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PubMed2002

Identification of Streptomyces odor spectrum.

The chemical composition of odors produced by nine strains of Streptomyces was determined. Strains Streptomyces aureofaciens, S. avermitilis, S. cinamomensis, S. coelicolor, S. griseus, S. lividans, S. rimosus, S. spectabilis, S. virginiae (as representatives of producers of biologically active compounds) were cultivated at the same time statically in dishes and in shaken flasks at similar cultivation conditions. According to the GC-MS analysis of odor compounds, more than twenty noteworthy volatile chemical individuals were identified. As the main component of odor spectrum geosmin and homologues of oxolones (dihydrofuranones) were found; the other compounds (pyrazine derivatives, acetoin and its homologues, aromatic esters, furan derivatives, etc.) were in minority.

Furans↗

PubMed1997

Special transformation processes using fungal spores and immobilized cells.

Although many microbial processes have been described which are able to produce interesting aroma compounds, the number of industrial applications are limited. Reasons for this are in most cases low final product yield, low biotransformation rates, substrates and/or end-products inhibition, toxicity towards the microorganisms themselves and difficulties of recovery from the bioreaction mixture. This means that the development of specific catalysts and processes is an important challenge for researchers in this field. This review presents two special kinds of catalysts, fungal spores and immobilized cells, with emphasis on their production and on their use in the production of aroma compounds. The production of fungal spores by solid state fermentation is described in greater detail. In the second part, this review also offers examples of development of three production processes, the production of methyl ketones of spores of Penicillium roquefortii, the hydroxylation of beta-ionone by immobilized Aspergillus niger cells, and the production of alkyl pyrazines by bacteria in liquid and solid media. For each of these processes, the analysis of limiting steps-biological and/or physico-chemical-is presented and the significant role of process conditions to increase aroma yield is discussed.

Alkylation↗

PubMed1997

In vitro and in vivo effects of SCA40 on guinea pig airways.

SCA40 (6-bromo-8-methylaminoimidazo [1,2-a]-pyrazine-2-carbonitrile), a compound which had been described as an opener of Ca(2+)-dependent large conductance potassium channels (BKCa channels), was investigated in comparison with salbutamol for in vitro and in vivo bronchospasmolytic effects and for the ability to reverse airways hyperreactivity in guinea pigs. SCA40 reduced the spontaneous tone of isolated guinea pig tracheal rings with a biphasic concentration-response curve (first phase: pD2 = 8.0, EMax = 29.7% of maximal effect; second phase: pD2 = 6.4, EMax = 72.6%). The salbutamol curve was monophasic (pD2 = 8.0, EMax = 100%). Total lung resistance (RL) was determined in anaesthetized, ventilated guinea pigs. Bronchoconstriction, measured as an increase in RL, was elicited in normoreactive animals by i.v. infusion of bombesin (100 ng/kg/min) or by i.v. injection of histamine (1.8-5.6 micrograms/kg). Airways hyperreactivity was induced by acute i.v. administration of pre-formed immune complexes. Intravenous bolus injections of histamine (2.4 micrograms/kg) were used to define the sensitivity of the airways prior to and after the exposure to immune complex. Following intratracheal (i.t.) administration, SCA40 reversed bombesin-induced bronchoconstriction with an ED50 of 43 micrograms/kg (EMax = 57%). The ED50 for salbutamol was 0.8 microgram/kg i.t. (EMax = 78%). Histamine-induced bronchoconstriction in hyperreactive guinea pigs was inhibited by SCA40 with an ED50 of 13 micrograms/ kg i.t. (EMax = 82%). Salbutamol completely inhibited histamine-induced bronchospasm with an ED50 of 9 ng/kg i.t. In normoreactive guinea pigs, SCA40 prevented histamine-induced bronchoconstriction with an ED50 of 100 micrograms/kg i.t.; for salbutamol the ED50 in this test was 0.48 microgram/kg i.t. Thus, for both SCA40 and salbutamol, the effects obtained at low doses in hyperreactive guinea pigs represent a true reversal of airways hyperreactivity, whereas at higher doses, anti-hyperreactive and bronchospasmolytic properties may account for the observed effects. In conclusion, SCA40 relaxes guinea pig airways smooth muscle in vitro and in vivo, and it partly reverses airways hyperreactivity. With respect to both potency and efficacy, SCA40 is markedly less active than the beta-adrenoceptor agonist salbutamol.

