Specific Synthetic Substrates Research Articles

The secretion of the pancreas upon intraduodenal administration of trypsin and trypsin with its specific synthetic substrate

Earlier in our laboratory, conclusions were drawn regarding the specific substrates alleviating the selective inhibitory effects of enzymes by interacting at the catalytic center of the enzyme with the substrate. This was evidenced by data using a specific trypsin inhibitor that blocked the inhibitory effect of intraduodenally and intravenously administered trypsin (1), along with our laboratory's findings of alleviating the selective inhibition of amylase secretion by intraduodenal administration of a specific pancreatic amylase inhibitor (2). However, both protein and lipid emulsion, as substrates, exhibited elements of nonspecific enzyme sorption in their alleviating action

CHARACTERISATION OF CHOLINESTERASE ACTIVITY FROM ASIAN SWAMP EEL; MONOPTERUS ALBUS, AND TESTING ITS CAPABILITIES AS A BIOSENSOR ON METAL ION CONTAMINATION / PENCIRIAN AKTIVITI KOLINESTERES DARI OTOT BELUT SAWAH; MONOPTERUS ALBUS, DAN PENGUJIAN KEUPAYAANNYA SEBAGAI BIOPENANDA TERHADAP PENCEMARAN ION LOGAM

The presence of high concentrations of heavy metals in the river impedes daily activities and damages the surrounding aquatic ecosystem. Continuous monitoring should be implemented to prevent continuous discharge resulting in increased levels of pollution over time. This study aims to determine the sensitivity of Monopterus albus muscle ChE to metal ions. Priorly, ChE was purified from M. albus muscle tissue using the ion exchange matrix, DEAE, with the yield percentage of 42.16% with the purification fold of 2.17. PTC was selected as a specific synthetic substrate with the highest concentration and lowest biomolecular constant at 145838 ± 7533 μM.min.-1mg.-1 and 0.26 mM, while optimal assay parameters were obtained at pH 7.5 at 20 ° C. Inhibition studies of metal ions involving Cr, Cd, Ag, As, Hg, Pb, Ni and Cu at the concentration of 10 mg/L. The activity of M. albus ChE was 56.61% inhibited by Hg2+ and the highest recorded compared to the other metals ion arranged in declining order; Cr <Cd = Ni <Cu <Pb <As <Ag. Overall, purified ChE from M. albus muscle has proven its ability to be applied as a biosensor that can be used for environmental monitoring programs.

Purification and biochemical characterization of fibrino(geno)lytic enzymes from tissues of Antarctic hydrobionts

Considering the continuing increase of morbidity and mortality rates associated with cardiovascular diseases, the search for novel compounds able to affect the hemostasis system is among the current trends of modern science and pharmacology. Fibrino(geno)lytic enzymes because of their role in dissolving blood clots as well as prevention of their formation attract special attention. The main goal of the current research was to develop the methodological approaches to obtain fibrino(geno)lytic enzymes from Antarctic hydrobionts and study their effects on the functioning of the hemostasis system. A complex approach which included affinity chromatography and size-exclusion chromatography was applied to isolate the fibrino(geno)lytic enzymes from the tissue of Antarctic nemertea (Parborlasia corrugatus), Antarctic sea urchin (Sterechinus neumayeri), and Antarctic sea star (Odontaster validus). The presence of proteolytic activity was monitored by zymographic technique. Fibrin(ogen)olytic activity was assessed by incubation of the samples with fibrinogen followed by 10% SDS-PAGE analysis. To test the substrate specificity of the enzymes, the chromogenic substrates such as H-D-Phe-Pip-Arg-pNA, pyroGlu-Pro-Arg-pNA, H-D-Val-Leu-Lys-pNA and Bz-IIe-Glu(γ-OR)-Gly-Arg-pNA were used. The influence of fib rino(geno)lytic enzymes on platelet aggregation was assessed in platelet-rich plasma. To analyze the effect of the fibrino(geno)lytic enzymes on coagulation the blood coagulation time was assessed. The obtained results clearly indicated the presence of enzymes with activity toward fibrinogen in the tissues of tested hydrobionts. Based on the results of SDS-PAGE and zymography the molecular weight of the fibrino(geno)lytic enzymes was in the range of 26–34 kDa. The fibrinogen cleavage pattern analyzed by SDS-PAGE revealed the susceptibility of fibrinogen chains to degradation by enzymes from tissues of Antarctic hydrobionts. The fibrino(geno)lytic enzymes from all tested hydrobionts cleaved preferentially the Aα-chain and more slowly the Bβ-chain of fibrinogen. The fibrino(geno)lytic enzymes mediated the significant prolongation of blood clotting time in chronometric tests and inhibition of ADP-induced platelet aggregation. The enzymes exhibit activity against chromogenic substrates, which was more expressed in case of pyroGlu-Pro-Arg-pNA — a specific synthetic substrate for activated protein C and factor XIa. The enzymes isolated from the tissues of Antarctic marine hydrobionts possess a fibrin(ogen)olytic activity and can be of medical interest as therapeutic agents in the treatment and prevention of thrombotic disorders.

