Computational study of novel natural inhibitors targeting aminopeptidase N(CD13).

OBJECTIVES: To screen and identify ideal leading compounds from a drug library (ZINC15 database) with potential inhibition of aminopeptidase N(CD13) to contribute to medication design and development. RESULTS: Two novel natural compounds, ZINC000000895551 and ZINC000014820583, from the ZINC15 database were found to have a higher binding affinity and more favorable interaction energy binding with CD13 with less rodent carcinogenicity, Ames mutagenicity, and non-inhibition with cytochrome P-450 2D6. Molecular dynamics simulation analysis suggested that the 2 complexes, ZINC000000895551-CD13 and ZINC000014820583-CD13, have favorable potential energy, and exist stably in the natural circumstances. CONCLUSION: This study discovered that ZINC000000895551 and ZINC000014820583 were ideal leading compounds to be inhibitions targeting to CD13. These compounds were selected as safe drug candidates as CD13 target medication design and improvement. MATERIALS AND METHOD: Potential inhibitors of CD13 were identified using a series of computer-aided structural and chemical virtual screening techniques. Structure-based virtual screening was carried out to calculate LibDock scores, followed by analyzing their absorption, distribution, metabolism, and excretion and toxicity predictions. Molecule docking was employed to reveal binding affinity between the selected compounds and CD13. Molecular dynamics simulation was applied to evaluate stability of the ligand-CD13 complex under natural environment.

Identification and Validation of VEGFR2 Kinase as a Target of Voacangine by a Systematic Combination of DARTS and MSI.

Although natural products are an important source of drugs and drug leads, identification and validation of their target proteins have proven difficult. Here, we report the development of a systematic strategy for target identification and validation employing drug affinity responsive target stability (DARTS) and mass spectrometry imaging (MSI) without modifying or labeling natural compounds. Through a validation step using curcumin, which targets aminopeptidase N (APN), we successfully standardized the systematic strategy. Using label-free voacangine, an antiangiogenic alkaloid molecule as the model natural compound, DARTS analysis revealed vascular endothelial growth factor receptor 2 (VEGFR2) as a target protein. Voacangine inhibits VEGFR2 kinase activity and its downstream signaling by binding to the kinase domain of VEGFR2, as was revealed by docking simulation. Through cell culture assays, voacangine was found to inhibit the growth of glioblastoma cells expressing high levels of VEGFR2. Specific localization of voacangine to tumor compartments in a glioblastoma xenograft mouse was revealed by MSI analysis. The overlap of histological images with the MSI signals for voacangine was intense in the tumor regions and showed colocalization of voacangine and VEGFR2 in the tumor tissues by immunofluorescence analysis of VEGFR2. The strategy employing DARTS and MSI to identify and validate the targets of a natural compound as demonstrated for voacangine in this study is expected to streamline the general approach of drug discovery and validation using other biomolecules including natural products.

Selenium nanoparticles enhanced thermal tolerance and maintain cellular stress protection of Pangasius hypophthalmus reared under lead and high temperature.

There is strong relation between nutrition and thermal tolerance of fish in terms of improved critical temperature minima (CTmin), lethal temperature minima (LTmin), critical temperature maxima (CTmax), and lethal temperature maxima (LTmax). Fishes act as quantifying indicators of the climate change due to their critical thermal limits in nature and ability to adjust thermal sensitivity to maintain their homeostasis. In the present study, biologically synthesised selenium nanoparticles (Se-NPs) from fisheries waste (fish gill) were used to evaluate its efficacy for enhancement of thermal tolerance of Pangasius hypophthalmus reared under lead (Pb) and high temperature (34°C) for 72days. At the end of the experimental period, CTmin, LTmin, and CTmax, LTmax and cellular metabolic stress were evaluated. The CTmin, LTmin were noticeably (p<0.01) reduced and CTmax, LTmax were enhanced in treatment groups fed with dietary Se-NPs. It was also observed that CTmax and LTmax were increased with acclimation temperature. The positive correlation was perceived between CTmin, LTmin (Y=-0.45+11.05x, R2, 0.768) and CTmax, LTmax (Y=-0.78+8.17x, R2, 0.858) fed with Se-NPs supplemented diet and exposed to Pb and high temperature. Significant improvements in the oxidative and metabolic enzymes were noticed post thermal tolerance experiment. Overall results indicated that incorporation of Se-NPs @ 1mg/kg in diet can confer protection against Pb and thermal stress in P. hypophthalmus.

