Organization of aerobactin, hemolysin, and antibacterial resistance genes in lactose negative Escherichia coli strains of serotype O4 isolated from children with dhiarrea

Epidemiologically related, non-lactose-fermenting (NLF) Escherichia coli strains of serotype O4 have been isolated at a high frequency from children with diarrhea in Somalia (M. Nicoletti, F. Superti, C. Conti, A. Calconi, and C. Zagaglia, J. Clin. Microbiol. 26:524-529, 1988). In order to define the virulence potential of these strains, we characterized the replication properties of their high-molecular-weight plasmids and studied the genetic locations and organization of the aerobactin (aer) and hemolysin (hly) determinants encoded by 23 NLF O4 E. coli strains. Southern blot hybridizations, mobilization assays of nonconjugative plasmids, and incompatibility-exclusion experiments conducted with a conjugative incompatibility group FI (IncFI) plasmid showed that (i) 20 out of the 23 strains examined harbor a 160- to 180-kb IncFI plasmid that shares homology with the basic replicons RepFIA, RepFIB, and (except for the plasmid of one strain) RepFIC, and 22 strains also contain a 40- to 140-kb IncFII plasmid sharing homology with the RepFIIA replicon; (ii) the IncFI plasmid is nonconjugative and carries antibiotic resistance genes; (iii) the aer system is located on the IncFI plasmids and/or the chromosomes in the three strains not harboring IncFI, and it is found in an inverted orientation; (iv) the hly determinants are located on the chromosome, and their genetic organization is well conserved and closely resembles that of the reference hemolytic plasmid pHly152; and (v) Hly- mutants obtained by transposon insertion mutagenesis are not cytotoxic to HeLa cell monolayers, indicating that hemolysin is responsible for the high cytotoxic activity we have previously reported for these strains. The structural organization of the plasmid-encoded aer operon, together with the finding that those plasmids also carry antibiotic resistance genes, indicates that the IncFI plasmid of the NLF O4 E. coli strains studied more closely resembles aer-encoding virulence IncFI Salmonella R plasmids than E. coli ColV plasmids. The data presented here cannot rule out whether the strains examined are potentially intestinal or extraintestinal pathogens. Nevertheless, the genetic organization of the virulence genes, together with the epidemiological behavior and the wide spectrum of antibiotic resistance of the NLF O4 E. coli strains, indicates that these strains are structured as typical E. coli pathogenic isolates of human origin

Studio della regolazione dell'espressione dei geni di virulenza in Escherichia coli enteroinvasivo (EIEC) e Shigella

Dottorato di ricerca in biologia cellulare e dello sviluppo. 8. ciclo. A.a. 1992-95. Docente guida B. ColonnaConsiglio Nazionale delle Ricerche - Biblioteca Centrale - P.le Aldo Moro, 7, Rome; Biblioteca Nazionale Centrale - P.za Cavalleggeri, 1, Florence / CNR - Consiglio Nazionale delle RichercheSIGLEITItal

Telomere length analysis and in vitro telomerase assay

Barring exceptional instances, the DNA contained in eukaryotic chromosomes is linear. Linearity of the chromosomal DNA and the compartmentalized architecture of the eukaryotic cell are the two principle features that distinguish the prokaryotes from the eukary- otes and that have facilitated the evolution of totally different strat- egies for interaction with other species and the environment. Major changes in strategy that were made possible by linearity of chromo- somes were an enormous increase in the information content of the genome and the development of sexuality as a means for efficient exchange of genetic information. But linear chromosomes have ends, and the presence of ends produced two major biological prob- lems. One problem was identified early by Muller and by McClintock, as the requirement to protect natural ends of chro- mosomes from fusion and recombination with other chromosomes and from exonucleolytic erosion. It was thus recognized that the ends of chromosomes must have specialized structural and func- tional features required for chromosome stability. We know today that another potential cause of chromosomal instability that must be dealt with is that unless it is protected, a DNA end will be recog- nized as damaged DNA that the cell will attempt to heal with ensu- ing loss of chromosome integrity and cell viability. The second problem was identified more recently, when it became evident that all known DNA polymerases are unable to start synthesis de novo but can only add a nucleotide onto an already existing 3'-OH group, normally provided by an RNA primer. Such a primer positioned at the very end of the chromosomal DNA would be degraded, shorten- ing the lagging DNA strand. Both these sets of problems have found a solution through the development of a unique chromosomal organelle, the telomere, comprised of particular telomeric DNA sequences complexed with particular non-histone proteins, that are organized at least in part in a non-nucleosomal heterochromatic form of chromatin, defined as the telosome

Telomeres and telomerase activity in pig tissues

The current state of the art concerning telomeres and telomerase sterns almost exclusively from the analysis of protozoa, yeast, and a small number of mammals. In the present study, we confirm that the pig telomeric sequence is indeed T(2)AG(3), as previously Suggested. By making use of sequence analysis of pig telorneric DNA variant telorneric repeats in the medial region of the telorneres, interspersed with canonical T(2)AG(3) repeats, were identified. This telomere organization is similar to the one present in humans. Analysis of terminal restriction fragments showed that the majority of telomeres from different pig tissues are longer than ill humans but shorter than in Mus musculus. Telomeres from spermatozoa were found to be longer, ranging in size between 13 and 44 kb. Most of the somatic pig tissues expressed significant levels of telomerase activity, a situation more similar to mouse and that contrasts with the one in humans and dog. Moreover, the analysis of sperm cells from different epididymal compartments of an adult animal showed that telomerase activity is absent in Maturing spematozoa, Suggesting that sperm telomere elongation is restricted during spermatogenesis. (C) 2003 Published by Elsevier SAS

Design and validation of siRNAs and shRNAs

RNAi is a highly conserved intracellular mechanism, whereby dsRNA strands conduct post-transcriptional modulation of gene expression through a degradation or inhibition of the translation of target mRNA. Since its discovery in 1998, RNAi has been identified in many different organisms, including mammals, and this mechanism has provided new approaches for studies in cellular and molecular biology, functional genomics and drug discovery. siRNAs can be predicted by sequence and thermodynamic features, and the wide and proficient application of RNAi relies on the ability to select the most active siRNAs from among numerous predicted molecules. Recently, the first-generation prediction algorithms based on the characteristics of siRNAs, short hairpin (sh)RNAs and micro-(mi)RNAs have been improved by the use of computational models that account for the experimentally determined activities of large numbers of siRNAs/shRNAs and miRNAs. These second-generation algorithms differ from the first-generation algorithms in the computational tools that are used for the prediction of siRNA efficacy; although these new algorithms improve the design of effective siRNAs, they do not eliminate the requirement for an experimental evaluation of the activities of siRNAs. This review reports on the most significant second-generation algorithms of siRNA and shRNA characteristics, as well as on recently designed systems for the experimental evaluation of siRNA activities. © Thomson Reuters (Scientific) Ltd

Downregulation of the human and murine ENaC channel by RNA interference

Presentazione oral

Endocarditis caused by Lactobacillus jensenii in an immunocompetent patient

Lactobacilli are Gram-positive rod-shaped bacteria that inhabit the oral cavity, gastrointestinal tract, vagina and nasal cavity In this report, a rare case of Lactobacillus Jensenii endocarditis in a 47-year-old immunocompetent patient is described. Blood cultures and a replaced mitral valve were positive for L Jensenii as assessed by 16S rRNA gene sequencing Based on susceptibility tests the patient was successfully treated with a mixture of teicoplanin and meropenem antimicrobial therap