Proteomic analysis of arginine adducts on glyoxal-modified ribonuclease

Accumulation of advanced glycation end-products (AGEs) on proteins is associated with the development of diabetic complications. Although the overall extent of modification of protein by AGEs is limited, localization of these modifications at a few critical sites might have a significant effect on protein structure and function. In the present study, we describe the sites of modification of RNase by glyoxal under physiological conditions. Arg(39) and Arg(85), which are closest to the active site of the enzyme, were identified as the primary sites of formation of the glyoxal-derived dihydroxyimidazolidine and hydroimidazolone adducts. Lower amounts of modification were detected at Arg(10), while Arg(33) appeared to be unmodified. We conclude that dihydroxyimidazolidine adducts are the primary products of modification of protein by glyoxal, that Arg(39) and Arg(85) are the primary sites of modification of RNase by glyoxal, and that modification of arginine residues during Maillard reactions of proteins is a highly selective process.

Electrophysiological correlates of spatial orienting towards angry faces: a source localization study

The goal of this study was to examine behavioral and electrophysiological correlates of involuntary orienting toward rapidly presented angry faces in non-anxious, healthy adults using a dot-probe task in conjunction with high-density event-related potentials and a distributed source localization technique. Consistent with previous studies, participants showed hypervigilance toward angry faces, as indexed by facilitated response time for validly cued probes following angry faces and an enhanced P1 component. An opposite pattern was found for happy faces suggesting that attention was directed toward the relatively more threatening stimuli within the visual field (neutral faces). Source localization of the P1 effect for angry faces indicated increased activity within the anterior cingulate cortex, possibly reflecting conflict experienced during invalidly cued trials. No modulation of the early C1 component was found for affect or spatial attention. Furthermore, the face-sensitive N170 was not modulated by emotional expression. Results suggest that the earliest modulation of spatial attention by face stimuli is manifested in the P1 component, and provide insights about mechanisms underlying attentional orienting toward cues of threat and social disapproval.

Functional analysis of a novel missense NBC1 mutation and of other mutations causing proximal renal tubular acidosis

Mutations in the Na+-HCO3- cotransporter NBC1 cause severe proximal tubular acidosis (pRTA) associated with ocular abnormalities. Recent studies have suggested that at least some NBC1 mutants show abnormal trafficking in the polarized cells. This study identified a new homozygous NBC1 mutation (G486R) in a patient with severe pRTA. Functional analysis in Xenopus oocytes failed to detect the G486R activity due to poor surface expression. In ECV304 cells, however, G486R showed the efficient membrane expression, and its transport activity corresponded to approximately 50% of wild-type (WT) activity. In Madin-Darby canine kidney (MDCK) cells, G486R was predominantly expressed in the basolateral membrane domain as observed for WT. Among the previously identified NBC1 mutants that showed poor surface expression in oocytes, T485S showed the predominant basolateral expression in MDCK cells. On the other hand, L522P was exclusively retained in the cytoplasm in ECV304 and MDCK cells, and functional analysis in ECV304 cells failed to detect its transport activity. These results indicate that G486R, like T485S, is a partial loss of function mutation without major trafficking abnormalities, while L522P causes the clinical phenotypes mainly through its inability to reach the plasma membranes. Multiple experimental approaches would be required to elucidate potential disease mechanism by NBC1 mutations.

Comparing detection and location performance of perpendicular and parallel broadside GPR antenna orientations

GPR (Ground Penetrating Radar) results are shown for perpendicular broadside and parallel broadside antenna orientations. Performance in detection and localization of concrete tubes and steel tanks is compared as a function of acquisition configuration. The comparison is done using 100 MHz and 200 MHz center frequency antennas. All tubes and tanks are buried at the geophysical test site of IAG/USP in Sao Paulo city, Brazil. The results show that the long steel pipe with a 38-mm diameter was well detected with the perpendicular broadside configuration. The concrete tubes were better detected with the parallel broadside configuration, clearly showing hyperbolic diffraction events from all targets up to 2-m depth. Steel tanks were detected with the two configurations. However, the parallel broadside configuration was generated to a much lesser extent an apparent hyperbolic reflection corresponding to constructive interference of diffraction hyperbolas of adjacent targets placed at the same depth. Vertical concrete tubes and steel tanks were better contained with parallel broadside antennas, where the apexes of the diffraction hyperbolas better corresponded to the horizontal location of the buried target disposition. The two configurations provide details about buried targets emphasizing how GPR multi-component configurations have the potential to improve the subsurface image quality as well as to discriminate different buried targets. It is judged that they hold some applicability in geotechnical and geoscientific studies. (C) 2009 Elsevier B.V. All rights reserved.

