Coupling 2D SDS−PAGE with CNBr Cleavage and MALDI-TOFMS:  A Strategy Applied to the Identification of Proteins Induced by a Hypochlorous Acid Stress inEscherichia coli

1998 ◽  
Vol 70 (20) ◽  
pp. 4433-4440 ◽  
Author(s):  
S. Dukan ◽  
E. Turlin ◽  
F. Biville ◽  
G. Bolbach ◽  
D. Touati ◽  
...  
Keyword(s):  
Hypochlorous Acid ◽  
Acid Stress ◽  
Inescherichia Coli ◽  
Sds Page ◽  
Cnbr Cleavage

Stress tolerance in Lactobacillus plantarum LMF6 isolated from human breast milk

2020 ◽  
Vol 9 (5) ◽  
pp. 173-184
Author(s):  
Fatima Zohra Baghdad Belhadj ◽  
Faiza Boublenza ◽  
Nour-Eddine Karam
Keyword(s):  
Breast Milk ◽  
Lactobacillus Plantarum ◽  
Food Industry ◽  
Stress Proteins ◽  
Acid Stress ◽  
Human Breast Milk ◽  
Human Breast ◽  
Beneficial Effects ◽  
Sds Page ◽  
Extreme Heat Stress

Lactobacillus plantarum is a lactic acid bacterium widely used in the food industry because of its beneficial effects on human health and its ability of adaptation to different stress conditions, hence the purpose of this work was to study the adaptation abilities of Lactobacillus plantarum LM6 and stress proteins involved during this adapta on. Lb. plantarum LMF6 was isolated from human breast milk and was exposed to acid, alkaline, thermal, oxidative, osmotic, detergent and nutritional stresses in order to determine their effects on growth, viability, tolerance and mortality. SDS-PAGE electrophoresis allowed us to compare the total proteins in the absence and in the presence of stress then the ImageJ® so ware analyzed the obtained pro les. The results show that Lb. plantarum LMF6 is highly tolerant to osmo c (at 9% NaCl, the UFC number is 3.4×1010 UFC/ml), alkaline (4.7×107UFC/ml at pH10), detergent (the UFC number is close to the control), oxydative (3.3×108 UFC/ml), nutri onnal (5.2×107 UFC/ml), acid (pH5, pH4 and pH3) and heat (40°C, 45°C and 50°C with 1.45×1011, 2.78×109 and 2.80×108UFC/ml respec vely) stresses, but sensi ve to extreme acid stress (pH1 and pH2 with mortality rate variable from 5log to 10log) and extreme heat stress (55°C and 60°C when mortality increases to 8log at 60°C). Comparison of proteins profiles allowed us to see quantitative and qualitative differences. Our results allowed to say that Lb. plantarum LMF6 showed interesting characteristics and could be used in food industry as probio c lactobacilli.

scholarly journals Potential Probiotic Pediococcus pentosaceus M41 Modulates Its Proteome Differentially for Tolerances Against Heat, Cold, Acid, and Bile Stresses

2021 ◽  
Vol 12 ◽  
Author(s):  
Mohd Affan Baig ◽  
Mark S. Turner ◽  
Shao-Quan Liu ◽  
Anas A. Al-Nabulsi ◽  
Nagendra P. Shah ◽  
...  
Keyword(s):  
Acid Stress ◽  
Stress Conditions ◽  
Lactate Oxidase ◽  
Glycerol Phosphate ◽  
Pediococcus Pentosaceus ◽  
Sds Page ◽  
Tyrosine Protein Kinase ◽  
Imidazole Glycerol Phosphate ◽  
Proteomic Responses ◽  
Related Proteins

