RESUMEN
In this study, several parameters affecting the toxin production of Corynebacterium diphtheriae Parke Williams 8 (PW8) were investigated in detail. The comparison studies of amino acid profile in NZ Amine A-based medium (NZ medium) and beef digest-based medium (BD medium) suggested that an insufficient supply of amino acids was not responsible for low toxin yield observed in NZ medium. Supplementation of additional amino acids and growth promoting nutrient (in a form of yeast extract) into NZ medium enhanced only cell growth but not toxin production. Thus, BD medium was selected as the most suitable base medium for toxin production as it gave a significantly higher limit of flocculation (93 ± 0 Lf/ml) than NZ medium (46 ± 0 Lf/ml). Interestingly, a supplementation of 0.2% YE into BD medium resulted in a significant increase in growth as well as toxin production (235 ± 5 Lf/ml). In conclusion, consistently high toxin titer (174-239 Lf/ml) could be obtained from BD medium at a 5 L-scale production as long as 1) the protein content of BD medium was at least 24 g/L, 2) the iron content was below 0.15 ppm and 3) 0.2% YE was supplemented into the medium.
Asunto(s)
Corynebacterium diphtheriae/crecimiento & desarrollo , Medios de Cultivo/química , Toxina Diftérica/biosíntesis , Toxina Diftérica/aislamiento & purificaciónRESUMEN
Toxigenic Corynebacterium ulcerans is a zoonotic pathogen that produces diphtheria toxin and causes a diphtheria-like illness in humans. The organism is known to infect and circulate among dogs, which can then transmit it to humans. Furthermore, previous studies have found that C. ulcerans is carried by wild animals, including game animals. In the present study, we tested hunting and companion dogs for the presence of toxigenic C. ulcerans and succeeded in isolating the bacterium from a hunting dog. Moreover, several hunting dogs had serum diphtheria antitoxin titers that were higher than the titers required for protection in humans, suggesting a history of exposure to toxigenic Corynebacterium strains. Notably, ribotyping, pulsed-field gel electrophoresis and tox gene sequencing demonstrated that the isolate from the hunting dog clustered with previously characterized C. ulcerans strains isolated from wild animals, as opposed to groups of isolates from humans and companion dogs. Interestingly, the wild animal cluster also contains an isolate from an outdoor breeding dog, which could have formed a bridge between isolates from wild animals and those from companion dogs. The results presented herein provide insight into the mechanism by which the zoonotic pathogen C. ulcerans circulates among wild animals, hunting and companion dogs, and humans.
Asunto(s)
Infecciones por Corynebacterium/veterinaria , Corynebacterium/inmunología , Corynebacterium/aislamiento & purificación , Toxina Diftérica/inmunología , Enfermedades de los Perros/microbiología , Animales , Animales Salvajes/microbiología , Anticuerpos Antibacterianos/sangre , Chlorocebus aethiops , Corynebacterium/genética , Infecciones por Corynebacterium/sangre , Infecciones por Corynebacterium/inmunología , Infecciones por Corynebacterium/microbiología , Girasa de ADN/genética , Antitoxina Diftérica/sangre , Toxina Diftérica/genética , Toxina Diftérica/aislamiento & purificación , Enfermedades de los Perros/sangre , Enfermedades de los Perros/inmunología , Perros , Electroforesis en Gel de Campo Pulsado/métodos , Femenino , Humanos , Japón , Masculino , Células Vero , Zoonosis/inmunología , Zoonosis/microbiologíaRESUMEN
A key step in the mode of cytotoxic action of diphtheria toxin (DT) is the transfer of its catalytic domain (Cd) from endosomes into the cytosol. The main activity in this process is performed by the transport domain (Td), but the molecular mechanism of its action remains unknown. We have previously shown that Td can have some influence on the endosomal transport of DT The aim of this work was to study the effect of diphtheria toxin on the toxin compartmentalization in the intracellular transporting pathway and endosomal pH. We used recombinant fragments of DT which differed only by the presence of Td in their structure, fused with fluorescent proteins. It was shown that the toxin fragment with Td moved slower by the pathway early-late endosomes-lysosomes, and had a slightly different pattern of colocalization with endosomal markers than DT fragment without Td. In addition, endosomes containing DT fragments with Td had a constant pH of about 6.5 from the 10th to 50th minute of observation, for the same time endosomes containing DT fragments without Td demonstrated a decrease in pH from 6.3 to 5.5. These results indicate that Td inhibits acidification of endosomal medium. One of possible explanations for this may be the effect of the ion channel formed by the T-domain on the process of the endosomal acidification. This property of Td may not only inhibit maturation of endosomes but also inhibit activation of endosomal pH-dependent proteases, and this promotes successful transport of Cd into the cell cytosol.
