RESUMEN
As falls and fall-related injuries remain a major challenge in the public health domain, reliable and immediate detection of falls is important so that adequate medical support can be delivered. Available home alarm systems are placed on the hip, but have several shortcomings. A fall detector based on accelerometers and placed at head level was developed, as well as an algorithm able to distinguish between activities of daily living and simulated falls. Accelerometers were integrated into a hearing-aid housing, which was fixed behind the ear. The sensitivity of the fall detection was assessed by investigation into the acceleration patterns of the head of a young volunteer during intentional falls. The specificity was assessed by investigation into activities of daily living of the same volunteer. In addition, a healthy elderly woman (83 years) wore the sensor during the day. Three trigger thresholds were identified so that a fall could be recognised: the sum-vector of acceleration in the xy-plane higher than 2 g; the sum-vector of velocity of all spatial components right before the impact higher than 0.7 m s(-1); and the sum-vector of acceleration of all spatial components higher than 6 g. The algorithm was able to discriminate activities of daily living from intentional falls. Thus high sensitivity and specificity of the algorithm could be demonstrated that was better than in other fall detectors worn at the hip or wrist at the same stage of development.
Asunto(s)
Accidentes por Caídas , Monitoreo Ambulatorio/instrumentación , Aceleración , Actividades Cotidianas , Adulto , Anciano de 80 o más Años , Femenino , Humanos , Proyectos PilotoRESUMEN
High-resolution two-dimensional gel electrophoresis of pulse-labeled Haemophilus influenzae extracts allows for the separation and quantification of more than five hundred protein spots. We have determined the changes in the protein synthesis patterns triggered by treatment with inhibitors of transcription, Rifampicin (Rif) and translation, Chloramphenicol (Chl), Erythromycin (Ery), Fusidate (Fus), Puromycin (Pur), Kanamycin (Kan), Streptomycin (Str), and Tetracycline (Tet) relative to the total protein synthesis rate. More than 200 spots changed in intensity under at least one condition. With the exception of the aminoglycosides, Kan and Str, all inhibitors triggered a clear increase in the synthesis rates of ribosomal proteins and RNA polymerase subunits. Northern analysis of rpoA, rpoB, rpoC, and six ribosomal protein genes indicated induction of transcription as well as antitermination as part of the mechanism of the regulation of gene expression. Total RNA synthesis was increased after exposure to Chl, Ery, Fus, and Tet, whereas Str had no effect. Rif led to an almost complete shutdown of RNA synthesis. Exposure to Chl, Ery, Fus, Rif, and Tet resulted in a decrease in the concentration of the stringent factor, guanosine 5',3'-bis-diphosphate (ppGpp) whereas Str again had no effect. Thus, as in Escherichia coli, the response of H. influenzae to translational inhibitors appears to be mediated by the regulatory nucleotide ppGpp.
Asunto(s)
Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Haemophilus influenzae/genética , Haemophilus influenzae/metabolismo , Proteínas Bacterianas/aislamiento & purificación , Secuencia de Bases , ADN Bacteriano/genética , ARN Polimerasas Dirigidas por ADN/biosíntesis , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/aislamiento & purificación , Electroforesis en Gel Bidimensional , Genes Bacterianos/efectos de los fármacos , Haemophilus influenzae/efectos de los fármacos , Humanos , Biosíntesis de Proteínas/efectos de los fármacos , Proteoma , ARN Bacteriano/biosíntesis , ARN Bacteriano/genética , Proteínas Ribosómicas/biosíntesis , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/aislamiento & purificación , Transcripción Genética/efectos de los fármacosRESUMEN
Streptococcus pneumoniae remains a major causative agent of serious human diseases. The worldwide increase of antibiotic resistant strains revealed the importance of horizontal gene transfer in this pathogen, a scenario that results in the modulation of the species-specific gene pool. We investigated genomic variation in 20 S. pneumoniae isolates representing major antibiotic-resistant clones and 10 different capsular serotypes. Variation was scored as decreased hybridization signals visualized on a high-density oligonucleotide array representing 1,968 genes of the type 4 reference strain KNR.7/87. Up to 10% of the genes appeared altered between individual isolates and the reference strain; variability within clones was below 2.1%. Ten gene clusters covering 160 kb account for half of the variable genes. Most of them are associated with transposases and are assumed to be part of a flexible gene pool within the bacterial population; other variable loci include mosaic genes encoding antibiotic resistance determinants and gene clusters related to bacteriocin production. Genomic comparison between S. pneumoniae and commensal Streptococcus mitis and Streptococcus oralis strains indicates distinct antigenic profiles and suggests a smooth transition between these species, supporting the validity of the microarray system as an epidemiological and diagnostic tool.
