RESUMEN
This was a three group randomized clinical trial of interim methadone and patient navigation involving 225 pre-trial detainees with opioid use disorder in Baltimore. The HIV Risk Assessment Battery (RAB) was administered at baseline (in jail), and at 6 and 12 months post-release. Generalized linear mixed model analyses indicated the condition × time interaction effect failed to reach significance (ps > .05) for both the drug risk and sex risk subscale scores. Therefore, findings suggest that there were no intervention effects on drug or sex risk behaviors. However, increased use of cocaine at baseline was associated with increases in drug- (b = .04, SE = .02) and sex-risk (b = .01, SE = .003) behaviors. These results suggest that interventions targeting cocaine use among pre-trial detainees may serve as a means of reducing HIV risk associated with drug- and sex-risk behaviors.Clinical Trials Registration: Clinicaltrials.gov NCT02334215.
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Infecciones por VIH , Trastornos Relacionados con Opioides , Adulto , Baltimore/epidemiología , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/epidemiología , Infecciones por VIH/prevención & control , Humanos , Metadona/uso terapéutico , Trastornos Relacionados con Opioides/epidemiología , Asunción de RiesgosRESUMEN
The use, misuse, and overuse of antimicrobials is one of the main public health threats of the 21st century. We investigated the risk factor of the presence of extended-spectrum, cephalosporin-resistant Enterobacterales in feces of non-domestic and domestic birds and other domestic animals in Piauí State, northeast Brazil. We collected a total of 387 cloacal and rectal swab samples of free-living birds, domestic birds, and domestic mammals in five municipalities: Amarante, Água Branca, Lagoa Alegre, Parnaíba, and Teresina. A total of 59/387 (15.2%) of these samples harbored extended spectrum beta-lactamase (ESBL)-producing Enterobacterales. Using the MALDI-TOF technique, we identified fifty-seven samples as Escherichia coli and two samples as Klebsiella pneumoniae. Teresina and Parnaíba had the highest prevalence of animals with resistant bacteria (32.1% and 27.1%, respectively) and highest exposure risk factor (OR of 16.06 and 8.58, respectively, and p < 0.001 for all). Multidrug-resistant, ESBL-producing Enterobacterales were observed in 72.8% of the samples (43/59). For the free-living birds, the positive samples belonged to a great kiskadee (Pitangus sulphuratus) and a semipalmated sandpiper (Calidris pusilla) in migratory and resident species, respectively. For domestic animals, the swine samples showed the highest prevalence of antimicrobial resistance. The lack of access to veterinary care and information regarding antimicrobial therapy, along with the easy access to antimicrobials without medical prescription, favors the inadequate use of antimicrobials in Piauí.
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OBJECTIVE: To compare the effects of weight loss by an energy-restricted low-fat diet vs low-carbohydrate diet on serum peptide YY (PYY) levels. DESIGN: 8-Week prospective study of 30 obese adults (mean age: 42.8+/-2.0 years, mean body mass index 35.5+/-0.6 kg m(-2)). RESULTS: After 8 weeks, subjects on the low-carbohydrate diet lost substantially more weight than those on the low-fat diet (5.8 vs 0.99 kg, P<0.001). Weight loss by either diet resulted in a 9% reduction in both mean fasting serum PYY levels (baseline: 103.5+/-8.8 pg ml(-1), after weight loss: 94.1+/-6.5 pg ml(-1), P<0.01) and postprandial area under the curve (AUC) PYY (baseline: (20.5+/-1.5) x 10(3) pg h(-1) ml(-1), after weight loss: mean AUC PYY (18.8+/-1.4) x 10(3) pg h(-1) ml(-1), P<0.001). There was a trend towards lower levels of PYY with greater degrees of weight loss. CONCLUSIONS: Reduced PYY levels after weight loss by an energy-restricted low-fat or low-carbohydrate diet likely represents a compensatory response to maintain energy homeostasis and contributes to difficulty in weight loss during energy-restricted diets.
