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Paromomycin

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Paromomycin is a broad-spectrum antibiotic that is used as a laboratory reagent. It inhibits protein synthesis in bacteria, protozoa, and some fungi.

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Lab products found in correlation

Amphotericin b, by Merck Group (7 mentions) Tobramycin, by Merck Group (6 mentions) Amikacin, by Merck Group (6 mentions) Miltefosine, by Merck Group (4 mentions) Neomycin, by Merck Group (4 mentions) Trivalent antimony, by Merck Group (4 mentions) L glutamine, by Thermo Fisher Scientific (4 mentions) Apramycin, by Merck Group (3 mentions) Gentamicin, by Merck Group (3 mentions) Usp grade, by Thermo Fisher Scientific (3 mentions) Triton x 100, by Merck Group (3 mentions) Hygromycin, by Merck Group (3 mentions) Bleomycin, by Thermo Fisher Scientific (3 mentions) Spectinomycin, by Merck Group (3 mentions) Kanamycin b, by Merck Group (3 mentions) Geneticin, by Merck Group (3 mentions) Hygromycin b, by Merck Group (3 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions) Potassium antimonyl tartrate, by Merck Group (2 mentions) Kanamycin, by Merck Group (2 mentions)

57 protocols using paromomycin

1

Determining ssh1 Gene Mutation by AFLP

Cited 2 times
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The gene mutated in ssh1 was determined by AFLP analysis (Kathir et al., 2003 (link)) in combination with the Chlamydomonas genome database (genome.jgi-psf.org/Chlre4/Chlre4.home.html) and flagella proteome database (Pazour et al., 2005 (link)). The primers used in this study are listed in Supplemental Table S2 of Kubo et al. (2014) (link).
To generate pf28pf30tpg1::TTLL9HA strains, pf28pf30tpg1 was cotransformed with a construct encoding TTLL9-HA (Kubo et al., 2014 (link)) and the paromomycin resistance gene and selected on a TAP agar plate containing 10 μg/ml paromomycin (Sigma, St. Louis, MO). Expression of TTLL9-HA was confirmed by Western blotting using anti–HA-tag antibody (3F10).
Kubo T., Hirono M., Aikawa T., Kamiya R, & Witman G.B. (2015). Reduced tubulin polyglutamylation suppresses flagellar shortness in Chlamydomonas. Molecular Biology of the Cell, 26(15), 2810-2822.
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2

Quantifying Stop Codon Readthrough and Amino Acid Misincorporation

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HuH-7 sgRNA-ctrl and HuH-7 sgRNA-24 cells were seeded in 12 well plates at 30,000 cells/well. 24 hrs later cells were transfected using Lipofectamine 2000 (Invitrogen) with 0.1 μg per well of the indicated luciferase reporter construct. Cells were lysed after 24 hrs in passive lysis buffer and Rluc and Fluc activity was assessed using the Dual-luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions and using a Glomax microplate luminometer (Promega). For stop codon readthrough experiments, performed in the presence of paromomycin, 1 mg/ml paromomycin (Sigma) was added to cells 8 hrs post-transfection. To measure stop codon readthrough (%), normalized Fluc activity (Fluc/Rluc) from UAG or UGA stop codon readthrough luciferase reporters was further normalized to a control construct, which does not have a stop codon between Rluc and Fluc as described (Jack et al., 2011 (link)). To measure amino acid misincorporation (%), normalized Fluc activity (Fluc/Rluc) from CGC or AAU amino acid misincorporation luciferase reporters was normalized to a control construct, which does not contain a point mutation in Fluc as described (Fujii et al., 2018 (link)). The amino acid misincorporation (%) or stop codon readthrough (%) values from the indicated number of independent experiments in HuH-7 sgRNA-ctrl and sgRNA-24 cells are shown.
McMahon M., Contreras A., Holm M., Uechi T., Forester C.M., Pang X., Jackson C., Calvert M.E., Chen B., Quigley D.A., Luk J.M., Kelley R.K., Gordan J.D., Gill R.M., Blanchard S.C, & Ruggero D. (2019). A single H/ACA small nucleolar RNA mediates tumor suppression downstream of oncogenic RAS. eLife, 8, e48847.
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3

