RESUMEN
Trypsin-modulating oostatic factor (TMOF), a mosquito decapeptide, terminates trypsin biosynthesis in the mosquito gut. The hormone is secreted from the ovary, starting 18 h after the blood meal, circulates in the hemolymph, binds to a gut receptor and stops trypsin biosynthesis by exerting a translational control on trypsin mRNA. Because of the unique primary amino acid sequence of the hormone (YDPAPPPPPP) and its stable three-dimensional conformation, TMOF is not degraded by gut proteolytic enzymes and can traverse the gut epithelial cells into the hemolymph of adults and larvae. Using this unique property, hormone fed to different species of mosquito larvae stops food digestion and causes larval mortality. To determine the shortest amino acid sequence that can bind to the gut receptor and still cause high larval mortality, 25 analogues of TMOF were synthesized and tested. The tetrapeptide (YDPA) was as effective as the decapeptide, indicating that the binding to the gut receptor is at the N-terminus of the molecule. Cloning and expressing the hormone on the coat protein of tobacco mosaic virus (TMV) in Chlorella sp. and Saccharomyces cerevisiae cells and feeding the recombinant cells to mosquito larvae caused larval mortality. These results indicate that TMOF can be used as a new biorational insecticide against mosquito larvae.
Asunto(s)
Culicidae/metabolismo , Digestión/efectos de los fármacos , Regulación de la Expresión Génica , Control de Mosquitos/métodos , Oligopéptidos/farmacología , Tripsina/biosíntesis , Secuencia de Aminoácidos , Animales , Proteínas de la Cápside/metabolismo , Chlorella , Culicidae/efectos de los fármacos , Hormonas de Insectos/metabolismo , Larva/efectos de los fármacos , Larva/metabolismo , Oligopéptidos/genética , Oligopéptidos/metabolismo , Conformación Proteica , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae , Virus del Mosaico del Tabaco/metabolismoRESUMEN
Proteolytic enzyme biosynthesis in the midgut of the 4th instar larva of Heliothis virescens is cyclical. Protease activity increases immediately after the molt from the 3rd to the 4th instar larvae and declines just before the molt into the 5th instar. Characterization of the midgut proteases using soybean tryspin inhibitor (SBTI) Bowman Birk Inhibitor (BBI) 4-(2-aminoethyl)benzensulfonylfluoride (AEBSF) and N-tosyl-L-phenylalanine chloromethylketone (TPCK) indicate that protease activity is mostly trypsinlike (80%) with a small amount of chymotrypsinlike activity (20%). Injections of late 3rd and 4th instar larval hemolymph into H. virescens larvae inhibited tryspin biosynthesis in the larval midgut. Similar results were obtained when highly purified 4th instar larval hemolymph that crossreacted with Aea-TMOF antisurm using ELISA was injected into 2nd instar larvae. Injections of Aea-TMOF and its analogues into 2nd instar, and Aea-TMOF alone into 4th instar larvae stopped trypsin biosynthesis 24 and 48 h after the injections, respectively. Injections of 4th instar H. virescens larval hemolymph into female Aedes aegypti that took a blood meal stopped trypsin biosynthesis and egg development. These results show that the biosynthesis of trypsin-like enzymes in the midgut of a lepidoptera is modulated with a hemolymph circulating TMOF-like factor that is closely related to Aea-TMOF. Arch.
Asunto(s)
Mariposas Nocturnas/enzimología , Oligopéptidos/metabolismo , Serina Endopeptidasas/biosíntesis , Animales , Hemolinfa/metabolismo , Larva/enzimología , Mariposas Nocturnas/crecimiento & desarrollo , Inhibidores de Serina Proteinasa/farmacología , Aumento de PesoRESUMEN
Trypsin modulating oostatic factor (TMOF) is a decapeptide that inhibits the biosynthesis of trypsin-like enzymes in the midgut of several insect species and, as such, serves as a dipteran oostatic hormone. In vitro incubation of lepidopteran prothoracic glands with Aedes aegypti TMOF revealed that this decapeptide, in the presence of brain extract, modulates ecdysteroid production. The modulatory effect was highly dependent on both the concentration of TMOF and brain extract. Typically, TMOF was stimulatory in the presence of lower concentrations of Lymantria dispar brain extract (0.01 and 0. 025 brain equivalent), and either neutral or inhibitory at higher concentrations (0.25, 0.5, and 1.0 brain equivalent) of extract. In the presence of European corn borer (Ostrinia nubilalis) brain extract, TMOF also exhibited modulatory effects, effects that again were dependent on the concentrations of both brain extract and TMOF present in the incubation medium. At 1.5 brain equivalents, TMOF was inhibitory at all but the highest concentration tested (5x10(-6) M), at 1.0 brain equivalent, TMOF was stimulatory at 10(-6) M and at 0. 5 brain equivalents, TMOF did not significantly affect PTG synthesis of ecdysteroids. Results suggest the presence of a modulatory peptide(s), which fine tunes the synthesis and release of ecdysteroids by PTGs in accordance with the insect's developmental/physiological requirements.
