Effect of Calmodulin-like Gene (CML) Overexpression on Stilbene Biosynthesis in Cell Cultures of Vitis amurensis Rupr.

Stilbenes are plant phenolics known to rapidly accumulate in grapevine and other plants in response to injury or pathogen attack and to exhibit a great variety of healing beneficial effects. It has previously been shown that several calmodulin-like protein (CML) genes were highly up-regulated in cell cultures of wild-growing grapevine Vitis amurensis Rupr. in response to stilbene-modulating conditions, such as stress hormones, UV-C, and stilbene precursors. Both CML functions and stilbene biosynthesis regulation are still poorly understood. In this study, we investigated the effect of overexpression of five VaCML genes on stilbene and biomass accumulation in the transformed cell cultures of V. amurensis. We obtained 16 transgenic cell lines transformed with the VaCML52, VaCML65, VaCML86, VaCML93, and VaCML95 genes (3–4 independent lines per gene) under the control of the double CaMV 35S promoter. HPLC-MS analysis showed that overexpression of the VaCML65 led to a considerable and consistent increase in the content of stilbenes of 3.8–23.7 times in all transformed lines in comparison with the control calli, while biomass accumulation was not affected. Transformation of the V. amurensis cells with other analyzed VaCML genes did not lead to a consistent and considerable effect on stilbene biosynthesis in the cell lines. The results indicate that the VaCML65 gene is implicated in the signaling pathway regulating stilbene biosynthesis as a strong positive regulator and can be useful in viticulture and winemaking for obtaining grape cultivars with a high content of stilbenes and stress resistance.

Download Full-text

The Influence of the Grapevine Bacterial and Fungal Endophytes on Biomass Accumulation and Stilbene Production by the In Vitro Cultivated Cells of Vitis amurensis Rupr.

Plants ◽

10.3390/plants10071276 ◽

2021 ◽

Vol 10 (7) ◽

pp. 1276

Author(s):

Olga A. Aleynova ◽

Andrey R. Suprun ◽

Nikolay N. Nityagovsky ◽

Alexandra S. Dubrovina ◽

Konstantin V. Kiselev

Keyword(s):

Cell Culture ◽

Cell Suspension ◽

Endophytic Fungi ◽

Endophytic Bacteria ◽

Fungal Endophytes ◽

Biomass Accumulation ◽

Vitis Amurensis ◽

Grape Cell ◽

Stilbene Biosynthesis

Plant endophytes are known to alter the profile of secondary metabolites in plant hosts. In this study, we identified the main bacterial and fungal representatives of the wild grape Vitis amurensis Rupr. microbiome and investigated a cocultivation effect of the 14 endophytes and the V. amurensis cell suspension on biomass accumulation and stilbene biosynthesis. The cocultivation of the V. amurensis cell culture with the bacteria Agrobacterium sp., Bacillus sp., and Curtobacterium sp. for 2 weeks did not significantly affect the accumulation of cell culture fresh biomass. However, it was significantly inhibited by the bacteria Erwinia sp., Pantoea sp., Pseudomonas sp., and Xanthomonas sp. and fungi Alternaria sp., Biscogniauxia sp., Cladosporium sp., Didymella sp. 2, and Fusarium sp. Cocultivation of the grapevine cell suspension with the fungi Didymella sp. 1 and Trichoderma sp. resulted in cell death. The addition of endophytic bacteria increased the total stilbene content by 2.2–5.3 times, while the addition of endophytic fungi was more effective in inducing stilbene accumulation by 2.6–16.3 times. The highest content of stilbenes in the grapevine cells cocultured with endophytic fungi was 13.63 and 13.76 mg/g of the cell dry weight (DW) after cultivation with Biscogniauxia sp. and Didymella sp. 2, respectively. The highest content of stilbenes in the grapevine cells cocultured with endophytic bacteria was 4.49 mg/g DW after cultivation with Xanthomonas sp. The increase in stilbene production was due to a significant activation of phenylalanine ammonia lyase (PAL) and stilbene synthase (STS) gene expression. We also analyzed the sensitivity of the selected endophytes to eight antibiotics, fluconazole, and trans-resveratrol. The endophytic bacteria were sensitive to gentamicin and kanamycin, while all selected fungal strains were resistant to fluconazole with the exception of Cladosporium sp. All endophytes were tolerant of trans-resveratrol. This study showed that grape endophytes stimulate the production of stilbenes in grape cell suspension, which could further contribute to the generation of a new stimulator of stilbene biosynthesis in grapevine or grape cell cultures.

Download Full-text

The effect of explant origin and collection season on stilbene biosynthesis in cell cultures of Vitis amurensis Rupr.

