Studies on trypsin inhibitors. Part I. Synthesis of protected peptides related to sequence 1-10 of porcine pancreatic secretory trypsin inhibitor II (Kazal).

The general strategy for the synthesis, by conventional procedures, of the entire sequence of porcine pancreatic secretory trypsin inhibitor II (Kazal) is discussed. The synthesis of two protected peptides corresponding to positions 2-10 and 1-10 of the proposed primary structure of the inhibitor is described. The heptapeptide free base threonyl-S-acetamidomethylcysteinylthreonylseryl-gamma-tert-butylglutamylvalylserine tert-butyloxycarbonylhydrazide (sequence 4-10) was acylated, by the azide procedure, with either the dipeptide benzyloxycarbonyl-gamma-tert-butylglutamylalanine hydrazide (sequence 2-3) or the tripeptide Nalpha-benzyloxycarbonyl-Nomega-nitroarginylglutamylala-nine hydrazide (sequence 1-3). The stereochemical homogeneity of the resulting peptides, benzyloxycarbonyl-gamma-tert-butylglutamylalanylthreonyl-S-acetamidomethyl-cysteinylthreonylseryl-gamma-tert-butylglutamylvalylserine tert-butyloxycarbonylhydrazide and Nalpha-benzyloxycarbonyl-Nomega-nitroarginylglutamylalanylthreonyl-S-acetamido-methylcysteinylthreonylseryl-gamma-tert-butylglutamylvalylserine tert-butyloxycarbonyl-hydrazide, was assessed, after partial deprotection with liquid hydrogen fluoride, by digestion with aminopeptidase M followed by quantitative amino acid analysis.     

Mapping the binding of monoclonal antibodies to histone H5

The binding sites of nine monoclonal antibodies along the polypeptide chain of histone H5 were mapped. Immunoblotting experiments with peptides generated from H5 by trypsin digestion, N-bromosuccinimide cleavage, and cyanogen bromide cleavage revealed that all of the monoclonal antibodies reacted with the globular region of H5 which is encompassed by amino acid residues 22-98. Within this globular segment, the epitopes could be subdivided into three regions. Monoclonals 1G11, 2E5, and 2H5 bind to residues 28-31. The close proximity of the epitopes was verified by a competitive enzyme-linked immunosorbent assay and by their binding pattern to a tryptic digest of H5. Monoclonals 4C6, 6E12, and 2E12 bind to a region encompassed by amino acids 28-53 while monoclonals 4H7, 1C3, and 3H9 bind to a region encompassed by residues 53-98. Precise localization of the epitopes in the primary sequence of H5 will allow detailed studies on the mode of binding of H5 to core particles in chromatin.     

Monoclonal anti-gonadotropin releasing hormone (GnRH) antibodies reacting to sequence of GnRH preferentially recognize to native hormone

Monoclonal anti-GnRH antibodies (MoAbs) P862, P778, P813 and P764 reacted optimally to native GnRH and poorly to GnRH(OH) of sequence 4-6, 7-10, 4-10 and 1-10. The heptapeptide 4-10 showed maximum reactivity amongst the four peptides tested for immunoreactivity. Sepharose 4B-GnRH(OH)4-10 (H-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-OH) column was therefore used to purify the fraction of MoAb reacting to this sequence. The affinity purified MoAbs (A-MoAbs) were further characterized for their binding to the different sequences and affinity with native GnRH. The binding, cross-reactivity and affinity characteristics of A-MoAbs were comparable with those of MoAbs. Immunoreactivity of A-MoAbs was also observed to be partly regained when GnRH(OH)1-10 was coupled to Lys, Lys-MDP or H-Ala-Ala-Thr-Lys-Pro-Arg-OH. These observations clearly demonstrate that MoAbs were neither contaminated nor were sequence specific but were directed against the conformation of the molecule.     

Novel ligands for the affinity labelling of luteinizing hormone releasing hormone receptors.

