Antimicrobial Peptides from Human Platelets

ABSTRACT Platelets share structural and functional similarities with granulocytes known to participate in antimicrobial host defense. To evaluate the potential antimicrobial activities of platelet proteins, normal human platelets were stimulated with human thrombin in vitro. Components of the stimulated-platelet supernatants were purified to homogeneity by reversed-phase high-performance liquid chromatography. Purified peptides with inhibitory activity against Escherichia coli ML35 in an agar diffusion antimicrobial assay were characterized by mass spectrometry, amino acid analysis, and sequence determination. These analyses enabled the identification of seven thrombin-releasable antimicrobial peptides from human platelets: platelet factor 4 (PF-4), RANTES, connective tissue activating peptide 3 (CTAP-3), platelet basic protein, thymosin β-4 (Tβ-4), fibrinopeptide B (FP-B), and fibrinopeptide A (FP-A). With the exception of FP-A and FP-B, all peptides were also purified from acid extracts of nonstimulated platelets. The in vitro antimicrobial activities of the seven released peptides were further tested against bacteria (E. coli and Staphylococcus aureus) and fungi (Candida albicans and Cryptococcus neoformans). Each peptide exerted activity against at least two organisms. Generally, the peptides were more potent against bacteria than fungi, activity was greater at acidic pHs, and antimicrobial activities were dose dependent. Exceptions to these observations were observed with PF-4, which displayed a bimodal dose-response relationship in microbicidal assays, and Tβ-4, which had greater activity at alkaline pHs. At concentrations at which they were individually sublethal, PF-4 and CTAP-3 exerted synergistic microbicidal activity against E. coli. Collectively, these findings suggest a direct antimicrobial role for platelets as they are activated to release peptides in response to trauma or mediators of inflammation.

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Synthesis of Human Bone Morphogenetic Protein-2 (hBMP-2) in E. coli Periplasmic Space: Its Characterization and Preclinical Testing

Cells ◽

10.3390/cells10123525 ◽

2021 ◽

Vol 10 (12) ◽

pp. 3525

Author(s):

João E. Oliveira ◽

Miriam F. Suzuki ◽

Renata Damiani ◽

Eliana R. Lima ◽

Kleicy C. Amaral ◽

...

Keyword(s):

High Performance ◽

Osmotic Shock ◽

Cytoplasmic Inclusion ◽

Reversed Phase ◽

C2c12 Cells ◽

Size Exclusion ◽

Bone Morphogenetic Protein 2 ◽

E Coli

Human BMP-2, a homodimeric protein that belongs to the TGF- β family, is a recognized osteoinductor due to its capacity of inducing bone regeneration and ectopic bone formation. The administration of its recombinant form is an alternative to autologous bone grafting. A variety of E. coli-derived hBMP-2 has been synthesized through refolding of cytoplasmic inclusion bodies. The present work reports the synthesis, purification, and characterization of periplasmic hBMP-2, obtained directly in its correctly folded and authentic form, i.e., without the initial methionine typical of the cytoplasmic product that can induce undesired immunoreactivity. A bacterial expression vector was constructed including the DsbA signal peptide and the cDNA of hBMP-2. The periplasmic fluid was extracted by osmotic shock and analyzed via SDS-PAGE, Western blotting, and reversed-phase high-performance liquid chromatography (RP-HPLC). The purification was carried out by heparin affinity chromatography, followed by high-performance size-exclusion chromatography (HPSEC). HPSEC was used for qualitative and quantitative analysis of the final product, which showed >95% purity. The classical in vitro bioassay based on the induction of alkaline phosphatase activity in myoblastic murine C2C12 cells and the in vivo bioassay consisting of treating calvarial critical-size defects in rats confirmed its bioactivity, which matched the analogous literature data for hBMP-2.

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Thrombin Stimulated Platelet Accumulation on Protein Coated Glass Capillaries: Role of Adhesive Platelet α-Granule Proteins

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651040 ◽

1989 ◽

Vol 62 (03) ◽

pp. 989-995 ◽

Cited By ~ 5

Author(s):

Juliette N Mulvihill ◽

J Andrew Davies ◽

Florence Toti ◽

Jean-Marie Freyssinet ◽

Jean-Pierre Cazenave

Keyword(s):

