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Thrombosis is the formation of abnormal blood clots in the blood vessels that obstruct blood flow and lead to thrombosis. Current treatments for thrombosis are associated with serious side effects. Therefore there is a need for alternative natural therapy. A fibrinolytic protease was isolated from fresh leaves of Moringa oleifera Lam. and characterized for its potential to solubilize blood clots and hydrolyse fibrin under in-vitro conditions. The isolated protease showed a single protein band on native-PAGE. It showed optimum fibrinolytic activity at pH 8.0, 37 oC with 50 µg protein. The fibrinolytic activity of isolated protease was also confirmed by fibrin zymography. Km and Vmax of isolated protease were determined by the Lineweaver Burk plot. The isolated protease could solubilize 96.41% of blood clots by 96 h under in-vitro conditions. In-vitro fibrin hydrolysis and blood clot solubilization activities shown by an isolated protease from leaves of Moringa oleifera Lam. suggest its fibrinolytic potential to dissolve blood clots. Being a natural molecule and from a dietary plant it can be explored as an alternative natural therapy against thrombosis. [ABSTRACT FROM AUTHOR]

Mannheimia haemolytica is the main etiological bacterial agent in ruminant respiratory disease. M. haemolytica secretes leukotoxin, lipopolysaccharides, and proteases, which may be targeted to treat infections. We recently reported the purification and in vivo detection of a 110 kDa Zn metalloprotease with collagenase activity (110-Mh metalloprotease) in a sheep with mannheimiosis, and this protease may be an important virulence factor. Due to the increase in the number of multidrug-resistant strains of M. haemolytica, new alternatives to antibiotics are being explored; one option is lactoferrin (Lf), which is a multifunctional iron-binding glycoprotein from the innate immune system of mammals. Bovine apo-lactoferrin (apo-bLf) possesses many properties, and its bactericidal and bacteriostatic effects have been highlighted. The present study was conducted to investigate whether apo-bLf inhibits the secretion and proteolytic activity of the 110-Mh metalloprotease. This enzyme was purified and sublethal doses of apo-bLf were added to cultures of M. haemolytica or co-incubated with the 110-Mh metalloprotease. The collagenase activity was evaluated using zymography and azocoll assays. Our results showed that apo-bLf inhibited the secretion and activity of the 110-Mh metalloprotease. Molecular docking and overlay assays showed that apo-bLf bound near the active site of the 110-Mh metalloprotease, which affected its enzymatic activity. [ABSTRACT FROM AUTHOR]

Objectives: To evaluate the effect of proanthocyanidin-functionalized hydroxyapatite nanoparticles (nHAp_PA) used as pretreatment at different concentrations on the microtensile bond strength (µTBS) and endogenous enzymatic activity (MMPs) on pH-cycled dentin after 24 h and 6 months of artificial aging. Materials and methods: Fifty human sound dentin blocks were randomly assigned to 5 groups (n = 10): (i) negative control (no treatment); (ii) positive control (pH-cycling); (iii) pH-cycling + 2% nHAp_PA for 60s; (iv) pH-cycling + 6.5% nHAp_PA for 60s; (v) pH-cycling + 15% nHAp_PA for 60s. A self-etch adhesive was used for bonding procedures before resin composite build-ups. Specimens were tested with the µTBS test after 24 h and 6 months of laboratory storage. The proteolytic activity in each group was evaluated with gelatin zymography and in situ zymography. Data were statistically analyzed (p < 0.05). Results: At 24 h, the µTBS of the experimental groups were significantly higher than the controls (p ≤ 0.001), and no differences were observed between different concentrations (p > 0.05). Artificial aging significantly decreased bond strength in all groups (p ≤ 0.008); however, nHAp_PA 2% still yielded higher bonding values than controls (p ≤ 0.007). The groups pretreated with nHAp_PA exhibited lower MMP-9 and MMP-2 activities compared to the positive control group and almost the same enzymatic activity as the negative control group. In situ zymography showed that after 6 months of aging, nHAp_PA 2% and nHAp_PA 6,5% decreased enzymatic activity as well as the negative control. Conclusions: Dentin pretreatment with nHAp_PA increased the bonding performance of a self-etch adhesive and decreased MMP-2 and MMP-9 activities after 6 months. [ABSTRACT FROM AUTHOR]

Simple Summary: Insect hemolymph transports, exchanges, and eliminates soluble compounds from the hemocoel. These processes play a critical role in many physiological processes, including development and immunity. Since matrix metalloproteases (MMPs) are the major circulating gelatinases in the blood of various species, including mammals, we used gel zymography to analyze gelatinase and caseinase activities in Drosophila larval hemolymph under normal and pathological conditions. Our investigations demonstrate that the major gelatinases and caseinases in Drosophila larval hemolymph are not MMPs but serine proteases (SPs). We identified more than 60 SPs in these proteolytic active bands. While a role in immunity has been suggested for some of these circulating SPs, the physiological functions of most of them remain to be elucidated. After separation on gel zymography, Drosophila melanogaster hemolymph displays gelatinase and caseinase bands of varying sizes, ranging from over 140 to 25 kDa. Qualitative and quantitative variations in these bands were observed during larval development and between different D. melanogaster strains and Drosophila species. The activities of these Drosophila hemolymph gelatinase and caseinase were strongly inhibited by serine protease inhibitors, but not by EDTA. Mass spectrometry identified over 60 serine proteases (SPs) in gel bands corresponding to the major D. melanogaster gelatinases and caseinases, but no matrix metalloproteinases (MMPs) were found. The most abundant proteases were tequila and members of the Jonah and trypsin families. However, the gelatinase bands did not show any change in the tequila null mutant. Additionally, no clear changes could be observed in D. melanogaster gel bands 24 h after injection of bacterial lipopolysaccharides (LPS) or after oviposition by Leptopilina boulardi endoparasitoid wasps. It can be concluded that the primary gelatinases and caseinases in Drosophila larval hemolymph are serine proteases (SPs) rather than matrix metalloproteinases (MMPs). Furthermore, the gelatinase pattern remains relatively stable even after short-term exposure to pathogenic challenges. [ABSTRACT FROM AUTHOR]

Zymography is an electrophoretic method in which proteins are separated in a polyacrylamide gel in the presence of sodium dodecyl sulfate (SDS-PAGE). This method is used for the detection of enzymatic activity and molecular characterization of proteins. In contrast to the standard SDS-PAGE method, a substrate is incorporated into the gel during zymography, which is subsequently cleaved by target proteases. Many studies have focused on the development and progression of inflammatory diseases affecting the gastrointestinal tract, emphasizing the role of the largest group of proteases, matrix metalloproteinases (MMPs). The most used classification of this group of enzymes (by researchers in MMP biology) is based in part on the historical evaluation of the substrate specificity of MMPs and in part on the cellular localization of MMPs. MMPs are thus classified into the groups of collagenases, gelatinases, stromelysins, matrilysins, membrane-type MMPs (MT-MMPs), and others. An important group of MMPs are gelatinases which are involved in the breakdown of collagen type IV and gelatin of extracellular matrix and participate in the regulation of various physiological or pathological processes such as morphogenesis, angiogenesis, tissue repair, cirrhosis, arthritis, and metastasis. The present study's objective was to determine the amount of active MMP-9 and MMP-2 forms in tissue samples using zymography. The patient group was according to histology findings divided into the benign tumor (control) group (8 patients), and the malignant tumor group (24 patients). The respondents in the malignant tumor group were further divided according to the standard TNM classification. The results of this study confirmed that MMP-2, unlike MMP-9, can be used as a prognostic biomarker of CRC, because only the expression of active MMP-2 confirmed statistically significant differences between individual stages of CRC. Moreover, MMP-2 seems to play a more important role in higher stages of CRC. Substantial disparities in the determination of active MMPs between the observed groups support the assumption for the integration of zymography into clinical diagnostics of CRC together with molecular and other studies. [ABSTRACT FROM AUTHOR]

Purification of amylases from digestive tracts of three freshwater fish species with Q-Sepharose Fast Flow and Sephacryl S-200 columns displayed two isoforms of amylases from Osteochilus hasselti (O1, O2) and three isoforms of those from both Hampala dispar (UB, H1, H2) and Puntioplites proctozystron (P1, P2, P3). The optimum pH values displayed at 7.0 and 8.0, while the optimum temperatures revealed at 40 and 50 °C. Almost isoenzyme activities were activated by NaCl and CaCl2, whereas EDTA and SDS strongly inhibited all enzymatic activities. Verification with an atomic absorption spectrophotometry exhibited the presence of Ca2+ ions in the range of 0.02–13.53 ppm per mg protein indicating that amylases are Ca2+ dependent. Molecular weight analysis revealed 12 to 147 kDa. The UB, O1, and H2 amylases with appropriate molecular masses of 64, 49, and 25 kDa validated with LC-MS/MS were selected. Three certain enzymes revealed high stability in a sample buffer after five cycles of freeze-thawing process upon storage at − 20 °C for 12 weeks. No protein degradation was observed on polyacrylamide gel, and the enzymes still displayed sharp and clear bands on zymograms. The result suggested that the purified fish amylases, which expressed high activities and stabilities, were potentially used as enzyme molecular weight markers for zymography. [ABSTRACT FROM AUTHOR]

