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(1) Background: The replacement of diets high in saturated fat (SAFA) with monounsaturated fatty acids (MUFA) is associated with better cardiovascular function and is related to the modulation of the activity of the local renin–angiotensin system (RAS) and the collagenase activity of dipeptidyl peptidase IV (DPP-IV). The objective of the work was to verify the capacity of different types of dietary fat on the regulatory activities of RAS and DPP-IV. (2) Methods: Male Wistar rats were fed for 24 weeks with three different diets: the standard diet (S), the standard diet supplemented with virgin olive oil (20%) (VOO), or with butter (20%) plus cholesterol (0.1%) (Bch). The proteolytic activities were determined by fluorometric methods in the soluble (sol) and membrane-bound (mb) fractions of the left ventricle and atrium, aorta, and plasma samples. (3) Results: With the VOO diet, angiotensinase values were significantly lower than with the Bch diet in the aorta (GluAP and ArgAP (mb)), ventricle (ArgAP (mb)) and atrium (CysAP (sol)). Significant decreases in DPP-IV (mb) activity occurred with the Bch diet in the atrium and aorta. The VOO diet significantly reduced the activity of the cardiac damage marker LeuAP (mb) in the ventricle and aorta, except for LeuAP (sol) in the ventricle, which was reduced with the Bch diet. (4) Conclusions: The introduction into the diet of a source rich in MUFA would have a beneficial cardiovascular effect on RAS homeostasis and cardiovascular functional stability., ACCIÓN 1 PAIUJA 2019 2020: BIO221.

The present study aimed at selecting non-starter lactic acid bacteria strains, with desirable technological and enzymatic activities, suitable as adjunct culture for the Provola dei Nebrodi cheese production. One hundred and twenty-one lactic acid bacteria, isolated from traditional Provola dei Nebrodi cheese samples, were genetically identified by Rep-PCR genomic fingerprinting, using the (GTG)5-primer, and by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS). Twenty-seven strains, included in the qualified presumption of safety (QPS) list, were tested for technological and proteinase/peptidase activities. Results showed that technological features and flavour formation abilities were strain-dependent. Among the selected strains, Lacticaseibacillus paracasei PN 76 and Limosilactobacillus fermentum PN 101 were used as adjunct culture in pilot-scale cheese-making trials. Data revealed that adjunct cultures positively affected the flavour development of cheese, starting from 30 days of ripening, contributing to the formation of key flavour compounds. The volatile organic compound profiles of experimental cheeses was significantly different from those generated in the controls, suggesting that the selected adjunct strains were able to accelerate the flavour development, contributing to a unique profile of Provola dei Nebrodi cheese

Technical and human support provided by CICT and CEPA of Universidad de Jaén (UJA, MINECO, Junta de Andalucía; FEDER) is gratefully acknowledged., High-fat diets (HFD) have been widely associated with an increased risk of metabolic disorders and overweight. However, a high intake of sources that are rich in monounsaturated fatty acids has been suggested as a dietary agent that is able to positively influence energy metabolism and vascular function. The main objective of this study was to analyze the role of dietary fats on hepatic peptidases activities and metabolic disorders. Three diets: standard (S), HFD supplemented with virgin olive oil (VOO), and HFD supplemented with butter plus cholesterol (Bch), were administered over six months to male Wistar rats. Plasma and liver samples were collected for clinical biochemistry and aminopeptidase activities (AP) analysis. The expression of inducible nitric oxide synthase (iNOS) was also determined by Western blot in liver samples. The diet supplement with VOO did not induce obesity, in contrast to the Bch group. Though the VOO diet increased the time that was needed to return to the basal levels of plasma glucose, the fasting insulin/glucose ratio and HOMA2-%B index (a homeostasis model index of insulin secretion and valuation of β-cell usefulness (% β-cell secretion)) were improved. An increase of hepatic membrane-bound dipeptidyl-peptidase 4 (DPP4) activity was found only in VOO rats, even if no differences in fasting plasma glucagon-like peptide 1 (GLP-1) were obtained. Both HFDs induced changes in hepatic pyroglutamyl-AP in the soluble fraction, but only the Bch diet increased the soluble tyrosyl-AP. Angiotensinase activities that are implicated in the metabolism of angiotensin II (AngII) to AngIV increased in the VOO diet, which was in agreement with the higher activity of insulin-regulated-AP (IRAP) in this group. Otherwise, the diet that was enriched with butter increased soluble gamma-glutamyl transferase (GGT) and Leucyl-AP, iNOS expression in the liver, and plasma NO. In summary, VOO increased the hepatic activity of AP that were related to glucose metabolism (DPP4, angiotensinases, and IRAP). However, the Bch diet increased activities that are implicated in the control of food intake (Tyrosine-AP), the index of hepatic damage (Leucine-AP and GGT), and the expression of hepatic iNOS and plasma NO. Taken together, these results support that the source of fat in the diet affects several peptidases activities in the liver, which could be related to alterations in feeding behavior and glucose metabolism., This work was supported by grant code: ACCIÓN 1 PAIUJA 2019 2020: BIO221.

