Noninvasive Biomarkers of Gut Barrier Function in Patients Suffering from Diarrhea Predominant-IBS: An Update.

The intestinal barrier plays a crucial role in the absorption of nutrients and in preventing the entry of pathogenic microorganisms and toxic molecules. Several studies have shown a compromised intestinal barrier associated with low-grade inflammation in the small intestinal mucosa in celiac disease, inflammatory bowel disease, and irritable bowel syndrome (IBS), particularly in IBS with diarrhea (IBS-D). In light of these new data, IBS is no longer considered a functional disease but rather a heterogeneous syndrome that has yet to be carefully studied. Therefore, investigating the integrity and function of the intestinal barrier is now essential to improving knowledge of the pathophysiology of IBS-D and to improving the management of IBS-D patients. However, the study of the intestinal barrier must clarify some still unsolved methodological aspects and propose standardised assays before becoming a useful diagnostic tool. In this framework, this review will discuss data about the tests that noninvasively evaluate the integrity and functionality of the human intestinal barrier, paying particular attention to patients with IBS-D, in both clinical and research situations.

The mechanism of freeze-thawing induced accumulation of γ-aminobutyric acid in germinated soybean.

BACKGROUND: γ-Aminobutyric acid (GABA) is a non-protein amino acid with several functions in the human body. Although freeze-thawing could effectively accumulate GABA in soybean sprouts, the mechanism has not been revealed. The mechanism by which freeze-thawing enhances GABA accumulation in germinated soybean was revealed by evaluating GABA content, the activity of related synthesis enzymes, and the microstructure of the tissues and cells of sprouts. The germinated soybeans were treated at different temperatures (from -196 °C to 25 °C) for 12 h and then thawed at 25 °C for 6 h. RESULTS: The results showed that GABA content in frozen soybean sprouts did not change significantly before thawing. After thawing, the GABA content of sprouts increased by 83.9% and 82.9% when treated by liquid nitrogen flash freeze at - 80 °C for 12 h compared with the control (4 °C treatment for 12 h). The results indicated that GABA formation mainly occurred during thawing. However, glutamate decarboxylase (GAD), diamine oxidase (DAO), and aminoaldehyde dehydrogenase (AMADH) activity decreased during thawing. Based on the malonaldehyde (MDA) content and microstructure of sprouts, it was suggested that freezing at lower temperatures (< -20 °C) maintained the integrity of the cell structure, while the tissues and cell membranes were broken during thawing. CONCLUSION: These results could provide evidence for the hypothesis that GABA formation resulted from full contact between enzymes and substrates during thawing, rather than the contribution of higher enzyme activity. © 2019 Society of Chemical Industry.

Vascular adhesion protein-1 as indicator of breast cancer tumor aggressiveness and invasiveness.

Recent studies suggest that vascular adhesion protein-1 (VAP-1), a 180-KDa homodimeric glycoprotein, may be associated with cancer-related events including tumor cell migration, motility, invasion, or metastasis. Therefore, this study applies VAP-1 immunohistochemical staining to demonstrate the invasiveness component of the breast cancer. The VAP-1 staining results were compared in 148 breast cancer cases to identify possible correlations with clinical status, including age, tumor size, tumor grade, TNM stage, lymphatic invasion, metastasis, recurrence, and survival rate. Immunohistochemical staining results showed VAP-1 negative or weak staining in normal ducts and ductal carcinoma in situ (DCIS), but these phenotypes were positively associated with a stiffened VAP-1 that presented at the invasive front of the lesion. Our data demonstrated that VAP-1 expression was positively associated with lymphatic invasion, distant metastasis, and patient survival in breast carcinoma. Notably, VAP-1 expression was found to be significantly correlated with the overall survival (p < 0.0001). Multivariate Cox analysis indicated that VAP-1 expression was a significant independent prognostic indicator of overall survival in breast carcinoma (p < 0.0001). In conclusion, this study suggests that VAP-1 is linked to progression of tumor invasion and metastasis in breast carcinoma. VAP-1 is shown to be a biomarker that can be predict invasive potential and clinical outcome in breast cancer.

