The influence of fulvic and ulmic acids from peat, on the spontaneous contractile activity of smooth muscles.

The aim of the presented studies was to evaluate which classes of compounds of peat ingredients could be responsible for the partial agonistic effect of aqueous peat extract on the alpha2 adreno and D2 dopamine receptors of smooth muscles, which we have reported from former investigations. Based on the different solubility of peat ingredients, water-soluble components of fulvic and ulmic acids were separated according to the pH-value and chemical structure of the solvent. The biological activity of these acids was examined in peat baths using smooth muscle fibers of guinea pig stomach. The results demonstrate that the water-soluble components of fulvic and ulmic acids have a partially agonistic effect on the alpha2 adreno and D2 dopamine receptors, but at the same time quite different effects in terms of their influence on the spontaneous contractile activity (SCA) of smooth muscles have to be noted. From these investigations, we can conclude that aqueous peat extract and the water-soluble components of fulvic acid exhibit similar partial agonistic effects on the alpha2 adreno and D2 dopamine receptors. Therefore it is likely that the mentioned effects of the peat extract derive from the fulvic- and ulmic acids only. The water-soluble component of ulmic acid also showed partial agonistic effects on alpha2 adreno and D2 dopamine receptors. In this case, another substance group is involved, which has a faster blocking effect on these receptors, but was barely soluble in water at a normal pH-value (pH 7).     

Effect of Thymol on the spontaneous contractile activity of the smooth muscles

Effects of Thymol on the spontaneous contractile activity (SCA) have been found in in vitro experiments with circular smooth-muscle strips (SMAs) from guinea pig stomach and vena portae. Thymol was found to possess an agonistic effect on the alpha(1)-, alpha(2)- and beta-adrenergic receptors. Its spasmolytic effect is registered at doses higher than 10(-6)M. Thymol in a dose of 10(-4)M inhibits 100% the SCA of the SMAs and reduces the excitatory effect of 10(-5)M ACH to 35%. It is assumed that Thymol has an analgesic effect through its action on the alpha(2)-adrenergic receptors of the nerve cells. By influencing the beta-adrenergic receptors in the adipose cells, it is possible to induce increased synthesis of fatty acids and glycerol, which is a prerequisite for increased heat release.     

The impact of metal-ions on the activity spectrum of aqueous peat extract on the smooth musculature

PROJECT: The results of our recent studies prove the stimulating effect of aqueous peat extract (APE) on the spontaneous contractile activity (SCA) of the smooth musculature. Only substances with a molecular weight of <3,000 Dalton are able to evoke any effect. As we know from the corresponding literature, trace elements as for instance copper, manganese, lead and cadmium do also influence the SCA even when they appear in low concentrations (micromol-range). The purpose of this study therefore is to examine the influence of the trace-elements on the inspected stimulating effects which aqueous peat extract has on the SCA. PROCEDURE: During in-vitro experiments with smooth muscles in organbaths, it has been examined--under application of a standardized method--if trace-elements are the cause for the stimulating effect of aqueous peat extract on the SCA of the smooth musculature. RESULTS: The results have shown that--independent from their concentration within the peat - the trace-elements do not influence the SCA of the smooth muscles. CONCLUSION: The results can be explained by the chelating capacity of the peat-components, that leads to the absorption of the trace-elements. Additionally we can conclude that organic substances are the exclusive reason for the described effects.     

Synthesis and biological activity of new series of N-modified analogues of the N/OFQ(1–13)NH2 with aminophosphonate moiety

New series of N-modified analogues of the N/OFQ(1-13)NH(2) with aminophosphonate moiety have been synthesized and investigated for biological activity. These peptides were prepared by solid-phase peptide synthesis-Fmoc-strategy. The N/OFQ(1-13)NH(2) analogues were tested for agonistic activity in vitro on electrically stimulated rat vas deferens smooth-muscle preparations isolated from Wistar albino rats. Our study has shown that the selectivity of the peptides containing 1-[(methoxyphosphono)methylamino]cycloalkanecarboxylic acids to the N-side of Phe is not changed-they remain selective agonists of NOP receptors. The derivative with the largest ring (NOC-6) demonstrated efficacy similar to that of N/OFQ(1-13)NH(2), but in a 10-fold higher concentration. The agonistic activity of newly synthesized N-modified analogues of N/OFQ(1-13)NH(2) with aminophosphonate moiety was investigated for the first time.     

