RESUMEN
Veterinary drugs play a crucial role in the treatment of various animal diseases. However, their residues, stemming from issues, such as withdrawal period lapses, overuse, or abuse, can jeopardize food safety and human health. This study addresses recent regulations in Korea concerning specific veterinary drugs (anacolin, ephedrine, menichlopholan, piperonyl butoxide, and etisazole HCl) and their ongoing discussions. This study aimed to validate two pre-developed methods for quantifying residues in livestock and fishery products using QuEChERS and liquid chromatography-tandem mass spectrometry. Both methods exhibited excellent linearity, recoveries (70.3-119%), and coefficient of variations (1.3-28%), along with low limits of detection and quantification (0.3-4 ng/g and 1-12 ng/g). This study is significant for its contribution to the detection of veterinary drugs in livestock and fishery products, given the limited research available on the methods for analyzing these substances.
RESUMEN
We report herein the potential of colony-stimulating factor-1 receptor (CSF1R) inhibitors as therapeutic agents in neuroinflammatory diseases, with a focus on Alzheimer's disease (AD). Employing a carefully modified scaffold, N-(4-heterocycloalkyl-2-cycloalkylphenyl)-5-methylisoxazole-3-carboxamide, we identify highly selective and potent CSF1R inhibitorsâ7dri and 7dsi. Molecular docking studies shed light on the binding modes of these key compounds within the CSF1R binding site. Remarkably, kinome-wide selectivity assessment underscores the impressive specificity of 7dri for CSF-1R. Notably, 7dri emerges as a potent CSF-1R inhibitor with favorable cellular activity and minimal cytotoxicity among the synthesized compounds. Demonstrating efficacy in inhibiting CSF1R phosphorylation in microglial cells and successfully mitigating neuroinflammation in an in vivo LPS-induced model, 7dri establishes itself as a promising antineuroinflammatory agent.
Asunto(s)
Enfermedades Neurodegenerativas , Humanos , Enfermedades Neurodegenerativas/tratamiento farmacológico , Factor Estimulante de Colonias de Macrófagos , Fosforilación , Simulación del Acoplamiento Molecular , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos , Proteínas Tirosina Quinasas Receptoras/metabolismoRESUMEN
The activation or inactivation of B-cell lymphoma-2 (Bcl-2) antagonist/killer (Bak) is critical for controlling mitochondrial outer membrane permeabilization-dependent apoptosis. Its pro-apoptotic activity is controlled by intermolecular interactions with the Bcl-2 homology 3 (BH3) domain, which is accommodated in the hydrophobic pocket of Bak. Bcl-2-interacting protein 5 (Bnip5) is a noncanonical BH3 domain-containing protein that interacts with Bak. Bnip5 is characterized by its controversial effects on the regulation of the pro-apoptotic activity of Bak. In the present study, we determined the crystal structure of Bak bound to Bnip5 BH3. The intermolecular association appeared to be typical at first glance, but we found that it is maintained by tight hydrophobic interactions together with hydrogen/ionic bonds, which accounts for their high binding affinity with a dissociation constant of 775 nM. Structural analysis of the complex showed that Bnip5 interacts with Bak in a manner similar to that of the Bak-activating pro-apoptotic factor peroxisomal testis-enriched protein 1, particularly in the destabilization of the intramolecular electrostatic network of Bak. Our structure is considered to reflect the initial point of drastic and consecutive conformational and stoichiometric changes in Bak induced by Bnip5 BH3, which helps in explaining the effects of Bnip5 in regulating Bak-mediated apoptosis.
