RESUMEN

The nitrilase superfamily enzymes from Pyrococcus abyssi and Pyrococcus horikoshii hydrolyze several different amides. No nitriles that we tested were hydrolyzed by either enzyme. Propionamide and acetamide were the most rapidly hydrolyzed of all the substrates tested. Amide substrate docking studies on the wild-type and C146A variant P. horikoshii enzymes suggest a sequence in which the incoming amide substrate initially hydrogen bonds to the amino group of Lys-113 and the backbone carbonyl of Asn-171. When steric hindrance is relieved by replacing the cysteine with alanine, the amide then docks such that the amino group of Lys-113 and the backbone amide of Phe-147 are hydrogen-bonded to the substrate carbonyl oxygen, while the backbone carbonyl oxygen of Asn-171 and the carboxyl oxygen of Glu-42 are hydrogen-bonded to the amino group of the substrate. Here, we confirm the location of the acetamide and glutaramide ligands experimentally in well-resolved crystal structures of the C146A mutant of the enzyme from P. horikoshii. This ligand location suggests that there is no direct interaction between the substrate amide and the other active site glutamate, Glu-120, and supports an active-site geometry leading to the formation of the thioester intermediate via an attack on the si-face of the amide by the sulfhydryl of the active site cysteine.

Asunto(s)

Pyrococcus horikoshii , Acetamidas , Amidas , Amidohidrolasas/química , Amidohidrolasas/genética , Cisteína/química , Hidrógeno , Ligandos , Oxígeno , Especificidad por Sustrato

RESUMEN

The development of effective vaccines is urgently needed to curb the spread of human immunodeficiency virus type 1 (HIV-1). A major focal point of current HIV vaccine research is the production of soluble envelope (Env) glycoproteins which reproduce the structure of the native gp160 trimer. These antigens are produced in mammalian cells, which requires a sophisticated infrastructure for manufacture that is mostly absent in developing countries. The production of recombinant proteins in plants is an attractive alternative for the potentially cheap and scalable production of vaccine antigens, especially for developing countries. In this study, we developed a transient expression system in Nicotiana benthamiana for the production of soluble HIV Env gp140 antigens based on two rationally selected virus isolates (CAP256 SU and Du151). The scalability of the platform was demonstrated and both affinity and size exclusion chromatography (SEC) were explored for recovery of the recombinant antigens. Rabbits immunized with lectin affinity-purified antigens developed high titres of binding antibodies, including against the V1V2 loop region, and neutralizing antibodies against Tier 1 viruses. The removal of aggregated Env species by gel filtration resulted in the elicitation of superior binding and neutralizing antibodies. Furthermore, a heterologous prime-boost regimen employing a recombinant modified vaccinia Ankara (rMVA) vaccine, followed by boosts with the SEC-purified protein, significantly improved the immunogenicity. To our knowledge, this is the first study to assess the immunogenicity of a near-full length plant-derived Env vaccine immunogen.