Human Platelets ◽

Capillary Perfusion ◽

Coated Glass ◽

Bovine Collagen ◽

Glass Capillaries ◽

Human Thrombin ◽

Platelet Accumulation ◽

Granule Proteins ◽

Von Willebrand

SummaryThe generation of trace amounts of thrombin at artificial surfaces in contact with blood is likely to be a contributing factor in thrombosis on biomaterials. Using an in vitro capillary perfusion system, platelet accumulation on glass surfaces, uncoated or precoated with purified bovine collagen or human plasma proteins, was determined in the presence or absence of preadsorbed purified human thrombin. Static adsorption for 15 min at 22° C from solutions of thrombin 100 NIH units (33 μg)/ml gave surface concentrations in the range 0.019-0.101 μg/cm2. Protein coated capillaries, thrombin treated or untreated, were perfused for 2 min at 37° C with suspensions of washed 111In-labeled human platelets in Tyrode's-albumin buffer containing 40% washed red blood cells, under conditions of controlled, non pulsatile laminar flow (50 s−1 or 2,000 s−1). Platelet accumulation was increased in the presence of surface adsorbed thrombin on uncoated and albumin or fibrinogen coated glass but little affected on fibronectin or collagen coated glass. On von Willebrand factor (vWF) coated glass, thrombin enhancement was observed only at high shear forces. In experiments using antibodies against human platelet α-granule proteins, thrombin stimulated platelet deposition in uncoated glass capillaries was inhibited at 2,000 s−1 by anti-vWF and to a lesser extent by anti-fibrinogen but not by antithrombospondin antibodies.

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Loss of 111Indium as indicator of platelet injury

Blood ◽

10.1182/blood.v58.2.350.350 ◽

1981 ◽

Vol 58 (2) ◽

pp. 350-353 ◽

Cited By ~ 18

Author(s):

JH Joist ◽

RK Baker

Keyword(s):

Small Molecules ◽

Adenine Nucleotides ◽

Human Platelets ◽

Metabolic Energy ◽

Platelet Factor ◽

Platelet Protein ◽

Threshold Rate ◽

Immune Injury ◽

Triton X

Abstract We previously demonstrated that platelets can be labeled with 111Inoxine with high labeling efficiency and that 111In is not liberated from labeled platelets during the platelet release reaction or prolonged in vitro storage. In view of these findings, we examined the potential usefulness of loss of 111In from labeled platelets as an indicator or platelet damage by comparing the loss of 111In with that of 51Cr and LDH (in some experiments also with platelet factor 3 availability) under different conditions of platelet injury. When washed human platelets labeled with either 51Cr-chromate or 111In-oxine were exposed to increasing concentrations of detergents (Triton X-100, lysolecithin), threshold, rate, and extent of loss of 111In, 51Cr and, LDH were similar. In contrast, when labeled platelets were depleted of metabolic energy by incubation in glucose-free Tyrode albumin solution or glucose-depleted plasma in the presence of antimycin A and 2-deoxy-D- glucose, loss of 51Cr (and PF3a) occurred earlier and progressed at a faster rate than that of 111In or LDH. Similar results were obtained when platelets were exposed to increasing concentrations of PlA1 antibody, causing complement-mediated immune injury. The findings indicate that with certain agents that cause rapid platelet disruption (lysis), different platelet constituents are lost at similar rates. However, under conditions of more subtle or slowly progressive platelet injury, small molecules such as adenine nucleotides (51Cr) may escape earlier and at faster rates than larger molecules such as LDH or 111In- binding platelet protein. Thus, neither 111In loss nor LDH loss appear to be suitable indicators for sublytic or prelytic platelet injury.

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Periplasmic synthesis and purification of the human prolactin antagonist Δ1-11-G129R-hPRL

AMB Express ◽

10.1186/s13568-021-01209-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Miriam F. Suzuki ◽

Larissa A. Almeida ◽

Stephanie A. Pomin ◽

Felipe D. Silva ◽

Renan P. Freire ◽

...

Keyword(s):

