Aminopeptidase N Activity Research Articles

Intestinal microbiota differentially affect brush border enzyme activity and gene expression in the neonatal gnotobiotic pig

To study microbial influence on intestinal development pertaining to nutrient digestion, two separate gnotobiotic experiments were performed, each with 16 piglets allocated to four treatment groups: germfree (GF), monoassociation with Escherichia coli, monoassociation with Lactobacillus fermentum or conventionalization with faecal bacteria (CV). Enzyme activity and gene expression of lactase phlorizin hydrolase (LPH) and aminopeptidase N (APN) were measured in isolated enterocytes, harvested on day 14, using specific substrates and quantitative PCR respectively. Enterocytes of CV pigs had reduced APN activity, but had increased gene expression relative to GF, making the specific activity:mRNA (A:G) ratio dramatically lower (p < 0.05). Similarly, LPH A:G ratio was significantly reduced (p < 0.05) in enterocytes of CV pigs as compared with GF. The results of co-incubation of L. fermentum, E. coli and faecal bacteria with APN indicate a direct relationship between enzyme inactivation and specific A:G ratio in enterocytes. We conclude that enterocyte up-regulation of APN expression occurs as either a direct response to microbial colonization or as a feedback mechanism in response to reduced enzyme activity through microbial degradation. This mechanism may play a role in ensuring effective competition of the host with the intestinal microbiota for available nutrients.

The effect of short- and long-term fasting on digestive and metabolic flexibility in the Andean toad, Bufo spinulosus

Hibernation in ectothermic animals was historically considered as a simple cold-induced torpor state resulting from the inability to maintain a high body temperature at low ambient temperatures. During the last decades this vision changed and nowadays there is a myriad of studies showing that hibernation implies different adjustments at the genetic, molecular, biochemical and cellular levels. However, studies oriented to evaluate changes of whole organism structure and physiology still are scarce, which is particularly true for amphibians that hibernate on land. Accordingly, in the Andean toad (Bufo spinulosus), we investigated the effect of short-term fasting and hibernation on the hydrolytic activity of digestive enzymes, histology of the small intestine, gross morphology of digestive and other internal organs and standard metabolic rate. Based on the pattern of size variation, internal organs may be grouped into those that were affected by both season and feeding condition (small intestine, stomach and liver), those that were only affected by season (fat bodies), those that were only affected by feeding condition (kidneys) and, finally, those that did not change between the three groups (large intestine, heart and lungs). Hydrolytic activity of maltase, trehalase and aminopeptidase-N followed the same pattern of variation (feeding > fasting > hibernating toads), although the change for the latter enzyme was less noticeable than for the disaccharidases. Enzymatic adjustments were correlated with changes in small intestine histology: villus and enterocyte height increased from hibernating to fasting and more markedly from fasting to feeding toads. Metabolic rate decreased during hibernation to 7.8% (at 5 degrees C) and 13.6% (at 15 degrees C) of summer values, which is one of the highest metabolic depressions reported for any ectothermic vertebrate. Our results suggest that amphibian persistence in highly seasonal environments is related to a large capacity of phenotypic flexibility at different organisational levels; an ability that may be related to the extensive ranges of temporal existence and geographic distribution of these vertebrates.

Specific aminopeptidases of excised human nasal epithelium and primary culture: a comparison of functional characteristics and gene transcripts expression

To investigate whether growing human nasal epithelium as primary cultures alters aminopeptidase B (APB), aminopeptidase N (APN) and dipeptidyldipeptidase (DPPIV) metabolic characteristics, and mRNA gene transcript expression. The formation of 7-amino-methyl coumarin from specific substrates for APN (L-alanine-4-methyl-coumaryl-7-amide, APB (L-arginine-4-methyl-coumaryl-7-amide) and DPPIV (glycyl-L-proline-4-methyl-coumaryl-7-amide) was used to estimate the KM, Vmax and the effect of aminopeptidases inhibitors on the enzymes. Polymerase chain reaction was used to investigate gene expression. Results of this study showed that: (1) both the excised tissues and primary cultures of human nasal epithelium expressed APN, APB and DPPIV activity; (2) the KM of APB, APN and DPPIV was not significantly different in cell and tissue homogenates; (3) except for APN, the Vmax was not significantly different in the two metabolism models; (4) there was no statistically significant difference in the behaviours of APB, APN and DPPIV in response to inhibition by puromycin and bestatin in the two models; (5) the mRNA transcripts that encode APB, APN and DPPIV were expressed in both cell culture and tissue homogenate. Based on the results of this study, it may be concluded that nasal primary culture system is suitable for investigating peptide and protein metabolism and enzymatic stability in human nasal epithelium. Except for APN, the tissue culture conditions did not significantly alter the functional and molecular expression of the aminopeptidases.

