Sample records for a1 iga1 protease

  1. Lack of cleavage of immunoglobulin A (IgA) from rhesus monkeys by bacterial IgA1 proteases.

    PubMed Central

    Reinholdt, J; Kilian, M

    1991-01-01

    Bacterial immunoglobulin A1 (IgA1) proteases cleaving IgA1 and secretory IgA1 molecules in the hinge region are believed to be important virulence factors. Previous studies have indicated that IgA of humans, gorillas, and chimpanzees are the exclusive substrates of these enzymes. In a recent study, IgA from the rhesus monkey was found to be susceptible to the IgA1 protease activity of Streptococcus pneumoniae. In an attempt to reproduce this observation, we found that neither five isolates of S. pneumoniae nor other IgA1 protease-producing bacteria representing different cleavage specificities caused cleavage of rhesus monkey IgA. Hence, the rhesus monkey does not appear to be a suitable animal model for studies of IgA1 proteases as virulence factors. Images PMID:2037384

  2. Serological Analysis of Immunogenic Properties of Recombinant Meningococcus IgA1 Protease-Based Proteins.

    PubMed

    Kotelnikova, O V; Zinchenko, A A; Vikhrov, A A; Alliluev, A P; Serova, O V; Gordeeva, E A; Zhigis, L S; Zueva, V S; Razgulyaeva, O A; Melikhova, T D; Nokel, E A; Drozhzhina, E Yu; Rumsh, L D

    2016-07-01

    Using the genome sequence of IgA1 protease of N. meningitidis of serogroup B, four recombinant proteins of different structure and molecular weight were constructed. These proteins were equal in inducing the formation of specific antibodies to IgA1 protease and had protective properties against meningococci. In the sera of immunized mice, anti-IgA1 protease antibodies were detected by whole-cell ELISA, which indicated the presence of IgA1 protease on the surface of these bacteria. We hypothesized that the protective properties of IgA1 protease-based antigens and IgA1 protease analogs could be realized not only via impairment of bacterium adhesion to the mucosa, but also via suppression of this pathogen in the organism. The presented findings seem promising for using these proteins as the basis for anti-meningococcus vaccine.

  3. Immunoglobulins in Nasal Secretions of Healthy Humans: Structural Integrity of Secretory Immunoglobulin A1 (IgA1) and Occurrence of Neutralizing Antibodies to IgA1 Proteases of Nasal Bacteria

    PubMed Central

    Kirkeby, Line; Rasmussen, Trine Tang; Reinholdt, Jesper; Kilian, Mogens

    2000-01-01

    Certain bacteria, including overt pathogens as well as commensals, produce immunoglobulin A1 (IgA1) proteases. By cleaving IgA1, including secretory IgA1, in the hinge region, these enzymes may interfere with the barrier functions of mucosal IgA antibodies, as indicated by experiments in vitro. Previous studies have suggested that cleavage of IgA1 in nasal secretions may be associated with the development and perpetuation of atopic disease. To clarify the potential effect of IgA1 protease-producing bacteria in the nasal cavity, we have analyzed immunoglobulin isotypes in nasal secretions of 11 healthy humans, with a focus on IgA, and at the same time have characterized and quantified IgA1 protease-producing bacteria in the nasal flora of the subjects. Samples in the form of nasal wash were collected by using a washing liquid that contained lithium as an internal reference. Dilution factors and, subsequently, concentrations in undiluted secretions could thereby be calculated. IgA, mainly in the secretory form, was found by enzyme-linked immunosorbent assay to be the dominant isotype in all subjects, and the vast majority of IgA (median, 91%) was of the A1 subclass, corroborating results of previous analyses at the level of immunoglobulin-producing cells. Levels of serum-type immunoglobulins were low, except for four subjects in whom levels of IgG corresponded to 20 to 66% of total IgA. Cumulative levels of IgA, IgG, and IgM in undiluted secretions ranged from 260 to 2,494 (median, 777) μg ml−1. IgA1 protease-producing bacteria (Haemophilus influenzae, Streptococcus pneumoniae, or Streptococcus mitis biovar 1) were isolated from the nasal cavities of seven subjects at 2.1 × 103 to 7.2 × 106 CFU per ml of undiluted secretion, corresponding to 0.2 to 99.6% of the flora. Nevertheless, α-chain fragments characteristic of IgA1 protease activity were not detected in secretions from any subject by immunoblotting. Neutralizing antibodies to IgA1 proteases of autologous

  4. [Isolation and characteristics of IgA1 and its use for detecting bacterial IgA1 proteases].

    PubMed

    Amelina, I P; Zakharova, N A

    1984-12-01

    Sufficiently purified IgA, subclass I, has been isolated from the defibrinated plasma of a myeloma patient by chromatography on columns packed with DEAE-Sephadex A-50 or Sephadex G-200, and rabbit antiserum to this immunoglobulin has been obtained. These preparations have been used for detecting specific protease in Bordetella pertussis. The tested B. pertussis strains have been shown to induce, as revealed by immunoelectrophoretic methods, the proteolysis of human IgA, subclass I.

  5. In vivo cleavage of immunoglobulin A1 by immunoglobulin A1 proteases from Prevotella and Capnocytophaga species.

    PubMed

    Frandsen, E V; Reinholdt, J; Kjeldsen, M; Kilian, M

    1995-10-01

    Immunoglobulin A1 (IgA1) proteases secreted by oral Prevotella and Capnocytophaga species specifically cleave IgA1 at the same peptide bond in the hinge region, leaving intact monomeric Fab and Fc fragments. Assuming that Prevotella- and Capnocytophaga-induced Fab fragments of IgA1 expose a specific immunogenic neoepitope at the cleavage site, we established an enzyme-linked immunosorbent assay to measure human serum antibodies to this neoepitope as indirect evidence of in vivo activity of Prevotella and Capnocytophaga IgA1 proteases. The assay used a monoclonal antibody with specificity for the neoepitope, and the ability to block binding of the monoclonal antibody to the neoepitope was investigated. Absorption of sera with Prevotella melaninogenica-induced Fab fragments of IgA1 resulted in removal of antibodies blocking binding of the monoclonal antibody, whereas absorption with Fab fragments induced by bacterial IgA1 proteases of other cleavage specificities did not remove blocking antibodies. Consequently, we assume that the antibodies detected had been induced by a neoepitope an the Fab fragment of IgA1 exposed exclusively after cleavage with IgA1 proteases from Prevotella and Capnocytophaga, indicating in vivo activity of these IgA1 proteases. Evidence, though indirect, of in vivo activity of Prevotella and Capnocytophaga IgA1 proteases was present in 42 of 92 sera examined and in a significantly higher proportion of sera from adults with periodontal disease compared with control individuals. No correlation with disease was observed for the juvenile periodontitis groups.

  6. Inhibition of Prevotella and Capnocytophaga immunoglobulin A1 proteases by human serum.

    PubMed

    Frandsen, E V; Kjeldsen, M; Kilian, M

    1997-07-01

    Oral Prevotella and Capnocytophaga species, regularly isolated from periodontal pockets and associated with extraoral infections, secret specific immunoglobulin A1 (IgA1) proteases cleaving human IgA1 in the hinge region into intact Fab and Fc fragments. To investigate whether these enzymes are subject to inhibition in vivo in humans, we tested 34 sera from periodontally diseased and healthy individuals in an enzyme-linked immunosorbent assay for the presence and titers of inhibition of seven Prevotella and Capnocytophaga proteases. All or nearly all of the sera inhibited the IgA1 protease activity of Prevotella buccae, Prevotella oris, and Prevotella loescheii. A minor proportion of the sera inhibited Prevotella buccalis, Prevotella denticola, and Prevotella melaninogenica IgA1 proteases, while no sera inhibited Capnocytophaga ochracea IgA1 protease. All inhibition titers were low, ranging from 5 to 55, with titer being defined as the reciprocal of the dilution of serum causing 50% inhibition of one defined unit of protease activity. No correlation between periodontal disease status and the presence, absence, or titer of inhibition was observed. The nature of the low titers of inhibition in all sera of the IgA1 proteases of P. buccae, P. oris, and P. loescheii was further examined. In size exclusion chromatography, inhibitory activity corresponded to the peak volume of IgA. Additional inhibition of the P. oris IgA1 protease was found in fractions containing both IgA and IgG. Purification of the IgG fractions of five sera by passage of the sera on a protein G column resulted in recovery of inhibitory IgG antibodies against all three IgA1 proteases, with the highest titer being for the P. oris enzyme. These finding indicate that inhibitory activity is associated with enzyme-neutralizing antibodies.

  7. Inhibition of Prevotella and Capnocytophaga immunoglobulin A1 proteases by human serum.

    PubMed Central

    Frandsen, E V; Kjeldsen, M; Kilian, M

    1997-01-01

    Oral Prevotella and Capnocytophaga species, regularly isolated from periodontal pockets and associated with extraoral infections, secret specific immunoglobulin A1 (IgA1) proteases cleaving human IgA1 in the hinge region into intact Fab and Fc fragments. To investigate whether these enzymes are subject to inhibition in vivo in humans, we tested 34 sera from periodontally diseased and healthy individuals in an enzyme-linked immunosorbent assay for the presence and titers of inhibition of seven Prevotella and Capnocytophaga proteases. All or nearly all of the sera inhibited the IgA1 protease activity of Prevotella buccae, Prevotella oris, and Prevotella loescheii. A minor proportion of the sera inhibited Prevotella buccalis, Prevotella denticola, and Prevotella melaninogenica IgA1 proteases, while no sera inhibited Capnocytophaga ochracea IgA1 protease. All inhibition titers were low, ranging from 5 to 55, with titer being defined as the reciprocal of the dilution of serum causing 50% inhibition of one defined unit of protease activity. No correlation between periodontal disease status and the presence, absence, or titer of inhibition was observed. The nature of the low titers of inhibition in all sera of the IgA1 proteases of P. buccae, P. oris, and P. loescheii was further examined. In size exclusion chromatography, inhibitory activity corresponded to the peak volume of IgA. Additional inhibition of the P. oris IgA1 protease was found in fractions containing both IgA and IgG. Purification of the IgG fractions of five sera by passage of the sera on a protein G column resulted in recovery of inhibitory IgG antibodies against all three IgA1 proteases, with the highest titer being for the P. oris enzyme. These finding indicate that inhibitory activity is associated with enzyme-neutralizing antibodies. PMID:9220164

  8. A Comprehensive Genetic Study of Streptococcal Immunoglobulin A1 Proteases: Evidence for Recombination within and between Species

    PubMed Central

    Poulsen, Knud; Reinholdt, Jesper; Jespersgaard, Christina; Boye, Kit; Brown, Thomas A.; Hauge, Majbritt; Kilian, Mogens

    1998-01-01

    An analysis of 13 immunoglobulin A1 (IgA1) protease genes (iga) of strains of Streptococcus pneumoniae, Streptococcus oralis, Streptococcus mitis, and Streptococcus sanguis was carried out to obtain information on the structure, polymorphism, and phylogeny of this specific protease, which enables bacteria to evade functions of the predominant Ig isotype on mucosal surfaces. The analysis included cloning and sequencing of iga genes from S. oralis and S. mitis biovar 1, sequencing of an additional seven iga genes from S. sanguis biovars 1 through 4, and restriction fragment length polymorphism (RFLP) analyses of iga genes of another 10 strains of S. mitis biovar 1 and 6 strains of S. oralis. All 13 genes sequenced had the potential of encoding proteins with molecular masses of approximately 200 kDa containing the sequence motif HEMTH and an E residue 20 amino acids downstream, which are characteristic of Zn metalloproteinases. In addition, all had a typical gram-positive cell wall anchor motif, LPNTG, which, in contrast to such motifs in other known streptococcal and staphylococcal proteins, was located in their N-terminal parts. Repeat structures showing variation in number and sequence were present in all strains and may be of relevance to the immunogenicities of the enzymes. Protease activities in cultures of the streptococcal strains were associated with species of different molecular masses ranging from 130 to 200 kDa, suggesting posttranslational processing possibly as a result of autoproteolysis at post-proline peptide bonds in the N-terminal parts of the molecules. Comparison of deduced amino acid sequences revealed a 94% similarity between S. oralis and S. mitis IgA1 proteases and a 75 to 79% similarity between IgA1 proteases of these species and those of S. pneumoniae and S. sanguis, respectively. Combined with the results of RFLP analyses using different iga gene fragments as probes, the results of nucleotide sequence comparisons provide evidence of

  9. Occurrence and Evolution of the Paralogous Zinc Metalloproteases IgA1 Protease, ZmpB, ZmpC, and ZmpD in Streptococcus pneumoniae and Related Commensal Species

    PubMed Central

    Bek-Thomsen, Malene; Poulsen, Knud; Kilian, Mogens

    2012-01-01

    ABSTRACT The distribution, genome location, and evolution of the four paralogous zinc metalloproteases, IgA1 protease, ZmpB, ZmpC, and ZmpD, in Streptococcus pneumoniae and related commensal species were studied by in silico analysis of whole genomes and by activity screening of 154 representatives of 20 species. ZmpB was ubiquitous in the Mitis and Salivarius groups of the genus Streptococcus and in the genera Gemella and Granulicatella, with the exception of a fragmented gene in Streptococcus thermophilus, the only species with a nonhuman habitat. IgA1 protease activity was observed in all members of S. pneumoniae, S. pseudopneumoniae, S. oralis, S. sanguinis, and Gemella haemolysans, was variably present in S. mitis and S. infantis, and absent in S. gordonii, S. parasanguinis, S. cristatus, S. oligofermentans, S. australis, S. peroris, and S. suis. Phylogenetic analysis of 297 zmp sequences and representative housekeeping genes provided evidence for an unprecedented selection for genetic diversification of the iga, zmpB, and zmpD genes in S. pneumoniae and evidence of very frequent intraspecies transfer of entire genes and combination of genes. Presumably due to their adaptation to a commensal lifestyle, largely unaffected by adaptive mucosal immune factors, the corresponding genes in commensal streptococci have remained conserved. The widespread distribution and significant sequence diversity indicate an ancient origin of the zinc metalloproteases predating the emergence of the humanoid species. zmpB, which appears to be the ancestral gene, subsequently duplicated and successfully diversified into distinct functions, is likely to serve an important but yet unknown housekeeping function associated with the human host. PMID:23033471

  10. A comparison of the binding of secretory component to immunoglobulin A (IgA) in human colostral S-IgA1 and S-IgA2

    PubMed Central

    Almogren, Adel; Senior, Bernard W; Kerr, Michael A

    2007-01-01

    A detailed investigation of the binding of secretory component to immunoglobulin A (IgA) in human secretory IgA2 (S-IgA2) was made possible by the development of a new method of purifying S-IgA1, S-IgA2 and free secretory component from human colostrum using thiophilic gel chromatography and chromatography on Jacalin-agarose. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis of unreduced pure S-IgA2 revealed that, unlike in S-IgA1, a significant proportion of the secretory component was bound non-covalently in S-IgA2. When S-IgA1 was incubated with a protease purified from Proteus mirabilis the secretory component, but not the α-chain, was cleaved. This is in contrast to serum IgA1, in which the α-chain was cleaved under the same conditions – direct evidence that secretory component does protect the α-chain from proteolytic cleavage in S-IgA. Comparisons between the products of cleavage with P. mirabilis protease of free secretory component and bound secretory component in S-IgA1 and S-IgA2 also indicated that, contrary to the general assumption, the binding of secretory component to IgA is different in S-IgA2 from that in S-IgA1. PMID:17156102

  11. β1,4-galactosyltransferase 1 is a novel receptor for IgA in human mesangial cells.

    PubMed

    Molyneux, Karen; Wimbury, David; Pawluczyk, Izabella; Muto, Masahiro; Bhachu, Jasraj; Mertens, Peter R; Feehally, John; Barratt, Jonathan

    2017-12-01

    IgA nephropathy is characterized by mesangial deposition of IgA, mesangial cell proliferation, and extracellular matrix production. Mesangial cells bind IgA, but the identity of all potential receptors involved remains incomplete. The transferrin receptor (CD71) acts as a mesangial cell IgA receptor and its expression is upregulated in many forms of glomerulonephritis, including IgA nephropathy. CD71 is not expressed in healthy glomeruli and blocking CD71 does not completely abrogate mesangial cell IgA binding. Previously we showed that mesangial cells express a receptor that binds the Fc portion of IgA and now report that this receptor is an isoform of β-1,4-galactosyltransferase. A human mesangial cell cDNA library was screened for IgA binding proteins and β-1,4-galactosyltransferase identified. Cell surface expression of the long isoform of β-1,4-galactosyltransferase was shown by flow cytometry and confocal microscopy and confirmed by immunoblotting. Glomerular β-1,4-galactosyltransferase expression was increased in IgA nephropathy. IgA binding and IgA-induced mesangial cell phosphorylation of spleen tyrosine kinase and IL-6 synthesis were inhibited by a panel of β-1,4-galactosyltransferase-specific antibodies, suggesting IgA binds to the catalytic domain of β-1,4-galactosyltransferase. Thus, β-1,4-galactosyltransferase is a constitutively expressed mesangial cell IgA receptor with an important role in both mesangial IgA clearance and the initial response to IgA deposition. Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

  12. Galactosylation of IgA1 Is Associated with Common Variation in C1GALT1.

    PubMed

    Gale, Daniel P; Molyneux, Karen; Wimbury, David; Higgins, Patricia; Levine, Adam P; Caplin, Ben; Ferlin, Anna; Yin, Peiran; Nelson, Christopher P; Stanescu, Horia; Samani, Nilesh J; Kleta, Robert; Yu, Xueqing; Barratt, Jonathan

    2017-07-01

    IgA nephropathy (IgAN), an important cause of kidney failure, is characterized by glomerular IgA deposition and is associated with changes in O -glycosylation of the IgA1 molecule. Here, we sought to identify genetic factors contributing to levels of galactose-deficient IgA1 (Gd-IgA1) in white and Chinese populations. Gd-IgA1 levels were elevated in IgAN patients compared with ethnically matched healthy subjects and correlated with evidence of disease progression. White patients with IgAN exhibited significantly higher Gd-IgA1 levels than did Chinese patients. Among individuals without IgAN, Gd-IgA1 levels did not correlate with kidney function. Gd-IgA1 level heritability (h 2 ), estimated by comparing midparental and offspring Gd-IgA1 levels, was 0.39. Genome-wide association analysis by linear regression identified alleles at a single locus spanning the C1GALT1 gene that strongly associated with Gd-IgA1 level ( β =0.26; P =2.35×10 -9 ). This association was replicated in a genome-wide association study of separate cohorts comprising 308 patients with membranous GN from the UK ( P <1.00×10 -6 ) and 622 controls with normal kidney function from the UK ( P <1.00×10 -10 ), and in a candidate gene study of 704 Chinese patients with IgAN ( P <1.00×10 -5 ). The same extended haplotype associated with elevated Gd-IgA1 levels in all cohorts studied. C1GALT1 encodes a galactosyltransferase enzyme that is important in O -galactosylation of glycoproteins. These findings demonstrate that common variation at C1GALT1 influences Gd-IgA1 level in the population, which independently associates with risk of progressive IgAN, and that the pathogenic importance of changes in IgA1 O -glycosylation may vary between white and Chinese patients with IgAN. Copyright © 2017 by the American Society of Nephrology.

  13. Serum under-O-glycosylated IgA1 level is not correlated with glomerular IgA deposition based upon heterogeneity in the composition of immune complexes in IgA nephropathy.

    PubMed

    Satake, Kenji; Shimizu, Yoshio; Sasaki, Yohei; Yanagawa, Hiroyuki; Suzuki, Hitoshi; Suzuki, Yusuke; Horikoshi, Satoshi; Honda, Shinichiro; Shibuya, Kazuko; Shibuya, Akira; Tomino, Yasuhiko

    2014-06-13

    Although serum under-O-glycosylated IgA1 in IgA nephropathy (IgAN) patients may deposit more preferentially in glomeruli than heavily-O-glycosylated IgA1, the relationship between the glomerular IgA deposition level and the O-glycan profiles of serum IgA1 remains obscure. Serum total under-O-glycosylated IgA1 levels were quantified in 32 IgAN patients by an enzyme-linked immunosorbent assay (ELISA) with Helix aspersa (HAA) lectin. Serum under-O-glycosylated polymeric IgA1 (pIgA1) was selectively measured by an original method using mouse Fcα/μ receptor (mFcα/μR) transfectant and flow cytometry (pIgA1 trap). The percentage area of IgA deposition in the whole glomeruli (Area-IgA) was quantified by image analysis on the immunofluorescence of biopsy specimens. Correlations were assessed between the Area-IgA and data from HAA-ELISA or pIgA1 trap. The relationships between clinical parameters and data from HAA-ELISA or pIgA1 trap were analyzed by data mining approach. While the under-O-glycosylated IgA1 levels in IgAN patients were significantly higher than those in healthy controls when measured (p<0.05), there was no significant difference in under-O-glycosylated pIgA1. There was neither a correlation observed between the data from HAA-ELISA and pIgA1 trap (r2=0.09) in the IgAN patients (r2=0.005) nor was there a linear correlation between Area-IgA and data from HAA-ELISA or the pIgA1 trap (r2=0.005, 0.03, respectively). Contour plots of clinical parameters versus data from HAA-ELISA and the pIgA1 trap revealed that patients with a high score in each clinical parameter concentrated in specific areas, showing that patients with specific O-glycan profiles of IgA1 have similar clinical parameters. A decision tree analysis suggested that dominant immune complexes in glomeruli were consisted of: 1) IgA1-IgG and complements, 2) pIgA1 and complements, and 3) monomeric IgA1-IgA or aggregated monomeric IgA1. Serum under-O-glycosylated IgA1 levels are not correlated with

  14. Naturally Occurring Structural Isomers in Serum IgA1 O-Glycosylation

    PubMed Central

    Takahashi, Kazuo; Smith, Archer D.; Poulsen, Knud; Kilian, Mogens; Julian, Bruce A.; Mestecky, Jiri; Novak, Jan; Renfrow, Matthew B.

    2013-01-01

    IgA is the most abundantly produced antibody and plays an important role in the mucosal immune system. Human IgA is represented by two isotypes, IgA1 and IgA2. The major structural difference between these two subclasses is the presence of nine potential sites of O-glycosylation in the hinge region between the first and second constant region domains of the heavy chain. Thr225, Thr228, Ser230, Ser232 and Thr236 have been identified as the predominant sites of O-glycan attachment. The range and distribution of O-glycan chains at each site within the context of adjacent sites in this clustered region create a complex heterogeneity of surface epitopes that is incompletely defined. We previously described the analysis of IgA1 O-glycan heterogeneity by use of high resolution LC/MS and electron capture dissociation tandem MS to unambiguously localize all amino acid attachment sites in IgA1 (Ale) myeloma protein. Here, we report the identification and elucidation of IgA1 O-glycopeptide structural isomers that occur based on amino acid position of the attached glycans (positional isomers) and the structure of the O-glycan chains at individual sites (glycan isomers). These isomers are present in a model IgA1 (Mce1) myeloma protein and occur naturally in normal human serum IgA1. Variable O-glycan chains attached to Ser230, Thr233 or Thr236 produce the predominant positional isomers, including O-glycans composed of a single GalNAc residue. These findings represent the first definitive identification of structural isomeric IgA1 O-glycoforms, define the single-site heterogeneity for all O-glycan sites in a single sample, and have implications for defining epitopes based on clustered O-glycan variability. PMID:22067045

  15. HIV-1-Specific IgA Monoclonal Antibodies from an HIV-1 Vaccinee Mediate Galactosylceramide Blocking and Phagocytosis

    PubMed Central

    2018-01-01

    ABSTRACT Vaccine-elicited humoral immune responses comprise an array of antibody forms and specificities, with only a fraction contributing to protective host immunity. Elucidation of antibody effector functions responsible for protective immunity against human immunodeficiency virus type 1 (HIV-1) acquisition is a major goal for the HIV-1 vaccine field. Immunoglobulin A (IgA) is an important part of the host defense against pathogens; however, little is known about the role of vaccine-elicited IgA and its capacity to mediate antiviral functions. To identify the antiviral functions of HIV-1-specific IgA elicited by vaccination, we cloned HIV-1 envelope-specific IgA monoclonal antibodies (MAbs) by memory B cell cultures from peripheral blood mononuclear cells from an RV144 vaccinee and produced two IgA clonal cell lines (HG129 and HG130) producing native, nonrecombinant IgA MAbs. The HG129 and HG130 MAbs mediated phagocytosis by monocytes, and HG129 blocked HIV-1 Env glycoprotein binding to galactosylceramide, an alternative HIV-1 receptor. These findings elucidate potential antiviral functions of vaccine-elicited HIV-1 envelope-specific IgA that may act to block HIV-1 acquisition at the portal of entry by preventing HIV-1 binding to galactosylceramide and mediating antibody Fc receptor-mediated virion phagocytosis. Furthermore, these findings highlight the complex and diverse interactions of vaccine-elicited IgA with pathogens that depend on IgA fine specificity and form (e.g., multimeric or monomeric) in the systemic circulation and mucosal compartments. IMPORTANCE Host-pathogen interactions in vivo involve numerous immune mechanisms that can lead to pathogen clearance. Understanding the nature of antiviral immune mechanisms can inform the design of efficacious HIV-1 vaccine strategies. Evidence suggests that both neutralizing and nonneutralizing antibodies can mediate some protection against HIV in animal models. Although numerous studies have characterized the

  16. Cloning and structural analysis of two highly divergent IgA isotypes, IgA1 and IgA2 from the duck billed platypus, Ornithorhynchus anatinus.

    PubMed

    Vernersson, M; Belov, K; Aveskogh, M; Hellman, L

    2010-01-01

    To trace the emergence of modern IgA isotypes during vertebrate evolution we have studied the immunoglobulin repertoire of a model monotreme, the platypus. Two highly divergent IgA-like isotypes (IgA1 and IgA2) were identified and their primary structures were determined from full-length cDNAs. A comparative analysis of the amino acid sequences for IgA from various animal species showed that the two platypus IgA isotypes form a branch clearly separated from their eutherian (placental) counterparts. However, they still conform to the general structure of eutherian IgA, with a hinge region and three constant domains. This indicates that the deletion of the second domain and the formation of a hinge region in IgA did occur very early during mammalian evolution, more than 166 million years ago. The two IgA isotypes in platypus differ in primary structure and appear to have arisen from a very early gene duplication, possibly preceding the metatherian eutherian split. Interestingly, one of these isotypes, IgA1, appears to be expressed in only the platypus, but is present in the echidna based on Southern blot analysis. The platypus may require a more effective mucosal immunity, with two highly divergent IgA forms, than the terrestrial echidna, due to its lifestyle, where it is exposed to pathogens both on land and in the water. Copyright 2010 Elsevier Ltd. All rights reserved.

  17. Novel lectin-independent approach to detect galactose-deficient IgA1 in IgA nephropathy.

    PubMed

    Yasutake, Junichi; Suzuki, Yusuke; Suzuki, Hitoshi; Hiura, Naoko; Yanagawa, Hiroyuki; Makita, Yuko; Kaneko, Etsuji; Tomino, Yasuhiko

    2015-08-01

    Galactose-deficient IgA1 (Gd-IgA1) is a critical effector molecule in the pathogenesis of IgA nephropathy (IgAN). Although many researchers have measured serum levels of Gd-IgA1 using snail helix aspersa agglutinin (HAA) lectin-based assay, the lectin-dependent assay has some serious problems in robustness. In this study, we aimed to establish a more robust and stable enzyme-linked immunosorbent assay (ELISA) method that uses a specific monoclonal antibody to recognize a hinge region in human Gd-IgA1 (Gd-IgA1 ELISA). Rats were immunized with human Gd-IgA1 hinge region peptide to obtain Gd-IgA1-specific monoclonal antibody KM55. Gd-IgA1 ELISA for specifically detecting serum Gd-IgA1 was consequently constructed. Serum Gd-IgA1 concentrations in human subjects were measured using KM55 ELISA assay. To further confirm specificity of the Gd-IgA1-specific antibody, KM55 was also applied for immunofluorescence staining of glomerular Gd-IgA1 in paraffin-embedded sections of renal biopsy specimens. Measurement of serum levels of Gd-IgA1 in human subjects by Gd-IgA1 ELISA revealed increased serum Gd-IgA1 level in patients with IgAN compared with patients with other renal diseases or non-renal diseases. Importantly, the results obtained from Gd-IgA1 ELISA positively correlated with those from the HAA lectin-based assay (R = 0.75). Immunofluorescence staining of renal biopsy specimens with KM55 detected glomerular co-localization of Gd-IgA1 and IgA. This novel lectin-independent method with KM55 for measuring serum levels of Gd-IgA1 can pave the way for more convincing diagnosis and activity assessment of IgAN, and can expedite clinical research to better understand this difficult disease. © The Author 2015. Published by Oxford University Press on behalf of ERA-EDTA.

  18. Novel lectin-independent approach to detect galactose-deficient IgA1 in IgA nephropathy

    PubMed Central

    Yasutake, Junichi; Suzuki, Yusuke; Suzuki, Hitoshi; Hiura, Naoko; Yanagawa, Hiroyuki; Makita, Yuko; Kaneko, Etsuji; Tomino, Yasuhiko

    2015-01-01

    Background Galactose-deficient IgA1 (Gd-IgA1) is a critical effector molecule in the pathogenesis of IgA nephropathy (IgAN). Although many researchers have measured serum levels of Gd-IgA1 using snail helix aspersa agglutinin (HAA) lectin-based assay, the lectin-dependent assay has some serious problems in robustness. In this study, we aimed to establish a more robust and stable enzyme-linked immunosorbent assay (ELISA) method that uses a specific monoclonal antibody to recognize a hinge region in human Gd-IgA1 (Gd-IgA1 ELISA). Methods Rats were immunized with human Gd-IgA1 hinge region peptide to obtain Gd-IgA1-specific monoclonal antibody KM55. Gd-IgA1 ELISA for specifically detecting serum Gd-IgA1 was consequently constructed. Serum Gd-IgA1 concentrations in human subjects were measured using KM55 ELISA assay. To further confirm specificity of the Gd-IgA1-specific antibody, KM55 was also applied for immunofluorescence staining of glomerular Gd-IgA1 in paraffin-embedded sections of renal biopsy specimens. Results Measurement of serum levels of Gd-IgA1 in human subjects by Gd-IgA1 ELISA revealed increased serum Gd-IgA1 level in patients with IgAN compared with patients with other renal diseases or non-renal diseases. Importantly, the results obtained from Gd-IgA1 ELISA positively correlated with those from the HAA lectin-based assay (R = 0.75). Immunofluorescence staining of renal biopsy specimens with KM55 detected glomerular co-localization of Gd-IgA1 and IgA. Conclusion This novel lectin-independent method with KM55 for measuring serum levels of Gd-IgA1 can pave the way for more convincing diagnosis and activity assessment of IgAN, and can expedite clinical research to better understand this difficult disease. PMID:26109484

  19. Impaired selection of IgA and intestinal dysbiosis associated with PD-1-deficiency

    PubMed Central

    Maruya, Mikako; Kawamoto, Shimpei; Kato, Lucia M.; Fagarasan, Sidonia

    2013-01-01

    A major function of immunoglobulin A (IgA) is to maintain balanced bacterial communities in the gut. We have previously shown that diversification of IgA upon somatic hypermutation (SHM) is critical for IgA function yet the principles governing the selection of IgA in the gut have remained elusive. Here we discuss recent progress in understanding this process as revealed by our studies in mice that lack the inhibitory co-receptor programmed cell death–1 (PD-1). We found that PD-1 affects the dynamics of germinal center (GC) B cells by controlling the number and the nature of T helper cells in the Peyer’s patches (PPs). Deregulation of the T cell compartment impacts the selection of IgA plasma cells leading to gut dysbiosis. When the PD-1-dependent checkpoint is missing, gut bacteria go beyond the mucosal barrier and induce systemic GCs that can generate antibodies with auto-reactive properties. PMID:23333864

  20. TBK1 controls IgA class switching by negatively regulating noncanonical NF-κB signaling

    PubMed Central

    Jin, Jin; Xiao, Yichuan; Chang, Jae-Hoon; Yu, Jiayi; Hu, Hongbo; Starr, Robyn; Brittain, George C.; Chang, Mikyoung; Cheng, Xuhong; Sun, Shao-Cong

    2012-01-01

    Immunoglobulin (Ig) class switching is crucial for generating antibody diversity in humoral immunity and, if deregulated, also has severe pathological consequences. How the magnitude of Ig isotype switching is controlled is still poorly understood. Here we identify TANK-binding kinase 1 (TBK1) as a pivotal negative regulator of IgA class switching. B cell-specific TBK1 ablation in mice resulted in uncontrolled production of IgA and development of nephropathy-like disease symptoms. TBK1 negatively regulated IgA class switching by attenuating noncanonical NF-κB signaling, an action that involved TBK1-mediated phosphorylation and subsequent degradation of the NF-κB-inducing kinase. These findings establish TBK1 as a pivotal negative regulator of the noncanonical NF-κB pathway and highlight a unique mechanism that controls IgA production. PMID:23023393

  1. GWAS for serum galactose-deficient IgA1 implicates critical genes of the O-glycosylation pathway

    PubMed Central

    Kiryluk, Krzysztof; Moldoveanu, Zina; Suzuki, Hitoshi; Reily, Colin; Hou, Ping; Xie, Jingyuan; Mladkova, Nikol; Prakash, Sindhuri; Fischman, Clara; Shapiro, Samantha; Bradbury, Drew; Ionita-Laza, Iuliana; Eitner, Frank; Rauen, Thomas; Maillard, Nicolas; Floege, Jürgen; Chen, Nan; Zhang, Hong; Scolari, Francesco; Wyatt, Robert J.; Julian, Bruce A.; Gharavi, Ali G.; Novak, Jan

    2017-01-01

    Aberrant O-glycosylation of serum immunoglobulin A1 (IgA1) represents a heritable pathogenic defect in IgA nephropathy, the most common form of glomerulonephritis worldwide, but specific genetic factors involved in its determination are not known. We performed a quantitative GWAS for serum levels of galactose-deficient IgA1 (Gd-IgA1) in 2,633 subjects of European and East Asian ancestry and discovered two genome-wide significant loci, in C1GALT1 (rs13226913, P = 3.2 x 10−11) and C1GALT1C1 (rs5910940, P = 2.7 x 10−8). These genes encode molecular partners essential for enzymatic O-glycosylation of IgA1. We demonstrated that these two loci explain approximately 7% of variability in circulating Gd-IgA1 in Europeans, but only 2% in East Asians. Notably, the Gd-IgA1-increasing allele of rs13226913 is common in Europeans, but rare in East Asians. Moreover, rs13226913 represents a strong cis-eQTL for C1GALT1 that encodes the key enzyme responsible for the transfer of galactose to O-linked glycans on IgA1. By in vitro siRNA knock-down studies, we confirmed that mRNA levels of both C1GALT1 and C1GALT1C1 determine the rate of secretion of Gd-IgA1 in IgA1-producing cells. Our findings provide novel insights into the genetic regulation of O-glycosylation and are relevant not only to IgA nephropathy, but also to other complex traits associated with O-glycosylation defects, including inflammatory bowel disease, hematologic disease, and cancer. PMID:28187132

  2. The role of the carbohydrate chains in complement (C3) fixation by solid-phase-bound human IgA.

    PubMed Central

    Nikolova, E B; Tomana, M; Russell, M W

    1994-01-01

    In contrast to antigen-antibody complexes containing native human IgA, solid-phase-deposited IgA activates the alternative complement pathway and binds C3b. To investigate the role of carbohydrate chains in this, various human IgA preparations were treated with neuraminidase alone or together with N-glycanase or O-glycanase, or with mixed glycosidases from the oral bacterium, Streptococcus mitis. Depletion of oligosaccharides was determined by carbohydrate analysis. Removal of sialic acid and N-linked glycan chains greatly increased the C3b-fixing properties of normal serum IgA1 and IgA2. Myeloma IgA1 and IgA2 proteins and secretory IgA had higher C3b-binding activity than normal serum IgA, and this was further increased by removal of sialic acid and N-linked glycans. Fc alpha and Fc alpha-SC fragments of myeloma and secretory IgA1, respectively, but not Fab alpha fragments, obtained by cleavage with bacterial IgA1 proteases and also free secretory component, fixed C3b by the alternative pathway. Images Figure 4 PMID:7927504

  3. Curcumin derivatives as HIV-1 protease inhibitors

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sui, Z.; Li, J.; Craik, C.S.

    1993-12-31

    Curcumin, a non-toxic natural compound from Curcuma longa, has been found to be an HIV-1 protease inhibitor. Some of its derivatives were synthesized and their inhibitory activity against the HIV-1 protease was tested. Curcumin analogues containing boron enhanced the inhibitory activity. At least of the the synthesized compounds irreversibly inhibits the HIV-1 protease.

  4. CagA, a major virulence factor of Helicobacter pylori, promotes the production and underglycosylation of IgA1 in DAKIKI cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yang, Man; Li, Fu-gang; Xie, Xi-sheng

    Highlights: • CagA stimulated cell proliferation and the production of IgA1 in DAKIKI cells. • CagA promoted the underglycosylation of IgA1 in DAKIKI cells. • CagA decreased the expression of C1GALT1 and its chaperone Cosmc in DAKIKI cells. • Helicobacter pylori infection may participate in the pathogenesis of IgAN via CagA. - Abstract: While Helicobacter pylori (Hp) infection is closely associated with IgA nephropathy (IgAN), the underlying molecular mechanisms remain to be elucidated. This study was to investigate the effect of cytotoxin associated gene A protein (CagA), a major virulence factor of Hp, on the production and underglycosylation of IgA1more » in the B cell line DAKIKI cells. Cells were cultured and treated with recombinant CagA protein. We found that CagA stimulated cell proliferation and the production of IgA1 in a dose-dependent and time-dependent manner. Moreover, CagA promoted the underglycosylation of IgA1, which at least partly attributed to the downregulation of β1,3-galactosyltransferase (C1GALT1) and its chaperone Cosmc. In conclusion, we demonstrated that Hp infection, at least via CagA, may participate in the pathogenesis of IgAN by influencing the production and glycosylation of IgA1 in B cells.« less

  5. Human High Temperature Requirement Serine Protease A1 (HTRA1) Degrades Tau Protein Aggregates*

    PubMed Central

    Tennstaedt, Annette; Pöpsel, Simon; Truebestein, Linda; Hauske, Patrick; Brockmann, Anke; Schmidt, Nina; Irle, Inga; Sacca, Barbara; Niemeyer, Christof M.; Brandt, Roland; Ksiezak-Reding, Hanna; Tirniceriu, Anca Laura; Egensperger, Rupert; Baldi, Alfonso; Dehmelt, Leif; Kaiser, Markus; Huber, Robert; Clausen, Tim; Ehrmann, Michael

    2012-01-01

    Protective proteases are key elements of protein quality control pathways that are up-regulated, for example, under various protein folding stresses. These proteases are employed to prevent the accumulation and aggregation of misfolded proteins that can impose severe damage to cells. The high temperature requirement A (HtrA) family of serine proteases has evolved to perform important aspects of ATP-independent protein quality control. So far, however, no HtrA protease is known that degrades protein aggregates. We show here that human HTRA1 degrades aggregated and fibrillar tau, a protein that is critically involved in various neurological disorders. Neuronal cells and patient brains accumulate less tau, neurofibrillary tangles, and neuritic plaques, respectively, when HTRA1 is expressed at elevated levels. Furthermore, HTRA1 mRNA and HTRA1 activity are up-regulated in response to elevated tau concentrations. These data suggest that HTRA1 is performing regulated proteolysis during protein quality control, the implications of which are discussed. PMID:22535953

  6. Insulin-like growth factor binding protein-1 levels are increased in patients with IgA nephropathy

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Tokunaga, Koki; Uto, Hirofumi, E-mail: hirouto@m2.kufm.kagoshima-u.ac.jp; Takami, Yoichiro

    2010-08-20

    Research highlights: {yields} IGFBP-1 mRNA over express in kidneys obtained from mice model of IgA nephropathy. {yields} Serum IGFBP-1 levels are high in patients with IgA nephropathy. {yields} Serum IGFBP-1 levels correlate with renal function and the severity of renal injury. -- Abstract: The mechanisms underlying the pathogenesis of immunoglobulin A (IgA) nephropathy (IgAN) are not well understood. In this study, we examined gene expression profiles in kidneys obtained from mice with high serum IgA levels (HIGA mice), which exhibit features of human IgAN. Female inbred HIGA, established from the ddY line, were used in these experiments. Serum IgA levels,more » renal IgA deposition, mesangial proliferation, and glomerulosclerosis were increased in 32-week-old HIGA mice in comparison to ddY animals. By microarray analysis, five genes were observed to be increased by more than 2.5-fold in 32-week-old HIGA in comparison to 16-week-old HIGA; these same five genes were decreased more than 2.5-fold in 32-week-old ddY in comparison to 16-week-old ddY mice. Of these five genes, insulin-like growth factor (IGF) binding protein (IGFBP)-1 exhibited differential expression between these mouse lines, as confirmed by quantitative RT-PCR. In addition, serum IGFBP-1 levels were significantly higher in patients with IgAN than in healthy controls. In patients with IgAN, these levels correlated with measures of renal function, such as estimated glomerular filtration rate (eGFR), but not with sex, age, serum IgA, C3 levels, or IGF-1 levels. Pathologically, serum IGFBP-1 levels were significantly associated with the severity of renal injury, as assessed by mesangial cell proliferation and interstitial fibrosis. These results suggest that increased IGFBP-1 levels are associated with the severity of renal pathology in patients with IgAN.« less

  7. IgA Enhances IGF-1 Mitogenic Activity Via Receptor Modulation in Glomerular Mesangial Cells: Implications for IgA-Induced Nephropathy.

    PubMed

    Al-Eisa, Amal; Dhaunsi, Gursev S

    2017-01-01

    Glomerulonephritis due to mesangial proliferation is responsible for renal dysfunction in IgA nephropathy (IgAN), however molecular mechanisms of pathogenesis are not well known. We examined the effect of IgA on Insulin-like Growth Factor-1 (IGF-1) activity, a potent mitogen with vital role in growth and development of children, and IGF-1 receptor (IGF-1R) in cultures of glomerular mesangial cells (GMC). GMC were isolated from rat kidneys using sieving and enzymatic digestion of tissue homogenates, and cultured in RPMI 1640 medium. GMC cultures were treated with IgA (0-10 µg/ml) in the presence or absence of IGF-1 and fetal bovine serum (FBS), and BrdU incorporation was measured. IGF-1 levels were assayed along with real-time PCR quantification of IGF-1R mRNA. Treatment of GMC with IgA (5 -10 µg/ml) significantly (p < 0.01) increased the BrdU incorporation in the presence or absence of FBS or IGF-1. IgA-mediated effects were more pronounced in IGF-1 treated cells that were significantly (p < 0.01) blocked by pretreatment of cells with IGF-1 receptor antibody or genistein. IgA significantly increased the levels of IGF-1 in culture supernatants and GMC homogenates. IGF-1R mRNA was significantly (p < 0.01) increased in IgA treated cells particularly by co-treatment with IGF-1. These findings show that IgA enhances the IGF-1 activity in GMC via stimulation of IGF-1R gene transcription and suggest a role for IGF-1 in pathogenesis of IgAN. © 2017 The Author(s). Published by S. Karger AG, Basel.

  8. HIV-1 protease-substrate coevolution in nelfinavir resistance.

    PubMed

    Kolli, Madhavi; Ozen, Ayşegül; Kurt-Yilmaz, Nese; Schiffer, Celia A

    2014-07-01

    Resistance to various human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) challenges the effectiveness of therapies in treating HIV-1-infected individuals and AIDS patients. The virus accumulates mutations within the protease (PR) that render the PIs less potent. Occasionally, Gag sequences also coevolve with mutations at PR cleavage sites contributing to drug resistance. In this study, we investigated the structural basis of coevolution of the p1-p6 cleavage site with the nelfinavir (NFV) resistance D30N/N88D protease mutations by determining crystal structures of wild-type and NFV-resistant HIV-1 protease in complex with p1-p6 substrate peptide variants with L449F and/or S451N. Alterations of residue 30's interaction with the substrate are compensated by the coevolving L449F and S451N cleavage site mutations. This interdependency in the PR-p1-p6 interactions enhances intermolecular contacts and reinforces the overall fit of the substrate within the substrate envelope, likely enabling coevolution to sustain substrate recognition and cleavage in the presence of PR resistance mutations. Resistance to human immunodeficiency virus type 1 (HIV-1) protease inhibitors challenges the effectiveness of therapies in treating HIV-1-infected individuals and AIDS patients. Mutations in HIV-1 protease selected under the pressure of protease inhibitors render the inhibitors less potent. Occasionally, Gag sequences also mutate and coevolve with protease, contributing to maintenance of viral fitness and to drug resistance. In this study, we investigated the structural basis of coevolution at the Gag p1-p6 cleavage site with the nelfinavir (NFV) resistance D30N/N88D protease mutations. Our structural analysis reveals the interdependency of protease-substrate interactions and how coevolution may restore substrate recognition and cleavage in the presence of protease drug resistance mutations. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  9. Association of IgG co-deposition with serum levels of galactose-deficient IgA1 in pediatric IgA nephropathy

    PubMed Central

    Eison, T. Matthew; Hastings, M. Colleen; Moldoveanu, Zina; Sanders, John T.; Gaber, Lillian; Walker, Patrick D.; Lau, Keith K; Julian, Bruce A.; Novak, Jan; Wyatt, Robert J.

    2012-01-01

    Objective: To determine whether the absence of mesangial IgG deposits is associated with the absence of elevated blood levels of galactose-deficient IgA1 (Gd-IgA1) in pediatric patients with IgA nephropathy (IgAN). Design and methods: Serum Gd-IgA1 levels were determined by ELISA using an N-acetylgalactosamine-specific lectin from Helix aspersa. Levels of Gd-IgA1 above the 90th percentile for healthy pediatric controls were considered to be elevated. Renal biopsy samples were examined by immunofluorescence for presence and intensity of staining for IgA, IgG, IgM, C3 and C1q and by light microscopy for histological changes. Findings were graded by a single pathologist (L. Gaber) at UTHSC until 2007 and by NephropathTM (Little Rock, AR, USA) thereafter. Staining for the mesangial deposits was considered negative when intensity was trace or less, and positive at greater intensity. Fisher’s exact-test was used to determine significance of 2 × 2 tables. Results: Serum samples were obtained from 30 patients with IgAN diagnosed before age 18 years. Male : female ratio was 2.3 : 1. Twenty were Caucasian and 10 were African-American. Blood was obtained within 3 months of biopsy (incident cases) for 12, while 18 provided blood > 3 months after biopsy (prevalent cases). Serum Gd-IgA1 level was elevated in 23 (77%) of cases and 20 (67%) had a biopsy positive for IgG. Of those 20 patients, 18 (90%) had an elevated serum Gd-IgA1 level, whereas 5 (50%) of patients with biopsies without IgG had a normal serum Gd-IgA1 level (p = 0.026). Summary: In this small study we found a weak association between the absence of IgG in the biopsy and normal serum Gd-IgA1 level. PMID:23006340

  10. Comprehensive mutagenesis of HIV-1 protease: a computational geometry approach.

    PubMed

    Masso, Majid; Vaisman, Iosif I

    2003-05-30

    A computational geometry technique based on Delaunay tessellation of protein structure, represented by C(alpha) atoms, is used to study effects of single residue mutations on sequence-structure compatibility in HIV-1 protease. Profiles of residue scores derived from the four-body statistical potential are constructed for all 1881 mutants of the HIV-1 protease monomer and compared with the profile of the wild-type protein. The profiles for an isolated monomer of HIV-1 protease and the identical monomer in a dimeric state with an inhibitor are analyzed to elucidate changes to structural stability. Protease residues shown to undergo the greatest impact are those forming the dimer interface and flap region, as well as those known to be involved in inhibitor binding.

  11. Induction of mucosal IgA by a novel jet delivery technique for HIV-1 DNA.

    PubMed

    Lundholm, P; Asakura, Y; Hinkula, J; Lucht, E; Wahren, B

    1999-04-09

    Novel ways of delivering plasmid DNA to elicit humoral IgA, IgG and cell-mediated immune responses in mice were investigated. Intraoral administration of DNA in the cheek, using a jet immunization technique, elicited the highest IgA mucosal responses. Intranasal immunization gave strong mucosal IgA responses and persistent systemic IgG. Immunoglobulin isotype analysis revealed an IgG1 profile for intramuscular tongue and gene gun immunizations and an IgG2a profile following oral jet injection and intranasal application. The route of delivery was of importance for the characteristics and quality of the mucosal immune response following DNA immunization. For DNA vaccine delivery, the intraoral jet injection technique has the advantages of being a simple and rapid way of administering the DNA in solution and of provoking specific mucosal IgA when administered in the mucosal associated lymphoid tissue.

  12. Separation of Two Distinct O-Glycoforms of Human IgA1 by Serial Lectin Chromatography Followed by Mass Spectrometry O-Glycan Analysis.

    PubMed

    Lehoux, S; Ju, T

    2017-01-01

    Human immunoglobulin A1 (IgA1), which carries four to six mucin-type O-glycans (O-glycans) on its hinge region (HR), is the most abundant O-glycoprotein in plasma or serum. While normal O-glycans from hematopoietic-originated cells are core 1-based complex structures, many reports showed that the IgA1 from patients with IgA nephropathy (IgAN) carries undergalactosylated or truncated O-glycans such as the Tn antigen and its sialylated version the SialylTn (STn) antigen on the HR. Yet, there is still a debate whether Tn/STn on the HR of IgA1 is specific to the IgA1 from patients with IgAN since these antigens have also been seen in serum IgA1 of healthy individuals. An additional question is whether the O-glycans at all sites on the two HRs of one IgA1 molecule are homogeneous (either all normal or all Tn/STn) or heterogeneous (both normal and Tn/STn O-glycans). To address these questions, we conducted a systematic study on the O-glycans of plasma IgA1 from both IgAN patients and healthy controls using serial HPA and PNA lectin chromatography followed by western blotting and further analysis of O-glycans from HPA-bound and PNA-bound IgA1 fractions by mass spectrometry. Unexpectedly, we found that a variable minor fraction of IgA1 from both IgAN patients and healthy controls had Tn/STn antigens, and that the O-glycoprotein IgA1 molecules from most samples had only two distinct O-glycoforms: one major glycoform with homogeneous normal core 1-based O-glycans and one minor glycoform with homogeneous Tn/STn antigens. These results raised a serious question about the role of Tn/STn antigens on IgA1 in pathogenesis of IgAN, and there is a demand for a practical methodology that any laboratory can utilize to analyze the O-glycans of IgA1. Herein, we describe the methodology we developed in more detail. The method could also be applied to the analysis of any other O-glycosylated proteins. © 2017 Elsevier Inc. All rights reserved.

  13. Evaluation of intra- and extra-epithelial secretory IgA in chlamydial infections

    PubMed Central

    Armitage, Charles W; O’Meara, Connor P; Harvie, Marina C G; Timms, Peter; Wijburg, Odilia L; Beagley, Kenneth W

    2014-01-01

    Immunoglobulin A is an important mucosal antibody that can neutralize mucosal pathogens by either preventing attachment to epithelia (immune exclusion) or alternatively inhibit intra-epithelial replication following transcytosis by the polymeric immunoglobulin receptor (pIgR). Chlamydia trachomatis is a major human pathogen that initially targets the endocervical or urethral epithelium in women and men, respectively. As both tissues contain abundant secretory IgA (SIgA) we assessed the protection afforded by IgA targeting different chlamydial antigens expressed during the extra- and intra-epithelial stages of infection. We developed an in vitro model using polarizing cells expressing the murine pIgR together with antigen-specific mouse IgA, and an in vivo model using pIgR−/− mice. Secretory IgA targeting the extra-epithelial chlamydial antigen, the major outer membrane protein, significantly reduced infection in vitro by 24% and in vivo by 44%. Conversely, pIgR-mediated delivery of IgA targeting the intra-epithelial inclusion membrane protein A bound to the inclusion but did not reduce infection in vitro or in vivo. Similarly, intra-epithelial IgA targeting the secreted protease Chlamydia protease-like activity factor also failed to reduce infection. Together, these data suggest the importance of pIgR-mediated delivery of IgA targeting extra-epithelial, but not intra-epithelial, chlamydial antigens for protection against a genital tract infection. PMID:24827556

  14. Label Free QCM Immunobiosensor for AFB1 Detection Using Monoclonal IgA Antibody as Recognition Element.

    PubMed

    Ertekin, Özlem; Öztürk, Selma; Öztürk, Zafer Ziya

    2016-08-11

    This study introduces the use of an IgA isotype aflatoxin (AF) specific monoclonal antibody for the development of a highly sensitive Quartz Crystal Microbalance (QCM) immunobiosensor for the detection of AF in inhibitory immunoassay format. The higher molecular weight of IgA antibodies proved an advantage over commonly used IgG antibodies in label free immunobiosensor measurements. IgA and IgG antibodies with similar affinity for AF were used in the comparative studies. Sensor surface was prepared by covalent immobilization of AFB1, using self assembled monolayer (SAM) formed on gold coated Quartz Crystal, with 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxy succinimide (EDC/NHS) method using a diamine linker. Nonspecific binding to the surface was decreased by minimizing the duration of EDC/NHS activation. Sensor surface was chemically blocked after AF immobilization without any need for protein blocking. This protein free sensor chip endured harsh solutions with strong ionic detergent at high pH, which is required for the regeneration of the high affinity antibody-antigen interaction. According to the obtained results, the detection range with IgA antibodies was higher than IgG antibodies in QCM immunosensor developed for AFB1.

  15. Label Free QCM Immunobiosensor for AFB1 Detection Using Monoclonal IgA Antibody as Recognition Element

    PubMed Central

    Ertekin, Özlem; Öztürk, Selma; Öztürk, Zafer Ziya

    2016-01-01

    This study introduces the use of an IgA isotype aflatoxin (AF) specific monoclonal antibody for the development of a highly sensitive Quartz Crystal Microbalance (QCM) immunobiosensor for the detection of AF in inhibitory immunoassay format. The higher molecular weight of IgA antibodies proved an advantage over commonly used IgG antibodies in label free immunobiosensor measurements. IgA and IgG antibodies with similar affinity for AF were used in the comparative studies. Sensor surface was prepared by covalent immobilization of AFB1, using self assembled monolayer (SAM) formed on gold coated Quartz Crystal, with 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxy succinimide (EDC/NHS) method using a diamine linker. Nonspecific binding to the surface was decreased by minimizing the duration of EDC/NHS activation. Sensor surface was chemically blocked after AF immobilization without any need for protein blocking. This protein free sensor chip endured harsh solutions with strong ionic detergent at high pH, which is required for the regeneration of the high affinity antibody-antigen interaction. According to the obtained results, the detection range with IgA antibodies was higher than IgG antibodies in QCM immunosensor developed for AFB1. PMID:27529243

  16. [Protease activity of microflora in the oral cavity of patients with periodontitis].

    PubMed

    Voropaeva, E A; Baĭrakova, A L; Bichucher, A M; D'iakov, V L; Kozlov, L V

    2008-01-01

    Microbial spectrum and non-specific as well as specific IgA1 protease activity of isolated microorganisms were investigated in gingival liquid of patients with periodontitis. Microorganisms from the gingival liqud of these patients belonged to conditional-pathogenic obligate and facultatively anaerobic bacteria. 24 strains of microorganisms have been identified. Nonspecific proteolytic activity was found in the following microorganisms: Actinomyces israelii, Actinomyces naeslundii, Aerococcus viridans, Bifidobacterium longum, Neisseria subflave, Streptococcus parvulus, Eubacterium alactolyticum, Lactobaccilus catenoforme, Bacillus spp. Specific IgA1-protease activity and lack of proteolytic activity towards IgG was found in Streptococcus acidominimus, Streptococcus hansenii, Streptococcus salivarius, Leptotrychia buccalis, Staphylococcus haemolyticus and Neisseria sicca. No proteolytic activity was found in cultivation medium of Eubacterium alactolyticum (1 strain), Prevotella buccalis, Aerococcus viridans and Streptococcus sanguis.

  17. Lack of galectin-3 up-regulates IgA expression by peritoneal B1 lymphocytes during B cell differentiation.

    PubMed

    Oliveira, Felipe L; Bernardes, Emerson S; Brand, Camila; dos Santos, Sofia N; Cabanel, Mariana P; Arcanjo, Kátia D; Brito, José M; Borojevic, Radovan; Chammas, Roger; El-Cheikh, Márcia C

    2016-02-01

    Galectin-3 is a β-galactoside-binding protein with an inhibitory role in B cell differentiation into plasma cells in distinct lymphoid tissues. We use a model of chronic schistosomiasis, a well-characterized experimental disease hallmarked by polyclonal B cell activation, in order to investigate the role of galectin-3 in controlling IgA production through peritoneal B1 cells. Chronically infected, galectin-3-deficient mice (Lgals3(-/-)) display peritoneal fluid hypercellularity, increased numbers of atypical peritoneal IgM(+)/IgA(+) B1a and B1b lymphocytes and histological disturbances in plasma cell niches when compared with Lgals3(+/+) mice. Similar to our infection model, peritoneal B1 cells from uninfected Lgals3(-/-) mice show enhanced switching to IgA after in vitro treatment with interleukin-5 plus transforming growth factor-β (IL-5 + TGF-β1). A higher number of IgA(+) B1a lymphocytes was found in the peritoneal cavity of Lgals3(-/-)-uninfected mice at 1 week after i.p. injection of IL-5 + TGF-β1; this correlates with the increased levels of secreted IgA detected in the peritoneal fluid of these mice after cytokine treatment. Interestingly, a higher number of degranulated mast cells is present in the peritoneal cavity of uninfected and Schistosoma mansoni-infected Lgals3(-/-) mice, indicating that, at least in part, mast cells account for the enhanced differentiation of B1 into IgA-producing B cells found in the absence of galectin-3. Thus, a novel role is revealed for galectin-3 in controlling the expression of surface IgA by peritoneal B1 lymphocytes; this might have important implications for manipulating the mucosal immune response.

  18. Serum anti-phenolic glycolipid-1 IgA correlates to IgM isotype in leprosy patients: a possible candidate for seroepidemiological surveys?

    PubMed

    de Macedo, Alexandre C; Guimarães, Juliana A; Rodrigues, Raphael O; Araújo, Thiago D V; Tavares, Clodis M; Cabral, Paula B; de Moraes-Pinto, Maria Isabel; Nagao-Dias, Aparecida T

    2018-03-01

    The aim of this study was to compare serum anti-phenolic glycolipid-1 IgA, IgG, and IgM levels in leprosy patients and controls. Analysis of anti-PGL-1 IgA, IgG, or IgM in serum samples from multibacillary (MB, n=32) and paucibacillary (PB, n=22) leprosy patients, and in non-endemic controls (n=17), using an indirect enzyme-linked immunosorbent assay. A strong correlation between serum IgM and IgA isotypes was found (r=.745, P<.0001) in MB patients. A moderate correlation was found in all analyses in PB patients. A moderate agreement was found between anti-PGL1 IgA and IgM tests. Based on the ROC curves, the cut-off values were selected and the parameters of validation were calculated. Considering the clinical forms altogether, the diagnostic sensitivities were 50.0% for IgA, 22.2% for IgG, and 74.1% for IgM. The positive (VPP) and negative (VPN) predictive values were estimated for each isotype. For IgA, the VPP and VPN were, respectively, 100.0% (87.0%-100.0%; 95% confidence interval) and 38.7% (24.4%-54.5%); for IgG, 100% (87.0%-100.0%) and 28.8% (17.8%-42.1%), respectively; and for IgM, 95.2% (83.8%-99.4%) and 51.7% (32.5%-70.6%), respectively. Despite the limiting factors, anti-PGL1 IgA correlates to IgM levels and it could be considered as a possible laboratorial tool to be also used, for instance, in serological follow-up studies. © 2017 Wiley Periodicals, Inc.

  19. Vaccine-Induced Plasma IgA Specific for the C1 Region of the HIV-1 Envelope Blocks Binding and Effector Function of IgG

    DTIC Science & Technology

    2013-05-28

    uninfected vaccine recipients in RV144. Moreover, Env-specific IgA antibodies from RV144 vaccinees blocked the binding of ADCC-mediating mAb to HIV-1 Env... vaccine re- cipients in the case control study. There was a significantly greater number of infected vaccinees with IgA/IgG ratio >1e-02 (A1 Congp140 Env... vaccine efficacy. Second, we demonstrated that IgA mAbs isolated from RV144 vaccinees can both inhibit Env binding and block ADCC function of vaccine

  20. Oral Administration of β-1,3/1,6-Glucan to Dogs Temporally Changes Total and Antigen-Specific IgA and IgM▿

    PubMed Central

    Stuyven, E.; Verdonck, F.; Van Hoek, I.; Daminet, S.; Duchateau, L.; Remon, J. P.; Goddeeris, B. M.; Cox, E.

    2010-01-01

    The effect of oral administration of β-1,3/1,6-glucans from Saccharomyces cerevisiae on humoral immunity in domestic dogs is not known. In this study, 15 beagle dogs were orally given MacroGard tablets, which contain 150 mg of this β-glucan, daily for 4 weeks. At the end of this period, the total serum immunoglobulin A (IgA) level decreased significantly in the group treated with the glucan compared to that in the control group as well as compared to the concentrations before supplementation. In contrast, the total serum IgM level rose significantly, whereas no effect on the IgG level occurred. Similar changes were seen in Bordetella-specific IgA and IgM titers following vaccination during the supplementation period. The IgA concentration also became significantly lower in the saliva and tears of the glucan group than in the placebo group. The effects disappeared 1 week after the cessation of the supplementation. In conclusion, the results showed a temporary change in the isotype profile during glucan supplementation. PMID:20032218

  1. Comparison of the Specificities of IgG, IgG-Subclass, IgA and IgM Reactivities in African and European HIV-Infected Individuals with an HIV-1 Clade C Proteome-Based Array

    PubMed Central

    Gallerano, Daniela; Ndlovu, Portia; Makupe, Ian; Focke-Tejkl, Margarete; Fauland, Kerstin; Wollmann, Eva; Puchhammer-Stöckl, Elisabeth; Keller, Walter; Sibanda, Elopy; Valenta, Rudolf

    2015-01-01

    A comprehensive set of recombinant proteins and peptides of the proteome of HIV-1 clade C was prepared and purified and used to measure IgG, IgG-subclass, IgA and IgM responses in HIV-infected patients from Sub-Saharan Africa, where clade C is predominant. As a comparison group, HIV-infected patients from Europe were tested. African and European patients showed an almost identical antibody reactivity profile in terms of epitope specificity and involvement of IgG, IgG subclass, IgA and IgM responses. A V3-peptide of gp120 was identified as major epitope recognized by IgG1>IgG2 = IgG4>IgG3, IgA>IgM antibodies and a C-terminal peptide represented another major peptide epitope for the four IgG subclasses. By contrast, gp41-derived-peptides were mainly recognized by IgG1 but not by the other IgG subclasses, IgA or IgM. Among the non-surface proteins, protease, reverse transcriptase+RNAseH, integrase, as well as the capsid and matrix proteins were the most frequently and strongly recognized antigens which showed broad IgG subclass and IgA reactivity. Specificities and magnitudes of antibody responses in African patients were stable during disease and antiretroviral treatment, and persisted despite severe T cell loss. Using a comprehensive panel of gp120, gp41 peptides and recombinant non-surface proteins of HIV-1 clade C we found an almost identical antibody recognition profile in African and European patients regarding epitopes and involved IgG-sublass, IgA- and IgM-responses. Immune recognition of gp120 peptides and non-surface proteins involved all four IgG subclasses and was indicative of a mixed Th1/Th2 immune response. The HIV-1 clade C proteome-based test allowed diagnosis and monitoring of antibody responses in the course of HIV-infections and assessment of isotype and subclass responses. PMID:25658330

  2. Interdependence of Inhibitor Recognition in HIV-1 Protease.

    PubMed

    Paulsen, Janet L; Leidner, Florian; Ragland, Debra A; Kurt Yilmaz, Nese; Schiffer, Celia A

    2017-05-09

    Molecular recognition is a highly interdependent process. Subsite couplings within the active site of proteases are most often revealed through conditional amino acid preferences in substrate recognition. However, the potential effect of these couplings on inhibition and thus inhibitor design is largely unexplored. The present study examines the interdependency of subsites in HIV-1 protease using a focused library of protease inhibitors, to aid in future inhibitor design. Previously a series of darunavir (DRV) analogs was designed to systematically probe the S1' and S2' subsites. Co-crystal structures of these analogs with HIV-1 protease provide the ideal opportunity to probe subsite interdependency. All-atom molecular dynamics simulations starting from these structures were performed and systematically analyzed in terms of atomic fluctuations, intermolecular interactions, and water structure. These analyses reveal that the S1' subsite highly influences other subsites: the extension of the hydrophobic P1' moiety results in 1) reduced van der Waals contacts in the P2' subsite, 2) more variability in the hydrogen bond frequencies with catalytic residues and the flap water, and 3) changes in the occupancy of conserved water sites both proximal and distal to the active site. In addition, one of the monomers in this homodimeric enzyme has atomic fluctuations more highly correlated with DRV than the other monomer. These relationships intricately link the HIV-1 protease subsites and are critical to understanding molecular recognition and inhibitor binding. More broadly, the interdependency of subsite recognition within an active site requires consideration in the selection of chemical moieties in drug design; this strategy is in contrast to what is traditionally done with independent optimization of chemical moieties of an inhibitor.

  3. Serine protease activity in m-1 cortical collecting duct cells.

    PubMed

    Liu, Lian; Hering-Smith, Kathleen S; Schiro, Faith R; Hamm, L Lee

    2002-04-01

    An apical serine protease, channel-activating protease 1 (CAP1), augments sodium transport in A6 cells. Prostasin, a novel serine protease originally purified from seminal fluid, has been proposed to be the mammalian ortholog of CAP1. We have recently found functional evidence for a similar protease activity in the M-1 cortical collecting duct cell line. The purposes of the present studies were to determine whether prostasin (or CAP1) is present in collecting duct cells by use of mouse M-1 cells, to sequence mouse prostasin, and to further characterize the identity of the serine protease activity and additional functional features in M-1 cells. Using mouse expressed sequence tag sequences that are highly homologous to the published human prostasin sequence as templates, reverse transcription-polymerase chain reaction and RACE (rapid amplification of cDNA ends) were used to sequence mouse prostasin mRNA, which shows 99% identical to published mouse CAP1 sequence. A single 1800-bp transcript was found by Northern analysis, and this was not altered by aldosterone. Equivalent short-circuit current (I(eq)), which represents sodium transport in these cells, dropped to 59+/-3% of control value within 1 hour of incubation with aprotinin, a serine protease inhibitor. Trypsin increased the I(eq) in aprotinin-treated cells to the value of the control group within 5 minutes. Application of aprotinin not only inhibited amiloride sensitive I(eq) but also reduced transepithelial resistance (R(te)) to 43+/-2%, an effect not expected with simple inhibition of sodium channels. Trypsin partially reversed the effect of aprotinin on R(te). Another serine protease inhibitor, soybean trypsin inhibitor (STI), decreased I(eq) in M-1 cells. STI inhibited I(eq) gradually over 6 hours, and the inhibition of I(eq) by 2 inhibitors was additive. STI decreased transepithelial resistance much less than did aprotinin. Neither aldosterone nor dexamethasone significantly augmented protease activity

  4. Interdependence of Inhibitor Recognition in HIV-1 Protease

    PubMed Central

    2017-01-01

    Molecular recognition is a highly interdependent process. Subsite couplings within the active site of proteases are most often revealed through conditional amino acid preferences in substrate recognition. However, the potential effect of these couplings on inhibition and thus inhibitor design is largely unexplored. The present study examines the interdependency of subsites in HIV-1 protease using a focused library of protease inhibitors, to aid in future inhibitor design. Previously a series of darunavir (DRV) analogs was designed to systematically probe the S1′ and S2′ subsites. Co-crystal structures of these analogs with HIV-1 protease provide the ideal opportunity to probe subsite interdependency. All-atom molecular dynamics simulations starting from these structures were performed and systematically analyzed in terms of atomic fluctuations, intermolecular interactions, and water structure. These analyses reveal that the S1′ subsite highly influences other subsites: the extension of the hydrophobic P1′ moiety results in 1) reduced van der Waals contacts in the P2′ subsite, 2) more variability in the hydrogen bond frequencies with catalytic residues and the flap water, and 3) changes in the occupancy of conserved water sites both proximal and distal to the active site. In addition, one of the monomers in this homodimeric enzyme has atomic fluctuations more highly correlated with DRV than the other monomer. These relationships intricately link the HIV-1 protease subsites and are critical to understanding molecular recognition and inhibitor binding. More broadly, the interdependency of subsite recognition within an active site requires consideration in the selection of chemical moieties in drug design; this strategy is in contrast to what is traditionally done with independent optimization of chemical moieties of an inhibitor. PMID:28358514

  5. Neutralization Takes Precedence Over IgG or IgA Isotype-related Functions in Mucosal HIV-1 Antibody-mediated Protection.

    PubMed

    Astronomo, Rena D; Santra, Sampa; Ballweber-Fleming, Lamar; Westerberg, Katharine G; Mach, Linh; Hensley-McBain, Tiffany; Sutherland, Laura; Mildenberg, Benjamin; Morton, Georgeanna; Yates, Nicole L; Mize, Gregory J; Pollara, Justin; Hladik, Florian; Ochsenbauer, Christina; Denny, Thomas N; Warrier, Ranjit; Rerks-Ngarm, Supachai; Pitisuttithum, Punnee; Nitayapan, Sorachai; Kaewkungwal, Jaranit; Ferrari, Guido; Shaw, George M; Xia, Shi-Mao; Liao, Hua-Xin; Montefiori, David C; Tomaras, Georgia D; Haynes, Barton F; McElrath, Juliana M

    2016-12-01

    HIV-1 infection occurs primarily through mucosal transmission. Application of biologically relevant mucosal models can advance understanding of the functional properties of antibodies that mediate HIV protection, thereby guiding antibody-based vaccine development. Here, we employed a human ex vivo vaginal HIV-1 infection model and a rhesus macaque in vivo intrarectal SHIV challenge model to probe the protective capacity of monoclonal broadly-neutralizing (bnAb) and non-neutralizing Abs (nnAbs) that were functionally modified by isotype switching. For human vaginal explants, we developed a replication-competent, secreted NanoLuc reporter virus system and showed that CD4 binding site bnAbs b12 IgG1 and CH31 IgG1 and IgA2 isoforms potently blocked HIV-1 JR-CSF and HIV-1 Bal26 infection. However, IgG1 and IgA nnAbs, either alone or together, did not inhibit infection despite the presence of FcR-expressing effector cells in the tissue. In macaques, the CH31 IgG1 and IgA2 isoforms infused before high-dose SHIV challenge were completely to partially protective, respectively, while nnAbs (CH54 IgG1 and CH38 mIgA2) were non-protective. Importantly, in both mucosal models IgG1 isotype bnAbs were more protective than the IgA2 isotypes, attributable in part to greater neutralization activity of the IgG1 variants. These findings underscore the importance of potent bnAb induction as a primary goal of HIV-1 vaccine development. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  6. The proteases HtrA2/Omi and UCH-L1 regulate TNF-induced necroptosis

    PubMed Central

    2013-01-01

    Background In apoptosis, proteolysis by caspases is the primary mechanism for both initiation and execution of programmed cell death (PCD). In contrast, the impact of proteolysis on the regulation and execution of caspase-independent forms of PCD (programmed necrosis, necroptosis) is only marginally understood. Likewise, the identity of the involved proteases has remained largely obscure. Here, we have investigated the impact of proteases in TNF-induced necroptosis. Results The serine protease inhibitor TPKC protected from TNF-induced necroptosis in multiple murine and human cells systems whereas inhibitors of metalloproteinases or calpain/cysteine and cathepsin proteases had no effect. A screen for proteins labeled by a fluorescent TPCK derivative in necroptotic cells identified HtrA2/Omi (a serine protease previously implicated in PCD) as a promising candidate. Demonstrating its functional impact, pharmacological inhibition or genetic deletion of HtrA2/Omi protected from TNF-induced necroptosis. Unlike in apoptosis, HtrA2/Omi did not cleave another protease, ubiquitin C-terminal hydrolase (UCH-L1) during TNF-induced necroptosis, but rather induced monoubiquitination indicative for UCH-L1 activation. Correspondingly, pharmacologic or RNA interference-mediated inhibition of UCH-L1 protected from TNF-induced necroptosis. We found that UCH-L1 is a mediator of caspase-independent, non-apoptotic cell death also in diseased kidney podocytes by measuring cleavage of the protein PARP-1, caspase activity, cell death and cell morphology. Indicating a role of TNF in this process, podocytes with stably downregulated UCH-L1 proved resistant to TNF-induced necroptosis. Conclusions The proteases HtrA2/Omi and UCH-L1 represent two key components of TNF-induced necroptosis, validating the relevance of proteolysis not only for apoptosis, but also for caspase-independent PCD. Since UCH-L1 clearly contributes to the non-apoptotic death of podocytes, interference with the necroptotic

  7. Hinnuliquinone, a C2-symmetric dimeric non-peptide fungal metabolite inhibitor of HIV-1 protease.

    PubMed

    Singh, Sheo B; Ondeyka, John G; Tsipouras, Nasios; Ruby, Carolyn; Sardana, Vinod; Schulman, Marvin; Sanchez, Manuel; Pelaez, Fernando; Stahlhut, Mark W; Munshi, Sanjeev; Olsen, David B; Lingham, Russell B

    2004-11-05

    HIV-1 protease is one of several key enzymes required for the replication and maturation of HIV-1 virus. An almost two-decade research effort by academic and pharmaceutical institutions resulted in the successful commercialization of seven drugs that are potent inhibitors of HIV-1 protease activity and which, if used correctly, are highly effective in managing viral load. However, identification of clinical viral isolates that are resistant to these drugs indicates that this is a significant problem and that new classes of inhibitors are continually needed. Screening of microbial extracts followed by bioassay-guided isolation led to the discovery of a natural hinnuliquinone, a C(2)-symmetric bis-indolyl quinone natural product that inhibited the wild-type and a clinically resistant (A44) strain of HIV-1 protease with K(i) values of 0.97 and 1.25microM, respectively. Crystallographic analysis of the inhibitor-bound HIV-1 protease helped explain the importance of the C(2)-symmetry of hinnuliquinone for activity. Details of the isolation, biological activity, and crystallographic analysis of the inhibitor-bound protease are herein described.

  8. HvPap-1 C1A Protease and HvCPI-2 Cystatin Contribute to Barley Grain Filling and Germination1

    PubMed Central

    Velasco-Arroyo, Blanca; Cambra, Ines; Gonzalez-Melendi, Pablo; Lopez-Gonzalvez, Angeles; Garcia, Antonia

    2016-01-01

    Proteolysis is an essential process throughout the mobilization of storage proteins in barley (Hordeum vulgare) grains during germination. It involves numerous types of enzymes, with C1A Cys proteases the most abundant key players. Manipulation of the proteolytic machinery is a potential way to enhance grain yield and quality, and it could influence the mobilization of storage compounds along germination. Transgenic barley plants silencing or over-expressing the cathepsin F-like HvPap-1 Cys protease show differential accumulation of storage molecules such as starch, proteins, and free amino acids in the grain. It is particularly striking that the HvPap-1 artificial microRNA lines phenotype show a drastic delay in the grain germination process. Alterations to the proteolytic activities in the over-expressing and knock-down grains associated with changes in the level of expression of several C1A peptidases were also detected. Similarly, down-regulating cystatin Icy-2, one of the proteinaceous inhibitors of the cathepsin F-like protease, also has important effects on grain filling. However, the ultimate physiological influence of manipulating a peptidase or an inhibitor cannot be always predicted, since the plant tries to compensate the modified proteolytic effects by modulating the expression of some other peptidases or their inhibitors. PMID:26912343

  9. IgE, IgG4 and IgA specific to Bet v 1-related food allergens do not predict oral allergy syndrome.

    PubMed

    Guhsl, E E; Hofstetter, G; Lengger, N; Hemmer, W; Ebner, C; Fröschl, R; Bublin, M; Lupinek, C; Breiteneder, H; Radauer, C

    2015-01-01

    Birch pollen-associated plant food allergy is caused by Bet v 1-specific IgE, but presence of cross-reactive IgE to related allergens does not predict food allergy. The role of other immunoglobulin isotypes in the birch pollen-plant food syndrome has not been investigated in detail. Bet v 1-sensitized birch pollen-allergic patients (n = 35) were diagnosed for food allergy by standardized interviews, skin prick tests, prick-to-prick tests and ImmunoCAP. Concentrations of allergen-specific IgE, IgG1, IgG4 and IgA to seven Bet v 1-related food allergens were determined by ELISA. Bet v 1, Cor a 1, Mal d 1 and Pru p 1 bound IgE from all and IgG4 and IgA from the majority of sera. Immunoglobulins to Gly m 4, Vig r 1 and Api g 1.01 were detected in <65% of the sera. No significant correlation was observed between plant food allergy and increased or reduced levels of IgE, IgG1, IgG4 or IgA specific to most Bet v 1-related allergens. Api g 1-specific IgE was significantly (P = 0.01) elevated in celeriac-allergic compared with celeriac-tolerant patients. Likewise, frequencies of IgE (71% vs 15%; P = 0.01) and IgA (86% vs 38%; P = 0.04) binding to Api g 1.01 were increased. Measurements of allergen-specific immunoglobulins are not suitable for diagnosing Bet v 1-mediated plant food allergy to hazelnut and Rosaceae fruits. In contrast, IgE and IgA to the distantly related allergen Api g 1 correlate with allergy to celeriac. © 2014 The Authors. Allergy Published by John Wiley & Sons Ltd.

  10. Revealing interaction mode between HIV-1 protease and mannitol analog inhibitor.

    PubMed

    Yan, Guan-Wen; Chen, Yue; Li, Yixue; Chen, Hai-Feng

    2012-06-01

    HIV protease is a key enzyme to play a key role in the HIV-1 replication cycle and control the maturation from HIV viruses to an infectious virion. HIV-1 protease has become an important target for anti-HIV-1 drug development. Here, we used molecular dynamics simulation to study the binding mode between mannitol derivatives and HIV-1 protease. The results suggest that the most active compound (M35) has more stable hydrogen bonds and stable native contacts than the less active one (M17). These mannitol derivatives might have similar interaction mode with HIV-1 protease. Then, 3D-QSAR was used to construct quantitative structure-activity models. The cross-validated q(2) values are found as 0.728 and 0.611 for CoMFA and CoMSIA, respectively. And the non-cross-validated r(2) values are 0.973 and 0.950. Nine test set compounds validate the model. The results show that this model possesses better prediction ability than the previous work. This model can be used to design new chemical entities and make quantitative prediction of the bioactivities for HIV-1 protease inhibitors before resorting to in vitro and in vivo experiment. © 2012 John Wiley & Sons A/S.

  11. Design of novel HIV-1 protease inhibitors incorporating isophthalamide-derived P2-P3 ligands: Synthesis, biological evaluation and X-ray structural studies of inhibitor-HIV-1 protease complex

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ghosh, Arun K.; Brindisi, Margherita; Nyalapatla, Prasanth R.

    Based upon molecular insights from the X-ray structures of inhibitor-bound HIV-1 protease complexes, we have designed a series of isophthalamide-derived inhibitors incorporating substituted pyrrolidines, piperidines and thiazolidines as P2-P3 ligands for specific interactions in the S2-S3 extended site. Compound 4b has shown an enzyme Ki of 0.025 nM and antiviral IC50 of 69 nM. An X-ray crystal structure of inhibitor 4b-HIV-1 protease complex was determined at 1.33 Å resolution. We have also determined X-ray structure of 3b-bound HIV-1 protease at 1.27 Å resolution. These structures revealed important molecular insight into the inhibitor–HIV-1 protease interactions in the active site.

  12. The higher barrier of darunavir and tipranavir resistance for HIV-1 protease

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wang, Yong; Liu, Zhigang; Brunzelle, Joseph S.

    2011-11-17

    Darunavir and tipranavir are two inhibitors that are active against multi-drug resistant (MDR) HIV-1 protease variants. In this study, the invitro inhibitory efficacy was tested against a MDR HIV-1 protease variant, MDR 769 82T, containing the drug resistance mutations of 46L/54V/82T/84V/90M. Crystallographic and enzymatic studies were performed to examine the mechanism of resistance and the relative maintenance of potency. The key findings are as follows: (i) The MDR protease exhibits decreased susceptibility to all nine HIV-1 protease inhibitors approved by the US Food and Drug Administration (FDA), among which darunavir and tipranavir are the most potent; (ii) the threonine 82more » mutation on the protease greatly enhances drug resistance by altering the hydrophobicity of the binding pocket; (iii) darunavir or tipranavir binding facilitates closure of the wide-open flaps of the MDR protease; and (iv) the remaining potency of tipranavir may be preserved by stabilizing the flaps in the inhibitor-protease complex while darunavir maintains its potency by preserving protein main chain hydrogen bonds with the flexible P2 group. These results could provide new insights into drug design strategies to overcome multi-drug resistance of HIV-1 protease variants.« less

  13. HIV-1 protease inhibitor mutations affect the development of HIV-1 resistance to the maturation inhibitor bevirimat.

    PubMed

    Fun, Axel; van Maarseveen, Noortje M; Pokorná, Jana; Maas, Renée Em; Schipper, Pauline J; Konvalinka, Jan; Nijhuis, Monique

    2011-08-24

    Maturation inhibitors are an experimental class of antiretrovirals that inhibit Human Immunodeficiency Virus (HIV) particle maturation, the structural rearrangement required to form infectious virus particles. This rearrangement is triggered by the ordered cleavage of the precursor Gag polyproteins into their functional counterparts by the viral enzyme protease. In contrast to protease inhibitors, maturation inhibitors impede particle maturation by targeting the substrate of protease (Gag) instead of the protease enzyme itself. Direct cross-resistance between protease and maturation inhibitors may seem unlikely, but the co-evolution of protease and its substrate, Gag, during protease inhibitor therapy, could potentially affect future maturation inhibitor therapy. Previous studies showed that there might also be an effect of protease inhibitor resistance mutations on the development of maturation inhibitor resistance, but the exact mechanism remains unclear. We used wild-type and protease inhibitor resistant viruses to determine the impact of protease inhibitor resistance mutations on the development of maturation inhibitor resistance. Our resistance selection studies demonstrated that the resistance profiles for the maturation inhibitor bevirimat are more diverse for viruses with a mutated protease compared to viruses with a wild-type protease. Viral replication did not appear to be a major factor during emergence of bevirimat resistance. In all in vitro selections, one of four mutations was selected: Gag V362I, A364V, S368N or V370A. The impact of these mutations on maturation inhibitor resistance and viral replication was analyzed in different protease backgrounds. The data suggest that the protease background affects development of HIV-1 resistance to bevirimat and the replication profiles of bevirimat-selected HIV-1. The protease-dependent bevirimat resistance and replication levels can be explained by differences in CA/p2 cleavage processing by the different

  14. Docking analysis of gallic acid derivatives as HIV-1 protease inhibitors.

    PubMed

    Singh, Anjali; Pal, Tapan Kumar

    2015-01-01

    HIV-1 Protease (HIV-1 PR) enzymes are essential for accurate assembly and maturation of infectious HIV retroviruses. The significant role of HIV-1 protease in viral replication has made it a potential drug target. In the recent past, phytochemical Gallic Acid (GA) derivatives have been screened for protease inhibitor activity. The present work aims to design and evaluate potential GA-based HIV-1 PR phytoinhibitors by docking approach. The ligands were prepared by ChemDraw and docking was performed in HEX software. In this present study, one of the GA analogues (GA4) emerged as a potent drug candidate for HIV-1 PR inhibition, and docking results showed it to be comparable with anti-HIV drugs, darunavir and amprenavir. The GA4 derivative provided a lead for designing more effective HIV-1 PR inhibitors.

  15. Learning and memory deficits in mice lacking protease activated receptor-1

    PubMed Central

    Almonte, Antoine G.; Hamill, Cecily E.; Chhatwal, Jasmeer P.; Wingo, Thomas S.; Barber, Jeremy A.; Lyuboslavsky, Polina N.; Sweatt, J. David; Ressler, Kerry J.; White, David A.; Traynelis, Stephen F.

    2007-01-01

    The roles of serine proteases and protease activated receptors have been extensively studied in coagulation, wound healing, inflammation, and neurodegeneration. More recently, serine proteases have been suggested to influence synaptic plasticity. In this context, we examined the role of protease activated receptor 1 (PAR1), which is activated following proteolytic cleavage by thrombin and plasmin, in emotionally-motivated learning. We were particularly interested in PAR1 because its activation enhances the function of NMDA receptors, which are required for some forms of synaptic plasticity. We examined several baseline behavioral measures, including locomotor activity, expression of anxiety-like behavior, motor task acquisition, nociceptive responses, and startle responses in C57Bl/6 mice in which the PAR1 receptor has been genetically deleted. In addition, we evaluated learning and memory in these mice using two memory tasks, passive avoidance and cued fear-conditioning. Whereas locomotion, pain response, startle, and measures of baseline anxiety were largely unaffected by PAR1 removal, PAR1−/− animals showed significant deficits in a passive avoidance task and in cued fear conditioning. These data suggest that PAR1 may play an important role in emotionally-motivated learning. PMID:17544303

  16. The Chloroplast Protease AMOS1/EGY1 Affects Phosphate Homeostasis under Phosphate Stress1

    PubMed Central

    Yu, Fang Wei; Zhu, Xiao Fang; Li, Guang Jie; Kronzucker, Herbert J.; Shi, Wei Ming

    2016-01-01

    Plastid intramembrane proteases in Arabidopsis (Arabidopsis thaliana) are involved in jasmonic acid biosynthesis, chloroplast development, and flower morphology. Here, we show that Ammonium-Overly-Sensitive1 (AMOS1), a member of the family of plastid intramembrane proteases, plays an important role in the maintenance of phosphate (P) homeostasis under P stress. Loss of function of AMOS1 revealed a striking resistance to P starvation. amos1 plants displayed retarded root growth and reduced P accumulation in the root compared to wild type (Col-0) under P-replete control conditions, but remained largely unaffected by P starvation, displaying comparable P accumulation and root and shoot growth under P-deficient conditions. Further analysis revealed that, under P-deficient conditions, the cell wall, especially the pectin fraction of amos1, released more P than that of wild type, accompanied by a reduction of the abscisic acid (ABA) level and an increase in ethylene production. By using an ABA-insensitive mutant, abi4, and applying ABA and ACC exogenously, we found that ABA inhibits cell wall P remobilization while ethylene facilitates P remobilization from the cell wall by increasing the pectin concentration, suggesting ABA can counteract the effect of ethylene. Furthermore, the elevated ABA level and the lower ethylene production also correlated well with the mimicked P deficiency in amos1. Thus, our study uncovers the role of AMOS1 in the maintenance of P homeostasis through ABA-antagonized ethylene signaling. PMID:27516532

  17. Extreme Entropy-Enthalpy Compensation in a Drug Resistant Variant of HIV-1 Protease

    PubMed Central

    King, Nancy M.; Prabu-Jeyabalan, Moses; Bandaranayake, Rajintha M.; Nalam, Madhavi N. L.; Nalivaika, Ellen A.; Özen, Ayşegül; Haliloglu, Türkan; Yılmaz, Neşe Kurt; Schiffer, Celia A.

    2012-01-01

    The development of HIV-1 protease inhibitors has been the historic paradigm of rational structure-based drug design, where structural and thermodynamic analyses have assisted in the discovery of novel inhibitors. While the total enthalpy and entropy change upon binding determine the affinity, often the thermodynamics are considered in terms of inhibitor properties only. In the current study, profound changes are observed in the binding thermodynamics of a drug resistant variant compared to wild-type HIV-1 protease, irrespective of the inhibitor bound. This variant (Flap+) has a combination of flap and active site mutations and exhibits extremely large entropy-enthalpy compensation compared to wild-type protease, 5–15 kcal/mol, while losing only 1–3 kcal/mol in total binding free energy for any of six FDA approved inhibitors. Although entropy-enthalpy compensation has been previously observed for a variety of systems, never have changes of this magnitude been reported. The co-crystal structures of Flap+ protease with four of the inhibitors were determined and compared with complexes of both the wildtype protease and another drug resistant variant that does not exhibit this energetic compensation. Structural changes conserved across the Flap+ complexes, which are more pronounced for the flaps covering the active site, likely contribute to the thermodynamic compensation. The finding that drug resistant mutations can profoundly modulate the relative thermodynamic properties of a therapeutic target independent of the inhibitor presents a new challenge for rational drug design. PMID:22712830

  18. New insights into the pathogenesis of IgA nephropathy.

    PubMed

    Yeo, See Cheng; Cheung, Chee Kay; Barratt, Jonathan

    2018-05-01

    IgA nephropathy is the most common form of glomerulonephritis in many parts of the world and remains an important cause of end-stage renal disease. Current evidence suggests that IgA nephropathy is not due to a single pathogenic insult, but rather the result of multiple sequential pathogenic "hits". An abnormally increased level of circulating poorly O-galactosylated IgA1 and the production of O-glycan-specific antibodies leads to the formation of IgA1-containing immune complexes, and their subsequent mesangial deposition results in inflammation and glomerular injury. While this general framework has formed the foundation of our current understanding of the pathogenesis of IgA nephropathy, much work is ongoing to try to precisely define the genetic, epigenetic, immunological, and molecular basis of IgA nephropathy. In particular, the precise origin of poorly O-galactosylated IgA1 and the inciting factors for the production of O-glycan-specific antibodies continue to be intensely evaluated. The mechanisms responsible for mesangial IgA1 deposition and subsequent renal injury also remain incompletely understood. In this review, we summarize the current understanding of the key steps involved in the pathogenesis of IgA nephropathy. It is hoped that further advances in our understanding of this common glomerulonephritis will lead to novel diagnostic and prognostic biomarkers, and targeted therapies to ameliorate disease progression.

  19. Comparison of Antiviral Activity between IgA and IgG Specific to Influenza Virus Hemagglutinin: Increased Potential of IgA for Heterosubtypic Immunity

    PubMed Central

    Yokoyama, Ayaka; Miyamoto, Hiroko; Kajihara, Masahiro; Maruyama, Junki; Nao, Naganori; Manzoor, Rashid; Takada, Ayato

    2014-01-01

    Both IgA and IgG antibodies are known to play important roles in protection against influenza virus infection. While IgG is the major isotype induced systemically, IgA is predominant in mucosal tissues, including the upper respiratory tract. Although IgA antibodies are believed to have unique advantages in mucosal immunity, information on direct comparisons of the in vitro antiviral activities of IgA and IgG antibodies recognizing the same epitope is limited. In this study, we demonstrate differences in antiviral activities between these isotypes using monoclonal IgA and IgG antibodies obtained from hybridomas of the same origin. Polymeric IgA-producing hybridoma cells were successfully subcloned from those originally producing monoclonal antibody S139/1, a hemaggulutinin (HA)-specific IgG that was generated against an influenza A virus strain of the H3 subtype but had cross-neutralizing activities against the H1, H2, H13, and H16 subtypes. These monoclonal S139/1 IgA and IgG antibodies were assumed to recognize the same epitope and thus used to compare their antiviral activities. We found that both S139/1 IgA and IgG antibodies strongly bound to the homologous H3 virus in an enzyme-linked immunosorbent assay, and there were no significant differences in their hemagglutination-inhibiting and neutralizing activities against the H3 virus. In contrast, S139/1 IgA showed remarkably higher cross-binding to and antiviral activities against H1, H2, and H13 viruses than S139/1 IgG. It was also noted that S139/1 IgA, but not IgG, drastically suppressed the extracellular release of the viruses from infected cells. Electron microscopy revealed that S139/1 IgA deposited newly produced viral particles on the cell surface, most likely by tethering the particles. These results suggest that anti-HA IgA has greater potential to prevent influenza A virus infection than IgG antibodies, likely due to increased avidity conferred by its multivalency, and that this advantage may be

  20. Development and evaluation of a phenotypic assay monitoring resistance formation to protease inhibitors in HIV-1-infected patients.

    PubMed

    Gehringer, Heike; Von der Helm, Klaus; Seelmeir, Sigrid; Weissbrich, Benedikt; Eberle, Josef; Nitschko, Hans

    2003-05-01

    A novel phenotypic assay, based on recombinant expression of the HIV-1-protease was developed and evaluated; it monitors the formation of resistance to protease inhibitors. The HIV-1 protease-encoding region from the blood sample of patients was amplified, ligated into the expression vector pBD2, and recombinantly expressed in Escherichia coli TG1 cells. The resulting recombinant enzyme was purified by a newly developed one-step acid extraction protocol. The protease activity was determined in presence of five selected HIV protease inhibitors and the 50% inhibitory concentration (IC(50)) to the respective protease inhibitors determined. The degree of resistance was expressed in terms of x-fold increase in IC(50) compared to the IC(50) value of an HIV-1 wild type protease preparation. The established test system showed a reproducible recombinant expression of each individual patients' HIV-1 protease population. Samples of nine clinically well characterised HIV-1-infected patients with varying degrees of resistance were analysed. There was a good correlation between clinical parameters and the results obtained by this phenotypic assay. For the majority of patients a blind genotypic analysis of the patients' protease domain revealed a fair correlation to the results of the phenotypic assay. In a minority of patients our phenotypic results diverged from the genotypic ones. This novel phenotypic assay can be carried out within 8-10 days, and offers a significant advantage in time to the current employed phenotypic tests.

  1. [A case of IgA2-lambda type M-protein that IgA concentration differs from the values of M-protein by serum protein electrophoresis].

    PubMed

    Fukushima, M; Sugano, M; Ichikawa, T; Honda, T; Totsuka, M; Katsuyama, T; Fujita, K

    2001-07-01

    We report an IgA-lambda type M-protein in which the IgA concentration differed from the values of M-protein by serum protein electrophoresis found in a 53-year-old man with multiple myeloma. The M-protein value as determined by serum protein electrophoresis was 6,170 mg/dl. However, the serum IgA concentration was 3,052 mg/dl by turbidimetric immunoassay. Immuno-fixation electrophoresis using IgA subclass antisera revealed that this M-protein was the IgA2-lambda type. Western blotting analysis showed that the IgA2 molecules were composed of two approximately 68 kDa alpha 2 chains and two 28 kDa lambda chains. In addition the free lambda chain band was detected at the position of 28 kDa without 2-mercaptoethanol(2-ME) even though the patient IgA was purified. Since it is known that IgA2m(1) allotype easily release light chains from the IgA molecules in SDS-PAGE without 2-ME, we speculated that in this patient the IgA was the IgA2m(1) allotype. After peripheral blood stem cell transplantation(PBSCT), immunofixation electrophoresis of the patient serum revealed not only the bands of IgA2-lambda type M-protein, but also three bands of IgG1-kappa type M-protein in the gamma region.

  2. IgA and IgG1 reactivities assessed by flow cytometry mirror clinical aspects of infants with ocular congenital toxoplasmosis.

