RESUMEN
Positive allosteric modulators (PAMs) of 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptors receive increasing interest as therapeutic drugs and have long served as important experimental tools in the study of the molecular mechanisms underlying glutamate-mediated neurotransmission. The aim of this study was to investigate functional and structural aspects of a novel analog of the AMPA receptor PAM cyclothiazide (CTZ) on recombinant and native glutamate receptors. We expressed rat GluA4flip and flop in Xenopus oocytes and characterized NS1376 and CTZ under two-electrode voltage-clamp. The dose-response analyses revealed dual effects of NS1376. The modulator induced 30-fold and 42-fold reductions in glutamate potency and increased the glutamate efficacy by 3.2-fold and 5.3-fold at GluA4flip and GluA4flop, respectively. Rapid application of glutamate to excised outside-out patches showed that NS1376 markedly attenuated desensitization, supporting the increased efficacy observed in the oocytes. Furthermore, when applied to acutely isolated mouse brain slices, NS1376 reduced the field excitatory postsynaptic potentials (fEPSPs) in the hippocampus to 51.6 ± 4.3% of baseline, likely as a consequence of reduced glutamate potency. However, the modulator displayed no effects on a sub-maximal long-term potentiation (LTP) protocol. We confirmed that CTZ increases presynaptic transmitter release, a property which was not shared by NS1376. Finally, we obtained detailed molecular information through X-ray structures, docking and molecular dynamics, which revealed that NS1376 interacts at the dimer interface of the ligand-binding domain in a manner overall similar to CTZ. NS1376 reveals that minor structural changes in CTZ can result in an altered modulatory profile, both enhancing agonist efficacy while markedly reducing agonist potency. These unique properties add new aspects to the complexity of allosteric modulations in neuronal systems.
Asunto(s)
Regulación Alostérica/efectos de los fármacos , Benzotiadiazinas/farmacología , Hipocampo/fisiología , Receptores AMPA/metabolismo , Sinapsis/fisiología , Animales , Benzotiadiazinas/química , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Ácido Glutámico/farmacología , Hipocampo/efectos de los fármacos , Potenciación a Largo Plazo/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Recombinantes/química , Proteínas Recombinantes/efectos de los fármacos , Sinapsis/efectos de los fármacos , Xenopus laevisRESUMEN
The human pathogen Mycoplasma pneumoniae can cause atypical pneumonia through adherence to epithelial cells in the respiratory tract. The major immunogenic protein, P1, participates in the attachment of the bacteria to the host cells. To investigate the adhesion properties of P1, a recombinant protein (rP1-II) covering amino acids 1107-1518 of the P1 protein was produced. This protein inhibited the adhesion of M. pneumoniae to human HEp-2 cells, as visualized in a competitive-binding assay using immunofluorescence microscopy. Previous studies have shown that mAbs that recognize two epitopes in this region of P1 also reduce M. pneumoniae adhesion. Therefore, peptides covering these epitopes, of 8 and 13 aa, respectively, were synthesized to further investigate the adhesion region. None of these synthetic peptides reduced the binding of M. pneumoniae to the receptors on the host cells. Instead, 10 overlapping synthetic peptides covering the whole of rP1-II were evaluated in the competitive-binding assay using immunofluorescence microscopy. A reduction in the number of M. pneumoniae microcolonies was seen, which was confirmed for five peptides using a POLARstar OPTIMA reader to measure fluorescence intensity. The number of M. pneumoniae microcolonies adhering to the host cells was significantly reduced by these five peptides. Further investigations showed that inhibiting peptide 7 (amino acids 1347-1396) of the major adhesin protein P1 bound directly to host receptors, suggesting that the observed M. pneumoniae-inhibiting peptides occupied HEp-2 receptors, which are otherwise available for P1-mediated M. pneumoniae adhesion.
