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 Toxicology of Drugs and Chemicals, Deichmann, W.B., New York, Academic Press, Inc., 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 Results in Cancer Research., 52(76), 1975 [PMID:796916]
220401 mouse LD50 oral 25530 ug/kg National Cancer Institute Screening Program Data Summary, Developmental Therapeutics Program., JAN1986
220401 mouse LD50 intraperitoneal 35 mg/kg Naunyn-Schmiedeberg's Archiv fuer Experimentelle Pathologie und Pharmakologie., 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.

(-)-demecolcine is a secondary amino compound that is (S)-colchicine in which the N-acetyl group is replaced by an N-methyl group. Isolable from the autumn crocus, Colchicum autumnale, it is less toxic than colchicine and is used as an antineoplastic. It has a role as an antineoplastic agent and a microtubule-destabilising agent. It is an alkaloid and a secondary amino compound.
Demecolcine has been reported in Colchicum arenarium, Gagea lutea, and other organisms with data available.
Demecolcine is a colchicine analog with potential antimitotic and antineoplastic activities. Demecolcine acid binds to the colchicine-binding site of tubulin, inhibiting its polymerization into microtubules, causing cell cycle arrest at metaphase and preventing cell division.
An alkaloid isolated from Colchicum autumnale L. and used as an antineoplastic.

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Few complete pathways have been established for the biosynthesis of medicinal compounds from plants. Accordingly, many plant-derived therapeutics are isolated directly from medicinal plants or plant cell culture1. A lead example is colchicine, a US Food and Drug Administration (FDA)-approved treatment for inflammatory disorders that is sourced from Colchicum and Gloriosa species2-5. Here we use a combination of transcriptomics, metabolic logic and pathway reconstitution to elucidate a near-complete biosynthetic pathway to colchicine without prior knowledge of biosynthetic genes, a sequenced genome or genetic tools in the native host. We uncovered eight genes from Gloriosa superba for the biosynthesis of N-formyldemecolcine, a colchicine precursor that contains the characteristic tropolone ring and pharmacophore of colchicine6. Notably, we identified a non-canonical cytochrome P450 that catalyses the remarkable ring expansion reaction that is required to produce the distinct carbon scaffold of colchicine. We further used the newly identified genes to engineer a biosynthetic pathway (comprising 16 enzymes in total) to N-formyldemecolcine in Nicotiana benthamiana starting from the amino acids phenylalanine and tyrosine. This study establishes a metabolic route to tropolone-containing colchicine alkaloids and provides insights into the unique chemistry that plants use to generate complex, bioactive metabolites from simple amino acids.[3]

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In conclusion, this study is the first to demonstrate that Demecolcine (DEM)/DEM-assisted enucleation of oocytes has marked advantages over other enucleation techniques. In DEM-assisted enucleation, MPF is retained in the cytoplast and the amount of cyclin B1 in the nuclear region is decreased. Following DEM-assisted enucleation of oocytes, cyclin B1 is homogenously distributed in the cytoplasm and the level of the protein is increased overall, which corresponds to increased MPF activity. MPF is crucial for the development of reconstructed embryos and for efficient SCNT. Thus, DEM-assisted enucleation appears to be the best method to produce cloned pigs by 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.)