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Dieta Baja en Carbohidratos , Dieta con Restricción de Grasas , Obesidad/sangre , Péptido YY/sangre , Pérdida de Peso/fisiología , Adulto , Índice de Masa Corporal , Dieta Reductora , Femenino , Humanos , Masculino , Obesidad/fisiopatología , Periodo Posprandial , Estudios ProspectivosRESUMEN
BACKGROUND: Opioid use disorder is common among detainees in US jails, yet methadone treatment is rarely initiated. METHODS: This is a three-group randomized controlled trial in which 225 detainees in Baltimore treated for opioid withdrawal were assigned to: (1) interim methadone (IM) with patient navigation (IMâ¯+â¯PN); (2) IM; or (3) enhanced treatment-as-usual (ETAU). Participants in both IM groups were able to enter standard methadone treatment upon release, while ETAU participants received an assessment/referral number. Follow-up assessments at 1, 3, 6, and 12 months post-release determined treatment enrollment, urine drug testing results, self-reported days of drug use, criminal activity, and overdose events. Generalized linear mixed modelling examined two planned contrasts: (1) IM groups combined vs. ETAU; and (2) IMâ¯+â¯PN vs. IM. RESULTS: On an intention-to-treat basis, compared to ETAU, significantly more participants in the combined IM groups were in treatment 30 days post-release, while the IMâ¯+â¯PN vs. IM groups did not significantly differ. By month 12, there were no significant differences in the estimated marginal means of enrollment in any kind of drug treatment (0.40 and 0.27 for IMâ¯+â¯PN and IM groups, respectively, compared to 0.29 for ETAU). There were no significant differences for either contrast in opioid-positive tests, although all groups reported a sharp decrease in heroin use from baseline to follow-up. There were five fatal overdoses, but none occurred during methadone treatment. CONCLUSION: Initiating methadone treatment in jail was effective in promoting entry into community-based drug abuse treatment but subsequent treatment discontinuation attenuated any potential impact of such treatment.
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Sobredosis de Droga/epidemiología , Metadona/uso terapéutico , Trastornos Relacionados con Opioides/tratamiento farmacológico , Prisioneros/estadística & datos numéricos , Adulto , Analgésicos Opioides/uso terapéutico , Baltimore/epidemiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Tratamiento de Sustitución de Opiáceos/métodos , Aceptación de la Atención de Salud/estadística & datos numéricos , Resultado del TratamientoRESUMEN
The 9th-10th type III fibronectin domain pair (9-10FNIII) has found widespread use as a biomimetic surface for cell adhesion. However, the effect of mutations to 9-10FNIII on its surface adsorption characteristics have not been investigated. Here we address this issue using total internal reflection fluorescence (TIRF) and circular dichroism spectroscopy, comparing two conformationally stable 9-10FNIII mutants against the wild type. Desorption of the 9-10FNIII mutants from the silica surface was minimal in comparison to desorption of 9-10FNIII. The extent and rate of protein desorption from silica was empirically matched by loss of secondary structure upon adsorption, with only the spectrum for 9-10FNIII showing extensive loss of the beta-sandwich fold. For the proteins adsorbed to hydrophobic surfaces, only the CD spectra for the 9-10FNIII mutant constrained via an interdomain disulphide bridge showed similarity with the corresponding solution structure. Since the binding of 9-10FNIII to integrin alpha5beta1 is highly dependent on the relative spatial arrangement of the two domains, we suggest that the observed differences in cell adhesion and spreading on wild type 9-10FNIII and mutants may in part be attributed to the extent of protein desorption and unfolding at the surface.
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Fibronectinas/química , Fibronectinas/fisiología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/fisiología , Conformación Proteica , Pliegue de Proteína , Adsorción , Sustitución de Aminoácidos , Fibronectinas/genética , Humanos , Fragmentos de Péptidos/genética , Estructura Terciaria de Proteína , Dióxido de Silicio , Propiedades de SuperficieRESUMEN
Small, spherical silver nanoclusters were synthesised on the surface of paper as a model cellulosic fibre substrate by a standard chemical reduction method. The concentration of the silver nanoclusters on the substrate surface is roughly proportional to the initial silver salt concentration. However, there is a noticeable degree of nanocluster aggregation to larger agglomerates. The addition of small amounts of α-cellulose, carboxymethyl cellulose or aminocellulose during the synthesis of the silver/cellulose nanocomposites suppresses this aggregation and significantly increases the concentration of the silver nanoclusters on the surface of the fibres of cellulose. These small, surface-stabilised silver nanoclusters, with the desired size and morphology, deposited from aqueous solutions on the surface of cellulosic cotton fibres, show enhanced antibacterial activity against MRSA compared to that of the corresponding silver/cotton nanocomposites prepared in the absence of a cellulosic surface stabiliser.
