RESUMO
Molting is induced in decapod crustaceans via multiple leg autotomy (MLA) or eyestalk ablation (ESA). MLA removes five or more walking legs, which are regenerated and become functional appendages at ecdysis. ESA eliminates the primary source of molt-inhibiting hormone (MIH) and crustacean hyperglycemic hormone (CHH), which suppress the production of molting hormones (ecdysteroids) from the molting gland or Y-organ (YO). Both MLA and ESA are effective methods for molt induction in Gecarcinus lateralis. However, some G. lateralis individuals are refractory to MLA, as they fail to complete ecdysis by 12weeks post-MLA; these animals are in the "blocked" condition. Quantitative polymerase chain reaction was used to quantify mRNA levels of neuropeptide and mechanistic target of rapamycin (mTOR) signaling genes in YO, eyestalk ganglia (ESG), thoracic ganglion (TG), and brain of intact and blocked animals. Six of the seven neuropeptide signaling genes, three of four mTOR signaling genes, and Gl-elongation factor 2 (EF2) mRNA levels were significantly higher in the ESG of blocked animals. Gl-MIH and Gl-CHH mRNA levels were higher in the TG and brain of blocked animals and levels increased in both control and blocked animals in response to ESA. By contrast, mRNA levels of Gl-EF2 and five of the 10 MIH signaling pathway genes in the YO were two to four orders of magnitude higher in blocked animals compared to controls. These data suggest that increased MIH and CHH synthesis in the ESG contributes to the prevention of molt induction by MLA in blocked animals. The up-regulation of MIH signaling genes in the YO of blocked animals suggests that the YO is more sensitive to MIH produced in the ESG, as well as MIH produced in brain and TG of ESA animals. Both the up-regulation of MIH signaling genes in the YO and of Gl-MIH and Gl-CHH in the ESG, TG, and brain appear to contribute to some G. lateralis individuals being refractory to MLA and ESA.
Assuntos
Proteínas de Artrópodes/metabolismo , Braquiúros/fisiologia , Glândulas Exócrinas/inervação , Gânglios dos Invertebrados/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Hormônios de Invertebrado/metabolismo , Modelos Neurológicos , Proteínas do Tecido Nervoso/metabolismo , Animais , Proteínas de Artrópodes/genética , Oceano Atlântico , Braquiúros/crescimento & desenvolvimento , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , República Dominicana , Ecdisteroides/biossíntese , Ecdisteroides/metabolismo , Glândulas Exócrinas/crescimento & desenvolvimento , Glândulas Exócrinas/metabolismo , Olho/crescimento & desenvolvimento , Olho/inervação , Olho/metabolismo , Gânglios dos Invertebrados/crescimento & desenvolvimento , Hormônios de Invertebrado/genética , Masculino , Muda , Proteínas do Tecido Nervoso/genética , Especificidade de Órgãos , Fatores de Alongamento de Peptídeos/genética , Fatores de Alongamento de Peptídeos/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Cavidade Torácica/crescimento & desenvolvimento , Cavidade Torácica/inervação , Cavidade Torácica/metabolismoRESUMO
Crustacean hyperglycemic hormones (CHHs) are multifunctional neuropeptides ubiquitous in crustaceans. In Litopenaeus vannamei, CHH-B2 is a CHH eyestalk isoform whose expression has been shown to vary with enviromental conditions, suggesting its relevance for ecophysiological performance of shrimp, controlling processes related to metabolism and osmo-ionic regulation. To study the involvement of CHH-B2 in these processes, we cloned and expressed a recombinant version with a free C-terminal glycine (rCHH-B2-Gly) in the methylotrophic yeast Pichia pastoris. The rCHH-B2-Gly peptide secreted to the culture medium was purified by RP-HPLC and used for in vivo glucose, triglyceride, and osmoregulation dose-response analyses with juvenile shrimp. The peptide was also amidated at the C-terminus using an α-amidating enzyme to produce rCHH-B2-amide. The shrimp showed a dose-dependent effect of rCHH-B2-Gly to hemolymph glucose and triglyceride levels, inducing maximal increases by injecting 500 and 1000pmol of hormone, respectively. Additionally, 10pmol of hormone was sufficient to reduce the hypo-osmoregulatory capacity of shrimp at 35. These findings suggest that CHH-B2 has regulatory roles in carbohydrate and lipid metabolism, and a potential involvement in osmoregulation of L. vannamei. Injection of 100pmol of rCHH-B2-amide increased glucose and triglyceride levels by 15 and 28%, respectively in comparison with rCHH-B2-Gly, suggesting an important role for the C-terminal amidation. Additionally, an in silico structural analysis done with the CHH-B1 and rCHH-B2-Gly peptides suggests that the C-terminal region may be relevant for the activity of the L. vannamei isoforms and explain the functional divergence from other crustacean CHH/CHH-like peptides.
