Inflammation-induced changes in expression and glycosylation of genetic variants of alpha 1-acid glycoprotein. Studies with human sera, primary cultures of human hepatocytes and transgenic mice.

The relative occurrence of genetic variants of human alpha 1-acid glycoprotein (AGP) in relation to changes in glycosylation was studied in sera of patients with burn injury, media of cytokine-treated primary cultures of human hepatocytes and Hep 3B cells, and sera of transgenic mice expressing the human AGP-A gene. It is concluded (i) that the glycosylation of AGP was not dependent on its genetic expression and (ii) that both the variants determined by the AGP-A gene as well as by the AGP-B/B' genes are increased after inflammation or treatment with interleukins 1 and 6.

Orosomucoid (alpha-1 acid glycoprotein) phenotyping by use of immobilized pH gradients with 8 M urea and immunoblotting. A new variant encountered in a population study.

Orosomucoid (ORM) phenotyping has been performed on 329 unrelated Swiss subjects, using immobilized pH gradients with 8 M urea and 2% v/v 2-mercaptoethanol followed by immunoblotting. After desialylation the band patterns of ORM confirmed that the polymorphism of the structural locus ORM1 is controlled by three codominant autosomal alleles (ORM1*F1, ORM1*S and ORM1*F2). One rare and one new allele were detected. The rare variant, tentatively assigned to the second structural locus ORM2, is observed in a cathodal position and named ORM2 B1. The new variant, tentatively assigned to the first structural locus ORM1, is observed in a region located between ORM1 S and ORM1 F2, and named ORM1 F3. Moreover, the pI values of the ORM variants have been measured accurately with Immobiline Dry Plates (LKB): they were found to be within the pH range 4.93-5.14.

alpha-Smooth muscle actin and proliferating cell nuclear antigen expression in focal segmental glomerulosclerosis: functional and structural parameters of renal disease progression

The aim of the present study was to investigate the expression of alpha-smooth muscle actin (alpha-SM-actin) and proliferating cell nuclear antigen (PCNA) in renal cortex from patients with focal segmental glomerulosclerosis (FSGS) and their correlations with parameters of renal disease progression. We analyzed renal biopsies from 41 patients with idiopathic FSGS and from 14 control individuals. The alpha-SM-actin immunoreaction was evaluated using a score that reflected the changes in the extent and intensity of staining in the glomerular or cortical area. The PCNA reaction was quantified by counting the labeled cells of the glomeruli or renal cortex. The results, reported as median ± percentile (25th; 75th), showed that the alpha-SM-actin scores in the glomeruli and tubulointerstitium from the renal cortex were 2.0 (2.0; 4.0) and 3.0 (3.0; 4.0), respectively, in patients with FSGS, and 0.5 (0.0; 1.0) and 0.0 (0.0; 0.5) in the controls. The number of PCNA-positive cells per glomerulus and graded field of tubulointerstitium from the renal cortex was 0.2 (0.0; 0.4) and 1.1 (0.3; 2.2), respectively, for patients with FSGS, and 0.0 (0.0; 0.5) and 0.0 (0.0; 0.0) for controls. The present data showed an increase of alpha-SM-actin and PCNA expression in glomeruli and renal cortex from FSGS patients. The extent of immunoreaction for alpha-SM-actin in the tubulointerstitial area was correlated with the intensity of proteinuria. However, there was no correlation between the kidney expression of these proteins and the reciprocal of plasma creatinine level or renal fibrosis. These findings suggest that the immunohistochemical alterations may be reversible.