[EN] The addition of aminopeptidase inhibitors (AMIs) to rabbit semen extenders could be a solution to decrease the hormone degradation (GnRH) by the aminopeptidases existing in the seminal plasma. Therefore, the quantity of GnRH needed to induce ovulation in doe would be comparable with the amount administered intramuscularly (i.m.). This study was conducted to evaluate the effects of two AMIs (bestatin and EDTA) on rabbit semen quality parameters, beta nerve growth factor ((beta-NGF) degradation and reproductive performance after artificial insemination. Results showed that seminal quality was not affected by the incubation with AMIs; the values of motility, acrosome integrity and sperm viability were not significantly different between the AMIs and the control groups (positive i.m. and negative intravaginally without AMIs). In addition, the aminopeptidase activity of seminal plasma was inhibited in a 55.5% by the AMIs as well as beta-NGF degradation. On the other hand, regarding the effect of AMIs on reproductive performance, our results showed that the presence of bestatin and EDTA did neither affect fertility (85.3 vs. 88.6%), nor the prolificacy rate (10.12 vs. 10.51 kits per delivery), comparing AMIs group to positive control group, respectively. We conclude that the addition of specific AMIs in the rabbit semen extender has no effect on reproductive performance. Therefore, due to the fact that AMIs inhibit part of the aminopeptidase activity that degrades the GnRH analogue and beta-NGF, they could be used to develop new extenders with less hormone concentration. (C) 2017 Elsevier Inc. All rights reserved., This research was supported in part by the RTA2013-00058-00-00 from INIA, the European Social Fund and the European FEDER Funds. L. Casares-Crespo is supported by a scholarship from Instituto Valenciano de Investigaciones Agrarias (IVIA) and the European Social Fund. P. Fernandez-Serrano is supported by funds from Instituto Valenciano de Investigaciones Agrarias (IVIA) and Ministerio de Empleo y Seguridad Social (Programa de Garantia Juvenil).

International audience; The aim of this work was to investigate in which phases of ripening of Parmigiano Reggiano cheese lactic acid bacteria aminopeptidases present in cheese extract could be involved in release of free amino acids and to better understand the behavior of these enzymes in physical-chemical conditions that are far from their optimum. In particular, we evaluated 6 different substrates to reproduce broad-specificity aminopeptidase N, broad-specificity aminopeptidase C, glutamyl aminopeptidase A, peptidase with high specificity for leucine and alanine, proline iminopeptidase, and X-prolyl dipeptidyl aminopeptidase activities releasing different N-terminal amino acids. The effects of pH, NaCl concentration, and temperature on the enzyme activities of amino acid β-naphthylamide (βNA)-substrates were determined by modulating the variables in 19 different runs of an experimental design, which allowed the building of mathematical models able to assess the effect on aminopeptidases activities over a range of values, obtained with bibliographic data, covering different environmental conditions in different zones of the cheese wheel at different aging times. The aminopeptidases tested in this work were present in cell-free Parmigiano Reggiano cheese extract after a 17-mo ripening and were active when tested in model system. The modeling approach shows that to highlight the individual and interactive effects of chemical-physical variables on enzyme activities, it is helpful to determine the true potential of an aminopeptidase in cheese. Our results evidenced that the 6 different lactic acid bacteria peptidases participate in cheese proteolysis and are induced or inhibited by the cheese production parameters that, in turn, depend on the cheese dimension. Generally, temperature and pH exerted the more relevant effects on the enzymatic activities, and in many cases, a relevant interactive effect of these variables was observed. Increasing salt concentration slowed down broad-specificity aminopeptidase C, glutamyl aminopeptidase A, proline iminopeptidase, and peptidase with high specificity for leucine and alanine. Interestingly, this variable did not affect broad-specificity aminopeptidase N and positively affected X-prolyl dipeptidyl aminopeptidase. The models elaborated varying pH, temperatures, and salt concentration and were a useful, low cost, and fast tool to understand the role of the main peptidases in the different phases of cheese ripening in relation to the major environmental factors influencing enzyme activity.