    PubMed

    de Jesus, Laura Néspoli Nassar Pansini; Tonini, Aline de Castro Zacche; Barros, Geisa Baptista; Coelho-dos-Reis, Jordana Grazziela A; Béla, Samantha Ribeiro; Antonelli, Lis Ribeiro do Valle; Machado, Anderson Silva; Carneiro, Ana Carolina Aguiar Vasconcelos; Andrade, Gláucia Manzan Queiroz; Vasconcelos-Santos, Daniel Vitor; Januário, José Nélio; Teixeira-Carvalho, Andréa; Vitor, Ricardo Wagner Almeida; Ferro, Eloísa A V; Mineo, José Roberto; Bahia-Oliveira, Lilian Maria Garcia; Martins-Filho, Olindo Assis; Lemos, Elenice Moreira

    2016-01-01

    This study intended to apply the flow cytometric analysis of IgA and IgG reactivity and intracytoplasmic cytokine analysis to understand and decode the clinical aspects of infants with ocular congenital toxoplasmosis. The Toxoplasma gondii-infected infants (TOXO) were subdivided according to their clinical aspects based on the absence (NRL), presence of active (ARL), active/cicatricial (ACRL) or cicatricial retinochoroidal lesions (CRL) and compared to non-infected controls (NI). The reactivity of anti-T. gondii IgG subclasses resembles the clinical aspects of ocular lesions. IgG and IgG1 discriminate infants with cicatricial lesions (ACRL and CRL) from both ARL and NLR. IgG2 and IgG3 are particularly higher in ACRL and CRL as compared to NLR. No differences were observed when IgG4 reactivity was evaluated. Thus, the results indicated that the reactivity patterns of IgA, IgG and IgG subclasses are able to discriminate ARL, ACRL and CRL from NLR or NI. IgA and IgG subclasses are relevant serological biomarkers with diagnostic and prognostic applicability, respectively. Moreover, IgA and IgG1 were closely related to cytokine production by innate/adaptive immunity cells. IgA reactivity was directly associated to TNF-α-derived from neutrophils, monocytes and CD8(+) T-cells, while IgG1 was inversely correlated with IFN-γ-producing CD4(+) and CD8(+) T-cells but positively correlated with IL-10(+) B-cells. These findings provide insights on the relationship between the cytokine production by innate/adaptive immunity and the antibody pattern of infants with ocular congenital toxoplasmosis. In addition, the present study supports the use of flow cytometric serology as a potential tool for the diagnosis and monitoring of ocular lesions in T. gondii-infected infants in the clinical setting. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Structural, kinetic, and thermodynamic studies of specificity designed HIV-1 protease.

    PubMed

    Alvizo, Oscar; Mittal, Seema; Mayo, Stephen L; Schiffer, Celia A

    2012-07-01

    HIV-1 protease recognizes and cleaves more than 12 different substrates leading to viral maturation. While these substrates share no conserved motif, they are specifically selected for and cleaved by protease during viral life cycle. Drug resistant mutations evolve within the protease that compromise inhibitor binding but allow the continued recognition of all these substrates. While the substrate envelope defines a general shape for substrate recognition, successfully predicting the determinants of substrate binding specificity would provide additional insights into the mechanism of altered molecular recognition in resistant proteases. We designed a variant of HIV protease with altered specificity using positive computational design methods and validated the design using X-ray crystallography and enzyme biochemistry. The engineered variant, Pr3 (A28S/D30F/G48R), was designed to preferentially bind to one out of three of HIV protease's natural substrates; RT-RH over p2-NC and CA-p2. In kinetic assays, RT-RH binding specificity for Pr3 increased threefold compared to the wild-type (WT), which was further confirmed by isothermal titration calorimetry. Crystal structures of WT protease and the designed variant in complex with RT-RH, CA-p2, and p2-NC were determined. Structural analysis of the designed complexes revealed that one of the engineered substitutions (G48R) potentially stabilized heterogeneous flap conformations, thereby facilitating alternate modes of substrate binding. Our results demonstrate that while substrate specificity could be engineered in HIV protease, the structural pliability of protease restricted the propagation of interactions as predicted. These results offer new insights into the plasticity and structural determinants of substrate binding specificity of the HIV-1 protease. Copyright © 2012 The Protein Society.

  4. Identification of Broad-Based HIV-1 Protease Inhibitors From Combinatorial Libraries

    PubMed Central

    Chang, Max W.; Giffin, Michael J.; Muller, Rolf; Savage, Jeremiah; Lin, Ying C.; Hong, Sukwon; Jin, Wei; Whitby, Landon R.; Elder, John H.; Boger, Dale L.; Torbett, Bruce E.

    2011-01-01

    Clinically approved inhibitors of HIV-1 protease function via a competitive mechanism. A particular vulnerability of competitive inhibitors is their sensitivity to increases in substrate concentration, as may occur during virion assembly, budding and processing into a mature, infectious viral particle. Advances in chemical synthesis have led to the development of new chemical libraries with high diversity using rapid in-solution syntheses. These libraries have been previously shown to be effective at disrupting protein-protein and protein-nucleic acid interfaces. We have screened 44,000 compounds from such a library to identify inhibitors of HIV-1 protease. One compound was identified that inhibits wild type protease, as well as a drug-resistant protease with 6 mutations. Moreover, analysis of this compound suggests an allosteric, non-competitive mechanism of inhibition and may represent a starting point for an additional strategy for anti-retroviral therapy. PMID:20507280

  5. Intracellular serine protease 1 of Bacillus subtilis is formed in vivo as an unprocessed, active protease in stationary cells.

    PubMed Central

    Sheehan, S M; Switzer, R L

    1990-01-01

    Western immunoblots and assays of Bacillus subtilis extracts showed that intracellular serine protease 1 is produced in a form larger than previously reported, appears not to have undergone N-terminal processing, and is active in the presence or absence of calcium. No evidence for an inactive precursor form of the protease was found. Images FIG. 1 PMID:2104610

  6. Human Plasma-derived Polymeric IgA and IgM Antibodies Associate with Secretory Component to Yield Biologically Active Secretory-like Antibodies*

    PubMed Central

    Longet, Stéphanie; Miled, Sarah; Lötscher, Marius; Miescher, Sylvia M.; Zuercher, Adrian W.; Corthésy, Blaise

    2013-01-01

    Immunotherapy with monoclonal and polyclonal immunoglobulin is successfully applied to improve many clinical conditions, including infection, autoimmune diseases, or immunodeficiency. Most immunoglobulin products, recombinant or plasma-derived, are based on IgG antibodies, whereas to date, the use of IgA for therapeutic application has remained anecdotal. In particular, purification or production of large quantities of secretory IgA (SIgA) for potential mucosal application has not been achieved. In this work, we sought to investigate whether polymeric IgA (pIgA) recovered from human plasma is able to associate with secretory component (SC) to generate SIgA-like molecules. We found that ∼15% of plasma pIgA carried J chain and displayed selective SC binding capacity either in a mixture with monomeric IgA (mIgA) or after purification. The recombinant SC associated covalently in a 1:1 stoichiometry with pIgA and with similar efficacy as colostrum-derived SC. In comparison with pIgA, the association with SC delayed degradation of SIgA by intestinal proteases. Similar results were obtained with plasma-derived IgM. In vitro, plasma-derived IgA and SIgA neutralized Shigella flexneri used as a model pathogen, resulting in a delay of bacteria-induced damage targeted to polarized Caco-2 cell monolayers. The sum of these novel data demonstrates that association of plasma-derived IgA or IgM with recombinant/colostrum-derived SC is feasible and yields SIgA- and SIgM-like molecules with similar biochemical and functional characteristics as mucosa-derived immunoglobulins. PMID:23250751

  7. Evaluation of retro-inverso modifications of HTLV-1 protease inhibitors containing a hydroxyethylamine isoster.

    PubMed

    Tatsumi, Tadashi; Awahara, Chiyuki; Naka, Hiromi; Aimoto, Saburo; Konno, Hiroyuki; Nosaka, Kazuto; Akaji, Kenichi

    2010-04-01

    Effects of retro-inverso (RI) modifications of HTLV-1 protease inhibitors containing a hydroxyethylamine isoster backbone were clarified. Construction of the isoster backbone was achieved by a stereoselective aldol reaction. Four diastereomers with different configurations at the isoster hydroxyl site and the scissile site substituent were synthesized. Inhibitory activities of the new inhibitors suggest that partially modified RI inhibitors would interact with HTLV-1 protease in the same manner as the parent hydroxyethylamine inhibitor. Copyright 2010 Elsevier Ltd. All rights reserved.

  8. Detection of IgA antibodies and quantification of IgA antibody-producing cells specific for ovalbumin or Trichinella spiralis in the rat.

    PubMed

    Van Loveren, H; Osterhaus, A D; Nagel, J; Schuurman, H J; Vos, J G

    1988-09-01

    This report describes procedures to quantify IgA responses in the rat sensitized to ovalbumin or infected with the parasite Trichinella spiralis: an ELISPOT detecting specific IgA antibody-producing cells in lymph nodes, and an ELISA demonstrating IgA antibody in serum and gut mucosal scrapings. For this purpose a mouse monoclonal anti-rat IgA antibody was produced. This IgG1-kappa 1 antibody recognized rat IgA but not rat IgM, IgG, or IgE. It proved very suitable in both assays. Using this reagent we could demonstrate large numbers of IgA anti-ovalbumin-producing cells in the mesenteric lymph nodes 15 days after sensitization to ovalbumin via the Peyer's patches. At 28 days after sensitization the numbers were much lower. IgA antibody titres to ovalbumin in serum were maximal between days 14 and 21 after immunization. Maximal numbers of IgA anti-T. spiralis-producing cells were found in the mesenteric lymph nodes 12 days after infection with muscle larvae, followed by a sharp decrease at 15 days. Maximal IgA anti-T. spiralis antibody titres in serum and mucus scrapings of small intestines were found on days 10 and 12 after oral infection with the parasite.

  9. Conformations of the HIV-1 protease: A crystal structure data set analysis.

    PubMed

    Palese, Luigi Leonardo

    2017-11-01

    The HIV protease is an important drug target for HIV/AIDS therapy, and its structure and function have been extensively investigated. This enzyme performs an essential role in viral maturation by processing specific cleavage sites in the Gag and Gag-Pol precursor polyproteins so as to release their mature forms. This 99 amino acid aspartic protease works as a homodimer, with the active site localized in a central cavity capped by two flexible flap regions. The dimer presents closed or open conformations, which are involved in the substrate binding and release. Here the results of the analysis of a HIV-1 protease data set containing 552 dimer structures are reported. Different dimensionality reduction methods have been used in order to get information from this multidimensional database. Most of the structures in the data set belong to two conformational clusters. An interesting observation that comes from the analysis of these data is that some protease sequences are localized preferentially in specific areas of the conformational landscape of this protein. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. HIV-1 Protease in the Fission Yeast Schizosaccharomyces pombe.

    PubMed

    Benko, Zsigmond; Elder, Robert T; Li, Ge; Liang, Dong; Zhao, Richard Y

    2016-01-01

    HIV-1 protease (PR) is an essential viral enzyme. Its primary function is to proteolyze the viral Gag-Pol polyprotein for production of viral enzymes and structural proteins and for maturation of infectious viral particles. Increasing evidence suggests that PR cleaves host cellular proteins. However, the nature of PR-host cellular protein interactions is elusive. This study aimed to develop a fission yeast (Schizosaccharomyces pombe) model system and to examine the possible interaction of HIV-1 PR with cellular proteins and its potential impact on cell proliferation and viability. A fission yeast strain RE294 was created that carried a single integrated copy of the PR gene in its chromosome. The PR gene was expressed using an inducible nmt1 promoter so that PR-specific effects could be measured. HIV-1 PR from this system cleaved the same indigenous viral p6/MA protein substrate as it does in natural HIV-1 infections. HIV-1 PR expression in fission yeast cells prevented cell proliferation and induced cellular oxidative stress and changes in mitochondrial morphology that led to cell death. Both these PR activities can be prevented by a PR-specific enzymatic inhibitor, indinavir, suggesting that PR-mediated proteolytic activities and cytotoxic effects resulted from enzymatic activities of HIV-1 PR. Through genome-wide screening, a serine/threonine kinase, Hhp2, was identified that suppresses HIV-1 PR-induced protease cleavage and cell death in fission yeast and in mammalian cells, where it prevented PR-induced apoptosis and cleavage of caspase-3 and caspase-8. This is the first report to show that HIV-1 protease is functional as an enzyme in fission yeast, and that it behaves in a similar manner as it does in HIV-1 infection. HIV-1 PR-induced cell death in fission yeast could potentially be used as an endpoint for mechanistic studies, and this system could be used for developing a high-throughput system for drug screenings.

  11. Sweet potato SPAP1 is a typical aspartic protease and participates in ethephon-mediated leaf senescence.

    PubMed

    Chen, Hsien-Jung; Huang, Yu-Hsuan; Huang, Guan-Jhong; Huang, Shyh-Shyun; Chow, Te-Jin; Lin, Yaw-Huei

    2015-05-15

    Plant aspartic proteases are generally divided into three categories: typical, nucellin-like, and atypical aspartic proteases based on their gene and protein structures. In this report, a full-length cDNA SPAP1 was cloned from sweet potato leaves, which contained 1515 nucleotides (504 amino acids) and exhibited high amino acid sequence identity (ca. 51-72%) with plant typical aspartic proteases, including tomato LeAspP, potato StAsp, and wheat WAP2. SPAP1 also contained conserved DTG and DSG amino acid residues within its catalytic domain and plant specific insert (PSI) at the C-terminus. The cDNA corresponding to the mature protein (starting from the 66th to 311th amino acid residues) without PSI domain was constructed with pET30a expression vector for fusion protein and antibody production. RT-PCR and protein blot hybridization showed that SPAP1 expression level was the highest in L3 mature leaves, then gradually declined until L5 completely yellow leaves. Ethephon, an ethylene-releasing compound, also enhanced SPAP1 expression at the time much earlier than the onset of leaf senescence. Exogenous application of SPAP1 fusion protein promoted ethephon-induced leaf senescence, which could be abolished by pre-treatment of SPAP1 fusion protein with (a) 95 °C for 5 min, (b) aspartic protease inhibitor pepstatin A, and (c) anti-SPAP1 antibody, respectively. Exogenous SPAP1 fusion protein, whereas, did not significantly affect leaf senescence under dark. These data conclude that sweet potato SPAP1 is a functional typical aspartic protease and participates in ethephon-mediated leaf senescence. The SPAP1-promoted leaf senescence and its activity are likely not associated with the PSI domain. Interaction of ethephon-inducible components for effective SPAP1 promotion on leaf senescence is also suggested. Copyright © 2015 Elsevier GmbH. All rights reserved.

  12. Levels and complexity of IgA antibody against oral bacteria in samples of human colostrum.

    PubMed

    Petrechen, L N; Zago, F H; Sesso, M L T; Bertoldo, B B; Silva, C B; Azevedo, K P; de Lima Pereira, S A; Geraldo-Martins, V R; Ferriani, V P L; Nogueira, R D

    2015-01-01

    Streptococcus mutans (SM) have three main virulence antigens: glucan binding protein B (gbpB), glucosyltransferase (Gtf) and antigens I/II (Ag I/II) envolved in the capacity of those bacteria to adhere and accumulate in the dental biofilm. Also, the glycosyltransferases 153 kDa of Streptococcus gordonii (SGO) and 170kDa of Streptococcus sanguinis (SSA) were important antigens associated with the accumulation of those bacterias. Streptococcus mitis (SMI) present IgA1 protease of 202 kDa. We investigated the specificity and levels IgA against those antigens of virulence in samples of human colostrum. This study involved 77 samples of colostrum that were analyzed for levels of immunoglobulian A, M and G by Elisa. The specificity of IgA against extracts of SM and initials colonizators (SSA, SMI, SGO) were analyzed by the Western blot. The mean concentration of IgA was 2850.2 (±2567.2) mg/100 mL followed by IgM and IgG (respectively 321.8±90.3 and 88.3±51.5), statistically different (p<0.05). Results showed that the majority of samples had detectable levels of IgA antibodies to extracts of bacteria antigens and theirs virulence antigens. To SM, the GbpB was significantly lower detected than others antigens of SM (p<0.05). High complexities of response to Ags were identified in the samples. There were no significant differences in the mean number of IgA-reactive Ags between the antigens (p>0.4). So, the breast milk from first hours after birth presented significant levels of IgA specific against important virulence of antigens those oral streptococci, which can disrupt the installation and accumulation process of these microorganisms in the oral cavity. Copyright © 2014 Elsevier GmbH. All rights reserved.

  13. HvPap-1 C1A Protease and HvCPI-2 Cystatin Contribute to Barley Grain Filling and Germination.

    PubMed

    Diaz-Mendoza, Mercedes; Dominguez-Figueroa, Jose D; Velasco-Arroyo, Blanca; Cambra, Ines; Gonzalez-Melendi, Pablo; Lopez-Gonzalvez, Angeles; Garcia, Antonia; Hensel, Goetz; Kumlehn, Jochen; Diaz, Isabel; Martinez, Manuel

    2016-04-01

    Proteolysis is an essential process throughout the mobilization of storage proteins in barley (Hordeum vulgare) grains during germination. It involves numerous types of enzymes, with C1A Cys proteases the most abundant key players. Manipulation of the proteolytic machinery is a potential way to enhance grain yield and quality, and it could influence the mobilization of storage compounds along germination. Transgenic barley plants silencing or over-expressing the cathepsin F-like HvPap-1 Cys protease show differential accumulation of storage molecules such as starch, proteins, and free amino acids in the grain. It is particularly striking that the HvPap-1 artificial microRNA lines phenotype show a drastic delay in the grain germination process. Alterations to the proteolytic activities in the over-expressing and knock-down grains associated with changes in the level of expression of several C1A peptidases were also detected. Similarly, down-regulating cystatin Icy-2, one of the proteinaceous inhibitors of the cathepsin F-like protease, also has important effects on grain filling. However, the ultimate physiological influence of manipulating a peptidase or an inhibitor cannot be always predicted, since the plant tries to compensate the modified proteolytic effects by modulating the expression of some other peptidases or their inhibitors. © 2016 American Society of Plant Biologists. All Rights Reserved.

  14. Structure Determination of Mycobacterium tuberculosis Serine Protease Hip1 (Rv2224c).

    PubMed

    Naffin-Olivos, Jacqueline L; Daab, Andrew; White, Andre; Goldfarb, Nathan E; Milne, Amy C; Liu, Dali; Baikovitz, Jacqueline; Dunn, Ben M; Rengarajan, Jyothi; Petsko, Gregory A; Ringe, Dagmar

    2017-05-02

    The Mycobacterium tuberculosis (Mtb) serine protease Hip1 (hydrolase important for pathogenesis; Rv2224c) promotes tuberculosis (TB) pathogenesis by impairing host immune responses through proteolysis of a protein substrate, Mtb GroEL2. The cell surface localization of Hip1 and its immunomodulatory functions make Hip1 a good drug target for new adjunctive immune therapies for TB. Here, we report the crystal structure of Hip1 to a resolution of 2.6 Å and the kinetic studies of the enzyme against model substrates and the protein GroEL2. The structure shows a two-domain protein, one of which contains the catalytic residues that are the signature of a serine protease. Surprisingly, a threonine is located within the active site close enough to hydrogen bond with the catalytic residues Asp463 and His490. Mutation of this residue, Thr466, to alanine established its importance for function. Our studies provide insights into the structure of a member of a novel family of proteases. Knowledge of the Hip1 structure will aid in designing inhibitors that could block Hip1 activity.

  15. Kinetic characterization of the critical step in HIV-1 protease maturation.

    PubMed

    Sadiq, S Kashif; Noé, Frank; De Fabritiis, Gianni

    2012-12-11

    HIV maturation requires multiple cleavage of long polyprotein chains into functional proteins that include the viral protease itself. Initial cleavage by the protease dimer occurs from within these precursors, and yet only a single protease monomer is embedded in each polyprotein chain. Self-activation has been proposed to start from a partially dimerized protease formed from monomers of different chains binding its own N termini by self-association to the active site, but a complete structural understanding of this critical step in HIV maturation is missing. Here, we captured the critical self-association of immature HIV-1 protease to its extended amino-terminal recognition motif using large-scale molecular dynamics simulations, thus confirming the postulated intramolecular mechanism in atomic detail. We show that self-association to a catalytically viable state requires structural cooperativity of the flexible β-hairpin "flap" regions of the enzyme and that the major transition pathway is first via self-association in the semiopen/open enzyme states, followed by enzyme conformational transition into a catalytically viable closed state. Furthermore, partial N-terminal threading can play a role in self-association, whereas wide opening of the flaps in concert with self-association is not observed. We estimate the association rate constant (k(on)) to be on the order of ∼1 × 10(4) s(-1), suggesting that N-terminal self-association is not the rate-limiting step in the process. The shown mechanism also provides an interesting example of molecular conformational transitions along the association pathway.

  16. Plasmodium subtilisin-like protease 1 (SUB1): insights into the active-site structure, specificity and function of a pan-malaria drug target.

    PubMed

    Withers-Martinez, Chrislaine; Suarez, Catherine; Fulle, Simone; Kher, Samir; Penzo, Maria; Ebejer, Jean-Paul; Koussis, Kostas; Hackett, Fiona; Jirgensons, Aigars; Finn, Paul; Blackman, Michael J

    2012-05-15

    Release of the malaria merozoite from its host erythrocyte (egress) and invasion of a fresh cell are crucial steps in the life cycle of the malaria pathogen. Subtilisin-like protease 1 (SUB1) is a parasite serine protease implicated in both processes. In the most dangerous human malarial species, Plasmodium falciparum, SUB1 has previously been shown to have several parasite-derived substrates, proteolytic cleavage of which is important both for egress and maturation of the merozoite surface to enable invasion. Here we have used molecular modelling, existing knowledge of SUB1 substrates, and recombinant expression and characterisation of additional Plasmodium SUB1 orthologues, to examine the active site architecture and substrate specificity of P. falciparum SUB1 and its orthologues from the two other major human malaria pathogens Plasmodium vivax and Plasmodium knowlesi, as well as from the rodent malaria species, Plasmodium berghei. Our results reveal a number of unusual features of the SUB1 substrate binding cleft, including a requirement to interact with both prime and non-prime side residues of the substrate recognition motif. Cleavage of conserved parasite substrates is mediated by SUB1 in all parasite species examined, and the importance of this is supported by evidence for species-specific co-evolution of protease and substrates. Two peptidyl alpha-ketoamides based on an authentic PfSUB1 substrate inhibit all SUB1 orthologues examined, with inhibitory potency enhanced by the presence of a carboxyl moiety designed to introduce prime side interactions with the protease. Our findings demonstrate that it should be possible to develop 'pan-reactive' drug-like compounds that inhibit SUB1 in all three major human malaria pathogens, enabling production of broad-spectrum antimalarial drugs targeting SUB1. Copyright © 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

  17. Gluten exacerbates IgA nephropathy in humanized mice through gliadin-CD89 interaction.

    PubMed

    Papista, Christina; Lechner, Sebastian; Ben Mkaddem, Sanae; LeStang, Marie-Bénédicte; Abbad, Lilia; Bex-Coudrat, Julie; Pillebout, Evangéline; Chemouny, Jonathan M; Jablonski, Mathieu; Flamant, Martin; Daugas, Eric; Vrtovsnik, François; Yiangou, Minas; Berthelot, Laureline; Monteiro, Renato C

    2015-08-01

    IgA1 complexes containing deglycosylated IgA1, IgG autoantibodies, and a soluble form of the IgA receptor (sCD89), are hallmarks of IgA nephropathy (IgAN). Food antigens, notably gluten, are associated with increased mucosal response and IgAN onset, but their implication in the pathology remains unknown. Here, an IgAN mouse model expressing human IgA1 and CD89 was used to examine the role of gluten in IgAN. Mice were given a gluten-free diet for three generations to produce gluten sensitivity, and then challenged for 30 days with a gluten diet. A gluten-free diet resulted in a decrease of mesangial IgA1 deposits, transferrin 1 receptor, and transglutaminase 2 expression, as well as hematuria. Mice on a gluten-free diet lacked IgA1-sCD89 complexes in serum and kidney eluates. Disease severity depended on gluten and CD89, as shown by reappearance of IgAN features in mice on a gluten diet and by direct binding of the gluten-subcomponent gliadin to sCD89. A gluten diet exacerbated intestinal IgA1 secretion, inflammation, and villous atrophy, and increased serum IgA1 anti-gliadin antibodies, which correlated with proteinuria in mice and patients. Moreover, early treatment of humanized mice with a gluten-free diet prevented mesangial IgA1 deposits and hematuria. Thus, gliadin-CD89 interaction may aggravate IgAN development through induction of IgA1-sCD89 complex formation and a mucosal immune response. Hence, early-stage treatment with a gluten-free diet could be beneficial to prevent disease.

  18. Monoclonal IgA Antibodies for Aflatoxin Immunoassays

    PubMed Central

    Ertekin, Özlem; Pirinçci, Şerife Şeyda; Öztürk, Selma

    2016-01-01

    Antibody based techniques are widely used for the detection of aflatoxins which are potent toxins with a high rate of occurrence in many crops. We developed a murine monoclonal antibody of immunoglobulin A (IgA) isotype with a strong binding affinity to aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2) and aflatoxin M1 (AFM1). The antibody was effectively used in immunoaffinity column (IAC) and ELISA kit development. The performance of the IACs was compatible with AOAC performance standards for affinity columns (Test Method: AOAC 991.31). The total binding capacity of the IACs containing our antibody was 111 ng, 70 ng, 114 ng and 73 ng for AFB1, AFB2, and AFG1 andAFG2, respectively. Furthermore, the recovery rates of 5 ng of each AF derivative loaded to the IACs were determined as 104.9%, 82.4%, 85.5% and 70.7% for AFB1, AFB2, AFG1 and AFG2, respectively. As for the ELISA kit developed using non-oriented, purified IgA antibody, we observed a detection range of 2–50 µg/L with 40 min total test time. The monoclonal antibody developed in this research is hitherto the first presentation of quadruple antigen binding IgA monoclonal antibodies in mycotoxin analysis and also the first study of their utilization in ELISA and IACs. IgA antibodies are valuable alternatives for immunoassay development, in terms of both sensitivity and ease of preparation, since they do not require any orientation effort. PMID:27187470

  19. Toxoplasma gondii serine-protease inhibitor-1: A new adjuvant candidate for asthma therapy.

    PubMed

    Soto, Ariadna S; Fenoy, Ignacio M; Sanchez, Vanesa R; March, Florencia; Perrone Sibilia, Matías D; Aldirico, María de Los Angeles; Picchio, Mariano S; Arcon, Nadia; Acosta, Patricio L; Polack, Fernando P; Martin, Valentina; Goldman, Alejandra

    2017-01-01

    Serine-proteases are important players in the pathogenesis of asthma, promoting inflammation and tissue remodeling. It's also known that many serine protease inhibitors display immunomodulatory properties. TgPI-1 is a Toxoplasma gondii protein that exhibits broad spectrum inhibitory activity against serine proteases. In view of the increased prevalence of atopic disorders and the need to develop new treatment strategies we sought to investigate the potential of TgPI-1 for treating respiratory allergies. For this purpose, we developed a therapeutic experimental model. BALB/c mice were rendered allergic by intraperitoneal ovalbumin-alum sensitization and airway-challenged. Once the asthmatic phenotype was achieved, mice were intranasally treated with rTgPI-1 alone or with a mixture of rTgPI-1 and ovalbumin (OVA). A week later mice were given a secondary aerosol challenge. Treatment with rTgPI-1 alone or co-administered with OVA diminished bronchoalveolar eosinophilia, mucus production and peribronchial lung infiltration. This effect was accompanied by a lung resistance reduction of 26.3% and 50.3% respectively. Both treatments resulted in the production of lower levels of IL-4, IL-5, IFN-γ and regulatory IL-10 by thoracic lymph node cells stimulated with OVA. Interestingly, significant decreases in OVA specific IgE and T cell proliferation, and increases in FoxP3+ T cells at local and systemic levels were only detected when the inhibitor was administered along with OVA. These results show that both rTgPI-1 treatments reduced asthma hallmarks. However, co-administration of the inhibitor with the allergen was more effective. Hence, rTgPI-1 emerges as a novel adjuvant candidate for asthma treatment.

  20. Toxoplasma gondii serine-protease inhibitor-1: A new adjuvant candidate for asthma therapy

    PubMed Central

    Soto, Ariadna S.; Fenoy, Ignacio M.; Sanchez, Vanesa R.; March, Florencia; Perrone Sibilia, Matías D.; Aldirico, María de los Angeles; Picchio, Mariano S.; Arcon, Nadia; Acosta, Patricio L.; Polack, Fernando P.; Martin, Valentina

    2017-01-01

    Serine-proteases are important players in the pathogenesis of asthma, promoting inflammation and tissue remodeling. It’s also known that many serine protease inhibitors display immunomodulatory properties. TgPI-1 is a Toxoplasma gondii protein that exhibits broad spectrum inhibitory activity against serine proteases. In view of the increased prevalence of atopic disorders and the need to develop new treatment strategies we sought to investigate the potential of TgPI-1 for treating respiratory allergies. For this purpose, we developed a therapeutic experimental model. BALB/c mice were rendered allergic by intraperitoneal ovalbumin-alum sensitization and airway-challenged. Once the asthmatic phenotype was achieved, mice were intranasally treated with rTgPI-1 alone or with a mixture of rTgPI-1 and ovalbumin (OVA). A week later mice were given a secondary aerosol challenge. Treatment with rTgPI-1 alone or co-administered with OVA diminished bronchoalveolar eosinophilia, mucus production and peribronchial lung infiltration. This effect was accompanied by a lung resistance reduction of 26.3% and 50.3% respectively. Both treatments resulted in the production of lower levels of IL-4, IL-5, IFN-γ and regulatory IL-10 by thoracic lymph node cells stimulated with OVA. Interestingly, significant decreases in OVA specific IgE and T cell proliferation, and increases in FoxP3+ T cells at local and systemic levels were only detected when the inhibitor was administered along with OVA. These results show that both rTgPI-1 treatments reduced asthma hallmarks. However, co-administration of the inhibitor with the allergen was more effective. Hence, rTgPI-1 emerges as a novel adjuvant candidate for asthma treatment. PMID:29073215

  1. Loss of hippocampal serine protease BSP1/neuropsin predisposes to global seizure activity.

    PubMed

    Davies, B; Kearns, I R; Ure, J; Davies, C H; Lathe, R

    2001-09-15

    Serine proteases in the adult CNS contribute both to activity-dependent structural changes accompanying learning and to the regulation of excitotoxic cell death. Brain serine protease 1 (BSP1)/neuropsin is a trypsin-like serine protease exclusively expressed, within the CNS, in the hippocampus and associated limbic structures. To explore the role of this enzyme, we have used gene targeting to disrupt this gene in mice. Mutant mice were viable and overtly normal; they displayed normal hippocampal long-term synaptic potentiation (LTP) and exhibited no deficits in spatial navigation (water maze). Nevertheless, electrophysiological studies revealed that the hippocampus of mice lacking this specifically expressed protease possessed an increased susceptibility for hyperexcitability (polyspiking) in response to repetitive afferent stimulation. Furthermore, seizure activity on kainic acid administration was markedly increased in mutant mice and was accompanied by heightened immediate early gene (c-fos) expression throughout the brain. In view of the regional selectivity of BSP1/neuropsin brain expression, the observed phenotype may selectively reflect limbic function, further implicating the hippocampus and amygdala in controlling cortical activation. Within the hippocampus, our data suggest that BSP1/neuropsin, unlike other serine proteases, has little effect on physiological synaptic remodeling and instead plays a role in limiting neuronal hyperexcitability induced by epileptogenic insult.

  2. Protease signaling through protease activated receptor 1 mediate nerve activation by mucosal supernatants from irritable bowel syndrome but not from ulcerative colitis patients

    PubMed Central

    Buhner, Sabine; Hahne, Hannes; Hartwig, Kerstin; Li, Qin; Vignali, Sheila; Ostertag, Daniela; Meng, Chen; Hörmannsperger, Gabriele; Braak, Breg; Pehl, Christian; Frieling, Thomas; Barbara, Giovanni; De Giorgio, Roberto; Demir, Ihsan Ekin; Ceyhan, Güralp Onur; Zeller, Florian; Boeckxstaens, Guy; Haller, Dirk; Kuster, Bernhard

    2018-01-01

    Background & aims The causes of gastrointestinal complaints in irritable bowel syndrome (IBS) remain poorly understood. Altered nerve function has emerged as an important pathogenic factor as IBS mucosal biopsy supernatants consistently activate enteric and sensory neurons. We investigated the neurally active molecular components of such supernatants from patients with IBS and quiescent ulcerative colitis (UC). Method Effects of supernatants from 7 healthy controls (HC), 20 IBS and 12 UC patients on human and guinea pig submucous neurons were studied with neuroimaging techniques. We identify differentially expressed proteins with proteome analysis. Results Nerve activation by IBS supernatants was prevented by the protease activated receptor 1 (PAR1) antagonist SCHE79797. UC supernatants also activated enteric neurons through protease dependent mechanisms but without PAR1 involvement. Proteome analysis of the supernatants identified 204 proteins, among them 17 proteases as differentially expressed between IBS, UC and HC. Of those the four proteases elastase 3a, chymotrypsin C, proteasome subunit type beta-2 and an unspecified isoform of complement C3 were significantly more abundant in IBS compared to HC and UC supernatants. Of eight proteases, which were upregulated in IBS, the combination of elastase 3a, cathepsin L and proteasome alpha subunit-4 showed the highest prediction accuracy of 98% to discriminate between IBS and HC groups. Elastase synergistically potentiated the effects of histamine and serotonin–the two other main neuroactive substances in the IBS supernatants. A serine protease inhibitor isolated from the probiotic Bifidobacterium longum NCC2705 (SERPINBL), known to inhibit elastase-like proteases, prevented nerve activation by IBS supernatants. Conclusion Proteases in IBS and UC supernatants were responsible for nerve activation. Our data demonstrate that proteases, particularly those signalling through neuronal PAR1, are biomarker candidates for

  3. Lower serum IgA is associated with COPD exacerbation risk in SPIROMICS

    PubMed Central

    Paul, Gabriel G.; Azar, Antoine; Wise, Robert A.; O’Neal, Wanda K.; Dransfield, Mark T.; Woodruff, Prescott G.; Curtis, Jeffrey L.; Comellas, Alejandro P.; Drummond, M. Bradley; Lambert, Allison A.; Paulin, Laura M.; Fawzy, Ashraf; Kanner, Richard E.; Paine, Robert; Han, MeiLan K.; Martinez, Fernando J.; Bowler, Russell P.; Barr, R. Graham; Hansel, Nadia N.

    2018-01-01

    Background Decreased but measurable serum IgA levels (≤70 mg/dL) have been associated with risk for infections in some populations, but are unstudied in COPD. This study tested the hypothesis that subnormal serum IgA levels would be associated with exacerbation risk in COPD. Methods Data were analyzed from 1,049 COPD participants from the observational cohort study SPIROMICS (535 (51%) women; mean age 66.1 (SD 7.8), 338 (32%) current smokers) who had baseline serum IgA measured using the Myriad RBM biomarker discovery platform. Exacerbation data was collected prospectively (mean 944.3 (SD 281.3) days), and adjusted linear, logistic and zero-inflated negative binomial regressions were performed. Results Mean IgA was 269.1 mg/dL (SD 150.9). One individual had deficient levels of serum IgA (<7 mg/dL) and 25 (2.4%) had IgA level ≤70 mg/dL. Participants with IgA ≤70 mg/dL were younger (62 vs. 66 years, p = 0.01) but otherwise similar to those with higher IgA. In adjusted models, IgA ≤70 mg/dL was associated with higher exacerbation incidence rates (IRR 1.71, 95% CI 1.01–2.87, p = 0.044) and greater risk for any severe exacerbation (OR 2.99, 95% CI 1.30–6.94, p = 0.010). In adjusted models among those in the lowest decile (<120 mg/dL), each 10 mg/dL decrement in IgA (analyzed continuously) was associated with more exacerbations during follow-up (β 0.24, 95% CI 0.017–0.46, p = 0.035). Conclusions Subnormal serum IgA levels were associated with increased risk for acute exacerbations, supporting mildly impaired IgA levels as a contributing factor in COPD morbidity. Additionally, a dose-response relationship between lower serum IgA and number of exacerbations was found among individuals with serum IgA in the lowest decile, further supporting the link between serum IgA and exacerbation risk. Future COPD studies should more comprehensively characterize immune status to define the clinical relevance of these findings and their potential for therapeutic correction

  4. The QSAR and docking calculations of fullerene derivatives as HIV-1 protease inhibitors

    NASA Astrophysics Data System (ADS)

    Saleh, Noha A.

    2015-02-01

    The inhibition of HIV-1 protease is considered as one of the most important targets for drug design and the deactivation of HIV-1. In the present work, the fullerene surface (C60) is modified by adding oxygen atoms as well as hydroxymethylcarbonyl (HMC) groups to form 6 investigated fullerene derivative compounds. These compounds have one, two, three, four or five O atoms + HMC groups at different positions on phenyl ring. The effect of the repeating of these groups on the ability of suggested compounds to inhibit the HIV protease is studied by calculating both Quantitative Structure Activity Relationship (QSAR) properties and docking simulation. Based on the QSAR descriptors, the solubility and the hydrophilicity of studied fullerene derivatives increased with increasing the number of oxygen atoms + HMC groups in the compound. While docking calculations indicate that, the compound with two oxygen atoms + HMC groups could interact and binds with HIV-1 protease active site. This is could be attributed to the active site residues of HIV-1 protease are hydrophobic except the two aspartic acids. So that, the increase in the hydrophilicity and polarity of the compound is preventing and/or decreasing the hydrophobic interaction between the compound and HIV-1 protease active site.

  5. Structure Determination of Mycobacterium tuberculosis Serine Protease Hip1 (Rv2224c)

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Naffin-Olivos, Jacqueline L.; Daab, Andrew; White, Andre

    The Mycobacterium tuberculosis (Mtb) serine protease Hip1 (hydrolase important for pathogenesis; Rv2224c) promotes tuberculosis (TB) pathogenesis by impairing host immune responses through proteolysis of a protein substrate, Mtb GroEL2. The cell surface localization of Hip1 and its immunomodulatory functions make Hip1 a good drug target for new adjunctive immune therapies for TB. Here, we report the crystal structure of Hip1 to a resolution of 2.6 Å and the kinetic studies of the enzyme against model substrates and the protein GroEL2. The structure shows a two-domain protein, one of which contains the catalytic residues that are the signature of a serinemore » protease. Surprisingly, a threonine is located within the active site close enough to hydrogen bond with the catalytic residues Asp463 and His490. Mutation of this residue, Thr466, to alanine established its importance for function. Our studies provide insights into the structure of a member of a novel family of proteases. Knowledge of the Hip1 structure will aid in designing inhibitors that could block Hip1 activity« less

  6. An efficient procedure for the expression and purification of HIV-1 protease from inclusion bodies.

    PubMed

    Nguyen, Hong-Loan Thi; Nguyen, Thuy Thi; Vu, Quy Thi; Le, Hang Thi; Pham, Yen; Trinh, Phuong Le; Bui, Thuan Phuong; Phan, Tuan-Nghia

    2015-12-01

    Several studies have focused on HIV-1 protease for developing drugs for treating AIDS. Recombinant HIV-1 protease is used to screen new drugs from synthetic compounds or natural substances. However, large-scale expression and purification of this enzyme is difficult mainly because of its low expression and solubility. In this study, we constructed 9 recombinant plasmids containing a sequence encoding HIV-1 protease along with different fusion tags and examined the expression of the enzyme from these plasmids. Of the 9 plasmids, pET32a(+) plasmid containing the HIV-1 protease-encoding sequence along with sequences encoding an autocleavage site GTVSFNF at the N-terminus and TEV plus 6× His tag at the C-terminus showed the highest expression of the enzyme and was selected for further analysis. The recombinant protein was isolated from inclusion bodies by using 2 tandem Q- and Ni-Sepharose columns. SDS-PAGE of the obtained HIV-1 protease produced a single band of approximately 13 kDa. The enzyme was recovered efficiently (4 mg protein/L of cell culture) and had high specific activity of 1190 nmol min(-1) mg(-1) at an optimal pH of 4.7 and optimal temperature of 37 °C. This procedure for expressing and purifying HIV-1 protease is now being scaled up to produce the enzyme on a large scale for its application. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Plasma Gelsolin Induced Glomerular Fibrosis via the TGF-β1/Smads Signal Transduction Pathway in IgA Nephropathy

    PubMed Central

    Zhang, Lei; Han, Changsong; Ye, Fei; He, Yan; Jin, Yinji; Wang, Tianzhen; Wu, Yiqi; Jiang, Yang; Zhang, Fengmin; Jin, Xiaoming

    2017-01-01

    Glomerular fibrosis has been shown to be closely related to the progression and prognosis of IgA nephropathy (IgAN). However, mechanism underlying IgAN glomerular fibrosis remains unclear. Recently, our study showed that plasma gelsolin (pGSN) was decreased in the serum of an IgAN mouse model and that pGSN deposition was found in the glomeruli. Another cytokine, TGF-β1, which is closely related to glomerular fibrosis, was also found to be highly expressed in the glomeruli. In the present study, we report that pGSN induces glomerular fibrosis through the TGF-β1/Smads signal transduction pathway. This is supported by the following findings: human mesangial cells (HMCs) show remarkable morphological changes and proliferation in response to co-stimulation with pGSN and polymeric IgA1 (pIgA1) from IgAN patients compared to other controls. Moreover, ELISA assays showed that more TGF-β1 secretion was found in HMCs supernatants in the co-stimulation group. Further experiments showed increased TGF-β1, Smad3, p-Smad2/3, Smad4, and collagen 1 and decreased Smad7 expression in the co-stimulation group. Our present study implied that the synergistic effect of pGSN and pIgA induced glomerular fibrosis via the TGF-β1/Smads signal transduction pathway. This might be a potential mechanism for the glomerular fibrosis observed in IgAN patients. PMID:28208683

  8. Protease nexin 1 is a potent urinary plasminogen activator inhibitor in the presence of collagen type IV.

    PubMed

    Crisp, Robert J; Knauer, Mary F; Knauer, Daniel J

    2002-12-06

    Protease nexin 1 (PN1) in solution forms inhibitory complexes with thrombin or urokinase, which have opposing effects on the blood coagulation cascade. An initial report provided data supporting the idea that PN1 target protease specificity is under the influence of collagen type IV (1). Although collagen type IV demonstrated no effect on the association rate between PN1 and thrombin, the study reported that the association rate between PN1 and urokinase was allosterically reduced 10-fold. This has led to the generally accepted idea that the primary role of PN1 in the brain is to act as a rapid thrombin inhibition and clearance mechanism during trauma and loss of vascular integrity. In studies to identify the structural determinants of PN1 that mediate the allosteric interaction with collagen type IV, we found that protease specificity was only affected after transient exposure of PN1 to acidic conditions that mimic the elution protocol from a monoclonal antibody column. Because PN1 used in previous studies was purified over a monoclonal antibody column, we propose that the allosteric regulation of PN1 target protease specificity by collagen type IV is a result of the purification protocol. We provide both biochemical and kinetic data to support this conclusion. This finding is significant because it implies that PN1 may play a much larger role in the modeling and remodeling of brain tissues during development and is not simply an extravasated thrombin clearance mechanism as previously suggested.

  9. Potential elucidation of a novel CTL epitope in HIV-1 protease by the protease inhibitor resistance mutation L90M.

    PubMed

    Smidt, Werner

    2013-01-01

    The combination of host immune responses and use of antiretrovirals facilitate partial control of human immunodeficiency virus type 1 (HIV-1) infection and result in delayed progression to Acquired Immunodeficiency Syndrome (AIDS). Both treatment and host immunity impose selection pressures on the highly mutable HIV-1 genome resulting in antiretroviral resistance and immune escape. Researchers have shown that antiretroviral resistance mutations can shape cytotoxic T-lymphocyte immunity by altering the epitope repertoire of HIV infected cells. Here it was discovered that an important antiretroviral resistance mutation, L90M in HIV protease, occurs at lower frequencies in hosts that harbor the B*15, B*48 or A*32 human leukocyte antigen subtypes. A likely reason is the elucidation of novel epitopes by L90M. NetMHCPan predictions reveal increased affinity of the peptide spanning the HIV protease region, PR 89-97 and PR 90-99 to HLA-B*15/B*48 and HLA-A*32 respectively due to the L90M substitution. The higher affinity could increase the chance of the epitope being presented and recognized by Cytotoxic T-lymphocytes and perhaps provide additional immunological pressures in the presence of antiretroviral attenuating mutations. This evidence supports the notion that knowledge of HLA allotypes in HIV infected individuals could augment antiretroviral treatment by the elucidation of epitopes due to antiretroviral resistance mutations in HIV protease.

  10. Lung inflammation promotes metastasis through neutrophil protease-mediated degradation of Tsp-1

    PubMed Central

    El Rayes, Tina; Catena, Raúl; Lee, Sharrell; Stawowczyk, Marcin; Joshi, Natasha; Fischbach, Claudia; Powell, Charles A.; Dannenberg, Andrew J.; Altorki, Nasser K.; Gao, Dingcheng; Mittal, Vivek

    2015-01-01

    Inflammation is inextricably associated with primary tumor progression. However, the contribution of inflammation to tumor outgrowth in metastatic organs has remained underexplored. Here, we show that extrinsic inflammation in the lungs leads to the recruitment of bone marrow-derived neutrophils, which degranulate azurophilic granules to release the Ser proteases, elastase and cathepsin G, resulting in the proteolytic destruction of the antitumorigenic factor thrombospondin-1 (Tsp-1). Genetic ablation of these neutrophil proteases protected Tsp-1 from degradation and suppressed lung metastasis. These results provide mechanistic insights into the contribution of inflammatory neutrophils to metastasis and highlight the unique neutrophil protease–Tsp-1 axis as a potential antimetastatic therapeutic target. PMID:26668367

  11. Altered Substrate Specificity of Drug-Resistant Human Immunodeficiency Virus Type 1 Protease

    PubMed Central

    Dauber, Deborah S.; Ziermann, Rainer; Parkin, Neil; Maly, Dustin J.; Mahrus, Sami; Harris, Jennifer L.; Ellman, Jon A.; Petropoulos, Christos; Craik, Charles S.

    2002-01-01

    Resistance to human immunodeficiency virus type 1 protease (HIV PR) inhibitors results primarily from the selection of multiple mutations in the protease region. Because many of these mutations are selected for the ability to decrease inhibitor binding in the active site, they also affect substrate binding and potentially substrate specificity. This work investigates the substrate specificity of a panel of clinically derived protease inhibitor-resistant HIV PR variants. To compare protease specificity, we have used positional-scanning, synthetic combinatorial peptide libraries as well as a select number of individual substrates. The subsite preferences of wild-type HIV PR determined by using the substrate libraries are consistent with prior reports, validating the use of these libraries to compare specificity among a panel of HIV PR variants. Five out of seven protease variants demonstrated subtle differences in specificity that may have significant impacts on their abilities to function in viral maturation. Of these, four variants demonstrated up to fourfold changes in the preference for valine relative to alanine at position P2 when tested on individual peptide substrates. This change correlated with a common mutation in the viral NC/p1 cleavage site. These mutations may represent a mechanism by which severely compromised, drug-resistant viral strains can increase fitness levels. Understanding the altered substrate specificity of drug-resistant HIV PR should be valuable in the design of future generations of protease inhibitors as well as in elucidating the molecular basis of regulation of proteolysis in HIV. PMID:11773410

  12. Antimalarial activity of HIV-1 protease inhibitor in chromone series.

    PubMed

    Lerdsirisuk, Pradith; Maicheen, Chirattikan; Ungwitayatorn, Jiraporn

    2014-12-01

    Increasing parasite resistance to nearly all available antimalarial drugs becomes a serious problem to human health and necessitates the need to continue the search for new effective drugs. Recent studies have shown that clinically utilized HIV-1 protease (HIV-1 PR) inhibitors can inhibit the in vitro and in vivo growth of Plasmodium falciparum. In this study, a series of chromone derivatives possessing HIV-1 PR inhibitory activity has been tested for antimalarial activity against P. falciparum (K1 multi-drug resistant strain). Chromone 15, the potent HIV-1 PR inhibitor (IC50=0.65μM), was found to be the most potent antimalarial compound with IC50=0.95μM while primaquine and tafenoquine showed IC50=2.41 and 1.95μM, respectively. Molecular docking study of chromone compounds against plasmepsin II, an aspartic protease enzyme important in hemoglobin degradation, revealed that chromone 15 exhibited the higher binding affinity (binding energy=-13.24kcal/mol) than the known PM II inhibitors. Thus, HIV-1 PR inhibitor in chromone series has the potential to be a new class of antimalarial agent. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. Analysis of correlated mutations in HIV-1 protease using spectral clustering.

    PubMed

    Liu, Ying; Eyal, Eran; Bahar, Ivet

    2008-05-15

    The ability of human immunodeficiency virus-1 (HIV-1) protease to develop mutations that confer multi-drug resistance (MDR) has been a major obstacle in designing rational therapies against HIV. Resistance is usually imparted by a cooperative mechanism that can be elucidated by a covariance analysis of sequence data. Identification of such correlated substitutions of amino acids may be obscured by evolutionary noise. HIV-1 protease sequences from patients subjected to different specific treatments (set 1), and from untreated patients (set 2) were subjected to sequence covariance analysis by evaluating the mutual information (MI) between all residue pairs. Spectral clustering of the resulting covariance matrices disclosed two distinctive clusters of correlated residues: the first, observed in set 1 but absent in set 2, contained residues involved in MDR acquisition; and the second, included those residues differentiated in the various HIV-1 protease subtypes, shortly referred to as the phylogenetic cluster. The MDR cluster occupies sites close to the central symmetry axis of the enzyme, which overlap with the global hinge region identified from coarse-grained normal-mode analysis of the enzyme structure. The phylogenetic cluster, on the other hand, occupies solvent-exposed and highly mobile regions. This study demonstrates (i) the possibility of distinguishing between the correlated substitutions resulting from neutral mutations and those induced by MDR upon appropriate clustering analysis of sequence covariance data and (ii) a connection between global dynamics and functional substitution of amino acids.

  14. Structural insights into the mechanisms of drug resistance in HIV-1 protease NL4-3.

    PubMed

    Heaslet, Holly; Kutilek, Victoria; Morris, Garrett M; Lin, Ying-Chuan; Elder, John H; Torbett, Bruce E; Stout, C David

    2006-03-03

    The development of resistance to anti-retroviral drugs targeted against HIV is an increasing clinical problem in the treatment of HIV-1-infected individuals. Many patients develop drug-resistant strains of the virus after treatment with inhibitor cocktails (HAART therapy), which include multiple protease inhibitors. Therefore, it is imperative that we understand the mechanisms by which the viral proteins, in particular HIV-1 protease, develop resistance. We have determined the three-dimensional structure of HIV-1 protease NL4-3 in complex with the potent protease inhibitor TL-3 at 2.0 A resolution. We have also obtained the crystal structures of three mutant forms of NL4-3 protease containing one (V82A), three (V82A, M46I, F53L) and six (V82A, M46I, F53L, V77I, L24I, L63P) point mutations in complex with TL-3. The three protease mutants arose sequentially under ex vivo selective pressure in the presence of TL-3, and exhibit fourfold, 11-fold, and 30-fold resistance to TL-3, respectively. This series of protease crystal structures offers insights into the biochemical and structural mechanisms by which the enzyme can overcome inhibition by TL-3 while recovering some of its native catalytic activity.

  15. Rapamycin ameliorates IgA nephropathy via cell cycle-dependent mechanisms

    PubMed Central

    Tian, Jihua; Wang, Yanhong; Liu, Xinyan; Zhou, Xiaoshuang

    2014-01-01

    IgA nephropathy is the most frequent type of glomerulonephritis worldwide. The role of cell cycle regulation in the pathogenesis of IgA nephropathy has been studied. The present study was designed to explore whether rapamycin ameliorates IgA nephropathy via cell cycle-dependent mechanisms. After establishing an IgA nephropathy model, rats were randomly divided into four groups. Coomassie Brilliant Blue was used to measure the 24-h urinary protein levels. Renal function was determined using an autoanalyzer. Proliferation was assayed via Proliferating Cell Nuclear Antigen (PCNA) immunohistochemistry. Rat mesangial cells were cultured and divided into the six groups. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) and flow cytometry were used to detect cell proliferation and the cell cycle phase. Western blotting was performed to determine cyclin E, cyclin-dependent kinase 2, p27Kip1, p70S6K/p-p70S6K, and extracellular signal-regulated kinase 1/2/p- extracellular signal-regulated kinase 1/2 protein expression. A low dose of the mammalian target of rapamycin (mTOR) inhibitor rapamycin prevented an additional increase in proteinuria, protected kidney function, and reduced IgA deposition in a model of IgA nephropathy. Rapamycin inhibited mesangial cell proliferation and arrested the cell cycle in the G1 phase. Rapamycin did not affect the expression of cyclin E and cyclin-dependent kinase 2. However, rapamycin upregulated p27Kip1 at least in part via AKT (also known as protein kinase B)/mTOR. In conclusion, rapamycin can affect cell cycle regulation to inhibit mesangial cell proliferation, thereby reduce IgA deposition, and slow the progression of IgAN. PMID:25349217

  16. Spontaneous remission of IgA nephropathy associated with resolution of hepatitis A.

    PubMed

    Han, Seung Hyeok; Kang, Ea Wha; Kie, Jeong Hae; Yoo, Tae Hyun; Choi, Kyu Hun; Han, Dae-Suk; Kang, Shin-Wook

    2010-12-01

    Although most cases of immunoglobulin A (IgA) nephropathy are idiopathic, several diseases are associated with IgA nephropathy. Of these, chronic liver disease resulting from hepatitis B or C virus infection has been reported as a secondary cause of IgA nephropathy. Recently, hepatitis A virus (HAV)-associated kidney disease has received attention because acute kidney injury can occur as a complication of HAV infection, generally caused by acute tubular necrosis or interstitial nephritis. However, unlike IgA nephropathy related to hepatitis B or C, HAV-associated IgA nephropathy is extremely rare and long-term outcomes have not been reported yet. We describe a case of spontaneous remission of IgA nephropathy associated with serologically documented HAV infection. The patient presented with microhematuria and moderate proteinuria, but acute kidney injury did not occur during active hepatic injury. Kidney biopsy specimens clearly showed mesangial IgA deposits with intact tubules and interstitium. Serum liver enzyme levels returned to reference values 1 month after the onset of acute hepatitis, but urinary protein excretion remained increased. Approximately 1 year later, urinary abnormalities were resolved and a second biopsy showed no mesangial IgA deposits. These findings suggest that IgA nephropathy can transiently accompany HAV infection, but may not progress to chronic glomerulonephritis after recovery from HAV. Copyright © 2010 National Kidney Foundation, Inc. Published by Elsevier Inc. All rights reserved.

  17. Enhancement of intestinal IgA production by Ajoene in mice.

    PubMed

    Washiya, Yuki; Nishikawa, Tomoaki; Fujino, Tsuchiyoshi

    2013-01-01

    We investigated the effects of ajoene on intestinal IgA production. Ajoene (1.35, 4.5, and 13.5 µg/kg/d) was administered to mice for 4 weeks. The fecal IgA level in the 13.5 µg/kg/d group increased after 3 weeks. The intestinal IgA level also increased in a dose-dependent manner upon ajoene administration. An oil-macerated garlic extract, with 1500 µg/g of ajoene, enhanced the intestinal IgA production.

  18. Rapamycin ameliorates IgA nephropathy via cell cycle-dependent mechanisms.

    PubMed

    Tian, Jihua; Wang, Yanhong; Liu, Xinyan; Zhou, Xiaoshuang; Li, Rongshan

    2015-07-01

    IgA nephropathy is the most frequent type of glomerulonephritis worldwide. The role of cell cycle regulation in the pathogenesis of IgA nephropathy has been studied. The present study was designed to explore whether rapamycin ameliorates IgA nephropathy via cell cycle-dependent mechanisms. After establishing an IgA nephropathy model, rats were randomly divided into four groups. Coomassie Brilliant Blue was used to measure the 24-h urinary protein levels. Renal function was determined using an autoanalyzer. Proliferation was assayed via Proliferating Cell Nuclear Antigen (PCNA) immunohistochemistry. Rat mesangial cells were cultured and divided into the six groups. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) and flow cytometry were used to detect cell proliferation and the cell cycle phase. Western blotting was performed to determine cyclin E, cyclin-dependent kinase 2, p27(Kip1), p70S6K/p-p70S6K, and extracellular signal-regulated kinase 1/2/p- extracellular signal-regulated kinase 1/2 protein expression. A low dose of the mammalian target of rapamycin (mTOR) inhibitor rapamycin prevented an additional increase in proteinuria, protected kidney function, and reduced IgA deposition in a model of IgA nephropathy. Rapamycin inhibited mesangial cell proliferation and arrested the cell cycle in the G1 phase. Rapamycin did not affect the expression of cyclin E and cyclin-dependent kinase 2. However, rapamycin upregulated p27(Kip1) at least in part via AKT (also known as protein kinase B)/mTOR. In conclusion, rapamycin can affect cell cycle regulation to inhibit mesangial cell proliferation, thereby reduce IgA deposition, and slow the progression of IgAN. © 2014 by the Society for Experimental Biology and Medicine.

  19. Streptomyces flavogriseus HS1: isolation and characterization of extracellular proteases and their compatibility with laundry detergents.

    PubMed

    Ghorbel, Sofiane; Kammoun, Maher; Soltana, Hala; Nasri, Moncef; Hmidet, Noomen

    2014-01-01

    The present study describes the isolation of a new protease producing Streptomyces strain HS1 and the biochemical characterization of the secreted proteases. By sequencing of its noted 16S rDNA, HS1 strain was found to have a 100% identity with Streptomyces flavogriseus. The highest protease production was found using FermII media. In these conditions maximum protease production (99 U/mL) was obtained after 96 h incubation at 30°C and 150 rpm. HS1 strain produced at least five proteases as revealed by zymogram technique. The enzyme preparation exhibited activity over a broad range of pH (5-11) and temperature (25-70°C). Optimum activity was observed at a pH of 7.0 and a temperature of 50°C. Proteolytic activity was significantly unaffected by Ca(2+) and Mg(2+). EDTA and PMSF highly decreased the original activity. The crude extracellular proteases showed high stability when used as a detergent additive. These properties offer an interesting potential for enzymatic hydrolysis at the industrial level.

  20. Streptomyces flavogriseus HS1: Isolation and Characterization of Extracellular Proteases and Their Compatibility with Laundry Detergents

    PubMed Central

    Kammoun, Maher; Soltana, Hala; Nasri, Moncef; Hmidet, Noomen

    2014-01-01

    The present study describes the isolation of a new protease producing Streptomyces strain HS1 and the biochemical characterization of the secreted proteases. By sequencing of its noted 16S rDNA, HS1 strain was found to have a 100% identity with Streptomyces flavogriseus. The highest protease production was found using FermII media. In these conditions maximum protease production (99 U/mL) was obtained after 96 h incubation at 30°C and 150 rpm. HS1 strain produced at least five proteases as revealed by zymogram technique. The enzyme preparation exhibited activity over a broad range of pH (5–11) and temperature (25–70°C). Optimum activity was observed at a pH of 7.0 and a temperature of 50°C. Proteolytic activity was significantly unaffected by Ca2+ and Mg2+. EDTA and PMSF highly decreased the original activity. The crude extracellular proteases showed high stability when used as a detergent additive. These properties offer an interesting potential for enzymatic hydrolysis at the industrial level. PMID:24804214

  1. Breast milk IgA to foods has different epitope specificity than serum IgA-Evidence for entero-mammary link for food-specific IgA?

    PubMed

    Seppo, A E; Savilahti, E M; Berin, M C; Sampson, H A; Järvinen, K M

    2017-10-01

    We have previously shown that maternal cow's milk (CM) elimination results in downregulation of CM-specific IgA antibody levels in BM, but not in serum, suggesting that an entero-mammary link may exist for food-specific antibody-secreting cells. We sought to investigate whether food-specific IgA epitope profiles differ intra-individually between mother's serum and BM. We also examined how infants' food epitope-specific IgA develops in early infancy and the relationship of IgA epitope recognition with development of cow's milk allergy (CMA). We measured specific IgA to a series of overlapping peptides in major CM allergens (α s1 -, α s2 -, β- and κ-caseins and β-lactoglobulin) in paired maternal and infant serum as well as BM samples in 31 mother-infant dyads within the first 15 post-partum months utilizing peptide microarray. There was significant discordance in epitope specificity between BM and maternal sera ranging from only 13% of sample pairs sharing at least one epitope in α s1 -casein to 73% in κ-casein. Epitope-specific IgA was detectable in infants' sera starting at less than 3 months of age. Sera of mothers with a CMA infant had increased binding of epitope-specific IgA to CM proteins compared to those with a non-CMA infant. These findings support the concept that mother's milk has a distinct antifood antibody repertoire when compared to the antibody repertoire of the peripheral blood. Increased binding of serum epitope-specific IgA to CM in mothers of infants with CMA may reflect inherited systemic immunogenicity of CM proteins in these families, although specific IgA in breast milk was not proportionally up-regulated. © 2017 John Wiley & Sons Ltd.

  2. Lumenal exposed regions of the D1 protein of PSII are long enough to be degraded by the chloroplast Deg1 protease.

    PubMed

    Knopf, Ronit Rimon; Adam, Zach

    2018-03-27

    Degradation of the D1 protein of photosystem II (PSII) reaction center is a pre-requisite for the repair cycle from photoinhibition. Two types of thylakoid proteases, FtsH and Deg, have been demonstrated to participate in this process. However, the location of the proteolytic sites of the lumenal Deg1 protease within its internal sphere raised the question whether the lumenal-exposed regions of D1 are indeed long enough to reach these sites. Implanting these regions into the stable GFP rendered it sensitive to the presence of Deg1 in vitro, demonstrating that the flexible regions of D1 that protrude into the lumen can penetrate through the three side-openings of Deg1 and reach its internal proteolytic sites. This mode of action, facilitating cooperation between proteases on both sides of the thylakoid membranes, should be applicable to the degradation of other integral thylakoid membrane proteins as well.

  3. Structure-Based Design of Novel HIV-1 Protease Inhibitors to Combat Drug Resistance

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ghosh,A.; Sridhar, P.; Leshchenko, S.

    2006-01-01

    Structure-based design and synthesis of novel HIV protease inhibitors are described. The inhibitors are designed specifically to interact with the backbone of HIV protease active site to combat drug resistance. Inhibitor 3 has exhibited exceedingly potent enzyme inhibitory and antiviral potency. Furthermore, this inhibitor maintains impressive potency against a wide spectrum of HIV including a variety of multi-PI-resistant clinical strains. The inhibitors incorporated a stereochemically defined 5-hexahydrocyclopenta[b]furanyl urethane as the P2-ligand into the (R)-(hydroxyethylamino)sulfonamide isostere. Optically active (3aS,5R,6aR)-5-hydroxy-hexahydrocyclopenta[b]furan was prepared by an enzymatic asymmetrization of meso-diacetate with acetyl cholinesterase, radical cyclization, and Lewis acid-catalyzed anomeric reduction as the key steps.more » A protein-ligand X-ray crystal structure of inhibitor 3-bound HIV-1 protease (1.35 Angstroms resolution) revealed extensive interactions in the HIV protease active site including strong hydrogen bonding interactions with the backbone. This design strategy may lead to novel inhibitors that can combat drug resistance.« less

  4. IgA Fc receptors.

    PubMed

    Monteiro, Renato C; Van De Winkel, Jan G J

    2003-01-01

    The IgA receptor family comprises a number of surface receptors including the polymeric Ig receptor involved in epithelial transport of IgA/IgM, the myeloid specific IgA Fc receptor (FcalphaRI or CD89), the Fcalpha/muR, and at least two alternative IgA receptors. These are the asialoglycoprotein receptor and the transferrin receptor, which have been implicated in IgA catabolism, and tissue IgA deposition. In this review we focus on the biology of FcalphaRI (CD89). FcalphaRI is expressed on neutrophils, eosinophils, monocytes/macrophages, dendritic cells, and Kupffer cells. This receptor represents a heterogeneously glycosylated transmembrane protein that binds both IgA subclasses with low affinity. A single gene encoding FcalphaRI has been isolated, which is located within the leukocyte receptor cluster on chromosome 19. The FcalphaRI alpha chain lacks canonical signal transduction domains but can associate with the FcR gamma-chain that bears an activation motif (ITAM) in the cytoplasmic domain, allowing activatory functions. FcalphaRI expressed alone mediates endocytosis and recyling of IgA. No FcalphaRI homologue has been defined in the mouse, and progress in defining the in vivo role of FcalphaRI has been made using human FcalphaRI transgenic (Tg) mice. FcalphaRI-Tg mice demonstrated FcalphaRI expression on Kupffer cells and so defined a key role for the receptor in mucosal defense. The receptor functions as a second line of antibacterial defense involving serum IgA rather than secretory IgA. Studies in FcalphaRI-Tg mice, furthermore, defined an essential role for soluble FcalphaRI in the development of IgA nephropathy by formation of circulating IgA-FcalphaRI complexes. Finally, recent work points out a role for human IgA in treatment of infectious and neoplastic diseases.

  5. A Randomized, Controlled Trial of Rituximab in IgA Nephropathy with Proteinuria and Renal Dysfunction

    PubMed Central

    Lafayette, Richard A.; Canetta, Pietro A.; Rovin, Brad H.; Appel, Gerald B.; Novak, Jan; Nath, Karl A.; Sethi, Sanjeev; Tumlin, James A.; Mehta, Kshama; Hogan, Marie; Erickson, Stephen; Julian, Bruce A.; Leung, Nelson; Enders, Felicity T.; Brown, Rhubell; Knoppova, Barbora; Hall, Stacy

    2017-01-01

    IgA nephropathy frequently leads to progressive CKD. Although interest surrounds use of immunosuppressive agents added to standard therapy, several recent studies have questioned efficacy of these agents. Depleting antibody–producing B cells potentially offers a new therapy. In this open label, multicenter study conducted over 1-year follow-up, we randomized 34 adult patients with biopsy–proven IgA nephropathy and proteinuria >1 g/d, maintained on angiotensin–converting enzyme inhibitors or angiotensin receptor blockers with well controlled BP and eGFR<90 ml/min per 1.73 m2, to receive standard therapy or rituximab with standard therapy. Primary outcome measures included change in proteinuria and change in eGFR. Median baseline serum creatinine level (range) was 1.4 (0.8–2.4) mg/dl, and proteinuria was 2.1 (0.6–5.3) g/d. Treatment with rituximab depleted B cells and was well tolerated. eGFR did not change in either group. Rituximab did not alter the level of proteinuria compared with that at baseline or in the control group; three patients in each group had ≥50% reduction in level of proteinuria. Serum levels of galactose-deficient IgA1 or antibodies against galactose-deficient IgA1 did not change. In this trial, rituximab therapy did not significantly improve renal function or proteinuria assessed over 1 year. Although rituximab effectively depleted B cells, it failed to reduce serum levels of galactose-deficient IgA1 and antigalactose–deficient IgA1 antibodies. Lack of efficacy of rituximab, at least at this stage and severity of IgA nephropathy, may reflect a failure of rituximab to reduce levels of specific antibodies assigned salient pathogenetic roles in IgA nephropathy. PMID:27821627

  6. A Randomized, Controlled Trial of Rituximab in IgA Nephropathy with Proteinuria and Renal Dysfunction.

    PubMed

    Lafayette, Richard A; Canetta, Pietro A; Rovin, Brad H; Appel, Gerald B; Novak, Jan; Nath, Karl A; Sethi, Sanjeev; Tumlin, James A; Mehta, Kshama; Hogan, Marie; Erickson, Stephen; Julian, Bruce A; Leung, Nelson; Enders, Felicity T; Brown, Rhubell; Knoppova, Barbora; Hall, Stacy; Fervenza, Fernando C

    2017-04-01

    IgA nephropathy frequently leads to progressive CKD. Although interest surrounds use of immunosuppressive agents added to standard therapy, several recent studies have questioned efficacy of these agents. Depleting antibody-producing B cells potentially offers a new therapy. In this open label, multicenter study conducted over 1-year follow-up, we randomized 34 adult patients with biopsy-proven IgA nephropathy and proteinuria >1 g/d, maintained on angiotensin-converting enzyme inhibitors or angiotensin receptor blockers with well controlled BP and eGFR<90 ml/min per 1.73 m 2 , to receive standard therapy or rituximab with standard therapy. Primary outcome measures included change in proteinuria and change in eGFR. Median baseline serum creatinine level (range) was 1.4 (0.8-2.4) mg/dl, and proteinuria was 2.1 (0.6-5.3) g/d. Treatment with rituximab depleted B cells and was well tolerated. eGFR did not change in either group. Rituximab did not alter the level of proteinuria compared with that at baseline or in the control group; three patients in each group had ≥50% reduction in level of proteinuria. Serum levels of galactose-deficient IgA1 or antibodies against galactose-deficient IgA1 did not change. In this trial, rituximab therapy did not significantly improve renal function or proteinuria assessed over 1 year. Although rituximab effectively depleted B cells, it failed to reduce serum levels of galactose-deficient IgA1 and antigalactose-deficient IgA1 antibodies. Lack of efficacy of rituximab, at least at this stage and severity of IgA nephropathy, may reflect a failure of rituximab to reduce levels of specific antibodies assigned salient pathogenetic roles in IgA nephropathy. Copyright © 2017 by the American Society of Nephrology.

  7. Insights into the mechanism of drug resistance. X-ray structure analysis of multi-drug resistant HIV-1 protease ritonavir complex

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Liu, Zhigang; Yedidi, Ravikiran S.; Wang, Yong

    2013-01-08

    Ritonavir (RTV) is a first generation HIV-1 protease inhibitor with rapidly emerging drug resistance. Mutations at residues 46, 54, 82 and 84 render the HIV-1 protease drug resistant against RTV. We report the crystal structure of multi-drug resistant (MDR) 769 HIV-1 protease (carrying resistant mutations at residues 10, 36, 46, 54, 62, 63, 71, 82, 84 and 90) complexed with RTV and the in vitro enzymatic IC50 of RTV against MDR HIV-1 protease. The structural and functional studies demonstrate significant drug resistance of MDR HIV-1 protease against RTV, arising from reduced hydrogen bonds and Van der Waals interactions between RTVmore » and MDR HIV-1 protease.« less

  8. Purification and characterization of an alkaline protease Prot 1 from Botrytis cinerea : biodetergent catalyst assay.

    PubMed

    Abidi, Ferid; Limam, Ferid; Marzouki, M Nejib

    2007-01-01

    Alkaline thiol protease named Prot 1 was isolated from a culture filtrate of Botrytis cinerea. The enzyme was purified by ammonium sulfate fractionation, gel filtration, and ion-exchange chromatography. Thus, the enzyme was purified to homogeneity with specific activity of 30-fold higher than that of the crude broth. The purified alkaline protease has an apparent molecular mass of 43 kDa under denaturing conditions as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native molecular mass (45 kDa), determined by gel filtration, indicated that the alkaline protease has a monomeric form. The purified protease was biochemically characterized. The enzyme is active at alkaline pH and has a suitable and high thermostability. The optimal pH and temperature for activity were 9.0-10.0 and 60 degrees C, respectively. This protease was stable between pH 5.0 and 12.0. The enzyme retained 85% of its activity by treatment at 50 degrees C over 120 min; it maintained 50% of activity after 60 min of heating at 60 degrees C. Furthermore, the protease retained almost complete activity after 4 wk storage at 25 degrees C. The activity was significantly affected by thiol protease inhibitors, suggesting that the enzyme belongs to the alkaline thiol protease family. With the aim on industrial applications, we focused on studying the stability of the protease in several conditions. Prot 1 activity was not affected by ionic strength and different detergent additives, and, thus, the protease shows remarkable properties as a biodetergent catalyst.

  9. Differential susceptibility of Dectin-1 isoforms to functional inactivation by neutrophil and fungal proteases.

    PubMed

    Griffiths, James S; Thompson, Aiysha; Stott, Matthew; Benny, Ankita; Lewis, Natalie A; Taylor, Philip R; Forton, Julian; Herrick, Sarah; Orr, Selinda J; McGreal, Eamon P

    2018-06-01

    Patients with cystic fibrosis (CF) experience chronic or recurrent bacterial and fungal lung infections. Many patients with CF cannot effectively clear Aspergillus from their lungs. This may result in IgE sensitization and the development of allergic bronchopulmonary aspergillosis, or invasive infections, such as Aspergillus bronchitis. Lung disease in patients with CF is associated with neutrophil-dominated inflammation and elevated levels of the serine protease, neutrophil elastase (NE). Various C-type lectin-like receptors (CLRs), including Dectin-1 and Dectin-2, are involved in the immune response to Aspergillus. Here, we show that purified NE cleaves Dectin-1 in an isoform-specific manner. Bronchoalveolar lavage fluid from patients with CF, which contains high NE activity, induces Dectin-1 cleavage. Similarly, filtrate from a protease-producing strain of Aspergillus fumigatus induces isoform-specific cleavage of Dectin-1. Dectin-1 knockout (KO) cells and NE-treated cells demonstrated reduced phagocytosis of zymosan, a fungal cell wall preparation. In addition, NE cleaves 2 other CLRs, Dectin-2 and Mincle, and fungal-induced cytokine production was reduced in Dectin-1 KO cells, Dectin-2 KO cells, and NE-treated cells. Thus, Dectin-1 and Dectin-2 cleavage by NE and/or A. fumigatus-derived proteases results in an aberrant antifungal immune response that likely contributes to disease pathology in patients with CF.-Griffiths, J. S., Thompson, A., Stott, M., Benny, A., Lewis, N. A., Taylor, P. R., Forton, J., Herrick, S., Orr, S. J., McGreal, E. P. Differential susceptibility of Dectin-1 isoforms to functional inactivation by neutrophil and fungal proteases.

  10. Design and Synthesis of Piperazine Sulfonamide Cores Leading to Highly Potent HIV-1 Protease Inhibitors.

    PubMed

    Bungard, Christopher J; Williams, Peter D; Schulz, Jurgen; Wiscount, Catherine M; Holloway, M Katharine; Loughran, H Marie; Manikowski, Jesse J; Su, Hua-Poo; Bennett, David J; Chang, Lehua; Chu, Xin-Jie; Crespo, Alejandro; Dwyer, Michael P; Keertikar, Kartik; Morriello, Gregori J; Stamford, Andrew W; Waddell, Sherman T; Zhong, Bin; Hu, Bin; Ji, Tao; Diamond, Tracy L; Bahnck-Teets, Carolyn; Carroll, Steven S; Fay, John F; Min, Xu; Morris, William; Ballard, Jeanine E; Miller, Michael D; McCauley, John A

    2017-12-14

    Using the HIV-1 protease binding mode of MK-8718 and PL-100 as inspiration, a novel aspartate binding bicyclic piperazine sulfonamide core was designed and synthesized. The resulting HIV-1 protease inhibitor containing this core showed an 60-fold increase in enzyme binding affinity and a 10-fold increase in antiviral activity relative to MK-8718 .

  11. Biomarkers of IgA vasculitis nephritis in children

    PubMed Central

    Pillebout, Evangeline; Jamin, Agnès; Ayari, Hamza; Housset, Pierre; Pierre, Melissa; Sauvaget, Virginia; Viglietti, Denis; Deschenes, Georges

    2017-01-01

    Henoch–Schönlein purpura is a systemic vasculitis characterized by IgA deposits, which target the skin, joints, and kidneys, among other organs. In children, prognosis is often good but little is known about biomarkers of pediatric nephritis. We hypothesized that biological markers, including cytokines, immunoglobulins, IgA-immune complexes, IgA glycosylation and neutrophil gelatinase-associated lipocalin (NGAL), may discriminate IgA vasculitis (IgAV) pediatric patients with renal involvement from those without renal involvement. Fifty children at the time of IgAV rash between 2010 and 2015 were prospectively enrolled and compared to 21 controls. All patients were assessed for clinical and biological parameters at the time of diagnosis, including the levels of cytokines, immunoglobulins, immune complexes, IgA glycosylation and NGAL in serum and urine. Among IgAV patients, 33 patients exhibited nephritis (IgAV-N) and 17 children were without nephritis (IgAV-woN). The serum level of galactose-deficient (Gd)-IgA1 (p<0.01) and the urinary concentrations of IgA, IgG, IgM, IL-6, IL-8, IL-10, IgA-IgG complexes and IgA-sCD89 complexes (p<0.001 for all) were higher in the IgAV-N patients than in the IgAV-woN patients. Among those markers, urinary IgA and IgM had the highest AUC (0.86 and 0.87 respectively, p<0.0001). This prospective cohort study furthers our understanding of the pathophysiology of IgAV. We identified biomarkers that are able to distinguish patients initially with or without nephritis. To conclude, serum Gd-IgA1 and urinary IgA, IgG, IgM, IL-6, IL-8, IL-10, and IgA-IgG and IgA-sCD89 complexes could identify IgAV pediatric patients with renal involvement at the time of diagnosis. PMID:29190714

  12. A novel serine protease, Sep1, from Bacillus firmus DS-1 has nematicidal activity and degrades multiple intestinal-associated nematode proteins.

    PubMed

    Geng, Ce; Nie, Xiangtao; Tang, Zhichao; Zhang, Yuyang; Lin, Jian; Sun, Ming; Peng, Donghai

    2016-04-27

    Plant-parasitic nematodes (PPNs) cause serious harm to agricultural production. Bacillus firmus shows excellent control of PPNs and has been produced as a commercial nematicide. However, its nematicidal factors and mechanisms are still unknown. In this study, we showed that B. firmus strain DS-1 has high toxicity against Meloidogyne incognita and soybean cyst nematode. We sequenced the whole genome of DS-1 and identified multiple potential virulence factors. We then focused on a peptidase S8 superfamily protein called Sep1 and demonstrated that it had toxicity against the nematodes Caenorhabditis elegans and M. incognita. The Sep1 protein exhibited serine protease activity and degraded the intestinal tissues of nematodes. Thus, the Sep1 protease of B. firmus is a novel biocontrol factor with activity against a root-knot nematode. We then used C. elegans as a model to elucidate the nematicidal mechanism of Sep1, and the results showed that Sep1 could degrade multiple intestinal and cuticle-associated proteins and destroyed host physical barriers. The knowledge gained in our study will lead to a better understanding of the mechanisms of B. firmus against PPNs and will aid in the development of novel bio-agents with increased efficacy for controlling PPNs.

  13. Malt1 protease inactivation efficiently dampens immune responses but causes spontaneous autoimmunity

    PubMed Central

    Jaworski, Maike; Marsland, Ben J; Gehrig, Jasmine; Held, Werner; Favre, Stéphanie; Luther, Sanjiv A; Perroud, Mai; Golshayan, Déla; Gaide, Olivier; Thome, Margot

    2014-01-01

    The protease activity of the paracaspase Malt1 has recently gained interest as a drug target for immunomodulation and the treatment of diffuse large B-cell lymphomas. To address the consequences of Malt1 protease inactivation on the immune response in vivo, we generated knock-in mice expressing a catalytically inactive C472A mutant of Malt1 that conserves its scaffold function. Like Malt1-deficient mice, knock-in mice had strong defects in the activation of lymphocytes, NK and dendritic cells, and the development of B1 and marginal zone B cells and were completely protected against the induction of autoimmune encephalomyelitis. Malt1 inactivation also protected the mice from experimental induction of colitis. However, Malt1 knock-in mice but not Malt1-deficient mice spontaneously developed signs of autoimmune gastritis that correlated with an absence of Treg cells, an accumulation of T cells with an activated phenotype and high serum levels of IgE and IgG1. Thus, removal of the enzymatic activity of Malt1 efficiently dampens the immune response, but favors autoimmunity through impaired Treg development, which could be relevant for therapeutic Malt1-targeting strategies. PMID:25319413

  14. Design, synthesis and evaluation of a potent substrate analog inhibitor identified by scanning Ala/Phe mutagenesis, mimicking substrate co-evolution, against multidrug-resistant HIV-1 protease

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yedidi, Ravikiran S.; Muhuhi, Joseck M.; Liu, Zhigang

    Highlights: •Inhibitors against MDR HIV-1 protease were designed, synthesized and evaluated. •Lead peptide (6a) showed potent inhibition (IC{sub 50}: 4.4 nM) of MDR HIV-1 protease. •(6a) Showed favorable binding isotherms against NL4-3 and MDR proteases. •(6a) Induced perturbations in the {sup 15}N-HSQC spectrum of MDR HIV-1 protease. •Molecular modeling suggested that (6a) may induce total flap closure inMDR protease. -- Abstract: Multidrug-resistant (MDR) clinical isolate-769, human immunodeficiency virus type-1 (HIV-1) protease (PDB ID: (1TW7)), was shown to exhibit wide-open flaps and an expanded active site cavity, causing loss of contacts with protease inhibitors. In the current study, the expanded activemore » site cavity of MDR769 HIV-1 protease was screened with a series of peptide-inhibitors that were designed to mimic the natural substrate cleavage site, capsid/p2. Scanning Ala/Phe chemical mutagenesis approach was incorporated into the design of the peptide series to mimic the substrate co-evolution. Among the peptides synthesized and evaluated, a lead peptide (6a) with potent activity (IC{sub 50}: 4.4 nM) was identified against the MDR769 HIV-1 protease. Isothermal titration calorimetry data showed favorable binding profile for 6aagainst both wild type and MDR769 HIV-1 protease variants. Nuclear magnetic resonance spectrum of {sup 15}N-labeled MDR769 HIV-1 protease in complex with 6a showed some major perturbations in chemical shift, supporting the peptide induced conformational changes in protease. Modeling analysis revealed multiple contacts between 6a and MDR769 HIV-1 protease. The lead peptide-inhibitor, 6a, with high potency and good binding profile can be used as the basis for developing potent small molecule inhibitors against MDR variants of HIV.« less

  15. The Inflammasome Drives GSDMD-Independent Secondary Pyroptosis and IL-1 Release in the Absence of Caspase-1 Protease Activity.

    PubMed

    Schneider, Katharina S; Groß, Christina J; Dreier, Roland F; Saller, Benedikt S; Mishra, Ritu; Gorka, Oliver; Heilig, Rosalie; Meunier, Etienne; Dick, Mathias S; Ćiković, Tamara; Sodenkamp, Jan; Médard, Guillaume; Naumann, Ronald; Ruland, Jürgen; Kuster, Bernhard; Broz, Petr; Groß, Olaf

    2017-12-26

    Inflammasomes activate the protease caspase-1, which cleaves interleukin-1β and interleukin-18 to generate the mature cytokines and controls their secretion and a form of inflammatory cell death called pyroptosis. By generating mice expressing enzymatically inactive caspase-1 C284A , we provide genetic evidence that caspase-1 protease activity is required for canonical IL-1 secretion, pyroptosis, and inflammasome-mediated immunity. In caspase-1-deficient cells, caspase-8 can be activated at the inflammasome. Using mice either lacking the pyroptosis effector gasdermin D (GSDMD) or expressing caspase-1 C284A , we found that GSDMD-dependent pyroptosis prevented caspase-8 activation at the inflammasome. In the absence of GSDMD-dependent pyroptosis, the inflammasome engaged a delayed, alternative form of lytic cell death that was accompanied by the release of large amounts of mature IL-1 and contributed to host protection. Features of this cell death modality distinguished it from apoptosis, suggesting it may represent a distinct form of pro-inflammatory regulated necrosis. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  16. Expression and characterization of plant aspartic protease nepenthesin-1 from Nepenthes gracilis.

    PubMed

    Kadek, Alan; Tretyachenko, Vyacheslav; Mrazek, Hynek; Ivanova, Ljubina; Halada, Petr; Rey, Martial; Schriemer, David C; Man, Petr

    2014-03-01

    Carnivorous plants of the genus Nepenthes produce their own aspartic proteases, nepenthesins, to digest prey trapped in their pitchers. Nepenthesins differ significantly in sequence from other aspartic proteases in the animal or even plant kingdoms. This difference, which also brings more cysteine residues into the structure of these proteases, can be a cause of uniquely high temperature and pH stabilities of nepenthesins. Their detailed structure characterization, however, has not previously been possible due to low amounts of protease present in the pitcher fluid and also due to limited accessibility of Nepenthes plants. In the present study we describe a convenient way for obtaining high amounts of nepenthesin-1 from Nepenthes gracilis using heterologous production in Escherichia coli. The protein can be easily refolded in vitro and its characteristics are very close to those described for a natural enzyme isolated from the pitcher fluid. Similarly to the natural enzyme, recombinant nepenthesin-1 is sensitive to denaturing and reducing agents. It also has maximal activity around pH 2.5, shows unusual stability at high pH and its activity is not irreversibly inhibited even after prolonged incubation in the basic pH range. On the other hand, temperature stability of the recombinant enzyme is lower in comparison with the natural enzyme, which can be attributed to missing N-glycosylation in the recombinant protein. Copyright © 2013 Elsevier Inc. All rights reserved.

  17. HIV-1 protease cleavage site prediction based on two-stage feature selection method.

    PubMed

    Niu, Bing; Yuan, Xiao-Cheng; Roeper, Preston; Su, Qiang; Peng, Chun-Rong; Yin, Jing-Yuan; Ding, Juan; Li, HaiPeng; Lu, Wen-Cong

    2013-03-01

    Knowledge of the mechanism of HIV protease cleavage specificity is critical to the design of specific and effective HIV inhibitors. Searching for an accurate, robust, and rapid method to correctly predict the cleavage sites in proteins is crucial when searching for possible HIV inhibitors. In this article, HIV-1 protease specificity was studied using the correlation-based feature subset (CfsSubset) selection method combined with Genetic Algorithms method. Thirty important biochemical features were found based on a jackknife test from the original data set containing 4,248 features. By using the AdaBoost method with the thirty selected features the prediction model yields an accuracy of 96.7% for the jackknife test and 92.1% for an independent set test, with increased accuracy over the original dataset by 6.7% and 77.4%, respectively. Our feature selection scheme could be a useful technique for finding effective competitive inhibitors of HIV protease.

  18. Molecular characterization of alkaline protease of Bacillus amyloliquefaciens SP1 involved in biocontrol of Fusarium oxysporum.

    PubMed

    Guleria, Shiwani; Walia, Abhishek; Chauhan, Anjali; Shirkot, C K

    2016-09-02

    An alkaline protease gene was amplified from genomic DNA of Bacillus amyloliquefaciens SP1 which was involved in effective biocontrol of Fusarium oxysporum. We investigated the antagonistic capacity of protease of B. amyloliquifaciens SP1, under in vitro conditions. The 5.62 fold purified enzyme with specific activity of 607.69U/mg reported 24.14% growth inhibition of F. oxysporum. However, no antagonistic activity was found after addition of protease inhibitor i.e. PMSF (15mM) to purified enzyme. An 1149bp nucleotide sequence of protease gene encoded 382 amino acids of 43kDa and calculated isoelectric point of 9.29. Analysis of deduced amino acid sequence revealed high homology (86%) with subtilisin E of Bacillus subtilis. The B. amyloliquefaciens SP1 protease gene was expressed in Escherichiax coli BL21. The expressed protease was secreted into culture medium by E. coli and exhibited optimum activity at pH8.0 and 60°C. The most reliable three dimensional structure of alkaline protease was determined using Phyre 2 server which was validated on the basis of Ramachandran plot and ERRAT value. The expression and structure prediction of the enzyme offers potential value for commercial application in agriculture and industry. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Design, synthesis, X-ray studies, and biological evaluation of novel macrocyclic HIV-1 protease inhibitors involving the P1'-P2' ligands

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ghosh, Arun K.; Sean Fyvie, W.; Brindisi, Margherita

    Design, synthesis, and evaluation of a new class of HIV-1 protease inhibitors containing diverse flexible macrocyclic P1'-P2' tethers are reported. Inhibitor 5a with a pyrrolidinone-derived macrocycle exhibited favorable enzyme inhibitory and antiviral activity (Ki = 13.2 nM, IC50 = 22 nM). Further incorporation of heteroatoms in the macrocyclic skeleton provided macrocyclic inhibitors 5m and 5o. These compounds showed excellent HIV-1 protease inhibitory (Ki = 62 pM and 14 pM, respectively) and antiviral activity (IC50 = 5.3 nM and 2.0 nM, respectively). Inhibitor 5o also remained highly potent against a DRV-resistant HIV-1 variant.

  20. Human milk IgA concentrations during the first year of lactation

    PubMed Central

    Weaver, L.; Arthur, H.; Bunn, J.; Thomas, J.

    1998-01-01

    AIMS—To measure the concentrations of total IgA in the milk secreted by both breasts, throughout the first year of lactation, in a cohort of Gambian mothers of infants at high risk of infection.
SUBJECTS AND METHODS—Sixty five women and their infants were studied monthly from the 4th to 52nd postpartum week. Samples of milk were obtained from each breast by manual expression immediately before the infant was suckled. Milk intakes were measured by test weighing the infants before and after feeds over 12 hour periods; IgA concentrations were determined by enzyme linked immunosorbent assay.
RESULTS—A total of 1590 milk samples was measured. The median (interquartile range) concentration of IgA for all samples was 0.708(0.422-1.105) g/l; that in milk obtained from the left breast was 0.785 (0.458-1.247) g/l, and that in milk obtained from the right breast was 0.645 (0.388-1.011) g/l (p < 0.0001). There was no significant change in milk or IgA intakes with advancing infant age, but there was a close concordance of IgA concentrations between the two breasts, with "tracking" of the output of the left and right breasts. There was a significant (p < 0.01) negative correlation between maternal age and parity, and weight of milk ingested by infants. During the dry season (December to May) the median (interquartile range) IgA concentration was significantly higher at 0.853 (0.571-1.254) g/l than during the rainy season (June to November), when it was 0.518 (0.311-0.909) g/l (p < 0.0001).
CONCLUSIONS—Sustained IgA secretion is likely to protect suckling infants from microbial infection.

 PMID:9613353

  1. Subtype-Specific Differences in Gag-Protease-Driven Replication Capacity Are Consistent with Intersubtype Differences in HIV-1 Disease Progression.

    PubMed

    Kiguoya, Marion W; Mann, Jaclyn K; Chopera, Denis; Gounder, Kamini; Lee, Guinevere Q; Hunt, Peter W; Martin, Jeffrey N; Ball, T Blake; Kimani, Joshua; Brumme, Zabrina L; Brockman, Mark A; Ndung'u, Thumbi

    2017-07-01

    There are marked differences in the spread and prevalence of HIV-1 subtypes worldwide, and differences in clinical progression have been reported. However, the biological reasons underlying these differences are unknown. Gag-protease is essential for HIV-1 replication, and Gag-protease-driven replication capacity has previously been correlated with disease progression. We show that Gag-protease replication capacity correlates significantly with that of whole isolates ( r = 0.51; P = 0.04), indicating that Gag-protease is a significant contributor to viral replication capacity. Furthermore, we investigated subtype-specific differences in Gag-protease-driven replication capacity using large well-characterized cohorts in Africa and the Americas. Patient-derived Gag-protease sequences were inserted into an HIV-1 NL4-3 backbone, and the replication capacities of the resulting recombinant viruses were measured in an HIV-1-inducible reporter T cell line by flow cytometry. Recombinant viruses expressing subtype C Gag-proteases exhibited substantially lower replication capacities than those expressing subtype B Gag-proteases ( P < 0.0001); this observation remained consistent when representative Gag-protease sequences were engineered into an HIV-1 subtype C backbone. We identified Gag residues 483 and 484, located within the Alix-binding motif involved in virus budding, as major contributors to subtype-specific replicative differences. In East African cohorts, we observed a hierarchy of Gag-protease-driven replication capacities, i.e., subtypes A/C < D < intersubtype recombinants ( P < 0.0029), which is consistent with reported intersubtype differences in disease progression. We thus hypothesize that the lower Gag-protease-driven replication capacity of subtypes A and C slows disease progression in individuals infected with these subtypes, which in turn leads to greater opportunity for transmission and thus increased prevalence of these subtypes. IMPORTANCE HIV-1 subtypes

  2. Screening and characterization of the alkaline protease isolated from PLI-1, a strain of Brevibacillus sp. collected from Indonesia's hot springs

    NASA Astrophysics Data System (ADS)

    Wang, Shuai; Lin, Xuezheng; Huang, Xiaohang; Zheng, Li; Zilda, Dewi Seswita

    2012-06-01

    A total of 69 strains of thermophilic bacteria were isolated from water, soil and sediment samples from three Indonesia's hot spring areas (Pantai cermin, Kalianda and Banyu wedang) by using Minimal Synthetic Medium (MSM). The extreme thermophile Brevibacillus sp. PLI-1 was found to produce extracellular thermophilic alkaline protease with optimal activity at 70° and pH 8.0-9.0. The molecular weight of the protease was estimated to be around 56 kD by SDS-PAGE. The maximum activity of the protease was 26.54 U mL-1. The protease activity did not decrease after 30 min and still retained more than 70% of relative activity after 60 min at 70°C and pH 8.0. The ion Mg2+ was found to promote protease activity at both low and high concentrations, whereas Cu2+ and Zn2+ could almost completely inhibit the activity. Divalent cation chelator EDTA inhibited the enzyme activity by 55.06% ± 0.27%, while the inhibition caused by PMSF, Leupeptin, Pepstain A and Benzamidine were 66.78% ± 3.25%, 52.37% ± 0.25%, 62.47% ± 2.96% and 50.99% ± 0.24%, respectively. Based on these observations, the enzyme activity was conspicuously sensitive to the serine and cysteine protease inhibitors. All these results indicated that the protease isolated from the strain PLI-1 was a thermophilic protease and had a high-temperature stability and a pH stability.

  3. Computational Study on Substrate Specificity of a Novel Cysteine Protease 1 Precursor from Zea mays

    PubMed Central

    Liu, Huimin; Chen, Liangcheng; Li, Quan; Zheng, Mingzhu; Liu, Jingsheng

    2014-01-01

    Cysteine protease 1 precursor from Zea mays (zmCP1) is classified as a member of the C1A family of peptidases (papain-like cysteine protease) in MEROPS (the Peptidase Database). The 3D structure and substrate specificity of the zmCP1 is still unknown. This study is the first one to build the 3D structure of zmCP1 by computer-assisted homology modeling. In order to determine the substrate specificity of zmCP1, docking study is used for rapid and convenient analysis of large populations of ligand–enzyme complexes. Docking results show that zmCP1 has preference for P1 position and P2 position for Arg and a large hydrophobic residue (such as Phe). Gly147, Gly191, Cys189, and Asp190 are predicted to function as active residues at the S1 subsite, and the S2 subsite contains Leu283, Leu193, Ala259, Met194, and Ala286. SIFt results indicate that Gly144, Arg268, Trp308, and Ser311 play important roles in substrate binding. Then Molecular Mechanics-Poisson-Boltzmann Surface Area (MM-PBSA) method was used to explain the substrate specificity for P1 position of zmCp1. This study provides insights into the molecular basis of zmCP1 activity and substrate specificity. PMID:24921705

  4. Bioprocess optimization for production of thermoalkali-stable protease from Bacillus subtilis K-1 under solid-state fermentation.

    PubMed

    Singh, Satbir; Bajaj, Bijender Kumar

    2016-10-02

    Cost-effective production of proteases, which are robust enough to function under harsh process conditions, is always sought after due to their wide industrial application spectra. Solid-state production of enzymes using agro-industrial wastes as substrates is an environment-friendly approach, and it has several advantages such as high productivity, cost-effectiveness, being less labor-intensive, and less effluent production, among others. In the current study, different agro-wastes were employed for thermoalkali-stable protease production from Bacillus subtilis K-1 under solid-state fermentation. Agricultural residues such as cotton seed cake supported maximum protease production (728 U ml(-1)), which was followed by gram husk (714 U ml(-1)), mustard cake (680 U ml(-1)), and soybean meal (653 U ml(-1)). Plackett-Burman design of experiment showed that peptone, moisture content, temperature, phosphates, and inoculum size were the significant variables that influenced the protease production. Furthermore, statistical optimization of three variables, namely peptone, moisture content, and incubation temperature, by response surface methodology resulted in 40% enhanced protease production as compared to that under unoptimized conditions (from initial 728 to 1020 U ml(-1)). Thus, solid-state fermentation coupled with design of experiment tools represents a cost-effective strategy for production of industrial enzymes.

  5. IgA deficiency in wolves.

    PubMed

    Frankowiack, Marcel; Hellman, Lars; Zhao, Yaofeng; Arnemo, Jon M; Lin, Miaoli; Tengvall, Katarina; Møller, Torsten; Lindblad-Toh, Kerstin; Hammarström, Lennart

    2013-06-01

    Low mean concentrations of serum immunoglobulin A (IgA) and an increased frequency of overt IgA deficiency (IgAD) in certain dog breeds raises the question whether it is a breeding-enriched phenomenon or a legacy from the dog's ancestor, the gray wolf (Canis lupus). The IgA concentration in 99 serum samples from 58 free-ranging and 13 captive Scandinavian wolves, was therefore measured by capture ELISA. The concentrations were markedly lower in the wolf serum samples than in the dog controls. Potential differences in the IgA molecule between dogs and wolves were addressed by sequencing the wolf IgA heavy chain constant region encoding gene (IGHA). Complete amino acid sequence homology was found. Detection of wolf and dog IgA was ascertained by showing identity using double immunodiffusion. We suggest that the vast majority of wolves, the ancestor of the dog, are IgA deficient. Copyright © 2013 Elsevier Ltd. All rights reserved.

