Reverse Zymography Research Articles

Characterization and kinetics of a cathepsin B-inhibiting protein from Musa acuminata Colla peel

Hyperexpression of cathepsin B caused by an imbalance of endogenous inhibitors is involved in multiple pathologies, hence making it a key therapeutic target. Protease inhibitors are effective biomolecules that regulate protease activities and are considered potential therapeutic agents in various diseases. Plant protease inhibitors have been reported as an effective complementary alternative drug. A proteinaceous cathepsin B inhibitor (CBI-BP) has been isolated from Musa acuminata Colla (banana) peel with a molecular weight of 27.9 kDa on SDS-PAGE. The purity of the CBI-BP was confirmed on the native- PAGE. The isolated CBI-BP showed an IC50 value of 8.14 μg and a Ki value of 10.59 μg (0.19 μM). Cathepsin B inhibition kinetics indicated that CBI-BP follows a mixed-type of cathepsin B inhibition. Its inhibition activity was also confirmed by reverse zymography. The inhibitor was stable from pH 2.6 - 10.0 with maximum activity at pH 7.2, temperature 25-100°C and exhibited thermostability for 60 min at 70°C. MALDI/TOF/MS analysis of CBI-BP showed 40% similarity to the GH18 domain-containing protein (A0A4S8JRM9) from Musa balbisiana. Although in-silico docking studies showed binding of A0A4S8JRM9 to cathepsin B affects the binding energy of the substrate to cathepsin B but is not reported for any anti-cathepsin B activity. This suggests that isolated CBI-BP might be a novel protein with anti-cathepsin B activity. Thus the isolated CBI-BP may be further explored as possible anti-cathepsin B drug.

Shatavarin-IV, a steroidal saponin from Asparagus racemosus, inhibits cell cycle progression and epithelial-to-mesenchymal transition in AGS cells under hyperglycemic conditions

Gastric cancer (GC)-diabetes co-morbidity is nowadays growing into a rising concern. However, no separate treatment procedures have been outlined for such patients. Phytochemicals and their derivatives can therefore be used as therapeutics as they have greater effectiveness, reduced toxicity, and a reduced likelihood of developing multi-drug resistance in cancer treatments. The present study intended to assess the therapeutic efficacy of Shatavarin-IV – a major steroidal saponin from the roots of Asparagus racemosus, in human gastric adenocarcinoma cell line under hyperglycemic conditions and explore its mechanism of action in controlling GC progression. For the present study, AGS cells were incubated in high glucose-containing media and the effects of Shatavarin-IV therein have been evaluated. Cell proliferation, confocal microscopic imaging, flow-cytometric analysis for cell cycle and apoptosis, immunoblotting, zymography, reverse zymography, wound-healing, colony formation, and invasion assays were performed. Shatavarin-IV has a prominent effect on AGS cell proliferation; with IC50 of 2.463 µ M under hyperglycemic conditions. Shatavarin-IV induces cell cycle arrest at the G0/G1 phase, thereby preventing hyperglycemia-induced excessive cell proliferation that later on leads to apoptotic cell death at 36 h of incubation. Shatavarin-IV further inhibits the migratory and invasive potential of AGS cells by altering the expression patterns of different EMT markers. It also inhibits MMP-9 while promoting TIMP-1 activity and expression; thereby regulating ECM turnover. This is the first report demonstrating the therapeutic efficacy of Shatavarin-IV against AGS cells grown in hyperglycemic conditions, implicating new insights into the treatment paradigm of patients with GC-diabetes co-morbidity.

Melatonin Inhibits AGS Cell Proliferation by Binding to the ATP Binding Site of CDK2 Under Hyperglycemic Conditions.