Asunto(s)
Citosol/metabolismo , Toxina Diftérica/metabolismo , Endosomas/metabolismo , Lisosomas/metabolismo , Animales , Transporte Biológico , Dominio Catalítico , Chlorocebus aethiops , Corynebacterium diphtheriae/química , Corynebacterium diphtheriae/metabolismo , Citosol/efectos de los fármacos , Toxina Diftérica/aislamiento & purificación , Toxina Diftérica/toxicidad , Endosomas/efectos de los fármacos , Colorantes Fluorescentes/metabolismo , Expresión Génica , Concentración de Iones de Hidrógeno , Lisosomas/efectos de los fármacos , Transporte de Proteínas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Células VeroRESUMEN
Many recombinant therapeutic proteins are purified from Escherichia coli. While expression in E. coli is easily achieved, some disadvantages such as protein aggregation, formation of inclusion bodies, and contamination of purified proteins with the lipopolysaccharides arise. Lipopolysaccharides have to be removed to prevent inflammatory responses in patients. Use of the Gram-positive Bacillus anthracis as an expression host offers a solution to circumvent these problems. Using the multiple protease-deficient strain BH460, we expressed a fusion of the N-terminal 254 amino acids of anthrax lethal factor (LFn), the N-terminal 389 amino acids of diphtheria toxin (DT389) and human transforming growth factor alpha (TGFα). The resulting fusion protein was constitutively expressed and successfully secreted by B. anthracis into the culture supernatant. Purification was achieved by anion exchange chromatography and proteolytic cleavage removed LFn from the desired fusion protein (DT389 fused to TGFα). The fusion protein showed the intended specific cytotoxicity to epidermal growth factor receptor-expressing human head and neck cancer cells. Final analyses showed low levels of lipopolysaccharides, originating most likely from contamination during the purification process. Thus, the fusion to LFn for protein secretion and expression in B. anthracis BH460 provides an elegant tool to obtain high levels of lipopolysaccharide-free recombinant protein.
Asunto(s)
Antígenos Bacterianos/aislamiento & purificación , Bacillus anthracis/metabolismo , Toxinas Bacterianas/aislamiento & purificación , Toxina Diftérica/aislamiento & purificación , Inmunotoxinas/aislamiento & purificación , Proteínas Recombinantes de Fusión/aislamiento & purificación , Factor de Crecimiento Transformador alfa/aislamiento & purificación , Antígenos Bacterianos/biosíntesis , Antígenos Bacterianos/farmacología , Bacillus anthracis/genética , Toxinas Bacterianas/biosíntesis , Toxinas Bacterianas/farmacología , Línea Celular Tumoral , Cromatografía por Intercambio Iónico , Toxina Diftérica/biosíntesis , Toxina Diftérica/farmacología , Humanos , Inmunotoxinas/farmacología , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/farmacología , Factor de Crecimiento Transformador alfa/biosíntesis , Factor de Crecimiento Transformador alfa/farmacologíaRESUMEN
We assessed the IgG levels anti-diphtheria (D-Ab) and T cell counts (CD4+ and CD8+) in HIV-1 infected subjects undergoing or not highly active antiretroviral therapy (HAART). Approximately 70% of all HIV-1 patients were unprotected against diphtheria. There were no differences in D-Ab according to CD4 counts. Untreated patients had higher D-Ab (geometric mean of 0.62 IU/ml) than HAART-patients (geometric mean of 0.39 IU/ml). The data indicated the necessity of keeping all HIV-1 patients up-to-date with their vaccination.
