Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Link between light and fatty acid synthesis: Thioredoxin-linked reductive activation of plastidic acetyl-CoA carboxylase

doi:

Figure Lengend Snippet: Effects of thiol reagents on the ACCase activity. ( A ) ACCase activity was measured in the indicated concentration of a thiol reagent as described in Materials and Methods . Plastidic ACCase from pea, continuous lines; cytosolic ACCase from pea, dashed line. β-SH, 2-mercaptoethanol; GSH, reduced form of glutathione. Lipoic acid was dissolved into 50 mM Tricine⋅KOH (pH 8) containing 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamide, and 5 mM ɛ-amino-n-caproic acid at 20 mM, and 0–7 μl of 20 mM solution was added to 13-μl reaction mixture. The activity without any thiol reagent was 11 nmol/20 min per mg of protein for pea plastidic ACCase and 210 nmol/20 min per mg of protein for pea cytosolic ACCase. The activity is relative to that without a thiol reagent, taken to be 1. Results are the means of duplicate determinations that differed less than 10%. ( B ) The effects of preincubation of plastidic ACCase with DTT on activity were examined. Changes with time are shown. After the enzyme was incubated at 30°C for 10 min with 2 mM DTT, the reaction was started by the addition of buffer, ATP, MgCl 2 , KCl, acetyl-CoA, and NaH 14 CO 3 . The final concentrations of these reagents were the same as above. The final concentration of DTT was 1 mM.

Article Snippet: The ammonium sulfate precipitates of the plastidic or the cytosolic ACCase fraction were dissolved in 50–100 μl of a 50 mM Tricine⋅KOH (pH 8) buffer containing 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamide, and 5 mM ɛ-amino-n-caproic acid, and desalted by centrifugation in a gel filtration column (Biospin 6, or Biospin 30, Bio-Rad).

Techniques: Activity Assay, Concentration Assay, Incubation

Journal: International Journal of Nanomedicine

Article Title: Inhibition of autophagy contributes to the toxicity of cadmium telluride quantum dots in Saccharomyces cerevisiae

doi: 10.2147/IJN.S108636

Figure Lengend Snippet: Effect of CdTe QDs of different sizes on autophagy in yeast at different time points. Notes: Cells transformed with a plasmid encoding GFP-Atg8 were treated with ( A ) 50 nmol/L CdTe QDs or 100 ng/mL rapamycin or ( B ) phenylmethanesulfonyl fluoride (PMSF), which was added at 0 hour and 16 hours to reach a final concentration of 2 mmol/L, and photographed at 4 hours, 8 hours, 16 hours, and 42 hours using fluorescence microscopy. Scale bars: 5 µm. Abbreviations: CdTe QDs, cadmium telluride quantum dots; G-CdTe, green-emitting CdTe; O-CdTe, orange-emitting CdTe.

Article Snippet: Phenylmethanesulfonyl fluoride was obtained from Amresco (Solon, OH, USA).

Techniques: Transformation Assay, Plasmid Preparation, Concentration Assay, Fluorescence, Microscopy

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Trypsin is a multifunctional factor in spermatogenesis

doi: 10.1073/pnas.0907631106

Figure Lengend Snippet: Effects of trypsin on spermatogenesis in the Japanese eel in vitro. ( A ) Effect of anti-trypsinogen antibody (Anti-TN) treatment on DHP-induced DNA replication of germ cells in a germ cell/somatic cell coculture system. IC, Initial control; KT, 10 ng/mL; DHP, 10 ng/mL; +, with anti-TN; -, without anti-TN. ( B ) Effect of serine protease inhibitor treatment on DHP-induced DNA replication of germ cells in a germ cell/somatic cell coculture system. PM, Phenylmethylsulfonyl fluoride (PMSF); AEB, 4-(2-Aminoethyl)-benzenesulfonyl fluoride (AEBSF). ( C ) Effect of various concentrations of porcine trypsin on germ cell/somatic cell cocultures.

Article Snippet: To further understand the function of trypsin during spermatogenesis, these cultures were exposed to porcine trypsin (Nacalai Tesque), Phenylmethylsulfonyl fluoride (PMSF; Nacalai Tesque), 4-(2-Aminoethyl)-benzenesulfonyl fluoride (AEBSF; Nacalai Tesque) or anti-eel trypsinogen antibodies.

Techniques: In Vitro, Protease Inhibitor

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Trypsin is a multifunctional factor in spermatogenesis

doi: 10.1073/pnas.0907631106

Figure Lengend Snippet: Localization of trypsin in eel sperm. ( A ) Immunohistochemical analysis of whole cells using an anti-eel trypsinogen antibody. Trypsin localizes at the sperm membrane and within the mitochondria in the caput end of the sperm head. (Scale bar, 10 μm.) ( B ) Gelatin zymography assay and Western blot analysis of the eel sperm membrane with an anti-trypsinogen antibody (Anti-TN). PMSF − and + indicate with or without Phenylmethylsulfonyl fluoride, respectively. Arrowheads indicate molecular markers ( Left ), trypsin activity assayed by zymography, or signal detection by Western blot analysis using the anti-eel trypsinogen antibody.

Article Snippet: To further understand the function of trypsin during spermatogenesis, these cultures were exposed to porcine trypsin (Nacalai Tesque), Phenylmethylsulfonyl fluoride (PMSF; Nacalai Tesque), 4-(2-Aminoethyl)-benzenesulfonyl fluoride (AEBSF; Nacalai Tesque) or anti-eel trypsinogen antibodies.

Techniques: Immunohistochemistry, Zymography Assay, Western Blot, Activity Assay, Zymography