Selected abstracts on intra-arterial chemotherapy in cancer treatment /

Includes indexes.

Directory of cancer related information resources : (review version) /

Sponsored by NIH/NCI contract N01-CO-35403.

Rinderpest bibliography /

Description based on: June 1981.

Special listing.

Description based on: NCI/ICRDB/SL-332.

Prediction of protein B-factor profiles

The polypeptide backbones and side chains of proteins are constantly moving due to thermal motion and the kinetic energy of the atoms. The B-factors of protein crystal structures reflect the fluctuation of atoms about their average positions and provide important information about protein dynamics. Computational approaches to predict thermal motion are useful for analyzing the dynamic properties of proteins with unknown structures. In this article, we utilize a novel support vector regression (SVR) approach to predict the B-factor distribution (B-factor profile) of a protein from its sequence. We explore schemes for encoding sequences and various settings for the parameters used in SVR. Based on a large dataset of high-resolution proteins, our method predicts the B-factor distribution with a Pearson correlation coefficient (CC) of 0.53. In addition, our method predicts the B-factor profile with a CC of at least 0.56 for more than half of the proteins. Our method also performs well for classifying residues (rigid vs. flexible). For almost all predicted B-factor thresholds, prediction accuracies (percent of correctly predicted residues) are greater than 70%. These results exceed the best results of other sequence-based prediction methods. (C) 2005 Wiley-Liss, Inc.

Intramolecular disulphide bond arrangements in nonhomologous proteins

The presence and location of intramolecular disulphide bonds are a key determinant of the structure and function of proteins. Intramolecular disulphide bonds in proteins have previously been analyzed under the assumption that there is no clear relationship between disulphide arrangement and disulphide concentration. To investigate this, a set of sequence nonhomologous protein chains containing one or more intramolecular disulphide bonds was extracted from the Protein Data Bank, and the arrangements of the bonds, Protein Data Bank header, and Structural Characterization of Proteins fold were analyzed as a function of intramolecular, containing proteins were disulphide bond concentration. Two populations of intramolecular disulphide bond-containing identified, with a naturally occurring partition at 25 residues per bond. These populations were named intramolecular disulphide bond-rich and -poor. Benefits of partitioning were illustrated by three results: (1) rich chains most frequently contained three disulphides, explaining the plateaux in extant disulphide frequency distributions; (2) a positive relationship between median chain length and the number of disulphides, only seen when the data were partitioned-, and (3) the most common bonding pattern for chains with three disulphide bonds was based on the most common for two, only when the data were partitioned. The two populations had different headers, folds, bond arrangements, and chain lengths. Associations between IDSB concentration, IDSB bonding pattern, loop sizes, SCOP fold, and PDB header were also found. From this, we found that intramolecular disulphide bond-rich and -poor proteins follow different bonding rules, and must be considered separately to generate meaningful models of bond formation.

Predicting the solvent accessibility of transmembrane residues from protein sequence

In this study, we propose a novel method to predict the solvent accessible surface areas of transmembrane residues. For both transmembrane alpha-helix and beta-barrel residues, the correlation coefficients between the predicted and observed accessible surface areas are around 0.65. On the basis of predicted accessible surface areas, residues exposed to the lipid environment or buried inside a protein can be identified by using certain cutoff thresholds. We have extensively examined our approach based on different definitions of accessible surface areas and a variety of sets of control parameters. Given that experimentally determining the structures of membrane proteins is very difficult and membrane proteins are actually abundant in nature, our approach is useful for theoretically modeling membrane protein tertiary structures, particularly for modeling the assembly of transmembrane domains. This approach can be used to annotate the membrane proteins in proteomes to provide extra structural and functional information.

O Conflito de Agência Exposto ao Efeito de Mercado: Um Estudo Empírico de Precificação de Ações da Bombril S/A

