Analysis of sequence dependent variations in secondary and tertiary structure of tRNA molecules

The double helical regions of the five tRNA(Phe) and two tRNA(Asp) crystal structures have been analyzed using the local basepair step parameters. The sequence dependent effects in the mini double helices of tRNA are very similar to those observed in the crystal structures of oligonucleotides in the A-form, the purine-pyrimidine and purine-purine steps have small roll angles when compared to the fiber models of A-DNA as well as A-RNA, while the pyrimidine-purine doublet steps have large roll angles. The orientation of the basepairs in the D-stem is unusual and invariant i.e. they are different from the other three stems but are very similar in all the five tRNA(Phe) crystal structures, presumably due to tertiary interaction of the Watson-Crick basepairs with other bases, with all bases being highly conserved. The origin of the differences between the tertiary structures of tRNA(Phe) and tRNA(Asp) from yeast has also been investigated. It is found that even though the angle between the acceptor arm and the D-stem is very similar in the two structures, the angle subtended by the acceptor arm and the anticodon arm is smaller in the tRNA(Phe) structure (by more than 10 degrees). This is due to differences in the orientation of the two mini helices constituting the anticodon arm, which are inclined to each other by approximately 25 degrees in tRNA(Phe) and 16 degrees in tRNA(Asp). In addition, the acceptor arm, the D-stem and the anticodon stem are nearly coplanar in tRNA(Phe), while in tRNA(Asp) the anticodon stem projects out of the plane defined by the acceptor arm and the anticodon stem. These two features together lead to a larger separation between the acceptor and anticodon ends in tRNA(Asp) and indicate that the junction between the D-stem and the anticodon stem is quite variable, with features characteristic of a ball-and-socket type joint and determined for each tRNA molecule by the base sequence at the junction.

Synthesis and Structure-Activity Correlation Studies of Secondary- and Tertiary-Amine-Based Glutathione Peroxidase Mimics

In this study, a series of seeondary- and tertiary-amino-substituted diaryl diselenides were synthesized and studied for their glutathione peroxidase (GPx) like antioxidant activities with H2O2, cumene hydroperoxide, or tBuOOH as substrates and with PhSH or glutathione (GSH) as thiol cosubstrates. This study reveals that replacement of the tert-amino groups in benzylamine-based diselenides by sec-amino moieties drastically enhances the catalytic activities in both the aromatic thiol (PhSH) and GSH assay systems. Particularly, the N-propyl- and N-isopropylamino-substituted diselenides are 8-18 times more active than the corresponding N,N-dipropyl- and N,N-diisopropylamine-based compounds in all three peroxide systems when GSH is used as the thiol cosubstrate. Although the catalytic mechanism of sec-amino-substituted disclenides is similar to that of the tert-amine-based compounds, differences in the stability and reactivity of some of the key intermediates account for the differences in the GPx-like activities. it is observed that the sec-amino groups are better than the tert-amino moieties for generating the catalytically active selenols. This is due to the absence of any significant thiol-exchange reactions in the selenenyl sulfides derived from sec-amine-based diselenides. Furthermore, the seleninic acids (RSeO2H) derived from the sec-amine-based compounds are more stable toward further reactions with peroxides than their tert-amine-based analogues.

A novel short neurotoxin, cobrotoxin c, from monocellate cobra (Naja kaouthia) venom: isolation and purification, primary and secondary structure determination, and tertiary structure modeling

A novel short neurotoxin, cobrotoxin c (CBT C) was isolated from the venom of monocellate cobra (Naja kaouthia) using a combination of ion-exchange chromatography and FPLC. Its primary structure was determined by Edman degradation. CBT C is composed of 61 amino acid residues. It differs from cobrotoxin b (CBT B) by only two amino acid substitutions, Thr/Ala11 and Arg/Thr56, which are not located on the functionally important regions by sequence similarity. However, the LD50 is 0.08 mg/g to mice, i.e. approximately five-fold higher than for CBT B. Strikingly, a structure-function relationship analysis suggests the existence of a functionally important domain on the outside of Loop III of CBT C. The functionally important basic residues on the outside of Loop III might have a pairwise interaction with alpha subunit, instead of gamma or delta subunits of the nicotinic acetylcholine receptor (nAChR). (C) 2002 Elsevier Science Inc. All rights reserved.

