Cis aconitate

Manufactured by Merck Group

Sourced in United States

Cis-aconitate is a chemical compound used as a laboratory reagent. It is a dicarboxylic acid that is an intermediate in the citric acid cycle, a key metabolic pathway. The compound can be used in various chemical and biochemical applications that require a source of this specific organic acid.

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22 protocols using cis aconitate

Comprehensive Cardiac Iron Homeostasis Analysis

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Heart tissue (50–100 mg) was homogenized in the buffer (10 µl/mg tissue) containing: 20 mM HEPES pH 7.9, 1.5 mM MgCl₂,10 mM KCl, 0.5 mM EDTA, 2% glycerol, 1% NP-40 (all chemicals from Sigma) and Complete Protease Inhibitor Cocktail (Thermo Scientific). Homogenate was centrifuged 20000 × g for 20 min. Supernatant was collected, portioned and frozen in liquid nitrogen. The total protein concentration were determined by Bradford method.
Aconitase activity was measured spectrophotometrically by monitoring the disappearance of cis-aconitate at 240 nm in 50 mM HEPES pH 7.4 (Sigma), 0.4 mM cis-aconitate (Sigma) and 80 µg protein extract at room temperature. Units represent nanomoles of substrate consumed per minute (ε_{240 nm} = 3.6 mM⁻¹ cm⁻¹).
Iron responsive element binding protein (IRP) 1 and 2, hepcidin, transferrin receptor 1 (TfR1), divalent metal transporter 1 (DMT1), ferroportin (Fpn) as well as ferritin light (l) chain, ferritin heavy (h) chain and mitochondrial (Mito) ferritin concentrations were assayed by ELISA according to the manufacturer’s instructions (all from Cloud-Clone).

Paterek A., Kępska M., Sochanowicz B., Chajduk E., Kołodziejczyk J., Polkowska-Motrenko H., Kruszewski M., Leszek P., Mackiewicz U, & Mączewski M. (2018). Beneficial effects of intravenous iron therapy in a rat model of heart failure with preserved systemic iron status but depleted intracellular cardiac stores. Scientific Reports, 8, 15758.

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Itaconic Acid Production Assay

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Enzyme activity was measured according to Dwiarti et al. (34 (link)) Assays were performed in triplicate. In general, 125 µL 0.2 M sodium phosphate buffer or sodium acetate buffer was mixed on ice with 15 µL enzyme in GF buffer and 10 µL cis-aconitate (Sigma-Aldrich #A3412), pH 6.5 (NaOH). Incubation for 10 min at 37 °C was immediately followed by heat inactivation of the enzyme at 95 °C for 15 min. The protein precipitate was pelleted by centrifugation. Supernatants were filtered using Spin-X 0.22-µm centrifuge tube filters and were acidified with 100 µL 100 mM H₃PO₄. Itaconic acid was measured by HPLC (Licrosphere C-8 reverse-phase column, 1 mL/min 10 mM H₃PO₄, detection at 210 nm).

Chen F., Lukat P., Iqbal A.A., Saile K., Kaever V., van den Heuvel J., Blankenfeldt W., Büssow K, & Pessler F. (2019). Crystal structure of cis-aconitate decarboxylase reveals the impact of naturally occurring human mutations on itaconate synthesis. Proceedings of the National Academy of Sciences of the United States of America, 116(41), 20644-20654.

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Induction of Differentiation in Trypanosomes

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L224, Bdf2^KO, or Bdf3^kd trypanosomes were treated for 23 d with I-BET151 and Dox, respectively. Cells were washed twice in media to eliminate I-BET151 or Dox, resuspended in HMI-9 and subjected to 24 h of treatment at 27°C, or in 6mM cis-aconitate (Sigma A3412). Control cells were kept at 37°C. Cells were stained with anti-EP1 (Cedarlane CLP001A) and an anti-mouse IgG FITC-labeled secondary antibody (BD 349031) and analyzed by flow cytometry.

Schulz D., Mugnier M.R., Paulsen E.M., Kim H.S., Chung C.W., Tough D.F., Rioja I., Prinjha R.K., Papavasiliou F.N, & Debler E.W. (2015). Bromodomain Proteins Contribute to Maintenance of Bloodstream Form Stage Identity in the African Trypanosome. PLoS Biology, 13(12), e1002316.

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Metabolic Enzyme Assay Reagents

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Nicotine amide adenine dinucleotide (NAD) disodium salt, 3-phosphoglycerate [D-(–)-3-phosphoglyceric acid disodium salt], l-homoserine, d-homoserine, l-homocysteine, 2-oxoglutarate (α-ketoglutaric acid sodium salt), oxaloacetate, cis-aconitate, molecular weight standards (Gel Filtration Markers Kit for Protein Molecular Weights) for size exclusion chromatography analyses, and Murashige–Skoog (MS) vitamin solution were purchased from Sigma–Aldrich Co., Ltd. (St. Louis, MO, USA). The other amino acids, the salt mixture of MS medium, Good’s buffers, NADH (β-diphosphopyridine nucleotide disodium salt, reduced form), fumarate, malate (l-malate), citrate (citric acid monohydrate), and isocitrate [trisodium ( ± )-isocitrate n-hydrate] were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

Okamura E, & Hirai M.Y. (2017). Novel regulatory mechanism of serine biosynthesis associated with 3-phosphoglycerate dehydrogenase in Arabidopsis thaliana. Scientific Reports, 7, 3533.

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Bloodstream Parasite Differentiation

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Bloodstream parasites were spun down and resuspended in differentiation media [21 (link)] supplemented with 6 mM cis-aconitate (Sigma-Aldrich A3412) at 27˚C for three days.

