The 20S proteasome alpha(5) subunit of Arabidopsis thaliana carries an RNase activity and interacts in planta with the Lettuce mosaic potyvirus HcPro protein

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Estudo de transições durante o processo de desenovelamento térmico da RNase A por meio da calorimetria (DSC) e espectroscopia de correlação bidimensional no infravermelho médio (2D-IR)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Clonagem, expressão e purificação de domínios da proteína AtRLI2 (RNase L Inhibitor), um supressor endógeno de silenciamento por RNA de Arabidopis thaliana, visando estudos estruturais

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Trypanosoma cruzi: establishment of permeable cells for RNA processing analysis with drugs

Pre-mRNA maturation in trypanosomatids occurs through a process called trans-splicing which involves excision of introns and union of exons in two independent transcripts. For the first time, we present the standardization of Trypanosoma cruzi permeable cells (Y strain) as a model for trans-splicing study of mRNAs in trypanosomes, following by RNase protection reaction, which localizes the SL exon and intron. This trans-splicing reaction in vitro was also used to analyze the influence of NFOH-121, a nitrofurazone-derivative, on this mechanism. The results suggested that the prodrug affects the RNA processing in these parasites, but the trans-splicing reaction still occurred.

Purification and physicochemical properties of a ribonuclease produced by Aspergillus flavipes (IZ : 1501)

A RNase of Aspergillus flavipes (IZ:1501) was purified from culture medium by chromatography on DEAE-cellulose and Sephadex G50 columns, after 96 h of cultivation. The molecular weight of the RNase was estimated to be 15 kD by gel filtration using Sephadex G100, and the optimum pH and temperature were 4.0 and 55 degrees C, respectively. Catalytic activity was inhibited by Hg2+, Ag+, Fe3+, Co2+ and Zn2+. The enzyme showed guanosine specificity producing only 3'-GMP from yeast RNA.

Three-dimensional structure of ribonuclease T-1 complexed with an isosteric phosphonate substrate analogue of GpU: Alternate substrate binding modes and catalysis

The X-ray crystal structure of a complex between ribonuclease T-1 and guanylyl(3'-6')-6'-deoxyhomouridine (GpcU) has been determined at 2.0 Angstrom resolution. This Ligand is an isosteric analogue of the minimal RNA substrate, guanylyl(3'-5')uridine (GpU), where a methylene is substituted for the uridine 5'-oxygen atom. Two protein molecules are part of the asymmetric unit and both have a GpcU bound at the active site in the same manner. The protein-protein interface reveals an extended aromatic stack involving both guanines and three enzyme phenolic groups. A third GpcU has its guanine moiety stacked on His92 at the active site on enzyme molecule A and interacts with GpcU on molecule B in a neighboring unit via hydrogen bonding between uridine ribose 2'- and 3'-OH groups. None of the uridine moieties of the three GpcU molecules in the asymmetric unit interacts directly with the protein. GpcU-active-site interactions involve extensive hydrogen bonding of the guanine moiety at the primary recognition site and of the guanosine 2'-hydroxyl group with His40 and Glu58. on the other hand, the phosphonate group is weakly bound only by a single hydrogen bond with Tyr38, unlike ligand phosphate groups of other substrate analogues and 3'-GMP, which hydrogen-bonded with three additional active-site residues. Hydrogen bonding of the guanylyl 2'-OH group and the phosphonate moiety is essentially the same as that recently observed for a novel structure of a RNase T-1-3'-GMP complex obtained immediately after in situ hydrolysis of exo-(S-p)-guanosine 2',3'-cyclophosphorothioate [Zegers et al. (1998) Nature Struct. Biol. 5, 280-283]. It is likely that GpcU at the active site represents a nonproductive binding mode for GpU [:Steyaert, J., and Engleborghs (1995) fur. J. Biochem. 233, 140-144]. The results suggest that the active site of ribonuclease T-1 is adapted for optimal tight binding of both the guanylyl 2'-OH and phosphate groups (of GpU) only in the transition state for catalytic transesterification, which is stabilized by adjacent binding of the leaving nucleoside (U) group.

Ribonuclease production by Aspergillus species

Ribonuclease production by Aspergillus flavipes. A sulphureus and A. fischeri in semisynthetic medium, after 24-144 hours at 30 degrees C under shaking, was studied. After cultivation, the medium was separated from micelia by filtration and the resultant solution was used as enzymatic extract. The highest amount of biomass and RNase was obtained after 96 hours of cultivation. The enzymes produced by three species presented similar characteristics, with optimum temperature at 55 degrees C and two peaks of activity at pH 4.5 and 7.0. A. flavipes RNases were more sensitive to temperature: 50% of the initial activity was lost after 1 hour at 70 degrees C. After this heat treatment, RNase of A. sulphureus lost 30% of this activity and that of A. fischeri only 16%. The nucleotides released by enzimatic hydrolysis of RNA were separated by ion exchange chromatography in a AG-1X8-formiate column and identified by paper chromatography. This procedure indicated that the raw enzymatic extract of Aspergillus flavipes is able to hydrolyze RNA, releasing 3'-nucleotides monophosphate at pH 4.5 and 3' and 5'-nucleotides monophosphate at pH 7.0 and 8.5. This result suggests that this strain produces two different types of RNase, one acidic and other alcaline, with different specificities.

