Commonality of molecular mechanisms of neuroplasticity and neuropathology: integrative approach

Analysis of our own data and of the results from other groups brings about a concept concerning community of the basic molecular mechanisms involved in neuroplasticity and neuropathology at different levels. Along with the fundamental significance of this idea for understanding of processes taking place both in normal brain and in neuropathological conditions, the concept is of principal importance for practical application. Failure in development of the "pathogenetically directed" approaches to treatment of neural diseases (e. g. stroke) are, in particular, related to the neglecting of identity of the basic molecular mechanism underlying both normal brain functioning and neuropathological conditions resulting in intervention into these common mechanisms (NMDA receptors blockade, inhibitor of the so called "apoptotic" proteases).     

Study of the reaction of grafting acrylamide onto xanthan gum

The present study aimed to study the reaction conditions of grafting of acrylamide on xanthan gum. It was analyzed the influence of reaction conditions, mainly type of initiator activation, initiator concentration and initiator/acrylamide ratio, on graft parameters and copolymer properties. Potassium persulfate was employed as an initiator and heating or N,N,N',N'-tetramethylethylenediamine was used to activate the initiator. Reaction time and initiator concentration were varied and final values for grafting percentage and grafting efficiency were the same for both methods, whereas speed in reaching these values differs from one technique to another. We found that reaction time was inversely proportional to intrinsic viscosity, likely due to main chain degradation promoted by potassium persulfate (KPS); furthermore, the increasing in the KPS concentration lowers grafting percentage, acrylamide conversion and chain degradation, possibly as a result of O(2) formation at high KPS concentrations.     

Fast and aimed delivery of voltage-sensitive dyes to mammalian brain slices by biolistic techniques

Fast voltage-sensitive dyes (VSD) are widely used in modern neuroscience for optical recording of electrical potentials at many levels, from single cell compartment to brain areas, containing populations of many neural cells. The more lipophilic a VSD, the better signal-to-noise ratio of the optical signal, but there are no effective ways to deliver a water-insoluble dye into the membrane of live cell. Here we report a new protocol based on rapid biolistic delivery of VSDs, which is optimal for further recordings of optical signals from live neurons of rat brain slices. This protocol allows us to stain locally (150 mkm) neural somata of brain structures with a Golgi-like pattern, and a VSD propagates even to distant neurites of stained cells very quickly. This technique also can be used for rapid local delivery of any lipophilic and water-insoluble substances into live cells, further optical recording of neural activity, and analysis of potential propagation in a nerve cell.     

Local delivery of chitosan/VEGF siRNA nanoplexes reduces angiogenesis and growth of breast cancer in vivo

Vascular endothelial growth factor (VEGF) is the important angiogenic factor associated with tumor growth and metastasis in a wide variety of solid tumors. The aim of this study is to investigate the tumor suppressive effect of chitosan/small interfering RNA (siRNA)-VEGF nanoplexes in the rat breast cancer model. Chitosan/siRNA nanoplexes (siVEGF-A, siVEGFR-1, siVEGFR-2) and NRP-1 were prepared in a 15 to1 ratio and injected (intratumorally) into the breast-tumor-bearing Sprague-Dawley rats. Tumor volumes were measured during 21 days. To investigate the effect of chitosan/siRNA nanoplexes on VEGF expression in tumors, VEGF was analyzed with immunohistochemistry and western blotting. The mRNA levels of VEGF in tumor samples were determined with real-time PCR (RT-PCR). After siRNA treatment, a marked reduction in tumor volumes was measured in complex-injected rats (97%). Free siRNA injection showed lower tumor inhibition. Reduction of VEGF protein was also shown with western blotting and immunohistochemistry. Similar results were obtained with RT-PCR also. These results indicate that the chitosan/siRNA targeting to VEGF nanoplexes have a remarkably suppressive effect on VEGF expression and tumor volume in breast cancer model of rats.     

