RESUMEN
The present study has been undertaken in order to highlight the healing effect of Zizyphus lotus vegetable oil. The seeds of this plant contain an oil rate of 30%. The obtained results on the main elements composing the vegetable oil have shown that Zizyphus lotus vegetable oil has a low value of acidity index and it presents a not negligible degree of unsaturation. The value of the peroxide index of Zizyphus lotus vegetable oil is less than 10 which characterizes the most of conventional oils. Furthermore, the spectral analysis by gas chromatography has shown the presence of 53 majority and minority molecules. Thus, the evaluation of the healing activity of Z. lotus seed vegetable oil has demonstrated a highly significant effect against the negative control and silver sulfadiazine was used as conventional treatment for burns. Based on the obtained results, we can suggest that the oil extracted from the seeds of the studied plant could be used to cure wounds.
Asunto(s)
Quemaduras/prevención & control , Aceites de Plantas/farmacología , Semillas/química , Cicatrización de Heridas/efectos de los fármacos , Ziziphus/química , Animales , Quemaduras/etiología , Quemaduras/fisiopatología , Cromatografía de Gases , Calor/efectos adversos , Ratones , Aceites de Plantas/análisis , Aceites de Plantas/aislamiento & purificaciónRESUMEN
Nearly full-length Circumsporozoite protein (CSP) from Plasmodium falciparum, the C-terminal fragments from both P. falciparm and P. yoelii CSP and a fragment comprising 351 amino acids of P.vivax MSPI were expressed in the slime mold Dictyostelium discoideum. Discoidin-tag expression vectors allowed both high yields of these proteins and their purification by a nearly single-step procedure. We exploited the galactose binding activity of Discoidin Ia to separate the fusion proteins by affinity chromatography on Sepharose-4B columns. Inclusion of a thrombin recognition site allowed cleavage of the Discoidin-tag from the fusion protein. Partial secretion of the protein was obtained via an ER independent pathway, whereas routing the recombinant proteins to the ER resulted in glycosylation and retention. Yields of proteins ranged from 0.08 to 3 mg l(-1) depending on the protein sequence and the purification conditions. The recognition of purified MSPI by sera from P. vivax malaria patients was used to confirm the native conformation of the protein expressed in Dictyostelium. The simple purification procedure described here, based on Sepharose-4B, should facilitate the expression and the large-scale purification of various Plasmodium polypeptides.
Asunto(s)
Dictyostelium/genética , Dictyostelium/metabolismo , Lectinas , Proteína 1 de Superficie de Merozoito/metabolismo , Plasmodium/metabolismo , Proteínas Protozoarias/metabolismo , Animales , Anticuerpos Antiprotozoarios/inmunología , Cromatografía Líquida de Alta Presión , Discoidinas , Vectores Genéticos , Humanos , Malaria Vivax/inmunología , Malaria Vivax/parasitología , Proteína 1 de Superficie de Merozoito/genética , Proteína 1 de Superficie de Merozoito/inmunología , Proteína 1 de Superficie de Merozoito/aislamiento & purificación , Plasmodium/genética , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Plasmodium vivax/genética , Plasmodium vivax/metabolismo , Plasmodium yoelii/genética , Plasmodium yoelii/inmunología , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , Proteínas Protozoarias/aislamiento & purificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
The circumsporozoite protein (CSP), a major antigen of Plasmodium falciparum, was expressed in the slime mold Dictyostelium discoideum. Fusion of the parasite protein to a leader peptide derived from Dictyostelium contact site A was essential for expression. The natural parasite surface antigen, however, was not detected at the slime mold cell surface as expected but retained intracellularly. Removal of the last 23 amino acids resulted in secretion of CSP, suggesting that the C-terminal segment of the CSP, rather than an ectoplasmic domain, was responsible for retention. Cell surface expression was obtained when the CSP C-terminal segment was replaced by the D. discoideum contact site A glycosyl phosphatidylinositol anchor signal sequence. Mice were immunized with Dictyostelium cells harboring CSP at their surface. The raised antibodies recognized two different regions of the CSP. Anti-sporozoite titers of these sera were equivalent to anti-peptide titers detected by enzyme-linked immunosorbent assay. Thus, cell surface targeting of antigens can be obtained in Dictyostelium, generating sporozoite-like cells having potentials for vaccination, diagnostic tests, or basic studies involving parasite cell surface proteins.
