The Relevance of Ribonuclease III in Pathogenic Bacteria

Dissertation presented to obtain the Ph.D degree in Biology.

Regulation of ribonuclease III processing by double-helical sequence antideterminants

The double helix is a ubiquitous feature of RNA molecules and provides a target for nucleases involved in RNA maturation and decay. Escherichia coli ribonuclease III participates in maturation and decay pathways by site-specifically cleaving double-helical structures in cellular and viral RNAs. The site of cleavage can determine RNA functional activity and half-life and is specified in part by local tertiary structure elements such as internal loops. The involvement of base pair sequence in determining cleavage sites is unclear, because RNase III can efficiently degrade polymeric double-stranded RNAs of low sequence complexity. An alignment of RNase III substrates revealed an exclusion of specific Watson–Crick bp sequences at defined positions relative to the cleavage site. Inclusion of these “disfavored” sequences in a model substrate strongly inhibited cleavage in vitro by interfering with RNase III binding. Substrate cleavage also was inhibited by a 3-bp sequence from the selenocysteine-accepting tRNASec, which acts as an antideterminant of EF-Tu binding to tRNASec. The inhibitory bp sequences, together with local tertiary structure, can confer site specificity to cleavage of cellular and viral substrates without constraining the degradative action of RNase III on polymeric double-stranded RNA. Base pair antideterminants also may protect double-helical elements in other RNA molecules with essential functions.

Ethidium-dependent uncoupling of substrate binding and cleavage by Escherichia coli ribonuclease III

Ethidium bromide (EB) is known to inhibit cleavage of bacterial rRNA precursors by Escherichia coli ribonuclease III, a dsRNA-specific nuclease. The mechanism of EB inhibition of RNase III is not known nor is there information on EB-binding sites in RNase III substrates. We show here that EB is a reversible, apparently competitive inhibitor of RNase III cleavage of small model substrates in vitro. Inhibition is due to intercalation, since (i) the inhibitory concentrations of EB are similar to measured EB intercalation affinities; (ii) substrate cleavage is not affected by actinomycin D, an intercalating agent that does not bind dsRNA; (iii) the EB concentration dependence of inhibition is a function of substrate structure. In contrast, EB does not strongly inhibit the ability of RNase III to bind substrate. EB also does not block substrate binding by the C-terminal dsRNA-binding domain (dsRBD) of RNase III, indicating that EB perturbs substrate recognition by the N-terminal catalytic domain. Laser photocleavage experiments revealed two ethidium-binding sites in the substrate R1.1 RNA. One site is in the internal loop, adjacent to the scissile bond, while the second site is in the lower stem. Both sites consist of an A-A pair stacked on a CG pair, a motif which apparently provides a particularly favorable environment for intercalation. These results indicate an inhibitory mechanism in which EB site-specifically binds substrate, creating a cleavage-resistant complex that can compete with free substrate for RNase III. This study also shows that RNase III recognition and cleavage of substrate can be uncoupled and supports an enzymatic mechanism of dsRNA cleavage involving cooperative but not obligatorily linked actions of the dsRBD and the catalytic domain.

Yeast RNase III triggers polyadenylation-independent transcription termination

Transcription termination of messenger RNA (mRNA) is normally achieved by polyadenylation followed by Rat1p-dependent 5'-3' exoribonuleolytic degradation of the downstream transcript. Here we show that the yeast ortholog of the dsRNA-specific ribonuclease III (Rnt1p) may trigger Rat1p-dependent termination of RNA transcripts that fail to terminate near polyadenylation signals. Rnt1p cleavage sites were found downstream of several genes, and the deletion of RNT1 resulted in transcription readthrough. Inactivation of Rat1p impaired Rnt1p-dependent termination and resulted in the accumulation of 3' end cleavage products. These results support a model for transcription termination in which cotranscriptional cleavage by Rnt1p provides access for exoribonucleases in the absence of polyadenylation signals.