Expression and regulation of the neutral amino acid transporter B0AT1 in rat small intestine.

Absorption of neutral amino acids across the luminal membrane of intestinal enterocytes is mediated by the broad neutral amino acid transporter B0AT1 (SLC6A19). Its intestinal expression depends on co-expression of the membrane-anchored peptidase angiotensin converting enzyme 2 (ACE2) and is additionally enhanced by aminopeptidase N (CD13). We investigated in this study the expression of B0AT1 and its auxiliary peptidases as well as its transport function along the rat small intestine. Additionally, we tested its possible short- and long-term regulation by dietary proteins and amino acids. We showed by immunofluorescence that B0AT1, ACE2 and CD13 co-localize on the luminal membrane of small intestinal villi and by Western blotting that their protein expression increases in distal direction. Furthermore, we observed an elevated transport activity of the neutral amino acid L-isoleucine during the nocturnal active phase compared to the inactive one. Gastric emptying was delayed by intragastric application of an amino acid cocktail but we observed no acute dietary regulation of B0AT1 protein expression and L-isoleucine transport. Investigation of the chronic dietary regulation of B0AT1, ACE2 and CD13 by different diets revealed an increased B0AT1 protein expression under amino acid-supplemented diet in the proximal section but not in the distal one and for ACE2 protein expression a reverse localization of the effect. Dietary regulation for CD13 protein expression was not as distinct as for the two other proteins. Ring uptake experiments showed a tendency for increased L-isoleucine uptake under amino acid-supplemented diet and in vivo L-isoleucine absorption was more efficient under high protein and amino acid-supplemented diet. Additionally, plasma levels of branched-chain amino acids were elevated under high protein and amino acid diet. Taken together, our experiments did not reveal an acute amino acid-induced regulation of B0AT1 but revealed a chronic dietary adaptation mainly restricted to the proximal segment of the small intestine.

Aryl Sulfonamide Inhibitors of Insulin-Regulated Aminopeptidase Enhance Spine Density in Primary Hippocampal Neuron Cultures.

The zinc metallopeptidase insulin regulated aminopeptidase (IRAP), which is highly expressed in the hippocampus and other brain regions associated with cognitive function, has been identified as a high-affinity binding site of the hexapeptide angiotensin IV (Ang IV). This hexapeptide is thought to facilitate learning and memory by binding to the catalytic site of IRAP to inhibit its enzymatic activity. In support of this hypothesis, low molecular weight, nonpeptide specific inhibitors of IRAP have been shown to enhance memory in rodent models. Recently, it was demonstrated that linear and macrocyclic Ang IV-derived peptides can alter the shape and increase the number of dendritic spines in hippocampal cultures, properties associated with enhanced cognitive performance. After screening a library of 10 500 drug-like substances for their ability to inhibit IRAP, we identified a series of low molecular weight aryl sulfonamides, which exhibit no structural similarity to Ang IV, as moderately potent IRAP inhibitors. A structural and biological characterization of three of these aryl sulfonamides was performed. Their binding modes to human IRAP were explored by docking calculations combined with molecular dynamics simulations and binding affinity estimations using the linear interaction energy method. Two alternative binding modes emerged from this analysis, both of which correctly rank the ligands according to their experimental binding affinities for this series of compounds. Finally, we show that two of these drug-like IRAP inhibitors can alter dendritic spine morphology and increase spine density in primary cultures of hippocampal neurons.

Inhibition of p38 MAPK Phosphorylation Is Critical for Bestatin to Enhance ATRA-Induced Cell Differentiation in Acute Promyelocytic Leukemia NB4 Cells.

