RESUMO
Human cytomegalovirus (HCMV) infects the majority of the human population and represents the leading viral cause of congenital birth defects. HCMV utilizes the glycoproteins gHgLgO (Trimer) to bind to platelet-derived growth factor receptor alpha (PDGFRα) and transforming growth factor beta receptor 3 (TGFßR3) to gain entry into multiple cell types. This complex is targeted by potent neutralizing antibodies and represents an important candidate for therapeutics against HCMV. Here, we determine three cryogenic electron microscopy (cryo-EM) structures of the trimer and the details of its interactions with four binding partners: the receptor proteins PDGFRα and TGFßR3 as well as two broadly neutralizing antibodies. Trimer binding to PDGFRα and TGFßR3 is mutually exclusive, suggesting that they function as independent entry receptors. In addition, Trimer-PDGFRα interaction has an inhibitory effect on PDGFRα signaling. Our results provide a framework for understanding HCMV receptor engagement, neutralization, and the development of anti-viral strategies against HCMV.
Assuntos
Citomegalovirus/química , Glicoproteínas de Membrana/química , Proteínas do Envelope Viral/química , Internalização do Vírus , Microscopia Crioeletrônica , Citomegalovirus/fisiologia , Glicoproteínas de Membrana/metabolismo , Modelos Moleculares , Proteoglicanas/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/química , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Proteínas do Envelope Viral/metabolismoRESUMO
PURPOSE: Wet age-related macular degeneration (AMD) is a blinding retinal disease. Monthly intravitreal anti-VEGF antibody injections of bevacizumab (off-label) and ranibizumab (FDA approved) are the standard of care. Antibody aggregation may interfere with ocular absorption/distribution. This study assessed topical delivery of dilute antibodies to the posterior segment of rabbit eyes using a novel anti-aggregation formula (AAF). METHODS: Bevacizumab, or biosimilar ranibizumab was diluted to 5 mg/ml in AAF. All rabbits were dosed twice daily. Substudy 1 rabbits (bevacizumab, 100 µl eye drops): Group 1 (bevacizumab/AAF, n = 6); Group 2 (bevacizumab/PBS, n = 7) and Vehicle control (AAF, n = 1). Substudy 2 rabbits (ranibizumab biosimilar/AAF, 50 µl eye drops): (ranibizumab biosimilar/AAF, n = 8). At 14.5 days, serum was drawn from rabbits. Aqueous, vitreous and retina samples were recovered from eyes and placed into AAF aliquots. Tissue analyzed using AAF as diluent. RESULTS: Bevacizumab in AAF permeated/accumulated in rabbit aqueous, vitreous and retina 10 times more, than when diluted in PBS. AAF/0.1% hyaluronic acid eye drops, dosed twice daily, provided mean tissue concentrations (ng/g) in retina (29.50), aqueous (12.34), vitreous (3.46), and serum (0.28 ng/ml). Additionally, the highest concentration (ng/g) of ranibizumab biosimilar was present in the retina (18.0), followed by aqueous (7.82) and vitreous (1.47). Serum concentration was negligible (< 0.04 ng/ml). No irritation was observed throughout the studies. CONCLUSIONS: Bevacizumab and ranibizumab, in an AAF diluent eye drop, can be delivered to the retina, by the twice daily dosing of a low concentration mAb formulation. This may prove to be an adjunct to intravitreal injections.
Assuntos
Bevacizumab , Soluções Oftálmicas , Ranibizumab , Retina , Animais , Ranibizumab/administração & dosagem , Ranibizumab/farmacocinética , Coelhos , Bevacizumab/administração & dosagem , Bevacizumab/farmacocinética , Soluções Oftálmicas/administração & dosagem , Retina/metabolismo , Retina/efeitos dos fármacos , Inibidores da Angiogênese/administração & dosagem , Inibidores da Angiogênese/farmacocinética , Corpo Vítreo/metabolismo , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Injeções Intravítreas , Medicamentos Biossimilares/administração & dosagem , Medicamentos Biossimilares/farmacocinética , Degeneração Macular Exsudativa/tratamento farmacológicoRESUMO
Pathological features of alkali concentration-associated burn were studied using non-invasive anterior segment optical coherence tomography (AS-OCT) and OCT angiography (OCTA). Alkali burn was induced in C57BL/6J mice (n = 20) by placing filter paper soaked in 0.1, 0.25, 0.5, and 1 M NaOH for 30s on the right eye (left eye control). Longitudinal imaging was performed with AS-OCT/OCTA and fluorescein angiography over 14 days, after which eyes were enucleated at 7 and 14 days for histology and immunofluorescence. Concentration-associated corneal swelling was maximal at 0.5M, increasing linearly in a concentration-dependent fashion at 0.1, 0.25, and 0.5 M NaOH, to levels of 50%, 100%, and 175% of control, respectively. At 0.1M, corneal swelling and surface erosions were prominent, while at 0.25M, deep tissue damage, limbal neovascularization, and stromal haze were evident at 7 days. At 0.5M and 1M, severe exacerbation of the corneal swelling, angle closure, Descemet's membrane detachment, hyphema, and profuse central neovascularization were noted as early as day 3, which further progressed to inflammation, fibrosis, and opacity by day 7. We conclude that alkali concentration-dependent burn intensity biomarkers can be assessed by non-invasive AS-OCT/OCTA, distinguishing between mild, moderate, and severe ocular injury, with potential relevance toward clinical utilization in human eyes.
