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Poultry Science Dec 2019The objective of this study was to assess the effect of dietary yeast products on broiler chickens challenged with salmonella lipopolysaccharide (LPS). The chicks were...
The objective of this study was to assess the effect of dietary yeast products on broiler chickens challenged with salmonella lipopolysaccharide (LPS). The chicks were divided into 8 treatments with 6 replicates and 9 birds per replicate. The treatments consisted of a positive control (PC) [without supplementation and not challenged]; negative control (NC) [without supplementation but challenged]; whole yeast and challenged; yeast cell wall and challenged; yeast glucan and challenged; yeast mannan and challenged; zinc bacitracin and challenged; and Salinomycin and challenged. Whole yeast or Yeast cell wall was included at 2.0 g/kg diet. Yeast glucan or mannan was added at 0.20 g/kg diet. Zinc bacitracin (ZNB) and Salinomycin (SAL) was included at 50 and 60 ppm, respectively. Dietary treatments had no effect (P > 0.05) on feed intake (FI) at day 10. Supplementation with yeast and its derivatives improved (P < 0.05) body weight gain (BWG) and feed conversion ratio (FCR) on day 10. On days 24 and 35, LPS challenge declined FI, BWG, FCR, and flock uniformity (day 28) in the NC group compared to the PC group. Yeast products and antibiotics improved (P < 0.05) FI, BWG, FCR, and flock uniformity in LPS-challenged birds. On day 24, spleen weight increased while bursa weight decreased in the NC group relative to the PC group; this effect was reversed (P < 0.05) by feeding all yeasts and antibiotics. On day 24, application of all the dietary treatments ameliorated the changes observed in white blood cell, lymphocyte and monocyte counts as well as albumin and immunoglobulin G of NC birds. On day 35, all yeasts additives, ZNB and SAL improved (P < 0.05) the meat yield of broilers challenged with LPS. In conclusion, supplementation of diets with yeast and its derivatives can ameliorate the negative effects of salmonella LPS challenge on broiler chicks, thus improving the performance, flock uniformity, and meat yield.
Topics: Animal Feed; Animal Nutritional Physiological Phenomena; Animals; Autolysis; Body Composition; Chickens; Diet; Dietary Supplements; Lipopolysaccharides; Salmonella; Weight Gain; Yeasts
PubMed: 31392341
DOI: 10.3382/ps/pez452 -
The Biochemical Journal Jan 1981The autolysis of trypsin and alpha-chymotrypsin is accelerated in the presence of colloidal silica and glass surfaces. It is proposed that adsorption of the enzymes...
The autolysis of trypsin and alpha-chymotrypsin is accelerated in the presence of colloidal silica and glass surfaces. It is proposed that adsorption of the enzymes (favoured by electrostatic factors) results in a conformational change that renders the adsorbed enzyme more susceptible to proteolytic attack. Although the adsorbed enzymes are more susceptible to proteolysis, their activity towards low-molecular-weight substrates is not affected, indicating a relatively minor conformational change on adsorption. The rates of autolysis in solution (i.e. in ;inert' vessels) are second-order for both trypsin and alpha -chymotrypsin, with rate constants of 13.0mol(-1).dm(3).s(-1) for trypsin (in 50mm-NaCl at pH8.0 at 25 degrees C) and 10.2mol(-1).dm(3).s(-1) for alpha-chymotrypsin (in 0.1m-glycine at pH9.2 at 30 degrees C). In glass vessels or in the presence of small areas of silica surface (as colloidal silica particles), the autolysis of both trypsin and alpha-chymotrypsin can show first-order kinetics. Under these conditions, saturation of the surface occurs and the fast surface proteolytic reaction controls the overall kinetic order. However, when greater areas of silica surface are present, saturation of the surface does not occur, and, since for a considerable portion of the adsorption isotherm the amount adsorbed is approximately proportional to the concentration in solution, second-order kinetics are again observed. A number of negatively charged macromolecules have been shown similarly to increase the rate of autolysis of trypsin: thus this effect, observed initially with glass and silica surfaces, is of more general occurrence when these enzymes adsorb on or interact with negatively charged surfaces and macromolecules. These observations explain the confusion in the literature with regard to the kinetics of autolysis of alpha-chymotrypsin, where first-order, second-order and intermediate kinetics have been reported. A further effect of glass surfaces and negatively charged macromolecules is to shift the pH-activity curve of trypsin to higher pH values, as a consequence of the effective decrease in pH in the ;microenvironment' of the enzyme associated with the negatively charged surface or macromolecule.