Asociación de polimorfismos de diaminoxidasa e histamina N metiltransferasa con la presencia, discapacidad y severidad de migraña en un grupo de madres mexicanas de niños alérgicos / Association of diamine oxidase and histamine N-methyltransferase polymorphisms with presence of migraine in a group of Mexican mothers of children with allergies

Introducción: Se ha sugerido que una degradación disminuida de histamina puede contribuir en la patogénesis de migraña y alergia. Este trabajo investiga una posible asociación entre 2 polimorfismos de un solo nucleótido (SNP) de 2 enzimas que degradan histamina, C314T para la histamina N-metil-transferasa (HNMT) y C2029G para diaminoxidasa (DAO), con la presencia, discapacidad y severidad de la migraña. Material y métodos: Se reclutó a 162 madres de niños alérgicos (80 con migraña y 82 sin migraña) determinando las variantes alélicas por PRC tiempo real usando un modelo de casos y controles. Mediante regresión logística se determinaron las OR para los genotipos y haplotipos. Resultados: El alelo mutado G para DAO fue significativamente más frecuente en el grupo de mujeres migrañosas que en los controles (OR = 1,6; IC del 95% = 1,1-2,1). No encontramos diferencias significativas para el alelo mutado T de la HNMT. Ambos alelos mutados estuvieron asociados a la discapacidad causada por la migraña. La coexistencia de ambas mutaciones (haplotipos) mostró una fuerte asociación con migraña. Los haplotipos que tenían ambos alelos mutados (ya sea como homocigotos o heterocigotos) estuvieron fuertemente asociados a la discapacidad por migraña grado iv (OR = 45,0, IC del 95% = 5,2-358). Esto sugiere que los alelos mutados T para HNMT y G para DAO pueden interactuar incrementando el riesgo y el impacto de la migraña. Conclusiones: Se sugiere una asociación sinérgica de polimorfismos de HNMT y DAO con migraña el cual debe ser confirmado en futuros estudios. La interpretación debe tomar en cuenta las características étnicas de la población estudiada (AU)

Background: Low histamine metabolism has been suggested to play a role in the pathogenesis of allergy and migraine. We investigated the possible association between 2 single-nucleotide polymorphisms (SNP), C314 T HNMT and C2029G DAO, and the presence and severity of migraine and migraine-related disability. Materials and methods: We studied the frequency of C314 T HNMT and C2029G DAO allelic variants in 162 mothers of children with allergies (80 with migraine and 82 without) using a TaqMan-based qPCR Assay and a case-control model. We conducted a logistic regression analysis to examine the association between migraine and the allelic and haplotype variants. Results: Mutant C2029G DAO SNP was found significantly more frequently in the group of women with migraine than in controls (OR, 1.6; 95% CI, 1.1–2.1). No significant differences were found in frequencies of genotypes or alleles in the case of C314T HNMT SNP. Both mutated alleles were associated with migraine-related disability. Coexistence of alleles for both SNPs (haplotypes) showed a strong association with migraine. Haplotypes containing both mutated alleles (either heterozygous or homozygous) were very strongly associated with MIDAS grade iv migraine (OR, 45.0; 95% CI, 5.2-358). This suggests that mutant alleles of C314 T for HNMT and C2029G for DAO polymorphisms may interact in a way that increases the risk and impact of migraine. Conclusions: We suggest a synergistic association between HNMT and DAO functional polymorphisms and migraine; this hypothesis must be further confirmed by larger studies. However, the characteristics and ethnic differences between analysed populations should be considered when interpreting the results (AU)

Association between two polymorphisms of histamine-metabolising enzymes and the severity of allergic rhinitis in a group of Mexican children

