RESUMEN
The comprehensive understanding of the orientation of antibodies on a solid surface is crucial for affinity-based sensing mechanisms. In this study, we demonstrated that the orientation of primary antibodies modified on carboxy-functionalized polystyrene (PS) particles can be analyzed using zeta potential behavior at different pH based on the combined Gouy-Chapman-Stern model and the acid dissociation of carboxy groups and antibodies. We observed that at low surface concentrations of the primary antibody, a side-on orientation was predominant. However, at higher concentrations (approximately 30000 antibodies per PS particle), the orientation shifted to an end-on type due to steric hindrance. Furthermore, the reaction mechanism of the secondary antibody exhibited pH-dependent behavior. At pH > 7, the zeta potential changes were attributed to the antibody-antibody reaction, whereas at pH < 7, adsorption of secondary antibody onto the PS particle was observed, leading to a change in the orientation of the primary antibody modified on the PS particle to an end-on type. The change in zeta potential due to secondary antibody binding indicated a detection limit of 37000 antibodies per PS particle. As a result, we revealed that the analysis of zeta potential behavior enables the evaluation of antibody orientation and the detection of zeptomole order antibodies. This study represents the first demonstration of this capability. We anticipate that the present concept and results will broaden the quantitative application of zeta potential measurements and have significant implications for research areas, including physical chemistry and analytical chemistry.
Asunto(s)
Anticuerpos , Poliestirenos , Poliestirenos/química , Concentración de Iones de Hidrógeno , Anticuerpos/química , Anticuerpos/inmunología , Propiedades de Superficie , Tamaño de la PartículaRESUMEN
Tetrahydrobiopterin (BH4) is an essential cofactor for dopamine and serotonin synthesis in monoaminergic neurons, phenylalanine metabolism in hepatocytes, and nitric oxide synthesis in endothelial and immune cells. BH4 is consumed as a cofactor or is readily oxidized by autooxidation. Quinonoid dihydropteridine reductase (QDPR) is an enzyme that reduces quinonoid dihydrobiopterin (qBH2) back to BH4, and we have previously demonstrated the significance of QDPR in maintaining BH4 in vivo using Qdpr-KO mice. In addition to the levels of BH4 in the cells, the ratios of oxidized to reduced forms of BH4 are supposed to be important for regulating nitric oxide synthase (NOS) via the so-called uncoupling of NOS. However, previous studies were limited due to the absence of specific and high-affinity inhibitors against QDPR. Here, we performed a high-throughput screening for a QDPR inhibitor and identified Compound 9b with an IC50 of 0.72 µM. To understand the inhibition mechanism, we performed kinetic analyses and molecular dynamics simulations. Treatment with 9b combined with methotrexate (MTX), an inhibitor of another BH4-reducing enzyme, dihydrofolate reductase (DHFR), significantly oxidized intracellular redox states in HepG2, Jurkat, SH-SY5Y, and PC12D cells. Collectively, these findings suggest that 9b may enhance the anticancer and anti-autoimmune effects of MTX.
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Biopterinas , Dihidropteridina Reductasa , Sinergismo Farmacológico , Metotrexato , Metotrexato/farmacología , Biopterinas/análogos & derivados , Biopterinas/metabolismo , Humanos , Dihidropteridina Reductasa/metabolismo , Inhibidores Enzimáticos/farmacología , Oxidación-Reducción/efectos de los fármacos , Animales , Simulación de Dinámica MolecularRESUMEN
Background/Aim: Synchronous colorectal cancer, which occurs in approximately 4.8-8.4% of all colorectal cancers, has a genetic profile with a higher rate of v-raf murine sarcoma viral oncogene homolog B1 (BRAF) mutation and microsatellite instability-high than solitary colorectal cancer. However, little information is available on heterogeneity among tumor lesions because of difficulty in performing genetic tests in all lesions in clinical practice. Case Report: A 44-year-old man presented with multiple recurrent lung metastases 42 months after the endoscopic resection of early stage synchronous ascending and sigmoid colon cancers. The genetic testing of sigmoid colon cancer tissue samples, their state being more advanced than that of ascending colon cancer, revealed a v-Ki-ras 2 Kirsten rat sarcoma viral oncogene homolog mutation (G13C) and BRAF wild type. However, the tumor was refractory to initial chemotherapy and rapidly progressed to new liver metastases. Therefore, we suspected that there may be biological heterogeneity between the primary sigmoid colon lesion and liver metastases. Next, we performed next-generation sequencing on circulating tumor DNA from the patient's plasma (Foundation One Liquid CDx®), which revealed the V600E mutation of BRAF, suggesting that there was genetic heterogeneity among the synchronized primary lesions, one of which was responsible for the chemo-refractory rapid-growing liver metastases. Conclusion: Genetic profiling with liquid biopsy at the time of recurrence and metastasis may be useful in patients with multiple synchronous cancers because there is less heterogeneity between primary and metastatic sites.
