Zymographic Analysis Research Articles

Cellulolytic potential of gut bacterial biomass in silkworm Bombyx mori. L

Abstract Exploring bacterial populations with cellulolytic activity from the midgut of Bombyx mori was proposed in this study. As the initial efforts, cellulose degrading bacteria was harvested from the midgut of Bombyx mori by using selective CMC (carboxymethylcellulose) agar medium. The gut bacterial biomass was categorized by biochemical and molecular identification; they were validated through 16S rDNA analysis. All the gut isolates were subjected to plate assay separately, to display the efficient cellulose fabricator. Among them, Bacillus aryabhattai and Bacillus sp. has shown the highest level of cellulolytic activity, when compared to the zone of clearance developed by the other isolates. Zymographic analysis also revealed same, while treated with cellulolytic substrate and the cellulolytic activity was visualized in the gut isolate Bacillus aryabhattai. For futher adjudication, cellulose degradation study was performed with Bacillus aryabhattai and Bacillus sp in HPLC and FTIR. Both the results of HPLC and FTIR conferred the status that Bacillus aryabhattai is the superior in cellulolytic activity. The Productivity of cellulase in Bacillus aryabhattai was detected and optimized at different pH, temperature, incubation time and different concentration of cellulose and identified their peak outputs. The gut microbiome based cellulase can act as a novel biocatalyst in the field biofuels recovery and also can be utilized as Phytobezoars in treatment of cattle and humans.

Extraction of Cathepsin D-Like Protease from Neon Flying Squid (Ommastrephes bartramii) Viscera and Application in Antioxidant Hydrolysate Production

A protease from neon flying squid (Ommastrephes bartramii) viscera (SVCE3(f)) was partially purified by isoelectric solubilization/precipitation combined with ultra-membrane filtration (ISP-UMF). Two protein bands of 45 and 27 KDa were determined by SDS-PAGE assay. The protease characteristic of the protein band of 45 KDa was confirmed using casein zymography analysis. The result of UPLC-ESI-MS/MS suggested that the band of 45 KDa could be a cathepsin D-like protease. This cathepsin D-like protease showed an optimum pH of 3.0 and optimum temperature of 60 °C when casein was used as s substrate. Furthermore, its protease activity was stable at 30–50 °C and under a pH range of 1.0–5.0, maintaining about 60% of its initial activity. SVCE3(f) can digest half-fin anchovy (Setipinna taty) to generate antioxidant hydrolysates (HAHp-SEs). The degree of hydrolysis (DH) of HAHp-SEs increased along with the hydrolysis time and reached stability after 60 min of digestion. HAHp-SEs(30) with relatively lower DH exhibited the highest DPPH radical scavenging activity as compared with other HAHp-SEs. However, a stronger hydroxyl radical scavenging activity and greater reducing power were observed for HAHp-SEs that underwent higher DH. Accordingly, the partially purified cathepsin D-like protease of neon flying squid viscera using ISP-UMF could have potential application in antioxidant hydrolysates production.

Viridicatol and viridicatin isolated from a shark-gill-derived fungus Penicilliumpolonicum AP2T1 as MMP-2 and MMP-9 inhibitors in HT1080 cells by MAPKs signaling pathway and docking studies

Matrix metalloproteinases (MMPs), the key enzymes in extracellular matrix degradation, seemed to increase in tumorigenesis, which has a relationship with metastasis and invasions. In this study, we investigated the MMP inhibitory effect of viridicatol and viridicatin, two compounds extracted from shark-gill-derived fungus Penicilliumpolonicum AP2T1, on MMP-2 and -9 in vitro and in silico. The MTT assay showed that viridicatol and viridicatin had no cytotoxicity at a concentration of 50 µM on HT1080 human fibrosarcoma cells. In addition, the studies by gelatin zymography and Western blot analysis confirmed that the two compounds effectively inhibited the protein activities and expressions of MMP-2 and -9. Moreover, molecular docking studies simulated and clarified the interaction between compounds and MMPs, respectively. Furthermore, the effect of those two compounds on mitogen activated protein kinases (MAPKs) was also assessed by Western blot analysis. Collectively, these results suggested that viridicatol and viridicatin could be potential inhibitors of MMPs and P.polonicum may be a rich source of bioactive compounds with MMP inhibitory property.

