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This experiment aimed to investigate the effect of Lonicerae flos and Turmeric extracts (LTE) added to diets on growth performance and intestinal health of broilers. A total of 720 healthy 21-day-old yellow-feathered broilers were randomly divided into 3 treatment groups, with 6 replicates and 40 broilers per replicate. These 3 dietary treatments included a basal diet + 0 g/t LTE (CON), a basal diet + 300 g/t LTE (LTE300), and a basal diet + 500 g/t LTE (LTE500). The results showed that dietary supplementation of LTE linearly increased (P < 0.05) average daily gain (d 21-38) and average daily feed intake (d 21-60). At d 60, LTE300 had the highest serum total antioxidant capacity and total superoxide dismutase (P < 0.05), and LTE500 had the lowest malondialdehyde level (P < 0.05) among the three groups. Moreover, compared to CON, LTE300 significantly (P < 0.05) reduced endotoxin (d 38 and d 60) and diamine oxidase activity (d 38); LTE500 significantly (P < 0.05) reduced endotoxin (d 38 and d 60) and diamine oxidase levels (d 60) in the serum. LTE groups significantly (P < 0.05) increased ileal the ratio of villus height to crypt depth and serum immunoglobulin G. Furthermore, dietary supplementation of LTE also improved the intestinal epithelial barrier by the up-regulated mRNA expression of Claudin-1, Occludin and zonula occludens-1, and decreased the mRNA expression of interleukin-2, interleukin-8, tumor necrosis factor-α, nuclear factor κB, myeloid differentiation factor 88 and toll-like receptor 4. Compared to CON, 16S rRNA sequencing analysis showed that LTE300 had a better effect on the microbial diversity and composition in the ileum, and Bacillus and Lactobacillus&#95;agilis were significantly enriched in LTE300. PICRUSt results showed that LTE300 was significantly (P < 0.05) enriched in four pathway pathways at KEGG level 2. In conclusion, dietary supplementation with LTE improved growth performance and intestinal health by enhancing antioxidant capacity, intestinal barrier and immune function, and regulating intestinal flora of yellow-feathered broilers., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The availability of more efficient analytical methods that answer the world's demands is a challenge and their development continues to be a difficult task. In this work the construction of an electrochemical cell, based on low-cost and accessible materials, that can be easily constructed and used for electroanalytical purposes, is described. Pencil leads were used as electrodes and a transparency sheet as the base. This cell was used as transducer for developing an amperometric biosensor for the quantification of histamine, which is the only biogenic amine regulated by law. The analysis was based on the use of diamine oxidase as biorecognition element, hexacyanoferrate(III) as electron-transfer mediator, and chronoamperometry, at +0.5 V during 100 s, to record the analytical signal. A linear relationship between histamine concentration and the analytical signal was established between 5.0 and 35 mg L -1 and a low limit of detection (1.0 mg L -1 ) was achieved. The analysis of different fish species (sardine and tuna) was performed, obtaining recovery values between 102% and 110%. The stability of the sensor is noteworthy: it maintained 95% of the initial analytical signal after 15 days., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Neonatal calf diarrhoea is one of the most important health challenges in cattle herds causing substantial economic losses and antimicrobial use. Due to the raising problem of antimicrobial resistance, effective alternatives are urgently required, in line with European policies. The aim of this study was to evaluate the effect of tributyrin supplementation in milk replacer on diarrhoea, performance and metabolic status in preweaning Holstein calves. Twelve newborn calves, after colostrum administration, were randomly allotted in two experimental groups for 42 days: control (CTRL) fed milk replacer, tributyrin (TRIB) fed milk replacer supplemented with 0.3% of liquid tributyrin on milk powder weight. Calves BW was recorded on a weekly basis from day 7 to day 42, and feed intake was recorded daily to calculate zootechnical performance. Faecal consistency was assessed daily through the faecal score (0-3 scale; considering diarrhoea moderate = 2 and severe = 3). Faecal samples were collected weekly from rectal ampulla for microbiological analysis by plate counting method evaluating the number of total bacteria, lactic acid bacteria and coliform bacteria. On day 0 and day 42, individual blood samples were collected from jugular vein for metabolic profile analysis. Serum samples of day 42 were also evaluated for the antioxidant barrier using a colorimetric test, while glucagon-like peptide 2 and diamine oxidase concentrations were measured through immunoenzymatic assays. Tributyrin supplementation did not influence the zootechnical performance of calves over 42 days of trial. Diarrhoea frequency was significantly lower in TRIB compared to CTRL group (27.91 and 38.37%; P < 0.01) considering the whole experimental period. In particular, the major effect was observed for moderate diarrhoea in TRIB group that showed a significantly reduced frequency compared to CTRL (P < 0.01) thus suggesting a preventive effect of tributyrin. Faecal total bacterial, lactic acid and coliform bacteria counts did not show differences between groups. Urea serum concentrations tended to be lower in TRIB compared to CTRL, indicating an efficient utilisation of dietary protein. Antioxidant barrier and glucagon-like peptide 2 were comparable between CTRL and TRIB on day 42. Diamine oxidase concentrations were significantly decreased in TRIB compared to CTRL group after 42 days of trial (P < 0.01), suggesting a higher gut epithelial integrity probably due to lower diarrhoea frequency and the nourish effect of tributyrin on enterocytes. In conclusion, tributyrin could be considered as a valuable bioactive feed additive to decrease the neonatal diarrhoea occurrence and support intestinal integrity in preweaning calves., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

