Phenylalanine Chloromethyl Ketone Research Articles

Effects of inhibitors on the protease profiles and degradation of activated Cry toxins in larval midgut juices of Cnaphalocrocis medinalis (Lepidoptera: Pyralidae)

Midgut juice plays an important role in food digestion and detoxification in insects. In order to understand the potential of midgut juice of Cnaphalocrocis medinalis (Guenée) to degrade Bt proteins, the enzymatic activity of midgut juice and its degradation of Bt proteins (Cry2A, Cry1C, Cry1Aa, and Cry1Ac) were evaluated in this study through protease inhibitor treatments. The activities of total protease in midgut juices were significantly inhibited by phenylmethylsulfonyl fluoride (PMSF), tosyl-L-lysine chloromethyl ketone (TLCK), pepstatin A and leupeptin. The enzymatic activity of chymotrypsin was significantly inhibited by PMSF, and enzymatic activity of trypsin was significantly inhibited by ethylenediaminetetraacetic acid (EDTA), PMSF, tosyl phenylalanine chloromethyl ketone (TPCK), TLCK and trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane (E-64). EDTA could significantly inhibit the degradation of Cry2A by C. medinalis. EDTA, PMSF, TPCK, and TLCK could inhibit the degradation of Cry1C and Cry1Aa. EDTA, PMSF, TPCK, TLCK, and E-64 could inhibit the degradation of Cry1Ac. Our results indicated that some protease inhibitors hindered various enzymatic activities in the larval midgut of C. medinalis, which may reduce the insect's ability to degrade Bt toxins. These findings may aid the application of protease inhibitors in the management of this insect pest in the future.

Inhibitory Effect of Protease Inhibitors on Larval Midgut Protease Activities and the Performance of Plutella xylostella (Lepidoptera: Plutellidae).

Plutella xylostella L. (diamondback moth) is a pest of cruciferous plants. To understand the relationship among protease inhibitors, protease activities and the growth and development of this insect, the activities of midgut proteases of P. xylostella larvae were determined in this study. Protease samples were extracted from the midguts of P. xylostella larvae, and the protease activities were determined using enzyme specific substrates. The results showed that CaCl2, EDTA, and EGTA inhibited only the trypsin. Among the common protease inhibitors, phenylmethyl sulfonyl fluorine (PMSF), Nα-p-methyl sulfonyl-L-lysine chloromethylketone (TLCK), Nα-methyl sulfonyl-L- phenylalanine chloromethyl ketone (TPCK), soybean trypsin inhibitor (STI), and PMSF inhibited the total protease, high-alkaline trypsin (a trypsin subtype with highly alkaline pH optimum), low-alkaline trypsin (another trypsin subtype with slightly alkaline pH optimum), and chymotrypsin; TLCK inhibited the total protease and high-alkaline trypsin, whereas TPCK only activated the high-alkaline trypsin activities. STI had an inhibitory effect on all the proteases. These results showed that protease inhibitors had a certain extent inhibition to protease activities in the larval midgut of P. xylostella and that STI can potentially be used for effective pest control. The development of P. xylostella was delayed in the presence of different inhibitors. These effects were also related to the concentration of the inhibitor. A higher STI concentration showed a longer lasting effect but lower effect in this study compared to that of TLCK. The protease inhibitors had some inhibitory effect on the synthesis and secretion of proteases, and interfered with the protease activity, thereby inhibiting the absorption of nutrients and delaying the growth and development of P. xylostella and reducing their ability to reproduce. These findings should provide the baseline information about using for effective pest management in the future.

