Journal: Journal of Assisted Reproduction and Genetics

Article Title: Identification and characterization of RSIY-11, a novel seminal peptide derived from semenogelin-1, which acts as a neutral endopeptidase inhibitor modulating sperm motility

doi: 10.1007/s10815-019-01524-8

Figure Lengend Snippet: Determination of the effect of pre-incubation of RSIY-11 with NEP on inhibition. Fifty microliters of recombinant human NEP enzyme (0.1 μg/ml) and 50 μl of RSIY-11 (8.2 μM) were incubated for 0, 3, or 24 h at 37 °C prior to the addition of 50 μl of fluorogenic NEP substrate to determine enzymatic activity, and then, activity was measured every minute over 15 min. A control was performed by incubating NEP for the indicated time without RSIY-11, which was used as the 100% for each time point. Assays were performed in triplicate and averaged. Enzymatic activity was expressed as a percentage of the activity of the control. Error bars represent standard deviation. Asterisks (*) = statistically different from the control, *p value < 0.05; **p value < 0.01. NS no significant difference

Article Snippet: Michaelis-Menten Constant A fluorometric assay was performed using recombinant human NEP enzyme at 0.1 μg/ml (R&D Systems, Minneapolis, USA) and fluorogenic NEP peptide substrate ((7-methoxycoumarin-4-yl)-acetyl-R-P-P-G-F-S-A-F-K-(2, 4-dinitrophenyl)-OH, R&D Systems).

Techniques: Incubation, Inhibition, Recombinant, Activity Assay, Standard Deviation

Journal: Journal of Cellular and Molecular Medicine

Article Title: The complex effects of the slow-releasing hydrogen sulfide donor GYY4137 in a model of acute joint inflammation and in human cartilage cells

doi: 10.1111/jcmm.12016

Figure Lengend Snippet: Effect of GYY4137 on LPS-induced expression of COX-2, iNOS and TNF-α-converting enzyme (TACE) expression and activity in human synoviocytes and human articular chondrocytes. HAC and HFLS were treated with GYY4137 at the concentrations stated for 1 hr prior to LPS (10 μg/ml) stimulation for 18 hr. After this time, cells were lysed and levels of COX-2 ( A ) and iNOS ( B ) determined by commercial ELISA. ( C ) Expression of pre- and mature TACE was determined in HFLS by Western blotting after 24 hr. ( D – F ) Effects of GYY4137 on isolated human recombinant COX-2 ( D ), iNOS ( E ) and ( F ) TACE activity. Recombinant enzyme activities are expressed as % control enzyme activity after 1 hr (iNOS and COX-2) and 4 hr (TACE). GYY4137 was added at the concentrations stated and enzyme activity determined. DuP697 (1 μM) was used as a positive control for COX-2 activity and L-NNA (supplied with the NOS activity kit; 100 μM) as positive control for iNOS activity. Western blots are representative of three separate determinations and data shown are mean ± SEM of at least three separate experiments. * P < 0.05 c.f . LPS-stimulated cells.

Article Snippet: TACE activity was determined using recombinant human TACE enzyme (R&D Systems) and the fluorogenic TACE substrate Mca-Pro-Leu-Ala-Gln-Ala-Val-DPA-Arg-Ser-Ser-Ser-Arg-NH 2 (R&D Systems).

Techniques: Expressing, Activity Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Isolation, Recombinant, Control, Positive Control