Assuntos
Proteínas de Artrópodes/genética , Hormônios de Invertebrado/genética , Proteínas do Tecido Nervoso/genética , Osmorregulação , Penaeidae/metabolismo , Amidas/química , Animais , Proteínas de Artrópodes/metabolismo , Sequência de Bases , Bioensaio , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Clonagem Molecular , Simulação por Computador , Vetores Genéticos/metabolismo , Hiperglicemia/metabolismo , Hormônios de Invertebrado/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de SequênciaRESUMO
Crustacean hyperglycemic hormone (CHH) is a neuropeptide that is synthesized, stored, and released by brain and eyestalk structures in decapods. CHH participates in the regulation of several mechanisms, including increasing the level of glucose in hemolymph. Although CHH mRNA levels have been quantified and the CHH protein has been localized in various structures of the crayfish P. clarkii, CHH synthesis has only been reported in the X-organ-sinus gland (XO-SG). Therefore, the aim of this study was to use in situ hybridization to determine whether CHH mRNA is located in other structures, including the putative pacemaker, eyestalk and brain, of crayfish P. clarkii at two times of day. CHH mRNA was observed in both the eyestalk and the brain of P. clarkii, indicating that CHH is synthesized in several structures in common with other crustaceans, possibly to provide metabolic support for these regions by increasing glucose levels.
Assuntos
Proteínas de Artrópodes/metabolismo , Astacoidea/metabolismo , Encéfalo/metabolismo , Olho/metabolismo , Hormônios de Invertebrado/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Proteínas de Artrópodes/genética , Relógios Circadianos/fisiologia , Ritmo Circadiano/fisiologia , Glucose/metabolismo , Hemolinfa/metabolismo , Hibridização In Situ , Hormônios de Invertebrado/genética , Proteínas do Tecido Nervoso/genética , Reação em Cadeia da Polimerase , RNA Mensageiro/análiseRESUMO
In decapod crustaceans, molting is controlled by the pulsatile release of molt-inhibiting hormone (MIH) from neurosecretory cells in the X-organ/sinus gland (XO/SG) complex in the eyestalk ganglia (ESG). A drop in MIH release triggers molting by activating the molting gland or Y-organ (YO). Post-transcriptional mechanisms ultimately control MIH levels in the hemolymph. Neurotransmitter-mediated electrical activity controls Ca2+-dependent vesicular release of MIH from the SG axon terminals, which may be modulated by nitric oxide (NO). In green shore crab, Carcinus maenas, nitric oxide synthase (NOS) protein and NO are present in the SG. Moreover, C. maenas are refractory to eyestalk ablation (ESA), suggesting other regions of the nervous system secrete sufficient amounts of MIH to prevent molting. By contrast, ESA induces molting in the blackback land crab, Gecarcinus lateralis. Double-label immunofluorescence microscopy and quantitative polymerase chain reaction were used to localize and quantify MIH and NOS proteins and transcripts, respectively, in the ESG, brain, and thoracic ganglion (TG) of C. maenas and G. lateralis. In ESG, MIH- and NOS-immunopositive cells were closely associated in the SG of both species; confocal microscopy showed that NOS was localized in cells adjacent to MIH-positive axon terminals. In brain, MIH-positive cells were located in a small number of cells in the olfactory lobe; no NOS immunofluorescence was detected. In TG, MIH and NOS were localized in cell clusters between the segmental nerves. In G. lateralis, Gl-MIH and Gl-crustacean hyperglycemic hormone (CHH) mRNA levels were ~105-fold higher in ESG than in brain or TG of intermolt animals, indicating that the ESG is the primary source of these