RESUMO
The present report shows a partial biochemical characterization and life cycle expression of N-ß-alanyldopamine hydrolase (Tan protein) in Ceratitis capitata and Drosophila melanogaster. This enzyme catalyzes the hydrolysis of N-ß-alanyldopamine (NBAD), the main tanning precursor of insect brown cuticles. It also plays an important role in the metabolism of brain neurotransmitters, recycling dopamine and histamine. In contrast to NBAD-synthase, Tan is expressed constitutively in epidermis and does not respond directly to microbial challenge. Immunodetection experiments showed the novel localization of NBAD-hydrolase in the embryo central neural system and in different regions of the adult brain, in addition to optic lobes. We sequenced and characterized Drosophila mutants tan¹ and tan³. The latter appears to be a mutant with normal expression in neural tissue but weak one in epidermis.
Assuntos
Ceratitis capitata/enzimologia , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Proteínas de Insetos/metabolismo , Animais , Sequência de Bases , Encéfalo/enzimologia , Ceratitis capitata/crescimento & desenvolvimento , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/genética , Derme/enzimologia , Proteínas de Drosophila/genética , Drosophila melanogaster/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Insetos/genética , Larva/enzimologia , Dados de Sequência Molecular , Fenótipo , Pupa/enzimologiaRESUMO
Dopamine (DA) and norepinephrine (NE) derivatives play an important role in the sclerotization and pigmentation of insect cuticles by serving as precursors for cuticular cross-linking. Protein preparations from prepupae of the medfly, Ceratitis capitata, were able to conjugate beta-alanine with DA producing N-beta-alanyldopamine (NBAD) or with NE, synthesizing N-beta-alanylnorepinephrine (NBANE). The latter reaction has been demonstrated for the first time. Apparent kinetic parameters were obtained for both substrates, DA (V(max)=30.7+/-6.0 pmol min(-1) mg(-1); K(m)=29.5+/-3.5 microM) and NE (V(max)=16.1+/-6.6 pmol min(-1)mg(-1); K(m)=89.0+/-8.3 microM). The same protein seems to be responsible for both enzymatic activities, judging from several criteria like identical behavior under heat inactivation as well as identical Mg2+ and Mn2+ dependent stimulation and Co2+ inhibition. Furthermore, the melanic mutants niger of C. capitata and ebony(4) of D. melanogaster, known to be defective for NBAD synthase, were also unable to synthesize NBANE. The protein preparation acylated tyrosine with much less efficiency, to produce sarcophagine (beta-alanyltyrosine). Strikingly, extracts from the melanic mutants were unable to synthesize sarcophagine. Our results strongly suggest that the enzymatic activity previously known as NBAD synthase is in fact a novel catalytic protein showing broad substrate specificity. We propose to identify it as catecholamine-beta-alanyl ligase.
Assuntos
Catecolaminas/metabolismo , Dípteros/enzimologia , Dopamina/análogos & derivados , Dopamina/biossíntese , Ligases/metabolismo , Norepinefrina/biossíntese , Animais , Extratos Celulares , Sistema Livre de Células , Dipeptídeos/biossíntese , Dípteros/genética , Dípteros/metabolismo , Dopa Descarboxilase/metabolismo , MelaninasRESUMO
There are no recent reports focusing on insect glycogen metabolism that take the advances made in mammalian and yeast systems into account. Moreover, little is known about glycogen synthesis and degradation during insect metamorphosis. The biosynthesis and mobilization of insect glycogen were measured during the larva to adult transition in the Medfly, Ceratitis capitata. The glycogen accumulated by larva decreased to reach almost undetectable levels at the beginning of the pupation process. Histological preparations of 40 h muscles and fat body confirmed a low glycogen content, in contrast with high glycogen images in third larva tissues. After 40 h, glycogen was synthesized de novo and accumulates up to adult ecdysis. We obtained the metamorphosis-dependent profiles of phosphorylase, glycogen synthase, and a glycogenin-like protein. This novel insect glycogen initiator protein (the first measured in an arthropod) appeared to be similar to the homologous enzymes from vertebrates and yeast. We have correlated these results with other biochemical events and anatomical landmarks to understand the use of storage carbohydrates during the sequence of metamorphosis events.
