RESUMO
The antihypertensive potential of a land snail by-product hydrolysate, obtained after an agri-food processing of the raw material, was studied in vitro and in vivo. First, the ACE inhibitory activity of hydrolysates obtained before and after an ultrafiltration step with a 10kDa membrane cutoff was assayed. The IC50 obtained were of 98.3µg·mL-1 and 23µg·mL-1, respectively, showing a 4.2 fold increase in ACE inhibitory capacity after the ultrafiltration step. Then, ACE inhibitory capacity of the hydrolysate was followed. No significant modification of the ACE inhibition rate was observed during an in vitro simulated human gastro-intestinal digestion. Moreover, the hydrolysate was not only safe for human intestinal cells but besides that it stimulated their metabolic activity. No toxicity of the hydrolysate was observed in vivo in Wistar rats regarding to the results of the acute toxicity study. The partial purification of the hydrolysate led to the obtention of an active fraction characterized by an IC50 of 0.007µg·mL-1. The sequences of the 17 most abundant peptides of the fraction were identified by LC/MS/MS analysis. Seven of these peptides (YG, YA, VY, FG/GF, DF, SF and VW) are known ACE inhibitory peptides. Finally, in vivo study on SHR rats showed that the hydrolysate reduced systolic blood pressure by 20mmHg after a single oral administration at both doses of 400 and 800mg·kg-1.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Anti-Hipertensivos/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Hidrolisados de Proteína/farmacologia , Animais , Humanos , Concentração Inibidora 50 , Ratos Endogâmicos SHR , CaramujosRESUMO
We propose a novel approach to the selection of Escherichia coli bacterial strains improved for the production of recombinant functional proteins. This approach is based on aggregation-induced toxicity of recombinant proteins. We show that selection of clones displaying a reduced toxicity is an efficient means of isolating bacteria producing recombinant protein with reduced aggregation in favour of correct folding. For an efficient selection, we found that time of toxicity induction must be precisely determined and recombinant protein must be expressed as a fusion with a protein whose activity is easily detectable on plates, thus allowing elimination of non-productive mutants. Choosing the expression to the periplasmic space of an scFv fragment fused to the N-terminus of alkaline phosphatase as a model, we selected chromosomal mutations that reduce aggregation-induced toxicity and showed that they concomitantly improve production of a functional recombinant hybrid. The effects of the mutations isolated could then be cumulated with those of other strategies used for recombinant scFv production. Thus, we could ensure a 6- to 16-fold increase in production of a functional scFv-PhoA hybrid. This is the first report demonstrating the possibility of directly selecting on agar plates E.coli strains improved for functional recombinant protein production from a large bacterial mutant library.
Assuntos
Clonagem Molecular , Escherichia coli/genética , Fragmentos de Imunoglobulinas/genética , Mutação , Proteínas Recombinantes/genética , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/genética , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/metabolismo , Ensaio de Imunoadsorção Enzimática , Escherichia coli/metabolismo , Fragmentos de Imunoglobulinas/imunologia , Proteínas Recombinantes/imunologiaRESUMO
Downy mildew of sunflower (Helianthus annuus L.), caused by the pathogen Plasmopara halstedii, is a potentially devastating disease. Seventy-seven isolates of P. halstedii collected in twelve countries from four continents were investigated for RAPD polymorphism with 21 primers. The study led to a binary matrix, which was subjected to various complementary analyses. This is the first report on the international genetic diversity of the pathogen. Similarity indices ranged from 89% to 100%. Neither a consensus unweighted pair group method with arithmetic means (UPGMA) tree constructed after bootstrap resampling of markers nor a principal component analysis based on distance matrix revealed very consistent clusterings of the isolates, and groups did not fit race or geographical origins. Phylogenies were probably obscured by limited diversity. Analysis of molecular variance (AMOVA) and Nei's genetic diversity statistics gave similar conclusions. Most of the genetic diversity was attributable to individual differences. The most differentiated races also had the lowest within-diversity indices, which suggest that they appeared recently with strong bottleneck effects. Our analyses suggest that this pathogen is probably homothallic or has an asexual mode of reproduction and that gene flow among countries can occur through commercial exchanges. Knowledge of the downy mildew populations' structure at the international level will help to devise strategies for controlling this potentially devastating disease.