Haloferax sulfurifontis GUMFAZ2 producing xylanase-free cellulase retrieved from Haliclona sp. inhabiting rocky shore of Anjuna, Goa-India.

Salt stable cellulases are implicated in detritic food webs of marine invertebrates for their role in the degradation of cellulosic material. A haloarchaeon, Haloferax sulfurifontis GUMFAZ2 producing cellulase was successfully isolated from marine Haliclona sp., a sponge inhabiting the rocky intertidal region of Anjuna, Goa. The culture produced extracellular xylanase-free cellulase with a maximum activity of 11.7 U/ml, using carboxymethylcellulose-Na (CMC-Na), as a sole source of carbon in 3.5 M NaCl containing medium, pH 7 at 40°C and produced cellobiose and glucose, detectable by thin-layer chromatography. Nondenaturing polyacrylamide gel electrophoresis of the crude enzyme, revealed a single protein band of 19.6 kDa which on zymographic analysis exhibited cellulase activity while corresponding sodium dodecyl sulfate polyacrylamidegelelectrophoresis revealed a molecular weight of 46 kDa. Unlike conventional cellulases, this enzyme is active in presence of 5 M NaCl and does not have accompanying xylanase activity, hence can be considered as xylanase-free cellulase. Such enzymes from haloarchaea offer great potential for biotechnological application because of their stability at high salinity and is therefore worth pursuing.

Use of Aqueous Two-Phase and Three-Phase Partitioning Systems for Purification of Lipase Obtained in Solid-State Fermentation by Rhizopus arrhizus

Background: Purification of enzymes by conventional methods such as precipitation and chromatographic techniques is a costly and time-consuming procedure and may lead to low yields of enzyme activity. Alternative liquid-liquid extraction methods such as Aqueous Two-Phase Systems (ATPS) and Three Phase Partitioning (TPP) are characterized by the high enzyme yields and purification degree. Objective: The objective of this study was the application of partitioning systems ATPS and TPP for purification of lipase produced in solid-state fermentation by Rhizopus arrhizus. Methods: ATPS and TPP were used for purification of lipase, obtained by solid state cultivation of Rhizopus arrhizus. Results: Lipase was isolated with PEG4000/potassium sodium tartrate ATPS and the effect of the system composition, including PEG 4000 and potassium sodium tartrate concentrations on lipase partitioning was studied. When using 30% PEG4000/21% potassium sodium tartrate, lipase was distributed in the top phase, and the highest recovery yield of 217% and purification fold of 6.1 were achieved. It was found that at PEG4000 concentration of or higher than 15%, the enzyme was present in the top polymer-rich phase with a partitioning yield of over 90%. Upon application of TPP for lipase isolation, the effect of t-butanol concentration, ammonium sulfate concentration and pH on enzyme partitioning was investigated. The highest lipase recovery yield of 71% and 19.1-fold purification were achieved in the interfacial phase in the presence of 30% ammonium sulfate saturation with 1.0:0.5 crude extract/t-butanol ratio at pH 7 in a single step. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis and zymographic analysis showed significant purification of lipase by TPP and the presence of two multiple forms of the enzyme. Conclusion: ATPS (PEG4000/ Potassium sodium tartrate) and TPP (1.0:0.5 crude extract/t-butanol ratio, 30% ammonium sulfate saturation, pH 7) proved to be rapid methods for the isolation and purification of lipase and they can be used in downstream processing for industrial preparation of the enzyme.

