scholarly journals CMT-308, a Nonantimicrobial Chemically-Modified Tetracycline, Exhibits Anti-Melanogenic Activity by Suppression of Melanosome Export

Biomedicines ◽  
2020 ◽  
Vol 8 (10) ◽  
pp. 411
Author(s):  
Shilpi Goenka ◽  
Sanford R. Simon
Keyword(s):  
Cell Cultures ◽  
Mammalian Cells ◽  
Selective Reduction ◽  
Mushroom Tyrosinase ◽  
Free System ◽  
Melanin Biosynthesis ◽  
Chemically Modified ◽  
B16f10 Cells ◽  
Chemically Modified Tetracycline

CMT-308 is a nonantimicrobial chemically-modified tetracycline (CMT), which we have previously shown exhibits antifungal activity and pleiotropic anti-inflammatory activities, including inhibition of the enzymatic activity of matrix metalloproteinases (MMPs). Based on its chemical structure, we hypothesized that CMT-308 could inhibit melanogenesis and might be a candidate for the treatment of skin hyperpigmentation disorders which occur due to unregulated melanin biosynthesis and/or transport. CMT-308 was first studied for any effects on activity of the enzyme tyrosinase in vitro using a purified preparation of mushroom tyrosinase; the mode of inhibition of the soluble fungal enzyme was evaluated by Lineweaver-Burk and Dixon plots as well as by non-linear least squares fitting. Next, the effects of CMT-308 were tested in mammalian cell cultures using B16F10 mouse melanoma cells and further validated in darkly-pigmented human melanocytes (HEMn-DP). Our results showed that micromolar concentrations of CMT-308 inhibited mushroom tyrosinase enzyme activity, using the first two substrates in the melanogenesis pathway (l-tyrosine and l-3,4-dihydroxyphenylalanine (l-DOPA)); CMT-308 inhibited mushroom tyrosinase primarily via a mixed mode of inhibition, with the major contribution from a competitive mode. In B16F10 cell cultures, CMT-308 (10 µM) significantly diminished total melanin levels with a selective reduction of extracellular melanin levels, under both basal and hormone-stimulated conditions without any cytotoxicity over a duration of 72 h. Studies of potential mechanisms of inhibition of melanogenesis in B16F10 cells showed that, in mammalian cells, CMT-308 did not inhibit intracellular tyrosinase activity or the activity of α-glucosidase, an enzyme that regulates maturation of tyrosinase. However, CMT-308 suppressed MITF protein expression in B16F10 cells and showed copper chelating activity and antioxidant activity in a cell-free system. The significantly lower extracellular melanin levels obtained at 10 µM indicate that CMT-308’s anti-melanogenic action may be attributed to a selective inhibition of melanosome export with the perinuclear aggregation of melanosomes, rather than a direct effect on the tyrosinase-catalyzed steps in melanin biosynthesis. These results were validated in HEMn-DP cells where CMT-308 suppressed dendricity in a fully reversible manner without affecting intracellular melanin synthesis. Furthermore, the capacity of CMT-308 to inhibit melanosome export was retained in cocultures of HEMn-DP and HaCaT. In summary, our results offer promise for therapeutic strategies to combat the effects of hyperpigmentation by use of CMT-308 at low micromolar concentrations.

Novel Amide Derivatives as Potent Tyrosinase Inhibitors; In-vitro, In-vivo Antimelanogenic Activity and Computational Studies

2019 ◽  
Vol 15 (7) ◽  
pp. 715-728 ◽  
Author(s):  
Anser Ali ◽  
Zaman Ashraf ◽  
Muhammad Rafiq ◽  
Ajeet Kumar ◽  
Farukh Jabeen ◽  
...  
Keyword(s):  
Molecular Docking ◽  
Tyrosinase Activity ◽  
Potential Candidate ◽  
Tyrosinase Inhibition ◽  
Free System ◽  
Melanin Biosynthesis ◽  
B16f10 Cells ◽  
Amide Derivatives