Plant Cell Tissue and Organ Culture (PCTOC) ◽

10.1007/s11240-018-1490-x ◽

2018 ◽

Vol 136 (1) ◽

pp. 189-196 ◽

Cited By ~ 2

Author(s):

A. P. Tyunin ◽

A. R. Suprun ◽

N. N. Nityagovsky ◽

A. Y. Manyakhin ◽

Y. A. Karetin ◽

...

Keyword(s):

Cell Cultures ◽

Vitis Amurensis ◽

Stilbene Biosynthesis

Download Full-text

The effect of stress hormones, UV-C, and stilbene precursors on calmodulin (CaM) and calmodulin-like gene (CML) expression in Vitis amurensis Rupr

Plant Cell Tissue and Organ Culture (PCTOC) ◽

10.1007/s11240-021-02042-1 ◽

2021 ◽

Author(s):

Konstantin V. Kiselev ◽

Olga A. Aleynova ◽

Zlata V. Ogneva ◽

Andrey R. Suprun ◽

Alexey A. Ananev ◽

...

Keyword(s):

Stress Hormones ◽

Vitis Amurensis ◽

Uv C

Download Full-text

Hepatoma Cell Cultures as In Vitro Models for the Hepatotoxicity of Xenobiotics

Alternatives to Laboratory Animals ◽

10.1177/026119298801600108 ◽

1988 ◽

Vol 16 (1) ◽

pp. 32-37

Author(s):

Margherita Ferro ◽

Anna Maria Bassi ◽

Giorgio Nanni

Keyword(s):

Cell Lines ◽

Cell Cultures ◽

Membrane Lipids ◽

Hepatoma Cell ◽

Positive Control ◽

In Vitro Models ◽

Low Concentrations ◽

Formation Efficiency ◽

Dose Dependent

Two hepatoma cell cultures were examined as in vitro models to be used in genotoxicity and cytotoxicity tests without the addition of bioactivating enzymes. The MH1C1, and HTC hepatoma lines were used in this study to establish their sensitivity to a number of xenobiotics, namely, cyclophosphamide (CP), the classical positive control in bioactivation tests; benzaldehyde (BA), a short-chain aldehyde; and 4-hydroxynonenal (HNE), a major toxic end-product of the peroxidative degradation of cell membrane lipids. As a first approach, we compared the following cytotoxicity tests: release of lactate dehydrogenase (LDH), and colony formation efficiency (CF). Colony-forming cells were exposed to the drugs according to different procedures, before or after the anchorage phase. The leakage of LDH into the medium following exposure of both cell lines to HNE, CP and BA for up to 24 hours was found not to be a good index of cytotoxicity. A better indicator of cytotoxicity was CF, as evaluated by exposure of the cells 24 hours after seeding. The effects were detectable at very low concentrations, corresponding to 10, 90 and 100μM for HNE, CP and BA, respectively. The impairment of CF efficiency was dose-dependent and time-dependent, and several differences between the two cell lines were observed.

Download Full-text

Differential heat shock response of primary human cell cultures and established cell lines

Biochemical and Biophysical Research Communications ◽

10.1016/s0006-291x(86)80332-1 ◽

1986 ◽

Vol 141 (1) ◽

pp. 46-52 ◽

Cited By ~ 11

Author(s):

Werner W. Richter ◽

Olaf-Georg Issinger

Keyword(s):

Heat Shock ◽

Cell Lines ◽

Cell Cultures ◽

Human Cell ◽

Heat Shock Response ◽

Differential Heat ◽

Established Cell Lines ◽

Human Cell Cultures ◽

Shock Response ◽

Established Cell

Download Full-text

Non-breeding feather concentrations of testosterone, corticosterone and cortisol are associated with subsequent survival in wild house sparrows

Proceedings of The Royal Society B Biological Sciences ◽

10.1098/rspb.2011.2062 ◽

2011 ◽

Vol 279 (1733) ◽

pp. 1560-1566 ◽

Cited By ~ 71

Author(s):

Lee Koren ◽

Shinichi Nakagawa ◽

Terry Burke ◽

Kiran K. Soma ◽

Katherine E. Wynne-Edwards ◽

...

Keyword(s):

Stress Hormones ◽

Passer Domesticus ◽

House Sparrows ◽

Long Term Study ◽

Darwinian Fitness ◽

Liquid Chromatography Tandem Mass ◽

In The Wild ◽

The Subject ◽

As Stress

Potential mechanistic mediators of Darwinian fitness, such as stress hormones or sex hormones, have been the focus of many studies. An inverse relationship between fitness and stress or sex hormone concentrations has been widely assumed, although empirical evidence is scarce. Feathers gradually accumulate hormones during their growth and provide a novel way to measure hormone concentrations integrated over time. Using liquid chromatography–tandem mass spectrometry, we measured testosterone, corticosterone and cortisol in the feathers of house sparrows ( Passer domesticus ) in a wild population which is the subject of a long-term study. Although corticosterone is considered the dominant avian glucocorticoid, we unambiguously identified cortisol in feathers. In addition, we found that feathers grown during the post-nuptial moult in autumn contained testosterone, corticosterone and cortisol levels that were significantly higher in birds that subsequently died over the following winter than in birds that survived. Thus, feather steroids are candidate prospective biomarkers to predict the future survival of individuals in the wild.