Esta dissertação apresenta um estudo exploratório que tem como objetivo a avaliação da reação do mercado frente aos problemas de agência e assimetria informacional entre os acionistas majoritários-controladores e os acionistas minoritários de uma empresa brasileira de capital aberto, no tocante ao valor das suas ações negociadas na Bolsa de Valores de São Paulo. O estudo se propõe a investigar o impacto de precificação dessas ações promovendo a análise do seu comportamento frente à divulgação de notícias de crise e fraude da empresa por meio da mídia especializada. A metodologia adotada consiste na aplicação de estudo de eventos para identificação de retornos anormais da empresa desde a divulgação da primeira notícia selecionada, datada de maio de 2001, até a última notícia em outubro de 2005, utilizando-se de procedimentos estatísticos como a regressão linear e aplicação do teste t de student para estimar e comparar os resultados. Os dados foram obtidos por meio do banco de dados da Economática Ltda, conforme acesso realizado na Universidade de São Paulo. Para objeto de pesquisa foi selecionada a empresa Bombril S/A, por estar em evidências quanto a problemas de agência no âmbito do mercado nacional. Os resultados obtidos apontaram que o mercado reagiu significantemente aos anúncios dos conflitos selecionados, apresentando um valor de p-value <0,05 para os blocos de eventos, o que significa a rejeição da hipótese nula, constatando que a evidencia estatística dos dados testados comprova retornos anormais acumulados diferentes de zero. Entretanto, sugere-se novas pesquisas com outros parâmetros de eventos na busca de mais evidências sobre o efeito das informações no preço das ações.

RECIPROCIDADE ENTRE INDIVÍDUO E ORGANIZAÇÃO E SEUS IMPACTOS SOBRE A SATISFAÇÃO E O COMPROMETIMENTO

Reciprocidade indivíduo-organização e afetividade são dois fenômenos presentes na vida organizacional e que se tornaram tópicos de pesquisa no campo de estudos do comportamento organizacional. Esse estudo teve como objetivo reforçar as evidências empíricas acerca das relações entre cognições de troca indivíduo-organização e afetividade no contexto de trabalho. Para tanto, foram submetidas à teste empírico cinco hipóteses inspiradas em um estudo inédito desenvolvido por Siqueira (2002b). Contou-se com a utilização de um questionário composto por cinco escalas validadas, referentes as variáveis do estudo, que avaliaram percepção de suporte organizacional (PSO), percepção de reciprocidade organizacional (PRO), comprometimento organizacional normativo (CON), satisfação no trabalho (STR) e comprometimento organizacional afetivo (COA). Participaram 275 profissionais, sendo 183 pertencentes ao setor administrativo e 92 ao setor administrativo-acadêmico de uma Instituição de Ensino Superior, situada na região do Grande ABCD Paulista e com inserção no estado de São Paulo. OS participantes do estudo tinham idade média de 32 anos, sendo a maioria (58,2%) do sexo feminino, com escolaridade em nível superior (39,6%) e tempo médio de trabalho na organização de quatro anos. Os dados coletados foram organizados em um banco de dados eletrônico para tratamento estatístico, quando se utilizou o aplicativo SPSS (Statistical Package for the Social Scienses). Foram realizadas análises descritivas das variáveis e análises de correlação e de regressão múltipla para os testes das cinco hipóteses. Todas as hipóteses foram confirmadas. Conclui-se então, que o presente estudo reforça as proposições de Siqueira (2002b) acerca da pertinência de se considerar os três conceitos analisados (PSO, PRO E CON) como integrantes do esquema mental de reciprocidade. Ainda, os resultados do estudo reafirmam as evidências acerca da capacidade preditiva do EMR sobre satisfação no trabalho e comprometimento organizacional afetivo. Por fim, a análise dos dados aponta percepção de suporte organizacional como sendo o componente cognitivo do EMR com maior poder de influência sobre satisfação no trabalho e comprometimento organizacional afetivo. Portanto, cognições acerca das relações de troca social com organizações antecedem satisfação e comprometimento afetivo que, por sua vez, são importantes fatores a favor da organização, contribuindo para sua efetividade.