Developmental adjustments of house sparrow (Passer domesticus)nestlings to diet composition

House sparrow nestlings are fed primarily on insects during the first 3 days of their life, and seeds become gradually more important afterwards. We tested whether developmental changes in size and functional capacity of the digestive tract in young house sparrows are genetically hard-wired and independent of diet, or can be modified by food type. Under laboratory conditions, we hand-fed young house sparrows with either a starch-free insect-like diet, based mainly on protein and fat, or a starch-containing diet with a mix of substrates similar to that offered to older nestlings in natural nests when they are gradually weaned from an insect to a seed diet. Patterns of overall development in body size and thermoregulatory ability, and in alimentary organ size increase, were relatively similar in house sparrow nestlings developing on both diets. However, total intestinal maltase activity, important in carbohydrate breakdown, was at least twice as high in house sparrow nestlings fed the starch-containing diet (P<0.001). The change in maltase activity of nestlings was specific, as no change occurred in aminopeptidase-N activity in the same tissues. There was no significant diet effect on digesta retention time, but assimilation efficiency for radiolabeled starch tended to be higher (P=0.054) in nestlings raised on starch-containing diet. Future studies must test whether the diet-dependent increase in maltase activity during development is irreversible or reversible, reflecting, respectively, a developmental plasticity or a phenotypic flexibility.

Seasonal effects on intestinal enzyme activity in the Australian agamid lizard, Lophognathus temporalis

The tropical agamid lizard, Lophognathus temporalis, has higher metabolic and feeding rates during the wet season compared to the dry season. Also, lizards from urban sites tend to be larger than those from natural sites, partly due to site differences in food availability. Therefore, we hypothesized that activity of membrane-bound intestinal enzymes and masses of organs related to digestion would differ both seasonally and between urban and natural sites. To test this, we measured activities of aminopeptidase-N (APN), maltase, and sucrase, as well as organ masses. APN activity (micromol min(-1) g(-1)) was highest in the middle portion of the intestine (section 2), followed by the proximal portion (section 1) and then the distal portion (section 3). Maltase activity was highest in section 1 and decreased distally. We detected some sucrase activity in section 1 but none in sections 2 or 3. We found similar enzyme activities within each section irrespective of site or season. However, total enzyme activities were higher during the wet season compared to the dry season for both urban and bush L. temporalis. Total wet season enzyme activity in urban and bush L. temporalis was greatest for APN (25.4; 15.8 micromol min(-1); respectively), then maltase (3.9; 3.6 micromol min(-1); respectively) and then sucrase (0.3; 0.2 micromol min(-1); respectively). The higher total enzyme activities was the result of an increase in intestinal mass during the wet season.

Colorimetric Assay of Aminopeptidase N Activity Based on Inhibition of the Disassembly of Gold Nano-composites Conjugated with a Thermo-responsive Copolymer

A colorimetric change of gold nano-composites conjugated with a thermo-responsive copolymer has been applied to a colorimetric assay to quantify the activity of aminopeptidase N (APN). Heating a solution of assembled gold nano-composites provokes the gold nano-composites to disassemble, leading to a colorimetric change in the solution from blue-purple to red. The disassembly is inhibited by cysteine, but not by cysteinylglycine, thus allowing us to monitor the progress of an enzymatic decomposition of cysteinylglycine into cysteine and glycine with APN. The activity of APN is estimated through a colorimetric change in a solution of gold nano-composites from blue-purple to red, after being heated (98 degrees C for 30 min), followed by being cooled. A good relationship between the a(*) value in L(*)a(*)b(*) color coordinates, which quantified the color of the solution, and the activity of APN was obtained in 3 h of the incubation time, indicating the potential of a colorimetric assay of APN activity.