Assuntos
Queimaduras Químicas , Edema da Córnea , Animais , Biomarcadores , Queimaduras Químicas/diagnóstico por imagem , Queimaduras Químicas/patologia , Modelos Animais de Doenças , Angiofluoresceinografia/métodos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Hidróxido de Sódio/toxicidade , Tomografia de Coerência Óptica/métodosRESUMO
Two new modified Bacillus thuringiensis (Bt) proteins, Cry1Da_7 and Cry1B.868, with activity against fall armyworms (FAW), Spodoptera frugiperda (J.E. Smith), were evaluated for their potential to bind new insect receptors compared to proteins currently deployed as plant-incorporated protectants (PIPs) in row crops. Results from resistant insect bioassays, disabled insecticidal protein (DIP) bioassays, and cell-based assays using insect cells expressing individual receptors demonstrate that receptor utilizations of the newly modified Cry1Da_7 and Cry1B.868 proteins are distinct from each other and from those of commercially available Bt proteins such as Cry1F, Cry1A.105, Cry2Ab, and Vip3A. Accordingly, these two proteins target different insect proteins in FAW midgut cells and when pyramided together should provide durability in the field against this economically important pest.IMPORTANCE There is increased concern with the development of resistance to insecticidal proteins currently expressed in crop plants, especially against high-resistance-risk pests such as fall armyworm (FAW), Spodoptera frugiperda, a maize pest that already has developed resistance to Bacillus thuringiensis (Bt) proteins such as Cry1F. Lepidopteran-specific proteins that bind new insect receptors will be critical in managing current Cry1F-resistant FAW and delaying future resistance development. Results from resistant insect assays, disabled insecticidal protein (DIP) bioassays, and cell-based assays using insect cells expressing individual receptors demonstrate that target receptors of the Cry1Da_7 and Cry1B.868 proteins are different from each other and from those of commercially available Bt proteins such as Cry1F, Cry1A.105, Cry2Ab, and Vip3A. Therefore, pyramiding these two new proteins in maize will provide durable control of this economically important pest in production agriculture.
Assuntos
Proteínas de Bactérias/metabolismo , Endotoxinas/metabolismo , Proteínas Hemolisinas/metabolismo , Proteínas de Insetos/metabolismo , Resistência a Inseticidas , Spodoptera/efeitos dos fármacos , Spodoptera/metabolismo , Animais , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacologia , Endotoxinas/genética , Endotoxinas/farmacologia , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/farmacologia , Proteínas de Insetos/genética , Inseticidas/metabolismo , Inseticidas/farmacologia , Doenças das Plantas/parasitologia , Plantas Geneticamente Modificadas/parasitologia , Ligação Proteica , Spodoptera/genética , Zea mays/parasitologiaRESUMO
SIGNIFICANCE: We investigated, for safety and awareness, ultraviolet and high-energy violet light-blocking protection provided by assorted types of eyewear. Ultraviolet and high-energy violet light-filtering efficiency varied and did not correlate with price or advertised claims. Standardization of methods and specifications for lens spectral transmission evaluation is recommended. PURPOSE: Studies have linked exposure of high-energy visible blue light to effect and damage on retinal epithelial cells, photoreceptors, and ganglion cells. "Blue light" is more accurately differentiated into "high-energy visible blue-violet light" and "circadian rhythm blue-turquoise light." This study measured and compared spectral transmission of ultraviolet and high-energy violet light of low-, medium-, and high-priced sunglasses. METHODS: Sunglasses and lens blanks were obtained from the University of Texas Medical Branch Optical Shop and vendors. Groups were based on promotional, retail, designer sunglasses, or "blue blocker" lenses. The percent transmittance of ultraviolet/visible spectral scans (800 to 350 nm) was measured using an Agilent Cary 50 spectrophotometer. High-energy violet/blue light was defined as 400 to 450 nm. RESULTS: Promotional sunglasses (tinted polycarbonate) blocked 100% ultraviolet and 67 to 99.8% high-energy violet blue light. Retail sunglasses filtered out 95 to 100% ultraviolet A and 67% high-energy violet light. The tested designer sunglasses varied widely in their optical transmissibility with respect to their ultraviolet A and high-energy violet light-blocking properties, with some not blocking ultraviolet A. Clear and colorless Kodak Total Blue provided maximal high-energy violet protection, whereas clear Essilor Crizal Prevencia provided less high-energy violet blocking between 400 and 450 nm. CONCLUSIONS: The ultraviolet and high-energy violet (400 to 450 nm) light-filtering efficiency varied between sunglasses and clear lenses and did not correlate with price or advertised claims. Standardization of methods and specifications for lens spectral transmission evaluation is recommended.