Asunto(s)
Humanos , Suero Antilinfocítico , Difteria , VIH , Infecciones por VIH , Linfocitos T/patología , Toxina Diftérica/análisis , Toxina Diftérica/aislamiento & purificación , Toxoide Diftérico/análisis , Vacunas Tifoides-Paratifoides/análisis , Inmunidad Celular , Métodos , Pacientes , VacunaciónRESUMEN
Infections caused by Corynebacterium diphtheriae frequently induce situations in which very small doses of antigens injected intradermally can cause strong inflammatory reactions. This bacterium secretes the diphtheria toxin (DT), a virulence factor that can be lethal to the human organism at doses below 0.1 µg/kg of body weight. The present work proposes alternative methods of DT purification using affinity chromatography and of DT detoxification through conjugating with the polymer methoxypolyethylene glycol activated (mPEG). Tests were performed to evaluate: the formation of edemas and the presence of dermonecrotic activity, in vitro cytotoxicity to Vero cells, the neutralizing activity of serum from guinea pigs immunized with the diphtheria toxoid inactivated with mPEG, and the immunogenic activity of the purified and modified toxin. The results indicated that purification with Blue Sepharose was an efficient method, yielding antigen purity equivalent to 2600 Lf/mg of protein nitrogen. The modification of the Purified Toxin with mPEG did not result in the formation of edema or necrosis although it was immunogenic and stimulated the formation of antibodies that could neutralize the Purified Toxin. The toxoid obtained from the purified toxin maintained its immunogenic characteristics, inducing antibodies with neutralizing activity; edema and necrosis were still observed, however.
Asunto(s)
Cromatografía de Afinidad/métodos , Citotoxinas/aislamiento & purificación , Toxina Diftérica/aislamiento & purificación , Desintoxicación por Sorción/métodos , Animales , Supervivencia Celular/efectos de los fármacos , Chlorocebus aethiops , Citotoxinas/química , Difteria/tratamiento farmacológico , Difteria/metabolismo , Difteria/patología , Toxina Diftérica/química , Toxoide Diftérico/inmunología , Toxoide Diftérico/farmacología , Relación Dosis-Respuesta a Droga , Cobayas , Humanos , Pruebas de Neutralización , Polietilenglicoles/química , Sefarosa/análogos & derivados , Sefarosa/química , Factores de Tiempo , Células VeroRESUMEN
Infections caused by Corynebacterium diphtheriae frequently induce situations in which very small doses of antigens injected intradermally can cause strong inflammatory reactions. This bacterium secretes the diphtheria toxin (DT), a virulence factor that can be lethal to the human organism at doses below 0.1 mg/kg of body weight. The present work proposes alternative methods of DT purification using affinity chromatography and of DT detoxification through conjugating with the polymer methoxypolyethylene glycol activated(mPEG). Tests were performed to evaluate: the formation of edemas and the presence of dermonecrotic activity, in vitro cytotoxicity to Vero cells, the neutralizing activity of serum from guinea pigs immunized with the diphtheria toxoid inactivated with mPEG, and the immunogenic activity of the purified and modified toxin. The results indicated that purification with Blue Sepharose was an efficient method, yielding antigen purity equivalent to 2600 Lf/mg of protein nitrogen. The modification of the Purified Toxin with mPEG did not result in the formation of edema or necrosis although it was immunogenic and stimulated the formation of antibodies that could neutralize the Purified Toxin. The toxoidobtained from the purified toxin maintained its immunogenic characteristics, inducing antibodies with neutralizing activity; edema and necrosis were still observed, however.
Asunto(s)
Cobayas , Difteria/microbiología , Toxina Diftérica/aislamiento & purificación , Toxina Diftérica/toxicidad , Toxoide Diftérico/administración & dosificación , Toxoide Diftérico/uso terapéutico , Cromatografía de Afinidad/métodos , Pruebas de Toxicidad/métodosRESUMEN
Fusion toxins are an emerging class of targeted therapeutics for the treatment of cancer. Diphtheria toxin-stem cell factor (DT-SCF) is one such novel fusion toxin designed to target malignancies expressing c-kit. Since, c-kit overexpression has been reported on many types of cancers, it appeared to be a reasonably good molecule to target. In the present study, we report construction, expression, purification, and characterization of DT-SCF. DT-SCF gene coding for 1-387 amino acids of diphtheria toxin, His-Ala linker, 2-141 amino acids of SCF was cloned into expression vector with C terminal His tag. The induced DT-SCF protein was exclusively expressed in insoluble fraction. Purification of DT-SCF was achieved by inclusion body isolation and metal affinity chromatography under denaturing and reducing conditions. Purified DT-SCF was renatured partially on-column by gradually reducing denaturant concentration followed by complete refolding through rapid dilution technique. Cell viability assay provided the evidence that DT-SCF is a potent cytotoxic agent selective to cells expressing c-kit. The novelty of this study lies in employing SCF as a ligand in construction of fusion toxin to target wide range of malignancies expressing c-kit. Efficacy of DT-SCF fusion toxin was demonstrated over a range of malignancies such as chronic myeloid leukemia (K562), acute lymphoblastic leukemia (MOLT4), pancreatic carcinoma (PANC-1), and cervical carcinoma (HeLa 229). This is the first study reporting specificity and efficacy of DT-SCF against tumor cells expressing c-kit. There was significant correlation (P = 0.007) between c-kit expression on cells and their sensitivity to DT-SCF fusion toxin.
