RESUMO
Monitoring the quality of medicines plays a crucial role in an integrated medicines quality assurance system. In a publicly available medicines quality database (MQDB), the U.S. Pharmacopeial Convention (USP) reports results of data collected from medicines quality monitoring (MQM) activities spanning the period of 2003-2013 in 17 countries of Africa, Asia, and South America. The MQDB contains information on 15,063 samples collected and tested using Minilab® screening methods and/or pharmacopeial methods. Approximately 71% of the samples reported came from Asia, 23% from Africa, and 6% from South America. The samples collected and tested include mainly antibiotic, antimalarial, and antituberculosis medicines. A total of 848 samples, representing 5.6% of total samples, failed the quality test. The failure proportion per region was 11.5%, 10.4%, and 2.9% for South America, Africa, and Asia, respectively. Eighty-one counterfeit medicines were reported, 86.4% of which were found in Asia and 13.6% in Africa. Additional analysis of the data shows the distribution of poor-quality medicines per region and by therapeutic indication as well as possible trends of counterfeit medicines.
Assuntos
Antibacterianos/normas , Antimaláricos/normas , Antituberculosos/normas , Preparações Farmacêuticas/normas , África , Antimaláricos/química , Antituberculosos/química , Ásia , Medicamentos Falsificados , Bases de Dados Factuais , América do Sul , Fatores de TempoRESUMO
BACKGROUND: The relationship between helminthiases and allergy is a matter of considerable interest and research. In the tropics, house dust mite exposure, a known risk factor for asthma, is frequently concurrent with helminth infections. It remains to be defined whether infection with the common roundworm Ascaris or its bystander immunological effects influence the prevalence and pathogenesis of asthma independently of mite sensitization. OBJECTIVE: To investigate the relationship between the IgE responses to Ascaris and its purified allergens and the risk of asthma in a tropical country. METHODS: A nested case-control study was performed in 356 subjects who reported current and past asthma symptoms (asthmatics) and 435 controls that had never experienced such symptoms. They were tested for serum levels of total IgE and specific IgE to Ascaris extract, Asc s 1 (ABA-1), Asc l 3 (tropomyosin) and GST (glutathione transferase). In addition, specific IgE to Dermatophagoides pteronyssinus, Blomia tropicalis and their tropomyosins Der p 10 and Blo t 10 was measured. Sensitization was defined as a positive specific IgE result to any extract or recombinant allergen. RESULTS: Sensitization to Ascaris and D. pteronyssinus was independently associated with asthma after adjustment for age, gender, socio-economic stratum, city and other IgE levels (adjusted ORs: 2.17; 95% CI 1.37-3.42 and 2.46; 95% CI 1.54-3.92), respectively. There was also a significant association with sensitization to the highly allergenic and cross-reactive tropomyosins Asc l 3, Blo t 10 and Der p10 (aORs: 1.76; 95% CI 1.21-2.57, 1.64; 95% CI 1.14-2.35 and 1.51; 95% CI 1.02-2.24), respectively. CONCLUSION AND CLINICAL RELEVANCE: IgE responses to Ascaris are associated with asthma symptoms in a population living in the tropics. Sensitization to the cross-reactive Ascaris and mite tropomyosins partially underlies this finding. These results have potential relevance in asthma diagnosis and management.