RESUMEN

BACKGROUND: Rift Valley fever virus (RVFV), the causative agent of Rift Valley fever, is an enveloped single-stranded negative-sense RNA virus in the genus Phlebovirus, family Bunyaviridae. The virus is spread by infected mosquitoes and affects ruminants and humans, causing abortion storms in pregnant ruminants, high neonatal mortality in animals, and morbidity and occasional fatalities in humans. The disease is endemic in parts of Africa and the Arabian Peninsula, but is described as emerging due to the wide range of mosquitoes that could spread the disease into non-endemic regions. There are different tests for determining whether animals are infected with or have been exposed to RVFV. The most common serological test is antibody ELISA, which detects host immunoglobulins M or G produced specifically in response to infection with RVFV. The presence of antibodies to RVFV nucleocapsid protein (N-protein) is among the best indicators of RVFV exposure in animals. This work describes an investigation of the feasibility of producing a recombinant N-protein in Nicotiana benthamiana and using it in an ELISA. RESULTS: The human-codon optimised RVFV N-protein was successfully expressed in N. benthamiana via Agrobacterium-mediated infiltration of leaves. The recombinant protein was detected as monomers and dimers with maximum protein yields calculated to be 500-558 mg/kg of fresh plant leaves. The identity of the protein was confirmed by liquid chromatography-mass spectrometry (LC-MS) resulting in 87.35% coverage, with 264 unique peptides. Transmission electron microscopy revealed that the protein forms ring structures of ~ 10 nm in diameter. Preliminary data revealed that the protein could successfully differentiate between sera of RVFV-infected sheep and from sera of those not infected with the virus. CONCLUSIONS: To the best of our knowledge this is the first study demonstrating the successful production of RVFV N-protein as a diagnostic reagent by Agrobacterium-mediated transient heterologous expression in N. benthamiana. Preliminary testing of the antigen showed its ability to distinguish RVFV-positive animal sera from RVFV negative animal sera when used in an enzyme linked immunosorbent assay (ELISA). The cost-effective, scalable and simple production method has great potential for use in developing countries where rapid diagnosis of RVFV is necessary.

Asunto(s)

Antígenos Virales/genética , Nicotiana/genética , Proteínas de la Nucleocápside/genética , Fiebre del Valle del Rift/diagnóstico , Virus de la Fiebre del Valle del Rift/genética , Virus de la Fiebre del Valle del Rift/metabolismo , Enfermedades de las Ovejas/diagnóstico , Animales , Antígenos Virales/sangre , Antígenos Virales/metabolismo , Ensayo de Inmunoadsorción Enzimática/métodos , Expresión Génica , Proteínas de la Nucleocápside/sangre , Proteínas de la Nucleocápside/metabolismo , Fiebre del Valle del Rift/sangre , Fiebre del Valle del Rift/virología , Ovinos , Enfermedades de las Ovejas/sangre , Enfermedades de las Ovejas/virología , Nicotiana/metabolismo

RESUMEN

Iron-sulphur (Fe-S) clusters are ubiquitous co-factors which require multi-protein systems for their synthesis. In Mycobacterium tuberculosis, the Rv1460-Rv1461-Rv1462-Rv1463-csd-Rv1465-Rv1466 operon (suf operon) encodes the primary Fe-S cluster biogenesis system. The first gene in this operon, Rv1460, shares homology with the cyanobacterial SufR, which functions as a transcriptional repressor of the sufBCDS operon. Rv1460's function in M. tuberculosis has however not been determined. In this study, we demonstrate that M. tuberculosis mutants lacking a functional Rv1460 protein are impaired for growth under standard culture conditions. Elevated expression of Rv1460 and Rv1461 was observed in the mutant, implicating Rv1460 in the regulation of the suf operon. Binding of an Fe-S cluster to purified recombinant Rv1460 was confirmed by UV-visible spectroscopy and circular dichroism. Furthermore, three conserved cysteine residues, C203, C216 and C244, proposed to provide ligands for the coordination of an Fe-S cluster, were shown to be required for the function of Rv1460 in M. tuberculosis. Rv1460 therefore seems to be functionally analogous to cyanobacterial SufR.

Asunto(s)

Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Genes Bacterianos , Proteínas Hierro-Azufre/genética , Proteínas Hierro-Azufre/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Sitios de Unión/genética , Secuencia Conservada , Cianobacterias/genética , Cianobacterias/metabolismo , Eliminación de Gen , Humanos , Proteínas Hierro-Azufre/química , Mutación , Mycobacterium tuberculosis/crecimiento & desarrollo , Operón , Regiones Promotoras Genéticas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Represoras/química