Asunto(s)
Cromosomas Bacterianos , Genoma Bacteriano , Streptococcus pneumoniae/genética , Alelos , Secuencia de Bases , Farmacorresistencia Microbiana , Variación Genética , Humanos , Datos de Secuencia Molecular , Mosaicismo , Streptococcus pneumoniae/efectos de los fármacos , Trimetoprim/farmacologíaRESUMEN
An assay was developed to determine the activity of peptide deformylase (PDF) inhibitors under conditions as close as possible to the physiological situation. The assay principle is the detection of N-terminal [35S]methionine labeling of a protein that contains no internal methionine. If PDF is active, the deformylation of the methionine renders the peptide a substrate for methionine aminopeptidase, resulting in the removal of the N-terminal methionine label. In the presence of a PDF inhibitor, the deformylation is blocked so that the N-formylated peptide is not processed and the label is detected. Using this assay, it is possible to determine the PDF activity under near-physiological conditions in a cell-free transcription-translation system as well as in intact bacterial cells.
Asunto(s)
Amidohidrolasas , Aminopeptidasas/antagonistas & inhibidores , Antibacterianos/farmacología , Inhibidores Enzimáticos/farmacología , Escherichia coli/efectos de los fármacos , Pruebas de Sensibilidad Microbiana/métodos , Proteínas Bacterianas/efectos de los fármacos , Extractos Celulares/análisis , Escherichia coli/enzimología , Metionina/metabolismo , Biosíntesis de Proteínas/efectos de los fármacos , Transcripción Genética/efectos de los fármacosRESUMEN
New inhibitors of peptide deformylase (PDF) which are very potent against the isolated enzyme and show a certain degree of antibacterial activity have recently been synthesized by our group. Several lines of experimental evidence indicate that these inhibitors indeed interfere with the target enzyme in the bacterial cell. (i) The inhibition of Escherichia coli growth could be counteracted by overexpression of PDF from different organisms, including E. coli, Streptococcus pneumoniae, and Haemophilus influenzae. Conversely, reduced expression of PDF in S. pneumoniae resulted in an increased susceptibility to the inhibitors. (ii) Proteome analysis on two-dimensional gels revealed a shift for many proteins towards lower pI in the presence of PDF inhibitors, as would be expected if the proteins still carry their N-formyl-Met terminus. (iii) PDF inhibitors show no antimicrobial activity against E. coli under conditions that make growth independent of formylation and deformylation. The antibacterial activity in E. coli was characterized as bacteriostatic. Furthermore, the development of resistance in E. coli was observed to occur with high frequency (10(-7)). Resistant mutants show a reduced growth rate, and DNA sequence analysis revealed mutations in their formyl transferase gene. Taking all these aspects into account, we conclude that PDF may not be an optimal target for broad-spectrum antibacterial agents.