Assuntos
Variação Genética , Helianthus/microbiologia , Oomicetos/genética , Oomicetos/isolamento & purificação , DNA/isolamento & purificação , Oomicetos/classificação , Oomicetos/fisiologia , Filogenia , Doenças das Plantas/etiologia , Polimorfismo Genético , Técnica de Amplificação ao Acaso de DNA Polimórfico/métodos , Reprodução AssexuadaRESUMO
We determined the distances separating five functionally important residues (Gln(10), Lys(27), Trp(29), Arg(33), and Lys(47)) of a three-fingered snake neurotoxin from the reduced disulfide bond alpha(Cys(192)-Cys(193)) located at the alphagamma interface of the Torpedo nicotinic acetylcholine receptor. Each toxin position was substituted individually for a cysteine, which was then linked to a maleimido moiety through three different spacers, varying in length from 10 to 22 A. We estimated the coupling efficiency between the 15 toxin derivatives and the reduced cystine alpha(192-193) by gel densitometry of Coomassie Blue-stained gels. A nearly quantitative coupling was observed between alphaCys(192) and/or alphaCys(193) and all probes introduced at the tip of the first (position 10) and second (position 33) loops of Naja nigricollis alpha-neurotoxin. These data sufficed to locate the reactive thiolate in a "croissant-shaped" volume comprised between the first two loops of the toxin. The volume was further restrained by taking into account the absence or partial coupling of the other derivatives. Altogether, the data suggest that alphaCys(192) and/or alphaCys(193), at the alphagamma interface of a muscular-type acetylcholine receptor, is (are) located in a volume located between 11.5 and 15.5 A from the alpha-carbons at positions 10 and 33 of the toxin, under the tip of the toxin first loop and close to the second one.
Assuntos
Cisteína/química , Neurotoxinas/química , Receptores Nicotínicos/química , Venenos de Serpentes/química , Acetilcolina/análogos & derivados , Acetilcolina/farmacologia , Animais , Membrana Celular/química , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Dissulfetos , Eletroforese em Gel de Poliacrilamida , Immunoblotting , Cinética , Espectrometria de Massas , Modelos Químicos , Mutagênese Sítio-Dirigida , Biossíntese Peptídica , Ligação Proteica , Estrutura Quaternária de Proteína , Compostos de Sulfidrila/química , Torpedo/metabolismoRESUMO
The obligate parasitic fungus-like organism Plasmopara halstedii (Farl.) Berl. et De Toni, is the causal agent of downy mildew disease in sunflower (Helianthus annuus). New races of this economically important parasite are regularly detected throughout the world. In addition, fungicide-resistant isolates have been reported in Europe and North America. These observations of parasite evolution, as well as the risk of propagation of the disease by infected seeds, means that it is necessary to guarantee the absence of Plasmopara halstedii in seed shipments. We report here the development of a rapid assay that can be used to detect infection by Plasmopara halstedii in plant tissues. Based on the nucleotide sequence information obtained from one cloned random amplified polymorphic DNA fragment, specific oligonucleotides were designed and used as primers for in vitro DNA amplification by polymerase chain reaction. An amplification product was detected on agarose gel stained with ethidium bromide when DNA from various Plasmopara halstedii races was tested, whereas no amplified DNA was detected when DNA from other origins was tested, including DNA from the host plant. The sensitivity of the technique was evaluated. The assay successfully reveals the presence of Plasmopara halstedii in infected sunflower plants prior to sporulation.
Assuntos
Helianthus/microbiologia , Oomicetos/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , DNA Fúngico/análise , Oomicetos/genética , Sensibilidade e Especificidade , Especificidade da EspécieRESUMO
We have investigated whether the nonpathogenic gram-positive bacteria Staphylococcus xylosus and S. carnosus can display a whole domain of a toxic protein on their surface and if such vectors are suitable for immunization of BALB/c mice. The nucleotide sequence encoding the receptor-binding domain (DTR; amino acids 382 to 535) of diphtheria toxin (DT) was inserted into plasmids pSE'mp18ABPXM and pSPPmABPXM, which were designed to display heterologous proteins on S. xylosus and S. carnosus cell surfaces, respectively. Western blot analysis of the resulting bacterial lysates indicates that DTR is produced by each expression system. However, analysis of rabbit anti-DTR antisera binding to the transformed live bacteria shows that DTR is not displayed on the surface of S. xylosus cells whereas it is efficiently exposed on S. carnosus. A significant anti-DT antibody response was raised in BALB/c mice immunized intraperitoneally with S. carnosus displaying DTR, and the antisera abolished DT cytotoxicity on Vero cells. Thus, only S. carnosus can display a whole domain of a toxic protein and represents a potential vector for humoral vaccination.
