems. We introduced recombinant DNA technology into the neuroendocrine system and established the concept that a single peptide precursor encompasses multiple biologically active peptides and brings about coordinate functions in various biological systems. We then developed a novel functional cloning of membrane receptors and ion channels by combining an oocyte expression system with electrophysiology. We molecularly elucidated not only various peptide receptors, including the first demonstration of the molecular entity of a G protein-coupled peptide receptor (GPCR), substance K receptor, and also diverse members of both G protein-coupled metabotropic type and NMDA type of neurotransmitter glutamate receptors. We demonstrated many novel synaptic mechanisms involving distinct types of glutamate receptors in brain function and dysfunction. These include the mechanisms underlying segregation of light-dark signals in visual transmission, discrimination and memory formation in olfactory transmission, and motor co-ordination in the cerebellum, basal ganglia and the retinal network. iubmb Life, 58: 349-357, 2006 [source] Forensic aspects of DNA-based human identity testingJOURNAL OF FORENSIC NURSING, Issue 4 2008Stephen M. Roper MS Abstract The forensic applications of DNA-based human identity laboratory testing are often underappreciated. Molecular biology has seen an exponential improvement in the accuracy and statistical power provided by identity testing in the past decade. This technology, dependent upon an individual's unique DNA sequence, has cemented the use of DNA technology in the forensic laboratory. This paper will discuss the state of modern DNA-based identity testing, describe the technology used to perform this testing, and describe its use as it relates to forensic applications. We will also compare individual technologies, including polymerase chain reaction (PCR) and Southern Blotting, that are used to detect the molecular differences that make all individuals unique. An increasing reliance on DNA-based identity testing dictates that healthcare providers develop an understanding of the background, techniques, and guiding principles of this important forensic tool. [source] Can glycans unveil the origin of glycoprotein hormones?,human chorionic gonadotrophin as an example,JOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 7 2008R. Ramírez-Llanelis Abstract Doping with (glyco)protein hormones represent an extremely challenging, analytical problem as nearly all are constitutively present at low concentrations that fluctuate according to circadian or alternative periodical, or external stimuli. Thus the mere concentration in a biological sample is only resolutive when this surpasses extreme values. As the vast majority of these molecules are produced by recombinant DNA technology it is believed that the exogenous molecules could bear the signature of the host cell. In particular, these could comprise structural differences originated from co or post-translational differences. In this study we have employed both proteomics and glycomics strategies to compare recombinant and urinary human chorionic gonadotrophin in order to evaluate this hypothesis. As anticipated the recombinant hormone could be shown to contain N -glycolyl neuraminic acid, a sialic acid that cannot be produced by humans. Furthermore, differences were observed in the overall glycosylation, in particular the presence of abundant hybrid-type glycans that were much less pronounced in the recombinant species. These differences were determined to occur predominantly in the ,-subunit for which antidoping strategies focussed on these elements could be used for both chorionic gonadotrophin and lutrophin as they share the same ,-subunit. Copyright © 2008 John Wiley & Sons, Ltd. [source] Association of allergic patients' phenotypes with IgE reactivity to recombinant pollen marker allergensALLERGY, Issue 3 2010A. Twardosz-Kropfmüller To cite this article: Twardosz-Kropfmüller A, Singh MB, Niederberger V, Horak F, Kraft D, Spitzauer S, Valenta R, Swoboda I. Association of allergic patients' phenotypes with IgE reactivity to recombinant pollen marker allergens. Allergy 2010; 65: 296,303. Abstract Background:, During the last decade allergen molecules from several allergen sources have been produced by recombinant DNA technology. The aim of this study was to investigate whether IgE reactivity to recombinant pollen allergens with broad and narrow cross-reactivity is associated with clinical phenotypes of allergic sensitization. Methods:, Serum IgE reactivity to a panel of six recombinant birch and grass pollen allergens was measured by ELISA in pollen sensitized patients from Central Europe