Quantitation of changes in gene expression of norepinephrine biosynthetic enzymes in rat stellate ganglia induced by stress

Enzymes involved in catecholamine synthesis are present in the highest concentration in the adrenal medulla, however they were found also in other, mainly nervous tissues. The aim of our study was to quantify the exact concentration of tyrosine hydroxylase (TH) and dopamine-ss-hydroxylase (DBH) mRNA in rat stellate ganglia under control conditions and at different intervals after exposure to immobilization stress (IMO). In rats immobilized once for 2h, we determined TH and DBH mRNA in different time intervals up to 22 h after the end of the stress stimulus. TH immunoreactive protein levels were also determined in stellate ganglia. TH and DBH mRNA levels were quantified by RT-competitive-PCR.In stellate ganglia, the concentration of TH mRNA was 17+/-1.6 amol/microg of total RNA, which is approximately 30-times lower than in the adrenal medulla. The concentration of DBH mRNA in the stellate ganglia was 2601+/-203 amol/microg of total RNA, which is the concentration similar to adrenal medulla, but is 150-times higher than concentration of TH mRNA in stellate ganglia. After a single 2-h immobilization the highest elevation of TH and DBH mRNA levels was measured 22 h after the termination of the stress stimulus. Repeated immobilization (7 days, 2h daily) did not produce further increase in TH and DBH mRNA levels compared to already elevated levels in adapted control group (immobilized for 6 days, 2h daily and decapitated 22 h later). Levels of TH protein were significantly changed only after the repeated immobilization.This study compared for the first time the precise amounts of TH and DBH mRNA in rat stellate ganglia under control conditions and after immobilization stress, and indicates large differences in their concentration. TH and DBH mRNA concentrations in stellate ganglia are markedly elevated for a prolonged period of time after termination of the stress stimuli.     

Analysis of silver binding nucleolar organizer regions in exfoliative cytology smears of potentially malignant and malignant oral lesions

Nucleolar organizer regions are nucleolar components that contain proteins that are stained selectively by silver methods; they can be identified as black dots throughout the nucleolus and are known as silver binding nucleolar organizer regions (AgNOR). The number of AgNOR is related to the cell cycle and the proliferative activity of the cells. We investigated AgNOR using exfoliative cytology smears of potentially malignant oral lesions. Eighty individuals were divided into four equal groups: healthy controls, oral leukoplakia, oral submucous fibrosis and oral squamous cell carcinoma. The mean number of AgNOR in each study group gradually increased from control to oral leukoplakia to oral submucous fibrosis to oral squamous cell carcinoma. The proliferative index was increased in the oral premalignant and malignant patients compared to normal subjects. The mean AgNOR size gradually increased from control to oral leukoplakia to oral submucous fibrosis to oral squamous cell carcinoma. Spherical shaped AgNOR were most common in controls, whereas large, clustered and kidney shapes were most common in oral squamous cell carcinoma. Multiparameter analysis of AgNOR in oral exfoliative smears is a simple, sensitive and cost-effective method for differentiating premalignant from malignant lesions and can be used in conjunction with routine cytomorphological evaluation.     

Protective Effect of Insulin on Rat Cortical Neurons in Oxidative Stress and Its Dependence on Modulation of Protein Kinase B (Akt) Activity

Clinical trials of insulin and experiments on its intranasal administration to animals suggest that this hormone can be efficient in treating human neurodegenerative and some other diseases associated with brain injury. However, the mechanism of the neuroprotective effect of intranasal insulin is far from being understood. The aim of the present work was to study the protective and antioxidative effects of insulin at various concentrations on rat brain cortical neurons under oxidative stress conditions and to estimate the contribution of protein kinase B (Akt) activity modulation to insulin-induced enhancement of neuronal viability in the rat brain cortex. The protective effect of insulin was shown to be dose-dependent within the nanomolar range (1 nM < 10 nM < 100 nM and/or 1 μM). A study of the antioxidative effect of insulin revealed the efficacy of such a low concentration as 1 nM. Immunoblot analysis showed that insulin at concentrations of 100 nM and 1 μM activates Akt both in neurons and control cells at different times after their exposure to a pro-oxidant agent. LY294002, a specific PI3K/Akt signaling pathway inhibitor, was shown to significantly reduce the protective and antioxidative effects of insulin. Insulin-induced upregulation both of Akt activity and antiapoptotic protein Bcl-2 appears to play an important role in the neuroprotective effect of insulin.     

