scholarly journals Sedolisins, a New Class of Secreted Proteases from Aspergillus fumigatus with Endoprotease or Tripeptidyl-Peptidase Activity at Acidic pHs

2006 ◽  
Vol 72 (3) ◽  
pp. 1739-1748 ◽  
Author(s):  
Utz Reichard ◽  
Barbara Léchenne ◽  
Abdul R. Asif ◽  
Frank Streit ◽  
Eric Grouzmann ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Signal Sequence ◽  
Gene Families ◽  
Peptidase Activity ◽  
New Class ◽  
Tripeptidyl Peptidase ◽  
Genes Encoding ◽  
Member Gene ◽  
Culture Supernatants ◽  
Hydrolysis Of

ABSTRACT The secreted proteolytic activity of Aspergillus fumigatus is of potential importance as a virulence factor and in the industrial hydrolysis of protein sources. The A. fumigatus genome contains sequences that could encode a five-member gene family that produces proteases in the sedolisin family (MEROPS S53). Four putative secreted sedolisins with a predicted 17- to 20-amino-acid signal sequence were identified and termed SedA to SedD. SedA produced heterologously in Pichia pastoris was an acidic endoprotease. Heterologously produced SedB, SedC, and SedD were tripeptidyl-peptidases (TPP) with a common specificity for tripeptide-p-nitroanilide substrates at acidic pHs. Purified SedB hydrolyzed the peptide Ala-Pro-Gly-Asp-Arg-Ile-Tyr-Val-His-Pro-Phe to Arg-Pro-Gly, Asp-Arg-Ile, and Tyr-Val-His-Pro-Phe, thereby confirming TPP activity of the enzyme. SedB, SedC, and SedD were detected by Western blotting in culture supernatants of A. fumigatus grown in a medium containing hemoglobin as the sole nitrogen source. A degradation product of SedA also was observed. A search for genes encoding sedolisin homologues in other fungal genomes indicates that sedolisin gene families are widespread among filamentous ascomycetes.

scholarly journals Intracellular α-Amylase ofStreptococcus mutans

1998 ◽  
Vol 180 (17) ◽  
pp. 4711-4717 ◽  
Author(s):  
Christine L. Simpson ◽  
Roy R. B. Russell
Keyword(s):  
Amino Acid ◽  
Amylase Activity ◽  
Signal Sequence ◽  
Amino Acid Identity ◽  
Reading Frame ◽  
Cell Extracts ◽  
Genes Encoding ◽  
Culture Supernatants ◽  
Produce Acid

ABSTRACT Sequencing upstream of the Streptococcus mutans gene for a CcpA gene homolog, regM, revealed an open reading frame, named amy, with homology to genes encoding α-amylases. The deduced amino acid sequence showed a strong similarity (60% amino acid identity) to the intracellular α-amylase of Streptococcus bovis and, in common with this enzyme, lacked a signal sequence. Amylase activity was found only in S. mutans cell extracts, with no activity detected in culture supernatants. Inactivation of amy by insertion of an antibiotic resistance marker confirmed that S. mutanshas a single α-amylase activity. The amylase activity was induced by maltose but not by starch, and no acid was produced from starch.S. mutans can, however, transport limit dextrins and maltooligosaccharides generated by salivary amylase, but inactivation of amy did not affect growth on these substrates or acid production. The amylase digested the glycogen-like intracellular polysaccharide (IPS) purified from S. mutans, but the amy mutant was able to digest and produce acid from IPS; thus, amylase does not appear to be essential for IPS breakdown. However, when grown on excess maltose, theamy mutant produced nearly threefold the amount of IPS produced by the parent strain. The role of Amy has not been established, but Amy appears to be important in the accumulation of IPS in S. mutans grown on maltose.

scholarly journals Arabidopsis Genes Essential for Seedling Viability: Isolation of Insertional Mutants and Molecular Cloning

Genetics ◽  
2001 ◽  
Vol 159 (4) ◽  
pp. 1765-1778
Author(s):  
Gregory J Budziszewski ◽  
Sharon Potter Lewis ◽  
Lyn Wegrich Glover ◽  
Jennifer Reineke ◽  
Gary Jones ◽  
...  
Keyword(s):  
Large Scale ◽  
Protein Translocation ◽  
Gene Families ◽  
Mutant Phenotype ◽  
Lethal Mutant ◽  
A Genome ◽  
Genes Encoding ◽  
High Level ◽  
Mutant Lines ◽  
Genome Scale

