scholarly journals Antibacterial Profile of a Microbicidal Agent Targeting Tyrosine Phosphatases and Redox Thiols, Novel Drug Targets

Antibiotics ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 1310
Author(s):  
Kylie White ◽  
Gina Nicoletti ◽  
Hugh Cornell
Keyword(s):  
Drug Development ◽  
Plasma Proteins ◽  
Tyrosine Phosphatase ◽  
Bacterial Species ◽  
In Vitro Assays ◽  
Antioxidant Systems ◽  
Thiol Oxidation ◽  
Tyrosine Phosphatases ◽  
Activity Profile

The activity profile of a protein tyrosine phosphatase (PTP) inhibitor and redox thiol oxidant, nitropropenyl benzodioxole (NPBD), was investigated across a broad range of bacterial species. In vitro assays assessed inhibitory and lethal activity patterns, the induction of drug variants on long term exposure, the inhibitory interactions of NPBD with antibiotics, and the effect of plasma proteins and redox thiols on activity. A literature review indicates the complexity of PTP and redox signaling and suggests likely metabolic targets. NPBD was broadly bactericidal to pathogens of the skin, respiratory, urogenital and intestinal tracts. It was effective against antibiotic resistant strains and slowly replicating and dormant cells. NPBD did not induce resistant or drug-tolerant phenotypes and showed low cross reactivity with antibiotics in synergy assays. Binding to plasma proteins indicated lowered in-vitro bioavailability and reduction of bactericidal activity in the presence of thiols confirmed the contribution of thiol oxidation and oxidative stress to lethality. This report presents a broad evaluation of the antibacterial effect of PTP inhibition and redox thiol oxidation, illustrates the functional diversity of bacterial PTPs and redox thiols, and supports their consideration as novel targets for antimicrobial drug development. NPBD is a dual mechanism agent with an activity profile which supports consideration of tyrosine phosphatases and bacterial antioxidant systems as promising targets for drug development.

Characterization of Serratia species and qualitative detection of Serratia marcescens in raw and pasteurized milk by an analytical method based on polymerase chain reaction

2021 ◽  
Vol 67 (2) ◽  
pp. 3453-3464
Author(s):  
Evelin Korcz ◽  
László Varga ◽  
Zoltán Kerényi
Keyword(s):  
Serratia Marcescens ◽  
Bacterial Species ◽  
Relevant Literature ◽  
Inhibitory Effect ◽  
Test Methods ◽  
In Vitro Assays ◽  
Pasteurized Milk ◽  
Milk Samples ◽  
Microbiological Methods

Serratia species are opportunistic pathogenic microorganisms primarily known as nosocomial infectious agents, which can also cause food quality problems. The appearance of the extracellular pigment-producing Serratia marcescens in cow’s milk causes its red discoloration, posing a challenge to the dairy industry and food certification laboratories. The detection of the bacterium by conventional procedures based on microbiological methods is time-consuming and labor-intensive, and in many cases does not lead to satisfactory results due to the competitive inhibitory effect of the accompanying microflora. Following the analysis of the relevant literature, the published endpoint PCR methods and the primers used for the detection of S. marcescens were evaluated in in silico and in vitro assays, and then the procedure was tested on farm milk samples. Using the method, a total of 60 raw and pasteurized milk samples were analyzed, more than half of which (i.e., 32) were identified as S. marcescens positive. The significance of our work is mainly represented by the application of the published test methods in food industry practice. Our results highlight to the importance of detecting this bacterial species.

scholarly journals Study on a Fermented Whole Wheat: Phenolic Content, Activity on PTP1B Enzyme and In Vitro Prebiotic Properties

Molecules ◽  
2019 ◽  
Vol 24 (6) ◽  
pp. 1120 ◽  
Author(s):  
Diletta Balli ◽  
Maria Bellumori ◽  
Paolo Paoli ◽  
Giuseppe Pieraccini ◽  
Monica Di Paola ◽  
...  
Keyword(s):  
Lactobacillus Reuteri ◽  
Tyrosine Phosphatase ◽  
Bacterial Species ◽  
Food Ingredient ◽  
Microbial Composition ◽  
Acidic Hydrolysis ◽  
Inhibitory Power ◽  
Methyl Ferulate ◽  
First Time