Assuntos
Ascaris/imunologia , Asma/imunologia , Imunoglobulina E/imunologia , Ácaros/imunologia , Tropomiosina/imunologia , Adolescente , Adulto , Fatores Etários , Alérgenos/imunologia , Animais , Especificidade de Anticorpos/imunologia , Asma/epidemiologia , Estudos de Casos e Controles , Criança , Feminino , Humanos , Imunoglobulina E/sangue , Masculino , Pessoa de Meia-Idade , Razão de Chances , Fatores de Risco , Fatores Sexuais , Adulto JovemRESUMO
The aim of the study is to assess the hypotensive properties of the hydro-ethanolic crude root extract (CRE), the n-butanol fraction (F(BtOH)) and nuatigenin-3-O-ß-chacotriose, from Solanum sisymbriifolium Lam., in adrenal regeneration hypertension+deoxycorticosterone acetate (ARH+DOCA) rats, following a chronic administration. The roots of S. sisymbriifolium Lam. (Solanaceae) were extracted by reflux with ethanol-water 7:3 and the active extract was fractionated by bioassay-guided liquid-liquid separation. Nuatigenin-3-O-ß-chacotriose (B(3-1)) was identified as the main hypotensive compound from the crude drug by spectroscopic methods. Immature Wistar rats of both sexes were submitted to both surgery and deoxycorticosterone acetate treatment to obtain adrenal regeneration hypertensive rats (ARH+DOCA). Different groups of experimentally induced hypertensive rats were randomly allotted and received during 16 weeks a daily oral administration of 1% saline solution (0.1 mL/100g body weigh), 100.0 mg/kg of CRE, 10.0, 30.0 and 50.0 mg/kg of F(BtOH), and 1.0 mg/kg of B(3-1), respectively. In addition, two groups of ARH+DOCA rats were randomly assigned to receive either B(3-1) (1.0 mg/kg/day) or 1% of saline solution (0.1 mL/100g body weight/day) for 7 weeks and then a cross over procedure was performed in order to complete the 16th-week treatment. After 16 weeks of oral administration of crude root extract (CRE), butanolic fraction (F(BtOH)) and nuatigenin-3-O-ß-chacotriose (B(3-1)) a significant reduction of blood pressure value was induced in hypertensive animals (ARH+DOCA) in comparison to the control group receiving 1% saline solution, at the end of experiment. Administration of B(3-1) (1.0 mg/kg/day p.o.) to ARH+DOCA rats provoked a significant reduction of blood pressure, observed gradually from 5th week (p<0.05) to the end of the 16th week period of treatment (p<0.01). Moreover, in a cross over design it was observed that the reduction of blood pressure to normotensive condition is associated to B(3-1). The latest demonstrated that the blood pressure-lowering effect, in clearly hypertensive animals, is reversible and depend upon administration of nuatigenin-3-O-ß-chacotriose (B(3-1)). Our results demonstrated that daily oral administration of CRE, F(BtOH) and nuatigenin-3-O-ß-chacotriose from S. sisymbriifolium for a 16-week period exhibits an antihypertensive effect in experimentally hypertensive (ARH+DOCA) rats.
Assuntos
Anti-Hipertensivos/farmacologia , Hipertensão/tratamento farmacológico , Saponinas/farmacologia , Solanum/química , Triterpenos/farmacologia , Tropanos/farmacologia , Animais , Pressão Sanguínea/efeitos dos fármacos , Esquema de Medicação , Hipertensão/fisiopatologia , Camundongos , Paraguai , Fitoterapia , Extratos Vegetais/farmacologia , Raízes de Plantas/química , RatosRESUMO
BACKGROUND: Analysis of cross-reactivity between the nematode Ascaris ssp. and dust mites, two important allergen sources in the tropics, will contribute in understanding their influence on asthma and atopy. The objective of this study was to investigate immunoglobulin E (IgE) cross-reactivity between Ascaris and two domestic mites in the tropics. METHODS: Sera from 24 asthmatic patients were used in ELISA and immunoblotting IgE-binding inhibition assays using Ascaris, Blomia tropicalis and Dermatophagoides pteronyssinus extracts and the recombinants Blo t 10, ABA-1 and Blo t 13 as competitors. Identification of Ascaris allergens was confirmed by mass spectrometry (LC-MS/MS). RESULTS: We detected at least 12 human IgE-binding components in Ascaris extract. Blomia tropicalis and D. pteronyssinus inhibited 83.3% and 79% of IgE-binding to Ascaris, while Ascaris inhibited 58.3% and 79.3% to B. tropicalis and D. pteronyssinus respectively. Mite tropomyosin inhibited 85% of IgE-binding to Ascaris. Affinity-purified human IgE to rBlo t 10 identified an allergen of 40 kDa in Ascaris extract, further confirmed as tropomyosin by LC-MS/MS. We found no evidence of IgE cross-reactivity between rABA-1 and any allergen component in mite extracts, including rBlo t 13. CONCLUSIONS: There is cross-reactivity between Ascaris and mites, determined by several allergens including tropomyosin and glutathione-S-transferase. In addition to its potential impact on asthma pathogenesis, Ascaris infection and mite allergy diagnosis relying on the determination of specific IgE could be affected by this cross-reactivity. ABA-1 has no cross-reactive counterpart in mite extracts, suggesting its usefulness as a more specific marker of Ascaris infection.