The disulfide-containing molecule cystamine and the thiosulfonate thiotaurine are of interest as therapeutics. Both are precursors of taurine, but the chemistry of their metabolism is not clear. The rates at which these molecules are metabolized is also unknown. The chemistry and rate constants have been determined for a process in which cystamine is converted in four reactions to thiotaurine. Cystamine is oxidized by diamine oxidase with a specificity constant comparable to other diamine substrates. The rapid hydrogen peroxide-mediated oxidation of cystaldimine yields reactive glyoxal and thiocysteamine, which quickly performs transsulfuration with hypotaurine. Thiotaurine reacts spontaneously with hydrogen peroxide to form taurine and sulfite, but it is 15-fold less reactive than hypotaurine as an antioxidant. An estimation of biological rates of reaction indicates that cystamine is likely to be oxidized by diamine oxidase in vivo, but its metabolic products will be diverted to molecules other than thiotaurine., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: Severe burns can cause a hypermetabolic response and organ damage. Glutamine is a conditionally essential amino acid with various pharmacological effects. In this study, whether glutamine could alleviate the hypermetabolic response and maintain organ function after burn injury was analyzed., Methods: A multicenter, randomized, single-blind, parallel controlled trial was conducted to evaluate the efficacy of glutamine in decreasing hypermetabolism after burn injury. Physiological and biochemical indexes, such as vital signs, metabolic hormones, metabolic rate, and organ damage, were recorded on the 7th and 14th days after treatment., Results: In total, 55 adult burn patients with a total burn surface area (TBSA) of 30-70% were included in this study and randomly divided into the burn control (B, 28 patients) and burn+glutamine (B+G, 27 patients) groups. Except for the glutamine administration, the groups did not differ in the other treatments and nutrition supplements. The levels of diamine oxidase (DAO), lactulose/mannitol (L/M), β 2 -microglobulin, lactate dehydrogenase (LDH), hydroxybutyrate dehydrogenase (HBD) and cardiac troponin l (cTnl) in the B+G group were significantly lower than those in the B group (p < 0.05 or 0.01). The levels of resting energy expenditure (REE), serum catecholamines, glucagon, lactate and Homeostasis model assessment (HOMA) in the B+G group were significantly lower than those in the B group (p < 0.05 or 0.01). No significant difference was found in the length of hospitalization or the mortality rate between the two groups (p > 0.05)., Conclusions: Glutamine moderately alleviates the hypermetabolic response and reduces organ damage after severe burns. Therefore, the early application of glutamine, which is effective and safe, should be used as an active intervention as early as possible., Competing Interests: Conflict of Interest The authors including Zi En Wang, Jian Jun Zheng, Jin Bin Feng, Dan Wu, Sen Su, Yong Jun Yang, Yan Wei, Zhao Hong Chen and Xi Peng declare no conflicts of interest., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The existing zymography method for the detection of diamine oxidase (DAO) activity has been improved by a new staining procedure with the aim to ameliorate its sensitivity. Both procedures used SDS-PAGE gels containing uniformly distributed entrapped peroxidase (that wouldn't migrate during electrophoresis). The new approach with 3,5-dichloro-2-hydroxybenzenesulfonate (DCHBS) as peroxidase substrate and with 4-amino-antipyrine as color stabilizer allows a more sensitive detection of DAO when compared to the previously reported o-phenylenediamine (o-PDA) as peroxidase substrate. The newly improved method appears faster, simple and environmentally friendly. It can be used for most of oxidases releasing hydrogen peroxide as reaction product., (Copyright © 2022 Elsevier Inc. All rights reserved.)

