Measurement of antimicrobial activity under reducing conditions in a modified radial diffusion assay

Background:: Urine is considered one of the biological fluids in which antimicrobial peptides are secreted or expressed. Cow urine has not been investigated for the presence of these peptides using MALDI-TOF-MS. Objective:: The aim of this study is to isolate, identify and assess the antimicrobial activity of urinary antimicrobial peptides from healthy normal cycling cows. Method:: We analyzed the urine sample using diafiltration, ion exchange chromatography, Reverse Phase High-Performance Liquid Chromatography (RP-HPLC), acid urea polyacrylamide gel electrophoresis (AU-PAGE) coupled with identification through Peptide Mass Fingerprinting (PMF) by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDITOF- MS). The in vitro antimicrobial effects of purified fractions were assessed using Radial Diffusion Assay (RDA) and microtitre broth dilution assay against Gram-positive and Gramnegative bacteria. Results: : Proteins corresponding to the peaks were identified using SWISSPROT protein database. This study revealed constitutive expression of β-Defensin-1 (DEFB1), β-Defensin-4A (DFB4A), Neutrophil Defensin-1 (DEF1), Neutrophil Defensin-3 (DEF3) in cow urine. The identified peptides are cationic antimicrobial peptides of the defensin family. The purified fractions exhibited antimicrobial effects in radial diffusion assay and MIC values in the range of 2.93-29.3 &*#181;M/L. Conclusion:: This study concludes that cow urine, previously unexplored with regard to antimicrobial peptides, would be a promising source of highly potent AMPs and an effective alternative to the resistant antibiotics.

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Radial diffusion assay of lipoamide dehydrogenase and its use to assess riboflavin deficiency

Analytical Biochemistry ◽

10.1016/0003-2697(78)90730-3 ◽

1978 ◽

Vol 89 (1) ◽

pp. 103-109 ◽

Cited By ~ 1

Author(s):

Jason C.H. Shih

Keyword(s):

Radial Diffusion ◽

Riboflavin Deficiency ◽

Lipoamide Dehydrogenase ◽

Radial Diffusion Assay ◽

Diffusion Assay

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Radial diffusion assay of tissue collagenase and its application in evaluation of collagenase inhibitors

Biochimica et Biophysica Acta (BBA) - Enzymology ◽

10.1016/0005-2744(77)90363-1 ◽

1977 ◽

Vol 482 (1) ◽

pp. 159-172 ◽

Cited By ~ 13

Author(s):

J.A. Yankellov ◽

W.B. Wacker ◽

M.M. Schweri

Keyword(s):

Radial Diffusion ◽

Radial Diffusion Assay ◽

Diffusion Assay

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The Measurement of Human Plasma Antiplasmin Activity by Radial Diffusion Assay

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646703 ◽

1978 ◽

Vol 39 (02) ◽

pp. 437-449 ◽

Cited By ~ 1

Author(s):

W A Andes

Keyword(s):

Human Plasma ◽

Antithrombin Iii ◽

Normal Subjects ◽

Radial Diffusion ◽

Physical Methods ◽

Α2 Macroglobulin ◽

Radial Diffusion Assay ◽

Diffusion Assay

SummaryAn assay of human antiplasmins has been developed utilizing radial diffusion of plasma from wells cut in plasmin-enriched, fibrinogen-agarose plates. After diffusion the fibrinogen is clotted. Zones of fibrin protected from background fibrinolysis develop as the result of plasma antiplasmin activity. A pooled plasma standard was taken to contain 100 % antiplasmin activity. Antiplasmin activity of 52 normal subjects varied from 64 to 132 %. Washed platelets contained 1-5 % antiplasmin activity. Using antisera to precipitate individual inhibitors, physical methods of separation, and electrophoresis of plasma in agarose, several different proteins were found to have antiplasmin activity in this assay. Thus, α2-macroglobulin contributed 56%, α1-antitrypsin 20%, antithrombin III 2%, and other proteins 22% of the total antiplasmin activity. 1 ml of whole plasma neutralized 7.0 CTA units of plasmin.

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Evaluation of susceptibility of gram-positive and -negative bacteria to human defensins by using radial diffusion assay.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.40.10.2280 ◽

1996 ◽

Vol 40 (10) ◽

pp. 2280-2284 ◽

Cited By ~ 37

Author(s):

H Takemura ◽

M Kaku ◽

S Kohno ◽

Y Hirakata ◽

H Tanaka ◽

...

