Liver glycogen phosphorylase is upregulated in glioblastoma and provides a metabolic vulnerability to high dose radiation

Channelling of glucose via glycogen, known as the glycogen shunt, may play an important role in the metabolism of brain tumours, especially in hypoxic conditions. We aimed to dissect the role of glycogen degradation in glioblastoma (GBM) response to ionising radiation (IR). Knockdown of the glycogen phosphorylase liver isoform (PYGL), but not the brain isoform (PYGB), decreased clonogenic growth and survival of GBM cell lines and sensitised them to IR doses of 10–12 Gy. Two to five days after IR exposure of PYGL knockdown GBM cells, mitotic catastrophy and a giant multinucleated cell morphology with senescence-like phenotype developed. The basal levels of the lysosomal enzyme alpha-acid glucosidase (GAA), essential for autolysosomal glycogen degradation, and the lipidated forms of gamma-aminobutyric acid receptor-associated protein-like (GABARAPL1 and GABARAPL2) increased in shPYGL U87MG cells, suggesting a compensatory mechanism of glycogen degradation. In response to IR, dysregulation of autophagy was shown by accumulation of the p62 and the lipidated form of GABARAPL1 and GABARAPL2 in shPYGL U87MG cells. IR increased the mitochondrial mass and the colocalisation of mitochondria with lysosomes in shPYGL cells, thereby indicating reduced mitophagy. These changes coincided with increased phosphorylation of AMP-activated protein kinase and acetyl-CoA carboxylase 2, slower ATP generation in response to glucose loading and progressive loss of oxidative phosphorylation. The resulting metabolic deficiencies affected the availability of ATP required for mitosis, resulting in the mitotic catastrophy observed in shPYGL cells following IR. PYGL mRNA and protein levels were higher in human GBM than in normal human brain tissues and high PYGL mRNA expression in GBM correlated with poor patient survival. In conclusion, we show a major new role for glycogen metabolism in GBM cancer. Inhibition of glycogen degradation sensitises GBM cells to high-dose IR indicating that PYGL is a potential novel target for the treatment of GBMs.Subject terms: Cancer metabolism, CNS cancer     

Distribution of lecithin-retinol acyltransferase activity in different types of rat liver cells and subcellular fractions

It is now well documented that lecithin-retinol acyltransferase (LRAT) is the physiologically important enzyme activity involved in the esterification of retinol in the liver. However, no information regarding the cellular distribution of this enzyme in the liver is presently available. This study characterizes the distribution of LRAT activity in the different types of rat liver cells. Purified preparations of isolated parenchymal, fat-storing, and Kupffer + endothelial cells were isolated from rat livers and the LRAT activity present in microsomes prepared from each of these cell fractions was determined. The fat-storing cells were found to contain the highest level of LRAT specific activity (383 +/- 54 pmol retinyl ester formed min-1.mg-1 versus 163 +/- 22 pmol retinyl ester formed min-1.mg-1 for whole liver microsomes). The level of LRAT specific activity in parenchymal cell microsomes (158 +/- 53 pmol retinyl ester formed min-1.mg-1) was very similar to LRAT levels in whole liver microsomes. The Kuppfer + endothelial cell microsome fractions were found to contain LRAT, at low levels of activity. These results indicate that the fat-storing cells are very enriched in LRAT but the parenchymal cells also posses significant levels of LRAT activity.     

Petunia peroxidase a: isolation, purification and characteristics

The fast-moving anionic peroxidase isoenzyme variant PRXa was purified from leaves of petunia (Petunia hybrida). Over 1300-fold purification was achieved by subjecting extracellular extracts to two sequential acetone precipitations and resuspending the pellets at pH 5.0 and pH 8.0, respectively, followed by gel filtration and chromatofocusing. The purified enzyme had an absorbance ratio (A405 nm/A280 nm) of 3.6, a molecular mass of about 37 kDa and a pI of 3.8. Three molecular forms with slightly different molecular masses were separated by concanavalin-A--Sepharose affinity chromatography, indicating that these three forms differ in their carbohydrate moieties. The absorption spectrum of PRXa had maxima at 496 and 636 nm and a Soret band at 405 nm. Spectra of compounds I and IV were obtained by titrating a batch of PRXa stored for several months at -20 degrees C with H2O2. The addition of 1 mol H2O2/mol freshly purified PRXa caused the formation of compound II, indicating that freshly isolated PRXa contains a bound hydrogen donor which is lost upon storage. Compound III was obtained from both preparations in the presence of excess H2O2. The pH optimum of PRXa for the reaction with H2O2 and guaiacol was 5.0 and its specific activity 61 mkat/g protein. Among various aromatic compounds, coniferyl alcohol was polymerized by PRXa to presumed lignin-like material. The extracellular localization and high affinity of PRXa for the cinnamic acid derivatives suggest that this isoenzyme functions in the polymerization or cross-linking of lignin in the plant cell wall.     

