RESUMEN
OBJECTIVE: To provide clinical clues to differential diagnosis in patients with chorea and other movement disorders with blood acanthocytes. METHODS: We present a long-term video accompanied follow-up of six Caucasian patients with neuroacanthocytosis from several centers, three diagnosed with chorea-acanthocytosis (ChAc): 34-y.o.(no.1), 36-y.o.(no.2), 43-y.o.(no.3), two diagnosed with McLeod Syndrome (MLS): 52-y.o.(no.4), 61-y.o.(no.5) and one 63-y.o.(no.6), a brother of no.5, with clinical suspicion of MLS. Additionally we report pathological findings of the mother of two brothers with MLS reported in our series with acanthocytes on peripheral blood smear RESULTS: The patients had an unremarkable family history and were asymptomatic until adulthood. Patients no. 1,2,4,5,6 developed generalized chorea and patient no. 3 had predominant bradykinesia. Patients no. 1,2,3 had phonic and motor tics, additionally patients no. 1 and 2 exhibited peculiar oromandibular dystonia with tongue thrusting. In patients no. 2 and 3 dystonic supination of feet was observed, patient no. 3 subsequently developed bilateral foot drop. Patients no. 2 and 4 had signs of muscle atrophy. Tendon reflexes were decreased or absent and electroneurography demonstrated sensorimotor neuropathy in patients no. 1,2,3,4,5, except no. 6. Generalized seizures were seen in patients no. 2,3,5,6 and myoclonic jerks in patient no. 1. Cognitive deterioration was reported in patients no. 1,2,3,5,6. Serum creatine kinase levels were elevated in all six patients. CONCLUSION: We highlight the variability of clinical presentation of neuroacanthocytosis syndromes and the long time from the onset to diagnosis with the need to screen the blood smears in uncertain cases, however, as in one of our cases acanthocytes may even be not found. Based on our observations and data from the literature we propose several red flags that should raise the suspicion of an NA syndrome in a patient with a movement disorder: severe orofacial dyskinesia with tongue and lip-biting (typical of ChAc), feeding dystonia, psychiatric and cognitive disturbances, seizures, peripheral neuropathy, elevation of creatine kinase, elevation of transaminases, hepatosplenomegaly, cardiomyopathy and arrhythmias, and an X-linked pattern of inheritance (McLeod Syndrome, MLS).
Asunto(s)
Neuroacantocitosis/diagnóstico , Adulto , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Neuroacantocitosis/fisiopatologíaRESUMEN
Phase-diverse Fresnel coherent diffractive imaging has been shown to reveal the structure and composition of biological specimens with high sensitivity at nanoscale resolution. However, the method has yet to be applied using X-ray illumination with energy in the so-called 'water-window' that lies between the carbon and oxygen K edges. In this range, differences in the strength of the X-ray interaction for protein based biological materials and water is increased. Here we demonstrate a proof-of-principle application of FCDI at an X-ray energy within the water-window to a dehydrated cellular sample composed of red blood cells infected with the trophozoite stage of the malaria parasite, Plasmodium falciparum. Comparison of the results to both optical and electron microscopy shows that the correlative imaging methods that include water-window FCDI will find utility in studying cellular architecture.