Neuropeptides are neurotransmitters and modulators distributed in the central nervous system (CNS) and peripheral nervous system. Their abnormalities cause neurological and mental diseases. Neuropeptidases are enzymes crucial for the biosynthesis and biodegradation of neuropeptides. We here focus on the peptidases involved in the metabolism of the well-studied opioid peptides. Bioactive enkephalins are formed from propeptides by processing enzymes—prohormone thiol protease, prohormone convertase 1 and 2 (PC 1 and 2), carboxypeptidase H/E, and Arg/Lys aminopeptidase. After they exert their biological effects, enkephalins are likely to be inactivated by degrading enzymes—angiotensin-converting enzyme (ACE), aminopeptidase N (APN), puromycin-sensitive aminopeptidase (PSA), and endopeptidase 24.11. Recently, a neuron-specific aminopeptidase (NAP), which was a putative enkephalin-inactivating enzyme at the synapses, was found. Neuropeptidases are useful drug targets and their inhibitors can be therapeutic. Synthetic anti-enkephalinases and anti-aminopeptidases are being developed. They are potent analgesics but have fewer side effects than the opiates.

[EN] The bioavailability of buserelin acetate when added to the seminal dose appears to be determined by the activity of the existing aminopeptidases. Thus, the addition of amino peptidase inhibitors to rabbit semen extenders could be a solution to decrease the hormone degradation. This study was conducted to evaluate the effect of the protease activity inhibition on rabbit semen quality parameters and reproductive performance after artificial insemination. Seminal quality was not affected by the incubation with protease inhibitors, being the values of motility, viability, and acrosome integrity not significantly different between the protease inhibitors and the control group. In addition, seminal plasma aminopeptidase activity was inhibited in a 55.1% by the protease inhibitors. On the other hand, regarding the effect of protease inhibitors on reproductive performance, our results showed that the presence of protease inhibitors affected the prolificacy rate (9.2 +/- 0.26 and 9.3 +/- 0.23 vs. 8.2 +/- 0.22 total born per litter for negative control, positive control, and aminopeptidase inhibitors group, respectively; P < 0.05), having this group one kit less per delivery. We conclude that the addition of a wide variety of protease inhibitors in the rabbit semen extender negatively affects prolificacy rate. Therefore, the development of new extenders with specific aminopeptidase inhibitors would be one of the strategies to increase the bioavailability of GnRH analogues without affecting the litter size. (C) 2016 Elsevier Inc. All rights reserved., This research was supported in part by the RTA2013-00058-00-00 from INIA, the European Social Fund and the European FEDER Funds. L Casares-Crespo is supported by a scholarship from Institute Valenciano de Investigaciones Agrarias (IVIA) and the European Social Fund.

