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Molecular cloning of Proteus morganii phenylalanine deaminase gene in Escherichia coli.

The gene for phenylalanine deaminase (PAD) of Proteus morganii strain 2815 has been isolated on a 6.3-kb HindIII restriction fragment and cloned within RP4-prime plasmids, pYB2321 and pYB2322, in both orientations. Expression of the cloned gene in Escherichia coli strains was comparable to that in P. morganii 2815. The hybrid plasmids mobilized the 2815 chromosome with trajectories in reverse directions from an origin between ser-2 and ade-1, suggesting the map location of the PAD gene.

Amino Acid Oxidoreductases↗

PubMed1989

Nucleotide sequence of the Erwinia chrysanthemi gene encoding 2-keto-3-deoxygluconate permease.

The phytopathogenic bacterium Erwinia chrysanthemi produces a group of pectolytic enzymes able to depolymerise the pectic compounds in plant cell walls. The resulting tissue maceration is known as soft rot disease. The degraded pectin products are transported by 2-keto-3-deoxygluconate permease into the bacterial cell, where they serve as carbon and energy sources. This H+ coupled transport system is encoded by the kdgT gene; we report the nucleotide sequence of kdgT. It is encoded by an open reading frame (ORF) of 1194 bp, which is preceded by an Escherichia coli-type promoter region. The ORF encodes a protein with 398 amino acid (aa) residues and a predicted Mr of 48,550. As would be expected for a membrane protein, it is very hydrophobic, containing 63% nonpolar aa. However, the kdgT gene has no apparent evolutionary relationship to other genes encoding sugar transport proteins, such as lacY, melB or the E. coli citrate transport gene. Southern hybridization experiments indicate a strong homology between the Er. chrysanthemi and E. coli kdgT genes; there is also a second region on the E. coli chromosome with homology to kdgT. The kdgT gene is located near the ade-377 marker on the Er. chrysanthemi chromosome (equivalent to the region between 20 and 30 min in E. coli), whereas the E. coli kdgT gene is located at 88 min. Thus, these two enterobacteria show some significant differences in their genomic organization.

Amino Acid Sequence↗

PubMed1991

A DNA fragment containing the ADE2 gene from Schwanniomyces occidentalis can be maintained as an extrachromosomal element.

A 4.05-kb DNA fragment containing the ADE2 gene from Schwanniomyces occidentalis was cloned into the pUC19 vector. When an ade2 strain of Sc. occidentalis was transformed with this plasmid, pADE-2 was found to integrate into the host chromosome and was also present in a variety of extrachromosomal species. These extrachromosomal elements were present in multiple copies, varied in molecular mass and were composed of polymerized forms of pADE-2. A fragment containing the ADE2 gene was used to transform a Sc. occidentalis ade2 mutant, as either a linear or circularized molecule. The linear form integrated into the host genome, whereas the circularized form was found as a stably maintained extrachromosomal element with no evidence of integration or detectable loss of the Ade+ phenotype upon subculturing of transformed yeast under nonselective conditions for 60 generations. The ratio of the number of extrachromosomal ADE2 genes to genomic ADE2 ranged from 3.8 to 6.6.

Blotting, Southern↗

PubMed1987

Profiles of antibody-dependent enhancement of dengue virus type 2 infection.

Antibody-dependent infection enhancement (ADE) was studied with P-388D1 mouse macrophage-like cells, 21 dengue virus type 2 (DEN-2) strains, and 8 monoclonal antibodies reactive with flavivirus group-specific or dengue serotype-specific determinants. Testing a constant number of virions against serial dilutions of antibody for their ability to infect P-388D1 cells, a reproducible 'enhancement profile' was observed. The profile was characterized by (1) appearance, peak, decline, and disappearance of infection enhancement when antibody-containing ascitic fluids were diluted beyond the neutralizing endpoint, and (2) evolution over an approximate 10,000-fold dilutional range. The profiles were similar regardless of whether viruses were complexed with antibody at flavivirus group or serotype determinants, but the antibody dilution at which infection enhancement was maximal varied with the neutralization titer of the antibody. Neutralization and antibody-dependent enhancement of dengue infection appear to be biological outcomes of interactions between antibodies and single viral epitopes at different antibody: virus ratios.

