The mechanism of action of phenylalanine ammonia-lyase: the role of prosthetic dehydroalanine.

Phenylalanine ammonia-lyase (EC 4.3.1.5) from parsley is posttranslationally modified by dehydrating its Ser-202 to the catalytically essential dehydroalanine prosthetic group. The codon of Ser-202 was changed to those of alanine and threonine by site-directed mutagenesis. These mutants and the recombinant wild-type enzyme, after treatment with sodium borohydride, were virtually inactive with L-phenylalanine as substrate but catalyzed the deamination of L-4-nitrophenylalanine, which is also a substrate for the wild-type enzyme. Although the mutants reacted about 20 times slower with L-4-nitrophenylalanine than the wild-type enzyme, their Vmax for L-4-nitrophenylalanine was two orders of magnitude higher than for L-phenylalanine. In contrast to L-tyrosine, which was a poor substrate, DL-3-hydroxyphenylalanine (DL-m-tyrosine) was converted by phenylalanine ammonia-lyase at a rate comparable to that of L-phenylalanine. These results suggest a mechanism in which the crucial step is an electrophilic attack of the prosthetic group at position 2 or 6 of the phenyl group. In the resulting carbenium ion, the beta-HSi atom is activated in a similar way as it is in the nitro analogue. Subsequent elimination of ammonia, concomitant with restoration of both the aromatic ring and the prosthetic group, completes the catalytic cycle.

The mode of action of aspirin-like drugs: effect on inducible nitric oxide synthase.

Nitric oxide synthesized by inducible nitric oxide synthase (iNOS) has been implicated as a mediator of inflammation in rheumatic and autoimmune diseases. We report that exposure of lipopolysaccharide-stimulated murine macrophages to therapeutic concentrations of aspirin (IC50 = 3 mM) and hydrocortisone (IC50 = 5 microM) inhibited the expression of iNOS and production of nitrite. In contrast, sodium salicylate (1-3 mM), indomethacin (5-20 microM), and acetaminophen (60-120 microM) had no significant effect on the production of nitrite at pharmacological concentrations. At suprapharmacological concentrations, sodium salicylate (IC50 = 20 mM) significantly inhibited nitrite production. Immunoblot analysis of iNOS expression in the presence of aspirin showed inhibition of iNOS expression (IC50 = 3 mM). Sodium salicylate variably inhibited iNOS expression (0-35%), whereas indomethacin had no effect. Furthermore, there was no significant effect of these nonsteroidal anti-inflammatory drugs on iNOS mRNA expression at pharmacological concentrations. The effect of aspirin was not due to inhibition of cyclooxygenase 2 because both aspirin and indomethacin inhibited prostaglandin E2 synthesis by > 75%. Aspirin and N-acetylimidazole (an effective acetylating agent), but not sodium salicylate or indomethacin, also directly interfered with the catalytic activity of iNOS in cell-free extracts. These studies indicate that the inhibition of iNOS expression and function represents another mechanism of action for aspirin, if not for all aspirin-like drugs. The effects are exerted at the level of translational/posttranslational modification and directly on the catalytic activity of iNOS.

Rapid local synchronization of action potentials: toward computation with coupled integrate-and-fire neurons.

The collective behavior of interconnected spiking nerve cells is investigated. It is shown that a variety of model systems exhibit the same short-time behavior and rapidly converge to (approximately) periodic firing patterns with locally synchronized action potentials. The dynamics of one model can be described by a downhill motion on an abstract energy landscape. Since an energy landscape makes it possible to understand and program computation done by an attractor network, the results will extend our understanding of collective computation from models based on a firing-rate description to biologically more realistic systems with integrate-and-fire neurons.

T-cell receptor (TCR) usage in Lewis rat experimental autoimmune encephalomyelitis: TCR beta-chain-variable-region V beta 8.2-positive T cells are not essential for induction and course of disease.

Predominant usage of V beta 8.2 gene segments, encoding a T-cell receptor (TCR) beta chain variable region, has been reported for pathogenic Lewis rat T cells reactive to myelin basic protein (MBP). However, up to 75% of the alpha/beta T cells in a panel of MBP-specific T-cell lines did not display TCR V beta 8.2, V beta 8.5, V beta 10, or V beta 16 elements. To further investigate TCR usage, we sorted the T-cell lines for V beta 8.2- and V beta 10-positive T cells or depleted the lines of cells with these TCRs. V beta 8.2-positive T cells and one of the depleted T-cell lines strongly reacted against the MBP peptide MBP-(68-88). The depleted T-cell line caused marked experimental autoimmune encephalomyelitis (EAE) even in Lewis rats in which endogenous V beta 8.2-positive T cells had been eliminated by neonatal treatment with anti-V beta 8.2 monoclonal antibodies. T-cell hybridomas generated from this line predominantly used V beta 3 TCR genes coexpressed with TCR V alpha 2 transcripts, which were also used by V beta 8.2-positive T cells. Furthermore, V beta 10-positive T cells reactive to MBP-(44-67) were encephalitogenic when injected immediately after positive selection. After induction of EAE by sorted V beta 8.2- or V beta 10-positive T-cell lines, immunocytochemical analysis of the spinal cord tissue showed a predominance of the injected TCR or of nontypable alpha/beta T cells after injection of the depleted line. Our results demonstrate heterogeneity of TCR beta-chain usage even for a single autoantigen in an inbred strain. Moreover, V beta 8.2-positive T cells are not essential for the induction and progression of adoptive-transfer EAE.