Asunto(s)
Amidohidrolasas , Aminopeptidasas/antagonistas & inhibidores , Antibacterianos/farmacología , Farmacorresistencia Microbiana , Inhibidores Enzimáticos/farmacología , Escherichia coli/efectos de los fármacos , Pruebas de Sensibilidad Microbiana/métodos , Aminopeptidasas/genética , Aminopeptidasas/metabolismo , Antiinfecciosos Urinarios/farmacología , Proteínas Bacterianas/efectos de los fármacos , Proteínas Bacterianas/metabolismo , Antagonismo de Drogas , Escherichia coli/enzimología , Escherichia coli/genética , Genes Bacterianos , Haemophilus influenzae/efectos de los fármacos , Haemophilus influenzae/metabolismo , Mutagénesis Insercional , Mutación , Streptococcus pneumoniae/efectos de los fármacos , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/metabolismo , Transformación Bacteriana , Trimetoprim/farmacologíaRESUMEN
The responses of Haemophilus influenzae to DNA gyrase inhibitors were analyzed at the transcriptional and the translational level. High-density microarrays based on the genomic sequence were used to monitor the expression levels of >80% of the genes in this bacterium. In parallel the proteins were analyzed by two-dimensional electrophoresis. DNA gyrase inhibitors of two different functional classes were used. Novobiocin, as a representative of one class, inhibits the ATPase activity of the enzyme, thereby indirectly changing the degree of DNA supercoiling. Ciprofloxacin, a representative of the second class, obstructs supercoiling by inhibiting the DNA cleavage-resealing reaction. Our results clearly show that different responses can be observed. Treatment with the ATPase inhibitor Novobiocin changed the expression rates of many genes, reflecting the fact that the initiation of transcription for many genes is sensitive to DNA supercoiling. Ciprofloxacin mainly stimulated the expression of DNA repair systems as a response to the DNA damage caused by the stable ternary complexes. In addition, changed expression levels were also observed for some genes coding for proteins either annotated as "unknown function" or "hypothetical" or for proteins not directly involved in DNA topology or repair.
Asunto(s)
Ciprofloxacina/farmacología , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Haemophilus influenzae/efectos de los fármacos , Haemophilus influenzae/genética , Novobiocina/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Proteínas Bacterianas/análisis , Proteínas Bacterianas/biosíntesis , Electroforesis en Gel Bidimensional , Inhibidores Enzimáticos/farmacología , Haemophilus influenzae/enzimología , ARN Bacteriano/biosíntesis , ARN Mensajero/biosíntesis , ARN Mensajero/metabolismo , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Inhibidores de Topoisomerasa IIRESUMEN
We have identified in the Streptococcus pneumoniae genome sequence a two-component system (TCS13, Blp [bacteriocin-like peptide]) which is closely related to quorum-sensing systems regulating cell density-dependent phenotypes such as the development of genetic competence or the production of antimicrobial peptides in lactic acid bacteria. In this study we present evidence that TCS13 is a peptide-sensing system that controls a regulon including genes encoding Blps. Downstream of the Blp TCS (BlpH R) we identified open reading frames (blpAB) that have the potential to encode an ABC transporter that is homologous to the ComA/B export system for the competence-stimulating peptide ComC. The putative translation product of blpC, a small gene located downstream of blpAB, has a leader peptide with a Gly-Gly motif. This leader peptide is typical of precursors processed by this family of transporters. Microarray-based expression profiling showed that a synthetic oligopeptide corresponding to the processed form of BlpC (BlpC*) induces a distinct set of 16 genes. The changes in the expression profile elicited by synthetic BlpC* depend on BlpH since insertional inactivation of its corresponding gene abolishes differential gene induction. Comparison of the promoter regions of the blp genes disclosed a conserved sequence element formed by two imperfect direct repeats upstream of extended -10 promoter elements. We propose that BlpH is the sensor for BlpC* and the conserved sequence element is a recognition sequence for the BlpR response regulator.