Asunto(s)
Aedes/metabolismo , Hormonas de Insectos/farmacología , Mariposas Nocturnas/efectos de los fármacos , Mariposas Nocturnas/metabolismo , Oligopéptidos/farmacología , Esteroides/biosíntesis , Animales , Encéfalo/metabolismo , Ecdisteroides , Glándulas Endocrinas/efectos de los fármacos , Glándulas Endocrinas/metabolismo , Técnicas In VitroRESUMEN
Trypsin mRNA from the citrus weevil, Diaprepes abbreviatus, was reverse transcribed and amplified by PCR. A cDNA species of 513 bp was cloned and sequenced. The 3' and 5' ends of the gene (262 bp and 237 bp, respectively) were amplified by rapid amplification of cDNA ends, cloned and sequenced. The deduced sequence of the trypsin cDNA (860 bp) encodes for 250 amino acids including 11 amino acids of activation and signal peptides and exhibited 16.8% identity to trypsin genes of selected Lepidoptera and Diptera. A three-dimensional model of Diaprepes trypsin contained two domains of beta-barrel sheets as has been found in Drosophila and Neobellieria. The catalytic active site is composed of the canonical triad of His41, Asp92 and Ser185 and a specificity pocket occupied by Asp179 with maximal activity at pH 10.4. Southern blot analysis indicated that at least two copies of the gene are encoded by Diaprepes midgut. Northern blot analysis detected a single RNA band below 1.35 kb at different larval ages (28-100 days old). The message increased with age and was most abundant at 100 days. Trypsin activity, on the other hand, reached a peak at 50 days and fell rapidly afterwards indicating that the trypsin message is probably regulated translationally. Feeding of soybean trypsin inhibitor and Aedes aegypti trypsin modulating oostatic factor affected trypsin activity and trypsin biosynthesis, respectively. These results indicate that Diaprepes regulates trypsin biosynthesis with a trypsin modulating oostatic factor-like signal.
Asunto(s)
Escarabajos/enzimología , ADN Complementario/química , ADN Complementario/genética , Tripsina/química , Tripsina/genética , Aedes/enzimología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Citrus/parasitología , Clonación Molecular , Escarabajos/genética , ADN Complementario/biosíntesis , Genes de Insecto , Concentración de Iones de Hidrógeno , Hormonas de Insectos/farmacología , Proteínas de Insectos/biosíntesis , Proteínas de Insectos/química , Proteínas de Insectos/genética , Larva/efectos de los fármacos , Larva/enzimología , Larva/genética , Larva/crecimiento & desarrollo , Datos de Secuencia Molecular , Oligopéptidos/farmacología , Proteínas de Plantas , ARN Mensajero/metabolismo , Proteínas de Soja , Glycine max , Tripsina/biosíntesis , Inhibidores de Tripsina/farmacologíaRESUMEN
The cDNA coding for a Ser-protease-related protein (Scg-SPRP) was cloned from desert locust (Schistocerca gregaria) midgut. The derived amino acid sequence consists of 260 residues and shows strong sequence similarity to insect trypsin-like molecules. It is, however, likely that Scg-SPRP is not a proteolytically active enzyme and that it plays another physiologically relevant role, since two out of three residues which are indispensable for catalytic activity of Ser-proteases are replaced. Northern analysis revealed that the Scg-SPRP gene is expressed in midgut tissue and that this expression is strongly induced in adult female locusts. Moreover, the occurrence of the transcript (1.2 kb) fluctuates during the molting cycle and during the female reproductive cycle. Juvenile hormone (JH III) dependence of transcription was investigated by chemical allatectomy (precocene I) of adult females. This resulted in inhibition of vitellogenesis and in disappearance of the Scg-SPRP transcript. Expression of Scg-SPRP in precocene-treated locusts could be reinduced by additional treatment with JH III or with 20-OH-ecdysone.
