Related homing endonucleases I-BmoI and I-TevI use different strategies to cleave homologous recognition sites

A typical homing endonuclease initiates mobility of its group I intron by recognizing DNA both upstream and downstream of the intron insertion site of intronless alleles, preventing the endonuclease from binding and cleaving its own intron-containing allele. Here, we describe a GIY-YIG family homing endonuclease, I-BmoI, that possesses an unusual recognition sequence, encompassing 1 base pair upstream but 38 base pairs downstream of the intron insertion site. I-BmoI binds intron-containing and intronless substrates with equal affinity but can nevertheless discriminate between the two for cleavage. I-BmoI is encoded by a group I intron that interrupts the thymidylate synthase (TS) gene (thyA) of Bacillus mojavensis s87-18. This intron resembles one inserted 21 nucleotides further downstream in a homologous TS gene (td) of Escherichia coli phage T4. I-TevI, the T4 td intron-encoded GIY-YIG endonuclease, is very similar to I-BmoI, but each endonuclease gene is inserted within a different position of its respective intron. Remarkably, I-TevI and I-BmoI bind a homologous stretch of TS-encoding DNA and cleave their intronless substrates in very similar positions. Our results suggest that each endonuclease has independently evolved the ability to distinguish intron-containing from intronless alleles while maintaining the same conserved recognition sequence centered on DNA-encoding active site residues of TS.

Identification of peptides specific for cerebrospinal fluid antibodies in multiple sclerosis by using phage libraries.

The study of the origin and pathogenetic relevance of the oligoclonal antibodies present in the cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients has been hampered by a lack of specific ligands. We recently reported a general strategy, based on phage-displayed random peptide libraries, to identify ligands for disease-specific antibodies even in the absence of any information on the nature of the pathologic antigen. With this procedure, we identified several peptides specifically recognized by antibodies present in the CSF of MS patients. Using these peptides as reagents, we demonstrated that they mimic different natural epitopes and react with antibodies enriched in the CSF of MS patients. Antibodies recognizing the selected peptides are commonly found with equal frequency in the sera of MS patients and of normal individuals. In contrast, the repertoire of CSF antibodies appears to be individual-specific and is probably the result of a nonspecific immunodysregulation rather than a stereotyped response to a single antigen/agent.

Conformational transitions monitored for single molecules in solution.

Phenomena that can be observed for a large number of molecules may not be understood if it is not possible to observe the events on the single-molecule level. We measured the fluorescence lifetimes of individual tetramethylrhodamine molecules, linked to an 18-mer deoxyribonucleotide sequence specific for M13 DNA, by time-resolved, single-photon counting in a confocal fluorescence microscope during Brownian motion in solution. When many molecules were observed, a biexponential fluorescence decay was observed with equal amplitudes. However, on the single-molecule level, the fraction of one of the amplitudes spanned from 0 to unity for a collection of single-molecule detections. Further analysis by fluorescence correlation spectroscopy made on many molecules revealed a process that obeys a stretched exponential relaxation law. These facts, combined with previous evidence of the quenching effect of guanosine on rhodamines, indicate that the tetramethylrhodamine molecule senses conformational transitions as it associates and dissociates to a guanosine-rich area. Thus, our results reveal conformational transitions in a single molecule in solution under conditions that are relevant for biological processes.

Cell density regulates cellular reversal frequency in Myxococcus xanthus.

Myxococcus xanthus is a Gram-negative bacterium that aggregates to form fruiting bodies when nutrients are limiting. Previous studies showed that the frz mutants that are defective in chemotaxis exhibited irregular and infrequent patterns of cellular reversal. In contrast, wild-type cells, when examined individually, reverse relatively frequently, about once every 6 min. It is not known how the change of reversal frequency effects cellular aggregation during fruiting body formation in M. xanthus. In this study, we stained cells with a tetrazolium dye so that we could track the reversal frequencies of single cells and cells in groups. We found that developmental cells in large groups reverse much less than cells in small groups or as single cells. This reduced cellular reversal frequency is related to the frz signal transduction system and correlated with the methylation of FrzCD (a methyl-accepting chemotaxis protein). Cells containing a mutation in the frz genes or in the genes required for social motility do not respond in this way. The reduction in cellular reversals as developmental cells accumulate in groups suggests a simple hypothesis for the aggregation of cells into discrete mounds during fruiting body formation. We also found that M. xanthus cells glide with equal frequency in the forward or reverse directions, indicating that cells do not contain a "head" or "tail."