[EN] Since ovulation in rabbit is induced by sensory stimulation associated with mating, an effective control of ovulation is required in the artificial insemination process. The intramuscular application of GnRH analogues is the most common practice in rabbit reproductive management; however, the process can be simplified replacing the intramuscular application by the addition of the GnRH analogue to the semen extender, thus reducing the time needed to carry out the insemination, the risks associated with the use of needles and animal distress. The efficiency of GnRH analogue when administered with the semen extender depends on the degradation of the hormone by aminopeptidases, a group of enzymes present in seminal plasma. In this final degree project, aminopeptidase activity present in seminal plasma of males belonged to the New Zealand White line from five months (beginning of the training of those males used in artificial insemination) to nine months old (adult males with a normal semen production) is determined. For this, semen samples from each male were collected weekly and aminopeptidase activity was measured using three different substrates: alanine, cysteine and leucine-β- naphthylamide. Regarding aminopeptidase activity, it can be distinguished a group of males with a high aminopeptidase activity (around 1000 pmol/mg protein/min) and another group with half of the activity approximately. Correlation between quality sperm parameters and aminopeptidase activity was found, concretely with acrosome status and sperm abnormalities. Significant differences for aminopeptidase activity and male age or kinetic sperm parameters were not found. Regarding the results, it can be concluded that aminopeptidase activity varies among males and is not dependent on male age; this new parameter could be used to choose the best males for the artificial insemination procedure which involves GnRH analogue application with the semen extender., [ES] En la inseminación artificial del conejo, al ser una especie de ovulación inducida, se requiere un control efectivo de la ovulación, por lo que en el momento de la inseminación hay que inducir la ovulación, lo que habitualmente se hace mediante la aplicación intramuscular de un análogo sintético de la GnRH. El proceso puede simplificarse sustituyendo la aplicación intramuscular de la GnRH mediante su incorporación en el diluyente, lo que reduce el tiempo de aplicación de la dosis seminal a la vez que evita el riesgo asociado al uso de jeringuillas del personal que aplica la dosis y el estrés animal asociado al pinchazo. La eficacia de la hormona en el diluyente depende de varios factores, entre los que destaca la degradación del análogo por parte de las aminopeptidasas, unas enzimas presentes en el plasma seminal. En el presente Proyecto de Fin de Grado se determinó la actividad aminopeptidasa presente en el plasma seminal en machos de origen neozelandés desde los cinco meses de edad (inicio del entrenamiento de los machos utilizados en inseminación artificial) hasta los nueve meses de edad (machos adultos en plena producción de semen). Para ello, se recuperaron muestras de semen semanalmente de los machos y se determinó la actividad aminopeptidasa usando tres sustratos diferentes: alanina, cisteína y leucina-β-naftilamida. En base a los resultados obtenidos, se distingue un grupo de machos con alta actividad aminopeptidasa (alrededor de 1000 pmol/mg protein/min) y otro con una actividad de aproximadamente la mitad. Se observó cierta correlación entre algunos parámetros de la calidad del semen y la actividad aminopeptidasa, concretamente con el estado del acrosoma y anormalidades en el esperma. La edad de los machos no afectó significativamente a la actividad aminopeptidasa. Con los resultados obtenidos se puede concluir que la actividad aminopeptidasa varía en función del macho y es independiente de su edad, pudiéndose utilizar este nuevo parámetro para seleccionar los mejores machos para la técnica de inseminación artificial que utiliza GnRH en el diluyente.

The potential aminopeptidase activity (APA) in surface sediment (0-1 cm), and important ecological factors of a eutrophicated inlet were monitored for one and a half years on a weekly to biweekly basis to elucidate the fluctuations in extracellular hydrolysis of protein during an annual cycle, and main regulating factors on an annual and/or a seasonal basis. APA was estimated in sediment slurry prepared with unamended bottom water, as the Vmax of the rate of the enzymatic hydrolysis of a coumarine derivative fluorophore at in situ temperatures. APA varied 5-6 fold during the study period with highest values (225-275 nmole AMC h-1 g-1 dry weight) during September-October or early June when the temperature was 22-28°C and dissolved oxygen concentrations (DO) (2-4 mg L-1) in overlying bottom water were high, and much lower values (ca. 50 nmoles AMC h-1 g -1 dry weight) in July-August or December-February. On an annual basis, APA correlated only marginally (p > 0.05; r = 0.25) with the protein content of the surface sediment and not at all with any other variables. However, it showed highly significant correlations (p > 0.001) with temperature (r = 0.81), and protein content (0.65) in mid December-early June, when temperatures were relatively low (ca. 12-22°C) and DO was abundant (ca. 2-6 mg L-1) in overlying bottom water.