Probiotics containing functional food confer health benefits in addition to their nutritional properties. In this study, we have evaluated the differential proteomic responses of a potential novel probiotic Pediococcus pentosaceus M41 under heat, cold, acid, and bile stress conditions. We identified stress response proteins that could provide tolerances against these stresses and could be used as probiotic markers for evaluating stress tolerance. Pediococcus pentosaceus M41 was exposed for 2 h to each condition: 50°C (heat stress), 4°C (cold stress), pH 3.0 (acid stress) and 0.05% bile (bile stress). Proteomic analysis was carried out using 2D-IEF SDS PAGE and LC-MS/MS. Out of 60 identified proteins, 14 upregulated and 6 downregulated proteins were common among all the stress conditions. These proteins were involved in different biological functions such as translation-related proteins, carbohydrate metabolism (phosphoenolpyruvate phosphotransferase), histidine biosynthesis (imidazole glycerol phosphate synthase) and cell wall synthesis (tyrosine-protein kinase CapB). Proteins such as polysaccharide deacetylase, lactate oxidase, transcription repressor NrdR, dihydroxyacetone kinase were upregulated under three out of the four stress conditions. The differential expression of these proteins might be responsible for tolerance and protection of P. pentosaceus M41 against different stress conditions.

scholarly journals Characterization of non-covalent oligomers of proteins treated with hypochlorous acid

2003 ◽  
Vol 375 (1) ◽  
pp. 33-40 ◽  
Author(s):  
Anna L. P. CHAPMAN ◽  
Christine C. WINTERBOURN ◽  
Stephen O. BRENNAN ◽  
T. William JORDAN ◽  
Anthony J. KETTLE
Keyword(s):  
Hypochlorous Acid ◽  
Tissue Injury ◽  
Convincing Evidence ◽  
Protein Carbonyls ◽  
Molar Ratio ◽  
Myeloperoxidase Activity ◽  
Reducing Conditions ◽  
Sds Page ◽  
Methionine Residues

Hypochlorous acid (HOCl) is a potent oxidant produced by myeloperoxidase that causes aggregation of many proteins. Treatment of apohaemoglobin and apomyoglobin with HOCl produced a regular series of oligomer bands when the proteins were separated by SDS/PAGE under reducing conditions. Aggregation was detectable at a HOCl/protein molar ratio of 0.5:1 and was maximal at ratios of 10:1–20:1. Dimers formed within 1 min of adding HOCl, and further aggregation occurred over the next 30 min. No convincing evidence for covalent cross-linking was obtained by amino acid analysis, peptide analysis or electrospray ionization-MS of HOCl-modified apomyoglobin. The latter showed an increase in mass consistent with conversion of the two methionine residues into sulphoxides. A 5-fold excess of HOCl generated approximately three chloramines on the apomyoglobin. These underwent slow decay. Protein carbonyls were formed and were almost entirely located only on the polymer bands. Conversion of positively into negatively charged groups on the protein by succinylation caused preformed aggregates to dissociate. Treatment of apomyoglobin with taurine chloramine generated methionine sulphoxides but few protein carbonyls, and did not result in aggregation. We conclude that aggregation was due to strong, non-covalent interactions between protein chains. We propose that formation of protein carbonyls and possibly chloramines, along with methionine oxidation, alters protein folding to expose hydrophobic areas on neighbouring molecules that associate to form dimers and higher-molecular-mass aggregates. This process could lead to the formation of aggregated proteins at sites of myeloperoxidase activity and contribute to inflammatory tissue injury.

scholarly journals Interplay between tolerance mechanisms to copper and acid stress inEscherichia coli

2017 ◽  
pp. 201620232 ◽  
Author(s):  
Karrera Y. Djoko ◽  
Minh-Duy Phan ◽  
Kate M. Peters ◽  
Mark J. Walker ◽  
Mark A. Schembri ◽  
...  
Keyword(s):  
Acid Stress ◽  
Inescherichia Coli ◽  
Tolerance Mechanisms

scholarly journals Purification and characterization of a novel phosphoenolpyruvate carboxylase from banana fruit

1995 ◽  
Vol 307 (3) ◽  
pp. 807-816 ◽  
Author(s):  
R D Law ◽  
W C Plaxton
Keyword(s):  
Phosphoenolpyruvate Carboxylase ◽  
Specific Activity ◽  
Immune Serum ◽  
Enzymic Activity ◽  
Banana Fruit ◽  
Native Molecular Mass ◽  
Apparent Homogeneity ◽  
Sds Page ◽  
Cnbr Cleavage