neuropeptides. Gl-NOS and Gl-elongation factor (EF2) mRNA levels were also higher in the ESG. Molt stage had little or no effect on CHH, NOS, NOS-interacting protein (NOS-IP), membrane Guanylyl Cyclase-II (GC-II), and NO-independent GC-III expression in the ESG of both species. By contrast, MIH and NO receptor GC-I beta subunit (GC-Iß) transcripts were increased during premolt and postmolt stages in G. lateralis, but not in C. maenas. MIH immunopositive cells in the brain and TG may be a secondary source of MIH; the release of MIH from these sources may contribute to the difference between the two species in response to ESA. The MIH-immunopositive cells in the TG may be the source of an MIH-like factor that mediates molt inhibition by limb bud autotomy. The association of MIH- and NOS-labeled cells in the ESG and TG suggests that NO may modulate MIH release. A model is proposed in which NO-dependent activation of GC-I inhibits Ca2+-dependent fusion of MIH vesicles with the nerve terminal membrane; the resulting decrease in MIH activates the YO and the animal enters premolt.
Assuntos
Proteínas de Artrópodes/metabolismo , Braquiúros/fisiologia , Sistema Nervoso Central/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Hormônios de Invertebrado/metabolismo , Neurônios/metabolismo , Óxido Nítrico Sintase/metabolismo , Animais , Aquicultura , Proteínas de Artrópodes/genética , Oceano Atlântico , Braquiúros/crescimento & desenvolvimento , California , Sistema Nervoso Central/citologia , Sistema Nervoso Central/enzimologia , República Dominicana , Olho , Gânglios dos Invertebrados/citologia , Gânglios dos Invertebrados/enzimologia , Gânglios dos Invertebrados/metabolismo , Hormônios de Invertebrado/genética , Masculino , Muda , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Neurônios/enzimologia , Óxido Nítrico Sintase/genética , Córtex Olfatório/citologia , Córtex Olfatório/enzimologia , Córtex Olfatório/metabolismo , Especificidade de Órgãos , Oceano Pacífico , Especificidade da Espécie , TóraxRESUMO
Bursicon is a heterodimeric neurohormone that acts through a G protein-coupled receptor named rickets (rk), thus inducing an increase in cAMP and the activation of tyrosine hydroxylase, the rate-limiting enzyme in the cuticular tanning pathway. In insects, the role of bursicon in the post-ecdysial tanning of the adult cuticle and wing expansion is well characterized. Here we investigated the roles of the genes encoding the bursicon subunits during the adult cuticle development in the honeybee, Apis mellifera. RNAi-mediated knockdown of AmBurs α and AmBurs ß bursicon genes prevented the complete formation and tanning (melanization/sclerotization) of the adult cuticle. A thinner, much less tanned cuticle was produced, and ecdysis toward adult stage was impaired. Consistent with these results, the knockdown of bursicon transcripts also interfered in the expression of genes encoding its receptor, AmRk, structural cuticular proteins, and enzymes in the melanization/sclerotization pathway, thus evidencing roles for bursicon in adult cuticle formation and tanning. Moreover, the expression of AmBurs α, AmBurs ß and AmRk is contingent on the declining ecdysteroid titer that triggers the onset of adult cuticle synthesis and deposition. The search for transcripts of AmBurs α, AmBurs ß and candidate targets in RNA-seq libraries prepared with brains and integuments strengthened our data on transcript quantification through RT-qPCR. Together, our results support our premise that bursicon has roles in adult cuticle formation and tanning, and are in agreement with other recent studies pointing for roles during the pharate-adult stage, in addition to the classical post-ecdysial ones.