The influence of the initial humidity of balneological peat on its pharmacological features]

The viscosity of balneological peat got special meaning according to its thermical features. Until now, there has been no answer on the question in how far balneological peat, which is prepared with different degrees of humidity, can vary the pharmacological character. Aim of this work was, on the one hand, to examine which initial degree of peat-humidity is necessary to get the wished viscosity in therapy when diluted with water. On the other hand, how far the pharmacological character of balneological is influenced by the initial degree of humidity. From peat, with initial different degrees of humidity, balneological peat is prepared, fulfilling the demands of the Quentin-Test and therefore the necessary consistence for therapy. Here in this work the in-vitro effect of aqueous peat extract (APE) on the spontaneous contractile activity of the smooth muscle fibres of guinea-pig stomachs was examined. The APE used got initial different degrees of humidity. It was found out, that the ability of peat according to a new water-uptake, with a initial humidity under 60%, was clearly reduced. Because of the reduced water content of balneological peat its thermical features were deteriorated. This goes for peat with a humidity factor < 60%. First, with the Quentin-Test, showing the optimal viscosity of peat before medical use, you can also make statements according its ability of maximum uptake of water, meaning the ability taking up water until it becomes gel-like. Second, the results show that the initial humidity of peat got no influence on the pharmacological features.     

Circular dichroism studies on lipid-protein complexes of a hydrophobic myelin protein

Circular dichroism spectra of lipophilin (a hydrophobic protein purified from human central nervous system myelin) were analyzed by the method of Chen et al. (1974) to obtain information on its secondary structure in aqueous and lipid environments. When introduced into phosphatidylcholine vesicles by dialysis from 2-chloroethanol, the protein possessed about 75% alpha helix. A new water-soluble form of lipophilin also containing over 70% alpha helix was obtained by a similar dialysis in the absence of lipid. This product had a higher helical content than two other water-soluble preparations derived by dialysis from phenol-acetic acid-urea. Interaction of all three aqueous forms of the protein with lysolecithin micelles resulted in increases in total helical content or in the average length of helical segments. The amount of beta sheet was at a minimum for lipophilin incorporated into vesicles, where the presence of lipid also provided some protection against thermal denaturation.     

Synthesis and biological activity of novel small peptides with aminophosphonates moiety as NOP receptor ligands

The aim of the present study was the synthesis and the biological screening of new analogs of Ac-RYYRWK-NH₂, modified at the N-terminal with 1-[(methoxyphosphono)methylamino]cycloalkanecarboxylic acids. The four newly synthesized ligands for the nociceptin/orphanin FQ (N/OFQ) receptor (NOP) have been prepared by solid-phase peptide synthesis—Fmoc-strategy. These compounds were tested for agonistic activity in vitro on electrically stimulated smooth-muscle preparations isolated from vas deferens of Wistar rats. Our data showed that substitution of Arg at position 1 with aminophosphonates moiety decreased significantly the affinity of ligands to the NOP receptor. Furthermore, the enlargement of the cycle (with 5–8 carbon atoms) additionally diminished both the activity and the selectivity for NOP-receptor.     

Modulation of medical prefrontal cortical D1 receptors on the excitatory firing activity of nucleus accumbens neurons elicited by (-)-Stepholidine

(-)-Stepholidine (SPD), with D1 agonistic action, elicited an excitatory firing activity of nucleus accumbens (NAc) neurons by intravenous administration, but this effect was hardly observed by iontophoresis of SPD into the NAc. The present study intends to determine whether D1 receptors in the medial prefrontal cortex (mPFC) are involved in the action of SPD on the firing activity of NAc neurons in the chloral hydrate-anesthetized male rats. The results showed that the intra-mPFC microinjected SCH-23390 (D1 antagonist, 30 mM), but not the D2 antagonist spiperone (30 mM), significantly attenuated the enhanced firing activity induced by intravenous injection of SPD (2 mg/kg). Similarly, the excitatory firing of NAc neurons was also exhibited by the microinjection of either SPD or D1 agonist SKF-38393 into the mPFC. The SPD-induced excitatory effect was in a dose-dependent way from 277.8 +/- 51.3% (10 mM) to 1105.4 +/- 283.5% (30 mM) of NAc basal firing, which was completely reversed by SCH-23390 (i.v.). Furthermore, the direct D1 agonistic action of SPD on the mPFC neuron was observed with microiontophoresis. These results indicate that SPD possesses a direct agonistic action on the mPFC D1 receptors, by which it modulates the firing activity of NAc neurons.     