Regulation of Multidrug Resistance-Associated Protein 2 by Calcium Signaling in Mouse Liver

Mu hiding resistance associated protein 2 (Mrp2) is a canalicular transporter responsible for organic anion secretion into bile. Mrp2 activity is regulated by insertion into the plasma membrane; however, the factors that control this are not understood. Calcium (Ca(2+)) signaling regulates exocytosis of vesicles in most cell types, and the type II inositol 1,4,5-triphosphate receptor (InsP(3)R2) regulates Ca(2+) release in the canalicular region of hepatocytes. However, the role of InsP(3)R2 and of Ca(2+) signals in canalicular insertion and function of Mrp2 is not known. The aim of this study was to determine the role of InsP(3)R2-mediated Ca(2+) signals in targeting Mrp2 to the canalicular membrane. Livers, isolated hepatocytes, and hepatocytes in collagen sandwich culture from wild-type (WT) and InsP(3)R2 knockout (KO) mice were used for western blots, confocal immunofluorescence, and time-lapse imaging of Ca(2+) signals and of secretion of a fluorescent organic anion. Plasma membrane insertion of green fluorescent protein (GFP)-Mrp2 expressed in HepG2 cells was monitored by total internal reflection microscopy. InsP(3)R2 was concentrated in the canalicular region of WT mice but absent in InsP(3)R2 KO livers, whereas expression and localization of InsP(3)R1 was preserved, and InsP(3)R3 was absent from both WT and KO livers. Ca(2+) signals induced by either adenosine triphosphate (ATP) or vasopressin were impaired in hepatocytes lacking InsP(3)R2. Canalicular secretion of the organic anion 5-chloromethylfluorescein diacetate (CMFDA) was reduced in KO hepatocytes, as well as in WT hepatocytes treated with 1,2-bis(o-aminophenoxy)ethane-N,N,N`,N`-tetra-acetic acid (BAPTA). Moreover, the choleretic effect of tauroursodeoxycholic acid (TUDCA) was impaired in InsP(3)R2 KO mice. Finally, ATP increased GFP-Mrp2 fluorescence in the plasma membrane of HepG2 cells, and this also was reduced by BAPTA. Conclusion: InsP(3)R2-mediated Ca(2+) signals enhance organic anion secretion into bile by targeting Mrp2 to the canalicular membrane. (HEPATOLOGY 2010;52:327-337)

Interaction of Mycoplasma genitalium with host cells: evidence for nuclear localization

Mycoplasma genitalium (Mg) is a mollicute that causes a range of human urogenital infections. A hallmark of these bacteria is their ability to establish chronic infections that can persist despite completion of appropriate antibiotic therapies and intact and functional immune systems. Intimate adherence and surface colonization of mycoplasmas to host cells are important pathogenic features. However, their facultative intracellular nature is poorly understood, partly due to difficulties in developing and standardizing cellular interaction model systems. Here, we characterize growth and invasion properties of two Mg strains (G37 and 1019V). Mg G37 is a high-passage laboratory strain, while Mg 1019V is a low-passage isolate recovered from the cervix. The two strains diverge partially in gene sequences for adherence-related proteins and exhibit subtle variations in their axenic growth. However, with both strains and consistent with our previous studies, a subset of adherent Mg organisms invade host cells and exhibit perinuclear targeting. Remarkably, intranuclear localization of Mg proteins is observed, which occurred as early as 30 min after infection. Mg strains deficient in adherence were markedly reduced in their ability to invade and associate with perinuclear and nuclear sites.

Structural modeling and mutational analysis of yeast eukaryotic translation initiation factor 5A reveal new critical residues and reinforce its involvement in protein synthesis

