Correlation between acidic potassium permanganate chemiluminescence and in vitro cell culture assay : physiologically meaningful antioxidant activity

There is great interest in the activity of antioxidant molecules, including polyphenols, from food and plant sources. Acidic potassium permanganate chemiluminescence signal intensity was shown to predict the ability of polyphenols to positively act on cellular redox state and attenuate oxidative stress in cultured skeletal muscle cells.

Assessing health risk of reclaimed water using human cell culture.

For risk analysis of reclaimed water, current animal and toxicity testing may not detect subtle effects such as interactions that could contribute to complex diseases such as cancers that develop over a long period of time. There is a need for assays that can be validated against known human physiological processes. We have previously validated sensitive human cell culture assays for their responsiveness to agents that induce carcinogenesis in vivo. In this initial study we analysed the effects of three batches of reclaimed water on human colonic cells. At concentrations of up to 10-fold, they had no significant effect on the cellular markers, indicating an overall lack of biological activity. The assay has potential but needs to be refined to maximise its sensitivity.

Electromagnetic field and other physical methods influencing cell growth in mammal cell culture systems

The growth rate of cultured mammalian cells can be influenced by chemical and physical methods such as electromagnetic fields (EMF), light, temperature and plasma. These physical methods have a number of well documented effects on mammalian cells including modification of gene expression, cell cycle, invasion, motility, cell viability, proliferation, apoptosis and mammosphere numbers. A study of the existing literature confirms that the impact of physical method on mammalian cells depends on the cell type, culture environment, exposure time, frequency, wave shape, and amount of dose. The modification of cell proliferation and apoptosis is necessary for cells products, tissue engineering, and therapy. In this article, we reviewed the impact of four physical methods on the growth rate and viability of cells. Plasma is the best method among fours because we can get desired result ranging from increasing cell proliferation to inducing apoptosis depending on the dose.

Bioconversion of α-Linolenic Acid into n-3 Long-Chain polyunsaturated fatty acid in hepatocytes and ad hoc cell culture optimisation

This study aimed to establish optimal conditions for a cell culture system that would allow the measurement of 18:3n-3 (ALA) bioconversion into n-3 long-chain polyunsaturated fatty acid (n-3 LC-PUFA), and to determine the overall pathway kinetics. Using rat hepatocytes (FaO) as model cells, it was established that a maximum 20:5n-3 (EPA) production from 50 mM ALA initial concentration was achieved after 3 days of incubation. Next, it was established that a gradual increase in the ALA concentration from 0 up to 125mM lead to a proportional increase in EPA, without concomitant increase in further elongated or desaturated products, such as 22:5n-3 (DPA) and 22:6n-3 (DHA) in 3 day incubations. Of interest, ALA bioconversion products were observed in the culture medium. Therefore, in vitro experiments disregarding the medium fatty acid content are underestimating the metabolism efficiency. The novel application of the fatty acid mass balance (FAMB) method on cell culture system (cells with medium) enabled quantifying the apparent enzymatic activities for the biosynthesis of n-3 LC-PUFA. The activity of the key enzymes was estimated and showed that, under these conditions, 50% (Km) of the theoretical maximal (Vmax = 3654 mmol.g21 of cell protein.hour21) Fads2 activity on ALA can be achieved with 81 mM initial ALA. Interestingly, the apparent activity of Elovl2 (20:5n-3 elongation) was the slowest amongst other biosynthesis steps. Therefore, the possible improvement of Elovl2 activity is suggested toward a more efficient DHA production from ALA. The present study proposed and described an ad hoc optimised cell culture conditions and methodology towards achieving a reliable experimental platform, using FAMB, to assist in studying the efficiency of ALA bioconversion into n-3 LC-PUFA in vitro. The FAMB proved to be a powerful and inexpensive method to generate a detailed description of the kinetics of n-3 LC-PUFA biosynthesis enzymes activities in vitro.