To overcome some of the disadvantages of conventional hatcheries, a new concept is being explored in the broiler industry: on-farm hatching. In on-farm hatching systems, broiler eggs are transported to the broiler house on day 18 of incubation. On-farm hatched chicks hatch in a low dust environment, are immediately exposed to light, and have instant access to nutrients and water. Previous studies reported that on-farm hatching systems provide birds with an improved intestinal health and a lower feed conversion rate; resulting in a reduced use of antibiotics. Although it is generally agreed that the intestinal health of on-farm hatched chicks is better, the causative factors remain largely unknown. To explore the effect of hatching system on intestinal development, a full factorial in vivo experiment was designed, taking into account commercial age (minus two days, D-1, D1 and D2) and hatching condition (hatchery-born, hatchery-born with Spectoliphen 100 (SL) treatment, and on-farm hatched using the NestBorn-system) as factors. To assess intestinal development, diamine oxidase (DAO) serum levels were measured. DAO, a highly active intracellular enzyme that is synthesised mainly by the intestinal mucosal cells, is generally used as an indicator for intestinal maturation and intestinal permeability (IP) in mammals and birds. Analysis of serum samples showed that serum DAO levels in hatchery-born chicks were significantly lower compared to their on-farm hatched counterparts on all four days, suggesting that the intestinal development in the latter took place earlier. However, the long-term effect was not explored in this study. An additional comparison between the hatching systems was made, not according to commercial age, but in reference to time of access to feed. In this comparison, no differences between the two groups were observed. Interestingly, in the hatchery-born chicks, no compensatory development of the intestines took place within the time span of this experiment. The effect of SL during the first days on intestinal development and IP of chicks remains poorly understood and requires further investigation., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

N1-Dansylspermine and related sulfonamides of the natural polyamines are very potent blockers of NMDA-type glutamate receptors. They exhibit pharmacological properties which were not predicted from the constituents of the conjugates. Cytotoxicity and calmodulin antagonism of N1-dansylspermine were especially impressive. Calmodulin antagonism implies that N1-dansylspermine prevents induction of ornithine decarboxylase and inhibits its own active uptake via the polyamine transport system. Structure-activity considerations demonstrated that an aromatic character of the substituent is not required; amide bond formation with an aliphatic sulfonic acid is sufficient to transform spermine into a highly toxic calmodulin antagonist. Cytotoxicity and calmodulin antagonism are properties which are intrinsic to spermine, but they are observed only at very high concentrations. Amide bond formation at N1 with a lipophilic residue appears to 'amplify' these normally latent properties. The use of polyamine conjugates structurally related to the amides described in this work for targeting tumours may be marred by their calmodulin antagonism.

This paper reports the use of proton NMR spectroscopy for the analysis of trimethylamine in the urine of a patient with trimethylaminuria. Analysis of this compound was also performed for other members of his family. Qualitative and quantitative determination of trimethylamine and trimethylamine-N-oxide was simultaneously performed on untreated urine within a few minutes. The application of the method is discussed.

The capacity of bovine serum amineoxidase (SAO) to oxidize free amino groups of nonconventional substrates, such as polylysine (up to 50 kDa) and some proteins as lysozyme and ribonuclease A, is described. The oxidation was quantified from the amount of H2O2 and NH3 enzymatically produced by SAO. Kinetic analysis indicated a stereospecific preference for L-configuration. Maximal oxidation rate was obtained with poly-L-lysine (9.6 kDa). After 10 h of incubation at 37 degrees C, the poly-L-lysine was partially oxidized generating 1.5 moles of H2O2 by one mole of polylysine. Denatured SAO presented very low oxidation rates with the mentioned substrates.

The influence of bovine serum amineoxidase (SAO), a circulating copper-enzyme, on neuronal K+ channels is described. Bovine SAO enhanced K+ channel currents in N1E-115 neuroblastoma cells in a time-dependent manner. Unlike ceruloplasmin (another copper-protein, shown as depolarizing factor in neurons), SAO had no effect on resting potential of neurons. However, pretreatment of cells with SAO inhibited ceruloplasmin-induced membrane depolarization. Although ceruloplasmin alone inhibited K+ channel currents, it further enhanced K+ channel currents in the presence of SAO. Therefore, SAO may be another endogenous modulator of neuronal K+ channels with effect and mechanisms different from those of ceruloplasmin.