Bestatin has been known as an immunomodulating agent in anti-leukemia treatment. The mechanism by which Bestatin enhances all-trans retinoic acid (ATRA)-induced cell differentiation of acute promyelocytic leukemia (APL) cells is generally attributed to inhibition of cell surface CD13/aminopeptidase N activity. Bestatin also exerts its biological activities besides its ability to inhibit aminopeptidase N enzymatic activity. This article provides data to support an alternative mechanism regarding an important role of inhibition of p38 mitogen-activated protein kinase (MAPK) signal pathway in Bestatin's anti-leukemia effect. Bestatin enhanced ATRA-induced differentiation and inhibited ATRA-driven phosphorylation of p38 MAPK in ATRA-sensitive APL NB4 cells. In contrast, Bestatin could not reverse the differentiation block in ATRA-resistant APL MR2 cells, in which ATRA was unable to induce phosphorylation of p38 MAPK. Moreover, CD13 ligation with anti-CD13 antibody WM-15 resulted in phosphorylation of p38 MAPK, reduced the inhibition of Bestatin on the phosphorylation of p38 MAPK, and completely abolished the enhancement of Bestatin on ATRA-inducing differentiation in NB4 cells. This study shows that inhibition of p38 MAPK phosphorylation is critical for Bestatin to enhance ATRA-induced cell differentiation in ATRA-sensitive APL NB4 cells. Results suggested that pharmacological inhibition of the p38 MAPK pathway might enhance ATRA-dependent differentiation.

Efficacy of parenteral administration of bee venom in experimental arthritis in the rat: a comparison with methotrexate.

The use of bee venom (BV) to treat inflammation and pain in arthritis has become increasingly common. This study aimed to compare the effects of BV and methotrexate (MTX), the most used disease-modifying anti-rheumatic drug, in arthritic rats. Edema, erythema, cyanosis, hyperalgesia, reduction of the body mass gain, high circulating tumor necrosis factor alpha (TNF-α) and anti-type II collagen antibodies (AACII), and altered activity of basic (APB) and neutral (APN) aminopeptidases and dipeptidyl peptidase IV (DPPIV) are present in arthritic rats. MTX and/or BV do not affect AACII in healthy or arthritic individuals, but restores TNF-α to normal levels in arthritic rats. BV restores body mass gain to normal levels and MTX ameliorates body mass gain. BV contains DPPIV. BV restores APN in synovial fluid (SF) and in soluble fraction (S) from synovial tissue (ST), and DPPIV in solubilized membrane-bound fraction (M) from peripheral blood mononuclear cells (PBMCs). MTX restores APN of SF, as well as ameliorates APB of S-PBMCs, APN of S-ST and DPPIV of M-PBMCs. The combination therapy does not overcome the effects of BV or MTX alone on the peptidase activities. Edema is ameliorated by MTX or BV alone. MTX, but not BV, is effective in reducing hyperalgesia. Data show that anti-arthritic effects of BV at non-acupoints are not negligible when compared with MTX.

Rapid screening of aminopeptidase N inhibitors by capillary electrophoresis with electrophoretically mediated microanalysis.

In this work, an electrophoretically mediated microanalysis (EMMA) method with a partial-filling technique was setup to evaluate the inhibitory potency of novel compounds toward aminopeptidase N (APN). It was necessary to optimize the electrophoretic conditions with respect to the kinetic constraints and for attaining high sensitivity. In our setup, a part of the capillary was filled with the incubation buffer for the enzyme reaction, whereas the rest was filled with a suitable BGE for the separation of substrates and products. To monitor the performance of the newly developed method, the kinetic constants (Km and Vmax) for the catalyzed dissociation of L-Leucine-p-nitroanilide in the presence of APN as well as the inhibition constant (IC50 ) of a known competitive inhibitor, that is bestatin, were determined and these results were compared with those obtained by a classical spectrophotometric assay. The developed EMMA method was subsequently applied to the screening of 30 APN inhibitors. Whereas the inhibition potency of these inhibitors (expressed in IC50 values) were significantly underestimated by the EMMA method, the order of the inhibitory potential of these various compounds was found in agreement with the literature.

Extensive regenerative plasticity among adult NG2-glia populations is exclusively based on self-renewal.