PERCEPÇÃO DE SUCESSO NA CARREIRA E RESILIÊNCIA: UM ESTUDO COM ADMINISTRADORES

Sucesso profissional está relacionado à satisfação do indivíduo com a sua carreira em longo prazo. Essa satisfação deriva de aspectos intrínsecos e extrínsecos, referentes a uma dimensão objetiva - aspectos mais visíveis do sucesso na carreira - que inclui: salários, progressão profissional, status e oportunidades de desenvolvimento de carreira, como promoção; e outra subjetiva, que se refere à interpretação pessoal do que seja sucesso, em especial na carreira: satisfação com o trabalho, orgulho, sentimentos de autorrealização, dentre outros. A percepção do sucesso com a carreira pode estar associada a características individuais como, por exemplo, a resiliência, que representa o processo dinâmico de adaptação positiva frente às adversidades. Na literatura, não foram localizados estudos que relacionem ambas as variáveis, isto é, sobre o quanto a resiliência pessoal pode contribuir para a percepção de sucesso na carreira. A fim de investigar essa influência, esta pesquisa tem como objetivo principal identificar se resiliência pessoal de administradores prediz sua percepção de sucesso na carreira. Participaram 137 administradores, formados em diversas instituições, sendo 56,1% do sexo feminino e 43,7% do sexo masculino, com idade média de 33 anos, divididos entre casados ou solteiros (44,5% para ambos). Os dados foram coletados por meio de um questionário sociodemográfico, baseado na Escala de Percepção de Sucesso na Carreira e da Connor-Davidson Resilience Scale (CD-RISC). As respostas compuseram um banco eletrônico de dados e foram analisados por meio do Statistical Package for the Social Sciences (SPSS). Resultados de análises de regressão hierárquica revelaram que resiliência prediz 5,5% da percepção do sucesso na carreira objetiva e 9% da percepção de sucesso na carreira subjetiva. Ao acrescentar a interação entre idade e tempo de trabalho, o poder de predição de ambos os modelos, tanto para sucesso objetivo, quanto para o subjetivo, elevou-se substancialmente, chegando ao dobro. Resiliência contribui para que os participantes percebam sucesso na carreira em ambas as dimensões, objetiva e subjetiva, e a predição é potencializada pela interação entre idade e tempo de trabalho. Os achados deste estudo confirmaram a hipótese levantada. O estudo trouxe contribuições para a área, mas também foram reconhecidas limitações, em função das quais foi proposta uma agenda de pesquisa para estudos futuros.

Overcoming barriers to membrane protein structure determination

After decades of slow progress, the pace of research on membrane protein structures is beginning to quicken thanks to various improvements in technology, including protein engineering and microfocus X-ray diffraction. Here we review these developments and, where possible, highlight generic new approaches to solving membrane protein structures based on recent technological advances. Rational approaches to overcoming the bottlenecks in the field are urgently required as membrane proteins, which typically comprise ~30% of the proteomes of organisms, are dramatically under-represented in the structural database of the Protein Data Bank.

Novel strategies to improve recombinant membrane protein production in yeast

Approximately 60% of pharmaceuticals target membrane proteins; 30% of the human genome codes for membrane proteins yet they represent less than 1% of known unique crystal structures deposited in the Protein Data Bank (PDB), with 50% of structures derived from recombinant membrane proteins having been synthesized in yeasts. G protein-coupled receptors (GPCRs) are an important class of membrane proteins that are not naturally abundant in their native membranes. Unfortunately their recombinant synthesis often suffers from low yields; moreover, function may be lost during extraction and purification from cell membranes, impeding research aimed at structural and functional determination. We therefore devised two novel strategies to improve functional yields of recombinant membrane proteins in the yeast Saccharomyces cerevisiae. We used human adenosine A2A receptor (hA2AR) as a model GPRC since it is functionally and structurally well characterised.In the first strategy, we investigated whether it is possible to provide yeast cells with a selective advantage (SA) in producing the fusion protein hA2AR-Ura3p when grown in medium lacking uracil; Ura3p is a decarboxylase that catalyzes the sixth enzymatic step in the de novo biosynthesis of pyrimidines, generating uridine monophosphate. The first transformant (H1) selected using the SA strategy gave high total yields of hA2AR-Ura3p, but low functional yields as determined by radio-ligand binding, leading to the discovery that the majority of the hA2AR-Ura3p had been internalized to the vacuole. The yeast deletion strain spt3Δ is thought to have slower translation rates and improved folding capabilities compared to wild-type cells and was therefore utilised for the SA strategy to generate a second transformant, SU1, which gave higher functional yields than H1. Subsequently hA2AR-Ura3p from H1 was solubilised with n-dodecyl-β-D-maltoside and cholesteryl hemisuccinate, which yielded functional hA2AR-Ura3p at the highest yield of all approaches used. The second strategy involved using knowledge of translational processes to improve recombinant protein synthesis to increase functional yield. Modification of existing expression vectors with an internal ribosome entry site (IRES) inserted into the 5ˊ untranslated region (UTR) of the gene encoding hA2AR was employed to circumvent regulatory controls on recombinant synthesis in the yeast host cell. The mechanisms involved were investigated through the use of yeast deletion strains and drugs that cause translation inhibition, which is known to improve protein folding and yield. The data highlight the potential to use deletion strains to increase IRES-mediated expression of recombinant hA2AR. Overall, the data presented in this thesis provide mechanistic insights into two novel strategies that can increase functional membrane protein yields in the eukaryotic microbe, S. cerevisiae.