Asunto(s)
Saltamontes/genética , Proteínas de Insectos/genética , Serina Endopeptidasas/genética , Vitelogeninas/biosíntesis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Benzopiranos/farmacología , Clonación Molecular , ADN Complementario/genética , Sistema Digestivo/enzimología , Femenino , Saltamontes/efectos de los fármacos , Saltamontes/enzimología , Hormonas Juveniles/antagonistas & inhibidores , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Reproducción , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Sesquiterpenos/farmacologíaRESUMEN
The hexapeptide Neb-TMOF (H-NPTNLH-OH, trypsin modulating oostatic factor of the gray fleshfly, Neobellieria bullata)2 occurs in vitellogenic ovaries and is involved in negative feedback regulation of trypsin biosynthesis in the gut of late vitellogenic females. Polyclonal antisera were raised against the synthetic peptide and were used to identify and immunolocalize Neb-TMOF epitopes in different fleshfly tissues. Neb-TMOF-immunoreactive material first appears in the cortical layer of young vitellogenic oocytes and later spreads over the yolk granules. This suggests a pinocytosis with the three yolk polypeptides (vitellogenins). Controls treated with the preimmune sera or with anti-Neb-TMOF antiserum preadsorbed to Neb-TMOF peptide coupled to a solid phase support did not stain. There was no immunostaining in the central nervous system (brain and ventral nerve cord), the retrocerebral complex, the fat body, or the testes. Western blot analysis showed that the anti-Neb-TMOF antisera specifically recognize a putative hormone precursor polypeptide (Mr 75 kDa) from vitellogenic ovaries. This protein is virtually absent from the hemolymph. It is not immunologically related to the three yolk polypeptides, since it is not recognized by yolk polypeptides antisera. In adult females the ovary appears to be the only site of synthesis of Neb-TMOF and of its precursor. Immunopositive staining is found in the apical areas of ovarian follicle cells, suggesting these cells as a site of hormone precursor biosynthesis. This is the first demonstration that a protein colocalized with yolk proteins might act as a precursor for a folliculostatic hormone.
Asunto(s)
Dípteros/química , Hormonas de Insectos/análisis , Oligopéptidos/análisis , Oocitos/química , Vitelogénesis/fisiología , Animales , Western Blotting , Drosophila melanogaster , Proteínas del Huevo/inmunología , Electroforesis en Gel de Poliacrilamida , Femenino , Saltamontes , Hemolinfa/inmunología , Moscas Domésticas , Sueros Inmunes/inmunología , Inmunohistoquímica , Hormonas de Insectos/inmunología , Oligopéptidos/inmunología , Oocitos/inmunología , Oocitos/ultraestructura , Sensibilidad y Especificidad , Membrana Vitelina/ultraestructura , Vitelogénesis/inmunologíaRESUMEN
Trypsin mRNA from the grey fleshfly (Neobellieria bullata) was reversed transcribed and amplified by means of PCR. Two cDNA species of 600 bp and 800 bp were cloned and sequenced. The 3' end of the gene (300 bp) was amplified by means of the rapid-amplification-of-cDNA-ends method, cloned and sequenced. The deduced protein sequence of 254 amino acids exhibited 46% identity to Drosophila trypsin and 32% identity to Anophiline trypsin and Aedes trypsin. Three-dimensional models of Neobellieria trypsin and Drosophilia trypsin were built and compared. Both models contain two domains of beta-barrel sheets as was shown by means of X-ray crystallography of mammalian trypsin. The catalytic active site is composed of the canonical triad of His42, Asp87 and Ser182 whereas Asp176 sits as the bottom of the specificity pocket. Southern blot analysis suggested that Neobellieria trypsin is encoded by one gene. Northern blot analysis showed that an early trypsin transcript is found in the midgut of sugar-fed females. This message disappeared after a liver meal, and was replaced by a late transcript. Injection of trypsin-modulating oostatic factor (TMOF) at 10(-9) M prevented the disappearance and the translation of the early transcript. TMOF did not prevent the appearance of the late transcript. However, in the presence of the hormone the late transcript was not translated. Thus, TMOF is the biological signal that terminates the translation of trypsin mRNA in the fleshfly gut and probably in the mosquito gut.
