l tarentolae parrot tarii strain atcc pra  (ATCC)

Journal: bioRxiv

Article Title: Novel Binding Partners of the Vacuolar Transporter Chaperone (VTC) complex in Acidocalcisomes of Leishmania tarentolae

doi: 10.1101/2025.09.23.677757

Figure Lengend Snippet: CRISPR/Cas9 tagging and expression of LtVtc1 and LtVtc4. (A) Schematic representation of LtVtc1 and LtVtc4. SPX: SYG1/Pho81/XPR domain, CD: Catalytic domain, TMH: Transmembrane helix domain. (B) PCR verification of mNG-tagged LtVtc1 and LtVtc4 showing a band at ∼2200 bp, absent in WT. (C) Fluorescent detection of mNG-LtLtVtc1, and LtVtc4 expressed in L. tarentolae ; Coomassie blue staining of WT L. tarentolae , mNG-LtVtc1, mNG-LtVtc4 confirms comparable total protein levels, ensuring consistent sample loading for fluorescent detection. (D) Fluorescence microscopy micrographs of mNG-LtLtVtc1 and mNG-LtVtc4 with mPPase in L.tarentolae (Pearson’s r = 0.85 and 0.83, respectively), scale bar: 5 µm.

Article Snippet: L. tarentolae P10 strain promastigotes (LEXSY host P10, Jena Biosciences) were cultured at 27 °C in ventilated tissue culture flasks in 10 ml of brain heart infusion (BHI) medium containing 37 mg/mL BHI powder (Lexsy Broth BHI, Jena Biosciences) supplemented with 5 μg/mL hemin as described by the manufacturer (Jena Biosciences).

Techniques: CRISPR, Expressing, Staining, Fluorescence, Microscopy

Journal: bioRxiv

Article Title: Novel Binding Partners of the Vacuolar Transporter Chaperone (VTC) complex in Acidocalcisomes of Leishmania tarentolae

doi: 10.1101/2025.09.23.677757

Figure Lengend Snippet: Colocalisation analysis of L. tarentolae expressing mNG-LtVtc4 and mCh-tagged by immunofluorescence microscopy. mNG-LtVtc4 colocalises with mCh-LtVtc1 (Pearson’s r = 0.83) as well as with mCh-LtPho91, mCh-LtCa²⁺-ATPase, mCh-LtV-H⁺-ATPase_A, and mCh-LtZnT, showing Pearson’s correlation coefficients of 0.87, 0.83, 0.87, and 0.84, respectively. A lower degree of colocalisation is observed with mCh-IP₃R (r = 0.67). Scale bar: 5 µm.

Article Snippet: L. tarentolae P10 strain promastigotes (LEXSY host P10, Jena Biosciences) were cultured at 27 °C in ventilated tissue culture flasks in 10 ml of brain heart infusion (BHI) medium containing 37 mg/mL BHI powder (Lexsy Broth BHI, Jena Biosciences) supplemented with 5 μg/mL hemin as described by the manufacturer (Jena Biosciences).

Techniques: Expressing, Immunofluorescence, Microscopy

Journal: bioRxiv

Article Title: Novel Binding Partners of the Vacuolar Transporter Chaperone (VTC) complex in Acidocalcisomes of Leishmania tarentolae

doi: 10.1101/2025.09.23.677757

Figure Lengend Snippet:

Article Snippet: L. tarentolae P10 strain promastigotes (LEXSY host P10, Jena Biosciences) were cultured at 27 °C in ventilated tissue culture flasks in 10 ml of brain heart infusion (BHI) medium containing 37 mg/mL BHI powder (Lexsy Broth BHI, Jena Biosciences) supplemented with 5 μg/mL hemin as described by the manufacturer (Jena Biosciences).