PPD v1.0 - an integrated, web-accessible database of experimentally determined protein pKa values

The Protein pKa Database (PPD) v1.0 provides a compendium of protein residue-specific ionization equilibria (pKa values), as collated from the primary literature, in the form of a web-accessible postgreSQL relational database. Ionizable residues play key roles in the molecular mechanisms that underlie many biological phenomena, including protein folding and enzyme catalysis. The PPD serves as a general protein pKa archive and as a source of data that allows for the development and improvement of pKa prediction systems. The database is accessed through an HTML interface, which offers two fast, efficient search methods: an amino acid-based query and a Basic Local Alignment Search Tool search. Entries also give details of experimental techniques and links to other key databases, such as National Center for Biotechnology Information and the Protein Data Bank, providing the user with considerable background information.

Editorial overview:new protein production tools for structural biology

Full text: The idea of producing proteins from recombinant DNA hatched almost half a century ago. In his PhD thesis, Peter Lobban foresaw the prospect of inserting foreign DNA (from any source, including mammalian cells) into the genome of a λ phage in order to detect and recover protein products from Escherichia coli [ 1 and 2]. Only a few years later, in 1977, Herbert Boyer and his colleagues succeeded in the first ever expression of a peptide-coding gene in E. coli — they produced recombinant somatostatin [ 3] followed shortly after by human insulin. The field has advanced enormously since those early days and today recombinant proteins have become indispensable in advancing research and development in all fields of the life sciences. Structural biology, in particular, has benefitted tremendously from recombinant protein biotechnology, and an overwhelming proportion of the entries in the Protein Data Bank (PDB) are based on heterologously expressed proteins. Nonetheless, synthesizing, purifying and stabilizing recombinant proteins can still be thoroughly challenging. For example, the soluble proteome is organized to a large part into multicomponent complexes (in humans often comprising ten or more subunits), posing critical challenges for recombinant production. A third of all proteins in cells are located in the membrane, and pose special challenges that require a more bespoke approach. Recent advances may now mean that even these most recalcitrant of proteins could become tenable structural biology targets on a more routine basis. In this special issue, we examine progress in key areas that suggests this is indeed the case. Our first contribution examines the importance of understanding quality control in the host cell during recombinant protein production, and pays particular attention to the synthesis of recombinant membrane proteins. A major challenge faced by any host cell factory is the balance it must strike between its own requirements for growth and the fact that its cellular machinery has essentially been hijacked by an expression construct. In this context, Bill and von der Haar examine emerging insights into the role of the dependent pathways of translation and protein folding in defining high-yielding recombinant membrane protein production experiments for the common prokaryotic and eukaryotic expression hosts. Rather than acting as isolated entities, many membrane proteins form complexes to carry out their functions. To understand their biological mechanisms, it is essential to study the molecular structure of the intact membrane protein assemblies. Recombinant production of membrane protein complexes is still a formidable, at times insurmountable, challenge. In these cases, extraction from natural sources is the only option to prepare samples for structural and functional studies. Zorman and co-workers, in our second contribution, provide an overview of recent advances in the production of multi-subunit membrane protein complexes and highlight recent achievements in membrane protein structural research brought about by state-of-the-art near-atomic resolution cryo-electron microscopy techniques. E. coli has been the dominant host cell for recombinant protein production. Nonetheless, eukaryotic expression systems, including yeasts, insect cells and mammalian cells, are increasingly gaining prominence in the field. The yeast species Pichia pastoris, is a well-established recombinant expression system for a number of applications, including the production of a range of different membrane proteins. Byrne reviews high-resolution structures that have been determined using this methylotroph as an expression host. Although it is not yet clear why P. pastoris is suited to producing such a wide range of membrane proteins, its ease of use and the availability of diverse tools that can be readily implemented in standard bioscience laboratories mean that it is likely to become an increasingly popular option in structural biology pipelines. The contribution by Columbus concludes the membrane protein section of this volume. In her overview of post-expression strategies, Columbus surveys the four most common biochemical approaches for the structural investigation of membrane proteins. Limited proteolysis has successfully aided structure determination of membrane proteins in many cases. Deglycosylation of membrane proteins following production and purification analysis has also facilitated membrane protein structure analysis. Moreover, chemical modifications, such as lysine methylation and cysteine alkylation, have proven their worth to facilitate crystallization of membrane proteins, as well as NMR investigations of membrane protein conformational sampling. Together these approaches have greatly facilitated the structure