RESUMO
Aberrant activity of the cysteine protease Cathepsin S (CTSS) has been implicated across a wide range of pathologies. Notably in cancer, CTSS has been shown to promote tumour progression, primarily through facilitating invasion and migration of tumour cells and augmenting angiogenesis. Whilst an attractive therapeutic target, more efficacious CTSS inhibitors are required. Here, we investigated the potential application of Variable New Antigen Receptors (vNARs) as a novel inhibitory strategy. A panel of potential vNAR binders were identified following a phage display panning process against human recombinant proCTSS. These were subsequently expressed, purified and binding affinity confirmed by ELISA and SPR based approaches. Selected lead clones were taken forward and were shown to inhibit CTSS activity in recombinant enzyme activity assays. Further assessment demonstrated that our lead clones functioned by a novel inhibitory mechanism, by preventing the activation of proCTSS to the mature enzyme. Moreover, using an intrabody approach, we exhibited the ability to express these clones intracellularly and inhibit CTSS activity whilst lead clones were also noted to impede cell invasion in a tumour cell invasion assay. Collectively, these findings illustrate a novel mechanistic approach for inhibiting CTSS activity, with anti-CTSS vNAR clones possessing therapeutic potential in combating deleterious CTSS activity. Furthermore, this study exemplifies the potential of vNARs in targeting intracellular proteins, opening a range of previously "undruggable" targets for biologic-based therapy.
RESUMO
Mice lacking functional neurokinin-1 receptors (NK1R-/-) display abnormal behaviours seen in Attention Deficit Hyperactivity Disorder (hyperactivity, impulsivity and inattentiveness). These abnormalities were evident when comparing the behaviour of separate (inbred: 'Hom') wildtype and NK1R-/- mouse strains. Here, we investigated whether the inbreeding protocol could influence their phenotype by comparing the behaviour of these mice with that of wildtype (NK1R+/+) and NK1R-/- progeny of heterozygous parents ('Het', derived from the same inbred strains). First, we recorded the spontaneous motor activity of the two colonies/genotypes, over 7 days. This continuous monitoring also enabled us to investigate whether the diurnal rhythm in motor activity differs in the two colonies/genotypes. NK1R-/- mice from both colonies were hyperactive compared with their wildtypes and their diurnal rhythm was also disrupted. Next, we evaluated the performance of the four groups of mice in the 5-Choice Serial Reaction-Time Task (5-CSRTT). During training, NK1R-/- mice from both colonies expressed more impulsive and perseverative behaviour than their wildtypes. During testing, only NK1R-/- mice from the Hom colony were more impulsive than their wildtypes, but NK1R-/- mice from both colonies were more perseverative. There were no colony differences in inattentiveness. Moreover, a genotype difference in this measure depended on time of day. We conclude that the hyperactivity, perseveration and, possibly, inattentiveness of NK1R-/- mice is a direct consequence of a lack of functional NK1R. However, the greater impulsivity of NK1R-/- mice depended on an interaction between a functional deficit of NK1R and other (possibly environmental and/or epigenetic) factors.
