Evenly tritium-labeled peptides and their in vivo and in vitro biodegradation

Biologically active peptides evenly labeled with tritium were used for studying the in vitro and in vivo biodegradation of the peptides. Tritium-labeled peptides with a specific radioactivity of 50-150 Ci/mmol were obtained by high temperature solid phase catalytic isotope exchange (HSCIE) with spillover tritium. The distribution of the isotope label among all amino acid residues of these peptides allows the simultaneous determination of practically all possible products of their enzymatic hydrolysis. The developed analytical method includes extraction of tritium-labeled peptides from organism tissues and chromatographic isolation of individual labeled peptides from the mixture of degradation products. The concentrations of a peptide under study and the products of its biodegradation were calculated from the results of liquid scintillation counting. This approach was used for studying the pathways of biodegradation of the heptapeptide TKPRPGP (Selank) and the tripeptide PGP in blood plasma. The pharmacokinetics of Selank, an anxiolytic peptide, was also studied in brain tissues using the intranasal in vivo administration of this peptide. The concentrations of labeled peptides were determined, and the pentapeptide TKPRP, tripeptide TKP, and dipeptides RP and GP were shown to be the major products of Selank biodegradation. The study of the biodegradation of the heptapeptide MEHFPGP (Semax) in the presence of nerve cells showed that the major products of its biodegradation are the pentapeptide HFPGP and tripeptide PGP. The enkephalinase activity of blood plasma was studied with the use of evenly tritium-labeled [Leu]enkephalin. A high inhibitory effect of Semax on blood plasma enkephalinases was shown to arise from its action on aminopeptidases. The method, based on the use of evenly tritium-labeled peptides, allows the determination of peptide concentrations and the activity of enzymes involved in their degradation on a tg scale of biological samples both in vitro and in vivo.     

Short peptide fragments with antiulcer activity from a collagen hydrolysate

A peptide acidic hydrolysate of collagen (PHC) was obtained under conditions (4 N HCl) ensuring the predominant formation of short peptides, glyprolines. They were separated and their antiulcer activity was studied. Thirty individual peptides with molecular masses of 174-420 amu were isolated from the PHC by HPLC. The PHC was shown to predominantly contain 2- to 4-aa peptides, including PG, GP, and PGP. Experiments on rats demonstrated that, on intragastric administration at a dose of 1 mg/kg, PHC enhances the stability of the gastric mucosa to the action of ulcerogenic factors, such as ethanol and stress, and exhibits a protecting antiulcer effect. Even a lesser dose (0.1 mg/kg), which reduced ulcer area twofold, was effective in the stress model of ulcer formation. The intraperitoneal and intragastric administration of PHC at a dose of 1 mg/kg was found to exhibit a therapeutic effect in the acetate model of ulcer formation.     

Evenly tritium labeled peptides in study of peptide in vivo and in vitro biodegradation

Biologically active peptides evenly labeled with tritium were used for studying the in vitro and in vivo biodegradation of the peptides. Tritium-labeled peptides with a specific radioactivity of 50–150 Ci/mmol were obtained by high temperature solid phase catalytic isotope exchange (HSCIE) with spillover tritium. The distribution of the isotope label among all amino acid residues of these peptides allows the simultaneous determination of practically all possible products of their enzymatic hydrolysis. The developed analytical method includes extraction of tritium-labeled peptides from organism tissues and chromatographic isolation of individual labeled peptides from the mixture of degradation products. The concentrations of a peptide under study and the products of its biodegradation were calculated from the results of liquid scintillation counting. This approach was used for studying the pathways of biodegradation of the heptapeptide TKPRPGP (Selank) and the tripeptide PGP in blood plasma. The pharmacokinetics of Selank, an anxiolytic peptide, was also studied in brain tissues using the intranasal in vivo administration of this peptide. The concentrations of labeled peptides were determined, and the pentapeptide TKPRP, tripeptide TKP, and dipeptides RP and GP were shown to be the major products of Selank biodegradation. The study of the biodegradation of the heptapeptide MEHFPGP (Semax) in the presence of nerve cells showed that the major products of its biodegradation are the pentapeptide HFPGP and tripeptide PGP. The enkephalinase activity of blood plasma was studied with the use of evenly tritium labeled [Leu]enkephalin. A high inhibitory effect of Semax on blood plasma enkephalinases was shown to arise from its action on aminopeptidases. The method, based on the use of evenly tritium-labeled peptides, allows the determination of peptide concentrations and the activity of enzymes involved in their degradation on a μg scale of biological samples both in vitro and in vivo.     

Short peptide fragments with antiulcer activity from a collagen hydrolysate

A peptide acidic hydrolysate of collagen (PHC) was obtained under conditions (4 N HCl) ensuring the predominant formation of short peptides, glyprolines. They were separated and their antiulcer activity was studied. Thirty individual peptides with molecular masses of 174–420 amu were isolated from the PHC by HPLC. The PHC was shown to predominantly contain 2-to 4-aa peptides, including PG, GP, and PGP. Experiments on rats demonstrated that, on intragastric administration at a dose of 1 mg/kg, PHC enhances the stability of the gastric mucosa to the action of ulcerogenic factors, such as ethanol and stress, and exhibits a protecting antiulcer effect. Even a lesser dose (0.1 mg/kg), which reduced ulcer area twofold, was effective in the stress model of ulcer formation. The intraperitoneal and intragastric administration of PHC at a dose of 1 mg/kg was found to exhibit a therapeutic effect in the acetate model of ulcer formation.     

