Vol 11, No 4 (2017)

Glioblastoma multiforme (GBM) is the most common and first leading cause of death in primary human brain cancers. Following current interventions, despite continuous advancements in medicine, the median survival time of patients is unfavorable regardless of radiotherapy, chemotherapy, or surgery. This poor outcome is referred to “cancer stem cell” (CSC) concept. According to it a minority of cells within the GBM mass originates from neural stem cells (NSCs) or alternatively hijack similar checkpoints in them that evolution has selected in order to prevent inappropriate neurogenic-to-gliogenic switch which is responsible for the growth of tumor and its resistance to existing therapies. New developments in non-surgical treatment of GBM are based on this knowledge, however, similarities in molecular pathways between GBM and glioma stem cells (GSCs) motive the race to identify and target GSC regulators with less and more harmful effect in normal adult NSC and GBM respectively. In it the implications of downstream stemness genes with modulatory roles for stemness state pathways are assumed importance. This review, embodies NSC-intrinsic genes by recent elucidated roles of importance for GBM to affix to the stemness. Therefor it could facilitate a holistic approach of molecular targeted therapy to compromise the formidable resistant GBM to eradication.

In the present study, we estimated the levels of various forms of glutathione and the state of the glutathione antioxidant system in the subcellular fractions of the rat brain and in the liver during early postnatal ontogeny. Several groups of animals were studied, including 10-, 20-, and 30-day-old male Wistar rats, which allowed us to study different periods of brain maturation. It was shown that during the postnatal development of the rat brain from day 10 to day 30 the contents of both reduced and oxidized forms of glutathione decreased. In early ontogeny, when the activity of most of antioxidant enzymes is low, reduced glutathione may perform an antioxidant function. On the other hand, despite the decrease in the absolute value, the portion of GSH in the total glutathione pool increased and modified the redox state of the cells toward a more reduced condition. The decrease in the GSH level may be related to lower activity of glutathione reductase, which reduces oxidized glutathione, or to increased activity of the antioxidant enzymes, such as glutathione peroxidase and glutathione-S-transferase, which use GSH as a cofactor in their reactions.

Molecular biological experiments sometimes require expression of two or more genes in a single cell with an accurate ratio between their expression levels. One of the methods to provide this control is the use of the internal ribosome entry site (IRES) from the encephalomyocarditis virus as a separating insert between two target genes in the expression vector. Previously, it was shown that the efficacy of translation of the gene after IRES varies considerably in a range from 6 to 100% depending on the cell type. In neurons, the exact ratio between the expression levels of genes that are located before and after the IRES in the expression vector is unknown. Here, we analyzed the ratio between the amounts of products of the first and second genes located before and after the IRES in a plasmid that was used to transfect neurons in a primary hippocampal culture. We created two plasmid vectors that contain genes of the yellow (Venus) and red (mCherry) fluorescent proteins in different orders, which are separated by the IRES. We found that the unmodified IRES sequence of the encephalomyocarditis virus decreases the expression in the second cistron by a factor of 2.7 in a primary culture of hippocampal neurons. These data will help us to use currently available libraries of mutant IRES sequences for accurate control of the relationships between the expression of different target genes in neurons.

The most important component of the pathogenesis of Parkinson’s disease is the degradation of the nigrostriatal dopaminergic system, which is a key link in the regulation of motor function. Impaired motor function, and, consequently, the possibility of diagnosing the disease, occur only 20–30 years after the onset of the pathological process with the death of 50–60% of dopaminergic neurons and a decrease in the level of synthesis of dopamine in the striatum by 70–80%. The lack of effective therapy is associated with the late diagnosis of the disease, when there are practically no targets for pharmacotherapy. Therefore, one of the most important priorities of modern neurology is the development of a method for the early diagnosis of Parkinson’s disease at the preclinical (asymptomatic) stage. In this work, a new approach was developed to assess the functional deficiency of the dopaminergic nigrostriatal system using an inhibitor of dopamine-α-methyl-p-tyrosine synthesis in a chronic model of the preclinical stage of Parkinson’s disease in mice. Administration of α-methyl-p-tyrosine to animals in a model of the preclinical stage of Parkinson’s disease led to the appearance of motor behavior disorders but not in the control group. These data point to the possibility of using this approach as a provocative test for the diagnosis of this disease at the preclinical stage, which will allow us to begin treatment aimed at slowing the death of dopaminergic neurons.

