Journal: PLoS ONE

Article Title: A Conserved Cysteine Motif Is Critical for Rice Ceramide Kinase Activity and Function

doi: 10.1371/journal.pone.0018079

Figure Lengend Snippet: (A) Schematic representation of the sequence organization of OsCERK . The sizes of the 5′-UTR (white), exons (black) and 3′-UTR (grey) are indicated by numbers below the boxes. The sizes of introns are shown above. (B) Phylogenetic analysis of CERKs and the related enzymes sphingosine kinase (SPK)s, and diacylglycerol kinase (DAGK)s. The architecture of each gene was obtained from SMART using the architecture analysis function, and conserved domains (letters a–d) are indicated as follows: a, Pleckstrin homology domain (PH domain; accession number SM00233); b, Diacylglycerol kinase catalytic domain (DAGKc; SMART accession number SM00046); c, segments of low compositional complexity determined by the program; d, diacylgycerol kinase catalytic domain (DAGK_cat; Pfam accession number PF00781). Detailed sequences information is described in . AtCERK is ACD5. (C) CLUSTAL W alignment of CERK from various organisms showing the highly conserved CXXXCXXC motif. (D) RT-PCR expression analysis of OsCERK in leaves, stem and roots of 2-week-old seedlings. β-actin was used as a control. (E) CERK activity of rice extracts. Crude plant extracts made from leaves and roots of rice, respectively, were incubated at 40°C for 30 min as described in . The reaction mixture (1 µl) was resolved on a TLC plate. Purified OsCERK recombinant protein was used as the positive control (+CK). For the negative control (−CK) no extract was added. (F) Comparison of recombinant OsCERK and rACD5. The recombinant protein (1.0 µg) was added in 100 µl reaction system, and incubated at 40°C for 30 min. –CK, no recombinant protein was added. Arrows in (E and F) indicated the formed product C6-NBD-ceramide-1-phosphate (C6-NBD-Cer-1P).

Article Snippet: The C6-NBD-ceramide-1-phosphate products were identified by comparison with C6-NBD-ceramide (Avanti Polar lipids, Alabaster, AL, USA) and quantified using IQTL software (GE Healthcare).

Techniques: Sequencing, Reverse Transcription Polymerase Chain Reaction, Expressing, Activity Assay, Incubation, Purification, Recombinant, Positive Control, Negative Control

Journal: PLoS ONE

Article Title: A Conserved Cysteine Motif Is Critical for Rice Ceramide Kinase Activity and Function

doi: 10.1371/journal.pone.0018079

Figure Lengend Snippet: (A) Effects of cycloheximide (CHX) on C6 ceramide-induced cell death. (B) Effects of K252a on C6 ceramide-induced cell death. (C) Effects of C2 ceramide-1-phosphate on C2 ceramide induced cell death. Protoplasts were treated with less than 0.15% solvent or the indicated reagent. Percentage of surviving cells was estimated by FDA staining. These assays were repeated three times with similar results. Letters indicate that values are different using Fisher's PLSD test ( P <0.05). Bars show standard deviations.

Article Snippet: The C6-NBD-ceramide-1-phosphate products were identified by comparison with C6-NBD-ceramide (Avanti Polar lipids, Alabaster, AL, USA) and quantified using IQTL software (GE Healthcare).

Techniques: Staining