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Neurological and psychiatric side effects accompany the high-dose interferon-alpha (IFNA) therapy. The primary genes responsible for these complications are mostly unknown. Our genome-wide search in mouse and rat genomes for the conservative genes containing IFN-stimulated response elements (ISRE) in their promoters revealed a new potential target gene of IFNA, Grin3α, which encodes the 3A subunit of NMDA receptor. This study aimed to explore the impact of IFNA on the expression of Grin3α and Ifnα genes and neurotransmitters endo/exocytosis in the mouse brain. We administered recombinant human IFN-alpha 2b (rhIFN-α2b) intracranially, and 24 h later, we isolated six brain regions and used the samples for RT-qPCR and western blot analysis. Synaptosomes were isolated from the cortex to analyze endo/exocytosis with acridine orange and L-[14C]glutamate. IFNA induced an increase in Grin3α mRNA and GRIN3A protein, but a decrease in Ifnα mRNA and protein. IFNA did not affect the accumulation and distribution of L-[14C]glutamate and acridine orange between synaptosomes and the extra-synaptosomal space. It caused the more significant acridine orange release activated by NMDA or glutamate than from control mice's synaptosomes. In response to IFNA, the newly discovered association between elevated Grin3α expression and NMDA- and glutamate-evoked neurotransmitters release from synaptosomes implies a new molecular mechanism of IFNA neurotoxicity.

Hyperhomocysteinemia is a medical condition characterized by an abnormally high level of homocysteine in the blood associated with multiple human pathologies including preeclampsia – the most feared complication of pregnancy, with placenta playing the central role in the pathogenesis of preeclampsia. The developing placenta is highly sensitive to different adverse factors but its response to hyperhomocysteinemia is not fully clear. Previously we have demonstrated the activation of reactions of methionine cycle and the transsulfuration pathway in placental explants cultivated with homocysteine. The reactions of the methionine cycle are tightly connected with reactions of the folate cycle, encompassing reactions ofde novopurine biosynthesis, which are crucial for the developing placenta, as they support rapid ATP generation to maintain energy status and increased biosynthesis of macromolecules. The sensitivity ofde novopurine biosynthesis to hyperhomocysteinemia is not known. The aim of this study was to evaluate the impact of homocysteine on placentalde novopurine biosynthesis.MethodsWe developed a simplified method to measure the level of all and newly formed purines by HPLC/ESI-MS, using a stable isotope glycine to label newly synthesized purines. The developed method proved to be highly sensitive, interday repeatable and intraday reproducible. We applied a method for placental explants from the first and third trimesters of gestation and MCF7 cells cultivated with 20 μM and 40 μM homocysteine corresponding to its concentrations at mild and intermediate hyperhomocysteinemia.ResultsThe developed method proved to be highly sensitive, interday repeatable and intraday reproducible. The content of total purines in placental explants from the first trimester of gestation was around 9.0 μmol/g wet tissues at specified conditions of cultivation. The newly formed purines comprised around 1 % of total purines, decreased steadily in explants cultivated with 20 μM and 40 μM homocysteine, and reached the values characteristic for explants from third trimester cultivated without homocysteine, 4.0 μmol/g wet tissues. The effect of homocysteine reproduced with MCF7 cells.ConclusionHomocysteine in concentrations characteristic of mild and intermediate hyperhomocysteinemia induces the down regulation ofde novopurine biosynthesis in placental explants, and implies the shift of metabolic pathway to homocysteine remethylation and transsulfuration at the expense ofde novopurine biosynthesis.

Water-soluble analogue of quercetin, corvitin is used in patients with myocardial infarction as blocker of 5-lipoxygenase. However, its effects on secretion, lipid content and physico-chemical properties of bile have not been understood yet. We investigated the effect of corvitin, applied in different doses, on the level of bile flow, the content of bile free and esterified cholesterol, phospholipids, triacylglycerols, and free fatty acids. In order to determine stability of the bile colloidal system, we examined the relationship between different lipid components. The rats were injected intraportally with a bolus of corvitin. At doses of 2.5, 5, and 10 mg/kg, the latter increased bile flow and concentration of total cholates, as well as free fatty acids. Corvitin (5 mg/kg) elevated phospholipids and cholesterol content, but at a dose of 10 mg/kg it increased the concentration of bile cholesterol esters and triacylglycerols. Corvitin applied at doses of 2.5 and 10 mg/kg increased total cholates/cholesterol ratio, but at a dose of 10 mg/kg, the drug reduced cholesterol / esterified cholesterol ratio. The results suggest that corvitin exerts choleretic effect and improves stability of bile colloidal system.

Purine nucleotides serve as immediate building blocks for the synthesis of RNAs and DNA, energy carriers and co-substrates in the activation of many metabolites, regulators in cellular metabolism, and signaling molecules. Knowledge of the actual tissue-specific concentration of these metabolites and their synthesis rate is essential to studies focused on a tissue-specific metabolic rewiring in the pathogenesis of different diseases, including carcinogenesis. The existing data mainly characterize the purine concentration in body fluids and malignant cells. The protocols for estimating de novo purine biosynthesis usually required the usage of radioactive precursors and costly facilities like HPLC with tandem mass spectrometry or nuclear magnetic resonance. We developed a new simplified method to measure the level of purines by HPLC/ESI-MS, using a stable isotope glycine to label and measure newly synthesized purines. The developed method proved to be highly sensitive, interday repeatable, and intraday reproducible. We applied the method to measure purine concentration and the rate of purine synthesis in placental explants from the first and third trimesters of gestation and MCF7 cells cultivated with 20 μM and 40 μM homocysteine. Corresponding homocysteine concentration is typical for breast cancer and pregnancy-associated complications affecting the human placenta. The content of total purines in placental explants from the first trimester of gestation was around 9.0 μmol/g wet tissues at specified cultivation conditions. The newly formed purines comprised approximately 1% of total purines and decreased steadily in explants cultivated with 20 mM and 40 μM homocysteine. They reached the values characteristic for explants from the third trimester grown without homocysteine, 4.0 μmol/g wet tissues. In MCF7 cells, the newly formed purines comprised around 50% of total purines and decreased under homocysteine treatment. A low-cost mass spectrometry-based approach may be used to estimate the level and the rate of purine synthesis in eukaryotes’ tissues in a wide range of purines concentrations.