Source of electrons for extracellular Fe(III) reduction in iron-deficient bean roots

Iron-deficient Phaseolus vulgaris L. cv. Prelude developed a high reducing capacity for extracellular Fe(III) at the root surface. This reduction was competitively inhibited by Nitro-Blue Tetrazolium salt (Nitro-BT) which was deposited as a blue precipitate within the epidermis cells of the youngest root parts. Root respiration was not influenced by Nitro-BT. The intracellular reduction of Nitro-BT could largely be prevented by excess extracellular Fe(III)EDTA. Iron-sufficient control plants reduced both extracellular Fe(III)EDTA and intracellular Nitro-BT at a much slower rate. A role of cytosolic NADH or NADPH as direct electron donors for the enhanced Fe(III) reduction at the plasmalemma is indicated. NAD⁺-3-phosphate dehydrogenase activity was higher in preparations from iron-deficient root parts than in preparations from control root parts. Ferricyanide, dichlorophenolindophenol and phenazine methosulfate were also reduced at an increased rate by iron-deficient roots. We conclude that a trans-plasma membrane electron transfer, mediated by a membrane-bound reductase, is responsible for the reduction of extracellular Fe(III).     

Acetoacetate enhancement of glucose mediated DNA glycation

Acetoacetate (AA) is a ketone body, which generates reactive oxygen species (ROS). ROS production is impacted by the formation of covalent bonds between amino groups of biomacromolecules and reducing sugars (glycation). Glycation can damage DNA by causing strand breaks, mutations, and changes in gene expression. DNA damage could contribute to the pathogenesis of various diseases, including neurological disorders, complications of diabetes, and aging. Here we studied the enhancement of glucose-mediated DNA glycation by AA for the first time. The effect of AA on the structural changes, Amadori and advanced glycation end products (AGEs) formation of DNA incubated with glucose for 4 weeks were investigated using various techniques. These included UV–Vis, circular dichroism (CD) and fluorescence spectroscopy, and agarose gel electrophoresis. The results of UV–Vis and fluorescence spectroscopy confirmed that AA increased the DNA-AGE formation. The NBT test showed that AA also increased Amadori product formation of glycated DNA. Based on the CD and agarose gel electrophoresis results, the structural changes of glycated DNA was increased in the presence of AA. The chemiluminescence results indicated that AA increased ROS formation. Thus AA has an activator role in DNA glycation, which could enhance the adverse effects of glycation under high glucose conditions.     

Extracellular organics from specific cultures of Heterosigma akashiwo (Raphidophyceae) irreversibly alter respiratory activity in mammalian cells

Raphidophyte blooms have been well documented in several coastal areas around the world. Centring raphidophyte-bloom research has been a focus evolving around issues of ichthyotoxicity, allelopathy and anti-predatory activity. However, the details of these phenomena such as the identity of the compounds and the mechanisms underlying these processes are poorly understood. One such raphidophyte, Heterosigma akashiwo (Hada) Hara et Chihara, has historically received much attention with regard to its ichthyotoxic and allelopathic properties. In this study, we collected extracellular organic compounds from cultures of nine H. akashiwo isolates and tested those exudates on two mammalian cell lines: rat osteoblastic sarcoma (UMR-106) and human embryonic kidney (HEK-293). A tetrazolium colourimetric assay was used to determine the activity of mitochondrial dehydrogenases. Exposure of the mammalian cell lines to exudates collected from cultures of H. akashiwo (strain 764) significantly increased activity in a concentration- and time-dependent manner. Exudate concentrations of as little as 0.3 mg ml⁻¹ elicited a stimulatory response in the mammalian cells. This is comparable to the range of exudate concentrations that were originally in the algal cultures (>0.1 mg ml⁻¹). Significant increases in activity were observed 12–24 h following continuous or 1 h (transient) exposure to the exudate. Production of the stimulatory bioactive exudate was not altered by nutrient-stressed H. akashiwo cultures (reduced iron, phosphate or nitrate). Collectively, these bioactive compound(s) consistently increased cellular activity 3–15-fold. Interestingly, of the nine isolates tested, four of them produced the stimulatory exudate, whereas four others did not produce the stimulatory compound(s) and isolate 560R produced a compound(s) that was inhibitory in nature. Thus, we have shown that cultures of H. akashiwo produce organic compounds that can alter the metabolic activity of mammalian cells. Future isolation and characterization of these bioactive compounds may determine them to have ecological relevance, potentially involved in the ichthyotoxic, allelopathic and/or anti-predatory behaviour of this alga.     

Cytotoxicity of mycotoxins evaluated by the MTT-cell culture assay

The application of a modified colorimetric bioassay for the evaluation of the biological effects of mycotoxins is reported. Using three different monolayer cell lines (swine kidney, Madin Darby canine kidney, HeLa) the influence of nine different mycotoxins on the cellular methylthiazoltetrazolium (MTT)-cleavage activity was evaluated. The yellow tetrazolium salt MTT is converted by mitochondrial dehydrogenases of metabolically active cells to an insoluble purple formazan product, which was then solubilized with dimethylsulfoxide. The optical density of this homogeneous solution was suitable for a precise spectrophotometric measurement by a plate reader at a wavelength of 510 nm. Nine mycotoxins were simultaneously tested in all three cell lines, from which the swine kidney cell line proved to be the most sensitive. The effects of additional 35 mycotoxins were therefore tested using swine kidney monolayers as target cells. A total of 28 toxins of the 44 mycotoxins tested proved to be cytotoxic in the MTT-bioassay. Most of them belong to the group of trichothecene mycotoxins. Concentrations ranged between 0.01 µg and 100 µg/ml of cell culture medium. The MTT cleavage assay was found to be a quick (24 hours) and easy to perform system for the evaluation of the biological activity of many different mycotoxins and may also provide a useful tool for the testing of a large variety of sample materials.     

A simple indirect colorimetric assay for measuring mitochondrial energy metabolism based on uncoupling sensitivity

PurposeCancer cells rapidly adjust their balance between glycolytic and mitochondrial ATP production in response to changes in their microenvironment and to treatments like radiation and chemotherapy. Reliable, simple, high throughput assays that measure the levels of mitochondrial energy metabolism in cells are useful determinants of treatment effects. Mitochondrial metabolism is routinely determined by measuring the rate of oxygen consumption (OCR). We have previously shown that indirect inhibition of plasma membrane electron transport (PMET) by the mitochondrial uncoupler, FCCP, may also be a reliable measure of mitochondrial energy metabolism. Here, we aimed to validate these earlier findings by exploring the relationship between stimulation of oxygen consumption by FCCP and inhibition of PMET.MethodsWe measured PMET by reduction of the cell impermeable tetrazolium salt WST-1/PMS. We characterised the effect of different growth conditions on the extent of PMET inhibition by FCCP. Next, we compared FCCP-mediated PMET inhibition with FCCP-mediated stimulation of OCR using the Seahorse XF96e flux analyser, in a panel of cancer cell lines.ResultsWe found a strong inverse correlation between stimulation of OCR and PMET inhibition by FCCP. PMET and OCR were much more severely affected by FCCP in cells that rely on mitochondrial energy production than in cells with a more glycolytic phenotype.ConclusionIndirect inhibition of PMET by FCCP is a reliable, simple and inexpensive high throughput assay to determine the level of mitochondrial energy metabolism in cancer cells.