Positive selection pressure introduces secondary mutations at Gag cleavage sites in human immunodeficiency virus type 1 harboring major protease resistance mutations

DEFF Research Database (Denmark)

Banke, S.; Lillemark, M.R.; Gerstoft, J.

2009-01-01

Human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) specifically target the HIV-1 protease enzyme. Mutations in the enzyme can result in PI resistance (termed PI mutations); however, mutations in the HIV-1 gag region, the substrate for the protease enzyme, might also lead to PI ...

Cellular Signaling and Production of Galactose-Deficient IgA1 in IgA Nephropathy, an Autoimmune Disease

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Colin Reily

2014-01-01

Full Text Available Immunoglobulin A (IgA nephropathy (IgAN, the leading cause of primary glomerulonephritis, is characterized by IgA1-containing immunodeposits in the glomeruli. IgAN is a chronic disease, with up to 40% of patients progressing to end-stage renal disease, with no disease-specific treatment. Multiple studies of the origin of the glomerular immunodeposits have linked elevated circulating levels of aberrantly glycosylated IgA1 (galactose-deficient in some O-glycans; Gd-IgA1 with formation of nephritogenic Gd-IgA1-containing immune complexes. Gd-IgA1 is recognized as an autoantigen in susceptible individuals by anti-glycan autoantibodies, resulting in immune complexes that may ultimately deposit in the kidney and induce glomerular injury. Genetic studies have revealed that an elevated level of Gd-IgA1 in the circulation of IgAN patients is a hereditable trait. Moreover, recent genome-wide association studies have identified several immunity-related loci that associated with IgAN. Production of Gd-IgA1 by IgA1-secreting cells of IgAN patients has been attributed to abnormal expression and activity of several key glycosyltransferases. Substantial evidence is emerging that abnormal signaling in IgA1-producing cells is related to the production of Gd-IgA1. As Gd-IgA1 is the key autoantigen in IgAN, understanding the genetic, biochemical, and environmental aspects of the abnormal signaling in IgA1-producing cells will provide insight into possible targets for future disease-specific therapy.

Detection of FMD virus type specific IgG1, IgG2 and IgA antibodies in milk and serum of buffaloes vaccinated with oil adjuvanted polyvalent FMD vaccine

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R. Sharma

2010-02-01

Full Text Available The present investigation was carried out on 15 randomly selected milch buffaloes divided into three groups on the basis of lactation at an organized farm, to study the foot and mouth disease virus type specific antibodies in milk and serum following FMD vaccination. Milk and serum samples collected before vaccination i.e. 0 day and on 7, 14, 28, 42 and 56 days post vaccination, were analyzed for the detection of FMD virus specific IgG1, IgG2 and IgA antibody response by indirect double antibody sandwich ELISA. Significant FMD virus type specific antibody titres (IgG1, IgG2 and IgA were detected in milk and serum of buffaloes on different days post vaccination, though the levels of antibodies were lower in milk as compared to serum. FMD virus type specific IgG1 was found to be the predominant subclass as compared to IgG2 and IgA both in milk and serum of vaccinated buffaloes. Milk and serum IgG1, IgG2 and IgA antibody titres were positively correlated with values of regression coefficient (R as 0.506, 0.434 and 0.396, respectively.

SjAPI, the first functionally characterized Ascaris-type protease inhibitor from animal venoms.

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Zongyun Chen

Full Text Available BACKGROUND: Serine protease inhibitors act as modulators of serine proteases, playing important roles in protecting animal toxin peptides from degradation. However, all known serine protease inhibitors discovered thus far from animal venom belong to the Kunitz-type subfamily, and whether there are other novel types of protease inhibitors in animal venom remains unclear. PRINCIPAL FINDINGS: Here, by screening scorpion venom gland cDNA libraries, we identified the first Ascaris-type animal toxin family, which contains four members: Scorpiops jendeki Ascaris-type protease inhibitor (SjAPI, Scorpiops jendeki Ascaris-type protease inhibitor 2 (SjAPI-2, Chaerilus tricostatus Ascaris-type protease inhibitor (CtAPI, and Buthus martensii Ascaris-type protease inhibitor (BmAPI. The detailed characterization of Ascaris-type peptide SjAPI from the venom gland of scorpion Scorpiops jendeki was carried out. The mature peptide of SjAPI contains 64 residues and possesses a classical Ascaris-type cysteine framework reticulated by five disulfide bridges, different from all known protease inhibitors from venomous animals. Enzyme and inhibitor reaction kinetics experiments showed that recombinant SjAPI was a dual function peptide with α-chymotrypsin- and elastase-inhibiting properties. Recombinant SjAPI inhibited α-chymotrypsin with a Ki of 97.1 nM and elastase with a Ki of 3.7 μM, respectively. Bioinformatics analyses and chimera experiments indicated that SjAPI contained the unique short side chain functional residues "AAV" and might be a useful template to produce new serine protease inhibitors. CONCLUSIONS/SIGNIFICANCE: To our knowledge, SjAPI is the first functionally characterized animal toxin peptide with an Ascaris-type fold. The structural and functional diversity of animal toxins with protease-inhibiting properties suggested that bioactive peptides from animal venom glands might be a new source of protease inhibitors, which will accelerate the

Analysis of O-glycan heterogeneity in IgA1 myeloma proteins by Fourier transform ion cyclotron resonance mass spectrometry: implications for IgA nephropathy

DEFF Research Database (Denmark)

Renfrow, MB; Mackay, CL; Chalmers, MJ

2007-01-01

deficiency in IgA1 proteins occurs randomly or preferentially at specific sites. We have previously demonstrated the first direct localization of multiple O-glycosylation sites on a single IgA1 myeloma protein by use of activated ion-electron capture dissociation (AI-ECD) Fourier transform ion cyclotron...... resonance (FT-ICR) tandem mass spectrometry. Here, we report the analysis of IgA1 O-glycan heterogeneity by use of FT-ICR MS and liquid chromatography FT-ICR MS to obtain unbiased accurate mass profiles of IgA1 HR glycopeptides from three different IgA1 myeloma proteins. Additionally, we report the first AI......-ECD fragmentation on an individual IgA1 O-glycopeptide from an IgA1 HR preparation that is reproducible for each IgA1 myeloma protein. These results suggest that future analysis of IgA1 HR from IgAN patients and normal healthy controls should be feasible....

Transient glyco-engineering to produce recombinant IgA1 with defined N- and O-glycans in plants

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Martina eDicker

2016-01-01

Full Text Available The production of therapeutic antibodies to combat pathogens and treat diseases such as cancer is of great interest for the biotechnology industry. The recent development of plant-based expression systems has demonstrated that plants are well-suited for the production of recombinant monoclonal antibodies with defined glycosylation. Compared to immunoglobulin G (IgG, less effort has been undertaken to express immunoglobulin A (IgA, which is the most prevalent antibody class at mucosal sites and a promising candidate for novel recombinant biopharmaceuticals with enhanced anti-tumour activity. Here, we transiently expressed recombinant human IgA1 against the VP8* rotavirus antigen in glyco-engineered deltaXT/FT Nicotiana benthamiana plants. Mass spectrometric analysis of IgA1 glycopeptides revealed the presence of complex biantennary N-glycans with terminal N-acetylglucosamine present on the N-glycosylation site of the CH2 domain in the IgA1 alpha chain. Analysis of the peptide carrying nine potential O-glycosylation sites in the IgA1 alpha chain hinge region showed the presence of plant-specific modifications including hydroxyproline formation and the attachment of pentoses. By co-expression of enzymes required for initiation and elongation of human O-glycosylation it was possible to generate disialylated mucin-type core 1 O-glycans on plant-produced IgA1. Our data demonstrate that deltaXT/FT Nicotiana benthamiana plants can be engineered towards the production of recombinant IgA1 with defined human-type N- and O-linked glycans.

Higher Desolvation Energy Reduces Molecular Recognition in Multi-Drug Resistant HIV-1 Protease

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Ladislau C. Kovari

2012-05-01

Full Text Available Designing HIV-1 protease inhibitors that overcome drug-resistance is still a challenging task. In this study, four clinical isolates of multi-drug resistant HIV-1 proteases that exhibit resistance to all the US FDA-approved HIV-1 protease inhibitors and also reduce the substrate recognition ability were examined. A multi-drug resistant HIV-1 protease isolate, MDR 769, was co-crystallized with the p2/NC substrate and the mutated CA/p2 substrate, CA/p2 P1’F. Both substrates display different levels of molecular recognition by the wild-type and multi-drug resistant HIV-1 protease. From the crystal structures, only limited differences can be identified between the wild-type and multi-drug resistant protease. Therefore, a wild-type HIV-1 protease and four multi-drug resistant HIV-1 proteases in complex with the two peptides were modeled based on the crystal structures and examined during a 10 ns-molecular dynamics simulation. The simulation results reveal that the multi-drug resistant HIV-1 proteases require higher desolvation energy to form complexes with the peptides. This result suggests that the desolvation of the HIV-1 protease active site is an important step of protease-ligand complex formation as well as drug resistance. Therefore, desolvation energy could be considered as a parameter in the evaluation of future HIV-1 protease inhibitor candidates.

THE IGA SYSTEM

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South, Mary Ann; Cooper, Max D.; Wollheim, Frank A.; Hong, Richard; Good, Robert A.

1966-01-01

1. Five patients with congenital or acquired agammaglobulinemia, lacking detectable IgA in serum or saliva, were transfused with 1 to 2 liters of normal plasma. In 2 of these patients IgA was demonstrated in parotid saliva collected after transfusion, but in none of the 5 was salivary IgG or IgM found. This observation indicates the selective transport of IgA into saliva. 2. The observation by others of an immunochemical difference between serum and sahvary IgA globulin was confirmed. In contrast to serum IgA, salivary IgA is attached to a protein having antigenicity which migrates as a gamma1 globulin. We have termed this protein component "transport piece". 3. The transport piece has been found in an unbound form in the saliva of persons completely lacking IgA: agammaglobulinemic patients, ataxia-telangiectasia patients, a healthy person lacking IgA, and a newborn infant. Free transport piece still occurs in the normal child's saliva after IgA production begins. By adulthood there is usually no free transport piece in the saliva. 4. Heat-aggregated salivary IgA, like heat-aggregated serum IgA, does not fix complement. 5. Our findings offer support for the view that there is a distinct local antibody system for the protection of the mucous surfaces. PMID:4160397

DNA methylation in Cosmc promoter region and aberrantly glycosylated IgA1 associated with pediatric IgA nephropathy.

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Qiang Sun

Full Text Available IgA nephropathy (IgAN is one of the most common glomerular diseases leading to end-stage renal failure. Elevation of aberrantly glycosylated IgA1 is a key feature of it. The expression of the specific molecular chaperone of core1ß1, 3galactosyl transferase (Cosmc is known to be reduced in IgAN. We aimed to investigate whether the methylation of CpG islands of Cosmc gene promoter region could act as a possible mechanism responsible for down-regulation of Cosmc and related higher secretion of aberrantly glycosylated IgA1in lymphocytes from children with IgA nephropathy. Three groups were included: IgAN children (n = 26, other renal diseases (n = 11 and healthy children (n = 13. B-lymphocytes were isolated and cultured, treated or not with IL-4 or 5-Aza-2'-deoxycytidine (AZA. The levels of DNA methylation of Cosmc promotor region were not significantly different between the lymphocytes of the three children populations (P = 0.113, but there were significant differences between IgAN lymphocytes and lymphocytes of the other two children populations after IL-4 (P<0.0001 or AZA (P<0.0001. Cosmc mRNA expression was low in IgAN lymphocytes compared to the other two groups (P<0.0001. The level of aberrantly glycosylated IgA1 was markedly higher in IgAN group compared to the other groups (P<0.0001. After treatment with IL-4, the levels of Cosmc DNA methylation and aberrantly glycosylated IgA1 in IgAN lymphocytes were remarkably higher than the other two groups (P<0.0001 with more markedly decreased Cosmc mRNA content (P<0.0001. After treatment with AZA, the levels in IgAN lymphocytes were decreased, but was still remarkably higher than the other two groups (P<0.0001, while Cosmc mRNA content in IgAN lymphocytes were more markedly increased than the other two groups (P<0.0001. The alteration of DNA methylation by IL-4 or AZA specifically correlates in IgAN lymphocytes with alterations in Cosmc mRNA expression and with the level of aberrantly glycosylated

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

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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.

Mutations in serine protease inhibitor Kazal type 1 are strongly associated with chronic pancreatitis

OpenAIRE

Drenth, J P H; te Morsche, R; Jansen, J B M J

2002-01-01

Background: Although chronic pancreatitis is associated with risk factors such as alcoholism, hyperparathyroidism, and hypertriglyceridaemia, little is known of the actual aetiology of the disease. It is thought that inappropriate activation of trypsinogen causes pancreatitis, and indeed in cases of hereditary pancreatitis mutations of cationic trypsinogen (PRSS1) have been described. As serine protease inhibitor Kazal type 1 (SPINK1) is a potent natural inhibitor of pancreatic trypsin activi...

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.

OpenAIRE

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 ritona...

The Occurrence of Type S1A Serine Proteases in Sponge and Jellyfish

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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.

Molecular cloning and tissue-specific expression analysis of mouse spinesin, a type II transmembrane serine protease 5

International Nuclear Information System (INIS)

Watanabe, Yoshihisa; Okui, Akira; Mitsui, Shinichi; Kawarabuki, Kentaro; Yamaguchi, Tatsuyuki; Uemura, Hidetoshi; Yamaguchi, Nozomi

2004-01-01

We have previously reported novel serine proteases isolated from cDNA libraries of the human and mouse central nervous system (CNS) by PCR using degenerate oligodeoxyribonucleotide primers designed on the basis of the serine protease motifs, AAHC and DSGGP. Here we report a newly isolated serine protease from the mouse CNS. This protease is homologous (77.9% identical) to human spinesin type II transmembrane serine protease 5. Mouse spinesin (m-spinesin) is also composed of (from the N-terminus) a short cytoplasmic domain, a transmembrane domain, a stem region containing a scavenger-receptor-like domain, and a serine protease domain, as is h-spinesin. We also isolated type 1, type 2, and type 3 variant cDNAs of m-spinesin. Full-length spinesin (type 4) and type 3 contain all the domains, whereas type 1 and type 2 variants lack the cytoplasmic, transmembrane, and scavenger-receptor-like domains. Subcellular localization of the variant forms was analyzed using enhanced green fluorescent protein (EGFP) fusion proteins. EGFP-type 4 fusion protein was predominantly localized to the ER, Golgi apparatus, and plasma membrane, whereas EGFP-type 1 was localized to the cytoplasm, reflecting differential classification of m-spinesin variants into transmembrane and cytoplasmic types. We analyzed the distribution of m-spinesin variants in mouse tissues, using RT-PCR with variant-specific primer sets. Interestingly, transmembrane-type spinesin, types 3 and 4, was specifically expressed in the spinal cord, whereas cytoplasmic type, type 1, was expressed in multiple tissues, including the cerebrum and cerebellum. Therefore, m-spinesin variants may have distinct biological functions arising from organ-specific variant expression

Detection of specific IgA antibodies against a novel deamidated 8-Mer gliadin peptide in blood plasma samples from celiac patients.

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Sara Vallejo-Diez

Full Text Available We studied whether celiac disease (CD patients produce antibodies against a novel gliadin peptide specifically generated in the duodenum of CD patients by a previously described pattern of CD-specific duodenal proteases. Fingerprinting and ion-trap mass spectrometry of CD-specific duodenal gliadin-degrading protease pattern revealed a new 8-mer gliadin-derived peptide. An ELISA against synthetic deamidated 8-mer peptides (DGP 8-mer was used to study the presence of IgA anti-DGP 8-mer antibodies in plasma samples from 81 children (31 active CD patients (aCD, 17 CD patients on a gluten-free diet (GFD, 10 healthy controls (C and 23 patients with other gastrointestinal pathology (GP and 101 adults (16 aCD, 12 GFD, 27 C and 46 GP-patients. Deamidation of the 8-mer peptide significantly increased the reactivity of the IgA antibodies from CD patients against the peptide. Significant IgA anti-DGP 8-mer antibodies levels were detected in 93.5% of aCD-, 11.8% of GFD- and 4.3% of GP-patients in children. In adults, antibodies were detected in 81.3% of aCD-patients and 8.3% of GFD-patients while were absent in 100% of C- and GP-patients. Duodenal CD-specific gliadin degrading proteases release an 8-mer gliadin peptide that once deamidated is an antigen for specific IgA antibodies in CD patients which may provide a new accurate diagnostic tool in CD.

Crystal structure of a novel cysteinless plant Kunitz-type protease inhibitor

International Nuclear Information System (INIS)

Hansen, Daiane; Macedo-Ribeiro, Sandra; Verissimo, Paula; Yoo Im, Sonia; Sampaio, Misako Uemura; Oliva, Maria Luiza Vilela

2007-01-01

Bauhinia bauhinioides Cruzipain Inhibitor (BbCI) is a cysteine protease inhibitor highly homologous to plant Kunitz-type inhibitors. However, in contrast to classical Kunitz family inhibitors it lacks cysteine residues and therefore disulfide bridges. BbCI is also distinct in the ability to inactivate enzymes belonging to two different classes, cysteine and serine proteases. Besides inhibiting the cysteine protease cruzipain, BbCI also inhibits cathepsin L and the serine proteases HNE (human neutrophil elastase) and PPE (porcine pancreatic elastase). Monoclinic crystals of the recombinant inhibitor that diffract to 1.7 A resolution were obtained using hanging drop method by vapor diffusion at 18 o C. The refined structure shows the conservative β-trefoil fold features of the Kunitz inhibitors. In BbCI, one of the two characteristic S-S bonds is replaced by the water-mediated interaction between Tyr125 and Gly132. In this work we explore the structural differences between Kunitz-type inhibitors and analyze the essential interactions that maintain the protein structural stability preserving its biological function

Primary breast cancer tumours contain high amounts of IgA1 immunoglobulin

DEFF Research Database (Denmark)

Welinder, Charlotte; Baldetorp, Bo; Blixt, Klas Ola

2013-01-01

seen in the percentage of stained cells and in the staining pattern in the different breast cancers analysed. Anti-Tn antibody and HPA were also shown to specifically bind to a number of possible constellations of the Tn antigen in the hinge region of IgA1. Both reagents could also detect the presence....... The short O-glycan that forms the antigen is carried by a number of different proteins. One potential carrier of the Tn antigen is immunoglobulin A1 (IgA1), which we surprisingly found in tumour cells of the invasive parts of primary breast carcinoma. Conventional immunohistochemical analysis of paraffin......-embedded sections from primary breast cancers showed IgA1 to be present in the cytoplasm and plasma membrane of 35 out of 36 individual primary tumours. The immunohistochemical staining of HPA and anti-Tn antibody (GOD3-2C4) did to some extent overlap with the presence of IgA1 in the tumours, but differences were...

[Analysis of Relationship between Serum Total Light Chain κ/λ Ratio and Proportion of Bone Marrow Plasma Cells in Patients with IgG type and IgA type Multiple Myeloma].

Science.gov (United States)

Zhu, An-You; Zhu, Fang-Bing; Wang, Feng-Chao; Zhang, Lun-Jun; Ma, Yue; Hu, Jian-Guo

2017-10-01

To explore the relationship between serum total light chain κ/λ ratio (sTLC-κ/λ) and proportion of bone marrow plasma cells (BMPC) in patients with IgG type and IgA type multiple myeloma (MM) and its clinical significance. The levels of serum IgG, IgA, κ type and λ type total light chain were detected in 79 newly diagnosed patients with IgG type (n=52) and IgA type (n=27) MM by immuno-nephelometric assay and the sTLC-κ/λ ratio was calculated. The proportion of BMPC was determined by bone marrow smears in the corresponding period, and the changes in sTLC-κ/λ ratio and the proportion of BMPC were observed in 19 patients with IgG type(n=16) and IgA type (n=3) MM undergoing treatment, 26 cases of non-phasmocytic proliferative diseases were enrolled in control group. In MM patients with IgGκ type and IgAκ type, the sTLC-κ/λ ratio was significantly higher than that in the control group (Pratio was significantly lower than that in the control group (Pratio was significantly higher than that in MM patients with IgAκ(Pratio in MM patients with IgGλ was significantly lower than that in MM patients with IgAλ. The sTLC-κ/λ ratios in MM patients with IgGκ and IgAκ were positively correlated with the concentrations of IgG (r=0.778,P=0.000) and IgA (r=0.601,P=0.039), while the sTLC-κ/λ ratios of patients with IgGλ and IgAλ were negativily correlated with the IgG(r=-0.586,P=0.01) and IgA level(r=-0.718,P=0.003). In addition, a correlation between each type MM was not found except the IgGκ type MM which had a positive correlation between the sTLC-κ/λ ratio and proportion of BMPC (r=0.579,P=0.002). Nonetheless, 18 of 19 patients with IgG type and IgA type MM undergoing treatment showed concordance between the sTLC-κ/λ ratio and proportion of BMPC change. There is a lower correlation between the sTLC-κ/λ ratio and the proportion of BMPC in MM patients with IgG type and IgA type, but there is a high concordance between the sTLC-κ/λ ratio and the

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

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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.

Autoprocessing of human immunodeficiency virus type 1 protease miniprecursor fusions in mammalian cells

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Chen Chaoping

2010-07-01

Full Text Available Abstract Background HIV protease (PR is a virus-encoded aspartic protease that is essential for viral replication and infectivity. The fully active and mature dimeric protease is released from the Gag-Pol polyprotein as a result of precursor autoprocessing. Results We here describe a simple model system to directly examine HIV protease autoprocessing in transfected mammalian cells. A fusion precursor was engineered encoding GST fused to a well-characterized miniprecursor, consisting of the mature protease along with its upstream transframe region (TFR, and small peptide epitopes to facilitate detection of the precursor substrate and autoprocessing products. In HEK 293T cells, the resulting chimeric precursor undergoes effective autoprocessing, producing mature protease that is rapidly degraded likely via autoproteolysis. The known protease inhibitors Darunavir and Indinavir suppressed both precursor autoprocessing and autoproteolysis in a dose-dependent manner. Protease mutations that inhibit Gag processing as characterized using proviruses also reduced autoprocessing efficiency when they were introduced to the fusion precursor. Interestingly, autoprocessing of the fusion precursor requires neither the full proteolytic activity nor the majority of the N-terminal TFR region. Conclusions We suggest that the fusion precursors provide a useful system to study protease autoprocessing in mammalian cells, and may be further developed for screening of new drugs targeting HIV protease autoprocessing.

Conserved hydrogen bonds and water molecules in MDR HIV-1 protease substrate complexes

Energy Technology Data Exchange (ETDEWEB)

Liu, Zhigang [Wayne State Univ., Detroit, MI (United States); Case Western Reserve Univ., Cleveland, OH (United States); Harbor Hospital Baltimore, MD (United States); Wang, Yong [Wayne State Univ., Detroit, MI (United States); Yedidi, Ravikiran S. [Wayne State Univ., Detroit, MI (United States); National Institutes of Health, Bethesda, MD (United States); Dewdney, Tamaria G. [Wayne State Univ., Detroit, MI (United States); Reiter, Samuel J. [Wayne State Univ., Detroit, MI (United States); Brunzelle, Joseph S. [Northwestern Univ. Feinberg School of Medicine, Chicago, IL (United States); Kovari, Iulia A. [Wayne State Univ., Detroit, MI (United States); Kovari, Ladislau C. [Wayne State Univ., Detroit, MI (United States)

2012-12-19

Success of highly active antiretroviral therapy (HAART) in anti-HIV therapy is severely compromised by the rapidly developing drug resistance. HIV-1 protease inhibitors, part of HAART, are losing their potency and efficacy in inhibiting the target. Multi-drug resistant (MDR) 769 HIV-1 protease (resistant mutations at residues 10, 36, 46, 54, 62, 63, 71, 82, 84, 90) was selected for the present study to understand the binding to its natural substrates. The nine crystal structures of MDR769 HIV-1 protease substrate hepta-peptide complexes were analyzed in order to reveal the conserved structural elements for the purpose of drug design against MDR HIV-1 protease. Our structural studies demonstrated that highly conserved hydrogen bonds between the protease and substrate peptides, together with the conserved crystallographic water molecules, played a crucial role in the substrate recognition, substrate stabilization and protease stabilization. Additionally, the absence of the key flap-ligand bridging water molecule might imply a different catalytic mechanism of MDR769 HIV-1 protease compared to that of wild type (WT) HIV-1 protease.

Structures of a bi-functional Kunitz-type STI family inhibitor of serine and aspartic proteases: Could the aspartic protease inhibition have evolved from a canonical serine protease-binding loop?

Science.gov (United States)

Guerra, Yasel; Valiente, Pedro A; Pons, Tirso; Berry, Colin; Rudiño-Piñera, Enrique

2016-08-01

Bi-functional inhibitors from the Kunitz-type soybean trypsin inhibitor (STI) family are glycosylated proteins able to inhibit serine and aspartic proteases. Here we report six crystal structures of the wild-type and a non-glycosylated mutant of the bifunctional inhibitor E3Ad obtained at different pH values and space groups. The crystal structures show that E3Ad adopts the typical β-trefoil fold of the STI family exhibiting some conformational changes due to pH variations and crystal packing. Despite the high sequence identity with a recently reported potato cathepsin D inhibitor (PDI), three-dimensional structures obtained in this work show a significant conformational change in the protease-binding loop proposed for aspartic protease inhibition. The E3Ad binding loop for serine protease inhibition is also proposed, based on structural similarity with a novel non-canonical conformation described for the double-headed inhibitor API-A from the Kunitz-type STI family. In addition, structural and sequence analyses suggest that bifunctional inhibitors of serine and aspartic proteases from the Kunitz-type STI family are more similar to double-headed inhibitor API-A than other inhibitors with a canonical protease-binding loop. Copyright © 2016. Published by Elsevier Inc.

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

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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 (pbacteria antigens and theirs virulence antigens. To SM, the GbpB was significantly lower detected than others antigens of SM (p0.4). 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.

Potent neutralizing serum immunoglobulin A (IgA) in human immunodeficiency virus type 2-exposed IgG-seronegative individuals

DEFF Research Database (Denmark)

Lizeng, Q; Nilsson, C; Sourial, S

2004-01-01

Links Potent neutralizing serum immunoglobulin A (IgA) in human immunodeficiency virus type 2-exposed IgG-seronegative individuals.Lizeng Q, Nilsson C, Sourial S, Andersson S, Larsen O, Aaby P, Ehnlund M, Bjorling E. Research Center, South Hospital, Stockholm, Sweden. The mechanisms behind...... the resistance to human immunodeficiency virus type 2 (HIV-2) infection are still not fully understood. In the present study, we explored the HIV-2-specific humoral serum immunoglobulin A (IgA) immune response in HIV-2-exposed IgG-seronegative (EGSN) individuals. Serum samples from heterosexual EGSN individuals...... and their known HIV-2-infected partners, as well as controls originating from Guinea-Bissau in Africa, were studied. Antibody reactivity to native and recombinant envelope glycoproteins was investigated, and the capacity of purified serum IgA to neutralize HIV-2(SBL6669) was tested. Our results showed that 16...

Differences in serum IgA responses to HIV-1 gp41 in elite controllers compared to viral suppressors on highly active antiretroviral therapy.

Directory of Open Access Journals (Sweden)

Rafiq Nabi

Full Text Available Mechanisms responsible for natural control of human immunodeficiency type 1 (HIV replication in elite controllers (EC remain incompletely defined. To determine if EC generate high quality HIV-specific IgA responses, we used Western blotting to compare the specificities and frequencies of IgA to HIV antigens in serum of gender-, age- and race-matched EC and aviremic controllers (HC and viremic noncontrollers (HN on highly active antiretroviral therapy (HAART. Concentrations and avidity of IgA to HIV antigens were measured using ELISA or multiplex assays. Measurements for IgG were performed in parallel. EC were found to have stronger p24- and V1V2-specific IgG responses than HN, but there were no IgG differences for EC and HC. In contrast, IgA in EC serum bound more frequently to gp160 and gag proteins than IgA in HC or HN. The avidity of anti-gp41 IgA was also greater in EC, and these subjects had stronger IgA responses to the gp41 heptad repeat region 1 (HR1, a reported target of anti-bacterial RNA polymerase antibodies that cross react with gp41. However, EC did not demonstrate greater IgA responses to E. coli RNA polymerase or to peptides containing the shared LRAI sequence, suggesting that most of their HR1-specific IgA antibodies were not induced by intestinal microbiota. In both EC and HAART recipients, the concentrations of HIV-specific IgG were greater than HIV-specific IgA, but their avidities were comparable, implying that they could compete for antigen. Exceptions were C1 peptides and V1V2 loops. IgG and IgA responses to these antigens were discordant, with IgG reacting to V1V2, and IgA reacting to C1, especially in EC. Interestingly, EC with IgG hypergammaglobulinemia had greater HIV-specific IgA and IgG responses than EC with normal total IgG levels. Heterogeneity in EC antibody responses may therefore be due to a more focused HIV-specific B cell response in some of these individuals. Overall, these data suggest that development of

Natural inhibitors of tumor-associated proteases

International Nuclear Information System (INIS)

Magdolen, U.; Krol, J.; Sato, S.; Schmitt, M.; Magdolen, V.; Krueger, A.; Mueller, M.M.; Sperl, S.

2002-01-01

The turnover and remodelling of extracellular matrix (ECM) is an essential part of many normal biological processes including development, morphogenesis, and wound healing. ECM turnover also occurs in severe pathological situations like artherosclerosis, fibrosis, tumor invasion and metastasis. The major proteases involved in this turnover are serine proteases (especially the urokinase-type plasminogen activator/plasmin system), matrix metalloproteases (a family of about 20 zinc-dependent endopeptidases including collagenases, gelatinases, stromelysins, and membrane-type metalloproteases), and cysteine proteases. In vivo, the activity of these proteases is tightly regulated in the extracellular space by zymogen activation and/or controlled inhibition. In the present review, we give an overview on the structure and biochemical properties of important tumor-associated protease inhibitors such as plasminogen activator inhibitor type 1 and type 2 (PAI-1, PAI-2), tissue inhibitors of metalloproteinases (TIMP-1, -2, -3, and -4), and the cysteine protease inhibitor cystatin C. Interestingly, some of these inhibitors of tumor-associated proteases display multiple functions which rather promote than inhibit tumor progression, when the presence of inhibitors in the tumor tissue is not balanced. (author)

Intra-tumour IgA1 is common in cancer and is correlated with poor prognosis in bladder cancer.

Directory of Open Access Journals (Sweden)

Charlotte Welinder

2016-08-01

Full Text Available A high frequency of IgA1-positive tumour cells was found in tissue micro-arrays of oesophagus, colon, testis, lung, breast, bladder and ovarian cancer. IgA1 was observed in the cytoplasm and the plasma membrane. A correlation was found between intra-tumour IgA1 and poor overall survival in a large cohort of bladder cancer patients (n = 99, p = 0.011, log-rank test. The number of IgA1-positive tumour cells was also found to be higher in female than male bladder cancer patients. The presence of IgA1 was confirmed in formalin-fixed paraffin-embedded ovarian carcinoma samples using LC-MS/MS analysis. Uptake of IgA1 was also observed in breast cancer and melanoma cell lines when cultivated in the presence of serum from healthy individuals, indicating a possible origin of the IgA1 antibodies in cancer cells.

Curcumin derivatives as HIV-1 protease inhibitors

Energy Technology Data Exchange (ETDEWEB)

Sui, Z.; Li, J.; Craik, C.S.; Ortiz de Montellano, P.R. [Univ. of California, San Francisco, CA (United States)

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.

Characterization of IgA response among women with incident HPV 16 infection

International Nuclear Information System (INIS)

Onda, Takashi; Carter, Joseph J.; Koutsky, Laura A.; Hughes, James P.; Lee, Shu-Kuang; Kuypers, Jane; Kiviat, Nancy; Galloway, Denise A.

2003-01-01

Previous studies have characterized the prevalence and duration of serum IgG antibodies to human papillomavirus type 16 (HPV 16) in a well-studied cohort of college women, using viruslike particle- (VLP) based ELISAs. In this study IgA antibodies in cervical secretions and sera were examined using a newly developed capsomer-based ELISA and the patterns observed for serum IgG, serum IgA, and cervical IgA antibodies were compared. The median time to antibody detection from the first detection of HPV 16 DNA was 10.5 months for IgA in cervical secretions and 19.1 months for serum IgA. Serum IgA antibody conversion was observed less frequently and occurred later than IgA conversion in cervical secretions (P = 0.011) or serum IgG conversion (P 0.051). The median time to antibody reversion, following seroconversion, was 12.0 months for IgA in cervical secretions and 13.6 months for serum IgA, whereas approximately 20% of women with serum IgG antibodies reverted within 36 months. Thus, the duration of IgA in cervical secretions and sera was shorter than the duration of serum IgG (P = 0.007 and 0.001)

Type-specific IgG and IgA antibodies in old lymphogranuloma venerum determined by solid-phase radioimmunoassay

Energy Technology Data Exchange (ETDEWEB)

Meurman, O.; Terho, P.; Sonck, C.E.

1981-01-01

A solid-phase radioimmunoassay (RIA) using egg-grown purified Chlamydia trachomatis lymphogranuloma venereum (LGV) serotypes L1, L2, and L3 as antigen was used to measure type-specific IgG and IgA antibodies in sera of 36 patients who had contracted LGV infection about 40 years ago. The RIA test gave compatible results with the standard microimmunofluorescence test, and by RIA it was possible to identify the infecting serotype in 30 out of 36 patients studied. In 28 cases this was L2 and in two cases L1. Each patient had IgG antibodies and most of them (80%) IgA antibodies to at least one of the LGV serotypes. The antibody titers were still high 40 years after the acute infection, being higher than in male patients with a recent chlamydial urethritis. Highest antibody titers were detected in LGV patients who had a severe disease with intestinal involvement.

Type-specific IgG and IgA antibodies in old lymphogranuloma venerum determined by solid-phase radioimmunoassay

International Nuclear Information System (INIS)

Meurman, O.; Terho, P.; Sonck, C.E.

1981-01-01

A solid-phase radioimmunoassay (RIA) using egg-grown purified Chlamydia trachomatis lymphogranuloma venereum (LGV) serotypes L1, L2, and L3 as antigen was used to measure type-specific IgG and IgA antibodies in sera of 36 patients who had contracted LGV infection about 40 years ago. The RIA test gave compatible results with the standard microimmunofluorescence test, and by RIA it was possible to identify the infecting serotype in 30 out of 36 patients studied. In 28 cases this was L2 and in two cases L1. Each patient had IgG antibodies and most of them (80%) IgA antibodies to at least one of the LGV serotypes. The antibody titers were still high 40 years after the acute infection, being higher than in male patients with a recent chlamydial urethritis. Highest antibody titers were detected in LGV patients who had a severe disease with intestinal involvement. (orig.)

Loss of second and sixth conserved cysteine residues from trypsin inhibitor-like cysteine-rich domain-type protease inhibitors in Bombyx mori may induce activity against microbial proteases.

Science.gov (United States)

Li, Youshan; Liu, Huawei; Zhu, Rui; Xia, Qingyou; Zhao, Ping

2016-12-01

Previous studies have indicated that most trypsin inhibitor-like cysteine-rich domain (TIL)-type protease inhibitors, which contain a single TIL domain with ten conserved cysteines, inhibit cathepsin, trypsin, chymotrypsin, or elastase. Our recent findings suggest that Cys 2nd and Cys 6th were lost from the TIL domain of the fungal-resistance factors in Bombyx mori, BmSPI38 and BmSPI39, which inhibit microbial proteases and the germination of Beauveria bassiana conidia. To reveal the significance of these two missing cysteines in relation to the structure and function of TIL-type protease inhibitors in B. mori, cysteines were introduced at these two positions (D36 and L56 in BmSPI38, D38 and L58 in BmSPI39) by site-directed mutagenesis. The homology structure model of TIL domain of the wild-type and mutated form of BmSPI39 showed that two cysteine mutations may cause incorrect disulfide bond formation of B. mori TIL-type protease inhibitors. The results of Far-UV circular dichroism (CD) spectra indicated that both the wild-type and mutated form of BmSPI39 harbored predominantly random coil structures, and had slightly different secondary structure compositions. SDS-PAGE and Western blotting analysis showed that cysteine mutations affected the multimerization states and electrophoretic mobility of BmSPI38 and BmSPI39. Activity staining and protease inhibition assays showed that the introduction of cysteine mutations dramaticly reduced the activity of inhibitors against microbial proteases, such as subtilisin A from Bacillus licheniformis, protease K from Engyodontium album, protease from Aspergillus melleus. We also systematically analyzed the key residue sites, which may greatly influence the specificity and potency of TIL-type protease inhibitors. We found that the two missing cysteines in B. mori TIL-type protease inhibitors might be crucial for their inhibitory activities against microbial proteases. The genetic engineering of TIL-type protease inhibitors may be

Three monoclonal antibodies against the serpin protease nexin-1 prevent protease translocation

DEFF Research Database (Denmark)

Kousted, Tina Mostrup; Skjoedt, K; Petersen, S V

2013-01-01

abolish the protease inhibitory activity of PN-1. In the presence of the antibodies, PN-1 does not form a complex with its target proteases, but is recovered in a reactive centre cleaved form. Using site-directed mutagenesis, we mapped the three overlapping epitopes to an area spanning the gap between...

Protease-activated receptor-1 impairs host defense in murine pneumococcal pneumonia: a controlled laboratory study

NARCIS (Netherlands)

Schouten, Marcel; van't Veer, Cornelis; Roelofs, Joris J. T. H.; Levi, Marcel; van der Poll, Tom

2012-01-01

Streptococcus pneumoniae is the most common causative pathogen in community-acquired pneumonia. Protease-activated receptor-1 (PAR-1) is expressed by multiple cell types present in the lungs and can be activated by various proteases generated during acute inflammation. The cellular effect of PAR-1

Salivary IgA concentration in diabetic patients compared to healthy controls

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Farimah Sardari

2015-01-01

Full Text Available Introduction: The alterations in salivary flow rate and its compositions could affect the development, symptoms, and severity of oral changes in diabetic patients. The aim of this study was to assess the concentration of salivary IgA in type I in comparison with type II diabetic patients and healthy controls. Materials and Methods: In this cross-sectional study, 25 patients with type I diabetes, 25 patients with type II diabetes, and 25 control subjects (12 subjects for the type I and 13 subjects for the type II were enrolled. Unstimulated saliva samples were collected by spitting method and the concentration of salivary IgA was measured byenzyme-linked immunosorbent assay (ELISA method. Results: The mean of salivary IgA in type I diabetic patients was 148.3 ± 38.7 μg/ml and in their controls was 65.8 ± 17.4 μg/ml (P < 0.001. In type II diabetic patients the mean of salivary IgA was 67.3 ± 20.6 μg/ml and in their controls was 63.3 ± 15.2 μg/ml. There was no significant difference between patients with type II diabetes and controls (P = 0.54. The mean of salivary IgA in patients with type I diabetes was significantly higher than in patients with type II diabetes (148.3 ± 38.7 versus 67.3 ± 20.6 μg/ml, respectively, P < 0.001. Conclusions: Level of salivary IgA in type II diabetic patients in comparison with their healthy control did not show any significant difference, but in type I diabetic patients was higher than that of healthy controls and type II diabetic patients.

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

International Nuclear Information System (INIS)

Tokunaga, Koki; Uto, Hirofumi; Takami, Yoichiro; Mera, Kumiko; Nishida, Chika; Yoshimine, Yozo; Fukumoto, Mayumi; Oku, Manei; Sogabe, Atsushi; Nosaki, Tsuyoshi; Moriuchi, Akihiro; Oketani, Makoto; Ido, Akio; Tsubouchi, Hirohito

2010-01-01

Research highlights: → IGFBP-1 mRNA over express in kidneys obtained from mice model of IgA nephropathy. → Serum IGFBP-1 levels are high in patients with IgA nephropathy. → 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, 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.

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

Energy Technology Data Exchange (ETDEWEB)

Tokunaga, Koki [Department of Digestive and Life-Style Related Disease, Health Research Course, Human and Environmental Sciences, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8520 (Japan); Uto, Hirofumi, E-mail: hirouto@m2.kufm.kagoshima-u.ac.jp [Department of Digestive and Life-Style Related Disease, Health Research Course, Human and Environmental Sciences, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8520 (Japan); Takami, Yoichiro; Mera, Kumiko; Nishida, Chika; Yoshimine, Yozo; Fukumoto, Mayumi; Oku, Manei; Sogabe, Atsushi; Nosaki, Tsuyoshi; Moriuchi, Akihiro; Oketani, Makoto; Ido, Akio; Tsubouchi, Hirohito [Department of Digestive and Life-Style Related Disease, Health Research Course, Human and Environmental Sciences, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8520 (Japan)

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, 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.