Assuntos
Comportamento Animal/fisiologia , Comportamento de Escolha/fisiologia , Comportamento Impulsivo/fisiologia , Receptores da Neurocinina-1/genética , Animais , Transtorno do Deficit de Atenção com Hiperatividade/genética , Camundongos Knockout , Fenótipo , Tempo de Reação/genética , Receptores da Neurocinina-1/deficiênciaRESUMO
OBJECTIVE: To compare factors influencing adequacy of endometrial samples obtained using two outpatient sampling devices--Pipelle and Tao Brush. DESIGN: Pragmatic unblinded trial with investigation schedule randomised separately within two groups according to endometrial cancer risk. SETTING: Gynaecology outpatient clinic of a large city hospital in Edinburgh, Scotland. POPULATION: All women referred to a gynaecology outpatient clinic during a 28-month period complaining of abnormal vaginal bleeding. METHODS: Women were assigned to two 'risk groups' for endometrial cancer ('high risk' for postmenopausal women and 'moderate risk' for premenopausal women aged over 40 years or with other risk factors). Women in each risk group had both types of biopsy and were randomised to two outpatient visualisations: hysteroscopy and/or transvaginal ultrasound scan. MAIN OUTCOME MEASURES: Completion of the investigation, adequacy of sample and acceptability of investigation to women. RESULTS: In 200 high-risk women, adequate samples were significantly more likely to be obtained by Tao Brush than Pipelle (P < 0.001). Nulliparity was strongly associated with failed insertion for both devices (P < 0.001). Inadequate samples were strongly associated with postmenopausal status only for Pipelle (P < 0.001), and among premenopausal women, for both samplers, with nulliparity (P < 0.001). A significantly greater proportion of women preferred the Tao Brush to the Pipelle endometrial sampler (P < 0.001). CONCLUSIONS: In postmenopausal women, Tao Brush sampling offers advantages over use of Pipelle, and the former should be considered as an alternative or additional sampling device in this group of women.
Assuntos
Biópsia/instrumentação , Endométrio/patologia , Manejo de Espécimes/instrumentação , Hemorragia Uterina/patologia , Biópsia/efeitos adversos , Hiperplasia Endometrial/patologia , Neoplasias do Endométrio/patologia , Feminino , Humanos , Pessoa de Meia-Idade , Satisfação do Paciente , Pós-MenopausaRESUMO
A colorimetric method, reverse transcriptase PCR with an enzyme-linked immunosorbent assay (RT-PCR-ELISA) was evaluated for ease of use, reliability, and sensitivity when detecting known human pathogenic virus present in shellfish, using a traditional polyethylene precipitation or immunocapture virus concentration method. The newly developed ELISA method could successfully detect enteroviruses and noroviruses in artificially and naturally contaminated shellfish. Overall, ELISA was shown to be a robust and sensitive method, which had a detection limit of 10 to 100 50% tissue culture infective dose enterovirus per gram of Crassostrea gigas (Pacific oyster) digestive gland and whole Mytilus edulis (common blue mussel). The technique was easily established in a new laboratory and required no specialized equipment. The method had a high sample throughput capable of screening 96 samples per run, making the technique extremely time efficient. RT-PCR-ELISA is a safe, quick, reliable technique, which has the potential for use as a standard virus detection method.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Contaminação de Alimentos/análise , Moluscos/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Frutos do Mar/virologia , Animais , Qualidade de Produtos para o Consumidor , Enterovirus/isolamento & purificação , Humanos , Norovirus/isolamento & purificação , Sensibilidade e EspecificidadeRESUMO
Eighty-four phyllodes tumours (71 benign, eight borderline and five malignant) diagnosed over a 16-year period were studied retrospectively, to assess the diagnostic value of the pre-operative modalities used. Mammography and ultrasound appearances were non-specific. The possibility of phyllodes tumour was raised in only 23% on fine needle aspiration cytology, and in 65% on core biopsy. Accuracy was better in smaller tumours, suggesting that larger tumours need more samples. For phyllodes tumours whose growth was measured, almost all had growth rates greater than for growing fibroadenomas. The pre-operative diagnosis of phyllodes tumours is difficult, and rapid growth and/or large size of apparent fibroadenomas may be the only imaging findings to suggest phyllodes tumour. It is important to review most fibroadenomas with ultrasound, to assess the rate of growth if any. Whole breast ultrasound showed that nearly one third of women with phyllodes tumours had concurrent fibroadenomas.
