scholarly journals Metabolic Regulation Analysis of Ajmalicine Biosynthesis Pathway in Catharanthus roseus (L.) G. Don Suspension Culture Using Nanosensor

Processes ◽  
2020 ◽  
Vol 8 (5) ◽  
pp. 589
Author(s):  
Ghazala Ambrin ◽  
Hayssam M. Ali ◽  
Altaf Ahmad
Keyword(s):  
Metabolic Engineering ◽  
Cell Lines ◽  
Metabolic Regulation ◽  
Antihypertensive Drugs ◽  
Antioxidant Activities ◽  
Regulatory Gene ◽  
Indole Alkaloid ◽  
Resonance Energy ◽  
Flux Analysis ◽  
Suspension Cells

Ajmalicine is one of the most popular antihypertensive drugs obtained from the root barks of Cathranthus roseus (L.) G. Don and Rauvolfia serpentine (L.) Benth. ex Kurz. It has also potential antimicrobial, cytotoxic, central depressant and antioxidant activities. As the demand for the alkaloid is significantly high, metabolic engineering approaches are being tried to increase its production in both homologous and heterologous systems. The metabolic engineering approach requires knowledge of the metabolic regulation of the alkaloid. For understanding the metabolic regulation, fluxomic analysis is important as it helps in understanding the flux of the alkaloid through the complicated metabolic pathway. The present study was conducted to analyse the flux analysis of the ajmalicine biosynthesis, using a genetically encoded Fluorescent Resonance Energy Transfer FRET-based nanosensor for ajmalicine (FLIP-Ajn). Here, we have silenced six important genes of terpenoid indole alkaloid (TIA), namely G10H, 10HGO, TDC, SLS, STR and SDG, through RNA-mediated gene silencing in different batches of C. roseus suspension cells, generating six silenced cell lines. Monitoring of the ajmalicine level was carried out using FLIP-Ajn in these silenced cell lines, with high spatial and temporal resolution. The study offers the rapid, high throughput real-time measurement of ajmalicine flux in response to the silenced TIA genes, thereby identifying the regulatory gene controlling the alkaloid flux in C. roseus suspension cells. We have reported that the STR gene encoding strictosidine synthase of the TIA pathway could be the regulatory gene of the ajmalicine biosynthesis.

New Spirocyclic Hydroxamic Acids as Effective Antiproliferative Agents

2020 ◽  
Vol 20 ◽  
Author(s):  
Margarita E. Neganova ◽  
Sergey G. Klochkov ◽  
Yulia R. Aleksandrova ◽  
Vladimir N. Osipov ◽  
Dmitry V. Avdeev ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Histone Deacetylase ◽  
Cell Lines ◽  
Anticancer Agents ◽  
Antioxidant Activities ◽  
Hydroxamic Acids ◽  
Cytotoxic Activities ◽  
Tumor Cell Lines ◽  
Hek 293 ◽  
Underlying Mechanisms

Aims: The main goal of this work where is to synthesize new original spirocyclic hydroxamic acids, investigate their cytotoxicity against to the panel of tumor cell lines and possible mechanism of action of these active compounds. Background: Hydroxamic acids are one of the promising classes of chemical compounds with proven has anticancer potential properties. This is manifested in the presence of metal chelating and antioxidant activities, the ability to inhibit histone deacetylase enzymes and a chemosensitizing effect against well known cytostatics. Objective: Original spirocyclic hydroxamic acids were synthesized and spectrums of their antiproliferative activities were investigated. Methods: The cytotoxic activities on different tumor lines (SH-SY5Y, HeLa and healthy cells HEK-293) were investigated and determined possible underlying mechanisms of their activity. Result: New original spirocyclic hydroxamic acids were synthesized. These compounds exhibit antiproliferative properties against of the various tumor cultures cells and also exhibits antioxidant activity, a depolarizing effect on the mitochondrial membrane, inhibit the activity of the histone deacetylase enzyme, and also decrease of basal glycolysis and glycolytic capacity reserve of HeLa and SH-SY5Y tumor cell lines. Conclusion: The most promising are compounds 5j-l containing two chlorine atoms as substituents in the quinazoline part of the molecule and hydroxamate function. Therefore, these compounds can be considered as hit compounds for the development on their basis multi-target anticancer agents.