Asunto(s)
Adhesinas Bacterianas/química , Adhesinas Bacterianas/metabolismo , Células Epiteliales/microbiología , Mycoplasma pneumoniae/fisiología , Receptores de Superficie Celular/metabolismo , Adhesinas Bacterianas/genética , Secuencia de Aminoácidos , Adhesión Bacteriana , Unión Competitiva , Línea Celular , Fluoresceína-5-Isotiocianato , Humanos , Microscopía Fluorescente , Datos de Secuencia Molecular , Mycoplasma pneumoniae/patogenicidad , Péptidos/síntesis química , Péptidos/química , Péptidos/metabolismoRESUMEN
Chlamydia trachomatis infection induces an inflammatory response that is crucial in resolving acute infection but may also play a key role in the pathogenesis of C trachomatis associated infertility. The immune response is linked to cytokine secretion pattern which is influenced by the host genetic background. To study a relationship between interleukin-10 (IL-10) promoter -1082 polymorphism and cell-mediated immune response during C trachomatis infection in vitro, lymphocyte proliferation and cytokine (IL-10, IFN-gamma, TNF-alpha, IL-2, IL-4 and IL-5) secretion were analysed in subjects with different IL-10 genotypes. Enhanced IL-10 secretion and reduced antigen-specific lymphocyte proliferative and IFN-gamma responses were found in subjects with IL-10 -1082 GG genotype when compared to those with -1082 AA genotype. CD14+ monocytes were main source of IL-10 indicating that these cells are important regulators of the antigen-specific cell-mediated responses during active C trachomatis infection. We conclude that impaired cell-mediated response to C trachomatis is associated with IL-10 genotype in subjects with high IL-10 producing capacity. A comparison of immune markers between subjects with a history of noncomplicated and complicated infection is needed to further understand the confounding factors associated with the development of C trachomatis associated sequelae.
Asunto(s)
Infecciones por Chlamydia/inmunología , Chlamydia trachomatis/inmunología , Inmunidad Celular/genética , Interleucina-10/genética , Polimorfismo Genético , Adolescente , Adulto , Anticuerpos Antibacterianos/sangre , Infecciones por Chlamydia/genética , Citocinas/metabolismo , Femenino , Humanos , Interferón gamma/metabolismo , Interleucina-10/metabolismo , Persona de Mediana Edad , Regiones Promotoras Genéticas/genéticaRESUMEN
BACKGROUND: To evaluate the role of Chlamydia trachomatis-induced humoral and cell-mediated immune (CMI) responses in predicting tubal factor infertility (TFI). METHODS: Blood samples were taken from 88 women with TFI and 163 control women. C. trachomatis and chlamydial heat shock protein 60 (CHSP60)-specific immunoglobulin G (IgG) antibodies were analysed using enzyme-linked immunosorbent assay (ELISA) kits. Proliferative reactivity of peripheral blood mononuclear cells was studied in vitro against Chlamydia elementary body (EB) and recombinant CHSP60 antigens. RESULTS: C. trachomatis-specific IgG antibodies were found more frequently (43.2 versus 13.5%), and the antibody levels were higher in the TFI cases than in the controls (P < 0.001). C. trachomatis EB-induced lymphocyte responses were positive in 81.8% of the TFI cases and 58.9% of the controls (P < 0.001). Similarly, CHSP60-induced lymphocyte responses were found in 45.5% of the TFI cases and 30.7% of the controls (P < 0.001). CHSP60 antibody test was the best single test predicting TFI. Compared to cases with all four markers negative, the estimated risk for TFI was 4.1 (95% CI 1.4-11.9) among those with one positive marker and 19.9 (95% CI 6.9-57.4) among those with three to four positive markers. CONCLUSION: Our results show that TFI prediction model can be improved by combining tests for humoral and CMI response to chlamydial antigens.