Asunto(s)
Dípteros/química , Hormonas de Insectos/fisiología , Oligopéptidos/fisiología , Biosíntesis de Proteínas/fisiología , Tripsina/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario , Femenino , Humanos , Masculino , Modelos Moleculares , Datos de Secuencia Molecular , ARN Mensajero/genética , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido , Tripsina/genética , Tripsina/aislamiento & purificaciónRESUMEN
Female Aedes aegypti that were given a blood meal by enema deposited yolk in their oocytes and synthesized trypsinlike enzymes in their midgut. When females were given an enema of Aea-TMOF (Trypsin Modulating Oostatic Factor) (NH2-YDPAPPPPPP-COOH) and blood both egg development and trypsin biosynthesis were inhibited. Similar results were observed if TMOF was mixed with the blood meal and fed to female mosquitoes through a membrane. Renin inhibitor (NH2-PHPFHFFVYK-COOH) or poly proline given by enema with the blood meal did not affect egg development or trypsin biosynthesis. Feeding of TMOF analogs P1 (NH2-YDPAP-COOH) or P4 (NH2-YDPAPPPP-COOH) inhibited trypsin biosynthesis in the midgut. Injecting or giving an enema of an amidated peptide (NH2-WRPGPPPPPP-CONH2) of HIV-2 X-ORF protein also inhibited egg development and trypsin biosynthesis in the mosquito gut. When [3H]TMOF was purified by high performance liquid chromatography (HPLC) and fed with the blood meal through a membrane to female mosquitoes, [3H]TMOF outside the gut increased linearly for the first 24 h and 28% of the hormone was found outside the gut at 72 h. These results suggest that TMOF and its active analogs traverse the gut epithelial cells into the hemolymph, bind TMOF gut receptor(s) and modulate trypsin biosynthesis.
Asunto(s)
Aedes/efectos de los fármacos , Hormonas de Insectos/farmacología , Oligopéptidos/farmacología , Óvulo/efectos de los fármacos , Tripsina/biosíntesis , Aedes/embriología , Secuencia de Aminoácidos , Animales , Transporte Biológico , Fenómenos Fisiológicos Sanguíneos , Enema , Femenino , Hemolinfa/metabolismo , Hormonas de Insectos/farmacocinética , Mucosa Intestinal/citología , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Datos de Secuencia Molecular , Oligopéptidos/farmacocinéticaRESUMEN
The sequences of two folliculostatic peptides of the fleshfly Neobellieria bullata have been determined recently. The first peptide (Neb-TMOF: H-NPTNLH-OH), originates from a 75 kDa precursor protein found in vitellogenic oocytes. The hexapeptide directly inhibits the synthesis of trypsin-like enzymes in the gut, and thus lowers the concentration of yolk polypeptides in the hemolymph. It also inhibits the biosynthesis of ecdysone in the larval ring gland. Therefore, it could also be named prothoracicostatic hormone (Neb-PTSH). The second peptide (Neb-colloostatin: H-SIV-PLGLPVPIGPIVVGPR-OH) acts on previtellogenic follicles and is a cleaved product of a collagen-like precursor molecule. Our results indicate that peptides that are cleaved from matrix proteins could act as growth-inhibiting factors. Gonadotropin releasing hormone (GnRH)-immunolike peptides were not identified, but progress is being made in the isolation and characterization of factors which stimulate cAMP production by the ovary. Using these results, a novel model of growth control in which matrix proteins play an important role as a potential source of growth regulators has been developed.
Asunto(s)
Dípteros/crecimiento & desarrollo , Gonadotropinas/fisiología , Inhibinas/fisiología , Hormonas de Insectos/fisiología , Proteínas de Insectos , Secuencia de Aminoácidos , Animales , Dípteros/fisiología , Femenino , Hormonas de Insectos/química , Larva/crecimiento & desarrollo , Masculino , Modelos Químicos , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/fisiología , Oligopéptidos/fisiología , VitelogénesisRESUMEN
During the purification of trypsin-modulating oostatic factor (TMOF) of the grey fleshfly Neobellieria bullata, a new factor with oostatic activity was discovered. We report herein its purification, primary structure and effects on oocyte development. Its amino acid sequence was determined as H-SIVPLGLPVPIGPIVVGPR-OH. Due to structural sequence similarities with parts of several known collagens and its oostatic activity, we named it Neb-colloostatin. The synthetic peptide inhibits yolk uptake by previtellogenic oocytes and might have a role in the absence of yolk deposition in penultimate oocytes. Neb-colloostatin does not inhibit trypsin biosynthesis in the gut or ecdysone biosynthesis by larval ring glands. It decreases vitellogenin concentrations in the hemolymph by an unknown mode of action. The role of extracellular matrix proteins in the feedback control of growth is discussed.
