RESUMEN
Infection with Zika virus (ZIKV), a member of the Flavivirus genus of the Flaviviridae family, typically results in mild self-limited illness, but severe neurological disease occurs in a limited subset of patients. In contrast, serious outcomes commonly occur in pregnancy that affect the developing fetus, including microcephaly and other major birth defects. The genetic similarity of ZIKV to other widespread flaviviruses, such as dengue virus (DENV), presents a challenge to the development of specific ZIKV diagnostic assays. Nonstructural protein 1 (NS1) is established for use in immunodiagnostic assays for flaviviruses. To address the cross-reactivity of ZIKV NS1 with proteins from other flaviviruses we used site-directed mutagenesis to modify putative epitopes. Goat polyclonal antibodies to variant ZIKV NS1 were affinity-purified to remove antibodies binding to the closely related NS1 protein of DENV. An antigen-capture ELISA configured with the affinity-purified polyclonal antibody showed a linear dynamic range between approximately 500 and 30 ng/mL, with a limit of detection of between 1.95 and 7.8 ng/mL. NS1 proteins from DENV, yellow fever virus, St. Louis encephalitis virus and West Nile virus showed significantly reduced reactivity in the ZIKV antigen-capture ELISA. Refinement of approaches similar to those employed here could lead to development of ZIKV-specific immunoassays suitable for use in areas where infections with related flaviviruses are common.
Asunto(s)
Antígenos Virales/inmunología , Inmunoensayo/métodos , Proteínas no Estructurales Virales/inmunología , Virus Zika/inmunología , Animales , Anticuerpos Antivirales/inmunología , Reacciones Cruzadas/inmunología , Dengue/virología , Virus del Dengue/inmunología , Ensayo de Inmunoadsorción Enzimática , Epítopos , Femenino , Flavivirus , Humanos , Pruebas Inmunológicas , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Embarazo , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genética , Virus del Nilo Occidental/inmunología , Virus de la Fiebre Amarilla/inmunología , Virus Zika/genética , Virus Zika/aislamiento & purificación , Infección por el Virus Zika/diagnóstico , Infección por el Virus Zika/virologíaRESUMEN
We report on a novel solution immersed silicon (SIS) sensor modified with bio-receptor to detect toluene. To perform this approach, bio-receptor PAS1 which specifically interacts with toluene was chosen as a capture agent for SIS ellipsometric sensing. We constructed wild PAS1 and mutant PAS1 (F46A and F79Y) which are toluene binding-defective. Especially, we utilized an easily accessible capturing approach based on silica binding peptide (SBP) for direct immobilization of PAS1 on the SiO2 surfaces. After the immobilization of SBP-tagged PAS1 to the sensing layers, PAS1-based SIS sensor was evaluated for its ability to recognize toluene. As a result, a significant up-shift in Psi (Ψ) was clearly observed with a low limit of detection (LOD) of 0.1⯵M, when treated with toluene on wild PAS1-surface, but not on mutant PAS1-sensing layers, indicating the selective interactions between PAS1 and toluene molecule. The PAS1-SIS sensor showed no changes in Psi (Ψ), if any, negligible, when exposed to benzene, phenol, xylene and 4-nitrophenol as negative controls, thereby demonstrating the specificity of interaction between PAS1 and toluene. Taken together, our results strongly indicate that PAS1-modified ellipsometry sensor can provide a high fidelity system for the accurate and selective detection of toluene.
Asunto(s)
Proteínas Bacterianas/química , Técnicas Biosensibles/métodos , Proteínas Quinasas/química , Pseudomonas putida/química , Silicio/química , Tolueno/análisis , Proteínas Inmovilizadas/química , Límite de Detección , Dominios Proteicos , Proteínas Recombinantes de Fusión/química , Contaminantes Químicos del Agua/análisisRESUMEN
Enzyme-based biosensing devices have been extensively developed over the last few decades, and have proven to be innovative techniques in the qualitative and quantitative analysis of a variety of target substrates over a wide range of applications. Distinct advantages that enzyme-based biosensors provide, such as high sensitivity and specificity, portability, cost-effectiveness, and the possibilities for miniaturization and point-of-care diagnostic testing make them more and more attractive for research focused on clinical analysis, food safety control, or disease monitoring purposes. Therefore, this review article investigates the operating principle of enzymatic biosensors utilizing electrochemical, optical, thermistor, and piezoelectric measurement techniques and their applications in the literature, as well as approaches in improving the use of enzymes for biosensors.
