Cationic Phenosafranin Photosensitizers Based on Polyhedral Oligomeric Silsesquioxanes for Inactivation of Gram-Positive and Gram-Negative Bacteria

The high photodynamic effect of the Newman strain of the S. aureus and of clinical strains of S. aureus MRSA 12673 and E. coli 12519 are observed for new cationic light-activated phenosafranin polyhedral oligomeric silsesquioxane (POSS) conjugates in vitro. Killing of bacteria was achieved at low concentrations of silsesquioxanes (0.38 µM) after light irradiation (λem. max = 522 nm, 10.6 mW/cm2) for 5 min. Water-soluble POSS-photosensitizers are synthesized by chemically coupling a phenosafranin dye (PSF) (3,7-diamino-5-phenylphenazine chloride) to an inorganic silsesquioxane cage activated by attachment of succinic anhydride rings. The chemical structure of conjugates is confirmed by 1H, 13C NMR, HRMS, IR, fluorescence spectroscopy and UV-VIS analyzes. The APDI and daunorubicin (DAU) synergy is investigated for POSSPSFDAU conjugates. Confocal microscopy experiments indicate a site of intracellular accumulation of the POSSPSF, whereas iBuPOSSPSF and POSSPSFDAU accumulate in the cell wall or cell membrane. Results from the TEM study show ruptured S. aureus cells with leaking cytosolic mass and distorted cells of E. coli. Bacterial cells are eradicated by ROS produced upon irradiation of the covalent conjugates that can kill the bacteria by destruction of cellular membranes, intracellular proteins and DNA through the oxidative damage of bacteria.

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The effect of a benzodiazepine, flurazepam, on the response of in vitro skeletal muscle preparations to muscle relaxants: are purines or their receptors involved?

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y82-124 ◽

1982 ◽

Vol 60 (7) ◽

pp. 877-884 ◽

Cited By ~ 10

Author(s):

John T. Hamilton ◽

Peggy A. Stone

Keyword(s):

Skeletal Muscle ◽

Phrenic Nerve ◽

Neuromuscular Blocking ◽

Muscle Relaxants ◽

Water Soluble ◽

Neuromuscular Activity ◽

Changing Trends ◽

Low Concentrations ◽

Partial Blockade

Changing trends in the use of anxiolytic agents and recent reassessment of their neuropharmacological activity has prompted this evaluation of the peripheral neuromuscular activity of the benzodiazepine, flurazepam. In previous reports we have documented peripheral neuromuscular activity of chlordiazepoxide and diazepam on the rat phrenic nerve diaphragm preparation. The water soluble benzodiazepine, flurazepam, has been studied on the rat phrenic nerve diaphragm and frog rectus abdominis in vitro. On the former preparation flurazepam enhanced and then blocked the response to indirect electrical stimulation (0.2 Hz) and readily blocked posttetanic potentiation and prevented the preparation from sustaining a tetanic contracture (30 Hz). On the later preparation, flurazepam blocked in a noncompetitive manner the response of the frog muscle to applied cholinergic agonists. Studies on the rat preparation with the neuromuscular blocking drug succinylcholine have shown an unexpected protection against blockade in preparations pretreated with low concentrations of flurazepam. This was not observed when flurazepam was given prior to d-tubocurarinc. The application of adenosine to rat diaphragms during steady-state partial blockade caused by flurazepam or d-tubocurarine showed an inhibiting action of adenosine which was reversed by theophylline. Pretreatment of rat preparations with dipyridamole significantly enhanced the blocking action of standard concentrations of succinylcholine.These results, along with those in the literature, encourage a reassessment of the action of purines and benzodiazepines on skeletal muscle and encourage a consideration of a possible involvement of purinergic neuromodulation of transmission which is unmasked when the safety factor for transmission is altered by muscle relaxants. The possible clinical significance of protection against succinylcholine by benzodiazepines is noted.