  6. Structural studies of FIV and HIV-1 proteases complexed with an efficient inhibitor of FIV protease.

    PubMed

    Li, M; Morris, G M; Lee, T; Laco, G S; Wong, C H; Olson, A J; Elder, J H; Wlodawer, A; Gustchina, A

    2000-01-01

    Three forms of feline immunodeficiency virus protease (FIV PR), the wild type (wt) and two single point mutants, V59I and Q99V, as well as human immunodeficiency virus type 1 protease (HIV-1 PR), were cocrystallized with the C2-symmetric inhibitor, TL-3. The mutants of FIV PR were designed to replace residues involved in enzyme-ligand interactions by the corresponding HIV-1 PR residues at the structurally equivalent position. TL-3 shows decreased (improved) inhibition constants with these FIV PR mutants relative to wt FIV PR. Despite similar modes of binding of the inhibitor to all PRs (from P3 to P3'), small differences are evident in the conformation of the Phe side chains of TL-3 at the P1 and P1' positions in the complexes with the mutated FIV PRs. The differences mimick the observed binding of TL-3 in HIV-1 PR and correlate with a significant improvement in the inhibition constants of TL-3 with the two mutant FIV PRs. Large differences between the HIV-1 and FIV PR complexes are evident in the binding modes of the carboxybenzyl groups of TL-3 at P4 and P4'. In HIV-1 PR:TL-3, these groups bind over the flap region, whereas in the FIV PR complexes, the rings are located along the major axis of the active site. A significant difference in the location of the flaps in this region of the HIV-1 and FIV PRs correlates with the observed conformational changes in the binding mode of the peptidomimetic inhibitor at the P4 and P4' positions. These findings provide a structural explanation of the observed Ki values for TL-3 with the different PRs and will further assist in the development of improved inhibitors.

  7. H1-A, a compound isolated from Fusarium oxysporum inhibits hepatitis C virus (HCV) NS3 serine protease.

    PubMed

    Yang, Li-Yuan; Lin, Jun; Zhou, Bin; Liu, Yan-Gang; Zhu, Bao-Quan

    2016-04-01

    The present study was aimed to isolate the active compounds from the fermentation products of Fusarium oxysporum, which had hepatitis C virus (HCV) NS3 protease inhibitory activity. A bioactive compound was isolated by reverse-phase silica-gel column chromatography, silica-gel column chromatography, semi-preparative reverse-phase High Performance Liquid Chromatography (HPLC), and then its molecular structure was elucidated based on the spectrosopic analysis. As a result, the compound (H1-A, 1) Ergosta-5, 8 (14), 22-trien-7-one, 3-hydroxy-,(3β, 22E) was isolated and identified. To the best of our knowledge, this was the first report on the isolation of H1-A from microorganisms with the inhibitory activity of NS3 protease. Copyright © 2016 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

  8. Low pretransplant IgA level is associated with early post-lung transplant seromucous infection.

    PubMed

    Murthy, Sudish C; Avery, Robin K; Budev, Marie; Gupta, Sandeep; Pettersson, Gösta B; Nowicki, Edward R; Mehta, Atul; Chapman, Jeffrey T; Rajeswaran, Jeevanantham; Blackstone, Eugene H

    2018-04-13

    Infection is an important cause of morbidity and mortality after lung transplantation. Immunoglobulins are part of both seromucous (IgA) and serum (IgG) infection defense mechanisms. We therefore hypothesized that lower pretransplant IgA levels would be associated with more early post-lung transplant seromucous infections and greater mortality independent of IgG. From January 2000 to July 2010, 538 patients undergoing primary lung transplantation had pretransplant IgA (n = 429) and IgG (n = 488) measured as a clinical routine. Median IgA was 200 mg·dL -1 (2% < 70 mg·dL -1 , lower limit of normal); median IgG was 970 mg·dL -1 (5% < 600 mg·dL -1 ). Intensive microbiology review was used to categorize infections and their causative organisms within the first posttransplant year. In total, 397 seromucous infections were observed in 247 patients, most bacterial. Although IgA and IgG were moderately correlated (r = 0.5, P < .0001), low pretransplant IgA was a strong risk factor (P = .01) for seromucous infections, but pretransplant IgG was not (P ≥ .6). As pretransplant IgA levels fell below 200 mg·dL -1 , the risk of these posttransplant infections rose nearly linearly. Lower pretransplant levels of IgA were associated with greater posttransplant mortality to end of follow-up (P = .004), but pretransplant IgG was not (P ≥ .3). Low levels of preoperative IgA, an important immunoglobulin involved in mucosal immunologic defense, but not IgG, are associated with seromucous infections in the year after lung transplantation and increased follow-up mortality. It would appear prudent to identify patients with relative IgA deficiency at listing and to increase vigilance of monitoring for, and prophylaxis against, seromucous infection in this high-risk population. Copyright © 2018. Published by Elsevier Inc.

  9. Purification and characterization of naturally occurring HIV-1 (South African subtype C) protease mutants from inclusion bodies.

    PubMed

    Maseko, Sibusiso B; Natarajan, Satheesh; Sharma, Vikas; Bhattacharyya, Neelakshi; Govender, Thavendran; Sayed, Yasien; Maguire, Glenn E M; Lin, Johnson; Kruger, Hendrik G

    2016-06-01

    Human immunodeficiency virus (HIV) infections in sub-Saharan Africa represent about 56% of global infections. Many studies have targeted HIV-1 protease for the development of drugs against AIDS. Recombinant HIV-1 protease is used to screen new drugs from synthetic compounds or natural substances. Along with the wild type (C-SA) we also over-expressed and characterized two mutant forms from patients that had shown resistance to protease inhibitors. Using recombinant DNA technology, we constructed three recombinant plasmids in pGEX-6P-1 and expressed them containing a sequence encoding wild type HIV protease and two mutants (I36T↑T contains 100 amino acids and L38L↑N↑L contains 101 amino acids). These recombinant proteins were isolated from inclusion bodies by using QFF anion exchange and GST trap columns. In SDS-PAGE, we obtained these HIV proteases as single bands of approximately 11.5, 11.6 and 11.7 kDa for the wild type, I36T↑Tand L38L↑N↑L mutants, respectively. The enzyme was recovered efficiently (0.25 mg protein/L of Escherichia coli culture) and had high specific activity of 2.02, 2.20 and 1.33 μmol min(-1) mg(-1) at an optimal pH of 5 and temperature of 37 °C for the wild type, I36T↑T and L38L↑N↑L, respectively. The method employed here provides an easy and rapid purification of the HIV-1(C-SA) protease from the inclusion bodies, with high yield and high specific activities. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Do we need to measure total serum IgA to exclude IgA deficiency in coeliac disease?

    PubMed Central

    Sinclair, D; Saas, M; Turk, A; Goble, M; Kerr, D

    2006-01-01

    Background Screening for IgA deficiency in patients with coeliac disease is essential because of the increased incidence of IgA deficiency associated with the disease, which usually relies on the estimation of IgA levels in each case. Aim To devise a method of excluding IgA deficiency without measuring total serum IgA in each case. Materials and methods The optical density readings on enzyme‐linked immunosorbent assay (ELISA) of 608 routine samples received for tissue transglutaminase (TTG) antibody testing for coeliac disease were compared with their total IgA concentrations. Dilution experiments were also carried out to ensure linear relationships between optical density on ELISA and IgA concentrations and to compare the sensitivities for TTG and endomysium antibodies in TTG‐positive samples. Results and discussion A clear relationship was shown between total IgA concentration and TTG optical density readings by ELISA. To ensure a positive TTG result if antibodies are present, it was possible to recommend an optical density level above which all samples have sufficient IgA. Samples with optical density <0.05 should be investigated further by estimating total IgA and, if low, samples should be subjected to immunofluorescence microscopy testing for IgA and IgG endomysium antibodies. Conclusions An easier, more cost‐effective and practical way of excluding IgA deficiency in the investigation on coeliac disease is reported. PMID:16489174

  11. Effectiveness of commercial inhibitors against subtype F HIV-1 protease.

    PubMed

    Krauchenco, Sandra; Martins, Nadia H; Sanches, Mario; Polikarpov, Igor

    2009-06-01

    Subtype F wild type HIV protease has been kinetically characterized using six commercial inhibitors (amprenavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir) commonly used for HIV/AIDS treatment, as well as inhibitor TL-3 and acetyl-pepstatin. We also obtained kinetic parameters for two multi-resistant proteases (one of subtype B and one of subtype F) harboring primary and secondary mutations selected by intensive treatment with ritonavir/nelfinavir. This newly obtained biochemical data shows that all six studied commercially available protease inhibitors are significantly less effective against subtype F HIV proteases than against HIV proteases of subtype B, as judged by increased K(i) and biochemical fitness (vitality) values. Comparison with previously reported kinetic values for subtype A and C HIV proteases show that subtype F wild type proteases are significantly less susceptible to inhibition. These results demonstrate that the accumulation of natural polymorphisms in subtype F proteases yields catalytically more active enzymes with a large degree of cross-resistance, which thus results in strong virus viability.

  12. HIV-1 evades virus-specific IgG2 and IgA class switching by targeting systemic and intestinal B cells via long-range intercellular conduits

    PubMed Central

    Xu, Weifeng; Santini, Paul A.; Sullivan, John S.; He, Bing; Shan, Meimei; Ball, Susan C.; Dyer, Wayne B.; Ketas, Thomas J.; Chadburn, Amy; Cohen-Gould, Leona; Knowles, Daniel M.; Chiu, April; Sanders, Rogier W.; Chen, Kang; Cerutti, Andrea

    2009-01-01

    Contact-dependent communication between immune cells generates protection, but also facilitates viral spread. We found that macrophages formed long-range actin-propelled conduits in response to negative factor (Nef), a human immunodeficiency virus type-1 (HIV-1) protein with immunosuppressive functions. Conduits attenuated immunoglobulin G2 (IgG2) and IgA class switching in systemic and intestinal lymphoid follicles by shuttling Nef from infected macrophages to B cells through a guanine exchange factor-dependent pathway involving the amino-terminal anchor, central core and carboxy-terminal flexible loop of Nef. By showing stronger virus-specific IgG2 and IgA responses in patients harboring Nef-deficient virions, our data suggest that HIV-1 exploits intercellular highways as a “Trojan horse” to deliver Nef to B cells and evade humoral immunity systemically and at mucosal sites of entry. PMID:19648924

  13. The crystal structure of the secreted aspartic protease 1 from Candida parapsilosis in complex with pepstatin A

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Dostál, Jiří; Brynda, Jiří; Hrušková-Heidingsfeldová, Olga

    2010-09-01

    Opportunistic pathogens of the genus Candida cause infections representing a major threat to long-term survival of immunocompromised patients. Virulence of the Candida pathogens is enhanced by production of extracellular proteolytic enzymes and secreted aspartic proteases (Saps) are therefore studied as potential virulence factors and possible targets for therapeutic drug design. Candida parapsilosis is less invasive than C. albicans, however, it is one of the leading causative agents of yeast infections. We report three-dimensional crystal structure of Sapp1p from C. parapsilosis in complex with pepstatin A, the classical inhibitor of aspartic proteases. The structure of Sapp1p was determined from protein isolatedmore » from its natural source and represents the first structure of Sap from C. parapsilosis. Overall fold and topology of Sapp1p is very similar to the archetypic fold of monomeric aspartic protease family and known structures of Sap isoenzymes from C. albicans and Sapt1p from C. tropicalis. Structural comparison revealed noticeable differences in the structure of loops surrounding the active site. This resulted in differential character, shape, and size of the substrate binding site explaining divergent substrate specificities and inhibitor affinities. Determination of structures of Sap isoenzymes from various species might contribute to the development of new Sap-specific inhibitors.« less

  14. Crystal structures of inhibitor complexes of human T-cell leukemia virus (HTLV-1) protease

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Satoh, Tadashi; Li, Mi; Nguyen, Jeffrey-Tri

    2010-09-28

    Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus associated with several serious diseases, such as adult T-cell leukemia and tropical spastic paraparesis/myelopathy. For a number of years, the protease (PR) encoded by HTLV-1 has been a target for designing antiviral drugs, but that effort was hampered by limited available structural information. We report a high-resolution crystal structure of HTLV-1 PR complexed with a statine-containing inhibitor, a significant improvement over the previously available moderate-resolution structure. We also report crystal structures of the complexes of HTLV-1 PR with five different inhibitors that are more compact and more potent. A detailedmore » study of structure-activity relationships was performed to interpret in detail the influence of the polar and hydrophobic interactions between the inhibitors and the protease.« less

  15. Crystal Structures of Inhibitir Complexes of Human T-Cell Leukemia Virus (HTLV-1) Protease

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Satoh, Tadashi; Li, Mi; Nguyen, Jeffrey-Tri

    2010-09-17

    Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus associated with several serious diseases, such as adult T-cell leukemia and tropical spastic paraparesis/myelopathy. For a number of years, the protease (PR) encoded by HTLV-1 has been a target for designing antiviral drugs, but that effort was hampered by limited available structural information. We report a high-resolution crystal structure of HTLV-1 PR complexed with a statine-containing inhibitor, a significant improvement over the previously available moderate-resolution structure. We also report crystal structures of the complexes of HTLV-1 PR with five different inhibitors that are more compact and more potent. A detailedmore » study of structure-activity relationships was performed to interpret in detail the influence of the polar and hydrophobic interactions between the inhibitors and the protease.« less

  16. Comparative one-factor-at-a-time, response surface (statistical) and bench-scale bioreactor level optimization of thermoalkaline protease production from a psychrotrophic Pseudomonas putida SKG-1 isolate.

    PubMed

    Singh, Santosh K; Singh, Sanjay K; Tripathi, Vinayak R; Khare, Sunil K; Garg, Satyendra K

    2011-12-28

    Production of alkaline protease from various bacterial strains using statistical methods is customary now-a-days. The present work is first attempt for the production optimization of a solvent stable thermoalkaline protease by a psychrotrophic Pseudomonas putida isolate using conventional, response surface methods, and fermentor level optimization. The pre-screening medium amended with optimized (w/v) 1.0% glucose, 2.0% gelatin and 0.5% yeast extract, produced 278 U protease ml(-1) at 72 h incubation. Enzyme production increased to 431 Uml(-1) when Mg2+ (0.01%, w/v) was supplemented. Optimization of physical factors further enhanced protease to 514 Uml(-1) at pH 9.0, 25°C and 200 rpm within 60 h. The combined effect of conventionally optimized variables (glucose, yeast extract, MgSO4 and pH), thereafter predicted by response surface methodology yielded 617 U protease ml(-1) at glucose 1.25% (w/v), yeast extract 0.5% (w/v), MgSO4 0.01% (w/v) and pH 8.8. Bench-scale bioreactor level optimization resulted in enhanced production of 882 U protease ml(-1) at 0.8 vvm aeration and 150 rpm agitation during only 48 h incubation. The optimization of fermentation variables using conventional, statistical approaches and aeration/agitation at fermentor level resulted in ~13.5 folds increase (882 Uml(-1)) in protease production compared to un-optimized conditions (65 Uml(-1)). This is the highest level of thermoalkaline protease reported so far by any psychrotrophic bacterium.

  17. The Occurrence of Type S1A Serine Proteases in Sponge and Jellyfish

    NASA Technical Reports Server (NTRS)

    Rojas, Ana; Doolittle, Russell F.

    2003-01-01

    Although serine proteases are found in all kinds of cellular organisms and many viruses, the classic "chymotrypsin family" (Group S1A by th e 1998 Barrett nomenclature) has an unusual phylogenetic distribution , being especially common in animals, entirely absent from plants and protists, and rare among fungi. The distribution in Bacteria is larg ely restricted to the genus Streptomyces, although a few isolated occ urrences in other bacteria have been reported. The family may be enti rely absent from Archaea. Although more than a thousand sequences have been reported for enzymes of this type from animals, none of them ha ve been from early diverging phyla like Porifera or Cnidaria, We now report the existence of Group SlA serine proteases in a sponge (phylu m Porifera) and a jellyfish (phylum Cnidaria), making it safe to conc lude that all animal groups possess these enzymes.

  18. T cell cytokine polarity as a determinant of immunoglobulin A (IgA) glycosylation and the severity of experimental IgA nephropathy.

    PubMed

    Chintalacharuvu, S R; Yamashita, M; Bagheri, N; Blanchard, T G; Nedrud, J G; Lamm, M E; Tomino, Y; Emancipator, S N

    2008-09-01

    Immunoglobulin A (IgA) glycosylation, recognized as an important pathogenic factor in IgA nephropathy (IgAN), is apparently controlled by the polarity of T helper (Th) cytokine responses. To examine the role of cytokine polarity in IgAN, inbred mice were immunized by intraperitoneal priming with inactivated Sendai virus (SeV) emulsified in either complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA), which promote Th1- or Th2-immune response, respectively, and then boosted identically twice orally with aqueous suspensions of inactivated virus. Next, some mice were challenged intranasally with infectious SeV. Mice primed with CFA or IFA had equal reductions in nasal viral titre relative to non-immune controls, and equally increased serum levels of SeV-specific IgA antibody. Mice primed with CFA showed higher SeV-specific IgG than those with IFA. Splenocytes from mice primed with IFA produced copious amounts of interleukin (IL)-4 and IL-5, but little interferon-gamma and IL-2; those primed with CFA had reciprocal cytokine recall responses. Total serum IgA and especially SeV-specific IgA from mice primed with IFA showed a selective defect in sialylation and galactosylation. Although the frequency and intensity of glomerular deposits and haematuria did not differ, glomerulonephritis in mice primed with IFA and challenged with infectious virus was more severe than in those given CFA, as judged by serum creatinine level. We conclude that the polarity of T cell cytokines controls the pattern of IgA glycosylation and exerts direct or indirect effects on functional glomerular responses to immune complex deposition.

  19. Cloning a Chymotrypsin-Like 1 (CTRL-1) Protease cDNA from the Jellyfish Nemopilema nomurai

    PubMed Central

    Heo, Yunwi; Kwon, Young Chul; Bae, Seong Kyeong; Hwang, Duhyeon; Yang, Hye Ryeon; Choudhary, Indu; Lee, Hyunkyoung; Yum, Seungshic; Shin, Kyoungsoon; Yoon, Won Duk; Kang, Changkeun; Kim, Euikyung

    2016-01-01

    An enzyme in a nematocyst extract of the Nemopilema nomurai jellyfish, caught off the coast of the Republic of Korea, catalyzed the cleavage of chymotrypsin substrate in an amidolytic kinetic assay, and this activity was inhibited by the serine protease inhibitor, phenylmethanesulfonyl fluoride. We isolated the full-length cDNA sequence of this enzyme, which contains 850 nucleotides, with an open reading frame of 801 encoding 266 amino acids. A blast analysis of the deduced amino acid sequence showed 41% identity with human chymotrypsin-like (CTRL) and the CTRL-1 precursor. Therefore, we designated this enzyme N. nomurai CTRL-1. The primary structure of N. nomurai CTRL-1 includes a leader peptide and a highly conserved catalytic triad of His69, Asp117, and Ser216. The disulfide bonds of chymotrypsin and the substrate-binding sites are highly conserved compared with the CTRLs of other species, including mammalian species. Nemopilema nomurai CTRL-1 is evolutionarily more closely related to Actinopterygii than to Scyphozoan (Aurelia aurita) or Hydrozoan (Hydra vulgaris). The N. nomurai CTRL1 was amplified from the genomic DNA with PCR using specific primers designed based on the full-length cDNA, and then sequenced. The N. nomurai CTRL1 gene contains 2434 nucleotides and four distinct exons. The 5′ donor splice (GT) and 3′ acceptor splice sequences (AG) are wholly conserved. This is the first report of the CTRL1 gene and cDNA structures in the jellyfish N. nomurai. PMID:27399771

  20. Cloning a Chymotrypsin-Like 1 (CTRL-1) Protease cDNA from the Jellyfish Nemopilema nomurai.

    PubMed

    Heo, Yunwi; Kwon, Young Chul; Bae, Seong Kyeong; Hwang, Duhyeon; Yang, Hye Ryeon; Choudhary, Indu; Lee, Hyunkyoung; Yum, Seungshic; Shin, Kyoungsoon; Yoon, Won Duk; Kang, Changkeun; Kim, Euikyung

    2016-07-05

    An enzyme in a nematocyst extract of the Nemopilema nomurai jellyfish, caught off the coast of the Republic of Korea, catalyzed the cleavage of chymotrypsin substrate in an amidolytic kinetic assay, and this activity was inhibited by the serine protease inhibitor, phenylmethanesulfonyl fluoride. We isolated the full-length cDNA sequence of this enzyme, which contains 850 nucleotides, with an open reading frame of 801 encoding 266 amino acids. A blast analysis of the deduced amino acid sequence showed 41% identity with human chymotrypsin-like (CTRL) and the CTRL-1 precursor. Therefore, we designated this enzyme N. nomurai CTRL-1. The primary structure of N. nomurai CTRL-1 includes a leader peptide and a highly conserved catalytic triad of His(69), Asp(117), and Ser(216). The disulfide bonds of chymotrypsin and the substrate-binding sites are highly conserved compared with the CTRLs of other species, including mammalian species. Nemopilema nomurai CTRL-1 is evolutionarily more closely related to Actinopterygii than to Scyphozoan (Aurelia aurita) or Hydrozoan (Hydra vulgaris). The N. nomurai CTRL1 was amplified from the genomic DNA with PCR using specific primers designed based on the full-length cDNA, and then sequenced. The N. nomurai CTRL1 gene contains 2434 nucleotides and four distinct exons. The 5' donor splice (GT) and 3' acceptor splice sequences (AG) are wholly conserved. This is the first report of the CTRL1 gene and cDNA structures in the jellyfish N. nomurai.

  1. A Molecular Sensor To Characterize Arenavirus Envelope Glycoprotein Cleavage by Subtilisin Kexin Isozyme 1/Site 1 Protease.

    PubMed

    Oppliger, Joel; da Palma, Joel Ramos; Burri, Dominique J; Bergeron, Eric; Khatib, Abdel-Majid; Spiropoulou, Christina F; Pasquato, Antonella; Kunz, Stefan

    2016-01-15

    Arenaviruses are emerging viruses including several causative agents of severe hemorrhagic fevers in humans. The advent of next-generation sequencing technology has greatly accelerated the discovery of novel arenavirus species. However, for many of these viruses, only genetic information is available, and their zoonotic disease potential remains unknown. During the arenavirus life cycle, processing of the viral envelope glycoprotein precursor (GPC) by the cellular subtilisin kexin isozyme 1 (SKI-1)/site 1 protease (S1P) is crucial for productive infection. The ability of newly emerging arenaviruses to hijack human SKI-1/S1P appears, therefore, to be a requirement for efficient zoonotic transmission and human disease potential. Here we implement a newly developed cell-based molecular sensor for SKI-1/S1P to characterize the processing of arenavirus GPC-derived target sequences by human SKI-1/S1P in a quantitative manner. We show that only nine amino acids flanking the putative cleavage site are necessary and sufficient to accurately recapitulate the efficiency and subcellular location of arenavirus GPC processing. In a proof of concept, our sensor correctly predicts efficient processing of the GPC of the newly emergent pathogenic Lujo virus by human SKI-1/S1P and defines the exact cleavage site. Lastly, we employed our sensor to show efficient GPC processing of a panel of pathogenic and nonpathogenic New World arenaviruses, suggesting that GPC cleavage represents no barrier for zoonotic transmission of these pathogens. Our SKI-1/S1P sensor thus represents a rapid and robust test system for assessment of the processing of putative cleavage sites derived from the GPCs of newly discovered arenavirus by the SKI-1/S1P of humans or any other species, based solely on sequence information. Arenaviruses are important emerging human pathogens that can cause severe hemorrhagic fevers with high mortality in humans. A crucial step in productive arenavirus infection of human

  2. Association of frailty with the serine protease HtrA1 in older adults.

    PubMed

    Lorenzi, Maria; Lorenzi, Teresa; Marzetti, Emanuele; Landi, Francesco; Vetrano, Davide L; Settanni, Silvana; Antocicco, Manuela; Bonassi, Stefano; Valdiglesias, Vanessa; Bernabei, Roberto; Onder, Graziano

    2016-08-01

    Frailty is a geriatric syndrome characterized by multi system dysregulation. It has been suggested that chronic inflammation may be involved in the pathogenesis of frailty. No study so far has identified accurate, specific and sensitive molecular biomarkers for frailty. High-temperature requirement serine protease A1 (HtrA1) is a secreted multidomain serine protease implicated in the inhibition of signaling of active transforming growth factor-β (TGF-β)1, a cytokine which has an important anti-inflammation role. The aim of the present study was to investigate the association of circulating levels of HtrA1 with frailty in a sample of older adults. The study was performed in 120 older adults aged >65years and admitted to a geriatric outpatient clinic. The frailty status of participants was assessed by both the Fried's criteria (physical frailty, PF) and a modified Rockwood's frailty index (FI). Plasma HtrA1 concentration was measured using commercial ELISA kit. Frailty was identified in 61/120 participants (50.8%) using PF, and in 60/118 subjects (50.8%) using FI. Plasma levels of HtrA1 were significantly higher in individuals classified as frail according to PF (75.9ng/mL, 95% CI 67.4-85.6) as compared with non-frail participants (48.4ng/mL, 95% CI 42.5-54.6, p<0.001). A significant association was also observed between frailty, assessed by FI, and HtrA1 levels (72.2ng/mL, 95% CI 63.4-82.3, vs. 50.4ng/mL, 95% CI 44.3-58.0, p<0.001). These associations were confirmed after adjusting for potential confounders. This study demonstrates for the first time the association of plasma levels of HtrA1 with frailty status. Future investigations are needed to validate the potential value of HtrA1 as possible biomarker for frailty. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. The cysteine protease CEP1, a key executor involved in tapetal programmed cell death, regulates pollen development in Arabidopsis.

    PubMed

    Zhang, Dandan; Liu, Di; Lv, Xiaomeng; Wang, Ying; Xun, Zhili; Liu, Zhixiong; Li, Fenglan; Lu, Hai

    2014-07-01

    Tapetal programmed cell death (PCD) is a prerequisite for pollen grain development in angiosperms, and cysteine proteases are the most ubiquitous hydrolases involved in plant PCD. We identified a papain-like cysteine protease, CEP1, which is involved in tapetal PCD and pollen development in Arabidopsis thaliana. CEP1 is expressed specifically in the tapetum from stages 5 to 11 of anther development. The CEP1 protein first appears as a proenzyme in precursor protease vesicles and is then transported to the vacuole and transformed into the mature enzyme before rupture of the vacuole. cep1 mutants exhibited aborted tapetal PCD and decreased pollen fertility with abnormal pollen exine. A transcriptomic analysis revealed that 872 genes showed significantly altered expression in the cep1 mutants, and most of them are important for tapetal cell wall organization, tapetal secretory structure formation, and pollen development. CEP1 overexpression caused premature tapetal PCD and pollen infertility. ELISA and quantitative RT-PCR analyses confirmed that the CEP1 expression level showed a strong relationship to the degree of tapetal PCD and pollen fertility. Our results reveal that CEP1 is a crucial executor during tapetal PCD and that proper CEP1 expression is necessary for timely degeneration of tapetal cells and functional pollen formation. © 2014 American Society of Plant Biologists. All rights reserved.

  4. Activities of the human immunodeficiency virus type 1 (HIV-1) protease inhibitor nelfinavir mesylate in combination with reverse transcriptase and protease inhibitors against acute HIV-1 infection in vitro.

    PubMed

    Patick, A K; Boritzki, T J; Bloom, L A

    1997-10-01

    Nelfinavir mesylate (formerly AG1343) is a potent and selective, nonpeptidic inhibitor of human immunodeficiency virus type 1 (HIV-1) protease that was discovered by protein structure-based design methodologies. We evaluated the antiviral and cytotoxic effects of two-drug combinations of nelfinavir with the clinically approved antiretroviral therapeutics zidovudine (ZDV), lamivudine (3TC), dideoxycytidine (ddC; zalcitabine), stavudine (d4T), didanosine (ddI), indinavir, saquinavir, and ritonavir and a three-drug combination of nelfinavir with ZDV and 3TC against an acute HIV-1 strain RF infection of CEM-SS cells in vitro. Quantitative assessment of drug interaction was evaluated by a universal response surface approach (W. R. Greco, G. Bravo, and J. C. Parsons, Pharm. Rev. 47:331-385, 1995) and by the method of M. N. Prichard and C. Shipman (Antiviral Res. 14:181-206, 1990). Both analytical methods yielded similar results and showed that the two-drug combinations of nelfinavir with the reverse transcriptase inhibitors ZDV, 3TC, ddI, d4T, and ddC and the three-drug combination with ZDV and 3TC resulted in additive to statistically significant synergistic interactions. In a similar manner, the combination of nelfinavir with the three protease inhibitors resulted in additive (ritonavir and saquinavir) to slightly antagonistic (indinavir) interactions. In all combinations, minimal cellular cytotoxicity was observed with any drug alone and in combination. These results suggest that administration of combinations of the appropriate doses of nelfinavir with other currently approved antiretroviral therapeutic agents in vivo may result in enhanced antiviral activity with no associated increase in cellular cytotoxicity.

  5. PAR-1 mediated apoptosis of breast cancer cells by V. cholerae hemagglutinin protease.

    PubMed

    Ray, Tanusree; Pal, Amit

    2016-05-01

    Bacterial toxins have emerged as promising agents in cancer treatment strategy. Hemagglutinin (HAP) protease secreted by Vibrio cholerae induced apoptosis in breast cancer cells and regresses tumor growth in mice model. The success of novel cancer therapies depends on their selectivity for cancer cells with limited toxicity for normal tissues. Increased expression of Protease Activated Receptor-1 (PAR-1) has been reported in different malignant cells. In this study we report that HAP induced activation and over expression of PAR-1 in breast cancer cells (EAC). Immunoprecipitation studies have shown that HAP specifically binds with PAR-1. HAP mediated activation of PAR-1 caused nuclear translocation of p50-p65 and the phosphorylation of p38 which triggered the activation of NFκB and MAP kinase signaling pathways. These signaling pathways enhanced the cellular ROS level in malignant cells that induced the intrinsic pathway of cell apoptosis. PAR-1 mediated apoptosis by HAP of malignant breast cells without effecting normal healthy cells in the same environment makes it a good therapeutic agent for treatment of cancer.

  6. Effects of drug-resistant mutations on the dynamic properties of HIV-1 protease and inhibition by Amprenavir and Darunavir

    PubMed Central

    Yu, Yuqi; Wang, Jinan; Shao, Qiang; Shi, Jiye; Zhu, Weiliang

    2015-01-01

    Molecular dynamics simulations are performed to investigate the dynamic properties of wild-type HIV-1 protease and its two multi-drug-resistant variants (Flap + (L10I/G48V/I54V/V82A) and Act (V82T/I84V)) as well as their binding with APV and DRV inhibitors. The hydrophobic interactions between flap and 80 s (80’s) loop residues (mainly I50-I84’ and I50’-I84) play an important role in maintaining the closed conformation of HIV-1 protease. The double mutation in Act variant weakens the hydrophobic interactions, leading to the transition from closed to semi-open conformation of apo Act. APV or DRV binds with HIV-1 protease via both hydrophobic and hydrogen bonding interactions. The hydrophobic interactions from the inhibitor is aimed to the residues of I50 (I50’), I84 (I84’), and V82 (V82’) which create hydrophobic core clusters to further stabilize the closed conformation of flaps, and the hydrogen bonding interactions are mainly focused with the active site of HIV-1 protease. The combined change in the two kinds of protease-inhibitor interactions is correlated with the observed resistance mutations. The present study sheds light on the microscopic mechanism underlying the mutation effects on the dynamics of HIV-1 protease and the inhibition by APV and DRV, providing useful information to the design of more potent and effective HIV-1 protease inhibitors. PMID:26012849

  7. Synthetic, structural mimetics of the β-hairpin flap of HIV-1 protease inhibit enzyme function.

    PubMed

    Chauhan, Jay; Chen, Shen-En; Fenstermacher, Katherine J; Naser-Tavakolian, Aurash; Reingewertz, Tali; Salmo, Rosene; Lee, Christian; Williams, Emori; Raje, Mithun; Sundberg, Eric; DeStefano, Jeffrey J; Freire, Ernesto; Fletcher, Steven

    2015-11-01

    Small-molecule mimetics of the β-hairpin flap of HIV-1 protease (HIV-1 PR) were designed based on a 1,4-benzodiazepine scaffold as a strategy to interfere with the flap-flap protein-protein interaction, which functions as a gated mechanism to control access to the active site. Michaelis-Menten kinetics suggested our small-molecules are competitive inhibitors, which indicates the mode of inhibition is through binding the active site or sterically blocking access to the active site and preventing flap closure, as designed. More generally, a new bioactive scaffold for HIV-1PR inhibition has been discovered, with the most potent compound inhibiting the protease with a modest K(i) of 11 μM. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. [Progress in understanding the pathogenesis of IgA nephropathy: new perspectives for the near future?].

    PubMed

    Segarra, A

    2010-01-01

    Progress in understanding the pathogenesis of IgA nephropathy has shown that probably there is no a single IgA nephropathy with the same pathogenic mechanism, clinical course and response to therapy. The evidence currently available suggests the existence of at least two possible mechanisms of IgA deposition in the renal mesangium. In a small percentage of patients, mesangial deposition of IgA1 colocalizes with secretory component, indicating that the deposited IgA1 in glomeruli originates completely or partly in the mucose-associated lymphoid tissue. This deposition pattern has been associated with activation of complement by the lectin pathway and has been associated with a worse prognosis, although this last statement needs to be confirmed in long-term studies. The mechanisms responsible for secretory IgA deposition are not known. In the majority of patients with IgA nephropathy secretory component is not detectable in the mesangium. In these cases, the presence of elevated circulating levels of galactose-deficient IgA, produced by bone marrow plasma cells would be a predisposing factor but not sufficient to induce nephropathy. To produce kidney disease, galactose-deficient IgA1 must be deposited in the renal mesangium, and once there, either by interaction with specific receptors (CD71?), by direct activation of complement or by being the target of an IgG autoimmune response anti-IgA, induce activation, proliferation and increased mesangial matrix synthesis and eventually cell injury. In parallel, galactose-deficient IgA, through interaction with the RR Fc alpha/gamma, may activate circulating lymphocytes and monocytes and enhance their response to chemoattractants produced by the mesangial cell, causing, thus, the inflammatory infiltrate to initiate and maintain the interstitial injury. In the next few years, advances recently added to the knowledge of the pathogenesis of nephropathy IgA1 could provide new variables that allow walking in the direction of

  9. Discovery of potent peptide-mimetic antagonists for the human thrombin receptor, protease-activated receptor-1 (PAR-1).

    PubMed

    Maryanoff, Bruce E; Zhang, Han-Cheng; Andrade-Gordon, Patricia; Derian, Claudia K

    2003-03-01

    Protease-activated receptors (PARs) represent a unique family of seven-transmembrane G-protein-coupled receptors, which are enzymatically cleaved to expose a new extracellular N-terminus that acts as a tethered activating ligand. PAR-1 is cleaved and activated by the serine protease alpha-thrombin, is expressed in various tissues (e.g. platelets and vascular cells), and is involved in cellular responses associated with hemostasis, proliferation, and tissue injury. By using a de novo design approach, we have discovered a series of potent heterocycle-based peptide-miimetic antagonists of PAR-1, exemplified by advanced leads RWJ-56110 (22) and RWJ-58259 (32). These compounds are potent, selective PAR-1 antagonists, devoid of PAR-1 agonist and thrombin inhibitory activity: they bind to PAR-1, interfere with calcium mobilization and cellular functions associated with PAR-1, and do not affect PAR-2, PAR-3, or PAR-4. RWJ-56110 was determined to be a direct inhibitor of PAR-1 activation and internalization, without affecting PAR-1 N-terminal cleavage. At high concentrations of alpha-thrombin, RWJ-56110 fully blocked activation responses in human vascular cells, but not in human platelets; whereas, at high concentrations of TRAP-6, RWJ-56110 blocked activation responses in both cell types. This result is consistent with the presence of another thrombin receptor on human platelets, namely PAR-4. RWJ-56110 and RWJ-58259 clearly interrupt the binding of a tethered ligand to its receptor. RWJ-58259 demonstrated antirestenotic activity in a rat balloon angioplasty model and antithrombotic activity in a cynomolgus monkey arterial injury model. Such PAR-1 antagonists should not only serve as useful tools to delineate the physiological and pathophysiological roles of PAR-1, but also may have therapeutic potential for treating thrombosis and restenosis in humans.

  10. Discovery of HIV Type 1 Aspartic Protease Hit Compounds through Combined Computational Approaches.

    PubMed

    Xanthopoulos, Dimitrios; Kritsi, Eftichia; Supuran, Claudiu T; Papadopoulos, Manthos G; Leonis, Georgios; Zoumpoulakis, Panagiotis

    2016-08-05

    A combination of computational techniques and inhibition assay experiments was employed to identify hit compounds from commercial libraries with enhanced inhibitory potency against HIV type 1 aspartic protease (HIV PR). Extensive virtual screening with the aid of reliable pharmacophore models yielded five candidate protease inhibitors. Subsequent molecular dynamics and molecular mechanics Poisson-Boltzmann surface area free-energy calculations for the five ligand-HIV PR complexes suggested a high stability of the systems through hydrogen-bond interactions between the ligands and the protease's flaps (Ile50/50'), as well as interactions with residues of the active site (Asp25/25'/29/29'/30/30'). Binding-energy calculations for the three most promising compounds yielded values between -5 and -10 kcal mol(-1) and suggested that van der Waals interactions contribute most favorably to the total energy. The predicted binding-energy values were verified by in vitro inhibition assays, which showed promising results in the high nanomolar range. These results provide structural considerations that may guide further hit-to-lead optimization toward improved anti-HIV drugs. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. A Modified P1 Moiety Enhances in vitro Antiviral Activity against Various Multi-Drug-Resistant HIV-1 Variants and in vitro CNS Penetration Properties of a Novel Nonpeptidic Protease Inhibitor, GRL-10413

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Amano, Masayuki; Salcedo-Gómez, Pedro Miguel; Zhao, Rui

    We here report that GRL-10413, a novel non-peptidic HIV-1 protease inhibitor (PI) containing a modified P1 moiety and a sulfonamide isostere, is highly active against laboratory HIV-1 strains and primary clinical isolates (EC 50: 0.00035 - 0.0018 μM) with minimal cytotoxicity (CC 50: 35.7 μM). GRL-10413 blocked the infectivity and replication of HIV-1 NL4-3variants selected by up to 5 μM concentrations of atazanavir, lopinavir, or amprenavir (EC 50: 0.0021 - 0.0023 μM). GRL-10413 also maintained its strong antiviral activity against multi-drug-resistant clinical HIV-1 variants isolated from patients, who no longer responded to various antiviral regimens after long-term antiretroviral therapy. Themore » development of resistance against GRL-10413 was significantly delayed compared to that of APV. In addition, GRL-10413 showed a favorable central nervous system (CNS) penetration property as assessed with anin vitroblood brain barrier (BBB) reconstruction system. Analysis of the crystal structure of HIV-1 protease in complex with GRL-10413 demonstrated that the modified P1 moiety of GRL-10413 has a greater hydrophobic surface area and makes greater van der Waals contacts with active-site amino acids of protease than in the case of darunavir. Moreover, the chlorine substituent in the P1 moiety interacts with protease in two distinct configurations. The present data demonstrate that GRL-10413 has desirable features for treating patients infected with wild-type and/or multi-drug-resistant HIV-1 variants with favorable CNS-penetration capability and that the newly modified P1-moiety may confer desirable features in designing novel anti-HIV-1 PIs.« less

  12. NS3 protease resistance-associated substitutions in liver tissue and plasma samples from patients infected by hepatitis C virus genotype 1A or 1B.

    PubMed

    Morsica, Giulia; Andolina, Andrea; Merli, Marco; Messina, Emanuela; Hasson, Hamid; Lazzarin, Adriano; Uberti-Foppa, Caterina; Bagaglio, Sabrina

    2017-08-01

    The presence of naturally occurring resistance-associated substitutions (RASs) in the HCV-protease domain has been poorly investigated in the liver, the main site of HCV replication. We evaluated the natural resistance of the virus to NS3 protease inhibitors in liver tissue and plasma samples taken from HCV-infected patients. RASs were investigated by means of viral population sequencing in liver tissue samples from 18 HCV-infected patients harbouring genotype 1a or genotype 1b; plasma samples from 12 of these patients were also available for virological investigation. A discordant genotype was found in two of the 12 patients (16.6%) who provided samples from both compartments. Sequence analysis of the NS3 protease domain showed the presence of RASs in four of the 18 liver tissue samples (22.2%), two of which showed cross-resistance to protease inhibitors in clinical use or phase 2-3 trials. The analysis of the 12 paired tissues and plasma samples excluded the presence of RASs in the plasma compartment. The dominance of discordant genotypes in the paired liver and plasma samples of some HCV-infected patients suggests mixed infection possibly leading to the selective advantage of different genotype in the two compartments. The presence of RASs at intra-hepatic level is not uncommon and may lead to the early emergence of cross-resistant strains.

  13. In vitro protease cleavage and computer simulations reveal the HIV-1 capsid maturation pathway

    NASA Astrophysics Data System (ADS)

    Ning, Jiying; Erdemci-Tandogan, Gonca; Yufenyuy, Ernest L.; Wagner, Jef; Himes, Benjamin A.; Zhao, Gongpu; Aiken, Christopher; Zandi, Roya; Zhang, Peijun

    2016-12-01

    HIV-1 virions assemble as immature particles containing Gag polyproteins that are processed by the viral protease into individual components, resulting in the formation of mature infectious particles. There are two competing models for the process of forming the mature HIV-1 core: the disassembly and de novo reassembly model and the non-diffusional displacive model. To study the maturation pathway, we simulate HIV-1 maturation in vitro by digesting immature particles and assembled virus-like particles with recombinant HIV-1 protease and monitor the process with biochemical assays and cryoEM structural analysis in parallel. Processing of Gag in vitro is accurate and efficient and results in both soluble capsid protein and conical or tubular capsid assemblies, seemingly converted from immature Gag particles. Computer simulations further reveal probable assembly pathways of HIV-1 capsid formation. Combining the experimental data and computer simulations, our results suggest a sequential combination of both displacive and disassembly/reassembly processes for HIV-1 maturation.

  14. Immunocytochemical localization of the ovine immunoglobulins IgA, IgG1, IgG1A and IgG2: effect of gastro-intestinal parasitism in the sheep

    PubMed Central

    Curtain, C. C.; Anderson, N.

    1971-01-01

    A study has been made of the immunocytochemical localization of IgG1, IgG2, IgG1A and IgA in the alimentary tract and associated lymph nodes of parasitized and parasite-free sheep. No immunoglobulin-containing cells were found in the abomasal mucosa of the parasite-free sheep. On the other hand, large numbers of IgG1 and IgG1A-containing cells were found in the lamina propria and at the base of the villi of the abomasum of the parasitized sheep. IgG1, IgG1A, and IgA-containing cells were found in mucosal sections from the jejunum and ileum of both parasitized and parasite-free sheep, the number of IgG1A-containing cells being sifnificantly greater in the former than in the latter. This increase was considered to be of some importance since the IgG1A subclass appears to be involved in the allergic response of the sheep to intestinal parasites. ImagesFIG. 1FIG. 2FIG. 3FIG. 4FIG. 5FIG. 6FIG. 7 PMID:4924939

  15. HTRA1, an age-related macular degeneration protease, processes extracellular matrix proteins EFEMP1 and TSP1.

    PubMed

    Lin, Michael K; Yang, Jin; Hsu, Chun Wei; Gore, Anuradha; Bassuk, Alexander G; Brown, Lewis M; Colligan, Ryan; Sengillo, Jesse D; Mahajan, Vinit B; Tsang, Stephen H

    2018-05-05

    High-temperature requirement protein A1 (HTRA1) is a serine protease secreted by a number of tissues including retinal pigment epithelium (RPE). A promoter variant of the gene encoding HTRA1 is part of a mutant allele that causes increased HTRA1 expression and contributed to age-related macular degeneration (AMD) in genomewide association studies. AMD is characterized by pathological development of drusen, extracellular deposits of proteins and lipids on the basal side of RPE. The molecular pathogenesis of AMD is not well understood, and understanding dysregulation of the extracellular matrix may be key. We assess the high-risk genotype at 10q26 by proteomic comparison of protein levels of RPE cells with and without the mutation. We show HTRA1 protein level is increased in high-risk RPE cells along with several extracellular matrix proteins, including known HTRA1 cleavage targets LTBP-1 and clusterin. In addition, two novel targets of HTRA1 have been identified: EFEMP1, an extracellular matrix protein mutated in Doyne honeycomb retinal dystrophy, a genetic eye disease similar to AMD, and thrombospondin 1 (TSP1), an inhibitor of angiogenesis. Our data support the role of RPE extracellular deposition with potential effects in compromised barrier to neovascularization in exudative AMD. © 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  16. Physiological and serological variation in S. mitis biovar 1 from the human oral cavity during the first year of life

    PubMed Central

    Kirchherr, Jennifer L.; Bowden, George H.; Cole, Michael F.; Kawamura, Yoshiaki; Richmond, Dorothy A.; Sheridan, Michael J.; Wirth, Katherine A.

    2007-01-01

    SUMMARY Objective The purpose of the study was to explore the physiological and antigenic diversity of a large number of S. mitis biovar 1 isolates in order to begin to determine whether these properties contribute to species persistence. Design S. mitis biovar 1 was collected from four infants from birth to one year of age. At each of 8–9 visits 60 isolates each were obtained from the cheeks, tongue and incisors (once erupted) yielding 4,440 in total. These were tested for production of neuraminidase, β1-N-acetylglucosaminidase, β1-N-acetylgalactosaminidase, IgA1 protease, and amylase-binding. Antigenic diversity was examined by ELISA and Western immunoblotting using antisera raised against S. mitis biovar 1 NCTC 12261T and SK145. Results 3,330 (75%) of the isolates were identified as S. mitis biovar 1 and 3,144 (94.4%) could be divided into four large phenotypic groups based on glycosidase production. 54% of the isolates produced IgA1 protease, but production was disproportionate among the phenotypes. Between 1/3 to 1/2 of the strains of each phenotype bound salivary α-amylase. Antisera against strains NCTC 12261T and SK145 displayed different patterns of reactivity with randomly selected representatives of the four phenotypes. Conclusions S. mitis biovar 1 is physiologically and antigenically diverse, properties which could aid strains in avoiding host immunity and promote re-colonization of a habitat or transfer to a new habitat. PMID:17045561

  17. Selective IgA Deficiency

    MedlinePlus

    ... immunoglobulins. Videos: Choosing Wisely » Selective IgA Deficiency Treatment & Management The underlying cause for Selective IgA Deficiency is ... the Evidence » Practice Parameter for the Diagnosis and Management of Primary Immunodefiency » 2017 Non-CME Recordings » Vaccination ...

  18. The role played by serine proteases in the development and worsening of vascular complications in type 1 diabetes mellitus.

    PubMed

    Finotti, Paola

    2006-08-01

    Much attention has been given to the role played by serine proteases in the development and worsening of vascular complications in Type 1 diabetes mellitus. A generalized increase in proteolytic activity, either due to a true increase in concentration of specific proteases or defects of their protease inhibitors, represents an early marker of diabetes. However, the precise molecular mechanism whereby an unopposed proteolytic activity leads to overt vascular alterations has not fully been elucidated as yet. The picture is further complicated by the fact that, although sharing the same function, serine proteases constitute a structurally heterogeneous class of molecules. Besides classical proteases, for most part belonging to coagulative and fibrinolytic systems, other unrelated molecules exhibit serine-like protease activity and are capable of triggering both inflammatory and immune reactions. The specific role of these non classical serine proteases in the complex pathogenesis of diabetes and its vascular complications is attracting a new investigative interest, as these molecules may represent additional therapeutic targets. This review will focus on most recent acquisitions on this issue relevant to Type 1 diabetes.

  19. A systematic review of anti-rotavirus serum IgA antibody titer as a potential correlate of rotavirus vaccine efficacy.

    PubMed

    Patel, Manish; Glass, Roger I; Jiang, Baoming; Santosham, Mathuram; Lopman, Ben; Parashar, Umesh

    2013-07-15

    Identifying an immunological correlate of protection for rotavirus vaccines (Rotarix [RV1] and RotaTeq [RV5]) would substantially facilitate testing of interventions for improving efficacy in developing countries and evaluating additional candidate rotavirus vaccines. We accessed PubMed and ClinicalTrials.gov to identify immunogenicity and efficacy trials for RV1 and RV5 to correlate anti-rotavirus serum immunoglobulin A (IgA) antibody titers vs efficacy in regions stratified by all-cause under-5 mortality rates (u5MR). We established a cutoff point for IgA geometric mean concentration or titer (GMC) that predicted lower efficacy and calculated pooled vaccine efficacy among countries with high vs low IgA titers. We observed an inverse correlation between u5MR and IgA titers for RV1 (r(2) = 0.72; P < .001 and RV5 (r(2) = 0.66; P < .001) and between efficacy and IgA titers for both vaccines (r(2) = 0.56; P = .005). Postimmunization anti-rotavirus IgA GMC <90 were associated with decline in vaccine efficacy. Efficacy during first 2 years of life was significantly lower among countries with IgA GMC < 90 (44%; 95% confidence interval [CI], 30-55) compared to countries with GMC > 90 (85%; 95% CI, 82-88). We observed a significant correlation between IgA titers and rotavirus vaccine efficacy and hypothesize that a critical level of IgA antibody titer is associated with a sufficient level of sustained protection after rotavirus vaccination.

  20. Targeting cysteine residues of human immunodeficiency virus type 1 protease by reactive free radical species.

    PubMed

    Basu, A; Sehajpal, P K; Ogiste, J S; Lander, H M

    1999-01-01

    Nitric oxide (NO) is a naturally occurring free radical with many functions. The oxidized form of NO, the nitrosonium ion, reacts with the thiol group of cysteine residues resulting in their modification to S-nitrosothiols. The human immunodeficiency virus type 1 (HIV-1) protease (HIV-PR) has two cysteine residues that are conserved amongst different viral isolates found in patients with acquired immunodeficiency syndrome (AIDS). In an active dimer, these residues are located near the surface of the protease. We have found that treatment of HIV-PR with different NO congeners results in loss of its proteolytic activity and simultaneous formation of S-nitrosothiols. Sodium nitroprusside inhibited HIV-PR up to 70% and S-nitroso-N-acetylpenicillamine completely inhibited the protease within 5 min of treatment. The pattern of inhibition by NO donors is comparable to its inhibition by N-acetyl pepstatin. Using electrospray ionization-mass spectrometry, we identified the modification of HIV-PR by NO as that of S-nitrosation. Our findings point towards a possible role of NO in mediating resistance to HIV-1 infection.

  1. Activities of the human immunodeficiency virus type 1 (HIV-1) protease inhibitor nelfinavir mesylate in combination with reverse transcriptase and protease inhibitors against acute HIV-1 infection in vitro.

    PubMed Central

    Patick, A K; Boritzki, T J; Bloom, L A

    1997-01-01

    Nelfinavir mesylate (formerly AG1343) is a potent and selective, nonpeptidic inhibitor of human immunodeficiency virus type 1 (HIV-1) protease that was discovered by protein structure-based design methodologies. We evaluated the antiviral and cytotoxic effects of two-drug combinations of nelfinavir with the clinically approved antiretroviral therapeutics zidovudine (ZDV), lamivudine (3TC), dideoxycytidine (ddC; zalcitabine), stavudine (d4T), didanosine (ddI), indinavir, saquinavir, and ritonavir and a three-drug combination of nelfinavir with ZDV and 3TC against an acute HIV-1 strain RF infection of CEM-SS cells in vitro. Quantitative assessment of drug interaction was evaluated by a universal response surface approach (W. R. Greco, G. Bravo, and J. C. Parsons, Pharm. Rev. 47:331-385, 1995) and by the method of M. N. Prichard and C. Shipman (Antiviral Res. 14:181-206, 1990). Both analytical methods yielded similar results and showed that the two-drug combinations of nelfinavir with the reverse transcriptase inhibitors ZDV, 3TC, ddI, d4T, and ddC and the three-drug combination with ZDV and 3TC resulted in additive to statistically significant synergistic interactions. In a similar manner, the combination of nelfinavir with the three protease inhibitors resulted in additive (ritonavir and saquinavir) to slightly antagonistic (indinavir) interactions. In all combinations, minimal cellular cytotoxicity was observed with any drug alone and in combination. These results suggest that administration of combinations of the appropriate doses of nelfinavir with other currently approved antiretroviral therapeutic agents in vivo may result in enhanced antiviral activity with no associated increase in cellular cytotoxicity. PMID:9333041

  2. Production and characterization of a novel protease from Bacillus sp. RRM1 under solid state fermentation.

    PubMed

    Rajkumar, Renganathan; Kothilmozhian, Jayappriyan; Ramasamy, Rengasamy

    2011-06-01

    A commercially important alkaline protease, produced by Bacillus sp. RRM1 isolated from the red seaweed Kappaphycus alvarezii (Doty) Doty ex Silva, was first recognized and characterized in the present study. Identification of the isolated bacterium was done using both biochemical characterization as well as 16S rRNA gene sequencing. The bacterial strain, Bacillus sp. RRM1, produced a high level of protease using easily available, inexpensive agricultural residues solid-state fermentation (SSF). Among them, wheat bran was found to be the best substrate. Influences of process parameters such as moistening agents, moisture level, temperature, inoculum concentration, and co-carbon and co-nitrogen sources on the fermentation were also evaluated. Under optimized conditions, maximum protease production (i.e., 2081 U/g) was obtained from wheat bran, which is about 2-fold greater than the initial conditions. The protease enzyme was stable over a temperature range of 30-60 degrees C and pH 6-12, with maximum activity at 50 degrees C and pH 9.0. Whereas the metal ions Na+, Ca2+, and K+ enhanced the activity of the enzyme, others such as Hg2+, Cu2+, Fe2+, Co2+, and Zn2+ had rendered negative effects. The activity of the enzyme was inhibited by EDTA and enhanced by Cu2+ ions, thus indicating the nature of the enzyme as a metalloprotease. The enzyme showed extreme stability and activity even in the presence of detergents, surfactants, and organic solvents. Moreover, the present findings opened new vistas in the utilization of wheat bran, a cheap, abundantly available, and effective waste as a substrate for SSF.

  3. Cytokine, chemokine and secretory IgA levels in human milk in relation to atopic disease and IgA production in infants.

    PubMed

    Böttcher, Malin F; Jenmalm, Maria C; Björkstén, Bengt

    2003-02-01

    The relationship between breast-feeding, IgA production and development of atopic disease in children is a matter of controversy. Some of this controversy might be due to individual differences in the composition of breast milk. The aim of this study was to relate the levels of cytokines, chemokines and secretory (S)-IgA antibodies in breast milk to the development of atopic manifestation and salivary IgA production in infants. Cytokine, chemokine and SIgA levels, as measured with enzyme-linked immunosorbent assay (ELISA), in colostrum and mature milk were analyzed in relation to the development of positive skin-prick tests (SPT), allergic symptoms and salivary IgA antibody production during the first 2 years of life in 53 infants. There was no association between levels of IL-4, -5, -6, -8, -10, -13, -16, IFN-gamma, TGF-beta1, -beta2, RANTES, eotaxin or SIgA levels in the breast milk with either SPT-positivity, development of allergic symptoms or salivary IgA levels during the first 2 years of life in the infants. Thus, differences in the composition of cytokines, chemokines and SIgA in breast milk did not, to any major degree, affect the development of a positive SPT, atopic symptoms, nor salivary IgA antibody production during the first 2 years of life.

  4. Transcriptional activation of a 37 kDa ethylene responsive cysteine protease gene, RbCP1, is associated with protein degradation during petal abscission in rose

    PubMed Central

    Tripathi, Siddharth Kaushal; Singh, Amar Pal; Sane, Aniruddha P.; Nath, Pravendra

    2009-01-01

    Cysteine proteases play an important role in several developmental processes in plants, particularly those related to senescence and cell death. A cysteine protease gene, RbCP1, has been identified that encodes a putative protein of 357 amino acids and is expressed in the abscission zone (AZ) of petals in rose. The gene was responsive to ethylene in petals, petal abscission zones, leaves, and thalamus. The expression of RbCP1 increased during both ethylene-induced as well as natural abscission and was inhibited by 1-MCP. Transcript accumulation of RbCP1 was accompanied by the appearance of a 37 kDa cysteine protease, a concomitant increase in protease activity and a substantial decrease in total protein content in the AZ of petals. Agro-injection of rose petals with a 2.0 kb region upstream of the RbCP1 gene could drive GUS expression in an abscission zone-specific manner and was blocked by 1-MCP. It is concluded that petal abscission is associated with a decrease in total protein content resulting from rapid transcription of RbCP1 and the expression of a 37 kDa protease. PMID:19346241

  5. Human milk lactoferrin inactivates two putative colonization factors expressed by Haemophilus influenzae.

    PubMed

    Qiu, J; Hendrixson, D R; Baker, E N; Murphy, T F; St Geme, J W; Plaut, A G

    1998-10-13

    Haemophilus influenzae is a major cause of otitis media and other respiratory tract disease in children. The pathogenesis of disease begins with colonization of the upper respiratory mucosa, a process that involves evasion of local immune mechanisms and adherence to epithelial cells. Several studies have demonstrated that human milk is protective against H. influenzae colonization and disease. In the present study, we examined the effect of human milk on the H. influenzae IgA1 protease and Hap adhesin, two autotransported proteins that are presumed to facilitate colonization. Our results demonstrated that human milk lactoferrin efficiently extracted the IgA1 protease preprotein from the bacterial outer membrane. In addition, lactoferrin specifically degraded the Hap adhesin and abolished Hap-mediated adherence. Extraction of IgA1 protease and degradation of Hap were localized to the N-lobe of the bilobed lactoferrin molecule and were inhibited by serine protease inhibitors, suggesting that the lactoferrin N-lobe may contain serine protease activity. Additional experiments revealed no effect of lactoferrin on the H. influenzae P2, P5, and P6 outer-membrane proteins, which are distinguished from IgA1 protease and Hap by the lack of an N-terminal passenger domain or an extracellular linker region. These results suggest that human milk lactoferrin may attenuate the pathogenic potential of H. influenzae by selectively inactivating IgA1 protease and Hap, thereby interfering with colonization. Future studies should examine the therapeutic potential of lactoferrin, perhaps as a supplement in infant formulas.

  6. Glomerular diseases: emerging tests and therapies for IgA nephropathy.

    PubMed

    Canetta, Pietro A; Kiryluk, Krzysztof; Appel, Gerald B

    2014-03-01

    The last decade has seen major progress in understanding the pathogenesis as well as the prognosis and treatment of patients with IgA nephropathy (IgAN). Although the diagnostic criterion of a kidney biopsy demonstrating dominant or codominant IgA deposition remains unchanged, much more is known about the genetic and environmental factors predisposing to disease development and progression. These advances have led to the identification of novel diagnostic and prognostic markers. Among the most promising clinically are genetic profiling, quantification of galactose-deficient IgA1 levels, and measurement of anti-IgA1 immunoglobulins. While targeted treatment for IgAN remains elusive, there is mounting evidence for therapeutic interventions that alter the disease course. The appropriate validation and integration of these discoveries into clinical care represent a major challenge, but one that holds tremendous promise for refining prognostication, guiding therapy, and improving the lives of patients with IgAN.

  7. Room temperature neutron crystallography of drug resistant HIV-1 protease uncovers limitations of X-ray structural analysis at 100K

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Gerlits, Oksana O.; Keen, David A.; Blakeley, Matthew P.

    HIV-1 protease inhibitors are crucial for treatment of HIV-1/AIDS, but their effectiveness is thwarted by rapid emergence of drug resistance. To better understand binding of clinical inhibitors to resistant HIV-1 protease, we used room-temperature joint X-ray/neutron (XN) crystallography to obtain an atomic-resolution structure of the protease triple mutant (V32I/I47V/V82I) in complex with amprenavir. The XN structure reveals a D+ ion located midway between the inner Oδ1 oxygen atoms of the catalytic aspartic acid residues. Comparison of the current XN structure with our previous XN structure of the wild-type HIV-1 protease-amprenavir complex suggests that the three mutations do not significantly altermore » the drug–enzyme interactions. This is in contrast to the observations in previous 100 K X-ray structures of these complexes that indicated loss of interactions by the drug with the triple mutant protease. These findings, thus, uncover limitations of structural analysis of drug binding using X-ray structures obtained at 100 K.« less

  8. Room temperature neutron crystallography of drug resistant HIV-1 protease uncovers limitations of X-ray structural analysis at 100K

    DOE PAGES

    Gerlits, Oksana O.; Keen, David A.; Blakeley, Matthew P.; ...

    2017-02-14

    HIV-1 protease inhibitors are crucial for treatment of HIV-1/AIDS, but their effectiveness is thwarted by rapid emergence of drug resistance. To better understand binding of clinical inhibitors to resistant HIV-1 protease, we used room-temperature joint X-ray/neutron (XN) crystallography to obtain an atomic-resolution structure of the protease triple mutant (V32I/I47V/V82I) in complex with amprenavir. The XN structure reveals a D+ ion located midway between the inner Oδ1 oxygen atoms of the catalytic aspartic acid residues. Comparison of the current XN structure with our previous XN structure of the wild-type HIV-1 protease-amprenavir complex suggests that the three mutations do not significantly altermore » the drug–enzyme interactions. This is in contrast to the observations in previous 100 K X-ray structures of these complexes that indicated loss of interactions by the drug with the triple mutant protease. These findings, thus, uncover limitations of structural analysis of drug binding using X-ray structures obtained at 100 K.« less

  9. Proteases in agricultural dust induce lung inflammation through PAR-1 and PAR-2 activation.

    PubMed

    Romberger, Debra J; Heires, Art J; Nordgren, Tara M; Souder, Chelsea P; West, William; Liu, Xiang-de; Poole, Jill A; Toews, Myron L; Wyatt, Todd A

    2015-08-15

    Workers exposed to aerosolized dust present in concentrated animal feeding operations (CAFOs) are susceptible to inflammatory lung diseases, such as chronic obstructive pulmonary disease. Extracts of dust collected from hog CAFOs [hog dust extract (HDE)] are potent stimulators of lung inflammatory responses in several model systems. The observation that HDE contains active proteases prompted the present study, which evaluated the role of CAFO dust proteases in lung inflammatory processes and tested whether protease-activated receptors (PARs) are involved in the signaling pathway for these events. We hypothesized that the damaging proinflammatory effect of HDE is due, in part, to the proteolytic activation of PARs, and inhibiting the proteases in HDE or disrupting PAR activation would attenuate HDE-mediated inflammatory indexes in bronchial epithelial cells (BECs), in mouse lung slices in vitro, and in a murine in vivo exposure model. Human BECs and mouse lung slice cultures stimulated with 5% HDE released significantly more of each of the cytokines measured (IL-6, IL-8, TNF-α, keratinocyte-derived chemokine/CXC chemokine ligand 1, and macrophage inflammatory protein-2/CXC chemokine ligand 2) than controls, and these effects were markedly diminished by protease inhibition. Inhibition of PARs also blunted the HDE-induced cytokine release from BECs. In addition, protease depletion inhibited HDE-induced BEC intracellular PKCα and PKCε activation. C57BL/6J mice administered 12.5% HDE intranasally, either once or daily for 3 wk, exhibited increased total cellular and neutrophil influx, bronchial alveolar fluid inflammatory cytokines, lung histopathology, and inflammatory scores compared with mice receiving protease-depleted HDE. These data suggest that proteases in dust from CAFOs are important mediators of lung inflammation, and these proteases and their receptors may provide novel targets for therapeutic intervention in CAFO dust-induced airways disease.

  10. Cysteine desulfurase Nfs1 and Pim1 protease control levels of Isu, the Fe-S cluster biogenesis scaffold.

    PubMed

    Song, Ji-Yoon; Marszalek, Jaroslaw; Craig, Elizabeth Anne

    2012-06-26

    Fe-S clusters are critical prosthetic groups for proteins involved in various critical biological processes. Before being transferred to recipient apo-proteins, Fe-S clusters are assembled on the highly conserved scaffold protein Isu, the abundance of which is regulated posttranslationally on disruption of the cluster biogenesis system. Here we report that Isu is degraded by the Lon-type AAA+ ATPase protease of the mitochondrial matrix, Pim1. Nfs1, the cysteine desulfurase responsible for providing sulfur for cluster formation, is required for the increased Isu stability occurring after disruption of cluster formation on or transfer from Isu. Physical interaction between the Isu and Nfs1 proteins, not the enzymatic activity of Nfs1, is the important factor in increased stability. Analysis of several conditions revealed that high Isu levels can be advantageous or disadvantageous, depending on the physiological condition. During the stationary phase, elevated Isu levels were advantageous, resulting in prolonged chronological lifespan. On the other hand, under iron-limiting conditions, high Isu levels were deleterious. Compared with cells expressing normal levels of Isu, such cells grew poorly and exhibited reduced activity of the heme-containing enzyme ferric reductase. Our results suggest that modulation of the degradation of Isu by the Pim1 protease is a regulatory mechanism serving to rapidly help balance the cell's need for critical iron-requiring processes under changing environmental conditions.

  11. Generation of an antibody that recognizes Plasmodium chabaudi cysteine protease (chabaupain-1) in both sexual and asexual parasite life cycle and evaluation of chabaupain-1 vaccine potential.

    PubMed

    Armada, Ana; Gazarini, Marcos L; Gonçalves, Lídia M; Antunes, Sandra; Custódio, Ana; Rodrigues, Armanda; Almeida, António J; Silveira, Henrique; Rosário, Virgílio do; Santos-Gomes, Gabriela; Domingos, Ana

    2013-09-01

    Malaria cysteine proteases have been shown to be immunogenic and are being exploited as serodiagnostic markers, drug and vaccine targets. Several Plasmodium spp. cysteine proteases have been described and the best characterized of these are the falcipains, a family of papain-family enzymes. Falcipain-2 and falcipain-3 act in concert with other proteases to hydrolyze host erythrocyte hemoglobin in the parasite food vacuole. Falcipain-1 has less similarity to the other falcipains and its physiological role in parasite asexual blood stage still remains uncertain. Immunolocalization studies using an antibody developed against the Plasmodium chabaudi recombinant chabaupain-1, the falcipain-1 ortholog, were performed confirming its cellular localization in both erythrocyte and mosquito ookinete stage. Immunostaining of chabaupain-1 preferentially in apical portion of parasite ookinete suggests that this protease may be related with parasite egression from mosquito midgut. Immune responses to chabaupain-1 were evaluated using two different adjuvants, chitosan nanoparticles and hydroxide aluminum. Mice immunized with the recombinant protein alone or in association with nanoparticles were challenged with P. chabaudi showing that immunization with the recombinant protein confers partial protection to blood stage infection in BALB/c animal model. Copyright © 2013 Elsevier Inc. All rights reserved.

  12. Cell entry by a novel European filovirus requires host endosomal cysteine proteases and Niemann-Pick C1

    PubMed Central

    Ng, Melinda; Ndungo, Esther; Jangra, Rohit K.; Cai, Yingyun; Postnikova, Elena; Radoshitzky, Sheli R.; Dye, John M.; de Arellano, Eva Ramírez; Negredo, Ana; Palacios, Gustavo; Kuhn, Jens H.; Chandran, Kartik

    2014-01-01

    Lloviu virus (LLOV), a phylogenetically divergent filovirus, is the proposed etiologic agent of die-offs of Schreiber’s long-fingered bats (Miniopterus schreibersii) in western Europe. Studies of LLOV remain limited because the infectious agent has not yet been isolated. Here, we generated a recombinant vesicular stomatitis virus expressing the LLOV spike glycoprotein (GP) and used it to show that LLOV GP resembles other filovirus GP proteins in structure and function. LLOV GP must be cleaved by endosomal cysteine proteases during entry, but is much more protease-sensitive than EBOV GP. The EBOV/MARV receptor, Niemann Pick C1 (NPC1), is also required for LLOV entry, and its second luminal domain is recognized with high affinity by a cleaved form of LLOV GP, suggesting that receptor binding would not impose a barrier to LLOV infection of humans and non-human primates. The use of NPC1 as an intracellular entry receptor may be a universal property of filoviruses. PMID:25310500

  13. Decreased IgA+ B cells population and IgA, IgG, IgM contents of the cecal tonsil induced by dietary high fluorine in broilers.

    PubMed

    Liu, Juan; Cui, Hengmin; Peng, Xi; Fang, Jing; Zuo, Zhicai; Deng, Junliang; Wang, Hesong; Wu, Bangyuan; Deng, Yuanxin; Wang, Kangping

    2013-05-02

    Fluoride is an environmental and industrial pollutant that affects various organs in humans and animals. The cecal tonsil is an important component of the mucosal immune system and performs important and unique immune functions. In the present study, we investigated the effects of dietary high fluorine on the quantities of IgA+ B cells in the cecal tonsil by immunohistochemistry, and the immunoglobulin A (IgA), immunoglobulin G (IgG) and immunoglobulin M (IgM) contents in the cecal tonsil by ELISA. A total of 280 one-day-old avian broilers were divided into four groups and fed on a corn-soybean basal diet as control diet (fluorine 22.6 mg/kg) or the same diet supplemented with 400, 800 and 1,200 mg/kg fluorine (high fluorine groups I, II and III) in the form of sodium fluoride, respectively, throughout a 42-day experimental period. The results showed that the quantities of IgA+ B cells were lower (p < 0.05 or p < 0.01) and the IgA, IgG, and IgM contents were decreased (p < 0.05 or p < 0.01) in high fluorine groups II and III in comparison with those of control group. It was concluded that dietary fluorine, in the 800-1,200 mg/kg range, could reduce the numbers of the IgA+ B cells and immunoglobulin contents in the cecal tonsil, implying the local mucosal immune function was ultimately impacted in broilers.

  14. Decreased IgA+ B Cells Population and IgA, IgG, IgM Contents of the Cecal Tonsil Induced by Dietary High Fluorine in Broilers

    PubMed Central

    Liu, Juan; Cui, Hengmin; Peng, Xi; Fang, Jing; Zuo, Zhicai; Deng, Junliang; Wang, Hesong; Wu, Bangyuan; Deng, Yuanxin; Wang, Kangping

    2013-01-01

    Fluoride is an environmental and industrial pollutant that affects various organs in humans and animals. The cecal tonsil is an important component of the mucosal immune system and performs important and unique immune functions. In the present study, we investigated the effects of dietary high fluorine on the quantities of IgA+ B cells in the cecal tonsil by immunohistochemistry, and the immunoglobulin A (IgA), immunoglobulin G (IgG) and immunoglobulin M (IgM) contents in the cecal tonsil by ELISA. A total of 280 one-day-old avian broilers were divided into four groups and fed on a corn-soybean basal diet as control diet (fluorine 22.6 mg/kg) or the same diet supplemented with 400, 800 and 1,200 mg/kg fluorine (high fluorine groups I, II and III) in the form of sodium fluoride, respectively, throughout a 42-day experimental period. The results showed that the quantities of IgA+ B cells were lower (p < 0.05 or p < 0.01) and the IgA, IgG, and IgM contents were decreased (p < 0.05 or p < 0.01) in high fluorine groups II and III in comparison with those of control group. It was concluded that dietary fluorine, in the 800–1,200 mg/kg range, could reduce the numbers of the IgA+ B cells and immunoglobulin contents in the cecal tonsil, implying the local mucosal immune function was ultimately impacted in broilers. PMID:23644827

  15. Relationship between frequency of pilocarpine administration and salivary IgA level.

    PubMed

    Smith, D J; Taubman, M A; Ebersole, J L; King, W

    1982-12-01

    The effect of repetitive administration of pilocarpine nitrate on the salivary volume and salivary IgA concentration was studied in the NIH white hamster. One and one-half to three-fold increases in salivary volume, coupled with decreases of 1/3 to 2/3 in IgA concentration, occurred as the frequency of administration of pilocarpine increased.

  16. Characterization of thermostable alkaline proteases from Bacillus infantis SKS1 isolated from garden soil.

    PubMed

    Saggu, Sandeep Kaur; Mishra, Prakash Chandra

    2017-01-01

    Proteases are one of the largest groups of hydrolytic enzymes constituting about 60% of total worldwide sales of industrial enzymes due to their wide applications in detergent, leather, textile, food and pharmaceutical industry. Microbial proteases have been preferred over animal and plant proteases because of their fundamental features and ease in production. Bacillus infantis SKS1, an alkaline protease producing bacteria has been isolated from garden soil of north India and identified using morphological, biochemical and molecular methods. 16S rDNA sequence amplified using universal primers has 99% sequence identity with corresponding gene sequence of Bacillus infantis strain FM 34 and Bacillus sp. Beige. The bacterial culture and its 16S rDNA gene sequence have been deposited to Microbial Culture Collection (Pune, India) with accession number MCC 3035 and GenBank with accession number KR092197 respectively. The partially purified extract of Bacillus infantis SKS1 was thermostable and active in presence of Mg2+, acetyl acetone and laundry detergents implicating its application in industry. Production of these enzymes using this strain was maximized by optimization of various parameters including temperature, pH, media components and other growth conditions. Our results show that fructose and dextrose serve as the best carbon sources for production of these enzymes, highlighting the use of this strain for enzyme production utilizing relatively inexpensive substrates like beet molasses and corn steep liquor. Additionally, this strain showed maximum production of enzymes at 40°C similar to bacterial species used for commercial production of alkaline proteases. Characterization of alkaline proteases from this strain of Bacillus infantis and optimization of parameters for its production would help in understanding its industrial application and large-scale production.

  17. Characterization of thermostable alkaline proteases from Bacillus infantis SKS1 isolated from garden soil

    PubMed Central

    Saggu, Sandeep Kaur

    2017-01-01

    Proteases are one of the largest groups of hydrolytic enzymes constituting about 60% of total worldwide sales of industrial enzymes due to their wide applications in detergent, leather, textile, food and pharmaceutical industry. Microbial proteases have been preferred over animal and plant proteases because of their fundamental features and ease in production. Bacillus infantis SKS1, an alkaline protease producing bacteria has been isolated from garden soil of north India and identified using morphological, biochemical and molecular methods. 16S rDNA sequence amplified using universal primers has 99% sequence identity with corresponding gene sequence of Bacillus infantis strain FM 34 and Bacillus sp. Beige. The bacterial culture and its 16S rDNA gene sequence have been deposited to Microbial Culture Collection (Pune, India) with accession number MCC 3035 and GenBank with accession number KR092197 respectively. The partially purified extract of Bacillus infantis SKS1 was thermostable and active in presence of Mg2+, acetyl acetone and laundry detergents implicating its application in industry. Production of these enzymes using this strain was maximized by optimization of various parameters including temperature, pH, media components and other growth conditions. Our results show that fructose and dextrose serve as the best carbon sources for production of these enzymes, highlighting the use of this strain for enzyme production utilizing relatively inexpensive substrates like beet molasses and corn steep liquor. Additionally, this strain showed maximum production of enzymes at 40°C similar to bacterial species used for commercial production of alkaline proteases. Characterization of alkaline proteases from this strain of Bacillus infantis and optimization of parameters for its production would help in understanding its industrial application and large-scale production. PMID:29190780

  18. The Cysteine Protease CEP1, a Key Executor Involved in Tapetal Programmed Cell Death, Regulates Pollen Development in Arabidopsis[W][OPEN

    PubMed Central

    Zhang, Dandan; Liu, Di; Lv, Xiaomeng; Wang, Ying; Xun, Zhili; Liu, Zhixiong; Li, Fenglan; Lu, Hai

    2014-01-01

    Tapetal programmed cell death (PCD) is a prerequisite for pollen grain development in angiosperms, and cysteine proteases are the most ubiquitous hydrolases involved in plant PCD. We identified a papain-like cysteine protease, CEP1, which is involved in tapetal PCD and pollen development in Arabidopsis thaliana. CEP1 is expressed specifically in the tapetum from stages 5 to 11 of anther development. The CEP1 protein first appears as a proenzyme in precursor protease vesicles and is then transported to the vacuole and transformed into the mature enzyme before rupture of the vacuole. cep1 mutants exhibited aborted tapetal PCD and decreased pollen fertility with abnormal pollen exine. A transcriptomic analysis revealed that 872 genes showed significantly altered expression in the cep1 mutants, and most of them are important for tapetal cell wall organization, tapetal secretory structure formation, and pollen development. CEP1 overexpression caused premature tapetal PCD and pollen infertility. ELISA and quantitative RT-PCR analyses confirmed that the CEP1 expression level showed a strong relationship to the degree of tapetal PCD and pollen fertility. Our results reveal that CEP1 is a crucial executor during tapetal PCD and that proper CEP1 expression is necessary for timely degeneration of tapetal cells and functional pollen formation. PMID:25035401

  19. Glomerular Diseases: Emerging Tests and Therapies for IgA Nephropathy

    PubMed Central

    Kiryluk, Krzysztof; Appel, Gerald B.

    2014-01-01

    Summary The last decade has seen major progress in understanding the pathogenesis as well as the prognosis and treatment of patients with IgA nephropathy (IgAN). Although the diagnostic criterion of a kidney biopsy demonstrating dominant or codominant IgA deposition remains unchanged, much more is known about the genetic and environmental factors predisposing to disease development and progression. These advances have led to the identification of novel diagnostic and prognostic markers. Among the most promising clinically are genetic profiling, quantification of galactose-deficient IgA1 levels, and measurement of anti-IgA1 immunoglobulins. While targeted treatment for IgAN remains elusive, there is mounting evidence for therapeutic interventions that alter the disease course. The appropriate validation and integration of these discoveries into clinical care represent a major challenge, but one that holds tremendous promise for refining prognostication, guiding therapy, and improving the lives of patients with IgAN. PMID:24071652

  20. Studies on a Novel Serine Protease of a ΔhapAΔprtV Vibrio cholerae O1 Strain and Its Role in Hemorrhagic Response in the Rabbit Ileal Loop Model

    PubMed Central

    Syngkon, Aurelia; Elluri, Sridhar; Koley, Hemanta; Rompikuntal, Pramod K.; Saha, Dhira Rani; Chakrabarti, Manoj K.; Bhadra, Rupak K.; Wai, Sun Nyunt; Pal, Amit

    2010-01-01

    Background Two well-characterized proteases secreted by Vibrio cholerae O1 strains are hemagglutinin protease (HAP) and V. cholerae protease (PrtV). The hapA and prtV knock out mutant, V. cholerae O1 strain CHA6.8ΔprtV, still retains residual protease activity. We initiated this study to characterize the protease present in CHA6.8ΔprtV strain and study its role in pathogenesis in rabbit ileal loop model (RIL). Methodology/Principal Findings We partially purified the residual protease secreted by strain CHA6.8ΔprtV from culture supernatant by anion-exchange chromatography. The major protein band in native PAGE was identified by MS peptide mapping and sequence analysis showed homology with a 59-kDa trypsin-like serine protease encoded by VC1649. The protease activity was partially inhibited by 25 mM PMSF and 10 mM EDTA and completely inhibited by EDTA and PMSF together. RIL assay with culture supernatants of strains C6709 (FA ratio 1.1+/−0.3 n = 3), CHA6.8 (FA ratio 1.08+/−0.2 n = 3), CHA6.8ΔprtV (FA ratio 1.02+/−0.2 n = 3) and partially purified serine protease from CHA6.8ΔprtV (FA ratio 1.2+/−0.3 n = 3) induced fluid accumulation and histopathological studies on rabbit ileum showed destruction of the villus structure with hemorrhage in all layers of the mucosa. RIL assay with culture supernatant of CHA6.8ΔprtVΔVC1649 strain (FA ratio 0.11+/−0.005 n = 3) and with protease incubated with PMSF and EDTA (FA ratio 0.3+/−0.05 n = 3) induced a significantly reduced FA ratio with almost complete normal villus structure. Conclusion Our results show the presence of a novel 59-kDa serine protease in a ΔhapAΔprtV V. cholerae O1 strain and its role in hemorrhagic response in RIL model. PMID:20927349

  1. Protease nexin-1 promotes secretory granule biogenesis by preventing granule protein degradation.

    PubMed

    Kim, Taeyoon; Loh, Y Peng

    2006-02-01

    Dense-core secretory granule (DCG) biogenesis is a prerequisite step for the sorting, processing, and secretion of neuropeptides and hormones in (neuro)endocrine cells. Previously, chromogranin A (CgA) has been shown to play a key role in the regulation of DCG biogenesis in vitro and in vivo. However, the underlying mechanism of CgA-mediated DCG biogenesis has not been explored. In this study, we have uncovered a novel mechanism for the regulation of CgA-mediated DCG biogenesis. Transfection of CgA into endocrine 6T3 cells lacking CgA and DCGs not only recovered DCG formation and regulated secretion but also prevented granule protein degradation. Genetic profiling of CgA-expressing 6T3 versus CgA- and DCG-deficient 6T3 cells, followed by real-time reverse transcription-polymerase chain reaction and Western blotting analyses, revealed that a serine protease inhibitor, protease nexin-1 (PN-1), was significantly up-regulated in CgA-expressing 6T3 cells. Overexpression of PN-1 in CgA-deficient 6T3 cells prevented degradation of DCG proteins at the Golgi apparatus, enhanced DCG biogenesis, and recovered regulated secretion. Moreover, depletion of PN-1 by antisense RNAs in CgA-expressing 6T3 cells resulted in the specific degradation of DCG proteins. We conclude that CgA increases DCG biogenesis in endocrine cells by up-regulating PN-1 expression to stabilize granule proteins against degradation.

  2. Protease Nexin-1 Promotes Secretory Granule Biogenesis by Preventing Granule Protein Degradation

    PubMed Central

    Kim, Taeyoon; Loh, Y. Peng

    2006-01-01

    Dense-core secretory granule (DCG) biogenesis is a prerequisite step for the sorting, processing, and secretion of neuropeptides and hormones in (neuro)endocrine cells. Previously, chromogranin A (CgA) has been shown to play a key role in the regulation of DCG biogenesis in vitro and in vivo. However, the underlying mechanism of CgA-mediated DCG biogenesis has not been explored. In this study, we have uncovered a novel mechanism for the regulation of CgA-mediated DCG biogenesis. Transfection of CgA into endocrine 6T3 cells lacking CgA and DCGs not only recovered DCG formation and regulated secretion but also prevented granule protein degradation. Genetic profiling of CgA-expressing 6T3 versus CgA- and DCG-deficient 6T3 cells, followed by real-time reverse transcription-polymerase chain reaction and Western blotting analyses, revealed that a serine protease inhibitor, protease nexin-1 (PN-1), was significantly up-regulated in CgA-expressing 6T3 cells. Overexpression of PN-1 in CgA-deficient 6T3 cells prevented degradation of DCG proteins at the Golgi apparatus, enhanced DCG biogenesis, and recovered regulated secretion. Moreover, depletion of PN-1 by antisense RNAs in CgA-expressing 6T3 cells resulted in the specific degradation of DCG proteins. We conclude that CgA increases DCG biogenesis in endocrine cells by up-regulating PN-1 expression to stabilize granule proteins against degradation. PMID:16319172

  3. Effects of yogurt fermented with Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1 on the IgA flow rate of saliva in elderly persons residing in a nursing home: A before-after non-randomised intervention study.

    PubMed

    Yamamoto, Yuko; Fujino, Kazuhiro; Saruta, Juri; Takahashi, Toru; To, Masahiro; Fuchida, Shinya; Shimizu, Tomoko; Kamata, Yohei; Misawa, Kyoko; Tsukinoki, Keiichi

    2017-12-01

    The aim of this study was to investigate the alterations in the salivary IgA levels of elderly persons administered yogurt fermented with Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) OLL1073R-1, which has been reported to reduce the risk of colds. Salivary immunoglobulin (Ig)A plays an important role in the defence of the oral cavity mucous membrane against foreign antigens and pathogens. Accordingly, low levels of salivary IgA are associated with an increased risk of upper respiratory tract infection. Furthermore, salivary IgA secretion has been reported to decrease with age. Recently, several studies have reported that certain strains of Lactobacillus and their products can modulate the immune response, but there are currently few studies on the effects of on the IgA level in human saliva. This was a before-after non-randomised intervention study. Thirty-seven elderly persons (mean age, 82.7 years) residing in a single nursing home ingested 112 g of the yogurt every morning for 12 weeks. The participants' saliva was collected before and after 4, 8 and 12 weeks of yogurt intake. Our results showed that yogurt intake affected the concentration of IgA in the saliva (P < .0001). Additionally, yogurt intake and the body weight of the participants affected the IgA flow rate of saliva (P = .0003 and .03, respectively). Continuous intake of yogurt fermented with L. bulgaricus OLL1073R-1 may help improve the mucosal immune function in elderly people with weakened immune systems. © 2017 John Wiley & Sons A/S and The Gerodontology Association. Published by John Wiley & Sons Ltd.

  4. Does low IgA in human milk predispose the infant to development of cow's milk allergy?

    PubMed

    Järvinen, K M; Laine, S T; Järvenpää, A L; Suomalainen, H K

    2000-10-01

    We sought a relationship between total and cow's milk-specific IgA levels in colostrum and human milk and subsequent development of cow's milk allergy (CMA) in the breast-fed infant. The study included 87 nursing mothers and their infants (age, 2 d to 7 mo), followed prospectively up to 1 y. At 1 y, 48 mothers (69% with an atopic constitution) had an infant with CMA, verified by clinical cow's milk challenge, eight (38% with an atopic constitution) had a baby who had had protracted infantile colic but no CMA (disease control group), and 31 (23% with an atopic constitution) had a healthy infant. Total breast-milk IgA was measured by radial immunodiffusion, and IgA antibodies to cow's milk were measured by ELISA during the breast-feeding period. The levels of total and cow's milk-specific IgA antibodies in colostrum and human milk were significantly lower in the mothers whose baby later developed CMA [estimated third day value, 0.38 g/L (95% confidence interval, 0. 24-0.82)] than in the ones whose infant remained healthy or had had infantile colic but not CMA [0.82 g/L (95% confidence interval, 0. 99-1.51); p < 0.05]. The infants developed CMA significantly more often if the concentration of total IgA antibodies in milk was <0.25 g/L, when measured between 6 d and 4 wk postpartum [sensitivity, 0. 55; specificity, 0.92; odds ratio, 14.7 (95% confidence interval, 3. 1-70.2); p < 0.001]. The levels of cow's milk-specific IgA positively correlated with the levels of total IgA but not with the development of CMA in the infant. The levels of total or cow's milk-specific IgA did not correlate with maternal atopy. IgA antibodies in colostrum and human milk may prevent antigen entry at the intestinal surface of the breast-fed infant. A low IgA content in human milk may lead to defective exclusion of food antigens and thus predispose an offspring to develop food allergies.

  5. Nasal IgA Provides Protection against Human Influenza Challenge in Volunteers with Low Serum Influenza Antibody Titre.

    PubMed

    Gould, Victoria M W; Francis, James N; Anderson, Katie J; Georges, Bertrand; Cope, Alethea V; Tregoning, John S

    2017-01-01

    In spite of there being a number of vaccines, influenza remains a significant global cause of morbidity and mortality. Understanding more about natural and vaccine induced immune protection against influenza infection would help to develop better vaccines. Virus specific IgG is a known correlate of protection, but other factors may help to reduce viral load or disease severity, for example IgA. In the current study we measured influenza specific responses in a controlled human infection model using influenza A/California/2009 (H1N1) as the challenge agent. Volunteers were pre-selected with low haemagglutination inhibition (HAI) titres in order to ensure a higher proportion of infection; this allowed us to explore the role of other immune correlates. In spite of HAI being uniformly low, there were variable levels of H1N1 specific IgG and IgA prior to infection. There was also a range of disease severity in volunteers allowing us to compare whether differences in systemic and local H1N1 specific IgG and IgA prior to infection affected disease outcome. H1N1 specific IgG level before challenge did not correlate with protection, probably due to the pre-screening for individuals with low HAI. However, the length of time infectious virus was recovered from the nose was reduced in patients with higher pre-existing H1N1 influenza specific nasal IgA or serum IgA. Therefore, IgA contributes to protection against influenza and should be targeted in vaccines.

  6. Activation of the classical complement pathway by mannose-binding protein in association with a novel C1s-like serine protease

    PubMed Central

    1992-01-01

    Serum mannose-binding protein (MBP) is a C-type lectin that binds to terminal mannose and N-acetylglucosamine moieties present on surfaces of certain pathogens and activates the classical complement pathway. In the present study, we describe the mechanism underlying the activation triggered by MBP. The human serum MBP fraction was obtained by sequential affinity chromatography on mannan-Sepharose, anti-IgM- Sepharose and anti-MBP-Sepharose in the presence of calcium ions. This fraction contained a C1s-like serine protease as assessed by C4 consumption. The C1s-like serine protease, designated MBP-associated serine protease (MASP), was separated from MBP by rechromatography on anti-MBP-Sepharose in the presence of ethylenediaminetetraacetic acid. MASP exhibited both C4- and C2-consuming activities. The molecular mass of MASP was estimated to be 83 kD with two polypeptides of heavy (66 kD) and light (L) (31 kD) chains linked by disulfide bonds. The serine residue responsible for protease activity is located on the L chain. Reconstitution experiments using MASP and MBP revealed that combination of the two components restores C4- and C2-activating capacity on mannan. Based on analyses of molecular size, antigenicity, and 11 NH2- terminal amino acid sequences of the L chain, we conclude that MASP is a novel protein different from C1r or C1s. Our findings are not in accord with a proposed mechanism by which MBP utilizes the C1r2-C1s2 complex to initiate the classical complement pathway. PMID:1460414

  7. Pressure-induced structural transition of mature HIV-1 protease from a combined NMR/MD simulation approach.

    PubMed

    Roche, Julien; Louis, John M; Bax, Ad; Best, Robert B

    2015-12-01

    We investigate the pressure-induced structural changes in the mature human immunodeficiency virus type 1 protease dimer, using residual dipolar coupling (RDC) measurements in a weakly oriented solution. (1)DNH RDCs were measured under high-pressure conditions for an inhibitor-free PR and an inhibitor-bound complex, as well as for an inhibitor-free multidrug resistant protease bearing 20 mutations (PR20). While PR20 and the inhibitor-bound PR were little affected by pressure, inhibitor-free PR showed significant differences in the RDCs measured at 600 bar compared with 1 bar. The structural basis of such changes was investigated by MD simulations using the experimental RDC restraints, revealing substantial conformational perturbations, specifically a partial opening of the flaps and the penetration of water molecules into the hydrophobic core of the subunits at high pressure. This study highlights the exquisite sensitivity of RDCs to pressure-induced conformational changes and illustrates how RDCs combined with MD simulations can be used to determine the structural properties of metastable intermediate states on the folding energy landscape. Published 2015. This article is a U.S. Government work and is in the public domain in the USA.

  8. Purification and functional characterization of mucosal IgA from vaccinated and SIV-infected rhesus macaques.

    PubMed

    Musich, Thomas; Demberg, Thorsten; Morgan, Ian L; Estes, Jacob D; Franchini, Genoveffa; Robert-Guroff, Marjorie

    2015-06-01

    Vaccine-induced mucosal antibodies are often evaluated using small volumes of secretory fluids. However, fecal matter containing mucosal IgA is abundant. We purified fecal IgA from five SIV-vaccinated and five SIV-infected rhesus macaques by sequential affinity chromatography. The purified IgA was dimeric by native PAGE, contained secretory component, and was analogous to IgA in colostrum and vaginal fluid by western blot. IgA from one infected and four vaccinated animals neutralized H9-derived SIV(mac)251 with IC(50)s as low as 1 μg/mL. Purified IgAs inhibited transcytosis and exhibited phagocytic activity, the latter significantly correlated with SIV(mac)251 Env-specific IgA in the purified samples. Among different affinity resins, peptide M was optimal compared to jacalin, anti-monkey IgA and SSL7 for IgA purification, as confirmed using tandem peptide M/anti-monkey IgA columns. Fecal IgA provided material sufficient for several assays relevant to protective efficacy, and was shown to be multifunctional. Our approach is potentially applicable to human clinical studies. Published by Elsevier Inc.

  9. Thymopentin treatment of selective IgA deficiency.

    PubMed

    Fiorilli, M; Quinti, I; Russi, G; Seminara, R; Ensoli, B; Aiuti, F

    1985-01-01

    Thymic hormones have been shown to modulate immunoglobulin production in a number of experiments and it is generally agreed that this action is mediated by modulation of helper and/or suppressor T cell activities. The possibility of upregulating the immunoglobulins is of particular relevance in patients with hypogammaglobulinemias and this paper reports on the results of thymopentin treatment in 9 patients with selective IgA deficiency. Two out of 4 patients responded positively in an open-label trial; in one the serum IgA values remained stable up to 8 weeks after discontinuation of treatment whereas there was a rapid fall in the other. Both responders had consistently normal T4/T8 ratios during the treatment, whereas the nonresponders revealed high ratios with large fluctuations of the T4/T8 ratio. In a subsequent (still ongoing) double-blind trial in 5 patients (3 thymopentin, 2 placebo) no significant change of serum or secretory IgA levels has been observed. Taken together, the data suggest that the tested dose regimen of thymopentin (i.e. daily i.m. injections of 1 mg/kg for 2 weeks, then same dose 3 time weekly for 10 weeks) may only work in a subset of patients with selective IgA deficiency. In the present study we did not attempt to distinguish to which of the three known subgroups the 9 patients belonged, nor did we try alternative dose regimens of thymopentin.

  10. Selective deficiency of IgA

    MedlinePlus

    ... Complications Autoimmune disorders such as rheumatoid arthritis , systemic lupus erythematosus , and celiac sprue may develop. People with IgA deficiency may develop antibodies to IgA. As a result, they can have severe, even life-threatening reactions ...

  11. Theoretical study on the mechanism of a ring-opening reaction of oxirane by the active-site aspartic dyad of HIV-1 protease.

    PubMed

    Kóna, Juraj

    2008-01-21

    Two possible mechanisms of the irreversible inhibition of HIV-1 protease by epoxide inhibitors are investigated on an enzymatic model using ab initio (MP2) and density functional theory (DFT) methods (B3LYP, MPW1K and M05-2X). The calculations predict the inhibition as a general acid-catalyzed nucleophilic substitution reaction proceeding by a concerted SN2 mechanism with a reaction barrier of ca. 15-21 kcal mol(-1). The irreversible nature of the inhibition is characterized by a large negative reaction energy of ca. -17-(-24) kcal mol(-1). A mechanism with a direct proton transfer from an aspartic acid residue of the active site onto the epoxide ring has been shown to be preferred compared to one with the proton transfer from the acid catalyst facilitated by a bridging catalytic water molecule. Based on the geometry of the transition state, structural data important for the design of irreversible epoxide inhibitors of HIV-1 protease were defined. Here we also briefly discuss differences between the epoxide ring-opening reaction in HIV-1 protease and epoxide hydrolase, and the accuracy of the DFT method used.

  12. Loss of Drosophila i-AAA protease, dYME1L, causes abnormal mitochondria and apoptotic degeneration.

    PubMed

    Qi, Y; Liu, H; Daniels, M P; Zhang, G; Xu, H

    2016-02-01

    Mitochondrial AAA (ATPases Associated with diverse cellular Activities) proteases i-AAA (intermembrane space-AAA) and m-AAA (matrix-AAA) are closely related and have major roles in inner membrane protein homeostasis. Mutations of m-AAA proteases are associated with neuromuscular disorders in humans. However, the role of i-AAA in metazoans is poorly understood. We generated a deletion affecting Drosophila i-AAA, dYME1L (dYME1L(del)). Mutant flies exhibited premature aging, progressive locomotor deficiency and neurodegeneration that resemble some key features of m-AAA diseases. dYME1L(del) flies displayed elevated mitochondrial unfolded protein stress and irregular cristae. Aged dYME1L(del) flies had reduced complex I (NADH/ubiquinone oxidoreductase) activity, increased level of reactive oxygen species (ROS), severely disorganized mitochondrial membranes and increased apoptosis. Furthermore, inhibiting apoptosis by targeting dOmi (Drosophila Htra2/Omi) or DIAP1, or reducing ROS accumulation suppressed retinal degeneration. Our results suggest that i-AAA is essential for removing unfolded proteins and maintaining mitochondrial membrane architecture. Loss of i-AAA leads to the accumulation of oxidative damage and progressive deterioration of membrane integrity, which might contribute to apoptosis upon the release of proapoptotic molecules such as dOmi. Containing ROS level could be a potential strategy to manage mitochondrial AAA protease deficiency.

