Up-regulation of leucine aminopeptidase-A in cadmium-treated tomato roots.

The effects of cadmium (Cd) on aminopeptidase (AP) activities and Leucine-AP (LAP) expression were investigated in the roots of tomato (Solanum lycopersicum L., var Ibiza) plants. Three-week-old plants were grown for 10 days in the presence of 0.3-300 µM Cd and compared to control plants grown in the absence of Cd. AP activities were measured using six different p-nitroanilide (p-NA) substrates. Leu, Met, Arg, Pro and Lys hydrolyzing activities increased in roots of Cd-treated plants, while Phe-pNA cleavage was not enhanced after Cd treatments. The use of peptidase inhibitors showed that most of the Leu-pNA hydrolyzing activity was related to one or several metallo-APs. Changes in Lap transcripts, protein and activities were measured in the roots of 0 and 30-µM Cd-treated plants. LapA transcript levels increased in Cd-treated roots, whereas LapN RNAs levels were not modified. To assess amount of Leu-pNA hydrolyzing activity associated with the hexameric LAPs, LAP activity was measured following immunoprecipitation with a LAP polyclonal antiserum. LAP activity increased in Cd-treated roots. There was a corresponding increase in LAP-A protein levels detected in 2D-immunoblots. The role of LAP-A in the proteolytic response to Cd stress is discussed.

Influence of dipeptidyl peptidase inhibitors on growth, peptidase activity, and ammonia production by ruminal microorganisms.

The aim was to investigate known and potential new inhibitiors of dipeptidyl peptidases (DPP) for their effects on ruminal microorganisms. Gly-Phe diazomethylketone (GPD), Ala-Ala chloromethylketone (AAC), benserazide (DL-serine 2-(2,3,4- trihydroxybenzyl) hydrazide), and diprotin A (Ile-Pro-Ile) inhibited DPP activities of Prevotella albensis, P. ruminicola, P. bryantii, P. brevis, and mixed ruminal microorganisms, though incompletely and, except for diprotin A, without absolute specificity for any of the peptidases. Leucine aminopeptidase activity of Streptococcus bovis was also inhibited by GPD and benserazide. The inhibitors had no effect on the growth of the bacteria, except for GPD, which inhibited growth of P. albensis when only peptides were available for growth. Benserazide had some inhibitory effects on the growth of Megasphaera elsdenii and Prevotella spp., even in the absence of peptides. The predatory activity of ciliate protozoa on bacteria was unaffected by DPP inhibitors. Ammonia production from casein by mixed ruminal microorganisms was inhibited significantly (P < 0.05) by AAC (29% inhibition) and benserazide (33%). It was concluded that DPP inhibitors can influence the rate of NH3 production in the rumen and may form the basis for developing protein-sparing feed additives for ruminants.

Piperine modulates permeability characteristics of intestine by inducing alterations in membrane dynamics: influence on brush border membrane fluidity, ultrastructure and enzyme kinetics.

Piperine (1-Piperoyl piperidine) is a major alkaloid of Piper nigrum Linn. and Piper longum Linn. It is shown to possess bioavailability-enhancing activity with various structurally and therapeutically diverse drugs. The mechanism of enhancing the bioavailability, is, however, not understood. We hypothesize that piperine's bioavailability-enhancing property may be attributed to increased absorption, which may be due to alteration in membrane lipid dynamics and change in the conformation of enzymes in the intestine. Results of membrane fluidity studies using an apolar fluorescent probe, pyrene (which measures the fluid properties of hydrocarbon core), showed an increase in intestinal brush border membrane (BBM) fluidity. Piperine also stimulated Leucine amino peptidase and Glycyl-glycine dipeptidase activity, due to the alteration in enzyme kinetics. This suggests that piperine could modulate the membrane dynamics due to its apolar nature by interacting with surrounding lipids and hydrophobic portions in the protein vicinity, which may decrease the tendency of membrane lipids to act as stearic constrains to enzyme proteins and thus modify enzyme conformation. Ultra structural studies with piperine showed an increase in microvilli length with a prominent increase in free ribosomes and ribosomes on the endoplasmic reticulum in enterocytes, suggesting that synthesis or turnover of cytoskeletal components or membrane proteins may be involved in the observed effect. In conclusion, it is suggested that piperine may be inducing alterations in membrane dynamics and permeation characteristics, along with induction in the synthesis of proteins associated with cytoskeletal function, resulting in an increase in the small intestine absorptive surface, thus assisting efficient permeation through the epithelial barrier.

Comparison of vaginal aminopeptidase enzymatic activities in various animals and in humans.

