Mutational analysis of active-site residues in the Mycobacterium leprae RecA intein, a LAGLIDADG homing endonuclease: Asp(122) and Asp(193) are crucial to the double-stranded DNA cleavage activity whereas Asp(218) is not

Mycobacterium leprae recA harbors an in-frame insertion sequence that encodes an intein homing endonuclease (PI-MleI). Most inteins (intein endonucleases) possess two conserved LAGLIDADG (DOD) motifs at their ctive center. A common feature of LAGLIDADG-type homing endonucleases is that they recognize and cleave the same or very similar DNA sequences. However, PI-MleI is distinctive from other members of the family of LAGLIDADG-type HEases for its modular structure with functionally separable domains for DNA-binding and cleavage, each with distinct sequence preferences. Sequence alignment analyses of PI-MleI revealed three putative LAGLIDADG motifs; however, there is conflicting bioinformatics data in regard to their identity and specific location within the intein polypeptide. To resolve this conflict and to determine the active-site residues essential for DNA target site recognition and double-stranded DNA cleavage, we performed site-directed mutagenesis of presumptive catalytic residues in the LAGLIDADG motifs. Analysis of target DNA recognition and kinetic parameters of the wild-type PI-MleI and its variants disclosed that the two amino acid residues, Asp(122) (in Block C) and Asp(193) (in functional Block E), are crucial to the double-stranded DNA endonuclease activity, whereas Asp(218) (in pseudo-Block E) is not. However, despite the reduced catalytic activity, the PI-MleI variants, like the wild-type PI-MleI, generated a footprint of the same length around the insertion site. The D122T variant showed significantly reduced catalytic activity, and D122A and D193A mutations although failed to affect their DNA-binding affinities, but abolished the double-stranded DNA cleavage activity. On the other hand, D122C variant showed approximately twofold higher double-stranded DNA cleavage activity, compared with the wild-type PI-MleI. These results provide compelling evidence that Asp(122) and Asp(193) in DOD motif I and II, respectively, are bona fide active-site residues essential for DNA cleavage activity. The implications of these results are discussed in this report.

Sapientia, 1976, Vol. XXXI, nº 122 (número completo)

Contenido: Verdad, historia y hermenéutica / Octavio N. Derisi – El lumen naturale en Santo Tomás y el essere ideale en Rosmini / William R. Darós – El constitutivo esencial de la persona / Octavio N. Derisi – Filosofía y desarrollo integral / Alberto Caturelli – La respuesta del hombre frente a lo existente : intuición y palabra / Osvaldo Francella – Notas y comentarios -- Bibliografía

Diário da Constituinte [gravação de vídeo] : [programa n. 122]

Líderes dos partidos reunidos com o Presidente da ANC decidem que não haverá recesso. Ulysses Guimarães(PMDB-SP) diz que seria danoso para o país e para as instituições a interrupção dos trabalhos da Constituinte. Bonifácio de Andrada(PMDB-MG) também é contra a paralisação dos trabalhos. Os líderes decidiram também que a Comissão de Sistematização não aceitará emendas que mudem o sentido das propostas já apresentadas pelas comissões. Bernardo Cabral(PMDB-AM) afirma que, segundo o Regimento, só cabem emendas de adequação. Mario Covas(PMDB-SP) explica que se for alterado o Regimento Interno, permitindo a apresentação de substitutivos, corre-se o risco de uma maioria parlamentar fazer a Constituição sozinha. Brandão Monteiro(PDT-RJ) diz que há uma distorção em relação aos trabalhos na Constituinte. Na sessão O Povo Pergunta, cidadão quer saber o que os constituintes estão fazendo em termos de justiça no Brasil. Paes Landim(PFL-PI) responde que, para acelerar os processos judiciais, a Assembleia Nacional Constituinte criou a figura dos juízes distritais que participarão dos julgamentos, com a presença de populares. Iram Saraiva(PMDB-GO) apresenta projeto de decisão a Comissão de Sistematização, para que seja possível a transmissão, pelas emissoras de televisão e de rádio, das votações da Constituinte. Terminou o prazo para apresentação de emendas, tendo sido recebidas mais de 5 mil.

Biology of the amphipod Gammarus pulex L. in water-bodies of the Leningrad district, and its piscicultural importance [Translation from: Trudy Vsesoyuznogo Gidrobiologicheskogo Obshchestva 7 99-122, 1956]

Observations and laboratory experiments are summarised that examine the widely discussed issue of what Gammarus pulex feeds on. On the basis of the observations of the authors and data from the literature it can be noted that G. pulex belongs to the omnivorous organisms, but, depending on the conditions of subsistence, in its rations either plant or animal food can predominate.

Void swelling in MA956 ODS steel irradiated with 122 MeV Ne-ions at elevated temperatures

An investigation of a commercial oxide dispersion-strengthened steel (MA9561) irradiated with high energy Ne-ions to high doses at elevated temperatures is presented. Specimens of MA956 oxide dispersion strengthened steel together with a 9% Cr ferritic/martensitic steel, e.g., Grade 92 steel were irradiated simultaneously with 20Ne-ions (with 122 MeV) to successively increasing damage levels of 1, 5 and 10 dpa at the damage peak at 440 C and 570 C, respectively. Cross-sectional microstructures of the specimens were investigated with transmission electron microscopy. MA956 oxide dispersion strengthened steel showed a higher resistance to void swelling especially to void growth at the grain boundaries than the ferritic/martensitic steel, e.g., Grade 92 steel did, and thus exhibited a prominence for an application in the situation of a high He production at high temperatures. The suppression of the growth of voids especially at the grain boundaries in MA956 is ascribed to an enhanced recombination of the point defects and a trapping of Ne atoms at the interfaces of the yttrium–aluminum oxide particles and the matrix.

Reintegração paisagística de infraestruturas rodoviárias: o caso da Estrada Nacional 122 e do Itinerário Complementar 27

Dissertação de Mestrado, Arquitetura Paisagista, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2015

Could concomitant radio-chemotherapy improve the outcomes of early-stage node negative anal canal cancer patients? A retrospective analysis of 122 patients.

One hundred twenty-two early-stage anal canal cancer patients (median age: 69 years) were treated with curative radiotherapy with (70 patients) or without (52 patients) concomitant chemotherapy. Median follow-up was 65 months (range: 4-238). At multivariate analysis, concomitant chemotherapy significantly improved local control (p = .007). Local control significantly influenced all considered endpoints, except the metastases free survival. The global rates of G3-G4 acute and late toxicity were 13.1% and 8.2%, respectively, and they were not increased by concomitant chemotherapy. Finally, concomitant chemotherapy is efficacious and safe in the treatment of T1-2N0 anal canal cancer patients and should be prospectively studied.