Establishment of potent and specific synthetic substrate for dipeptidyl-peptidase 7

Bacterial dipeptidyl-peptidase (DPP) 7 liberates a dipeptide with a preference for aliphatic and aromatic penultimate residues from the N-terminus. Although synthetic substrates are useful for activity measurements, those currently used are problematic, because they are more efficiently degraded by DPP5. We here aimed to develop a potent and specific substrate and found that the kcat/Km value for Phe-Met-methylcoumaryl-7-amide (MCA) (41.40 ± 0.83 μM−1 s−1) was highest compared to Met-Leu-, Leu-Leu-, and Phe-Leu-MCA (1.06–3.77 μM−1 s−1). Its hydrolyzing activity was abrogated in a Porphyromonas gingivalis dpp7-knockout strain. Conclusively, we propose Phe-Met-MCA as an ideal synthetic substrate for DPP7.

Measurement of the Caspase-1-Like Activity of Vacuolar Processing Enzyme in Plants.

Caspase-like activities are essential to regulate programed cell death in plants. Although no caspase orthologous enzymes with aspartic acid specificity have been identified in plants, vacuolar processing enzyme (VPE) exhibits a caspase-1-like activity. In this chapter, we introduce two methods for the measurement of the caspase-1-like/VPE activity. These methods are based on the cleavage of caspase-1 specific synthetic substrates and on monitoring the active forms of VPE using a biotinylated-inhibitor blot analysis. Both methods are also adaptable to other plant caspase-like activities.

Trypsin from unicorn leatherjacket (Aluterus monoceros) pyloric caeca: purification and its use for preparation of fish protein hydrolysate with antioxidative activity

Fish proteases, especially trypsin, could be used to prepare fish protein hydrolysates with antioxidative activities. In this study, trypsin from the pyloric caeca of unicorn leatherjacket was purified by ammonium sulfate precipitation and soybean trypsin inhibitor (SBTI)-Sepharose 4B affinity chromatography. Hydrolysate from Indian mackerel protein isolate with different degrees of hydrolysis (20, 30 and 40% DH) was prepared using the purified trypsin, and antioxidative activities (1,1-diphenyl-2-picrylhydrazyl and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical-scavenging activities, ferric-reducing antioxidant power and ferrous-chelating activity) of the hydrolysate were determined. Trypsin was purified 26.43-fold with a yield of 13.43%. The purified trypsin had a molecular weight (MW) of 23.5 kDa and optimal activity at pH 8.0 and 55 °C. It displayed high stability in the pH range of 6.0-11.0 and was thermally stable up to 50 °C. Both SBTI (0.05 mmol L(-1)) and N-p-tosyl-L-lysine-chloromethylketone (5 mmol L(-1)) completely inhibited trypsin activity. Antioxidative activities of the hydrolysate from Indian mackerel protein isolate increased with increasing DH up to 40% (P < 0.05). Based on sodium dodecyl sulfate polyacrylamide gel electrophoresis, the hydrolysate with 40% DH had a MW lower than 6.5 kDa. The purified protease from unicorn leatherjacket pyloric caeca was identified as trypsin based on its ability to hydrolyze a specific synthetic substrate and the response to specific trypsin inhibitors. The purified trypsin could hydrolyze Indian mackerel protein isolate, and the resulting hydrolysate exhibited antioxidative activity depending on its DH.

