Brain Protease Activated Receptor 1 Pathway: A Therapeutic Target in the Superoxide Dismutase 1 (SOD1) Mouse Model of Amyotrophic Lateral Sclerosis.

Glia cells are involved in upper motor neuron degeneration in amyotrophic lateral sclerosis (ALS). Protease activated receptor 1 (PAR1) pathway is related to brain pathologies. Brain PAR1 is located on peri-synaptic astrocytes, adjacent to pyramidal motor neurons, suggesting possible involvement in ALS. Brain thrombin activity in superoxide dismutase 1 (SOD1) mice was measured using a fluorometric assay, and PAR1 levels by western blot. PAR1 was localized using immunohistochemistry staining. Treatment targeted PAR1 pathway on three levels; thrombin inhibitor TLCK (N-Tosyl-Lys-chloromethylketone), PAR1 antagonist SCH-79797 and the Ras intracellular inhibitor FTS (S-trans-trans-farnesylthiosalicylic acid). Mice were weighed and assessed for motor function and survival. SOD1 brain thrombin activity was increased (p < 0.001) particularly in the posterior frontal lobe (p = 0.027) and hindbrain (p < 0.01). PAR1 levels were decreased (p < 0.001, brain, spinal cord, p < 0.05). PAR1 and glial fibrillary acidic protein (GFAP) staining decreased in the cerebellum and cortex. SOD1 mice lost weight (≥17 weeks, p = 0.047), and showed shorter rotarod time (≥14 weeks, p < 0.01). FTS 40mg/kg significantly improved rotarod scores (p < 0.001). Survival improved with all treatments (p < 0.01 for all treatments). PAR1 antagonism was the most efficient, with a median survival improvement of 10 days (p < 0.0001). Our results support PAR1 pathway involvement in ALS.

MG132 exerts anti-viral activity against HSV-1 by overcoming virus-mediated suppression of the ERK signaling pathway.

Herpes simplex virus 1 (HSV-1) causes a number of clinical manifestations including cold sores, keratitis, meningitis and encephalitis. Although current drugs are available to treat HSV-1 infection, they can cause side effects such as nephrotoxicity. Moreover, owing to the emergence of drug-resistant HSV-1 strains, new anti-HSV-1 compounds are needed. Because many viruses exploit cellular host proteases and encode their own viral proteases for survival, we investigated the inhibitory effects of a panel of protease inhibitors (TLCK, TPCK, E64, bortezomib, or MG132) on HSV-1 replication and several host cell signaling pathways. We found that HSV-1 infection suppressed c-Raf-MEK1/2-ERK1/2-p90RSK signaling in host cells, which facilitated viral replication. The mechanism by which HSV-1 inhibited ERK signaling was mediated through the polyubiquitination and proteasomal degradation of Ras-guanine nucleotide-releasing factor 2 (Ras-GRF2). Importantly, the proteasome inhibitor MG132 inhibited HSV-1 replication by reversing ERK suppression in infected cells, inhibiting lytic genes (ICP5, ICP27 and UL42) expression, and overcoming the downregulation of Ras-GRF2. These results indicate that the suppression of ERK signaling via proteasomal degradation of Ras-GRF2 is necessary for HSV-1 infection and replication. Given that ERK activation by MG132 exhibits anti-HSV-1 activity, these results suggest that the proteasome inhibitor could serve as a novel therapeutic agent against HSV-1 infection.

Activation of Bt Protoxin Cry1Ac in Resistant and Susceptible Cotton Bollworm.

Crystalline (Cry) proteins from Bacillus thuringiensis (Bt) are used extensively for insect control in sprays and transgenic plants, but their efficacy is reduced by evolution of resistance in pests. Here we evaluated reduced activation of Cry1Ac protoxin as a potential mechanism of resistance in the invasive pest Helicoverpa armigera. Based on the concentration killing 50% of larvae (LC50) for a laboratory-selected resistant strain (LF120) divided by the LC50 for its susceptible parent strain (LF), the resistance ratio was 1600 for Cry1Ac protoxin and 1200 for trypsin-activated Cry1Ac toxin. The high level of resistance to activated toxin as well as to protoxin indicates reduced activation of protoxin is not a major mechanism of resistance to Cry1Ac in LF120. For both insect strains, treatment with either the trypsin inhibitor N-a-tosyl-L-lysine chloromethyl ketone (TLCK) or the chymotrypsin inhibitor N-a-tosyl-L-phenylalanine chloromethyl ketone (TPCK) did not significantly affect the LC50 of Cry1Ac protoxin. Enzyme activity was higher for LF than LF120 for trypsin-like proteases, but did not differ between strains for chymotrypsin-like proteases. The results here are consistent with previous reports indicating that reduced activation of protoxin is generally not a major mechanism of resistance to Bt proteins.

