Neurotropic peptide HLDF-6-amide reduces age-related decline in sexual activity in old male rats.

Aging is associated with a decline in the erectile capacity and sexual motivation. Emerging new therapy for the treatment of these age-related pathologies in men is the use of the regulatory peptides. We validated the use of HLDF-6-amide (Thr-Gly-Glu-Hse-His-Arg-NH2) as a potential modulator of sexual performance in aged male rats. Behavioral tests, including the standard parameters of sexual behavior, were performed longitudinally at 20 and 26 months of age. The effects of HLDF-6-amide administered daily at 300 µg/kg for 3 week on the levels of sex hormones and the activity of antioxidant enzymes and indicators of inflammation were evaluated. HLDF-6-amide administration increased the copulative activity of the 20-month-old male rats. This effect of HLDF-6-amide was more pronounced in the 26-month-old rats. Although HLDF-6-amide did not have the effect on the levels of circulating testosterone and estradiol, it reduced the activity of leukocyte elastase and glutathione-S-peroxidase, suggesting that the peptide has anti-inflammatory and antioxidant properties. Therefore, this study shows that HLDF-6-amide has the positive impact on sexual activity in this rodent model, representing a new therapeutic approach for improving sexual performance in older men.

[The Qualitative Analysis of the Amide Derivative of HLDF-6 Peptide and Its Metabolites with the Use of Tritium- and Deuterium-Labeled Derivatives].

The goal of the study was to elaborate the pharmacokinetics methods of the amide derivative of peptide HLDF-6 (TGENHR-NH2) and its range of nootropic and neuroprotective activity is wide. The hexapeptide 41TGENHR46 is a fragment of the HDLF differentiation factor. It forms the basis for the development of preventive and therapeutic preparations for treating cerebrovascular and neurodegenerative conditions. Pharmacokinetic and molecular mechanisms of the action of the HLDF-6 peptide were studied using tritium- and deuterium-labeled derivatives of this peptide, produced with the use of the high-temperature solid-state catalytic isotope exchange reaction (HSCIE). This reaction was employed to produce the tritium-labeled peptide [3H]TGENHR-NH2 with a molar radioactivity of 230 Ci/mmol and the deuterium-labeled peptide [2H]TGENHR-NH2 with an average deuterium incorporation equal to 10.5 atoms. It was shown by the NMR spectroscopy that the isotope label distribution over the labeled peptide's molecule was uniform, which allowed qualitative analysis ofboth the peptide itself and its fragments in the organism's tissues to be conducted. The newly developed pharmacokinetics method makes it possible to avoid almost completely losses of the peptides under study due to biodegradation during the analysis of tissues. These labeled peptides were used in mice, rats and rabbits to study the pharmacokinetics of the peptide and to calculate the values of its principal pharmacokinetic parameters. Characteristics of its pharmacokinetic profile in the blood were obtained, the hypothesis of pharmacokinetics linearity tested, its metabolism analyzed and its bioavailability value, 34%, calculated. It has been shown that the studied TGENHR-NH2 peptide shows high resistance to hydrolysis in the blood plasma, with dipeptidyl aminopeptidases making the largest contribution to its hydrolysis.

[Solid state isotope hydrogen exchange for deuterium and tritium in human gene-engineered insulin].

The reaction of high temperature solid state catalytic isotope exchange in peptides and proteins under the action of catalyst-activated spillover hydrogen was studied. The reaction of human gene-engineered insulin with deuterium and tritium was conducted at 120-140° C to produce insulin samples containing 2-6 hydrogen isotope atoms. To determine the distribution of the isotope label over tritium-labeled insulin's amino acid residues, oxidation of the S-S bonds of insulin by performic acid was performed and polypeptide chains isolated; then their acid hydrolysis, amino acid analysis and liquid scintillation counts of tritium in the amino acids were conducted. The isotope label was shown to be incorporated in all amino acids of the protein, with the peptide fragment FVNQHLCGSHLVE of the insulin ß-chain showing the largest incorporation. About 45% of the total protein isotope label was incorporated in His5 and His10 of this fragment. For the analysis of isotope label distribution in labeled insulin's peptide fragments, the recovery of the S-S bonds by mercaptoethanol, the enzymatic hydrolysis by glutamyl endopeptidase from Bacillus intermedius and HPLC division of the resulting peptides were carried out. Attribution of the peptide fragments formed due to hydrolysis at the Glu-X bond in the ß-chain was accomplished by mass spectrometry. Mass spectrometry analysis data of the deuterium-labeled insulin samples' isotopomeric composition showed that the studied solid state isotope exchange reaction equally involved all the protein molecules. Biological studying of tritium-labeled insulin showed its physiological activity to be completely retained.