Structure and Mechanism of Cysteine Peptidase Gingipain K (Kgp), a Major Virulence Factor of Porphyromonas gingivalis in Periodontitis

Cysteine peptidases are key proteolytic virulence factors of the periodontopathogen Porphyromonas gingivalis, which causes chronic periodontitis, the most prevalent dysbiosis-driven disease in humans. Two peptidases, gingipain K (Kgp) and R (RgpA and RgpB), which differ in their selectivity after lysines and arginines, respectively, collectively account for 85% of the extracellular proteolytic activity of P. gingivalis at the site of infection. Therefore, they are promising targets for the design of specific inhibitors. Although the structure of the catalytic domain of RgpB is known, little is known about Kgp, which shares only 27% sequence identity. We report the high resolution crystal structure of a competent fragment of Kgp encompassing the catalytic cysteine peptidase domain and a downstream immunoglobulin superfamily-like domain, which is required for folding and secretion of Kgp in vivo. The structure, which strikingly resembles a tooth, was serendipitously trapped with a fragment of a covalent inhibitor targeting the catalytic cysteine. This provided accurate insight into the active site and suggested that catalysis may require a catalytic triad, Cys(477)-His(444)-Asp(388), rather than the cysteine-histidine dyad normally found in cysteine peptidases. In addition, a 20-Å-long solvent-filled interior channel traverses the molecule and links the bottom of the specificity pocket with the molecular surface opposite the active site cleft. This channel, absent in RgpB, may enhance the plasticity of the enzyme, which would explain the much lower activity in vitro toward comparable specific synthetic substrates. Overall, the present results report the architecture and molecular determinants of the working mechanism of Kgp, including interaction with its substrates.

The assessment of cholinesterase from the liver of Puntius javanicus as detection of metal ions.

Crude extract of ChE from the liver of Puntius javanicus was purified using procainamide-sepharyl 6B. S-Butyrylthiocholine iodide (BTC) was selected as the specific synthetic substrate for this assay with the highest maximal velocity and lowest biomolecular constant at 53.49 µmole/min/mg and 0.23 mM, respectively, with catalytic efficiency ratio of 0.23. The optimum parameter was obtained at pH 7.5 and optimal temperature in the range of 25 to 30°C. The effect of different storage condition was assessed where ChE activity was significantly decreased after 9 days of storage at room temperature. However, ChE activity showed no significant difference when stored at 4.0, 0, and −25°C for 15 days. Screening of heavy metals shows that chromium, copper, and mercury strongly inhibited P. javanicus ChE by lowering the activity below 50%, while several pairwise combination of metal ions exhibited synergistic inhibiting effects on the enzyme which is greater than single exposure especially chromium, copper, and mercury. The results showed that P. javanicus ChE has the potential to be used as a biosensor for the detection of metal ions.

Using a Caesalpinia echinata Lam. protease inhibitor as a tool for studying the roles of neutrophil elastase, cathepsin G and proteinase 3 in pulmonary edema