Size Exclusion Chromatography ◽

High Performance ◽

Signal Sequence ◽

Osmotic Shock ◽

Size Exclusion ◽

Specific Expression ◽

E Coli ◽

Human Prolactin ◽

Exclusion Chromatography

AbstractThe human prolactin antagonist Δ1-11-G129R-hPRL is a 21.9 kDa recombinant protein with 188 amino acids that downregulates the proliferation of a variety of cells expressing prolactin receptors. Periplasmic expression of recombinant proteins in E. coli has been considered an option for obtaining a soluble and correctly folded protein, as an alternative to cytoplasmic production. The aim of this work was, therefore, to synthesize for the first time, the Δ1-11-G129R-hPRL antagonist, testing different activation temperatures and purifying it by classical chromatographic techniques. E. coli BL21(DE3) strain was transformed with a plasmid based on the pET25b( +) vector, DsbA signal sequence and the antagonist cDNA sequence. Different doses of IPTG were added, activating under different temperatures, and extracting the periplasmic fluid via osmotic shock. The best conditions were achieved by activating at 35 °C for 5 h using 0.4 mM IPTG, which gave a specific expression of 0.157 ± 0.015 μg/mL/A600 at a final optical density of 3.43 ± 0.13 A600. Purification was carried out by nickel-affinity chromatography followed by size-exclusion chromatography, quantification being performed via high-performance size-exclusion chromatography (HPSEC). The prolactin antagonist was characterized by SDS-PAGE, Western blotting, reversed-phase high-performance liquid chromatography (RP-HPLC) and MALDI-TOF–MS. The final product presented > 95% purity and its antagonistic effects were evaluated in vitro in view of potential clinical applications, including inhibition of the proliferation of cancer cells overexpressing the prolactin receptor and specific antidiabetic properties, taking also advantage of the fact that this antagonist was obtained in a soluble and correctly folded form and without an initial methionine.

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Synthesis and In Vitro Antimicrobial Evaluation of Photoactive Multi—Block Chalcone Conjugate Phthalimide and 1,8-Naphthalimide Novolacs

Polymers ◽

10.3390/polym13111859 ◽

2021 ◽

Vol 13 (11) ◽

pp. 1859

Author(s):

Periyan Durairaju ◽

Chinnasamy Umarani ◽

Govindasami Periyasami ◽

Perumberkandigai Adikesavan Vivekanand ◽

Mostafizur Rahaman

Keyword(s):

Spectroscopic Analysis ◽

Photophysical Properties ◽

Gel Permeation Chromatography ◽

13C Nmr Spectroscopy ◽

Antimicrobial Activities ◽

Microbial Activities ◽

E Coli ◽

Molecular Weights ◽

Antimicrobial Evaluation

Herein we report new multiblock chalcone conjugate phthalimide and naphthalimide functionalized copolymers with a topologically novel architecture synthesis using nucleophilic substitution and polycondensation methodology. The structures of the synthesized novolacs were elucidated on the basis of their spectroscopic analysis including FTIR, 1H NMR, and 13C NMR spectroscopy. Further, the number-average and weight-average molecular weights of the novolac polymers were determined by gel permeation chromatography (GPC). We examined the solubility of the synthesized polymers in various organic solvents including CHCl3, CH3CN, THF, H2O, CH3OH, DMSO, and DMF and found they are insoluble in both methanol and water. The novolac polymers were evaluated for their photophysical properties and microbial activities. The investigation of the antimicrobial activities of these polymers reveals significant antimicrobial activity against the pathogens E. coli, S. aureus, C. albicans, and A. niger.

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Chemical Analysis and Evaluation of Antioxidant, Antimicrobial, and Photoprotective Activities of Schinopsis brasiliensis Engl. (Anacardiaceae)

The Scientific World JOURNAL ◽

10.1155/2017/1713921 ◽

2017 ◽

Vol 2017 ◽

pp. 1-10 ◽

Cited By ~ 3

Author(s):

Sarah Raquel Gomes de Lima-Saraiva ◽

Fernanda Granja da Silva Oliveira ◽

Raimundo Gonçalves de Oliveira Junior ◽

Camila de Souza Araújo ◽

Ana Paula de Oliveira ◽

...

Keyword(s):

High Performance ◽

Ethanolic Extract ◽

Antimicrobial Activities ◽

Physicochemical Analysis ◽

Minimal Bactericidal Concentration ◽

Native Plant ◽

Protection Factor ◽

Dpph Method ◽

High Concentrations

Schinopsis brasiliensis Engl. is a native plant of Caatinga which has high concentrations of compounds capable of absorbing ultraviolet light, suggesting its potential application for the development of sunscreen preparations. After its identification and collection, this vegetable drug was submitted to a physicochemical analysis through the preparation of ethanolic extract. The phytochemical screening and analysis of extracts were carried out by high-performance liquid chromatography (HPLC) evaluation. The antioxidant activity of the extract was evaluated by 2,2-diphenyl-1-picrylhydrazyl (DPPH) method and β-carotene bleaching test. Inhibitory hemolytic activity and morphological deformation of erythrocytes induced by H2O2 were also demonstrated and the antimicrobial activity was analyzed by the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) method. For the in vitro determination of the sun protection factor (SPF), the spectrophotometric method was used. From the analyses carried out with this species, this plant showed significant results for the antioxidant and antimicrobial activities, as well as sunscreen action. Important flavonoids were identified. These data are an important step for the development of new photoprotective cosmetic with Caatinga species, revealing importance and representing another incentive for the preservation of the species involved and analyzed in the study.