Background: Tyrosinase is involved in the melanin biosynthesis and the abnormal accumulation of melanin pigments leading to hyperpigmentation disorders. Controlling the melanogenesis could be an important strategy for treating abnormal pigmentation. Methods: In the present study, a series of amide derivatives (3a-e and 5a-e) were synthesized aiming to inhibit tyrosinase activity and melanin production. All derivatives were screened for tyrosinase inhibition in a cell-free system. The possible interactions of amide derivatives with tyrosinase enzyme and effect of these interactions on tyrosinase structure were checked by molecular docking in silico and by Circular Dichroism (CD) studies, respectively. The most potent amide derivative (5c) based on cell-free experiments, was further tested for cellular ROS inhibition and for tyrosinase activity using mouse skin melanoma (B16F10) cells. Results: The tyrosinase inhibitory concentration (IC50) for tested compounds was observed between the range of 68 to 0.0029 µg/ml with a lowest IC50 value of compound 5c which outperforms the reference arbutin and kojic acid. The cellular tyrosinase activity and melanin quantification assay demonstrate that 15µg/ml of 5c attenuates 36% tyrosinase, 24% melanin content of B16F10 cells without significant cell toxicity. Moreover, the zebrafish in vivo assay reveals that 5c effectively reduces melanogenesis without perceptible toxicity. Furthermore, the molecular docking demonstrates that compound 5c interacts with copper ions and multiple amino acids in the active site of tyrosinase with best glide score (-5.387 kcal/mol), essential for mushroom tyrosinase inhibition and the ability to diminish the melanin synthesis in-vitro and in-vivo. Conclusion: Thus, we propose compound 5c as a potential candidate to control tyrosinase rooted hyperpigmentation in the future.

scholarly journals Transcription of 4′-thioDNA templates to natural RNA in vitro and in mammalian cells

2015 ◽  
Vol 51 (37) ◽  
pp. 7887-7890 ◽  
Author(s):  
Hideto Maruyama ◽  
Kazuhiro Furukawa ◽  
Hiroyuki Kamiya ◽  
Noriaki Minakawa ◽  
Akira Matsuda
Keyword(s):  
Nucleic Acids ◽  
Mammalian Cells ◽  
Genetic Material ◽  
Rna Polymerases ◽  
Chemically Modified ◽  
Biological Studies ◽  
Modified Nucleic Acids

Synthetic chemically modified nucleic acids, which are compatible with DNA/RNA polymerases, have great potential as a genetic material for synthetic biological studies.

scholarly journals Electroporated recombinant proteins as tools for in vivo functional complementation, imaging and chemical biology

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Amal Alex ◽  
Valentina Piano ◽  
Soumitra Polley ◽  
Marchel Stuiver ◽  
Stephanie Voss ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
Mammalian Cells ◽  
Cellular Localization ◽  
Mitotic Checkpoint ◽  
Transferase Activity ◽  
Checkpoint Signaling ◽  
Chemically Modified ◽  
Promising Tool

Delivery of native or chemically modified recombinant proteins into mammalian cells shows promise for functional investigations and various technological applications, but concerns that sub-cellular localization and functional integrity of delivered proteins may be affected remain high. Here, we surveyed batch electroporation as a delivery tool for single polypeptides and multi-subunit protein assemblies of the kinetochore, a spatially confined and well-studied subcellular structure. After electroporation into human cells, recombinant fluorescent Ndc80 and Mis12 multi-subunit complexes exhibited native localization, physically interacted with endogenous binding partners, and functionally complemented depleted endogenous counterparts to promote mitotic checkpoint signaling and chromosome segregation. Farnesylation is required for kinetochore localization of the Dynein adaptor Spindly. In cells with chronically inhibited farnesyl transferase activity, in vitro farnesylation and electroporation of recombinant Spindly faithfully resulted in robust kinetochore localization. Our data show that electroporation is well-suited to deliver synthetic and chemically modified versions of functional proteins, and, therefore, constitutes a promising tool for applications in chemical and synthetic biology.