RESUMEN

Horseradish peroxidase (HRP) is a commercially important reagent enzyme used in molecular biology and in the diagnostic product industry. It is typically purified from the roots of the horseradish (Armoracia rusticana); however, this crop is only available seasonally, yields are variable and often low, and the product is a mixture of isoenzymes. Engineering high-level expression in transiently transformed tobacco may offer a solution to these problems. In this study, a synthetic Nicotiana benthamiana codon-adapted full-length HRP isoenzyme gene as well as C-terminally truncated and both N- and C-terminally truncated versions of the HRP C gene were synthesized, and their expression in N. benthamiana was evaluated using an Agrobacterium tumefaciens-mediated transient expression system. The influence on HRP C expression levels of co-infiltration with a silencing suppressor (NSs) construct was also evaluated. Highest HRP C levels were consistently obtained using either the full length or C-terminally truncated HRP C constructs. HRP C purification by ion exchange chromatography gave an overall yield of 54% with a Reinheitszahl value of >3 and a specific activity of 458 U/mg. The high level of HRP C production in N. benthamiana in just five days offers an alternative, viable, and scalable system for production of this commercially significant enzyme.

Asunto(s)

Peroxidasa de Rábano Silvestre/genética , Nicotiana/genética , Codón/genética , Peroxidasa de Rábano Silvestre/química , Peroxidasa de Rábano Silvestre/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Nicotiana/enzimología , Nicotiana/metabolismo

RESUMEN

High-risk human papillomaviruses (hr-HPVs) cause cervical cancer, the fourth most common cancer in women worldwide. A HPV-16 candidate therapeutic vaccine, LALF32-51-E7, was developed by fusing a modified E7 protein to a bacterial cell-penetrating peptide (LALF): this elicited both tumour protection and regression in pre-clinical immunization studies. In the current study, we investigated the potential for producing LALF32-51-E7 in a plant expression system by evaluating the effect of subcellular localization and usage of different expression vectors and gene silencing suppressors. The highest expression levels of LALF32-51-E7 were obtained by using a self-replicating plant expression vector and chloroplast targeting, which increased its accumulation by 27-fold compared to cytoplasmic localization. The production and extraction of LALF32-51-E7 was scaled-up and purification optimized by affinity chromatography. If further developed, this platform could potentially allow for the production of a more affordable therapeutic vaccine for HPV-16. This would be extremely relevant in the context of developing countries, where cervical cancer and other HPV-related malignancies are most prevalent, and where the population have limited or no access to preventative vaccines due to their typical high costs.

Asunto(s)

Papillomavirus Humano 16/química , Nicotiana/genética , Proteínas E7 de Papillomavirus/biosíntesis , Vacunas contra Papillomavirus/biosíntesis , Péptidos/metabolismo , Proteínas Recombinantes/biosíntesis , Agrobacterium/genética , Agrobacterium/metabolismo , Cloroplastos/genética , Cloroplastos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Femenino , Expresión Génica , Silenciador del Gen/inmunología , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Papillomavirus Humano 16/inmunología , Humanos , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/inmunología , Infecciones por Papillomavirus/inmunología , Infecciones por Papillomavirus/prevención & control , Infecciones por Papillomavirus/virología , Vacunas contra Papillomavirus/genética , Vacunas contra Papillomavirus/inmunología , Péptidos/genética , Péptidos/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Nicotiana/metabolismo , Neoplasias del Cuello Uterino/inmunología , Neoplasias del Cuello Uterino/prevención & control , Neoplasias del Cuello Uterino/virología

RESUMEN

A GC-vacuum ultraviolet (UV) method to perform group-type separations of diesel range fuels was developed. The method relies on an ionic liquid column to separate diesel samples into saturates, mono-, di-, and polyaromatics by gas chromatography, with selective detection via vacuum UV absorption spectroscopy. Vacuum UV detection was necessary to solve a coelution between saturates and monoaromatics. The method was used to measure group-type composition of 10 oilsands-derived Synfuel light diesel samples, 3 Syncrude light gas oils, and 1 quality control sample. The gas chromatography (GC)-vacuum UV results for the Synfuel samples were similar (absolute % error of 0.8) to historical results from the supercritical fluid chromatography (SFC) analysis. For the light gas oils, discrepancies were noted between SFC results and GC-vacuum UV results; however, these samples are known to be challenging to quantify by SFC-flame ionization detector (FID) due to incomplete resolution between the saturate/monoaromatic and/or monoaromatic/diaromatic group types when applied to samples heavier than diesel (i.e., having a larger fraction of higher molecular weight species). The quality control sample also performed well when comparing both methods (absolute % error of 0.2) and the results agreed within error for saturates, mono- and polyaromatics.

