leupeptins and ubenimex

Toxoplasmosis is a zoonotic disease and a global food and water-borne infection. The disease is caused by the parasite Toxoplasma gondii, which is a highly successful and remarkable pathogen because of its ability to infect almost any nucleated cell in warm-blooded animals. The present study was done to demonstrate the effect of protease inhibitors cocktail (PIC), which inhibit both cysteine and serine proteases, on in vitro cultured T. gondii tachyzoites on HepG2 cell line. This was achieved by assessing its effect on the invasion of the host cells and the intracellular development of T.gondii tachyzoites through measuring their number and viability after their incubation with PIC. Based on the results of the study, it was evident that the inhibitory action of the PIC was effective when applied to tachyzoites before their cultivation on HepG2 cells. Pre-treatment of T.gondii tachyzoites with PIC resulted in failure of the invasion of most of the tachyzoites and decreased the intracellular multiplication and viability of the tachyzoites that succeeded in the initial invasion process. Ultrastructural studies showed morphological alteration in tachyzoites and disruption in their organelles. This effect was irreversible till the complete lysis of cell monolayer in cultures. It can be concluded that PIC, at in vitro levels, could prevent invasion and intracellular multiplication of Toxoplasma tachyzoites. In addition, it is cost effective compared to individual protease inhibitors. It also had the benefit of combined therapy as it lowered the concentration of each protease inhibitor used in the cocktail. Other in vivo experiments are required to validate the cocktail efficacy against toxoplasmosis. Further studies may be needed to establish the exact mechanism by which the PIC exerts its effect on Toxoplasma tachyzoites behavior and its secretory pathway.

Topics: Analysis of Variance; Animals; Aprotinin; Culture Media, Serum-Free; Cysteine Proteinase Inhibitors; Drug Combinations; Hep G2 Cells; Humans; Leucine; Leupeptins; Mice; Microscopy, Electron, Transmission; Organelles; Pilot Projects; Protease Inhibitors; Serine Proteinase Inhibitors; Statistics, Nonparametric; Sulfones; Toxoplasma

A cleavage product of alpha-fodrin may be an important organ-specific autoantigen in the pathogenesis of Sjogren's syndrome (SS), but the mechanisms of alpha-fodrin cleavage remain unclear. Since EBV has been implicated in the pathogenesis of SS, we determined whether EBV activation could induce the SS-specific 120-kDa autoantigen alpha-fodrin. ZEBRA mRNA expression, a marker for activation of the lytic cycle of EBV, was found in the salivary gland tissues from SS patients, but not in those from control individuals. ZEBRA-expressing lymphoid cells were also found in the SS glands in double-stained immunohistochemistry. Furthermore, a significant link between production of Abs against 120-kDa alpha-fodrin and reactivated EBV Ag was found in sera from patients with SS, but not in those from control individuals. EBV-activated lymphoid cells showed specific alpha-fodrin cleavage to the expected 120-kDa fragments in vitro. Pretreatment with caspase inhibitors inhibited cleavage of alpha-fodrin. Thus, an increase in apoptotic protease activities induced by EBV reactivation may be involved in the progression of alpha-fodrin proteolysis in the development of SS.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Aprotinin; Autoantigens; Carrier Proteins; Caspases; Cysteine Proteinase Inhibitors; DNA-Binding Proteins; Herpesvirus 4, Human; Humans; Hydrolysis; Leucine; Leupeptins; Microfilament Proteins; Molecular Weight; Organ Specificity; Pepstatins; Sjogren's Syndrome; Trans-Activators; Tumor Cells, Cultured; Viral Proteins; Virus Activation

Porphyromonas gingivalis is an asaccharolytic bacterium that requires nitrogen substrates as carbon and energy sources. The aims of this study were to investigate the aminopeptidase activities of P. gingivalis and to evaluate the effect of aminopeptidase inhibitors on bacterial growth. Only arginine aminopeptidase and dipeptidyl aminopeptidase IV activities were detected. Experimental evidence was obtained suggesting that the Arg-gingipains of P. gingivalis can function as both an endopeptidase and an aminopeptidase. Firstly, the arginine aminopeptidase activity was found to be inhibited by leupeptin, a well-known inhibitor of Arg-gingipain activity. Secondly, a preparation of Arg-gingipain activity could hydrolyze the chromogenic substrate for arginine aminopeptidase. Lastly, a mutant of P. gingivalis constructed via gene disruption by use of suicide plasmids and deficient in both Arg-gingipain A and B was also devoid of arginine aminopeptidase activity. To investigate the key role of aminopeptidase activities in growth of P. gingivalis, aminopeptidase inhibitors were incorporated in the culture medium prior to inoculation. Bestatin and actinonin were the only ones to inhibit growth of P. gingivalis. Their mechanism of growth inhibition appears to be different but does not involve inhibition of the two major aminopeptidase activities (arginine aminopeptidase and dipeptidyl aminopeptidase IV).