determination of more than 40 membrane proteins to date. It may be an advantage to produce a target protein in mammalian cells, especially if authentic post-translational modifications such as glycosylation are required for proper activity. Chinese Hamster Ovary (CHO) cells and Human Embryonic Kidney (HEK) 293 cell lines have emerged as excellent hosts for heterologous production. The generation of stable cell-lines is often an aspiration for synthesizing proteins expressed in mammalian cells, in particular if high volumetric yields are to be achieved. In his report, Buessow surveys recent structures of proteins produced using stable mammalian cells and summarizes both well-established and novel approaches to facilitate stable cell-line generation for structural biology applications. The ambition of many biologists is to observe a protein's structure in the native environment of the cell itself. Until recently, this seemed to be more of a dream than a reality. Advances in nuclear magnetic resonance (NMR) spectroscopy techniques, however, have now made possible the observation of mechanistic events at the molecular level of protein structure. Smith and colleagues, in an exciting contribution, review emerging ‘in-cell NMR’ techniques that demonstrate the potential to monitor biological activities by NMR in real time in native physiological environments. A current drawback of NMR as a structure determination tool derives from size limitations of the molecule under investigation and the structures of large proteins and their complexes are therefore typically intractable by NMR. A solution to this challenge is the use of selective isotope labeling of the target protein, which results in a marked reduction of the complexity of NMR spectra and allows dynamic processes even in very large proteins and even ribosomes to be investigated. Kerfah and co-workers introduce methyl-specific isotopic labeling as a molecular tool-box, and review its applications to the solution NMR analysis of large proteins. Tyagi and Lemke next examine single-molecule FRET and crosslinking following the co-translational incorporation of non-canonical amino acids (ncAAs); the goal here is to move beyond static snap-shots of proteins and their complexes and to observe them as dynamic entities. The encoding of ncAAs through codon-suppression technology allows biomolecules to be investigated with diverse structural biology methods. In their article, Tyagi and Lemke discuss these approaches and speculate on the design of improved host organisms for ‘integrative structural biology research’. Our volume concludes with two contributions that resolve particular bottlenecks in the protein structure determination pipeline. The contribution by Crepin and co-workers introduces the concept of polyproteins in contemporary structural biology. Polyproteins are widespread in nature. They represent long polypeptide chains in which individual smaller proteins with different biological function are covalently linked together. Highly specific proteases then tailor the polyprotein into its constituent proteins. Many viruses use polyproteins as a means of organizing their proteome. The concept of polyproteins has now been exploited successfully to produce hitherto inaccessible recombinant protein complexes. For instance, by means of a self-processing synthetic polyprotein, the influenza polymerase, a high-value drug target that had remained elusive for decades, has been produced, and its high-resolution structure determined. In the contribution by Desmyter and co-workers, a further, often imposing, bottleneck in high-resolution protein structure determination is addressed: The requirement to form stable three-dimensional crystal lattices that diffract incident X-ray radiation to high resolution. Nanobodies have proven to be uniquely useful as crystallization chaperones, to coax challenging targets into suitable crystal lattices. Desmyter and co-workers review the generation of nanobodies by immunization, and highlight the application of this powerful technology to the crystallography of important protein specimens including G protein-coupled receptors (GPCRs). Recombinant protein production has come a long way since Peter Lobban's hypothesis in the late 1960s, with recombinant proteins now a dominant force in structural biology. The contributions in this volume showcase an impressive array of inventive approaches that are being developed and implemented, ever increasing the scope of recombinant technology to facilitate the determination of elusive protein structures. Powerful new methods from synthetic biology are further accelerating progress. Structure determination is now reaching into the living cell with the ultimate goal of observing functional molecular architectures in action in their native physiological environment. We anticipate that even the most challenging protein assemblies will be tackled by recombinant technology in the near future.

The synthesis of recombinant membrane proteins in yeast for structural studies

Historically, recombinant membrane protein production has been a major challenge meaning that many fewer membrane protein structures have been published than those of soluble proteins. However, there has been a recent, almost exponential increase in the number of membrane protein structures being deposited in the Protein Data Bank. This suggests that empirical methods are now available that can ensure the required protein supply for these difficult targets. This review focuses on methods that are available for protein production in yeast, which is an important source of recombinant eukaryotic membrane proteins. We provide an overview of approaches to optimize the expression plasmid, host cell and culture conditions, as well as the extraction and purification of functional protein for crystallization trials in preparation for structural studies.