Differences in the RNA binding sites of iron regulatory proteins and potential target diversity.

Posttranscriptional regulation of genes of mammalian iron metabolism is mediated by the interaction of iron regulatory proteins (IRPs) with RNA stem-loop sequence elements known as iron-responsive elements (IREs). There are two identified IRPs, IRP1 and IRP2, each of which binds consensus IREs present in eukaryotic transcripts with equal affinity. Site-directed mutagenesis of IRP1 and IRP2 reveals that, although the binding affinities for consensus IREs are indistinguishable, the contributions of arginine residues in the active-site cleft to the binding affinity are different in the two RNA binding sites. Furthermore, although each IRP binds the consensus IRE with high affinity, each IRP also binds a unique alternative ligand, which was identified in an in vitro systematic evolution of ligands by exponential enrichment procedure. Differences in the two binding sites may be important in the function of the IRE-IRP regulatory system.

Detection and localization of individual antibody-antigen recognition events by atomic force microscopy.

A methodology has been developed for the study of molecular recognition at the level of single events and for the localization of sites on biosurfaces, in combining force microscopy with molecular recognition by specific ligands. For this goal, a sensor was designed by covalently linking an antibody (anti-human serum albumin, polyclonal) via a flexible spacer to the tip of a force microscope. This sensor permitted detection of single antibody-antigen recognition events by force signals of unique shape with an unbinding force of 244 +/- 22 pN. Analysis revealed that observed unbinding forces originate from the dissociation of individual Fab fragments from a human serum albumin molecule. The two Fab fragments of the antibody were found to bind independently and with equal probability. The flexible linkage provided the antibody with a 6-nm dynamical reach for binding, rendering binding probability high, 0.5 for encounter times of 60 ms. This permitted fast and reliable detection of antigenic sites during lateral scans with a positional accuracy of 1.5 nm. It is indicated that this methodology has promise for characterizing rate constants and kinetics of molecular recognition complexes and for molecular mapping of biosurfaces such as membranes.

Modification of the substrate specificity of an acyl-acyl carrier protein thioesterase by protein engineering.

The plant acyl-acyl carrier protein (ACP) thioesterases (TEs) are of biochemical interest because of their roles in fatty acid synthesis and their utilities in the bioengineering of plant seed oils. When the FatB1 cDNA encoding a 12:0-ACP TE (Uc FatB1) from California bay, Umbellularia californica (Uc) was expressed in Escherichia coli and in developing oilseeds of the plants Arabidopsis thaliana and Brassica napus, large amounts of laurate (12:0) and small amounts of myristate (14:0) were accumulated. We have isolated a TE cDNA from camphor (Cinnamomum camphorum) (Cc) seeds that shares 92% amino acid identity with Uc FatB1. This TE, Cc FatB1, mainly hydrolyzes 14:0-ACP as shown by E. coli expression. We have investigated the roles of the N- and C-terminal regions in determining substrate specificity by constructing two chimeric enzymes, in which the N-terminal portion of one protein is fused to the C-terminal portion of the other. Our results show that the C-terminal two-thirds of the protein is critical for the specificity. By site-directed mutagenesis, we have replaced several amino acids in Uc FatB1 by using the Cc FatB1 sequence as a guide. A double mutant, which changes Met-197 to an Arg and Arg-199 to a His (M197R/R199H), turns Uc FatB1 into a 12:0/14:0 TE with equal preference for both substrates. Another mutation, T231K, by itself does not effect the specificity. However, when it is combined with the double mutant to generate a triple mutant (M197R/R199H/T231K), Uc FatB1 is converted to a 14:0-ACP TE. Expression of the double-mutant cDNA in E. coli K27, a strain deficient in fatty acid degradation, results in accumulation of similar amounts of 12:0 and 14:0. Meanwhile the E. coli expressing the triple-mutant cDNA produces predominantly 14:0 with very small amounts of 12:0. Kinetic studies indicate that both wild-type Uc FatB1 and the triple mutant have similar values of Km,app with respect to 14:0-ACP. Inhibitory studies also show that 12:0-ACP is a good competitive inhibitor with respect to 14:0-ACP in both the wild type and the triple mutant. These results imply that both 12:0- and 14:0-ACP can bind to the two proteins equally well, but in the case of the triple mutant, the hydrolysis of 12:0-ACP is severely impaired. The ability to modify TE specificity should allow the production of additional "designer oils" in genetically engineered plants.