Serine protease inhibitors interact with IFN-γ through up-regulation of FasR; a novel therapeutic strategy against cancer.

Among the many immunomodulatory and anti-tumor activities, IFN-γ up-regulates tumor cell death mediated by Fas receptor (FasR). Our and several other studies have demonstrated the involvement of trypsin-like proteases (TLPs) in the mode of action of IFN-γ. In the present study, we tried to unravel the role of serine proteases in IFN-γ induced Fas-mediated cell death. Our present results show that both tosyl-l-Lysine chloromethylketone (TLCK), a trypsin like protease inhibitor and tosyl-l-phenylalanine chloromethylketone (TPCK) - a chymotrypsin like protease (CLP) inhibitor, sensitize HeLa cells to Fas-mediated cell death. The combined effect of these protease inhibitors with anti-Fas was stronger than additive. In contrast, elastase inhibitor III (EI), which also contains the chloromethyl ketone moiety, was not active. Furthermore, co-addition of TLCK or TPCK with IFN-γ markedly enhanced Fas-induced cell death. IFN-γ led to up-regulation of FasR on its own, which was further enhanced by the co-addition of TLCK or TPCK. This was evident both by increased expression of Fas receptor on cell surface and by elevated Fas mRNA level. This study may provide the basis for the design of a novel combinatory therapeutic strategy that could enhance the eradication of tumors.

The serine protease inhibitor TLCK attenuates intrinsic death pathways in neurons upstream of mitochondrial demise.

It is well-established that activation of proteases, such as caspases, calpains and cathepsins are essential components in signaling pathways of programmed cell death (PCD). Although these proteases have also been linked to mechanisms of neuronal cell death, they are dispensable in paradigms of intrinsic death pathways, e.g. induced by oxidative stress. However, emerging evidence implicated a particular role for serine proteases in mechanisms of PCD in neurons. Here, we investigated the role of trypsin-like serine proteases in a model of glutamate toxicity in HT-22 cells. In these cells glutamate induces oxytosis, a form of caspase-independent cell death that involves activation of the pro-apoptotic protein BH3 interacting-domain death agonist (Bid), leading to mitochondrial demise and ensuing cell death. In this model system, the trypsin-like serine protease inhibitor Nα-tosyl-l-lysine chloromethyl ketone hydrochloride (TLCK) inhibited mitochondrial damage and cell death. Mitochondrial morphology alterations, the impairment of the mitochondrial membrane potential and ATP depletion were prevented and, moreover, lipid peroxidation induced by glutamate was completely abolished. Strikingly, truncated Bid-induced cell death was not affected by TLCK, suggesting a detrimental activity of serine proteases upstream of Bid activation and mitochondrial demise. In summary, this study demonstrates the protective effect of serine protease inhibition by TLCK against oxytosis-induced mitochondrial damage and cell death. These findings indicate that TLCK-sensitive serine proteases play a crucial role in cell death mechanisms upstream of mitochondrial demise and thus, may serve as therapeutic targets in diseases, where oxidative stress and intrinsic pathways of PCD mediate neuronal cell death.

Protease in sturgeon sperm and the effects of protease inhibitors on sperm motility and velocity.