Inactivation of cdc2 kinase during mitosis requires regulated and constitutive proteins in a cell-free system

1993 ◽  
Vol 104 (3) ◽  
pp. 873-881
Author(s):  
F.A. Suprynowicz
Keyword(s):  
Mammalian Cells ◽  
Protein Phosphatase 1 ◽  
Cdc2 Kinase ◽  
Free System ◽  
Culture Cells ◽  
Anaphase Onset ◽  
Tissue Culture Cells ◽  
A Cell ◽  
Protein Components

Inactivation of the cyclin-p34cdc2 protein kinase complex is a major requirement for anaphase onset and exit from mitosis. To facilitate identification of specific molecules that regulate this event in mammalian cells, I have developed a cell-free assay in which cdc2 kinase associated with a chromosomal fraction from metaphase tissue culture cells is inactivated by a cell-cycle-regulated cytosolic system. In vitro kinase inactivation requires ATP, Mg2+ and the dephosphorylation of one or more sites in the chromosomal fraction by protein phosphatase 1 and/or 2A. Cyclin B is destroyed during inactivation, while the level of p34cdc2 remains constant. Ammonium sulfate fractionation resolves the cytosolic inactivating system into at least two distinct protein components that are both required for inactivation and are differentially regulated during mitosis.

scholarly journals GENETICS OF SOMATIC MAMMALIAN CELLS

1958 ◽  
Vol 108 (6) ◽  
pp. 945-956 ◽  
Author(s):  
Theodore T. Puck ◽  
Steven J. Cieciura ◽  
Arthur Robinson
Keyword(s):  
Cell Cultures ◽  
Mammalian Cells ◽  
Human Subjects ◽  
Calf Serum ◽  
Active Growth ◽  
Genetic Changes ◽  
Large Numbers ◽  
Normal Human

A methodology designed to eliminate mitotic inhibitor action and involving use of pretested fetal calf serum and careful pH and temperature control has been described by which cells from normal human and animal tissue can be maintained in active growth for long periods in vitro without development of aneuploidy. By means of this procedure, it is possible reliably to establish cell cultures from minute skin biopsies which can be taken from any individual. Clones of mammalian cells with chromosomal markers have been isolated by this means from x-irradiated non-irradiated cell cultures. Application of these techniques to chromosome delineation in large numbers of human subjects; determination of chromosomal sex in patients; spontaneuos and induced genetic changes in somatic mammalian cells in vivo and in vitro; comparison of metabolic differences between normal and cancerous cells and other problems have been indicated.

scholarly journals Inositol acylation of glycosylphosphatidylinositols in the pathogenic fungus Cryptococcus neoformansz and the model yeast Saccharomyces cerevisiae

1999 ◽  
Vol 340 (1) ◽  
pp. 25-32 ◽  
Author(s):  
Sarah P. FRANZOT ◽  
Tamara L. DOERING
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fatty Acids ◽  
Fatty Acid ◽  
Mammalian Cells ◽  
Pathogenic Fungus ◽  
Sugar Residue ◽  
Yeast Saccharomyces Cerevisiae ◽  
Free System ◽  
Life Threatening

Cryptococcus neoformans, an opportunistic fungus responsible for life-threatening infection in immunocompromised patients, is able to synthesize glycosylphosphatidylinositol (GPI) structures. Radiolabelling experiments in vitro with the use of a cryptococcal cell-free system showed that the pathway begins as in other eukaryotes, with the addition of N-acetylglucosamine to phosphatidylinositol, followed by deacetylation of the sugar residue. The third step, acylation of the inositol ring, seemed to involve a fatty acid other than palmitate, in contrast with previous findings in Saccharomyces cerevisiae and mammalian GPI pathways. A systematic study of inositol acylation in C. neoformans and S. cerevisiae showed that both organisms used a variety of fatty acids in this step; these were transferred directly from acyl-CoA to inositol without modification. However, the specificity of fatty acid utilization was quite distinct in the two fungi, with the pathogen being substantially more restrictive. In mammalian cells fatty acids added exogenously as acyl-CoAs are not transferred directly to inositol. These results suggest significant differences in the GPI biosynthetic pathway between mammalian and C. neoformans cells that could represent targets for anti-cryptococcal therapy.