Growth media in anaerobic fermentative processes: The underestimated potential of thermophilic fermentation and anaerobic digestion

Fermentation and anaerobic digestion of organic waste and wastewater is broadly studied and applied. Despite widely available results and data for these processes, comparison of the generated results in literature is difficult. Not only due to the used variety of process conditions, but also because of the many different growth media that are used. Composition of growth media can influence biogas production (rates) and lead to process instability during anaerobic digestion. To be able to compare results of the different studies reported, and to ensure nutrient limitation is not influencing observations ascribed to process dynamics and/or reaction kinetics, a standard protocol for creating a defined growth medium for anaerobic digestion and mixed culture fermentation is proposed. This paper explains the role(s) of the different macro- and micronutrients, as well as the choices for a growth medium formulation strategy. In addition, the differences in nutrient requirements between mesophilic and thermophilic systems are discussed as well as the importance of specific trace metals regarding specific conversion routes and the possible supplementary requirement of vitamins. The paper will also give some insight into the bio-availability and toxicity of trace metals. A remarkable finding is that mesophilic and thermophilic enzymes are quite comparable at their optimum temperatures. This has consequences for the trace metal requirements of thermophiles under certain conditions. Under non-limiting conditions, the trace metal requirement of thermophilic systems is about 3 times higher than for mesophilic systems.     

Screening for mutations causing X-linked severe combined immunodeficiency in the IL-2Rγ chain gene by single-strand conformation polymorphism analysis

Mutations in the common gamma chain (_c or IL2RG) of the interleukin-2, –4, –7, –9 and –15 receptors have been found to cause X-linked severe combined immunodeficiency (SCIDX1). We report here on the mutations identified in a further ten families. Two of the mutations identified have occurred twice in unrelated families, indicating two possible mutational hotspots. Seven of the mutations, which were identified by single-strand conformational polymorphism (SSCP) analysis, are point mutations, and the eighth is a small deletion. We also report on the first use of assays based on these mutations within IL2RG for unambiguous carrier determination. The consequences for the _c proteins produced as a result of these mutations are discussed.     

Water cytotoxicity and dioxins bioaccumulation in an Egyptian delta wetland ecosystem

Manzala Lake, as one of the main Egyptian wetland ecosystems, is facing risks of pollution. An in vitro cytotoxicity test using a mammalian cell line was employed to determine the toxicity of multiple pollutants in the water and Tilapia zillii fish sampled from the lake. The concentrations of seven polychlorinated dibenzo-p-dioxins and ten polychlorinated dibenzofurans were investigated in water and muscle of the fish in 2014. Cytotoxicity testing showed that the percentage inhibition of cell viability in the studied sites ranged between 56.16% and 83.22%. Dioxin analysis indicated that the average concentrations of 1,2,3,4,6,7,8,9-octachlorodibenzo-p-dioxin, 1,2,3,4,7,8-hexachlorodibenzofuran, 1,2,3,4,6,7,8-heptachlorodibenzofuran and 1,2,3,4,6,7,8,9-octachlorodibenzofuran were higher than the toxic equivalence quotients (TEQs) set by the World Health Organization (WHO) in all water and fish muscle samples; however, the average concentration of 2,3,7,8-tetrachlorodibenzofuran was higher only in fish muscle samples. The bioaccumulation factor (BAF) ranged dramatically between 2 and 58.5 for the detected dioxins. Adverse human health effects through the consumption of fish are not expected, because dioxin levels in fish muscle are deemed safe for human consumption. Implementation of a strategic multidisciplinary action plan is strongly recommended to sustain this delta wetland ecosystem.     

HLA-DRw6 and renal allograft rejection.

HLA-DRw6-positive patients are "high responders" to certain renal allograft antigens. A study was therefore conducted of the outcome of 247 first renal allografts in 74 DRw6-positive and 173 DRw6-negative recipients. The effectiveness of matching for HLA-DR determinants in both groups was also analysed. The one-year graft survival in DRw6-positive patients was 59% as compared with 75% in DRw6-negative recipients (p = 0.012). A striking difference between the two groups was that HLA-DR matching significantly improved renal allograft survival only in the DRw6-positive patients. In those patients the one-year survival of HLA-DR-identical grafts was 95% as compared with only 38% for 2-DR mismatched grafts (p = 0.009). In DRw6-negative patients only a slight beneficial effect of HLA-DR matching was observed (83% versus 72% at one year for the 0-DR and 2-DR mismatched grafts, respectively) (p greater than 0.05). These findings are clear evidence that DRw6-positive patients (about a quarter of the patients on the waiting list of Eurotransplant) should be given HLA-DR-identical kidney transplants only.     

Hormonal Interactions in Mucor mucedo and Blakeslea trispora

Evidence is presented that progametangia in both the plus and the minus mating types of Mucor mucedo can be induced by one substance, namely (-)-trisporic acid B. A method is described for the determination of the concentration of the sex factors (trisporone, trisporic acid B, trisporic acid C) in mated cultures of Mucorales by polarography. It can be demonstrated that the amount of plus mycelium is limiting for the production of the sex factors in Blakeslea trispora. It is shown that the minus type of this organism is able to synthesize the sex factors when incubated in the filtered medium of a mated culture. Cycloheximide and 5-fluorouracil inhibit strongly the sex factor production in a mated culture of B. trispora at any time. This result suggests that sexual activity comprises the synthesis of proteins which are involved in the production of the sex factors.