Assuntos
Toxina Diftérica/biossíntese , Vetores Genéticos , Soros Imunes/imunologia , Proteínas Recombinantes/biossíntese , Staphylococcus/genética , Animais , Western Blotting , Chlorocebus aethiops , Toxina Diftérica/genética , Toxina Diftérica/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Fragmentos de Peptídeos/imunologia , Coelhos , Células VeroRESUMO
Fusion of antigenic proteins to Ig-binding proteins such as protein A from Staphylococcus aureus and its derived ZZ fragment is known to increase immunogenicity of the fused Ag in vivo. To shed light on the origin of this effect, we used snake toxins as Ags and observed that 1) fusion of toxins to ZZ enhanced their presentation to a toxin-specific T cell hybridoma (T1B2), using A20 B lymphoma cells, splenocytes, or peritoneal exudate cells as APCs; 2) this enhancement further increased when the number of fused Ig-binding domains varied from two with ZZ to five with protein A; and 3) the phenomenon vanished when the fusion protein was preincubated with an excess of free ZZ or when P388D1 monocytes cells were used as APCs. Therefore, ZZ-fused toxins are likely to be targeted to surface Igs of APCs by their ZZ moiety. Furthermore, ZZ-alpha and toxin alpha stimulated similar profiles of toxin-specific T cells in BALB/c mice, suggesting a comparable processing and presentation in vivo for both toxin forms. To improve the targeting efficiency, ZZ-alpha was noncovalently complexed to various Igs directed to different cell surface components of APCs. The resulting complexes were up to 10(3)-fold more potent than the free toxin at stimulating T1B2. Also, they elicited both a T cell and an Ab response in BALB/c mice, without the need of any adjuvant. This simple approach may find practical applications by increasing the immunogenicity of recombinant proteins without the use of adjuvant.
Assuntos
Antígenos/metabolismo , Proteínas de Transporte/imunologia , Proteínas de Transporte/metabolismo , Imunoglobulinas/metabolismo , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Animais , Formação de Anticorpos , Apresentação de Antígeno , Células Apresentadoras de Antígenos/imunologia , Membrana Celular/imunologia , Erabutoxinas/imunologia , Hibridomas , Imunização , Técnicas In Vitro , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Proteína Estafilocócica A/imunologia , Proteína Estafilocócica A/metabolismo , Linfócitos T/imunologiaRESUMO
Two recombinant fragments of diphtheria toxin (DT) were fused to an engineered tandem repeat of the immunoglobulin (Ig) binding domain of protein A, called ZZ. These fragments are (i) the receptor binding domain (DTR), which comprises amino acids 382 to 535 of DT, and (ii) a linear peptide (DT(168-220)) which comprises residues 168 to 220 of the loop between fragment A and fragment B of DT. The fusion proteins were produced in Escherichia coli and purified by affinity chromatography. In vitro experiments showed that the DTR domain is responsible for the capacity of ZZ-DTR to bind to Vero cells and is capable of inhibiting the cytotoxicity of DT for these cells. These findings suggest that DTR binds to the cell surface receptors of DT and hence adopts a conformation that is similar to that of the receptor binding domain of DT. We compared the capacities of ZZ-DTR, ZZ-DT(168-220), and a chemically detoxified form of DT currently used for vaccination to elicit antibodies in rabbits. The toxoid was more immunogenic than ZZ-DT(168-220), which in turn was more immunogenic than ZZ-DTR. However, ZZ-DT(168-220) antiserum was poorly efficient at neutralizing DT cytotoxicity on Vero cells, whereas ZZ-DTR antiserum was only 15-fold less potent than anti-DT antisera.