RESUMEN

First- and second-dimension retention times for a series of alkyl phosphates were predicted for multiple column combinations in GC×GC. This was accomplished through the use of a three-parameter thermodynamic model where the analytes' interactions with the stationary phases in both dimensions are known. Ionic liquid columns were employed to impart unique selectivity for alkyl phosphates, and it was determined that for alkyl phosphate compounds, ionic liquid columns are best used in the primary dimension. Retention coordinates for unknown phosphates are predicted from the thermodynamic parameters of a set standard alkyl phosphates. Additionally, we present changing retention properties of alkyl phosphates on some ionic liquid columns, due to suspected reaction between the analyte and column. This makes it difficult to accurately predict their retention properties, and in general poses a problem for ionic liquid columns with these types of analytes.

RESUMEN

Bacteroides fragilis is an opportunistic pathogen which can cause life threatening infections in humans and animals. The ability to adhere to components of the extracellular matrix, including collagen, is related to bacterial host colonisation. Collagen Far Western analysis of the B. fragilis outer membrane protein (OMP) fraction revealed the presence two collagen adhesin bands of ∼ 31 and ∼ 34 kDa. The collagen adhesins in the OMP fraction were separated and isolated by two-dimensional SDS-PAGE and also purified by collagen affinity chromatography. The collagen binding proteins isolated by both these independent methods were subjected to tandem mass spectroscopy for peptide identification and matched to a single hypothetical protein encoded by B. fragilis NCTC 9343 (BF0586), conserved in YCH46 (BF0662) and 638R (BF0633) and which is designated in this study as cbp1 (collagen binding protein). Functionality of the protein was confirmed by targeted insertional mutagenesis of the cbp1 gene in B. fragilis GSH18 which resulted in the specific loss of both the ∼ 31 kDa and the ∼ 34 kDa adhesin bands. Purified his-tagged Cbp1, expressed in a B. fragilis wild-type and a glycosylation deficient mutant, confirmed that the cbp1 gene encoded the observed collagen adhesin, and showed that the 34 kDa band represents a glycosylated version of the ∼ 31 kDa protein. Glycosylation did not appear to be required for binding collagen. This study is the first to report the presence of collagen type I adhesin proteins in B. fragilis and to functionally identify a gene encoding a collagen binding protein.

Asunto(s)

Adhesinas Bacterianas/metabolismo , Bacteroides fragilis/metabolismo , Adhesinas Bacterianas/química , Adhesinas Bacterianas/genética , Secuencia de Aminoácidos , Adhesión Bacteriana/genética , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Bacteroides fragilis/genética , Colágeno Tipo I , Glicosilación , Datos de Secuencia Molecular , Mutación , Unión Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo

RESUMEN

All known nitrilase superfamily amidase and carbamoylase structures have an additional glutamate that is hydrogen bonded to the catalytic lysine in addition to the Glu, Lys, Cys "catalytic triad." In the amidase from Geobacillus pallidus, mutating this glutamate (Glu-142) to a leucine or aspartate renders the enzyme inactive. X-ray crystal structure determination shows that the structural integrity of the enzyme is maintained despite the mutation with the catalytic cysteine (Cys-166), lysine (Lys-134), and glutamate (Glu-59) in positions similar to those of the wild-type enzyme. In the case of the E142L mutant, a chloride ion is located in the position occupied by Glu-142 O(ε1) in the wild-type enzyme and interacts with the active site lysine. In the case of the E142D mutant, this site is occupied by Asp-142 O(δ1.) In neither case is an atom located at the position of Glu-142 O(ε2) in the wild-type enzyme. The active site cysteine of the E142L mutant was found to form a Michael adduct with acrylamide, which is a substrate of the wild-type enzyme, due to an interaction that places the double bond of the acrylamide rather than the amide carbonyl carbon adjacent to the active site cysteine. Our results demonstrate that in the wild-type active site a crucial role is played by the hydrogen bond between Glu-142 O(ε2) and the substrate amino group in positioning the substrate with the correct stereoelectronic alignment to enable the nucleophilic attack on the carbonyl carbon by the catalytic cysteine.