The synthesis and preliminary activity assay In Vitro of peptide-like derivatives as APN inhibitors

Both the aminopeptidase N (APN) and matrix metalloproteinase (MMP) are essential metallopeptidases in the development of tumor invasion and angiogenesis. A series of novel peptide-like derivatives were designed and synthesized as antitumor agents. Their structures were confirmed by IR, MS, and (1)H-NMR. These compounds exhibited potent inhibitory activities against APN and low activity against MMP in vitro. The derivatives with methoxy group show better activities than those with other substituted group and could be used as lead compounds for exploring new APN inhibitors in the future.

Novel Biological Properties of Oenothera paradoxa Defatted Seed Extracts: Effects on Metallopeptidase Activity

In this study, for the first time, we used the in vitro metallopeptidase model for the identification of a potential novel activity of defatted evening primrose seed extracts. Prepared extracts of different polarity (aqueous, 60% ethanolic, isopropanolic, and 30% isopropanolic) at concentrations of 1.5-100 microg/mL exhibited a significant and dose dependent inhibition of three tested enzymes. The 50% inhibition of enzymes activity showed that aminopeptidase N (APN) was the enzyme affected to the greatest extent with IC50 at the level of 2.8 microg/mL and 2.9 microg/mL for aqueous and 30% isopropanolic extracts, respectively. The activity of neutral endopeptidase (NEP) was quite strongly inhibited by the extracts as well. The HPLC-DAD analysis and bioguided fractionation led to the identification of four active compounds: (-)-epicatechin gallate, proanthocyanidin B3, oenothein B, and penta-O-galloyl-beta-D-glucose (PGG). Oenothein B has been shown previously to inhibit metallopeptidases. The three other compounds are known to inhibit angiotensin-converting enzyme (ACE), but they have not been previously reported to inhibit the NEP and APN activity. PGG and procyanidins with different degrees of polymerization, as the dominating compounds in O. paradoxa seeds, seemed to play a role in the crude extract activity.

The Significance of Urinary Markers in the Evaluation of Diabetic Nephropathy

The Significance of Urinary Markers in the Evaluation of Diabetic Nephropathy Oxidative stress is considered to be a unifying link between diabetes mellitus (DM) and its complications, including nephropathy (DN). The aim of this study was to determine the parameters of oxidative injury of lipids and proteins as well as the activity of ectoenzymes in the urine of DN patients. The study included 40 individuals: 10 patients with type 2 diabetes mellitus and microalbuminuria (DMT2-MIA), 10 type 2 diabetic patients with macroalbuminuria (DMT2-MAA), 10 patients with type 1 diabetes and microalbuminuria (DMT1-MIA) and 10 age- and sex-matched healthy subjects (control). In the urine we determined TBA reactive substances (TBARS), reactive carbonyl groups (RCG), and the activity of ectoenzymes N-acetyl-β-d-glucosaminidase (NAG), plasma cell differentiation antigen (PC-1), aminopeptidase N (APN) and dipeptidyl peptidase IV (DPP IV). A higher concentration of TBARS in the urine was found in DMT2-MIA and DMT1-MIA, compared to the control group (p&lt;0.001 and P&lt;0.05). The urine concentration of RCD shows similar results with a significant elevation in the groups with DMT2-MAA and DMT1-MIA, compared to the DMT2-MIA (p&lt;0.001) and control group (p&lt;0.001). Activities of NAG, APN and DPPIV were significantly higher in the urine of DMT2-MAA, compared to the control (p&lt;0.01). The activity of PC-1 was slightly increased in that group, but not significantly. In conclusion, the level of oxidative stress markers and activities of brush border ectoenzymes in the urine may be a useful non-invasive and easily repeatable test in DN.