Assuntos
Abelhas/genética , Ecdisteroides/genética , Hormônios de Invertebrado/genética , Metamorfose Biológica/genética , Animais , Abelhas/crescimento & desenvolvimento , AMP Cíclico/genética , Ecdisteroides/biossíntese , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Hormônios de Invertebrado/antagonistas & inibidores , Muda/genética , Interferência de RNA , Receptores Acoplados a Proteínas G/genética , Asas de Animais/crescimento & desenvolvimento , Asas de Animais/metabolismoRESUMO
This study was aimed at determining the role of the crustacean hyperglycemic hormone (CHH) in the physiological compensation to both saline and thermal stress, in the freshwater crayfish Cherax quadricarinatus. By determining the expression of the CHH gene in the eyestalk of juvenile crayfish, we found that maximal induction of CHH was induced at high salinity (10 g/L) and low temperature (20 °C). In order to investigate the role of CHH in the physiological compensation to such stressful conditions, recombinant CHH was supplied to stressed animals. CHH-injected crayfish showed increased hemolymphatic levels of glucose, in accordance with a significant utilization of glycogen reserves from the hepatopancreas. Furthermore, CHH administration allowed stressed animals to regulate hemolymphatic sodium and potassium at more constant levels than controls. Taken together, these results suggest a relevant role of CHH in increasing the energy available intended for processes involved in the physiological compensation of C. quadricarinatus to both saline and thermal stress.
Assuntos
Proteínas de Artrópodes/metabolismo , Astacoidea/metabolismo , Temperatura Baixa , Metabolismo Energético , Água Doce/química , Hormônios de Invertebrado/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Salinidade , Tolerância ao Sal , Estresse Fisiológico , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/administração & dosagem , Proteínas de Artrópodes/genética , Astacoidea/genética , Clonagem Molecular , Ecossistema , Metabolismo Energético/efeitos dos fármacos , Glucose/metabolismo , Glicogênio/metabolismo , Hemolinfa/metabolismo , Hepatopâncreas/metabolismo , Hormônios de Invertebrado/administração & dosagem , Hormônios de Invertebrado/genética , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/administração & dosagem , Proteínas do Tecido Nervoso/genética , Potássio/metabolismo , Proteínas Recombinantes/administração & dosagem , Transdução de Sinais , Sódio/metabolismoRESUMO
The method usually employed to stimulate gonadal maturation and spawning of captive shrimp involves unilateral eyestalk ablation, which results in the removal of the endocrine complex responsible for gonad-inhibiting hormone (GIH) synthesis and release. In the present study, RNAi technology was used to inhibit transcripts of GIH in Litopenaeus vannamei females. The effect of gene silencing on gonad development was assessed by analyzing the expression of GIH and vitellogenin, respectively, in the eyestalk and ovaries of L. vannamei females, following ablation or injection with dsRNA-GIH, dsRNA-IGSF4D (non-related dsRNA), or saline solution. Histological analyses were performed to determine the stage of gonadal development and to assess the diameter of oocytes throughout the experimental procedure. Only oocytes at pre-vitellogenesis and primary vitellogenesis stages were identified in females injected with dsRNA-GIH, dsRNA-IGSF4D, or saline solution. Oocytes at all developmental stages were observed in eyestalk-ablated females, with predominance of later stages, such as secondary vitellogenesis and mature oocytes. Despite achieving 64, 73, and 71% knockdown of eyestalk GIH mRNA levels by 15, 30, and 37 days post-injection (dpi), respectively, in dsRNA-GIH-injected females, the expected increase in ovary vitellogenin mRNA expression was only observed on the 37th dpi. This is the first report of the use of RNAi technology to develop an alternative method to eyestalk ablation in captive L. vannamei shrimps.