Asunto(s)
Genes Bacterianos , Proteínas Quinasas/genética , Regulón , Streptococcus pneumoniae/enzimología , Alelos , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Bacteriocinas/genética , Secuencia de Bases , ADN Bacteriano , Regulación Bacteriana de la Expresión Génica , Variación Genética , Genoma Bacteriano , Histidina Quinasa , Datos de Secuencia Molecular , Familia de Multigenes , Análisis de Secuencia por Matrices de Oligonucleótidos , Péptidos/genética , Péptidos/metabolismo , Proteínas Quinasas/metabolismo , Streptococcus pneumoniae/genética , Transcripción Genética , Activación TranscripcionalRESUMEN
BACKGROUND: Lytic transglycosylases are bacterial muramidases that catalyse the cleavage of the beta- 1,4-glycosidic bond between N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) in peptidoglycan with concomitant formation of a 1,6-anhydrobond in the MurNAc residue. These muramidases play an important role in the metabolism of the bacterial cell wall and might therefore be potential targets for the rational design of antibacterial drugs. One of the lytic transglycosylases is Slt35, a naturally occurring soluble fragment of the outer membrane bound lytic transglycosylase B (MltB) from Escherichia coli. RESULTS: The crystal structure of Slt35 has been determined at 1.7 A resolution. The structure reveals an ellipsoid molecule with three domains called the alpha, beta and core domains. The core domain is sandwiched between the alpha and beta domains. Its fold resembles that of lysozyme, but it contains a single metal ion binding site in a helix-loop-helix module that is surprisingly similar to the eukaryotic EF-hand calcium-binding fold. Interestingly, the Slt35 EF-hand loop consists of 15 residues instead of the usual 12 residues. The only other prokaryotic proteins with an EF-hand motif identified so far are the D-galactose-binding proteins. Residues from the alpha and core domains form a deep groove where the substrate fragment GlcNAc can be bound. CONCLUSIONS: The three-domain structure of Slt35 is completely different from the Slt70 structure, the only other lytic transglycosylase of known structure. Nevertheless, the core domain of Slt35 closely resembles the fold of the catalytic domain of Slt70, despite the absence of any obvious sequence similarity. Residue Glu162 of Slt35 is in an equivalent position to Glu478, the catalytic acid/base of Slt70. GlcNAc binds close to Glu162 in the deep groove. Moreover, mutation of Glu162 into a glutamine residue yielded a completely inactive enzyme. These observations indicate the location of the active site and strongly support a catalytic role for Glu162.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli/enzimología , Glicósido Hidrolasas , Glicosiltransferasas/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sitios de Unión , Calcio/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Escherichia coli/genética , Glicosiltransferasas/genética , Glicosiltransferasas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Muramidasa/química , Peptidoglicano/metabolismo , Conformación Proteica , Estructura Secundaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
In bacteria, adaptive responses to environmental stimuli are often initiated by two-component signal transduction systems (TCS). The prototypical TCS comprises two proteins: a histidine kinase (HK, hk) and a response regulator (RR rr). Recent research has suggested that compounds that inhibit two-component systems might have good antibacterial activity. In order to identify TCS that are crucial for growth or virulence of Streptococcus pneumoniae, we have examined the genomic sequence of a virulent S. pneumoniae strain for genes that are related to known histidine kinases or response regulators. Altogether 13 histidine kinases and 13 response regulators have been identified. The protein sequences encoded by these genes were compared with sequences deposited in public databases. This analysis revealed that two of the 13 pneumococcal TCSs have been described before (ciaRH and comDE) and two are homologous to the yycFG and the phoRP genes of Bacillus subtilis. All the pneumococcal response regulators contain putative DNA binding motifs within the C-terminal output domain, implying that they are involved in transcriptional control. Two of these response regulators are obviously the first representatives of a new subfamily containing an AraC-type DNA-binding effector domain. To assess the regulatory role of these transcription factors, we disrupted each of the 13 response regulator genes by insertional mutagenesis. All the viable mutant strains with disrupted response regulator genes were further characterized with regard to growth in vitro, competence, and experimental virulence. Two response regulator genes could not be inactivated, indicating that they may regulate essential cellular functions. The possibility of using these systems as targets for the development of novel antibacterials will be discussed.
Asunto(s)
Proteínas Quinasas/genética , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/patogenicidad , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/genética , Femenino , Histidina Quinasa , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas Quimiotácticas Aceptoras de Metilo , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Proteínas Quinasas/metabolismo , Transducción de Señal , Streptococcus pneumoniae/metabolismo , VirulenciaRESUMEN
Plasmids for the tetracycline regulated gene expression in Streptococcus pneumoniae have been developed. The plasmids were used for the tetracycline-dependent production of firefly luciferase in streptococci. The production of luciferase can be induced fivefold by the addition of tetracycline. By using two promoters of different strength and depending on the presence or absence of tetracycline, an 80-fold range of luciferase activities can be covered. The system was also used to construct strains that depend on the addition of tetracycline for the production of the A subunit of DNA gyrase, an essential streptococcal protein. The growth of such a strain depends on the addition of tetracycline to the medium. In the absence of tetracycline, the cells cease to grow and are not viable. The system presented in this report should be useful for the characterization of gene networks in S. pneumoniae. It especially allows one to study the function of essential genes that can not be investigated by standard knock-out techniques.