Cancer cells utilize glucose as their primary energy source. The aggressive nature of cancer cells is therefore enhanced in hyperglycemic conditions. This study has been adopted to investigate the therapeutic potential of melatonin against such aggressive proliferation of AGS cells-a human gastric cancer cell line, under hyperglycemic conditions. AGS cells were incubated with high glucose-containing media, and the effects of melatonin have been evaluated, therein. Cell proliferation, ROS generation, flow-cytometric analysis for cell cycle and apoptosis, wound healing, immunoblotting, zymography, reverse zymography assays, in-silico analysis, and kinase activity assays were performed to evaluate the effects of melatonin. We observed that melatonin inhibited the hyperglycemia-induced cell proliferation in a dose-dependent manner. It further altered the expression and activity of MMP-9 and TIMP-1. Moreover, melatonin inhibited AGS cell proliferation by arresting AGS cells in the G0/G1 phase after binding in the ATP binding site of CDK-2, thereby inhibiting its kinase activity. In association, a significant decrease in the expression of cyclin D1, cyclin E, CDK-4, and CDK-2 were observed. In conclusion, these findings suggest that melatonin has anti-gastric cancer potential. Melatonin could therefore be included in future drug designs for gastric cancer-hyperglycemia co-morbidity treatment.

Zymograms as a Tool to Detect PPIs in Host plants of Antheraea Assamensis

Antheraea assamensis (vernacular: ‘muga’) larvae is commercially reared on two tree species of the Lauraceae family, Persea bombycina and Litsea monopetala for its golden yellow, lustrous cocoon silk. Biochemical and molecular studies suggested that the midgut digestive enzymes and their transcripts in larvae feeding on P. bombycina differ from those found in larvae feeding on L. monopetala indicating that host plant 'choice' affects the digestive physiology of this insect. Ingestion of plant proteinase inhibitors is known to influence expression of digestive enzymes. Using reverse zymography technique trypsin and chymotrypsin inhibitors were detected in herbivore-induced leaves of L. monopetala and P. bombycina that could inhibit midgut proteinases of A. assamensis. Such interactions may affect proteolytic digestion in larvae reared on different host plant species. This work may have significance in quality of silk produced by muga silkworm, ultimately benefiting the silkworm rearers/industry.

Perfil das metaloproteínas de matriz extracelular em cabras Toggenburg sadias e infectadas por lentivírus de pequenos ruminantes no Sudeste brasileiro

Small ruminant lentiviruses (SRLV) are difficult to diagnose due to their escape mechanisms. Therefore, proteomics is an alternative in the search for biomarkers through extracellular matrix metalloproteinases (MMPs), enzymes related to the immune response. In this sense, this study aimed to analyze the profile of MMPs in healthy and infected Toggenburg goats with chronic SRLV infection in Southeast Brazil. Five positive and five negative goats for SRLV were selected using the agar gel immunodiffusion (AGID) microtechnique, western blot (WB), and nested polymerase chain reaction (nPCR). All animals were submitted to blood collection by puncture of the jugular vein, followed by centrifugation to obtain blood plasma, protein quantification by the Bradford method, one-dimensional electrophoretic separation (1D), and identification of protease activity by zymography and confirmation via reverse zymography in the presence of MMP-2 through the action of tissue inhibitors (TIMP-2). The analysis of protein bands was performed using descriptive statistics and densitometry values for zymography were subjected to the Shapiro-Wilk test to determine normality. Little difference was observed in the occurrence of protein bands between groups. Regarding MMPs, no differences were observed in the expression of proMMP-9, MMP-9, and MMP-2 in animals affected by SRLV. TIMP-2 inhibited proMMP-2 and MMP-2 in all animals. Thus, the profile of protein bands does not change in healthy goats with chronic SRLV infection. The TIMP-2 expression allowed proving the existence of MMP-2 in animals chronically infected by SRLV via reverse zymography.