Albuterol↗

PubMed1997

Bronchodilator and anti-inflammatory activities of SCA40: studies in human isolated bronchus, human eosinophils, and in the guinea-pig in vivo.

There is currently interest in the use of inhibitors of cyclic nucleotide phosphodiesterases (PDE) as potential anti-asthma agents. In this study we examined the effects of SCA40 (6-bromo-8-methylaminoimidazol-[1,2-a] pyrazine-2-carbonitrile), a preferential inhibitor of PDE 3 also endowed with PDE 4 and 5 inhibitory activities, on isolated bronchus and eosinophil functions and in an animal model of asthma. SCA40 (1 nM-0.1 mM) produced concentration-dependent inhibition of spontaneous and stimulated tone of human isolated bronchus and reached a maximal relaxation similar to that of theophylline (3 mM). The potency (-log EC50 values) of SCA40 against spontaneous tone (6.52 +/- 0.10) was greater than against tone raised by equieffective concentrations (approximately 70%) of histamine (5.76 +/- 0.06), leukotriene C4 (5.44 +/- 0.11), and acetylcholine (4.98 +/- 0.09). In the presence of cytochalasin B, the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP; 0.5 microM) induced leukotriene C4 production in human eosinophils isolated in discontinuous metrizamide gradients. The production of leukotriene C4 was inhibited by SCA40 in a concentration-related fashion (-log IC50 = 6.04 +/- 0.20; n = 6). Rolipram, a selective PDE 4 inhibitor, was also effective (-log IC50 = 7.29 +/- 0.32) but the selective PDE 3 inhibitor SKF94120 was scarcely effective (< 10% inhibition for 10 microM). In ovalbumin sensitized guinea-pigs, SCA40 (1 mg kg(-1), i.p.) given 30 min before antigen challenge significantly inhibited the acute bronchoconstriction produced by aerosol antigen (5 mg ml(-1), 30 s) (antigen response was 185 +/- 13 and 91 +/- 21 cmH2O l(-1) s(-1) in control and SCA40-treated animals, respectively, P < 0.05). Pretreatment with SCA40 (1 mg kg(-1), i.p., 30 min pre- and 3 h post-antigen exposure) prevented airway hyperreactivity to histamine which developed 24 h after exposure of conscious guinea-pigs to aerosol antigen. Eosinophil lung accumulation that accompanied airway hyperreactivity was also inhibited by SCA40 (from 6.15 +/- 0.86 in control to 1.27 +/- 0.27 in treated animals; expressed as eosinophils x 10(6); P < 0.05). SCA40 (1 mg kg(-1), i.p.) also inhibited the microvascular leakage produced after inhaled antigen (5 mg ml(-1), 30 s) at all airway levels. The haemodynamic effects of SCA40 (1 mg kg(-1), i.p.) consisted of a rapid decrease (peak at 5 min) in mean arterial blood pressure (-39.4 +/- 2.4%) and tracheal mucosal blood flow (-13.5 +/- 2.0%) that slowly recovered with time. These data support previous work showing that PDE inhibition results in antispasmogenic and anti-inflammatory effects. SCA40 was effective in vitro and in vivo and these effects are probably related to its activity as a mixed PDE inhibitor.

Animals↗

PubMed2004

Attenuation of nicotine's discriminative stimulus effects in rats and its locomotor activity effects in mice by serotonergic 5-HT2A/2C receptor agonists.