NG2-glia are known to proliferate in the adult brain, however the extent of their mitotic and regenerative capacity and particularly their adult origin is uncertain. By employing a paradigm of mitotic blockade in conjunction with genetic fate tracing we demonstrate that intracerebroventricular mitotic blocker infusion leads to wide-spread and complete ablation of NG2-glial cells in the hypothalamus and other periventricular brain regions. However, despite the extensive glia loss, parenchymal NG2-glia coverage is fully restored to pretreatment levels within two weeks. We further reveal that in response to mitotic blocker treatment, NG2-glia bordering the ablated territories start to express the stem cell marker nestin, divide and migrate to replace the lost cells. Importantly, the migration front of repopulating NG2-glia invariably proceeds from the distal parenchyma towards the ventricles, ruling out contributions of the subventricular zone neurogenic niche or the corresponding area of the third ventricle as source of new NG2-glia. NG2-CreER-based fate tracing further substantiates that NG2-glia which have been spared from mitotic blockade are the sole source of regenerating NG2-glia. Collectively, our data reveals that all adult NG2-glia retain the ability to divide and that they are capable of fully restoring parenchymal NG2-glia coverage after wide-spread NG2 cell loss, indicating complete self-sufficiency in maintaining NG2-glia population levels in the adult brain.

Discovery of a synthetic Aminopeptidase N inhibitor LB-4b as a potential anticancer agent.

APN inhibitors have been considered as potential anticancer agents for years. LB-4b is the first synthetic APN inhibitor to be evaluated for both of its anti-invasion and anti-angiogenesis effects. As a potent synthetic APN inhibitor (IC50=850 nM, versus bestatin of 8.1 µM), LB-4b was determined to have more significant block effects to cancer cell invasion and angiogenesis than bestatin. Besides, it is able to be easily synthesized with a high total yield, while the reported synthetic methods of bestatin are much more complex.

A novel aminopeptidase N inhibitor developed by virtual screening approach.

A virtual screening was performed to discover novel lead structures as potent aminopeptidase N(APN) inhibitors. A commercial database containing about 1,60,000 molecules in SPECS was filtered by rule of five, zinc binding groups, pharmacophore models and binding pattern analysis. At last, 24 molecules were selected for enzyme inhibition assay and compound 2 exhibited the inhibition constant (K(i)) of 2.79±0.32 µM against APN compared with Bestatin (K(i)= 3.37±0.24 µM). Our results indicated that compound 2 exhibited good antiproliferative activities against a broad spectrum of human cancer cell lines, and induced cell cycle arrest at G1 phase and eventual apoptosis. Moreover, compound 2 can inhibit the invasion of MDA-MB-231 cells. In summary, our results suggest that compound 2, a potent APN inhibitor, is worthy of further development.

Compatibility of garlic (Allium sativum L.) leaf agglutinin and Cry1Ac δ-endotoxin for gene pyramiding.

δ-Endotoxins produced by Bacillus thuringiensis (Bt) have been used as bio-pesticides for the control of lepidopteran insect pests. Garlic (Allium sativum L.) leaf agglutinin (ASAL), being toxic to several sap-sucking pests and some lepidopteran pests, may be a good candidate for pyramiding with δ-endotoxins in transgenic plants for enhancing the range of resistance to insect pests. Since ASAL shares the midgut receptors with Cry1Ac in Helicoverpa armigera, there is possibility of antagonism in their toxicity. Our study demonstrated that ASAL increased the toxicity of Cry1Ac against H. armigera while Cry1Ac did not alter the toxicity of ASAL against cotton aphids. The two toxins interacted and increased binding of each other to brush border membrane vesicle (BBMV) proteins and to the two important receptors, alkaline phosphatase (ALP) and aminopeptidase N (APN). The results indicated that the toxins had different binding sites on the ALP and APN but influenced mutual binding. We conclude that ASAL can be safely employed with Cry1Ac for developing transgenic crops for wider insect resistance.

Molecular modeling of Bt Cry1Ac (DI-DII)-ASAL (Allium sativum lectin)-fusion protein and its interaction with aminopeptidase N (APN) receptor of Manduca sexta.