RESUMEN
Here we report an effective method for protein immobilization on a surface plasmon resonance (SPR) gold chip, describing the combination of cysteine- and oligomerization domain-mediated immobilization of enhanced green fluorescent protein (EGFP) as a model protein for the purpose of orientation-controlled surface density packing. In order to facilitate the oligomerization of EGFP, the dimeric and trimeric constructs derived from GCN4- leucine zipper domain were chosen for multimeric EGFP assembly. For orientation-controlled immobilization of the protein, EGFP modified with cysteine residues showing excellent orientation on a gold chip was used as a starting protein, as previously reported in our earlier study (Anal. Chem., 2007, 79, 2680-2687). Constructs of EGFP with oligomerization domains were genetically engineered, and corresponding fusion proteins were purified, applied to a gold chip, and then analyzed under SPR. The immobilized EGFP density on a gold chip increased according to the states of protein oligomerization, as dimeric and trimeric EGFPs displayed better adsorption capability than monomeric and dimeric forms, respectively. Fluorescence measurement corroborated the SPR results. Taken together, our findings indicated that the combination of cysteine- and oligomerization domain-mediated immobilization of protein could be used in SPR biosensor applications, allowing for an excellent orientation and high surface density simultaneously.
Asunto(s)
Cisteína/química , Oro/química , Proteínas Fluorescentes Verdes/química , Resonancia por Plasmón de Superficie/métodos , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/química , Técnicas Biosensibles/métodos , Proteínas Inmovilizadas/química , Leucina Zippers , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/químicaRESUMEN
BACKGROUND: HIV-1 mediated perturbation of the plasma membrane can produce an alteration in the transmembrane gradients of cations and other small molecules leading to cell death. Several HIV-1 proteins have been shown to perturb membrane permeability and ion transport. Xenopus laevis oocytes have few functional endogenous ion channels, and have proven useful as a system to examine direct effects of exogenously added proteins on ion transport. RESULTS: HIV-1 Nef induces alterations in the intracellular potassium concentration in CD4+ T-lymphoblastoid cells, but not intracellular pH. Two electrode voltage-clamp recording was used to determine that Nef did not form ion channel-like pores in Xenopus oocytes. CONCLUSION: These results suggest that HIV-1 Nef regulates intracellular ion concentrations indirectly, and may interact with membrane proteins such as ion channels to modify their electrical properties.
Asunto(s)
Permeabilidad de la Membrana Celular/efectos de los fármacos , Líquido Intracelular/metabolismo , Potasio/metabolismo , Proteínas Reguladoras y Accesorias Virales/farmacología , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/farmacología , Animales , Línea Celular Tumoral , Humanos , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Líquido Intracelular/efectos de los fármacos , Transporte Iónico/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Proteínas Recombinantes/farmacología , Xenopus laevisRESUMEN
BACKGROUND: CXC chemokine receptor 4 (CXCR4), a member of the G-protein-coupled chemokine receptor family, can serve as a co-receptor along with CD4 for entry into the cell of T-cell tropic X4 human immunodeficiency virus type 1 (HIV-1) strains. Productive infection of T-lymphoblastoid cells by X4 HIV-1 markedly reduces cell-surface expression of CD4, but whether or not the co-receptor CXCR4 is down-regulated has not been conclusively determined. RESULTS: Infection of human T-lymphoblastoid cell line RH9 with HIV-1 resulted in down-regulation of cell surface CXCR4 expression. Down-regulation of surface CXCR4 correlated temporally with the increase in HIV-1 protein expression. CXCR4 was concentrated in intracellular compartments in H9 cells after HIV-1 infection. Immunofluorescence microscopy studies showed that CXCR4 and HIV-1 glycoproteins were co-localized in HIV infected cells. Inducible expression of HIV-1 envelope glycoproteins also resulted in down-regulation of CXCR4 from the cell surface. CONCLUSION: These results indicated that cell surface CXCR4 was reduced in HIV-1 infected cells, whereas expression of another membrane antigen, CD3, was unaffected. CXCR4 down-regulation may be due to intracellular sequestering of HIV glycoprotein/CXCR4 complexes.
