Search

Background: Diagnosis of acute kidney injury (AKI) relies on serum creatinine (SCr) changes. This study investigated if urinary aminopeptidases are early and predictive biomarkers of cardiac surgery-associated AKI (CSA-AKI)., Methods: Glutamyl aminopeptidase (GluAp), alanyl aminopeptidase (AlaAp), dipeptidyl peptidase-4 (DPP4), proteinuria, albuminuria, N-acetyl-β- D -glucosaminidase (NAG), and neutrophile gelatinase-associated lipocalin (NGAL) were measured in urine samples from 44 patients at arrival in the intensive care unit (ICU) after cardiac surgery. Sensitivity, specificity, and positive and negative predictive value for diagnosis of stages 1, 2, and 3 of AKI were analyzed for the highest quartile of each marker. We also studied the relationship with SCr after surgery, 6- and 12-month glomerular filtration rates (GFRs), and other long-term events over the next 5 years., Results: GluAp diagnosed the maximal number of patients that developed stage 2 or 3 of AKI, increasing diagnostic sensitivity from 0% to 75%. In addition, GluAp and DPP4 were related to the decrease in GFR at 6 or 12 months after surgery., Conclusions: Urinary aminopeptidases are a potential tool for the early diagnosis of CSA-AKI, with GluAp being the most effective marker for diagnosing stage 2 or 3 of AKI at ICU admission. GluAp and DPP4 serve as predictive biomarkers for a decrease in GFR.

Background: Fibromyalgia (FM) is a complex syndrome of uncertain etiology, characterized by the presence of widespread pain. Both nitric oxide and enkephalinases modulate pain perception., Objectives: The aim of this study was to evaluate the relationships among serum nitric oxide levels, oxytocinase activity, and enkephalin-degrading aminopeptidase (EDA) activity with pain-related clinical manifestations in women with FM., Methods: We performed an observational case study in a population of 58 women diagnosed with FM. Serum nitric oxide levels were analyzed by an ozone chemiluminescence-based assay. Both serum oxytocinase and EDA activities were fluorometrically determined. Pain threshold and pain magnitude were evaluated using the PainMatcher. The pressure pain thresholds were measured using a digital pressure algometer. We used a visual analog scale, the Central Sensitization Inventory, the Revised Fibromyalgia Impact Questionnaire, and the Beck Anxiety Inventory to assess the global level of pain, the symptoms associated with the central sensitization syndrome, the severity of FM, and the anxiety level, respectively., Results: Multiple linear regression analysis adjusted by age, body mass index, and menopause status revealed significant associations between nitric oxide levels and dominant occiput pressure pain thresholds, nondominant occiput pressure pain thresholds, and FM effects. Significant associations of oxytocinase activity with the visual analog scale and dominant knee pressure pain thresholds were also found. Moreover, results showed a significant association between high EDA activity levels and dominant second-rib pressure pain thresholds., Discussion: Our data have shown significant relationships of serum nitric oxide levels and oxytocinase and EDA activities with some body pressure pain thresholds, the daily activity level, and the global intensity of pain in women with FM. These results suggest that pain, which is the main symptom of this syndrome, may be related to alterations in nitric oxide levels and in oxytocinase and EDA activities in patients with FM., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Aminopeptidases (APs) are metalloenzymes that hydrolyze peptides and polypeptides by scission of the N-terminus amino acid and that also participate in the intracellular final digestion of proteins. APs play an important role in protein maturation, signal transduction, and cell-cycle control, among other processes. These enzymes are especially relevant in the control of cardiovascular and renal functions. APs participate in the regulation of the systemic and local renin-angiotensin system and also modulate the activity of neuropeptides, kinins, immunomodulatory peptides, and cytokines, even contributing to cholesterol uptake and angiogenesis. This review focuses on the role of four key APs, aspartyl-, alanyl-, glutamyl-, and leucyl-cystinyl-aminopeptidases, in the control of blood pressure (BP) and renal function and on their association with different cardiovascular and renal diseases. In this context, the effects of AP inhibitors are analyzed as therapeutic tools for BP control and renal diseases. Their role as urinary biomarkers of renal injury is also explored. The enzymatic activities of urinary APs, which act as hydrolyzing peptides on the luminal surface of the renal tubule, have emerged as early predictive renal injury biomarkers in both acute and chronic renal nephropathies, including those induced by nephrotoxic agents, obesity, hypertension, or diabetes. Hence, the analysis of urinary AP appears to be a promising diagnostic and prognostic approach to renal disease in both research and clinical settings.

Background: The neuronal ceroid lipofuscinosis 2 (NCL2) or classic late-infantile neuronal ceroid lipofuscinosis (LINCL) is a neurogenetic disorder caused by mutations in the TPPI gene, which codes for the lysosomal tripeptidyl peptidase 1 (TPPI) EC 3.4.14.9. Loss of functional TPPI activity results in progressive visual and neurological symptoms starting at around 1-2 years of age causing early death., Methods: We report a DBS-based TPPI assay that cleaves a synthetic tetrapeptide substrate generating a product that is detected by HPLC. Probands and carriers were identified with 100% accuracy (7 probands, 30 carriers, 13 controls)., Results: The assay detected a single TPPI activity at a lower pH towards the substrate tested. TPPI activity measurable when extracted at lower pH while inactive at neutral pH showed steady increase for at least 8 h incubation. No loss in TPPI activity was observed when DBS were stored for at least 2 weeks either in freezer, refrigerator, room temperature or 42 °C., Conclusion: A sequence variant causing Arg339Gln substitution in a proband had 12% TPPI. TPPI activity can be reliably measured in DBS, giving an opportunity to diagnose NCL2 at birth and refer patients for enzyme replacement or other therapies for earliest intervention, or alternatively offers a second-tier confirmatory test., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The search for novel chemical classes of anti-malarial compounds to cope with the current state of chemoresistance of malaria parasites has led to the identification of Plasmodium falciparum aminopeptidase 1 (PfA-M1) as a new therapeutic target. PfA-M1, known to be involved in the hemoglobin digestion cascade which helps to provide most of the amino acids necessary to the parasite's metabolism, is currently considered as a promising target for anti-malarial chemotherapy. However, its immunogenic properties have not yet been tested in the Gabonese population. In Gabon, the prevalence of malaria remains three times higher in semi-urban areas (60·12%) than in urban areas (17·06%). We show that malaria-specific PfA-M1 antibodies are present in children and increase with the level of infection. Children living in semi-urban areas have higher anti-PfA-M1 antibody titers (0·14 ± 0·02 AU) than those living in urban areas (0·08 ± 0·02 AU, P = 0·03), and their antibody titers increase with age (P < 0·0001). Moreover, anti-PfA-M1 antibody titers decrease in children with hyperparasitemia (0·027 ± 0·055 AU) but they remain high in children with low parasite density (0·21 ± 0·034 AU, P = 0·034). In conclusion, our results suggest that malaria-specific PfA-M1 antibodies may play an important role in the immune response of the host against P. falciparum in Gabonese children. Further studies on the role of PfA-M1 during anemia are needed., (© 2020 British Society for Immunology.)

Objectives: Fibromyalgia (FM) is a chronic pain condition of unclear etiology. We have analyzed, for the first time, the activity of a broad spectrum of aminopeptidases (APs) in patients with FM and controls to investigate whether they are involved in the pathophysiology of this syndrome., Method: In this case-control study, we fluorometrically measured specific AP activities in serum samples of 75 patients with FM and 29 healthy controls. The predictive value of AP activities in FM was determined by receiver operating characteristic (ROC) analysis., Results: Oxytocinase activity was higher in patients with FM than in controls ( p < .001). A subgroup of patients with FM ( n = 18; 24%) showed low levels of enkephalin-degrading aminopeptidase (EDA) activity when compared with the healthy controls ( p < .001) and with the rest of FM patients ( p < .001). There were no significant differences in the activity levels of aminopeptidase A, aminopeptidase B, aspartyl aminopeptidase, insulin-regulated aminopeptidase, pyroglutamyl aminopeptidase, or aminopeptidase N between FM patients and controls. According to ROC analysis, oxytocinase activity may be a good marker for differentiating individuals with FM from healthy subjects., Conclusions: Our findings show that serum oxytocinase activity is increased in patients with FM, which could alter the metabolism of peptides with analgesic effects such as oxytocin and enkephalins. The determination of serum oxytocinase activity may aid in FM diagnosis. Additionally, we have identified a subpopulation of FM patients with abnormally low serum EDA activity.

