The protective and immunomodulatory effects of hypothalamic proline-rich polypeptide galarmin against methicillin-resistant Staphylococcus aureus infection in mice.

The present research summarizes the protective and immunomodulatory activity of hypothalamic proline-rich polypeptide galarmin against methicillin-resistant Staphylococcus aureus (MRSA). The protective effect of galarmin was shown on MRSA-infected animals' survival and weight loss recovery. The immunological impact of galarmin was evaluated in terms of immunocompetent cell recruitment, serum immunoglobulins, complement components C3 and C4, and pro- and anti-inflammatory cytokines (IL-6, IL-8, IL-10, IL-1b, TNFa, and KC) secretion. Galarmin efficiently protects mice against lethal MRSA infection (100% of survival vs. 0% in the untreated group) when intramuscularly injected 24 h before infection and during the 1-h post-infection period at a concentration of 1 µg per mouse, while its higher concentrations (5 and 10 µg) were protective when injected in parallel to the infection process. The protective effect of galarmin was not due to a direct effect on MRSA, but should be attributed to an action on the host response to infection. Galarmin significantly increased and modulated the levels of IL-6, IL-8, IL-1b, IL-10, and KC in both peritoneal lavages and blood, leukocyte and platelet counts, lymphocytes percentage, serum IgM and IgG, and complement C3 and C4 components secretion. The experimental results allow concluding that galarmin is a powerful immunomodulatory and protective agent for the in vivo prophylaxis and treatment of MRSA-induced infection.

Proline rich polypeptide (PRP-1) increases the superoxide-producing and ferrihemoglobin reducing activities of cytochrome B(558) isoforms from human lymphosarcoma tissue cells.

The two cytochromes (cyt) b(558) of acidic nature, one-95-100 kDa and another one, 60-70 kDa were isolated for the first time from the human's lymphosarcoma tissue cells using gel filtration and ion exchange chromatography. These hemoproteins possess NADPH dependent O(2)(-)-producing and ferrihemoglobin-reducing activities. The incubation of neuropeptide PRP-1 (5 µg) with cytochrome b(558), caused elevation of these activities. The gel filtration results indicated possible binding of PRP-1 to these cytochromes b(558). PRP-1 activated both NADPH dependent O(2)(-)-producing and ferriHb-reducing activities of the cyt b(1)(558) and cyt b(2)(558), obtained from human lymphosarcoma tissue cells. One can assume that PRP-1 associated with cyt b(558) on the surface of the tumor cells by increasing both NADPH dependent O(2)(-)-producing and ferriHb-reducing activities of cyt b(558), increases the oxidation- reduction status. Changing the oxidation-reduction status and oxygen homeostasis of the tumor cells by PRP-1 can serve as one of the possible explanation of antitumorigenic effect of this cytokine.

Vitamin B-complex initiates growth and development of human embryonic brain cells in vitro.

We studied a combined effect of subcomponents of vitamin B complex on the growth, development, and death of human embryonic brain-derived cells (E90) cultured using a modified method of Matson. Cell death was detected by trypan blue staining. According to our results, vitamin B-complex in low-doses promote the development, maturation, and enlargement of human embryonic brain cells, on the one hand, and increases the percent of cell death, which attests to accelerated maturation and metabolism, on the other.

Antioxidant and electron donating function of hypothalamic polypeptides: galarmin and Gx-NH2.

Chemical mechanisms of antioxidant and electron donating function of the hypothalamic proline-rich polypeptides have been clarified on the molecular level. The antioxidant-chelating property of Galarmin and Gx-NH(2) was established by their capability to inhibit copper(II) dichloride catalyzed H(2)O(2) decomposition, thus preventing formation of HO(*) and HOO(*) radicals. The antiradical activity of Galarmin and Gx-NH(2) was determined by their ability to react with 2,2-diphenyl-1-picrylhydrazyl radical applying differential pulse voltammetry and UV-Vis spectrophotometry methods. Galarmin manifest antiradical activity towards 2,2-diphenyl-1-picrylhydrazyl radical, depending on the existence of phenolic OH group in tyrosine residue at the end of the molecule. The presence of antiradical activity and reduction properties of Galarmin are confirmed by the existence of an oxidation specific peak in voltammograms made by differential pulse voltammetry at E ( composite function) = 0.795 V vs. Ag/Ag(+) aq.

