Identification of two subtilisin-like serine proteases engaged in the degradation of recombinant proteins in Nicotiana benthamiana.

The tobacco variant Nicotiana benthamiana has recently emerged as a versatile host for the manufacturing of protein therapeutics, but the fidelity of many recombinant proteins generated in this system is compromised by inadvertent proteolysis. Previous studies have revealed that the anti-HIV-1 antibodies 2F5 and PG9 as well as the protease inhibitor α1 -antitrypsin (A1AT) are particularly susceptible to N. benthamiana proteases. Here, we identify two subtilisin-like serine proteases (NbSBT1 and NbSBT2) whose combined action is sufficient to account for all major cleavage events observed upon expression of 2F5, PG9 and A1AT in N. benthamiana. We propose that downregulation of NbSBT1 and NbSBT2 activities could constitute a powerful means to optimize the performance of this promising platform for the production of biopharmaceuticals. DATABASES: NbSBT sequence data are available in the DDBJ/EMBL/GenBank databases under the accession numbers MN534996 to MN535005.

Ultrasound-Assisted Enzyme-Catalyzed Hydrolysis of Collagen to Produce Peptides With Biomedical Potential: Collagenase From Aspergillus terreus UCP1276.

Ultrasound has been applied for varied purposes as it provides additional mechanical energy to a system, and is still profitable and straightforward, which are advantages for industrial applications. In this work, ultrasonic treatments were applied to purified collagenase fractions from a fermented extract by Aspergillus terreus UCP 1276 aiming to evaluate the potential effect on collagen hydrolysis. The physical agent was evaluated as an inductor of collagen degradation and consequently as a producer of peptides with anticoagulant activity. The sodium dodecyl sulphate-polyacrylamide gel electrophoresis analyses were also carried out to compare the hydrolysis techniques. The ultrasound (40 kHz, 47.4 W/L) processing was conducted under the same conditions of pH and temperature at different times. The ultrasound-assisted reaction was accelerated in relation to conventional processing. Collagenolytic activity was enhanced and tested in the presence of phenylmethanesulfonyl fluoride inhibitor. Underexposure, the activity was enhanced, reaching more than 72.0% of improvement in relation to the non-exposed enzyme. A period of 30 min of incubation under ultrasound exposure was enough to efficiently produce peptides with biological activity, including anticoagulation and effect on prothrombin time at about 60%. The results indicate that low-frequency ultrasound is an enzymatic inducer with likely commercial applicability accelerating the enzymatic reaction. Bioelectromagnetics. 2020;41:113-120. © 2019 Bioelectromagnetics Society.

Specific inhibitors of lysozyme and peptidases inhibit the growth of the rumen protozoan Entodinium caudatum without decreasing feed digestion or fermentation in vitro.

AIMS: Experiments were designed to determine the effects of different chemical inhibitors of lysozyme and peptidases on rumen protozoa and the associated prokaryotes, and in vitro fermentation using Entodinium caudatum as a model protozoan species. METHODS AND RESULTS: Imidazole (a lysozyme inhibitor), phenylmethylsulphonyl fluoride (PMSF, a serine peptidase inhibitor) and iodoacetamide (IOD, a cysteine peptidase inhibitor) were evaluated in vitro both individually and in two- and three-way combinations using E. caudatum monocultures with respect to their ability to inhibit the protozoan and their effect on feed digestion, fermentation and the microbiota. All the three inhibitors, both individually and in combination, decreased E. caudatum counts (P < 0·001), and IOD and its combinations with the other inhibitors significantly (P < 0·01) decreased ammonia concentration, with the two- and three-way combinations showing additive effective. Feed digestion was not affected, but fermentation and microbial diversity were affected mostly by PMSF, IOD and their combinatorial treatments potentially due to the overgrowth of Streptococcus luteciae accompanying with the disappearance of host ciliates. CONCLUSIONS: Entodinium caudatum depends on lysozyme and peptidase for digestion and utilization of the engulfed microbes and specific inhibition of these enzymes can inhibition E. caudatum without adversely affecting feed digestion or fermentation even though they changed the microbiota composition in the cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: The peptidase inhibitors may have the potential to be used in controlling rumen protozoa to improve ruminal nitrogen utilization efficiency.