Distribution of Dipeptidyl Peptidase IV in Patients with Chronic Tonsillitis

In the pathogeneses of recurrent tonsillitis (RT) and tonsillar hypertrophy (TH), different immunological mechanisms are involved. Dipeptidyl peptidase IV (DPP IV) and aminopeptidase N (APN) participate in the regulation of the immune response during inflammation. In this study, the localization of DPP IV and the enzymatic activities of DPP IV and APN in 32 patients, 13 with RT and 19 with TH, who underwent tonsillectomy were investigated. The localization of DPP IV in tonsils was studied using histochemical and immunohistochemical methods. The enzymatic activities of DPP IV and APN in tonsillar lymphocytes and the patients' sera were determined kinetically at 37 degrees C using Gly-Pro-p-nitroanilide (for DPP IV) and Ala-p-nitroanilide (for APN) as chromogenic substrates. In samples from both RT and TH patients, DPP IV was found to localize mainly in extrafollicular areas of tonsillar tissue in a pattern corresponding to the T-cell distribution. Significantly higher (P < 0.001) levels of DPP IV and APN activities in sera from patients with TH than in sera from patients with RT were found. A correlation of DPP IV activities in sera and tonsillar lymphocytes from patients with TH was also found (r = 0.518; P < 0.05). Moreover, the results show that DPP IV and APN activities in sera decreased significantly with age. Tonsillar lymphocytes demonstrated a wide range of DPP IV and APN activities, without significant differences between the investigated groups. The results of this study show that the localization of DPP IV does not depend on the type of tonsillitis, whereas the variety in levels of DPP IV and APN activities in sera of patients with TH and RT suggests different patterns of participation of antigen-stimulated tonsils in the immune system.

Matched regulation of gastrointestinal performance in the Burmese python,Python molurus

In Burmese pythons fasting and feeding cause dramatic regulation of gastric acid production and intestinal nutrient uptake. Predictably, other components of their gastrointestinal tract are similarly regulated with each meal. We therefore assessed the matched regulation of gastrointestinal performance by comparing the postprandial activities and capacities of gastric (pepsin), pancreatic (amylase and trypsin) and intestinal (aminopeptidase-N and maltase) enzymes, and intestinal nutrient uptake. Tissue samples were collected from pythons fasted and at 0.25, 0.5, 1, 2, 3, 4, 6, 10 and 15 days following their consumption of rodent meals equaling 25% of snake body mass. With feeding, pythons experience no significant change in stomach mass, whereas both the pancreas and small intestine doubled in mass. Feeding also triggered a depletion of gastric mucosal pepsinogen, a respective 5.7- and 20-fold increase in the peak activities of pancreatic trypsin and amylase, and a respective 2.3- and 5.5-fold increase in the peak activities of intestinal maltase and aminopeptidase-N. Enzyme activities peaked between 2 and 4 days postfeeding and returned to fasting levels by day 10. Independent of digestive stage, python intestine exhibited a proximal to distal decline in enzyme activity. For both sugars and proteins, intestinal capacities for enzyme activity were significantly correlated with nutrient uptake capacities. The concomitant postprandial upregulation of tissue morphology, intestinal nutrient transport rates and enzyme activities illustrate, for the python, the matched regulation of their gastrointestinal performance with each meal.

High glucose alters tubular epithelial cell expression of molecules in the renin‐angiotensin axis

Purpose: Aminopeptidase N (APN), a type II membrane ectoenzyme also known as CD13, is a major component of Glut‐4 containing vesicles. It has been postulated to regulate secretory function in general and glucose transport in particular. The present study was performed to determine the effect of glucose and insulin on the expression and activity of APN in renal epithelial cells.Methods: A heightened glucose milieu was created by culturing immortalized HK‐2 cells in 25 mM glucose as compared to the normal 5 mM glucose and a control for hyperosmolality (cells grown in 5 mM glucose with 20 mM mannitol)(OC). The model was validated by studying the altered growth and adhesion characteristics of the HK‐2 cells in the three conditions. Assays for APN activity and APN transcript abundance were performed on cells cultured in the three conditions. The effect of insulin and lisinopril on glucose‐induced changes APN activity was examined.Results: APN transcript, protein, and activity were greater in cells treated with 25 mM vs 5 mM glucose and the same in cells treated with OC vs 5 mM glucose. Insulin reduced APN activity to that observed in low glucose (5mM) levels (p&lt;0.05).Conclusions: Glucose is capable of stimulating APN in renal epithelial cells. This stimulation was blocked by insulin, raising the possibilities that APN plays a role in glucose homeostasis in general and in the pathogenesis of diabetes specifically.