Fermented cereals, staple foods in Asia and Africa, are recently receiving a growing interest in Western countries. The object of this work is the characterization of a fermented wheat used as a food ingredient and dietary supplement. To this aim, the phenolic composition, the activity on protein tyrosine phosphatase 1B (PTP1B), an enzyme overexpressed in type-II diabetes, the in vitro prebiotic properties on Lactobacillus reuteri and the microbial composition were investigated. Basic and acidic hydrolysis were tested for an exhaustive recovery of bound phenols: the acidic hydrolysis gave best yields. Methyl ferulate and neocarlinoside were identified for the first time in wheat. The inhibitory power of the extracts of several batches were investigated on PTP1B enzyme. The product was not able to inhibit the enzyme, otherwise, for the first time, a complete inhibition was observed for schaftoside, a major C-flavonoid of wheat. The microbial composition was assessed identifying Lactobacillus, Enterococcus, and Pediococcus as the main bacterial species. The fermented wheat was a suitable substrate for the grown of L. reuteri, recognized for its health properties in the human gut. The proposed method for phenols is easier compared to those based on strong basic hydrolysis; our results assessed the bound phenols as the major fraction, differently from that suggested by the literature for fermented cereals.

Effect of sorafenib on cathepsin B-dependent BID-mediated apoptosis in cancer cells.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. e15515-e15515
Author(s):  
Giorgio Santoni ◽  
Consuelo Amantini ◽  
Matteo Santoni ◽  
Maria Beatrice Morelli ◽  
Valerio Farfariello ◽  
...  
Keyword(s):  
Western Blot ◽  
Cell Lines ◽  
Cathepsin B ◽  
Tyrosine Phosphatase ◽  
Ros Generation ◽  
Tyrosine Phosphatases ◽  
Multikinase Inhibitor ◽  
Acridine Orange Staining ◽  
Induced Apoptosis

e15515 Background: Several tyrosine kinase inhibitors (TKIs), have been developed and approved for clinical use in multi-targeted cancer therapy. Among these, sorafenib is an orally available multikinase inhibitor approved for the treatment of the advanced renal cell carcinoma (RCC) and hepatocellular carcinoma (HCC). Aim of our study was to evaluate the mechanisms responsible for the cytotoxic effects induced by in vitro use of µM doses of sorafenib in 5637 and J82 bladder cancer (BC) cell lines. Methods: The viability of BC cell lines were tested by MTT assay. Autophagy was evaluated by western blot analysis with the anti-LC3 and anti-p62 antibodies, acridine orange staining and cytofluorimetric analysis. Apoptosis, (ΔΨm) dissipation and ROS generation were determined by Annexin-V/PI, JC-1 and DCFDA staining, respectively and cytofluorimetric analysis. The cathepsin B activation was evaluated by western blot using an anti-cathepsin B antibody; the cathepsin B proteolytic activity was determined using the fluorogenic Z-Arg-Arg-AMC peptide and the fluorescence of the hydrolyzed 7-amino-4-methyl-coumarin was detected by a SpectraMax Gemini XPS microplate reader. Results: We found that sorafenib markedly reduced at µM dose the viability of BC cells. Treatment for 24h with 20µM of sorafenib, triggered “Incomplete autophagy”, that induced apoptosis of BC cells. Sorafenib by inducing an increased cathepsin B activity and pro-apoptotic protein BID activation, triggered a ROS-mediated-mitochondrial-dependent apoptosis of BC cells. Moreover, the increase of cathepsin B activity induced by sorafenib was inhibited by a specific tyrosine phosphatase inhibitor (e.g., orthovanadate) strongly suggesting for a contribute of tyrosine-phosphatases in sorafenib-induced apoptosis. Conclusions: Sorafenib by triggering incomplete autophagy, stimulates a cathepsin B-induced-BID-mediated-ROS- and mitochondrial-dependent apoptosis of BC cells, which is likely regulated by tyrosine-phosphatases.

scholarly journals Porphyromonas gingivalis Tyrosine Phosphatase Php1 Promotes Community Development and Pathogenicity

mBio ◽  
2019 ◽  
Vol 10 (5) ◽  
Author(s):  
Young-Jung Jung ◽  
Daniel P. Miller ◽  
John D. Perpich ◽  
Zackary R. Fitzsimonds ◽  
Daonan Shen ◽  
...  
Keyword(s):  
Community Development ◽  
Phosphatase Activity ◽  
Tyrosine Phosphorylation ◽  
Porphyromonas Gingivalis ◽  
Tyrosine Phosphatase ◽  
Protein Tyrosine Phosphorylation ◽  
Tyrosine Phosphatases ◽  
Content Type ◽  
Protein Tyrosine