  13. Variants in Complement Factor H and Complement Factor H-Related Protein Genes, CFHR3 and CFHR1, Affect Complement Activation in IgA Nephropathy.

    PubMed

    Zhu, Li; Zhai, Ya-Ling; Wang, Feng-Mei; Hou, Ping; Lv, Ji-Cheng; Xu, Da-Min; Shi, Su-Fang; Liu, Li-Jun; Yu, Feng; Zhao, Ming-Hui; Novak, Jan; Gharavi, Ali G; Zhang, Hong

    2015-05-01

    Complement activation is common in patients with IgA nephropathy (IgAN) and associated with disease severity. Our recent genome-wide association study of IgAN identified susceptibility loci on 1q32 containing the complement regulatory protein-encoding genes CFH and CFHR1-5, with rs6677604 in CFH as the top single-nucleotide polymorphism and CFHR3-1 deletion (CFHR3-1∆) as the top signal for copy number variation. In this study, to explore the clinical effects of variation in CFH, CFHR3, and CFHR1 on IgAN susceptibility and progression, we enrolled two populations. Group 1 included 1178 subjects with IgAN and available genome-wide association study data. Group 2 included 365 subjects with IgAN and available clinical follow-up data. In group 1, rs6677604 was associated with mesangial C3 deposition by genotype-phenotype correlation analysis. In group 2, we detected a linkage between the rs6677604-A allele and CFHR3-1∆ and found that the rs6677604-A allele was associated with higher serum levels of CFH and lower levels of the complement activation split product C3a. Furthermore, CFH levels were positively associated with circulating C3 levels and negatively associated with mesangial C3 deposition. Moreover, serum levels of the pathogenic galactose-deficient glycoform of IgA1 were also associated with the degree of mesangial C3 deposition in patients with IgAN. Our findings suggest that genetic variants in CFH, CFHR3, and CFHR1 affect complement activation and thereby, predispose patients to develop IgAN. Copyright © 2015 by the American Society of Nephrology.

  14. Activation of mas-related G-protein-coupled receptors by the house dust mite cysteine protease Der p1 provides a new mechanism linking allergy and inflammation.

    PubMed

    Reddy, Vemuri B; Lerner, Ethan A

    2017-10-20

    Cysteine and serine proteases function via protease-activated and mas-related G-protein-coupled receptors (Mrgprs) to contribute to allergy and inflammation. Der p1 is a cysteine protease and major allergen from the house dust mite and is associated with allergic rhinitis and allergic asthma. Der p1 activates protease-activated receptor 2 and induces the release of the pro-inflammatory cytokine IL-6 from cells. However, the possibility that Der p1 acts on Mrgprs has not been considered. We report here that ratiometric calcium imaging reveals that Der p1 activates the human receptor MRGPRX1 and the mouse homolog MrgprC11, implicated previously in itch. Der p1 cleavage of N-terminal receptor peptides followed by site-directed mutagenesis of the cleavage sites links receptor activation to specific amino acid residues. Der p1 also induced the release of IL-6 from heterologous cells expressing MRGPRX1. In summary, activation of Mrgprs by the allergen Der p1 may contribute to inflammation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Purification and molecular characterization of subtilisin-like alkaline protease BPP-A from Bacillus pumilus strain MS-1.

    PubMed

    Miyaji, T; Otta, Y; Nakagawa, T; Watanabe, T; Niimura, Y; Tomizuka, N

    2006-03-01

    The present study was conducted by screening zein-degrading bacteria in an attempt to obtain zein-degrading protease. Soil bacteria were screened by formation of a clear zone on zein plates. Characterization of a zein-degrading bacterium indicated a taxonomic affiliation to Bacillus pumilus, and was named MS-1 strain. The strain produced two different types of extracellular proteases, BPP-A and BPP-B. In this study, we purified and characterized BPP-A because it exhibited a higher ability to hydrolyze zein than BPP-B. When casein was used as the substrate, the optimal pH for BPP-A was 11.0. In BPP-A, zein was better substrate than casein at pH 13.0, whereas casein was better one than zein at pH 11.0. The bppA gene encoded a 383-amino acid pre-pro form of BPP-A, and mature BPP-A contained 275 amino acid residues. It was concluded that BPP-A belonged to the subtilisin family. A zein-degrading bacterium assigned to B. pumilus produced two different types of extracellular proteases, BPP-A and BPP-B. BPP-A exhibited an ability to hydrolyze zein in an extreme alkaline condition. This is a first report on screening for zein-degrading micro-organisms. The subtilisin-like protease BPP-A is possible to utilize as an industrial enzyme for the production of zein hydrolysates.

  16. Serpin1 and WSCP differentially regulate the activity of the cysteine protease RD21 during plant development in Arabidopsis thaliana

    PubMed Central

    Rustgi, Sachin; Boex-Fontvieille, Edouard; Reinbothe, Christiane; von Wettstein, Diter; Reinbothe, Steffen

    2017-01-01

    Proteolytic enzymes (proteases) participate in a vast range of physiological processes, ranging from nutrient digestion to blood coagulation, thrombosis, and beyond. In plants, proteases are implicated in host recognition and pathogen infection, induced defense (immunity), and the deterrence of insect pests. Because proteases irreversibly cleave peptide bonds of protein substrates, their activity must be tightly controlled in time and space. Here, we report an example of how nature evolved alternative mechanisms to fine-tune the activity of a cysteine protease dubbed RD21 (RESPONSIVE TO DESICCATION-21). One mechanism in the model plant Arabidopsis thaliana studied here comprises irreversible inhibition of RD21’s activity by Serpin1, whereas the other mechanism is a result of the reversible inhibition of RD21 activity by a Kunitz protease inhibitor named water-soluble chlorophyll-binding protein (WSCP). Activity profiling, complex isolation, and homology modeling data revealed unique interactions of RD21 with Serpin1 and WSCP, respectively. Expression studies identified only partial overlaps in Serpin1 and WSCP accumulation that explain how RD21 contributes to the innate immunity of mature plants and arthropod deterrence of seedlings undergoing skotomorphogenesis and greening. PMID:28179567

  17. Serpin1 and WSCP differentially regulate the activity of the cysteine protease RD21 during plant development in Arabidopsis thaliana.

    PubMed

    Rustgi, Sachin; Boex-Fontvieille, Edouard; Reinbothe, Christiane; von Wettstein, Diter; Reinbothe, Steffen

    2017-02-28

    Proteolytic enzymes (proteases) participate in a vast range of physiological processes, ranging from nutrient digestion to blood coagulation, thrombosis, and beyond. In plants, proteases are implicated in host recognition and pathogen infection, induced defense (immunity), and the deterrence of insect pests. Because proteases irreversibly cleave peptide bonds of protein substrates, their activity must be tightly controlled in time and space. Here, we report an example of how nature evolved alternative mechanisms to fine-tune the activity of a cysteine protease dubbed RD21 (RESPONSIVE TO DESICCATION-21). One mechanism in the model plant Arabidopsis thaliana studied here comprises irreversible inhibition of RD21's activity by Serpin1, whereas the other mechanism is a result of the reversible inhibition of RD21 activity by a Kunitz protease inhibitor named water-soluble chlorophyll-binding protein (WSCP). Activity profiling, complex isolation, and homology modeling data revealed unique interactions of RD21 with Serpin1 and WSCP, respectively. Expression studies identified only partial overlaps in Serpin1 and WSCP accumulation that explain how RD21 contributes to the innate immunity of mature plants and arthropod deterrence of seedlings undergoing skotomorphogenesis and greening.

  18. Molecular Basis for the Relative Substrate Specificity of Human Immunodeficiency Virus Type 1 and Feline Immunodeficiency Virus Proteases

    PubMed Central

    Beck, Zachary Q.; Lin, Ying-Chuan; Elder, John H.

    2001-01-01

    We have used a random hexamer phage library to delineate similarities and differences between the substrate specificities of human immunodeficiency virus type 1 (HIV-1) and feline immunodeficiency virus (FIV) proteases (PRs). Peptide sequences were identified that were specifically cleaved by each protease, as well as sequences cleaved equally well by both enzymes. Based on amino acid distinctions within the P3-P3′ region of substrates that appeared to correlate with these cleavage specificities, we prepared a series of synthetic peptides within the framework of a peptide sequence cleaved with essentially the same efficiency by both HIV-1 and FIV PRs, Ac-KSGVF↓VVNGLVK-NH2 (arrow denotes cleavage site). We used the resultant peptide set to assess the influence of specific amino acid substitutions on the cleavage characteristics of the two proteases. The findings show that when Asn is substituted for Val at the P2 position, HIV-1 PR cleaves the substrate at a much greater rate than does FIV PR. Likewise, Glu or Gln substituted for Val at the P2′ position also yields peptides specifically susceptible to HIV-1 PR. In contrast, when Ser is substituted for Val at P1′, FIV PR cleaves the substrate at a much higher rate than does HIV-1 PR. In addition, Asn or Gln at the P1 position, in combination with an appropriate P3 amino acid, Arg, also strongly favors cleavage by FIV PR over HIV PR. Structural analysis identified several protease residues likely to dictate the observed specificity differences. Interestingly, HIV PR Asp30 (Ile-35 in FIV PR), which influences specificity at the S2 and S2′ subsites, and HIV-1 PR Pro-81 and Val-82 (Ile-98 and Gln-99 in FIV PR), which influence specificity at the S1 and S1′ subsites, are residues which are often involved in development of drug resistance in HIV-1 protease. The peptide substrate KSGVF↓VVNGK, cleaved by both PRs, was used as a template for the design of a reduced amide inhibitor, Ac-GSGVFΨ(CH2NH)VVNGL-NH2

  19. Distribution of IgA subclass response to Coxiella burnetii in patients with acute and chronic Q fever.

    PubMed

    Camacho, M T; Outschoorn, I; Echevarría, C; Kovácová, E; Yebra, M; Maté, I; Auffray, P; Téllez, A

    1998-07-01

    The progression of Coxiella burnetii infection to acute or chronic Q fever has been attributed to biological characteristics of the bacterium and to the host immune response. We measured whether serum levels of total and specific subclasses IgA1 and IgA2 could be correlated with the course of disease in acute and chronic Q fever infections, and with the occurrence of endocarditis. In patients with chronic infection, total IgA2 levels were significantly increased. Q-fever-specific IgA1 antibodies were detectable in both acute and chronic infections, but only patients with endocarditis had IgA2 antibodies to C. burnetii phase II antigens. These findings indicate that the measurement of IgA subclasses may be a useful aid in the serological diagnosis of Q fever. Our results reinforce the idea that immunologically mediated host factors are important in the pathogenesis of Q fever and in the disease outcome of this infection. Copyright 1998 Academic Press.

  20. Screening of phytochemicals against protease activated receptor 1 (PAR1), a promising target for cancer.

    PubMed

    Kakarala, Kavita Kumari; Jamil, Kaiser

    2015-02-01

    Drug resistance and drug-associated toxicity are the primary causes for withdrawal of many drugs, although patient recovery is satisfactory in many instances. Interestingly, the use of phytochemicals in the treatment of cancer as an alternative to synthetic drugs comes with a host of advantages; minimum side effects, good human absorption and low toxicity to normal cells. Protease activated receptor 1 (PAR1) has been established as a promising target in many diseases including various cancers. Strong evidences suggest its role in metastasis also. There are no natural compounds known to inhibit its activity, so we aimed to identify phytochemicals with antagonist activity against PAR1. We screened phytochemicals from Naturally Occurring Plant-based Anticancer Compound-Activity-Target database (NPACT, http://crdd.osdd.net/raghava/npact/ ) against PAR1 using virtual screening workflow of Schrödinger software. It analyzes pharmaceutically relevant properties using Qikprop and calculates binding energy using Glide at three accuracy levels (high-throughput virtual screening, standard precision and extra precision). Our study led to the identification of phytochemicals, which showed interaction with at least one experimentally determined active site residue of PAR1, showed no violations to Lipinski's rule of five along with predicted high human absorption. Furthermore, structural interaction fingerprint analysis indicated that the residues H255, D256, E260, S344, V257, L258, L262, Y337 and S344 may play an important role in the hydrogen bond interactions of the phytochemicals screened. Of these residues, H255 and L258 residues were experimentally proved to be important for antagonist binding. The residues Y183, L237, L258, L262, F271, L332, L333, Y337, L340, A349, Y350, A352, and Y353 showed maximum hydrophobic interactions with the phytochemicals screened. The results of this work suggest that phytochemicals Reissantins D, 24,25-dihydro-27-desoxywithaferin A, Isoguaiacin

  1. Serological testing for coeliac disease in Type 1 diabetes mellitus: is immunoglobulin A level measurement necessary?

    PubMed

    Kurien, M; Leeds, J S; Hopper, A D; Wild, G; Egner, W; Tesfaye, S; Hadjivassiliou, M; Sanders, D S

    2013-07-01

    Immunoglobulin A (IgA) measurement is advocated when case finding for coeliac disease in patients with Type 1 diabetes mellitus. Currently, there is a paucity of contemporary studies assessing IgA deficiency in Type 1 diabetes. This study evaluates the prevalence of IgA deficiency in individuals with Type 1 diabetes, compared with patients with coeliac disease and control subjects. In addition, we evaluate whether routine IgA measurement is justifiable when case finding for coeliac disease in patients with Type 1 diabetes. All patients were assessed using IgA endomysial antibodies, IgA anti-tissue transglutaminase antibodies and total IgA levels. Altogether, 2434 individuals were tested: 1000 patients with Type 1 diabetes, 234 patients with coeliac disease and 1200 population control subjects. Definitive IgA deficiency was defined as total IgA levels < 0.07 g/l. The prevalence of IgA deficiency was significantly more common in patients with Type 1 diabetes (0.9%, n = 9/1000; P = 0.036) and coeliac disease (1.29%, n = 3/234; P = 0.041) when compared with population control subjects (prevalence of 0.17%, 2/1200). No statistical difference between Type 1 diabetes and coeliac disease for IgA deficiency was identified (P = 0.87). Of patients in the group with Type 1 diabetes, 3.3% (33/1000) had coeliac disease, and of those only one patient had IgA deficiency leading to an antibody-negative presentation. Both IgA-deficient individuals within the population control subjects had normal duodenal biopsies and no relevant symptoms. IgA deficiency is more common in Type 1 diabetes compared with population control subjects. Despite this, very few individuals with Type 1 diabetes and IgA deficiency appear to have villous atrophy on biopsy. These outcomes question the practice of routine IgA measurement when case finding for coeliac disease in patients with Type 1 diabetes. © 2013 The Authors. Diabetic Medicine © 2013 Diabetes UK.

  2. Cutting Edge: Active TGF-β1 Released from GARP/TGF-β1 Complexes on the Surface of Stimulated Human B Lymphocytes Increases Class-Switch Recombination and Production of IgA.

    PubMed

    Dedobbeleer, Olivier; Stockis, Julie; van der Woning, Bas; Coulie, Pierre G; Lucas, Sophie

    2017-07-15

    Production of active TGF-β is regulated at a posttranslational level and implies release of the mature cytokine dimer from the inactive, latent TGF-β precursor. There are several cell-type specific mechanisms of TGF-β activation. We identified a new mechanism operating on the surface of human regulatory T cells and involving membrane protein GARP, which binds latent TGF-β1. The paracrine activity of regulatory T cell-derived TGF-β1 contributes to immunosuppression and can be inhibited with anti-GARP Abs. Whether other immune cell types use surface GARP to activate latent TGF-β1 was not known. We show in this study that stimulated, human B lymphocytes produce active TGF-β1 from surface GARP/latent TGF-β1 complexes with isotype switching to IgA production. Copyright © 2017 by The American Association of Immunologists, Inc.

  3. Design, Synthesis, and Biological and Structural Evaluations of Novel HIV-1 Protease Inhibitors To Combat Drug Resistance

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Parai, Maloy Kumar; Huggins, David J.; Cao, Hong

    2012-09-11

    A series of new HIV-1 protease inhibitors (PIs) were designed using a general strategy that combines computational structure-based design with substrate-envelope constraints. The PIs incorporate various alcohol-derived P2 carbamates with acyclic and cyclic heteroatomic functionalities into the (R)-hydroxyethylamine isostere. Most of the new PIs show potent binding affinities against wild-type HIV-1 protease and three multidrug resistant (MDR) variants. In particular, inhibitors containing the 2,2-dichloroacetamide, pyrrolidinone, imidazolidinone, and oxazolidinone moieties at P2 are the most potent with Ki values in the picomolar range. Several new PIs exhibit nanomolar antiviral potencies against patient-derived wild-type viruses from HIV-1 clades A, B, and Cmore » and two MDR variants. Crystal structure analyses of four potent inhibitors revealed that carbonyl groups of the new P2 moieties promote extensive hydrogen bond interactions with the invariant Asp29 residue of the protease. These structure-activity relationship findings can be utilized to design new PIs with enhanced enzyme inhibitory and antiviral potencies.« less

  4. Polymorphisms and resistance mutations in the protease and reverse transcriptase genes of HIV-1 F subtype Romanian strains.

    PubMed

    Paraschiv, Simona; Otelea, Dan; Dinu, Magdalena; Maxim, Daniela; Tinischi, Mihaela

    2007-03-01

    To evaluate the prevalence of resistance mutations in the genome of HIV-1 F subtype strains isolated from Romanian antiretroviral (ARV) treatment-naïve patients and to assess the phylogenetic relatedness of these strains with other HIV-1 strains. Twenty-nine HIV-1 strains isolated from treatment-naïve adolescents (n=15) and adults (n=14) were included in this study. Resistance genotyping was performed by using Big Dye Terminator chemistry provided by the ViroSeq Genotyping System. The sequences of the protease and reverse transcriptase genes were aligned (ClustalW) and a phylogenetic tree was built (MEGA 3 software). For subtyping purposes, all the nucleotide sequences were submitted to the Stanford database. All the studied strains were found to harbor accessory mutations in the protease gene. The most frequent mutation was M36I (29 of 29 strains), followed by L63T, K20R, and L10V. The number of polymorphisms associated with protease inhibitor resistance was different for the two age groups. Intraphylogenetic divergence was greater for adults than for adolescents infected in childhood. All the strains were found to belong to the F1 subtype. The phylogenetic analysis revealed that Romanian strains clustered together, but distinctly from F1 HIV-1 strains isolated in other parts of the world (Brazil, Finland, and Belgium). Protease secondary mutations are present with high frequency in the HIV-1 F subtype strains isolated from Romanian ARV treatment-naïve patients, but no major resistance mutations were found.

  5. The dimer interfaces of protease and extra-protease domains influence the activation of protease and the specificity of GagPol cleavage.

    PubMed

    Pettit, Steven C; Gulnik, Sergei; Everitt, Lori; Kaplan, Andrew H

    2003-01-01

    Activation of the human immunodeficiency virus type 1 (HIV-1) protease is an essential step in viral replication. As is the case for all retroviral proteases, enzyme activation requires the formation of protease homodimers. However, little is known about the mechanisms by which retroviral proteases become active within their precursors. Using an in vitro expression system, we have examined the determinants of activation efficiency and the order of cleavage site processing for the protease of HIV-1 within the full-length GagPol precursor. Following activation, initial cleavage occurs between the viral p2 and nucleocapsid proteins. This is followed by cleavage of a novel site located in the transframe domain. Mutational analysis of the dimer interface of the protease produced differential effects on activation and specificity. A subset of mutations produced enhanced cleavage at the amino terminus of the protease, suggesting that, in the wild-type precursor, cleavages that liberate the protease are a relatively late event. Replacement of the proline residue at position 1 of the protease dimer interface resulted in altered cleavage of distal sites and suggests that this residue functions as a cis-directed specificity determinant. In summary, our studies indicate that interactions within the protease dimer interface help determine the order of precursor cleavage and contribute to the formation of extended-protease intermediates. Assembly domains within GagPol outside the protease domain also influence enzyme activation.

  6. Highly Potent HIV-1 Protease Inhibitors with Novel Tricyclic P2-ligands: Design, Synthesis, and Protein-ligand X-Ray Studies

    PubMed Central

    Ghosh, Arun K.; Parham, Garth L.; Martyr, Cuthbert D.; Nyalapatla, Prasanth R.; Osswald, Heather L.; Agniswamy, Johnson; Wang, Yuan-Fang; Amano, Masayuki; Weber, Irene T.; Mitsuya, Hiroaki

    2013-01-01

    The design, synthesis, and biological evaluation of a series of HIV-1 protease inhibitors incorporating stereochemically defined fused tricyclic P2-ligands are described. Various substituent effects were investigated in order to maximize the ligand-binding site interactions in the protease active site. Inhibitors 16a and 16f showed excellent enzyme inhibitory and antiviral activity while incorporation of sulfone functionality resulted in a decrease in potency. Both inhibitors 16a and 16f have maintained activity against a panel of multidrug resistant HIV-1 variants. A high-resolution X-ray crystal structure of 16a-bound HIV-1 protease revealed important molecular insights into the ligand-binding site interactions which may account for the inhibitor’s potent antiviral activity and excellent resistance profiles. PMID:23947685

  7. Enhanced enteroviral infectivity via viral protease-mediated cleavage of Grb2-associated binder 1

    PubMed Central

    Deng, Haoyu; Fung, Gabriel; Shi, Junyan; Xu, Suowen; Wang, Chen; Yin, Meimei; Hou, Jun; Zhang, Jingchun; Jin, Zheng-Gen; Luo, Honglin

    2015-01-01

    Coxsackievirus B3 (CVB3), an important human causative pathogen for viral myocarditis, pancreatitis, and meningitis, has evolved different strategies to manipulate the host signaling machinery to ensure successful viral infection. We previously revealed a crucial role for the ERK1/2 signaling pathway in regulating viral infectivity. However, the detail mechanism remains largely unknown. Grb2-associated binder 1 (GAB1) is an important docking protein responsible for intracellular signaling assembly and transduction. In this study, we demonstrated that GAB1 was proteolytically cleaved after CVB3 infection at G175 and G436 by virus-encoded protease 2Apro, independent of caspase activation. Knockdown of GAB1 resulted in a significant reduction of viral protein expression and virus titers. Moreover, we showed that virus-induced cleavage of GAB1 is beneficial to viral growth as the N-terminal proteolytic product of GAB1 (GAB1-N1–174) further enhances ERK1/2 activation and promotes viral replication. Our results collectively suggest that CVB3 targets host GAB1 to generate a GAB1-N1–174 fragment that enhances viral infectivity, at least in part, via activation of the ERK pathway. The findings in this study suggest a novel mechanism that CVB3 employs to subvert the host signaling and facilitate consequent viral replication.—Deng, H., Fung, G., Shi, J., Xu, S., Wang, C., Yin, M., Hou, J., Zhang, J., Jin, Z.-G., Luo, H. Enhanced enteroviral infectivity via viral protease-mediated cleavage of Grb2-associated binder 1. PMID:26183772

  8. [In vitro function of outer membrane protease T of Escherichia coli K1 pathogenic strain].

    PubMed

    Hui, Changye; Guo, Yan; Wu, Shuchi; Peng, Liang; Cao, Hong; Huang, Shenghe

    2010-01-01

    Plasminogen activation and antimicrobial peptide hydrolysis contribute to pathogens invasion and survival in vivo. To demonstrate the expression of outer membrane protease T in E. coli K1 pathogenic strain E44, its activity of plasminogen activator and protamine hydrolysis. After Benzamidine Sepharose Fast Flow and SOURCE 30Q chromatography, we got E44 outer membrane mixed fraction, and examined its activity of plasminogen activation with chromogenic substrate S-2251 method. An ompT deletion mutant of E44 was constructed by using the suicide vector pCVD442, termed as E44ompT. We examined 0.1 mg/mL cationic antimicrobial peptide protamine susceptibility of E44, ompT mutant strain E44ompT and E44ompT harboring pUCT, which was constructed by inserting complete ompT open reading frame into pUC13. We got about 37 kDa E44 membrane extract, which could activate plasminogen, and activation was membrane extract dose dependent. This confirmed the expression of outer membrane protease T in the outer membrane of E44. E44ompT was, more susceptible to 0.1 mg/mL protamine than E44, and E440mpT was partially complemented by pUCT. Outer membrane protease T is expressed in E. coli K1 pathogenic strain E44, and can activate plasminogen and hydrolyze protamine.

  9. Cytokine profile of NALT during acute stress and its possible effect on IgA secretion.

    PubMed

    Gutiérrez-Meza, Juan Manuel; Jarillo-Luna, Rosa Adriana; Rivera-Aguilar, Victor; Miliar-García, Angel; Campos-Rodríguez, Rafael

    2017-08-01

    Stress stimuli affect the immune system responses that occur at mucosal membranes, particularly IgA secretion. It has been suggested that acute stress increases the levels of IgA and that sympathetic innervation plays an important role in this process. We herein explore in a murine model how acute stress affects the Th1/Th2/Treg cytokine balance in NALT, and the possible role of glucocorticoids in this effect. Nine-week-old male CD1 mice were divided into three groups: unstressed (control), stressed (subjected to 4h of immobilization), and stressed after pretreatment with a single dose of the corticosterone receptor antagonist RU-486. The parameters evaluated included plasma corticosterone and epinephrine, IgA levels in nasal fluid (by ELISA), the percentage of CD19 + B220 + IgA + lymphocytes and CD138 + IgA + plasma cells, and the mRNA expression of heavy α chain, J chain and pIgR. Moreover, the gene and protein expression of Th1 cytokines (TNFα, IL-2 and INF-γ), Th2 cytokines (IL-4 and IL-5) and Treg cytokines (IL-10 and TGFβ) were determined in nasal mucosa. The results show that acute stress generated a shift towards the dominance of an anti-inflammatory immune response (Th2 and Treg cytokines), evidenced by a significant rise in the amount of T cells that produce IL4, IL-5 and IL-10. This immune environment may favor IgA biosynthesis by CD138 + IgA + plasma cells, a process mediated mostly by glucocorticoids. Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

  10. Free energy component analysis for drug design: a case study of HIV-1 protease-inhibitor binding.

    PubMed

    Kalra, P; Reddy, T V; Jayaram, B

    2001-12-06

    A theoretically rigorous and computationally tractable methodology for the prediction of the free energies of binding of protein-ligand complexes is presented. The method formulated involves developing molecular dynamics trajectories of the enzyme, the inhibitor, and the complex, followed by a free energy component analysis that conveys information on the physicochemical forces driving the protein-ligand complex formation and enables an elucidation of drug design principles for a given receptor from a thermodynamic perspective. The complexes of HIV-1 protease with two peptidomimetic inhibitors were taken as illustrative cases. Four-nanosecond-level all-atom molecular dynamics simulations using explicit solvent without any restraints were carried out on the protease-inhibitor complexes and the free proteases, and the trajectories were analyzed via a thermodynamic cycle to calculate the binding free energies. The computed free energies were seen to be in good accord with the reported data. It was noted that the net van der Waals and hydrophobic contributions were favorable to binding while the net electrostatics, entropies, and adaptation expense were unfavorable in these protease-inhibitor complexes. The hydrogen bond between the CH2OH group of the inhibitor at the scissile position and the catalytic aspartate was found to be favorable to binding. Various implicit solvent models were also considered and their shortcomings discussed. In addition, some plausible modifications to the inhibitor residues were attempted, which led to better binding affinities. The generality of the method and the transferability of the protocol with essentially no changes to any other protein-ligand system are emphasized.

  11. The dog as a genetic model for immunoglobulin A (IgA) deficiency: identification of several breeds with low serum IgA concentrations.

    PubMed

    Olsson, Mia; Frankowiack, Marcel; Tengvall, Katarina; Roosje, Petra; Fall, Tove; Ivansson, Emma; Bergvall, Kerstin; Hansson-Hamlin, Helene; Sundberg, Katarina; Hedhammar, Ake; Lindblad-Toh, Kerstin; Hammarström, Lennart

    2014-08-15

    Immunoglobulin A (IgA) serves as the basis of the secretory immune system by protecting the lining of mucosal sites from pathogens. In both humans and dogs, IgA deficiency (IgAD) is associated with recurrent infections of mucosal sites and immune-mediated diseases. Low concentrations of serum IgA have previously been reported to occur in a number of dog breeds but no generally accepted cut-off value has been established for canine IgAD. The current study represents the largest screening to date of IgA in dogs in terms of both number of dogs (n=1267) and number of breeds studied (n=22). Serum IgA concentrations were quantified by using capture ELISA and were found to vary widely between breeds. We also found IgA to be positively correlated with age (p<0.0001). Apart from the two breeds previously reported as predisposed to low IgA (Shar-Pei and German shepherd), we identified six additional breeds in which ≥ 10% of all tested dogs had very low (<0.07 g/l) IgA concentrations (Hovawart, Norwegian elkhound, Nova Scotia duck tolling retriever, Bullterrier, Golden retriever and Labrador retriever). In addition, we discovered low IgA concentrations to be significantly associated with canine atopic dermatitis (CAD, p<0.0001) and pancreatic acinar atrophy (PAA, p=0.04) in German shepherds. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. Integrase inhibitor versus protease inhibitor based regimen for HIV-1 infected women (WAVES): a randomised, controlled, double-blind, phase 3 study

    PubMed Central

    Squires, Kathleen; Kityo, Cissy; Hodder, Sally; Johnson, Margaret; Voronin, Evgeny; Hagins, Debbie; Avihingsanon, Anchalee; Koenig, Ellen; Jiang, Shuping; White, Kirsten; Cheng, Andrew; Szwarcberg, Javier; Cao, Huyen

    2018-01-01

    Summary Background Women are under-represented in HIV antiretroviral therapy (ART) studies. Guidelines for selection of ART as initial therapy in patients with HIV-1 infection do not contain sex-specific treatment. We aimed to assess the safety and efficacy of the single tablet integrase inhibitor regimen containing elvitegravir, cobicistat, emtricitabine, and tenofovir disoproxil fumarate compared with a boosted protease inhibitor regimen of ritonavir-boosted atazanavir with emtricitabine and tenofovir disoproxil fumarate. Methods In this international, randomised, controlled, double-blind, phase 3 study (Women AntiretroViral Efficacy and Safety study [WAVES]), we recruited treatment-naive HIV-infected women with an estimated creatinine clearance of 70 mL/min or higher from 80 centres in 11 countries. Women were randomly assigned (1:1) to receive elvitegravir, cobicistat, emtricitabine, and tenofovir disoproxil fumarate (integrase inhibitor regimen) or ritonavir-boosted atazanavir with emtricitabine and tenofovir disoproxil fumarate (protease inhibitor based regimen); regimens were masked with matching placebos. Randomisation was done by a computer-generated allocation sequence (block size four) and was stratified by HIV-1 RNA viral load and race. Investigators, patients, study staff, and those assessing outcomes were masked to treatment group. All participants who received one dose of study drug were included in the primary efficacy and safety analyses. The main outcome was the proportion of patients with plasma HIV-1 RNA less than 50 copies per mL at week 48 as defined by US Food and Drug Administration snapshot algorithm (prespecified non-inferiority margin of 12%). This study is registered with ClinicalTrials.gov, number NCT01705574. Findings Between Nov 28, 2012, and March 12, 2014, 575 women were enrolled. 289 were randomly assigned to receive the integrase inhibitor regimen and 286 to receive the protease inhibitor based regimen. 252 (87%) women in the

  13. A cathepsin L-like protease from Strongylus vulgaris: an orthologue of Caenorhabditis elegans CPL-1.

    PubMed

    Ultaigh, Sinéad Nic An; Carolan, James C; Britton, Collette; Murray, Linda; Ryan, Michael F

    2009-04-01

    Cathespin L-like proteases (CPLs), characterized from a wide range of helminths, are significant in helminth biology. For example, in Caenorhabditis elegans CPL is essential for embryogenesis. Here, we report a cathepsin L-like gene from three species of strongyles that parasitize the horse, and describe the isolation of a cpl gene (Sv-cpl-1) from Strongylus vulgaris, the first such from equine strongyles. It encodes a protein of 354 amino acids with high similarity to other parasitic Strongylida (90-91%), and C.elegans CPL-1 (87%), a member of the same Clade. As S.vulgaris cpl-1 rescued the embryonic lethal phenotype of the C.elegans cpl-1 mutant, these genes may be orthologues, sharing the same function in each species. Targeting Sv-CPL-1 might enable novel control strategies by decreasing parasite development and transmission.

  14. Novel Mycosin Protease MycP1 Inhibitors Identified by Virtual Screening and 4D Fingerprints

    PubMed Central

    2015-01-01

    The rise of drug-resistant Mycobacterium tuberculosis lends urgency to the need for new drugs for the treatment of tuberculosis (TB). The identification of a serine protease, mycosin protease-1 (MycP1), as the crucial agent in hydrolyzing the virulence factor, ESX-secretion-associated protein B (EspB), potentially opens the door to new tuberculosis treatment options. Using the crystal structure of mycobacterial MycP1 in the apo form, we performed an iterative ligand- and structure-based virtual screening (VS) strategy to identify novel, nonpeptide, small-molecule inhibitors against MycP1 protease. Screening of ∼485 000 ligands from databases at the Genomics Research Institute (GRI) at the University of Cincinnati and the National Cancer Institute (NCI) using our VS approach, which integrated a pharmacophore model and consensus molecular shape patterns of active ligands (4D fingerprints), identified 81 putative inhibitors, and in vitro testing subsequently confirmed two of them as active inhibitors. Thereafter, the lead structures of each VS round were used to generate a new 4D fingerprint that enabled virtual rescreening of the chemical libraries. Finally, the iterative process identified a number of diverse scaffolds as lead compounds that were tested and found to have micromolar IC50 values against the MycP1 target. This study validated the efficiency of the SABRE 4D fingerprints as a means of identifying novel lead compounds in each screening round of the databases. Together, these results underscored the value of using a combination of in silico iterative ligand- and structure-based virtual screening of chemical libraries with experimental validation for the identification of promising structural scaffolds, such as the MycP1 inhibitors. PMID:24628123

  15. Computational design and experimental study of tighter binding peptides to an inactivated mutant of HIV-1 protease

    PubMed Central

    Altman, Michael D.; Nalivaika, Ellen A.; Prabu-Jeyabalan, Moses; Schiffer, Celia A.; Tidor, Bruce

    2009-01-01

    Drug resistance in HIV-1 protease, a barrier to effective treatment, is generally caused by mutations in the enzyme that disrupt inhibitor binding but still allow for substrate processing. Structural studies with mutant, inactive enzyme, have provided detailed information regarding how the substrates bind to the protease yet avoid resistance mutations; insights obtained inform the development of next generation therapeutics. Although structures have been obtained of complexes between substrate peptide and inactivated (D25N) protease, thermodynamic studies of peptide binding have been challenging due to low affinity. Peptides that bind tighter to the inactivated protease than the natural substrates would be valuable for thermodynamic studies as well as to explore whether the structural envelope observed for substrate peptides is a function of weak binding. Here, two computational methods — namely, charge optimization and protein design — were applied to identify peptide sequences predicted to have higher binding affinity to the inactivated protease, starting from an RT–RH derived substrate peptide. Of the candidate designed peptides, three were tested for binding with isothermal titration calorimetry, with one, containing a single threonine to valine substitution, measured to have more than a ten-fold improvement over the tightest binding natural substrate. Crystal structures were also obtained for the same three designed peptide complexes; they show good agreement with computational prediction. Thermodynamic studies show that binding is entropically driven, more so for designed affinity enhanced variants than for the starting substrate. Structural studies show strong similarities between natural and tighter-binding designed peptide complexes, which may have implications in understanding the molecular mechanisms of drug resistance in HIV-1 protease. PMID:17729291

  16. The Dimer Interfaces of Protease and Extra-Protease Domains Influence the Activation of Protease and the Specificity of GagPol Cleavage

    PubMed Central

    Pettit, Steven C.; Gulnik, Sergei; Everitt, Lori; Kaplan, Andrew H.

    2003-01-01

    Activation of the human immunodeficiency virus type 1 (HIV-1) protease is an essential step in viral replication. As is the case for all retroviral proteases, enzyme activation requires the formation of protease homodimers. However, little is known about the mechanisms by which retroviral proteases become active within their precursors. Using an in vitro expression system, we have examined the determinants of activation efficiency and the order of cleavage site processing for the protease of HIV-1 within the full-length GagPol precursor. Following activation, initial cleavage occurs between the viral p2 and nucleocapsid proteins. This is followed by cleavage of a novel site located in the transframe domain. Mutational analysis of the dimer interface of the protease produced differential effects on activation and specificity. A subset of mutations produced enhanced cleavage at the amino terminus of the protease, suggesting that, in the wild-type precursor, cleavages that liberate the protease are a relatively late event. Replacement of the proline residue at position 1 of the protease dimer interface resulted in altered cleavage of distal sites and suggests that this residue functions as a cis-directed specificity determinant. In summary, our studies indicate that interactions within the protease dimer interface help determine the order of precursor cleavage and contribute to the formation of extended-protease intermediates. Assembly domains within GagPol outside the protease domain also influence enzyme activation. PMID:12477841

  17. A unique serpin P1' glutamate and a conserved β-sheet C arginine are key residues for activity, protease recognition and stability of serpinA12 (vaspin).

    PubMed

    Ulbricht, David; Pippel, Jan; Schultz, Stephan; Meier, René; Sträter, Norbert; Heiker, John T

    2015-09-15

    SerpinA12 (vaspin) is thought to be mainly expressed in adipose tissue and has multiple beneficial effects on metabolic, inflammatory and atherogenic processes related to obesity. KLK7 (kallikrein 7) is the only known protease target of vaspin to date and is inhibited with a moderate inhibition rate. In the crystal structure, the cleavage site (P1-P1') of the vaspin reactive centre loop is fairly rigid compared with the flexible residues before P2, possibly supported by an ionic interaction of P1' glutamate (Glu(379)) with an arginine residue (Arg(302)) of the β-sheet C. A P1' glutamate seems highly unusual and unfavourable for the protease KLK7. We characterized vaspin mutants to investigate the roles of these two residues in protease inhibition and recognition by vaspin. Reactive centre loop mutations changing the P1' residue or altering the reactive centre loop conformation significantly increased inhibition parameters, whereas removal of the positive charge within β-sheet C impeded the serpin-protease interaction. Arg(302) is a crucial contact to enable vaspin recognition by KLK7 and it supports moderate inhibition of the serpin despite the presence of the detrimental P1' Glu(379), which clearly represents a major limiting factor for vaspin-inhibitory activity. We also show that the vaspin-inhibition rate for KLK7 can be modestly increased by heparin and demonstrate that vaspin is a heparin-binding serpin. Noteworthily, we observed vaspin as a remarkably thermostable serpin and found that Glu(379) and Arg(302) influence heat-induced polymerization. These structural and functional results reveal the mechanistic basis of how reactive centre loop sequence and exosite interaction in vaspin enable KLK7 recognition and regulate protease inhibition as well as stability of this adipose tissue-derived serpin. © 2015 Authors; published by Portland Press Limited.

  18. Stability of thermostable alkaline protease from Bacillus licheniformis RP1 in commercial solid laundry detergent formulations.

    PubMed

    Sellami-Kamoun, Alya; Haddar, Anissa; Ali, Nedra El-Hadj; Ghorbel-Frikha, Basma; Kanoun, Safia; Nasri, Moncef

    2008-01-01

    The stability of crude extracellular protease produced by Bacillus licheniformis RP1, isolated from polluted water, in various solid laundry detergents was investigated. The enzyme had an optimum pH and temperature at pH 10.0-11.0 and 65-70 degrees C. Enzyme activity was inhibited by PMSF, suggesting that the preparation contains a serine-protease. The alkaline protease showed extreme stability towards non-ionic (5% Tween 20% and 5% Triton X-100) and anionic (0.5% SDS) surfactants, which retained 100% and above 73%, respectively, of its initial activity after preincubation 60 min at 40 degrees C. The RP1 protease showed excellent stability and compatibility with a wide range of commercial solid detergents at temperatures from 40 to 50 degrees C, suggesting its further application in detergent industry. The enzyme retained 95% of its initial activity with Ariel followed by Axion (94%) then Dixan (93.5%) after preincubation 60 min at 40 degrees C in the presence of 7 mg/ml of detergents. In the presence of Nadhif and New Det, the enzyme retained about 83.5% of the original activity. The effects of additives such as maltodextrin, sucrose and PEG 4000 on the stability of the enzyme during spray-drying and during subsequent storage in New Det detergent were also examined. All additives tested enhanced stability of the enzyme.

  19. Disruption of Smad4 Expression in T Cells Leads to IgA Nephropathy-Like Manifestations

    PubMed Central

    Yamashita, Michifumi; Choi, Sung Hee; Tomino, Yasuhiko; Letterio, John J.; Emancipator, Steven N.

    2013-01-01

    The link between glomerular IgA nephropathy (IgAN) and T helper 2 (Th2) response has been implicated, however, the mechanisms are poorly defined because of the lack of an appropriate model. Here we report a novel murine model characterized by lineage-restricted deletion of the gene encoding MAD homologue 4 (Smad4) in T cells (Smad4co/co;Lck-cre). Loss of Smad4 expression in T cells results in overproduction of Th2 cytokines and high serum IgA levels. We found that Smad4co/co;Lck-cre mice exhibited massive glomerular IgA deposition, increased albumin creatinine ratio, aberrant glycosylated IgA, IgA complexed with IgG1 and IgG2a, and polymeric IgA, all known features of IgAN in humans. Furthermore, we examined the β1, 4-galactosyltransferases (β4GalT) enzyme which is involved in the synthesis of glycosylated murine IgA, and we found reduced β4GalT2 and β4GalT4 mRNA levels in B cells. These findings indicate that Smad4co/co;Lck-cre mice could be a useful model for studying the mechanisms between IgAN and Th2 response, and further, disruption of Smad4-dependent signaling in T cells may play an important role in the pathogenesis of human IgAN and contributing to a Th2 T cell phenotype. PMID:24223846

  20. Understanding the basis of I50V-induced affinity decrease in HIV-1 protease via molecular dynamics simulations using polarized force field.

    PubMed

    Duan, Rui; Lazim, Raudah; Zhang, Dawei

    2015-09-30

    Human immunodeficiency virus (HIV)-1 protease is one of the most promising drug target commonly utilized to combat Acquired Immune Deficiency Syndrome (AIDS). However, with the emergence of drug resistance arising from mutations, the efficiency of protease inhibitors (PIs) as a viable treatment for AIDS has been greatly reduced. I50V mutation as one of the most significant mutations occurring in HIV-1 protease will be investigated in this study. Molecular dynamics (MD) simulation was utilized to examine the effect of I50V mutation on the binding of two PIs namely indinavir and amprenavir to HIV-1 protease. Prior to the simulations conducted, the electron density distributions of the PI and each residue in HIV-1 protease are derived by combining quantum fragmentation approach molecular fractionation with conjugate caps and Poisson-Boltzmann solvation model based on polarized protein-specific charge scheme. The atomic charges of the binding complex are subsequently fitted using delta restrained electrostatic potential (delta-RESP) method to overcome the poor charge determination of buried atom. This way, both intraprotease polarization and the polarization between protease and the PI are incorporated into partial atomic charges. Through this study, the mutation-induced affinity variations were calculated and significant agreement between experiments and MD simulations conducted was observed for both HIV-1 protease-drug complexes. In addition, the mechanism governing the decrease in the binding affinity of PI in the presence of I50V mutation was also explored to provide insights pertaining to the design of the next generation of anti-HIV drugs. © 2015 Wiley Periodicals, Inc.

  1. Evolution of inhibitor-resistant natural mutant forms of HIV-1 protease probed by pre-steady state kinetic analysis.

    PubMed

    Zakharova, Maria Yu; Kuznetsova, Alexandra A; Kaliberda, Elena N; Dronina, Maria A; Kolesnikov, Alexander V; Kozyr, Arina V; Smirnov, Ivan V; Rumsh, Lev D; Fedorova, Olga S; Knorre, Dmitry G; Gabibov, Alexander G; Kuznetsov, Nikita A

    2017-11-01

    Pre-steady state kinetic analysis of mechanistic features of substrate binding and processing is crucial for insight into the evolution of inhibitor-resistant forms of HIV-1 protease. These data may provide a correct vector for rational drug design assuming possible intrinsic dynamic effects. These data should also give some clues to the molecular mechanism of protease action and resistance to inhibitors. Here we report pre-steady state kinetics of the interaction of wild type or mutant forms of HIV-1 protease with a FRET-labeled peptide. The three-stage "minimal" kinetic scheme with first and second reversible steps of substrate binding and with following irreversible peptide cleavage step adequately described experimental data. For the first time, a set of "elementary" kinetic parameters of wild type HIV-1 protease and its natural mutant inhibitor-resistant forms MDR-HM, ANAM-11 and prDRV4 were compared. Inhibitors of the first and second generation were used to estimate the inhibitory effects on HIV-1 protease activity. The resulting set of kinetic data supported that the mutant forms are kinetically unaffected by inhibitors of the first generation, proving their functional resistance to these compounds. The second generation inhibitor darunavir inhibited mutant forms MDR-HM and ANAM-11, but was ineffective against prDRV4. Our kinetic data revealed that these inhibitors induced different conformational changes in the enzyme and, thereby they have different mode of binding in the enzyme active site. These data confirmed hypothesis that the driving force of the inhibitor-resistance evolution is disruption of enzyme-inhibitor complex by changing of the contact network in the inhibitor binding site. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  2. Dysregulation of Protease and Protease Inhibitors in a Mouse Model of Human Pelvic Organ Prolapse

    PubMed Central

    Budatha, Madhusudhan; Silva, Simone; Montoya, Teodoro Ignacio; Suzuki, Ayako; Shah-Simpson, Sheena; Wieslander, Cecilia Karin; Yanagisawa, Masashi; Word, Ruth Ann; Yanagisawa, Hiromi

    2013-01-01

    Mice deficient for the fibulin-5 gene (Fbln5−/−) develop pelvic organ prolapse (POP) due to compromised elastic fibers and upregulation of matrix metalloprotease (MMP)-9. Here, we used casein zymography, inhibitor profiling, affinity pull-down, and mass spectrometry to discover additional protease upregulated in the vaginal wall of Fbln5−/− mice, herein named V1 (25 kDa). V1 was a serine protease with trypsin-like activity similar to protease, serine (PRSS) 3, a major extrapancreatic trypsinogen, was optimum at pH 8.0, and predominantly detected in estrogenized vaginal epithelium of Fbln5−/− mice. PRSS3 was (a) localized in epithelial secretions, (b) detected in media of vaginal organ culture from both Fbln5−/− and wild type mice, and (c) cleaved fibulin-5 in vitro. Expression of two serine protease inhibitors [Serpina1a1-antitrypsin) and Elafin] was dysregulated in Fbln5−/− epithelium. Finally, we confirmed that PRSS3 was expressed in human vaginal epithelium and that SERPINA1 and Elafin were downregulated in vaginal tissues from women with POP. These data collectively suggest that the balance between proteases and their inhibitors contributes to support of the pelvic organs in humans and mice. PMID:23437119

  3. Prevalence, mutation patterns, and effects on protease inhibitor susceptibility of the L76V mutation in HIV-1 protease.

    PubMed

    Young, Thomas P; Parkin, Neil T; Stawiski, Eric; Pilot-Matias, Tami; Trinh, Roger; Kempf, Dale J; Norton, Michael

    2010-11-01

    Patterns of HIV-1 protease inhibitor (PI) resistance-associated mutations (RAMs) and effects on PI susceptibility associated with the L76V mutation were studied in a large database. Of 20,501 sequences with ≥1 PI RAM, 3.2% contained L76V; L76V was alone in 0.04%. Common partner mutations included M46I, I54V, V82A, I84V, and L90M. L76V was associated with a 2- to 6-fold decrease in susceptibility to lopinavir, darunavir, amprenavir, and indinavir and a 7- to 8-fold increase in susceptibility to atazanavir and saquinavir.

  4. Clinical and pathological analysis of IgA nephropathy with chronic renal failure.

    PubMed

    Liu, Yuyuan; Hu, Qinfeng; Shen, Ping; Tang, Li; Yuan, Gang; Zhou, Yongmei; Chai, Huaqi

    2016-10-01

    To investigative clinical and pathological characteristics of IgA nephropathy with chronic renal failure. Clinical and pathological findings from 65 cases of IgA nephropathy with chronic renal failure were reviewed. Pathological characteristics of all the cases were analyzed according to WHO definition and Oxford Classification. Evaluating the severity of pathological lesions by the Katafuchi R semiquantitative scoring system, and analyzing their relationship with clinical indexes of renal function. Of all 65 cases the male and female ratio was 1.4, and the mean age was 37 ± 13 years old. Levels of systolic pressure, mean arterial pressure (MAP), blood urea nitrogen (BUN), serum creatinine (Scr), uric acid (UA), album (Alb), serum IgG and 24 h urinary protein were related with eGRF level (p < 0.05, respectively). The most common pathological type was proliferative sclerosis glomerulonephritis (PSGN) and M1S1E0T0 according to WHO definition and Oxford Classification, respectively, and most of the 65 cases had glomerulosclerosis. Simple IgA deposition was the most common immunopathologic type. Of all the cases, 44.6% accompanied with C3 while 4.6% with C1q. Further analysis revealed there were no relationships between severity of pathological lesion and levels of clinical indexes (Scr and eGRF) (p > 0.05). IgA nephropathy with chronic renal failure usually occurred in young adults, and it had severe clinical condition and pathological changes, while there was no significant relationship between them.

  5. Dynamics of Preferential Substrate Recognition in HIV-1 Protease: Redefining the Substrate Envelope

    PubMed Central

    Özen, Ayşegül; Haliloğlu, Türkan; Schiffer, Celia A.

    2011-01-01

    HIV-1 protease (PR) permits viral maturation by processing the Gag and Gag-Pro-Pol polyproteins. Though HIV-1 PR inhibitors (PIs) are used in combination antiviral therapy, the emergence of drug resistance has limited their efficacy. The rapid evolution of HIV-1 necessitates the consideration of drug resistance in novel drug-design strategies. Drug-resistant HIV-1 PR variants, while no longer efficiently inhibited, continue to efficiently hydrolyze the natural viral substrates. Though highly diverse in sequence, the HIV-1 PR substrates bind in a conserved three-dimensional shape we defined as the “substrate envelope”. We previously showed that resistance mutations arise where PIs protrude beyond the substrate envelope, as these regions are crucial for drug binding but not for substrate recognition. Here, we extend this model by considering the role of protein dynamics in the interaction of HIV-1 PR with its substrates. Seven molecular dynamics simulations of PR-substrate complexes were performed to estimate the conformational flexibility of substrates in their complexes. Interdependency of the substrate-protease interactions may compensate for the variations in cleavage-site sequences, and explain how a diverse set of sequences can be recognized as substrates by the same enzyme. This diversity may be essential for regulating sequential processing of substrates. We also define a dynamic substrate envelope as a more accurate representation of PR-substrate interactions. This dynamic substrate envelope, described by a probability distribution function, is a powerful tool for drug design efforts targeting ensembles of resistant HIV-1 PR variants with the aim of developing drugs that are less susceptible to resistance. PMID:21762811

  6. Degradation of recombinant proteins by CHO host cell proteases is prevented by Matriptase-1 knock-out.

    PubMed

    Laux, Holger; Romand, Sandrine; Nuciforo, Sandro; Farady, Christopher J; Tapparel, Joel; Buechmann-Moeller, Stine; Sommer, Benjamin; Oakeley, Edward J; Bodendorf, Ursula

    2018-05-19

    An increasing number of non-antibody format proteins are entering the clinical development. However, one of the major hurdles for the production of non-antibody glycoproteins is host cell-related proteolytic degradation, which can drastically impact developability and timelines of pipeline projects. Chinese hamster ovary (CHO) cells are the preferred production host for recombinant therapeutic proteins. Using protease inhibitors, transcriptomics and genetic knockdowns we have identified, out of the more than 700 known proteases in rodents, Matriptase-1 as the major protease involved in degradation of recombinant proteins expressed in CHO-K1 cells. Subsequently Matriptase-1 was deleted in CHO-K1 cells using "Transcription Activator-Like Effector Nucleases" (TALENs) as well as zinc-finger nucleases (ZFNs). This resulted in a superior CHO-K1 matriptase knockout (KO) cell line with strongly reduced or no proteolytic degradation activity towards a panel of recombinantly-expressed proteins. The matriptase KO cell line was evaluated in spike-in experiments, and showed little or no degradation of proteins incubated in culture supernatant derived from the KO cells. This effect was confirmed when the same proteins were recombinantly expressed in the KO cell line. In summary, the combination of novel cell line engineering tools, next generation sequencing screening methods and the recently published Chinese hamster genome has enabled the development of this novel matriptase KO CHO cell line capable of improving expression yields of intact therapeutic proteins. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  7. The effect of desolvation on the binding of inhibitors to HIV-1 protease and cyclin-dependent kinases: Causes of resistance.

    PubMed

    Fong, Clifford W

    2016-08-01

    Studies of the cyclin-dependent kinase inhibitors and HIV-1 protease inhibitors have confirmed that ligand-protein binding is dependent on desolvation effects. It has been found that a four parameter linear model incorporating desolvation energy, lipophilicity, dipole moment and molecular volume of the ligands is a good model to describe the binding between ligands and kinases or proteases. The resistance shown by MDR proteases to the anti-viral drugs is multi-faceted involving varying changes in desolvation, lipophilicity and dipole moment interaction compared to the non-resistant protease. Desolvation has been shown to be the dominant factor influencing the effect of inhibitors against the cyclin-dependent kinases, but lipophilicity and dipole moment are also significant factors. The model can differentiate between the inhibitory activity of CDK2/cycE, CDK1/cycB and CDK4/cycD enzymes. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Comparison of interferon and bovine herpesvirus-1-specific IgA levels in nasal secretions of dairy cattle administered an intranasal modified live viral vaccine prior to calving or on the day of calving.

    PubMed

    Cortese, Victor S; Woolums, Amelia; Hurley, David J; Berghaus, Roy; Bernard, John K; Short, Thomas H

    2017-05-01

    Thirty-two Holstein cows were allocated to receive intranasal vaccination with modified live bovine herpesvirus-1 (BHV-1), bovine respiratory syncytial virus (BRSV) and parainfluenza type 3 virus (PI3V) vaccine either two weeks prior to their projected calving date, or within 24h after calving. Nasal secretions were collected twice at a 12-h interval on the day prior to vaccination (day 0) and at 2, 4, 7, 10 and 14days post vaccination to measure interferon (IFN) alpha, IFN-beta, IFN-gamma, and BHV-1-specific IgA by ELISA. Serum neutralizing antibody titers to BHV-1 and BRSV were measured on days 0, 7, and 14. There was a significant treatment effect (p<0.0004) and interaction (p<0.05) on nasal BHV-1 IgA levels, with higher IgA levels in cows vaccinated within 24h after calving. There was a significant treatment effect on nasal IFN-gamma concentration (p<0.05) and on nasal total IFN concentration (p<0.05), with higher IFN-gamma and total IFN concentrations seen in cows vaccinated within 24h after calving. There was no significant treatment or interaction effect on nasal IFN-alpha or IFN-beta concentrations, or on serum neutralizing titers to BRSV. In spite of prior viral vaccination during the previous lactation, cows vaccinated on the day of calving responded to an intranasal viral vaccination with increased concentrations of IFN-gamma and increased titers of IgA following vaccination which was significantly higher than cows vaccinated precalving. This study is the first to examine respiratory mucosal responses in immunologically mature dairy cattle vaccinated intranasally before and after calving. Copyright © 2017. Published by Elsevier B.V.

  9. Saliva secretory IgA antibodies against molds and mycotoxins in patients exposed to toxigenic fungi.

    PubMed

    Vojdani, Aristo; Kashanian, Albert; Vojdani, Elroy; Campbell, Andrew W

    2003-11-01

    Upper respiratory exposure to different environmental antigens results first in the activation of mucosal immunity and production of IgA antibodies in different secretions including saliva. Despite this there is no study, which addresses secretory antibodies against molds and mycotoxins. The purpose of this study was to evaluate mold-specific salivary IgA in individuals exposed to molds and mycotoxins in a water-damaged building environment. Saliva IgA antibody levels against seven different molds and two mycotoxins were studied in 40 patients exposed to molds and in 40 control subjects. Mold-exposed patients showed significantly higher levels of salivary IgA antibodies against one or more mold species. A majority of patients with high IgA antibodies against molds exhibited elevation in salivary IgA against mycotoxins, as well. These IgA antibodies against molds and mycotoxins are specific, since using molds and mycotoxins in immune absorption could reduce antibody levels, significantly. Detection of high counts of molds in water-damaged buildings, strongly suggests the existence of a reservoir of mold spores in the environment. This viable microbial activity with specific mold and mycotoxin IgA in saliva may assist in the diagnosis of mold exposure. Whether mold and mycotoxin specific IgA antibodies detected in saliva are indicative of the role of IgA antibodies in the late phase of type-1 hypersensitivity reaction or in type-2 and type-3 delayed sensitivities is a matter that warrants further investigation.

  10. Crystal structure of a complex of HIV-1 protease with a dihydroxyethylene-containing inhibitor: comparisons with molecular modeling.

    PubMed Central

    Thanki, N.; Rao, J. K.; Foundling, S. I.; Howe, W. J.; Moon, J. B.; Hui, J. O.; Tomasselli, A. G.; Heinrikson, R. L.; Thaisrivongs, S.; Wlodawer, A.

    1992-01-01

    The structure of a crystal complex of recombinant human immunodeficiency virus type 1 (HIV-1) protease with a peptide-mimetic inhibitor containing a dihydroxyethylene isostere insert replacing the scissile bond has been determined. The inhibitor is Noa-His-Hch psi [CH(OH)CH(OH)]Vam-Ile-Amp (U-75875), and its Ki for inhibition of the HIV-1 protease is < 1.0 nM (Noa = 1-naphthoxyacetyl, Hch = a hydroxy-modified form of cyclohexylalanine, Vam = a hydroxy-modified form of valine, Amp = 2-pyridylmethylamine). The structure of the complex has been refined to a crystallographic R factor of 0.169 at 2.0 A resolution by using restrained least-squares procedures. Root mean square deviations from ideality are 0.02 A and 2.4 degrees, for bond lengths and angles, respectively. The bound inhibitor diastereomer has the R configurations at both of the hydroxyl chiral carbon atoms. One of the diol hydroxyl groups is positioned such that it forms hydrogen bonds with both the active site aspartates, whereas the other interacts with only one of them. Comparison of this X-ray structure with a model-built structure of the inhibitor, published earlier, reveals similar positioning of the backbone atoms and of the side-chain atoms in the P2-P2' region, where the interaction with the protein is strongest. However, the X-ray structure and the model differ considerably in the location of the P3 and P3' end groups, and also in the positioning of the second of the two central hydroxyl groups. Reconstruction of the central portion of the model revealed the source of the hydroxyl discrepancy, which, when corrected, provided a P1-P1' geometry very close to that seen in the X-ray structure. PMID:1304383

  11. The higher frequency of IgA deficiency among Swedish twins is not explained by HLA haplotypes.

    PubMed

    Frankowiack, M; Kovanen, R-M; Repasky, G A; Lim, C K; Song, C; Pedersen, N L; Hammarström, L

    2015-01-01

    Serum immunoglobulin A (IgA) concentrations were determined in 12 600 adult Swedish twins, applying a high-throughput reverse-phase protein microarray technique. The prevalence of IgA deficiency (IgAD) was found to be 1:241 in monozygotic (MZ) twins and 1:198 in dizygotic (DZ) twins. Hence, the prevalence in twins is markedly elevated as compared with the normal Swedish adult population (1:600). The twins did not show a difference in the frequency of HLA haplotypes in comparison with almost 40 000 healthy Swedish controls. As expected, the risk-conveying HLA alleles A*01, B*08 and DRB1*01 were overrepresented among the IgAD twins and were also associated with significantly lower mean serum IgA concentrations in the twin cohort. In contrast, significantly higher mean IgA concentrations were found among individuals carrying the protective HLA alleles B*07 and DRB1*15. Exome sequencing data from two MZ twin pairs discordant for the deficiency showed no differences between the siblings. Model fitting analyses derived a heritability of 35% and indicate that genetic influences are modestly important for IgAD. The probandwise concordance rates for IgAD were found to be 31% for MZ and 13% for DZ twins.

  12. Vertically transmitted fecal IgA levels distinguish extra-chromosomal phenotypic variation

    PubMed Central

    Wallace, Meghan A.; D, Carey-Ann; Burnham; Virgin, Herbert W.; Stappenbeck, Thaddeus S.

    2014-01-01

    Summary The proliferation of genetically modified mouse models has exposed phenotypic variation between investigators and institutions that has been challenging to control1-5. In many cases, the microbiota is the presumed culprit of the variation. Current solutions to account for phenotypic variability include littermate and maternal controls or defined microbial consortia in gnotobiotic mice6,7. In conventionally raised mice, the microbiome is transmitted from the dam2,8,9. Here we show that microbially–driven dichotomous fecal IgA levels in WT mice within the same facility mimic the effects of chromosomal mutations. We observed in multiple facilities that vertically-transmissible bacteria in IgA-Low mice dominantly lowered fecal IgA levels in IgA-High mice after cohousing or fecal transplantation. In response to injury, IgA-Low mice showed increased damage that was transferable by fecal transplantation and driven by fecal IgA differences. We found that bacteria from IgA-Low mice degraded the secretory component (SC) of SIgA as well as IgA itself. These data indicate that phenotypic comparisons between mice must take into account the non-chromosomal hereditary variation between different breeders. We propose fecal IgA as one marker of microbial variability and conclude that cohousing and/or fecal transplantation enables analysis of progeny from different dams. PMID:25686606

  13. Protease-activated receptor (PAR)-2 is required for PAR-1 signalling in pulmonary fibrosis

    PubMed Central

    Lin, Cong; von der Thüsen, Jan; Daalhuisen, Joost; ten Brink, Marieke; Crestani, Bruno; van der Poll, Tom; Borensztajn, Keren; Spek, C Arnold

    2015-01-01

    Idiopathic pulmonary fibrosis is the most devastating diffuse fibrosing lung disease of unknown aetiology. Compelling evidence suggests that both protease-activated receptor (PAR)-1 and PAR-2 participate in the development of pulmonary fibrosis. Previous studies have shown that bleomycin-induced lung fibrosis is diminished in both PAR-1 and PAR-2 deficient mice. We thus have been suggested that combined inactivation of PAR-1 and PAR-2 would be more effective in blocking pulmonary fibrosis. Human and murine fibroblasts were stimulated with PAR-1 and PAR-2 agonists in the absence or presence of specific PAR-1 or PAR-2 antagonists after which fibrotic markers like collagen and smooth muscle actin were analysed by Western blot. Pulmonary fibrosis was induced by intranasal instillation of bleomycin into wild-type and PAR-2 deficient mice with or without a specific PAR-1 antagonist (P1pal-12). Fibrosis was assessed by hydroxyproline quantification and (immuno)histochemical analysis. We show that specific PAR-1 and/or PAR-2 activating proteases induce fibroblast migration, differentiation and extracellular matrix production. Interestingly, however, combined activation of PAR-1 and PAR-2 did not show any additive effects on these pro-fibrotic responses. Strikingly, PAR-2 deficiency as well as pharmacological PAR-1 inhibition reduced bleomycin-induced pulmonary fibrosis to a similar extent. PAR-1 inhibition in PAR-2 deficient mice did not further diminish bleomycin-induced pulmonary fibrosis. Finally, we show that the PAR-1-dependent pro-fibrotic responses are inhibited by the PAR-2 specific antagonist. Targeting PAR-1 and PAR-2 simultaneously is not superior to targeting either receptor alone in bleomycin-induced pulmonary fibrosis. We postulate that the pro-fibrotic effects of PAR-1 require the presence of PAR-2. PMID:25689283

  14. Protease-activated receptor (PAR)-2 is required for PAR-1 signalling in pulmonary fibrosis.

    PubMed

    Lin, Cong; von der Thüsen, Jan; Daalhuisen, Joost; ten Brink, Marieke; Crestani, Bruno; van der Poll, Tom; Borensztajn, Keren; Spek, C Arnold

    2015-06-01

    Idiopathic pulmonary fibrosis is the most devastating diffuse fibrosing lung disease of unknown aetiology. Compelling evidence suggests that both protease-activated receptor (PAR)-1 and PAR-2 participate in the development of pulmonary fibrosis. Previous studies have shown that bleomycin-induced lung fibrosis is diminished in both PAR-1 and PAR-2 deficient mice. We thus have been suggested that combined inactivation of PAR-1 and PAR-2 would be more effective in blocking pulmonary fibrosis. Human and murine fibroblasts were stimulated with PAR-1 and PAR-2 agonists in the absence or presence of specific PAR-1 or PAR-2 antagonists after which fibrotic markers like collagen and smooth muscle actin were analysed by Western blot. Pulmonary fibrosis was induced by intranasal instillation of bleomycin into wild-type and PAR-2 deficient mice with or without a specific PAR-1 antagonist (P1pal-12). Fibrosis was assessed by hydroxyproline quantification and (immuno)histochemical analysis. We show that specific PAR-1 and/or PAR-2 activating proteases induce fibroblast migration, differentiation and extracellular matrix production. Interestingly, however, combined activation of PAR-1 and PAR-2 did not show any additive effects on these pro-fibrotic responses. Strikingly, PAR-2 deficiency as well as pharmacological PAR-1 inhibition reduced bleomycin-induced pulmonary fibrosis to a similar extent. PAR-1 inhibition in PAR-2 deficient mice did not further diminish bleomycin-induced pulmonary fibrosis. Finally, we show that the PAR-1-dependent pro-fibrotic responses are inhibited by the PAR-2 specific antagonist. Targeting PAR-1 and PAR-2 simultaneously is not superior to targeting either receptor alone in bleomycin-induced pulmonary fibrosis. We postulate that the pro-fibrotic effects of PAR-1 require the presence of PAR-2. © 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  15. Synthesis and disulfide bond connectivity-activity studies of a kalata B1-inspired cyclopeptide against dengue NS2B-NS3 protease.

    PubMed

    Gao, Yaojun; Cui, Taian; Lam, Yulin

    2010-02-01

    Kalata B1 is a plant protein with remarkable thermal, chemical and enzymatic stability. Its potential applications could be centered on the possibility of using its cyclic structure and cystine knot motif as a scaffold for the design of stable pharmaceuticals. To discover potent dengue NS2B-NS3 protease inhibitors, we have prepared various kalata B1 analogues by varying the amino acid sequence. Mass spectrometric and biochemical investigations of these analogues revealed a cyclopeptide whose two fully oxidized forms are substrate-competitive inhibitors of the dengue viral NS2B-NS3 protease. Both oxidized forms showed potent inhibition with K(i) of 1.39+/-0.35 and 3.03+/-0.75 microM, respectively. Copyright (c) 2009 Elsevier Ltd. All rights reserved.

  16. Inflammation-induced IgA+ cells dismantle anti-liver cancer immunity

    PubMed Central

    Shalapour, Shabnam; Lin, Xue-Jia; Bastian, Ingmar N.; Brain, John; Burt, Alastair D.; Aksenov, Alexander A.; Vrbanac, Alison F.; Li, Weihua; Perkins, Andres; Matsutani, Takaji; Zhong, Zhenyu; Dhar, Debanjan; Navas-Molina, Jose A.; Xu, Jun; Loomba, Rohit; Downes, Michael; Yu, Ruth T.; Evans, Ronald M.; Dorrestein, Pieter C.; Knight, Rob; Benner, Christopher; Anstee, Quentin M.; Karin, Michael

    2018-01-01

    The role of adaptive immunity in early cancer development is controversial. Here we show that chronic inflammation and fibrosis in humans and mice with non-alcoholic fatty liver disease is accompanied by accumulation of liver-resident immunoglobulin-A-producing (IgA+) cells. These cells also express programmed death ligand 1 (PD-L1) and interleukin-10, and directly suppress liver cytotoxic CD8+ T lymphocytes, which prevent emergence of hepatocellular carcinoma and express a limited repertoire of T-cell receptors against tumour-associated antigens. Whereas CD8+ T-cell ablation accelerates hepatocellular carcinoma, genetic or pharmacological interference with IgA+ cell generation attenuates liver carcinogenesis and induces cytotoxic T-lymphocyte-mediated regression of established hepatocellular carcinoma. These findings establish the importance of inflammation-induced suppression of cytotoxic CD8+ T-lymphocyte activation as a tumour-promoting mechanism. PMID:29144460

  17. In Vitro Evolution of the Human Immunodeficiency Virus Type 1 Gag-Protease Region and Maintenance of Reverse Transcriptase Resistance following Prolonged Drug Exposure†

    PubMed Central

    La Seta Catamancio, Simona; De Pasquale, Maria Pia; Citterio, Paola; Kurtagic, Semir; Galli, Massimo; Rusconi, Stefano

    2001-01-01

    We studied the human immunodeficiency virus type 1 phenotypic and genotypic profiles of a dual drug-resistant isolate (isolate 14aPost-DR) selected for zidovudine (ZDV) and lamivudine (3TC) resistance and then cultured in the presence of 3TC and a protease inhibitor: indinavir (IDV), ritonavir, or KNI-272. The IDV-treated virus was highly resistant to 3TC, ZDV, and IDV and accumulated protease mutations at positions M46I and V82F. A change from alanine to valine was observed in 4 of 10 clones in the P2 position of the p7-p1 Gag-protease cleavage site, linked to position M46I in the dominant viral quasispecies. Previous 3TC resistance did not impair the development of additional mutations in the protease and Gag-protease cleavage regions. PMID:11230439

  18. Borrelia burgdorferi-specific IgA in Lyme Disease.

    PubMed

    D'Arco, Christina; Dattwyler, Raymond J; Arnaboldi, Paul M

    2017-05-01

    The laboratory diagnosis of Lyme disease is currently dependent on the detection of IgM and IgG antibodies against Borrelia burgdorferi, the causative agent of the disease. The significance of serum IgA against B. burgdorferi remains unclear. The production of intrathecal IgA has been noted in patients with the late Lyme disease manifestation, neuroborreliosis, but production of antigen-specific IgA during early disease has not been evaluated. In the current study, we assessed serum IgA binding to the B. burgdorferi peptide antigens, C6, the target of the FDA-cleared C6 EIA, and FlaB(211-223)-modVlsE(275-291), a peptide containing a Borrelia flagellin epitope linked to a modified VlsE sequence, in patients with early and late Lyme disease. Specific IgA was detected in 59 of 152 serum samples (38.8%) from early Lyme disease patients. Approximately 50% of early Lyme disease patients who were seropositive for peptide-specific IgM and/or IgG were also seropositive for peptide-specific IgA. In a subpopulation of patients, high peptide-specific IgA could be correlated with disseminated disease, defined as multiple erythema migrans lesions, and neurological disease complications. These results suggest that there may be an association between elevated levels of antigen-specific IgA and particular disease manifestations in some patients with early Lyme disease. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  19. Functional analyses of the three simian hemorrhagic fever virus nonstructural protein 1 papain-like proteases.

    PubMed

    Vatter, Heather A; Di, Han; Donaldson, Eric F; Radu, Gertrud U; Maines, Taronna R; Brinton, Margo A

    2014-08-01

    The N-terminal region of simian hemorrhagic fever virus (SHFV) nonstructural polyprotein 1a is predicted to encode three papain-like proteases (PLP1α, PLP1β, and PLP1γ). Catalytic residues and cleavage sites for each of the SHFV PLP1s were predicted by alignment of the SHFV PLP1 region sequences with each other as well as with those of other arteriviruses, and the predicted catalytic residues were shown to be proximal by homology modeling of the SHFV nsp1s on porcine respiratory and reproductive syndrome virus (PRRSV) nsp1 crystal structures. The functionality of the predicted catalytic Cys residues and cleavage sites was tested by analysis of the autoproteolytic products generated in in vitro transcription/translation reactions done with wild-type or mutant SHFV nsp1 constructs. Cleavage sites were also analyzed by mass spectroscopy analysis of selected immunoprecipitated cleavage products. The data showed that each of the three SHFV PLP1s is an active protease. Cys63 was identified as the catalytic Cys of SHFV PLP1α and is adjacent to an Ala instead of the canonical Tyr observed in other arterivirus PLP1s. SHFV PLP1γ is able to cleave at both downstream and upstream nsp1 junction sites. Although intermediate precursor polyproteins as well as alternative products generated by each of the SHFV PLP1s cleaving at sites within the N-terminal region of nsp1β were produced in the in vitro reactions, Western blotting of SHFV-infected, MA104 cell lysates with SHFV nsp1 protein-specific antibodies detected only the three mature nsp1 proteins. SHFV is unique among arteriviruses in having three N-terminal papain-like protease 1 (PLP1) domains. Other arteriviruses encode one or two active PLP1s. This is the first functional study of the SHFV PLP1s. Analysis of the products of in vitro autoprocessing of an N-terminal SHFV nonstructural 1a polypeptide fragment showed that each of the three SHFV PLP1s is active, and the predicted catalytic Cys residues and cleavage sites

  20. Energetic basis for drug resistance of HIV-1 protease mutants against amprenavir

    NASA Astrophysics Data System (ADS)

    Kar, Parimal; Knecht, Volker

    2012-02-01

    Amprenavir (APV) is a high affinity (0.15 nM) HIV-1 protease (PR) inhibitor. However, the affinities of the drug resistant protease variants V32I, I50V, I54V, I54M, I84V and L90M to amprenavir are decreased 3 to 30-fold compared to the wild-type. In this work, the popular molecular mechanics Poisson-Boltzmann surface area method has been used to investigate the effectiveness of amprenavir against the wild-type and these mutated protease variants. Our results reveal that the protonation state of Asp25/Asp25' strongly affects the dynamics, the overall affinity and the interactions of the inhibitor with individual residues. We emphasize that, in contrast to what is often assumed, the protonation state may not be inferred from the affinities but requires pKa calculations. At neutral pH, Asp25 and Asp25' are ionized or protonated, respectively, as suggested from pKa calculations. This protonation state was thus mainly considered in our study. Mutation induced changes in binding affinities are in agreement with the experimental findings. The decomposition of the binding free energy reveals the mechanisms underlying binding and drug resistance. Drug resistance arises from an increase in the energetic contribution from the van der Waals interactions between APV and PR (V32I, I50V, and I84V mutant) or a rise in the energetic contribution from the electrostatic interactions between the inhibitor and its target (I54M and I54V mutant). For the V32I mutant, also an increased free energy for the polar solvation contributes to the drug resistance. For the L90M mutant, a rise in the van der Waals energy for APV-PR interactions is compensated by a decrease in the polar solvation free energy such that the net binding affinity remains unchanged. Detailed understanding of the molecular forces governing binding and drug resistance might assist in the design of new inhibitors against HIV-1 PR variants that are resistant against current drugs.

  1. Biomarkers for IgA nephropathy on the basis of multi-hit pathogenesis.

    PubMed

    Suzuki, Hitoshi

    2018-05-08

    IgA nephropathy (IgAN) is the most prevalent glomerular disease worldwide and is associated with a poor prognosis. Development of curative treatment strategies and approaches for early diagnosis is necessary. Renal biopsy is the gold standard for the diagnosis and assessment of disease activity. However, reliable biomarkers are needed for the noninvasive diagnosis of this disease and to more fully delineate the risk of progression. With regard to the pathogenesis of IgAN, the multi-hit hypothesis, including production of galactose-deficient IgA1 (Gd-IgA1; Hit 1), IgG or IgA autoantibodies that recognize Gd-IgA1 (Hit 2), and their subsequent immune complexes formation (Hit 3) and glomerular deposition (Hit 4), has been widely supported by many studies. Although the prognostic values of several biomarkers have been discussed, we recently developed a highly sensitive and specific diagnostic method by measuring serum levels of Gd-IgA1 and Gd-IgA1-containing immune complexes. In addition, urinary Gd-IgA1 may represent a disease-specific biomarker for IgAN. We also confirmed that there is a significant correlation between serum levels of these effector molecules and disease activity, suggesting that each can be considered a practical surrogate marker of therapeutic response. Thus, these disease-oriented specific serum and urine biomarkers may be useful for screening of potential IgAN with isolated hematuria, earlier diagnosis, disease activity, and eventually, response to treatment. In this review, we discuss these concepts, with a focus on potential clinical applications of these biomarkers.

  2. Drug resistance conferred by mutations outside the active site through alterations in the dynamic and structural ensemble of HIV-1 protease.

    PubMed

    Ragland, Debra A; Nalivaika, Ellen A; Nalam, Madhavi N L; Prachanronarong, Kristina L; Cao, Hong; Bandaranayake, Rajintha M; Cai, Yufeng; Kurt-Yilmaz, Nese; Schiffer, Celia A

    2014-08-27

    HIV-1 protease inhibitors are part of the highly active antiretroviral therapy effectively used in the treatment of HIV infection and AIDS. Darunavir (DRV) is the most potent of these inhibitors, soliciting drug resistance only when a complex combination of mutations occur both inside and outside the protease active site. With few exceptions, the role of mutations outside the active site in conferring resistance remains largely elusive. Through a series of DRV-protease complex crystal structures, inhibition assays, and molecular dynamics simulations, we find that single and double site mutations outside the active site often associated with DRV resistance alter the structure and dynamic ensemble of HIV-1 protease active site. These alterations correlate with the observed inhibitor binding affinities for the mutants, and suggest a network hypothesis on how the effect of distal mutations are propagated to pivotal residues at the active site and may contribute to conferring drug resistance.

  3. IgA Function in Relation to the Intestinal Microbiota.

    PubMed

    Macpherson, Andrew J; Yilmaz, Bahtiyar; Limenitakis, Julien P; Ganal-Vonarburg, Stephanie C

    2018-04-26

    IgA is the dominant immunoglobulin isotype produced in mammals, largely secreted across the intestinal mucosal surface. Although induction of IgA has been a hallmark feature of microbiota colonization following colonization in germ-free animals, until recently appreciation of the function of IgA in host-microbial mutualism has depended mainly on indirect evidence of alterations in microbiota composition or penetration of microbes in the absence of somatic mutations in IgA (or compensatory IgM). Highly parallel sequencing techniques that enable high-resolution analysis of either microbial consortia or IgA sequence diversity are now giving us new perspectives on selective targeting of microbial taxa and the trajectory of IgA diversification according to induction mechanisms, between different individuals and over time. The prospects are to link the range of diversified IgA clonotypes to specific antigenic functions in modulating the microbiota composition, position and metabolism to ensure host mutualism.

  4. Nationwide epidemiological survey of childhood IgA vasculitis associated hospitalization in the USA.

    PubMed

    Okubo, Yusuke; Nochioka, Kotaro; Sakakibara, Hiroshi; Hataya, Hiroshi; Terakawa, Toshiro; Testa, Marcia; Sundel, Robert P

    2016-11-01

    At the national level, IgA vasculitis-related hospitalizations among children in the USA are scarce. Furthermore, nationwide epidemiology and hospital course of children with IgA vasculitis have not been fully described in the USA, and disparities by race/ethnicity remain unknown. Hospital discharge records of patients aged 19 years or younger were obtained from the 2003, 2006, 2009, and 2012 Kids' Inpatient Database, and they were weighted to estimate the annual hospitalization rates with respect to age, gender, and race/ethnicity in the USA. Annual hospitalization rates were calculated using weighted case estimates and US census data. Negative binomial regression was used to ascertain the factors associated with length of hospital stay. Total annual hospitalization rates showed a significant decreasing trend, ranging from 2.45 per 100,000 children in 2003 to 1.89 per 100,000 children in 2012 (p < 0.001). The peak ages of the hospitalized children with IgA vasculitis were 2 and 7 years, and male-to-female ratios were 1.38-1.44. Factors associated with length of hospital stay were patients' ages (10-14 and 15-19 years), race/ethnicity (Hispanic, Asian, and Pacific Islander), comorbid electrolyte abnormality, GI hemorrhage, intussusception, renal symptoms, and GI symptoms. The annual hospitalization rates for IgA vasculitis are declining in the USA across multiple age groups. GI and renal manifestations are associated with increased length of hospital stay.

  5. Self perceived work related stress and the relation with salivary IgA and lysozyme among emergency department nurses

    PubMed Central

    Yang, Y; Koh, D; Ng, V; Lee, C; Chan, G; Dong, F; Goh, S; Anantharaman, V; Chia, S

    2002-01-01

    Aims: To assess and compare the self perceived work related stress among emergency department (ED) and general ward (GW) nurses, and to investigate its relation with salivary IgA and lysozyme. Methods: One hundred and thirty two of 208 (63.5%) registered female ED and GW nurses participated in the study. A modified mental health professional stress scale (PSS) was used to measure self perceived stress. ELISA methods were used to determine the salivary IgA and lysozyme levels. Results: On PSS, ED nurses had higher scores (mean 1.51) than GW nurses (1.30). The scores of PSS subscales such as organisational structure and processes (OS), lack of resources (RES), and conflict with other professionals (COF) were higher in ED than in GW nurses. ED nurses had lower secretion rates of IgA (geometric mean (GM) 49.1 µg/min) and lysozyme (GM 20.0 µg/min) than GW nurses (68.2 µg/min, 30.5 µg/min). Significant correlations were observed between PSS and log IgA and lysozyme secretion rates. OS, RES, and COF were correlated with log IgA and lysozyme levels. Conclusion: ED nurses, who reported a higher level of professional stress, showed significantly lower secretion rates of salivary IgA and lysozyme compared to GW nurses. Salivary IgA and lysozyme were inversely correlated with self perceived work related stress. As these salivary biomarkers are reflective of the mucosal immunity, results support the inverse relation between stress and mucosal immunity. PMID:12468751

  6. GRL-09510, a Unique P2-Crown-Tetrahydrofuranylurethane -Containing HIV-1 Protease Inhibitor, Maintains Its Favorable Antiviral Activity against Highly-Drug-Resistant HIV-1 Variants in vitro

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Amano, Masayuki; Miguel Salcedo-Gómez, Pedro; Yedidi, Ravikiran S.

    We report that GRL-09510, a novel HIV-1 protease inhibitor (PI) containing a newly-generated P2-crown-tetrahydrofuranylurethane (Crwn-THF), a P2'-methoxybenzene, and a sulfonamide isostere, is highly active against laboratory and primary clinical HIV-1 isolates (EC50: 0.0014–0.0028 μM) with minimal cytotoxicity (CC50: 39.0 μM). Similarly, GRL-09510 efficiently blocked the replication of HIV-1NL4-3 variants, which were capable of propagating at high-concentrations of atazanavir, lopinavir, and amprenavir (APV). GRL-09510 was also potent against multi-drug-resistant clinical HIV-1 variants and HIV-2ROD. Under the selection condition, where HIV-1NL4-3 rapidly acquired significant resistance to APV, an integrase inhibitor raltegravir, and a GRL-09510 congener (GRL-09610), no variants highly resistant against GRL-09510more » emerged over long-term in vitro passage of the virus. Crystallographic analysis demonstrated that the Crwn-THF moiety of GRL-09510 forms strong hydrogen-bond-interactions with HIV-1 protease (PR) active-site amino acids and is bulkier with a larger contact surface, making greater van der Waals contacts with PR than the bis-THF moiety of darunavir. The present data demonstrate that GRL-09510 has favorable features for treating patients infected with wild-type and/or multi-drug-resistant HIV-1 variants, that the newly generated P2-Crwn-THF moiety confers highly desirable anti-HIV-1 potency. The use of the novel Crwn-THF moiety sheds lights in the design of novel PIs.« less

  7. Complement factor H-related proteins in IgA nephropathy-sometimes a gentle nudge does the trick.

    PubMed

    Thurman, Joshua M; Laskowski, Jennifer

    2017-10-01

    Complement activation probably contributes to glomerular inflammation and damage in IgA nephropathy. In this issue, 2 groups report that levels of factor H-related protein 1 are elevated in patients with IgA nephropathy and correlate with disease progression. These studies provide new evidence that the complement cascade is important to the pathogenesis of this disease. These results also suggest that factor H-related protein 1 levels may be useful for identifying those patients at high risk of disease progression. Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

  8. Serine protease activity of Cur l 1 from Curvularia lunata augments Th2 response in mice.

    PubMed

    Tripathi, Prabhanshu; Kukreja, Neetu; Singh, B P; Arora, Naveen

    2009-05-01

    Studies with mite allergens demonstrated that proteolytic activity augments allergic airway inflammation. This knowledge is limited to few enzyme allergens. The objective of this study is to investigate the effect of serine protease Cur l 1 from Curvularia lunata in airway inflammation/hyper-responsiveness. Cur l 1 was purified and inactivated using a serine protease inhibitor. Balb/c mice were sensitized with enzymatically active Cur l 1 or C. lunata extract. Sensitized mice were given booster dose on day 14 with active or inactivated Cur l 1. Intranasal challenge was given on day 28, 29, and 30. Airway hyper-responsiveness was measured by plethysmography. Blood, bronchoalveolar lavage fluid (BALF), spleen, and lungs from mice were analyzed for cellular infiltration, immunoglobulins, and cytokine levels. Mice challenged with enzymatically active Cur l 1 demonstrated significantly higher airway inflammation than inactive Cur l 1 group mice (p < 0.01). There was a significant difference in serum IgE and IgG1 levels among mice immunized with active Cur l 1 and inactive Cur l 1 (p < 0.01). IL-4 and IL-5 were higher in BALF and splenocyte culture supernatant of active Cur l 1 than inactive Cur l 1 mice. Lung histology revealed increased eosinophil infiltration, goblet cell hyperplasia and mucus secretion in active group. Proteolytic activity of Cur l 1 plays an important role in airway inflammation and the inactivated Cur l 1 has potential to be explored for immunotherapy.

  9. A New Method for the Characterization of Strain-Specific Conformational Stability of Protease-Sensitive and Protease-Resistant PrPSc

    PubMed Central

    Pirisinu, Laura; Di Bari, Michele; Marcon, Stefano; Vaccari, Gabriele; D'Agostino, Claudia; Fazzi, Paola; Esposito, Elena; Galeno, Roberta; Langeveld, Jan; Agrimi, Umberto; Nonno, Romolo

    2010-01-01

    Although proteinacious in nature, prions exist as strains with specific self-perpetuating biological properties. Prion strains are thought to be associated with different conformers of PrPSc, a disease-associated isoform of the host-encoded cellular protein (PrPC). Molecular strain typing approaches have been developed which rely on the characterization of protease-resistant PrPSc. However, PrPSc is composed not only of protease-resistant but also of protease-sensitive isoforms. The aim of this work was to develop a protocol for the molecular characterization of both, protease-resistant and protease-sensitive PrPSc aggregates. We first set up experimental conditions which allowed the most advantageous separation of PrPC and PrPSc by means of differential centrifugation. The conformational solubility and stability assay (CSSA) was then developed by measuring PrPSc solubility as a function of increased exposure to GdnHCl. Brain homogenates from voles infected with human and sheep prion isolates were analysed by CSSA and showed strain-specific conformational stabilities, with mean [GdnHCl]1/2 values ranging from 1.6 M for MM2 sCJD to 2.1 for scrapie and to 2.8 M for MM1/MV1 sCJD and E200K gCJD. Interestingly, the rank order of [GdnHCl]1/2 values observed in the human and sheep isolates used as inocula closely matched those found following transmission in voles, being MM1 sCJD the most resistant (3.3 M), followed by sheep scrapie (2.2 M) and by MM2 sCJD (1.6 M). In order to test the ability of CSSA to characterise protease-sensitive PrPSc, we analysed sheep isolates of Nor98 and compared them to classical scrapie isolates. In Nor98, insoluble PrPSc aggregates were mainly protease-sensitive and showed a conformational stability much lower than in classical scrapie. Our results show that CSSA is able to reveal strain-specified PrPSc conformational stabilities of protease-resistant and protease-sensitive PrPSc and that it is a valuable tool for strain typing in natural

  10. Microbial ecology perturbation in human IgA deficiency.

    PubMed

    Fadlallah, Jehane; El Kafsi, Hela; Sterlin, Delphine; Juste, Catherine; Parizot, Christophe; Dorgham, Karim; Autaa, Gaëlle; Gouas, Doriane; Almeida, Mathieu; Lepage, Patricia; Pons, Nicolas; Le Chatelier, Emmanuelle; Levenez, Florence; Kennedy, Sean; Galleron, Nathalie; de Barros, Jean-Paul Pais; Malphettes, Marion; Galicier, Lionel; Boutboul, David; Mathian, Alexis; Miyara, Makoto; Oksenhendler, Eric; Amoura, Zahir; Doré, Joel; Fieschi, Claire; Ehrlich, S Dusko; Larsen, Martin; Gorochov, Guy

    2018-05-02

    Paradoxically, loss of immunoglobulin A (IgA), one of the most abundant antibodies, does not irrevocably lead to severe infections in humans but rather is associated with relatively mild respiratory infections, atopy, and autoimmunity. IgA might therefore also play covert roles, not uniquely associated with control of pathogens. We show that human IgA deficiency is not associated with massive quantitative perturbations of gut microbial ecology. Metagenomic analysis highlights an expected pathobiont expansion but a less expected depletion in some typically beneficial symbionts. Gut colonization by species usually present in the oropharynx is also reminiscent of spatial microbiota disorganization. IgM only partially rescues IgA deficiency because not all typical IgA targets are efficiently bound by IgM in the intestinal lumen. Together, IgA appears to play a nonredundant role at the forefront of the immune/microbial interface, away from the intestinal barrier, ranging from pathobiont control and regulation of systemic inflammation to preservation of commensal diversity and community networks. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  11. Immobilization of Bacillus amyloliquefaciens SP1 and its alkaline protease in various matrices for effective hydrolysis of casein.

    PubMed

    Guleria, Shiwani; Walia, Abhishek; Chauhan, Anjali; Shirkot, C K

    2016-12-01

    An extracellular alkaline protease producing B. amyloliquefaciens SP1 was isolated from apple rhizosphere having multifarious plant growth-promoting activities. B. amyloliquefaciens SP1 protease was immobilized using various concentrations of calcium alginate, agar and polyacrylamide to determine the optimum concentration for formation of the beads. Enzyme activity before immobilization (at 60 °C, pH 8.0 for 5 min) was 3580 µg/ml/min. The results of immobilization with various matrices revealed that 3 % calcium alginate (2829.92 µg/ml/min), 2 % agar (2600 µg/ml/min) and 10 % polyacrylamide (5698.99 µg/ml/min) were optimum concentrations for stable bead formation. Immobilized enzyme reusability results indicated that calcium alginate, agar and polyacrylamide beads retained 25.63, 22.05 and 34.04 % activity in their fifth repeated cycle, respectively. In cell immobilization technique, the free movement of microorganisms is restricted in the process, and a semi-continuous system of fermentation can be used. In the present work, this technique has been used for alkaline protease production using different matrices. Polyacrylamide (10 %) was found with the highest total alkaline protease titer, i.e., 24,847 µg/ml/min semi-continuously for 18 days as compared to agar (total enzyme titer: 5800 in 10 days) and calcium alginate (total enzyme titer: 13,010 in 15 days). This present study reported that polyacrylamide (10 %) among different matrices has maximum potential of immobilization of B. amyloliquefaciens SP1 and its detergent stable alkaline protease with effective application in bloodstain removal.

  12. IgA and neutralizing antibodies to influenza a virus in human milk: a randomized trial of antenatal influenza immunization.

    PubMed

    Schlaudecker, Elizabeth P; Steinhoff, Mark C; Omer, Saad B; McNeal, Monica M; Roy, Eliza; Arifeen, Shams E; Dodd, Caitlin N; Raqib, Rubhana; Breiman, Robert F; Zaman, K

    2013-01-01

    Antenatal immunization of mothers with influenza vaccine increases serum antibodies and reduces the rates of influenza illness in mothers and their infants. We report the effect of antenatal immunization on the levels of specific anti-influenza IgA levels in human breast milk. (ClinicalTrials.gov identifier NCT00142389; http://clinicaltrials.gov/ct2/show/NCT00142389). The Mother's Gift study was a prospective, blinded, randomized controlled trial that assigned 340 pregnant Bangladeshi mothers to receive either trivalent inactivated influenza vaccine, or 23-valent pneumococcal polysaccharide vaccine during the third trimester. We evaluated breast milk at birth, 6 weeks, 6 months, and 12 months, and serum at 10 weeks and 12 months. Milk and serum specimens from 57 subjects were assayed for specific IgA antibody to influenza A/New Caledonia (H1N1) using an enzyme-linked immunosorbent assay (ELISA) and a virus neutralization assay, and for total IgA using ELISA. Influenza-specific IgA levels in breast milk were significantly higher in influenza vaccinees than in pneumococcal controls for at least 6 months postpartum (p = 0.04). Geometric mean concentrations ranged from 8.0 to 91.1 ELISA units/ml in vaccinees, versus 2.3 to 13.7 ELISA units/mL in controls. Virus neutralization titers in milk were 1.2 to 3 fold greater in vaccinees, and correlated with influenza-specific IgA levels (r = 0.86). Greater exclusivity of breastfeeding in the first 6 months of life significantly decreased the expected number of respiratory illness with fever episodes in infants of influenza-vaccinated mothers (p = 0.0042) but not in infants of pneumococcal-vaccinated mothers (p = 0.4154). The sustained high levels of actively produced anti-influenza IgA in breast milk and the decreased infant episodes of respiratory illness with fever suggest that breastfeeding may provide local mucosal protection for the infant for at least 6 months. Studies are needed to determine the

  13. C1q Protein Binds to the Apoptotic Nucleolus and Causes C1 Protease Degradation of Nucleolar Proteins*

    PubMed Central

    Cai, Yitian; Teo, Boon Heng Dennis; Yeo, Joo Guan; Lu, Jinhua

    2015-01-01

    In infection, complement C1q recognizes pathogen-congregated antibodies and elicits complement activation. Among endogenous ligands, C1q binds to DNA and apoptotic cells, but whether C1q binds to nuclear DNA in apoptotic cells remains to be investigated. With UV irradiation-induced apoptosis, C1q initially bound to peripheral cellular regions in early apoptotic cells. By 6 h, binding concentrated in the nuclei to the nucleolus but not the chromatins. When nucleoli were isolated from non-apoptotic cells, C1q also bound to these structures. In vivo, C1q exists as the C1 complex (C1qC1r2C1s2), and C1q binding to ligands activates the C1r/C1s proteases. Incubation of nucleoli with C1 caused degradation of the nucleolar proteins nucleolin and nucleophosmin 1. This was inhibited by the C1 inhibitor. The nucleoli are abundant with autoantigens. C1q binding and C1r/C1s degradation of nucleolar antigens during cell apoptosis potentially reduces autoimmunity. These findings help us to understand why genetic C1q and C1r/C1s deficiencies cause systemic lupus erythematosus. PMID:26231209

  14. C1q protein binds to the apoptotic nucleolus and causes C1 protease degradation of nucleolar proteins.

    PubMed

    Cai, Yitian; Teo, Boon Heng Dennis; Yeo, Joo Guan; Lu, Jinhua

    2015-09-11

    In infection, complement C1q recognizes pathogen-congregated antibodies and elicits complement activation. Among endogenous ligands, C1q binds to DNA and apoptotic cells, but whether C1q binds to nuclear DNA in apoptotic cells remains to be investigated. With UV irradiation-induced apoptosis, C1q initially bound to peripheral cellular regions in early apoptotic cells. By 6 h, binding concentrated in the nuclei to the nucleolus but not the chromatins. When nucleoli were isolated from non-apoptotic cells, C1q also bound to these structures. In vivo, C1q exists as the C1 complex (C1qC1r2C1s2), and C1q binding to ligands activates the C1r/C1s proteases. Incubation of nucleoli with C1 caused degradation of the nucleolar proteins nucleolin and nucleophosmin 1. This was inhibited by the C1 inhibitor. The nucleoli are abundant with autoantigens. C1q binding and C1r/C1s degradation of nucleolar antigens during cell apoptosis potentially reduces autoimmunity. These findings help us to understand why genetic C1q and C1r/C1s deficiencies cause systemic lupus erythematosus. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Purification, Cloning and Immuno-Biochemical Characterization of a Fungal Aspartic Protease Allergen Rhi o 1 from the Airborne Mold Rhizopus oryzae

    PubMed Central

    Sircar, Gaurab; Saha, Bodhisattwa; Mandal, Rahul Shubhra; Pandey, Naren; Saha, Sudipto; Gupta Bhattacharya, Swati

    2015-01-01

    Background Fungal allergy is considered as serious health problem worldwide and is increasing at an alarming rate in the industrialized areas. Rhizopus oyzae is a ubiquitously present airborne pathogenic mold and an important source of inhalant allergens for the atopic population of India. Here, we report the biochemical and immunological features of its 44 kDa sero-reactive aspartic protease allergen, which is given the official designation ‘Rhi o 1’. Method The natural Rhi o 1 was purified by sequential column chromatography and its amino acid sequence was determined by mass spectrometry and N-terminal sequencing. Based on its amino acid sequence, the cDNA sequence was identified, cloned and expressed to produce recombinant Rhi o 1. The allergenic activity of rRhi o 1 was assessed by means of its IgE reactivity and histamine release ability. The biochemical property of Rhi o 1 was studied by enzyme assay. IgE-inhibition experiments were performed to identify its cross-reactivity with the German cockroach aspartic protease allergen Bla g 2. For precise characterization of the cross-reactive epitope, we used anti-Bla g 2 monoclonal antibodies for their antigenic specificity towards Rhi o 1. A homology based model of Rhi o 1 was built and mapping of the cross-reactive conformational epitope was done using certain in silico structural studies. Results The purified natural nRhi o 1 was identified as an endopeptidase. The full length allergen cDNA was expressed and purified as recombinant rRhi o 1. Purified rRhi o 1 displayed complete allergenicity similar to the native nRhi o 1. It was recognized by the serum IgE of the selected mold allergy patients and efficiently induced histamine release from the sensitized PBMC cells. This allergen was identified as an active aspartic protease functional in low pH. The Rhi o 1 showed cross reactivity with the cockroach allergen Bla g 2, as it can inhibit IgE binding to rBla g 2 up to certain level. The rBla g 2 was also found

  16. Singlet Oxygen-Induced Membrane Disruption and Serpin-Protease Balance in Vacuolar-Driven Cell Death1[OPEN

    PubMed Central

    Carmieli, Raanan; Mor, Avishai; Fluhr, Robert

    2016-01-01

    Singlet oxygen plays a role in cellular stress either by providing direct toxicity or through signaling to initiate death programs. It was therefore of interest to examine cell death, as occurs in Arabidopsis, due to differentially localized singlet oxygen photosensitizers. The photosensitizers rose bengal (RB) and acridine orange (AO) were localized to the plasmalemma and vacuole, respectively. Their photoactivation led to cell death as measured by ion leakage. Cell death could be inhibited by the singlet oxygen scavenger histidine in treatments with AO but not with RB. In the case of AO treatment, the vacuolar membrane was observed to disintegrate. Concomitantly, a complex was formed between a vacuolar cell-death protease, RESPONSIVE TO DESSICATION-21 and its cognate cytoplasmic protease inhibitor ATSERPIN1. In the case of RB treatment, the tonoplast remained intact and no complex was formed. Over-expression of AtSerpin1 repressed cell death, only under AO photodynamic treatment. Interestingly, acute water stress showed accumulation of singlet oxygen as determined by fluorescence of Singlet Oxygen Sensor Green, by electron paramagnetic resonance spectroscopy and the induction of singlet oxygen marker genes. Cell death by acute water stress was inhibited by the singlet oxygen scavenger histidine and was accompanied by vacuolar collapse and the appearance of serpin-protease complex. Over-expression of AtSerpin1 also attenuated cell death under this mode of cell stress. Thus, acute water stress damage shows parallels to vacuole-mediated cell death where the generation of singlet oxygen may play a role. PMID:26884487

  17. Natural polyreactive IgA antibodies coat the intestinal microbiota

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Bunker, Jeffrey J.; Erickson, Steven A.; Flynn, Theodore M.