Eukaryotic translation initiation factor 5A (eIF5A) is a protein that is highly conserved and essential for cell viability. This factor is the only protein known to contain the unique and essential amino acid residue hypusine. This work focused on the structural and functional characterization of Saccharomyces cerevisiae eIF5A. The tertiary structure of yeast eIF5A was modeled based on the structure of its Leishmania mexicana homologue and this model was used to predict the structural localization of new site-directed and randomly generated mutations. Most of the 40 new mutants exhibited phenotypes that resulted from eIF-5A protein-folding defects. Our data provided evidence that the C-terminal alpha-helix present in yeast eIF5A is an essential structural element, whereas the eIF5A N-terminal 10 amino acid extension not present in archaeal eIF5A homologs, is not. Moreover, the mutants containing substitutions at or in the vicinity of the hypusine modification site displayed nonviable or temperature-sensitive phenotypes and were defective in hypusine modification. Interestingly, two of the temperature-sensitive strains produced stable mutant eIF5A proteins - eIF5A(K56A) and eIF5A(Q22H,L93F)- and showed defects in protein synthesis at the restrictive temperature. Our data revealed important structural features of eIF5A that are required for its vital role in cell viability and underscored an essential function of eIF5A in the translation step of gene expression.

Structural modeling and mutational analysis of yeast eukaryotic translation initiation factor 5A reveal new critical residues and reinforce its involvement in protein synthesis

Eukaryotic translation initiation factor 5A (eIF5A) is a protein that is highly conserved and essential for cell viability. This factor is the only protein known to contain the unique and essential amino acid residue hypusine. This work focused on the structural and functional characterization of Saccharomyces cerevisiae eIF5A. The tertiary structure of yeast eIF5A was modeled based on the structure of its Leishmania mexicana homologue and this model was used to predict the structural localization of new site-directed and randomly generated mutations. Most of the 40 new mutants exhibited phenotypes that resulted from eIF-5A protein-folding defects. Our data provided evidence that the C-terminal alpha-helix present in yeast eIF5A is an essential structural element, whereas the eIF5A N-terminal 10 amino acid extension not present in archaeal eIF5A homologs, is not. Moreover, the mutants containing substitutions at or in the vicinity of the hypusine modification site displayed nonviable or temperature-sensitive phenotypes and were defective in hypusine modification. Interestingly, two of the temperature-sensitive strains produced stable mutant eIF5A proteins - eIF5A(K56A) and eIF5A(Q22H,L93F)- and showed defects in protein synthesis at the restrictive temperature. Our data revealed important structural features of eIF5A that are required for its vital role in cell viability and underscored an essential function of eIF5A in the translation step of gene expression.

Wannier-function approach to electron states in superlattices under an electric field

Electron quasi-stationary states in a periodic semiconductor superlattice are calculated, as linear combinations of Wannier-Kohn functions, for different values of an electric field applied along the heterostructure. A comparison with an alternative approach, which is based on the localization of quasi-stationary states, is performed. (C) 2004 Elsevier Ltd. All rights reserved.

Towards the prediction of essential genes by integration of network topology, cellular localization and biological process information

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

Somatic cell nuclear transfer is associated with altered expression of angiogenic factor systems in bovine placentomes at term

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

Influence of different doses of retinoic acid on cardiac remodeling

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Conservation of the 5S-bearing chromosome pair and co-localization with major rDNA clusters in five species of Astyanax (Pisces, Characidae)

Major and 5S ribosomal genes have been localized in chromosomes from five fish species, genus Aslyanax, using in situ hybridization (FISH) with 28S and 5S rDNA probes. In situ signals for the major rDNA co-localized with the 5S rDNA clusters in the pericentromeric region of one marker chromosome in all five species analyzed. The conserved localization of these two rDNA clusters in the five related Astyanax species was considered as indicative of a close relationship among them. The use of these molecular markers for elucidating evolutionary relationships among closely related taxa is discussed. Copyright (C) 2002 S. Karger AG, Basel.

Topological K-equivalence of analytic function-germs

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Rhipicephalus (Boophilus) microplus (Canestrini, 1887) (Acari: Ixodidae): Acid phosphatase and ATPase activities localization in salivary glands of females during the feeding period

This study investigates the presence and the localization of acid phosphatase and ATPase in the salivary glands of Rhipicephalus (Boophilus) microplus female ticks during feeding. Semi-engorged females showed a larger amount of acid phosphatase compared to those at beginning of feeding, localized mainly in the apical portion of the secretory cells, and in the basal labyrinth of the interstitial cells. Ultrastructural observations also demonstrated its presence in secretion granules and inside some nuclei of secretory cells at beginning of feeding. Acid phosphatase in a free form probably has a hemolymph and/or ribosomal origin and participates in salivary gland secretion control. ATPase was detected in basal membrane of all types of acini and/or in the cytoplasm of the secretory cells at both feeding stages. The enzyme activities found strongly suggests that cell death by apoptosis occurs during the degenerative process. © 2006 Elsevier Inc. All rights reserved.