ABSTRACT Protein-tyrosine phosphorylation in bacteria plays a significant role in multiple cellular functions, including those related to community development and virulence. Metal-dependent protein tyrosine phosphatases that belong to the polymerase and histindinol phosphatase (PHP) family are widespread in Gram-positive bacteria. Here, we show that Porphyromonas gingivalis, a Gram-negative periodontal pathogen, expresses a PHP protein, Php1, with divalent metal ion-dependent tyrosine phosphatase activity. Php1 tyrosine phosphatase activity was attenuated by mutation of conserved histidine residues that are important for the coordination of metal ions and by mutation of a conserved arginine residue, a key residue for catalysis in other bacterial PHPs. The php1 gene is located immediately downstream of the gene encoding the bacterial tyrosine (BY) kinase Ptk1, which was a substrate for Php1 in vitro. Php1 rapidly caused the conversion of Ptk1 to a state of low tyrosine phosphorylation in the absence of discernible intermediate phosphoforms. Active Php1 was required for P. gingivalis exopolysaccharide production and for community development with the antecedent oral biofilm constituent Streptococcus gordonii under nutrient-depleted conditions. In contrast, the absence of Php1 had no effect on the ability of P. gingivalis to form monospecies biofilms. In vitro, Php1 enzymatic activity was resistant to the effects of the streptococcal secreted metabolites pABA and H2O2, which inhibited Ltp1, an enzyme in the low-molecular-weight (LMW) phosphotyrosine phosphatase family. Ptk1 reciprocally phosphorylated Php1 on tyrosine residues 159 and 161, which independently impacted phosphatase activity. Loss of Php1 rendered P. gingivalis nonvirulent in an animal model of periodontal disease. Collectively, these results demonstrate that P. gingivalis possesses active PHP and LMW tyrosine phosphatases, a unique configuration in Gram-negatives which may allow P. gingivalis to maintain phosphorylation/dephosphorylation homeostasis in multispecies communities. Moreover, Php1 contributes to the pathogenic potential of the organism. IMPORTANCE Periodontal diseases are among the most common infections of humans and are also associated with systemic inflammatory conditions. Colonization and pathogenicity of P. gingivalis are regulated by signal transduction pathways based on protein tyrosine phosphorylation and dephosphorylation. Here, we identify and characterize a novel component of the tyrosine (de)phosphorylation axis: a polymerase and histindinol phosphatase (PHP) family enzyme. This tyrosine phosphatase, designated Php1, was required for P. gingivalis community development with other oral bacteria, and in the absence of Php1 activity P. gingivalis was unable to cause disease in a mouse model of periodontitis. This work provides significant insights into the protein tyrosine (de)phosphorylation network in P. gingivalis, its adaptation to heterotypic communities, and its contribution to colonization and virulence.

scholarly journals CD45 and the immune response.

1993 ◽  
Vol 4 (4) ◽  
pp. 976-985 ◽  
Author(s):  
J A Donovan ◽  
G A Koretzky
Keyword(s):  
Signal Transduction ◽  
Amino Acids ◽  
Immune System ◽  
Single Gene ◽  
Tyrosine Phosphatase ◽  
Consensus Sequence ◽  
Extracellular Domain ◽  
In Vitro Assays

CD45 is a major transmembrane glycoprotein expressed on all nucleated hematopoietic cells. Eight isoforms of CD45 are distributed through the immune system according to cell type and degree of cellular differentiation. Heterogeneity among the isoforms is found entirely in the extracellular domain, arising from the differential splicing of up to four exons of a single gene. The control of isoform expression suggests that the extracellular domain may participate in protein-protein interactions with isoform-specific ligands. The intracellular domain of CD45 is large (approximately 700 amino acids), identical for all isoforms, and highly conserved across species. Two nonidentical intracellular sequences of about 240 amino acids that are homologous with a tyrosine phosphatase consensus sequence have been identified. Studies with purified CD45 have shown that all isoforms possess enzymatic activity in in vitro assays. In several T and B cell lines and in natural killer cells, it appears that CD45 is required for optimal signal transduction after stimulation through a number of surface receptors. Although an in vivo substrate has not been identified conclusively, one model suggests that CD45 functions to dephosphorylate a negative-regulatory tyrosine residue on one or more protein tyrosine kinases involved in receptor-mediated second messenger formation. In T cells, the src family kinases, lck and fyn, are candidates for this regulated kinase. In this review, some of the structural and functional aspects of CD45 and its role in signal transduction in the immune system are discussed.