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Novel Binding Partners of the Vacuolar Transporter Chaperone (VTC) complex in Acidocalcisomes of Leishmania tarentolae

doi: 10.1101/2025.09.23.677757

Figure Lengend Snippet: Colocalisation analysis of L. tarentolae expressing mNG-LtVtc4 and mCh-tagged LtVBP1, LtVBP2, and LtVBP3 by immunofluorescence microscopy. All three proteins show colocalisation with LtVtc4 in acidocalcisomes. Pearson’s correlation coefficients with LtVtc4 are 0.83 for LtVBP1, 0.85 for LtVBP2, and 0.82 for LtVBP3. Scale bar: 5 µm.

Article Snippet: L. tarentolae P10 strain promastigotes (LEXSY host P10, Jena Biosciences) were cultured at 27 °C in ventilated tissue culture flasks in 10 ml of brain heart infusion (BHI) medium containing 37 mg/mL BHI powder (Lexsy Broth BHI, Jena Biosciences) supplemented with 5 μg/mL hemin as described by the manufacturer (Jena Biosciences).

Techniques: Expressing, Immunofluorescence, Microscopy

Journal: bioRxiv

Article Title: Novel Binding Partners of the Vacuolar Transporter Chaperone (VTC) complex in Acidocalcisomes of Leishmania tarentolae

doi: 10.1101/2025.09.23.677757

Figure Lengend Snippet: Pulldown assay of novel VTC binding partners with mNG-LtVtc4 in L. tarentolae. (A-C) SDS-PAGE with fluorescence detection shows capture of mCh-LtVBP1, mCh-LtVBP2, and mCh-LtVBP3 by mNG-LtVtc4 using mNeonGreen-Trap agarose beads, as indicated by signal in the bound fraction. (D) Pulldown of mNG-LtVtc4 with mCh-LtVBP3 using ChromoTek RFP-Trap beads confirms interaction. Lane labels: I – input; FT – flow-through; W3 – third wash; B – bound fraction. Green bands correspond to mNG-LtVtc4; some I and B lines appear as yellowish due to partial detection of the mNG tag in the 532 nm channel ( S6 Fig ). Red bands correspond to mCherry-tagged proteins. The fluorescence signal marked by a white asterisk in the gel represents free mCh tag, visible only in the input due to partial degradation.

Article Snippet: L. tarentolae P10 strain promastigotes (LEXSY host P10, Jena Biosciences) were cultured at 27 °C in ventilated tissue culture flasks in 10 ml of brain heart infusion (BHI) medium containing 37 mg/mL BHI powder (Lexsy Broth BHI, Jena Biosciences) supplemented with 5 μg/mL hemin as described by the manufacturer (Jena Biosciences).

Techniques: Binding Assay, SDS Page, Fluorescence

Journal: bioRxiv

Article Title: A conserved mechanism for the retrieval of polyubiquitinated proteins from cilia

doi: 10.1101/2025.04.24.650332

Figure Lengend Snippet: (A) Schematic workflow of pulldown experiment from flagellar lysates using immobilized K48- and K63-linked ubiquitin chains and subsequent quantitative multiplexed mass spectrometry through tandem mass tagging (TMT-MS) analysis. (B, C) Heatmaps showing quantification of significantly enriched ubiquitin readers in C. reinhardtii (B) and L. tarentolae (C) flagellar lysates identified in triplicate or quadruplicate pulldowns (R1-R4). A one-way ANOVA was performed for each protein across experimental groups, followed by Ben-jamini-Hochberg false discovery rate (FDR) correction for multiple testing. Proteins with q-values below 0.01 were considered statistically significant, and those exhibiting a fold change greater than 2 compared to the empty beads control group were retained. (D) Venn diagram of proteins identified in the TMT-MS analysis of C. reinhardtii and L. tarentolae ubiquitin pulldowns. Only homologs of CFAP36, TOM1L2 and USP5 were identified in pulldowns from both organisms.

Article Snippet: Briefly, L. tarentolae strain P10 (Jena Bioscience, #LT-101) were grown in BHI medium (HIMEDIA, #N210) supplemented with 5 mg/mL hemin chloride (Sigma, #3741) and 10 U/mL Penicillin-Streptomycin (Gibco, #15070063) at 26 °C.