Topics: Adhesins, Bacterial; Aminopeptidases; Anti-Bacterial Agents; Carbon Radioisotopes; Cathepsins; Chromogenic Compounds; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Dipeptidyl Peptidase 4; Gingipain Cysteine Endopeptidases; Guanidines; Hemagglutinins; Humans; Hydroxamic Acids; Leucine; Leupeptins; Mutation; Oligopeptides; Peptides; Porphyromonas gingivalis; Protease Inhibitors; Radiopharmaceuticals

The purpose of these investigations was to determine whether the aminopeptidase B and leucine aminopeptidase inhibitor bestatin, the chymase inhibitor chymostatin, the calpain inhibitor E-64, and the neutral serine protease inhibitor leupeptin affect the angiotensin converting enzyme (ACE) activity in T-lymphocytes. ACE activity in homogenates of T-lymphocytes or in intact T-lymphocytes in suspension was measured by determining fluorimetrically histidyl-leucine, formed from the conversion of hippuryl-histidyl-leucine, coupled with ophtaldialdehyde. The effect of various concentrations (10(-9) to 10(-3) mol/L) of the angiotensin-converting enzyme inhibitors lisinopril and captopril and of the various protease inhibitors on ACE activity was studied. Lisinopril and captopril reduced the ACE activity in homogenates of T-lymphocytes in a concentration-dependent manner. Lisinopril exhibited a more pronounced inhibition of ACE in T-lymphocytes than did captopril. Chymostatin and E-64 had no effect on the ACE activity in T-lymphocytes, whereas leupeptin inhibited its activity in a dose-dependent fashion. Bestatin, on the contrary, increased the ACE activity in homogenates of T-lymphocytes as well as in intact T-lymphocytes in proportion to the concentration. Our data showed that the ACE activity in T-lymphocytes was stimulated by bestatin and inhibited by leupeptin, whereas chymostatin and E-64 did not affect the ACE activity in T-lymphocytes.

Topics: Adult; Aminopeptidases; Angiotensin-Converting Enzyme Inhibitors; Calpain; Captopril; Cathepsins; Chymotrypsin; Cysteine Proteinase Inhibitors; Humans; Leucine; Leupeptins; Lisinopril; Lymphocyte Activation; Male; Oligopeptides; Peptidyl-Dipeptidase A; Protease Inhibitors; T-Lymphocytes

The cyclic expression of the period (PER) and timeless (TIM) proteins is critical for the molecular circadian feedback loop in Drosophila. The entrainment by light of the circadian clock is mediated by a reduction in TIM levels. To elucidate the mechanism of this process, the sensitivity of TIM regulation by light was tested in an in vitro assay with inhibitors of candidate proteolytic pathways. The data suggested that TIM is degraded through a ubiquitin-proteasome mechanism. In addition, in cultures from third-instar larvae, TIM degradation was blocked specifically by inhibitors of proteasome activity. Degradation appeared to be preceded by tyrosine phosphorylation. Finally, TIM was ubiquitinated in response to light in cultured cells.