Regulated intramembrane proteolysis contributes to control of the osmoprotective transcription factor, NFAT5

Nuclear Factor of Activated T‐Cells 5 (NFAT5, also TonEBP or OREBP) increases transcription of osmoprotective genes in response to hypertonicity. Regulated intramembrane proteolysis (RIP) is involved in a number of intracellular and extracellular signaling pathways. The present studies were aimed at testing for a possible role of RIP in controlling NFAT5 activity. RIP is activated by sheddases and intramembrane cleaving proteases (iCLiPs). We studied the effects of inhibitors of those proteases on NFAT5 transcriptional and transactivating activities, using luciferase reporters in HEK293 cells. We screened inhibitors of sheddases and iCLiPS, namely ADAM, MMP, β‐secretase, γ‐secretase, SPP, zinc metalloprotease, and rhomboid. Zinc metalloprotease inhibitor (1, 10 phenanthroline monohydrate) and rhomboid inhibitor (tosyl phenylalanine chloromethyl ketone: TPCK) inhibit hypertonicity induced NFAT5 transcriptional and transactivating activity in a dose dependent manner. Further, zinc metalloprotease inhibitor, but not rhomboid inhibitor, reduces hypertonicity‐induced increase of NFAT5 protein abundance. We conclude that RIP contributes to hypertonicity‐induced increase of NFAT5 activity. The research is funded by The Intramural Research Program of NHLBI, NIH.

Enzymatic extract from Ecklonia cava induces the activation of lymphocytes by IL-2 production through the classical NF-κB pathway.

Activated nuclear factor-kappa B (NF-κB), a well-known transcription factor, leads to the development, differentiation, and proliferation of T and B lymphocytes and the secretion of cytokines by the classical pathway. We have examined here whether an enzymatic extract (ECK) from the brown seaweed, Alariaceae Laminariales Ecklonia cava may contribute to activating lymphocytes through the NF-κB pathway for participation in immune responses. In our study, ECK dose-dependently enhanced the proliferation of lymphocytes. ECK significantly increased the phosphorylation of inhibitors of κB at 0.25 and 0.5 h of exposure, followed by its gradual decrease. In addition, NF-κB p65 was gradually activated, and its binding to nuclear deoxyribonucleic acid was observed from 0.25 h after stimulation (up to 0.5 h). Further experiments showed that the application of N-p-tosyl-L: -phenylalanine chloromethyl ketone, an NF-κB inhibitor, significantly blocked ECK-induced lymphocyte's proliferation and the interleukin (IL)-2 productions. Accordingly, our results suggest that ECK increases the production of IL-2 through the activation of NF-κB then induces the proliferation of lymphocytes with the coordinated stimulation of IL-2.

Soluble expression, purification, and characterization of Gloydius shedaoensis venom gloshedobin in Escherichia coli by using fusion partners

Gloshedobin, a thrombin-like enzyme from the venom of Gloydius shedaoensis, is usually produced as inclusion bodies in Escherichia coli cell. In this work, gloshedobin was separately fused with three fusion partners NusA, GST, and TrxA at its N terminus and then was expressed as fusion proteins in E. coli. The results showed that the NusA was the most efficient fusion partner to improve the solubility of recombinant gloshedobin. The purified NusA-fused gloshedobin with an overall yield of 64.6% was resolved as one band in the SDS-PAGE gel with molecular mass of about 90 kDa. Both fibrinogen clotting and fibrinogenolytic activities were found for the recombinant product. The purified NusA-fused gloshedobin exhibited amidolytic activity of 506 U/mg under optimal conditions of pH of 8.0 and 40 degrees C. The inhibition study of NusA-fused gloshedobin by various inhibitors showed that serine protease inhibitors, phenylmethylsulphonyl fluoride, and N-tosyl-L: -phenylalanine chloromethyl ketone, strongly inhibited its admidolytic activity, whereas ethylenediaminetetraacetic acid as well as heparin and hirudin did not, suggesting that NusA-fused gloshedobin exhibited the same characteristics as the native form of gloshedobin. The strategy of this work may contribute to improve the soluble expression level of other thrombin-like enzymes from snake venom in E. coli.

