Zymogram Assay Research Articles

Experimental Strategy to Discover Microbes with Gluten-degrading Enzyme Activities.

Gluten proteins contained in the cereals barley, rye and wheat cause an inflammatory disorder called celiac disease in genetically predisposed individuals. Certain immunogenic gluten domains are resistant to degradation by mammalian digestive enzymes. Enzymes with the ability to target such domains are potentially of clinical use. Of particular interest are gluten-degrading enzymes that would be naturally present in the human body, e.g. associated with resident microbial species. This manuscript describes a selective gluten agar approach and four enzyme activity assays, including a gliadin zymogram assay, designed for the selection and discovery of novel gluten-degrading microorganisms from human biological samples. Resident and harmless bacteria and/or their derived enzymes could potentially find novel applications in the treatment of celiac disease, in the form of a probiotic agent or as a dietary enzyme supplement.

Cloning, expression, and biochemical characterization of a novel GH16 β-agarase AgaG1 from Alteromonas sp. GNUM-1

Alteromonas sp. GNUM-1 is known to degrade agar, the main cell wall component of red macroalgae, for their growth. A putative agarase gene (agaG1) was identified from the mini-library of GNUM-1, when extracellular agarase activity was detected in a bacterial transformant. The nucleotide sequence revealed that AgaG1 had significant homology to GH16 agarases. agaG1 encodes a primary translation product (34.7 kDa) of 301 amino acids, including a 19-amino-acid signal peptide. For intracellular expression, a gene fragment encoding only the mature form (282 amino acids) was cloned into pGEX-5X-1 in Escherichia coli, where AgaG1 was expressed as a fusion protein with GST attached to its N-terminal (GST-AgaG1). GST-AgaG1 purified on a glutathione sepharose column had an apparent molecular weight of 59 kDa on SDS-PAGE, and this weight matched with the estimated molecular weight (58.7 kDa). The agarase activity of the purified protein was confirmed by the zymogram assay. GST-AgaG1 could hydrolyze the artificial chromogenic substrate, p-nitrophenyl-β-D-galactopyranoside but not p-nitrophenyl-α-D-galactopyranoside. The optimum pH and temperature for GST-AgaG1 activity were identified as 7.0 and 40 °C, respectively. GST-AgaG1 was stable up to 40 °C (100 %), and it retained more than 70 % of its initial activity at 45 °C after heat treatment for 30 min. The K m and V max for agarose were 3.74 mg/ml and 23.8 U/mg, respectively. GST-AgaG1 did not require metal ions for its activity. Thin layer chromatography analysis, mass spectrometry, and (13)C-nuclear magnetic resonance spectrometry of the GST-AgaG1 hydrolysis products revealed that GST-AgaG1 is an endo-type β-agarase that hydrolyzes agarose and neoagarotetraose into neoagarobiose.

Proteolytic activity regarding Sarconesiopsis magellanica (Diptera: Calliphoridae) larval excretions and secretions

Sarconesiopsis magellanica (Diptera: Calliphoridae) is a medically important necrophagous fly which is used for establishing the post-mortem interval. Diptera maggots release proteolytic enzymes contained in larval excretion and secretion (ES) products playing a key role in digestion. Special interest in proteolytic enzymes has also been aroused regarding understanding their role in wound healing since they degrade necrotic tissue during larval therapy. This study was thus aimed at identifying and characterising S. magellanica proteolytic enzyme ES products for the first time. These products were obtained from first-, second- and third-instar larvae taken from a previously-established colony. ES proteins were separated by SDS-PAGE and their proteolytic activity was characterised by zymograms and inhibition assays involving BAPNA (Nα-benzoyl-dl-Arg-p-nitroanilide) and SAPNA substrates, using synthetic inhibitors. The protein profile ranged from ∼69kDa to ∼23kDa; several of them coincided with the Lucilia sericata ES protein profile. Serine-protease hydrolysis activity (measured by zymogram) was confirmed when a ∼25kDa band disappeared upon ES incubation with PMSF inhibitor at pH 7.8. Analysis of larval ES proteolytic activity on BAPNA and SAPNA substrates (determined by using TLCK and TPCK specific inhibitors) suggested a greater amount of trypsin-like protease. These results support the need for further experiments aimed at validating S. magellanica use in larval therapy.