Walsh M.E., Naudzius E.M., Diaz S.J., Wismar T.W., Martchenko Shilman M, & Schulz D. (2020). Identification of clinically approved small molecules that inhibit growth and affect transcript levels of developmentally regulated genes in the African trypanosome. PLoS Neglected Tropical Diseases, 14(3), e0007790.

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Procyclic Differentiation Assays for Trypanosomes

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Procyclic differentiation assays refer to the previous literature with modifications as described (McDonald et al., 2018 (link)). Pleomorphic T. brucei (Tbp1) parasites, laboratory-adapted monomorphic passages (includingTbp40, Tbp80, Tbp120) and monomorphic T. evansi and T. equiperdum parasites, were purified from whole blood and resuspended at 2 × 10⁶ cell ml^–1 in SDM-79 + 10% FBS (v/v) media supplemented with 6 mM cis-aconitate (Sigma) to induce procyclic differentiation. These were then incubated at 27°C in 5% CO₂. After 1, 6, 12, 24, or 48 h cultivation (depending on the strains), 1 ml of the cell culture was concentrated at 1000 g, and then air-dried smears were prepared from the parasite pellets and fixed in 1% paraformaldehyde for stage-specific procyclin coat expression analysis by immunofluorescence. Meanwhile, CA was removed from the medium after 6- or 24-h treatments and the parasites were used for subculture in vitro.

Cai X.L., Li S.J., Zhang P., Li Z., Hide G., Lai D.H, & Lun Z.R. (2022). The Occurrence of Malignancy in Trypanosoma brucei brucei by Rapid Passage in Mice. Frontiers in Microbiology, 12, 806626.

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Trypanosome Differentiation Monitoring

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Differentiation from slender to insect procyclic form was induced by 3 mM cis‐Aconitate (Sigma‐Aldrich), with a temperature shift from 37°C to 28°C and switching the medium to Differentiating Trypanosome Medium (DTM) as previously described 34. The assessment of the differentiation process was monitored by a double IF to detect the expression of the surface glycoproteins using anti‐procyclin and anti‐VSG221 antibodies.

Saura A., Iribarren P.A., Rojas‐Barros D., Bart J.M., López‐Farfán D., Andrés‐León E., Vidal‐Cobo I., Boehm C., Alvarez V.E., Field M.C, & Navarro M. (2019). SUMOylated SNF2PH promotes variant surface glycoprotein expression in bloodstream trypanosomes. EMBO Reports, 20(12), e48029.

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Transformation of Bloodstream Form Trypanosoma brucei into Procyclic Culture Forms

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Bloodstream form (BSF) Trypanosoma brucei brucei strain MSUS/CI/78/TSW 196 [26] (link) was transformed into procyclic culture forms (PFs) by suspension in DTM medium with a final cis-aconitate (Sigma) concentration of 3 mM and cultured at 26°C [27] (link). Transformed trypanosomes were initially subcultured and maintained in a 1∶1 mix of DTM and SDM-79 media (Gibco), but subsequently cultured with SDM-79.

Ooi C.P., Haines L.R., Southern D.M., Lehane M.J, & Acosta-Serrano A. (2015). Tsetse GmmSRPN10 Has Anti-complement Activity and Is Important for Successful Establishment of Trypanosome Infections in the Fly Midgut. PLoS Neglected Tropical Diseases, 9(1), e3448.

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Mitochondrial Aconitase Assay

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Primaquine, sodium l-ascorbate, DL-isocitric acid, l-malic acid, n-acetyl-l-cysteine, mitochondrial aconitase from porcine heart and cis-aconitate were purchased from Sigma Aldrich.

Lalève A., Vallières C., Golinelli-Cohen M.P., Bouton C., Song Z., Pawlik G., Tindall S.M., Avery S.V., Clain J, & Meunier B. (2015). The antimalarial drug primaquine targets Fe–S cluster proteins and yeast respiratory growth. Redox Biology, 7, 21-29.

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Assay for Isocitrate Dehydrogenase Activity

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Protocol was adapted from (7 (link)). Cells are washed with cold PBS and lysed by addition of lysis buffer [40 mM KCl, 25 mM tris-Cl (pH 7.5), 1% Triton X-100, 0.1 M fresh DTT, 1 M Na citrate, and 1 M MnCl₂] with a protease inhibitor cocktail (Sigma-Aldrich, 5892791001). After 10-min incubation on ice, lysates are collected and centrifuged at 4°C at 21,000g for 10 min. Supernatant is collected, and protein concentration is quantified using a BCA protein assay kit (Pierce, 23225). Twelve micrograms of protein was loaded into 8% polyacrylamide/TBE gel supplemented with 1 M Na citrate and run at 175 V for 4 hours in running buffer [25 mM tris base, 192 mM glycine, and 3.6 mM citrate (pH 8)]. Gel was washed with distilled water and incubated at 37°C for 5 to 45 min in reaction solution [100 mM tris (pH 8.0), 1 mM nicotinamide adenine dinucleotide phosphate (Sigma-Aldrich, 10128031001), 2.5 mM cis-aconitate (Sigma-Aldrich, A3412), 5 mM MgCl₂, 1.2 mM MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Thermo Fisher Scientific, M6494), 0.3 mM phenazine methosulfate (Sigma-Aldrich, P9625), and isocitrate dehydrogenase (5 U/ml; Sigma-Aldrich, I2002)]. Gel is washed three times with distilled water for 5 min to remove background and gel is imaged.

Terzi E.M., Sviderskiy V.O., Alvarez S.W., Whiten G.C, & Possemato R. (2021). Iron-sulfur cluster deficiency can be sensed by IRP2 and regulates iron homeostasis and sensitivity to ferroptosis independent of IRP1 and FBXL5. Science Advances, 7(22), eabg4302.

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