The role of Bcl-xL and nuclear factor-κB in the effect of taxol on the viability of dendritic cells

Taxol has been used effectively in cancer therapies. Our previous study demonstrated that taxol induced altered maturation and improved viability of dendritic cells (DCs). However, the effects of taxol on DC viability have not been fully elucidated. In the present study, flow cytometric analyses revealed that taxol treatment significantly increased the number of viable DCs and the expression levels of a representative anti-apoptotic protein Bcl-xL. Furthermore, mobilization of the p65 subunit of nuclear factor-κB (NF-κB) from the cytosol to the nucleus in DCs was observed by confocal microscopy. An inhibition assay using N-p-tosyl-L-phenylalanine chloromethyl ketone confirmed that NF-κB was intimately involved in the effects of taxol on DC viability. In addition, we investigated the mechanisms of taxol enhancement of DC viability. Since taxol is a popular anticancer agent used in clinic, this study may provide a rationale for the use of taxol in DC immunotherapy to treat cancer patients. Taken together, these results confirm that taxol increases DC viability, and this information may provide new insights for new clinical applications of both taxol and DCs.

Effects of serine protease inhibitors on viability and morphology of Leishmania (Leishmania) amazonensis promastigotes

To investigate the importance of serine proteases in Leishmania amazonensis promastigotes, we analyzed the effects of classical serine protease inhibitors and a Kunitz-type inhibitor, obtained from sea anemone Stichodactyla helianthus (ShPI-I), on the viability and morphology of parasites in culture. Classical inhibitors were selected on the basis of their ability to inhibit L. amazonensis serine proteases, previously described. The N-tosyl-L: -phenylalanine chloromethyl ketone (TPCK) and benzamidine (Bza) inhibitors, which are potential Leishmania proteases inhibitors, in all experimental conditions reduced the parasite viability, with regard to time dependence. On the other hand, N-tosyl-lysine chloromethyl ketone (TLCK) did not significantly affect the parasite viability, as it was poor Leishmania enzymes inhibitor. Ultrastructural analysis demonstrated that both Bza and TPCK induced changes in the flagellar pocket region with membrane alteration, including bleb formation. However, TPCK effects were more pronounced than those of Bza in Leishmania flagellar pocket in plasma membrane, and intracellular vesicular bodies was visualized. ShPI-I proved to be a powerful inhibitor of L. amazonensis serine proteases and the parasite viability. The ultrastructural alterations caused by ShPI-I were more dramatic than those induced by the classical inhibitors. Vesiculation of the flagellar pocket membrane, the appearance of a cytoplasmic vesicle that resembles an autophagic vacuole, and alterations of promastigotes shape resulted.

Isolation and Characterization of a Novel Elastase Inhibitor, AFLEI from Aspergillus flavus

A novel elastase inhibitor from Aspergillus flavus (AFLEI) was isolated, and biochemical properties of AFLEI were examined. Column chromatography using diethylaminoethyl (DE) 52-Cellulose and Sephadex G-75 was used to purify the inhibitor. The final preparation was found to be homogeneous as indicated by a single band after disc polyacrylamide gel (PAGE) and isoelectric focusing electrophoreses. AFLEI had a molecular weight of 7,525.8 as determined by TOF-MS (time of flight mass spectrometry). The elastolytic activity of elastases from A. flavus, A. fumigatus and human leukocytes were inhibited by AFLEI. However, this activity from porcine pancreas elastase, trypsin, chymotrypsin, thrombin, and Ac1-Proteinase from snake venom was not affected by AFLEI. The fibrinogenase activity of the elastase from A. flavus was inhibited by AFLEI. AFLEI was inhibited by alpha2-macroglobulin. However, ethylenediaminetetraacetic acid (EDTA-2Na), benzamidine, chymostatin, tosyl phenylalanine chloromethyl ketone (TPCK) and dithiothreitol (DTT) did not show any inhibitory effect on the elastase inhibitory activity of AFLEI.

Human immunodeficiency virus type 1 Tat protein decreases cyclic AMP synthesis in rat microglia cultures.