In mammals, proteases are present in sperm acrosome and play key role in fertilization. Sturgeon sperm has an acrosome, but its physiology, biochemistry, and potential role in fertilization are unknown. In the present study, we have observed high protease activity in acidic extract of intact sperm compared to that of seminal plasma in sterlet (Acipenser ruthenus). The protease activity was decreased and increased in acidic extract of motility-activated sperm and in the activation medium, respectively. Molecular analysis revealed total protease and serine (acrosin) protease activities in sperm acidic extract which was accumulated in a protein band with relative molecular mass of 35 kDa. Immunoelectron microscopy using an affinity-purified polyclonal antibody for boar acrosin localized the protease at the acrosome region. Moreover, initiation of sperm motility was inhibited after activation in the presence of inhibitors for both trypsin-like and chymotrypsin-like proteases, while the effects of protease inhibitors on sperm velocity were uncertain. Our results indicate similarities in physiology and biochemistry of acrosome between sturgeon and mammals and suggest potential role of protease in the initiation of sperm motility in sturgeon.

[Expression of Bim, Bax and Bak in the process of gingipain-induced osteoblast apoptosis].

OBJECTIVE: To establish osteoblast apoptosis model induced by gingipains, and to examine the expression of pro-apoptotic protein Bcl-2 interacting mediator (Bim), Bcl-2 associated X protein (Bax) and Bcl-2 antagonist/killer (Bak). METHODS: Gingipain and gingipain acticity were extracted and measured. Mouse osteoblast cell line MC3T3-E1 cells were cultured in the presence of 0.453, 0.906, 1.812 U/L gingipains for 0, 16, 24 and 48 h. Apoptosis was examined by 4',6-diamidino-2-phenylindole (DAPI) staining or annexin V/propidine iodide (PI) staining.Protein expression of Bim, Bax and Bak was determined by Western blotting after osteoblasts were cultured with 1.812 U/L gingipain for 0, 4, 8, 16, 24 and 48 h. Osteoblasts were cultured with 1.812 U/L gingipain which had been inhibited with N-alpha-tosyl L-lysyl-chlorom ethylketone (TLCK). Western blotting was used to detect Bim expression and DAPI staining to measure apoptosis. RESULTS: Arginine-specific proteinases (Rgp) activity was (18.11 ± 2.11) U/L and specific proteinases (Kgp) was (1.02 ± 0.25) U/L. Percentage of osteoblast apoptosis induced by 1.812 U/L gingipain rose to (6.31 ± 0.37)% after 16 h, and reached (11.20 ± 0.35)% at 24 h and (10.80 ± 0.46)% after 48 h with DAPI staining. Annexin V/PI staining supported the result from DAPI staining.Bim protein level increased during osteoblast apoptosis, the relative fold rose to (0.31 ± 0.03) after 4 h (about 2 fold compared to control), peaking at 24 h (0.57 ± 0.05, 3-4 fold compared to control). Proteinase inhibitor TLCK effectively blocked the activity of gingipain and inhibited up-regulation of Bim induced by gingipains from (0.58 ± 0.04) to (0.14 ± 0.03). The percentage of osteoblast apoptosis decreased from (11.20 ± 0.35)% to (4.31 ± 0.38)% in the presence of TLCK. Expression of Bax remained unchanged when cells were cultured with or without gingipains. Bak was under the detectable level in MC3T3-E1. CONCLUSIONS: 1.812 U/L gingipains induced osteoblast apoptosis. Protein expression of Bim was up-regulated during cell apoptosis and was down-regulated when gingipain inhibited with TLCK, suggesting that Bim was involved in osteoblast apoptosis induced by gingipain. Inhibition of Bim protein expression protected osteoblast from apoptosis.

LL-37 in periodontal health and disease and its susceptibility to degradation by proteinases present in gingival crevicular fluid.

AIM: To determine the levels of LL-37 in and its susceptibility to degradation by components of gingival crevicular fluid (GCF) in periodontal health and disease. MATERIALS AND METHODS: Levels of LL-37 in GCF from periodontitis patients and periodontally healthy subjects were determined by ELISA. In addition, degradation of synthetic/exogenous LL-37 by components of GCF in the presence and absence of inhibitors was determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry. RESULTS: The concentration of native LL-37 in GCF from Porphyromonas gingivalis positive (Pg+) and P. gingivalis negative (Pg-) sites in periodontitis patients was significantly higher than in GCF from healthy subjects. When synthetic LL-37 was added to healthy GCF, the peptide was not degraded. Conversely, GCF from Pg+ sites rapidly degraded synthetic LL-37 which was prevented in the presence of Arg- and Lys- gingipain inhibitors. Synthetic LL-37 was degraded more slowly by GCF from Pg- sites. CONCLUSIONS: LL-37 is detectable in GCF in periodontal health and disease. The rapid degradation of synthetic LL-37 in periodontitis GCF, particularly in Pg+ sites, limits its role as a potential therapeutic in the gingival crevice. These results highlight the need to design stable peptide mimetics of LL-37 as future therapeutics in periodontitis.

