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We examined the effect of amphotericin B (AmB) on the following enzymatic markers: aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and on non-enzymatic markers: glucose, triglycerides, and proteins in the haemolymph of a model organism, Apis mellifera. AmB is an antifungal antibiotic. Despite its toxicity, it is used to treat disease conditions. The haemolymph of honey bees is considered as an analogue of human blood, and changes in marker values indicate pathological states, both in humans and honey bees. Three groups of caged bees were fed sugar syrup (the control group). The syrup was supplemented with AmB at concentrations of 0.25 mg/ml (AmB-25) and 0.50 mg/ml (AmB-50). The authors observed that the biochemical markers were age-related in the control group. Decreased values of the enzymatic markers in the AmB-treated groups confirm that AmB has a negative effect on the organism. The higher the dose of the antibiotic, the greater the increase in the concentration of the non-enzymatic markers. Our research shows that honey bees are an important model for studying the effects of AmB.
Insect hemolymph, like vertebrate serum, contains several different types of polypeptides that are able to inhibit the catalytic function of proteolytic enzymes, however studies on proteins possessing this capability have been limited to a rela­tively few species. A comparative examination of the inhibition of trypsin, chymo- trypsin, neutrophil elastase and cathepsin G and pancreatic elastase by the hemo­lymph of 14 insect species belonging to six orders showed great diversity in terms of both total proteinase inhibitory capacity and specificity. Most of the inhibitors exa­mined fall into two groups: low molecular mass proteins (below 10 kDa) related to Kunitz type inhibitors, and proteins of about 45 kDa which belong to the serpin superfamily of serine proteinase inhibitors. This minireview describes the properties, characteristics and possible biological significance of selected inhibitors.
In developing Gallería mellonella larvae (reared at 30°C) three proteins of 74/ 76 and 81/82 kDa were identified. They represent a group of storage proteins (LHP proteins). In Galleria larvae, the development of which is arrested by low temperature (18°C)/ accumulation of the 74, 76 and 81/82 kDa proteins was detected in the hemolymph. The synthesis of 74 kDa and 76 kDa proteins started after 24 h, and that of about 80 kDa after 96 h following the transfer of larvae from 30°C to 18°C. 20-Hydroxyecdysone inhibited synthesis of the 74 and 76 kDa proteins in larvae exposed to low temperature. The arrest of development of Galleria larvae is associated with the synthesis and accumulation of storage proteins, and ecdysteroids are involved in these processes.
Juvenile hormone (JH) is essential for multiple physiological processes: it controls larval development, metamorphosis and adult reproduction. In insect hemolymph more than 99 % of JH is bound to juvenile hormone binding protein (JHBP), which protects JH from degradation by nonspecific hydrolases and serves as a carrier to supply the hormone to the target tissues. In Galleria mellonella hemolymph, JHBP is found in a complex with lipid-binding high molecular weight proteins (HMWP) and this interaction is enhanced in the presence of JH. In this report, we present studies on the interaction of JHBP with low molecular weight proteins (LMWP) in the hemolymph. Using ligand blotting we found that JHBP interacts with a protein of about 44 kDa. To identify the protein that preferentially binds JHBP, a LMWP fraction was applied to a Sepharose-bound JHBP and, after washing, the column was eluted with free JHBP acting as a specific competitor or with carbonic anhydrase as a negative control. The eluted proteins were separated by SDS/PAGE and analyzed by mass spectrometry. Isocitrate dehydrogenase was identified as a component of the supramolecular complex of JHBP with hemolymph proteins.
The conotoxin-like (ctx) gene encodes a small cysteine-rich polypeptide in various baculoviruses. Previous research has demonstrated that the product of the ctx gene could be purified from insect cells infected by Autographa californica nuclear polyhedrosis virus (AcMNPV), but its function was unknown. In this paper, we compared the conserved cysteine motif structure (CX3GX2CX5CCX3CX6C) of the ctx gene in baculoviruses and generated recombinant Bombyx mori nuclear polyhedrosis virus (BmNPV) with the BmNPV bacmid system. !e recombinant BmNPV contained the ctx gene from AcMNPV or a fusion gene of ctx with eGFP, respectively. Fluorescence in CTX-eGFPpositive cells was mainly observed on the cell membrane. To gain insight into CTX function, two methods were used to elucidate the affect CTX had on hemolymph melanization in vivo and in vitro in insect larvae and pupae. The results indicated that CTX abrogates hemolymph melanization; however, the mechanisms require further evaluation.
The influence of extracts from Varroa destructor, a parasitic mite of the honeybee Apis mellifera, on the proteinase activity of worker bee haemolymph was analysed in vitro, along with the influence of bee haemolymph on the proteolytic activity of V. destructor extract. The study was conducted in three different environments: pH 7.5 (high activity of bee enzymes and very low activity of parasite enzymes), pH 5 (moderate activity of enzymes from both sources) and pH 3.5 (limited activity of bee proteinases and high activity of mite proteinases). Based on electrophoretic studies, the inhibition of the activity of bee haemolymph proteinases by V. destructor extracts was observed at each pH. The study at pH 7.5 with commercial inhibitors of the 4 main classes of proteinases (pepstatin A, ethylenediaminetetraacetic acid (EDTA), E-64 (trans-epoxysuccinyl-L-leucylamido-(4-guanidino)-butane), soybean trypsin inhibitor and Kunitz inhibitor) suggested that parasite extracts mainly inhibited serine proteinases and, to a lower degree, cysteine and aspartyl proteinases. At pH 3.5 and pH 5, a decrease of approximately 40% in parasite proteinase activity was also observed in the presence of bee haemolymph. The result points to the presence of aspartyl proteinase inhibitors in bee haemolymph, which may be an important defence element for bees during food intake by a mite. It was demonstrated that trypsin and trypsin inhibitors are active in the excretion/secretion products of V. destructor, the proteinases of which may assist the parasite in food suckling by preventing haemolymph coagulation, among other things.
The work describes purification and biochemical characterization of two inducible antimicrobial peptides from the hemolymph of Galleria mellonella. The peptides were isolated by a sequence of reversed-phase chromatography steps from the hemolymph of larvae immunized with viable bacteria. The first peptide is a member of the cecropin family while the second one is rich in proline residues and has a unique se­quence.
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