Asunto(s)

Amidohidrolasas/genética , Amidohidrolasas/metabolismo , Biocatálisis , Geobacillus/enzimología , Ácido Glutámico/genética , Mutación/genética , Acrilamida/metabolismo , Amidohidrolasas/química , Dominio Catalítico , Cristalografía por Rayos X , Cisteína/metabolismo , Activación Enzimática , Estabilidad de Enzimas , Espectrometría de Masas , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Oxidación-Reducción , Especificidad por Sustrato

RESUMEN

Retention behaviors of alkyl phosophates were studied on a series of ionic liquid gas chromatography columns. The selectivity of the IL columns for alkyl phosphates were compared with a 5% phenyl column as a route to evaluating the potential use of IL columns in the analysis of alkyl phosphates in petroleum samples in both one- and multi-dimensional GC. Most interestingly, we demonstrate for the first time the dependence of elution order on separation temperature for members of a homologous series of compounds. At low temperatures it was found that trihexyl phosphate eluted before trioctyl phosphate, while at higher temperatures this pattern was reversed.

Asunto(s)

Cromatografía de Gases/métodos , Líquidos Iónicos/química , Organofosfatos/química , Cromatografía de Gases/instrumentación , Organofosfatos/análisis , Organofosfatos/aislamiento & purificación , Petróleo , Temperatura

RESUMEN

MshB, a zinc-based deacetylase, catalyses a step in the mycothiol biosynthetic pathway that involves the deacetylation of 1-O-(2-acetamido-2-deoxy-α-D-glucopyranosyl)-D-myo-inositol (GlcNAc-Ins), via cleavage of an amide bond, to 1-O-(2-amino-2-deoxy-α-D-glucopyranosyl)-D-myo-inositol (GlcN-Ins) and acetate. In this study, MshB was expressed, purified and crystallized. A new crystal form was encountered in 0.1 M sodium acetate, 0.2 M ammonium sulfate, 25% PEG 4000 pH 4.6. The crystals diffracted to 1.95 Å resolution and the resulting electron-density map revealed glycerol and the reaction product, acetate, in the active site. These ligands enabled the natural substrate GlcNAc-Ins to be modelled in the active site with some certainty. One acetate O atom is hydrogen bonded to Tyr142 and is located 2.5 Å from the catalytic zinc. The other acetate O atom is located 2.7 Å from a carboxylate O atom of Asp15. This configuration strongly suggests that Asp15 acts both as a general base catalyst in the nucleophilic attack of water on the amide carbonyl C atom and in its protonated form acts as a general acid to protonate the amide N atom. The configuration of Tyr142 differs from that observed previously in crystal structures of MshB (PDB entries 1q74 and 1q7t) and its location provides direct structural support for recently published biochemical and mutational studies suggesting that this residue is involved in a conformational change on substrate binding and contributes to the oxyanion hole that stabilizes the tetrahedral intermediate.

Asunto(s)

Acetatos/química , Amidohidrolasas/química , Proteínas Bacterianas/química , Dominio Catalítico , Glicerol/química , Mycobacterium tuberculosis/química , Tuberculosis/microbiología , Acetatos/metabolismo , Amidohidrolasas/metabolismo , Sulfato de Amonio/química , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Glicerol/metabolismo , Simulación de Dinámica Molecular , Mycobacterium tuberculosis/metabolismo , Polietilenglicoles/química , Conformación Proteica