The aim of this study was to determine the activity of matrix metalloproteinase-2 and -9 in blood and tumour tissue of female dogs with benign and malignant mammary gland neoplasias. The increased activity of matrix metalloproteinase-2 and -9 was registered in 3.8–8.9% of canines with benign neoplasias (adenoma, mixed tumour) and in 32.5–63.5% of those with malignant neoplasias (carcinoma: mixed type, ductal, tubular, tubulopapillary). Neoplastic transformation is accompanied by blood level increase of both latent and active enzyme forms. The dogs with malignant mammary gland neoplasias were diagnosed with credible increase of matrix metalloproteinases-2 activity up to 1.59–1.96 in blood plasma and up to 21.57–24.84 in tumour tissue and the increase of matrix metalloproteinases-9 activity up to 2.16–2.67 and 29.53–35.26 respectively. For benign mammary neoplasms, the proportion of dogs with enhanced expression of matrix metalloproteinase-2 was higher than the number of patients in whom an increase in the level of matrix metalloproteinase-9 or both of these enzymes was registered by 1.7 times, for dogs with malignant tumours – in blood plasma – by 1.4–1.6 times, dogs with neoplastic changed functional tissue – 1.7 and 1.9 times, respectively. Histological type and metastatic foci presence did not correlate with enzymes’ activity. The enzymes’ activity figures in benign neoplasias fluctuated within those in clinically healthy animals. A positive characteristic of determining the plasma and tissue expression level of matrix metalloproteinases in dogs with mammary tumours is the low degree of invasiveness of the method against the background of the high informativeness of the results obtained in the preoperative period. The obtained results prove the possibility of using matrix metalloproteinases-2 and -9 to predict the course and to control the treatment of mammary neoplasia.

Purpose: To investigate the long-term effect of 0.05% or 0.1% caffeic acid phenethyl ester (CAPE) on dentin matrix stability and hybrid layer stability, using an etch-and-rinse (Adper Scotchbond Multipurpose/ASB) or a self-etch adhesive (Clearfil SE Bond/CSE). Materials and Methods: Dentin matrix specimens were assigned to five groups: 0.05% or 0.1% CAPE, green tea (GT), and the controls distilled water (DW) and dimethyl sulfoxide (DMSO). Following immersion of specimens for 1 h, modulus of elasticity (ME) and dentin mass change (MG) were determined at 3 post-treatment time points: immediately afterwards and at 3 and 6 months. Collagen solubilization (CS) was estimated by hydroxyproline (HYP) quantification. Resin-dentin interfaces with both adhesives were assessed with in situ zymography tests to evaluate gelatinolytic activity (GA). The dentin pretreatments were actively applied for 60 s. The sealing ability of aged resin-bonded slices was assessed by nanoleakage tests. Results: GT increased immediate ME, which decreased significantly after 3 months (p < 0.0001). The CAPE groups did not differ from the control groups. GT provided a significant increase in dentin matrix mass after treatment (p < 0.0001). No significant differences regarding MG were observed for CAPE 0.1%, CAPE 0.05%, DW, and DMSO groups after 3 and 6 months. Cumulative HYP release revealed that CAPE groups and GT were statistically similar to DW and DMSO; the GT group exhibited statistically significantly less HYP release than did CAPE groups (p = 0.0073). Treatment with 0.05% or 0.1% CAPE presented lower GA when applied to ASB before acid conditioning (p < 0.05), but no differences were detected when the CAPE groups were applied to CSE. CAPE at 0.1% significantly reduced nanoleakage for CSE, and 0.05% CAPE with CSE presented levels of nanoleakage similar to those of the CSE control group. Conclusion: CAPE at 0.05% or 0.01% did not influence ME, MG, or CS, but reduced GA when applied to ASB before acid conditioning. CAPE at 0.1% with CSE promoted adhesive layer integrity. [ABSTRACT FROM AUTHOR]

Understanding biophysical and biochemical processes in detritusphere is critical to quantifying and modeling plant residue decomposition dynamics and subsequent soil organic carbon (C) accrual. The objectives of the study were to explore (i) micro-environmental conditions within the detritusphere formed around decomposing corn and soybean leaves, and (ii) the relationships between moisture distribution, enzyme activity, and C dynamics during decomposition. We assessed spatial and temporal dynamics of moisture distribution using X-ray and neutron computed tomography, and activities of β-glucosidase and chitinase, two enzymes involved in soil C and N processing, using zymography. We used 13C labeled residue to track residue contribution to atmospheric CO2 and soil organic C. Moisture redistribution pattern varied depending on the residue type. While the water was immediately absorbed by the corn leaves and maintained afterward, switchgrass leaves absorbed water more slowly and created water-deficient zones within ∼1mm from the residue. This initial moisture depletion led to lower chitinase activity and residue-derived CO2 emissions in switchgrass. In contrast to chitinase, β-glucosidase activity was influenced by a combination of vegetation history and residue type, and it was higher when the origin of the residue matched the vegetation history of the soil. Pore size had an opposite impact on the studied enzymes, supporting the notion that contrasting soil pore architecture can stimulate activities of different enzymes through a selection of dominant enzyme producers. We concluded that the decomposition dynamics of plant residues is not only a simple function of residue chemistry, but rather a combined effect of the vegetation history, in part through its effect on microbial community composition, the plant residue chemical and likely physical characteristics, and the soil pore structure in the detritusphere. Together, they create temporally dynamic micro-environmental conditions influencing decomposition. Specifically, our study demonstrated that the initial micro-environment formulated in detritusphere can play an important role in enzyme activities and consequent C dynamics.

Gelatin zymography is widely used to detect gelatinase activity, which is performed on unfixed tissue because it is assumed that fixation inactivates enzymes. However, using fixed tissues has several advantages over using fresh tissues for such prevention of tissue decay, thereby preserving the proteins as well as the morphology and structure of the specimens. In this study, we investigated the effects of the four commonly used fixatives (ethanol, acetone, zinc-based fixative (ZBF), and paraformaldehyde (PFA)) on the gelatinolytic activity in mouse brain tissue. Multiple protocols were employed to extract proteins from the fixed brain tissue. Western blotting and in-gel zymography (IGZ) were used to detect the gelatinase proteins and gelatinolytic activity of the extractions, respectively. In situ zymography (ISZ) revealed that ethanol, acetone, ZBF, and short-time PFA fixation did not inhibit gelatinolytic activity. Neither 1% Triton + 1 M NaCl nor 10% DMSO + 1 M NaCl was effective in extracting proteins from ethanol-, acetone-, ZBF-, or PFA-fixed brain tissues. However, 8 M urea + 4% CHAPS effectively extracted gelatinase proteins from ethanol- and acetone-fixed tissues while retaining the gelatinolytic activity. 2% SDS effectively extracted gelatinase proteins from ethanol-, acetone-, and ZBF-fixed tissues while retaining the gelatinolytic activity. Although 2% SDS + heating extracted gelatinase proteins from ethanol-, acetone-, ZBF-, and even long-term PFA-fixed tissues, the gelatinolytic activity was not retained. Our findings suggest that both ISZ and IGZ can be performed on fixed brain tissue, which is anticipated to be an improvement over the conventionally used gelatin zymography methods. (J Histochem Cytochem 71: 481–493, 2023) [ABSTRACT FROM AUTHOR]

Matrix metalloproteinase (MMP) is a type of protease that degrades and removes the extracellular matrix of the cell. Only matrix metalloproteinase-1 (MMP-1) and matrix metalloproteinase-2 (MMP-2) are found in Drosophila, playing different roles in the fruit fly. Malathion is a lethal insecticide which mostly affects the neurological system of Drosophila. Hence, the objective of the study was to evaluate the toxicological effect of the organophosphate - Malathion - on MMP-1 and MMP-2 in 3rd instar larvae of Drosophila melanogaster using appropriate techniques. A total of 80 third instar larvae of Drosophila melanogaster were taken for our experiment (40 larvae for MMP-1 and 40 larvae for MMP-2). For each assessment with 40 larvae, 20 larvae were treated with LC50 dose of Malathion and 20 larvae were taken as control sample. Casein and gelatin zymography were run at room temperature to detect MMP-1 and MMP-2 expression in both control and treated larvae sample. Expressions of both matrix metalloproteinases in treated larvae changed substantially. The level of MMP-1 increased from 35.38% to 64.62%. MMP-2 level also rose from 38.37% to 61.63%. Our result implies that an LC50 dose of Malathion may activate oxidative stress and upregulate MMP-1 and MMP-2 levels in 3rd instar larvae of Drosophila. The deleterious outcome of Malathion could be seen in other non-target organisms, including pollinators and humans. Hence, the use of such compounds should be checked to minimize the risk of insecticide toxicity. [ABSTRACT FROM AUTHOR]