The calcium-sensing receptor regulates protein tyrosine phosphorylation through PDK1 in boar spermatozoa.

Regulation of protein tyrosine phosphorylation is required for sperm capacitation and oocyte fertilization. The objective of the present work was to study the role of the calcium-sensing receptor (CaSR) on protein tyrosine phosphorylation in boar spermatozoa under capacitating conditions. To do this, boar spermatozoa were incubated in Tyrode's complete medium for 4 hr and the specific inhibitor of the CaSR, NPS2143, was used. Also, to study the possible mechanism(s) by which this receptor exerts its function, spermatozoa were incubated in the presence of specific inhibitors of the 3-phosphoinositide dependent protein kinase 1 (PDK1) and protein kinase A (PKA). Treatment with NPS2143, GSK2334470, an inhibitor of PDK1 and H-89, an inhibitor of PKA separately induced an increase in tyrosine phosphorylation of 18 and 32 kDa proteins, a decrease in the serine/threonine phosphorylation of the PKA substrates together with a drop in sperm motility and viability. The present work proposes a new signalling pathway of the CaSR, mediated by PDK1 and PKA in boar spermatozoa under capacitating conditions. Our results show that the inhibition of the CaSR induces the inhibition of PDK1 that blocks PKA activity resulting in a rise in tyrosine phosphorylation of p18 and p32 proteins. This novel signalling pathway has not been described before and could be crucial to understand boar sperm capacitation within the female reproductive tract.

Proteases of Sporothrix schenckii: Cytopathological effects on a host-cell model.

BACKGROUND: Sporotrichosis is a fungal infection caused by the Sporothrix schenckii complex. The adhesion of the fungus to the host tissue has been considered the key step in the colonization and invasion, but little is known about the early events in the host-parasite interaction. AIMS: To evaluate the proteolytic activity of S. schenckii on epithelial cells. METHODS: The proteolytic system (at pH 5 and 7) was evaluated using azocoll and zymograms. The host-parasite interaction and epithelial cell response were also analyzed by examining the microfilament cytoskeleton using phalloidin-FITC and transmission electron microscopy. Finally, the metabolic activity was determined using an XTT assay. RESULTS: The zymograms showed that S. schenckii yeast cells possess high intracellular and extracellular proteolytic activities (Mr≥200, 116, 97, and 70kDa) that are pH dependent and are inhibited by PMSF and E64, which act on serine and cysteine-type proteases. During the epithelial cell-protease interaction, the cells showed alterations in the microfilament distribution, as well as in the plasma membrane structure. Moreover, the metabolic activity of the epithelial cells decreased 60% without a protease inhibitor. CONCLUSIONS: Our data demonstrate the complexity of the cellular responses during the infection process. This process is somehow counteracted by the action of proteases inhibitors. Furthermore, the results provide critical information for understanding the nature of host-fungus interactions and for searching a new effective antifungal therapy, which includes protease inhibitors.

Involvement of bilitranslocase and beta-glucuronidase in the vascular protection by hydroxytyrosol and its glucuronide metabolites in oxidative stress conditions.