Topics: Acetylcysteine; Animals; Biological Clocks; Cells, Cultured; Circadian Rhythm; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Darkness; Drosophila; Drosophila Proteins; Feedback; Insect Proteins; Leucine; Leupeptins; Light; Multienzyme Complexes; Neurons; Phosphorylation; Phosphotyrosine; Protease Inhibitors; Proteasome Endopeptidase Complex; Ubiquitins

Leupeptin-inactivating enzyme (LIE) was purified from Streptomyces exfoliatus SMF13 by ammonium sulphate fractionation of cell-free culture broth, ultrafiltration, anion-exchange chromatography on DEAE-Sephadex A-50 and gel filtration chromatography on Sephadex G-75. The molecular mass of the purified enzyme was measured as 34700 Da and the N-terminal amino acid sequence was APTPPDIPLANVPA. Acetyl-leucine, leucine and argininal were identified as the products of leupeptin inactivated by the LIE, indicating that leupeptin is inactivated by hydrolysis of peptide bond between leucine and leucine and between leucine and argininal of leupeptin (acetyl-leucine-leucine-argininal). Synthetic-peptide substrates specificity of LIE showed that LIE has absolute specificity for peptide bonds with leucine in the P1 position, suggesting that LIE is a leucine-specific protease. The optimum pH and temperature were pH 9.0 and 45 degrees C, respectively. LIE activity was inhibited by metalloprotease inhibitors such as EDTA, EGTA, o-phenanthroline and bestatin, but activated by Mg2+ and Ca2+, suggesting that the enzyme is a metalloprotease. Aerial-mycelium growth and aerial spore formation of S. exfoliatus SMF13 were inhibited by the addition of bestatin, an inhibitor of LIE. The inhibition of morphological differentiation was due to the inhibition of trypsin-like protease (TLP) activity, which is essential for aerial-mycelium formation and is inhibited specifically by remaining leupeptin that was not inactivated. These results show that LIEs play a role in controlling the amount of leupeptin during colony development. Therefore, it is suggested that the physiological function of LIE is to inactivate leupeptin when or where TLP activity is required for aerial-mycelium formation.

Topics: Amino Acid Sequence; Chromatography; Edetic Acid; Egtazic Acid; Enzyme Activation; Enzyme Inhibitors; Hydrolysis; Leucine; Leupeptins; Metalloendopeptidases; Microscopy, Electron, Scanning; Molecular Weight; Peptide Fragments; Phenanthrolines; Streptomyces; Substrate Specificity

A method was investigated for monitoring the activity of protease(s) in cytosol of a single starfish oocyte using succinyl-Phe-Leu-Arg-coumarylamido-4-methanesulfonic acid as the substrate, which was injected into the cell. After preincubation of immature oocytes with a proteasome inhibitor, N-carbobenzoxy-L-leucinyl-L-leucinyl-L-norvalinal, the initial hydrolysis of the substrate was remarkably inhibited. The inhibitor blocked 1-methyladenine-triggered cyclin degradation, which is known to be mediated by proteasome. However, calpain inhibitor E-64 did not inhibit the hydrolysis of the substrate. These results suggested that the protease activity measured by this method is mainly attributable to cytoplasmic proteasome. The hydrolysis of the substrate was partially inhibited by bestatin, suggesting that the substrate was cleaved by aminopeptidase. Thus, the initial velocity of hydrolysis of the substrate (V0) by proteasome was assayed in a living oocyte after preinjection of bestatin. The values of V0 increased gradually after 1-methyladenine addition and reached the maximum level at the time corresponding to cyclin degradation. The calculated maximum velocity of hydrolysis by a mature oocyte was approximately three times higher than that by an immature oocyte. The Michaelis-Menten constant value was also higher in mature than immature oocytes. These results suggest that proteasome-dependent proteolysis is regulated not only by ubiquitination of substrates, as is generally believed, but also by the proteasome activity itself.

Topics: Adenine; Aminopeptidases; Animals; Cell Membrane Permeability; Coumarins; Cyclins; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Cytosol; Fluorescent Dyes; Hydrolysis; Kinetics; Leucine; Leupeptins; Meiosis; Mesylates; Multienzyme Complexes; Oligopeptides; Oocytes; Proteasome Endopeptidase Complex; Spectrometry, Fluorescence; Starfish