Asunto(s)
Dípteros/química , Inhibinas/aislamiento & purificación , Oocitos/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Colágeno/metabolismo , Proteínas de la Matriz Extracelular/fisiología , Femenino , Inhibinas/química , Inhibinas/farmacología , Masculino , Datos de Secuencia Molecular , Oocitos/crecimiento & desarrollo , Vitelogeninas/análisisRESUMEN
A sensitive and specific colorimetric dot assay following polymerase chain reaction (PCR) method has been developed to detect 0.1 pg of eastern equine encephalomyelitis viral (EEEV) RNA. The assay is 250-fold more sensitive than analysis by electrophoresis and is based on converting a 291-nucleotide sequence of the viral coat protein amino terminus into a double-stranded DNA (dsDNA) and amplifying the DNA using a specific primer pair and PCR. The amplified complementary DNA (cDNA) is denatured adsorbed onto a nylon strip, baked, and detected with a digoxigenin-labeled probe. Dots with viral cDNA are stained dark red, whereas controls do not stain or stain lightly. The assay is very specific and sensitive and detects only EEEV. RNA of Venezuelan equine encephalitis, St. Louis encephalitis, Keystone, Flanders, Tensaw, and western equine encephalitis viruses were not detected. EEEV (Ten Broeck) RNA was detected at the 10-ng level, indicating that the prototype we used may have different nucleotides in the region where the primer pair binds. The PCR amplified EEEV cDNA that was 92% homologous to the consensus sequence of EEEV. The detection of EEEV in the liver of an infected Emu bird and in field-collected mosquitoes from Florida and Massachusetts that were analyzed concurrently as blind samples by tissue culture plaque assay and by PCR dot analysis proved that the assay is sensitive and can be used to detect infected mosquitoes. The assay can detect at least 1 infected mosquito in a pool of 1,000 uninfected mosquitoes.
Asunto(s)
Culicidae/virología , Virus de la Encefalitis Equina del Este/aislamiento & purificación , ARN Viral/análisis , Animales , Secuencia de Bases , Cromatografía Líquida de Alta Presión/veterinaria , Colorimetría/métodos , Colorimetría/veterinaria , ADN Complementario/química , Electroforesis en Gel de Agar/veterinaria , Virus de la Encefalitis Equina del Este/genética , Immunoblotting/veterinaria , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/veterinariaRESUMEN
The role of the male accessory glands (MAG) in reproduction was investigated in the mosquito Aedes aegypti. MAG incubated with [14C]acetate synthesized radioactively labeled JH III, JH III bisepoxide and methyl farnesoate. MAG incubated with L-[methyl-3H]methionine synthesized [3H]JH III and a molecule that chromatographed on HPLC with JH I. Analysis of MAG and whole males extract by glass capillary combined gas-chromatography-selected ion monitoring mass spectrometry identified JH III and I as the main analogs that were synthesized by male mosquitoes. MAG of Culex nigripalpus, Anopheles rangeli and Anopheles trinkae also synthesized JH III from L-[methyl-3H]methionine, which indicates that the male mosquito has a complete JH III biosynthetic pathway. Unfed and unmated Culex quinquefasciatus do not develop their ovaries to the resting stage. Females injected with one MAG extract equivalent or implanted with A. aegypti MAG developed their ovaries to the resting previtellogenic stage, whereas females that were injected with saline did not. These results indicate that MAG synthesize and secrete JH III. The corpora allata (CA) of the male Aedes aegypti also synthesize JH III from L-[methyl-3H]methionine. This observation may suggest that JH synthesized by the male's CA is used for internal regulation, whereas JH synthesized by the MAG is transferred with the sperm into the female.