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.

Science.gov (United States)

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.

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

Science.gov (United States)

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; Schemann, Michael

2018-01-01

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). 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. 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. 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 IBS, and protease profiling may be used to

Recombinant human immunoglobulin (Ig)A1 and IgA2 anti-D used for detection of IgA deficiency and anti-IgA

DEFF Research Database (Denmark)

Nielsen, Leif K; Dziegiel, Morten Hanefeld

2008-01-01

To avoid anaphylactic reactions, immunoglobulin (Ig)A-deficient patients with anti-IgA should be transfused with IgA-deficient blood components. There is a need for fast and robust assays for demonstration of IgA deficiency and for detection of anti-IgA.......To avoid anaphylactic reactions, immunoglobulin (Ig)A-deficient patients with anti-IgA should be transfused with IgA-deficient blood components. There is a need for fast and robust assays for demonstration of IgA deficiency and for detection of anti-IgA....

Structure of HIV-1 protease determined by neutron crystallography

International Nuclear Information System (INIS)

Adachi, Motoyasu; Kuroki, Ryota

2009-01-01

HIV-1 protease is an aspartic protease, and plays an essential role in replication of HIV. To develop HIV-1 protease inhibitors through structure-based drug design, it is necessary to understand the catalytic mechanism and inhibitor recognition of HIV-1 protease. We have determined the crystal structure of HIV-1 protease in complex with KNI-272 to 1.9 A resolution by neutron crystallography in combination with 1.4 A resolution X-ray diffraction data. The results show that the carbonyl group of hydroxymethylcarbonyl (HMC) in KNI-272 forms a hydrogen bonding interaction with protonated Asp 25 and the hydrogen atom from the hydroxyl group of HMC forms a hydrogen bonding interaction with the deprotonated Asp125. This is the first neutron report for HIV-1/inhibitor complex and shows directly the locations of key hydrogen atoms in catalysis and in the binding of a transition-state analog. The results confirm key aspect of the presumed catalytic mechanism of HIV-1 protease and will aid in the further development of protease inhibitors. (author)

Cell-based fluorescence assay for human immunodeficiency virus type 1 protease activity

Czech Academy of Sciences Publication Activity Database

Lindsten, K.; Kondrová, Taťána; Konvalinka, Jan; Masucci, M. G.; Dantuma, N. P.

2001-01-01

Roč. 45, č. 9 (2001), s. 2616-2622 ISSN 0066-4804 R&D Projects: GA ČR GA303/98/1559 Grant - others:HHMI(US) 75195-540801; ECTM(XE) ERBFMRXCT960026 Institutional research plan: CEZ:AV0Z4055905 Keywords : HIV-1 * protease activity Subject RIV: CE - Biochemistry Impact factor: 4.562, year: 2001

Reactivities of N-acetylgalactosamine-specific lectins with human IgA1 proteins

DEFF Research Database (Denmark)

Moore, J.S.; Kulhavy, R.; Tomana, M.

2007-01-01

, VV reacted with sugars of both IgA subclasses and IgG, indicating that it also recognized N-linked glycans without GalNAc. Furthermore, HAA and HPA from several manufacturers differed in their ability to bind various IgA1 myeloma proteins and other GalNAc-containing glycoproteins in ELISA and Western...

Circulating immune complexes, immunoglobulin classes (IgG, IgA ...

African Journals Online (AJOL)

Objective:- To evaluate serum levels of circulating immune complexes (CICs), immunoglobulin classes (IgG, IgA and IgM) and Complement Components (C3c, C4 and Factor B) in Nigerians with Type 1 or Type 2 diabetes mellitus. Design:- Case control study. Setting:- University College Hospital, Ibadan, Oyo State, Nigeria.

Uncoupling of glomerular IgA deposition and disease progression in alymphoplasia mice with IgA nephropathy.

Directory of Open Access Journals (Sweden)

Masashi Aizawa

Full Text Available Previous clinical and experimental studies have indicated that cells responsible for IgA nephropathy (IgAN, at least in part, are localized in bone marrow (BM. Indeed, we have demonstrated that murine IgAN can be experimentally reconstituted by bone marrow transplantation (BMT from IgAN prone mice in not only normal mice, but also in alymphoplasia mice (aly/aly independent of IgA+ cells homing to mucosa or secondary lymphoid tissues. The objective of the present study was to further assess whether secondary lymph nodes (LN contribute to the progression of this disease. BM cells from the several lines of IgAN prone mice were transplanted into aly/aly and wild-type mice (B6. Although the transplanted aly/aly showed the same degree of mesangial IgA and IgG deposition and the same serum elevation levels of IgA and IgA-IgG immune-complexes (IC as B6, even in extent, the progression of glomerular injury was observed only in B6. This uncoupling in aly/aly was associated with a lack of CD4+ T cells and macrophage infiltration, although phlogogenic capacity to nephritogenic IC of renal resident cells was identical between both recipients. It is suggested that secondary LN may be required for the full progression of IgAN after nephritogenic IgA and IgA/IgG IC deposition.

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

Science.gov (United States)

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.

Enterovirus type 71 2A protease functions as a transcriptional activator in yeast

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Lai Meng-Jiun

2010-08-01

Full Text Available Abstract Enterovirus type 71 (EV71 2A protease exhibited strong transcriptional activity in yeast cells. The transcriptional activity of 2A protease was independent of its protease activity. EV71 2A protease retained its transcriptional activity after truncation of 40 amino acids at the N-terminus but lost this activity after truncation of 60 amino acids at the N-terminus or deletion of 20 amino acids at the C-terminus. Thus, the acidic domain at the C-terminus of this protein is essential for its transcriptional activity. Indeed, deletion of amino acids from 146 to 149 (EAME in this acidic domain lost the transcriptional activity of EV71 2A protein though still retained its protease activity. EV71 2A protease was detected both in the cytoplasm and nucleus using confocal microscopy analysis. Coxsackie virus B3 2A protease also exhibited transcriptional activity in yeast cells. As expected, an acidic domain in the C-terminus of Coxsackie virus B3 2A protease was also identified. Truncation of this acidic domain resulted in the loss of transcriptional activity. Interestingly, this acidic region of poliovirus 2A protease is critical for viral RNA replication. The transcriptional activity of the EV71 or Coxsackie virus B3 2A protease should play a role in viral replication and/or pathogenesis.

Interleukin-6-deficient mice refractory to IgA dysregulation but not anorexia induction by vomitoxin (deoxynivalenol) ingestion.

Science.gov (United States)

Pestka, J J; Zhou, H R

2000-07-01

Dietary exposure to the trichothecene vomitoxin (VT) causes feed refusal and elevates IgA production in the mouse. Based on the observations that IL-6 can cause anorexia and promote IgA production and that gene expression of this cytokine is increased in vivo and ex vivo on VT exposure, we hypothesized that IL-6 is an essential cytokine in VT-induced feed refusal and IgA dysregulation. To test this hypothesis, the effects of dietary VT on feed intake, weight gain, serum IgA levels and kidney mesangial IgA deposition in an IL-6-"knockout" mouse (B6129-IL6(tmi Kopf)) were compared to those in both a corresponding "wildtype" (B6129F2) and a previously characterized "sentinel" strain (B6C3F1) that possess the intact gene for this cytokine. IL-6 deficiency did not alter the capacity of VT to cause feed refusal or impair weight gain. VT-fed B6129F2 and B6C3F1 mice had significantly higher serum IgA concentrations than did their corresponding controls fed clean diet, whereas significant differences were not observed between IL-6 KO mice fed VT or control diets. Kidneys taken from VT-fed wild-type and sentinel mice had significantly increased mesangial IgA deposition as compared to controls. While slight increases in mesangial IgA were observed in VT-fed IL-6 KO mice, mean fluorescence intensities were significantly less than that found in the corresponding wild-type and sentinel strains. IL-6 KO mice appeared to be less prone to the development of microscopic haematuria following VT exposure than were the corresponding wild-type and sentinel strains. In total, the results suggested that IL-6-deficient mice were refractory to VT-induced dysregulation of IgA production and development of IgA nephropathy, whereas chronic VT-mediated nutritional effects related to feed intake and weight gain were unaffected.

Identification of an archaeal presenilin-like intramembrane protease.

Science.gov (United States)

Torres-Arancivia, Celia; Ross, Carolyn M; Chavez, Jose; Assur, Zahra; Dolios, Georgia; Mancia, Filippo; Ubarretxena-Belandia, Iban

2010-09-29

The GXGD-type diaspartyl intramembrane protease, presenilin, constitutes the catalytic core of the γ-secretase multi-protein complex responsible for activating critical signaling cascades during development and for the production of β-amyloid peptides (Aβ) implicated in Alzheimer's disease. The only other known GXGD-type diaspartyl intramembrane proteases are the eukaryotic signal peptide peptidases (SPPs). The presence of presenilin-like enzymes outside eukaryots has not been demonstrated. Here we report the existence of presenilin-like GXGD-type diaspartyl intramembrane proteases in archaea. We have employed in vitro activity assays to show that MCMJR1, a polytopic membrane protein from the archaeon Methanoculleus marisnigri JR1, is an intramembrane protease bearing the signature YD and GXGD catalytic motifs of presenilin-like enzymes. Mass spectrometry analysis showed MCMJR1 could cleave model intramembrane protease substrates at several sites within their transmembrane region. Remarkably, MCMJR1 could also cleave substrates derived from the β-amyloid precursor protein (APP) without the need of protein co-factors, as required by presenilin. Two distinct cleavage sites within the transmembrane domain of APP could be identified, one of which coincided with Aβ40, the predominant site processed by γ-secretase. Finally, an established presenilin and SPP transition-state analog inhibitor could inhibit MCMJR1. Our findings suggest that a primitive GXGD-type diaspartyl intramembrane protease from archaea can recapitulate key biochemical properties of eukaryotic presenilins and SPPs. MCMJR1 promises to be a more tractable, simpler system for in depth structural and mechanistic studies of GXGD-type diaspartyl intramembrane proteases.

IGA/SCC propagation rate measurements on alloy 600 steam generator tubing using a side stream model boiler

International Nuclear Information System (INIS)

Takamatsu, H.; Matsueda, K.; Matsunaga, T.; Kitera, T.; Arioka, K.; Tsuruta, T.; Okamoto, S.

1993-01-01

IGA/SCC crack propagation rate measurements using various types of IGA/SCC predefected ALloy 600 tubing were tested in model boilers, a side stream model boiler at Ohi Unit 1 and similar model boilers in the laboratory. Types of IGA/SCC predefects introduced from the outside of the tubing were as follows. (1) Actual IGA/SCC predefect introduced by high temperature caustic environments; (2) Longitudinal predefect by electrodischarge machining (EDM) method, and then crack tip fatigue was introduced to serve as the marker on the fractured surface (EDM slit + fatigue). IGA/SCC crack propagation rate was measured after the destructive examination by Cr concentration profile on fracture surface for (1), and observation of intergranular fractured surface propagated from the marked fatigue was employed for (2) and (3) after the model boiler tests. As for the water chemistry conditions, mainly AVT (high N 2 H 4 ) + boric acid (5-10ppm as B in SGs) treatment for both model boilers, and some of the tests for the model boiler in the laboratory employed AVT (high N 2 H 4 ) without boric acid. The results of IGA/SCC crack propagation rate measurements were compared with each other, and the three methods employed showed a good coincidence with the rate of ca. 1 x 10 -5 mm/Hr for AVT (high N 2 H 4 ) + boric acid treatment condition, in the case that crack tip boron intensity (B/O value by IMMA analysis) of more than 1 was observed

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

Science.gov (United States)

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 (Pdiabetic patients than the controls. Determination of salivary constituents may be useful in the description and management of oral findings in diabetic patients.

GS-8374, a Prototype Phosphonate-Containing Inhibitor of HIV-1 Protease, Effectively Inhibits Protease Mutants with Amino Acid Insertions

Czech Academy of Sciences Publication Activity Database

Grantz Šašková, Klára; Kožíšek, Milan; Stray, K.; Jong de, D.; Řezáčová, Pavlína; Brynda, Jiří; Maarseveen van, N. M.; Nijhuis, M.; Cihlář, T.; Konvalinka, Jan

2014-01-01

Roč. 88, č. 6 (2014), s. 3586-3590 ISSN 0022-538X R&D Projects: GA ČR GAP207/11/1798 Grant - others:OPPC(XE) CZ.2.16/3.1.00/24016 Institutional support: RVO:61388963 ; RVO:68378050 Keywords : virus type-1 protease * antiviral activity * drug resistance Subject RIV: EE - Microbiology, Virology Impact factor: 4.439, year: 2014

Identification of an archaeal presenilin-like intramembrane protease.

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Celia Torres-Arancivia

Full Text Available BACKGROUND: The GXGD-type diaspartyl intramembrane protease, presenilin, constitutes the catalytic core of the γ-secretase multi-protein complex responsible for activating critical signaling cascades during development and for the production of β-amyloid peptides (Aβ implicated in Alzheimer's disease. The only other known GXGD-type diaspartyl intramembrane proteases are the eukaryotic signal peptide peptidases (SPPs. The presence of presenilin-like enzymes outside eukaryots has not been demonstrated. Here we report the existence of presenilin-like GXGD-type diaspartyl intramembrane proteases in archaea. METHODOLOGY AND PRINCIPAL FINDINGS: We have employed in vitro activity assays to show that MCMJR1, a polytopic membrane protein from the archaeon Methanoculleus marisnigri JR1, is an intramembrane protease bearing the signature YD and GXGD catalytic motifs of presenilin-like enzymes. Mass spectrometry analysis showed MCMJR1 could cleave model intramembrane protease substrates at several sites within their transmembrane region. Remarkably, MCMJR1 could also cleave substrates derived from the β-amyloid precursor protein (APP without the need of protein co-factors, as required by presenilin. Two distinct cleavage sites within the transmembrane domain of APP could be identified, one of which coincided with Aβ40, the predominant site processed by γ-secretase. Finally, an established presenilin and SPP transition-state analog inhibitor could inhibit MCMJR1. CONCLUSIONS AND SIGNIFICANCE: Our findings suggest that a primitive GXGD-type diaspartyl intramembrane protease from archaea can recapitulate key biochemical properties of eukaryotic presenilins and SPPs. MCMJR1 promises to be a more tractable, simpler system for in depth structural and mechanistic studies of GXGD-type diaspartyl intramembrane proteases.

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

Energy Technology Data Exchange (ETDEWEB)

Yang, Man [Department of Nephrology, The First Affiliated Hospital of Chengdu Medical College, Chengdu City 610500 (China); Li, Fu-gang [Department of Nephrology, Affiliated Hospital of Luzhou Medical College, Luzhou City 646000 (China); Xie, Xi-sheng [Department of Nephrology, Second Clinical Medical Institution of North Sichuan Medical College (Nanchong Central Hospital), Nanchong City 637400 (China); Wang, Shao-qing [Department of Nephrology, The First Affiliated Hospital of Chengdu Medical College, Chengdu City 610500 (China); Fan, Jun-ming, E-mail: junmingfan@163.com [Department of Nephrology, The First Affiliated Hospital of Chengdu Medical College, Chengdu City 610500 (China); Department of Nephrology, Affiliated Hospital of Luzhou Medical College, Luzhou City 646000 (China)

2014-02-07

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 IgA1 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.

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

International Nuclear Information System (INIS)

Yang, Man; Li, Fu-gang; Xie, Xi-sheng; Wang, Shao-qing; Fan, Jun-ming

2014-01-01

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 IgA1 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

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

Science.gov (United States)

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

Effect of sorghum type and malting on production of free amino nitrogen in conjunction with exogenous protease enzymes.

Science.gov (United States)

Dlamini, Bhekisisa C; Buys, Elna M; Taylor, John R N

2015-01-01

Sorghum types suitable for brewing and bioethanol production are required. The effect of sorghum type (white non-tannin versus white type II tannin) on free amino nitrogen (FAN) production from sorghum grain and malt using exogenous protease enzymes was investigated over extended incubation at moderate temperature (45 °C). With grain in the absence of exogenous proteases, white non-tannin sorghum produced substantially higher levels of FAN than white type II tannin sorghum, due to the tannins in the latter. Incubating sorghum grain with neutral proteinase and amino-peptidase in combination improved FAN production. The two sorghum types produced similar FAN levels when malted and incubated in the absence of the exogenous proteases. When both sorghums were malted and incubated with neutral proteinase alone substantially more FAN yield (124-126 mg 100 g(-1)) occurred than with grains (61-84 mg 100 g(-1)). The combination of amino-peptidase and proteinase did not improve FAN further. Neither, did malting influence wort free amino acid profile. Group B amino acids constituted the highest percentage (42-47%). With grain, white non-tannin sorghum plus proteinase and amino-peptidase yields the highest FAN, with malt both white non-tannin and white type II tannin sorghums plus proteinase yield the highest FAN. © 2014 Society of Chemical Industry.

Cold denaturation of the HIV-1 protease monomer

DEFF Research Database (Denmark)

Rösner, Heike Ilona; Caldarini, Martina; Prestel, Andreas

2017-01-01

The HIV-1-protease is a complex protein which in its active form adopts a homodimer dominated by -sheet structures. We have discovered a cold-denatured state of the monomeric subunit of HIV-1-protease which is populated above 0ºC and therefore directly accessible to various spectroscopic approac...

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

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Özlem Ertekin

2016-08-01

Full Text Available 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.

Evaluation of Salivary Glucose, IgA and Flow Rate in Diabetic Patients: A Case-Control Study

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P. Bakianian Vaziri

2010-03-01

Full Text Available Objective: 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.Materials and Methods: 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. Nephelometricand Pars method were used to measure salivary IgA and salivary glucose concentrations,respectively. Statistical analysis was performed by Chi-square and t test.Results: 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.Conclusion: Determination of salivary constituents may be useful in the description and management of oral findings in diabetic patients.

The kinetics of glomerular deposition of nephritogenic IgA.

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Kenji Yamaji

Full Text Available Whether IgA nephropathy is attributable to mesangial IgA is unclear as there is no correlation between intensity of deposits and extent of glomerular injury and no clear mechanism explaining how these mesangial deposits induce hematuria and subsequent proteinuria. This hinders the development of a specific therapy. Thus, precise events during deposition still remain clinical challenge to clarify. Since no study assessed induction of IgA nephropathy by nephritogenic IgA, we analyzed sequential events involving nephritogenic IgA from IgA nephropathy-prone mice by real-time imaging systems. Immunofluorescence and electron microscopy showed that serum IgA from susceptible mice had strong affinity to mesangial, subepithelial, and subendothelial lesions, with effacement/actin aggregation in podocytes and arcade formation in endothelial cells. The deposits disappeared 24-h after single IgA injection. The data were supported by a fluorescence molecular tomography system and real-time and 3D in vivo imaging. In vivo imaging showed that IgA from the susceptible mice began depositing along the glomerular capillary from 1 min and accumulated until 2-h on the first stick in a focal and segmental manner. The findings indicate that glomerular IgA depositions in IgAN may be expressed under the balance between deposition and clearance. Since nephritogenic IgA showed mesangial as well as focal and segmental deposition along the capillary with acute cellular activation, all glomerular cellular elements are a plausible target for injury such as hematuria.

Kinetic intermediates en route to the final serpin-protease complex: studies of complexes of α1-protease inhibitor with trypsin.

Science.gov (United States)

Maddur, Ashoka A; Swanson, Richard; Izaguirre, Gonzalo; Gettins, Peter G W; Olson, Steven T

2013-11-01

Serpin protein protease inhibitors inactivate their target proteases through a unique mechanism in which a major serpin conformational change, resulting in a 70-Å translocation of the protease from its initial reactive center loop docking site to the opposite pole of the serpin, kinetically traps the acyl-intermediate complex. Although the initial Michaelis and final trapped acyl-intermediate complexes have been well characterized structurally, the intermediate stages involved in this remarkable transformation are not well understood. To better characterize such intermediate steps, we undertook rapid kinetic studies of the FRET and fluorescence perturbation changes of site-specific fluorophore-labeled derivatives of the serpin, α1-protease inhibitor (α1PI), which report the serpin and protease conformational changes involved in transforming the Michaelis complex to the trapped acyl-intermediate complex in reactions with trypsin. Two kinetically resolvable conformational changes were observed in the reactions, ascribable to (i) serpin reactive center loop insertion into sheet A with full protease translocation but incomplete protease distortion followed by, (ii) full conformational distortion and movement of the protease and coupled serpin conformational changes involving the F helix-sheet A interface. Kinetic studies of calcium effects on the labeled α1PI-trypsin reactions demonstrated both inactive and low activity states of the distorted protease in the final complex that were distinct from the intermediate distorted state. These studies provide new insights into the nature of the serpin and protease conformational changes involved in trapping the acyl-intermediate complex in serpin-protease reactions and support a previously proposed role for helix F in the trapping mechanism.

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

Science.gov (United States)

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.

HIV-1 protease-induced apoptosis

Czech Academy of Sciences Publication Activity Database

Rumlová, Michaela; Křížová, Ivana; Keprová, Alena; Hadravová, Romana; Doležal, Michal; Strohalmová, Karolína; Pichová, Iva; Hájek, Miroslav; Ruml, T.

2014-01-01

Roč. 11, May 20 (2014), 37/1-37/15 ISSN 1742-4690 R&D Projects: GA ČR GA204/09/1388 Institutional support: RVO:61388963 Keywords : HIV protease * BCA3 * AKIP-1 * apoptosis * mitochondria Subject RIV: EE - Microbiology, Virology Impact factor: 4.185, year: 2014 http://www.retrovirology.com/content/11/1/37

Interaction of protein C inhibitor with the type II transmembrane serine protease enteropeptidase.

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Thomas A Prohaska

Full Text Available The serine protease inhibitor protein C inhibitor (PCI is expressed in many human tissues and exhibits broad protease reactivity. PCI binds glycosaminoglycans and certain phospholipids, which modulate its inhibitory activity. Enteropeptidase (EP is a type II transmembrane serine protease mainly found on the brush border membrane of epithelial cells in the duodenum, where it activates trypsinogen to initiate the digestion of food proteins. Some active EP is also present in duodenal fluid and has been made responsible for causing pancreatitis in case of duodeno-pancreatic reflux. Together with its substrate trypsinogen, EP is furthermore present in the epidermis and in some cancer cells. In this report, we show that PCI inhibited EP with an apparent 2nd order rate constant of 4.48 × 10(4 M(-1 s(-1. Low molecular weight (LMWH and unfractionated heparin (UFH slightly reduced the inhibitory effect of PCI. The SI (stoichiometry of inhibition value for the inhibition of EP by PCI was 10.8 in the absence and 17.9 in the presence of UFH (10 U/ml. By inhibiting trypsin, chymotrypsin, and additionally EP, PCI might play a role in the protection of the pancreas from autodigestion. Furthermore the interaction of PCI with EP may influence the regulation of epithelial differentiation.

Isolated lymphoid follicles are not IgA inductive sites for recombinant Salmonella

International Nuclear Information System (INIS)

Hashizume, Tomomi; Momoi, Fumiki; Kurita-Ochiai, Tomoko; Kaminogawa, Shuichi; Hosono, Akira; Kataoka, Kosuke; Shinozaki-Kuwahara, Noriko; Kweon, Mi-Na; Yamamoto, Masafumi

2007-01-01

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-α-deficient (LTα -/- ) 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. Interestingly, no IgA anti-LPS Abs were detected in the fecal extracts of LTα -/- 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

Interdependence of Inhibitor Recognition in HIV-1 Protease.

Science.gov (United States)

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.

Molecular Basis for Drug Resistance in HIV-1 Protease

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Celia A. Schiffer

2010-11-01

Full Text Available HIV-1 protease is one of the major antiviral targets in the treatment of patients infected with HIV-1. The nine FDA approved HIV-1 protease inhibitors were developed with extensive use of structure-based drug design, thus the atomic details of how the inhibitors bind are well characterized. From this structural understanding the molecular basis for drug resistance in HIV-1 protease can be elucidated. Selected mutations in response to therapy and diversity between clades in HIV-1 protease have altered the shape of the active site, potentially altered the dynamics and even altered the sequence of the cleavage sites in the Gag polyprotein. All of these interdependent changes act in synergy to confer drug resistance while simultaneously maintaining the fitness of the virus. New strategies, such as incorporation of the substrate envelope constraint to design robust inhibitors that incorporate details of HIV-1 protease’s function and decrease the probability of drug resistance, are necessary to continue to effectively target this key protein in HIV-1 life cycle.

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

Science.gov (United States)

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.

Development of bioassay for pathogenecity testing of Ureaplasma urealyticum as part of host-pathogen communication

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Purnomo Soeharso

2005-12-01

Full Text Available Bioassay of Ureaplasma urealyticum is necessary for detection as well as determination of pathogenic factors in order to understand the pathogenesis of diseases associate with ureaplasma infection. Cultivation and verification of ureaplasma is the first step of this study in the purpose of discovering sensitive method for ureaplasma detection. Cultivation of ureaplasma either in liquid or in solid media are able to detect the existence of ureaplasma in samples analyzed. However, application of PCR using specific primers to be compatible with urease gene (ure would confirm the presence of ureaplasma. The pathogenicity of ureaplasma is potentially monitored using reporter gene as a marker for gene expression. IceC was chosen as reporter gene for ureaplasma pathogenic determination as the gene has great sensitivity, easily detectable and quantitated in simple method of ice nucleation assay. Transposon 916 (Tn916 was selected as a vector for iceC gene to transform ureaplasma. The application of recombinant Tn916-iceC which is considered as pUI, allow detection of ureaplasma activities when transform ureaplasma is tested by ice nucleation assay. It was expected that ureaplasma transformation is the manifestation of mutagenesis which interfere genes responsible for bacterial pathogenicity, in order pathogenesis of bacterial infection to be analyzed accurately. IgA1 protease is considered to be an important factor for ureaplasma pathogenicity as the enzyme is required for successful colonization. Identification of iga gene and  determination of IgA1 protease activity are important for understanding the pathogenesis of ureaplasma infection. Putative iga gene of Mycoplasma genitalium was used as a reference to identify the presence of iga nucleotide sequence in U. urealyticum. Convincing evidence were obtained after PCR amplification of ureaplasma DNA using primers designed to be compatible with putative iga gene of M. genitalium followed by the

Suboptimal inhibition of protease activity in human immunodeficiency virus type 1: Effects on virion morphogenesis and RNA maturation

International Nuclear Information System (INIS)

Moore, Michael D.; Fu, William; Soheilian, Ferri; Nagashima, Kunio; Ptak, Roger G.; Pathak, Vinay K.; Hu, Wei-Shau

2008-01-01

Protease activity within nascently released human immunodeficiency virus type 1 (HIV-1) particles is responsible for the cleavage of the viral polyproteins Gag and Gag-Pol into their constituent parts, which results in the subsequent condensation of the mature conical core surrounding the viral genomic RNA. Concomitant with viral maturation is a conformational change in the packaged viral RNA from a loosely associated dimer into a more thermodynamically stable form. In this study we used suboptimal concentrations of two protease inhibitors, lopinavir and atazanavir, to study their effects on Gag polyprotein processing and on the properties of the RNA in treated virions. Analysis of the treated virions demonstrated that even with high levels of inhibition of viral infectivity (IC 90 ), most of the Gag and Gag-Pol polyproteins were processed, although slight but significant increases in processing intermediates of Gag were detected. Drug treatments also caused a significant increase in the proportion of viruses displaying either immature or aberrant mature morphologies. The aberrant mature particles were characterized by an electron-dense region at the viral periphery and an electron-lucent core structure in the viral center, possibly indicating exclusion of the genomic RNA from these viral cores. Intriguingly, drug treatments caused only a slight decrease in overall thermodynamic stability of the viral RNA dimer, suggesting that the dimeric viral RNA was able to mature in the absence of correct core condensation

Proteases and protease inhibitors of urinary extracellular vesicles in diabetic nephropathy.

Science.gov (United States)

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.

Proteases and Protease Inhibitors of Urinary Extracellular Vesicles in Diabetic Nephropathy

Directory of Open Access Journals (Sweden)

Luca Musante

2015-01-01

Full Text Available 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.

Selective excretion of IgA in rat bronchial secretions: lack of significant contribution from plasma IgA

International Nuclear Information System (INIS)

Lemaitre-Coelho, I.; Yamakido, M.; Montgomery, P.C.; Langendries, A.E.; Vaerman, J.P.

1982-01-01

Concentrated rat bronchial washings (BW) were analyzed by gel-filtration and immunochemical methods. BW contained mainly albumin, transferrin and IgG. Free secretory component and secretory IgA were identified in BW; the BW-IgA had the same three sedimentation coefficients, i.e. +/- 11 S, 13 S, 15 S by sucrose density gradient ultracentrifugation, as rat milk and rat bile IgA; the three peaks were secretory IgA. Compared to serum, and relatively to albumin, BW were significantly enriched in IgA, although much less than rat bile. Purified polyclonal rat polymeric 125 I-IgA was injected intravenously into normal rats, and into rats with bile duct ligation or partial hepatectomy, which decrease the liver plasma-to-bile transfer of IgA. BW were then collected, one or four hours later, to assess the recovery of the 125 I-IgA in BW and to estimate the contribution of serum IgA to BW-IgA. Very little 125 I-IgA (less than 0.2%) was recovered in all BW. The specific activity, measured only in the rat with the highest recovery in BW, was 20 times lower in BW than in serum. The data demonstrate that rat serum IgA does not contribute significantly to IgA in BW

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

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Werner Smidt

Full Text Available 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.

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

Science.gov (United States)

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.

Encapsulation of protease from Aspergillus oryzae and lipase from Thermomyces lanuginoseus using alginate and different copolymer types

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Truong Thi Mong Thu

2016-05-01

Full Text Available Although the application of enzymes in food as a food processing aid and enzyme supplement is of interest and widely used, the enzymes can be easily deactivated or lose their activity due to many causes such as pH and moisture as well as through the introduction of incompatible ingredients during food processing and storage. These problems can be solved by the encapsulation technique, especially in a gel matrix. The influences were studied of the alginate concentration, types of copolymer and their concentrations on the bead size, encapsulation yield (EY, encapsulation efficiency (EE, leakage and the retention of enzyme activity during storage period of encapsulated protease from Aspergillus oryzae and lipase from Thermomyces lanuginosus beads. A solution of purified protease or lipase was encapsulated in calcium alginate-chitosan beads (CACB, calcium alginate-xanthan gum beads (CAXB and calcium alginate-maltodextrin beads (CAMB using the extrusion method. Increasing the alginate and copolymer concentrations in the solution increased the bead size, EY, EE and the retention of enzyme activity during the storage period and reduced leakage of both the encapsulated protease and lipase. In addition, different types of copolymer significantly (p ≤ 0.05 affected these properties of both encapsulated enzymes. Furthermore, protease encapsulated using 2.0% alginate and 0.2% chitosan provided the highest EY (81.7% and EE (77.2% with a bead size of 1.85 mm and 8.1% leakage. The retention of encapsulated protease activity and the shelf-life of encapsulated enzyme which was expressed as half-life, the time required for the enzyme activity to decrease by half (thalf life were 75.8% and 27.2 wk, respectively after storage at 4 °C for 10 wk. For lipase, encapsulation using 2.0% alginate and 0.4% xanthan gum provided the highest EY (42.5% and EE (43.9% and the bead size and leakage were 1.81 mm and 6.2%, respectively. The retention of encapsulated

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

Science.gov (United States)

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.

IgA nephropathy enigma

Czech Academy of Sciences Publication Activity Database

Městecký, Jiří; Novák, J.; Moldoveanu, Z.; Raška, M.

2016-01-01

Roč. 172, NOV 2016 SI (2016), s. 72-77 ISSN 1521-6616 R&D Projects: GA MZd(CZ) NV15-33686A Institutional support: RVO:61388971 Keywords : IgA nephropathy * IgA subclasses * Autoimmunity Subject RIV: EE - Microbiology, Virology Impact factor: 3.990, year: 2016

Isogeometric Analysis of Hyperelastic Materials Using PetIGA

KAUST Repository

Bernal, L.M.

2013-06-01

In this work different nonlinear hyperelastic models for slightly compressible materials are implemented in an isogeometric finite element model. This is done within the recently developed computational framework called PetIGA, which uses isoge- ometric analysis and modern computational tools to solve systems of equations directly and iteratively. A flexible theoretical background is described to implement other hyperelastic models and possibly transient problems in future work. Results show quadratic convergence of the nonlinear solution consistent with the Newton-Raphson method that was used. Finally, PetIGA proves to be a powerful and versatile tool to solve these types of problems efficiently.

Isogeometric Analysis of Hyperelastic Materials Using PetIGA

KAUST Repository

Bernal, L.M.; Calo, Victor M.; Collier, Nathan; Espinosa, G.A.; Fuentes, F.; Mahecha, J.C.

2013-01-01

In this work different nonlinear hyperelastic models for slightly compressible materials are implemented in an isogeometric finite element model. This is done within the recently developed computational framework called PetIGA, which uses isoge- ometric analysis and modern computational tools to solve systems of equations directly and iteratively. A flexible theoretical background is described to implement other hyperelastic models and possibly transient problems in future work. Results show quadratic convergence of the nonlinear solution consistent with the Newton-Raphson method that was used. Finally, PetIGA proves to be a powerful and versatile tool to solve these types of problems efficiently.

Cleavage specificity analysis of six type II transmembrane serine proteases (TTSPs using PICS with proteome-derived peptide libraries.

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Olivier Barré

Full Text Available Type II transmembrane serine proteases (TTSPs are a family of cell membrane tethered serine proteases with unclear roles as their cleavage site specificities and substrate degradomes have not been fully elucidated. Indeed just 52 cleavage sites are annotated in MEROPS, the database of proteases, their substrates and inhibitors.To profile the active site specificities of the TTSPs, we applied Proteomic Identification of protease Cleavage Sites (PICS. Human proteome-derived database searchable peptide libraries were assayed with six human TTSPs (matriptase, matriptase-2, matriptase-3, HAT, DESC and hepsin to simultaneously determine sequence preferences on the N-terminal non-prime (P and C-terminal prime (P' sides of the scissile bond. Prime-side cleavage products were isolated following biotinylation and identified by tandem mass spectrometry. The corresponding non-prime side sequences were derived from human proteome databases using bioinformatics. Sequencing of 2,405 individual cleaved peptides allowed for the development of the family consensus protease cleavage site specificity revealing a strong specificity for arginine in the P1 position and surprisingly a lysine in P1' position. TTSP cleavage between R↓K was confirmed using synthetic peptides. By parsing through known substrates and known structures of TTSP catalytic domains, and by modeling the remainder, structural explanations for this strong specificity were derived.Degradomics analysis of 2,405 cleavage sites revealed a similar and characteristic TTSP family specificity at the P1 and P1' positions for arginine and lysine in unfolded peptides. The prime side is important for cleavage specificity, thus making these proteases unusual within the tryptic-enzyme class that generally has overriding non-prime side specificity.

Optimizing HIV-1 protease production in Escherichia coli as fusion protein

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Piubelli Luciano

2011-06-01

Full Text Available Abstract Background Human immunodeficiency virus (HIV is the etiological agent in AIDS and related diseases. The aspartyl protease encoded by the 5' portion of the pol gene is responsible for proteolytic processing of the gag-pol polyprotein precursor to yield the mature capsid protein and the reverse transcriptase and integrase enzymes. The HIV protease (HIV-1Pr is considered an attractive target for designing inhibitors which could be used to tackle AIDS and therefore it is still the object of a number of investigations. Results A recombinant human immunodeficiency virus type 1 protease (HIV-1Pr was overexpressed in Escherichia coli cells as a fusion protein with bacterial periplasmic protein dithiol oxidase (DsbA or glutathione S-transferase (GST, also containing a six-histidine tag sequence. Protein expression was optimized by designing a suitable HIV-1Pr cDNA (for E. coli expression and to avoid autoproteolysis and by screening six different E. coli strains and five growth media. The best expression yields were achieved in E. coli BL21-Codon Plus(DE3-RIL host and in TB or M9 medium to which 1% (w/v glucose was added to minimize basal expression. Among the different parameters assayed, the presence of a buffer system (based on phosphate salts and a growth temperature of 37°C after adding IPTG played the main role in enhancing protease expression (up to 10 mg of chimeric DsbA:HIV-1Pr/L fermentation broth. GST:HIVPr was in part (50% produced as soluble protein while the overexpressed DsbA:HIV-1Pr chimeric protein largely accumulated in inclusion bodies as unprocessed fusion protein. A simple refolding procedure was developed on HiTrap Chelating column that yielded a refolded DsbA:HIV-1Pr with a > 80% recovery. Finally, enterokinase digestion of resolubilized DsbA:HIV-1Pr gave more than 2 mg of HIV-1Pr per liter of fermentation broth with a purity ≤ 80%, while PreScission protease cleavage of soluble GST:HIVPr yielded ~ 0.15 mg of pure HIV-1

Dysregulation of protease and protease inhibitors in a mouse model of human pelvic organ prolapse.

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Madhusudhan Budatha

Full Text Available 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 [Serpina1a (α1-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.

Activation of the Arabidopsis membrane-bound transcription factor bZIP28 is mediated by site-2 protease, but not site-1 protease.

Science.gov (United States)

Iwata, Yuji; Ashida, Makoto; Hasegawa, Chisa; Tabara, Kazuki; Mishiba, Kei-Ichiro; Koizumi, Nozomu

2017-08-01

The unfolded protein response (UPR) is a homeostatic cellular response conserved in eukaryotic cells to alleviate the accumulation of unfolded proteins in the endoplasmic reticulum (ER). Arabidopsis bZIP28 is a membrane-bound transcription factor activated by proteolytic cleavage in response to ER stress, thereby releasing its cytosolic portion containing the bZIP domain from the membrane to translocate into the nucleus where it induces the transcription of genes encoding ER-resident molecular chaperones and folding enzymes. It has been widely recognized that the proteolytic activation of bZIP28 is mediated by the sequential cleavage of site-1 protease (S1P) and site-2 protease (S2P). In the present study we provide evidence that bZIP28 protein is cleaved by S2P, but not by S1P. We demonstrated that wild-type and s1p mutant plants produce the active, nuclear form of bZIP28 in response to the ER stress inducer tunicamycin. In contrast, tunicamycin-treated s2p mutants do not accumulate the active, nuclear form of bZIP28. Consistent with these observations, s2p mutants, but not s1p mutants, exhibited a defective transcriptional response of ER stress-responsive genes and significantly higher sensitivity to tunicamycin. Interestingly, s2p mutants accumulate two membrane-bound bZIP28 fragments with a shorter ER lumen-facing C-terminal domain. Importantly, the predicted cleavage sites are located far from the canonical S1P recognition motif previously described. We propose that ER stress-induced proteolytic activation of bZIP28 is mediated by the sequential actions of as-yet-unidentified protease(s) and S2P, and does not require S1P. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

CD44 expression in IgA nephropathy

NARCIS (Netherlands)

Florquin, Sandrine; Nunziata, Raffaele; Claessen, Nike; van den Berg, Frank M.; Pals, Steven T.; Weening, Jan J.

2002-01-01

Immunoglobulin A (IgA) nephropathy is a frequent, chronic renal disease characterized by a broad spectrum of clinical presentations and pathologic findings. CD44, a family of type I transmembrane glycoproteins: involved in cell-cell and cell-matrix interactions, may orchestrate partially the cascade

Immunohistochemical analysis of IgA expression differentiates IgG4-related disease from plasma cell-type Castleman disease.

Science.gov (United States)

Manabe, Akihiro; Igawa, Takuro; Takeuchi, Mai; Gion, Yuka; Yoshino, Tadashi; Sato, Yasuharu

2017-03-01

Plasma cell-type Castleman disease (PCD) is often encountered when differentiating IgG4-related disease (IgG4-RD). Given that serum IgA is often elevated in Castleman disease, we investigated whether IgA expression levels in histological specimens can be used to differentiate between the two diseases. Lymph node lesions obtained from 12 IgG4-RD and 11 PCD patients were analysed by immunohistochemistry with anti-IgG, -IgG4, and -IgA antibodies. In addition to all 12 cases of IgG4-RD, 8/11 cases (72.7 %) of PCD also met the diagnostic criteria of IgG4-RD (serum IgG4 ≥135 mg/dl and IgG4/IgG-positive cells ≥40 %). IgA-positive cells were sparsely and densely distributed in IgG4-RD and PCD cases, respectively. The median number of IgA-positive cells ± SD in all 12 cases of IgG4-RD was 31 ± 37 cells per three high-powered fields (3HPFs) (range 4-118 cells/3HPFs). In contrast, the median number of IgA-positive cells, which was significantly higher in all 11 cases of PCD, was 303 ± 238 cells/3HPFs (range 74-737 cells/3HPFs) (P IgG4-RD.