Adenylate kinase and GDP-kinase activity of rod outer segments in the frog retina. Possible functional role of the T beta subunit of transducin

Pure frog retina rod outer segments (ROS) preparations (A280/A500 = 2,1-2,3) catalyze the synthesis of ATP from ADP in the presence of Mg2+. Adenylate kinase (AK) (ATP:AMP phosphotransferase, EC 2.7.4.3) specific activities for ROS preparations are within the range 2-4 mumole per hour for mg protein. The enzymatic activity of investigated preparations is due to intact, but not destroyed ROS. The component which possesses AK is found in water-soluble, but not in membranous ROS fractions and seems to be a part of the predominant ROS plasma protein--GTP-binding complex of transducin. It has been shown, that this component is the T beta subunit of transducin and its enzymatic activity is controlled by other subunits of the transducin complex. The obtained results indicate that GDP kinase (ATP:GDP phosphotransferase, EC 2.7.4.6) activity of transducin depends on the work of both of T beta and T alpha subunits. It has been shown that in the ROS preparations synthesis of the ATP from ADP and GDP phosphorylation are stimulated by a lowering of Ca2+ concentration (less than 10(-5)-10(-7) M). T beta component is suggested to play the role of phosphotransferase which phosphorylates GDP associated with the T alpha subunits and it leads to formation of a complex T alpha X GTP which is an activator of vertebrate retina ROS phosphodiesterase.     

A process to prepare a synthetic filter material containing nutrients for biofiltration

In this study, an optimal process to prepare a synthetic filter material (poly(vinyl alcohol) (PVA)/peat/KNO(3) composite bead) containing nutrients was developed for biofiltration. The optimal preparing condition was that each of the peat and PVA aqueous solutions contains 6.4 g KNO(3) and the nitrogen content in the boric and phosphate aqueous solutions must retain higher than 3.94 and 1.52 g N/l, respectively. The equilibrium amount of water-soluble nitrogen dissolved out of the prepared composite bead was between 7.95 and 8.21mg N/g dry solid. The path of water-soluble nitrogen dissolving out of the A-type bead was the water-soluble nitrogen dispersed in the peat phase initially diffused into the outer PVA phase and then it diffused out of the bead surface. And the path of water-soluble nitrogen dissolving out of the H-type bead was the water-soluble nitrogen dispersed in both the peat and PVA phases simultaneously diffused into the outer PVA phase and out of the bead surface, respectively. The microbial growth rate k(g) of the H-type composite bead was higher than that of the A-type composite bead approximately 1.09-1.58 times, and its value was between 0.100 and 0.417 day(-1) as the composite bead was immersed in 0-0.896 M KNO(3) solution. The maximum value of k(g) appeared at the composite bead immersed in 0.384 M KNO(3) solution and was higher than that of the compost by a factor approximately 1.49. The percentage of removed volatile organic compounds (VOCs) remained at more than 98% during the biofilter operating 230 days as the composite bead was immersed in KNO(3) aqueous solution before packing. This composite bed was without the further addition of nutrients during this operating period. It was proved that this composite bead was superior to the compost as a filter material.     

The complete chirospectroscopic signature of the peptide 3(10)-helix in aqueous solution

We synthesized by solution methods a water-soluble, terminally blocked heptapeptide based on five markedly helicogenic, C(alpha)-tetrasubstituted alpha-amino acids C(alpha)-methyl-L-norvalines and two strongly hydrophilic 2-amino-3-[1-(1,4,7-triazacyclononane)]-L-propanoic acid residues at positions 2 and 5. A Fourier transform infrared absorption and NMR analysis in deuterated chloroform and aqueous solutions of the heptapeptide and two side-chain protected synthetic precursors confirmed our working hypothesis that all oligomers are folded in the 3(10)-helical conformation. Based on these findings, we exploited this heptapeptide as a chiral reference compound for detailed electronic CD, vibrational CD, and Raman optical activity characterizations of the 3(10)-helix in aqueous solution.     