Assuntos
Óculos , Filtração/instrumentação , Luz , Proteção Radiológica/instrumentação , Raios Ultravioleta , Humanos , Cristalino/efeitos da radiação , Lesões por Radiação/prevenção & controle , Proteção Radiológica/normas , Retina/efeitos da radiação , Análise EspectralRESUMO
PURPOSE: Permeation studies, with near infrared (NIR) light and anti-aggregation antibody formulation, were used to investigate the in vitro permeation of bevacizumab, ranibizumab and aflibercept through human sclera. METHODS: A vertical, spherical Franz cell diffusion apparatus was used for this scleral tissue permeation model. A photokinetic ocular drug delivery (PODD) testing device accommodated the placement of NIR LEDs above the donor chambers. An adjustable LED driver/square wave generator provided electrical energy with a variable pulse rate and pulse width modulation (duty cycle). RESULTS: Exposure to non-thermal NIR light had no effect on mAbs with regard to monomer concentration or antibody binding potential, as determined by SE-HPLC and ELISA. The optimal LED wavelength was found to be 950 nm. Duty cycle power of 5% vs 20% showed no difference in permeation. When compared to controls, the combination of non-aggregating antibody formulation and NIR illumination provided an average transscleral drug flux enhancement factor of 3X. CONCLUSION: Narrow wavelength incoherent (non-laser) light from an NIR LED source is not harmful to mAbs and can be used to enhance drug permeation through scleral tissue. The topical formulation, combined with pulsed NIR light irradiation, significantly improved scleral permeation of three anti-VEGF antibody drugs.
Assuntos
Inibidores da Angiogênese/farmacocinética , Raios Infravermelhos , Esclera/metabolismo , Administração Oftálmica , Inibidores da Angiogênese/administração & dosagem , Bevacizumab/administração & dosagem , Bevacizumab/farmacocinética , Humanos , Degeneração Macular/tratamento farmacológico , Degeneração Macular/patologia , Permeabilidade/efeitos da radiação , Ranibizumab/administração & dosagem , Ranibizumab/farmacocinética , Receptores de Fatores de Crescimento do Endotélio Vascular/administração & dosagem , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/farmacocinética , Esclera/efeitos da radiação , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
PURPOSE: Studies were conducted to investigate dilute solutions of the monoclonal antibody (mAb) bevacizumab, mAb fragment ranibizumab and fusion protein aflibercept, develop common procedures for formulation of low concentration mAbs and identify a stabilizing formulation for anti-VEGF mAbs for use in in vitro permeation studies. METHODS: Excipient substitutions were screened. The most stabilizing formulation was chosen. Standard dilutions of bevacizumab, ranibizumab and aflibercept were prepared in PBS, manufacturer's formulation, and the new formulation. Analysis was by SE-HPLC and ELISA. Stability, disaggregation and pre-exposure tests were studied. RESULTS: When Avastin, Lucentis and Eylea are diluted in PBS or manufacturer's formulation, there is a 40-50% loss of monomer concentration and drug activity. A formulation containing 0.3% NaCl, 7.5% trehalose, 10 mM arginine and 0.04% Tween 80 at a pH of 6.78 stabilized the mAbs and minimized the drug loss. The formulation also disaggregates mAb aggregation while preserving the activity. Degassing the formulation increases recovery. CONCLUSIONS: We developed a novel formulation that significantly stabilizes mAbs under unfavorable conditions such as low concentration or body temperature. The formulation allows for tissue permeation experimentation. The formulation also exhibits a disaggregating effect on mAbs, which can be applied to the manufacture/packaging of mAbs and bioassay reagents.