Asunto(s)
Anticuerpos Antiprotozoarios/biosíntesis , Antígenos de Protozoos/inmunología , Proteínas de la Membrana/inmunología , Plasmodium falciparum/inmunología , Proteínas Protozoarias/inmunología , Secuencia de Aminoácidos , Animales , Antígenos de Protozoos/biosíntesis , Antígenos de Protozoos/genética , Dictyostelium/genética , Expresión Génica , Vectores Genéticos , Glicosilfosfatidilinositoles , Inmunización , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Plasmodium falciparum/genética , Señales de Clasificación de Proteína/genética , Proteínas Protozoarias/biosíntesis , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Proteínas Recombinantes de Fusión/biosíntesisRESUMEN
We have studied the immunogenicity of Plasmodium falciparum circumsporozoite (CS) protein-derived synthetic polypeptides in mice. These synthetic peptides correspond to the N- and the C-terminal domains 22-125 and 289-390, respectively of the P. falciparum 7G8 isolate CS protein expressed on the sporozoite surface. They comprise what is believed to be the mature protein, except for the central repetitive B cell domain. BALB/c (H-2d) mice were immunized s.c. with 50 micrograms soluble CS polypeptides emulsified in IFA. After a single immunization, CS-specific helper and cytotoxic T lymphocytes (CTLs) could be obtained. The resultant CTLs obtained by in vitro restimulation of primed lymph node (LN) cells recognized H-2Kd target cells in the presence of short synthetic peptides defined in the present study. These epitopes are contained within the N- and C-terminal regions of the CS protein, and correspond to sequences 39-47 and 333-342. In addition, these CTLs can specifically lyse H-2d target cells transfected with the CS gene. These results suggest that, by immunization of mice with large soluble CS synthetic polypeptides in IFA, it is possible to obtain MHC class I-restricted T cell responses specific for the CS protein. This approach might be advantageous in the formulation of efficient malaria subunit vaccines.
Asunto(s)
Vacunas contra la Malaria/inmunología , Fragmentos de Péptidos/inmunología , Proteínas Protozoarias/inmunología , Linfocitos T Citotóxicos/inmunología , Secuencia de Aminoácidos , Animales , Antígenos H-2/inmunología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Fragmentos de Péptidos/biosíntesis , Proteínas Protozoarias/biosíntesis , Proteínas Recombinantes/biosíntesis , TransfecciónRESUMEN
We designed a trap system to isolate different amino acid sequences which could target proteins to the cell surface via GPI anchor transfer. This selection procedure is based on the insertion of various sequences which regenerate a functional GPI anchor signal sequence and therefore provoke re-expression at the surface of a reporter molecule. Using this trap for cell surface targeting sequences, we could show the importance of the defined elements essential for GPI anchor addition. Such a system could be used for an exhaustive analysis of the carboxyl terminus structural requirements for GPI membrane anchoring.
Asunto(s)
Antígenos de Superficie/metabolismo , Glicosilfosfatidilinositoles/biosíntesis , Glicoproteínas de Membrana/metabolismo , Secuencia de Aminoácidos , Antígenos de Superficie/química , Células Cultivadas , Citometría de Flujo , Vectores Genéticos , Humanos , Glicoproteínas de Membrana/química , Péptidos/metabolismo , Señales de Clasificación de Proteína , TransfecciónRESUMEN
We designed a trap system to isolate different amino acid sequences which could target proteins to the cell surface via GPI anchor transfer. This selection procedure is based on the insertion of various sequences which regenerate a functional GPI anchor signal sequence and therefore provoke re-expression at the surface of a reporter molecule. Using this trap for cell surface targeting sequences, we could show the importance of the defined elements essential for GPI anchor addition. Such a system could be used for an exhaustive analysis of the carboxyl terminus structural requeriments for GPI membrane anchoring
Asunto(s)
Fosfatidilinositoles/metabolismo , Glucolípidos/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Citometría de Flujo , Glicoproteínas de Membrana , Datos de Secuencia Molecular , Retículo Endoplásmico/metabolismoRESUMEN
Gelatinous drop-like corneal dystrophy is a rare disease, described for the first time by Nakaizumi (Japan) in 1914. We report 5 cases of this primary corneal amyloidosis. Three of our patients are brothers, the two other cases are isolated cases. Photophobia and progressive loss of vision are the first presenting signs. Corneal lesions are typically bilateral white nodular deposits beneath the epithelium. Patients underwent repeated keratoplasties with a mean interval of 5 years because of the recurrence of the disease on the corneal graft. Diagnosis of corneal amyloidosis was confirmed in all cases by histology. We compared our results with the data reported in the literature.