Prostacyclin synthesis elicited by cholinergic agonists is linked to activation of M2 alpha and M2 beta muscarinic receptors in the rabbit aorta

This study was conducted to investigate the subtypes of muscarinic receptors involved in the action of cholinergic agents on prostacyclin synthesis in the rabbit aorta. Prostacyclin production measured as 6-keto-PGF1 alpha was assessed after exposing the aortic rings to different cholinergic agents. Acetylcholine (ACh) (M1 and M2 agonist) (1-10 microM) and arecaidine proparagyl ester (APE) (M2 selective agonist) (1-10 microM) enhanced 6-keto-PGF1 alpha output in a concentration-dependent manner. A selective M1 receptor agonist, McN-A-343, at 1 microM-1 mM did not alter 6-keto-PGF1 alpha output. ACh- and APE induced increases in 6-keto-PGF1 alpha output were attenuated by the M1/M2 antagonist atropine (0.1 microM), M2 alpha antagonist (AF-DX 116), (0.1-1.0 microM), and by selective M2 beta antagonist, hexahydro-sila-difendiol (HHSiD) (0.1-1.0 microM), but not by the M1 antagonist pirenzepine (1.0 microM). 6-Keto-PGF1 alpha output elicited by ACh- or APE was not altered by the adrenergic receptor antagonists phentolamine and propranolol or by the nicotinic receptor blocker hexamethonium. Similarly, the arachidonic acid- or norepinephrine induced 6-keto-PGF1 alpha accumulation was not altered by these muscarinic receptor antagonists. Indomethacin, a cyclooxygenase inhibitor, prevented arachidonic acid, ACh- or APE induced 6-keto-PGF1 alpha output. Removal of the endothelium abolished the production of 6-keto-PGF1 alpha elicited by ACh, APE, bradykinin, and calcium ionophore A 23187, but not that induced by angiotensin II, K+ or norepinephrine. These data suggest that vascular prostaglandin generation elicited by cholinergic agonists is mediated via activation of M2 alpha and M2 beta but not M1 muscarinic receptors, which are most likely located on the endothelium.     

Bcl2 inhibits abasic site repair by down-regulating APE1 endonuclease activity

Bcl2 not only prolongs cell survival but also suppresses the repair of abasic (AP) sites of DNA lesions. Apurinic/apyrimidinic endonuclease 1 (APE1) plays a central role in the repair of AP sites via the base excision repair pathway. Here we found that Bcl2 down-regulates APE1 endonuclease activity in association with inhibition of AP site repair. Exposure of cells to nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone results in accumulation of Bcl2 in the nucleus and interaction with APE1, which requires all of the BH domains of Bcl2. Deletion of any of the BH domains from Bcl2 abrogates the ability of Bcl2 to interact with APE1 as well as the inhibitory effects of Bcl2 on APE1 activity and AP site repair. Overexpression of Bcl2 in cells reduces formation of the APE1.XRCC1 complex, and purified Bcl2 protein directly disrupts the APE1.XRCC1 complex with suppression of APE1 endonuclease activity in vitro. Importantly, specific knockdown of endogenous Bcl2 by RNA interference enhances APE1 endonuclease activity with accelerated AP site repair. Thus, Bcl2 inhibition of AP site repair may occur in a novel mechanism by down-regulating APE1 endonuclease activity, which may promote genetic instability and tumorigenesis.     

Agonistic and antagonistic activity of glutamate analogs on neuromuscular excitation in the walking limbs of lobsters.