Asunto(s)
Anticuerpos/química , Chaperonina 60/inmunología , Infecciones por Chlamydia/complicaciones , Chlamydia trachomatis/metabolismo , Enfermedades de las Trompas Uterinas/microbiología , Infertilidad/microbiología , Adulto , Estudios de Casos y Controles , Chaperonina 60/química , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Sistema Inmunológico , Inmunoglobulina G/química , Sensibilidad y EspecificidadRESUMEN
AIMS: The aim of this study was to investigate toxicological differences between strains of the cyanobacterium Microcystis aeruginosa isolated from a potable water supply in the north of Portugal over a 2-month period. METHODS AND RESULTS: Twenty-six strains of M. aeruginosa were isolated, grown in pure culture, and tested using a range of techniques including matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), ELISA and a PCR procedure targeting the genes implicated in the production of toxic microcystins. There was considerable variation with respect to the amounts of microcystin produced by each of the strains as measured by ELISA, with values ranging from 0.02 to 0.53% dry weight. The results of the MALDI-TOF MS analysis demonstrated the presence of several chemically distinct forms of microcystin as well as aeruginosins, anabaenopeptins and several other unidentified peptide-like compounds. CONCLUSIONS: The growth of individual strains that comprise bloom populations, with unique 'chemotypes' can potentially be an important factor affecting the toxicity of bloom populations. Molecular probes, targeting the genes responsible for microcystin production were shown to be useful for distinguishing between toxic and nontoxic strains and showed good agreement with the results obtained from the other analyses. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study show that the analysis of cyanobacterial bloom populations at the subspecies (strain) level can potentially provide important information regarding the toxin-producing potential of a cyanobacterial bloom and could be used as an 'early warning' for toxic bloom development.
Asunto(s)
Microcystis/fisiología , Ríos/microbiología , Microbiología del Agua , Toxinas Bacterianas/análisis , Medios de Cultivo , ADN Bacteriano/análisis , Inhibidores Enzimáticos/análisis , Ensayo de Inmunoadsorción Enzimática/métodos , Microcistinas , Microcystis/aislamiento & purificación , Oligopéptidos/análisis , Péptidos/análisis , Péptidos Cíclicos/análisis , Reacción en Cadena de la Polimerasa/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodosRESUMEN
Mycoplasma hominis has been previously described as a heterogeneous species, and in the present study intraspecies diversity of 20 M. hominis isolates from different individuals was analyzed using parts of the unlinked gyrase B (gyrB), elongation factor Tu (tuf), SRalpha homolog (ftsY), hitB-hitL, excinuclease ABC subunit A (uvrA) and glyceraldehyde-3-phosphate dehydrogenase (gap) genes. The level of variability of these M. hominis genes was low compared with the housekeeping genes from Helicobacter pylori and Neisseria meningitidis, but only few M. hominis isolates had identical sequences in all genes indicating the presence of recombination. In order to test for intergenic recombination, phylogenetic trees were reconstructed for each of the genes but no well-supported bifurcating phylogenetic trees could be obtained. The genes were tested for intragenic recombination using the correlation between linkage disequilibrium and distance between the segregating sites, by the homoplasy ratio (H ratio), and by compatibility matrices. The gap gene showed well-supported evidence for high levels of recombination, whereas recombination was less frequent and not significant within the other genes. The analysis revealed intergenic and intragenic recombination in M. hominis and this may explain the high intraspecies variability. The results obtained in the present study may be of importance for future population studies of Mycoplasma species.