Genetic engineering of Bacillus thuringiensis (Bt) Cry proteins has resulted in the synthesis of various novel toxin proteins with enhanced insecticidal activity and specificity towards different insect pests. In this study, a fusion protein consisting of the DI-DII domains of Cry1Ac and garlic lectin (ASAL) has been designed in silico by replacing the DIII domain of Cry1Ac with ASAL. The binding interface between the DI-DII domains of Cry1Ac and lectin has been identified using protein-protein docking studies. Free energy of binding calculations and interaction profiles between the Cry1Ac and lectin domains confirmed the stability of fusion protein. A total of 18 hydrogen bonds was observed in the DI-DII-lectin fusion protein compared to 11 hydrogen bonds in the Cry1Ac (DI-DII-DIII) protein. Molecular mechanics/Poisson-Boltzmann (generalized-Born) surface area [MM/PB (GB) SA] methods were used for predicting free energy of interactions of the fusion proteins. Protein-protein docking studies based on the number of hydrogen bonds, hydrophobic interactions, aromatic-aromatic, aromatic-sulphur, cation-pi interactions and binding energy of Cry1Ac/fusion proteins with the aminopeptidase N (APN) of Manduca sexta rationalised the higher binding affinity of the fusion protein with the APN receptor compared to that of the Cry1Ac-APN complex, as predicted by ZDOCK, Rosetta and ClusPro analysis. The molecular binding interface between the fusion protein and the APN receptor is well packed, analogously to that of the Cry1Ac-APN complex. These findings offer scope for the design and development of customized fusion molecules for improved pest management in crop plants.

In vivo priming and ex vivo activation of equine neutrophils in black walnut extract-induced equine laminitis is not attenuated by systemic lidocaine administration.

Laminitis is a crippling disease of horses characterized by an inflammatory response in the tissue that suspends the axial skeleton within the hoof. Pain is a common feature of laminitic pathology and its management is an important component of the treatment regime for this disease. Systemic lidocaine administration is commonly utilized to manage pain in equine laminitis; however, the potential anti-inflammatory effects of this drug during the treatment of equine laminitis have not been investigated. Here, we sought to determine if lidocaine concentrations achieved in the plasma (therapeutic concentrations) of horses systemically administered lidocaine are capable of attenuating neutrophil activation and associated inflammation. To identify markers of activation, purified neutrophils were stimulated in vitro with LPS or recombinant equine IL-8 (reqIL-8) and surface expression of CD13 and CD18 was ascertained by immunofluorescent staining. Activation with LPS or reqIL-8 in vitro induced an elevated expression of CD13 as well as a putative conformational change in CD18 detected by elevated staining with a sub-saturating concentration of anti-CD18 mAb. Lidocaine attenuated the activation-induced changes in CD13 and CD18 expression only when used at 30-70 times therapeutic concentrations. For in vivo analyses, horses were administered black walnut extract (BWE) to induce laminitis and either systemic lidocaine (n=6) or saline (n=6) as a control. Whole blood was collected and incubated with or without reqIL-8. Following which, leukocytes were stained for CD13 and CD18. Protein was extracted from laminar tissue and subjected to gelatin zymography to measure matrix metalloproteinase-9 (MMP-9) accumulation. Results obtained show that changes in neutrophil size, granularity/complexity, CD13 surface expression and CD18 staining intensity occurred over time post BWE administration irrespective of lidocaine treatment in response to incubation alone or with 100 ng/ml of reqIL-8. The mean fluorescence intensities of neutrophils stained for either CD13 or CD18 did not differ between lidocaine treated and saline controls, nor did lamellar MMP-9 content measured by gelatin zymography. Thus, using changes in surface expression of CD13 and CD18 as markers of neutrophil activation in the horse we have shown that BWE treatment activates neutrophils in vivo and this is not affected by systemic administration of lidocaine at levels used to manage pain.

Indices of inflammation in the lung and liver in the early stages of the black walnut extract model of equine laminitis.