[EN] Ovulation induction in artificially inseminated rabbits by adding GnRH synthetic analogues in the seminal doses is a welfare-orientated method to induce ovulation in rabbits and could have some advantages in field practice. This study was conducted to determine the effect of male genotype on the aminopeptidase activity in rabbit seminal plasma and the effects of dilution rate of semen on availability and reproductive performance when buserelin acetate is added to the seminal dose. To study the aminopeptidase activity, 12 mature bucks belonging to a paternal line and 12 from a maternal line were used. The bucks from the paternal line were used to study the effect of dilution rate on the availability of buserelin acetate after 2 hours of dilution and on the reproductive performance of the doses after artificial insemination of 389 commercial crossbreed does. Aminopeptidase activity in seminal plasma is dependent on the male genotype. The paternal line resulted 27% more aminopeptidase activity than the maternal line (P < 0.05). On the other hand, semen diluted 1:20 exhibited a marked increase in the availability of buserelin acetate and the fertility in this group was significantly higher than females from dilution rate 1:5 group, which showed similar results to that of the negative control group (does inseminated with semen diluted 1:20 in non-GnRH-supplemented extender). We conclude that the bioavailability of buserelin acetate when added to the seminal dose appears to be determined by the activity of the existing aminopeptidases and is consequently affected by the dilution rate used to prepare the artificial insemination doses. (C) 2014 Elsevier Inc. All rights reserved., This research was supported in part by the RTA2010-00117-00-00 from INIA and the European FEDER Funds. E. Moce is supported by funds from Ministerio de Ciencia e Innovacion from Subprograma Ramon y Cajal and the European Social Fund (ref. RYC-2010-06162; Madrid, Spain).

The potential aminopeptidase activity (APA) vis-a-vis important ecological parameters of surface and bottom waters of Uranouchi Inlet (Japan), a semi-enclosed eutrophic coastal ecosystem, were studied on a weekly to biweekly basis from mid-August 1996 to early November 1997. The aim of this study was to investigate the main factors regulating the annual and seasonal dynamics of APA in the inlet. APA was estimated in short-term incubations as the V, of the rate of enzymatic hydrolysis of a fluorophore 7-L-leucyl-4-methyl couomarinylarnide under conditions close to those of in situ. The water column structure at the study site changed considerably during the study period as the ecological parameters such as temperature, dissolved oxygen (DO), nutrient contents, and bacterial abundance exhibited pronounced seasonal stratification (May to September). When the water column was mixed (October to March), surface and bottom waters possessed similar APA . But when the water column became stratified, pronounced differences became apparent, with surface water showing considerably higher APA. On extreme days, surface water possessed as much as -20 times higher APA than that of bottom water. Thus, APA of surface water demonstrated strong seasonality and varied by almost 40-fold compared to that of bottom water, which varied only about 12-fold during the study period. Notably, bacterial abundances of both waters showed limited seasonality and varied only -3-fold during the same period. These results imply that changes in APA are not merely a function of bacterial abundance but rather more of per-cell activity. On an annual basis, APA in both waters were positively correlated (p 0.05) with DO, chlorophyll a (p 0.05) content. In contrast, when the water column became stratified, APA correlated only marginally with temperature (0.01 c p 0.05) with APA regardless of whether the water column was mixed or stratified. These correlations suggest that temperature played a critical role in regulating the APA when the water column was mixed (October to March), and DO or inorganic nutrient contents (May to September) when the water colun~n was stratified.