DELAYED URIC ACID ACCUMULATION IN PLASMA PROVIDES ADDITIONAL ANTI-OXIDANT PROTECTION AGAINST IRON-TRIGGERED OXIDATIVE STRESS AFTER A WINGATE TEST

Reactive oxygen species are produced during anaerobic exercise mostly by Fe ions released into plasma and endothelial/muscle xanthine oxidase activation that generates uric acid (UA) as the endpoint metabolite. Paradoxically, UA is considered a major antioxidant by virtue of being able to chelate pro-oxidative iron ions. This work aimed to evaluate the relationship between UA and plasma markers of oxidative stress following the exhaustive Wingate test. Plasma samples of 17 male undergraduate students were collected before, 5 and 60 min after maximal anaerobic effort for the measurement of total iron, haem iron, UA, ferric-reducing antioxidant activity in plasma (FRAP), and malondialdehyde (MDA, biomarker of lipoperoxidation). Iron and FRAP showed similar kinetics in plasma, demonstrating an adequate pro-/antioxidant balance immediately after exercise and during the recovery period (5–60 min). Slight variations of haem iron concentrations did not support a relevant contribution of rhabdomyolysis or haemolysis for iron overload following exercise. UA concentration did not vary immediately after exercise but rather increased 29% during the recovery period. Unaltered MDA levels were concomitantly measured. We propose that delayed UA accumulation in plasma is an auxiliary antioxidant response to post-exercise (iron-mediated) oxidative stress, and the high correlation between total UA and FRAP in plasma (R-Square = 0.636; p = 0.00582) supports this hypothesis.     

Dependence of protective effect of α-tocopherol on its concentration and time of action on pc12 cells under conditions of oxidative stress

At the short-term incubation (0.5 and 1.5 h) of cells of the PC12 neuronal line with α-tocopherol, its protective effect against the cytotoxic hydrogen peroxide action was increased with rise of its concentration in samples; the protection was practically absent at action of nanomolar antioxidant concentrations, but was well expressed at its micromolar concentrations. These data agree with the concept that α-tocopherol increases the cell viability by reacting directly with free radicals, which leads to formation of the less reactive compounds deprived of non-paired electron. The evidence is obtained that at the long-term action on PC12 cells, α-tocopherol not only in micro-, but also in nanomolar concentrations increases significantly the cell viability under conditions of oxidative stress. As follows from the obtained data, an important role in realization of the α-tocopherol protective effect at the long-term incubation seems to be played by modulation by this antioxidant of activity of protein kinase activated by extracellular signaling, phosphatidylinosite 3-kinase, and protein kinase C.     

Antioxidant Status in Adolescents of Small Siberian Ethnoses

The components of the antioxidant defense (AOD) system were analyzed in 74 male and female adolescents representing small ethnic groups that inhabit the territory of Eastern Siberia. Our findings indicate a dependence of the antioxidant status indicators on gender and ethnic identity of test subjects. More adaptive variants of the AOD system’s functioning were found in Tofalar boys (elevated levels of fat-soluble vitamins, increased glutathione system activity), whereas Evenk boys were characterized by a decreased activity of antioxidant factors (low α-tocopherol and retinol supply, glutathione system imbalance), as compared both to girls and Tofalar boys. The changes revealed in this group were also supported by increased values of the oxidative stress index.