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

Energy Technology Data Exchange (ETDEWEB)

Yedidi, Ravikiran S. [Department of Biochemistry and Molecular Biology, School of Medicine, Wayne State University, Detroit, MI 48201 (United States); Muhuhi, Joseck M. [Department of Chemistry, Wayne State University, Detroit, MI 48202 (United States); Liu, Zhigang [Department of Biochemistry and Molecular Biology, School of Medicine, Wayne State University, Detroit, MI 48201 (United States); Bencze, Krisztina Z. [Department of Chemistry, Fort Hays State University, Hays, KS 67601 (United States); Koupparis, Kyriacos [Department of Biochemistry and Molecular Biology, School of Medicine, Wayne State University, Detroit, MI 48201 (United States); Department of Chemistry, Wayne State University, Detroit, MI 48202 (United States); O’Connor, Carrie E.; Kovari, Iulia A. [Department of Biochemistry and Molecular Biology, School of Medicine, Wayne State University, Detroit, MI 48201 (United States); Spaller, Mark R. [Department of Chemistry, Wayne State University, Detroit, MI 48202 (United States); Kovari, Ladislau C., E-mail: kovari@med.wayne.edu [Department of Biochemistry and Molecular Biology, School of Medicine, Wayne State University, Detroit, MI 48201 (United States)

2013-09-06

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 active 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.

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

International Nuclear Information System (INIS)

Yedidi, Ravikiran S.; Muhuhi, Joseck M.; Liu, Zhigang; Bencze, Krisztina Z.; Koupparis, Kyriacos; O’Connor, Carrie E.; Kovari, Iulia A.; Spaller, Mark R.; Kovari, Ladislau C.

2013-01-01

Highlights: •Inhibitors against MDR HIV-1 protease were designed, synthesized and evaluated. •Lead peptide (6a) showed potent inhibition (IC 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 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 active 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 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 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

A new method for the characterization of strain-specific conformational stability of protease-sensitive and protease-resistant PrPSc.

Directory of Open Access Journals (Sweden)

Laura Pirisinu

Full Text Available 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 PrP(Sc, a disease-associated isoform of the host-encoded cellular protein (PrP(C. Molecular strain typing approaches have been developed which rely on the characterization of protease-resistant PrP(Sc. However, PrP(Sc 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 PrP(Sc aggregates. We first set up experimental conditions which allowed the most advantageous separation of PrP(C and PrP(Sc by means of differential centrifugation. The conformational solubility and stability assay (CSSA was then developed by measuring PrP(Sc 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 PrP(Sc, we analysed sheep isolates of Nor98 and compared them to classical scrapie isolates. In Nor98, insoluble PrP(Sc 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 PrP(Sc conformational stabilities of protease-resistant and protease-sensitive PrP(Sc and that it is a valuable tool

Nefropatia por IgA nas espondiloartrites IgA nephropathy in spondyloarthritis

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Daniela Castelo Azevedo

2011-02-01

Full Text Available Pacientes com espondiloartrites poderiam ser mais acometidos pela nefropatia por IgA do que a população geral, havendo, possivelmente, um mecanismo etiopatogênico comum. O seguinte artigo relaciona quatro casos que exemplificam essa possível associaçãoSpondyloarthritis patients can be more frequently affected by IgA nephropathy than the general population, and a common etiopathogenic mechanism can be involved. We report four cases that may exemplify that association

Human Cell Line-Derived Monoclonal IgA Antibodies for Cancer Immunotherapy

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Felix Hart

2017-05-01

Full Text Available IgA antibodies have great potential to improve the functional diversity of current IgG antibody-based cancer immunotherapy options. However, IgA production and purification is not well established, which can at least in part be attributed to the more complex glycosylation as compared to IgG antibodies. IgA antibodies possess up to five N-glycosylation sites within their constant region of the heavy chain as compared to one site for IgG antibodies. The human GlycoExpress expression system was developed to produce biotherapeutics with optimized glycosylation and used here to generate a panel of IgA isotype antibodies directed against targets for solid (TA-mucin 1, Her2, EGFR, Thomsen–Friedenreich and hematological (CD20 cancer indications. The feasibility of good manufacturing practice was shown by the production of 11 g IgA within 35 days in a one liter perfusion bioreactor, and IgA antibodies in high purity were obtained after purification. The monoclonal IgA antibodies possessed a high sialylation degree, and no non-human glycan structures were detected. Kinetic analysis revealed increased avidity antigen binding for IgA dimers as compared to monomeric antibodies. The IgA antibodies exhibited potent Fab- and Fc-mediated functionalities against cancer cell lines, whereby especially granulocytes are recruited. Therefore, for patients who do not sufficiently benefit from therapeutic IgG antibodies, IgA antibodies may complement current regiment options and represent a promising strategy for cancer immunotherapy. In conclusion, a panel of novel biofunctional IgA antibodies with human glycosylation was successfully generated.

A Kunitz-type cysteine protease inhibitor from cauliflower and Arabidopsis

DEFF Research Database (Denmark)

Halls, C.E.; Rogers, S. W.; Ouffattole, M.

2006-01-01

proaleurain maturation protease and of papain when assayed at pH 4.5 but not at pH 6.3. In a pull-down assay, the inhibitor bound tightly to papain, but only weakly to the aspartate protease pepsin. When the cauliflower protease inhibitor was transiently expressed in tobacco suspension culture protoplasts...

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

Energy Technology Data Exchange (ETDEWEB)

Ghosh, Arun K.; Brindisi, Margherita; Nyalapatla, Prasanth R.; Takayama, Jun; Ella-Menye, Jean-Rene; Yashchuk, Sofiya; Agniswamy, Johnson; Wang, Yuan-Fang; Aoki, Manabu; Amano, Masayuki; Weber, Irene T.; Mitsuya, Hiroaki

2017-10-01

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.

PARP-1 cleavage fragments: signatures of cell-death proteases in neurodegeneration

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Alexander Jonathan S

2010-12-01

Full Text Available Abstract The normal function of poly (ADP-ribose polymerase-1 (PARP-1 is the routine repair of DNA damage by adding poly (ADP ribose polymers in response to a variety of cellular stresses. Recently, it has become widely appreciated that PARP-1 also participates in diverse physiological and pathological functions from cell survival to several forms of cell death and has been implicated in gene transcription, immune responses, inflammation, learning, memory, synaptic functions, angiogenesis and aging. In the CNS, PARP inhibition attenuates injury in pathologies like cerebral ischemia, trauma and excitotoxicity demonstrating a central role of PARP-1 in these pathologies. PARP-1 is also a preferred substrate for several 'suicidal' proteases and the proteolytic action of suicidal proteases (caspases, calpains, cathepsins, granzymes and matrix metalloproteinases (MMPs on PARP-1 produces several specific proteolytic cleavage fragments with different molecular weights. These PARP-1 signature fragments are recognized biomarkers for specific patterns of protease activity in unique cell death programs. This review focuses on specific suicidal proteases active towards PARP-1 to generate signature PARP-1 fragments that can identify key proteases and particular forms of cell death involved in pathophysiology. The roles played by some of the PARP-1 fragments and their associated binding partners in the control of different forms of cell death are also discussed.

Plasma Gelsolin Promotes Proliferation of Mesangial Cell in IgA Nephropathy

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Lei Zhang

2016-12-01

Full Text Available Background/Aims: Plasma gelsolin (pGSN is an actin-binding protein that plays a critical role in the pathogenesis of rheumatoid arthritis. However, whether pGSN is involved in other immunological diseases remains unknown. This study focused on the relationship between pGSN and immunoglobulin A (IgA nephropathy (IgAN. Methods: Two hundred patients with IgAN, 200 patients each with several other types of nephropathy and healthy controls (HCs who underwent kidney biopsies between 2000 and 2014 were enrolled in the study. The Oxford classification system was used to predict the risk of disease progression. Serum and renal tissue were used to detect pGSN, and the correlations between pGSN and IgA, galactose-deficient IgA1 (Gd-IgA1, transforming growth factor beta1 (TGF-β1, fibronectin (FN content, clinical symptoms, and kidney function were analyzed. Results: We found that the pGSN levels were significantly decreased in sera from IgAN patients compared to sera from patients with other forms of glomerular nephritis and HCs. Furthermore, the serum pGSN levels were negatively correlated with the serum IgA1, FN, and TGF-β1 levels, and positively correlated with the estimated glomerular filtration rate. Conversely, the glomerular pGSN content was significantly elevated in the IgAN patients and was positively correlated with TGF-β1 and FN levels. In renal tissue, the pGSN levels were significantly higher in IgAN patients with M1 and S1 compared to patients with M0 and S0 (p in vitro. pGSN also promoted integrin α2β1 expression in HMCs and enhanced the integrin α2β1-pGSN interaction. Conclusion: Our study suggested that pGSN may play an important role in the development of IgAN by promoting the proliferation of mesangial cells and that serum and glomerular pGSN levels may be new markers for predicting IgAN progression and prognosis.

Elucidating heterogeneity of IgA1 hinge-region O-glycosylation by use of MALDI-TOF/TOF mass spectrometry: role of cysteine alkylation during sample processing.

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Franc, Vojtěch; Řehulka, Pavel; Raus, Martin; Stulík, Jiří; Novak, Jan; Renfrow, Matthew B; Šebela, Marek

2013-10-30

Determining disease-associated changes in protein glycosylation provides a better understanding of pathogenesis. This work focuses on human immunoglobulin A1 (IgA1), where aberrant O-glycosylation plays a key role in the pathogenesis of IgA nephropathy (IgAN). Normal IgA1 hinge region carries 3 to 6 O-glycans consisting of N-acetylgalactosamine (GalNAc) and galactose (Gal); both sugars may be sialylated. In IgAN patients, some O-glycans on a fraction of IgA1 molecules are Gal-deficient. Here we describe a sample preparation protocol with optimized cysteine alkylation of a Gal-deficient polymeric IgA1 myeloma protein prior to in-gel digestion and analysis of the digest by MALDI-TOF/TOF mass spectrometry (MS). Following a novel strategy, IgA1 hinge-region O-glycopeptides were fractionated by reversed-phase liquid chromatography using a microgradient device and identified by MALDI-TOF/TOF tandem MS (MS/MS). The acquired MS/MS spectra were interpreted manually and by means of our own software. This allowed assigning up to six O-glycosylation sites and demonstration, for the first time, of the distribution of isomeric O-glycoforms having the same molecular mass, but a different glycosylation pattern. The most abundant Gal-deficient O-glycoforms were GalNAc4Gal3 and GalNAc5Gal4 with one Gal-deficient site and GalNAc5Gal3 and GalNAc4Gal2 with two Gal-deficient sites. The most frequent Gal-deficient sites were at Ser230 and/or Thr236. In this work, we studied the O-glycosylation in the hinge region of human immunoglobulin A1 (IgA1). Aberrant glycosylation of the protein plays a key role in the pathogenesis of IgA nephropathy. Thus identification of the O-glycan composition of IgA1 is important for a deeper understanding of the disease mechanism, biomarker discovery and validation, and implementation and monitoring of disease-specific therapies. We developed a new procedure for elucidating the heterogeneity of IgA1 O-glycosylation. After running a polyacrylamide gel

Human antibodies to immunoglobulin A (IgA)

International Nuclear Information System (INIS)

Munster, P.J.J. van; Nadorp, J.H.S.M.; Shuurman, H.J.

1978-01-01

In the sera of 12 out of 27 individuals with IgA deficiency (serum level below 0.02 mg IgA/m) class-specific anti-IgA antibodies were demonstrated by haemagglutination. These sera showed false-positive results in a solid-phase inhibition radioimmunoassay (RIST) (apparent IgA concentration between 0.6 and 13.7 μg IgA/ml) indicating that the RIST is not an appropriate test for the analysis of serum of IgA seficient individuals. A modification of the RIST is proposed (titration RIA) that permits differentiation between low levels of IgA and class-specific anti-IgA antibodies. With this test IgA deficient individuals could be classified as those with low but detectable levels of IgA and those with class-specific anti-IgA antibodies. A computer procedure was developed to calculate both the amount and the avidity (K) of the anti-IgA antibodies and to simulate the assay system. The K value calculated from experimental points proved to be an overestimation of the K value which fitted most adequately in the simulation. The comparison of the results with clinical findings indicated a possible correlation between the amount and the avidity of the anti-IgA antibodies and the appearence of anaphylactic reactions after transfusion of IgA. (Auth.)

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

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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 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.

Chlamydial serum IgG, IgA and local IgA antibodies in patients with genital-tract infections measured by solid-phase radioimmunoassay

International Nuclear Information System (INIS)

Terho, P.; Meurman, O.

1981-01-01

A solid-phase radioimmunoassay (RIA) for IgG and IgA class antibodies to Chlamydia trachomatis was developed with C. trachomatis serotype L2 as antigen. The assay was sensitive, reproducible and correlated well with an immunofluorescence test (r = 0.85). Serum IgG antibodies were detected in 79% of Chlamydia isolation-positive versus 43% of isolation-negative male patients with urethritis and serum IgA antibodies in 53% and 21%, respectively. Urethral IgA antibodies, measured from specimens taken for chlamydial isolation, could be detected in 94% and 38%, respectively. From 737 male urethral and 909 female cervical secretions screened for the presence of IgA antibodies, about half were isolation and IgA negative. Only 4% (6/151) of male and 5.4% (2/37) of female isolation-positive specimens were IgA negative. The determination of local IgA antibodies may be used as a screening test in chlamydial genital infections. (author)

The binding mechanism of a peptidic cyclic serine protease inhibitor

DEFF Research Database (Denmark)

Jiang, Longguang; Svane, Anna Sigrid P.; Sørensen, Hans Peter

2011-01-01

Serine proteases are classical objects for studies of catalytic and inhibitory mechanisms as well as interesting as therapeutic targets. Since small-molecule serine protease inhibitors generally suffer from specificity problems, peptidic inhibitors, isolated from phage-displayed peptide libraries......, have attracted considerable attention. Here, we have investigated the mechanism of binding of peptidic inhibitors to serine protease targets. Our model is upain-1 (CSWRGLENHRMC), a disulfide-bond-constrained competitive inhibitor of human urokinase-type plasminogen activator with a noncanonical...... inhibitory mechanism and an unusually high specificity. Using a number of modified variants of upain-1, we characterised the upain-1-urokinase-type plasminogen activator complex using X-ray crystal structure analysis, determined a model of the peptide in solution by NMR spectroscopy, and analysed binding...

Sequence Quality Analysis Tool for HIV Type 1 Protease and Reverse Transcriptase

OpenAIRE

DeLong, Allison K.; Wu, Mingham; Bennett, Diane; Parkin, Neil; Wu, Zhijin; Hogan, Joseph W.; Kantor, Rami

2012-01-01

Access to antiretroviral therapy is increasing globally and drug resistance evolution is anticipated. Currently, protease (PR) and reverse transcriptase (RT) sequence generation is increasing, including the use of in-house sequencing assays, and quality assessment prior to sequence analysis is essential. We created a computational HIV PR/RT Sequence Quality Analysis Tool (SQUAT) that runs in the R statistical environment. Sequence quality thresholds are calculated from a large dataset (46,802...

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

Science.gov (United States)

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.

IGA resistance of TT Alloy 690 and concentration behavior of Broached Egg Crate tube support configuration

International Nuclear Information System (INIS)

Suzuki, S.; Kusakabe, T.; Yamamoto, H.; Arioka, K.; Ochi, T.

1992-01-01

In order to improve the reliability of the Steam Generator (SG), TT Alloy 690 and BEC (Broached Egg Crate) type tube support plate has been developed. Some tests are carried out to heighten the reliability for these improvements all the more and the following results are obtained. (1) SERT test (Slow Extension Rate Test) made clear that TT690 has less IGA susceptibility in comparison with MA600. (2) The alkaline susceptibility on the occurrence of IGA/SCC on TT690 and MA600 obtained by SERT corresponds to that obtained by Model Boiler test. (3) By model boiler test, superior concentration behaviors for BEC type tube support plate configuration have been recognized in comparison with Drill type. This result is obtained by the joint research of the five utilities (Kansai Epco, Hokkaido Epco, Shikoku Epco, Kyushu Epco, JAPCO) and MHI

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

Science.gov (United States)

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.

Shared IgG Infection Signatures vs. Hemorrhage-Restricted IgA Clusters in Human Dengue: A Phenotype of Differential Class-Switch via TGFβ1

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Chung-Hao Huang

2017-12-01

Full Text Available Phenotypic manifestations of infectious diseases are closely related to individual immune responses. Methods to extract information from patients’ own immune reactions would be of great use for both diagnosis and treatment. Dengue fever is one of the diseases that clinical aggravations could occur paradoxically after humoral immunity appears. This property makes dengue fever an excellent disease model to explore. A principal component analyses (PCAs-based framework derived from a prior vaccination study was developed. The framework was verified by successful demonstrations of known IgG signatures from a Mexico Dengue data set. Afterward the pipeline was tested upon de novo IgG and IgA libraries of Dengue patients from southern Taiwan. We discovered four infection signatures within IgG repertoires, two of which were identical to previous reports. However, it was IgA but not IgG that could differentiate hemorrhagic from non-hemorrhagic patients. IgA repertoires were found more diversified among bleeders, from whom seven signature clusters were characterized. The expressions of transforming growth factor beta 1 (TGFβ1 and accordingly mediated class-switch activity of IgA were distinct only among the PCA-segregated bleeding group. In sum, intercontinental sharing of IgG signatures in dengue fever was demonstrated via a unified working flow. Differential regulation of IgA class-switch with associated diversity expansion plus existences of hemorrhage-restricted clusters were shown. The ability of the framework to find common IgG signatures would implicate applications to infections even from unknown pathogens. The clusters within IgA repertoires could offer perspectives to other IgA-related bleeding disorders such as Henoch-Schönlein purpura or IgA nephropathy. Substantiated grounds for IgA-specific effector function via TGFβ1-mediated class-switch would be a new factor to consider for infectious diseases.

Borrelia burgdorferi-specific IgA in Lyme Disease

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Christina D'Arco

2017-05-01

Full Text Available 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.

Borrelia burgdorferi-specific IgA in Lyme Disease.

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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.

Developing HIV-1 Protease Inhibitors through Stereospecific Reactions in Protein Crystals.

Science.gov (United States)

Olajuyigbe, Folasade M; Demitri, Nicola; De Zorzi, Rita; Geremia, Silvano

2016-10-31

Protease inhibitors are key components in the chemotherapy of HIV infection. However, the appearance of viral mutants routinely compromises their clinical efficacy, creating a constant need for new and more potent inhibitors. Recently, a new class of epoxide-based inhibitors of HIV-1 protease was investigated and the configuration of the epoxide carbons was demonstrated to play a crucial role in determining the binding affinity. Here we report the comparison between three crystal structures at near-atomic resolution of HIV-1 protease in complex with the epoxide-based inhibitor, revealing an in-situ epoxide ring opening triggered by a pH change in the mother solution of the crystal. Increased pH in the crystal allows a stereospecific nucleophile attack of an ammonia molecule onto an epoxide carbon, with formation of a new inhibitor containing amino-alcohol functions. The described experiments open a pathway for the development of new stereospecific protease inhibitors from a reactive lead compound.

Developing HIV-1 Protease Inhibitors through Stereospecific Reactions in Protein Crystals

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Folasade M. Olajuyigbe

2016-10-01

Full Text Available Protease inhibitors are key components in the chemotherapy of HIV infection. However, the appearance of viral mutants routinely compromises their clinical efficacy, creating a constant need for new and more potent inhibitors. Recently, a new class of epoxide-based inhibitors of HIV-1 protease was investigated and the configuration of the epoxide carbons was demonstrated to play a crucial role in determining the binding affinity. Here we report the comparison between three crystal structures at near-atomic resolution of HIV-1 protease in complex with the epoxide-based inhibitor, revealing an in-situ epoxide ring opening triggered by a pH change in the mother solution of the crystal. Increased pH in the crystal allows a stereospecific nucleophile attack of an ammonia molecule onto an epoxide carbon, with formation of a new inhibitor containing amino-alcohol functions. The described experiments open a pathway for the development of new stereospecific protease inhibitors from a reactive lead compound.

Ninety-nine is not enough: molecular characterization of inhibitor-resistant human immunodeficiency virus type 1 protease mutants with insertions in the flap region

Czech Academy of Sciences Publication Activity Database

Kožíšek, Milan; Grantz Šašková, Klára; Řezáčová, Pavlína; Brynda, Jiří; Maarseveen van, N. M.; De Jongh, D.; Boucher, Ch. A. B.; Kagan, R. M.; Nijhuis, M.; Konvalinka, Jan

2008-01-01

Roč. 82, č. 12 (2008), s. 5869-5878 ISSN 0022-538X R&D Projects: GA MŠk 1M0508; GA MZd NR8571 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50520514 Keywords : HIV protease inhibitors * aspartic proteases * viral resistance * insertions Subject RIV: CE - Biochemistry Impact factor: 5.308, year: 2008

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

Science.gov (United States)

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.

Anti Helicobacter pylori IgG and IgA response in patients with gastric cancer and chronic gastritis.

Science.gov (United States)

Manojlovic, Nebojsa; Babic, Dragana; Filipovic-Ljeshovic, Ivana; Pilcevic, Dijana

2008-01-01

Immune response against Helicobacter pylori is important for the course and outcome of infection. We conducted study looking for the difference in anti H. pylori IgG and IgA between patients with intestinal type of gastric cancer, superficial and atrophic gastritis. For this study, 133 patients infected with H. pylori were enrolled: 50 with superficial gastritis, 42 with atrophic gastritis and 41 with gastric cancer. Anti H. pylori IgG and IgA ELISA tests were performed. The difference in antibody titers of IgG and IgA, frequency of IgA > IgG ratio and combination of low IgG and IgA > IgG ratio were analyzed. The patients with gastritis had higher titer of IgG that the patients with gastric cancer (p gastritis had higher titer of IgA than the patients with gastric cancer (p IgG ratio is more frequent in patients with gastric cancer than in the patients with superficial gastritis (p IgG is more frequent in the patients with gastric cancer than in the patients with gastritis (p cancer elicit different anti H. pylori IgG and IgA response than the patients with superficial and atrophic gastritis. Low IgG and IgA predominance seems characteristic for gastric cancer.

[The effect of enzymatic treatment using proteases on properties of persistent sodium current in CA1 pyramidal neurons of rat hippocampus].

Science.gov (United States)

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).

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

Science.gov (United States)

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.

House dust mite major allergens Der p 1 and Der p 5 activate human airway-derived epithelial cells by protease-dependent and protease-independent mechanisms

Directory of Open Access Journals (Sweden)

Timmerman J André B

2006-03-01

Full Text Available Abstract House dust mite allergens (HDM cause bronchoconstriction in asthma patients and induce an inflammatory response in the lungs due to the release of cytokines, chemokines and additional mediators. The mechanism how HDM components achieve this is largely unknown. The objective of this study was to assess whether HDM components of Dermatophagoides pteronissinus with protease activity (Der p 1 and unknown enzymatic activity (Der p 2, Der p 5 induce biological responses in a human airway-derived epithelial cell line (A549, and if so, to elucidate the underlying mechanism(s of action. A549 cells were incubated with HDM extract, Der p 1, recombinant Der p 2 and recombinant Der p 5. Cell desquamation was assessed by microscopy. The proinflammatory cytokines, IL-6 and IL-8, were measured by ELISA. Intracellular Ca2+ levels were assessed in A549 cells and in mouse fibroblasts expressing the human protease activated receptor (PAR1, PAR2 or PAR4. HDM extract, Der p 1 and Der p 5 dose-dependently increased the production of IL-6 and IL-8. Added simultaneously, Der p 1 and Der p 5 further increased the production of IL-6 and IL-8. The action of Der p 1 was blocked by cysteine-protease inhibitors, while that of Der p 5 couldn't be blocked by either serine- or cysteine protease inhibitors. Der p 5 only induced cell shrinking, whereas HDM extract and Der p1 also induced cell desquamation. Der p 2 had no effect on A549 cells. Der p 1's protease activity causes desquamation and induced the release of IL6 and IL-8 by a mechanism independent of Ca2+ mobilisation and PAR activation. Der p 5 exerts a protease-independent activation of A549 that involves Ca2+ mobilisation and also leads to the production of these cytokines. Together, our data indicate that allergens present in HDM extracts can trigger protease-dependent and protease-independent signalling pathways in A549 cells.

Chimeric exchange of coronavirus nsp5 proteases (3CLpro) identifies common and divergent regulatory determinants of protease activity.

Science.gov (United States)

Stobart, Christopher C; Sexton, Nicole R; Munjal, Havisha; Lu, Xiaotao; Molland, Katrina L; Tomar, Sakshi; Mesecar, Andrew D; Denison, Mark R

2013-12-01

Human coronaviruses (CoVs) such as severe acute respiratory syndrome CoV (SARS-CoV) and Middle East respiratory syndrome CoV (MERS-CoV) cause epidemics of severe human respiratory disease. A conserved step of CoV replication is the translation and processing of replicase polyproteins containing 16 nonstructural protein domains (nsp's 1 to 16). The CoV nsp5 protease (3CLpro; Mpro) processes nsp's at 11 cleavage sites and is essential for virus replication. CoV nsp5 has a conserved 3-domain structure and catalytic residues. However, the intra- and intermolecular determinants of nsp5 activity and their conservation across divergent CoVs are unknown, in part due to challenges in cultivating many human and zoonotic CoVs. To test for conservation of nsp5 structure-function determinants, we engineered chimeric betacoronavirus murine hepatitis virus (MHV) genomes encoding nsp5 proteases of human and bat alphacoronaviruses and betacoronaviruses. Exchange of nsp5 proteases from HCoV-HKU1 and HCoV-OC43, which share the same genogroup, genogroup 2a, with MHV, allowed for immediate viral recovery with efficient replication albeit with impaired fitness in direct competition with wild-type MHV. Introduction of MHV nsp5 temperature-sensitive mutations into chimeric HKU1 and OC43 nsp5 proteases resulted in clear differences in viability and temperature-sensitive phenotypes compared with MHV nsp5. These data indicate tight genetic linkage and coevolution between nsp5 protease and the genomic background and identify differences in intramolecular networks regulating nsp5 function. Our results also provide evidence that chimeric viruses within coronavirus genogroups can be used to test nsp5 determinants of function and inhibition in common isogenic backgrounds and cell types.

Targeting Neutrophil Protease-Mediated Degradation of Tsp-1 to Induce Metastatic Dormancy

Science.gov (United States)

2017-10-01

AWARD NUMBER: W81XWH-16-1-0615 TITLE: Targeting Neutrophil Protease-Mediated Degradation of Tsp-1 to Induce Metastatic Dormancy PRINCIPAL...29 Sep 2017 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Targeting Neutrophil Protease-Mediated Degradation of Tsp-1 to Induce Metastatic Dormancy...infection or cigarette smoke enhanced pulmonary metastasis from breast cancer in humans and mice. Similarly, autoimmune arthritis, characterized by

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

Directory of Open Access Journals (Sweden)

Kangping Wang

2013-05-01

Full Text Available 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.

Association of IgA multiple myeloma with pre-existing disease

Energy Technology Data Exchange (ETDEWEB)

Schafer, A.I.; Miller, J.B.

1979-01-01

A retrospective analysis of 153 patients with multiple myeloma was performed for evaluation of the possible significance of pre-existing disease. 37% of the group had no significant antecedent disorder. The most common prior illnesses were peptic ulcer disease and gallbladder disease. Of 12 patients in the group who had prior biliary tract disease and for whom immunoelectrophoretic studies were available, eight (66.7%) had IgA paraproteins. This figure is statistically higher than the 14.1% of prevalence of IgA paraproteins in those myeloma patients without biliary disease. We conclude that prior inflammatory gastrointestinal, pulmonary, and, particularly, biliary disease may be implicated in the pathogenesis of the IgA subset of multiple myeloma.

An antibiotic, heavy metal resistant and halotolerant Bacillus cereus SIU1 and its thermoalkaline protease

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Vikram Surendra

2010-07-01

Full Text Available Abstract Background Many workers have reported halotolerant bacteria from saline conditions capable of protease production. However, antibiotic resistance and heavy metal tolerance pattern of such organisms is not documented very well. Similarly, only a few researchers have reported the pattern of pH change of fermentation medium during the course of protease production. In this study, we have isolated a halotolerant Bacillus cereus SIU1 strain from a non-saline environment and studied its antibiotic and heavy metal resistance pattern. The isolate produces a thermoalkaline protease and changes the medium pH during the course of fermentation. Thermostability of protease was also studied for 30 min. Results Seventy bacterial strains isolated from the soils of Eastern Uttar Pradesh, India were screened for protease production. All of them exhibited protease activity. However, 40% bacterial isolates were found good protease producers as observed by caseinolytic zones on milk agar plates. Among them, culture S-4 was adjudged as the best protease producer, and was identified as Bacillus cereus by morphological, biochemical and 16 S rDNA sequence analyses. The isolate was resistant to heavy metals (As2+, Pb2+, Cs1+ and antibiotics (penicillin, lincomycin, cloxacillin, pefloxacin. Its growth behavior and protease production was studied at 45°C and pH 9.0. The protease units of 88 ml-1 were noted in unoptimized modified glucose yeast extract (GYE medium during early stationary phase at 20 h incubation period. The enzyme was stable in the temperature range of 35°-55°C. Conclusions An antibiotic and heavy metal resistant, halotolerant Bacillus cereus isolate is capable of producing thermoalkaline protease, which is active and stable at pH 9.0 and 35°-55°C. This isolate may be useful in several industrial applications owing to its halotolerance and antibiotic and heavy metal resistance characteristics.

Anti-actin IgA antibodies in severe coeliac disease.

Science.gov (United States)

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

2004-08-01

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.

Estimation of the Binding Free Energy of AC1NX476 to HIV-1 Protease Wild Type and Mutations Using Free Energy Perturbation Method.

Science.gov (United States)

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.

Structure of the Enterovirus 71 3C Protease in Complex with NK-1.8k and Indications for the Development of Antienterovirus Protease Inhibitor.

Science.gov (United States)

Wang, Yaxin; Cao, Lin; Zhai, Yangyang; Yin, Zheng; Sun, Yuna; Shang, Luqing

2017-07-01

Hand-foot-and-mouth disease (HFMD), caused by enterovirus, is a threat to public health worldwide. To date, enterovirus 71 (EV71) has been one of the major causative agents of HFMD in the Pacific-Asia region, and outbreaks with EV71 cause millions of infections. However, no drug is currently available for clinical therapeutics. In our previous works, we developed a set of protease inhibitors (PIs) targeting the EV71 3C protease (3C pro ). Among these are NK-1.8k and NK-1.9k, which have various active groups and high potencies and selectivities. In the study described here, we determined the structures of the PI NK-1.8k in complex with wild-type (WT) and drug-resistant EV71 3C pro Comparison of these structures with the structure of unliganded EV71 3C pro and its complex with AG7088 indicated that the mutation of N69 to a serine residue destabilized the S2 pocket. Thus, the mutation influenced the cleavage activity of EV71 3C pro and the inhibitory activity of NK-1.8k in an in vitro protease assay and highlighted that site 69 is an additional key site for PI design. More information for the optimization of the P1' to P4 groups of PIs was also obtained from these structures. Together with the results of our previous works, these in-depth results elucidate the inhibitory mechanism of PIs and shed light to develop PIs for the clinical treatment of infections caused by EV71 and other enteroviruses. Copyright © 2017 American Society for Microbiology.

Earthworm Protease

Directory of Open Access Journals (Sweden)

Rong Pan

2010-01-01

Full Text Available The alimentary tract of earthworm secretes a group of proteases with a relative wide substrate specificity. In 1983, six isozymes were isolated from earthworm with fibrinolytic activities and called fibriniolytic enzymes. So far, more isozymes have been found from different earthworm species such as Lumbricus rubellus and Eisenia fetida. For convenience, the proteases are named on the basis of the earthworm species and the protein function, for instance, Eisenia fetida protease (EfP. The proteases have the abilities not only to hydrolyze fibrin and other protein, but also activate proenzymes such as plasminogen and prothrombin. In the light of recent studies, eight of the EfPs contain oligosaccharides chains which are thought to support the enzyme structure. Interestingly, EfP-II has a broader substrate specificity presenting alkaline trypsin, chymotrypsin and elastase activities, but EfP-III-1 has a stricter specificity. The protein crystal structures show the characteristics in their specificities. Earthworm proteases have been applied in several areas such as clinical treatment of clotting diseases, anti-tumor study, environmental protection and nutritional production. The current clinical utilizations and some potential new applications of the earthworm protease will be discussed in this paper.

Earthworm Protease

International Nuclear Information System (INIS)

Pan, R.; Zhang, Z.; He, R.

2010-01-01

The alimentary tract of earthworm secretes a group of proteases with a relative wide substrate specificity. In 1983, six isozymes were isolated from earthworm with fibrinolytic activities and called fibrinolytic enzymes. So far, more isozymes have been found from different earthworm species such as Lumbricus rubellus and Eisenia fetida. For convenience, the proteases are named on the basis of the earthworm species and the protein function, for instance, Eisenia fetida protease (EfP). The proteases have the abilities not only to hydrolyze fibrin and other protein, but also activate pro enzymes such as plasminogen and prothrombin. In the light of recent studies, eight of the EfPs contain oligosaccharides chains which are thought to support the enzyme structure. Interestingly, EfP-II has a broader substrate specificity presenting alkaline trypsin, chymotrypsin and elastase activities, but EfP-III-1 has a stricter specificity. The protein crystal structures show the characteristics in their specificities. Earthworm proteases have been applied in several areas such as clinical treatment of clotting diseases, anti-tumor study, environmental protection and nutritional production. The current clinical utilizations and some potential new applications of the earthworm protease will be discussed in this paper.

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

Science.gov (United States)

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.

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

Science.gov (United States)

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.

Intravenous IgA complexed with antigen reduces primary antibody response to the antigen and anaphylaxis upon antigen re-exposure by inhibiting Th1 and Th2 activation in mice.

Science.gov (United States)

Yamaki, Kouya; Miyatake, Kenji; Nakashima, Takayuki; Morioka, Ayumi; Yamamoto, Midori; Ishibashi, Yuki; Ito, Ayaka; Kuranishi, Ayu; Yoshino, Shin

2014-10-01

Serum IgG, IgE and IgM have been shown to enhance the primary antibody responses upon exposure to the soluble antigens recognized by those antibodies. However, how IgA affects these responses remains unknown. We investigated the effects of intravenously administered monoclonal IgA on the immune responses in mice. DBA/1J mice were immunized with ovalbumin in the presence or absence of anti-ovalbumin monoclonal IgA. The Th1 and Th2 immune responses to ovalbumin and the anaphylaxis induced by re-exposure to ovalbumin were measured. IgA complexed with antigen attenuated the primary antibody responses to the antigen in mice, in contrast to IgG2b and IgE. The primary antibody responses, i.e. the de novo synthesis of anti-ovalbumin IgG2a, IgG1 and IgE in the serum, and the subsequent anaphylaxis induced with re-exposure to ovalbumin were reduced by the co-injection of anti-ovalbumin monoclonal IgA at ovalbumin immunization. The Th1, Th2 and Tr1 cytokines interferon-γ, interleukin-4 and interleukin-10, respectively, released from ovalbumin-restimulated cultured splenocytes collected from allergic mice were also reduced by the treatment. The induction of interferon-γ and interleukin-4 secretion by splenocytes from ovalbumin-immunized mice stimulated in vitro with ovalbumin was also significantly reduced by the antigen complexed with anti-ovalbumin IgA. These data suggest that the direct inhibition of Th1 and Th2 activation by anti-ovalbumin monoclonal IgA participates in the inhibition of the primary antibody responses. IgA plays important immunosuppressive roles under physiological and pathological conditions and is a promising candidate drug for the treatment of immune disorders.

Recurrence and graft loss after renal transplantation in adults with IgA vasculitis.

Science.gov (United States)

Kawabe, Mayuko; Yamamoto, Izumi; Komatsuzaki, Yo; Yamakawa, Takafumi; Katsumata, Haruki; Katsuma, Ai; Mafune, Aki; Nakada, Yasuyuki; Kobayashi, Akimitsu; Tanno, Yudo; Ohkido, Ichiro; Tsuboi, Nobuo; Yokoyama, Keitaro; Horita, Shigeru; Okumi, Masayoshi; Ishida, Hideki; Yamamoto, Hiroyasu; Yokoo, Takashi; Tanabe, Kazunari

2017-08-01

IgA vasculitis, a rare condition resulting in end-stage renal disease, is a small-vessel vasculitis that affects the kidney in 49-83 % of adults. The reported recurrence rate of IgA vasculitis in renal transplant recipients is 11.5-60 %, leading to graft loss in 0-50 % of these patients. However, limited data are available on recurrence and graft loss after renal transplantation. We evaluated renal transplant recipients seen from 1987 to 2015 at the Jikei University School of Medicine and the Department of Urology, Tokyo Women's Medical University. Using a 1:2 match, 21 patients with IgA vasculitis and 42 controls were selected. The mean post-transplant follow-up was 121 ± 69 months for IgA vasculitis and 147 ± 66 months for the controls. The 15-year patient survival was 100 % in IgA vasculitis and 97.6 % in the controls (p = 0.22). The 5-, 10-, and 15-year graft survival rates were 95.2, 90.5, and 81 % in IgA vasculitis and 100, 90.5, and 88.1 % in the controls, respectively (p = 0.63). The recurrence rate was 28.6 % (6 of 21 cases) and half of them (3 of 6 cases) showed histological activity (ISKDC III). We treated them with methylprednisolone pulse therapy and/or tonsillectomy. None of the recurrence cases lost the allograft. The long-term patient and graft survival of IgA vasculitis in renal transplantation were comparable with the previous reports. The recurrence rate was 28.6 %, but none of the recurrent cases showed allograft loss in this study. We speculate that methylprednisolone pulse therapy and/or tonsillectomy prevent the progression of recurrent IgA vasculitis.

Glomerular diseases: emerging tests and therapies for IgA nephropathy.

Science.gov (United States)

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.

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

Czech Academy of Sciences Publication Activity Database

Kádek, Alan; Tretyachenko, V.; Mrázek, Hynek; Ivanova, Ljubina; Halada, Petr; Rey, M.; Schriemer, D. C.; Man, Petr

2014-01-01

Roč. 95, MAR 2014 (2014), s. 121-128 ISSN 1046-5928 R&D Projects: GA ČR GAP206/12/0503; GA MŠk(CZ) EE2.3.30.0003 Institutional support: RVO:61388971 Keywords : Plant aspartic protease * Nepenthesin * Protease characterization Subject RIV: CE - Biochemistry Impact factor: 1.695, year: 2014

Inducement of IGA/SCC in Inconel 600 steam generator tubing during unit outages

Energy Technology Data Exchange (ETDEWEB)

Durance, D.; Sedman, K. [Bruce Power, Tiverton, Ontario (Canada); Roberts, J. [CANTECH Associates Ltd., Burlington, Ontario (Canada); King, P. [Babcock and Wilcox Canada, Cambridge, Ontario (Canada); Gorman, J. [Dominion Engineering, Reston, VA (United States); Allen, R. [Kinectrics, Inc., Toronto, Ontario (Canada)

2008-07-01

The degradation of Unit 4 SG tubing by IGA/SCC has limited both the operating period and end of life predictions for Unit 4 since restart in late 2003. The circumferential IGA/SCC has been most significant in SG4 with substantial increases in both initiation and growth rates from 2005 through the spring of 2007. A detailed review of the occurrence of circumferential OD IGA/SCC at the RTZ in the HL TTS region of Bruce 4 steam generator tubes has led a conclusion that it is probable that the IGA/SCC has been the result of attack by partially reduced sulfur species such as tetrathionates and thiosulfates during periods of low temperature exposure. It is believed that attack of this type has mostly likely occurred during startup evolutions following outages as the result the development of aggressive reduced sulfur species in the TTS region during periods when the boilers were fully drained for maintenance activities. The modification of outage practices to limit secondary side oxygen ingress in the spring of 2007 has apparently arrested the degradation and has had significant affects on the allowable operating interval and end of life predictions for the entire unit. (author)

Inducement of IGA/SCC in Inconel 600 steam generator tubing during unit outages

International Nuclear Information System (INIS)

Durance, D.; Sedman, K.; Roberts, J.; King, P.; Gorman, J.; Allen, R.