A number of novel luteinizing hormone releasing hormone (LHRH) analogues incorporating biotin together with potential covalent attachment sites have been synthesized. Those based on the des-Gly10-[D-Lys6]-LHRH ethylamide peptide backbone resulted in the most useful characteristics of binding to the LHRH receptor in rat anterior pituitary gland membranes. Of these, des-Gly10-[biotinyl-aminoethylglycyl-D-Lys6]-LHRH ethylamide (XBAL) gave the best specific: non-specific binding ratio, with 44 +/- 6% (+/- S.E.M.) of total binding being specific with a Kd of 131 +/- 16 pM (+/- S.E.M., n = 4) as determined by Scatchard analysis. Two methods have been used to covalently crosslink these analogues with the LHRH receptor; photoaffinity labelling and the use of homobifunctional N-hydroxysuccinimide ester crosslinkers. The photoaffinity analogues gave poor specific: non-specific binding ratios. Of the chemical crosslinkers tested, ethylene glycolbis(succinimidylsuccinate) (EGS) was found to be the most efficient at covalently linking the 125I-XBAL bound to the LHRH receptor site. At an EGS concentration of 5 mM, 23 +/- 3% (+/- S.E.M.) of the specific binding of 125I-XBAL was covalently crosslinked.     

Monoclonal antibody recognition of abscisic Acid analogs

Specificities of three monoclonal antibodies (15-I-C5, DBPA 1, and MAC 62) raised against the plant hormone (S)-(+)-abscisic acid (ABA) have been compared. Immunological cross-reactivities against fifteen biologically active analogs of ABA were measured. The ABA analogs were altered at one or more of four positions: the double bonds in the ring, at C-2 C-3 and at C-4 C-5, and in the oxidation level at C-1. Several analogs were optically active with chiral centers at C-1′ and C-2′. For cross-reactivity, all three monoclonal antibodies required the carboxylic acid group, and the cis configuration of the double bond at C-2 C-3 of the ABA molecule. Monoclonals 15-I-C5 and DBPA 1 required the entire ABA sidechain from the C-1 to C-1′, but these monoclonals did cross-react with analogs with the ring double bond reduced and the C-2′ methyl cis to the sidechain. Only MAC 62 recognized analogs containing an acetylene at C-4 C-5. MAC 62 had more strict requirements for the ring double bond, but gave some cross-reactivity with acetylenic analogs having a saturated ring. All three monoclonals had higher specificity for analogs having the same absolute configuration at C-1′ as (S)-(+)-ABA. This work provides new information about the spatial regions of the ABA molecule that elicit immunological recognition, and serves as a basis for future investigations of the ABA receptor using ABA analogs and anti-idiotypic antibodies.     

Purification, refolding, and characterization of recombinant LHRH-T multimer

To make the native LHRH immunogenic, a multimer of LHRH interspersed with T non-B peptides (r-LHRH-d2) was expressed as recombinant protein in Escherichia coli. The expression level of the recombinant protein was around 15% of the total cellular protein and it aggregated as inclusion bodies. Inclusion bodies from the bacterial cells were isolated and purified to homogeneity. Instead of high concentrations of chaotropic agents, r-LHRH- d2 was solubilized in 50 mM citrate buffer at pH 3 containing 2 M urea. The protein was refolded by 5-fold dilution (pulsatile) with cold 10 mM citrate buffer at pH 6 in presence of 0.3 M L-arginine. Purification of r-LHRH-d2 was carried out by successive passages on CM-Sepharose column at pH 6.0 which retained extraneous proteins and pH 4.8 at which r-LHRH-d2 bound to the resin. The elution was carried out by using linear salt gradient (0.1-1 M NaCl). The overall yield of the purified r-LHRH-d2 was 40% of the initial inclusion body proteins. The purity and homogeneity were confirmed by a single homogeneous peak on analytical HPLC eluting out at 29.51 min and by single band on SDS-PAGE reactive with polyvalent anti-LHRH antibodies. Mass spectroscopic analysis indicated the protein to be of 16.6 kDa which equals the theoretically expected mass. The N-terminal amino acid analysis of r-LHRH-d2 showed the sequence which corresponded to the designed protein. The CD spectrum of the refolded r-LHRH-d2 showed that the multimer has considerable beta sheet structure like the monomeric LHRH protein.     