BACKGROUND: It has been suggested that polymorphisms of histamine metabolising enzymes can be a risk factor for developing histamine-involving diseases. The aim of the present study is to research the possible association between two functional single nucleotide polymorphisms (SNPs): C314T in the Histamine-N-Methyl Transferase gene and C2029G in the Diamine Oxidase gene, with the severity of allergic rhinitis and the number of allergic diseases, in a group of allergic Mexican children. METHODS: We studied 154 unrelated allergic children. SNPs were analysed by RT-PCR. The total serum IgE was measured by chemiluminescence and the serum histamine by ELISA. We used logistic regression analysis to determine OR. RESULTS: Patients carrying the mutant allele for any SNP had more risk to develop higher rhinitis severity or a bigger number of allergic diseases. Haplotype analysis revealed that this effect is synergistic. In patients carrying one or two mutant alleles, serum histamine levels were higher than those of patients carrying only wild alleles. Serum IgE levels were not associated with the presence of mutant alleles. CONCLUSION: The presence of these SNPs in patients with allergic rhinitis can lead to higher serum histamine, therefore to a higher risk of developing more severe symptoms or more associated allergic diseases, even if the serum IgE remains low

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Overexpression of vascular adhesion protein-1 is associated with poor prognosis of astrocytomas.

Vascular adhesion protein-1 (VAP-1) is one of the endothelial adhesion molecules that is believed to play a role in tumor progression and metastasis, supporting cancer cell extravasation. Very few studies have been performed on analyzing the contribution of VAP-1 in brain tumor. Astrocytomas are the most common type of brain tumors, which are classified by World Health Organization (WHO) into four grades according to the degree of malignancy. This study was designed to investigate VAP-1 expression level in different astrocytoma grades and its correlation with clinicopathological features as well as prognosis of astrocytoma patients. Eighty-seven patients with different grades of astrocytoma (WHO Grade I-Grade IV) were enrolled in this study. The expression of VAP-1 was assayed by immunohistochemistry. The correlation between VAP-1 expression and clinicopathological features was evaluated by Chi-square test, and overall survival was analyzed by Kaplan-Meier method. Cox regression analysis was applied to analyze the independent influence of each parameter on overall survival. The expression level of VAP-1 was significantly higher in diffuse astrocytoma than those of pilocytic astrocytoma (p < 0.0001). In the subgroup analysis, upregulated VAP-1 expression was frequently found in older age patients (≥50 years). The VAP-1 expression was found to be significantly correlated with the overall survival (p = 0.0002). There was a statistical correlation between VAP-1(high) tumors in diffuse astrocytoma and VAP-1(low) tumors in pilocytic astrocytoma (p < 0.0001). Multivariate Cox analysis indicated VAP-1 was an independent predictive marker for poorer prognosis (p = 0.0036). Therefore, VAP-1 could be a promising prognostic biomarker in astrocytoma.

Enzymatic assay to test diamines produced by vaginal bacteria.

An enzymatic assay was developed to determine the concentration of diamines (DA) in clinical samples of vaginal fluids. Putrescine and cadaverine are DA produced by anaerobic bacteria and are typically present in the vaginal fluids of women with an abnormal microbiota, as occurs in bacterial vaginosis. The vaginal DA (VADA) assay is based on the enzyme diamine oxidase which reacts with putrescine and cadaverine to produce H2O2 in a quantitative manner. H2O2 concentration is measured spectrophotometrically by a chromogenic reaction catalyzed by horseradish peroxidase. The VADA assay proved to be capable of detecting DA concentrations as low as 4 mM and showed a dose-response relationship which was linear over DA concentrations ranging from 4 to 256 mM. Using clinical samples it was possible to show that the VADA assay can be performed on human vaginal swabs and that the mean DA concentration is significantly higher in samples positive for microbial pathogens.

Soluble vascular adhesion protein-1 predicts incident major adverse cardiovascular events and improves reclassification in a finnish prospective cohort study.

