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1.
FIG. 7.

FIG. 7. From: Characterization of Metacaspases with Trypsin-Like Activity and Their Putative Role in Programmed Cell Death in the Protozoan Parasite Leishmania .

Sensitivity of LdMC enzyme activity to protease inhibitors. Effects of different protease inhibitors on LdMC activity from lysates of axenic amastigotes using Boc-GKR-AMC as the substrate. The results are shown as percentages of the LdMC activity measured without inhibitor (control) (black bar). Results represent the averages of three independent assays. Standard deviations (error bars) are shown.

Nancy Lee, et al. Eukaryot Cell. 2007 October;6(10):1745-1757.
2.
FIG. 4.

FIG. 4. From: Characterization of Metacaspases with Trypsin-Like Activity and Their Putative Role in Programmed Cell Death in the Protozoan Parasite Leishmania .

Processing of the LdMCs in L. donovani. Autoradiogram showing the radiolabeled LdMCs immunoprecipitated with the anti-LdMC antibody (anti-LdMC) or preimmune serum (NRS) from lysates of radiolabeled promastigotes (Pro) and axenic amastigotes (AxAm). Parasites were pulse-labeled with [35S]methionine for 10 min and chased for 0 and 60 min in culture medium supplemented with amphotericin B (1 μg/ml) (+) or not supplemented with amphotericin B (−). The molecular masses (in kilodaltons) of protein standards are shown to the left of the gel.

Nancy Lee, et al. Eukaryot Cell. 2007 October;6(10):1745-1757.
3.
FIG. 3.

FIG. 3. From: Characterization of Metacaspases with Trypsin-Like Activity and Their Putative Role in Programmed Cell Death in the Protozoan Parasite Leishmania .

Expression of LdMC1 and LdMC2 in promastigotes and axenic amastigotes. (A) Western blot showing the reactivity of the anti-LdMC antibody (anti-LdMC) or preimmune serum (NRS) with lysates of log-phase promastigotes (P) and axenic amastigotes (Ax). The Western blot membrane was reprobed with an antitubulin antibody (bottom gels). The positions of the LdMC proteins is indicated by an arrow. (B) Western blot showing the reactivity of the anti-LdMC or anti-HA antibodies with lysates of log-phase promastigotes transfected with either the control pKS NEO (KS) or pKS NEO LdMC1 (MC1) or pKS NEO LdMC2 (MC2) expression plasmid. The positions of HA-tagged proteins LdMC1-HA and LdMC2-HA and the endogenous LdMC proteins (LdMCs) are indicated by arrows. The molecular masses (in kilodaltons) of protein standards are shown to the left of the gels.

Nancy Lee, et al. Eukaryot Cell. 2007 October;6(10):1745-1757.
4.
FIG. 8.

FIG. 8. From: Characterization of Metacaspases with Trypsin-Like Activity and Their Putative Role in Programmed Cell Death in the Protozoan Parasite Leishmania .

Stage expression and pH optimum of LdMC enzyme activity. (A) LdMC enzyme activity in lysates of axenic amastigotes (AxAm) and promastigotes (Pro) of L. donovani. The LdMC enzyme activity is expressed in relative fluorescence units (RFU) (black bars). Control enzymatic assays were done using the preimmune serum (NRS) during the immunoprecipitation step (white bars). (B) LdMC enzyme activity in lysates of axenic amastigotes at different pHs. Relative activity is expressed as a percentage of the activity at optimal pH (7.5). The results in panels A and B were obtained using Boc-GKR-AMC as the substrate and represent the averages of three independent assays. Standard deviations (error bars) are shown.

Nancy Lee, et al. Eukaryot Cell. 2007 October;6(10):1745-1757.
5.
FIG. 5.

FIG. 5. From: Characterization of Metacaspases with Trypsin-Like Activity and Their Putative Role in Programmed Cell Death in the Protozoan Parasite Leishmania .

Immunolocalization of LdMCs in L. donovani. L. donovani promastigotes (Pro) and axenic amastigotes (AxAm) were processed for immunofluorescence and reacted with both anti-LdMC and anti-TcPPase antibodies. The reactivity with the anti-LdMC is shown in the red channel (panels A and E), and that of the anti-TcPPase antibody is shown in the green channel (panels B and F). Merging of the red and green channels (yellow) is shown in panels C and G. The corresponding phase-contrast images are shown in panels D and H. Bars in panels C and G, 10 μm. Panel K shows the reactivity of the anti-TcPPase antibody with lysates of L. donovani promastigotes (Ld) in Western blots. The molecular masses (in kilodaltons) of protein standards are shown to the left of the blot.

Nancy Lee, et al. Eukaryot Cell. 2007 October;6(10):1745-1757.
6.
FIG. 1.

FIG. 1. From: Characterization of Metacaspases with Trypsin-Like Activity and Their Putative Role in Programmed Cell Death in the Protozoan Parasite Leishmania .