Animals↗

PubMed2004

Phospholipid membrane bending as assessed by the shape sequence of giant oblate phospholipid vesicles.

Vesicle shape transformations caused by decreasing the difference between the equilibrium areas of membrane monolayers were studied on phospholipid vesicles with small volume to membrane area ratios. Slow transformations of the vesicle shape were induced by lowering of the concentration of lipid monomers in the solution outside the vesicle. The complete sequence of shapes consisted of a string of pearls, and wormlike, starfish, discocyte and stomatocyte shapes. The transformation from discocyte to stomatocyte vesicle shapes was analyzed theoretically to see whether these observations accord with the area difference elasticity (ADE) model. The membrane shape equation and boundary conditions were derived for axisymmetrical shapes for low volume vesicles, part of whose membranes are in contact. Calculated shapes were arranged into a phase diagram. The theory predicts that the transition between discocyte and stomatocyte shapes is discontinuous for relatively high volumes and continuous for low volumes. The calculated shape sequences matched well with the observed ones. By assuming a linear decrease of the equilibrium area difference with time, the ratio between the nonlocal and local bending constants is in agreement with reported values.

Elasticity↗

PubMed2006

Structure-activity relationship for nucleoside analogs as inhibitors or substrates of adenosine kinase from Mycobacterium tuberculosis. I. Modifications to the adenine moiety.

Adenosine kinase (Ado kinase, EC 2.7.1.20) is a purine salvage enzyme that phosphorylates adenosine (Ado) to AMP. Ado kinase from Mycobacterium tuberculosis also catalyzes an essential step in the conversion of 2-methyl-Ado to a compound with selective antimycobacterial activity. In order to aid in the design of more potent and selective Ado analogs, eighty nucleoside analogs with modifications to the adenine (Ade) moiety of Ado were evaluated as both substrates and inhibitors of Ado kinase from M. tuberculosis, and a subset was further tested with human Ado kinase for the sake of comparison. The best substrates were 2-aza-Ado, 8-aza-9-deaza-Ado, and 2-fluoro-Ado and the most potent inhibitors were N1-benzyl-Ado (Ki=0.19 microM), 2-fluoro-Ado (Ki=0.5 microM), 6-cyclopentyloxy-purine riboside (Ki=0.15 microM), and 7-iodo-7-deaza-Ado (Ki=0.21 microM). These studies revealed the presence of a hydrophobic pocket near the N6- and N1-positions that can accommodate substitutions at least as large as a benzyl group. The ability to fit into this pocket increased the likelihood that a compound would be an inhibitor and not a substrate. The 2-position was able to accommodate exocyclic substitutions as large as a methoxy group, although substrate activity was low. Similarly, the 7-position could bind an exocyclic group as large as a carboxamido moiety. However, all of the compounds tested with modifications at the 7-position were much better inhibitors than substrates. MIC studies performed with selected compounds have yielded several Ado analogs with promising antitubercular activity. Future studies will utilize this information for the design of new analogs that may be selective antitubercular agents.

Adenine↗

PubMed2006

Formation of depurinating N3adenine and N7guanine adducts after reaction of 1,2-naphthoquinone or enzyme-activated 1,2-dihydroxynaphthalene with DNA. Implications for the mechanism of tumor initiation by naphthalene.

