Application of Capillary and Free-Flow Zone Electrophoresis for Analysis and Purification of Antimicrobial β-Alanyl-tyrosine from Hemolymph of Fleshfly Neobellieria bullata

The problem of a growing resistance of bacteria and other microorganisms to conventional antibiotics gave rise to a search for new potent antimicrobial agents. Insect antimicrobial peptides (AMPs) seem to be promising novel potential anti-infective therapeutics. The dipeptide β-alanyl-tyrosine (β-Ala-Tyr) is one of the endogenous insect toxins exhibiting antibacterial activity against both Gram-negative and Gram-positive bacteria. Prior to testing its other antimicrobial activities, it has to be prepared in a pure form. In this study, we have developed a capillary zone electrophoresis (CZE) method for analysis of β‑Ala‑Tyr isolated from the extract of the hemolymph of larvae of the fleshfly Neobellieria bullata by reversed-phase high-performance liquid chromatography (RP-HPLC). Based on our previously described correlation between CZE and free-flow zone electrophoresis (FFZE), analytical CZE separation of β‑Ala‑Tyr and its admixtures have been converted into preparative purification of β‑Ala‑Tyr by FFZE with preparative capacity of 45.5 mg per hour. The high purity degree of the β‑Ala‑Tyr obtained by FFZE fractionation was confirmed by its subsequent CZE analysis.

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The Design of Alapropoginine, a Novel Conjugated Ultrashort Antimicrobial Peptide with Potent Synergistic Antimicrobial Activity in Combination with Conventional Antibiotics

Antibiotics ◽

10.3390/antibiotics10060712 ◽

2021 ◽

Vol 10 (6) ◽

pp. 712

Author(s):

Ali Salama ◽

Ammar Almaaytah ◽

Rula M. Darwish

Keyword(s):

Antimicrobial Activity ◽

Hemolytic Activity ◽

Antimicrobial Agents ◽

Antimicrobial Activities ◽

Resistant Bacteria ◽

Human Red Blood Cells ◽

Drug Resistant Bacteria ◽

Different Strains ◽

Conventional Antibiotics

(1) Background: Antimicrobial resistance represents an urgent health dilemma facing the global human population. The development of novel antimicrobial agents is needed to face the rising number of resistant bacteria. Ultrashort antimicrobial peptides (USAMPs) are considered promising antimicrobial agents that meet the required criteria of novel antimicrobial drug development. (2) Methods: Alapropoginine was rationally designed by incorporating arginine (R), biphenylalanine (B), and naproxen to create an ultrashort hexapeptide. The antimicrobial activity of alapropoginine was evaluated against different strains of bacteria. The hemolytic activity of alapropoginine was also investigated against human erythrocytes. Finally, synergistic studies with antibiotics were performed using the checkerboard technique and the determination of the fractional inhibitory index. (3) Results: Alapropoginine displayed potent antimicrobial activities against reference and multi-drug-resistant bacteria with MIC values of as low as 28.6 µg/mL against methicillin-resistant S. aureus. Alapropoginine caused negligible toxicity toward human red blood cells. Moreover, the synergistic studies showed improved activities for the combined conventional antibiotics with a huge reduction in their antimicrobial concentrations. (4) Conclusions: The present study indicates that alapropoginine exhibits promising antimicrobial activity against reference and resistant strains of bacteria with negligible hemolytic activity. Additionally, the peptide displays synergistic or additive effects when combined with several antibiotics.

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Loxosceles gaucho Spider Venom: An Untapped Source of Antimicrobial Agents

Toxins ◽

10.3390/toxins10120522 ◽

2018 ◽

Vol 10 (12) ◽

pp. 522 ◽

Cited By ~ 5

Author(s):

Paula Segura-Ramírez ◽

Pedro Silva Júnior

Keyword(s):

Bacterial Infections ◽

High Performance ◽

Antimicrobial Agents ◽

Chemical Properties ◽

Carcinoma Cells ◽

Promising Candidate ◽

Human Red Blood Cells ◽

New Antibiotics ◽

Conventional Antibiotics ◽

Human Cervical Carcinoma Cells

The remarkable ability of microorganisms to develop resistance to conventional antibiotics is one of the biggest challenges that the pharmaceutical industry currently faces. Recent studies suggest that antimicrobial peptides discovered in spider venoms may be useful resources for the design of structurally new anti-infective agents effective against drug-resistant microorganisms. In this work, we found an anionic antibacterial peptide named U1-SCRTX-Lg1a in the venom of the spider Loxosceles gaucho. The peptide was purified using high-performance liquid chromatography (HPLC), its antimicrobial activity was tested through liquid growth inhibition assays, and its chemical properties were characterized using mass spectrometry. U1-SCRTX-Lg1a was found to show a monoisotopic mass of 1695.75 Da, activity against Gram-negative bacteria, a lack of hemolytic effects against human red blood cells, and a lack of cytotoxicity against human cervical carcinoma cells (HeLa). Besides this, the sequence of the peptide exhibited great similarity to specific regions of phospholipases D from different species of Loxosceles spiders, leading to the hypothesis that U1-SCRTX-Lg1a may have originated from a limited proteolytic cleavage. Our data suggest that U1-SCRTX-Lg1a is a promising candidate for the development of new antibiotics that could help fight bacterial infections and represents an exciting discovery for Loxosceles spiders.