RESUMEN

Heterocapsa circularisquama RNA virus is a non-enveloped icosahedral ssRNA virus infectious to the harmful bloom-forming dinoflagellate, H. circularisquama, and which is assumed to be the major natural agent controlling the host population. The viral capsid is constructed from a single gene product. Electron cryo-microscopy revealed that the virus has a diameter of 34 nm and T = 3 symmetry. The 180 quasi-equivalent monomers have an unusual arrangement in that each monomer contributes to a 'bump' on the surface of the protein. Though the capsid protein probably has the classic 'jelly roll' ß-sandwich fold, this is a new packing arrangement and is distantly related to the other positive-sense ssRNA virus capsid proteins. The handedness of the structure has been determined by a novel method involving high resolution scanning electron microscopy of the negatively stained viruses and secondary electron detection.

Asunto(s)

Cápside , Microscopía por Crioelectrón/métodos , Virus ARN/ultraestructura , Cápside/química , Cápside/ultraestructura , Dinoflagelados/virología , Procesamiento de Imagen Asistido por Computador , Conformación Proteica , Virus ARN/química , Virus ARN/aislamiento & purificación , Propiedades de Superficie

RESUMEN

Geobacillus pallidus RAPc8 (NRRL: B-59396) is a moderately thermophilic gram-positive bacterium, originally isolated from Australian lake sediment. The G. pallidus RAPc8 gene encoding an inducible nitrilase was located and cloned using degenerate primers coding for well-conserved nitrilase sequences, coupled with inverse PCR. The nitrilase open reading frame was cloned into an expression plasmid and the expressed recombinant enzyme purified and characterized. The protein had a monomer molecular weight of 35,790 Da, and the purified functional enzyme had an apparent molecular weight of approximately 600 kDa by size exclusion chromatography. Similar to several plant nitrilases and some bacterial nitrilases, the recombinant G. pallidus RAPc8 enzyme produced both acid and amide products from nitrile substrates. The ratios of acid to amide produced from the substrates we tested are significantly different to those reported for other enzymes, and this has implications for our understanding of the mechanism of the nitrilases which may assist with rational design of these enzymes. Electron microscopy and image classification showed complexes having crescent-like, "c-shaped", circular and "figure-8" shapes. Protein models suggested that the various complexes were composed of 6, 8, 10 and 20 subunits, respectively.

Asunto(s)

Aminohidrolasas/genética , Aminohidrolasas/metabolismo , Geobacillus/enzimología , Nitrilos/metabolismo , Secuencia de Aminoácidos , Aminohidrolasas/química , Cromatografía en Gel , Clonación Molecular , Análisis por Conglomerados , Cartilla de ADN/genética , Electroforesis en Gel de Poliacrilamida , Expresión Génica , Vectores Genéticos , Calor , Datos de Secuencia Molecular , Peso Molecular , Filogenia , Plásmidos , Reacción en Cadena de la Polimerasa/métodos , Multimerización de Proteína , Subunidades de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia

RESUMEN

The fungal cyanide hydratases form a functionally specialized subset of the nitrilases which catalyze the hydrolysis of cyanide to formamide with high specificity. These hold great promise for the bioremediation of cyanide wastes. The low resolution (3.0 nm) three-dimensional reconstruction of negatively stained recombinant cyanide hydratase fibers from the saprophytic fungus Neurospora crassa by iterative helical real space reconstruction reveals that enzyme fibers display left-handed D(1) S(5.4) symmetry with a helical rise of 1.36 nm. This arrangement differs from previously characterized microbial nitrilases which demonstrate a structure built along similar principles but with a reduced helical twist. The cyanide hydratase assembly is stabilized by two dyadic interactions between dimers across the one-start helical groove. Docking of a homology-derived atomic model into the experimentally determined negative stain envelope suggests the location of charged residues which may form salt bridges and stabilize the helix.