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Eritrocitos/parasitología , Eritrocitos/ultraestructura , Aumento de la Imagen/métodos , Malaria Falciparum/patología , Malaria/patología , Refractometría/métodos , Difracción de Rayos X/métodos , Malaria/diagnóstico por imagen , Malaria/parasitología , Malaria Falciparum/diagnóstico por imagen , Malaria Falciparum/parasitología , Microscopía de Contraste de Fase/métodos , Radiografía , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
BACKGROUND AND OBJECTIVES: Rh-associated glycoprotein (RhAG) is closely associated with the Rh proteins in the red cell membrane. Two high frequency antigens (Duclos and DSLK) and one low frequency antigen (Ol(a)) have serological characteristics suggestive of expression on RhAG. MATERIALS AND METHODS: RHAG was sequenced from the DNA of one Duclos-negative, one DSLK-negative, and two Ol(a+) individuals. Recombinant protein was expressed in HEK 293 cells. Protein models with RhAG subunits were constructed. RESULTS: The original Duclos-negative patient was homozygous for RHAG 316C>G, encoding Gln106Glu. HEK 293 cells expressing Gln106Glu mutant RhAG did not react with anti-Duclos. An individual with DSLK-negative red cells was homozygous for 490A>C, encoding Lys164Gln. Two Ol(a+) members of the original Norwegian family were heterozygous for 680C>T, encoding Ser227Leu. A Japanese donor with Rh(mod) phenotype had Ol(a+) red cells and was homozygous for 680C>T. CONCLUSION: The three red cell antigens encoded by RHAG form the RHAG blood group system: Duclos is RHAG1 (030001); Ol(a) is RHAG2 (030002); and DSLK is provisionally RHAG3 (030003).
Asunto(s)
Proteínas Sanguíneas/genética , Glicoproteínas de Membrana/genética , Sistema del Grupo Sanguíneo Rh-Hr/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Proteínas Sanguíneas/inmunología , Citometría de Flujo , Humanos , Glicoproteínas de Membrana/inmunología , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Sistema del Grupo Sanguíneo Rh-Hr/inmunologíaRESUMEN
BACKGROUND AND OBJECTIVES: RH48 (JAL) is a low-incidence Rh antigen of unknown molecular background associated with weakened expression of RhCE antigens. The objective of this study was to establish the molecular basis of JAL. MATERIALS AND METHODS: Seventeen JAL+ samples, from seven black (one of them a Brazilian of mixed race: black/Caucasian), nine European Caucasians and one Asian individuals, were typed with anti-D, -C, -c, -E and -e. Some samples were also tested for V/VS and ce (f). Titration studies and flow cytometry were used to analyse the expression of the JAL antigen and genomic DNA sequencing of all RHCE exons was conducted on all samples. Routine genotyping for RHCE was carried out on all samples. Screening of RHD exons 1-10, which included detection of the DAU allele, was carried out on all except one of the black samples. The Caucasian samples and remaining black sample were screened for the DAU mutation 1136C>T (T379M). RESULTS: Six black individuals had the Dce haplotype with RHCE mutations 340C>T (R114W) and 733C>G (L245V) [V/VS] and the RHD mutation T379M [DAU]. One mixed race individual had the Dce haplotype with the RHCE mutation 340C>T (R114W) but without the V/VS or DAU mutation. Eight Caucasians had the DCe haplotype with the 340C>T mutation. One Caucasian and one Asian had the Dce haplotype with a different mutation in an adjacent nucleotide, 341G>A (R114Q). All Caucasian individuals were negative for the DAU mutation 1136C>T (T379M). Previously described weakness of CE-related Rh antigens when present in single dose on JAL+ samples of DCe and Dce haplotypes was observed. Weak expression of V/VS was observed in the three black samples tested and weakness of JAL was observed in the black samples compared to the Caucasian samples. CONCLUSION: The same mutation (340C>T, R114W) in two different haplotypes (DCe and Dce) and another mutation (341G>A, R114Q) in one of these haplotypes (Dce) are associated with expression of the JAL antigen. One of the RHCE mutations detected in our samples (340C>T) has been previously described but not in association with the JAL antigen. Our results indicate that the previously described RhCeMA and ce(s)(340) alleles encode the JAL antigen. Expression of V/VS antigen is weakened in the presence of JAL and expression of JAL is usually weaker when associated with the Dce haplotype compared to DCe.