Techniques: Mass Spectrometry, Control

Journal: bioRxiv

Article Title: A conserved mechanism for the retrieval of polyubiquitinated proteins from cilia

doi: 10.1101/2025.04.24.650332

Figure Lengend Snippet: Related to . (A)SDS-PAGE analysis of biotinylated K48- and K63-linked polyubiquitin (pUb) chains before and after addition of streptavidin (SA) beads. (B-C) SDS-PAGE analysis of elution from empty and K48- or K63-linked ubiquitin-coated beads incubated with flagellar extracts from C. reinhardtii (B)and L. tarentolae (C). Each elution was analyzed by quantitative mass spectrometry. Replicate number is shown above each lane. (D)Volcano plots generated from TMT-MS analysis of flagellar extracts from C. reinhardtii comparing protein binding to empty beads versus beads coated with K48-linked (left) or K63-linked (right) polyubiquitin chains. Each plot shows significance versus enrichment. Individual proteins are shown as gray dots except for ARL3, CFAP36, TOM1L2, and USP5, which are colored and labeled. (E)Volcano plots generated from TMT-MS analysis of flagellar extracts from L. tarentolae comparing protein binding to empty beads versus beads coated with K48-linked (left) or K63-linked (right) ubiquitin chains in the absence (top) or presence (bottom) of GTP.

Article Snippet: Briefly, L. tarentolae strain P10 (Jena Bioscience, #LT-101) were grown in BHI medium (HIMEDIA, #N210) supplemented with 5 mg/mL hemin chloride (Sigma, #3741) and 10 U/mL Penicillin-Streptomycin (Gibco, #15070063) at 26 °C.

Techniques: SDS Page, Incubation, Mass Spectrometry, Generated, Protein Binding, Labeling

Journal: bioRxiv

Article Title: A conserved mechanism for the retrieval of polyubiquitinated proteins from cilia

doi: 10.1101/2025.04.24.650332

Figure Lengend Snippet: Related to . (A)A simplified phylogenetic tree illustrating the evolutionary relationships among various species, with indicators for the presence of cilia/flagella, ubiquitin, intraflagellar transport (IFT) proteins, and CFAP36. CFAP36 is only present in organisms with cilia, ubiquitin and IFT. (B)A multiple sequence alignment of CFAP36 protein sequences from the five organisms used in this study: Homo sapiens (human), Sus scrofa (porcine), Mus musculus (murine), Chlamydomonas reinhardtii (green algae), and Leishmania tarentolae (protozoan parasite). Secondary structure elements predicted from the human CFAP36 sequence are annotated above the alignment, with α-helices (α) indicated. The BART-like domain and predicted ubiquitin interacting motifs (UIMs) are labeled. Invariant residues, conserved across all five species, are highlighted in rust. Similar residues, sharing conserved properties, are highlighted in yellow.

Article Snippet: Briefly, L. tarentolae strain P10 (Jena Bioscience, #LT-101) were grown in BHI medium (HIMEDIA, #N210) supplemented with 5 mg/mL hemin chloride (Sigma, #3741) and 10 U/mL Penicillin-Streptomycin (Gibco, #15070063) at 26 °C.

Techniques: Sequencing, Algae, Labeling

Journal: Microbial Cell

Article Title: A Modular Cloning Toolkit for the production of recombinant proteins in Leishmania tarentolae

