Synthesis, cardiostimulatory, and cardioinhibitory effects of selected insect peptides on Tenebrio molitor.

The subject of these studies was a search for proctolin antagonists among peptides originating from insect species because the proctolin antagonists constantly pose a problem. During these studies we performed the synthesis of the following peptides: a native decapeptide from Manduca sexta Mas-MT-I and its 11 analogs with shortened sequences at the N-end as well as a growth suppressor, a pentapeptide isolated from Antheraea yamamai, Any-GS and its 10 analogs, modified at position 1 and with a shortened peptide chain. Biological effects were evaluated by the cardiotropic test on the semi-isolated heart of the insect species Tenebrio molitor. Mas-MT-I and six analogs stimulate the heartbeat frequency, especially [6-10]-Mas-MT-I, whereas the [4-10]-Mas-MT-I analog shows a strong inhibition of the heartbeat frequency, if insect. The Any-GS and the analogs [Gln(1)]- and [Gly(1)]-Any-GS also show a strong cardioinhibitory effect.

Biological activity of structural analogs and effect of oil as a carrier of trypsin modulating oostatic factor of the gray fleshfly Neobellieria bullata.

The trypsin modulating oostatic factor from the gray fleshfly Neobellieria bullata (Neb-TMOF) is released from the ovary at the end of vitellogenesis and inhibits trypsin biosynthesis in the midgut. This inhibition indirectly results in an arrest of oocyte growth. Additional experiments with N. bullata were performed to characterize its trypsin modulating and oostatic properties in more detail. After suspending the peptide in wheat germ oil, the threshold dose for oostatic activity was lowered one thousand times (2.10(-5) in oil versus 2.10(-2) pmoles per fly in Ringer). By use of the Neobellieria trypsin biosynthesis assay, 17 analogs of the hexapeptide were tested for inhibitory activity. The following structural elements were demonstrated to be critical for biological activity: the alcohol function at position 3 (Thr residue); a positively charged basic group at the C terminus (His residue); and the Asn side chain at positions 1 and 4.

Pharmacological studies of proctolin receptors on foregut and hindgut of Blaberus craniifer.

Proctolin (Arg-Tyr-Leu-Pro-Thr) and proctolin analogs modified at position 1, 2, or 5 caused dose dependent contractions of Blaberus fore- and hindgut. The varying contractile effects between both tissues revealed the possible presence of receptor subtypes as identified by [GABA1]-proctolin. A single population of binding sites (Kd approximately 100 nM) was deduced from Scatchard analysis. In addition, nanomolar concentrations of proctolin induced a dose-dependent hydrolysis of phosphoinositides (PIns) augmented by GTPgammaS (1 microM) on foregut membranes but no accumulation of cAMP. Proctolin induced contractions are likely mediated via a phospholipase C linked to a heptahelical receptor bound to heterotrimeric G-proteins.

Antiovulatory antagonists of LHRH related to antide.

We report 104 analogues of the potent antiovulatory antagonist of LHRH, N-Ac-D-Nal-D-Cpa-D-Pal-Ser-Lys(Nic)-D-Lys(Nic)-Leu-Ilys-Pro-D-Ala- NH2, Antide. We replaced the Nic group in Antide with other acyl substituents to modulate size, hydrophilicity or basicity of the molecule, we also replaced the Lys residues with shorter basic amino acids, and made cyclic 5/6 analogues as well as position 5 or 6 dimers. We substituted Ilys8 with other alkyl groups and acyl derivatives. When injected in 0.1% DMSO in water in a typical antiovulatory (AO) assay. Antide gives six rats ovulating out of eight (6/8) at 2 micrograms, 4/8 at 4 micrograms, and in the histamine release assay (HRA). ED50 is > 300 micrograms/ml; [Lys(N-Isobutyl)8]Antide gave 2/8 at 2 micrograms/rat; [Lys (8-Qis)5]Antide gave 1/8 at 1 microgram, and 0/8 at 2 micrograms, and in the HRA ED50. 22 micrograms/ml; [D-Lys(8-Qis)5]Antide gave 4/8 at 1 microgram and 0/8 at 2 micrograms, and in the HRA, ED50 was 27 micrograms/ml; [Lys(8-Qic)8] gave 5/8 at 1 microgram 1/8 at 2 micrograms/ [Lys(2-Pyc)6]Antide gave 3/8 at 1 microgram, and 0/8 at 2 micrograms, and in the HRA ED50 was 116 micrograms/ml; [D-Lys (2-Pyc)5]Antide gave 5/8 at 1 microgram and in the HRA, ED50 was 100- > 300 micrograms/ml; [Lys(2-Pyc)5.D-Lys(2-Pyc)6]Antide gave 2/8 at 1 microgram. The substitutions of the Nic groups of Antide at Lys5 or D-Lys6 with 8-Qis or with 2-Pyc groups seem to give highly potent antiovulatory antagonists of LHRH and constitute significant new leads to generate potent antiovulatory compounds endowed with moderate or low histamine release.

