Ming J. Wu

Rat pancreatic stellate cells secrete matrix metalloproteinases: implications for extracellular matrix turnover

Background: Pancreatic fibrosis is a characteristic feature of chronic pancreatic injury and is thought to result from a change in the balance between synthesis and degradation of extracellular matrix (ECM) proteins. Recent studies suggest that activated pancreatic stellate cells (PSCs) play a central role in pancreatic fibrogenesis via increased synthesis of ECM proteins. However, the role of these cells in ECM protein degradation has not been fully elucidated. Aims: To determine: (i) whether PSCs secrete matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) and, if so (ii) whether MMP and TIMP secretion by PSCs is altered in response to known PSC activating factors such as tumour necrosis factor α (TNF-α), transforming growth factor β1 (TGF-β1), interleukin 6 (IL-6), ethanol, and acetaldehyde. Methods: Cultured rat PSCs (n=3–5 separate cell preparations) were incubated at 37°C for 24 hours with serum free culture medium containing TNF-α (5–25 U/ml), TGF-β1 (0.5–1 ng/ml), IL-6 (0.001–10 ng/ml), ethanol (10–50 mM), or acetaldehyde (150–200 μM), or no additions (controls). Medium from control cells was examined for the presence of MMPs by zymography using a 10% polyacrylamide-0.1% gelatin gel. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to examine gene expression of MMP9 and the tissue inhibitors of metalloproteinases TIMP1 and TIMP2. Western blotting was used to identify a specific MMP, MMP2 (a gelatinase that digests basement membrane collagen and the dominant MMP observed on zymography) and a specific TIMP, TIMP2. Reverse zymography was used to examine functional TIMPs in PSC secretions. The effect of TNF-α, TGF-β1, and IL-6 on MMP2 secretion was assessed by densitometry of western blots. The effect of ethanol and acetaldehyde on MMP2 and TIMP2 secretion was also assessed by this method. Results: Zymography revealed that PSCs secrete a number of MMPs including proteinases with molecular weights consistent with MMP2, MMP9, and MMP13. RT-PCR demonstrated the presence of mRNA for metalloproteinase inhibitors TIMP1 and TIMP2 in PSCs while reverse zymography revealed the presence of functional TIMP2 in PSC secretions. MMP2 secretion by PSCs was significantly increased by TGF-β1 and IL-6, but was not affected by TNF-α. Ethanol and acetaldehyde induced secretion of both MMP2 and TIMP2 by PSCs. Conclusions: Pancreatic stellate cells have the capacity to synthesise a number of matrix metalloproteinases, including MMP2, MMP9, and MMP13 and their inhibitors TIMP1 and TIMP2. MMP2 secretion by PSCs is significantly increased on exposure to the proinflammatory cytokines TGF-β1 and IL-6. Both ethanol and its metabolite acetaldehyde increase MMP2 as well as TIMP2 secretion by PSCs. Implication: The role of pancreatic stellate cells in extracellular matrix formation and fibrogenesis may be related to their capacity to regulate the degradation as well as the synthesis of extracellular matrix proteins.

Antioxidant and anti-inflammatory activities of selected medicinal plants containing phenolic and flavonoid compounds

The antioxidant, anti-inflammatory, and cytotoxic activities of water and ethanol extracts of 14 Chinese medicinal plants were investigated and also their total phenolics and flavonoid contents measured. The antioxidant activity was evaluated in a biological assay using Saccharomyces cerevisiae , whereas the radical scavenging activity was measured using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method. Total phenolics and flavonoid contents were estimated by Folin-Ciocalteu and aluminum chloride methods, respectively. The anti-inflammatory activities of the plant extracts were determined by measuring the inhibition of production of nitric oxide (NO) and TNF-α in LPS and IFN-γ activated RAW 264.7 macrophages. Their cytotoxic activities against macrophages were determined by Alamar Blue assay. Four plants, namely, Scutellaria baicalensis , Taxillus chinensis , Rheum officinale , and Sophora japonica , showed significant antioxidant activity in both yeast model and also free radical scavenging methods. The ethanol extract of S. japonica showed highest levels of phenolics and flavonoids (91.33 GAE mg/g and 151.86 QE mg/g, respectively). A positive linear correlation between antioxidant activity and the total phenolics and flavonoid contents indicates that these compounds are likely to be the main antioxidants contributing to the observed activities. Five plant extracts (S. baicalensis, T. chinensis, S. japonica, Mahonia fortunei , and Sophora flavescens ) exhibited significant anti-inflammatory activity by in vitro inhibition of the production of NO and TNF-α with low IC(50) values. These findings suggest that some of the medicinal herbs studied in this paper are good sources of antioxidants.

