Cryopreservation of seeds and protocorms of rare temperate orchids

The efficiency of cryopreservation of seeds of five rare and endangered species of temperate orchids belonging to Platanthera and Dactylorhiza genera followed by their asymbiotic culture in vitro, as well as of in vitro cultured D. fuchsii protocorms (specific stage of orchid embryo development after release from the seed coat) was investigated. Germination rates of seeds after their exposure to liquid nitrogen were species-depended and could be either higher or lower than in the unfrozen control. There was no significant difference between growth rates of protocorms of the same species obtained from seeds collected in various Russia regions and cultured for 5 months. After vitrification, 9% of D. fuchsii protocorms with a larger diameter of 1200 μm survived cryopreservation; however, their growth was retarded for three months when compared to control protocorms.     

Creatinyl amino acids—new hybrid compounds with neuroprotective activity

Prolonged oral creatine administration resulted in remarkable neuroprotection in experimental models of brain stroke. However, because of its polar nature creatine has poor ability to penetrate the blood–brain barrier (BBB) without specific creatine transporter (CRT). Thus, synthesis of hydrophobic derivatives capable of crossing the BBB by alternative pathway is of great importance for the treatment of acute and chronic neurological diseases including stroke, traumatic brain injury and hereditary CRT deficiency. Here we describe synthesis of new hybrid compounds—creatinyl amino acids, their neuroprotective activity in vivo and stability to degradation in different media. The title compounds were synthesized by guanidinylation of corresponding sarcosyl peptides or direct creatine attachment using isobutyl chloroformate method. Addition of lipophilic counterion (p‐toluenesulfonate) ensures efficient creatine dissolution in DMF with simultaneous protection of guanidino group towards intramolecular cyclization. It excludes the application of expensive guanidinylating reagents, permits to simplify synthetic procedure and adapt it to large‐scale production. The biological activity of creatinyl amino acids was tested in vivo on ischemic stroke and NaNO₂‐induced hypoxia models. One of the most effective compounds—creatinyl‐glycine ethyl ester increases life span of experimental animals more than two times in hypoxia model and has neuroprotective action in brain stroke model when applied both before and after ischemia. These data evidenced that creatinyl amino acids can represent promising candidates for the development of new drugs useful in stroke treatment. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis of GnRH analogues and their application in targeted gene delivery systems

A set of GnRH analogues containing the nuclear localization signal (NLS) of the SV-40 virus was synthesized using solid phase peptide synthesis and chemical ligation techniques. Selective chemical ligation was achieved through hydrazone formation upon the interaction of NLS hydrazide and GnRH analogue modified with pyruvic acid. The efficiency of the synthesized compounds was demonstrated in experiments on transfection of various human cancer cell lines with reporter luciferase and β-galactosidase genes, as well as suicide thymidine kinase gene of HSV-1. Selectivity of the peptide-DNA complex effect on cancer cells is achieved as a result of its penetration through the cell membrane via GnRH receptor-mediated endocytosis pathway.     

The effect of pharmacologic substances on intraspecies aggression]

It is found, that aggression which occures in rats under inevitable painful stimulation is coursed as excessive excitation of the rats. This aggression may be used for revealing the sedative effect of drugs. The method of fighting for the stool which is caused by motivated fighting for the territory was worked out. This motivated aggression may be used for revealing the tranquillizing effect of drugs. Using these methods it is found that stelasine, haloperidol, amitriptyline, imipramine, chlordiazepoxide, diazepam and benactyzine in small doses have a tranquillizing effect, while pentobarbital and chlorpromazine have primarily a sedative effect.     

Possible role of positive reinforcement zones in the mechanisms of pain regulation and their relation to the endogenous opiate system

The consequences of self-stimulation reaction (RSS) to pain threshold in tail withdrawal test (55 degrees C) and naloxone effect have been investigated in tests, using male rats with chronically implanted electrodes into the hypothalamus (AP = 1.5, L = 1.5, H = 8.5) and suture dorsal nucleus (AP = 7.0, L = 0, H = 7.0) (coordinates according to Fifková atlas). It was established that right after RSS, pain threshold in both zones increased 2-2.5-fold and 30 min later reached the initial level. Naloxone injected before RSS increased pain thresholds and decreased RSS frequency from hypothalamus but failed to change these RSS parameters from suture dorsal nucleus. However, naloxone did not affect the increase in pain thresholds caused by RSS from both zones. Taking into account the fact that analgesia appearing after RSS from the anterior hypothalamus as well as from suture dorsal nucleus is not reversed by naloxone, it is suggested that positive reward zones activation partially realized by opioidergic mechanisms or having no connection with them may lead to the development of non-opiate type analgesia.     

Reduction of Selenite by Azospirillum brasilense with the Formation of Selenium Nanoparticles

The ability to reduce selenite (SeO₃ ^2?) ions with the formation of selenium nanoparticles was demonstrated in Azospirillum brasilense for the first time. The influence of selenite ions on the growth of A. brasilense Sp7 and Sp245, two widely studied wild-type strains, was investigated. Growth of cultures on both liquid and solid (2 % agar) media in the presence of SeO₃ ^2? was found to be accompanied by the appearance of the typical red colouration. By means of transmission electron microscopy (TEM), electron energy loss spectroscopy (EELS) and X-ray fluorescence analysis (XFA), intracellular accumulation of elementary selenium in the form of nanoparticles (50 to 400 nm in diameter) was demonstrated for both strains. The proposed mechanism of selenite-to-selenium (0) reduction could involve SeO₃ ^2? in the denitrification process, which has been well studied in azospirilla, rather than a selenite detoxification strategy. The results obtained point to the possibility of using Azospirillum strains as endophytic or rhizospheric bacteria to assist phytoremediation of, and cereal cultivation on, selenium-contaminated soils. The ability of A. brasilense to synthesise selenium nanoparticles may be of interest to nanobiotechnology for “green synthesis” of bioavailable amorphous red selenium nanostructures.