Assuntos
Toxina Diftérica/imunologia , Toxoide Diftérico/imunologia , Fragmentos de Peptídeos/imunologia , Vacinas Sintéticas/imunologia , Animais , Chlorocebus aethiops , Masculino , Testes de Neutralização , Coelhos , Células VeroRESUMO
Long chain and short chain curaremimetic toxins from snakes possess 66-74 residues with five disulfide bonds and 60-62 residues with four disulfide bonds, respectively. Despite their structural differences all of these toxins bind with high affinity to the peripheral nicotinic acetylcholine receptors (AChR). Binding experiments have now revealed that long chain toxins only, like the neuronal kappa-bungarotoxin, have a high affinity for a chimeric form of the neuronal alpha7 receptor, with Kd values ranging from about 1 to 12 nM. In contrast, all other toxins bind to the chimeric alpha7 receptor with a low affinity, with Kd values ranging between 3 and 22 microM. These results are supported by electrophysiological recordings on both the wild-type and chimeric alpha7 receptors. Amino acid sequence analyses have suggested that high affinities for the neuronal receptor are associated with the presence of the fifth disulfide at the tip of the toxin second loop. In agreement with this conclusion, we show that a long chain toxin whose fifth disulfide is reduced and then dithiopyridylated has a low affinity (Kd = 12 microM) for the neuronal alpha7 receptor, whereas it retains a high affinity (Kd = 0.35 nM) for the peripheral AChR. Thus, a long chain curaremimetic toxin having a reduced fifth disulfide bond behaves like a short chain toxin toward both the peripheral and neuronal AChR. Therefore, functional classification of toxins that bind to AChRs should probably be done by considering their activities on both peripheral and neuronal receptors.
Assuntos
Dissulfetos/metabolismo , Neurônios/metabolismo , Neurotoxinas/metabolismo , Receptores Nicotínicos/metabolismo , Venenos de Serpentes/química , Sequência de Aminoácidos , Animais , Ligação Competitiva , Bungarotoxinas/metabolismo , Epitopos/metabolismo , Humanos , Radioisótopos do Iodo , Mimetismo Molecular , Dados de Sequência Molecular , Neurotoxinas/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , TorpedoRESUMO
The aim of this work was to produce and to label snake neurotoxins, disulfide-rich proteins. A mutant of a snake toxin, erabutoxin a, was used as a model. Its N-terminal part was fused to ZZ, a synthetic IgG-binding domain of protein A (B. Nilsson et al., 1987, Protein Eng. 1, 107-113), thus preventing degradation in the bacterial cytoplasm and providing a simple affinity-purification method on IgG Sepharose. A soluble fusion protein was obtained with a yield of 60 mg/L, corresponding to 20 mg/L toxin. The toxin moiety was folded on the column while the hybrid was still bound. The oxidoreducing conditions for the refolding were optimized and were found to be oxidative but with a need for reducing molecules. The concentration of the hybrid bound to the column could be increased up to 3.3 mg/ml without significantly altering the folding process. CNBr cleavage of the fusion protein followed by a purification step yielded about 2 mg of biologically active toxin mutant per gram of dry cell weight. This procedure was applied to produce 55 mg of a toxin uniformly labeled with 15N.
Assuntos
Venenos Elapídicos/química , Erabutoxinas/biossíntese , Neurotoxinas/biossíntese , Ligação Competitiva , Cromatografia de Afinidade , Dicroísmo Circular , Brometo de Cianogênio , Eletroforese em Gel de Poliacrilamida , Erabutoxinas/química , Erabutoxinas/genética , Erabutoxinas/metabolismo , Escherichia coli/genética , Expressão Gênica , Vetores Genéticos , Marcação por Isótopo , Espectroscopia de Ressonância Magnética , Neurotoxinas/química , Neurotoxinas/genética , Neurotoxinas/metabolismo , Isótopos de Nitrogênio , Ligação Proteica , Dobramento de Proteína , Receptores Colinérgicos/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteína Estafilocócica A/química , Proteína Estafilocócica A/genética , Transformação Genética , Fosfolipases Tipo C/metabolismoRESUMO
Identification of some economically important Tuber species using classical morphological characteristics is sometimes difficult. We report here the molecular characterization of a species coming from China, Tuber indicum, mistaken with Tuber melanosporum species. Using restriction analysis of the amplified internal transcribed spacer (ITS) of rDNA, ITS sequence analysis, and sequence characterized amplified region markers, with DNA from fruit bodies or mycorrhizae, genetic variation was found between these two species, allowing to differentiate and characterize them.