Asunto(s)
Aedes/metabolismo , Hormonas Juveniles/biosíntesis , Sesquiterpenos/metabolismo , Aedes/crecimiento & desarrollo , Animales , Anopheles/metabolismo , Corpora Allata , Culex/crecimiento & desarrollo , Culex/metabolismo , Ácidos Grasos Insaturados/biosíntesis , Femenino , Genitales Masculinos/metabolismo , Técnicas In Vitro , Hormonas Juveniles/aislamiento & purificación , Masculino , Oocitos/crecimiento & desarrollo , Ovario/crecimiento & desarrollo , Sesquiterpenos/aislamiento & purificaciónRESUMEN
The gut receptor of trypsin-modulating oostatic factor (TMOF), a decapeptide hormone that regulates trypsin biosynthesis in the mosquito gut, has been characterized. The binding of TMOF to mosquito gut membranes reached maximum at pH 7.4 and 24 degrees C. No binding was observed at pH 2.5 and the binding to the membranes declined rapidly at pH 8.0. At equilibrium, maximum binding to the receptor was observed at 60 min and 24 degrees C. A synthetic complementary decapeptide NH2-Ile-Leu-Gly-Arg-Gly-Gly-Gly-Gly-Gly-Gly-COOH (FOMT) for TMOF successfully competed with the gut receptor, and specifically bound TMOF (Kd = 4 microM and Kassoc = 2.5 x 10(5) M-1). TMOF binding to gut membranes was characterized with FOMT and a specific ELISA to the hormone at 24 and 72 h after blood feeding. Two classes of binding sites were found on the gut membrane; high affinity (Kd1 = 4.6 +/- 0.7 x 10(-7) M; Kassoc = 2.2 x 10(6) M-1 Bmax = 0.1 pmol/gut) and low affinity (Kd2 = 4.43 +/- 1 x 10(-6) M; Kassoc = 2.3 x 10(5) M-1; Bmax = 0.2 pmol/gut). The total binding sites for high and low affinity classes of TMOF per gut were estimated as 6.3 x 10(10) and 1.1 x 10(11) sites, respectively. Specific binding sites on the gut increased after the blood meal and were visualized by immunocytochemical staining. These results suggest that TMOF regulates trypsin biosynthesis by binding to specific receptor sites that are located on the mosquito gut, and that this receptor can be studied using a complementary peptide approach.
Asunto(s)
Culicidae/química , Mucosa Intestinal/metabolismo , Oligopéptidos/metabolismo , Receptores de Péptidos/análisis , Secuencia de Aminoácidos , Animales , Femenino , Inmunohistoquímica , Datos de Secuencia Molecular , Receptores de Péptidos/fisiologíaRESUMEN
Injection of crude extracts of late vitellogenic ovaries into staged females of the grey fleshfly Neobellieria (Sarcophaga) bullata inhibited oocyte development and biosynthesis of trypsin-like enzymes in the gut. Trypsin synthesis in N. bullata is cyclic and is correlated with egg development, which is discontinuous. A trypsin modulating oostatic factor (Neb-TMOF) was purified from 10,000 vitellogenic ovaries and sequenced by mass spectrometry. Neb-TMOF is a hexapeptide (NH2-NPTNLH-COOH). Injection of the hormone at physiological concentrations (10(-9) M), inhibited trypsin-like synthesis by the midgut of liver-fed female flies, and caused a reduction of the vitellogenin concentration in the hemolymph and of oocyte growth. The role of Neb-TMOF in controlling egg development and the physiological similarities with Aedes-TMOF are discussed.
