Oxygen Insensitivity of the Histochemical Assay of Glucose-6-Phosphate Dehydrogenase Activity for the Discrimination Between Nonmalignant and Malignant Cells

Driel, B.E.M. van; Noorden, C. J. F. van

doi:10.1177/002215549904700501

Cited by 17 publications

(16 citation statements)

References 39 publications

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“…These electrons can then be picked up by water‐soluble colourless NBT. Reduction of the tetrazolium salt generates a water‐insoluble, intensely coloured formazan that precipitates at the site of dehydrogenase activity (Van Driel and Van Noorden, 1999). In Figure 1, the enzymatic reactions considered in our study are placed in the framework of carbon metabolism in maturing embryos.…”

Section: Resultsmentioning

confidence: 99%

A spatiotemporal analysis of enzymatic activities associated with carbon metabolism in wild‐type and mutant embryos of Arabidopsis using in situ histochemistry

Baud

Graham²

2006

The Plant Journal

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SummaryArabidopsis as a molecular genetic model offers many advantages for the study of seed development, but these do not extend to biochemical and enzymatic studies, which are often compromised by the limited amount of material available from the small developing embryos. A set of assays based on the coupling of an enzymatic reaction to the reduction of NAD, NADP or FAD, and subsequent reduction and precipitation of a tetrazolium salt, have been adapted to investigate 18 enzyme activities associated with carbon metabolism in developing Arabidopsis embryos. The use of organelle-specific marker enzymes demonstrates the utility of the method for detection of activities in mitochondria, plastids and peroxisomes as well as the cytosol. The temporal staining patterns obtained allow classification of the activities into three main categories based on whether they peak in the early, intermediate or late stages of maturation. An interesting switch from ATP to pyrophosphate consuming pathways occurs at the onset of the maturation phase, which involves key steps in primary carbon metabolism such as phosphofructokinase. This spatiotemporal characterization of carbon metabolism has also been applied to various mutants disrupted in embryo development including gnom (gn), acetyl-CoA carboxylase1 (acc1), schlepperless (slp), and wrinkled1 (wri1). The data obtained demonstrate that the extent to which carbon metabolism is affected in mutants is not necessarily correlated to the severity of the mutation considered. Through the advanced characterization of trehalose-6-P synthase1 (tps1) embryos, this approach finally provides new insight into the regulatory role played by trehalose metabolism in embryo development.

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Section: Resultsmentioning

confidence: 99%

A spatiotemporal analysis of enzymatic activities associated with carbon metabolism in wild‐type and mutant embryos of Arabidopsis using in situ histochemistry

Baud

Graham²

2006

The Plant Journal

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“…The method makes use of the NADPH produced by G6PD to reduce water-soluble tetrazolium salt via electron carriers to colored, insoluble formazan, which precipitates inside cells and is detectable, in situ, in regions where G6PD is localized. But whereas this technique has been used numerous times to localize G6PD activity in cells and tissues, as well as for quantitative histochemical enzyme analyses (Van Noorden 1984;Van Driel and Van Noorden 1999), there is, nonetheless, very little available information on the ultrastructural localization of G6PD. This is due in part to problems associated with electron microscopic detection of G6PD using tetrazolium salt: with few exceptions, formazans are lipophilic and non-osmiphilic, and, moreover, they are unstable in conventional electron microscopic preparations during postosmification processing (for reviews see Altman 1976;Van Noorden 1984).…”

Section: Introductionmentioning

confidence: 99%

Glucose-6-phosphate dehydrogenase cytochemistry using a copper ferrocyanide method and its application to rapidly frozen cells