BACKGROUND: Vascular adhesion protein-1 (VAP-1) associates to subclinical atherosclerotic manifestations in young people, but its association to incident major adverse cardiovascular events (MACEs) and cardiovascular mortality in a general population is not known. METHODS AND RESULTS: We used a newly developed ELISA to measure soluble VAP-1 (sVAP-1) levels in 2775 participants (mean age, 60 years) from a prospective cohort study (the FINRISK 2002). During a mean follow-up of 9 years, 265 participants underwent a MACE, and these participants had higher levels of sVAP-1 than those without MACE (868 ng/mL and 824 ng/mL, respectively, P<0.001). In multivariate-adjusted Cox proportional hazard model including traditional Framingham risk factors (age, sex, systolic blood pressure, cholesterol, high-density lipoprotein cholesterol, smoking, prevalent diabetes mellitus, and antihypertensive treatment), sVAP-1 independently predicted incident MACE (P=0.0046) and MACE mortality (P=0.026). The impact of sVAP-1 in predicting the 9-year absolute risk of MACE was analyzed using integrated discrimination improvement and net reclassification improvement with 10-fold cross-validation. Inclusion of sVAP-1 in the Framingham model improved integrated discrimination improvement (P=0.042), and the clinical net reclassification improvement by correctly reclassifying 9% (P=0.0019) of people in the intermediate risk (5%-20%) group. CONCLUSIONS: sVAP-1 associated with increased risk of MACE and MACE mortality in people aged >50 years without prior MACE, and inclusion of sVAP-1 in the risk prediction model improved the clinical net reclassification improvement of incident MACE. Thus, sVAP-1 may be a potential new biomarker for cardiovascular diseases.

Identification, prioritization, and evaluation of glycoproteins for aggressive prostate cancer using quantitative glycoproteomics and antibody-based assays on tissue specimens.

Prostate cancer is highly heterogeneous in nature; while the majority of cases are clinically insignificant, some cases are lethal. Currently, there are no reliable screening methods for aggressive prostate cancer. Since most established serum and urine biomarkers are glycoproteins secreted or leaked from the diseased tissue, the current study seeks to identify glycoprotein markers specific to aggressive prostate cancer using tissue specimens. With LC-MS/MS glycoproteomic analysis, we identified 350 glycopeptides with 17 being altered in aggressive prostate cancer. ELISA assays were developed/purchased to evaluate four candidates, that is, cartilage oligomeric matrix protein (COMP), periostin, membrane primary amine oxidase (VAP-1), and cathepsin L, in independent tissue sets. In agreement with the proteomic analysis, we found that COMP and periostin expressions were significantly increased in aggressive prostate tumors while VAP-1 expression was significantly decreased in aggressive tumor. In addition, the expression of these proteins in prostate metastases also follows the same pattern observed in the proteomic analysis. This study provides a workflow for biomarker discovery, prioritization, and evaluation of aggressive prostate cancer markers using tissue specimens. Our data suggest that increase in COMP and periostin and decrease in VAP-1 expression in the prostate may be associated with aggressive prostate cancer.

Analysis of the glycosylation pattern of plant copper amine oxidases by MALDI-TOF/TOF MS coupled to a manual chromatographic separation of glycans and glycopeptides.

The N-glycosylation in pea seedling amine oxidase and lentil seedling amine oxidase was analyzed in the present work. For that purpose, the enzymes were purified as native proteins from their natural sources. An enzymatic deglycosylation of pea seedling amine oxidase by endoglycosidase H under denaturing conditions combined with its proteolytic digestion by trypsin was carried out in order to analyze both N-glycans and "trimmed" N-glycopeptides with a residual N-acetylglucosamine attached at the originally occupied N-glycosylation sites. The released N-glycans were subjected to a manual chromatographic purification followed by MALDI-TOF/TOF MS. MS and MS/MS analyses were also performed directly on peptides and N-glycopeptides generated by proteolytic digestion of the studied enzymes. Sequencing of glycopeptides by MALDI-TOF/TOF MS/MS after their separation on a RP using a microgradient chromatographic device clearly demonstrated binding of paucimannose and hybrid N-glycan structures at Asn558. Such carbohydrates have been reported to exist in many plant N-glycoproteins, e.g. in peroxidases. Although high-mannose glycan structures were identified after the enzymatic deglycosylation, they could not be assigned to a particular N-glycosylation site. The presence of unoccupied glycosylation sites in several peptides was also confirmed from MS/MS results.