PARP-1 inhibition provides protection against elastase-induced emphysema by mitigating the expression of matrix metalloproteinases.

In our previous study, we have shown that PARP-1 inhibition (genetic or pharmacological) ameliorates elastase-induced inflammation and emphysema. Since matrix metalloproteinases (MMPs) particularly MMP-2 and MMP-9 are known to play a critical role in emphysema development, the present work was designed to evaluate the effects of PARP-1 inhibition on their expression utilizing elastase-induced mouse model of emphysema. Our data show that olaparib administration at a dose of 5mg/kg b.wt. (daily) significantly prevented the elastase-induced inflammation as indicated by decreased inflammatory cells particularly macrophages in BALF at 1 week post-injury. In addition, the drug restored the altered redox balance in the lungs of elastase-treated mice toward normal. Further, PCR data show that olaparib administration ameliorates the elastase-induced expression of MMP-2 and MMP-9 without having much effect on the expressions of their inhibitors TIMP-1 and TIMP-2. Next, our data on immunoblot, gelatin zymography, and immunohistochemical analysis indeed confirm that olaparib reduced the elastase-induced expression of MMP-2 and MMP-9. Reduction in the expression of metalloproteinases correlate well with the PARP activity as olaparib treatment suppressed the elastase-induced expression of PAR modified proteins markedly. Overall, our data strongly suggest that PARP-1 inhibition blunts elastase-induced MMP-2 and MMP-9 expression, which may be partly responsible for prevention of emphysema.

Axenic amastigote cultivation and in vitro development of Leishmania orientalis

Leishmania (Mundinia) orientalis is a recently described new species that causes leishmaniasis in Thailand. To facilitate characterization of this new species, an in vitro culture system to generate L. orientalis axenic amastigotes was developed. In vitro culture conditions of the axenic culture-derived amastigotes were optimized by manipulation of temperature and pH. Four criteria were used to evaluate the resulting L. orientalis axenic amastigotes, i.e., morphology, zymographic analysis of nucleases, cyclic transformation, and infectivity to the human monocytic cell line (THP-1) cells. Results revealed that the best culture condition for L. orientalis axenic amastigotes was Grace's insect medium supplemented with FCS 20%, 2% human urine, 1% BME vitamins, and 25μg/ml gentamicin sulfate, pH5.5 at 35°C. For promastigotes, the condition was M199 medium, 10% FCS supplemented with 2% human urine, 1% BME vitamins, and 25μg/ml gentamicin sulfate, pH6.8 at 26°C. Morphological characterization revealed six main stages of the parasites including amastigotes, procyclic promastigotes, nectomonad promastigotes, leptomonad promastigotes, metacyclic promastigotes, and paramastigotes. Also, changes in morphology during the cycle were accompanied by changes in zymographic profiles of nucleases. The developmental cycle of L. orientalis in vitro was complete in 12days using both culture systems. The infectivity to THP-1 macrophages and intracellular growth of the axenic amastigotes was similar to that of THP-1 derived intracellular amastigotes. These results confirmed the successful axenic cultivation of L. orientalis amastigotes. The axenic amastigotes and promastigotes can be used for further study on infection in permissive vectors and animals.

In Vitro Study of the Combination of Doxorubicin, Curcuma xanthorrhiza, Brucea javanica, and Ficus septica as a Potential Novel Therapy for Metastatic Breast Cancer