scholarly journals Cost-Effective Real-Time Metabolic Profiling of Cancer Cell Lines for Plate-Based Assays

Chemosensors ◽  
2021 ◽  
Vol 9 (6) ◽  
pp. 139
Author(s):  
Wiktoria Blaszczak ◽  
Zhengchu Tan ◽  
Pawel Swietach
Keyword(s):  
Real Time ◽  
Cell Lines ◽  
Acid Production ◽  
Metabolic Regulation ◽  
Cost Effective ◽  
Culture Conditions ◽  
Metabolic Inhibitors ◽  
Metabolic Phenotype ◽  
Ductal Adenocarcinoma ◽  
Oxygen Utilization

A fundamental phenotype of cancer cells is their metabolic profile, which is routinely described in terms of glycolytic and respiratory rates. Various devices and protocols have been designed to quantify glycolysis and respiration from the rates of acid production and oxygen utilization, respectively, but many of these approaches have limitations, including concerns about their cost-ineffectiveness, inadequate normalization procedures, or short probing time-frames. As a result, many methods for measuring metabolism are incompatible with cell culture conditions, particularly in the context of high-throughput applications. Here, we present a simple plate-based approach for real-time measurements of acid production and oxygen depletion under typical culture conditions that enable metabolic monitoring for extended periods of time. Using this approach, it is possible to calculate metabolic fluxes and, uniquely, describe the system at steady-state. By controlling the conditions with respect to pH buffering, O2 diffusion, medium volume, and cell numbers, our workflow can accurately describe the metabolic phenotype of cells in terms of molar fluxes. This direct measure of glycolysis and respiration is conducive for between-runs and even between-laboratory comparisons. To illustrate the utility of this approach, we characterize the phenotype of pancreatic ductal adenocarcinoma cell lines and measure their response to a switch of metabolic substrate and the presence of metabolic inhibitors. In summary, the method can deliver a robust appraisal of metabolism in cell lines, with applications in drug screening and in quantitative studies of metabolic regulation.

scholarly journals Antioxidant and cytotoxic studies of Acacia nilotica twig extract and their green synthesized silver nanoparticles

2020 ◽  
Vol 9 (2) ◽  
pp. 975-980 ◽  
Keyword(s):  
Antioxidant Activity ◽  
Silver Nanoparticles ◽  
Cell Lines ◽  
Antioxidant Activities ◽  
Reducing Power ◽  
Acacia Nilotica ◽  
Total Phenolic ◽  
Hek 293 ◽  
Selected Area Electron Diffraction ◽  
Wide Range

Acacia nilotica (L.) Delile is well known as “Desi Kikar”or Babul in India that possesses a wide range of pharmacological activities. In the present study, Acacia nilotica twig extract and its synthesized silver nanoparticles (AgNPs) were evaluated for total phenolic content (TPC), antioxidant activity and cytotoxic effects. Characterization of AgNPs was done by UV-Visible spectroscopy, Fourier-transform infrared spectroscopy (FTIR), Transmission electron microscopy (TEM) and Selected area electron diffraction (SAED) techniques. Antioxidant potential was determined using different assays including 2,2-diphenyl-1-picrylhydrazyl (DPPH), reducing power and β-carotene linoleic acid. Cytotoxicity was tested by 3-(4,5-dimethyl-2-yl)-2,5-diphynyl tetrazolium bromide (MTT) assay on Human embryonic kidney (HEK)-293 cell lines. The results indicated that AgNPs exhibited higher antioxidant activity (81.11 %) and TPC (57.35 mg of GAE/mL of extract) as compare to plant extract. A positive correlation was observed between the TPC and antioxidant activities. The inhibitory concentration (IC50) of A. nilotica extract and AgNPs was 52.08µg/mL and 56.82µg/mL respectively. Cytotoxicity against HEK-293 cell lines was dose dependent. Accordingly, it is summarized that A. nilotica based AgNPs could serve as a potential antioxidant for therapeutic purposes.

scholarly journals Designing and Development of FRET-Based Nanosensor for Real Time Analysis of N-Acetyl-5-Neuraminic Acid in Living Cells