Topics: Adsorption; Chemical Phenomena; Chemistry; Chymotrypsin; Colloids; Hydrogen-Ion Concentration; Kinetics; Macromolecular Substances; Silicon Dioxide; Surface Properties; Trypsin Inhibitors
PubMed: 6272706
DOI: 10.1042/bj1930285 -
The Biochemical Journal Sep 1996The kinetics of autolysis and activation of mu-calpain were measured with microtubule-associated protein 2 (MAP2) as a very sensitive substrate. The initial rate of MAP2...
The kinetics of autolysis and activation of mu-calpain were measured with microtubule-associated protein 2 (MAP2) as a very sensitive substrate. The initial rate of MAP2 hydrolysis was found to be a linear function of the autolysed 76 kDa form of mu-calpain large subunit at both 10 and 300 microM Ca2+, and both straight lines intersected the origin. This finding supports the view that native mu-calpain is an inactive proenzyme and that activation is accompanied by autolysis. The first-order rate constant of autolysis, K1(aut), was determined at different Ca2+ concentrations: the half-maximal value was at pCa2+ = 3.7 (197 microM Ca2+), whereas the maximal value was 1.52 s-1, at 30 degrees C. The Ca(2+)-induced activation process was then monitored by using our novel, continuous fluorimetric assay with labelled MAP2 as substrate. The first-order rate constant of activation, k1(act), was derived as the reciprocal of the lag phase ('transit time') at the initial part of the progress curve: half-maximum was at pCa2+ = 3.8 (158 microM Ca2+) and the maximum value was 2.15 s-1. The good agreement between the kinetic parameters of mu-calpain autolysis and activation is remarkable. We claim that this is the first kinetically correct determination of the rate constant of autolysis of mu-calpain. Pre-activated mu-calpain has a Ca2+ requirement that is almost three orders of magnitude smaller [half-maximal activation at pCa2+ = 6.22 (0.6 microM Ca2+)]. We cannot exclude the possibility that the activation process involves other mechanistic steps, e.g. the rapid dissociation of the mu-calpain heterodimer, but we state that in our conditions in vitro autolysis and activation run in close parallel.
Topics: Autolysis; Calpain; Enzyme Activation; Enzyme Precursors; Erythrocytes; Humans; In Vitro Techniques; Kinetics; Microtubule-Associated Proteins; Molecular Structure; Protein Conformation
PubMed: 8836135
DOI: 10.1042/bj3180897 -
BMC Microbiology Nov 2016Streptococcus pneumoniae is one of the leading causes of community acquired pneumonia and acute otitis media. Certain aspects of S. pneumoniae's virulence are dependent...
BACKGROUND
Streptococcus pneumoniae is one of the leading causes of community acquired pneumonia and acute otitis media. Certain aspects of S. pneumoniae's virulence are dependent upon expression and release of the protein toxin pneumolysin (PLY) and upon the activity of the peroxide-producing enzyme, pyruvate oxidase (SpxB). We investigated the possible synergy of these two proteins and identified that release of PLY is enhanced by expression of SpxB prior to stationary phase growth.
RESULTS
Mutants lacking the spxB gene were defective in PLY release and complementation of spxB restored PLY release. This was demonstrated by cytotoxic effects of sterile filtered supernatants upon epithelial cells and red blood cells. Additionally, peroxide production appeared to contribute to the mechanism of PLY release since a significant correlation was found between peroxide production and PLY release among a panel of clinical isolates. Exogenous addition of HO failed to induce PLY release and catalase supplementation prevented PLY release in some strains, indicating peroxide may exert its effect intracellularly or in a strain-dependent manner. SpxB expression did not trigger bacterial cell death or LytA-dependent autolysis, but did predispose cells to deoxycholate lysis.