RESUMEN
The ZOT N-terminal domain was expressed and refolded, yielding a soluble protein with defined secondary structure. Although distantly related to protein I of filamentous phages, no evidence of ATPase activity was found. It is therefore unlikely that the ZOT N-terminal domain is involved in cholera toxin phage packaging in Vibrio cholerae. The ZOT C-terminal domain caused delocalisation of occludin and ZO-1 from Caco-2 cell-cell contacts, irrespective of disulfide bridge formation in its putative binding domain. However, the C-terminal domain did not cause actin reorganisation and this may explain the absence of a concomitant reduction in the transepithelial electrical resistance across cell monolayers.
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Toxina del Cólera/química , Toxina del Cólera/farmacología , Uniones Estrechas/efectos de los fármacos , Actinas/metabolismo , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Células CACO-2 , Endotoxinas , Humanos , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Ocludina , Fosfoproteínas/metabolismo , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Proteína de la Zonula Occludens-1RESUMEN
The 9th-10th type III fibronectin domain pair shows promise in tissue engineering and tumour vasculature targeting. Calorimetry and structure-function analysis were used to investigate the effects of solution formulation and lyophilisation of a mutant ((9-10)FNIII-P). A single endothermic transition for (9-10)FNIII-P in solution was observed at pH<8, irrespective of addition of sucrose or PEG. The temperature at the maximum heat capacity (T(m)) and enthalpy (deltaH) of the transition increased for increasing sucrose concentrations but decreased for increasing PEG concentrations. The transition was fitted to a single two-state unfolding mechanism (in contrast to unfolding in guanidine. x HCl) and was partially reversible only at pH 4, with increasing concentrations of sucrose causing a marked fall in deltaH between scans. Circular dichroism spectra for the thermal unfolding of (9-10)FNIII-P at pH 4 showed loss of native beta-sheet structure and loss of aromatic contributions to the peak centred around 226 nm yielding an intermediate conformation, which in the presence of sucrose was more disordered. Despite a glass transition (T(g)') for (9-10)FNIII-P(aq) of -70 degrees C, primary drying at -30 degrees C did not perturb its conformation upon reconstitution or its biological activity following lyophilisation; the addition of sucrose or PEG had no influence on structure or activity. The main consideration in the formulation of (9-10)FNIII-P was therefore pH.
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Fibronectinas/química , Proteínas Recombinantes de Fusión/química , Animales , Calorimetría , Rastreo Diferencial de Calorimetría , Adhesión Celular , Dicroismo Circular , Cricetinae , Calor , Concentración de Iones de Hidrógeno , Polietilenglicoles/química , Espectrometría de Fluorescencia , Relación Estructura-Actividad , Sacarosa/química , Ingeniería de TejidosRESUMEN
The unfolding of the dimeric enzyme citrate synthase from pig heart in solutions of guanidinium chloride (GdnHCl) was studied. Data from fluorescence, circular dichroism (CD) and thiol group reactivity studies indicated that the enzyme was almost completely unfolded at GdnHCl concentrations greater than or equal to 4 M. On dilution of GdnHCl, essentially no reactivation of the enzyme occurred. The implications of this finding for the process of folding and assembly in vivo of this and other mitochondrial enzymes are discussed. Exposure of the enzyme to high pH (9-10) led to only a small loss of secondary structure and partial reactivation could be observed on readjustment of the pH to 8.0.