Less optimized therapeutic effects constrain the use of doxorubicin as the main agent of chemotherapy for metastatic breast cancer, resistance and side effects. Therefore we need a combination of more than one chemopreventive agent which has different molecular targets to solve that problem. The aims of this study is to prove the inhibitory effect of ethanolic extract of rhizome of Curcuma xanthorrhiza (ECx), fruit of Brucea javanica (EBj), leave of Ficus septica (EFs) and doxorubicin (Dox) alone and its combination on migration and invasion of a highly metastatic 4T1 breast cancer cell line. Cytotoxic activity of single and combination treatment was evaluated by MTT assay, followed by an experiment of apoptosis induction by using flow cytometry. The inhibitory effect on migration was observed by the scratch wound-healing assay. Furthermore, the observation of the activity of matrix metalloproteinase-9 (MMP-9) was analyzed by gelatin zymography. The results showed that ECx, EBj, EFs, and Dox has cytotoxic activity on 4T1 cells with the value of IC 50 respectively 49.7±1.53mg/mL, 59.9±1.79mg/mL, 15.2±2.12mg/mL and 1.2±0.23mM. Furthermore, combination of ECx-EBj-Dox and ECx-EBj-EFs revealed synergistic effect on 4T1 cells and decrease cell viability through the induction of apoptosis and necrosis. Based on wound healing assay, 24 hours incubation of this combination inhibited 4T1 cells migration compared to single treatment. Gelatin zymography analysis showed that this combination also inhibited the activity of MMP-9 greater than a single use. Curcuma xanthorrhiza , Brucea javanica , and Ficus septica may have potential to be developed as a combination with or without doxorubicin for metastatic breast cancer treatment.

A complex of novel protease inhibitor, ovostatin homolog, with its cognate proteases in immature mice uterine luminal fluid

A predominant gelatinolytic enzyme with approximately 26 kDa was observed in gelatin zymogram of immature mice uterine luminal fluid (ULF). Size exclusion analysis revealed that the native size of this enzyme was close to that of human α2-macroglobulin (α2-MG), a 725 kDa protein. This large protease was isolated by a series of chromatographic steps on the Sephacryl S-400 and DEAE-Sepharose columns. The results from gelatin zymography and SDS-PAGE analysis supported that this large protease consists of gelatinolytic enzyme and a 360 kDa protein. Through tandem mass spectrometry analysis followed by MASCOT database search, the 360 kDa protein was identified as ovostatin homolog (accession: NP_001001179.2) assigned as a homolog of chicken ovostatin, a protease inhibitor. The co-fractionation analysis by gel filtration and mouse ovostatin homolog (mOH) co-immunoprecipitation experiments demonstrated that the mOH formed a complex with three gelatinolytic enzymes in immature mice ULF. Substrate zymography analysis revealed that the mOH-associated gelatinolytic enzymes were suitable to digest type I collagen rather than type IV collagen. In addition, the refolded mOH-associated 26 kDa gelatinolytic enzyme displayed the type I collagen-digesting activity in the assay, but the other two enzymes did not have this function. RT-PCR analysis showed that mOH gene was abundantly expressed in brain, spinal cord, lung, uterus, and in 17-day embryo. Taken together, our data suggest that mOH/cognate protease system may play a potential role in regulation of tissue remodeling and fetal development.

Involvement of matrix metalloproteinases in capillary degeneration following NMDA-induced neurotoxicity in the neonatal rat retina

Interactions between neuronal cells and vascular cells in the retina are critical for maintaining retinal tissue homeostasis. Impairment of cellular interactions contributes to development and progression of retinal diseases. Previous studies demonstrated that neuronal cell damage leads to capillary degeneration in an N-methyl-D-aspartic acid (NMDA)-induced retinal degeneration model. However, the mechanisms underlying this phenomenon are not fully understood. In this study, we examined the possible role of matrix metalloproteinase (MMP)-9 in neuronal cell loss and capillary degeneration in NMDA-treated retinas of neonatal rats. Intravitreal injection of NMDA (50 or 200 nmol) was performed on postnatal day (P) 7 and morphological changes in retinal neurons and vasculature were examined on P14. The MMP inhibitor CP101537 (100 nmol) or vehicle (dimethyl sulfoxide) was intravitreally injected simultaneously with, or 2 days after, NMDA injection. CP101537 protected against neurovascular degeneration in a time-dependent manner as follows: 1) simultaneous injection of CP101537 with NMDA prevented morphological changes in retinal neurons induced by NMDA (50 nmol); and 2) reduction in capillary density and number of vertical sprouts induced by NMDA (200 nmol) was prevented when CP101537 was injected 2 days after NMDA injection. Gelatin zymography and western blot analyses indicated that activity and protein levels of MMP-9 were enhanced from 4 h to 2 days after NMDA injection. Increased activity and protein levels of MMP-9 were suppressed by MMP inhibitors (CP101537 and GM6001). In situ zymography revealed that MMP activity was enhanced throughout the retinal vasculature in NMDA-treated retinas. These results indicate that MMP-9 plays an important role in neurovascular degeneration in the injured retina. Inhibition of MMP-9 may be an effective strategy for preventing and reducing neurovascular degeneration.