CONCLUSIONS
Here we demonstrate a novel link between spxB expression and PLY release. These findings link liberation of PLY toxin to oxygen availability and pneumococcal metabolism.
Topics: Autolysis; Bacterial Proteins; Catalase; DNA, Bacterial; Epithelial Cells; Erythrocytes; Genes, Bacterial; Hydrogen Peroxide; Oxygen; Pyruvate Oxidase; Sequence Deletion; Streptococcus pneumoniae; Streptolysins; Virulence
PubMed: 27829373
DOI: 10.1186/s12866-016-0881-6 -
International Journal of Clinical and... Jan 2008We investigated the dynamics of autolytic damage of the cortical neurons in adult brains for 24 hours at room temperature (+20 degrees C) after cardiac arrest. The...
We investigated the dynamics of autolytic damage of the cortical neurons in adult brains for 24 hours at room temperature (+20 degrees C) after cardiac arrest. The progressive histological and ultrastructural changes were documented using routine and immunohistochemical staining as well as electron microscopy. Our results demonstrated that there were no autolytic damages in the ultrastructure of cerebral neurons in the first 6 hours after warm cardiac arrest, in agreement with previous studies in other mammals. Interestingly, the activation of caspase-3 was observed in a significant number of neurons of the cerebellum and neocortex 9 hours following cardiac arrest. No significant changes related to autolysis were observed using amnio-cupric acid and Nissl (thionine) staining.
PubMed: 18784829
DOI: No ID Found -
Blood Jul 2000The anticoagulant human plasma serine protease, activated protein C (APC), inactivates blood coagulation factors Va (FVa) and VIIIa. The so-called autolysis loop of APC...
The anticoagulant human plasma serine protease, activated protein C (APC), inactivates blood coagulation factors Va (FVa) and VIIIa. The so-called autolysis loop of APC (residues 301-316, equivalent to chymotrypsin [CHT] residues 142-153) has been hypothesized to bind FVa. In this study, site-directed mutagenesis was used to probe the role of the charged residues in this loop in interactions between APC and FVa. Residues Arg306 (147 CHT), Glu307, Lys308, Glu309, Lys311, Arg312, and Arg314 were each individually, or in selected combinations, mutated to Ala. The purified recombinant protein C mutants were characterized using activated partial thromboplastin time (APTT) clotting assays and FVa inactivation assays. Mutants 306A, 308A, 311A, 312A, and 314A had mildly reduced anticoagulant activity. Based on FVa inactivation assays and APTT assays using purified Gln506-FVa and plasma containing Gln506-FV, it appeared that these mutants were primarily impaired for cleavage of FVa at Arg506. Studies of the quadruple APC mutant (306A, 311A, 312A, and 314A) suggested that the autolysis loop provides for up to 15-fold discrimination of the Arg506 cleavage site relative to the Arg306 cleavage site. This study shows that the loop on APC of residues 306 to 314 defines an FVa binding site and accounts for much of the difference in cleavage rates at the 2 major cleavage sites in FVa. (Blood. 2000;96:585-593)
Topics: Arginine; Autolysis; Binding Sites; Blood Coagulation; Blotting, Western; Factor Va; Humans; Mutagenesis, Site-Directed; Partial Thromboplastin Time; Protein C; Recombinant Proteins; Structure-Activity Relationship
PubMed: 10887122
DOI: No ID Found -
The Journal of Biological Chemistry Jun 1989A recent hypothesis suggests that proteolytic activity of the micromolar and millimolar Ca2+-requiring forms of the Ca2+-dependent proteinases (mu- and m-calpain,...