We have studied the modulation of cyclic AMP (cAMP) accumulation by the human immunodeficiency virus type 1 (HIV 1) protein Tat in microglia and astrocyte cultures obtained from neonatal rat brain. Pretreatment of microglia with recombinant Tat resulted in a dose- and time-dependent decrease of cAMP accumulation induced by subsequent exposure to isoproterenol (1 microM). The inhibitory action of 100 ng/mL Tat approached 50% after 4 h of preincubation and reached a maximum of 70% after 24 h. The Tat-induced time- and dose-dependent decrease of cAMP accumulation was observed also when microglial cultures were stimulated with the adenylyl cyclase activator forskolin (100 microM). In both cases, Tat inhibitory action was 70% reverted by a specific monoclonal anti-Tat antibody, but was not prevented either by the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xantine (100 microM) or by a 16-h pretreatment of microglial cultures with the Gi protein inhibitor pertussis toxin (10 ng/mL). All these results suggested that the viral protein acts at a step of the cAMP transduction pathway other than receptors, G proteins and phosphodiesterases. The target of Tat appeared to be adenylyl cyclase, whose activity was markedly reduced (up to 60%) in membranes prepared from Tat-treated microglial cells, both in basal conditions and after stimulation with isoproterenol and forskolin. The inability of the competitive inhibitor of nitric oxide synthase N(G)-monometyl- L-arginine (20 and 200 microM) to revert Tat action on forskolin-induced cAMP accumulation, and of two potent nitric oxide donors, PAPA and DETA (0.1-2 m M), to alter forskolin-induced cAMP accumulation, excluded an involvement of nitric oxide in Tat-induced adenylyl cyclase inhibition. On the contrary, two inhibitors of nuclear factor kappaB activation, N-tosyl-( L)-phenylalanine chloromethyl ketone (10 microM) and SN50 (25 microM), markedly prevented the reduction of forskolin-evoked cAMP accumulation by Tat, suggesting a possible role for this nuclear transcriptional factor in the regulation of adenylyl cyclase by Tat in microglia. This assumption was strengthened by the ability of lipopolysaccharide (100 ng/mL, 4 h) to mimic the inhibitory effect of the viral protein. Conversely, astrocyte cAMP accumulation was unaffected by the viral protein, as tested at various concentrations and time points. Finally, Tat inhibition of microglial adenylyl cyclase was not due to non-specific cytotoxicity. As cAMP has been reported to exert a neuroprotective role in several in vivo and in vitro models of brain pathologies, and microglia is believed to mediate Tat-induced neurotoxicity, these results suggest that the ability of Tat to inhibit cAMP synthesis in microglia may contribute to neuronal degeneration and cell death associated with HIV infection.

Studies on Trypsin-modified Bovine and Human Lens Acylpeptide Hydrolase

Acylpeptide hydrolase removes the N -acetylated amino acids from the peptide substrates but not from intact proteins. Cleavage between amino acid residues 203–204 of the native acylpeptide hydrolase results in the formation of a 55kDa truncated active enzyme in the bovine lens, in vivo. In this study we explored the hydrolytic properties of the truncated enzyme using lens β- and γ-crystallins as substrates. SDS–PAGE analysis indicated that the βB2-crystallin was cleaved by truncated acylpeptide hydrolase into several protein fragments (10–26kDa). No cleavage of the γ-crystallins was observed under similar conditions. Both the acylpeptide hydrolase activity and the protease activity of the 55kDa enzyme were completely inhibited by diisopropylfluorophosphate, p -chloromercuribenzoate and ebelactone, and moderately inhibited by N -tosyl phenylalanine chloromethyl ketone. SDS–PAGE analysis followed by fluorography of (3H) diisopropylfluorophosphate labeled human lens acylpeptide hydrolase preparation showed the presence of the 55kDa truncated form of the enzyme, as observed in the bovine lens. The peptide (d)-AIKGDQFL-NH2—the amino acid sequence 200–207 of the native bovine acylpeptide hydrolase with an in vivo cleavage site of native protein—was hydrolysed by the lens protease(s) suggesting that the in vivo generation of the 55kDa acylpeptide hydrolase may be mediated through a proteolytic processing. The protease(s) responsible for the cleavage of this peptide was inhibited by diisopropylfluorophosphate and p -chloromercuribenzoate.