2021 ◽  
Vol 8 ◽  
Author(s):  
Ruphi Naz ◽  
Mohammad K. Okla ◽  
Urooj Fatima ◽  
Mohd. Mohsin ◽  
Walid H. Soufan ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Metabolic Engineering ◽  
Sialic Acid ◽  
Real Time ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Neuraminic Acid ◽  
Yeast Cells ◽  
Metabolite Level ◽  
Health Maintenance

N-acetyl-5-neuraminic acid (NeuAc) plays crucial role in improving the growth, brain development, brain health maintenance, and immunity enhancement of infants. Commercially, it is used in the production of antiviral drugs, infant milk formulas, cosmetics, dietary supplements, and pharmaceutical products. Because of the rapidly increasing demand, metabolic engineering approach has attracted increasing attention for NeuAc biosynthesis. However, knowledge of metabolite flux in biosynthetic pathways is one of the major challenges in the practice of metabolic engineering. So, an understanding of the flux of NeuAc is needed to determine its cellular level at real time. The analysis of the flux can only be performed using a tool that has the capacity to measure metabolite level in cells without affecting other metabolic processes. A Fluorescence Resonance Energy Transfer (FRET)-based genetically-encoded nanosensor has been generated in this study to monitor the level of NeuAc in prokaryotic and eukaryotic cells. Sialic acid periplasmic binding protein (SiaP) from Haemophilus influenzae was exploited as a sensory element for the generation of nanosensor. The enhanced cyan fluorescent protein (ECFP) and Venus were used as Fluroscence Resonance Energy Transfer (FRET) pair. The nanosensor, which was termed fluorescent indicator protein for sialic acid (FLIP-SA), was successfully transformed into, and expressed in Escherichia coli BL21 (DE3) cells. The expressed protein of the nanosensor was isolated and purified. The purified nanosensor protein was characterized to assess the affinity, specificity, and stability in the pH range. The developed nanosensor exhibited FRET change after addition to NeuAc. The developed nanosensor was highly specific, exhibited pH stability, and detected NeuAc levels in the nanomolar to milimolar range. FLIP-SA was successfully introduced in bacterial and yeast cells and reported the real-time intracellular levels of NeuAc non-invasively. The FLIP-SA is an excellent tool for the metabolic flux analysis of the NeuAc biosynthetic pathway and, thus, may help unravel the regulatory mechanism of the metabolic pathway of NeuAc. Furthermore, FLIP-SA can be used for the high-throughput screening of E. coli mutant libraries for varied NeuAc production levels.

scholarly journals Assessment of Antioxidant and Cytotoxicity Activities of Saponin and Crude Extracts ofChlorophytum borivilianum

2013 ◽  
Vol 2013 ◽  
pp. 1-7 ◽  
Author(s):  
Mehdi Farshad Ashraf ◽  
Maheran Abd Aziz ◽  
Johnson Stanslas ◽  
Ismanizan Ismail ◽  
Mihdzar Abdul Kadir
Keyword(s):  
Cell Lines ◽  
Cancer Cell ◽  
Crude Extract ◽  
Antioxidant Activities ◽  
Radical Scavenging Activity ◽  
Radical Scavenging ◽  
Ferrous Ion ◽  
Total Saponin ◽  
Saponin Fraction ◽  
Mcf 7

The present paper focused on antioxidant and cytotoxicity assessment of crude and total saponin fraction ofChlorophytum borivilianumas an important medicinal plant. In this study, three different antioxidant activities (2,2-diphenyl-1-picrylhydrazyl radical scavenging (DPPH), ferrous ion chelating (FIC), andβ-carotene bleaching (BCB) activity) of crude extract and total saponin fraction ofC. borivilianumtubers were performed. Crude extract was found to possess higher free radical scavenging activity (ascorbic acid equivalents 2578 ± 111 mg AA/100 g) and bleaching activity (IC50= 0.7 mg mL−1), while total saponin fraction displayed higher ferrous ion chelating (EC50= 1 mg mL−1). Cytotoxicity evaluation of crude extract and total saponin fraction against MCF-7, PC3, and HCT-116 cancer cell lines using 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) cell viability assay indicated a higher cytotoxicity activity of the crude extract than the total saponin fraction on all cell lines, being most effective and selective on MCF-7 human breast cancer cell line.