A recent hypothesis suggests that proteolytic activity of the micromolar and millimolar Ca2+-requiring forms of the Ca2+-dependent proteinases (mu- and m-calpain, respectively) is regulated in vivo by their association with a phosphatidylinositol-containing site on the plasma membrane followed by autolysis of the proteinases. Phosphatidylinositol association lowers the Ca2+ concentration needed for autolysis, and autolysis, in turn, lowers the Ca2+ concentration needed for proteolytic activity. To test this hypothesis, we have compared the Ca2+ concentrations needed for autolysis and for proteolytic activity of the calpains both in the presence and the absence of phosphatidylinositol. Bovine skeletal muscle mu-calpain required 40-50 microM Ca2+ for half-maximal rate of proteolysis of a casein substrate, 140-150 microM Ca2+ for half-maximal autolysis in the presence of 80 microM phosphatidylinositol, and 190-210 microM Ca2+ for half-maximal autolysis in the absence of phosphatidylinositol. Consequently, mu-calpain is an active proteinase and does not require autolysis for activation. Bovine skeletal muscle m-calpain required 700-740 microM Ca2+ for half-maximal rate of proteolysis of a casein substrate, 370-400 microM Ca2+ for half-maximal autolysis in the presence of 80 microM phosphatidylinositol, and 740-780 microM Ca2+ for half-maximal autolysis in the absence of phosphatidylinositol. These results are consistent with the idea that m-calpain functions in its autolyzed form, but the results do not demonstrate that unautolyzed m-calpain is inactive. 80 microM phosphatidylinositol had no effect on the Ca2+ requirement of the autolyzed forms of either mu- or m-calpain but lowered the specific activity of mu-calpain to 20% of its activity in the absence of phosphatidylinositol. Of the four forms of the calpains, unautolyzed m-calpain, autolyzed m-calpain, and unautolyzed mu-calpain would not be proteolytically active at the free Ca2+ concentrations of 300-1200 nM present inside normal cells, and neither mu- nor m-calpain would undergo autolysis at these Ca2+ concentrations, even in the presence of phosphatidylinositol. Cells must contain a mechanism other than or in addition to membrane association and autolysis to activate the calpains.
Topics: Animals; Calcium; Calpain; Cattle; Hydrolysis; Isoenzymes; Kinetics; Muscles; Phosphatidylinositols
PubMed: 2542320
DOI: No ID Found -
Antimicrobial Agents and Chemotherapy Jan 2013The growing prevalence of antibiotic-resistant infections underscores the need to discover new antibiotics and to use them with maximum effectiveness. In response to...
The growing prevalence of antibiotic-resistant infections underscores the need to discover new antibiotics and to use them with maximum effectiveness. In response to these needs, we describe a screening protocol for the discovery of autolysis-inducing agents that uses two Bacillus subtilis reporter strains, SH-536 and BAU-102. To screen chemical libraries, autolysis-inducing agents were first identified with a BAU-102-based screen and then subdivided with SH-536 into two major groups: those that induce autolysis by their direct action on the cell membrane and those that induce autolysis secondary to inhibition of cell wall synthesis. SH-536 distinguishes between the two groups of autolysis-inducing agents by synthesizing and then releasing β-galactosidase (β-Gal) in late stationary phase at a time that cells have nearly stopped growing and are therefore tolerant of cell wall synthesis inhibitors. Four hits, named compound 2, compound 3, compound 5, and compound 24, obtained previously as inducers of autolysis by screening a 10,080-compound discovery library with BAU-102, were probed with SH-536 and found to release β-Gal, indicating that their mode of action was to permeabilize the B. subtilis cell membrane. The four primary hits inhibited growth in Staphylococcus aureus, Enterococcus faecium, Bacillus subtilis, and Bacillus anthracis, with MICs in the 12.5- to 25-μg/ml (20 to 60 μM) range. The four primary hits were further used to probe B. subtilis, and their action was partially characterized with respect to the dependence of induced autolysis on specific autolysins.
Topics: Anti-Bacterial Agents; Bacillus anthracis; Bacillus subtilis; Bacterial Proteins; Bacteriolysis; Cell Membrane; Cell Membrane Permeability; Cell Wall; Drug Discovery; Enterococcus faecium; High-Throughput Screening Assays; Microbial Sensitivity Tests; N-Acetylmuramoyl-L-alanine Amidase; Small Molecule Libraries; Staphylococcus aureus; Structure-Activity Relationship; beta-Galactosidase
PubMed: 23089762
DOI: 10.1128/AAC.00741-12 -
Malaria Journal Oct 2009Between 10,000 and 12,000 cases of imported malaria are notified in the European Union each year. Despite an excellent health care system, fatalities do occur. In case... (Comparative Study)
Comparative Study
BACKGROUND
Between 10,000 and 12,000 cases of imported malaria are notified in the European Union each year. Despite an excellent health care system, fatalities do occur. In case of advanced autolysis, the post-mortem diagnostic is impaired. Quicker diagnosis could be achieved by using rapid diagnostic malaria tests.