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Proteínas Proto-Oncogénicas c-bcl-2 , Proteína Destructora del Antagonista Homólogo bcl-2 , Proteínas Proto-Oncogénicas c-bcl-2/química , Proteína Destructora del Antagonista Homólogo bcl-2/química , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Dominios Proteicos , Proteína bcl-X/metabolismo , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/metabolismo , Apoptosis/fisiologíaRESUMEN
The N-end rule pathway is a proteolytic system involving the destabilization of N-terminal amino acids, known as N-degrons, which are recognized by N-recognins. Dysregulation of the N-end rule pathway results in the accumulation of undesired proteins, causing various diseases. The E3 ligases of the UBR subfamily recognize and degrade N-degrons through the ubiquitin-proteasome system. Herein, we investigated UBR4, which has a distinct mechanism for recognizing type-2 N-degrons. Structural analysis revealed that the UBR box of UBR4 differs from other UBR boxes in the N-degron binding sites. It recognizes type-2 N-terminal amino acids containing an aromatic ring and type-1 N-terminal arginine through two phenylalanines on its hydrophobic surface. We also characterized the binding mechanism for the second ligand residue. This is the report on the structural basis underlying the recognition of type-2 N-degrons by the UBR box with implications for understanding the N-end rule pathway.
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Ubiquitina-Proteína Ligasas , Ubiquitina , Ubiquitina-Proteína Ligasas/metabolismo , Proteolisis , Ubiquitina/metabolismo , Unión Proteica , Aminoácidos/metabolismoRESUMEN
Human papillomaviruses (HPVs) can increase the proliferation of infected cells during HPV-driven abnormalities, such as cervical cancer or benign warts. To date, more than 200 HPV genotypes have been identified, most of which are classified into three major genera: Alphapapillomavirus, Betapapillomavirus, and Gammapapillomavirus. HPV genomes commonly encode two structural (L1 and L2) and seven functional (E1, E2, E4-E7, and E8) proteins. L2, the minor structural protein of HPVs, not only serves as a viral capsid component but also interacts with various human proteins during viral infection. A recent report revealed that L2 of HPV16 recruits polo-like kinase 1 (Plk1), a master regulator of eukaryotic mitosis and cell cycle progression, for the delivery of viral DNA to mitotic chromatin during HPV16 infection. In this study, we verified the direct and potent interactions between the polo-box domain (PBD) of Plk1 and PBD-binding motif (S-S-pT-P)-containing phosphopeptides derived from L2 of HPV16/HPV18 (high-risk alphapapillomaviruses), HPV5b (low-risk betapapillomavirus), and HPV4 (low-risk gammapapillomavirus). Subsequent structural determination of the Plk1 PBD bound to the HPV18 or HPV4 L2-derived phosphopeptide demonstrated that they interact with each other in a canonical manner, in which electrostatic interactions and hydrogen bonds play key roles in sustaining the complex. Therefore, our structural and biochemical data imply that Plk1 is a broad binding target of L2 of various HPV genotypes belonging to the Alpha-, Beta-, and Gammapapillomavirus genera.
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Virus del Papiloma Humano , Infecciones por Papillomavirus , Humanos , Proteínas de la Cápside/genética , Fosfopéptidos/química , Fosfopéptidos/metabolismo , Quinasa Tipo Polo 1RESUMEN
A sensitive and accurate analytical method was developed and validated to detect bambermycin, a commonly used antibiotic in animal feed and livestock. The presence of bambermycin residues in food products can pose health risks to consumers, emphasizing the need for a sensitive and accurate analytical method. A reversed-phase analytical column was utilized with a mobile phase comprising 0.005 mol/L ammonium acetate in 5% acetonitrile (A) and 0.005 mol/L ammonium acetate in 95% acetonitrile (B) to achieve effective chromatographic separation. Quantitative determination of bambermycin in various samples, including beef, pork, chicken, milk, eggs, flatfish, eel, and shrimp, was performed using ultra-high-performance liquid chromatography-tandem mass spectrometry. Sample extraction involved a mixture of methanol and a 25% ammonium hydroxide solution, followed by low-temperature purification and phospholipid removal utilizing a Phree cartridge. The method exhibited a satisfactory recovery rate ranging from 69% to 100%. Validation results demonstrated the reliability, robustness, and accuracy of the method, exhibiting good linearity, precision, and recovery. This validated method can be applied for routine analysis of bambermycin residues, assisting in the development of effective monitoring and control measures to ensure the safety of livestock and aquatic products.
