Identification and characterization of Staphylococcus spp. and their susceptibility to insect apolipophorin III.

Aim: This study investigated the effect of an insect antimicrobial protein, apolipophorin III (apoLp-III), against two newly isolated, identified and characterized clinical strains of Staphylococcus spp. Materials & methods: Both strains were identified by 16S rRNA sequencing and metabolic and phenotypic profiling. The antibacterial activity of apoLp-III was tested using a colony counting assay. ApoLp-III interaction with bacterial cell surface was analyzed by Fourier transform IR spectroscopy. Results:Staphylococcus epidermidis and Staphylococcus capitis were identified. ApoLp-III exerted a dose-dependent bactericidal effect on the tested strains. The differences in the Staphylococcus spp. surface components may contribute to the various sensitivities of these strains to apoLp-III. Conclusion: ApoLp-III may provide a baseline for development of antibacterial preparations against Staphylococcus spp. involved in dermatological problems.

Cooking Has Variable Effects on the Fermentability in the Large Intestine of the Fraction of Meats, Grain Legumes, and Insects That Is Resistant to Digestion in the Small Intestine in an in Vitro Model of the Pig's Gastrointestinal Tract.

This study aimed to evaluate the fermentation in the large intestine of indigestible dietary protein sources from animal, insect, and plant origin using an in vitro model of the pig's gastrointestinal tract. Protein sources were used raw and after a cooking treatment. Results showed that the category of the ingredient (meats, insects, or grain legumes) exerts a stronger impact on enzymatic digestibility, fermentation patterns, and bacterial metabolites such as short-chain fatty acids (SCFA) and hydrogen sulfide (H2S) than the cooking treatment. The digestibility and the fermentation characteristics of insects were more affected by the cooking procedure than the other categories. Per gram of consumed food, ingredients from animal origin, namely, meats and insects, were associated with fewer fermentation end-products (gas, H2S, SCFA) than ingredients from plant origin, which is related to their higher small intestinal digestibility.

PLGA microspheres containing bee venom proteins for preventive immunotherapy.

Bee venom (BV) allergy is potentially dangerous for allergic individuals because a single bee sting may induce an anaphylactic reaction, eventually leading to death. Currently, venom immunotherapy (VIT) is the only treatment with long-lasting effect for this kind of allergy and its efficiency has been recognized worldwide. This therapy consists of subcutaneous injections of gradually increasing doses of the allergen. This causes patient lack of compliance due to a long time of treatment with a total of 30-80 injections administered over years. In this article we deal with the characterization of different MS-PLGA formulations containing BV proteins for VIT. The PLGA microspheres containing BV represent a strategy to replace the multiple injections, because they can control the solute release. Physical and biochemical methods were used to analyze and characterize their preparation. Microspheres with encapsulation efficiencies of 49-75% were obtained with a BV triphasic release profile. Among them, the MS-PLGA 34kDa-COOH showed to be best for VIT because they presented a low initial burst (20%) and a slow BV release during lag phase. Furthermore, few conformational changes were observed in the released BV. Above all, the BV remained immunologically recognizable, which means that they could continuously stimulate the immune system. Those microspheres containing BV could replace sequential injections of traditional VIT with the remarkable advantage of reduced number of injections.

In situ imaging of honeybee (Apis mellifera) venom components from aqueous and aluminum hydroxide-adsorbed venom immunotherapy preparations.

BACKGROUND: Treatment with aqueous and aluminum hydroxide (Al[OH](3))-adsorbed purified honeybee (Apis mellifera) venom (HBV) preparations can reduce the incidence of side effects associated with venom immunotherapy. OBJECTIVE: The aim of the present study was to assess these purified HBV immunotherapy preparations in situ. METHODS: Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was used to visualize the distribution of HBV components. The preparations were administered on the back legs of naive Wistar rats. The rats were killed, and cryosectioned tissue sections were subjected to hematoxylin and eosin staining and MALDI-MSI analyses. RESULTS: Low-density maps of tissue distribution of HBV peptides, such as secapin, mast cell degranulating peptide, and melittin (Api m 4) were detected in the tissue after administration of HBV immunotherapy preparations. In addition, release of biogenic amines, cytokines, and leukotrienes was observed, and the distribution of HBV allergens, such as Api m 1 and Api m 2, was shown. At the 24-hour time point, the major HBV allergen Api m 1 was still detected at the site of Al(OH)(3)-adsorbed HVB injection, whereas in the case of aqueous HBV preparation, all the allergens, as well as most of the biogenic amines, were cleared at the 24-hour time point. CONCLUSION: The present study shows that the majority of low-molecular-weight HBV components are rapidly removed from the site of venom immunotherapy administration. Furthermore, Al(OH)(3)-adsorbed HBV preparation demonstrated a depot effect, prolonging the availability of bee venom allergens at the site of administration.