Assuntos
Neoplasias da Mama/diagnóstico , Tumor Filoide/diagnóstico , Adolescente , Adulto , Idoso , Biópsia por Agulha Fina , Neoplasias da Mama/cirurgia , Feminino , Fibroadenoma/diagnóstico , Humanos , Pessoa de Meia-Idade , Tumor Filoide/cirurgia , Estudos RetrospectivosRESUMO
A new method, termed RT-PCR-ELISA, was evaluated for ease of use, reliability and sensitivity when detecting infectious pancreatic necrosis virus (IPNV) present in trout kidney tissue. The method had comparable sensitivity to existing PCR assays and could successfully detect 1.5 x 10(4) pfu IPNV in artificially contaminated trout kidney samples. The technique was easily established in a new laboratory and required no specialised equipment. The method had a high sample throughput capable of screening 96 samples per run, making the technique extremely time efficient. The RT-PCR-ELISA is a safe, quick, reliable technique, which has the potential for use as a standard virus detection method.
Assuntos
Infecções por Birnaviridae/veterinária , Ensaio de Imunoadsorção Enzimática/métodos , Doenças dos Peixes/diagnóstico , Vírus da Necrose Pancreática Infecciosa/isolamento & purificação , Oncorhynchus mykiss/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Animais , Infecções por Birnaviridae/diagnóstico , Infecções por Birnaviridae/virologia , Doenças dos Peixes/virologia , Rim/virologia , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
In the current study, we investigated human leukocyte antigen (HLA) class II alleles in Caucasian women with primary biliary cirrhosis (PBC), a disease that preferentially affects women. Alleles of DRB1, DQA1, and DQB1 were determined by DNA-based HLA typing for women with PBC (n = 72) and healthy women (n = 381). All study subjects were Caucasian. HLA DRB1*08 was significantly increased in women with PBC compared to healthy women. The increase was primarily due to the DRB1*0801 allele, also the most common DRB1*08 allele among controls. DQB1*04 and DQA1*0401 were significantly increased. DRB1*1501, DQA1*0102, and DQB1*0602 were associated with decreased risk. Analyses conducted comparing parous women with PBC to parous healthy women (n = 68 and n = 282, respectively) yielded similar significant results. Although the DRB1*08-DQA1*0401-DQB1*04 haplotype was significantly associated with PBC, consistent with other studies, this haplotype nevertheless represented only 19% (14/72) of all PBC patients and can account for only a minority of the risk of PBC.
Assuntos
Alelos , Haplótipos/genética , Antígenos de Histocompatibilidade Classe II/genética , Cirrose Hepática Biliar/genética , Adulto , Idoso , Estudos de Casos e Controles , Feminino , Antígenos HLA-DQ/genética , Cadeias alfa de HLA-DQ , Cadeias beta de HLA-DQ , Antígenos HLA-DR/genética , Cadeias HLA-DRB1 , Humanos , Cirrose Hepática Biliar/imunologia , Glicoproteínas de Membrana/genética , Pessoa de Meia-Idade , Fatores de Risco , População BrancaRESUMO
OBJECTIVE: We investigated HLA class II alleles in women with systemic sclerosis (SSc), a rare disease that preferentially affects women. METHODS: Specific alleles of DRB1, DQA1 and DQB1 were determined by DNA-based HLA typing for women with SSc (n = 102) and healthy women (n = 533). All study subjects were Caucasian. DRB1, DQA1 and DQB1 allele frequencies of women with SSc were compared with those of healthy women. RESULTS: Among women with SSc, 29.4% (30/102) and among healthy women 10.7% (57/533) had DRB1*11. Allele frequencies were compared for women with SSc and healthy women (each woman has two alleles). The allele frequency of DRB1*11 was 15.7% (32/204 alleles) in SSc women and 5.8% (62/1066 alleles) in healthy women (P = 0.000002). The increase of DRB1*11 was found both in diffuse (P = 0.0001) and limited SSc (P = 0.002) (allele frequencies 15.0 and 17.2%, respectively). Among women with diffuse SSc, there was a disproportionate increase of the DRB1*1104 allele (P = 0.0004) with no increase of DRB1*1101 (P = 1.00). In contrast, in limited SSc the strongest association was with DRB1*1101 (P = 0.008), with a less significant increase of DRB1*1104 (P = 0.04). CONCLUSIONS: An increase of DRB1*11 in SSc is consistent with other reports. Although present in both diffuse and limited SSc disease subsets, the increase was predominantly due to over-representation of DRB1*1104 in women with diffuse SSc. Women with limited SSc had a preponderance of DRB1*1101, the most common allele in healthy women. DRB1*1104 and DRB1*1101 differ by a single amino acid at position 86, where the former has valine and the latter glycine.