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Bambermicinas , Animales , Bovinos , Cromatografía Líquida de Alta Presión/métodos , Ganado , Espectrometría de Masas en Tándem/métodos , Reproducibilidad de los Resultados , Contaminación de Alimentos/análisis , Inocuidad de los Alimentos , Acetonitrilos/química , Extracción en Fase SólidaRESUMEN
Bak is a critical executor of apoptosis belonging to the Bcl-2 protein family. Bak contains a hydrophobic groove where the BH3 domain of proapoptotic Bcl-2 family members can be accommodated, which initiates its activation. Once activated, Bak undergoes a conformational change to oligomerize, which leads to mitochondrial destabilization and the release of cytochrome c into the cytosol and eventual apoptotic cell death. In this study, we investigated the molecular aspects and functional consequences of the interaction between Bak and peroxisomal testis-specific 1 (Pxt1), a noncanonical BH3-only protein exclusively expressed in the testis. Together with various biochemical approaches, this interaction was verified and analyzed at the atomic level by determining the crystal structure of the Bak-Pxt1 BH3 complex. In-depth biochemical and cellular analyses demonstrated that Pxt1 functions as a Bak-activating proapoptotic factor, and its BH3 domain, which mediates direct intermolecular interaction with Bak, plays a critical role in triggering apoptosis. Therefore, this study provides a molecular basis for the Pxt1-mediated novel pathway for the activation of apoptosis and expands our understanding of the cell death signaling coordinated by diverse BH3 domain-containing proteins.
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Proteínas Proto-Oncogénicas c-bcl-2 , Humanos , Masculino , Apoptosis/fisiología , Proteína X Asociada a bcl-2 , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/metabolismo , Proteínas Portadoras/metabolismo , Mitocondrias/metabolismoRESUMEN
Antiapoptotic B-cell lymphoma-2 (Bcl-2) proteins suppress apoptosis by interacting with proapoptotic regulators. They commonly contain a hydrophobic groove where the Bcl-2 homology 3 (BH3) domain of Bcl-2 family members or BH3 domain-containing non-Bcl-2 family proteins can be accommodated. Peroxisomal testis-specific 1 (Pxt1) was previously identified as a male germ cell-specific protein whose overexpression causes germ cell apoptosis and infertility in male mice. Sequence and biochemical analyses also showed that human Pxt1, which is composed of 134 amino acids and is longer than mouse Pxt1 consisting of only 51 amino acids, has a BH3 domain that interacts with antiapoptotic Bcl-2 proteins, including Bcl-2 and Bcl-xL. In this study, we determined the crystal structure of Bcl-xL bound to the human Pxt1 BH3 domain. The five BH3 consensus residues are well conserved in the human Pxt1 BH3 domain and make a critical contribution to the complex formation in a canonical manner. Structural and biochemical analyses also demonstrated that Bcl-xL interacts with the BH3 domain of human Pxt1 but not with that of mouse Pxt1, and that residues 76-83 of human Pxt1, absent in mouse Pxt1, play a pivotal role in the intermolecular binding to Bcl-xL. While Bcl-xL consistently colocalized with human Pxt1 in mitochondria, it did not do so with mouse Pxt1, when expressed in HeLa cells. Collectively, these data verified that human and mouse Pxt1 differ in their binding ability to the antiapoptotic regulator Bcl-xL, which might affect their functionality in controlling apoptosis.