Assuntos
Proteínas de Transporte/genética , Hormônios de Invertebrado/genética , Ovário/crescimento & desenvolvimento , Indução da Ovulação/métodos , Penaeidae/genética , Interferência de RNA , Vitelogênese/genética , Animais , Feminino , Técnicas de Silenciamento de Genes/métodos , Inativação Gênica , Ovário/citologia , Penaeidae/crescimento & desenvolvimento , Fatores de Transcrição/genéticaRESUMO
The gonad-inhibiting hormone (GIH) belongs to a neuropeptide family synthesized and released in an X-organ sinus gland complex of crustacean eyestalks. GIH inhibits crustacean ovarian maturation by suppressing vitellogenin (Vtg) synthesis, whereas estrogen is responsible for the stimulation of vitellogenesis (not established). In this study, the effects of 17ß-estradiol (E2, 10(-6) M), estrogen receptor antagonist tamoxifen (TAM, 10(-6), 10(-7), and 10(-8) M), and the environmental estrogen nonylphenol (NP, 1 µg/L and 100 µg/L) on LvGIH expression in the eyestalks of shrimp were determined by quantitative real-time PCR. Results showed that LvGIH expression decreased significantly during the L. vannamei ovarian maturation cycle. E2 and NP significantly reduced LvGIH transcripts in vivo, but TAM neutralized the inhibitory action of E2 in a dose-dependent manner (P < 0.05). In addition, the LvGIH expression levels decreased significantly in a time-dependent manner (P < 0.05) when ovary fragments were cultured in vitro with E2. The results of this study suggested that estrogen regulates GIH expression in L. vannamei eyestalks. E2 promoted ovarian development not only by directly upregulating vitellogenesis in the hepatopancreas, but it was also capable of downregulating LvGIH expression, which indirectly resulted in the stimulation of L. vannamei vitellogenesis.
Assuntos
Proteínas de Transporte/biossíntese , Estradiol/farmacologia , Hormônios de Invertebrado/biossíntese , Penaeidae/efeitos dos fármacos , Fenóis/farmacologia , Animais , Proteínas de Transporte/genética , Antagonistas de Estrogênios/farmacologia , Estrogênios/metabolismo , Feminino , Expressão Gênica/efeitos dos fármacos , Hormônios de Invertebrado/genética , Ovário/efeitos dos fármacos , Ovário/metabolismo , Penaeidae/genética , Penaeidae/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Tamoxifeno/farmacologia , Vitelogênese/efeitos dos fármacos , Vitelogeninas/metabolismoRESUMO
Melatonin has been identified in a variety of crustacean species, but its function is not as well understood as in vertebrates. The present study investigates whether melatonin has an effect on crustacean hyperglycemic hormone (CHH) gene expression, oxygen consumption (VO2) and circulating glucose and lactate levels, in response to different dissolved-oxygen concentrations, in the crab Neohelice granulata, as well as whether these possible effects are eyestalk- or receptor-dependent. Melatonin decreased CHH expression in crabs exposed for 45 min to 6 (2, 200 or 20,000 pmol·crab-1) or 2 mgO2·L-1 (200 pmol·crab-1). Since luzindole (200 nmol·crab-1) did not significantly (p > 0.05) alter the melatonin effect, its action does not seem to be mediated by vertebrate-typical MT1 and MT2 receptors. Melatonin (200 pmol·crab-1) increased the levels of glucose and lactate in crabs exposed to 6 mgO2·L-1, and luzindole (200 nmol·crab-1) decreased this effect, indicating that melatonin receptors are involved in hyperglycemia and lactemia. Melatonin showed no effect on VO2. Interestingly, in vitro incubation of eyestalk ganglia for 45 min at 0.7 mgO2·L-1 significantly (p < 0.05) increased melatonin production in this organ. In addition, injections of melatonin significantly increased the levels of circulating melatonin in crabs exposed for 45 min to 6 (200 or 20,000 pmol·crab-1), 2 (200 and 20,000 pmol·crab-1) and 0.7 (200 or 20,000 pmol·crab-1) mgO2·L-1. Therefore, melatonin seems to have an effect on the metabolism of N. granulata. This molecule inhibited the gene expression of CHH and caused an eyestalk- and receptor-dependent hyperglycemia, which suggests that melatonin may have a signaling role in metabolic regulation in this crab.
