Microtubule/Tubulin

In mice, germ cells, and frog spermatocytes, colcemid (Demecolcine) (0.1-0.25 μg/ml, 1 hour) lowers the frequency of hypoploidy of phase II complement [3].

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Demecolcine (DEM) treatment of oocytes induces formation of a membrane protrusion containing a mass of condensed maternal chromosomes, which can be removed with minimal damage prior to somatic cell nuclear transfer (SCNT). However, the effect of this method on the distribution of maturation-promoting factor (MPF) in porcine oocytes has not been reported. Here, the level of MPF and the distribution of cyclin B1 were assessed in porcine oocytes following DEM treatment. In addition, the efficiencies of DEM-assisted and mechanical enucleation were compared, as were the development (in vitro and in vivo) of these oocytes following SCNT. MPF was uniformly distributed in oocytes that had been treated with 0.4 μg/ml DEM for 1 h. Immunofluorescence microscopy showed that in untreated oocytes, cyclin B1, the regulatory subunit of MPF, accumulated around the spindle, and was lowly detected in the cytoplasm. DEM treatment disrupted spindle microtubules, induced chromosome condensation, and reduced the level of cyclin B1 in the nuclear region. Cyclin B1 was uniformly distributed in DEM-treated oocytes and the level of MPF was increased. The potential of embryos generated from DEM-treated oocytes to develop in vivo was significantly greater than that of embryos generated from mechanically enucleated oocytes. This is the first study to report the effects of DEM-assisted enucleation of porcine oocytes on the distribution of cyclin B1. MPF in mature oocytes is important for the development of reconstructed embryos and for efficient SCNT.[6]

The incidence of poor ploidy in stage II division is increased in mice treated with intraperitoneal injection of Demecolcine (Colcemid; 0.3 mg/kg) [3].

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Colcemid/Demecolcine was found to induce a dose and schedule dependent marrow magakaryocytosis and peripheral thrombocytosis. The response could be divided into early and late components. The early component appears to have been due to a direct stimulatory effect, probably by enhancement of endoreduplication in metaphase arrested megakaryocyte precursors. The ealy stimulatory response was blunted on toxic drug schedules. In contrast, the late component of the thrombopoietic response was demonstrated best on the most toxic drug schedules. It coincided temporally with the reactive restoration of the mononuclear marrow and blood cell elements, respectively. Thus, the late component appears to be a nonspecific rebound phenomenon. On comparing the thrombopoietic properties of Colcemid with those of the vinca alkaloids in experimental systems, the former appears to have a more favorable therapeutic index. The data suggest that colchicine and its derivatives may be useful agents in the treatment of clinical thrombocytopenic states. [The effects of colcemid on hematopoiesis in the mouse. [J Clin Invest. 1976 Nov;58(5):1280-5. https://pubmed.ncbi.nlm.nih.gov/993346/]

Experiment 1: Effects of Demecolcine (DEM) Treatment on the Level of MPF in MII Oocytes [6]
The effects of Demecolcine (DEM) treatment for various amounts of time on the level of MPF were examined. Oocytes were cultured in NCSU-37 containing 0.6 mM cysteine, 4 mg/ml bovine serum albumin (BSA), and DEM for 0.5, 1, 2, or 3 h. The optimal concentration of DEM to induce ooplasmic protrusions in porcine oocytes is 0.4 µg/ml [11], [22]. Control oocytes were cultured in medium lacking DEM. The level of MPF in oocytes (30 per treatment group) was determined.

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Experiment 2: Effects of Demecolcine (DEM) Treatment on the Distribution of Cyclin B1 in MII Oocytes [6]
Oocyte bisection [6]
Mature oocytes that had formed the first polar body were cultured in medium supplemented with 0.4 μg/ml Demecolcine (DEM) and 0.05 M sucrose for the optimal amount of time (determined in experiment 1). Sucrose was used to enlarge the perivitelline space. Oocytes were bisected by the extrusion method using compression with a blunt pipette tip. To determine whether chromosomes were aligned at the metaphase plate, oocytes were stained with Hoechst 33342, photographed, and washed three times in PBS. The oocyte halves were collected less than 30 min after bisection. Control oocytes were cultured in medium without supplements and bisected by removing half the volume of cytoplasm. For each treatment, the level of MPF was assayed in 25 whole oocytes or 50 oocytes halves.