Pseudomonas aeruginosa alkaline protease exhibits a high renaturation capability.

Thermally induced unfolding and renaturation capability of alkaline proteases (AprA) of three Pseudomonas aeruginosa strains, i.e. ATCC 27853 and two clinical isolates, was examined. Sequence analyses demonstrated a high level of aprA genes identity (99.24-99.8%) in these bacterial strains. The proteases retained 45-60% and 15% of their activity after pre-treatment at 60oC and 80oC, respectively, whereas pre-incubation at 90-95oC resulted in a higher level of activity than at 80oC. Zymography analyses and immunoblotting with AprA antiserum suggested a high thermostability and renaturation capability of the studied enzymes in comparison to another P. aeruginosa protease, elastase B. An intrinsic capability of renaturation of P. aeruginosa AprA was confirmed by fluorescence spectra of the native, thermally denatured, and renatured enzyme. The value of the fluorescence intensity of the denatured and subsequently cooled enzyme recovered to about 80% of the value of the native protein fluorescence intensity. Moreover, pre-incubation of the enzyme at 60oC and 90oC exerted only a slight effect on the intensity of absorbance and the shape of the amide I band, as demonstrated by Fourier transform infrared (FTIR) spectroscopy performed after subsequent cooling of the pre-treated enzyme. The results indicated a high renaturation capability of the P. aeruginosa AprA proteins.

Cellulase-Free Xylanase by Halococcus thailandensis GUMFAS7 and Halorubrum saccharovorum GUMFAS1—Bionts of a Sponge Cinachyrella cavernosa

Intertidal sponges, during tidal flux and filter feeding cycle, often get exposed to an influx of water of varying salinities containing halophilic microbes and suspended agricultural detritus. Microbes get established in the lumen of the host during its filter feeding process and might participate in the degradation of incoming particulate matter. We successfully isolated two haloarchaeal bionts, strains GUMFAS1 and GUMFAS7, producing cellulase-free xylanases from one such sponge, identified as Cinachyrella cavernosa inhabiting the intertidal rocky region of Anjuna, Goa-India. The bionts exhibited 4.791 and 8.714 U/mg of cellulase-free xylanase activity, respectively. Xylanase from GUMFAS7 (XS7) showed maximum activity at 37°C, pH 5 and 20% NaCl concentration. Zymographic analysis of XS7 revealed a presence of three bands with xylanase activity of molecular weight 238, 60, and 18 kDa, respectively. The strains were identified by 16S rRNA gene sequencing as Halococcus thailandensis (GUMFAS7) and Halorubrum saccharovorum (GUMFAS1). The latter was identical to another isolate obtained from the waters surrounding the host. These findings reflect a unique type of interaction between haloarchaea and sponge. An in-depth study on this cellulase-free xylanase is underway and expected to be of significance in the field of extremozymes.

