Presence of a coordinated metal ion in a trans-acting antigenomic delta ribozyme.

We have investigated the cleavage induced by metal ions in an antigenomic form of a trans-acting delta ribozyme. A specific Mg(2+)-induced cleavage at position G(52)at the bottom of the P2 stem was observed to occur solely within catalytically active ribozyme-substrate complexes (i.e. those that performed the essential conformational transition step). Only the divalent cations which support catalytic activity permitted the detection of specific induced cleavages in this region. Using various mutant ribozymes and substrates, we demonstrated a correlation between enzymatic activity and the Mg(2+)-induced cleavage pattern. We show that the efficiency of the coordination of the magnesium to its binding site is related to the nature of the base pair in the middle of the P1 stem (i.e. Rz(23)-S(8)). Together with additional evidence from nuclease probing experiments that indicates the occurrence of a structural rearrangement involving the bottom of the P2 stem upon formation of the P1 helix, these results show that an intimate relationship exists between the folding and the catalytic activity of the delta ribozyme.

Important 2'-hydroxyl groups in model substrates for M1 RNA, the catalytic RNA subunit of RNase P from Escherichia coli.

The role of 2'-hydroxyl groups in a model substrate for RNase P from Escherichia coli was studied using mixed DNA/RNA derivatives of such a substrate. The presence of the 2'-hydroxyl groups of nucleotides at positions -1 and -2 in the leader sequence and at position 1, as well as at the first C in the 3'-terminal CCA sequence, are important but not absolutely essential for efficient cleavage of the substrate by RNase P or its catalytic RNA subunit, M1 RNA. The 2'-hydroxyl groups in the substrate that are important for efficient cleavage also participate in the binding of Mg2+. An all-DNA external guide sequence (EGS) can efficiently render a potential substrate, derived from the model substrate, susceptible to cleavage by the enzyme or its catalytic RNA subunit. Furthermore, both DNA and RNA EGSs turn over during the reaction with RNase P in vitro. The identity of the nucleotide at position 1 in the substrate, the adjacent Mg(2+)-binding site in the leader sequence, and the junction of the single and double-stranded regions are the important elements in the recognition of model substrates, as well as in the identification of the sites of cleavage in those model substrates.

Pathway of activation by magnesium ions of substrates for the catalytic subunit of RNase P from Escherichia coli.

The pathway is described for activation by Mg2+ of substrates for M1 RNA, the catalytic subunit of the RNase P from Escherichia coli. The dissociation constants are reported for binding of Mg2+ to the substrate and for the binding of the metal ion-substrate complex to the enzyme. The enzyme binds the substrate with the same affinity whether or not Mg2+ is already bound to the substate. However, only substrates with bound Mg2+ can make a productive ternary complex when combined with the enzyme. The presence of certain 2'-hydroxyl groups in the substrate is required to stabilize the binding of Mg2+ and, thereby, to increase the lifetime of the ternary complex. An energy profile for the reaction of M1 RNA with a small model substrate is presented and the role of Mg2+ bound to the substrate is discussed.

Peach latent mosaic viroid is locked by a 2',5'-phosphodiester bond produced by in vitro self-ligation.

Although some viroid-like satellite RNAs possess self-cleavage and self-ligation activities, we show that the peach latent mosaic viroid (PLMVd) is unique among all known viroids since it also has such activities. These catalytic activities should have important roles in the rolling circle replication of PLMVd. According to this proposed mechanism, self-cleavage of the multimeric strands occurs via hammerhead structures producing monomers possessing 2',3'-cyclic phosphate and 5'-hydroxyl termini. In the most stable predicted secondary structure for PLMVd these two extremities are juxtaposed, in order for self-ligation to occur. To establish the nature of the phosphodiester bond produced by self-ligation, we followed the classical procedure of complete enzymatic RNA hydrolysis coupled with thin layer chromatography fractionation. Using this procedure, we report that the self-ligation of PLMVd transcripts produces almost exclusively the 2',5' isomer (>96%). Primer extension assays also revealed that reverse transcriptase can read througth this 2', 5' linkage, suggesting that it does not prevent further replication of the viroid. Moreover, we have observed that this 2',5' linkage is resistant to the debranching activity contained in nuclear extracts, as well as being capable of preventing further viroid self-cleavage. Thus, if viroids do indeed self-ligate in vivo, the resulting 2', 5'-phosphodiester bond could contribute to the stability of these RNA species. Finally, an analysis of both the sequence and the structural requirements for hammerhead self-cleavage and self-ligation suggests that these two RNA processes may be interrelated. We hypothesize that the intramolecular self-ligation which produces circular conformers may contribute to the circularization step of the rolling circle replication, while the intermolecular non-enzymatic ligation is a potential mechanism for the sequence reassortment of viroids and viroid-like species.