Acute lung injury (ALI) is characterized by neutrophil infiltration and the release of proteases, mainly elastase (NE), cathepsin G (Cat G) and proteinase 3 (PR3), which can be controlled by specific endogenous inhibitors. However, inhibitors of these proteases have been isolated from different sources, including plants. For this study, CeEI, or Caesalpinia echinata elastase inhibitor, was purified from C. echinata (Brazil-wood) seeds after acetone fractionation, followed by ion exchange and reversed phase chromatographic steps. Characterization with SDS-PAGE, stability assays, amino acid sequencing and alignment with other protein sequences confirmed that CeEI is a member of the soybean Kunitz trypsin inhibitor family. Like other members of this family, CeEI is a 20 kDa monomeric protein; it is stable within a large pH and temperature range, with four cysteine residues forming two disulfide bridges, conserved amino acid residues and leucine-isoleucine residues in the reactive site. CeEI was able to inhibit NE and Cat G at a nanomolar range (with K(i)s of 1.9 and 3.6 nM, respectively) and inhibited PR3 within a micromolar range (K(i) 3.7 μM), leading to hydrolysis of specific synthetic substrates. In a lung edema model, CeEI reduced the lung weight and pulmonary artery pressure until 180 min after the injection of zymosan-activated polymorphonuclear neutrophils. In experiments performed in the presence of a Cat G and PR3, but not an NE inhibitor, lung edema was reduced only until 150 min and pulmonary artery pressure was similar to that of the control. These results confirm that NE action is crucial to edema establishment and progression. Additionally, CeEI appears to be a useful tool for studying the physiology of pulmonary edema and provides a template for molecular engineering and drug design for ALI therapy.

Acute myocardial infarction elevates serine protease activity in saliva of patients with periodontitis

There are indications that acute myocardial infarction (AMI) may have an effect on the oral environment, which is reflected in the expression of salivary and gingival proteinases. According to our knowledge, no studies have been carried out to investigate the effect of AMI on the activities of two major tissue-destructive serine protease and microbial effectors, elastase and cathepsin G, produced by oral fluid polymorphonuclear granulocytes (PMN). Therefore, we compared the activities of elastase and cathepsin G in saliva from patients with AMI and from systemically healthy subjects (non-AMI) with similar periodontal conditions. A total of 92 patients (47 AMI and 28 non-AMI patients with gingivitis or periodontitis, and 17 systemically and periodontally healthy subjects as a control group) were recruited. Clinical periodontal measurements were recorded, and stimulated whole-saliva samples were collected. The patients with AMI were clinically examined within 3-4 d after admission to the coronary care unit. The activities of saliva neutrophil elastase and cathepsin G were measured after collection, at specific time-points during incubation (from baseline to 23 h) by specific synthetic peptide substrate assays. The saliva of patients with AMI and periodontitis had a significant trend for the highest elastase activities among the study groups. Elastase and cathepsin G activities correlated significantly with each other in the AMI periodontitis group (r = 0.8, p < 0.01). In a logistic regression analysis, the level of salivary elastase activity associated significantly with periodontitis. AMI may be reflected in PMN serine protease elastase activity in saliva, despite its strong association with periodontitis.

Practical and theoretical characterization of Inga laurina Kunitz inhibitor on the control of Homalinotus coriaceus

Digestive endoprotease activities of the coconut palm weevil, Homalinotus coriaceus (Coleoptera: Curculionidae), were characterized based on the ability of gut extracts to hydrolyze specific synthetic substrates, optimal pH, and hydrolysis sensitivity to protease inhibitors. Trypsin-like proteinases were major enzymes for H. coriaceus, with minor activity by chymotrypsin proteinases. More importantly, gut proteinases of H. coriaceus were inhibited by trypsin inhibitor from Inga laurina seeds. In addition, a serine proteinase inhibitor from I. laurina seeds demonstrated significant reduction of growth of H. coriaceus larvae after feeding on inhibitor incorporated artificial diets. Dietary utilization experiments show that 0.05% I. laurina trypsin inhibitor, incorporated into an artificial diet, decreases the consumption rate and fecal production of H. coriaceus larvae. Dietary utilization experiments show that 0.05% I. laurina trypsin inhibitor, incorporated into an artificial diet, decreases the consumption rate and fecal production of H. coriaceus larvae. We have constructed a three-dimensional model of the trypsin inhibitor complexed with trypsin. The model was built based on its comparative homology with soybean trypsin inhibitor. Trypsin inhibitor of I. laurina shows structural features characteristic of the Kunitz type trypsin inhibitor. In summary, these findings contribute to the development of biotechnological tools such as transgenic plants with enhanced resistance to insect pests.