Purpose: CLN2 disease is a genetic disorder caused by dysfunction of the lysosomal enzyme tripeptidyl peptidase 1 (TPP1) that belongs to the neuronal ceroid lipofuscinoses (NCL) and leads to epilepsy, dementia, and death in young persons. CLN2 disease has recently become treatable by enzyme replacement, which can only be effective when the disease is diagnosed early. We have investigated the reliability of a test for TPP1 deficiency in dried blood specimens (DBS) to detect CLN2 disease., Results: During a 12-year period we have received 3882 samples for testing TPP1. Quality of samples was checked by measuring two additional lysosomal enzyme activities. For 50 samples with subnormal TPP1 activity and good sample quality, we obtained adequate clinical and molecular genetic data. All 50 patients had doubtless evidence of CLN2 disease (including seven atypical patients) as shown by clinical findings and the presence of known pathogenic CLN2 variants. Our institution is a major reference center for NCL, and we have never received information that a patient with a normal DBS test was later diagnosed with CLN2 disease., Conclusions: We consider our TPP1 test on DBS to be a reliable, convenient and inexpensive tool for a first diagnostic step in suspected CLN2 disease., (Copyright © 2019. Published by Elsevier B.V.)

Endoplasmic reticulum aminopeptidase 1 (ERAP1) is a multi-functional enzyme. In this study, we analysed its role in lipopolysaccharide-induced inflammatory response in wild-type and ERAP1-knockout mice. Following lipopolysaccharide injection, ERAP1 was secreted into the blood, increasing leucine aminopeptidase activity and NO synthesis therein. Among the amino acids tested, arginine concentration was significantly increased in wild-type mice compared to ERAP1-knockout mice. These results suggest that ERAP1 behaves similar to acute-phase proteins, which are secreted into the blood in response to infectious/inflammatory stimuli and are involved in enhancing NO synthesis as a host defense mechanism.

We first characterized PPT1 and TPP1 enzymes in dried blood spots (DBS), plasma/serum, and leukocytes/lymphocytes using neuronal ceroid lipofuscinosis (NCL) 1 and 2 patients and control subjects. PPT1 enzyme had only one acid form in control DBS, plasma/serum, and leukocytes/lymphocytes and showed deficient activities in these samples from NCL 1 patients. Conversely, TPP1 enzymes in control DBS and leukocytes/lymphocytes consisted of two forms, an acidic form and a neutral form, whereas serum TPP1 enzyme had only a neutral form. In control subjects, the optimal pH of PPT1 enzyme in DBS, plasma/serum, and leukocytes/lymphocytes was 4.5 to 5.0 in the acidic form, whereas TPP1 enzyme in control DBS and leukocytes/lymphocytes was pH 4.5 and 6.5, respectively. In NCL 1 and 2, both PPT1 and TPP1 enzyme activities in DBS, plasma, and leukocytes/lymphocytes were markedly reduced in acidic pH, whereas heterozygotes of NCL 1 and 2 in the acidic form showed intermediate activities between patients and control subjects. In neutral conditions, pH 6.0, the PPT1 enzyme activities in NCL 1 patients showed rather higher residual activities and intermediate activities in heterozygotes in NCL 1, which was probably caused by mutated proteins in three cases with NCL 1 patients. TPP1 enzyme activities at neutral pH 6.5 to 7.0 in DBS and leukocytes/lymphocytes showed higher enzyme activities in NCL 2 patients and heterozygotes. The reason for the increases of neutral TPP1 enzyme activities at pH 6.5 to 7.0 in NCL 2 DBS and leukocytes/lymphocytes, is obscure, but possibly caused by secondary activation of neutral TPP1 enzyme due to the absence of the acidic form. Interestingly, TPP1 activity in serum only consisted of a neutral form, no acidic form, and was not deficient in any NCL 2 patient. Therefore, we can diagnose NCL 1 patients by plasma/serum enzyme assay of PPT1, but not diagnose NCL 2 by serum TPP1 enzyme assay. A pilot study of newborn screening of NCL 1 and 2 has been established by more than 1000 newborn DBS assays. Using this assay system, we will be able to perform newborn screening of NCL 1 and 2 by DBS., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Aims: Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) differ in their renin-angiotensin system function and sympathetic tone. The metabolism of angiotensins and vasopressin depends on the action of certain aminopeptidases whose activity may be influenced by the autonomic nervous system. Their regulation may differ between WKY and SHR in hypothalamus and plasma according to the sympathetic tone. We analyzed aminopeptidases responsible for the hydrolysis of certain angiotensins, vasopressin, cholecystokinin or enkephalins in hypothalamus and plasma of WKY and SHR in untreated controls rats and under beta-adrenoceptor blockade. Systolic blood pressure, food intake, water intake and diuresis were measured as parameters modulated by the autonomic nervous system and the above mentioned peptides., Main Methods: Glutamyl-, aspartyl-, cystinyl- and alanyl-aminopeptidase activities were analyzed fluorimetrically in plasma and hypothalamus of control and propranolol-treated (100mg/kg/day administered in drinking water for 1month) WKY and SHR, using arylamide derivatives as substrates., Key Findings: An opposite response of aminopeptidases to propranolol treatment between plasma and hypothalamus was observed in either WKY and SHR. Furthermore, the behavior of aminopeptidases was inversed between WKY and SHR either in hypothalamus and plasma: while the activity increased in hypothalamus and decreased in plasma of WKY, it decreased in hypothalamus and increased in plasma of SHR., Significance: These results revealed an inverse response of aminopeptidases between hypothalamus and plasma and also an opposite behavior of these enzymes between WKY and SHR in hypothalamus and plasma. These observations support the involvement of the sympathetic system in the modulation of aminopeptidase activities., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Activities of aminopeptidases A, B, and N (ApA, ApB & ApN) and insulin-regulated aminopeptidase (IRAP) have been seen to be decreased amongst patients with Alzheimer's disease (AD). All of these enzymes are involved with the brain renin-angiotensin system which is believed to be involved with learning and memory. This study aimed to explore the time course and the mechanisms underlying these changes. Serum samples were collected from 45 AD patients at the start of the study, and again 13months later (n=37). The control group was 22 healthy, older, adults. Enzyme activity was determined at two substrate concentrations to allow Michaelis-Menten analysis of the enzyme activity. The results indicated that there was decreased activity of ApA, ApB and ApN amongst AD patients but no difference in serum IRAP activity. There were no associations between enzyme activity and age, gender nor scores on psychomotor tests. Consideration of the data for the two time points for AD patients showed that the changes in ApB occurred at an early stage of the disease and persisted, whilst those of ApA and ApN only became apparent at later stages of the disease. Although differences in Michaelis-Menten parameters were not statistically significant, consideration of the values suggested that the decrease in ApB activity may be a result of changes in enzyme protein conformation, whilst that of ApN may be a consequence of decreased enzyme expression. Importantly, the different time courses of the effects and the differential changes in enzyme affinity and expression indicated that the observed changes with progression of AD were not a 'class effect' for serum aminopeptidases but were idiosyncratic for the individual enzymes., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Serum or plasma proteases have been associated with various diseases including cancer, inflammation, or reno-cardiovascular diseases. We aimed to investigate whether the enzymatic activities of serum proteases are associated with the estimated glomerular filtration rate (eGFR) in patients with different stages of chronic kidney disease (CKD). Our study population comprised 268 participants of the "Greifswald Approach to Individualized Medicine" (GANI&#95;MED) cohort. Enzymatic activity of aminopeptidase A, aminopeptidase B, alanyl (membrane) aminopeptidase, insulin-regulated aminopeptidase, puromycin-sensitive aminopeptidase, leucine aminopeptidase 3, prolyl-endopeptidase (PEP), dipeptidyl peptidase 4 (DPP4), angiotensin I-converting enzyme, and angiotensin I-converting enzyme 2 (ACE2) proteases was measured in serum. Linear regression of the respective protease was performed on kidney function adjusted for age and sex. Kidney function was modeled either by the continuous Modification of Diet in Renal Disease (MDRD)-based eGFR or dichotomized by eGFR < 15 mL/min/1.73 m 2 or <45 mL/min/1.73 m 2 , respectively. Results with a false discovery rate below 0.05 were deemed statistically significant. Among the 10 proteases investigated, only the activities of ACE2 and DPP4 were correlated with eGFR. Patients with lowest eGFR exhibited highest DPP4 and ACE2 activities. DPP4 and PEP were correlated with age, but all other serum protease activities showed no associations with age or sex. Our data indicate that ACE2 and DPP4 enzymatic activity are associated with the eGFR in patients with CKD. This finding distinguishes ACE2 and DPP4 from other serum peptidases analyzed and clearly indicates that further analyses are warranted to identify the precise role of these serum ectopeptidases in the pathogenesis of CKD and to fully elucidate underlying molecular mechanisms. Impact statement • Renal and cardiac diseases are very common and often occur concomitantly, resulting in increased morbidity and mortality. Understanding of molecular mechanisms linking both diseases is limited, available fragmentary data point to a role of the renin-angiotensin system (RAS) and, in particular, Ras-related peptidases. • Here, a comprehensive analysis of serum peptidase activities in patients with different stages of chronic kidney disease (CKD) is presented, with special emphasis given to RAS peptidases • The serum activities of the peptidases angiotensin I-converting enzyme 2 and dipeptidyl peptidase 4 were identified as closely associated with kidney function, specifically with the estimated glomerular filtration rate. The findings are discussed in the context of available data suggesting protective roles for both enzymes in reno-cardiac diseases. • The data add to our understanding of pathomechanisms underlying development and progression of CKD and indicate that both enzymes might represent potential pharmacological targets for the preservation of renal function.