Asunto(s)
Biotecnología/métodos , Toxina Diftérica/aislamiento & purificación , Toxina Diftérica/metabolismo , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Factor de Células Madre/aislamiento & purificación , Factor de Células Madre/metabolismo , Ciclo Celular , Línea Celular , Clonación Molecular , Toxina Diftérica/química , Técnica del Anticuerpo Fluorescente , Humanos , Immunoblotting , Proteínas Proto-Oncogénicas c-kit/metabolismo , Proteínas Recombinantes de Fusión/química , Factor de Células Madre/químicaRESUMEN
Latex diagnostic kit with high specificity and sensitivity of diphtheria toxin and toxoid detection has been developed on the basis of protective monoclonal antibodies to diphtheria toxin and polyacrolein microspheres. Diagnostic kit was stable during 1 year of shelf-life (time of the study).
Asunto(s)
Antitoxina Diftérica/aislamiento & purificación , Toxina Diftérica/aislamiento & purificación , Pruebas de Fijación de Látex/métodos , Juego de Reactivos para Diagnóstico , Anticuerpos Monoclonales , Toxina Diftérica/inmunología , Microesferas , Sensibilidad y EspecificidadRESUMEN
Urotensin II is a neuropeptide first isolated from fish and later found in mammals: where it has potent cardiovascular, endocrine and behavioral effects. In rat brain the urotensin II receptor (UII-R) is predominately expressed in the cholinergic neurons of the pedunculopontine (PPTg) and laterodorsal tegmental nuclei. Typically, the function of the PPTg has been examined using excitotoxins, destroying both cholinergic and non-cholinergic neurons, which confounds interpretation. We took advantage of UII-R's unique expression profile, by combining UII with diphtheria toxin, to engineer a toxin specific for cholinergic neurons of the PPTg. In vitro, two different toxin constructs were shown to selectively activate UII-R (average EC50 approximately 30 nmol/L; calcium mobility assay) and to be 10,000-fold more toxic to UII-R expressing CHO cells, than wildtype cells (average LD50 approximately 2 nmol/L; cell viability). In vivo, pressure injection into the PPTg of rats, resulted in specific loss of choline transporter and NADPH diaphorase positive neurons known to express the UII-R. The lesions developed over time, resulting in the loss of over 80% of cholinergic neurons at 21 days, with little damage to surrounding neurons. This is the first highly selective molecular tool for the depletion of mesopontine cholinergic neurons. The toxin will help to functionally dissect the pedunculopontine and laterodorsal tegmental nuclei, and advance the understanding of the functions of these structures.
Asunto(s)
Toxina Diftérica/química , Toxina Diftérica/toxicidad , Neuronas/efectos de los fármacos , Síndromes de Neurotoxicidad/patología , Neurotoxinas/química , Neurotoxinas/toxicidad , Sistema Nervioso Parasimpático/efectos de los fármacos , Puente/patología , Tegmento Mesencefálico/patología , Urotensinas/química , Urotensinas/toxicidad , Animales , Células CHO , Supervivencia Celular/efectos de los fármacos , Cricetinae , Cricetulus , Toxina Diftérica/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Masculino , NADPH Deshidrogenasa/metabolismo , NADPH Deshidrogenasa/fisiología , Neuronas/patología , Sistema Nervioso Parasimpático/patología , Plásmidos/genética , RatasRESUMEN
OBJECTIVE: To construct a novel recombinant immunotoxin (IT) expression vector by fusing mouse macrophage inflammatory protein-1alpha gene (mMIP-1alpha) into a truncated diphtheria toxin gene (DT390), and examine the expression of mMIP-1alpha-DT390 fusion protein in NIH3T3 cells. METHODS: mMIP-1alpha cDNA was cloned from mouse liver tissue through reverse transcription-polymerase chain reaction (RT-PCR), and inserted in the expression plasmid SRalpha, that includes the DT390 gene, to form the recombinant vector SRa-mMIP-1alpha-DT390. The positive recombinant plasmid was identified by PCR, the restriction endonucleases digestion and DNA sequencing, and then by liposome protocol, the identified positive plasmid was transferred into NIH3T3 cells for observing the fusion protein expression by immunofluorescence, with detecting the activities of the immunotoxin in vitro through MTT. RESULTS: We have successfully constructed a recombinant immunotoxin expression vector named as SRalpha-mMIP-1alpha-DT390. The immunofluorescence photograph sourced from fluorescence immunocytochemical method showed that the fusion gene could express in the cytomembrane and cytoplasm of NIH3T3 cells. In the bioactivity detection assay, the supernatant of the transfected cell culture was observed to have the obvious cytotoxic activity to activated T-lymphocyte. CONCLUSION: The SRalpha-mMIP-1alpha-DT390 recombinant immunotoxin expression vector will provide the basis of studying the targeted cytotoxic activity to inflammatory cells, and may have some potential value for clinical application.