Abstract We have undertaken a large-scale genetic screen to identify genes with a seedling-lethal mutant phenotype. From screening ~38,000 insertional mutant lines, we identified >500 seedling-lethal mutants, completed cosegregation analysis of the insertion and the lethal phenotype for >200 mutants, molecularly characterized 54 mutants, and provided a detailed description for 22 of them. Most of the seedling-lethal mutants seem to affect chloroplast function because they display altered pigmentation and affect genes encoding proteins predicted to have chloroplast localization. Although a high level of functional redundancy in Arabidopsis might be expected because 65% of genes are members of gene families, we found that 41% of the essential genes found in this study are members of Arabidopsis gene families. In addition, we isolated several interesting classes of mutants and genes. We found three mutants in the recently discovered nonmevalonate isoprenoid biosynthetic pathway and mutants disrupting genes similar to Tic40 and tatC, which are likely to be involved in chloroplast protein translocation. Finally, we directly compared T-DNA and Ac/Ds transposon mutagenesis methods in Arabidopsis on a genome scale. In each population, we found only about one-third of the insertion mutations cosegregated with a mutant phenotype.

scholarly journals The Yeast Signal Sequence Trap Identifies Secreted Proteins of the Hemibiotrophic Corn Pathogen Colletotrichum graminicola

2008 ◽  
Vol 21 (10) ◽  
pp. 1325-1336 ◽  
Author(s):  
Jorrit-Jan Krijger ◽  
Ralf Horbach ◽  
Michael Behr ◽  
Patrick Schweizer ◽  
Holger B. Deising ◽  
...  
Keyword(s):  
Cell Walls ◽  
Leaf Extract ◽  
Signal Sequence ◽  
Stem Rot ◽  
Colletotrichum Graminicola ◽  
In Planta ◽  
Chain Reaction ◽  
Genes Encoding ◽  
Polymerase Chain

The hemibiotroph Colletotrichum graminicola is the causal agent of stem rot and leaf anthracnose on Zea mays. Following penetration of epidermal cells, the fungus enters a short biotrophic phase, followed by a destructive necrotrophic phase of pathogenesis. During both phases, secreted fungal proteins are supposed to determine progress and success of the infection. To identify genes encoding such proteins, we constructed a yeast signal sequence trap (YSST) cDNA-library from RNA extracted from mycelium grown in vitro on corn cell walls and leaf extract. Of the 103 identified unigenes, 50 showed significant similarities to genes with a reported function, 25 sequences were similar to genes without a known function, and 28 sequences showed no similarity to entries in the databases. Macroarray hybridization and quantitative reverse-transcriptase polymerase chain reaction confirmed that most genes identified by the YSST screen are expressed in planta. Other than some genes that were constantly expressed, a larger set showed peaks of transcript abundances at specific phases of pathogenesis. Another set exhibited biphasic expression with peaks at the biotrophic and necrotrophic phase. Transcript analyses of in vitro-grown cultures revealed that several of the genes identified by the YSST screen were induced by the addition of corn leaf components, indicating that host-derived factors may have mimicked the host milieu.

scholarly journals Immune Responses of Human Immature Dendritic Cells Can Be Modulated by the Recombinant Aspergillus fumigatus Antigen Aspf1

2009 ◽  
Vol 16 (10) ◽  
pp. 1485-1492 ◽  
Author(s):  
Michael Ok ◽  
Jean Paul Latgé ◽  
Carina Baeuerlein ◽  
Frank Ebel ◽  
Markus Mezger ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Aspergillus Fumigatus ◽  
Organ Transplant ◽  
Solid Organ ◽  
Transplant Recipients ◽  
Cytokines And Chemokines ◽  
Expression Of Genes ◽  
Immature Dendritic Cells ◽  
Genes Encoding ◽  
Solid Organ Transplant Recipients

ABSTRACT Invasive aspergillosis is a significant cause of morbidity and mortality in patients after stem cell transplantation, in solid organ transplant recipients, and in patients with hematological malignancies. The interactions between human immature dendritic cells (iDCs) and Aspergillus fumigatus antigens are widely uncharacterized. We analyzed the immune response of iDCs to different recombinant A. fumigatus antigens (Aspf1 and Crf1). One of these antigens, the 18-kDa RNase Aspf1, triggered the increased level of expression of genes encoding proinflammatory cytokines and chemokines, and augmented the activation of NFκB and the apoptosis of iDCs. Furthermore, by fluorescence microscopy, we could demonstrate that in the first 3 h a major portion of Aspf1 accumulates on the cell surface. Finally, we could show an increased segregation of cytokines and chemokines after the stimulation of iDCs by an Aspf1 deletion mutant strain of A. fumigatus.