Copper and lactational hormones influence the CTR1 copper transporter in PMC42-LA mammary epithelial cell culture models

Adequate amounts of copper in milk are critical for normal neonatal development, however the mechanisms regulating copper supply to milk have not been clearly defined. PMC42-LA cell cultures representative of resting, lactating and suckled mammary epithelia were used to investigate the regulation of the copper uptake protein, CTR1. Both the degree of mammary epithelial differentiation (functionality) and extracellular copper concentration greatly impacted upon CTR1 expression and its plasma membrane association. In all three models (resting, lactating and suckling) there was an inverse correlation between extracellular copper concentration and the level of CTR1. Cell surface biotinylation studies demonstrated that as extracellular copper concentration increased membrane associated CTR1 was reduced. There was a significant increase in CTR1 expression (total and membrane associated) in the suckled gland model in comparison to the resting gland model, across all copper concentrations investigated (0-50 μM). Regulation of CTR1 expression was entirely post-translational, as quantitative real-time PCR analyses showed no change to CTR1 mRNA between all models and culture conditions. X-ray fluorescence microscopy on the differentiated PMC42-LA models revealed that organoid structures distinctively accumulated copper. Furthermore, as PMC42-LA cell cultures became progressively more specialised, successively more copper accumulated in organoids (resting

In vitro response to functionalized self-assembled peptide scaffolds for three-dimensional cell culture

Nanomaterials are rich in potential, particularly for the formation of scaffolds that mimic the landscape of the host environment of the cell. This niche arises from the spatial organization of a series of biochemical and biomechanical signals. Self-assembling peptides have emerged as an important tool in the development of functional (bio-)nanomaterials; these simple, easily synthesized subunits form structures which present the properties of these larger, more complex systems. Scaffolds based upon these nanofibrous matrices are promising materials for regenerative medicine as part of a new methodology in scaffold design where a "bottom-up" approach is used in order to simulate the native cellular milieu. Importantly, SAPs hold the potential to be bioactive through the presentation of biochemical and biomechanical signals in a context similar to the natural extracellular matrix, making them ideal targets for providing structural and chemical support in a cellular context. Here, we discuss a new methodology for the presentation of biologically relevant epitopes through their effective presentation on the surface of the nanofibers. Here, we demonstrate that these signals have a direct effect on the viability of cells within a three-dimensional matrix as compared with an unfunctionalized, yet mechanically and morphologically similar system. © 2014 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 102: 197-205, 2014.

Ceruloplasmin is regulated by copper and lactational hormones in PMC42-LA mammary epithelial cell culture models

Ceruloplasmin (Cp) is a multicopper ferroxidase that is considered to be an important source of copper in milk for normal neonatal development. We investigated the expression, subcellular localization and secretion of Cp in PMC42-LA cell culture models representative of resting, lactating and suckled human mammary epithelia. Both secreted Cp (sCp) and plasma membrane associated glycosylphosphatidylinositol-linked Cp (GPI-Cp) were expressed in PMC42-LA cells. In all three epithelial models (resting, lactating and suckled), the expression and secretion of copper-bound, ferroxidase active, Cp (holo-Cp) was dependent on media copper concentration. In low copper (bathocuproinedisulphonic acid/d-penicillamine treated models) there was greater than a 2-fold decrease in holo-Cp expression and secretion, which was mirrored by a 2-fold increase in the expression and secretion of copper-free Cp protein (apo-Cp). Cell surface biotinylation studies revealed that the state of PMC42-LA cell differentiation (functionality), and the level of extracellular copper, had no significant effect on the level of plasma membrane bound GPI-Cp. Quantitative real time PCR analyses determined that there was no significant (P > 0.05) difference in Cp mRNA levels across all copper conditions investigated (0, 5, 50 μM). However, there was a significant (P < 0.05) increase (∼2-fold) in Cp mRNA in both the lactating and suckled models in comparison to the resting model. Furthermore, the Cp mRNA increase in response to PMC42-LA differentiation corresponded with more secreted Cp protein, both apo and holo forms, indicating a link between function and Cp requirement. Our results provide significant insight on the regulation of Cp expression and secretion in lactation and copper incorporation into milk.