Alkaline sphingomyelinase (Alk-SMase) and neutral ceramidase (N-CDase) in the intestinal microvillar membrane are responsible for dietary sphingomyelin digestion. The activities of the enzymes require the presence of bile salt, and the enzymes can be released into the gut lumen in active forms by bile salts and trypsin. It is unclear to what extent that the intestinal presence of bile salts is critical for the intraluminal activity of these enzymes. We compared the activities of Alk-SMase, N-CDase, and other types of SMases in control and permanently bile diverted rats. In the intestinal tract of control rats, the activity of Alk-SMase was profoundly higher than those of acid and neutral Wases. Bile diversion reduced Alk-SMase activity by 85% in the small intestinal content, and by 68% in the faeces, but did not significantly change the activity in the intestinal mucosa. Western blot showed a marked reduction of the enzyme in the intestinal lumen but not mucosa. N-CDase activities both in the intestinal mucosa and content were reduced by bile diversion. Bile diversion also decreased aminopeptidase N activity in the content and increased that in the mucosa, but had no effects on that of alkaline phosphatase. In conclusion, the presence of bile salts is important for maintaining high intraluminal levels of Alk-SMase and N-CDase, two key enzymes for hydrolysis of sphingomyelin in the gut. We speculate that the sphingomyelin hydrolysis in cholestatic conditions is impaired not only by reduced hydrolytic activity but also by deficient dissociation of the enzymes from the membrane. (c) 2007 Elsevier B.V. All rights reserved.

Strains of Lactobacillus plantarum originally isolated from sausages were screened for proteinase and aminopeptidase activities toward synthetic substrates; on the basis of that screening, L. plantarum CRT, 681 was selected for further assays on muscle proteins. The activities of whole cells, cell extracts (CE), and a combination of both on sarcoplasmic and myofibrillar protein extracts were determined by protein, peptide, and free-amino-acid analyses. Proteinase from whole cells initiated the hydrolysis of sarcoplasmic proteins. The addition of CE intensified the proteolysis. Whole cells generated hydrophilic peptides from both sarcoplasmic and myofibrillar proteins. Other peptides of a hydrophobic nature resulted from the combination of whole cells and CE, The action of both enzymatic sources on myofibrillar proteins caused maximal increases in lysine, arginine, and leucine, while the action of those on sarcoplasmic proteins mainly released alanine, In general, pronounced hydrolysis of muscle proteins required enzyme activities from whole cells in addition to those supplied by CE., grant ALI98-0890 from Comisión Interministerial de Ciencia y Tecnología (CICYT, Madrid, Spain)

The malarial parasite utilises amino acids drawn from the host haemoglobin for it's intraerythrocytic development. The endopeptidases described to date will only produce peptides from host haemoglobin. In order to obtain free aminoacids for incorporation in malarial proteins the malarial parasite requires exopeptidases (i.e. aminopeptidases and carboxypeptidases). Aminopeptidase activity was described in human malarial parasites Plasmodium falciparum rodent malarial parasites Plasmodium chabaudi chabaudi and Plasmodium berghei. These enzymes were shown to be inhibited by the aminopeptidase inhibitor bestatin. In light of these findings, the antimalarial effects of bestatin were investigated both in vitro and in vivo. Bestatin inhibited the in vitro growth of P. c. chabaudi, of chloroquine sensitive P. falciparum clone FCH5.C2 and chloroquine resistant P. falciparum clone K 1/Thailand. P. chabaudi infection is naturally lethal to male mice whereas female mice develop protective immunity against a similar infection. In vivo investigation of the effects of bestatin showed that this aminopeptidase inhibitor could induce self-healing of P. c. chabaudi infection in male CD1 mice. The aminopeptidase was purified by a combination of sizeexclusion HPLC and affinity chromatography and, alternatively, by a combination of size-exclusion HPLC and anion exchange chromatography. The aminopeptidase activity appears to be composed of four different aminopeptidases with an apparent monomeric molecular weight range of 60 to 70 kDa. Comparative studies of the inhibition of the P. c. chabaudi and porcine kidney aminopeptidase by bestatin indicate that the active site of the malarial aminopeptidase could differ to the mammalian aminopeptidase. Nucleotide probes/primers to clone this aminopeptidase(s) have been designed from the sequences of bestatin and metal ions binding site of several leucine aminopeptidases. This thesis shows that the malarial aminopeptidase is a potential chemotherapeutic target with bestatin and/or bestatin analogues as potential antimalarial drugs. The difference between the binding site of bestatin on mammalian and malarial aminopeptidase could be exploited for the design of drugs that would be specific to the malarial parasite enzyme.