Journal: The Journal of Biological Chemistry

Article Title: 4-Methylumbelliferyl glucuronide contributes to hyaluronan synthesis inhibition

doi: 10.1074/jbc.RA118.006166

Figure Lengend Snippet: 4-MUG, a metabolite of 4-MU, inhibits HA synthesis. A, molecular structures for 4-MU and its primary metabolites, 4-MUG and 4-MUS. B, concentrations of 4-MU and its metabolites in plasma of animals fed 4-MU chow for 2 weeks, measured via HPLC. n = 3 animals per group. C, different concentrations of 4-MU and 4-MUG in the serum of mice fed 4-MU for 2 weeks measured via HPLC. n = 3 animals per group. D and E, HA production by B16F10 cells cultured for 48 h in (D) 4-MU or (E) 4-MUG. F, representative images of HA staining in B16F10 cells cultured in DMSO as control (left), 4-MU (middle), or 4-MUG (right). Scale bar = 50 μm. Data represent mean ± S.E.; *, p < 0.05 by unpaired t test or one way analysis of variance (ANOVA) with Bonferroni post test.

Article Snippet: We previously determined that this chow formulation delivers 250 mg/mouse/day, yielding a serum drug concentration of 640.3 ± 17.2 nmol/liter in mice, as measured by HPLC-MS. 4-MUG (ChemImpex) was distributed in the drinking water at a concentration of 2 mg/ml, delivering 10 mg/mouse/day, yielding a serum drug concentration of 357.1 ± 72.6 ng/ml in mice, as measured by LC-MS/MS.

Techniques: Cell Culture, Staining

Journal: The Journal of Biological Chemistry

Article Title: 4-Methylumbelliferyl glucuronide contributes to hyaluronan synthesis inhibition

doi: 10.1074/jbc.RA118.006166

Figure Lengend Snippet: 4-MUG is converted into 4-MU in vitro. A, fluorescence visualization in wells of a 96-well plate which was filled with 200 μl PBS and 10% FCS. In some wells 4-MU (middle) and 4-MUG (right) were added; control wells remained untreated (left). B, 4-MU and 4-MUG were separately added to DMEM and their fluorescent signal over time was measured as mean fluorescent intensity (MFI). Fluorescent values of 4-MUG were normalized to the 4-MU fluorescence. C, fluorescence of 4-MU and 4-MUG from B16F10 cells incubated for 24, 48, or 72 h with 4-MU and 4-MUG examined using flow cytometry. D, fluorescence of 4-MU and 4-MUG signal from 4-MU– and 4-MUG–treated B16F10 cells pre- and post-permeabilization was examined by flow cytometry.

Article Snippet: We previously determined that this chow formulation delivers 250 mg/mouse/day, yielding a serum drug concentration of 640.3 ± 17.2 nmol/liter in mice, as measured by HPLC-MS. 4-MUG (ChemImpex) was distributed in the drinking water at a concentration of 2 mg/ml, delivering 10 mg/mouse/day, yielding a serum drug concentration of 357.1 ± 72.6 ng/ml in mice, as measured by LC-MS/MS.

Techniques: In Vitro, Fluorescence, Incubation, Flow Cytometry

Journal: The Journal of Biological Chemistry

Article Title: 4-Methylumbelliferyl glucuronide contributes to hyaluronan synthesis inhibition

doi: 10.1074/jbc.RA118.006166

Figure Lengend Snippet: 4-MUG fluorescence can be detected in tissues via 2-photon imaging. A and B, representative 2-photon images of muscle tissue from 4-MU–treated mice (A) and 4-MUG–treated mice (B) show a specific signal in the 4-MU channel at a wavelength of 810 nm. C, representative 2-photon images of muscle tissue from untreated control mice (upper part) and 4-MU treated mice (lower part). D, representative 2-photon images of muscle tissue from 4-MU treated mice, where the muscle tissue from one mouse (upper part) was hyaluronidase digested. Untreated muscle tissue from a 4-MU treated mouse (lower part) serves as control. Upper and lower parts are indicated via a dashed line drawn in the picture. In each of those tissues 4-MU has a specific distribution as shown at 810 nm wavelength. Collagen was visualized at 920 nm, the 4-MU and collagen channel were merged for better structural orientation in the tissue. Scale bar = 100 μm.

