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The influenza virus haemagglutinin has an important role in the infectious cycle of the virus and carries multiple B and T cell epitopes. It is synthesized as a single polypeptide chain but viral infectivity depends on its post-translational enzymatic cleavage. The cleavage site of a trypsin-like enzyme responsible for this modification is found in the most conserved intersubunit region of the molecule. In this study the role of this region in antibody recognition was investigated. Synthetic peptides comprising the intact and cleaved forms of the intersubunit segment were used to examine the specificity of virus- or peptide-induced antibodies. The immune response elicited by viral infection resulted in the appearance of antibodies capable of neutralizing the virus without interfering with its binding to the receptor. A monoclonal antibody (MoAb) of such functional properties was shown to recognize the intact intersubunit region both in the uncleaved haemagglutinin molecule and in a 25-mer synthetic peptide comprising the intact intersubunit region. Specificity and functional studies revealed the conformation-dependent recognition of the C-terminal segment of the haemagglutinin 1 subunit by this MoAb. The binding of the antibody was shown to inhibit the trypsin-mediated cleavage of the haemagglutinin molecule and the membrane fusion event. The enzymatic cleavage of the haemagglutinin was demonstrated to abolish antibody recognition of the infective virus suggesting an escape mechanism mediated by the functional destruction of this highly conserved region. The synthetic peptide corresponding to the intact intersubunit region is characterized by an ordered structure and is able to elicit an antibody response in BALB/c mice while its subfragments are nonimmunogenic. Furthermore, this peptide elicited a protective immune response demonstrated by in vivo experiments.

The impact of glucocorticoids on beta-amyloid(1-42) (Abeta(1-42)) and NMDA-induced neurodegeneration was investigated in vivo. Abeta(1-42) or NMDA was injected into the cholinergic magnocellular nucleus basalis in adrenalectomized (ADX) rats, ADX rats supplemented with 25%, 100%, 2x100% corticosterone pellets, or sham-ADX controls. Abeta(1-42)- or NMDA-induced damage of cholinergic nucleus basalis neurones was assessed by quantitative acetylcholinesterase histochemistry. Plasma concentrations of corticosterone and cholinergic fibre loss after Abeta(1-42) or NMDA injection showed a clear U-shaped dose-response relationship. ADX and subsequent loss of serum corticosterone potentiated both the Abeta(1-42) and NMDA-induced neurodegeneration. ADX+25% corticosterone resulted in a 10-90 nM plasma corticosterone concentration, which significantly attenuated the Abeta(1-42) and NMDA neurotoxicity. ADX+100% corticosterone (corticosterone concentrations of 110-270 nM) potently decreased both Abeta(1-42)- and NMDA-induced neurotoxic brain damage. In contrast, high corticosterone concentrations of 310-650 nM potentiated Abeta(1-42)- and NMDA-triggered neurodegeneration. In conclusion, chronic low or high corticosterone concentrations increase the vulnerability of cholinergic cells to neurotoxic insult, while slightly elevated corticosterone levels protect against neurotoxic injury. Enhanced neurotoxicity of NMDA in the presence of high concentrations of specific glucocorticoid receptor agonists suggests that the corticosterone effects are mediated by glucocorticoid receptors.

Sensitive, specific and reproducible radioimmunoassay (RIA) was developed for the measurement of oxytocin (OXT) in rat blood plasma after various extraction methods. The assay is based on an antiserum raised against OXT in rabbit. The sensitivity, affinity constant, and cross-reactivity of the antiserum were determined. The 125I-labelled OXT for RIA was produced by chloramine-T method and purified with high pressure liquid chromatography (HPLC). Two extraction procedures were employed: 1. adsorption to an artificial silicate, Lichroprep Si 60 (Merck); 2. immunoextraction of the hormone applying a magnetic bearer covered with purified antibodies against OXT. The specificity of the extraction methods was characterized in comparative HPLC/RIA studies of specimens extracted from blood plasma in different ways. The basal level of the peptide measured after the extraction with thermally activated Lichroprep Si 60 or after the immunoextraction method was found to be 9.6 +/- 2.3 pg/ml (mean +/- S.E.) and 15.3 +/- 0.9 pg/ml (mean +/- S.E.), respectively. Various well known factors (ether exposure, hyperosmotic stress and suckling) appeared to be potent peripheral stimuli of OXT release, and thus indicated the suitability of the RIA method for the measurement of OXT in blood plasma.