2008-01-01

The degradation of Unit 4 SG tubing by IGA/SCC has limited both the operating period and end of life predictions for Unit 4 since restart in late 2003. The circumferential IGA/SCC has been most significant in SG4 with substantial increases in both initiation and growth rates from 2005 through the spring of 2007. A detailed review of the occurrence of circumferential OD IGA/SCC at the RTZ in the HL TTS region of Bruce 4 steam generator tubes has led a conclusion that it is probable that the IGA/SCC has been the result of attack by partially reduced sulfur species such as tetrathionates and thiosulfates during periods of low temperature exposure. It is believed that attack of this type has mostly likely occurred during startup evolutions following outages as the result the development of aggressive reduced sulfur species in the TTS region during periods when the boilers were fully drained for maintenance activities. The modification of outage practices to limit secondary side oxygen ingress in the spring of 2007 has apparently arrested the degradation and has had significant affects on the allowable operating interval and end of life predictions for the entire unit. (author)

PetIGA: A framework for high-performance isogeometric analysis

KAUST Repository

Dalcin, Lisandro; Collier, N.; Vignal, Philippe; Cortes, Adriano Mauricio; Calo, Victor M.

2016-01-01

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 of PetIGA, we solve some challenging nonlinear partial differential equations that include problems in both solid and fluid mechanics. We show strong scaling results on up to 40964096 cores, which confirm the suitability of PetIGA for large scale simulations.

PetIGA: A framework for high-performance isogeometric analysis

KAUST Repository

Dalcin, L.

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 of PetIGA, we solve some challenging nonlinear partial differential equations that include problems in both solid and fluid mechanics. We show strong scaling results on up to 40964096 cores, which confirm the suitability of PetIGA for large scale simulations.

Activation of Protease-Activated Receptor 2 Induces VEGF Independently of HIF-1

DEFF Research Database (Denmark)

Rasmussen, J.G.; Riis, Simone Elkjær; Frøbert, O.

2012-01-01

Human adipose stem cells (hASCs) can promote angiogenesis through secretion of proangiogenic factors such as vascular endothelial growth factor (VEGF). In other cell types, it has been shown that induction of VEGF is mediated by both protease activated receptor 2 (PAR2) and hypoxia inducible fact...

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

Science.gov (United States)

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.

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

Science.gov (United States)

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 efficacy and IgA titers for both vaccines (r(2) = 0.56; P = .005). Postimmunization anti-rotavirus IgA GMC vaccine efficacy. Efficacy during first 2 years of life was significantly lower among countries with IgA 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.

Imunocitoma IgA: A propósito de um caso clínico Immunocytoma IgA: Case report

Directory of Open Access Journals (Sweden)

Bebiana Conde

2009-01-01

Full Text Available O imunocitoma é um linfoma não Hodgkin (LNHde células B, com evolução habitualmente indolente. Representa aproximadamente 1 -3% dos LNH e atinge habitualmente adultos com mais de 50 anos, podendo manifestar-se por adenomegalias, hepatomegalia, esplenomegalia e linfocitose em 15 a 30% dos casos. Raramente tem envolvimento pulmonar. Com frequência ocorrendo picos monoclonais de imunoglobulinas, sericos, frequentemente IgM e raramente IgA. Como exemplo desta patologia apresentamos o caso clinico de um doente do sexo masculino, 52 anos, com clinica de infecções respiratórias bacterianas de repetição, com necessidade de internamentos sucessivos, cuja investigação identificou um imunocitoma IgA, estádio IV. Assumindo-se o diagnóstico de um linfoma indolente, decidiu-se iniciar terapêutica profiláctica com imunoglobulinas humanas poliespecÍficas, tendo havido diminuição das infecçoes respiratórias. Posteriormente, a evidência de progressão do linfoma condicionou o inicio de poliquimioterapia, com o esquema ciclofosfamida, vincristina, prednisolona (CVP e rituximabR, tendo-se alcançado uma resposta parcial, que se manteve durante dois anos.Immunocytoma is a non-Hodgkin’s indolent evolution B cell lymphoma. It accounts for approximately 1-3% of non-Hodgkin's limphomas and usually onsets in adults aged over 50 years old. It manifests as lymphadenopathy, splenomegaly, hepatomegaly and lymphcytosis in 15 -30% of cases and is rarely seen with pulmonary involvement. Monocloncal peaks of serum immunoglobulin often occur. These are IgM and rarely IgA. We present as an example a male patient aged 52 years old, with recurrent respiratory infections. Clinical work -up identified an immunocytoma IgA stage IV. Diagnosing an indolent lymphoma, we prophylactic polyspecific human immunoglobulin to treat the respiratory infection. Evidence of lymphoma progression leads us to prescribe combined cyclophosphamide (C, vincristine (V, prednisone (P

Construction of dengue virus protease expression plasmid and in vitro protease assay for screening antiviral inhibitors.

Science.gov (United States)

Lai, Huiguo; Teramoto, Tadahisa; Padmanabhan, Radhakrishnan

2014-01-01

Dengue virus serotypes 1-4 (DENV1-4) are mosquito-borne human pathogens of global significance causing ~390 million cases annually worldwide. The virus infections cause in general a self-limiting disease, known as dengue fever, but occasionally also more severe forms, especially during secondary infections, dengue hemorrhagic fever and dengue shock syndrome causing ~25,000 deaths annually. The DENV genome contains a single-strand positive sense RNA, approximately 11 kb in length. The 5'-end has a type I cap structure. The 3'-end has no poly(A) tail. The viral RNA has a single long open reading frame that is translated by the host translational machinery to yield a polyprotein precursor. Processing of the polyprotein precursor occurs co-translationally by cellular proteases and posttranslationally by the viral serine protease in the endoplasmic reticulum (ER) to yield three structural proteins (capsid (C), precursor membrane (prM), and envelope (E) and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The active viral protease consists of both NS2B, an integral membrane protein in the ER, and the N-terminal part of NS3 (180 amino acid residues) that contains the trypsin-like serine protease domain having a catalytic triad of H51, D75, and S135. The C-terminal part of NS3, ~170-618 amino acid residues, encodes an NTPase/RNA helicase and 5'-RNA triphosphatase activities; the latter enzyme is required for the first step in 5'-capping. The cleavage sites of the polyprotein by the viral protease consist of two basic amino acid residues such as KR, RR, or QR, followed by short chain amino acid residues, G, S, or T. Since the cleavage of the polyprotein by the viral protease is absolutely required for assembly of the viral replicase, blockage of NS2B/NS3pro activity provides an effective means for designing dengue virus (DENV) small-molecule therapeutics. Here we describe the screening of small-molecule inhibitors against DENV2 protease.

Functional characterization of the mammalian iAAA protease subunit, YME1L

OpenAIRE

Majczak, Joanna

2008-01-01

The iAAA protease is an ATP-dependent proteolytic complex in the mitochondrial inner membrane and belongs to the highly conserved family of AAA proteins. In the yeast Saccharomyces cerevisiae, the iAAA protease is a homo-oligomeric complex composed of Yme1p subunits which are active in the intermembrane space and mediate protein quality control. Yeast cells lacking Yme1p are characterized by pleiotropic phenotypes including a respiratory deficiency at elevated temperature and an aberrant mito...

Indispensable Role of Proteases in Plant Innate Immunity.

Science.gov (United States)

Balakireva, Anastasia V; Zamyatnin, Andrey A

2018-02-23

Plant defense is achieved mainly through the induction of microbe-associated molecular patterns (MAMP)-triggered immunity (MTI), effector-triggered immunity (ETI), systemic acquired resistance (SAR), induced systemic resistance (ISR), and RNA silencing. Plant immunity is a highly complex phenomenon with its own unique features that have emerged as a result of the arms race between plants and pathogens. However, the regulation of these processes is the same for all living organisms, including plants, and is controlled by proteases. Different families of plant proteases are involved in every type of immunity: some of the proteases that are covered in this review participate in MTI, affecting stomatal closure and callose deposition. A large number of proteases act in the apoplast, contributing to ETI by managing extracellular defense. A vast majority of the endogenous proteases discussed in this review are associated with the programmed cell death (PCD) of the infected cells and exhibit caspase-like activities. The synthesis of signal molecules, such as salicylic acid, jasmonic acid, and ethylene, and their signaling pathways, are regulated by endogenous proteases that affect the induction of pathogenesis-related genes and SAR or ISR establishment. A number of proteases are associated with herbivore defense. In this review, we summarize the data concerning identified plant endogenous proteases, their effect on plant-pathogen interactions, their subcellular localization, and their functional properties, if available, and we attribute a role in the different types and stages of innate immunity for each of the proteases covered.

Eosinophils may play regionally disparate roles in influencing IgA(+) plasma cell numbers during large and small intestinal inflammation.

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Forman, Ruth; Bramhall, Michael; Logunova, Larisa; Svensson-Frej, Marcus; Cruickshank, Sheena M; Else, Kathryn J

2016-05-31

Eosinophils are innate immune cells present in the intestine during steady state conditions. An intestinal eosinophilia is a hallmark of many infections and an accumulation of eosinophils is also observed in the intestine during inflammatory disorders. Classically the function of eosinophils has been associated with tissue destruction, due to the release of cytotoxic granule contents. However, recent evidence has demonstrated that the eosinophil plays a more diverse role in the immune system than previously acknowledged, including shaping adaptive immune responses and providing plasma cell survival factors during the steady state. Importantly, it is known that there are regional differences in the underlying immunology of the small and large intestine, but whether there are differences in context of the intestinal eosinophil in the steady state or inflammation is not known. Our data demonstrates that there are fewer IgA(+) plasma cells in the small intestine of eosinophil-deficient ΔdblGATA-1 mice compared to eosinophil-sufficient wild-type mice, with the difference becoming significant post-infection with Toxoplasma gondii. Remarkably, and in complete contrast, the absence of eosinophils in the inflamed large intestine does not impact on IgA(+) cell numbers during steady state, and is associated with a significant increase in IgA(+) cells post-infection with Trichuris muris compared to wild-type mice. Thus, the intestinal eosinophil appears to be less important in sustaining the IgA(+) cell pool in the large intestine compared to the small intestine, and in fact, our data suggests eosinophils play an inhibitory role. The dichotomy in the influence of the eosinophil over small and large intestinal IgA(+) cells did not depend on differences in plasma cell growth factors, recruitment potential or proliferation within the different regions of the gastrointestinal tract (GIT). We demonstrate for the first time that there are regional differences in the requirement of

Evaluation of Salivary Glucose, IgA and Flow Rate in Diabetic Patients: A Case-Control Study

OpenAIRE

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

2010-01-01

Objective: 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. Materials and Methods: 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. Nephelomet...

Radioimmunoassay for the detection of virus-specific IgA antibodies in saliva

International Nuclear Information System (INIS)

Friedman, M.G.

1982-01-01

The use of a sensitive and versatile radioimmunoassay (RIA) for detection of mumps-specific IgA and measles-specific IgA in unconcentrated saliva samples is described. The samples were obtained either by expectoration or by swabbing of the oral cavity, with or without stimulation of secretion, and were inactivated and clarified before testing. Mumps-specific IgA antibodies were detected as early as one day after onset of illness and peaked at 1-2 weeks after onset. Measles-specific salivary IgA antibodies were detected in 15-month old children 2-3 weeks after immunization. These results suggest that the RIA technique may be useful for early diagnosis of viral infections and for confirmation of response to immunization without the need for a blood sample, as well as for the study of the secretory immune response in very young and older subjects. (Auth.)

Low-dose oral tolerance due to antigen in the diet suppresses differentially the cholera toxin-adjuvantized IgE, IgA and IgG response

DEFF Research Database (Denmark)

Christensen, Hanne Risager; Kjær, Tanja; Frøkiær, Hanne

2003-01-01

Background: Cholera toxin (CT) is used as a mucosal adjuvant amongst other applications for studying food allergy because oral administration of antigen with CT induces an antigen-specific type 2 response, including IgE and IgA production. Priorly established oral tolerance due to antigen...... soy-trypsin inhibitor (KSTI) (F0 mice) and mice fed a soy-free diet (F2 mice) were orally immunized with KSTI and CT. KSTI-specific serum IgG1, IgG2a, IgA and IgE and fecal IgA were monitored. KSTI-stimulated cell proliferation and interleukin (IL)-6 production were determined. Results: The anti...... immunizations. However, cell proliferation and IL-6 production were clearly suppressed even after five immunizations. Conclusions: Priorly established low-dose oral tolerance considerably suppressed the CT-adjuvantized KSTI-specific IgE, IgA and cellular immune response but only weakly and transiently the Ig...

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

Science.gov (United States)

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.

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

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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.

HvPap-1 C1A Protease Participates Differentially in the Barley Response to a Pathogen and an Herbivore

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Mercedes Diaz-Mendoza

2017-09-01

Full Text Available Co-evolutionary processes in plant–pathogen/herbivore systems indicate that protease inhibitors have a particular value in biotic interactions. However, little is known about the defensive role of their targets, the plant proteases. C1A cysteine proteases are the most abundant enzymes responsible for the proteolytic activity during different processes like germination, development and senescence in plants. To identify and characterize C1A cysteine proteases of barley with a potential role in defense, mRNA and protein expression patterns were analyzed in response to biotics stresses. A barley cysteine protease, HvPap-1, previously related to abiotic stresses and grain germination, was particularly induced by flagellin or chitosan elicitation, and biotic stresses such as the phytopathogenic fungus Magnaporthe oryzae or the phytophagous mite Tetranychus urticae. To elucidate the in vivo participation of this enzyme in defense, transformed barley plants overexpressing or silencing HvPap-1 encoding gene were subjected to M. oryzae infection or T. urticae infestation. Whereas overexpressing plants were less susceptible to the fungus than silencing plants, the opposite behavior occurred to the mite. This unexpected result highlights the complexity of the regulatory events leading to the response to a particular biotic stress.

Early pre-eclampsia unmasks underlying IgA nephropathy

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Mona Singh

2010-12-01

Full Text Available Mona Singh, Akhenaton Pappoe, Burl R DonDivision of Nephrology, University of California Davis Medical Center, Sacramento, CA, USAAbstract: Pre-eclampsia is the most ominous complication of pregnancy, and primary glomerular diseases can mimic pre-eclampsia in presentation. A patient presented at 21 weeks gestation with signs and symptoms of both pre-eclampsia and primary glomerular nephropathy. A critical clinical decision whether to continue or terminate the pregnancy was dependent on results of a renal biopsy. The biopsy noted the presence of both pre-eclampsia and immunoglobulin A (IgA nephropathy. Thus, the onset of pre-eclampsia unmasked the presence of unrecognized IgA nephropathy, and the IgA nephropathy was a risk factor for this patient developing pre-eclampsia. The results of a renal biopsy are key in distinguishing pre-eclampsia from other kidney diseases and instituting appropriate clinical management.Keywords: proteinuria, IgA nephropathy, renal biopsy, pre-eclampsia

Mosaic serine proteases in the mammalian central nervous system.

Science.gov (United States)

Mitsui, Shinichi; Watanabe, Yoshihisa; Yamaguchi, Tatsuyuki; Yamaguchi, Nozomi

2008-01-01

We review the structure and function of three kinds of mosaic serine proteases expressed in the mammalian central nervous system (CNS). Mosaic serine proteases have several domains in the proenzyme fragment, which modulate proteolytic function, and a protease domain at the C-terminus. Spinesin/TMPRSS5 is a transmembrane serine protease whose presynaptic distribution on motor neurons in the spinal cord suggests that it is significant for neuronal plasticity. Cell type-specific alternative splicing gives this protease diverse functions by modulating its intracellular localization. Motopsin/PRSS12 is a mosaic protease, and loss of its function causes mental retardation. Recent reports indicate the significance of this protease for cognitive function. We mention the fibrinolytic protease, tissue plasminogen activator (tPA), which has physiological and pathological functions in the CNS.

A sparse version of IGA solvers

KAUST Repository

Beck, Joakim; Sangalli, Giancarlo; Tamellini, Lorenzo

2017-01-01

Isogeometric Analysis (IGA) typically adopts tensor-product splines and NURBS as a basis for the approximation of the solution of PDEs. In this work, we investigate to which extent IGA solvers can benefit from the so-called sparse-grids construction in its combination technique form, which was first introduced in the early 90s in the context of the approximation of high-dimensional PDEs. The tests that we report show that, in accordance to the literature, a sparse grids construction can indeed be useful if the solution of the PDE at hand is sufficiently smooth. Sparse grids can also be useful in the case of non-smooth solutions when some a-priori knowledge on the location of the singularities of the solution can be exploited to devise suitable non-equispaced meshes. Finally, we remark that sparse grids can be seen as a simple way to parallelize pre-existing serial IGA solvers in a straightforward fashion, which can be beneficial in many practical situations.

A sparse version of IGA solvers

KAUST Repository

Beck, Joakim

2017-07-30

Isogeometric Analysis (IGA) typically adopts tensor-product splines and NURBS as a basis for the approximation of the solution of PDEs. In this work, we investigate to which extent IGA solvers can benefit from the so-called sparse-grids construction in its combination technique form, which was first introduced in the early 90s in the context of the approximation of high-dimensional PDEs. The tests that we report show that, in accordance to the literature, a sparse grids construction can indeed be useful if the solution of the PDE at hand is sufficiently smooth. Sparse grids can also be useful in the case of non-smooth solutions when some a-priori knowledge on the location of the singularities of the solution can be exploited to devise suitable non-equispaced meshes. Finally, we remark that sparse grids can be seen as a simple way to parallelize pre-existing serial IGA solvers in a straightforward fashion, which can be beneficial in many practical situations.

A cyclic peptidic serine protease inhibitor

DEFF Research Database (Denmark)

Zhao, Baoyu; Xu, Peng; Jiang, Longguang

2014-01-01

Peptides are attracting increasing interest as protease inhibitors. Here, we demonstrate a new inhibitory mechanism and a new type of exosite interactions for a phage-displayed peptide library-derived competitive inhibitor, mupain-1 (CPAYSRYLDC), of the serine protease murine urokinase...... pocket, its carbonyl group aligning improperly relative to Ser195 and the oxyanion hole, explaining why the peptide is an inhibitor rather than a substrate. Substitution of the P1 Arg with novel unnatural Arg analogues with aliphatic or aromatic ring structures led to an increased affinity, depending......, in spite of a less favorable binding entropy and loss of a polar interaction. We conclude that increased flexibility of the peptide allows more favorable exosite interactions, which, in combination with the use of novel Arg analogues as P1 residues, can be used to manipulate the affinity and specificity...

Microarray analysis of thioacetamide-treated type 1 diabetic rats

International Nuclear Information System (INIS)

Devi, Sachin S.; Mehendale, Harihara M.

2006-01-01

It is well known that diabetes imparts high sensitivity to numerous hepatotoxicants. Previously, we have shown that a normally non-lethal dose of thioacetamide (TA, 300 mg/kg) causes 90% mortality in type 1 diabetic (DB) rats due to inhibited tissue repair allowing progression of liver injury. On the other hand, DB rats exposed to 30 mg TA/kg exhibit delayed tissue repair and delayed recovery from injury. The objective of this study was to investigate the mechanism of impaired tissue repair and progression of liver injury in TA-treated DB rats by using cDNA microarray. Gene expression pattern was examined at 0, 6, and 12 h after TA challenge, and selected mechanistic leads from microarray experiments were confirmed by real-time RT-PCR and further investigated at protein level over the time course of 0 to 36 h after TA treatment. Diabetic condition itself increased gene expression of proteases and decreased gene expression of protease inhibitors. Administration of 300 mg TA/kg to DB rats further elevated gene expression of proteases and suppressed gene expression of protease inhibitors, explaining progression of liver injury in DB rats after TA treatment. Inhibited expression of genes involved in cell division cycle (cyclin D1, IGFBP-1, ras, E2F) was observed after exposure of DB rats to 300 mg TA/kg, explaining inhibited tissue repair in these rats. On the other hand, DB rats receiving 30 mg TA/kg exhibit delayed expression of genes involved in cell division cycle, explaining delayed tissue repair in these rats. In conclusion, impaired cyclin D1 signaling along with increased proteases and decreased protease inhibitors may explain impaired tissue repair that leads to progression of liver injury initiated by TA in DB rats

Gluten sensitivity in patients with IgA nephropathy.

Science.gov (United States)

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.

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

Energy Technology Data Exchange (ETDEWEB)

Ghosh,A.; Sridhar, P.; Leshchenko, S.; Hussain, A.; Li, J.; Kovalevsky, A.; Walters, D.; Wedelind, J.; Grum-Tokars, V.; et al.

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. 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.

IgA attenuates anaphylaxis and subsequent immune responses in mice: possible application of IgA to vaccines.

Science.gov (United States)

Yamaki, Kouya; Nakashima, Takayuki; Miyatake, Kenji; Ishibashi, Yuki; Ito, Ayaka; Kuranishi, Ayu; Taguchi, Akihito; Morioka, Ayumi; Yamamoto, Midori; Yoshino, Shin

2014-01-01

Administration of the influenza vaccination to patients with an egg allergy is major health concern. Contaminating egg antigens occasionally induce severe anaphylactic shock in these patients following administration of the vaccination; therefore, the development of a safer vaccination is needed. In the present study, we investigated whether a mixture of four newly and previously generated anti-ovalbumin (OVA) IgA monoclonal antibodies (mAbs) could inhibit both anaphylactic shock upon a subcutaneous OVA challenge and subsequent further sensitization against OVA in passively anti-OVA IgE-sensitized mice and actively sensitized mice with an injection of OVA. The prevention of anaphylaxis by anti-OVA IgA mAbs was suggested to be mediated through the inhibition of OVA binding to allergenic antibodies such as anti-OVA IgE on mast cells and deceleration of the rate of OVA penetration from the injected site into the systemic circulation. Anti-OVA IgA mAbs inhibited further sensitization against OVA in mice actively sensitized with OVA, but did not affect sensitization against the unrelated antigen, phosphorylcholine-keyhole limpet hemocyanin co-injected with OVA. Our findings indicate that adding the anti-egg antigen IgA to the influenza vaccine should reduce not only the risk of inducing anaphylactic shock, but also undesired further sensitization against egg antigens following the vaccination without affecting the intended beneficial effect of the vaccine, namely the upregulation of immune responses to influenza viruses.

Linear IgA bullous dermatosis in a neonate.

Science.gov (United States)

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.

Pathological Role of Tonsillar B Cells in IgA Nephropathy

Directory of Open Access Journals (Sweden)

Yusuke Suzuki

2011-01-01

Full Text Available Although impaired immune regulation along the mucosa-bone marrow axis has been postulated to play an important role, the pathogenesis of IgA nephropathy (IgAN is unknown; thus, no disease-specific therapy for this disease exists. The therapeutic efficacy of tonsillectomy or tonsillectomy in combination with steroid pulse therapy for IgAN has been discussed. Although randomized control trials for these therapies are ongoing in Japan, the scientific rationale for these therapies remains obscure. It is now widely accepted that abnormally glycosylated IgA1 and its related immune complex (IC are probably key molecules for the pathogenesis, and are thus considered possible noninvasive biomarkers for this disease. Emerging evidence indicates that B cells in mucosal infections, particularly in tonsillitis, may produce the nephritogenic IgA. In this paper, we briefly summarize characteristics of the nephritogenic IgA/IgA IC, responsible B cells, and underlying mechanisms. This clinical and experimental information may provide important clues for a therapeutic rationale.

IgA against gut-derived endotoxins: does it contribute to suppression of hepatic inflammation in alcohol-induced liver disease?

DEFF Research Database (Denmark)

Parlesak, Alexandr; Schäfer, C.; Bode, C.

2002-01-01

Endotoxins of intestinal origin are supposed to play an important role in the development of alcoholic hepatitis in man. To estimate the role of immunoglobulin response to gut-derived endotoxin in the development of alcohol-induced liver disease, serum levels of IgA and IgG against fecal endotoxin......, endotoxin, and acute-phase proteins were measured in patients with different stages of alcoholic liver disease and in healthy controls. Antibodies of type IgA, but not IgG, against fecal endotoxins were significantly increased in patients with alcohol-induced liver disease. IgA antibodies against fecal...... endotoxin were found to be closely correlated with the plasma concentrations of alanine aminotransferase, gamma-glutamyl transferase, and C-reactive protein in patients with alcoholic liver disease. In conclusion, as IgA located in body tissue was shown to suppress the inflammatory process, enhanced...

Processing sites in the human immunodeficiency virus type 1 (HIV-1) Gag-Pro-Pol precursor are cleaved by the viral protease at different rates.

Science.gov (United States)

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.

Prevalence of serological markers for celiac disease (IgA and IgG class antigliadin antibodies and IgA class antiendomysium antibodies in patients with autoimmune rheumatologic diseases in Belo Horizonte, MG, Brazil Pesquisa de anticorpos antigliadina (classes IgA e IgG e anticorpos antiendomísio classe IgA, em pacientes com doenças reumatológicas autoimunes em Belo Horizonte, Brasil

Directory of Open Access Journals (Sweden)

Victor de Barros Koehne

2010-09-01

Full Text Available CONTEXT: Patients with autoimmune rheumatologic conditions and celiac disease tend to have a variety of autoantibodies, many of which have no clear pathogenic role. The literature contains frequent reports of celiac disease being more prevalent in patients with rheumatologic diseases, although this remains controversial. OBJECTIVES: To investigate the prevalence of positive serum tests for celiac disease, particularly IgA and IgG antigliadin (AGA antibodies and IgA antiendomysium antibodies (EmA in patients with autoimmune rheumatologic diseases. A second aim was to correlate positive serum tests with prednisone and immunosuppressant medication. METHODS: A total of 190 adults and pediatric patients with a variety of autoimmune rheumatologic diseases (systemic lupus erythematosus, rheumatoid arthritis, juvenile rheumatoid arthritis and spondyloarthrophathies were evaluated and tested for IgA and IgG antigliadin-antibodies and IgA antiendomysium antibodies. Patients with positive serum tests underwent endoscopic duodenal biopsies for pathology studies. RESULTS: There were four positive sera (2.1% for AGA IgA, all of which tested negative for AGA IgG and EmA. Three sera (1.6% tested positive for AGA IgG; all were negative for AGA IgA and EmA. The EmA test at a 1:2.5 serum dilution tested positive in 94 patients (49.5%; at a 1:5 serum dilution it was positive in 41 patients (21.6%. Eleven subjects tested positive for EmA at 1:40 dilution; and all of these tested negative for IgA tissue antitransglutaminase (tTG antibodies. Nine of the 11 EmA-positive patients and all 7 patients with positive antigliadin antibodies tests underwent duodenal endoscopic biopsies, and no significant changes were demonstrated in their duodenal mucosa. A positive EmA was associated with elevated optical density AGA IgA readings; however, there was no relationship between positive EmA and AGA IgG optical density readings. Prednisone and immunosuppressant use were unrelated

Vibrio Type III Effector VPA1380 Is Related to the Cysteine Protease Domain of Large Bacterial Toxins

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Calder, Thomas; Kinch, Lisa N.; Fernandez, Jessie; Salomon, Dor; Grishin, Nick V.; Orth, Kim

2014-01-01

Vibrio parahaemolyticus is a Gram-negative halophilic bacterium and one of the leading causes of food-borne gastroenteritis. Its genome harbors two Type III Secretion Systems (T3SS1 and T3SS2), but only T3SS2 is required for enterotoxicity seen in animal models. Effector proteins secreted from T3SS2 have been previously shown to promote colonization of the intestinal epithelium, invasion of host cells, and destruction of the epithelial monolayer. In this study, we identify VPA1380, a T3SS2 effector protein that is toxic when expressed in yeast. Bioinformatic analyses revealed that VPA1380 is highly similar to the inositol hexakisphosphate (IP6)-inducible cysteine protease domains of several large bacterial toxins. Mutations in conserved catalytic residues and residues in the putative IP6-binding pocket abolished toxicity in yeast. Furthermore, VPA1380 was not toxic in IP6 deficient yeast cells. Therefore, our findings suggest that VPA1380 is a cysteine protease that requires IP6 as an activator. PMID:25099122

Vibrio type III effector VPA1380 is related to the cysteine protease domain of large bacterial toxins.

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Thomas Calder

Full Text Available Vibrio parahaemolyticus is a Gram-negative halophilic bacterium and one of the leading causes of food-borne gastroenteritis. Its genome harbors two Type III Secretion Systems (T3SS1 and T3SS2, but only T3SS2 is required for enterotoxicity seen in animal models. Effector proteins secreted from T3SS2 have been previously shown to promote colonization of the intestinal epithelium, invasion of host cells, and destruction of the epithelial monolayer. In this study, we identify VPA1380, a T3SS2 effector protein that is toxic when expressed in yeast. Bioinformatic analyses revealed that VPA1380 is highly similar to the inositol hexakisphosphate (IP6-inducible cysteine protease domains of several large bacterial toxins. Mutations in conserved catalytic residues and residues in the putative IP6-binding pocket abolished toxicity in yeast. Furthermore, VPA1380 was not toxic in IP6 deficient yeast cells. Therefore, our findings suggest that VPA1380 is a cysteine protease that requires IP6 as an activator.

A sensitive fluorescence reporter for monitoring quorum sensing regulated protease production in Vibrio harveyi.

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Rajamani, Sathish; Sayre, Richard T

2011-02-01

Many bacteria produce and secrete proteases during host invasion and pathogenesis. Vibrio harveyi, an opportunistic pathogen of shrimp, is known to use a two-component quorum sensing (QS) mechanism for coordination of gene expression including protease secretion at high population densities. We examined the role of V. harveyi's QS signaling molecules, N-(3-hydroxybutanoyl)-L-homoserine lactone (AI-1) and the boron derivative of autoinducer-2 (BAI-2) in extracellular protease production. A fusion protein, M3CLPY (Rajamani et al., 2007), consisting of a large protease sensitive BAI-2 mutant receptor LuxP (~38kDa) flanked by two protease insensitive cyan and yellow variants of GFP (~28kDa each) was utilized as a substrate to detect secreted protease activity. The M3CLPY fusion, with the addition of wild-type V. harveyi (BB120) cell-free culture filtrate showed a time-dependent loss in fluorescence resonance energy transfer (FRET) associated with the cleavage of the LuxP linker protein and hence separation of the two fluorophores. This cleavage of LuxP linker protein leading to decreased FRET efficiency was further confirmed by immunoblotting using anti-GFP antibody. The addition of cell-free filtrates from strains defective in one or both of the two-component QS pathways: luxN(-) (defective in AI-1), luxS(-) (defective in BAI-2), and luxN(-)/luxS(-) (defective in both AI-1/BAI-2) showed differential levels of protease production. The observed protease activities were most pronounced in wild-type, followed by the AI-1 defective mutant (BB170) and the least for luxS(-) mutant (MM30) and luxN(-)/luxS(-) double mutant (MM32) strains. Incidentally, the lowest protease producing strains MM30 and MM32 were both defective in BAI-2 production. This observation was validated by addition of synthetic BAI-2 to MM30 and MM32 strains to restore protease production. Our results indicate that BAI-2 signaling in the two-component QS pathway plays the key role in regulating

HIV-1 specific IgA detected in vaginal secretions of HIV uninfected women participating in a microbicide trial in Southern Africa are primarily directed toward gp120 and gp140 specificities.

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Kelly E Seaton

Full Text Available Many participants in microbicide trials remain uninfected despite ongoing exposure to HIV-1. Determining the emergence and nature of mucosal HIV-specific immune responses in such women is important, since these responses may contribute to protection and could provide insight for the rational design of HIV-1 vaccines.We first conducted a pilot study to compare three sampling devices (Dacron swabs, flocked nylon swabs and Merocel sponges for detection of HIV-1-specific IgG and IgA antibodies in vaginal secretions. IgG antibodies from HIV-1-positive women reacted broadly across the full panel of eight HIV-1 envelope (Env antigens tested, whereas IgA antibodies only reacted to the gp41 subunit. No Env-reactive antibodies were detected in the HIV-negative women. The three sampling devices yielded equal HIV-1-specific antibody titers, as well as total IgG and IgA concentrations. We then tested vaginal Dacron swabs archived from 57 HIV seronegative women who participated in a microbicide efficacy trial in Southern Africa (HPTN 035. We detected vaginal IgA antibodies directed at HIV-1 Env gp120/gp140 in six of these women, and at gp41 in another three women, but did not detect Env-specific IgG antibodies in any women.Vaginal secretions of HIV-1 infected women contained IgG reactivity to a broad range of Env antigens and IgA reactivity to gp41. In contrast, Env-binding antibodies in the vaginal secretions of HIV-1 uninfected women participating in the microbicide trial were restricted to the IgA subtype and were mostly directed at HIV-1 gp120/gp140.

Evolution of IgA nephropathy into anaphylactoid purpura in six cases--further evidence that IgA nephropathy and Henoch-Schonlein purpura nephritis share common pathogenesis.

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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.

Production, purification and characterization of an aspartic protease from Aspergillus foetidus.

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Souza, Paula Monteiro; Werneck, Gabriela; Aliakbarian, Bahar; Siqueira, Felix; Ferreira Filho, Edivaldo Ximenes; Perego, Patrizia; Converti, Attilio; Magalhães, Pérola Oliveira; Junior, Adalberto Pessoa

2017-11-01

An acidic thermostable protease was extracellularly produced either in shake flask or in stirred tank bioreactor by an Aspergillus foetidus strain isolated from the Brazilian savanna soil using different nitrogen sources. Its maximum activity (63.7 U mL -1 ) was obtained in a medium containing 2% (w/v) peptone. A cultivation carried out in a 5.0 L stirred-tank bioreactor provided a maximum protease activity 9% lower than that observed in Erlenmeyer flasks, which was obtained after a significantly shorter (by 16-29%) time. Protease purification by a combination of gel-filtration chromatography resulted in a 16.9-fold increase in specific activity (248.1 U g -1 ). The estimated molecular weight of the purified enzyme was 50.6 kDa, and the optimal pH and temperature were 5.0 and 55 °C, respectively. The enzyme was completely inhibited by pepstatin A, and its activity enhanced by some metals. According to the inhibition profiles, it was confirmed that the purified acid protease belongs to the aspartic protease type. These results are quite promising for future development of large-scale production of such protease, which can be useful in biotechnological applications requiring high enzyme activity and stability under acidic conditions. Copyright © 2017 Elsevier Ltd. All rights reserved.

Recombinant IgA Is Sufficient To Prevent Influenza Virus Transmission in Guinea Pigs

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Seibert, Christopher W.; Rahmat, Saad; Krause, Jens C.; Eggink, Dirk; Albrecht, Randy A.; Goff, Peter H.; Krammer, Florian; Duty, J. Andrew; Bouvier, Nicole M.; García-Sastre, Adolfo

2013-01-01

A serum hemagglutination inhibition (HAI) titer of 40 or greater is thought to be associated with reduced influenza virus pathogenesis in humans and is often used as a correlate of protection in influenza vaccine studies. We have previously demonstrated that intramuscular vaccination of guinea pigs with inactivated influenza virus generates HAI titers greater than 300 but does not protect vaccinated animals from becoming infected with influenza virus by transmission from an infected cage mate. Only guinea pigs intranasally inoculated with a live influenza virus or a live attenuated virus vaccine, prior to challenge, were protected from transmission (A. C. Lowen et al., J. Virol. 83:2803–2818, 2009.). Because the serum HAI titer is mostly determined by IgG content, these results led us to speculate that prevention of viral transmission may require IgA antibodies or cellular immune responses. To evaluate this hypothesis, guinea pigs and ferrets were administered a potent, neutralizing mouse IgG monoclonal antibody, 30D1 (Ms 30D1 IgG), against the A/California/04/2009 (H1N1) virus hemagglutinin and exposed to respiratory droplets from animals infected with this virus. Even though HAI titers were greater than 160 1 day postadministration, Ms 30D1 IgG did not prevent airborne transmission to passively immunized recipient animals. In contrast, intramuscular administration of recombinant 30D1 IgA (Ms 30D1 IgA) prevented transmission to 88% of recipient guinea pigs, and Ms 30D1 IgA was detected in animal nasal washes. Ms 30D1 IgG administered intranasally also prevented transmission, suggesting the importance of mucosal immunity in preventing influenza virus transmission. Collectively, our data indicate that IgG antibodies may prevent pathogenesis associated with influenza virus infection but do not protect from virus infection by airborne transmission, while IgA antibodies are more important for preventing transmission of influenza viruses. PMID:23698296

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

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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.

PROTEOLYTIC PROCESSING OF VON WILLEBRAND FACTOR BY ADAMTS13 AND LEUKOCYTE PROTEASES

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Stefano Lancellotti

2013-09-01

Full Text Available ADAMTS13 is a 190 kDa zinc protease encoded by a gene located on chromosome 9q34.   This protease specifically hydrolyzes von Willebrand factor (VWF multimers, thus causing VWF size reduction. ADAMTS13 belongs to the A Disintegrin And Metalloprotease with ThromboSpondin type 1 repeats (ADAMTS family, involved in proteolytic processing of many matrix proteins. ADAMTS13 consists of numerous domains including a metalloprotease domain, a disintegrin domain, several thrombospondin type 1 (TSP1 repeats, a cysteine-rich domain, a spacer domain and 2 CUB (Complement c1r/c1s, sea Urchin epidermal growth factor, and Bone morphogenetic protein domains. ADAMTS13 cleaves a single peptide bond (Tyr1605-Met1606 in the central A2 domain of the VWF molecule. This proteolytic cleavage is essential to reduce the size of ultra-large VWF polymers, which, when exposed to high shear stress in the microcirculation, are prone to form with platelets clumps, which cause severe syndromes called thrombotic microangiopathies (TMAs. In this review, we a discuss the current knowledge of structure-function aspects of ADAMTS13 and its involvement in the pathogenesis of TMAs, b address the recent findings concerning proteolytic processing of VWF multimers by different proteases, such as the leukocyte-derived serine and metallo-proteases and c indicate the direction of future investigations

Dendritic cells support production of IgA and other non-IgM isotypes in clonal microculture.

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Schrader, C E; George, A; Kerlin, R L; Cebra, J J

1990-01-01

Microcultures of helper T (Th) cells and a few appropriately primed murine B cells can be used to detect cognate T-B interactions which lead to clonal production of IgM, IgG1, and IgE. However, IgG2, IgG3, and IgA are very rarely expressed. We have found that the addition of dendritic cells to such cultures creates an extremely supportive environment for clones expressing IgA with other isotypes, as well as clones expressing only detectable IgA. Typically, 400 dendritic cells were added to 3000 conalbumin-specific Th cells (D10.G4.1) and 30 hapten-specific Peyer's patch (PP) B cells with antigen in 15 microliters. The response was antigen dependent and clonal. Almost half of the clones expressed only non-IgM isotypes, 43% expressed some IgA, and 14% expressed some IgG3; isotype diversity increased over time. Dendritic cells from PP and spleen were found to be equally supportive, and allowed the number of T cells required in microculture to be decreased from 3000 to 400. However, T cell proliferation was not required for the supportive effect of dendritic cells. Surface IgD-bearing cells were also found to switch to IgA production in microculture as judged by their generating clones expressing IgM along with IgA and other isotypes. Again, IgA was usually expressed only in the presence of dendritic cells. The mechanism may involve dendritic cell-induced T cell activation and/or dendritic cell factors, and is under investigation.

High-level expression of alkaline protease using recombinant ...

African Journals Online (AJOL)

AJL

2012-02-16

Feb 16, 2012 ... compared with that of wild-type B. licheniformis CICIM B5102. Key word: Alkaline protease, Bacillus amyloliquefaciens, Bacillus licheniformis. INTRODUCTION. Proteases are one of the most important industrial enzyme groups, accounting for approximately 60% of the total enzyme sales (Beg et al., 2003).

Igaühele hea haridus!? / Olav Aarna

Index Scriptorium Estoniae

Aarna, Olav, 1942-

2005-01-01

Ilmunud ka: Järva Teataja 1. september lk. 2, Meie Maa 1. september lk. 2, Koit 1. september lk. 6, Severnoje Poberezhje 1. september lk. 2, Vali Uudised 2. september lk. 2, Sakala 2. september lk. 2, Hiiu Leht 2. september lk. 2, Kuulutaja 2. september lk. 4, Nädaline 8. september lk. 4. Riigikogu kultuurikomisjoni esimehe sõnul peaks igaüks sõnastama enda jaoks rahuldava vastuse küsimusele, mis on hea haridus

ReFlexIn: a flexible receptor protein-ligand docking scheme evaluated on HIV-1 protease.