Partial purification and some properties of a T2 ribonuclease from Aspergillus flavipes

A ribonuclease was partially purified from the culture medium of Aspergillus flavipes (IZ:1501), after 96 h of cultivation by chromatography on DEAE-cellulose and Sephadex G100 columns. The molecular weight of the RNase was estimated to be 40 kD by gel filtration using Sephadex G100, and the optimum pH and temperature were 4.0 and 50-55 degrees C, respectively. Catalytic activity was inhibited by Zn+2, Fe+3, Hg+2 and Ag+ ions. The enzyme did not show an exact base specificity and produced four kinds of 3'-nucleotides from yeast RNA.

Alkaline ribonuclease activity in maternal serum and in serum of newborns from birth up to thirty days of age. A study made with full-term appropriate-, full-term small- and preterm appropriate-for-gestational-age infants

We have studied the alkaline ribonuclease (RNase) activity in maternal serum and serum of full-term small- (T-SGA), full-term appropriate- (T-AGA) and preterm appropriate-for-gestational age (PT-AGA) newborns. A significantly lower level of RNase was observed in T-AGA and T-SGA newborns on the 30th day of age and in PT-AGA newborns on the 15th and 30th days of age, as compared to other T-AGA, T-SGA and PT-AGA groups of infants at birth. RNase activity was significantly higher in cord blood than in the maternal blood in all categories studied. Moreover, in preterm newborns, RNase activity in cord blood was significantly higher in those presenting a lower gestational age. We did not observe any significant difference in RNase levels in the cord blood of newborns from the 3 categories studied. The same results were observed concerning maternal blood. We, therefore, conclude that RNase activity in cord blood or in maternal blood is not a very statisfactory indicator of fetal malnutrition.

Age related obesity-induced shortening of GLUT4 mRNA poly(A) tail length in rat gastrocnemius skeletal muscle

Obese insulin resistant animals and humans have shown reduced GLUT4 gene expression. Yet, in skeletal muscle, discrepancy between mRNA and protein regulation has been frequently observed, suggesting a post-transcriptional modulation. We investigated the GLUT4 expression in adipose tissue and muscle of obese 12-month-old (12-mo) rats, comparing with lean 2-month-old (2-mo) animals. Obesity was accompanied by insulin resistance, and 65% reduction (P < 0.01) in GLUT4 mRNA and protein in adipose tissue. However, in muscle, despite increased (P < 0.05) mRNA content, GLUT4 protein was unchanged. RNase H and poly(A) test assays showed a reduction (P < 0.01) of ∼80 adenines in the GLUT4 mRNA poly(A) tail of muscle from 12-mo rats, recognizing that the poly(A) tail length correlates with translation efficiency. Concluding, age related obesity of 12-mo rats involves suppression of GLUT4 expression in adipose tissue; however, in muscle, GLUT4 mRNA content increases, but with a shorter poly(A) tail, thus unchanging the protein content. © 2007 Elsevier B.V. All rights reserved.

Trypanosoma cruzi: Evaluation of (-)-cubebin derivatives activity in the messenger RNAs processing

No fully effective treatment has been developed since the discovery of Chagas' disease. Since drug-resistant Trypanosoma cruzi strains are occurring and the current therapy is effective in the acute phase but with various adverse side effects, more studies are needed to characterize the susceptibility of T. cruzi to new drugs. Pre-mRNA maturation in trypanosomatids occurs through a process called trans-splicing, which is unusual RNA processing reaction, and it implies the processing of polycistronic transcription units into individual mRNAs; a short transcript spliced leader (SL RNA) is trans-spliced to the acceptor pre-mRNA, giving origin to the mature mRNA. Cubebin derivatives seem to provide treatments with less collateral effects than benznidazole and showed similar or better trypanocidal activities than benznidazole. Therefore, the cubebin derivatives ((-)-6,6′-dinitrohinokinin (DNH) and (-)-hinokinin (HQ)) interference in the mRNA processing was evaluated using T. cruzi permeable cells (Y and BOL (Bolivia) strains) following by RNase protection reaction. These substances seem to intervene in any step of the RNA transcription, promoting alterations in the RNA synthesis, even though the RNA processing mechanism still occurs. Furthermore, HQ presented better activity against the parasites than DNH, meaning that BOL strain seems to be more resistant than Y. © 2011 Springer-Verlag.

Modelo experiemtal de neotrquéia em coelho utilizando técnicas de engenharia de tecidos

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Produção e sensibilidade de isolados de Xanthomonas axonopodis pv. citri a bacteriocinas

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Light-emitting diode effects on combined decellularization of tracheae. A novel approach to obtain biological scaffolds

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)