Asunto(s)

Proteínas Fúngicas/química , Hidroliasas/química , Neurospora crassa/enzimología , Secuencia de Aminoácidos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/aislamiento & purificación , Proteínas Fúngicas/metabolismo , Expresión Génica , Hidroliasas/genética , Hidroliasas/aislamiento & purificación , Hidroliasas/metabolismo , Modelos Moleculares , Conformación Molecular , Datos de Secuencia Molecular , Neurospora crassa/química , Alineación de Secuencia

RESUMEN

Development of vaccine strategies against human papillomavirus (HPV), which causes cervical cancer, is a priority. We investigated the use of virus-like particles (VLPs) of the most prevalent type, HPV-16, as carriers of foreign proteins. Green fluorescent protein (GFP) was fused to the N or C terminus of both L1 and L2, with L2 chimeras being co-expressed with native L1. Purified chimaeric VLPs were comparable in size ( approximately 55 nm) to native HPV VLPs. Conformation-specific monoclonal antibodies (Mabs) bound to the VLPs, thereby indicating that they possibly retain their antigenicity. In addition, all of the VLPs encapsidated DNA in the range of 6-8 kb.

Asunto(s)

Proteínas de la Cápside/genética , Vectores Genéticos , Papillomavirus Humano 16/genética , Proteínas Oncogénicas Virales/genética , Cápside/inmunología , Proteínas de la Cápside/inmunología , Proteínas de la Cápside/metabolismo , Proteínas Fluorescentes Verdes , Papillomavirus Humano 16/inmunología , Papillomavirus Humano 16/ultraestructura , Humanos , Microscopía Electrónica de Transmisión , Proteínas Oncogénicas Virales/inmunología , Proteínas Oncogénicas Virales/metabolismo , Proteínas Recombinantes de Fusión/genética , Vacunas de ADN/inmunología

RESUMEN

Nitrilases convert nitriles to the corresponding carboxylic acids and ammonia. The nitrilase from Rhodococcus rhodochrous J1 is known to be inactive as a dimer but to become active on oligomerization. The recombinant enzyme undergoes post-translational cleavage at approximately residue 327, resulting in the formation of active, helical homo-oligomers. Determining the 3D structure of these helices using electron microscopy, followed by fitting the stain envelope with a model based on homology with other members of the nitrilase superfamily, enables the interacting surfaces to be identified. This also suggests that the reason for formation of the helices is related to the removal of steric hindrance arising from the 39 C-terminal amino acids from the wild-type protein. The helical form can be generated by expressing only residues 1-327.

Asunto(s)

Aminohidrolasas/metabolismo , Rhodococcus/enzimología , Secuencia de Aminoácidos , Aminohidrolasas/química , Estabilidad de Enzimas , Imagenología Tridimensional , Datos de Secuencia Molecular , Procesamiento Proteico-Postraduccional , Estructura Secundaria de Proteína , Proteínas Recombinantes/metabolismo

RESUMEN

Endocytosis is a fundamental process of eukaryotic cells and fulfills numerous functions, most notably, that of macromolecular nutrient uptake. Malaria parasites invade red blood cells and during their intracellular development endocytose large amounts of host cytoplasm for digestion in a specialized lysosomal compartment, the food vacuole. In the present study we have examined the effects of artemisinin and the quinoline drugs chloroquine and mefloquine on endocytosis in Plasmodium falciparum. By using novel assays we found that mefloquine and artemisinin inhibit endocytosis of macromolecular tracers by up to 85%, while the latter drug also leads to an accumulation of undigested hemoglobin in the parasite. During 5-h incubations, chloroquine inhibited hemoglobin digestion but had no other significant effect on the endocytic pathway of the parasite, as assessed by electron microscopy, the immunofluorescence localization of hemoglobin, and the distribution of fluorescent and biotinylated dextran tracers. By contrast, when chloroquine was added to late ring stage parasites, followed by a 12-h incubation, macromolecule endocytosis was inhibited by more than 40%. Moreover, there is an accumulation of transport vesicles in the parasite cytosol, possibly due to a disruption in vacuole-vesicle fusion. This fusion block is not observed with mefloquine, artemisinin, quinine, or primaquine but is mimicked by the vacuole alkalinizing agents ammonium chloride and monensin. These results are discussed in the light of present theories regarding the mechanisms of action of the antimalarials and highlight the potential use of drugs in manipulating and studying the endocytic pathway of malaria parasites.