Assuntos
Dípteros/crescimento & desenvolvimento , Dípteros/metabolismo , Glicogênio/biossíntese , Amilases/metabolismo , Animais , Glucosiltransferases , Glicogênio/metabolismo , Glicogênio Sintase/metabolismo , Glicoproteínas/química , Glicoproteínas/isolamento & purificação , Glicoproteínas/metabolismo , Proteínas de Insetos/química , Proteínas de Insetos/isolamento & purificação , Proteínas de Insetos/metabolismo , Cinética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Metamorfose Biológica , Fosforilases/metabolismo , Pupa/crescimento & desenvolvimento , Pupa/metabolismo , Especificidade da Espécie , Distribuição TecidualRESUMO
A random amplified polymorphic DNA polymerase chain reaction method using 10-base long commercial random primers was used to identify polymorphisms in Argentine populations of 2 fruit fly pests. A fast and reliable discrimination between Mediterranean fruit fly, Ceratitis capitata (Wiedemann), and Anastrepha fraterculus (Wiedemann) immature or mature stages was obtained using as few as 1 insect per assay. The population of origin of immature individuals can be identified unambiguously using 2 primers.
Assuntos
Dípteros/genética , Técnica de Amplificação ao Acaso de DNA Polimórfico , Animais , Dípteros/classificaçãoRESUMO
A shadow moving over head elicits an escape response in the crab Chasmagnathus that habituates promptly and for a long period. The effect of the protein synthesis inhibitor cycloheximide (CY) on this long-term memory was analyzed. Two hours after injection, 10 micrograms CY inhibited [14C]-amino amino acid incorporation into cerebral plus thoracic ganglia by 88% and 20 micrograms by 92%, but no inhibition was found at 24 h. A single injection of 10-20 micrograms CY given 30 min before training, failed to affect the short-term habituation. Similar doses impaired both context memory (CM) and long-term habituation (LTH) when tested at 72 and 120 h but only CM at 24 h. Such a disparity was explained by an unspecific depressing effect upon the response, attributed to an interaction between CY and training. The hypothesis was confirmed, because CY injected immediately after training disclosed amnestic effect at 24 h on both CM and LTH. A similar effect was proven when animals were injected at 2 h but not at 6 h after training. Results from experiments with pretraining and pretesting injections put aside a state-dependence or retrieval deficit effects of the drug. Taken together, findings of this article argue strongly for de novo protein synthesis as a mechanism of LTH and for the close relation between CM and LTH.
Assuntos
Braquiúros/fisiologia , Cicloeximida/farmacologia , Habituação Psicofisiológica/efeitos dos fármacos , Memória/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Aminoácidos/metabolismo , Amnésia/induzido quimicamente , Amnésia/psicologia , Animais , Condicionamento Operante/efeitos dos fármacos , Reação de Fuga/efeitos dos fármacos , Gânglios dos Invertebrados/efeitos dos fármacos , Gânglios dos Invertebrados/metabolismo , MasculinoRESUMO
Cuticle proteins of an insect pest, the Medfly Ceratitis capitata, were resolved in polyacrylamide gels and partially characterized. The pupal cuticle was found to be different from cuticles of other insects since more than 80% w/w of the protein is a single mannose-containing polypeptide (PCG-100). The temporally-regulated in vivo biosynthesis and deposition of cuticle proteins was studied by microinjection of [35S]methionine followed by hand dissection of pupal cuticles. The major pupal glycoprotein, PCG-100, is cuticle- and stage-specific and was the earliest to be labeled and deposited. Its synthesis was maximal at around 46 hours after pupariation and then it decreased. The deposited PCG-100 and other minor pupal cuticle proteins become non-extractable at the end of the instar (7 days after pupariation) probably by sclerotization phenomena. These results provide insight into the temporal control of gene expression programs involved in cuticle deposition during medfly metamorphosis.