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Preparation and Hydro-Lipophilic Properties of Selected Novel Chlorinated and Brominated N-Arylcinnamamides

Proceedings ◽

10.3390/ecsoc-23-06595 ◽

2019 ◽

Vol 41 (1) ◽

pp. 11

Author(s):

Tomas Strharsky ◽

Timotej Jankech ◽

Jiri Kos ◽

Kristina Maricakova ◽

Andrea Pramukova ◽

...

Keyword(s):

High Performance ◽

Chemical Structure ◽

Antimicrobial Agents ◽

Reversed Phase ◽

Log P ◽

Organic Modifier ◽

Chromatography Method ◽

P Values ◽

Liquid Chromatography Method ◽

Derivatives Of

A series of six di- and tri-halogenated N-arylcinnamanilides designed as anti-inflammatory and antimicrobial agents was prepared and characterized. Since it is known that lipophilicity significantly influences the biological activity of compounds, the hydro-lipophilic properties of these di- and tri-substituted N-arylcinnamanilides were investigated in the study. All the discussed derivatives of cinnamic acid were analyzed using the reversed-phase high performance liquid chromatography method to measure lipophilicity. The procedure was performed under isocratic conditions with methanol as an organic modifier in the mobile phase using an end-capped non-polar C18 stationary reversed-phase column. In the present study, the correlations between the logarithm of the capacity factor k and log P/Clog P values calculated in various ways as well as the relationships between the lipophilicity and the chemical structure of the studied compounds are discussed.

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Simultaneous analysis of three antimicrobial agents in feed premixes by reversed-phase high-performance liquid chromatography

Journal of Chromatography A ◽

10.1016/s0021-9673(01)90412-1 ◽

1985 ◽

Vol 323 (2) ◽

pp. 447-450 ◽

Cited By ~ 4

Author(s):

J. Torel ◽

J. Cillard ◽

P. Cillard ◽

M. Vie

Keyword(s):

High Performance Liquid Chromatography ◽

Liquid Chromatography ◽

High Performance ◽

Antimicrobial Agents ◽

Reversed Phase ◽

Simultaneous Analysis

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Haemoglobinopathies that occur with decreased HbA2 levels: a gene mutation set involving the δ gene at a Spanish centre

Journal of Clinical Pathology ◽

10.1136/jclinpath-2016-203879 ◽

2016 ◽

Vol 70 (1) ◽

pp. 75-80 ◽

Cited By ~ 2

Author(s):

Ana Villegas ◽

Fernando Ataúlfo González ◽

Jorge M Nieto ◽

Félix de la Fuente-Gonzalo ◽

Rafael Martínez ◽

...

Keyword(s):

High Performance ◽

Globin Gene ◽

Reversed Phase ◽

Globin Genes ◽

Reversed Phase Hplc ◽

Structural Variants ◽

Globin Chains ◽

Zone Electrophoresis ◽

Essential Parameter ◽

Capillary Zone

AimsHaemoglobin A2 (HbA2) consists of two globin chains, α and β. Alterations in any of these genes influences the level of HbA2. Here, we present cases of structural Hb variants and thalassaemias which present either alone or together and reduce the level of HbA2 at varying degrees. Furthermore, we present a novel structural mutation in the δ globin gene, called Hb A2-Madrid.MethodsThe levels of HbA2 and HbF and the different haemoglobin variants were measured and analysed by ion exchange high performance liquid chromatography (HPLC, VARIANT II), the types of haemoglobins were determined by capillary zone electrophoresis (CZE) (Sebia) and the globin chains were determined by reversed-phase HPLC. Genetic analysis was performed by automatic sequencing of the α and δ genes as well as by multiple PCRs for the α globin genes.ResultsIn α thalassaemia (n=94), the HbA2 levels ranged from 1.39% to 2.43%. Among individuals with δ thalassaemia (n=5), the HbA2 level of those with δ+ thalassaemia was 1.77%, and that of those with δ0 thalassaemia was 1.70%. Among the individuals with δβ thalassaemia (n=13), those who were homozygous lacked HbA2. All structural haemoglobinopathies (n=97) were heterozygous; the α chain variants (n=84) presented with an HbA2 level of 1.76%, while the δ chain variants (n=13) presented with a level of 1.75%.ConclusionHbA2 is an essential parameter in the diagnostics of haemoglobinopathies. HPLC-EC and CZE allow the quantification of HbA2. Here, we show that quantification of HbA2 is critical for the identification of α, δ and βδ thalassaemias. Structural variants are discovered by HPLC. Molecular genetics is required for the proper identification of the mutations. Only with this knowledge is genetic counselling possible.

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