Analysis of peptidic epitopes recognized by the three monoclonal antibodies specific for the same region of glycophorin A but showing different properties.

Analysis of epitopes for the three monoclonal antibodies (GPA105, GPA33, OSK4-1) against glycophorin A (GPA) was performed with the use of proteolytic fragments of GPA, the synthetic nonapeptide with the sequence of amino acid residues 35-43 of GPA, and a series of peptides synthesized on plastic pins. The antibodies were specific for a short peptide sequence RAHE (a.a. 39-42 of GPA, MAbs GPA105 and OSK4-1) or RAHEV (a.a. 39-43 of GPA, MAb GPA33). Despite recognizing the same fragment of GPA, the three antibodies showed differences in fine specificity and in response to antigen desialylation. Reactions with single replacement analogs of the RAHEV sequence showed that immunodominant (unreplaceable) residues for the MAbs GPA33 and OSK4-1 were His and Glu, respectively, whereas no such residue was found for the MAb GPA105. Desialylation of the antigen gave strong enhancement of reactivity with the MAb GPA33, moderate--with the MAb GPA105, and weak or no enhancement of reaction with the MAb OSK4-1. The results showed that monoclonal antibodies directed against the same fragment of the polypeptide chain of densely glycosylated antigen may recognize different subsites which are masked at different degree by sialic acid residues.

Insect peptide hormones, an overview of the present literature.

A comprehensive overview of the recent state of the art of insect peptide hormones with chemical structures is presented. An increased interest in insect neuropeptides and dynamic development of that research area has been influenced by a rapid improvement of instrumentation necessary for isolation and structural characterization. Several research teams have studied the relationships between biological properties of insect and vertebrate peptide hormones. Thus hormones from the AKH family can be considered glucagon counterparts, whereas the myotropic hormones such as proctolin and Lem-PK (LPK) are a substance P equivalent. Insect melanization hormones Bom-MRCH in their structural characteristics and properties resemble those of mammal MSH, and leucosulfakinins Lem-SK-I and -II show some similarities with gastrin II and cholecystokinin. Bombyxin-II (Bom-PTTH-II) reveals a structural homology with human insulin and similar biological properties to adenocorticotropic mammal hormone. Allatostatin (Dip-JHS-I) may be compared to somatostatin as it can be inferred from the observations that this peptide modulates JH secretion in cockroach, Blattella germanica. Determination of the primary structure of eclosion hormones Mas-EH and Bom-EH-II as well as the amino acid sequence of allatotropin and allatostatin is a significant contribution to the understanding of the molecular mechanisms of metamorphosis and insect development.

Further studies on proctolin analogues modified in position 2 of the peptide chain and their influence on heart-beat frequency of insects.

Seven proctolin analogues (I-VII) modified in position 2 of the peptide chain by Phe (p-guanidino) (I), Phe (p-OEt) (II), Tyr (3'-NH2) (III), Tyr (3'-NO2) (IV), Afb (p-OH) (V) (Afb = 3-amino-4-phenyl-L-butyric acid), Afb (p-NH2) (VI), Afb (p-NO2) (VII), and the tetrapeptide Tyr (3'-NH2)-Leu-Pro-Thr (VIII) were synthesized by the classic liquid-phase method. The biological effects of the peptides were investigated in cardioexcitatory tests on two insect species, the cockroach Periplaneta americana L., and the yellow mealworm, Tenebrio molitor L. Within physiological concentrations (10(-9)-10(-7) M) peptides II, III, and IV stimulated the heart action of P. americana like proctolin itself. Under identical conditions, in the case of T. molitor, only peptide III showed cardiostimulatory properties, whereas other compounds (including II and IV) were inactive at concentrations up to 10(-7) M. Results reported here reflect, with reference to the analogues I-VII, selective recognition of receptors on myocardium of both insect species. The tetrapeptide VIII revealed a weak deacceleratory effect on P. americana and T. molitor heart action.

