Direct evidence for activation and desensitization of the capsaicin receptor by N-oleoyldopamine on TRPV1-transfected cell, line in gene deleted mice and in the rat.

Effects of the endogenous lipid N-oleoyldopamine (OLDA) were analyzed on the rTRPV1-expressing HT1080 human fibrosarcoma cell line (HT5-1), on cultured rat trigeminal neurons, on the noxious heat threshold of rats and on nocifensive behavior of TRPV1 knockout mice. The EC(50) of capsaicin and OLDA on (45)Ca accumulation of rTRPV1-expressing HT5-1 cells was 36 nM and 1.8 microM, respectively. The efficacy of OLDA was 60% as compared to the maximum response of capsaicin. OLDA (330 nM to 3.3 microM) caused a transient increase in fluorescence of fura-2 loaded cultured small trigeminal neurons of the rat and rTRPV1-transfected HT5-1 cells measured with a ratiometric technique. Repeated application of OLDA and capsaicin caused similar desensitization in the Ca(2+) transients both in cultured neurons and rTRPV1-transfected HT5-1 cells. In the rat intraplantar injection of OLDA (5 nmol) decreased the noxious heat threshold by 6-9 degrees C and this response was strongly inhibited by the TRPV1 antagonist iodoresiniferatoxin (0.05 nmol intraplantarly (i.pl.)). In wild-type mice OLDA (50 nmol i.pl.) evoked paw lifting/licking which was significantly less sustained in TRPV1 knockout mice. It is concluded that on TRPV1 capsaicin receptors OLDA is 50 times less potent than capsaicin and it might serve as an endogenous ligand for TRPV1 in the rat, but more likely in humans.

Ellipticine analogues and related compounds as inhibitors of reverse transcriptase and as inhibitors of the efflux pump.

Ten polycyclic derivatives related to ellipticine have been synthesised and tested for their intercalating, reverse transcriptase (RT) inhibitory and multidrug resistance efflux pump inhibitory properties. The intercalating activity and the RT inhibitory activity of the derivatives suggest that ellipticine analogues bind at an allosteric binding site on RT or that this inhibition could be controlled at the DNA level. The MDR efflux pump inhibitory activities of these derivatives, however, appears to be unrelated to the DNA binding ability.

Synthesis of two new heteroaromatic beta-carboline-fused pentacycles. Observation of a new intercalating agent.

Five-step synthetic routes of two new polyfused heterocycles: indazolo[3,2-a]-beta-carboline (3) and benzo[4',5'][1,2,3]triazino[6,1-a]-beta-carbolinium salt (10) applying Pd(0)-catalyzed cross-coupling reaction have been elaborated.

Effect of methionine enrichment on the biological activities of food proteins.

A special enzymatic technique, the enzymatic peptide modification (EPM) reactions have been worked out in our laboratory, by which proteins can be modified with respect to their biological activities. Enzymatic modification methods provide several advantages in producing proteins of desired nutritional properties. Proteolytic modification, particularly when combined with methionine enrichment, significantly altered the biological activities and leads to increased nutritional value.

Novel dietary strategy for overcoming the antinutritional effects of soyabean whey of high agglutinin content.

A diet-switching experiment, which aimed to improve the utilization of soyabean whey was carried out for 61 d with young rats. Feeding was arranged in such a way that after a few days on the soyabean diet, the rats were switched to a high-quality lactalbumin diet for a short period, after which the cycle was repeated several times. The weights of the rats at the end of the soyabean phases were significantly less than those of animals pair-fed on a high-quality diet throughout. However, the test group regained the weight loss after switching to the lactalbumin diet. After three cycles there were no significant differences between the weights of the test rats fed on a poor soyabean diet for over a third of the experiment and those fed on the lactalbumin diet throughout. Feed conversion was always significantly higher with test rats in the lactalbumin period than with continually pair-fed controls. Similarly, faecal N losses were significantly higher for test rats in the soyabean phase, but these differences disappeared after switching to the lactalbumin diet. At the end of the experiment there were no significant differences in body protein or lipids between the groups although the pancreas was significantly heavier while the liver was lighter in soyabean-fed rats. The high destruction of trypsin inhibitors in the gut suggests that they probably had little effect on protein digestion in the gut. In contrast, as selective depletion of the agglutinin from soyabean whey removed the nutritional benefit in the lactalbumin part of the cycle, the improved feed conversion in this period must have been the result mainly of the survival and functionality of soyabean agglutinin and the benefits due to the hyperplastic growth and faster renewal of the gut surface it induced. As processing is unnecessary, this novel method is cheap and can be easily adapted for the use of soyabean whey, regarded as a waste product.

[The effect of keeping condition and genotype on the electrophoretic properties of pork proteins]. / Einfluss der Haltungsbedingungen und des Genotyps auf das Elektrophoreseverhalten von Schweinefleischproteinen.