doi: 10.15698/mic2024.04.821

Figure Lengend Snippet: (A) Map of the destination vector for the L. tarentolae MoClo kit based on the pLEXSY_I-blecherry3 plasmid from Jena Bioscience. BsaI restriction sites were removed and the coding region for lacZα flanked by BsaI restriction sites was introduced to yield CCAT and GCTT fusion sites upon digestion with BsaI. (B) List of 34 Level 0 MoClo parts for positions B2 to B5 that are compatible with the MoClo syntax for plants and algae. The color code for fusion sites was adopted from the Chlamydomonas MoClo kit . Nucleotides used in codons are underlined in white. SP, signal peptide; CDS, coding sequence; RS, retention signal; sAP1, secreted acid phosphatase 1; HA, human influenza hemagglutinin; Met, methionine; Strep, streptavidin; PP, PreScission protease cleavage site; GST, glutathione transferase; RBD, receptor binding domain of SARS-CoV-2; SV40, simian-virus 40; TEV, tobacco etch virus protease cleavage site; SP, serine-proline repeat; ER, endoplasmic reticulum.

Article Snippet: L. tarentolae strain T7-TR promastigotes (Jena Bioscience) were cultured at 27°C in ventilated tissue culture flasks in an upright position on a Rotamax 120 shaker at 50 rpm in 10 mL brain heart infusion (BHI) medium according to standard protocols [ , , ].

Techniques: Plasmid Preparation, Algae, Sequencing, Binding Assay, Virus

Journal: Microbial Cell

Article Title: A Modular Cloning Toolkit for the production of recombinant proteins in Leishmania tarentolae

doi: 10.15698/mic2024.04.821

Figure Lengend Snippet: List of level 0 parts for the L. tarentolae MoClo system.

Article Snippet: L. tarentolae strain T7-TR promastigotes (Jena Bioscience) were cultured at 27°C in ventilated tissue culture flasks in an upright position on a Rotamax 120 shaker at 50 rpm in 10 mL brain heart infusion (BHI) medium according to standard protocols [ , , ].

Techniques: Sequencing, Protein Purification, Strep-tag, Binding Assay, Amplification, Luciferase, Purification

Journal: Microbial Cell

Article Title: A Modular Cloning Toolkit for the production of recombinant proteins in Leishmania tarentolae

doi: 10.15698/mic2024.04.821

Figure Lengend Snippet: (A) Schematic overview and expected mass of the secreted RBD fusion proteins. (B) Comparative Western blot analysis with antibodies against the RBD domain (left) and the GST domain (right) of RBD fusion proteins in the cell-containing pellet fraction (P) and supernatant fraction (S) following tetracycline induction of the according L. tarentolae liquid cultures. Recombinant RBD served as positive control (p.c.) and an induced culture without plasmid as negative control (n.c.). The calculated masses from panel A are indicated.

Article Snippet: L. tarentolae strain T7-TR promastigotes (Jena Bioscience) were cultured at 27°C in ventilated tissue culture flasks in an upright position on a Rotamax 120 shaker at 50 rpm in 10 mL brain heart infusion (BHI) medium according to standard protocols [ , , ].

Techniques: Western Blot, Recombinant, Positive Control, Plasmid Preparation, Negative Control

Journal: Microbial Cell

Article Title: A Modular Cloning Toolkit for the production of recombinant proteins in Leishmania tarentolae

doi: 10.15698/mic2024.04.821

Figure Lengend Snippet: (A) Schematic overview and expected mass of the secreted RBD variants with swapped C-terminal peptide tags. Variant 2 with an N-terminal 3xHA tag instead of the N-terminal signal peptide served as a cytosolic control. Variant 4 also contains an SP20 sequence for extensive O -glycosylation. (B) Comparative Western blot analysis with antibodies against the RBD domain (left) or the 8xHis tag (right) in the cell-containing pellet fraction (P) and supernatant fraction (S) following tetracycline induction of the according L. tarentolae liquid cultures. An induced culture without plasmid served as negative control (n.c.) and recombinant His-tagged RBD with an expected size of 35 kDa as positive control (p.c.). The asterisk labels a band that was caused by a contamination of the supernatant fraction in the experiment shown. Both membranes were stripped and redecorated with an antibody against the HA tag. The calculated masses from panel A are indicated.