A cystatin C similar protein from Musa acuminata that inhibits cathepsin B involved in rheumatoid arthritis using in silico approach and in vitro cathepsin B inhibition by protein extract

Rheumatoid arthritis (RA) is an auto-immune disease that affects the synovial lining of the joints, causes synovitis and culminates to joint destruction. Cathepsin B is responsible for digesting unwanted proteins in extracellular matrix but its hyper expression could implicate in pathological diseases like RA. Available treatments for RA are classified into non-steroidal anti-inflammatory drugs (NSAIDs), disease-modifying anti-rheumatic drugs (DMARDs), and steroids, but the severe side effects associated with these drugs is one of concerns and cannot be ignored. Thus, any alternative therapy with minimum or no side effects would be a cornerstone. In our in silico studies a cystatin C similar protein (CCSP) has been identified from Musa acuminata that could effectively inhibit the cathepsin B activity. In silico and molecular dynamics studies showed that the identified CCSP and cathepsin B complex has binding energy −66.89 kcal/mol as compared to cystatin C - cathepsin B complex with binding energy of −23.38 kcal/mol. These results indicate that CCSP from Musa acuminata has better affinity towards cathepsin B as compared to its natural inhibitor cystatin C. Hence, CCSP may be suggested as an alternative therapeutic in combating RA by inhibiting its one of the key proteases cathepsin B. Further, in vitro experiments with fractionated protein extracts from Musa sp. peel inhibited cathepsin B to 98.30% at 300 µg protein concentration and its IC50 was found to be 45.92 µg indicating the presence of cathepsin B inhibitor(s) in protein extract of peel which was further confirmed by reverse zymography. Communicated by Ramaswamy H. Sarma

Identification of thrombin inhibiting antithrombin-III like protein from Punica granatum using in silico approach and in vitro validation of thrombin inhibition activity in crude protein

Thrombosis is characterized by the formation of clots in the blood vessels. Antithrombin-III deficiency in the blood causes thrombus formation. Supplementing antithrombin-III may serve as anticoagulant therapy. In the present studies, an antithrombin like Protein from Punica granatum has been identified and characterized using in silico approach. Based on sequence homology, an ALPP was selected depending upon its highest binding affinity of −41.28 kcal/mol with thrombin. Thrombin structure complexed with ALPP was docked with TAME using AutoDock Vina. No binding was observed for TAME at Ser195 of thrombin. MD simulation (50 ns) was performed to evaluate the flexibility and stability of docked complexes. In vitro assays with crude protein showed 78% thrombin inhibition at 5 µg and calculated IC50 value was 0.188 µg. The presence of thrombin inhibitors in crude protein was also confirmed by reverse zymography. Thus, it is very likely that the protein identified from P. granatum may act as thrombin inhibitor.

Salvianolic Acid B Strikes Back: New Evidence in the Modulation of Expression and Activity of Matrix Metalloproteinase 9 in MDA-MB-231 Human Breast Cancer Cells

Salvianolic acid B (SalB) is a bioactive compound from Salviae miltiorrhizae, one of the most important traditional herbal medicines widely used in several countries for the treatment of cardiovascular diseases. The aim of this study was to evaluate the in vitro effect of SalB on the expression and the activity of matrix metalloproteinase 9 (MMP-9), a zinc-dependent proteolytic enzyme, in human MDA-MB-231 breast cancer cells. This cellular model is characterized by a marked invasive phenotype, supported by a high constitutive expression of MMPs, especially gelatinases. SalB was first of all evaluated by in silico approaches primarily aimed at predicting the main pharmacokinetic parameters. The most favorable interaction between the natural compound and MMP-9 was instead tested by molecular docking analysis that was subsequently verified by an enzymatic inhibition assay. MDA-MB-231 cells were treated with SalB 5 µM and 50 µM for 24 h and 48 h. The conditioned media obtained from treated cells were then analyzed by gelatin zymography and reverse zymography to, respectively, evaluate the MMP-9 activity and the presence of TIMP-1. The expression of the enzyme was then evaluated by Western blot on conditioned media and by analysis of transcripts through reverse transcriptase-polymerase chain reaction (RT-PCR). The in silico approach showed the ability of SalB to interact with the catalytic zinc ion of the enzyme, with a plausible competitive mode of action. The analysis of conditioned culture media showed a reduction in MMP-9 activity and the concomitant decrease in the enzyme concentration, partially confirmed by analysis of transcripts. SalB showed the ability to modulate the function of MMP-9 in MDA-MB-231 cells. To our knowledge, this is the first time in which the role of SalB on MMP-9 in a highly invasive cellular model is investigated. The obtained results impose further and more specific evaluations in order to obtain a better understanding of the biochemical mechanisms that regulate the interaction between this natural compound and the MMP-9.