RATIONALE: Reports have indicated that administration of nicotine inhibits, while withdrawal of chronically administered nicotine augments effects of serotonergic 5HT2A/2C agonists. OBJECTIVE: It was our objective to determine whether 5HT2A/2C agonists can modulate the discriminative stimulus effects of nicotine in rats or its locomotor activity effects in mice. METHODS: Adult male Sprague-Dawley rats were trained to discriminate 0.3 mg/kg nicotine base from saline in a two-lever, fixed-ratio (FR10), food-reinforced, operant-conditioning task during daily (Monday-Friday) 15-min experimental sessions. After characterizing a dose-response curve for nicotine, we tested the ability of the 5HT(2A/2C) agonists (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCL (DOI; 0.18-1.0 mg/kg) and 1-(4-bromo-2, 5-dimethoxyphenyl)-2-aminopropane (DOB; 0.1-1.0 mg/kg), the 5HT2C agonist 6-chloro-2-(1-piperazinyl)pyrazine hydrochloride (MK 212; 0.1 mg/kg-1.0 mg/kg), and the 5HT1A agonist (+/-)-8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide (8-OH-DPAT; 0.01 mg/kg-1.0 mg/kg) to modulate nicotine's discriminative stimulus effects. After finding that DOI was able to attenuate the percentage nicotine lever responding (%NLR), we tested for it to also reverse nicotine's effects on locomotor activity in mice. RESULTS: The 5HT2A/2C agonists-in particular DOI-dose dependently attenuated %NLR. The effects of DOI were reversed by the 5HT2A/2C antagonist ketanserin. MK 212 and 8-OH-DPAT had irregular effects among rats and only reduced %NLR to below 50% levels at doses markedly suppressing responding. DOI also dose dependently blocked nicotine's acute rate-lowering locomotor activity effects. CONCLUSIONS: These results indicate that activation of serotonin 5HT2A/2C receptors can blunt the discriminative stimulus and locomotor activity effects of nicotine and presents the possibility that activation of these receptors might also be able to attenuate other effects of nicotine.

8-Hydroxy-2-(di-n-propylamino)tetralin↗

PubMed2005

Behavioral effects of systemically administered MK-212 are prevented by ritanserin microinfusion into the basolateral amygdala of rats exposed to the elevated plus-maze.

RATIONALE: Although 5-HT2 receptors seem to play an important role in anxiety, results from numerous studies are still highly variable. Moreover, little is known about the behavioral effects of centrally administered 5-HT2 compounds in animal models of anxiety. OBJECTIVE: The current study was performed to: (1) further investigate the effects of 5-HT2 receptor activation in rats exposed to the elevated plus-maze (EPM) and the open-field arena, two widely used animal models for studying anxiety and locomotor activity; and (2) evaluate the involvement of the 5-HT2 receptors within the basolateral nucleus of the amygdala (BLA) in the modulation of such effects. METHODS: In the first experiment, male Wistar rats were exposed for 5 min to the EPM 27 min following intraperitoneal (i.p.) (1.0 ml/kg) injections of the preferential 5-HT2C receptor agonist 6-chloro-2[1-piperazinyl]pyrazine (MK-212) at doses of 1.0, 2.0, or 4.0 mg/kg. Control animals were injected with saline. The percentage of open-arm entries and the percentage of time spent in these arms were employed as anxiety indexes, whereas the number of closed-arm entries was calculated as indicative of locomotor activity. In the second experiment, rats were exposed for 10 min in an open-field arena to further assess the interference of the same MK-212 doses upon locomotor activity. In Experiment 3, rats were microinjected (0.2 microl) either with the mixed 5-HT 2A/2C receptor antagonist ritanserin (0.5, 1.25, 2.5, and 5.0 microg) or its vehicle into the BLA 12 min following i.p. injections of saline or the intermediate dose of MK-212 (2.0 mg/kg). Fifteen minutes later, each animal was exposed to the EPM as before. RESULTS: Whereas the highest dose of MK-212 (4.0 mg/kg) induced motor-suppressant effects in both EPM and open-field arena, the intermediate dose of the drug (2.0 mg/kg) reduced open-arm exploration without significantly affecting the number of closed-arm entries. This behavioral profile, consistent with selective anxiogenic effect in the EPM, was dose-dependently prevented by ritanserin microinfusion into the BLA. In saline-pretreated animals, however, ritanserin (all doses) was ineffective. CONCLUSIONS: MK-212 increases anxiety and decreases locomotor activity. The anxiogenic-like profile of 5-HT2 receptor activation is prevented by the blockade of 5-HT2 receptors within the BLA, which does not have an effect by itself upon basal anxiety levels triggered by the EPM.

