Search Results (9)

Aminopeptidase A (APA) cleaves a single aspartate residue from the amino terminus of peptides within the renin angiotensin system (RAS). Since several RAS peptides contain an N-terminal aspartate, we developed an assay to evaluate the effect of recombinant APA on the cleavage of Ang I, Ang II, Ang-(1-7), Ang-(1-9), and Ang-(1-12). The latter peptide has been proposed to be a functional Ang II-forming substrate with a hypertensive action attributable to the formed Ang II acting on AT1 receptors. Here we investigated the following: (a) the hydrolytic action of APA on Ang-(1-12), Ang I (1-10), Ang-(1-9), Ang II and Ang-(1-7) and (b) whether Ang-(1-12) pressor activity is altered by recombinant APA (r-APA) or genetic APA deficiency. We found that (a) r-APA cleaves the N-terminal aspartate of not only Ang II but also [Ang-(1-12), Ang I (1-10), Ang-(1-9)] and [Ang-(1-7)]; (b) the pressor activity of Ang-(1-12) was abolished in the presence of Lisinopril or Telmisartan; (c) r-APA significantly attenuated the pressor activities of infused Ang I and Ang II but not Ang-(1-12); and (d) r-ACE2 also did not attenuate the pressor effect of infused Ang-(1-12). Thus, in addition to increasing blood pressure indirectly via the formation of Ang II, Ang-(1-12) increases blood pressure by an Ang II-independent mechanism. We conclude that APA has an antihypertensive effect attributable to rapid degradation of Ang II, and this action may have a therapeutic potential in forms of hypertension that are Ang II-dependent. In addition, APA metabolizes Ang-(1-12), a peptide that has a prohypertensive action, in part, as a source of Ang II formation but also by a yet to be determined action independent of Ang II. Full article

This work aimed to evaluate the effects of dried apple pomace (DAP) on the fermentation characteristics and proteolysis of alfalfa silages. The alfalfa was ensiled with (1) no additives (control), (2) 5% DAP, (3) 10% DAP and (4) 15% DAP based on fresh weight (FW) for 1, 3, 7, 14, 30 and 60 days, respectively. With the increasing proportion of DAP, lactic acid bacteria (LAB) count, lactic acid (LA) and dry matter (DM) content linearly (p < 0.05) increased, while the pH, the content of acetic acid (AA), propionic acid (PA), butyric acid (BA) and ammonia nitrogen (NH₃-N) linearly (p < 0.05) decreased during ensiling. The 10% and 15% DAP silages had significantly (p < 0.05) lower aerobic bacteria (AB), yeast and enterobacteria counts than the control during ensiling. The contents of nonprotein nitrogen (NPN), peptide nitrogen (peptide-N) and free amino acid nitrogen (FAA-N) and activities of carboxypeptidase, aminopeptidase and acid proteinase linearly (p < 0.05) decreased as DAP proportion increased during ensiling. On day 60, the addition of DAP significantly (p < 0.05) decreased the contents of tryptamine, phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine, spermine and total biogenic amines compared with the control. As the DAP ratio increased, the contents of threonine, valine, isoleucine, leucine, phenylalanine, lysine, histidine, arginine, aspartic acid, serine, glutamic, total amino acids, crude protein (CP) and water-soluble carbohydrates (WSCs) linearly (p < 0.05) increased, while the contents of glycine, alanine, cysteine, and proline linearly (p < 0.05) decreased on day 60. Overall, the addition of 15% DAP was optimal as indicated by better fermentation quality and less proteolysis than other treatments. Full article

Background: N-methyl-D-aspartate type glutamate receptors (NMDARs) are fundamental to neuronal physiology and pathophysiology. The prefrontal cortex (PFC), a key region for cognitive function, is heavily implicated in neuropsychiatric disorders, positioning the modulation of its glutamatergic neurotransmission as a promising therapeutic target. Our recently published findings indicate that AT₁ receptor activation enhances NMDAR activity in layer V pyramidal neurons of the rat PFC. At the same time, it suggests that alternative angiotensin pathways, presumably involving AT₄ receptors (AT4Rs), might exert inhibitory effects. Angiotensin IV (Ang IV) and its analogs have demonstrated cognitive benefits in animal models of learning and memory deficits. Methods: Immunohistochemistry and whole-cell patch-clamp techniques were used to map the cell-type-specific localization of AT4R, identical to insulin-regulated aminopeptidase (IRAP), and to investigate the modulatory effects of Ang IV on NMDAR function in layer V pyramidal cells of the rat PFC. Results: AT4R/IRAP expression was detected in pyramidal cells and GABAergic interneurons, but not in microglia or astrocytes, in layer V of the PFC in 9–12-day-old and 6-month-old rats. NMDA (30 μM) induced stable inward cation currents, significantly inhibited by Ang IV (1 nM–1 µM) in a subset of pyramidal neurons. This inhibition was reproduced by the IRAP inhibitor LVVYP-H7 (10–100 nM). Synaptic isolation of pyramidal neurons did not affect the Ang IV-mediated inhibition of NMDA currents. Conclusions: Ang IV/IRAP-mediated inhibition of NMDA currents in layer V pyramidal neurons of the PFC may represent a way of regulating cognitive functions and thus a potential pharmacological target for cognitive impairments and related neuropsychiatric disorders. Full article

