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Resultados filtrados por Publicador: The National Academy of Sciences

‣ The C-terminal domain of the regulatory protein NOVH is sufficient to promote interaction with fibulin 1C: A clue for a role of NOVH in cell-adhesion signaling

Perbal, Bernard; Martinerie, Cécile; Sainson, Richard; Werner, Michel; He, Bin; Roizman, Bernard
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 02/02/1999 Português
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The NOVH protein belongs to the emerging CCN [Connective tissue growth factor (CTGF), Cyr61/Cef10, nephroblastoma overexpressed gene] family of growth regulators sharing a strikingly conserved multimodular organization but exhibiting distinctive functional features. Two members of the family (CYR61 and CTGF) are positive regulators of cell proliferation, whereas NOVH and two other members (ELM1 and RCOP-1) exhibit features of negative regulators of growth. The multimodular structure of these proteins suggests that their biological role(s) may depend on interactions with several factors as well as proteins constitutive of the extracellular matrix. To gain insight into the functionality of these domains, we have used a two-hybrid system to identify proteins interacting with NOVH. We report here that the C-terminal domain confers on the full-length NOVH protein the capacity to bind fibulin 1C, a protein of the extracellular matrix that interacts with several other regulators of cell adhesion. Furthermore, we show that a natural N-truncated isoform of NOVH produced by cells expressing the full-length NOVH protein also binds fibulin 1C with a high affinity, and we hypothesize that the production of truncated isoforms of NOVH (and probably of other CCN proteins) may be a critical aspect in the modulation of their biological activity. These results set the stage for a study of NOVH–fibulin 1C interactions and their potential significance in cell-adhesion signaling in normal and pathological conditions.

‣ Structure-activity analysis of synthetic autoinducing thiolactone peptides from Staphylococcus aureus responsible for virulence

Mayville, Patricia; Ji, Guangyong; Beavis, Ronald; Yang, Hongmei; Goger, Michael; Novick, Richard P.; Muir, Tom W.
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 16/02/1999 Português
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451.23344%
The synthesis of virulence factors and other extracellular proteins responsible for pathogenicity in Staphylococcus aureus is under the control of the agr locus. A secreted agr-encoded peptide, AgrD, processed from the AgrD gene product, is known to be an effector of self-strain activation and cross-strain inhibition of the agr response. Biochemical analysis of AgrD peptides isolated from culture supernatants has suggested that they contain an unusual thiol ester-linked cyclic structure. In the present work, chemical synthesis is used to confirm that the mature AgrD peptides contain a thiolactone structure and that this feature is absolutely necessary for full biological activity. The AgrD synthetic thiolactone peptides exhibited biological activity in vivo in a mouse protection test. Structure-activity studies have allowed key aspects of the peptide structure involved in the differential activation and inhibition functions to be identified. Accordingly, we propose a model for activation and inhibition of the agr response in which the former, but not the latter, involves specific acylation of the agr transmembrane receptor, AgrC.

‣ On the mechanism by which vascular endothelial cells regulate their oxygen consumption

Clementi, Emilio; Brown, Guy Charles; Foxwell, Neale; Moncada, Salvador
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 16/02/1999 Português
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451.23344%
Two enzymes, soluble guanylyl cyclase and cytochrome c oxidase, have been shown to be exquisitely sensitive to nitric oxide (NO) at low physiological concentrations. Activation of the soluble guanylyl cyclase by endogenous NO and the consequent increase in the second messenger cyclic GMP are now known to control a variety of biological functions. Cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain, is inhibited by NO. However, it is not clear whether NO produced by the constitutive NO synthase interacts with cytochrome c oxidase, nor is it known what the biological consequences of such an interaction might be. We now show that NO generated by vascular endothelial cells under basal and stimulated conditions modulates the respiration of these cells in response to acute changes in oxygen concentration. This action occurs at the cytochrome c oxidase and depends on influx of calcium. Thus, NO plays a physiological role in adjusting the capacity of this enzyme to use oxygen, allowing endothelial cells to adapt to acute changes in their environment.

‣ A strategy for rapid sequencing of heparan sulfate and heparin saccharides

Turnbull, Jeremy E.; Hopwood, John J.; Gallagher, John T.
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 16/03/1999 Português
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Sulfated glycosaminoglycans (GAGs) are linear polysaccharides of repeating disaccharide sequences on which are superimposed highly complex and variable patterns of sulfation, especially in heparan sulfate (HS). HS and the structurally related heparin exert important biological functions, primarily by interacting with proteins and regulating their activities. Evidence is accumulating that these interactions depend on specific saccharide sequences, but the lack of simple, direct techniques for sequencing GAG saccharides has been a major obstacle to progress. We describe how HS and heparin saccharides can be sequenced rapidly by using an integrated strategy with chemical and enzymic steps. Attachment of a reducing-end fluorescent tag establishes a reading frame. Partial selective chemical cleavage at internal N-sulfoglucosamine residues with nitrous acid then creates a set of fragments of defined sizes. Subsequent digestion of these fragments with combinations of exosulfatases and exoglycosidases permits the selective removal of specific sulfates and monosaccharides from their nonreducing ends. PAGE of the products yields a pattern of fluorescent bands from which the saccharide sequence can be read directly. Data are presented on sequencing of heparin tetrasaccharides and hexasaccharides of known structure; these data show the accuracy and versatility of this sequencing strategy. Data also are presented on the application of the strategy to the sequencing of an HS decasaccharide of unknown structure. Application and further development of this sequencing strategy...

