The use of antioxidants to prevent glutamate-induced derangement of calcium ion metabolism in rat cerebral cortex synaptosomes.

Glutamate is shown to induce increases in intracellular Ca2+ concentrations ([Ca2+]i), increases in 45Ca2+ influx, decreases in the activity of Na+,K+-ATPase activity, and activation of the Na+/Ca2+ exchanger in rat cerebral cortex synaptosomes. NMDA receptor antagonists virtually prevented these effects. Preincubation of synaptosomes with alpha-tocopherol, superoxide dismutase, and ganglioside GM1 normalized [Ca2+]i, 45Ca2+ influx, and Na+,K+-ATPase activity in rat cerebral cortex synaptosomes exposed to glutamate. Glutamate and GM1 activated the Na+/K+ exchanger, and their effects were additive. Calcium ions entering cerebral cortex nerve cells via NMDA receptors during exposure to high glutamate concentrations appeared to be only the trigger for the processes activating free-radical reactions. Activation of these reactions led to increases in Ca2+ influx into cells, decreases in Na+,K+-ATPase activity, and significant increases in [Ca2+]i, though this could be prevented by antioxidants and gangliosides.

[The antioxidant prevention of disorders in calcium ion metabolism under the action of glutamate on the synaptosomes of the rat cerebral cortex]. / Predotvrashchenie antioksidantami narushenii obmena ionov kal'tsiia pri deistvii glutamata na sinaptosomy kory mozga krys.

An increase of intracellular calcium ion concentration and of the 45Ca2+ entry, a decrease in Na+,K(+)-ATPase activity, and activation of Na+/Ca2+ exchange were shown to be initiated by glutamate in the rat brain cortex synaptosomes. These effects could be prevented with antagonists and blocking agents of the NMDA receptors. Pre-incubation of the synaptosomes with alpha-tocopherol, superoxide dismutase, and ganglioside GM1 was shown to normalise [45Ca2+], the rate of 45Ca2+ entry, and the activity of Na+,K(+)-ATPase in the synaptosomes. The data obtained suggest that calcium ions entering the brain cortex neurones via the NMDA receptors in presence of excessive glutamate, trigger activation of free radical reactions damaging the neurones in ischemia, cerebral lesions, and other pathological conditions.

The difference in the effect of glutamate and NO synthase inhibitor on free calcium concentration and Na+, K+-ATPase activity in synaptosomes from various brain regions.

The significant increase of free calcium concentration ([Ca2+]i) was found in rat cerebral cortex synaptosomes and hippocampal crude synaptosomal fraction after their exposure to glutamate. But no change of [Ca2+]i was revealed in cerebellar synaptosomes, the slight increase of [Ca2+]i in striatal synaptosomes was not significant. The presence of Ng-nitro-L-arginine methyl ester (L-NAME) in the incubation medium practically prevented the increase of [Ca2+]i initiated by glutamate in cerebral cortex synaptosomes, but not in hippocampal ones. The significant diminution of [Ca2+]i in the presence of this inhibitor was shown in striatal synaptosomes exposed to glutamate. Na+,K+-ATPase activity is significantly lower in cerebral cortex, striatal and hippocampal synaptosomes exposed to glutamate. L-NAME prevented the inactivation of this enzyme by glutamate. In cerebellar synaptosomes the tendency to the decrease of enzymatic activity in the presence of L-NAME was on the contrary noticed. Thus, the data obtained provide evidence of the protective effect of NO synthase inhibitor in brain cortex and striatal synaptosomes, but not in cerebellar synaptosomes. Synaptosomes appear to be an adequate model to study the regional differences in the mechanism of toxic effect of excitatory amino acids.

[Analysis of new opiate-like peptides using a radioreceptor method]. / Analiz novykh opiatopodobnykh peptidov radioretseptornym metodom.

A screening of new synthetic opioid-like peptides has been carried out by the radioreceptor assay using selective labeled ligands to mu-, delta- and gamma-opioid receptors of the rat brain membranes. With this aim peptides from sequences of the following proteins were used: kapporphin-Tyr-Ser-Phe-Gly-Gly and its analogues-Tyr-Ser-Phe-Gly-Gly-NH2, Tyr-D-Ser-Phe-Gly-Gly, Tyr-D-Ser-Phe-Gly-Gly-NH2, myelorphin-Phe-Gly-Tyr-Gly-Gly, interenkephalin B-Arg-Arg-Gln-Phe-Lys and chimeric peptide IEPhBin 1-Tyr-Gly-Gly-Phe-Leu-Arg-Pro-Tyr-Ile-Leu consisting of leu-enkephalin and pentaneurotensin. It has been found that myelorphin has a prevalent affinity to mu-receptor, while the kapporphin analogues both to mu- and delta-receptors. The presence of pentaneurotensin in chimeric peptide does not affect the specificity of binding to opioid receptors, but decreases affinity to mu- and delta-receptors approximately by an order as compared to leu-enkephalin. Kapprorphin and interenkephalin B displace neither of the selective labeled opioid ligands under study.

[Highly active analogs of opiate-like peptides historphin and kapporphin]. / Vysokoaktivnye analogi opiatopodobnykh peptidov gistorfina i kapporfina.