Differential expression of isozymes enables fish to tolerate temperature fluctuations in their environment. The present study explores the modulation of lactate dehydrogenase (LDH) and cytoplasmic malate dehydrogenase (sMDH) isozyme expression in the heart, muscle, brain, liver, gill, and kidney of juvenile Horabagrus brachysoma after 30 days of acclimation at 26, 31, 33, and 36℃. LDH and sMDH zymography were performed using native polyacrylamide gel electrophoresis. The zymography revealed five distinct bands of LDH isoenzymes (labelled from cathode to anode as LDHA4, LDH-A3B¹, LDH-A2B2, LDH-A1B3, and LDH-B4) and three distinct bands of sMDH isoenzymes (labelled from cathode to anode as sMDH-A2, sMDH-AB, and sMDH-B2), with considerable variation in their expression in the tissues. Acclimation to the test temperatures did not influence the expression patterns of LDH or sMDH isozymes. Densitometric analysis of individual isozyme bands revealed a reduction in the densities of bands containing the LDH-B and sMDH-B molecules, while the densities of bands containing the LDH-A and sMDH-A molecules increased in the gills and muscle, indicating the role of these organs in adaptive responses to thermal acclimation. However, the total densities of the LDH and sMDH isozymes increased with higher acclimation temperatures, indicating that adaptation to increased temperatures in H. brachysoma is primarily characterised by quantitative changes in isozyme expression. [ABSTRACT FROM AUTHOR]

Background: The aim of the study was to longitudinally evaluate the association between MMP-2, MMP-9, TIMP-1 and chest radiological findings in COVID-19 patients. Methods: COVID-19 patients were evaluated based on their hospital admission (baseline) and three months after hospital discharge (T post) and were stratified into ARDS and non-ARDS groups. As a control group, healthy donors (HD) were enrolled. Results: At the baseline, compared to HD (n = 53), COVID-19 patients (n = 129) showed higher plasma levels of MMP-9 (p < 0.0001) and TIMP-1 (p < 0.0001) and the higher plasma activity of MMP-2 (p < 0.0001) and MMP-9 (p < 0.0001). In the ARDS group, higher plasma levels of MMP-9 (p = 0.0339) and TIMP-1 (p = 0.0044) and the plasma activity of MMP-2 (p = 0.0258) and MMP-9 (p = 0.0021) compared to non-ARDS was observed. A positive correlation between the plasma levels of TIMP-1 and chest computed tomography (CT) score (ρ = 0.2302, p = 0.0160) was observed. At the T post, a reduction in plasma levels of TIMP-1 (p < 0.0001), whereas an increase in the plasma levels of MMP-9 was observed (p = 0.0088). Conclusions: The positive correlation between TIMP-1 with chest CT scores highlights its potential use as a marker of fibrotic burden. At T post, the increase in plasma levels of MMP-9 and the reduction in plasma levels of TIMP-1 suggested that inflammation and fibrosis resolution were still ongoing. [ABSTRACT FROM AUTHOR]

Previous studies have found that C turnover is bound to hotspots of microbial activity. The objective of this study was to analyze the effects of pure energy substrate (glucose), nutrient (mineral N or P) and combined substrate and nutrient (glucose + N, glucose + P, sterile DOC, artificial root exudate extract) additions to enzyme activity inside and outside hotspots as a proxy for microbial C turnover in a subsoil. By means of different substrate and nutrient additions, we tested how the limitations of our site were distributed on a small scale and depth-dependently to contribute to an increase in knowledge of subsoil mechanistics. The study site is a sandy Dystric Cambisol under an over 100-year-old beech forest stand in Lower Saxony, Germany. Forty-eight undisturbed soil samples from two depth increments (15–27 cm and 80–92 cm) of three profiles were sprayed homogeneously with easily available C, N and P sources to investigate the impacts of substrates and nutrients on three enzyme activities (acid phosphatase, β-glucosidase and N-acetylglucosaminidase) by using the soil zymography approach. Comparisons of upper and lower subsoils showed significantly fewer and smaller hotspots in the lower subsoil but with a high degree of spatial variation in comparison to the upper subsoil. Different patterns of enzyme distribution between upper and lower subsoil suggest microbial communities with a lower diversity are found in deeper soil regions of the site. Both substrate and nutrient additions stimulated enzyme activities significantly more outside the initial hotspots than within. Because of this, we conclude that microorganisms in the initial hotspots are less limited than in the surrounding bulk soil. Changes in enzyme activities owing to both substrate and nutrient addition were stronger in the lower subsoil than in the upper subsoil, showing differences in limitations and possible changes in microbial community structure with increasing depth. The results of our study emphasize the need to consider spatial factors in microbial turnover processes, especially in lower subsoil regions where stronger substrate and nutrient limitations occur. [ABSTRACT FROM AUTHOR]

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The aim of this study was to determine the activity of matrix metalloproteinase-2 and -9 in blood and tumour tissue of female dogs with benign and malignant mammary gland neoplasias. The increased activity of matrix metalloproteinase-2 and -9 was registered in 3.8–8.9% of canines with benign neoplasias (adenoma, mixed tumour) and in 32.5–63.5% of those with malignant neoplasias (carcinoma: mixed type, ductal, tubular, tubulopapillary). Neoplastic transformation is accompanied by blood level increase of both latent and active enzyme forms. The dogs with malignant mammary gland neoplasias were diagnosed with credible increase of matrix metalloproteinases-2 activity up to 1.59–1.96 in blood plasma and up to 21.57–24.84 in tumour tissue and the increase of matrix metalloproteinases-9 activity up to 2.16–2.67 and 29.53–35.26 respectively. For benign mammary neoplasms, the proportion of dogs with enhanced expression of matrix metalloproteinase-2 was higher than the number of patients in whom an increase in the level of matrix metalloproteinase-9 or both of these enzymes was registered by 1.7 times, for dogs with malignant tumours – in blood plasma – by 1.4–1.6 times, dogs with neoplastic changed functional tissue – 1.7 and 1.9 times, respectively. Histological type and metastatic foci presence did not correlate with enzymes’ activity. The enzymes’ activity figures in benign neoplasias fluctuated within those in clinically healthy animals. A positive characteristic of determining the plasma and tissue expression level of matrix metalloproteinases in dogs with mammary tumours is the low degree of invasiveness of the method against the background of the high informativeness of the results obtained in the preoperative period. The obtained results prove the possibility of using matrix metalloproteinases-2 and -9 to predict the course and to control the treatment of mammary neoplasia. [ABSTRACT FROM AUTHOR]

Featured Application: Porphyromonas gingivalis is an oral bacterium that uses gingipains as its primary invasion factor to cause periodontitis and certain systemic diseases in humans. To study the underlying mechanisms employed by gingipains, a simpler and quick approach is required to examine the protease activity. In the present study, passive diffusion-mediated protein elution and gelatin zymography methods were demonstrated to isolate and characterize the gingipains. This study found that an eluted protein with a molecular weight of about 180 kDa exhibited both Rgp and Kgp activity. While in the gelatin zymography, the proteins with a molecular weight of approximately 50 kDa, and above 245 kDa were found to be arginine-X gingipains when specific selective inhibitors, Leupeptin and Cathepsin B inhibitor II were used. The present study protocol will allow a more efficient separation of targeted protein samples with a greater difference in isoelectric point. Gingipains (RgpA, RgpB, and Kgp) are major virulence factors of the periodontitis-causing bacterium Porphyromonas gingivalis. Isolation of gingipains from the crude protein sample of P. gingivalis is critical for studying the underlying invasion mechanism that contributes to periodontitis, Alzheimer's disease, and cardiovascular disease (CVD). Chromatographic processes and molecular cloning are two standard techniques often used for gingipains isolation, which are time-consuming and costly. In this study, considerably easier methods based on passive-mediated diffusion gel elution and gelatin zymogram were used to isolate and characterize gingipains. Importantly, proteins eluted from Native-PAGE showed enzymatic activity for both Rgp and Kgp. In gelatin zymography, the proteins with a molecular size of ~50 kDa and above 245 kDa were suggested as arginine-specific gingipains. The passive diffusion-mediated gel elution method is a simpler technique to isolate gingipains from crude protein samples of P. gingivalis. By using covalent and highly specific gingipain inhibitors, gelatin zymography enabled an individual characterization of gingipain activity and inhibition. Finally, this protocol can be easily extended by adding the isoelectric focusing to further improve the protein separation and characterization. [ABSTRACT FROM AUTHOR]

Matrix metalloproteinase (MMP) is a type of protease that degrades and removes the extracellular matrix of the cell. Only matrix metalloproteinase-1 (MMP-1) and matrix metalloproteinase-2 (MMP-2) are found in Drosophila, playing different roles in the fruit fly. Malathion is a lethal insecticide which mostly affects the neurological system of Drosophila. Hence, the objective of the study was to evaluate the toxicological effect of the organophosphate ‒ Malathion ‒ on MMP-1 and MMP-2 in 3rd instar larvae of Drosophila melanogaster using appropriate techniques. A total of 80 third instar larvae of Drosophila melanogaster were taken for our experiment (40 larvae for MMP-1 and 40 larvae for MMP-2). For each assessment with 40 larvae, 20 larvae were treated with LC50 dose of Malathion and 20 larvae were taken as control sample. Casein and gelatin zymography were run at room temperature to detect MMP-1 and MMP-2 expression in both control and treated larvae sample. Expressions of both matrix metalloproteinases in treated larvae changed substantially. The level of MMP-1 increased from 35.38% to 64.62%. MMP-2 level also rose from 38.37% to 61.63%. Our result implies that an LC50 dose of Malathion may activate oxidative stress and upregulate MMP-1 and MMP-2 levels in 3rd instar larvae of Drosophila. The deleterious outcome of Malathion could be seen in other non-target organisms, including pollinators and humans. Hence, the use of such compounds should be checked to minimize the risk of insecticide toxicity.