Forty-six analogs of L-glutamate were tested for activity on muscle fibers in the walking limbs of lobsters. Effects on the membrane potential, input resistance, and amplitude of neurally evoked EPSPs and IPSPs were studied as well as effects on applied L-glutamate. Seventeen of the compounds studied depolarized the muscle fibers in a manner indicative of an agonistic action on receptors in the neuromuscular excitatory membrane. Six analogs selectively reduced the amplitude of evoked EPSPs, and at least three of these (kainic acid, D-glutamate, and D-aspartate) antagonized the excitatory action of applied L-glutamate. Kainic acid was the most potent of the blockers of neuromuscular excitation, but even it was relatively weak since a concentration of 1 mM was required for an apparent effect. Generally those analogs in the L-configuration which possessed activity, had agonistic actions, whereas those in the D-configuration were usually antagonistic. These observations provide pharmacological evidence for the concept that L-glutamate is the transmitter agent which mediates neuromuscular excitation in the walking limbs of lobsters. In addition, our results are consistent with recent studies which indicate that L-aspartate may also function in this neuromuscular excitatory process.     

Pigeon oesophageal EMG activity: analysis of intramural neural control

The effects of agonist and antagonist cholinergic and adrenergic drugs on spontaneous electrical activity of transverse muscular strips of pigeon cervical oesophagus were examined. Tetrodotoxin failed to affect EMG activity. Cholinomimetics produced excitatory effects. The response to carbachol was enhanced by hexamethonium and reversed into an inhibitory effect by atropine. Noradrenaline evoked a concentration-dependent, biphasic effect (inhibition at low and excitation at high concentrations). Isoproterenol induced inhibitory response unaffected by tetrodotoxin. Phenylephrine induced excitatory response completely antagonized by tetrodotoxin and partially opposed by atropine. It is concluded that: i) the oesophageal spontaneous EMG activity is myogenic; ii) the intramural neurons have no tonic influence on the spontaneous EMG activity; iii) in the intramural plexuses there are cholinergic excitatory-, non-cholinergic excitatory- and inhibitory neurons, with unknown neurotransmitter; iv) excitatory alpha-adrenoceptors, located on the nervous elements and inhibitory beta-adrenoceptors, located on the smooth-muscle cells, are present.     

Vascular smooth muscle influences the release of endothelium-derived relaxing factor

Conditioned medium was collected from vascular smooth-muscle cells grown in culture to determine if these cells synthesize vasoactive substances. The medium caused a short-acting endothelium-independent constriction of rat aorta, followed by a prolonged, endothelium-dependent relaxation. This relaxation was mediated through the release of endothelium-derived relaxing factor (EDRF) as it was abolished by the addition of methylene blue (5 x 10(-6) M), haemoglobin (10(-6) M) or methyl arginine, but was not affected by indomethacin (10(-5) M). Smooth-muscle medium stimulated the production of EDRF from both rat and rabbit thoracic aortic rings as well as from cultured bovine pulmonary artery endothelial cells. The prolonged stimulation of EDRF by smooth-muscle medium was not mimicked by known physiological stimuli to EDRF release; EDRF-stimulating activity was not affected when smooth-muscle cells were grown in the presence of indomethacin (10(-5) M), although serum in the medium was required. The EDRF-stimulating substance(s) in the smooth-muscle medium was heat stable and associated with a high molecular mass (30,000 greater than Mr greater than 3500) water-soluble species that is as yet unidentified.     

Neurons Efficiently Repair Glutamate-induced Oxidative DNA Damage by a Process Involving CREB-mediated Up-regulation of Apurinic Endonuclease 1

Glutamate, the major excitatory neurotransmitter in the brain, activates receptors coupled to membrane depolarization and Ca²⁺ influx that mediates functional responses of neurons including processes such as learning and memory. Here we show that reversible nuclear oxidative DNA damage occurs in cerebral cortical neurons in response to transient glutamate receptor activation using non-toxic physiological levels of glutamate. This DNA damage was prevented by intracellular Ca²⁺ chelation, the mitochondrial superoxide dismutase mimetic MnTMPyP (Mn-5,10,15,20-tetra(4-pyridyl)-21H,23H-porphine chloride tetrakis(methochloride)), and blockade of the permeability transition pore. The repair of glutamate-induced DNA damage was associated with increased DNA repair activity and increased mRNA and protein levels of apurinic endonuclease 1 (APE1). APE1 knockdown induced accumulation of oxidative DNA damage after glutamate treatment, suggesting that APE1 is a key repair protein for glutamate-induced DNA damage. A cAMP-response element-binding protein (CREB) binding sequence is present in the Ape1 gene (encodes APE1 protein) promoter and treatment of neurons with a Ca²⁺/calmodulin-dependent kinase inhibitor (KN-93) blocked the ability of glutamate to induce CREB phosphorylation and APE1 expression. Selective depletion of CREB using RNA interference prevented glutamate-induced up-regulation of APE1. Thus, glutamate receptor stimulation triggers Ca²⁺- and mitochondrial reactive oxygen species-mediated DNA damage that is then rapidly repaired by a mechanism involving Ca²⁺-induced, CREB-mediated APE1 expression. Our findings reveal a previously unknown ability of neurons to efficiently repair oxidative DNA lesions after transient activation of glutamate receptors.     