In Vitro Evaluation of Antileishmanial Compounds

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Miltefosine (MIL), paromomycin (PMM) and potassium antimonyl tartrate (SbIII) were purchased from Sigma Aldrich (Diegem, Belgium); sodium stiboglucanate (SbV) was obtained from Calbiochem (EMD Millipore Corporation, MA, USA); amphotericin B (AmB, Fungizone®) was obtained from Bristol-Myers-Squib (Gilead Sciences, CA, USA). The lead compounds (Table S1) in the nitroimidazole, oxaborole and aminopyrazole chemical series were selected after a SAR analysis and provided by DNDi (Geneva, Switzerland). Stock solutions for the in vitro assays were prepared in PBS for MIL (20 mM) and Sb (5.12 mg/mL) and in demineralized water for PMM (20 mM). AmB stock solutions (5.4 mM) were freshly prepared in 5% dextrose. Stock solutions (20 mM) of the DNDi lead compounds were prepared in DMSO and stored at 4 °C until use. Stock solutions were further diluted in demineralized water, except for DNDI-6148 that was diluted in 0.25% methylcellulose in water. In the in vitro assays, the final in-test concentration of DMSO was <1%.
Van den Kerkhof M., Mabille D., Chatelain E., Mowbray C.E., Braillard S., Hendrickx S., Maes L, & Caljon G. (2018). In vitro and in vivo pharmacodynamics of three novel antileishmanial lead series. International Journal for Parasitology: Drugs and Drug Resistance, 8(1), 81-86.
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4

Antibiotics for Pharmacological Experiments

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Paromomycin, neomycin, gentamicin, tobramycin, kanamycin, amikacin, and apramycin were obtained from Sigma.
Perez-Fernandez D., Shcherbakov D., Matt T., Leong N.C., Kudyba I., Duscha S., Boukari H., Patak R., Dubbaka S.R., Lang K., Meyer M., Akbergenov R., Freihofer P., Vaddi S., Thommes P., Ramakrishnan V., Vasella A, & Böttger E.C. (2014). 4′-O-substitutions determine selectivity of aminoglycoside antibiotics. Nature Communications, 5, 3112.
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5

Transfection and antibiotic treatment of HeLa cells

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HeLa cells were cultured at 37 °C and 5% CO2 in low glucose Dulbecco’s minimal essential medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum (FCS), 1% glutamine, 100 units/mL penicillin and 100 µg/mL streptomycin. 1 × 105 cells were seeded per well in a 12-well plate and transfected using Effectene transfection reagent (Qiagen) according to the manufacturer’s instructions. Transfection reagent was removed six hours after transfection. The cells were treated with the respective TRIDs and incubated for another 18 h. Amikacin, erythromycin, josamycin, paromomycin, tobramycin and tylosin were obtained from Sigma-Aldrich, gentamicin and G418 from Carl Roth. Stock solutions were made in water, except for erythromycin (70% ethanol).
Schilff M., Sargsyan Y., Hofhuis J, & Thoms S. (2021). Stop Codon Context-Specific Induction of Translational Readthrough. Biomolecules, 11(7), 1006.
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6

Characterization of Chlamydomonas RSP6 Mutant

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The parental strain CC-5325 and an insertional mutant in RSP6 (CLiP ID: LMJ.RY0402.248886) from the Chlamydomonas Library Project (CLiP; https://www.chlamylibrary.org)32 (link) were obtained from the Chlamydomonas resource center and maintained on Tris-Acetate-Phosphate (TAP) solid media (1.6% agar, USP grade, Thermo Fisher Scientific) with Hutner’s trace elements68 and 20 mg/mL paromomycin (Sigma). After isolation of single colonies and confirmation of the insertion site in the RSP6 mutant by PCR (Extended Data Fig. 9b), antibiotics was eliminated from the media. The resemblance in swimming phenotype of the CLiP RSP6 mutant and other reported RSP6 mutants33 (link), as well as the ability to restore motility in the CLiP RSP6 by complementation with wild-type RSP6 (Fig. 6a–b and Extended Data Fig. 9d), were considered as indications that the examined swimming phenotype of the CLiP RSP6 mutant was largely due to disruption of the RSP6 gene. Prior to motility analysis cells were grown in liquid TAP under constant illumination.
Grossman-Haham I., Coudray N., Yu Z., Wang F., Zhang N., Bhabha G, & Vale R.D. (2020). Structure of the radial spoke head and insights into its role in mechanoregulation of ciliary beating. Nature structural & molecular biology, 28(1), 20-28.
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7