Assuntos
Antígenos de Histocompatibilidade Classe II/genética , Escleroderma Sistêmico/genética , Adolescente , Adulto , Idoso , Feminino , Frequência do Gene , Antígenos HLA-DQ/genética , Cadeias alfa de HLA-DQ , Cadeias beta de HLA-DQ , Antígenos HLA-DR/genética , Cadeias HLA-DRB1 , Humanos , Desequilíbrio de Ligação , Pessoa de Meia-Idade , Esclerodermia Difusa/genética , Esclerodermia Limitada/genéticaRESUMO
AIMS: The purpose of this study was to comprehensively compare the response of nine biosensors capable of being induced by Hg. Induction by Hg was based upon the insertion of merR, merB, zntA and zntR promoter genes. LuxCDABE or lucFF reporter genes expressed luminescence, and host organisms were Escherichia coli, Vibrio anguillarum and Pseudomonas fluorescens. The role of transcriptional switches, reporter mechanism and host organism was to be investigated. METHODS AND RESULTS: All biosensors were subjected to the same assay conditions. Sensors had their own individual growth characteristics and response to the doses of Hg tested. Maximum bioluminescence response was induced by concentrations of Hg between 2.5 nm and 5 microM. E. coli pRB28 was found to detect levels of Hg as low as 1.6 nm and yet was capable of operating in a concentration range of up to 12.5 microM. CONCLUSIONS: The response of the sensors demonstrated their suitability for analysis under environmentally relevant concentrations. The sensitivity of the sensors, the optimum range and the expediency of the assay could not be related to a single sensor trait. It may be concluded that biosensor performance is dependent on more than one of the single factors studied. SIGNIFICANCE AND IMPACT OF THE STUDY: The results show that comparative testing of sensors is an important step in evaluating the relevance and performance of biosensors prior to routine environmental application.
Assuntos
Genes Bacterianos/genética , Genes Reporter/genética , Bactérias Gram-Negativas/genética , Mercúrio , Transcrição Gênica/genética , Disponibilidade Biológica , Técnicas Biossensoriais/métodos , Elementos de DNA Transponíveis/genética , Escherichia coli/genética , Medições Luminescentes , Mercúrio/análise , Mercúrio/farmacocinética , Regiões Promotoras Genéticas/genética , Pseudomonas fluorescens/genética , Vibrio/genéticaRESUMO
This study examined how the diagnosis of breast cancer is different in young women. Records were retrieved for 239 women diagnosed with breast cancer before age 40 and compared with 2101 women aged 40 and over with breast cancer. On mammography, lesions in the younger women were more likely to be undetected or interpreted as benign, especially in women with dense breasts. However, there were 10 young women where impalpable cancers with microcalcification under 10 mm would not have been diagnosed without mammography. An abnormality was detected on ultrasound in 92.2% of cancers in young women, but was more likely to be considered benign than in older women. If ultrasound alone had been used in the young women, at least 18 cancers would have been missed. Ultrasound was useful for predicting the ultimate tumour size at pathology, and for detecting multifocality. There were 14 cases where the ultrasound appearance was indistinguishable from fibroadenoma. The importance of fine needle aspiration cytology in the diagnosis of focal lesions in young women (over 20 years) was confirmed. For symptomatic women, the proportion of breast malignancies under 10 mm was similar in the two groups. However, the younger group had significantly more poorly differentiated tumours.