Article Snippet: L. tarentolae strain T7-TR promastigotes (Jena Bioscience) were cultured at 27°C in ventilated tissue culture flasks in an upright position on a Rotamax 120 shaker at 50 rpm in 10 mL brain heart infusion (BHI) medium according to standard protocols [ , , ].

Techniques: Variant Assay, Sequencing, Western Blot, Plasmid Preparation, Negative Control, Recombinant, Positive Control

Journal: Microbial Cell

Article Title: A Modular Cloning Toolkit for the production of recombinant proteins in Leishmania tarentolae

doi: 10.15698/mic2024.04.821

Figure Lengend Snippet: (A) Schematic overview and expected mass of the secreted RBD fusion proteins. (B) Western blot analysis with an antibody against the RBD domain of RBD fusion variants 1 and 2 in the cell-containing pellet fraction (P) and supernatant fraction (S) following tetracycline induction of the according L. tarentolae liquid cultures for the indicated time periods. An induced culture without plasmid served as negative control (n.c.) and recombinant RBD as positive control (p.c.). The calculated masses from panel A are indicated. (C) Western blot analysis with antibodies against the C-terminal His tag (left) or the RBD domain (right) of fusion variant 3.

Article Snippet: L. tarentolae strain T7-TR promastigotes (Jena Bioscience) were cultured at 27°C in ventilated tissue culture flasks in an upright position on a Rotamax 120 shaker at 50 rpm in 10 mL brain heart infusion (BHI) medium according to standard protocols [ , , ].

Techniques: Western Blot, Plasmid Preparation, Negative Control, Recombinant, Positive Control, Variant Assay

Journal: Microbial Cell

Article Title: A Modular Cloning Toolkit for the production of recombinant proteins in Leishmania tarentolae

doi: 10.15698/mic2024.04.821

Figure Lengend Snippet: (A) Schematic overview and expected mass of the secreted RBD fusion proteins. (B) Western blot analysis with antibodies against the RBD domain (left) and the GST domain (right) of RBD fusion variant 1 in the cell-containing pellet fraction (P) and supernatant fraction (S) following tetracycline induction of the according L. tarentolae liquid cultures containing the indicated amounts of antibiotics. An induced culture without plasmid served as negative control (n.c.) and recombinant RBD as positive control (p.c.). The asterisk labels the TCA precipitate of the 50% supernatant fraction which was lost in the experiment shown. The calculated mass from panel A is indicated. (C) Western blot analysis with antibodies against the HA tag (left), the RBD domain (right), and the His tag (bottom) of RBD fusion variant 2.

Article Snippet: L. tarentolae strain T7-TR promastigotes (Jena Bioscience) were cultured at 27°C in ventilated tissue culture flasks in an upright position on a Rotamax 120 shaker at 50 rpm in 10 mL brain heart infusion (BHI) medium according to standard protocols [ , , ].

Techniques: Western Blot, Variant Assay, Plasmid Preparation, Negative Control, Recombinant, Positive Control

Journal: The Journal of Biological Chemistry

Article Title: Resolving the polycistronic aftermath: Essential role of topoisomerase IA in preventing R-loops in L eishmania

doi: 10.1016/j.jbc.2024.107162

Figure Lengend Snippet: Topoisomerase IA, an essential protein of L. donovani . A , sequence comparison of E. coli TOPIA, M. tuberculosis TOPIA, T. brucei TOPIA and L. donovani TOPIA showing start and end residue of TOPRIM domain, Active site tyrosine and DNA binding domain region. B , homology modeled structure of LdTOPIA with the active site residues Tyr357, Glu135, Asp131, and Asp 133 exhibited in the zoomed image. C , microscopic images of (−Tet) tetracycline uninduced (top) and (+Tet) induced ( bottom ) LtT7TR parasites expressing antisense LdTOPIA construct, Scale bar: 25 μm (ii) Graphical representation of percentage viable LtT7TR parasites in (−Tet) and (+Tet) condition for indicated time points. (n = 5 mean ± SD, 3 biological replicates. p vs. respective control (0h)). D , relative quantitation of LtTOPIA, LtTOPIL, and Ltβ-Tub mRNA expression levels in (+Tet) parasites measured by qPCR and plotted as normalized values over 24 h (n = 3 mean ± SD, 3 biological replicates. p versus tetracycline treated for 24 h). E , flow cytometric analysis of cell-cycle arrest in antisense LtTOPIA transfected LtT7TR parasites without (−Tet, green ) or with (+Tet, red ) induction at indicated timepoints (representative image of n = 3). F , graphical representation of the cell cycle phases (G 0 -G 1 , S, G 2- M, and 4N) for antisense LtTOPIA transfected LtT7TR parasites without (−Tet) or with (+Tet) induction for indicated time points (n = 5, mean ± SD, 3 biological replicates for each time).