Role of guanidine group at the N-terminal proctolin chain in cardioexcitatory effects in insects.

Six proctolin analogues (I-VI) modified in position 1 of the peptide chain by the following amino acids: homo-Arg, Gac, Gav, Gap, Phe (p-guanidino) and Orn, were synthesized by conventional liquid phase method. The myotropic activity of the obtained peptides was investigated in cardioexcitatory test on two insect species, cockroach, Periplaneta americana L., and yellow mealworm, Tenebrio molitor.

The influence of synthetic tuftsin and its analogs on the function of granulocytes of children with acute lymphoblastic leukemia.

The effect of tuftsin, its synthetic analogs and arginine on phagocytosis of Staphylococcus aureus by granulocytes from leukemic children was investigated in vitro. The high stimulatory effect of tuftsin and arginine was shown. The decrease of phagocytosis of PMN from healthy subjects after preincubation with its analogs and arginine was observed, suggesting the regulatory effect of these peptides.

Influence of the peptide chain length of new elongated tuftsin analogs on phagocytosis process.

In this paper the synthesis of the following elongated tuftsin analogs: Arg-Thr-Lys-Pro-Arg (1), Pro-Arg-Thr-Lys-Pro-Arg(II), Lys-Pro-Arg-Thr-Lys-Pro-Arg(III) and Thr-Lys-Pro-Arg-Thr-Lys-Pro-Arg(IV) by classical and solid-phase methods are described. The obtained peptides were tested for their biological activity: restoration of the phagocytosis of defected granulocytes from blood of children with acute lymphoblastic leukemia (ALL).

Synthesis and biological investigations of new tuftsin analogs with elongated peptide chain.

In this paper the synthesis of the following elongated tuftsin analogs: Thr-Lys-Pro-Lys-Thr-Lys-Pro-Lys (I), Thr-Lys-Pro-Lys-Thr-Lys-Pro-Arg (II) and Ala-Lys-Thr-Lys-Pro-Arg-Glu-Gln (III) by the classical method is described. The compound II markedly inhibited the growth of murine sarcoma viruses (MSV).

The antineoplastic effects of tuftsin and tuftsinyltuftsin on B16/5B melanoma and L1210 cells.

The intraperitoneal (i.p.) injection of tuftsin, Thr-Lys-Pro-Arg, into C57BL/6 mice that were injected with B16/5B melanoma cells, resulted in a considerable suppression and elimination of solid tumor growth. While 100% of control animals exhibited tumor growth, 38% of the treated animals failed to show tumor formation for the duration of the experiment, 60-80 days. The octapeptide, tuftsinyltuftsin, was effective at 3 ng per mouse as was a dose of 2 and 20 micrograms per mouse. In each case there was a significant number of mice free of tumors. The octapeptide was also quite effective against L1210 cells resulting in the survival of 35-40% of the treated animals. The lethal effect of increased superoxide, O X 2, production by tuftsin treatment may explain the antineoplastic effect of the tetrapeptide. This may result not only from higher concentrations of O X 2 but also from the potentially lethal effects of H2O2 and OH X radical, both of which are products of O X 2 metabolism.

Tuftsin, a natural activator of phagocytic functions including tumoricidal activity.

Some of the properties of the tetrapeptide tuftsin, Thr-Lys-Pro-Arg, are discussed. We describe three phases of tuftsin activation of the macrophage. Tuftsinyltuftsin, the octapeptide Thr-Lys-Pro-Arg-Thr-Lys-Pro-Arg, was synthesized with a view of minimizing the formation of Lys-Pro-Arg, from tuftsin by tissue aminopeptidases. The tripeptide is a tuftsin inhibitor. The octapeptide proved to be quite effective in prolonging the life of syngeneic mice injected with L1210 leukemia cells. Its effect in our laboratory, was considerably better than we could obtain with tuftsin. A simple method for purifying tuftsin by high performance liquid chromatography is described using 0.75% trifluoroacetic acid in water. The tuftsin sequence Thr-Lys-Pro-Arg is present in P12 protein of Rausher murine leukemia virus. A close analog Thr-Arg-Pro-Lys appears in yet another virus protein the haemagglutinin of influenza virus. A second close analog Thr-Arg-Pro-Arg forms the penultimate carboxyterminal of a pancreatic polypeptide found in human and several animals.