Assuntos
Braquiúros/metabolismo , Melatonina/metabolismo , Transdução de Sinais , Anaerobiose , Animais , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Braquiúros/genética , Regulação da Expressão Gênica , Glucose/metabolismo , Hormônios de Invertebrado/genética , Hormônios de Invertebrado/metabolismo , Ácido Láctico/metabolismo , Masculino , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Consumo de Oxigênio , Transdução de Sinais/genéticaRESUMO
Anti-lipopolysaccharide factors (ALFs) are antimicrobial peptides found in limulids and crustaceans that have a potent and broad range of antimicrobial activity. We report here the identification and molecular characterisation of new sequences encoding for ALFs in the haemocytes of the freshwater prawn Macrobrachium olfersi and also in two Brazilian penaeid species, Farfantepenaeus paulensis and Litopenaeus schmitti. All obtained sequences encoded for highly cationic peptides containing two conserved cysteine residues flanking a putative LPS-binding domain. They exhibited a significant amino acid similarity with crustacean and limulid ALF sequences, especially with those of penaeid shrimps. This is the first identification of ALF in a freshwater prawn.
Assuntos
Clonagem Molecular , DNA Complementar/genética , Hormônios de Invertebrado/genética , Hormônios de Invertebrado/metabolismo , Palaemonidae/metabolismo , Penaeidae/metabolismo , Sequência de Aminoácidos , Animais , Brasil , Regulação da Expressão Gênica/fisiologia , Dados de Sequência MolecularRESUMO
The open reading frame (ORF) of the gene for the precursor of the octapeptide Red Pigment Concentrating Hormone (RPCH) from the blue crab Callinectes sapidus was cloned by PCR with oligonucleotides targeted to the initiation and the end of the translation coding sequences. A 272 bp intron was characterized between nucleotides 343 and 344 of the reported cDNA, present in the region coding for the last amino acids of the precursor related peptide of RPCH. The intron genomic structure here described is similar to that reported for the gene coding for the Adipokinetic Hormone (AKH) of the grasshopper Schistocerca nitans.
Assuntos
Braquiúros/genética , Hormônios de Invertebrado/genética , Oligopeptídeos/genética , Fases de Leitura Aberta/genética , Precursores de Proteínas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Íntrons/genética , Hormônios de Invertebrado/química , Dados de Sequência Molecular , Oligopeptídeos/química , Reação em Cadeia da Polimerase , Precursores de Proteínas/metabolismo , Ácido Pirrolidonocarboxílico/análogos & derivados , Alinhamento de SequênciaRESUMO
A PCR-based genomic DNA walking technique was used to clone the gene for the molt-inhibiting hormone of the crab, Charybdis feriatus. Several overlapping genomic clones were isolated, and the MIH gene for the crab was reconstructed. DNA sequence determination of the overlapping clone reveals that the MIH gene spans 4.3kb and consists of three exons and two introns. Exons 1 and 2 carry a coding sequence for the signal peptide, and exons 2 and 3 consist of coding sequence for the mature peptide. The exon-intron boundary of the crab MIH gene also follows the 'GT-AG rule' for the splice donor and acceptor. The deduced amino acid sequence of MIH shows the highest overall similarity to those of the crabs, Callinectes sapidus and Carcinus maenas, and the gonad-inhibiting hormone (GIH) of the lobster. The putative polyadenylation signal is approximately 1.0kb 3' downstream of the termination codon (TGA). Genomic Southern blot analysis indicates that few genomic fragments were hybridized to the cDNA probe. The 5' flanking region contains a putative promoter with several putative cis elements similar to some vertebrate neuropeptide genes. The 530-bp flanking region was subcloned separately to two promoterless reporter plasmids carrying either the Green Fluorescent Protein gene (GFP) or the Choramphenicol Acetyltransferase gene (CAT). The DNA constructs were transfected into insect cells (Sf21) and mouse pituitary cells (GH4ZR7), respectively. Green fluorescent protein was detected in some of the transfected insect cells, and expression of the CAT was detected in cells transfected with DNA constructs containing the crab promoter. By RT-PCR, MIH transcripts can be detected in the eyestalk of shrimp in intermolt, early premolt, late premolt stages and females that brood their eggs. It can also be found in the brain, but not in the ovary, hepatopancreas, muscle and epidermis. During early larval development, MIH mRNA can be detected in the pre-hatched and the newly hatched larvae. Unlike the adult, the expression of the MIH in the larvae is exclusively in the brain.
