Assessment of the Cytotoxicity of Peptide Modifications of Doxorubicin on Tetrahymena pyriformis.

The cytotoxicity of doxorubicin (Dox) and its peptide modifications Z-Gly-Pro-Dox and Boc-Gly-Pro-Dox were studied. Tetrahymena pyriformis was used as a test system, which made it possible, due to the short life cycle and high reproduction rate of ciliates, to trace their response to the effects of toxicants over several generations. It was found that peptide modification of the Dox molecule markedly reduces its cytotoxic and cytostatic effect. The Z-Gly-Pro-Dox modification has less cytotoxic and cytostatic effect compared to Boc-Gly-Pro-Dox. When determining the ability of drugs (at a concentration of 100 µM) to prevent bacterial contamination of samples, it was shown that the smallest degree of overgrowth was recorded in the presence of Dox (OD600nm 81.1). Boc-Gly-Pro-Dox also had a bacteriostatic effect, though less pronounced (OD600nm 93.8). The degree of overgrowth in the presence of Z-Gly-Pro-Dox was close to that of distilled water. The results obtained on ciliates did not contradict the data obtained in similar studies on mice.

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.

Haloperidol Reduces the Activity of Complement and Induces the Anti-Inflammatory Transformation of Peritoneal Macrophages in Rats.

It is known that psychotropic substances affect the immune system. Unfortunately, chronic antipsychotic administration causes side toxicological effects, associated with oxidative stress. The mechanisms of these effects are still unclear. We investigated the impact of sub-chronic administration of haloperidol (Hal) on parameters of innate immunity and related systems in healthy rats and compared them with Hal content. Hal administration (0.5 mg/kg, 3 weeks) resulted in two-fold decrease of the activity of the complement system and hemostasis. Hal content correlated with the activity of the complement (r = -0.71), phagocytic activity of peritoneal macrophages (r = 0.78), leukocyte elastase (r = -0.71) and glutathione-S-transferase activity (r = -0.67). Hal fully blocked in vitro PMA-induced iNOS expression in macrophages and changed their morphology to "anti-inflammatory" phenotype. The comparison of in vivo and in vitro data showed that Hal has a direct effect on phagocytic component of innate immunity and an indirect effect on leukocyte elastase and antioxidant enzymes. The results obtained in the present study indicated that Hal significantly affects homeostasis and causes a number of complex biological transformations. Graphical Abstract.

Dipeptidylpeptidase 4 (DPP4, CD26) activity in the blood serum of term and preterm neonates with cerebral ischemia.

BACKGROUND: To investigate the mechanisms of inflammation in neonates after cerebral ischemia (CI), we evaluated the DPP4 activity in their blood sera and compared these values with clinical indicators. METHODS: The activity of DPP4 was determined in blood serum by a fluorescent method. We studied the correlation between the blood serum DPP4 activity and clinical, neurological and biochemical parameters in neonates with CI. RESULTS: No correlation between the DPP4 activity in umbilical blood and the venous blood of mothers was discovered. Increased blood serum DPP4 activity in full-term and pre-term newborns with CI is demonstrated. The interrelation between serum DPP4 activity and the functional disturbances of CNS (such as depression or excitement) was found in mature but not in premature newborns. Enzyme activity was still elevated at 2-3weeks after birth. CONCLUSION: It is possible that in neonates this enzymatic system operates independently from mothers. It is assumed that increased DPP4 activity in newborns with CI is apparently connected with immune system activation in response to hypoxic stress. The obtained data support the participation of DPP4 in adaptive reactions of newborns and its regulating influence during hypoxemic damage of the CNS due to inflammation and neurodegeneration.

[Regulatory peptides and psychomotor development in infants].