Naphthalene is considered by the US Environmental Protection Agency to be a carcinogenic compound based on inhalation studies in rats. The primary metabolite of naphthalene is naphthalene 1,2-arene oxide. This unstable intermediate can lead to formation of 1-naphthol and naphthalene-1,2-dihydrodiol. Secondary metabolites include 1,2-dihydroxynaphthalene (1,2-DHN), which can be further oxidized to 1,2-naphthoquinone (1,2-NQ). Based on the metabolism of naphthalene and its similarity to the metabolic activation of carcinogenic natural estrogens, synthetic estrogens and benzene, we hypothesize that naphthalene is activated to initiate cancer by reaction of 1,2-NQ with DNA to form the depurinating adducts 1,2-DHN-4-N3Ade and 1,2-DHN-4-N7Gua. These adducts were synthesized by reaction of 1,2-NQ with Ade or dG in acetic acid/water/DMF (1:1:1). 1,2-NQ was reacted with DNA, and the depurinating 1,2-DHN-4-N3Ade and 1,2-DHN-4-N7Gua adducts were analyzed by ultraperformance liquid chromatography/tandem mass spectrometry and HPLC with electrochemical detection. After the reaction of 1,2-NQ with DNA, the N3Ade and N7Gua adducts were found. Similarly, when 1,2-DHN was activated by tyrosinase in the presence of DNA, higher amounts of the N3Ade and N7Gua adducts were detected. These same adducts were also formed when 1,2-DHN was activated by prostaglandin H synthase or 3-methylcholanthrene-induced rat liver microsomes in the presence of DNA. These depurinating adducts are analogous to those obtained from the ortho-quinones of natural estrogens, synthetic estrogens and benzene. These results suggest that reaction of ortho-quinones with DNA by 1,4-Michael addition is a general mechanism of weak carcinogenesis that occurs with naphthalene and a number of other aromatic compounds.

Adenine↗

PubMed2006

Medication reconciliation: developing and implementing a program.

During the past 5 years since the medication reconciliation process was formalized and automated, it has become an independent redundancy. The patient intervention rates are maintained at 30% to 35%, with ADE rates related to medication reconciliation at zero. The medication process takes into account the accuracy and appropriateness of restarting prehospital medications and current ICU medications. It includes the omission of important home medications along with inaccuracies of dosages and frequencies. This form assures that the patient is receiving continuity of care ad decreases complications of the patients health related to the changing of medications. Until recently this concept was disseminated by the staff without consistent administrative support. It was a process developed by nurses and perpetuated by nurses. Recently the administration has mandated that the process be implemented throughout the institution. A Hopkins health care-based collaborative is working to implement medication reconciliation hospital wide. The challenge exists in standardizing a process that is now specific to each functional unit. Multidisciplinary monthly meetings provided a forum for working through the barriers to incorporate these changes. This low-cost, high-impact safely initiative, if planned and performed strategically, can have a significant effect on patient safety.

Academic Medical Centers↗

PubMed2005

Reduction of adverse drug events and medication errors in a community hospital setting.

Keys to success in reducing ADEs have included the support of administrative leaders through their visibility and emphasis on safety as an organizational priority, and financial support for safety projects. Administrative participation was also helpful in promoting safety efforts through the reinforcement of expectations when progress was sluggish. The use of rapid cycle change provided enough early success to serve in motivating staff to push ahead. It allowed staff the opportunity to analyze changes, make adjustments, and retest on a slightly larger scale. Other key success factors included the motivation of teams through continual sharing of progress and success stories; celebrations for achievements are held routinely. As an organization, SJMC has shared its success strategies with other organizations and promoted networking with other organizations to determine what strategies have worked elsewhere. This is helpful as it prevents time from being wasted on solutions that have been tried without success. Within the OSF Healthcare System, the following phrase has been adopted in regard to patient safety, "Safety is like peeling an onion; the more you look, the more you find, and each layer makes you cry."

Adverse Drug Reaction Reporting Systems↗

PubMed2006

Evidence of one-dimensional scale-dependent fractional advection-dispersion.

A semi-analytical inverse method and the corresponding program FADEMain for parameter estimation of the fractional advection-dispersion equation (FADE) were developed in this paper. We have analyzed Huang et al.'s [Huang, K., Toride, N., van Genuchten, M.Th., 1995. Experimental investigation of solute transport in large homogeneous and heterogeneous saturated soil columns. Trans. Porous Media 18, 283-302.] laboratory experimental data of conservative solute transport in 12.5-m long homogeneous and heterogeneous soil columns to test the non-Fickian dispersion theory of FADE. The dispersion coefficient was calculated by fitting the analytical solution of FADE to the measured data at different transport scales. We found that the dispersion coefficient increased exponentially with transport scale for the homogeneous column, whereas it increased with transport scale in a power law function for the heterogeneous column. The scale effect of the dispersion coefficient in the heterogeneous soil was much more significant comparing to that in the homogeneous soil. The increasing rate of dispersion coefficient versus transport distance was smaller for FADE than that for the advection-dispersion equation (ADE). Finite difference numerical approximations of the scale-dependent FADE were established to interpret the experimental results. The numerical solutions were found to be adequate for predicting scale-dependent transport in the homogeneous column, while the prediction for the heterogeneous column was less satisfactory.