Assuntos
Alérgenos/imunologia , Antígenos de Dermatophagoides/imunologia , Ascaris/imunologia , Asma , Hipersensibilidade Imediata/imunologia , Imunoglobulina E , Ácaros/imunologia , Tropomiosina/imunologia , Adolescente , Adulto , Animais , Antígenos de Plantas , Asma/imunologia , Asma/fisiopatologia , Criança , Reações Cruzadas , Feminino , Glutationa Transferase/imunologia , Proteínas de Helminto/imunologia , Humanos , Hipersensibilidade Imediata/fisiopatologia , Imunoglobulina E/imunologia , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
The 13q33-34 region harbours a susceptibility locus to Ascaris lumbricoides, although the underlying genes are unknown. Immunoglobulin (Ig)E and IgG confer protective immunity and here we sought to investigate in an endemic population whether LIG4, TNFSF13B and IRS2 genes influence IgE and IgG levels against Ascaris and the ABA-1 allergen as a putative resistance marker. Mite-allergic asthmatic patients were analysed for potential relationships between Ascaris predisposition and allergy. One thousand and sixty-four subjects from Cartagena, Colombia, were included. Single nucleotide polymorphisms (SNPs) were genotyped using TaqMan assays. Antibody levels were measured by enzyme-linked immunosorbent assay. Linear and logistic regressions were used to model effects of genotypes on antibody levels. The GG genotype of LIG4 (rs1805388) was associated with higher IgE levels to Ascaris compared with other genotypes. TNFSF13B (rs10508198) was associated positively with IgG levels against Ascaris extract and IgE levels against ABA-1. In asthmatics, IRS2 (rs2289046) was associated with high total IgE levels. Associations held up after correction by population stratification using a set of 52 ancestry markers, age, sex and disease status. There was no association with asthma or mite sensitization. In a tropical population, LIG4 and TNFSF13B polymorphisms are associated with specific IgE and IgG to Ascaris, supporting previous linkage studies implicating the 13q33 region. Our results suggest that genes protecting against parasite infections can be different to those predisposing to asthma and atopy.
Assuntos
Anticorpos Anti-Helmínticos/sangue , Ascaríase/imunologia , Ascaris lumbricoides , Asma/genética , Imunoglobulina E/sangue , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Ascaríase/genética , Ascaris lumbricoides/imunologia , Asma/imunologia , Asma/microbiologia , Fator Ativador de Células B/genética , Estudos de Casos e Controles , Criança , DNA Ligase Dependente de ATP , DNA Ligases/genética , Feminino , Predisposição Genética para Doença , Genótipo , Humanos , Proteínas Substratos do Receptor de Insulina/genética , Modelos Lineares , Masculino , Pessoa de Meia-Idade , Polimorfismo Genético , Adulto JovemRESUMO
The aim of the present work is to evaluate the putative antidepressant-like effects of a hydro-ethanolic extract (CEAp) and their fractions from the aerial parts of Aloysia polystachya (Griseb.) Moldenke (Verbenaceae) on the performance of male mice in the forced swimming test (FST). A single dose (100.0mg/kgp.o.) of CEAp, in male mice provoked a significant reduction of the immobility time (p<0.01). Such effect was also observed with short-term treatment (7 days) with single doses of 1.0 (p<0.01), 10.0 (p<0.05) and 100.0 (p<0.05)mg/kg/day of CEAp. Additionally, in a different set of experiments, repeated administration in a 24-h period (24, 18 and 1h before swimming test) with doses of 1.0 (p<0.05) and 10.0 (p<0.05)mg/kg p.o., of CEAp and 10.0mg/kgp.o., (p<0.05) of ethyl acetate fraction, provoked significant reduction of the immobility time of male mice in the FST. Moreover, it was noted important differences in the onset of the antidepressant-like effect in the FST, depending on the modality of treatment with CEAp (acute, short-term or repeated). Both, efficacy and potency were higher when repeated administration of CEAp was used, and surprisingly the dose of 10mg/kg (24, 18 and 1h before swimming test) was more effective than imipramine. In the same way, the short term administration (7 days) improved significantly efficacy and potency of the CEAp in comparison to a single dose treatment. The ethyl acetate fraction submitted to TLC demonstrated that main and minor components are phenolics and terpenes, respectively. In addition, this fraction gives a negative Shinoda's test for flavonoids. These results indicate an antidepressant-like profile of action for the hydro-ethanolic extract and the component(s) of the ethyl acetate fraction obtained from A. polystachya, which deserve further investigation.