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Formulation, characterization, evaluation and in vitro study of transfersomal gel medroxyprogesterone acetate for transdermal drug delivery

International Journal of Research in Pharmaceutical Sciences ◽

10.26452/ijrps.v11i4.3159 ◽

2020 ◽

Vol 11 (4) ◽

pp. 5373-5381

Author(s):

Iskandarsyah ◽

Camelia Dwi Putri Masrijal ◽

Harmita

Keyword(s):

Drug Delivery ◽

Medroxyprogesterone Acetate ◽

Transdermal Drug Delivery ◽

High Performance ◽

Hormonal Contraception ◽

In Vitro Study ◽

Entrapment Efficiency ◽

Tween 80 ◽

Reversed Phase

A hormonal contraception progestin such as medroxyprogesterone acetate (MPA) is used to helps regulate ovulation thus as a part of contraception hormone therapy as a method of birth control. This study aimed to formulate, characterized, evaluated transfersomal gel containing medroxyprogesterone acetate and to increased subcutaneous penetration of medroxyprogesterone acetate. In this research, three transfersomes formulas were prepared and optimized, e.g. F1, F2 and F3 with phosphatidylcholine: tween 80 concentration were 90:10; 85:15; and 75:25, respectively. F2 was the best formula with the highest entrapment efficiency 81.20±0.42 %, Average 81.35 ±0.78 nm, morphology of vesicles were spheres, indeks polidispersity 0.198±0.012 and zeta potential was -34.83±0.64 mV. The transpersonal gel (FGT) containing F2, and non-transpersonal gel containing MPA in methanol(FG) were prepared. In vitro penetration test were conducted to both of them using Franz Diffusion cells. Analysis of medroxyprogesterone acetate used a high performance liquid chromatographic (HPLC) method with an ultraviolet detector on reversed-phase C18, 5µm; 150 x 4.6 mmcolumn; using acetonitrile-0.1% formic acid (60:40/v:v) and was detected at a wavelength of 240 nm with flow rate at 1.0 mL/min. Gel stability evaluation results showed that FGT was better than FG on pH stability, viscosity and rheological properties. Based on in vitro penetration study, cumulative subcutaneous penetration of medroxyprogesterone acetate from FGT was 2356.45 ± 197.73 ng.cm-2 and from FG 359.15 ± 13.60 ng.cm-2, respectively. Flux value for FGT and FG were 112.77 ± 6,47 ng.cm-2.hr-1and 17.99 ± 4.81 ng.cm-2.hr-1, respectively. It could be concluded that transfersomal gel medroxyprogesterone acetate for transdermal drug delivery increased cumulative transdermal penetration of medroxyprogesterone acetate by six times more than non-transfersomal gel dosage form.

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Preventive antimicrobial action and tissue architecture ameliorations of Bacillus subtilis in challenged broilers

Veterinary World ◽

10.14202/vetworld.2021.523-536 ◽

2021 ◽

Vol 14 (2) ◽

pp. 523-536

Author(s):

Essam S. Soliman ◽

Rania T. Hamad ◽

Mona S. Abdallah

Keyword(s):

Bacillus Subtilis ◽

Broiler Chickens ◽

Trichophyton Mentagrophytes ◽

Antimicrobial Activities ◽

Lymphoid Hyperplasia ◽

Bursa Of Fabricius ◽

Tissue Architecture ◽

E Coli

Background and Aim: Probiotics improve intestinal balance through bacterial antagonism and competitive exclusion. This study aimed to investigate the in vitro antimicrobial activity, as well as the in vivo preventive, immunological, productive, and histopathological modifications produced by probiotic Bacillus subtilis. Materials and Methods: The in vitro antimicrobial activities of B. subtilis (5×106 CFU/g; 0.5, 1.0*, 1.5, and 2.0 g/L) were tested against Escherichia coli O157: H7, Salmonella Typhimurium, Candida albicans, and Trichophyton mentagrophytes after exposure times of 0.25, 0.5, 1, and 2 h using minimal inhibitory concentration procedures. A total of 320 1-day-old female Ross broiler chickens were divided into five groups. Four out of the five groups were supplemented with 0.5, 1.0*, 1.5, and 2.0 g/L probiotic B. subtilis from the age of 1 day old. Supplemented 14-day-old broiler chickens were challenged with only E. coli O157: H7 (4.5×1012 CFU/mL) and S. Typhimurium (1.2×107 CFU/mL). A total of 2461 samples (256 microbial-probiotic mixtures, 315 sera, 315 duodenal swabs, and 1575 organs) were collected. Results: The in vitro results revealed highly significant (p<0.001) killing rates at all-time points in 2.0 g/L B. subtilis: 99.9%, 90.0%, 95.6%, and 98.8% against E. coli, S. Typhimurium, C. albicans, and T. mentagrophytes, respectively. Broilers supplemented with 1.5 and 2.0 g/L B. subtilis revealed highly significant increases (p<0.01) in body weights, weight gains, carcass weights, edible organs' weights, immune organs' weights, biochemical profile, and immunoglobulin concentrations, as well as highly significant declines (p<0.01) in total bacterial, Enterobacteriaceae, and Salmonella counts. Histopathological photomicrographs revealed pronounced improvements and near-normal pictures of the livers and hearts of broilers with lymphoid hyperplasia in the bursa of Fabricius, thymus, and spleen after supplementation with 2.0 g/L B. subtilis. Conclusion: The studies revealed that 1.5-2.0 g of probiotic B. subtilis at a concentration of 5×106 CFU/g/L water was able to improve performance, enhance immunity, and tissue architecture, and produce direct antimicrobial actions.