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The Escherichia coli cysG promoter belongs to the ‘extended −10’ class of bacterial promoters

Biochemical Journal ◽

10.1042/bj2960851 ◽

1993 ◽

Vol 296 (3) ◽

pp. 851-857 ◽

Cited By ~ 23

Author(s):

T Belyaeva ◽

L Griffiths ◽

S Minchin ◽

J Cole ◽

S Busby

Keyword(s):

Escherichia Coli ◽

Point Mutations ◽

Growth Conditions ◽

Upstream Region ◽

E Coli ◽

Run Off ◽

A Site ◽

Specific Sequences

The Escherichia coli cysG promoter has been subcloned and shown to function constitutively in a range of different growth conditions. Point mutations identify the -10 hexamer and an important 5′-TGN-3′ motif immediately upstream. The effects of different deletions suggest that specific sequences in the -35 region are not essential for the activity of this promoter in vivo. This conclusion was confirmed by in vitro run-off transcription assays. The DNAase I footprint of RNA polymerase at the cysG promoter reveals extended protection upstream of the transcript start, and studies with potassium permanganate as a probe suggest that the upstream region is distorted in open complexes. Taken together, the results show that the cysG promoter belongs to the ‘extended -10’ class of promoters, and the base sequence is similar to that of the P1 promoter of the E. coli galactose operon, another promoter in this class. In vivo, messenger initiated at the cysG promoter appears to be processed by cleavage at a site 41 bases downstream from the transcript start point.

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STUDY OF ANTIBACTERIAL ACTIVITY OF THE PREPARATIONS WITH DIOXIDINE AND LEVOFLOXACIN ON MAIN PURULENT-INFLAMMATORY PROCESSES PATHOGENS

Wiadomości Lekarskie ◽

10.36740/wlek202109115 ◽

2021 ◽

Vol 74 (9) ◽

pp. 2109-2111

Author(s):

Evheniia A. Shtaniuk ◽

Oleksandra O. Vovk ◽

Larisa V. Krasnikova ◽

Yuliia I. Polyvianna ◽

Tetiana I. Kovalenko

Keyword(s):

Antibacterial Activity ◽

Water Soluble ◽

Diffusion Method ◽

E Coli ◽

Mueller Hinton ◽

Microbiological Methods ◽

Pure Cultures ◽

Inflammatory Processes ◽

Definition Of

The aim: Study of antibacterial activity of the preparations, containing antiseptic dioxidine and antibiotic levofloxacin in vitro on standard strains of main optional-anaerobic pathogens of purulent-inflammatory processes of surgical wounds S. aureus, E. coli, P. aeruginosa and definition of more effective ones on them. Materials and methods: Solutions of dioxidine 1.2 %, dioxidine 1.2% with decamethaxin, Dioxisole, water soluble ointment with dioxidine 1.2% and levofloxacin 0.1% with decamethaxin were used in experiment. Antibacterial activity was studied on standard strains of S. aureus АТСС 25923, E. coli АТСС 25922, P. aeruginosa АТСС 27853. Distinguishing and identification of pure cultures of bacteria was done according to generally accepted microbiological methods. Determination of purulent-inflammatory processes pathogens sensitivity was done by disco-diffuse method on Mueller-Hinton medium. Antibacterial activity of solutions and ointments was studied with the help of agar diffusion method (“well” method) according to methodic recommendations. Each investigation was repeated 6 times. Method of variation statistics was used for the research results analysis. Results: All antibacterial preparations under study are effective and highly effective on S. aureus АТСС 25923, E. coli АТСС 25922, P. aeruginosa АТСС 27853. Solution with 1.2 % dioxidine with decamethaxin and ointment with 0.1 % levofloxacin and decamethaxin have larger growth retardation zones towards S. aureus and P. aeruginosa. E. coli strains are more sensitive to the solution of Dioxisole and ointment with 1.2 % dioxidine. Conclusions: All strains are sensitive, most of them are highly sensitive, up to 5 antibacterial preparations under study in vitro.