Amygdala↗

PubMed1999

Serotonin(2C) receptors appear to mediate genetic sensitivity to cocaine-induced convulsions.

RATIONALE: C57BL/6ByJ (6ByJ) and C57BL/6J (6J) mice differ in their sensitivity to cocaine-induced convulsions, with CD(50) values being 100 and 70 mg/kg, respectively. This genetic sensitivity to cocaine-induced convulsions is probably related to 5-HT(2) receptors, since the density of these sites and the concentration of 5-HT(2) antagonists required to block cocaine-induced convulsions is lower in 6J mice relative to 6ByJ mice. OBJECTIVE: Although 5-HT(2) receptors appear to play a role in mediating genetic sensitivity to cocaine-induced convulsions, the role of 5-HT(2) receptor subtypes in this effect of cocaine has not been examined. METHODS: The present study compared the effects of the preferential 5-HT(2C) agonists m-chlorophenylpiperazine (mCPP) and 6-chloro-2-(1-piperazinyl)pyrazine (MK212) on cocaine-induced convulsions in 6ByJ and 6J mice. General activity was also measured following pretreatment with mCPP and MK212. RESULTS: Both mCPP and MK212 potentiated cocaine-induced convulsions and the effect of these agonists was more robust in 6ByJ mice relative to 6J mice. CONCLUSION: The findings from this study support previous research suggesting that 5-HT(2) receptors play a role in mediating cocaine-induced convulsions, and extend previous research by suggesting that the 5-HT(2C) receptor subtype mediates cocaine-induced convulsions and genetic sensitivity to this toxic effect of cocaine.

Animals↗

PubMed2001

Detection and cytotoxicity of cisplatin-induced superoxide anion in monolayer cultures of a human ovarian cancer cell line.

Superoxide anions (O2-) generated by cisplatin [cis-diamminedichloroplatinum (II), DDP] were determined by measuring the chemiluminescence from the luminescence probe, 2-methyl-6-[p-methoxyphenyl]-3,7-dihydroimidazo[1,2-a]pyrazin-3-one (methyl Cypridina luciferin analog, MCLA), in monolayer cultures of a human ovarian cancer cell line (A2780) in physiological saline at pH 7.0. In a time-course study, chemiluminescence of MCLA (C-MCLA) showed a peak level at 10 min and a background level at 60 min after the addition of DDP. The intensity of C-MCLA increased with increasing concentrations of DDP or MCLA in a limited concentration range, and was significantly correlated (r = 0.960) with the number of A2780 cells. DDP-induced C-MCLA was completely inhibited by the addition of the O2- scavenger, superoxide dismutase (SOD). However, SOD did not decrease DDP cytotoxicity in terms of clonogenic cell survival. These findings suggest that DDP generates extracellular O2-, probably by interaction with the cellular membrane in A2780 cells, and O2- does not lead to cellular damage.

Antineoplastic Agents↗

PubMed1999

Protective effect of nitric oxide against iron-induced neuronal damage.