Efeito de diferentes intensidades do treinamento de força nos ganhos de força máxima e hipertrofia muscular

O presente estudo investigou o efeito de diferentes intensidades do treinamento de força (TF), aplicadas com volume total de treino (VTT) equalizado, nos ganhos de força dinâmica máxima (1RM) e massa muscular dos membros superiores e inferiores. Trinta voluntários do sexo masculino, com idade entre 18 e 30 anos, participaram de 12 semanas de TF com uma frequência semanal de duas sessões. Foi utilizado um protocolo de treinamento unilateral com um dos lados do corpo realizando o exercício com intensidade equivalente a 20% 1RM (G20) e o lado contralateral utilizando uma das três intensidades 40%, 60% ou 80% 1RM (G40, G60 e G80, respectivamente). O grupo G20 realizava três séries compostas de repetições até a falha concêntrica e o VTT era calculado e replicado para os demais grupos. A força dinâmica máxima e a área de secção transversa (AST) dos músculos flexores do cotovelo e do vasto lateral foram avaliadas nos momentos pré, 6 semanas e pós-treinamento. Os resultados demonstraram que os grupos G40, G60 e G80 apresentaram ganhos similares de AST (25%, 25,1% e 25%, flexores do cotovelo e 20,5%, 20,4% e 19,5% vasto lateral, respectivamente, p<0,05). Somente o grupo G80 demonstrou diferença significante com o grupo G20 na comparação do período pós-treinamento (25% e 14,4%, respectivamente para os flexores do cotovelo e 19,5% e 7,9%, respectivamente para vasto lateral, p<0,05). Para os ganhos de 1RM o grupo G80 demonstrou maiores aumentos após 12 semanas de TF para a flexão unilateral do cotovelo na posição em pé (54,2% p<0,05) e para o leg press 45º os grupos G60 e G80 demonstraram os maiores aumentos (55,4% e 45,7%, respectivamente, p<0,05). Assim, quando o VTT foi equalizado entre diferentes intensidades (40, 60 e 80% 1RM) os ganhos da AST tanto dos flexores do cotovelo quanto o vasto lateral foram semelhantes e a intensidade de 20% 1RM não causou aumento significante da AST. No que diz respeito a força muscular as intensidades mais elevadas (60% e 80% 1RM) foram superiores em promover ganhos de força do que as demais intensidades utilizadas. Esses dados sugerem que ao equalizar o VTT os ganhos de massa muscular são semelhantes para as intensidades de treinamento entre 40- 80% 1RM. Além disso, a intensidade de 20% 1RM, mesmo com o VTT equalizado com as intensidades maiores, não promove aumentos de massa muscular para ambos os segmentos corporais. Por outro lado, intensidades altas de treinamento produzem os maiores ganhos de força máxima em membros superiores e inferiores

Uma formulação por média-variância multi-período para o erro de rastreamento em carteiras de investimento.

Neste trabalho, deriva-se uma política de escolha ótima baseada na análise de média-variância para o Erro de Rastreamento no cenário Multi-período - ERM -. Referindo-se ao ERM como a diferença entre o capital acumulado pela carteira escolhida e o acumulado pela carteira de um benchmark. Assim, foi aplicada a metodologia abordada por Li-Ng em [24] para a solução analítica, obtendo-se dessa maneira uma generalização do caso uniperíodo introduzido por Roll em [38]. Em seguida, selecionou-se um portfólio do mercado de ações brasileiro baseado no fator de orrelação, e adotou-se como benchmark o índice da bolsa de valores do estado de São Paulo IBOVESPA, além da taxa básica de juros SELIC como ativo de renda fixa. Dois casos foram abordados: carteira composta somente de ativos de risco, caso I, e carteira com um ativo sem risco indexado à SELIC - e ativos do caso I (caso II).

Synthesis of γ-, δ-, and ε-Lactams by Asymmetric Transfer Hydrogenation of N-(tert-Butylsulfinyl)iminoesters

Highly enantiomerically enriched γ- and δ-lactams have been prepared by a simple and very efficient procedure that involves the asymmetric transfer hydrogenation of N-(tert-butylsulfinyl)iminoesters followed by desulfinylation of the nitrogen atom and spontaneous cyclization to the desired lactams during the basic workup procedure. Five- and six-membered ring lactams bearing aromatic, heteroaromatic, and aliphatic substituents have been obtained in very high yields and ee’s up to >99%. A slight modification of the procedure also allowed the preparation of ε-lactams in good yields and very high enantioselectivities. Both enantiomers of the final lactams could be prepared with equal efficiency by changing the absolute configuration of the sulfinyl chiral auxiliary.