Ishibashi

Tajima

Iwasaki

et al. 1999

Histochemistry and Cell Biology

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We describe an improved copper ferrocyanide-based method for cytochemical detection of glucose-6-phosphate dehydrogenase (G6PD), which was used to localize the enzyme within the ultrastructure of rat hepatocytes and adrenocortical cells. With this method, glutaraldehyde fixation and the addition of exogenous electron carriers (for example, phenazine methosulfate) to the cytochemical reaction medium were essential. Copper ferrocyanide reaction product showing the distribution of G6PD was readily recognized at the light microscopic level as Hatchett's brown staining and at the electron microscopic level as electron-dense deposits. Within stained regions, enzyme cytochemical G6PD activity was found to be associated with ribosome-like structures. Because G6PD is a soluble, cytosolic enzyme, its displacement or extraction may occur during conventional fixation. We, therefore, combined a rapid-freezing technique with G6PD enzyme cytochemistry. The resultant rapid-freezing enzyme cytochemistry enabled us to show the subcellular distribution of G6PD in a more life-like state; the localization of G6PD in rapidly frozen cells was in substantial agreement with that in conventionally fixed cells.

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“…To discriminate between malignant cells and nonmalignant cells, the histochemical assay of G6PD is performed with the tetrazolium salt neotetrazolium chloride (NT) (Van Noorden and Frederiks 1992;Van Driel and Van Noorden 1999). The method is based on the capture of electrons that are produced by the enzyme when it oxidizes its specific substrate.…”

mentioning

confidence: 99%

“…Certain metabolic changes in cancer cells are held responsible for the oxygen insensitivity phenomenon, such as a combination of elevated G6PD activity, decreased superoxide dismutase activity, and decreased lipid peroxidation capacity (Best et al 1990;Griffini et al 1994;Van Driel and Van Noorden 1999).…”

mentioning

confidence: 99%

Oxygen Insensitivity of the Histochemical Assay of Glucose-6-phosphate Dehydrogenase Activity for the Detection of (Pre)Neoplasm in Rat Liver

Jong

Frederiks

Noorden

2001

J Histochem Cytochem.

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S U M M A R YOxygen insensitivity of the histochemical assay to detect glucose-6-phosphate dehydrogenase (G6PD) activity with NT as tetrazolium salt has been proved to be a powerful tool to discriminate various types of adenocarcinoma from normal tissues. Here we investigated whether this phenomenon can also be applied to differentiate between chemically induced hepatocellular (pre)neoplasms and normal liver tissue in rats. Residual activity (percentage of the amount of final reaction product that is generated in oxygen and that is generated in nitrogen) was 60% in (pre)neoplastic cells and 6% in normal liver parenchymal cells. This means that the oxygen insensitivity test is a useful tool to distinguish (pre)neoplasms from normal rat liver tissue. N -Ethylmaleimide, a blocker of SH groups, did not affect G6PD activity in (pre)neoplastic cells, whereas activity in normal cells was reduced by half. Therefore, the absence of essential SH groups in G6PD in (pre)neoplastic cells is held responsible for the oxygen insensitivity phenomenon. We conclude that oxygen insensitivity of the histochemical assay for G6PD activity is a fast, easy, and cheap tool to diagnose (pre)neoplasms in rat liver. Discrimination is likely to be based on altered properties of the enzyme in (pre)neoplastic cells.

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Oxygen Insensitivity of the Histochemical Assay of Glucose-6-Phosphate Dehydrogenase Activity for the Discrimination Between Nonmalignant and Malignant Cells

Cited by 17 publications

References 39 publications

A spatiotemporal analysis of enzymatic activities associated with carbon metabolism in wild‐type and mutant embryos of Arabidopsis using in situ histochemistry

A spatiotemporal analysis of enzymatic activities associated with carbon metabolism in wild‐type and mutant embryos of Arabidopsis using in situ histochemistry

Glucose-6-phosphate dehydrogenase cytochemistry using a copper ferrocyanide method and its application to rapidly frozen cells

Oxygen Insensitivity of the Histochemical Assay of Glucose-6-phosphate Dehydrogenase Activity for the Detection of (Pre)Neoplasm in Rat Liver

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