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Proteínas Reguladoras de la Apoptosis , Testículo , Secuencia de Aminoácidos , Aminoácidos/metabolismo , Animales , Apoptosis , Proteínas Reguladoras de la Apoptosis/metabolismo , Células HeLa , Humanos , Masculino , Ratones , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Testículo/metabolismo , Proteína bcl-X/metabolismoRESUMEN
Aberrant activation of the Nod-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome plays an essential role in multiple diseases, including Alzheimer's disease (AD) and psoriasis. We report a novel small-molecule inhibitor, NLRP3-inhibitory compound 7 (NIC7), and its derivative, which inhibit NLRP3-mediated activation of caspase 1 along with the secretion of interleukin (IL)-1ß, IL-18, and lactate dehydrogenase. We examined the therapeutic potential of NIC7 in a disease model of AD by analyzing its effect on cognitive impairment as well as the expression of dopamine receptors and neuronal markers. NIC7 significantly reversed the associated disease symptoms in the mice model. On the other hand, NIC7 did not reverse the disease symptoms in the imiquimod (IMQ)-induced disease model of psoriasis. This indicates that IMQ-based psoriasis is independent of NLRP3. Overall, NIC7 and its derivative have therapeutic prospects to treat AD or NLRP3-mediated diseases.
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Enfermedad de Alzheimer , Disfunción Cognitiva , Psoriasis , Enfermedad de Alzheimer/tratamiento farmacológico , Animales , Disfunción Cognitiva/tratamiento farmacológico , Inflamasomas , Interleucina-1beta , Ratones , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Psoriasis/inducido químicamenteRESUMEN
High-risk genotypes of human papillomaviruses (HPVs) are directly implicated in various abnormalities associated with cellular hyperproliferation, including cervical cancer. E6 is one of two oncoproteins encoded in the HPV genome, which recruits diverse PSD-95/Dlg/ZO-1 (PDZ) domain-containing human proteins through its C-terminal PDZ-binding motif (PBM) to be degraded by means of the proteasome pathway. Among the three PDZ domain-containing protein tyrosine phosphatases, protein tyrosine phosphatase non-receptor type 3 (PTPN3) and PTPN13 were identified to be recognized by HPV E6 in a PBM-dependent manner. However, whether HPV E6 associates with PTPN4, which also has a PDZ domain and functions as an apoptosis regulator, remains undetermined. Herein, we present structural and biochemical evidence demonstrating the direct interaction between the PBM of HPV16 E6 and the PDZ domain of human PTPN4 for the first time. X-ray crystallographic structure determination and binding measurements using isothermal titration calorimetry demonstrated that hydrophobic interactions in which Leu158 of HPV16 E6 plays a key role and a network of intermolecular hydrogen bonds sustain the complex formation between PTPN4 PDZ and the PBM of HPV16 E6. In addition, it was verified that the corresponding motifs from several other high-risk HPV genotypes, including HPV18, HPV31, HPV33, and HPV45, bind to PTPN4 PDZ with comparable affinities, suggesting that PTPN4 is a common target of various pathogenic HPV genotypes.
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Alphapapillomavirus , Proteínas Oncogénicas Virales , Papillomaviridae , Proteína Tirosina Fosfatasa no Receptora Tipo 4 , Proteínas Represoras , Alphapapillomavirus/química , Alphapapillomavirus/metabolismo , Humanos , Proteínas Oncogénicas Virales/química , Proteínas Oncogénicas Virales/metabolismo , Dominios PDZ , Papillomaviridae/metabolismo , Unión Proteica , Proteína Tirosina Fosfatasa no Receptora Tipo 4/química , Proteína Tirosina Fosfatasa no Receptora Tipo 4/metabolismo , Proteínas Represoras/química , Proteínas Represoras/metabolismoRESUMEN
A successful passage of the blood-brain barrier (BBB) is an essential prerequisite for the drug molecules designed to act on the central nervous system. The logarithm of blood-brain partitioning (LogBB) has served as an effective index of molecular BBB permeability. Using the three-dimensional (3D) distribution of the molecular electrostatic potential (ESP) as the numerical descriptor, a quantitative structure-activity relationship (QSAR) model termed AlphaQ was derived to predict the molecular LogBB values. To obtain the optimal atomic coordinates of the molecules under investigation, the pairwise 3D structural alignments were conducted in such a way to maximize the quantum mechanical cross correlation between the template and a target molecule. This alignment method has the advantage over the conventional atom-by-atom matching protocol in that the structurally diverse molecules can be analyzed as rigorously as the chemical derivatives with the same scaffold. The inaccuracy problem in the 3D structural alignment was alleviated in a large part by categorizing the molecules into the eight subsets according to the molecular weight. By applying the artificial neural network algorithm to associate the fully quantum mechanical ESP descriptors with the extensive experimental LogBB data, a highly predictive 3D-QSAR model was derived for each molecular subset with a squared correlation coefficient larger than 0.8. Due to the simplicity in model building and the high predictability, AlphaQ is anticipated to serve as an effective computational screening tool for molecular BBB permeability.