The effects of enzyme inhibitors on vasoactive intestinal peptide (VIP)-induced decreases in airway opening pressure (PaO) and VIP-like immunoreactivity (VIP-LI) recovery were studied in isolated tracheal superfused guinea pig lungs. In the absence of inhibitors, VIP 0.38 (95% CI 0.33-0.54) nmol/kg animal, resulted in a 50% decrease in PaO and 33% of a 1 nmol/kg VIP dose was recovered as intact VIP. In the presence of two combinations of enzyme inhibitors, SCH 32615 (S, 10 microM) and aprotinin (A, 500 tyrpsin inhibitor units [TIU]/kg) or S and soybean trypsin inhibitor (T, 500 TIU/kg), VIP caused a significantly greater decrease in PaO and greater quantities of VIP were recovered from lung effluent (both P < 0.001). The addition of captopril, (3 microM), leupeptin (4 microM), or bestatin (1 microM) failed to further increase pulmonary relaxation or recovery of VIP-LI. When given singly, A, T, and S did not augment the effects or recovery of VIP. The efficacy of S (a specific inhibitor of neutral endopeptidase [NEP]) and A and T (serine protease inhibitors) thus implicated NEP and at least one serine protease as primary modulators of VIP activity in the guinea pig lung. We sought to corroborate this finding by characterizing the predominant amino acid sites at which VIP is hydrolized in the lung. When [mono(125I)iodo-Tyr10]VIP was offered to the lung, in the presence and absence of the active inhibitors, cleavage products consistent with activity by NEP and a tryptic enzyme were recovered. These data demonstrate that NEP and a peptidase with an inhibitor profile and cleavage pattern compatible with a tryptic enzyme inactivate VIP in a physiologically competitive manner.

Topics: Animals; Aprotinin; Captopril; Dose-Response Relationship, Drug; Endopeptidases; Guinea Pigs; Leucine; Leupeptins; Lung; Male; Muscle Relaxation; Muscle, Smooth; Perfusion; Protease Inhibitors; Time Factors; Trachea; Trypsin Inhibitors; Vasoactive Intestinal Peptide

Protease of carp retina were examined by electrophoresis and fluorogenic assays. A 70 kD serine protease with an alkaline pH optimum was detected in gelatin-containing polyacrylamide gels. A similar enzyme was found in carp brain and muscle, but not in lens. Using aminomethylcoumarin (MCA) substrates, activities that hydrolysed Z-Phe-Arg-MCA, Boc-Ala-Gly-Pro-Arg-MCA and various aminoacyl-MCAs were detected. The Z-Phe-Arg-MCA hydrolase was an acidic cysteine protease, whereas the Boc-Ala-Gly-Pro-Arg-MCA hydrolase was an alkaline cysteine protease. All aminoacyl hydrolase activities tested were inhibited by bestatin and o-phenanthroline, but not by inhibitors of serine, cysteine and aspartic proteases, suggesting they are metalloaminopeptidases. Of the substrates tested, Tyr-MCA was the most readily hydrolysed aminoacyl substrate. Preliminary evidence was obtained suggesting that levels of these activities do not differ between light- and dark-adapted retinae. The proteases have a potential involvement in retinal functioning and show similarities to other proteases known to act in the central nervous system. In particular, the Tyr-MCA hydrolase may be related to an enzyme known to remove the N-terminal tyrosine residue from enkephalin.

Topics: Amino Acid Sequence; Animals; Carps; Coumarins; Endopeptidases; Hydrogen-Ion Concentration; Leucine; Leupeptins; Molecular Sequence Data; Peptides; Phenanthrolines; Protease Inhibitors; Retina; Serine Endopeptidases; Tosyllysine Chloromethyl Ketone

To examine the mechanisms of changes in alveolar macrophage (AM) activities caused by phagocytic stimulus, we studied the effect of opsonized zymosan (OZ) on cytoplasmic motility (CM) of AM from dog lungs in vitro. Four days after the instillation of ferrimagnetic particles (Fe3O4, 3 mg/kg) into the lower lobe bronchus, AM were harvested by broncho-alveolar lavage. AM were adhered to the bottom of plastic vials (10(6) cells of AM per each vial). Remanent field strength (RFS) from the AM containing Fe3O4 particles was measured immediately after magnetization. RFS decreased with time due to particle rotation (relaxation), which is related to cytoplasmic motility of AM. OZ (1-500 micrograms) decreased lambda 0 (the relaxation rate for the first min) in a concentration-dependent fashion. Neither BW755C (10(-5) M), indomethacin (10(-6) M), leupeptin (10(-5) M), bestatin (10(-5) M), nor superoxide dismutase (1000 U/ml) inhibited OZ (500 micrograms)-induced inhibitory effects on lambda 0, suggesting that cyclooxygenase and lipoxygenase products, serine, thiol enzymes, aminopeptidase and superoxide anion wer not responsible for OZ-induced effects. OZ (500 micrograms) significantly increased the intracellular concentration of Ca2+ (P less than 0.01). Likewise, OZ (500 micrograms)-induced effects on lambda 0 of AM were significantly inhibited by replacement of the medium with a Ca2+ free solution (P less than 0.01). These results imply that opsonized zymosan inhibits cytoplasmic motility of AM via external calcium influx.