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Microencapsulation of Enteric Bacteriophages in a pH-Responsive Solid Oral Dosage Formulation Using a Scalable Membrane Emulsification Process

Pharmaceutics ◽

10.3390/pharmaceutics11090475 ◽

2019 ◽

Vol 11 (9) ◽

pp. 475 ◽

Cited By ~ 6

Author(s):

Vinner ◽

Richards ◽

Leppanen ◽

Sagona ◽

Malik

Keyword(s):

Epithelial Cells ◽

Prolonged Exposure ◽

Oral Dosage ◽

Bacterial Cells ◽

Ph Responsive ◽

Ion Milling ◽

Membrane Emulsification ◽

E Coli ◽

Emulsification Process

A scalable low-shear membrane emulsification process was used to produce microencapsulated Escherichia coli-phages in a solid oral dosage form. Uniform pH-responsive composite microparticles (mean size ~100 µm) composed of Eudragit® S100 and alginate were produced. The internal microstructure of the gelled microcapsules was studied using ion-milling and imaging, which showed that the microparticles had a solid internal core. The microencapsulation process significantly protected phages upon prolonged exposure to a simulated gastric acidic environment. Encapsulated phages that had been pre-exposed to simulated gastric acid were added to actively growing bacterial cells using in vitro cell cultures and were found to be effective in killing E. coli. Encapsulated phages were also shown to be effective in killing actively growing E. coli in the presence of human epithelial cells. Confocal microscopy images showed that the morphology of encapsulated phage-treated epithelial cells was considerably better than controls without phage treatment. The encapsulated phages were stable during refrigerated storage over a four-week period. The process of membrane emulsification is highly scalable and is a promising route to produce industrial quantities of pH-responsive oral solid dosage forms suitable for delivering high titres of viable phages to the gastrointestinal tract.

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Np4A alarmones function in bacteria as precursors to RNA caps

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1914229117 ◽

2020 ◽

Vol 117 (7) ◽

pp. 3560-3567 ◽

Cited By ~ 6

Author(s):

Daniel J. Luciano ◽

Joel G. Belasco

Keyword(s):

Rna Polymerase ◽

Transcription Initiation ◽

Promoter Sequence ◽

Rna Degradation ◽

Initiation Site ◽

Bacterial Cells ◽

E Coli ◽

N Incorporation ◽

Nascent Transcripts

Stresses that increase the cellular concentration of dinucleoside tetraphosphates (Np4Ns) have recently been shown to impact RNA degradation by inducing nucleoside tetraphosphate (Np4) capping of bacterial transcripts. However, neither the mechanism by which such caps are acquired nor the function of Np4Ns in bacteria is known. Here we report that promoter sequence changes upstream of the site of transcription initiation similarly affect both the efficiency with which Escherichia coli RNA polymerase incorporates dinucleoside polyphosphates at the 5′ end of nascent transcripts in vitro and the percentage of transcripts that are Np4-capped in E. coli, clear evidence for Np4 cap acquisition by Np4N incorporation during transcription initiation in bacterial cells. E. coli RNA polymerase initiates transcription more efficiently with Np4As than with ATP, particularly when the coding strand nucleotide that immediately precedes the initiation site is a purine. Together, these findings indicate that Np4Ns function in bacteria as precursors to Np4 caps and that RNA polymerase has evolved a predilection for synthesizing capped RNA whenever such precursors are abundant.

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Inactivation of the Elongation Factor Tu by Mosquitocidal Toxin-Catalyzed Mono-ADP-Ribosylation

Applied and Environmental Microbiology ◽

10.1128/aem.68.10.4894-4899.2002 ◽

2002 ◽

Vol 68 (10) ◽

pp. 4894-4899 ◽

Cited By ~ 24

Author(s):

Jörg Schirmer ◽

Hans-Joachim Wieden ◽

Marina V. Rodnina ◽

Klaus Aktories

Keyword(s):