Proline-rich polypeptide-1 protects the cells in vitro from genotoxic effects of mitomycin C.

A new proline-rich polypeptide (PRP-1) has been earlier shown to possess a broad spectrum of biological activities and seems to be a potential medicine. The potential genotoxic properties of PRP-1 and protective effect of PRP-1 against genotoxic action of Mitomycin C (MMC) were analyzed in details in the present work. DNA and chromosome damages were studied in KCL-22 cell line of human myeloid leukemia by the Comet assay and micronucleus induction test, respectively. The results suggest that DNA damages are, at least partly, transient and reparable. PRP-1 at the doses 0.5-2.0 microg/ml does not possess genotoxic activity. Moreover, this peptide expresses both preventive and therapeutic effects against MMC-induced DNA damage. Pre-treatment of cells with PRP-1 also prevents the appearance of daughter cells bearing as heavy MMC-induced DNA/chromosome damages as MNs. Thus, the polypeptide studied is able to protect the cells from genotoxic action of MMC. This defense includes not only DNA but also heritable chromosome damage in post-mitotic cells. Possible mechanisms of PRP-1 protective action are discussed.

Cardioactive protein-hormonal complexes of brain and heart.

New cardioactive protein-hormone complexes (PHC) are identified in magnocellular nuclei of hypothalamus. It was proved that they are specific for nervous tissues and are involved in the regulation of metabolic processes of brain and visceral organs, including the heart. PHC dissociate into high-molecular forms which are new specific glycoproteins and the low-molecular cardioactive neurohormones. Results of our own studies on the functional activities of PHC as well as cardioactive peptides in the precardiac and auricular regions of the heart with respect to the parameters of haemostasis system are reviewed.

Hypothalamic proline-rich polypeptide enhances bone marrow colony-forming cell proliferation and stromal progenitor cell differentiation.

The AGAPEPAEPAQPGVY proline-rich peptide (PRP-1) was isolated from neurosecretory granules of the bovine neurohypophysis; it is produced by N. supraopticus and N. paraventricularis. It has been shown that PRP-1 has many potentially beneficial biological effects, including immunoregulatory, hematopoietic, antimicrobial, and antineurodegenerative properties. Here we showed that PRP increased colony-forming cell (CFC) proliferation in rat bone marrow (BM) cells in vivo. In PRP-treated rat BM, the CFU number at day 7 and day 14 was considerably increased in comparison with untreated rat BM and no difference was found at day 21 and day 28. The related peptide [arg8]vasopressin did not reveal CFC proliferation. PRP failed to farther increase CFC proliferation in vitro in BM obtained from PRP-treated or untreated rats. After 3-4 days of human BM stromal cell cultivation in the presence of 2-20 microg/ml PRP the appearance of cells expressing CD15, CD10, CD11a, CD11b, CD3, CD4, and CD16 surface antigens did not differ from the untreated cells. PRP increased the appearance of CD14-positive cells upon 3-4-day incubation with both adult and fetal BM stromal cells. Our results suggest a previously undescribed role for the hypothalamic peptide within neurosecretory hypothalamus-bone marrow humoral axis, because PRP enhances BM colony-forming cell proliferation and stromal cell differentiation.

Treatment and prophylaxis of anthrax by new neurosecretory cytokines.