Preliminary analysis of miRNA pathway in Schistosoma mansoni

RNA silencing refers to a series of nuclear and cytoplasmatic processes involved in the post-transcriptional regulation of gene expression or post-transcriptional gene silencing (PTGS), either by sequence-specific mRNA degradation or by translational at-rest. The best characterized small RNAs are microRNAs (miRNAs), which predominantly perform gene silencing through post-transcriptional mechanisms. in this work we used bioinformatic approaches to identify the parasitic trematode Schistosoma Mansoni sequences that are similar to enzymes involved in the post-transcriptional gene silencing mediated by miRNA pathway. We used amino acid sequences of well-known proteins involved in the miRNA pathway against S. mansoni genome and transcriptome databases identifying a total of 13 Putative proteins in the parasite. In addition, the transcript levels of SinDicer1 and SmAgo2/3/4 were identified by qRT-PCR using cercariae, adult worms, eggs and in vitro Cultivated schistosomula. Our results showed that the SmDicer1 and SmAgo2/3/4 are differentially expressed during schistosomula development, suggesting that the miRNA pathway is regulated at the transcript level and therefore may control gene expression during the life cycle of S. mansoni. (C) 2008 Published by Elsevier Ireland Ltd.

A human ribonuclease induces apoptosis associated with p21WAF1/CIP1 induction and JNK inactivation

Ribonucleases are promising agents for use in anticancer therapy. Among the different ribonucleases described to be cytotoxic, a paradigmatic example is onconase which manifests cytotoxic and cytostatic effects, presents synergism with several kinds of anticancer drugs and is currently in phase II/III of its clinical trial as an anticancer drug against different types of cancer. The mechanism of cytotoxicity of PE5, a variant of human pancreatic ribonuclease carrying a nuclear localization signal, has been investigated and compared to that of onconase. Methods: Cytotoxicity was measured by the MTT method and by the tripan blue exclusion assay. Apoptosis was assessed by flow cytometry, caspase enzymatic detection and confocal microscopy. Cell cycle phase analysis was performed by flow cytometry. The expression of different proteins was analyzed by western blot.n Results: We show that the cytotoxicity of PE5 is produced through apoptosis, that it does not require the proapoptotic activity of p53 and is not prevented by the multiple drug resistance phenotype. We also show that PE5 and onconase induce cell death at the same extent although the latter is also able to arrest the cell growth. We have compared the cytotoxic effects of both ribonucleases in the NCI/ADR-RES cell line by measuring their effects on the cell cycle, on the activation of different caspases and on the expression of different apoptosis- and cell cycle-related proteins. PE5 increases the number of cells in S and G2/M cell cycle phases, which is accompanied by the increased expression of cyclin E and p21WAF1/CIP1 together with the underphosphorylation of p46 forms of JNK. Citotoxicity of onconase in this cell line does not alter the cell cycle phase distribution and it is accompanied by a decreased expression of XIAP. Conclusions: We conclude that PE5 kills the cells through apoptosis associated with the p21WAF1/CIP1 induction and the inactivation of JNK. This mechanism is significantly different from that found for onconase

Urothelial Carcinogen Resistance Driven By Stronger Toll-like Receptor 2 (tlr2) And Uroplakin Iii (up Iii) Defense Mechanisms: A New Model.

The objective of the study was to illustrate the applicability and significance of the novel Lewis urothelial cancer model compared to the classic Fisher 344. Fischer 344 and Lewis females rats, 7 weeks old, were intravesical instilled N-methyl-N-nitrosourea 1.5 mg/kg every other week for a total of four doses. After 15 weeks, animals were sacrificed and bladders analyzed: histopathology (tumor grade and stage), immunohistochemistry (apoptotic and proliferative indices) and blotting (Toll-like receptor 2-TLR2, Uroplakin III-UP III and C-Myc). Control groups received placebo. There were macroscopic neoplastic lesions in 20 % of Lewis strain and 70 % of Fischer 344 strain. Lewis showed hyperplasia in 50 % of animals, normal bladders in 50 %. All Fischer 344 had lesions, 20 % papillary hyperplasia, 30 % dysplasia, 40 % neoplasia and 10 % squamous metaplasia. Proliferative and apoptotic indices were significantly lower in the Lewis strain (p < 0.01). The TLR2 and UP III protein levels were significantly higher in Lewis compared to Fischer 344 strain (70.8 and 46.5 % vs. 49.5 and 16.9 %, respectively). In contrast, C-Myc protein levels were significantly higher in Fischer 344 (22.5 %) compared to Lewis strain (13.7 %). The innovative Lewis carcinogen resistance urothelial model represents a new strategy for translational research. Preservation of TLR2 and UP III defense mechanisms might drive diverse urothelial phenotypes during carcinogenesis in differently susceptible individuals.