Angiogenic changes in co-cultures of mast cells and myocardial microvascular endothelial cells under hyperglycemic conditions.

The aim of the present study was to determine the correlation between angiogenesis and the differential expression of growth factors and their receptors when myocardial microvascular endothelial cells (MMVECs) were co-cultured with mast cell granules (MCGs) under hyperglycemic conditions. MMVECs and mast cells (MCs) were isolated from Wistar rats. An in vitro angiogenesis assay was used to observe any differences when MMVECs were co-cultured with MCGs in normal or hyperglycemic medium. The mRNA and protein expression of growth factors and their receptors were analyzed by real-time reverse transcription (RT)-PCR and western blot analysis. Real-time RT-PCR analysis demonstrated the upregulated mRNA and protein expression of vascular endothelial growth factor (VEGF) in the MMVECs; however, the expression of its receptor, fms-like tyrosine kinase-1 (Flt-1) and fetal liver kinase-1 (Flk­1), decreased significantly, and the angiogenic ability of the MMVECs decreased under hyperglycemic conditions. The angiogenic ability of the MMVECs cultured under hyperglycemic conditions (even after the addition of MCGs) was inferior to that of the MMVECs cultured under normal glucose conditions. The specific inhibitor of tryptase, N-tosyl-L-lysine chloromethyl ketone (TLCK), suppressed angiogenesis regardless of the glucose concentration, and the specific inhibitor of chymase, N-tosyl-L-phenylalanyl chloromethyl ketone (TPCK), was not as effective as TLCK, which was mainly detected under hyperglycemic conditions. High glucose levels have a profound effect on angiogenesis; this effect may be more pronounced than the effects of MCGs on angiogenesis.

[Expression of integrin α5 and ß1 in osteoblast in the process of gingipains-induced apoptosis].

OBJECTIVE: To investigate the regulatory mechanisms of integrin α5 and ß1 in osteoblast in the process of gingipains-induced apoptosis. METHODS: Gingipains were isolated and purified from supernatants of Porphyromonas gingivalis W83 which was cultured under standard anaerobic conditions. MC3T3-E1 was challenged with or without 8.3480 U/L gingipains for 48 h and apoptosis was examined by transferase-mediated deoxyuridine triphosphate-biotin nick end labeling-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (TUNEL-DAPI) staining. The expression of integrin α5 and ß1 was analyzed by Western blotting after MC3T3-E1 was treated under different conditions. RESULTS: Arginine-specific proteinases(Rgp) activity was (41.74 ± 2.11) U/L and lysine-specific proteinase(Kgp) was (1.02 ± 0.25) U/L.Gingipains induced MC3T3-E1 cells apoptosis after 48 h. Compared with control group, expression of integrin α5 and ß1 was down-regulated by gingipains in a time-dependent manner within short periods ( ≤ 72 h), integrin α5 and ß1 relative expression was (0.485 ± 0.039),(0.504 ± 0.002) at 48 h,(0.398 ± 0.058),(0.179 ± 0.001) at 72 h respectively (P < 0.05). After 72 h, integrin α5 expression in MC3T3-E1 cells was stable compared with control group while integrin ß1 was still lower(control group:1.000 ± 0.000, 96 h:0.604 ± 0.003, 120 h: 0.357 ± 0.002) (P < 0.05). Proteinase inhibitor tosyl- L- lysine-chloromethyl-ketone(TLCK) effectively blocked the activity of gingipain and inhibited down-regulation of integrin α5 and ß1 induced by gingipains from (0.398 ± 0.058,0.179 ± 0.001 ) to (0.781 ± 0.012, 0.857 ± 0.060) (P < 0.05). TLCK alone did not have any effect on integrin α5 and ß1(P > 0.05). Gingipains also decreased integrin α5 and ß1 in a dose-dependent manner.When cells were treated with 20.8700 U/L gingipains, integrin α5 and ß1 relative expression reached to the lowest(0.105 ± 0.004,0.020 ± 0.000) (P < 0.05). CONCLUSIONS: Gingipains inhibited the expression of integrin α5 and ß1 in a time- and dose- dependent manner in osteoblasts in the process of apoptosis, which may not be mediated by direct proteolytic effect.