Asunto(s)
Dípteros/química , Hormonas de Insectos/química , Oligopéptidos/química , Aedes , Secuencia de Aminoácidos , Animales , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Femenino , Hormonas de Insectos/genética , Hormonas de Insectos/aislamiento & purificación , Hormonas de Insectos/farmacología , Masculino , Espectrometría de Masas , Datos de Secuencia Molecular , Oligopéptidos/genética , Oligopéptidos/aislamiento & purificación , Oligopéptidos/farmacología , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Ovario/química , Análisis de Secuencia , Tripsina/biosíntesis , Vitelogeninas/biosíntesisRESUMEN
Trypsin modulating oostatic factor (TMOF), a decapeptide that directly inhibits the biosynthesis of trypsin- and chymotrypsin-like enzymes in epithelial cells of mosquito midgut and indirectly inhibits vitellogenesis in anautogenous females, has been sequenced by Fourier transform mass spectrometry analysis. The peptide has a primary amino acid sequence of NH2-Tyr-Asp-Pro-Ala-(Pro)6-COOH and probably exhibits left-handed helical conformation as was shown by computer stereoview simulation. The factor is metabolized very rapidly (half-life of 1.6 h) in intact mosquitoes when injected after the blood meal. Inhibition of trypsin biosynthesis was followed in ligated abdomens, which synthesize trypsin but do not metabolise TMOF. At concentrations of 3 x 10(-9) M and 6.8 x 10(-6) M, TMOF inhibited 50 and 90% of trypsin-like enzyme biosynthesis, respectively. Several analogs of varying chain lengths were synthesized and evaluated for biological activity using dose-response curves. Switching the positions of Tyr and Asp at the N-terminus reduced the activity of the hormone, indicating that the N-terminus is important for biological activity. Removal of two to five prolines at the C-terminus also reduced activity, indicating that both the N- and C-termini are important. Synthesis of trypsin-like isozyme was followed in several insect species using [1,3-3H]diisopropyl-fluorophosphate (DFP) in the presence of tosylamide-2-phenylethyl chloromethyl ketone. Marked reduction of [1,3-3H]diisopropyl-phosphoryl-trypsin-like derivatives was noted after TMOF treatment, as assessed by polyacrylamide gel electrophoresis. These results indicate that the biosynthesis of trypsin-like enzyme in mosquitoes and other insects may be regulated by sequence-related TMOFs.
Asunto(s)
Aedes/química , Hormonas de Insectos/química , Oligopéptidos/química , Secuencia de Aminoácidos , Animales , Quimotripsina/biosíntesis , Dípteros , Femenino , Análisis de Fourier , Hormonas de Insectos/fisiología , Espectrometría de Masas/métodos , Modelos Moleculares , Datos de Secuencia Molecular , Oligopéptidos/fisiología , Tripsina/biosíntesisRESUMEN
Changes in the biosynthesis of serine proteases in adult Lutzomyia anthophora Addis were followed and compared with the larval and pupal stages. More chymotrypsinlike than trypsinlike enzyme was synthesized by 2-d-old and 3-d-old sugar-fed females and females that were fed blood 72 h earlier. A small increase in the amount of chymotrypsinlike enzyme occurred within the first 48 h after blood feeding, whereas trypsinlike enzyme activity increased rapidly after the blood meal and peaked at 72 h. [1,3-3H]DIP trypsinlike and chymotrypsinlike derivatives of sugar-fed and blood-fed females were compared using polyacrylamide gel electrophoresis.
Asunto(s)
Sistema Digestivo/enzimología , Isoenzimas/biosíntesis , Psychodidae/enzimología , Serina Endopeptidasas/biosíntesis , Animales , Quimotripsina/biosíntesis , Electroforesis en Gel de Poliacrilamida , Conducta Alimentaria , Femenino , Factores de Tiempo , Tripsina/biosíntesisRESUMEN
The solution structure of trypsin modulating oostatic factor (TMOF), a decapeptide (H-YDPAPPPPPP-OH) hormone that signals the termination of trypsin-like biosynthesis in mosquito midgut epithelial cells, was determined by 2-D 1H nuclear magnetic resonance spectroscopy and molecular modeling. The peptide forms a rod-shaped left-handed helix about 30 A long. No evidence was found to support a poly-L-proline beta-turn model. Hydrophobic contacts between the rings of tyrosine 1 and proline 3 may enhance the stability of the N-terminal segment. This peptide provides an interesting exception to the normal chemical shift index (csi) rules. Our results suggest that a sequence of positive csi indices, normally expected for a beta-strand structure, could also describe a left-handed poly-L-proline-like helix.