Peptide Mapping ◽

Elongation Factor ◽

Bacillus Sphaericus ◽

Elongation Factor Tu ◽

Bacterial Cells ◽

Single Chain ◽

E Coli ◽

Adp Ribosylation ◽

Flight Mass Spectrometry

ABSTRACT The mosquitocidal toxin (MTX) produced by Bacillus sphaericus strain SSII-1 is an ∼97-kDa single-chain toxin which contains a 27-kDa enzyme domain harboring ADP-ribosyltransferase activity and a 70-kDa putative binding domain. Due to cytotoxicity toward bacterial cells, the 27-kDa enzyme fragment cannot be produced in Escherichia coli expression systems. However, a nontoxic 32-kDa N-terminal truncation of MTX can be expressed in E. coli and subsequently cleaved to an active 27-kDa enzyme fragment. In vitro the 27-kDa enzyme fragment of MTX ADP-ribosylated numerous proteins in E. coli lysates, with dominant labeling of an ∼45-kDa protein. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry combined with peptide mapping identified this protein as the E. coli elongation factor Tu (EF-Tu). ADP ribosylation of purified EF-Tu prevented the formation of the stable ternary EF-Tuaminoacyl-tRNAGTP complex, whereas the binding of GTP to EF-Tu was not altered. The inactivation of EF-Tu by MTX-mediated ADP-ribosylation and the resulting inhibition of bacterial protein synthesis are likely to play important roles in the cytotoxicity of the 27-kDa enzyme fragment of MTX toward E. coli.

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Novel Plasmid-Mediated 16S rRNA m1A1408 Methyltransferase, NpmA, Found in a Clinically Isolated Escherichia coli Strain Resistant to Structurally Diverse Aminoglycosides

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00926-07 ◽

2007 ◽

Vol 51 (12) ◽

pp. 4401-4409 ◽

Cited By ~ 113

Author(s):

Jun-ichi Wachino ◽

Keigo Shibayama ◽

Hiroshi Kurokawa ◽

Kouji Kimura ◽

Kunikazu Yamane ◽

...

Keyword(s):

Escherichia Coli ◽

16S Rrna ◽

Recombinant Plasmid ◽

Coli Strain ◽

Methyltransferase Activity ◽

Ribosomal Subunits ◽

E Coli ◽

Rrna Molecule ◽

A Site

ABSTRACT We have isolated a multiple-aminoglycoside-resistant Escherichia coli strain, strain ARS3, and have been the first to identify a novel plasmid-mediated 16S rRNA methyltransferase, NpmA. This new enzyme shared a relatively low level of identity (30%) to the chromosomally encoded 16S rRNA methyltransferase (KamA) of Streptomyces tenjimariensis, an actinomycete aminoglycoside producer. The introduction of a recombinant plasmid carrying npmA could confer on E. coli consistent resistance to both 4,6-disubstituted 2-deoxystreptamines, such as amikacin and gentamicin, and 4,5-disubstituted 2-deoxystreptamines, including neomycin and ribostamycin. The histidine-tagged NpmA elucidated methyltransferase activity against 30S ribosomal subunits but not against 50S subunits and the naked 16S rRNA molecule in vitro. We further confirmed that NpmA is an adenine N-1 methyltransferase specific for the A1408 position at the A site of 16S rRNA. Drug footprinting data indicated that binding of aminoglycosides to the target site was apparently interrupted by methylation at the A1408 position. These observations demonstrate that NpmA is a novel plasmid-mediated 16S rRNA methyltransferase that provides a panaminoglycoside-resistant nature through interference with the binding of aminoglycosides toward the A site of 16S rRNA through N-1 methylation at position A1408.

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In vitro activity of water-soluble quaternary chitosan chloride salt against E. coli

World Journal of Microbiology and Biotechnology ◽

10.1007/s11274-010-0378-7 ◽

2010 ◽

Vol 26 (11) ◽

pp. 2089-2092 ◽

Cited By ~ 16

Author(s):

Leandro Prezotto da Silva ◽

Douglas de Britto ◽

Mirna Helena Regali Seleghim ◽

Odilio B. G. Assis

Keyword(s):

Water Soluble ◽

Vitro Activity ◽

Chloride Salt ◽

In Vitro Activity ◽

E Coli

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Conserved mechanism of cell-wall synthase regulation revealed by the identification of a new PBP activator inPseudomonas aeruginosa