Asunto(s)
Variación Genética , Mycoplasma hominis/genética , Recombinación Genética , Proteínas Bacterianas/genética , Girasa de ADN/genética , Endodesoxirribonucleasas/genética , Proteínas de Escherichia coli/genética , Genes Bacterianos , Gliceraldehído 3-Fosfato Deshidrogenasa (NADP+)/genética , Humanos , Modelos Genéticos , Datos de Secuencia Molecular , Mycoplasma hominis/aislamiento & purificación , Factor Tu de Elongación Peptídica/genética , Filogenia , Receptores Citoplasmáticos y Nucleares/genética , Análisis de Secuencia de ADNRESUMEN
Previous studies largely carried out with environmental samples or axenic and non-axenic cultures suggested that cyanobacteria may be a rich source of hitherto unexplored bioactive compounds. This has been confirmed in the present study by a screening of 146 axenic strains from the Pasteur Culture Collection (PCC) of cyanobacteria. Use of degenerate PCR primers, designed on the basis of conserved sequence motifs in the aminoacyl-adenylation domain of peptide synthetases, revealed the presence of the corresponding genes in the majority (75.3%) of the strains examined. Among unicellular cyanobacteria, only Chamaesiphon sp. strain PCC 6605, two strains of Gloeocapsa and most Microcystis isolates (22 out of 24) contained these genes; no amplicons were detected for any members of the genera Cyanothece, Gloeobacter and Gloeothece and the genetically diverse representatives of Synechococcus and Synechocystis. By contrast, eight out of ten pleurocapsalean members, 16 out of 25 oscillatorian strains, and all but two of the 63 filamentous heterocystous cyanobacteria tested gave positive amplification results. This information will be highly valuable for further exploring the corresponding cyanobacterial peptides and for elucidating the bioactivity of such non-ribosomally synthesized molecules.
Asunto(s)
Cianobacterias/genética , Péptido Sintasas/genética , Cianobacterias/enzimología , ADN Bacteriano/análisis , ADN Bacteriano/genéticaRESUMEN
The variable adherence-associated (Vaa) adhesin of the opportunistic human pathogen Mycoplasma hominis is a surface-exposed, membrane-associated protein involved in the attachment of the bacterium to host cells. The molecular masses of recombinant 1 and 2 cassette forms of the protein determined by a light-scattering (LS) method were 23.9 kD and 36.5 kD, respectively, and corresponded to their monomeric forms. Circular dichroism (CD) spectroscopy of the full-length forms indicated that the Vaa protein has an alpha-helical content of approximately 80%. Sequence analysis indicates the presence of coiled-coil domains in both the conserved N-terminal and antigenic variable C-terminal part of the Vaa adhesin. Experimental results obtained with recombinant proteins corresponding to the N- or C-terminal parts of the shortest one-cassette form of the protein were consistent with the hypothesis of two distinct coiled-coil regions. The one-cassette Vaa monomer appears to be an elongated protein with a axial shape ratio of 1:10. Analysis of a two-cassette Vaa type reveals a similar axial shape ratio. The results are interpreted in terms of the topological organization of the Vaa protein indicating the localization of the adherence-mediating structure.
Asunto(s)
Adhesinas Bacterianas , Proteínas Bacterianas/química , Proteínas de la Membrana , Mycoplasma hominis/química , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Cromatografía de Afinidad , Dicroismo Circular , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Luz , Modelos Moleculares , Datos de Secuencia Molecular , Plásmidos/metabolismo , Reacción en Cadena de la Polimerasa , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Dispersión de Radiación , Programas Informáticos , AguaRESUMEN
Autoaggregation is a phenomenon thought to contribute to colonization of mammalian hosts by pathogenic bacteria. Type 1 fimbriae are surface organelles of Escherichia coli that mediate d-mannose-sensitive binding to various host surfaces. This binding is conferred by the minor fimbrial component FimH. In this study, we have used random mutagenesis to identify variants of the FimH adhesin that confer the ability of E. coli to autoaggregate and settle from liquid cultures. Three separate autoaggregating clones were identified, all of which contained multiple amino acid changes located within the N-terminal receptor-binding domain of FimH. Autoaggregation could not be inhibited by mannose, but was inhibited by growth at temperatures at or below 30 degrees C. Using green fluorescent protein (GFP) as a reporter, we show that the autoaggregating clones do not mix with wild-type fimbriated cells. Electron microscopy shows that autoaggregating cells produce fimbriae with a twisted and entangled appearance. We present evidence that autoaggregating versions of FimH also occur in nature. Our results stress the highly adaptive nature of the ubiquitous FimH adhesin.