Cambios en la estructura metropolitana durante la última década. El caso del Área Metropolitana de Valencia

La realidad metropolitana de los países desarrollados está experimentando cambios importantes asociados a la creciente separación entre los espacios de residencia y trabajo. En las áreas metropolitanas se dan los procesos de este tipo más complejos cuyas consecuencias territoriales inmediatas son la potenciación de ciertas centralidades a la vez que crecen centros nuevos o tradicionales en sus áreas de influencia. La estructura territorial (la red urbana y de transportes, fundamentalmente) en el Área Metropolitana de Valencia (AMV) incide claramente en los problemas asociados a la equidad social y a la sostenibilidad ambiental. La comunicación que aquí se presenta se centra en el análisis de dichos cambios en la última década, a partir del comportamiento demográfico de los municipios, las pautas generales de movilidad y los cambios en la ocupación del suelo en el AMV.

Annotated almanac, 1766

Almanac containing three laid-in pages with handwritten notes and a few sporadic corrections to the rising and setting times of the moon on the calendar pages. The three laid-in pages consist of a small scrap of paper with the weights of the family, and two folded pages with notes about a lunar eclipse on August 20, 1766.

O tempo e o direito na sociedade de informação : o limite temporal para a manutenção de informações pessoais como corolário de um direito da personalidade

O tempo sempre desempenhou relevante função de estabilização das relações jurídicas e decantação da memória individual, fazendo com que a divulgação de fatos desagradáveis ou sacrificantes aos direitos da personalidade tivessem, salvo em eventos excepcionais de ordem histórica, por força de um processo natural de erosão da sua relevância e atualidade, como destino o esquecimento. Esse mecanismo natural, capaz de assegurar o exercício de novas escolhas e o livre desenvolvimento da personalidade, restou substancialmente mitigado pelo surgimento da sociedade de informação, com a expansão dos veículos de comunicação de massa e a rede mundial de computadores, com sua memória infalível, a permitir a divulgação e o amplo acesso, com idêntica facilidade, a informações atuais e do passado. Releva, portanto, discutir a existência de um direito ao esquecimento, como forma de estabelecer, salvo em situações de inequívoco interesse público, uma limitação temporal para a manutenção e para a divulgação de fatos passados e referências pessoais, fora de um contexto de atualidade, capazes de macular a honra, o bom nome, a privacidade e a integridade psicológica das pessoas, bem como a possibilidade de que a ofensa injustificada a um direito da personalidade protegido pelo esquecimento, praticada com abuso do direito de informar, seja considerada ilícita.

Revisiting the synthetic control estimator

The synthetic control (SC) method has been recently proposed as an alternative to estimate treatment effects in comparative case studies. The SC relies on the assumption that there is a weighted average of the control units that reconstruct the potential outcome of the treated unit in the absence of treatment. If these weights were known, then one could estimate the counterfactual for the treated unit using this weighted average. With these weights, the SC would provide an unbiased estimator for the treatment effect even if selection into treatment is correlated with the unobserved heterogeneity. In this paper, we revisit the SC method in a linear factor model where the SC weights are considered nuisance parameters that are estimated to construct the SC estimator. We show that, when the number of control units is fixed, the estimated SC weights will generally not converge to the weights that reconstruct the factor loadings of the treated unit, even when the number of pre-intervention periods goes to infinity. As a consequence, the SC estimator will be asymptotically biased if treatment assignment is correlated with the unobserved heterogeneity. The asymptotic bias only vanishes when the variance of the idiosyncratic error goes to zero. We suggest a slight modification in the SC method that guarantees that the SC estimator is asymptotically unbiased and has a lower asymptotic variance than the difference-in-differences (DID) estimator when the DID identification assumption is satisfied. If the DID assumption is not satisfied, then both estimators would be asymptotically biased, and it would not be possible to rank them in terms of their asymptotic bias.

Abhandlungen der Naturforschenden Gesellschaft zu Görlitz.

Maps for v. 8 in portfolio (with the society's publication).