RESUMO
Recently we have shown that BhSGAMP-1 is a developmentally regulated reiterated gene that encodes an antimicrobial peptide (AMP) and is expressed exclusively in the salivary glands, at the end of the larval stage. We show, for the first time, that a gene for an AMP is directly activated by 20-OH ecdysone. This control probably involves the participation of short-lived repressor(s). We also found that the promoter of BhSGAMP-1 is not equipped with elements that respond to infection, provoked by the injection of microorganisms, in the salivary glands or in the fat body. We produced polyclonal antibodies against the synthetic peptide and found that the BhSGAMP-1 peptide is secreted in the saliva. The BhSGAMP-1 gene was also activated during the third larval molt. These facts confirm our hypothesis that this preventive system of defense was selected to produce an environment free of harmful microorganisms in the insect's immediate vicinity, during molts.
Assuntos
Dípteros/genética , Ecdisterona/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Proteínas de Insetos/genética , Glândulas Salivares/efeitos dos fármacos , Proteínas e Peptídeos Salivares/genética , Animais , Western Blotting , Dípteros/metabolismo , Dípteros/microbiologia , Ecdisterona/fisiologia , Eletroforese em Gel de Poliacrilamida , Escherichia coli/fisiologia , Interações Hospedeiro-Patógeno , Proteínas de Insetos/metabolismo , Larva/efeitos dos fármacos , Larva/genética , Larva/microbiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saccharomyces cerevisiae/fisiologia , Glândulas Salivares/metabolismo , Proteínas e Peptídeos Salivares/metabolismo , Staphylococcus aureus/fisiologiaRESUMO
Phenoloxidase (monophenol, l-dopa: oxygen oxidoreductase, EC 1.14.18.1) is a multicopper oxidase, which plays an important role in melanin synthesis, necessary for defense against intruding microorganisms and parasites, wound healing and cuticle pigmentation. A phenoloxidase from the hemolymph of honey bee pupae exhibited an apparent molecular mass of 70 kDa, as estimated by gel filtration and SDS-PAGE. Optimal pH and temperature were 6.5 and 20 degrees C, respectively. Activity was fully stable for 30 min at 50 degrees C. Like phenoloxidases from the hemolymph of other insects, the honey bee enzyme was activated by trypsin and inhibited by protease inhibitors and phenylthiourea. Only high concentrations of sodium azide effectively inhibited the detected activity. A low concentration (5 microM) of Ca2+, Mg2+, and Mn2+ had a stimulatory effect on the activity. Single Michaelis-Menten curves were observed for l-dopa and dopamine oxidation, but the affinity of the enzyme for dopamine was greater than for L-dopa. Semiquantitative RT-PCR and Southern blot analysis using a 359 bp labeled probe, and quantification of the prophenoloxidase mRNA levels by real-time PCR showed increased amounts of transcripts in hemocytes and integument from young pupae injected with 20-hydroxyecdysone.