Regulatory peptides (RP) are an important homeostatic factor. The maternal organism and placenta are substantial sources of RP for fetus during the prenatal period. Not only endogenous, but also exogenous RP play an important role during early postnatal period. In this study, the concentration of exogenous RP (casomorphins-7) and the activity of peptidases (enkephalinases) in the serum of breastfed and bottle-fed infants were estimated. Possible interrelation between these two parameters and the psychomotor development (PMD) of infants were evaluated. Using specially developed RIA, the investigators estimated the presence of human and bovine casomorphins immunoreactivity (CMir) in the serum of breastfed and bottle-fed infants. A distinct correlation of CMir with PMD was demonstrated. The activity of RP-degrading serum enzymes also correlated with PMD level. The role of endo- and exogenous peptides in normal PMD process and in the pathogenesis of early child autism is discussed in the article.

[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.

[Effect of new peptide bioregulators livagen and epitalon on enkephalin-degrading enzymes in human serum]. / Vliianie novykh peptidnykh bioreguliatorov livagena i épitalona na énkefalindegradiruiushchie fermenty syvorotki krovi cheloveka.

The effect of new peptide bioregulators--Livagen (Lys-Glu-Asp-Ala) and Epitalon (Ala-Glu-Asp-Gly)--on endogenous opioid system was studied, particularly, their ability to change the activity of enkephalin-degrading enzymes from serum and interact with opioid receptors of the brain membrane fraction. Enkephalinase activity was assayed in vitro by the rate of 3H-Leu-enkephalin hydrolysis in the presence of the tested peptides. Livagen and Epitalon inhibited enkephalin-degrading enzymes from human serum. Livagen proved to be more efficient also as compared to well-known peptidase inhibitors such as puromycin, leupeptin, and D-PAM. The dose-inhibitory effect curves for Livagen and Epitalon were plotted; their IC50 equaled 20 and 500 microM, respectively. The interaction between the peptides and opioid receptors was estimated using a radioreceptor method with [3H][D-Ala2, D-Leu5]-enkephalin. No interaction was observed between the tested peptides and mu- or delta-opioid receptors of the membrane fraction from the rat brain.

Effects of Selank on behavioral reactions and activities of plasma enkephalin-degrading enzymes in mice with different phenotypes of emotional and stress reactions.

Comparative study of plasma activities of enkephalin-degrading enzymes in mice with different phenotypes of emotional and stress reactions revealed significant differences between intact BALB/c and C57Bl/6 mice by the half-life of plasma leu-enkephalin. Selank in a dose of 100 micrograms/kg produced an anxiolytic effect in the open-field test and increased the half-life of plasma leu-enkephalin in BALB/c mice, but had no effect on behavioral reactions and enkephalinase activities in C57Bl/6 mice. Our results suggest that anxiolytic activity of Selank is associated with inhibition of enkephalin-degrading enzymes.

The inhibitory effect of Selank on enkephalin-degrading enzymes as a possible mechanism of its anxiolytic activity.

Examination of patients with various forms of anxiety and phobic disorders (according to DSM-4 criteria) demonstrated a considerable shortening of enkephalin half-life and reduced total enkephalinase activity in the blood during generalized anxiety, but not during panic disorders and agoraphobia. This was probably related to low blood concentration of endogenous inhibitors of enkephalin-degrading enzymes in patients with generalized anxiety disorders. Heptapeptide Selank (Thr-Lys-Pro-Arg-Pro-Gly-Pro), which attenuates behavioral anxiety reactions and does not cause side effects typical of most anxiolytics, dose-dependently inhibited enzymatic hydrolysis of plasma enkephalin (IC50 15 microM). Selank was more potent than peptidase inhibitors bacitracin and puromycin in inhibiting enkephalinases. These results suggest that high efficiency of Selank in the therapy of anxiety and phobic disorders, including generalized anxiety, is due to its ability to inhibit enkephalin hydrolysis.

[Semax and selank inhibit the enkephalin-degrading enzymes from human serum]]. / Ingibiruiushchee deistvie semaksa i selanka na énkefalindegradiruiushchie fermenty syvorotki krovi cheloveka.