The effect of incubation temperature on the activity of bovine serum amine oxidase was studied using natural substrates of this enzyme, namely spermine and spermidine. The activity behavior was found to be rather complex and different from that observed using benzylamine as substrate. The enzyme is fully active after 3 min incubation at temperatures up to 60 degrees C, while at higher temperatures it shows an S-shaped irreversible decrease of the activity with a T50 of about 70 degrees C. The dependence of the kinetic parameters Kcat and Km on temperature was also studied in the range 5-66 degrees C, measuring the initial rates of oxidation of spermidine. The Arrhenius plot of kcat shows a continuous bend in the thermal stability range of the enzyme. This nonlinearity is due to the dramatic change of kcat activation entropy (from +11 eu at 8 degrees C, to -31 eu at 50 degrees C) and indicates that kcat is a composite constant, involving two or more successive steps characterized by different kinetic parameters. In the case of the ratio kcat/Km, which for natural substrates of bovine serum amine oxidase was shown that its dependence on ionic strength gives information on the influence of electrostatic field on k1 (Stevanato et al. (1994) Biochem. J. 299, 317-320), where k1 is the kinetic constant of interaction between the polyamine and the enzyme, the Arrhenius plot is linear in the range 12-45 degrees C, and an activation entropy of 18.8 eu was calculated at 37 degrees C. This value is in accord with a mechanism involving the formation of a polyamine-enzyme complex which is accompanied by neutralization of charges between the polycationic substrate and negatively charged groups of the enzyme.

The kinetic characterization of soybean seedling amine oxidase (SSAO) and the effect of ionic strength and pH on the enzyme activity have been studied. A strong dependence of the activity on the carbon chain length of alpha-omega diamines, peaking at C8 and at C5, for Km and kc, respectively, was found. The analysis of ionic strength effects on activity showed a high sensitivity of Km and an insensitivity of kc to this parameter. This behavior and the different dependence of Km and kc on the carbon chain length suggest a two-stage equilibrium model for the oxidative deamination of polyamines by SSAO. The formation of the first intermediate (complex amine-enzyme) is controlled mainly by ionic interactions and by the structure of the polyamine, while the formation of the second intermediate depends only on the length of the carbon chain of the diamine. The molecular dissociation constants of the system soybean seedling amine oxidase-cadaverine were obtained from the dependence of kc, Km, and kc/Km on pH. A mechanism of the initial steps of reaction, involving a two-stage equilibrium model, is proposed.

We examined subcellular histamine localizations in purified human basophils that were stimulated to degranulate with FMLP using an ultrastructural enzyme-affinity technique. Basophils were collected at early (0, 20 seconds, 1 minute) and late (10 minutes to 6 hours) time points poststimulation and were prepared for routine ultrastructural and diamine oxidase-gold (DAO-gold) cytochemical analysis. Histamine was present in unaltered cytoplasmic secretory granules (30.77 gold particles per square micrometer; P < .001 compared with background); specificity controls (histamine absorption, diamine oxidase digestion) abrogated granule labeling for histamine. Altered granules in stimulated cells were not significantly labeled for histamine, as compared with background (P = not significant); unaltered granules in the same cells contained more histamine than altered granules (P < .05). During recovery times, spanning 10 minutes to 6 hours, granules again appeared to be electron-dense and contained histamine (33.49/microns2; P = not significant as compared with unaltered granules in 1-minute FMLP-stimulated cells, and P < .05 as compared with altered granules in 1-minute FMLP-stimulated samples). Other structures devoid of histamine in actively secreting cells included extruded granules and intragranular and extruded Charcot-Leyden crystals. Recovering basophils displayed morphologic evidence of material and membrane conservation, granule content condensation, and biosynthesis. Subcellular histamine-rich sites in actively recovering basophils included condensing granules and collections of cytoplasmic vesicles in three locations: beneath the plasma membrane, adjacent to granules, and in the Golgi region. These studies show that unaltered granules of actively releasing human basophils, as well as similar granules that are reconstituted after FMLP-stimulated degranulation, contain histamine, but that altered granules in stimulated cells undergoing degranulation are devoid of histamine. Reconstitution of histamine-rich granules is associated with DAO-gold-positive cytoplasmic vesicles, suggesting transport of histamine derived from either new synthesis, re-uptake of released histamine, or both, to reconstituted granules.

The preparation and some properties of a hydroxyquinone showing an amine oxidase-like activity are described. The compound, 2-hydroxy-5-methyl-1,4-benzoquinone, oxidizes a wide range of primary amines, both benzylic and non-benzylic, at the expenses of molecular oxygen in the presence of Cu2+ ions, and reacts with hydrazine derivatives leading to stable, inactive adducts. This behaviour is similar to that of 2,4,5-trihydroxyphenylalanine quinone-containing amine oxidases and therefore the present compound could be a useful model to understand the reaction mechanism of those enzymes.