Hydrangenol suppresses VEGF-stimulated angiogenesis by targeting p27KIP1-dependent G1-cell cycle arrest, VEGFR-2-mediated signaling, and MMP-2 expression

ABSTRACTWe previously reported that hydrangenol has potent antitumor activity against human bladder cancer EJ cells. Here, we investigated the antiangiogenic activity of hydrangenol using in vitro and ex vivo models. Treatment with hydrangenol significantly inhibited the proliferation of vascular endothelial growth factor (VEGF)-induced HUVECs in a concentration-dependent manner (EC50 = 10 μM). Flow cytometry analysis revealed that hydrangenol suppressed the VEGF-induced inhibition of G1-cell cycle phase and also decreased cyclin D1, cyclin E, CDK2, and CDK4 levels. Hydrangenol-mediated arrest in the G1-cell cycle phase was associated with p27KIP1 level, but not p21WAF1 or p53 level. Hydrangenol also significantly inhibited VEGFR-2-mediated signaling pathways including ERK1/2, AKT, and endothelial nitric oxide synthase. Interestingly, immunoprecipitation assay demonstrated that the inhibition of VEGFR-2 activation was independent of VEGF binding, thereby suggesting an allosteric regulation of hydrangenol against VEGFR-2. Additionally, hydrangenol inhibited migration, invasion, and capillary-like tubular formation in VEGF-stimulated HUVECs. Zymography and immunoblot analyses revealed that these inhibitory activities were partially owing to the VEGF-induced inhibition of matrix metalloproteinase-2 activity. Finally, VEGF-mediated microvessel sprouting was inhibited in the presence of hydrangenol in ex vivo aortic ring assay. Taken together, hydrangenol possesses a potent antiangiogenesis potential; thus we believe that hydrangenol may be developed as a therapeutic reagent to treat angiogenesis-mediated diseases.

Characterization of a mycelial fructosyltransferase from Aspergillus tamarii NKRC 1229 for efficient synthesis of fructooligosaccharides

An efficient system for biotransformation of sucrose to fructooligosaccharides (FOS) was obtained using Aspergillus tamarii NKRC 1229 mycelial fructosyltransferase (m-FTase). Zymographic analysis confirmed mycelial localization of the FTase (36 U/g) and lyophilized fungal pellets were used for bioconversion. m-FTase had molecular weight ∼75 kDa with optimum activity at pH 7.0 and 20 °C. FOS production after parametric optimization (sucrose − 50% w/v, m-FTase dose − 4.5% w/v, inoculum age − 48 h and incubation time − 24 h) reached 325 g/L (55% yield) with 14% residual sucrose, 25% glucose and 6% fructose. FTase activity was enhanced after pre-treatment with organic solvents and SDS. FOS was purified in a single step using gel filtration matrix, Bio-Gel P2. FOS was characterized using Diffusion ordered spectroscopy-Nuclear Magnetic Resonance (1H DOSY-NMR) and Fourier-transform infrared spectroscopy (FTIR). Continuous generation of FOS was achieved using recyclable mycelia upto 10 consecutive cycles.

Talaromyces verruculosus tannase production, characterization and application in fruit juices detannification

Abstract A potential tannase producing fungus S3-07 was isolated from the tannin-rich soil sample and screened as better tannase producer with 38 ± 3 mm diameter of hydrolysis zone formation. It was identified as T. verruculosus (accession number KX863699) on the basis of the internal transcribed spacer ribosomal DNA (ITS-rDNA) sequence analysis. Tannase was produced with 8.22 ± 0.35 Unit per gram dry substrate (U/gds) activity in the unoptimized condition through solid state fermentation (SSF) using Babul (Acacia nilotica) bark as a solid substrate. Central composite design (CCD) was used to maximize the production and achieved 3.91 fold increments (32.18 ± 1.31 U/gds). Ammonium sulfate precipitation with 70% saturation and DEAE fractionation obtained 13.289 specific activity, 47.489% yield and 6.896 purification fold. SDS-PAGE and zymographic analysis showed two bands of tannase with ∼75 kDa and 58 kDa pH optima of tannase was measured at 8.00, whereas pH stability was recorded between 4.0 and 8.0. Tannase had temperature optima at 60 °C, however, stability was observed between 30 and 60 °C. In presence of ethanol, SDS, Mn2+ ions and gallic acid increased tannase activity, whereas ethyl acetate, tween 20, Ba2+ ion and urea inhibited maximum activity. Most of tannin reduction (93.27%) was measured in case of orange juice after 15 min incubation and 89.36% reduction in pomegranate juice after 30 min, whereas aonla juice was measured with 75.49% tannin reduction after 45 min incubation.