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Barrera Hematoencefálica/metabolismo , Redes Neurales de la Computación , Relación Estructura-Actividad Cuantitativa , Transporte Biológico , Modelos Moleculares , Preparaciones Farmacéuticas/química , Preparaciones Farmacéuticas/metabolismo , Teoría CuánticaRESUMEN
UBR box E3 ligases, also called N-recognins, are integral components of the N-degron pathway. Representative N-recognins include UBR1, UBR2, UBR4, and UBR5, and they bind destabilizing N-terminal residues, termed N-degrons. Understanding the molecular bases of their substrate recognition and the biological impact of the clearance of their substrates on cellular signaling pathways can provide valuable insights into the regulation of these pathways. This review provides an overview of the current knowledge of the binding mechanism of UBR box N-recognin/N-degron interactions and their roles in signaling pathways linked to G-protein-coupled receptors, apoptosis, mitochondrial quality control, inflammation, and DNA damage. The targeting of these UBR box N-recognins can provide potential therapies to treat diseases such as cancer and neurodegenerative diseases.
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Apoptosis , Daño del ADN , Inflamación/patología , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Humanos , Inflamación/metabolismoRESUMEN
Parabens are widely used preservatives in cosmetics and pharmaceutical products and are approved as food additives. These chemicals have been considered safe for many years. However, the literature classifies parabens as endocrine-disrupting chemicals, and an assessment of their influence on the endocrine system and systemic toxicity is important. This study explored long-term systemic toxicity, effects on the endocrine system, and toxicokinetic behavior after repeated subcutaneous administration of butylparaben to Sprague-Dawley rats. Rats were treated with vehicle (4% Tween 80) or butylparaben at dose levels of 2, 10, and 50 mg/kg/day for 13 weeks. Assessment of systemic toxicity and endocrine-disrupting effects was based on mortality; clinical signs; body weight; food and water consumption; ophthalmological findings; urinalysis; hematology and clinical biochemistry; organ weights; necropsy and histopathological findings; regularity and length of the estrous cycle; semen quality; and toxicokinetic behavior. Female uterine weight and estrous cycle, and male semen quality indicated no estrogenic effects. Butylparaben induced local irritation at the injection site in both sexes at a dose of 50 mg/kg/day, but systemic toxicity was not observed. Therefore, the no-observed-adverse-effect level of butylparaben is set at 50 mg/kg/day in rats of both sexes. Butylparaben was without endocrine system effects at this dose. Butylparaben displays dose-dependent systemic exposure up to the maximum dose of 50 mg/kg/day and repeated administration of butylparaben for 13 weeks shows no bioaccumulation.