scholarly journals The Antiaging Effect of Active Fractions and Ent-11α-Hydroxy-15-Oxo-Kaur-16-En-19-Oic Acid Isolated from Adenostemma lavenia (L.) O. Kuntze at the Cellular Level

Antioxidants ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 719
Author(s):  
Irmanida Batubara ◽  
Rika Indri Astuti ◽  
Muhammad Eka Prastya ◽  
Auliya Ilmiawati ◽  
Miwa Maeda ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Biological Activities ◽  
Antioxidant Properties ◽  
In Vitro Assays ◽  
Mitochondrial Activity ◽  
Related Factor ◽  
Dependent Manner ◽  
B16f10 Cells ◽  
Anti Inflammatory

Background: The extract of Adenostemma lavenia (L.) O. Kuntze leaves has anti-inflammatory activities and is used as a folk medicine to treat patients with hepatitis and pneumonia in China and Taiwan. The diterpenoid ent-11α-hydroxy-15-oxo-kaur-16-en-19-oic acid (11αOH-KA) is the major ingredient in the extract and has wide-spectrum biological activities, such as antitumor and antimelanogenic activities, as well as anti-inflammatory activity. However, the physical and biological properties of this compound as an antioxidant or antiaging agent have not been reported yet. Methods: In addition to in vitro assays, we monitored antioxidative and antiaging signals in Schizosaccharomyces pombe (yeast) and mouse melanoma B16F10 cells. Results: A. lavenia water and chloroform fractions showed antioxidant properties in vitro. The A. lavenia extracts and 11αOH-KA conferred resistance to H2O2 to S. pombe and B16F10 cells and extended the yeast lifespan in a concentration-dependent manner. These materials maintained the yeast mitochondrial activity, even in a high-glucose medium, and induced an antioxidant gene program, the transcriptional factor pap1+ and its downstream ctt1+. Accordingly, 11αOH-KA activated the antioxidative transcription factor NF-E2-related factor 2, NRF2, the mammalian ortholog of pap1+, in B16F10 cells, which was accompanied by enhanced hemeoxygenase expression levels. These results suggest that 11αOH-KA and A. lavenia extracts may protect yeast and mammalian cells from oxidative stress and aging. Finally, we hope that these materials could be helpful in treating COVID-19 patients, because A. lavenia extracts and NRF2 activators have been reported to alleviate the symptoms of pneumonia in model animals.

A Non-antibacterial Chemically-modified Tetracycline Inhibits Mammalian Collagenase Activity

1987 ◽  
Vol 66 (8) ◽  
pp. 1310-1314 ◽  
Author(s):  
L.M. Golub ◽  
T.F. McNamara ◽  
G. D'Angelo ◽  
R.A. Greenwald ◽  
N.S. Ramamurthy
Keyword(s):  
Male Rats ◽  
Toxicity Tests ◽  
Collagenolytic Activity ◽  
Collagenase Activity ◽  
Chemically Modified ◽  
Chemically Modified Tetracycline ◽  
Skin Collagen ◽  
Clinical Advantage