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Simon Leis

Full Text Available For many targets of pharmaceutical importance conformational changes of the receptor protein are relevant during the ligand binding process. A new docking approach, ReFlexIn (Receptor Flexibility by Interpolation, that combines receptor flexibility with the computationally efficient potential grid representation of receptor molecules has been evaluated on the retroviral HIV-1 (Human Immunodeficiency Virus 1 protease system. An approximate inclusion of receptor flexibility is achieved by using interpolation between grid representations of individual receptor conformations. For the retroviral protease the method was tested on an ensemble of protease structures crystallized in the presence of different ligands and on a set of structures obtained from morphing between the unbound and a ligand-bound protease structure. Docking was performed on ligands known to bind to the protease and several non-binders. For the binders the ReFlexIn method yielded in almost all cases ligand placements in similar or closer agreement with experiment than docking to any of the ensemble members without degrading the discrimination with respect to non-binders. The improved docking performance compared to docking to rigid receptors allows for systematic virtual screening applications at very small additional computational cost.

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

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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 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 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 are unevenly distributed globally, and there are reported differences in their rates of disease progression and epidemic spread. The biological determinants underlying these differences have not been fully elucidated. Here, we show that

Correlation between saliva IgA level and T cell CD4+ in HIV/AIDS patients

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Irna Sufiawati

2007-07-01

Full Text Available Background: HIV infection appears to have direct effects on oral mucosal immunity, cellular and humoral. Antibody secretion, especially salivary immunoglobulin A (IgA, is a useful indicator of mucosal immune function. This immune system component is recognized as an important first-line of defence against pathogens which colonize and invade mucosal surfaces in the oral cavity. Objectives: The purpose of this study was to investigate salivary IgA levels and to determine its correlation with CD4+ T-cell counts among HIV-infected patients in Pokdisus AIDS Cipto Mangunkusomo Hospital Jakarta. Methods: The design study was using a cross-sectional study. Whole paraffin-wax-stimulated saliva was collected from 103 HIV-infected patients and 30 healthy individuals. Saliva was collected using the spitting method. Salivary IgA levels were determined by the immunoturbidimetry method using the Behring Turbitimer Analyser. CD4+ T-cell counts were analyzed by flow cytometry. Results: Salivary IgA levels were 141.55 ± 83.23 (HIV group and 97.24 ± 38.25 (healthy individuals. The Mann-Whitney U test showed salivary IgA levels were significantly higher in HIV/AIDS subjects compared with healthy individuals (p0.1. Conclusion: This study indicates that total salivary IgA levels were significantly higher in the HIV-infected patients compared to control, and salivary IgA level seems not to be related significantly to CD4+ T-cell counts.

Correlation of Serum Anti- Helicobacter pylori Immunoglobulin A (IGA)

African Journals Online (AJOL)

Background: The seroprevalence of anti-H. pylori IgA antibodies has been reported to vary among populations and in relation to strains of Helicobacter pylori bacterium. However, there has been conflicting reports on the association between IgA serological status and the histological variables of chronic gastritis. This study ...

Large Scale Generation and Characterization of Anti-Human IgA Monoclonal Antibody in Ascitic Fluid of Balb/c Mice

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Ezzatifar, Fatemeh; Majidi, Jafar; Baradaran, Behzad; Aghebati Maleki, Leili; Abdolalizadeh, Jalal; Yousefi, Mehdi

2015-01-01

Purpose: Monoclonal antibodies are potentially powerful tools used in biomedical research, diagnosis, and treatment of infectious diseases and cancers. The monoclonal antibody against Human IgA can be used as a diagnostic application to detect infectious diseases. The aim of this study was to improve an appropriate protocol for large-scale production of mAbs against IgA. Methods: For large-scale production of the monoclonal antibody, hybridoma cells that produce monoclonal antibodies against Human IgA were injected intraperitoneally into Balb/c mice that were previously primed with 0.5 ml Pristane. After ten days, ascitic fluid was harvested from the peritoneum of each mouse. The ELISA method was carried out for evaluation of the titration of produced mAbs. The ascitic fluid was investigated in terms of class and subclass by a mouse mAb isotyping kit. MAb was purified from the ascitic fluid by ion exchange chromatography. The purity of the monoclonal antibody was confirmed by SDS-PAGE, and the purified monoclonal antibody was conjugated with HRP. Results: Monoclonal antibodies with high specificity and sensitivity against Human IgA were prepared by hybridoma technology. The subclass of antibody was IgG1 and its light chain was the kappa type. Conclusion: This conjugated monoclonal antibody could have applications in designing ELISA kits in order to diagnose different infectious diseases such as toxoplasmosis and H. Pylori. PMID:25789225

Large Scale Generation and Characterization of Anti-Human IgA Monoclonal Antibody in Ascitic Fluid of Balb/c Mice

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Fatemeh Ezzatifar

2015-03-01

Full Text Available Purpose: Monoclonal antibodies are potentially powerful tools used in biomedical research, diagnosis, and treatment of infectious diseases and cancers. The monoclonal antibody against Human IgA can be used as a diagnostic application to detect infectious diseases. The aim of this study was to improve an appropriate protocol for large-scale production of mAbs against IgA. Methods: For large-scale production of the monoclonal antibody, hybridoma cells that produce monoclonal antibodies against Human IgA were injected intraperitoneally into Balb/c mice that were previously primed with 0.5 ml Pristane. After ten days, ascitic fluid was harvested from the peritoneum of each mouse. The ELISA method was carried out for evaluation of the titration of produced mAbs. The ascitic fluid was investigated in terms of class and subclass by a mouse mAb isotyping kit. MAb was purified from the ascitic fluid by ion exchange chromatography. The purity of the monoclonal antibody was confirmed by SDS-PAGE, and the purified monoclonal antibody was conjugated with HRP. Results: Monoclonal antibodies with high specificity and sensitivity against Human IgA were prepared by hybridoma technology. The subclass of antibody was IgG1 and its light chain was the kappa type. Conclusion: This conjugated monoclonal antibody could have applications in designing ELISA kits in order to diagnose different infectious diseases such as toxoplasmosis and H. Pylori.

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

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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.

Processing sites in the human immunodeficiency virus type 1 (HIV-1 Gag-Pro-Pol precursor are cleaved by the viral protease at different rates

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Lindquist Jeffrey N

2005-11-01

Full Text Available Abstract 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.

Epigenetic silencing of serine protease HTRA1 drives polyploidy

International Nuclear Information System (INIS)

Schmidt, Nina; Irle, Inga; Ripkens, Kamilla; Lux, Vanda; Nelles, Jasmin; Johannes, Christian; Parry, Lee; Greenow, Kirsty; Amir, Sarah; Campioni, Mara; Baldi, Alfonso; Oka, Chio; Kawaichi, Masashi; Clarke, Alan R.; Ehrmann, Michael

2016-01-01

Increased numbers and improperly positioned centrosomes, aneuploidy or polyploidy, and chromosomal instability are frequently observed characteristics of cancer cells. While some aspects of these events and the checkpoint mechanisms are well studied, not all players have yet been identified. As the role of proteases other than the proteasome in tumorigenesis is an insufficiently addressed question, we investigated the epigenetic control of the widely conserved protease HTRA1 and the phenotypes of deregulation. Mouse embryonal fibroblasts and HCT116 and SW480 cells were used to study the mechanism of epigenetic silencing of HTRA1. In addition, using cell biological and genetic methods, the phenotypes of downregulation of HTRA1 expression were investigated. HTRA1 is epigenetically silenced in HCT116 colon carcinoma cells via the epigenetic adaptor protein MBD2. On the cellular level, HTRA1 depletion causes multiple phenotypes including acceleration of cell growth, centrosome amplification and polyploidy in SW480 colon adenocarcinoma cells as well as in primary mouse embryonic fibroblasts (MEFs). Downregulation of HTRA1 causes a number of phenotypes that are hallmarks of cancer cells suggesting that the methylation state of the HtrA1 promoter may be used as a biomarker for tumour cells or cells at risk of transformation. The online version of this article (doi:10.1186/s12885-016-2425-8) contains supplementary material, which is available to authorized users

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

Science.gov (United States)

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.

Structure Determination of Mycobacterium tuberculosis Serine Protease Hip1 (Rv2224c)

Energy Technology Data Exchange (ETDEWEB)

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

2017-04-07

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

Evaluating the relationship between dental caries number and salivary level of IgA in adults

Science.gov (United States)

Haeri-Araghi, Hesam; Safarzadeh-Khosroshahi, Shadab; Mirzadeh, Monirsadat

2018-01-01

Background Dental caries are the most common mouth infectious disease and also chronic disease of childhood. Saliva plays different roles in oral cavity; for example, salivary immunoglobulins play significant role in body and oral immunity. Various studies were conducted on the different effects of IgA on oral cavity, especially dental caries, and reported controversial results. The current study aimed to compare salivary IgA level at different stages of dental caries in adults. Material and Methods A total of 40 adults, aged 20 to 40 years, referred to the department of oral medicine at Qazvin Faculty of Dentistry, were selected voluntarily based on the number of decayed teeth. Their unstimulated saliva was collected by the spitting method. The cases were assigned to 4 groups each of 10, based on the number of decayed teeth, as follows: Group 1: Caries free, Group 2: With 1 or 2 decayed teeth, Group 3: With 3 or 4 decayed teeth, and Group 4: With 5 or more decayed teeth. None of the cases had systemic diseases or the history of using medicines which affect the quality or quantity of saliva. The salivary IgA level of the cases was measured immunoturbidometrically and analyzed by ANOVA and t test. Results Significant difference was observed between the groups 1 and 4, but there was no significant difference between the other groups. Conclusions According to the results of the current study, the salivary IgA can be considered as an index for the function of immune system, which may be increased by the number of decayed teeth. In fact, the increase of salivary IgA is just the response of immune system to the accumulation of microorganisms and may be the attempt of body to control them. Key words:Saliva, IgA, Dental caries. PMID:29670718

Protease and protease inhibitory activity in pregnant and postpartum involuting uterus

International Nuclear Information System (INIS)

Milwidsky, A.; Beller, U.; Palti, Z.; Mayer, M.

1982-01-01

The presence of two distinct proteolytic activities in the rat uterus was confirmed with 14 C-labeled globin used as a sensitive protein substrate and following release of label into the trichloroacetic acid-soluble supernatant fraction. Protease I is a cytoplasmic acid protease while protease II is associated with the pellet fraction, can be extracted by 0.6 M sodium chloride, and is active at pH 7.0. Protease I activity is low during pregnancy and markedly increases at term achieving maximal activity at day 3 post partum with a subsequent decline to preterm activity values. Lactation did not affect the uterine protease I activity. Protease II activity is not significantly different during pregnancy, at term, and post partum. The presence of an inhibitor of protease I was suggested by a decrease in enzyme activity with an increased cytosolic protein concentration. The inhibitor also lessened bovine trypsin activity but had no effect on protease II. Although its inhibitory potency on trypsin fluctuated during the various uterine physiologic stages, these changes appeared to be statistically insignificant. Human uterine samples were also found to contain the two protease activities with similar changes in protease I post partum. It is suggested that, both in the rat and in man, uterine involution post partum is associated with a marked increase in activity of acid cytosolic protease, while a particulate neutral protease and a soluble inhibitor of trypsin, which are also present in uterine cells, do not appear to play a significant role in the dissolution of uterine tissues after parturition

Sequence quality analysis tool for HIV type 1 protease and reverse transcriptase.

Science.gov (United States)

Delong, Allison K; Wu, Mingham; Bennett, Diane; Parkin, Neil; Wu, Zhijin; Hogan, Joseph W; Kantor, Rami

2012-08-01

Access to antiretroviral therapy is increasing globally and drug resistance evolution is anticipated. Currently, protease (PR) and reverse transcriptase (RT) sequence generation is increasing, including the use of in-house sequencing assays, and quality assessment prior to sequence analysis is essential. We created a computational HIV PR/RT Sequence Quality Analysis Tool (SQUAT) that runs in the R statistical environment. Sequence quality thresholds are calculated from a large dataset (46,802 PR and 44,432 RT sequences) from the published literature ( http://hivdb.Stanford.edu ). Nucleic acid sequences are read into SQUAT, identified, aligned, and translated. Nucleic acid sequences are flagged if with >five 1-2-base insertions; >one 3-base insertion; >one deletion; >six PR or >18 RT ambiguous bases; >three consecutive PR or >four RT nucleic acid mutations; >zero stop codons; >three PR or >six RT ambiguous amino acids; >three consecutive PR or >four RT amino acid mutations; >zero unique amino acids; or 15% genetic distance from another submitted sequence. Thresholds are user modifiable. SQUAT output includes a summary report with detailed comments for troubleshooting of flagged sequences, histograms of pairwise genetic distances, neighbor joining phylogenetic trees, and aligned nucleic and amino acid sequences. SQUAT is a stand-alone, free, web-independent tool to ensure use of high-quality HIV PR/RT sequences in interpretation and reporting of drug resistance, while increasing awareness and expertise and facilitating troubleshooting of potentially problematic sequences.

Oxidative Stress: Promoter of Allergic Sensitization to Protease Allergens?

NARCIS (Netherlands)

van Rijt, Leonie S.; Utsch, Lara; Lutter, René; van Ree, Ronald

2017-01-01

Allergies arise from aberrant T helper type 2 responses to allergens. Several respiratory allergens possess proteolytic activity, which has been recognized to act as an adjuvant for the development of a Th2 response. Allergen source-derived proteases can activate the protease-activated receptor-2,

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

Directory of Open Access Journals (Sweden)

Sandeep Kaur Saggu

Full Text Available 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.

Effect of hydrolysis enzymatic process of corn using protease crude (Rhizopus oligosporus-C1) to produce corn hydrolisate rich folic acid

Science.gov (United States)

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.

Genome-Wide Analyses Suggest Mechanisms Involving Early B-Cell Development in Canine IgA Deficiency.

Directory of Open Access Journals (Sweden)

Mia Olsson

Full Text Available Immunoglobulin A deficiency (IgAD is the most common primary immune deficiency disorder in both humans and dogs, characterized by recurrent mucosal tract infections and a predisposition for allergic and other immune mediated diseases. In several dog breeds, low IgA levels have been observed at a high frequency and with a clinical resemblance to human IgAD. In this study, we used genome-wide association studies (GWAS to identify genomic regions associated with low IgA levels in dogs as a comparative model for human IgAD. We used a novel percentile groups-approach to establish breed-specific cut-offs and to perform analyses in a close to continuous manner. GWAS performed in four breeds prone to low IgA levels (German shepherd, Golden retriever, Labrador retriever and Shar-Pei identified 35 genomic loci suggestively associated (p <0.0005 to IgA levels. In German shepherd, three genomic regions (candidate genes include KIRREL3 and SERPINA9 were genome-wide significantly associated (p <0.0002 with IgA levels. A ~20kb long haplotype on CFA28, significantly associated (p = 0.0005 to IgA levels in Shar-Pei, was positioned within the first intron of the gene SLIT1. Both KIRREL3 and SLIT1 are highly expressed in the central nervous system and in bone marrow and are potentially important during B-cell development. SERPINA9 expression is restricted to B-cells and peaks at the time-point when B-cells proliferate into antibody-producing plasma cells. The suggestively associated regions were enriched for genes in Gene Ontology gene sets involving inflammation and early immune cell development.

Two Kazal-type protease inhibitors from Macrobrachium nipponense and Eriocheir sinensis: comparative analysis of structure and activities.

Science.gov (United States)

Qian, Ye-Qing; Li, Ye; Yang, Fan; Yu, Yan-Qin; Yang, Jin-Shu; Yang, Wei-Jun

2012-03-01

Kazal-type inhibitors (KPIs) play important roles in many biological and physiological processes, such as blood clotting, the immune response and reproduction. In the present study, two male reproductive tract KPIs, termed Man-KPI and Ers-KPI, were identified in Macrobrachium nipponense and Eriocheir sinensis, respectively. The inhibitory activities of recombinant Man-KPI and Ers-KPI against chymotrypsin, elastase, trypsin and thrombin were determined. The results showed that both of them strongly inhibit chymotrypsin and elastase. Kinetic studies were performed to elucidate their inhibition mechanism. Furthermore, individual domains were also expressed to learn further which domain contributes to the inhibitory activities of intact KPIs. Only Man-KPI_domain3 is active in the inhibition of chymotrypsin and elastase. Meanwhile, Ers-KPI_domain2 and 3 are responsible for inhibition of chymotrypsin, and Ers-KPI_domains2, 3 and 4 are responsible for the inhibition of elastase. Meanwhile, the inhibitory activities of these two KPIs toward Macrobrachium rosenbergii, M. nipponense and E. sinensis sperm were compared with that of the Kazal-type peptidase inhibitor (MRPINK) characterized from the M. rosenbergii reproductive tract in a previous study. The results demonstrated that KPIs can completely inhibit the gelatinolytic activities of sperm proteases from their own species, while different levels of cross-inhibition were observed between KPI and proteases from different species. These results may provide new perspective to further clarify the mechanism of KPI-proteases interaction in the male reproductive system. Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.

[Diabetes and autoimmune diseases: prevalence of celiac disease in children and adolescents with type 1 diabetes].

Science.gov (United States)

Mont-Serrat, Camila; Hoineff, Claudio; Meirelles, Ricardo M R; Kupfer, Rosane

2008-12-01

Determine the prevalence of celiac disease in children and adolescents with type 1 diabetes mellitus (DM1) in attendance in Instituto Estadual de Diabetes e Endocrinologia Luiz Capriglione (IEDE). Blood samples were analyzed in 120 children and adolescents with DM1 from IEDE Diabetes Clinic for the IgA antitissue-transglutaminase antibody and dosage of the seric IgA. Those with positive serology were guided for upper endoscopy with small-bowel biopsy to confirm the celiac disease. The antibody was positive in 3 of the 120 patients. The small-bowel biopsy was confirmatory in all of the positive patients, leading to a prevalence of celiac disease of 2.5% in the studied group. The prevalence of celiac disease is increased in children and adolescents with DM1 when compared with normality. As most are asymptomatic, it is recommended periodical screening of celiac disease in children with DM1.

Toxicological Effects of Nickel Chloride on IgA+ B Cells and sIgA, IgA, IgG, IgM in the Intestinal Mucosal Immunity in Broilers

Directory of Open Access Journals (Sweden)

Bangyuan Wu

2014-08-01

Full Text Available 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.

Razlikovnost kao pretpostavka za registraciju žiga

OpenAIRE

Matanovac Vučković, Romana

2012-01-01

U radu se obrazlaže stupnjevanje razlikovnosti u ispitivanju apsolutnih smetnji za registraciju nekog znaka kao žiga nakon harmonizacije žigovnoga prava u Europskoj uniji. Obrazlaže se i nerazdruživost između funkcije označivanja podrijetla i razlikovnosti žiga. S time u svezi obrađuju se Direktiva 2008/95/EZ Europskog parlamenta i Vijeća od 22. listopada 2008. za usklađivanje propisa država članica koji se odnose na žigove (kodificirani tekst), Uredba Vijeća (EZ) 207/2009 od 26. veljače 2009...

"Iga päev..." : [luuletused] / Doris Kareva

Index Scriptorium Estoniae

Kareva, Doris, 1958-

2001-01-01

Tekst eesti ja inglise k. D. Kareva lühibiograafia eesti ja inglise k. lk. 175. Sisu: "Iga päev..." = "Every day..." ; "Ma nägin unes - Saatan kõneles..." = "I dream that I heard Satan speak..." ; "Viib sünnieelsest unest surmaunne..." = "Rainbow-coloured confusion bears us..." ; "Vaadeldes vikerkaarlevat maailma..." = "Viewing the rainbowing world..." ; "Ei jõua kirjutada puhtandit..." = "No time to write the final draft..." ; "Põletatud luuletused..." = ""Burnt poems..." ; Fraktalia ; Müsteerium 1-5 = Enigma 1-5 ; "Jumal juhtub..." = "God happens..." ; Moira 1-7 = Wishing well 1-7 ; Concerto strumenti e voce

Pre- and Posttransplant IgA Anti-Fab Antibodies to Predict Long-term Kidney Graft Survival.

Science.gov (United States)

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.

The Oxford IgA nephropathy clinicopathological classification is valid for children as well as adults

NARCIS (Netherlands)

Coppo, Rosanna; Troyanov, Stéphan; Camilla, Roberta; Hogg, Ronald J.; Cattran, Daniel C.; Cook, H. Terence; Feehally, John; Roberts, Ian S. D.; Amore, Alessandro; Alpers, Charles E.; Barratt, Jonathan; Berthoux, Francois; Bonsib, Stephen; Bruijn, Jan A.; D'Agati, Vivette; D'Amico, Giuseppe; Emancipator, Steven N.; Emma, Francesco; Ferrario, Franco; Fervenza, Fernando C.; Florquin, Sandrine; Fogo, Agnes B.; Geddes, Colin C.; Groene, Hermann J.; Haas, Mark; Herzenberg, Andrew M.; Hill, Prue A.; Hsu, Stephen I.; Jennette, J. Charles; Joh, Kensuke; Julian, Bruce A.; Kawamura, Tetsuya; Lai, Fernand M.; Li, Lei S.; Li, Philip K.; Liu, Zhi H.; Mezzano, Sergio; Schena, F. Paolo; Tomino, Yasuhiko; Walker, Patrick D.; Wang, Haiyan; Weening, Jan J.; Yoshikawa, Norishige; Zhang, Hong

2010-01-01

To study the predictive value of biopsy lesions in IgA nephropathy in a range of patient ages we retrospectively analyzed the cohort that was used to derive a new classification system for IgA nephropathy. A total of 206 adults and 59 children with proteinuria over 0.5 g/24h/1.73 m(2) and an eGFR of

Targeting protease activated receptor-1 with P1pal-12 limits bleomycin-induced pulmonary fibrosis

NARCIS (Netherlands)

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

2014-01-01

Idiopathic pulmonary fibrosis is the most devastating fibrotic diffuse parenchymal lung disease which remains refractory to pharmacological therapies. Therefore, novel treatments are urgently required. Protease-activated receptor (PAR)-1 is a G-protein-coupled receptor that mediates critical

Efficient inhibition of cathepsin B by a secreted type 1 cystatin of Fasciola gigantica.

Science.gov (United States)

Siricoon, Sinee; Grams, Suksiri Vichasri; Grams, Rudi

2012-12-01

Cysteine proteases are important antigens in the liver fluke genus Fasciola, essential for infection, protection and nutrition. While their biochemistry, biological roles and application as vaccines have been thoroughly studied there is a lack of data concerning their regulation. In the present study we have continued our investigation of cysteine protease inhibitors in Fasciola gigantica and demonstrate, in comparison with FgStefin-1 and human cystatin C, that a second type 1 cystatin of the parasite, FgStefin-2, has been evolutionary adapted to block cathepsin B. The protein, which unusually for a type 1 cystatin carries a signal peptide, is expressed from the metacercarial to adult stage and located in the epithelial cells of the intestinal tract in all stages and in the prostate gland cells in adults. Both cell types may contribute to the released FgStefin-2 observed in the ES product of the parasite. Distinct isoforms of cathepsin B are essential for host tissue penetration during the early infection process and FgStefin-2 may act as key regulator, required to protect the minute juvenile from autoproteolysis. Expression in the prostate gland in the adult stage suggests an additional regulative role of cysteine protease activity in the reproductive system. Copyright © 2012 Elsevier B.V. All rights reserved.

Deletion of aprA and nprA genes for alkaline protease A and neutral protease A from bacillus thuringiensis: effect on insecticidal crystal proteins.

Science.gov (United States)

Tan, Y; Donovan, W P

2001-11-17

The aprA gene encoding alkaline protease A (AprA) was cloned from Bacillus thuringiensis subsp. kurstaki, and the cloned gene was used to construct aprA-deleted (aprA1) strains of B. thuringiensis. An aprA1 strain of B. thuringiensis that contained the wild-type gene for neutral protease A (nprA(+)) displayed levels of extracellular proteolytic activity that were similar to those of an aprA(+)nprA(+) strain. However, when EDTA was included in the protease assay to inhibit NprA activity the aprA1nprA(+) strain displayed only 2% of the extracellular proteolytic activity of the aprA(+)nprA(+) strain. A strain that was deleted for both aprA and nprA (aprA1nprA3 strain) failed to produce detectable levels of proteolytic activity either in the presence or absence of EDTA in the assay. Compared with the aprA(+)nprA(+) strain the aprA1nprA(+) strain yielded 10% more full-length Cry1Bb crystal protein and the aprA1nprA3 strain yielded 25% more full-length Cry1Bb protein. No significant differences were seen in the 50% lethal dose of Cry1Bb protein from aprA(+)nprA(+) and aprA1nprA3 strains against three species of lepidopteran insects. These results suggest that enhanced yield of certain crystal proteins can be obtained by deletion of the genes aprA and nprA which are the major extracellular proteases of B. thuringiensis.

Protein energy malnutrition alters mucosal IgA responses and reduces mucosal vaccine efficacy in mice.

Science.gov (United States)

Rho, Semi; Kim, Heejoo; Shim, Seung Hyun; Lee, Seung Young; Kim, Min Jung; Yang, Bo-Gie; Jang, Myoung Ho; Han, Byung Woo; Song, Man Ki; Czerkinsky, Cecil; Kim, Jae-Ouk

2017-10-01

Oral vaccine responsiveness is often lower in children from less developed countries. Childhood malnutrition may be associated with poor immune response to oral vaccines. The present study was designed to investigate whether protein energy malnutrition (PEM) impairs B cell immunity and ultimately reduces oral vaccine efficacy in a mouse model. Purified isocaloric diets containing low protein (1/10 the protein of the control diet) were used to determine the effect of PEM. PEM increased both nonspecific total IgA and oral antigen-specific IgA in serum without alteration of gut permeability. However, PEM decreased oral antigen-specific IgA in feces, which is consistent with decreased expression of polymeric Immunoglobulin receptor (pIgR) in the small intestine. Of note, polymeric IgA was predominant in serum under PEM. In addition, PEM altered B cell development status in the bone marrow and increased the frequency of IgA-secreting B cells, as well as IgA secretion by long-lived plasma cells in the small intestinal lamina propria. Moreover, PEM reduced the protective efficacy of the mucosally administered cholera vaccine and recombinant attenuated Salmonella enterica serovar Typhimurium vaccine in a mouse model. Our results suggest that PEM can impair mucosal immunity where IgA plays an important role in host protection and may partly explain the reduced efficacy of oral vaccines in malnourished subjects. Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

Structure of the Unbound Form of HIV-1 Subtype A Protease: Comparison with Unbound Forms of Proteases from other HIV Subtypes

Energy Technology Data Exchange (ETDEWEB)

Robbins, Arthur H.; Coman, Roxana M.; Bracho-Sanchez, Edith; Fernandez, Marty A.; Gilliland, C.Taylor; Li, Mi; Agbandje-McKenna, Mavis; Wlodawer, Alexander; Dunn, Ben M.; McKenna, Robert (NCI); (Florida)

2010-03-12

The crystal structure of the unbound form of HIV-1 subtype A protease (PR) has been determined to 1.7 {angstrom} resolution and refined as a homodimer in the hexagonal space group P6{sub 1} to an R{sub cryst} of 20.5%. The structure is similar in overall shape and fold to the previously determined subtype B, C and F PRs. The major differences lie in the conformation of the flap region. The flaps in the crystal structures of the unbound subtype B and C PRs, which were crystallized in tetragonal space groups, are either semi-open or wide open. In the present structure of subtype A PR the flaps are found in the closed position, a conformation that would be more anticipated in the structure of HIV protease complexed with an inhibitor. The amino-acid differences between the subtypes and their respective crystal space groups are discussed in terms of the differences in the flap conformations.

Cocoa and cocoa fibre differentially modulate IgA and IgM production at mucosal sites.

Science.gov (United States)

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.

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

Science.gov (United States)

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). 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.

Linear IgA Bullous Dermatosis

DEFF Research Database (Denmark)

Lings, Kristina; Bygum, Anette

2015-01-01

Linear IgA bullous dermatosis (LAD) is an autoimmune, chronic bullous disease affecting primarily young children and adults. Studies on LAD are relatively sparse and from Scandinavia we could only find a few case reports. Therefore we decided to conduct a retrospective investigation of patients...

Extracellular acid protease from Aspergillus niger I1: purification and ...

African Journals Online (AJOL)

STORAGESEVER

2009-09-15

Sep 15, 2009 ... A new strain of Aspergillus niger producing acid protease was isolated and identified by universal primers NL1 and .... Media were autoclaved at 120°C for 20 min. ... molecular weight calibration kit as markers consisting of bovine ... then removed by washing the gel three times with 100 mM ..... New York.

The Evaluation of Psychological Factor and Salivary Cortisol and IgA Levels in

Directory of Open Access Journals (Sweden)

Fateme Arbabi-Kalati

2014-07-01

Full Text Available Background: Oral lichen planus (OLP is a chronic immunological disorder with unknown etiology. The aim of this study was to determine psychological factors and salivary cortisol, IgA level in patients with oral lichen planus. Materials and Methods: In this experimental study 20 patients with OLP and healthy person were admitted to this study. Saliva samples were collected between - Am. saliva cortisol, IgA level was detected by ELIZA method. In this study, patients with anxiety and depression were measured using the SCL-90 questionnaire. Data analyzed by t-test. Results: The mean salivary cortisol level in patients with OLP was 3.2±1.9 ng/mL and the mean saliva cortisol level in healthy person was 3.5±1.9 ng/mL. Significant difference was observed in the salivary cortisol levels in the 2 study groups (p=0.04. The mean salivary IgA level in patients with OLP was 0.69±0.29 ng/mL and the mean saliva IgA level in healthy person was 0.9±0.43 ng/mL but no significant difference was observed in the salivary cortisol levels in the 2 study groups. Results showed that anxiety levels in patients with oral lichen planus were slightly higher than controls but there was no significant difference between healthy subjects. Conclusion: Finding revealed the mean salivary cortisol level in patient with OLP less than healthy persons. Significant difference was observed in the salivary cortisol levels in the 2 study groups. Based on the t-student test, no significant difference was observed in the salivary IgA levels in the 2 study groups. Anxiety levels in patients with oral lichen planus were slightly higher than controls.

The crystal structure of protease Sapp1p from Candida parapsilosis in complex with the HIV protease inhibitor ritonavir

Czech Academy of Sciences Publication Activity Database

Dostál, Jiří; Brynda, Jiří; Hrušková-Heidingsfeldová, Olga; Pachl, Petr; Pichová, Iva; Řezáčová, Pavlína

2012-01-01

Roč. 27, č. 1 (2012), s. 160-165 ISSN 1475-6366 R&D Projects: GA MŠk(CZ) LC531; GA ČR GA310/09/1945; GA ČR GA203/09/0820 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50520514 Keywords : secreted aspartic protease * virulence factor * X-ray structure * candidiasis Subject RIV: CE - Biochemistry Impact factor: 1.495, year: 2012

Radioimmunoassay of IgM, IgG, and IgA brucella antibodies

International Nuclear Information System (INIS)

Parrett, D.; Nielson, K.H.; White, R.G.; Payne, D.J.H.

1977-01-01

A radioimmunoassay (R.I.A.) has been devised to measure the serum antibody against Brucella abortus in each of the immunoglobulin classes IgM, IgG, and IgA. This test was applied to 46 sera from individuals with various clinical types of brucellosis, and the results were compared with the results of conventional direct and indirect agglutination and complement-fixation tests. The R.I.A. provided a highly sensitive primary-type assay which avoided the difficulties with blocking or non-agglutinating antibody, and thus has many advantages in the diagnosis of acute and chronic stages of brucella infection in man. The R.I.A. was successful in detection of antibody in many instances in which conventional serological tests were negative, and such antibody could (if IgM) be associated with acute or (if IgG or IgA) with chronic cases of brucellosis. One case in which B.abortus was isolated by blood culture but which failed to yield antibody by conventional tests, nevertheless showed substantial levels of IgM and IgG antibody by R.I.A. In other cases the R.I.A. test helped to eliminate the diagnosis of brucellosis by revealing absent or low antibody levels. (author)

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

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Lei Zhang

2017-02-01

Full Text Available 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.

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

Science.gov (United States)

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

Iga viies SVH on ennetatav

Index Scriptorium Estoniae

2009-01-01

ONTARGET uuring tõestab, et telmisartaan kaitseb kõrge kardiovaskulaarse riskiga patsiente sama tõhusalt kui ramipriil, kuid on paremini talutav. Uuringust võib järeldada, et telmisartaaniga saab ennetada iga viiendat tõsist kardiovaskulaarset juhtumit. Eesti arstidele tutvustati uuringut 6. mail toimunud sümpoosiumil

Giardia muris trophozoite antigenic targets for mouse intestinal IgA antibody.

Science.gov (United States)

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.

Co-evolution of insect proteases and plant protease inhibitors.

Science.gov (United States)

Jongsma, Maarten A; Beekwilder, Jules

2011-08-01

Plants are at the basis of the food chain, but there is no such thing as a "free lunch" for herbivores. To promote reproductive success, plants evolved multi-layered defensive tactics to avoid or discourage herbivory. To the detriment of plants, herbivores, in turn, evolved intricate strategies to find, eat, and successfully digest essential plant parts to raise their own offspring. In this battle the digestive tract is the arena determining final victory or defeat as measured by growth or starvation of the herbivore. Earlier, specific molecular opponents were identified as proteases and inhibitors: digestive proteases of herbivores evolved structural motifs to occlude plant protease inhibitors, or alternatively, the insects evolved proteases capable of specifically degrading the host plant inhibitors. In response plant inhibitors evolved hyper-variable and novel protein folds to remain active against potential herbivores. At the level of protease regulation in herbivorous insects, it was shown that inhibition-insensitive digestive proteases are up-regulated when sensitive proteases are inhibited. The way this regulation operates in mammals is known as negative feedback by gut-luminal factors, so-called 'monitor peptides' that are sensitive to the concentration of active enzymes. We propose that regulation of gut enzymes by endogenous luminal factors has been an open invitation to plants to "hijack" this regulation by evolving receptor antagonists, although yet these plant factors have not been identified. In future research the question of the co-evolution of insect proteases and plant inhibitors should, therefore, be better approached from a systems level keeping in mind that evolution is fundamentally opportunistic and that the plant's fitness is primarily improved by lowering the availability of essential amino acids to an herbivore by any available mechanism.

Protease-sensitive synthetic prions.

Directory of Open Access Journals (Sweden)

David W Colby

2010-01-01

Full Text Available Prions arise when the cellular prion protein (PrP(C undergoes a self-propagating conformational change; the resulting infectious conformer is designated PrP(Sc. Frequently, PrP(Sc is protease-resistant but protease-sensitive (s prions have been isolated in humans and other animals. We report here that protease-sensitive, synthetic prions were generated in vitro during polymerization of recombinant (rec PrP into amyloid fibers. In 22 independent experiments, recPrP amyloid preparations, but not recPrP monomers or oligomers, transmitted disease to transgenic mice (n = 164, denoted Tg9949 mice, that overexpress N-terminally truncated PrP. Tg9949 control mice (n = 174 did not spontaneously generate prions although they were prone to late-onset spontaneous neurological dysfunction. When synthetic prion isolates from infected Tg9949 mice were serially transmitted in the same line of mice, they exhibited sPrP(Sc and caused neurodegeneration. Interestingly, these protease-sensitive prions did not shorten the life span of Tg9949 mice despite causing extensive neurodegeneration. We inoculated three synthetic prion isolates into Tg4053 mice that overexpress full-length PrP; Tg4053 mice are not prone to developing spontaneous neurological dysfunction. The synthetic prion isolates caused disease in 600-750 days in Tg4053 mice, which exhibited sPrP(Sc. These novel synthetic prions demonstrate that conformational changes in wild-type PrP can produce mouse prions composed exclusively of sPrP(Sc.

Intermittent fasting promotes bacterial clearance and intestinal IgA production in Salmonella typhimurium-infected mice.

Science.gov (United States)

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.

Changes in γ-secretase activity and specificity caused by the introduction of consensus aspartyl protease active motif in Presenilin 1

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Zhou Xiangdong

2008-05-01

Full Text Available Abstract Presenilin (PS1 or PS2 is an essential component of the active γ-secretase complex that liberates the Aβ peptides from amyloid precursor protein (APP. PS1 is regarded as an atypical aspartyl protease harboring two essential aspartic acids in the context of the sequence D257LV and D385FI, respectively, rather than the typical DTG...DTG catalytic motif of classical aspartyl proteases. In the present studies, we introduced the sequence DTG in PS1 at and around the catalytic D257 and D385 residues to generate three PS1 mutants: D257TG, D385TG, and the double-mutant D257TG/D385TG. The effects of these changes on the γ-secretase activity in the presence or absence of γ-secretase inhibitors and modulators were investigated. The results showed that PS1 mutants having D385TG robustly enhanced Aβ42 production compared to the wild type (wt, and were more sensitive than wt to inhibition by a classical aspartyl protease transition state mimic, and fenchylamine, a sulfonamide derivative. Unlike wt PS1 and some of its clinical mutants, all three PS1 artificial mutants decreased cleavage of Notch S3-site, suggesting that these artificial mutations may trigger conformational changes at the substrate docking and catalytic site that cause alteration of substrate specificity and inhibition pattern. Consistent with this notion, we have found that NSAID enzymatic inhibitors of COX, known modulators of the γ-secretase activity, cause PS1 mutants containing D385TG to produce higher levels of both Aβ38 and Aβ42, but to reduce levels of Aβ39, showing a pattern of Aβ formation different from that observed with wild type PS1 and its clinical mutants. This study provides an important structural clue for the rational design of drugs to inhibit processing of APP at the γ-site without interfering with Notch processing.

The role of protease-activated receptor type 2 in nociceptive signaling and pain

Czech Academy of Sciences Publication Activity Database

Mrózková, Petra; Paleček, Jiří; Špicarová, Diana

2016-01-01

Roč. 65, č. 3 (2016), s. 357-367 ISSN 0862-8408 R&D Projects: GA MŠk(CZ) LH12058; GA ČR(CZ) GBP304/12/G069; GA ČR(CZ) GA15-11138S; GA MŠk(CZ) LH15279; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:67985823 Keywords : protease-activated receptor (PAR2) * signaling pathways * nociception * pain * spinal cord Subject RIV: FH - Neurology Impact factor: 1.461, year: 2016

Arabidopsis SUMO protease ASP1 positively regulates flowering time partially through regulating FLC stability 

KAUST Repository

Kong, Xiangxiong; Luo, Xi; Qu, Gao Ping; Liu, Peng; Jin, Jing Bo

2016-01-01

The initiation of flowering is tightly regulated by the endogenous and environment signals, which is crucial for the reproductive success of flowering plants. It is well known that autonomous and vernalization pathways repress transcription of FLOWERING LOCUS C (FLC), a focal floral repressor, but how its protein stability is regulated remains largely unknown. Here, we found that mutations in a novel Arabidopsis SUMO protease 1 (ASP1) resulted in a strong late-flowering phenotype under long-days, but to a lesser extent under short-days. ASP1 localizes in the nucleus and exhibited a SUMO protease activity in vitro and in vivo. The conserved Cys-577 in ASP1 is critical for its enzymatic activity, as well as its physiological function in the regulation of flowering time. Genetic and gene expression analyses demonstrated that ASP1 promotes transcription of positive regulators of flowering, such as FT, SOC1 and FD, and may function in both CO-dependent photoperiod pathway and FLC-dependent pathways. Although the transcription level of FLC was not affected in the loss-of-function asp1 mutant, the protein stability of FLC was increased in the asp1 mutant. Taken together, this study identified a novel bona fide SUMO protease, ASP1, which positively regulates transition to flowering at least partly by repressing FLC protein stability.

Arabidopsis SUMO protease ASP1 positively regulates flowering time partially through regulating FLC stability 

KAUST Repository

Kong, Xiangxiong

2016-12-07

The initiation of flowering is tightly regulated by the endogenous and environment signals, which is crucial for the reproductive success of flowering plants. It is well known that autonomous and vernalization pathways repress transcription of FLOWERING LOCUS C (FLC), a focal floral repressor, but how its protein stability is regulated remains largely unknown. Here, we found that mutations in a novel Arabidopsis SUMO protease 1 (ASP1) resulted in a strong late-flowering phenotype under long-days, but to a lesser extent under short-days. ASP1 localizes in the nucleus and exhibited a SUMO protease activity in vitro and in vivo. The conserved Cys-577 in ASP1 is critical for its enzymatic activity, as well as its physiological function in the regulation of flowering time. Genetic and gene expression analyses demonstrated that ASP1 promotes transcription of positive regulators of flowering, such as FT, SOC1 and FD, and may function in both CO-dependent photoperiod pathway and FLC-dependent pathways. Although the transcription level of FLC was not affected in the loss-of-function asp1 mutant, the protein stability of FLC was increased in the asp1 mutant. Taken together, this study identified a novel bona fide SUMO protease, ASP1, which positively regulates transition to flowering at least partly by repressing FLC protein stability.