Characterization of a chemically synthesized RNA having the sequence of the yeast initiator tRNA(Met).

A 75-unit long oligoribonucleotide corresponding to the sequence of the Saccharomyces cerevisiae initiator tRNA was synthesized chemically. The crude RNA was purified, and the sequence was verified by RNA sequencing techniques. A particularly useful purification step involved hydrophobic chromatography on BND-cellulose. The purified RNA could be aminoacylated to 28% of a bona fide initiator tRNA(Met) sample and threonylated to 76% of the level observed with native tRNA(fMet) from E. coli.

Staghorn calculi treated by percutaneous nephrolithotomy: risk factors for recurrence.

Since 1985 our primary mode of therapy for staghorn calculi has been by percutaneous nephrolithotomy. Between January 1985 and June 1988 we have treated 57 cases using this method. We reviewed the rate of recurrence at a minimum of one-year follow-up and observed a 17 percent recurrence rate. Factors identified that were associated with an increased rate of recurrence were: positive urine cultures during follow-up (55% recurrence vs 12%); stone remnant greater than 5 mm (27.3% recurrence vs 13.8%); and stone complexity (25% recurrence for complex or complete staghorn vs 9.7% for noncomplex or partial staghorn). By identifying these risk factors we think that stone recurrence can be reduced and, with close follow-up, detected earlier to permit less invasive therapy if needed.

Electromyographic study of bulbocavernosus muscle in 15 patients with postmicturition dribbling.

Fifteen patients with postmicturition dribbling were studied with cystourethroscopy and electromyography of the bulbocavernosus muscle. In all patients the bulbocavernosus reflex and activity of the muscle during and after micturition were normal. No abnormality was found to explain the clinical symptoms.

Efficacy of finasteride is maintained in patients with benign prostatic hyperplasia treated for 5 years. The North American Finasteride Study Group.

OBJECTIVES: The purpose of this open-label study extension was to assess the long-term safety and efficacy of finasteride in the treatment of men with benign prostatic hyperplasia (BPH). METHODS: A Phase III North American BPH trial originally enrolled 895 men, 297 of whom were randomized to receive finasteride 5 mg. An enlarged prostate gland by digital rectal examination, symptoms of urinary obstruction, and a maximal urinary flow rate of less than 15 mL/s were required for entry. Patients who completed the initial 12-month, double-blind, placebo-controlled study were invited to participate in an open-label extension for 4 additional years. RESULTS: Of the 297 patients initially randomized to receive finasteride 5 mg, 259 completed 12 months in the double-blind period and 186 completed 48 months of open-label therapy. Prostate volume reached a nadir of -24.6% at month 24, and the effect was maintained through month 60. Compared with baseline values, month 60 prostate volume was decreased by 22.7% (P<0.001), the quasi-American Urological Association symptom score was decreased by 4.3 points, and maximal urinary flow was increased by 2.3 mL/s (P<0.001) on average. Finasteride was well tolerated, with no significant increase in the prevalence of sexual adverse events over time. CONCLUSIONS: Patients treated with finasteride 5 mg maintained an initial decrease in prostate volume and improvement in symptom score and maximal urinary flow rate over 5 years.

A Canadian isolate of hepatitis D (delta) virus.

Hepatitis D (delta) virus (HDV) is an infectious agent that propagates in hepatocytes only in the presence of hepatitis B virus, causing fulminant or chronic hepatitis with liver cirrhosis. HDV is a 36 nm particle that includes a circular RNA genome of 1.7 kilobases with an extensive internal complementary that allows it to fold into a rod-like structure. The relationships among genotypes, sequence variability, geographical distribution and disease severity of HDV remain unknown. Consequently, in the present study, the complete nucleotide sequence of an HDV isolated from a Canadian patient was determined. The viral RNA from serum was amplified using reverse transcription coupled to polymerase chain reaction amplification. The resulting complementary DNA was cloned and sequenced. Sequence analysis revealed that this new isolate contained 1672 nucleotides corresponding to genotype 1, which has a worldwide distribution. Sequencing of four independent clones revealed 17 substitutions, corresponding to an overall sequence variability of 1%. Surprisingly, seven mutations were found in the 48-nucleotide region located between the two highly conserved self-catalytic motifs. This is the first demonstration that many substitutions are identified in this region of HDV, and prompts the present authors to define it as a hypervariable region.