Copper amine oxidases (EC 1.4.3.6) exhibit stereochemical heterogeneity in their reaction specificities. Enzymes isolated from different sources have previously been shown to catalyze the deamination of tyramine and dopamine with abstraction of the pro-R hydrogen at C-1, the pro-S hydrogen, and net nonstereo-specific protein abstraction. In this study we report on the stereochemical course of proton removal from benzylamine for six copper amine oxidases using stereo-specifically deuterated benzylamines prepared by a combined chemical-enzymatic method. The product benzaldehydes from the amine oxidase reactions were reduced in situ with alcohol dehydrogenase and NADH providing benzyl alcohols which were analyzed by 1H NMR spectroscopy. The amine oxidases isolated from pea seedlings and bovine, horse, porcine, rabbit, and sheep plasma all react with abstraction of the pro-S hydrogen of benzylamine, irrespective of the stereochemical course of the oxidation of tyramine or dopamine. We also report that the enzymes isolated from horse and rabbit plasma contain 2,4,5-trihydroxyphenylalanine (topaquinone) as an organic cofactor based on visible absorbance spectroscopy of p-nitrophenylhydrazine-derivatized enzymes.

We report here a simple and sensitive method for the measurement of semicarbazide-sensitive amine oxidase (SSAO) activity in human plasma. Benzaldehyde, generated during a 1-h incubation of plasma with benzylamine, is derivatized with the specific aldehyde reagent dimedone after prior deproteinization. Quantitation of the derivatization product is done by automated injection onto an isocratic high-performance liquid chromatographic system with fluorimetric detection. The assay shows good linearity and reproducibility (intra-assay C.V. 7%). Detection limit is 25 mU/l (= pmol/ml/min). In 51 healthy controls (age 49 +/- 13 yr, 20 males) the measured SSAO activity was 352 +/- 102 mU/l (mean +/- S.D.). A large number of samples (70-80) can easily be processed in one day by one technician.

Although the existence of a membrane-bound (probably plasmalemmal) semicarbazide-sensitive amine oxidase (SSAO) is well established in various mammalian tissues, and especially within vascular smooth muscle, its importance and the possible consequences of its metabolism of certain physiological and xenobiotic amines in vivo are under continuing investigation. In this respect, there are major species-related differences in substrate specificity determined in vitro, not only towards the synthetic amine benzylamine, but also towards some other aromatic amines (e.g. tyramine, tryptamine, 2-phenylethylamine, dopamine, histamine) which are possible endogenous substrates. Inhibition of SSAO can potentiate the pharmacological activity of some amines in isolated tissue (e.g. blood vessel) preparations from some species. Recent evidence has accumulated that SSAO may also be involved in metabolizing endogenous aliphatic amines such as methylamine and aminoacetone, focussing attention on the fact that the aldehyde products (formaldehyde and methylglyoxal, respectively) are potentially cytotoxic agents. Indeed, SSAO has been implicated in experimental models of cardiovascular toxicity involving conversion of the industrial aliphatic amine allylamine to acrolein. In summary, metabolism by SSAO may reduce the physiological/pharmacological effects of some amines, but the resulting metabolites (aldehydes, H2O2) may also have important actions.

Semicarbazide-sensitive amine oxidases with a high affinity for benzylamine (Bz.SSAO) (E.C.1.4.3.6) have been biochemically described in many mammalian tissues (adipose tissue, lung, heart, blood vessels). The enzymic activity appears to be expressed by mesenchymal cells (fibroblasts, adipocytes, smooth muscles). Although the physiological role of this enzymic activity is still unclear, some possible physiological substrates such as histamine are discussed. Some enzymes of this class (SSAO) have been purified. They share many similarities, among which are that they contain copper and a carbonyl active site. The nature of the organic cofactor of these enzymes is discussed and data are presented which have identified pyridoxal in pig kidney diamine oxidase and in pig plasma benzylamine oxidase by gas chromatography-mass spectrometry.

The widespread distribution of enzymes classed as semicarbazide-sensitive amine oxidases (SSAO enzymes) throughout a very wide range of eukaryotic as well as prokaryotic organisms encourages the aspirations of those who wish to demonstrate physiological, pathological or pharmacological importance. Such enzymes are found in several tissues of mammals, both freely soluble, as in blood plasma, and membrane-bound, for example, in smooth muscle and adipose tissue. While they are capable of deaminating many amines with the production of an aldehyde and hydrogen peroxide, doubt still surrounds the identity of the most important endogenous substrates for these enzymes. At present, methylamine and aminoacetone appear to head the list of candidates. The possibility that SSAO enzymes can convert amine substrates to highly toxic metabolites is illustrated by the production of acrolein from the xenobiotic amine, allylamine and formaldehyde and methylglyoxal from methylamine and aminoacetone, respectively. Activities of SSAO enzymes may be influenced by physiological changes, such as pregnancy or pathologically by disease states, including diabetes, tumours and burns. Increased deamination of aminoacetone by tissue and plasma SSAO enzymes as a result of its increased production from L-threonine in conditions such as exhaustion, starvation and diabetes mellitus may be harmful. Such dangers could be mitigated either physiologically by a compensatory reduction in SSAO activity or pharmacologically by treatment with inhibitors of SSAO.