Neuronal ceroid lipofuscinoses (NCLs) are a heterogeneous group of lysosomal storage disorders. NCLs include the rare autosomal recessive neurodegenerative disorder neuronal ceroid lipofuscinosis type 2 (CLN2) disease, caused by mutations in the tripeptidyl peptidase 1 (TPP1)/CLN2 gene and the resulting TPP1 enzyme deficiency. CLN2 disease most commonly presents with seizures and/or ataxia in the late-infantile period (ages 2-4), often in combination with a history of language delay, followed by progressive childhood dementia, motor and visual deterioration, and early death. Atypical phenotypes are characterized by later onset and, in some instances, longer life expectancies. Early diagnosis is important to optimize clinical care and improve outcomes; however, currently, delays in diagnosis are common due to low disease awareness, nonspecific clinical presentation, and limited access to diagnostic testing in some regions. In May 2015, international experts met to recommend best laboratory practices for early diagnosis of CLN2 disease. When clinical signs suggest an NCL, TPP1 enzyme activity should be among the first tests performed (together with the palmitoyl-protein thioesterase enzyme activity assay to rule out CLN1 disease). However, reaching an initial suspicion of an NCL or CLN2 disease can be challenging; thus, use of an epilepsy gene panel for investigation of unexplained seizures in the late-infantile/childhood ages is encouraged. To confirm clinical suspicion of CLN2 disease, the recommended gold standard for laboratory diagnosis is demonstration of deficient TPP1 enzyme activity (in leukocytes, fibroblasts, or dried blood spots) and the identification of causative mutations in each allele of the TPP1/CLN2 gene. When it is not possible to perform both analyses, either demonstration of a) deficient TPP1 enzyme activity in leukocytes or fibroblasts, or b) detection of two pathogenic mutations in trans is diagnostic for CLN2 disease., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Objectives: Glucocorticoids (GC) remain a cornerstone of rheumatoid arthritis (RA) therapy, although a third of patients do not respond adequately. In order to find potential predictors for clinical response, the gene expression profile of CD4+T-cells as important players in the pathogenesis of RA was analysed before pulse therapy with 1000 mg methylprednisolone., Methods: Patients were treated with 3x1000 mg methylprednisolone in 5 days; hereafter response was determined by the European League Against Rheumatism (EULAR) response criteria. Before start of treatment, CD4+T-cells (and CD14+monocytes) were separated by MACS sorting. Labelled cRNA from CD4+T-cells from 5 responders and 5 non-responders was hybridised to Agilent 4x44K microarray chips and differentially expressed genes were identified via mixed-model analysis of variance based on permutation-based false discovery rates. Selected genes were validated by quantitative real-time PCR (qPCR)., Results: Four genes were significantly increased in CD4+T-cells of GC-responders; expression of ERAP2 (endoplasmic reticulum aminopeptidase 2), LST1 (leucocyte-specific transcript 1) and FAM26F (Family With Sequence Similarity 26, Member F) was confirmed by quantitative PCR (qPCR); their expression was inversely correlated with DAS28 at day 5 (LST1 and FAM26F p<0.05; ERAP2: p=0.07). Elevated expression of ERAP2 was also detected by qPCR in CD14+monocytes and after 24 hours in both cell types (all p<0.02)., Conclusions: The increased expression of ERAP2, LST1 and FAM26F in GC-responders before therapy warrants further investigation into their role as potential predictors for the response to GC, and in the inflammatory process of RA.

Background/aims: To establish the prevalence of the single nucleotide polymorphisms (SNPs) of endoplasmic reticulum aminopeptidase 1 (ERAP1), IL-23 receptor (IL-23R), signal transducer and activator of transcription 3 (STAT-3) and Janus kinase 2 (JAK-2) in ankylosing spondylitis (AS) and inflammatory bowel disease (IBD) in a Turkish population., Materials and Methods: A total of 562 subjects who presented at the Ankara University internal medicine departments of rheumatology and gastroenterology outpatient clinics were recruited in this study, including 365 patients with AS, 197 patients with IBD and 230 healthy controls. ERAP1, IL-23R, STAT-3 and JAK-2) were genotyped in competitive allele-specific polymerase chain reactions., Results: The ERAP1 (rs26653) polymorphism was found to increase the disease risk in patients with AS and IBD compared with the control group (p=0.02 and p=0.01, respectively). In addition, this polymorphism revealed a significant relationship with the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) and the Bath AS Functional Index (BASFI) in patients with AS (r=0.829, p < 0.001 and r=0.731, p < 0.001, respectively)., Conclusion: The ERAP1 gene polymorphism might be a risk factor in the pathogenesis of AS and IBD. In contrast, IL-23R gene polymorphisms may serve a protective role in AS and IBD.

The plasma activity of nine aminopeptidases was monitored over a year in first-episode psychotic patients. We observed significant differences in aminopeptidase B (APB), aminopeptidase N (APN) and dipeptidyl peptidase IV (DPPIV), but not in puromycin-sensitive aminopeptidase (PSA), prolyl endopeptidase (PEP), cysteine aminopeptidase (Cys-AP), aspartate aminopeptidase (Asp-AP), glutamate aminopeptidase (Glu) or piroglutamate aminopeptidase (PGI) in these patients compared to controls, and also a progressive increase in plasma activity, correlated to changes in scores on clinical scales, Global Assessment of Functioning scale (GAF) and Hamilton Depression Rating Scale (HDRS), at 1 month of follow-up. At 1 month after diagnosis, the median score obtained by patients on the GAF was negatively associated with the plasma activity of APB and PEP measured at the beginning of the psychotic episode, indicating a role as a negative prognostic factor that can predict psychiatric symptomatology. In the case of HDRS, scores at 1 month after diagnosis were found to be positively associated with the initial plasma activity of DPPIV, APN and PSA, indicating that their initial elevation is a negative prognostic factor that can predict subsequent depressive symptomatology. Taken together, these results suggest a pathophysiological involvement of plasma peptidases and indicate that aminopeptidase activity can predict the course of first-episode psychosis patients, acting as a prognostic indicator., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Cancer is responsible for many deaths and is a major source of healthcare expenditures. The identification of new, non-invasive biomarkers might allow improvement of the direct diagnostic or prognostic ability of already available tools. Here, we took the innovative approach of interrogating the activity of exopeptidases in the serum of cancer patients with the aim of establishing a distinction based on enzymatic function, instead of simple protein levels, as a means to biomarker discovery. We first analyzed two well-characterized mouse models of prostate cancer, each with a distinct genetic lesion, and established that broad exopeptidase and targeted aminopeptidase activity tests reveal proteolytic changes associated with tumor development. We also describe new peptide-based freeze-frame reagents uniquely suited to probe the altered balance of selected aminopeptidases, as opposed to the full array of exopeptidases, and/or their modulators in patient serum or plasma. One particular proteolytic activity was impaired in animals with aggressive disease relative to cancer-free littermates. We identified the protease in question as dipeptidyl peptidase 4 (DPP4) by analyzing selected knockout mice and evaluating the effect of specific inhibitors. DPP4 activity was also reduced in the sera of patients with metastatic prostate cancer relative to patients with localized disease or healthy controls. However, no significant differences in DPP4 serum levels were observed, which established the loss of activity as the result of impaired enzymatic function. Biochemical analysis indicated that reduced activity was the result not of post-translational modifications or allosteric changes, but instead of a low-molecular-weight inhibitor. After we adjusted for age and total prostate-specific antigen, reduced DPP4 activity remained a significant predictor of cancer status. The results of this proof-of-principle study suggest that DPP4 activity might be a potential blood-based indicator of the presence of metastatic cancer of prostatic origin, either by itself or, more likely, as a means to improve the sensitivity and specificity of existing markers., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