Amide-Based Glutathione Peroxidase Mimics: Effect of Secondary and Tertiary Amide Substituents on Antioxidant Activity

A series of secondary and tertiary amide-substituted diselenides were synthesized and studied for their GPx-like antioxidant activities using H2O2 Cum-OOH, and tBuOOH as substrates and PhSH as thiol co-substrate.The effect of substitution at the free -NH group of the amide moiety in the sec-amide-based diselenides on GPx activity was analyzed by detailed experimental and theoretical methods. It is observed that substitution at the free -NH group significantly enhances the GPx-like activities of the sec-amide-based diselenides, mainly by reducing the Se center dot center dot center dot O nonbonded interactions. The reduction in strength of the Se center dot center dot center dot O interaction upon introduction of N,N-dialkyl substituents not only prevents the undesired thiol exchange reactions, but also reduces the stability of selenenyl sulfide intermediates. This leads to a facile disproportionation of the selenenyl sulfide to the corresponding diselenide, which enhances the catalytic activity. The mechanistic investigations indicate that the reactivity of diselenides having sec-or tert-amide moieties with PhSH is extremely slow; indicating that the first step of the catalytic cycle involves the reaction between the diselenides and peroxide to produce the corresponding selenenic and seleninic acids.

Reorganization of secondary and tertiary health care levels: impact on the outcomes of oral cancer screening in the São Paulo State, Brazil

This study presents the strategies for prevention and early detection of oral cancer by means of screening in the elderly population of São Paulo, the richest and the most populous state of Brazil. This research was a retrospective longitudinal study based on the analysis of secondary data. The variables - number of participating cities, coverage of screening, and number of suspicious and confirmed cases of oral cancer - were divided into two periods: 2001-2004 and 2005-2008. Data were analyzed statistically by the chi-square test at 5% significance level. The implementation of a nationwide public oral health policy in 2004 and the reorganization of the secondary and tertiary health care were evaluated as mediator factors able to interfere in the achieved outcomes. From 2001 to 2008, 2,229,273 oral examinations were performed. There was an addition of 205 participating cities by the end of the studied period (p<0.0001). The coverage of oral cancer screening increased from 4.1% to 16% (p<0.0001). There was a decrease in the number of suspicious lesions (from 9% in 2005 to 5% in 2008) (p<0.0001) and in the rate of confirmed oral cancer cases per 100,000 examinations (from 20.89 in 2001 to 10.40 in 2008) (p<0.0001). After 8 years of screening, there was a decrease in the number of suspicious lesions and confirmed cases of oral cancer in the population. The reorganization of secondary and tertiary health care levels of oral care seems to have contributed to modify these numbers, having a positive impact on the outcomes of oral cancer screening in the São Paulo State.

Segmented filamentous bacterium uses secondary and tertiary lymphoid tissues to induce gut IgA and specific T helper 17 cell responses.

Segmented filamentous bacterium (SFB) is a symbiont that drives postnatal maturation of gut adaptive immune responses. In contrast to nonpathogenic E. coli, SFB stimulated vigorous development of Peyer's patches germinal centers but paradoxically induced only a low frequency of specific immunoglobulin A (IgA)-secreting cells with delayed accumulation of somatic mutations. Moreover, blocking Peyer's patch development abolished IgA responses to E. coli, but not to SFB. Indeed, SFB stimulated the postnatal development of isolated lymphoid follicles and tertiary lymphoid tissue, which substituted for Peyer's patches as inductive sites for intestinal IgA and SFB-specific T helper 17 (Th17) cell responses. Strikingly, in mice depleted of gut organized lymphoid tissue, SFB still induced a substantial but nonspecific intestinal Th17 cell response. These results demonstrate that SFB has the remarkable capacity to induce and stimulate multiple types of intestinal lymphoid tissues that cooperate to generate potent IgA and Th17 cell responses displaying only limited target specificity.