Immunohistochemical study of semicarbazide-sensitive amine oxidase/vascular adhesion protein-1 in the hippocampal vasculature: pathological synergy of Alzheimer's disease and diabetes mellitus.

Semicarbazide-sensitive amine oxidase/vascular adhesion protein-1 (SSAO/VAP-1) is involved in vascular endothelial damage as well as in the vascular degeneration underlying diabetes mellitus and Alzheimer's disease (AD). Recent evidence suggests that classic pathological features of AD are more pronounced in diabetic mellitus patients. To investigate the expression and distribution of SSAO/VAP-1 in the two pathologies, we have performed an immunohistochemical study in human hippocampal vessels of AD, AD with diabetic mellitus (ADD), diabetic mellitus (DM), and nondemented (ND) patients. The present results demonstrate major vessel accumulation of both SSAO/VAP-1 and amyloid-ß immunolabeling intensity in ADD compared with AD patients. Interestingly, nearly damaged vessels with high levels of SSAO/VAP-1 also showed increased oxidative damage markers (AGE, RAGE, and SOD-1) and glial activation (GFAP and HLA). Overall, this work suggests that high vascular SSAO/VAP-1 levels in human hippocampus may contribute to vascular degeneration, which can explain the severe progression in patients with both pathologies.

Usefulness of post mortem determination of serum tryptase, histamine and diamine oxidase in the diagnosis of fatal anaphylaxis.

The diagnosis of fatal anaphylaxis can be difficult for clinical features may not always be evident in necropsy. Therefore post mortem determination of tryptase and other blood parameters can be helpful in verifying the diagnosis. We compared post mortem tryptase, histamine and diamine oxidase (DAO) serum levels of two patients who had died after a Hymenoptera sting and one patient who died of bronchospasm during anaesthesia with data obtained from 55 control subjects who had died from other causes than anaphylaxis. In the three anaphylactic cases, serum tryptase level was 880, 68 and 200 µg/l (normal range in living subjects: <11.4 µg/l), histamine was 37.5, 8.5 and 23.2 ng/ml (normal range: <0.3 ng/ml) and DAO was 1, 30 and 4 U/ml (normal range 10-30 U/ml), respectively. Values in the control group were as follows: tryptase 1-340 µg/l (mean 24.2 ± 58.2), histamine 5.0-22.0 ng/ml (mean 14.7 ± 3.9) and DAO 0-114 U/ml (mean 21.1 ± 27.8). 19/55 (34.5%) of the controls had elevated tryptase levels >11.4 µg/l, with four of them showing values >45 µg/ml. Significantly higher histamine levels were seen in blood samples taken more than 24h post mortem (p<0.05), whereas the timing of blood collection had no effect on tryptase and DAO levels. While moderately elevated tryptase levels are common in post mortem sera, values above 45 µg/l may support the diagnosis of fatal anaphylaxis. Strongly elevated histamine levels might give an additional clue on fatal anaphylaxis, whereas DAO does not seem to be helpful.

Inhibition of semicarbazide-sensitive amine oxidase attenuates myocardial ischemia-reperfusion injury in an in vivo rat model.

AIMS: This study tested the hypothesis that the inhibition of semicarbazide-sensitive amine oxidase (SSAO) after ischemia could attenuate myocardial ischemia-reperfusion (I/R) injury. MAIN METHODS: Anesthetized male Sprague-Dawley rats underwent myocardial I/R injury. Saline, semicarbazide (SCZ, 30 mg/kg), hydralazine (HYD, 10mg/kg), or LJP 1207 (30 mg/kg) was administered intraperitoneally 3 min before reperfusion. After 30 min of ischemia and 180 min of reperfusion, the myocardial infarct size was determined using nitroblue tetrazolium staining. Myocardial myeloperoxidase activity was determined through biochemical assay. HE staining was used for histopathological evaluation. Myocardial SSAO activity was assayed with high performance liquid chromatography analysis. Additionally, the endothelial expression of P-selectin was evaluated using immunohistochemistry after 30 min of ischemia and 20 min of reperfusion. KEY FINDINGS: Myocardial SSAO activity was increased in myocardial I/R injury. Administration of SCZ, HYD, or LJP 1207 reduced the myocardial infarct size and decreased leukocyte infiltration and endothelial P-selectin expression in myocardial I/R injury in vivo. SIGNIFICANCE: These data suggest that myocardial I/R injury up-regulates myocardial SSAO activity, and the inhibition of SSAO prior to reperfusion is able to attenuate acute myocardial I/R injury.