The natural product fumagillin exhibits potent antiproliferative and antiangiogenic properties. The semisynthetic analog PPI-2458, [(3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methylbut-2-enyl)oxiran-2-yl]-1-oxaspiro[2.5]octan-6-yl] N-[(2R)-1-amino-3-methyl-1-oxobutan-2-yl]carbamate, demonstrates rapid inactivation of its molecular target, methionine aminopeptidase-2 (MetAP2), and good efficacy in several rodent models of cancer and inflammation with oral dosing despite low apparent oral bioavailability. To probe the basis of its in vivo efficacy, the metabolism of PPI-2458 was studied in detail. Reaction phenotyping identified CYP3A4/5 as the major source of metabolism in humans. Six metabolites were isolated from liver microsomes and characterized by mass spectrometry and nuclear resonance spectroscopy, and their structures were confirmed by chemical synthesis. The synthetic metabolites showed correlated inhibition of MetAP2 enzymatic activity and vascular endothelial cell growth. In an ex vivo experiment, MetAP2 inhibition in white blood cells, thymus, and lymph nodes in rats after single dosing with PPI-2458 and the isolated metabolites was found to correlate with the in vitro activity of the individual species. In a phase 1 clinical study, PPI-2458 was administered to patients with non-Hodgkin lymphoma. At 15 mg administered orally every other day, MetAP2 in whole blood was 80% inactivated for up to 48 hours, although the exposure of the parent compound was only ∼10% that of the summed cytochrome P450 metabolites. Taken together, the data confirm the participation of active metabolites in the in vivo efficacy of PPI-2458. The structures define a metabolic pathway for PPI-2458 that is distinct from that of TNP-470 ([(3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methylbut-2-enyl)oxiran-2-yl]-1-oxaspiro[2.5]octan-6-yl] N-(2-chloroacetyl)carbamate). The high level of MetAP2 inhibition achieved in vivo supports the value of fumagillin-derived therapeutics for angiogenic diseases.

Essential hypertension is one of the major contributors to premature morbidity and mortality due to the incresased risk for coronary heart disease, stroke, renal disease, peripheral vascular disease and vascular dementia for both men and women. However, its basic causes remain unknown. In the present work we studied the activity of several proteolytic regulatory enzymes related to renin-angiotensin-system (RAS) (aminopeptidase A, APA; aminopeptidase N, APN; aminopeptidase B, APB; and insulin-regulated aminopeptidase, IRAP); with oxytocin regulation (oxytocinase); with the metabolism of GnRH and TRH (pyrrolidone carboxypeptidase, Pcp); and with enkephalins metabolism (enkephalindegrading activity, EDA), to elucidate their role in the mechanisms responsible of essential hypertension and to discuss the possible gender differences. Serum samples of 53 individuals with essential hypertension and 60 healthy volunteers were collected and used to assay enzyme activities, gonad hormones testosterone and estradiol, TSH and free thyroxin (fT4). Differences were observed in APA, APN, Pcp and EDA specific activities, and in serum gonad hormone levels between hypertensive and control groups. Only Pcp activity showed gender differences. Regarding the RAS, APA is reduced while APN is increased, suggesting increased levels of angiotensin II and a facilitation of the conversion of angiotensin III in angiotensin IV. Thus, the changes in several RAS-regulating specific activities and other enzyme activities involved in the neuroendocrine modulation of gonad and stress-related functions are related to essential hypertension with minor gender differences. Therefore, aminopeptidases constitute new elements for the knowledge of the causes of essential hypertension and an alternative as therapeutic targets against the illness.

Purpose: The aim of this study was to investigate the putative changes in serum angiotensinase activities (aminopeptidase N, APN; aminopeptidase B, APB; aminopeptidase A, APA; aspartyl aminopeptidase, ASAP) involved in the renin-angiotensin system (RAS) in women with breast cancer treated or not with a neoadjuvant therapy of paclitaxel and anthracycline and in healthy women volunteers., Methods: We fluorometrically analysed serum APN, APB, APA and ASAP activities using their corresponding aminoacyl-β-naphthylamides as substrates in women with breast cancer treated with a neoadjuvant therapy of paclitaxel and anthracycline., Results: When compared with healthy controls, women with breast cancer not treated with neoadjuvant chemotherapy, showed a decrease in angiotensinase activity, which support the putative increase of angiotensin II (Ang II) levels, indicating that the tumour process would favour the development of the disease. Also, an increase in APN and APB activities was observed, which support a role for angiotensin IV (Ang IV). In women treated with a neoadjuvant therapy, we described an increase in ASAP and APA activities, supporting the idea that this treatment increases Ang II catabolism. The resulting decrease in Ang II level could lead to an inhibition of the tumour growth., Conclusion: Present results show changes in serum angiotensinase activities in women with breast cancer and in women with breast cancer treated with a neoadjuvant therapy of paclitaxel and anthracycline. Therefore, considerable attention should be focused on the development of RAS blockade therapy as a new strategy for breast cancer treatment., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Angiotensin I-converting enzyme inhibitors (ACEi) are widely used antihypertensive agents that are associated with a potentially life-threatening reaction, ACEi-angioedema. Impaired metabolism of bradykinin and des-Arg(9) -bradykinin by aminopeptidase P (APP) is a key contributor to ACEi-angioedema. This study aimed to characterize the genetic regulation of the XPNPEP2 gene and identify the genetic factors contributing to variance in plasma APP activity and ACEi-angioedema. Additive genetic factors accounted for 47.3% of variance in plasma APP activity in healthy individuals. Nested deletion analysis identified the minimal promoter (-338 bp to -147 bp) and an enhancer region (-2,502 bp to -2,238 bp). Three polymorphisms (c.-2399C>A, c.-1612G>T, and c.-393G>A) were significantly associated with plasma APP activity. Haplotype ATG was significantly associated with reduced reporter gene activity and with reduced plasma APP activity. The c.-2399C>A polymorphism was located in an enhancer region and was predicted to differentially bind hepatic nuclear factor 4 (HNF4). Over expression of HNF4 increased the activation of haplotype ATG compared with haplotype CGG. In a case control study of subjects with a history of ACEi-angioedema, haplotype ATG was significantly associated with ACEi-angioedema (OR 4.87 [1.78-13.35] P = 0.002). The ATG haplotype is functional and contributes to ACEi-angioedema through a reduction in APP., (© 2011 Wiley Periodicals, Inc.)

Late infantile neuronal ceroid lipofuscinosis (LINCL) is caused by mutations in the gene encoding tripeptidyl-peptidase 1 (TPP1). LINCL patients accumulate lysosomal storage materials in the CNS accompanied by neurodegeneration, blindness, and functional decline. Dachshunds homozygous for a null mutation in the TPP1 gene recapitulate many symptoms of the human disease. The objectives of this study were to determine whether intrathecal (IT) TPP1 treatment attenuates storage accumulation and functional decline in TPP1-/- Dachshunds and to characterize the CNS distribution of TPP1 activity. TPP1 was administered to one TPP1-/- and one homozygous wild-type (WT) dog. An additional TPP1-/- and WT dog received vehicle. Four IT administrations of 32 mg TPP1 formulated in 2.3 mL of artificial cerebrospinal fluid (aCSF) or vehicle were administered monthly via the cerebellomedullary cistern from four to seven months of age. Functional decline was assessed by physical and neurological examinations, electrophysiology, and T-maze performance. Neural tissues were collected 48 h after the fourth administration and analyzed for TPP1 activity and autofluorescent storage material. TPP1 was distributed at greater than WT levels in many areas of the CNS of the TPP1-/- dog administered TPP1. The amount of autofluorescent storage was decreased in this dog relative to the vehicle-treated affected control. No improvement in overall function was observed in this dog compared to the vehicle-treated TPP1-/- littermate control. These results demonstrate for the first time in a large animal model of LINCL widespread delivery of biochemically active TPP1 to the brain after IT administration along with a decrease in lysosomal storage material. Further studies with this model will be necessary to optimize the dosing route and regimen to attenuate functional decline., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Angiotensin peptides regulate vascular tone and natriohydric balance through the renin angiotensin system (RAS) and are related with the angiogenesis which plays an important role in the metastatic pathway. Estrogen influences the aminopeptidases (APs) involved in the metabolism of bioactive peptides of RAS through several pathways. We analyze RAS-regulating AP activities in serum of pre- and postmenopausal women with breast cancer to evaluate the putative value of these activities as biological markers of the development of breast cancer. We observed an increase in aminopeptidase N (APN) and aminopeptidase B (APB) activities in women with breast cancer; however, a decrease in aspartyl-aminopeptidase (AspAP) activity in premenopausal women. These results suggest a slow metabolism of angiotensin II (Ang II) to angiotensin III (Ang III) in premenopausal women and a rapid metabolism of Ang III to angiotensin IV (Ang IV) in pre- and postmenopausal women with breast cancer. An imbalance in the signals activated by Ang II may produce abnormal vascular growth with different response between pre- and postmenopausal women depending on the hormonal profile and the development of the disease., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The objective of this study was to investigate the catalytic activity of basic aminopeptidase (APB) and its association with periarticular edema and circulating tumor necrosis factor (TNF)-alpha and type II collagen (CII) antibodies (AACII) in a rat model of rheumatoid arthritis (RA) induced by CII (CIA). Edema does not occur in part of CII-treated, even when AACII is higher than in control. TNF-alpha is detectable only in edematous CII-treated. APB in synovial membrane is predominantly a membrane-bound activity also present in soluble form and with higher activity in edematous than in non-edematous CII-treated or control. Synovial fluid and blood plasma have lower APB in non-edematous than in edematous CII-treated or control. In peripheral blood mononuclear cells (PBMCs) the highest levels of APB are found in soluble form in control and in membrane-bound form in non-edematous CII-treated. CII treatment distinguishes two categories of rats: one with arthritic edema, high AACII, detectable TNF-alpha, high soluble and membrane-bound APB in synovial membrane and low APB in the soluble fraction of PBMCs, and another without edema and with high AACII, undetectable TNF-alpha, low APB in the synovial fluid and blood plasma and high APB in the membrane-bound fraction of PBMCs. Data suggest that APB and CIA are strongly related., (2011 Elsevier B.V. All rights reserved.)