Cloning eleven midgut trypsin cDNAs and evaluating the interaction of proteinase inhibitors with Cry1Ac against the tobacco budworm, Heliothis virescens (F.) (Lepidoptera: Noctuidae).

Midgut trypsins are associated with Bt protoxin activation and toxin degradation. Proteinase inhibitors have potential insecticidal toxicity against a wide range of insect species. This study was conducted to evaluate the interaction of proteinase inhibitors with Bt toxin and to examine midgut trypsin gene profile of Heliothis virescens. A sublethal dose (15 ppb) of Cry1Ac, 0.75% soybean trypsin inhibitor, and 0.1% and 0.2% N-α-tosyl-L-lysine chloromethyl ketone significantly suppressed midgut proteinase activities, and resulted in reductions in larval and pupal size and mass. The treatment with inhibitor+Bt suppressed approximately 65% more larval body mass and 21% more enzymatic activities than the inhibitor-only or Bt-only. Eleven trypsin-like cDNAs were sequenced from the midgut of H. virescens. All trypsins contained three catalytic center residues (H(73), D(153), and S(231)), substrate specificity determinant residues (D(225), G(250), and G(261)), and six cysteines for disulfide bridges. These putative trypsins were separated into three distinct groups, indicating the diverse proteinases evolved in this polyphagous insect. These results indicated that the insecticidal activity of proteinase inhibitors may be used to enhance Bt toxicity and delay resistance development.

Leishmania (L.) amazonensis peptidase activities inside the living cells and in their lysates.

In this study we investigated the peptidase activity in Leishmania (L.) amazonensis live amastigote by confocal microscopy using peptidyl-MCA as substrates, the hydrolysis of which releases the MCA fluorophore inside the cells. Cell pre-treatment with peptidase inhibitors indicated the presence of cysteine and serine peptidases. It was noteworthy that Leishmania amastigotes incorporate only substrates (Z-FR-MCA, Z-RR-MCA) or inhibitors (E64, TLCK) containing positively charged groups. The peptidase activities in the supernatants of amastigotes and promastigotes lysates were also evaluated with the same peptidyl-MCA substrates and inhibitors in the pH range 4.5-9.0. The effects of temperature and different salts were also included in this study. The hydrolytic activities of supernatants on Z-FR-MCA clearly indicate the presence of different cysteine peptidases that adapted to work in different environment conditions. Intact Leishmania cells incorporated Z-RR-MCA, the hydrolysis of which was inhibited only by TLCK indicating the presence of at least one serine peptidase. The pH profile of Z-RR-MCA hydrolysis by amastigotes and promastigotes lysate supernatants, and the hydrolysis time course of the FRET peptide Abz-AGRRRAQ-EDDnp at RA bond, followed by removal of the two C-termini R to yield Abz-AGR-OH that is a unique characteristic of oligopeptidase B, indicate its presence in the parasite.

Synthesis and acrosin inhibitory activities of substituted ethyl 5-(4-aminophenyl)-1H-pyrazole-3-carboxylate derivatives.

A series of novel ethyl 5-(4-aminophenyl)-1H-pyrazole-3-carboxylate derivatives were designed and synthesized and their in vitro acrosin inhibitory activities were evaluated. Most of the compounds exhibited acrosin inhibitory activities. Among them, three compounds (5l, 5n, and 5v) were more potent than that of the control TLCK. These provide a new structural type for the development of novel contraceptive acrosin inhibitory agents.

Expression of trypsin-like serine peptidases in pre-imaginal stages of Aedes aegypti (Diptera: Culicidae).