Hydrothermal synthesis and structural characterization of novel alpha-Keggin unit-supported Cu(II)- and Mn(II)-bipyridine complexes from a tri-lacunary precursor

Blue [{Cu(2,2'-bipy)(2)}(2){alpha-SiW12O40}] (bipy = bipyridyl) (1) and pale yellow [Mn(2,2'-bipy)(3)](2)[alpha-SiW12O40] (2) have been synthesized hydrothermally and characterized by IR spectroscopy and single crystal X-ray structure analysis. In 1, the [alpha-SiW12O40](4-) ion acts as a bridge between the two [{Cu(2,2'-bipy)(2)](2+) moieties via coordination through the terminal oxygen atoms, while in 2, the [Mn(2,2'-bipy)(3)](2+) ion balances the charge on the polyoxo anion without forming any covalent bond. To the best of our knowledge, this is the first example of transition metal-mediated transformation of [alpha-SiW9O34](10-) to [alpha-SiW12O40](4-).

Structural basis for the specificity of bipartite nuclear localization sequence binding by importin-alpha

Importin-alpha is the nuclear import receptor that recognizes cargo proteins carrying conventional basic monopartite and bipartite nuclear localization sequences (NLSs) and facilitates their transport into the nucleus. Bipartite NLSs contain two clusters of basic residues, connected by linkers of variable lengths. To determine the structural basis of the recognition of diverse bipartite NLSs by mammalian importin-alpha, we co-crystallized a non-autoinhibited mouse receptor protein with peptides corresponding to the NLSs from human retinoblastoma protein and Xenopus laevis phosphoprotein N1N2, containing diverse sequences and lengths of the linker. We show that the basic clusters interact analogously in both NLSs, but the linker sequences adopt different conformations, whereas both make specific contacts with the receptor. The available data allow us to draw general conclusions about the specificity of NLS binding by importin-alpha and facilitate an improved definition of the consensus sequence of a conventional basic/bipartite NLS (KRX10-12KRRK) that can be used to identify novel nuclear proteins.

Structural Basis of Importin-alpha-Mediated Nuclear Transport for Ku70 and Ku80

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

Molecular models of NS3 protease variants of the Hepatitis C virus

Background: Hepatitis C virus (HCV) currently infects approximately three percent of the world population. In view of the lack of vaccines against HCV, there is an urgent need for an efficient treatment of the disease by an effective antiviral drug. Rational drug design has not been the primary way for discovering major therapeutics. Nevertheless, there are reports of success in the development of inhibitor using a structure-based approach. One of the possible targets for drug development against HCV is the NS3 protease variants. Based on the three-dimensional structure of these variants we expect to identify new NS3 protease inhibitors. In order to speed up the modeling process all NS3 protease variant models were generated in a Beowulf cluster. The potential of the structural bioinformatics for development of new antiviral drugs is discussed.Results: the atomic coordinates of crystallographic structure 1CU1 and 1DY9 were used as starting model for modeling of the NS3 protease variant structures. The NS3 protease variant structures are composed of six subdomains, which occur in sequence along the polypeptide chain. The protease domain exhibits the dual beta-barrel fold that is common among members of the chymotrypsin serine protease family. The helicase domain contains two structurally related beta-alpha-beta subdomains and a third subdomain of seven helices and three short beta strands. The latter domain is usually referred to as the helicase alpha-helical subdomain. The rmsd value of bond lengths and bond angles, the average G-factor and Verify 3D values are presented for NS3 protease variant structures.Conclusions: This project increases the certainty that homology modeling is an useful tool in structural biology and that it can be very valuable in annotating genome sequence information and contributing to structural and functional genomics from virus. The structural models will be used to guide future efforts in the structure-based drug design of a new generation of NS3 protease variants inhibitors. All models in the database are publicly accessible via our interactive website, providing us with large amount of structural models for use in protein-ligand docking analysis.

Initial structural analysis of an alpha(4)beta(4) C-type lectin from the venom of Crotalus durissus terrificus

Convulxin, an alphabeta C-type lectin, is a potent platelet activator isolated from the venom of the South American rattlesnake Crotalus durissus terrificus. It is a 26.5 kDa alphabeta heterodimer consisting of two homologous disulfide-linked chains. The crystals belong to space group I4, with unit-cell parameters a = b = 131.61, c = 121.85 Angstrom, and diffraction data were collected to 2.7 Angstrom. The structure was solved by molecular replacement and the asymmetric unit contains two alphabeta heterodimers, each of which forms a disulfide-linked cyclic alpha(4)beta(4) tetramer in the unit cell. These alpha(4)beta(4) tetramers are stacked to form a large solvent channel.