After separation on gel zymography, Drosophila melanogaster hemolymph displays gelatinase and caseinase bands of varying sizes, ranging from over 140 to 25 kDa. Qualitative and quantitative variations in these bands were observed during larval development and between different D. melanogaster strains and Drosophila species. The activities of these Drosophila hemolymph gelatinase and caseinase were strongly inhibited by serine protease inhibitors, but not by EDTA. Mass spectrometry identified over 60 serine proteases (SPs) in gel bands corresponding to the major D. melanogaster gelatinases and caseinases, but no matrix metalloproteinases (MMPs) were found. The most abundant proteases were tequila and members of the Jonah and trypsin families. However, the gelatinase bands did not show any change in the tequila null mutant. Additionally, no clear changes could be observed in D. melanogaster gel bands 24 h after injection of bacterial lipopolysaccharides (LPS) or after oviposition by Leptopilina boulardi endoparasitoid wasps. It can be concluded that the primary gelatinases and caseinases in Drosophila larval hemolymph are serine proteases (SPs) rather than matrix metalloproteinases (MMPs). Furthermore, the gelatinase pattern remains relatively stable even after short-term exposure to pathogenic challenges.

ObjectiveThe mechanisms underlying cell and organ dysfunctions in hypertension are uncertain. The spontaneously hypertensive rat (SHR) has elevated levels of unchecked degrading proteases compared to the control Wistar Kyoto (WKY) rat. The extracellular proteases destroy membrane receptors leading to cell dysfunctions, including arteriolar constriction and elevated blood pressure. Our goal was to identify potential sources of the uncontrolled enzymatic activity.MethodsZymographic and digital immunohistochemical measurements in SHR pancreas and intestine were obtained as part of the digestive system with high levels of degrading enzymes.ObjectiveThe results showed that SHRs have significantly higher protease activity than WKY in pancreas (22.04 ± 9.01 vs 13.02 ± 3.92 casein fluorescence intensity unit; P < 0.05) and pancreatic venules (0.011 ± 0.003 vs 0.005 ± 0.003 trypsin absorbance; P < 0.05) as well as in venous blood (71.07 ± 13.92 vs 36.44 ± 16.59 casein fluorescence intensity unit; P < 0.05). The enzymatic activity is contributed by trypsin and chymotrypsin. Furthermore, a decrease of these enzyme activity levels achieved during a short-term fasting period is associated with a reduction in systolic blood pressurein SHR (135 ± 8 mm Hg vs 124 ± 7 mm Hg; P < 0.05).ConclusionsThe results suggest the pancreas of the SHR is a potential source for serine proteases leaking into the circulation and contributing to its protease activity.

Snake venom fibrinogenolytic enzymes have diagnostic and therapeutic value and are important for snakebite pathology. In the present study, the fibrinogenolytic activity of Montivipera raddei venom was investigated. Crude venom was incubated with human fibrinogen for different time periods at 37°C. An inhibition study was carried out using different protease inhibitors. The fibrinogenolytic activity was assessed by SDS-PAGE and fibrinogen zymography. An HPLCbased method was used to obtain confirmatory data. Montivipera raddei venom predominantly cleaved the Aα chain of fibrinogen in a time-dependent manner. A very slight decrease in band intensity of the Bβ chain was observable after a longer incubation time. Cleavage of fibrinogen was confirmed by HPLC. Zymography revealed that the venom contained 50 and 75 kDa fibrinogenolytic enzymes. Ethylenediaminetetraacetic acid (EDTA) and 1,10-phenanthroline inhibited the overall fibrinogenolytic activity, while phenylmethylsulfonyl fluoride (PMSF) only inhibited the degradation of the Bβ chain. These results indicated that metalloproteinases were major fibrinogenolytic enzymes in the venom. The inhibitor study suggested the presence of serine proteinases that broke down the Bβ chain. With this study, the fibrinogenolytic activity of M. raddei venom was shown for the first time. The results will be useful for further isolation and characterization studies.

The occurrence of the honeybee caste polyphenism arises when a change in diet is transduced into cellular metabolic responses, resulting in a developmental shift mediated by gene expression. The aim of this investigation was to detect and describe the expression profile of water-soluble proteases during the ontogenesis of honeybee worker-fate larvae. The extraction of insect homogenates was followed by the electrophoretic separation of the protein extract in polyacrylamide gels under semi-denaturing condition, precast with gelatin, pollen, or royal jelly protein extracts. The worker-fate honeybee larva showed a proteolytic pattern that varied with aging, and a protease with the highest activity at 72 h after hatching was named PS4. PS4 has a molecular weight of 45 kDa, it remained active until cell sealing, and its enzymatic properties suggest a serine-proteinase nature. To define the process that originates a queen-fate larvae, royal jelly and pollen were analysed, but PS4 was not detected in either of them. The effect of food on the PS4 was investigated by mixing crude extracts of queen and worker-fate larvae with pollen and royal jelly, respectively. Only royal jelly inhibited PS4 in worker-fate larvae. Taken together, our data suggest that PS4 could be involved in caste differentiation. [ABSTRACT FROM AUTHOR]

The aim of the present study was to evaluate the in situ/in vivo effect of quercetin on dentin erosion and abrasion. Human dentin blocks (2 × 2 × 2 mm) were embedded and assigned to 6 groups: 75 μg/mL, 150 μg/mL and 300 μg/mL quercetin (Q75, Q150, Q300); 120 μg/mL chlorhexidine (CHX, positive control); and deionized water and ethanol (the negative controls). The specimens were treated with the respective solutions for 2 min and then subjected to in situ /in vivo erosive/abrasive challenge for 7 d as follows: in vivo erosion 4 times a day and then in vivo toothbrush abrasion after the first and last erosive challenges of each day. Dentin loss was assessed by profilometry. An additional dentin specimen was used to evaluate the penetration depth of quercetin into dentin by tracking the spatial distribution of its characteristic Raman peak. Moreover, dentin blocks (7 × 1.7 × 0.7 mm) were used to detect the impact of quercetin on dentin-derived matrix metalloproteinase (MMP) inhibition by in situ zymography, and the inhibition percentage (%) was calculated. Additionally, the potential collagen crosslinking interactions with quercetin were detected by Raman spectroscopy, and the crosslinking degree was determined with a ninhydrin assay. Fully demineralized dentin beams (0.5 × 0.5 × 10 mm) were used to evaluate the impact of quercetin on the mechanical properties of dentin collagen fibre by the ultimate micro-tensile strength test (μUTS). The data were analysed by one-way analysis of variance and Tukey's test (α = 0.05). Compared to the negative controls, all treatment solutions significantly reduced dentin loss. The dentin loss of Q150 and Q300 was significantly less than that of CHX (all P < 0.05). The amount of quercetin decreased with increasing dentin depth, and the maximum penetration depth was approximately 25–30 µm. In situ zymography showed that quercetin significantly inhibited the activities of dentin-derived MMPs. The inhibitory percentages of Q75 and Q150 were significantly lower than that of CHX (all P < 0.05), but no significant difference was found between Q300 and CHX (P = 0.58). The collagen crosslinking interactions with quercetin primarily involved hydrogen bonding and the degree of crosslinking increased in a concentration-dependent manner. Statistically significant increases in μUTS values were observed for demineralized dentin beams after quercetin treatment compared with those of the control treatments (all P < 0.05). This study provides the first direct evidence that quercetin could penetrate approximately 25–30 µm into dentin and further prevent dentin erosion and abrasion by inhibiting dentin-derived MMP activity as well as crosslinking collagen of the demineralized organic matrix. [ABSTRACT FROM AUTHOR]