Long term follow-up of intravesical Bacillus Calmette-Guerin for the treatment of bladder transitional cell carcinoma.

OBJECTIVE: To review the long-term follow-up, in terms of recurrence and progression, of transitional cell carcinoma of the bladder treated with intravesical BCG with the following indications: CIS, Ta and T1. MATERIALS AND METHODS: Ninety-two patients who had received complete course of BCG between 1987 and 1993 were included in the study and followed for an average of 59 months (range 12 to 102). RESULTS: The recurrence and progression were looked at. Patients treated with BCG for Carcinoma in situ, 11 of 19 (53%) remained tumor-free after 1 or 2 courses of BCG for the duration of the follow-up (mean 4.9 years, range 1.5 to 8.5 years). For patients treated for recurring tumors, 17 of 50 (34%) had no recurrences after 1 or 2 courses of BCG with the same follow-up. When facing multiple tumors, 10 of 23 (43%) patients did not experience recurrences. Therefore, in the 92 patients treated, 38 presented no recurrences after 1 or 2 courses of BCG, for a success rate of 41%. In terms of progression, of the 19 patients treated with BCG for CIS, 4 (21%) went on to develop muscle invasive disease. Of the 50 patients treated for recurrent tumors, 2 (4%) eventually developed lamina propria invasion (initial lesion was a Ta tumor), 4 (8%) carcinoma in situ and 7 (14%) muscle invasive disease, for an overall progression rate of 26% in this group. Of the 25 patients treated for multiple tumors, 1 (4%) developed CIS and 3 (12%) presented with muscle invasive disease, for an overall progression rate of 16% for the duration of the follow-up. Therefore, 21 of 92 (23%) patients had progression of their disease following BCG therapy. No prognostic factors for recurrence or progression could be identified in these tumors. CONCLUSION: When indications warrant its use, BCG is effective in reducing recurrences and limiting progression in TCC of the bladder. Recurrence within 2 years of treatment is, however, a sign of poor prognosis and other therapeutic options should be sought.

[Perineal retraining in urinary stress incontinence]. / La rééducation périnéale dans l'incontinence urinaire d'effort.

Twenty patients suffering from urinary stress incontinence were treated by perineal reeducation. The assessment included a medical and urological questionnaire, a physical examination, a urine analysis and culture, a cystoscopy, urinary flow and cystometry, a urethral pressure profile and a subjective evaluation of the perineal musculature. The 20 patients selected had documented stress incontinence, had never been operated on for incontinence and had a stable bladder at urodynamic assessment. Treatment was identical for all patients and included 12 biofeedback and electrostimulation sessions over a 4 to 6 week period. The questionnaire, urodynamic and perineal assessment were repeated at the end of treatment. No complication occurred. Micturition frequency decreased in all patients. Clinical correction of incontinence was observed in ten patients, improvement in nine and no change in one for an overall cure or improvement rate of 95%. The urethrocystocele evaluation did not change. Perineal evaluation and urodynamic parameters were only slightly improved. At follow-up evaluation 6 to 9 months post treatment, a 75% cure or improvement rate was still present. Perineal reeducation is a non morbid and effective modality to correct urinary stress incontinence. Its long term efficacy and its use for other types of incontinence has to be demonstrated.

[Ureteroscopy versus in situ extracorporeal shockwave lithotripsy in the treatment of calculi of the distal ureter]. / Urétéroscopie versus lithotripsie par onde de choc extracorporelle in situ dans le traitement des calculs du tiers distal de l'uretère.

In a retrospective study from a unique center (St. Luc Hospital, Montreal) stone clearance of 88 consecutive distal ureteral calculi (below pelvic brim) treated by extracorporeal shock wave lithotripsy in situ were compared to a group of 94 distal ureteral calculi treated by ureteroscopy during the same period. Our results show 84% success rate for ureteroscopy which is clearly superior than 58% stone clearance rate at 3 month follow-up for ESWL Success rate was influenced by stone size in the ESWL group but not in the ureteroscopy group. This study reveals similar success rate for calculi smaller than 6 mm but for larger calculi, success rate of ureteroscopy is significantly superior.

Characterization of the siRNAs associated with peach latent mosaic viroid infection.

