[Psychosocial determinants of drug users to use a new syringe]. / Déterminants psychosociaux d'utilisateurs de drogues d'utiliser une seringue neuve.

BACKGROUND: A limited number of injection drug users are hard to reach through needle-exchange programs. They obtain their injection material from drug-using peers. This dependence on others can make them more at risk of contracting HIV through sharing non-sterile syringes. The aim of this study is to identify determinants of the intention to systematically resort to use of a new syringe by injection drug users rarely or never involved in needle-exchange programs in Quebec. METHODS: A cross-sectional survey was conducted in Québec city by means of a questionnaire measuring variables from the theory of planned behavior and past behavior. Participants (n=97) were recruited by acquaintances who kept regular contacts with the local needle-exchange programs. Multiple linear regression was used to identify the psychosocial determinants of the intention, and beliefs underlying those determinants were identified using multiple logistic regression. RESULTS: Half of participants reported using a new syringe for each injection in the last month. In multivariate analyses, this past behavior together with theory of planned behavior constructs explained 70% of the variation in participants intent to use a new syringe for each injection (control beliefs: beta=0.39; past behavior: beta=0.27; attitude: beta=0.21; perceived behavioral control: beta=0.20; subjective norm: beta=0.12). In logistic regression, six important beliefs were identified. CONCLUSION: This study resulted in the development of a predictive model of intention to use always a new syringe for each injection among a population of injection drug users in Quebec. Study results could serve as the foundation for the development of interventions to promote this behavior.

Kinetic analysis of delta ribozyme cleavage.

The ability of delta ribozyme to catalyze the cleavage of an 11-mer RNA substrate was examined under both single- and multiple-turnover conditions. In both cases only small differences in the kinetic parameters were observed in the presence of either magnesium or calcium as cofactor. Under multiple-turnover conditions, the catalytic efficiency of the ribozyme (kcat/KM) was higher at 37 degreesC than at 56 degreesC. The cleavage reaction seems to be limited by the product release step at 37 degreesC and by the chemical cleavage step at 56 degreesC. We observed substrate inhibition at high concentrations of the 11-mer substrate. Cleavage rate constants were determined with a structural derivative characterized by an ultrastable L4 tetraloop. The kinetic parameters (kcat and KM) and dissociation constant (Kd) were almost identical for both ribozymes, suggesting that the stability of the L4 loop has a negligible impact on the catalytic activities of the examined ribozymes. Various cleavage inhibition and gel-shift assays with analogues, substrate, and both active and inactive ribozymes were performed. The 2'-hydroxyl group adjacent to the scissile phosphate was shown to be involved in binding with the ribozyme, while the essential cytosine residue of the J4/2 junction was shown to contribute to substrate association. We clearly show that substrate binding to the delta ribozyme is not restricted to the formation of a helix located downstream of the cleavage site. Using these results, we postulate a kinetic pathway involving a conformational transition step essential for the formation of the active ribozyme/substrate complex.

Identification of a Crohn's disease specific transcript with potential as a diagnostic marker.

BACKGROUND: A long time goal of the medical research community has been the identification of a reliable and valid marker for Crohn's disease. AIM: To identify differences in the genetic expression patterns of healthy and diseased tissues. METHOD: The RNA arbitrarily primed polymerase chain reaction (RAP-PCR) procedure was modified to improve its potential to identify clinical markers in heterogeneous RNA populations. RESULTS: With this procedure, a 1065 bp PCR product associated with the inflammation that occurs in Crohn's disease was identified, cloned and sequenced. Northern blot hybridisations showed that this novel sequence originates from a unique RNA species of 3.1 kb. Dot blot hybridisations clearly showed that this RNA species was specific to Crohn's disease. Moreover, its abundance seemed to correlate with the severity of inflammation. Finally, this RNA species was also detected in macroscopically normal areas from Crohn's disease specimens, suggesting that it appears either early during the disease or at least before severe manifestations. CONCLUSION: This finding of a 3.1 kb RNA species permits the discrimination of Crohn's disease manifestations. Although further clinical work is required, this transcript appears to have definite potential as a diagnostic marker.

The viroid and viroid-like RNA database.