Background/objective: Mitochondrial dysfunction, oxidative stress and protein metabolism impairment are the main molecular events underlying the pathogenesis of Parkinson's disease (PD). However, only few studies have addressed the changes produced by these phenomena in the blood of PD patients. Our purpose was to compare oxidative stress between newly diagnosed PD patients (ntPD) and PD patients under treatment (tPD). We also analyzed changes in plasma activity of several aminopeptidases (AP) involved in the metabolism of various active peptides., Methods: Plasma lipid peroxide (LPO) and lactate (LAC) concentrations were measured by colorimetric methods, and plasma AP activities were determined by fluorometric assay., Results: LPO and LAC concentrations were significantly elevated in ntPD and tPD patients versus controls, but there were no differences between the PD groups. Alanine-, cystine- and aspartate-AP activities were significantly lower in tPD versus ntPD patients. Nondenaturing electrophoresis and Western blot results confirmed these findings., Conclusions: The plasma LPO and LAC levels were high in both PD groups, indicating that they are elevated at an early stage of PD and are not affected by anti-PD treatment. The higher AP activities in ntPD versus tPD patients suggest that anti-PD treatment may improve protein metabolism while not altering oxidative stress. A therapy directed to reduce oxidative stress and normalize AP activity may be useful in the treatment of PD., (Copyright © 2010 S. Karger AG, Basel.)

In order to study the interaction between the renin-angiotensin system (RAS) and nitric oxide (NO), we analyzed the activity of aspartyl- (AspAP), glutamyl- (GluAP), alanyl- (AlaAP), and cystinylaminopeptidase (CysAP) enzymes involved in the RAS cascade, in the hypothalamus, and plasma of normotensive adult male rats after the inhibition of NO production with the NO synthase inhibitor L-NAME (L-N (G)-nitroarginine methyl ester). L-NAME treatment produced a significant increase of systolic blood pressure (SBP). In plasma, while GluAP activity decreased significantly, suggesting a lower Ang III formation, the other aminopeptidases did not change after L-NAME treatment. In hypothalamus, the activities of AspAP and CysAP were not affected after L-NAME treatment. In contrast, GluAP and AlaAP increased significantly. These results suggested mainly a higher formation of Ang III, but also higher levels of Ang IV in the hypothalamus of L-NAME treated rats. Both peptides have hypertensive properties at central level. On the contrary, Ang III may counteract the hypertensive action of Ang II at the periphery. Therefore, the increased SBP in L-NAME treated rats may be due in part to the increased activity of GluAP and AlaAP in hypothalamus and to a decreased activity of GluAP in plasma.

Huntington's disease (HD) is a genetic neurodegenerative disorder. Oxidative stress, mitochondrial dysfunction, and protein metabolism impairment have been implicated in its pathogenesis. However, the contribution of these phenomena to HD onset or progression is not well known, and they have been less studied in peripheral blood. We analyzed plasma lipid peroxide (LPO) and lactate (LAC) concentrations as indicators of oxidative stress and mitochondrial dysfunction in symptomatic HD patients (sHD) and asymptomatic HD gene carriers (aHD). We also measured the plasma activity of aminopeptidases (APs), an important group of proteolytic enzymes. LPO and LAC concentrations were significantly elevated in sHD patients but not in aHD carriers. Aspartate and glutamate AP activities were significantly reduced in sHD patients and aHD carriers. These findings demonstrate that sHD patients are under oxidative stress, which may favor progression of the disease. Plasma AP activity was decreased before the appearance of HD symptoms and oxidative stress and may be related to protein metabolism impairment. These results indicate that therapy directed to improve oxidative stress and normalize AP activity may be useful in the treatment of HD. They also suggest that decreased plasma AP activity in aHD carriers may predict the future onset of HD symptoms.

Neurotensin(8-13) and two related analogues were used as model systems to directly compare various N-terminal peptide modifications representing both commonly used and novel capping groups. Each N-terminal modification prevented aminopeptidase cleavage but surprisingly differed in its ability to inhibit cleavage at other sites, a phenomenon attributed to long-range conformational effects. None of the capping groups were inherently detrimental to human neurotensin receptor 1 (hNTR1) binding affinity or receptor agonism. Although the most stable peptides exhibited the lowest binding affinities and were the least potent receptor agonists, they produced the largest in vivo effects. Of the parameters studied only stability significantly correlated with in vivo efficacy, demonstrating that a reduction in binding affinity at NTR1 can be countered by increased in vivo stability.

CCK and apolipoprotein AIV (apo AIV) are gastrointestinal satiety signals whose synthesis and secretion by the gut are stimulated by fat absorption. Intraperitoneally administered CCK-8 is more potent in suppressing food intake than a similar dose administered intravenously, but the reason for this disparity is unclear. In contrast, both intravenous and intraperitoneally administered apo AIV are equally as potent in inhibiting food intake. When we compared the lymphatic concentration of CCK-8 and apo AIV, we found that neither intraperitoneally nor intravenously administered CCK-8 or apo AIV altered lymphatic flow rate. Interestingly, intraperitoneal administration of CCK-8 produced a significantly higher lymphatic concentration at 15 min than did intravenous administration. Intraperitoneal injection of apo AIV also yielded a higher lymphatic concentration at 30 min than did intravenous administration. Intraperitoneal administration of CCK-8 and apo AIV also resulted in a much longer period of elevated CCK-8 and apo AIV peptide concentration in lymph than intravenous administration. Furthermore, enzymatic activity of dipeptidyl peptidase IV (DPPIV) and aminopeptidase was higher in plasma than in lymph during fasting, and so, satiation peptides, such as CCK-8 and apo AIV in the lymph, are protected from degradation by the significantly lower DPPIV and aminopeptidase activity levels in lymph than in plasma. Therefore, the higher potency of intraperitoneally administered CCK-8 compared with intravenously administered CCK-8 in inhibiting food intake may be explained by both its higher concentration in lymph and the prolonged duration of its presence in the lamina propria.

Introduction: The renin-angiotensin and kininogen-kinin hormonal systems are critically involved in regulating blood pressure and are candidates in contributing to oral contraceptive pill (OCP)-induced hypertension. Angiotensin-converting enzyme (ACE) and aminopeptidase P (AP-P) are key enzymes in these systems and are both involved in the degradation of the vasodilator bradykinin., Methods: Circulating ACE and AP-P levels were measured by activity assay using selective fluorogenic peptide substrates in plasma samples from the Leeds Family Study. In addition, the effect of progesterone on the expression of AP-P and ACE was examined in cells., Results: Women on the OCP had higher age-adjusted plasma AP-P (mean [95% confidence interval]) (0.27 [0.23-0.32] nmol/min/ml (n = 53)) compared with women not on the OCP (0.17 [0.16-0.19] nmol/min/ml (n = 133), p < 0.001) or males (0.19 [0.17-0.20] nmol/min/ml (n = 209), p<0.001). There were no differences in the age-adjusted plasma ACE levels among the three groups. In HepG2 cells, progesterone treatment increased the AP-P protein and mRNA expression, whereas no effect of progesterone treatment was observed for ACE., Conclusion: Increased AP-P may result in increased breakdown of bradykinin. These data suggest that progesterone-induced increases in AP-P may contribute to the development of OCP-induced hypertension in susceptible Women.