    Large quantities of immunoglobulin A (IgA) are constitutively secreted by intestinal plasma cells to coat and contain the commensal microbiota, yet the specificity of these antibodies remains elusive. In this paper, we profiled the reactivities of single murine IgA plasma cells by cloning and characterizing large numbers of monoclonal antibodies. IgAs were not specific to individual bacterial taxa but rather polyreactive, with broad reactivity to a diverse, but defined, subset of microbiota. These antibodies arose at low frequencies among naïve B cells and were selected into the IgA repertoire upon recirculation in Peyer’s patches. This selection process occurred independent ofmore » microbiota or dietary antigens. Furthermore, although some IgAs acquired somatic mutations, these did not substantially influence their reactivity. In conclusion, these findings reveal an endogenous mechanism driving homeostatic production of polyreactive IgAs with innate specificity to microbiota.« less

  18. Natural polyreactive IgA antibodies coat the intestinal microbiota

    DOE PAGES

    Bunker, Jeffrey J.; Erickson, Steven A.; Flynn, Theodore M.; ...

    2017-09-28

    Large quantities of immunoglobulin A (IgA) are constitutively secreted by intestinal plasma cells to coat and contain the commensal microbiota, yet the specificity of these antibodies remains elusive. In this paper, we profiled the reactivities of single murine IgA plasma cells by cloning and characterizing large numbers of monoclonal antibodies. IgAs were not specific to individual bacterial taxa but rather polyreactive, with broad reactivity to a diverse, but defined, subset of microbiota. These antibodies arose at low frequencies among naïve B cells and were selected into the IgA repertoire upon recirculation in Peyer’s patches. This selection process occurred independent ofmore » microbiota or dietary antigens. Furthermore, although some IgAs acquired somatic mutations, these did not substantially influence their reactivity. In conclusion, these findings reveal an endogenous mechanism driving homeostatic production of polyreactive IgAs with innate specificity to microbiota.« less

  19. Engineering Human Immunodeficiency Virus 1 Protease Heterodimers as Macromolecular Inhibitors of Viral Maturation

    NASA Astrophysics Data System (ADS)

    McPhee, Fiona; Good, Andrew C.; Kuntz, Irwin D.; Craik, Charles S.

    1996-10-01

    Dimerization of human immunodeficiency virus type 1 protease (HIV-1 PR) monomers is an essential prerequisite for viral proteolytic activity and the subsequent generation of infectious virus particles. Disruption of the dimer interface inhibits this activity as does formation of heterodimers between wild-type and defective monomers. A structure-based approach was used to identify amino acid substitutions at the dimer interface of HIV-1 PR that facilitate preferential association of heterodimers and inhibit self-association of the defective monomers. Expression of the designed PR monomers inhibits activity of wild-type HIV-1 PR and viral infectivity when assayed in an ex vivo model system. These results show that it is possible to design PR monomers as macromolecular inhibitors that may provide an alternative to small molecule inhibitors for the treatment of HIV infection.

  20. Flap Conformations in HIV-1 Protease are Altered by Mutations

    NASA Astrophysics Data System (ADS)

    Fanucci, Gail; Blackburn, Mandy; Veloro, Angelo; Galiano, Luis; Fangu, Ding; Simmerling, Carlos

    2009-03-01

    HIV-1 protease (PR) is an enzyme that is a major drug target in the treatment of AIDS. Although the structure and function of HIV-1 PR have been studied for over 20 years, questions remain regarding the conformations and dynamics of the β-hairpin turns (flaps) that cover the active site cavity. Distance measurements with pulsed EPR spectroscopy of spin labeled constructs of HIV-1 PR have been used to characterize the flap conformations in the apo and inhibitor bound states. From the most probably distances and the breadth of the distance distribution profiles from analysis of the EPR data, insights regarding the flap conformations and flexibility are gained. The EPR results clearly show how drug pressure selected mutations alter the average conformation of the flaps and the degree of opening of the flaps. Molecular dynamics simulations successfully regenerate the experimentally determined distance distribution profiles, and more importantly, provide structural models for full interpretation of the EPR results. By combining experiment and theory to understand the role that altered flap flexibility/conformations play in the mechanism of drug resistance, key insights are gained toward the rational development of new inhibitors of this important enzyme.

  1. HIV-1 Protease Function and Structure Studies with the Simplicial Neighborhood Analysis of Protein Packing (SNAPP) Method

    PubMed Central

    Zhang, Shuxing; Kaplan, Andrew H.; Tropsha, Alexander

    2009-01-01

    The Simplicial Neighborhood Analysis of Protein Packing (SNAPP) method was used to predict the effect of mutagenesis on the enzymatic activity of the HIV-1 protease (HIVP). SNAPP relies on a four-body statistical scoring function derived from the analysis of spatially nearest neighbor residue compositional preferences in a diverse and representative subset of protein structures from the Protein Data Bank. The method was applied to the analysis of HIVP mutants with residue substitutions in the hydrophobic core as well as at the interface between the two protease monomers. Both wild type and tethered structures were employed in the calculations. We obtained a strong correlation, with R2 as high as 0.96, between ΔSNAPP score (i.e., the difference in SNAPP scores between wild type and mutant proteins) and the protease catalytic activity for tethered structures. A weaker but significant correlation was also obtained for non-tethered structures as well. Our analysis identified residues both in the hydrophobic core and at the dimeric interface (DI) that are very important for the protease function. This study demonstrates a potential utility of the SNAPP method for rational design of mutagenesis studies and protein engineering. PMID:18498108

  2. Fine Mapping Implicates a Deletion of CFHR1 and CFHR3 in Protection from IgA Nephropathy in Han Chinese

    PubMed Central

    Xie, Jingyuan; Kiryluk, Krzysztof; Li, Yifu; Mladkova, Nikol; Zhu, Li; Hou, Ping; Ren, Hong; Wang, Weiming; Zhang, Hong; Chen, Nan

    2016-01-01

    An intronic variant at the complement factor H (CFH) gene on chromosome 1q32 (rs6677604) associates with risk of IgA nephropathy (IgAN), but the association signal has not been uniformly replicated in Han Chinese populations. We investigated whether the causal sequence variant resides in the CFH gene or the neighboring complement factor H–related 1 (CFHR1) gene and CFHR3, which harbor an 84-kb combined deletion (CFHR3,1Δ) in linkage disequilibrium with rs6677604. Imputation of 1000 Genomes Project data did not suggest new causal single–nucleotide variants within the CFH cluster. We next performed copy number analysis across the CFH locus in two independent Han Chinese case-control cohorts (combined n=3581). The CFHR3,1Δ and rs6677604-A alleles were rare (4.4% in patients and 7.1% in controls) and in strong linkage disequilibrium with each other (r2=0.95); of these alleles, CFHR3,1Δ associated more significantly with decreased risk of IgAN (odds ratio [OR], 0.56; 95% confidence interval [95% CI], 0.46 to 0.70; P=8.5 × 10−8 versus OR, 0.61; 95% CI, 0.50 to 0.75; P=1.6 × 10−6 for rs6677604-A). Moreover, CFHR3,1Δ explained all of the association signal at rs6677604 and remained significant after conditioning on rs6677604 genotype (P=0.01). Exploratory analyses of clinical and histopathologic parameters using the Oxford classification criteria revealed a suggestive association of CFHR3,1Δ with reduced tubulointerstitial injury (OR, 0.46; 95% CI, 0.25 to 0.79). These data indicate that dysregulated activity of the alternative complement pathway contributes to IgAN pathogenesis in both Asians and Europeans and implicate CFHR3,1Δ as the functional allele at this locus. PMID:26940089

  3. Protection of scaffold protein Isu from degradation by the Lon protease Pim1 as a component of Fe-S cluster biogenesis regulation.

    PubMed

    Ciesielski, Szymon J; Schilke, Brenda; Marszalek, Jaroslaw; Craig, Elizabeth A

    2016-04-01

    Iron-sulfur (Fe-S) clusters, essential protein cofactors, are assembled on the mitochondrial scaffold protein Isu and then transferred to recipient proteins via a multistep process in which Isu interacts sequentially with multiple protein factors. This pathway is in part regulated posttranslationally by modulation of the degradation of Isu, whose abundance increases >10-fold upon perturbation of the biogenesis process. We tested a model in which direct interaction with protein partners protects Isu from degradation by the mitochondrial Lon-type protease. Using purified components, we demonstrated that Isu is indeed a substrate of the Lon-type protease and that it is protected from degradation by Nfs1, the sulfur donor for Fe-S cluster assembly, as well as by Jac1, the J-protein Hsp70 cochaperone that functions in cluster transfer from Isu. Nfs1 and Jac1 variants known to be defective in interaction with Isu were also defective in protecting Isu from degradation. Furthermore, overproduction of Jac1 protected Isu from degradation in vivo, as did Nfs1. Taken together, our results lead to a model of dynamic interplay between a protease and protein factors throughout the Fe-S cluster assembly and transfer process, leading to up-regulation of Isu levels under conditions when Fe-S cluster biogenesis does not meet cellular demands. © 2016 Ciesielski et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  4. Evaluation of salivary glucose, IgA and flow rate in diabetic patients: a case-control study.

    PubMed

    Bakianian Vaziri, P; Vahedi, M; Mortazavi, H; Abdollahzadeh, Sh; Hajilooi, M

    2010-01-01

    An association between diabetes mellitus and alterations in the oral cavity has been noted. In this study, we evaluated differences between salivary IgA, glucose and flow rate in diabetic patients compared with healthy controls. Forty patients with type 1 diabetes, 40 patients with type 2 diabetes and 40 healthy controls were selected. Whole unstimulated saliva samples were collected by the standard method and the salivary flow rate was determined. Nephelometric and Pars method were used to measure salivary IgA and salivary glucose concentrations, respectively. Statistical analysis was performed by Chi-square and t test. There were no significant differences in salivary IgA and glucose concentrations between type 1 and type 2 diabetic patients and their matched control subjects (P>0.05). Salivary flow rate was significantly lower in diabetic patients (P<0.05). In addition, DMFT was higher in diabetic patients than the controls. Determination of salivary constituents may be useful in the description and management of oral findings in diabetic patients.

  5. IgA antibodies against endomysium and transglutaminase: a comparison of methods.

    PubMed

    Jaskowski, T D; Schroder, C; Martins, T B; Litwin, C M; Hill, H R

    2001-01-01

    Recently, the endomysial antigen has been identified as the protein cross-linking enzyme known as tissue transglutaminase (tTG). Our objective was to compare a novel enzyme immunoassay (EIA) that detects IgA antibody against tTG to two standard IFA methods utilizing thin tissue sections of rat kidney/rat stomach (KS) and distal primate esophagus (PE) as substrates to detect IgA antibody against endomysium (EMA). Sera from 100 patients suspected of having gluten-sensitive enteropathy (GSE) and 23 sera possessing various antibodies used for EIA cross-reactivity studies were included. Additional tests, performed routinely in our laboratory, were utilized to further assess sera from patients suspected having GSE. These tests include anti-gliadin IgA antibody (AGA) and anti-reticulin IgA antibody (ARA) and are part of the European Society for Pediatric Gastroenterology and Nutrition (ESPGAN) revised criteria for diagnosing GSE. When compared to IFA using KS, the tTG EIA had a sensitivity of 87.5%, was 97.1% specific, and had an overall agreement of 94.0%. When compared to IFA using PE, the tTG EIA had a sensitivity of 92.6%, was 93.2% specific, and had an overall agreement of 93.0%. When the KS IFA was compared to the PE IFA for EMA, the KS IFA had a sensitivity of 96.3%, was 91.8% specific, and had an overall agreement of 93.0%. The majority of sera that were positive for tTG but were negative by IFA (KS, n = 2/PE, n = 5) possessed IgA antibodies against gliadin and/or reticulin. Five of six sera with negative results by PE IFA were positive by the KS IFA and possessed one or more antibodies to tTG and/or gliadin and/or reticulin. We conclude that the tTG EIA compares well to both KS and PE IFAs when detecting IgA antibody against endomysium. We do not recommend the use of PE to detect EMA primarily because of the inconsistencies (i.e., tissue selection, quality, and preparation) and limited availability of commercially prepared PE tissue.

  6. Monitoring the impact of an aspartic protease (MpAPr1) on grape proteins and wine properties.

    PubMed

    Theron, Louwrens Wiid; Bely, Marina; Divol, Benoit

    2018-04-23

    The perception of haze in wine is brought about when pathogenesis-related proteins become unstable and aggregate, subsequently resulting in crosslinking until it develops into light-dispersing particles. Elimination of these proteins is usually achieved via bentonite fining, which, although effective, suffers from several drawbacks. The utilization of proteases has been proposed as an ideal alternative. In a previous study, an aspartic protease (MpAPr1) from the yeast Metschnikowia pulcherrima was purified and shown to be partially active against grape proteins in synthetic medium. In this study, the effects of pure MpAPr1 supplemented to Sauvignon Blanc juice on subsequent fermentation were investigated. The juice was incubated for 48 h and thereafter inoculated with Saccharomyces cerevisiae. Results revealed that the enzyme had no observable effects on fermentation performance and retained activity throughout. Protein degradation could be detected and resulted in a significant modification of the wine composition and an increase in the presence of certain volatile compounds, especially those linked to amino acid metabolism.

  7. Anti-actin IgA antibodies in severe coeliac disease

    PubMed Central

    Granito, A; Muratori, P; Cassani, F; Pappas, G; Muratori, L; Agostinelli, D; Veronesi, L; Bortolotti, R; Petrolini, N; Bianchi, F B; Volta, U

    2004-01-01

    Anti-actin IgA antibodies have been found in sera of coeliacs. Our aim was to define the prevalence and clinical significance of anti-actin IgA in coeliacs before and after gluten withdrawal. One hundred and two biopsy-proven coeliacs, 95 disease controls and 50 blood donors were studied. Anti-actin IgA were evaluated by different methods: (a) antimicrofilament positivity on HEp-2 cells and on cultured fibroblasts by immunofluorescence; (b) anti-actin positivity by enzyme-linked immuosorbent assay (ELISA); and (c) presence of the tubular/glomerular pattern of anti-smooth muscle antibodies on rat kidney sections by immunofluorescence. Antimicrofilament IgA were present in 27% of coeliacs and in none of the controls. Antimicrofilament antibodies were found in 25 of 54 (46%) coeliacs with severe villous atrophy and in three of 48 (6%) with mild damage (P < 0·0001). In the 20 patients tested, antimicrofilaments IgA disappeared after gluten withdrawal in accordance with histological recovery. Our study shows a significant correlation between antimicrofilament IgA and the severity of intestinal damage in untreated coeliacs. The disappearance of antimicrofilament IgA after gluten withdrawal predicts the normalization of intestinal mucosa and could be considered a useful tool in the follow-up of severe coeliac disease. PMID:15270857

  8. Engineering human immunodeficiency virus 1 protease heterodimers as macromolecular inhibitors of viral maturation.

    PubMed Central

    McPhee, F; Good, A C; Kuntz, I D; Craik, C S

    1996-01-01

    Dimerization of human immunodeficiency virus type 1 protease (HIV-1 PR) monomers is an essential prerequisite for viral proteolytic activity and the subsequent generation of infectious virus particles. Disruption of the dimer interface inhibits this activity as does formation of heterodimers between wild-type and defective monomers. A structure-based approach was used to identify amino acid substitutions at the dimer interface of HIV-1 PR that facilitate preferential association of heterodimers and inhibit self-association of the defective monomers. Expression of the designed PR monomers inhibits activity of wild-type HIV-1 PR and viral infectivity when assayed in an ex vivo model system. These results show that it is possible to design PR monomers as macromolecular inhibitors that may provide an alternative to small molecule inhibitors for the treatment of HIV infection. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8876160

  9. Scarcity of autoreactive human blood IgA+ memory B cells

    PubMed Central

    Prigent, Julie; Lorin, Valérie; Kök, Ayrin; Hieu, Thierry; Bourgeau, Salomé

    2016-01-01

    Class‐switched memory B cells are key components of the “reactive” humoral immunity, which ensures a fast and massive secretion of high‐affinity antigen‐specific antibodies upon antigenic challenge. In humans, IgA class‐switched (IgA+) memory B cells and IgA antibodies are abundant in the blood. Although circulating IgA+ memory B cells and their corresponding secreted immunoglobulins likely possess major protective and/or regulatory immune roles, little is known about their specificity and function. Here, we show that IgA+ and IgG+ memory B‐cell antibodies cloned from the same healthy humans share common immunoglobulin gene features. IgA and IgG memory antibodies have comparable lack of reactivity to vaccines, common mucosa‐tropic viruses and commensal bacteria. However, the IgA+ memory B‐cell compartment contains fewer polyreactive clones and importantly, only rare self‐reactive clones compared to IgG+ memory B cells. Self‐reactivity of IgAs is acquired following B‐cell affinity maturation but not antibody class switching. Together, our data suggest the existence of different regulatory mechanisms for removing autoreactive clones from the IgG+ and IgA+ memory B‐cell repertoires, and/or different maturation pathways potentially reflecting the distinct nature and localization of the cognate antigens recognized by individual B‐cell populations. PMID:27469325

  10. Optimization studies on production of a salt-tolerant protease from Pseudomonas aeruginosa strain BC1 and its application on tannery saline wastewater treatment

    PubMed Central

    Sivaprakasam, Senthilkumar; Dhandapani, Balaji; Mahadevan, Surianarayanan

    2011-01-01

    Treatment and safe disposal of tannery saline wastewater, a primary effluent stream that is generated by soaking salt-laden hides and skin is one of the major problems faced by the leather manufacturing industries. Conventional treatment methods like solar evaporation ponds and land composting are not eco-friendly as they deteriorate the ground water quality. Though, this waste stream is comprised of high concentration of dissolved proteins the presence of high salinity (1–6 % NaCl by wt) makes it non-biodegradable. Enzymatic treatment is one of the positive alternatives for management of such kind of waste streams. A novel salt-tolerant alkaline protease obtained from P.aeruginosa (isolated from tannery saline wastewater) was used for enzymatic degradation studies. The effect of various physical factors including pH, temperature, incubation time, protein source and salinity on the activity of identified protease were investigated. Kinetic parameters (Km , Vmax) were calculated for the identified alkaline protease at varying substrate concentrations. Tannery saline wastewater treated with identified salt tolerant protease showed 75 % protein removal at 6 h duration and 2 % (v/v) protease addition was found to be the optimum dosage value. PMID:24031785

  11. Evaluation of pre-rigor injection of beef with proteases on cooked meat volatile profile after 1 day and 21 days post-mortem storage.

    PubMed

    Ma, Q L; Hamid, N; Bekhit, A E D; Robertson, J; Law, T F

    2012-12-01

    This research was carried out to determine the effects of pre-rigor injection of beef semimembranosus muscle with nine proteases from plant and microbial sources, on the volatile profile of cooked beef after 1 day and 21 days post-mortem (PM) storage using Solid-phase microextraction gas chromatography mass spectrometry analysis. A total of 23 aldehydes, 5 ketones, 3 furans, 8 nitrogen and sulphur compounds, 4 alkanes, 7 alcohols and 6 terpenes were detected. Eleven volatile compounds characteristic of ginger flavour were detected in zingibain-treated meat. Benzaldehyde significantly increased (p<0.05) only in kiwifruit juice (KJ), fungal 31 protease and Asparagus protease (ASP) treated samples from 1 day to 21 days PM storage. A significant increase (p<0.05) in 3-methylbutanal was observed in KJ, bacterial and fungal protease treated samples at 21 days PM storage. Treatments with bromelain, papain, ASP, actinidin, and KJ (except KJ 21 days) proteases resulted in flavour profiles closer to that of the control beef sample. Copyright © 2012 Elsevier Ltd. All rights reserved.

  12. Assessment of the value of detecting specific IgA antibodies for the diagnosis of a recently acquired primary Toxoplasma infection.

    PubMed

    Nascimento, Fernanda Santos; Suzuki, Lisandra Akemi; Rossi, Cláudio Lúcio

    2008-08-01

    To assess the value of detecting IgA antibodies for the diagnosis of a recently acquired primary Toxoplasma infection. IgA antibodies were screened in sera from 87 women with different serological profiles of Toxoplasma gondii IgM and IgG antibodies and Toxoplasma-specific IgG avidity. The IgM and IgG antibodies and the IgG avidity were measured with an automated Vitek Immuno Diagnostic Assay System (VIDAS). Anti-T.gondii IgA was measured with Platelia Toxo IgA TMB kits. All 12 sera obtained from women with clinical and/or serological evidence of a recently acquired Toxoplasma infection were positive for IgA. In 42 serum samples obtained more than 6 months after T. gondii infection from women with no clinical evidence of infection, but who had a positive IgM test and a high IgG avidity index, the IgA-enzyme linked immunosorbent assay (ELISA) test results were positive, negative, and doubtful in 16 (38.1%), 23 (54.8%), and 3 (7.1%) sera, respectively. In eight women, IgA was detected in sera collected more than 9 months after the onset of infection. The IgA test result was also positive in 11 of 12 sera (91.7%) obtained from women with no clinical evidence of toxoplasmosis, but who had a positive IgM test and a borderline IgG avidity index. The IgA-ELISA was negative in 21 sera obtained more than 2 years after the onset of T. gondii infection from women with no clinical evidence of toxoplasmosis, but who had a negative IgM test and a positive IgG test. These results show that IgA is not a dependable marker for a recently acquired primary Toxoplasma infection. Copyright (c) 2008 John Wiley & Sons, Ltd.

  13. Linear IgA bullous dermatosis in a neonate.

    PubMed

    Hruza, L L; Mallory, S B; Fitzgibbons, J; Mallory, G B

    1993-06-01

    A newborn black boy had two facial blisters at birth that progressed to bullous lesions over the trunk, genitals, extremities, and oral and tracheal mucosa. A biopsy specimen demonstrated a subepidermal bulla with mixed eosinophilic and neutrophilic, inflammatory infiltrate. Direct immunofluorescence showed linear IgA, IgG, and C3 depositions along the basement membrane zone, consistent with a diagnosis of childhood linear IgA bullous dermatosis (chronic bullous dermatosis of childhood). The skin disease was controlled with combined prednisone and dapsone. This is the youngest reported patient with the disease. Linear IgA bullous dermatosis should be considered in the differential diagnosis of blistering diseases of the newborn, and immunofluorescence should be performed on a skin biopsy specimen.

  14. A sensitive assay using a native protein substrate for screening HIV-1 maturation inhibitors targeting the protease cleavage site between the matrix and capsid.

    PubMed

    Lee, Sook-Kyung; Cheng, Nancy; Hull-Ryde, Emily; Potempa, Marc; Schiffer, Celia A; Janzen, William; Swanstrom, Ronald

    2013-07-23

    The matrix/capsid processing site in the HIV-1 Gag precursor is likely the most sensitive target to inhibit HIV-1 replication. We have previously shown that modest incomplete processing at the site leads to a complete loss of virion infectivity. In the study presented here, a sensitive assay based on fluorescence polarization that can monitor cleavage at the MA/CA site in the context of the folded protein substrate is described. The substrate, an MA/CA fusion protein, was labeled with the fluorescein-based FlAsH (fluorescein arsenical hairpin) reagent that binds to a tetracysteine motif (CCGPCC) that was introduced within the N-terminal domain of CA. By limiting the size of CA and increasing the size of MA (with an N-terminal GST fusion), we were able to measure significant differences in polarization values as a function of HIV-1 protease cleavage. The sensitivity of the assay was tested in the presence of increasing amounts of an HIV-1 protease inhibitor, which resulted in a gradual decrease in the fluorescence polarization values demonstrating that the assay is sensitive in discerning changes in protease processing. The high-throughput screening assay validation in 384-well plates showed that the assay is reproducible and robust with an average Z' value of 0.79 and average coefficient of variation values of <3%. The robustness and reproducibility of the assay were further validated using the LOPAC(1280) compound library, demonstrating that the assay provides a sensitive high-throughput screening platform that can be used with large compound libraries for identifying novel maturation inhibitors targeting the MA/CA site of the HIV-1 Gag polyprotein.

  15. Predominate HIV1-specific IgG activity in various mucosal compartments of HIV1-infected individuals.

    PubMed

    Lü, F X

    2000-10-01

    Evaluating mucosal humoral immunity is important for understanding local immunity induced by HIV infection or vaccination and designing prophylactic strategies. To characterize the mucosal humoral immunity following HIV infection, the levels of immunoglobulins (Igs), antibodies (Abs), and HIV1-specific Ab activity were evaluated in cervicovaginal secretions (CVS), saliva, breast milk, and sera of HIV-infected individuals. HIV1-specific IgG activity was significantly higher than that of IgA in CVS, saliva, and breast milk. The highest HIV1-specific IgG activity was found in breast milk. The data suggest that anti-HIV1 Abs in CVS were most likely serum derived. However, HIV1-specific Abs in saliva and breast milk were mainly locally produced. The prevalence of HIV1-specific Abs in seropositive subjects was 97% for IgG and 95% for IgA in CVS, 100% for IgG and 80% for IgA in saliva, and 59% for IgG and 94% for IgA in breast milk. These data provide evidence for both a better understanding of the nature of humoral mucosal responses after HIV1 infection and the development of strategies to induce desirable functional mucosal immunity for preventing HIV transmission. Copyright 2000 Academic Press.

  16. A rare case of Addison's disease, hepatitis, thyreoiditis, positive IgG anti-tissue transglutaminase antibodies and partial IgA deficiency.

    PubMed

    Baleva, Marta P; Mihaylova, Snejina; Yankova, Petja; Atanasova, Iliana; Nikolova-Vlahova, Milena; Naumova, Elissaveta

    2016-01-01

    Selective IgA deficiency (IgAD) is the most prevalent type of primary immune deficiencies, but partial IgA deficiency is even more common. Addison's disease is a rare condition associated with primary adrenal insufficiency due to infection or autoimmune destruction of the adrenals. The association between IgA deficiency and Addison's disease is very rare. We observed a 22-year-old male patient with marked darkening of the skin, especially on the palms and areolae, jaundice on the skin and sclera, astheno-adynamia, hypotension (80/50 mm Hg), and pain in the right hypochondrium. The laboratory investigations revealed increased serum levels of total and indirect bilirubin, AST, ALT, GGT and LDH, negative HBsAg, anti-HBc IgM, anti-HCV and anti-HAV IgM, very low serum IgA levels (0.16 g/l) with normal IgG and IgM, negative ANA, ANCA, AMA, LKM-1, anti-GAD-60, anti-IA-2, anti-thyroglobulin antibodies, a mild increase in anti-TPO antibodies titer, a marked increase in IgG anti-tissue transglutaminase antibodies, with no typical changes in cellular immunity, negative T-SPOT-TB test, HLA - A*01; B*08; DRB1*03; DQB1*02, karyotype - 46, XY. We present a rare case of partial IgA deficiency with Addison's disease, hepatitis, thyroiditis and positive anti-tissue transglutaminase antibodies. IgAD and some autoimmune disorders share several predisposing HLA genes, thus explaining the increased prevalence of IgAD in certain patient groups.

  17. Shear stress reduces protease activated receptor-1 expression in human endothelial cells

    NASA Technical Reports Server (NTRS)

    Nguyen, K. T.; Eskin, S. G.; Patterson, C.; Runge, M. S.; McIntire, L. V.

    2001-01-01

    Shear stress has been shown to regulate several genes involved in the thrombotic and proliferative functions of endothelial cells. Thrombin receptor (protease-activated receptor-1: PAR-1) increases at sites of vascular injury, which suggests an important role for PAR-1 in vascular diseases. However, the effect of shear stress on PAR-1 expression has not been previously studied. This work investigates effects of shear stress on PAR-1 gene expression in both human umbilical vein endothelial cells (HUVECs) and microvascular endothelial cells (HMECs). Cells were exposed to different shear stresses using a parallel plate flow system. Northern blot and flow cytometry analysis showed that shear stress down-regulated PAR-1 messenger RNA (mRNA) and protein levels in both HUVECs and HMECs but with different thresholds. Furthermore, shear-reduced PAR-1 mRNA was due to a decrease of transcription rate, not increased mRNA degradation. Postshear stress release of endothelin-1 in response to thrombin was reduced in HUVECs and HMECs. Moreover, inhibitors of potential signaling pathways applied during shear stress indicated mediation of the shear-decreased PAR-1 expression by protein kinases. In conclusion, shear stress exposure reduces PAR-1 gene expression in HMECs and HUVECs through a mechanism dependent in part on protein kinases, leading to altered endothelial cell functional responses to thrombin.

  18. Binding of novel fullerene inhibitors to HIV-1 protease: insight through molecular dynamics and molecular mechanics Poisson-Boltzmann surface area calculations

    NASA Astrophysics Data System (ADS)

    Tzoupis, Haralambos; Leonis, Georgios; Durdagi, Serdar; Mouchlis, Varnavas; Mavromoustakos, Thomas; Papadopoulos, Manthos G.

    2011-10-01

    The objectives of this study include the design of a series of novel fullerene-based inhibitors for HIV-1 protease (HIV-1 PR), by employing two strategies that can also be applied to the design of inhibitors for any other target. Additionally, the interactions which contribute to the observed exceptionally high binding free energies were analyzed. In particular, we investigated: (1) hydrogen bonding (H-bond) interactions between specific fullerene derivatives and the protease, (2) the regions of HIV-1 PR that play a significant role in binding, (3) protease changes upon binding and (4) various contributions to the binding free energy, in order to identify the most significant of them. This study has been performed by employing a docking technique, two 3D-QSAR models, molecular dynamics (MD) simulations and the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method. Our computed binding free energies are in satisfactory agreement with the experimental results. The suitability of specific fullerene derivatives as drug candidates was further enhanced, after ADMET (absorption, distribution, metabolism, excretion and toxicity) properties have been estimated to be promising. The outcomes of this study revealed important protein-ligand interaction patterns that may lead towards the development of novel, potent HIV-1 PR inhibitors.

  19. Drug-resistant molecular mechanism of CRF01_AE HIV-1 protease due to V82F mutation

    NASA Astrophysics Data System (ADS)

    Liu, Xiaoqing; Xiu, Zhilong; Hao, Ce

    2009-05-01

    Human immunodeficiency virus type 1 protease (HIV-1 PR) is one of the major targets of anti-AIDS drug discovery. The circulating recombinant form 01 A/E (CRF01_AE, abbreviated AE) subtype is one of the most common HIV-1 subtypes, which is infecting more humans and is expanding rapidly throughout the world. It is, therefore, necessary to develop inhibitors against subtype AE HIV-1 PR. In this work, we have performed computer simulation of subtype AE HIV-1 PR with the drugs lopinavir (LPV) and nelfinavir (NFV), and examined the mechanism of resistance of the V82F mutation of this protease against LPV both structurally and energetically. The V82F mutation at the active site results in a conformational change of 79's loop region and displacement of LPV from its proper binding site, and these changes lead to rotation of the side-chains of residues D25 and I50'. Consequently, the conformation of the binding cavity is deformed asymmetrically and some interactions between PR and LPV are destroyed. Additionally, by comparing the interactive mechanisms of LPV and NFV with HIV-1 PR we discovered that the presence of a dodecahydroisoquinoline ring at the P1' subsite, a [2-(2,6-dimethylphenoxy)acetyl]amino group at the P2' subsite, and an N2 atom at the P2 subsite could improve the binding affinity of the drug with AE HIV-1 PR. These findings are helpful for promising drug design.

  20. Can foreign proteins imported into yeast mitochondria interfere with PIM1p protease and/or chaperone function?

    PubMed

    Saveliev, A S; Kovaleva, I E; Novikova, L A; Isaeva, L V; Luzikov, V N

    1999-03-15

    When studying the fate of mammalian apocytochrome P450scc (apo-P450scc) imported in small amounts into isolated yeast mitochondria, we found that it undergoes degradation, this process being retarded if recipient mitochondria are preloaded in vivo (to about 0.2% of total organelle protein) with a fusion protein composed of mammalian adrenodoxin reductase and adrenodoxin (AdR-Ad); in parallel we observed aggregation of apo-P450scc. These effects suggest some overload of Pim1p protease and/or mtHsp70 system by AdR-Ad, as both of them are involved in the degradation of apo-P450scc (see Savel'ev et al. J. Biol. Chem. 273, 20596-20602, 1998). However, under the same conditions AdR-Ad was not able to impede the import of proteins into mitochondria and the development of the mitochondrial respiratory machinery in yeast, the processes requiring the mtHsp70 system and Pim1p, respectively. These data imply that chaperones and Pim1p protease prefer their natural targets in mitochondria to imported foreign proteins. Copyright 1999 Academic Press.

  1. Detection of IgA-class circulating immune complexes (CIC) in sera from patients with IgA nephropathy using a solid-phase anti-C3 Facb enzyme immunoassay (EIA).

    PubMed Central

    Yagame, M; Tomino, Y; Miura, M; Tanigaki, T; Suga, T; Nomoto, Y; Sakai, H

    1987-01-01

    The detection of circulating immune complexes (CIC) in sera from patients with IgA nephropathy is described. A solid-phase anti-C3 Facb enzyme immunoassay (EIA) was employed for detection of IgA-, IgG- and IgM-CIC in sera. The C1q-binding enzyme assay was also used for the detection of CIC in sera from these patients and healthy adults. Twenty-two patients with IgA nephropathy, 14 patients with other glomerular diseases and 19 healthy adults were examined by anti-C3 Facb EIA. The levels of IgA-CIC in sera from patients with IgA nephropathy were significantly higher than those in sera from patients with other glomerular diseases and healthy adults. CIC measured by the C1q-binding enzyme assay was detected in some patients with IgA nephropathy. The levels of serum IgA in patients with IgA nephropathy were significantly higher than those in patients with other glomerular diseases and healthy adults. However, there was no significant correlation between the levels of IgA-CIC in sera and those of serum IgA in patients with IgA nephropathy. There was also no significant correlation between the levels of IgA-CIC in sera and the degree of histopathological injuries in the patients. It is concluded that the solid-phase anti-C3 Facb EIA is useful for the detection of IgA-CIC in sera from patients with IgA nephropathy. PMID:3301093

  2. IgA antibasement membrane nephritis with pulmonary hemorrhage.

    PubMed

    Border, W A; Baehler, R W; Bhathena, D; Glassock, R J

    1979-07-01

    Goodpasture's syndrome has characteristically been described as being mediated by IgG antibodies. We have recently seen a 55-year-old man who developed renal failure and hemoptysis; a renal biopsy showed linear deposits of IgA and C3 involving glomerular and tubular basement membrane. Serologic tests for detecting (IgG) antiglomerular basement membrane antibodies were negative. Elution studies of kidney and lung showed the presence of an IgA antibasement membrane antibody only. The patient's serum contained IgA, but not IgG, antibodies reactive with glomerular and tubular basement membrane of normal human kidney and alveolar basement membrane of normal human lung. Attempts to transfer disease with the patient's IgA antibody to a monkey and to Lewis and Brown-Norway rats were unsuccessful. Immunoglobulin A antibasement membrane antibody must be considered in the design of immunoserologic procedures for the diagnosis of Goodpasture's syndrome.

  3. PetIGA: A framework for high-performance isogeometric analysis

    DOE PAGES

    Dalcin, Lisandro; Collier, Nathaniel; Vignal, Philippe; ...

    2016-05-25

    We present PetIGA, a code framework to approximate the solution of partial differential equations using isogeometric analysis. PetIGA can be used to assemble matrices and vectors which come from a Galerkin weak form, discretized with Non-Uniform Rational B-spline basis functions. We base our framework on PETSc, a high-performance library for the scalable solution of partial differential equations, which simplifies the development of large-scale scientific codes, provides a rich environment for prototyping, and separates parallelism from algorithm choice. We describe the implementation of PetIGA, and exemplify its use by solving a model nonlinear problem. To illustrate the robustness and flexibility ofmore » PetIGA, we solve some challenging nonlinear partial differential equations that include problems in both solid and fluid mechanics. Lastly, we show strong scaling results on up to 4096 cores, which confirm the suitability of PetIGA for large scale simulations.« less

  4. A new class of HIV-1 protease inhibitor: the crystallographic structure, inhibition and chemical synthesis of an aminimide peptide isostere.

    PubMed

    Rutenber, E E; McPhee, F; Kaplan, A P; Gallion, S L; Hogan, J C; Craik, C S; Stroud, R M

    1996-09-01

    The essential role of HIV-1 protease (HIV-1 PR) in the viral life cycle makes it an attractive target for the development of substrate-based inhibitors that may find efficacy as anti-AIDS drugs. However, resistance has arisen to potent peptidomimetic drugs necessitating the further development of novel chemical backbones for diversity based chemistry focused on probing the active site for inhibitor interactions and binding modes that evade protease resistance. AQ148 is a potent inhibitor of HIV-1 PR and represents a new class of transition state analogues incorporating an aminimide peptide isostere. A 3-D crystallographic structure of AQ148, a tetrapeptide isostere, has been determined in complex with its target HIV-1 PR to a resolution of 2.5 A and used to evaluate the specific structural determinants of AQ148 potency and to correlate structure-activity relationships within the class of related compounds. AQ148 is a competitive inhibitor of HIV-1 PR with a Ki value of 137 nM. Twenty-nine derivatives have been synthesized and chemical modifications have been made at the P1, P2, P1', and P2' sites. The atomic resolution structure of AQ148 bound to HIV-1 PR reveals both an inhibitor binding mode that closely resembles that of other peptidomimetic inhibitors and specific protein/inhibitor interactions that correlate with structure-activity relationships. The structure provides the basis for the design, synthesis and evaluation of the next generation of hydroxyethyl aminimide inhibitors. The aminimide peptide isostere is a scaffold with favorable biological properties well suited to both the combinatorial methods of peptidomimesis and the rational design of potent and specific substrate-based analogues.

  5. Distribution of immunoglobulin G (IgG) and A (IgA) subclasses following Q fever vaccination with soluble phase I Coxiella burnetii extract.

    PubMed

    Camacho, M T; Outschoorn, I; Kovácová, E; Téllez, A

    2000-03-06

    High levels of IgG1, IgG3 and IgA2 antibodies have been observed in patients with Q fever following Coxiella burnetii infection. This IgG subclass distribution is more typical of viral and autoimmune diseases than of bacterial infections. It seemed, therefore, of interest to carry out a prospective study of the distribution of immunoglobulin subclasses after vaccination with phase I C. burnetii tricloroacetic soluble extracts to detect possible differences with respect to natural infection. The antibody response found in vaccinees was mainly restricted to the IgG1, IgG2 and IgA1 subclasses. These findings confirm differences in isotype distribution when compared to those of patients with acute or chronic Coxiella infections and opens an area of interest with respect to the role of IgA subclasses.

  6. Identification of cysteine protease inhibitors that belong to cystatin family 1 in the cellular slime mold Dictyostelium discoideum.

    PubMed

    El-Halawany, Medhat S; Ohkouchi, Susumu; Shibata, Hideki; Hitomi, Kiyotaka; Maki, Masatoshi

    2004-06-01

    Family 1 cystatins are cytosolic inhibitors of cysteine proteases, and they are conserved in higher eukaryotes. We characterized two newly identified family 1 cystatins of the cellular slime mold Dictyostelium discoideum, cystatin A1 and A2. Their recombinant proteins showed specific inhibitory activity against papain and cathepsin B, respectively. Using specific polyclonal antibodies, we found that cystatin A1 is stably expressed throughout the life cycle of Dictyostelium, whereas cystatin A2 expression is up-regulated during the course of development.

  7. Serum IgG and IgA levels in polio and non-polio acute flaccid paralysis cases in western Uttar Pradesh, India.

    PubMed

    Mohanty, Madhu C; Nalavade, Uma P; Deshpande, Jagadish M

    2015-03-08

    IgG and IgA immunocompetence of children with wild poliovirus poliomyelitis and non-polio acute flaccid paralysis. 932 cases of acute flaccid paralysis, reported in 2008-2009, were tested for presence of polio and non-polio enteroviruses according to the WHO standards. Serum IgA and IgG levels were determined by sandwich ELISA. Mean (SD) IgA levels [0.87 (0.62)g/L; n=28] of virologically confirmed poliomyelitis cases were lower than those of virus negative [1.21 (0.83)g/L; n=612] and non-polio Enterovirus positive [1.22 (0.79)g/L; n=240] cases of acute flaccid paralysis. No significant difference was observed in the concentration of IgG among these groups. IgA plays an important role in protection against poliomyelitis.

  8. Development of the gut microbiota and mucosal IgA responses in twins and gnotobiotic mice

    PubMed Central

    Planer, Joseph D.; Peng, Yangqing; Kau, Andrew L.; Blanton, Laura V.; Ndao, I. Malick; Tarr, Phillip I.; Warner, Barbara B.; Gordon, Jeffrey I.

    2016-01-01

    Immunoglobulin A (IgA), the major class of antibody secreted by the gut mucosa, is an important contributor to gut barrier function1–3. The repertoire of IgA bound to gut bacteria reflects both T cell-dependent and -independent pathways4,5, plus glycans present on the antibody’s secretory component6. Human gut bacterial taxa targeted by IgA in the setting of intestinal barrier dysfunction are capable of producing intestinal pathology when isolated and transferred to gnotobiotic mice7,8. A complex reorientation of gut immunity occurs as infants transition from passively acquired IgA present in breast milk to host-derived IgA9–11. How IgA responses co-develop with assembly of the microbiota during this period remains poorly understood. Here, we (i) identify a set of age-discriminatory bacterial taxa whose representations define a program of microbiota assembly/maturation during the first 2 postnatal years that is shared across 40 healthy USA twin pairs; (ii) describe a pattern of progression of gut mucosal IgA responses to bacterial members of the microbiota that is highly distinctive for family members (twin pairs) during the first several postnatal months then generalizes across pairs in the second year; and (iii) assess the effects of zygosity, birth mode and breast feeding. Age-associated differences in these IgA responses can be recapitulated in young germ-free mice, colonized with fecal microbiota obtained from two twin pairs at 6 and 18 months of age, and fed a sequence of human diets that simulate the transition from milk feeding to complementary foods. The majority of these responses were robust to diet suggesting that ‘intrinsic’ properties of community members play a dominant role in dictating IgA responses. The approach described can be used to define gut mucosal immune development in health and disease states and help discover ways for repairing or preventing perturbations in this facet of host immunity. PMID:27279225

  9. Development of the gut microbiota and mucosal IgA responses in twins and gnotobiotic mice.

    PubMed

    Planer, Joseph D; Peng, Yangqing; Kau, Andrew L; Blanton, Laura V; Ndao, I Malick; Tarr, Phillip I; Warner, Barbara B; Gordon, Jeffrey I

    2016-06-09

    Immunoglobulin A (IgA), the major class of antibody secreted by the gut mucosa, is an important contributor to gut barrier function. The repertoire of IgA bound to gut bacteria reflects both T-cell-dependent and -independent pathways, plus glycans present on the antibody's secretory component. Human gut bacterial taxa targeted by IgA in the setting of barrier dysfunction are capable of producing intestinal pathology when isolated and transferred to gnotobiotic mice. A complex reorientation of gut immunity occurs as infants transition from passively acquired IgA present in breast milk to host-derived IgA. How IgA responses co-develop with assembly of the microbiota during this period remains poorly understood. Here, we (1) identify a set of age-discriminatory bacterial taxa whose representations define a program of microbiota assembly and maturation during the first 2 postnatal years that is shared across 40 healthy twin pairs in the USA; (2) describe a pattern of progression of gut mucosal IgA responses to bacterial members of the microbiota that is highly distinctive for family members (twin pairs) during the first several postnatal months then generalizes across pairs in the second year; and (3) assess the effects of zygosity, birth mode, and breast feeding. Age-associated differences in these IgA responses can be recapitulated in young germ-free mice, colonized with faecal microbiota obtained from two twin pairs at 6 and 18 months of age, and fed a sequence of human diets that simulate the transition from milk feeding to complementary foods. Most of these responses were robust to diet, suggesting that 'intrinsic' properties of community members play a dominant role in dictating IgA responses. The approach described can be used to define gut mucosal immune development in health and disease states and to help discover ways of repairing or preventing perturbations in this facet of host immunity.

  10. An oxidant- and solvent-stable protease produced by Bacillus cereus SV1: application in the deproteinization of shrimp wastes and as a laundry detergent additive.

    PubMed

    Manni, Laila; Jellouli, Kemel; Ghorbel-Bellaaj, Olfa; Agrebi, Rym; Haddar, Anissa; Sellami-Kamoun, Alya; Nasri, Moncef

    2010-04-01

    The current increase in amount of shrimp wastes produced by the shrimp industry has led to the need in finding new methods for shrimp wastes disposal. In this study, an extracellular organic solvent- and oxidant-stable metalloprotease was produced by Bacillus cereus SV1. Maximum protease activity (5,900 U/mL) was obtained when the strain was grown in medium containing 40 g/L shrimp wastes powder as a sole carbon source. The optimum pH, optimum temperature, pH stability, and thermal stability of the crude enzyme preparation were pH 8.0, 60 degrees C, pH 6-9.5, and <55 degrees C, respectively. The crude protease was extremely stable toward several organic solvents. No loss of activity was observed even after 60 days of incubation at 30 degrees C in the presence of 50% (v/v) dimethyl sulfoxide and ethyl ether; the enzyme retained more than 70% of its original activity in the presence of ethanol and N,N-dimethylformamide. The protease showed high stability toward anionic (SDS) and non-ionic (Tween 80, Tween 20, and Triton X-100) surfactants. Interestingly, the activity of the enzyme was significantly enhanced by oxidizing agents. In addition, the enzyme showed excellent compatibility with some commercial liquid detergents. The protease of B. cereus SV1, produced under the optimal culture conditions, was tested for shrimp waste deproteinization in the preparation of chitin. The protein removal with a ratio E/S of 20 was about 88%. The novelties of the SV1 protease include its high stability to organic solvents and surfactants. These unique properties make it an ideal choice for application in detergent formulations and enzymatic peptide synthesis. In addition, the enzyme may find potential applications in the deproteinization of shrimp wastes to produce chitin.

  11. Effect of hydrolysis enzymatic process of corn using protease crude (Rhizopus oligosporus-C1) to produce corn hydrolisate rich folic acid

    NASA Astrophysics Data System (ADS)

    Maryati, Yati; Susilowati, Agustine; Melanie, Hakiki; Lotulung, Puspa D.

    2017-11-01

    Corn hydrolyzate (Zea mays L) as a functional food fortificant derived from natural folic acid has been evaluated through a hydrolysis process using protease enzyme Rhizopus oligosporus strain C1. Enzymatic hydrolysis was carried out on two types of corn; yellow and white pearl variety corn, at concentration of protease enzyme (rough) 0.025; 0.125; and 0.225% (v/w of soluble nixtamal corn protein) with a hydrolysis time of 24 h at 30 °C, and pH 5.0. The results showed that the concentration of protease enzymes can increase the folic acid to the optimum condition, from the beginning to the end of the process time. Folic acid optimization of hydrolysis results in each corn was at the concentration of protease enzyme 0.225% (v/w of soluble nixtamal corn protein) in white corn and yellow corn at 24 hours hydrolysis, with folic acid composition, 283.56 µg/mL and 412.52 µg/mL, 1.07 and 1.04 mg/mL of soluble proteins, proteolytic activity 2.09 and 2.06 U/mL, total solids of 21.74 and 17.85%, total sugars of 0.56 and 2.22 mg/mL, and reducing sugar 91.72 and 48.47 mg/mL. In this condition, the increase of optimum folic acid for white corn was 33.57% and for yellow corn was 71.60% after hydrolysis.

  12. Importance of polar solvation and configurational entropy for design of antiretroviral drugs targeting HIV-1 protease.

    PubMed

    Kar, Parimal; Lipowsky, Reinhard; Knecht, Volker

    2013-05-16

    Both KNI-10033 and KNI-10075 are high affinity preclinical HIV-1 protease (PR) inhibitors with affinities in the picomolar range. In this work, the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method has been used to investigate the potency of these two HIV-1 PR inhibitors against the wild-type and mutated proteases assuming that potency correlates with the affinity of the drugs for the target protein. The decomposition of the binding free energy reveals the origin of binding affinities or mutation-induced affinity changes. Our calculations indicate that the mutation I50V causes drug resistance against both inhibitors. On the other hand, we predict that the mutant I84V causes drug resistance against KNI-10075 while KNI-10033 is more potent against the I84V mutant compared to wild-type protease. Drug resistance arises mainly from unfavorable shifts in van der Waals interactions and configurational entropy. The latter indicates that neglecting changes in configurational entropy in the computation of relative binding affinities as often done is not appropriate in general. For the bound complex PR(I50V)-KNI-10075, an increased polar solvation free energy also contributes to the drug resistance. The importance of polar solvation free energies is revealed when interactions governing the binding of KNI-10033 or KNI-10075 to the wild-type protease are compared to the inhibitors darunavir or GRL-06579A. Although the contributions from intermolecular electrostatic and van der Waals interactions as well as the nonpolar component of the solvation free energy are more favorable for PR-KNI-10033 or PR-KNI-10075 compared to PR-DRV or PR-GRL-06579A, both KNI-10033 and KNI-10075 show a similar affinity as darunavir and a lower binding affinity relative to GRL-06579A. This is because of the polar solvation free energy which is less unfavorable for darunavir or GRL-06579A relative to KNI-10033 or KNI-10075. The importance of the polar solvation as revealed here

  13. Evaluation of seven assays detecting serum immunoglobulin classes and subclasses and salivary and faecal secretory IgA against Fasciola excretory/secretory (ES) antigens in diagnosing fascioliasis.

    PubMed

    Noureldin, Mohamed S; el-Ganaini, Goman A; Abou El-Enin, Ahmed M; el-Nemr, Hosam-Eldin I; Hussin, Eman M; Sultan, Doaa M

    2004-08-01

    Seven assays detecting serum IgM, IgG, IgG1, IgG4, IgA and salivary and fecal excretory IgA against Fasciola excretory/secretory (ES) antigens were evaluated in diagnosing fascioliasis, for cross reactivity with Schistosoma mansoni sera and for evaluation of cure of Fasciola infection after treatment. Assays detecting sera IgM, IgG1, IgG4 and IgA against Fasciola ES antigens showed 100% specificity and sensitivity. Assays detecting IgM and IgG showed 98% and 96% sensitivity and 100% and 94.6% specificity respectively. Assays detecting salivary and faecal IgA showed 92% & 96% sensitivity and 100% & 100% specificity respectively. Assays detecting IgM and IgG4 were the best in evaluation of cure and assays detecting IgG4 & IgA showed the lowest cross-reactivity with sera from S. mansoni infected patients. So, assays detecting serum IgA, IgG1 & IgG4 against Fasciola ES antigens were highly sensitive and specific for diagnosis of fascioliasis and assays detecting salivary and faecal IgA were promising and of great help in diagnosis of fascioliasis especially in epidemiologic studies.

  14. Effect of α1-acid glycoprotein on the intracellular accumulation of the HIV protease inhibitors saquinavir, ritonavir and indinavir in vitro

    PubMed Central

    Jones, K; Hoggard, P G; Khoo, S; Maher, B; Back, D J

    2001-01-01

    Aims Since α1-acid glycoprotein (AGP) levels may be raised during HIV infection, we have examined in vitro the effect of increasing the concentration of AGP on the intracellular accumulation of the HIV protease inhibitors saquinavir (SQV), ritonavir (RTV) and indinavir (IDV). Methods U937 cells (5 × 106 cells in 5 ml RPMI growth medium) were incubated at 37 °C for 18 h with [14C]-SQV (0.1 µCi), [3H]-RTV and [3H]-IDV (0.135 µCi) to a final concentration of 1 µm in the presence of 0, 0.5 and 2.0 mg ml−1 AGP. Following extraction in 60% methanol the intracellular drug concentration was determined by liquid scintillation counting. Results Binding to AGP (2.0 mg ml−1) reduced the mean intracellular concentration of SQV from 31.5 µm to 7.4 µm (P < 0.0001; 95% CI 19.4–28.8). RTV concentration was also reduced (8.8 µm to 1.6 µm; P < 0.0001; 95% CI 5.4–9.0) as was the concentration of IDV (3.0 µm to 1.5 µm; P < 0.0001; 95% CI 1.11.9). Conclusions Reduced intracellular protease inhibitor concentrations in the presence of increasing concentrations of AGP will certainly impact on the antiviral activity in vitro. However, since protease inhibitors are high clearance drugs, free drug concentration will likely remain unaffected in the presence of elevated AGP during chronic oral dosing although there will be an increase in total plasma drug concentration. PMID:11167671

  15. Gluten sensitivity in patients with IgA nephropathy.

    PubMed

    Smerud, Hilde Kloster; Fellström, Bengt; Hällgren, Roger; Osagie, Sonia; Venge, Per; Kristjánsson, Gudjón

    2009-08-01

    Coeliac disease is more frequent in IgA nephropathy (IgAN) patients compared to the healthy population. Several hypotheses postulate that food antigens like gluten may be involved in the onset of IgAN. In this study, we used a recently developed mucosal patch technique to evaluate the rectal mucosal inflammatory reaction to gluten in patients with IgAN (n = 27) compared to healthy subjects (n = 18). The rectal mucosal production of nitric oxide (NO) and release of myeloperoxidase (MPO) and eosinophil cationic protein (ECP) were measured. Serum samples were analysed for IgA and IgG antigliadin antibodies (AGA), IgA antibodies against tissue transglutaminase and IgA endomysium antibodies. Gluten reactivity, defined as increase in MPO and/or NO after gluten exposure, was observed in 8 of 27 IgAN patients. The prevalence of HLA-DQ2 and DQ8 was not increased among gluten-sensitive patients, and the total prevalence among IgAN patients was the same as for the normal population. An elevated serum IgA AGA response was seen in 9 of 27 IgAN patients. The increase in IgA AGA did not correlate with the gluten sensitivity as measured by NO and/or MPO. A specific serum IgG AGA response was seen in one patient only. Antibodies against tissue transglutaminase and endomysium were not observed. It is concluded that approximately one-third of our IgAN patients have a rectal mucosal sensitivity to gluten, but without signs of coeliac disease, and we hypothesize that such sub-clinical inflammation to gluten might be involved in the pathogenesis of IgAN in a subgroup of patients.

  16. Frequency of anti- Toxoplasma gondii IgA, IgM, and IgG antibodies in high-risk pregnancies, in Brazil.

    PubMed

    Murata, Fernando Henrique Antunes; Ferreira, Marina Neves; Camargo, Natália Sahyoun; Santos, Gabriela Soria; Spegiorin, Lígia Cosentino Junqueira Franco; Silveira-Carvalho, Aparecida Perpétuo; Pereira-Chioccola, Vera Lucia; Mattos, Luiz Carlos de; Mattos, Cinara Cássia Brandão de

    2016-01-01

    Toxoplasmosis during pregnancy can be severe; thus, it is essential to diagnose the disease via serological tests. An enzyme-linked immunosorbent assay (ELISA) was used to investigate anti-Toxoplasma gondii immunoglobulin A (IgA), M (IgM) and G (IgG) antibodies in 62 high-risk pregnant women. Forty-three (69.4%) women were positive for IgA, 31 (50%) for IgG, and 57 (91.9%) for IgM; 4 (6,5%) were positive for IgA but negative for IgM; 10 (16.1%) were negative for IgA and IgM but positive for IgG. Testing for these antibodies can help diagnose infection in pregnant women, thereby contributing to clinical management.

  17. Primary genotypic resistance of HIV-1 to the maturation inhibitor PA-457 in protease inhibitor-experienced patients.

    PubMed

    Malet, Isabelle; Wirden, Marc; Derache, Anne; Simon, Anne; Katlama, Christine; Calvez, Vincent; Marcelin, Anne-Geneviève

    2007-04-23

    Sequences from 82 protease inhibitors (PI)-experienced patients were analysed for the presence of previously described in-vitro resistance mutations to PA-457 located in the C-terminal capside (H226Y, L231F, L231M) and in the N-terminal SP1 (A1V, A3T, A3V) within the CA-SP1 boundary domain. Overall, the CA-SP1 cleavage site was highly conserved in PI pre-treated patients and only one patient showed an L231M mutation. The impact of this mutation should be further addressed in vivo.

  18. Pre- and Posttransplant IgA Anti-Fab Antibodies to Predict Long-term Kidney Graft Survival.

    PubMed

    Amirzargar, M A; Amirzargar, A; Basiri, A; Hajilooi, M; Roshanaei, G; Rajabi, G; Solgi, G

    2015-05-01

    Immunologic factors are reliable markers for allograft monitoring, because of their seminal role in rejection process. One of these factors is the immunoglobulin (Ig)A anti-Fab of the IgG antibody. This study aimed to evaluate the predictive value of pre- and posttransplant levels of this marker for kidney allograft function and survival. Sera samples of 59 living unrelated donor kidney recipients were collected before and after transplantation (days 7, 14, and 30) and investigated for IgA anti-Fab of IgG antibody levels using enzyme-linked immunosorbent assay in relation with allograft outcome. Among 59 patients, 15 cases (25%) including 10 with acute rejection and 5 with chronic rejection episodes showed graft failure during a mean of 5 years of follow-up. High posttransplant levels of IgA anti-Fab antibodies were observed more frequently in patients with stable graft function (SGF) compared with patients with graft failure (P = 2 × 10(-6)). None of patients with acute or chronic rejection episodes had high levels of IgA anti-Fab antibodies at day 30 posttransplant compared with the SGF group (P = 10(-6) and P = .01, respectively). In addition, high levels of IgA anti-Fab antibody correlated with lesser concentration of serum creatinine at 1 month posttransplantation (P = .01). Five-year graft survival was associated with high levels of pre- and posttransplant IgA anti-Fab antibodies (P = .02 and P = .003, respectively). Our findings indicate the protective effect of higher levels of IgA anti-Fab antibodies regarding to kidney allograft outcomes and long-term graft survival. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Specific IgA and IgG antibodies in paired serum and breast milk samples in human strongyloidiasis.

    PubMed

    Mota-Ferreira, Daniela M L; Gonçalves-Pires, Maria do Rosário F; Júnior, Alvaro Ferreira; Sopelete, Mônica C; Abdallah, Vânia O S; Costa-Cruz, Julia M

    2009-02-01

    Strongyloidiasis, caused by the nematode Strongyloides stercoralis, is one of the major worldwide parasitic infections in humans. Breastfeeding may offer a potential protection against this infection. Feces, serum and milk samples were obtained from 90 lactating women from Clinical Hospital of Universidade Federal de Uberlândia, Brazil. The fecal samples were collected for parasitological diagnosis and the serum and milk samples were examined for specific S. stercoralis IgA and IgG antibodies using the indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA). Fecal examination showed that the rate of prevalence of S. stercoralis infection in the lactating women was 4.4%. IFAT manifested a 16.7% positivity rate for specific IgA antibody in serum and a 28.9% rate in milk samples; specific IgG was 41.1% in serum and 25.5% in milk samples. According to ELISA the positivity rate for specific IgA antibody was 21.1% in serum and 42.2% in milk samples; specific IgG was 40% in serum and 18.9% in milk samples. In serum samples, these immunological tests showed a concurrence of 91.1% and 94.4%, respectively, in detecting specific IgA and IgG antibodies. In milk samples, they showed a concurrence of 70% and 78.9%, respectively, in detecting specific IgA and IgG antibodies. There was a statistically significant difference between concordant and discordant results of immunological tests (P<0.0001). IFAT and ELISA highly concurred in their detection of specific S. stercoralis IgA and IgG antibodies in serum and in milk samples reconfirming prior studies that the serological method is a complement to the direct diagnosis of the parasite, and suggesting that immunological methods using milk samples can also be helpful. Furthermore, in endemic areas, infants may acquire antibodies to S. stercoralis from breast milk, possibly, contributing to the enhancement of specific mucosal immunity against this parasite.

  20. An enantioselective enzymatic desymmetrization route to hexahydro-4H-furopyranol, a high-affinity ligand for HIV-1 protease inhibitors.

    PubMed

    Ghosh, Arun K; Sarkar, Anindya

    2017-08-16

    An enantioselective synthesis of ( 3 a S , 4S , 7 a R )-hexahydro-4 H -furo[2,3- b ]pyran-4-ol, a high-affinity nonpeptide ligand for a variety of potent HIV-1 protease inhibitors is described. The key steps involved a highly enantioselective enzymatic desymmetrization of meso -diacetate, an efficient transacetalization, and a highly diastereoselective reduction of a ketone. This route is amenable to large-scale synthesis using readily available starting materials.

  1. Pretreatment resistance to hepatitis C virus protease inhibitors boceprevir/telaprevir in hepatitis C virus subgenotype 1a-infected patients from Manitoba.

    PubMed

    Andonov, Anton; Kadkhoda, Kamran; Osiowy, Carla; Kaita, Kelly

    2013-07-01

    Traditional therapy with pegylated interferon and ribavirin combined with the new protease inhibitors boceprevir or telaprevir has demonstrated improved outcomes in hepatitis C virus (HCV)-infected patients. Prevalence data regarding pre-existing drug-resistant variants to these two new virus inhibitors in the Canadian population are not available. To detect pre-existing mutations conferring resistance to boceprevir and⁄or telaprevir in Canadian patients infected with HCV genotype 1a. Resistance-associated mutations (RAMs) were evaluated in 85 patients infected with HCV genotype 1a who had not yet received antiviral therapy. The NS3 protease gene was sequenced and common RAMs were identified based on a recently published list. The overall prevalence of pre-existing RAMs to boceprevir and telaprevir was higher compared with other similar studies. All of the observed RAMs were associated with a low level of resistance. A surprisingly high proportion of patients had the V55A RAM (10.6%). None of the mutations associated with a high level of resistance were observed. The simultaneous presence of two low-level resistance mutations (V36L and V55A) was observed in only one patient. Three other patients had both T54S RAM and V55I mutations, which may require a higher concentration of the protease drugs. The prevalence of various mutations in Aboriginal Canadian patients was higher (37.5%) compared with Caucasians (16.39%) (P=0.038). The present study was the first to investigate pre-existing drug resistance to boceprevir⁄telaprevir in Canadian HCV-infected patients. A relatively high proportion of untreated HCV genotype 1a patients in Manitoba harbour low-level RAMs, especially patients of Aboriginal descent, which may contribute to an increased risk of treatment failure.

  2. Breastfeeding linked to the reduction of both rotavirus shedding and IgA levels after Rotarix® immunization in Mexican infants.

    PubMed

    Bautista-Marquez, Aurora; Velasquez, Daniel E; Esparza-Aguilar, Marcelino; Luna-Cruz, Maria; Ruiz-Moran, Tatiana; Sugata, Ken; Jiang, Baoming; Parashar, Umesh; Patel, Manish; Richardson, Vesta

    2016-10-17

    We examined potential risk factors on vaccine virus shedding and antibody seroresponse to human rotavirus vaccine (Rotarix) in Mexican infants. Two doses of Rotarix were administered to infants during the first two visits for their routine childhood immunization (∼8 and 15weeks of age) in Mexico City. Infant's characteristics and socioeconomic indicators were obtained, including history of long-term feeding practices (exclusively/predominantly breastfed and exclusively/predominantly non-breastfed). Two serum specimens were collected, one during the second rotavirus vaccine visit and one 7weeks later. Stool specimens were collected between days 4-7 after each of the two rotavirus vaccine doses. Rotavirus IgA and IgG titers in serum were determined by enzyme immunoassays (EIA) and rotavirus shedding in stool was assessed by EIA and confirmed by RT-PCR. The overall rotavirus IgA geometric mean titers (GMT) increased significantly post dose 2 from post dose 1 [176 (95%CI: 113-273) to 335 (238-471); p=0.020). Infants who were exclusively/predominantly breastfed were less likely to shed vaccine virus in stool than those who were formula-fed (22% vs. 43%, p=0.016). Infants who were breastfed had lower rotavirus IgA titers than those who were formula-fed after dose 1 [GMT: 145 (84-250) vs. 267 (126-566) p=0.188] and dose 2 [236 (147-378) vs.578 (367-910), p=0.007]. Infants who shed vaccine virus post dose 1 had significantly higher serum IgA GMT than those who did not shed [425 (188-965) vs. 150 (84-266), p=0.038]. Breastfeeding was linked with the reduction of both stool vaccine shedding, and IgA seroresponse. The reduced rotavirus replication in the gut and shedding after dose 1 may explain in part the lower IgA response in serum. Published by Elsevier Ltd.

  3. Plasmodium vivax rhomboid-like protease 1 gene diversity in Thailand.

    PubMed

    Mataradchakul, Touchchapol; Uthaipibull, Chairat; Nosten, Francois; Vega-Rodriguez, Joel; Jacobs-Lorena, Marcelo; Lek-Uthai, Usa

    2017-10-01

    Plasmodium vivax infection remains a major public health problem, especially along the Thailand border regions. We examined the genetic diversity of this parasite by analyzing single-nucleotide polymorphisms (SNPs) of the P. vivax rhomboid-like protease 1 gene (Pvrom1) in parasites collected from western (Tak province, Thai-Myanmar border) and eastern (Chanthaburi province, Thai-Cambodia border) regions. Data were collected by a cross-sectional survey, consisting of 47 and 45 P. vivax-infected filter paper-spotted blood samples from the western and eastern regions of Thailand, respectively during September 2013 to May 2014. Extracted DNA was examined for presence of P. vivax using Plasmodium species-specific nested PCR. Pvrom1 gene was PCR amplified, sequenced and the SNP diversity was analyzed using F-STAT, DnaSP, MEGA and LIAN programs. Comparison of sequences of the 92 Pvrom1 831-base open reading frames with that of a reference sequence (GenBank acc. no. XM001615211) revealed 17 samples with a total of 8 polymorphic sites, consisting of singleton (exon 3, nt 645) and parsimony informative (exon 1, nt 22 and 39; exon 3, nt 336, 537 and 656; and exon 4, nt 719 and 748) sites, which resulted in six different deduced Pvrom1 variants. Non-synonymous to synonymous substitutions ratio estimated by the DnaSP program was 1.65 indicating positive selection, but the Z-tests of selection showed no significant deviations from neutrality for Pvrom1 samples from western region of Thailand. In addition McDonald Kreitman test (MK) showed not significant, and Fst values are not different between the two regions and the regions combined. Interestingly, only Pvrom1 exon 2 was the most conserved sequences among the four exons. The relatively high degree of Pvrom1 polymorphism suggests that the protein is important for parasite survival in face of changes in both insect vector and human populations. These polymorphisms could serve as a sensitive marker for studying plasmodial

  4. Purification and Characterization of a Protease Produced by a Planomicrobium sp. L-2 from Gut of Octopus vulgaris

    PubMed Central

    Liu, Qing; Sun, Shujing; Piao, Meizi; Yang, Ji Young

    2013-01-01

    Protease widely exists in the digestive tract of animals and humans, playing a very important role in protein digestion and absorption. In this study, a high protease-producing strain Planomicrobium sp. L-2 was isolated and identified from the digestive tract of Octopus variabilis. The strain was identified by physiological and biochemical experiments and 16S rDNA sequences analysis. A protease was obtained from the strain Planomicrobium sp. L-2 through ammonium sulfate precipitation, dialysis and enrichment, DEAE-Sephadex A50 anion-exchange chromatography, and Sephadex G-100 gel chromatography. The molecular weight and properties of the protease were characterized, including optimum temperature and pH, thermal stability, protease inhibitions and metal ions. According to our results, the protease from Planomicrobium sp. L-2 strain designated as F1-1 was obtained by three-step separation and purification from crude enzyme. The molecular weight of the protease was 61.4 kDa and its optimum temperature was 40°C. The protease F1-1 showed a broad pH profile for casein hydrolysis between 5.0~11.0. No residual activity was observed after incubation for 40 min at 60°C and 60 min at 50°C. F1-1 protease was inhibited by Mn2+, Hg2+, Pb2+, Zn2+, and Cu2+ ions, as well as PMSF, indicating that the protease F1-1 was a serine protease. Additionally, research basis provided by this study could be considered for industrial application of octopus intestinal proteases. PMID:24551830

  5. Reformatting palivizumab and motavizumab from IgG to human IgA impairs their efficacy against RSV infection in vitro and in vivo.

    PubMed

    Jacobino, Shamir R; Nederend, Maaike; Reijneveld, J Frederiek; Augustijn, Daan; Jansen, J H Marco; Meeldijk, Jan; Reiding, Karli R; Wuhrer, Manfred; Coenjaerts, Frank E J; Hack, C Erik; Bont, Louis J; Leusen, Jeanette H W

    2018-04-01

    Respiratory syncytial virus (RSV) infection is a leading cause of hospitalization and mortality in young children. Protective therapy options are limited. Currently, palivizumab, a monoclonal IgG1 antibody, is the only licensed drug for RSV prophylaxis, although other IgG antibody candidates are being evaluated. However, at the respiratory mucosa, IgA antibodies are most abundant and act as the first line of defense against invading pathogens. Therefore, it would be logical to explore the potential of recombinant human IgA antibodies to protect against viral respiratory infection, but very little research on the topic has been published. Moreover, it is unknown whether human antibodies of the IgA isotype are better suited than those of the IgG isotype as antiviral drugs to combat respiratory infections. To address this, we generated various human IgA antibody formats of palivizumab and motavizumab, two well-characterized human IgG1 anti-RSV antibodies. We evaluated their efficacy to prevent RSV infection in vitro and in vivo and found similar, but somewhat decreased efficacy for different IgA subclasses and formats. Thus, reformatting palivizumab or motavizumab into IgA reduces the antiviral potency of either antibody. Moreover, our results indicate that the efficacy of intranasal IgA prophylaxis against RSV infection in human FcαRI transgenic mice is independent of Fc receptor expression.

  6. Allergenic proteases cleave the chemokine CX3CL1 directly from the surface of airway epithelium and augment the effect of rhinovirus.

    PubMed

    Loxham, M; Smart, D E; Bedke, N J; Smithers, N P; Filippi, I; Blume, C; Swindle, E J; Tariq, K; Howarth, P H; Holgate, S T; Davies, D E

    2018-03-01

    CX3CL1 has been implicated in allergen-induced airway CD4 + T-lymphocyte recruitment in asthma. As epidemiological evidence supports a viral infection-allergen synergy in asthma exacerbations, we postulated that rhinovirus (RV) infection in the presence of allergen augments epithelial CX3CL1 release. Fully differentiated primary bronchial epithelial cultures were pretreated apically with house dust mite (HDM) extract and infected with rhinovirus-16 (RV16). CX3CL1 was measured by enzyme-linked immunosorbent assay and western blotting, and shedding mechanisms assessed using inhibitors, protease-activated receptor-2 (PAR-2) agonist, and recombinant CX3CL1-expressing HEK293T cells. Basolateral CX3CL1 release was unaffected by HDM but stimulated by RV16; inhibition by fluticasone or GM6001 implicated nuclear factor-κB and ADAM (A Disintegrin and Metalloproteinase) sheddases. Conversely, apical CX3CL1 shedding was stimulated by HDM and augmented by RV16. Although fluticasone or GM6001 reduced RV16+HDM-induced apical CX3CL1 release, heat inactivation or cysteine protease inhibition completely blocked CX3CL1 shedding. The HDM effect was via enzymatic cleavage of CX3CL1, not PAR-2 activation, yielding a product mitogenic for smooth muscle cells. Extracts of Alternaria fungus caused similar CX3CL1 shedding. We have identified a novel mechanism whereby allergenic proteases cleave CX3CL1 from the apical epithelial surface to yield a biologically active product. RV16 infection augmented HDM-induced CX3CL1 shedding-this may contribute to synergy between allergen exposure and RV infection in triggering asthma exacerbations and airway remodeling.

  7. Autocatalytic Activity of the Ubiquitin-Specific Protease Domain of Herpes Simplex Virus 1 VP1-2▿†

    PubMed Central

    Bolstad, M.; Abaitua, F.; Crump, C. M.; O'Hare, P.

    2011-01-01

    The herpes simplex virus (HSV) tegument protein VP1-2 is essential for virus entry and assembly. VP1-2 also contains a highly conserved ubiquitin-specific protease (USP) domain within its N-terminal region. Despite conservation of the USP and the demonstration that it can act on artificial substrates such as polyubiquitin chains, identification of the relevance of the USP in vivo to levels or function of any substrate remains limited. Here we show that HSV VP1-2 USP can act on itself and is important for stability. VP1-2 N-terminal variants encompassing the core USP domain itself were not affected by mutation of the catalytic cysteine residue (C65). However, extending the N-terminal region resulted in protein species requiring USP activity for accumulation. In this context, C65A mutation resulted in a drastic reduction in protein levels which could be stabilized by proteosomal inhibition or by the presence of normal C65. The functional USP domain could increase abundance of unstable variants, indicating action at least in part, in trans. Interestingly, full-length variants containing the inactive USP, although unstable when expressed in isolation, were stabilized by virus infection. The catalytically inactive VP1-2 retained complementation activity of a VP1-2-negative virus. Furthermore, a recombinant virus expressing a C65A mutant VP1-2 exhibited little difference in single-step growth curves and the kinetics and abundance of VP1-2 or a number of test proteins. Despite the absence of a phenotype for these replication parameters, the USP activity of VP1-2 may be required for function, including its own stability, under certain circumstances. PMID:21715485

  8. Evaluation of the Oxford Classification of IgA nephropathy: a systematic review and meta-analysis.

    PubMed

    Lv, Jicheng; Shi, Sufang; Xu, Damin; Zhang, Hong; Troyanov, Stéphan; Cattran, Daniel C; Wang, Haiyan

    2013-11-01

    The Oxford Classification of the pathology of immunoglobulin A (IgA) nephropathy, developed in 2009, is highly predictive of renal prognosis. It has been validated in different populations, but the results remain inconsistent. Systematic review and meta-analysis. Patients with biopsy-proven primary IgA nephropathy. Studies assessing the Oxford Classification of IgA nephropathy published between January 2009 and December 2012 were included following systematic searching of the MEDLINE and EMBASE databases. 4 pathologic lesions of the Oxford Classification: mesangial hypercellularity (M), endocapillary hypercellularity (E), segmental glomerulosclerosis (S), and tubular atrophy/interstitial fibrosis (T). Kidney failure defined as doubled serum creatinine level, 50% decline in estimated glomerular filtration rate, or end-stage kidney disease. 16 retrospective cohort studies with 3,893 patients and 570 kidney failure events were included. In a multivariate model, HRs for kidney failure were 0.6 (95% CI, 0.5-0.8; P < 0.001), 1.8 (95% CI, 1.4-2.4; P < 0.001), and 3.2 (95% CI, 1.8-5.6; P < 0.001) for scores of M0 (mesangial hypercellularity score ≤0.5), S1 (presence of segmental glomerulosclerosis), and T1/2 (>25% tubular atrophy/interstitial fibrosis), respectively, without evidence of heterogeneity. Pooled results showed that E lesions were not associated with kidney failure (HR, 1.4; 95% CI, 0.9-2.0; P = 0.1), with evidence of heterogeneity (I(2) = 54.1%; P = 0.01). Crescent (C) lesions were associated with kidney failure (HR, 2.3; 95% CI, 1.6-3.4; P < 0.001), with no evidence of heterogeneity (I(2) = 14.7%; P = 0.3). All studies were retrospective. This was not an individual-patient-data meta-analysis. This study suggests that M, S, T, and C lesions, but not E lesions, are associated strongly with progression to kidney failure and thus should be included in the Oxford Classification system. Copyright © 2013 National Kidney Foundation, Inc. Published by Elsevier Inc

  9. Contrasting Roles of the Apoplastic Aspartyl Protease APOPLASTIC, ENHANCED DISEASE SUSCEPTIBILITY1-DEPENDENT1 and LEGUME LECTIN-LIKE PROTEIN1 in Arabidopsis Systemic Acquired Resistance1,2[W

    PubMed Central

    Breitenbach, Heiko H.; Wenig, Marion; Wittek, Finni; Jordá, Lucia; Maldonado-Alconada, Ana M.; Sarioglu, Hakan; Colby, Thomas; Knappe, Claudia; Bichlmeier, Marlies; Pabst, Elisabeth; Mackey, David; Parker, Jane E.; Vlot, A. Corina

    2014-01-01

    Systemic acquired resistance (SAR) is an inducible immune response that depends on ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1). Here, we show that Arabidopsis (Arabidopsis thaliana) EDS1 is required for both SAR signal generation in primary infected leaves and SAR signal perception in systemic uninfected tissues. In contrast to SAR signal generation, local resistance remains intact in eds1 mutant plants in response to Pseudomonas syringae delivering the effector protein AvrRpm1. We utilized the SAR-specific phenotype of the eds1 mutant to identify new SAR regulatory proteins in plants conditionally expressing AvrRpm1. Comparative proteomic analysis of apoplast-enriched extracts from AvrRpm1-expressing wild-type and eds1 mutant plants led to the identification of 12 APOPLASTIC, EDS1-DEPENDENT (AED) proteins. The genes encoding AED1, a predicted aspartyl protease, and another AED, LEGUME LECTIN-LIKE PROTEIN1 (LLP1), were induced locally and systemically during SAR signaling and locally by salicylic acid (SA) or its functional analog, benzo 1,2,3-thiadiazole-7-carbothioic acid S-methyl ester. Because conditional overaccumulation of AED1-hemagglutinin inhibited SA-induced resistance and SAR but not local resistance, the data suggest that AED1 is part of a homeostatic feedback mechanism regulating systemic immunity. In llp1 mutant plants, SAR was compromised, whereas the local resistance that is normally associated with EDS1 and SA as well as responses to exogenous SA appeared largely unaffected. Together, these data indicate that LLP1 promotes systemic rather than local immunity, possibly in parallel with SA. Our analysis reveals new positive and negative components of SAR and reinforces the notion that SAR represents a distinct phase of plant immunity beyond local resistance. PMID:24755512

  10. Deficiency of a alpha-1-antitrypsin influences systemic iron homeostasis

    EPA Science Inventory

    Abstract Background: There is evidence that proteases and anti-proteases participate in the iron homeostasis of cells and living systems. We tested the postulate that alpha-1 antitrypsin (A1AT) polymorphism and the consequent deficiency of this anti-protease in humans are asso...

  11. Understanding the HIV-1 protease reactivity with DFT: what do we gain from recent functionals?

    PubMed

    Garrec, J; Sautet, P; Fleurat-Lessard, P

    2011-07-07

    The modeling of HIV-1 plays a crucial role in the understanding of its reactivity and its interactions with specific drugs. In this work, we propose a medium sized model to test the ability of a variety of quantum chemistry approaches to provide reasonable geometric parameters and energetics for this system. Although our model is large enough to include the main polarizing groups of the active site, it is small enough to be used within full quantum studies up to the second order Møller-Plesset (MP2) level with extrapolations to coupled cluster CCSD(T) level. These high level calculations are used as reference to assess the ability of electronic structure methods (semiempirical and DFT) to provide accurate geometries and energies for the HIV-1 protease reaction. All semiempirical methods fail to describe the geometry of the protease active site. Within DFT, pure generalized gradient approximation (GGA) functionals have difficulty in reproducing the reaction energy and underestimate the barrier. Hybrid and/or meta GGA approaches do not yield a consistent improvement. The best results are obtained with hybrid GGA B3LYP or X3LYP and with hybrid meta GGA functionals with a fraction of exact exchange around 30-40%, such as M06, B1B95, or BMK functionals. On the basis of these results, we propose an accurate and computationally efficient strategy, employing quantum chemistry methods. This is applied here to study the protonation state of the reaction intermediate and could be easily used in further QM/MM studies.

  12. Intermittent fasting promotes bacterial clearance and intestinal IgA production in Salmonella typhimurium-infected mice.

    PubMed

    Godínez-Victoria, M; Campos-Rodriguez, R; Rivera-Aguilar, V; Lara-Padilla, E; Pacheco-Yepez, J; Jarillo-Luna, R A; Drago-Serrano, M E

    2014-05-01

    The impact of intermittent fasting versus ad libitum feeding during Salmonella typhimurium infection was evaluated in terms of duodenum IgA levels, bacterial clearance and intestinal and extra-intestinal infection susceptibility. Mice that were intermittently fasted for 12 weeks or fed ad libitum were infected with S. typhimurium and assessed at 7 and 14 days post-infection. Next, we evaluated bacterial load in the faeces, Peyer's patches, spleen and liver by plate counting, as well as total and specific intestinal IgA and plasmatic corticosterone levels (by immunoenzymatic assay) and lamina propria IgA levels in plasma cells (by cytofluorometry). Polymeric immunoglobulin receptor, α- and J-chains, Pax-5 factor, pro-inflammatory cytokine (tumour necrosis factor-α and interferon-γ) and anti-inflammatory cytokine (transforming growth factor-β) mRNA levels were assessed in mucosal and liver samples (by real-time PCR). Compared with the infected ad libitum mice, the intermittently fasted infected animals had (1) lower intestinal and systemic bacterial loads; (2) higher SIgA and IgA plasma cell levels; (3) higher mRNA expression of most intestinal parameters; and (4) increased or decreased corticosterone levels on day 7 and 14 post-infection, respectively. No contribution of liver IgA was observed at the intestinal level. Apparently, the changes following metabolic stress induced by intermittent fasting during food deprivation days increased the resistance to S. typhimurium infection by triggering intestinal IgA production and presumably, pathogen elimination by phagocytic inflammatory cells. © 2014 John Wiley & Sons Ltd.

  13. Proposal and validation of prognostic scoring systems for IgG and IgA monoclonal gammopathies of undetermined significance.

    PubMed

    Rossi, Francesca; Petrucci, Maria Teresa; Guffanti, Andrea; Marcheselli, Luigi; Rossi, Davide; Callea, Vincenzo; Vincenzo, Federico; De Muro, Marianna; Baraldi, Alessandra; Villani, Oreste; Musto, Pellegrino; Bacigalupo, Andrea; Gaidano, Gianluca; Avvisati, Giuseppe; Goldaniga, Maria; Depaoli, Lorenzo; Baldini, Luca

    2009-07-01

    The presenting clinico-hematologic features of 1,283 patients with IgG and IgA monoclonal gammopathies of undetermined significance (MGUS) were correlated with the frequency of evolution into multiple myeloma (MM). Two IgG MGUS populations were evaluated: a training sample (553 patients) and a test sample (378 patients); the IgA MGUS population consisted of 352 patients. Forty-seven of the 553 training group patients and 22 of 378 test group IgG patients developed MM after a median follow-up of 6.7 and 3.6 years, respectively. Multivariate analysis showed that serum monoclonal component (MC) levels of < or =1.5 g/dL, the absence of light-chain proteinuria and normal serum polyclonal immunoglobulin levels defined a prognostically favorable subset of patients, and could be used to stratify the patients into three groups at different 10-year risk of evolution (hazard ratio, 1.0, 5.04, 11.2; P < 0.001). This scoring system was validated in the test sample. Thirty of the 352 IgA patients developed MM after a median follow-up of 4.8 years, and multivariate analysis showed that hemoglobin levels of <12.5 g/dL and reduced serum polyclonal immunoglobulin correlated with progression. A pooled statistical analysis of all of the patients confirmed the validity of Mayo Clinic risk model showing that IgA class, serum MC levels, and light-chain proteinuria are the most important variables correlated with disease progression. Using simple variables, we validated a prognostic model for IgG MGUS. Among the IgA cases, the possible prognostic role of hemoglobin emerged in addition to a decrease in normal immunoglobulin levels.

  14. A study of proteases and protease-inhibitor complexes in biological fluids

    PubMed Central

    Granelli-Piperno, A; Reich, E

    1978-01-01

    We have (a) screened a variety of cell lines and body fluids for plasminogen activators and (b) studied the activity of proteases bound to α2- macroglobulin after exposing the complexes to partial degradation and/or denaturing procedures to unmask proteolytic activity. The respective results show (a) that the plasminogen activators in urine and cell culture media are generally of lower molecular weight than those in plasma; and (b) that proteases bound to α2-macroglobulin recover the ability to attack macromolecular substrates after exposure to sodium dodecyl sulfate while retaining the electrophoretic mobility of the protease inhibitor complex. This indicates that the protease and inhibitor are probably linked by covalent bonds. In contrast, other complexes formed between proteases and inhibitors of lower molecular weight (such as soybean or Kunitz inhibitors) are fully dissociated by sodium dodecyl sulfate (SDS). The experiments described were based on a new procedure for detecting proteolytic enzyme activity in SDS-polyacrylamide gels. The method relies on solutions of nonionic detergents for extracting SDS, after which the electrophoretic gel is applied to an indicator gel consisting of a fibrin- agar mixture. The method is sensitive, permitting the detection of proteinases in less than 1 μl of fresh plasma, and it is effective for resolving small differences in molecular weight. The procedure can be quantitated and, with minor modifications appropriate to each particular system, it has been applied to a broad spectrum of serine enzymes and proenzymes, including some that function in the pathways of fibrinolysis, coagulation and kinin-generation. Other potential applications appear likely. PMID:78958

  15. [Comparison of two rat models of IgA nephropathy].

    PubMed

    Peng, Wei; Liu, Zheng-rong

    2008-10-01

    To study the methods for rapid establishment of rat models of IgA nephropathy. Forty female SD rats weighing 160-200 g were randomized into 3 groups. In group A, the rats received intravenous injection of staphylococcal enterotoxin B (SEB) and oral bovine serum albumin (BSA), and in group B, CCl4 was injected subcutaneously in addition to the above treatments; the rats in group C received no treatments to serve as the normal control group. The rats were sacrificed 10 and 14 weeks after the treatment for biochemical testing of the arterial blood and histopathological and IgA immunofluorescence examination of the renal tissues. The twenty-four-hour urine was collected at 10, 12, and 14 weeks after the treatments for detecting the urine proteins. Compared with the control group, the rats in groups A and B showed significantly increased serum creatinine, urine nitrogen and protein levels. Pathological examination of the renal tissue showed mild to moderate mesangial expansion and mesangial cell proliferation in groups A and B, without obvious difference between the two groups; but hematuria and proteinuria occurred earlier in group B with stronger IgA immunofluorescence than in group A. Both of the methods used in group A and group B can successfully induce IgA nephropathy in rats, but in group B, hematuria and urineprotein occurs earlier and IgA immunofluorescence is more stronger. Therefore intravenous SEB injection combined with oral BSA and subcutaneous CCl4 administration is a better method for time-efficient establishment of rat models of IgA nephropathy.

  16. Sera of IgA nephropathy patients contain a heterogeneous population of relatively cationic alpha-heavy chains.

    PubMed

    Shuib, A S; Chua, C T; Hashim, O H

    1998-01-01

    Sera of IgA nephropathy (IgAN) patients and normal subjects were analysed by two-dimensional (2-D) gel electrophoresis. Densitometric analysis of the 2-D gels of IgAN patients and normal subjects revealed that their protein maps were comparable. There was no shift of pI values in the major alpha-heavy chain spots. However, the volume of the alpha-heavy chain bands were differently distributed. Distribution was significantly lower at the anionic region in IgAN patients (mean anionic:cationic ratio of 1.184 +/- 0.311) as compared to normal healthy controls (mean anionic:cationic ratio of 2.139 +/- 0.538). Our data are in support of the previously reported findings that IgA1 of IgAN patients were lacking in sialic acid residues.

  17. Exploration of the effect of sequence variations located inside the binding pocket of HIV-1 and HIV-2 proteases.

    PubMed

    Triki, Dhoha; Billot, Telli; Visseaux, Benoit; Descamps, Diane; Flatters, Delphine; Camproux, Anne-Claude; Regad, Leslie

    2018-04-10

    HIV-2 protease (PR2) is naturally resistant to most FDA (Food and Drug Administration)-approved HIV-1 protease inhibitors (PIs), a major antiretroviral class. In this study, we compared the PR1 and PR2 binding pockets extracted from structures complexed with 12 ligands. The comparison of PR1 and PR2 pocket properties showed that bound PR2 pockets were more hydrophobic with more oxygen atoms and fewer nitrogen atoms than PR1 pockets. The structural comparison of PR1 and PR2 pockets highlighted structural changes induced by their sequence variations and that were consistent with these property changes. Specifically, substitutions at residues 31, 46, and 82 induced structural changes in their main-chain atoms that could affect PI binding in PR2. In addition, the modelling of PR1 mutant structures containing V32I and L76M substitutions revealed a cooperative mechanism leading to structural deformation of flap-residue 45 that could modify PR2 flexibility. Our results suggest that substitutions in the PR1 and PR2 pockets can modify PI binding and flap flexibility, which could underlie PR2 resistance against PIs. These results provide new insights concerning the structural changes induced by PR1 and PR2 pocket variation changes, improving the understanding of the atomic mechanism of PR2 resistance to PIs.

  18. Cyclic strain increases protease-activated receptor-1 expression in vascular smooth muscle cells

    NASA Technical Reports Server (NTRS)

    Nguyen, K. T.; Frye, S. R.; Eskin, S. G.; Patterson, C.; Runge, M. S.; McIntire, L. V.

    2001-01-01

    Cyclic strain regulates many vascular smooth muscle cell (VSMC) functions through changing gene expression. This study investigated the effects of cyclic strain on protease-activated receptor-1 (PAR-1) expression in VSMCs and the possible signaling pathways involved, on the basis of the hypothesis that cyclic strain would enhance PAR-1 expression, reflecting increased thrombin activity. Uniaxial cyclic strain (1 Hz, 20%) of cells cultured on elastic membranes induced a 2-fold increase in both PAR-1 mRNA and protein levels. Functional activity of PAR-1, as assessed by cell proliferation in response to thrombin, was also increased by cyclic strain. In addition, treatment of cells with antioxidants or an NADPH oxidase inhibitor blocked strain-induced PAR-1 expression. Preincubation of cells with protein kinase inhibitors (staurosporine or Ro 31-8220) enhanced strain-increased PAR-1 expression, whereas inhibitors of NO synthase, tyrosine kinase, and mitogen-activated protein kinases had no effect. Cyclic strain in the presence of basic fibroblast growth factor induced PAR-1 mRNA levels beyond the effect of cyclic strain alone, whereas no additive effect was observed between cyclic strain and platelet-derived growth factor-AB. Our findings that cyclic strain upregulates PAR-1 mRNA expression but that shear stress downregulates this gene in VSMCs provide an opportunity to elucidate signaling differences by which VSMCs respond to different mechanical forces.

  19. Processing sites in the human immunodeficiency virus type 1 (HIV-1) Gag-Pro-Pol precursor are cleaved by the viral protease at different rates.

    PubMed

    Pettit, Steve C; Lindquist, Jeffrey N; Kaplan, Andrew H; Swanstrom, Ronald

    2005-11-01

    We have examined the kinetics of processing of the HIV-1 Gag-Pro-Pol precursor in an in vitro assay with mature protease added in trans. The processing sites were cleaved at different rates to produce distinct intermediates. The initial cleavage occurred at the p2/NC site. Intermediate cleavages occurred at similar rates at the MA/CA and RT/IN sites, and to a lesser extent at sites upstream of RT. Late cleavages occurred at the sites flanking the protease (PR) domain, suggesting sequestering of these sites. We observed paired intermediates indicative of half- cleavage of RT/RH site, suggesting that the RT domain in Gag-Pro-Pol was in a dimeric form under these assay conditions. These results clarify our understanding of the processing kinetics of the Gag-Pro-Pol precursor and suggest regulated cleavage. Our results further suggest that early dimerization of the PR and RT domains may serve as a regulatory element to influence the kinetics of processing within the Pol domain.

  20. Determinants of the VP1/2A junction cleavage by the 3C protease in foot-and-mouth disease virus-infected cells.

    PubMed

    Kristensen, Thea; Normann, Preben; Gullberg, Maria; Fahnøe, Ulrik; Polacek, Charlotta; Rasmussen, Thomas Bruun; Belsham, Graham J

    2017-03-01

    The foot-and-mouth disease virus (FMDV) capsid precursor, P1-2A, is cleaved by FMDV 3C protease to yield VP0, VP3, VP1 and 2A. Cleavage of the VP1/2A junction is the slowest. Serotype O FMDVs with uncleaved VP1-2A (having a K210E substitution in VP1; at position P2 in cleavage site) have been described previously and acquired a second site substitution (VP1 E83K) during virus rescue. Furthermore, introduction of the VP1 E83K substitution alone generated a second site change at the VP1/2A junction (2A L2P, position P2' in cleavage site). These virus adaptations have now been analysed using next-generation sequencing to determine sub-consensus level changes in the virus; this revealed other variants within the E83K mutant virus population that changed residue VP1 K210. The construction of serotype A viruses with a blocked VP1/2A cleavage site (containing K210E) has now been achieved. A collection of alternative amino acid substitutions was made at this site, and the properties of the mutant viruses were determined. Only the presence of a positively charged residue at position P2 in the cleavage site permitted efficient cleavage of the VP1/2A junction, consistent with analyses of diverse FMDV genome sequences. Interestingly, in contrast to the serotype O virus results, no second site mutations occurred within the VP1 coding region of serotype A viruses with the blocked VP1/2A cleavage site. However, some of these viruses acquired changes in the 2C protein that is involved in enterovirus morphogenesis. These results have implications for the testing of potential antiviral agents targeting the FMDV 3C protease.