The liver and lung are not only described as "target organs" in sepsis in most species, but are purported to be sources of circulating inflammatory mediators central to the systemic inflammatory response syndrome (SIRS). As we have recently reported an inflammatory response in the laminar tissue in laminitis similar to that described in "target organs" in human sepsis, we investigated the inflammatory response of the lung and liver in the black walnut extract (BWE) model of equine laminitis to determine (1) if a similar systemic inflammatory response occurs in this laminitis model as described for these organs in human sepsis, and (2) if these organs may be an important source of the inflammatory mediators leading to laminar inflammation. Real-time quantitative PCR (RT-qPCR) was used to measure hepatic and pulmonary mRNA concentrations of IL-1beta, IL-4, IL-6, IL-8, IL-10, TNF-alpha, COX-1 and COX-2. Hepatic samples were assessed from two time points in the developmental/prodromal period: (1) 1.5h post-BWE administration (BWE-1.5H, n = 5), and (2) the "developmental time point" (onset of leukopenia, approximately 3h post-BWE administration, BWE-DEV, n = 5). Pulmonary samples were only assessed for the BWE-DEV group. One control group (CON-3H, n = 5) was used for both the 1.5H and DEV groups. Finally, CD13 immunohistochemistry was performed to assess leukocyte emigration into hepatic and pulmonary parenchyma. Hepatic and pulmonary mRNA concentrations of the proinflammatory cytokines IL-6, IL-8 and TNF-alpha were significantly increased (P < 0.05) in BWE-1.5H and BWE-DEV groups compared to the control group; IL-1beta mRNA concentrations were only increased in the lung. The "anti-inflammatory" cytokines, IL-10 and IL-4, underwent transient decreases at different time points. Significant increases in parenchymal leukocyte numbers occurred in both the lung and liver at the BWE-DEV time point. Hepatic and pulmonary proinflammatory cytokine expression differ from that previously reported for the laminae in that TNF-alpha was increased in the hepatic and pulmonary tissues, the increases in expression of IL-6 and IL-8 are dramatically smaller for the liver and lung compared to those reported for the laminae, and the peak changes appear to occur later in the disease process in the liver than in the laminae (BWE-DEV in liver vs. 1.5H in the laminae).

The insecticidal activity of recombinant garlic lectins towards aphids.

The heterodimeric and homodimeric garlic lectins ASAI and ASAII were produced as recombinant proteins in the yeast Pichia pastoris. The proteins were purified as functional dimeric lectins, but underwent post-translational proteolysis. Recombinant ASAII was a single homogenous polypeptide which had undergone C-terminal processing similar to that occurring in planta. The recombinant ASAI was glycosylated and subject to variable and heterogenous proteolysis. Both lectins showed insecticidal effects when fed to pea aphids (Acyrthosiphon pisum) in artificial diet, ASAII being more toxic than ASAI at the same concentration. Acute toxicity (mortality at < or =48 h exposure; similar timescale to starvation) was only apparent at the highest lectin concentrations tested (2.0 mg ml(-)1), but dose-dependent chronic toxicity (mortality at >3d exposure) was observed over the concentration range 0.125-2.0 mg ml(-1). The recombinant lectins caused mortality in both symbiotic and antibiotic-treated aphids, showing that toxicity is not dependent on the presence of the bacterial symbiont (Buchnera aphidicola), or on interaction with symbiont proteins, such as the previously identified lectin "receptor" symbionin. A pull-down assay coupled with peptide mass fingerprinting identified two abundant membrane-associated aphid gut proteins, alanyl aminopeptidase N and sucrase, as "receptors" for lectin binding.

Effects of alpha1-adrenergic receptor blockade by doxazosin on renin-angiotensin system-regulating aminopeptidase and vasopressin-degrading activities in male and female rat thalamus.

The thalamus has connections with central autonomic centers involved in cardiovascular control and is enervated by noradrenergic fibers. The excitability of thalamic neurons is due to a reduction of ionic currents mediated by alpha(1)-adrenoceptors. The brain renin- angiotensin system (RAS) and the peptide hormone arginine-vasopressin (AVP) are also involved in the central control of blood pressure, and fluid and electrolyte homeostasis. It has been extensively reported that aminopeptidase A (APA), aminopeptidase B (APB), aminopeptidase N (APN), and vasopressin-degrading cystyl aminopeptidase activity (AVP-DA) play an important role in the regulation of the activity of angiotensins and AVP. We have analyzed the effect of alpha(1)-adrenoceptor blockade by doxazosin on RAS-regulating aminopeptidase activities and AVP-DA in soluble and membrane-bound fractions of male and female rat thalamus. Our results show that alpha(1)-adrenoceptors blockade by doxazosin does not modify the RAS through its degrading peptidases at thalamic level either in male or female rats. However, alpha(1)-adrenoceptors blockade shows gender differences in AVP-DA, increasing in males but not in females, supporting an increased capacity of males against females to degrade AVP and, therefore, to regulate cardiovascular homeostasis, under this pharmacological manipulation.