Preparation of Highly Conductive Polyaniline in Carbamide Aqueous Solution Using SnCl4 as Initiator

2012 ◽  
Vol 476-478 ◽  
pp. 2205-2208 ◽  
Author(s):  
Guan Nan Lin ◽  
Qun Yu ◽  
Wei Wang ◽  
Gui Bao Wang
Keyword(s):  
Aqueous Solution ◽  
Ammonium Persulfate ◽  
Conductivity Measurements ◽  
X Ray ◽  
New Class ◽  
Vis Spectroscopy ◽  
Polymerization System ◽  
Novel Method ◽  
Conductive Polyaniline ◽  
Hydrolysis Of

In this paper, we demonstrated a novel method for the preparation of highly conductive polyaniline (PANI) compounded with Sn(OH)4. We obtained the PANI directly in the oxidation polymerization system via simultaneous reaction of aniline (using ammonium persulfate, APS as oxidant) and SnCl4 in carbamide aqueous solution. The resulting PANI was compounded with Sn(OH)4 had been characterized by FTIR, UV-Vis spectroscopy, X-ray diffractometry, thermal analysis, scanning electron microscope and conductivity measurements, and the results showed that PANI was in well doped state due to the hydrolysis of APS and the complex between PANI and Sn(OH)4. We are sure this alkali-guided polymerization to obtain conductive PANI will lead to the preparation of a new class of PANI composites.

scholarly journals Antiidiotypic DNA vaccination induces serum bactericidal activity and protection against group B meningococci

2006 ◽  
Vol 203 (1) ◽  
pp. 111-118 ◽  
Author(s):  
Concetta Beninati ◽  
Angelina Midiri ◽  
Giuseppe Mancuso ◽  
Carmelo Biondo ◽  
Milena Arigò ◽  
...  
Keyword(s):  
Bactericidal Activity ◽  
Capsular Polysaccharide ◽  
Signal Sequence ◽  
Developed Countries ◽  
Single Chain ◽  
Serum Bactericidal Activity ◽  
Scfv Gene ◽  
Genes Encoding ◽  
Encapsulated Bacteria ◽  
Group B

No vaccine is available for preventing infections by serogroup B Neisseria meningitidis (MenB), which accounts for a major portion of meningococcal cases in developed countries, because of the poor immunogenicity of the capsular polysaccharide (CP) even after protein conjugation. We have previously induced anticapsular antibodies by immunization with a single chain variable fragment (scFv), which mimics a protective CP epitope. This surrogate antigen, however, was ineffective at inducing serum bactericidal activity, an accepted marker of protection in humans. Serum bactericidal activity was consistently achieved by immunizing mice with the scFv-encoding gene. Immunization with vectors without a secretory signal sequence before the scFv resulted in markedly higher bactericidal activity relative to those with such a sequence. The induced antibodies were capsule specific, as shown by complete inhibition of bactericidal activity by purified MenB CP and by resistance to killing of MenA or MenC. Moreover, these antibodies were predominantly of the IgG2a isotype, reflecting a T helper type 1 response. Administration of sera from scFv gene–vaccinated animals protected infant rats against MenB bacteremia. These data illustrate the potential of vaccination with genes encoding capsular mimics in providing protection against MenB and other encapsulated bacteria.

scholarly journals Peptidase activity of β-lactamases

1999 ◽  
Vol 341 (2) ◽  
pp. 409-413 ◽  
Author(s):  
Noureddine RHAZI ◽  
Moreno GALLENI ◽  
Michael I. PAGE ◽  
Jean-Marie FRÈRE
Keyword(s):  
Molecular Mass ◽  
Enterobacter Cloacae ◽  
Peptidase Activity ◽  
Rate Enhancement ◽  
Enhancement Factors ◽  
Lactam Ring ◽  
Class C ◽  
Hydrolysis Of

Although β-lactamases have generally been considered as being devoid of peptidase activity, a low but significant hydrolysis of various N-acylated dipeptides was observed with representatives of each class of β-lactamases. The kcat/Km values were below 0.1 M-1˙s-1, but the enzyme rate enhancement factors were in the range 5000-20000 for the best substrates. Not unexpectedly, the best ‘peptidase’ was the class C β-lactamase of Enterobacter cloacae P99, but, more surprisingly, the activity was always higher with the phenylacetyl- and benzoyl-D-Ala-D-Ala dipeptides than with the diacetyl- and α-acetyl-L-Lys-D-Ala-D-Ala tripeptides, which are the preferred substrates of the low-molecular-mass, soluble DD-peptidases. A comparison between the β-lactamases and DD-peptidases showed that it might be as difficult for a DD-peptidase to open the β-lactam ring as it is for the β-lactamases to hydrolyse the peptides, an observation which can be explained by geometric and stereoelectronic considerations.