Deglycosylation Increases the Aggregation and Angiogenic Properties of Mutant Tissue Inhibitor of Metalloproteinase 3 Protein: Implications for Sorsby Fundus Dystrophy.

Sorsby fundus dystrophy (SFD) is an autosomal dominant macular disorder caused by mutations in tissue Inhibitor of the metalloproteinase-3 (TIMP3) gene with the onset of symptoms including choroidal neovascularization as early as the second decade of life. We have previously reported that wild-type TIMP3 is an endogenous angiogenesis inhibitor that inhibits Vascular Endothelial Growth Factor (VEGF)-mediated signaling in endothelial cells. In contrast, SFD-related S179C-TIMP3 when expressed in endothelial cells, does not have angiogenesis-inhibitory properties. To evaluate if this is a common feature of TIMP3 mutants associated with SFD, we examined and compared endothelial cells expressing S179C, Y191C and S204C TIMP3 mutants for their angiogenesis-inhibitory function. Western blot analysis, zymography and reverse zymography and migration assays were utilized to evaluate TIMP3 protein, Matrix Metalloproteinase (MMP) and MMP inhibitory activity, VEGF signaling and in vitro migration in endothelial cells expressing (VEGF receptor-2 (VEGFR-2) and wild-type TIMP3 or mutant-TIMP3. We demonstrate that mutant S179C, Y191C- and S204C-TIMP3 all show increased glycosylation and multimerization/aggregation of the TIMP3 protein. In addition, endothelial cells expressing TIMP3 mutations show increased angiogenic activities and elevated VEGFR-2. Removal of N-glycosylation by mutation of Asn184, the only potential N-glycosylation site in mutant TIMP3, resulted in increased aggregation of TIMP3, further upregulation of VEGFR-2, VEGF-induced phosphorylation of VEGFR2 and VEGF-mediated migration concomitant with reduced MMP inhibitory activity. These results suggest that even though mutant TIMP3 proteins are more glycosylated, post-translational deglycosylation may play a critical role in the aggregation of mutant TIMP3 and contribute to the pathogenesis of SFD. The identification of factors that might contribute to changes in the glycome of patients with SFD will be useful. Future studies will evaluate whether variations in the glycosylation of mutant TIMP3 proteins are contributing to the severity of the disease.

Abstract 3837: The physiological relevance of TIMP dimer formation

Abstract Tissue inhibitors of metalloproteinases (TIMPs) are a family of four paralogous genes that modulate the activity of matrix metalloproteinases (MMPs) and are vital for tissue homeostasis. Additionally, TIMPs display activities that are independent of their MMP inhibitory activities. Dysregulation and perturbations within this system can lead to complex tissue-specific pathologies. TIMP2 is the most abundantly expressed member of the TIMP family and has been shown to display potential therapeutic uses in cancer and other diseases. We have noted the persistent presence of SDS-stable TIMP2 dimers/multimers in SDS-PAGE at a level of ~5% total protein. The goal of this study is to determine the stability and biological relevance of SDS-stable dimer formation of TIMP2 in in vitro and in vivo. We found that TIMP2 dimers display an enhanced ability to inhibit MMP2 activity in reverse zymography assays. Immunoblotting revealed gel extracted TIMP2 dimer entered an equilibrium with monomeric TIMP2, while the extracted monomer did not form new dimer. In addition, MMP2 activity assays do not corroborate the enhanced MMP2 inhibitory activity observed in reverse zymograms but are consistent with the observation that isolated TIMP2 dimers re-equilibrate with monomers. This implies that structural or sequence difference mediates the formation of dimers. Dimer/multimer formation is observed across the TIMP family. Mass spectrometry analysis of gel extracted TIMP monomers and dimers show that monomer and dimer forms of TIMPs display unique post-translation modification (PTM) profiles. We propose that understanding the interplay between TIMP multimers and PTMs will reveal new biological functions within this intriguing family of proteins. We are currently working to resolve the functional differences between monomer and dimer and reveal the underlying mechanisms at play. Citation Format: Carolyn Lazaroff, David Peeney, Sadeechya Gurung, William G. Stetler-Stevenson. The physiological relevance of TIMP dimer formation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3837.