Regulation of aminopeptidase N (EC 3.4.11.2; APN; CD13) on the HL-60 cell line by TGF-β1

Membrane-bound peptidases interfere with cellular growth, differentiation, activation and death by fine-tuning local concentrations of various signaling peptides such as the growth factors, hormones, chemokines and cytokines. We examined the effects of anti-inflammatory cytokine transforming growth factor-beta1 (TGF-β1) on the expression and activity of aminopeptidase N (APN), an ectoenzyme processing several signaling peptides. Myelo-monocytic HL-60 cell line having high basal APN activity corresponding to the membrane CD13 marker served as a model. Regulation of CD13/APN was assayed at the levels of mRNA and at the membrane marker CD13. Functional properties of CD13/APN were examined by measuring the enzyme activity, and the signal transduction ability, followed as Ca++ mobilization triggered by APN-blocking WM-15 antibody. TGF-β1 at physiological concentrations (0.16 to 2.5 ng/mL) increased expression of CD13 both at mRNA and membrane protein level in a time- and concentration-dependent manner. Transcriptional activation of CD13 by TGF-β1 is suggested as actinomycin-D, an inhibitor of RNA synthesis, abrogated the TGF-β1-induced up-regulation of CD13. Increased membrane CD13 expression was associated with an increase of its enzyme (APN) activity and with a decrease of its signal transduction ability. Anti-inflammatory cytokine TGF-β1 counteracted the effects of pro-inflammatory cytokine IFN-γ on membrane CD13 expression in a time- and concentration-dependent fashion, suggesting a cytokine-regulated role of CD13/APN in inflammation. This is the first report on regulation of CD13/APN expression by TGF-β1 on immature cells of myelo-monocytic origin. As obtained with physiological concentrations of TGF-β1 these findings may be relevant for cytokine-regulated CD13/APN expression on mature myeloid cells in the course of inflammation.

Diagnostic Value of the Aminopeptidase N, N-Acetyl-β-D-Glucosaminidase and Dipeptidylpeptidase IV in Evaluating Tubular Dysfunction in Patients with Glomerulopathies

Aim. The aim of the present study was to investigate the value of the urine cell glycoprotein 1 (PC-1), aminopeptidase N (APN), N-acetyl-β-D-glucosaminidase (NAGA), and dipeptidylpeptidase IV (DPP IV) in the evaluation of tubular damage in patients with primary glomerulonephritis, diabetic nephropathy, and lupus nephritis. Subjects and Methods. PC-1, APN, NAGA, and DPP IV activities were determined in serum, urine, and lymphocytes of 178 subjects, including 10 patients with membranous nephropathy, 38 with IgA nephropathy, 29 with lupus nephritis, 51 with diabetic nephropathy, and 50 control subjects. Results. Urinary PC-1 excretion in IgA nephropathy group was significantly higher (p < 0.05) than in controls. Urinary NAGA excretion was markedly (p < 0.01) higher in membranous nephropathy group, and APN excretion in diabetic nephropathy group was significantly higher (p < 0.01) than in healthy controls. Urinary APN activity was significantly (p < 0.01) higher in both type 1 and type 2 diabetic patients with microalbuminuria, as well as urinary NAGA and DPP IV activities in type 2 diabetics with microalbuminuria (p < 0.01 and p < 0.05, respectively) compared to controls. Serum PC-1 and APN activities were significantly higher than the control level in membranous nephropathy group, as well as serum PC-1 and DPP IV activities in IgA nephropathy patients (p < 0.05). However, significantly lower serum DPP IV and APN activity was observed in type 2 diabetics with microalbuminuria compared to controls (p < 0.05). Conclusion. Damage of tubules in primary glomerulonephritis, lupus nephritis, and diabetic nephropathy is accompanied by a release of several tubular enzymes, with possible diagnostic and prognostic significance. Increased serum PC-1, APN, and DPP IV activities could be also of diagnostic significance.