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Parabenos/toxicidad , Conservadores Farmacéuticos/toxicidad , Toxicocinética , Animales , Relación Dosis-Respuesta a Droga , Ciclo Estral/efectos de los fármacos , Femenino , Inyecciones Subcutáneas , Masculino , Nivel sin Efectos Adversos Observados , Parabenos/administración & dosificación , Conservadores Farmacéuticos/administración & dosificación , Ratas , Ratas Sprague-Dawley , Semen/efectos de los fármacos , Factores SexualesRESUMEN
In this study, we developed a simple screening procedure for the determination of 18 anthelmintics (including benzimidazoles, macrocyclic lactones, salicylanilides, substituted phenols, tetrahydropyrimidines, and imidazothiazoles) in five animal-derived food matrices (chicken muscle, pork, beef, milk, and egg) using liquid chromatography-tandem mass spectrometry. Analytes were extracted using acetonitrile/1% acetic acid (milk and egg) and acetonitrile/1% acetic acid with 0.5 mL of distilled water (chicken muscle, pork, and beef), and purified using saturated n-hexane/acetonitrile. A reversed-phase analytical column and a mobile phase consisting of (A) 10 mM ammonium formate in distilled water and (B) methanol were used to achieve optimal chromatographic separation. Matrix-matched standard calibration curves (R 2 ≥0.9752) were obtained for concentration equivalent to ×1/2, ×1, ×2, ×3, ×4, and ×5 fold the maximum residue limit (MRL) stipulated by the Korean Ministry of Food and Drug Safety. Recoveries of 61.2-118.4%, with relative standard deviations (RSDs) of ≤19.9% (intraday and interday), were obtained for each sample at three spiking concentrations (×1/2, ×1, and ×2 the MRL values). Limits of detection, limits of quantification, and matrix effects were 0.02-5.5 µg/kg, 0.06-10 µg/kg, and -98.8 to 13.9% (at 20 µg/kg), respectively. In five samples of each food matrix (chicken muscle, pork, beef, milk, and egg) purchased from large retailers in Seoul that were tested, none of the target analytes were detected. It has therefore been shown that this protocol is adaptable, accurate, and precise for the quantification of anthelmintic residues in foods of animal origin.
RESUMEN
Protein arginylation is a critical regulator of a variety of biological processes. The ability to uncover the global arginylation pattern and its associated signaling pathways would enable us to identify novel disease targets. Here, we report the development of a tool able to capture the N-terminal arginylome. This tool, termed R-catcher, is based on the ZZ domain of p62, which was previously shown to bind N-terminally arginylated proteins. Mutating the ZZ domain enhanced its binding specificity and affinity for Nt-Arg. R-catcher pulldown coupled to LC-MS/MS led to the identification of 59 known and putative arginylated proteins. Among these were a subgroup of novel ATE1-dependent arginylated ER proteins that are linked to diverse biological pathways, including cellular senescence and vesicle-mediated transport as well as diseases, such as Amyotrophic Lateral Sclerosis and Alzheimer's disease. This study presents the first molecular tool that allows the unbiased identification of arginylated proteins, thereby unlocking the arginylome and provide a new path to disease biomarker discovery.
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Aminoaciltransferasas/metabolismo , Arginina/metabolismo , Retículo Endoplásmico/metabolismo , Vectores Genéticos/genética , Proteínas de la Membrana/metabolismo , Procesamiento Proteico-Postraduccional , Aminoaciltransferasas/química , Aminoaciltransferasas/genética , Arginina/química , Arginina/genética , Células HeLa , Humanos , Proteínas de la Membrana/genética , Especificidad por SustratoRESUMEN
Zaire ebolavirus, commonly called Ebola virus (EBOV), is an RNA virus that causes severe hemorrhagic fever with high mortality. Viral protein 35 (VP35) is a virulence factor encoded in the EBOV genome. VP35 inhibits host innate immune responses and functions as a critical cofactor for viral RNA replication. EBOV VP35 contains a short conserved motif that interacts with dynein light chain 8 (LC8), which serves as a regulatory hub protein by associating with various LC8-binding proteins. Herein, we present the crystal structure of human LC8 bound to the peptide comprising residues 67-76 of EBOV VP35. Two VP35 peptides were found to interact with homodimeric LC8 by extending the central ß-sheets, constituting a 2:2 complex. Structural analysis demonstrated that the intermolecular binding between LC8 and VP35 is mainly sustained by a network of hydrogen bonds and supported by hydrophobic interactions in which Thr73 and Thr75 of VP35 are involved. These findings were verified by binding measurements using isothermal titration calorimetry. Biochemical analyses also verified that residues 67-76 of EBOV VP35 constitute a core region for interaction with LC8. In addition, corresponding motifs from other members of the genus Ebolavirus commonly bound to LC8 but with different binding affinities. Particularly, VP35 peptides originating from pathogenic species interacted with LC8 with higher affinity than those from noninfectious species, suggesting that the binding of VP35 to LC8 is associated with the pathogenicity of the Ebolavirus species.