Besides differentiation of animal species the electrophoretic methods are efficient also in determinations of the factors effecting the protein patterns. The goal of this study is to compare the effect of the genotype and the keeping conditions on the electrophoretic behaviour of the pork meat proteins. Isoelectric focusing was found to be more efficient than SDS-PAGE concerning studies of the effect of these factors. An optimal separation has been achieved with an ampholyte-mixture at pH ranges 2-11, 5-7 and 7-9. Peroxidase activity of the separated myoglobines of the meat samples was stained for detecting the differences in protein zones according to the keeping conditions. The myoglobin zones stained with o-dianisidin were evaluated by densitometry. The protein zones of the organic meat samples showed higher intensity than those of the control-samples.

Differences in the electrophoretic patterns of soluble pork proteins as a consequence of pig rearing conditions.

In recent years the importance of 'organic meat' has increased. Rigorous rearing conditions has raised the emphasis on meat quality problems. Pigs (genotype: Hungarian Large White 75% and Mangalica, pigs with curly bristles, 25%) were kept in a natural farming system, and did not receive growth promoters, hormones or antibiotics. The meat of these pigs is called 'organic meat', or 'bio meat'. Longissimus dorsi muscles of pigs kept under natural circumstances and under traditional conditions were compared using SDS polyacrylamide gel electrophoresis and isoelectric focusing. Electrophoretic separation, protein and esterase enzyme staining showed definite differences between the proteolysates of organic and control meat samples.

Selective restoration of immunosuppressive effect of cytotoxic agents by thymopoietin fragments.

Swiss male mice were immunosuppressed by cyclophosphamide, cisplatinum, vincristine and methotrexate. The ability of the thymopoietin (TP) fragments TP-3, TP-4 and TP-5 to restore antibody production and phagocytosis was studied. Impaired antibody production after vincristine treatment was partially or totally restored by TP-3, TP-4 or TP-5. Only TP-3 or TP-5 interfered with the antibody-production-damaging effect of cisplatinum. The same effect of methotrexate could not be influenced by any of the TP fragments. The phagocytic capacity of peritoneal macrophages was reduced by vincristine, methotrexate and cyclophosphamide treatment. In this respect, TP-3 protected the function of macrophages against vincristine and cyclophosphamide treatment. TP-4 was active in the case of damage caused by vincristine and methotrexate, and TP-5 interfered with the phagocytosis-inhibiting effect of methotrexate. Each TP fragment seems to have a specific target orientation within the immune system. This also means that the proper TP fragment should always be chosen for combination therapy with various types of cytotoxic drugs.

The effect of thymopoietin 32-34 (TP3) on suramin-induced inhibition on delayed type hypersensitivity in guinea pigs.

Suramin administration inhibits virus replication and produces impaired immunoregulation. Delayed type hypersensitivity (DTH)-reaction was inhibited by Suramin (200 mg/kg, i.p.) injection in guinea-pigs immunized with BCG vaccine. Suramin-inhibited DTH-reaction was restored to normal level after 4 days of treatment with thymopoietin 32-34 (TP 3) and TP5 (1 mg/kg, i.p.), TP3 greater than TP5. TP3 produced the strongest restoration when applied together with Suramin, but a single treatment with TP3 or TP5 oligopeptide inhibited the development of DTH-reaction. TP3 with Suramin treatment might be advantageous to eliminate defective side effects of Suramin on immune system and TP3 with indirect inducing action on IL-2, IFN-gamma production may be useful in therapy of AIDS patients.

Therapeutic possibilities of thymopoietin fragments (TP3 and TP4) based on experimental animal models.

The effects of thymic hormones are focused on the induction of T-cell subpopulations and restoration of the reactivity of an impaired immune system. TP3 and TP4 (corresponding to thymopoietin 32-34 and 32-35) exert a thymic hormone substitution effect. These peptides elicit dissimilar quantitative and qualitative effects. The aim of the present experiments was to investigate: (a) the effect of thymopoietin fragments in mice with unbalanced immune systems caused by experimental manipulation; and (b) the ratio of target cells after treatment. The distribution of Thy1, Lyt1, Lyt2 positivity was determined in a direct complement mediated cytotoxicity test. Autoantibody production was measured by Coombs' test. A count of Lewis Lung Tumour (LLT) metastases was made after two weeks of inoculation. Groups of mice were thymectomized and/or injected with cyclophosphamide (CY) (240 mg/kg) 96 h before tumour cell inoculation. The number of LLT metastases was decreased by treatment with peptides (TP3 = 72, TP4 = 97, TP5[thymopoietin 32-36] = 83.1 in %) and immunosuppression produced by CY was partly restored. After thymectomy, however, only TP3 treatment caused a decreasing effect (97.4%) on CY immunotoxicity independently of thymectomy. Inhibition of autoantibody production was detected with TP3 (5-6 weeks earlier than in mice treated with TP5). The ratio of Thy1+ and Lyt2+ cells was increased by treatment with TP3 and TP4, but the ratio of Lyt1+ cells was decreased by application of TP5. After TP3 treatment of nude mice the Lyt1+/Lyt2+ ratio increased both in bone marrow and spleen. No effect of TP4 was observed on Lyt 1+ cells, but the number of Lyt2+ increased in bone marrow.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunoregulating peptides II. In vitro effects of TP5 analogs on E-rosette formation and cell division.