Asunto(s)
Alelos , Regulación de la Expresión Génica/fisiología , Haplotipos/genética , Mutación Missense , Sistema del Grupo Sanguíneo Rh-Hr/biosíntesis , Sistema del Grupo Sanguíneo Rh-Hr/genética , Femenino , Humanos , Masculino , Grupos RacialesRESUMEN
Bone cements produced by different manufacturers vary in their mechanical properties and antibiotic elution characteristics. Small changes in the formulation of a bone cement, which may not be apparent to surgeons, can also affect these properties. The supplier of Palacos bone cement with added gentamicin changed in 2005. We carried out a study to examine the mechanical characteristics and antibiotic elution of Schering-Plough Palacos, Heraeus Palacos and Depuy CMW Smartset bone cements. Both Heraeus Palacos and Smartset bone cements performed significantly better than Schering-Plough Palacos in terms of mechanical characteristics, with and without additional vancomycin (p < 0.001). All cements show a deterioration in flexural strength with increasing addition of vancomycin, albeit staying above ISO minimum levels. Both Heraeus Palacos and Smartset elute significantly more gentamicin cumulatively than Schering-Plough Palacos. Smartset elutes significantly more vancomycin cumulatively than Heraeus Palacos. The improved antibiotic elution characteristics of Smartset and Heraeus Palacos are not associated with a deterioration in mechanical properties. Although marketed as the 'original' Palacos, Heraeus Palacos has significantly altered mechanical and antibiotic elution characteristics compared with the most commonly-used previous version.
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Antibacterianos/análisis , Cementos para Huesos/análisis , Ensayo de Materiales , Polimetil Metacrilato/análisis , Estrés Mecánico , Soporte de Peso/fisiología , Artroplastia de Reemplazo , Gentamicinas/análisis , Pruebas de Dureza , Humanos , Estadística como Asunto , Vancomicina/análisisRESUMEN
The most deadly of the human malaria parasites, Plasmodium falciparum, invades the erythrocytes of its host and initiates a remarkable series of morphological rearrangements within the host cell cytoplasm. The mature erythrocyte is effectively a floating sack of haemoglobin with no endogenous protein synthesis or protein trafficking machinery. In order to colonise and remodel its extracellular space, the parasite generates a series of novel structures that are involved in the export of virulence factors to the surface of the host cell. These include extensions of the parasite's vacuolar membrane, known as the tubulovesicular network, and structures referred to as Maurer's clefts. Maurer's clefts are convoluted collections of distorted discs that are tethered to the red blood cell membrane by structures with stalk-like profiles. Recently electron tomography has enabled visualisation--in three dimensions and at unprecedented resolution--the complexity of the membrane systems within the infected RBC cytoplasm.
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Eritrocitos/parasitología , Eritrocitos/ultraestructura , Malaria Falciparum/patología , Plasmodium falciparum/ultraestructura , Animales , Microscopía Electrónica/métodos , Transporte de Proteínas , Proteínas Protozoarias/análisis , Proteínas Protozoarias/metabolismoRESUMEN
BACKGROUND AND OBJECTIVE: The gene responsible for the P1 polymorphism of the P blood-group system remains unidentified, although the A4GALT gene, whose product is responsible for the production of P(k), has been implicated. No coding differences in A4GALT account for the P1 polymorphism, but homozygosity for two polymorphisms (-551_-550insC and -160A>G) in the 5' untranslated region of the gene has been reported to be unique to Japanese P1- individuals. This study aimed to confirm this correlation in a larger number of British individuals. MATERIALS AND METHODS: Serologically confirmed P1+ (n = 35) and P1- (n = 15) individuals were genotyped for polymorphisms in the 5' untranslated region of A4GALT. RESULTS: In addition to those previously reported, a further polymorphism, -164C>T, was identified. All P1- individuals were homozygous for -551_-550insC and -160G as compared with 10 of 35 (29%) P1+ individuals (P = 0.000003, two-tailed Fisher's exact test). Allele frequencies for all polymorphisms and estimated haplotype frequencies across the region differed significantly between P1+ and P1- groups. CONCLUSIONS: Homozygosity for the A4GALT-551_-550insC and -160G allele is significantly associated with, but not restricted to, the P1- phenotype. No single A4GALT genotype or haplotype was unique to P1- individuals. Thus, A4GALT cannot be unequivocally confirmed as the gene responsible for the P1 phenotype.