This study reports the biochemical characterization and comparative analyses of highly active serine proteases in the larval and pupal developmental stages of Aedes aegypti (Linnaeus) using substrate-SDS-PAGE. Zymographic analysis of larval stadia detected proteolytic activity in 6-8 bands with apparent molecular masses ranging from 20 to 250 kDa, with activity observed from pH 5.5 to 10.0. The pupal stage showed a complex proteolytic activity in at least 11 bands with apparent Mr ranging from 25 to 250 kDa, and pH optimum at 10.0. The proteolytic activities of both larval and pupal stages were strongly inhibited by phenyl-methyl sulfonyl-fluoride and N-α-Tosyl-L-lysine chloromethyl ketone hydrochloride, indicating that the main proteases expressed by these developmental stages are trypsin-like serine proteases. The enzymes were active at temperatures ranging from 4 to 85°C, with optimal activity between 37 and 60°C, and low activity at 85°C. Comparative analysis between the proteolytic enzymes expressed by larvae and pupae showed that substantial changes in the expression of active trypsin-like serine proteases occur during the developmental cycle of A. aegypti.

Collagenolytic activity in sonic extracts of Tannerella forsythia.

OBJECTIVE: The purpose of the present study was to characterize the collagenolytic activity in a sonicated extract of Tannerella forsythia and to investigate the activation of proMMMP-2 and -9 by the T. forsythia extract. METHODS: The T. forsythia extract was incubated with type I collagen. The cleaved products were then analyzed by SDS-PAGE using the method of Laemmli. We studied the effects of cysteine, DTT, CaCl(2), and various proteinase inhibitors on collagenolytic activity. A HT1080 cell culture supernatant containing proMMP-2 and -9 was incubated with the T. forsythia extract and analyzed for the activation of proMMP-2 and -9 by gelatin zymography. RESULTS: The T. forsythia extract degraded type I collagen. Cysteine increased the collagenolytic activity of the extract, and 5mM CaCl(2) was required for this activity. The collagenolytic activity of T. forsythia was inhibited by N-ethylmaleimide, iodoacetamide, iodoacetic acid, EDTA, and leupeptin, but not by PMSF, E-64, TLCK, or TPCK. When proMMP-2 and -9 were incubated with the T. forsythia extract, gelatinases with the relative molecular masses of MMP-2 and -9 were produced. CONCLUSION: The present study suggests that T. forsythia extract is able to degrade type I collagen and activate proMMP-2 and -9.

Purification, characterization and substrate specificity of a trypsin from the Amazonian fish tambaqui (Colossoma macropomum).

An enzyme was purified from the pyloric caecum of tambaqui (Colossoma macropomum) through heat treatment, ammonium sulfate fractionation, Sephadex G-75 and p-aminobenzamidine-agarose affinity chromatography. The enzyme had a molecular mass of 23.9 kDa, NH(2)-terminal amino acid sequence of IVGGYECKAHSQPHVSLNI and substrate specificity for arginine at P1, efficiently hydrolizing substrates with leucine and lysine at P2 and serine and arginine at P1'. Using the substrate z-FR-MCA, the enzyme exhibited greatest activity at pH 9.0 and 50 degrees C, whereas, with BAPNA activity was higher in a pH range of 7.5-11.5 and at 70 degrees C. Moreover, the enzyme maintained ca. 60% of its activity after incubated for 3h at 60 degrees C. The enzymatic activity significantly decreased in the presence of TLCK, benzamidine (trypsin inhibitors) and PMSF (serine protease inhibitor). This source of trypsin may be an attractive alternative for the detergent and food industry.

Okadaic acid induces DNA fragmentation via caspase-3-dependent and caspase-3-independent pathways in Chinese hamster ovary (CHO)-K1 cells.