Asunto(s)
Adhesinas Bacterianas/genética , Adhesinas Bacterianas/fisiología , Adhesinas de Escherichia coli , Escherichia coli/genética , Escherichia coli/fisiología , Proteínas Fimbrias , Adhesión Bacteriana/genética , Adhesión Bacteriana/fisiología , Escherichia coli/patogenicidad , Escherichia coli/ultraestructura , Fimbrias Bacterianas/genética , Fimbrias Bacterianas/fisiología , Fimbrias Bacterianas/ultraestructura , Mutación del Sistema de Lectura , Genes Bacterianos , Humanos , Microscopía Electrónica , Mutagénesis , Fenotipo , Eliminación de Secuencia , VirulenciaRESUMEN
The complete genome of Chlamydia pneumoniae contains a total of 21 genes encoding polymorphic membrane proteins (Pmp). From this large Pmp family three genes, pmp8, pmp10 and pmp11, were cloned and antibodies against recombinant full-length Pmp proteins were produced. Indirect immunofluorescence microscopy of HEp-2 cells infected with C. pneumoniae CWL029 was performed with the Pmp antibodies in combination with a Chlamydia-specific anti-lipopolysaccharide (LPS) antibody. This double staining technique clearly showed that expression of Pmp10 was differential. Additional double staining with monoclonal antibodies to the surface of C. pneumoniae elementary bodies and the anti-LPS antibody resulted in identification of seven monoclonal antibodies that reacted identically to the Pmp10 antibody indicating that Pmp10 is an immunodominant protein. Finally, the molecular mechanism responsible for differential expression is suggested to be variation in the guanine residues in the polyG tract of pmp10.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Chlamydophila pneumoniae/crecimiento & desarrollo , Anticuerpos Monoclonales , Proteínas de la Membrana Bacteriana Externa/genética , Línea Celular , Chlamydophila pneumoniae/metabolismo , Clonación Molecular , ADN Bacteriano/genética , Genes Bacterianos , Humanos , Microscopía Fluorescente , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADNRESUMEN
Chlamydophila pneumoniae displays surprisingly little genomic variation, as seen by comparisons of the published genomes from three different isolates and sequencing of four different genes from different isolates. We have in the present study, however, demonstrated genomic variation between 10 C. pneumoniae isolates in the 11690-bp region between the two outer membrane protein genes pmp1 and pmp2. This region of the C. pneumoniae CWL-029 isolate contains seven C. pneumoniae-specific open reading frames (hb1-7, encoding hydrophobic beta-sheet-containing proteins). We identified additionally 12 open reading frames in the C. pneumoniae CWL-029 genome encoding hypothetical proteins with similarity to the seven hypothetical Hb-proteins. Compared to other isolates, genomic variation is seen to cause frame-shifting of three of the 19 hb-open reading frames, which are proposed to be three full-length genes and eight frame-shifted pseudogenes. The hypothetical proteins encoded by these proposed genes contain an N-terminally located highly hydrophobic stretch of 50-60 residues. A similar motif is found in all identified Chlamydia inclusion membrane proteins and therefore the Hb-proteins are candidate inclusion proteins.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/genética , Chlamydophila pneumoniae/clasificación , Chlamydophila pneumoniae/genética , ADN Intergénico/genética , Variación Genética/genética , Genoma Bacteriano , Secuencia de Bases , Enzimas de Restricción del ADN/metabolismo , Humanos , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADNRESUMEN
BACKGROUND: The role of Mycoplasma genitalium in the pathogenesis of pelvic inflammatory disease has not been characterized. METHODS: Sera from 308 infertile women were investigated for antibodies to M. genitalium by immunoblotting. Women with tubal factor infertility (TFI) made up 132 of the patients, 67 of the women had an infertile male partner and 109 were infertile for unknown reasons. RESULTS: Of the TFI patients 29 (22.0%) were seropositive to the major adhesin, MgPa, of M. genitalium versus 11 (6.3%) in the group of women with normal tubes. No cross-reactions between MgPa and P1 of the related Mycoplasma pneumoniae were found. Besides, MgPa positive sera were confirmed by immunoblotting using a cloned fragment of the C-terminal part of MgPa specific to M. genitalium. Chlamydia trachomatis is known to be able to cause infertility as a result of salpingitis. Therefore, the sera were tested against C. trachomatis using a commercial ELISA test. Seventy-five (56.8%) of the TFI patients were seropositive to C. trachomatis. Eight (27.6%) TFI patients seropositive to MgPa were negative to C. trachomatis. CONCLUSIONS: This study indicates that M. genitalium may be an independent risk factor in the development of an inflammatory process leading to scarring of the uterine tubes in women and thereby causing infertility.