Anti-inflammatory property of quercetin through downregulation of ICAM-1 and MMP-9 in TNF-α-activated retinal pigment epithelial cells

Quercetin is a flavonoid polyphenolic compound present in fruits and vegetables that has proven anti-inflammatory activity. The goal of the present investigation was to investigate the effects of quercetin on tumor necrosis factor-α (TNF-α)-induced inflammatory responses via the expression of ICAM-1 and MMP-9 in human retinal pigment epithelial cells (ARPE-19 cells). Real-time PCR, gelatin zymography, and Western blot analysis showed that TNF-α induced the expression of ICAM-1 and MMP-9 protein and mRNA in a time-dependent manner. These effects were attenuated by pretreatment of ARPE-19 cells with quercetin. Quercetin inhibited the TNF-α-induced phosphorylation of PKCδ, JNK1/2, ERK1/2. Quercetin, rottlerin, SP600125 and U0126 attenuated TNF-α-stimulated c-Jun phosphorylation and AP-1–Luc activity. Pretreatment with quercetin, rottlerin, SP600125, or Bay 11-7082 attenuated TNF-α-induced NF-κB (p65) phosphorylation, translocation and RelA/p65–Luc activity. TNF-α significantly increased MMP-9 promoter activity and THP-1 cell adherence, and these effects were attenuated by pretreatment with quercetin, rottlerin, SP600125, U0126, tanshinone IIA or Bay 11-7082. These results suggest that quercetin attenuates TNF-α-induced ICAM-1 and MMP-9 expression in ARPE-19 cells via the MEK1/2–ERK1/2 and PKCδ–JNK1/2–c-Jun or NF-κB pathways.

Deficiency of mouse mast cell protease 4 mitigates cardiac dysfunctions in mice after myocardium infarction

Mouse mast cell protease-4 (mMCP4) is a chymase that has been implicated in cardiovascular diseases, including myocardial infarction (MI). This study tested a direct role of mMCP4 in mouse post-MI cardiac dysfunction and myocardial remodeling. Immunoblot and immunofluorescent double staining demonstrated mMCP4 expression in cardiomyocytes from the infarct zone from mouse heart at 28 day post-MI. At this time point, mMCP4-deficient Mcpt4−/− mice showed no difference in survival from wild-type (WT) control mice, yet demonstrated smaller infarct size, improved cardiac functions, reduced macrophage content but increased T-cell accumulation in the infarct region compared with those of WT littermates. mMCP4-deficiency also reduced cardiomyocyte apoptosis and expression of TGF-β1, p-Smad2, and p-Smad3 in the infarct region, but did not affect collagen deposition or α-smooth muscle actin expression in the same area. Gelatin gel zymography and immunoblot analysis revealed reduced activities of matrix metalloproteinases and expression of cysteinyl cathepsins in the myocardium, macrophages, and T cells from Mcpt4−/− mice. Immunoblot analysis also found reduced p-Smad2 and p-Smad3 in the myocardium from Mcpt4−/− mice, yet fibroblasts from Mcpt4−/− mice showed comparable levels of p-Smad2 and p-Smad3 to those of WT fibroblasts. Flow cytometry, immunoblot analysis, and immunofluorescent staining demonstrated that mMCP4-deficiency reduced the expression of proapoptotic cathepsins in cardiomyocytes and protected cardiomyocytes from H2O2-induced apoptosis. This study established a role of mMCP4 in mouse post-MI dysfunction by regulating myocardial protease expression and cardiomyocyte death without significant impact on myocardial fibrosis or survival post-MI in mice.