Monoamine oxidase and semicarbazide-sensitive amine oxidase kinetic analysis in mesenteric arteries of patients with type 2 diabetes.

Monoamine oxidase (MAO, type A and B) and semicarbazide-sensitive amine oxidase (SSAO) metabolize biogenic amines, however, the impact of these enzymes in arteries from patients with type 2 diabetes remains poorly understood. We investigated the kinetic parameters of the enzymes to establish putative correlations with noradrenaline (NA) content and patient age in human mesenteric arteries from type 2 diabetic patients. The kinetic parameters were evaluated by radiochemical assay and NA content by high-performance liquid chromatography (HPLC). The activity of MAO-A and SSAO in type 2 diabetic vascular tissues was significantly lower compared to the activity obtained in non-diabetic tissues. In the correlation between MAO-A (K(m)) and NA content, we found a positive correlation for both the diabetic and non-diabetic group, but no correlation was established for patient age. In both groups, MAO-B (V(max)) showed a negative correlation with age. The results show that MAO-A and SSAO activities and NA content of type 2 diabetic tissues are lower compared to the non-diabetic tissues, while MAO-B activity remained unchanged. These remarks suggest that MAO-A and SSAO may play an important role in vascular tissue as well as in the vascular pathophysiology of type 2 diabetes.

Absolute quantification of semicarbazide-sensitive amine oxidase in human umbilical artery by single-reaction monitoring with electrospray tandem mass spectrometry.

Semicarbazide-sensitive amine oxidase (SSAO; E.C.1.4.3.6.) is widely distributed in different tissues, particularly in vascular smooth muscle and adipose tissue. Its physiological function remains unclear. Up to now, the common method to determine SSAO is based on enzymatic activity measurement. However, enzymatic activity could be easily influenced by the temperature, pH, and circumstance. In the present study, we have developed the single-reaction monitoring (SRM) approach for measuring the absolute amount of SSAO expression in human umbilical artery based on LC-ESI-MS/MS. The measurement of protein was converted to the measurement of a unique peptide of SSAO from Homo sapiens. The peptide (YQLAVTQR) was confirmed to be unique to the SSAO in human using the ExPasy blast tools, and thus the synthetic peptide was used as the standard which can produce abundant parent ion (m/z = 490.0) and daughter ion (m/z = 687.4) in the mass spectrometry. Trap drive and fragmentation energy of MS/MS of the unique peptide was 60 V and 0.6 V, respectively. The calibration curve was linear over the range of 1.99-127.8 fmol/microL, with 1.99 fmol/microl of the lower limit of quantification. The inter- and intra-day precisions and recoveries for all samples were satisfactory. The results demonstrated SSAO protein concentration was 7.75 fmol/g wet weight. It proved that the novel assay was sensitive and selective to measure the amount of SSAO protein originated from H. sapiens.

Zymographic assay of plant diamine oxidase on entrapped peroxidase polyacrylamide gel electrophoresis. A study of stability to proteolysis.