scholarly journals Bioactive Properties and Metabolite Profiles of Endolichenic Fungi in Mangrove Ecosystem of Negombo Lagoon, Sri Lanka

2021 ◽  
Vol 16 (10) ◽  
pp. 1934578X2110486
Author(s):  
Ramani H. Weerasinghe ◽  
Kasun Maduranga ◽  
Renuka N. Attanayake ◽  
Chaitrali Shevkar ◽  
Abhijeet S. Kate ◽  
...  
Keyword(s):  
Sri Lanka ◽  
Radical Scavenging Activity ◽  
Bacterial Species ◽  
Radical Scavenging ◽  
Mangrove Ecosystem ◽  
In Vitro Assays ◽  
Curvularia Lunata ◽  
Endolichenic Fungi ◽  
Anti Inflammatory

Endolichenic fungi (ELF) serve as a novel source of natural products with distinctive bioactivities. A total of 58 ELF isolated from 29 lichens collected from mangrove and mangrove-associated plants of Negombo lagoon, Sri Lanka were identified using morphological and DNA barcoding techniques. Ethyl acetate extracts of 18 such identified ELF isolates were subjected to in vitro assays to determine antioxidant, anti-inflammatory, tyrosinase inhibitory and antibacterial potency. Liquid chromatography–mass spectrometry (LC–MS) dereplication was conducted on the crude extracts in order to detect the secondary metabolites present. The extracts of Daldinia eschscholtzii and Hypoxylon lividipigmentum had the highest radical scavenging activity with SC50 values 14.27 ± 0.24 µg/mL and 18.34 ± 1.37 µg/mL, respectively. D. eschscholtzii also exhibited remarkable anti-inflammatory activity (IC50 7.97 ± 0.09 µg/mL). Tyrosinase inhibitory activity was highest in Cytospora xylocarpi (IC50 68.50 ± 0.34 µg/mL), while the highest activity against aerobic bacterial species Escherichia coli, Bacillus subtilis, Staphylococcus aureus and the anaerobic bacterial strain Streptococcus mutans was observed in the extracts of Xylaria feegenesis and Curvularia lunata. After a thorough study of the LC–MS profiles, it was found that the chemical profiles of Neofusicoccum occulatum, H. lividipigmentum and Myramaececium rubricosum were previously poorly explored in the literature.

Activité antimicrobienne d'antioxydants phénoliques

1978 ◽  
Vol 24 (11) ◽  
pp. 1306-1320 ◽  
Author(s):  
Pierre Turcotte ◽  
Samir A. Saheb
Keyword(s):  
Culture Media ◽  
Osmotic Shock ◽  
Bacterial Species ◽  
Butylated Hydroxytoluene ◽  
In Vitro Assays ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
The Family ◽  
Increased Sensitivity

The antimicrobial activity of three antioxydants, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA) and ethoxyquin (ETO) was studied. In vitro assays showed that when these antioxydants are added to the culture media at concentrations lower or equal to that used in nutrition, they inhibit or decrease the growth of certain microorganisms. BHT showed the most marked effect, affecting Gram-positive bacteria at a higher degree than the Gram-negative bacteria belonging to the family Enterobacteriaceae. Inactivation study of different bacterial species by BHT revealed differences in sensitivity among a single genus and between strains of the same species. The association of ETO with BHT results in an increase of the inhibitory activity. The increased sensitivity to BHT resulting from the osmotic shock of Escherichia coli cells suggests that the resistance to BHT of the Gram-negative bacteria belonging to the family Enterobacteriaceae might be due in part to the structure of their cell wall.

scholarly journals 1341. Development of a Series of High-Throughput Screens to Identify Leads for Nontuberculous Mycobacteria Drug Design