Secretory IgA as a diagnostic tool for Pseudomonas aeruginosa respiratory colonization

DEFF Research Database (Denmark)

Aanaes, Kasper; Johansen, Helle Krogh; Poulsen, Steen Seier

2012-01-01

BACKGROUND: Pseudomonas aeruginosa sinusitis may be the focus for intermittent lung colonization in patients with cystic fibrosis (CF). The sinusitis may induce elevated IgA levels in nasal secretion and saliva against P. aeruginosa. METHODS: 120 CF patients chronically infected, intermittently...... colonized or without P. aeruginosa in the lungs participated in this cross-sectional study. IgA and IgG against P. aeruginosa sonicate and alginate were measured in nasal secretions, saliva, and in serum by ELISA. RESULTS: The intermittently colonized patients had significantly higher IgA levels in nasal...... secretions and saliva than those without P. aeruginosa in the lungs, indicating that P. aeruginosa sinusitis may precede intermittent colonization and chronic infection of the lungs. CONCLUSIONS: Specific IgA against P. aeruginosa in nasal secretions and saliva can contribute to differentiation between...

Coxsackievirus B3 2A protease promotes encephalomyocarditis virus replication.

Science.gov (United States)

Song, Qin-Qin; Lu, Ming-Zhi; Song, Juan; Chi, Miao-Miao; Sheng, Lin-Jun; Yu, Jie; Luo, Xiao-Nuan; Zhang, Lu; Yao, Hai-Lan; Han, Jun

2015-10-02

To determine whether 2A protease of the enterovirus genus with type I internal ribosome entry site (IRES) effect on the viral replication of type II IRES, coxsackievirus B3(CVB3)-encoded protease 2A and encephalomyocarditis virus (EMCV) IRES (Type II)-dependent or cap-dependent report gene were transiently co-expressed in eukaryotic cells. We found that CVB3 2A protease not only inhibited translation of cap-dependent reporter genes through the cleavage of eIF4GI, but also conferred high EMCV IRES-dependent translation ability and promoted EMCV replication. Moreover, deletions of short motif (aa13-18 RVVNRH, aa65-70 KNKHYP, or aa88-93 PRRYQSH) resembling the nuclear localization signals (NLS) or COOH-terminal acidic amino acid motif (aa133-147 DIRDLLWLEDDAMEQ) of CVB3 2A protease decreased both its EMCV IRES-dependent translation efficiency and destroy its cleavage on eukaryotic initiation factor 4G (eIF4G) I. Our results may provide better understanding into more effective interventions and treatments for co-infection of viral diseases. Copyright © 2015 Elsevier B.V. All rights reserved.

Gene Expression Analysis in Tubule Interstitial Compartments Reveals Candidate Agents for IgA Nephropathy

Directory of Open Access Journals (Sweden)

Jinling Wang

2014-09-01

Full Text Available Background/Aims: Our aim was to explore the molecular mechanism underlying development of IgA nephropathy and discover candidate agents for IgA nephropathy. Methods: The differentially expressed genes (DEGs between patients with IgA nephropathy and normal controls were identified by the data of GSE35488 downloaded from GEO (Gene Expression Omnibus database. The co-expressed gene pairs among DEGs were screened to construct the gene-gene interaction network. Gene Ontology (GO enrichment analysis was performed to analyze the functions of DEGs. The biologically active small molecules capable of targeting IgA nephropathy were identified using the Connectivity Map (cMap database. Results: A total of 55 genes involved in response to organic substance, transcription factor activity and response to steroid hormone stimulus were identified to be differentially expressed in IgA nephropathy patients compared to healthy individuals. A network with 45 co-expressed gene pairs was constructed. DEGs in the network were significantly enriched in response to organic substance. Additionally, a group of small molecules were identified, such as doxorubicin and thapsigargin. Conclusion: Our work provided a systematic insight in understanding the mechanism of IgA nephropathy. Small molecules such as thapsigargin might be potential candidate agents for the treatment of IgA nephropathy.

Intracellular alkaline proteases produced by thermoacidophiles: detection of protease heterogeneity by gelatin zymography and polymerase chain reaction (PCR)

Energy Technology Data Exchange (ETDEWEB)

Kocab, S.; Erdem, B. [Middle East Technical University, Ankara (Turkey). Dept. of Biological Sciences

2002-08-01

In this study 24 thermoacidophilic archeal and bacterial strains isolated from hot-springs and hot-soils were screened for their ability to produce intracellular alkaline proteases. The protease activities of the strains, based on azocasein hydrolysis, showed a variation from 0.6 to 5.1 U. The cell extracts of three most potent producers were further examined and it was found that their proteases exhibited maximum activity at 60-70{sup o}C and showed a pH optimum over a range of pH 7.0-8.5. Gelatin zymography revealed that two of the selected archeal strains produced multiple active SDS-resistant proteases. On the other hand, PCR amplification of alkaline serine protease gene sequences of total DNA from all isolates yielded four distinct amplification fragments of 650, 450, 400 and 300 bp, which might have been derived from different serine protease genes. (author)

Differential Response of Extracellular Proteases of Trichoderma Harzianum Against Fungal Phytopathogens.

Science.gov (United States)

Sharma, Vivek; Salwan, Richa; Sharma, Prem N

2016-09-01

In the present study, production of extracellular proteases by Trichoderma harzianum was evaluated based on the relative gene expression and spectrophotometric assay. The fungal isolates were grown in Czapek Dox Broth medium supplemented with deactivated mycelium of plant fungal pathogens such as Fusarium oxysporum, Colletotrichum capsici, Gloeocercospora sorghi, and Colletotrichum truncatum. The maximum protease activity was detected after 48 h of incubation against Colletotrichum spp. Similarly in qRT-PCR, the relative gene expression of four proteases varied from 48 to 96 h against host pathogens in a time-independent manner. Among proteases, statistically significant upregulation of asp, asp, and srp was observed against Colletotrichum spp., followed by F. oxysporum. But in the case of pepM22, maximum upregulation was observed against F. oxysporum. The variation in enzyme assay and qRT-PCR of proteases at different time intervals against various fungal phytopathogens could be due to the limitation of using casein as a substrate for all types of proteases or protease-encoding transcripts selected for qRT-PCR, which may not be true representative of total protease activity.

A novel protease activity assay using a protease-responsive chaperone protein

International Nuclear Information System (INIS)

Sao, Kentaro; Murata, Masaharu; Fujisaki, Yuri; Umezaki, Kaori; Mori, Takeshi; Niidome, Takuro; Katayama, Yoshiki; Hashizume, Makoto

2009-01-01

Protease activity assays are important for elucidating protease function and for developing new therapeutic agents. In this study, a novel turbidimetric method for determining the protease activity using a protease-responsive chaperone protein is described. For this purpose, a recombinant small heat-shock protein (sHSP) with an introduced Factor Xa protease recognition site was synthesized in bacteria. This recombinant mutant, FXa-HSP, exhibited chaperone-like activity at high temperatures in cell lysates. However, the chaperone-like activity of FXa-HSP decreased dramatically following treatment with Factor Xa. Protein precipitation was subsequently observed in the cell lysates. The reaction was Factor Xa concentration-dependent and was quantitatively suppressed by a specific inhibitor for Factor Xa. Protein aggregation was detected by a simple method based on turbidimetry. The results clearly demonstrate that this assay is an effective, easy-to-use method for determining protease activities without the requirement of labeling procedures and the use of radioisotopes.

A novel protease activity assay using a protease-responsive chaperone protein

Energy Technology Data Exchange (ETDEWEB)

Sao, Kentaro [Graduate School of Systems Life Sciences, Kyushu University, 744 Motooka Nishi-ku, Fukuoka 819-0395 (Japan); Murata, Masaharu, E-mail: m-murata@dem.med.kyushu-u.ac.jp [Department of Advanced Medical Initiatives, Faculty of Medical Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku Fukuoka 812-8582 (Japan); Fujisaki, Yuri; Umezaki, Kaori [Department of Advanced Medical Initiatives, Faculty of Medical Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku Fukuoka 812-8582 (Japan); Mori, Takeshi; Niidome, Takuro; Katayama, Yoshiki [Graduate School of Systems Life Sciences, Kyushu University, 744 Motooka Nishi-ku, Fukuoka 819-0395 (Japan); Department of Applied Chemistry, Faculty of Engineering, Kyushu University, Nishi-ku Fukuoka 819-0395 (Japan); Center for Future Chemistry, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395 (Japan); Hashizume, Makoto [Department of Advanced Medical Initiatives, Faculty of Medical Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku Fukuoka 812-8582 (Japan)

2009-06-05

Protease activity assays are important for elucidating protease function and for developing new therapeutic agents. In this study, a novel turbidimetric method for determining the protease activity using a protease-responsive chaperone protein is described. For this purpose, a recombinant small heat-shock protein (sHSP) with an introduced Factor Xa protease recognition site was synthesized in bacteria. This recombinant mutant, FXa-HSP, exhibited chaperone-like activity at high temperatures in cell lysates. However, the chaperone-like activity of FXa-HSP decreased dramatically following treatment with Factor Xa. Protein precipitation was subsequently observed in the cell lysates. The reaction was Factor Xa concentration-dependent and was quantitatively suppressed by a specific inhibitor for Factor Xa. Protein aggregation was detected by a simple method based on turbidimetry. The results clearly demonstrate that this assay is an effective, easy-to-use method for determining protease activities without the requirement of labeling procedures and the use of radioisotopes.

PetIGA-MF: a multi-field high-performance toolbox for structure-preserving B-splines spaces

KAUST Repository

Sarmiento, Adel; Cô rtes, A.M.A.; Garcia, D.A.; Dalcin, Lisandro; Collier, N.; Calo, V.M.

2016-01-01

We describe a high-performance solution framework for isogeometric discrete differential forms based on B-splines: PetIGA-MF. Built on top of PetIGA, an open-source library we have built and developed over the last decade, PetIGA-MF is a general

Role of disulphide bonds in a thermophilic serine protease aqualysin I from Thermus aquaticus YT-1.

Science.gov (United States)

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.

Antiviral Potential of a Novel Compound CW-33 against Enterovirus A71 via Inhibition of Viral 2A Protease

Directory of Open Access Journals (Sweden)

Ching-Ying Wang

2015-06-01

Full Text Available Enterovirus A71 (EV-A71 in the Picornaviridae family causes hand-foot-and-mouth disease, aseptic meningitis, severe central nervous system disease, even death. EV-A71 2A protease cleaves Type I interferon (IFN-α/β receptor 1 (IFNAR1 to block IFN-induced Jak/STAT signaling. This study investigated anti-EV-A7l activity and synergistic mechanism(s of a novel furoquinoline alkaloid compound CW-33 alone and in combination with IFN-β Anti-EV-A71 activities of CW-33 alone and in combination with IFN-β were evaluated by inhibitory assays of virus-induced apoptosis, plaque formation, and virus yield. CW-33 showed antiviral activities with an IC50 of near 200 µM in EV-A71 plaque reduction and virus yield inhibition assays. While, anti-EV-A71 activities of CW-33 combined with 100 U/mL IFN-β exhibited a synergistic potency with an IC50 of approximate 1 µM in plaque reduction and virus yield inhibition assays. Molecular docking revealed CW-33 binding to EV-A71 2A protease active sites, correlating with an inhibitory effect of CW33 on in vitro enzymatic activity of recombinant 2A protease IC50 = 53.1 µM. Western blotting demonstrated CW-33 specifically inhibiting 2A protease-mediated cleavage of IFNAR1. CW-33 also recovered Type I IFN-induced Tyk2 and STAT1 phosphorylation as well as 2',5'-OAS upregulation in EV-A71 infected cells. The results demonstrated CW-33 inhibiting viral 2A protease activity to reduce Type I IFN antagonism of EV-A71. Therefore, CW-33 combined with a low-dose of Type I IFN could be applied in developing alternative approaches to treat EV-A71 infection.

Thermodynamic and structural analysis of HIV protease resistance to darunavir - analysis of heavily mutated patient- derived HIV-1 proteases

Czech Academy of Sciences Publication Activity Database

Kožíšek, Milan; Lepšík, Martin; Grantz Šašková, Klára; Brynda, Jiří; Konvalinka, Jan; Řezáčová, Pavlína

2014-01-01

Roč. 281, č. 7 (2014), s. 1834-1847 ISSN 1742-464X R&D Projects: GA ČR GAP207/11/1798 Grant - others:OPPC(XE) CZ.2.16/3.1.00/24016 Institutional support: RVO:61388963 ; RVO:68378050 Keywords : enthropic contribution * HIV protease inhibitors * isothermal titration calorimetry * resistance mutation * X-ray crystallography Subject RIV: CE - Biochemistry Impact factor: 4.001, year: 2014

HIV-1 protease inhibitory substances from Cassia garrettiana

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Jindaporn Puripattanvong

2007-01-01

Full Text Available Cassia garrettiana Craib, a Thai medicinal plant locally known as Samae-sarn, was investigated for its active constituents against HIV-1 protease (HIV-1 PR. Bioassay-guided fractionation of the heart woodof this plant led to the isolation of a stilbene derivative (1, piceatannol and an anthraquinone derivative (2, chrysophanol. Piceatannol exhibited appreciable inhibitory effect against HIV-1 PR with an IC50 value of25.4 μg/ml, whereas that of chrysophanol was 73.5 μg/ml. In addition, other two stilbenoids together with three anthraquinone derivatives were also investigated for their anti-HIV-1 PR activities. The resultindicated that resveratrol possessed anti-HIV-1 PR activity with an IC50 value of 85.0 μg/ml, whereas other stilbenoid (oxyresveratrol and anthraquinone derivatives (emodin, aloe-emodin, rhein were inactive (IC50 > 100 μg/ml.

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

Science.gov (United States)

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.

Gut TFH and IgA: key players for regulation of bacterial communities and immune homeostasis.

Science.gov (United States)

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

2014-01-01

The main function of the immune system is to protect the host against pathogens. However, unlike the systemic immune system, the gut immune system does not eliminate, but instead nourishes complex bacterial communities and establishes advanced symbiotic relationships. Immunoglobulin A (IgA) is the most abundant antibody isotype in mammals, produced mainly in the gut. The primary function of IgA is to maintain homeostasis at mucosal surfaces, and studies in mice have demonstrated that IgA diversification has an essential role in the regulation of gut microbiota. Dynamic diversification and constant adaptation of IgA responses to local microbiota require expression of activation-induced cytidine deaminase by B cells and control from T follicular helper and Foxp3(+) T cells in germinal centers (GCs). We discuss the finely tuned regulatory mechanisms for IgA synthesis in GCs of Peyer's patches and emphasize the roles of CD4(+) T cells for IgA selection and the maintenance of appropriate gut microbial communities required for immune homeostasis.

Presença de Candida nas mucosas vaginal e bucal e sua relação com IgA salivar Relationship between Candida in vaginal and oral mucosae and salivary IgA

Directory of Open Access Journals (Sweden)

Célia Regina Gonçalves e Silva

2008-06-01

Full Text Available OBJETIVO: correlacionar a presença de leveduras do gênero Candida na cavidade bucal e vaginal de mulheres com e sem candidíase vulvovaginal (CVV com os níveis de IgA secretora (IgAs presentes na saliva. MÉTODOS: cinqüenta e uma mulheres foram incluídas; 13 apresentaram CVV e 38 formaram o Grupo Controle. De cada paciente, foram coletados 2,0 mL de saliva sem estimulação e secreção vaginal com o auxílio de swab, que foi imerso a seguir em 2,0 mL de solução fisiológica. As amostras foram semeadas em ágar Sabouraud dextrose com cloranfenicol para isolamento e contagem de colônias, e os isolados foram identificadas fenotipicamente. Na saliva de ambos os grupos foi quantificada IgA pela técnica ELISA. RESULTADOS: nas 13 pacientes com diagnóstico clínico e micológico de CVV, a média de unidades formadoras de colônias de Candida por mililitro de secreção vaginal (ufc/mL foi de 52.723 e 23,8% dos pacientes apresentaram colonização na mucosa bucal com menor quantidade de ufc/mL (6.030. Os níveis de IgAs na saliva foram mais baixos no grupo com CVV (média de densidade: 0,3 quando comparados aos níveis de IgA do Grupo Controle (média de DO: 0,6. Onze pacientes (37% do Grupo Controle apresentaram colonização por Candida na cavidade bucal, com média de ufc/mL mais baixa quando comparada ao grupo com CVV. O Grupo Controle também apresentou menor quantidade de ufc/mL (1.973 na cavidade vaginal quando comparado com o Grupo CVV (52.942. CONCLUSÕES: os resultados demonstraram que os pacientes com diagnóstico clínico de candidíase vulvovaginal apresentaram maior quantidade de Candida, tanto na cavidade vaginal quanto na bucal, e apresentaram menores níveis de IgA anti-Candida na saliva.PURPOSE: to correlate the presence of yeast from the Candida genus in the oral and vaginal cavity of women with and without vulvovaginal candidiasis (VVC, with secretor IgA levels (IgAs present in the saliva. METHODS: among the 51 women

Intervention with Serine Protease Activity with Small Peptides

DEFF Research Database (Denmark)

Xu, Peng

2015-01-01

Serine proteases perform proteolytic reactions in many physiological and metabolic processes and have been certified as targets for therapeutics. Small peptides can be used as potent antagonists to target serine proteases and intervene with their activities. Urokinase-type plasminogen activator (u......PA) plays an important role in plasminogen activation system, which has many physiological and pathological functions and is closely associated with the metastasis of tumor cells. Based on a mono-cyclic peptidic inhibitor of murine uPA (muPA), mupain-1, which was screened out from a phage-display library...... before, we elucidated the binding and inhibitory mechanism by using multiple techniques, like X-ray crystallography, site-directed mutagenesis, isothermal titration calorimetry and surface plasmon resonance analysis. By studying the peptide-enzyme interaction, we discovered an unusual inhibitor...

Linear IgA bullous dermatosis in a patient with renal cell carcinoma

NARCIS (Netherlands)

Van der Waal, RIF; Van de Scheur, MR; Pas, HH; Jonkman, MF; Van Groeningen, CJ; Nieboer, C; Starink, TM

Linear IgA bullous dermatosis (LABD) is an autoimmune subepidermal bullous disease with heterogeneous clinical manifestations, characterized by linear deposition of IgA along the epidermal basement membrane zone. We report a patient with a metastasized renal cell carcinoma who developed an extensive

Serum IgG and IgA levels in polio and non-polio acute flaccid paralysis cases in western Uttar Pradesh, India.

Science.gov (United States)

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.

Proteases in Escherichia coli and Staphylococcus aureus confer reduced susceptibility to lactoferricin B.

Science.gov (United States)

Ulvatne, Hilde; Haukland, Hanne Husom; Samuelsen, Ørjan; Krämer, Manuela; Vorland, Lars H

2002-10-01

Lactoferricin B is a cationic antimicrobial peptide derived from the N-terminal part of bovine lactoferrin. The effect of bacterial proteases on the antibacterial activity of lactoferricin B towards Escherichia coli and Staphylococcus aureus was investigated using various protease inhibitors and protease-deficient E. coli mutants. Sodium-EDTA, a metalloprotease inhibitor, was the most efficient inhibitors in both species, but combinations of sodium-EDTA with other types of protease inhibitor gave a synergic effect. The results indicate that several groups of proteases are involved in resistance to lactoferricin B in both E. coli and S. aureus. We also report that genetic inactivation of the heat shock-induced serine protease DegP increased the susceptibility to lactoferricin B in E. coli, suggesting that this protease, at least, is involved in reduced susceptibility to lactoferricin B.

Identification and characterization of fusolisin, the Fusobacterium nucleatum autotransporter serine protease.

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Lior Doron

Full Text Available Fusobacterium nucleatum is an oral anaerobe associated with periodontal disease, adverse pregnancy outcomes and colorectal carcinoma. A serine endopeptidase of 61-65 kDa capable of damaging host tissue and of inactivating immune effectors was detected previously in F. nucleatum. Here we describe the identification of this serine protease, named fusolisin, in three oral F. nucleatum sub-species. Gel zymogram revealed fusobacterial proteolytic activity with molecular masses ranging from 55-101 kDa. All of the detected proteases were inhibited by the serine protease inhibitor PMSF. analysis revealed that all of the detected proteases are encoded by genes encoding an open reading frame (ORF with a calculated mass of approximately 115 kDa. Bioinformatics analysis of the identified ORFs demonstrated that they consist of three domains characteristic of autotransporters of the type Va secretion system. Our results suggest that the F. nucleatum fusolisins are derived from a precursor of approximately 115 kDa. After crossing the cytoplasmic membrane and cleavage of the leader sequence, the C-terminal autotransporter domain of the remaining 96-113 kDa protein is embedded in the outer membrane and delivers the N-terminal S8 serine protease passenger domain to the outer cell surface. In most strains the N-terminal catalytic 55-65 kDa domain self cleaves and liberates itself from the autotransporter domain after its transfer across the outer cell membrane. In F. nucleatum ATCC 25586 this autocatalytic activity is less efficient resulting in a full length membrane-anchored serine protease. The mature serine protease was found to cleave after Thr, Gly, Ala and Leu residues at the P1 position. Growth of F. nucleatum in complex medium was inhibited when serine protease inhibitors were used. Additional experiments are needed to determine whether fusolisin might be used as a target for controlling fusobacterial infections.

Mechanism of Folding and Activation of Subtilisin Kexin Isozyme-1 (SKI-1)/Site-1 Protease (S1P)*

Science.gov (United States)

da Palma, Joel Ramos; Cendron, Laura; Seidah, Nabil Georges; Pasquato, Antonella; Kunz, Stefan

2016-01-01

The proprotein convertase subtilisin kexin isozyme-1 (SKI-1)/site-1 protease (S1P) is implicated in lipid homeostasis, the unfolded protein response, and lysosome biogenesis. The protease is further hijacked by highly pathogenic emerging viruses for the processing of their envelope glycoproteins. Zymogen activation of SKI-1/S1P requires removal of an N-terminal prodomain, by a multistep process, generating the mature enzyme. Here, we uncover a modular structure of the human SKI-1/S1P prodomain and define its function in folding and activation. We provide evidence that the N-terminal AB fragment of the prodomain represents an autonomous structural and functional unit that is necessary and sufficient for folding and partial activation. In contrast, the C-terminal BC fragment lacks a defined structure but is crucial for autoprocessing and full catalytic activity. Phylogenetic analysis revealed that the sequence of the AB domain is highly conserved, whereas the BC fragment shows considerable variation and seems even absent in some species. Notably, SKI-1/S1P of arthropods, like the fruit fly Drosophila melanogaster, contains a shorter prodomain comprised of full-length AB and truncated BC regions. Swapping the prodomain fragments between fly and human resulted in a fully mature and active SKI-1/S1P chimera. Our study suggests that primordial SKI-1/S1P likely contained a simpler prodomain consisting of the highly conserved AB fragment that represents an independent folding unit. The BC region appears as a later evolutionary acquisition, possibly allowing more subtle fine-tuning of the maturation process. PMID:26645686

Treatment of IgA nephropathy.

Science.gov (United States)

Barratt, J; Feehally, J

2006-06-01

IgA nephropathy (IgAN) is an important cause of progressive kidney disease with 25-30% of patients developing end-stage renal disease within 20 years of diagnosis. There is still no treatment to modify mesangial IgA deposition and available treatments are those extrapolated from the management of other patterns of chronic glomerulonephritis. There remains no consensus on the use of immunosuppressive agents for treatment of progressive IgAN and this is compounded by the relative lack in IgAN of randomized controlled trials relevant to current clinical practice. Patients with recurrent macroscopic hematuria or isolated microscopic hematuria and proteinuria renal biopsy should be managed as for minimal change nephropathy. There is no evidence to support the use of corticosteroids for nephrotic IgAN outside this group of patients. Patients presenting with acute renal failure require evaluation to distinguish acute tubular necrosis, which requires supportive therapy only, from crescentic IgAN, for which treatment with cyclophosphamide and corticosteroids in a regimen similar to that for renal small vessel vasculitis is indicated in the absence of significant chronic histologic injury. Patients at greatest risk of progressive renal impairment are those with hypertension, proteinuria >1 g/24 h, and reduced glomerular filtration rate at diagnosis. All such patients should be treated to a blood pressure of 125/75 mm Hg with dual blockade of the renin-angiotensin system with angiotensin-converting enzyme inhibition and angiotensin receptor blockade. At present, there is insufficient evidence for the additional use of immunosuppressive agents, antiplatelet agents, or anticoagulants.

The Plasmodium serine-type SERA proteases display distinct expression patterns and non-essential in vivo roles during life cycle progression of the malaria parasite.

Science.gov (United States)

Putrianti, Elyzana D; Schmidt-Christensen, Anja; Arnold, Iris; Heussler, Volker T; Matuschewski, Kai; Silvie, Olivier

2010-06-01

Parasite proteases play key roles in several fundamental steps of the Plasmodium life cycle, including haemoglobin degradation, host cell invasion and parasite egress. Plasmodium exit from infected host cells appears to be mediated by a class of papain-like cysteine proteases called 'serine repeat antigens' (SERAs). A SERA subfamily, represented by Plasmodium falciparum SERA5, contains an atypical active site serine residue instead of a catalytic cysteine. Members of this SERAser subfamily are abundantly expressed in asexual blood stages, rendering them attractive drug and vaccine targets. In this study, we show by antibody localization and in vivo fluorescent tagging with the red fluorescent protein mCherry that the two P. berghei serine-type family members, PbSERA1 and PbSERA2, display differential expression towards the final stages of merozoite formation. Via targeted gene replacement, we generated single and double gene knockouts of the P. berghei SERAser genes. These loss-of-function lines progressed normally through the parasite life cycle, suggesting a specialized, non-vital role for serine-type SERAs in vivo. Parasites lacking PbSERAser showed increased expression of the cysteine-type PbSERA3. Compensatory mechanisms between distinct SERA subfamilies may thus explain the absence of phenotypical defect in SERAser disruptants, and challenge the suitability to develop potent antimalarial drugs based on specific inhibitors of Plasmodium serine-type SERAs.

Targeted-release budesonide versus placebo in patients with IgA nephropathy (NEFIGAN)

DEFF Research Database (Denmark)

Fellström, Bengt C.; Barratt, Jonathan; Cook, Heather

2017-01-01

Background IgA nephropathy is thought to be associated with mucosal immune system dysfunction, which manifests as renal IgA deposition that leads to impairment and end-stage renal disease in 20–40% of patients within 10–20 years. In this trial (NEFIGAN) we aimed to assess safety and efficacy...... at 62 nephrology clinics across ten European countries. We recruited patients aged at least 18 years with biopsy-confirmed primary IgA nephropathy and persistent proteinuria despite optimised renin-angiotensin system (RAS) blockade. We randomly allocated patients with a computer algorithm, with a fixed...

Degradation of Uniquely Glycosylated Secretory Immunoglobulin A in Tears From Patients With Pseudomonas aeruginosa Keratitis

DEFF Research Database (Denmark)

Lomholt, Jeanet Andersen; Kilian, Mogens

2008-01-01

PURPOSE. To investigate the integrity of secretory IgA (S-IgA) in tear fluid during bacterial keratitis and to evaluate the significance of specific Pseudomonas aeruginosa extracellular proteases in the observed degradation of S-IgA. METHODS. The integrity of component chains of S-IgA in tear fluid...... from patients with keratitis caused by P. aeruginosa, Streptococcus group G, Moraxella catarrhalis, Staphylococcus aureus, coagulase-negative staphylococci, and the IgA1 protease-producing Streptococcus pneumoniae were compared with S-IgA in tear fluid, colostrum, and saliva from healthy individuals......, and with tear S-IgA incubated with clinical isolates and genetically engineered P. aeruginosa strains with different protease profiles. Degradation of S-IgA and the significance of its glycosylation were analyzed in Western blots developed with antibodies against individual chains of S-IgA. RESULTS. Secretory...

Influence of startup oxidizing transients of IGA/SCC in PWR steam generators

International Nuclear Information System (INIS)

Gorman, J.A.; McIlree, A.R.; Gaudreau, T.; Bjornkvist, L.; Andersson, P.-O.

1998-01-01

There is a considerable amount of evidence oxidizing conditions during and following startups are an important factor in the intergranular corrosion/stress corrosion cracking (IGA/SCC) of mill annealed alloy 600 steam generator tubes. This evidence includes plant data that indicate that the growth of IGA/SCC correlates better in some cases with numbers of startups than with time at power, laboratory tests in several plausible crevice environments that show that small amounts of copper oxides accelerate the rate of IGA/SCC, laboratory tests that show that elevating the electrochemical potential (ECP) increases the rates of IGA/SCC in many chemical environments, and laboratory tests that show that copper oxides, hematite, and other oxidized corrosion products can raise the ECP of several solution chemistries into aggressive ranges. Some preliminary data also exist that show that some amounts of oxidized species are produced during typical layup and startup conditions, but data for the subsequent reduction of these oxides are largely lacking. The purpose of this paper is to review the available evidence, to arrive at conclusions regarding the probable importance of oxidizing conditions during startup on occurrence of IGA/SCC, and to identify needed research to better quantify the situation. (author)

Duration of secretory IgM and IgA antibodies to respiratory syncytial virus in a community study in Guinea-Bissau

DEFF Research Database (Denmark)

Stensballe, L G; Kofoed, P E; Nante, E J

2000-01-01

phase of infection. A secondary response may be more likely in children with low IgM responses in the acute phase (RR = 2.08 (95% confidence interval (CI) 0.92-4.70)). The IgA response was highest on days 28 and 42 after antigen detection, 72% having a detectable IgA response within the first 1.5 mo...... of these children had a simultaneous IgM-positive response. When 29 of the children were tested after an epidemic when they were 1-3-y-old, >80% again had high IgM (24/29, 82%) and IgA (28/29, 94%) levels. Among samples collected over a 1-y period from infants with LRI in a community morbidity surveillance...

Phase Field Modeling Using PetIGA

KAUST Repository

Vignal, Philippe; Collier, Nathan; Calo, Victor M.

2013-01-01

, and having a highly efficient and parallel framework to solve them is necessary. In this work, a brief review on phase field models is given, followed by a short analysis of the Phase Field Crystal Model solved with Isogeometric Analysis us- ing PetIGA. We

Intranasal IFNγ extends passive IgA antibody protection of mice against Mycobacterium tuberculosis lung infection

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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

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

Science.gov (United States)

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.

Sumo-dependent substrate targeting of the SUMO protease Ulp1

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Westerbeck Jason W

2011-10-01

Full Text Available Abstract Background In the yeast Saccharomyces cerevisiae, the essential small ubiquitin-like modifier (SUMO protease Ulp1 is responsible for both removing SUMO/Smt3 from specific target proteins and for processing precursor SUMO into its conjugation-competent form. Ulp1 localizes predominantly to nuclear pore complexes but has also been shown to deconjugate sumoylated septins at the bud-neck of dividing cells. How Ulp1 is directed to bud-neck localized septins and other cytoplasmic deconjugation targets is not well understood. Results Using a structure/function approach, we set out to elucidate features of Ulp1 that are required for substrate targeting. To aid our studies, we took advantage of a catalytically inactive mutant of Ulp1 that is greatly enriched at the septin ring of dividing yeast cells. We found that the localization of Ulp1 to the septins requires both SUMO and specific structural features of Ulp1's catalytic domain. Our analysis identified a 218-amino acid, substrate-trapping mutant of the catalytic domain of Ulp1, Ulp1(3(C580S, that is necessary and sufficient for septin localization. We also used the targeting and SUMO-binding properties of Ulp1(3(C580S to purify Smt3-modified proteins from cell extracts. Conclusions Our study provides novel insights into how the Ulp1 SUMO protease is actively targeted to its substrates in vivo and in vitro. Furthermore, we found that a substrate-trapping Ulp1(3(C580S interacts robustly with human SUMO1, SUMO2 and SUMO2 chains, making it a potentially useful tool for the analysis and purification of SUMO-modified proteins.

The Oxford classification of IgA nephropathy: rationale, clinicopathological correlations, and classification

NARCIS (Netherlands)

Cattran, Daniel C.; Coppo, Rosanna; Cook, H. Terence; Feehally, John; Roberts, Ian S. D.; Troyanov, Stéphan; Alpers, Charles E.; Amore, Alessandro; Barratt, Jonathan; Berthoux, Francois; Bonsib, Stephen; Bruijn, Jan A.; D'Agati, Vivette; D'Amico, Giuseppe; Emancipator, Steven; Emma, Francesco; Ferrario, Franco; Fervenza, Fernando C.; Florquin, Sandrine; Fogo, Agnes; Geddes, Colin C.; Groene, Hermann-Josef; Haas, Mark; Herzenberg, Andrew M.; Hill, Prue A.; Hogg, Ronald J.; Hsu, Stephen I.; Jennette, J. Charles; Joh, Kensuke; Julian, Bruce A.; Kawamura, Tetsuya; Lai, Fernand M.; Leung, Chi Bon; Li, Lei-Shi; Li, Philip K. T.; Liu, Zhi-Hong; Mackinnon, Bruce; Mezzano, Sergio; Schena, F. Paolo; Tomino, Yasuhiko; Walker, Patrick D.; Wang, Haiyan; Weening, Jan J.; Yoshikawa, Nori; Zhang, Hong

2009-01-01

IgA nephropathy is the most common glomerular disease worldwide, yet there is no international consensus for its pathological or clinical classification. Here a new classification for IgA nephropathy is presented by an international consensus working group. The goal of this new system was to

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

Science.gov (United States)

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.

Current and Novel Inhibitors of HIV Protease

Czech Academy of Sciences Publication Activity Database

Pokorná, Jana; Machala, L.; Řezáčová, Pavlína; Konvalinka, Jan

2009-01-01

Roč. 1, č. 3 (2009), s. 1209-1239 ISSN 1999-4915 R&D Projects: GA MŠk 1M0508 Grant - others:GA AV ČR(CZ) IAAX00320901 Program:IA Institutional research plan: CEZ:AV0Z40550506 Keywords : HIV protease * protease inhibitor * HAART Subject RIV: CE - Biochemistry

IgA LINEAR BULLOUS DERMATOSIS IN CHILDHOOD.

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Ivelina Yordanova

2015-12-01

Full Text Available IgA linear bullous dermatosis, also known as chronic bullous dermatosis of childhood, is an autoimmune disease which may be idiopathic or drug-induced. The disease affects children and adults. We present a 4 years old girl with itchy polymorphic eruptions. The skin rash was presented by bullous-erosive rosette-like lesions with reddish-brown crust in the center, distributed on the skin of the face, trunk and extremities. The vesicles were filled with serous and hemorrhagic content. Laboratory examinations were within normal values according the age. Histopathological examination of the lesional skin revealed sub epidermal blister. Direct immunofluorescence of perilesional skin demonstrated linear deposition of IgA in the basement membrane. Systemic treatment with Methylprednisolon and Claritromycin was applied with satisfactory effect. The patient is under observation.

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.

Science.gov (United States)

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

Aspartic Protease Zymography Case Study: Detection of Fungal Acid Proteases by Zymography.

Science.gov (United States)

Kernaghan, Gavin; Mayerhofer, Michael

2017-01-01

This chapter describes a method for the production and characterization of fungal acid proteases. Protease production is induced by growth on BSA media over a pH gradient and protein levels are monitored over time with the Bradford assay. Once protein is depleted, the media is purified and proteases are characterized by gelatin zymography using acrylamide and buffers at near-neutral pH. Maintaining pH levels below those found in traditional zymographic systems avoids the potential loss of activity that may occur in aspartic proteases under alkaline conditions.

A Novel Aspartic Protease with HIV-1 Reverse Transcriptase Inhibitory Activity from Fresh Fruiting Bodies of the Wild Mushroom Xylaria hypoxylon

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Qing-Xiu Hu

2012-01-01

Full Text Available A novel aspartic protease with HIV-1 RT inhibitory activity was isolated and characterized from fruiting bodies of the wild mushroom Xylaria hypoxylon. The purification protocol comprised distilled water homogenization and extraction step, three ion exchange chromatographic steps (on DEAE-cellulose, Q-Sepharose, and CM-cellulose in succession, and final purification was by FPLC on Superdex 75. The protease was adsorbed on all the three ion exchangers. It was a monomeric protein with a molecular mass of 43 kDa as estimated by SDS-PAGE and FPLC. Its N-terminal amino acid sequence was HYTELLSQVV, which exhibited no sequence homology to other proteases reported. The activity of the protease was adversely affected by Pepstatin A, indicating that it is an aspartic protease. The protease activity was maximal or nearly so in the pH range 6–8 and in the temperature range 35–60°C. The purified enzyme exhibited HIV-1 RT inhibitory activity with an IC50 value of 8.3 μM, but was devoid of antifungal, ribonuclease, and hemagglutinating activities.

Lack of serologic evidence to link IgA nephropathy with celiac disease or immune reactivity to gluten.

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Sina Moeller

Full Text Available 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.

Lack of serologic evidence to link IgA nephropathy with celiac disease or immune reactivity to gluten.

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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.

Nucleic Acid Aptamers Against Proteases

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Dupont, D M; Andersen, L M; Bøtkjær, Kenneth Alrø

2011-01-01

, directed against blood coagulation factors, are in clinical trials as anticoagulant drugs. Several of the studies on protease-binding aptamers have been pioneering and trend-setting in the field. The work with protease-binding aptamers also demonstrates many interesting examples of non-standard selection......Proteases are potential or realized therapeutic targets in a wide variety of pathological conditions. Moreover, proteases are classical subjects for studies of enzymatic and regulatory mechanisms. We here review the literature on nucleic acid aptamers selected with proteases as targets. Designing...... small molecule protease inhibitors of sufficient specificity has proved a daunting task. Aptamers seem to represent a promising alternative. In our review, we concentrate on biochemical mechanisms of aptamer selection, proteinaptamer recognition, protease inhibition, and advantages of aptamers...

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

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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.

Effects of eye rubbing on the levels of protease, protease activity and cytokines in tears: relevance in keratoconus.

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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.

CTA1-DD adjuvant promotes strong immunity against human immunodeficiency virus type 1 envelope glycoproteins following mucosal immunization.

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Sundling, Christopher; Schön, Karin; Mörner, Andreas; Forsell, Mattias N E; Wyatt, Richard T; Thorstensson, Rigmor; Karlsson Hedestam, Gunilla B; Lycke, Nils Y

2008-12-01

Strategies to induce potent and broad antibody responses against the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (Env) at both systemic and mucosal sites represent a central goal for HIV-1 vaccine development. Here, we show that the non-toxic CTA1-DD adjuvant promoted mucosal and systemic humoral and cell-mediated immune responses following intranasal (i.n.) immunizations with trimeric or monomeric forms of HIV-1 Env in mice and in non-human primates. Env-specific IgG subclasses in the serum of immunized mice reflected a balanced Th1/Th2 type of response. Strikingly, i.n. immunizations with Env and the CTA1-DD adjuvant induced substantial levels of mucosal anti-Env IgA in bronchial alveolar lavage and also detectable levels in vaginal secretions. By contrast, parenteral immunizations of Env formulated in Ribi did not stimulate mucosal IgA responses, while the two adjuvants induced a similar distribution of Env-specific IgG-subclasses in serum. A single parenteral boost with Env in Ribi adjuvant into mice previously primed i.n. with Env and CTA1-DD, augmented the serum anti-Env IgG levels to similar magnitudes as those observed after three intraperitoneal immunizations with Env in Ribi. The augmenting potency of CTA1-DD was similar to that of LTK63 or CpG oligodeoxynucleotides (ODN). However, in contrast to CpG ODN, the effect of CTA1-DD and LTK63 appeared to be independent of MyD88 and toll-like receptor signalling. This is the first demonstration that CTA1-DD augments specific immune responses also in non-human primates, suggesting that this adjuvant could be explored further as a clinically safe mucosal vaccine adjuvant for humoral and cell-mediated immunity against HIV-1 Env.

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

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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.

Crystal Structure of Human Taspase1, a Crucial Protease Regulating the Function of MLL

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Khan,J.; Dunn, B.; Tong, L.

2005-01-01

Taspase1 catalyzes the proteolytic processing of the mixed lineage leukemia (MLL) nuclear protein, which is required for maintaining Hox gene expression patterns. Chromosomal translocations of the MLL gene are associated with leukemia in infants. Taspase1, a threonine aspartase, is a member of the type 2 asparaginase family, but is the only protease in this family. We report here the crystal structures of human activated Taspase1 and its proenzyme, as well as the characterization of the effects of mutations in the active site region using a newly developed fluorogenic assay. The structure of Taspase1 has significant differences from other asparaginases, especially near the active site. Mutation of the catalytic nucleophile, Thr234, abolishes autocatalytic processing in cis but does not completely block proteolysis in trans. The structure unexpectedly showed the binding of a chloride ion in the active site, and our kinetic studies confirm that chlorides ions are inhibitors of this enzyme at physiologically relevant concentrations.