Degradation of the ACTH(4-10) analog Semax in the presence of rat basal forebrain cell cultures and plasma membranes.

Here a new approach of the elucidation of paths of proteolytic biodegradation of physiologically active peptides, based on the use of a peptide with isotopic label at all amino acid residues and the enrichment of HPLC samples with unlabeled peptide fragments in UV-detectable concentration, has been proposed. The method has been applied for the investigation of degradation dynamics of the neuroactive heptapeptide MEHFPGP (Semax) in the presence of plasma membranes, and cultures of glial and neuronal cells obtained from the rat basal forebrain. The splitting away of ME and GP, and formation of pentapeptides are the predominant processes in the presence of all tested objects, whereas the difference in patterns of resulting peptide products for glial and neuronal cells has been detected. In conclusion, the approach applied allows analyzing physiologically active peptide concentrations in biological tissues and degradation pathways of peptides in the presence of targets of their action.

Isotopic effects in the electronic spectra of tryptophan.

No influence of isotopic substitution in deuterium-substituted tryptophan on the florescence excitation spectrum has previously been found out. Here, the isotopic effects of electronic excitation of deuterium-substituted tryptophan were experimentally and theoretically analyzed for first time. It was shown a short-wave shift of the UV-absorption maximum at 220 nm corresponding to the 360 cal/mol and short-wave shift for fluorescence spectrum corresponding to the 210 cal/mol. To account for this effect, the quantum chemical calculations of the geometric and electron structure, frequencies of normal vibrations and transition energies have been performed. The isotopic effects originate from the zero-point energies of ground and excited states. It was found that isotopic shifts depend on the position of isotope in the molecule and kind of transition. So, it can be utilized in the analysis of proteins structure and complexation.

The effect of three-dimensional structure on the solid state isotope exchange of hydrogen in polypeptides with spillover hydrogen.

The effect of the three-dimensional structure of polypeptides and proteins on their ability to undergo isotopic exchange under the action of spillover hydrogen (SH) in the high temperature solid state catalytic isotope exchange reaction (HSCIE) was theoretically and experimentally studied. The HSCIE reaction in the beta-galactosidase protein from Thermoanaerobacter ethanolicus (83kDa) was studied. The influence of the beta-galactosidase structure on isotopic exchange as peptide fragments with spillover tritium was studied. The most accessible peptide fragment, which does not contribute to alpha-helix and beta-strand formations (KEMQKE215-220), had the largest relative reactivity. The one located in the contact area between the subunits (YLRDSE417-422) showed the smallest relative reactivity. The relative reactivities of these peptides differ more than 150 times. Data collected during a study devoted to the HSCIE reaction of the beta-galactosidase protein indicate that the HSCIE reaction might be employed for acquiring information about their three-dimensional structure and protein-protein interactions. The results of ab initio calculations have shown that alpha-helix formation in polypeptides decreases the reactivity in HSCIE. Hydrogen exchange in the alpha-helical fragment Trp1-Leu8 of zervamycin IIB was also analyzed using theoretical methods. It was shown by ab initio quantum-chemical calculations that the high degree of substitution of C(alpha)H for tritium in Gln3 might be associated with the participation of electron donor O and N atoms in transition state stabilization in the HSCIE reaction.

New development in the tritium labelling of peptides and proteins using solid catalytic isotopic exchange with spillover-tritium.

The mechanism of the reaction of high temperature solid state catalytic isotope exchange (HSCIE) of hydrogen in peptides with spillover-tritium at 140-180 degrees C was analyzed. This reaction was used for preparing [(3)H]enkephalins such as [(3)H]DALG with specific activity of 138 Ci/mmol and [(3)H]LENK with specific activity of 120 Ci/mmol at 180 degrees C. The analogues of [(3)H]ACTG(4-10) with specific activity of 80 Ci/mmol, [(3)H]zervamicin IIB with specific activity of 70 Ci/mmol and [(3)H]conotoxin G1 with specific activity 35 Ci/mmol were produced. The obtained preparations completely retained their biological activity. [(3)H]Peptide analysis using (3)H NMR spectroscopy on a Varian UNITY-600 spectrometer at 640 MHz was carried out. The reaction ability of amino fragments in HSCIE was shown to depend both of their structures and on the availability and the mobility of the peptide chain. The reaction of HSCIE with the beta-galactosidase from Termoanaerobacter ethanolicus was studied. The selected HSCIE conditions allow to prepare [(3)H] beta-galactosidase with specific activity of 1440 Ci/mmol and completely retained its the enzymatic activity.