scholarly journals Oncorhyncin III: a potent antimicrobial peptide derived from the non-histone chromosomal protein H6 of rainbow trout, Oncorhynchus mykiss

2003 ◽  
Vol 373 (2) ◽  
pp. 621-628 ◽  
Author(s):  
Jorge M. O. FERNANDES ◽  
Nathalie SAINT ◽  
Graham D. KEMP ◽  
Valerie J. SMITH
Keyword(s):  
Rainbow Trout ◽  
Antimicrobial Peptide ◽  
Lipid Bilayers ◽  
Solid Phase ◽  
Reversed Phase ◽  
Exchange Chromatography ◽  
Haemolytic Activity ◽  
Acid Extraction ◽  
Chromosomal Protein ◽  
Bacterial Cells

A 6.7 kDa antimicrobial peptide was isolated from trout skin secretions using acid extraction followed by cation-exchange chromatography, tC18 solid-phase extraction, and C18 reversed-phase HPLC. The molecular mass of this peptide, which is tentatively named oncorhyncin III, is 6671 Da, as determined by matrix-assisted laser-desorption ionization MS. N-terminal amino acid sequencing revealed that the first 13 residues of oncorhyncin III are identical with those of the non-histone chromosomal protein H6 from rainbow trout. Hence these data combined with the MS results indicate that oncorhyncin III is likely to be a cleavage product of the non-histone chromosomal protein H6 (residues 1–66) and that it probably contains two methylated residues or one double methylation. The purified peptide exhibits potent antibacterial activity against both Gram-positive and Gram-negative bacteria, with minimal inhibitory concentrations in the submicromolar range. The peptide is sensitive to NaCl, and displays no haemolytic activity towards trout erythrocytes at concentrations below 1 μM. Scanning electron microscopy revealed that oncorhyncin III does not cause direct disruption of bacterial cells. Reconstitution of the peptide in planar lipid bilayers strongly disturbs the membranes, but does not induce the formation of stable ion channels. Taken together, these results support the hypothesis that oncorhyncin III plays a role in mucosal innate host defence.

Synthesis of modified human C-peptide and its fragments

1988 ◽  
Vol 53 (11) ◽  
pp. 2637-2644 ◽  
Author(s):  
Běla Bendlová ◽  
Michal Lebl ◽  
Pavel Štolba ◽  
Luboslav Stárka
Keyword(s):  
Solid Phase ◽  
Gel Filtration ◽  
Reversed Phase ◽  
Exchange Chromatography ◽  
Phase Method ◽  
Reversed Phase Hplc ◽  
Radioactive Label ◽  
C Peptide ◽  
Solid Phase Method ◽  
Internal Marker

Syntheses of the modified human C-peptide containing residues suitable for the introduction of the radioactive label (tyrosine) and internal marker for monitoring binding to carrier (norvaline) and five of its fragments are described. The syntheses were performed by solid phase method using either 9-fluorenylmethoxycarbonyl or tert-butyloxycarbonyl protecting groups. The products were purified by gel filtration, ion exchange chromatography and reversed phase HPLC. The reactivity of prepared peptides with antisera was determined and the modified C-peptide was found fully reactive.

scholarly journals Anti-microbial properties of histone H2A from skin secretions of rainbow trout, Oncorhynchus mykiss

2002 ◽  
Vol 368 (2) ◽  
pp. 611-620 ◽  
Author(s):  
Jorge M.O. FERNANDES ◽  
Graham D. KEMP ◽  
M. Gerard MOLLE ◽  
Valerie J. SMITH
Keyword(s):  
Rainbow Trout ◽  
Amino Acid ◽  
Reversed Phase ◽  
Bacterial Activity ◽  
Bacterial Invasion ◽  
Haemolytic Activity ◽  
Histone H2a ◽  
Yeast Saccharomyces Cerevisiae ◽  
Microbial Properties ◽  
Peptide Mass

Skin exudates of rainbow trout contain a potent 13.6kDa anti-microbial protein which, from partial internal amino acid sequencing, peptide mass fingerprinting, matrix-associated laser desorption/ionization MS and amino acid analysis, seems to be histone H2A, acetylated at the N-terminus. The protein, purified to homogeneity by ion-exchange and reversed-phase chromatography, exhibits powerful anti-bacterial activity against Gram-positive bacteria, with minimal inhibitory concentrations in the submicromolar range. Kinetic analysis revealed that at a concentration of 0.3μM all test bacteria lose viability after 30min incubation. Weaker activity is also displayed against the yeast Saccharomyces cerevisiae. The protein is salt-sensitive and has no haemolytic activity towards trout erythrocytes at concentrations below 0.3μM. Reconstitution of the protein in a planar lipid bilayer strongly disturbs the membrane but does not form stable ion channels, indicating that its anti-bacterial activity is probably not due to pore-forming properties. This is the first report to show that, in addition to its classical function in the cell, histone H2A has extremely strong anti-microbial properties and could therefore help contribute to protection against bacterial invasion.