to define groups of patients with exclusive IgE reactivity to rBet v 1, with exclusive reactivity to major grass pollen allergens (rPhl p 1, rPhl p 2, rPhl p 5) and with IgE reactivity to cross-reactive pollen allergens (rBet v 2, rPhl p 7). Patients' clinical phenotypes were recorded. IgE responses to tree, grass and weed pollen as well as plant food extracts were evaluated in vitro by CAP-FEIA and clinical sensitivities were confirmed in vivo by skin prick testing. Results:, IgE reactivity to the recombinant major birch pollen allergen, rBet v 1, was associated with sensitization to pollen from birch, taxonomically related trees and to certain plant-derived food. Reactivity to the recombinant timothy grass pollen allergens, rPhl p 1, rPhl p 2, rPhl p 5, indicated sensitization to pollen from grasses. Patients reacting with the highly cross-reactive allergen rPhl p 7 were polysensitized to pollen from unrelated trees, grasses and weeds and rBet v 2-positive patients were polysensitized to pollen and plant-derived food from unrelated plants. Conclusions:, IgE reactivity to recombinant marker allergens is associated with clinical phenotypes of allergic sensitization and may be useful for the selection of treatment strategies. [source] Molecular characterization and corrosion behavior of thermophilic (55,°C) SRB Desulfotomaculum kuznetsovii isolated from cooling tower in petroleum refineryMATERIALS AND CORROSION/WERKSTOFFE UND KORROSION, Issue 9 2009B. Anandkumar Abstract Desulfotomaculum kuznetsovii (D. kuznetsovii), a thermophilic sulfate-reducing bacterium (SRB), was identified in a cooling tower of a petroleum refinery by 16S rRNA gene sequencing and its functional gene encoding dissimilatory sulfite reductase (dsrAB). The thermophilic sulfate-reducing bacterial species have been reported for the first time in the cooling towers of an Indian petroleum refinery. The protein coded by dsrAB gene was cloned, expressed, and identified using recombinant DNA technology. Weight loss method, electrochemical and surface analysis showed the corrosion behavior of the isolate. In the presence of D. kuznetsovii, the corrosion rate was higher when compared to control at 55,°C. It suppresses the anodic reaction and enhances the cathodic reaction by the production of organic complex and iron sulfide, respectively. Numerous pitting were noticed on mild steel which is due to the presence of D. kuznetsovii and its role in the corrosion process has been discussed. [source] Twenty years of phylogeography: the state of the field and the challenges for the Southern HemisphereMOLECULAR ECOLOGY, Issue 17 2008LUCIANO B. BEHEREGARAY Abstract Phylogeography is a young, vigorous and integrative field of study that uses genetic data to understand the history of populations. This field has recently expanded into many areas of biology and also into several historical disciplines of Earth sciences. In this review, I present a numerical synthesis of the phylogeography literature based on an examination of over 3000 articles published during the first 20 years of the field (i.e. from 1987 to 2006). Information from several topics needed to evaluate the progress, tendencies and deficiencies of the field is summarized for 10 major groups of organisms and at a global scale. The topics include the geography of phylogeographic surveys, comparative nature of studies, temporal scales and major environments investigated, and genetic markers used. I also identify disparities in research productivity between the developing and the developed world, and propose ways to reduce some of the challenges faced by phylogeographers from less affluent countries. Phylogeography has experienced explosive growth in recent years fuelled by developments in DNA technology, theory and statistical analysis. I argue that the intellectual maturation of the field will eventually depend not only on these recent developments, but also on syntheses of comparative information across different regions of the globe. For this to become a reality, many empirical phylogeographic surveys in regions of the Southern Hemisphere (and in developing countries of the Northern Hemisphere) are needed. I expect the information and views presented here will assist in promoting international collaborative work in phylogeography and in guiding research efforts at both regional and global levels. [source] Physicochemical consequences of the perdeuteriation of glutathione S -transferase from S. japonicumPROTEIN SCIENCE, Issue 3 2001David Brockwell Abstract Glutathione S -transferase (GST) from Schistosoma japonicum has been prepared in both normal protiated (pGST) and fully deuteriated (dGST) form by recombinant DNA technology. Electrospray mass spectrometry showed that the level of deuteriation in dGST was 96% and was homogeneous across the sample. This result is attributed to the use of a deuterium-tolerant host Escherichia coli strain in the preparation of the protein. 