Asunto(s)
Regiones no Traducidas 5'/genética , Frecuencia de los Genes , Globósidos/genética , Polimorfismo Genético , Haplotipos , Humanos , Reino UnidoRESUMEN
There is growing epidemiological evidence for statistical associations between increases in air pollution, especially particulate matter, and increases in cardiovascular morbidity and mortality. Laboratory studies have shown that transition metals contribute strongly to the effects of high lung doses of model particles on changes in the electrocardiograms of animals. The present study evaluated the effects of short-term inhalation exposure to respirable particles of specific oxide and sulfate forms of transition metals on heart rate and the electrocardiogram of old dogs having preexisting cardiac abnormalities. Conscious beagle dogs were exposed by oral inhalation for 3 h on each of 3 successive days to aerosols of manganese, nickel, vanadium, iron, and copper oxides, and nickel and vanadium sulfates as single compounds at concentrations of 0.05 mg/m(3). Electrocardiograms were recorded and evaluated for exposure-related changes in heart rate, heart rate variability, and abnormalities of waveforms. Although the electrocardiograms of this population of dogs having potential age and cardiovascular susceptibility factors reflected their underlying clinical abnormalities, no significant effect of exposure to the transition metal aerosols was observed.
Asunto(s)
Administración por Inhalación , Contaminantes Atmosféricos/análisis , Anomalías Cardiovasculares/fisiopatología , Electrocardiografía , Elementos de Transición/administración & dosificación , Aire/análisis , Animales , Cobre/administración & dosificación , Perros , Electrocardiografía/efectos de los fármacos , Femenino , Compuestos Férricos/administración & dosificación , Frecuencia Cardíaca/efectos de los fármacos , Masculino , Compuestos de Manganeso/administración & dosificación , Níquel/administración & dosificación , Óxidos/administración & dosificación , Factores de Tiempo , Compuestos de Vanadio/administración & dosificación , Complejos Prematuros Ventriculares/tratamiento farmacológicoRESUMEN
Alpha1-antichymotrypsin (ACT: new identification SERPINA3) is a member of the serine proteinase inhibitor (serpin) gene family and biochemically has been shown to be a constituent of the senile plaques of Alzheimer's disease. We describe a polymorphism (G-->T) in the promoter region of the ACT gene with the T allele being associated with a 22% increase in the mean plasma ACT concentrations. By reporter gene studies, the T allele is consistently associated with higher mean basal expression in both the human liver cell-line Hep G2 (32%) and in a human glial cell-line T98G (30%). Following 6-h stimulation with the cytokine oncostatin-M, there was a 30-fold increase in Hep G2 and a four-fold increase in T98G cells. The T allele in the promoter region is also in almost complete linkage disequilibrium with the T allele in the signal peptide region of the ACT gene with a standardised disequilibrium coefficient (D') of 0.97; P<0.001. This is the first description of a polymorphism in the ACT gene promoter directly associated with altered gene expression.