Early treatment of Class III malocclusion: 10-year clinical follow-up

Angle Class III malocclusion has been a challenge for researchers concerning diagnosis, prognosis and treatment. It has a prevalence of 5% in the Brazilian population, and may have a genetic or environmental etiology. This malocclusion can be classified as dentoalveolar, skeletal or functional, which will determine the prognosis. Considering these topics, the aim of this study was to describe and discuss a clinical case with functional Class III malocclusion treated by a two-stage approach (interceptive and corrective), with a long-term follow-up. In this case, the patient was treated with a chincup and an Eschler arch, used simultaneously during 14 months, followed by corrective orthodontics. It should be noticed that, in this case, initial diagnosis at the centric relation allowed visualizing the anterior teeth in an edge-to-edge relationship, thereby favoring the prognosis. After completion of the treatment, the patient was followed for a 10-year period, and stability was observed. The clinical treatment results showed that it is possible to achieve favorable outcomes with early management in functional Class III malocclusion patients.

Segmental LeFort I osteotomy for treatment of a class III malocclusion with temporomandibular disorder

This article reports the case of a 19-year-old young man with Class III malocclusion and posterior crossbite with concerns about temporomandibular disorder (TMD), esthetics and functional problems. Surgical-orthodontic treatment was carried out by decompensation of the mandibular incisors and segmentation of the maxilla in 4 pieces, which allowed expansion and advancement. Remission of the signs and symptoms occurred after surgical-orthodontic intervention. The maxillary dental arch presented normal transverse dimension. Satisfactory static and functional occlusion and esthetic results were achieved and remained stable. Three years after the surgical-orthodontic treatment, no TMD sign or symptom was observed and the occlusal results had not changed. When vertical or horizontal movements of the maxilla in the presence of moderate maxillary constriction are necessary, segmental LeFort I osteotomy can be an important part of treatment planning.

Orthodontic-surgical treatment of class III malocclusion with mandibular asymmetry

Class III skeletal malocclusion may present several etiologies, among which maxillary deficiency is the most frequent. Bone discrepancy may have an unfavorable impact on esthetics, which is frequently aggravated by the presence of accentuated facial asymmetries. This type of malocclusion is usually treated with association of Orthodontics and orthognathic surgery for correction of occlusion and facial esthetics. This report presents the treatment of a patient aged 15 years and 1 month with Class III skeletal malocclusion, having narrow maxilla, posterior open bite on the left side, anterior crossbite and unilateral posterior crossbite, accentuated negative dentoalveolar discrepancy in the maxillary arch, and maxillary and mandibular midline shift. Clinical examination also revealed maxillary hypoplasia, increased lower one third of the face, concave bone and facial profiles and facial asymmetry with mandibular deviation to the left side. The treatment was performed in three phases: presurgical orthodontic preparation, orthognathic surgery and orthodontic finishing. In reviewing the patient's final records, the major goals set at the beginning of treatment were successfully achieved, providing the patient with adequate masticatory function and pleasant facial esthetics.

Modified silicas covalently bounded to 5,10,15,20-tetrakis(2-hydroxy-5-nitrophenyl)porphyrinato iron(III): synthesis, spectroscopic and EPR characterization. Catalytic studies