Download Full-text

Growth of Nosema cuniculi in established cell lines

Laboratory Animals ◽

10.1258/002367775780994835 ◽

1975 ◽

Vol 9 (1) ◽

pp. 61-68 ◽

Cited By ~ 9

Author(s):

T. Waller

Keyword(s):

Cell Lines ◽

Cell Cultures ◽

Growth Patterns ◽

Rabbit Kidney ◽

Kidney Cells ◽

Scale Production ◽

Simple Method ◽

Established Cell Lines ◽

Established Cell ◽

Canine Kidney

Growth patterns of Nosema cuniculi ( Encephalitozoon cuniculi) in cell cultures of bovine kidney, canine kidney, feline lung, and rabbit kidney were studied. All cell cultures used were easy to manage and the last 3 are commercially-available established cell lines. The dog kidney cells were the most suitable for large-scale production of Nosema. When grown in plastic flasks with a bottom area of 75 cm2, the weekly yield from Nosema-infected canine kidney cells during the 10th to 17th week after inoculation was between 4·1 x 107 and 9·9 x 107 spores per flask. An equilibrium was obtained between the Nosema infection and the kidney cells during this time. A simple method for estimating the numbel of harvested spores is also described.

Download Full-text

Antimigratory effect of berberine in T98G, U87MG and primary glioma cell culture.

Journal of Clinical Oncology ◽

10.1200/jco.2021.39.15_suppl.e15045 ◽

2021 ◽

Vol 39 (15_suppl) ◽

pp. e15045-e15045

Author(s):

Irina V. Mezhevova ◽

Svetlana Yu. Filippova ◽

Sofia V. Timofeeva ◽

Anastasia O. Sitkovskaya ◽

Tatiana V. Shamova ◽

...

Keyword(s):

Cell Culture ◽

Cell Lines ◽

Cell Cultures ◽

Glioma Cell ◽

Glioma Cells ◽

Surgical Removal ◽

Experimental Sample ◽

Wound Area ◽

Migratory Activity ◽

Primary Glioma

e15045 Background: Berberine is an alkaloid compound with a structure that is highly similar to that of intercalating agents. It affects numerous cell signaling pathways and is widely studied as potential anticancer drug. It is known that berberine affects cancer cells migration through metalloproteinase-2 inhibition, but this effect was never studied on glioma cells. Anti-migratory drugs are of special interest in brain cancer therapy since glioma's highly invasive nature makes total surgical removal of tumor practically impossible. The aim of the study was to evaluate berberine anti-migratory activity on glioma cells. Methods: Cell migration capacity of T98G and U87MG cell lines, as well as primary glioma cell culture established in our laboratory, was assessed via standard wound healing assay with automated image acquisition and analysis on Lionheart FX (BioTek) cell imager. Prior to assay setting up cell cultures were maintained in DMEM medium with L-glutamine (1 μM) (Gibco) and 10% FBS (Gibco) at 37C0 and 5.0% CO2. Cells were seeded at 250 000 cells per well on 24-well plates and incubated overnight in order to attach to plate bottom. After that a vertical wound was made manually in each well, and berberine was added to experimental wells to final concentration 50 mg/L. Plates with cells were continuously incubated and photographed in cell imager at 37C0 and 5.0% CO2. The extent of cells migration was measured as the percent of wound area decrease after 24 hours of incubation in relation to starting time point. Data are given as: Mean ± 95% confidence interval. Results: In our study we berberine exhibited anti-migratory activity in all cell cultures under study. In rather fast growing primary cell culture wound area decrease was 99.23%±0.62% in control sample and 91.75%±0.28% in experimental sample. The difference was small but significant at p < 0.001 level (df = 30). Popular permanent glioma cell lines T98G and U87MG showed more prominent decrease in studied parameter with higher degree of variance at the same time. In T98G wound area decrease was 71.6%±12.3% in control and 48.8%± 7.6% in experimental samples after 24 hours of cultivation in presence of 50 mg/L berberine. While U87MG demonstrated 60.28%±5.13% and 37.5%± 8.34% wound area decrease accordingly. The obtained difference between control and experimental groups in permanent cell cultures was statistically significant at the 0.05 level (df = 30). Conclusions: Our preliminary research proved berberine to be potent anti-migratory agent in glioma treatment. Further investigations are needed to evaluate its ability to inhibit glioma cell expansion in vivo.

Download Full-text