Assuntos
Antidepressivos/análise , Resposta de Imobilidade Tônica/efeitos dos fármacos , Fitoterapia , Extratos Vegetais/administração & dosagem , Verbenaceae/química , Animais , Depressão/tratamento farmacológico , Masculino , Camundongos , Estresse Psicológico/tratamento farmacológico , Natação/psicologiaRESUMO
The aim of the present work is to demonstrate the putative sedative and anxiolytic-like effects of a hydro-ethanolic extract obtained from the aerial parts of Aloysia polystachya (Verbenaceae) in male mice using several behavioural assays. Groups of male mice orally treated with doses of 1.0, 10.0 and 100.0 mg/kg of the extract did not show any significant alteration of their locomotor activity, body temperature or motor coordination. The same treatment increased the duration of the sleeping time induced by 30.0 mg/kg i.p. of sodium pentobarbital. However, the sleeping time induced by ethyl ether was not modified by the oral administration of the extract, not confirming the putative sedative effect of the plant. The ethanolic extract also significantly increased the percentage of both entries (1.0 and 100.0 mg/kg) and the time spent (10.0 and 100.0 mg/kg) into the open arms of the elevated plus maze (EPM). Nevertheless, the binding of (3)H-flunitrazepam ((3)H-FNZ) to the benzodiazepine binding site (BDZ-bs), in washed crude synaptosomal membranes from rat cerebral cortex, was not affected by the semi-purified components from Aloysia polystachya. These results indicate an anxiolytic-like profile of action for the extract of Aloysia polystachya without sedative side effect, being this activity probably mediated by other mechanism than BDZ-bs modulation at the GABA(A) receptors.
Assuntos
Ansiolíticos/farmacologia , Extratos Vegetais/farmacologia , Verbenaceae , Animais , Comportamento Animal/efeitos dos fármacos , Temperatura Corporal/efeitos dos fármacos , Flunitrazepam/metabolismo , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Atividade Motora/efeitos dos fármacos , Sono/efeitos dos fármacosRESUMO
The ABA-1 protein of Ascaris lumbricoides (of humans) and Ascaris suum (of pigs) is abundant in the pseudocoelomic fluid of the parasites and also appears to be released by the tissue-parasitic larvae and the adult stages. The genes encoding the polyprotein precursor of ABA-1 (aba-1) were found to be arranged similarly in the two taxa, comprising tandemly repeating units encoding a large polyprotein which is cleaved to yield polypeptides of approximately 15 kDa which fall into 2 distinct classes, types A and B. The polyprotein possibly comprises only 10 units. The aba-1 gene of A. lumbricoides is polymorphic, and the majority of substitutions observed occur in or near predicted loop regions in the encoded proteins. mRNA for ABA-1 is present in infective larvae within the egg, and in all parasitic stages, but was not detectable in unembryonated eggs. ABA-1 mRNA was confined to the gut of adult parasites, and not in body wall or reproductive tissues. Recombinant protein representing a single A-type unit for the A. lumbricoides aba-1 gene was produced and found to bind retinol (Vitamin A) and a range of fatty acids, including the pharmacologically active lipids lysophosphatidic acid, lysoplatelet activating factor, and there was also evidence of binding to leukotrienes. It failed to bind to any of the anthelmintics screened. Differential Scanning Calorimetry showed that the recombinant protein was highly stable, and unfolded in a single transition at 90.4 degrees C. Analysis of the transition indicated that the protein occurs as a dimer and that the dimer dissociates simultaneously with the unfolding of the monomer units.