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Dineínas Citoplasmáticas/química , Ebolavirus/química , Proteínas de la Nucleocápside/química , Secuencia de Aminoácidos , Calorimetría , Simulación por Computador , Cristalización , Cristalografía por Rayos X , Fiebre Hemorrágica Ebola/virología , Interacciones Microbiota-Huesped , Humanos , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Mapeo de Interacción de Proteínas , Multimerización de Proteína , Proteínas Virales/química , Factores de Virulencia/químicaRESUMEN
Herein, an analytical method was developed for simultaneous determination of 12 anthelmintics (closantel, niclosamide, nitroxynil, rafoxanide, cymiazole, fluazuron, levamisole, morantel, praziquantel, pyrantel, thiophanate, and trichlorfon) in fishery products (eel, flatfish, and shrimp) using liquid-liquid extraction coupled with liquid chromatography-tandem mass spectrometry. A reversed-phase analytical column was then used to separate the analytes from various matrices. Linear matrix-matched calibration curves were generated with coefficients of determination ≥ 0.9935. Recovery rates at three spiking levels (5, 10, and 20 µg/kg) ranged between 61.58% and 119.37% with relative standard deviations ≤ 19.05%. Limits of detection were in the range of 0.3-1.6 µg/kg, whereas limits of quantification ranged between 1.0 and 5.0 µg/kg. The matrix effect was moderate with values ranging from -99.47% to 51.98%. Matrices procured from large markets tested negative for the 12 anthelmintics. The developed method proved amenable to real sample testing and can be used for simultaneous determination of target analytes in aquatic products.
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Cromatografía Líquida de Alta Presión , Explotaciones Pesqueras , Análisis de los Alimentos/métodos , Contaminación de Alimentos/análisis , Espectrometría de Masas en Tándem , Drogas Veterinarias/análisis , Residuos de Medicamentos/análisis , Límite de Detección , Extracción Líquido-Líquido , Alimentos Marinos/análisis , Factores de TiempoRESUMEN
Human papillomaviruses (HPVs) cause cellular hyperproliferation-associated abnormalities including cervical cancer. The HPV genome encodes two major viral oncoproteins, E6 and E7, which recruit various host proteins by direct interaction for proteasomal degradation. Recently, we reported the structure of HPV18 E7 conserved region 3 (CR3) bound to the protein tyrosine phosphatase (PTP) domain of PTPN14, a well-defined tumor suppressor, and found that this intermolecular interaction plays a key role in E7-driven transformation and tumorigenesis. In this study, we carried out a molecular analysis of the interaction between CR3 of HPV18 E7 and the PTP domain of PTPN21, a PTP protein that shares high sequence homology with PTPN14 but is putatively oncogenic rather than tumor-suppressive. Through the combined use of biochemical tools, we verified that HPV18 E7 and PTPN21 form a 2:2 complex, with a dissociation constant of 5 nM and a nearly identical binding manner with the HPV18 E7 and PTPN14 complex. Nevertheless, despite the structural similarities, the biological consequences of the E7 interaction were found to differ between the two PTP proteins. Unlike PTPN14, PTPN21 did not appear to be subjected to proteasomal degradation in HPV18-positive HeLa cervical cancer cells. Moreover, knockdown of PTPN21 led to retardation of the migration/invasion of HeLa cells and HPV18 E7-expressing HaCaT keratinocytes, which reflects its protumor activity. In conclusion, the associations of the viral oncoprotein E7 with PTPN14 and PTPN21 are similar at the molecular level but play different physiological roles.