Affinity 2009

Affinity 2009

Characterization of the proteases in the midgut of the xylophagous larvae of Oemona hirta (Coleoptera: Cerambycidae)

Abstract The protein digestive capability of the larvae of the longhorn beetle (Oemona hirta, Coleoptera: Cerambycidae, Fabricius, 1775) was investigated. This species feeds only on wood where there is a high proportion of vascular tissue. The pH of the midgut, the major digestive organ, was alkaline and protein hydrolysis was maximal at alkaline pH. Use of specific synthetic peptide substrates showed that the major protease activities were the endopeptidases, trypsin and chymotrypsin‐like activity, and the exopeptidase, leucine aminopeptidase and the pH curves corresponded to that with protein substrate. Studies using a range of serine protease inhibitors as well as specific inhibitors of metalloproteases, cysteine proteases and aspartate proteases confirmed a serine protease‐based digestive system similar to earlier reports of sapwood‐feeding Cerambycids. Control of these insect pests using protease inhibitors is discussed.

Identification of a cysteine proteinase from Jumbo squid ( Dosidicus gigas) hepatopancreas as cathepsin L

A cysteine proteinase from Jumbo squid ( Dosidicus gigas) hepatopancreas was partially purified by a two step procedure involving ammonium sulfate precipitation and gel filtration chromatography and further by SDS–PAGE. The molecular weight of the proteinase was 24 kDa determined by SDS–PAGE and 23.7 kDa with mass spectrometry. The activity had an optimum pH of 4.5 and optimum temperature of 55 °C under the assay for cathepsin L specific synthetic substrate Z-PAAFC. The cathepsin B and H specific synthetic substrates Z-AAAFC and H-AMC did not show any hydrolysis with the partially purified enzyme. Peptide mapping of trypsin digests of the 24 kDa band from SDS–PAGE showed the squid cysteine proteinase was homologous to cathepsin L from different animal sources. The activity of the partially purified fraction with the cathepsin L specific substrate Z-PAAFC was inhibited 75–89% by enzyme inhibitors specific for cysteine proteinases but was also significantly inhibited by serine and aspartate proteinase inhibitors.

CONTINUOUS INFUSION OF SIVELESTAT SODIUM HYDRATE PREVENTS LIPOPOLYSACCHARIDE‐INDUCED INTESTINAL PARALYSIS AND HYPOTENSION IN CONSCIOUS GUINEA‐PIGS

1. Sivelestat sodium hydrate (sivelestat), a neutrophil elastase inhibitor, is used to treat acute lung injury associated with systemic inflammatory response syndrome, but its effects have not been described for endotoxaemia. In the present study, we examined the effects of a continuous infusion of sivelestat on intestinal mechanical activity and blood pressure using an endotoxaemic model in conscious, unrestrained guinea-pigs. 2. Guinea-pigs underwent laparotomy while anaesthetized and were implanted with a force transducer sutured onto the taenia caecum. With this transducer, changes in tension in the intestinal longitudinal muscle were measured continuously via telemetry. Catheters were inserted into the carotid artery and jugular vein, were tunnelled subcutaneously and were accessed from the back of the neck. These catheters were connected to a cannula swivel and were used to monitor arterial pressure as well as to administer drugs i.v. in conscious, unrestrained guinea-pigs. Twenty hours after surgery, guinea-pigs received a single dose of lipopolysaccharide (LPS; 0.3 mg/kg, i.p.) 10 min after the start of a continuous 2 h i.v. infusion of sivelestat (30 mg/kg per h) or vehicle (saline). Elastase activity before and after sivelestat or vehicle administration was measured spectrometrically using a specific synthetic substrate. 3. We confirmed that intestinal longitudinal muscle tension decreased 2-3 h after LPS administration in the control group, with a concurrent decline in blood pressure. In guinea-pigs treated with sivelestat, the LPS-induced decreases in muscle tension and blood pressure were significantly reduced. In LPS-treated control guinea-pigs, serum elastase activity was elevated and this increase was significantly attenuated by administration of sivelestat. 4. The findings from the present study suggest that sivelstat can effectively reduce intestinal dysfunction and attenuate LPS-induced decreases in blood pressure in endotoxaemia.