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Immunofluorescence microscopy [6]
Immunofluorescence microscopy was performed on whole mounts. Demecolcine (DEM)-treated or untreated MII oocytes were fixed with PBS containing 4% paraformaldehyde for at least 30 min at room temperature. Cells were permeabilized with PBS containing 1% Triton X-100 for 10 min at 4°C, blocked in 1% BSA for 1 h, and incubated overnight at 4°C with an anti-cyclin B1 antibody diluted 1∶50 in blocking solution. After three washes in PBS containing 0.1% Tween 20 and 0.01% Triton X-100 for 5 min each, oocytes were labeled with FITC-conjugated goat anti-rabbit IgG diluted 1∶100 in blocking solution for 45 min at 37°C in the dark. Samples were incubated with 10 µg/ml Hoechst 33342 for 10 min to label DNA. Stained oocytes were mounted beneath a coverslip using antifade mounting medium to retard photo-bleaching. Slides were examined using laser-scanning confocal microscopy (Leica TCS SP5) and the appropriate filters to simultaneously excite FITC (cyclin B1 labeling) and Hoechst 33342 (DNA labeling).

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Experiment 3: Efficiencies of Demecolcine (DEM)-assisted and Mechanical Enucleation of Porcine Oocytes, and the Development of these Enucleated Oocytes Following SCNT [6]
Demecolcine (DEM)-assisted enucleation [6]
Demecolcine (DEM)-treated oocytes with a protruding membrane were moved to medium supplemented with 5 μg/ml cytochalasin B and 0.4 μg/ml DEM for the optimal amount of time (determined in experiment 1). The protrusion was removed using a beveled pipette, and the first polar body and a portion of the directly underlying cytoplasm were aspirated (Figure 1).

Animal/Disease Models: Mice [3]
Doses: 0.3 mg/kg
Route of Administration: intraperitoneal (ip) injection
Experimental Results: The frequency of hypoploidy and hyperploidy in metaphase II cells increased 7.8-8 times compared to the control.

220401 Human TDLo Oral 200 ug/kg Skin and appendages (skin): hair: Other drug and chemical toxicology, Deichmann, WB, New York, Academic Press, 1969, -(184), 1969
220401 Rat LD50 Intravenous 1700 ug/kg Arzneimittel-Forschung. Drug Research, 20(1467), 1970 [PMID:4991432]
220401 Rat LD50 Parenteral 1700 ug/kg Recent advances in cancer research, 52(76), 1975 [PMID:796916]
220401 Mouse LD50 Oral 25530 ug/kg Summary of data from the National Cancer Institute Screening Program, Developmental Therapy Program, January 1986
220401 Mouse LD50, intraperitoneal injection 35 mg/kg. Nauen-Schmidberg, Laboratory Pathology and Pharmacology, 230(559), 1957 [PMID:13526771]

[1]. Colcemid-induced apoptosis of cultured human glioma: electron microscopic and confocal laser microscopic observation of cells sorted in different phases of cell cycle. Cytometry. 1998 Apr 1;31(4):295-9.

[2]. Ultra-Low Colcemid Doses Induce Microtubule Dysfunction as Revealed by Super-Resolution Microscopy. Bioexiv. doi.org/10.1101/2020.08.13.249664

[3]. Discovery and engineering of colchicine alkaloid biosynthesis. Nature. 2020 Aug;584(7819):148-153. Erratum in: Nature. 2020 Jul 30.

[4]. Antitubulin effects of derivatives of 3-demethylthiocolchicine, methylthio ethers of natural colchicinoids, and thioketones derived from thiocolchicine. Comparison with colchicinoids. J Med Chem. 1990 Feb;33(2):567-71.

[5]. An improved method for cytogenetic analysis of meiotic aneuploidy in rodent and frog spermatocytes. Mutat Res. 1990 Dec;234(6):361-8.

[6]. Effect of demecolcine-assisted enucleation on the MPF level and cyclin B1 distribution in porcine oocytes. PLoS One. 2014 Mar 13;9(3):e91483.