Olive oil vascular benefits have been attributed to hydroxytyrosol (HT). However, HT biological actions are still debated because it is extensively metabolized into glucuronides (GCs). The aim of this study was to test HT and GC vasculoprotective effects and the underlying mechanisms using aorta rings from 8-week-old male Wistar rats. In the absence of oxidative stress, incubation with 100 µM HT or GC for 5 min did not exert any vasorelaxing effect and did not influence the vascular function. Conversely, in condition of oxidative stress [upon incubation with 500 µM tert-butylhydroperoxide (t-BHP) for 30 min], preincubation with HT or GC improved acetylcholine-induced vasorelaxation compared with untreated samples (no t-BHP). This protective effect was lost for GC, but not for HT, when a washing step (15 min) was introduced between preincubation with HT or GC and t-BHP addition, suggesting that only HT enters the cells. In agreement, bilitranslocase inhibition with 100 µM phenylmethanesulfonyl fluoride for 20 min reduced significantly HT, but not GC, effect on the vascular function upon stress induction. Moreover, GC protective effect (improvement of endothelium-dependent relaxation in response to acetylcholine) in oxidative stress conditions was reduced by preincubation of aorta rings with 300 µM D-saccharolactone to inhibit ß-glucuronidase, which can deconjugate polyphenols. Finally, only HT was detected by high-pressure liquid chromatography in aorta rings incubated with GC and t-BHP. These results suggest that, in conditions of oxidative stress, GC can be deconjugated into HT that is transported through the cell membrane by bilitranslocase to protect vascular function.

Secreted Compounds of the Probiotic Bacillus clausii Strain O/C Inhibit the Cytotoxic Effects Induced by Clostridium difficile and Bacillus cereus Toxins.

Although the use of probiotics based on Bacillus strains to fight off intestinal pathogens and antibiotic-associated diarrhea is widespread, the mechanisms involved in producing their beneficial effects remain unclear. Here, we studied the ability of compounds secreted by the probiotic Bacillus clausii strain O/C to counteract the cytotoxic effects induced by toxins of two pathogens, Clostridium difficile and Bacillus cereus, by evaluating eukaryotic cell viability and expression of selected genes. Coincubation of C. difficile and B. cereus toxic culture supernatants with the B. clausii supernatant completely prevented the damage induced by toxins in Vero and Caco-2 cells. The hemolytic effect of B. cereus was also avoided by the probiotic supernatant. Moreover, in these cells, the expression of rhoB, encoding a Rho GTPase target for C. difficile toxins, was normalized when C. difficile supernatant was pretreated using the B. clausii supernatant. All of the beneficial effects observed with the probiotic were abolished by the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF). Suspecting the involvement of a secreted protease in this protective effect, a protease was purified from the B. clausii supernatant and identified as a serine protease (M-protease; GenBank accession number Q99405). Experiments on Vero cells demonstrated the antitoxic activity of the purified protease against pathogen supernatants. This is the first report showing the capacity of a protease secreted by probiotic bacteria to inhibit the cytotoxic effects of toxinogenic C. difficile and B. cereus strains. This extracellular compound could be responsible, at least in part, for the protective effects observed for this human probiotic in antibiotic-associated diarrhea.

Treponema denticola invasion into human gingival epithelial cells.

Host cell invasion is important for periodontal pathogens in evading host defenses and spreading into deeper areas of the periodontal tissue. Treponema denticola has been implicated in a number of potentially pathogenic processes, including periodontal tissue penetration. Here we tested the ability of T. denticola strains to invade human gingival epithelial cells (HGEC). After 2 h infection, intracellular location of T. denticola cells was confirmed by confocal laser scanning microscopy (CLSM). Results from an antibiotic protection assay following [(3)H]uridine labeling indicated that invasion efficiency reached a maximum at 2 h after infection. Internalized T. denticola cells were still observed in HGEC at 24 h by CLSM. A dentilisin deficient mutant exhibited significantly decreased invasion (p < 0.05) compared with the wild-type strain. In inhibition assays, phenylmethylsulfonyl fluoride and metabolic inhibitors such as methyl-ß-cyclodextrin and staurosporine significantly reduced T. denticola invasion. Under CLSM, T. denticola colocalized with GM-1 ganglioside-containing membrane microdomains in a cholesterol-dependent manner. These results indicated that T. denticola has the ability to invade into and survive within HGECs. Dentilisin activity of T. denticola and lipid rafts on HGEC appear to play important roles in this process.

Phosphorylation of Fe65 amyloid precursor protein-binding protein in response to neuronal differentiation.