Article Snippet: The conditional antisense strategy uses a commercially available L. tarentolae (LtT7TR) strain (Jena Bioscience) wherein the tetracycline repressor (TR) and the T7 RNA polymerase (T7) is integrated into the genome of this strain under the control of hygromycin and nourseothricin selectable markers, respectively ( E -i) ( , ).

Techniques: Sequencing, Comparison, Residue, Binding Assay, Expressing, Construct, Quantitation Assay, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Resolving the polycistronic aftermath: Essential role of topoisomerase IA in preventing R-loops in L eishmania

doi: 10.1016/j.jbc.2024.107162

Figure Lengend Snippet: Functional characterization of purified LdTOPIA. A , SDS-PAGE (10%) analysis of purified LdTOPIA, LdTOPIA Y357A , and LdTOPIA E135A from tetracycline induced, pLew100v5 cloned LdTOPIA, LdTOPIA Y357A , and LdTOPIA E135A transfected LtT7TR conditional expression system, stained with Coomassie G-250. Plasmid DNA relaxation assay using (−SC) pBluescript and ( B ) LdTOPIA or its active site mutants LdTOPIA Y357A and LdTOPIA E135A or ( C ) LdTOPIA along with Camptothecin (CPT) or Etoposide (Etop) and in presence of Mg 2+ at 37 °C for 25 min followed by electrophoresis in 1% agarose gel and thereafter EtBr staining for visualization. D , plasmid DNA relaxation assay using (−SC) pBluescript, increasing concentration of Mg 2+ and purified LdTOPIA at 37 °C for 15 min. E , bidirectional agarose gel electrophoresis using (−SC) pBluescript and reverse gyrase generated (+SC) pBluescript plasmid DNA incubated with Human TOPII and purified LdTOPIA in order to differentiate the relaxation of negative and positive topoisomers. F , electrophoretic mobility shift assay (EMSA) using 100 nM γ-32P labeled (i) single-stranded and (ii) double-stranded oligonucleotide substrates incubated with increasing concentrations of LdTOPIA (5–200) nM ( G ) DNA binding affinity was measured by fluorescence polarization using 5′ FAM tagged ssDNA and dsDNA substrate incubated with increasing concentration of LdTOPIA. Fraction-bound values were plotted against LdTOPIA concentration (2–200) nM and K D values of LdTOPIA were calculated for ssDNA and dsDNA (n = 5, mean ± SD, 3 biological replicates).

Article Snippet: The conditional antisense strategy uses a commercially available L. tarentolae (LtT7TR) strain (Jena Bioscience) wherein the tetracycline repressor (TR) and the T7 RNA polymerase (T7) is integrated into the genome of this strain under the control of hygromycin and nourseothricin selectable markers, respectively ( E -i) ( , ).