The Summary of a Course of Experimental Philosophical Lectures, 1746-1747

Leather hardcover notebook containing a handwritten copy of John Winthrop's course of experimental and philosophical lectures presented between March 10, 1746 and June 16, 1746. The first one-hundred pages of the volume are divided into twenty chapters which were presented in thirty-three lectures. The chapters contain text and diagrams on mechanical powers, the lever, the pulley, the axis in peritrochio, the inclined plane, the wedge, the screw, compound engines, the laws of motion, gravity, attraction of cohesion, the power of repulsion, magnetism, fluids, electricity, opticks, and astronomy. There is a five-page addenda to the course summary added in 1747, and a sixty-page text titled "The Method of Astronomical calculations" containing thirteen problems related to calculating distances with a list of astronomical characters, and followed with charts related to the eclipse of Jupiter's satellites.

The summary of a course of experimental lectures in natural philosophy by Dr. John Winthrop (manuscript copy), 1772-1774

Notebook with a handwritten copy of lecture summaries for a physics course given by Harvard Professor John Winthrop. The notes were made by Timothy Foster in 1772 and 1773. The volume contains twenty-five lectures with some diagrams.

Preventing the rise of sovereign borrowing costs in the Eurozone: what can the ESM and the ECB achieve? Egmont Paper No. 56, November 2012

This paper focuses on the possible instruments for ‘ex-ante’/’preventive’/’precautionary’ interventions which can be deployed by the ESM and the ECB in order to prevent a debt crisis in a eurozone country. The potential of Eurobonds will also be discussed in this crisis management perspective. The first part of this paper traces the underlying trends of the evolution of interest rates in eurozone countries over the last decades. The second part discusses the principles of a preventive intervention in sovereign bond markets for the purpose of lowering borrowing costs of countries facing refinancing constraints; the limits and main issues of an ex-ante intervention will be underlined. In the third part, the properties of the ESM’s precautionary financial assistance and secondary market support facility will be discussed in details. The ECB preemptive intervention policies and, in particular, the OMT will be analyzed in the fourth part of the paper. The most likely course of action – a combined intervention by the ESM and the ECB – will be discussed in the fifth part. Finally, I will point out the core challenges of introducing Eurobonds as additional instruments to mitigate the rise of borrowing costs in the short term.

The differential evolution of hot- and cold-dust in galaxies in the last 7.5 GYR

Tese de mestrado em Física, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2016

A ação direta da vítima contra a seguradora nos seguros facultativos de responsabilidade civil na dogmática brasileira

A investigação parte do surgimento histórico do contrato de seguro, abordando em sequencia sua teoria geral e a dogmática brasileira pertinente para, após, traçar os contornos específicos do contrato de seguro de responsabilidade civil, tanto pelo viés doutrinário quanto da legislação vigente no Brasil, dando ênfase maior ao seu conceito e finalidade social, às peculiaridades de seu sinistro, assim como à pluralidade de interesses nele albergado, afastando-se a ideia de se tratar de um seguro de reembolso, e adotando-se sua conceituação como de um seguro de garantia. Após, são fixados os conceitos básicos de processo civil aplicáveis ao objeto do estudo: jurisdição, processo, ação e seus elementos, para somente então expor o surgimento e desenvolvimento teórico da ação direta da vítima contra a seguradora na doutrina e jurisprudência francesas, passando pelas fases exegética, legalista e doutrinária de sua análise. Com base neste desenvolvimento histórico, adota-se concepção da ação direta da vítima contra a seguradora como instituto jurídico de conformação própria, oriundo dos princípios de equidade e justiça, e destinado ao afastamento dos princípios gerais de direito civil da relatividade contratual e da igualdade entre credores, constituindo meio de exercício do direito próprio da vítima contra a seguradora do causador do dano. Delimitados os conceitos em estudo, são expostas algumas experiências estrangeiras acerca do uso da ação direta e, partindo-se do embasamento teórico do seguro de responsabilidade civil e da ação direta da vítima por este protegida, ingressa-se na análise de seu desenvolvimento na dogmática brasileira. Para tanto, volta-se à ideia do seguro de responsabilidade civil como seguro de reembolso, e aos argumentos dela decorrentes para afastar o cabimento da ação direta, tais como o princípio da relatividade contratual, a ausência de previsão de solidariedade entre segurado e seguradora, e as dificuldades de exercício da ação e também de defesa por parte da seguradora e do segurado. Expostos e criticados os argumentos contrários ao cabimento da ação direta, passa-se ao estudo das tentativas doutrinárias e jurisprudenciais, no direito brasileiro, de se fornecer à vítima um meio de exercício de sua ação contra a seguradora, inicialmente por instrumentos de processo civil, e terminando por se reconhecer a possibilidade de acionamento conjunto de segurado e seguradora pelo Superior Tribunal de Justiça na sua Súmula 529. Por último, são tratados os argumentos doutrinários e legislativos favoráveis ao cabimento da ação direta da vítima contra a seguradora em qualquer seguro de responsabilidade civil, com e sem a participação inicial do segurado no processo, com destaque à função social do contrato de seguro de responsabilidade civil facultativo e ao direito próprio da vítima perante a seguradora. Conclui-se, assim, que a ação direta da vítima contra a seguradora, em qualquer seguro de responsabilidade civil, é instrumento apto e cabível na dogmática brasileira para dar vazão aos preceitos de equidade e justiça, despersonalizando a responsabilidade civil, ao levar seu foco da imputação para a indenidade, respeitando ao duplo interesse do moderno contrato de seguro de responsabilidade civil, e solucionando pela via mais apta e simples situação complexa.