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Citrato (si)-Sintasa , Miocardio/enzimología , Oxo-Ácido-Liasas , Animales , Dicroismo Circular , Activación Enzimática , Reactivadores Enzimáticos , Guanidina , Guanidinas , Concentración de Iones de Hidrógeno , Conformación Proteica , Soluciones , Espectrometría de Fluorescencia , Compuestos de Sulfhidrilo , PorcinosRESUMEN
The unfolding and refolding of flavocytochrome P-450 BM3 and its constituent haem and flavin domains have been analysed, using guanidinium chloride (GdnHCl) as a denaturant. Enzyme activities are lost at GdnHCl concentrations too low to cause major changes in secondary structure (0.1-0.5 M). The losses are primarily due to time-dependent FMN removal. Fluorescence and visible CD spectroscopies show that FMN dissociation is complete by 0.7 M GdnHCl, whereas FAD removal is complete by 1.5 M GdnHCl. Limited regain of activity is achieved by dilution of enzyme from solutions of < or = 0.75 M GdnHCl into fresh buffer. Supplementation of GdnHCl-free assay media with flavins (FAD and FMN) causes small additional regains in flavin domain (cytochrome-c reductase) activity lost at low [GdnHCl]. However, flavin addition during the denaturation step affords greater protection against inactivation, suggesting that conformational changes may occur subsequent to flavin loss and that these changes are not readily reversed on dilution of GdnHCl. Loss of catalytically competent haem ligation occurs over the same [GdnHCl] range for P-450 BM3 and its haem domain. In both cases, the 'denatured' P-420 form accumulates in the reduced/carbon monoxide-bound visible spectrum from 0.5 to 2 M GdnHCl. Secondary structure loss also occurs over similar [GdnHCl] ranges for P-450 BM3 and its two domains (80-90% lost from 0.5-3 M GdnHCl), indicating that there is little mutual stabilisation of domains in the holoenzyme. Differential scanning calorimetry measurements support this conclusion, but show that the haem domain is more thermostable than the flavin domain.
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Proteínas Bacterianas/química , Sistema Enzimático del Citocromo P-450/química , Oxigenasas de Función Mixta/química , Bacillus megaterium/enzimología , Proteínas Bacterianas/efectos de los fármacos , Sitios de Unión , Dicroismo Circular , Sistema Enzimático del Citocromo P-450/efectos de los fármacos , Mononucleótido de Flavina/metabolismo , Flavina-Adenina Dinucleótido/metabolismo , Guanidina , Guanidinas/farmacología , Oxigenasas de Función Mixta/efectos de los fármacos , NADPH-Ferrihemoproteína Reductasa , Unión Proteica , Desnaturalización Proteica , Pliegue de Proteína , Proteínas Recombinantes de Fusión/química , Espectrometría de Fluorescencia , Análisis EspectralRESUMEN
Cytochrome P-450 BM3 from Bacillus megaterium catalyses NADPH oxidation in the absence of added substrate. This activity is also associated with the independently expressed flavin-containing reductase domain of the protein. The rates of these activities are more than two orders of magnitude lower than those in the presence of fatty acid P-450 substrates or artificial electron acceptors. Electrons derived from NADPH in this fashion are transferred onto oxygen, generating superoxide (O2-) anions. The formation of these active oxygen species is detectable by luminometry and the chemiluminescence can be inhibited through the addition of superoxide dismutase (but not catalase). This activity is reminiscent of the microbicidal NADPH oxidase activity associated with neutrophils and other leukocyte blood cell types. Diphenyliodonium, a potent inhibitor of the neutrophil NADPH oxidase, effectively inhibits fatty acid hydroxylase and electron transferase activities catalysed by P-450 BM3 and its reductase domain. CD studies on the native and NADPH-reduced P-450 BM3 and BM3 reductase indicate that no secondary structural alteration is caused by pre-incubation with the reductant. Therefore, the previously recognised reversible time-dependent inactivation of P-450 BM3 by NADPH may be attributed to the NADPH oxidase activity associated with the reductase domain of the enzyme.
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Proteínas Bacterianas , Sistema Enzimático del Citocromo P-450/metabolismo , Oxigenasas de Función Mixta/metabolismo , NADH NADPH Oxidorreductasas/metabolismo , Oxidorreductasas/metabolismo , Bacillus megaterium/enzimología , Compuestos de Bifenilo/farmacología , Catálisis , Dicroismo Circular , Clonación Molecular , Sistema Enzimático del Citocromo P-450/genética , Inhibidores Enzimáticos/farmacología , Escherichia coli/genética , Mediciones Luminiscentes , Oxigenasas de Función Mixta/genética , NADH NADPH Oxidorreductasas/antagonistas & inhibidores , NADP/metabolismo , NADPH Oxidasas , NADPH-Ferrihemoproteína Reductasa , Compuestos Onio/farmacología , Oxidación-Reducción , Especies Reactivas de OxígenoRESUMEN
The small, monomeric, phosphoglycerate mutase (PGAM) from Schizosaccharomyces pombe has been overexpressed in a strain of Saccharomyces cerevisiae in which the gene encoding PGAM has been deleted, with a yield of purified enzyme of 10-15 mg per litre cell culture. Three mutants in which histidine residues in S. pombe PGAM have been substituted by glutamine have been purified and characterised. Two mutants (H151Q and H196Q) have kinetic and structural properties very similar to wild-type enzyme, consistent with the proposed location of these (non-conserved) histidines on the surface of the enzyme. The third mutant (H163Q) involving a histidine thought to be part of the active site has greatly reduced mutase and phosphatase activities. Mass spectrometry shows that the phosphorylated form of the H163Q is several 100-times more stable towards hydrolysis than the phosphorylated form of wild-type enzyme. The H163Q mutant appears to be structurally quite distinct from wild-type enzyme. 600 MHz 1D proton NMR spectra of good quality have been obtained for wild-type enzyme and the H151Q and H196Q mutants.