[Preparation and identification of recombinant PTD-maxadilan].

In order to construct a novel fusion protein PTD-maxadilan (PTD-MAX) that can enter the blood-brain barrier (BBB) efficiently, a new gene encoding PTD-MAX was synthesized and cloned into the expression vector pKYB. The recombinant vector pKYB-PTD-MAX was transformed into Escherichia coli ER2566. The expression of fusion protein consisting of PTD-MAX, intein and chitin binding domain was induced by IPTG and the target PTD-MAX protein was purified using Intein Mediated Purification with an Affinity Chitin-binding Tag system. The molecular weight of PTD-MAX determined by the laser flight mass spectrometry was coherent with its theoretical value. The results of the experiment in vivo indicated that the recombinant PTD-MAX can permeate into BBS and inhibitory effects on the food intake were more significantly than maxadilan (P<0.05). The preparation of PTD-MAX lay the foundation for its further application.

In vitro and in vivo evaluation of recombinant silk-elastinlike hydrogels for cancer gene therapy.

The objectives of this study were to evaluate: (i). the influences of hydrogel geometry, DNA molecular weight, and DNA conformation on DNA release from a silk-elastinlike protein polymer (SELP) hydrogel, (ii). the bioactivity and transfection efficiency of encapsulated DNA over time in vitro, (iii). the delivery and transfection of a reporter gene in a murine model of human breast cancer in vivo, and (iv). the in vitro release and bioactivity of adenovirus containing the green fluorescent protein (gfp) gene as a marker of gene transfer. Plasmid DNA was released from SELP hydrogels in a size-dependent manner, with the average effective diffusivity ranging from 1.70+/-0.52 x 10(-12) cm(2)/s for a larger plasmid (11 kbp) to 2.55+/-0.51 x 10(-10) cm(2)/s for a smaller plasmid (2.6 kbp). Plasmid conformation also influenced the rate of release, with the rank order linear>supercoiled>open-circular. DNA retained bioactivity in vitro, after encapsulation in a SELP hydrogel for up to 28 days. Delivery of pRL-CMV from a SELP hydrogel resulted in increased transfection in a murine model of human breast cancer by 1-3 orders of magnitude, as compared to naked DNA. The release of a bioactive adenoviral vector was related to the concentration of the polymer in the hydrogel. These studies indicate that genetically engineered SELP hydrogels have potential as matrices for controlled nonviral and viral gene delivery.

Uptake of atmospheric carbon dioxide into silk fiber by silkworms.

The relation between the uptake of atmospheric CO(2) and insect's production of silk fiber has not yet been reported. Here, we provide the first quantitative demonstrations that four species of silkworms (Bombyx mori, Samia cynthia ricini, Antheraea pernyi, and Antheraea yamamai) and a silk-producing spider (Nephila clavata) incorporate atmospheric CO(2) into their silk fibers. The abundance of (13)C incorporated from the environment was determined by mass spectrometry and (13)C NMR measurements. Atmospheric CO(2) was incorporated into the silk fibers in the carbonyl groups of alanine, aspartic acid, serine, and glycine and the C(gamma) of aspartic acid. We show a simple model for the uptake of atmospheric CO(2) by silkworms. These results will demonstrate that silkworm has incorporated atmospheric CO(2) into silk fiber via the TCA cycle; however, the magnitude of uptake into the silk fibers is smaller than that consumed by the photosynthesis in trees and coral reefs.

Synthesis and characterization of bactericidal oligopeptides designed on the basis of an insect anti-bacterial peptide.