The multiple antigenic peptide (MAP) method was applied to improve the immunogenicity of synthetic peptides representing distinct regions of the influenza virus hemagglutinin (HA). A tetrameric MAP with multiply incorporated overlapping B- and T-cell epitopes was combined with a particular HA sequence representing the slightly modified fusion peptide on the C-terminus of the Lys core (MAP-1). As a result of repeated injections of BALB/c mice with MAP-1 but not with the monomeric HA1C[Arg] peptide, the appearance of MAP-1-specific antibodies crossreactive with the acid-pretreated virus could be observed. In vitro studies revealed the potency of the MAP-1 structure to induce proliferation of HA1C[Arg]-primed T-cells, and in vivo studies demonstrated the protective feature of the immune response elicited by MAP-1 and to a lesser extent by the monomeric HA1C[Arg]. The increased level of MAP-1 specific antibodies upon viral challenge shows the activation of MAP-1-specific B- and/or T-cells. The advantage of the previously verified FP3 helper T-cell epitope included in MAP-1 was further utilized to synthesize chimeric structures comprising short fragments of the zeta of (MAP-2) or delta (MAP-3) subunits of the T-cell antigen receptor (TCR) complex. The selected peptides of the zeta and delta-chain regions failed to elicit an antibody response in BALB/c mice as tetra- or octamers, but the inclusion of the modified fusion region resulted in an immunogenic construction.(ABSTRACT TRUNCATED AT 250 WORDS)

Porcine cholecystokinin-33 has been synthesized on solid phase and characterized both chemically and biologically. In order to develop a successful synthetic strategy, a new anchor molecule (4-succinylamido-2,2',4'-trimethoxybenzhydrylamine) was designed and coupled to aminomethyl-polystyrene. The resulting 4-succinylamido-2,2',4'-trimethoxybenzhydrylamine resin was successfully used for the synthesis of cholecystokinin-33 using N-fluorenylmethoxycarbonyl amino acid symmetric anhydrides. Tyrosine-O-sulfate has been synthesized by direct sulfation of tyrosine with chlorosulfonic acid and incorporated into the peptide sequence by coupling as N-fluorenylmethoxy-O-sulfatotyrosine-pentafluorophenyl ester. Side chains of the trifunctional amino acids were protected mostly by t-butyl-type protecting groups. The guanidino function of arginine was protected by the 2,2,5,7,8-pentamethylchromane-6-sulfonyl group. After completion of the synthesis, the peptide was cleaved off the support with 50% trifluoroacetic acid (15 min); this treatment cleaved the side-chain protecting groups simultaneously. Preparative high-performance liquid chromatography resulted in pure cholecystokinin-33 of full biological activity. The structure of the peptide was proved by amino acid analysis, IR and UR spectroscopy, fast atomic bombardment mass spectroscopy and comparative high-performance liquid chromatography of the synthetic and native cholecystokinin-33.

Intraperitoneal (ip) injection of cholecystokinin octapeptide sulfate ester (CCK; 5, 10 and 50 micrograms/kg) reduced the hypothermic response to 8 mg/kg and 32 mg/kg systemic morphine in restrained and freely moving rats, respectively. The hyperthermia elicited by a subcutaneous (sc) injection of 8 mg/kg morphine to freely moving rats was not diminished by CCK pretreatment. CCK (5 micrograms/kp ip) completely prevented the restraint stress-induced hyperthermia. Naloxone (1 mg/gk sc) was effective in antagonizing both the hyperthermic and the hypothermic effects of morphine and the stress-induced emotional hyperthermia. These results support the hypothesis that CCK may contribute to regulation of the endogenous opioid system.

The effects of several doses of cholecystokinin octapeptide sulphate ester (CCK-8-SE) and nonsulphated cholecystokinin octapeptide (CCK-8-NS), and two CCK-related peptide analogues Ac-Thr5-caerulein, and nonsulphated Ac-Thr5-caerulein were investigated on electroshock-(ES)-induced seizures after intraperitoneal administration in mice. As parameters, the duration of the tonic and clonic phase of the fit, and those of postictal coma and behavioural depression were measured. CCK-8-SE decreased the duration of the clonic phase; its highest dose, 3.2 mumol/kg, shortened the coma. CCK-8-NS antagonized only slightly the clonic phase of seizure. Ac-Thr5-caerulein did not influence ES-induced seizures in any dose, only increased the duration of behavioural depression. Similarly to CCK-8-NS, the nonsulphated form of Ac-Thr5-caerulein inhibited selectively the clonic phase of seizures. The reference drugs, diazepam and phenobarbital, antagonized dose-dependently and most effectively the tonic phase of ES-induced seizures, but in much higher doses than did the CCK-related peptides. Besides, diazepam increased and phenobarbital decreased the duration of postictal coma. The results showed that the tested CCK-related peptides inhibit prevalently the clonic phase of ES-induced seizures after peripheral administration.