Asunto(s)
Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Plásmidos , Streptococcus pneumoniae/genética , Tetraciclina/farmacología , Secuencia de Bases , ADN-Topoisomerasas de Tipo II/genética , ADN Bacteriano , Luciferasas/genética , Datos de Secuencia Molecular , Streptococcus pneumoniae/enzimología , Streptococcus pneumoniae/crecimiento & desarrolloRESUMEN
The prenyltransferase undecaprenyl pyrophosphate synthetase (di-trans,poly-cis-decaprenylcistransferase; EC 2.5.1.31) was purified from the soluble fraction of Escherichia coli by TSK-DEAE, ceramic hydroxyapatite, TSK-ether, Superdex 200, and heparin-Actigel chromatography. The protein was labeled with the photolabile analogue of the farnesyl pyrophosphate analogue (E, E)-[1-3H]-(2-diazo-3-trifluoropropionyloxy)geranyl diphosphate and was detected on a sodium dodecyl sulfate-polyacrylamide gel as a protein with an apparent molecular mass of 29 kDa. This protein band was cut out from the gel, trypsin digested, and subjected to matrix-assisted laser desorption ionization mass spectrometric analysis. Comparison of the experimental data with computer-simulated trypsin digest data for all E. coli proteins yielded a single match with a protein of unassigned function (SWISS-PROT Q47675; YAES_ECOLI). Sequences with strong similarity indicative of homology to this protein were identified in 25 bacterial species, in Saccharomyces cerevisiae, and in Caenorhabditis elegans. The homologous genes (uppS) were cloned from E. coli, Haemophilus influenzae, and Streptococcus pneumoniae, expressed in E. coli as amino-terminal His-tagged fusion proteins, and purified over a Ni2+ affinity column. An untagged version of the E. coli uppS gene was also cloned and expressed, and the protein purified in two chromatographic steps. We were able to detect Upp synthetase activity for all purified enzymes. Further, biochemical characterization revealed no differences between the recombinant untagged E. coli Upp synthetase and the three His-tagged fusion proteins. All enzymes were absolutely Triton X-100 and MgCl2 dependent. With the use of a regulatable gene disruption system, we demonstrated that uppS is essential for growth in S. pneumoniae R6.
Asunto(s)
Transferasas Alquil y Aril/genética , Transferasas Alquil y Aril/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Transferasas Alquil y Aril/química , Secuencia de Aminoácidos , Animales , Bacterias/enzimología , Caenorhabditis elegans/enzimología , Cromatografía de Afinidad , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Mapeo Cromosómico , Cromosomas Bacterianos , Clonación Molecular , Cinética , Datos de Secuencia Molecular , Peso Molecular , Mutagénesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimología , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
The requirement for novel classes of antibiotics to combat the emergence of resistant and multi-resistant bacteria has coincided with the completion sequencing of a number of bacterial genomes. The in silico analysis of these genomes coupled with innovative genetic manipulation has already led to the identification of conserved essential (either in vitro or in vivo, depending on the methodology) genes that are potential targets for antibacterial research. New technologies, made possible by access to the genomic sequences, are capable of simultaneously quantifying almost the entire complement of gene products synthesised by bacterial cells. These technologies are opening up the way for the analysis of expression patterns elicited in cells in response to changes in their environment. The integration of these technologies into the drug discovery process is still in its infancy and the potential wealth of information, some of it already available, has yet to be fully realised.