Effects of Zhuhong ointment on MMPs activities and production by HSF

HuaFu Shengji is the primary traditional Chinese medicine (TCM) therapy for treating chronic skin ulcer. The high activities of the protein enzyme in the wound fluids is one of the main cause of healing delay. In order to investigate the effect of TCM Zhuhong ointment for promoting wound healing. This research focused on its influence on matrix metalloproteinase (MMP) activities in wound fluids with TCM Yang syndromes, directly on the activated MMP-1,2 activities in vitro and on MMP-1,-2,-9 production by HSF. 8 wound fluid samples were collected, which were diagnosed Yang Syndromes in TCM. Wound fluid activities of MMP-2 and MMP-9 were measured by gelatin zymogram assay. MMP-1 and MMP-2 activities in vitro were measured by substrate cleavage. CCK-8 was used to observe the toxicity of Zhuhong ointment on HSF. MMP-1,-2,-9 production by HSF were detected by confocal microscope. Zhuhong ointment from 1 to 25 g x L(-1) obviously inhibited MMP-2 activity in wound fluid. When Zhuhong ointment was over 5 g x L(-1), it showed significantly inhibitory effect on wound fluid MMP-9 activity. In vitro study, when the mercury concentration was 320 mg x L(-1), Zhuhong ointment solution directly inhibited both MMP-1 activity and MMP-2. But mercury concentration from 0.51-2.56 mg x L(-1), it could activate MMP-1 activity, and from 0.51-64 mg x L(-1), activate MMP-2 activity instead. The mercury concentration when Zhuhong ointment saturated in DMEM was 39.6 mg x L(-1). When the mercury concentration was over 1.23 mg x L(-1), Zhuhong ointment showed toxicity to HSF. At 1.23, 0.62, 0.31 mg x L(-1) of mercury concentration, it increased MMP-1 expression by HSF, and at 1.23, 0.62 mg x L(-1), decreased MMP-2 expression. However, at 1.23, 0.62, 0.31 mg x L(-1), it decreased MMP-9 expression. At higher concentration, Zhuhong ointment can inhibit MMP-2, MMP-9 activities in wound fluid with dose-dependent way and show a direct inhibitory effect on activated MMP-1 and MMP-2 in vitro. But at a lower concentration, it showed two-way adjustment, with increased MMP-1, MMP-2 activities and its expression by HSF and decreased MMP-9 activity.

Three Pseudomonas aeruginosa strains with different protease profiles.

The proteolytic activity of three Pseudomonas aeruginosa strains, ATCC 27853 - a reference strain, and two clinical isolates was tested. The activity was examined after culturing the bacteria in two different growth media: the minimal M9 medium and rich Luria-Bertani broth (LB). Based on zymograms and protease activity specific assays, it was concluded that the reference strain produced three proteolytic enzymes in the LB medium: protease IV, elastase B and elastase A, while alkaline protease was only produced in the M9 medium. The clinical isolates of P. aeruginosa produced elastase B and alkaline protease when grown in the LB medium and the minimal M9 medium, respectively. PCR analysis confirmed the presence of both the lasB gene encoding elastase B and aprA coding for alkaline protease in the genomes of the three P. aeruginosa strains analyzed. The expression of these genes coding for two important P. aeruginosa virulence factors was dependent on the growth conditions in all the strains studied. The contribution of the extracellular proteinases to the virulence of P. aeruginosa strains used in this study was investigated using an insect model, the greater wax moth Galleria mellonella.

Arabinose is metabolized via a phosphoketolase pathway in Clostridium acetobutylicum ATCC 824

In this report, a novel zymogram assay and coupled phosphoketolase assay were employed to demonstrate that Clostridium acetobutylicum gene CAC1343 encodes a bi-functional xylulose-5-P/fructose-6-P phosphoketolase (XFP). The specific activity of purified recombinant XFP was 6.9U/mg on xylulose-5-P and 21U/mg on fructose-6-P, while the specific activity of XFP in concentrated C. acetobutylicum whole-cell extract was 0.094 and 0.52U/mg, respectively. Analysis of crude cell extracts indicated that XFP activity was present in cells grown on arabinose but not glucose and quantitative PCR was used to show that CAC1343 mRNA expression was induced 185-fold during growth on arabinose when compared to growth on glucose. HPLC analysis of metabolites revealed that during growth on xylose and glucose more butyrate than acetate was formed with final acetate:butyrate ratios of 0.72 and 0.83, respectively. Growth on arabinose caused a metabolic shift to more oxidized products with a final acetate:butyrate ratio of 1.95. The shift towards more oxidized products is consistent with the presence of an XFP, suggesting that arabinose is metabolized via a phosphoketolase pathway while xylose is probably metabolized via the pentose phosphate pathway.