Recent policies and silvicultural management call for forest regeneration that involve the selection of tree species able to cope with low soil nutrient availability in forest ecosystems. Understanding the impact of different tree species on the rhizosphere processes (e.g., enzyme activities) involved in nutrient mobilisation is critical in selecting suitable species to adapt forests to environmental change. Here, we visualised and investigated the rhizosphere distribution of enzyme activities (cellobiohydrolase, leucine-aminopeptidase, and acid phosphomonoesterase) using zymography. We related the distribution of enzyme activities to the seedling root morphological traits of European beech (Fagus sylvatica) and Norway spruce (Picea abies), the two most cultivated temperate tree species that employ contrasting strategies in soil nutrient acquisition. We found that spruce showed a higher morphological heterogeneity along the roots than beech, resulting in a more robust relationship between rhizoplane-associated enzyme activities and the longitudinal distance from the root apex. The rhizoplane enzyme activities decreased in spruce and increased in beech with the distance from the root apex over a power-law equation. Spruce revealed broader rhizosphere extents of all three enzymes, but only acid phosphomonoesterase activity was higher compared with beech. This latter result was determined by a larger root system found in beech compared with spruce that enhanced cellobiohydrolase and leucine-aminopeptidase activities. The root hair zone and hair lengths were significant variables determining the distribution of enzyme activities in the rhizosphere. Our findings indicate that spruce has a more substantial influence on rhizosphere enzyme production and diffusion than beech, enabling spruce to better mobilise nutrients from organic sources in heterogeneous forest soils. [ABSTRACT FROM AUTHOR]

Viperidae snakes are the most important agents of snakebites in Brazil. The protein composition of snake venoms has been frequently analyzed by means of electrophoretic techniques, but the interaction of proteins in venoms has barely been addressed. An electrophoretic technique that has gained prominence to study this type of interaction is blue native polyacrylamide gel electrophoresis (BN-PAGE), which allows for the high-resolution separation of proteins in their native form. These protein complexes can be further discriminated by a second-dimension gel electrophoresis (SDS-PAGE) from lanes cut from BN-PAGE. Once there is no study on the use of bidimensional BN/SDS-PAGE with snake venoms, this study initially standardized the BN/SDS-PAGE technique in order to evaluate protein interactions in Bothrops atrox, Bothrops erythromelas, and Bothrops jararaca snake venoms. Results of BN/SDS-PAGE showed that native protein complexes were present, and that snake venom metalloproteinases and venom serine proteinases maintained their enzymatic activity after BN/SDS-PAGE. C-type lectin-like proteins were identified by Western blotting. Therefore, bidimensional BN/SDS-PAGE proved to be an easy, practical, and efficient method for separating functional venom proteins according to their assemblage in complexes, as well as to analyze their biological activities in further details. [ABSTRACT FROM AUTHOR]

Polyamide 4 (PA4) is expected to solve the issue of marine plastic pollution due to its excellent mechanical properties and biodegradability. In this study, to reveal the mechanism of PA4 biodegradation in the marine environment, we isolated 5 strains of PA4-degrading bacteria belonging to Aliiglaciecola, Dasania, and Pseudophaeobacter from a marine environment. The isolated 5 strains are novel PA4-degrading bacteria that are phylogenetically distinct from those isolated in previous studies. In addition, we compared the PA4-degrading activities and structures of the PA4-degrading enzymes secreted by the 5 strains and PA4-degrading strains isolated in our previous study. The PA4-degrading activity in the supernatant of the cultivation solutions differed among the strains. Native-PAGE and zymography using a polyacrylamide gel containing a PA4 emulsion demonstrated that PA4-degrading enzymes are classified into no less than three types of structures. These results suggested that marine PA4-degrading bacteria have multiple PA4-degrading enzymes. Our findings will contribute to a better understanding of the microbial degradation of PA4 in the marine environment.

The objective was to evaluate the effect of different root canal irrigating solutions on the activity of matrix metalloproteinases (MMPs), and correlation to the push-out bond strength (PBS) and nanoleakage expression (NL) in the root dentin. Seventy-two single-rooted teeth were treated endodontically and distributed into four groups (n = 6 for in-situ zymography, n = 10 for PBS, and n = 2 for NL per group) according to the irrigating solutions used: (I) saline (S); (II) 5.25% sodium hypochlorite (SH); (III) 5.25% SH + 10% citric acid (CA); and (IV) 5.25% SH + 10% CA + 0.2% chlorhexidine (CHX). After root canal obturation, post space was prepared to receive the glass fiber post. Dual-cure resin was used for luting and light polymerization was performed. The root/fiber post assemblies were sectioned and subjected to in situ zymography, and PBS and NL expression analysis tests. The enzymatic activity was quantified and expressed as a percentage of the green fluorescence, while fractographic evaluation was performed after PBS with a stereomicroscope, and data were statistically analyzed at p < 0.05. The zymography analysis shows high expression of MMPs in the middle third of the root in all groups, while the most abundant activity of MMPs following the irrigating solutions is observed in groups I and III, where saline and citric acid are used, respectively. Inversely, group IV, where chlorhexidine is the final rinse, records the lowest MMP activity with the highest PBS, and the statistical analysis of the groups are ranked as: IV > II > III > I (p < 0.05). The combination of SH, CA, and CHX results in lower expression of MMPs and higher push-out bond strength of fiber posts to root dentin, with no difference seen in the nanoleakage expression (p > 0.05); hence, this irrigation regime with chlorhexidine as a final rinse is more favorable than other combinations in ensuring optimal adhesion to root dentine. [ABSTRACT FROM AUTHOR]

Neurofilament light chain (NfL) is a specific biomarker of neuro-axonal damage. Matrix metalloproteinases (MMPs) are zinc-dependent enzymes involved in blood–brain barrier (BBB) integrity. We explored neuro-axonal damage, alteration of BBB integrity and SARS-CoV-2 RNA presence in COVID-19 patients with severe neurological symptoms (neuro-COVID) as well as neuro-axonal damage in COVID-19 patients without severe neurological symptoms according to disease severity and after recovery, comparing the obtained findings with healthy donors (HD). Overall, COVID-19 patients (n = 55) showed higher plasma NfL levels compared to HD (n = 31) (p < 0.0001), especially those who developed ARDS (n = 28) (p = 0.0005). After recovery, plasma NfL levels were still higher in ARDS patients compared to HD (p = 0.0037). In neuro-COVID patients (n = 12), higher CSF and plasma NfL, and CSF MMP-2 levels in ARDS than non-ARDS group were observed (p = 0.0357, p = 0.0346 and p = 0.0303, respectively). SARS-CoV-2 RNA was detected in four CSF and two plasma samples. SARS-CoV-2 RNA detection was not associated to increased CSF NfL and MMP levels. During COVID-19, ARDS could be associated to CNS damage and alteration of BBB integrity in the absence of SARS-CoV-2 RNA detection in CSF or blood. CNS damage was still detectable after discharge in blood of COVID-19 patients who developed ARDS during hospitalization. [ABSTRACT FROM AUTHOR]

Abstract Background Lignocellulolytic enzymes are essential for agricultural waste disposal and production of renewable bioenergy. Many commercialized cellulase mixtures have been developed, mostly from saprophytic or endophytic fungal species. The cost of complete cellulose digestion is considerable because a wide range of cellulolytic enzymes is needed. However, most fungi can only produce limited range of highly bioactive cellulolytic enzymes. We aimed to investigate a simple yet specific method for discovering unique enzymes so that fungal species producing a diverse group of cellulolytic enzymes can be identified. Results The culture medium of an endophytic fungus, Daldinia caldariorum D263, contained a complete set of cellulolytic enzymes capable of effectively digesting cellulose residues into glucose. By taking advantage of the unique product inhibition property of β-glucosidases, we have established an improved zymography method that can easily distinguish β-glucosidase and exoglucanase activity. Our zymography method revealed that D263 can secrete a wide range of highly bioactive cellulases. Analyzing the assembled genome of D263, we found over 100 potential genes for cellulolytic enzymes that are distinct from those of the commercially used fungal species Trichoderma reesei and Aspergillus niger. We further identified several of these cellulolytic enzymes by mass spectrometry. Conclusions The genome of Daldinia caldariorum D263 has been sequenced and annotated taking advantage of a simple yet specific zymography method followed by mass spectrometry analysis, and it appears to encode and secrete a wide range of bioactive cellulolytic enzymes. The genome and cellulolytic enzyme secretion of this unique endophytic fungus should be of value for identifying active cellulolytic enzymes that can facilitate conversion of agricultural wastes to fermentable sugars for the industrial production of biofuels.

The aim of this work was to study the composition and functions of intracellular and extracellular proteases of the production Vibrio cholerae strains 569B serovar Inaba and M41 serovar Ogawa using zymographic and proteomic analysis.Materials and methods. Samples of intracellular proteases were obtained from cell lysates by ultrasonication of bacterial cells in a 9 M urea solution. The extracellular protease fraction was precipitated from the culture liquid by adding 50 % trichloroacetic acid to a final concentration of 10 % and incubating on ice. Lyophilized preparations of proteinase K and proteovibrin enzyme complex were used as a control of proteolytic activity. Proteases were detected by substrate gel electrophoresis in 12.5 % polyacrylamide gel impregnated with 0.1 % gelatin, followed by identification of the composition of protein fractions of lysates and exoproteins of both strains using molecular mass spectrometric scanning.Results and discussion. A comparative study of the production strains of V. cholerae 569B serovar Inaba and M41 serovar Ogawa using zymographic and proteomic analysis showed that the greatest enzymatic activity was detected in the fraction of extracellular proteases sample of V. cholerae M41 strain, where five major and four minor zones of gelatin hydrolysis were identified, and high-intensity zones with MW 20–23 and 37–40 kDa were also found in the preparation of proteovibrin isolated from the culture fluid of that strain. As a result of proteomic analysis of the studied strains, 66 enzymes of V. cholerae with different functional activity were reliably identified, among which 15 enzymes had protease activity. The high information content of the complex of modern methods provided for the possibility of identifying qualitative and quantitative differences in the composition of intracellular and extracellular proteases in production strains of V. cholerae, which offers an effective means of screening inter-strain differences in the protease spectrum in production strains.