Molecular architecture of Aβ fibrils grown in cerebrospinal fluid solution and in a cell culture model of Aβ plaque formation

OBJECTIVES: The detailed structure of brain-derived Aβ amyloid fibrils is unknown. To approach this issue, we investigate the molecular architecture of Aβ(1-40) fibrils grown in either human cerebrospinal fluid solution, in chemically simple phosphate buffer in vitro or extracted from a cell culture model of Aβ amyloid plaque formation. METHODS: We have used hydrogen-deuterium exchange (HX) combined with nuclear magnetic resonance, transmission electron microscopy, seeding experiments both in vitro and in cell culture as well as several other spectroscopic measurements to compare the morphology and residue-specific conformation of these different Aβ fibrils. RESULTS AND CONCLUSIONS: Our data reveal that, despite considerable variations in morphology, the spectroscopic properties and the pattern of slowly exchanging backbone amides are closely similar in the fibrils investigated. This finding implies that a fundamentally conserved molecular architecture of Aβ peptide fold is common to Aβ fibrils.

Differential regulation of Adiponectin receptor gene expression by Adiponectin and Leptin in Myotubes derived from obese and diabetic individuals

Objective: This study aimed to investigate the regulation of adiponectin receptors 1 (AdipoR1) and 2 (AdipoR2) gene expression in primary skeletal muscle myotubes, derived from human donors, after exposure to globular adiponectin (gAd) and leptin. Research Methods and Procedures: Four distinct primary cell culture groups were established [ Lean, Obese, Diabetic, Weight Loss (Wt Loss); n = 7 in each] from rectus abdominus muscle biopsies obtained from surgical patients. Differentiated myotube cultures were exposed to gAd (0.1 mug/mL) or leptin (2.5 mug/mL) for 6 hours. AdipoR1 and AdipoR2 gene expression was measured by real-time polymerase chain reaction analysis. Results: AdipoR1 mRNA expression in skeletal muscle myotubes derived from Lean subjects (p < 0.05) was stimulated 1.8-fold and 2.5-fold with gAd and leptin, respectively. No increase in AdipoR1 gene expression was measured in myotubes derived from Obese, Diabetic, or Wt Loss subjects. AdipoR2 mRNA expression was unaltered after gAd and leptin exposure in all myotube groups. Discussion: Adiponectin and leptin are rapid and potent stimulators of AdipoR1 in myotubes derived from lean healthy individuals. This effect was abolished in myotubes derived from obese, obese diabetic subjects, and obese-prone individuals who had lost significant weight after bariatric surgery. The incapacity of skeletal muscle of obese and diabetic individuals to respond to exogenous adiponectin and leptin may be further suppressed as a result of impaired regulation of the AdipoR1 gene.

Leptin stimulation of COXIV is impaired in obese skeletal muscle myotubes

Objective
To investigate the effects of leptin on the mRNA abundance of key genes involved in fatty acid oxidation and mitochondrial biogenesis in cultured skeletal muscle myotubes derived from lean and obese individuals.

Research methods and procedures
Rectus abdominus muscle biopsies were obtained from surgical patients to establish primary skeletal muscle cell cultures. Two distinct primary cell culture groups were established (Lean and Obese) n = 7 in each group. Differentiated cultures were then exposed to leptin (2.5 μg/ml) for 6 h. mRNA expression was subsequently measured by real-time PCR analysis.

Results
Basal mRNA expression of βHAD, COXIII, COXIV, PGC-1α and SOCS3 in the cultured human skeletal muscle myotubes were similar, however, PDK4 mRNA was elevated (P < 0.05) in the myotubes derived from obese individuals. The addition of leptin resulted in a 2.5-fold increase in COXIV mRNA expression in the myotubes derived from Lean individuals only (P < 0.05). There was also a tendency for leptin to increase COXIII, βHAD and PDK4 mRNA expression in this same group. Leptin had no impact on the gene expression of all measured transcripts in myotubes derived from obese individuals.

Conclusion
Short-term exposure of human skeletal muscle myotubes to leptin stimulated the expression of the mitochondrial enzyme COXIV in myotubes derived from lean individuals, an effect that was abrogated in myotubes derived from obese individuals. These data demonstrate a novel capacity for leptin to increase mitochondrial biogenesis and thus, a possible increased capacity for lipid oxidation and the persistence of a defect in leptin signalling in human myotubes cultured from obese individuals.