Response of Fibroblasts to Transforming Growth Factor-β1 on Two-Dimensional and in Three-Dimensional Hyaluronan Hydrogels

Transforming growth factor-β1 (TGF-β1), an important cytokine with multiple functions, is secreted during wound healing. Previous studies have utilized two-dimensional (2D) cell culture to elucidate the functions of TGF-β1; however, 2D culture does not represent the complex three-dimensional (3D) in vivo environment. Using a synthetic hyaluronan (HA) extracellular matrix (ECM) hydrogel, we investigated the effect of TGF-β1 on fibroblasts cultured in three conditions--on tissue culture polystyrene (TCP), on HA (2D), and in HA (3D). After TGF-β1 treatment (0.1 to 20 ng/mL), morphological features and ECM regulation were analyzed by immunocytochemistry, Western blot, quantitative polymerase chain reaction, and zymogram assays. On TCP, cells showed the typical spindle shape with strong alpha smooth muscle actin (α-SMA) staining of cytoplasmic myofilaments along the cell axes after TGF-β1 treatment; on HA (2D), spindle-shape cells showed little α-SMA staining; in HA (3D), cells were smaller and rounded with less α-SMA deposition. The α-SMA gene and protein expression on TCP were significantly upregulated by TGF-β1, but TGF-β1 did not induce α-SMA expression in the presence of HA (both 2D and 3D). 3D HA culture significantly downregulated collagen I, III, and fibronectin expression, increased matrix metalloproteinase 1 and 2 (MMP1/MMP2) activity, upregulated MMP1 mRNA and downregulated TIMP3 mRNA expression. This study suggested that exogenous HA, particularly in 3D culture, appears to suppress ECM production, enhances ECM degradation and remodeling, and inhibits myofibroblast differentiation without decreasing TGF-β receptor expression.

Comprehensive survey of redox sensitive starch metabolising enzymes in Arabidopsis thaliana

In chloroplasts, the ferredoxin/thioredoxin pathway regulates enzyme activity in response to light by reduction of regulatory disulfides in target enzymes, ensuring coordination between photosynthesis and diurnal metabolism. Although earlier studies have suggested that many starch metabolic enzymes are similarly regulated, redox regulation has only been verified for a few of these invitro. Using zymograms and enzyme assays, we performed a comprehensive analysis of the redox sensitivity of known starch metabolising enzymes in extracts of Arabidopsis thaliana. Manipulation of redox potentials revealed that several enzymatic activities where activated by reduction at physiologically relevant potentials. Among these where the isoamylase complex AtISA1/AtISA2, the limit dextrinase AtLDA, starch synthases AtSS1 and AtSS3, and the starch branching enzyme AtBE2. The reversibility of the redox reaction was confirmed by enzyme assays for AtLDA, AtSS1 and AtSS3. Analysis of an AtBAM1 knock-out mutant identified an additional redox sensitive β-amylase activity, which was most likely AtBAM3. A similar requirement for reducing conditions was observed for recombinant chloroplastic α-amylase (AtAMY3) activity. This study adds further candidates to the list of reductively activated starch metabolising enzymes and supports the view that redox regulation plays a role in starch metabolism.

Role of c-Jun N-Terminal Protein Kinase 1/2 (JNK1/2) in Macrophage-Mediated MMP-9 Production in Response to Moraxella catarrhalis Lipooligosaccharide (LOS)

Moraxella catarrhalis is a Gram negative bacterium and a leading causative agent of otitis media (OM) in children. Recent reports have provided strong evidence for the presence of high levels of matrix metalloproteinase (MMPs) in effusion fluids from children suffering with OM, however, the precise mechanisms by which MMPs are generated are currently unknown. We hypothesized that MMPs are secreted from macrophages in the presence of M. catarrhalis lipooligosaccharide (LOS). In this report, we demonstrate that in vitro stimulation of murine macrophage RAW 264.7 cells with LOS leads to secretion of MMP-9 as determined by ELISA and zymogram assays. We have also shown that inhibition of ERK1/2 and p38 kinase completely blocked LOS induced MMP-9 production. In contrast, inhibition of JNK1/2 by the specific inhibitor SP600125 actually increased the level of expression and production of MMP-9 at both mRNA and protein levels, respectively by almost five fold. This latter result was confirmed by knocking down JNK1/2 using siRNA. Similar results have been observed in murine bone marrow derived macrophages in vitro. In contrast to and in parallel with the LOS-induced increased levels of MMP-9 in the presence of SP600125, we found a corresponding dose-dependent inhibition of TIMP-1 (tissue inhibitor of matrix metalloproteinase-1) secretion. Results of subsequent in vitro studies provided evidence that when JNK1/2 was inhibited prior to stimulation with LOS, it significantly increased both the extent of macrophage cell migration and invasion compared to control cells or cells treated with LOS alone. The results of these studies contribute to an increased understanding of the underlying pathophysiology of OM with effusion in children.