Assuntos
Neoplasias da Mama/diagnóstico por imagem , Adulto , Fatores Etários , Idade de Início , Biópsia por Agulha , Mama/anatomia & histologia , Doenças Mamárias/diagnóstico por imagem , Calcinose , Diagnóstico Diferencial , Feminino , Humanos , Mamografia , Estudos Retrospectivos , Sensibilidade e Especificidade , UltrassonografiaRESUMO
The aim of this work was to generate a cyanobacterial biosensor that could be used to detect herbicides and other environmental pollutants. A representative freshwater cyanobacterium, Synechocystis sp. strain PCC6803, was chromosomally marked with the luciferase gene luc (from the firefly Photinus pyralis) to create a novel bioluminescent cyanobacterial strain. Successful expression of the luc gene during growth of Synechocystis sp. strain PCC6803 cultures was characterized by measuring optical density and bioluminescence. Bioluminescence was optimized with regard to uptake of the luciferase substrate, luciferin, and the physiology of the cyanobacterium. Bioassays demonstrated that a novel luminescent cyanobacterial biosensor has been developed which responded to a range of compounds including different herbicide types and other toxins. This biosensor is expected to provide new opportunities for the rapid screening of environmental samples or for the investigation of potential environmental damage.
Assuntos
Técnicas Biossensoriais/métodos , Cianobactérias/fisiologia , Herbicidas/análise , Microbiologia da Água , Poluentes Químicos da Água/análise , Cianobactérias/efeitos dos fármacos , Cianobactérias/genética , Genes Reporter , Herbicidas/química , Herbicidas/metabolismo , Luciferases/genética , Luminescência , Plasmídeos/genética , Reação em Cadeia da Polimerase , Poluentes Químicos da Água/metabolismoRESUMO
BACKGROUND: Myenteric interneurones are involved in the reflexes that control the motility of the human colon. AIMS: The distribution of choline acetyltransferase (ChAT) and nitric oxide synthase (NOS) immunoreactivity in myenteric interneurones was investigated in this study. METHODS: DiI (1,1'- didodecyl 3,3,3',3'-indocarbocyanine perchlorate) was applied to the myenteric plexus of the human colon followed by organotypic culture. Retrogradely labelled neurones, with projections longer than motor neurones (>10 mm), were studied to exclude motor neurone populations. ChAT and NOS immunoreactivity was then determined in the interneurones. RESULTS: We found that 90% of interneurones projecting orally contained ChAT and none contained NOS. Ninety five per cent of descending interneurones were labelled with ChAT and/or NOS antisera; 46% contained NOS immunoreactivity alone, 20% contained ChAT immunoreactivity alone, and 29% contained both ChAT and NOS. Anally directed interneurones had significantly longer projections than orally projecting interneurones. CONCLUSIONS: Nearly all interneurones contain either NOS or ChAT immunoreactivity. Orally projecting interneurones are of two types: 90% contain ChAT alone and the remainder contain immunoreactivity for neither ChAT nor NOS. There are three main types of anally projecting interneurones: the largest, which contains NOS but not ChAT, and the two smaller classes which contain ChAT and NOS, and CHAT alone.