Assuntos
Braquiúros/genética , Genes/genética , Hormônios de Invertebrado/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Braquiúros/química , Células COS/citologia , Células COS/metabolismo , Linhagem Celular , Clonagem Molecular , DNA/química , DNA/genética , Éxons , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Íntrons , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
The primary structure of the neurohormone crustacean hyperglycemic hormone (CHH-II) was determined by means of enzymatic digestions, manual Edman degradation, and mass spectrometry. CHH-II is a 72 residue peptide (molecular mass 8388 Da), with six cysteines forming three disulfide bridges that connect residues 7-43, 23-39, and 26-52. The peptide has blocked N- and C-termini, and lacks tryptophan, histidine, and methionine. The CHH-I and CHH-II of Procambarus bouvieri have identical sequences and elicit levels of hyperglycemia that are not distinguishable. The difference between the two isomorphs consists in a posttranslational modification of a L-Phe in CHH-I to a D-Phe in CHH-II at the third position from the N-terminus.
Assuntos
Astacoidea/química , Hormônios de Invertebrado/química , Proteínas do Tecido Nervoso/química , Sequência de Aminoácidos , Aminoácidos/química , Animais , Proteínas de Artrópodes , Astacoidea/genética , Astacoidea/metabolismo , Cromatografia Líquida de Alta Pressão , Cisteína/química , Ensaio de Imunoadsorção Enzimática , Hormônios de Invertebrado/genética , Hormônios de Invertebrado/metabolismo , Espectrometria de Massas , Dados de Sequência Molecular , Estrutura Molecular , Peso Molecular , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Fragmentos de Peptídeos/química , Processamento de Proteína Pós-Traducional , EstereoisomerismoRESUMO
Antistasin is a Factor Xa inhibitor that is present in the salivary glands of the Mexican leech Haementeria officinalis. The antistasin protein consists of 119 amino acids, of which residues 1-55 (domain I) are 56% similar to residues 56-110 (domain II). Of the nine C-terminal amino acids (residues 111-119; domain III), four are positively charged. The reactive site for Factor Xa is located in domain I. In this study we assessed the role of separate domains and of individual amino acids in the reactive site for the inhibition of Factor Xa. A series of mutants was constructed and expressed in Chinese hamster ovary (CHO) cells. In vitro chromogenic assays for Factor Xa show that domain I is sufficient for inhibition of Factor Xa. Domains II and III neither contain any intrinsic Factor Xa inhibitory activity, nor contribute to the activity of domain I. Furthermore, domain II does not become a Factor Xa inhibitor by partially adaptating its sequence towards that of the reactive site in domain I. Mutation of the cysteine at position 33 is not crucial for Factor Xa inhibition, suggesting a relatively rigid reactive site loop structure.
Assuntos
Anticoagulantes/isolamento & purificação , Inibidores do Fator Xa , Hormônios de Invertebrado/genética , Hormônios de Invertebrado/isolamento & purificação , Sanguessugas/química , Sequência de Aminoácidos , Animais , Células CHO/metabolismo , Cricetinae , Análise Mutacional de DNA , Sondas de DNA , Sanguessugas/genética , Dados de Sequência MolecularRESUMO
As a factor Xa inhibitor, antistasin is a potent anti-coagulant and anti-metastatic agent that is found in the salivary gland of the Mexican leech Haementaria officinalis. cDNA clones that encode antistasin have been isolated. Subsequent sequence analysis and comparison with the amino acid sequence of the mature protein indicates that antistasin is produced as a pre-protein containing a 17-amino acid signal peptide. Antistasin exists as at least two variants. By sequence analysis of multiple cDNA clones, we found two additional sites for amino acid substitutions, confirming variants that differ from each other by amino acid changes at a minimum of four residues. These sequence variations appear to be the result of allelic variation rather than gene duplication as deduced from DNA blot analyses. Sequence data suggest that antistasin may have evolved from a smaller ancestral gene by a duplication event giving rise to a two-fold structural homology between the N- and C-terminal halves of the molecule. Insect cells transfected with a recombinant baculovirus expressed antistasin which was biologically active and had an electrophoretic mobility identical to that of the native molecule.