Asunto(s)
Antibacterianos/aislamiento & purificación , Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Bacterias/genética , Genoma Bacteriano , Bacterias/crecimiento & desarrollo , Bacterias/patogenicidad , Biología Computacional/métodos , Perfilación de la Expresión Génica , Genes Bacterianos/genética , Genes Esenciales/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteoma , Programas Informáticos , Transcripción Genética , Virulencia/genéticaRESUMEN
Two-dimensional electrophoresis was applied to the global analysis of the cellular response of Haemophilus influenzae to sulfamethoxazole and trimethoprim, both inhibitors of tetrahydrofolate synthesis. Deregulation of the synthesis rate of 118 proteins, involved in different metabolic pathways, was observed. The regulation of the genes involved in the metabolism of the amino acids methionine, threonine, serine, glycine, and aspartate was investigated in detail by analysis of protein synthesis and Northern hybridization. The results suggested that the synthesis of methionine biosynthetic enzymes in H. influenzae is regulated in a similar fashion as in Escherichia coli. A good correlation between the results obtained by Northern hybridization and quantification of protein synthesis was observed. In contrast to trimethoprim, sulfamethoxazole triggered the increased synthesis of the heat shock proteins DnaK, GroEL, and GroES.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/análisis , Electroforesis en Gel Bidimensional , Haemophilus influenzae/efectos de los fármacos , Sulfametoxazol/farmacología , Tetrahidrofolatos/biosíntesis , Trimetoprim/farmacología , 5-Metiltetrahidrofolato-Homocisteína S-Metiltransferasa/metabolismo , Aminoácidos/biosíntesis , Electroforesis en Gel Bidimensional/métodos , Antagonistas del Ácido Fólico , Glicina Hidroximetiltransferasa/metabolismo , Haemophilus influenzae/química , Haemophilus influenzae/metabolismo , Metionina/biosíntesis , ARN MensajeroRESUMEN
Penicillin-resistant isolates of Streptococcus pneumoniae generally contain mosaic genes encoding the low-affinity penicillin-binding proteins (PBPs) PBP2x, PBP2b, and PBP1a. We now present evidence that PBP2a and PBP1b also appear to be low-affinity variants and are encoded by distinct alleles in beta-lactam-resistant transformants of S. pneumoniae obtained with chromosomal donor DNA from a Streptococcus mitis isolate. Different lineages of beta-lactam-resistant pneumococcal transformants were analyzed, and transformants with low-affinity variants of all high-molecular-mass PBPs, PBP2x, -2a, -2b, -1a, and -1b, were isolated. The MICs of benzyl-penicillin, oxacillin, and cefotaxime for these transformants were up to 40, 100, and 50 microg/ml, respectively, close to the MICs for the S. mitis donor strain. Recruitment of low-affinity PBPs was accompanied by a decrease in cross-linked muropeptides as revealed by high-performance liquid chromatography of muramidase-digested cell walls, but no qualitative changes in muropeptide chemistry were detected. The growth rates of all transformants were identical to that of the parental S. pneumoniae strain. The results stress the potential for the acquisition by S. pneumoniae of high-level beta-lactam resistance by interspecies gene transfer.
Asunto(s)
Aminoaciltransferasas , Antibacterianos/farmacología , Proteínas Bacterianas , Proteínas Portadoras/genética , Hexosiltransferasas , Muramoilpentapéptido Carboxipeptidasa/genética , Péptido Sintasas , Peptidil Transferasas , Streptococcus pneumoniae/efectos de los fármacos , Streptococcus/efectos de los fármacos , Transformación Bacteriana , Secuencia de Aminoácidos , Proteínas Portadoras/análisis , Proteínas Portadoras/química , Cefotaxima/farmacología , Farmacorresistencia Microbiana , Datos de Secuencia Molecular , Peso Molecular , Muramoilpentapéptido Carboxipeptidasa/análisis , Muramoilpentapéptido Carboxipeptidasa/química , Resistencia a las Penicilinas , Proteínas de Unión a las Penicilinas , Streptococcus/genética , Streptococcus pneumoniae/genéticaRESUMEN
We have used high-density oligonucleotide probe arrays (chips) for bacterial transcript imaging. We designed a chip containing probes representing 106 Hemophilus influenzae genes and 100 Streptococcus pneumoniae genes. The apparent lack of polyadenylated transcripts excludes enrichment of mRNA by affinity purification and we thus used total, chemically biotinylated RNA as hybridization probe. We show that hybridization of Streptococcus RNA to a chip allows simultaneous quantification of the transcript levels. The sensitivity was found to be in the range of one to five transcripts per cell. The quantitative chip results were in good agreement with conventional Northern blot analysis of selected genes. This technology allows simultaneous and quantitative measurement of the transcriptional activity of entire bacterial genomes on a single oligonucleotide probe array.