Asunto(s)
Quimiocina CCL3/genética , Toxina Diftérica/genética , Células Eucariotas , Inmunotoxinas/genética , Plásmidos/genética , Proteínas Recombinantes de Fusión/genética , Animales , Quimiocina CCL3/inmunología , Quimiocina CCL3/aislamiento & purificación , Quimiocina CCL3/toxicidad , Toxina Diftérica/inmunología , Toxina Diftérica/aislamiento & purificación , Toxina Diftérica/toxicidad , Células Eucariotas/citología , Expresión Génica , Vectores Genéticos/genética , Inmunotoxinas/inmunología , Inmunotoxinas/aislamiento & purificación , Inmunotoxinas/toxicidad , Ratones , Células 3T3 NIH , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/toxicidad , Análisis de Secuencia de ADN , Linfocitos T/inmunología , TransfecciónRESUMEN
Previous studies have demonstrated that the covalent modification of target protein and polysaccharide antigens with the activated complement product C3d results in dramatically enhanced immunogenicity of the target antigens. In this paper, we describe our attempts to enhance the immunogenicity of the non-toxic B fragment of diphtheria toxin (DT-B) by genetic fusion to polypeptides derived from the C3d coding sequence. Contrary to expectations, we found that the antibody responses elicited by immunizing mice with DT-B genetically linked to three tandem copies of C3d-derived sequences were markedly reduced relative to the antibody responses elicited by immunizing mice with DT-B alone. These results demonstrate levels of complexity in the immunomodulatory effects of the complement system that were not apparent in earlier reports on the adjuvant effects of C3d administered to mice as genetic fusions to target antigens, such as hen egg lysozyme, human immunodeficiency virus (HIV) gp120 or influenza virus hemaglutinnin. The data presented herein suggest that C3d may act as a negative regulator, in some immunological contexts, for antibody production in the mammalian host.
Asunto(s)
Formación de Anticuerpos/inmunología , Especificidad de Anticuerpos , Complemento C3d/genética , Complemento C3d/inmunología , Toxina Diftérica/genética , Toxina Diftérica/inmunología , Secuencia de Aminoácidos , Animales , Antígenos/inmunología , Complemento C3d/química , Complemento C3d/aislamiento & purificación , Toxina Diftérica/aislamiento & purificación , Toxina Diftérica/metabolismo , Femenino , Expresión Génica/genética , Fusión Génica/genética , Ratones , Datos de Secuencia Molecular , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/aislamiento & purificación , Fragmentos de Péptidos/metabolismoRESUMEN
Diphtheria toxin consists of an A-fragment that inactivates elongation factor 2 and a B-fragment that both binds to the toxin receptor and mediates translocation of the A-fragment across cellular membranes to the cytosol. Several fragments of the toxin and an inactive version of the holotoxin have been expressed in Escherichia coli, but the B-fragment alone has proven difficult to express. Only low levels of expression have been achieved. We have designed a hexahistidine tagged version of a modified diphtheria toxin B-fragment (DT-BHis) that can be expressed to high levels in E. coli. DT-BHis contains the entire diphtheria toxin B-fragment preceded by an alanine and succeeded by a leucine, a glutamic acid and a hexahistidine tag and could be purified in a single step using nickel-coated agarose beads to 85% homogeneity. DT-BHis bound specifically to the diphtheria toxin receptor and was able to compete out the effect of the wild type diphtheria toxin. Furthermore, DT-BHis was able to form pores in cellular membranes in a manner similar to the wild type B-fragment. The high yield makes DT-BHis a suitable tool in studies of diphtheria toxin interaction with cells or liposomes since functional diphtheria toxin was easily formed upon addition of A-fragment. The reconstituted diphtheria toxin showed toxicity in the same range as the wild type.