The relationship between the soluble copper topaquinone amine oxidases, the membrane bound semicarbazide-sensitive amine oxidases and lysyl oxidase remains unclear. The stereochemical course of substrate oxidation has been determined for each enzyme type and these studies suggest that SSAO and lysyl oxidase are closely related mechanistically, and that they are distinct from the copper amine oxidases. Both lysyl oxidase and SSAO catalyze the oxidation of tyramine with removal of the pro-S hydrogen from C-1 of this substrate. The copper amine oxidase enzymes that react with abstraction of the pro-S hydrogen from C-1 of substrates do not exhibit a solvent exchange pathway. In contrast, this exchange occurs in lysyl oxidase and SSAO reactions. The organic cofactor in all three enzyme types is a quinone; however, the spectral features of phenylhydrazine and p-nitrophenylhydrazine-derivatized SSAO differ from those reported for all known topaquinone-containing enzymes. Cofactor identification is further complicated by the lack of the characteristic topa motif, Asn-Tyr-Asp/Glu, in lysyl oxidase and the absence of any sequence information for SSAO.

An improved purification scheme for an amine oxidase from equine plasma (EPAO), a nonruminant source, is described and the protein's active-site is characterized. EPAO is dimeric and contains one Type-2 Cu(II) ion per monomer. The EPAO Cu(II) site is spectroscopically very similar to the Cu(II) sites in other amine oxidases. Unlike the extensively investigated nonruminant amine oxidase from porcine plasma, EPAO does not display half-of-the-sites reactivity; titrations with p-nitrophenylhydrazine and phenylhydrazine indicate two active cofactors per dimer. This cofactor is determined to be the same as that of other copper-containing amine oxidases, 6-hydroxydopa quinone (topa quinone). Upon anaerobic reduction with substrate at ambient temperature, the EPR spectrum of EPAO exhibits a sharp signal at g congruent to 2, attributable to the topa semiquinone. Equine plasma amine oxidase possesses novel in vitro substrate specificity; while other mammalian amine oxidases oxidize norepinephrine only slowly or not at all, EPAO displays significant activity toward this biogenic amine.

We used light and electron microscopy to analyze the eyelid inflammation that develops in transgenic mice that overexpress interleukin-4 (IL-4; Tepper et al, Cell 62:457, 1990). Analysis of alkaline Giemsa-stained plastic sections examined by light microscopy (Dvorak et al, J Exp Med 132:558, 1970), as well as by routine transmission electron microscopy, indicated that the mast cells in the inflammatory eyelid lesions were undergoing piecemeal degranulation, a form of secretion in which the cells' cytoplasmic granules exhibit characteristic morphologic changes that are thought to be associated with the prolonged, vesicle-mediated release of the granules' constituents. Moreover, by using a newly reported enzyme affinity-gold method, which stains histamine based on binding to diamine oxidase-gold (Dvorak et al, J Histochem Cytochem 41:787, 1993), we show that these activated mast cells had released much of their histamine content. The eyelid lesions also exhibited increased numbers of mast cells; interstitial fibrosis, particularly around cutaneous nerves and blood vessels; activated fibroblasts; focal axonal damage; venules with endothelial cells containing numerous vesiculo-vacuolar organelles; and infiltrates of neutrophils and eosinophils. Our findings illustrate that overexpression of the IL-4 gene in vivo can result in eyelid lesions associated with piecemeal degranulation of mast cells, as well as tissue fibrosis and a variety of other pathologic changes. These results also represent the first direct morphologic evidence for histamine secretion by mast cells in vivo.

We report a rapid breakdown of dopamine and especially of 3,4-dihydroxybenzylamine, the frequently-used internal standard in catecholamine determinations, in plasma of many but not all animal species. Species investigated were cow, sheep, goat, pig, horse, rabbit, dog, guinea pig, mouse, chicken, rat and man. In some species 3,4-dihydroxybenzylamine nearly completely disappeared at 4 degrees C within 15 min after addition to the plasma. Added dopamine, but not norepinephrine and epinephrine, also rapidly disappeared at 4 degrees C. Disappearance rates were increased at higher temperatures, and at 20 degrees C also norepinephrine showed some breakdown. The breakdown is caused by a semicarbazide-sensitive amine oxidase in the plasma, and can be completely blocked by the addition of the inhibitor semicarbazide. Measurement of plasma catecholamine concentrations in animal species can thus lead to erroneous results, especially when 3,4-dihydroxybenzylamine is used as an internal standard. Only when blood is collected in tubes containing an inhibitor of semicarbazide-sensitive amine oxidase like semicarbazide can reliable plasma catecholamine measurements be performed.