Analgetic activity of analogues of new opioid peptides historphine Tyr-Gly-Phe-Gly-Gly and capporphine Tyr-Ser-Phe-Gly-Gly was studied. Analogues of historphine Tyr-D-Ala-Phe-Gly-Gly and of capporphine Tyr-D-Ser-Phe-Gly-Gly exceeded 10(3)-fold the Leu-encephaline efficiency using a tail-jerk test and their derivatives containing amide group in the C-end position--10(4)-fold. All the four analogues increased the resistance period in the heat plate test. Naloxone removed or decreased the effect of these peptides studied. After repeated administration peptides Tyr-D-Ala-Phe-Gly-Gly-NH2 and Tyr-D-Ser-Phe-Gly-NH2 were similar in the activity to the most strong native opioid peptide desmorphine. Considering that analogues of historphine and capporphine exhibited high analgetic activity, they may be used as promising structures in more complicated modifications in order to produce highly effective analogues of opioid peptides.

[Opiate-like peptides with primary structure different from that of enkephalins]. / Printsipial'no otlichnye po pervichnoi strukture ot énkefalinov opiatopodobnye peptidy.

Some new opiate-like peptides originating from opioid peptide precursors, dinorphine, histone H2b, major myeline protein, natriuretic atriopeptide and from the immunomodulating protein splenin whose primary structure differs essentially from that of enkephalins are described. Being intracysternally injected to mice, all the peptides under study caused a naloxone-sensitive analgetic effect as could be judged from the tail pinch tests. The effects of some opiate-like peptides were much stronger than that of leu-enkephalin. According to their primary structure, the opiate-like peptides can randomly be allocated into two families. Dipeptide Lys-Arg and free arginine also possess a marked analgetic activity which is abolished by naloxone. It seems likely that the epiate-like activity of the peptides under study is due to the similarity of their secondary and ternary structure to that of enkephalins of to their involvement in the regulation of opioid peptide metabolism.

[Structural organization of the bacterial nucleoid using endonucleolysis]. / Issledovanie struktyrnoi organizatsii bakterial'nogo nukleoida pospedstvom endonukleoliza.

The fragmentation of bacterial deoxyribonucleoprotein (bDNP) in the spheroplasts of Escherichia coli, Serratia marcescens, Pseudomonas fluorescens and Micrococcus luteus by bacterial intracellular Ca2+ or Ca2+, Mg2+-dependent endonucleases in situ was studied. An electrophoresis of the extracted nuclease-split bDNP revealed the presence of high molecular weight (nuclease-resistant) and low molecular weight multiple fragments (100--120 nucleotide pairs). The electrophoretic mobility of the smallest nuclease-split DNA fragments in all bacterial species under study was similar, indicating the orderly structure of bDNP. Two total fractions whose electrophoretic mobility corresponded to that of the histones H2a and H2b from calf thymus were prevalent in the spectrum of acid-soluble bDNP proteins of gram-negative species. The heterogeneity of DNP with respect to its sensitivity to nucleases, is interaction with membranes and protein distribution pattern were revealed by treatment of the bacterial nucleoid with endogenous endonucleases, which probably reflects differences in the functional state of individual sites of the genome.

[Structural heterogeneity of chromatin as revealed by electrophoretic methods]. / Strukturnaia geterogennost' khromatina, vyiavliaemaia elektroforeticheskimi metodami.

Electrophoresis of endonuclease-split rat liver chromatin in polyacrylamide and agarose gels of a monotonous concentration revealed different separation patterns. The first pattern exhibited a spectrum of chromatin fragments of discrete sizes, while the second one -- two fractions, i. e. DNP and DNA. The polyacrylamide gel concentration gradient allowed to achieve the best resolution of individual classes of chromation fragments with individual subfractions in sub-, mono- and trinucleosomes. A comparison of micrococcal nuclease-split chromatin fragments of vertebrates revealed nucleosomes of varying sizes. The heterogeneity of the nucleosomes was also found during isotachophoresis and free flow electrophoresis. An analysis of individual subfractions of the mononucleosomes revealed differences in the composition and number of proteins as well as in the kinetics of basic and acidic titration. The chromatin fractions obtained by discontinuous endonucleolysis differed in their composition from the individual subfractions of histones.

[Probing the structure of bacterial deoxyribonucleoproteins with exogenous and endogenous nucleases]. / Zondirovanie struktury bakterial'nykh dezoksiribonukleoproteidov ékzogennoi i éndogennoi nukleazami.

During digestion of deoxyribonucleoproteins (DNP) of gram-negative bacteria by micrococcal nuclease and Ca2+, Mg2+-dependent endonuclease in situ regular series fragments-and large nuclease-resistent fragments of DNP were revealed by electrophoresis. The DNP length of the smallest DNP-fragment was tentatively 120-140 base pairs. In investigated bacterial species DNP contained at least two basic proteins which had electrophoretic mobility similar to that of histone H4 of eucaryot. It is suggested that bacterial DNPs have common regular structure.

[Fractionation of chromatin in acrylamide gel with immobilized non-histone proteins and DNA]. / Fraktsionrovanie khromatina na akrilamidnom gele s immobilizovannymi negistonovymi belkami i DNK.

Liver and kidney chromatin and DNA were fractionated in acrylamide columns with immobilized readily extracted non-histone chromosomal proteins (RE-NHCP). Chromatin was fractionated in two fractions on the column with RE-NHCP, one of the fractions is sorbing and has a poor protein contents, as compared to the initial chromatin preparation. Fractionation of chromatin in two fractions (sorbing and non-sorbing) was also observed under its chromatography in the column with immobilized DNA. Both chromatin fractions are stripped in the protein content after the elution, which is probably due to the transfer of some proteins from chromatin to immobilized DNA. Chromatography of DNA on gel with homologous RE-NHCP also results in DNA fractionation into sorbing and non-sorbing fractions. The data obtained suggest the irregularity of the distribution and the restriction of "binding sites" for exogenous homologous and heterologous RE-NHCP and of the number of chromatin proteins, which are capable of additional binding with exogenous homologous DNA.