‣ A synthetic inhibitor of histone deacetylase, MS-27-275, with marked in vivo antitumor activity against human tumors

Saito, Akiko; Yamashita, Takashi; Mariko, Yukiyasu; Nosaka, Yasuhito; Tsuchiya, Katsutoshi; Ando, Tomoyuki; Suzuki, Tsuneji; Tsuruo, Takashi; Nakanishi, Osamu
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 13/04/1999 Português
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451.23344%
Synthetic benzamide derivatives were investigated for their ability to inhibit histone deacetylase (HDA). In this study, one of the most active benzamide derivatives, MS-27-275, was examined with regard to its biological properties and antitumor efficacy. MS-27-275 inhibited partially purified human HDA and caused hyperacetylation of nuclear histones in various tumor cell lines. It behaved in a manner similar to other HDA inhibitors, such as sodium butyrate and trichostatin A; MS-27-275 induced p21WAF1/CIP1 and gelsolin and changed the cell cycle distribution, decrease of S-phase cells, and increase of G1-phase cells. The in vitro sensitivity spectrum of MS-27-275 against various human tumor cell lines showed a pattern different than that of a commonly used antitumor agent, 5-fluorouracil, and, of interest, the accumulation of p21WAF1/CIP1 tended to be faster and greater in the cell lines sensitive to MS-27-275. MS-27-275 administered orally strongly inhibited the growth in seven of eight tumor lines implanted into nude mice, although most of these did not respond to 5-fluorouracil. A structurally analogous compound to MS-27-275 without HDA-inhibiting activity showed neither the biological effects in cell culture nor the in vivo therapeutic efficacy. These results suggest that MS-27-275 acts as an antitumor agent through HDA inhibition and may provide a novel chemotherapeutic strategy for cancers insensitive to traditional antitumor agents.

‣ Assembly and activation of site-specific recombination complexes

Peña, Carol E. A.; Kahlenberg, J. Michelle; Hatfull, Graham F.
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
451.23344%
Site-specific recombination is responsible for a broad range of biological phenomena, including DNA inversion, resolution of transposition intermediates, and the integration and excision of bacteriophage genomes. Integration of mycobacteriophage L5 is catalyzed by a phage-encoded integrase with recombination occurring between specific attachment sites on the phage and mycobacterial chromosomes (attP and attB, respectively). Although some site-specific recombination systems simply involve binding of the recombinase to the sites of strand exchange, synapsis, and recombination, phage systems typically require the assembly of higher-order structures within which the recombinational potential of integrase is activated. The requirement for these structures derives from the necessity to regulate the directionality of recombination—either integration or excision—which must be closely coordinated with other aspects of the phage growth cycles. We show herein that there are multiple pathways available for the assembly of L5 recombination complexes, including the early synapsis of the attP and attB DNAs. This process is in contrast to the model for lambda integration and illustrates the different usage of molecular machineries to accomplish the same biological outcome.

‣ Demonstration of the in vivo interaction of key cell death regulators by structure-based design of second-site suppressors

Parrish, Jay; Metters, Helen; Chen, Lin; Xue, Ding
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
451.23344%
Demonstrating in vivo interaction of two important biomolecules and the relevance of the interaction to a biological process have been difficult issues in biomedical research. Here, we report the use of a homology modeling approach to establish the significance of protein interactions in governing the activation of programmed cell death in Caenorhabditis elegans. A protein interaction cascade has been postulated to mediate activation of cell death in nematodes, in which the BH3-domain-containing (Bcl-2 homology region 3) protein EGL-1 binds the cell-death inhibitor CED-9 and induces release of the death-activating protein CED-4 from inhibitory CED-4/CED-9 complexes. We show here that an unusual gain-of-function mutation in ced-9 (substitution of glycine 169 to glutamate) that results in potent inhibition of most nematode cell deaths impairs the binding of EGL-1 to CED-9 and EGL-1-induced release of CED-4 from CED-4/CED-9 complexes. Based on a modeled EGL-1/CED-9 complex structure, we generated second-site compensatory mutations in EGL-1 that partially restore the binding of EGL-1 to CED-9(G169E) and EGL-1-induced release of CED-4 from CED-4/CED-9(G169E) complexes. Importantly, these mutations also significantly suppress the death-protective activity of CED-9(G169E) in vivo. These results establish that direct physical interaction between EGL-1 and CED-9 is essential for the release of CED-4 and the activation of cell death. The structure-based design of second-site suppressors via homology modeling should be widely applicable for probing important molecular interactions that are implicated in fundamental biological processes.

‣ Coupled two-way clustering analysis of gene microarray data

Getz, Gad; Levine, Erel; Domany, Eytan
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Português
Relevância na Pesquisa
451.23344%
We present a coupled two-way clustering approach to gene microarray data analysis. The main idea is to identify subsets of the genes and samples, such that when one of these is used to cluster the other, stable and significant partitions emerge. The search for such subsets is a computationally complex task. We present an algorithm, based on iterative clustering, that performs such a search. This analysis is especially suitable for gene microarray data, where the contributions of a variety of biological mechanisms to the gene expression levels are entangled in a large body of experimental data. The method was applied to two gene microarray data sets, on colon cancer and leukemia. By identifying relevant subsets of the data and focusing on them we were able to discover partitions and correlations that were masked and hidden when the full dataset was used in the analysis. Some of these partitions have clear biological interpretation; others can serve to identify possible directions for future research.