In this work we have studied cyclooctene epoxidation with PhIO, using a new iron porphyrin, 5,10,15,20-tetrakis(2-hydroxy-5-nitrophenyl)porphyrinato iron(III), supported on silica matrices via eletrostatic interaction and / or covalent bonds as catalyst. These catalysts were obtained and immobilized on the solid supports propyltrimethylammonium silica (SiN+); propyltrimethylammonium and propylimidazole silica [SiN+(IPG)] and chloropropylsilica (CPS) via elestrostatic interactions and covalent binding. Characterization of the supported catalysts by UV-Vis spectroscopy and EPR (Electron paramagnetic resonance) indicated the presence of a mixture of FeII and FeIII species in all of the three obtained catalysts. In the case of (Z)-cyclooctene epoxidation by PhIO the yields observed for cis-epoxycyclooctane were satisfactory for the reactions catalyzed by the three materials (ranging from 68% to 85%). Such results indicate that immobilization of metalloporphyrins onto solid supports via groups localized on the ortho positions of their mesophenyl rings can lead to efficient catalysts for epoxidation reactions. The catalyst 1-CPS is less active than 1-SiN and 1-SiN(IPG), this argues in favour of the immobilization of this metalloporphyrin onto solids via electrostatic interactions, which is easier to achieve and results in more active oxidation catalysts. Interestingly, the activity of the supported catalysts remained the same even after three successive recyclings; therefore, they are stable under the oxidizing conditions.

Metal-free synthesis of indanes by iodine(III)-mediated ring contraction of 1, 2-dihydronaphthalenes

A metal-free protocol was developed to synthesize indanes by ring contraction of 1, 2-dihydronaphthalenes promoted by PhI(OH)OTs (HTIB or Koser's reagent). This oxidative rearrangement can be performed in several solvents (MeOH, CH3CN, 2 , 2, 2-trifluoroethanol (TFE), 1 , 1, 1, 3, 3, 3-hexafluoroisopropanol (HFIP), and a 1:4 mixture of TFE:CH2Cl2) under mild conditions. The ring contraction diastereoselectively gives functionalized trans-1, 3-disubstituted indanes, which are difficult to obtain in synthetic organic chemistry

Cerambycidae (Coleoptera) do estado do Maranhão, Brasil: III

Oitenta e quatro espécies são registradas para o Estado do Maranhão: Prioninae (uma), Disteniinae (uma), Cerambycinae (49) e Lamiinae (33). O número total de espécies assinaladas para o Estado eleva-se a 367. Nove novas espécies são descritas em Cerambycinae: Chlorida inexpectata sp. nov. (Bothriospilini); Jupoata germana sp. nov. (Cerambycini); Eburodacrys separatus sp. nov. e Eburodacrys cincora sp. nov. (Eburiini); Ectenessa affinis sp. nov. (Ectenessini); Minibidion bicolor sp. nov. (Ibidionini, Tropidiina); Compsibidion maculatum sp. nov. e Compsibidion ytu sp. nov. (Ibidionini,Ibidionina); Ideratus beatus sp. nov. (Ideratini). Três espécies são descritas em Lamiinae: Aerenea gibba sp. nov. (Compsosomatini); Mimasyngenes barbozai sp. nov. (Desmiphorini); Cacostola exilis sp. nov. (Onciderini). Uma variação no colorido de Adesmus paradiana Galileo & Martins, 2004 é observada. Corrigenda de Piruana pulchra Martins et al., 2009 para Piruauna pulchra (Desmiphorini).

Contribuição para o estudo dos Rhinotragini (Coleoptera, Cerambycidae). III: Oxyommata Zajciw, 1970 e novo gênero oriundo da divisão de Xenocrasis Bates, 1873

Oxyommata Zajciw, 1970 e O. collaris (Audinet-Serville, 1833) são redescritos e comentados. Um novo gênero proveniente da divisão de Xenocrasis Bates, 1873 é descrito e três espécies alocadas nele: X. fulvicollis(Lacordaire, 1868); X. pubipennis (Fisher, 1952); e X. vestitipennis Zajciw, 1963. É apresentada chave para as espécies do novo gênero.

Novos cerambycidae (coleoptera) da coleção Odette Morvan, Kaw, Guiana Francesa: III

Novas espécies da Guiana Francesa são descritas e figuradas: Trestonia bilineta sp. nov. (Onciderini); Ischnolea odettae sp. nov. (Desmiphorini); Oreodera mageia sp. nov. (Acanthoderini) e Icupima ampliata sp. nov. (Hemilophini). Faz-se um novo registro de Plocaederus fragosoi (Cerambycini) para a Guiana Francesa.