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Mutación , Fosfoglicerato Mutasa/química , Fosfoglicerato Mutasa/genética , Schizosaccharomyces/enzimología , Secuencia de Bases , Dicroismo Circular , Histidina/genética , Cinética , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fosfoglicerato Mutasa/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Schizosaccharomyces/genética , Espectrometría de FluorescenciaRESUMEN
The unfolding of the bacterial chaperone protein groEL (cpn60) in solutions of guanidinium chloride (GdnHCl) has been studied. From the results of CD, fluorescence and light scattering, it is clear that major structural transitions in the protein occur over the range 1.0-1.5 M GdnHCl. The ATPase activity of the protein is lost at lower concentrations (0.75 M). After denaturation in concentrations of GdnHCl above 1.5 M, removal of the denaturing agent by dialysis results in very nearly complete regain of secondary structure (as judged by CD), but not the regain of correct tertiary or quaternary structure, or ATPase activity. The product was shown to be very sensitive to proteolysis by thermolysin, unlike the native protein, and not to show enhanced binding of ANS, a characteristic property of the 'molten globule' state of proteins. The results are discussed in relation to current information concerning the assembly of the groEL protein.
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Proteínas Bacterianas/química , Proteínas de Choque Térmico/química , Adenosina Trifosfatasas/química , Chaperonina 60 , Dicroismo Circular , Guanidina , Guanidinas , Conformación Proteica , Desnaturalización Proteica , Pliegue de Proteína , Dispersión de Radiación , Espectrofotometría Ultravioleta , TermolisinaRESUMEN
The interaction of one of the two DNA methyltransferases encoded by the HgaI restriction and modification system, M.HgaI-2, with substrates and substrate analogues is described. Circular dichroism spectroscopy has been used to demonstrate that addition of the methyl donor, S-adenosyl-L-methionine and the inhibitory substrate analogue sinefungin, both induce conformational transitions in the protein in the absence of DNA. Moreover, the addition of DNA is shown to enhance the apparent secondary structure of M.HgaI-2 whilst addition of sinefungin or S-adenosyl-L-methionine reduces apparent secondary structure. The circular dichroism spectrum of the abortive complex between the enzyme, DNA and sinefungin is dominated by the conformational properties of the binary complex of enzyme and sinefungin alone. Addition of a specific oligodeoxynucleotide duplex in which the target cytosine is replaced by a pyrimidinone, leads to a further ligand induced conformational transition as determined by electrophoretic analysis. The addition of sinefungin, or S-adenosyl-L-methionine, to M.HgaI-2 bound to the reactive oligodeoxynucleotide duplex, leads to yet another conformational transition in the protein as determined by the differential susceptibility of ternary and binary complexes to proteolysis. These experiments identify at least six ligand-inducible conformational states of M.HgaI-2 and, in view of the sequence similarity amongst this class of enzymes, suggest that conformational flexibility is a general feature of C-5 cytosine-specific DNA methyltransferases. Moreover, the substitution of the target cytosine by a pyrimidinone mimics the effect of 5-azacytosine incorporation into DNA.