Characterization of Chlamydomonas RSP6 Mutant

Cited 2 times
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The parental strain CC-5325 and an insertional mutant in RSP6 (CLiP ID: LMJ.RY0402.248886) from the Chlamydomonas Library Project (CLiP; https://www.chlamylibrary.org)32 (link) were obtained from the Chlamydomonas resource center and maintained on Tris-Acetate-Phosphate (TAP) solid media (1.6% agar, USP grade, Thermo Fisher Scientific) with Hutner’s trace elements68 and 20 mg/mL paromomycin (Sigma). After isolation of single colonies and confirmation of the insertion site in the RSP6 mutant by PCR (Extended Data Fig. 9b), antibiotics was eliminated from the media. The resemblance in swimming phenotype of the CLiP RSP6 mutant and other reported RSP6 mutants33 (link), as well as the ability to restore motility in the CLiP RSP6 by complementation with wild-type RSP6 (Fig. 6a–b and Extended Data Fig. 9d), were considered as indications that the examined swimming phenotype of the CLiP RSP6 mutant was largely due to disruption of the RSP6 gene. Prior to motility analysis cells were grown in liquid TAP under constant illumination.
Grossman-Haham I., Coudray N., Yu Z., Wang F., Zhang N., Bhabha G, & Vale R.D. (2020). Structure of the radial spoke head and insights into its role in mechanoregulation of ciliary beating. Nature structural & molecular biology, 28(1), 20-28.
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8

Diverse Compound Collection for Drug Discovery

Cited 1 time
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Amphotericin B, miltefosine, and paromomycin were purchased from Sigma-Aldrich (St. Louis, MO). Pentostam was obtained from Calbiochem (EMD Millipore, Billericca, MA). Sitamaquine and pentamidine were obtained from the Walter Reed Army Institute of Research Chemical Repository. The Malaria Box, a set of 400 diverse compounds assembled by MMV, was acquired from MMV. This collection consists of 200 diverse drug-like compounds suitable for oral administration and 200 diverse probe-like compounds for use as biological tools for drug discovery and development for neglected diseases. This collection was distilled from 20,000 hits generated by an extensive screening campaign of 4 million compounds conducted by St. Judes Children's Research Hospital, Novartis, and Glaxo-Smith Kline.9 (link)–12 (link) The selection was made to provide the broadest cross section of structural diversity and, in the case of the drug-like compounds, properties commensurate with oral absorption and the minimum presence of toxicophores.
Khraiwesh M., Leed S., Roncal N., Johnson J., Sciotti R., Smith P., Read L., Paris R., Hudson T., Hickman M, & Grogl M. (2016). Antileishmanial Activity of Compounds Derived from the Medicines for Malaria Venture Open Access Box against Intracellular Leishmania major Amastigotes. The American Journal of Tropical Medicine and Hygiene, 94(2), 340-347.
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9

Antimony-Based Leishmania Treatment Assay

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Trivalent antimony (SbIII), amphotericin B (AmB), paromomycin, Triton X-100, paraformaldehyde, 4′,6-diamidino-2-phenylindole dilactate (DAPI), n-dodecyl-β-D-maltoside (DDM), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and phorbol 12-myristate 13-acetate (PMA) were obtained from Sigma-Aldrich (St. Louis, USA). Miltefosine was purchased from Æterna Zentaris (Frankfurt, Germany). Glucantime® was purchased from Sanofi-Aventis (Paris, France). L-glutamine and penicillin/streptomycin were obtained from Gibco. All chemicals were of the highest quality available.
Gómez Pérez V., García-Hernandez R., Corpas-López V., Tomás A.M., Martín-Sanchez J., Castanys S, & Gamarro F. (2016). Decreased antimony uptake and overexpression of genes of thiol metabolism are associated with drug resistance in a canine isolate of Leishmania infantum. International Journal for Parasitology: Drugs and Drug Resistance, 6(2), 133-139.
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10

Cultivation and Screening of Chlamydomonas Transformants

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Chlamydomonas reinhardtii strains 137c cells were grown in Tris-acetate-phosphate (TAP) medium. To screen transformants, cells were grown on TAP agar supplemented with paromomycin (10 μg/ml; Sigma-Aldrich, St. Louis, MO). C. reinhardtii strains used for this study are listed in Table 1.
Oda T., Yanagisawa H, & Kikkawa M. (2015). Detailed structural and biochemical characterization of the nexin-dynein regulatory complex. Molecular Biology of the Cell, 26(2), 294-304.
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