The viroid and viroid-like RNA database is a compilation of all natural sequences published in journals or available from the GenBank and EMBL nucleotide sequence libraries. Several information regarding these RNA species such as the position of their self-catalytic domains and the open reading frame of the human hepatitits delta virus are provided. The database also includes a determination of the likely ancestral sequence of most species and a prediction of the most stable secondary structures of these sequences. This online database is available on the World Wide Web (http://www.callisto.si.usherb.ca/[symbol: see text]jpperra ). It should provide an excellent reference point for further phylogenetic and structure-function studies of these RNA species.

Update of the viroid and viroid-like sequence database: addition of a hepatitis delta virus RNA section.

Recently, we developed and made available an online database that includes all the reported (to our knowledge) viroid and viroid-like RNA sequences [Bussire,F., Lafontaine,D. and Perreault,J.-P. (1996)Nucleic Acids Res.24, 1793-1798]. We report here an update of this catalogue which includes the addition of a new section devoted to human hepatitis delta virus (vHDV) sequences. This new section comprises all available vHDV sequences, irrespective of their completeness, which have been either published or were available from nucleic acid libraries. Additional structural characteristics of the vHDV genome, such as the positions of the self-catalytic domains, the antigen open reading frames, etc., are also included. The catalogue is available on the World Wide Web (see text) in a user-friendly form. It should provide an excellent reference point for further molecular studies of these small circular pathogenic RNAs.

Expression of a reporter gene interrupted by the Candida albicans group I intron is inhibited by base analogs.

We previously reported the identification of an intron (CaLSU) in the 25S ribosomal RNA of some Candida albicans yeast strains. CaLSU was shown to self-splice and has the potential to adopt a secondary structure typical of group I introns. The presence of CaLSU inC. albicans strains correlates with a high degree of susceptibility to base analog antifungal agents, 5-fluorocytosine (5-FC) or 5-fluorouracil (5-FU). Cell death, resulting from addition of base analogs to growing cultures, precluded demonstration of a causal relationship between CaLSU presence and susceptibility to base analogs. In the present study, CaLSU was inserted in a non-essential lacZ reporter gene and expression was examined in Saccharomyces cerevisiae. Different mutations affecting in vitro self-splicing also had similar effects on reporter gene expression in vivo. This indicates that in vivo removal of CaLSU from the reporter gene occurs through the typical self-splicing mechanism of group I introns. Base analogs inhibited expression of the reporter gene product in a concentration-dependent manner upon their addition to the cultures. This supports a model in which disruption of intron secondary structure, consecutive to the incorporation of nucleotide analogs, is a major factor determining the susceptibility of C.albicans cells to base analogs.

Characterization of stable RNAs from the resected intestinal tissues of individuals with either Crohn's disease or ulcerative colitis.

Circular RNAs reminiscent of viroids and the human hepatitis delta virus have been proposed as possible nonconventional pathogens responsible for Crohn's disease and ulcerative colitis, two inflammatory bowel diseases. Consequently, RNA was extracted from various areas of intestinal tissues from individuals with either Crohn's disease or ulcerative colitis as well as several appropriate control diseases, and analyzed by two-dimensional gel electrophoresis. No circular viroid-like RNAs (< 1500 nucleotides) were detected, confirming a previous report that was limited to the investigation of small RNAs (< 300 nucleotides). However, three small, unusually stable, linear RNAs were shown to be associated to both Crohn's disease and ulcerative colitis tissues: a specific 28S ribosomal RNA cleavage product characterized previously; a 5.8S ribosomal RNA conformer; and a fragment homologous to transcripts from DNA CpG islands. The two last RNAs were detected prior to visible morphological tissue alterations, suggesting that they are produced early during the inflammation and that they have value as molecular diagnostic tools for the inflammatory bowel diseases. The potential cellular mechanisms producing these RNAs and their involvement in inflammatory bowel disease are discussed.

A novel group I intron in Candida dubliniensis is homologous to a Candida albicans intron.