Acute renal failure is a serious condition of Crotalus bites, which could be treated with statins. The effects of Crotalus durissus terrificus venom (vCdt) and simvastatin on renal function, oxidative stress and representative plasma, urinary and renal aminopeptidase (AP) activities were evaluated in mice. Eighty percent LD50 of vCdt caused hyperuricemia and urinary hypoosmolality (100%) and hypercreatinemia (60%). Plasma neutral, pyroglutamyl and dipeptidyl IV and renal soluble and membrane-bound APs were susceptible to vCdt. Cortical and medullar oxidative stress (GSSG/GSH ratio) was increased by vCdt. Simvastatin (3mg/kg body wt.) altered urinary creatinine and urea, membranal protein in cortex and medulla, plasma neutral and dipeptidyl IV APs and most of renal APs in nonenvenomed, and exacerbated hypercreatinemia, but mitigated uricosuria, renal oxidative stress and protein increase in envenomed. Hyperuricemia and urinary hypoosmolality are early signs of indirect myotoxicity of vCdt with diagnostic significance. In kidney, oxidative stress and alteration of protein content and AP activities suggest membrane destruction, enzyme release and protein loss, which may be due to direct tissue damage. Plasma AP activities might be nephrotoxicity markers of C. d. terrificus envenomation. The deleterious effects of simvastatin on creatinemia and APs constitute important restrictions to its use within the antivenom therapy.

The cleavage of peptides by aminopeptidase enzyme types could be among the mechanisms related to certain disruptions on mediator and modulatory functions in diabetes mellitus. In order to examine this hypothesis, we measured representative aminopeptidase activities in tissues of peripheral organs of control and streptozotocin-diabetic rats. None of the examined aminopeptidase activities differed between diabetics and controls in plasma, ileum, stomach or lung. Soluble and membrane-associated alanyl, and membrane-associated cystyl aminopeptidase activities were higher in the kidney of diabetics. Decreased activity was observed in soluble and membrane-associated aspartyl and soluble dipeptidyl-peptidase IV, while increased activity was observed in soluble alanyl, arginyl, and cystyl aminopeptidases in the pancreas of diabetics. In the jejunum, soluble cystyl aminopeptidase increased in diabetics. Soluble arginyl and type-1-pyroglutamyl aminopeptidase and membrane-associated dipeptidyl-peptidase IV activities increased in the liver of diabetics. Membrane-associated dipeptidyl-peptidase IV and alanyl aminopeptidase activities in the spleen were higher in diabetics than in controls. Membrane-associated alanyl aminopeptidase activity also increased in the heart of diabetics. All these changes in streptozotocin-treated rats were avoided by the administration of insulin. Our comparative analysis of a diverse array of aminopeptidase activities supported the proposal that the regulation of peptide cleavage by these enzyme types is associated with the effects of streptozotocin-diabetes mellitus on peripheral organs.

Acute hypotensive transfusion reactions are newly characterized transfusion reactions in which hypotension is the prominent feature. The pathophysiology of acute hypotensive transfusion reactions is related to the bradykinin function and its metabolism. A liver transplant recipient on treatment with an angiotensin converting enzyme inhibitor developed sudden hypotension, that is, systolic pressure of 60 mm Hg, after receiving 200 mL of a blood product mixture without significant surgical blood loss. He responded to the resuscitation measure, although hypotension developed again after a challenge transfusion of 200 mL of the blood mixture. A severe hypotensive reaction to the blood transfusion and diffuse bleeding from the dissection surfaces forced the transplantation to be aborted after the common bile duct had been divided. We hypothesized that the patient had an acute hypotensive transfusion reaction due to disordered bradykinin metabolism. Analysis of his blood showed low levels of both angiotensin converting enzyme and aminopeptidase P enzyme activity, confirming that the patient experienced an acute hypotensive transfusion reaction that was due to the use of the angiotensin converting enzyme inhibitor and was precipitated by an abnormality in the metabolic enzyme pathway. It is recommended to discontinue angiotensin converting enzyme inhibitors and switch to a different class of antihypertensive medications for patients with a high Model for End-Stage Liver Disease score on the waiting list for liver transplantation.

Background: Although acute allergic reactions after ingestion of peanuts and tree nuts are common, fatalities are rare. Other than patients with coexisting asthma, it is currently not possible to predict which patients are most likely to develop severe reactions., Objective: The aim of this study was to determine which clinical and laboratory parameters best predict the likelihood of severe allergic reactions., Methods: From 1992 to 2004, we collected detailed information on the clinical severity and allergy test results of 1094 patients with peanut and tree nut allergy attending a regional allergy center. In a subgroup of 122 patients, sera were assayed for activity of enzymes involved in the catabolism of bradykinin., Results: Severe pharyngeal edema was 3.8 (2.1-6.9) times more common in patients with severe rhinitis and 2.6 (1.8-3.7) more common after ingestion of tree nuts compared with peanuts. Patients with serum angiotensin-converting enzyme concentrations <37.0 mmol/L had a 9.6 (1.6-57)-fold risk of severe pharyngeal edema. Life-threatening bronchospasm was most likely in patients with severe asthma (relative risk, 6.8 [4.1-11.3]) and less so in patients with milder asthma (2.7 [1.7-4.0]). Altered levels of consciousness were more likely in patients with severe eczema (3.1 [1.1-8.4])., Conclusion: Severity of coexisting atopic diseases predicted which patients attending a tertiary referral clinic were most likely to develop life-threatening allergic reactions to peanuts and tree nuts. Patients with the lowest serum angiotensin-converting enzyme concentrations were more likely to develop life-threatening pharyngeal edema, suggesting that this complication may be partly mediated by bradykinin.

Background: Aminopeptidase P (APP) plays an important role in the catabolism of kinins in human plasma, mostly for des-Arg(9)-bradykinin. Impaired degradation of this active bradykinin metabolite was found to be associated with a decreased APP activity in hypertensive patients who experienced angioedema while being treated with angiotensin I-converting enzyme inhibitors. The pathophysiology of hereditary angioedema is presently attributed only to a quantitative/qualitative C1 inhibitor (CI-INH) defect with increased bradykinin release., Objectives: In the context of androgen prophylaxis, increased CI-INH function cannot fully explain protection from angioedema attacks alone because of the limited reversion of the CI-INH defects. Therefore we hypothesized that androgen prophylaxis could enhance plasma APP activity., Methods: Patients with hereditary angioedema were investigated for plasma metallopeptidase activities responsible for kinin catabolism (APP, angiotensin I-converting enzyme, and carboxypeptidase N) and for CI-INH function in treated and untreated patients., Results: APP activity was asymmetrically distributed in untreated patients (n = 147): the mean value was significantly lower than the value in a reference healthy and unmedicated population (n = 116; P < or = .001). Prophylaxis with androgen induced a significant increase in APP activity (P < or = .001), whereas it did not affect the other metallopeptidase activities. In both patient groups, APP activity showed a significant inverse relationship to disease severity (P < or = .001)., Conclusion: In addition to the effect on circulating CI-INH levels, the increase in APP levels brought on by androgens could contribute to a more effective control of the kinin accumulation considered to be responsible for the symptoms of angioedema.

Parkinson's disease (PD) is an age-related neurodegenerative disease characterized by a progressive motor disorder, but frequently is accompanied by autonomic symptoms such as hypotension. Together with the decrease of dopamine, significant decreases in aminopeptidase activities have been reported in PD brains. However, up to date there are no studies about changes of aminopeptidase activities in plasma of PD patients. We studied plasma activities of alanyl-, aspartyl-(AspAP), cystinyl-(CysAP) and glutamyl-aminopeptidase (GluAP) in two groups of subjects: control (n=41) and PD (n=48). Plasma activities of AspAP, CysAP, and GluAP showed significant decreases of 24.9% (p<0.05), 39.4% (p<0.01) and 33.3% (p<0.01), respectively, in PD group. These aminopeptidases are involved in the metabolism of circulating peptides such as the ones of the renin-angiotensin system. The importance of aminopeptidases in striatal dopamine content and in neuroendocrine system in PD is discussed.