The mechanism of nutrients dissolved out of a synthetic composite bead filter material in a biofilter

In this study, an optimal process to prepare a synthetic material having nutrient (PVA/peat/KNO₃ composite bead) is developed. The equilibrium water-soluble nitrogen content in the composite bead prepared by this process is 8.25–10.06 mg N/g dry solid. The mass-transport process for the water-soluble nitrogen dissolved out of the composite bead was also investigated. The dissolved out process occurs in two stages: external mass transport occurs in the early stage and the intraparticle diffusion process occurs in the long-term stage. The rate of water-soluble nitrogen dissolved out in both stages is concentration dependent. The path of nitrogen dissolved out is that the nitrogen dispersed in the peat and PVA phases simultaneously diffused into the outer PVA phase and out of the bead surface. The moisture holding capacity of the composite bead bed is better than the compost bed. The percentage of removed volatile organic compounds (VOCs) can remain at levels higher than 99% for a longer time (about 230 d) as the composite bead immersed in a KNO₃ aqueous solution before packing with an optimal concentration of KNO₃ aqueous solution of 0.384 M. The rate of nitrogen dissolved out in the intraparticle diffusion process could be used as an index to predict the microbial growth rate in the biofilter.     

Agonistic and antagonistic activity of glutamate analogs on neuromuscular excitation in the walking limbs of lobsters

Forty-six analogs of L -glutamate were tested for activity on muscle fibers in the walking limbs of lobsters. Effects on the membrane potential, input resistance, and amplitude of neurally evoked EPSPs and IPSPs were studied as well as effects on applied L -glutamate. Seventeen of the compounds studied depolarized the muscle fibers in a manner indicative of an agonistic action on receptors in the neuromuscular excitatory membrane. Six analogs selectively reduced the amplitude of evoked EPSPs, and at least three of these (kainic acid, D -glutamate, and D -aspartate) antagonized the excitatory action of applied L -glutamate. Kainic acid was the most potent of the blockers of neuromuscular excitation, but even it was relatively weak since a concentration of 1 mM was required for an apparent effect. Generally those analogs in the l-configuration which possessed activity, had agonistic actions, whereas those in the d-configuration were usually antagonistic. These observations provide pharmacological evidence for the concept that L -glutamate is the transmitter agent which mediates neuromuscular excitation in the walking limbs of lobsters. In addition, our results are consistent with recent studies which indicate that l-aspartate may also function in this neuromuscular excitatory process.     

The preparation and partial characterization of antigenic fractions obtained from the mycelial walls of several Aspergillus species

Extracts with immunological activity were prepared from Aspergillus fumigatus, A. flavus, A. terreus, A. niger and A. nidulans. In each case crude mycelial wall was extracted with an aqueous solution of Triton X-100 giving detergent-soluble material. Further fractionation was achieved by removing the detergent from this solution; the resultant precipitate was removed by centrifugation, and the aqueous supernatant was used as a source of soluble antigens. The sensitivity of these preparations was compared with that of water-soluble antigenic material, prepared from whole macerated mycelium, by double diffusion and counterimmunoelectrophoresis using homologous antisera and sera from patients suffering from aspergilloma and allergic bronchopulmonary aspergillosis. The selectivity of these antigenic preparations was monitored with heterologous antisera raised in rabbits. Batch variability was analysed for one strain of A. fumigatus using chemical and immunological methods. The nature of the antigenic sites involved in these reactions was investigated by studying the susceptibility of the preparations to proteolytic hydrolysis, periodate oxidation and concanavalin A treatment. The total protein and carbohydrate content of each fraction was determined and the constituent sugars analysed in an attempt to correlate chemical composition with antigenic activity.