(-)-Desmethylcolchicine is a secondary amino compound, a compound in which the N-acetyl group of (S)-colchicine is replaced by an N-methyl group. It can be isolated from Colchicum autumnale, is less toxic than colchicine, and is used as an antitumor drug. It has dual effects of antitumor activity and microtubule depolymerization. It is an alkaloid and also a secondary amino compound. Desmethylcolchicine has been reported to exist in Colchicum arenarium, Gagea lutea, and other organisms with relevant data. Desmethylcolchicine is a colchicine analog with potential antimitotic and antitumor activity. Norcolchic acid binds to the colchicine binding site of tubulin, inhibiting its polymerization to form microtubules, leading to cell cycle arrest at metaphase, thereby preventing cell division. Norcolchic acid is an alkaloid isolated from Colchicum autumnale L. and used as an antitumor drug. Currently, the biosynthetic pathways of plant-derived medicinal compounds are rarely fully elucidated. Therefore, many plant-derived therapeutics are directly isolated from medicinal plants or plant cell cultures.¹ Colchicine is a typical example; it is an FDA-approved drug for treating inflammatory diseases, derived from plants in the genera Colchicum and Gloriosa.^2-5 This article, combining transcriptomics, metabolic logic, and pathway reconstruction techniques, elucidates a nearly complete biosynthetic pathway of colchicine without prior knowledge of biosynthetic genes, genome sequences, or natural host genetic tools. We identified eight genes involved in the biosynthesis of N-formylcolchicine from Gloriosa superba. N-formylcolchicine is a precursor of colchicine, containing the colchicine-specific cyclophenol ring and pharmacophore⁶. Notably, we identified a non-classical cytochrome P450 that catalyzes a significant ring-expansion reaction necessary for the formation of the unique carbon skeleton of colchicine. We further utilized these newly discovered genes to construct an N-formylcolchicine biosynthetic pathway in tobacco (Nicotiana benthamiana) using phenylalanine and tyrosine as starting materials (containing a total of 16 enzymes). This study established a metabolic pathway for the synthesis of colchicine alkaloids containing tyrosine and revealed the unique chemical mechanism by which plants utilize simple amino acids to generate complex bioactive metabolites. [3] In summary, this study demonstrates for the first time that the norecolchicine (DEM)/DEM-assisted enucleation method has significant advantages over other enucleation techniques. During DEM-assisted enucleation, MPF is retained in the cytoplasm, while the content of cyclin B1 in the nucleus is reduced. After DEM-assisted enucleation, cyclin B1 is evenly distributed in the cytoplasm, and the overall level of this protein is increased, which corresponds to the enhancement of MPF activity. MPF is crucial for the development of reconstructed embryos and efficient somatic cell nuclear transfer (SCNT). Therefore, DEM-assisted enucleation appears to be the best method for producing cloned pigs using SCNT. [6]

C21H25NO5

371.43

371.173

C, 67.91; H, 6.78; N, 3.77; O, 21.54

477-30-5

477-30-5; 1246817-95-7 (HBr)

220401

White to off-white solid powder

1.2±0.1 g/cm3

625.5±55.0 °C at 760 mmHg

73-75ºC

332.1±31.5 °C

0.0±1.8 mmHg at 25°C

1.582

1.52

1

6

5

27

653

1

O(C([H])([H])[H])C1C(=C(C([H])=C2C=1C1=C([H])C([H])=C(C(C([H])=C1[C@]([H])(C([H])([H])C2([H])[H])N([H])C([H])([H])[H])=O)OC([H])([H])[H])OC([H])([H])[H])OC([H])([H])[H]

NNJPGOLRFBJNIW-HNNXBMFYSA-N

InChI=1S/C21H25NO5/c1-22-15-8-6-12-10-18(25-3)20(26-4)21(27-5)19(12)13-7-9-17(24-2)16(23)11-14(13)15/h7,9-11,15,22H,6,8H2,1-5H3/t15-/m0/s1

(7S)-1,2,3,10-tetramethoxy-7-(methylamino)-6,7-dihydro-5H-benzo[a]heptalen-9-one

C 12669; C-12669; Colcemid; Demecolcine; C12669; (-)-Demecolcine; Colchamine; Demecolcine; colcemid; 477-30-5; Colchamine; (-)-Demecolcine; Reichstein's F; Desmecolcine; Demecolcin; Colchamin

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~75 mg/mL (~201.9 mM)
Ethanol: ~75 mg/mL (~201.9 mM)
Water: ~75 mg/mL (~201.9 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.73 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.73 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.73 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)