Characterization of peptidases of adult Trichuris muris

Parasitology ◽  
1994 ◽  
Vol 109 (5) ◽  
pp. 623-630 ◽  
Author(s):  
L. J. Drake ◽  
A. E. Bianco ◽  
D. A. P. Bundy ◽  
F. Ashall
Keyword(s):  
Mammalian Cells ◽  
Culture Medium ◽  
Matrix Protein ◽  
Sodium Dodecyl ◽  
Time Dependent ◽  
Extracellular Matrix Protein ◽  
Peptidase Activity ◽  
Trichuris Muris ◽  
Hydrolysis Of

Excretory/secretory (E/S) material of Trichuris muris was found to contain 2 major peptidases, Mr 85 and 105 kDa, which degrade gelatin optimally at pH 6·0 in sodium dodecyl sulphate–polyacrylamide gels. The peptidases were inactivated diisopropylfluorophosphate, leupeptin and soybean trypsin inhibitor, but were unaffected by inhibitors of aspartic-, cysteine- and metallo-peptidases, indicating that they are serine peptidases. Both enzymes were detectable within 5 h after incubation of worms in culture medium and showed a time-dependent increase in levels. Neither peptidase was detected in worm extracts, suggesting that they are activated during or following secretion from worms. Live worms degraded radio-isotope labelled extracellular matrix protein substratum derived from mammalian cells. Aminopeptidase activities capable of catalysing hydrolysis of amino acyl aminomethylcoumarin (MCA) substrates and a Z-Phe-Arg-MCA-hydrolysing cysteine peptidase activity, were detected in extracts of adult worms but not in E/S material.

Detection of esterase activity in susceptible and organophosphate resistant strains of the cattle tick Boophilus microplus (Acari: Ixodidae)

1997 ◽  
Vol 87 (2) ◽  
pp. 197-202 ◽  
Author(s):  
R. Rosario-Cruz ◽  
E. Miranda-Miranda ◽  
Z. Garcia-Vasquez ◽  
M. Ortiz-Estrada
Keyword(s):  
Esterase Activity ◽  
Boophilus Microplus ◽  
Cattle Tick ◽  
Molecular Weights ◽  
Sds Page ◽  
Genes Encoding ◽  
Resistant Strains ◽  
Hydrolysis Of ◽  
Coomassie Brilliant ◽  
Naphthyl Acetate

AbstractTwo organophosphate (OP) resistant strains of the cattle tick Boophilus microplus (Canestrini) from Mexico and Costa Rica were used to analyse the presence of esterase activity associated with resistance. The concentrations of six major proteins in both resistant strains were increased compared to the susceptible Morelos strain, both when stained with Coomassie Brilliant Blue after SDS-PAGE, and when analysed for esterase activity by the hydrolysis of naphthyl acetate esters. Esterases were named A or B in relation to the substrate preference for alpha or beta naphthyl acetate and numbered according to their position on the SDS—PAGE. The molecular weights of these proteins were: 125, 115, 108, 77, 43 and 67 Kd for Est-Bl, Est-B2, Est-B3, Est-B4, Est-B5 and Est-A respectively. Est-B3 showed cholinesterase (ChE) activity. This study strengthens the hypothesis that the mechanism associated with OP resistance found in many other insects includes an increase of esterase activity, probably as a result of gene amplification. The genes encoding these enzymes could be potentially used as molecular markers to detect resistance in the cattle tick B. microplus using a DNA probe.

scholarly journals Frequency of BKC-1-Producing Klebsiella Species Isolates

2016 ◽  
Vol 60 (8) ◽  
pp. 5044-5046 ◽  
Author(s):  
Willames M. B. S. Martins ◽  
Adriana G. Nicoletti ◽  
Silvia R. Santos ◽  
Jorge L. M. Sampaio ◽  
Ana C. Gales
Keyword(s):  
Clonal Complex ◽  
Pulsed Field Gel Electrophoresis ◽  
Beta Lactam ◽  
Klebsiella Species ◽  
Content Type ◽  
Beta Lactamases ◽  
New Class ◽  
Class A ◽  
Genes Encoding ◽  
Extended Spectrum Beta Lactamases

ABSTRACTBKC-1 is a new class A serine carbapenemase that was recently identified inKlebsiella pneumoniaeclinical isolates. The principal objective of this study was to evaluate the frequency ofblaBKC-1by testing a collection ofKlebsiellaisolates. Only 2 of 635Klebsiellaisolates (0.3%) carriedblaBKC-1. The two BKC-1-producing isolates belonged to clonal complex 442 and possessed identical pulsed-field gel electrophoresis patterns. TheblaBKC-1gene was inserted into a 10-kb plasmid that was identical to the previously reported plasmid, p60136. The BKC-producingK. pneumoniaeisolates presented also possessed other mechanisms for beta-lactam resistance, such as genes encoding extended-spectrum beta-lactamases and mutations in the genesompK35andompK36, encoding the major porins.

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