Assuntos
Colina O-Acetiltransferase/análise , Colo/inervação , Motilidade Gastrointestinal/fisiologia , Interneurônios/enzimologia , Plexo Mientérico/ultraestrutura , Óxido Nítrico Sintase/análise , Idoso , Contagem de Células , Extensões da Superfície Celular/ultraestrutura , Feminino , Humanos , Imuno-Histoquímica/métodos , Interneurônios/ultraestrutura , Masculino , Pessoa de Meia-IdadeRESUMO
Antibody phage display libraries (Griffin and Tomlinson I) displaying antibody genes and maintained and amplified in Escherichia coli were used to isolate antibodies to the hapten target microcystin LR (1000 Da) conjugated to either bovine serum albumin or keyhole limpet haemocyanin. In competition enzyme-linked immunosorbent assay, bacterially expressed antibodies selected via the Griffin library showed at least 300 times greater sensitivity than those isolated from the Tomlinson library, for free microcystin. Bacterially expressed phage antibody libraries provide a rapid and relatively easy route for the selection of monoclonal antibodies specific for even the most difficult of antigenic targets such as free haptens.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Especificidade de Anticorpos , Escherichia coli/genética , Haptenos/imunologia , Biblioteca de Peptídeos , Peptídeos Cíclicos/imunologia , Anticorpos Monoclonais/genética , Ligação Competitiva , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática , Haptenos/química , Hemocianinas , Humanos , Toxinas Marinhas , Microcistinas , Peptídeos Cíclicos/química , Soroalbumina BovinaRESUMO
This study determined that the bacterial luciferase fusion gene (luxAB) was not a suitable in vivo gene reporter in the model eukaryotic organisms Saccharomyces cerevisiae and Caenorhabditis elegans. LuxAB expressing S. cerevisiae strains displayed distinctive rapid decays in luminescence upon addition of the bacterial luciferase substrate, n-decyl aldehyde, suggesting a toxic response. Growth studies and toxicity bioassays have subsequently confirmed, that the aldehyde substrate was toxic to both organisms at concentrations well tolerated by Escherichia coli. As the addition of aldehyde is an integral part of the bacterial luciferase activity assay, our results do not support the use of lux reporter genes for in vivo analyses in these model eukaryotic organisms.
Assuntos
Aldeídos/farmacologia , Caenorhabditis elegans/efeitos dos fármacos , Luciferases/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Animais , Caenorhabditis elegans/fisiologia , Genes Reporter/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/fisiologiaRESUMO
The complexity and expense of producing anti-hapten monoclonals via the traditional hybridoma route and the preferential selection of antibodies that recognise the conjugated form of the hapten, over antibodies that specifically recognise free hapten, are two of the more important problems that have limited the development and application of anti-hapten antibodies. The advent of phage display technology allows the rapid isolation of monoclonal antibody fragments from libraries of different antibodies (>10(8)) displayed on the surface of filamentous bacteriophages. Much of the power of this new approach lies in the flexibility with which these libraries can be screened for suitable binders. Using an optimised selection procedure, we have isolated from a sheep antibody phage display library, super-sensitive anti-hapten antibodies specific for the herbicide and environmental pollutant, atrazine. In particular, two phage clones have been isolated that can be expressed cheaply and in quantity in Escherichia coli, demonstrate excellent stability in nonphysiological conditions and are exciting prospects for immunoassay applications including ELISA, dip-stick formats, on-line monitoring and biosensor technologies. In ELISA formats they show low levels of cross reactivity with related molecules and a limit of detection of a 1-2 parts per trillion (p.p.t.), well within the 100 p.p.t. required by EC legislation.
Assuntos
Anticorpos Monoclonais/isolamento & purificação , Animais , Anticorpos Monoclonais/genética , Especificidade de Anticorpos , Atrazina/imunologia , Técnicas Biossensoriais , Ensaio de Imunoadsorção Enzimática , Haptenos , Herbicidas/imunologia , Camundongos , Biblioteca de Peptídeos , OvinosRESUMO
The dose response relationship between seven commonly used herbicides and four luminescence-based bacterial biosensors was characterised. As herbicide concentration increased the light emitted by the test organism declined in a concentration dependent manner. These dose responses were used to compare the predicted vs. observed response of a biosensor in the presence of multiple contaminants. For the majority of herbicide interactions, the relationship was not additive but primarily antagonistic and sometimes synergistic. These biosensors provide a sensitive test and are able to screen a large volume and wide range of samples with relative rapidity and ease of interpretation. In this study biosensor technology has been successfully applied to interpret the interactive effects of herbicides in freshwater environments.