Defensin from a beetle, Allomyrina dichotoma, is known to have anti-bacterial activity against Gram-positive bacteria. This peptide, which comprises 43 amino acid residues, was effective against methicillin-resistant Staphylococcus aureus. We identified the active site of beetle defensin by measuring anti-bacterial activity against S. aureus of 64 overlapping 12-mer peptides with either a free carboxylate or a free amide group at their C-termini. An LCAAHCLAIGRR-NH2 (19L-30R-NH2) fragment showed the greatest activity of the synthetic oligopeptides. The 19L-30R-NH2 fragment was effective against both Gram-positive and Gram-negative bacteria. CD spectra showed that the 19L-30R-NH2 fragment formed an alpha-helical structure in the lipidic environment. The anti-bacterial effect of the 19L-30R-NH2 fragment was due to its interaction with bacterial membranes, judging from the leakage of liposome-entrapped glucose. Its anti-bacterial activity was increased when certain amino acid residues were replaced. Truncated peptides having had some amino acids removed from the N-terminus of the 19L-30R-NH2 fragment (8-10-mer peptides) still had strong anti-bacterial activity. Deleting some amino acids from the C-terminal region of the fragment dramatically reduced activity, indicating that the C-terminal region of the 19L-30R-NH2 fragment, i.e. RR-NH2, is important for exerting anti-bacterial activity. The AHCLAIGRR-NH2 (22A-30R-NH2) fragment and its analogues exhibited about 3-fold and 9-12-fold higher activity against S. aureus than did the 19L-30R-NH2 fragment, and these analogues were effective against methicillin-resistant S. aureus and Pseudomonas aeruginosa isolated from patients. These oligopeptides showed no haemolytic activity and did not inhibit the growth of murine fibroblast cells.

Fungicidal and binding properties of the natural peptides cecropin B and dermaseptin.

In vitro fungicidal properties of cecropin B and dermaseptin were explored using non-germinating and germinating conidia from Aspergillus flavus, A. fumigatus, A. niger, Fusarium n2oniliforme and F oxysporum. Cecropin B produced LD50 values for germinating A. flavus, A. fumigatus and A. niger conidia of 30, 0.5 and 2.0 microM, respectively, while dermaseptin gave LD50 values of 4.0, 0.05 and 2.0 microM, respectively. Cecropin B produced an LD50 value of 0.2 microM for non-germinating F. moniliforme and F. oxysporum conidia, while dermaseptin did not reduce either as much as 50% at any level tested. LD50 levels for CB were 0.2 and 0.1 microM, respectively, for germinating F. moniliforme and F. oxysporum conidia. Dermaseptin was less effective, giving LD50 values for germinating F. moniliforme and F. oxysporum conidia of 0.3 and 0.8 microM, respectively. Neither peptide reduced conidial viabilities of non-germinating Aspergillus spp. Physicochemical studies indicated cecropin B and dermaseptin bound to ergosterol and cholesterol, conidial wall constituents, but not to chitin or beta-1,3-glucan.

Differential accumulation and tissue distribution of mosquito hexamerins during metamorphosis.

The pupal hexamerins were characterized for two mosquitoes representative of the culicine and anopheline families, Aedes aegypti and Anopheles gambiae. Like higher Diptera, both mosquito species express two types of hexamerins, Hex-1 and Hex-2, whose subunits are distinguished by different levels of methionine and aromatic amino acids. In A. aegypti there are two heterohexamers, AaHex-1 and AaHex-2. In A. gambiae there are two homohexamers, AgHex-1.1 and AgHex-1.2, and one heterohexamer, AgHex-2. These hexamerins are rich in aromatic residues, with 18-23% Phe + Tyr for Hex-1 subunits and 13-17% Phe + Tyr for Hex-2 subunits. In addition, both mosquito species synthesize methionine-rich Hex-1 subunits: Aedes AaHex-1 gamma (8% met) and Anopheles AgHex-1.1 (3.9% met). Aedes Hex-1 and Hex-2 proteins exhibit different, stage-specific tissue distributions: AaHex-2 is the primary hexamerin of late larval hemolymph whereas AaHex-1 is the most important non-hemolymph protein of early pupae. Although both proteins are stored in the pupal fat body, peak AaHex-1 levels are 2-fold higher. Both pupal protein levels decline rapidly between 25 and 36 h after pupation. Furthermore, AaHex-1 not only reaches peak values in female Aedes pupae later than in males, but the methionine-rich AaHex-1 gamma subunit level is specifically higher in females. These observations suggest different roles for Hex-1 and Hex-2 during mosquito development.