Adaptability of the digestive function according to age at weaning in the rabbit: I. Effect on feed intake and digestive functionality

The functional adaptability of the digestive system to the level of feed intake was investigated in the young rabbits by comparing two groups of 12 litters each, weaned at 21 (W21) or 35 (W35) days of age. From 14 days onwards, rabbits were fed a pelleted feed (NDF: 332 g/kg, CP: 177 g/kg, starch: 98 g/kg, as-fed basis). Until 49 days of age, the profile of digestive enzymes was weekly determined in the small intestinal content and mucosa, as well as caecal fermentation traits and fibrolytic activities. In the W21 group, the solid feed intake was increased by 57% between 21 and 35 days (P < 0.01), while the daily body growth was lower from 21 till 42 days (−17%, P < 0.05) when compared with the W35 group. Activities of enzymes of pancreatic origin were only scarcely influenced by the weaning age. In the W21 group, amylase activity tended to be lower at 28 days of age (−36%, P = 0.064), and trypsin activity was decreased by 31% at 49 days of age (P < 0.01). Lipase activity was similar in both weaning groups. Duodenal and jejunal activities of maltase and aminopeptidase N (APN) were higher on day 28 in the W21 group as compared with the W35 group (×1.4 to ×2.4, respectively, P < 0.05). On day 35, duodenal APN activity was twice as higher in the W21 group than in the W35 group (P < 0.01). In caecum, major differences between both weaning groups were observed at 28 days of age with a decrease in ammonia concentration (−43%, P < 0.01) in W21 compared with W35 rabbits. Conversely, the acetate proportion was 5% higher in the W21 group (P < 0.01) on day 28. In conclusion, the digestive tract of early-weaned rabbits showed some adaptative properties in response to nutritional environment changes, but they were insufficient to maintain their growth rate.

Aminopeptidase N in arterial hypertension

Aminopeptidase N (APN) or CD13 is a conserved type II integral membrane zinc-dependent metalloprotease in the M1 family of ectoenzymes. APN is abundant in the kidneys and central nervous system. Identified substrates include Angiotensin III (Ang III); neuropeptides, including enkephalins and endorphins; and homones, including kallidan and somatostatin. It is developmentally expressed, a myelomonocytic marker for leukemias, and a receptor for coronovirus. There is evolving support for APN in the regulation of arterial blood pressure and the pathogenesis of hypertension. In rodent strains, intracerebraventricular (i.c.v.) infusions of APN reduces, while inhibitors of APN activity have a pressor effect on blood pressure. Dysregulation of central APN has been linked to the pathogenesis of hypertension in the spontaneously hypertensive rat. There is evidence that renal tubule APN inhibits Na flux and plays a mechanistic role in salt-adaptation. A functional polymorphism of the ANP gene has been identified in the Dahl salt-sensitive rat. Signaling by APN impacting on blood pressure is likely mediated by regulation of the metabolism of Ang III to Ang IV. Whether APN regulates arterial blood pressure in humans or is a therapeutic target for hypertension are subjects for future exploration.

Metabolic and digestive response to food ingestion in a binge-feeding lizard, the Gila monster (Heloderma suspectum)

The gastrointestinal tract possesses the capacity to change in form and function in response to fasting and feeding. Such plasticity can be dramatic for species that naturally experience long episodes of fasting between large meals (e.g. sit-and-wait foraging snakes, estivating anurans). By contrast, for active foraging species that feed more frequently on smaller meals, gastrointestinal responses are more modest in magnitude. The Gila monster Heloderma suspectum is an active foraging lizard that feeds infrequently on meals weighing up to one-third of its body mass. Additionally, Gila monsters possess a species-specific salivary peptide, exendin-4, which may be involved in the regulation of metabolic and digestive performance. To investigate the adaptive postprandial response of Gila monsters and the potential regulatory role of exendin-4, we measured metabolic and intestinal responses to feeding in the presence or absence of circulating exendin-4. Following the consumption of rodent or egg meals equivalent to 10% of lizard body mass, metabolic rates peaked at 4.0- to 4.9-fold of standard metabolic rates and remained elevated for 5-6 days. Specific dynamic action of these meals (43-60 kJ) was 13-18% of total meal energy. Feeding triggered significant increases in mucosal mass, enterocyte width and volume, and the upregulation of D-glucose uptake rates and aminopeptidase-N activity. Total intestinal uptake capacity for L-leucine, L-proline and D-glucose were significantly elevated within 1-3 days after feeding. Whereas the absence of circulating exendin-4 had no impact on postprandial metabolism or the postprandial response of intestinal structure and nutrient uptake, it significantly increased intestinal aminopeptidase-N activity. Within the continuum of physiological responses to feeding and fasting, Gila monsters occupy an intermediate position in experiencing moderate, though significant, regulation of intestinal performance with feeding.