Effects of intraperitoneal administration of cholecystokinin (CCK)-related peptides were studied on retention of single-trial learning passive avoidance behaviour. The COOH-terminal octapeptide of CCK (CCK-1-8-SE), the unsulfated octapeptide (CCK-1-8-NS), as well as the COOH-terminal tetrapeptide of CCK (CCK-5-8), administered immediately after the learning trial, facilitated passive avoidance behaviour. The data indicate that these peptides may influence memory consolidation processes.

Antibodies were raised in rabbits and sheep with antigens prepared by coupling NE or E to bovine serum albumin. Tissues were fixed with either 4% paraformaldehyde or 4% paraformaldehyde plus 0.5% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.3). Cryostat sections were stained by the indirect immunofluorescence technique. Two parenchymal cell types, representing the NE and E-storing cells, were observed in the adrenal medulla. In the central nervous system, fibers and perikarya could be visualized by the antiserum to the NE antigen. In the peripheral nervous system, NE immunoreactive fibers were demonstrated. There was no detectable cross reactivity of NE antibodies with regard to E and 5-HT. Cross reactivity of E antibodies to NE and 5-HT equally could not be demonstrated.

The possible cataleptogenic or antlcataleptic effects of cholecystokinin octapeptide sulphate ester (CCK-8-SE) and desulphated CCK-8 (CCK-8-NS) were evaluated in rats after intracerebroventricular (i.c.v.) administration. Neither CCK-8-SE nor CCK-8-NS induced any sign of catalepsy. When haloperidol, in a dose od 1.o mg/kg (i.p.), and either CCK-8-SE or CCK-8-NS, in doses of 8, 80 or 800 pmol (i.c.v.), were applied at the same time, the peptides significantly decreased the total duration of catalepsy, the 80 pmol dose being the most effective. For the inhibition of catalepsy CCK-8-NS was the more active as it decreased catalepsy scores even 120 min after administration. Both peptides showed only transient effects on the fully developed catalepsy induced by haloperidol.

In addition to the classical microtubule inhibitors (antimitotic agents), transganglionic degenerative atrophy of central terminals of primary sensory neurons can be induced also by means of applying to a peripheral nerve basic polypeptides (Polymyxin B and Colimycin) and two basic derivatives of glutamic acid that do not exert any microtubule inhibition. This effect is independent of other pharmacological effects (histamine liberation, Ca2+ -binding, etc.) of the applied compounds, and probably it is based on a competitive reaction with nerve growth factor.

Intraperitoneal administration of cholecystokinin octapeptide sulphate ester (CCK-8-SE) and nonsulphated cholecystokinin octapeptide (CCK-8-NS) enhanced the latency of seizures induced by picrotoxin in mice. Experiments with N- and C-terminal fragments revealed that the C-terminal tetrapeptide (CCK-5-8) was the active centre of the CCK octapeptide molecule. The analogues CCK-8-SE and CCK-8-NS (dose range 0.2-6.4 mumol/kg) and caerulein dose range 0.1-0.8 mumol/kg) showed bell-shaped dose-effect curves, with the greatest maximum inhibition for CCK-8-NS. The peptide CCK-5-8 had weak anticonvulsant activity in comparison to the octapeptides, 3.2 mumol/kg and larger doses of the reference drug, diazepam, totally prevented picrotoxin-induced seizures and mortality. The maximum effect of the peptides tested was less than that of diazepam. Experiments with analogues and derivatives of CCK-5-8 demonstrated that the effectiveness of the beta-alanyl derivatives of CCK-5-8 were enhanced and that they were equipotent with CCK-8-SE. Of the CCK-2-8 analogues, Ser(SO3H)7-Ac-CCK-2-8-SE and Thr(SO3H)7-Ac-CCK-2-8-SE and Hyp(SO3H)-Ac-CCK-2-8-SE were slightly more active than CCK-8-SE.

Subcutaneous injection of cholecystokinin octapeptide (CCK-8) (0.005–1.25 mg/kg) elicited dose-dependent hypothermia in rats. The threshold of the response was between 0.01 and 0.05 mg/kg and the dose-response curve levelled off at doses larger than 0.2–0.5 mg/kg. Warm and cold ambient temperatures decreased and increased the response, respectively. Pretreatment with capsaicin, morphine, naloxone, atropine, haloperidol or propranolol did not affect the response to CCK-8, whereas pre-treatment with phenoxybenzamine and a large dose of proglumide, an antagonist for CCK-receptors, attenuated the hypothermia. It seems that neither capsaicin-sensitive thermal and non-thermal afferents, nor opiate mechanisms are involved in the response, but alpha-adrenoceptors might be of some importance in the hypothermia. Non-sulphated-CCK-8, the C-terminal tetrapeptide and hexapeptide, [ D -Ala4]-CCK-8 and [ D -Met6]-CCK-8 were ineffective. Chronic treatment with CCK-8 resulted in the development of tolerance to the thermoregulatory effect, while the hypothermie responses to apomorphine and capsaicin were not affected. It seems that the tolerance cannot be attributed to conditioned homeostatic reactions.