Improved methods in the identification and screening for antimicrobial resistance: chromogenic screening for Staphylococcus aureus and methicillin resistance

New enzyme specific synthetic chromogenic substrates offer an advance in the laboratory screening and detection of Staphylococcus aureus and methicillin-resistant S. aureus (MRSA), with increased efficiency, reduced labour costs and decreased turn around times, especially when incorporated into existing laboratory workflows, to support current methods of microbial identification and the detection of resistance.

Expression and biochemical characterization of nsP2 cysteine protease of Chikungunya virus

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes epidemic fever, rash and polyarthralgia in Africa and Asia. Although it is known since the 1950s, new epidemiological and clinical features reported during the recent outbreak in the Indian Ocean can be regarded as the emergence of a new disease. Numerous severe forms of the infection have been described that put emphasis on the lack of efficient antiviral therapy. Among the virus-encoded enzymes, nsP2 constitutes an attractive target for the development of antiviral drugs. It is a multifunctional protein of approximately 90kDa with a helicase motif in the N-terminal portion of the protein while the papain-like protease activity resides in the C-terminal portion. The nsP2 proteinase is an essential enzyme whose proteolytic activity is critical for virus replication.In this work, a recombinant CHIKV nsP2pro and a C-terminally truncated variant were expressed in Escherichia coli and purified by metal–chelate chromatography. The enzymatic properties of the proteinase were then determined using specific synthetic fluorogenic substrates. This study constitutes the first characterization of a recombinant CHIKV nsP2 cysteine protease, which may be useful for future drug screening.

Mechanism of Activation of Caspase-9 and Caspase-3 during Hypoxia in the Cerebral Cortex of Newborn Piglets: The Role of Nuclear Ca2+-Influx

In previous studies, we have shown that cerebral hypoxia results in increased activity of caspase-9, the initiator caspase, and caspase-3, the executioner of programmed cell death. We have also shown that cerebral hypoxia results in high affinity Ca2+-ATPase-dependent increase in nuclear Ca2+ -influx in the cerebral cortex of newborn piglets. The present study tests the hypothesis that inhibiting nuclear Ca2+ -influx by pretreatment with clonidine, an inhibitor of high affinity Ca2+ -ATPase, will prevent the hypoxia-induced increase in caspase-9 and caspase-3 activity in the cerebral cortex of newborn piglets. Thirteen newborn piglets were divided into three groups, normoxic (Nx, n=4), hypoxic (Hx, n=4), and hypoxic treated with clonidine (100 mg/kg) (Hx-Cl, n=5). Anesthetized, ventilated animals were exposed to an FiO2 of 0.21 (Nx) or 0.07 (Hx) for 60 min. Cerebral tissue hypoxia was documented biochemically by determining levels of ATP and phosphocreatine (PCr). Caspase-9 and -3 activity were determined spectrofluoro-metrically using specific fluorogenic synthetic substrates. ATP (micromoles/g brain) was 4.6 +/- 0.3 in Nx, 1.7 +/- 0.4 in Hx (P < 0.05 vs. Nx), and 1.5 +/- 0.2 in Hx-Cl (P < 0.05 vs. Nx). PCr (micromoles/g brain) was 3.6 +/- 0.4 in Nx, 1.1 +/- 0.3 in Hx (P < 0.05 vs. Nx), and 1.0 +/- 0.2 in Hx-Cl (P < 0.05 vs. Nx). Caspase-9 activity (nmoles/mg protein/h) was 0.548 +/- 0.0642 in Nx and increased to 0.808 +/- 0.080 (P < 0.05 vs. Nx and Hx-Cl) in the Hx and 0.562 +/- 0.050 in the Hx-Cl group (p = NS vs. Nx). Caspase-3 activity (nmoles/mg protein/h) was 22.0 +/- 1.3 in Nx and 32 +/- 6.3 in Hx (P < 0.05 vs. Nx) and 18.8 +/- 3.2 in the Hx-Cl group (P < 0.05 vs. Hx). The data demonstrate that clonidine administration prior to hypoxia prevents the hypoxia-induced increase in the activity of caspase-9 and caspase-3. We conclude that the high afinity Ca2+ -ATPase-dependent increased nuclear Ca2+ during hypoxia results in increased caspase-9 and caspase-3 activity.