N-tosyl-L-phenylalanine chloromethyl ketone, a serine protease inhibitor, identifies glutamate 398 at the coenzyme-binding site of human aldehyde dehydrogenase. Evidence for a second "naked anion" at the active site.

Human aldehyde dehydrogenase isozymes were inactivated by N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), an inhibitor of chymotrypsin. The inactivation was a first-order process that followed saturation kinetics. NAD and chloral when used together protected against inactivation. In steady-state kinetics, TPCK produced only slope effects versus varied NAD, both slope and intercept effects versus varied glycolaldehyde were produced, indicating that TPCK reacted with the same enzyme form with which NAD reacted. Ki values from steady-state kinetics and saturation kinetics were comparable. Use of [3H]-labeled TPCK showed that inactivation was associated with the incorporation of two molecules of TPCK per molecule of enzyme. The label incorporation occurred into a single tryptic peptide and also into a single chymotryptic peptide of the E1 isozyme. Purification of labeled peptides, followed by sequencing, demonstrated that E398 of aldehyde dehydrogenase was labeled. Reaction of a haloketone, TPCK, with a carboxyl group of E398 indicates that E398 occurs as a "naked anion" within the molecule. This paper constitutes identification of the second (after E268) "naked anion" at the active site of aldehyde dehydrogenase.

Ragweed pollen proteolytic enzymes: Possible roles in allergies and asthma

Extracts from ragweed pollen grains contain novel trypsin and chymotrypsin-like serine peptidases which are described in this report. The molecular mass of the chymotrypsin-like enzyme was 82 kDa, had a pH optimum near 9.0, and its activity was unaffected by chelating or reducing agents. It was inhibited by diisopropyl fluorophosphate (DFP), a general serine class inhibitor, and more specifically N-p- tosyl- l - phenylalanine chloromethyl ketone (TPCK), a chymotrypsin-like proteinase inhibitor. In addition to various synthetic substrates, the neuropeptides, vasoactive intestinal peptide (VIP) and substance P, which are required for normalized lung functions, were also rapidly hydrolysed. Activity toward protein substrates was not detected with the exception of the inactivation of α-1-proteinase inhibitor (α-1-PI) which occurred through cleavage within the reactive site loop. The ‘trypsin-like’ enzyme has a molecular mass near 80 kDa, a blocked N-terminus, a pH optimum near 9.0, and requires Ca ++ for stability and activity, but not reducing agents. It is inhibited by DFP, and more specifically the trypsin-like proteinase inhibitor, N-p- tosyl- l - lysine chloromethyl ketone (TLCK). Again, activity toward protein substrates was not detected, but various synthetic substrates and biologically active peptides were efficiently cleaved. Significantly, atrial natriuretic peptide (ANP) and angiotensin 2 (ATII), whose degradation would amplify kinin activity and influence inflammatory diseases of the respiratory tract and nasal passages, were also rapidly hydrolyzed.

Chloromethyl ketones are insulin-like stimulators of lipid synthesis in locust fat body.

N alpha-tosyl-L-lysine chloromethyl ketone (TLCK) stimulates lipid synthesis in locust fat body in vitro, and is able to reverse the inhibitory effects of AKH-I on lipid synthesis. Effective stimulatory concentrations of TLCK were in the range of 0.2-1.0 mM. Similar stimulatory effects were also achieved with phenylalanine chloromethyl ketone (PheCK) and leucine chloromethyl ketone (LeuCK), but not with tosyl-phenylalanine chloromethyl ketone (TPCK), dansyl-glu-gly-arg-CK, chloroacetone, chloroacetic acid, chloroacetamide, chloroacetaldehyde, chloroacetyl-L-leucine or acetylated or fluorescamine-labelled TLCK, PheCK, and LeuCK. The level of stimulation caused by TLCK was dependent on incubation time, so that after a 5-h preincubation of fat body tissue with TLCK the stimulated rate was severalfold higher than the control. TLCK also increased the rate of uptake of trehalose and uridine, but not glucose, deoxyglucose or glycine. Increasing concentrations of bovine serum albumin (BSA) in the incubation medium caused a reduction in the rate of TLCK-stimulated acetate uptake, such that levels of uptake were no higher with 1% BSA than in the controls. A range of more specific protease and kinase inhibitors was tested, but none caused stimulation; thus the mode of action of TLCK on the stimulation of acetate uptake has yet to be identified. Elucidation of the mode of action of TLCK may facilitate the development of novel compounds for insect pest control.