Synthesis of hepatitis B surface antigen pre-S2 region fragments and studies on their immunogenicity

1988 ◽  
Vol 53 (11) ◽  
pp. 2952-2956 ◽  
Author(s):  
Bernard Lammek ◽  
Zbigniew Maćkiewicz ◽  
Izabela Derdowska ◽  
Hanna Świderska ◽  
Adam Nowosławski ◽  
...  
Keyword(s):  
Hepatitis B ◽  
Surface Antigen ◽  
Solid Phase ◽  
Hepatitis B Surface Antigen ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Antigenic Determinants ◽  
Phase Method ◽  
Peptide Fragments ◽  
Humoral Immune

Two peptide fragments of hepatitis B surface antigen pre-S2 region were synthesized by the solid phase method. The peptides were purified by gel filtration or ion-exchange chromatography on Sephadex SP-C-25. Both peptides induced a cellular and humoral immune response in rabbits. The results showed that fragment 14-22 of pre-S2 region contains one of the antigenic determinants.

LC–MS-MS Method for the Determination of Antidepressants and Benzodiazepines in Meconium

2020 ◽  
Vol 44 (6) ◽  
pp. 580-588
Author(s):  
A López-Rabuñal ◽  
E Lendoiro ◽  
M Concheiro ◽  
M López-Rivadulla ◽  
A Cruz ◽  
...  
Keyword(s):  
Extraction Efficiency ◽  
Solid Phase ◽  
Gradient Methods ◽  
Reversed Phase ◽  
Process Efficiency ◽  
Limit Of Quantification ◽  
Mode 2 ◽  
Positive Mode ◽  
Compound Method

Abstract An LC–MS-MS method for the determination of 14 benzodiazepines (BZDs) (alprazolam, α-hydroxyalprazolam, clonazepam, bromazepam, diazepam, nordiazepam, lorazepam, lormetazepam, oxazepam, flunitrazepam, 7-aminoflunitrazepam, triazolam, midazolam and zolpidem) and 15 antidepressants (ADs) (amitriptyline, nortriptyline, imipramine, desipramine, clomipramine, norclomipramine, fluoxetine, norfluoxetine, sertraline, norsertraline, paroxetine, venlafaxine, desmethylvenlafaxine, citalopram and desmethylcitalopram) in meconium was developed and validated. Meconium samples (0.25 ± 0.02 g) were homogenized in methanol and subjected to mixed-mode cation exchange solid-phase extraction. Chromatographic separation was performed in reversed phase, with a gradient of 0.1% formic acid in 2 mM ammonium formate and acetonitrile. Two different chromatographic gradient methods were employed, one for the separation of ADs and another for BZDs. Analytes were monitored by tandem mass spectrometry employing electrospray positive mode in MRM mode (2 transitions per compound). Method validation included: linearity [n = 5, limit of quantification (LOQ) to 400 ng/g], limits of detection (n = 6, 1–20 ng/g), LOQ (n = 9, 5–20 ng/g), selectivity (no endogenous or exogenous interferences), accuracy (n = 15, 90.6–111.5%), imprecision (n = 15, 0–14.6%), matrix effect (n = 10, −73 to 194.9%), extraction efficiency (n = 6, 35.9–91.2%), process efficiency (n = 6, 20.1–188.2%), stability 72 h in the autosampler (n = 3, −8.5 to 9%) and freeze/thaw stability (n = 3, −1.2 to −47%). The method was applied to four meconium specimens, which were analyzed with and without hydrolysis (enzymatic and alkaline). The authentic meconium samples tested positive for alprazolam, α-hydroxyalprazolam, clonazepam, diazepam, nordiazepam, fluoxetine, norfluoxetine, clomipramine and norclomipramine. Therefore, the present LC–MS-MS method allows a high throughput determination of the most common BZDs and ADs in meconium, which could be useful in clinical and forensic settings.

scholarly journals Metabolic Evaluation of Urine from Patients Diagnosed with High Grade (HG) Bladder Cancer by SPME-LC-MS Method

Molecules ◽  
2021 ◽  
Vol 26 (8) ◽  
pp. 2194
Author(s):  
Kamil Łuczykowski ◽  
Natalia Warmuzińska ◽  
Sylwia Operacz ◽  
Iga Stryjak ◽  
Joanna Bogusiewicz ◽  
...  
Keyword(s):  
Thin Film ◽  
Bladder Cancer ◽  
High Performance ◽  
Biomarker Discovery ◽  
Solid Phase ◽  
Biological Fluids ◽  
Reversed Phase ◽  
Control Group ◽  
Metabolic Evaluation ◽  
Metabolomic Profiling