Characterization of the opsonic and monocyte adherence functions of the specific fibronectin fragment that enhances phagocytosis of particulate activators

The functional opsonic and monocyte adherence domains within the 180,000 m.w. opsonic fibronectin fragment (180K-opFnf) that selectively augments human monocyte phagocytosis of particulate activators of the alternative complement pathway were analyzed with Fab fragments of monoclonal anti-fibronectin antibodies BC7, CE9, BD4, AB3, and CPG1, and with fragments of intact human plasma fibronectin derived by cathepsin cleavage and isolated by affinity chromatography. Monoclonals AB3 and CPG1, which recognize epitopes within 40,000 daltons of the carboxy terminus of intact fibronectin, and the cathepsin D-derived, disulfide-linked fragments that contain these epitopes each inhibited the opsonic function of 180K-opFnf. Monoclonals AB3 and CPG1 inhibited monocyte ingestion of rabbit erythrocytes (Er) by 60 and 50%, respectively, when 180K-opFnf was pretreated with 20 micrograms of these monoclonals, but neither monoclonal affected the enhanced monocyte ingestion of Er pretreated with the fibronectin fragment. The pretreatment of Er with 5 micrograms and 40 micrograms of the disulfide-linked, cathepsin D derivatives isolated from high and low affinity heparin fractions, respectively, inhibited the proportion of ingesting monocytes by 60%, but these types of fragments had little effect when concurrently incubated with the opsonic fragment and Er. Monoclonals CE9 and BD4, which recognize epitopes located adjacent to or within the cell-adhesive domain of intact fibronectin, respectively, inhibited the monocyte adherence function of 180K-opFnf, as evidence by their comparable inhibitory effects when present before or after Er were opsonized with 180K-opFnf. When 20 micrograms of monoclonals CE9 and BD4 were each introduced before and after Er were opsonized with 180K-opFnf, monocyte ingestion was inhibited by 60 and 65% and by 51 and 60%, respectively. At 42 micrograms, cathepsin D-derived, non-gelatin-binding, low affinity heparin fragments that contained both BD4 and CE9 determinants or only the BD4 determinant inhibited monocyte ingestion by 53 and 74%, respectively, when concurrently incubated with 180K-opFnf and target Er, but were without effect when used to pretreat Er before the addition of 180K-opFnf. Thus, the inhibitory effects produced by monoclonals AB3 and CPG1 and by cathepsin D-derived, disulfide-linked fragments containing their corresponding epitopes demonstrated that the opsonic domain within 180K-opFnf is immunologically similar to regions within the carboxy terminus of intact plasma fibronectin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Active immunization of male pigs against gonadoliberin: effect on the secretion of gonadotropic hormones and on 5 alpha-androst-16-ene-one levels in adipose tissue

27 boars aged 100 days were allocated to 3 experimental groups. The control group was untreated. The 2 other groups were given injections of LHRH, bound to BSA, with either Freund complete adjuvant or alumina gel. 4 immunisations were achieved at weekly intervals and a fifth injection was performed after 4 weeks. In animals treated with alumina gel, no specific antibodies were obtained. In the other hand, all 9 boars treated with FCA exhibited significant titers of specific anti-LHRH antibodies. In these animals, the neutralisation of blood LHRH induced a significant decline in both LH and FSH concentrations. Consequently, fat androstenone levels dropped dramatically, down to low levels, similar to those observed in surgically castrated males.     

Use of blocking ELISA additivity test and antibody-antibody competition technique for analysis of recognition ability of monoclonal anti-gonadotropin releasing hormone antibodies

Monoclonal anti-GnRH antibodies reacting to the heptapeptide 4-10 (H-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-OH) were isolated by affinity chromatography using Sepharose 4B-heptapeptide (4-10) column. The ELISA additivity test and antibody-antibody competition techniques were used to study whether the affinity purified MoAb (A-MoAb) fraction recognize the sequence or the conformation of the native hormone. All four A-MoAbs, P862, P778, P764 and P813, were able to recognize the common epitope and did not allow to bind the conventional anti-GnRH antibodies (CoAbs) indicating that the CoAbs were conformation specific. Similarly in antibody-antibody competition technique, all A-MoAbs were able to compete with CoAbs, indicating that MoAbs were generated against the conformation of GnRH involving the entire molecule.     