Study of relation between the onset age of type 1 diabetes and celiac disease in children andadole scents

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robabe Ghergherehchi

2010-02-01

Full Text Available Celiac disease (CD is a chronic enteropathy caused by hypersensitivity to gluten. Most studies have showed more prevalence of CD in the patients with diabetes mellitus type 1. Both diseases are autoimmune and their incidence is related to inheritance and environmental factors. The aim of this research is the study of relation between CD prevalence and diabetic age onset. Materials and Methods: In this descriptive cross-sectional study, 135 children with diabetes mellitus type 1 referring to Tabriz children hospital endocrine department and clinic between 2006-2008 were selected. After filling individual identity of the patients and the measurement of weight and height, the serumic level of anti-tissue Trans glutaminase IgA antibody (A-tTG-A-IgA, anti endomisial IgA antibody (AEA-IgA and anti-gliadin IgG antibody (AGA-IgG were measured. In the case that A-tTG-A either AEA alone or with AGA was high, small intestinal biopsy was preformed. The data was analysed using SPSS ver 16 software. Results: 28 of 135 patients with diabetes mellitus type 1, were serologically positive for celiac. Confirmed celiac prevalence based on biopsy was 6. 8%. From diabetic age onset and celiac incidence point of view there was not any significant relation (P=0. 996. Conclusion: Celiac disease in type1 diabetic patients dose not have correlation with the onset age of type 1 diabetes and diabetic patients should be followed up from celiac point of view during treatment and prevention.

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

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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

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

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Amaal M. Alhelli

2016-11-01

Full Text Available 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.

Bilateral Testicular Infarction from IgA Vasculitis of the Spermatic Cords

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Mazen Toushan

2017-01-01

Full Text Available A 51-year-old man with type 2 diabetes mellitus and chronic obstructive pulmonary disease presented to the emergency room with increasing bilateral leg pain, rash, and scrotal swelling with pain. Skin biopsy from his thigh revealed IgA-associated vasculitis. Due to hematuria, a renal biopsy was performed and showed an IgA glomerulonephritis with focal fibrinoid necrosis and neutrophil accumulation. Bilateral orchiectomies were performed in two separate procedures ten and thirteen days after the renal biopsy, as a result of uncontrolled abscess formation in testicles. Microscopically, both testicles revealed large abscess formation destroying almost the entire testicular parenchyma without tumor cells. Spermatic cord margins were further scrutinized microscopically to show bilateral vasculitis in many small size vessels, confirmed by positive endothelial staining for IgA. Some of the affected arteries revealed central organizing thrombi with recanalization features, highly suggestive of vasculitis-associated thrombi formation, resulting in testicular ischemic infarction and abscess formation. We conclude that this adult patient developed a severe form of Henoch-Schönlein purpura, with vasculitis affecting multiple organs, including the most serious and unusual complication of bilateral testicular infarction.

Efficient expression and purification of a protease from the common cold virus, human rhinovirus type 14

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Leong, L. E.-C.; Walker, P. A.; Porter, A. G.

1992-08-01

The protease (3C pro) from human rhinovirus serotype-14 (HRV-14) has been cloned and efficiently expressed in E. coli. A straightforward single-step purification of the recombinant 3C pro has been achieved by fusing the protein to the car☐y-terminus of the glutathione-S-transferase from Schistosoma japonicum. Modifications made to the 5' end of the PCR fragment coding for the 3C pro have allowed the specific cleavage of the fusion protein using thrombin to yield mature 3C pro with the correct amino-terminal amino acid. This protease has been shown to be active when assayed using synthetic peptides corresponding to the natural cleavage recognition sequences within the polyprotein. Other substrates are being developed for this protease for possible use in the screening of inhibitors of 3C pro. Sufficient protease 3C pro has been purified for initial attempts at crystallization.

Minimal Change Disease and IgA Deposition: Separate Entities or Common Pathophysiology?

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Brandon S. Oberweis

2013-01-01

Full Text Available Introduction. Minimal Change Disease (MCD is the most common cause of nephrotic syndrome in children, while IgA nephropathy is the most common cause of glomerulonephritis worldwide. MCD is responsive to glucocorticoids, while the role of steroids in IgA nephropathy remains unclear. We describe a case of two distinct clinical and pathological findings, raising the question of whether MCD and IgA nephropathy are separate entities or if there is a common pathophysiology. Case Report. A 19-year old man with no medical history presented to the Emergency Department with a 20-day history of anasarca and frothy urine, BUN 68 mg/dL, Cr 2.3 mg/dL, urinalysis 3+ RBCs, 3+ protein, and urine protein : creatinine ratio 6.4. Renal biopsy revealed hypertrophic podocytes on light microscopy, podocyte foot process effacement on electron microscopy, and immunofluorescent mesangial staining for IgA. The patient was started on prednisone and exhibited dramatic improvement. Discussion. MCD typically has an overwhelming improvement with glucocorticoids, while the resolution of IgA nephropathy is rare. Our patient presented with MCD with the uncharacteristic finding of hematuria. Given the improvement with glucocorticoids, we raise the question of whether there is a shared pathophysiologic component of these two distinct clinical diseases that represents a clinical variant.

Mechanism of Folding and Activation of Subtilisin Kexin Isozyme-1 (SKI-1)/Site-1 Protease (S1P).

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da Palma, Joel Ramos; Cendron, Laura; Seidah, Nabil Georges; Pasquato, Antonella; Kunz, Stefan

2016-01-29

The proprotein convertase subtilisin kexin isozyme-1 (SKI-1)/site-1 protease (S1P) is implicated in lipid homeostasis, the unfolded protein response, and lysosome biogenesis. The protease is further hijacked by highly pathogenic emerging viruses for the processing of their envelope glycoproteins. Zymogen activation of SKI-1/S1P requires removal of an N-terminal prodomain, by a multistep process, generating the mature enzyme. Here, we uncover a modular structure of the human SKI-1/S1P prodomain and define its function in folding and activation. We provide evidence that the N-terminal AB fragment of the prodomain represents an autonomous structural and functional unit that is necessary and sufficient for folding and partial activation. In contrast, the C-terminal BC fragment lacks a defined structure but is crucial for autoprocessing and full catalytic activity. Phylogenetic analysis revealed that the sequence of the AB domain is highly conserved, whereas the BC fragment shows considerable variation and seems even absent in some species. Notably, SKI-1/S1P of arthropods, like the fruit fly Drosophila melanogaster, contains a shorter prodomain comprised of full-length AB and truncated BC regions. Swapping the prodomain fragments between fly and human resulted in a fully mature and active SKI-1/S1P chimera. Our study suggests that primordial SKI-1/S1P likely contained a simpler prodomain consisting of the highly conserved AB fragment that represents an independent folding unit. The BC region appears as a later evolutionary acquisition, possibly allowing more subtle fine-tuning of the maturation process. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Extracellular Neutrophil Proteases Are Efficient Regulators of IL-1, IL-33, and IL-36 Cytokine Activity but Poor Effectors of Microbial Killing.

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Clancy, Danielle M; Sullivan, Graeme P; Moran, Hannah B T; Henry, Conor M; Reeves, Emer P; McElvaney, Noel G; Lavelle, Ed C; Martin, Seamus J

2018-03-13

Neutrophil granule proteases are thought to function as anti-microbial effectors, cooperatively hydrolyzing microorganisms within phagosomes, or upon deployment into the extracellular space. However, evidence also suggests that neutrophil proteases play an important role in the coordination and escalation of inflammatory reactions, but how this is achieved has been obscure. IL-1 family cytokines are important initiators of inflammation and are typically released via necrosis but require proteolytic processing for activation. Here, we show that proteases liberated from activated neutrophils can positively or negatively regulate the activity of six IL-1 family cytokines (IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and IL-36γ) with exquisite sensitivity. In contrast, extracellular neutrophil proteases displayed very poor bactericidal activity, exhibiting 100-fold greater potency toward cytokine processing than bacterial killing. Thus, in addition to their classical role as phagocytes, neutrophils play an important immunoregulatory role through deployment of their granule proteases into the extracellular space to process multiple IL-1 family cytokines. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

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

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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.

Escherichia coli contains a soluble ATP-dependent protease (Ti) distinct from protease La

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Hwang, B.J.; Park, W.J.; Chung, C.H.; Goldberg, A.L.

1987-08-01

The energy requirement for protein breakdown in Escherichia coli has generally been attributed to the ATP-dependence of protease La, the lon gene product. The authors have partially purified another ATP-dependent protease from lon/sup -/ cells that lack protease La (as shown by immunoblotting). This enzyme hydrolyzes (/sup 3/H)methyl-casein to acid-soluble products in the presence of ATP and Mg/sup 2 +/. ATP hydrolysis appears necessary for proteolytic activity. Since this enzyme is inhibited by diisopropyl fluorophosphate, it appears to be a serine protease, but it also contains essential thiol residues. They propose to name this enzyme protease Ti. It differs from protease La in nucleotide specificity, inhibitor sensitivity, and subunit composition. On gel filtration, protease Ti has an apparent molecular weight of 370,000. It can be fractionated by phosphocellulose chromatography or by DEAE chromatography into two components with apparent molecular weights of 260,000 and 140,000. When separated, they do not show preteolytic activity. One of these components, by itself, has ATPase activity and is labile in the absence of ATP. The other contains the diisopropyl fluorophosphate-sensitive proteolytic site. These results and the similar findings of Katayama-Fujimura et al. indicate that E. coli contains two ATP-hydrolyzing proteases, which differ in many biochemical features and probably in their physiological roles.

Escherichia coli contains a soluble ATP-dependent protease (Ti) distinct from protease La

International Nuclear Information System (INIS)

Hwang, B.J.; Park, W.J.; Chung, C.H.; Goldberg, A.L.

1987-01-01

The energy requirement for protein breakdown in Escherichia coli has generally been attributed to the ATP-dependence of protease La, the lon gene product. The authors have partially purified another ATP-dependent protease from lon - cells that lack protease La (as shown by immunoblotting). This enzyme hydrolyzes [ 3 H]methyl-casein to acid-soluble products in the presence of ATP and Mg 2+ . ATP hydrolysis appears necessary for proteolytic activity. Since this enzyme is inhibited by diisopropyl fluorophosphate, it appears to be a serine protease, but it also contains essential thiol residues. They propose to name this enzyme protease Ti. It differs from protease La in nucleotide specificity, inhibitor sensitivity, and subunit composition. On gel filtration, protease Ti has an apparent molecular weight of 370,000. It can be fractionated by phosphocellulose chromatography or by DEAE chromatography into two components with apparent molecular weights of 260,000 and 140,000. When separated, they do not show preteolytic activity. One of these components, by itself, has ATPase activity and is labile in the absence of ATP. The other contains the diisopropyl fluorophosphate-sensitive proteolytic site. These results and the similar findings of Katayama-Fujimura et al. indicate that E. coli contains two ATP-hydrolyzing proteases, which differ in many biochemical features and probably in their physiological roles

Extracellular proteases of Trichoderma species. A review.

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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.

Role of Proteases in Chronic Obstructive Pulmonary Disease

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Kailash C. Pandey

2017-08-01

Full Text Available Chronic obstructive pulmonary disease (COPD is generally associated with progressive destruction of airways and lung parenchyma. Various factors play an important role in the development and progression of COPD, like imbalance of proteases, environmental and genetic factors and oxidative stress. This review is specifically focused on the role of proteases and their imbalance in COPD. There are three classes (serine, mettalo, and cysteine of proteases involved in COPD. In serine proteases, neutrophil elastase, cathepsin G, and proteinase-3 are involved in destruction of alveolar tissue. Matrix-mettaloproteinase-9, 12, 13, plays an influential role in severity of COPD. Among cysteine proteases, caspase-3, caspases-8 and caspase-9 play an important role in controlling apoptosis. These proteases activities can be regulated by inhibitors like α-1-antitrypsin, neutrophil elastase inhibitor, and leukocyte protease inhibitor. Studies suggest that neutrophil elastase may be a therapeutic target for COPD, and specific inhibitor against this enzyme has potential role to control the disease. Current study suggests that Dipeptidyl Peptidase IV is a potential marker for COPD. Since the expression of proteases and its inhibitors play an important role in COPD pathogenesis, therefore, it is worth investigating the role of proteases and their regulation. Understanding the biochemical basis of COPD pathogenesis using advanced tools in protease biochemistry and aiming toward translational research from bench-to-bedside will have great impact to deal with this health problem.

Characterization of Fibrinolytic Proteases from Gloydius blomhoffii siniticus Venom

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Suk Ho Choi

2011-09-01

Full Text Available Objectives : This study was undertaken to identify fibrinolytic proteases from Gloydius blomhoffii siniticus venom and to characterize a major fibrinolytic protease purified from the venom. Methods: The venom was subjected to chromatography using columns of Q-Sepharose and Sephadex G-75. The molecular weights of fibrinolytic proteases showing fibrinolytic zone in fibrin plate assay were determined in SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis The effects of inhibitors and metal ions on fibrinolytic protease and the proteolysis patterns of fibrinogen, gelatin, and bovine serum albumin were investigated. Results : 1 The fibrinolytic fractions of the three peaks isolated from Gloydius blomhoffii siniticus venom contained two polypeptides of 46 and 59 kDa and three polypeptides of 32, 18, and 15 kDa and a major polypeptide of 54 kDa, respectively. 2 The fibrinolytic activity of the purified protease of 54 kDA was inhibited by metal chelators, such as EDTA, EGTA, and 1,10-phenanthroline, and disulfhydryl-reducing compounds, such as dithiothreitol and cysteine. 3 Calcium chloride promoted the fibrinolytic activity of the protease, but mercuric chloride and cobalt(II chloride inhibited it. 4 The fibrinolytic protease cleaved preferentially A-chain and slowly B-chain of fibrinogen. It also hydrolyzed gelatin but not bovine serum albumin. Conclusions: The Gloydius blomhoffii siniticus venom contained more than three fibrinolytic proteases. The major fibrinolytic protease was a metalloprotease which hydrolyzed both fibrinogen and gelatin, but not bovine serum albumin.

Lysine sulfonamides as novel HIV-protease inhibitors: Nepsilon-acyl aromatic alpha-amino acids.

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Stranix, Brent R; Lavallée, Jean-François; Sévigny, Guy; Yelle, Jocelyn; Perron, Valérie; LeBerre, Nicholas; Herbart, Dominik; Wu, Jinzi J

2006-07-01

A series of lysine sulfonamide analogues bearing Nepsilon-acyl aromatic amino acids were synthesized using an efficient synthetic route. Evaluation of these novel protease inhibitors revealed compounds with high potency against wild-type and multiple-protease inhibitor-resistant HIV viruses.

Evaluation of Serum IgA level in nontreated and treated oral squamous cell carcinoma patients

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Richa Mishra

2018-01-01

Full Text Available Introduction: Research in early cancer detection has led to discovery of many immunological tumor markers that contribute considerably to supplement the method of diagnosis. High serum immunoglobulin A (IgA values in patients with cancer have been used as tumor markers. Aims and Objectives: To evaluate and compare the serum IgA levels in nontreated, treated oral squamous cell carcinoma (SCC patients, and control group. Materials and Methods: A total of 60 patients were included in the study. 20 biopsy confirmed oral SCC patients, who have received no medical treatment, 20 oral SCC patients treated with surgery and/or radiotherapy and 20 normal healthy individuals. Venous blood samples were collected from anterior cubital vein and were delivered to the biochemistry laboratory for the estimation of serum IgA level by nephelometry method. Statistical Analysis Used: Statistical method employed were the Pearson's Chi-square test and One-way analysis of variance (Welch followed by Games-Howell post-hoc test. Results: We observed significant difference for serum IgA between study subjects in control, nontreated and treated oral SCC patients (P < 0.001. Serum IgA level in nontreated group was significantly higher than treated group and there was an approximately two-fold increase in serum IgA level in nontreated oral SCC patients when compared to that of the normal healthy individuals. Conclusion: Serum level of IgA might be employed as diagnostic and prognostic indicators in oral cancer.

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

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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.

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

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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

The ATP-Dependent Protease ClpP Inhibits Biofilm Formation by Regulating Agr and Cell Wall Hydrolase Sle1 in Staphylococcus aureus

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Liu, Qian; Wang, Xing; Qin, Juanxiu; Cheng, Sen; Yeo, Won-Sik; He, Lei; Ma, Xiaowei; Liu, Xiaoyun; Li, Min; Bae, Taeok

2017-01-01

Biofilm causes hospital-associated infections on indwelling medical devices. In Staphylococcus aureus, Biofilm formation is controlled by intricately coordinated network of regulating systems, of which the ATP-dependent protease ClpP shows an inhibitory effect. Here, we demonstrate that the inhibitory effect of ClpP on biofilm formation is through Agr and the cell wall hydrolase Sle1. Biofilm formed by clpP mutant consists of proteins and extracellular DNA (eDNA). The increase of the protein was, at least in part, due to the reduced protease activity of the mutant, which was caused by the decreased activity of agr. On the other hand, the increase of eDNA was due to increased cell lysis caused by the higher level of Sle1. Indeed, as compared with wild type, the clpP mutant excreted an increased level of eDNA, and showed higher sensitivity to Triton-induced autolysis. The deletion of sle1 in the clpP mutant decreased the biofilm formation, the level of eDNA, and the Triton-induced autolysis to wild-type levels. Despite the increased biofilm formation capability, however, the clpP mutant showed significantly reduced virulence in a murine model of subcutaneous foreign body infection, indicating that the increased biofilm formation capability cannot compensate for the intrinsic functions of ClpP during infection. PMID:28555174

The ATP-Dependent Protease ClpP Inhibits Biofilm Formation by Regulating Agr and Cell Wall Hydrolase Sle1 in Staphylococcus aureus

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Qian Liu

2017-05-01

Full Text Available Biofilm causes hospital-associated infections on indwelling medical devices. In Staphylococcus aureus, Biofilm formation is controlled by intricately coordinated network of regulating systems, of which the ATP-dependent protease ClpP shows an inhibitory effect. Here, we demonstrate that the inhibitory effect of ClpP on biofilm formation is through Agr and the cell wall hydrolase Sle1. Biofilm formed by clpP mutant consists of proteins and extracellular DNA (eDNA. The increase of the protein was, at least in part, due to the reduced protease activity of the mutant, which was caused by the decreased activity of agr. On the other hand, the increase of eDNA was due to increased cell lysis caused by the higher level of Sle1. Indeed, as compared with wild type, the clpP mutant excreted an increased level of eDNA, and showed higher sensitivity to Triton-induced autolysis. The deletion of sle1 in the clpP mutant decreased the biofilm formation, the level of eDNA, and the Triton-induced autolysis to wild-type levels. Despite the increased biofilm formation capability, however, the clpP mutant showed significantly reduced virulence in a murine model of subcutaneous foreign body infection, indicating that the increased biofilm formation capability cannot compensate for the intrinsic functions of ClpP during infection.

Bacterial proteases and virulence

DEFF Research Database (Denmark)

Frees, Dorte; Brøndsted, Lone; Ingmer, Hanne

2013-01-01

signalling to short-circuit host cell processes. Common to both intra- and extracellular proteases is the tight control of their proteolytic activities. In general, substrate recognition by the intracellular proteases is highly selective which is, in part, attributed to the chaperone activity associated...... tolerance to adverse conditions such as those experienced in the host. In the membrane, HtrA performs similar functions whereas the extracellular proteases, in close contact with host components, pave the way for spreading infections by degrading host matrix components or interfering with host cell...... with the proteases either encoded within the same polypeptide or on separate subunits. In contrast, substrate recognition by extracellular proteases is less selective and therefore these enzymes are generally expressed as zymogens to prevent premature proteolytic activity that would be detrimental to the cell...

Identification of SlpB, a Cytotoxic Protease from Serratia marcescens.

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Shanks, Robert M Q; Stella, Nicholas A; Hunt, Kristin M; Brothers, Kimberly M; Zhang, Liang; Thibodeau, Patrick H

2015-07-01

The Gram-negative bacterium and opportunistic pathogen Serratia marcescens causes ocular infections in healthy individuals. Secreted protease activity was characterized from 44 ocular clinical isolates, and a higher frequency of protease-positive strains was observed among keratitis isolates than among conjunctivitis isolates. A positive correlation between protease activity and cytotoxicity to human corneal epithelial cells in vitro was determined. Deletion of prtS in clinical keratitis isolate K904 reduced, but did not eliminate, cytotoxicity and secreted protease production. This indicated that PrtS is necessary for full cytotoxicity to ocular cells and implied the existence of another secreted protease(s) and cytotoxic factors. Bioinformatic analysis of the S. marcescens Db11 genome revealed three additional open reading frames predicted to code for serralysin-like proteases noted here as slpB, slpC, and slpD. Induced expression of prtS and slpB, but not slpC and slpD, in strain PIC3611 rendered the strain cytotoxic to a lung carcinoma cell line; however, only prtS induction was sufficient for cytotoxicity to a corneal cell line. Strain K904 with deletion of both prtS and slpB genes was defective in secreted protease activity and cytotoxicity to human cell lines. PAGE analysis suggests that SlpB is produced at lower levels than PrtS. Purified SlpB demonstrated calcium-dependent and AprI-inhibited protease activity and cytotoxicity to airway and ocular cell lines in vitro. Lastly, genetic analysis indicated that the type I secretion system gene, lipD, is required for SlpB secretion. These genetic data introduce SlpB as a new cytotoxic protease from S. marcescens. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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.

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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.

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

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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

The Comparison of Salivary IgA and IgE Levels in Children with Breast- and Formula- Feeding During Infancy Period

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A. Jafarzadeh

2007-01-01

Full Text Available Introduction: Oral local immune factors may play a protective role against oral diseases and defend against microbial agents. Salivary immunoglobulin A (IgA is a major factor for the local host defence against caries and periodontal disease. The aims of this study were to determine the concentrations of salivary IgA and IgE levels in breast-fed and formula-fed children in infancy period.Methods and Materials: Totally, 80 healthy 5 years old children were included in the study. According to type of feeding in infancy period, the children divided into two groups: 50 breast-fed and 30 formula-fed. One milliliter of saliva was collected from each participant, centrifuged, and stored at -70 C. The salivary IgA and IgE concentrations were measured, using ELISA technique.Results: In breast-fed children, the salivary IgA level (39.6 mg/l ± 17.3 was significantly higher than that in formula-fed children (26.9 mg/l ± 14 (P=0.0001. However, the salivary IgE level was significantly lower in breast-fed children, comparing with formula fed ones (5.01 IU/ml ± 19.70 vs. 11.74 IU/ml ± 39.40 (P=0.047.Discussion: These results suggest that breast feeding enhances salivary IgA level in the early period of life which may contribute in oral cavity immunity. Higher salivary IgE level observed in formula-fed subjects may have a potential role in development of allergic or inflammatory reactions.

Effects of HIV-1 protease on cellular functions and their potential applications in antiretroviral therapy

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Yang Hailiu

2012-09-01

Full Text Available Abstract 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

Mature and progenitor endothelial cells perform angiogenesis also under protease inhibition: the amoeboid angiogenesis.

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Chillà, Anastasia; Margheri, Francesca; Biagioni, Alessio; Del Rosso, Mario; Fibbi, Gabriella; Laurenzana, Anna

2018-04-03

Controlling vascular growth is a challenging aim for the inhibition of tumor growth and metastasis. The amoeboid and mesenchymal types of invasiveness are two modes of migration interchangeable in cancer cells: the Rac-dependent mesenchymal migration requires the activity of proteases; the Rho-ROCK-dependent amoeboid motility is protease-independent and has never been described in endothelial cells. A cocktail of physiologic inhibitors (Ph-C) of serine-proteases, metallo-proteases and cysteine-proteases, mimicking the physiological environment that cells encounter during their migration within the angiogenesis sites was used to induce amoeboid style migration of Endothelial colony forming cells (ECFCs) and mature endothelial cells (ECs). To evaluate the mesenchymal-ameboid transition RhoA and Rac1 activation assays were performed along with immunofluorescence analysis of proteins involved in cytoskeleton organization. Cell invasion was studied in Boyden chambers and Matrigel plug assay for the in vivo angiogenesis. In the present study we showed in both ECFCs and ECs, a decrease of activated Rac1 and an increase of activated RhoA upon shifting of cells to the amoeboid conditions. In presence of Ph-C inhibitors both cell lines acquired a round morphology and Matrigel invasion was greatly enhanced with respect to that observed in the absence of protease inhibition. We also observed that the urokinase-plasminogen-activator (uPAR) receptor silencing and uPAR-integrin uncoupling with the M25 peptide abolished both mesenchymal and amoeboid angiogenesis of ECFCs and ECs in vitro and in vivo, indicating a role of the uPAR-integrin-actin axis in the regulation of amoeboid angiogenesis. Furthermore, under amoeboid conditions endothelial cells seem to be indifferent to VEGF stimulation, which induces an amoeboid signaling pattern also in mesenchymal conditions. Here we first provide a data set disclosing that endothelial cells can move and differentiate into vascular

Urinary uromodulin excretion predicts progression of chronic kidney disease resulting from IgA nephropathy.

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Jingjing Zhou

Full Text Available BACKGROUND: Uromodulin, or Tamm-Horsfall protein, is the most abundant urinary protein in healthy individuals. Recent studies have suggested that uromodulin may play a role in chronic kidney diseases. We examined an IgA nephropathy cohort to determine whether uromodulin plays a role in the progression of IgA nephropathy. METHODS: A total of 344 IgA nephropathy patients were involved in this study. Morphological changes were evaluated with the Oxford classification of IgA nephropathy. Enzyme Linked Immunosorbent Assay (ELISA measured the urinary uromodulin level on the renal biopsy day. Follow up was done regularly on 185 patients. Time-average blood pressure, time-average proteinuria, estimated glomerular filtration rate (eGFR and eGFR decline rate were caculated. Association between the urinary uromodulin level and the eGFR decline rate was analyzed with SPSS 13.0. RESULTS: We found that lower baseline urinary uromodulin levels (P = 0.03 and higher time-average proteinuria (P = 0.04 were risk factors for rapid eGFR decline in a follow-up subgroup of the IgA nephropathy cohort. Urinary uromodulin level was correlated with tubulointerstitial lesions (P = 0.016. Patients that had more tubular atrophy/interstitial fibrosis on the surface had lower urinary uromodulin levels (P = 0.02. CONCLUSIONS: Urinary uromodulin level is associated with interstitial fibrosis/tubular atrophy and contributes to eGFR decline in IgA nephropathy.

Mast cells limit extracellular levels of IL-13 via a serglycin proteoglycan-serine protease axis.

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Waern, Ida; Karlsson, Iulia; Thorpe, Michael; Schlenner, Susan M; Feyerabend, Thorsten B; Rodewald, Hans-Reimer; Åbrink, Magnus; Hellman, Lars; Pejler, Gunnar; Wernersson, Sara

2012-12-01

Mast cell (MC) granules contain large amounts of proteases of the chymase, tryptase and carboxypeptidase A (MC-CPA) type that are stored in complex with serglycin,a proteoglycan with heparin side chains. Hence, serglycinprotease complexes are released upon MC degranulation and may influence local inflammation. Here we explored the possibility that a serglycin-protease axis may regulate levels of IL-13, a cytokine involved in allergic asthma. Indeed, we found that wild-type MCs efficiently degraded exogenous or endogenously produced IL-13 upon degranulation,whereas serglycin −/− MCs completely lacked this ability.Moreover, MC-mediated IL-13 degradation was blocked both by a serine protease inhibitor and by a heparin antagonist,which suggests that IL-13 degradation is catalyzed by serglycin-dependent serine proteases and that optimal IL-13 degradation is dependent on both the serglycin and the protease component of the serglycin-protease complex.Moreover, IL-13 degradation was abrogated in MC-CPA −/−MC cultures, but was normal in cultures of MCs with an inactivating mutation of MC-CPA, which suggests that the IL-13-degrading serine proteases rely on MC-CPA protein.Together, our data implicate a serglycin-serine protease axis in the regulation of extracellular levels of IL-13. Reduction of IL-13 levels through this mechanism possibly can provide a protective function in the context of allergic inflammation.

Detection of H. Pylori infection on dyspepsia patients with IgA H. Pylori antibody

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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.

Gut proteases target Yersinia invasin in vivo

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Freund Sandra

2011-04-01

Full Text Available Abstract Background Yersinia enterocolitica is a common cause of food borne gastrointestinal disease. After oral uptake, yersiniae invade Peyer's patches of the distal ileum. This is accomplished by the binding of the Yersinia invasin to β1 integrins on the apical surface of M cells which overlie follicle associated lymphoid tissue. The gut represents a barrier that severely limits yersiniae from reaching deeper tissues such as Peyer's patches. We wondered if gut protease attack on invasion factors could contribute to the low number of yersiniae invading Peyer's patches. Findings Here we show that invasin is rapidly degraded in vivo by gut proteases in the mouse infection model. In vivo proteolytic degradation is due to proteolysis by several gut proteases such as trypsin, α-chymotrypsin, pancreatic elastase, and pepsin. Protease treated yersiniae are shown to be less invasive in a cell culture model. YadA, another surface adhesin is cleaved by similar concentrations of gut proteases but Myf was not cleaved, showing that not all surface proteins are equally susceptible to degradation by gut proteases. Conclusions We demonstrate that gut proteases target important Yersinia virulence factors such as invasin and YadA in vivo. Since invasin is completely degraded within 2-3 h after reaching the small intestine of mice, it is no longer available to mediate invasion of Peyer's patches.

Suppressing IL-36-driven inflammation using peptide pseudosubstrates for neutrophil proteases.

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Sullivan, Graeme P; Henry, Conor M; Clancy, Danielle M; Mametnabiev, Tazhir; Belotcerkovskaya, Ekaterina; Davidovich, Pavel; Sura-Trueba, Sylvia; Garabadzhiu, Alexander V; Martin, Seamus J

2018-03-07

Sterile inflammation is initiated by molecules released from necrotic cells, called damage-associated molecular patterns (DAMPs). Members of the extended IL-1 cytokine family are important DAMPs, are typically only released through necrosis, and require limited proteolytic processing for activation. The IL-1 family cytokines, IL-36α, IL-36β, and IL-36γ, are expressed as inactive precursors and have been implicated as key initiators of psoriatic-type skin inflammation. We have recently found that IL-36 family cytokines are proteolytically processed and activated by the neutrophil granule-derived proteases, elastase, and cathepsin G. Inhibitors of IL-36 processing may therefore have utility as anti-inflammatory agents through suppressing activation of the latter cytokines. We have identified peptide-based pseudosubstrates for cathepsin G and elastase, based on optimal substrate cleavage motifs, which can antagonize activation of all three IL-36 family cytokines by the latter proteases. Human psoriatic skin plaques displayed elevated IL-36β processing activity that could be antagonized by peptide pseudosubstrates specific for cathepsin G. Thus, antagonists of neutrophil-derived proteases may have therapeutic potential for blocking activation of IL-36 family cytokines in inflammatory conditions such as psoriasis.

A consistency-based feature selection method allied with linear SVMs for HIV-1 protease cleavage site prediction.

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Orkun Oztürk

Full Text Available BACKGROUND: Predicting type-1 Human Immunodeficiency Virus (HIV-1 protease cleavage site in protein molecules and determining its specificity is an important task which has attracted considerable attention in the research community. Achievements in this area are expected to result in effective drug design (especially for HIV-1 protease inhibitors against this life-threatening virus. However, some drawbacks (like the shortage of the available training data and the high dimensionality of the feature space turn this task into a difficult classification problem. Thus, various machine learning techniques, and specifically several classification methods have been proposed in order to increase the accuracy of the classification model. In addition, for several classification problems, which are characterized by having few samples and many features, selecting the most relevant features is a major factor for increasing classification accuracy. RESULTS: We propose for HIV-1 data a consistency-based feature selection approach in conjunction with recursive feature elimination of support vector machines (SVMs. We used various classifiers for evaluating the results obtained from the feature selection process. We further demonstrated the effectiveness of our proposed method by comparing it with a state-of-the-art feature selection method applied on HIV-1 data, and we evaluated the reported results based on attributes which have been selected from different combinations. CONCLUSION: Applying feature selection on training data before realizing the classification task seems to be a reasonable data-mining process when working with types of data similar to HIV-1. On HIV-1 data, some feature selection or extraction operations in conjunction with different classifiers have been tested and noteworthy outcomes have been reported. These facts motivate for the work presented in this paper. SOFTWARE AVAILABILITY: The software is available at http

Kinetics Study of Extracellular Detergent Stable Alkaline Protease from Rhizopus oryzae

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Zareena Mushtaq

2015-04-01

Full Text Available In this study, extracellular alkaline protease was produced from Rhizopus oryzae in submerged fermentation using dairy waste (whey as a substrate. Fermentation kinetics was studied and various parameters were optimized. The strain produced maximum protease at initial medium pH of 6.0 medium depth of 26 mm, inoculum size of 2% at incubation temperature of 35ºC for 168 h of fermentation. Alkaline protease was purified to homogeneity by ammonium sulphate fractionation followed by sephadex G-100 chromatography. The molecular mass of alkaline protease was 69 kDa determined by 10% SDS-PAGE. The optimum pH and temperature of alkaline protease was 9.0 and 40ºC, respectively. Metal profile of the enzyme showed that the enzyme was non-metallic in nature. The Km , Kcat , Vmax and Kcat/Km values of purified protease were 7.0 mg/mL, 3.8 x102S-1, 54.30 µmol/min and 54.28 s-1mg -1.mL respectively, using casein as substrate. The purified alkaline protease had stability with commercial detergents.

Structural Characterization of the Loop at the Alpha-Subunit C-Terminus of the Mixed Lineage Leukemia Protein Activating Protease Taspase1.

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Johannes van den Boom

Full Text Available Type 2 asparaginases, a subfamily of N-terminal nucleophile (Ntn hydrolases, are activated by limited proteolysis. This activation yields a heterodimer and a loop region at the C-terminus of the α-subunit is released. Since this region is unresolved in all type 2 asparaginase crystal structures but is close to the active site residues, we explored this loop region in six members of the type 2 asparaginase family using homology modeling. As the loop model for the childhood cancer-relevant protease Taspase1 differed from the other members, Taspase1 activation as well as the conformation and dynamics of the 56 amino acids loop were investigated by CD and NMR spectroscopy. We propose a helix-turn-helix motif, which can be exploited as novel anticancer target to inhibit Taspase1 proteolytic activity.

Prediction of mutational tolerance in HIV-1 protease and reverse transcriptase using flexible backbone protein design.

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Elisabeth Humphris-Narayanan

Full Text Available Predicting which mutations proteins tolerate while maintaining their structure and function has important applications for modeling fundamental properties of proteins and their evolution; it also drives progress in protein design. Here we develop a computational model to predict the tolerated sequence space of HIV-1 protease reachable by single mutations. We assess the model by comparison to the observed variability in more than 50,000 HIV-1 protease sequences, one of the most comprehensive datasets on tolerated sequence space. We then extend the model to a second protein, reverse transcriptase. The model integrates multiple structural and functional constraints acting on a protein and uses ensembles of protein conformations. We find the model correctly captures a considerable fraction of protease and reverse-transcriptase mutational tolerance and shows comparable accuracy using either experimentally determined or computationally generated structural ensembles. Predictions of tolerated sequence space afforded by the model provide insights into stability-function tradeoffs in the emergence of resistance mutations and into strengths and limitations of the computational model.

Exposure to ozone modulates human airway protease/antiprotease balance contributing to increased influenza A infection.

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Matthew J Kesic

Full Text Available Exposure to oxidant air pollution is associated with increased respiratory morbidities and susceptibility to infections. Ozone is a commonly encountered oxidant air pollutant, yet its effects on influenza infections in humans are not known. The greater Mexico City area was the primary site for the spring 2009 influenza A H1N1 pandemic, which also coincided with high levels of environmental ozone. Proteolytic cleavage of the viral membrane protein hemagglutinin (HA is essential for influenza virus infectivity. Recent studies suggest that HA cleavage might be cell-associated and facilitated by the type II transmembrane serine proteases (TTSPs human airway trypsin-like protease (HAT and transmembrane protease, serine 2 (TMPRSS2, whose activities are regulated by antiproteases, such as secretory leukocyte protease inhibitor (SLPI. Based on these observations, we sought to determine how acute exposure to ozone may modulate cellular protease/antiprotease expression and function, and to define their roles in a viral infection. We utilized our in vitro model of differentiated human nasal epithelial cells (NECs to determine the effects of ozone on influenza cleavage, entry, and replication. We show that ozone exposure disrupts the protease/antiprotease balance within the airway liquid. We also determined that functional forms of HAT, TMPRSS2, and SLPI are secreted from human airway epithelium, and acute exposure to ozone inversely alters their expression levels. We also show that addition of antioxidants significantly reduces virus replication through the induction of SLPI. In addition, we determined that ozone-induced cleavage of the viral HA protein is not cell-associated and that secreted endogenous proteases are sufficient to activate HA leading to a significant increase in viral replication. Our data indicate that pre-exposure to ozone disrupts the protease/antiprotease balance found in the human airway, leading to increased influenza susceptibility.

Linear IgA dermatosis associated with ulcerative colitis: complete and sustained remission after total colectomy

OpenAIRE

Vargas,Thiago Jeunon de Sousa; Fialho,Mônica; Santos,Luiza Tavares dos; Rodrigues,Palmira Assis de Jesus Barreto; Vargas,Ana Luisa Bittencourt Sampaio Jeunon; Sousa,Maria Auxiliadora Jeunon

2013-01-01

Linear IgA dermatosis has been increasingly associated with inflammatory bowel diseases, particularly ulcerative colitis. A 13-year-old male patient with an 11-month history of ulcerative colitis developed vesicles, pustules and erosions on the skin of the face, trunk and buttocks and in the oral mucosa. The work-up revealed a neutrophil-rich sub-epidermal bullous disease and linear deposition of IgA along the dermoepidermal junction, establishing the diagnosis of linear IgA dermatosis. The p...

Human Kunitz-type protease inhibitor engineered for enhanced matrix retention extends longevity of fibrin biomaterials.

Science.gov (United States)

Briquez, Priscilla S; Lorentz, Kristen M; Larsson, Hans M; Frey, Peter; Hubbell, Jeffrey A

2017-08-01

Aprotinin is a broad-spectrum serine protease inhibitor used in the clinic as an anti-fibrinolytic agent in fibrin-based tissue sealants. However, upon re-exposure, some patients suffer from hypersensitivity immune reactions likely related to the bovine origin of aprotinin. Here, we aimed to develop a human-derived substitute to aprotinin. Based on sequence homology analyses, we identified the Kunitz-type protease inhibitor (KPI) domain of human amyloid-β A4 precursor protein as being a potential candidate. While KPI has a lower intrinsic anti-fibrinolytic activity than aprotinin, we reasoned that its efficacy is additionally limited by its fast release from fibrin material, just as aprotinin's is. Thus, we engineered KPI variants for controlled retention in fibrin biomaterials, using either covalent binding through incorporation of a substrate for the coagulation transglutaminase Factor XIIIa or through engineering of extracellular matrix protein super-affinity domains for sequestration into fibrin. We showed that both engineered KPI variants significantly slowed plasmin-mediated fibrinolysis in vitro, outperforming aprotinin. In vivo, our best engineered KPI variant (incorporating the transglutaminase substrate) extended fibrin matrix longevity by 50%, at a dose at which aprotinin did not show efficacy, thus qualifying it as a competitive substitute of aprotinin in fibrin sealants. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

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

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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.

Evolutionary dynamics of hepatitis C virus NS3 protease domain during and following treatment with narlaprevir, a potent NS3 protease inhibitor

NARCIS (Netherlands)

de Bruijne, J.; Thomas, X. V.; Rebers, S. P.; Weegink, C. J.; Treitel, M. A.; Hughes, E.; Bergmann, J. F.; de Knegt, R. J.; Janssen, H. L. A.; Reesink, H. W.; Molenkamp, R.; Schinkel, J.

2013-01-01

Narlaprevir, a hepatitis C virus (HCV) NS3/4A serine protease inhibitor, has demonstrated robust antiviral activity in a placebo-controlled phase 1 study. To study evolutionary dynamics of resistant variants, the NS3 protease sequence was clonally analysed in thirty-two HCV genotype 1-infected

IgA response in serum and gut secretion in sensitized mice fed with the dust mite Dermatophagoides pteronyssinus extract

Directory of Open Access Journals (Sweden)

Maciel M.