Matrix metalloproteinase-2 and matrix metalloproteinase-9 in mice with ocular toxocariasis.

In ocular toxocariasis, Toxocara canis-induced inflammatory reaction can lead to eye destruction and granuloma, which is formed by immune cell infiltration and concurrent extensive remodeling tissue. Herein, the histomorphology of granuloma and proteinase production in the eye of T. canis-infected BALB/c mice were investigated. Pathological effects substantially increased after the infection culminated in a severe leukocyte infiltration and granuloma formation from days 4 to 56 post-inoculation. The matrix metalloproteinase (MMP)-2 and MMP-9 activities remarkably increased, compared with those of uninfected control, by gelatin zymography and Western blot analysis in ocular toxocariasis. Granuloma formation had a remarkably positive correlation with MMP-2 and MMP-9 levels. We suggested that T. canis larvae and leukocytes infiltrated from blood vessel both migrated into corpus adiposum orbitae. Activated leukocytes secreted MMP-2 and MMP-9, leading to fibronectin degradation. The imbalance of MMP-2/TIMP-2 and MMP-9/TIMP-1 may play a role in inflammatory cell infiltration and extracellular matrix degradation, forming granuloma, in ophthalmological pathogenesis of T. canis infection.

Visualizing the Distribution of Matrix Metalloproteinases in Ischemic Brain Using In Vivo 19F-Magnetic Resonance Spectroscopic Imaging.

Matrix metalloproteinases (MMPs) damage the neurovascular unit, promote the blood-brain barrier (BBB) disruption following ischemic stroke, and play essential roles in hemorrhagic transformation (HT), which is one of the most severe side effects of thrombolytic therapy. However, no biomarkers have presently been identified that can be used to track changes in the distribution of MMPs in the brain. Here, we developed a new 19F-molecular ligand, TGF-019, for visualizing the distribution of MMPs in vivo using 19F-magnetic resonance spectroscopic imaging (19F-MRSI). We demonstrated TGF-019 has sufficient sensitivity for the specific MMPs suspected in evoking HT during ischemic stroke, i.e., MMP2, MMP9, and MMP3. We then utilized it to assess those MMPs at 22 to 24 hours after experimental focal cerebral ischemia on MMP2-null mice, as well as wild-type mice with and without the systemic administration of the recombinant tissue plasminogen activator (rt-PA). The 19F-MRSI of TGN-019-administered mice showed high signal intensity within ischemic lesions that correlated with total MMP2 and MMP9 activity, which was confirmed by zymographic analysis of ischemic tissues. Based on the results of this study, 19F-MRSI following TGN-019 administration can be used to assess potential therapeutic strategies for ischemic stroke.

Assessment of Proteolytic Activities in the Bone Marrow Microenvironment.

During cytokine- or chemotherapy-induced hematopoietic stem cell (HSC) mobilization, a highly proteolytic microenvironment can be observed in the bone marrow that has a strong influence on adhesive and chemotactic interactions of HSC with their niches. The increase of proteases during mobilization goes along with a decrease of endogenous protease inhibitors. Prominent members of the proteases involved in HSC mobilization belong to the families of matrix metalloproteinases and cathepsins, which are able to degrade chemokines/cytokines, extracellular matrix components, and membrane-bound adhesion receptors. To determine the functional activity of different proteolytic enzymes, zymographic analyses with different substrates and pH conditions can be employed. An involvement of cysteine cathepsins can be determined by the "active site labeling" technique using a modified inhibitor irreversibly binding to the active center of the enzymes. Intact or degraded chemokines and cytokines, which fall into the range between 1000 and 20,000Da, can readily be detected by MALDI-TOF analysis. These three methods can help to detect proteolytic activities directly involved in the mobilization process.