Assuntos
Inibidores da Angiogênese/química , Produtos Biológicos/química , Composição de Medicamentos/métodos , Excipientes/química , Bevacizumab/química , Bioensaio/métodos , Estabilidade de Medicamentos , Agregados Proteicos , Ranibizumab/química , Receptores de Fatores de Crescimento do Endotélio Vascular/química , Proteínas Recombinantes de Fusão/química , Soluções , TemperaturaRESUMO
Amino acids serve as transport forms for organic nitrogen in the plant, and multiple transport steps are involved in cellular import and export. While the nature of the export mechanism is unknown, overexpression of GLUTAMINE DUMPER1 (GDU1) in Arabidopsis (Arabidopsis thaliana) led to increased amino acid export. To gain insight into GDU1's role, we searched for ethyl-methanesulfonate suppressor mutants and performed yeast-two-hybrid screens. Both methods uncovered the same gene, LOSS OF GDU2 (LOG2), which encodes a RING-type E3 ubiquitin ligase. The interaction between LOG2 and GDU1 was confirmed by glutathione S-transferase pull-down, in vitro ubiquitination, and in planta coimmunoprecipitation experiments. Confocal microscopy and subcellular fractionation indicated that LOG2 and GDU1 both localized to membranes and were enriched at the plasma membrane. LOG2 expression overlapped with GDU1 in the xylem and phloem tissues of Arabidopsis. The GDU1 protein encoded by the previously characterized intragenic suppressor mutant log1-1, with an arginine in place of a conserved glycine, failed to interact in the multiple assays, suggesting that the Gdu1D phenotype requires the interaction of GDU1 with LOG2. This hypothesis was supported by suppression of the Gdu1D phenotype after reduction of LOG2 expression using either artificial microRNAs or a LOG2 T-DNA insertion. Altogether, in accordance with the emerging bulk of data showing membrane protein regulation via ubiquitination, these data suggest that the interaction of GDU1 and the ubiquitin ligase LOG2 plays a significant role in the regulation of amino acid export from plant cells.
Assuntos
Aminoácidos/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Proteínas de Membrana/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Substituição de Aminoácidos/genética , Proteínas de Arabidopsis/química , Membrana Celular/enzimologia , DNA Bacteriano/genética , Genes Supressores , Glucuronidase/metabolismo , Proteínas de Membrana/genética , MicroRNAs/metabolismo , Microssomos/enzimologia , Mutação/genética , Ácido Mirístico/metabolismo , Fenótipo , Feixe Vascular de Plantas/enzimologia , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Frações Subcelulares/enzimologia , Supressão Genética , Ubiquitina-Proteína Ligases/química , UbiquitinaçãoRESUMO
Human cytomegalovirus (HCMV) represents the viral leading cause of congenital birth defects and uses the gH/gL/UL128-130-131A complex (Pentamer) to enter different cell types, including epithelial and endothelial cells. Upon infection, Pentamer elicits the most potent neutralizing response against HCMV, representing a key vaccine candidate. Despite its relevance, the structural basis for Pentamer receptor recognition and antibody neutralization is largely unknown. Here, we determine the structures of Pentamer bound to neuropilin 2 (NRP2) and a set of potent neutralizing antibodies against HCMV. Moreover, we identify thrombomodulin (THBD) as a functional HCMV receptor and determine the structures of the Pentamer-THBD complex. Unexpectedly, both NRP2 and THBD also promote dimerization of Pentamer. Our results provide a framework for understanding HCMV receptor engagement, cell entry, antibody neutralization, and outline strategies for antiviral therapies against HCMV.