A novel aminopeptidase in the fat body of the moth Achaea janata as a receptor for Bacillus thuringiensis Cry toxins and its comparison with midgut aminopeptidase

Bacillus thuringiensis insecticidal crystal proteins bind to cell-surface receptors which represent a family of aminopeptidases [APN (aminopeptidase N)] present on the brush border membrane of insect midgut cells of susceptible insects leading to pore formation and death of the insect. We report here for the first time the presence of a novel APN in the fat body of the moth Achaea janata. Northern blotting detected at least one APN-specific transcript in the fat body, whereas two transcripts of different sizes were detected in the midgut. We have cloned two full-length APN cDNAs of 3015 bp and 2850 bp from fat body and midgut respectively, which encode proteins of 1004 and 950 amino acids. These two APNs share only 33% amino acid sequence identity, but both display the typical APN features, such as the N-terminal signal peptide, several putative glycosylation sites, C-terminal glycosylphosphatidylinositol anchor signal, the APN-specific zinc-binding/gluzincin motif HEXXHX(18)E and gluzincin motif GAMENWG. The fat body APN manifested a variation in its expression with respect to tissue and developmental stage. In spite of the abundance of the APN transcript in the fat body, fairly low APN activity was detected in this tissue. The fat-body- and midgut-specific APNs showed differential interaction with various Cry1A toxins. Besides, the level of toxicity of different Cry subtypes varied enormously with mode/site of delivery, such as intrahaemocoelic injections and feeding bioassays. These data indicate that the fat body might be a potential alternative Cry toxin target site in the moth.

Small potent ligands to the insulin‐regulated aminopeptidase (IRAP)/AT4 receptor

Angiotensin IV analogs encompassing aromatic scaffolds replacing parts of the backbone of angiotensin IV have been synthesized and evaluated in biological assays. Several of the ligands displayed high affinities to the insulin-regulated aminopeptidase (IRAP)/AT(4) receptor. Displacement of the C-terminal of angiotensin IV with an o-substituted aryl acetic acid derivative delivered the ligand 4, which exhibited the highest binding affinity (K(i) = 1.9 nM). The high affinity of this ligand provides support to the hypothesis that angiotensin IV adopts a gamma-turn in the C-terminal of its bioactive conformation. Ligand (4) inhibits both human IRAP and aminopeptidase N-activity and induces proliferation of adult neural stem cells at low concentrations. Furthermore, ligand 4 is degraded considerably more slowly in membrane preparations than angiotensin IV. Hence, it might constitute a suitable research tool for biological studies of the (IRAP)/AT(4) receptor.

Ethanol Inhibitory Effect on Rat Kidney Brush Border Aminopeptidases

Background/Aim: Confusing data have been reported about the effect of ethanol or its metabolic products on blood pressure. The pressor agent, angiotensin II (Ang II), is found to be susceptible to degradation by different enzymes known as angiotensinases. We have studied the effects of ethanol and L-NAME, an inhibitor of nitric oxide synthase, consumption on rat serum and kidney ectoenzymes: aminopeptidase N (APN) and aminopeptidase A (APA). Methods: Enzymatic activity of both enzymes was determined spectrophotometrically in serum and 10% homogenates of the rat kidney cortex using appropriate chromogenic substrates. Results: After 2 weeks of treatment with ethanol and L-NAME, blood urea and creatinine levels were significantly increased. The activities of APN (EC 3.4.11.2) and APA (EC 3.4.11.7) were reduced in serum as well as in kidney tissue during this period. L-NAME significantly attenuated activities of both enyzmes. Ethanol and L-NAME given simultaneously did not have an additional effect on the activity of investigated enzymes. Conclusion: Hypertension caused by chronic ethanol treatment as well as L-NAME administration could be associated with the reduction of APN and APA activity. Possible ethanol- and L-NAME-mediated inhibition of angiotensins degrading aminopeptidases could potentiate their effects on blood pressure.