Teresa Kubiak

Differential Activation of “Social” and “Solitary” Variants of the Caenorhabditis elegans G Protein-coupled Receptor NPR-1 by Its Cognate Ligand AF9

Natural variations of wild Caenorhabditis elegans isolates having either Phe-215 or Val-215 in NPR-1, a putative orphan neuropeptide Y-like G protein-coupled receptor, result in either “social” or “solitary” feeding behaviors (de Bono, M., and Bargmann, C. I. (1998) Cell 94, 679–689). We identified a nematode peptide, GLGPRPLRF-NH2 (AF9), as a ligand activating the cloned NPR-1 receptor heterologously expressed in mammalian cells. Shifting cell culture temperatures from 37 to 28 °C, implemented 24 h after transfections, was essential for detectable functional expression of NPR-1. AF9 treatments linked both cloned receptor variants to activation of Gi/Go proteins and cAMP inhibition, thus allowing for classification of NPR-1 as an inhibitory G protein-coupled receptor. The Val-215 receptor isoform displayed higher binding and functional activity than its Phe-215 counterpart. This finding parallels the in vivo observation of a more potent repression of social feeding by the npr-1 gene encoding the Val-215 form of the receptor, resulting in dispersing (solitary) animals. Since neuropeptide Y shows no sequence homology to AF9 and was functionally inactive at the cloned NPR-1, we propose to rename NPR-1 and refer to it as an AF9 receptor, AF9-R1.

Luteinizing Hormone-Releasing Hormone Involvement in the Reproductive Behavior of a Male Amphibian

The present study demonstrates that an intracerebroventricular injection of luteinizing hormone-releasing hormone (LHRH) activates sexual behaviors and elevates the plasma androgen concentrations in rough-skinned newts (Taricha granulosa). The stimulatory effect of LHRH on male behavior may be a seasonal phenomenon, because LHRH stimulation of sexual behavior was only observed during the early part of the breeding season (November and December). When an active LHRH antagonist was injected into male newts, sexual behavior was suppressed. These studies indicate that endogenous LHRH is involved in regulating sexual behavior in this amphibian.

Antagonists of the luteinizing hormone releasing hormone with pyridyl-alanines which completely inhibit ovulation at nanogram dosage

[N-Ac-D-2-Nal1,pCl-D-Phe2,D-3-Pal3,D-Arg6,D-Ala10]-LHRH caused 100% and 57% inhibition of ovulation in rats, s.c., at 500 and 250 ng, respectively, and 56%, per os at 500 micrograms. [N-Ac-3,4-diC1-D-Phe1,pC1-D-Phe2,D-3-Pal3,D-Arg6,D-Ala10]-LHRH inhibited ovulation, s.c., 82% at 500 ng, and 63%, per os at 500 micrograms. These analogs are the most effective reported inhibitors of ovulation. The new introduction of pyridyl-alanines can be superior substituents. For pairs of analogs, relationships are: D-3-Pal (beta-(3-pyridyl)-D-alpha-alanine) in position 3 is superior to D-Trp3, D-2-Pal3 and D-4-Pal3. D-Arg6 was superior to D-3-Pal6 and D-4-Pal6 was superior by 2-fold to D-Arg6. D-Ala10 was superior to Gly10 and D-Abu10.

Activities of Antagonists of the Luteinizing Hormone Releasing Hormone with Emphasis on Positions 1, 5 and 6 and on Positions 1, 2 and 3

Abstract Analogs of the luteinizing hormone releasing hormone (LHRH) which are antagonists for controlling ovulation require potency and negligible release of histamine as a side effect. Forty analogs were designed, synthesized and bioassayed in two groups with emphasis upon positions 1, 5 and 6 and upon positions 1, 2 and 3. N-Ac-D-2-Nal1, D-pClPhe2, D-3-Pal3, Ser4, Tyr5, D-Lys6, Leu7, Arg8, Pro9, D-Ala10-NH2 and N-Ac-D-CL2Phe1, D-α-Me-pCIPhe2, D-3-Pal3, Ser4, Tyr5 D-Arg6, Leu7, Arg8, Pro9, D-Ala10-NH2 caused 100% inhibition of ovulation at 0.5 μg in rats. The former analog showed 12.5% anh-ovulatory activity (AOA) and the latter analog showed 40% AOA at 0.25 (μg. The neutral Cit6 moiety is unique since it and the basic D-NicLys6 moiety provided peptides comparable in activity. Frequently, D-2-Nal, D-pClPhe and D-CL2Phe are comparable in position 1. Histamine release was substantially low for a D-NicLys6 analog.

Antiovulatory Potency and Conformation of an Antagonist of the Luteinizing Hormone-Releasing Hormone Having Six D-Amino Acids

Abstract [N-Ac-Thr 1 ,D-Phe 2,D-Trp 3,6]-LHRH was the model antagonist of LHRH, which was the basis for tho design, synthesis and bioassay of seven peptides having four, five and six D-amino acids, which resulted from three single, three double, and one triple introductions of D-amino acids in positions 4, 5 and 8 of the model. Only the analog with six D-amino acids, [N-Ac-Thr 1,D-Phe 2 ,D-Trp 3,D-Ser 4, D-Tyr 5 ,D-Trp 6 ,D-Arg 8]-LHRH, had antiovulatory activity which was higher than that of the model antagonist, i.e., 70% antiovulatory activity at 25 μg/rat compared with 50% activity at 50 μg/rat, respectively. Empirical energy calculations gave a conformational structure for [N-Ac-Thr 1,D-Phe 2,D-Trp 3, D-Ser 4,D-Tyr 5,D-Arg 6,D-Arg 8]-LHRH which is similar to that calculated for previous potent antagonists. These results are a basis of new designs of antagonists having D-sub-stituents in up to ten positions toward effective inhibitors of ovulation by the parenteral and oral routes of administration.