Asunto(s)
Polimorfismo Genético , Regiones Promotoras Genéticas , alfa 1-Antiquimotripsina/sangre , alfa 1-Antiquimotripsina/genética , Anciano , Alelos , Enfermedad de Alzheimer/sangre , Enfermedad de Alzheimer/genética , Secuencia de Bases , Línea Celular , ADN/genética , Femenino , Expresión Génica , Genes Reporteros , Hepatocitos/metabolismo , Humanos , Masculino , Datos de Secuencia Molecular , Neuroglía/metabolismo , Polimorfismo de Nucleótido Simple , TransfecciónRESUMEN
We describe an approach for the rapid mapping of epitopes within a malaria antigen using a combination of phage display techniques. Phage display of antigen fragments identifies the location of the epitopes, then random peptide libraries displayed on phage are employed to identify accurately amino acids involved in the epitope. Finally, phage display of mutant fragments confirms the role of each residue in the epitope. This approach was applied to the apical membrane antigen-1 (AMA1), which is a leading candidate for inclusion in a vaccine directed against the asexual blood stages of Plasmodium falciparum. As part of the effort both to understand the function of AMA1 in the parasite life cycle and to define the specificity of protective immune responses, a panel of monoclonal antibodies (MAbs) was generated to obtain binding reagents to the various domains within the molecule. There is a pressing need to determine rapidly the regions recognized by these antibodies and the structural requirements required within AMA1 for high affinity binding of the MAbs. Using phage displaying random AMA1 fragments, it was shown that MAb5G8 recognizes a short linear epitope within the pro-domain of AMA1 whereas the epitope recognized by MAb 1F9 is reduction sensitive and resides within a disulphide-bonded 57 amino acid sub-domain of domain-1. Phage displaying random peptide libraries and mutant AMA1 fragments were employed for fine mapping of the MAb5G8 core epitope to a three-residue sequence in the AMA1 prodomain.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos de Protozoos/inmunología , Mapeo Epitopo/métodos , Proteínas de la Membrana/inmunología , Plasmodium falciparum/inmunología , Proteínas Protozoarias/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/genética , Especificidad de Anticuerpos , Antígenos de Protozoos/química , Antígenos de Protozoos/genética , Bacteriófagos/genética , Disulfuros/química , Epítopos/inmunología , Eritrocitos/parasitología , Proteínas de la Membrana/química , Datos de Secuencia Molecular , Mutación , Biblioteca de Péptidos , Péptidos/genética , Péptidos/inmunología , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Protozoarias/química , Alineación de SecuenciaRESUMEN
The malaria parasite, Plasmodium falciparum, spends part of its life cycle inside the enucleated erythrocytes of its human host. The parasite modifies the cytoplasm and plasma membrane of its host cell by exporting proteins beyond the confines of its own plasma membrane. We have previously provided evidence that a plasmodial homologue of the COPII protein, Sar1p, is involved in the trafficking of proteins across the erythrocyte cytoplasm. We have now characterised an additional plasmodial COPII protein homologue, namely Sec31p. Recombinant proteins corresponding to the WD-40 and the intervening domains of the PfSec31p sequence were used to raise antibodies. The affinity-purified antisera recognised a protein with an apparent relative molecular mass of 1.6 x 10(5) on western blots of malaria parasite-infected erythrocytes but not on blots of uninfected erythrocytes. PfSec31p was shown to be largely insoluble in nonionic detergent, suggesting cytoskeletal attachment. Confocal immunofluorescence microscopy of malaria parasite-infected erythrocytes was used to show that PfSec31p is partly located within the parasite and partly exported to structures outside the parasite in the erythrocyte cytoplasm. We have also shown that PfSec31p and PfSar1p occupy overlapping locations. Furthermore, the location of PfSec31p overlaps that of the cytoadherence-mediating protein PfEMP1. These data support the suggestion that the malaria parasite establishes a vesicle-mediated trafficking pathway outside the boundaries of its own plasma membrane - a novel paradigm in eukaryotic biology.