10 heteroatom-bound deuteriums (in addition to the carbon-bound deuteriums) were resistant to exchange when dGST was incubated in protiated buffer. The physicochemical and biological properties of the two proteins were compared. dGST was relatively less stable to heat denaturation and to proteolytic cleavage than was pGST. The midpoint transition temperature for pGST was 54.9°C, whereas that for dGST was 51.0°C. Static light-scattering measurements revealed that the association behavior of dGST is also different from that of pGST. The perdeuteriated enzyme shows a tendency to associate into dimers of the fundamental dimer. This is in contrast with results that have been obtained for other perdeuteriated proteins in which perdeuteriation has been shown to promote dissociation of aggregates. dGST showed a similar Km to pGST; similar results had been obtained previously with bacterial alkaline phosphatase. However, whereas the alkaline phosphatase showed a reduced rate of catalysis on deuteriation, dGST exhibited a slightly higher rate of catalysis than pGST. It is clear that the bulk substitution of deuterium for protium has significant effects on the properties of proteins. Until many more examples have been studied, it will be difficult to predict these effects for any given protein. Nevertheless, deuteriation represents an intriguing method of preparing functional analogs of recombinant proteins. [source] Bioengineering nitrogen acquisition in rice: can novel initiatives in rice genomics and physiology contribute to global food security?BIOESSAYS, Issue 6 2004Dev T. Britto Rice is the most important crop species on earth, providing staple food for 70% of the world's human population. Over the past four decades, successes in classical breeding, fertilization, pest control, irrigation and expansion of arable land have massively increased global rice production, enabling crop scientists and farmers to stave off anticipated famines. If current projections for human population growth are correct, however, present rice yields will be insufficient within a few years. Rice yields will have to increase by an estimated 60% in the next 30 years, or global food security will be in danger. The classical methods of previous green revolutions alone will probably not be able to meet this challenge, without being coupled to recombinant DNA technology. Here, we focus on the promise of these modern technologies in the area of nitrogen acquisition in rice, recognizing that nitrogen deficiency compromises the realization of rice yield potential in the field more than any other single factor. We summarize rice-specific advances in four key areas of research: (1) nitrogen fixation, (2) primary nitrogen acquisition, (3) manipulations of internal nitrogen metabolism, and (4) interactions between nitrogen and photosynthesis. We develop a model for future plant breeding possibilities, pointing out the importance of coming to terms with the complex interactions among the physiological components under manipulation, in the context of ensuring proper targeting of intellectual and financial resources in this crucial area of research. BioEssays 26:683,692, 2004. © 2004 Wiley Periodicals, Inc. [source] Crystallization and preliminary X-ray crystallographic analysis of PhoK, an extracellular alkaline phosphatase from Sphingomonas sp.ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2009BSAR- Alkaline phosphatases (APs) are widely distributed from microbes to humans and are involved in several important biological processes such as phosphate nutrition, signal transduction and pathogenesis. Alkaline phosphatases are also useful in various industrial applications and in recombinant DNA technology. A new AP enzyme from Sphingomonas sp. strain BSAR-1, termed PhoK, has been shown to be useful in uranium bioprecipitation. PhoK was expressed, purified and crystallized. The crystals belonged to space group P43212 or P41212, with unit-cell parameters a = b = 87.37, c = 168.16,Å, and contained one enzyme molecule in the asymmetric unit. Native diffraction data have been collected to 1.95,Å resolution at the ESRF. [source] Improving Glucose and Glutamine Metabolism of Human HEK 293 and Trichoplusiani Insect Cells Engineered To Express a Cytosolic Pyruvate Carboxylase EnzymeBIOTECHNOLOGY PROGRESS, Issue 1 2003Cynthia B. Elias Metabolic engineering has been defined as a directed improvement of product formation or cellular properties by modification of