Keyword(s):

Bacterial Species ◽

Antibacterial Activities ◽

Radial Diffusion ◽

The Other ◽

Gram Positive ◽

Methicillin Resistant ◽

Rp Hplc ◽

Radial Diffusion Assay ◽

Human Defensins ◽

Diffusion Assay

Defensins are small cationic bactericidal peptides present abundantly in the granules of polymorphonuclear neutrophils (PMNs). Human PMNs contain four defensins termed HNP-1 to HNP-4. We used a new assay system in agar plates, the radial diffusion assay, to evaluate the effects of human defensins against gram-positive and -negative bacteria. A crude mixture of HNP-1, -2, and -3 (crude HNPs) was purified from human PMN extracts by reversed-phase high-pressure liquid chromatography (RP-HPLC). The different components were later separated by RP-HPLC and gel permeation chromatography. We compared the antibacterial activities of purified HNP-1, -2, and -3 against Escherichia coli, Pseudomonas aeruginosa, methicillin-susceptible Staphylococcus aureus, and methicillin-resistant S. aureus strains using the radial diffusion assay. The antibacterial activities of HNP-1 and HNP-2 against all strains tested were similar to those of the crude HNPs, but the activity of HNP-3 was less than those of the other defensins. To quantitate the activities of HNPs against different bacteria, we defined the minimal dose of crude HNPs forming a detectable clear zone around the bacteria as the minimal inhibitory dose (MID) and determined the MIDs for 10 strains of E. coli, 12 strains of P. aeruginosa, 10 strains of methicillin-susceptible S. aureus, and 12 strains of methicillin-resistant S. aureus isolates, including clinical isolates. In general, the MIDs of the HNPs were similar against similar bacterial species. However, the MIDs for P. aeruginosa were higher than those for the other organisms tested. The radial diffusion assay is suitable as a screening test for measuring the susceptibilities of isolates to defensins, because it is sensitive and simple and has good reproducibility.

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Sensitive detection of specific repair endonucleases: Radial diffusion assay utilizing differential alkaline denaturation of supercoiled and nicked PM2 DNA in agarose gels

Analytical Biochemistry ◽

10.1016/0003-2697(81)90596-0 ◽

1981 ◽

Vol 118 (2) ◽

pp. 371-381 ◽

Cited By ~ 3

Author(s):

David L. Hurley ◽

Reginald A. Deering

Keyword(s):

Radial Diffusion ◽

Sensitive Detection ◽

Agarose Gels ◽

Alkaline Denaturation ◽

Radial Diffusion Assay ◽

Diffusion Assay

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Determination of Plant Proteinase Inhibitors by Agar Gel Radial Diffusion Assay 1

Environmental Entomology ◽

10.1093/ee/12.1.255 ◽

1983 ◽

Vol 12 (1) ◽

pp. 255-259 ◽

Cited By ~ 3

Author(s):

R. H. Shukle ◽

L. L. Murdock

Keyword(s):

Proteinase Inhibitors ◽

Radial Diffusion ◽

Agar Gel ◽

Radial Diffusion Assay ◽

Diffusion Assay

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A study of antimicrobial activity of polyphenols derived from wood

Bulletin of Russian State Medical University ◽

10.24075/brsmu.2018.043 ◽

2018 ◽

pp. 46-49 ◽

Cited By ~ 1

Author(s):

A. B. Shevelev ◽

E. P. Isakova ◽

E. V. Trubnikova ◽

N. La Porta ◽

S. Martens ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Pseudomonas Aeruginosa ◽

Antimicrobial Activity ◽

Drug Resistance ◽

Bacterial Infections ◽

In Vivo Studies ◽

Radial Diffusion Assay ◽

Diffusion Assay ◽

Novel Antibiotics

Due to the spreading and increasing drug resistance of pathogens, the search for novel antibiotics is becoming ever more important. Plant-derived polyphenols are a vast and promising class of compounds with a potential to fight infectious diseases. Still, they are not routinely used in clinical practice. No reports on the in vivo studies of these compounds have been presented. The aim of our work was to compare the antimicrobial activity of resveratrol (stilbene), dihydroquercetin and dihydromyricetin (flavonols) extracted from the bark and wood of conifers against the dermatophytes Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans. Using the radial diffusion assay, we established that dihydroquercetin, resveratrol and dihydromyricetin exhibit high activity against S. aureus even at the smallest possible concentrations of 0.22, 0.15, and 0.15 mM, respectively. In contrast, the highest achievable concentrations of these compounds in the solutions (21.5, 15.5 and 15.0 mM for dihydroquercetin, resveratrol and dihydromyricetin, respectively) have no effect on the growth of P. aeruginosa and C. albicans. These findings suggest that polyphenols derived from conifers could have a potential to be used as a medicine for topical application to treat bacterial infections of the skin caused by S. aureus.

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