Lentil seedling amine oxidase catalyzes the oxidation of putrescine and in the presence of tetranitromethane gives rise to the formation of nitroform anion. The initial rate of substrate and enzyme-dependent nitroform production is linearly related to the functional active site content and is proportional to the tetranitromethane concentration. Diethylpyrocarbonate modifies two histidyl residues on the lentil amine oxidase. Incubation of the enzyme with diethylpyrocarbonate at 25 degrees C and pH 7.0 irreversibly inhibits enzyme activity by a pseudo first-order kinetics process. The data obtained are consistent with the enzyme-dependent abstraction of an alpha-proton from the substrate to form an intermediate enzyme bound carbanion and indicate a functional role for histidine in lentil amine oxidase catalysis consistent with that of a general base in proton abstraction.

A simple and rapid procedure for purification of soybean seedling amine oxidase is reported. The crude enzyme, obtained by ammonium sulfate fractionation was purified by ion-exchange chromatography on a cellulose phosphate column and batch affinity chromatography on 6-aminohexyl-Sepharose. Cyclohexylamine, a competitive inhibitor, was utilized to elute the enzyme. A homogeneous enzyme was obtained with a yield higher than 25%, the content of minor components being < or = 2%. The M(r) estimated by gel filtration is 113,000 and 77,000 by sodium lauryl sulfate-polyacrylamide gel electrophoresis. The enzyme is a dimer and contains two Cu2+ ion per molecule. Its EPR spectrum is typical of Cu2+ in a tetragonal symmetry. The enzyme oxidizes cadaverine at high rate, the specific activity being 4.3 mukat/mg. Molecular, spectroscopic, and kinetic properties of this enzyme are reported.

Allylamine is a specific cardiovascular toxin that causes vascular and myocardial lesions. Previous studies showed that allylamine-induced chronic lesions are markedly reduced by semicarbazide, an inhibitor of semicarbazide-sensitive amine oxidase (SSAO), and that allylamine is metabolized to the aldehyde, acrolein, by SSAO. We hypothesized that inhibitors of SSAO might reduce the acute cardiovascular toxicity of allylamine. To test our hypothesis, we fed 150 mg/kg allylamine to semicarbazide-pretreated (3 h; 98 mg/kg) rats. Animals were sacrificed 1 h after allylamine treatment. Aorta, epicardium, and endocardium were assayed for SSAO, glutathione peroxidase, catalase, thiol status and lipid peroxidation. SSAO activity was decreased significantly in aorta, epicardium and endocardium. Activity was 30-times higher in aorta than in epicardium and endocardium. A striking decrease in malonaldehyde level (lipid peroxidation) was found in aorta of pretreated rats as compared to allylamine-only treated rats. The reduction of free-SH content in aortic mitochondria was also attenuated in pretreated rats. Changes were not so marked in epicardium and endocardium. These results suggest that in vivo pretreatment with semicarbazide at least partially protects aortic mitochondria from allylamine toxicity. The mechanism can be explained on the basis of the fact that semicarbazide inhibits acrolein formation in allylamine-treated rats.

Bovine serum amine oxidase (BSAO, EC 1.4.3.6) catalyzes the oxidative deamination of polyamines giving rise to the corresponding aldehydes, ammonia and hydrogen peroxide (H2O2). This study demonstrates that amine oxidase (BSAO) purified from bovine serum and exogenous spermine caused cytotoxicity in Chinese hamster ovary (CHO) cells. Cytotoxicity occurred when cells were exposed to BSAO (0.0164-16.4 micrograms/ml) in the presence of spermine (1.9-340 microM). BSAO and spermine alone were not toxic at these concentrations. Cytotoxicity was dependent on the concentration of spermine and on the incubation time, and was also accelerated at 42 degrees C relative to 37 degrees C. Kinetic analysis of the enzymatic reaction, as a function of spermine concentration, showed Michaelis-Menten saturation kinetics. The apparent Vmax increased from 19.1 +/- 0.4 microM min-1 at 37 degrees C to 23.0 +/- 0.3 microM min-1 at 42 degrees C. The apparent Km decreased from 25.5 +/- 2.6 microM at 37 degrees C to 17.7 +/- 1.3 microM at 42 degrees C. Catalase inhibited cytotoxicity, suggesting that H2O2 was partially responsible for cytotoxicity. This work shows that the oxidation products of polyamines, rather than the polyamines themselves, are responsible for cytotoxicity in mammalian cells. The significance of this study is that amine oxidases could have therapeutic potential in cancer treatment regimens and a beneficial effect is likely when the enzyme is used together with clinical hyperthermia.