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ADN-Citosina Metilasas/química , Estructura Secundaria de Proteína , Adenosina/análogos & derivados , Adenosina/farmacología , Secuencia de Aminoácidos , Secuencia de Bases , Dicroismo Circular , ADN/metabolismo , ADN-Citosina Metilasas/efectos de los fármacos , ADN-Citosina Metilasas/genética , Ligandos , Datos de Secuencia Molecular , S-Adenosilmetionina/farmacologíaRESUMEN
DNA duplexes in which the target cytosine base is replaced by 2-H pyrimidinone have previously been shown to bind with a significantly greater affinity to C5-cytosine DNA methyltransferases than unmodified DNA. Here, it is shown that 2-H pyrimidinone, when incorporated into DNA duplexes containing the recognition sites for M.HgaI-2 and M.MspI, elicits the formation of inhibitory covalent nucleoprotein complexes. We have found that although covalent complexes are formed between 2-H pyrimidinone-modified DNA and both M.HgaI-2 and M.MspI, the kinetics of complex formation are quite distinct in each case. Moreover, the formation of a covalent complex is still observed between 2-H pyrimidinone DNA and M.MspI in which the active-site cysteine residue is replaced by serine or threonine. Covalent complex formation between M.MspI and 2-H pyrimidinone occurs as a direct result of nucleophilic attack by the residue at the catalytic position, which is enhanced by the absence of the 4-amino function in the base. The substitution of the catalytic cysteine residue by tyrosine or chemical modification of the wild-type enzyme with N-ethylmaleimide, abolishes covalent interaction. Nevertheless the 2-H pyrimidinone-substituted duplex still binds to M.MspI with a greater affinity than a standard cognate duplex, since the 2-H pyrimidinone base is mis-paired with guanine.
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Citidina/análogos & derivados , ADN (Citosina-5-)-Metiltransferasas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Sitios de Unión , Catálisis , Dicroismo Circular , Citidina/química , Citidina/farmacología , Citosina/química , Citosina/metabolismo , Espectroscopía de Resonancia Magnética , Mutagénesis Sitio-Dirigida , Unión Proteica , Especificidad por SustratoRESUMEN
The means to attenuate Salmonella and to endow such avirulent strains with the ability to express colonization and virulence antigens from other pathogens has achieved considerable progress during the past several years. One can therefore begin to design and construct strains with specificity to a given animal host and to express in a defined way specific colonization and virulence antigens in a manner to stimulate long-lasting immunity to the Salmonella and to the pathogen supplying the genetic information for the colonization and virulence antigens. Since most pathogens colonize on or invade through mucosal surfaces, the use of recombinant bivalent Salmonella vaccine strains to stimulate a mucosal immune response would induce the development of a first line of defense against a diversity of pathogens. Mucosal immunity should therefore reduce contagious spread of many pathogens since the dose to overcome the mucosal immune barrier would be increased to result in a diminished likelihood of infection. The fact that the recombinant Salmonella vaccine strains also induce humoral and cellular immune responses justifies their use for induction of long-lasting immunity. Although considerable progress has been made in targeting antigens to the GALT by use of avirulent Salmonella, a similar strategy for delivery of antigens to the BALT has yet to be discovered and developed. In addition to constituting a system for induction of immunity against a diversity of pathogens, the recombinant avirulent Salmonella system should provide a means to explore parameters of the mucosal immune response. This would include investigation of the location and duration of memory, the age dependence of induction of mucosal immunity, and the means for the possible induction of oral tolerance with regard to either the mucosal or humoral response to an antigen expressed by the recombinant Salmonella. It is also possible to contemplate using the avirulent Salmonella to target expression of various modulators of the immune system such as interleukin-2 and interferon-gamma to the GALT and thus further enhance the immune response. Lastly, one can introduce into avirulent Salmonella strains genes for putative colonization antigens in order to investigate whether induction of an immune response against the putative colonization antigen does or does not interfere with infection. This system, therefore, permits another means to analyze the relative importance of various bacterial surface attributes in conferring pathogenicity to the microbe.