Techniques: Functional Assay, Purification, SDS Page, Clone Assay, Transfection, Expressing, Staining, Plasmid Preparation, Electrophoresis, Agarose Gel Electrophoresis, Concentration Assay, Generated, Incubation, Electrophoretic Mobility Shift Assay, Labeling, Binding Assay, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Resolving the polycistronic aftermath: Essential role of topoisomerase IA in preventing R-loops in L eishmania

doi: 10.1016/j.jbc.2024.107162

Figure Lengend Snippet: LdTOPIA prevents R-loop formation during polycistronic transcription. A , LtT7TR strain transfected with anti-LtTOPIA construct was either untreated or treated with tetracycline for indicated time points to observe the extent of R-loop formation. Parasites were stained with anti-LdTOPIA-AlexaFluor488, anti-DNA-RNA (S9.6) Alexa Fluor568 antibodies, and counterstained with DRAQ5. Scale Bar, 5 μm. B , graphical representation of the extent of R-loop formation estimated from the fluorescence intensity. [n = 60 (20 nuclei of 3 biological replicates), mean ± SD. p versus 0h]. C , DRIB assay was carried out from genomic DNA isolated from untreated or tetracycline-treated antisense LtTOPIA transfected LtT7TR parasites after indicated time points. D , densitometry of the same samples. [n = 3 and 3 biological replicates, mean ± SD, p values for (+Tet) versus (+Tet +RNaseH) and p versus 0h of (+Tet +RNaseH)]. E , DRIB assay was carried out using genomic DNA isolated from untreated or tetracycline-treated antisense LtTOPIA transfected LtT7TR parasites and complemented with LdTOPIA, LdTOPIAΔNLS or LdTOPIAΔNLS-SV40NLS for indicated time points. F , densitometric analysis of the same samples [n = 3 and 3 biological replicates, mean ± SD, p values for (+Tet) versus (+Tet +LdTOPIA) and p versus 0h of (+Tet +LdTOPIA)].

Article Snippet: The conditional antisense strategy uses a commercially available L. tarentolae (LtT7TR) strain (Jena Bioscience) wherein the tetracycline repressor (TR) and the T7 RNA polymerase (T7) is integrated into the genome of this strain under the control of hygromycin and nourseothricin selectable markers, respectively ( E -i) ( , ).

Techniques: Transfection, Construct, Staining, Fluorescence, Isolation

Journal: Nature Communications

Article Title: Nucleoside analogue activators of cyclic AMP-independent protein kinase A of Trypanosoma

doi: 10.1038/s41467-019-09338-z

Figure Lengend Snippet: Activators of trypanosome PKA. a Hit compounds with their EC 50 of in vivo PKA activity (VASP reporter assay, mean ± SD, n = 3 independent replicates, representative western blots as inset) and their EC 50 of in vitro kinase activity (kemptide phosphorylation by T. brucei PKAR-PKAC1 holoenzyme expressed in L. tarentolae ). A representative dose response for Toyo ( n = 5), 5-I-Tu ( n = 4), 5-Br-Tu ( n = 2), tubercidin ( n = 2), and sangivamycin ( n = 1) is shown with SD of technical duplicates or triplicates. Binding parameters to T. brucei PKAR(199–499) expressed in E. coli were determined by isothermal titration calorimetry (ITC). The power differential (DP) between the reference and sample cells upon injection was measured as a function of time (inset). The main plot presents the total heat exchange per mole of injectant (integrated peak areas from inset) as a function of the molar ratio of ligand to protein. One out of three independent replicates is shown. b Data for 7-CN-7-C-Ino as in a ; for number of independent replicates see Table . The EC 50 and K D values given in a and b are rounded values from Table . c Thermodynamic signature (Δ G in blue, Δ H in green, and − T Δ S in red) compiled from ITC measurements (mean ± SD of n = 3 independent replicates) in a and b . Source data are provided as a file

Article Snippet: The L. tarentolae strain LEXSY T7-TR (Jena Biosciences) was cultivated at 26.5 °C in BHI medium supplemented with 10 µg ml –1 hemin, 100 U L −1 streptomycin and 100 mg L −1 penicillin according to the protocols provided by Jena Biosciences.