Asunto(s)
Reactores Biológicos , Toxina Diftérica/aislamiento & purificación , Toxina Diftérica/metabolismo , Histidina/metabolismo , Oligopéptidos/metabolismo , Animales , Western Blotting , Línea Celular Tumoral , Chlorocebus aethiops , Clonación Molecular , Cartilla de ADN , Toxina Diftérica/toxicidad , Escherichia coli , Humanos , Fragmentos de Péptidos/aislamiento & purificación , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/toxicidad , Pruebas de Toxicidad , Células VeroAsunto(s)
Anticuerpos Antibacterianos/sangre , Corynebacterium diphtheriae/aislamiento & purificación , Toxina Diftérica/aislamiento & purificación , Difteria/diagnóstico , Animales , Técnicas Bacteriológicas , Biomarcadores/sangre , Corynebacterium diphtheriae/genética , Corynebacterium diphtheriae/inmunología , Difteria/microbiología , Toxina Diftérica/genética , Toxina Diftérica/inmunología , Ensayo de Inmunoadsorción Enzimática , Humanos , Pruebas Inmunológicas/métodos , Reacción en Cadena de la Polimerasa , Valores de ReferenciaRESUMEN
Bacterial protein toxins are the most powerful human poisons known, exhibiting an LD(50) of 0.1-1 ng kg(-)(1). A major subset of such toxins is the NAD(+)-dependent ADP-ribosylating exotoxins, which include pertussis, cholera, and diphtheria toxin. Diphtheria toxin catalyzes the ADP ribosylation of the diphthamide residue of eukaryotic elongation factor 2 (eEF-2). The transition state of ADP ribosylation catalyzed by diphtheria toxin has been characterized by measuring a family of kinetic isotope effects using (3)H-, (14)C-, and (15)N-labeled NAD(+) with purified yeast eEF-2. Isotope trapping experiments yield a commitment to catalysis of 0.24 at saturating eEF-2 concentrations, resulting in suppression of the intrinsic isotope effects. Following correction for the commitment factor, intrinsic primary kinetic isotope effects of 1.055 +/- 0.003 and 1.022 +/- 0.004 were observed for [1(N)'-(14)C]- and [1(N)-(15)N]NAD(+), respectively; the double primary isotope effect was 1.066 +/- 0.004 for [1(N)'-(14)C, 1(N)-(15)N]NAD(+). Secondary kinetic isotope effects of 1.194 +/- 0.002, 1.101 +/- 0.003, 1.013 +/- 0.005, and 0.988 +/- 0.002 were determined for [1(N)'-(3)H]-, [2(N)'-(3)H]-, [4(N)'-(3)H]-, and [5(N)'-(3)H]NAD(+), respectively. The transition state structure was modeled using density functional theory (B1LYP/6-31+G) as implemented in Gaussian 98, and theoretical kinetic isotope effects were subsequently calculated using Isoeff 98. Constraints were varied in a systematic manner until the calculated kinetic isotope effects matched the intrinsic isotope effects. The transition state model most consistent with the intrinsic isotope effects is characterized by the substantial loss in bond order of the nicotinamide leaving group (bond order = 0.18, 1.99 A) and weak participation of the attacking imidazole nucleophile (bond order = 0.03, 2.58 A). The transition state structure imparts strong oxacarbenium ion character to the ribose ring even though significant bond order remains to the nicotinamide leaving group. The transition state model presented here is asymmetric and consistent with a dissociative S(N)1 type mechanism in which attack of the diphthamide nucleophile lags behind departure of the nicotinamide.
Asunto(s)
Adenosina Difosfato Ribosa/química , Toxina Diftérica/química , Factor 2 de Elongación Peptídica/química , Proteínas de Saccharomyces cerevisiae/química , ADP Ribosa Transferasas/química , ADP Ribosa Transferasas/metabolismo , Adenosina Difosfato Ribosa/metabolismo , Unión Competitiva , Radioisótopos de Carbono/metabolismo , Catálisis , Dominio Catalítico , Toxina Diftérica/aislamiento & purificación , Toxina Diftérica/metabolismo , Hidrólisis , Cinética , NAD+ Nucleosidasa/metabolismo , Isótopos de Nitrógeno/metabolismo , Factor 2 de Elongación Peptídica/aislamiento & purificación , Factor 2 de Elongación Peptídica/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Fragmentos de Péptidos/metabolismo , Proteínas de Saccharomyces cerevisiae/aislamiento & purificación , Proteínas de Saccharomyces cerevisiae/metabolismo , Tritio/metabolismoRESUMEN
PCR standardization was performed in order to detect a fragment and a whole tox gene which is presented in all toxigenic strains of Corynebacterium diphtheriae. PCR is one of the least time-consuming methods for detection toxigenicity of C. diphtheriae strains near EIA (enzyme immunoassay) and ICS (immunochromatographic strip) tests.