The system bovine plasma amine oxidase-polyamine-phosphate ion was investigated by activity measurements and 31P NMR spectroscopy. Lineweaver-Burk plots showed that phosphate ion, under physiological conditions, is an apparent competitive inhibitor of bovine plasma amine oxidase. While NMR measurements of the T1 of 31P do not suggest the binding of phosphate to/or near the paramagnetic Cu(II) sites of bovine plasma amine oxidase, the chemical shift dependence of 31P on spermidine concentration indicates the formation of a spermidine-phosphate complex. The value of the dissociation constant of this complex was found 18.5 +/- 1.4 mM, at pH 7.2, by NMR, in good agreement with the value 17.0 +/- 0.8 mM calculated from activity measurements, assuming the enzyme activity is proportional to the free amine concentration, under second order conditions. Our data suggest that the decrease of the free spermidine, due to the binding of phosphate ion, is responsible of the observed inhibition of bovine plasma amine oxidase.

Cultured hepatocytes express binding sites for bovine serum amine oxidase on their membrane surfaces as evaluated at the electron microscopic level by using enzyme-gold complexes. Hepatocytes show binding sites as small clusters of gold granules, not bound in a specialized region of the plasma membrane. The binding competition of enzyme-gold ligand to cells was achieved by preincubation with uncoupled bovine serum amine oxidase. In addition, N-acetyl-galactosamine, N-acetyl-glucosamine and Mannose, at the final concentration of 80 mM, partially inhibit the binding.

The carbonyl cofactor of bovine plasma amine oxidase (EC 1.4.3.6), recently shown to be 6-hydroxydopa (also known as topa), has been spin labeled to the extent of one label per enzyme dimer molecule, using 4-amino-2,2,6,6-tetramethylpiperidine-N-oxyl (4-amino-TEMPO) and 4-hydrazino-TEMPO followed by reduction with borohydride. By studying the EPR spectra of the labeled enzyme, it has been deduced that there is no magnetic interaction between the copper and the spin label, and that the spin label is at least 1.3 nm distant from the copper(II) ion in the resting enzyme. The bound label is strongly immobilized, is in a sterically constricted environment, and is not accessible to small anions. Removal of the copper does not alter the EPR spectrum of the label. The results are similar to results for porcine plasma amine oxidase, and show that the copper is not close to, and does not directly interact with, the topa-bound substrate.

The presence of covalently bound pyrroloquinoline quinone (PQQ) in bovine plasma amine oxidase (BPAO) was examined by the use of gas chromatography/mass spectrometry. The enzyme was subjected to proteolysis with proteinase in the presence of [U-13C]PQQ as an internal standard. After isolation and derivatization of PQQ with phenyltrimethylammonium hydroxide, molecular peaks at m/z 448 and 462 were used for detection of PQQ and [U-13C]PQQ, respectively, by selected ion monitoring (SIM). In the SIM profile, although the sample extract obtained from BPAO treated with proteinase clearly showed the peak at m/z 462 for the internal standard, there were no peaks detectable at m/z 448, showing the absence of PQQ in the proteolysis digest of BPAO. Thus, our results do not support the claim that BPAO contains covalently bound PQQ in its structure.

An oxygraphic method for the assay of 3,4-dihydroxyphenylalanine (Dopa) decarboxylase activity which makes use of the coupled reactions of Dopa decarboxylase with serum amine oxidase is presented. Both free and immobilized amine oxidases were utilized. The assay is simple, rapid, and allows a continuous monitoring of the reaction. The kinetic parameters for Dopa decarboxylase obtained with the coupled assay do not significantly differ from those obtained by standard methods.

Amine oxidase activity has been identified in commercial samples of bovine serum albumin (BSA). Benzylamine, phenylethylamines and to a lesser extent, indoleamines, were found to be substrates. The amine oxidase activity was inhibited by semicarbazide and was virtually absent in electrophoretically purified samples. Kinetic analysis of benzylamine deamination and experiments utilizing mixed substrates indicate that more than one catalytic activity may be involved. The results show that amine deamination should be considered as a potential source of error in experiments employing high concentrations of commercially available BSA preparations. This would be of particular importance for in vitro studies with dopamine since this amine was found to be deaminated at a rapid rate.

Analysis of the thermal unfolding of bovine serum amine oxidase by differential scanning calorimetry reveals for the dimeric protein a four domain structure consisting of two sets of domains. Each set contains two domains of similar size. The two smaller domains, in contrast with the larger ones, greatly differ in thermostability. Removal of copper changes the calorimetric pattern dramatically. The findings confirm that the metal cofactor plays a structural role. Since the enzyme contains two copper atoms and only one titratable carbonyl group, the calorimetric pattern suggests that the difference in thermostability of the two small domains might be due to the presence of a single organic cofactor.