specific biochemical pathways or introduction of new enzymatic reactions by recombinant DNA technology. The use of metabolic flux analysis (MFA) has helped in the understanding of the key limitation in the metabolic pathways of cultured animal cells. The MFA of the major nutrients glucose and glutamine showed that the flux of glucose to the TCA cycle and its subsequent utilization is limited as a result of the lack of certain key enzymes in the pathway. One of the key enzymes controlling this flux is pyruvate carboxylase. Introduction of this enzyme into mammalian cells has been shown to improve the utilization of glucose and limit the production of lactate and ammonia, which are deleterious to cell growth. In the present work a yeast pyruvate carboxylase gene has been introduced into mammalian (HEK 293) and insect ( Trichoplusiani High-Five) cells, resulting in the cytosolic expression of the enzyme. In both cases the resulting transfected cells were able to utilize glucose and glutamine more efficiently and produce lower amounts of lactate and ammonia. Differences in the amino acid utilization pattern were also observed, indicating changes in the basic metabolism of the cells. The performance of the transfected cells as expression systems for adenovirus and baculovirus vectors, respectively, has also been examined. The results obtained and their impact on the process development for protein and viral vector production are discussed. [source] New strategies for cancer gene therapy: Progress and opportunitiesCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 1 20102nd Australia, China Biomedical Research Conference (ACBRC2009) Summary 1.,To date, cancer persists as one of the most devastating diseases worldwide. Problems such as metastasis and tumour resistance to chemotherapy and radiotherapy have seriously limited the therapeutic effects of existing clinical treatments. 2.,To address these problems, cancer gene therapy has been developing over the past two decades, specifically designed to deliver therapeutic genes to treat cancers using vector systems. So far, a number of genes and delivery vehicles have been evaluated and significant progress has been made with several gene therapy modalities in clinical trials. However, the lack of an ideal gene delivery system remains a major obstacle for the successful translation of regimen to the clinic. 3.,Recent understanding of hypoxic and necrotic regions within solid tumours and rapid development of recombinant DNA technology have reignited the idea of using anaerobic bacteria as novel gene delivery systems. These bacterial vectors have unique advantages over other delivery systems and are likely to become the vector of choice for cancer gene therapy in the near future. 4.,Meanwhile, complicated tumour pathophysiology and associated metastasis make it hard to rely on a single therapeutic modality for complete tumour eradication. Therefore, the combination of cancer gene therapy with other conventional treatments has become paramount. 5.,The present review introduces important cancer gene therapy strategies and major vector systems that have been studied so far with an emphasis on bacteria-mediated cancer gene therapy. In addition, exemplary combined therapies are briefly reviewed. [source] Congenic Rats For Hypertension: How Useful Are They For The Hunting Of Hypertension Genes?CLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 4 2000Toru Nabika SUMMARY 1. Linkage studies have revealed quantitative trait loci (QTL) for blood pressure in the rat genome using genetic hypertensive rat models. To identify the genes responsible for hypertension, the construction of congenic rats is essential. 2. To date, several congenic strains have been obtained from spontaneously hypertensive or Dahl salt-sensitive rats. The results of these studies should be interpreted according to whether the rats carry the whole QTL region or not. 3. After establishing congenic strains, three strategies are possible: (i) an orthodox positional cloning in which, using subcongenic strains, the QTL region is cut down to smaller fragments suitable for physical mapping; (ii) a positional candidate strategy in which candidate genes in the QTL regions are studied; or (iii) physiological studies in which intermediate phenotypes directly associated with the hypertension gene are explored. Several other experimental strategies are also available using congenic strains as new animal models for hypertension. 4. To make the most of advances in DNA technology, the precise evaluation of the phenotypic difference between congenic strains carrying different QTL or between a congenic and parental strain is critical. [source] | |||||||||||||||||||