A zymographic assay of diamine oxidase (DAO, histaminase, EC 1.4.3.6), based on a coupled peroxidase reaction, and its behavior at proteolysis in simulated gastric and intestinal conditions, are described. The DAO activity from a vegetal extract of Lathyrus sativus seedlings was directly determined on sodium dodecyl sulfate polyacrylamide electrophoretic gels containing entrapped horseradish peroxidase, with putrescine as substrate of histaminase and ortho-phenylenediamine as co-substrate of peroxidase. The accumulation of azo-aniline, as peroxidase-catalyzed oxidation product, led to well-defined yellow-brown bands on gels, with intensities corresponding to the enzymatic activity of DAO. After image analysis of gels, a linear dependency of DAO content (Coomassie-stained protein bands) and of its enzymatic activity (zymographic bands) with the concentration of the vegetal extract was obtained. In simulated gastric conditions (pH 1.2, 37 degrees C), the DAO from the vegetal extract lost its enzymatic activity before 15 min of incubation, either in the presence or absence of pepsin. The protein pattern (Coomassie-stained) revealed that the DAO content from the vegetal extract was kept almost constant in the simulated intestinal fluid (containing pancreatin or not), with a slight diminution in the presence of pancreatic proteases. After 10 h of incubation at 37 degrees C, the DAO enzymatic activity from the vegetal extract was 44.7% in media without pancreatin and 13.6% in the presence of pancreatin, whereas the purified DAO retained only 4.65% of its initial enzymatic activity in the presence of pancreatin.

Substrate-selective supramolecular tandem assays: monitoring enzyme inhibition of arginase and diamine oxidase by fluorescent dye displacement from calixarene and cucurbituril macrocycles.

A combination of moderately selective host-guest binding with the impressive specificity of enzymatic transformations allows the real-time monitoring of enzymatic reactions in a homogeneous solution. The resulting enzyme assays ("supramolecular tandem assays") exploit the dynamic binding of a fluorescent dye with a macrocyclic host in competition with the binding of the substrate and product. Two examples of enzymatic reactions were investigated: the hydrolysis of arginine to ornithine catalyzed by arginase and the oxidation of cadaverine to 5-aminopentanal by diamine oxidase, in which the substrates have a higher affinity to the macrocycle than the products ("substrate-selective assays"). The depletion of the substrate allows the fluorescent dye to enter the macrocycle in the course of the enzymatic reaction, which leads to the desired fluorescence response. For arginase, p-sulfonatocalix[4]arene was used as the macrocycle, which displayed binding constants of 6400 M(-1) with arginine, 550 M(-1) with ornithine, and 60,000 M(-1) with the selected fluorescent dye (1-aminomethyl-2,3-diazabicyclo[2.2.2]oct-2-ene); the dye shows a weaker fluorescence in its complexed state, which leads to a switch-off fluorescence response in the course of the enzymatic reaction. For diamine oxidase, cucurbit[7]uril (CB7) was used as the macrocycle, which showed binding constants of 4.5 x 10(6) M(-1) with cadaverine, 1.1 x 10(5) M(-1) with 1-aminopentane (as a model for the thermally unstable 1-aminopentanal), and 2.9 x 10(5) M(-1) with the selected fluorescent dye (acridine orange, AO); AO shows a stronger fluorescence in its complexed state, which leads to a switch-on fluorescence response upon enzymatic oxidation. It is demonstrated that tandem assays can be successfully used to probe the inhibition of enzymes. Inhibition constants were estimated for the addition of known inhibitors, i.e., S-(2-boronoethyl)-L-cysteine and 2(S)-amino-6-boronohexanoic acid for arginase and potassium cyanide for diamine oxidase. Through the sequential coupling of a "product-selective" with a "substrate-selective" assay it was furthermore possible to monitor a multistep biochemical pathway, namely the decarboxylation of lysine to cadaverine by lysine decarboxylase followed by the oxidation of cadaverine by diamine oxidase. This "domino tandem assay" was performed in the same solution with a single reporter pair (CB7/AO).

Alteration of serum semicarbazide-sensitive amine oxidase activity in chronic renal failure.

Despite recent intensive investigations, physiological and pathological role of semicarbazide-sensitive amine oxidase (SSAO) is far from clear. In this study, serum SSAO activity was determined, radiochemically, in various groups of uremic patients: haemodialysed (HD), peritoneally dialysed (PD) and those receiving conservative treatment but still not dialysed (ND), as well as in controls. Reduced enzyme activity was found in HD uremic patients before and after dialysis treatment, compared to controls (5260 +/- 862 and 6011 +/- 958 pmol/h/ml vs. 8601 +/- 283 pmol/h/ml, p < 0.01 and p < 0.05, respectively). The activity was slightly lower in PD, and normal in ND patients. In HD patients SSAO activity was also determined by an assay based on the formation of hydrogen peroxide, and was found to be elevated compared to controls (2384 +/- 323 pmol/h/ml vs. 1437 +/- 72 pmol/h/ml, p < 0.05). The elevated serum SSAO activity measured through the detection of the enzyme-generated hydrogen peroxide in HD patients might indicate its contribution to the accelerated atherosclerotic disease observed in uremia.