SAXS Studies of the Endoglucanase Cel12A from Gloeophyllum trabeum Show Its Monomeric Structure and Reveal the Influence of Temperature on the Structural Stability of the Enzyme

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Apocynin: Chemical and biophysical properties of a NADPH oxidase inhibitor

Apocynin is the most employed inhibitor of NADPH oxidase (NOX), a multienzymatic complex capable of catalyzing the one-electron reduction of molecular oxygen to the superoxide anion. Despite controversies about its selectivity, apocynin has been used as one of the most promising drugs in experimental models of inflammatory and neurodegenerative diseases. Here, we aimed to study the chemical and biophysical properties of apocynin. The oxidation potential was determined by cyclic voltammetry (Epa = 0.76V), the hydrophobicity index was calculated (logP = 0.83) and the molar absorption coefficient was determined (ε275nm = 1.1 × 104 M-1 cm-1). Apocynin was a weak free radical scavenger (as measured using the DPPH, peroxyl radical and nitric oxide assays) when compared to protocatechuic acid, used here as a reference antioxidant. On the other hand, apocynin was more effective than protocatechuic acid as scavenger of the non-radical species hypochlorous acid. Apocynin reacted promptly with the non-radical reactive species H2O2 only in the presence of peroxidase. This finding is relevant, since it represents a new pathway for depleting H2O2 in cellular experimental models, besides the direct inhibition of NADPH oxidase. This could be relevant for its application as an inhibitor of NOX4, since this isoform produces H 2O2 and not superoxide anion. The binding parameters calculated by fluorescence quenching showed that apocynin binds to human serum albumin (HSA) with a binding affinity of 2.19 × 104 M -1. The association did not alter the secondary and tertiary structure of HSA, as verified by synchronous fluorescence and circular dichroism. The displacement of fluorescent probes suggested that apocynin binds to site I and site II of HSA. Considering the current biomedical applications of this phytochemical, the dissemination of these chemical and biophysical properties can be very helpful for scientists and physicians interested in the use of apocynin.

Chemical and thermal influence of the [4Fe-4S](2+) cluster of A/G-specific adenine glycosylase from Corynebacterium pseudotuberculosis

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

From residue matching patterns to protein folding topographies: General model and bovine pancreatic trypsin inhibitor

A coarse-grained model for protein-folding dynamics is introduced based on a discretized representation of torsional modes. The model, based on the Ramachandran map of the local torsional potential surface and the class (hydrophobic/polar/neutral) of each residue, recognizes patterns of both torsional conformations and hydrophobic-polar contacts, with tolerance for imperfect patterns. It incorporates empirical rates for formation of secondary and tertiary structure. The method yields a topological representation of the evolving local torsional configuration of the folding protein, modulo the basins of the Ramachandran map. The folding process is modeled as a sequence of transitions from one contact pattern to another, as the torsional patterns evolve. We test the model by applying it to the folding process of bovine pancreatic trypsin inhibitor, obtaining a kinetic description of the transitions between the contact patterns visited by the protein along the dominant folding pathway. The kinetics and detailed balance make it possible to invert the result to obtain a coarse topographic description of the potential energy surface along the dominant folding pathway, in effect to go backward or forward between a topological representation of the chain conformation and a topographical description of the potential energy surface governing the folding process. As a result, the strong structure-seeking character of bovine pancreatic trypsin inhibitor and the principal features of its folding pathway are reproduced in a reasonably quantitative way.

Structural studies of p21Waf1/Cip1/Sdi1 in the free and Cdk2-bound state: conformational disorder mediates binding diversity.

The cyclin-dependent kinase (Cdk) inhibitor p21Waf1/Cip1/Sdi1, important for p53-dependent cell cycle control, mediates G1/S arrest through inhibition of Cdks and possibly through inhibition of DNA replication. Cdk inhibition requires a sequence of approximately 60 amino acids within the p21 NH2 terminus. We show, using proteolytic mapping, circular dichroism spectropolarimetry, and nuclear magnetic resonance spectroscopy, that p21 and NH2-terminal fragments that are active as Cdk inhibitors lack stable secondary or tertiary structure in the free solution state. In sharp contrast to the disordered free state, however, the p21 NH2 terminus adopts an ordered stable conformation when bound to Cdk2, as shown directly by NMR spectroscopy. We have, thus, identified a striking disorder-order transition for p21 upon binding to one of its biological targets, Cdk2. This structural transition has profound implications in light of the ability of p21 to bind and inhibit a diverse family of cyclin-Cdk complexes, including cyclin A-Cdk2, cyclin E-Cdk2, and cyclin D-Cdk4. Our findings suggest that the flexibility, or disorder, of free p21 is associated with binding diversity and offer insights into the role for structural disorder in mediating binding specificity in biological systems. Further, these observations challenge the generally accepted view of proteins that stable secondary and tertiary structure are prerequisites for biological activity and suggest that a broader view of protein structure should be considered in the context of structure-activity relationships.