Asunto(s)
Genoma Bacteriano , Haemophilus influenzae/genética , Sondas de Oligonucleótidos , ARN Bacteriano , Streptococcus pneumoniae/genética , Transcripción Genética/genética , Biotinilación , Northern Blotting , Colorantes Fluorescentes , Hibridación de Ácido NucleicoRESUMEN
The inevitable emergence and spread of resistance to new antibiotics entering the market necessitates a new approach in drug discovery. Novel classes of antimicrobial compounds are required that are not enfeebled by widespread resistance mechanisms. The favoured approach is to gain a better understanding of the essential pathways and cellular functions and then to select new unexploited targets. This strategy has coincided with the deposition of fully assembled genomic sequences of several bacteria in the public databases. Various technologies have been reported that, when optimised, will enable the analysis of global cellular processes at the molecular level thus greatly contributing to our understanding of cellular physiology. In this article, some of the major advances in technology, which are expected in the future to be essential for the optimal use of the information contained within the genomic sequences, will be outlined.
RESUMEN
A key step in the supercoiling reaction is the DNA gyrase-mediated cleavage and religation step of double-stranded DNA. Footprinting studies suggest that the DNA gyrase binding site is 100-150 bp long and that the DNA is wrapped around the enzyme with the cleavage site located near the center of the fragment. Subunit A inhibitors interrupt this cleavage and resealing cycle and result in cleavage occurring at preferred sites. We have been able to show that even a 30 bp DNA fragment containing a 20 bp preferred cleavage sequence from the pBR322 plasmid was a substrate for the DNA gyrase-mediated cleavage reaction in the presence of inhibitors. This DNA fragment was cleaved, although with reduced efficiency, at the same sites as a 122 bp DNA fragment. A 20 bp DNA fragment was cleaved with low efficiency at one of these sites and a 10 bp DNA fragment was no longer a substrate. We therefore propose that subunit A inhibitors interact with DNA at inhibitor-specific positions, thus determining cleavage sites by forming ternary complexes between DNA, inhibitors and DNA gyrase.
Asunto(s)
Plásmidos/metabolismo , Inhibidores de Topoisomerasa II , Bencimidazoles/química , Bencimidazoles/farmacología , Sitios de Unión , Calcio/farmacología , Ciprofloxacina/química , Ciprofloxacina/farmacología , Fleroxacino/química , Fleroxacino/farmacología , Magnesio/farmacología , Estructura Molecular , Pirimidinas/química , Pirimidinas/farmacologíaRESUMEN
PURPOSE: To evaluate volumetric CT data as a guide for indication and assessment of prognosis for transarterial chemo-embolisation (TAE) of hepatocellular carcinomas (HCC). MATERIAL AND METHOD: 74 patients with HCC were treated with repeated TAE. 50 mg Adriblastin/m2 body surface, 50 mg cisplatin/m2 body surface and 10 ml lipiodol were combined with 2-10 ml Spherex and injected selectively (25 cases) or superselectively (49 cases); in 28 patients a single injection and in 46 patients multiple injections were used. RESULTS: CT findings before and after the procedure showed a solitary lesion in 17 patients, two lesions in 18 patients and in 39 patients there were three or more lesions. Mean expectation of life was 523 days (median = 372 days; 57% of one year survival probability). In 29 patients with > 75% lipiodol retention, mean survival was 819 days; in 17 patients with < 75% lipiodol retention it was 382 days; lower lipiodol retention of < 50% it was 231 days (< 25% 192 and < 10% 152 days). A statistically significant relationship (p < 0.0001) could be established between survival time and tumour volume, relationship of tumour to liver volume, intratumoral lipiodol retention, the type of tumour growth and the number of liver segments involved. CONCLUSION: TAE provides best survival rates after repeated injections of solitary HCC with tumour volumes < 50 ml and > 75% intra-tumoral lipiodol retention.