pr-39 and protegrin-1

Qualitative and quantitative mass spectrometric studies of biomolecules for example proteins, peptides, or lipids contained in biological samples like physiologic fluids are very important for many fields of science such as medicine, veterinary medicine, biology, biochemistry, molecular biology, or environmental sciences. In the last two decades, MALDI TOF MS - matrix-assisted laser desorption mass spectrometry, proved to be an especially convenient tool for these analyses. The main advantages of this method are its rapidity and high sensitivity which is particularly appreciated in the case of studies of complex biological specimen. A major challenge for many researchers is to maximize this sensitivity, among others, by appropriate procedures of sample preparation for the measurement. The objective of this work was to optimize these procedures, selecting the optimal matrix and optimum proportions of the sample and the matrix solution in a mixture of both solutions, aiming at the achievement of the maximum intensity of ion current. In this respect, five low molecular mass cathelicidins were studied: prophenin-2, protegrins 1-3, PR-39. All of them were obtained directly from the porcine blood. As a result of studies, the authors determined such experimental conditions when the intensity of investigated ionic current had the highest value.

Topics: Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Neutrophils; Proteins; Reference Standards; Specimen Handling; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Swine

To implement coexpression of antimicrobial peptides PR-39 and Protegrin-1 (PG-1) in prokaryotic expression system, a tandem gene fragment encoding PR-39 and PG-1 has been synthesized chemically. The cleavage site (Asn-Gly) of hydroxylamine hydrochloride was introduced between PR-39 and PG-1. The fragment was inserted into vector pGEX-4T-1 and expressed in Escherichia coli. The fusions of single peptides to GST were created at the same time. The fusion protein GST-PR-39-PG-1, purified by affinity chromatography, was cleaved first by hydroxylamine hydrochloride to release recombinant PG-1 and then by enterokinase to release PR-39. Purification of recombinant PR-39 and PG-1 was achieved. About 1.9 mg/l recombinant PR-39 and 1.1 mg/l PG-1 were obtained. The recombinant antimicrobial peptides showed antibacterial activities that were similar to those released from fusions of single peptides to GST.

Topics: Amino Acid Sequence; Antimicrobial Cationic Peptides; Chromatography, Affinity; Escherichia coli; Genetic Vectors; Glutathione Transferase; Molecular Sequence Data; Peptide Fragments; Recombinant Fusion Proteins

Innate immunity plays an important role in protection against respiratory infections in humans and animals. Host defense peptides such as beta-defensins represent major components of innate immunity. We recently developed a novel porcine model of pertussis, an important respiratory disease of young children and infants worldwide. Here, we investigated the role of porcine beta-defensin 1 (pBD-1), a porcine defensin homologue of human beta-defensin 2, in conferring protection against respiratory infection with Bordetella pertussis. In this model, newborn piglets were fully susceptible to infection and developed severe bronchopneumonia. In contrast, piglets older than 4 weeks of age were protected against infection with B. pertussis. Protection was associated with the expression of pBD-1 in the upper respiratory tract. In fact, pBD-1 expression was developmentally regulated, and the absence of pBD-1 was thought to contribute to the increased susceptibility of newborn piglets to infection with B. pertussis. Bronchoalveolar lavage specimens collected from older animals as well as chemically synthesized pBD-1 displayed strong antimicrobial activity against B. pertussis in vitro. Furthermore, in vivo treatment of newborn piglets with only 500 mug pBD-1 at the time of challenge conferred protection against infection with B. pertussis. Interestingly, pBD-1 displayed no bactericidal activity in vitro against Bordetella bronchiseptica, a closely related natural pathogen of pigs. Our results demonstrate that host defense peptides play an important role in protection against pertussis and are essential in modulating innate immune responses against respiratory infections.

Topics: Animals; Animals, Newborn; Antimicrobial Cationic Peptides; beta-Defensins; Bordetella pertussis; Bronchoalveolar Lavage Fluid; Female; Immunity, Innate; Lactoferrin; Lung; Muramidase; Pertussis Vaccine; Proteins; Respiratory Mucosa; Sodium Chloride; Swine; Whooping Cough

A total of 90 weaned female pigs (Duroc x Landrace x Yorkshire) were used in a 15-d growth experiment to investigate the effect of lactoferrin on growth performance, intestinal morphology, and expression of PR-39 and protegrin-1 genes. The pigs were allocated on the basis of BW and litter to 3 dietary treatments in a randomized complete block design. There were 3 replicate pens per treatment, and the pigs were grouped with 10 pigs per pen. The dietary treatments were (1) basal diet; (2) basal diet + 20 mg of flavomycin/kg + 110 mg of aureomycin/kg; (3) basal diet + 1.0 g of lactoferrin/kg. Six pigs, randomly selected from each treatment (2 piglets/pen) were slaughtered for intestinal morphology and expression of PR-39 and protegrin-1 genes at the end of the experiment. Supplementation with lactoferrin improved growth performance; it increased ADG by 41.80% (P < 0.01) and efficiency of gain (G:F) by 17.20% (P < 0.05). Intestinal villus height was increased by 15.30% (P < 0.05), and crypt depth was decreased by 9.60% (P < 0.05). Supplemental lactoferrin increased the relative abundance of mRNA for PR-39 and protegrin-1 by 143% (P < 0.01) and 217% (P < 0.01), respectively. The use of lactoferrin as an additive to improve nonspecific immunity and strengthen host defenses would be good a method of defending weaned pigs from infections and weanling stress.

Topics: Actins; Animal Feed; Animals; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Bambermycins; Bone Marrow; Chlortetracycline; Dietary Supplements; DNA Primers; Female; Gene Expression; Growth; Intestine, Small; Lactoferrin; Proteins; Random Allocation; Reverse Transcriptase Polymerase Chain Reaction; Swine