We investigated the effect of nitric oxide (NO) on iron-induced neuronal damage. Incubation of PC12 cells after the addition of FeCl2 induced rapid increases (within 1 hr) in lipid peroxidation and a concentration (0.1-2 mM)-dependent decrease in cell viability at 48 hr, both of which were blocked by deferoxamine and 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazine-3-o ne hydrochloride (MCLA) (a superoxide scavenger) but not by mannitol (a hydroxyl radical scavenger). Iron-induced cytotoxicity was also antagonized by superoxide dismutase with catalase. On the other hand, the NO donors S-nitroso-N-acetylpenicillamine (SNAP), 3-¿(+/-)-(E)-ethyl-2'-[(E)-hydroxylamino]-5-nitro-3-hexenecarbo moyl¿-pyridine (NOR-4), and 2,2'-(hydroxynitrosohydrazono)bis-ethanamine (NOC-18) decreased cell viability 48 hr after addition without increasing lipid peroxidation. However, when added with 1 mM FeCl2, NO donors including NOC-18, SNAP and NOR-4 (0.1-1 mM) inhibited lipid peroxidation in a concentration-dependent manner and suppressed cell death at lower concentrations. Addition of MCLA and NOC-18 also suppressed decreases in iron-induced [3H]thymidine incorporation. In rat brain homogenate, NOC-18 and SNAP both suppressed iron-induced lipid peroxidation. These findings suggest that NO has a dual effect on neuronal viability and can act as an antioxidant which protects neurons from iron-induced damage.

Animals↗

PubMed2006

Degradation of 2,5-dimethylpyrazine by Rhodococcus erythropolis strain DP-45 isolated from a waste gas treatment plant of a fishmeal processing company.

A bacterium, strain DP-45, capable of degrading 2,5-dimethylpyrazine (2,5-DMP) was isolated and identified as Rhodococcus erythropolis. The strain also grew on many other pyrazines found in the waste gases of food industries, like 2,3-dimethylpyrazine (2,3-DMP), 2,6-dimethylpyrazine (2,6-DMP), 2-ethyl-5(6)-dimethylpyrazine (EMP), 2-ethylpyrazine (EP), 2-methylpyrazine (MP), and 2,3,5-trimethylpyrazine (TMP). The strain utilized 2,5-DMP as sole source of carbon and nitrogen and grew optimally at 25 degrees C with a doubling time of 7.6 h. The degradation of 2,5-DMP was accompanied by the growth of the strain and by the accumulation of a first intermediate, identified as 2-hydroxy-3,6-dimethylpyrazine (HDMP). The disappearance of HDMP was accompanied by the release of ammonium into the medium. No other metabolite was detected. The degradation of 2,5-DMP and HDMP by strain DP-45 required molecular oxygen. The expression of the first enzyme in the pathway was induced by 2,5-DMP and HDMP whereas the second enzyme was constitutively expressed. The activity of the first enzyme was inhibited by diphenyliodonium (DPI), a flavoprotein inhibitor, methimazole, a competitive inhibitor of flavin-containing monooxygenases, and by cytochrome P450 inhibitors, 1-aminobenzotriazole (ABT) and phenylhydrazine (PHZ). The activity of the second enzyme was inhibited by DPI, ABT, and PHZ. Sodium tungstate, a specific antagonist of molybdate, had no influence on growth and consumption of 2,5-DMP by strain DP-45. These results led us to propose that a flavin-dependent monooxygenase or a cytochrome P450-dependent monooxygenase rather than a molybdenum hydroxylase catalyzed the initial hydroxylation step and that a cytochrome P450 enzyme is responsible for the transformation of HDMP in the second step.

Animals↗

PubMed1990

A highly sensitive method for determining both Mn- and Cu-Zn superoxide dismutase activities in tissues and blood cells.

A highly sensitive method for determining the superoxide dismutase (EC 1.15.1.1) in various tissues and blood cells is described. This method involves inhibition of a cypridina luciferin analog that is chemiluminescence dependent upon O2- generated by hypoxanthine-xanthine oxidase. Manganeous superoxide dismutase, which is sensitive to sodium dodecyl sulfate, was determined and calculated by subtraction of superoxide dismutase activity in tissue extract treated with this detergent (Cu-Zn superoxide dismutase) from that in untreated tissue extract (total superoxide dismutase). Both Mn- and Cu-Zn superoxide dismutase activities were expressed as equivalent nanograms of bovine erythrocyte superoxide dismutase per milliliter. Sensitivity limits of the chemiluminescence methods with 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-alpha]pyraz in-3-one and 2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-alpha]pyrazin-3-one as cypridina luciferin analogs were 1 ng and 2-3 ng of superoxide dismutase/ml, respectively.