  1. Autocatalytic maturation, physical/chemical properties, and crystal structure of group N HIV-1 protease: relevance to drug resistance.

    PubMed

    Sayer, Jane M; Agniswamy, Johnson; Weber, Irene T; Louis, John M

    2010-11-01

    The mature protease from Group N human immunodeficiency virus Type 1 (HIV-1) (PR1(N)) differs in 20 amino acids from the extensively studied Group M protease (PR1(M)) at positions corresponding to minor drug-resistance mutations (DRMs). The first crystal structure (1.09 Å resolution) of PR1(N) with the clinical inhibitor darunavir (DRV) reveals the same overall structure as PR1(M), but with a slightly larger inhibitor-binding cavity. Changes in the 10s loop and the flap hinge propagate to shift one flap away from the inhibitor, whereas L89F and substitutions in the 60s loop perturb inhibitor-binding residues 29-32. However, kinetic parameters of PR1(N) closely resemble those of PR1(M), and calorimetric results are consistent with similar binding affinities for DRV and two other clinical PIs, suggesting that minor DRMs coevolve to compensate for the detrimental effects of drug-specific major DRMs. A miniprecursor (TFR 1-61-PR1(N)) comprising the transframe region (TFR) fused to the N-terminus of PR1(N) undergoes autocatalytic cleavage at the TFR/PR1(N) site concomitant with the appearance of catalytic activity characteristic of the dimeric, mature enzyme. This cleavage is inhibited at an equimolar ratio of precursor to DRV (∼6 μM), which partially stabilizes the precursor dimer from a monomer. However, cleavage at L34/W35 within the TFR, which precedes the TFR 1-61/PR1(N) cleavage at pH ≤ 5, is only partially inhibited. Favorable properties of PR1(N) relative to PR1(M) include its suitability for column fractionation by size under native conditions and >10-fold higher dimer dissociation constant (150 nM). Exploiting these properties may facilitate testing of potential dimerization inhibitors that perturb early precursor processing steps.

  2. IGA: A Simplified Introduction and Implementation Details for Finite Element Users

    NASA Astrophysics Data System (ADS)

    Agrawal, Vishal; Gautam, Sachin S.

    2018-05-01

    Isogeometric analysis (IGA) is a recently introduced technique that employs the Computer Aided Design (CAD) concept of Non-uniform Rational B-splines (NURBS) tool to bridge the substantial bottleneck between the CAD and finite element analysis (FEA) fields. The simplified transition of exact CAD models into the analysis alleviates the issues originating from geometrical discontinuities and thus, significantly reduces the design-to-analysis time in comparison to traditional FEA technique. Since its origination, the research in the field of IGA is accelerating and has been applied to various problems. However, the employment of CAD tools in the area of FEA invokes the need of adapting the existing implementation procedure for the framework of IGA. Also, the usage of IGA requires the in-depth knowledge of both the CAD and FEA fields. This can be overwhelming for a beginner in IGA. Hence, in this paper, a simplified introduction and implementation details for the incorporation of NURBS based IGA technique within the existing FEA code is presented. It is shown that with little modifications, the available standard code structure of FEA can be adapted for IGA. For the clear and concise explanation of these modifications, step-by-step implementation of a benchmark plate with a circular hole under the action of in-plane tension is included.

  3. Extracellular proteases of Trichoderma species. A review.

    PubMed

    Kredics, L; Antal, Zsuzsanna; Szekeres, A; Hatvani, L; Manczinger, L; Vágvölgyi, Cs; Nagy, Erzsébet

    2005-01-01

    Cellulolytic, xylanolytic, chitinolytic and beta-1,3-glucanolytic enzyme systems of species belonging to the filamentous fungal genus Trichoderma have been investigated in details and are well characterised. The ability of Trichoderma strains to produce extracellular proteases has also been known for a long time, however, the proteolytic enzyme system is relatively unknown in this genus. Fortunately, in the recent years more and more attention is focused on the research in this field. The role of Trichoderma proteases in the biological control of plant pathogenic fungi and nematodes has been demonstrated, and it is also suspected that they may be important for the competitive saprophytic ability of green mould isolates and may represent potential virulence factors of Trichoderma strains as emerging fungal pathogens of clinical importance. The aim of this review is to summarize the information available about the extracellular proteases of Trichoderma. Numerous studies are available about the extracellular proteolytic enzyme profiles of Trichoderma strains and about the effect of abiotic environmental factors on protease activities. A number of protease enzymes have been purified to homogeneity and some protease encoding genes have been cloned and characterized. These results will be reviewed and the role of Trichoderma proteases in biological control as well as their advantages and disadvantages in biotechnology will be discussed.

  4. Serine proteases in rodent hippocampus.

    PubMed

    Davies, B J; Pickard, B S; Steel, M; Morris, R G; Lathe, R

    1998-09-04

    Brain serine proteases are implicated in developmental processes, synaptic plasticity, and in disorders including Alzheimer's disease. The spectrum of the major enzymes expressed in brain has not been established previously. We now present a systematic study of the serine proteases expressed in adult rat and mouse hippocampus. Using a combination of techniques including polymerase chain reaction amplification and Northern blotting we show that tissue-type plasminogen activator (t-PA) is the major species represented. Unexpectedly, the next most abundant species were RNK-Met-1, a lymphocyte protease not reported previously in brain, and two new family members, BSP1 (brain serine protease 1) and BSP2. We report full-length sequences of the two new proteases; homologies indicate that these are of tryptic specificity. Although BSP2 is expressed in several brain regions, BSP1 expression is strikingly restricted to hippocampus. Other enzymes represented, but at lower levels, included elastase IV, proteinase 3, complement C2, chymotrypsin B, chymotrypsin-like protein, and Hageman factor. Although thrombin and urokinase-type plasminogen activator were not detected in the primary screen, low level expression was confirmed using specific polymerase chain reaction primers. In contrast, and despite robust expression of t-PA, the usual t-PA substrate plasminogen was not expressed at detectable levels.

  5. Evolution of IgA nephropathy into anaphylactoid purpura in six cases--further evidence that IgA nephropathy and Henoch-Schonlein purpura nephritis share common pathogenesis.

    PubMed

    Kamei, Koichi; Ogura, Masao; Sato, Mai; Ito, Shuichi; Ishikura, Kenji

    2016-05-01

    As the morphological and immunohistochemical manifestations of immunoglobulin A (IgA) nephropathy and Henoch-Schonlein purpura nephritis (HSPN) are very similar, they are considered to share a common pathogenesis. Although HSPN usually develops after the appearance of anaphylactoid purpura, we have encountered patients whose renal symptoms preceded purpura. We reviewed the clinical courses of patients who were first diagnosed with IgA nephropathy, but developed purpura later, at the National Center for Child Health and Development in Tokyo, Japan. Of the 53 patients who were diagnosed with primary IgA nephropathy at our institute during the study period (March 2002 to July 2015), six (11 %) developed anaphylactoid purpura after the diagnosis of primary IgA nephropathy and therefore met the inclusion criteria. Duration between the onset of nephritis and subsequent appearance of purpura ranged from 5 months to 14 years. One patient reached end-stage renal failure due to IgA nephropathy and developed purpura after renal transplantation. All renal biopsies performed before the appearance of purpura showed mesangial proliferation with predominant IgA deposits. Urinary findings deteriorated in three patients after the appearance of purpura, including one patient who developed rapidly progressive glomerulonephritis. Renal biopsy findings worsened in two patients. At the last observation, two patients showed mild renal insufficiency. Our clinical experience and previous reports support the argument that IgA nephropathy and HSPN are different manifestations of a single disease. Hence, it is acceptable to consider that they are variants of a single disease.

  6. Ablation of the stress protease OMA1 protects against heart failure in mice.

    PubMed

    Acin-Perez, Rebeca; Lechuga-Vieco, Ana Victoria; Del Mar Muñoz, Maria; Nieto-Arellano, Rocío; Torroja, Carlos; Sánchez-Cabo, Fátima; Jiménez, Concepción; González-Guerra, Andrés; Carrascoso, Isabel; Benincá, Cristiane; Quiros, Pedro M; López-Otín, Carlos; Castellano, José María; Ruíz-Cabello, Jesús; Jiménez-Borreguero, Luis Jesús; Enríquez, José Antonio

    2018-03-28

    Heart failure (HF) is a major health and economic burden in developed countries. It has been proposed that the pathogenesis of HF may involve the action of mitochondria. We evaluate three different mouse models of HF: tachycardiomyopathy, HF with preserved left ventricular (LV) ejection fraction (LVEF), and LV myocardial ischemia and hypertrophy. Regardless of whether LVEF is preserved, our results indicate that the three models share common features: an increase in mitochondrial reactive oxygen species followed by ultrastructural alterations in the mitochondrial cristae and loss of mitochondrial integrity that lead to cardiomyocyte death. We show that the ablation of the mitochondrial protease OMA1 averts cardiomyocyte death in all three murine HF models, and thus loss of OMA1 plays a direct role in cardiomyocyte protection. This finding identifies OMA1 as a potential target for preventing the progression of myocardial damage in HF associated with a variety of etiologies. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  7. Rapid discovery and structure-activity profiling of novel inhibitors of human immunodeficiency virus type 1 protease enabled by the copper(I)-catalyzed synthesis of 1,2,3-triazoles and their further functionalization.

    PubMed

    Whiting, Matthew; Tripp, Jonathan C; Lin, Ying-Chuan; Lindstrom, William; Olson, Arthur J; Elder, John H; Sharpless, K Barry; Fokin, Valery V

    2006-12-28

    Building from the results of a computational screen of a range of triazole-containing compounds for binding efficiency to human immunodeficiency virus type 1 protease (HIV-1-Pr), a novel series of potent inhibitors has been developed. The copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), which provides ready access to 1,4-disubstituted-1,2,3-triazoles, was used to unite a focused library of azide-containing fragments with a diverse array of functionalized alkyne-containing building blocks. In combination with direct screening of the crude reaction products, this method led to the rapid identification of a lead structure and readily enabled optimization of both azide and alkyne fragments. Replacement of the triazole with a range of alternative linkers led to greatly reduced protease inhibition; however, further functionalization of the triazoles at the 5-position gave a series of compounds with increased activity, exhibiting Ki values as low as 8 nM.

  8. Translocalized IgA mediates neutralization and stimulates innate immunity inside infected cells.

    PubMed

    Bidgood, Susanna R; Tam, Jerry C H; McEwan, William A; Mallery, Donna L; James, Leo C

    2014-09-16

    IgA is the most prevalent antibody type on mucosal surfaces and the second most prevalent antibody in circulation, yet its role in immune defense is not fully understood. Here we show that IgA is carried inside cells during virus infection, where it activates intracellular virus neutralization and innate immune signaling. Cytosolic IgA-virion complexes colocalize with the high-affinity antibody receptor tripartite motif-containing protein 21 (TRIM21) and are positive for lysine-48 ubiquitin chains. IgA neutralizes adenovirus infection in a TRIM21- and proteasome-dependent manner in both human and mouse cells. Translocated IgA also potently activates NF-κB signaling pathways in cells expressing TRIM21, whereas viral infection in the absence of antibody or TRIM21 is undetected. TRIM21 recognizes an epitope in IgG Fc that is not conserved in IgA; however, fluorescence anisotropy experiments demonstrate that direct binding to IgA is maintained. We use molecular modeling to show that TRIM21 forms a nonspecific hydrophobic seal around a β-loop structure that is present in IgG, IgM, and IgA, explaining how TRIM21 achieves such remarkable broad antibody specificity. The findings demonstrate that the antiviral protection afforded by IgA extends to the intracellular cytosolic environment.

  9. Translocalized IgA mediates neutralization and stimulates innate immunity inside infected cells

    PubMed Central

    Bidgood, Susanna R.; Tam, Jerry C. H.; McEwan, William A.; Mallery, Donna L.; James, Leo C.

    2014-01-01

    IgA is the most prevalent antibody type on mucosal surfaces and the second most prevalent antibody in circulation, yet its role in immune defense is not fully understood. Here we show that IgA is carried inside cells during virus infection, where it activates intracellular virus neutralization and innate immune signaling. Cytosolic IgA–virion complexes colocalize with the high-affinity antibody receptor tripartite motif-containing protein 21 (TRIM21) and are positive for lysine-48 ubiquitin chains. IgA neutralizes adenovirus infection in a TRIM21- and proteasome-dependent manner in both human and mouse cells. Translocated IgA also potently activates NF-κB signaling pathways in cells expressing TRIM21, whereas viral infection in the absence of antibody or TRIM21 is undetected. TRIM21 recognizes an epitope in IgG Fc that is not conserved in IgA; however, fluorescence anisotropy experiments demonstrate that direct binding to IgA is maintained. We use molecular modeling to show that TRIM21 forms a nonspecific hydrophobic seal around a β-loop structure that is present in IgG, IgM, and IgA, explaining how TRIM21 achieves such remarkable broad antibody specificity. The findings demonstrate that the antiviral protection afforded by IgA extends to the intracellular cytosolic environment. PMID:25169018

  10. Intranasal IFNγ extends passive IgA antibody protection of mice against Mycobacterium tuberculosis lung infection

    PubMed Central

    Reljic, R; Clark, S O; Williams, A; Falero-Diaz, G; Singh, M; Challacombe, S; Marsh, P D; Ivanyi, J

    2006-01-01

    Intranasal inoculation of mice with monoclonal IgA against the α-crystallin (acr1) antigen can diminish the tuberculous infection in the lungs. As this effect has been observed only over a short-term, we investigated if it could be extended by inoculation of IFNγ 3 days before infection, and further coinoculations with IgA, at 2 h before and 2 and 7 days after aerosol infection with Mycobacterium tuberculosis H37Rv. This treatment reduced the lung infection at 4 weeks more than either IgA or IFNγ alone (i.e. 17-fold, from 4·2 × 107 to 2·5 × 106 CFU, P = 0·006), accompanied also by lower granulomatous infiltration of the lungs. IFNγ added prior to infection of mouse peritoneal macrophages with IgA-opsonized bacilli resulted in a synergistic increase of nitric oxide and TNFα production and a 2–3 fold decrease in bacterial counts. Our improved results suggest, that combined treatment with IFNγ and IgA could be developed towards prophylactic treatment of AIDS patients, or as an adjunct to chemotherapy. PMID:16487246

  11. Evaluation of three fully automated immunoassay systems for detection of IgA anti-beta 2-glycoprotein I antibodies.

    PubMed

    Pérez, D; Martínez-Flores, J A; Serrano, M; Lora, D; Paz-Artal, E; Morales, J M; Serrano, A

    2016-10-01

    In recent years, we have been witnessing increased clinical interest in the determination of IgA anti-beta 2-glycoprotein I (aB2GPI) antibodies as well as increased demand for this test. Some ELISA-based diagnostic systems for IgA aB2GPI antibodies detection are suboptimal to detect it. The aim of our study was to determine whether the diagnostic yield of modern detection systems based on automatic platforms to measure IgA aB2GPI is equivalent to that of the well-optimized ELISA-based assays. In total, 130 patients were analyzed for IgA aB2GPI by three fully automated immunoassays using an ELISA-based assay as reference. The three systems were also analyzed for IgG aB2GPI with 58 patients. System 1 was able to detect IgA aB2GPI with good sensitivity and kappa index (99% and 0.72, respectively). The other two systems had also poor sensitivity (20% and 15%) and kappa index (0.10 and 0.07), respectively. On the other hand, kappa index for IgG aB2GPI was >0.89 in the three systems. Some analytical methods to detect IgA aB2GPI are suboptimal as well as some ELISA-based diagnostic systems. It is important that the scientific community work to standardize analytical methods to determine IgA aB2GPI antibodies. © 2016 John Wiley & Sons Ltd.

  12. Serine proteases SP1 and SP13 mediate the melanization response of Asian corn borer, Ostrinia furnacalis, against entomopathogenic fungus Beauveria bassiana.

    PubMed

    Chu, Yuan; Liu, Yang; Shen, Dongxu; Hong, Fang; Wang, Guirong; An, Chunju

    2015-06-01

    Exposure to entomopathogenic fungi is one approach for insect pest control. Little is known about the immune interactions between fungus and its insect host. Melanization is a prominent immune response in insects in defending against pathogens such as bacteria and fungi. Clip domain serine proteases in insect plasma have been implicated in the activation of prophenoloxidase, a key enzyme in the melanization. The relationship between host melanization and the infection by a fungus needs to be established. We report here that the injection of entomopathogenic fungus Beauveria bassiana induced both melanin synthesis and phenoloxidase activity in its host insect, the Asian corn borer, Ostrinia furnacalis (Guenée). qRT-PCR analysis showed several distinct patterns of expression of 13 clip-domain serine proteases in response to the challenge of fungi, with seven increased, two decreased, and four unchanged. Of special interest among these clip-domain serine protease genes are SP1 and SP13, the orthologs of Manduca sexta HP6 and PAP1 which are involved in the prophenoloxidase activation pathway. Recombinant O. furnacalis SP1 was found to activate proSP13 and induce the phenoloxidase activity in corn borer plasma. Additionally, SP13 was determined to directly cleave prophenoloxidase and therefore act as the prophenoloxidase activating protease. Our work thus reveals a biochemical mechanism in the melanization in corn borer associated with the challenge by B. bassiana injection. These insights could provide valuable information for better understanding the immune responses of Asian corn borer against B. bassiana. Copyright © 2015 Elsevier Inc. All rights reserved.

  13. A Pseudomonas aeruginosa strain isolated from a contact lens-induced acute red eye (CLARE) is protease-deficient.

    PubMed

    Estrellas, P S; Alionte, L G; Hobden, J A

    2000-03-01

    Pseudomonas aeruginosa proteases are thought to be important virulence factors in the pathogenesis of corneal disease. This study examined protease production from two strains of P. aeruginosa responsible for two very distinct clinical diseases: strain Paer1, isolated from a Contact Lens-induced Acute Red Eye (CLARE), and strain KEI 1025, isolated from a corneal ulcer. Strains were compared to a laboratory strain (ATCC 19660) known to produce severe keratitis in experimentally infected mice for protease production and for ocular virulence. Protease production was examined with colorimetric assays, gelatin zymography and western blots. Elastase A activity was quantitated with a staphylolytic assay. Ocular virulence was examined using a mouse scratch model of keratitis. In contrast to strains KEI 1025 or ATCC 19660, Paer1 was unable to produce enzymatically active elastase A, elastase, and protease IV. All three strains produced active alkaline protease. Strains KEI 1025 and ATCC 19660 produced a fulminant keratitis in mice whereas Paer1 produced a mild transient infection. Restoration of elastase activity in Paer1 via genetic complementation did not result in a virulent phenotype. Co-infection of mouse eyes with strains Paer1 and ATCC 19660 resulted in the eventual loss of Paer1 from corneal tissue. These studies suggest that P. aeruginosa elastase A and/or protease IV, but not alkaline protease or elastase, contribute to the ocular virulence of this organism.

  14. Hepatitis A Virus Capsid Protein VP1 Has a Heterogeneous C Terminus

    PubMed Central

    Graff, Judith; Richards, Oliver C.; Swiderek, Kristine M.; Davis, Michael T.; Rusnak, Felicia; Harmon, Shirley A.; Jia, Xi-Yu; Summers, Donald F.; Ehrenfeld, Ellie

    1999-01-01

    Hepatitis A virus (HAV) encodes a single polyprotein which is posttranslationally processed into the functional structural and nonstructural proteins. Only one protease, viral protease 3C, has been implicated in the nine protein scissions. Processing of the capsid protein precursor region generates a unique intermediate, PX (VP1-2A), which accumulates in infected cells and is assumed to serve as precursor to VP1 found in virions, although the details of this reaction have not been determined. Coexpression in transfected cells of a variety of P1 precursor proteins with viral protease 3C demonstrated efficient production of PX, as well as VP0 and VP3; however, no mature VP1 protein was detected. To identify the C-terminal amino acid residue of HAV VP1, we performed peptide sequence analysis by protease-catalyzed [18O]H2O incorporation followed by liquid chromatography ion-trap microspray tandem mass spectrometry of HAV VP1 isolated from purified virions. Two different cell culture-adapted isolates of HAV, strains HM175pE and HM175p35, were used for these analyses. VP1 preparations from both virus isolates contained heterogeneous C termini. The predominant C-terminal amino acid in both virus preparations was VP1-Ser274, which is located N terminal to a methionine residue in VP1-2A. In addition, the analysis of HM175pE recovered smaller amounts of amino acids VP1-Glu273 and VP1-Thr272. In the case of HM175p35, which contains valine at amino acid position VP1-273, VP1-Thr272 was found in addition to VP1-Ser274. The data suggest that HAV 3C is not the protease responsible for generation of the VP1 C terminus. We propose the involvement of host cell protease(s) in the production of HAV VP1. PMID:10364353

  15. Additive In Vitro Antiplasmodial Effect of N-Alkyl and N-Benzyl-1,10-Phenanthroline Derivatives and Cysteine Protease Inhibitor E64

    PubMed Central

    Wijayanti, Mahardika Agus; Sholikhah, Eti Nurwening; Hadanu, Ruslin; Jumina, Jumina; Supargiyono, Supargiyono; Mustofa, Mustofa

    2010-01-01

    Potential new targets for antimalarial chemotherapy include parasite proteases, which are required for several cellular functions during the Plasmodium falciparum life cycle. Four new derivatives of N-alkyl and N-benzyl-1,10-phenanthroline have been synthesized. Those are (1)-N-methyl-1,10-phenanthrolinium sulfate, (1)-N-ethyl-1,10-phenanthrolinium sulfate, (1)-N-benzyl-1,10-phenanthrolinium chloride, and (1)-N-benzyl-1,10-phenanthrolinium iodide. Those compounds had potential antiplasmodial activity with IC50 values from 260.42 to 465.38 nM. Cysteine proteinase inhibitor E64 was used to investigate the mechanism of action of N-alkyl and N-benzyl-1,10-phenanthroline derivatives. A modified fixed-ratio isobologram method was used to study the in vitro interactions between the new compounds with either E64 or chloroquine. The interaction between N-alkyl and N-benzyl-1,10-phenanthroline derivatives and E64 was additive as well as their interactions with chloroquine were also additive. Antimalarial mechanism of chloroquine is mainly on the inhibition of hemozoin formation. As the interaction of chloroquine and E64 was additive, the results indicated that these new compounds had a mechanism of action by inhibiting Plasmodium proteases. PMID:22332022

  16. Modified Clp Protease Complex in the ClpP3 Null Mutant and Consequences for Chloroplast Development and Function in Arabidopsis1[C][W][OA

    PubMed Central

    Kim, Jitae; Olinares, Paul Dominic; Oh, Soo-hyun; Ghisaura, Stefania; Poliakov, Anton; Ponnala, Lalit; van Wijk, Klaas J.

    2013-01-01

    The plastid ClpPRT protease consists of two heptameric rings of ClpP1/ClpR1/ClpR2/ClpR3/ClpR4 (the R-ring) and ClpP3/ClpP4/ClpP5/ClpP6 (the P-ring) and peripherally associated ClpT1/ClpT2 subunits. Here, we address the contributions of ClpP3 and ClpP4 to ClpPRT core organization and function in Arabidopsis (Arabidopsis thaliana). ClpP4 is strictly required for embryogenesis, similar to ClpP5. In contrast, loss of ClpP3 (clpp3-1) leads to arrest at the hypocotyl stage; this developmental arrest can be removed by supplementation with sucrose or glucose. Heterotrophically grown clpp3-1 can be transferred to soil and generate viable seed, which is surprising, since we previously showed that CLPR2 and CLPR4 null alleles are always sterile and die on soil. Based on native gels and mass spectrometry-based quantification, we show that despite the loss of ClpP3, modified ClpPR core(s) could be formed, albeit at strongly reduced levels. A large portion of ClpPR subunits accumulated in heptameric rings, with overaccumulation of ClpP1/ClpP5/ClpP6 and ClpR3. Remarkably, the association of ClpT1 to the modified Clp core was unchanged. Large-scale quantitative proteomics assays of clpp3-1 showed a 50% loss of photosynthetic capacity and the up-regulation of plastoglobules and all chloroplast stromal chaperone systems. Specific chloroplast proteases were significantly up-regulated, whereas the major thylakoid protease (FtsH1/FtsH2/FtsH5/FtsH8) was clearly unchanged, indicating a controlled protease network response. clpp3-1 showed a systematic decrease of chloroplast-encoded proteins that are part of the photosynthetic apparatus but not of chloroplast-encoded proteins with other functions. Candidate substrates and an explanation for the differential phenotypes between the CLPP3, CLPP4, and CLPP5 null mutants are discussed. PMID:23548781

  17. Toxicological Effects of Nickel Chloride on IgA+ B Cells and sIgA, IgA, IgG, IgM in the Intestinal Mucosal Immunity in Broilers

    PubMed Central

    Wu, Bangyuan; Cui, Hengmin; Peng, Xi; Fang, Jing; Zuo, Zhicai; Deng, Junliang; Huang, Jianying

    2014-01-01

    The objective of this study was to investigate the toxicological effects of dietary NiCl2 on IgA+ B cells and the immunoglobulins including sIgA, IgA, IgG and IgM in the small intestine and cecal tonsil of broilers by the methods of immunohistochemistry and enzyme-linked immunosorbent assay (ELISA). Two hundred and forty one-day-old avian broilers were randomly divided into four groups and fed on a control diet and three experimental diets supplemented with 300, 600, and 900 mg/kg NiCl2 for 42 days. Compared with the control group, the IgA+ B cell number and the sIgA, IgA, IgG, and IgM contents in the NiCl2-treated groups were significantly decreased (p < 0.05 or p < 0.01). It was concluded that dietary NiCl2 in the excess of 300 mg/kg had negative effects on the IgA+ B cell number and the abovementioned immunoglobulin contents in the small intestine and the cecal tonsil. NiCl2-reduced sIgA, IgA, IgG and IgM contents is due to decrease in the population and/or the activation of B cell. The results suggest that NiCl2 at high levels has intestinal mucosal humoral immunotoxicity in animals. PMID:25116637

  18. Mucosal IgA increase in rats by continuous CLA feeding during suckling and early infancy.

    PubMed

    Pérez-Cano, Francisco J; Ramírez-Santana, Carolina; Molero-Luís, Marta; Castell, Margarida; Rivero, Montserrat; Castellote, Cristina; Franch, Angels

    2009-03-01

    The aim of this work was to establish the effect of the cis9,trans11 conjugated linoleic acid (CLA) isomer on mucosal immunity during early life in rats, a period when mucosal immunoglobulin production is poorly developed, as is also the case in humans. CLA supplementation was performed during three life periods: gestation, suckling, and early infancy. The immune status of supplemented animals was evaluated at two time points: at the end of the suckling period (21-day-old rats) and 1 week after weaning (28-day-old rats). Secretory IgA was quantified in intestinal washes from 28-day-old rats by ELISA technique. IgA, TGFbeta, and PPARgamma mRNA expression was measured in small intestine and colon by real time PCR, using Taqman specific probes and primers. IgA mucosal production was enhanced in animals supplemented with CLA during suckling and early infancy: in 28-day-old rats, IgA mRNA expression was increased in small intestine and colon by approximately 6- and 4-fold, respectively, and intestinal IgA protein by approximately 2-fold. TGFbeta gene expression was independent of age and type of tissue considered, and was not modified by dietary CLA. Gene expression of PPARgamma, a possible mediator of CLA's effects was also upregulated in animals receiving CLA during early life. In conclusion, dietary supplementation with CLA during suckling and extended to early infancy enhances development of the intestinal immune response in rats.

  19. Defect of Hepatocyte Growth Factor Activator Inhibitor Type 1/Serine Protease Inhibitor, Kunitz Type 1 (Hai-1/Spint1) Leads to Ichthyosis-Like Condition and Abnormal Hair Development in Mice

    PubMed Central

    Nagaike, Koki; Kawaguchi, Makiko; Takeda, Naoki; Fukushima, Tsuyoshi; Sawaguchi, Akira; Kohama, Kazuyo; Setoyama, Mitsuru; Kataoka, Hiroaki

    2008-01-01

    Hepatocyte growth factor activator inhibitor type 1 (HAI-1)/serine protease inhibitor, Kunitz type 1 (SPINT1) is a membrane-bound, serine proteinase inhibitor initially identified as an inhibitor of hepatocyte growth factor activator. It also inhibits matriptase and prostasin, both of which are membrane-bound serine proteinases that have critical roles in epidermal differentiation and function. In this study, skin and hair phenotypes of mice lacking the Hai-1/Spint1 gene were characterized. Previously, we reported that the homozygous deletion of Hai-1/Spint1 in mice resulted in embryonic lethality attributable to impaired placental development. To test the role of Hai-1/Spint1 in mice, the placental function of Hai-1/Spint1-mutant mice was rescued. Injection of Hai-1/Spint1+/+ blastocysts with Hai-1/Spint1−/− embryonic stem cells successfully generated high-chimeric Hai-1/Spint1−/− embryos (B6Hai-1−/−High) with normal placentas. These embryos were delivered without apparent developmental abnormalities, confirming that embryonic lethality of Hai-1/Spint1−/− mice was caused by placental dysfunction. However, newborn B6Hai-1−/−High mice showed growth retardation and died by 16 days. These mice developed scaly skin because of hyperkeratinization, reminiscent of ichthyosis, and abnormal hair shafts that showed loss of regular cuticular septation. The interfollicular epidermis showed acanthosis with enhanced Akt phosphorylation. Immunoblot analysis revealed altered proteolytic processing of profilaggrin in Hai-1/Spint1-deleted skin with impaired generation of filaggrin monomers. These findings indicate that Hai-1/Spint1 has critical roles in the regulated keratinization of the epidermis and hair development. PMID:18832587

  20. Production of interleukin-2 (IL-2) and expression of IL-2 receptor in patients with IgA nephropathy.

    PubMed

    Lee, T W; Kim, M J

    1992-01-01

    IL-2 production has been measured in several disease including type I diabetes mellitus, systemic lupus erythematosus, acquired immunodeficiency syndrome and active pulmonary sarcoidosis and its pathogenetic role was suggested. In IgA nephropathy, altered T cell subsets were reported to be associated with increased synthesis of IgA. The altered IL-2 production and the expression of IL-2 receptor might be involved in the pathogenesis of IgA nephropathy. To investigate the role of T cell mediated immunity in the pathogenesis of IgA nephropathy, the immune parameters such as T cell subsets, NK cell activity, interleukin-2 (IL-2) production and IL-2 receptor expression on peripheral blood mononuclear cells (PBMC) were measured before and/or after phytohemagglutinin (PHA) stimulation in 15 patients with IgA nephropathy. Age and sex matched 15 healthy controls and the correlations between the IL-2 production and immune parameters were evaluated. The mean percentages of T helper/inducer cells (CD4), T suppressor/cytotoxic cells (CD8) and the CD4/CD8 ratio of the patients were not different from those of controls and the proportions of CD8 CD11b cell in the patients (21.0 +/- 3.6%) were significantly lower than those in controls (30.5 +/- 5.3%) (p < 0.005). The production of IL-2 by fresh PBMC of both patients and controls was in undetectable ranges. The production of IL-2 by PHA stimulated PBMC of patients was significantly higher than that of controls (140.03 +/- 43.2 U/ml vs 106.5 +/- 42.1 U/ml, p < 0.05). The proportions of lymphocytes expressing the IL-2 receptor (CD25) before the stimulation with PHA in patients were 1.22 +/- 1.00 percent and were not different from those in controls (1.12 +/- 0.78 percent). The correlations between the production of IL-2 and the concentrations of serum IgA, the degrees of histologic alterations and the proportions of CD8 and CD8CD11b cells were not significant. There was a weak tendency of a positive correlation (p < 0.1) between

  1. The sites of catabolism of murine monomeric IgA.

    PubMed

    Moldoveanu, Z; Epps, J M; Thorpe, S R; Mestecky, J

    1988-07-01

    The tissue sites of monomeric IgA (mIgA) catabolism were determined in a BALB/c mouse model. Mouse mIgA myeloma proteins were labeled either by direct iodination or by coupling the residualizing label, dilactitol-125I-tyramine (125I-DLT) to the proteins; catabolites from protein labeled with 125I-DLT accumulate at the site of protein degradation, allowing identification of the tissue and cellular sites involved in catabolism of the protein. The circulating half-lives of 125I- and 125I-DLT-mIgA were the same. The distribution of radioactivity in tissues was measured at 1, 3, 24, and 96 h after iv. injection of 125I-DLT-labeled mIgA, dimeric IgA (dIgA), IgG, or mouse serum albumin. The greatest uptake of 125I-DLT-mIgA was attributable to the liver. This organ accounted for more internal catabolism of mIgA than all other tissues combined. In contrast, 125I-DLT-IgG was catabolized equally in skin, muscle, and liver. These data indicate that, in mice, the liver is the major site of mIgA catabolism. To determine the cell types involved, collagenase digestion was used to isolate parenchymal and non-parenchymal cells from perfused liver of animals injected with 125-DLT-mIgA. Most of the radioactivity was associated with the hepatocyte fraction, even though both cell types showed uptake of 125I-DLT-mIgA. Inhibition studies, with asialofetuin and mouse IgA demonstrated that the uptake of mIgA by liver cells was mediated primarily by the asialoglycoprotein receptor.

  2. Recognition of Epidermal Transglutaminase by IgA and Tissue Transglutaminase 2 Antibodies in a Rare Case of Rhesus Dermatitis

    PubMed Central

    Sestak, Karol; Mazumdar, Kaushiki; Midkiff, Cecily C.; Dufour, Jason; Borda, Juan T.; Alvarez, Xavier

    2011-01-01

    Tissue transglutaminase 2 (tTG2) is an intestinal digestive enzyme which deamidates already partially digested dietary gluten e.g. gliadin peptides. In genetically predisposed individuals, tTG2 triggers autoimmune responses that are characterized by the production of tTG2 antibodies and their direct deposition into small intestinal wall 1,2. The presence of such antibodies constitutes one of the major hallmarks of the celiac disease (CD). Epidermal transglutaminase (eTG) is another member of the transglutaminase family that can also function as an autoantigen in a small minority of CD patients. In these relatively rare cases, eTG triggers an autoimmune reaction (a skin rash) clinically known as dermatitis herpetiformis (DH). Although the exact mechanism of CD and DH pathogenesis is not well understood, it is known that tTG2 and eTG share antigenic epitopes that can be recognized by serum antibodies from both CD and DH patients 3,4. In this study, the confocal microscopy examination of biopsy samples from skin lesions of two rhesus macaques (Macaca mulatta) with dermatitis (Table 1, Fig. 1 and 2) was used to study the affected tissues. In one animal (EM96) a spectral overlap of IgA and tTG2 antibodies (Fig. 3) was demonstrated. The presence of double-positive tTG2+IgA+ cells was focused in the deep epidermis, around the dermal papillae. This is consistent with lesions described in DH patients 3. When EM96 was placed on a gluten-free diet, the dermatitis, as well as tTG2+IgA+ deposits disappeared and were no longer detectable (Figs. 1-3). Dermatitis reappeared however, based on re-introduction of dietary gluten in EM96 (not shown). In other macaques including animal with unrelated dermatitis, the tTG2+IgA+ deposits were not detected. Gluten-free diet-dependent remission of dermatitis in EM96 together with presence of tTG2+IgA+ cells in its skin suggest an autoimmune, DH-like mechanism for the development of this condition. This is the first report of DH

  3. A novel method for detecting IgA endomysial antibodies by using human umbilical vein endothelial cells.

    PubMed

    Castellino, F; Scaglione, N; Grosso, S B; Sategna-Guidetti, C

    2000-01-01

    Although tissue transglutaminase was recently identified as the main autoantigen recognized by endomysial antibodies in coeliac patients, anti-endomysium antibody detection still persists as the gold standard for coeliac disease screening and diagnosis. (1) To evaluate human umbilical vein cells (HUVEC) as an alternative source of endomysial antigen and to assess their suitability in the diagnosis of coeliac disease. (2) To verify whether tissue transglutaminase is one target antigen eliciting the endomysial antibody fraction of coeliac serum IgA. University teaching hospital. Sera from 123 untreated adults with biopsy-proven coeliac disease and 84 controls (40 healthy and 44 diseased) were assessed by indirect immunofluorescence, using HUVEC on glass slides prepared by cytocentrifugation and permeabilized by using Triton X (0.5%). Indirect immunofluorescence was performed: (1) using coeliac disease serum samples on HUVEC with or without prior incubation with tissue transglutaminase; and (2) incubating both HUVEC and monkey oesophagus with goat anti-guinea pig tissue transglutaminase antibody. All the coeliac patients, who were also positive on monkey oesophagus, showed the typical fluorescent homogeneous cytoplasmic stain on HUVEC. All control sera were negative both on HUVEC and on monkey oesophagus. IgA antibodies did not react with non-permeabilized cells, with intact membrane. Preincubation of coeliac sera with tissue transglutaminase abolished the typical fluorescent pattern. The incubation of anti-tissue transglutaminase antibody with monkey oesophagus and HUVEC resulted in an immunofluorescence staining pattern identical to that obtained with positive coeliac sera. (1) As a substrate for anti-endomysial antibody, HUVEC may provide the same diagnostic accuracy as monkey oesophagus, thus bypassing economical and ethical problems. The HUVEC antigen reacting with IgA from coeliac disease sera is an intracellular rather than a cell-surface antigen, as IgA

  4. Dual renin-angiotensin system blockade plus oral methylprednisone for the treatment of proteinuria in IgA nephropathy.

    PubMed

    Trimarchi, Hernán; Muryan, Alexis; Young, Pablo; Forrester, Mariano; Iotti, Alejandro; Pereyra, Horacio; Lombi, Fernando; Seminario, Omar; Alonso, Mirta; Iotti, Roberto

    2007-01-01

    Renin-angiotensin system inhibition is a widely accepted approach to initially deal with proteinuria in IgA nephropathy, while the role of immunosuppressants remains controversial in many instances. A prospective, uncontrolled, open-label trial was undertaken in patients with biopsy-proven IgA nephropathy with proteinuria > 0.5 g/day and normal renal function to assess the efficacy of a combination treatment of angiotensin converting enzyme inhibitors plus angiotensin receptor blockers enalapril valsartan coupled with methylprednisone to decrease proteinuria to levels below 0.5 g/day. Twenty patients were included: Age 37.45 +/- 13.26 years (50% male); 7 patients (35%) were hypertensive; proteinuria 2.2 +/- 1.86 g/day; serum creatinine 1.07 +/- 0.29 mg/dl; mean follow-up 60.10 +/- 31.47 months. IgA nephropathy was subclassified according to Haas criteria. Twelve patients (60%) were class II; seven (35%) were class III and one (5%) class V. All patients received dual renin-angiotensin system blockade as tolerated. Oral methylprednisone was started at 0.5 mg/kg/day for the initial 8 weeks and subsequently tapered bi-weekly until the maintenance dose of 4 mg was reached. Oral steroids were discontinued after 24 weeks (6 months) of therapy but renin-angiotensin inhibition remained unchanged. At 10 weeks of therapy proteinuria decreased to 0.15 +/- 0.07 g/day (P < 0.001) while serum creatinine did not vary: 1.07 +/- 0.28 mg/dl (P = ns). After a mean follow-up of 42.36 +/- 21.56 months urinary protein excretion (0.12 +/- 0.06 g/day) and renal function (serum creatinine 1.06 +/- 0.27 mg/dl) remained stable. No major side effects were reported during the study. Renin-angiotensin blockade plus oral steroids proved useful to significantly decrease proteinuria to < 0.5 g/day in patients with IgA nephropathy without changes in renal function.

  5. Design and Synthesis of Highly Potent HIV-1 Protease Inhibitors Containing Tricyclic Fused Ring Systems as Novel P2 Ligands: Structure-Activity Studies, Biological and X-ray Structural Analysis.

    PubMed

    Ghosh, Arun K; R Nyalapatla, Prasanth; Kovela, Satish; Rao, Kalapala Venkateswara; Brindisi, Margherita; Osswald, Heather L; Amano, Masayuki; Aoki, Manabu; Agniswamy, Johnson; Wang, Yuan-Fang; Weber, Irene T; Mitsuya, Hiroaki

    2018-05-24

    The design, synthesis, and biological evaluation of a new class of HIV-1 protease inhibitors containing stereochemically defined fused tricyclic polyethers as the P2 ligands and a variety of sulfonamide derivatives as the P2' ligands are described. A number of ring sizes and various substituent effects were investigated to enhance the ligand-backbone interactions in the protease active site. Inhibitors 5c and 5d containing this unprecedented fused 6-5-5 ring system as the P2 ligand, an aminobenzothiazole as the P2' ligand, and a difluorophenylmethyl as the P1 ligand exhibited exceptional enzyme inhibitory potency and maintained excellent antiviral activity against a panel of highly multidrug-resistant HIV-1 variants. The umbrella-like P2 ligand for these inhibitors has been synthesized efficiently in an optically active form using a Pauson-Khand cyclization reaction as the key step. The racemic alcohols were resolved efficiently using a lipase catalyzed enzymatic resolution. Two high resolution X-ray structures of inhibitor-bound HIV-1 protease revealed extensive interactions with the backbone atoms of HIV-1 protease and provided molecular insight into the binding properties of these new inhibitors.

  6. Network Analyses Reveal Pervasive Functional Regulation Between Proteases in the Human Protease Web

    PubMed Central

    Fortelny, Nikolaus; Cox, Jennifer H.; Kappelhoff, Reinhild; Starr, Amanda E.; Lange, Philipp F.; Pavlidis, Paul; Overall, Christopher M.

    2014-01-01

    Proteolytic processing is an irreversible posttranslational modification affecting a large portion of the proteome. Protease-cleaved mediators frequently exhibit altered activity, and biological pathways are often regulated by proteolytic processing. Many of these mechanisms have not been appreciated as being protease-dependent, and the potential in unraveling a complex new dimension of biological control is increasingly recognized. Proteases are currently believed to act individually or in isolated cascades. However, conclusive but scattered biochemical evidence indicates broader regulation of proteases by protease and inhibitor interactions. Therefore, to systematically study such interactions, we assembled curated protease cleavage and inhibition data into a global, computational representation, termed the protease web. This revealed that proteases pervasively influence the activity of other proteases directly or by cleaving intermediate proteases or protease inhibitors. The protease web spans four classes of proteases and inhibitors and so links both recently and classically described protease groups and cascades, which can no longer be viewed as operating in isolation in vivo. We demonstrated that this observation, termed reachability, is robust to alterations in the data and will only increase in the future as additional data are added. We further show how subnetworks of the web are operational in 23 different tissues reflecting different phenotypes. We applied our network to develop novel insights into biologically relevant protease interactions using cell-specific proteases of the polymorphonuclear leukocyte as a system. Predictions from the protease web on the activity of matrix metalloproteinase 8 (MMP8) and neutrophil elastase being linked by an inactivating cleavage of serpinA1 by MMP8 were validated and explain perplexing Mmp8 −/− versus wild-type polymorphonuclear chemokine cleavages in vivo. Our findings supply systematically derived and

  7. Value of Isolated IgA anti-β2GPI Positivity in the Diagnosis of the Antiphospholipid Syndrome

    PubMed Central

    Murthy, Vijaya; Willis, Rohan; Romay-Penabad, Zurina; Ruiz-Limón, Patricia; Martínez-Martínez, Laura A.; Jatwani, Shraddha; Jajoria, Praveen; Seif, Alan; Alarcón, Graciela S.; Papalardo, Elizabeth; Liu, Jigna; Vilá, Luis M.; McGwin, Gerald; McNearney, Terry A.; Maganti, Rashmi; Sunkureddi, Prashanth; Parekh, Trisha; Tarantino, Michael; Akhter, Ehtisham; Fang, Hong; Gonzalez, Emilio B.; Binder, Walter R.; Norman, Gary L.; Shums, Zakera; Teodorescu, Marius; Reveille, John D.; Petri, Michelle; Pierangeli, Silvia S.

    2014-01-01

    Purpose To examine the prevalence of isolated IgA anti-β2Glycoprotein I (anti-β2GPI) positivity and the association of these antibodies, and a subgroup that bind specifically to domain IV/V of β2GPI, with clinical manifestations of the Antiphospholipid Syndrome (APS) in three patients groups. The pathogenicity of IgA anti-β2GPI was also evaluated in a mouse model of thrombosis. Methods Patients with systemic lupus erythematosus (SLE) from a multiethnic, multicenter cohort (LUpus in MInorities, NAture versus nurture [LUMINA]) (n=558), patients with SLE from the Hopkins Lupus Cohort (n=215), and serum samples referred to the Antiphospholipid Standardization Laboratory (APLS) (n=5,098) were evaluated. IgA anti-β2GPI titers and binding to domain IV/V of β2GPI were examined by enzyme-linked immunosorbent assay (ELISA). CD1 mice were inoculated with purified IgA anti- β2GPI antibodies, and surgical procedures and ELISAs were performed to evaluate thrombus development and tissue factor (TF) activity. Results A total of 198 patients were found to be positive for IgA anti-β2GPI isotype, and 57 patients were positive exclusively for IgA anti-β2GPI antibodies. Of these, 13 of 23 patients (56.5%) in the LUMINA cohort, 17 of 17 patients (100%) in the Hopkins cohort, and 10 of 17 patients (58.9%) referred to APLS had at least one APS-related clinical manifestation. Fifty-four percent of all the IgA anti-β2GPI positive serum samples reacted with domain IV/V of anti-β2GPI, and 77% of those had clinical features of APS. Isolated IgA anti-β2GPI positivity was associated with an increased risk for arterial thrombosis (p<0.001), venous thrombosis (p=0.015) and all thrombosis (p<0.001). The association between isolated IgA anti-β2GPI and arterial thrombosis (p=0.0003) and all thrombosis (p=0.0003) remained significant after adjusting for other risk factors for thrombosis. In vivo mouse studies demonstrated that IgA anti-β2GPI antibodies induced significantly larger

  8. Dysfunctions of the Iga system: a common link between intestinal and renal diseases

    PubMed Central

    Papista, Christina; Berthelot, Laureline; Monteiro, Renato C

    2011-01-01

    Immunoglobulin A (Iga)-isotype antibodies play an important role in immunity owing to their structure, glycosylation, localization and receptor interactions. Dysfunctions in this system can lead to multiple types of pathology. This review describes the characteristics of Iga and discusses the involvement of abnormalities in the Iga system on the development of celiac disease and Iga nephropathy. PMID:21278767

  9. Protease-Activated Receptor 4 Induces Bladder Pain through High Mobility Group Box-1

    PubMed Central

    Kouzoukas, Dimitrios E.; Ma, Fei; Meyer-Siegler, Katherine L.; Westlund, Karin N.; Hunt, David E.; Vera, Pedro L.

    2016-01-01

    Pain is the significant presenting symptom in Interstitial Cystitis/Painful Bladder Syndrome (IC/PBS). Activation of urothelial protease activated receptor 4 (PAR4) causes pain through release of urothelial macrophage migration inhibitory factor (MIF). High Mobility Group Box-1 (HMGB1), a chromatin-binding protein, mediates bladder pain (but not inflammation) in an experimental model (cyclophosphamide) of cystitis. To determine if PAR4-induced bladder hypersensitivity depends on HMGB1 downstream, we tested whether: 1) bladder PAR4 stimulation affected urothelial HMGB1 release; 2) blocking MIF inhibited urothelial HMGB1 release; and 3) blocking HMGB1 prevented PAR4-induced bladder hypersensitivity. HMGB1 release was examined in immortalized human urothelial cultures (UROtsa) exposed to PAR4-activating peptide (PAR4-AP; 100 μM; 2 hours) or scrambled control peptide. Female C57BL/6 mice, pretreated with a HMGB1 inhibitor (glycyrrhizin: 50 mg/kg; ip) or vehicle, received intravesical PAR4-AP or a control peptide (100 μM; 1 hour) to determine 1) HMGB1 levels at 1 hour in the intravesical fluid (released HMGB1) and urothelium, and 2) abdominal hypersensitivity to von Frey filament stimulation 24 hours later. We also tested mice pretreated with a MIF blocker (ISO-1: 20 mg/kg; ip) to determine whether MIF mediated PAR4-induced urothelial HMGB1 release. PAR4-AP triggered HMGB1 release from human (in vitro) and mice (in vivo) urothelial cells. Intravesical PAR4 activation elicited abdominal hypersensitivity in mice that was prevented by blocking HMGB1. MIF inhibition prevented PAR4-mediated HMGB1 release from mouse urothelium. Urothelial MIF and HGMB1 represent novel targets for therapeutic intervention in bladder pain conditions. PMID:27010488

  10. Effect of Oral Methylprednisolone on Clinical Outcomes in Patients With IgA Nephropathy: The TESTING Randomized Clinical Trial.

    PubMed

    Lv, Jicheng; Zhang, Hong; Wong, Muh Geot; Jardine, Meg J; Hladunewich, Michelle; Jha, Vivek; Monaghan, Helen; Zhao, Minghui; Barbour, Sean; Reich, Heather; Cattran, Daniel; Glassock, Richard; Levin, Adeera; Wheeler, David; Woodward, Mark; Billot, Laurent; Chan, Tak Mao; Liu, Zhi-Hong; Johnson, David W; Cass, Alan; Feehally, John; Floege, Jürgen; Remuzzi, Giuseppe; Wu, Yangfeng; Agarwal, Rajiv; Wang, Hai-Yan; Perkovic, Vlado

    2017-08-01

    Guidelines recommend corticosteroids in patients with IgA nephropathy and persistent proteinuria, but the effects remain uncertain. To evaluate the efficacy and safety of corticosteroids in patients with IgA nephropathy at risk of progression. The Therapeutic Evaluation of Steroids in IgA Nephropathy Global (TESTING) study was a multicenter, double-blind, randomized clinical trial designed to recruit 750 participants with IgA nephropathy (proteinuria greater than 1 g/d and estimated glomerular filtration rate [eGFR] of 20 to 120 mL/min/1.73 m2 after at least 3 months of blood pressure control with renin-angiotensin system blockade] and to provide follow-up until 335 primary outcomes occurred. Patients were randomized 1:1 to oral methylprednisolone (0.6-0.8 mg/kg/d; maximum, 48 mg/d) (n = 136) or matching placebo (n = 126) for 2 months, with subsequent weaning over 4 to 6 months. The primary composite outcome was end-stage kidney disease, death due to kidney failure, or a 40% decrease in eGFR. Predefined safety outcomes were serious infection, new diabetes, gastrointestinal hemorrhage, fracture/osteonecrosis, and cardiovascular events. The mean required follow-up was estimated to be 5 years. After randomization of 262 participants (mean age, 38.6 [SD, 11.1] years; 96 [37%] women; eGFR, 59.4 mL/min/1.73 m2; urine protein excretion, 2.40 g/d) and 2.1 years' median follow-up, recruitment was discontinued because of excess serious adverse events. Serious events occurred in 20 participants (14.7%) in the methylprednisolone group vs 4 (3.2%) in the placebo group (P = .001; risk difference, 11.5% [95% CI, 4.8%-18.2%]), mostly due to excess serious infections (11 [8.1%] vs 0; risk difference, 8.1% [95% CI, 3.5%-13.9%]; P < .001), including 2 deaths. The primary renal outcome occurred in 8 participants (5.9%) in the methylprednisolone group vs 20 (15.9%) in the placebo group (hazard ratio, 0.37 [95% CI, 0.17-0.85]; risk difference, 10.0% [95% CI, 2

  11. Simultaneous Subcutaneous and Intranasal Administration of a CAF01-Adjuvanted Chlamydia Vaccine Elicits Elevated IgA and Protective Th1/Th17 Responses in the Genital Tract

    PubMed Central

    Wern, Jeanette Erbo; Sorensen, Maria Rathmann; Olsen, Anja Weinreich; Andersen, Peter; Follmann, Frank

    2017-01-01

    The selection of any specific immunization route is critical when defining future vaccine strategies against a genital infection like Chlamydia trachomatis (C.t.). An optimal Chlamydia vaccine needs to elicit mucosal immunity comprising both neutralizing IgA/IgG antibodies and strong Th1/Th17 responses. A strategic tool to modulate this immune profile and mucosal localization of vaccine responses is to combine parenteral and mucosal immunizations routes. In this study, we investigate whether this strategy can be adapted into a two-visit strategy by simultaneous subcutaneous (SC) and nasal immunization. Using a subunit vaccine composed of C.t. antigens (Ags) adjuvanted with CAF01, a Th1/Th17 promoting adjuvant, we comparatively evaluated Ag-specific B and T cell responses and efficacy in mice following SC and simultaneous SC and nasal immunization (SIM). We found similar peripheral responses with regard to interferon gamma and IL-17 producing Ag-specific splenocytes and IgG serum levels in both vaccine strategies but in addition, the SIM protocol also led to Ag-specific IgA responses and increased B and CD4+ T cells in the lung parenchyma, and in lower numbers also in the genital tract (GT). Following vaginal infection with C.t., we observed that SIM immunization gave rise to an early IgA response and IgA-secreting plasma cells in the GT in contrast to SC immunization, but we were not able to detect more rapid recruitment of mucosal T cells. Interestingly, although SIM vaccination in general improved mucosal immunity we observed no improved efficacy against genital infection compared to SC, a finding that warrants for further investigation. In conclusion, we demonstrate a novel vaccination strategy that combines systemic and mucosal immunity in a two-visit strategy. PMID:28567043

  12. Effects of HIV-1 protease on cellular functions and their potential applications in antiretroviral therapy

    PubMed Central

    2012-01-01

    Human Immunodeficiency Virus Type 1 (HIV-1) protease inhibitors (PIs) are the most potent class of drugs in antiretroviral therapies. However, viral drug resistance to PIs could emerge rapidly thus reducing the effectiveness of those drugs. Of note, all current FDA-approved PIs are competitive inhibitors, i.e., inhibitors that compete with substrates for the active enzymatic site. This common inhibitory approach increases the likelihood of developing drug resistant HIV-1 strains that are resistant to many or all current PIs. Hence, new PIs that move away from the current target of the active enzymatic site are needed. Specifically, allosteric inhibitors, inhibitors that prohibit PR enzymatic activities through non-competitive binding to PR, should be sought. Another common feature of current PIs is they were all developed based on the structure-based design. Drugs derived from a structure-based strategy may generate target specific and potent inhibitors. However, this type of drug design can only target one site at a time and drugs discovered by this method are often associated with strong side effects such as cellular toxicity, limiting its number of target choices, efficacy, and applicability. In contrast, a cell-based system may provide a useful alternative strategy that can overcome many of the inherited shortcomings associated with structure-based drug designs. For example, allosteric PIs can be sought using a cell-based system without considering the site or mechanism of inhibition. In addition, a cell-based system can eliminate those PIs that have strong cytotoxic effect. Most importantly, a simple, economical, and easy-to-maintained eukaryotic cellular system such as yeast will allow us to search for potential PIs in a large-scaled high throughput screening (HTS) system, thus increasing the chances of success. Based on our many years of experience in using fission yeast as a model system to study HIV-1 Vpr, we propose the use of fission yeast as a possible

  13. Lack of a significant impact of Gag-Protease-mediated HIV-1 replication capacity on clinical parameters in treatment-naive Japanese individuals.

    PubMed

    Sakai, Keiko; Chikata, Takayuki; Brumme, Zabrina L; Brumme, Chanson J; Gatanaga, Hiroyuki; Gatanag, Hiroyuki; Oka, Shinichi; Takiguchi, Masafumi

    2015-11-19

    HLA class I-associated escape mutations in HIV-1 Gag can reduce viral replication, suggesting that associated fitness costs could impact HIV-1 disease progression. Previous studies in North American and African cohorts have reported reduced Gag-Protease mediated viral replication capacity (Gag-Pro RC) in individuals expressing protective HLA class I alleles including HLA-B*57:01, B*27:05, and B*81:01. These studies also reported significant positive associations between Gag-Pro RCs and plasma viral load (pVL). However, these HLA alleles are virtually absent in Japan, and the importance of Gag as an immune target is not clearly defined in this population. We generated chimeric NL4-3 viruses carrying patient-derived Gag-Protease from 306 treatment-naive Japanese individuals chronically infected with HIV-1 subtype B. We analyzed associations between Gag-Pro RC and clinical markers of HIV-1 infection and host HLA expression. We observed no significant correlation between Gag-Pro RC and pVL in Japan in the overall cohort. However, upon exclusion of individuals expressing Japanese protective alleles HLA-B*52:01 and B*67:01, Gag-Pro RC correlated positively with pVL and negatively with CD4 T-cell count. Our results thus contrast with studies from other global cohorts reporting significantly lower Gag-Pro RC among persons expressing protective HLA alleles, and positive relationships between Gag-Pro RC and pVL in the overall study populations. We also identified five amino acids in Gag-Protease significantly associated with Gag-Pro RC, whose effects on RC were confirmed by site-directed mutagenesis. However, of the four mutations that decreased Gag-Pro RC, none were associated with reductions in pVL in Japan though two were associated with lower pVL in North America. These data indicate that Gag fitness does not affect clinical outcomes in subjects with protective HLA class I alleles as well as the whole Japanese population. Moreover, the impact of Gag fitness costs on HIV

  14. The role of protease-activated receptors PAR-1 and PAR-2 in the repair of 16HBE 14o(-) epithelial cell monolayers in vitro.

    PubMed

    Ewen, D; Clarke, S L; Smith, J R; Berger, C; Salmon, G; Trevethick, M; Shute, J K

    2010-03-01

    We recently reported that repair following mechanical wounding of epithelial cell layers in vitro is dependent on fibrin formation and the activity of locally expressed coagulation cascade proteins. Serine proteases of the coagulation cascade are an important group of protease-activated receptor (PAR) activators and PAR-1 to 4 are expressed by the normal bronchial epithelium. We tested the hypothesis that activation of PAR-1 and PAR-2 by coagulation cascade proteases stimulates epithelial repair via effects on fibrin formation. Using mechanically wounded 16HBE 14o(-) epithelial cell layers in culture, we investigated the effect of PAR-1 and PAR-2 agonist peptides, control partially scrambled peptides and PAR-neutralizing antibodies on the rate of repair and fibrin formation. Coagulation factors in culture supernatants were measured by immunoblot. RT-PCR was used to investigate PAR-1, PAR-2 and PGE2 receptor (EP-1 to EP-4) expression in this model and qRT-PCR to quantify responses to wounding. Additionally, we investigated the effect of exogenously added factor Xa (FXa) and neutrophil elastase and the influence of PGE2 and indomethacin on the repair response. PAR-1 and PAR-2 peptide agonists stimulated the rate of repair and enhanced the formation of a fibrin provisional matrix to support the repair process. Conversely, PAR-neutralizing antibodies inhibited repair. Under serum-free culture conditions, 16HBE 14o(-) cells expressed EP-2 and EP-3, but not EP-1 or EP-4, receptors. Wounding induced an increased expression of EP-3 but did not alter EP-2, PAR-1 or PAR-2 expression. In the absence of PAR agonists, there was no evidence for a role for PGE2 in fibrin formation or the repair process. Indomethacin attenuated fibrin formation in wounded cultures only in the presence of the PAR-2 peptide. FXa stimulated epithelial repair while neutrophil elastase reduced the levels of coagulation factors and inhibited repair. Locally expressed serine proteases of the coagulation

  15. Processing sites in the human immunodeficiency virus type 1 (HIV-1) Gag-Pro-Pol precursor are cleaved by the viral protease at different rates

    PubMed Central

    Pettit, Steve C; Lindquist, Jeffrey N; Kaplan, Andrew H; Swanstrom, Ronald

    2005-01-01

    We have examined the kinetics of processing of the HIV-1 Gag-Pro-Pol precursor in an in vitro assay with mature protease added in trans. The processing sites were cleaved at different rates to produce distinct intermediates. The initial cleavage occurred at the p2/NC site. Intermediate cleavages occurred at similar rates at the MA/CA and RT/IN sites, and to a lesser extent at sites upstream of RT. Late cleavages occurred at the sites flanking the protease (PR) domain, suggesting sequestering of these sites. We observed paired intermediates indicative of half- cleavage of RT/RH site, suggesting that the RT domain in Gag-Pro-Pol was in a dimeric form under these assay conditions. These results clarify our understanding of the processing kinetics of the Gag-Pro-Pol precursor and suggest regulated cleavage. Our results further suggest that early dimerization of the PR and RT domains may serve as a regulatory element to influence the kinetics of processing within the Pol domain. PMID:16262906

  16. Defective anti-polysaccharide IgG vaccine responses in IgA deficient mice.

    PubMed

    Furuya, Yoichi; Kirimanjeswara, Girish S; Roberts, Sean; Racine, Rachael; Wilson-Welder, Jennifer; Sanfilippo, Alan M; Salmon, Sharon L; Metzger, Dennis W

    2017-09-05

    We report that IgA -/- mice exhibit specific defects in IgG antibody responses to various polysaccharide vaccines (Francisella tularensis LPS and Pneumovax), but not protein vaccines such as Fluzone. This defect further included responses to polysaccharide-protein conjugate vaccines (Prevnar and Haemophilus influenzae type b-tetanus toxoid vaccine). In agreement with these findings, IgA -/- mice were protected from pathogen challenge with protein- but not polysaccharide-based vaccines. Interestingly, after immunization with live bacteria, IgA +/+ and IgA -/- mice were both resistant to lethal challenge and their IgG anti-polysaccharide antibody responses were comparable. Immunization with live bacteria, but not purified polysaccharide, induced production of serum B cell-activating factor (BAFF), a cytokine important for IgG class switching; supplementing IgA -/- cell cultures with BAFF enhanced in vitro polyclonal IgG production. Taken together, these findings show that IgA deficiency impairs IgG class switching following vaccination with polysaccharide antigens and that live bacterial immunization can overcome this defect. Since IgA deficient patients also often show defects in antibody responses following immunization with polysaccharide vaccines, our findings could have relevance to the clinical management of this population. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Design, Synthesis, Biological Evaluation, and X-ray Studies of HIV-1 Protease Inhibitors with Modified P2′ Ligands of Darunavir

    PubMed Central

    Fyvie, W. Sean; Brindisi, Margherita; Steffey, Melinda; Agniswamy, Johnson; Wang, Yuan-Fang; Aoki, Manabu; Amano, Masayuki; Weber, Irene T.; Mitsuya, Hiroaki

    2018-01-01

    The structure-based design, synthesis, and biological evaluation of a series of nonpeptidic HIV-1 protease inhibitors with rationally designed P2′ ligands are described. The inhibitors are designed to enhance backbone binding interactions, particularly at the S2′ subsite. Synthesis of inhibitors was carried out efficiently. The stereochemistry of alcohol functionalities of the P2′ ligands was set by asymmetric reduction of the corresponding ketone using (R,R)- or (S,S)-Noyori catalysts. A number of inhibitors displayed very potent enzyme inhibitory and antiviral activity. Inhibitors 3g and 3h showed enzyme Ki values of 27.9 and 49.7 pM and antiviral activity of 6.2 and 3.9 nM, respectively. These inhibitors also remained quite potent against darunavir-resistant HIV-1 variants. An X-ray structure of inhibitor 3g in complex with HIV-1 protease revealed key interactions in the S2′ subsite. PMID:29110408

  18. Boosted protease inhibitor monotherapy versus boosted protease inhibitor plus lamivudine dual therapy as second-line maintenance treatment for HIV-1-infected patients in sub-Saharan Africa (ANRS12 286/MOBIDIP): a multicentre, randomised, parallel, open-label, superiority trial.

    PubMed

    Ciaffi, Laura; Koulla-Shiro, Sinata; Sawadogo, Adrien Bruno; Ndour, Cheik Tidiane; Eymard-Duvernay, Sabrina; Mbouyap, Pretty Rosereine; Ayangma, Liliane; Zoungrana, Jacques; Gueye, Ndeye Fatou Ngom; Diallo, Mohamadou; Izard, Suzanne; Bado, Guillaume; Kane, Coumba Toure; Aghokeng, Avelin Fobang; Peeters, Martine; Girard, Pierre Marie; Le Moing, Vincent; Reynes, Jacques; Delaporte, Eric

    2017-09-01

    Despite satisfactory efficacy of WHO-recommended second-line antiretroviral treatment for patients with HIV in low-income countries, the need for simplified, low-cost, and less-toxic maintenance strategies remains high. We compared boosted protease inhibitor monotherapy with dual therapy with boosted protease inhibitor plus lamivudine in patients on second-line antiretrovial therapy (ART). We did a multicentre, randomised, parallel, open-label, superiority, trial in the HIV services of five hospitals in sub-Saharan Africa (Yaoundé, Cameroon; Dakar, Senegal; and Bobo Dioulasso, Burkina Faso). We recruited patients from the long-term, post-trial cohort of the ANRS 12169/2LADY study that compared the efficacy of three second-line combinations based on boosted protease inhibitors. Participants for our study were HIV-1 infected with multiple mutations including M184V, at first-line failure, aged 18 years and older, on boosted protease inhibitor plus two nucleoside reverse transcriptase inhibitors (NRTI) for at least 48 weeks with at least 48 weeks follow-up in the 2LADY trial, with two viral load measurements of less than 200 copies per mL in the previous 6 months, CD4 counts of more than 100 cells per μL, adherence of at least 90%, and no change to ART in the past 3 months. We randomly assigned participants (1:1) to receive either monotherapy with their boosted protease inhibitor (once-daily darunavir 800 mg [two 400 mg tablets] boosted with ritonavir 100 mg [one tablet] or coformulation of lopinavir 200 mg with ritonavir 50 mg [two tablets taken twice per day]) or to boosted protease inhibitor plus once-daily lamivudine 300 mg (one 300 mg tablet or two 150 mg tablets). Computer-generated randomisation was stratified by study site and viral load at screening (< 50 copies per mL, and 50-200 copies per mL), and concealed from study personnel throughout the inclusion period. After randomisation, treatment allocation was not masked from clinicians or patients]. Patients

  19. Adaptor Protein Complex-2 (AP-2) and Epsin-1 Mediate Protease-activated Receptor-1 Internalization via Phosphorylation- and Ubiquitination-dependent Sorting Signals*

    PubMed Central

    Chen, Buxin; Dores, Michael R.; Grimsey, Neil; Canto, Isabel; Barker, Breann L.; Trejo, JoAnn

    2011-01-01

    Signaling by protease-activated receptor-1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, is regulated by desensitization and internalization. PAR1 desensitization is mediated by β-arrestins, like most classic GPCRs. In contrast, internalization of PAR1 occurs through a clathrin- and dynamin-dependent pathway independent of β-arrestins. PAR1 displays two modes of internalization. Constitutive internalization of unactivated PAR1 is mediated by the clathrin adaptor protein complex-2 (AP-2), where the μ2-adaptin subunit binds directly to a tyrosine-based motif localized within the receptor C-tail domain. However, AP-2 depletion only partially inhibits agonist-induced internalization of PAR1, suggesting a function for other clathrin adaptors in this process. Here, we now report that AP-2 and epsin-1 are both critical mediators of agonist-stimulated PAR1 internalization. We show that ubiquitination of PAR1 and the ubiquitin-interacting motifs of epsin-1 are required for epsin-1-dependent internalization of activated PAR1. In addition, activation of PAR1 promotes epsin-1 de-ubiquitination, which may increase its endocytic adaptor activity to facilitate receptor internalization. AP-2 also regulates activated PAR1 internalization via recognition of distal C-tail phosphorylation sites rather than the canonical tyrosine-based motif. Thus, AP-2 and epsin-1 are both required to promote efficient internalization of activated PAR1 and recognize discrete receptor sorting signals. This study defines a new pathway for internalization of mammalian GPCRs. PMID:21965661

  20. Two Membrane-Anchored Aspartic Proteases Contribute to Pollen and Ovule Development1[OPEN

    PubMed Central

    Gao, Hui; Zhang, Yinghui; Wang, Wanlei; Zhao, Keke; Liu, Chunmei; Bai, Lin; Li, Rui

    2017-01-01

    Aspartic proteases are a class of proteolytic enzymes with conserved aspartate residues, which are implicated in protein processing, maturation, and degradation. Compared with yeast and animals, plants possess a larger aspartic protease family. However, little is known about most of these enzymes. Here, we characterized two Arabidopsis (Arabidopsis thaliana) putative glycosylphosphatidylinositol (GPI)-anchored aspartic protease genes, A36 and A39, which are highly expressed in pollen and pollen tubes. a36 and a36 a39 mutants display significantly reduced pollen activity. Transmission electron microscopy and terminal-deoxynucleotidyl transferase-mediated nick end labeling assays further revealed that the unviable pollen in a36 a39 may undergo unanticipated apoptosis-like programmed cell death. The degeneration of female gametes also occurred in a36 a39. Aniline Blue staining, scanning electron microscopy, and semi in vitro guidance assays indicated that the micropylar guidance of pollen tubes is significantly compromised in a36 a39. A36 and A39 that were fused with green fluorescent protein are localized to the plasma membrane and display punctate cytosolic localization and colocalize with the GPI-anchored protein COBRA-LIKE10. Furthermore, in a36 a39, the abundance of highly methylesterified homogalacturonans and xyloglucans was increased significantly in the apical pollen tube wall. These results indicate that A36 and A39, two putative GPI-anchored aspartic proteases, play important roles in plant reproduction in Arabidopsis. PMID:27872247

  1. Role of disulphide bonds in a thermophilic serine protease aqualysin I from Thermus aquaticus YT-1.

    PubMed

    Sakaguchi, Masayoshi; Takezawa, Makoto; Nakazawa, Rie; Nozawa, Kazutaka; Kusakawa, Taro; Nagasawa, Takeshi; Sugahara, Yasusato; Kawakita, Masao

    2008-05-01

    A thermophilic serine protease, Aqualysin I, from Thermus aquaticus YT-1 has two disulphide bonds, which are also found in a psychrophilic serine protease from Vibrio sp. PA-44 and a proteinase K-like enzyme from Serratia sp. at corresponding positions. To understand the significance of these disulphide bonds in aqualysin I, we prepared mutants C99S, C194S and C99S/C194S (WSS), in which Cys69-Cys99, Cys163-Cys194 and both of these disulphide bonds, respectively, were disrupted by replacing Cys residues with Ser residues. All mutants were expressed stably in Escherichia coli. The C99S mutant was 68% as active as the wild-type enzyme at 40 degrees C in terms of k(cat) value, while C194S and WSS were only 6 and 3%, respectively, as active, indicating that disulphide bond Cys163-Cys194 is critically important for maintaining proper catalytic site conformation. Mutants C194S and WSS were less thermostable than wild-type enzyme, with a half-life at 90 degrees C of 10 min as compared to 45 min of the latter and with transition temperatures on differential scanning calorimetry of 86.7 degrees C and 86.9 degrees C, respectively. Mutant C99S was almost as stable as the wild-type aqualysin I. These results indicate that the disulphide bond Cys163-Cys194 is more important for catalytic activity and conformational stability of aqualysin I than Cys67-Cys99.

  2. IgA measurements in over 12 000 Swedish twins reveal sex differential heritability and regulatory locus near CD30L

    PubMed Central

    Viktorin, Alexander; Frankowiack, Marcel; Padyukov, Leonid; Chang, Zheng; Melén, Erik; Sääf, Annika; Kull, Inger; Klareskog, Lars; Hammarström, Lennart; Magnusson, Patrik K.E.

    2014-01-01

    In a broad attempt to improve the understanding of the genetic regulation of serum IgA levels, the heritability was estimated in over 12 000 Swedish twins, and a genome-wide association study was conducted in a subsample of 9617. Using the classical twin model the heritability was found to be significantly larger among females (61%) compared with males (21%), while contribution from shared environment (20%) was only seen for males. By modeling the genetic relationship matrix with IgA levels, we estimate that a substantial proportion (31%) of variance in IgA levels can ultimately be explained by the investigated SNPs. The genome-wide association study revealed significant association to two loci: (i) rs6928791 located on chromosome 6, 22 kb upstream of the gene SAM and SH3 domain containing 1 (SASH1) and (ii) rs13300483 on chromosome 9, situated 12 kb downstream the CD30 ligand (CD30L) encoding gene. The association to rs13300483 was replicated in two additional independent Swedish materials. The heritability of IgA levels is moderate and can partly be attributable to common variation in the CD30L locus. PMID:24676358

  3. PL-100, a novel HIV-1 protease inhibitor displaying a high genetic barrier to resistance: an in vitro selection study.

    PubMed

    Dandache, Serge; Coburn, Craig A; Oliveira, Maureen; Allison, Timothy J; Holloway, M Katharine; Wu, Jinzi J; Stranix, Brent R; Panchal, Chandra; Wainberg, Mark A; Vacca, Joseph P

    2008-12-01

    The development of new HIV inhibitors with distinct resistance profiles is essential in order to combat the development of multi-resistant viral strains. A drug discovery program based on the identification of compounds that are active against drug-resistant viruses has produced PL-100, a novel potent protease inhibitor (PI) that incorporates a lysine-based scaffold. A selection for resistance against PL-100 in cord blood mononuclear cells was performed, using the laboratory-adapted IIIb strain of HIV-1, and it was shown that resistance appears to develop slower against this compound than against amprenavir, which was studied as a control. Four mutations in protease (PR) were selected after 25 weeks: two flap mutations (K45R and M46I) and two novel active site mutations (T80I and P81S). Site-directed mutagenesis revealed that all four mutations were required to develop low-level resistance to PL-100, which is indicative of the high genetic barrier of the compound. Importantly, these mutations did not cause cross-resistance to currently marketed PIs. In contrast, the P81S mutation alone caused hypersensitivity to two other PIs, saquinavir (SQV) and nelfinavir (NFV). Analysis of p55Gag processing showed that a marked defect in protease activity caused by mutation P81S could only be compensated when K45R and M46I were present. These data correlated well with the replication capacity (RC) of the mutant viruses as measured by a standard viral growth assay, since only viruses containing all four mutations approached the RC of wild type virus. X-ray crystallography provided insight on the structural basis of the resistance conferred by the identified mutations.

  4. Interaction of human, rat, and mouse immunoglobulin A (IgA) with Staphylococcal superantigen-like 7 (SSL7) decoy protein and leukocyte IgA receptor.

    PubMed

    Wines, Bruce D; Ramsland, Paul A; Trist, Halina M; Gardam, Sandra; Brink, Robert; Fraser, John D; Hogarth, P Mark

    2011-09-23

    Host survival depends on an effective immune system and pathogen survival on the effectiveness of immune evasion mechanisms. Staphylococcus aureus utilizes a number of molecules to modulate host immunity, including the SSL family of which SSL7 binds IgA and inhibits Fcα receptor I (FcαRI)-mediated function. Other Gram-positive bacterial pathogens produce IgA binding proteins, which, similar to SSL7, also bind the Fc at the CH2/CH3 interface (the junction between constant domains 2 and 3 of the heavy chain). The opposing activities of the host FcαRI-IgA receptor ligand pair and the pathogen decoy proteins select for host and pathogen variants, which exert stronger protection or evasion, respectively. Curiously, mouse but not rat IgA contains a putative N-linked glycosylation site in the center of this host receptor and pathogen-binding site. Here, we demonstrate that this site is glycosylated and that the effect of amino acid changes and glycosylation of the CH2/CH3 interface inhibits interaction with the pathogen IgA binding protein SSL7, while maintaining binding of pIgR, essential to the biosynthesis and transport of SIgA.

  5. Isolated lymphoid follicles are not IgA inductive sites for recombinant Salmonella

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hashizume, Tomomi; Momoi, Fumiki; Kurita-Ochiai, Tomoko

    2007-08-24

    In this study, we investigated whether isolated lymphoid follicles (ILF) play a role in the regulation of intestinal IgA antibody (Ab) responses. The transfer of wild type (WT) bone marrow (BM) to lymphotoxin-{alpha}-deficient (LT{alpha}{sup -/-}) mice resulted in the formation of mature ILF containing T cells, B cells, and FDC clusters in the absence of mesenteric lymph nodes and Peyer's patches. Although the ILF restored total IgA Abs in the intestine, antigen (Ag)-specific IgA responses were not induced after oral immunization with recombinant Salmonella expressing fragment C of tetanus toxin. Moreover, Ag-specific cell proliferation was not detected in the ILF.more » Interestingly, no IgA anti-LPS Abs were detected in the fecal extracts of LT{alpha}{sup -/-} mice reconstituted with WT BM. On the basis of these findings, ILF can be presumed to play a role in the production of IgA Abs, but lymphoid nodules are not inductive sites for the regulation of Ag-specific intestinal IgA responses to recombinant Salmonella.« less

  6. Peptidyl aldehyde NK-1.8k suppresses enterovirus 71 and enterovirus 68 infection by targeting protease 3C.

    PubMed

    Wang, Yaxin; Yang, Ben; Zhai, Yangyang; Yin, Zheng; Sun, Yuna; Rao, Zihe

    2015-05-01

    Enterovirus (EV) is one of the major causative agents of hand, foot, and mouth disease in the Pacific-Asia region. In particular, EV71 causes severe central nervous system infections, and the fatality rates from EV71 infection are high. Moreover, an outbreak of respiratory illnesses caused by an emerging EV, EV68, recently occurred among over 1,000 young children in the United States and was also associated with neurological infections. Although enterovirus has emerged as a considerable global public health threat, no antiviral drug for clinical use is available. In the present work, we screened our compound library for agents targeting viral protease and identified a peptidyl aldehyde, NK-1.8k, that inhibits the proliferation of different EV71 strains and one EV68 strain and that had a 50% effective concentration of 90 nM. Low cytotoxicity (50% cytotoxic concentration, >200 μM) indicated a high selective index of over 2,000. We further characterized a single amino acid substitution inside protease 3C (3C(pro)), N69S, which conferred EV71 resistance to NK-1.8k, possibly by increasing the flexibility of the substrate binding pocket of 3C(pro). The combination of NK-1.8k and an EV71 RNA-dependent RNA polymerase inhibitor or entry inhibitor exhibited a strong synergistic anti-EV71 effect. Our findings suggest that NK-1.8k could potentially be developed for anti-EV therapy. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  7. [The effect of enzymatic treatment using proteases on properties of persistent sodium current in CA1 pyramidal neurons of rat hippocampus].

    PubMed

    Lun'ko, O O; Isaiev, D S; Maxymiuk, O P; Kryshtal', O O; Isaieva, O V

    2014-01-01

    We investigated the effect of proteases, widely used for neuron isolation in electrophysiological studies, on the amplitude and kinetic characteristics of persistent sodium current (I(NaP)) in hippocampal CA1 pyramidal neurons. Properties of I(NaP) were studied on neurons isolated by mechanical treatment (control group) and by mechanical and enzymatic treatment using pronase E (from Streptomyces griseus) or protease type XXIII (from Aspergillus oryzae). We show that in neurons isolated with pronase E kinetic of activation and density of I(NaP) was unaltered. Enzymatic treatment with protease type XXIII did not alter I(NaP) activation but result in significant decrease in I(NaP) density. Our data indicates that enzymatic treatment using pronase E for neuron isolation is preferable for investigation of I(NaP).

  8. Rapamycin slows IgA nephropathy progression in the rat.

    PubMed

    Tian, Jihua; Wang, Yanhong; Zhou, Xiaoshuang; Li, Yanjiao; Wang, Chen; Li, Jiaming; Li, Rongshan

    2014-01-01

    IgA nephropathy (IgAN) is the most frequent glomerulonephritis worldwide. Different therapeutic approaches have been tested against IgAN. The present study was designed to explore the renoprotective potential of low-dose mammalian target of rapamycin (mTOR) inhibitor rapamycin in an IgAN rat model and the possible mechanism of action. After establishing an IgAN model, the rats were randomly divided into four groups: control, control with rapamycin treatment, IgAN model, and IgAN model with rapamycin treatment. Coomassie Brilliant Blue was utilized to measure 24-hour urinary protein levels. Hepatic and renal function was determined with an autoanalyzer. Proliferation was assayed via 5-bromo-2'-deoxyuridine incorporation. Real-time PCR and immunohistochemistry were utilized to detect the expression of α-SMA, collagen I, collagen III, TGF-β1 and platelet-derived growth factor. Western blotting and immunohistochemistry were performed to determine p-S6 protein levels. Low-dose mTOR inhibitor rapamycin prevented an additional increase in proteinuria and protected kidney function in a model of IgAN. Rapamycin directly or indirectly interfered with multiple key pathways in the progression of IgAN to end-stage renal disease: (1) reduced the deposition of IgA and inhibited cell proliferation; (2) decreased the expression of fibrosis markers α-SMA and type III collagen, and (3) downregulated the expression of the profibrotic growth factors platelet-derived growth factor and TGF-β1. The expression of p-S6 was significantly elevated in IgAN rats. The mTOR pathway was activated in IgAN rats and the early application of low-dose mTOR inhibitor rapamycin may slow the renal injury of IgAN in rats.

  9. Coexistence of Fabry disease and IgA nephropathy: a report of two cases.

    PubMed

    Yin, G; Wu, Y; Zeng, C-H; Chen, H-P; Liu, Z-H

    2014-12-01

    Coexistence of Fabry disease and IgA nephropathy is rare. Moreover, the coexisting Fabry disease may be unrecognized due to unapparent clinical manifestations. We described two cases with coexisting Fabry disease and IgA nephropathy. The clinicopathological features of these two patients were studied. A 54-year-old male presented with proteinuria, hematuria, and hypertension, and a 33-year-old male presented with proteinuria without clinical signs or family history of Fabry disease. Both of them were diagnosed with IgA nephropathy at admission, whereas Fabry disease was not suspected. Subsequent immunofluorescent study confirmed the diagnosis of IgA nephropathy by showing positive staining for IgA and complement C3 in the mesangium. Meanwhile, light microscopy showed remarkable vacuolation of podocytes with mild mesangial expansion, which was characteristic of Fabry nephropathy. Further examination of toluidine blue-stained semi-thin sections and electron microscopy demonstrated blue bodies and myelin figures in the cytoplasm of podocytes, respectively. The diagnosis of coexisting Fabry disease was finally established based on deficient α-galactosidase A activity in both patients. This case study is an important reminder of the role of kidney biopsy as an indicator of Fabry disease and its rare coexistence with IgA nephropathy.

  10. Thrombin enhances herpes simplex virus infection of cells involving protease-activated receptor 1.

    PubMed

    Sutherland, M R; Friedman, H M; Pryzdial, E L G

    2007-05-01

    We have previously shown that the surface of purified herpes family viruses can initiate thrombin production by expressing host-encoded and virus-encoded procoagulant factors. These enable the virus to bypass the normal cell-regulated mechanisms for initiating coagulation, and provide a link between infection and vascular disease. In the current study we investigated why these viruses may have evolved to generate thrombin. Using cytolytic viral plaque assays, the current study examines the effect of thrombin on human umbilical vein endothelial cell (HUVEC) or human foreskin fibroblast (HFF) infection by purified herpes simplex virus type 1 (HSV1) and type 2 (HSV2). Demonstrating that the availability of thrombin is an advantage to the virus, purified thrombin added to serum-free inoculation media resulted in up to a 3-fold enhancement of infection depending on the virus strain and cell type. The effect of thrombin on HUVEC infection was generally greater than its effect on HFF. To illustrate the involvement of thrombin produced during inoculation, hirudin was shown to inhibit the infection of each HSV strain, but only when serum containing clotting factors for thrombin production was present in media. The involvement of protease-activated receptor 1 (PAR1) was supported using PAR1-activating peptides in place of thrombin and PAR1-specific antibodies to inhibit the effects of thrombin. These data show that HSV1 and HSV2 initiate thrombin production to increase the susceptibility of cells to infection through a mechanism involving PAR1-mediated cell modulation.

  11. Potentiation of C1-esterase inhibitor by heparin and interactions with C1s protease as assessed by surface plasmon resonance.

    PubMed

    Rajabi, Mohsen; Struble, Evi; Zhou, Zhaohua; Karnaukhova, Elena

    2012-01-01

    Human C1-esterase inhibitor (C1-INH) is a multifunctional plasma protein with a wide range of inhibitory and non-inhibitory properties, mainly recognized as a key down-regulator of the complement and contact cascades. The potentiation of C1-INH by heparin and other glycosaminoglycans (GAGs) regulates a broad spectrum of C1-INH activities in vivo both in normal and disease states. SCOPE OF RESEARCH: We have studied the potentiation of human C1-INH by heparin using Surface Plasmon Resonance (SPR), circular dichroism (CD) and a functional assay. To advance a SPR for multiple-unit interaction studies of C1-INH we have developed a novel (consecutive double capture) approach exploring different immobilization and layout. Our SPR experiments conducted in three different design versions showed marked acceleration in C1-INH interactions with complement protease C1s as a result of potentiation of C1-INH by heparin (from 5- to 11-fold increase of the association rate). Far-UV CD studies suggested that heparin binding did not alter C1-INH secondary structure. Functional assay using chromogenic substrate confirmed that heparin does not affect the amidolytic activity of C1s, but does accelerate its consumption due to C1-INH potentiation. This is the first report that directly demonstrates a significant acceleration of the C1-INH interactions with C1s due to heparin by using a consecutive double capture SPR approach. The results of this study may be useful for further C-INH therapeutic development, ultimately for the enhancement of current C1-INH replacement therapies. Published by Elsevier B.V.

  12. Determination of IL-1B (rs16944) and IL-6 (rs1800796) genetic polymorphisms in IgA nephropathy in a northwest Chinese Han population.

    PubMed

    Zhang, Daofa; Xie, Maowei; Yang, Xiaohong; Zhang, Yin; Su, Yan; Wang, Yanni; Huang, Haiyang; Han, Hui; Li, Wenning; Fu, Keying; Su, Huiluan; Xu, Wentan; Han, Yeguang; Wang, Ru; Zhang, Pei; Wu, Wei; Huang, Yun; Chen, Daojun; Jin, Tianbo; Wei, Jiali

    2017-09-22

    IgA nephropathy (IgAN) is the most common form of primary glomerulonephritis worldwide, but etiology and pathogenesis continue to be poorly understood. Polymorphisms in the cytokine genes may play a role in the etiology and pathogenesis of IgAN. The incidence of different between diverse ethnic groups suggested important genetic influences on its pathogenesis. We genotype 10 single nucleotide polymorphisms (SNPs) in IL-1B and IL-6 gene using Sequenom Mass-ARRAY technology from 417 IgAN patients and 463 healthy controls of the Chinese Han population. We evaluated these SNPs associated with IgAN utilising the chi-square tests and genetic model analysis. We identified that the minor alleles of rs16944 ("A"), rs1800796 ("G") in IL-1B, IL-6 were involved in an increasingly risk of IgAN in allelic model analysis, respectively. The rs16944 in IL-1B and rs1800796 in IL-6 were associated with 1.23-fold (95% CI, 1.02-1.48, P = 0.031) and 1.33-fold (95% CI, 1.11-1.66, P = 0.003) increases in the risk of developing IgAN, respectively. There was only rs1800796 still correlated with IgAN in the allelic model after adjustment by age and gender and the Bonferroni correction. In addition, Haplotype G rs1800796 A rs2069837 G rs2069840 ( P = 0.037) and G rs1800796 A rs2069837 C rs2069840 ( P = 0.042) in IL-6 were considered to be associated with increased IgAN risk. This study verified the IL-6, IL-1B genetic variants polymorphisms contributed to IgAN susceptibility in a Chinese Han population. Although we identified SNPs susceptibility, however, replication studies and functional research are required to confirm the genetic contribution in IgAN.

  13. Accurate ensemble molecular dynamics binding free energy ranking of multidrug-resistant HIV-1 proteases.

    PubMed

    Sadiq, S Kashif; Wright, David W; Kenway, Owain A; Coveney, Peter V

    2010-05-24

    Accurate calculation of important thermodynamic properties, such as macromolecular binding free energies, is one of the principal goals of molecular dynamics simulations. However, single long simulation frequently produces incorrectly converged quantitative results due to inadequate sampling of conformational space in a feasible wall-clock time. Multiple short (ensemble) simulations have been shown to explore conformational space more effectively than single long simulations, but the two methods have not yet been thermodynamically compared. Here we show that, for end-state binding free energy determination methods, ensemble simulations exhibit significantly enhanced thermodynamic sampling over single long simulations and result in accurate and converged relative binding free energies that are reproducible to within 0.5 kcal/mol. Completely correct ranking is obtained for six HIV-1 protease variants bound to lopinavir with a correlation coefficient of 0.89 and a mean relative deviation from experiment of 0.9 kcal/mol. Multidrug resistance to lopinavir is enthalpically driven and increases through a decrease in the protein-ligand van der Waals interaction, principally due to the V82A/I84V mutation, and an increase in net electrostatic repulsion due to water-mediated disruption of protein-ligand interactions in the catalytic region. Furthermore, we correctly rank, to within 1 kcal/mol of experiment, the substantially increased chemical potency of lopinavir binding to the wild-type protease compared to saquinavir and show that lopinavir takes advantage of a decreased net electrostatic repulsion to confer enhanced binding. Our approach is dependent on the combined use of petascale computing resources and on an automated simulation workflow to attain the required level of sampling and turn around time to obtain the results, which can be as little as three days. This level of performance promotes integration of such methodology with clinical decision support systems for

  14. Determination of IL-1B (rs16944) and IL-6 (rs1800796) genetic polymorphisms in IgA nephropathy in a northwest Chinese Han population

    PubMed Central

    Zhang, Yin; Su, Yan; Wang, Yanni; Huang, Haiyang; Han, Hui; Li, Wenning; Fu, Keying; Su, Huiluan; Xu, Wentan; Han, Yeguang; Wang, Ru; Zhang, Pei; Wu, Wei; Huang, Yun; Chen, Daojun; Jin, Tianbo; Wei, Jiali

    2017-01-01

    IgA nephropathy (IgAN) is the most common form of primary glomerulonephritis worldwide, but etiology and pathogenesis continue to be poorly understood. Polymorphisms in the cytokine genes may play a role in the etiology and pathogenesis of IgAN. The incidence of different between diverse ethnic groups suggested important genetic influences on its pathogenesis. We genotype 10 single nucleotide polymorphisms (SNPs) in IL-1B and IL-6 gene using Sequenom Mass-ARRAY technology from 417 IgAN patients and 463 healthy controls of the Chinese Han population. We evaluated these SNPs associated with IgAN utilising the chi-square tests and genetic model analysis. We identified that the minor alleles of rs16944 (“A”), rs1800796 (“G”) in IL-1B, IL-6 were involved in an increasingly risk of IgAN in allelic model analysis, respectively. The rs16944 in IL-1B and rs1800796 in IL-6 were associated with 1.23-fold (95% CI, 1.02-1.48, P = 0.031) and 1.33-fold (95% CI, 1.11-1.66, P = 0.003) increases in the risk of developing IgAN, respectively. There was only rs1800796 still correlated with IgAN in the allelic model after adjustment by age and gender and the Bonferroni correction. In addition, Haplotype Grs1800796A rs2069837G rs2069840 (P = 0.037) and G rs1800796A rs2069837C rs2069840 (P = 0.042) in IL-6were considered to be associated with increased IgAN risk. This study verified the IL-6, IL-1B genetic variants polymorphisms contributed to IgAN susceptibility in a Chinese Han population. Although we identified SNPs susceptibility, however, replication studies and functional research are required to confirm the genetic contribution in IgAN. PMID:29069743

  15. WIDE AND THICK GRAIN 1, which encodes an otubain-like protease with deubiquitination activity, influences grain size and shape in rice.

    PubMed

    Huang, Ke; Wang, Dekai; Duan, Penggen; Zhang, Baolan; Xu, Ran; Li, Na; Li, Yunhai

    2017-09-01

    Grain size and shape are two crucial traits that influence grain yield and grain appearance in rice. Although several factors that affect grain size have been described in rice, the molecular mechanisms underlying the determination of grain size and shape are still elusive. In this study we report that WIDE AND THICK GRAIN 1 (WTG1) functions as an important factor determining grain size and shape in rice. The wtg1-1 mutant exhibits wide, thick, short and heavy grains and also shows an increased number of grains per panicle. WTG1 determines grain size and shape mainly by influencing cell expansion. WTG1 encodes an otubain-like protease, which shares similarity with human OTUB1. Biochemical analyses indicate that WTG1 is a functional deubiquitinating enzyme, and the mutant protein (wtg1-1) loses this deubiquitinating activity. WTG1 is expressed in developing grains and panicles, and the GFP-WTG1 fusion protein is present in the nucleus and cytoplasm. Overexpression of WTG1 results in narrow, thin, long grains due to narrow and long cells, further supporting the role of WTG1 in determining grain size and shape. Thus, our findings identify the otubain-like protease WTG1 to be an important factor that determines grain size and shape, suggesting that WTG1 has the potential to improve grain size and shape in rice. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  16. Effects of protease-activated receptor 1 inhibition on anxiety and fear following status epilepticus.

    PubMed

    Bogovyk, Ruslan; Lunko, Oleksii; Fedoriuk, Mihail; Isaev, Dmytro; Krishtal, Oleg; Holmes, Gregory L; Isaeva, Elena

    2017-02-01

    Protease-activated receptor 1 (PAR1) is an important contributor to the pathogenesis of a variety of brain disorders associated with a risk of epilepsy development. Using the lithium-pilocarpine model of temporal lobe epilepsy (TLE), we recently showed that inhibition of this receptor during the first ten days after pilocarpine-induced status epilepticus (SE) results in substantial anti-epileptogenic and neuroprotective effects. As PAR1 is expressed in the central nervous system regions of importance for processing emotional reactions, including amygdala and hippocampus, and TLE is frequently associated with a chronic alteration of the functions of these regions, we tested the hypothesis that PAR1 inhibition could modulate emotionally driven behavioral responses of rats experiencing SE. We showed that SE induces a chronic decrease in the animals' anxiety-related behavior and an increase of locomotor activity. PAR1 inhibition after SE abolished the alteration of the anxiety level but does not affect the increase of locomotor activity in the open field and elevated plus maze tests. Moreover, while PAR1 inhibition produces an impairment of memory recall in the context fear conditioning paradigm in the control group, it substantially improves contextual and cued fear learning in rats experiencing SE. These data suggest that PAR1-dependent signaling is involved in the mechanisms underlying emotional disorders in epilepsy. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Response Surface Methodology Modelling of an Aqueous Two-Phase System for Purification of Protease from Penicillium candidum (PCA 1/TT031) under Solid State Fermentation and Its Biochemical Characterization.

    PubMed

    Alhelli, Amaal M; Abdul Manap, Mohd Yazid; Mohammed, Abdulkarim Sabo; Mirhosseini, Hamed; Suliman, Eilaf; Shad, Zahra; Mohammed, Nameer Khairulla; Meor Hussin, Anis Shobirin

    2016-11-11

    Penicillium candidum (PCA 1/TT031) synthesizes different types of extracellular proteases. The objective of this study is to optimize polyethylene glycol (PEG)/citrate based on an aqueous two-phase system (ATPS) and Response Surface Methodology (RSM) to purify protease from Penicillium candidum (PCA 1/TT031). The effects of different PEG molecular weights (1500-10,000 g/mol), PEG concentration (9%-20%), concentrations of NaCl (0%-10%) and the citrate buffer (8%-16%) on protease were also studied. The best protease purification could be achieved under the conditions of 9.0% ( w / w ) PEG 8000, 5.2% NaCl, and 15.9% sodium citrate concentration, which resulted in a one-sided protease partitioning for the bottom phase with a partition coefficient of 0.2, a 6.8-fold protease purification factor, and a yield of 93%. The response surface models displayed a significant ( p ≤ 0.05) response which was fit for the variables that were studied as well as a high coefficient of determination (R²). Similarly, the predicted and observed values displayed no significant ( p > 0.05) differences. In addition, our enzyme characterization study revealed that Penicillium candidum (PCA 1/TT031) produced a slight neutral protease with a molecular weight between 100 and 140 kDa. The optimal activity of the purified enzyme occurred at a pH of 6.0 and at a temperature of 50 ° C. The stability between different pH and temperature ranges along with the effect of chemical metal ions and inhibitors were also studied. Our results reveal that the purified enzyme could be used in the dairy industry such as in accelerated cheese ripening.