Asunto(s)
Infertilidad Femenina/microbiología , Infecciones por Mycoplasma/complicaciones , Infecciones por Mycoplasma/diagnóstico , Pruebas Serológicas , Adhesinas Bacterianas/química , Adhesinas Bacterianas/genética , Adhesinas Bacterianas/inmunología , Secuencia de Aminoácidos/genética , Anticuerpos Antibacterianos/análisis , Proteínas Bacterianas/inmunología , Chlamydia trachomatis/inmunología , Reacciones Cruzadas , Ensayo de Inmunoadsorción Enzimática , Enfermedades de las Trompas Uterinas/complicaciones , Femenino , Humanos , Immunoblotting , Infertilidad Femenina/etiología , Datos de Secuencia Molecular , Mycoplasma/inmunología , Mycoplasma hominis/inmunología , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Neumonía por Mycoplasma/inmunología , Proteínas Recombinantes , Valores de ReferenciaRESUMEN
Chlamydia pneumoniae is a common cause of community-acquired pneumonia and it has been associated with atherosclerosis. C. pneumoniae has usually been diagnosed by serology using a microimmunofluorescence test, but more recently polymerase chain reaction (PCR) has been viewed as an advantageous alternative. We developed a quantitative real-time PCR for detection of C. pneumoniae. Primers were targeted for the pmp4 gene, and the PCR fragment was detected real-time with a fluorescence resonance energy transfer probe set using a LightCycler instrument. The PCR was used on DNA released from 50 microm sections of paraffin-embedded formalin-fixed lung tissue from experimentally infected mice. Thereby, the number of C. pneumoniae genomes was determined. To our knowledge this is the first time quantification of C. pneumoniae DNA has been attempted on paraffin-embedded formalin-fixed tissue. C. pneumoniae-specific immunohistochemistry (IHC) was done on 5 microm sections adjacent to the sections used in PCR, and the number of inclusions were counted in each section. Good correlation was found when comparing results from PCR and IHC, which is in contrast to many previous studies.
Asunto(s)
Chlamydophila pneumoniae/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Animales , Infecciones por Chlamydophila/diagnóstico , Infecciones por Chlamydophila/microbiología , Chlamydophila pneumoniae/genética , ADN Bacteriano/análisis , Células HeLa , Humanos , Inmunohistoquímica , Ratones , Mycoplasma pneumoniae/genética , Sensibilidad y EspecificidadRESUMEN
Chlamydia pneumoniae is an obligate intracellular human pathogen infecting epithelial cells of the upper respiratory tract. It is a Gram-negative bacteria and has a unique biphasic developmental cycle. In this study, we use two-dimensional gel electrophoresis in combination with radioactive labeling to investigate time-dependent expression and processing of C. pneumoniae proteins. We report on (i) the identification of a hypothetical protein which is expressed late in the developmental cycle and subsequently processed; we speculate that this protein may be of importance for the developmental cycle of Chlamydia; (ii) the identification of the major outer membrane protein in three different variants, which may all be present in vivo.