Computer Simulation↗

PubMed2005

Structural snapshots of MTA/AdoHcy nucleosidase along the reaction coordinate provide insights into enzyme and nucleoside flexibility during catalysis.

MTA/AdoHcy nucleosidase (MTAN) irreversibly hydrolyzes the N9-C1' bond in the nucleosides, 5'-methylthioadenosine (MTA) and S-adenosylhomocysteine (AdoHcy) to form adenine and the corresponding thioribose. MTAN plays a vital role in metabolic pathways involving methionine recycling, biological methylation, polyamine biosynthesis, and quorum sensing. Crystal structures of a wild-type (WT) MTAN complexed with glycerol, and mutant-enzyme and mutant-product complexes have been determined at 2.0A, 2.0A, and 2.1A resolution, respectively. The WT MTAN-glycerol structure provides a purine-free model and in combination with the previously solved thioribose-free MTAN-ADE structure, we now have separate apo structures for both MTAN binding subsites. The purine and thioribose-free states reveal an extensive enzyme-immobilized water network in their respective binding subsites. The Asp197Asn MTAN-MTA and Glu12Gln MTAN-MTR.ADE structures are the first enzyme-substrate and enzyme-product complexes reported for MTAN, respectively. These structures provide representative snapshots along the reaction coordinate and allow insight into the conformational changes of the enzyme and the nucleoside substrate. A "catalytic movie" detailing substrate binding, catalysis, and product release is presented.

Amino Acid Substitution↗

PubMed2006

Structure and substrate recognition of the Escherichia coli DNA adenine methyltransferase.

The structure of the Escherichia coli Dam DNA-(adenine-N6)-methyltransferase in complex with cognate DNA was determined at 1.89 A resolution in the presence of S-adenosyl-L-homocysteine. DNA recognition and the dynamics of base-flipping were studied by site-directed mutagenesis, DNA methylation kinetics and fluorescence stopped-flow experiments. Our data illustrate the mechanism of coupling of DNA recognition and base-flipping. Contacts to the non-target strand in the second (3') half of the GATC site are established by R124 to the fourth base-pair, and by L122 and P134 to the third base-pair. The aromatic ring of Y119 intercalates into the DNA between the second and third base-pairs, which is essential for base-flipping to occur. Compared to previous published structures of bacteriophage T4 Dam, three major new observations are made in E.coli Dam. (1) The first Gua is recognized by K9, removal of which abrogates the first base-pair recognition. (2) The flipped target Ade binds to the surface of EcoDam in the absence of S-adenosyl-L-methionine, which illustrates a possible intermediate in the base-flipping pathway. (3) The orphaned Thy residue displays structural flexibility by adopting an extrahelical or intrahelical position where it is in contact to N120.

DNA Methylation↗

PubMed2004

Recent advances in animal models of alcohol craving and relapse.

Animal models designed to examine different facets of alcohol-related behaviors have been developed to study genetic and neurobiological factors underlying alcoholism and alcohol abuse. One goal has been to develop valid, congruent, complementary animal models of alcohol craving and relapse, with the ultimate objective of assessing the effectiveness of pharmacological agents with these models. Animal models of alcohol craving include drug-induced responding (drug reinstatement), cue-induced responding, Pavlovian Spontaneous Recovery (PSR), and appetitive/consummatory responding. A primary experimental approach to study alcohol relapse has been through expression of the Alcohol Deprivation Effect (ADE) following a single deprivation or multiple deprivations. To date, five selectively bred lines of rats have been developed to study alcohol-drinking behavior. These are the ALKO/Alcohol (AA), alcohol-preferring (P), high alcohol-drinking (HAD-1 and HAD-2 replicates), and the Sardinian alcohol-preferring (sP) lines of rats. Findings thus far indicate that only the P line of rats meets all the criteria established for a valid animal model of alcoholism, with progress having been made in characterizing the AA, HAD and sP lines of rats. The focus of the current review will be to analyze the various models of alcohol craving, emphasizing the use of the Indiana University selected rat lines (P and HADs). Overall, the findings indicate substantial progress has been made in developing animal models of alcohol abuse, relapse and craving using these selectively bred rat lines, as well as outbred rats.