Tetracyclines (including the semi-synthetic analogues, minocycline and doxycycline) are considered useful adjuncts in periodontal therapy because they suppress Gram-negative periodontopathogens. Recently, these antibiotics were found to inhibit mammalian collagenase activity, a property which may also be of therapeutic value. It has been suggested that the anti-collagenase properties of the tetracyclines are independent of their antibiotic efficacy. To advance this hypothesis further, we chemically converted tetracycline hydrochloride to its non-antimicrobial analogue, de-dimethylaminotetracycline. This chemically-modified tetracycline (CMT), although no longer an effective antibiotic, was found to inhibit the in vitro activity of collagenase from partially purified extracts of human rheumatoid synovial tissue and rachitic rat epiphysis. In a preliminary in vivo study, pathologically-excessive collagenase in skin and gingiva was induced by rendering adult male rats diabetic, and the oral administration of CMT to these rats significantly reduced the excessive collagenase activity in both tissues. Moreover, CMT administration did not affect the severe hyperglycemia in these rats but did prevent, at least in part, the diabetes-induced loss of body weight, skin weight, and skin collagen mass; these effects suggest a lack of toxicity in this animal model. A proposed clinical advantage of CMT over conventional tetracyclines, in the treatment of diseases characterized by excessive collagenolytic activity, is the lack of development of antibiotic-resistant micro-organisms during prolonged use. However, the consideration of clinical trials to support this hypothesis must await further laboratory and extensive toxicity tests.

scholarly journals Ku-Dependent Nonhomologous DNA End Joining inXenopus Egg Extracts

1999 ◽  
Vol 19 (4) ◽  
pp. 2585-2593 ◽  
Author(s):  
Paul Labhart
Keyword(s):  
Mammalian Cells ◽  
High Efficiency ◽  
End Joining ◽  
Free System ◽  
Egg Extract ◽  
Egg Extracts ◽  
Dependent Protein ◽  
Immunological Assays ◽  
Dna Ends

ABSTRACT An extract from activated Xenopus eggs joins both matching and nonmatching ends of exogenous linear DNA substrates with high efficiency and fidelity (P. Pfeiffer and W. Vielmetter, Nucleic Acids Res. 16:907–924, 1988). In mammalian cells, such nonhomologous end joining (NHEJ) is known to require the Ku heterodimer, a component of DNA-dependent protein kinase. Here I investigated whether Ku is also required for the in vitro reaction in the egg extract. Immunological assays indicate that Ku is very abundant in the extract. I found that all NHEJ was inhibited by autoantibodies against Ku and that NHEJ between certain combinations of DNA ends was also decreased after immunodepletion of Ku from the extract. The formation of a joint between a DNA end with a 5′-protruding single strand (PSS) and an end with a 3′-PSS, between two ends with 3′-PSS, and between two blunt ends was most Ku dependent. On the other hand, NHEJ between two DNA ends bearing 5′-PSS was Ku independent. These results show that theXenopus cell-free system will be useful to biochemically dissect the role of Ku in eukaryotic NHEJ.

scholarly journals Mushroom tyrosinase inhibitory activity and major fatty acid constituents of Amazonian native flora oils

2012 ◽  
Vol 48 (3) ◽  
pp. 399-404 ◽  
Author(s):  
Raquel da Silva Teixeira ◽  
Paula Rafaela Rocha ◽  
Hudson Caetano Polonini ◽  
Marcos Antônio Fernandes Brandão ◽  
Maria das Graças Afonso Miranda Chaves ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Inhibitory Activity ◽  
Major Fatty Acid ◽  
Mushroom Tyrosinase ◽  
Melanin Biosynthesis ◽  
Tyrosinase Inhibitory Activity ◽  
Native Flora ◽  
Global Trend ◽  
Main Components

In order to treat hyperpigmentation-related problems, there has been a global trend in developing cosmetics claiming to have skin-whitening properties, which act by inhibiting melanin biosynthesis. The objective of this work was to evaluate the in vitro mushroom tyrosinase inhibitory activity of five Amazonian native flora oils, and so to verify the possibility of their incorporation into cosmetic products. In addition, the fatty acid composition of the essential oils was determined by gas chromatography-flame ionisation detection in order to determine the main components of these oils. The tyrosinase inhibitory activity of the tested oils was found to be in the following order: açaí (IA50 = 66.08 µg mL-1) > tucumã > patauá > pracaxi > castanha do Brasil. This study suggests that açaí oil has great potential in the treatment of hyperpigmentation and other related disorders, due to its considerable tyrosinase inhibitory activity.

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