Leydig cell receptors for luteinizing hormone releasing hormone and its agonists and their modulation by administration or deprivation of the releasing hormone

Leydig cells isolated from adult rat testes bound ¹²⁵I-labelled luteinizing hormone releasing hormone (LHRH) agonist with high affinity (K_A=1.2 × 10⁹M) and specificity. LHRH and the 3–9 and 4–9 fragments of LHRH agonist competed for binding sites with ¹²⁵I-LHRH agonist but with reduced affinities, whereas fragments of LHRH, and oxytocin and TRH were largely inactive. Somatostatin inhibited binding at high (10^?4M) concentrations but was inactive at 10^?6M and less. Pretreatment of rats for 7 days with 5 μg/day of LHRH agonist reduced binding of ¹²⁵I-LHRH agonist to Leydig cells $\text{in vitro}$ by 25%, whilst inhibition of endogenous LHRH by antibodies for 7 days caused a 40% decrease.     

Leukotriene C4-induced release of LHRH into the hypophyseal portal blood and of LH into the peripheral blood

Intracerebroventricular (i.c.v.) administration of leukotriene (LT) C4 at doses of 2, 0.5 and 0.2 micrograms/rat significantly stimulated (3-12 fold) the release of LH into the peripheral blood of male rats. Injection of anti-LHRH serum had no effect on LTC4-stimulated LH release, but did block PGE2- stimulated LH release. I.c.v.- infused LTC4 also stimulated the release of LHRH into the hypophyseal portal blood. This is the first report of an in vivo action of LTC4 on the release of a hypothalamic releasing factor (LHRH) and a pituitary hormone (LH). These observations, plus in vitro results, clearly show that LTC4 stimulates LH release by acting on both the hypothalamus, causing LHRH release, and on the pituitary. Then the action of LTC4 on LH release in vivo is quite different from the indirect action of PGE2.     

Absence of specific luteinizing hormone releasing hormone receptors in ovine, bovine and porcine ovaries

Crude and membrane-enriched homogenates of unfrozen follicular and luteal tissue from cows, ewes and sows were assayed for the presence of specific luteinizing hormone releasing hormone (LHRH) receptors by one-point saturation analysis using [D-Ser-(TBU)6, des-Gly-NH2(10)] LHRH-EA as the labeled and unlabeled ligand. Pituitaries from cows, ewes, sows and rats, and rat ovaries served as positive controls and were assayed with each ovarian tissue assay. Scatchard analysis was used to determine binding affinity of pools of ovarian and pituitary tissue. Specific high-affinity LHRH receptors were found in the pituitaries of cows, ewes, sows and rats and in the rat ovary. In contrast, no specific LHRH binding was detected in follicular or luteal tissue of cows, ewes or sows. Thus, unlike the rat ovary which contains LHRH receptors, ovaries from these domestic species lack specific LHRH receptors.     

Fasciola hepatica: development of monoclonal antibodies against somatic antigens and their characterization by ultrastructural localization of antibody binding

A series of monoclonal antibodies was prepared against tegumental and internal antigens of Fasciola hepatica by immunizing mice with whole adult-fluke homogenates prior to harvesting the splenic lymphocytes for fusion. Preliminary screening by the Indirect Fluorescent Antibody technique indicated the occurrence of discrete groups of monoclonals differing from one another in tissue-specificity but within which IFA labelling patterns were fairly consistent. Representative hybridomas for 5 of these groups were stabilized and used to produce ascites fluid in mice. By application of an immunogold labelling technique it was possible to map the distribution of antigens for which each monoclonal antibody had affinity throughout the tissues of 4-week and 12-week flukes. Several monoclonals specifically labelled antigenic determinants on the important tegumental antigen T1. However the distribution of gold colloid labelling suggested that epitopes other than that normally exposed to the infected host were recognized; and several monoclonals specifically attached to T1 antigen in the tegument of juvenile worms only. The glycocalyx of the gut and excretory system of flukes shared T1 antigenicity with the tegument. Monoclonal antibodies were produced against an internal immunogen associated with ribosomes and heterochromatin in active protein-producing cells, and against interstitial material of adult flukes. Monoclonals against antigens in parenchymal cell cytoplasm and in mature vitelline cells were recognized but the corresponding hybridomas were not stabilized.     