Asunto(s)
Corynebacterium diphtheriae/aislamiento & purificación , Toxina Diftérica/aislamiento & purificación , Difteria/microbiología , Reacción en Cadena de la Polimerasa , Cromatografía/métodos , Toxina Diftérica/biosíntesis , Humanos , Técnicas para Inmunoenzimas , Reacción en Cadena de la Polimerasa/métodos , Sensibilidad y EspecificidadRESUMEN
Diphtheria toxin is produced by growing Corinebacterium diphtheriae either in a semisynthetic casein-based medium or in the Pope-Lingood meat extract based medium. The World Health Organization advises the use of the semisynthetic one, as it has important advantages. Data on the composition of casein-based media and their ability to support high toxin production are not freely available. Important factors affecting toxin production during C. diphtheriae cultivation are the pH of the culture medium and the concentration of casein hydrolysate and of Fe2+. We established that the optimal pH for toxin production is 7.2. The highest yield of toxin was obtained using a casein hydrolysate concentration of 35.0 g/L and a Fe2+ concentration of 0.05-0.41 microg/mL. Under these conditions, diphtheria toxin with higher purity and yield compared with the batches obtained using the meat-based medium of Pope-Lingood was produced.
Asunto(s)
Caseínas/química , Corynebacterium diphtheriae/crecimiento & desarrollo , Medios de Cultivo/química , Toxina Diftérica/biosíntesis , Corynebacterium diphtheriae/metabolismo , Toxina Diftérica/aislamiento & purificación , Fermentación , Compuestos Ferrosos , Concentración de Iones de HidrógenoRESUMEN
A genetically engineered fusion toxin targeted to acute myeloid leukemia (AML) blasts was designed with the first 388 amino acid residues of diphtheria toxin with an H-M linker fused to human interleukin-3. The cDNA was subcloned in the pRK bacterial expression plasmid and used to transform BLR (DE3) Escherichia coli. A single transformed colony was grown in Superbroth with ampicillin; bacteria were centrifuged at an OD(650) of 1.3; master cell bank aliquots of bacteria in 30% glycerol/Superbroth were frozen and stored at -80 degrees C. Master cell bank bacteria were diluted 1500-fold into Superbroth and recombinant protein was induced with 1 mM IPTG at an OD(650) of 0.6. After two additional hours of fermentation, inclusion bodies were isolated, washed, and denatured in guanidine hydrochloride and dithioerythritol. Recombinant protein was refolded by diluted 100-fold in cold buffer with arginine and oxidized glutathione. After dialysis, purified protein was obtained after anion-exchange, size exclusion on FPLC, and polymyxin B affinity chromatography. The final material was filter sterilized, aseptically vialed, and stored at -80 degrees C. Seventy-five 3-L bacterial culture preparations were made and pooled for the AT-1 batch (568 mL) and twenty-four 3-L bacterial culture preparations were made and pooled for the AT-2 batch (169 mL). The final product was characterized by Coomassie Plus protein assay, Coomassie-stained SDS-PAGE, limulus amebocyte lysate endotoxin assay, human AML TF/H-ras cell cytotoxicity assay, sterility, tandem mass spectroscopy, IL3 receptor binding affinity, ADP ribosylation activity, inhibition of normal human CFU-GM, disulfide bond analysis, immunoblots, peptide mapping, stability, HPLC TSK3000, N-terminal sequencing, E. coli DNA contamination, C57BL/6 mouse toxicity, cynomolgus monkey toxicity, and immunohistochemistry. Yields were 25.7+/-5.6 mg/L bacterial culture of denatured fusion toxin. After refolding and chromatography, final yields were 20+/-11% or 5 mg/L. Vialed product was sterile. Batches were in 0.25 M sodium chloride/5 mM Tris, pH 8, and had protein concentrations of 1.8-1.9 mg/mL. Purity by SDS-PAGE was 99+/-1%. Aggregates by HPLC were <1 %. Potency revealed a 48 h IC(50) of 6-8 pM on TF/H-ras cells. Endotoxin levels were 1 eu/mg. The remaining chemical and biologic assays confirmed the purity, composition, and functional activities of the molecule. The LD(10) in mice was 250 microg/kg/day every other day for six doses. The MTD in monkeys was 60 microg/kg/day every other day for six doses. Drug did not react with tested frozen human tissue sections by immunohistochemistry. There was no evidence of loss of solubility, proteolysis aggregation, or loss of potency over 6 months at -80 and -20 degrees C. Further, the drug was stable at 4 and 25 degrees C in the plastic syringe and administration tubing for 24 h and at 37 degrees C in human serum for 24 h. The synthesis of this protein drug should be useful for production for clinical phase I/II clinical trials and may be suitable for other diphtheria fusion toxins indicated for clinical development.