RESUMO
Purpose: Chemical burns due to alkalis cause extensive damage to the ocular surface leading to blindness. Assessment of ocular burn could be challenging due to severe opacity, inflammation, and angiogenesis. Anterior segment optical coherence tomography (AS-OCT) and OCT angiography (OCTA) may provide fast, non-invasive deep tissue visualization of pathology with high sensitivity in conjunction with slit-lamp analysis. Methods: C57-BL/6J mice were anesthetized with ketamine/dexmedetomidine, and corneal alkali burn was induced (n = 6) by placing filter paper soaked in 1-M sodium hydroxide for 30 seconds on the right eye while the left eye was kept as control. Longitudinal imaging was done with AS-OCT/OCTA and fluorescein angiography at various time intervals for 14 days. Results: AS-OCT showed characteristic pathological changes in alkali-burned eyes with high sensitivity. Although OCT/OCTA showed three-dimensional and cross-sectional views of the anterior chamber and angiogenesis, fluorescein angiography showed nascent vessels with active leakage. Corneal swelling progressively increased by 125.26% on day 12 with a high prevalence of epithelial bullae, stromal cysts, stromal splitting, and Descemet's membrane detachment. Neovascularization was noted as early as day 4 in the burned eyes by both methods. Severe corneal opacity and anterior chamber inflammation were also detected by AS-OCT/OCTA. Conclusions: AS-OCT/OCTA is a promising, noninvasive, high-resolution imaging modality that can provide both qualitative and quantitative information regarding deep tissue pathology at a structural level. Translational Relevance: Noninvasive AS-OCT/OCTA and fluorescein methods show promise in clinical pathology evaluation for ocular injury management and prognostic indications, as the early presence of Descemet's membrane detachment and corneal swelling appears to be correlated with the severity and localization of corneal neovascularization.
Assuntos
Álcalis , Tomografia de Coerência Óptica , Animais , Córnea/diagnóstico por imagem , Estudos Transversais , Angiofluoresceinografia , CamundongosRESUMO
Epigenetic gene silencing suppresses transposon activity and is critical for normal development . Two common epigenetic gene-silencing marks are DNA methylation and histone H3 lysine 9 dimethylation (H3K9me2). In Arabidopsis thaliana, H3K9me2, catalyzed by the methyltransferase KRYPTONITE (KYP/SUVH4), is required for maintenance of DNA methylation outside of the standard CG sequence context. Additionally, loss of DNA methylation in the met1 mutant correlates with a loss of H3K9me2. Here we show that KYP-dependent H3K9me2 is found at non-CG methylation sites in addition to those rich in CG methylation. Furthermore, we show that the SRA domain of KYP binds directly to methylated DNA, and SRA domains with missense mutations found in loss-of-function kyp mutants have reduced binding to methylated DNA in vitro. These data suggest that DNA methylation is required for the recruitment or activity of KYP and suggest a self-reinforcing loop between histone and DNA methylation. Lastly, we found that SRA domains from two Arabidopsis SRA-RING proteins also bind methylated DNA and that the SRA domains from KYP and SRA-RING proteins prefer methylcytosines in different sequence contexts. Hence, unlike the methyl-binding domain (MBD), which binds only methylated-CpG sequences, the SRA domain is a versatile new methyl-DNA-binding motif.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Metilação de DNA , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Estrutura Terciária de Proteína , Sequência de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Sequência de Bases , Imunoprecipitação da Cromatina , Imunofluorescência , Histona-Lisina N-Metiltransferase/genética , Dados de Sequência Molecular , Mutação de Sentido Incorreto/genética , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
OBJECTIVES: Inhalation injury contributes to the morbidity and mortality of burn victims. In humans and in an ovine model of combined smoke inhalation and burn injury, bronchospasm and acute airway obstruction contribute to progressive pulmonary insufficiency. This study tests the hypothesis that muscarinic receptor antagonist therapy with tiotropium bromide, an M1 and M3 muscarinic receptor antagonist, will decrease the airway constrictive response and acute bronchial obstruction to improve pulmonary function compared to injured animals without treatment. DESIGN: Randomized, prospective study involving 32 sheep. SETTING: Large-animal intensive care research laboratory. INTERVENTIONS: The study consisted of six groups: a sham group (n=4, instrumented noninjured), a control group (n=6, injured and not treated), and tiotropium bromide-treated groups, including both preinjury and postinjury nebulization protocols. Treatments for these groups included nebulization with 36 µg of tiotropium bromide 1 hr before injury (n=6) and postinjury nebulization protocols of 18 µg (n=6), 36 µg (n=6), and 72 µg (n=4) administered 1 hr after injury. All treated groups received an additional 14.4 µg every 4 hrs for the 24-hr study period. MAIN RESULTS: Pretreatment with tiotropium bromide significantly attenuated the increases in ventilatory pressures, pulmonary dysfunction, and upper airway obstruction that occur after combined smoke inhalation and burn injury. Postinjury treatments with tiotropium bromide were as effective as pretreatment in preventing pulmonary insufficiency, although a trend toward decreased obstruction was present only in all post-treatment conditions. There was no improvement noted in pulmonary function in animals that received a higher dose of tiotropium bromide. CONCLUSIONS: This study describes a contribution of acetylcholine to the airway constrictive and lumenal obstructive response after inhalation injury and identifies low-dose nebulization of tiotropium bromide as a potentially efficacious therapy for burn patients with severe inhalation injury.