Effect of SPARC Suppression in Mice, Perfused Human Anterior Segments, and Trabecular Meshwork Cells

PurposeSecreted protein, acidic and rich in cysteine (SPARC) elevates intraocular pressure (IOP), increases certain structural extracellular matrix (ECM) proteins in the juxtacanalicular trabecular meshwork (JCT), and decreases matrix metalloproteinase (MMP) protein levels in trabecular meshwork (TM) endothelial cells. We investigated SPARC as a potential target for lowering IOP. We hypothesized that suppressing SPARC will decrease IOP, decrease structural JCT ECM proteins, and alter the levels of MMPs and/or their inhibitors.MethodsA lentivirus containing short hairpin RNA of human SPARC suppressed SPARC in mouse eyes and perfused cadaveric human anterior segments with subsequent IOP measurements. Immunohistochemistry determined structural correlates. Human TM cell cultures were treated with SPARC suppressing lentivirus. Quantitative reverse transcriptase polymerase chain reaction (PCR), immunoblotting, and zymography determined total RNA, relative protein levels, and MMP enzymatic activity, respectively.ResultsSuppressing SPARC decreased IOP in mouse eyes and perfused human anterior segments by approximately 20%. Histologically, this correlated to a decrease in collagen I, IV, and VI in both the mouse TM and human JCT regions; in the mouse, fibronectin was also decreased but not in the human. In TM cells, collagen I and IV, fibronectin, MMP-2, and tissue inhibitor of MMP-1 were decreased. Messenger RNA of the aforementioned genes was not changed. Plasminogen activator inhibitor 1 (PAI-1) was upregulated in vitro by quantitative PCR and immunoblotting. MMP-1 activity was reduced in vitro by zymography.ConclusionsSuppressing SPARC decreased IOP in mice and perfused cadaveric human anterior segments corresponding to qualitative structural changes in the JCT ECM, which do not appear to be the result of transcription regulation.

Seed Protein from Artocarpus hirsutus Lam. with Trypsin Inhibitory, Micro-bicidal and Antioxidant Activities Induces Depletion of Human Skin Cancer (A431) and Colon Cancer (HT29) Cells

: Natural trypsin inhibitors are increasingly recognised as putative anticancer or antimicrobial therapeutics. In the present study, we isolated a proteinaceous trypsin inhibitor from the seeds of Artocarpus hirsutus to assess its anticancer, antioxidant and microbicidal effects. A trypsin inhibitory protein (AhTI) was isolated from the seeds of A. hirsutus using ion exchange and gel filtration chromatographic methods. The trypsin inhibitory activity of AhTI was confirmed by activity staining using Reverse Zymography. The effect of AhTI on human cancer cell lines (A431 and HT29) was studied by microscopic examination, MTT assay and LDH leakage analysis. Antioxidant activity of AhTI was analysed by ferric reducing power and DPPH radical scavenging assays. An agar well diffusion method was used to check the antimicrobial activity of AhTI. The purification protocols used in the study significantly increased the specific activity of the target protein from 72.65 ± 0.86 to 2048 ± 27.3. AhTI offers significant activity against the proliferation of the A431 and HT29 cancer cell lines. In vitro AhTI shows potent antioxidant activity and exhibits antimicrobial activity against all the bacteria and fungi isolates used in this study. Of particular note, AhTI was particularly effective against Staphylococcus aureus and Escherichia coli. This in vitro study shows that AhTIis a promising candidate for further development novel therapeutics targeting cancer, microbial infections and oxidative stress.