Techniques: In Vivo, Activity Assay, Reporter Assay, Western Blot, In Vitro, Binding Assay, Isothermal Titration Calorimetry, Injection

Journal: The Journal of Biological Chemistry

Article Title: Flavone-resistant Leishmania donovani Overexpresses LdMRP2 Transporter in the Parasite and Activates Host MRP2 on Macrophages to Circumvent the Flavone-mediated Cell Death *

doi: 10.1074/jbc.M113.539742

Figure Lengend Snippet: A, analysis of MRP transporter levels associated with the resistant phenotype. Gene expression of LdABCC2 and LdABCC1 in WT and BLN-resistant promastigotes (Prom) and axenic amastigotes (Amas) was analyzed by RT-PCR. The products were run in a 1.5% agarose gel. B, relative expression of MRP2 gene in resistant parasites. Real time RT-PCR was carried out for 35 cycles using RNA isolated from the above stated parasites as well as 8-week BLN-unexposed parasites. The threshold cycle values were taken, and relative expression was calculated using GAPDH as internal control. The -fold expression of MRP2 transcript in each parasite was calculated using the 2−ΔΔCT method. The -fold expression in promastigotes and axenic amastigotes was plotted as means ± S.D. C, association of MRP2 gene with other resistant Leishmania strains. Gene expression of LdABCC2 in WT, SAG-resistant (GE1), CPT-resistant (CPTR), DIM-resistant (DIMR), BLN-resistant (pB25R), and BLN-treated BLN-resistant (pB25R+BLN) parasites and at the indicated weeks (wk) of BLN-unexposed BLN-resistant parasites was analyzed by RT-PCR. D, baicalein sensitivity of wild type, BLN-resistant, empty vector-transfected, and LdABCC2-transfected promastigotes. Parasites (2 × 106 cells/ml) were grown in M199 medium without phenol red and incubated with 25 μm CPT. Optical density was measured at 600 nm, and the percentage of live promastigotes over that of control was plotted. Results represent the means ± S.D. of experiments performed in triplicate. E, involvement of MRP2 gene in baicalein-resistant phenomenon. Baicalein-resistant (25 μm) L. tarentolae (LT.T7.TR) were developed in the laboratory and termed LTB25R. A 300-bp antisense construct of LdMRP2 was cloned in pLew82v4 vector (pLew/aMRP2). The empty vector (pLew82v4) or antisense construct (pLew/aMRP2) was transfected in LTB25R parasites and selected using phleomycin. The transformed parasites were grown in the presence or absence of BLN (20 μm) for 4 h, and the apoptotic population was quantified using flow cytometry in a BD FACSAria II cytometer and analyzed using BD FACScan software. PI, propidium iodide. F, enhanced DNA fragmentation in MRP2-down-regulated parasites. The above stated parasites were treated with 20 μm BLN for the indicated time periods. As a negative control, LTB25R parasites were also treated with 0.2% DMSO. Values were obtained from the Multiskan EX readings at 405 nm. The percentage was plotted as units of time. Data represent means ± S.D. (n = 3). G, decreased efflux of BLN in antisense construct (aMRP2)-transfected resistant L. tarentolae strains (LTB25R/aMRP2). Wild type L. tarentolae (LT.T7.TR), resistant parasites (LTB25R), and MRP2-down-regulated L. tarentolae (LTB25R/aMRP2) were loaded under conditions that yielded similar amounts of intracellular BLN, and the amount of BLN retained in parasites maintained after that in drug-free culture medium was measured at different time points up to 150 min. The data represent means ± S.D. The fitted lines (asymmetric) from these data points (n = 3) have R2 values of 0.9915, 0.9987, and 0.9841, respectively. Error bars represent S.D.

Article Snippet: Antisense Constructs The L. tarentolae T7.TR strain was procured from Jena Bioscience.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Quantitative RT-PCR, Isolation, Plasmid Preparation, Transfection, Incubation, Construct, Clone Assay, Transformation Assay, Flow Cytometry, Cytometry, Software, Negative Control