The effects of seventeen synthetic analogs of thymopentin (TP-5) have been studied in the active and azathioprine-inhibited E-rosette tests. Thymopentin was gradually shortened from the C terminus to peptides and single amino acids. Thymopoietin 32-34 (Arg-Lys-Asp-RGH-0205-TP-3) (II) and thymopoietin 32-35 (Arg-Lys-Asp-Val-RGH-0206-TP-4) (I) were the most active peptides. Dipeptide Arg-Lys produced significant stimulatory effect on azathioprine (ED75) inhibited E-receptor. Treatment of azathioprine (ED75)-inhibited E-rosette forming cells (ERFC) with arginine or especially lysine increased the number of ERFC. Some of TP-4 analogs decreased further the number of ERFC decreased by azathioprine ED30. These "suppressive" peptides as well as TP-3 caused a partial arrest of K 562 cell proliferation up to 96 hours. Results suggest that TP-5 is not the smallest active fragment of thymopoietins, since peptides (TP-3 and TP-4) exhibit similar or higher T-cell membrane activation on E-receptor. Arginine, lysine, and acidic aspartyl residue seem to be a necessary basic structure to produce a cumulative chemical signal on the activity of T-lymphocytes.

The effect of TP-5 and its analogs on skin grafts in mice.

The immunostimulatory action of oligopeptides RGH-0205 (Arg-Lys-Asp), RGH-0206 (Arg-Lys-Asp-Val) and TP-5 (Arg-Lys-Asp-Val-Try) was measured using the B10LP to C57BL skin graft system and the determination of splenic T cell ratio. While thymectomy increased the period between skin grafting and rejection, each of the oligopeptides increased the number of splenic T cells and in some extent restored the rejection capacity of thymectomised C57Bl mice, presumably by restoration of thymic hormone function.

Structural investigation of radiation-induced aggregates of ribonuclease.

Following irradiation of bovine pancreatic ribonuclease in aqueous solution with 60Co gamma-rays protein aggregates are formed. The nature of the bonds linking these radiation-induced aggregates together has been investigated by chromatographic and electrophoretic methods. Thin-layer gel filtration and polyacrylamide gel electrophoresis, both in the presence of sodium dodecyl sulphate, demonstrated the existence of covalent crosslinks between the aggregates. However, non-covalent crosslinking also plays a role in the radiolysis of ribonuclease. Thin-layer gel filtration with and without 6 M urea and 2 per cent beta-mercaptoethanol added to the gel, revealed that only part of the covalent bonds between the aggregates consisted of disulphide linkages. By separation of the reduced aggregates by thin-layer gel filtration and electrophoresis, both with SDS, this finding was substantiated. Densitometric measurements indicated for example that the percentage of covalently linked dimers held together by disulphide bridges amounted to about 40-45 per cent, whereas the remaining 55-60 per cent of the dimers must be linked by other covalent bonds. The existence of covalent crosslinks other than disulphide bonds was also confirmed by isoelectric focusing. By this method definite differences were established between the proteolytic hydrolysates of the reduced aggregates and the reduced monomer of gamma-irradiated ribonuclease.

Immuno-regulating peptides, I. Synthesis and structure-activity relationships of thymopentin analogs.

Seventeen peptides, related to thymopoietin pentapeptide, L-arginyl-L-lysyl-L-aspartyl-L-valyl-L-tyrosine (thymopentin) were synthesized by the stepwise strategy in solution. Of these, L-arginyl-L-lysyl-L-aspartic acid and L-arginyl-L-lysyl-L-aspartyl-L-valine, shortened from the C-terminus of the pentapeptide, exhibit significant immuno-stimulating potencies, exceeding those of thymopentin, both in vitro and in vivo immunological tests. Studies on the structure-activity relationships suggest that the potencial active site of thymopoietins is very sensitive to the N- and C-terminal elongations of the peptide chain. For thymic hormones, an active site carrying cumulative chemical signals is proposed instead of well-defined active centers.

Comparative pharmacology of a 1:10 combination of indomethacin-sodium salicylate.

It has been found that gastrointestinal side-effects of indomethacin could be abolished when administered in combination indomethacin:sodium salicylate (ratio 1:10). In this paper comparative pharmacological data of this combination and its basal compounds are presented. Acute toxicity of the combined preparation was 72 times less in rats than that of indomethacin alone. All therapeutic indexes were markedly increased in the combination of indomethacin-sodium salicylate compared with the separate drug treatments.