Inhibition of testicular and Vipera russelli snake venom hyaluronidase activity by Butea monosperma (Lam) Kuntze stem bark

This study was carried out to investigate the anti-fertility and anti-venom activities of the extract of the stem bark of Butea monosperma by inhibiting hyaluronidase, which is a spreading factor and plays a role in fertilisation. Among ethanol, methanol and water extracts, the ethanol extract dose-dependently inhibited the ovine, mouse testicular and Vipera russelli snake venom hyaluronidase enzyme activities, with IC50 values 12.00 ± 0.45, 49.40 ± 1.58 µg and 125.42 ± 2.82 µg mL–1, respectively. In a zymogram assay, the extract showed differential inhibition towards hyaluronidase isoform preferentially with low-molecular weight isoforms. The V. russelli snake venom-induced hemorrhage was significantly reduced at 1:05 ratio of venom-to-extract in mouse. The high antioxidant activity and total phenolic content in the ethanolic extract strongly correlated with the hyaluronidase inhibition. The above results justify the traditional use of the stem bark of B. monosperma as a contraceptive and a strong antidote to snake venom.

Isolation and characterization of a psychrophilic bacterium producing cold active lactose hydrolyzing enzyme from soil of Mt. Himalaya in Nepal

Psychrophilic bacteria, which grew on lactose as a carbon source, were isolated from soil of Island Peak area at Mt. Himalaya in Nepal, and identified. 148 bacterial isolates were isolated via X-gal hydrolysis on plate. Among these, three isolates, referred to as KNOUC 401, 402 and 403, displayed good cold-adapted growth and activity for hydrolysis of o-nitrophenyl-β-D-galactopyranoside (ONPG) and lactose in cell free extracts. Strain KNOUC403 showing negative activity in β-hemolysis was selected and identified. The strain was gram-positive bacilli, non-motile, catalase-positive and aerobic. Optimum growth was done at 20°C and pH 6.5~6.8. Anteiso-C15:0 (68.14%) was predominant in the fatty acid composition of cell. The physiological and biochemical properties, cellular fatty acid composition and sequence of 16S rDNA, suggest that strain KNOUC403 indicate a taxonomic affiliation to Arthrobacter psychrolactophilus. The optimal conditions for ONPG hydrolysis of cell free extracts from the strain were 15°C and pH 6.0, and the enzyme was stable at 4°C for 42 h. Zymogram assay of cell free extract from the strain in nondenaturing polyacrylamide gel showed a distinct band of X-gal hydrolysis. Key words: Psychrophilic bacteria, Arthrobacter psychrolactophilus, cold-active lactose hydrolyzing enzyme, Mt. Himalaya.

In Vitro antimetastatic effect of Changweiqing (肠胃清) through antiinvasion of hypoxic colorectal carcinoma LoVo cells