Background: The aim of the study was to longitudinally evaluate the association between MMP-2, MMP-9, TIMP-1 and chest radiological findings in COVID-19 patients. Methods: COVID-19 patients were evaluated based on their hospital admission (baseline) and three months after hospital discharge (T post) and were stratified into ARDS and non-ARDS groups. As a control group, healthy donors (HD) were enrolled. Results: At the baseline, compared to HD (n = 53), COVID-19 patients (n = 129) showed higher plasma levels of MMP-9 (p < 0.0001) and TIMP-1 (p < 0.0001) and the higher plasma activity of MMP-2 (p < 0.0001) and MMP-9 (p < 0.0001). In the ARDS group, higher plasma levels of MMP-9 (p = 0.0339) and TIMP-1 (p = 0.0044) and the plasma activity of MMP-2 (p = 0.0258) and MMP-9 (p = 0.0021) compared to non-ARDS was observed. A positive correlation between the plasma levels of TIMP-1 and chest computed tomography (CT) score (ρ = 0.2302, p = 0.0160) was observed. At the T post, a reduction in plasma levels of TIMP-1 (p < 0.0001), whereas an increase in the plasma levels of MMP-9 was observed (p = 0.0088). Conclusions: The positive correlation between TIMP-1 with chest CT scores highlights its potential use as a marker of fibrotic burden. At T post, the increase in plasma levels of MMP-9 and the reduction in plasma levels of TIMP-1 suggested that inflammation and fibrosis resolution were still ongoing.

Previous studies have found that C turnover is bound to hotspots of microbial activity. The objective of this study was to analyze the effects of pure energy substrate (glucose), nutrient (mineral N or P) and combined substrate and nutrient (glucose + N, glucose + P, sterile DOC, artificial root exudate extract) additions to enzyme activity inside and outside hotspots as a proxy for microbial C turnover in a subsoil. By means of different substrate and nutrient additions, we tested how the limitations of our site were distributed on a small scale and depth-dependently to contribute to an increase in knowledge of subsoil mechanistics. The study site is a sandy Dystric Cambisol under an over 100-year-old beech forest stand in Lower Saxony, Germany. Forty-eight undisturbed soil samples from two depth increments (15–27 cm and 80–92 cm) of three profiles were sprayed homogeneously with easily available C, N and P sources to investigate the impacts of substrates and nutrients on three enzyme activities (acid phosphatase, β-glucosidase and N-acetylglucosaminidase) by using the soil zymography approach. Comparisons of upper and lower subsoils showed significantly fewer and smaller hotspots in the lower subsoil but with a high degree of spatial variation in comparison to the upper subsoil. Different patterns of enzyme distribution between upper and lower subsoil suggest microbial communities with a lower diversity are found in deeper soil regions of the site. Both substrate and nutrient additions stimulated enzyme activities significantly more outside the initial hotspots than within. Because of this, we conclude that microorganisms in the initial hotspots are less limited than in the surrounding bulk soil. Changes in enzyme activities owing to both substrate and nutrient addition were stronger in the lower subsoil than in the upper subsoil, showing differences in limitations and possible changes in microbial community structure with increasing depth. The results of our study emphasize the need to consider spatial factors in microbial turnover processes, especially in lower subsoil regions where stronger substrate and nutrient limitations occur.

Abstract Laccases belong to a family of multicopper enzymes able to oxidize a broad spectrum of organic compounds. Despite the well‐known property of laccases to carry out bleaching and degradation of industrial dyes and polyphenolic compounds, their industrial use is often limited by the high cost, low efficiency, or instability of these enzymes. To look for new microorganisms which produce laccases that are potentially suitable for industrial applications, we have isolated several fungal strains from a cave in northern Spain. Their phenotypic analysis on agar plates supplemented with ABTS (2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonic acid)) disclosed two laccase‐positive strains. Further genotyping revealed that they belonged to the Gliomastix murorum and Conidiobolus thromboides species. The secretion of G. murorum and C. thromboides laccase‐like enzymes was then confirmed by zymography. Further identification of these polypeptides by mass‐spectroscopy revealed the nature of the laccases and made it possible to predict their functional domains and other features. In addition, plate assays revealed that the laccases secreted by both G. murorum and C. thromboides were capable of degrading industrial dyes (Congo Red, Indigo, and Eriochrome Black T). Homology modeling and substrate docking predicted the putative structure of the currently uncrystallized G. murorum enzyme as well as its amino acid residues potentially involved in interactions with these dyes. In summary, new biochemical and structural insights into decolorization mediated by G. murorum laccase as well as identification of laccase‐like oxidase in C. thromboides point to a promising future for these enzymes in biotechnology.

This study aimed to investigate matrix metalloproteinase (MMP) activity in human dentin using in-situ and gelatin zymography, after at-home and in-office bleaching, related to their clinical exposure times. Dentin specimens (n = 5) were treated with 35% hydrogen peroxide (50 min per session/4 sessions), 10% carbamide peroxide (180 min/21 sessions), or no treatment. All were subjected to in-situ zymography. Dentin slices were, subsequently, obtained, covered with fluoresceinconjugated gelatin, and examined with confocal laser-scanning microscopy. The fluorescence intensity was quantified and statistically analyzed using one-way ANOVA and Bonferroni tests (α = 0.05). Furthermore, gelatin zymography was performed on protein extracts obtained from dentin powder (N = 8 teeth), treated with hydrogen peroxide or carbamide peroxide, with different exposure times (10/50 min for hydrogen peroxide; 252/1260 min for carbamide peroxide). The results of the in-situ zymography showed no statistical differences between the bleached specimens and the control group, with a medium level of gelatinolytic activity expressed in the dentin tubules. The results of gelatin zymography showed an increased expression of pro-MMP-9 in carbamide peroxide groups. The expression of pro-MMP-2 decreased in all the experimental groups. The bleaching treatments performed on the enamel of sound teeth do not influence dentinal enzymatic activity. However, when unprotected dentin tissue is bleached, matrix metalloproteinases are more expressed, particularly when carbamide peroxide is used, proportional to the exposure time. [ABSTRACT FROM AUTHOR]

In recent years, a lack of stability of dairy products with extended shelf life (e.g., yoghurt products, UHT desserts) has occurred, with the corresponding products liquefying significantly after days or weeks. This project aimed to identify the enzymes responsible for the liquefaction of the affected products based on differential proteomic analyses. No evidence was found for the presence of starch-degrading bacteria in the affected products. With zymography and proteome analysis, we detected the cause of liquefaction in a pudding by contamination of its aroma component with an engineered amylolytic enzyme, cyclomaltodextrin glucanotransferase (CGTase) from Thermoanaerobacterium thermosulfurigenes. In addition, we detected contamination with Pseudomonas-derived proteolytic ATP-dependent Clp protease in one pudding batch and proteases in technically used amylases, which degraded β-caseins in another batch. Identification of these agents with liquefying properties in dairy products are useful for adjustment of production protocols and/or composition of additives, and thus shelf life extension. [ABSTRACT FROM AUTHOR]

Gingipains (RgpA, RgpB, and Kgp) are major virulence factors of the periodontitis-causing bacterium Porphyromonas gingivalis. Isolation of gingipains from the crude protein sample of P. gingivalis is critical for studying the underlying invasion mechanism that contributes to periodontitis, Alzheimer’s disease, and cardiovascular disease (CVD). Chromatographic processes and molecular cloning are two standard techniques often used for gingipains isolation, which are time-consuming and costly. In this study, considerably easier methods based on passive-mediated diffusion gel elution and gelatin zymogram were used to isolate and characterize gingipains. Importantly, proteins eluted from Native-PAGE showed enzymatic activity for both Rgp and Kgp. In gelatin zymography, the proteins with a molecular size of ~50 kDa and above 245 kDa were suggested as arginine-specific gingipains. The passive diffusion-mediated gel elution method is a simpler technique to isolate gingipains from crude protein samples of P. gingivalis. By using covalent and highly specific gingipain inhibitors, gelatin zymography enabled an individual characterization of gingipain activity and inhibition. Finally, this protocol can be easily extended by adding the isoelectric focusing to further improve the protein separation and characterization.