Assuntos
Ascaríase/parasitologia , Ascaris lumbricoides/genética , Ascaris suum/genética , Proteínas de Helminto/genética , Alérgenos/química , Alérgenos/genética , Alérgenos/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Anti-Helmínticos/química , Antígenos de Helmintos/química , Antígenos de Helmintos/genética , Antígenos de Helmintos/imunologia , Antígenos de Plantas , Ascaríase/sangue , Ascaris lumbricoides/química , Ascaris lumbricoides/imunologia , Ascaris suum/química , Ascaris suum/imunologia , Sequência de Bases , Varredura Diferencial de Calorimetria , China , DNA de Helmintos/química , DNA de Helmintos/isolamento & purificação , Regulação da Expressão Gênica , Guatemala , Proteínas de Helminto/química , Proteínas de Helminto/imunologia , Humanos , Ligantes , Dados de Sequência Molecular , Plasmídeos , Polimorfismo Genético/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
BACKGROUND: We have previously described a cDNA (clone Bt6) encoding a novel allergen from Blomia tropicalis, which showed sequence similarities to the FABP/P2/cellular retinoic acid binding protein/cellular retinol binding protein, a family of cytosolic lipid transport proteins (cLTPs). This work was planned to better characterize this allergen to which the official name Blo t 13 had been assigned. METHODS: Fluorescence-based lipid ligand binding assays and secondary structure analysis by circular dicroism were carry out using recombinant Blo t 13 (rBlo t 13) protein. Structural predictions and molecular modelling were performed based on the amino acid sequence inferred from the open reading frame of Bt6 cDNA sequence. RESULTS: rBlot t 13 binds the natural fluorescent fatty acid cis-parinaric acid and oleic acid by competition, but not retinol, retinoic acid, cholesterol, dansylated or anthroxylated fatty acids such as dansyl-DL-aminocaprylic acid and 12-(9-anthroyloxy)-stereate. Circular dichroism analysis indicated that rBlo t 13 comprises 45% beta-sheet and 13% alpha-helix. The amino acid sequence of Blo t 13 modelled well to known crystal structures of cLTPs providing a tertiary structural model comprising ten beta-strands organized into two beta-sheets, and two short alpha-helices. CONCLUSION: Blo t 13 is a fatty acid-specific member of the beta-rich cLTP family of proteins.
Assuntos
Alérgenos/química , Proteínas de Transporte/química , Ácaros , Alérgenos/genética , Alérgenos/metabolismo , Animais , Antígenos de Plantas , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ligação a Ácido Graxo , Ácidos Graxos/metabolismo , Ligantes , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
It has recently been shown using genetic markers that Ascaris in humans and pigs in Central America comprise reproductively isolated populations. We present a similar analysis for a region of China in which close association between pigs and humans has been the norm for thousands of years, and agricultural practices will result in frequent exposure to eggs from both sources. DNA fragments from selected regions of mitochondrial and ribosomal DNA were amplified by PCR and allelic forms identified following digestion with a panel of restriction enzymes, using DNA from a total of 115 individual worms from both people and pigs from 2 neighbouring villages. Significant frequency differences in both mtDNA haplotypes and the rDNA spacer were found between the 2 host-associated populations, indicating that they represented reproductively isolated populations. Mitochondrial haplotype frequencies were different from those observed in Guatemala and also from other Asian Ascaris populations, suggesting low levels of gene flow between populations. However, we found no evidence for significant heterogeneity in the genetic composition of Ascaris infrapopulations in either humans or pigs, possibly indicative of agricultural practices in China which have resulted in a random distribution of alleles within the parasite populations.