  18. High-affinity monoclonal IgA regulates gut microbiota and prevents colitis in mice.

    PubMed

    Okai, Shinsaku; Usui, Fumihito; Yokota, Shuhei; Hori-I, Yusaku; Hasegawa, Makoto; Nakamura, Toshinobu; Kurosawa, Manabu; Okada, Seiji; Yamamoto, Kazuya; Nishiyama, Eri; Mori, Hiroshi; Yamada, Takuji; Kurokawa, Ken; Matsumoto, Satoshi; Nanno, Masanobu; Naito, Tomoaki; Watanabe, Yohei; Kato, Tamotsu; Miyauchi, Eiji; Ohno, Hiroshi; Shinkura, Reiko

    2016-07-04

    Immunoglobulin A (IgA) is the main antibody isotype secreted into the intestinal lumen. IgA plays a critical role in the defence against pathogens and in the maintenance of intestinal homeostasis. However, how secreted IgA regulates gut microbiota is not completely understood. In this study, we isolated monoclonal IgA antibodies from the small intestine of healthy mouse. As a candidate for an efficient gut microbiota modulator, we selected a W27 IgA, which binds to multiple bacteria, but not beneficial ones such as Lactobacillus casei. W27 could suppress the cell growth of Escherichia coli but not L. casei in vitro, indicating an ability to improve the intestinal environment. Indeed W27 oral treatment could modulate gut microbiota composition and have a therapeutic effect on both lymphoproliferative disease and colitis models in mice. Thus, W27 IgA oral treatment is a potential remedy for inflammatory bowel disease, acting through restoration of host-microbial symbiosis.

  19. Value of isolated IgA anti-β2 -glycoprotein I positivity in the diagnosis of the antiphospholipid syndrome.

    PubMed

    Murthy, Vijaya; Willis, Rohan; Romay-Penabad, Zurina; Ruiz-Limón, Patricia; Martínez-Martínez, Laura A; Jatwani, Shraddha; Jajoria, Praveen; Seif, Alan; Alarcón, Graciela S; Papalardo, Elizabeth; Liu, Jigna; Vilá, Luis M; McGwin, Gerald; McNearney, Terry A; Maganti, Rashmi; Sunkureddi, Prashanth; Parekh, Trisha; Tarantino, Michael; Akhter, Ehtisham; Fang, Hong; Gonzalez, Emilio B; Binder, Walter R; Norman, Gary L; Shums, Zakera; Teodorescu, Marius; Reveille, John D; Petri, Michelle; Pierangeli, Silvia S

    2013-12-01

    To examine the prevalence of isolated IgA anti-β2 -glycoprotein I (anti-β2 GPI) positivity and the association of these antibodies, and a subgroup that bind specifically to domain IV/V of β2 GPI, with clinical manifestations of the antiphospholipid syndrome (APS) in 3 patient groups and to evaluate the pathogenicity of IgA anti-β2 GPI in a mouse model of thrombosis. Patients with systemic lupus erythematosus (SLE) from a multiethnic, multicenter cohort (LUpus in MInorities, NAture versus nurture [LUMINA]) (n = 558), patients with SLE from the Hopkins Lupus Cohort (n = 215), and serum samples referred to the Antiphospholipid Standardization Laboratory (APLS) (n = 5,098) were evaluated. IgA anti-β2 GPI titers and binding to domain IV/V of β2 GPI were examined by enzyme-linked immunosorbent assay (ELISA). CD1 mice were inoculated with purified IgA anti-β2 GPI antibodies, and surgical procedures and ELISAs were performed to evaluate thrombus development and tissue factor (TF) activity. A total of 198 patients were found to be positive for IgA anti-β2 GPI isotype, and 57 patients were positive exclusively for IgA anti-β2 GPI antibodies. Of these, 13 of 23 patients (56.5%) in the LUMINA cohort, 17 of 17 patients (100%) in the Hopkins cohort, and 10 of 17 patients (58.9%) referred to APLS had at least one APS-related clinical manifestation. Fifty-four percent of all the IgA anti-β2 GPI-positive serum samples reacted with domain IV/V of anti-β2 GPI, and 77% of those had clinical features of APS. Isolated IgA anti-β2 GPI positivity was associated with an increased risk of arterial thrombosis (P < 0.001), venous thrombosis (P = 0.015), and all thrombosis (P < 0.001). The association between isolated IgA anti-β2 GPI and arterial thrombosis (P = 0.0003) and all thrombosis (P = 0.0003) remained significant after adjusting for other risk factors for thrombosis. In vivo mouse studies demonstrated that IgA anti-β2 GPI antibodies induced significantly larger thrombi

  20. Expression, purification, and characterization of the Necator americanus aspartic protease-1 (Na-APR-1 (M74)) antigen, a component of the bivalent human hookworm vaccine.

    PubMed

    Seid, Christopher A; Curti, Elena; Jones, R Mark; Hudspeth, Elissa; Rezende, Wanderson; Pollet, Jeroen; Center, Lori; Versteeg, Leroy; Pritchard, Sonya; Musiychuk, Konstantin; Yusibov, Vidadi; Hotez, Peter J; Bottazzi, Maria Elena

    2015-01-01

    Over 400 million people living in the world's poorest developing nations are infected with hookworms, mostly of the genus Necator americanus. A bivalent human hookworm vaccine composed of the Necator americanus Glutathione S-Transferase-1 (Na-GST-1) and the Necator americanus Aspartic Protease-1 (Na-APR-1 (M74)) is currently under development by the Sabin Vaccine Institute Product Development Partnership (Sabin PDP). Both monovalent vaccines are currently in Phase 1 trials. Both Na-GST-1 and Na-APR-1 antigens are expressed as recombinant proteins. While Na-GST-1 was found to express with high yields in Pichia pastoris, the level of expression of Na-APR-1 in this host was too low to be suitable for a manufacturing process. When the tobacco plant Nicotiana benthamiana was evaluated as an expression system, acceptable levels of solubility, yield, and stability were attained. Observed expression levels of Na-APR-1 (M74) using this system are ∼300 mg/kg. Here we describe the achievements and obstacles encountered during process development as well as characterization and stability of the purified Na-APR-1 (M74) protein and formulated vaccine. The expression, purification and analysis of purified Na-APR-1 (M74) protein obtained from representative 5 kg reproducibility runs performed to qualify the Na-APR-1 (M74) production process is also presented. This process has been successfully transferred to a pilot plant and a 50 kg scale manufacturing campaign under current Good Manufacturing Practice (cGMP) has been performed. The 50 kg run has provided a sufficient amount of protein to support the ongoing hookworm vaccine development program of the Sabin PDP.

  1. Enhanced stability of monomer fold correlates with extreme drug resistance of HIV-1 protease.

    PubMed

    Louis, John M; Tözsér, József; Roche, Julien; Matúz, Krisztina; Aniana, Annie; Sayer, Jane M

    2013-10-29

    During treatment, mutations in HIV-1 protease (PR) are selected rapidly that confer resistance by decreasing affinity to clinical protease inhibitors (PIs). As these unique drug resistance mutations can compromise the fitness of the virus to replicate, mutations that restore conformational stability and activity while retaining drug resistance are selected on further evolution. Here we identify several compensating mechanisms by which an extreme drug-resistant mutant bearing 20 mutations (PR20) with >5-fold increased Kd and >4000-fold decreased affinity to the PI darunavir functions. (1) PR20 cleaves, albeit poorly, Gag polyprotein substrates essential for viral maturation. (2) PR20 dimer, which exhibits distinctly enhanced thermal stability, has highly attenuated autoproteolysis, thus likely prolonging its lifetime in vivo. (3) The enhanced stability of PR20 results from stabilization of the monomer fold. Both monomeric PR20(T26A) and dimeric PR20 exhibit Tm values 6-7.5 °C higher than those for their PR counterparts. Two specific mutations in PR20, L33F and L63P at sites of autoproteolysis, increase the Tm of monomeric PR(T26A) by ~8 °C, similar to PR20(T26A). However, without other compensatory mutations as seen in PR20, L33F and L63P substitutions, together, neither restrict autoproteolysis nor significantly reduce binding affinity to darunavir. To determine whether dimer stability contributes to binding affinity for inhibitors, we examined single-chain dimers of PR and PR(D25N) in which the corresponding identical monomer units were covalently linked by GGSSG sequence. Linking of the subunits did not appreciably change the ΔTm on inhibitor binding; thus stabilization by tethering appears to have little direct effect on enhancing inhibitor affinity.

  2. A lead discovery strategy driven by a comprehensive analysis of proteases in the peptide substrate space

    PubMed Central

    Sukuru, Sai Chetan K; Nigsch, Florian; Quancard, Jean; Renatus, Martin; Chopra, Rajiv; Brooijmans, Natasja; Mikhailov, Dmitri; Deng, Zhan; Cornett, Allen; Jenkins, Jeremy L; Hommel, Ulrich; Davies, John W; Glick, Meir

    2010-01-01

    We present here a comprehensive analysis of proteases in the peptide substrate space and demonstrate its applicability for lead discovery. Aligned octapeptide substrates of 498 proteases taken from the MEROPS peptidase database were used for the in silico analysis. A multiple-category naïve Bayes model, trained on the two-dimensional chemical features of the substrates, was able to classify the substrates of 365 (73%) proteases and elucidate statistically significant chemical features for each of their specific substrate positions. The positional awareness of the method allows us to identify the most similar substrate positions between proteases. Our analysis reveals that proteases from different families, based on the traditional classification (aspartic, cysteine, serine, and metallo), could have substrates that differ at the cleavage site (P1–P1′) but are similar away from it. Caspase-3 (cysteine protease) and granzyme B (serine protease) are previously known examples of cross-family neighbors identified by this method. To assess whether peptide substrate similarity between unrelated proteases could reliably translate into the discovery of low molecular weight synthetic inhibitors, a lead discovery strategy was tested on two other cross-family neighbors—namely cathepsin L2 and matrix metallo proteinase 9, and calpain 1 and pepsin A. For both these pairs, a naïve Bayes classifier model trained on inhibitors of one protease could successfully enrich those of its neighbor from a different family and vice versa, indicating that this approach could be prospectively applied to lead discovery for a novel protease target with no known synthetic inhibitors. PMID:20799349

  3. P1-Substituted Symmetry-Based Human Immunodeficiency Virus Protease Inhibitors with Potent Antiviral Activity against Drug-Resistant Viruses

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    DeGoey, David A.; Grampovnik, David J.; Chen, Hui-Ju

    2013-03-07

    Because there is currently no cure for HIV infection, patients must remain on long-term drug therapy, leading to concerns over potential drug side effects and the emergence of drug resistance. For this reason, new and safe antiretroviral agents with improved potency against drug-resistant strains of HIV are needed. A series of HIV protease inhibitors (PIs) with potent activity against both wild-type (WT) virus and drug-resistant strains of HIV was designed and synthesized. The incorporation of substituents with hydrogen bond donor and acceptor groups at the P1 position of our symmetry-based inhibitor series resulted in significant potency improvements against the resistantmore » mutants. By this approach, several compounds, such as 13, 24, and 29, were identified that demonstrated similar or improved potencies compared to 1 against highly mutated strains of HIV derived from patients who previously failed HIV PI therapy. Overall, compound 13 demonstrated the best balance of potency against drug resistant strains of HIV and oral bioavailability in pharmacokinetic studies. X-ray analysis of an HIV PI with an improved resistance profile bound to WT HIV protease is also reported.« less

  4. Proteases and protease inhibitors of urinary extracellular vesicles in diabetic nephropathy.

    PubMed

    Musante, Luca; Tataruch, Dorota; Gu, Dongfeng; Liu, Xinyu; Forsblom, Carol; Groop, Per-Henrik; Holthofer, Harry

    2015-01-01

    Diabetic nephropathy (DN) is one of the major complications of diabetes mellitus (DM), leads to chronic kidney disease (CKD), and, ultimately, is the main cause for end-stage kidney disease (ESKD). Beyond urinary albumin, no reliable biomarkers are available for accurate early diagnostics. Urinary extracellular vesicles (UEVs) have recently emerged as an interesting source of diagnostic and prognostic disease biomarkers. Here we used a protease and respective protease inhibitor array to profile urines of type 1 diabetes patients at different stages of kidney involvement. Urine samples were divided into groups based on the level of albuminuria and UEVs isolated by hydrostatic dialysis and screened for relative changes of 34 different proteases and 32 protease inhibitors, respectively. Interestingly, myeloblastin and its natural inhibitor elafin showed an increase in the normo- and microalbuminuric groups. Similarly, a characteristic pattern was observed in the array of protease inhibitors, with a marked increase of cystatin B, natural inhibitor of cathepsins L, H, and B as well as of neutrophil gelatinase-associated Lipocalin (NGAL) in the normoalbuminuric group. This study shows for the first time the distinctive alterations in comprehensive protease profiles of UEVs in diabetic nephropathy and uncovers intriguing mechanistic, prognostic, and diagnostic features of kidney damage in diabetes.

  5. Proteases and Protease Inhibitors of Urinary Extracellular Vesicles in Diabetic Nephropathy

    PubMed Central

    Tataruch, Dorota; Gu, Dongfeng; Liu, Xinyu; Forsblom, Carol; Groop, Per-Henrik; Holthofer, Harry

    2015-01-01

    Diabetic nephropathy (DN) is one of the major complications of diabetes mellitus (DM), leads to chronic kidney disease (CKD), and, ultimately, is the main cause for end-stage kidney disease (ESKD). Beyond urinary albumin, no reliable biomarkers are available for accurate early diagnostics. Urinary extracellular vesicles (UEVs) have recently emerged as an interesting source of diagnostic and prognostic disease biomarkers. Here we used a protease and respective protease inhibitor array to profile urines of type 1 diabetes patients at different stages of kidney involvement. Urine samples were divided into groups based on the level of albuminuria and UEVs isolated by hydrostatic dialysis and screened for relative changes of 34 different proteases and 32 protease inhibitors, respectively. Interestingly, myeloblastin and its natural inhibitor elafin showed an increase in the normo- and microalbuminuric groups. Similarly, a characteristic pattern was observed in the array of protease inhibitors, with a marked increase of cystatin B, natural inhibitor of cathepsins L, H, and B as well as of neutrophil gelatinase-associated Lipocalin (NGAL) in the normoalbuminuric group. This study shows for the first time the distinctive alterations in comprehensive protease profiles of UEVs in diabetic nephropathy and uncovers intriguing mechanistic, prognostic, and diagnostic features of kidney damage in diabetes. PMID:25874235

  6. IgA Vasculitis: Genetics and Clinical and Therapeutic Management.

    PubMed

    González-Gay, Miguel A; López-Mejías, Raquel; Pina, Trinitario; Blanco, Ricardo; Castañeda, Santos

    2018-04-02

    The purpose of the study is to perform an update on the current knowledge on genetics, clinical manifestations, and therapy in immunoglobulin A vasculitis (IgAV) (Henoch-Schönlein purpura). A strong genetic predisposition in individuals with IgAV was confirmed. It was due to the association with the HLA class II region that in people of European background is mainly related to HLA-DRB1*01 allele. Recent reports support the claim that kidney disease is more common in adults than in children with IgAV. The clinical spectrum and outcome of adults with IgAV depends on the age of onset. Relapses are not uncommon in IgAV. The presence of renal impairment or proteinuria excretion exceeding 1 g/24 h at the time of disease diagnosis and the degree of renal damage on the kidney biopsy are the best predictors of end-stage renal failure in adults with IgAV. The levels of urinary IgA at the onset of the disease may predict a poor renal outcome. The use of prednisone does not seem to prevent persistent kidney disease in children with IgAV. No additional benefit of adding cyclophosphamide to glucocorticoids in adults with IgAV was found. Rituximab seems to be a promising therapy in the management of adults with IgAV. In this overview, we focus on the genetics, clinical manifestations, and therapy of IgA vasculitis, emphasizing the main differences in the clinical expression of the disease between children and adults.

  7. IgA measurements in over 12 000 Swedish twins reveal sex differential heritability and regulatory locus near CD30L.

    PubMed

    Viktorin, Alexander; Frankowiack, Marcel; Padyukov, Leonid; Chang, Zheng; Melén, Erik; Sääf, Annika; Kull, Inger; Klareskog, Lars; Hammarström, Lennart; Magnusson, Patrik K E

    2014-08-01

    In a broad attempt to improve the understanding of the genetic regulation of serum IgA levels, the heritability was estimated in over 12 000 Swedish twins, and a genome-wide association study was conducted in a subsample of 9617. Using the classical twin model the heritability was found to be significantly larger among females (61%) compared with males (21%), while contribution from shared environment (20%) was only seen for males. By modeling the genetic relationship matrix with IgA levels, we estimate that a substantial proportion (31%) of variance in IgA levels can ultimately be explained by the investigated SNPs. The genome-wide association study revealed significant association to two loci: (i) rs6928791 located on chromosome 6, 22 kb upstream of the gene SAM and SH3 domain containing 1 (SASH1) and (ii) rs13300483 on chromosome 9, situated 12 kb downstream the CD30 ligand (CD30L) encoding gene. The association to rs13300483 was replicated in two additional independent Swedish materials. The heritability of IgA levels is moderate and can partly be attributable to common variation in the CD30L locus. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  8. Naringin prevents HIV-1 protease inhibitors-induced metabolic complications in vivo.

    PubMed

    Nzuza, Sanelisiwe; Zondi, Sindiswa; Owira, Peter M O

    2017-01-01

    Insulin resistance, glucose intolerance and overt diabetes are known metabolic complications associated with chronic use of HIV-Protease Inhibitors. Naringin is a grapefruit-derived flavonoid with anti-diabetic, anti-dyslipidemia, anti-inflammatory and anti-oxidant activities. The study investigated the protective effects of naringin on glucose intolerance and impaired insulin secretion and signaling in vivo. Male Wistar rats were divided into six groups (n = 6) and were daily orally treated with distilled water {3.0 ml/kg body weight (BW)}, atazanavir (133 mg/kg BW), saquinavir (333 mg/kg BW) with or without naringin (50 mg/kg BW), respectively for 56 days. Body weights and water consumption were recorded daily. Glucose tolerance tests were carried out on day 55 of the treatment and thereafter, the rats were sacrificed by halothane overdose. Atazanavir (ATV)- or saquinavir (SQV)-treated rats exhibited significant weight loss, polydipsia, elevated Fasting blood glucose (FBG), reduced Fasting Plasma Insulin (FPI) and expression of phosphorylated, Insulin Receptor Substrate-1 (IRS-1) and Akt proteins, hepatic and pancreatic glucokinase levels, and also increasing pancreatic caspase-3 and -9 as well as UCP2 protein expressions compared to controls, respectively. These effects were completely reversed by naringin treatment. Naringin prevents PI-induced glucose intolerance and impairment of insulin signaling and as nutritional supplement it could therefore alleviate metabolic complications associated with antiretroviral therapy.

  9. Naringin prevents HIV-1 protease inhibitors-induced metabolic complications in vivo

    PubMed Central

    Nzuza, Sanelisiwe; Zondi, Sindiswa; Owira, Peter M. O.

    2017-01-01

    Background Insulin resistance, glucose intolerance and overt diabetes are known metabolic complications associated with chronic use of HIV-Protease Inhibitors. Naringin is a grapefruit-derived flavonoid with anti-diabetic, anti-dyslipidemia, anti-inflammatory and anti-oxidant activities. Objectives The study investigated the protective effects of naringin on glucose intolerance and impaired insulin secretion and signaling in vivo. Methods Male Wistar rats were divided into six groups (n = 6) and were daily orally treated with distilled water {3.0 ml/kg body weight (BW)}, atazanavir (133 mg/kg BW), saquinavir (333 mg/kg BW) with or without naringin (50 mg/kg BW), respectively for 56 days. Body weights and water consumption were recorded daily. Glucose tolerance tests were carried out on day 55 of the treatment and thereafter, the rats were sacrificed by halothane overdose. Results Atazanavir (ATV)- or saquinavir (SQV)-treated rats exhibited significant weight loss, polydipsia, elevated Fasting blood glucose (FBG), reduced Fasting Plasma Insulin (FPI) and expression of phosphorylated, Insulin Receptor Substrate-1 (IRS-1) and Akt proteins, hepatic and pancreatic glucokinase levels, and also increasing pancreatic caspase-3 and -9 as well as UCP2 protein expressions compared to controls, respectively. These effects were completely reversed by naringin treatment. Conclusion Naringin prevents PI-induced glucose intolerance and impairment of insulin signaling and as nutritional supplement it could therefore alleviate metabolic complications associated with antiretroviral therapy. PMID:29121676

  10. Structural Dynamics Investigation of Human Family 1 & 2 Cystatin-Cathepsin L1 Interaction: A Comparison of Binding Modes.

    PubMed

    Nandy, Suman Kumar; Seal, Alpana

    2016-01-01

    Cystatin superfamily is a large group of evolutionarily related proteins involved in numerous physiological activities through their inhibitory activity towards cysteine proteases. Despite sharing the same cystatin fold, and inhibiting cysteine proteases through the same tripartite edge involving highly conserved N-terminal region, L1 and L2 loop; cystatins differ widely in their inhibitory affinity towards C1 family of cysteine proteases and molecular details of these interactions are still elusive. In this study, inhibitory interactions of human family 1 & 2 cystatins with cathepsin L1 are predicted and their stability and viability are verified through protein docking & comparative molecular dynamics. An overall stabilization effect is observed in all cystatins on complex formation. Complexes are mostly dominated by van der Waals interaction but the relative participation of the conserved regions varied extensively. While van der Waals contacts prevail in L1 and L2 loop, N-terminal segment chiefly acts as electrostatic interaction site. In fact the comparative dynamics study points towards the instrumental role of L1 loop in directing the total interaction profile of the complex either towards electrostatic or van der Waals contacts. The key amino acid residues surfaced via interaction energy, hydrogen bonding and solvent accessible surface area analysis for each cystatin-cathepsin L1 complex influence the mode of binding and thus control the diverse inhibitory affinity of cystatins towards cysteine proteases.

  11. Structural Dynamics Investigation of Human Family 1 & 2 Cystatin-Cathepsin L1 Interaction: A Comparison of Binding Modes

    PubMed Central

    Nandy, Suman Kumar; Seal, Alpana

    2016-01-01

    Cystatin superfamily is a large group of evolutionarily related proteins involved in numerous physiological activities through their inhibitory activity towards cysteine proteases. Despite sharing the same cystatin fold, and inhibiting cysteine proteases through the same tripartite edge involving highly conserved N-terminal region, L1 and L2 loop; cystatins differ widely in their inhibitory affinity towards C1 family of cysteine proteases and molecular details of these interactions are still elusive. In this study, inhibitory interactions of human family 1 & 2 cystatins with cathepsin L1 are predicted and their stability and viability are verified through protein docking & comparative molecular dynamics. An overall stabilization effect is observed in all cystatins on complex formation. Complexes are mostly dominated by van der Waals interaction but the relative participation of the conserved regions varied extensively. While van der Waals contacts prevail in L1 and L2 loop, N-terminal segment chiefly acts as electrostatic interaction site. In fact the comparative dynamics study points towards the instrumental role of L1 loop in directing the total interaction profile of the complex either towards electrostatic or van der Waals contacts. The key amino acid residues surfaced via interaction energy, hydrogen bonding and solvent accessible surface area analysis for each cystatin-cathepsin L1 complex influence the mode of binding and thus control the diverse inhibitory affinity of cystatins towards cysteine proteases. PMID:27764212

  12. Alteration of Substrate and Inhibitor Specificity of Feline Immunodeficiency Virus Protease

    PubMed Central

    Lin, Ying-Chuan; Beck, Zachary; Lee, Taekyu; Le, Van-Duc; Morris, Garrett M.; Olson, Arthur J.; Wong, Chi-Huey; Elder, John H.

    2000-01-01

    Feline immunodeficiency virus (FIV) protease is structurally very similar to human immunodeficiency virus (HIV) protease but exhibits distinct substrate and inhibitor specificities. We performed mutagenesis of subsite residues of FIV protease in order to define interactions that dictate this specificity. The I37V, N55M, M56I, V59I, and Q99V mutants yielded full activity. The I37V, N55M, V59I, and Q99V mutants showed a significant increase in activity against the HIV-1 reverse transcriptase/integrase and P2/nucleocapsid junction peptides compared with wild-type (wt) FIV protease. The I37V, V59I, and Q99V mutants also showed an increase in activity against two rapidly cleaved peptides selected by cleavage of a phage display library with HIV-1 protease. Mutations at Q54K, I98P, and L101I dramatically reduced activity. Mutants containing a I35D or I57G substitution showed no activity against either FIV or HIV substrates. FIV proteases all failed to cut HIV-1 matrix/capsid, P1/P6, P6/protease, and protease/reverse transcriptase junctions, indicating that none of the substitutions were sufficient to change the specificity completely. The I37V, N55M, M56I, V59I, and Q99V mutants, compared with wt FIV protease, all showed inhibitor specificity more similar to that of HIV-1 protease. The data also suggest that FIV protease prefers a hydrophobic P2/P2′ residue like Val over Asn or Glu, which are utilized by HIV-1 protease, and that S2/S2′ might play a critical role in distinguishing FIV and HIV-1 protease by specificity. The findings extend our observations regarding the interactions involved in substrate binding and aid in the development of broad-based inhibitors. PMID:10775609

  13. Properties of an ionic liquid-tolerant Bacillus amyloliquefaciens CMW1 and its extracellular protease.

    PubMed

    Kurata, Atsushi; Senoo, Humiya; Ikeda, Yasuyuki; Kaida, Hideaki; Matsuhara, Chiaki; Kishimoto, Noriaki

    2016-07-01

    An ionic liquid-tolerant bacterium, Bacillus amyloliquefaciens CMW1, was isolated from a Japanese fermented soybean paste. Strain CMW1 grew in the presence of 10 % (v/v) 1-butyl-3-methylimidazolium chloride ([BMIM]Cl), a commonly used ionic liquid. Additionally, strain CMW1 grew adequately in the presence of the hydrophilic ionic liquids 10 % (v/v) 1-ethyl-3-methylimidazolium trifluoromethanesulfonate ([EMIM]CF3SO3) or 2.5 % (v/v) 1-butyl-3-methylimidazolium trifluoromethanesulfonate ([BMIM]CF3SO3). Strain CMW1 produced an extracellular protease (BapIL) in the culture medium. BapIL was stable in the presence of 80 % (v/v) ionic liquids, [EMIM]CF3SO3, [BMIM]Cl, [BMIM]CF3SO3, 1-butyl-3-methylimidazolium tetrafluoroborate, 1-butyl-3-methylimidazolium hexafluorophosphate, and 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide, and functioned in 10 % (v/v) these ionic liquids. BapIL was stable at pH 4.0-12.6 or in 4004 mM NaCl solution, and exhibited activity in the presence of 50 % (v/v) hydrophilic or hydrophobic organic solvents. BapIL was completely inhibited by 1 mM PMSF and partially by 5 mM EDTA. BapIL belongs to the true subtilisins according to analysis of the deduced amino acid sequence. We showed that BapIL from the ionic liquid-tolerant B. amyloliquefaciens CMW1 exhibited tolerance to ionic liquid and halo, alkaline, and organic solvents.

  14. Response Surface Methodology Modelling of an Aqueous Two-Phase System for Purification of Protease from Penicillium candidum (PCA 1/TT031) under Solid State Fermentation and Its Biochemical Characterization

    PubMed Central

    Alhelli, Amaal M.; Abdul Manap, Mohd Yazid; Mohammed, Abdulkarim Sabo; Mirhosseini, Hamed; Suliman, Eilaf; Shad, Zahra; Mohammed, Nameer Khairulla; Meor Hussin, Anis Shobirin

    2016-01-01

    Penicillium candidum (PCA 1/TT031) synthesizes different types of extracellular proteases. The objective of this study is to optimize polyethylene glycol (PEG)/citrate based on an aqueous two-phase system (ATPS) and Response Surface Methodology (RSM) to purify protease from Penicillium candidum (PCA 1/TT031). The effects of different PEG molecular weights (1500–10,000 g/mol), PEG concentration (9%–20%), concentrations of NaCl (0%–10%) and the citrate buffer (8%–16%) on protease were also studied. The best protease purification could be achieved under the conditions of 9.0% (w/w) PEG 8000, 5.2% NaCl, and 15.9% sodium citrate concentration, which resulted in a one-sided protease partitioning for the bottom phase with a partition coefficient of 0.2, a 6.8-fold protease purification factor, and a yield of 93%. The response surface models displayed a significant (p ≤ 0.05) response which was fit for the variables that were studied as well as a high coefficient of determination (R2). Similarly, the predicted and observed values displayed no significant (p > 0.05) differences. In addition, our enzyme characterization study revealed that Penicillium candidum (PCA 1/TT031) produced a slight neutral protease with a molecular weight between 100 and 140 kDa. The optimal activity of the purified enzyme occurred at a pH of 6.0 and at a temperature of 50 °C. The stability between different pH and temperature ranges along with the effect of chemical metal ions and inhibitors were also studied. Our results reveal that the purified enzyme could be used in the dairy industry such as in accelerated cheese ripening. PMID:27845736

  15. Using Hierarchical Virtual Screening To Combat Drug Resistance of the HIV-1 Protease.

    PubMed

    Li, Nan; Ainsworth, Richard I; Ding, Bo; Hou, Tingjun; Wang, Wei

    2015-07-27

    Human immunodeficiency virus (HIV) protease inhibitors (PIs) are important components of highly active anti-retroviral therapy (HAART) that block the catalytic site of HIV protease, thus preventing maturation of the HIV virion. However, with two decades of PI prescriptions in clinical practice, drug-resistant HIV mutants have now been found for all of the PI drugs. Therefore, the continuous development of new PI drugs is crucial both to combat the existing drug-resistant HIV strains and to provide treatments for future patients. Here we purpose an HIV PI drug design strategy to select candidate PIs with binding energy distributions dominated by interactions with conserved protease residues in both wild-type and various drug-resistant mutants. On the basis of this strategy, we have constructed a virtual screening pipeline including combinatorial library construction, combinatorial docking, MM/GBSA-based rescoring, and reranking on the basis of the binding energy distribution. We have tested our strategy on lopinavir by modifying its two functional groups. From an initial 751 689 candidate molecules, 18 candidate inhibitors were selected using the pipeline for experimental validation. IC50 measurements and drug resistance predictions successfully identified two ligands with both HIV protease inhibitor activity and an improved drug resistance profile on 2382 HIV mutants. This study provides a proof of concept for the integration of MM/GBSA energy analysis and drug resistance information at the stage of virtual screening and sheds light on future HIV drug design and the use of virtual screening to combat drug resistance.

  16. Label-free Quantitative Proteomics of Mouse Cerebrospinal Fluid Detects β-Site APP Cleaving Enzyme (BACE1) Protease Substrates In Vivo*

    PubMed Central

    Dislich, Bastian; Wohlrab, Felix; Bachhuber, Teresa; Müller, Stephan A.; Kuhn, Peer-Hendrik; Hogl, Sebastian; Meyer-Luehmann, Melanie; Lichtenthaler, Stefan F.

    2015-01-01

    Analysis of murine cerebrospinal fluid (CSF) by quantitative mass spectrometry is challenging because of low CSF volume, low total protein concentration, and the presence of highly abundant proteins such as albumin. We demonstrate that the CSF proteome of individual mice can be analyzed in a quantitative manner to a depth of several hundred proteins in a robust and simple workflow consisting of single ultra HPLC runs on a benchtop mass spectrometer. The workflow is validated by a comparative analysis of BACE1−/− and wild-type mice using label-free quantification. The protease BACE1 cleaves the amyloid precursor protein (APP) as well as several other substrates and is a major drug target in Alzheimer's disease. We identified a total of 715 proteins with at least 2 unique peptides and quantified 522 of those proteins in CSF from BACE1−/− and wild-type mice. Several proteins, including the known BACE1 substrates APP, APLP1, CHL1 and contactin-2 showed lower abundance in the CSF of BACE1−/− mice, demonstrating that BACE1 substrate identification is possible from CSF. Additionally, ectonucleotide pyrophosphatase 5 was identified as a novel BACE1 substrate and validated in cells using immunoblots and by an in vitro BACE1 protease assay. Likewise, receptor-type tyrosine-protein phosphatase N2 and plexin domain-containing 2 were confirmed as BACE1 substrates by in vitro assays. Taken together, our study shows the deepest characterization of the mouse CSF proteome to date and the first quantitative analysis of the CSF proteome of individual mice. The BACE1 substrates identified in CSF may serve as biomarkers to monitor BACE1 activity in Alzheimer patients treated with BACE inhibitors. PMID:26139848

  17. IgA modulates respiratory dysfunction as a sequela to pulmonary chlamydial infection as neonates

    PubMed Central

    Lanka, Gopala Krishna Koundinya; Yu, Jieh-Juen; Gong, Siqi; Gupta, Rishein; Mustafa, Shamimunisa B.; Murthy, Ashlesh K.; Zhong, Guangming; Chambers, James P.; Guentzel, M. Neal; Arulanandam, Bernard P.

    2016-01-01

    Neonatal Chlamydia lung infections are associated with serious sequelae such as asthma and airway hyper-reactivity in children and adults. Our previous studies demonstrated the importance of Th-1 type cytokines, IL-12 and IFN-γ in protection against neonatal pulmonary chlamydial challenge; however, the role of the humoral arm of defense has not been elucidated. We hypothesized that B-cells and IgA, the major mucosal antibody, play a protective role in newborns against development of later life respiratory sequelae to Chlamydia infection. Our studies using neonatal mice revealed that all WT and IgA-deficient (IgA−/−) animals survived a sublethal pulmonary Chlamydia muridarum challenge at one day after birth with similar reduction in bacterial burdens over time. In contrast, all B-cell-deficient (μMT) mice succumbed to infection at the same challenge dose correlating to failure to control bacterial burdens in the lungs. Although IgA may not be important for bacterial clearance, we observed IgA−/− mice displayed greater respiratory dysfunction 5 weeks post challenge. Specifically, comparative respiratory functional analyses revealed a significant shift upward in P–V loops, and higher dynamic resistance in IgA−/− animals. This study provides insight(s) into the protective role of IgA in neonates against pulmonary chlamydial infection induced respiratory pathological sequelae observed later in life. PMID:26755533

  18. Proteases in doping control analysis.

    PubMed

    Thevis, M; Maurer, J; Kohler, M; Geyer, H; Schänzer, W

    2007-07-01

    Urine manipulation in sports drug testing has become a serious problem for doping control laboratories, and recent scandals in elite endurance sports have revealed the problem of urine manipulation presumably using proteases, which will impede the detection of drugs such as erythropoietin (EPO) or other peptide hormones. Using commonly accepted analytical strategies, a protocol was developed enabling the determination of elevated protease activities in doping control specimens followed by the visualization of protein degradation and identification of proteases such as chymotrypsin, trypsin and papain. Therefore, protease detection kits based on fluorescein isothiocyanate-labeled casein were employed, and protease concentrations greater than 15 microg/mL of urine entailed subsequent 1-dimensional gel electrophoretic visualization of urinary proteins. The presence of 20 microg of proteases per mL of urine caused a complete degradation of proteins usually observed in urinary matrices ("trace of burning"), while respective proteases were still detected in spiked urine samples after 10 days of storage at + 4 and - 20 degrees C. Identification of target proteases at respective molecular weights was accomplished using bottom-up sequencing approaches based on in-gel digestion of separated enzymes followed by capillary liquid chromatography--Orbitrap tandem mass spectrometry.

  19. Purification and characterisation of a salt-stable protease from the halophilic archaeon Halogranum rubrum.

    PubMed

    Gao, Ruichang; Shi, Tong; Liu, Xiangdong; Zhao, Mengqin; Cui, Henglin; Yuan, Li

    2017-03-01

    Because proteases play an important role in the fermentation of fish sauce, the purification and characterisation of an extracellular protease from the halophilic archaeon Halogranum rubrum was investigated. The molecular mass of the protease was estimated to be approximately 47 kDa based on sodium dodecyl sulfate-polyacrylamide gel electropheresis (SDS-PAGE) and native-PAGE analysis. The optimum conditions for catalytic activity were pH 8.0 and 50°C. The protease showed alkaline stability (pH 7.0-10.0). The protease also exhibited novel catalytic ability over a broad range of salinity (NaCl 0-3 mol L -1 ). Calcium ion enhanced the proteolytic activity of the enzyme. The K m and V max values of the purified protease for casein were calculated to be 4.89 mg mL -1 and 1111.11 U mL -1 , respectively. The protease was strongly inhibited by ethylenediamine tetraacetic acid (EDTA) and phenylmethanesulfonyl fluoride (PMSF). Meanwhile, the protease was stable in the presence of Triton X-100, isopropanol, ethanol or dithio-bis-nitrobenzoic (DTNB), but was inhibited by sodium dodecyl sulfate (SDS), dimethyl sulfoxide (DMSO) or methanol. MALDI -TOF/TOF MS analysis revealed that the protease shared some functional traits with protease produced by Halogranum salarium. Furthermore, it exhibited high hydrolytic activity on silver carp myosin protein. The protease is an alkaline and salt-tolerant enzyme that hydrolyses silver carp myosin with high efficiency. These excellent characteristics make this protease an attractive candidate for industrial use in low-salt fish sauce fermentation. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  20. Giardia muris trophozoite antigenic targets for mouse intestinal IgA antibody.

    PubMed

    Heyworth, M F; Vergara, J A

    1994-02-01

    The aim of this work was to characterize Giardia muris trophozoite proteins that are targets for intestinal anti-trophozoite IgA in G. muris-infected mice. Intestinal secretions were obtained from immunocompetent BALB/c mice that had been infected with G. muris cysts 4-5 weeks previously and from control uninfected BALB/c mice. Flow cytometry of G. muris trophozoites that had been incubated with intestinal secretions and with fluorescein isothiocyanate-conjugated anti-mouse IgA showed that anti-trophozoite IgA was present in intestinal secretions obtained from infected BALB/c mice. By immunoblotting on G. muris trophozoite proteins separated by one-dimensional gel electrophoresis, this IgA recognized at least one trophozoite protein of molecular mass of approximately 80 kDa. The 80-kDa G. muris protein(s) has a molecular mass similar to that described for cysteine-rich surface proteins of the human parasite Giardia lamblia.

  1. A comparison of salivary IgA in children with Down syndrome and their family members.

    PubMed

    Balaji, Karthika; Milne, Trudy J; Drummond, Bernadette K; Cullinan, Mary P; Coates, Dawn E

    2016-07-01

    The aim of this study was to compare total IgA in the whole saliva of children with Down syndrome with levels in sibling and parent groups. IgA measurements were presented as the concentration in saliva (μg/ml) and also adjusted for salivary flow rate (SFR; μg/min). Twenty children with Down syndrome, ten siblings and twenty parents were recruited. Stimulated whole saliva was collected from the participants and SFR calculated. The measurement of salivary IgA (sIgA) was carried out using an indirect competitive Enzyme-Linked Immunosorbent Assay. The difference in the mean SFR between children with Down syndrome, parents and siblings were not statistically significant. The mean salivary concentration of IgA was higher in children with Down syndrome (95.1 μg/ml) compared with siblings (48.3 μg/ml; p=0.004). When adjusted for SFR children with Down syndrome had mean sIgA levels of 98.8 μg/min and the siblings 48.6 μg/min (p=0.008). The children with Down syndrome had sIgA levels similar to those of the parents (92.5 μg/ml; 93.2 μg/min). There was a positive correlation between age and sIgA concentration in the siblings (p=0.008) but not for children with Down syndrome (p=0.363). This suggests that under similar environmental influences, the levels of sIgA in children with Down syndrome are higher than in the siblings, from a very young age. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Heme oxygenase-1-derived bilirubin counteracts HIV protease inhibitor-mediated endothelial cell dysfunction

    PubMed Central

    Liu, Xiao-Ming; Durante, Zane E.; Peyton, Kelly J.; Durante, William

    2016-01-01

    The use of HIV protease inhibitors (PIs) has extended the duration and quality of life for HIV-positive individuals. However there is increasing concern that this antiviral therapy may promote premature cardiovascular disease by impairing endothelial cell (EC) function. In the present study, we investigated the effect of HIV PIs on EC function and determined if the enzyme heme oxygenase (HO-1) influences the biological action of these drugs. We found that three distinct PIs, including ritonavir, atazanavir, and lopinavir, stimulated the expression of HO-1 protein and mRNA. The induction of HO-1 was associated with an increase in NF-E2-related factor-2 (Nrf2) activity and reactive oxygen species (ROS). PIs also stimulated HO-1 promoter activity and this was prevented by mutating the antioxidant responsive element or by overexpressing dominant-negative Nrf2. In addition, the PI-mediated induction of HO-1 was abolished by N-acetyl-L-cysteine and rotenone. Furthermore, PIs blocked EC proliferation and migration and stimulated the expression of intercellular adhesion molecule-1 and the adhesion of monocytes on ECs. Inhibition of HO-1 activity or expression potentiated the anti-proliferative and inflammatory actions of PIs which was reversed by bilirubin but not carbon monoxide. Alternatively, adenovirus-mediated overexpression of HO-1 attenuated the growth-inhibitory and inflammatory effect of PIs. In contrast, blocking HO-1 activity failed to modify the anti-migratory effect of the PIs. Thus, induction of HO-1 via the ROS–Nrf2 pathway in human ECs counteracts the anti-proliferative and inflammatory actions of PIs by generating bilirubin. Therapeutic approaches targeting HO-1 may provide a novel approach in preventing EC dysfunction and vascular disease in HIV-infected patients undergoing antiretroviral therapy. PMID:26968795

  3. Prohibitin is involved in the activated internalization and degradation of protease-activated receptor 1.

    PubMed

    Wang, Yan-Jie; Guo, Xiao-Long; Li, Sheng-An; Zhao, Yu-Qi; Liu, Zi-Chao; Lee, Wen-Hui; Xiang, Yang; Zhang, Yun

    2014-07-01

    The protease-activated receptor 1 (PAR1) is a G-protein-coupled receptor that is irreversibly activated by either thrombin or metalloprotease 1. Due this irrevocable activation, activated internalization and degradation are critical for PAR1 signaling termination. Prohibitin (PHB) is an evolutionarily conserved, ubiquitously expressed, pleiotropic protein and belongs to the stomatin/prohibitin/flotillin/HflK/C (SPFH) domain family. In a previous study, we found that PHB localized on the platelet membrane and participated in PAR1-mediated human platelet aggregation, suggesting that PHB likely regulates the signaling of PAR1. Unfortunately, PHB's exact function in PAR1 internalization and degradation is unclear. In the current study, flow cytometry revealed that PHB expressed on the surface of endothelial cells (HUVECs) but not cancer cells (MDA-MB-231). Further confocal microscopy revealed that PHB dynamically associates with PAR1 in a time-dependent manner following induction with PAR1-activated peptide (PAR1-AP), though differently between HUVECs and MDA-MB-231 cells. Depletion of PHB by RNA interference significantly inhibited PAR1 activated internalization and led to sustained Erk1/2 phosphorylation in the HUVECs; however, a similar effect was not observed in MDA-MB-231 cells. For both the endothelial and cancel cells, PHB repressed PAR1 degradation, while knockdown of PHB led to increased PAR1 degradation, and PHB overexpression inhibited PAR1 degradation. These results suggest that persistent PAR1 signaling due to the absence of membrane PHB and decreased PAR1 degradation caused by the upregulation of intracellular PHB in cancer cells (such as MDA-MB-231 cells) may render cells highly invasive. As such, PHB may be a novel target in future anti-cancer therapeutics, or in more refined cancer malignancy diagnostics. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. Multiple receptor conformation docking and dock pose clustering as tool for CoMFA and CoMSIA analysis - a case study on HIV-1 protease inhibitors.

    PubMed

    Sivan, Sree Kanth; Manga, Vijjulatha

    2012-02-01

    Multiple receptors conformation docking (MRCD) and clustering of dock poses allows seamless incorporation of receptor binding conformation of the molecules on wide range of ligands with varied structural scaffold. The accuracy of the approach was tested on a set of 120 cyclic urea molecules having HIV-1 protease inhibitory activity using 12 high resolution X-ray crystal structures and one NMR resolved conformation of HIV-1 protease extracted from protein data bank. A cross validation was performed on 25 non-cyclic urea HIV-1 protease inhibitor having varied structures. The comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) models were generated using 60 molecules in the training set by applying leave one out cross validation method, r (loo) (2) values of 0.598 and 0.674 for CoMFA and CoMSIA respectively and non-cross validated regression coefficient r(2) values of 0.983 and 0.985 were obtained for CoMFA and CoMSIA respectively. The predictive ability of these models was determined using a test set of 60 cyclic urea molecules that gave predictive correlation (r (pred) (2) ) of 0.684 and 0.64 respectively for CoMFA and CoMSIA indicating good internal predictive ability. Based on this information 25 non-cyclic urea molecules were taken as a test set to check the external predictive ability of these models. This gave remarkable out come with r (pred) (2) of 0.61 and 0.53 for CoMFA and CoMSIA respectively. The results invariably show that this method is useful for performing 3D QSAR analysis on molecules having different structural motifs.

  5. Multicentric Castleman's disease associated with IgA vasculitis (Henoch-Schönlein purpura) responding well to tocilizumab: a case report.

    PubMed

    Oshima, Yoichi; Hoshino, Junichi; Suwabe, Tatsuya; Hayami, Noriko; Yamanouchi, Masayuki; Sekine, Akinari; Ueno, Toshiharu; Mizuno, Hiroki; Yabuuchi, Junko; Imafuku, Aya; Kawada, Masahiro; Hiramatsu, Rikako; Hasegawa, Eiko; Sawa, Naoki; Takaichi, Kenmei; Hayashi, Nobukazu; Fujii, Takeshi; Ubara, Yoshifumi

    2017-03-01

    A 41-year-old man was referred to our hospital for the evaluation of hypergammaglobulinemia (IgG 2898 mg/dL and IgA 587 mg/dL), inflammation (CRP 6.7 mg/dL and serum interleukin-6 (IL-6) 15.1 ng/L), and anemia (Hb 10.9 mg/dL). Castleman's disease (CD) was diagnosed by axillary lymph node biopsy. Five months later, painful purpura (multiple palpable 5 mm lesions) developed on his legs, gradually spreading to the upper limbs, thighs, and trunk, accompanied by arthralgia of the wrists, ankles, and knees. Skin biopsy revealed leukocytoclastic vasculitis with IgA deposits in dermal vessels. Accordingly, IgA vasculitis (Henoch-Schönlein purpura) was diagnosed. Tocilizumab (an anti-IL-6 receptor antibody) was administered intravenously at 8 mg/kg and treatment was repeated at monthly intervals. His purpura and clinical findings specific to CD improved rapidly. CD is well known to cause various skin lesions. The findings in this case indicate that overproduction of IL-6 contributes to IgA vasculitis (Henoch-Schönlein purpura) as well as to the pathogenesis of CD.

  6. Structural basis for norovirus neutralization by an HBGA blocking human IgA antibody.

    PubMed

    Shanker, Sreejesh; Czakó, Rita; Sapparapu, Gopal; Alvarado, Gabriela; Viskovska, Maria; Sankaran, Banumathi; Atmar, Robert L; Crowe, James E; Estes, Mary K; Prasad, B V Venkataram

    2016-10-04

    Human noroviruses (HuNoVs) cause sporadic and epidemic gastroenteritis worldwide. They are classified into two major genogroups (GI and GII), with each genogroup further divided into multiple genotypes. Susceptibility to these viruses is influenced by genetically determined histo-blood group antigen (HBGA) expression. HBGAs function as cell attachment factors by binding to a surface-exposed region in the protruding (P) domain of the capsid protein. Sequence variations in this region that result in differential HBGA binding patterns and antigenicity are suggested to form a basis for strain diversification. Recent studies show that serum antibodies that block HBGA binding correlate with protection against illness. Although genogroup-dependent variation in HBGA binding specificity is structurally well characterized, an understanding of how antibodies block HBGA binding and how genotypic variations affect such blockade is lacking. Our crystallographic studies of the GI.1 P domain in complex with the Fab fragment of a human IgA monoclonal antibody (IgA 5I2) with HBGA blocking activity show that the antibody recognizes a conformational epitope formed by two surface-exposed loop clusters in the P domain. The antibody engulfs the HBGA binding site but does not affect its structural integrity. An unusual feature of the antigen recognition by IgA 5I2 is the predominant involvement of the CDR light chain 1 in contrast to the commonly observed CDR heavy chain 3, providing a unique perspective into antibody diversity in antigen recognition. Identification of the antigenic site in the P domain shows how genotypic variations might allow escape from antibody neutralization and exemplifies the interplay between antigenicity and HBGA specificity in HuNoV evolution.

  7. Prediction of ingredient quality and the effect of a combination of xylanase, amylase, protease and phytase in the diets of broiler chicks. 1. Growth performance and digestible nutrient intake.

    PubMed

    Cowieson, A J; Singh, D N; Adeola, O

    2006-08-01

    1. A total of 2208 broiler chicks were used in two growth experiments (8 treatments and 12 replicate pens in each experiment) to assess the effects of xylanase, amylase, protease and phytase in maize-based diets. 2. A positive control diet was formulated containing adequate nutrient concentrations. A negative control diet was formulated to contain approximately 628 kJ/kg, 0.13%, 0.12% and 1 to 2% less metabolisable energy (ME), phosphorus (P), calcium (Ca) and amino acids, respectively, than the positive control. In addition, two further negative control diets that contained 167 or 334 kJ/kg more ME, respectively, than negative control 1 were formulated. 3. A further 4 dietary treatments were made by supplementing each of the 4 negative control diets with a combination of xylanase, amylase, protease and phytase, resulting in 8 dietary treatments in a 4 by 2 factorial arrangement. 4. The scale of the removal of energy, P, Ca and amino acids from the positive control diet was determined using least square models based on in vivo data for both the xylanase/amylase/protease cocktail and for phytase and it was predicted that performance of birds fed on negative control 1 would be returned by supplemental enzymes to that of those fed on the positive control. 5. In both experiments there was a significantly poorer performance in birds fed on the negative control 1 than in those fed on the positive control. The poorer weight gain and feed conversion ratio could be attributed in part to a reduced intake of digestible energy, P, nitrogen (N) and amino acids associated with birds fed on the negative control diet. 6. Supplementation of the negative control diets with the enzyme combination returned performance to that of the positive control in both experiments. 7. These data indicate that exogenous xylanase, amylase, protease and phytase can be used successfully in a strategically formulated low nutrient density diet to maintain performance to that of birds fed on a

  8. Correlation Between Expression of High Temperature Requirement Serine Protease A1 (HtrA1) in Nucleus Pulposus and T2 Value of Magnetic Resonance Imaging.

    PubMed

    Li, Dapeng; Yue, Jiawei; Jiang, Lu; Huang, Yonghui; Sun, Jifu; Wu, Yan

    2017-04-22

    BACKGROUND Degrading enzymes play an important role in the process of disc degeneration. The objective of this study was to investigate the correlation between the expression of high temperature requirement serine protease A1 (HtrA1) in the nucleus pulposus and the T2 value of the nucleus pulposus region in magnetic resonance imaging (MRI). MATERIAL AND METHODS Thirty-six patients who had undergone surgical excision of the nucleus pulposus were examined by MRI before surgery. Pfirrmann grading of the target intervertebral disc was performed according to the sagittal T2-weighted imaging, and the T2 value of the target nucleus pulposus was measured according to the median sagittal T2 mapping. The correlation between the Pfirrmann grade and the T2 value was analyzed. The expression of HtrA1 in the nucleus pulposus was analyzed by RT-PCR and Western blot. The correlation between the expression of HtrA1 and the T2 value was analyzed. RESULTS The T2 value of the nucleus pulposus region was 33.11-167.91 ms, with an average of 86.64±38.73 ms. According to Spearman correlation analysis, there was a rank correlation between T2 value and Pfirrmann grade (P<0.0001), and the correlation coefficient (rs)=-0.93617. There was a linear correlation between the mRNA level of HtrA1 and T2 value in nucleus pulposus tissues (a=3.88, b=-0.019, F=112.63, P<0.0001), normalized regression coefficient=-0.88. There was a linear correlation between the expression level of HtrA1 protein and the T2 value in the nucleus pulposus tissues (a=3.30, b=-0.016, F=93.15, P<0.0001) and normalized regression coefficient=-0.86. CONCLUSIONS The expression of HtrA1 was strongly related to the T2 value, suggesting that HtrA1 plays an important role in the pathological process of intervertebral disc degeneration.

  9. Cocoa and cocoa fibre differentially modulate IgA and IgM production at mucosal sites.

    PubMed

    Massot-Cladera, Malen; Franch, Àngels; Pérez-Cano, Francisco J; Castell, Margarida

    2016-05-01

    Previous studies have shown that a 10 % cocoa (C10) diet, containing polyphenols and fibre among others, modifies intestinal and systemic Ig production. The present study aimed at evaluating the impact of C10 on IgA and IgM production in the intestinal and extra-intestinal mucosal compartments, establishing the involvement of cocoa fibre (CF) in such effects. Mechanisms by which C10 intake may affect IgA synthesis in the salivary glands were also studied. To this effect, rats were fed either a standard diet, a diet containing C10, CF or inulin. Intestinal (the gut wash (GW), Peyer's patches (PP) and mesenteric lymph nodes (MLN)) and extra-intestinal (salivary glands) mucosal tissues and blood samples were collected for IgA and IgM quantification. The gene expressions of IgA production- and homing-related molecules were studied in the salivary glands. The C10 diet decreased intestinal IgA and IgM production. Although the CF diet decreased the GW IgA concentration, it increased PP, MLN and serum IgA concentrations. Both the C10 and the CF diets produced a down-regulatory effect on IgA secretion in the extra-intestinal tissues. The C10 diet interacted with the mechanisms involved in IgA synthesis, whereas the CF showed particular effects on the homing and transcytosis of IgA across the salivary glands. Overall, CF was able to up-regulate IgA production in the intestinal-inductor compartments, whereas it down-regulated its production at the mucosal-effector ones. Further studies must be directed to ascertain the mechanisms involved in the effect of particular cocoa components on gut-associated lymphoid tissue.

  10. SARS coronavirus papain-like protease induces Egr-1-dependent up-regulation of TGF-β1 via ROS/p38 MAPK/STAT3 pathway.

    PubMed

    Li, Shih-Wein; Wang, Ching-Ying; Jou, Yu-Jen; Yang, Tsuey-Ching; Huang, Su-Hua; Wan, Lei; Lin, Ying-Ju; Lin, Cheng-Wen

    2016-05-13

    SARS coronavirus (SARS-CoV) papain-like protease (PLpro) has been identified in TGF-β1 up-regulation in human promonocytes (Proteomics 2012, 12: 3193-205). This study investigates the mechanisms of SARS-CoV PLpro-induced TGF-β1 promoter activation in human lung epithelial cells and mouse models. SARS-CoV PLpro dose- and time-dependently up-regulates TGF-β1 and vimentin in A549 cells. Dual luciferase reporter assays with TGF-β1 promoter plasmids indicated that TGF-β1 promoter region between -175 to -60, the Egr-1 binding site, was responsible for TGF-β1 promoter activation induced by SARS-CoV PLpro. Subcellular localization analysis of transcription factors showed PLpro triggering nuclear translocation of Egr-1, but not NF-κB and Sp-1. Meanwhile, Egr-1 silencing by siRNA significantly reduced PLpro-induced up-regulation of TGF-β1, TSP-1 and pro-fibrotic genes. Furthermore, the inhibitors for ROS (YCG063), p38 MAPK (SB203580), and STAT3 (Stattic) revealed ROS/p38 MAPK/STAT3 pathway involving in Egr-1 dependent activation of TGF-β1 promoter induced by PLpro. In a mouse model with a direct pulmonary injection, PLpro stimulated macrophage infiltration into lung, up-regulating Egr-1, TSP-1, TGF-β1 and vimentin expression in lung tissues. The results revealed that SARS-CoV PLpro significantly triggered Egr-1 dependent activation of TGF-β1 promoter via ROS/p38 MAPK/STAT3 pathway, correlating with up-regulation of pro-fibrotic responses in vitro and in vivo.

  11. Comparison of combined leflunomide and low-dose corticosteroid therapy with full-dose corticosteroid monotherapy for progressive IgA nephropathy.

    PubMed

    Min, Lulin; Wang, Qin; Cao, Liou; Zhou, Wenyan; Yuan, Jiangzi; Zhang, Minfang; Che, Xiajing; Mou, Shan; Fang, Wei; Gu, Leyi; Zhu, Mingli; Wang, Ling; Yu, Zanzhe; Qian, Jiaqi; Ni, Zhaohui

    2017-07-18

    IgA nephropathy is the most common primary glomerulonephritis and one of the leading causes of end-stage renal disease. We performed a randomized, controlled, prospective, open-label trial to determine whether leflunomide combined with low-dose corticosteroid is safe and effective for the treatment of progressive IgA nephropathy, as compared to full-dose corticosteroid monotherapy. Biopsy-proved primary IgA nephropathy patients with an estimated glomerular filtration rate ≥ 30 ml/min/1.73m2 and proteinuria ≥1.0 g/24h were randomly assigned to receive leflunomide+low-dose corticosteroid (leflunomide group; n = 40) or full-dose corticosteroid (corticosteroids group; n = 45). The primary outcome was renal survival; secondary outcomes were proteinuria and adverse events. After 12 months of treatment and an average follow-up of 88 months, 11.1% vs. 7.5% of patients reached end-stage renal disease and 20% versus 10% of patients had a ≥ 50% increase in serum creatinine in the corticosteroids and leflunomide groups, respectively. Kaplan-Meier analysis did not reveal a between-group difference in these outcomes. Decreases in 24-hour proteinuria were similar in the two groups during the treatment period, but a more marked reduction was observed during follow-up in the leflunomide group. Although the incidence of adverse events was similar in the two groups, serious adverse events were observed only in the corticosteroid group. Thus, leflunomide combined with low-dose corticosteroid is at least as effective as corticosteroid alone for the treatment of progressive IgA nephropathy, and showed a greater reduction of proteinuria during long-term follow-up and fewer severe adverse events.

  12. High Degree of Interlaboratory Reproducibility of Human Immunodeficiency Virus Type 1 Protease and Reverse Transcriptase Sequencing of Plasma Samples from Heavily Treated Patients

    PubMed Central

    Shafer, Robert W.; Hertogs, Kurt; Zolopa, Andrew R.; Warford, Ann; Bloor, Stuart; Betts, Bradley J.; Merigan, Thomas C.; Harrigan, Richard; Larder, Brendon A.

    2001-01-01

    We assessed the reproducibility of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and protease sequencing using cryopreserved plasma aliquots obtained from 46 heavily treated HIV-1-infected individuals in two laboratories using dideoxynucleotide sequencing. The rates of complete sequence concordance between the two laboratories were 99.1% for the protease sequence and 99.0% for the RT sequence. Approximately 90% of the discordances were partial, defined as one laboratory detecting a mixture and the second laboratory detecting only one of the mixture's components. Only 0.1% of the nucleotides were completely discordant between the two laboratories, and these were significantly more likely to occur in plasma samples with lower plasma HIV-1 RNA levels. Nucleotide mixtures were detected at approximately 1% of the nucleotide positions, and in every case in which one laboratory detected a mixture, the second laboratory either detected the same mixture or detected one of the mixture's components. The high rate of concordance in detecting mixtures and the fact that most discordances between the two laboratories were partial suggest that most discordances were caused by variation in sampling of the HIV-1 quasispecies by PCR rather than by technical errors in the sequencing process itself. PMID:11283081

  13. The cholesterol, fatty acid and triglyceride synthesis pathways regulated by site 1 protease (S1P) are required for efficient replication of severe fever with thrombocytopenia syndrome virus.

    PubMed

    Urata, Shuzo; Uno, Yukiko; Kurosaki, Yohei; Yasuda, Jiro

    2018-06-12

    Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by the SFTS virus (SFTSV), which has a high mortality rate. Currently, no licensed vaccines or therapeutic agents have been approved for use against SFTSV infection. Here, we report that the cholesterol, fatty acid, and triglyceride synthesis pathways regulated by S1P is involved in SFTSV replication, using CHO-K1 cell line (SRD-12B) that is deficient in site 1 protease (S1P) enzymatic activity, PF-429242, a small compound targeting S1P enzymatic activity, and Fenofibrate and Lovastatin, which inhibit triglyceride and cholesterol synthesis, respectively. These results enhance our understanding of the SFTSV replication mechanism and may contribute to the development of novel therapies for SFTSV infection. Copyright © 2018. Published by Elsevier Inc.

  14. Heterocyclic HIV-protease inhibitors.

    PubMed

    Calugi, C; Guarna, A; Trabocchi, A

    2013-01-01

    In the panorama of HIV protease inhibitors (HIV PIs), many efforts have been devoted to the development of new compounds with reduced peptidic nature in order to improve pharmacokinetics and pharmacodynamics features. The introduction of cyclic scaffolds in the design of new chemical entities reduces flexibility and affords more rigid inhibitors. Specifically, common dipeptide isosteres are replaced by a central cyclic scaffold designed to address the key interactions with catalytic aspartic acids and residues belonging to the flap region of the active site. The current interest in cyclic chemotypes addressing key interactions of HIV protease is motivated by the different nature of interactions formed with the enzyme, although maintaining key structural resemblance to a peptide substrate, hopefully giving rise to novel HIV-1 PIs displaying an improved profile towards multidrug resistant strains. This approach has been demonstrated for Tipranavir, which is a potent FDA approved HIV-1 PI representing the most famous example of heterocyclic aspartic protease inhibitors.

  15. Reishi mushroom Ganoderma lucidum Modulates IgA production and alpha-defensin expression in the rat small intestine.

    PubMed

    Kubota, Atsuhito; Kobayashi, Masaki; Sarashina, Sota; Takeno, Reiko; Okamoto, Keisuke; Narumi, Katsuya; Furugen, Ayako; Suzuki, Yuji; Takahashi, Natsuko; Iseki, Ken

    2018-03-25

    Immunoglobulin A (IgA) secretion and alpha-defensins play a role in the innate immune system to protect against infection. Ganoderma lucidum (W.Curt.: Fr.) P. Karst. (Reishi) is a well-known mushroom in traditional Chinese medicine. This study aimed to determine the effects of Reishi on IgA secretion from Peyer's patch (PP) cells and alpha-defensin-5 (RD-5) and RD-6 expression in the rat small intestine. The rats received an oral injection of 0.5-5mg/kg of Reishi powder (1mL/kg) by sonde. All animals were euthanized 24h after Reishi administration. We examined RD-5, RD-6, and Toll-like receptor (TLR) 4 mRNA levels in the jejunum, ileum, and in Peyer's patches (PP) through quantitative real-time PCR analysis. IgA secretion from PP was measured through enzyme-linked immunosorbent assay of the supernatant after primary culture. Reishi increased IgA secretion in the presence of lipopolysaccharide (LPS) and increased TLR4 mRNA levels, but had no effect on the viability of PP cells. Moreover, Reishi increased RD-5, RD-6, and TLR4 mRNA levels significantly in the ileum in a concentration-dependent manner. Reishi can induce IgA secretion and increase the mRNA levels of RD-5 and RD-6 in the rat small intestine, through a TLR4-dependent pathway. The present results indicate that Reishi might reduce the risk of intestinal infection. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Clinical efficacy of anti-glycopeptidolipid-core IgA test for diagnosing Mycobacterium avium complex infection in lung.

    PubMed

    Numata, Takanori; Araya, Jun; Yoshii, Yutaka; Shimizu, Kenichiro; Hara, Hiromichi; Nakayama, Katsutoshi; Kuwano, Kazuyoshi

    2015-11-01

    It is difficult to verify the bacteriological diagnosis of Mycobacterium avium complex (MAC) infection. The anti-glycopeptidolipid (GPL)-core IgA antibody test was recently developed as a diagnostic method for MAC pulmonary disease. Only a few studies evaluate its clinical efficacy. We conducted retrospective evaluations of clinical characteristics of patients suspected of MAC infection to explore the usefulness of the anti-GPL-core IgA antibody test. We retrospectively evaluated 296 patients who were suspected to have MAC infection and underwent anti-GPL-core IgA antibody test between March 2013 and July 2014 in Jikei University hospital. A total of 29 patients were diagnosed with 'definite MAC' based on the American Thoracic Society (ATS) criteria with multiple identifications of MAC. On the other hand, 106 patients were diagnosed with other pulmonary diseases than MAC. The sensitivity and specificity of anti-GPL-core IgA antibody test for MAC diagnosis were 58.6% and 98.1%, respectively. The definite MAC group showed no significant differences in strains, treatment history or number of segments involved. The duration of MAC disease in the positive-antibody group was significantly longer than in the negative-antibody group (P = 0.046). A significant increase in the false-negative rate was observed in patients with malignant disease (P = 0.029). The anti-GPL-core IgA antibody test demonstrated high sensitivity and specificity for the diagnosis of MAC infection especially in patients without malignant diseases. © 2015 Asian Pacific Society of Respirology.

  17. Discovery of the 3-Imino-1,2,4-thiadiazinane 1,1-Dioxide Derivative Verubecestat (MK-8931)–A β-Site Amyloid Precursor Protein Cleaving Enzyme 1 Inhibitor for the Treatment of Alzheimer’s Disease

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Scott, Jack D.; Li, Sarah W.; Brunskill, Andrew P. J.

    Verubecestat 3 (MK-8931), a diaryl amide-substituted 3-imino-1,2,4-thiadiazinane 1,1-dioxide derivative, is a high-affinity β-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitor currently undergoing Phase 3 clinical evaluation for the treatment of mild to moderate and prodromal Alzheimer’s disease. Although not selective over the closely related aspartyl protease BACE2, verubecestat has high selectivity for BACE1 over other key aspartyl proteases, notably cathepsin D, and profoundly lowers CSF and brain Aβ levels in rats and nonhuman primates and CSF Aβ levels in humans. In this annotation, we describe the discovery of 3, including design, validation, and selected SAR around the novel iminothiadiazinanemore » dioxide core as well as aspects of its preclinical and Phase 1 clinical characterization.« less

  18. Possible mechanism of acute effect of ethanol on intestinal IgA expression in rat.

    PubMed

    Budec, Mirela; Koko, Vesna; Todorović, Vera; Marković, Dragana; Postić, Marija; Drndarević, Neda; Spasić, Andelka; Mitrović, Olivera

    2007-06-01

    The purpose of this study was to investigate the possible mechanism of acute effect of ethanol on IgA expression in rat intestine. To this end, adult female Wistar rats showing diestrus day 1 were treated with (a) ethanol (2 or 4 g/kg, i.p.); (b) N omega-nitro-L-arginine-methyl ester (L-NAME), which inhibits the activity of all isoforms of nitric oxide synthase, (30 mg/kg, s.c.) followed by ethanol 3 h later; and (c) L-NAME (30 mg/kg, s.c.) followed by saline 3 h later. Saline-injected and untreated rats were used as controls. The animals were sacrificed 0.5 h after ethanol administration. Intestinal expression of IgA was evaluated by both immunohistochemistry and Western immunoblotting. Morphometric analysis showed that acute ethanol treatment increased the number of IgA-immunoreactive cells in a dose-dependent manner. Pretreatment with L-NAME abolished this action of alcohol. Injection of L-NAME followed by saline had no influence on the number of IgA+cells. The results, obtained by Western immunoblotting, paralleled our immunohistochemical findings. Taken together, these data suggest that acute effect of ethanol on intestinal IgA might be mediated by endogenous nitric oxide.

  19. Activities of Vacuolar Cysteine Proteases in Plant Senescence.

    PubMed

    Martínez, Dana E; Costa, Lorenza; Guiamét, Juan José

    2018-01-01

    Plant senescence is accompanied by a marked increase in proteolytic activities, and cysteine proteases (Cys-protease) represent the prevailing class among the responsible proteases. Cys-proteases predominantly locate to lytic compartments, i.e., to the central vacuole (CV) and to senescence-associated vacuoles (SAVs), the latter being specific to the photosynthetic cells of senescing leaves. Cellular fractionation of vacuolar compartments may facilitate Cys-proteases purification and their concentration for further analysis. Active Cys-proteases may be analyzed by different, albeit complementary approaches: (1) in vivo examination of proteolytic activity by fluorescence microscopy using specific substrates which become fluorescent upon cleavage by Cys-proteases, (2) protease labeling with specific probes that react irreversibly with the active enzymes, and (3) zymography, whereby protease activities are detected in polyacrylamide gels copolymerized with a substrate for proteases. Here we describe the three methods mentioned above for detection of active Cys-proteases and a cellular fractionation technique to isolate SAVs.

  20. A Functional Interplay between Human Immunodeficiency Virus Type 1 Protease Residues 77 and 93 Involved in Differential Regulation of Precursor Autoprocessing and Mature Protease Activity

    PubMed Central

    Counts, Christopher J.; Ho, P. Shing; Donlin, Maureen J.; Tavis, John E.; Chen, Chaoping

    2015-01-01

    HIV-1 protease (PR) is a viral enzyme vital to the production of infectious virions. It is initially synthesized as part of the Gag-Pol polyprotein precursor in the infected cell. The free mature PR is liberated as a result of precursor autoprocessing upon virion release. We previously described a model system to examine autoprocessing in transfected mammalian cells. Here, we report that a covariance analysis of miniprecursor (p6*-PR) sequences derived from drug naïve patients identified a series of amino acid pairs that vary together across independent viral isolates. These covariance pairs were used to build the first topology map of the miniprecursor that suggests high levels of interaction between the p6* peptide and the mature PR. Additionally, several PR-PR covariance pairs are located far from each other (>12 Å Cα to Cα) relative to their positions in the mature PR structure. Biochemical characterization of one such covariance pair (77–93) revealed that each residue shows distinct preference for one of three alkyl amino acids (V, I, and L) and that a polar or charged amino acid at either of these two positions abolishes precursor autoprocessing. The most commonly observed 77V is preferred by the most commonly observed 93I, but the 77I variant is preferred by other 93 variances (L, V, or M) in supporting precursor autoprocessing. Furthermore, the 77I93V covariant enhanced precursor autoprocessing and Gag polyprotein processing but decreased the mature PR activity. Therefore, both covariance and biochemical analyses support a functional association between residues 77 and 93, which are spatially distant from each other in the mature PR structure. Our data also suggests that these covariance pairs differentially regulate precursor autoprocessing and the mature protease activity. PMID:25893662

  1. Protein-ligand binding free energy estimation using molecular mechanics and continuum electrostatics. Application to HIV-1 protease inhibitors

    NASA Astrophysics Data System (ADS)

    Zoete, V.; Michielin, O.; Karplus, M.

    2003-12-01

    A method is proposed for the estimation of absolute binding free energy of interaction between proteins and ligands. Conformational sampling of the protein-ligand complex is performed by molecular dynamics (MD) in vacuo and the solvent effect is calculated a posteriori by solving the Poisson or the Poisson-Boltzmann equation for selected frames of the trajectory. The binding free energy is written as a linear combination of the buried surface upon complexation, SAS bur, the electrostatic interaction energy between the ligand and the protein, Eelec, and the difference of the solvation free energies of the complex and the isolated ligand and protein, ΔGsolv. The method uses the buried surface upon complexation to account for the non-polar contribution to the binding free energy because it is less sensitive to the details of the structure than the van der Waals interaction energy. The parameters of the method are developed for a training set of 16 HIV-1 protease-inhibitor complexes of known 3D structure. A correlation coefficient of 0.91 was obtained with an unsigned mean error of 0.8 kcal/mol. When applied to a set of 25 HIV-1 protease-inhibitor complexes of unknown 3D structures, the method provides a satisfactory correlation between the calculated binding free energy and the experimental pIC 50 without reparametrization.

  2. Quantification of Pelvic Organ Prolapse in Mice: Vaginal Protease Activity Precedes Increased MOPQ Scores in Fibulin 5 Knockout Mice1

    PubMed Central

    Wieslander, Cecilia K.; Rahn, David D.; McIntire, Donald D.; Acevedo, Jesús F.; Drewes, Peter G.; Yanagisawa, Hiromi; Word, R. Ann

    2008-01-01

    Two mouse models of pelvic organ prolapse have been generated recently, both of which have null mutations in genes involved in elastic fiber synthesis and assembly (fibulin 5 and lysyl oxidase-like 1). Interestingly, although these mice exhibit elastinopathies early in life, pelvic organ prolapse does not develop until later in life. In this investigation we developed and validated a tool to quantify the severity of pelvic organ prolapse in mice, and we used this tool prospectively to study the role of fibulin 5, aging, and vaginal proteases in the development of pelvic organ prolapse. The results indicate that >90% of Fbln5−/− mice develop prolapse by 6 mo of age, even in the absence of vaginal delivery, and that increased vaginal protease activity precedes the development of prolapse. PMID:18987327

  3. A single intranasal immunization with a subunit vaccine formulation induces higher mucosal IgA production than live respiratory syncytial virus

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Garg, Ravendra

    Respiratory syncytial virus (RSV) causes serious respiratory illness in infants and elderly. RSV infection induces short-lived immunity, which leaves people prone to re-infection. In contrast, the RSV fusion (F) protein formulated with a novel adjuvant (∆F/TriAdj) elicits long term protective immunity. A comparison of RSV-immunized mice to mice vaccinated with a single dose of ∆F/TriAdj showed no difference in IgG1 and IgG2a production; however, local IgA secreting memory B cell development and B cell IgA production were significantly lower in RSV vaccinated mice than in ∆F/TriAdj-immunized mice. This indicates a potential reason as to why long-term immunity is not inducedmore » by RSV infection. The comparison also revealed that germinal center lymphocyte populations were higher in ∆F/TriAdj-vaccinated mice. Furthermore, ∆F/TriAdj induced higher gene expression of activation-induced cytidine deaminase (AID), as well as IL-6, IL-21, TGF-β cytokines, which are key players in IgA class switch recombination, ultimately leading to a sustained long-term memory response. - Highlights: •Immune responses to adjuvanted RSV F protein, ∆F/TriAdj, and RSV were compared. •∆F/TriAdj stimulates more local IgA production than RSV. •∆F/TriAdj induces more local IgA secreting memory B cells than RSV. •Germinal center lymphocyte populations are higher in ∆F/TriAdj-vaccinated mice. •∆F/TriAdj induces higher gene expression of AID, IL-6, IL-21, and TGF-β than RSV.« less

  4. Evidence that the recA441 (tif-1) mutant of Escherichia coli K-12 contains a thermosensitive intragenic suppressor of RecA constitutive protease activity.

    PubMed

    Wang, W B; Tessman, E S

    1985-07-01

    The recA441 mutant of Escherichia coli, which has been thought to have thermoinducible constitutive RecA protease activity, is known to have two mutations within recA. We show here that the mutation that alters codon 38 actually confers temperature-independent constitutive protease activity; the second mutation in recA441, which is at codon 298, appears to be acting as a temperature-sensitive suppressor of the protease activity.

  5. Efficacy and safety of switching to fixed-dose bictegravir, emtricitabine, and tenofovir alafenamide from boosted protease inhibitor-based regimens in virologically suppressed adults with HIV-1: 48 week results of a randomised, open-label, multicentre, phase 3, non-inferiority trial.

    PubMed

    Daar, Eric S; DeJesus, Edwin; Ruane, Peter; Crofoot, Gordon; Oguchi, Godson; Creticos, Catherine; Rockstroh, Jürgen K; Molina, Jean-Michel; Koenig, Ellen; Liu, Ya-Pei; Custodio, Joseph; Andreatta, Kristen; Graham, Hiba; Cheng, Andrew; Martin, Hal; Quirk, Erin

    2018-06-15

    Switching from therapy based on a boosted protease inhibitor to bictegravir, emtricitabine, and tenofovir alafenamide could avoid drug interactions and unwanted side-effects in virologically suppressed adults with HIV-1 infection, while maintaining a high barrier to resistance and providing a simplified once-daily, single-tablet regimen. Here, we report 48 week results of a phase 3 study investigating this switch. In this multicentre, randomised, open-label, active-controlled, non-inferiority, phase 3 trial, adults with HIV-1 infection were enrolled at 121 outpatient centres in ten countries. Eligible participants were aged 18 years or older, had an estimated glomerular filtration rate of 50 mL per min or higher, had been virologically suppressed (plasma HIV-1 RNA <50 copies per mL) for 6 months or more before screening, and were on a regimen consisting of boosted atazanavir or darunavir plus either emtricitabine and tenofovir disoproxil fumarate or abacavir and lamivudine. We randomly assigned participants (1:1), using a computer-generated randomisation sequence, to switch to co-formulated once-daily bictegravir (50 mg), emtricitabine (200 mg), and tenofovir alafenamide (25 mg), herein known as the bictegravir group, or to remain on their baseline boosted protease inhibitor regimen, herein known as the boosted protease inhibitor group, for 48 weeks. Randomisation was stratified by use of tenofovir disoproxil fumarate or abacavir at screening. The primary endpoint was the proportion of participants with plasma HIV-1 RNA of 50 copies per mL or higher at week 48 (by US Food and Drug Administration snapshot algorithm), with a prespecified non-inferiority margin of 4%. Efficacy and safety analyses included all participants who received at least one dose of study drug. This study is ongoing but not actively recruiting patients and is registered with ClinicalTrials.gov, number NCT02603107. Between Dec 2, 2015, and July 15, 2016, 578 participants were randomly assigned

  6. Prevention of Streptococcus mutans colonization by salivary IgA antibodies.

    PubMed

    Gregory, R L; Michalek, S M; Filler, S J; Mestecky, J; McGhee, J R

    1985-01-01

    The levels of salivary and serum IgA, IgG, and IgM antibodies to the seven serotypes (a-g) of Streptococcus mutans were established in 12 laboratory volunteers using a sensitive enzyme-linked immunosorbent assay. Salivary IgA antibody levels to the serotype c organism were significantly lower (P less than 0.005) than antibody levels to the other six serotypes of S. mutans. Similar results were found with a purified S. mutans serotype c carbohydrate. Serum IgG and IgM antibody titers to the serotype c whole cells were significantly higher (P less than 0.05) than to four other S. mutans serotypes (a, e-g). The abilities of S. mutans serotypes c and d to colonize molar tooth surfaces were examined in eight volunteers. S. mutans serotype d was cleared from the tooth surfaces within 24 hr of challenge, whereas S. mutans serotype c was detected in six of the eight volunteers after 2 weeks and in three of eight after 3 weeks. These results provide additional evidence for the role of salivary IgA antibodies in regulating S. mutans infection and suggest that the low levels of salivary IgA antibodies to S. mutans serotype c may contribute to the predominance of this serotype in the U.S. population.

  7. SARS coronavirus papain-like protease induces Egr-1-dependent up-regulation of TGF-β1 via ROS/p38 MAPK/STAT3 pathway

    PubMed Central

    Li, Shih-Wein; Wang, Ching-Ying; Jou, Yu-Jen; Yang, Tsuey-Ching; Huang, Su-Hua; Wan, Lei; Lin, Ying-Ju; Lin, Cheng-Wen

    2016-01-01

    SARS coronavirus (SARS-CoV) papain-like protease (PLpro) has been identified in TGF-β1 up-regulation in human promonocytes (Proteomics 2012, 12: 3193-205). This study investigates the mechanisms of SARS-CoV PLpro-induced TGF-β1 promoter activation in human lung epithelial cells and mouse models. SARS-CoV PLpro dose- and time-dependently up-regulates TGF-β1 and vimentin in A549 cells. Dual luciferase reporter assays with TGF-β1 promoter plasmids indicated that TGF-β1 promoter region between −175 to −60, the Egr-1 binding site, was responsible for TGF-β1 promoter activation induced by SARS-CoV PLpro. Subcellular localization analysis of transcription factors showed PLpro triggering nuclear translocation of Egr-1, but not NF-κB and Sp-1. Meanwhile, Egr-1 silencing by siRNA significantly reduced PLpro-induced up-regulation of TGF-β1, TSP-1 and pro-fibrotic genes. Furthermore, the inhibitors for ROS (YCG063), p38 MAPK (SB203580), and STAT3 (Stattic) revealed ROS/p38 MAPK/STAT3 pathway involving in Egr-1 dependent activation of TGF-β1 promoter induced by PLpro. In a mouse model with a direct pulmonary injection, PLpro stimulated macrophage infiltration into lung, up-regulating Egr-1, TSP-1, TGF-β1 and vimentin expression in lung tissues. The results revealed that SARS-CoV PLpro significantly triggered Egr-1 dependent activation of TGF-β1 promoter via ROS/p38 MAPK/STAT3 pathway, correlating with up-regulation of pro-fibrotic responses in vitro and in vivo. PMID:27173006

  8. Mitochondrial enzymes are protected from stress-induced aggregation by mitochondrial chaperones and the Pim1/LON protease

    PubMed Central

    Bender, Tom; Lewrenz, Ilka; Franken, Sebastian; Baitzel, Catherina; Voos, Wolfgang

    2011-01-01

    Proteins in a natural environment are constantly challenged by stress conditions, causing their destabilization, unfolding, and, ultimately, aggregation. Protein aggregation has been associated with a wide variety of pathological conditions, especially neurodegenerative disorders, stressing the importance of adequate cellular protein quality control measures to counteract aggregate formation. To secure protein homeostasis, mitochondria contain an elaborate protein quality control system, consisting of chaperones and ATP-dependent proteases. To determine the effects of protein aggregation on the functional integrity of mitochondria, we set out to identify aggregation-prone endogenous mitochondrial proteins. We could show that major metabolic pathways in mitochondria were affected by the aggregation of key enzyme components, which were largely inactivated after heat stress. Furthermore, treatment with elevated levels of reactive oxygen species strongly influenced the aggregation behavior, in particular in combination with elevated temperatures. Using specific chaperone mutant strains, we showed a protective effect of the mitochondrial Hsp70 and Hsp60 chaperone systems. Moreover, accumulation of aggregated polypeptides was strongly decreased by the AAA-protease Pim1/LON. We therefore propose that the proteolytic breakdown of aggregation-prone polypeptides represents a major protective strategy to prevent the in vivo formation of aggregates in mitochondria. PMID:21209324

  9. Design of HIV-1 Protease Inhibitors with Amino-bis-tetrahydrofuran Derivatives as P2-Ligands to Enhance Backbone-Binding Interactions. Synthesis, Biological Evaluation, and Protein-Ligand X-ray Studies

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ghosh, Arun K.; Martyr, Cuthbert D.; Osswald, Heather L.

    Structure-based design, synthesis, and biological evaluation of a series of very potent HIV-1 protease inhibitors are described. In an effort to improve backbone ligand–binding site interactions, we have incorporated basic-amines at the C4 position of the bis-tetrahydrofuran (bis-THF) ring. We speculated that these substituents would make hydrogen bonding interactions in the flap region of HIV-1 protease. Synthesis of these inhibitors was performed diastereoselectively. A number of inhibitors displayed very potent enzyme inhibitory and antiviral activity. Inhibitors 25f, 25i, and 25j were evaluated against a number of highly-PI-resistant HIV-1 strains, and they exhibited improved antiviral activity over darunavir. Two high resolutionmore » X-ray structures of 25f- and 25g-bound HIV-1 protease revealed unique hydrogen bonding interactions with the backbone carbonyl group of Gly48 as well as with the backbone NH of Gly48 in the flap region of the enzyme active site. These ligand–binding site interactions are possibly responsible for their potent activity.« less

  10. Evidence that the recA441 (tif-1) mutant of Escherichia coli K-12 contains a thermosensitive intragenic suppressor of RecA constitutive protease activity.

    PubMed Central

    Wang, W B; Tessman, E S

    1985-01-01

    The recA441 mutant of Escherichia coli, which has been thought to have thermoinducible constitutive RecA protease activity, is known to have two mutations within recA. We show here that the mutation that alters codon 38 actually confers temperature-independent constitutive protease activity; the second mutation in recA441, which is at codon 298, appears to be acting as a temperature-sensitive suppressor of the protease activity. Images PMID:3891740

  11. Idiopathic linear IgA bullous dermatosis: prognostic factors based on a case series of 72 adults.

    PubMed

    Gottlieb, J; Ingen-Housz-Oro, S; Alexandre, M; Grootenboer-Mignot, S; Aucouturier, F; Sbidian, E; Tancrede, E; Schneider, P; Regnier, E; Picard-Dahan, C; Begon, E; Pauwels, C; Cury, K; Hüe, S; Bernardeschi, C; Ortonne, N; Caux, F; Wolkenstein, P; Chosidow, O; Prost-Squarcioni, C

    2017-07-01

    Linear IgA bullous dermatosis (LABD) is a clinically and immunologically heterogeneous, subepidermal, autoimmune bullous disease (AIBD), for which the long-term evolution is poorly described. To investigate the clinical and immunological characteristics, follow-up and prognostic factors of adult idiopathic LABD. This retrospective study, conducted in our AIBD referral centre, included adults, diagnosed between 1995 and 2012, with idiopathic LABD, defined as pure or predominant IgA deposits by direct immunofluorescence. Clinical, histological and immunological findings were collected from charts. Standard histology was systematically reviewed, and indirect immunofluorescence (IIF) on salt-split skin (SSS) and immunoblots (IBs) on amniotic membrane extracts using anti-IgA secondary antibodies were performed, when biopsies and sera obtained at diagnosis were available. Prognostic factors for complete remission (CR) were identified using univariate and multivariate analyses. Of the 72 patients included (median age 54 years), 60% had mucous membrane (MM) involvement. IgA IIF on SSS was positive for 21 of 35 patients tested; 15 had epidermal and dermal labellings. Immunoelectron microscopy performed on the biopsies of 31 patients labelled lamina lucida (LL) (26%), lamina densa (23%), anchoring-fibril zone (AFz) (19%) and LL+AFz (23%). Of the 34 IgA IBs, 22 were positive, mostly for LAD-1/LABD97 (44%) and full-length BP180 (33%). The median follow-up was 39 months. Overall, 24 patients (36%) achieved sustained CR, 19 (29%) relapsed and 35% had chronic disease. CR was significantly associated with age > 70 years or no MM involvement. No prognostic immunological factor was identified. Patients with LABD who are < 70 years old and have MM involvement are at risk for chronic evolution. © 2017 British Association of Dermatologists.

  12. Determination of the absolute binding free energies of HIV-1 protease inhibitors using non-equilibrium molecular dynamics simulations

    NASA Astrophysics Data System (ADS)

    Ngo, Son Tung; Nguyen, Minh Tung; Nguyen, Minh Tho

    2017-05-01

    The absolute binding free energy of an inhibitor to HIV-1 Protease (PR) was determined throughout evaluation of the non-bonded interaction energy difference between the two bound and unbound states of the inhibitor and surrounding molecules by the fast pulling of ligand (FPL) process using non-equilibrium molecular dynamics (NEMD) simulations. The calculated free energy difference terms help clarifying the nature of the binding. Theoretical binding affinities are in good correlation with experimental data, with R = 0.89. The paradigm used is able to rank two inhibitors having the maximum difference of ∼1.5 kcal/mol in absolute binding free energies.

  13. A New Subtilase-Like Protease Deriving from Fusarium equiseti with High Potential for Industrial Applications.

    PubMed

    Juntunen, Kari; Mäkinen, Susanna; Isoniemi, Sari; Valtakari, Leena; Pelzer, Alexander; Jänis, Janne; Paloheimo, Marja

    2015-09-01

    A gene encoding a novel extracellular subtilisin-like protease was cloned from the ascomycete Fusarium equiseti and expressed in Trichoderma reesei. The F. equiseti protease (Fe protease) showed excellent performance in stain removal and good compatibility with several commercial laundry detergent formulations, suggesting that it has high potential for use in various industrial applications. The recombinant enzyme was purified and characterized. The temperature optimum of the Fe protease was 60 °C and it showed high activity in the pH range of 6-10, with a sharp decline in activity at pH above 10. The amino acid specificity of the Fe protease was studied using casein, cytochrome c, and ubiquitin as substrates. The Fe protease had broad substrate specificity: almost all amino acid residues were accepted at position P1, even though it showed some preference for cleavage at the C-terminal side of asparagine and histidine residues. The S4 subsite of Fe protease favors aspartic acid and threonine. The other well-characterized proteases from filamentous fungi, Proteinase K from Engyodontium album, Thermomycolin from Malbranchea sulfurea, and alkaline subtilisins from Bacillus species prefer hydrophobic amino acids in both the S1 and S4 subsites. Due to its different specificity compared to the members of the S8 family of clan SB of proteases, we consider that the Fe protease is a new protease. It does not belong to any previously defined IUBMB groups of proteases.

  14. Foliar extracts from transgenic tomato plants expressing the structural polyprotein, P1-2A, and protease, 3C, from foot-and-mouth disease virus elicit a protective response in guinea pigs.

    PubMed

    Pan, Li; Zhang, Yongguang; Wang, Yonglu; Wang, Baoqin; Wang, Wenxiu; Fang, Yuzhen; Jiang, Shoutian; Lv, Jianliang; Wang, Wei; Sun, Yuan; Xie, Qingge

    2008-01-15

    The expression of recombinant antigens in transgenic plants is increasingly used as an alternative method of producing experimental immunogens. In this report, we describe the production of transgenic tomato plants that express the structural polyprotein, P1-2A, and protease, 3C, from foot-and-mouth disease (FMDV). P1-2A3C was inserted into the plant binary vector, pBin438, and transformed into tomato plants using Agrobacterium tumefaciens strain, GV3101. The presence of P1-2A3C was confirmed by PCR, transcription was verified by RT-PCR, and recombinant protein expression was confirmed by sandwich-ELISA and Western blot analyses. Guinea pigs immunized intramuscularly with foliar extracts from P1-2A3C-transgenic tomato plants were found to develop a virus-specific antibody response against FMDV. Vaccinated guinea pigs were fully protected against a challenge infection, while guinea pigs injected with untransformed plant extracts failed to elicit an antibody response and were not protected against challenge. These results demonstrate that transgenic tomato plants expressing the FMDV structural polyprotein, P1-2A, and the protease, 3C, can be used as a source of recombinant antigen for vaccine production.

  15. Effects of eye rubbing on the levels of protease, protease activity and cytokines in tears: relevance in keratoconus.

    PubMed

    Balasubramanian, Sivaraman A; Pye, David C; Willcox, Mark D P

    2013-03-01

    Proteases, protease activity and inflammatory molecules in tears have been found to be relevant in the pathogenesis of keratoconus. We sought to determine the influence of eye rubbing on protease expression, protease activity and concentration of inflammatory molecules in tears. Basal tears were collected from normal volunteers before and after 60 seconds of experimental eye rubbing. The total amount of matrix metalloproteinase (MMP)-13 and inflammatory molecules interleukin (IL)-6 and tumour necrosis factor (TNF)-α in the tear samples were measured using specific enzyme-linked immunosorbent assays (ELISA). Tear collagenase activity was investigated using a specific activity assay. The concentrations of MMP-13 (51.9 ± 34.3 versus 63 ± 36.8 pg/ml, p = 0.006), IL-6 (1.24 ± 0.98 versus 2.02 ± 1.52 pg/ml, p = 0.004) and TNF-α (1.16 ± 0.74 versus 1.44 ± 0.66 pg/ml, p = 0.003) were significantly increased in normal subjects after eye rubbing. The experimental eye rub did not alter significantly the collagenase activity (5.02 ± 3 versus 7.50 ± 3.90 fluorescent intensity units, p = 0.14) of tears. Eye rubbing for 60 seconds increased the level of tear MMP-13, IL-6 and TNF-α in normal study subjects. This increase in protease, protease activity and inflammatory mediators in tears after eye rubbing may be exacerbated even further during persistent and forceful eye rubbing seen in people with keratoconus and this in turn may contribute to the progression of the disease. © 2013 The Authors. Clinical and Experimental Optometry © 2013 Optometrists Association Australia.

  16. Role of maternal elimination diets and human milk IgA in development of cow’s milk allergy in the infants

    PubMed Central

    Järvinen, Kirsi M.; Westfall, Jennifer E.; Seppo, Max S.; James, Aisha K.; Tsuang, Angela J.; Feustel, Paul J.; Sampson, Hugh A.; Berin, Cecilia

    2014-01-01

    Background The role of maternal avoidance diets in the prevention of food allergies is currently under debate. Little is known regarding the effects of such diets on human milk (HM) composition or induction of infant humoral responses. Objective To assess the association of maternal cow’s milk (CM) avoidance during breastfeeding with specific IgA levels in HM and development of cow’s milk allergy (CMA) in infants. Methods We utilized HM and infant serum samples from a prospective birth cohort of 145 dyads. Maternal serum and HM samples were assessed for casein and beta-lactoglobulin (BLG)-specific IgA and IgG by ELISA; 21 mothers prophylactically initiated a strict maternal CM avoidance diet due to a sibling’s history of food allergy and 16 due to atopic eczema or regurgitation/vomiting seen in their infants within the first 3 months of life. Infants’ sera were assessed for casein and BLG-specific IgG, IgA and IgE; CMA was confirmed by an oral food challenge. The impact of HM on BLG uptake was assessed in transcytosis assays utilizing Caco-2 intestinal epithelial cell line. Results Mothers avoiding CM had lower casein- and BLG-specific IgA in HM than mothers with no CM restriction (p=0.019 and p=0.047). Their infants had lower serum casein- and BLG-specific IgG1 (p=0.025 and p<0.001) and BLG-specific IgG4 levels (p=0.037) and their casein- and BLG-specific IgA levels were less often detectable than those with no CM elimination diet (p=0.003 and p=0.007). Lower CM-specific IgG4 and IgA levels in turn were associated with infant CMA. Transcytosis of BLG was impaired by HM with high, but not low levels of specific IgA. Conclusions Maternal CM avoidance was associated with lower levels of mucosal specific IgA levels and development of CMA in infants. Clinical relevance HM IgA may play a role in preventing excessive, uncontrolled food antigen uptake in the gut lumen and thereby in the prevention of CMA. PMID:24164317

  17. Role of maternal elimination diets and human milk IgA in the development of cow's milk allergy in the infants.

    PubMed

    Järvinen, K M; Westfall, J E; Seppo, M S; James, A K; Tsuang, A J; Feustel, P J; Sampson, H A; Berin, C

    2014-01-01

    The role of maternal avoidance diets in the prevention of food allergies is currently under debate. Little is known regarding the effects of such diets on human milk (HM) composition or induction of infant humoral responses. To assess the association of maternal cow's milk (CM) avoidance during breastfeeding with specific IgA levels in HM and development of cow's milk allergy (CMA) in infants. We utilized HM and infant serum samples from a prospective birth cohort of 145 dyads. Maternal serum and HM samples were assessed for casein and beta-lactoglobulin (BLG)-specific IgA and IgG by ELISA; 21 mothers prophylactically initiated a strict maternal CM avoidance diet due to a sibling's history of food allergy and 16 due to atopic eczema or regurgitation/vomiting seen in their infants within the first 3 months of life. Infants' sera were assessed for casein and BLG-specific IgG, IgA and IgE; CMA was confirmed by an oral food challenge. The impact of HM on BLG uptake was assessed in transcytosis assays utilizing Caco-2 intestinal epithelial cell line. Mothers avoiding CM had lower casein- and BLG-specific IgA in HM than mothers with no CM restriction (P = 0.019 and P = 0.047). Their infants had lower serum casein- and BLG-specific IgG(1) (P = 0.025 and P < 0.001) and BLG-specific IgG(4) levels (P = 0.037), and their casein- and BLG-specific IgA levels were less often detectable than those with no CM elimination diet (P = 0.003 and P = 0.007). Lower CM-specific IgG4 and IgA levels in turn were associated with infant CMA. Transcytosis of BLG was impaired by HM with high, but not low levels of specific IgA. Maternal CM avoidance was associated with lower levels of mucosal-specific IgA levels and the development of CMA in infants. HM IgA may play a role in preventing excessive, uncontrolled food antigen uptake in the gut lumen and thereby in the prevention of CMA. © 2013 John Wiley & Sons Ltd.

  18. Defective anti-polysaccharide IgG vaccine responses in IgA deficient mice

    USDA-ARS?s Scientific Manuscript database

    IgA deficient patients often show defects in antibody responses following immunization with polysaccharide vaccines. We now show that IgA-/- mice exhibit specific defects in IgG antibody responses to various polysaccharide vaccines, but not protein vaccines. Defects in anti-polysaccharide IgG resp...

  19. Anal lymphogranuloma venereum infection screening with IgA anti-Chlamydia trachomatis-specific major outer membrane protein serology.