Asunto(s)

Antimaláricos/farmacología , Artemisininas/farmacología , Endocitosis/efectos de los fármacos , Plasmodium falciparum/efectos de los fármacos , Quinolonas/farmacología , Sesquiterpenos/farmacología , Cloruro de Amonio/farmacología , Animales , Biotina/farmacología , Western Blotting , Cloroquina/farmacología , Dextranos/farmacología , Fluoresceína-5-Isotiocianato , Técnica del Anticuerpo Fluorescente , Colorantes Fluorescentes , Hemoglobinas/metabolismo , Mefloquina/farmacología , Microscopía Electrónica , Microscopía Fluorescente , Monensina/farmacología , Plasmodium falciparum/ultraestructura , Vesículas Transportadoras/efectos de los fármacos , Vacuolas/efectos de los fármacos , Vacuolas/ultraestructura

RESUMEN

BACKGROUND: Apoptotic propensity is currently viewed as an important parameter in drug-induced toxicity. But other cell death pathways exist e.g. micronucleation, intermitotic cell death, abnormal nuclear morphology and necrosis. This investigation explores the onset of apoptosis and abnormal morphology in response to 3 drugs i.e. Cisplatin, a novel Ferrocene (fctfa) and a novel Rhodium-Ferrocene [Rh(fctfa)(cod)] complex. MATERIALS AND METHODS: A pair of prostate cell lines from normal human prostate epithelium (1542N) and malignant human prostate epithelium (1542T) were exposed to increasing concentrations of the drugs for 24 hours, double-stained with FITC-Annexin V and with Propidium Iodide and analysed by dual parameter flow cytometry to quantitate viable cells in quadrant I, early apoptotic cells in quadrant IV and late apoptotic/necrotic cells in quadrant III. Apoptosis was also scored by microscopy after Acridine Orange staining, by Western blots for caspase 3 induction and for caspase 8 induction using a colorimetric assay. RESULTS: The toxicity of Cisplatin and the Ferrocene and Rhodium-Ferrocene complexes was found to be 0.9-1.3 microM; 4.1-4.5 microM and 10.1-13.2 microM, respectively. Apoptotic propensity scored after 24 hours was found to be dose-dependent and in the range of 7-19% for Cisplatin and 1-4.1% for the Ferrocene and Rhodium-Ferrocene complexes. Cisplatin produces a distinct apoptotic response followed by a necrotic response, whereas the Ferrocene and the Rhodium-Ferrocene complexes produce a massive necrotic reaction in the region of 3-19% and very little if any apoptosis. Absence of apoptosis was corroborated by lack of caspase 3 activation, absence of typical apoptotic morphology and by lack of caspase 8 activation. CONCLUSION: The 3 drugs Cisplatin, the novel Ferrocene and the novel Rhodium-Ferrocene complexes show similar toxicities in the 1-10 micro-molar range in prostate cell lines. However the drugs differ significantly in the activation of death pathways. While Cisplatin predominantly induces apoptosis documented by morphology, Annexin V staining and caspase 8 activation, the Ferrocene and Rhodium-Ferrocene complexes induce late necrosis and abnormal nuclear morphology. Unlike Cisplatin-treated cells which enter apoptosis and necrosis sequentially, the 2 Ferrocene drugs invoke direct entry of cells into late necrosis without first entering the early apoptotic compartment.

Asunto(s)

Antineoplásicos/farmacología , Compuestos Ferrosos/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Rodio/farmacología , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Cisplatino/farmacología , Compuestos Ferrosos/química , Humanos , Concentración 50 Inhibidora , Masculino , Metalocenos , Compuestos Organometálicos/química , Compuestos Organometálicos/farmacología , Neoplasias de la Próstata/patología , Rodio/química