Assuntos
Técnicas Biossensoriais/métodos , Água Doce/química , Herbicidas/análise , Herbicidas/toxicidade , Testes de Toxicidade/métodos , Poluentes Químicos da Água/análise , Poluentes Químicos da Água/toxicidade , Bactérias/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Medições Luminescentes , Fatores de TempoRESUMO
Murine monoclonal antibody 206 (MAb mu206) binds to gH, the varicella-zoster virus (VZV) fusogen, neutralizing the virus in vitro in the absence of complement and inhibiting cell-to-cell spread and egress of VZV in cultured cells. We have humanized this antibody to generate MAb hu206 by complementarity determining region grafting. MAb hu206 retained binding and in vitro neutralizing activity, as well as cross-reactivity with ten different VZV strains. Single-chain antibody fragments (scAb) derived from MAb hu206 were produced in Escherichia coli. These scAb retained the binding properties of the whole antibody. However, monomeric scAb exhibited markedly reduced neutralizing activity compared to the bivalent parental MAb hu206. Shortening the peptide linker joining the V(H) to the V(kappa) domain from 14 to 5 or even 0 residues encouraged multimerization and increased neutralizing efficacy. The fact that Fab fragments enzymatically generated from whole MAb hu206 lost their neutralizing potency lent support to the proposal that valency is important for VZV neutralization at this epitope.
Assuntos
Anticorpos Antivirais/imunologia , Herpesvirus Humano 3/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/genética , Linhagem Celular , Relação Dose-Resposta Imunológica , Escherichia coli/genética , Vetores Genéticos , Glicoproteínas/imunologia , Herpes Zoster/imunologia , Herpesvirus Humano 3/química , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Camundongos , Testes de Neutralização , Engenharia de Proteínas , Proteínas do Envelope Viral/imunologiaRESUMO
We describe a novel approach to assess toxicity to the free-living nematode Caenorhabditis elegans that relies on the ability of firefly luciferase to report on endogenous ATP levels. We have constructed bioluminescent C. elegans with the luc gene under control of a constitutive promoter. Light reduction was observed in response to increasing temperature, concentrations of copper, lead and 3,5-dichlorophenol. This was due to increased mortality coupled with decreased metabolic activity in the surviving animals. The light emitted by the transgenic nematodes gave a rapid, real-time indication of metabolic status. This forms the basis of rapid and biologically relevant toxicity tests.
Assuntos
Caenorhabditis elegans/metabolismo , Biologia Molecular/métodos , Testes de Toxicidade/métodos , Trifosfato de Adenosina/metabolismo , Animais , Bioensaio , Clorofenóis/toxicidade , Besouros , Cobre/toxicidade , Relação Dose-Resposta a Droga , Poluentes Ambientais/toxicidade , Temperatura Alta , Chumbo/toxicidade , Luz , Luciferases/metabolismo , Luminescência , Regiões Promotoras Genéticas , Estresse Fisiológico , TemperaturaRESUMO
Single-chain antibody fragments against the cyanobacterial hepatotoxin microcystin-LR were isolated from a naive human phage display library and expressed in Escherichia coli. In competition enzyme-linked immunosorbent assay (ELISA), the most sensitive antibody clone selected from the library detected free microcystin-LR with an IC(50) value of 4 microM. It was found to cross react with three other microcystin variants - microcystin-RR, microcystin-LW and microcystin-LF - and detected microcystins in extracts of the cyanobacterium Microcystis aeruginosa, found to contain the toxins by high-performance liquid chromatography (HPLC). The quantification of microcystins in these extracts by ELISA and HPLC showed good correlation. Although the antibody isolated in this study was considerably less sensitive than the polyclonal and monoclonal antibodies already available for microcystin detection, phage display technology represents a cheaper, more rapid alternative for the production of anti-microcystin antibodies than the methods currently in use.