Growth decline linked to warming-induced water limitation in hemi-boreal forests

Hemi-boreal forests, which make up the transition from temperate deciduous forests to boreal forests in southern Siberia, have experienced significant warming without any accompanying increase in precipitation during the last 80 years. This climatic change could have a profound impact on tree growth and on the stability of forest ecosystems in this region, but at present evidence for these impacts is lacking. In this study, we report a recent dramatic decline in the growth of hemi-boreal forests, based on ring width measurements from three dominant tree-species (Pinus sylvestris, Larix sibirica and Larix gmelinii), sampled from eight sites in the region. We found that regional tree growth has become increasingly limited by low soil water content in the pre- and early-growing season (from October of the previous year to July of the current year) over the past 80 years. A warming-induced reduction in soil water content has also increased the climate sensitivity of these three tree species. Beginning in the mid-1980s, a clear decline in growth is evident for both the pine forests and the larch forests, although there are increasing trends in the proxy of soil water use efficiencies. Our findings are consistent with those from other parts of the world and provide valuable insights into the regional carbon cycle and vegetation dynamics, and should be useful for devising adaptive forest management strategies.     

Reculatory fragments of collagen in gastric mucosa homeostasis

The new glyproline family was distinguished from the regulatory peptides recently. It includes the simplest proline- and glycine-containing peptides: PG, GP, PGP, and respective peptides with hydroxylated proline residues. Glyproline's bioactivity covers many important systems of the body including suppression of some reaction in the blood coagulation and platelet aggregation and gastric mucosal maintenance. It was shown that PGP, PG and GP have a wide spectrum of antiulcer activity with respect to gastric mucosal damages of various aetiology. GHyp and HypGP show also antiulcer action. In vivo glyprolines being fragments of collagen may be generated during synthesis and catabolism of collagen. It is well known that approximately 10-60% of newly synthesized collagen degrade intracellularly with succeeding secretion of small peptides composed of less than 5 aminoacid residues out of cells. Different simplest proline and hydroxyproline fragments of glyprolines are revealed in various type of collagen: GP, GHyp, PG, PPG, PGP, PHypG., GPHyp, GPP, GPG, GHypP, HypGP. It is possible that these fragments may be also secreted out of cells during the stage of degradation of newly synthesized collagen. We showed that the intragastric (per oral) introduction of hydrolyzed gelatin, having 20 small peptide fragments, including PGP and HypGP, also increase gastric stability showing protective and therapeutic antiulcer effect. The corresponding receptors for glyprolines are not completely identified yet but it may be supposed that PGP, GP and other glyprolines interact with the same receptors with which the III type collagen is binding with platelet's receptors. It is supposed that octapeptide sequence KPGGluPGPK of collagen is rather important for binding with receptor. When this sequence in the structure of collagen's molecule binds with the receptor, platelet aggregation is induced. Free octapeptide blocks the receptor and inhibits platelet aggregations. Qualitatve characteristics of parameters of inhibition with intact octapeptide and glyproline, as well as the receptor's structure--that's our concern for the nearest future.     

Antiulcer Effects of the Tripeptide PGP and Its Possible Metabolites (PG, GP, Glycine, and Proline) in Different Models of Ulcer Induction in Rats

The study of the effect of equimolar concentrations (3.7 mol/kg) of the tripeptide PGP and its possible metabolites—PG, GP, proline, and glycine—on ethanol-, stress-, and indomethacin-induced ulcer development in rats showed that only PGP exhibited consistent antiulcer effects on all three ulceration models. Glycine and proline tended to increase the area of indomethacin-induced lesions in the stomach. PG reduced the ethanol- and indomethacin-induced lesions approximately twofold but was considerably less effective in stress-induced ulcer. GP decreased the stress- and indometacin-induced ulcer but tended to stimulate the ethanol-induced lesions. The results of this study indicate that the antiulcer activity of PGP may be related to the effect of this tripeptide itself and to proteolysis of PGP to dipeptides and amino acids as well.     

The effect of glyprolines on stressogenic disorders in the rat behaviour

Protective and antistress effects of three glyprolines--PGP, PG and GP, were studied. Stress influences produced typical changes of behavioural activity of rats in the elevated pluz-maze and the hole-board tests. These changes suggest a significant enhancement of anxiety and a drop of the level of orientation-investigative activity. A preliminary (15 minutes before stress agent) intraperitoneal administration of PGP or GP in doses of 3.7 microm/kg significantly decreased stress disturbances of behaviour. Analysis of these data shows to the possibility of PGP and GP influences on the CNS structures, which take part in the organism reciprocal reactions to stress factor. The peptide GP at equimolar dose didn't possess pronounced protective properties and just slightly decreased stress disturbance of behavioural activity of rats.     

Rapid warming accelerates tree growth decline in semi‐arid forests of Inner Asia

Forests around the world are subject to risk of high rates of tree growth decline and increased tree mortality from combinations of climate warming and drought, notably in semi‐arid settings. Here, we assess how climate warming has affected tree growth in one of the world's most extensive zones of semi‐arid forests, in Inner Asia, a region where lack of data limits our understanding of how climate change may impact forests. We show that pervasive tree growth declines since 1994 in Inner Asia have been confined to semi‐arid forests, where growing season water stress has been rising due to warming‐induced increases in atmospheric moisture demand. A causal link between increasing drought and declining growth at semi‐arid sites is corroborated by correlation analyses comparing annual climate data to records of tree‐ring widths. These ring‐width records tend to be substantially more sensitive to drought variability at semi‐arid sites than at semi‐humid sites. Fire occurrence and insect/pathogen attacks have increased in tandem with the most recent (2007–2009) documented episode of tree mortality. If warming in Inner Asia continues, further increases in forest stress and tree mortality could be expected, potentially driving the eventual regional loss of current semi‐arid forests.