Animals↗

PubMed1991

5,5-dimethyl-1-pyrroline-N-oxide alone enhances the spontaneous superoxide generation by primaquine.

Primaquine (PQ), a well-known antimalarial drug, has been reported to generate superoxide (O2-) in the presence of reducing agents such as NADPH. In the present study, chemiluminescence was detected by adding only PQ to aqueous 2-methyl-6-[p-methoxyphenyl]-3,7-dihydroimidazo-[1,2-alpha] pyrazin-3-one (MCLA), which is a specific chemiluminescent probe for O2-, and was quenched by superoxide dismutase (SOD), indicating that PQ alone can generate O2- in aerobic conditions. Furthermore, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) enhanced the O2- generation by PQ. Superoxide spin adduct, DMPO-OOH, was also detected by ESR both in aqueous solutions and in dimethyl sulfoxide with DMPO. The level of O2- generation showed a linear correlation with the DMPO concentration, and SOD competitively inhibited the DMPO-OOH formation. The results suggested that in aerobic conditions PQ is autoxidized to 5-hydroxy-PQ, which generates O2-, and DMPO accelerates the autoxidation process by trapping O2-. DMPO or M4PO alone enhances the spontaneous O2- generation by PQ, therefore cautious evaluation is necessary in all studies using the ESR/spin trapping technique to elucidate the mechanism of PQ-related radical generation.

Cyclic N-Oxides↗

PubMed1991

Mechanism for the generation of superoxide anion and singlet oxygen during heme compound-catalyzed linoleic acid hydroperoxide decomposition.

Heme compound, hematin or cytochrome c, catalyzes the decomposition of 13-hydroperoxy linoleic acid yielding both O2- and 1O2 under aerobic conditions. No 1O2 is produced when hydrogen peroxide and cumene hydroperoxide are used as substrates. In these experiments, both O2- and 1O2 could be precisely detected by a chemiluminescence method using a cypridina luciferin analog, 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazin++ +-3-one, as a chemiluminescence probe, in the absence and presence of Cu-Zn superoxide dismutase in catalytic amounts. The reduction and oxidation cycle of ferric heme compound and the bimolecular reaction of peroxyl radicals are plausible reaction mechanisms for O2- and 1O2 production, respectively, in the systems studied.

Catalysis↗

PubMed1980

Inhibition by amiloride of 22Na+ transport into toad bladder microsomes.

The diuretic, amiloride, inhibited 22Na+ accumulation by microsomes from toad (Bufo marinus) bladder by between 40 and 70%. Accumulated 22Na+ was separated from free isotope by ion-exchange chromatography. The amiloride-sensitive process was directly proportional to the protein concentration and a hyperbolic function of the Na+ concentration. The inhibition was competitive. At least 9 times more activity was found in the microsomal fraction than the mitochondrial fraction, and none at all was foumd in the cytosol. Amiloride inhibition could not be reproduced by similar pyrazine and guanidine compounds, such as sulfaguanidine. Amiloride-sensitive Na+ accumulation was totally reversed by nigericin. The intravesicular volume of the lbadder microsomes as determined by the [3H]H2O/[14C]inulin method was shown to be nearly equal to the volume occupied by the amiloride-sensitive portion of the 22Na+ accumulation. The amiloride-sensitive Na+ accumulation most likely represents transport into vesicles. Since amiloride does not affect gramicidin-mediated 22Na+ uptake into phospholipid vesicles, the amiloride inhibition seen with bladder microsomes probably represents a specific channel blockade and not simply interference with cation movement that might be produced by any cationic hydrophobic molecule.

Amiloride↗

PubMed1992

A high involvement of O2- possibly generated in inner membranes for iron-induced microsomal lipid peroxidation.

