SUI1 protein, human

Plasma concentrations of sunitinib and its active metabolite, SU12662, were determined on days 1 and 28 of cycles 1 to 4. Plasma concentrations of both were determined predose by a liquid chromatography/mass spectrometry method at BASi (West Lafayette, IN), with a lower limit of detection of 0.1 ng/mL [23 (link)].
Predose plasma samples were collected on days 1 and 28 of each cycle for assessment of soluble proteins that may be correlates of angiogenic activity and/or pharmacodynamic inhibition of VEGF receptor-mediated signaling [24 -26 (link)]. Each of the soluble proteins was analyzed with enzyme-linked immunosorbent assay (ELISA) kits (all manufactured by R&D Systems, Minneapolis, MN). The (VEGF)-A ELISA assay measures the VEGF-A165 and VEGF-A121 isoforms. The PlGF assay primarily measures PlGF-1. sVEGFR-2 was quantified with an ELISA that measures the extracellular (soluble) domain of VEGFR-2 [27 (link)]. Similarly, an ELISA component kit that measures the extracellular (soluble) domain of VEGFR-3 was employed. Both the sVEGFR-2 and sVEGFR-3 assays are calibrated against recombinant proteins consisting of the full-length extracellular domain of the respective receptors. No cross-reactivity or interference is detected between the two receptors in the ELISA assays. Though the structural details of sVEGFR-2 and sVEGFR-3 remain to be established, plasma-derived sVEGFR-2 has been reported to be heavily glycosylated and to have a molecular weight of ~90 kDa when de-glycosylated; the size is similar to that or insect-derived recombinant sVEGFR-2, implying that endogenous sVEGFR-2 may be similar in structure to recombinant versions of the VEGFR-2 extraceullar domain [27 (link)]. All ELISA assays were run under Good Laboratory Practice conditions, and performance specifications of each ELISA were validated for their intended purpose, as per established guidelines [28 (link)].

The sensitivity of the isolates with single or/and double sdh mutations to several SDHIs (fluxapyroxad, boscalid, penthiopyrad, isofetamid, and fluopyram) was tested by measuring the inhibition of the spores’ germ tube length. In total, four (4) isolates (A137, A127, B7, and B9) with the H270Y mutation in the sdhB subunit, five (5) isolates (B14, A67, A69, A121, and B144) possessing the H65Q/P66S mutations in the sdhD subunit, three (3) isolates (B10, B11, and B12), possessing all three sdh mutations, and three (3) isolates that were sensitive to all of the tested fungicides were included in the study. The fungicide concentrations used were 0, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, and 50 mg L⁻¹. Aliquots (30 μL) of the conidia suspension of each isolate (1 × 10⁴ conidia mL⁻¹) were plated with a sterile colony spreader on RPMI-1640 agar medium amended or not with each fungicide concentration. Cultures were incubated for 18 h in a growth chamber (27 °C, dark) and after the end of the incubation period, the length of the germination tube was measured in 50 conidia per isolate and fungicide concentration.

The reaction mixtures (210 μl) contained 4 or 7 mM magnesium acetate, 150 mM potassium chloride, 20 mM Tris-HCl pH 7.6, 300 μM spermidine, 15 mM putrescine, 1 mM dithiothreitol, 1 mM GTP, 2 mM ATP, 10 mM phosphoenolpyruvate (PEP), 0.07 mg/ml PEP Kinase, 5 μM EF-G, 1 μM of each initiation factor (IF1, IF2 and IF3), 10 μM EF-Tu, 5 μM EF-Ts, 140 U of aa-tRNA synthetase, 1 μM formyl methionine transferase, 850 μM 10-formyltetrahydrofolate, 2 μM RF1, 2 μM RF3, 20 μM of each amino acid required for the translation of 027mRNA [45 (link)], 10 μM tRNAs mixture, 0.5 μM ribosome, 2.5 μM 027mRNA and 5 μM RRF, when present. The reaction mixtures were incubated at 37°C and after 0, 5, 10, 15, 20 and 40 minutes of incubation, 30 μl of the reaction mixture was spotted on a 1 cm x 1 cm 3MM filter and dropped in 5% TCA and 0.25% sodium tungstate for at least 1 hour at room temperature. The TCA/sodium tungstate mixture was changed and boiled for 10 minutes, washed three times with the same mixture, one time with ethanol/ether (v/v) and one time with ether. The filters were dried and the radioactivity was measured with a scintillation counter. For measuring phenylalanine incorporation, unlabeled phenylalanine was replaced in the above reaction mixture with 0.2 μM [³H]-phenylalanine and 19.8 μM of unlabeled phenylalanine (final concentration was 20 μM). 20 pmols of phenylalanine gave 2,650 CPM. This value was used to calculate the pmol of phenylalanine incorporated. For measuring leucine incorporation, [¹⁴C]-leucine was added in the reaction mixture to a final concentration of 20 μM. Twenty pmols of leucine corresponded to 13,447 CPM. For measuring valine incorporation, [¹⁴C]-valine was added in the reaction mixture to a final concentration of 25 nM. Twenty pmols of valine corresponded to 15,600 CPM.