Tosylphenylalanine chloromethyl ketone inhibits TNF-alpha mRNA synthesis in the presence of activated NF-kappa B in RAW 264.7 macrophages.

Serine proteinase inhibitors such as N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and N alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) were shown to inhibit production of tumour necrosis factor-alpha (TNF-alpha) in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages. The proteinase inhibitors were also reported to inhibit activation of the transcription factor nuclear factor-kappa B (NF-kappa B) by blocking the signalling pathway for stimuli-induced phosphorylation of the inhibitory subunit (I kappa B alpha) and thus preventing its degradation. In RAW 264.7 cells TPCK and TLCK significantly suppressed LPS-induced increase in TNF-alpha mRNA, induction of nuclear kappa B-binding activity and degradation of I kappa B alpha. TPCK and TLCK effectively blocked TNF-alpha mRNA synthesis even when they were added after LPS stimulation. In these cells, however, the inhibitory modes of the two inhibitors were found to be different: while addition of TLCK suppressed I kappa B alpha degradation and reduced NF-kappa B activity, a comparable decrease in the nuclear kappa B-binding activity or in I kappa B alpha degradation was not observed in cells treated with TPCK. Our results show that TPCK inhibits LPS-induced TNF-alpha mRNA synthesis in the presence of activated NF-kappa B and suggests that mechanisms other than NF-kappa B activation are involved in the transcriptional regulation of the TNF-alpha gene.

Neisseria gonorrhoeae epithelial cell interaction leads to the activation of the transcription factors nuclear factor kappaB and activator protein 1 and the induction of inflammatory cytokines.

We have studied the effect of human bacterial pathogen Neisseria gonorrhoeae (Ngo) on the activation of nuclear factor (NF)-kappaB and the transcriptional activation of inflammatory cytokine genes upon infection of epithelial cells. During the course of infection, Ngo, the etiologic agent of gonorrhea, adheres to and penetrates mucosal epithelial cells. In vivo, localized gonococcal infections are often associated with a massive inflammatory response. We observed upregulation of several inflammatory cytokine messenger RNAs (mRNAs) and the release of the proteins in Ngo-infected epithelial cells. Moreover, infection with Ngo induced the formation of a NF-kappaB DNA-protein complex and, with a delay in time, the activation of activator protein 1, whereas basic leucine zipper transcription factors binding to the cAMP-responsive element or CAAT/enhancer-binding protein DNA-binding sites were not activated. In supershift assays using NF-kappaB-specific antibodies, we identified a NF-kappaB p50/p65 heterodimer. The NF-kappaB complex was formed within 10 min after infection and decreased 90 min after infection. Synthesis of tumor necrosis factor alpha and interluekin (IL)-1beta occurred at later times and therefore did not account for NF-kappaB activation. An analysis of transiently transfected IL-6 promoter deletion constructs suggests that NF-kappaB plays a crucial role for the transcriptional activation of the IL-6 promoter upon Ngo infection. Inactivation of NF-kappaB conferred by the protease inhibitor N-tosyl--phenylalanine chloromethyl ketone inhibited mRNA upregulation of most, but not all, studied cyctokine genes. Activation of NF-kappaB and cytokine mRNA upregulation also occur in Ngo-infected epithelial cells that were treated with cytochalasin D, indicating an extracellular signaling induced before invasion.

Stimulation of Fc gamma receptors in rat peritoneal macrophages induces the expression of nitric oxide synthase and chemokines by mechanisms showing different sensitivities to antioxidants and nitric oxide donors.