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S485-S485
Author(s):  
Sarah McGuffin ◽  
Steven Mullen ◽  
Julie Early ◽  
Tanya Parish
Keyword(s):  
Drug Discovery ◽  
Drug Development ◽  
High Throughput ◽  
Nontuberculous Mycobacteria ◽  
Human Infection ◽  
Attrition Rate ◽  
In Vitro Assays ◽  
Vitro Assay

Abstract Background Nontuberculous mycobacteria (NTM), particularly Mycobacterium avium complex and Mycobacterium abscessus complex, cause significant morbidity and mortality in patients with impaired host immunity or pre-existing structural lung conditions. NTM infections are increasing at an alarming rate worldwide and there is a dearth of progress in regard to the development of efficacious and tolerable drugs to treat such infections. Traditional drug discovery screens do not account for the diverse physiological conditions, microenvironments, and compartments that the bacilli encounter during human infection. In order to help populate the NTM drug pipeline, and explore the disconnect between in vitro activity, in vivo activity, and clinical outcomes, we are developing a high throughput in vitro assay platform that will more closely model the unique infection-relevant conditions encountered by NTM. Methods We are developing and validating a suite of in vitro assays that screen compounds for activity against extracellular planktonic bacteria, extracellular bacteria within biofilms, intracellular bacteria, and nutrient-starved non-replicating bacteria. Results We are using both the smooth and rough morphotypes of M. abscessus and M. avium. We have validated high throughput assays to pharmaceutical standards for replicating and non-replicating M. abscessus. We have also tested a panel of 18 known anti-mycobacterial compounds. Assay development is currently underway to test compounds for activity against NTM in biofilm and inside macrophages as well. Conclusion To enhance hit identification for scaffolds to use as starting points for NTM drug development, focused libraries of compounds that have undergone significant preclinical profiling and/or compounds with known activity against M. tuberculosis (TB) will be screened. Such a “piggyback” approach usurps advances made in TB drug development and leverages them for NTM drug discovery. This will help expedite novel drug development, reduce attrition rate, and offer a shorter route to clinical use as it exploits the prior investment in medicinal chemistry, pharmacology, and toxicology. Disclosures All authors: No reported disclosures.

scholarly journals CD45 and RPTPα display different protein tyrosine phosphatase activities in T lymphocytes

1997 ◽  
Vol 327 (3) ◽  
pp. 867-876 ◽  
Author(s):  
H. W. David NG ◽  
D. Mojgan JABALI ◽  
Arpita MAITI ◽  
Peter BORODCHAK ◽  
W. Kenneth HARDER ◽  
...  
Keyword(s):  
T Cells ◽  
Tyrosine Phosphatase ◽  
Protein Tyrosine Phosphatases ◽  
Cell Receptor ◽  
Artificial Substrates ◽  
Receptor Protein ◽  
Tyrosine Phosphatases ◽  
Recombinant Enzymes ◽  
Protein Tyrosine

To examine the substrate specificity and function of two receptor protein tyrosine phosphatases, CD45 and RPTPα, RPTPα was expressed in a CD45-, T-cell receptor (TCR)+, BW5147 T-lymphoma cell. High levels of expression of RPTPα did not fully restore either proximal or distal TCR-mediated signalling events. RPTPα was unable to reconstitute the phosphorylation of CD3ζ and did not increase the expression of the activation marker, CD69, on stimulation with TCR/CD3. RPTPα did not significantly alter the phosphorylation state or kinase activity of two CD45 substrates, p56lck or p59fyn, suggesting that RPTPα does not have the same specificity or function as CD45 in T-cells. Further comparison of the two phosphatases indicated that immunoprecipitated RPTPα was approx. one-seventh to one-tenth as active as CD45 when tested against artificial substrates. This difference in activity was also observed in vitro with purified recombinant enzymes at physiological pH. Additional analysis with Src family phosphopeptides and recombinant p56lck as substrates indicated that CD45 was consistently more active than RPTPα, having both higher Vmax and lower Km values. Thus CD45 is intrinsically a much more active phosphatase than RPTPα, which provides one reason why RPTPα cannot effectively dephosphorylate p56lck and substitute for CD45 in T-cells. This work establishes that these two related protein tyrosine phosphatases are not interchangeable in T-cells and that this is due, at least in part, to quantitative differences in phosphatase activity.

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