[Display omitted] • Purification and characterisation of methanol tolerant lipase from Bacillus mycoides. • Algal lipid transesterification employing cross-linked lipase enzyme aggregate. • Analysis of algal biodiesel (FAME) by TLC and FTIR. • Comparative FTIR analysis of algal biodiesel, algal lipid, and petroleum. Methanol induced lipase inactivation is the major hindrance in commercialization of enzyme catalyzed biodiesel production. Thus, methanol tolerance is highly needed for the lipase to be used in transesterification reaction. Hence, by virtue of such desirability, a methanol tolerant, extracellular lipase FDS8(L) was extracted from Bacillus mycoides strain CV18 and purified to homogeneity with 22.3 % yield. It was a non-position specific, alkaline lipase with 44 kDa of relative molecular mass and had no isoforms (confirmed by zymography). It showed optimal activity at pH 9.0 and 30 °C temperature, while stability in the pH range 7.0–10.0 and temperature upto 50 °C. Organic solvents (hexane, acetone and methanol) as well as metal ions (Mg2+, Mn2+ and Na+) had stimulatory effects on lipase activity. Lipase immobilization was done using glutaraldehyde to form cross linked lipase enzyme aggregate (CLEA) which further enhanced its stability in the presence of methanol. Owing to its methanol tolerance property, it was employed heterogeneously for catalyzing transesterification of algal lipids in methanol. The qualitative efficacy of the FDS8 lipase in converting algal lipids into biodiesel was confirmed after analyzing the hexane extracted transesterification products by TLC and FTIR spectroscopy. [ABSTRACT FROM AUTHOR]

Bacterial L-asparaginase is the key therapeutic enzyme in cancer therapy and is also witnessing demand as a food processing aid. In this study, L-asparaginase of newly isolated Bacillus subtilis ETMC-2 was cloned and over-expressed in Escherichia coli as an active soluble protein using ligation independent cloning strategy. The molecular mass was estimated to be 40 kDa and was optimally active at 50 °C. Zymography revealed that the enzyme was active in homo-tetramer state (~160 KDa). The encoded protein after BLASTp analysis on NCBI showed 99.73% similarity with L-ASNase that of Bacillus sp. Physico-chemical properties were predicted using Protparam leading to categorization of the enzyme as a stable protein with an instability index (II) of 19.02. The calculated aliphatic index (85.44) indicated the high thermal stability of the protein with GRAVY value of −0.317. Protein-Ligand docking revealed that the residues Thr 89 , Thr 121 , and Asp 122 were fundamental in protein–ligand complexation. After homology modelling, model validation was performed using Ramachandran plot, VERIFY3D, and RMSD. The paper describes cloning, heterologous expression, catalytic characteristics and physico-chemical properties of the type II B. subtilis L-ASNase. • Cloning of B. subtilis L-asparaginase using ligation independent cloning • Characterization and zymography of B. subtilis L-ASNase • In silico structural modelling of B. subtilis L-ASNase • Physico-chemical properties of B. subtilis L- ASNase type II revealed [ABSTRACT FROM AUTHOR]

Purpose: Increasing the pH of acid soils is a well-recognized means of improving their fertility; however, the effects that plants impose on rhizosphere soils in response to this change are not well understood. This research sought to investigate changes in phosphorus (P) availability in the rhizosphere of blue lupin (Lupinus angustifolius) in response to an increase in pH. Methods: Blue lupin plants were grown in rhizoboxes using two contrasting acid soils at their native pH (5.3 and 4.7) and treated with lime to increase their pH to 6.3 in a replicated trial. Measurements of localized acid phosphomonoesterase activity and P flux were made next to lateral root segments using a combination of zymography and diffusive gradient in thin films (DGT). Rhizosphere and bulk soils were tested for pH, organic anions, exchangeable aluminium (Al), labile P and phosphomonoesterase activity. Root morphological traits, root and shoot yields and shoot nutrient concentrations were also recorded. Results: Profiles of DGT-P fluxes across lateral roots showed mobilization of P in the soil with the higher organic P content, and depletion in the other. The extent of acid phosphomonoesterase activity in the rhizosphere decreased with soil pH increase. Shoot P uptake was strongly correlated with fine root length and total organic anions in the rhizosphere. The proportion of thin roots decreased at pH 6.3 compared to the native pH, whereas that of thick roots increased. Conclusion: Soil pH increase to 6.3 using lime negatively affects the P acquisition by blue lupin due to the reduction of organic anion exudation, fine root length and the extent of acid phosphomonoesterase activity in the rhizosphere. [ABSTRACT FROM AUTHOR]

The study aimed to evaluate the effect of dietary inclusions of Moringa oleifera on the growth, haematology, antioxidant activities, immunity, and protection against Aeromonas hydrophila in Oreochromis niloticus. Diets supplemented with 0.5% or 1% of either crude moringa (CM) powder or alcoholic moringa extracts (AME) were used. Weight gain (WG), specific growth rate (SGR), and haematological parameters (RBCs count, Hb content and HCT %) were significantly improved (p < 0.05) with feeding CM than control fish, meanwhile they decreased significantly (p < 0.05) in AME treatments. The feeding of CM significantly improved WBCs and lymphocyte counts (p < 0.05). Cholesterol and triglycerides showed no significant changes (p > 0.05) in CM treatments, but they significantly increased in fish fed 0.5% AME. Liver and kidney functions (ALT, AST, uric acid and creatinine) were not relatively affected by moringa treatments. The non‐specific immune responses of fish fed CM showed significant (p < 0.05) improvement, including serum proteins, lysozyme activity, and antiprotease activity. Peroxidase activity (POX) and Cytochrome P450 (CYP) were increased significantly (p ˂ 0.05) with CM and in 0.5% AME treatment. Superoxide dismutase (SOD) and Catalase (CAT) showed insignificant changes (p > 0.05). Antioxidant activities were also analysed by staining on polyacrylamide gel electrophoresis. Challenge experiment indicated higher survivability of fish fed moringa‐supplemented diets. The survivability was found the highest (80%) in the group fed with 0.5% CM. These results indicate that dietary supplementation with crude M. oleifera enhances the growth, antioxidant activities, immune competence and potentiates resistance against A. hydrophila in O. niloticus, with the greatest effects of 0.5% crude M. oleifera. [ABSTRACT FROM AUTHOR]

Abstract Lipases are currently used in food technology for the modification of fats and oils. The thermal stability of lipase is an essential characteristic for this application. This study compares four individual single-step methods (heat treatment, ethanol precipitation, ammonium sulfate precipitation, and size-exclusion chromatography) to enrich lipase concentrations from thermophilic bacterial (Geobacillius stearothermophilus and Anoxybacillus flavithermus) cell lysates. SDS-PAGE and size exclusion chromatography were used to determine the molecular weights of the lipases and the enrichment efficiencies were determined using specific enzyme activities. The molecular weight of G. stearothermophilus lipase was approximately 42 kDa, and approximately 33 kDa for A. flavithermus lipase. For each organism, ethanol precipitation resulted in the highest enrichment fold, followed by ammonium sulfate precipitation, gel filtration and heat treatment respectively. The highest yields for G. stearothermophilus lipase were obtained with ammonium sulfate precipitation, followed by get filtration, and ethanol precipitation respectively. The highest yields for A. flavithermus lipase were obtained from heat treatment followed by ammonium sulfate precipitation, gel filtration and ethanol precipitation respectively. Ethanol precipitation and heat treatment are simple methods for enzyme enrichment from cell lysates and can result in high enzyme yields with moderate enrichment folds compared to complex multi-step purification methods.

Abstract Ananas Comosus (also known as pineapple) is a part of Bromeliaceae family and it is consumed as food as well as folk medicine for the treatment of various diseases. It is reported that pineapple is a rich source of bromelain, a cysteine protease and it is considered as an important enzyme in different industries due to its significant therapeutic and industrial applications such as anticancer, anti-inflammatory and meat tenderizing. Bromelain is mostly present in fruit and stem of pineapple, but it is reported that crown, core, and peels, which constitute the waste of the pineapple plant, also contain bromelain but limited data is available. Therefore, the proposed study aimed at utilizing pineapple waste for the extraction and characterization of bromelain. Firstly, crude bromelain was extracted with phosphate buffer (pH 7), then it was subjected to partial purification using different fractions of ammonium sulphate (NH4)2SO4 such as 30, 40, 50 and 60% followed by desalting and concentration. Enzyme activity was calculated by using casein digesting unit (CDU) method. The results demonstrated that the crown bromelain showed highest purification of 4.34-fold at 30% (NH4)2SO4 saturation, whereas core and peel bromelain showed highest purification of 2.75 and 2.59-fold at 40% (NH4)2SO4 saturation. The molecular weight of crude and partially purified bromelain was determined by SDS-PAGE analysis and found to be 26 KDa. The pH and thermal stability of all the parts of pineapple showed maximum stability at pH 7 and at 35oC temperature.