Asunto(s)
Infecciones por VIH/virología , VIH-1/metabolismo , Receptores CXCR4/metabolismo , Línea Celular , Regulación hacia Abajo , Humanos , Membranas Intracelulares/metabolismoRESUMEN
The thyroid gland is afflicted in several endocrine, autoimmune, and malignant diseases. Previous studies detected immunoreactivity against proteins of a human intracisternal A-type retroviral particle type-I (HIAP-I) in serum samples from the majority of patients with Graves' disease, an autoimmune disease of the thyroid that can also affect other organs, most prominently the eyes. To determine whether hyperthyroid animals might provide a model for the retroviral involvement in thyroid autoimmunity, serum samples from 32 cats (21 hyperthyroid and 11 controls) and 10 hypothyroid dogs were examined for immunoreactivity with HIAP-I using a Western blot technique. Of the 21 hyperthyroid cats 15 (71.4%) were HIAP-I positive, while only 2 of 11 (11.8%) control animals without endocrine pathology were positive. No significant correlations were seen between HIAP seroreactivity and serum thyroid hormone levels (T3 and T4), age, gender, treatment history, vaccination status, or weight. No seroreactivity to HIAP-I was detected in hypothyroid dogs. An examination of HIAP-I reactivity in feline leukemia virus (FeLV)-seroconverting cats found that 7/9 (78%) animals viremic for FeLV-A showed an alteration in HIAP serology, whereas only 1/7 (14%) nonviremic animals showed a change in HIAP-I serology. These results suggest that it may be possible to develop an animal (feline) model for the role of retroviruses in thyroid autoimmune diseases.
Asunto(s)
Genes de Partícula A Intracisternal/inmunología , Hipertiroidismo/inmunología , Proteínas de los Retroviridae/inmunología , Animales , Anticuerpos Antivirales/análisis , Gatos , Perros , HumanosRESUMEN
Prior studies have linked retroviruses to various arthropathies and autoimmune diseases. Sjögren's syndrome (SS), a systemic autoimmune disease, is characterized by aggressive infiltration of lymphocytes into the salivary and lacrimal glands, resulting in destruction of the glands and dry mouth and eyes (sicca syndrome). The infiltrating lymphocytes in SS may become overtly malignant, and thus, the incidence of lymphoma is greatly increased in SS patients. A human intracisternal A-type retroviral particle type I (HIAP-I) has been isolated from persons with SS. HIAP-I shares a limited number of antigenic epitopes with human immunodeficiency virus (HIV), but is distinguishable from HIV by morphological, physical, and biochemical criteria. A substantial majority of patients with SS or systemic lupus erythematosus (SLE) have serum antibodies to the proteins of this human retrovirus. Fewer than 3% of the normal blood donor population have antibodies to any HIAP-associated proteins. A second type of a human intracisternal A-type retrovirus, HIAP-II, was detected in a subset of patients with idiopathic CD4 lymphocytopenia (ICL), an AIDS-like immunodeficiency disease. Most HIAP-II positive ICL patients were also antinuclear antibody positive. Reviewed here are additional studies from several laboratories suggesting that HIAP or related viruses may be involved in SLE and other autoimmune conditions. Additionally, results of comprehensive surveys of autoimmune patients to determine seroreactivity to HIAP, and other human retroviruses, including HIV and human T-lymphotropic virus type I, are reported.