Affinity-driven selection of tripeptide inhibitors of ribonucleotide reductase

Tripeptide libraries of the type Fmoc(W/F)XF were screened for binding to the large subunit of mouse ribonucleotide reductase (mRR), using a new, affinity chromatography method. A high-affinity tripeptide, FmocWFF, was found that inhibited mRR activity with a K(i) equal to that of AcFTLDADF, the heptapeptide corresponding to the C-terminus of the small subunit of mRR.     

Leptin acts centrally to induce the prepubertal secretion of luteinizing hormone in the female rat

Recent data generated from adult male and female rats indicates that leptin is capable of stimulating luteinizing hormone (LH) secretion via a hypothalamic action. Consequently, we hypothesized that this peptide may similarly play a role in controlling LH secretion during late juvenile and peripubertal development; hence, contributing to hypothalamic-pituitary function during sexual maturation. Therefore, this study was conducted to determine if leptin is capable of stimulating LH release during this critical time of development and, if so, to determine whether this action is due to an effect at the hypothalamic level. Results showed that leptin, when administered directly into the brain third ventricle (3V), can stimulate (P < 0. 01) LH release in late juvenile animals at doses of 0.01-1.0 microg. A higher dose of 10 microg was ineffective in stimulating LH release. Immunoneutralization of luteinizing hormone-releasing hormone (LHRH) via 3V administration of LHRH antiserum to late juvenile animals indicated a hypothalamic site of action, since the leptin-induced LH release was blocked in the animals that received anti-LHRH, but not in the control animals that received normal rabbit serum. Leptin did not significantly stimulate LH release from animals in first proestrus, estrus, or diestrus. We also report that the serum levels of leptin increase (P < 0.05) during the late juvenile period of development, then decrease (P < 0.05) once the animal enters the peripubertal period. Collectively, our results show that leptin is capable of acting centrally to stimulate LH release, but only during late juvenile development; thus, we suggest the peptide likely plays a facilitatory role on late juvenile LH secretion, but does not drive the LHRH/LH releasing system to first ovulation and hence, sexual maturity.     

Simultaneous measurement of luteinizing hormone-releasing hormone and luteinizing hormone during estradiol-induced luteinizing hormone surges in the ovariectomized ewe

Sequential bleeding and push-pull perfusion of the hypothalamus were used to characterize luteinizing hormone (LH) and LH-releasing hormone (LHRH) release in ovariectomized (OVX) ewes after injection of corn oil or estradiol benzoate (EB). Push-pull cannulae were surgically implanted into the stalk median eminences of 24 OVX ewes. Seven to 14 days later each of 20 animals was given an i.m. injection of 50 micrograms EB. Blood samples and push-pull perfusate were collected at 10-min intervals for 6-12 h beginning 12-15 h after EB injection. Four OVX ewes were given i.m. injections of corn oil 7 days after implantation of push-pull cannulae. Blood samples and push-pull perfusate were collected at 10-min intervals for 4 h between 18 and 22 h after injection of corn oil. Luteinizing hormone remained below 2 ng/ml throughout most of the sampling periods in 9 of 20 EB-treated ewes. In 5 of these 9 LHRH also was undetectable, whereas in 4 LHRH was detectable (1.84 +/- 0.29 pg/10 min), but did not increase with time. Preovulatory-like surges of LH occurred in 11 EB-treated ewes, but LHRH was undetectable in 5. In 4 of 6 ewes showing LH surges and detectable LHRH, sampling occurred during the onset of the LH surge.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of modification of the carboxyl group in short D-arginine 2-containing enkephalin analogs on their affinity and selectivity for opiate receptors