Assuntos
Antagonistas Muscarínicos/farmacologia , Síndrome do Desconforto Respiratório/tratamento farmacológico , Derivados da Escopolamina/farmacologia , Lesão por Inalação de Fumaça/tratamento farmacológico , Obstrução das Vias Respiratórias/tratamento farmacológico , Obstrução das Vias Respiratórias/etiologia , Obstrução das Vias Respiratórias/patologia , Animais , Líquido da Lavagem Broncoalveolar , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Escala de Gravidade do Ferimento , Troca Gasosa Pulmonar , Distribuição Aleatória , Valores de Referência , Síndrome do Desconforto Respiratório/etiologia , Testes de Função Respiratória , Fatores de Risco , Ovinos , Carneiro Doméstico , Lesão por Inalação de Fumaça/complicações , Lesão por Inalação de Fumaça/fisiopatologia , Estatísticas não Paramétricas , Brometo de Tiotrópio , Resultado do TratamentoRESUMO
Cluster of differentiation 20 (CD20) is a B cell membrane protein that is targeted by monoclonal antibodies for the treatment of malignancies and autoimmune disorders but whose structure and function are unknown. Rituximab (RTX) has been in clinical use for two decades, but how it activates complement to kill B cells remains poorly understood. We obtained a structure of CD20 in complex with RTX, revealing CD20 as a compact double-barrel dimer bound by two RTX antigen-binding fragments (Fabs), each of which engages a composite epitope and an extensive homotypic Fab:Fab interface. Our data suggest that RTX cross-links CD20 into circular assemblies and lead to a structural model for complement recruitment. Our results further highlight the potential relevance of homotypic Fab:Fab interactions in targeting oligomeric cell-surface markers.
Assuntos
Antígenos CD20/química , Rituximab/química , Antígenos CD20/imunologia , Proteínas do Sistema Complemento/imunologia , Microscopia Crioeletrônica , Humanos , Fragmentos Fab das Imunoglobulinas/química , Conformação Proteica , Multimerização Proteica , Rituximab/imunologiaRESUMO
Appropriate methylation of genomes is essential for gene regulation. Here, we describe the six-member ORTHRUS (ORTH) gene family of Arabidopsis thaliana that plays a role in DNA methylation in vivo. ORTH1- ORTH5 are predicted to encode proteins that contain one plant homeodomain (PHD), two really interesting new gene (RING) domains, and one set ring associated (SRA) domain, whereas ORTHlike-1 encodes a protein with only one RING and SRA domain. cDNAs for ORTH1, ORTH2, ORTH5 and ORTHlike-1 were isolated, and when expressed as glutathione-S-transferase (GST) fusion proteins, were capable of promoting ubiquitylation in vitro with the E2 AtUBC11. ORTH1 promotes ubiquitylation when paired with additional AtUBC8 family members. ORTH1 proteins with substitutions in metal-ligand binding residues in each ORTH1 RING domain individually, and ORTH1 truncation derivatives lacking one or both RING domains, were tested for their ability to catalyze ubiquitylation in vitro. In these assays, either ORTH1 RING domain is capable of promoting ubiquitylation. The PHD alone is not active as an E3 ligase, nor is it required for ligase activity. GFP-ORTH1 and GFP-ORTH2 are nuclear-localized in transgenic Arabidopsis plants. Overexpression of ORTH1 or ORTH2 in Arabidopsis leads to an altered flowering time. Inspection of DNA methylation at FWA and Cen180 repeats revealed hypomethylation when ORTH proteins were overexpressed. Once initiated, a late-flowering phenotype persisted in the absence of the ORTH transgene, consistent with epigenetic effects at FWA. We conclude that ORTH proteins are E3 ligases mediating DNA methylation status in vivo.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Clonagem Molecular , DNA Complementar/genética , Proteínas de Ligação a DNA/genética , Eucromatina/metabolismo , Flores/genética , Flores/crescimento & desenvolvimento , Flores/metabolismo , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Heterocromatina/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Família Multigênica , Mutagênese Sítio-Dirigida , Fenótipo , Filogenia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo , Domínios RING Finger , RNA de Plantas/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
The expression analysis of recombinant proteins is a challenging step in any high-throughput protein production pipeline. Often multiple expression systems and a variety of expression construct designs are considered for the production of a protein of interest. There is a strong need to triage constructs rapidly and systematically. This chapter describes a semiautomated method for the simultaneous purification and characterization of proteins expressed from multiple samples of expression cultures from the E. coli, baculovirus expression vector system, and mammalian transient expression systems. This method assists in the selection of the most promising expression construct(s) or the most favorable expression condition(s) to move forward into large-scale protein production.