Zymography and Reverse Zymography for Testing Proteases and Their Inhibitors.

Zymography is a powerful technique for the assay of different hydrolases that act upon any biological macromolecule. In particular, zymography is used to assay the activities of serine proteases, e.g., matrix metalloproteinases (MMPs), and reverse zymography is used for tissue inhibitors of metalloproteinases (TIMPs) in multifarious experimental samples. Zymography is a method of electrophoretic separation of proteases under non-reducing conditions in a polyacrylamide gel containing substrate. The resolved proteins are renatured by exchange of the anionic detergent with a nonionic one, and the gel is incubated in a specific buffer for the specific proteases. After staining the gel by Coomassie blue staining solution, the proteolytic activities are visualized as clear colorless bands against a dark background. In contrast, reverse zymography is a parallel technique to detect protease inhibitors. In addition to substrate gelatin, proteases (i.e., MMPs) are also incorporated in proper ratio into the polyacrylamide gel. After electrophoresis, during the developing step, the MMPs specifically digest the substrate in regions where TIMPs are absent. Thus, inhibitors/TIMP is represented as dark zones of inhibition against a transparent background after staining. In this chapter, common troubleshoots during sample preparation, running zymography, and data interpretation are discussed. Notes are specified to enhance the sensitivity of the methods. In conclusion, zymography could be crucial for enzyme assay at the nanogram level and for the improvement of new investigative techniques for diseases such as endometriosis, rheumatoid arthritis, osteoarthritis, tumor invasion, and inflammation.

Changes in the levels of MMP-9, MMP-2, and their inhibitors in the serum of dairy cows with laminitis

Laminitis is regarded as an important underlying cause of lameness disorders, yet its specific pathogenesis remains unclear. Consistent histological changes in cows with laminitis consist in the degradation of the basement membrane (BM). The breakdown of BM is accomplished by numerous proteases, especially MMP-9 and MMP-2. This study recruited 45 cows according to veterinary diagnostic criteria and divided them into three groups: subclinical laminitis cows (SCL, n = 15), chronic laminitis cows (CL, n = 15), and healthy cows (CON, n = 15). After blood samples were collected from the jugular vein, the serum was separated and frozen at –80°C. The serum samples were analyzed by gelatin zymography and reverse zymography to evaluate the activities of MMP-9, MMP-2, TIMP-1, and TIMP-2. In the SCL group, the activity of MMP-9 significantly increased (P <0.01) and the activity of TIMP-1 significantly decreased (P <0.05), compared with those in the CON group, while the activities of MMP-2 and TIMP-2 were not significantly different. In the CL group, the activities of MMP-9 (P <0.001) and MMP-2 (P <0.05) significantly increased, and the activities of TIMP-1 (P <0.01) and TIMP-2 (P <0.05) significantly decreased compared with those in the CON group. This is the first study to report changes in the content of MMP-2 and MMP-9 and their inhibitors, TIMP-2 and TIMP-1, in the serum of dairy cows with laminitis. These results indicate that inadequate regulation of the activities of MMPs and TIMPs in serum may play a role in the development of laminitis in dairy cows.

Unraveling a natural protease inhibitor from marine Streptomyces griseoincarnatus HK12 active against Chikungunya virus

Proteases play an indispensable role in the life cycles of several life-threatening organisms such as the ones causing malaria, cancer and AIDS. A targeted blockade of these enzymes could be an efficient approach for drug modeling against these causative agents. Our study was directed towards the extraction and characterization of a protease inhibitor having activity against Chikungunya virus (CHIKV). A protein-based protease inhibitor (PI) in Streptomyces griseoincarnatus HK12 with anti-viral activity against CHIKV was revealed when screened against two major proteases, papain and trypsin. The PI was efficiently extracted at 60 % ammonium sulfate saturation and purified by ion-exchange chromatography (CM-Sepharose) at 300 mM NaCl elution followed by SDS-PAGE (10 %). The protein was characterized by denaturing SDS-PAGE, reverse zymography, and MALDI-TOF peptide mass fingerprinting. The protein-based PI was studied to have a high molecular weight of 66−70 kDA. The PI was tested to supress the supress cytopathic effects (CPE) exerted by the clinically isolated virus in BHK21 cells. This was used as a measure to determine the antiviral activity. The PI exerted significant effects with an effective concentration calculated as EC50 11.21 μg/mL. The protein was found to be reported as the first of its kind which also stands out to be the first a natural protease inhibitor against the treatment of the chikungunya virus.