Background: In previous reports, changes in oxytocinase activity in human breast cancer tissue and in the serum of N-methyl-nitrosourea (NMU)-induced rat mammary tumors were described. Insulin-regulated aminopeptidase (IRAP) has been identified with oxytocinase and has also been referred to as placental leucine aminopeptidase (P-LAP)., Materials and Methods: The IRAP/P-LAP activity in rat serum was assayed to analyze the putative role that IRAP/P-LAP may play in regulating mammary gland carcinogenesis induced by NMU. Furthermore, as it has been recently described that IRAP/P-LAP is the angiotensin IV (Ang IV) receptor AT4, the activities of Ang IV-forming aminopeptidase N (APN) and aminopeptidase B (APB) were also assayed., Results: Changes in serum IRAP/P-LAP and Ang IV-forming APB activities were found in rats with mammary tumors induced by NMU. Both activities were greatly increased, although the Ang IV-forming APN activity was not modified., Conclusion: These changes in aminopeptidase activities may reflect the local functional status of their substrates, which can be selectively activated or inhibited in the affected tissue as a result of specific conditions brought about by the tumor. Thus, these enzymatic activities may be involved in the promotion and progression of breast cancer through oxytocin (OT), vasopressin (AVP) and/or renin-angiotensin system (RAS) misregulation.

Biologically active peptides evenly labeled with tritium were used for studying the in vitro and in vivo biodegradation of the peptides. Tritium-labeled peptides with a specific radioactivity of 50-150 Ci/mmol were obtained by high temperature solid phase catalytic isotope exchange (HSCIE) with spillover tritium. The distribution of the isotope label among all amino acid residues of these peptides allows the simultaneous determination of practically all possible products of their enzymatic hydrolysis. The developed analytical method includes extraction of tritium-labeled peptides from organism tissues and chromatographic isolation of individual labeled peptides from the mixture of degradation products. The concentrations of a peptide under study and the products of its biodegradation were calculated from the results of liquid scintillation counting. This approach was used for studying the pathways of biodegradation of the heptapeptide TKPRPGP (Selank) and the tripeptide PGP in blood plasma. The pharmacokinetics of Selank, an anxiolytic peptide, was also studied in brain tissues using the intranasal in vivo administration of this peptide. The concentrations of labeled peptides were determined, and the pentapeptide TKPRP, tripeptide TKP, and dipeptides RP and GP were shown to be the major products of Selank biodegradation. The study of the biodegradation of the heptapeptide MEHFPGP (Semax) in the presence of nerve cells showed that the major products of its biodegradation are the pentapeptide HFPGP and tripeptide PGP. The enkephalinase activity of blood plasma was studied with the use of evenly tritium-labeled [Leu]enkephalin. A high inhibitory effect of Semax on blood plasma enkephalinases was shown to arise from its action on aminopeptidases. The method, based on the use of evenly tritium-labeled peptides, allows the determination of peptide concentrations and the activity of enzymes involved in their degradation on a tg scale of biological samples both in vitro and in vivo.

Background & Objective: Serum fast band of glycylproline dipeptidyl aminopeptidase isoenzyme (GPDA-F) is useful to the diagnosis of hepatocellular carcinoma, especially for the cases without expression of alpha-fetoprotein (AFP). Polyacrylamide electrophoresis for detection of GPDA-F is relatively complicated and has limitation in its clinical use. This study was to establish a simple and easy method of immunoelectrophoresis to detect serum GPDA-F, and evaluate clinical value of GPDA-F in the diagnosis of hepatocelluar carcinoma., Methods: Serum GPDA-F was purified to raise polyclonal GPDA-F antibody, and immunoelectrophoresis was established for the detection of serum GPDA-F. Serum GPDA-F in 99 specimens of hepatocellular carcinoma and 115 specimens of benign liver diseases (36 cases of liver cirrhosis, 23 cases of acute hepatitis, 38 cases of chronic hepatitis, and 18 cases of benign liver space-occupying lesions) was simultaneously detected by both polyacrylamide electrophoresis and immunoelectrophoresis. The clinical value of serum GPDA-F detected by immunoelectrophoresis was compared with that by polyacrylamide electrophoresis for the diagnosis of hepatocellular carcinoma., Results: If the cut-off was set at 71 u/L, the diagnostic sensitivity, specificity, and accuracy of immunoelectrophoresis in detecting GPDA-F in hepatocellular carcinoma were 83.8%, 85.2%, and 84.6%, respectively; while those of polyacrylamide electrophoresis were 81.8%, 77.3%, and 79.4%, respectively., Conclusions: Serum GPDA-F detected by immunoelectrophoresis is useful to the diagnosis of hepatocellular carcinoma. Compared with polyacrylamide electrophoresis, this method is cheap, time-saving, and easy.

Mammalian sera contain enzymes that catalyze the hydrolytic degradation of peptidoglycans and molecules of related structure and are relevant for the metabolism of peptidoglycans. We now report on a novel L,(L/D)-aminopeptidase found in human and mammalian sera. The enzyme hydrolyses the pentapeptide L-Ala-D-iso-Gln-meso-DAP(omegaNH(2))-D-Ala-D-Ala yielding the free L-alanine and the respective tetrapeptide (K(M) 18 mM). L,(L/D)-aminopeptidase from guinea pig serum was highly purified in four chromatographic steps, up to 700-fold. Molecular weight of the enzyme was estimated by HPLC to be approximately 175,000. The configuration of alanine obtained by hydrolysis of the pentapeptide was determined by oxidation with L-amino acid oxidase. The amino acids sequence in the respective tetrapeptide was deduced from the results of mass spectrometry. The novel L,(L/D)-aminopeptidase also hydrolyzed alanine-4-nitroanilide (K(M)=0.6 mM) and several peptides comprising L-amino acids. Peptides containing D-amino acid at the amino end and L-Asp-L-Asp were not the substrates for this enzyme. The purified enzyme also exhibited enkephalin degrading activity, hydrolyzing enkephalins comprising L,L- and L,D-peptide bonds. The enzyme was inhibited strongly by metal chelating agents, bestatin and amastatin.

Angiotensin I-converting enzyme inhibitors (ACEi), which are used to treat common cardiovascular diseases, are associated with a potentially life-threatening adverse reaction known as angioedema (AE-ACEi). We have previously documented a significant association between AE-ACEi and low plasma aminopeptidase P (APP) activity. With eight large pedigrees, we hereby demonstrate that this quantitative trait is partially regulated by genetic factors. We tested APP activity using a variance-component QTL analysis of a 10-cM genomewide microsatellite scan enriched with seven markers over two candidate regions. We found significant linkage (LOD = 3.75) to a locus that includes the XPNPEP2 candidate gene encoding membrane-bound APP. Mutation screening of this QTL identified a large coding deletion segregating in one pedigree and an upstream single-nucleotide polymorphism (C-2399A SNP), which segregates in the remaining seven pedigrees. Measured genotype analysis strongly suggests that the linkage signal for APP activity at this locus is accounted for predominantly by the SNP association. In a separate case-control study (20 cases and 60 controls), we found significant association of this SNP to ACEi-induced AE (P=.0364). In conclusion, our findings provide supporting evidence that the C-2399A variant in XPNPEP2 is associated with reduced APP activity and a higher incidence of AE-ACEi.

APP (aminopeptidase P) has the unique ability to cleave the N-terminal amino acid residue from peptides exhibiting a proline at P(1)'. Despite its putative involvement in the processing of bioactive peptides, among them the kinins, little is known about the physiological roles of both human forms of APP. The purpose of the present study is first to engineer and characterize a secreted form of hmAPP (human membrane-bound APP). Our biochemical analysis has shown that the expressed glycosylated protein is fully functional, and exhibits enzymic parameters similar to those described previously for mAPP purified from porcine or bovine lungs or expressed from a porcine clone. This soluble form of hmAPP cross-reacts with a polyclonal antiserum raised against a 469-amino-acid hmAPP fragment produced in Escherichia coli. Secondly, we synthesized three internally quenched fluorescent peptide substrates that exhibit a similar affinity for the enzyme than its natural substrates, the kinins, and a higher affinity compared with the tripeptide Arg-Pro-Pro used until now for the quantification of APP in biological samples. These new substrates represent a helpful analytical tool for rapid and reliable screening of patients susceptible to adverse reactions associated with angiotensin-converting enzyme inhibitors or novel vasopeptidase (mixed angiotensin-converting enzyme/neprilysin) inhibitors.