Alcohol Drinking↗

PubMed2004

[Medical practice analysis in internal medicine: a national descriptive study].

PURPOSE: This descriptive and epidemiological study was conducted in Mars 2002 in Internal Medicine in order to (1) participate in elaborating a White Book about the speciality, (2) analyse the post-university formation needs of the specialists in Internal Medicine. METHODS: A questionnaire was sent to all specialists in Internal Medicine listed on the ADELI file (n = 2155). For the first three patients seen in consultation and during hospital stay, questioned specialists had to mention the age, sex, origin, motive of the visit, nature of symptoms, complexity of the problem and the nature of the required abilities. They also had to precise the main diagnosis of all patients seen in the same day. RESULTS: Three hundred and sixty answers have been received. Three hundred and thirty two were exploitable. Five thousand six hundred and eleven main diagnosis were listed. Fifteen percent of the questioned specialists did practise in other specialities than Internal Medicine. Orphaned diseases were the most common pathologies carried out in consultation (17%). Patients seen during their hospital stay suffered more frequently from infectious, haematological and malignant diseases. In 55% of the cases, patients were seen in second or third line after a visit to a general practitioner or another specialist. The abilities of the Internal Medicine specialist alone were sufficient in 70% of the cases to solve the problem. Complexity of the problem was evaluated by the specialists themselves at about 45/100 on an analogical scale. CONCLUSIONS: This study inform the medical community about the type of patients treated by the specialists in Internal Medicine, precise the exact nature of their professional exercise and their real need in medical post-university formation.

France↗

PubMed2005

Dengue virus: molecular basis of cell entry and pathogenesis, 25-27 June 2003, Vienna, Austria.

Multivalent dengue vaccines now in late stage development pose unique vaccine safety challenges in that primary or secondary vaccine failures might place vaccines at risk to antibody-dependent enhanced (ADE) wild-type dengue infections. This conference was organized to address this unique vaccine safety issue. New data were presented on the structure of dengue and other flaviviruses, the cellular receptors of dengue virus for biologically relevant cells, dengue viral cell entry mechanisms and mechanisms underlying in vivo protection, neutralization and enhancement of dengue virus infection. It was concluded that a targeted research program should aim to develop an in vitro test to characterize persons immunized with dengue vaccines as completely or partially protected. Achievement of this aim will require a better understanding of the basic mechanisms by which dengue viruses recognize, attach, enter and infect relevant human cells and how antibodies protect against dengue infections.

Antibodies, Viral↗

PubMed2005

Immunogenicity and protective efficacy in monkeys of purified inactivated Vero-cell SARS vaccine.

BACKGROUND: In 2003, severe acute respiratory syndrome (SARS) resulted in hundreds of infections and deaths globally. We aim to assess immunogenicity and protective efficacy of purified inactivated Vero-cell SARS vaccine in monkeys. METHODS: The cultures of SARS coronavirus (SARS-CoV) BJ-01 strain infected Vero cells were inactivated with beta-propiolactone. Sequential procedures, including ultrafiltration, gel filtration and ion exchange chromatography, were performed to obtain purified inactivated SARS vaccine. The purified SARS vaccine was analyzed with electron microscope, HPLC and Western blotting. We immunized three groups of cynomolgus macaques fascicularis with adjuvant-containing purified vaccine, purified vaccine and unpurified vaccine, respectively, and a fourth group served as a control. Antibody titers were measured by plaque reduction neutralization test. The vaccinated monkeys were challenged with SARS-CoV BJ-01 strain to observe protective efficacy. Additionally, three groups of rhesus monkeys were immunized with different doses of the purified inactivated SARS vaccine (0.5, 1 and 2mug/time/monkey) on days 0 and 7, and the monkeys were challenged with SARS-CoV GZ-01 strain. We assessed the safety of the SARS vaccine and observed whether the antibody dependent enhancement (ADE) occurred under low levels of neutralizing antibody in rhesus. FINDINGS: The purity of SARS vaccine was 97.6% by HPLC identification and reacted with convalescent sera of SARS patients. The purified SARS vaccine induced high levels of neutralizing antibodies and prevented the replication of SARS-CoV in monkeys. Under low levels of neutralizing antibody, no exacerbation of clinical symptoms was observed when the immunized monkeys were challenged with SARS-CoV. In this preliminary animal trial, no side effects were detected when monkeys were immunized with purified SARS vaccine either at normal or large doses. INTERPRETATION: The purified inactivated SARS vaccine could induce high levels of neutralizing antibody, and protect the monkeys from the challenge of SARS-CoV. The SARS vaccine prepared in the study appeared to be safe in monkeys.