Overexpressed HGF promotes metastasis of squamous cell carcinoma of the head and neck through the PI3K/Akt and JNK signaling pathways.

Background: The role of HGF in squamous cell carcinoma of the head and neck (SCCHN) is not clear. Methods: Reverse transcription PCR, western blotting, gelatin zymography, immunohistochemistry, actin polymerization, chemotaxis and migration assays were used in the authors' study. Results: HGF expression level was upregulated in SCCHN cells, which was associated with clinical stage; tumor, node, metastasis classification; and lymphatic invasion. SCCHN cells with high Met expression were sensitive to cell invasion, which was blocked by inhibiting PI3K/Akt and JNK. HGF induced MMP9 expression and enhanced its activity. Akt induced the activation of JNK through the PI3K/Akt and JNK signaling pathways. Conclusion: HGF upregulates MMP9 through the activation of the PI3K/Akt and JNK signaling pathways in SCCHN cells.

Zymography-types, advantages, uses and troubleshooting - A detailed review

There are many methods for the study and quantification of enzymes in the body systems. Off-late, the method of zymography has assumed immense importance in diagnostic medicine. It is a method where enzymes are visualized using the substrate conversion technique. The product of reaction appears, or the substrate disappears and the mechanisms specific for the recognition of this measure the biochemical reaction. This procedure has many advantages, the most important ones being providing both qualitative and measurable data, such as molecular zymography methods for visualising hydrolytic enzymes and differentiating among whole molecules, degradation and complexes. There are three main types of zymography, namely- In-Gel zymography (IGZ), In-Situ zymography (ISZ) and In-vivo zymography (IVZ). There are many variations of this technique like transfer 2D and reverse zymography. This method is mainly used to study the expression of matrix metalloproteinases. Alternate to the conventional zymography techniques have been suggested, like usage of the new Ponceau S staining protocol that provides significant benefits in terms of assay usability and cost reduction and is comparatively faster and easier. Mild alterations in the actual procedure of zymography can take care of minor issues arising during the procedure.

Description of a serpin toxin in Loxosceles (Brown spider) venoms: Cloning, expression in baculovirus-infected insect cells and functional characterization

Several classes of toxins are present in the venom of Brown spiders (Loxosceles genus), some of them are highly expressed and others are less expressed. In this work, we aimed to clone the sequence of a little expressed novel toxin from Loxosceles venom identified as a serine protease inhibitor (serpin), as well as to express and characterize its biochemical and biological properties. It was named LSPILT, derived from Loxoscelesserine protease inhibitor-like toxin. Multiple alignment analysis revealed high identity between LSPILT and other serpin molecules from spiders and crab. LSPILT was produced in baculovirus-infected insect cells, resulting in a 46-kDa protein fused to a His-tag. Immunological assays showed epitopes in LSPILT that resemble native venom toxins of Loxosceles spiders. The inhibitory activity of LSPILT on trypsin was found both by reverse zymography and fluorescent gelatin-degradation assay. Additionally, LSPILT inhibited the complement-dependent lysis of Trypanosoma cruzi epimastigotes, reduced thrombin-dependent clotting and suppressed B16-F10 melanoma cells migration. Results described herein prove the existence of conserved serpin-like toxins in Loxosceles venoms. The availability of a recombinant serpin enabled the determination of its biological and biochemical properties and indicates potential applications in future studies regarding the pathophysiology of the envenoming or for biotechnological purposes.