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Inhibitory effect of the glucosinolate–myrosinase system on Phytophthora cinnamomi and Pythium spiculum

Plant Protection Science ◽

10.17221/98/2018-pps ◽

2019 ◽

Vol 55 (No. 2) ◽

pp. 93-101 ◽

Cited By ~ 2

Author(s):

Francisco Teodoro Arroyo Cordero ◽

Rocío Rodríguez-Arcos ◽

Ana Jiménez-Araujo ◽

Rafael Guillén-Bejarano ◽

María José Basallote ◽

...

Keyword(s):

Mycelial Growth ◽

High Performance ◽

Phytophthora Cinnamomi ◽

Inhibitory Effect ◽

Reversed Phase ◽

Field Application ◽

Additional Information ◽

Array Detection ◽

Important Additional Information

Glucosinolate extracts from sprouts of common Brassica nigra, B. juncea cv. Scala, B. carinata cv. Eleven, and Sinapis alba cv. Ludique were analysed by reversed phase high-performance liquid chromatography-diode array detection-mass spectrometry. The effect of the glucosinolate–myrosinase system on in vitro mycelial growth of Phytophthora cinnamomi Rands and Pythium spiculum B. Paul was assessed. Likewise, sinigrin and sinalbin monohydrate commercial standards were also tested. The extracts from B. carinata, which contained 159 mmol/g plant DW equivalent (85% sinigrin, 5% gluconapin, and 3% glucotropaeolin), were the most effective against Phytophthora and Pythium isolates used in this study. However, the extract from S. alba, which contained 1 180 mmol/g (100% sinalbin), did not inhibit the mycelial growth of the isolates tested. The use of the glucosinolate-myrosinase system provides important additional information to advance in the implementation of field application of brassicaceous amendments for the control of soil-borne pathogens.

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Expression of Hybrid Peptide EF-1 in Pichia pastoris, Its Purification, and Antimicrobial Characterization

Molecules ◽

10.3390/molecules25235538 ◽

2020 ◽

Vol 25 (23) ◽

pp. 5538

Author(s):

Zhongxuan Li ◽

Qiang Cheng ◽

Henan Guo ◽

Rijun Zhang ◽

Dayong Si

Keyword(s):

Pichia Pastoris ◽

High Performance ◽

Large Scale ◽

Expression System ◽

Reversed Phase ◽

E Coli ◽

Cell Membrane Permeabilization ◽

Bactericidal Activities ◽

The Individual ◽

Industrial Level

EF-1 is a novel peptide derived from two bacteriocins, plantaricin E and plantaricin F. It has a strong antibacterial activity against Escherichia coli and with negligible hemolytic effect on red blood cells. However, the chemical synthesis of EF-1 is limited by its high cost. In this study, we established a heterologous expression of EF-1 in Pichia pastoris. The transgenic strain successfully expressed hybrid EF-1 peptide, which had a molecular weight of ~5 kDa as expected. The recombinant EF-1 was purified by Ni2+ affinity chromatography and reversed-phase high performance liquid chromatography (RP-HPLC), which achieved a yield of 32.65 mg/L with a purity of 94.9%. The purified EF-1 exhibited strong antimicrobial and bactericidal activities against both Gram-positive and -negative bacteria. Furthermore, propidium iodide staining and scanning electron microscopy revealed that EF-1 can directly induce cell membrane permeabilization of E. coli. Therefore, the hybrid EF-1 not only preserves the individual properties of the parent peptides, but also acquires the ability to disrupt Gram-negative bacterial membrane. Meanwhile, such an expression system can reduce both the time and cost for large-scale peptide production, which ensures its potential application at the industrial level.

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