Assuntos
Ascomicetos/classificação , DNA Ribossômico/análise , Ascomicetos/genética , Sequência de Bases , China , Clonagem Molecular , DNA Fúngico/análise , DNA Fúngico/genética , Genes Fúngicos , Genoma Fúngico , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Técnica de Amplificação ao Acaso de DNA Polimórfico , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência do Ácido NucleicoRESUMO
Toxin alpha from Naja nigricollis (61 amino acids, four disulfide bridges) belongs to the "three finger" fold family, which contains snake toxins with various biological activities and nontoxic proteins from different origins. In this paper, we report an extensive 1H and 15N NMR study of the dynamics of toxin alpha in solution. 15N relaxation, 1H off-resonance ROESY, and H-D exchange experiments allowed us to probe picosecond to hour motions in the protein. Analysis of these NMR measurements demonstrates that toxin alpha exhibits various time scale motions, i.e., particularly large amplitude picosecond to nanosecond motions at the tips of the loops, observable microsecond to millisecond motions around two disulfide bridges, second time scale motions around the C-N bonds of asparagine and glutamine side chains which are more or less rapid depending on their amino acid solvent accessibility, and minute to hour motions in the beta-sheet structure. The less well-defined regions of toxin alpha solution structures are subject to important picosecond to nanosecond motions. The toxic site is organized around residues belonging to the rigid core of the molecule but also comprises residues exhibiting dynamics on various time scales. The Malpha1 epitope is subject to large picosecond to millisecond motions, which are probably modified by the interaction with the antibody. This phenomenon could be linked to the neutralizing properties of the antibody.
Assuntos
Proteínas Neurotóxicas de Elapídeos/química , Conformação Proteica , Animais , Anisotropia , Anticorpos/imunologia , Asparagina/química , Sítios de Ligação , Proteínas Neurotóxicas de Elapídeos/imunologia , Proteínas Neurotóxicas de Elapídeos/toxicidade , Deutério , Dissulfetos/química , Epitopos/imunologia , Glutamina/química , Cinética , Modelos Moleculares , Isótopos de Nitrogênio , Ressonância Magnética Nuclear Biomolecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , SoftwareRESUMO
Curaremimetic toxins are typical non-enzymatic toxins that bind to their target [the nicotinic acetylcholine receptor (AChR)] through multiple residues. Nevertheless, we show that the concomitant substitutions of only three of the ten functionally important residues of such a toxin sufficed to cause an affinity decrease of the toxin for AChR that is higher than four orders of magnitude. Despite these triple mutations, the overall conformation of the mutated protein remains similar to that of a related recombinant toxin, as judged from both circular dichroism analysis and investigation of antigenicity, using monoclonal and polyclonal antibodies. Furthermore, we show that the detoxified toxin is capable of eliciting antibodies that neutralize the binding of a wild-type toxin to AChR. Therefore, transformation of a non-enzymatic toxin into a toxoid can be achieved, like in the case of enzymatic toxins, by introducing a small number of mutations at positions identified to be critical for expression of toxicity.
Assuntos
Erabutoxinas/química , Mutagênese Sítio-Dirigida/genética , Receptores Nicotínicos/metabolismo , Toxoides/química , Animais , Afinidade de Anticorpos , Sequência de Bases , Dicroísmo Circular , Primers do DNA/química , Elapidae , Ensaio de Imunoadsorção Enzimática , Erabutoxinas/genética , Erabutoxinas/imunologia , Soros Imunes/imunologia , Soros Imunes/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Concentração Osmolar , Ligação Proteica , Coelhos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Toxoides/genética , Toxoides/imunologiaRESUMO
In several instances, a monoclonal antibody raised against a receptor ligand has been claimed to mimic the ligand receptor. Thus, a specific monoclonal antibody (Malpha2-3) raised against a short-chain toxin from snake was proposed to mimic the nicotinic acetylcholine receptor (AChR) (). Further confirming this mimicry, we show that (i) like AChR, Malpha2-3 elicits anti-AChR antibodies, which in turn elicit anti-toxin antibodies; and (ii) the region 106-122 of the alpha-chain of AChR shares 66% primary structure identity with complementarity-determining regions of Malpha2-3. Also, a mutational analysis of erabutoxin a reveals that the epitope recognized by Malpha2-3 consists of 10 residues, distributed within the three toxin loops. Eight of these residues also belong to the 10-residue epitope recognized by AChR, a result that offers an explanation as to the functional similarities between the receptor and the antibody. Strikingly, however, most of the residues common to the two epitopes contribute differentially to the energetic formation of the antibody-toxin and the receptor-toxin complexes. Together, the data suggest that the mimicry between AChR and Malpha2-3 is partial only.