Animals↗

PubMed2006

Immunity induced with a Salmonella enterica serovar Enteritidis live vaccine is regulated by Th1-cell-dependent cellular and humoral effector mechanisms in susceptible BALB/c mice.

The objective of this study was to characterize the immune response induced by a live attenuated Salmonella Enteritidis (SE; ade(-)/his(-)) vaccine using an intraperitoneal immunization/challenge model in susceptible wild-type and cytokine-deficient BALB/c mice. In wild-type mice, inoculation of the SE live vaccine induced a protective immune response characterized by both cellular (production of interleukin(IL)-12 and interferon(IFN)-gamma, granuloma formation in liver and spleen, DTH response) and humoral effector mechanisms (high antigen-specific IgG2a titers). IL-12- and IL-4-deficient mice were immunized to study the individual roles of Th1 and Th2 cells, respectively. Protective immunity in wild-type mice required inoculation of >5 x 10(3)CFU of the attenuated live SE vaccine strain used. While IL-4-deficient mice developed a protective immune response similar to that found in wild-type mice, it was not possible to induce protective immunity in the highly susceptible IL-12-deficient mice due to severe disease symptoms and death following inoculation of the SE vaccine strain (doses >or=5 x 10(2)CFU were lethal for IL-12-deficient mice). Interestingly, persistence of the vaccine strain was observed in IL-4-deficient mice, indicating a role of IL-4 for clearance which, however, did not interfere with protective immunity. Together, the data indicate that the SE live vaccine activates a cellular and a humoral immune response, which are both regulated by Th1 cells via the secretion of IFN-gamma, whereas Th2 cells did not contribute essentially to the SE live-vaccine-induced immunity.

Animals↗

PubMed2006

Longitudinal mixing in meandering channels: new experimental data set and verification of a predictive technique.

Evaluation of longitudinal mixing processes in open channel flows is important in environmental management, requiring the quantification of mixing coefficients. Estimates of these coefficients sufficiently accurate for environmental impact assessments cannot be achieved using current theoretical or semi-empirical methods for natural channels. This inaccuracy is caused by a limited understanding and quantification of the interaction of the dominant mechanisms resulting from natural channel features, such as plan form curvature and changes in cross-sectional shape. Experimental results are presented here from studies conducted in three self-formed channels, developed by known discharges. Longitudinal mixing was investigated at various flow rates within each of the channels by monitoring the development of tracer plumes during transit through the channels. Using an optimisation procedure, coefficients required for solution of the one-dimensional advection dispersion equation (1D-ADE) were found in the range 0.02-0.2m(2)/s. The coefficients were found to vary as functions of longitudinal meander location, channel form and discharge. Predictions of these longitudinal mixing coefficients were made using a mathematical technique requiring only channel form properties and flow rate as inputs. Predicted values were typically within 20% of the measured values, although deviation of up to 50% was found for the lowest discharge in each channel. This large error is likely to have been caused by increased dead zone effects associated with channel bathymetry at low discharges that are not captured by the method. The method was shown to be capable of capturing the variation in the longitudinal mixing coefficient with longitudinal meander location, with channel form and with discharge.

Diffusion↗