Using effector analysis, we studied the mechanism of anti-seizure activity of the alcoxy-derived benzodiazepine (propoxazepam). In models of chemically induced seizures we determined the average molar and weight effective doses (ED₅₀) of propoxazepam as an antagonist of picrotoxin (4.10 ± 0.21 μmol/kg, 1.67 ± 0.09 mg/kg), pentylenetetrazole (2.24 ± 0.93 μmol/kg, 0.9 ± 0.04 mg/kg), and strychnine (40.33 ± 14.91 μmol/kg, 14.24 ± 0.47 mg/kg), which reflect the high activity level of the substance. On the basis of dose–effect curves, using comparative quantile analysis for chemoconvulsants with different mechanisms of action, we showed different stages of interaction of propoxazepam with GABA and glycine receptors under in vivo conditions. We evaluated the partial contribution of myoclonic and toxic components to the general structure of seizures induced by various chemoconvulsants. We believe that the results we obtained indicate that the anti-seizure action of propoxazepam is predominantly mediated by a GABAergic mechanism. Glycinergic components of the inhibition of strychnine-induced seizures by propoxazepam occur at doses that exceed the ED₅₀ and seem to be an additional means of anti-seizure action.

We studied peak-wave activity in WAG/Rij rats at ages of 2 and 6 months. We found age-dependent enhancement of peak-wave discharges. At 2 months, the discharges were rare and weak, whereas 6-month-old rats had robust peak-wave discharges. We measured the concentrations of monoamines and their metabolites in 2- and 6-month-old WAG/Rij and Wistar rats in five brain structures: the prefrontal cortex, nucleus accumbens, hypothalamus, striatum, and hippocampus. In 2-month-old WAG/Rij rats, we found a decrease in the concentration of dopamine (DA) metabolites (HVA) in the prefrontal cortex and striatum compared to Wistar rats. In 6-month-old WAG/Rij rats, we found a considerable decrease in the activity of DA system compared to Wistar rats in all five studied brain structures. Concentrations of both DA and its metabolites decreased. We found impairments of learning and memory of WAG/Rij rats compared to Wistar rats. Impairment of learning and memory were more pronounced in 6-month-old WAG/Rij rats compared to 2-month-old WAG/Rij rats. We believe that the DA system not only responds to emotionally positive states but is also a reward and reinforcement system. An increase in the concentration of DA and its metabolites by madopar prevents disturbances of learning and memory. Our general conclusion is that enhancement of absence epilepsy induces a strong deficit of activity of the mesocorticolimbic and nigrostriatal DA system, which induces depression-like behavior and disturbance of learning and memory in WAG/Rij rats. These are the mechanisms of the development of impairments and the integration of these impairments, as well as the aggravation and widening of pathological states.

The method of radioligand binding ex vivo has been used to evaluate the involvement of the ionotropic and metabotropic glutamate and GABA receptors in the mechanism of anticonvulsant action of levetiracetam and the original compound GIZh-290 (2-oxo-4-phenylpyrrolidin-1-yl) acetic acid) in the rat brain. We found that at the peak of lithium-pilocarpine seizures, the B_max value (the density of specific binding sites) decreases for [³H]-SR 95531, [³H](–)baclofen, [³Н](+)MK-801, and [³H]LY 354740 by 30–50% from the control level. The pre-treatment with levetiracetam (600 mg/kg) 40 minutes before pilocarpine kept the number of GABA- and NMDA-type receptors at the control level without affecting the metabotropic receptors. The administration of GIZh-290 (5.0 mg/kg), in contrast, maintained the density of GABA_A receptors at the control level, while the density of other types of receptors remained decreased at the peak of seizures. Thus, the anticonvulsant effect of levetiracetam and GIZh-290 has different mechanisms.

The development of oxidative stress is considered as one of the key molecular mechanisms that lead to the death of dopamine-producing neurons in Parkinson’s disease (PD). Here, we characterized the oxidative status (OS) of patients depending on the severity of the disease, the time course of the disease, and treatment with antiparkinsonian drugs. The OS was evaluated using Fe²⁺-induced chemiluminescence of serum lipoproteins (LPs) on the basis of informative parameters, including preformed lipid hydroperoxides, the maximum intensity of lipid peroxidation (LPO) and LP resistance to oxidation, due to the activity of the endogenous antioxidant system. In all patients, regardless of the disease stage (second, third, or fourth), we observed a comparable unidirectional increase in the level of lipid hydroperoxides and maximum intensity of (LPO). However, the activity of the endogenous antioxidant system decreased proportionally to the disease severity: second stage > third stage > fourth stage. In patients at the second and third stages of the disease who were treated with antiparkinsonian drugs we found a disruption of the OS in comparison with untreated patients of the same stage. In general, the deterioration of the OS in patients at the second and third stages of the disease, which included an increase in the level of lipid hydroperoxides and the maximum intensity of LPO, was detected in both patients receiving levodopa at a daily dose of more than 300 mg and in patients treated with drugs that did not contain levodopa. The decrease in the activity of the endogenous antioxidant system in these patients did not depend on the method of treatment and the dose of levodopa. The duration of treatment (up to 5 years) did not influence the OS of patients at the second and third stages of the disease. These data confirm the concept of the significance of oxidative stress in the pathogenesis of PD and indicate the advisability of using antioxidant drugs in complex therapy of PD.