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1717925115 ◽

2018 ◽

Vol 115 (12) ◽

pp. 3150-3155 ◽

Cited By ~ 20

Author(s):

Neil G. Greene ◽

Coralie Fumeaux ◽

Thomas G. Bernhardt

Keyword(s):

Cell Wall ◽

Drug Targets ◽

Bacterial Cells ◽

Acinetobacter Baylyi ◽

Gram Negative ◽

E Coli ◽

Outer Membrane Lipoprotein ◽

Synthase Regulation

Penicillin-binding proteins (PBPs) are synthases required to build the essential peptidoglycan (PG) cell wall surrounding most bacterial cells. The mechanisms regulating the activity of these enzymes to control PG synthesis remain surprisingly poorly defined given their status as key antibiotic targets. Several years ago, the outer-membrane lipoproteinEcLpoB was identified as a critical activator ofEscherichia coliPBP1b (EcPBP1b), one of the major PG synthases of this organism. Activation ofEcPBP1b is mediated through the association ofEcLpoB with a regulatory domain onEcPBP1b called UB2H. Notably,Pseudomonas aeruginosaalso encodes PBP1b (PaPBP1b), which possesses a UB2H domain, but this bacterium lacks an identifiable LpoB homolog. We therefore searched for potentialPaPBP1b activators and identified a lipoprotein unrelated to LpoB that is required for the in vivo activity ofPaPBP1b. We named this protein LpoP and found that it interacts directly withPaPBP1b in vitro and is conserved in many Gram-negative species. Importantly, we also demonstrated thatPaLpoP-PaPBP1b as well as an equivalent protein pair fromAcinetobacter baylyican fully substitute forEcLpoB-EcPBP1b inE. colifor PG synthesis. Furthermore, we show that amino acid changes inPaPBP1b that bypass thePaLpoP requirement map to similar locations in the protein as changes promotingEcLpoB bypass inEcPBP1b. Overall, our results indicate that, although different Gram-negative bacteria activate their PBP1b synthases with distinct lipoproteins, they stimulate the activity of these important drug targets using a conserved mechanism.

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Identification of residues required for stalled-ribosome rescue in the codon-independent release factor YaeJ

Nucleic Acids Research ◽

10.1093/nar/gkt1280 ◽

2013 ◽

Vol 42 (5) ◽

pp. 3152-3163 ◽

Cited By ~ 21

Author(s):

Hiroyuki Kogure ◽

Yoshihiro Handa ◽

Masahiro Nagata ◽

Naoto Kanai ◽

Peter Güntert ◽

...

Keyword(s):

Solution Structure ◽

Mutational Analysis ◽

Single Amino Acid ◽

Resonance Spectroscopy ◽

Amino Acid Substitutions ◽

Release Factor ◽

E Coli ◽

Ribosome Binding ◽

A Site

Abstract The YaeJ protein is a codon-independent release factor with peptidyl-tRNA hydrolysis (PTH) activity, and functions as a stalled-ribosome rescue factor in Escherichia coli. To identify residues required for YaeJ function, we performed mutational analysis for in vitro PTH activity towards rescue of ribosomes stalled on a non-stop mRNA, and for ribosome-binding efficiency. We focused on residues conserved among bacterial YaeJ proteins. Additionally, we determined the solution structure of the GGQ domain of YaeJ from E. coli using nuclear magnetic resonance spectroscopy. YaeJ and a human homolog, ICT1, had similar levels of PTH activity, despite various differences in sequence and structure. While no YaeJ-specific residues important for PTH activity occur in the structured GGQ domain, Arg118, Leu119, Lys122, Lys129 and Arg132 in the following C-terminal extension were required for PTH activity. All of these residues are completely conserved among bacteria. The equivalent residues were also found in the C-terminal extension of ICT1, allowing an appropriate sequence alignment between YaeJ and ICT1 proteins from various species. Single amino acid substitutions for each of these residues significantly decreased ribosome-binding efficiency. These biochemical findings provide clues to understanding how YaeJ enters the A-site of stalled ribosomes.

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