Tertiary structural changes of the alpha-hemolysin from Staphylococcus aureus on association with liposome membranes

The interaction of alpha-hemolysin (also called alpha-toxin) from Staphylococcus aureus with mixed egg-yolk phosphatidylcholine/cholesterol liposomes has been investigated using the intrinsic tryptophan fluorescence emission (ITFE) signal. The ITFE intensity of alpha-hemolysin, which was obtained using a novel purification protocol, showed a triphasic increase on incubation with liposomes at low protein/lipid ratios. The first, rapid phase results in an increase in ITFE of 10%, which reflects rapid conformation changes in the alpha-hemolysin on association with the liposome membrane, the second phase of the ITFE increase is associated with a red shift from 334 to 339 nm in the maximum emission wavelength, suggesting the transition to a partially unfolded intermediate in the oligomerization process. The third phase of the ITFE intensity change demonstrates a temporal correlation with the appearance of SDS-stable oligomers. The results demonstrate the feasibility of identification of intermediate protein conformations in complex membrane-associated processes by manipulation of the liposomal membrane composition. (C) 1998 Academic Press.

Structural studies of BmooMP alpha-I, a non-hemorrhagic metalloproteinase from Bothrops moojeni venom

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Structural and spectroscopic analysis of gamma-Al2O3 to alpha-Al2O3-CoAl2O4 phase transition

Co-doped alumina powders were synthesized by means of the polymeric precursor method to obtain ceramic pigments. The effect of different contents of Co2+ on phase transition gamma to alpha-Al2O3 and appearing of CoAl2O4 spinel were studied by means of X-ray diffraction. A partial phase diagram of the system CoAl2O3 was proposed from these data by means of determination of the percentages of these phases according to the calcining temperature. Critical particle size to phase transition was determined by means of calculations of crystallite size and determination of superficial area through the BET method. UV-vis spectroscopy of the samples allow to compare the band shift with the phase transition. Besides, a study of thermal stability and intensity of the blue coloration of the synthesized powders with the presence of cobalt in relation to the calcining temperature was accomplished and compared to the phase transition. The results show that the higher blue color intensity was obtained for the powders with Co-doped gamma-Al2O3 closest of phase transition to alpha-Al2O3 + CoAl2O4. (c) 2005 Elsevier B.V. All rights reserved.

Functional and Structural Adaptations in the Pancreatic alpha-Cell and Changes in Glucagon Signaling During Protein Malnutrition

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

Structural studies of Helicase NS3 variants from Hepatitis C virus genotype 3 in virological sustained responder and non-responder patients

Background. About 130 million people are infected with the hepatitis C virus (HCV) worldwide, but effective treatment options are not yet available. One of the most promising targets for antiviral therapy is nonstructural protein 3 (NS3). To identify possible changes in the structure of NS3 associated with virological sustained response or non-response of patients, a model was constructed for each helicase NS3 protein coding sequence. From this, the goal was to verify the interaction between helicases variants and their ligands. Findings. Evidence was found that the NS3 helicase portion of non-responder patients contained substitutions in its ATP and RNA binding sites. K210E substitution can cause an imbalance in the distribution of loads, leading to a decrease in the number of ligations between the essential amino acids required for the hydrolysis of ATP. W501R substitution causes an imbalance in the distribution of loads, leading and forcing the RNA to interact with the amino acid Thr269, but not preventing binding of ribavirin inhibitor. Conclusions. Useful information is provided on the genetic profiling of the HCV genotype 3, specifically the coding region of the NS3 protein, improving our understanding of the viral genome and the regions of its protein catalytic site. © 2010 Rahal et al; licensee BioMed Central Ltd.