Assuntos
Anticorpos Monoclonais/metabolismo , Receptores Colinérgicos/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Proteínas Neurotóxicas de Elapídeos/imunologia , Proteínas Neurotóxicas de Elapídeos/metabolismo , Reações Cruzadas , Curare/imunologia , Curare/metabolismo , Epitopos/química , Epitopos/imunologia , Erabutoxinas/imunologia , Erabutoxinas/metabolismo , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Receptores Colinérgicos/imunologia , Alinhamento de Sequência , TorpedoRESUMO
We have designed an expression vector permitting the production in the periplasm of Escherichia coli of a fusion protein comprising a dimer of bacterial alkaline phosphatase (PhoA) and two Fab or scFv fragments of a monoclonal antibody directed against human IgG. Each hybrid protein expressed both high specificity for the antigen and full PhoA activity. We show that crude periplasmic extracts containing these conjugates can be used as such in enzyme immunoassays for the detection of human IgG, as exemplified in the case of anti-hepatitis B immunoglobulin.
Assuntos
Fosfatase Alcalina , Anticorpos Anti-Hepatite B/análise , Antígenos de Superfície da Hepatite B/imunologia , Imunoglobulina G/análise , Fosfatase Alcalina/genética , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Sequência de Bases , Escherichia coli , Humanos , Dados de Sequência Molecular , Sondas de Oligonucleotídeos/química , Proteínas Recombinantes de FusãoRESUMO
Using site-directed mutagenesis, we previously identified some residues that probably belong to the site by which Erabutoxin a (Ea), a sea snake toxin, recognizes the nicotinic acetylcholine receptor (AcChoR) (Pillet, L., Trémeau, O., Ducancel, F. Drevet, P., Zinn-Justin, S., Pinkasfeld, S., Boulain, J.-C., and Ménez, A. (1993) J. Biol. Chem. 268, 909-916). We have now studied the effect of mutating 26 new positions on the affinity of Ea for AcChoR. The mutations are F4A, N5V, H6A, Q7L, S9G, Q10A, P11N, Q12A, T13V, T14A, K15A, T16A, delta S18, E21A, Y25F, Q28A, S30A, T35A, I36R, P44V, T45A, V46A, K47A, P48Q, I50Q, and S53A. Binding affinity decreases upon mutation at Gln-7, Gln-10 and to a lesser extent at His-6, Ser-9 and Tyr-25 whereas it increases upon mutation at Ile-36. Other mutations have no effect on Ea affinity. In addition, new mutations of the previously explored Ser-8, Asp-31, Arg-33, and Glu-38 better explain the functional role of these residues in Ea. The previous and present mutational analysis suggest that the "functional" site of Ea covers a homogeneous surface of at least 680 A2, encompassing the three toxin loops, and includes both conserved and variant residues. The variable residues might contribute to the selectivity of Ea for some AcChoRs, including those from fish, the prey of sea snakes.
Assuntos
Erabutoxinas/química , Erabutoxinas/genética , Neurotoxinas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Erabutoxinas/fisiologia , Engenharia Genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Receptores Nicotínicos/metabolismo , Relação Estrutura-Atividade , TorpedoRESUMO
The Pl1 locus in sunflower, Helianthus annuus L., conferring resistance to downy mildew, Plasmopara halstedii, race 1 has been located in linkage group 1 of the consensus RFLP map of the cultivated sunflower. Bulked segregant analyses were used on 135 plants of an F2 progeny from a cross between a downy mildew susceptible line, GH, and RHA266, a line carrying Pl1. Two RFLP markers and one RAPD marker linked to the Pl1 locus have been identified. The RFLP markers are located at 5.6 cM and 7.1 cM on either side of Pl1. The RAPD marker is situated at 43.7 cM from Pl1. The significance and applications of these markers in sunflower breeding are discussed.