The induction of nitric oxide (NO) production and the expression of cytokine-induced neutrophil chemoattractant (CINC-1) were studied in rat peritoneal adherent cells stimulated with insoluble immune complexes containing rabbit IgG Ab and OVA as the cognate Ag (IC). Incubation with IC at concentrations as low as 10 microg/ml induced NO production and the expression of inducible NO synthase (iNOS) protein. This was accompanied by the expression of CINC-1 mRNA and the activation of nuclear factor-kappaB (NF-kappaB). However, the expression of iNOS and CINC-1 mRNA induced by IC showed a different temporal pattern and a different sensitivity to both the antioxidant agent pyrrolidine dithiocarbamate (PDTC) and modulation by NO itself. Whereas iNOS mRNA and protein expression were blunted by PDTC and NO-generating compounds, CINC-1 mRNA expression was either enhanced or not affected by PDTC and NO donors. The time course of NF-kappaB activation was parallel to that of iNOS induction and was influenced in the same sense as iNOS induction by antioxidants, NO donors, the protease inhibitor N-tosyl phenylalanine chloromethyl ketone, and inhibitors of protein tyrosine phosphorylation reactions. These data indicate the existence in rat macrophages of a signaling mechanism triggered by Fc gammaR occupancy that leads to nuclear signaling, is initiated by protein tyrosine phosphorylation reactions, and shows specific sensitivities to antioxidants and NO. Whereas trans-activation of the iNOS gene can be fully explained by the stimulation of NF-kappaB, induction of CINC-1 mRNA expression seems influenced by additional regulatory elements.

Effects of protease inhibitors on LPS-mediated activation of a mouse macrophage cell line (J774)

Pretreatment (1 h) of a mouse macrophage-like cell line, J774, with the protease inhibitor, phenylalanine-chloromethyl ketone (PCK) or its structural analogue, tosylphenylalanine chloromethyl ketone (TPCK) was found to cause substantial inhibition of LPS-triggered activation of NF-κB. Pretreatment of cells with other types of protease inhibitors or their various structural analogues had no effect. PCK or TPCK appeared to exert its inhibitory effect by: (i) partially preventing LPS-triggered degradation of IκBα protein; (ii) preventing LPS-triggered nuclear translocation of NF-κB proteins (p50, RelA and Rel); and (iii) inhibiting the DNA-binding activities of NF-κB proteins. Pretreatment of cells with PCK or TPCK also resulted in the total or partial inhibition of LPS activatable (AP-1 or CREB) or constitutively-existing (Oct-1) transcription factors, but not of another constitutively-expressed transcription factor (SP-1). Pretreatment of J774 cells with PCK was found to substantially suppress LPS-induced expression of mRNAs specific for cytokine genes (TNFα, IL-1α and β, and IL-6), inducible nitric oxide synthase (iNOS) gene and IκBα gene, but not NF-κB1 p105 gene or β-actin gene. Furthermore, PCK pretreatment inhibited, in a dose-dependent manner, LPS-triggered production of nitric oxide production and tumoricidal activity.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is expressed in mouse skin in response to tumor-promoting agents and modulates dermal inflammation and epidermal dark cell numbers.

In mouse dorsal skin multistage carcinogenesis models, tumor promotion can be mediated by chemical agents, but also by wounding or abrasion of the epidermis, suggesting that endogenous growth factors mediate this process. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is one such factor that has been reported to be produced by keratinocytes in vitro, and has been suggested both to stimulate keratinocyte proliferation, and also to be a chemoattractant for neutrophils and macrophages. In this study we examined the expression and function of GM-CSF in mouse skin following the application of tumor-promoting agents. Both single and multiple applications of 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in accumulation of GM-CSF mRNA in the epidermis. Various phorbol and non-phorbol ester tumor promoters were found to induce increases in epidermal GM-CSF mRNA levels commensurate with their relative tumor promoting capabilities. Fluocinolone acetonide (FA) and tosyl phenylalanine chloromethyl ketone (TPCK), inhibitors of tumor promotion, inhibited tumor promoter-mediated GM-CSF accumulation, whereas all-trans-retinoic acid (RA) enhanced the TPA-induced increase. The retinoic acid analogue RO-109359 which, unlike RA, does not have tumor promoting activity per se, inhibited the TPA-induced increase in epidermal GM-CSF mRNA levels. When an antibody specific to GM-CSF was administered prior to TPA, the promoter-induced dermal inflammation and increase in epidermal dark cell number were reduced, yet promoter-induced epidermal hyperplasia was not. These findings implied that elevation of GM-CSF levels plays an important role in chemically-mediated mouse skin tumor promotion and principally via effects on promoter-induced inflammation and increased epidermal dark cell number.