In 1881, Louis Pasteur described the Bacillus anthracis vaccine, which plays an important role for the treatment and prophylaxis of anthrax. Currently, treatment for anthrax infection involves the use of several different antibiotics, used in combination with vaccines, which possess potential virulence in white mice and guinea pigs. We discovered several new immunomodulators cytokines (polypeptides) produced by the neurosecretory cells of hypothalamus, some of which can be used as drugs for the treatment and prophylaxis of the anthrax. The proline-rich polypeptides, which consist from 10 to 15 amino acids and four proline residues, are of the special interest; one of them (PRP-1), which consist of 15 amino acids and has the following primary structure ALa-GLy-ALa-Pro-GLu-Pro-Ala-GLu-Pro-Ala-GLn-Pro-GLy-Val-Tyr (AGAPEPAEPAQPGVY) possesses antibacterial activity, and a new proline-rich peptide described by Galoyan and called Gx-NH2. Both were tested for treatment against the anthrax bacillus or anthrax strain N55 vaccine in guinea pigs and mice in vivo, and in vitro preparations. The results of experiments show that these hypothalamic neurosecretory cytokines have a strong prophylaxis and therapeutic properties towards animals infected by episodic strain of anthrax and anthrax vaccine N55. The conventional concepts concerning the function of hypothalamic neurosecretion and hypothalamic mechanisms of adaptation have to be reconsidered.

Hypothalamic proline-rich polypeptide protects brain neurons in aluminum neurotoxicosis.

The damaging effect of aluminum ions (Al3+) on the organism is widely investigated in clinics and experiments that indicate its role as a participant in the synthesis of precursors for amyloid proteins and as a potential agent in the ethiology of Alzheimer's disease. It has been shown that Al produces neurotoxic effects. We established that AlCl3 produces degenerative changes in the ultrastructure of Hasserian neurinoma cells in vitro and in L929 fibroblast cells. Proline-rich peptide-1 (PRP-1) isolated from neurosecretory granules of bovine neurohypophysis is a potent antineurodegenerative agent against spinal cord hemisection and crush syndrome-induced neurodegeneration of brain and spinal cord neurons. PRP-1 is one of the neurotrophic brain factors. By electron microscopic study of the rat hippocampus and other tissues, we succeeded in visualizing the epithelioprotectory effect of PRP-1, contributing as a powerful agent in removal of aluminum accumulation in different tissues in experimental aluminum neurotoxicosis.

Protective effect of a new hypothalamic peptide against cobra venom and trauma-induced neuronal injury.

A study of separate and combined actions of cobra venom (CV) and a new hypothalamic proline-rich polypeptide (PRP) isolated from magnocellular cells (NPV and NSO) on intoxication- and trauma-induced neuronal injury (during 3-4 weeks after hemisection with and without PRP treatment) was carried out. The registration of background and evoked impulse activity flow, changes in spinal cord (SC) inter- and motoneurons, responding to flexor, extensor, and mixed nerve stimulation in both acute and chronic experimental neurodegeneration was performed. The facilitating effect of PRP on the abovementioned neurons was revealed. High doses of CV that evoked the neurodegenerative changes demonstrated an inhibitory effect. In this case PRP treatment both before and after intoxication restored electrical neuronal activity to baseline level and higher. These results are evidence of protective action of PRP. The low doses of CV induced a facilitating effect. The combination of CV and PRP displayed an additive facilitating effect; in a number of cases the repeated administration of CV led to decrease of significant PRP effect till baseline level (for example, the inhibition after primary response prior to secondary late discharge). Greater liability of the secondary early and late long-time discharges of poststimulus responses, differently expressed in various neuron types of SC to chemical influences is of interest. PRP-induced inhibition of the paroxysmal activity related with CV action is also very interesting. Morpho-functional experiments with SC injury demonstrated the abolition of difference in the background and evoked SC neuronal activity below the section and on intact symmetric side after daily PRP administration for 3 weeks. PRP hindered the scar formation and activated neuroglia proliferation; it promoted white matter element growth, hampered the degeneration of cellular elements, and protected against tissue stress. Our results favor the combined use of PRP and CV in clinical practice for the treatment of neurodegeneration of toxic and traumatic origin, as well as specific neurodegenerative diseases such as Alzheimer's.