Asunto(s)
Oligopéptidos/química , Estructura Secundaria de Proteína , Secuencia de Aminoácidos , Hidrógeno , Espectroscopía de Resonancia Magnética , Modelos Estructurales , Datos de Secuencia Molecular , Oligopéptidos/síntesis química , SolucionesRESUMEN
Biosynthesis and metabolism of juvenile hormone (JH) III in vivo and in vitro were studied in female Aedes aegypti (L.). [12-3H]Methyl farnesoate was used to follow the synthesis and [12-3H]-(10R)-JH III to study metabolism. The rate of biosynthesis of [12-3H]JH III in vivo after adult eclosion increased from 9 fmol/h per female at 1 h to 22 fmol/h per female at day 6. The rate of biosynthesis by exposed corpora allata (CA) in vitro was 23 fmol/h per CA during the 1st d after adult eclosion, then dropped to 4.8 fmol/h per CA on day 3, then increased again to a constant level of synthesis (12 fmol/h per CA) at days 4-6. Immediately after blood feeding, the rate of synthesis of [12-3H]JH III in vivo and in vitro increased to 27 fmol/h per female and to 23 fmol/h per CA, respectively. The rate of synthesis then decreased in vivo to 12 fmol/h per female at 4 h and in vitro to 6 fmol/h per CA 10 h after the blood meal. After this decrease, the rate of synthesis of [12-3H]JH III increased again reaching a peak of 25 fmol/h per female at 48-96 h in vivo and 12 fmol/h per CA at 72 h in vitro. These results indicated that the CA of sugar-fed and blood-fed female A. aegypti synthesized JH III in vivo and in vitro from [12-3H]methyl farnesoate. When [12-3H]-(10R)-JH III metabolism was followed in vivo in female A. aegypti, the ratio between JH III diol acid:JH III acid:JH III diol was 17:4:1, indicating that JH III was first hydrolyzed by JH III esterase to the acid form, then hydrated to the diol acid by JH III epoxide hydrase. Females treated with [12-3H]JH III acid converted 46% of the JH III acid in 60 min to the diol acid. These results indicated that the enzyme epoxide hydrase acted on JH III acid 17 times faster than JH III.
Asunto(s)
Aedes/metabolismo , Corpora Allata/metabolismo , Ácidos Grasos Insaturados/biosíntesis , Hormonas Juveniles/biosíntesis , Sesquiterpenos/metabolismo , Animales , FemeninoRESUMEN
The biosynthesis of trypsinlike and chymotrypsinlike enzymes was followed in the four instars and pupa of Lutzomyia anthophora Addis. A 32-fold increase in the biosynthesis of trypsinlike enzymes was observed from the first to the fourth instar. Trypsinlike and chymotrypsinlike isozymes were also synthesized by pupae 1-8 d old. Similarly, a 29-fold increase in the biosynthesis of chymotrypsinlike isozymes also was observed from first to fourth instars. Several different [1,3-3H]DIP trypsinlike and chymotrypsinlike derivatives of first to fourth instars and pupae (1 and 8 d old) were studied using polyacrylamide gel electrophoresis and fluorography.
Asunto(s)
Quimotripsina/biosíntesis , Insectos Vectores/enzimología , Isoenzimas/biosíntesis , Psychodidae/enzimología , Tripsina/biosíntesis , Animales , Larva/metabolismo , Pupa/metabolismoRESUMEN
The in-vitro biosynthesis of [12-3H] juvenile hormone (JH) III by exposed corpora allata (CA) of teneral, sugar-fed, and blood-fed female Lutzomyia anthophora (Addis) was followed by incubating the CA for 4 h with [12-3H]methyl farnesoate. Synthesis of [12-3H]H III was determined by C18 reversed-phase high-pressure liquid chromatography (HPLC) and preparative gas chromatography. The rate of synthesis of JH III by teneral females was 5.6 fmol/h per CA. The CA of 1-d-old females synthesized 17 fmol/h per CA, whereas 3-d-old females synthesized 5.4 fmol/h per CA. The rate of synthesis of JH III 4 h after the blood meal increased to 17.3 fmol/h per CA and then declined to reach a minimum of 1.6 fmol/h per CA at 30 h before increasing again to reach 21.6 fmol/h per CA at 96 h. The concentration of methyl farnesoate in the tissue culture medium during incubation of CA from sugar- and blood-fed females was compared with the rate of synthesis of JH III and its metabolites diol-acid, diol, acid, and bisepoxide. The rate of synthesis of JH III and its metabolites from methyl farnesoate indicated a steady-state equilibrium of synthesis and metabolism of JH III by the exposed CA. The rapid increase in JH III synthesis immediately after the blood meal confirmed that in sand flies, like mosquitoes, there is an increase in the rate of synthesis of JH III immediately after the female takes blood. The role of the hormone in vitellogenin biosynthesis is also discussed.