A dose-dependent effect of synthetic heptapeptides Semax (Met-Glu-His-Phe-Pro-Gly-Pro) and Selank (Thr-Lys-Pro-Arg-Pro-Gly-Pro) on the enkephalin-degrading enzymes of human serum was demonstrated. The inhibitory effects of Semax (IC50 10 microM) and Selank (IC50 20 microM) are more pronounced than those of puromycin (IC50 10 mM), bacitracin, and some other inhibitors of peptidases. Beside the heptapeptides, their pentapeptide fragments also possessed an inhibitory effect; tri-, tetra-, and hexapeptide fragments did not display such an effect. As the above enzymes take part in degradation of not only enkephalins but also other regulatory peptides, it can be assumed that one of the mechanisms of biological activity of Semax and Selank is related to this inhibitory activity of theirs.

Identification of delta- and mu-type opioid receptors on human and murine dendritic cells.

The purpose of this study was to evaluate mu- and delta-opioid receptors (OR) on human and murine dendritic cells (DC). Expression of mu- and delta-OR mRNA on DC was demonstrated by RT-PCR. The immunocytochemical and Western blot analyses revealed the expression of OR protein in DC. Radioreceptor assay demonstrated the specific saturated temperature-dependent binding of [3H]-labeled opioid ligand on DC and B(max)=2.8+/-0.3 fmol/10(6) cells and K(D)=4.8+/-1.0 nM were calculated by a Scatchard analysis. Finally, OR ligands DADLE and DAGO dose-dependently modulated the capacity of DC to induce T cell proliferation in an MLR assay. Importantly, expression of functional OR on DC was significantly increased upon TNF-alpha-induced DC maturation. Thus, these data suggest a new mechanism of opioid-dependent neuroendocrine immunomodulation.

[Effect of beta-endorphin and myelopeptides on the cAMP level and proliferation of lymphocytes in vitro]. / Issledovanie vliianiia beta-éndorfina i mielopeptidov na uroven' tsAMF i proliferatsiiu limfotsitov in vitro.

Beta-endorphin (10(-11)-10(-9) M) has been shown to induce naloxone-independent depression of the proliferative activity of human peripheral lymphocytes (HL), stimulated by pokeweed mitogen without affecting PHA-stimulated HL proliferation. Beta-endorphin (10(-10)-10(-7) M) also caused changes in HL cAMP level, that were blocked by naloxone. Marked individual sensitivity to beta-endorphin effects has been noted. It has been also shown that a bone marrow preparation, stimulating antibody production (myelopeptides), causes naloxone-independent depression in the proliferative activity of HL, stimulated by PHA and pokeweed mitogen, as well as naloxone-blocked decrease in cAMP HL level. It has been concluded that beta-endorphin interacts with several types of opiate lymphocyte receptors and that opioids, contained in myelopeptides, are involved in the realization of myelopeptide effect on lymphocytes.

[Opiate activity of a bone marrow humoral factor which stimulates antibody production]. / Opiatnaia aktivnost' gumoral'nogo faktora kostnogo mozga, stimuliruiushchego produktsiiu antitel.

The radioceptor method was used to demonstrate that humoral factor of the bone marrow, a stimulant of antibody production (SAP), contains substances that competitively remove 3H-morphine and 3H-met-enkephalin from opiate receptors of the rat brain, and 3H-met-enkephalin from specific binding sites on human lymphocytes. Comparison of the magnitudes EC50 SAP obtained during the removal of labeled opiates allows a suggestion to be made that opiates contained by the preparation under study have a greater capability to interact with delta-type opiate receptors rather than with those of the mu-type.

[Effect of morphine on the cAMP level of lymphocytes]. / Vliianie morfina na uroven' tsAMF v limfotsitakh.

A study was made of the effect of morphine on the cAMP level in peripheral blood lymphocytes in tobacco smoking and non-smoking donors. Morphine was shown to produce the naloxone-removable activation of adenylate cyclase, the relationship between enzymatic activity and opiate concentration in the medium being complex in nature. In tobacco smoking donors, the maximal effect on cAMP was produced by morphine at concentrations that were one order of magnitude higher than those in non-smoking ones. On the basis of the data obtained the assumptions are made (1) about the presence on the lymphocytes of opiate receptors whose action mode is associated with adenylate cyclase control, and (2) about the development during tobacco smoking of morphine tolerance at the level of opiate-dependent adenylate cyclase of lymphocytes.