Aspartyl, arginyl and alanyl aminopeptidase activities in the hippocampus and hypothalamus of streptozotocin-induced diabetic rats.

Acid (aspartyl), basic (arginyl) and neutral (alanyl) aminopeptidases degrade angiotensins, vasopressin, oxytocin, bradykinin and enkephalins. These peptides regulate memory, energy homeostasis, water-salt balance and blood pressure, functions that are mainly exerted in the hippocampus and hypothalamus, and that can be affected by diabetes mellitus. To evaluate the relationship between the diabetes mellitus and processing and inactivation roles of these representative aminopeptidases, we measured their activities in both brain structures of control and streptozotocin-diabetic rats. Hypothalamic soluble aspartyl and arginyl aminopeptidases presented significant decreased activity levels in diabetic rats, which were mitigated by insulin therapy. In addition to membrane-bound puromycin sensitive and insensitive alanyl aminopeptidases, its soluble puromycin sensitive form did not differ between diabetic and control rats in both brain structures. Glucose and/or insulin did not seem to alter in vitro the hypothalamic activities of soluble aspartyl and arginyl aminopeptidases. The implied hypothalamic control of regulatory peptide activity by aspartyl and arginyl aminopeptidases supports the hypothesis that the hydrolytic ability of these enzyme types could be a common link for the disruptions of water-salt balance, blood pressure and energy homeostasis in diabetes mellitus.

The protein fraction of Phyllanthus niruri plays a protective role against acetaminophen induced hepatic disorder via its antioxidant properties.

The aim of this study was to investigate the hepatoprotective action of the protein fraction of Phyllanthus niruri against acetaminophen (APAP) hepatotoxicity. The partially purified protein fraction of P. niruri was injected intraperitoneally in mice either prior to (preventive) or after the induction of toxicity (curative). Levels of different liver marker enzymes in serum and different antioxidant enzymes, as well as lipid peroxidation in total liver homogenates were measured in normal, control (toxicity induced) and P. niruri protein fraction-treated mice. P. niruri significantly reduced the elevated glutamate pyruvate transaminase (GPT) and alkaline phosphatase (ALP) levels in the sera of toxicity induced mice, compared with the control group. Lipid peroxidation levels were also reduced in mice treated with P. niruri protein fraction compared with the APAP treated control group. Among the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST) levels were restored to almost normal levels compared with the control group. P. niruri treatment also enhanced reduced hepatic glutathione (GSH) levels caused by APAP administration. The results demonstrated that the protein fraction of P. niruri protected liver tissues against oxidative stress in mice, probably acting by increasing antioxidative defense.

Ethanol modulates neuropeptide-degrading aminopeptidases at synapse level in calcium-dependent conditions.

AIMS: To investigate the role of aminopeptidases in the pathways to peptides neurotransmission/neuromodulation ending in the actions of ethanol (EtOH) on the brain. METHODS: The effects of EtOH on alanyl-, arginyl-, cystyl-, leucyl- and tyrosyl-aminopeptidase activities were studied under basal/resting and K+-stimulated conditions at the synapse level, using mouse frontal cortex synaptosomes and their incubation supernatant in a Ca2+-containing or Ca2+-free medium. RESULTS: Under basal conditions, synaptosome aminopeptidase activities showed an inhibitory or biphasic response depending on the concentration of EtOH used and the aminopeptidase assayed, whereas supernatant activities showed a more complex response. Under K+-stimulated conditions, EtOH inhibited all synaptosome aminopeptidases assayed in presence of Ca2+. However, in absence of Ca2+, different responses were obtained depending on the concentration of EtOH used. In the supernatant, the highest concentration of EtOH inhibited the K+-stimulated increase on aminopeptidase activities, although the lowest concentration enhanced the release in presence of Ca2+. In absence of it, EtOH blocked the K+-stimulated decrease or increased the activity depending on the concentration of EtOH used. CONCLUSIONS: The changes on aminopeptidase activities induced by EtOH may reflect the functional status of their corresponding endogenous substrates. EtOH may influence opioid peptides, oxytocin, vasopressin and the brain renin-angiotensin system through their degrading enzymes.