Influence of hypothalamic proline-rich peptide on the level of [14C]glucose utilization during crush syndrome.

The number of publications on the investigation of crush syndrome (CS) pathogenesis at traumatic toxicosis is rather limited. The influence of some pharmacological preparations on the development of CS pathogenesis is not very well clarified. Proline-rich peptide (PRP) is a fragment of a glycopeptide comprising the carboxyterminus of the neurohypophyseal vasopressin-neurophysin precursor isolated from the bovine neurohypophysis neurosecretory granules. The polypeptide possesses stimulating activity on differentiation and proliferation of T-lymphocytes and Interleukin-2 (Il-2) biosynthesis. The experimental model of CS of white rats was induced by 2-h of compression followed by 2, 24, and 48-h of decompression of femoral muscle tissue. The influence of PRP on [14C]glucose utilization was investigated in brain, heart, and kidney tissues. The level of [14C]glucose utilization decreased in brain during compression followed by 2-h and 24-h of decompression, while it increased under the influence of PRP at all decompression periods. The influence of PRP on the myocardium and kidneys differs, depending on its nature and on the periods of decompression.

Immunohistochemical study of immunophilin 1-15 fragment in intact frog brain, and in the brain and spinal cord of intact and spinal cord hemisectioned rats.

Previously by immunohistochemical technique the distribution of immunophilin 1-15 fragment (IphF) isolated from bovine hypothalamus was examined in various tissues (heart, lung), including immune system organs (spleen and thymus) of intact rats. IphF-like immunoreactivity (IphF-LI) was revealed in several cell types: lymphocytes, monocytes, macrophages and mast cells. In the present study the immunohistochemical localization of IphF was examined in intact rat and frog brains. In rat brain several cell groups concentrated particularly in the supraoptic nucleus (SON) of hypothalamus, medulla oblongata (reticular formation, olives, hypoglossal and facial motor nuclei) and cerebellum (lateral cerebellar nucleus) demonstrated IphF-LI. In frog hypothalamus (SON) the same working dilution (1:5000) of IphF-antiserum revealed very strong immunoreactivity. In the paraventricular nucleus (PVN) IphF-LI varicosities were scattered around the immunonegative cells. The second cell groups showing IphF-LI in the frog brain were gliocytes (mainly the astrocytes). Besides, IphF distribution was investigated in rats subjected to hemisection of spinal cord (SC) with and without administration of proline-rich polypeptide (PRP). PRP was isolated from bovine neurohypophysis neurosecretory granules, produced by magnocellular nuclei of hypothalamus. Hemisection of SC led to changes of IphF distribution in the hypothalamus. In PRP treated animals IphF showed no immunoreactivity. PRP is suggested to act as a neurotransmitter and neuroregulator.

Identification of macrophage migration inhibitory factor isoforms in bovine brain.

In the course of the study of the primary structures and molecular mechanisms of action of immunologically active compounds of the nervous system we have isolated from the soluble fraction of total bovine brain two heat-stable proteins. The purification procedure was mainly based on DEAE-Servacel ion-exchange chromatography and reversed-phase HPLC. The proteins were identified by the N-terminal Edman microsequence analysis and database searching as macrophage migration inhibitory factor (MIF). The N-terminal sequences for MIF1 and MIF2 were found to be identical. According to mass spectral analysis, the molecular masses for MIF1 and MIF2 were determined respectively as 12,369.21 and 12,299.7 Da. In addition, we have also isolated a third peptide having the same N-terminal sequence and Mr 9,496.2 that seems to be a proteolytic fragment of MIF. Using p-hydroxyphenylpyruvate as a substrate, we have not revealed tautomerase activity of either MIF1 or MIF2. As both the immunologic and enzymatic activities were reported to be expressed by the oligomeric structure of MIF, we suggest that the present study may give additional information on MIF in terms of structural properties of this protein. A comparatively simple purification procedure is presented that may be widely used for simultaneous isolation in one run of MIF isoforms.