METHODS
In order to evaluate different methods for the post-mortem diagnosis of Plasmodium falciparum malaria in non-immunes, a study was performed on the basis of forensic autopsies of corpses examined at variable intervals after death in five cases of fatal malaria (with an interval of four hours to five days), and in 20 cases of deaths unrelated to malaria. Detection of parasite DNA by PCR and an immunochromatographic test (ICT) based upon the detection of P. falciparum histidine-rich protein 2 (PfHRP2) were compared with the results of microscopic examination of smears from cadaveric blood, histopathological findings, and autopsy results.
RESULTS
In all cases of fatal malaria, post-mortem findings were unsuspicious for the final diagnosis, and autoptic investigations, including histopathology, were only performed because of additional information by police officers and neighbours. Macroscopic findings during autopsy were unspecific. Histopathology confirmed sequestration of erythrocytes and pigment in macrophages in most organs in four patients (not evaluable in one patient due to autolysis). Microscopy of cadaveric blood smears revealed remnants of intraerythrocytic parasites, and was compromised or impossible due to autolysis in two cases. PCR and ICT performed with cadaveric blood were positive in all malaria patients and negative in all controls.
CONCLUSION
In non-immune fatalities with unclear anamnesis, ICT can be recommended as a sensitive and specific tool for post-mortem malaria diagnosis, which is easier and faster than microscopy, and also applicable when microscopic examination is impossible due to autolysis. PCR is more expensive and time-consuming, but may be used as confirmatory test. In highly endemic areas where asymptomatic parasitaemia is common, confirmation of the diagnosis of malaria as the cause of death has to rely on histopathological findings.
Topics: Adult; Aged; Aged, 80 and over; Antigens, Protozoan; Cadaver; Chromatography; Female; Humans; Immunoassay; Malaria, Falciparum; Male; Microscopy; Middle Aged; Plasmodium falciparum; Polymerase Chain Reaction; Predictive Value of Tests; Protozoan Proteins; Reagent Kits, Diagnostic; Reproducibility of Results; Sensitivity and Specificity; Time Factors
PubMed: 19863789
DOI: 10.1186/1475-2875-8-244 -
PloS One Apr 2010Staphylococcus aureus is a potent biofilm former on host tissue and medical implants, and biofilm growth is a critical virulence determinant for chronic infections....
Staphylococcus aureus is a potent biofilm former on host tissue and medical implants, and biofilm growth is a critical virulence determinant for chronic infections. Recent studies suggest that many clinical isolates form polysaccharide-independent biofilms. However, a systematic screen for defective mutants has not been performed to identify factors important for biofilm formation in these strains. We created a library of 14,880 mariner transposon mutants in a S. aureus strain that generates a proteinaceous and extracellular DNA based biofilm matrix. The library was screened for biofilm defects and 31 transposon mutants conferred a reproducible phenotype. In the pool, 16 mutants overproduced extracellular proteases and the protease inhibitor alpha(2)-macroglobulin restored biofilm capacity to 13 of these mutants. The other 15 mutants in the pool displayed normal protease levels and had defects in genes involved in autolysis, osmoregulation, or uncharacterized membrane proteins. Two transposon mutants of interest in the GraRS two-component system and a putative inositol monophosphatase were confirmed in a flow cell biofilm model, genetically complemented, and further verified in a community-associated methicillin-resistant S. aureus (CA-MRSA) isolate. Collectively, our screen for biofilm defective mutants identified novel loci involved in S. aureus biofilm formation and underscored the importance of extracellular protease activity and autolysis in biofilm development.
Topics: Autolysis; Bacterial Proteins; Biofilms; DNA Transposable Elements; Extracellular Matrix Proteins; Gene Library; Genes, Bacterial; Mutation; Peptide Hydrolases; Polysaccharides; Staphylococcus aureus
PubMed: 20418950
DOI: 10.1371/journal.pone.0010146