Article Snippet: We previously determined that this chow formulation delivers 250 mg/mouse/day, yielding a serum drug concentration of 640.3 ± 17.2 nmol/liter in mice, as measured by HPLC-MS. 4-MUG (ChemImpex) was distributed in the drinking water at a concentration of 2 mg/ml, delivering 10 mg/mouse/day, yielding a serum drug concentration of 357.1 ± 72.6 ng/ml in mice, as measured by LC-MS/MS.

Techniques: Fluorescence, Imaging

Journal: The Journal of Biological Chemistry

Article Title: 4-Methylumbelliferyl glucuronide contributes to hyaluronan synthesis inhibition

doi: 10.1074/jbc.RA118.006166

Figure Lengend Snippet: 4-MU and 4-MUG concentrations in serum and organs from 4-MU and 4-MUG treated mice. A, C, E, G, and I, 4-MU and 4-MUG concentrations were analyzed in the serum (A), pancreas (C), fat (E), liver (G), and muscle (I) from untreated control mice and 4-MU– and 4-MUG–treated mice using LC-MS/MS. n = 3–5 mice per group. B, D, F, H, and J, calculated molar ratio of 4-MU and 4-MUG from serum (B), pancreas (D), fat (F), liver (H), and muscle (J) in the different treatment groups. Data represent mean ± S.E.

Article Snippet: We previously determined that this chow formulation delivers 250 mg/mouse/day, yielding a serum drug concentration of 640.3 ± 17.2 nmol/liter in mice, as measured by HPLC-MS. 4-MUG (ChemImpex) was distributed in the drinking water at a concentration of 2 mg/ml, delivering 10 mg/mouse/day, yielding a serum drug concentration of 357.1 ± 72.6 ng/ml in mice, as measured by LC-MS/MS.

Techniques: Liquid Chromatography with Mass Spectroscopy

Journal: The Journal of Biological Chemistry

Article Title: 4-Methylumbelliferyl glucuronide contributes to hyaluronan synthesis inhibition

doi: 10.1074/jbc.RA118.006166

Figure Lengend Snippet: 4-MU and 4-MUG treatment prevents progression in autoimmune diabetes and increases Treg numbers. A, representative HA staining of pancreatic tissue from untreated DORmO mice (control), DORmO mice fed 4-MU, and DORmO mice fed 4-MUG, at 12 weeks of age. B, blood glucose of untreated DORmO mice, and DORmO mice fed 4-MU and 4-MUG, beginning at 5 weeks of age, and maintained on 4-MU and 4-MUG for 15 weeks. n = 5–10 mice per group. Data represent mean ± S.E. C, representative FoxP3 staining (brown) of pancreatic islet tissue from untreated (control) and 4-MU treated DORmO mice. Original magnification, ×40. D–G, numbers of CD3+ cells, CD4+ among CD3+ cells, and Foxp3+ among CD3+/CD4+ cells, in splenocytes isolated from mice that were treated with 4-MU (0.5 mg i.p.) or 4-MUG (1 mg i.p.) daily for 14 days, as analyzed by flow cytometry. Scale bar = 20 μm. *, p < 0.05; **, p <0.01 by unpaired t test with Welch's correction. Data represent mean ± S.E.

Article Snippet: We previously determined that this chow formulation delivers 250 mg/mouse/day, yielding a serum drug concentration of 640.3 ± 17.2 nmol/liter in mice, as measured by HPLC-MS. 4-MUG (ChemImpex) was distributed in the drinking water at a concentration of 2 mg/ml, delivering 10 mg/mouse/day, yielding a serum drug concentration of 357.1 ± 72.6 ng/ml in mice, as measured by LC-MS/MS.

Techniques: Staining, Isolation, Flow Cytometry