RESUMEN
Monoclonal antibodies coupled to highly toxic molecules (immunoconjugates) are currently being developed for cancer therapy. We have used an in silico procedure for evaluating some physicochemical properties of two tumor-targeting anti-HER2 immunoconjugates: (a) the single-chain antibody scFv(FRP5) linked to a bacterial toxin, that has been recently progressed to phase I clinical trial in human cancer; (b) the putative molecule formed by the intrinsically stable scFv(800E6), which has been proposed as toxin carrier to cancer cells in human therapy, joined to the same toxin of (a). Structural models of the immunoconjugates have been built by homology modeling and assessed by molecular dynamics simulations. The trajectories have been analyzed to extract some biochemical properties and to assess the potential effects of the toxin on the structure and dynamics of the anti-HER2 antibodies. The results of the computational approach indicate that the antibodies maintain their correct folding even in presence of the toxin, whereas a certain stiffness in correspondence of some structural regions is observed. Furthermore, the toxin does not seem to affect the antibody solubility, whereas it enhances the structural stability. The proposed computational approach represent a promising tool for analyzing some physicochemical properties of immunoconjugates and for predicting the effects of the linked toxin on structure, dynamics, and functionality of the antibodies.
Asunto(s)
Vacunas contra el Cáncer/uso terapéutico , Simulación por Computador , Exotoxinas/uso terapéutico , Inmunoconjugados/química , Inmunoconjugados/farmacología , Neoplasias/inmunología , Neoplasias/terapia , Receptor ErbB-2/inmunología , Proteínas Recombinantes de Fusión/uso terapéutico , Secuencia de Aminoácidos , Humanos , Enlace de Hidrógeno , Datos de Secuencia Molecular , Neoplasias/genética , Estructura Secundaria de Proteína , Receptor ErbB-2/genética , Homología de Secuencia de Aminoácido , Anticuerpos de Cadena ÚnicaRESUMEN
There is an ever-growing interest in plant molecular farming as a system for producing valuable recombinant pharmaceutical molecules, such as single-chain variable fragments, on an industrial/agricultural scale and it appears that it is going to become a reality. Human epidermal growth factor receptor-2 (HER2) is an oncogene involved in abnormal cell growth in breast cancer and is considered for the development of new cancer therapies. We describe here the cloning and expression of a scFv-alpha HER2 that has been produced in Escherichia coli and in plants using both stable and transient systems in tobacco and Nicotiana benthamiana. Single-chain antibodies (ScFvs) extracted and purified from E. coli and plant tissues were tested for functionality and specificity by flow cytometry analysis on several cell lines and showed positive results when used on breast cancer slides coming from human frozen tissues. As a result, scFv-alpha HER2 represents a good opportunity for application and use in diagnosis and therapy.
Asunto(s)
Formación de Anticuerpos/genética , Inmunotoxinas/genética , Inmunotoxinas/inmunología , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Animales , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Escherichia coli/metabolismo , Femenino , Genes erbB-2 , Humanos , Hibridomas , Ratones , Plásmidos/genética , Potexvirus/genética , Potexvirus/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , NicotianaRESUMEN
Typical ECG of that described for Brugada syndrome was elicited in a patient diagnosed with labyrinthopathy during infusion of dimenhydrinate, a first-generation antihistamine usually used to treat motion sickness. Although the patient had no history of syncope or palpitations, and there was no family history of cardiac disease or sudden death, the ECG abnormality was reproduced later with intravenous flecainide, and an asymptomatic Brugada syndrome was diagnosed.
Asunto(s)
Bloqueo de Rama/inducido químicamente , Dimenhidrinato/administración & dosificación , Dimenhidrinato/efectos adversos , Antagonistas de los Receptores Histamínicos H1/administración & dosificación , Antagonistas de los Receptores Histamínicos H1/efectos adversos , Bombas de Infusión , Taquicardia Ventricular/inducido químicamente , Adulto , Bloqueo de Rama/diagnóstico , Electrocardiografía , Humanos , Masculino , Síndrome , Taquicardia Ventricular/diagnósticoRESUMEN
The influence of isolation medium on the biodiversity of Burkholderia cepacia strains recovered from the rhizosphere of Zea mays was evaluated by comparing the genetic diversity of isolates obtained by plating serial dilutions of root macerates on the two selective media TB-T and PCAT. From each medium, 50 randomly chosen colonies were isolated. On the basis of the restriction patterns of DNA coding for 16S rRNA (16S rDNA) amplified by means of PCR (ARDRA), all strains isolated from TB-T medium were assigned to the B. cepacia species, whereas among PCAT isolates only 74% were assigned to the B. cepacia species. Genetic diversity among the PCAT and TB-T isolates was evaluated by the random amplified polymorphic DNA (RAPD) technique. The analysis of molecular variance (AMOVA) method was applied to determine the variance component for RAPD patterns. Most of the genetic diversity (90.59%) was found within the two groups of isolates, but an appreciable amount (9.41%) still separated the two groups (P < 0.001). Mean genetic distances among PCAT isolates (10.39) and TB-T isolates (9.36) were significantly different (P < 0.0001). The results indicate that the two different isolation media select for B. cepacia populations with a different degree of genetic diversity. Moreover, a higher degree of genetic diversity was observed among strains isolated from PCAT medium than among those isolated from TB-T medium.
RESUMEN
Abstract Burkholderia cepacia populations associated with the Zea mays root system were investigated to assess the influence of soil type, maize cultivar, and root localization on the degree of their genetic diversity. A total of 180 B. cepacia isolates were identified by restriction analysis of the amplified 16S rDNA (ARDRA technique). The genetic diversity among B. cepacia isolates was analyzed by the random amplified polymorphic DNA (RAPD) technique, using the 10-mer primer AP5. The analysis of molecular variance (AMOVA) method was applied to estimate the variance components for the RAPD patterns. The results indicated that, among the factors studied, the soil was clearly the dominant one in affecting the genetic diversity of maize root-associated B. cepacia populations. In fact, the percentage of variation among populations was significantly higher between B. cepacia populations recovered from maize planted in different soils than between B. cepacia populations isolated from different maize cultivars and from distinct root compartments such as rhizoplane and rhizosphere. The analysis of the genetic relationships among B. cepacia isolates resulted in dendrograms showing bacterial populations with frequent recombinations and a nonclonal genetic structure. The dendrograms were also in agreement with the AMOVA results. We were able to group strains obtained from distinct soils on the basis of their origin, confirming that soil type had the major effect on the degree of genetic diversity of the maize root-associated B. cepacia populations analyzed. On the other hand, strains isolated from distinct root compartments exhibited a random distribution which confirmed that the rhizosphere and rhizoplane populations analyzed did not significantly differ in their genetic structure.http://link.springer-ny.com/link/service/journals/00248/bibs/38n3p273.html