Isolation, partial chemical and biological characterization of thymone B

Abstract A peptide, thymone B, was isolated from bovine thymus by sequences of solvent extractions, gel filtrations, ion exchange chromatography and micromanipulation. Paucity of sample restricted all assays. Thymone B showed essentially single spots by TLC in 3 systems and by electrophoresis in one system. Levels of 100 nanograms (lower?) and 10 micrograms (lowers?) of thymone B stimulated incorporation of [3H]-thymidine into DNA, and the synthesis of cGMP, respectively; 100 micrograms did not stimulate cAMP. Trypsin destroyed activity indicating an active peptide. Analysis indicated up to 13 individual amino acids: Asp, Glu, Gly, Ala, Val, Ile, Leu, Ser, Pro, Thr, His, Lys and Arg, and a molecule considerably smaller than that of thymone A.

Isolation of thymone A from bovine thymus partial chemical and biological characterization

A peptide, designated thymone A, was isolated from bovine thymus by a sequence of eight procedures prior to micromanipulation. Trypsin destroyed the activity indicating the absence of a non-peptidic but active component. The peptide showed essentially single spots under three conditions of electrophoresis. Analysis revealed up to 14 individual amino acids: Asp, Glu, Gly, Ala, Val, Ile, Leu, Pro, Ser, Thr, Met, Lys, Arg and His, and a composition of > 68–71 amino acids (MW 7291–7677). The MW by behavior over Bio-Gel P-6 was ca. 8000. A level between 10–100 ng of thymone A stimulated incorporation of [3H]-thymidine into DNA; a level of 1 μg (lower?) stimulated the synthesis of cAMP. Thymone A may have functional activity or be an active pro-hormone.

Analogs of the Luteinizing Hormone Releasing Hormone having the Azagly10 Moiety with Antiovulatory Activity

Twenty-four new analogs of the luteinizing hormone releasing hormone (LHRH) were synthesized and bioassayed for antiovulatory activity in rats. [N-Ac-3⊿Pro1, pF-D-Phe2, D-Trp3,6, Azagly10]-LHRH com pletely inhibited ovulation at 6 μg and was the most potent of the 24, and is a relatively potent antagonist in the field. The Azagly- and Ac-NHNH- and D-Alamoieties in position 10, and D-Arg in position 6, and diverse substitutions in position 1 were emphasized. D-Arg6 was inferior to D-Trp6, and pCl-D-Phe6 appeared superior to D-Trp6. D-Trp3,6 was superior to D-2-Nal3,6 and D-His3,6.

The finding and partial purification and characterization of thymone C

Abstract A fraction, isolated from bovine thymus by sequences of ca. ten steps of extractions, gel filtrations, ion exchange chromatography, etc., stimulated the synthesis of both cAMP and cGMP at a level of 100 μg/ml. Thymone A stimulated cAMP, but is chromatographically remote from the fraction. Essentially pure thymone B, which stimulates cGMP, did not stimulate cAMP even at a high level of 100 μg/ml. The presence of thymone B in this fraction is supported by TLC data. This fraction also stimulated the mixed lymphocyte reaction. The cAMP-stimulating entity is designated thymone C until it is characterized and related to or differentiated from products of other investigators.

Synthesis and Bioassay of Antagonists of the Luteinizing Hormone Releasing Hormone Having the Azagly10 Moiety

Abstract Seven new analogs of the luteinizing hormone releasing hormone (LHRH), having an Azagly10 moiety, and three corresponding Gly10 -analogs were synthesized for bioassay and comparison of inhibitory potencies. This study was toward a possible advantage of the Azagly10 moiety to minimize C-terminal degradation, in vivo. Of the three procedures which were studied to achieve Azagly10-peptides, the reaction of cyanate ion with hydrazides was the most favorable. Variations of substitution in position 1 were also studied. The data from the antiovulatory assay showed that an Azagly10 moiety may not depress activity, and may allow equal or even higher activity than the Gly10 moiety, depending on the analog. [N-Ac-D-Thr1 , D-p-Cl-Phe2 , D-Trp3,6, Azagly10]-LHRH was more inhibitory than the corresponding Gly10-analog. Based on pairs of analogs, the following relationships appeared: (1) N-Ac-D-Thr1 was more effective than N-Ac-p-Cl-Phe1 ; (2) The L-configuration of Ala as N-Ac-Ala1- was more effective than the D-; (3) N-Ac-Ala1 appeared more effective than the N-Ac-D-Thr1 ; (4) D-Trp6 appeared more effective than D-Phe6. In an ultimate clinical use of an antagonist of LHRH to block ovulation, the Azagly10 moiety may be advantageous for limitation of enzymatic degradation.