The specific enzymatic activity of four different aminopeptidases (aminopeptidase N, leucine aminopeptidase, aminopeptidase A and aminopeptidase B) in vaginal homogenates from rabbit, rat, guinea-pig, sheep and humans was compared. The purpose of the study was to find an appropriate animal model that can be used in degradation studies of protein and peptide drugs. Different substrates were used as the relative specific substrates for the determination of aminopeptidase enzymatic activity: 4-methoxy-2-naphthylamide of L-alanine for aminopeptidase N, 4-methoxy-2-naphthylamide of L-leucine for leucine aminopeptidase, 4-methoxy-2-naphthylamide of L-glutamic acid for aminopeptidase A and 4-methoxy-2-naphthylamide of L-arginine for aminopeptidase B. The vaginal aminopeptidase enzymatic activity of different species was determined spectrofluorometrically. The inhibition of aminopeptidase activity in the presence of bestatin and puromycin inhibitors was also investigated. The results showed the presence of aminopeptidase enzymatic activity in all vaginal homogenates in the order: sheep > guinea-pig > rabbit > or = human > or = rat. Based on the results of the hydrolysis and inhibition of the 4-methoxy-2-naphthylamide substrates, it was difficult to have an exact decision on the aminopeptidase type in the vaginal homogenates from the species studied. It was found that the aminopeptidase activity in rat, rabbit and humans was not statistically different. Therefore, we suggest that rats and rabbits could be used as model animals for vaginal enzymatic activity studies and for determination of the degradation of protein and peptide drugs in the vagina.

Effect of sodium tauroursodeoxycholate on phalloidin-induced cholestasis in rats.

We investigated the therapeutic effect of tauroursodeoxycholate on phalloidin-induced cholestasis in rats. Intrahepatic cholestasis was induced by administration of phalloidin (500 microg/kg, i.p.) for 7 days. From the day of the last phalloidin injection, tauroursodeoxycholate (60-360 micromol/kg) was given intravenously twice a day for 4 days. On the next day after the last tauroursodeoxycholate administration, bile flow, serum biochemical parameters and biliary lipid excretion rates were determined. Tauroursodeoxycholate significantly suppressed the decrease in bile flow and increases in serum alkaline phosphatase, leucine aminopeptidase and glutamic pyruvic transaminase activities, cholesterol, phospholipid and bile acid concentrations observed in phalloidin-induced cholestasis in rats. Furthermore, tauroursodeoxycholate significantly improved the biliary cholesterol and phospholipid excretion rates in phalloidin-induced cholestasis in rats. These results demonstrate the usefulness of tauroursodeoxycholate as a therapeutic agent in intrahepatic cholestasis.

Ketone potentiation of haloalkane-induced hepatotoxicity: CCl4 and ketone treatment on hepatic membrane integrity.

Previous results in male Sprague-Dawley rats indicate that acetone (A), methyl ethyl ketone (MEK), and methyl isobutyl ketone (MiBK) pretreatments (3 d, p.o.) at a dosage of 6.8 mmol/kg potentiate CCl4 hepatotoxicity. The potentiation potency profile observed was MiBK > A > MEK. In the present study, male Sprague-Dawley rats were treated for 3 d with 6.8 mmol/kg (p.o.) of A, MEK, or MiBK using Emulphor as vehicle (10 ml/kg). Rats were either killed 18 h after the last pretreatment or treated with CCl4 (prepared in corn oil) and then killed 48 h later. Livers were perfused; purified plasma membrane (PM), sinusoidal (SM) and basal canalicular membrane (BCM) fractions were prepared. Membrane fluidity was monitored by fluorescence polarization using 1,6-diphenyl-1,3,5-hexatriene (DPH) or 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH). The following membrane enzymes were measured to monitor membrane purity and treatment effects: 5'-nucleotidase (5N), leucine aminopeptidase (LAP), and alkaline phosphatase (AP). Our results suggest that CCl4 modifies membrane integrity as indicated by a decrease in liver membrane 5N, LAP, and AP activity. CCl4 also increased the fluidity of the lipid and protein portions of the liver membranes as measured by the DPH and TMA-DPH fluorescence probes, respectively. Of the three ketones, only A altered CCl4 effects on plasma membrane enzymes and decreased BCM fluidity. The data only partially support increased susceptibility of liver membranes by ketone pretreatment as a factor implicated in the mechanism of potentiation of CCl4-induced hepatotoxicity.

Effects of iodinated x-ray contrast media on renal epithelial cells in culture.

RATIONALE AND OBJECTIVES: To study cellular mechanisms that cause contrast media nephropathy, an in vitro system for proximal and distal tubular cells has been established to evaluate the influence of x-ray contrast media on tubular function. METHODS: Confluent cell cultures of the two renal cell lines, proximal tubule (LLC-PK1) and distal tubule (MDCK), were exposed for 20 hours to 0 to 100 mg iodine/mL of the ionic monomer metrizoate, the ionic dimer ioxaglate, and the non-ionic monomer iohexol. Toxicity was assessed by electron microscopy, cell viability, and biochemical assays of brush-border and lysosomal marker enzymes. RESULTS: The results demonstrated a concentration-dependent toxic effect from the contrast media on cellular appearance consisting of an increased vacuolization and on the activity of brush-border and lysosomal marker enzymes in cells and in culture media. CONCLUSION: The results, in which the nonionic x-ray contrast media iohexol appeared to be less toxic than the ionic x-ray contrast media investigated, demonstrated that defined renal cells in culture are valuable tools in studies regarding renal toxicity of x-ray contrast media.