Assuntos
Proteínas Recombinantes/metabolismo , Animais , Baculoviridae/genética , Baculoviridae/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Recombinantes/genéticaRESUMO
Fire accident victims who sustain both thermal injury to skin and smoke inhalation have gross evidence of systemic and pulmonary oxidant damage and acute lung injury. We hypothesized that gamma-tocopherol (gT), a reactive O(2) and N(2) scavenger, when delivered into the airway, would attenuate lung injury induced by burn and smoke inhalation. Acute lung injury was induced in chronically prepared, anesthetized sheep by 40% total burn surface area, third-degree skin burn and smoke insufflation (48 breaths of cotton smoke, <40 degrees C). The study groups were: (1) Sham (not injured, flaxseed oil (FO)-nebulized, n=6); (2) SA-neb (injured, saline-nebulized, n=6); (3) FO-neb (injured, FO-nebulized, n=6); and (4) gT+FO-neb (injured, gT and FO-nebulized, n=6). Nebulization was started 1 h postinjury, and 24 ml of FO with or without gT (51 mg/ml) was delivered into airways over 47 h using our newly developed lipid aerosolization device (droplet size: 2.5-5 microm). The burn- and smoke inhalation-induced pathological changes seen in the saline group were attenuated by FO nebulization; gT addition further improved pulmonary function. Pulmonary gT delivery along with a FO source may be a novel effective treatment strategy in management of patients with acute lung injury.
Assuntos
Encéfalo/fisiopatologia , Testes de Função Respiratória , Ovinos/fisiologia , Lesão por Inalação de Fumaça/fisiopatologia , gama-Tocoferol/administração & dosagem , Aerossóis , Animais , Encéfalo/enzimologia , Encéfalo/metabolismo , Feminino , Imuno-Histoquímica , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Malondialdeído/metabolismo , Nebulizadores e Vaporizadores , Poli(ADP-Ribose) Polimerases/metabolismo , RNA Mensageiro/genética , Lesão por Inalação de Fumaça/enzimologia , Lesão por Inalação de Fumaça/metabolismoRESUMO
We have developed a simple method for isolating and purifying plasma membrane proteins from various cell types. This one-step affinity-chromatography method uses the property of the lectin concanavalin A (ConA) and the technique of magnetic bead separation to obtain highly purified plasma membrane proteins from crude membrane preparations or cell lines. ConA is immobilized onto magnetic beads by binding biotinylated ConA to streptavidin magnetic beads. When these ConA magnetic beads were used to enrich plasma membranes from a crude membrane preparation, this procedure resulted in 3.7-fold enrichment of plasma membrane marker 5'-nucleotidase activity with 70% recovery of the activity in the crude membrane fraction of rat liver. In agreement with the results of 5'-nucleotidase activity, immunoblotting with antibodies specific for a rat liver plasma membrane protein, CEACAM1, indicated that CEACAM1 was enriched about threefold relative to that of the original membranes. In similar experiments, this method produced 13-fold enrichment of 5'-nucleotidase activity with 45% recovery of the activity from a total cell lysate of PC-3 cells and 7.1-fold enrichment of 5'-nucleotidase activity with 33% recovery of the activity from a total cell lysate of HeLa cells. These results suggest that this one-step purification method can be used to isolate total plasma membrane proteins from tissue or cells for the identification of membrane biomarkers.