The peach latent mosaic viroid (PLMVd) is a small, circular RNA species that has been shown to induce RNA silencing in plants. With the goal of better understanding the biological mechanism underlying this process, the siRNAs found in PLMVd infected peach leaves were isolated and sequenced. Analysis of the resulting data prompted several conclusions, including: i. PLMVd strands of both polarities are substrates for the Dicer-like enzymes found in peach leaves; ii. the more highly structured regions of PLMVd trigger the activity of the Dicer-like enzymes; and, iii. the circular PLMVd conformers appear to be favored for transport into the cytoplasm.

Molecular features of new Peach Latent Mosaic Viroid variants suggest that recombination may have contributed to the evolution of this infectious RNA.

Nucleotide sequences of a broad range of Peach Latent Mosaic Viroid (PLMVd) variants were determined. The variants were isolated from peach, pear, and almond tree samples collected in Tunisia. Sequence analysis confirmed the high variability of PLMVd, as no less than 119 new variants were identified. Variations included new polymorphic positions, insertions of 11 to 14 nucleotides, and new mutations within the hammerhead self-cleavage motifs. We provide the first covariation-based evidence for certain stems within the proposed secondary structure. Our covariation analysis also strengthens the view that a pseudoknot closes the replication domain. On the basis of phylogenetic tree studies and informative positions, PLMVd variants are proposed to cluster into groups and subgroups likely to have resulted from recombination events. PLMVd thus emerges as a suitable viroid for retracing the evolution of an RNA genome.

The kinetics and magnesium requirements for the folding of antigenomic delta ribozymes.

Using an oligonucleotide hybridization assay to gain insight into the folding of delta ribozymes, we demonstrate a correlation between their folding and catalytic behavior. Together with recent structural information on the crystal structure of self-cleaved genomic delta ribozyme, in which the L3 loop interacts with J1/4 to form the newly proposed stem P1.1, we conclude that it is likely that the P1.1 stem forms only in the presence of Mg(2+). This stem can be detected in both the self-cleaved and trans-acting delta ribozymes. When the trans-acting version of antigenomic delta ribozyme was studied, it is demonstrated that its L3 loop requires magnesium and, apparently, formation of the P1 stem for the subsequently formation of the P1.1 stem. Most importantly, the kinetics were monitored, and provide a significant addition to our understanding of ribozyme tertiary structure formation prior to the chemical cleavage step. Using previous kinetic data and our new findings, we discuss the rate-limiting characteristics of delta ribozyme folding.

Substrate specificity of delta ribozyme cleavage.

The specificity of delta ribozyme cleavage was investigated using a trans-acting antigenomic delta ribozyme. Under single turnover conditions, the wild type ribozyme cleaved the 11-mer ribonucleotide substrate with a rate constant of 0.34 min-1, an apparent Km of 17.9 nM and an apparent second-order rate constant of 1.89 x 10(7) min-1 M-1. The substrate specificity of the delta ribozyme was thoroughly investigated using a collection of substrates that varied in either the length or the nucleotide sequence of their P1 stems. We observed that not only is the base pairing of the substrate and the ribozyme important to cleavage activity, but also both the identity and the combination of the nucleotide sequence in the substrates are essential for cleavage activity. We show that the nucleotides in the middle of the P1 stem are essential for substrate binding and subsequent steps in the cleavage pathway. The introduction of any mismatches at these positions resulted in a complete lack of cleavage by the wild type ribozyme. Our findings suggest that factors more complex than simple base pairing interactions, such as tertiary structure interactions, could play an important role in the substrate specificity of delta ribozyme cleavage.

Natural 2',5'-phosphodiester bonds found at the ligation sites of peach latent mosaic viroid.

Peach latent mosaic viroid (PLMVd) is a circular RNA pathogen that replicates in a DNA-independent fashion via a rolling circle mechanism. PLMVd has been shown to self-ligate in vitro primarily via the formation of 2',5'-phosphodiester bonds; however, in vivo the occurrence and necessity of this nonenzymatic mechanism are not evident. Here, we unequivocally report the presence of 2', 5'-phosphodiester bonds at the ligation site of circular PLMVd strands isolated from infected peach leaves. These bonds serve to close the linear conformers (i.e., intermediates), yielding circular ones. Furthermore, these bonds are shown to stabilize the replicational circular templates, resulting in a significant advantage in terms of viroid viability. Although the mechanism responsible for the formation of these 2',5'-phosphodiester bonds remains to be elucidated, a hypothesis describing in vivo nonenzymatic self-ligation is proposed. Most significantly, our results clearly show that 2',5'-phosphodiester bonds are still present in nature and that they are of biological importance.