Topics: 4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyrazol-3-amine; Aminopeptidases; Animals; Calcium; Captopril; Cells, Cultured; Cytoplasm; Dogs; Indomethacin; Leucine; Leupeptins; Macrophages; Phagocytosis; Pulmonary Alveoli; Superoxide Dismutase; Zymosan

Any effect of serum on the antigenicity of peptides is potentially relevant to their use as immunogens in vivo. Here we demonstrate that serum contains distinct proteases that can increase or decrease the antigenicity of peptides. By using a functional assay, we show that a serum component other than beta 2-microglobulin enhances the presentation of ovalbumin peptides produced by cyanogen bromide cleavage. Three features of this serum activity implicate proteolysis: it is temperature dependent, it results in increased antigenicity in a low molecular weight peptide fraction, and it is inhibited by the protease inhibitor leupeptin. Conversely, presentation of the synthetic peptide OVA-(257-264) is inhibited by serum. This inhibition is unaffected by leupeptin but is blocked by bestatin, a protease inhibitor with distinct substrate specificities. Implications for peptide-based vaccine design and immunotherapy are discussed.

Topics: Animals; Antigen-Presenting Cells; Cells, Cultured; Cyanogen Bromide; Endopeptidases; In Vitro Techniques; Leucine; Leupeptins; Mice; Molecular Weight; Ovalbumin; Peptides

An antitumor antibiotic C-1027, a complex protein consisting of an apoprotein and a non-covalently bound chromophore, showed some aminopeptidase activity, 1/15 (on the basis of activity per mg protein) that of porcine kidney enzyme [E.C. 3.4.11.2] by use of L-phenylalanyl 4-methyl-coumaryl-7-amide as the substrate. Neither the apoprotein alone nor the chromophore alone were active. Amastatin and bestatin but not leupeptin inhibited the activity. The enzyme activity of the holo-antibiotic, as opposed to that of the porcine kidney enzyme, was readily lost by UV irradiation, indicating that the intact structure of the chromophore was needed to maintain the native conformation of the holo-antibiotic. The cytotoxicity of the holo-antibiotic, but not that of the chromophore, to Ehrlich carcinoma cells in vitro was reduced to 1/5 by 1 microgram/ml of amastatin which alone had no effect on cell growth. The porcine aminopeptidase was not cytotoxic at all even at higher concentrations (higher enzyme activities/ml). Amastatin possibly occupied the catalytic domain of the holo-antibiotic, interfering with the binding of the holo-antibiotic with some cell-surface protein(s). Amastatin did not inhibit the holo-antibiotic to cleave isolated DNA.

Topics: Aminoglycosides; Aminopeptidases; Animals; Anti-Bacterial Agents; Antibiotics, Antineoplastic; Apoproteins; Carcinoma, Ehrlich Tumor; Cell Survival; DNA, Neoplasm; Enediynes; Leucine; Leupeptins; Microsomes; Oligopeptides; Peptides; Proteins; Substrate Specificity; Swine; Tumor Cells, Cultured; Ultraviolet Rays

We studied the effect of substance P (SP) on the electric properties of cultured canine tracheal epithelium and its possible modulation by neutral endopeptidase (NEP) by Ussing's short-circuited technique in vitro. Addition of SP (5 x 10(-6) M) to the mucosal side increased short-circuit current (SCC) from 5.1 +/- 0.9 to 10.3 +/- 2.2 microA/cm2 (mean +/- SE; p less than 0.01), which was accompanied by increases in transepithelial potential difference and conductance. The effect of the mucosal SP on SCC was dose-dependent, with the maximal increase from the baseline value being 5.8 +/- 1.0 microA/cm2 observed at 5 x 10(-5) M. The NEP inhibitor phosphoramidon (10(-5) M) did not affect these responses. On the other hand, SCC was not altered by the addition of SP to the submucosal side. However, it was increased dose-dependently in the presence of phosphoramidon (10(-5) M) but not in the presence of captopril, bestatin or leupeptin. This stimulatory effect of submucosal SP was abolished by furosemide, diphenylamine-2-carboxylate and Cl-free medium, but not by amiloride. These results suggest that SP may selectively stimulate Cl secretion across the airway epithelium and that this effect may be modulated by submucosal NEP.