RESUMEN
Quinonoid dihydropteridine reductase (QDPR) catalyses the reduction of quinonoid-form dihydrobiopterin (qBH2) to tetrahydrobiopterin (BH4). BH4 metabolism is a drug target for neglected tropical disorders because trypanosomatid protozoans, including Leishmania and Trypanosoma, require exogenous sources of biopterin for growth. Although QDPR is a key enzyme for maintaining intracellular BH4 levels, the precise catalytic properties and reaction mechanisms of QDPR are poorly understood due to the instability of quinonoid-form substrates. In this study, we analysed the binding profile of qBH2 to human QDPR in combination with in silico and in vitro methods. First, we performed docking simulation of qBH2 to QDPR to obtain possible binding modes of qBH2 at the active site of QDPR. Then, among them, we determined the most plausible binding mode using molecular dynamics simulations revealing its atomic-level interactions and confirmed it with the in vitro assay of mutant enzymes. Moreover, it was found that not only qBH2 but also quinonoid-form dihydrofolate (qDHF) could be potential physiological substrates for QDPR, suggesting that QDPR may be a bifunctional enzyme. These findings in this study provide important insights into biopterin and folate metabolism and would be useful for developing drugs for neglected tropical diseases.
Asunto(s)
Biopterinas , Dihidropteridina Reductasa , Humanos , Dihidropteridina Reductasa/metabolismoRESUMEN
BACKGROUND: Olanzapine treatment prevents chemotherapy-induced nausea and vomiting (CINV) in patients receiving highly emetogenic chemotherapy (HEC). However, its role in the secondary prevention of breakthrough CINV in real-world cancer care should be further evaluated. METHOD: We conducted a retrospective study on patients receiving olanzapine to prevent breakthrough CINV refractory to standard antiemetic therapy. The major outcome was improvement in CINV, defined as any downgrade in CINV symptoms, according to the Common Terminology Criteria for Adverse Events. Comprete response was defined as no symptoms in CINV, i.e., Grade 0. These outcomes were compared in the HEC versus non-HEC groups and the standard- (5 or 10 mg/day) versus low- (2.5 mg/day) dose groups. The other outcome measurement was adverse events (AEs). RESULTS: We analyzed 127 patients, including 92 women, with a median age of 50 years (range: 19-89 years). Baseline CINV severity was grade 1, 2, and 3 in 18%, 69%, and 13% of the patients, respectively. After prophylaxis with olanzapine at doses of 2.5, 5, or 10 mg/day, improvement was observed in 105 (83%) patients, with a complete response in 42 patients (33%). The improvement and complete remission rates for the HEC (n = 96) and non-HEC (n = 31) groups were 86% and 71% (p = 0.048) versus 38% and 19% (p = 0.062), respectively. The rates for the standard- (n = 98) and low- (n = 29) dose groups were 86% and 82% (p = 0.568) versus 28% and 52% (p = 0.015), respectively. Thirty-four patients (27%) experienced olanzapine-related AEs, mainly somnolence (n = 28). Grade 3 or higher AEs were not observed. CONCLUSION: Our study results support the clinical application of olanzapine for the secondary prevention of breakthrough CINV.