To investigate the in vitro effects and the primary mechanisms of Changweiqing (, CWQ) on antimetastasis and antiinvasion of hypoxic colon carcinoma cells. In addition, to provide experimental evidence for the Chinese medicinal theory of "strengthening the body's resistance to eliminate pathogenic factors" in the treatment of colorectal cancer, including its invasion and metastasis. First, CWQ sera were prepared with serum-pharmacology methods. Then, the modified hypoxic chamber was designed and flushed with 5% CO(2) and 95% N(2) at 37 °C to induce a hypoxic environment. The effect of CWQ serum on the viability of LoVo cells was tested with MTT cytotoxicity assay. The wound model and chamber model were established to estimate the effects of CWQ serum on migration and invasion of LoVo cells. The model for cell adhesion was established to evaluate the effect of CWQ serum on LoVo cells' adhesion. The gelatin zymography model was performed to determine the effects of CWQ serum on the activities of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9). The effects of CWQ serum on the hypoxia-inducible factor 1 α (HIF-1α) nuclear translocation and the mRNA level of vascular endothelial growth factor (VEGF) in LoVo cells were determined by Western blot and reverse transcription-polymerase chain reaction (RT-PCR) analyses, respectively. CWQ inhibited LoVo cells' migration based on wound healing assay. The inhibitive effect could reach about 68.00% under hypoxic culture and about 29.87% under normoxic culture when cells were treated with 10% CWQ serum for 24 h. The results from both cell invasion and adhesion assays showed that CWQ serum could dose-dependently repress the invasion of LoVo cells and inhibit cells from adhering to extra cellular matrix (ECM). Under the hypoxic culture condition, RT-PCR analysis showed that 10% CWQ serum had down-regulated the expression of VEGF by 45.87%, and the result of Western blot analysis provided further evidence. The HIF-1α amount in the nucleus of the LoVo cells was also diminished in a dose-dependent manner, as shown by the Western blot. Gel zymogram assay revealed that CWQ serum could suppress the activities of MMP-2 and MMP-9. CWQ could effectively inhibit tumor metastasis in vitro The antimetastatic effects of CWQ were associated with the inhibition of cell motility, which was evidenced by inhibition of cell invasion and adhesion. The molecular mechanisms of the inhibition of tumor invasion by CWQ were due to the reduced expression of both HIF-1α and VEGF and the suppression of MMP-2 and MMP-9 expression.

Urine matrix metalloproteinases (MMPs) as biomarkers for the progression of fracture healing

Whilst the majority of fractures heal normally, it is estimated that ∼10% of fractures exhibit some level of delayed or impaired healing. Although radiography is the primary diagnostic tool to assess the progression of fracture healing, radiographic features only qualitatively correlate with tissue level increases in mineral content and do not quantitatively measure underlying biological processes that are associated with the progression of healing. Specific metaloproteinases have been shown to be essential to processes of both angiogenesis and mineralised cartilage resorption and bone remodelling at different phases of fracture healing. The aim of this study was to determine the potential of using a simple urine based assay of the activity of two MMPs as a means of assessing the biological progression of fracture healing through the endochondral phase of healing. Using a standard mid-diaphyseal murine model of femoral fracture, MMP9 and MMP13 proteins and enzymatic activity levels were quantified in the urine of mice across the time-course of fracture healing and compared to the mRNA and protein expression profiles in the calluses. Both urinary MMP9 and MMP13 protein and enzymatic activity levels, assessed by Western blot, zymogram and specific MMP fluorometric substrate assays, corresponded to mRNA expression and immunohistologic assays of the proteins within callus tissues. These studies suggest that urinary levels of MMP9 and MMP13 may have potential as metabolic markers to monitor the progression of fracture healing.

Anti-Metalloproteinase-9 Activities of Selected Indonesian Zingiberaceae Rhizome Extracts in Lipopolysaccharide-Induced Human Vascular Endothelial Cells <i>In Vitro</i>

Problem statement: Atherosclerosis is associated with chronic inflammation triggered by bacterial infection that activates the breakdown of extracellular matrix protein by matrix Metalloproteinases (MMPs). Zingiberaceae, a group of tropical food crops grown in Indonesia and other Southeast Asia regions, has been traditionally used for food coloring, seasoning, culinary and medicinal purposes. However, its efficacy as natural vascular protection has not been explored. Approach: The research was aimed to investigate the effects of 10 Indonesian Zingiberaceae rhizome extracts on inhibition of MMP-9 expression in human vascular endothelial cells treated with Lipopolysaccharide (LPS) in vitro by conducting gelatin zymogram, Western blotting and RT-PCR assays. Results: LPS (2 μg mL−1) significantly elevated the expression of MMP-9 secretion, protein and mRNA in the vascular endothelial cells. Selected Zingiberaceae exctracts (5 μg mL−1), i.e., Curcuma xanthorrhiza, C. aeruginosa, C. mangga, C. longa, Kaempferia galanga, Alpinia galanga and Zingiberaceae officinale, effectively attenuated the expression of MMP-9 secretion, protein and mRNA in LPS-induced vascular endothelial cells. Furthermore, MMP-9 expression was specifically blocked by MAPK inhibitors, i.e., PD98059 (ERK1/2 inhibitor), SB203580 (p38 inhibitor), SP600125 (JNK inhibitor) and PI3K inhibitor (LY294002), indicating that MAPK and PI3K signaling pathways are involved in regulation of MMP-9 gene expression in LPS-induced vascular endothelial cells. Conclusion: These results suggest that selected Indonesian Zingiberaceae rhizomes with potent MMP- 9 inhibitory activity may scientifically offer the promising therapeutic target in vascular diseases, particularly atherosclerosis.