Asunto(s)
Enfermedades Autoinmunes/virología , Autoinmunidad/inmunología , Genes de Partícula A Intracisternal/inmunología , Proteínas de los Retroviridae/inmunología , Síndrome de Sjögren/inmunología , Enfermedades Autoinmunes/etiología , Genes de Partícula A Intracisternal/fisiología , Humanos , Lupus Eritematoso Sistémico/patología , Lupus Eritematoso Sistémico/virología , Linfocitos/inmunología , Linfocitos/patología , Síndrome de Sjögren/sangre , Síndrome de Sjögren/patología , Síndrome de Sjögren/virologíaRESUMEN
The transmissible spongiform encephalopathies (TSE) that manifest as Creutzfeldt-Jakob disease in humans, as scrapie in sheep and goats, mad cow disease in cattle, or chronic wasting disease in cervids (deer) represent a serious human health crisis and a significant economical problem. Despite much research, the nature of the elusive pathogen directly involved with TSE is currently unresolved. This article reviews current pathogen-cell plasma membrane properties, showing that the primary biochemical marker of the prion disease is used as a receptor by the intracellular bacterium Brucella abortus. Such observation makes plausible the role for the prion in the pathogenesis of TSE, and supports the concept that Spiroplasma, a wall-less bacterium, may be a transmissible agent of TSE. Over the past three decades, we have published convincing evidence that Spiroplasma infection is associated with TSE. The bacterial-prion-receptor concept by other laboratories support a model for TSE wherein a Spiroplasma bacterium can bind to prion receptors (alone or with anchors) on the cell surface lipid raft, allowing entry of the microbe into the cell to initiate infection. The relevance of this new concept is that it offers a new window for future research involving a bacterium in the pathogenesis of TSE. Data from the bacterial-prion-receptor model will aid in the development diagnostic tests and/or treatment protocols for TSE.
Asunto(s)
Síndrome de Creutzfeldt-Jakob/virología , Enfermedades por Prión/virología , Priones/metabolismo , Scrapie/microbiología , Spiroplasma/metabolismo , Animales , Bovinos , Síndrome de Creutzfeldt-Jakob/metabolismo , Humanos , Enfermedades por Prión/transmisión , Priones/química , Priones/genética , Priones/patogenicidad , Conformación Proteica , Scrapie/metabolismoRESUMEN
The Dominican Republic (DR) has experienced tremendous increase in the number of cases of dengue virus (DENV) reported in the past few years. There are four serotypes of DENV (1-4), and each can cause classic dengue fever (DF), dengue hemorrhagic fever (DHF), or dengue shock syndrome (DSS). DENV-1-4 are currently circulating in the DR; however, the Department of Epidemiology of the Dominican Republic (DIEGPI) has been able to isolate only DENV-2 and DENV-4. In the DR, the development of DENV infection occurred primarily in the second semester of the year. Since the DR instituted a vigilant approach to dengue infections, there have been three major outbreaks detected: one in 1998, one in 2000, and one in 2002. DF is the clinical presentation most currently seen at clinics, accounting for about 75% of cases, while patients with DHF account for about 19% of cases seen. Currently, there are seven provinces in which the total number of dengue cases per 100,000 inhabitants is higher than 32. With a vigilant approach, the DR should continue to see success in controlling the DENV outbreak.
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Virus del Dengue/genética , Dengue/epidemiología , ADN Viral/análisis , Dengue/virología , Virus del Dengue/clasificación , Virus del Dengue/patogenicidad , Brotes de Enfermedades , República Dominicana/epidemiología , Humanos , Epidemiología MolecularRESUMEN
Certain genotypes of hepatitis C virus (HCV) respond less often than others to treatment with interferon (IFN). The mechanisms for this differential response are not known. In this report antiviral effects of IFN-alpha2b on translation were examined in a hepatic cell line using chimeric clones of internal ribosome entry site (IRES) sequences from six different HCV genotypes and the green fluorescence protein (GFP) gene. As a control, IFN action at the level of the IRES was examined in the presence of different cytokines. It was determined that IFN-alpha2b specifically inhibited the translation of GFP mediated by IRES sequences from six major HCV genotypes in a concentration-dependent manner. Other cytokines including tumour necrosis factor alpha, transforming growth factor beta 1, interleukin 1 and interleukin 6 have no inhibitory effect. The inhibition of translation in these experiments was not due to extensive intracellular degradation of IRES-GFP mRNA. These results suggest that the antiviral action of IFN-alpha2b blocks IRES-mediated translation and this effect is the same among HCVs of other genotypes.
