Toyonobu Usuki

BACKGROUND: Desmosine and isodesmosine are crosslinking amino acids found in extracellular matrix protein elastin, which imparts elasticity to tissues such as those of the lungs and arteries. These compounds are promising biomarkers for diseases involving elastin degradation, such as chronic obstructive pulmonary disease. RESEARCH DESIGN AND METHOD: This study examined the correlation between isotope-dilution LC-MS/MS and a newly established ELISA for in vitro diagnosis using a variety of samples. RESULTS: Results of ELISA and LC-MS/MS analyses exhibited a high correlation coefficient (0.9941). However, whereas the LC-MS/MS measurements deviated approximately 2-fold from the theoretical values, the ELISA measurements ranged from 0.83 to 1.06 (avg 0.94) times the theoretical values. Precise measurement of the absorbance of synthetic desmosine revealed a molar extinction coefficient of 2403, which differed markedly from the previously reported value of 4900 in 1963. Using this value to recalculate the amount of added desmosine, the LC-MS/MS measurements were 0.68 to 0.99 (avg 0.87) times the theoretical values. CONCLUSION: Thus, the developed ELISA enables highly accurate determination of desmosine concentrations, comparable to LC-MS/MS, suggesting that ELISA is a potentially useful in vitro diagnostic tool.

BACKGROUND: Gynostemma pentaphyllum (Thunb.), a traditional adaptogenic herb, is known for its bioactive components with potential anti-cancer properties. Acute myeloid leukemia (AML) progression is significantly influenced by Feline McDonough Sarcoma (FMS)-like tyrosine kinase 3 (FLT3) signaling, while Wilms' tumor 1 (WT1) serves as a key prognostic marker. This study investigates the anti-leukemia activities of active G. pentaphyllum leaf extracts and their components, focusing on the inhibition of FLT3 and WT1 activity. METHODS: G. pentaphyllum extracts were prepared through maceration, yielding three crude fractional extracts. The cytotoxicity of the extracts was screened against various leukemia cell lines using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The most cytotoxic extract was further fractionated and purified via column chromatography. The anti-proliferative and apoptotic induction activities of the active extract and its fraction were evaluated through cell cycle and apoptosis analyses using flow cytometry. Changes in mitochondrial membrane potential (ΔΨm) were assessed by spectrofluorometry. To confirm anti-leukemia activity, the expression levels of FLT3, WT1 and apoptotic-related protein were analyzed using Western blotting. The major active compounds within the active fractions were identified and characterized using Electrospray Ionization Mass Spectrometry (ESI-MS) and Nuclear Magnetic Resonance (NMR) spectroscopy. RESULTS: The ethyl acetate fractional extract (F-EtOAc) demonstrated the highest cytotoxicity, particularly against FLT3-overexpressing EoL-1 (IC50 = 40.82 ± 0.8 µg/mL) and MV4-11 (IC50 = 35.54 ± 4.1 µg/mL) AML cell lines. Fraction F10 was identified as the most active fraction, significantly inhibited FLT3 and WT1 protein expression and induced G0/G1 cell cycle arrest in a dose-dependent manner. Additionally, F10 induced dose-dependent apoptosis through disruption of ΔΨm, p53 up-regulation and caspase-3 activation. Further purification of F10 identified dehydrovomifoliol as its major bioactive compound. CONCLUSION: These findings suggest that the ethyl acetate extract of G. pentaphyllum contains bioactive compounds with anti-leukemia potential, warranting further investigation to evaluate its efficacy against AML. CLINICAL TRIAL NUMBER: Not applicable.