Asunto(s)
Proteínas Bacterianas/análisis , Chlamydophila pneumoniae/fisiología , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Electroforesis en Gel Bidimensional/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodosRESUMEN
PURPOSE: To evaluate the prevalence, severity, and visual significance of glistenings seen in the AcrySof intraocular lens (IOL) and determine whether a large in-depth study of this lens is warranted. SETTING: John Moran Eye Center, University of Utah, Salt Lake City, Utah, USA. METHODS: Patients who had the AcrySof IOL implanted at 1 institution over the past 4 years were serially selected. Forty-two eyes that had phacoemulsification with implantation of the AcrySof IOL were evaluated. The examination consisted of visual acuity, glare, contrast sensitivity, and dilated fundus and dilated slitlamp evaluations. Glistenings in the IOL were graded at the slitlamp from trace to 4+. RESULTS: All 42 IOLs had some degree of lenticular glistenings. The glistenings were graded as trace in 27 IOLs (64%), 1+ in 5 (12%), 2+ in 5 (12%), 3+ in 3 (7%), and 4+ in 2 (5%). The Snellen acuity in eyes with severe glistenings (> or = 2+) was one-half line lower than in eyes with mild glistenings (+) (P =.01). In all eyes, the Brightness Acuity Tester score was a little more than one-half line lower than the Snellen acuity, and the difference was 1 full line in eyes with glistenings graded above 2+. Fourteen of 15 IOLs (93%) with glistenings graded greater than trace had been in the eye for more than 1 year. There was no evidence that contrast sensitivity was affected by the glistenings. CONCLUSIONS: Glistenings occurred frequently in AcrySof IOLs, with most cases mild. A larger study of this lens is needed to determine whether severe presentations affect visual function and to understand how glistenings change over time.
Asunto(s)
Acrilatos , Lentes Intraoculares/efectos adversos , Falla de Prótesis , Trastornos de la Visión/etiología , Anciano , Sensibilidad de Contraste , Femenino , Deslumbramiento , Humanos , Implantación de Lentes Intraoculares , Masculino , Facoemulsificación , Proyectos Piloto , Prevalencia , Factores de Tiempo , Agudeza VisualRESUMEN
Chlamydia pneumoniae is an obligate intracellular human pathogen that causes acute and chronic respiratory tract diseases and that has been implicated as a possible risk factor in the development of atherosclerotic heart disease. C. pneumoniae cultivated in Hep-2 cells were 35S-labeled and infectious elementary bodies (EB) were purified. The EB proteins were separated by two-dimensional gel electrophoresis. Excised protein spots were in-gel digested with trypsin and peptides were concentrated on reverse-phase chromatographic beads for identification analysis by matrix-assisted laser desorption/ionization-mass spectrometry. In the pH range from 3-11, 263 C. pneumoniae protein spots encoded from 167 genes were identified. These genes constitute 15% of the genome. The identified proteins include 31 hypothetical proteins. It has recently been suggested that EB should be able to synthesize ATP. This view may be strengthened by the identification of several proteins involved in energy metabolism. Furthermore, proteins have been found which are involved in the type III secretion apparatus important for pathogenesis of intracellular bacteria. Proteome maps and a table of all identified proteins have been made available on the world wide web at www.gram.au.dk.