Fe65 is a brain enriched multi domain adaptor protein involved in diverse cellular functions. One of its binding partners is the amyloid-ß (Aß) precursor protein (APP), which after sequential proteolytic processing by secretases gives rise to the Alzheimer's Aß peptide. Fe65 binds to the APP intracellular domain (AICD). Several studies have indicated that Fe65 binding promotes the amyloidogenic processing of APP. It has previously been shown that expression of APP increases concomitantly with a shift of its processing to the non-amyloidogenic pathway during neuronal differentiation. In this study we wanted to investigate the effects of neuronal differentiation on Fe65 expression. We observed that differentiation of SH-SY5Y human neuroblastoma cells induced by retinoic acid (RA), the phorbol ester PMA, or the γ-secretase inhibitor DAPT resulted in an electrophoretic mobility shift of Fe65. Similar effects were observed in rat PC6.3 cells treated with nerve growth factor. The electrophoretic mobility shift was shown to be due to phosphorylation. Previous studies have shown that Fe65 phosphorylation can prevent the APP-Fe65 interaction. We propose that phosphorylation is a way to modify the functions of Fe65 and to promote the non-amyloidogenic processing of APP during neuronal differentiation.

Microbial degradation of linear peptides by strain B-9 of Sphingosinicella and its application in peptide quantification using liquid chromatography-mass spectrometry.

The bacterial strain Sphingosinicella sp. B-9 was originally discovered to have the ability to degrade cyanobacterial cyclic peptides (microcystins), and has three hydrolytic enzymes (MlrA, MlrB, and MlrC). The purpose of this study was to examine in detail the degradation of glucagon/vasoactive intestinal polypeptide (VIP) family peptides by B-9, and to investigate the substrate specificity of B-9 proteases and the possibility of using a B-9 protease as a novel protease for peptide quantification by using a surrogate peptide and mass spectrometry (MS). The effective use of inhibitors revealed the following hydrolytic capability of B-9: One of the B-9 proteases (presumably MlrB) that was not inhibited by ethylenediaminetetraacetic acid (EDTA) cleaved bioactive peptides into medium-sized peptides with broad selectivity, similar to neutral endopeptidase, and another protease that was not inhibited by phenylmethylsulfonyl fluoride (PMSF) corresponded to MlrC and cleaved the resulting medium-sized peptides to smaller peptides or amino acids. The former property was desirable to obtain a suitable surrogate peptide, which was used successfully to quantify peptide using liquid chromatography (LC)-MS. Thus, the present study verified that one of the B-9 proteases has broad cleavage selectivity and cleavage sites, not seen in commercially available proteases, and is applicable to protein and peptide quantification using LC-MS.

Neutrophil cathepsin G, but not elastase, induces aggregation of MCF-7 mammary carcinoma cells by a protease activity-dependent cell-oriented mechanism.

We previously found that a neutrophil serine protease, cathepsin G, weakens adherence to culture substrates and induces E-cadherin-dependent aggregation of MCF-7 human breast cancer cells through its protease activity. In this study, we examined whether aggregation is caused by degradation of adhesion molecules on the culture substrates or through an unidentified mechanism. We compared the effect of treatment with cathepsin G and other proteases, including neutrophil elastase against fibronectin- (FN-) coated substrates. Cathepsin G and elastase potently degraded FN on the substrates and induced aggregation of MCF-7 cells that had been subsequently seeded onto the substrate. However, substrate-bound cathepsin G and elastase may have caused cell aggregation. After inhibiting the proteases on the culture substrates using the irreversible inhibitor phenylmethylsulfonyl fluoride (PMSF), we examined whether aggregation of MCF-7 cells was suppressed. PMSF attenuated cell aggregation on cathepsin G-treated substrates, but the effect was weak in cells pretreated with high concentrations of cathepsin G. In contrast, PMSF did not suppress cell aggregation on elastase-treated FN. Moreover, cathepsin G, but not elastase, induced aggregation on poly-L-lysine substrates which are not decomposed by these enzymes, and the action of cathepsin G was nearly completely attenuated by PMSF. These results suggest that cathepsin G induces MCF-7 aggregation through a cell-oriented mechanism.