Topics: Amiloride; Animals; Anti-Bacterial Agents; Biological Transport, Active; Captopril; Cells, Cultured; Complement C1; Dogs; Dose-Response Relationship, Drug; Electric Conductivity; Female; Furosemide; Glycopeptides; In Vitro Techniques; Ion Channel Gating; Leucine; Leupeptins; Male; Membrane Potentials; Neprilysin; Substance P; Time Factors; Trachea

To study the effect of atrial natriuretic factor (ANF) on airway ciliary motility, we measured ciliary beat frequency by a photoelectric method in response to ANF in cultured tracheal epithelial cells from rabbits. Addition of ANF but not [Tyr8]ANF-(5-27) decreased ciliary beat frequency in a dose-dependent fashion; the maximal decrease from the baseline value was 24.1 +/- 1.5% (+/- SE, P less than 0.001), and a half-maximal inhibitory concentration (IC50) was 3 x 10(-12) M. Inhibition of neutral endopeptidase activity by phosphoramidon (10(-6) M) or thiorphan (10(-6) M) potentiated the effect of ANF so that the dose-response curve for ANF was shifted to lower concentrations by approximately 0.5 log units (P less than 0.05, in each case). The inhibition of ciliary motility induced by ANF was not affected by the blockade of arachidonic acid metabolism with indomethacin, piroxicam, or nordihydroguaiaretic acid, but it was blocked by methylene blue, a soluble guanylate cyclase inhibitor, and was potentiated by M & B 22948, a guanosine 3',5'-cyclic monophosphate (cGMP) phosphodiesterase inhibitor. The intracellular cGMP levels were increased by ANF, an effect that was further potentiated by phosphoramidon or thiorphan. These results suggest that ANF inhibits ciliary motility presumably through a guanylate cyclase-dependent regulatory pathway and that neutral endopeptidase may play a role in modulating the ANF effect on airway mucociliary transport function.

Topics: Animals; Atrial Natriuretic Factor; Captopril; Cells, Cultured; Cilia; Enzyme Inhibitors; Epithelium; Glycopeptides; Indomethacin; Kinetics; Leucine; Leupeptins; Male; Masoprocol; Methylene Blue; Movement; Piroxicam; Purinones; Rabbits; Thiorphan; Trachea

17 different proteinase inhibitors were screened for their effect on the erythrocyte invasion by the malaria parasite Plasmodium falciparum. The effect was tested when the inhibitors were present in the culture medium and when they were trapped into erythrocyte ghosts. A very strong inhibition of invasion was observed in the presence of calpain inhibitors, with IC50 in the order of 10(-7) M. Chymostatin, leupeptin, pepstatin A and bestatin also caused inhibition of the invasion, but with IC50 in the order of 10(-5) M. The results suggest that participation of various proteinases in the process and point to the possibility of a calpain-mediated proteolytic event. This study may explain previous observations on the role of calcium in the invasion of the human erythrocyte by Plasmodium falciparum.

Topics: Animals; Calpain; Cysteine Proteinase Inhibitors; Erythrocytes; Humans; Leucine; Leupeptins; Oligopeptides; Pepstatins; Plasmodium falciparum

1. The effects of various metabolic and enzyme inhibitors on the degradation of endogenous proteins and internalized asialofetuin were examined in the primary cultured hepatocytes of rats. 2. The results showed the important physiological role of bestatin-sensitive aminopeptidases in the degradation of endogenous and internalized proteins as well as in the degradation of both long- and short-lived proteins.