Asunto(s)
Proteínas Portadoras/metabolismo , Citoplasma/metabolismo , Membrana Eritrocítica/metabolismo , Eritrocitos/parasitología , Fosfoproteínas/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Saccharomyces cerevisiae , Animales , Brefeldino A/farmacología , Proteínas Portadoras/genética , Humanos , Malaria/metabolismo , Datos de Secuencia Molecular , Proteínas de Unión al GTP Monoméricas/genética , Fosfoproteínas/genética , Plasmodium falciparum/genética , Plasmodium falciparum/parasitología , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiología , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Proteínas de Transporte VesicularRESUMEN
After invading human erythrocytes, the malarial parasite Plasmodium falciparum, initiates a remarkable process of secreting proteins into the surrounding erythrocyte cytoplasm and plasma membrane. One of these exported proteins, the knob-associated histidine-rich protein (KAHRP), is essential for microvascular sequestration, a strategy whereby infected red cells adhere via knob structures to capillary walls and thus avoid being eliminated by the spleen. This cytoadherence is an important factor in many of the deaths caused by malaria. Green fluorescent protein fusions and fluorescence recovery after photobleaching were used to follow the pathway of KAHRP deployment from the parasite endomembrane system into an intermediate depot between parasite and host, then onwards to the erythrocyte cytoplasm and eventually into knobs. Sequence elements essential to individual steps in the pathway are defined and we show that parasite-derived structures, known as Maurer's clefts, are an elaboration of the canonical secretory pathway that is transposed outside the parasite into the host cell, the first example of its kind in eukaryotic biology.
Asunto(s)
Eritrocitos/parasitología , Péptidos/metabolismo , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismo , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Brefeldino A/farmacología , Adhesión Celular , Citosol/química , Eritrocitos/metabolismo , Eritrocitos/ultraestructura , Aparato de Golgi/efectos de los fármacos , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Sustancias Macromoleculares , Microscopía Confocal , Microscopía Fluorescente , Datos de Secuencia Molecular , Fotoquímica , Señales de Clasificación de Proteína/fisiología , Estructura Terciaria de Proteína , Transporte de Proteínas , Proteínas Recombinantes de Fusión/metabolismo , Vacuolas/metabolismo , Vacuolas/parasitologíaRESUMEN
The synthesis of novel bisquinoline compounds comprising 4-(4-diethylamino-1-methylbutyl)aminoquinoline units joined through the 2-position by a (CH(2))(n) linker is described. Their ability to inhibit the growth of both chloroquine-sensitive (D10) and chloroquine-resistant (K1) strains of Plasmodium falciparum, the hydrogen peroxide-mediated pathway for decomposition of haem, and the conversion of haem to beta-haematin have been measured. The activity was affected by the length of the linker and the most active (6c, n=12) showed effects similar to chloroquine in three of the assays. However, it was even more active against the resistant strain [IC(50), 17 nM (K1); 43 nM (D10)], much superior to chloroquine (IC(50), 540 nM) and slightly better than mefloquine (IC(50), 30 nM) in this regard.
Asunto(s)
Antimaláricos/síntesis química , Quinolinas/síntesis química , Animales , Antimaláricos/química , Antimaláricos/farmacología , Cloroquina/farmacología , Inactivación Metabólica , Pruebas de Sensibilidad Parasitaria , Plasmodium falciparum/efectos de los fármacos , Quinolinas/farmacologíaAsunto(s)
Telemedicina , Medicina Veterinaria , Animales , Computadores , Humanos , Competencia Profesional , TelecomunicacionesRESUMEN
The histidine-rich protein 2 (PfHRP2) of Plasmodium falciparum has been implicated in the detoxification of ferriprotoporphyrin IX (FP) moieties that are produced as by-products of the digestion of haemoglobin. In this work, we have used a spectroscopic analysis to confirm that recombinant PfHRP2 binds FP. A monoclonal antibody that recognises both recombinant and authentic PfHRP2 was used in immunofluorescence microscopy studies. We found that PfHRP2 is mainly located in the erythrocyte cytosol of infected erythrocytes, however, dual labelling studies suggest that the location of a sub-population of the PfHRP2 molecules overlaps with that of the food vacuole-associated protein, P-glycoprotein homologue (Pgh-1). A semi-quantitative analysis of the level of PfHRP2 in infected erythrocytes suggests a concentration of a few micromolar in the food vacuole. Under conditions designed to mimic the parasite food vacuole, we found that 1.2 microM PfHRP2 is sufficient to catalyse the conversion of about 30% of a 100 microM sample of FP to beta-haematin within 24 h. Moreover, PfHRP2 is capable of promoting the H(2)O(2)-induced degradation of FP at pH 5.2. PfHRP2 also efficiently enhances the ability of FP to catalyse the H(2)O(2)-mediated oxidation of the model co-factor, ortho-phenylene diamine (OPD). These data suggest that PfHRP2 may promote the detoxification of FP and reactive oxygen species within the food vacuole. By contrast, PfHRP2 inhibits the destruction of FP by glutathione (GSH) at pH 7.4. This suggests that PfHRP2 is not a catalyst of FP degradation outside the food vacuole.