Leaf senescence is a developmental process allowing nutrient remobilization to sink organs. Previously cysteine proteases have been found to be highly expressed during leaf senescence in different plant species. Using biochemical and immunoblotting approaches, we characterized developmental senescence of barley (Hordeum vulgare L. var. ‘GemCraft’) leaves collected from 0 to 6 weeks after the onset of flowering. A decrease in total protein and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunits occurred in parallel with an increase in proteolytic activity measured using the fluorogenic substrates Z-RR-AMC, Z-FR-AMC, and casein labeled with fluorescein isothiocyanate (casein-FITC). Aminopeptidase activity detected with R-AMC peaked at week 3 and then decreased, reaching a low level by week 6. Maximal proteolytic activity with Z-FR-AMC and Z-RR-AMC was detected from pH 4.0 to pH 5.5 and pH 6.5 to pH 7.4, respectively, while two pH optima (pH 3.6 to pH 4.5 and pH 6.5 to pH 7.4) were found for casein-FITC. Compound E-64, an irreversible cysteine protease inhibitor, and CAA0225, a selective cathepsin L inhibitor, effectively inhibited proteolytic activity with IC₅₀ values in the nanomolar range. CA-074, a selective cathepsin B inhibitor, was less potent under the same experimental conditions, with IC₅₀ in the micromolar range. Inhibition by leupeptin and phenylmethylsulfonyl fluoride (PMSF) was weak, and pepstatin A, an inhibitor of aspartic acid proteases, had no effect at the concentrations studied (up to 0.2 mM). Maximal proteolytic activity with the aminopeptidase substrate R-AMC was detected from pH 7.0 to pH 8.0. The pH profile of DCG-04 (a biotinylated activity probe derived from E-64) binding corresponded to that found with Z-FR-AMC, suggesting that the major active proteases are related to cathepsins B and L. Moreover, immunoblotting detected increased levels of barley SAG12 orthologs and aleurain, confirming a possible role of these enzymes in senescing leaves. Full article

The A. japonicus industry has expanded significantly, but no research has focused on determining the age of A. japonicus during farming. Correctly estimating the age of A. japonicus can provide a decision-making basis for the breeding process and data for the protection of A. japonicus aquatic germplasm resources. DNA methylation levels in the body wall of Apostichopus japonicus at 4 months, 1 year, 2 years, and 3 years old were determined using MethylRAD-Seq, and differentially methylated genes were screened. A total of 441 and 966 differentially methylated genes were detected at the CCGG and CCWGG sites, respectively. Aspartate aminotransferase, succinate semialdehyde dehydrogenase, isocitrate dehydrogenase, the histone H2AX, heat shock protein Hsp90, aminopeptidase N, cell division cycle CDC6, Ras GTPase activating protein (RasGAP), slit guidance ligand slit1, integrin-linked kinase ILK, mechanistic target of rapamycin kinase Mtor, protein kinase A Pka, and autophagy-related 3 atg3 genes may play key roles in the growth and aging process of A. japonicus. This study provides valuable information regarding age-related genes for future research, and these candidate genes can be used to create an “epigenetic clock”. Full article

To deepen the understanding of the effect of potassium lactate on the taste of Rugao ham, proteolysis index, enzyme activities and protein degradation of Rugao ham salted with potassium lactate (0%, 0.5%, 1%, 2%) were investigated. Metabolites of Rugao ham were identified by ¹H nuclear magnetic resonance (NMR) spectroscopy and the metabolic pathways of the key metabolites were enriched by the Kyoto Encyclopedia of Genes and Genomes (KEGG); the relationship between taste and metabolites was assessed by partial least square discriminant analysis (PLS-DA). The hams with 2% potassium lactate showed lower cathepsin B and L activities, and higher aminopeptidase activities than that of the control group. The contents of free amino acids and organic acids significantly increased from the control to the treatment of 2% potassium lactate. PLS-DA further demonstrated that aspartate, glutamate, alanine, serine, threonine, acetate, lactate, succinate, carnosine, β-glucose and glycerol were the key metabolites to improve the taste of Rugao ham in the treatment of 2% potassium lactate. Metabolic pathways analysis further demonstrated that amino acids metabolism was the main pathway for the taste development of Rugao ham. Full article