The matrix metalloproteinases (MMPs) are a family of enzymes involved in extracellular matrix remodeling. MMPs are secreted in a latent form and activated by local and infiltrating cells. MMP-2 and -9 are the most studied in reproduction and have been detected in bovine, ovine, equine and human placenta. There is only one study on MMPs in the equine amniotic fluid (AF) reporting a decrease in the activity of MMP-2 in case of premature delivery. The aim of this study was focused on MMP-2 and -9 activity in AF collected at parturition from mares with normal or high-risk pregnancy. High-risk pregnancy was defined as a history of premature udder development/lactation, increase of combined thickness of the uterus and placenta, vulvar discharge and/or mare's systemic illness. The diagnosis of placental insufficiency was confirmed retrospectively after macroscopic and histopatologic examination of the placenta. AF was collected by needle puncture of the amnion within 5 min after its appearance through the vulva. The activity of MMP-2 and -9 was analyzed by in-gel zymography allowing the evaluation of both latent and active forms. Twenty mares with normal pregnancy (group 1) and 8 mares with high-risk pregnancy (group 2) were included. All mares in group 2 had a high-risk pregnancy with a diagnosis of placental insufficiency associated with placental villous hypoplasia, placentitis or placental edema. The bands relative to latent and active forms of MMP-2 were clearly visible in both groups and the activity of latent (P = 0.010) and active (P = 0.004) forms was lower in the AF samples of group 2. The band of the latent form of MMP-9 was visible in 17/20 samples of group 1, while it was completely absent in all samples of group 2. In contrast, the band of the active form was clearly visible and with a greater activity in AF samples of group 2 (P = 0.002). Placental dysfunction seems to induce a lower MMP-2 activity and a higher MMP-9 activity through the release of pro-inflammatory cytokines. Because fetal pulmonary secretions are a likely source of gelatinases in AF during late gestation, the increased MMP-9 activity could be related to fetal distress. These data provide a starting point to better understand the role of MMPs in equine pregnancy, although it should be confirmed in a larger and more homogeneous population of mares with high-risk pregnancy. • Zymography represents a functional assay for MMPs analysis in equine amniotic fluid. • The study confirms the activity of MMPs in amniotic fluid collected at parturition. • MMPs might have a role in the pathogenesis of equine neonatal illness. [ABSTRACT FROM AUTHOR]

Insects of the taxonomic order Coleoptera are recognised for considerable cellulolytic activity in their digestive fluid. The cellulolytic activity of the gut fluid in Hoplasoma unicolor, a member of Coleoptera, however, remains unexplored. In this study, we, for the first time, report the qualitative and quantitative analysis of cellulolytic activity in the digestive fluid of this insect. The cellulolytic endo-1,4-β-D-glucanase activity was confirmed in the supernatant of the insect's digestive fluid by agar plate assay using carboxymethyl cellulose as the substrate. To determine the optimum pH, enzyme activity was further assessed in an acidic pH range of 5 to 6, and the highest activity was observed at pH 5.3. For quantitative analysis, endoglucanase activity was measured using 3,5-dinitrosalicylic acid method which revealed that the specific activity of the gut sample was 0.69 (±0.01) units per mg of protein. For further characterisation of the cellulases in the sample, SDS-PAGE and zymogram analysis were carried out. Two active cellulolytic bands were detected on the zymogram suggesting the presence of two distinct endoglucanases which completely disappeared upon heating the sample at 55°C. Our study, therefore, highlights prospect of the gut fluid of H. unicolor as an important source of cellulase enzymes that merits further investigations into their extensive characterisation for potential industrial applications. [ABSTRACT FROM AUTHOR]

Aims: Soil organic carbon (C) efflux is tightly linked to the rhizosphere, where soil microorganisms rapidly decompose organic compounds released from roots. Recently, imaging approaches have greatly improved our understanding of small-scale C-turnover heterogeneity and promoted the term 'rhizosphere hotspots' for highly active areas. However, despite often assumed, the effect of these hotspots on total soil C balances is still unknown. We aim to bridge this gap by correlating rhizosphere imaging data to soil respiration on individual plant scale. Methods: We grew 17 maize (Zea mays L.) plants in rhizoboxes filled with sandy arable soil. After four weeks, the plants were labelled with 14CO2 and root exudation was visualized and quantified by 14C-imaging one day after labeling. The evolved CO2 was trapped in NaOH and 14CO2 as well as total CO2 was quantified before and after labelling. Enzyme activity (β-glucosidase) was quantified by soil zymography. Results: Bulk soil β-glucosidase activitiy negatively correlated to total CO2 efflux, and was the most important predictor (R2 = 0.55). Total and rhizosphere specific 14C-activity were solely correlated to 14CO2 efflux (r = 0.51, r = 0.58). A combination of bulk soil β-glucosidase activity, rhizosphere-14C activity and root biomass, explained about 75% of variance in CO2 efflux. Conclusions: This indicates that root exudation and enzyme-activity hotspots are suitable predictors for total soil respiration, particularly when combined with root biomass to account for three-dimensional variation, and that hotspots on the rhizosphere scale are directly linked to larger scale C balances. [ABSTRACT FROM AUTHOR]

Abstract Background MT1-MMP is a cell-surface enzyme whose regulation of pro-MMP-2 and ERK activation position it as a key facilitator of ECM remodelling and cell migration. These processes are modulated by endogenous MMP inhibitors, such as RECK, a GPI-anchored protein which has been shown to inhibit both MT1-MMP and MMP-2 activity. Our previous studies have revealed a link between MT1-MMP levels, and pro-MMP-2 and ERK activation in mammalian cells, as well as MT1-MMP and RECK co-localization in Xenopus embryos. We here investigated how modulation of RECK would impact MT1-MMP and MMP-2 levels, as well as ERK signalling in Xenopus A6 cells. Results We used a Morpholino approach to knockdown RECK, plasmid transfection to overexpress RECK, and PI-PLC treatment to shed RECK from the cell surface of Xenopus A6 cells. RECK reduction did not alter pERK or MT1-MMP levels, nor MMP-2 activity as measured by zymography; thus RECK-knockdown cells maintained the ability to remodel the ECM. RECK overexpression and PI-PLC treatment both increased ECM remodelling potential through increased MT1-MMP protein and relative MMP-2 activation levels. Conclusions RECK changes that reduce the ability of the cell to remodel the ECM (overexpression and cell surface shedding) are compensated for by increases in MT1-MMP, and MMP-2 levels as seen by zymography.

Viperidae snakes are the most important agents of snakebites in Brazil. The protein composition of snake venoms has been frequently analyzed by means of electrophoretic techniques, but the interaction of proteins in venoms has barely been addressed. An electrophoretic technique that has gained prominence to study this type of interaction is blue native polyacrylamide gel electrophoresis (BN-PAGE), which allows for the high-resolution separation of proteins in their native form. These protein complexes can be further discriminated by a second-dimension gel electrophoresis (SDS-PAGE) from lanes cut from BN-PAGE. Once there is no study on the use of bidimensional BN/SDS-PAGE with snake venoms, this study initially standardized the BN/SDS-PAGE technique in order to evaluate protein interactions in Bothrops atrox, Bothrops erythromelas, and Bothrops jararaca snake venoms. Results of BN/SDS-PAGE showed that native protein complexes were present, and that snake venom metalloproteinases and venom serine proteinases maintained their enzymatic activity after BN/SDS-PAGE. C-type lectin-like proteins were identified by Western blotting. Therefore, bidimensional BN/SDS-PAGE proved to be an easy, practical, and efficient method for separating functional venom proteins according to their assemblage in complexes, as well as to analyze their biological activities in further details.

Background: Lignocellulolytic enzymes are essential for agricultural waste disposal and production of renewable bioenergy. Many commercialized cellulase mixtures have been developed, mostly from saprophytic or endophytic fungal species. The cost of complete cellulose digestion is considerable because a wide range of cellulolytic enzymes is needed. However, most fungi can only produce limited range of highly bioactive cellulolytic enzymes. We aimed to investigate a simple yet specific method for discovering unique enzymes so that fungal species producing a diverse group of cellulolytic enzymes can be identified. Results: The culture medium of an endophytic fungus, Daldinia caldariorum D263, contained a complete set of cellulolytic enzymes capable of effectively digesting cellulose residues into glucose. By taking advantage of the unique product inhibition property of β-glucosidases, we have established an improved zymography method that can easily distinguish β-glucosidase and exoglucanase activity. Our zymography method revealed that D263 can secrete a wide range of highly bioactive cellulases. Analyzing the assembled genome of D263, we found over 100 potential genes for cellulolytic enzymes that are distinct from those of the commercially used fungal species Trichoderma reesei and Aspergillus niger. We further identified several of these cellulolytic enzymes by mass spectrometry. Conclusions: The genome of Daldinia caldariorum D263 has been sequenced and annotated taking advantage of a simple yet specific zymography method followed by mass spectrometry analysis, and it appears to encode and secrete a wide range of bioactive cellulolytic enzymes. The genome and cellulolytic enzyme secretion of this unique endophytic fungus should be of value for identifying active cellulolytic enzymes that can facilitate conversion of agricultural wastes to fermentable sugars for the industrial production of biofuels. [ABSTRACT FROM AUTHOR]