The lipid peroxidation of and the O2- generation by rat liver microsomes in the presence of NADPH or both NADPH and Fe3+ were determined by thiobarbituric acid-reacting substance formation and by chemiluminescence intensities with a cypridina luciferin analog, 2-methyl-6-(p-methoxyphenyl)-3, 7-dihydroimidazo[1,2-a]pyrazin-3-one(MCLA), as a chemiluminescence probe. Judging from the experiments with various inhibitors on the O2- generation and the lipid peroxidation, O2- generated, at intramembranous site, by cytochrome P-450 system is considered to be highly involved in the iron-induced lipid peroxidation.

Animals↗

PubMed1992

Identification of a processed protein related to the human chaperonins (hsp 60) protein in mammalian kidney.

The chaperonin family of proteins, which includes GroEL protein of E. coli, yeast heat shock protein (hsp-60) and the ribulose-1-5-bisphosphate carboxylase (Rubis Co.) subunit binding protein of plant chloroplasts, shows strong sequence homology to the Chinese hamster ovary (CHO) mitochondrial P1 protein. We have identified a 60 kDa protein from bovine kidney which by N-terminal sequencing gives the amino acid sequence AKDVKFGADARALLMLQGVDLLADA. Bovine whole kidney membranes were delipidated, solubilized with octyl glucoside and fractionated over an affinity column using the amiloride analog 5-N pyrazine amiloride as the ligand. After extensive washing with 200 mM NaCl, the column was eluted with pH 4.0 buffer. Analysis of column fractions on a 7.5% polyacrylamide gel revealed 3-4 bands with a predominant band at 60,000 Da. Amino acid analysis after transfer to immobilon membranes demonstrated sequence identity to the human HSP (60), extending 24 amino acids from the N-terminus, but lacking the leader sequence. These data indicate that a processed form of a protein related to the human HSP (60) chaperonin is associated with a membrane fraction in the mammalian kidney, and that the processed form of the protein binds strongly to an amiloride affinity support.

Amiloride↗

PubMed1984

Theophylline-like properties of xanthine analogs.

Theophylline and other methylxanthines display a large number of biological effects, some of which are clinically important. The effects of these compounds are commonly ascribed to an inhibition of cyclic AMP breakdown. However, it becomes actually evident that another mechanism, namely adenosine receptor antagonism, could be responsible for certain methylxanthine effects. It could be of interest to find new compounds displaying only one of these mechanisms, either phosphodiesterase inhibition or adenosine receptor antagonism. We have studied several synthetic imidazol[1,2a]pyrazines, some of which display theophylline-like pharmacological properties at lower doses than theophylline. We showed that some of these compounds inhibited mitogen-induced [3H]-thymidine uptake by human lymphocytes, which is consistent with increases in cyclic AMP levels: the most efficient compounds were those which were better phosphodiesterase inhibitors than theophylline and poorer adenosine receptor antagonists.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1994

Inhibition of free radical generation by biotin.

To assess the inhibitory effect of biotin on free radical generation, we used a spectrophotometric assay of cytochrome c reduction and determined the 2-methyl-6-phenyl-3,7-dihydroimidazo[1,3-a]-pyrazin-3-one (CLA)-dependent chemiluminescence response of human neutrophils or a hypoxanthine-xanthine oxidase (XOD) system. In the spectrophotometric assay of cytochrome c reduction, superoxide (O2-) generation by neutrophils stimulated with N-formyl-methionyl-leucyl-phenylalanine (f-MLP) was reduced significantly when biotin was added. In the CLA-dependent chemiluminescence test of neutrophils stimulated by f-MLP, biotin significantly reduced the generation of free radical species, including O2-, in a concentration-dependent manner, the concentration corresponding to 50% inhibition (IC50) of biotin for free radical generation was 1.12 x 10(-7) mol. However, biotin did not exert an inhibitory effect on oxidative metabolism by directly scavenging superoxide anion, as shown by the study using the hypoxanthine-XOD system.

Adult↗