    PubMed

    de Vries, Henry J C; Smelov, Vitaly; Ouburg, Sander; Pleijster, Jolein; Geskus, Ronald B; Speksnijder, Arjen G C L; Fennema, Johannes S A; Morré, Servaas A

    2010-12-01

    Anal lymphogranuloma venereum (LGV) infections, caused by Chlamydia trachomatis biovar L (Ct+/LGV+), are endemic among men who have sex with men (MSM). Anal non-LGV biovar Ct infections (Ct+/LGV-) can be eradicated with 1 week doxycycline, whereas Ct+/LGV+ infections require 3-week doxycycline. To differentiate Ct+/LGV+ from Ct+/LGV- infections, biovar-specific Nucleic Acid Amplification Test (NAAT) are standard, but also expensive and laborious. A chlamydia-specific serological assay could serve as an alternative test. MSM were screened for anal Ct+/LGV+ and Ct+/LGV- infections with a commercial nonspecific NAAT and an in house biovar L-specific NAAT. Serum samples were evaluated with chlamydia-specific anti-Major Outer Membrane Protein (MOMP) and antilipopolysaccharide assays of IgA and IgG classes. Asymptomatic patients were identified as: (1) no anal complaints or (2) no microscopic inflammation (i.e., <10 leucocytes per high power field in anal smears). The best differentiating assay was subsequently evaluated in 100 Ct+/LGV+ and 100 Ct+/LGV- MSM using different cut-off points. The anti-MOMP IgA assay was the most accurate to differentiate Ct+/LGV+ (n = 42) from Ct+/LGV- (n = 19) with 85.7% sensitivity (95% confidence interval [CI], 72.2-93.3) and 84.2% specificity (95% CI, 62.4-94.5), even among asymptomatic patients. In a population comprising 98 Ct+/LGV+ and 105 Ct+/LGV- patients, the anti-MOMP IgA assay scored most accurate when the cut-off point was set to 2.0 with 75.5% (95% CI, 65.8-83.6) sensitivity and 74.3% (95% CI, 64.8-82.3) specificity. The IgA anti-MOMP assay can identify a considerable proportion of the (asymptomatic) anal LGV infections correctly. Yet, biovar L-specific NAAT are still the preferred diagnostic tests in clinical settings.

  20. Protease-activated receptor-1 antagonists in long-term antiplatelet therapy. Current state of evidence and future perspectives.

    PubMed

    Moschonas, I C; Goudevenos, J A; Tselepis, A D

    2015-04-15

    Atherothrombosis and its clinical manifestations are among the leading causes of death in the developed world. The current standard-of-care antiplatelet therapy for the treatment of such events comprises aspirin and a thienopyridine or ticagrelor. However, recurrent ischemic events due to residual cardiovascular risk are a common phenomenon in these patients. It is believed that this residual risk is caused, at least in part, by thrombin, which signals through protease-activated receptors (PARs) and especially PAR-1. Thus, PAR-1 antagonism could represent an effective approach in the treatment of atherothrombotic disease. In this context, two potent and selective agents have been developed, vorapaxar and atopaxar. However, only vorapaxar has completed phase 3 clinical trials. In the present review, the main pharmacodynamic and pharmacokinetic properties of the PAR-1 antagonists are briefly described and the latest clinical data on vorapaxar are presented. Copyright © 2015. Published by Elsevier Ireland Ltd.

  1. Predicting Post-transplant Recurrence of IgA Nephropathy: The Importance of Crescents

    PubMed Central

    Avasare, Rupali S.; Rosenstiel, Paul E.; Zaky, Ziad S.; Tsapepas, Demetra; Appel, Gerald B.; Markowitz, Glen S.; Bomback, Andrew S.; Canetta, Pietro A.

    2017-01-01

    Background Most studies assessing the predictors of recurrent IgA nephropathy in the renal allograft have focused on post-transplant features. Identifying high risk pre-transplant features of IgA nephropathy is useful for counseling patients and may help tailor immunosuppression post-transplant. Methods We investigated the pre-transplant clinical and biopsy features of 62 patients with IgAN who received transplants at Columbia University Medical Center from 2001 to 2012 and compared the characteristics and outcomes of patients with IgAN recurrence to those without recurrence. The primary outcome was time to recurrent IgAN. Secondary outcomes were a composite of doubling of creatinine or allograft failure, and recurrent IgAN as a cause of allograft dysfunction. Results Of the 62 patients, 14 had recurrent IgAN in the allograft. Mean time to recurrence was 2.75 years. Those with recurrent disease were younger at time of native kidney biopsy (29 years vs. 41 years, P < 0.0009). Black race and Hispanic ethnicity composed a higher proportion of the recurrent disease group. On multivariable analysis, significant predictors of recurrent IgAN included age at diagnosis (HR 0.911, 95% CI 0.85 to 0.98), burden of crescents on native biopsy (HR 1.21 per 10% increase in crescents, 95% CI 1.00 to 1.47), and allograft rejection (HR 3.59, 95% CI 1.10 to 11.7) Conclusions Features of native IgAN can help predict the risk of recurrent disease in the renal allograft. In particular, immunologically active disease represented by earlier age of onset and greater burden of crescents on native biopsy is more likely to recur post-transplant. PMID:28056461

  2. Description of the L76V resistance protease mutation in HIV-1 B and "non-B" subtypes.

    PubMed

    Charpentier, Charlotte; Lambert-Niclot, Sidonie; Alteri, Claudia; Storto, Alexandre; Flandre, Philippe; Svicher, Valentina; Perno, Carlo-Federico; Brun-Vézinet, Françoise; Calvez, Vincent; Marcelin, Anne-Geneviève; Ceccherini-Silberstein, Francesca; Descamps, Diane

    2013-01-01

    To describe the prevalence of the L76V protease inhibitors resistance-associated mutation (PI-RAM) in relation with patients' characteristics and protease genotypic background in HIV-1 B- and "non-B"-infected patients. Frequency of the L76V mutation between 1998 and 2010 was surveyed in the laboratory database of 3 clinical centers. Major PI-RAMs were identified according to the IAS-USA list. Fisher's and Wilcoxon tests were used to compare variables. Among the overall 29,643 sequences analyzed, the prevalence of L76V was 1.50%, while was 5.42% in PI-resistant viruses. Since 2008 the prevalence of L76V was higher in "non-B"-infected than in B-infected patients each year. Median time since diagnosis of HIV-1 infection and median time under antiretroviral-based regimen were both shorter in "non-B"- than in B-infected patients (8 vs 11 years, P<0.0001; and 7 vs 8 years, P = 0.004). In addition, "non-B"-infected patients had been pre-exposed to a lower number of PI (2 vs 3, P = 0.016). The L76V was also associated with a lower number of major PI-RAMs in "non-B" vs B samples (3 vs 4, P = 0.0001), and thus it was more frequent found as single major PI-RAM in "non-B" vs B subtype (10% vs 2%, P = 0.014). We showed an impact of viral subtype on the selection of the L76V major PI-RAM with a higher prevalence in "non-B" subtypes observed since 2008. In addition, in "non-B"-infected patients this mutation appeared more rapidly and was associated with less PI-RAM.

  3. Identification of a Degradation Signal Sequence within Substrates of the Mitochondrial i-AAA Protease.

    PubMed

    Rampello, Anthony J; Glynn, Steven E

    2017-03-24

    The i-AAA protease is a component of the mitochondrial quality control machinery that regulates respiration, mitochondrial dynamics, and protein import. The protease is required to select specific substrates for degradation from among the diverse complement of proteins present in mitochondria, yet the rules that govern this selection are unclear. Here, we reconstruct the yeast i-AAA protease, Yme1p, to examine the in vitro degradation of two intermembrane space chaperone subunits, Tim9 and Tim10. Yme1p degrades Tim10 more rapidly than Tim9 despite high sequence and structural similarity, and loss of Tim10 is accelerated by the disruption of conserved disulfide bonds within the substrate. An unstructured N-terminal region of Tim10 is necessary and sufficient to target the substrate to the protease through recognition of a short phenylalanine-rich motif, and the presence of similar motifs in other small Tim proteins predicts robust degradation by the protease. Together, these results identify the first specific degron sequence within a native i-AAA protease substrate. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Truncation of a P1 leader proteinase facilitates potyvirus replication in a non-permissive host.

    PubMed

    Shan, Hongying; Pasin, Fabio; Tzanetakis, Ioannis E; Simón-Mateo, Carmen; García, Juan Antonio; Rodamilans, Bernardo

    2018-06-01

    The Potyviridae family is a major group of plant viruses that includes c. 200 species, most of which have narrow host ranges. The potyvirid P1 leader proteinase self-cleaves from the remainder of the viral polyprotein and shows large sequence variability linked to host adaptation. P1 proteins can be classified as Type A or Type B on the basis, amongst other things, of their dependence or not on a host factor to develop their protease activity. In this work, we studied Type A proteases from the Potyviridae family, characterizing their host factor requirements. Our in vitro cleavage analyses of potyvirid P1 proteases showed that the N-terminal domain is relevant for host factor interaction and suggested that the C-terminal domain is also involved. In the absence of plant factors, the N-terminal end of Plum pox virus P1 antagonizes protease self-processing. We performed extended deletion mutagenesis analysis to define the N-terminal antagonistic domain of P1. In viral infections, removal of the P1 protease antagonistic domain led to a gain-of-function phenotype, strongly increasing local infection in a non-permissive host. Altogether, our results shed new insights into the adaptation and evolution of potyvirids. © 2017 BSPP AND JOHN WILEY & SONS LTD.

  5. A real-time fluorescence assay for protease activity and inhibitor screening based on the aggregation-caused quenching of a perylene probe.

    PubMed

    Wang, Yan; Zhang, Zhifang; Zhang, Ya; Yu, Cong

    2018-06-01

    We have established a real-time and label-free fluorescence turn-on strategy for protease activity detection and inhibitor screening via peptide-induced aggregation-caused quenching of a perylene probe. Because of electrostatic interactions and high hydrophilicity, poly-l-glutamic acid sodium salt (PGA; a negatively charged peptide) could induce aggregation of a positively charged perylene probe (probe 1) and the monomer fluorescence of probe 1 was effectively quenched. After a protease was added, PGA was enzymatically hydrolyzed into small fragments and probe 1 disaggregated. The fluorescence recovery of probe 1 was found to be proportional to the concentration of protease in the range from 0 to 1 mU/ml. The detection limit was down to 0.1 mU/ml. In the presence of a protease inhibitor, protease activity was inhibited and fluorescence recovery reduced. Moreover, we demonstrated the potential application of our method in a complex mixture sample including 1% human serum. Our method is simple, fast and cost effective. Copyright © 2018 John Wiley & Sons, Ltd.

  6. RpA, an extracellular protease similar to the metalloprotease of serralysin family, is required for pathogenicity of Ralstonia pickettii.

    PubMed

    Chen, C-M; Liu, J-J; Chou, C-W; Lai, C-H; Wu, L-T

    2015-10-01

    To investigate the biochemical and functional properties of an extracellular protease, RpA, in Ralstonia pickettii WP1 isolated from water supply systems. An extracellular protease was identified and characterized from R. pickettii WP1. A mutant strain WP1M2 was created from strain WP1 by mini-Tn5 transposition. The culture filtrates from WP1M2 had a lower cytotoxic effect than the parental WP1 on several mammalian cell lines. Cloning and sequence analysis revealed the Tn5 transposon inserted at a protease gene (rpA) which is 81% homologous to prtA and aprX genes of Pseudomonas fluorescens. The rpA gene encodes a 482-residue protein showing sequence similarity to metalloproteases of the serralysin family. The RpA protein was expressed in Escherichia coli using a pET expression vector and purified as a 55 kDa molecular weight protein. Furthermore, the protease activity of RpA was inhibited by protease inhibitor and heat treatment. The in vitro cytotoxic activity of R. pickettii culture filtrates was attributed to RpA protease. An extracellular protease, RpA, was identified from R. pickettii WP1 isolated from water supply system. The RpA metalloproteases is required for the pathogenicity of R. pickettii to mammalian cell lines. © 2015 The Society for Applied Microbiology.

  7. Clinicopathological Features to Predict Progression of IgA Nephropathy with Mild Proteinuria.

    PubMed

    Chen, Ding; Liu, Jian; Duan, Shuwei; Chen, Pu; Tang, Li; Zhang, Li; Feng, Zhe; Cai, Guangyan; Wu, Jie; Chen, Xiangmei

    2018-03-06

    In the past, little attention has been paid to patients with IgA nephropathy (IgAN) who had minimal proteinuria upon the onset. The aim of this study was to analyze the clinicopathological features and the prognostic factors in patients with IgA nephropathy. Data of patients that had their first renal biopsy in our hospital and were diagnosed with primary IgAN with proteinuria <1 g/d from January 1995 to December 2014 were retrospectively examined. Clinical records of the clinicopathological features, renal function, and proteinuria were collected and investigated. The factors affecting the renal function and proteinuria were analyzed by Cox regression. The predictive efficiencies of clinical and pathological models were evaluated by Harrell concordance index (C-index). A total of 506 patients with IgA nephropathy were included in this study. (1) Baseline proteinuria greater than 0.5 g/d was positively associated with Oxford M, S, and T lesions. eGFR less than 90 mL/min/1.73 m2 were positively associated with Oxford T. (2) In the follow-up with a median of 50 months, 82 patients (16.2%) achieved complete clinical remission (CCR), whereas 54 patients (10.6%) showed an increase in creatinine by more than 50% (not progressing to end-stage renal disease). The cumulative proportion of creatinine increased >50%, and the values obtained by life-table analysis in 10, 15, and 20 years were 15%, 21%, and 22%, respectively. Significant differences were found in baseline age, proteinuria, and Oxford T between the group of creatinine increase >50% and the CCR group. (4) Multivariate COX regression showed that baseline age and proteinuria > 0.5 g/d were independent risk factors of adverse outcome. C-index suggested that the clinical model was more effective than the pathological models in predicting endpoint events. (5) Effect of the mean value during the follow-up on adverse endpoint events: Multivariate COX regression found that the mean proteinuria during follow-up was an

  8. Nanoplatforms for highly sensitive fluorescence detection of cancer-related proteases.

    PubMed

    Wang, Hongwang; Udukala, Dinusha N; Samarakoon, Thilani N; Basel, Matthew T; Kalita, Mausam; Abayaweera, Gayani; Manawadu, Harshi; Malalasekera, Aruni; Robinson, Colette; Villanueva, David; Maynez, Pamela; Bossmann, Leonie; Riedy, Elizabeth; Barriga, Jenny; Wang, Ni; Li, Ping; Higgins, Daniel A; Zhu, Gaohong; Troyer, Deryl L; Bossmann, Stefan H

    2014-02-01

    Numerous proteases are known to be necessary for cancer development and progression including matrix metalloproteinases (MMPs), tissue serine proteases, and cathepsins. The goal of this research is to develop an Fe/Fe3O4 nanoparticle-based system for clinical diagnostics, which has the potential to measure the activity of cancer-associated proteases in biospecimens. Nanoparticle-based "light switches" for measuring protease activity consist of fluorescent cyanine dyes and porphyrins that are attached to Fe/Fe3O4 nanoparticles via consensus sequences. These consensus sequences can be cleaved in the presence of the correct protease, thus releasing a fluorescent dye from the Fe/Fe3O4 nanoparticle, resulting in highly sensitive (down to 1 × 10(-16) mol l(-1) for 12 proteases), selective, and fast nanoplatforms (required time: 60 min).

  9. Halobacterium sp. SP1(1) as a starter culture for accelerating fish sauce fermentation.

    PubMed

    Akolkar, A V; Durai, D; Desai, A J

    2010-07-01

    Application of Halobacterium sp. SP1(1) for the acceleration of fish sauce fermentation. Traditional fish sauce fermentation was mimicked using Halobacterium sp. SP1(1) as starter culture. Protease activity, peptide release and α-amino content (parameters used to monitor the progress of the fermentation) were high at day 10 in tests and day 20 in un-inoculated controls. The total protein and nitrogen contents were also high in tests compared with controls. The amino acid profile observed at the end of fermentation in experimental samples, when compared with the commercial sauce preparation, was found to be better with respect to flavour and aroma contributing amino acids as well as essential amino acid lysine. Microflora analysis of the final fish sauce revealed the absence of any nonhalophilic or halotolerant micro-organisms. The protease-producing halophilic isolates obtained from the fish sauce of eviscerated and uneviscerated controls were identified as Halobacterium sp. F1 and F2, respectively, by 16S rDNA sequence analysis. Exogenous augmentation of Halobacterium sp. SP1(1) accelerated the fish sauce fermentation process with an additive effect on the existing natural microflora present in the fish during fermentation. Halobacterium sp SP1(1), therefore, can be used as an important starter culture for accelerating the fish fermentation process, which is attributed to its extracellular protease. The present study is the first report on use of Halobacterium species as a starter culture for accelerating fish sauce fermentation. Use of halobacterial starter cultures may revolutionize the process in fish sauce industries by reducing the fermentation time and making the process more economical with improved nutritive value of product. Journal compilation © 2009 The Society for Applied Microbiology. No claim to Indian Government works.

  10. Antiacanthain A: New proteases isolated from Bromelia antiacantha Bertol. (Bromeliaceae).

    PubMed

    Vallés, Diego; Cantera, Ana M B

    2018-07-01

    Crude extract (CE) from pulp of Bromelia antiacantha Bertol. mature fruit, contains at least 3 cysteine proteases with proteolytic activity. By single step cation exchange chromatography (Hi-trap SP-HP) of partially purified CE, the protease with the lowest pI, Antiacanthain A (AntA), was isolated. It showed maximum activity at pH9, and 75% of remaining activity was maintained over a wide pH range (pH6-10). The AntA activity exhibits a constant increase up to 70°C. Maintains almost 100% of its activity at 45 at pH6 and 9. A 60% of AntA was active by titration with specific inhibitor, E64. Amidasic activity was studied with pyroglutamyl-phenyl-leucyl-paranitroaniline (PFLNA) substrate having higher AntA catalytic efficiency of (k cat /K m =470s -1 M -1 ) relative to stem bromelain (k cat /K m =305s -1 M -1 ). Esterase activity using p-nitrophenyl esters of N-α-CBZ-l-Lysine (z-L-LysONp) showed a 10-fold higher catalytic efficiency for AntA (k cat /K m =6376s -1 M -1 ) relative to stem bromelain (k cat /K m =688s -1 M -1 ). Incubation with 8M Urea did not affect AntA activity and remained unchanged for 18h, with 6M GndHCl resulted in a 41% decrease in activity after 30min incubation, maintained this activity 18h. AntA exhibits high sequence identity with proteases of the Bromeliaceae family. Copyright © 2018 Elsevier B.V. All rights reserved.

  11. Resistant mechanism against nelfinavir of subtype C human immunodeficiency virus type 1 proteases

    NASA Astrophysics Data System (ADS)

    Liu, Xiaoqing; Dai, Qi; Li, Lihua; Xiu, Zhilong

    2011-02-01

    Human immunodeficiency virus type 1 protease (HIV-1 PR) is one of the major targets of anti-AIDS drug discovery, and the subtype C HIV-1 is infecting more and more humans. In this work, we executed computational simulations of subtype B (labeled B(WT)) HIV-1 PR, D30N mutant B (labeled B(D30N)) HIV-1 PR, subtype C (labeled C(Ref)) HIV-1 PR, D30N mutant C (labeled C(D30N)) HIV-1 PR, and D30N/N83T mutant C (labeled C(D30N/N83T)) HIV-1 PR with drug nelfinavir (NFV), aiming at clarifying (1) the resistant mechanism against NFV due to D30N mutation in subtype C HIV-1 PR; (2) the reason that the emergence rate of N83T mutation in C(D30N) HIV-1 PR is higher than that in B(D30N) HIV-1 PR; (3) the affinity of NFV with C(D30N/N83T) HIV-1 PR is higher than B(D30N) and C(D30N) HIV-1 PRs. The results indicate: (1) D30N mutation in subtype C HIV-1 PR reduces the hydrogen bond between the 30th residue and NFV, and the binding free energy contributions of some residues decrease; (2) the hydrogen bonds between the 83th/83'th residue and the 34th/34'th residue exist in both B(D30N) and C(D30N/N83T) HIV-1 PRs, while they disappear in C(D30N) HIV-1 PR. Meanwhile, the binding free energy contribution of N30 in C(D30N) is lower than that in B(D30N) and C(D30N/N83T); (3) N83T mutation makes some residues dislocate, and the contributions of these residues to binding free energy in C(D30N/N83T) increase comparing to those in B(D30N) and C(D30N). Our findings suggest that despite the nonactive site mutations, the polymorphisms regulate the emergence rates of these drug-resistant mutants.

  12. Reverse enzyme-linked immunosorbent assay using monoclonal antibodies against SAG1-related sequence, SAG2A, and p97 antigens from Toxoplasma gondii to detect specific immunoglobulin G (IgG), IgM, and IgA antibodies in human sera.

    PubMed

    Carvalho, Fernando R; Silva, Deise A O; Cunha-Júnior, Jair P; Souza, Maria A; Oliveira, Taísa C; Béla, Samantha R; Faria, Gabriele G; Lopes, Carolina S; Mineo, José R

    2008-08-01

    The present study aimed to evaluate the performance of three monoclonal antibodies (MAbs) in reverse enzyme-linked immunosorbent assays (ELISAs) for detecting immunoglobulin G (IgG), IgM, and IgA antibodies against Toxoplasma gondii in 175 serum samples from patients at different stages of T. gondii infection, as defined by both serological and clinical criteria, as follows: recent (n = 45), transient (n = 40), and chronic (n = 55) infection as well as seronegative subjects (n = 35). The results were compared with those obtained by indirect ELISA using soluble Toxoplasma total antigen (STAg). Our data demonstrated that MAb A3A4 recognizes a conformational epitope in SAG1-related-sequence (SRS) antigens, while A4D12 and 1B8 recognize linear epitopes defined as SAG2A surface antigen and p97 cytoplasmatic antigen, respectively. Reverse ELISA for IgG with A3A4 or A4D12 MAbs was highly correlated with indirect ELISA for anti-STAg IgG, whereas only A4D12 reverse ELISA showed high correlation with indirect ELISA for IgM and IgA isotypes. To our knowledge, this is the first report analyzing the performance of a reverse ELISA for simultaneous detection of IgG, IgM, and IgA isotypes active toward native SAG2A, SRS, and p97 molecules from STAg, using a panel of human sera from patients with recent and chronic toxoplasmosis. Thus, reverse ELISA based on the capture of native SAG2A and SRS antigens of STAg by MAbs could be an additional approach for strengthening the helpfulness of serological tests assessing the stage of infection, particularly in combination with highly sensitive and specific assays that are frequently used nowadays for diagnosis of toxoplasmosis during pregnancy or congenital infection in newborns.

  13. Elevated Peritoneal Fluid TNF-α Incites Ovarian Early Growth Response Factor 1 Expression and Downstream Protease Mediators

    PubMed Central

    Birt, Julie A.; Nabli, Henda; Stilley, Julie A.; Windham, Emma A.; Frazier, Shellaine R.

    2013-01-01

    Endometriosis-associated infertility manifests itself via multiple, poorly understood mechanisms. Our goal was to characterize signaling pathways, between peritoneal endometriotic lesions and the ovary, leading to failed ovulation. Genome-wide microarray analysis comparing ovarian tissue from an in vivo endometriosis model in the rat (Endo) with controls (Sham) identified 22 differentially expressed genes, including transiently expressed early growth response factor 1 (Egr1). The Egr1 regulates gene requisites for ovulation. The Egr1 promoter is responsive to tumor necrosis factor-alpha (TNF-α) signaling. We hypothesized that altered expression of ovarian EGR1 is induced by elevated peritoneal fluid TNF-α which is upregulated by the presence of peritoneal endometriosis. Endo rats, compared to controls, had more peritoneal fluid TNF-α and quantitative, spatial differences in Egr1 mRNA and EGR1 protein localization in follicular compartments. Interactions between elevated peritoneal fluid TNF-α and overexpression of follicular Egr1/EGR1 expression may affect downstream protease pathways impeding ovulation in endometriosis. Preliminary studies identified similar patterns of EGR1 protein localization in human ovaries from women with endometriosis and compared to those without endometriosis. PMID:23427178

  14. Antibody-Mediated Internalization of Infectious HIV-1 Virions Differs among Antibody Isotypes and Subclasses.

    PubMed

    Tay, Matthew Zirui; Liu, Pinghuang; Williams, LaTonya D; McRaven, Michael D; Sawant, Sheetal; Gurley, Thaddeus C; Xu, Thomas T; Dennison, S Moses; Liao, Hua-Xin; Chenine, Agnès-Laurence; Alam, S Munir; Moody, M Anthony; Hope, Thomas J; Haynes, Barton F; Tomaras, Georgia D

    2016-08-01

    Emerging data support a role for antibody Fc-mediated antiviral activity in vaccine efficacy and in the control of HIV-1 replication by broadly neutralizing antibodies. Antibody-mediated virus internalization is an Fc-mediated function that may act at the portal of entry whereby effector cells may be triggered by pre-existing antibodies to prevent HIV-1 acquisition. Understanding the capacity of HIV-1 antibodies in mediating internalization of HIV-1 virions by primary monocytes is critical to understanding their full antiviral potency. Antibody isotypes/subclasses differ in functional profile, with consequences for their antiviral activity. For instance, in the RV144 vaccine trial that achieved partial efficacy, Env IgA correlated with increased risk of HIV-1 infection (i.e. decreased vaccine efficacy), whereas V1-V2 IgG3 correlated with decreased risk of HIV-1 infection (i.e. increased vaccine efficacy). Thus, understanding the different functional attributes of HIV-1 specific IgG1, IgG3 and IgA antibodies will help define the mechanisms of immune protection. Here, we utilized an in vitro flow cytometric method utilizing primary monocytes as phagocytes and infectious HIV-1 virions as targets to determine the capacity of Env IgA (IgA1, IgA2), IgG1 and IgG3 antibodies to mediate HIV-1 infectious virion internalization. Importantly, both broadly neutralizing antibodies (i.e. PG9, 2G12, CH31, VRC01 IgG) and non-broadly neutralizing antibodies (i.e. 7B2 mAb, mucosal HIV-1+ IgG) mediated internalization of HIV-1 virions. Furthermore, we found that Env IgG3 of multiple specificities (i.e. CD4bs, V1-V2 and gp41) mediated increased infectious virion internalization over Env IgG1 of the same specificity, while Env IgA mediated decreased infectious virion internalization compared to IgG1. These data demonstrate that antibody-mediated internalization of HIV-1 virions depends on antibody specificity and isotype. Evaluation of the phagocytic potency of vaccine

  15. Antibody-Mediated Internalization of Infectious HIV-1 Virions Differs among Antibody Isotypes and Subclasses

    PubMed Central

    McRaven, Michael D; Sawant, Sheetal; Gurley, Thaddeus C; Xu, Thomas T.; Dennison, S. Moses; Liao, Hua-Xin; Chenine, Agnès-Laurence; Alam, S. Munir; Haynes, Barton F.; Tomaras, Georgia D.

    2016-01-01

    Emerging data support a role for antibody Fc-mediated antiviral activity in vaccine efficacy and in the control of HIV-1 replication by broadly neutralizing antibodies. Antibody-mediated virus internalization is an Fc-mediated function that may act at the portal of entry whereby effector cells may be triggered by pre-existing antibodies to prevent HIV-1 acquisition. Understanding the capacity of HIV-1 antibodies in mediating internalization of HIV-1 virions by primary monocytes is critical to understanding their full antiviral potency. Antibody isotypes/subclasses differ in functional profile, with consequences for their antiviral activity. For instance, in the RV144 vaccine trial that achieved partial efficacy, Env IgA correlated with increased risk of HIV-1 infection (i.e. decreased vaccine efficacy), whereas V1-V2 IgG3 correlated with decreased risk of HIV-1 infection (i.e. increased vaccine efficacy). Thus, understanding the different functional attributes of HIV-1 specific IgG1, IgG3 and IgA antibodies will help define the mechanisms of immune protection. Here, we utilized an in vitro flow cytometric method utilizing primary monocytes as phagocytes and infectious HIV-1 virions as targets to determine the capacity of Env IgA (IgA1, IgA2), IgG1 and IgG3 antibodies to mediate HIV-1 infectious virion internalization. Importantly, both broadly neutralizing antibodies (i.e. PG9, 2G12, CH31, VRC01 IgG) and non-broadly neutralizing antibodies (i.e. 7B2 mAb, mucosal HIV-1+ IgG) mediated internalization of HIV-1 virions. Furthermore, we found that Env IgG3 of multiple specificities (i.e. CD4bs, V1-V2 and gp41) mediated increased infectious virion internalization over Env IgG1 of the same specificity, while Env IgA mediated decreased infectious virion internalization compared to IgG1. These data demonstrate that antibody-mediated internalization of HIV-1 virions depends on antibody specificity and isotype. Evaluation of the phagocytic potency of vaccine

  16. Protease digestion from wheat stillage within a dry grind ethanol facility.

    PubMed

    Bals, Bryan; Brehmer, Ben; Dale, Bruce; Sanders, Johan

    2011-01-01

    As the current starch based ethanol market increases at its rapid pace, finding new markets for the primary coproduct, distiller's grains, has gained considerable interest. One possibility is to isolate the protein-rich fraction for use as precursors to biochemicals and bioplastics, further decreasing fossil fuel consumption. This research focuses on enzymatic extraction of protein peptides from wheat heavy stillage using commercially available proteases. The energy saved due to this process ranged from ∼ 1.5 to 3.0 GJ/ton wheat stillage compared to fossil fuel-based chemicals. Using Protex 6L (Genencor), ∼ 57% of the protein in the stillage was soluble 24 h after protease addition at 0.1% w/w loading. Of these proteins, ∼ 32% were already soluble, indicating the importance of using wet heavy stillage as the feedstock rather than dried distiller's grains. Peptide size was less than 6 kDa. Further improvements in protein removal may be obtained through a fed batch addition of protease and improved protease cocktails. Copyright © 2011 American Institute of Chemical Engineers (AIChE).

  17. Overexpression of SerpinE2/protease nexin-1 Contribute to Pathological Cardiac Fibrosis via increasing Collagen Deposition

    PubMed Central

    Li, Xuelian; Zhao, Dandan; Guo, Zhenfeng; Li, Tianshi; Qili, Muge; Xu, Bozhi; Qian, Ming; Liang, Haihai; E, Xiaoqiang; Chege Gitau, Samuel; Wang, Lu; Huangfu, Longtao; Wu, Qiuxia; Xu, Chaoqian; Shan, Hongli

    2016-01-01

    Although increases in cardiovascular load (pressure overload) are known to elicit ventricular remodeling including cardiomyocyte hypertrophy and interstitial fibrosis, the molecular mechanisms of pressure overload or AngII -induced cardiac interstitial fibrosis remain elusive. In this study, serpinE2/protease nexin-1 was over-expressed in a cardiac fibrosis model induced by pressure-overloaded via transverse aortic constriction (TAC) in mouse. Knockdown of serpinE2 attenuates cardiac fibrosis in a mouse model of TAC. At meantime, the results showed that serpinE2 significantly were increased with collagen accumulations induced by AngII or TGF-β stimulation in vitro. Intriguingly, extracellular collagen in myocardial fibroblast was reduced by knockdown of serpinE2 compared with the control in vitro. In stark contrast, the addition of exogenous PN-1 up-regulated the content of collagen in myocardial fibroblast. The MEK1/2- ERK1/2 signaling probably promoted the expression of serpinE2 via transcription factors Elk1 in myocardial fibroblast. In conclusion, stress-induced the ERK1/2 signaling pathway activation up-regulated serpinE2 expression, consequently led accumulation of collagen protein, and contributed to cardiac fibrosis. PMID:27876880

  18. KLK14 interactions with HAI-1 and HAI-2 serine protease inhibitors: A molecular dynamics and relative free-energy calculations study.

    PubMed

    Solís-Calero, Christian; Carvalho, Hernandes F

    2017-11-01

    Kallikrein 14 (KLK14) is a serine protease linked to several pathologies including prostate cancer and positively correlates with Gleason score. Though KLK14 functioning in cancer is poorly understood, it has been implicated in HGF/Met signaling, given that KLK14 proteolytically inhibits HGF activator-inhibitor 1 (HAI-1), which strongly inhibits pro-HGF activators, thereby contributing to tumor progression. In this work, KLK14 binding to either hepatocyte growth factor activator inhibitor type-1 (HAI-1) or type-2 (HAI-2) was essayed using homology modeling, molecular dynamic simulations and free-energy calculations through MM/PBSA and MM/GBSA. KLK14 was successfully modeled. Calculated free energies suggested higher binding affinity for the KLK14/HAI-1 interaction than for KLK14/HAI-2. This difference in binding affinity is largely explained by the higher stability of the hydrogen-bond networks in KLK14/HAI-1 along the simulation trajectory. A key arginine residue in both HAI-1 and HAI-2 is responsible for their interaction with the S1 pocket in KLK14. Additionally, MM/GBSA free-energy decomposition postulates that KLK14 Asp174 and Trp196 are hotspots for binding HAI-1 and HAI-2. © 2017 International Federation for Cell Biology.

  19. Poly(ADP-ribose) polymerase-1 (Parp-1)-deficient mice demonstrate abnormal antibody responses

    PubMed Central

    Ambrose, Helen E; Willimott, Shaun; Beswick, Richard W; Dantzer, Françoise; de Murcia, Josiane Ménissier; Yelamos, José; Wagner, Simon D

    2009-01-01

    Poly(ADP-ribosylation) of acceptor proteins is an epigenetic modification involved in DNA strand break repair, recombination and transcription. Here we provide evidence for the involvement of poly(ADP-ribose) polymerase-1 (Parp-1) in antibody responses. Parp-1−/− mice had increased numbers of T cells and normal numbers of total B cells. Marginal zone B cells were mildly reduced in number, and numbers of follicular B cells were preserved. There were abnormal levels of basal immunoglobulins, with reduced levels of immunoglobulin G2a (IgG2a) and increased levels of IgA and IgG2b. Analysis of specific antibody responses showed that T cell-independent responses were normal but T cell-dependent responses were markedly reduced. Germinal centres were normal in size and number. In vitro purified B cells from Parp-1−/− mice proliferated normally and showed normal IgM secretion, decreased switching to IgG2a but increased IgA secretion. Collectively our results demonstrate that Parp-1 has essential roles in normal T cell-dependent antibody responses and the regulation of isotype expression. We speculate that Parp-1 forms a component of the protein complex involved in resolving the DNA double-strand breaks that occur during class switch recombination. PMID:18778284

  20. Intramuscular Priming and Intranasal Boosting Induce Strong Genital Immunity Through Secretory IgA in Minipigs Infected with Chlamydia trachomatis

    PubMed Central

    Lorenzen, Emma; Follmann, Frank; Bøje, Sarah; Erneholm, Karin; Olsen, Anja Weinreich; Agerholm, Jørgen Steen; Jungersen, Gregers; Andersen, Peter

    2015-01-01

    International efforts in developing a vaccine against Chlamydia trachomatis have highlighted the need for novel immunization strategies for the induction of genital immunity. In this study, we evaluated an intramuscular (IM) prime/intranasal boost vaccination strategy in a Göttingen Minipig model with a reproductive system very similar to humans. The vaccine was composed of C. trachomatis subunit antigens formulated in the Th1/Th17 promoting CAF01 adjuvant. IM priming immunizations with CAF01 induced a significant cell-mediated interferon gamma and interleukin 17A response and a significant systemic high-titered neutralizing IgG response. Following genital challenge, intranasally boosted groups mounted an accelerated, highly significant genital IgA response that correlated with enhanced bacterial clearance on day 3 post infection. By detecting antigen-specific secretory component (SC), we showed that the genital IgA was locally produced in the genital mucosa. The highly significant inverse correlation between the vaginal IgA SC response and the chlamydial load suggests that IgA in the minipig model is involved in protection against C. trachomatis. This is important both for our understanding of protective immunity and future vaccination strategies against C. trachomatis and genital pathogens in general. PMID:26734002

  1. Nematode sperm maturation triggered by protease involves sperm-secreted serine protease inhibitor (Serpin)

    PubMed Central

    Zhao, Yanmei; Sun, Wei; Zhang, Pan; Chi, Hao; Zhang, Mei-Jun; Song, Chun-Qing; Ma, Xuan; Shang, Yunlong; Wang, Bin; Hu, Youqiao; Hao, Zhiqi; Hühmer, Andreas F.; Meng, Fanxia; L'Hernault, Steven W.; He, Si-Min; Dong, Meng-Qiu; Miao, Long

    2012-01-01

    Spermiogenesis is a series of poorly understood morphological, physiological and biochemical processes that occur during the transition of immotile spermatids into motile, fertilization-competent spermatozoa. Here, we identified a Serpin (serine protease inhibitor) family protein (As_SRP-1) that is secreted from spermatids during nematode Ascaris suum spermiogenesis (also called sperm activation) and we showed that As_SRP-1 has two major functions. First, As_SRP-1 functions in cis to support major sperm protein (MSP)-based cytoskeletal assembly in the spermatid that releases it, thereby facilitating sperm motility acquisition. Second, As_SRP-1 released from an activated sperm inhibits, in trans, the activation of surrounding spermatids by inhibiting vas deferens-derived As_TRY-5, a trypsin-like serine protease necessary for sperm activation. Because vesicular exocytosis is necessary to create fertilization-competent sperm in many animal species, components released during this process might be more important modulators of the physiology and behavior of surrounding sperm than was previously appreciated. PMID:22307610

  2. Amprenavir, new protease inhibitor, approved.

    PubMed

    James, J S

    1999-05-07

    A new protease inhibitor, amprenavir (Agenerase), has received FDA marketing approval. The approval was based on two 24-week controlled trials and safety data in more than 1,400 patients under FDA accelerated-approval rules. Amprenavir is approved for patients 4 years of age and older. The drug is taken twice daily, with or without food. Side effects include gastrointestinal disturbances, rashes, and oral paresthesia. Severe or life-threatening rashes have occurred in 1 percent of all patients. Pregnant women should not use the drug unless necessary. The drug was developed by Vertex Pharmaceuticals Inc. and is being marketed by Glaxo Wellcome. Some studies suggest that amprenavir is less likely than other protease inhibitors to be associated with lipid metabolism problems. It may have a resistance profile different from that of other protease inhibitors, and therefore may cause different cross resistance problems. Amprenavir appears to be synergistic with abacavir (Ziagen) in laboratory tests.

  3. Comparison of mucosal lining fluid sampling methods and influenza-specific IgA detection assays for use in human studies of influenza immunity.

    PubMed

    de Silva, Thushan I; Gould, Victoria; Mohammed, Nuredin I; Cope, Alethea; Meijer, Adam; Zutt, Ilse; Reimerink, Johan; Kampmann, Beate; Hoschler, Katja; Zambon, Maria; Tregoning, John S

    2017-10-01

    We need greater understanding of the mechanisms underlying protection against influenza virus to develop more effective vaccines. To do this, we need better, more reproducible methods of sampling the nasal mucosa. The aim of the current study was to compare levels of influenza virus A subtype-specific IgA collected using three different methods of nasal sampling. Samples were collected from healthy adult volunteers before and after LAIV immunization by nasal wash, flocked swabs and Synthetic Absorptive Matrix (SAM) strips. Influenza A virus subtype-specific IgA levels were measured by haemagglutinin binding ELISA or haemagglutinin binding microarray and the functional response was assessed by microneutralization. Nasosorption using SAM strips lead to the recovery of a more concentrated sample of material, with a significantly higher level of total and influenza H1-specific IgA. However, an equivalent percentage of specific IgA was observed with all sampling methods when normalized to the total IgA. Responses measured using a recently developed antibody microarray platform, which allows evaluation of binding to multiple influenza strains simultaneously with small sample volumes, were compared to ELISA. There was a good correlation between ELISA and microarray values. Material recovered from SAM strips was weakly neutralizing when used in an in vitro assay, with a modest correlation between the level of IgA measured by ELISA and neutralization, but a greater correlation between microarray-measured IgA and neutralizing activity. In conclusion we have tested three different methods of nasal sampling and show that flocked swabs and novel SAM strips are appropriate alternatives to traditional nasal washes for assessment of mucosal influenza humoral immunity. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Serum concentrations of IgG, IgA, and IgM in retired racing Greyhound dogs.

    PubMed

    Clemente, Mónica; Marín, Liliana; Iazbik, M Cristina; Couto, C Guillermo

    2010-12-01

    Greyhound dogs have significant physiologic, hematologic, and biochemical differences when compared with other breeds, including significantly lower serum globulin concentration owing to decreases in the α- and β-globulin fractions. The specific proteins that account for differences in globulin concentrations are not known, but IgA and IgM, both β-globulins, are potential candidates. The aims of this study were to measure serum IgG, IgA, and IgM in clinically healthy retired racing Greyhounds and compare the results with those of age- and sex-matched non-Greyhound dogs. Study animals included 25 Greyhound and 20 non-Greyhound dogs. Total protein, albumin, and total globulin concentrations were determined. IgG, IgA, and IgM concentrations were measured using a commercially available radial immunodiffusion kit. The Student t-test assuming equal variances was used to compare concentrations of immunoglobulins between groups. Serum concentrations of IgA and IgM in Greyhounds (IgA=49±20 mg/dL; IgM=132±47 mg/dL) were significantly lower than concentrations in non-Greyound dogs (IgA=70±39 mg/dL; Ig M=212±78 mg/dL). Concentrations of IgG did not differ between groups. Mean serum IgA and IgM concentrations in Greyhounds were lower than those in non-Greyhound dogs. This may contribute to low serum concentrations of β-globulins in Greyhounds. Specific reference intervals are recommended for Greyhounds to avoid possible misdiagnosis of IgA or IgM deficiency. ©2010 American Society for Veterinary Clinical Pathology.

  5. Analysis of Milk from Mothers Who Delivered Prematurely Reveals Few Changes in Proteases and Protease Inhibitors across Gestational Age at Birth and Infant Postnatal Age.

    PubMed

    Demers-Mathieu, Veronique; Nielsen, Søren Drud; Underwood, Mark A; Borghese, Robyn; Dallas, David C

    2017-06-01

    Background: Peptidomics research has demonstrated that protease activity is higher in breast milk from preterm-delivering mothers than from term-delivering mothers. However, to our knowledge, the effect of the degree of prematurity and postnatal age on proteases and protease inhibitors in human milk remains unknown. Objective: We aimed to determine the change of proteases and protease inhibitors in milk from mothers who delivered prematurely across gestational age (GA) and postnatal age. Methods: Milk samples were collected from 18 mothers aged 26-40 y who delivered preterm infants and who lacked mastitis. For analysis, samples were separated into 2 groups: 9 from early GA (EGA) (24-26 wk GA)-delivering mothers and 9 from late GA (LGA) (27-32 wk GA)-delivering mothers. Within the 9 samples in each group, the collection time ranged from postnatal days 2 to 47. The activity and predicted activity of proteases in preterm milk were determined with the use of fluorometric and spectrophotometric assays and peptidomics, respectively. Protease and protease inhibitor concentrations were determined with the use of ELISA. Linear mixed models were applied to compare enzymes across GA and postnatal age. Results: Carboxypeptidase B2, kallikrein, plasmin, elastase, thrombin, and cytosol aminopeptidase were present and active in the milk of preterm-delivering mothers. Most milk protease and antiprotease concentrations did not change with GA or postnatal age. However, the concentration and activity of kallikrein, the most abundant and active protease in preterm milk, increased by 25.4 ng · mL -1 · d -1 and 0.454 μg · mL -1 · d -1 postnatally, respectively, in EGA milk samples while remaining stable in LGA milk samples. Conclusions: This research demonstrates that proteases are active in human milk and begin to degrade milk protein within the mammary gland before consumption by infants. Proteases and protease inhibitors in milk from mothers of premature infants mostly did not

  6. Isotype- and subclass-specific responses to infection and reinfection with parainfluenza-3 virus: comparison of the diagnostic potential of ELISAs detecting seroconversion and specific IgM and IgA.

    PubMed

    Graham, D A; Mawhinney, K A; German, A; Foster, J C; Adair, B M; Merza, M

    1999-03-01

    Isotype- and subclass-specific indirect enzyme-linked immunosorbent assays were developed to detect parainfluenza-3 virus-specific IgG1, IgG2, IgM, and IgA responses. Sera were treated with protein G-agarose prior to testing for specific IgM and IgA to eliminate the possibility of false-positive results due to IgM-rheumatoid factor and to remove interisotypic competition due to specific IgG. IgM and IgA absorbance values were expressed as a percentage of the absorbance values of positive reference sera included on each plate (S/P%), and respective positive/negative threshold values of 15.0% and 28.0% were determined. The mean interval between experimental infection of 3 calves and initial detection of specific IgG1 and IgG2 responses was 8.0 and 9.3 days respectively, rising rapidly to an initial plateau 13.7 and 11.0 days postinfection (dpi). Reinfection of these calves at 30 dpi resulted in further rapid increases, with higher plateau values reached 13.0 (IgG1) and 13.7 (IgG2) days later. The mean interval between infection and the first positive IgM and IgA responses was 6.7 and 12.3 days, respectively. IgM S/P% values peaked at 13.0 dpi, with all 3 calves showing a secondary anamnestic response to reinfection, peaking 4.7 days later. The IgA response to initial infection was weak, with only 2 calves showing an obvious peak response at 15.0 dpi. A strong anamnestic IgA response to reinfection occurred in 2 calves, with a peak response 9.5 days later. Apparent biphasic and triphasic IgM and IgA responses were evident in some calves. Acute and convalescent serum samples from 80 calves involved in 17 outbreaks of respiratory disease were tested for specific IgM and IgA. Positive IgM results were detected in 15 outbreaks, with 71 sera from 44 calves testing positive. Although IgA-positive results were detected in the same 15 outbreaks, only 42 sera from 31 calves were positive. In a previous study, seroconversion was detected in 21 of these calves from 10 outbreaks

  7. Purification and characterization of a cysteine protease from corms of freesia, Freesia reflacta.

    PubMed

    Kaneda, M; Yonezawa, H; Uchikoba, T

    1997-09-01

    A protease (freesia protease B) has been purified to electrophoretic homogeneity from corms of freesia, Freesia reflacta by five steps of chromatography. Its M(r) was estimated to be about 26,000 by SDS-PAGE. The optimum pH of the enzyme was 6.0-7.0 at 30 degrees C using casein as a substrate. The enzyme was strongly inhibited by p-chloromercuribenzoic acid but not by phenylmethanesulphonylfluoride and EDTA. These results indicate that freesia protease B is a cysteine protease. Nine sites of oxidized insulin B-chain were cleaved by freesia protease B in 24 h of hydrolysis. The four cleavage sites among them resembled those of papain. From the digestion of five peptidyl substrates the specificity of freesia protease B was found to be approximately broad, but the preferential cleavage sites were negatively charged residues at P1 positions. Freesia protease B preferred also the large hydrophobic amino acid residues at the P2 position, in a similar manner to papain. The amino terminal sequence of freesia protease B was identical with those of papain in regard to the conservative residues of cysteine protease.

  8. Comparison of methods for profiling O-glycosylation: Human Proteome Organisation Human Disease Glycomics/Proteome Initiative multi-institutional study of IgA1.

    PubMed

    Wada, Yoshinao; Dell, Anne; Haslam, Stuart M; Tissot, Bérangère; Canis, Kévin; Azadi, Parastoo; Bäckström, Malin; Costello, Catherine E; Hansson, Gunnar C; Hiki, Yoshiyuki; Ishihara, Mayumi; Ito, Hiromi; Kakehi, Kazuaki; Karlsson, Niclas; Hayes, Catherine E; Kato, Koichi; Kawasaki, Nana; Khoo, Kay-Hooi; Kobayashi, Kunihiko; Kolarich, Daniel; Kondo, Akihiro; Lebrilla, Carlito; Nakano, Miyako; Narimatsu, Hisashi; Novak, Jan; Novotny, Milos V; Ohno, Erina; Packer, Nicolle H; Palaima, Elizabeth; Renfrow, Matthew B; Tajiri, Michiko; Thomsson, Kristina A; Yagi, Hirokazu; Yu, Shin-Yi; Taniguchi, Naoyuki

    2010-04-01

    The Human Proteome Organisation Human Disease Glycomics/Proteome Initiative recently coordinated a multi-institutional study that evaluated methodologies that are widely used for defining the N-glycan content in glycoproteins. The study convincingly endorsed mass spectrometry as the technique of choice for glycomic profiling in the discovery phase of diagnostic research. The present study reports the extension of the Human Disease Glycomics/Proteome Initiative's activities to an assessment of the methodologies currently used for O-glycan analysis. Three samples of IgA1 isolated from the serum of patients with multiple myeloma were distributed to 15 laboratories worldwide for O-glycomics analysis. A variety of mass spectrometric and chromatographic procedures representative of current methodologies were used. Similar to the previous N-glycan study, the results convincingly confirmed the pre-eminent performance of MS for O-glycan profiling. Two general strategies were found to give the most reliable data, namely direct MS analysis of mixtures of permethylated reduced glycans in the positive ion mode and analysis of native reduced glycans in the negative ion mode using LC-MS approaches. In addition, mass spectrometric methodologies to analyze O-glycopeptides were also successful.

  9. Mapping, Complementation, and Targets of the Cysteine Protease Actinidin in Kiwifruit1[C][W][OA

    PubMed Central

    Nieuwenhuizen, Niels J.; Maddumage, Ratnasiri; Tsang, Gianna K.; Fraser, Lena G.; Cooney, Janine M.; De Silva, H. Nihal; Green, Sol; Richardson, Kim A.; Atkinson, Ross G.

    2012-01-01

    Cysteine proteases (CPs) accumulate to high concentration in many fruit, where they are believed to play a role in fungal and insect defense. The fruit of Actinidia species (kiwifruit) exhibit a range of CP activities (e.g. the Actinidia chinensis variety YellowA shows less than 2% of the activity of Actinidia deliciosa variety Hayward). A major quantitative trait locus for CP activity was mapped to linkage group 16 in a segregating population of A. chinensis. This quantitative trait locus colocated with the gene encoding actinidin, the major acidic CP in ripe Hayward fruit encoded by the ACT1A-1 allele. Sequence analysis indicated that the ACT1A locus in the segregating A. chinensis population contained one functional allele (A-2) and three nonfunctional alleles (a-3, a-4, and a-5) each containing a unique frameshift mutation. YellowA kiwifruit contained two further alleles: a-6, which was nonfunctional because of a large insertion, and a-7, which produced an inactive enzyme. Site-directed mutagenesis of the act1a-7 protein revealed a residue that restored CP activity. Expression of the functional ACT1A-1 cDNA in transgenic plants complemented the natural YellowA mutations and partially restored CP activity in fruit. Two consequences of the increase in CP activity were enhanced degradation of gelatin-based jellies in vitro and an increase in the processing of a class IV chitinase in planta. These results provide new insight into key residues required for CP activity and the in vivo protein targets of actinidin. PMID:22039217

  10. MLF1 is a proapoptotic antagonist of HOP complex-mediated survival.

    PubMed

    Sun, Yi; Chao, Jyh-Rong; Xu, Wu; Pourpak, Alan; Boyd, Kelli; Moshiach, Simon; Qi, Guo-Yan; Fu, Amina; Shao, Hua-Rong; Pounds, Stanley; Morris, Stephan W

    2017-04-01

    In the HAX1/HtrA2-OMI/PARL (HOP) mitochondrial protein complex, anti-apoptotic signals are generated by cleavage and activation of the serine protease HtrA2/OMI by the rhomboid protease PARL upon recruitment of both proteases to inner mitochondrial membrane protein HAX1 (HS1-associated protein X-1). Here we report the negative regulation of the HOP complex by human leukemia-associated myeloid leukemia factor 1 (MLF1). We demonstrate that MLF1 physically and functionally associates with HAX1 and HtrA2. Increased interaction of MLF1 with HAX1 and HtrA2 displaces HtrA2 from the HOP complex and inhibits HtrA2 cleavage and activation, resulting in the apoptotic cell death. Conversely, over-expressed HAX1 neutralizes MLF1's effect and inhibits MLF1-induced apoptosis. Importantly, Mlf1 deletion reverses B- and T-cell lymphopenia and significantly ameliorates the progressive striatal and cerebellar neurodegeneration observed in Hax1 -/- mice, with a doubling of the lifespan of Mlf1 -/- /Hax1 -/- animals compared to Hax1 -/- animals. Collectively, these data indicate that MLF1 serves as a proapoptotic antagonist that interacts with the HOP mitochondrial complex to modulate cell survival. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. IgA, IgM and IgG anti-M. leprae antibodies in babies of leprosy mothers during the first 2 years of life.

    PubMed Central

    Melsom, R; Harboe, M; Duncan, M E

    1982-01-01

    IgA, IgM and IgG anti-M. leprae antibody activity was estimated by solid phase radioimmunoassay in repeated serum samples from cord sera to sera taken 2 years after birth from 29 babies of mothers with lepromatous leprosy (Group 1) and 16 babies of mothers with tuberculoid leprosy and non-leprosy control mothers (Group 2). IgA anti-M. leprae antibody activity could be detected in 30% and IgM anti-M. leprae antibody activity in 50% of cord sera from Group 1, but not in any of the cord sera from Group 2. After birth, there was a significantly higher increase of IgA and IgM anti-M. leprae antibody activity in sera taken 3-6 months after birth from babies of Group 1 compared to Group 2, but the IgA and IgM activity in sera taken after 6 months of age showed the same increase in the two groups. IgG anti-M. leprae antibody activity showed a marked decrease in sera from both Groups 1 and 2 taken 3-6 and 6-9 months after birth compared to the activity in the cord sera. No increase of the IgG activity could be demonstrated even in sera taken 15-24 months after birth in any of the two groups. These findings are discussed in relation to possible transfer of M. leprae bacilli across the placenta, the influence of M. leprae and other mycobacteria exposure on the antibody activity, the poor IgG anti-M. leprae antibody response and subclinical leprosy infection in babies exposed to leprosy below 2 years of age. PMID:6756719

  12. Losartan and Dexamethasone may inhibit chemotaxis to reduce the infiltration of Th22 cells in IgA nephropathy.

    PubMed

    Xiao, Chenggen; Zhou, Qiaoling; Li, Xiaozhao; Li, Hui; Zhong, Yong; Meng, Ting; Zhu, Mengyuan; Sun, Hong; Liu, Shuang; Tang, Rong; Pu, Jiaxi; Xu, Yan; Xiao, Ping

    2017-01-01

    Angiotensin II is considered a major profibrotic factor that is involved in tissue remodeling processes, as the inhibition of Angiotensin II can halt renal inflammatory processes. Dexamethasone, an important anti-inflammatory and immunosuppressive agent, has been widely used to treat renal disease for decades. In this study, we explored the frequency of Th22 cells in a mouse model of IgA nephropathy and compared the possible effects of Losartan and Dexamethasone on Th22 cells. The experiments were performed using 6-week-old BALB/c female mice in an established IgA nephropathy model. The mice were randomly separated into 4 groups, which were administered Losartan (30mg/kg/d) or Dexamethasone (10mg/kg/d) and subjected to IgA nephropathy or the normal control treatment for 1month. The frequency of Th22 cells was measured via flow cytometry, and the relative pathological changes in renal morphology were measured with different pathological staining methods. Immunohistochemistry was performed to verify the expression of CCR10 and CCL27, which is specialized receptor on Th22 cells and its corresponding chemokine, respectively. The concentrations of CCL27 and IL-22 in renal tissue homogenates and sera were detected using ELISAs. Losartan and Dexamethasone differentially decreased the frequency of Th22 cells after 1month, and mesangial cell proliferation was also improved. Moreover, the expression of CCR10, CCL27 and IL-22 was reduced by treatment with either drug. However, significant differences between Losartan and Dexamethasone were not observed. Based on these findings, Losartan and Dexamethasone may suppress inflammatory responses by inhibiting the chemotaxis of Th22 cells in IgA nephropathy. Copyright © 2016. Published by Elsevier B.V.

  13. IgA Antibodies in Rett Syndrome

    ERIC Educational Resources Information Center

    Reichelt, K. L.; Skjeldal, O.

    2006-01-01

    The level of IgA antibodies to gluten and gliadin proteins found in grains and to casein found in milk, as well as the level of IgG to gluten and gliadin, have been examined in 23 girls with Rett syndrome and 53 controls. Highly statistically significant increases were found for the Rett population compared to the controls. The reason for this…

  14. Lack of serologic evidence to link IgA nephropathy with celiac disease or immune reactivity to gluten.

    PubMed

    Moeller, Sina; Canetta, Pietro A; Taylor, Annette K; Arguelles-Grande, Carolina; Snyder, Holly; Green, Peter H; Kiryluk, Krzysztof; Alaedini, Armin

    2014-01-01

    IgA nephropathy is the most common form of primary glomerulonephritis worldwide. Mucosal infections and food antigens, including wheat gluten, have been proposed as potential contributing environmental factors. Increased immune reactivity to gluten and/or association with celiac disease, an autoimmune disorder triggered by ingestion of gluten, have been reported in IgA nephropathy. However, studies are inconsistent about this association. We aimed to evaluate the proposed link between IgA nephropathy and celiac disease or immune reactivity to gluten by conducting a comprehensive analysis of associated serologic markers in cohorts of well-characterized patients and controls. Study participants included patients with biopsy-proven IgA nephropathy (n = 99), unaffected controls of similar age, gender, and race (n = 96), and patients with biopsy-proven celiac disease (n = 30). All serum specimens were tested for IgG and IgA antibodies to native gliadin and deamidated gliadin, as well as IgA antibody to transglutaminase 2 (TG2). Anti-TG2 antibody-positive nephropathy patients and unaffected controls were subsequently tested for IgA anti-endomysial antibody and genotyped for celiac disease-associated HLA-DQ2 and -DQ8 alleles. In comparison to unaffected controls, there was not a statistically significant increase in IgA or IgG antibody reactivity to gliadin in individuals with IgA nephropathy. In addition, the levels of celiac disease-specific serologic markers, i.e., antibodies to deamidated gliadin and TG2, did not differ between IgA nephropathy patients and unaffected controls. Results of the additional anti-endomysial antibody testing and HLA genotyping were corroborative. The data from this case-control study do not reveal any evidence to suggest a significant role for celiac disease or immune reactivity to gluten in IgA nephropathy.

  15. Lack of Serologic Evidence to Link IgA Nephropathy with Celiac Disease or Immune Reactivity to Gluten

    PubMed Central

    Moeller, Sina; Canetta, Pietro A.; Taylor, Annette K.; Arguelles-Grande, Carolina; Snyder, Holly; Green, Peter H.; Kiryluk, Krzysztof; Alaedini, Armin

    2014-01-01

    IgA nephropathy is the most common form of primary glomerulonephritis worldwide. Mucosal infections and food antigens, including wheat gluten, have been proposed as potential contributing environmental factors. Increased immune reactivity to gluten and/or association with celiac disease, an autoimmune disorder triggered by ingestion of gluten, have been reported in IgA nephropathy. However, studies are inconsistent about this association. We aimed to evaluate the proposed link between IgA nephropathy and celiac disease or immune reactivity to gluten by conducting a comprehensive analysis of associated serologic markers in cohorts of well-characterized patients and controls. Study participants included patients with biopsy-proven IgA nephropathy (n = 99), unaffected controls of similar age, gender, and race (n = 96), and patients with biopsy-proven celiac disease (n = 30). All serum specimens were tested for IgG and IgA antibodies to native gliadin and deamidated gliadin, as well as IgA antibody to transglutaminase 2 (TG2). Anti-TG2 antibody-positive nephropathy patients and unaffected controls were subsequently tested for IgA anti-endomysial antibody and genotyped for celiac disease-associated HLA-DQ2 and -DQ8 alleles. In comparison to unaffected controls, there was not a statistically significant increase in IgA or IgG antibody reactivity to gliadin in individuals with IgA nephropathy. In addition, the levels of celiac disease-specific serologic markers, i.e., antibodies to deamidated gliadin and TG2, did not differ between IgA nephropathy patients and unaffected controls. Results of the additional anti-endomysial antibody testing and HLA genotyping were corroborative. The data from this case-control study do not reveal any evidence to suggest a significant role for celiac disease or immune reactivity to gluten in IgA nephropathy. PMID:24732864

  16. Structural determinants of tobacco vein mottling virus protease substrate specificity

    PubMed Central

    Sun, Ping; Austin, Brian P; Tözsér, József; Waugh, David S

    2010-01-01

    Tobacco vein mottling virus (TVMV) is a member of the Potyviridae, one of the largest families of plant viruses. The TVMV genome is translated into a single large polyprotein that is subsequently processed by three virally encoded proteases. Seven of the nine cleavage events are carried out by the NIa protease. Its homolog from the tobacco etch virus (TEV) is a widely used reagent for the removal of affinity tags from recombinant proteins. Although TVMV protease is a close relative of TEV protease, they exhibit distinct sequence specificities. We report here the crystal structure of a catalytically inactive mutant TVMV protease (K65A/K67A/C151A) in complex with a canonical peptide substrate (Ac-RETVRFQSD) at 1.7-Å resolution. As observed in several crystal structures of TEV protease, the C-terminus (∼20 residues) of TVMV protease is disordered. Unexpectedly, although deleting the disordered residues from TEV protease reduces its catalytic activity by ∼10-fold, an analogous truncation mutant of TVMV protease is significantly more active. Comparison of the structures of TEV and TVMV protease in complex with their respective canonical substrate peptides reveals that the S3 and S4 pockets are mainly responsible for the differing substrate specificities. The structure of TVMV protease suggests that it is less tolerant of variation at the P1′ position than TEV protease. This conjecture was confirmed experimentally by determining kinetic parameters kcat and Km for a series of oligopeptide substrates. Also, as predicted by the cocrystal structure, we confirm that substitutions in the P6 position are more readily tolerated by TVMV than TEV protease. PMID:20862670

  17. Effects of astaxanthin-enriched yeast on mucosal IgA induction in the jejunum and ileum of weanling mice.

    PubMed

    Nagayama, Tatsuhiko; Sugimoto, Miki; Ikeda, Shuntaro; Kume, Shinichi

    2014-04-01

    The present study was conducted to clarify the effects of astaxanthin-enriched yeast on the concentration of immunoglobulin A (IgA), the numbers of IgA antibody-secreting cells (ASC) and the messenger RNA (mRNA) expression of IgA C-region in the jejunum and ileum of weanling mice. Weanling mice were fed rodent feed or astaxanthin-enriched yeast-supplemented rodent feed for 7, 14 or 21 days. Supplemental astaxanthin-enriched yeast increased the numbers of IgA ASC in the jejunum and ileum after 7, 14 and 21 days of treatment. Supplemental astaxanthin-enriched yeast increased IgA concentrations in the jejunum after 21 days of treatment, but IgA concentrations in the ileum were not affected by the treatment. The mRNA expressions of IgA C-region in the jejunum after 14 and 21 days of treatment and the ileum after 14 days of treatment were enhanced by supplementation of astaxanthin-enriched yeast. These results indicate that supplementation of astaxanthin-enriched yeast is effective to enhance the numbers of IgA ASC in the jejunum and ileum and IgA concentrations in the ileum of weanling mice. © 2013 Japanese Society of Animal Science.

  18. Prominent role for plasmacytoid dendritic cells in mucosal T cell-independent IgA induction.

    PubMed

    Tezuka, Hiroyuki; Abe, Yukiko; Asano, Jumpei; Sato, Taku; Liu, Jiajia; Iwata, Makoto; Ohteki, Toshiaki

    2011-02-25

    Although both conventional dendritic cells (cDCs) and plasmacytoid dendritic cells (pDCs) are present in the gut-associated lymphoid tissues (GALT), the roles of pDCs in the gut remain largely unknown. Here we show a critical role for pDCs in T cell-independent (TI) IgA production by B cells in the GALT. When pDCs of the mesenteric lymph nodes (MLNs) and Peyer's patches (PPs) (which are representative GALT) were cultured with naive B cells to induce TI IgA class switch recombination (CSR), IgA production was substantially higher than in cocultures of these cells with cDCs. IgA production was dependent on APRIL and BAFF production by pDCs. Importantly, pDC expression of APRIL and BAFF was dependent on stromal cell-derived type I IFN signaling under steady-state conditions. Our findings provide insight into the molecular basis of pDC conditioning to induce mucosal TI IgA production, which may lead to improvements in vaccination strategies and treatment for mucosal-related disorders. Copyright © 2011 Elsevier Inc. All rights reserved.

  19. [Analysis of salivary protease spectrum in chronic periodontitis].

    PubMed

    Qian, Li; Xuedong, Zhou; Yaping, Fan; Tengyu, Yang; Songtao, Wu; Yu, Yu; Jiao, Chen; Ping, Zhang; Yun, Feng

    2017-02-01

    This study aimed to investigate the difference in salivary protease expression in patients with chronic periodontitis and normal individuals. The stimulating saliva in patients with chronic periodontitis and normal individuals were collected. Protein chip technology was adapted to analyze salivary protease spectrum. Among the 34 proteases in the chip, disintegrin and metalloproteinase (ADAM)8, matrix metalloproteinase (MMP)-8, MMP-12, neprilysin/CD10, and uridylyl phosphate adenosine/urokinase showed a significantly increased concentration in the saliva of chronic periodontitis patients compared with those in the saliva of normal individuals (P<0.01). By contrast, the concentrations of ADAM9, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)1, ADAMTS13, cathepsin B, E, L, V, X/Z/P, kallikrein 6, 7, 11, 13, MMP-9, proteinase 3, presenilin-1, and proprotein convertase 9 sharply decreased (P<0.05). The results demonstrated that protease spectrum in the saliva of chronic periodontitis patients and normal individuals significantly differed. Analysis of salivary protease spectrum is a potential clinical method to examine, diagnose, and monitor chronic periodontitis.

  20. A new fusion protein platform for quantitatively measuring activity of multiple proteases

    PubMed Central

    2014-01-01

    Background Recombinant proteins fused with specific cleavage sequences are widely used as substrate for quantitatively analyzing the activity of proteases. Here we propose a new fusion platform for multiple proteases, by using diaminopropionate ammonia-lyase (DAL) as the fusion protein. It was based on the finding that a fused His6-tag could significantly decreases the activities of DAL from E. coli (eDAL) and Salmonella typhimurium (sDAL). Previously, we have shown that His6GST-tagged eDAL could be used to determine the activity of tobacco etch virus protease (TEVp) under different temperatures or in the denaturant at different concentrations. In this report, we will assay different tags and cleavage sequences on DAL for expressing yield in E. coli, stability of the fused proteins and performance of substrate of other common proteases. Results We tested seven different protease cleavage sequences (rhinovirus 3C, TEV protease, factor Xa, Ssp DnaB intein, Sce VMA1 intein, thrombin and enterokinase), three different tags (His6, GST, CBD and MBP) and two different DALs (eDAL and sDAL), for their performance as substrate to the seven corresponding proteases. Among them, we found four active DAL-fusion substrates suitable for TEVp, factor Xa, thrombin and DnaB intein. Enterokinase cleaved eDAL at undesired positions and did not process sDAL. Substitution of GST with MBP increase the expression level of the fused eDAL and this fusion protein was suitable as a substrate for analyzing activity of rhinovirus 3C. We demonstrated that SUMO protease Ulp1 with a N-terminal His6-tag or MBP tag displayed different activity using the designed His6SUMO-eDAL as substrate. Finally, owing to the high level of the DAL-fusion protein in E. coli, these protein substrates can also be detected directly from the crude extract. Conclusion The results show that our designed DAL-fusion proteins can be used to quantify the activities of both sequence- and conformational-specific proteases, with

  1. Protease-Mediated Suppression of DRG Neuron Excitability by Commensal Bacteria.

    PubMed

    Sessenwein, Jessica L; Baker, Corey C; Pradhananga, Sabindra; Maitland, Megan E; Petrof, Elaine O; Allen-Vercoe, Emma; Noordhof, Curtis; Reed, David E; Vanner, Stephen J; Lomax, Alan E

    2017-11-29

    Peripheral pain signaling reflects a balance of pronociceptive and antinociceptive influences; the contribution by the gastrointestinal microbiota to this balance has received little attention. Disorders, such as inflammatory bowel disease and irritable bowel syndrome, are associated with exaggerated visceral nociceptive actions that may involve altered microbial signaling, particularly given the evidence for bacterial dysbiosis. Thus, we tested whether a community of commensal gastrointestinal bacteria derived from a healthy human donor (microbial ecosystem therapeutics; MET-1) can affect the excitability of male mouse DRG neurons. MET-1 reduced the excitability of DRG neurons by significantly increasing rheobase, decreasing responses to capsaicin (2 μm) and reducing action potential discharge from colonic afferent nerves. The increase in rheobase was accompanied by an increase in the amplitude of voltage-gated K + currents. A mixture of bacterial protease inhibitors abrogated the effect of MET-1 effects on DRG neuron rheobase. A serine protease inhibitor but not inhibitors of cysteine proteases, acid proteases, metalloproteases, or aminopeptidases abolished the effects of MET-1. The serine protease cathepsin G recapitulated the effects of MET-1 on DRG neurons. Inhibition of protease-activated receptor-4 (PAR-4), but not PAR-2, blocked the effects of MET-1. Furthermore, Faecalibacterium prausnitzii recapitulated the effects of MET-1 on excitability of DRG neurons. We conclude that serine proteases derived from commensal bacteria can directly impact the excitability of DRG neurons, through PAR-4 activation. The ability of microbiota-neuronal interactions to modulate afferent signaling suggests that therapies that induce or correct microbial dysbiosis may impact visceral pain. SIGNIFICANCE STATEMENT Commercially available probiotics have the potential to modify visceral pain. Here we show that secretory products from gastrointestinal microbiota derived from a human

  2. Purification of a factor from the granules of a rat natural killer cell line (RNK) that reduces tumor cell growth and changes tumor morphology. Molecular identity with a granule serine protease (RNKP-1).

    PubMed

    Sayers, T J; Wiltrout, T A; Sowder, R; Munger, W L; Smyth, M J; Henderson, L E

    1992-01-01

    We have purified a protein from the granules of the rat NK leukemia cell line (RNK) that is cytostatic to a variety of tumor cells. This protein shows no species specificity because certain tumor cell lines of mouse, rat, and human origin were equally sensitive to its growth inhibitory effects. Treatment of sensitive cells resulted in a rounding of the cells followed by homotypic aggregation into large aggregates. The granule protein was distinct from cytolysin, Na-Cbz-Lys-thiobenzylester-esterase, or leukolexin. It had a molecular mass of 29 to 31 kDa, bound strongly to heparin, was inactivated by heating at 70 degrees C for 5 min or reduction, but was stable to trypsin treatment. By using molecular sieve chromatography, heparin agarose chromatography, and reverse phase HPLC, this protein was purified to homogeneity. The first 33 amino acids of the N-terminal amino acid sequence showed complete identity to the sequence predicted from a rat serine protease gene recently cloned and designated RNKP-1. Therefore we have purified a novel serine protease and demonstrated that it has effects on the growth and morphology of certain tumor cells. Other serine proteases that were structurally related and have substantial homology with RNKP-1 at the amino acid level showed neither growth inhibitory properties nor affected the morphology of the tumor target cells we used.

  3. Insights into the functional role of protonation states in the HIV-1 protease-BEA369 complex: molecular dynamics simulations and free energy calculations.

    PubMed

    Chen, Jianzhong; Yang, Maoyou; Hu, Guodong; Shi, Shuhua; Yi, Changhong; Zhang, Qinggang

    2009-10-01

    The molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method combined with molecular dynamics (MD) simulations were used to investigate the functional role of protonation in human immunodeficiency virus type 1 (HIV-1) protease complexed with the inhibitor BEA369. Our results demonstrate that protonation of two aspartic acids (Asp25/Asp25') has a strong influence on the dynamics behavior of the complex, the binding free energy of BEA369, and inhibitor-residue interactions. Relative binding free energies calculated using the MM-PBSA method show that protonation of Asp25 results in the strongest binding of BEA369 to HIV-1 protease. Inhibitor-residue interactions computed by the theory of free energy decomposition also indicate that protonation of Asp25 has the most favorable effect on binding of BEA369. In addition, hydrogen-bond analysis based on the trajectories of the MD simulations shows that protonation of Asp25 strongly influences the water-mediated link of a conserved water molecule, Wat301. We expect that the results of this study will contribute significantly to binding calculations for BEA369, and to the design of high affinity inhibitors.

  4. Analysis of Milk from Mothers Who Delivered Prematurely Reveals Few Changes in Proteases and Protease Inhibitors across Gestational Age at Birth and Infant Postnatal Age123

    PubMed Central

    Demers-Mathieu, Veronique; Nielsen, Søren Drud; Underwood, Mark A; Borghese, Robyn

    2017-01-01

    Background: Peptidomics research has demonstrated that protease activity is higher in breast milk from preterm-delivering mothers than from term-delivering mothers. However, to our knowledge, the effect of the degree of prematurity and postnatal age on proteases and protease inhibitors in human milk remains unknown. Objective: We aimed to determine the change of proteases and protease inhibitors in milk from mothers who delivered prematurely across gestational age (GA) and postnatal age. Methods: Milk samples were collected from 18 mothers aged 26–40 y who delivered preterm infants and who lacked mastitis. For analysis, samples were separated into 2 groups: 9 from early GA (EGA) (24–26 wk GA)-delivering mothers and 9 from late GA (LGA) (27–32 wk GA)-delivering mothers. Within the 9 samples in each group, the collection time ranged from postnatal days 2 to 47. The activity and predicted activity of proteases in preterm milk were determined with the use of fluorometric and spectrophotometric assays and peptidomics, respectively. Protease and protease inhibitor concentrations were determined with the use of ELISA. Linear mixed models were applied to compare enzymes across GA and postnatal age. Results: Carboxypeptidase B2, kallikrein, plasmin, elastase, thrombin, and cytosol aminopeptidase were present and active in the milk of preterm-delivering mothers. Most milk protease and antiprotease concentrations did not change with GA or postnatal age. However, the concentration and activity of kallikrein, the most abundant and active protease in preterm milk, increased by 25.4 ng · mL−1 · d−1 and 0.454 μg · mL−1 · d−1 postnatally, respectively, in EGA milk samples while remaining stable in LGA milk samples. Conclusions: This research demonstrates that proteases are active in human milk and begin to degrade milk protein within the mammary gland before consumption by infants. Proteases and protease inhibitors in milk from mothers of premature infants mostly

  5. Prevalence, Mutation Patterns, and Effects on Protease Inhibitor Susceptibility of the L76V Mutation in HIV-1 Protease▿ †

    PubMed Central

    Young, Thomas P.; Parkin, Neil T.; Stawiski, Eric; Pilot-Matias, Tami; Trinh, Roger; Kempf, Dale J.; Norton, Michael

    2010-01-01

    Patterns of HIV-1 protease inhibitor (PI) resistance-associated mutations (RAMs) and effects on PI susceptibility associated with the L76V mutation were studied in a large database. Of 20,501 sequences with ≥1 PI RAM, 3.2% contained L76V; L76V was alone in 0.04%. Common partner mutations included M46I, I54V, V82A, I84V, and L90M. L76V was associated with a 2- to 6-fold decrease in susceptibility to lopinavir, darunavir, amprenavir, and indinavir and a 7- to 8-fold increase in susceptibility to atazanavir and saquinavir. PMID:20805393

  6. Genome expression analyses revealing the modulation of the Salmonella Rcs regulon by the attenuator IgaA.

    PubMed

    Mariscotti, Javier F; García-del Portillo, Francisco

    2009-03-01

    Intracellular growth attenuator A (IgaA) was identified as a Salmonella enterica regulator limiting bacterial growth inside fibroblasts. Genetic evidence further linked IgaA to repression of the RcsCDB regulatory system, which responds to envelope stress. How IgaA attenuates this system is unknown. Here, we present genome expression profiling data of S. enterica serovar Typhimurium igaA mutants grown at high osmolarity and displaying exacerbated Rcs responses. Transcriptome data revealed that IgaA attenuates gene expression changes requiring phosphorylated RcsB (RcsB~P) activity. Some RcsB-regulated genes, yciGFE and STM1862 (pagO)-STM1863-STM1864, were equally expressed in wild-type and igaA strains, suggesting a maximal expression at low levels of RcsB ~P. Other genes, such as metB, ypeC, ygaC, glnK, glnP, napA, glpA, and nirB, were shown for the first time and by independent methods to be regulated by the RcsCDB system. Interestingly, IgaA-deficient strains with reduced RcsC or RcsD levels exhibited different Rcs responses and distinct virulence properties. spv virulence genes were differentially expressed in most of the analyzed strains. spvA expression required RcsB and IgaA but, unexpectedly, was also impaired upon stimulation of the RcsC-->RcsD-->RcsB phosphorelay. Overproduction of either RcsB(+) or a nonphosphorylatable RcsB(D56Q) variant in strains displaying low spvA expression unveiled that both dephosphorylated RcsB and RcsB~P are required for optimal spvA expression. Taken together, our data support a model with IgaA attenuating the RcsCDB system by favoring the switch of RcsB~P to the dephosphorylated state. This role of IgaA in constantly fine-tuning the RcsB~P/RcsB ratio may ensure the proper expression of important virulence factors, such as the Spv proteins.

  7. Impact of HIV-1 subtype and antiretroviral therapy on protease and reverse transcriptase genotype: results of a global collaboration.

    PubMed

    Kantor, Rami; Katzenstein, David A; Efron, Brad; Carvalho, Ana Patricia; Wynhoven, Brian; Cane, Patricia; Clarke, John; Sirivichayakul, Sunee; Soares, Marcelo A; Snoeck, Joke; Pillay, Candice; Rudich, Hagit; Rodrigues, Rosangela; Holguin, Africa; Ariyoshi, Koya; Bouzas, Maria Belen; Cahn, Pedro; Sugiura, Wataru; Soriano, Vincent; Brigido, Luis F; Grossman, Zehava; Morris, Lynn; Vandamme, Anne-Mieke; Tanuri, Amilcar; Phanuphak, Praphan; Weber, Jonathan N; Pillay, Deenan; Harrigan, P Richard; Camacho, Ricardo; Schapiro, Jonathan M; Shafer, Robert W

    2005-04-01

    The genetic differences among HIV-1 subtypes may be critical to clinical management and drug resistance surveillance as antiretroviral treatment is expanded to regions of the world where diverse non-subtype-B viruses predominate. To assess the impact of HIV-1 subtype and antiretroviral treatment on the distribution of mutations in protease and reverse transcriptase, a binomial response model using subtype and treatment as explanatory variables was used to analyze a large compiled dataset of non-subtype-B HIV-1 sequences. Non-subtype-B sequences from 3,686 persons with well characterized antiretroviral treatment histories were analyzed in comparison to subtype B sequences from 4,769 persons. The non-subtype-B sequences included 461 with subtype A, 1,185 with C, 331 with D, 245 with F, 293 with G, 513 with CRF01_AE, and 618 with CRF02_AG. Each of the 55 known subtype B drug-resistance mutations occurred in at least one non-B isolate, and 44 (80%) of these mutations were significantly associated with antiretroviral treatment in at least one non-B subtype. Conversely, of 67 mutations found to be associated with antiretroviral therapy in at least one non-B subtype, 61 were also associated with antiretroviral therapy in subtype B isolates. Global surveillance and genotypic assessment of drug resistance should focus primarily on the known subtype B drug-resistance mutations.

  8. Deletion of BCG Hip1 protease enhances dendritic cell and CD4 T cell responses.

    PubMed

    Bizzell, Erica; Sia, Jonathan Kevin; Quezada, Melanie; Enriquez, Ana; Georgieva, Maria; Rengarajan, Jyothi

    2018-04-01

    Dendritic cells (DCs) play a key role in the generation of CD4 T cell responses to pathogens. Mycobacterium tuberculosis (Mtb) harbors immune evasion mechanisms that impair DC responses and prevent optimal CD4 T cell immunity. The vaccine strain Mycobacterium bovis Bacille Calmette-Guérin (BCG) shares many of the immune evasion proteins utilized by Mtb, but the role of these proteins in DC and T cell responses elicited by BCG is poorly understood. We previously reported that the Mtb serine protease, Hip1, promotes sub-optimal DC responses during infection. Here, we tested the hypothesis that BCG Hip1 modulates DC functions and prevents optimal antigen-specific CD4 T cell responses that limit the immunogenicity of BCG. We generated a strain of BCG lacking hip1 (BCGΔhip1) and show that it has superior capacity to induce DC maturation and cytokine production compared with the parental BCG. Furthermore, BCGΔhip1-infected DCs were more effective at driving the production of IFN-γ and IL-17 from antigen-specific CD4 T cells in vitro. Mucosal transfer of BCGΔhip1-infected DCs into mouse lungs induced robust CD4 T cell activation in vivo and generated antigen-specific polyfunctional CD4 T cell responses in the lungs. Importantly, BCGΔhip1-infected DCs enhanced control of pulmonary bacterial burden following Mtb aerosol challenge compared with the transfer of BCG-infected DCs. These results reveal that BCG employs Hip1 to impair DC activation, leading to attenuated lung CD4 T cell responses with limited capacity to control Mtb burden after challenge. ©2017 Society for Leukocyte Biology.

  9. Dysregulated LIGHT expression on T cells mediates intestinal inflammation and contributes to IgA nephropathy

    PubMed Central

    Wang, Jing; Anders, Robert A.; Wu, Qiang; Peng, Dacheng; Cho, Judy H.; Sun, Yonglian; Karaliukas, Reda; Kang, Hyung-Sik; Turner, Jerrold R.; Fu, Yang-Xin

    2004-01-01

    Whether and how T cells contribute to the pathogenesis of immunoglobulin A nephropathy (IgAN) has not been well defined. Here, we explore a murine model that spontaneously develops T cell–mediated intestinal inflammation accompanied by pathological features similar to those of human IgAN. Intestinal inflammation mediated by LIGHT, a ligand for lymphotoxin β receptor (LTβR), not only stimulates IgA overproduction in the gut but also results in defective IgA transportation into the gut lumen, causing a dramatic increase in serum polymeric IgA. Engagement of LTβR by LIGHT is essential for both intestinal inflammation and hyperserum IgA syndrome in our LIGHT transgenic model. Impressively, the majority of patients with inflammatory bowel disease showed increased IgA-producing cells in the gut, elevated serum IgA levels, and severe hematuria, a hallmark of IgAN. These observations indicate the critical contributions of dysregulated LIGHT expression and intestinal inflammation to the pathogenesis of IgAN. PMID:15067315

  10. Proteases and caspase-like activity in the yeast Saccharomyces cerevisiae.

    PubMed

    Wilkinson, Derek; Ramsdale, Mark

    2011-10-01

    A variety of proteases have been implicated in yeast PCD (programmed cell death) including the metacaspase Mca1 and the separase Esp1, the HtrA-like serine protease Nma111, the cathepsin-like serine carboxypeptideases and a range of vacuolar proteases. Proteasomal activity is also shown to have an important role in determining cell fate, with both pro- and anti-apoptotic roles. Caspase 3-, 6- and 8-like activities are detected upon stimulation of yeast PCD, but not all of this activity is associated with Mca1, implicating other proteases with caspase-like activity in the yeast cell death response. Global proteolytic events that accompany PCD are discussed alongside a consideration of the conservation of the death-related degradome (both at the level of substrate choice and cleavage site). The importance of both gain-of-function changes in the degradome as well as loss-of-function changes are highlighted. Better understanding of both death-related proteases and their substrates may facilitate the design of future antifungal drugs or the manipulation of industrial yeasts for commercial exploitation.

  11. Activated protein C (APC) can increase bone anabolism via a protease-activated receptor (PAR)1/2 dependent mechanism.

    PubMed

    Shen, Kaitlin; Murphy, Ciara M; Chan, Ben; Kolind, Mille; Cheng, Tegan L; Mikulec, Kathy; Peacock, Lauren; Xue, Meilang; Park, Sang-Youel; Little, David G; Jackson, Chris J; Schindeler, Aaron

    2014-12-01

    Activated Protein C (APC) is an anticoagulant with strong cytoprotective properties that has been shown to promote wound healing. In this study APC was investigated for its potential orthopedic application using a Bone Morphogenetic Protein 2 (rhBMP-2) induced ectopic bone formation model. Local co-administration of 10 µg rhBMP-2 with 10 µg or 25 µg APC increased bone volume at 3 weeks by 32% (N.S.) and 74% (p<0.01) compared to rhBMP-2 alone. This was associated with a significant increase in CD31+ and TRAP+ cells in tissue sections of ectopic bone, consistent with enhanced vascularity and bone turnover. The actions of APC are largely mediated by its receptors endothelial protein C receptor (EPCR) and protease-activated receptors (PARs). Cultured pre-osteoblasts and bone nodule tissue sections were shown to express PAR1/2 and EPCR. When pre-osteoblasts were treated with APC, cell viability and phosphorylation of ERK1/2, Akt, and p38 were increased. Inhibition with PAR1 and sometimes PAR2 antagonists, but not with EPCR blocking antibodies, ameliorated the effects of APC on cell viability and kinase phosphorylation. These data indicate that APC can affect osteoblast viability and signaling, and may have in vivo applications with rhBMP-2 for bone repair. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  12. A recombinant fusion protein-based, fluorescent protease assay for high throughput-compatible substrate screening.

    PubMed

    Bozóki, Beáta; Gazda, Lívia; Tóth, Ferenc; Miczi, Márió; Mótyán, János András; Tőzsér, József

    2018-01-01

    In connection with the intensive investigation of proteases, several methods have been developed for analysis of the substrate specificity. Due to the great number of proteases and the expected target molecules to be analyzed, time- and cost-efficient high-throughput screening (HTS) methods are preferred. Here we describe the development and application of a separation-based HTS-compatible fluorescent protease assay, which is based on the use of recombinant fusion proteins as substrates of proteases. The protein substrates used in this assay consists of N-terminal (hexahistidine and maltose binding protein) fusion tags, cleavage sequences of the tobacco etch virus (TEV) and HIV-1 proteases, and a C-terminal fluorescent protein (mApple or mTurquoise2). The assay is based on the fluorimetric detection of the fluorescent proteins, which are released from the magnetic bead-attached substrates by the proteolytic cleavage. The protease assay has been applied for activity measurements of TEV and HIV-1 proteases to test the suitability of the system for enzyme kinetic measurements, inhibition studies, and determination of pH optimum. We also found that denatured fluorescent proteins can be renatured after SDS-PAGE of denaturing conditions, but showed differences in their renaturation abilities. After in-gel renaturation both substrates and cleavage products can be identified by in-gel UV detection. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Structural determinants of tobacco vein mottling virus protease substrate specificity

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sun, Ping; Austin, Brian P.; Tozer, Jozsef

    2010-10-28

    Tobacco vein mottling virus (TVMV) is a member of the Potyviridae, one of the largest families of plant viruses. The TVMV genome is translated into a single large polyprotein that is subsequently processed by three virally encoded proteases. Seven of the nine cleavage events are carried out by the NIa protease. Its homolog from the tobacco etch virus (TEV) is a widely used reagent for the removal of affinity tags from recombinant proteins. Although TVMV protease is a close relative of TEV protease, they exhibit distinct sequence specificities. We report here the crystal structure of a catalytically inactive mutant TVMVmore » protease (K65A/K67A/C151A) in complex with a canonical peptide substrate (Ac-RETVRFQSD) at 1.7-{angstrom} resolution. As observed in several crystal structures of TEV protease, the C-terminus ({approx}20 residues) of TVMV protease is disordered. Unexpectedly, although deleting the disordered residues from TEV protease reduces its catalytic activity by {approx}10-fold, an analogous truncation mutant of TVMV protease is significantly more active. Comparison of the structures of TEV and TVMV protease in complex with their respective canonical substrate peptides reveals that the S3 and S4 pockets are mainly responsible for the differing substrate specificities. The structure of TVMV protease suggests that it is less tolerant of variation at the P1{prime} position than TEV protease. This conjecture was confirmed experimentally by determining kinetic parameters k{sub cat} and K{sub m} for a series of oligopeptide substrates. Also, as predicted by the cocrystal structure, we confirm that substitutions in the P6 position are more readily tolerated by TVMV than TEV protease.« less

  14. Alpha 1-protease inhibitor moderates human neutrophil elastase-induced emphysema and secretory cell metaplasia in hamsters.

    PubMed

    Stone, P J; Lucey, E C; Virca, G D; Christensen, T G; Breuer, R; Snider, G L

    1990-06-01

    A study was undertaken to determine whether emphysema and airway secretory cell metaplasia, induced in hamsters by intratracheal treatment with human neutrophil elastase (HNE), could be moderated by pretreatment with human alpha 1-protease inhibitor (API). API (4.9 mg) was given intratracheally to hamsters 1 h before 0.3 mg HNE. Eight weeks later, lung volumes and pressure-volume relationships were measured in the anaesthetized animals. Mean linear intercepts and secretory cell indices were measured in lung sections. API given 1 h before HNE moderated the development of bronchial secretory cell metaplasia. The severity of emphysema was reduced by 75%. Clearance studies indicated that 80% of the functional activity of instilled API could be lavaged from the lungs after 1 h, indicating a 4 h half-life in the lavageable compartment of the lungs. We calculate that for 50% protection from emphysema the molar ratio of lavageable API to HNE at the time of HNE instillation was 4.8 as compared with 0.78 for 50% inhibition of elastolytic activity in vitro, indicating that API is only 16% as efficient in vivo as compared with its in vitro HNE inhibitory effectiveness. Nevertheless, we conclude that human API given intratracheally is efficacious against HNE-induced emphysema and secretory cell metaplasia.

  15. Detection of H. Pylori infection on dyspepsia patients with IgA H. Pylori antibody

    NASA Astrophysics Data System (ADS)

    Loesnihari, R.

    2018-03-01

    Helicobacter pylori (H. pylori) has a big role in the relapse and pathogenesis of the upper gastrointestinal disease. Dyspepsia is characterized by uncomfortable feeling at the upper gastrointestinal area. IgA H. pylori antibody was in two-thirds of H. pylori infected patients, but about 7.2% of IgA H. Pylori antibody became the only positive result of the test between the two serology test (IgG and IgA). A cross-sectional study was conducted in 38 patients with dyspepsia. The IgA antibody test for H. pylori in the serum of dyspepsia patient conducted through the ELISA test. The hemoglobin levels, leukocytes, platelets number, and H. pylori infection via IgA antibody test on ulcer and non-ulcer dyspepsia patient had no significant difference. There was a relation between the number of platelets in the infected H. pylori patients compared to the non-infected patients. H. pylori infection in the ulcer and non-ulcer dyspepsia patient with serology method was 18%. H. pylori infection number on ulcer dyspepsia was not higher than the non-ulcer dyspepsia, all ulcer dyspepsia patients who were with H. pylori found with a lesion on the antrum.

  16. Neuronal nitric oxide synthase mediates the effect of ethanol on IgA.

    PubMed

    Budec, Mirela; Markovic, Dragana; Vignjevic, Sanja; Mitrovic, Olivera; Dikic, Dragoslava; Koko, Vesna; Cokic, Vladan P

    2013-01-01

    We showed previously that the acute effect of ethanol on intestinal immunoglobulin A (IgA) expression might be mediated by endogenous nitric oxide (NO). To extend these findings, this study was designed to investigate a possible role of neuronal NO synthase (nNOS) in the observed phenomenon, using 7-nitroindazole (7-NI), a selective inhibitor of its activity. Adult male Wistar rats were treated with: (a) ethanol (4 g/kg, intraperitoneally, i.p.), (b) 7-NI (25 mg/kg, i.p.) followed by ethanol (4 g/kg, i.p.) 30 min later and (c) 7-NI (25 mg/kg, i.p.) followed by saline 30 min later. Untreated rats were used as controls. The concentrations of serum and intestinal IgA were measured by enzyme-linked immunosorbent assay, while the expression of nNOS was determined using western blot and immunohistochemistry. Acute ethanol treatment significantly increased the concentration of IgA in the ileal extracts, whereas it decreased its serum level. Inhibition of nNOS activity by 7-NI abolished this action of alcohol on IgA. Additionally, western blot analysis revealed that the acute alcohol administration induced an increase in the expression of intestinal nNOS. Furthermore, nNOS-immunoreactive cells, observed within the lamina propria of small intestine, were numerous in ethanol-treated rats. Taken together, these results extended our previous findings suggesting that nNOS mediates the acute effect of ethanol on IgA and supported an immunomodulatory role of this enzyme isoform.

  17. Estimation of the Binding Free Energy of AC1NX476 to HIV-1 Protease Wild Type and Mutations Using Free Energy Perturbation Method.

    PubMed

    Ngo, Son Tung; Mai, Binh Khanh; Hiep, Dinh Minh; Li, Mai Suan

    2015-10-01

    The binding mechanism of AC1NX476 to HIV-1 protease wild type and mutations was studied by the docking and molecular dynamics simulations. The binding free energy was calculated using the double-annihilation binding free energy method. It is shown that the binding affinity of AC1NX476 to wild type is higher than not only ritonavir but also darunavir, making AC1NX476 become attractive candidate for HIV treatment. Our theoretical results are in excellent agreement with the experimental data as the correlation coefficient between calculated and experimentally measured binding free energies R = 0.993. Residues Asp25-A, Asp29-A, Asp30-A, Ile47-A, Gly48-A, and Val50-A from chain A, and Asp25-B from chain B play a crucial role in the ligand binding. The mutations were found to reduce the receptor-ligand interaction by widening the binding cavity, and the binding propensity is mainly driven by the van der Waals interaction. Our finding may be useful for designing potential drugs to combat with HIV. © 2015 John Wiley & Sons A/S.

  18. Interaction between the macrophage system and IgA immune complexes in IgA nephropathy.

    PubMed

    Roccatello, D; Coppo, R; Basolo, B; Martina, G; Rollino, C; Cordonnier, D; Busquet, G; Picciotto, G; Sena, L M; Piccoli, G

    1983-01-01

    In nine patients with IgA nephropathy, the function of the mononuclear phagocyte system was assessed by measuring in vivo clearance of anti-D coated red blood cells (RBC) and in vitro phagocytosis of sensitised RBC by monocytes. A strict correlation was found between in vivo macrophage function and in vitro monocyte phagocytosis. Statistical correlations were also found between in vivo clearance values and IgAIC and C3d values. A defective macrophage and monocyte function affects patients with major signs of clinical activity, highest IgAIC values, signs of complement activation and the most unfavourable clinical course.

  19. Saquinavir inhibits early events associated with establishment of HIV-1 infection: potential role for protease inhibitors in prevention.

    PubMed

    Stefanidou, Martha; Herrera, Carolina; Armanasco, Naomi; Shattock, Robin J

    2012-08-01

    The maturation of newly formed human immunodeficiency virus type 1 (HIV-1) virions is a critical step for the establishment of productive infection. We investigated the potential of saquinavir (SQV), a protease inhibitor (PI) used in highly active antiretroviral therapy (HAART), as a candidate microbicide. SQV inhibited replication of clade B and clade C isolates in a dose-dependent manner in all cellular models tested: PM-1 CD4 T cells, peripheral blood mononuclear cells (PBMCs), monocyte-derived macrophages (MDMs), and immature monocyte-derived dendritic cells (iMDDCs). SQV also inhibited production of infectious virus in cervical, penile, and colorectal explants cocultured with T cells. Moreover, SQV demonstrated inhibitory potency against trans infection of T cells by in vitro-derived dendritic cells and by primary dendritic cells that emigrate from penile and cervical tissue explants. No cellular or tissue toxicity was detected in the presence of SQV, suggesting that this drug could be considered for development as a component of an effective microbicide, capable of blocking viral maturation and transmission of HIV-1 at mucosal surfaces.

  20. Evaluation of cysteine proteases of Plasmodium vivax as antimalarial drug targets: sequence analysis and sensitivity to cysteine protease inhibitors.

    PubMed

    Na, Byoung-Kuk; Kim, Tong-Soo; Rosenthal, Philip J; Lee, Jong-Koo; Kong, Yoon

    2004-10-01

    Cysteine proteases perform critical roles in the life cycles of malaria parasites. In Plasmodium falciparum, treatment of cysteine protease inhibitors inhibits hemoglobin hydrolysis and blocks the parasite development in vitro and in vivo, suggesting that plasmodial cysteine proteases may be interesting targets for new chemotherapeutics. To determine whether sequence diversity may limit chemotherapy against Plasmodium vivax, we analyzed sequence variations in the genes encoding three cysteine proteases, vivapain-1, -2 and -3, in 22 wild isolates of P. vivax. The sequences were highly conserved among wild isolates. A small number of substitutions leading to amino acid changes were found, while they did not modify essential residues for the function or structure of the enzymes. The substrate specificities and sensitivities to synthetic cysteine protease inhibitors of vivapain-2 and -3 from wild isolates were also very similar. These results support the suggestion that cysteine proteases of P. vivax are promising antimalarial chemotherapeutic targets.