2004-01-01

Full Text Available Induced oral tolerance to mucosal-exposed antigens in immunized animals is of particular interest for the development of immunotherapeutic approaches to human allergic diseases. This is a unique feature of mucosal surfaces which represent the main contact interface with the external environment. However, the influence of oral tolerance on specific and natural polyreactive IgA antibodies, the major defense mechanism of the mucosa, is unknown. We have shown that oral administration of an extract of the dust mite Dermatophagoides pteronyssinus (Dp to primed mice caused down-regulation of IgE responses and an increase in tumor growth factor-ß secretion. In the present study, we observed that primed inbred female A/Sn mice (8 to 10 weeks old fed by gavage a total weight of 1.0-mg Dp extract on the 6th, 7th and 8th days post-immunization presented normal secretion of IL-4 and IL-10 in gut-associated lymphoid tissue and a decreased production of interferon gamma induced by Dp in the draining lymph nodes (13,340 ± 3,519 vs 29,280 ± 2,971 pg/ml. Mice fed the Dp extract also showed higher levels of serum anti-Dp IgA antibodies and an increase of IgA-secreting cells in mesenteric lymph nodes (N = 10, reflecting an increase in total fecal IgA antibodies (N = 10. The levels of secretory anti-Dp IgA antibodies increased after re-immunization regardless of Dp extract feeding. Oral tolerance did not interfere with serum or secretory IgA antibody reactivity related to self and non-self antigens. These results suggest that induction of oral tolerance to a Dp extract in sensitized mice triggered different regulatory mechanisms which inhibited the IgE response and stimulated systemic and secretory IgA responses, preserving the natural polyreactive IgA antibody production.

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

NARCIS (Netherlands)

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

The threonine protease activity of testes-specific protease 50 (TSP50 is essential for its function in cell proliferation.

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Yu-Yin Li

Full Text Available BACKGROUND: Testes-specific protease 50 (TSP50, a newly discovered threonine enzyme, has similar amino acid sequences and enzymatic structures to those of many serine proteases. It may be an oncogene. TSP50 is up-regulated in breast cancer epithelial cells, and ectopic expression of TSP50 in TSP50-deficient Chinese hamster ovary (CHO cells has been found to promote cell proliferation. However, the mechanisms by which TSP50 exerts its growth-promoting effects are not yet fully understood. METHODOLOGY/PRINCIPAL FINDINGS: To delineate whether the threonine protease activity of TSP50 is essential to its function in cell proliferation, we constructed and characterized a mutant TSP50, called TSP50 T310A, which was identified as a protease-dead mutant of TSP50. By a series of proliferation analyses, colony formation assays and apoptosis analyses, we showed that T310A mutation significantly depresses TSP50-induced cell proliferation in vitro. Next, the CHO stable cell line expressing either wild-type or T310A mutant TSP50 was injected subcutaneously into nude mice. We found that the T310A mutation could abolish the tumorigenicity of TSP50 in vivo. A mechanism investigation revealed that the T310A mutation prevented interaction between TSP50 and the NF-κBIκBα complex, which is necessary for TSP50 to perform its function in cell proliferation. CONCLUSION: Our data highlight the importance of threonine 310, the most critical protease catalytic site in TSP50, to TSP50-induced cell proliferation and tumor formation.

Modulation of HIV-1 Gag NC/p1 cleavage efficiency affects protease inhibitor resistance and viral replicative capacity

Czech Academy of Sciences Publication Activity Database

Maarseveen van, N. M.; Andersson, Dan; Lepšík, Martin; Fun, A.; Schipper, P. J.; Jong de, D.; Boucher, Ch. A. B.; Nijhuis, M.

2012-01-01

Roč. 9, č. 29 (2012), s. 1-7 ISSN 1742-4690 EU Projects: European Commission(XE) 37693 - HIV PI RESISTANCE Grant - others:Dutch AIDS Fund(XE) 2006028; (NWO) VIDI(XE) 91796349 Institutional research plan: CEZ:AV0Z40550506 Keywords : HIV-1 * protease * Gag * resistance * cleavage Subject RIV: CE - Biochemistry Impact factor: 5.657, year: 2012

Evaluation of natural regulatory T cells in subjects with selective IgA deficiency: from senior idea to novel opportunities.

Science.gov (United States)

Soheili, Habib; Abolhassani, Hassan; Arandi, Narges; Khazaei, Hossein Ali; Shahinpour, Shervin; Hirbod-Mobarakeh, Armin; Rezaei, Nima; Aghamohammadi, Asghar

2013-01-01

Selective IgA deficiency (SIgAD) is the most common primary immunodeficiency disorder, which is characterized by significantly decreased serum levels of IgA. Abnormalities of CD4+CD25(high)forkhead box P3 (FoxP3)+ regulatory T cells (T(reg)) have been shown in association with autoimmune and inflammatory disorders. In order to evaluate the relationship between autoimmunity and T(reg) in SIgAD, we studied 26 IgA-deficient patients (aged 4-17 years) with serum IgA levels 2.36%. Autoimmunity was recorded in 9 patients (53.3%) of G1 and only 1 patient of G2, respectively (p = 0.034). Although a defect in class switching recombination was observed in 40% of the patients in G1, none of the G2 patients had such a defect (p = 0.028). This study showed decreased proportions of T(reg) in SIgAD patients, particularly in those with signs of chronic inflammation. Copyright © 2012 S. Karger AG, Basel.

The protease inhibitor HAI-2, but not HAI-1, regulates matriptase activation and shedding through prostasin

DEFF Research Database (Denmark)

Friis, Stine; Sales, Katiuchia Uzzun; Schafer, Jeffrey Martin

2014-01-01

cells. Whereas ablation of HAI-1 did not affect matriptase in epithelial cells of the small or large intestine, ablation of HAI-2 resulted in the loss of matriptase from both tissues. Gene silencing studies in intestinal Caco-2 cell monolayers revealed that this loss of cell-associated matriptase......The membrane-anchored serine proteases, matriptase and prostasin, and the membrane-anchored serine protease inhibitors, hepatocyte growth factor activator inhibitor (HAI)-1 and HAI-2, are critical effectors of epithelial development and postnatal epithelial homeostasis. Matriptase and prostasin...... form a reciprocal zymogen activation complex that results in the formation of active matriptase and prostasin that are targets for inhibition by HAI-1 and HAI-2. Conflicting data, however, have accumulated as to the existence of auxiliary functions for both HAI-1 and HAI-2 in regulating...

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

Science.gov (United States)

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.

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.

Science.gov (United States)

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.

Insecticide resistance and intracellular proteases.

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Wilkins, Richard M

2017-12-01

Pesticide resistance is an example of evolution in action with mechanisms of resistance arising from mutations or increased expression of intrinsic genes. Intracellular proteases have a key role in maintaining healthy cells and in responding to stressors such as pesticides. Insecticide-resistant insects have constitutively elevated intracellular protease activity compared to corresponding susceptible strains. This increase was shown for some cases originally through biochemical enzyme studies and subsequently putatively by transcriptomics and proteomics methods. Upregulation and expression of proteases have been characterised in resistant strains of some insect species, including mosquitoes. This increase in proteolysis results in more degradation products (amino acids) of intracellular proteins. These may be utilised in the resistant strain to better protect the cell from stress. There are changes in insect intracellular proteases shortly after insecticide exposure, suggesting a role in stress response. The use of protease and proteasome inhibitors or peptide mimetics as synergists with improved application techniques and through protease gene knockdown using RNA interference (possibly expressed in crop plants) may be potential pest management strategies, in situations where elevated intracellular proteases are relevant. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Role of maternal elimination diets and human milk IgA in the development of cow's milk allergy in the infants.

Science.gov (United States)

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.

Prevalence of antigliadin IgA antibodies in psoriasis vulgaris and response of seropositive patients to a gluten-free diet

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Kolchak NA

2017-12-01

Full Text Available Nikolai A Kolchak,1 Maria K Tetarnikova,2 Maria S Theodoropoulou,3 Alexandra P Michalopoulou,4 Demetrios S Theodoropoulos5 1Department of Hematology, Omsk State Medical Academy, Omsk, Russia; 2Dermatology Private Practice, Chelyabinsk, Russia; 3Department of Pharmacy, Trikala General Hospital, Trikala, Greece; 4Department of Philosophy and Social Studies, School of Philosophy, University of Crete, Rethymnon, Greece; 5Allergy Associates of La Crosse, Onalaska, WI, USA Introduction: The course of psoriasis relies on a variety of metabolic and immunological parameters. Identification of underlying pro-inflammatory conditions and their control is desired for optimal management. Background: Increased prevalence of serum markers for celiac disease has been reported among patients with psoriasis. The likelihood of occult celiac disease in a subpopulation of patients has been postulated and gluten-free diets have been reported to be effective. Patients and methods: The prevalence of gliadin IgA antibodies was assessed among patients with psoriasis in an urban population. The clinical effects of a strict gluten-free diet were followed. Results: Over a 2-year period, 97 patients with Psoriasis Area and Severity Index greater than 2.4 were recruited from a population followed in a dermatology clinic. Gliadin IgA antibodies were assessed in all participants and in 91 controls. Elevated gliadin IgA antibodies were found in 13 patients (14% and two controls (2%. Values in five patients were assessed as greater than 30.0 U/mL or “strong positive” according to the manufacturer of the assay. All 13 patients were placed on a strict gluten-free diet without any other modifications in their ongoing treatment of psoriasis. Improvement of psoriatic lesions was observed in all patients with positive gliadin IgA antibodies but the decline in the Psoriasis Area and Severity Index score and the scaling down of pharmaceutical treatment was more pronounced in the five

The HIV-1 protease resistance mutation I50L is associated with resistance to atazanavir and susceptibility to other protease inhibitors in multiple mutational contexts.

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Sista, P; Wasikowski, B; Lecocq, P; Pattery, T; Bacheler, L

2008-08-01

The HIV-1 protease mutation I50 L causes atazanavir resistance but increases susceptibility to other PIs. Predicted phenotypic FC values were obtained from viral genotypes, using the virtual Phenotype-LM bioinformatics tool (powering vircoTYPE). To evaluate I50 L's effect on susceptibility to 8 PIs, in a large genotype database. I50 L containing routine clinical isolate samples in Virco's genotype database were paired with samples having like patterns (or profiles) of IAS-USA-defined primary PI mutations, but lacking I50 L. Using vircoTYPE (version 4.1), the median predicted FC for each mutational profile was determined. I50 L-associated shifts in FC were evaluated using drug-specific CCOs. We selected 307 and 37098 samples with and without I50 L. These corresponded to 31 mutation patterns of > or =3 samples each. I50 L caused resistance to atazanavir in all 31 mutation contexts, but was associated with higher susceptibility for other PIs. The largest I50 L-associated shifts in median predicted FC were: 1.2 to 42.4 (atazanavir), 10.2 to 3.2 (amprenavir), 3.3 to 0.5 (darunavir), 13 to 0.5 (indinavir), 34.9 to 1.3 (lopinavir), 22.3 to 1.3 (nelfinavir), 5.2 to 0.3 (saquinavir) and 29.9 to 5.2 (tipranavir). The PI mutation I50 L causes clinically relevant resistance and increased susceptibility to atazanavir and other PIs respectively.

Detection of circulating immune complexes of human IgA and beta 2 glycoprotein I in patients with antiphospholipid syndrome symptomatology.

Science.gov (United States)

Martínez-Flores, José A; Serrano, Manuel; Pérez, Dolores; Lora, David; Paz-Artal, Estela; Morales, José M; Serrano, Antonio

2015-07-01

Patients with antiphospholipid syndrome (APS) have a hypercoagulable condition associated with the presence of antiphospholipid autoantibodies (aPL). Consensus antibodies for diagnosis are lupus anticoagulant, anti-beta2 glycoprotein I (B2GPI) and anticardiolipin (IgG or IgM). Circulating immunocomplexes (CIC) of B2GPI associated with IgM or IgG were reported. Isolated IgA aB2GPI antibodies have achieved high diagnostic value although specific CIC of B2GPI bounded to IgA (B2A-CIC) has still not been described. CIC detection assays are mainly based on interaction with complement and are not appropriate to detect B2A-CIC because IgA does not fix complement using the classical pathway. Sera from healthy blood donors (N= 247) and from patients with thrombosis background and isolate positive for IgA aB2GPI (N = 68) were studied in a case-control study. Two methods were applied, these being a capture ELISA to quantify specific B2A-CIC and quantification of total IgA anti-B2GPI after dissociating CIC. B2A-CIC values in APS-patients were 19.27 ± 2.6 AU vs 6.1 ± 0.4 AU in blood donors (p < 0.001). There were 36.4% B2A-CIC positive patients (cutoff 21 AU) versus 5.5% in blood donors (p < 0.001). Dissociated IgA aB2GPI levels (total IgA aB2GPI) were 146.8 ± 10.8 IU/mL in patients vs. 22.4 IU/mL in controls (p < 0.001). B2A-CIC was independent of B2GPI and autoantibodies IgA aB2GPI serum levels. B2A-CIC can be identified and quantified in an easy and reproducible manner using two complement-independent methods. The use of these tests in prospective studies will allow better understanding of the prognosis and outcome of patients. Copyright © 2015 Elsevier B.V. All rights reserved.

Structural basis for the ATP-independent proteolytic activity of LonB proteases and reclassification of their AAA+ modules.

Science.gov (United States)

An, Young Jun; Na, Jung-Hyun; Kim, Myung-Il; Cha, Sun-Shin

2015-10-01

Lon proteases degrade defective or denature proteins as well as some folded proteins for the control of cellular protein quality. There are two types of Lon proteases, LonA and LonB. Each consists of two functional components: a protease component and an ATPase associated with various cellular activities (AAA+ module). Here, we report the 2.03 -resolution crystal structure of the isolated AAA+ module (iAAA+ module) of LonB from Thermococcus onnurineus NA1 (TonLonB). The iAAA+ module, having no bound nucleotide, adopts a conformation virtually identical to the ADP-bound conformation of AAA+ modules in the hexameric structure of TonLonB; this provides insights into the ATP-independent proteolytic activity observed in a LonB protease. Structural comparison of AAA+ modules between LonA and LonB revealed that the AAA+ modules of Lon proteases are separated into two distinct clades depending on their structural features. The AAA+ module of LonB belongs to the -H2 & Ins1 insert clade (HINS clade)- defined for the first time in this study, while the AAA+ module of LonA is a member of the HCLR clade.

Changes in protein metabolism after irradiation. Pt. 1. Protease activity, protease pattern, protein and free amino acids in cytoplasm and cell organelles of the rat spleen after 600 R whole body x irradiation

Energy Technology Data Exchange (ETDEWEB)

Valet, G [Max-Planck-Institut fuer Biochemie, Muenchen (F.R. Germany). Abt. fuer Experimentelle Medizin

1975-12-01

The protease activity of cytoplasm and cell organelles of the rat spleen against spleen protein and hemoglobin as a substrate increases during a initial reaction phase of the organism on the first day after 600 R whole body X-irradiation. The alkaline protease in the cytoplasm and the acid protease in the cell organelles increase, whereas the protease activity against externally added hemoglobin as substrate decreases below the initial values. The protein, the protease activity and the free amino acids of the cytoplasm and the cell organelles decrease during the disease phase on day 3 and 4 after irradiation. The protein loss of the spleen is therefore not explained by an increased protease activity. Acid proteases appear in the cytoplasm which derive probably from the cell organelles. The protease activity and the free amino acids are increased in the cytoplasm and the cell organelles during the regeneration phase of the organism between day 15 and 18 after irradiation.

Thioredoxin is involved in endothelial cell extracellular transglutaminase 2 activation mediated by celiac disease patient IgA.

Directory of Open Access Journals (Sweden)

Cristina Antonella Nadalutti

Full Text Available PURPOSE: To investigate the role of thioredoxin (TRX, a novel regulator of extracellular transglutaminase 2 (TG2, in celiac patients IgA (CD IgA mediated TG2 enzymatic activation. METHODS: TG2 enzymatic activity was evaluated in endothelial cells (HUVECs under different experimental conditions by ELISA and Western blotting. Extracellular TG2 expression was studied by ELISA and immunofluorescence. TRX was analysed by Western blotting and ELISA. Serum immunoglobulins class A from healthy subjects (H IgA were used as controls. Extracellular TG2 enzymatic activity was inhibited by R281. PX12, a TRX inhibitor, was also employed in the present study. RESULTS: We have found that in HUVECs CD IgA is able to induce the activation of extracellular TG2 in a dose-dependent manner. Particularly, we noted that the extracellular modulation of TG2 activity mediated by CD IgA occurred only under reducing conditions, also needed to maintain antibody binding. Furthermore, CD IgA-treated HUVECs were characterized by a slightly augmented TG2 surface expression which was independent from extracellular TG2 activation. We also observed that HUVECs cultured in the presence of CD IgA evinced decreased TRX surface expression, coupled with increased secretion of the protein into the culture medium. Intriguingly, inhibition of TRX after CD IgA treatment was able to overcome most of the CD IgA-mediated effects including the TG2 extracellular transamidase activity. CONCLUSIONS: Altogether our findings suggest that in endothelial cells CD IgA mediate the constitutive activation of extracellular TG2 by a mechanism involving the redox sensor protein TRX.

PetIGA-MF: a multi-field high-performance toolbox for structure-preserving B-splines spaces

KAUST Repository

Sarmiento, Adel

2016-10-01

We describe a high-performance solution framework for isogeometric discrete differential forms based on B-splines: PetIGA-MF. Built on top of PetIGA, an open-source library we have built and developed over the last decade, PetIGA-MF is a general multi-field discretization tool. To test the capabilities of our implementation, we solve different viscous flow problems such as Darcy, Stokes, Brinkman, and Navier-Stokes equations. Several convergence benchmarks based on manufactured solutions are presented assuring optimal convergence rates of the approximations, showing the accuracy and robustness of our solver.

2-D zymographic analysis of Broccoli (Brassica oleracea L. var. Italica) florets proteases: follow up of cysteine protease isotypes in the course of post-harvest senescence.

Science.gov (United States)

Rossano, Rocco; Larocca, Marilena; Riccio, Paolo

2011-09-01

Zymographic analysis of Broccoli florets (Brassica oleracea L. var. Italica) revealed the presence of acidic metallo-proteases, serine proteases and cysteine proteases. Under conditions which were denaturing for the other proteases, the study was restricted to cysteine proteases. 2-D zymography, a technique that combines IEF and zymography was used to show the presence of 11 different cysteine protease spots with molecular mass of 44 and 47-48kDa and pIs ranging between 4.1 and 4.7. pI differences could be ascribed to different degrees of phosphorylation that partly disappeared in the presence of alkaline phosphatase. Post-harvest senescence of Broccoli florets was characterized by decrease in protein and chlorophyll contents and increase of protease activity. In particular, as determined by 2-D zymography, the presence of cysteine protease clearly increased during senescence, a finding that may represent a useful tool for the control of the aging process. Copyright © 2011 Elsevier GmbH. All rights reserved.

Clinical efficacy of anti-glycopeptidolipid-core IgA test for diagnosing Mycobacterium avium complex infection in lung.

Science.gov (United States)

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.

IgA anti-Streptococcus mutans em crianças com e sem cárie dentária Anti-Streptococcus mutans IgA in children with and without dental caries

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Suzete Cristina YAZAKI

1999-07-01

Full Text Available A cárie dentária é uma doença infecciosa crônica que necessita pelo menos quatro componentes para desenvolver-se: hospedeiro suscetível, microbiota patogênica, dieta rica em sacarose e tempo. Este trabalho estuda as correlações existentes entre estreptococos salivares do grupo mutans, placa bacteriana e anticorpos IgA anti-Streptococcus mutans em crianças com e sem experiência de cárie. Para tanto, utilizou-se o meio Mitis Salivarius (DIFCO para determinar o número de Unidades Formadoras de Colônias (UFC/ml, o Índice de Higiene Oral Simplificado (IHOS para mensurar a quantidade de placa bacteriana e a técnica ELISA para detectar anticorpos anti-S. mutans. Os resultados obtidos mostraram que não existe uma correlação entre os níveis salivares de estreptococos (UFC/ml e IgA anti-S. mutans na população estudada. No grupo com cárie, uma correlação positiva, estatisticamente significante, foi observada entre o Índice de placa e IgA específica.Dental caries is a chronic infectious disease that needs at least four components to develop. As a susceptible host, a pathogenic microbiota, a high-sucrose diet and time. This work assess the relationships between Streptococcus mutans and dental plaque; Streptococcus mutans and IgA antibodies in children with and without caries experience. In order to achieve this goal we have used Mitis Salivarius bacitracin agar (DIFCO to determine the colony forming units (CFU/mL, the simplified oral hygiene index (SOHI for measuring bacterial plaque and ELISA for antibody detection. The results obtained have not shown any correlations between colony forming units of S. mutans and IgA antibodies. A significant correlation was found between bacterial plaque index and specific IgA in children with carious lesions.

Distorted secretory granule composition in mast cells with multiple protease deficiency.

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Grujic, Mirjana; Calounova, Gabriela; Eriksson, Inger; Feyerabend, Thorsten; Rodewald, Hans-Reimer; Tchougounova, Elena; Kjellén, Lena; Pejler, Gunnar

2013-10-01

Mast cells are characterized by an abundance of secretory granules densely packed with inflammatory mediators such as bioactive amines, cytokines, serglycin proteoglycans with negatively charged glycosaminoglycan side chains of either heparin or chondroitin sulfate type, and large amounts of positively charged proteases. Despite the large biological impact of mast cell granules and their contents on various pathologies, the mechanisms that regulate granule composition are incompletely understood. In this study, we hypothesized that granule composition is dependent on a dynamic electrostatic interrelationship between different granule compounds. As a tool to evaluate this possibility, we generated mice in which mast cells are multideficient in a panel of positively charged proteases: the chymase mouse mast cell protease-4, the tryptase mouse mast cell protease-6, and carboxypeptidase A3. Through a posttranslational effect, mast cells from these mice additionally lack mouse mast cell protease-5 protein. Mast cells from mice deficient in individual proteases showed normal morphology. In contrast, mast cells with combined protease deficiency displayed a profound distortion of granule integrity, as seen both by conventional morphological criteria and by transmission electron microscopy. An assessment of granule content revealed that the distorted granule integrity in multiprotease-deficient mast cells was associated with a profound reduction of highly negatively charged heparin, whereas no reduction in chondroitin sulfate storage was observed. Taken together with previous findings showing that the storage of basic proteases conversely is regulated by anionic proteoglycans, these data suggest that secretory granule composition in mast cells is dependent on a dynamic interrelationship between granule compounds of opposite electrical charge.

Ion specific effects of alkali cations on the catalytic activity of HIV-1 protease

Czech Academy of Sciences Publication Activity Database

Pokorná, Jana; Heyda, J.; Konvalinka, Jan

2013-01-01

Roč. 160, č. 1 (2013), s. 359-370 ISSN 1359-6640 R&D Projects: GA ČR GBP208/12/G016; GA ČR GAP207/11/1798 Institutional support: RVO:61388963 Keywords : HIV -1 protease * ion-protein interaction * Hofmeister series * enzyme kinetics * molecular dynamics Subject RIV: CE - Biochemistry Impact factor: 4.194, year: 2013

Hydroxyethylamine derivatives as HIV-1 protease inhibitors: a predictive QSAR modelling study based on Monte Carlo optimization.

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Bhargava, S; Adhikari, N; Amin, S A; Das, K; Gayen, S; Jha, T

2017-12-01

Application of HIV-1 protease inhibitors (as an anti-HIV regimen) may serve as an attractive strategy for anti-HIV drug development. Several investigations suggest that there is a crucial need to develop a novel protease inhibitor with higher potency and reduced toxicity. Monte Carlo optimized QSAR study was performed on 200 hydroxyethylamine derivatives with antiprotease activity. Twenty-one QSAR models with good statistical qualities were developed from three different splits with various combinations of SMILES and GRAPH based descriptors. The best models from different splits were selected on the basis of statistically validated characteristics of the test set and have the following statistical parameters: r 2 = 0.806, Q 2 = 0.788 (split 1); r 2 = 0.842, Q 2 = 0.826 (split 2); r 2 = 0.774, Q 2 = 0.755 (split 3). The structural attributes obtained from the best models were analysed to understand the structural requirements of the selected series for HIV-1 protease inhibitory activity. On the basis of obtained structural attributes, 11 new compounds were designed, out of which five compounds were found to have better activity than the best active compound in the series.

Molecular characterization of protease activity in Serratia sp. strain SCBI and its importance in cytotoxicity and virulence.

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Petersen, Lauren M; Tisa, Louis S

2014-11-01

A newly recognized Serratia species, termed South African Caenorhabditis briggsae isolate (SCBI), is both a mutualist of the nematode Caenorhabditis briggsae KT0001 and a pathogen of lepidopteran insects. Serratia sp. strain SCBI displays high proteolytic activity, and because secreted proteases are known virulence factors for many pathogens, the purpose of this study was to identify genes essential for extracellular protease activity in Serratia sp. strain SCBI and to determine what role proteases play in insect pathogenesis and cytotoxicity. A bank of 2,100 transposon mutants was generated, and six SCBI mutants with defective proteolytic activity were identified. These mutants were also defective in cytotoxicity. The mutants were found defective in genes encoding the following proteins: alkaline metalloprotease secretion protein AprE, a BglB family transcriptional antiterminator, an inosine/xanthosine triphosphatase, GidA, a methyl-accepting chemotaxis protein, and a PIN domain protein. Gene expression analysis on these six mutants showed significant downregulation in mRNA levels of several different types of predicted protease genes. In addition, transcriptome sequencing (RNA-seq) analysis provided insight into how inactivation of AprE, GidA, and a PIN domain protein influences motility and virulence, as well as protease activity. Using quantitative reverse transcription-PCR (qRT-PCR) to further characterize expression of predicted protease genes in wild-type Serratia sp. SCBI, the highest mRNA levels for the alkaline metalloprotease genes (termed prtA1 to prtA4) occurred following the death of an insect host, while two serine protease and two metalloprotease genes had their highest mRNA levels during active infection. Overall, these results indicate that proteolytic activity is essential for cytotoxicity in Serratia sp. SCBI and that its regulation appears to be highly complex. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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

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Amano, Masayuki; Salcedo-Gómez, Pedro Miguel; Zhao, Rui; Yedidi, Ravikiran S.; Das, Debananda; Bulut, Haydar; Delino, Nicole S.; Sheri, Venkata Reddy; Ghosh, Arun K.; Mitsuya, Hiroaki (Kumamoto); (NIH); (Purdue)

2016-09-12

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 (EC₅₀: 0.00035 - 0.0018 μM) with minimal cytotoxicity (CC₅₀: 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 (EC₅₀: 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. The 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.

Production of a novel wheat gluten hydrolysate containing dipeptidyl peptidase-IV inhibitory tripeptides using ginger protease.

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Taga, Yuki; Hayashida, Osamu; Kusubata, Masashi; Ogawa-Goto, Kiyoko; Hattori, Shunji

2017-09-01

Wheat gluten is a Pro-rich protein complex comprising glutenins and gliadins. Previous studies have reported that oral intake of enzymatic hydrolysates of gluten has beneficial effects, such as suppression of muscle injury and improvement of hepatitis. Here, we utilized ginger protease that preferentially cleaves peptide bonds with Pro at the P 2 position to produce a novel type of wheat gluten hydrolysate. Ginger protease efficiently hydrolyzed gluten, particularly under weak acidic conditions, to peptides with an average molecular weight of ginger protease can be used as a functional food for patients with type 2 diabetes.

Autoimmune responses in patients with linear IgA bullous dermatosis: both autoantibodies and T lymphocytes recognize the NC16A domain of the BP180 molecule.

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Lin, Mong-Shang; Fu, Chang-Ling; Olague-Marchan, Monica; Hacker, Mary K; Zillikens, Detlef; Giudice, George J; Fairley, Janet A

2002-03-01

Linear IgA bullous disease (LABD) is an autoimmune skin disease characterized by subepidermal blisters and IgA autoantibodies directed against the epidermal basement membrane zone (BMZ) of the skin. Various antigens have been identified as targets of IgA autoantibodies including BP180, a type II glycoprotein that spans the BMZ and lamina lucida. Previously, we have identified a subset of LABD patients whose sera contained IgA antibodies against the 16th noncollagenous (NC16A) domain of BP180. NC16A was previously shown to harbor epitopes that are recognized by both autoantibodies and T cells from patients with bullous pemphigoid and herpes gestationis and is thought to be associated with the development of these immunobullous diseases. The aim of this study was to determine whether T lymphocytes from LABD patients with anti-NC16A IgA autoantibodies respond to epitopes in the same region of the BP180 protein. Indeed, of the four LABD patients in our study, all had T cells that specifically proliferated in response to NC16A. Moreover, two subfragments of NC16A were identified as the predominant targets of LABD T cells. Further analysis of T cell lines and clones derived from these patients revealed that these cells express a CD4 memory T cell phenotype and secrete a Th1/Th2 mixed-cytokine profile, characteristics similar to those of T cells in bullous pemphigoid patients. Our data suggest that the BP180 protein, typically the NC16A region, is the common target of both cellular and humoral immune responses in some LABD patients. This information helps to further elucidate the autoimmune mechanisms in this disease.

Diagnosis and follow-up of genital chlamydial infection by direct methods and by detection of serum IgG, IgA and secretory IgA

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Fresse A

2010-01-01

Full Text Available Purpose: To determine the prevalence of Chlamydia trachomatis infection in a high-risk population by direct and indirect methods and to evaluate the diagnosis of secretory immunoglobulin A (sIgA. Patients and Methods: Urethral or endocervical specimens from 78 patients (48 females and 30 males were examined by cell culture, direct fluorescence assay, PCR Cobas Amplicor (Roche Molecular Diagnostics, and sIgA was detected by the recombinant lipopolysaccharide (LPS-enzyme-linked immunoassay (rELISA. Serum from each patient was also obtained and analysed for the presence of IgG and IgA antibody by in-house microimmunofluorescence (MIF and by the rELISA method (Medac, Hamburg, Germany. Results: The overall C. trachomatis prevalence determined by direct methods was 28%. The detection of sIgA antibodies was significantly higher in the group of patients with a positive direct detection (50% than in the group of negative direct detection (10.7%. The Chlamydia-specific IgA antibodies were detected by the rELISA in 40.9 and 53.6% of group I (positive direct detection and group II patients (negative direct detection, respectively. The species-specific IgA antibodies were detected by the MIF method in 18.2 and 16.1% of group I and II patients, respectively. Chlamydia genus-specific IgG antibodies were detected by the rELISA in 86.4 and 83.9% of group I and group II patients and, C. trachomatis specific IgG were present in 81.8 and 73.2% of group I and group II patients, respectively, as assessed by the MIF test. Conclusion: Combining the positive direct methods and/or positive sIgA antibody results from cervical or urethral specimens had an indication of current C. trachomatis infection.

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

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Dostál, Ji& #345; í; Brynda, Ji& #345; í; Hrušková-Heidingsfeldová, Olga; Sieglová, Irena; Pichová, Iva; & #344; ezá& #269; ová, Pavlína; (ASCR-ICP)

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 isolated 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.

Linear IgA dermatosis associated with ulcerative colitis: complete and sustained remission after total colectomy.

Science.gov (United States)

Vargas, Thiago Jeunon de Sousa; Fialho, Mônica; Santos, Luiza Tavares dos; Rodrigues, Palmira Assis de Jesus Barreto; Vargas, Ana Luisa Bittencourt Sampaio Jeunon; Sousa, Maria Auxiliadora Jeunon

2013-01-01

Linear IgA dermatosis has been increasingly associated with inflammatory bowel diseases, particularly ulcerative colitis. A 13-year-old male patient with an 11-month history of ulcerative colitis developed vesicles, pustules and erosions on the skin of the face, trunk and buttocks and in the oral mucosa. The work-up revealed a neutrophil-rich sub-epidermal bullous disease and linear deposition of IgA along the dermoepidermal junction, establishing the diagnosis of linear IgA dermatosis. The patient experienced unsatisfactory partial control of skin and intestinal symptoms despite the use of adalimumab, mesalazine, prednisone and dapsone for some months. After total colectomy, he presented complete remission of skin lesions, with no need of medications during two years of follow-up. A review of previously reported cases of the association is provided here and the role of ulcerative colitis in triggering linear IgA dermatosis is discussed.

Proteolytic crosstalk in multi-protease networks

Science.gov (United States)

Ogle, Curtis T.; Mather, William H.

2016-04-01

Processive proteases, such as ClpXP in E. coli, are conserved enzyme assemblies that can recognize and rapidly degrade proteins. These proteases are used for a number of purposes, including degrading mistranslated proteins and controlling cellular stress response. However, proteolytic machinery within the cell is limited in capacity and can lead to a bottleneck in protein degradation, whereby many proteins compete (‘queue’) for proteolytic resources. Previous work has demonstrated that such queueing can lead to pronounced statistical relationships between different protein counts when proteins compete for a single common protease. However, real cells contain many different proteases, e.g. ClpXP, ClpAP, and Lon in E. coli, and it is not clear how competition between proteins for multiple classes of protease would influence the dynamics of cellular networks. In the present work, we theoretically demonstrate that a multi-protease proteolytic bottleneck can substantially couple the dynamics for both simple and complex (oscillatory) networks, even between substrates with substantially different affinities for protease. For these networks, queueing often leads to strong positive correlations between protein counts, and these correlations are strongest near the queueing theoretic point of balance. Furthermore, we find that the qualitative behavior of these networks depends on the relative size of the absolute affinity of substrate to protease compared to the cross affinity of substrate to protease, leading in certain regimes to priority queue statistics.

Proteases and caspase-like activity in the yeast Saccharomyces cerevisiae.

Science.gov (United States)

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.

Proteases from Latex of Euphorbia spp. and Its Application on Milk Clot Formation

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Fidia Fibriana

2015-09-01

Full Text Available Crude proteases were extracted from Euphorbiaceae family, i.e. E. milii var imperata, E. trigona, and E. maculata. Among those three crude proteases, the activity of protease from E. trigona was the highest (812.50 U/ml, whereas E. milii and E. maculata crude proteases activity were 298.60 U/ml and 95.80 U/ml, respectively. E. maculata protein concentration was the highest among those three crude enzymes (1.206 mg/ml. The optimum pH and temperature of the enzymes were pH 7.0, pH 6.0, pH 6.5 and 60 °C, 50 °C, and 50 °C, respectively. Crude protease from E. milii var imperata, E. trigona, and E. maculata retained proteolytic activity over a wide range of pH (5.0–9.0 and temperature (up to 65 °C with casein as substrate. All crude proteases showed milk clotting activity ranged from 0.58 U/ml to 1.01 U/ml. Thus, these crude proteases are potential to be applied in dairy industries. However, further study on enzyme purification and characterization are necessary to obtain high purity of proteases before its application.Protease kasar berhasil diekstrak dari tanaman family Euphorbiaceae, yaitu E. milii var imperata, E. trigona, dan E. maculata. Diantara ketiga protease tersebut, aktivitas protease tertinggi diperoleh dari E. trigona (812,50 U/ml, sedangkan aktivitas protease dari E. milii dan E. maculata adalah 298,60 U/ml dan 95,80 U/ml, berturut-turut. Konsentrasi total protein tertinggi terdapat pada protease kasar E. maculata (1,206 mg/ml. pH dan suhu optimum ketiga enzim tersebut adalah pH 7.0, pH 6.0, pH 6.5 dan suhu 60 °C, 50 °C, and 50 °C, berturut-turut. Protease kasar dari E. milii var imperata, E. trigona, dan E. maculata menunjukkan aktivitas proteolitik pada rentang pH 5.0–9.0 dan rentang suhu sampai 65 °C menggunakan kasein sebagai substrat. Semua protease kasar menunjukkan aktivitas penggumpalan susu dengan rentang dari 0,58 U/ml sampai 1,01 U/ml. Berdasarkan hasil yang diperoleh, protease kasar dari ketiga jenis tanaman ini

Hereditary chronic pancreatitis in a patient with type 1 diabetes mellitus

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Varalakshmi Muthukrishnan

2018-01-01

Full Text Available In this case report, we present a young patient with type 1 diabetes who had hereditary chronic pancreatitis. We evaluated him for the cause of pancreatitis, but it was inconclusive and finally the genetic testing was done for him, which revealed heterozygous missense mutation in exon 3 of the PRSS1 gene (protease serine 1 gene on chromosome 7. Hence, we were able to make the diagnosis of hereditary chronic pancreatitis. Chronic pancreatitis secondary to any cause can lead to permanent diabetes, which is typically difficult to control. However, in this case, the episodes of recurrent pancreatitis were present after the onset of type 1 diabetes as compared to the usual presentation of diabetes after the advancement of chronic pancreatitis.

The p2 domain of human immunodeficiency virus type 1 Gag regulates sequential proteolytic processing and is required to produce fully infectious virions.

OpenAIRE

Pettit, S C; Moody, M D; Wehbie, R S; Kaplan, A H; Nantermet, P V; Klein, C A; Swanstrom, R

1994-01-01

The proteolytic processing sites of the human immunodeficiency virus type 1 (HIV-1) Gag precursor are cleaved in a sequential manner by the viral protease. We investigated the factors that regulate sequential processing. When full-length Gag protein was digested with recombinant HIV-1 protease in vitro, four of the five major processing sites in Gag were cleaved at rates that differ by as much as 400-fold. Three of these four processing sites were cleaved independently of the others. The CA/p...

Protease-Mediated Suppression of DRG Neuron Excitability by Commensal Bacteria.

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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

Effectiveness of sal deoiled seed cake as an inducer for protease production from Aeromonas sp. S1 for its application in kitchen wastewater treatment.

Science.gov (United States)

Saini, Vandana; Bhattacharya, Amrik; Gupta, Anshu

2013-08-01

The present study is an attempt to demonstrate the feasibility of sal (Shorea robusta) deoiled cake--a forest-based industrial by-product--as a cheaper media supplement for augmented protease production from Aeromonas sp. S1 and application of protease in the treatment of kitchen wastewater. Under optimized conditions, protease production could successfully be enhanced to 5.13-fold (527.5 U mL(-1)) on using sal deoiled seed cake extract (SDOCE), as medium additive, compared to an initial production of 102.7 U mL(-1) in its absence. The culture parameters for optimum production of protease were determined to be incubation time (48 h), pH (7.0), SDOCE concentration (3 % (v/v)), inoculum size (0.3-0.6 % (v/v)), and agitation rate (100 rpm). The enzyme was found to have an optimum pH and temperature of 8.0 and 60 °C, respectively. The protease preparation was tested for treatment of organic-laden kitchen wastewater. After 96 h of wastewater treatment under static condition, enzyme preparation was able to reduce 74 % biological oxygen demand, 37 % total suspended solids, and 41 % oil and grease. The higher and improved level of protease obtained using sal deoiled seed cake-based media hence offers a new approach for value addition to this underutilized biomass through industrial enzyme production. The protease produced using this biomass could also be used as pretreatment tool for remediation of organic-rich food wastewater.

Genome-wide identification and structure-function studies of proteases and protease inhibitors in Cicer arietinum (chickpea).

Science.gov (United States)

Sharma, Ranu; Suresh, C G

2015-01-01

Proteases are a family of enzymes present in almost all living organisms. In plants they are involved in many biological processes requiring stress response in situations such as water deficiency, pathogen attack, maintaining protein content of the cell, programmed cell death, senescence, reproduction and many more. Similarly, protease inhibitors (PIs) are involved in various important functions like suppression of invasion by pathogenic nematodes, inhibition of spores-germination and mycelium growth of Alternaria alternata and response to wounding and fungal attack. As much as we know, no genome-wide study of proteases together with proteinaceous PIs is reported in any of the sequenced genomes till now. Phylogenetic studies and domain analysis of proteases were carried out to understand the molecular evolution as well as gene and protein features. Structural analysis was carried out to explore the binding mode and affinity of PIs for cognate proteases and prolyl oligopeptidase protease with inhibitor ligand. In the study reported here, a significant number of proteases and PIs were identified in chickpea genome. The gene expression profiles of proteases and PIs in five different plant tissues revealed a differential expression pattern in more than one plant tissue. Molecular dynamics studies revealed the formation of stable complex owing to increased number of protein-ligand and inter and intramolecular protein-protein hydrogen bonds. The genome-wide identification, characterization, evolutionary understanding, gene expression, and structural analysis of proteases and PIs provide a framework for future analysis when defining their roles in stress response and developing a more stress tolerant variety of chickpea. Copyright © 2014 Elsevier Ltd. All rights reserved.