Phenylmethanesulfonyl fluoride, a serine protease inhibitor, suppresses naloxone-precipitated withdrawal jumping in morphine-dependent mice.

We have previously shown that intracerebroventricular (i.c.v.) administration of cysteine protease inhibitors suppresses naloxone-precipitated withdrawal jumping in morphine-dependent mice, presumably through the inhibition of dynorphin degradation (see (Tan-No, K., Sato, T., Shimoda, M., Nakagawasai, O., Niijima, F., Kawamura, S., Furuta, S., Sato, T., Satoh, S., Silberring, J., Terenius, L., Tadano, T., 2010. Suppressive effects by cysteine protease inhibitors on naloxone-precipitated withdrawal jumping in morphine-dependent mice. Neuropeptides 44, 279-283)). In the present study, we examined the effect of phenylmethanesulfonyl fluoride (PMSF), a serine protease inhibitor, on naloxone-precipitated withdrawal jumping in morphine-dependent mice. The doses of morphine (mg/kg per injection) were subcutaneously given twice daily for 2 days [day 1 (30) and day 2 (60)]. On day 3, naloxone (8 mg/kg) was intraperitoneally administered 3h after the final injection of morphine (60 mg/kg), and the number of jumps was immediately recorded for 20 min. Naloxone-precipitated withdrawal jumping was significantly suppressed by i.c.v. administration of PMSF (4 nmol), given 5 min before each morphine treatment during the induction phase, with none given on the test day. The expression of tissue plasminogen activator (tPA), a serine protease that converts plasminogen to plasmin, in the prefrontal cortex was significantly increased in morphine-dependent and -withdrawal mice, as compared with saline-treated mice. Moreover, trans-4-(aminomethyl)-cyclohexanecarboxylic acid (300 pmol), an antiplasmin agent, and (Tyr(1))-thrombin receptor activating peptide 7 (0.45 and 2 nmol), an antagonist of protease activated receptor-1 (PAR-1), significantly suppressed naloxone-precipitated withdrawal jumping. The present results suggest that PMSF suppresses naloxone-precipitated withdrawal jumping in morphine-dependent mice, presumably through the inhibition of activities of tPA and plasmin belonging to the serine proteases family, which subsequently activates PAR-1.

Cathepsin G induces cell aggregation of human breast cancer MCF-7 cells via a 2-step mechanism: catalytic site-independent binding to the cell surface and enzymatic activity-dependent induction of the cell aggregation.

Neutrophils often invade various tumor tissues and affect tumor progression and metastasis. Cathepsin G (CG) is a serine protease secreted from activated neutrophils. Previously, we have shown that CG induces the formation of E-cadherin-mediated multicellular spheroids of human breast cancer MCF-7 cells; however, the molecular mechanisms involved in this process are unknown. In this study, we investigated whether CG required its enzymatic activity to induce MCF-7 cell aggregation. The cell aggregation-inducing activity of CG was inhibited by pretreatment of CG with the serine protease inhibitors chymostatin and phenylmethylsulfonyl fluoride. In addition, an enzymatically inactive S195G (chymotrypsinogen numbering) CG did not induce cell aggregation. Furthermore, CG specifically bound to the cell surface of MCF-7 cells via a catalytic site-independent mechanism because the binding was not affected by pretreatment of CG with serine protease inhibitors, and cell surface binding was also detected with S195G CG. Therefore, we propose that the CG-induced aggregation of MCF-7 cells occurs via a 2-step process, in which CG binds to the cell surface, independently of its catalytic site, and then induces cell aggregation, which is dependent on its enzymatic activity.

Protease inhibitors decrease the resistance of Vitaceae to Plasmopara viticola.