Effect of E. faecalis on the release of serine proteases elastase and cathepsin G, and collagenase‐2 (MMP‐8) by human polymorphonuclear leukocytes (PMNs)

To measure the release of hydrolytic enzymes [elastase, cathepsin G and collagenase-2 (MMP-8)] from human polymorphonuclear leukocytes (PMNs) during interaction with strains of Enterococcus faecalis isolated from endodontic infections. Six E. faecalis strains isolated from treatment resistant cases of apical periodontitis were included in the study. Overnight cultures of the microbes were used for the experiments. PMNs were isolated using the Ficoll Paque technique, and their vitality was assessed throughout the experiments by the Trypan Blue exclusion test. A known amount of microbes and PMNs were mixed in PBS at +37 degrees C in air, and the release of elastase, cathepsin G and MMP-8 was measured at 0, 20, 60 and 120 min after initiation of incubation. The activities of elastase and cathepsin G were analysed by spectrophotometer assays using specific synthetic peptide substrates, and MMP-8 by western immunoblotting quantitated by computer densitometry. PMNs incubated in buffer without any added microbes served as negative controls, cells incubated with 5 ng mL(-1) phorbol myristic acetate (PMA) served as a positive control. The 95% confidence interval was used to compare the relative amount of elastase and cathepsin G released from the samples. One E. faecalis strain induced a similar or higher elastase, cathepsin G and MMP-8 release than the positive control, whereas the other five strains induced only moderate or no release of the three enzymes examined as compared with the negative and positive controls. Western immunoblot revealed that released MMP-8 had molecular sizes of 60 and 75 kDa representing active and latent forms of MMP-8. In addition, >110 kDa high molecular size and a fragmented 20-30 kDa MMP-8 species could be observed. The majority of the E. faecalis strains induced little or no release of hydrolytic enzymes from the PMN cells. The finding may partly explain the clinical observation that root canal infections dominated by E. faecalis are usually symptom free.

Lipase production by yeasts from extra virgin olive oil

Newly produced olive oil has an opalescent appearance due to the presence of solid particles and micro-drops of vegetation water from the fruits. Some of our recent microbiological research has shown that a rich micro-flora is present in the suspended fraction of the freshly produced olive oil capable of improving the quality of the oil through the hydrolysis of the oleuropein. Present research however has, for the first time, demonstrated the presence of lipase-positive yeasts in some samples of extra virgin olive oil which can lower the quality of the oil through the hydrolysis of the triglycerides. The tests performed with yeasts of our collection, previously isolated from olive oil, demonstrated that two lipase-producing yeast strains named Saccharomyces cerevisiae 1525 and Williopsis californica 1639 were able to hydrolyse different specific synthetic substrates represented by p-nitrophenyl stearate, 4-nitrophenyl palmitate, tripalmitin and triolein as well as olive oil triglycerides. The lipase activity in S. cerevisiae 1525 was confined to the whole cells, whereas in W. californica 1639 it was also detected in the extracellular fraction. The enzyme activity in both yeasts was influenced by the ratio of the aqueous to the organic phase reaching its maximum value in S. cerevisiae 1525 when the water added to the olive oil was present in a ratio of 0.25% (v/v), whereas in W. californica 1639 the optimal ratio was 1% (v/v). Furthermore, the free fatty acids of olive oil proved to be good inducers of lipase activity in both yeasts. The microbiological analysis carried out on commercial extra virgin olive oil, produced in four different geographic areas, demonstrated that the presence of lipase-producing yeast varied from zero to 56% of the total yeasts detected, according to the source of oil samples. The discovery of lipase-positive yeasts in some extra virgin olive oils leads us to believe that yeasts are able to contribute in a positive or negative way towards the organological quality of the olive oil.