Identification of an RNase J ortholog in Sulfolobus solfataricus: Implications for 5′-to-3′ directional decay and 5′-end protection of mRNA in Crenarchaeota

In both Bacteria and Eukaryotes, degradation is known to start at the 5' and at the 3' extremities of mRNAs. Until the recent discovery of 5'-to-3' exoribonucleases in hyperthermophilic Euryarchaeota, the exosome was assumed to be the key enzyme in mRNA degradation in Archaea. By means of zymogram assays and bioinformatics, we have identified a 5'-to-3' exoribonuclease activity in the crenarchaeum Sulfolobus solfataricus (Sso), which is affected by the phosphorylation state of the 5'-end of the mRNA. The protein comprises typical signature motifs of the β-CASP family of metallo-β-lactamases and was termed Sso-RNAse J. Thus, our study provides the first evidence for a 5'-to-3' directional mRNA decay pathway in the crenarchaeal clade of Archaea. In Bacteria the 5'-end of mRNAs is often protected by a tri-phosphorylated 5'-terminus and/or by stem-loop structures, while in Eukaryotes the cap-binding complex is responsible for this task. Here, we show that binding of translation initiation factor a/eIF2(γ) to the 5'-end of mRNA counteracts the 5'-to-3' exoribonucleolytic activity of Sso-RNase J in vitro. Hence, 5'-to-3' directional decay and 5'-end protection appear to be conserved features of mRNA turnover in all kingdoms of life.

Streptococcus pyogenes Ser/Thr Kinase-regulated Cell Wall Hydrolase Is a Cell Division Plane-recognizing and Chain-forming Virulence Factor

Cell division and cell wall synthesis are closely linked complex phenomena and play a crucial role in the maintenance and regulation of bacterial virulence. Eukaryotic-type Ser/Thr kinases reported in prokaryotes, including that in group A Streptococcus (GAS) (Streptococcus pyogenes Ser/Thr kinase (SP-STK)), regulate cell division, growth, and virulence. The mechanism of this regulation is, however, unknown. In this study, we demonstrated that SP-STK-controlled cell division is mediated under the positive regulation of secretory protein that possesses a cysteine and histidine-dependent aminohydrolases/peptidases (CHAP) domain with functionally active cell wall hydrolase activity (henceforth named as CdhA (CHAP-domain-containing and chain-forming cell wall hydrolase). Deletion of the CdhA-encoding gene resulted in severe cell division and growth defects in GAS mutants. The mutant expressing the truncated CdhA (devoid of the CHAP domain), although displayed no such defects, it became attenuated for virulence in mice and highly susceptible to cell wall-acting antibiotics, as observed for the mutant lacking CdhA. When CdhA was overexpressed in the wild-type GAS as well as in heterologous strains, Escherichia coli and Staphylococcus aureus, we observed a distinct increase in bacterial chain length. Our data reveal that CdhA is a multifunctional protein with a major function of the N-terminal region as a cell division plane-recognizing domain and that of the C-terminal CHAP domain as a virulence-regulating domain. CdhA is thus an important therapeutic target.

Transforming Growth Factor-β1 Bioassay Involving Matrix Metalloproteinase-2 Induction

The transforming growth factor-β1 (TGF-β1) bioassay developed in this study monitors increased luciferase activity in MCF10A cells containing the matrix metalloproteinase-2 (MMP-2) promoter with a luciferase reporter and treated with increasing TGF-β1 concentrations. The response was linear in the concentration range from 75 to 2,500 pg/mL. The abilities of 3 types of TGF-β in inducing MMP-2 were different. The luciferase activity induced by TGF-β1 was about 2 times more than that by TGF-β2 and TGF-β3. The MMP-2 promoter bioassay showed greater reproducibility (coefficient of variation [CV] 10%) than the previously developed anticell proliferation assay of TF-1 cell (CV 16%) and the MMP-2 zymogram assay (CV 40%).