Asunto(s)
Distrofias Hereditarias de la Córnea/genética , Adulto , Amiloidosis/etiología , Amiloidosis/patología , Amiloidosis/cirugía , Distrofias Hereditarias de la Córnea/patología , Distrofias Hereditarias de la Córnea/cirugía , Trasplante de Córnea , Femenino , Humanos , MasculinoRESUMEN
Glycosyl phosphatidylinositol (GPI)-anchored proteins contain in their COOH-terminal region a peptide segment that is thought to direct glycolipid addition. This signal has been shown to require a pair of small amino acids positioned 10-12 residues upstream of an hydrophobic C-terminal domain. We analysed the contribution of the region separating the anchor acceptor site and the C-terminal hydrophobic segment by introducing amino acid deletions and substitutions in the spacer element of the GPI-anchored Thy-1 glycoprotein. Deletions of 7 amino acids in this region, as well as the introduction of 2 charged residues, prevented the glycolipid addition to Thy-1, suggesting that the length and the primary sequence of the spacer domain are important determinants in the signal directing GPI anchor transfer onto a newly synthesized polypeptide. Furthermore, we tested these rules by creating a truncated form of the normally transmembranous Herpes simplex virus I glycoprotein D (gDI) and demonstrating that when its C-terminal region displays all the features of a GPI-anchored protein, it is able to direct glycolipid addition onto another cell surface molecule.
Asunto(s)
Glicosilfosfatidilinositoles/química , Glicosilfosfatidilinositoles/metabolismo , Proteínas de la Membrana/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos de Superficie/genética , Antígenos de Superficie/metabolismo , Secuencia de Bases , ADN Complementario/genética , Glicoproteínas/metabolismo , Células HeLa , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Datos de Secuencia Molecular , Estructura Molecular , Mutación , Fosfatidilinositol Diacilglicerol-Liasa , Hidrolasas Diéster Fosfóricas/farmacología , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Simplexvirus/metabolismo , Antígenos Thy-1RESUMEN
We have used the cellular slime mold, Dictyostelium discoideum (Dd), to express the Plasmodium falciparum circumsporozoite protein (CS), a potential component of a subunit vaccine against malaria. This was accomplished via an expression vector based on the discoidin I-encoding gene promoter, in which we linked a sequence coding for a Dd leader peptide to the almost complete CS coding region (pEDII-CS). CS production at both the mRNA and protein levels is induced by starving cells in a simple phosphate buffer. Variation in pH or cell density does not seem to influence CS synthesis. CS-producing cells can be grown either on their normal substrate, bacteria, or on a semi-synthetic media, without affecting CS accumulation level. The CS produced in Dd seems similar to the natural parasite protein as judged by its size and epitope recognition by a panel of monoclonal antibodies. We constructed a second expression vector in which the CS is under the control of a Dd ras promoter. CS accumulation can then be induced by external addition of cAMP. Such a tightly regulated promoter may allow expression of proteins potentially toxic to the cell. Thus, Dd could be a useful eukaryotic system to produce recombinant proteins, in particular from human or animal parasites like P. falciparum.
Asunto(s)
Antígenos de Protozoos/genética , Dictyostelium/genética , Lectinas , Plasmodium falciparum/genética , Proteínas Protozoarias/genética , Secuencia de Aminoácidos , Animales , Antígenos de Protozoos/química , Western Blotting , Clonación Molecular , Discoidinas , Proteínas Fúngicas/genética , Regulación de la Expresión Génica/efectos de los fármacos , Genes ras/genética , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Proteínas Protozoarias/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMEN
Many cell surface glycoproteins are anchored in the lipid bilayer by a glycosylphosphatidyl-inositol (GPI) structure. Recently, a number of cell lines which are deficient in the biosynthesis and/or addition of this anchor have been described. In this report, we summarize the current knowledge on these lines and discuss their potential use to isolate the genes involved in the GPI anchor biosynthetic pathway with a specific emphasis on L cell fibroblasts.