Plasmopara viticola must successfully infect susceptible grapevine cultivars to complete its biological cycle. In resistant grapevine varieties, P. viticola is blocked by the activation of defense mechanisms; these defense mechanisms produce hypersensitive reactions, which are related to programmed cell death. In animals, programmed cell death is dependent on caspase activities. In plants, different caspase-like proteases assume the same functions. To examine the roles of caspase-like proteases in P. viticola-grapevine interactions, three varieties of grapevine with different levels of P. viticola resistance were chosen. These grapevine varieties were treated with either PMSF, a serine protease inhibitor, or E-64, a cysteine protease inhibitor. The development of the pathogen was followed microscopically, and the plant defense reactions were estimated through stilbene quantification. Both protease inhibitor treatments increased the infection rate in the resistant and immune varieties, diminished the production of toxic stilbenes and changed the level of the plants' susceptibility to the pathogen. In particular, after either protease treatment, the cultivar that was originally immune became resistant (hyphae and haustoria were observed), the resistant cultivar reached the level of a susceptible cultivar (sporulation was observed) and the susceptible cultivar became more sensitive (P. viticola colonized the entirety of the leaf mesophyll).

Effect of cholinergic drugs on the activity of basic carboxypeptidases in rat nervous tissue.

Effects of a single administration of cholinergic drugs (arecoline, atropine, nicotine, mecamylamine) on the activity of carboxypeptidase H and of phenylmethylsulfonyl fluoride-inhibited carboxypeptidase, which are involved in metabolism of neuropeptides, were studied in brain parts and the adrenal glands of rats. The enzyme activities were determined fluorimetrically using specific inhibitors and substrates. In the majority of cases the enzyme activities decreased, and this decrease was retained for at least 72 h. Changes in the activities of the studied enzymes depended on the type of cholinergic action, the nervous system part, and the time after the injection. The changes in activities of the studied carboxypeptidases are supposed to be a possible mechanism responsible for changes in the levels of neuropeptides under the influence of high doses of the drugs.

Serine protease inhibitor mediated peptide bond re-synthesis in diverse protein molecules.

Protease inhibitors have been extensively used in research to prevent unwanted degradation of proteins during purification and analysis. Here, we report a remarkable discovery of protease inhibitor mediated reformation of peptide bonds by the serine protease inhibitor, PMSF in a diverse set of proteolyzed molecules. Interestingly, the religation reaction in the presence of PMSF occurs in a very short time period and with very high yields of the religated product. We also investigate the plausible mechanism of such a reaction and demonstrate through biochemical studies and X-ray crystallography that proximity of reacting termini is essential for the feasibility of this reaction.

Polyisoprenylation potentiates the inhibition of polyisoprenylated methylated protein methyl esterase and the cell degenerative effects of sulfonyl fluorides.

The polyisoprenylation pathway incorporates a reversible step that metabolizes polyisoprenylated methylated proteins from the ester to the carboxylate form. Polyisoprenylated protein methyl transferase (PPMTase) catalyses the esterification whereas polyisoprenylated methylated protein methyl esterase (PMPMEase) hydrolyzes them. Significant changes in the balance between the two enzymes may alter polyisoprenylated protein function possibly resulting in disease. Previous studies show that PMPMEase is the serine hydrolase, Sus scrofa carboxylesterase. Its susceptibility to the nonspecific serine hydrolase inhibitor, phenylmethylsulfonyl fluoride (PMSF) paved the way for its use as a prototypical compound to design and synthesize a series of putative high affinity specific inhibitors of PMPMEase. Pseudo first-order kinetics revealed an over 680-fold increase in k(obs)/[I] values from PMSF (6 M(-1)-1s(-1)), S-phenyl (L-50, 180 M(-1)s(-1)), S-benzyl (L-51, 350 M(-1)s(-1)), S-trans, trans-farnesyl (L-28, 2000 M(-1)s(-1)), to S-trans-geranylated (L-23, 4100 M(-1)s(-1)) 2-thioethanesulfonyl fluorides. C10 S-alkyl substitution revealed a k(obs)/[I] value (1800 M(-1)s(-1)) that was 298 times greater than that for PMSF. The compounds induced the degeneration of human neuroblastoma SH-SY5Y cells with EC(50) values of 49, 130 and >1000 µM for L-28, L-23 and PMSF, respectively. The increased affinity with the polyisoprenyl derivatization is consistent with the observed substrate specificity and the reported hydrophobic nature of the active site. These results suggest that (1) PMPMEase is a key enzyme for polyisoprenylated protein metabolism, (2) regulation of its activity is essential for maintaining normal cell viability, (3) abnormal activities may be involved in degenerative diseases and cancers and (4) its specific inhibitors may be useful in combating cancers.