Bladder cancer (BC) is a common malignancy of the urinary system and a leading cause of death worldwide. In this work, untargeted metabolomic profiling of biological fluids is presented as a non-invasive tool for bladder cancer biomarker discovery as a first step towards developing superior methods for detection, treatment, and prevention well as to further our current understanding of this disease. In this study, urine samples from 24 healthy volunteers and 24 BC patients were subjected to metabolomic profiling using high throughput solid-phase microextraction (SPME) in thin-film format and reversed-phase high-performance liquid chromatography coupled with a Q Exactive Focus Orbitrap mass spectrometer. The chemometric analysis enabled the selection of metabolites contributing to the observed separation of BC patients from the control group. Relevant differences were demonstrated for phenylalanine metabolism compounds, i.e., benzoic acid, hippuric acid, and 4-hydroxycinnamic acid. Furthermore, compounds involved in the metabolism of histidine, beta-alanine, and glycerophospholipids were also identified. Thin-film SPME can be efficiently used as an alternative approach to other traditional urine sample preparation methods, demonstrating the SPME technique as a simple and efficient tool for urinary metabolomics research. Moreover, this study’s results may support a better understanding of bladder cancer development and progression mechanisms.

scholarly journals Determination of Antiviral Drugs and Their Metabolites Using Micro-Solid Phase Extraction and UHPLC-MS/MS in Reversed-Phase and Hydrophilic Interaction Chromatography Modes

Molecules ◽  
2021 ◽  
Vol 26 (8) ◽  
pp. 2123
Author(s):  
Luboš Fical ◽  
Maria Khalikova ◽  
Hana Kočová Vlčková ◽  
Ivona Lhotská ◽  
Zuzana Hadysová ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Tandem Mass Spectrometry ◽  
Solid Phase Extraction ◽  
Solid Phase ◽  
Gradient Elution ◽  
Reversed Phase ◽  
Tandem Mass ◽  
Phase Extraction ◽  
Hydrophilic Interaction

Two new ultra-high performance liquid chromatography (UHPLC) methods for analyzing 21 selected antivirals and their metabolites were optimized, including sample preparation step, LC separation conditions, and tandem mass spectrometry detection. Micro-solid phase extraction in pipette tips was used to extract antivirals from the biological material of Hanks balanced salt medium of pH 7.4 and 6.5. These media were used in experiments to evaluate the membrane transport of antiviral drugs. Challenging diversity of physicochemical properties was overcome using combined sorbent composed of C18 and ion exchange moiety, which finally allowed to cover the whole range of tested antivirals. For separation, reversed-phase (RP) chromatography and hydrophilic interaction liquid chromatography (HILIC), were optimized using extensive screening of stationary and mobile phase combinations. Optimized RP-UHPLC separation was carried out using BEH Shield RP18 stationary phase and gradient elution with 25 mmol/L formic acid in acetonitrile and in water. HILIC separation was accomplished with a Cortecs HILIC column and gradient elution with 25 mmol/L ammonium formate pH 3 and acetonitrile. Tandem mass spectrometry (MS/MS) conditions were optimized in both chromatographic modes, but obtained results revealed only a little difference in parameters of capillary voltage and cone voltage. While RP-UHPLC-MS/MS exhibited superior separation selectivity, HILIC-UHPLC-MS/MS has shown substantially higher sensitivity of two orders of magnitude for many compounds. Method validation results indicated that HILIC mode was more suitable for multianalyte methods. Despite better separation selectivity achieved in RP-UHPLC-MS/MS, the matrix effects were noticed while using both chromatographic modes leading to signal enhancement in RP and signal suppression in HILIC.

scholarly journals The use of LC tandem mass spectrometry as part of a workflow for the screening and identification of hemoglobin variants. Characterization of Hb Ullevaal as an example

2017 ◽  
Vol 41 (3) ◽  
Author(s):  
Matthias Weber ◽  
Julia J.M. Eekels
Keyword(s):  
Mass Spectrometry ◽  
Tandem Mass Spectrometry ◽  
Cation Exchange ◽  
Peripheral Blood ◽  
Globin Gene ◽  
Reversed Phase ◽  
Exchange Chromatography ◽  
South Florida ◽  
Tandem Mass ◽  
Genetic Sequencing

AbstractBackground:About 2/3 of the hemoglobin (Hb) variants do not show a charge difference to the wildtype entity but most of them differ in hydrophobicity. In addition to cation exchange chromatography, globin differentiation by liquid chromatography-tandem mass spectrometry (MS) was introduced. Hb Ullevaal was chosen as one example to demonstrate the performance of the approach.Methods:Screening for Hb variants was performed using cation exchange HPLC. For globin separation reversed phase-LC/MS was performed. Tryptic digests of variants were separated on RP-HPLC with or without CID-fragmentation and database search for identification of mutation bearing fragments. Sequencing of the β-globin gene has been performed.Results:HbS, HbC, HbE, Hb South Florida and Hb Ullevaal show typical and distinct patterns in the globin LC/MS according to the theoretical protein data. The tryptic digest of Hb Ullevaal resulted in the identification of the respective mutated peptide βT9, which was confirmed by genetic sequencing.Conclusions:By the application of globin-LC/MS two more dimensions for the Hb identification are added, hydropathicity and protein mass. With this workflow as screening procedure for Hb variants it is expected to be able to detect and identify the majority of variants with the exception of highly unstable variants, which cannot be determined in the peripheral blood at all. A negative result makes the presence of a significant Hb variant in the peripheral blood improbable.

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