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Antígenos Bacterianos/administración & dosificación , Salmonella/inmunología , Animales , Antígenos Bacterianos/genética , Vacunas Bacterianas/aislamiento & purificación , Tejido Linfoide/inmunología , Tejido Linfoide/microbiología , Mutación , Recombinación Genética , Salmonella/genética , Salmonella/patogenicidad , VirulenciaRESUMEN
Shikimate kinase, despite low sequence identity, has been shown to be structurally a member of the nucleoside monophosphate (NMP) kinase family, which includes adenylate kinase. In this paper we have explored the roles of residues in the P-loop of shikimate kinase, which forms the binding site for nucleotides and is one of the most conserved structural features in proteins. In common with many members of the P-loop family, shikimate kinase contains a cysteine residue 2 amino acids upstream of the essential lysine residue; the side chains of these residues are shown to form an ion pair. The C13S mutant of shikimate kinase was found to be enzymatically active, whereas the K15M mutant was inactive. However, the latter mutant had both increased thermostability and affinity for ATP when compared to the wild-type enzyme. The structure of the K15M mutant protein has been determined at 1.8 A, and shows that the organization of the P-loop and flanking regions is heavily disturbed. This indicates that, besides its role in catalysis, the P-loop lysine also has an important structural role. The structure of the K15M mutant also reveals that the formation of an additional arginine/aspartate ion pair is the most likely reason for its increased thermostability. From studies of ligand binding it appears that, like adenylate kinase, shikimate kinase binds substrates randomly and in a synergistic fashion, indicating that the two enzymes have similar catalytic mechanisms.
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Lisina/química , Lisina/fisiología , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , 2,2'-Dipiridil/análogos & derivados , 2,2'-Dipiridil/farmacología , Adenosina Trifosfato/metabolismo , Arginina/química , Ácido Aspártico/química , Sitios de Unión , Rastreo Diferencial de Calorimetría , Catálisis , Dicroismo Circular , Cristalografía por Rayos X , Dickeya chrysanthemi/química , Disulfuros/farmacología , Escherichia coli/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Ligandos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Oxidantes/farmacología , Unión Proteica , Espectrometría de Fluorescencia , Relación Estructura-Actividad , Reactivos de Sulfhidrilo/farmacología , Temperatura , Ácido Trinitrobencenosulfónico/farmacología , Rayos UltravioletaRESUMEN
The paper describes the equilibrium dependant reaction for the formation of enamines in a microchemical system utilising electroosmotic flow (EOF) for fluid mobilisation. The authors have shown that the reaction can be carried out without the presence of a Lewis acid catalyst, in addition the enamine intermediate was synthesised at room temperature using mild solvent conditions. A 42% conversion of cyclohexanone into the enamine has been achieved to date.
RESUMEN
Circular Dichroism (CD) relies on the differential absorption of left and right circularly polarised radiation by chromophores which either possess intrinsic chirality or are placed in chiral environments. Proteins possess a number of chromophores which can give rise to CD signals. In the far UV region (240-180 nm), which corresponds to peptide bond absorption, the CD spectrum can be analysed to give the content of regular secondary structural features such as alpha-helix and beta-sheet. The CD spectrum in the near UV region (320-260 nm) reflects the environments of the aromatic amino acid side chains and thus gives information about the tertiary structure of the protein. Other non-protein chromophores such as flavin and haem moieties can give rise to CD signals which depend on the precise environment of the chromophore concerned. Because of its relatively modest resource demands, CD has been used extensively to give useful information about protein structure, the extent and rate of structural changes and ligand binding. In the protein design field, CD is used to assess the structure and stability of the designed protein fragments. Studies of protein folding make extensive use of CD to examine the folding pathway; the technique has been especially important in characterising molten globule intermediates which may be involved in the folding process. CD is an extremely useful technique for assessing the structural integrity of membrane proteins during extraction and characterisation procedures. The interactions between chromophores can give rise to characteristic CD signals. This is well illustrated by the case of the light harvesting complex from photosynthetic bacteria, where the CD spectra can be analysed to indicate the extent of orbital overlap between the rings of bacteriochlorophyll molecules. It is therefore evident that CD is a versatile technique in structural biology, with an increasingly wide range of applications.
Asunto(s)
Dicroismo Circular , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas/química , Aminoácidos/química , Disulfuros/química , Diseño de Fármacos , Ligandos , Modelos Moleculares , Pliegue de ProteínaRESUMEN
The latent precursors of the matrix metalloproteinases (MMPs) are converted by (4-aminophenylmercuric)acetate to active forms that lose their propeptide as a result of autolysis. C.D. and an active site mutant of progelatinase A (MMP2) were used to demonstrate that, although propeptide removal is accompanied by a decrease in the enzyme's beta-sheet content, the initial activation is achieved with only minor modifications to the conformation. Mixing activated gelatinase A with the natural inhibitor, TIMP-1, resulted in conformational changes that were absent when a synthetic inhibitor was used. The relevance of these results to MMP activation and inhibition is discussed.