The effects of peripherally administered cholecystokinin octapeptide sulphate ester and its unsulphated form on the active avoidance behaviour of rats were studied. The acquisition of avoidance behaviour was impaired, while extinction was facilitated, following cholecystokinin octapeptide sulphate ester or unsulphated cholecystokinin octapeptide treatment. These peptides had no action on open-field activity. It is concluded that peripherally administered cholecystokinin octapeptide influences acquisition and extinction of active avoidance behaviour and this effect is unrelated to general motor activity of the animals.

The effect of different doses of intracerebro-ventricularly administered cholecystokinin octapeptide sulphate ester (CCK-8-SE) was studied on dopamine (DA), norepinephrine (NE) and serotonin (5-HT) contents in the hypothalamus, mesencephalon, amygdala, septum and striatum, 10, 20 and 60 min following administration. The DA and NE content increased and the 5-HT content decreased in the hypothalamus and mesencephalon. A biphasic action was observed in the amygdala of DA, NE and 5-HT depending upon the time and doses used. Similar action was seen on DA and NE in the septum. In the striatum, the DA and 5-HT content decreased while the NE level first increased and then decreased. The data indicate that the CCK-8-SE is able to modify the activity of DA, NE and 5-HT in different brain regions in a time and dose-dependent manner, with a local specific action.

The effect of somatostatin and its two tetrapeptide fragments was investigated on turning activity induced by unilateral substantia nigra lesion in rats. Somatostatin in a dose of 0.6 nM had no action on the turning behavior, while a dose of 6 nM increased slightly while a dose of 12 nM significantly the contralateral turning. Cys-Lys-Asn-Phe and Phe-Trp-Lys-Thr had no action in low or high doses on the turning activity of the animals. The results suggest that somatostatin has a direct postsynaptic dopamine receptor stimulating effect. It seems that for dopamine receptor stimulating action the complete somatostatin molecule is needed.

The catabolism of the 14C-labelled pentagastrin was investigated in rats. The following results were found: -- the labelled BOC-glycine fragment is split off the pentagastrin in the small intestine -- it is absorbed through the intestinal wall, and -- enters first the portal and then the systemic circulation.

The effects of cholecystokinin octapeptide sulfate ester (CCK-8-SE) and unsulfated cholecystokinin (CCK-8-NS) were studied following intraventricular administration on active avoidance and conditioned feeding behaviour of rats. In the CCK-8-NS and CCK-8-SE treated animals the acquisition of active avoidance and conditioned feeding behaviour were considerably impaired compared to the control; furthermore, these peptides caused a facilitated extinction of active avoidance and conditioned feeding behaviour. The data suggest that cholecystokinin octapeptide is capable of modifying the fear and hunger motivated behaviours of rats.

The anticonvulsive activity of cholecystokinin octapeptide sulphate ester (CCK-8-SE), non-sulphated cholecystokinin octapeptide (CCK-8-NS) and three different N- and C-terminal fragments were investigated against seizures induced by picrotoxin and electroshock in rats after intracerebroventricular administration. Doses of 0.8 and 80 pmol of CCK-8-SE and CCK-8-NS significantly enhanced the latency of seizures induced by picrotoxin and shortened the duration of the clonic phase of the seizures induced by electroshock. Only CCK-8-SE shortened the recovery time and only 0.8 pmol of CCK-8-SE could shorten the duration of the tonic phase of convulsions induced by electroshock. Doses of the octapeptides of 8000 pmol were ineffective, with the exception of CCK-8-NS in the picrotoxin test. Of the fragments tested, the C-terminal tetrapeptide, CCK-5-8, enhanced the latency of seizures induced by picrotoxin in a dose of 0.8 pmol, and had a dose-dependent biphasic effect on the duration of the clonic phase of seizures induced by electroshock. Intracerebroventricular administration of diazepam enhanced only the latency of tremor and clonic seizures induced with picrotoxin in a dose of 40 nmol. Twelve nmole of diazepam shortened the clonic phase of convulsions induced by electroshock. The peptides tested were much more active than diazepam, and their effective doses were comparable to the amounts of cholecystokinin octapeptide found in brain structures.