Introducao: O receptor sensor de calcio (CASR)pertence a familiados receptores que acoplam a proteinaG e possui homologiacom a subfamiliados receptores metabotropicos do glutamato.Ele e importante no processo de homeostase do calcio,e quando a concentracao de calcio se eleva,ele inibe a secrecao de paratonnonio pelas paratireoidese a reabsorcao renal de calcio. O receptor sensor de calcio possui 1078 aminoacidos, com 612 aminoacidos no dominio extracelular (Em), 250 aminoacidos em sete regioes ttansmembrana (TM) conectadas com 3 alcas extracelularese intracelularese uma longa cauda intracitoplasmatica com 216 aminoacidos.A regiao ttansmembrana tem um papel fundamental na ativacao do receptor e analise da distnbuicaodas mutacoes ativadoras demonstra que a sua porcao distal
(IM 5, 6, 7 e alca extracelular 3) e a mais critica no processo de ativacao do receptor. O estudo de receptores mutados auxilia a elucidar a imponância da polaridade de detenninados aminoacidos presentes na regiao ttansmembrana na expressao do CASR A producao da proteina recombinante do dominio extracelulardo CASRproporciona novas expectativaspara uma analiseestruturaldesta regiaotao importante para a ligacaocom o agonista. Objetivos: Os objetivos do presente estudo foram: 1-investigaro grau de expressao de receptores mutados na regiao ttansmembrana. 2- Produzir a proteina recombinante do
dominio extracelular do CASR em sistema de E.. Resultados e Conclusoes: Nos resultados de analise da expressao, observamos que todos os receptores mutados e ttansfectados em celulas HEK 293 foram expressos, no entanto os receptores mutados nas posicoesF806C,A824S,A835D e A843Tapresentaram diminuicao na expressao quando comparados com o receptor nativo, sugerindo que a mutacao de arninoacidoshidrofobicos para hidrofilicosna porcao transmembrana pode diminuir o grau de expressao do CASR Obtivemos alta producao da proteina recombinante correspondente ao dominio extracelular do CASR, entretanto nao conseguimos quantidades suficientes de proteina soluveLdevido a fonnacao de cOIposde inclusoes.Isto sugere que o sistemade E. nao e eficiente para produzir proteinas com estruturas mais complexas como o dominio extracelulardo receptor sensor de calcio
Abstract
Abstract Background To date, oil-rich plants are the main source of biodiesel products. Because concerns have been voiced about the impact of oil-crop cultivation on the price of food commodities, the interest in oil plants not used for food production and amenable to cultivation on non-agricultural land has soared. As a non-food, drought-resistant and oil-rich crop, Jatropha curcas L. fulfils many of the requirements for biofuel production. Results We have generated 13,249 expressed sequence tags (ESTs) from developing and germinating Jatropha seeds. This strategy allowed us to detect most known genes related to lipid synthesis and degradation. We have also identified ESTs coding for proteins that may be involved in the toxicity of Jatropha seeds. Another unexpected finding is the high number of ESTs containing transposable element-related sequences in the developing seed library (800) when contrasted with those found in the germinating seed library (80). Conclusions The sequences generated in this work represent a considerable increase in the number of sequences deposited in public databases. These results can be used to produce genetically improved varieties of Jatropha with increased oil yields, different oil compositions and better agronomic characteristics.
Abstract Background The genus Bothrops is widespread throughout Central and South America and is the principal cause of snakebite in these regions. Transcriptomic and proteomic studies have examined the venom composition of several species in this genus, but many others remain to be studied. In this work, we used a transcriptomic approach to examine the venom gland genes of Bothrops alternatus , a clinically important species found in southeastern and southern Brazil, Uruguay, northern Argentina and eastern Paraguay. Results A cDNA library of 5,350 expressed sequence tags (ESTs) was produced and assembled into 838 contigs and 4512 singletons. BLAST searches of relevant databases showed 30% hits and 70% no-hits, with toxin-related transcripts accounting for 23% and 78% of the total transcripts and hits, respectively. Gene ontology analysis identified non-toxin genes related to general metabolism, transcription and translation, processing and sorting, (polypeptide) degradation, structural functions and cell regulation. The major groups of toxin transcripts identified were metalloproteinases (81%), bradykinin-potentiating peptides/C-type natriuretic peptides (8.8%), phospholipases A 2 (5.6%), serine proteinases (1.9%) and C-type lectins (1.5%). Metalloproteinases were almost exclusively type PIII proteins, with few type PII and no type PI proteins. Phospholipases A 2 were essentially acidic; no basic PLA 2 were detected. Minor toxin transcripts were related to L-amino acid oxidase, cysteine-rich secretory proteins, dipeptidylpeptidase IV, hyaluronidase, three-finger toxins and ohanin. Two non-toxic proteins, thioredoxin and double-specificity phosphatase Dusp6, showed high sequence identity to similar proteins from other snakes. In addition to the above features, single-nucleotide polymorphisms, microsatellites, transposable elements and inverted repeats that could contribute to toxin diversity were observed. Conclusions Bothrops alternatus venom gland contains the major toxin classes described for other Bothrops venoms based on trancriptomic and proteomic studies. The predominance of type PIII metalloproteinases agrees with the well-known hemorrhagic activity of this venom, whereas the lower content of serine proteases and C-type lectins could contribute to less marked coagulopathy following envenoming by this species. The lack of basic PLA 2 agrees with the lower myotoxicity of this venom compared to other Bothrops species with these toxins. Together, these results contribute to our understanding of the physiopathology of envenoming by this species.
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