Assuntos
Proteínas de Insetos , Insetos/crescimento & desenvolvimento , Metamorfose Biológica , Biossíntese de Proteínas , Animais , Eletroforese em Gel de Poliacrilamida , Regulação da Expressão Gênica , Glicoproteínas/análise , Insetos/genética , Proteínas/genética , Proteínas/isolamento & purificaçãoRESUMO
In vivo and in vitro synthesis of chitin in Triatoma infestans was studied. For in vivo experiments, [14C] sugars were injected through the abdominal wall. Maximal incorporation of radioactivity into the cuticle was attained immediately after the ecdysis. The identification of in vivo synthesized chitin was performed by the enzymatic hydrolysis of the alkali-insoluble material from the cuticle with Helix chitinase. The main water-soluble compound obtained was N-acetylglucosamine as demonstrated by chromatographic procedures. In vitro synthesis of chitin was carried out with enzymatic crude extracts from Triatoma infestans, and UDP-N-acetylglucosamine was used as "source" of glycosyl moieties. Higher amounts of [14C] N-acetylglucosamine incorporation to chitin than those previously reported by others, were obtained. The identity of the product was confirmed in a similar way as that from in vivo synthesis. Radioactivity was also found in a liposoluble fraction concomitantly with chitin synthesis. This compound had an anionic behavior, was acid labile and had similar chromatographic properties as dolichol pyrophosphate N-acetylglucosamine obtained with pig liver extracts. Knowledge about dolichol phosphate sugars mediated glycoprotein synthesis in eukaryotes, suggests the involvement of this type of N-acetylglucosaminyl-phospholipid in macromolecule "building" even in insects.
Assuntos
Quitina/biossíntese , Triatoma/metabolismo , Triatominae/metabolismo , Uridina Difosfato N-Acetilglicosamina/metabolismo , Açúcares de Uridina Difosfato/metabolismo , Animais , Baratas/metabolismo , Técnicas In Vitro , Fígado/metabolismoRESUMO
In vivo and in vitro synthesis of chitin in Triatoma infestans was studied. For in vivo experiments, [14C] sugars were injected through the abdominal wall. Maximal incorporation of radioactivity into the cuticle was attained immediately after the ecdysis. The identification of in vivo synthesized chitin was performed by the enzymatic hydrolysis of the alkali-insoluble material from the cuticle with Helix chitinase. The main water-soluble compound obtained was N-acetylglucosamine as demonstrated by chromatographic procedures. In vitro synthesis of chitin was carried out with enzymatic crude extracts from Triatoma infestans, and UDP-N-acetylglucosamine was used as [quot ]source[quot ] of glycosyl moieties. Higher amounts of [14C] N-acetylglucosamine incorporation to chitin than those previously reported by others, were obtained. The identity of the product was confirmed in a similar way as that from in vivo synthesis. Radioactivity was also found in a liposoluble fraction concomitantly with chitin synthesis. This compound had an anionic behavior, was acid labile and had similar chromatographic properties as dolichol pyrophosphate N-acetylglucosamine obtained with pig liver extracts. Knowledge about dolichol phosphate sugars mediated glycoprotein synthesis in eukaryotes, suggests the involvement of this type of N-acetylglucosaminyl-phospholipid in macromolecule [quot ]building[quot ] even in insects.
RESUMO
In vivo and in vitro synthesis of chitin in Triatoma infestans was studied. For in vivo experiments, [14C] sugars were injected through the abdominal wall. Maximal incorporation of radioactivity into the cuticle was attained immediately after the ecdysis. The identification of in vivo synthesized chitin was performed by the enzymatic hydrolysis of the alkali-insoluble material from the cuticle with Helix chitinase. The main water-soluble compound obtained was N-acetylglucosamine as demonstrated by chromatographic procedures. In vitro synthesis of chitin was carried out with enzymatic crude extracts from Triatoma infestans, and UDP-N-acetylglucosamine was used as [quot ]source[quot ] of glycosyl moieties. Higher amounts of [14C] N-acetylglucosamine incorporation to chitin than those previously reported by others, were obtained. The identity of the product was confirmed in a similar way as that from in vivo synthesis. Radioactivity was also found in a liposoluble fraction concomitantly with chitin synthesis. This compound had an anionic behavior, was acid labile and had similar chromatographic properties as dolichol pyrophosphate N-acetylglucosamine obtained with pig liver extracts. Knowledge about dolichol phosphate sugars mediated glycoprotein synthesis in eukaryotes, suggests the involvement of this type of N-acetylglucosaminyl-phospholipid in macromolecule [quot ]building[quot ] even in insects.