Biochemical and immunohistochemical analysis of tissue inhibitor of metalloproteinases-1 in human sound dentin

ObjectivesMatrix metalloproteases (MMPs) are a family of enzymes that operate a proteolytic activity at the level of the extracellular matrix. MMPs are regulated by tissue inhibitors of metalloproteinases (TIMPs) that can ubiquitously bind different enzyme forms. The study aims to identify a morfo-functional association between TIMP-1 and MMP-2 and -9 in human dentin.Materials and methodsProteins were extracted from demineralized human sound dentin powder and centrifuged to separate two aliquots with different molecular weights of proteins, higher and lower than 30 kDa. In each aliquot, the evaluation of the presence of TIMP-1/MMP-2 and TIMP-1/MMP-9 was performed using co-immunoprecipitation/immunoblotting analysis. The distribution of TIMP-1, in association with MMP-2 and -9, was investigated using a double immunohistochemical technique. Furthermore, the activity of TIMP-1 was measured by reverse zymography, where acrylamide gel was copolymerized with gelatin and recombinant MMP-2.ResultsCo-immunoprecipitation/immunoblotting analysis showed the association TIMP-1/MMP-2 and TIMP-1/MMP-9 in human sound dentin. Electron microscopy evaluation revealed a diffuse presence of TIMP-1 tightly associated with MMP-2 and -9. Reverse zymography analysis confirmed that TIMP-1 present in human dentin is active and can bind different MMPs isoforms.ConclusionsThe strict association of TIMP-1 with MMP-2 and -9 in situ appeared a constant finding in the human sound dentin.Clinical relevanceConsidering the role of TIMP-1, MMP-2, and MMP-9 within the connective tissues, clinically applicable protocols could be developed in the future to increase or decrease the level of TIMPs in human dentin to regulate the activity of MMPs, contributing to reduce caries progression and collagen degradation.

Change in Proteolytic Profile in Heifers After Oligofructose Overload.

Laminitis in cattle is an important underlying cause of lameness, which leads to a significant reduction in economic and animal welfare. Nevertheless, the disordered pathological processes of laminitis remain unclear. Several proteinases are probably involved in the disorder of basement membrane (BM) metabolism in laminitis, for instance, matrix metalloproteinases (MMPs), neutrophil elastase (NE), and myeloperoxidase (MPO). This study aimed to investigate the change in proteolytic profile in circulating and lamellar tissues using an oligofructose (OF) overload-induced laminitis model in heifers. Twelve clinically healthy and nonlame Chinese Holstein heifers were recruited and randomly divided into two groups: OF-induced and control (CON). The OF-induced heifers group (n = 6) was administered 17 g/kg of body weight (BW) of OF dissolved in 2 L/100 kg of BW of tap water via the oral-rumen tube. The CON group (n = 6) was given an equal volume of tap water. The plasma samples were collected 0, 6, 12, 18, 24, 36, 48, 60, and 72 h after administration, and the lamellar samples were collected 72 h after euthanasia. The plasma samples were analyzed by zymography and reverse zymography. Histological examination, zymography, reverse zymography, and Western blot of lamellar samples were conducted. In the plasma of the OF-induced group, the pro-MMP9 activity increased from 36 h (P < 0.001) to 60 h (P < 0.05). Moreover, the plasma tissue inhibitors of metalloproteinase 1 (TIMP1) activity decreased after 18 h (P < 0.05), while the ratio of pro-MMP9 to TIMP1 and TIMP2 increased after 18 h (P < 0.001) and 48 h (P < 0.05), respectively. The act-MMP2, pro-MMP9, and act-MMP9 activities increased in the lamellar tissue of the OF-induced group compared with the CON group (P < 0.05). In addition, the expression of lamellar NE protein was higher in the OF-induced group (P < 0.01), while no change was found in lamellar MPO protein compared with the CON group. In conclusion, increased pro-MMP9 combined with decreased TIMP1 activity in the circulation might have caused the activation of blood neutrophils, while the activation of proteolytic enzymes in lamellar tissue probably led to the dysfunction of BM in the OF-induced group.