Topics: alpha-Fetoproteins; Amino Acids; Animals; Anti-Bacterial Agents; Asialoglycoproteins; Cells, Cultured; Fetuins; Glucagon; Insulin; Leucine; Leupeptins; Liver; Male; Peptides; Proteins; Rats; Rats, Inbred Strains; Scintillation Counting

Ferret tracheal segments were infected with human influenza virus A/Taiwan/86 (H1N1) in vitro. After 4 days, the smooth muscle contractile responses to acetylcholine and to substance P were measured. The response to substance P was markedly accentuated, with a threefold increase in force of contraction at a substance P concentration of 10(-5) M, the highest concentration tested. In contrast, the response to acetylcholine was not affected by viral infection. Histological examination of tissues revealed extensive epithelial desquamation. Activity of enkephalinase (neutral metallo-endopeptidase, EC.3.4.24.11), an enzyme that degrades substance P, was decreased by 50% in infected tissues. Inhibiting enkephalinase activity by pretreating with thiorphan (10(-5) M) increased the response to substance P to the same final level in both infected and control tissues. Inhibiting other substance P-degrading enzymes including kininase II (angiotensin-converting enzyme), serine proteases, and aminopeptidases did not affect the response to substance P. Inhibiting cyclooxygenase and lipoxygenase activity using indomethacin and BW 755c did not affect hyperresponsiveness to substance P. Pretreating tissues with antagonists of alpha-adrenoceptors, beta-adrenoceptors, and H1 histamine receptors (phentolamine 10(-5) M, propranolol 5 X 10(-6) M, and pyrilamine 10(-5) M, respectively) had no effect on substance P-induced contraction. These results demonstrate that infection of ferret airway tissues with influenza virus increases the contractile response of airway smooth muscle to substance P. This effect is caused by decreased enkephalinase activity in infected tissues.

Topics: Animals; Captopril; Disease Models, Animal; Ferrets; In Vitro Techniques; Influenza A virus; Leucine; Leupeptins; Metalloendopeptidases; Muscle Contraction; Muscle, Smooth; Neprilysin; Orthomyxoviridae Infections; Thiorphan; Tiopronin; Trachea

The chemical properties of acid soluble peptides accumulated in liver or excreted into urine after administration of bestatin or leupeptin to rats were investigated extensively. At the same time, the effects of glucagon on the bestatin-induced accumulation of acid soluble peptides were studied. The results show the important role of bestatin- and leupeptin-sensitive proteases in the degradation pathway of intracellular proteins in vivo.

Topics: Aminopeptidases; Animals; Glucagon; Kinetics; Leucine; Leupeptins; Liver; Male; Models, Biological; Oligopeptides; Peptide Mapping; Peptides; Protease Inhibitors; Rats; Rats, Inbred Strains; Solubility

The effect of large doses of methylprednisolone sodium succinate (MPSS) and two protease inhibitors, leupeptin and bestatin, on experimental acute spinal cord injury was evaluated by morphometric analysis of degenerating axons with the aid of an automated image analyzer. Spinal cord injury was produced by epidural compression with a surgical clip on the T-11 segment in rats. The extent of axonal damage was assessed in Rexed's lamina VIII in the L-6 segment by measuring the amount of silver grains, representing degenerating axons and their terminals, using the Fink-Heimer method. The severity of axonal damage was expressed as the degeneration index: that is, the amount of silver grains in experimental animals/the amount of silver grains in cord-transected animals. When examined on the 7th postoperative day, axonal degeneration in MPSS-treated rats was significantly decreased, with an average degeneration index difference of 6 (p less than 0.05). Increased preservation of axons was seen in the leupeptin-treated rats sacrificed 7, 10, and 14 days after trauma. The difference in the degeneration index between the leupeptin-treated and untreated groups was 16 on Day 7 (p less than 0.001), 12 on Day 10 (p less than 0.001), and 13 on Day 14 (p less than 0.01). Bestatin had no beneficial effect. The implications for the use of calcium-activated neutral protease inhibitors in acute spinal cord injury are discussed.

Topics: Animals; Axons; Leucine; Leupeptins; Male; Methylprednisolone; Methylprednisolone Hemisuccinate; Rats; Rats, Inbred Strains; Spinal Cord Injuries

Leucine aminopeptidase (LAP) activity has been measured in extracts of eggs, miracidia, cercariae and adult worms of Schistosoma mansoni. Activity measured at pH 7.2 using L-leu-7-amino-4-trifluoro-methylcoumarin as substrate is 6- to 17-fold greater in eggs than in other life stages. LAP activity is also high in soluble egg antigen preparations and in hatching fluid. The release of LAP from eggs parallels hatching, and inhibitors of LAP also inhibit hatching. The possible role of LAP in the hatching process of S. mansoni eggs is discussed.