Phosphoenolpyruvate carboxylase (PEPC) from ripened banana (Musa cavendishii L.) fruits has been purified 127-fold to apparent homogeneity and a final specific activity of 32 mumol of oxaloacetate produced/min per mg of protein. Non-denaturing PAGE of the final preparation resolved a single protein-staining band that co-migrated with PEPC activity. Polypeptides of 103 (alpha-subunit) and 100 (beta-subunit) kDa, which stain for protein with equal intensity and cross-react strongly with anti-(maize leaf PEPC) immune serum, were observed following SDS/PAGE of the final preparation. CNBr cleavage patterns of the two subunits were similar, but not identical, suggesting that these polypeptides are related, but distinct, proteins. The enzyme's native molecular mass was estimated to be about 425 kDa. These data indicate that in contrast to the homotetrameric PEPC from most other sources, the banana fruit enzyme exists as an alpha 2 beta 2 heterotetramer. Monospecific rabbit anti-(banana PEPC) immune serum effectively immunoprecipitated the activity of the purified enzyme. Immunoblotting studies established that the 100 kDa subunit did not arise via proteolysis of the 103 kDa subunit after tissue extraction, and that the subunit composition of banana PEPC remains uniform throughout the ripening process. PEPC displayed a typical pH activity profile with an alkaline optimum and activity rapidly decreasing below pH 7.0. Enzymic activity was absolutely dependent on the presence of a bivalent metal cation, with Mg2+ or Mn2+ fulfilling this requirement. The response of the PEPC activity to PEP concentration and to various effectors was greatly influenced by pH and glycerol addition to the assay. The enzyme was activated by hexose-monophosphates and potently inhibited by malate, succinate, aspartate and glutamate at pH 7.0, whereas the effect of these metabolites was considerably diminished or completely abolished at pH 8.0. The significance of metabolite regulation of PEPC is discussed in relation to possible functions of this enzyme in banana fruit metabolism.

scholarly journals Viability and Effects on Bacterial Proteins by Oral Rinses with Hypochlorous Acid as Active Ingredient

2015 ◽  
Vol 26 (5) ◽  
pp. 519-524 ◽  
Author(s):  
Diana Marcela Castillo ◽  
Yormaris Castillo ◽  
Nathaly Andrea Delgadillo ◽  
Yineth Neuta ◽  
Johana Jola ◽  
...  
Keyword(s):  
Hypochlorous Acid ◽  
Similar Efficacy ◽  
Molecular Probes ◽  
P Value ◽  
Bonferroni Correction ◽  
Total Proteins ◽  
Bacterial Viability ◽  
Bacterial Proteins ◽  
Sds Page ◽  
Live Bacteria

Abstract: This study investigated the effect of hypochlorous acid (HOCl) rinses and chlorhexidine (CHX) on the bacterial viability of S. mutans, A. israelii, P. gingivalis, A. actinomycetemcomitans, E. corrodens, C. rectus, K. oxytoca, K. pneumoniae and E. cloacae. The percentage of live bacteria was tested by fluorescence method using Live/Dead kit(r) and BacLight (Molecular Probes(r)) and compared between groups by the Kruskal-Wallis and U Mann-Whitney tests with Bonferroni correction (p value<0.012). The effect of HOCl and CHX on total proteins of P. gingivalis and S. mutans was determined by SDS-PAGE. CHX showed a higher efficacy than HOCl against S. mutans, A. israelii, E. corrodens and E. cloacae (p<0.001) while HOCl was more effective than CHX against P. gingivalis, A. actinomycetemcomitans, C. rectus and K. oxytoca (p=0.001). CHX and HOCl had similar efficacy against K. pneumoniae. Proteins of P. gingivalis and S. mutans were affected similarly by HOCl and CHX. HOCl reduced the bacterial viability especially in periodontopathic bacteria, which may support its use in the control of subgingival biofilm in periodontal patients.

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