A new insight into mechanisms of interferon alpha neurotoxicity: Expression of GRIN3A subunit of NMDA receptors and NMDA-evoked exocytosis.

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.

Expression of genes, encoding the enzymes of cysteine metabolism in human placenta in the first and third trimesters of uncomplicated pregnancy.

The cellular cysteine is highly regulated in a narrow range of concentrations due to its cyto- and neurtoxicity when it is overwhelmed or its deficiency for protein synthesis and other vital metabolic reactions when its amount is restricted. The regulation of cysteine content and its metabolic products, glutathione, taurine and inorganic sulfur compounds, is scarcely explored in human placenta though cysteine metabolism is closely related to the maintenance of redox status and protection from free radical oxidation, elimination of homocysteine and detoxification. These processes are particularly important for placenta which meets substantial changes of oxygen supply during its development, and is the last metabolically active organ between mother and fetus. The abundance of CDO , CSAD , ADO , SUOX, GCLC and GCLM mRNAs was estimated by RT -qPCR and compared with the computationally analyzed microarray gene expression data from GEO , while the level of individual protein ­ by western-blot analysis, both in placental samples from first and third trimesters of uncomplicated pregnancies. The abundance of CDO mRNA is significantly up-regulated at term compared to the first trimester, the level of GCLM and GCLC mRNAs remains almost unchanged while the abundance of other mRNAs reduces to varying degrees. Overall, the changes of gene expression in third trimester in comparison to the first one estimated by RT-qPCR and microarray coincide while the former data are more informative for the limited group of genes. The data provide the basis for further research of these genes expression and phenotype of human placenta in health and disease

Placental markers of folate-related metabolism in preeclampsia.

The etiology and degree of clinical symptoms of preeclampsia depend on genotypic and phenotypic maternal and trophoblast factors, and elevated levels of plasma homocysteine (Hcy) are one of the pathogenetic factors of preeclampsia. To assess the impact of the folate-related metabolism, we characterized the indices of this metabolism in 40 samples from uncomplicated term placentas and 28 samples from preeclamptic pregnancies by quantifying the total content of folate, methionine (Met), Hcy and related cysteine, and glutathione (GSH) in compliance with the 677 C/T genotype of methylene tetrahydrofolate reductase (MTHFR). The prevalence of MTHFR genotypes was not significantly different between the two groups. The polymorphism of MTHFR was not unambiguously connected with the content of total placental Met, Hcy and related cysteine, and GSH either in uncomplicated or in complicated pregnancies. By contrast, the combination of the heterozygous MTHFR genotype with folate deficiency in the samples from preeclamptic pregnancies was characterized by a statistically significant decrease in the Met content, a trend toward increased Hcy levels and a tight association between metabolically directly and indirectly related compounds, e.g. positive relation between Hcy versus cysteine and folate versus GSH and negative relation between folate versus Hcy and both Hcy and cysteine versus GSH. We demonstrated the expression of cystathionine-ß-synthase (CBS) in human placenta at term by RT-PCR and western blot analysis, for the first time, and confirmed its catalytic activity and the accumulation of cysteine and CBS in placental explants cultivated in the presence of elevated Hcy concentrations. We suggest that disturbance in placental folate-related metabolism may be one of the pathogenetic factors in preeclampsia.

Low-density lipoproteins isolated from the blood of patients with coronary heart disease induce the accumulation of lipids in human aortic cells.

Smooth muscle cells cultured from the intima of unaffected human aorta accumulate lipids during incubation with the blood serum of patients with coronary heart disease (CHD). Blood sera of most healthy subjects fail to induce the deposition of lipids in cultured cells. Very-low-density lipoproteins (VLDL), low-density lipoproteins (LDL), and high-density lipoproteins of two subclasses (HDL2 and HDL3) were isolated from the blood of healthy subjects and CHD patients. LDL from the blood of healthy individuals did not raise intracellular lipid levels within 24 hr of cultivation (the maximal concentration used, 1000 micrograms/ml). During the same incubation period, LDL obtained from the blood of CHD patients (200 to 1000 micrograms/ml) caused a 2- to 5-fold rise in cholesteryl esters as well as a 1.5- to 3-fold rise in free cholesterol and triglycerides, whereas intracellular phospholipid levels remained unchanged. There was a direct correlation (r = 0.95) between cholesterol accumulation in the cells incubated with whole sera of CHD patients and cholesterol level in the cells incubated with LDL isolated from these sera. In one of the three cases, the ability to raise the intracellular level of cholesteryl esters was demonstrated by VLDL (500 micrograms/ml) derived from CHD patients' blood. HDL2 and HDL3 did not affect lipid levels in smooth muscle cells cultured from unaffected intima. HDL3 from the blood of CHD patients and healthy subjects (50 to 250 micrograms/ml) reduced cholesteryl ester levels in cells cultured from atherosclerotic plaques 1.5- to 2-fold. HDL2 also decreased the content of cholesteryl esters in plaque cells, though less effectively than HDL3. The data obtained suggest that circulating LDL and, possibly, VLDL in the blood of CHD patients are capable of inducing the accumulation of fat in vascular wall cells.

Blood serum atherogenicity associated with coronary atherosclerosis. Evidence for nonlipid factor providing atherogenicity of low-density lipoproteins and an approach to its elimination.

To reveal the presence of atherogenic potential in the blood serum obtained from patients with angiographically assessed coronary atherosclerosis we used primary cultures of subendothelial cells isolated by collagenase from unaffected human aortic intima. Earlier, we have demonstrated that such cultures are made up mostly of typical and modified smooth muscle cells. Within 24 hours of cultivation with a 40% sera of patients suffering from coronary atherosclerosis, the total intracellular cholesterol level increased twofold to fivefold. Cultivation with the sera of healthy subjects had no effect on the intracellular cholesterol level. The sera of patients were separated by ultracentrifugation into two fractions: total lipoprotein fraction containing the main classes of lipoproteins and a lipoprotein-deficient fraction. The former, but not the lipoprotein-deficient fraction, was characterized by atherogenicity (i.e., the ability to induce the accumulation of intracellular cholesterol). Lipoproteins of the patients' serum were separated into main classes: low density lipoproteins (LDL), very low density lipoproteins (VLDL), and high density lipoproteins (HDL2 and HDL3). An atherogenic component of the serum capable of stimulating the deposition of intracellular cholesterol was represented by LDL and, in one case, by VLDL, but not by other classes of lipoproteins. LDL and other lipoproteins isolated from the blood serum of healthy subjects failed to raise the cholesterol content in cultured cells; that is, they were nonatherogenic.(ABSTRACT TRUNCATED AT 250 WORDS)