Asunto(s)
Proteínas Bacterianas/análisis , Chlamydophila pneumoniae/química , ADN Bacteriano/análisis , Proteoma/análisis , Aminoácidos/biosíntesis , Proteínas de la Membrana Bacteriana Externa/análisis , Reparación del ADN , Replicación del ADN , Bases de Datos Factuales , Electroforesis en Gel Bidimensional/métodos , Metabolismo Energético , Humanos , Nucleótidos/metabolismo , Biosíntesis de Proteínas , Pliegue de Proteína , Procesamiento Proteico-Postraduccional , Recombinación Genética , Transcripción Genética , Células Tumorales CultivadasAsunto(s)
Chlamydiales/clasificación , Animales , Proteínas Bacterianas/genética , Infecciones por Chlamydiaceae/microbiología , Chlamydiales/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Genes de ARNr , Humanos , Filogenia , Reacción en Cadena de la Polimerasa , ARN Ribosómico/genética , Especificidad de la EspecieRESUMEN
The gene p75 encoding a 75-kDa surface-exposed membrane protein P75 was cloned and sequenced from Mycoplasma hominis type strain PG21T. To investigate the intraspecies variability, sequences were obtained from an additional two isolates 7488 and 183, and the three sequences were compared. The nucleotide and amino acid differences were not confined to specific regions of the gene/protein, but when comparing the three sequences, differences were present as single site substitutions or small insertions or deletions of nucleotides/amino acids. The intraspecies variability was further investigated by restriction enzyme analysis with two restriction enzymes (Alul and MboII) of PCR products amplified from p75 from 28 M. hominis isolates. On the basis of band patterns produced by the two restriction enzymes, the isolates could be divided into five and six groups. These groups neither matched categories of the M. hominis vaa gene nor the M. hominis p120 gene classes, indicating that the three genes vary by different mechanisms and possibly indicating horizontal gene transfer. Federation of European Microbiological Societies.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Bacterianas/genética , Variación Genética , Proteínas de la Membrana/genética , Infecciones por Mycoplasma/microbiología , Mycoplasma hominis/genética , Secuencia de Aminoácidos , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Clonación Molecular , Femenino , Humanos , Masculino , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Mycoplasma hominis/crecimiento & desarrollo , Mycoplasma hominis/metabolismo , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
The human pathogen Chlamydia trachomatis is an obligate intracellular bacterium, characterized by a developmental cycle that alternates between the infectious, extracellular elementary bodies and intracellular, metabolically active reticulate bodies. The cellular immune effector interferon gamma (IFN-gamma) inhibits chlamydial multiplication in human epithelial cells by induction of the tryptophan degrading enzyme indoleamine 2,3 dioxygenase. IFN-gamma causes persistent C. trachomatis serovar A infections with atypical reticulate bodies that are unable to redifferentiate into elementary bodies and show diminished expression of important immunogens, but not of GroEL. However, the sensitivity to IFN-gamma varies among serovars of C. trachomatis. In our previous study significant IFN-gamma-specific, but tryptophan reversible, induction of proteins in C. trachomatis A and L2 with molecular masses of approximately 30 and 40 kDa was observed on 2D-gels. The 30-kDa protein from C. trachomatis L2 migrated with a significantly lower molecular weight in C. trachomatis A. In this paper we include C. trachomatis B, C and D in our investigations and identify the proteins as alpha- and beta-subunits of the chlamydial tryptophan synthase using matrix-assisted laser desorption/ionization mass spectrometry. DNA sequencing of the trpA genes from C. trachomatis A and C shows that the TrpA in these serovars is a 7.7-kDa truncated version of C. trachomatis D and L2 TrpA. The truncation probably impairs the TrpA activity, thus elucidating a possible molecular mechanism behind variations in the pathogenesis of C. trachomatis serovars.
Asunto(s)
Chlamydia trachomatis/enzimología , Chlamydia trachomatis/patogenicidad , Porinas , Triptófano Sintasa/genética , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Línea Celular , Chlamydia trachomatis/clasificación , Chlamydia trachomatis/genética , Electroforesis en Gel Bidimensional , Variación Genética , Células HeLa , Humanos , Interferón gamma/farmacología , Ratones , Microscopía Fluorescente , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Triptófano Sintasa/química , Triptófano Sintasa/metabolismoRESUMEN
There is no universally accepted nonsurgical adjunct for management of the difficult airway in the emergency department. The gum elastic bougie is widely accepted in the British anesthesia literature. One model of endotracheal tube introducer, the Flex-Guide ET Tube Introducer (GreenField Medical Sourcing, Inc, Northborough, MA), is a less expensive plastic version of the gum elastic bougie with the same properties, available in the United States. We present 3 cases of its use in obtaining airway control in difficult airways in the ED. The bougie facilitates intubation where the cords are not visualized or neck movement is contraindicated, allows verification of correct placement before placing the endotracheal tube, is simple to use, and inexpensive to obtain. Reports of 100% first-attempt intubation success in difficult airways are published in the anesthesia literature. We advocate use of this device in the emergency department as a nonsurgical adjunct for difficult airway management.