Human mast cell tryptase attenuates the vasodilator activity of calcitonin generelated peptide

Calcitonin gene-related peptide (CGRP) is localized in and released from sensory nerves. It is a potent and long acting vasodilator which has been suggested to play a role in the control of blood flow. Using HPLC and trichloroacetic acid precipitation techniques, we have examined the ability of human mast cell lysates and a purified preparation of mast cell tryptase to degrade CGRP. We found that CGRP is effectively cleaved by tryptase ( K m = 6.8 10 −6 mol/L at 37°). Enzymatic activity was inhibited by antipain, leupeptin, N-α-p- tosyl- l - lysine chloromethyl ketone , benzamidine or aprotinin, but not by soybean trypsin inhibitor or N- tosyl- l - phenylalanine chloromethyl ketone . The degradation of CGRP by lysates of purified skin mast cells showed a similar pattern of inhibition suggesting that tryptase may be the major enzyme involved. The activity of tryptase was not affected by the presence of heparin. Incubation of CGRP with tryptase resulted in a loss of its vasodilator activity as observed by intravital microscopy of the hamster cheek pouch microvasculature. CGRP preincubated with tryptase failed to relax arterioles when added topically. It is suggested that the catalysis of CGRP by tryptase could represent an important means by which the activity of this neuropeptide is regulated in vivo.

Inhibitor studies of purified haemopoietic (myeloid) cell esterases: Evidence for the existence of distinct enzyme species

Human myeloid cells synthesize and express two major species of esterase, defined by isoelectric focusing (IEF). The first of these (MonEst) is specifically associated with haemopoietic cells of monocytic lineage, whereas the other species (ComEst) is common to all myeloid cells (granulocytes and monocytes) irrespective of lineage affiliation. Having recently purified these two species of human myeloid cell esterase, this present study extensively investigated the effects of 17 different inhibitors on their ability to hydrolyse the synthetic substrate alpha-naphthyl acetate (αNA). Significant inhibition of both ComEst and MonEst was exerted by 1% sodium dodecyl sulphate (SDS) and 1.0 mM diethyl pyrocarbonate (DEPC), but the patterns of inhibition for the two esterase species with the remaining compounds studied differed considerably; for example, 0.2 mM phenylmethylsulphonyl fluoride (PMSF), 5.0 × 10 −3 M dichloroisocoumarin (DCIC) and 0.1 mM N- tosyl- l- phenylalanine chloromethyl ketone (TPCK) all inhibited MonEst but not ComEst. Mechanisms of inhibition were also examined and these studies established that SDS, PMSF, DCIC and TPCK irreversibly inactivated MonEst whilst the inhibition of ComEst by SDS was reversible. Analysis of inhibition kinetics further showed that (a) the reversible inhibition of both ComEst and MonEst by sodium fluoride (NaF) was noncompetitive (with K i , values of 1.28 and 0.01 mM, respectively, indicating a marked difference in sensitivity); (b) the inhibition of MonEst by PMSF was of ‘mixed’ noncompetitive-competitive type; and (c) that DEPC exerted noncompetitive inhibition with similar K i values (0.05 mM) for both esterase species. These observations unequivocably demonstrate that ComEst and MonEst are unrelated enzyme species, with a common ability to hydrolyse αNA, and that these esterases show marked differences with respect to their active sites as adjudged by inhibitor sensitivities. These observations are particularly relevant to the histochemical analysis of these enzymes and to the elucidation of their in vivo functions.