Biochemical and molecular characterization of Coriolopsis rigida laccases involved in transformation of the solid waste from olive oil production

Two laccase isoenzymes were purified and characterized from the basidiomycete Coriolopsis rigida during transformation of the water-soluble fraction of "alpeorujo" (WSFA), a solid residue derived from the olive oil production containing high levels of toxic compounds. Zymogram assays of laccases secreted by the fungus growing on WSFA and WSFA supplemented with glucose showed two bands with isoelectric points of 3.3 and 3.4. The kinetic studies of the two purified isoenzymes showed similar affinity on 2,6-dimethoxyphenol and 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid), used as phenolic and non-phenolic model substrate, respectively. The molecular mass of both proteins was 66 kDa with 9% N-linked carbohydrate. Physico-chemical properties of the purified laccases from media containing WSFA were similar to those obtained from medium with glucose as the main carbon source. In-vitro studies performed with the purified laccases revealed a 42% phenol reduction of WSFA, as well as changes in the molecular mass distribution. These findings indicate that these laccases are involved in the process of transformation, via polymerization by the oxidation of phenolic compounds present in WSFA. A single laccase gene, containing an open reading frame of 1,488 bp, was obtained in PCR amplifications performed with cDNA extracted from mycelia grown on WSFA. The product of the gene shares 90% identity (95% similarity) with a laccase from Trametes trogii and 89% identity (95% similarity) with a laccase from Coriolopsis gallica. This is the first report on purification and molecular characterization of laccases directly involved in the transformation of olive oil residues.

LysK CHAP endopeptidase domain is required for lysis of live staphylococcal cells

LysK is a staphylococcal bacteriophage endolysin composed of three domains: an N-terminal cysteine, histidine-dependent amidohydrolases/peptidases (CHAP) endopeptidase domain, a midprotein amidase 2 domain, and a C-terminal SH3b_5 (SH3b) cell wall-binding domain. Both catalytic domains are active on purified peptidoglycan by positive-ion electrospray ionization MS. The cut sites are identical to LytA (phi11 endolysin), with cleavage between d-alanine of the stem peptide and glycine of the cross-bridge peptide, and N-acetylmuramoyl-l-alanine amidase activity. Truncations of the LysK containing just the CHAP domain lyse Staphylococcus aureus cells in zymogram analysis, plate lysis, and turbidity reduction assays but have no detectable activity in a minimal inhibitory concentration (MIC) assay. In contrast, truncations harboring just the amidase lytic domain show faint activity in both the zymogram and turbidity reduction assays, but no detectable activity in either plate lysis or MIC assays. A fusion of the CHAP domain to the SH3b domain has near full-length LysK lytic activity, suggesting the need for a C-terminal binding domain. Both LysK and the CHAP-SH3b fusion were shown to lyse untreated S. aureus and the coagulase-negative strains. In the checkerboard assay, the CHAP-SH3b fusion achieves the same level of antimicrobial synergy with lysostaphin as the full-length LysK.

Kaempferia pandurata Roxb. inhibits Porphyromonas gingivalis supernatant-induced matrix metalloproteinase-9 expression via signal transduction in human oral epidermoid cells

Ethnopharmacological relevance Kaempferia pandurata Roxb., an edible tropical medicinal plant (Zingiberaceae), has been traditionally used to treat dental caries. However, its efficacy on inhibition of matrix metalloproteinase (MMP)-9 for preventing periodontal inflammation has been rarely studied to date. Aim of the study This present study was focused to investigate the effects of the ethanolic extract of Kaempferia pandurata Roxb. on inhibition of Porphyromonas gingivalis supernatant-induced MMP-9 expression through activation of mitogen-activated protein kinase (MAPK) signaling pathways in human oral epidermoid KB cells. Materials and methods MMP-9 expression and its signaling pathways in KB cells induced by Porphyromonas gingivalis supernatant were determined by gelatin zymogram, Western blotting and reporter gene assays. Results Kaempferia pandurata significantly decreased MMP-9 expression at both protein and mRNA levels in a dose-dependent manner. Kaempferia pandurata interfered Porphyromonas gingivalis supernatant-induced MMP-9 expression in KB cells by downregulating MAPK phosphorylation (extracellular signal-related kinase 1/2, p38 kinase and c-Jun N-terminal kinase), inhibiting transcriptional expression (Elk1, c-Jun and c-Fos), and blocking AP-1 and NF-κB activities. Conclusions Kaempferia pandurata could be employed as a candidate for MMP-9 inhibitor with therapeutic potential for treatment of periodontal inflammation.