Asunto(s)
Toxina Diftérica/biosíntesis , Toxina Diftérica/aislamiento & purificación , Interleucina-3/biosíntesis , Interleucina-3/aislamiento & purificación , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/aislamiento & purificación , Enfermedad Aguda , Adenosina Difosfato Ribosa/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular Tumoral , Cromatografía Liquida/métodos , Toxina Diftérica/genética , Toxina Diftérica/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Estabilidad de Medicamentos , Femenino , Expresión Génica , Haplorrinos , Humanos , Interleucina-3/genética , Interleucina-3/metabolismo , Interleucina-3/farmacología , Leucemia Mieloide/tratamiento farmacológico , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Plásmidos/genética , Receptores de Interleucina-3/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacologíaRESUMEN
A stable liquid candidate Biological Reference Preparation (BRP) for diphtheria toxin was prepared in peptone buffer (nominal content of diphtheria toxin: 1 Lf/ml, 0.4 micro g/ml), filled in ampoules (filling volume: 1 ml) and characterised in a collaborative study. The toxin is to be used in the test "Absence of toxin and irreversibility of toxoid" as described in the current European Pharmacopoeia (Ph. Eur.) monograph Diphtheria Vaccine (Adsorbed) (2002:0443). Eleven laboratories assessed the specific activity of the preparation by in vivo and in vitro assays. The material is assumed to have satisfactory stability with a calculated predicted loss of activity of <1% per year at 4-8 degrees C. From the collaborative study, the specific activity was calculated as 77.6 (45-113) LD( 50)/ml (lethal challenge) and >75 000 Lr/Lf (intradermal challenge). The candidate BRP was successfully used in nine laboratories and confirmed suitable for use in the Vero cell test for "Absence of toxin and irreversibility of toxoid" as described in the Ph. Eur. monograph 2002:0443; i.e., concentrations of 5 x 10( -5) Lf/ml and below caused cytotoxic effects in the Vero cell test. Due to its liquid nature, the stability of the material will be monitored at regular intervals and preparation of a stable freeze-dried formulation will be considered for long-term use. Additional studies will be performed to confirm suitability of this BRP for other applications. The candidate BRP was adopted as the Ph. Eur. reference material for Diphtheria Toxin Batch 1 by the Ph. Eur. Commission at its session in March 2003.
Asunto(s)
Toxina Diftérica/normas , Estudios Multicéntricos como Asunto , Farmacopeas como Asunto/normas , Estándares de Referencia , Vacunas/biosíntesis , Animales , Chlorocebus aethiops , Difteria/prevención & control , Antitoxina Diftérica/administración & dosificación , Antitoxina Diftérica/aislamiento & purificación , Toxina Diftérica/administración & dosificación , Toxina Diftérica/aislamiento & purificación , Europa (Continente) , Femenino , Cobayas , Técnicas In Vitro , Inyecciones Intradérmicas , Inyecciones Subcutáneas , Masculino , Proyectos de Investigación , Tecnología Farmacéutica , Células VeroRESUMEN
In addition to the Eastern European resurgence of diphtheria during the last decade, there has also been an emergence of infections caused by non-toxigenic Corynebacterium diphtheriae and non-toxigenic, toxin gene bearing C. diphtheriae. Given that these strains may manifest as symptomatic infections of differing degrees of severity, their clinical and epidemiological significance need to be assessed. The persistence of toxigenic and non-toxigenic C. diphtheriae in circulation, together with genotypic and biotype variability means that innovative measures to vaccinate populations are pertinent. The most effective method of protecting the currently most vulnerable population group (adults) is to implement a booster dose of vaccine amongst the adult populations. Furthermore, in combination with an efficient surveillance system, effective antibiotic prophylaxis and an up-to-date vaccination programme, serological studies needs to be maintained to monitor the immunity status of the population.