Protection against snake venom-induced neuronal injury by the new hypothalamic neurohormone.

The action of PRP is characterized by the pronounced activation of the background activity (BA) of the brain spinal cord, and the degree of the activity depends on BA initial level. The typical peculiarity of Vipera raddei venom influence is the initial increase in frequency of BA with subsequent depression. A preliminary injection of PRP has a protective effect at subsequent influence of venom. In animals with hemisection the PRP increases the decreased activity of neurons on injury side. Taking into consideration the protective peculiarities of PRP in the relationship to snake venom and the possibility of the latter to stabilize and prolong the action of drugs (in the case of PRP) combined with them, it is supposed that the mentioned use of the combination in clinical practice will be perspective. The data obtained testify the PRP to be a neuroprotector against many toxic compounds formed in organism (glutamate, ceramid, beta-amyloid neurotoxisity, etc.). Investigations in this aspect are still in the process.

Comparison of the protection against neuronal injury by hypothalamic peptides and by dexamethasone.

The comparative study has been carried out on hypothalamic neurohormone (proline-rich polypeptides-PRP) and synthetic glucocorticoid dexamethasone (DEX) protective properties at the systemic (i/m) administration. Both background and evoked electrical activity (on n.ischiadicus stimulation) of single neurons in the lumbo-sacral part (laminae II-VI and VII-VIII by Rexed) and field potentials (FP) of spinal cord were recorded during acute experiments on intact spinal rats, subjected to Vipera Raddei (VR) venom intoxication, and chronic spinal cord trauma (hemisection). The action of PRP was characterized by the pronounced activation of the background activity (BA) with adaptive effect, depending on dose and initial level of BA, by results of the statistical analysis. A high effect is received from comparatively small doses. For comparison it was used strong glucocorticoid DEX, possessing single-directed but less expressed excitative action on investigated spinal cord neurons. The initial increase of BA frequency with subsequent depression was the typical symptom of venom influence. A protective effect of preliminary PRP injection is revealed on the succeeding VR venom influence. Use of PRP and DEX causes the increase of reduced activity of neurons on the injury side of animals with spinal cord hemisection. It provides the possibility of the therapeutic utilization. It was revealed considerably more expressed PRP action on neurodegenerative process connected to spinal cord injury (in comparison with DEX). The influence of hormones was compared in identical conditions of experiments on non-injured (control) and injured sides. Taking into consideration revealed protection characteristic of PRP and also the ability of snake venom to stabilize and to prolong its action combined with these preparations, the assumption is made on prospective use of the specified combination in clinical practice.

Identification of parvalbumin alpha in bovine hypothalamus: a partial primary structure.

In the course of the study of structure-functional properties and molecular mechanisms of neuropeptides and of low molecular weight proteins of the central nervous system we succeeded in isolating from the soluble fraction of bovine hypothalamus a protein having M(r) 11897.3, according to mass spectral analysis. The purification procedure was mainly based on reversed phase HPLC. As the N-terminus of the molecule was found to be blocked, we have subjected it to CNBr degradation. By Edman microsequence analysis of the peptide fragments and by data base searching the isolated substance was identified as parvalbumin alpha (PRVA)-one of the calcium-binding proteins. However, its primary structure was found not to be identical to that of the known PRVAs from other sources. One of the features of PRVA is its stability. Being subjected to an exhausting purification procedure it retains its complete structure. As neuropeptides and low molecular weight proteins are found to be polyfunctional, a central question concerns the biological role of PRVAs in terms of "where and when" they express their action.

Primary structure and biological activity of hemoglobin-related hypothalamic peptides.