Background: Breast cancer is the most frequent spontaneous malignancy diagnosed in women in the western world, although no specific etiological agent(s) or the mechanism responsible for the initiation of the disease has been identified as yet. Enkephalins (Leu5-enkephalin and Met5-enkephalin) (ENK) act in the breast in different ways such as modulating esteroid receptors and proteases secretion. ENK are hydrolyzed by specific enzymes, leading to their inactivation, such as the enkephalin-degrading tyrosyl aminopeptidase (EDA). Breast tumours induced in rats by administration of N-methyl-nitrosourea (NMU) constitute a useful tool for dissecting the multistep process of carcinogenesis, which involves initiation, promotion and progression. The aim of the present work was to analyse EDA activity (E.C: 3.4.11.-) in serum of rats with mammary tumours induced by NMU, to evaluate the potential value of this activity as a biological marker of the carcinogenesis process, and the putative role of ENK in the promotion and progression of the disease., Materials and Methods: Tumours were induced by intraperitoneal injection of three doses of NMU at 50, 80 and 110 days after birth. Serum EDA was measured fluorimetrically using tyrosyl-beta-naphthylamide as substrate., Results: The increase found in EDA activity suggests the existence of decreased serum circulating levels of ENK in rat with mammary tumours induced by NMU., Conclusion: Although the exact role of ENK in breast cancer initiation, promotion and/or progression remains unknown, our results suggest that changes in EDA activity might play an important role in the origin and evolution of breast cancer.

We identified a number of clinically used drugs and biologically active endogenous peptides able to significantly decrease the rate of human plasmatic aminopeptidase (AP) leucine-enkephalin (LEU) degradation. Bacitracin, bestatin, fluvoxamine, and each of 4 peptides tested significantly increased, in a dose-dependent manner (10-10 M), LEU degradation half-life (t1/2) in each of 5 plasma samples studied. Each sample was obtained by pooling equal volume of 6 randomly selected, individual plasmas (4 male and 2 female healthy, drug-free volunteers). Thirty subjects (20 females and 10 males) participated in this study. With the exception of fluvoxamine, this inhibitory effect was lacking in various other commonly used drugs with widely different chemical structures and pharmacological profiles, eg, antidepressants (SSRIs, imipramine-like tricyclics, MAOIs), acute antimigraine agents (triptan class drugs), the nonselective beta-adrenergic antagonist propranolol, and serotonin receptor agonists and antagonists. Agents (concentration 10 M used as illustration), listed in decreasing order of LEU-AP inhibitory activity: substance P > angiotensin III > methionine-enkephalin > angiotensin II > fluvoxamine > bestatin gave t1/2 values (+/- SD) of 39.3 +/- 1.1, 29.4 +/- 0.8, 28.3 +/- 0.8, 27.4 +/- 0.7, 24.5 +/- 1.5, and 23.6 +/- 0.9 minutes, respectively. Control, bacitracin, and fluphenazine (known LEU-AP inhibitors were used for comparison) values of 11.8 +/- 1.0, 31.3 +/- 0.7, and 19.6 +/- 1.0 minutes, respectively. As expected, these drugs significantly decreased the initial velocity of peptide degradation; Iv values (+/- SD) of: 0.17 +/- 0.1 (0.02 +/- 0.01), 0.23 +/- 0.2 (0.02 +/- 0.01), 0.25 +/- 0.2 (0.02 +/- 0.01), 0.26 +/- 0.2 (0.03 +/- 0.01), 0.31 +/- 0.1 (0.03 +/- 0.01), and 0.33 +/- 0.1 (0.03 +/- 0.01), respectively; control, bacitracin, and fluphenazine: 1.10 +/- 0.3 (0.12 +/- 0.03), 0.20 +/- 0.1 (0.02 +/- 0.01), and 0.82 +/- 0.2 (0.08 +/- 0.02) pg LEU/min (pg LEU/mg protein/min), respectively. Results emphasize the selective nature of chemical structures required to significantly inhibit AP activity and provided information that could help the rational design of agents with high specificity in a biologic milieu containing multiple peptidases. In this case, targeted modulation of the bioavailability of LEU and other endogenous AP-degraded hormonal and nonhormonal peptides could be useful in the treatment of the pathophysiology associated with various disease conditions. Whether their development could find useful pharmacological applications remains to be explored.

Background: Leukoreduction before storage, rather than bedside white blood cell filtration, is recommended to prevent hypotensive transfusion reactions., Study Design and Methods: Investigation of hypotensive transfusion reactions during radical prostatectomy in two patients on angiotensin-converting enzyme inhibitors. In Patient A, hypotension occurred during the transfusion of each of the following blood products: 2 units of autologous blood deposited and leukoreduced (LR) before storage; 3 units of allogeneic red cells LR before storage; and 2 units of non-LR acute normovolemic hemodilution (ANH) whole blood. When each of the transfusions was stopped, the blood pressure recovered. In Patient B, hypotension occurred during the transfusion of non-LR ANH whole blood. All implicated units were administered rapidly using a blood infuser at 37 degrees C. Bradykinin (BK) and des-Arg9-BK formation and degradation and the activity of kinin-degrading metallopeptidases were measured in plasma samples from both patients., Results: Degradation of des-Arg9-BK was severely impaired and the activity of aminopeptidase P severely reduced in Patient A, but not in Patient B. BK degradation was mildly impaired in both patients., Conclusion: Hypotensive reactions can occur with blood products that are LR before storage and non-LR ANH. An inherent defect in the metabolism of kinins may be a risk factor for the development of hypotensive transfusion reactions.

Asymmetries in the neuroendocrine system extend from central structures to paired endocrine glands and their innervation. In addition to the well-known asymmetry in the function of brain dopamine, there are also asymmetries in the peripheral response to experimental hemi-parkinsonism, performed by means of lesions of the nigrostriatal system with 6-hydroxydopamine (6-OHDA) injections into the left or right hemisphere. Therefore, it is speculated that the neuroendocrine system would also be asymmetrically affected in experimental hemi-parkinsonism. Aminopeptidases (AP) play a major role in the control of peptide concentration at both central and peripheral levels in tissues and blood, thus reflecting the functional status of their endogenous substrates. Therefore, to evaluate the peripheral response of hemi-parkinsonism, we have performed a comprehensive study of plasma AP activities after lesions of the nigrostriatal system with 6-OHDA administered into either left or right striatum of adult male rats. Saline was injected into control groups. AlaAP, CysAP, AspAP and GluAP activities were determined in plasma, using specific arylamides as substrates. Plasma AlaAP activity increased 3-fold (p < 0.001) whereas AspAP activity decreased by 30% (p < 0.05) after lesion of the right hemisphere. In contrast, CysAP and GluAP activities increased significantly after lesion of the left hemisphere by 200 and 50%, respectively (p < 0.05). The main discovery of the present results demonstrates that experimental hemi-parkinsonism affects differentially the plasma AP activities depending on the hemisphere in which the lesion is performed. This suggests that the circulating hormones, susceptible to be hydrolyzed by these enzymatic activities, are also modified.

We evaluated the effect of long-cycle structured intermittent therapy (SIT; 4 weeks without highly active antiretroviral therapy [HAART] followed by 8 weeks with HAART) versus continuous HAART. The study was prematurely terminated to new enrollment because of the emergence of genetic mutations associated with resistance to antiretroviral drugs in 5 patients. After 48 weeks, there was no significant difference between groups in lipid, hepatic transaminase, and C-reactive protein levels in 41 patients. Although there were no differences in CD4(+) or CD8(+) T cell counts or the percentage of cells that were CD4(+)CD25(+), CD8(+)CD25(+), or CD4(+)DR(+), patients who received SIT had a significantly higher percentage of CD8(+)CD38(+) and CD8(+)DR(+) cells. There was no clear autoimmunization effect by immunologic or virologic parameters. There was no benefit to long-cycle SIT versus continuous HAART with regard to certain toxicity, immunologic, or virologic parameters.

Background: Because non-denaturing electrophoresis and aminopeptidase activity staining often detect noncovalent multi-enzyme complexes, we adopted procedures to specifically detect the aminopeptidase N (APN) molecule itself in liver disease serum., Methods: Sera or their immunoprecipitate with anti-APN monoclonal antibody were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or two-dimensional electrophoresis and subsequent Western blotting with rabbit anti-APN serum., Results: In all the patient sera examined, the 140-kDa APN isoform was predominant. In all the sera from 10 patients with cholestatic diseases (8 with extra-hepatic cholestasis and 2 with primary biliary cirrhosis), we observed the 260-kDa isoform that was immunoprecipitated with monoclonal APN antibodies and had a similar isoelectric point to the 140-kDa isoform. However, the 260-kDa isoform was observed faintly in 2 out of 12 patients with other liver diseases, including chronic hepatitis and cirrhosis., Conclusions: We found a novel high-molecular-mass APN isoform (260-kDa) in serum, which is highly likely to be a homodimer of APNs bound covalently and a promising marker of cholestasis. This suggests increased cross-linking reaction between two APN molecules in cholestatic patients.