Assuntos
Bioquímica/métodos , Membrana Celular/metabolismo , Concanavalina A/metabolismo , Magnetismo , Proteínas de Membrana/isolamento & purificação , Microesferas , Animais , Linhagem Celular Tumoral , Humanos , Extratos Hepáticos/metabolismo , Proteínas de Membrana/metabolismo , Ratos , Estreptavidina/metabolismoRESUMO
The use of Bt proteins in crops has revolutionized insect pest management by offering effective season-long control. However, field-evolved resistance to Bt proteins threatens their utility and durability. A recent example is field-evolved resistance to Cry1Fa and Cry1A.105 in fall armyworm (Spodoptera frugiperda). This resistance has been detected in Puerto Rico, mainland USA, and Brazil. A S. frugiperda population with suspected resistance to Cry1Fa was sampled from a maize field in Puerto Rico and used to develop a resistant lab colony. The colony demonstrated resistance to Cry1Fa and partial cross-resistance to Cry1A.105 in diet bioassays. Using genetic crosses and proteomics, we show that this resistance is due to loss-of-function mutations in the ABCC2 gene. We characterize two novel mutant alleles from Puerto Rico. We also find that these alleles are absent in a broad screen of partially resistant Brazilian populations. These findings confirm that ABCC2 is a receptor for Cry1Fa and Cry1A.105 in S. frugiperda, and lay the groundwork for genetically enabled resistance management in this species, with the caution that there may be several distinct ABCC2 resistances alleles in nature.
Assuntos
Controle de Insetos , Inseticidas/química , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Spodoptera/química , Animais , Bacillus thuringiensis/química , Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/genética , Brasil , Endotoxinas/genética , Proteínas Hemolisinas/genética , Humanos , Resistência a Inseticidas/genética , Inseticidas/efeitos adversos , Proteína 2 Associada à Farmacorresistência Múltipla , Mutação , Proteômica , Porto Rico , Spodoptera/genética , Estados UnidosRESUMO
PURPOSE: To investigate light-enhanced molecular movement as a potential technology for drug delivery. To do this, we developed an in vitro eye model while representing similar concentration gradient conditions and compositions found in the eye. METHODS: The eye model unit was fabricated by inserting a cross-linked type I collagen membrane in a spectrophotometer cuvette with 1% hyaluronic acid as the drug recipient medium. Photokinetic delivery was studied by illuminating 1 mg/mL methotrexate (MTX) placed in the drug donor compartment on top of the membrane, with noncoherent 450 nm light at 8.2 mW from an LED source pulsed at 25 cycles per second, placed in contact with the solution. A modified UV-visual spectrophotometer was employed to rapidly determine the concentration of MTX, at progressive 1 mm distances away from the membrane, within the viscous recipient medium of the model eye after 1 h. RESULTS: A defined, progressive concentration gradient was observed within the nonagitated drug recipient media, diminishing with greater distances from the membrane. Transport of MTX through the membrane was significantly enhanced (ranging from 2 to 3 times, P < 0.05 to P ≤ 0.001) by photokinetic methods compared with control conditions by determining drug concentrations at 4 defined distances from the membrane. According to scanning electron microscopy images, no structural damage or shunts were created on the surface of the cross-linked gelatin membrane. CONCLUSION: The application of pulsed noncoherent visible light significantly enhances the permeation of MTX through a cross-linked collagen membrane and hyaluronic acid recipient medium without causing structural damage to the membrane.
Assuntos
Sistemas de Liberação de Medicamentos , Olho/metabolismo , Imunossupressores/administração & dosagem , Metotrexato/administração & dosagem , Transporte Biológico , Colágeno Tipo I/metabolismo , Gelatina/metabolismo , Ácido Hialurônico/metabolismo , Imunossupressores/farmacocinética , Cinética , Luz , Metotrexato/farmacocinética , Microscopia Eletrônica de Varredura , Permeabilidade , Fotoquímica , Espectrofotometria Ultravioleta/métodosRESUMO
Plant LOSS OF GDU 2 (LOG2) and Mammalian Mahogunin Ring Finger 1 (MGRN1) proteins are RING-type E3 ligases sharing similarity N-terminal to the RING domain. Deletion of this region disrupts the interaction of LOG2 with the plant membrane protein GLUTAMINE DUMPER1 (GDU1). Phylogenetic analysis identified two clades of LOG2/MGRN1-like proteins in vertebrates and plants. The ability of MGRN1 to functionally replace LOG2 was tested. MGRN1 ubiquitylates GDU1 in vitro and can partially substitute for LOG2 in the plant, partially restoring amino acid resistance to a GDU1-myc over-expression, log2-2 background. Altogether, these results suggest a conserved function for the N-terminal domain in evolution.