RESUMO
We constructed a synthetic gene encoding the published amino acid sequence of DTx from Dendroaspis angusticeps, a ligand of voltage-dependent postassium channels that facilitates neurotransmitter release. We expressed it in Escherichia coli as a fusion protein secreted in the culture medium. The recombinant DTx was generated in vitro by chemical treatment and recovered as two isoforms. One of them (rDTx), like the venom toxin, has an N-terminal pyroglutamate whereas the other (rQDTx) has a free N-terminal glutamine. Chromatographic differences between rDTx and natural DTx led us to re-examine the amino acid sequence of natural DTx. In contrast to what was previously published, position 12 was an Asp and not Asn. Despite this difference, rDTx and DTx had similar toxicity in mice and binding affinity to synaptosomes, suggesting that residue 12 is not important for DTx function. Nor is the N-terminal residue implicated in DTx function since rDTx and rQDTx also had similar biological activities. We also synthesized and expressed a mutant of the DTx gene in which the lysine triplet 28-30 was changed into Ala-Ala-Gly. The two resulting recombinant isoforms exhibited only small decreases in biological activity, excluding the possibility that the positively charged lysine triplet 28-30 of DTx is directly involved in the toxin functional site.
Assuntos
Venenos Elapídicos/química , Venenos Elapídicos/metabolismo , Lisina , Neurotoxinas/metabolismo , Canais de Potássio/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Ligação Competitiva , Encéfalo/metabolismo , Ventrículos Cerebrais/efeitos dos fármacos , Ventrículos Cerebrais/patologia , Galinhas , Venenos Elapídicos/toxicidade , Feminino , Genes Sintéticos , Glutamina , Técnicas In Vitro , Injeções Intraventriculares , Cinética , Dose Letal Mediana , Camundongos , Modelos Estruturais , Dados de Sequência Molecular , Contração Muscular/efeitos dos fármacos , Mutagênese Sítio-Dirigida , Conformação Proteica , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/toxicidade , Sinaptossomos/metabolismoRESUMO
LMWP is the low molecular weight protein which copurifies with alpha-latrotoxin, the main neurotoxin from the black widow venom. It contains 70 residues and three disulfides. We found that its primary structure, including its 6 half-cystines, can be aligned with the amino acid sequences of crustacean hyperglycemic hormones (CHHs) which contain 72-73 residues and three disulfides. To further investigate this structural relationship, we produced a recombinant analog of LMWP in which the unique Met was changed in Leu (LMWPM35L). LMWPM35L was produced as a folded fusion protein in the periplasm of Escherichia coli and was generated in vitro by treating the fusion protein with cyanogen bromide. We showed that LMWPM35L and CHHs have an identical disulfide pairing pattern and possess some alpha-helical structure, as deduced from a comparison of their circular dichroism spectra. In addition, LMWPM35L and CHHs are consensually predicted to possess a helical structure within the region 13-17. Together, the data indicate that CHHs are structurally related to LMWPM35L and presumably also to LMWP. Finally, preliminary studies showed that LMWPM35L is not toxic to mice and does not form channels in lipid bilayers, two well-known properties of alpha-latrotoxin preparations.
Assuntos
Hormônios de Invertebrado/química , Proteínas do Tecido Nervoso/química , Proteínas/isolamento & purificação , Venenos de Aranha/isolamento & purificação , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes , Sequência de Bases , Dicroísmo Circular , DNA Recombinante , Dissulfetos/química , Feminino , Bicamadas Lipídicas , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Peso Molecular , Estrutura Secundária de Proteína , Proteínas/química , Proteínas/toxicidade , Homologia de Sequência de AminoácidosRESUMO
A competitive enzyme immunoassay for rat prolactin using an immunogen and a tracer obtained by recombinant DNA technology is described. Polyclonal antibodies were raised in rabbits immunized with a purified chimeric protein consisting of rat prolactin fused with two synthetic immunoglobulin G binding domains derived from staphylococcal Protein A. The enzymatic tracer was obtained using an expression system which permits insertion of rPRL DNA sequence in the alkaline phosphatase gene. Antibodies and tracer were used to develop a solid-phase competitive immunoassay for the measurement of rat prolactin in plasma with a minimal detectable concentration of 0.5 ng/ml. The mean intra- and inter-assay coefficients of variation were 7.8 and 13.2%, respectively. Rat plasma concentrations measured with this assay correlated well with those obtained with a conventional enzyme immunoassay (r = 0.993, slope = 1.037, n = 24).