Start codon in the serine proteinase gene from Bacillus intermedius

The translation initiation site in the extracellular serine subtilisin-like proteinase gene from Bacillus intermedius (aprBi) (AN AY754946) secreting at the stationary growth phase was established. The analysis of aprBi open reading frame revealed three putative translation start sites (TTG, GTG, ATG). Using SignalP online freeware program we have determined the functional activity probability of each of them. To identify the translation start point the modified subtilisin-like protease genes carrying nucleotide replacements in supposed start codons were developed using oligonucleotide-directed mutagenesis. We have investigated the expression of these genetic constructions in protease-deficient strain B. subtilis AJ73. According our results it was concluded that the translation in aprBi gene starts from GTG kodon.     

P-glycoprotein polymorphism in hypo- and hyper-thyroidism patients

P-glycoprotein (Pgp) is encoded by the multidrug resistance gene (MDR1) in humans and is the product of MDR1. It is expressed in various tissues and is related to drug distribution in intestinal erythrocytes, capillary endotel of brain, proximal tubules cells of kidneys and liver canalicular cells. Expression of Pgp is affected by Pgp polymorphism, and exon 26 C3435T polymorphism is the most common one. It has been thought that expression of Pgp is high in C-allele subjects and this situation is responsible for the resistance against some drugs and substances. Pgp may have a role in the distribution of thyroid hormones, drugs used for hypo- and hyperthyroidism and the resistance occurred. For this purpose possible relationship between T and C alleles and frequency of Pgp polymorphism as well as thyroid hormone distribution in patients with hypo- and hyperthyroidism was investigated. Thirty five hyperthyroidism patients diagnosed as Graves’ disease, 78 hypothyroidism patients diagnosed as Hashimoto’s thyroiditis and 100 healthy volunteers were included in the study. According to the results obtained no statistically significant difference was found in Pgp C3435T polymorphism between hypo- and hyperthyroidism patients. In addition, the serum free T3 levels of hyperthyroidism patients with C alleles was higher than those of subjects with T alleles. No statistically significant difference was seen in the CC, CT and TT genotype frequencies between the patients and control groups. In conclusion, it seems that Pgp polymorphism is not a predictor factor for the occurrence of hypo- and hyperthyroidism. There is a significant relationship between Pgp and the elevated serum free T3 levels of hyperthyroidism patients, and further research will help understand this situation.     

Changes in pyridine nucleotide levels alter oxygen consumption and extra-mitochondrial phosphates in isolated mitochondria: a 31P-NMR and NAD(P)H fluorescence study

Isolated rat-liver mitochondria were used to study the relation between mitochondrial NADH levels, oxygen consumption (QO2), and extra-mitochondrial phosphates. Alterations in NADH and QO2 were accomplished by incubating mitochondria with different substrates or varying amounts of exogenous ATPase while monitoring QO2 and NAD(P)H fluorescence. Two sets of conditions were studied: (1) in the presence of excess ADP and inorganic phosphate, an increase in NAD(P)H fluorescence was associated with a linear increase in QO2; (2) when QO2 was driven by the steady-state hydrolysis of ATP by exogenous ATPase, increases in QO2 were associated with proportional decreases in NAD(P)H fluorescence. For all substrates tested this relation was linear; however, the slope was substrate dependent. Different substrates were able to maintain different NAD(P)H levels at the same QO2. To investigate this further, effects of changing substrates at constant QO2 on NAD(P)H and extra-mitochondrial phosphates were determined. Addition of glutamate + malate to mitochondria respiring on citrate caused a 50% increase in NAD(P)H fluorescence, a 41% decrease in ADP, and a 30% decrease in inorganic phosphate. Similar changes for the substrate jump, pyruvate + malate to glutamate + malate were found. Finally, it was determined that a linear relation holds between increases in NAD(P)H fluorescence and increases in QO2 when substrates were varied at constant, physiologic levels of extra-mitochondrial ADP. These results indicate that QO2 depends on NAD(P)H levels as well as on extra-mitochondrial phosphates over a wide range of respiratory rates.