DNA fragmentation is a hallmark of apoptosis that occurs in a variety of cell types; however, it remains unclear whether caspase-3 is required for its induction. To investigate this, we produced caspase-3 knockout Chinese hamster ovary (CHO)-K1 cells and examined the effects of gene knockout and treatment with caspase-3 inhibitors. Okadaic acid (OA) is a potent inhibitor of the serine/threonine protein phosphatases (PPs) PP1 and PP2A, which induce apoptotic cellular reactions. Treatment of caspase-3(-/-) cells with OA induced DNA fragmentation, indicating that caspase-3 is not an essential requirement. However, in the presence of benzyloxycarbonyl-Asp-Glu-Val-Asp (OMe) fluoromethylketone (z-DEVD-fmk), DNA fragmentation occurred in CHO-K1 cells but not in caspase-3(-/-) cells, suggesting that caspase-3 is involved in OA-induced DNA fragmentation that does not utilize DEVDase activity. In the absence of caspase-3, DEVDase activity may play an important role. In addition, OA-induced DNA fragmentation was reduced but not blocked in CHO-K1 cells, suggesting that caspase-3 is involved in caspase-independent OA-induced DNA fragmentation. Furthermore, OA-induced cleavage of caspase-3 and DNA fragmentation were blocked by pretreatment with the wide-ranging serine protease inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride. These results suggest that serine proteases regulate DNA fragmentation upstream of caspase-3.

Determination of the dissociation constants for Ca2+ and calmodulin from the plasma membrane Ca2+ pump by a lipid probe that senses membrane domain changes.

The purpose of this work was to obtain information about conformational changes of the plasma membrane Ca(2+)-pump (PMCA) in the membrane region upon interaction with Ca(2+), calmodulin (CaM) and acidic phospholipids. To this end, we have quantified labeling of PMCA with the photoactivatable phosphatidylcholine analog [(125)I]TID-PC/16, measuring the shift of conformation E(2) to the auto-inhibited conformation E(1)I and to the activated E(1)A state, titrating the effect of Ca(2+) under different conditions. Using a similar approach, we also determined the CaM-PMCA dissociation constant. The results indicate that the PMCA possesses a high affinity site for Ca(2+) regardless of the presence or absence of activators. Modulation of pump activity is exerted through the C-terminal domain, which induces an apparent auto-inhibited conformation for Ca(2+) transport but does not modify the affinity for Ca(2+) at the transmembrane domain. The C-terminal domain is affected by CaM and CaM-like treatments driving the auto-inhibited conformation E(1)I to the activated E(1)A conformation and thus modulating the transport of Ca(2+). This is reflected in the different apparent constants for Ca(2+) in the absence of CaM (calculated by Ca(2+)-ATPase activity) that sharply contrast with the lack of variation of the affinity for the Ca(2+) site at equilibrium. This is the first time that equilibrium constants for the dissociation of Ca(2+) and CaM ligands from PMCA complexes are measured through the change of transmembrane conformations of the pump. The data further suggest that the transmembrane domain of the PMCA undergoes major rearrangements resulting in altered lipid accessibility upon Ca(2+) binding and activation.

Properties of a Kunitz-type trypsin inhibitor from Delonix regia seeds against digestive proteinases of Anagasta kuehniella (Z.) and Corcyra cephalonica (S.) (Lepidoptera: Pyralidae).

DrTI was effective against trypsin-like enzymes from A. kuehniella and C. cephalonica, however an artificial diet was insufficient to affect the survival and body weight of either insect. The inhibitor stimulated chymotrypsin-like enzymes and probably induced the synthesis of enzymes insensitive to TLCK in neonate larvae.

[Nandeshi: a powerful inhibitor of human acrosin activity].

OBJECTIVE: To evaluate the inhibitory effect of Nandeshi, an acrosin inhibitor, on human acrosin activity. METHODS: We collected sperm samples from 10 healthy fertile men and cultured them with Nandeshi at 30 degrees C for 5 minutes at the concentrations of 0. 100, 0.120, 0.144, 0.173, 0.207, 0.249, 0.299, 0.358 and 0.430 mmol/L, with the controls treated with a well-known acrosin inhibitor N-alpha-p-tosyl-L-lysine chloromethylketone (TLCK) at 150.0, 189.8, 213.6, 240.3, 270.3, 304.1 and 342.1 mmol/L. Then we determined the residual activity of human acrosin by improved Kennedy assay. RESULTS: The residual activity of acrosin was negatively correlated with the Nandeshi concentration, and Nandeshi exhibited an inhibition rate about 800 times that of TLCK. CONCLUSION: Nandeshi has a powerful inhibitory effect on human acrosin, and improved Kennedy assay is a simple, practical and highly sensitive technique for the detection of human acrosin activity.