scholarly journals Megakaryocytic cell line-specific hyperploidy by cytotoxic necrotizing factor bacterial toxins

Blood ◽  
1996 ◽  
Vol 88 (9) ◽  
pp. 3465-3473 ◽  
Author(s):  
KM Hudson ◽  
NC Denko ◽  
E Schwab ◽  
E Oswald ◽  
A Weiss ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Phorbol Ester ◽  
Dna Content ◽  
Nuclear Dna ◽  
Nuclear Dna Content ◽  
Adherent Cell ◽  
Target Cells ◽  
Suspension Cells ◽  
Cytotoxic Necrotizing Factor

Cytotoxic necrotizing factor (CNF) toxins, isolated from certain Escherichia coli strains known to cause intestinal and extra intestinal infections, induce reorganization of the actin cytoskeleton and generate hyperploidy in adherent cell lines. We have examined the effect of CNF toxin on one of the few cell types that naturally increase nuclear DNA content, megakaryocytes. Our studies show that only hematopoietic cells capable of differentiating along the megakaryocyte lineage responded to the CNF2 toxin by becoming polyploid and by reorganizing actin. The K562, HEL, and CHRF-288–11 cell lines can be induced with phorbol ester to differentiate along the megakaryocyte lineage, and these cells also respond to the toxin with increased DNA content and actin cytoskeletal rearrangements. Interestingly, treatment of the K562 and HEL cell lines with CNF2 does not result in an increase in production of the megakaryocytic marker glycoprotein IIIa, unlike phorbol ester treatment. Conversely, two T-cell leukemic cell lines, CEM and Molt4, and the promyelocytic HL-60 cell line, which do not differentiate along the megakaryocyte lineage in response to phorbol myristate acetate, do not respond to CNF2, by increased expression of gpIIIa, increased nuclear DNA content, or actin reorganization. A potential target of these toxins, RhoA, is expressed by both megakaryocytic and nonmegakaryocytic cell lines, as shown by reverse transcription-polymerase chain reaction and Western blot. Although it is clear that the CNF toxins can affect a wide variety of adherent nonhematopoietic cell lines, we propose that the response to CNF, in terms of reorganizing actin structure and increase in DNA content in hematologic suspension cells, correlates with the capability of these target cells to differentiate along the megakaryocytic lineage.

Comparison of Various Strategies Designed to Optimize Indole Alkaloid Accumulation of a Cell Suspension Culture of Catharanthus roseus

1987 ◽  
Vol 42 (9-10) ◽  
pp. 1101-1108 ◽  
Author(s):  
Jochen Berlin ◽  
Christiane Mollenschott ◽  
Frank DiCosmo
Keyword(s):  
Cell Suspension ◽  
Cell Lines ◽  
Growth Medium ◽  
Catharanthus Roseus ◽  
Indole Alkaloid ◽  
Dry Mass ◽  
Culture Conditions ◽  
Production Medium ◽  
Initial Inoculum ◽  
Time Required

The cell line CP-3 of Catharanthus roseus produces low levels of indole alkaloids during station­ary phase of growth, but increased levels when transferred to growth-limiting production media. Highest specific yields of alkaloids were usually achieved 8-15 days after transfer of the cells to the production medium. We attempted to reduce the time required for alkaloid production by altering preculture conditions, and by adding an elicitor preparation known to stimulate alkaloid accumulation in other cells. Reduction of 2,4-D in the last growth medium before transfer to production medium and a high initial inoculum load into the production medium resulted in rapid and greatest alkaloid accumulation (up to 2 mg ajmalicine/g dry mass or 40 mg/1 within 7 days). The addition of elicitor to the cell suspension stimulated high levels of tryptamine biosynthesis and accumulation under all culture conditions. Additionally, two cell lines derived from CP-3 cells and maintained on NAA/kinetin containing medium were selected for rapid growth in the absence of 2,4-D and were characterized with respect to their ajmalicine production. At a high inoculum load these cells produced up to 1.3 mg/g or 20 mg ajmalicine/1 when cultivated on the growth medium. The biotechnological utility of such cell lines is discussed.

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