Five individual fractions from bovine hypothalamic extract, displaying coronary constrictory activity, were isolated and sequenced. All of them belong to the hemorphin group of hemoglobin-derived peptides. These peptides bind calmodulin and activate calmodulin-dependent enzymes. The relationship of isolated peptides with other members of the hemorphin group is discussed. Several new fragments of hemoglobin alpha- and beta-chains with yet unidentified activity were obtained from the same source. Their amino acid sequences have considerable overlap with the known sequences of hemoglobin fragments isolated from other tissues.

Antiarrhythmic action of the new calcium antagonist [1,2,5-trimethyl-4-phenyl-4-beta-(N-cyanoethyl-N-4'-methoxybenzyl) -ethylamino]piperidine dihydrochloride.

Arrhythmias induced by coronary artery ligation in cats, by CaCl2 and epinephrine in rats, and by ouabain in guinea-pigs were used as experimental models for studying the effects of a new calcium antagonist AR-1 ([1,2,5-trimethyl-4-phenyl-4-beta-(N-cyanoethyl-N-4'-methoxybenzyl) -ethylamino]piperidine, calcium channel blocker and calmodulin antagonist) on ventricular arrhythmias. Coronary ligation caused 90% lethality (ventricular fibrillation) with 12.5 min in untreated control cats, which was prevented by administration of AR-1 (4 mg/kg body weight (b.w.) before or after arrhythmia induction. Pretreatment with AR-1 afforded protection in a dose-related fashion. A dose of 1.5 mg/KG b.w. increased survival to 45%, and all cats dosed with 3 to 5 mg/Kg b.w. survived. CaCl2 (180 mg/Kg b.w., i.v.) induced ventricular fibrillation and 100% lethality. These effects were completely prevented by an anti-arrhythmic dose of AR-1 (3 mg/kg b.w.). Epinephrine-induced ventricular arrhythmias were also prevented by the same dose of AR-1. AR-1 (5 mg/kg b.w.) did not prevent ouabain (0.5 mg/kg b.w.)-induced arrhythmias that caused death within 17 +/- 3.7 min, but displayed protective effects during 67 +/- 7.7 min. The results from these animal studies, in conjunction with previously studies demonstrating coronarodilatory and anti-platelet efficacy of this compound, collectively suggest that AR-1 has a potential to become a useful antianginal and antiarrhythmic therapeutic agent.

The effect of N-terminal fragments of immunophilin on phospholipid composition of rat brain.

Rat brain slices have been incubated in the presence of water-soluble synthetic peptide fragments corresponding to residues 1-9 and 1-15 of the N-terminus of immunophilin and the effects on the phospholipid composition examined. During a 2 h incubation in the presence of 1 nM, 0-1 mu M and 10 mu M concentrations of the peptides there were significant and dose-dependent decreases in the amounts of phosphatidylcholine and phosphatidylethanolamine and increases in the amounts of phosphatidylserine and, to a lesser extent, phosphatidylinositol, cardiolipin and lysophosphatidylcholine. The overall decrease in the neutral phospholipids and increase in the acidic phospholipids tended to counteract any change in the phospholipid composition of the tissue. The results are discussed in terms of the possible effects of immunophilin on modulating phospholipid turnover in brain cell membranes.

Calcium- and calmodulin-independent modulation of calmodulin-sensitive hypothalamic cyclic nucleotide phosphodiesterase activity by the (11-19) fragment of thymosin beta 4.

A fragment (11-19) of thymosin beta 4 was found to stimulate phosphodiesterase activity even in the absence of calcium and calmodulin. Half-maximal enzyme activation occurred with 10 nM peptide, and was further increased by phospholipids such as phosphatidylserine. The mechanism of stimulation is an increase in the Vmax of cAMP degradation without a substantial change in the Km for the substrate. In the presence of calcium ions and calmodulin the peptide was also stimulatory.