Amino acid sequences of L. donovani LdMC1 and LdMC2. (A) Clustal W sequence alignment of LdMC1 (GenBank accession no. DQ367530), LdMC2 (GenBank accession no. DQ367531), and Leishmania major metacaspase (LmjMC) (GenBank accession no. AAZ14381) amino acid sequences. Asterisks indicate the difference in amino acid residues between LdMC1 and LdMC2. The solid square indicates the single amino acid residue difference between LdMC2 and LmjMC. The putative catalytic His147 and Cys202 residues are boxed. Solid bars above the sequences indicate the regions of repetitive sequences (PQGYYPPP and YPAP/QG) present in LdMC1, and arrows indicate the proline-rich domains. The gaps introduced to maximize alignment are indicated by dots.

Nancy Lee, et al. Eukaryot Cell. 2007 October;6(10):1745-1757.
7.
FIG. 9.

FIG. 9. From: Characterization of Metacaspases with Trypsin-Like Activity and Their Putative Role in Programmed Cell Death in the Protozoan Parasite Leishmania .

Sensitivity of L. donovani wild type and LdMC transfectants to H2O2. (A) Percentages of TUNEL-positive cells in L. donovani axenic amastigotes either treated with 2 mM H2O2 (+ H2O2) for 60 min or untreated (− H2O2) and analyzed by flow cytometry. (B) LdMC enzyme activity in parasites treated as described above for panel A, using Boc-GRR-AMC as the substrate and expressed in relative fluorescence units (RFU) (black bars). Control enzymatic assays were done using the preimmune serum during the immunoprecipitation step (white bars). (C) Percentages of TUNEL-positive cells in L. donovani axenic amastigote transfectants (KS, MC1, and MC1-mut [Mut]) either treated with 2 mM H2O2 (black bars) for 60 min or untreated (white bars) and analyzed by flow cytometry. Significant differences (P < 0.05) (*) and nonsignificant differences (P > 0.05) (**) are indicated. Results in panels A, B, and C represent the averages of three independent assays. Standard deviations (error bars) are shown.

Nancy Lee, et al. Eukaryot Cell. 2007 October;6(10):1745-1757.
8.
FIG. 2.

FIG. 2. From: Characterization of Metacaspases with Trypsin-Like Activity and Their Putative Role in Programmed Cell Death in the Protozoan Parasite Leishmania .

RT-PCR of LdMC1 and LdMC2. (A) Map of the LdMC1 and LdMC2 genes and flanking sequences. The start (ATG) and stop (TAA) codons of each open reading frame (open boxes) are indicated. The two regions of repetitive sequences (PQGYYPPP and YPAP/QG) present in LdMC1 are indicated by solid boxes. Vertical arrows indicate the location of the spliced leader acceptor site at position −151. Splice leader (SL) sequence is illustrated as a hatched box. Horizontal arrows (black, gray, and white) indicate the locations of the PCR primers (Table 1). (B) Ethidium bromide-stained agarose gel of the RT-PCR products obtained with LdMC1 (top gel) LdMC2 (middle gel), and tubulin (bottom gel) gene-specific primer sets (Table 1). The reverse transcription reaction was done using mRNA isolated from either log-phase (L) or stationary-phase (S) promastigotes (Pro) or axenic amastigotes (Ax) and with reverse transcriptase (+ RT) or without reverse transcriptase (−RT). The molecular sizes (in kilobases) of protein standards are shown to the left of the gel.

Nancy Lee, et al. Eukaryot Cell. 2007 October;6(10):1745-1757.
9.
FIG. 6.

FIG. 6. From: Characterization of Metacaspases with Trypsin-Like Activity and Their Putative Role in Programmed Cell Death in the Protozoan Parasite Leishmania .

Substrate specificity of LdMC enzyme activity. (A) LdMCs were immunoprecipitated from axenic amastigote lysates using the anti-LdMC antibody and incubated with either trypsin substrates (GKR, GRR, and AFK) or caspase substrates (DEVD, VEID, and LEHD) under appropriate reaction conditions (see Materials and Methods for details). The LdMC enzymatic activity is expressed in relative fluorescence units (RFU) (black bars). Control enzymatic assays were done using the preimmune serum during the immunoprecipitation step (white bars). Human recombinant caspase-3 was spiked into parallel assays containing the caspase substrates (gray bars). (B) Metacaspase activities of HA-tagged proteins (MC1, MC1-mut, and MC2) immunoprecipitated using the anti-HA antibody from axenic amastigote lysates of transfected parasites, and using Boc-GRR-AMC as the substrate (black bars). Parasites transfected with the pKS NEO expression plasmid was used as control (KS). Control enzymatic assays were done using the rabbit anti-mouse antibodies during the immunoprecipitation step (white bars). Results in panels A and B represent the averages of three independent assays. Standard deviations (error bars) are shown. (C) Parasites used in panel B were radiolabeled with [35S]methionine, radiolabeled HA-tagged proteins were immunoprecipitated with an anti-HA antibody or control rabbit anti-mouse antibodies (RAM), and material was analyzed by SDS-PAGE and fluorography. The molecular masses (in kilodaltons) of protein standards are shown to the left of the gel.

Nancy Lee, et al. Eukaryot Cell. 2007 October;6(10):1745-1757.

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