Assuntos
Ascaríase/parasitologia , Ascaris/genética , Variação Genética/genética , Doenças dos Suínos/parasitologia , Animais , Antinematódeos/uso terapêutico , Ascaríase/epidemiologia , Ascaris/imunologia , Criança , Pré-Escolar , China/epidemiologia , Enzimas de Restrição do DNA/química , DNA de Helmintos/química , DNA Mitocondrial/química , DNA Ribossômico/química , Eletroforese em Gel de Ágar , Fezes/parasitologia , Genética Populacional , Guatemala/epidemiologia , Haplótipos , Humanos , Reação em Cadeia da Polimerase , Prevalência , Pamoato de Pirantel/uso terapêutico , População Rural , Suínos , Doenças dos Suínos/epidemiologiaRESUMO
The translation of ferritin mRNA and degradation of transferrin receptor mRNA are regulated by the interaction of an RNA-binding protein, the iron-responsive element binding protein (IRE-BP), with RNA stem-loop structures known as iron-responsive elements (IREs) contained within these transcripts. IRE-BP produced in iron-replete cells has aconitase (EC 4.2.1.3) activity. The protein shows extensive sequence homology with mitochondrial aconitase, and sequences of peptides prepared from cytosolic aconitase are identical with peptides of IRE-BP. As an active aconitase, IRE-BP is expected to have an Fe-S cluster, in analogy to other aconitases. This Fe-S cluster has been implicated as the region of the protein that senses intracellular iron levels and accordingly modifies the ability of the IRE-BP to interact with IREs. Expression of the IRE-BP in cultured cells has revealed that the IRE-BP functions either as an active aconitase, when the cells are iron-replete, or as an active RNA-binding protein, when the cells are iron-depleted. We compare properties of purified authentic cytosolic aconitase from beef liver with those of IRE-BP from tissue culture cells and establish that characteristics of the physiologically relevant form of the protein from iron-depleted cells resemble those of cytosolic aconitase apoprotein. We demonstrate that loss of the labile fourth iron atom of the Fe-S cluster results in loss of aconitase activity, but that more extensive cluster alteration is required before the IRE-BP acquires the capacity to bind RNA with the affinity seen in vivo. These results are consistent with a model in which the cubane Fe-S cluster is disassembled when intracellular iron is depleted.
Assuntos
Aconitato Hidratase/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Ferro/metabolismo , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Animais , Apoproteínas/metabolismo , Desferroxamina/química , Ferricianetos/química , Ferritinas/genética , Hemina/química , Humanos , Técnicas In Vitro , Proteínas Reguladoras de Ferro , Camundongos , Oxirredução , Relação Estrutura-Atividade , Células Tumorais CultivadasAssuntos
Serviços de Saúde do Adolescente , Emigração e Imigração , Infecções por HIV/epidemiologia , Hispânico ou Latino , Refugiados , Serviço Social , Adolescente , América Central/etnologia , Feminino , Infecções por HIV/prevenção & controle , Humanos , Masculino , México/etnologia , São Francisco/epidemiologiaRESUMO
It might be expected that infections with transmissible agents will elicit an immune response to all of their exoantigens and that immune response (Ir) gene control of responses to individual epitopes on a given parasite component would be obscured by reaction to the molecule as a whole. Humans infected with parasitic nematodes, however, mount antibody responses which are selective for certain parasite components. This was modelled in inbred rats infected with the parasitic nematode Nippostrongylus brasiliensis and their responses to secreted antigens analysed by immunoprecipitation and SDS-PAGE. No strain responded to all the potential antigens and only those of identical major histocompatibility complex (MHC) had similar recognition profiles. This MHC-restricted response applied to whole molecules synthesized by the parasite, rather than merely to epitopes thereon and is, therefore, contrary to expectation. Moreover, the response patterns of F1 hybrid animals were not merely summations of parental responses. This suggests defective antigen presentation of particular parasite components by certain MHC class II molecules and/or cross-tolerance with background gene products.