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Hemina/metabolismo , Proteínas/metabolismo , Animales , Eritrocitos/parasitología , Técnica del Anticuerpo Fluorescente , Hemoglobinas/metabolismo , Humanos , Malaria Falciparum , Ratones , Microscopía Confocal , Oxidación-Reducción , Plasmodium falciparum/metabolismo , Proteínas/genética , ConejosRESUMEN
A series of terpene isonitriles, isolated from marine sponges, have previously been shown to exhibit antimalarial activities. Molecular modeling studies employing 3D-QSAR with receptor modeling methodologies performed with these isonitriles showed that the modeled molecules could be used to generate a pharmacophore hypothesis consistent with the experimentally derived biological activities. It was also shown that one of the modeled compounds, diisocyanoadociane (4), as well as axisonitrile-3 (2), both of which have potent antimalarial activity, interacts with heme (FP) by forming a coordination complex with the FP iron. Furthermore, these compounds were shown to inhibit sequestration of FP into beta-hematin and to prevent both the peroxidative and glutathione-mediated destruction of FP under conditions designed to mimic the environment within the malaria parasite. By contrast, two of the modeled diterpene isonitriles, 7-isocyanoamphilecta-11(20),15-diene (12) and 7-isocyano-15-isothiocyanatoamphilecta-11(20)-ene (13), that displayed little antimalarial activity also showed little inhibitory activity in these FP detoxification assays. These studies suggest that the active isonitrile compounds, like the quinoline antimalarials, exert their antiplasmodial activity by preventing FP detoxification. Molecular dynamics simulations performed with diisocyanoadociane (4) and axisonitrile-3 (2) allowed their different binding to FP to be distinguished.
Asunto(s)
Antimaláricos/química , Hemo/química , Nitrilos/química , Poríferos/química , Pirenos/química , Compuestos de Espiro/química , Terpenos/química , Animales , Antimaláricos/aislamiento & purificación , Antimaláricos/farmacología , Hemo/metabolismo , Hemoproteínas/química , Hemoproteínas/metabolismo , Espectrometría de Masas , Modelos Moleculares , Nitrilos/aislamiento & purificación , Nitrilos/farmacología , Océanos y Mares , Pirenos/farmacología , Relación Estructura-Actividad Cuantitativa , Compuestos de Espiro/farmacología , Terpenos/aislamiento & purificación , Terpenos/farmacologíaRESUMEN
Multiple single nucleotide polymorphisms (SNP) in the promoter region of the human interleukin-10 (IL-10) gene and in the signal/leader sequence of the human transforming growth factor beta 1 (TGF-beta1) gene, have been associated with susceptibility, severity and clinical outcome for a number of diseases. One common explanation for this, is that different haplotypes of these SNPs regulate the expression of the respective cytokines. Therefore, accurate determination of haplotypes by physical linkage analysis represents an important tool in investigating the pathogenesis of such diseases. Here, we demonstrate that the use of induced heteroduplex generators (IHGs) may be used to identify haplotypes within target sequences in the IL-10 and TGF-beta1 genes. Four haplotypes were observed within the IL-10 promoter region, consisting of -1082, -851, -819 and -592 SNPs. For the TGF-beta1 signal/leader sequence, we observed three haplotypes of the T869C (Leu10Pro) and G915C (Arg25Pro) SNPs. In both cases, all combinations of these haplotypes could be resolved unequivocally with a single IHG reagent.