RESUMEN
An α-l-fucosidase (Pap-Alf) was purified from the pancreas of a starfish Patiria pectinifera by ammonium sulfate precipitation followed by several column chromatographies. The molecular mass of the purified enzyme was estimated to be 52.6â¯kDa by SDS-PAGE, although gel filtration analysis of the native enzyme suggests it exists as a homodimer in solution. The purified enzyme showed maximal activity at pH 5.0 and 70⯰C. The enzyme was highly specific toward a fucosyl-monosaccharide (Fuc-α-pNP), but it also showed activity toward 2-sulfo-Fuc-α-pNP and fucosyl-α-lactosides (Fuc-α-Galß1â4Glc-ß-pNP). We determined the primary structure of the α-l-fucosidase and validated its expression level in starfish tissue. Whole genome sequence analysis of P. pectinifera was also performed in the present study. Detailed primary structural analysis using bioinformatics tools revealed Pap-Alf lacks the C-terminal region that is otherwise conserved in all previously described α-l-fucosidases. Quantitative gene expression analysis of Pap-Alf in each tissue indicated that the expression of Pap-Alf gene in pancreas was 5-fold higher than in ovary.
Asunto(s)
Clonación Molecular , Páncreas/enzimología , alfa-L-Fucosidasa/química , alfa-L-Fucosidasa/genética , Animales , Regulación Enzimológica de la Expresión Génica/genética , Estrellas de Mar , alfa-L-Fucosidasa/metabolismoRESUMEN
A novel substrate {Galß1,4GlcNAcß1,4GlcNAc-ß-pNP [Gal(GlcNAc)2-ß-pNP]} for assaying lysozyme activity has been designed using docking simulations and enzymatic synthesis via ß-1,4-galactosyltransferase-mediated transglycosylation from UDP-Gal as the donor to (GlcNAc)2-ß-pNP as the acceptor. Hydrolysis of the synthesized Gal(GlcNAc)2-ß-pNP and related compounds using hen egg-white lysozyme (HEWL) demonstrated that the substrate was specifically cleaved to Gal(GlcNAc)2 and p-nitrophenol (pNP). A combination of kinetic studies and docking simulation was further conducted to elucidate the mode of substrate binding. The results demonstrate that Gal(GlcNAc)2-ß-pNP selectively binds to a subsite of lysozyme to liberate the Gal(GlcNAc)2 and pNP products. The work therefore describes a new colorimetric method for quantifying lysozyme on the basis of the determination of pNP liberated from the substrate.
RESUMEN
An end-modified ß-d-galactosyl chitotetraose derivative [44-O-ß-d-galactosyl-ß-tri-N-acetylchitotriosyl 2-acetamide-2,3-dideoxy-glucopyranose; Gal(GlcN)3D] was designed and synthesized from chitin tetrasaccharide. The derivative was chemically modified by dehydration of the reducing end GlcN and enzymatic addition of a Gal group to the non-reducing end GlcN. Hydrolysis of Gal(GlcN)3D and related compounds using hen egg-white lysozyme was then examined. Gal(GlcN)3D was specifically cleaved to Gal(GlcN)2 and GlcND. Kinetic studies and docking simulations were further conducted to elucidate its mode of binding to lysozyme. These analyses revealed the binding of Gal(GlcN)3D to lysozyme is more favorable than that of (GlcN)4D. We conclude the 4-O-substituted Gal group at the non-reducing end of Gal(GlcN)3D does not prohibit the action of lysozyme, but gives some affinity to the subsite (i.e. equivalent to GlcN). From these results, a new assay method for quantifying lysozyme was established by utilizing the Morgan-Elson reaction based on the generation of product D (2-acetamide-2,3-dideoxy-glucopyranose), which serves as a chromophore, formed from Gal(GlcN)3D by lysozyme through a conjugated reaction involving ß-N-acetylhexosaminidase. The assay system gave a linear dose-response curve in the range of 2-31 µg of lysozyme during a 15 min incubation. This novel assay method for the quantification of lysozyme is highly specific, sensitive, accurate and reproducible.