Assuntos
Anticorpos Anti-Helmínticos/análise , Genes MHC da Classe II/imunologia , Infecções por Nematoides/imunologia , Nippostrongylus/imunologia , Animais , Antígenos de Helmintos/imunologia , Eletroforese em Gel de Poliacrilamida , Peso Molecular , Testes de Precipitina , Ratos , Ratos EndogâmicosRESUMO
The reactivity of cysteines following cluster destruction by iron chelation was investigated for [4Fe-4S]2+ and cubane [3Fe-4S]+ beef heart aconitase. When the chelator orthobathophenanthroline disulfonate was used, the formation of sulfur-sulfur bonds and the retention of inorganic sulfur from the cluster was observed. For both the 4Fe and 3Fe forms of aconitase, the two cysteines in peptide 7, the cysteine in peptide 3, and the cysteine in peptide 2 were found as the primary constituents of sulfur-sulfur bonds (the peptide sequences and nomenclature are from Plank, D. W., and Howard, J. B. (1988) J. Biol. Chem. 263, 8184-8189). Three of these four cysteines (peptides 3 and 7) correlated with those proposed to be cluster ligands recently determined by x-ray crystallography (Robbins, A. H. and Stout, C. D. (1989) Proteins, in press; Robbins, A. H., and Stout, C. D.,, (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 3639-3643) for pig heart aconitase. A mechanism is proposed whereby the greater affinity of orthobathophenanthroline disulfonate for Fe2+ relative to Fe3+ shifts the equilibrium toward reduction of ferric iron through sulfur-sulfur bond formation at the cluster site. Aconitase which has been oxidized with ferricyanide and from which the cluster iron has been removed by EDTA has been shown to have two di- or polysulfides (Kennedy, M. C., and Beinert, H. (1988) J. Biol. Chem. 263, 8194-8198). The cysteines found in the sulfur-sulfur bonds generated by this treatment also were predominantly those from peptides 3 and 7. In addition, the putative thiol ligands for the linear [3Fe-4S]+ cluster of aconitase are reported. The four cysteines of peptides 7 and 9 (two in each peptide) were found to be protected by the cluster from alkylation when the protein was denatured. The difference in the ligands between the cubane and linear forms indicates that a specific thiol exchange occurs during the conversion.
Assuntos
Aconitato Hidratase/metabolismo , Cisteína , Ferro/análise , Miocárdio/metabolismo , Alquilação , Sequência de Aminoácidos , Animais , Bovinos , Dissulfetos/análise , Ácido Edético/farmacologia , Ferricianetos/farmacologia , Iodoacetatos/metabolismo , Ácido Iodoacético , Cinética , Dados de Sequência Molecular , Oxirredução , Homologia de Sequência do Ácido Nucleico , SuínosRESUMO
Interactions between infections of Trichinella spiralis and Nippostrongylus brasiliensis were studied in the NIH strain of mouse which is known to react strongly to T. spiralis. The course of N. brasiliensis infection in this strain of mouse is described and expulsion is shown to be accelerated in immunized mice and inhibited in cortisone-treated mice. There was no evidence of inter-specific competition between the two species of worm in concurrent infections; the number and location of adults of both species were normal and T. spiralis was able to grow and reproduce normally. No evidence was found of direct immunological cross-reaction between N. brasiliensis and T. spiralis as assessed by the kinetics of adult worm numbers on heterologous challenge of immunized mice 90 days after the initiation of the last of 3 immunizing infections. Interaction was observed only when the timing of concurrent infections was such that one species was established in the intestine immediately before the beginning of expulsion of the second species. Interaction was manifested as a premature loss of worms and, in addition, as impairment of growth and fecundity of T. spiralis. These effects on T. spiralis were similar to those observed as a consequence of a specific immune response to T. spiralis. The rapidity of appearance of these effects and the lack of direct cross-immunity between the two species of worm suggest that the events involved in interaction were non-specific in action and possibly due to environmental changes in the gut caused by the immune response. These non-specific effects are therefore analogous, but not necessarily homologous, to the expulsion of these parasites in single species infections.