Asunto(s)
Citocinas/genética , ADN/análisis , Haplotipos , Secuencia de Bases , Citocinas/inmunología , ADN/genética , Electroforesis , Humanos , Datos de Secuencia Molecular , Sensibilidad y EspecificidadRESUMEN
Cardiac disease is one of the major causes of morbidity and mortality worldwide and is the leading cause of death in the United States. Epidemiologic studies have shown a close association between morbidity and mortality from cardiac disease, primarily in persons already affected, and with modest increases in levels of air pollution. At present, no mechanisms are known by which inhaled air pollutants interact with the heart to cause death. Thus, animal models of cardiac disease are needed to study possible interactions between inhaled pollutants and the heart and the resultant morbidity and mortality. Very little research in animals has been conducted in this area, and appropriate animal models must be carefully selected. The purpose of this review is to examine several potential animal models and to discuss their advantages and disadvantages in the study of cardiac disease and air pollution.
Asunto(s)
Contaminantes Atmosféricos/efectos adversos , Modelos Animales de Enfermedad , Cardiopatías/etiología , Exposición por Inhalación , Animales , Enfermedad Crónica , Cardiopatías/patología , HumanosRESUMEN
The Molecular Approaches to Malaria (MAM2000) conference, Lorne, Australia, 2-5 February 2000, brought together world-class malaria research scientists. The development of new tools and technologies - transfection, DNA microarrays and proteomic analysis - and the availability of DNA sequences generated by the Malaria Genome Project, along with more classic approaches, have facilitated the identification of novel drug targets, the development of new antimalarials and the generation of a deeper understanding of the molecular mechanism(s) of drug resistance in malaria. It is hoped that combinations of these technologies could lead to strategies that enable the development of effective, efficient and affordable new drugs to overcome drug-resistant malaria, as discussed at MAM2000 and outlined here by Ian Macreadie and colleagues.
Asunto(s)
Antimaláricos/farmacología , Antagonistas del Ácido Fólico/farmacología , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/enzimología , Quinolinas/farmacología , Animales , Antimaláricos/uso terapéutico , Diseño de Fármacos , Resistencia a Medicamentos , Antagonistas del Ácido Fólico/uso terapéutico , Humanos , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/parasitología , Quinolinas/uso terapéuticoRESUMEN
We produced a fluorescent antibody as a single recombinant protein in Escherichia coli by fusing a red-shifted mutant of green fluorescent protein (EGFP) to a single-chain antibody variable fragment (scFv) specific for hepatitis B surface antigen (HepBsAg). GFP is a cytoplasmic protein and it was not previously known whether it would fold correctly to form a fluorescent protein in the periplasmic space of E.COLI: In this study we showed that EGFP alone or fused to the N'- and C'-termini of the scFv resulted in fusion proteins that were in fact highly fluorescent in the periplasmic space of E.COLI: cells. Further characterization revealed that the periplasmic N'-terminal EGFP-scFv fusion was the most stable form which retained the fluorescent properties of EGFP and the antigen binding properties of the native scFv; thus representing a fully functional chimeric molecule. We also demonstrated the utility of EGFP-scFv in immunofluorescence studies. The results showed positive staining of COS-7 cells transfected with HepBsAg, with comparable sensitivity to a monoclonal antibody or the scFv alone, probed with conventional fluorescein-labelled second antibodies. In this study, we developed a simple technique to produce fluorescent antibodies which can potentially be applied to any scFv. We demonstrated the utility of an EGFP-scFv fusion protein for immunofluorescence studies, but there are many biological systems to which this technology may be applied.