Radioreceptor binding assay using a membrane fraction from the rat brain was applied to study [D-Arg2, Leu5] enkephalin and two series of its analogues truncated at the C-terminus with a free or modified carboxyl group: tetra- and tripeptide amides and ethyl esters. The affinity to mu-specific opiate receptor subtype of the N-terminal [D-Arg2] tetrapeptide ethyl ester was 44 times as high as that of the tripeptide with a free carboxyl, and thus the ester retained up to 10% of leucine-enkephalin binding potency. However, a comparable esterification of the carboxyl group in the N-terminal [D-Arg2] tripeptide led to a 6-fold reduction in its affinity to mu-receptors. Consequently, identical modifications of the C-terminal carboxyl group in enkephalin analogues of various length can have completely different effects. Substitution of the natural glycine residue by D-arginine residue in position 2 of the enkephalin molecule truncated at the C-terminus increased the mu-receptor binding potency of the tetrapeptide, whereas its delta receptor binding potency declined by more than one order of magnitude. Simultaneous replacement of glycine2 by D-arginine2 and carboxyl amidation resulted in the short enkephalin analogue Tyr--D--Arg--Gly--Phe--NH2, whose affinity to mu receptors was four times as high as that of leucine--enkephalin, the tetrapeptide being 284 times more selective for the mu vs. delta opiate receptors.     

Fractionation of polyclonal antibody by isoelectric focusing and chromatofocusing: separation of high-affinity rabbit clonotype anti-thyroxine antibody

Immunoglobulin G fractions prepared from conventional rabbit anti-thyroxine (T4) antisera were fractionated by agarose gel isoelectric focusing (IEF) in the range of pH 3 to 10, and by chromatofocusing using a Fast Protein Liquid Chromatography (FPLC) system. The clonotype antibodies were recovered from the fractions and subjected to Scatchard plot analysis. The highest affinity constants of the initial antibody (shown in parentheses) and those of the antibodies recovered were IEF, 1.8 X 10(9) to 8.3 X 10(9) M-1 (2.2 X 10(9) M-1); FPLC, 2.4 X 10(9) to 6.0 X 10(9) M-1 (2.5 X 10(9) M-1). A sensitive radioimmunoassay of T4 was achieved with the isolated high-affinity anti-T4 antibody. The minimum detectable concentration of T4 was 6.3 X 10(-15) to 1.5 X 10(-14) mol/tube, which was three to five times lower than detectable with the initial antibodies.     

Isocratic liquid chromatographic assay for monitoring the degradation of luteinizing hormone releasing hormone by extracts from the gastrointestinal tract of possums

A simple HPLC method to separate human luteinizing hormone releasing hormone (LHRH) from its metabolites using an isocratic elution is described. Intact LHRH and five metabolites were separated in 11.4 min. The calibration curve (peak area versus concentration) was linear over the concentration range 1.25-35 microg/ml (r(2)=0.99) with the intercept not significantly different from zero (P>0.05). Intra-day and inter-day variability of the assay was less than 5% for repeat injections of 5, 14.5 and 29 microg/ml. The method was applied to evaluate the susceptibility of LHRH to enzymes present in the lumen and mucosal extracts of the gastrointestinal tract of possums. The major degradation products of LHRH were identified by HPLC separation, amino acid analysis and mass spectrometry as LHRH (1-5), LHRH (1-4), LHRH (1-3) and LHRH (3-4).     

Studies on trypsin inhibitors. Part V. Synthesis of the protected octapeptide (sequence 36-43) of porcine pancreatic secretory trypsin inhibitor II (Kazal).

Synthesis is described of the partially protected octapeptide tert-butyloxycarbonyl-gamma-tert-butylglutamylasparaginyl-N-trifluoroacetyllysyl-N-trifluoracetyllysylarginyl-Ngamma-4,4'-dimethoxybenzhydryglutaminylthreonylproline corresponding to positions 36-43 of the amino acid sequence of porcine pancreatic secretory trypsin inhibitor II. The tetrapeptide free base arginyl-Ngamma-4,4'-dimethoxybenzhydrylglutaminylthreonylproline was acylated, by the azide proceedure, with the tripeptide benzyloxycarbonyl-asparaginyl-N-trifluoroacetyllsyl-N-trifluoroacetyllysine hydrazide. The resulting protected heptapeptide was partially deblocked by catalytic hydrogenation and reacted with alpha-1-succinimidyl-gamma-tert-butyl tert-butyloxycarbonylglutamate. The stereochemical homogeneity of the ensuing octapeptide was assessed, after partial deprotection with aqueous 90% trifluoroacetic acid, by digestion with papain and aminopeptidase M followed by quantitative amino acid analysis.