Mechanisms of copper loading on the Schizosaccharomyces pombe copper amine oxidase 1 expressed in Saccharomyces cerevisiae.

Copper amine oxidases (CAOs) are found in almost every living kingdom. Although Saccharomyces cerevisiae is one of the few yeast species that lacks an endogenous CAO, heterologous gene expression of CAOs from other organisms produces a functional enzyme. To begin to characterize their function and mechanisms of copper acquisition, two putative cao(+) genes from Schizosaccharomyces pombe were expressed in S. cerevisiae. Expression of spao1(+) resulted in the production of an active enzyme capable of catalysing the oxidative deamination of primary amines. On the other hand, expression of spao2(+) failed to produce an active CAO. Using a functional spao1(+)-GFP fusion allele, the SPAO1 protein was localized in the cytosol. Under copper-limiting conditions, yeast cells harbouring deletions of the MAC1, CTR1 and CTR3 genes were defective in amine oxidase activity. Likewise, atx1Delta null cells exhibited no CAO activity, while ccc2Delta mutant cells exhibited decreased levels of amine oxidase activity, and mutations in cox17Delta and ccs1Delta did not cause any defects in this activity. Copper-deprived S. cerevisiae cells expressing spao1(+) required a functional atx1(+) gene for growth on minimal medium containing ethylamine as the sole nitrogen source. Under these conditions, the inability of the atx1Delta cells to utilize ethylamine correlated with the lack of SPAO1 activity, in spite of the efficient expression of the protein. Cells carrying a disrupted ccc2Delta allele exhibited only weak growth on ethylamine medium containing a copper chelator. The results of these studies reveal that expression of the heterologous spao1(+) gene in S. cerevisiae is required for its growth in medium containing ethylamine as the sole nitrogen source, and that expression of an active Schiz. pombe SPAO1 protein in S. cerevisiae depends on the acquisition of copper through the high-affinity copper transporters Ctr1 and Ctr3, and the copper chaperone Atx1.

Histamine inactivation in the colon of pigs in relationship to abundance of catabolic enzymes.

OBJECTIVE: Catabolism of histamine plays a crucial role in the intestine in preventing intoxication by luminal histamine. Two enzymes are involved, namely histamine N-methyltransferase (HMT) and diamine oxidase (DAO). The purpose of this study was to find a link between histamine catabolism and the activities of HMT and DAO. MATERIAL AND METHODS: Epithelia of porcine proximal colon were mounted in Ussing chambers. After mucosal addition of (3)H-histamine (100 micromol x l(-1)) and (14)C-mannitol, the appearance of non-catabolized histamine, (3)H-histamine label (hist-rad) and (14)C-mannitol label were measured in parallel on the serosal side. Activities of HMT and DAO were determined in the proximal colon and proximal jejunum. RESULTS: Differences between the fluxes of histamine and hist-rad indicated catabolic conversion of 81.4+/-1.6% histamine during epithelial transit. Fluxes of hist-rad and histamine increased linearly with increasing mannitol fluxes but the percentage of catabolized histamine was not related to either mannitol or hist-rad fluxes. However, the percentage of catabolized histamine rose with increasing DAO activity. Given a negative correlation between DAO and HMT activities, the fraction of catabolized histamine decreased with increasing HMT activity. HMT activity was comparable in the colon and jejunum, but DAO activity was approximately nine times higher in the jejunum. CONCLUSIONS: Permeation, but not the relative efficiency of catabolism, of histamine depends on epithelial/paracellular tightness. While previous studies have shown that colonic HMT essentially catabolizes the bulk of histamine during permeation, DAO activity seems to be more variable and limiting for the overall efficiency of the catabolic process.