Lys- and Leu-aminopeptidase activity after acute toluene exposure in the rat brain.

Changes in Lys- and Leu-aminopeptidase activities in several brain regions of the rat, after acute toluene administration, are described in this research. Aminopeptidase activity has been suggested as a candidate regulator of the degradation of several neuroactive peptides. Lys-aminopeptidase activity was significantly decreased in the thalamus, amygdala, and medulla oblongata. Leu-aminopeptidase activity was significantly decreased in the thalamus and cerebellum. It is suggested that these aminopeptidase activities could play a part in the mechanism of toluene neurotoxicity.

Brain aminopeptidase activity after subacute xylene exposure.

Xylene is a neurotoxic aromatic hydrocarbon widely used in industry. In this article, the effect of subacute xylene exposure on neutral and basic aminopeptidase activities in several regions of the rat brain is described. Neutral aminopeptidase activity only decreased significantly in the thalamus. There were no changes in basic aminopeptidase activity after the solvent administration. Thus, these brain aminopeptidase activities are largely unaffected by subacute exposure to xylene, which is not the case with benzene or carbon disulfide. This could be in line with its recognized lesser toxicity at the central nervous system level.

Potent inhibition of endopeptidase 24.16 and endopeptidase 24.15 by the phosphonamide peptide N-(phenylethylphosphonyl)-Gly-L-Pro-L-aminohexanoic acid.

A phosphonamide peptide, N-(phenylethylphosphonyl)-Gly-L-Pro-L-aminohexanoic acid, previously shown to block Clostridium histolyticum collagenases, was examined as a putative inhibitor of endopeptidase 24.16 and endopeptidase 24.15. Hydrolysis of two endopeptidase 24.16 substrates, i.e. 3-carboxy-7-methoxycoumarin (Mcc)-Pro-Leu-Gly-Pro-D-Lys-dinitrophenyl (Dnp) and neurotensin, were completely and dose-dependently inhibited by the phosphonamide inhibitor with KI values of 0.3 and 0.9 nM respectively. In addition, the phosphonamide peptide inhibited the hydrolysis of benzoyl (Bz)-Gly-Ala-Ala-Phe-(pAB) p-aminobenzoate and neurotensin by endopeptidase 24.15 with about a 10-fold lower potency (KI values of 5 and 7.5 nM respectively). The selectivity of this inhibitor towards several exo- and endo-peptidases belonging to the zinc-containing metallopeptidase family established that a 1 microM concentration of this inhibitor was unable to affect leucine aminopeptidase, carboxypeptidase A, angiotensin-converting enzyme and endopeptidase 24.11. The present paper therefore reports on the first hydrophilic highly potent endopeptidase 24.16 inhibitor and describes the most potent inhibitory agent directed towards endopeptidase 24.15 developed to date. These tools should allow one to assess the contribution of endopeptidase 24.16 and endopeptidase 24.15 to the physiological inactivation of neurotensin as well as other neuropeptides.

Biochemical response to cadmium. Dose-time effect.

Dose- and time-related effects of Cd (II) (0.5 or 1.0 mg/kg, Cd as CdCl2.H2O, subcutaneously, daily for 48 h, 1, 3, or 6 wk) were investigated in rats. A dose-related increase in the activity of plasma alkaline phosphatase (ALP), lactate dehydrogenase (LDH), aspartate aminotransferase (GOT), and alanine aminotransferase (GPT) was evident only at 6 wk, whereas an early rise in ALP and LDH was seen at 3 wk in 1.0 mg Cd group only. The hepatic and renal metallothionein (MT) induction displayed a dose- as well as time-related increase with Cd accumulation. A significant increase in hepatic Zn and renal Cu, no change in hepatic Cu, and a slight increase in renal Zn was observed. Urinary ALP and leucine aminopeptidase (LAP) showed an initial increase at 48 h, thereafter returned to near normal. A second phase of enzymuria (ALP, LAP, GOT, GPT, gamma-glutamyl transpeptidase), proteinuria, and aminoaciduria occurred at 6 wk in a dose-related manner. The urinary excretion of specific renal enzymes appeared closely related to the MT induction and organ Cd levels.

[Cerebral aminopeptidase activity: effect of the administration of imipramine]. / Actividad aminopeptidásica cerebral: efecto de la administración de imipramina.

Imipramine is widely accepted as an effective antidepressant treatment. In this paper, changes in Lys- and Leu-aminopeptidase activities in several rat brain regions after imipramine treatment are described. Decreases of Lys-aminopeptidase activity in the olphactory bulbus, cerebellum, hypothalamus and pituitary gland were observed. In the same way, there were a decreases in Leu-aminopeptidase activity in the parietal and occipital cortices, cerebellum thalamus, hypothalamus and pituitary gland. It is suggested that these enzyme activities play a part in the imipramine action mechanism, possibly by regulating the activity of several neuroactive peptides.