In the present study, we determined the sequence of group I self-splicing introns found in the large ribosomal RNA subunit of Candida albicans, Candida stellatoidea and the recently-described species Candida dubliniensis. It was found that both the intron and ribosomal RNA nucleotide sequences are almost perfectly identical between different C. albicans strains as well as between C. albicans and C. stellatoidea strains. Comparisons of ribosomal RNA sequences suggest that local isolates of atypical C. albicans from individuals infected with human immunodeficiency virus can be assigned to the C. dubliniensis species. C. dubliniensis strains also harbor a group I intron in their ribosomal RNA, as observed in about 40% of C. albicans strains and all C. stellatoidea strains. This novel C. dubliniensis group I intron is identical to the C. albicans and C. stellatoidea intron, except for two widely divergent stem-loop regions. Despite these differences, the C. dubliniensis intron possesses self-splicing ability in an in vitro assay. Taken together, these data support the idea that C. albicans and C. stellatoidea should be joined together as variants of the same species while C. dubliniensis is a distinct but closely related microorganism. To our knowledge, the C. albicans and C. dubliniensis introns are the first example of a pair of homologous group I introns differing only by the presence of apparently facultative sequences in some stem-loops suspected to be involved in stabilization of tertiary structure.

Candida albicans serotype analysis by flow cytometry.

Candida albicans strains can be assigned to either of two major serogroups, A or B. Antigenic surface determinants present only in serotype A strains allow such a distinction, which has epidemiologic relevance. Reports have established that the relative distributions of the two serotypes can vary depending on the geographic origin of the isolates. A prevalence of susceptibility to an antifungal agent, flucytosine, was also observed with isolates of serotype A. More recently, it was suggested that the occurrence of serotype B isolates in various clinical forms of candidiasis is increasing. However, this latest finding remains controversial since serotyping results vary widely from one laboratory to another because of the lack of standardized methodologies. Difficulty in interpretation of results, which may lead to erroneous serotype identification, is the major setback associated with current methods. For this study, we thus devised a procedure that relies on flow cytometry and that may eliminate ambiguities in serotype determination. The validation of results was achieved with two types of serotype A-specific antisera, Iatron Factor 6 antiserum and an anti-C. albicans antiserum adsorbed on serotype B yeast cells. Agreement between results obtained with these two reagents was 100% with a wide array of Candida strains. These results confirmed the potential of the flow cytometric procedure as a reliable and reproducible method to establish the serotypes of C. albicans strains. Furthermore, some applications of this procedure to the epidemiological study of this human pathogen are presented.

Correlation between the presence of a self-splicing intron in the 25S rDNA of C.albicans and strains susceptibility to 5-fluorocytosine.

Candida albicans presents a well characterized EcoRI RFLP pattern of intensely staining bands. One of these bands, the dimorphic 3.7/4.2 kbp fragment shown to originate from the ribosomal RNA-encoding regions (rDNA), has been used by several investigators to subdivide C. albicans strains in two distinct subtypes. In the present manuscript, we report that an epidemiological study of 120 C.albicans strains revealed a significant correlation between these subtypes and susceptibility to 5-fluorocytosine, an antifungal agent extensively used for biotyping C.albicans. The 4.2 kbp strains being generally more susceptible than their counterparts to this agent and one of its metabolic by-product, 5-fluorouracil. A 379 nucleotides insertion in the 25S rRNA-encoding gene of 4.2 kbp type strains was shown to be responsible for the 3.7/4.2 size difference. This intervening sequence is typical of a group I intron by its site of insertion, its predicted secondary structure, and its self-splicing capability. Assuming there is a genuine causal relationship between presence of the intron and resistance to 5-fluorocytosine, one possible mechanism suggests that inhibition of self-splicing by the insertion of 5-fluorouracil residues in the 25S rRNA precursor might be responsible for the higher susceptibility of 4.2 kbp type strains.

Application of biotyping and DNA typing of Candida albicans to the epidemiology of recurrent vulvovaginal candidiasis.

One-hundred and five Candida albicans isolates from various anatomic sites of 28 patients, obtained at the onset of two consecutive episodes of well-documented recurrent vulvovaginitis, were typed by methods relying on physiologic or genomic markers. The isolates represented a wide variety of types, and neither a single biotype nor genotype was associated with recurrent vaginitis or a particular body site. Patients generally carried similar strains at various anatomic sites that persisted over time. Genomic methods indicated an 86% rate of relapse, which suggested that most recurrent vaginal infections are of endogenous origin. A similar evaluation with biotyping methods was inconclusive because of a lack of reproducibility, resulting from clonal variation or switching, and difficulties in establishing the number of phenotypic tests necessary to distinguish between identical and different strains. Therefore, Southern hybridization was considered the ideal reference method to study the epidemiology of C. albicans infections.