Topics: Animals; Cold Temperature; Darkness; Leucine; Leucyl Aminopeptidase; Leupeptins; Ovum; Schistosoma mansoni; Sodium Chloride

Two forms of a neutral protease that catalyzed the hydrolysis of succinyl-Leu-Leu-Val-Tyr-MCA were isolated from porcine skeletal muscle cytosol by fractionation on DEAE-cellulose, hydroxyapatite and Sephadex G-100. The native enzyme had a molecular mass of above 1000 kDa. Peak A, which was eluted from hydroxyapatite at 50 mM phosphate, was activated 37-fold by the detergent, SDS, while peak B which was eluted at 150 mM phosphate, was activated only 2-fold. After dialysis against water, the B form showed restored ability to be activated by SDS (9.6-fold with 0.04% SDS). The activated peak B was extremely sensitive to divalent and monovalent cations such as Ca2+, Mg2+, Na+, K+ and NH+4 as well as protease inhibitors such as leupeptin, chymostatin and DFP. These results suggest that these proteases are generally latent in the cells and may be regulated by changes in the concentrations of cations in the cytosol. We call this new type of protease, ingensin.

Topics: Animals; Antipain; Chromatography, DEAE-Cellulose; Chromatography, Gel; Chromatography, High Pressure Liquid; Coumarins; Cysteine Endopeptidases; Endopeptidases; Isoenzymes; Leucine; Leupeptins; Multienzyme Complexes; Muscles; Oligopeptides; Pepstatins; Proteasome Endopeptidase Complex; Sodium Dodecyl Sulfate; Swine

We compared the therapeutic effects of various low-molecular-weight enzyme inhibitors on dystrophic mice. Leupeptin, bestatin, forphenicinol and forphenicine significantly affected the enzymatic activities in the dystrophic muscles. The pattern of enzymatic changes in the muscles of forelimb and hindlimb caused by these inhibitors were similar in spite of the variety of their inhibitory spectra in vitro. However, comparing the pattern of enzymatic changes in spleen, forphenicinol differed from the other inhibitors tested. This may be related to the peculiar effects of this inhibitor on immunologically responsive cells.

Topics: Alkaline Phosphatase; Aminopeptidases; Animals; Esterases; Forelimb; Glutamyl Aminopeptidase; Glycine; Hindlimb; Leucine; Leucyl Aminopeptidase; Leupeptins; Male; Mice; Mice, Mutant Strains; Molecular Weight; Muscles; Muscular Dystrophy, Animal; Oligopeptides; Spleen

For the purpose of observing the therapeutic benefit of protease inhibitors for progressive muscular dystrophy, a large quantity of doses of leupeptin of 10 mg/kg/day and 50 mg/kg/day were administered i.p. to male chickens afflicted with hereditary muscular dystrophy (line 413) for 4 months starting on the 7th day ex ovo. No clinical improvement was identified in physical ability as a result of the examination by flip test, and creatine kinase (CK) values. The number of necrotic fibers in the pectoralis superficialis (PS) muscle which is known to be preferentially damaged in dystrophic chicken, did not decrease significantly in the birds treated with 10 mg leupeptin/kg/day (number of necrotic fibers; 47.7/mm2) and 50 mg/kg/day (46.4/mm2) as compared to that of the untreated ones (43.2/mm2). A morphometric analysis of fiber diameter distribution also showed no statistical difference between the treated and untreated birds. In the second group, 10 mg leupeptin/kg and a combination of leupeptin and bestatin of 10 mg/kg each were injected directly into the left lower half of the PS muscle three times a week for 4 months. Necrotic fibers were still present in the injected site, remote area of the left upper PS muscle treated with leupeptin (52.7/mm2), leupeptin and bestatin (52.2/mm2), and contralateral right upper PS muscle (41.6 and 53.5/mm2, respectively). The number of necrotic fibers in treated muscles was again not significantly different from that in untreated dystrophic ones (39.6/mm2). In fiber diameter analysis, no statistical difference was recognized between the treated and untreated dystrophic muscles.

Topics: Aminopeptidases; Animals; Chickens; Creatine Kinase; Drug Therapy, Combination; Leucine; Leupeptins; Male; Muscles; Muscular Dystrophy, Animal; Necrosis; Oligopeptides