This study isolates and identifies Pichia anomala (P. anomala) AR₂₀₁₆, and studies its effect on the growth and health of weaned pigs. A P. anomala strain from solid wine koji is isolated and identified using 26S rDNA analysis, and its culture conditions are optimized. Heat tolerance, bile salt tolerance, artificial gastric, and intestinal juice tolerance are evaluated. In our methodology, thirty 28 d Large White × Landrace × Rongchang weaned pigs were randomly divided into three groups with 10 barrows in each, and fed a maize-soybean meal diet and orally administered 0.85% saline (CK), 1 mL 1 × 10⁹ cfu/mL Candida utilis (C. utilis), and 1 mL 1 × 10⁹ cfu/mL P. anomala once daily for 28 days. A P. anomala strain was identified and named P. anomala AR₂₀₁₆. P. anomala AR₂₀₁₆ grew best in yeast extract peptone dextrose medium with pH 5.0 at 28 °C, 180 r/min and could tolerate 45 °C for 0.5 h, 0.2% pig bile salts, simulated gastric fluid, and 1.0% simulated intestinal fluid. Our results show that compared with the CK group, orally administered P. anomala AR₂₀₁₆ increases average daily gain, the ileal villus height, the ileal mucosal concentrations of occludin and zonula occluens-1, the serum glucose and total protein concentration, total superoxide dismutase, glutathione peroxidase, and total antioxidative capacity activity, the trypsin and lipase activity in jejunal and ileal contents, the jejunal and ileal mucosa mRNA levels of ALP, TNF-α, and TLR-2, and the relative abundance of Bacteroidetes, Actinobacteria, Succinivibrionaceae, Lachnospiraceae, and Prevotellaceae (p < 0.05). Compared with the CK group, oral administration of P. anomala AR₂₀₁₆ decreased the incidence of diarrhea, aspartate aminotransferase activity, alanine amino-transferase-activity, malondialdehyde, D-lactic acid and endotoxin content in serum, the mRNA level of aminopeptidase N of ileum mucosa, and the relative abundance of Proteobacteria, Clostridiaceae, Campylobacteraceae, Vibrionaceae, Bacillus, and Pseudon (p < 0.05). Collectively, the study indicates that P. anomala AR₂₀₁₆ can tolerate high acidity and high bile salts, and has high survivability in the artificial gastric intestinal juice environment. Oral administration of P. anomala AR₂₀₁₆ improves the growth performance, reduces the incidence of diarrhea, enhances intestinal barrier function, and improves microflora in weaned pigs. Full article

Treatment of chronic lymphocytic leukemia has advanced substantially as our understanding of the kinase signal transduction pathways driven by the B cell receptor (BcR) has developed. Particularly, understanding the role of Bruton tyrosine kinase and phosphatidyl inositol 3 kinase delta in driving prosurvival signal transduction in chronic lymphocytic leukemia (CLL) cells and their targeting with pharmacological inhibitors (ibrutinib and idelalisib, respectively) has improved patient outcomes significantly. The kinase signaling pathway induced by the BcR is highly complex and has multiple interconnecting branches mediated by tyrosine and serine/threonine kinases activated downstream of the BcR. There is a high level of redundancy in the biological responses, with several BcR-signaling kinases driving nuclear factor kappa B activation or inducing antiapoptotic Bcl-2 genes. Accordingly, common gene targets of BcR-signaling kinases may serve as biomarkers indicating enhanced BCR-signaling and aggressive disease progression. This study used a gene expression correlation analysis of malignant B cell lines and primary CLL cells to identify genes whose expression correlated with BCR-signaling kinases overexpressed and/or overactivated in CLL, namely: AKT1, AKT2, BTK, MAPK1, MAPK3, PI3KCD and ZAP70. The analysis identified a 32-gene signature with a strong prognostic potential and DNPEP, the gene coding for aspartic aminopeptidase, as a predictor of aggressive CLL. DNPEP gene expression correlated with MAPK3, PI3KCD, and ZAP70 expression and, in the primary CLL test dataset, showed a strong prognostic potential. The inhibition of DNPEP with a pharmacological inhibitor enhanced the cytotoxic potential of idelalisib and ibrutinib, indicating a biological functionality of DNPEP in CLL. DNPEP, as an aminopeptidase, contributes to the maintenance of the free amino acid pool in CLL cells found to be an essential process for the survival of many cancer cell types, and thus, these results warrant further research into the exploitation of aminopeptidase inhibitors in the treatment of drug-resistant CLL. Full article

The synthesis of racemic substituted 7-amino-5,7,8,9-tetrahydrobenzocyclohepten-6-one hydrochlorides was optimized to enhance reproducibility and increase the overall yield. In order to investigate their specificity, series of enzyme inhibition assays were carried out against a diversity of proteases, covering representative members of aspartic, cysteine, metallo and serine endopeptidases and including eight members of the monometallic M1 family of aminopeptidases as well as two members of the bimetallic M17 and M28 aminopeptidase families. This aminobenzosuberone scaffold indeed demonstrated selective inhibition of M1 aminopeptidases to the exclusion of other tested protease families; it was particularly potent against mammalian APN and its bacterial/parasitic orthologues EcPepN and PfAM1. Full article