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Hepacivirus/efectos de los fármacos , Interferón Tipo I/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , Ribonucleoproteínas/metabolismo , Ribosomas/efectos de los fármacos , Genotipo , Hepacivirus/crecimiento & desarrollo , Hepatitis C/tratamiento farmacológico , Hepatitis C/virología , Pruebas de Sensibilidad Microbiana , Ribonucleoproteínas/genética , Ribosomas/química , Ribosomas/metabolismoRESUMEN
The chemotherapeutic drug bleomycin causes DNA damage and apoptosis in the lungs of mice within hours of endotracheal instillation followed by inflammation and fibrosis weeks later. The p53 tumor suppressor protein mediates cellular responses to DNA damage, including induction of apoptosis, but the effects of p53 activation in the various cell types of the lung during bleomycin-induced pulmonary fibrosis remain unclear. We show here that a transgene with a dominant-negative mutant form of human p53 expressed from the surfactant protein C promoter sensitizes mice to bleomycin-induced lung injury. The bleomycin-exposed transgenic animals display more severe lung pathology with associated collagen deposition and more pronounced lung eosinophilia than simultaneously exposed nontransgenic littermates. These observations suggest that compromising p53 function in the alveolar epithelium impairs recovery of the lung from bleomycin-induced injury.
Asunto(s)
Antibióticos Antineoplásicos/toxicidad , Bleomicina/toxicidad , Genes p53/genética , Ratones Transgénicos/genética , Mutación , Fibrosis Pulmonar/inducido químicamente , Animales , Daño del ADN , Interpretación Estadística de Datos , Eosinófilos , Epitelio/metabolismo , Epitelio/patología , Femenino , Genes Dominantes , Humanos , Inmunohistoquímica , Pulmón/metabolismo , Pulmón/patología , Ratones , Fibrosis Pulmonar/genética , Fibrosis Pulmonar/patología , ARN/análisisRESUMEN
Dark cells (DCs) of mammalian and non-mammalian species help to maintain the homeostasis of the inner ear fluids in vivo. Although the avian cochlea is straight and the mammalian cochlea is coiled, no significant difference in the morphology and/or function of mammalian and avian DCs has been reported. The mammalian equivalent of avian DCs are marginal cells and are located in the stria vascularis along a bony sheet. Avian DCs hang free from the tegmentum vasculosum (TV) of the avian lagena between the perilymph and endolymph. Frame averaging was used to image the fluorescence emitted by several fluorochromes applied to freshly isolated dark cells (iDCs) from chickens (Gallus domesticus) inner ears. The viability of iDCs was monitored via trypan blue exclusion at each isolation step. Sodium Green, BCECF-AM, Rhodamine 123 and 9-anthroyl ouabain molecules were used to test iDC function. These fluorochromes label iDCs ionic transmembrane trafficking function, membrane electrogenic potentials and Na+/K+ ATPase pump's activity. Na+/K+ ATPase pump sites, were also evaluated by the p-nitrophenyl phosphatase reaction. These results suggest that iDCs remain viable for several hours after isolation without special culturing requirements and that the number and functional activity of Na+/K+ ATPase pumps in the iDCs were indistinguishable from in vivo DCs. Primary cultures of freshly iDCs were successfully maintained for 28 days in plastic dishes with RPMI 1640 culture medium. The preparation of iDCs overcomes the difficulty of DCs accessability in vivo and the unavoidable contamination that rupturing the inner ear microenvironments induces.