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.

Effect of serine-type protease of Candida spp. isolated from linear gingival erythema of HIV-positive children: critical factors in the colonization.

BACKGROUND: There are several kinds of oral soft tissue lesions that are common manifestations observed in human immunodeficiency virus (HIV)-infected children; for example, linear gingival erythema (LGE) that is a distinctive fiery red band along the margin of the gingivae. The etiology and pathogenesis of LGE are questionable, but a candidal origin has been suggested. Proteases are key virulence attributes produced by a variety of pathogenic fungi, including Candida. The objective of the present study is to identify the protease production in Candida species including, C. albicans (n=5), C. dubliniensis (n=1) and C. tropicalis (n=1), isolated directly from typical LGE lesions observed in six HIV-positive children, and also to test the effect of a serine protease inhibitor on the interaction of Candida spp. and epithelial cells in vitro. METHODS: The ability of Candida strains to release proteases in the culture supernatant fluids was visualized by gelatin-SDS-PAGE. Gel strips containing 30-fold concentrated supernatant (1.5×10(8) yeasts) were incubated at 37°C for 48 h in 50 mM sodium phosphate buffer, pH 5.5. The concentrated supernatants were also incubated with fibronectin, laminin, immunoglobulin G, bovine serum albumin and human serum albumin. The effect of serine protease inhibitor on the interaction of Candida spp. and epithelial cells (MA 104) was measured after pre-treatment of fungi with the inhibitor (phenylmethylsulphonyl fluoride, PMSF). RESULTS: All the extracellular proteases were completely inhibited by PMSF, identifying these activities as serine-type proteases. Interestingly, a common 62-kDa serine protease was observed in all Candida strains. The culture supernatants, rich in serine protease activities, cleaved several soluble proteinaceous substrates. Additionally, we demonstrated that pre-treatment of C. albicans, C. dubliniensis and C. tropicalis with PMSF diminished the interaction with epithelial cells. CONCLUSIONS: Collectively, our results show that Candida spp. isolated from LGE lesions produced and secreted serine proteases and these enzymes may be involved in the initial colonization events.

Discriminative stimulus properties of delta9-tetrahydrocannabinol (THC) in C57Bl/6J mice.

Primarily, rats have served as subjects in Delta(9)-tetrahydrocannabinol's (THC) discrimination studies although other species such as monkeys and pigeons have been used. While the introduction of the knockout and transgenic mice has vastly stimulated the study of the discriminative stimulus effects of drugs there is only a single published report of mice trained to discriminate THC. Thus, this study extended those results by providing a systematic replication that THC serves as an effective discriminative stimulus in mice and by further investigating the mechanisms of action involved in the THC discrimination model in the mouse. Male C57BL/6J mice were trained to discriminate 10 mg/kg THC from vehicle in 2-lever drug discrimination. THC fully and dose dependently substituted for itself. Cannabinoid indoles, except one with low cannabinoid CB(1) receptor affinity, substituted for THC. Anandamide failed to substitute for THC when administered alone but completely substituted when administered with the non-specific fatty acid amide hydrolase inhibitor, phenylmethylsulphonyl fluoride. As expected, nicotine failed to substitute for THC. Lastly, the cannabinoid CB(1) receptor antagonist rimonabant blocked THC's discriminative stimulus effects. Taken together these studies demonstrate THC's ability to produce discriminative stimulus effects as well as demonstrate its pharmacological specificity and mechanism of action in a two-lever drug discrimination mouse model.