Cell migration: a novel aspect of pancreatic stellate cell biology

Background: Pancreatic stellate cells (PSCs), implicated as key mediators of pancreatic fibrogenesis, are found in increased numbers in areas of pancreatic injury. This increase in PSC number may be due to increased local proliferation and/or migration of these cells from adjacent areas. The ability of PSCs to proliferate has been well established but their potential for migration has not been examined. Aims: Therefore, the aims of this study were to determine whether cultured rat PSCs have the capacity to migrate and, if so, to characterise this migratory capacity with respect to the influence of basement membrane components and the effect of platelet derived growth factor (PDGF, a known stimulant for migration of other cell types). Methods: Migration of freshly isolated (quiescent) and culture activated (passaged) rat PSCs was assessed across uncoated or Matrigel (a basement membrane-like substance) coated porous membranes (pore size 8 μm) in the presence or absence of PDGF (10 and 20 ng/ml) in the culture medium. A checkerboard assay was performed to assess whether the effect of PDGF on PSC migration was chemotactic or chemokinetic. Results: Cell migration was observed with both freshly isolated and passaged PSCs. However, compared with passaged (culture activated) cells, migration of freshly isolated cells was delayed, occurring only at or after 48 hours of incubation when the cells displayed an activated phenotype. PSC migration through Matrigel coated membranes was delayed but not prevented by basement membrane components. PSC migration was increased by PDGF and this effect was predominantly chemotactic (that is, in the direction of a positive concentration gradient). Conclusions: (i) PSCs have the capacity to migrate. (ii) Activation of PSCs appears to be a prerequisite for migration. (iii) PDGF stimulates PSC migration and this effect is predominantly chemotactic. Implication: Chemotactic factors released during pancreatic injury may stimulate the migration of PSCs through surrounding basement membrane towards affected areas of the gland.

Antioxidant and anti-inflammatory activities of selected Chinese medicinal plants and their relation with antioxidant content

BackgroundThe main aim of this study is to evaluate the antioxidant and anti-inflammatory properties of forty four traditional Chinese medicinal herbal extracts and to examine these activities in relation to their antioxidant content.MethodsThe antioxidant activities were investigated using DPPH radical scavenging method and yeast model. The anti-inflammatory properties of the herbal extracts were evaluated by measuring their ability to inhibit the production of nitric oxide and TNF-α in RAW 264.7 macrophages activated by LPS and IFN- γ, respectively. The cytotoxic effects of the herbal extracts were determined by Alomar Blue assay by measuring cell viability. In order to understand the variation of antioxidant activities of herbal extracts with their antioxidant contents, the total phenolics, total flavonoids and trace metal (Mg, Mn, Cu, Zn, Se and Mo) quantities were estimated and a correlation analysis was carried out.ResultsResults of this study show that significant levels of phenolics, flavonoids and trace metal contents were found in Ligustrum lucidum, Paeonia suffuticosa, Salvia miltiorrhiza, Sanguisorba officinalis, Spatholobus suberectus, Tussilago farfara and Uncaria rhyncophylla, which correlated well with their antioxidant and anti-inflammatory activities. Some of the plants displayed high antioxidant and anti-inflammatory activities but contained low levels of phenolics and flavonoids. Interestingly, these plants contained significant levels of trace metals (such as Zn, Mg and Se) which are likely to be responsible for their activities.ConclusionsThe results indicate that the phenolics, flavonoids and trace metals play an important role in the antioxidant activities of medicinal plants. Many of the plants studied here have been identified as potential sources of new antioxidant compounds.

The antimicrobial properties of some copper(II) and platinum(II) 1,10-phenanthroline complexes

Copper(II) (1(Cu)-21(Cu)) and previously established experimental anticancer platinum(II) metallointercalator complexes (1(Pt)-16(Pt)) have been prepared and investigated for their antimicrobial properties. These complexes are of the general structure [M(I(L))(A(L))](2+) where I(L) represents functionalised 1,10-phenanthrolines (1(IL)-10(IL)), and A(L) represents 1,2-diaminoethane, 1S,2S- or 1R,2R-diaminocyclohexane. The structures of synthesised complexes were confirmed using a combination of elemental analysis, UV spectrometry, circular dichroism, (1)H and [(1)H-(195)Pt]-HMQC NMR, X-ray crystallography, and electrospray ionisation mass spectrometry and where appropriate. Crystallisation attempts yielded single crystals of [Cu(4-methyl-1,10-phenanthroline)(1R,2R-diaminocyclohexane)](ClO(4))(2) (4(Cu)), [Cu(5,6-dimethyl-1,10-phenanthroline)(1R,2R-diaminocyclohexane)(H(2)O)](ClO(4))(2)·1.5H(2)O (10(Cu)) and [Cu(5,6-dimethyl-1,10-phenanthroline)(3)](ClO(4))(2)·5,6-dimethyl-1,10-phenanthroline·2H(2)O (21(Cu)). Growth inhibition of liquid cultures of bacteria (Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa), and yeast (Saccharomyces cerevisiae) discerned the most antimicrobially potent metal complexes ≤20 μM, as well as that of their intercalating ligands alone. To further investigate their mode of antimicrobial activity, membrane permeabilisation caused by selected complexes was visualised by means of a cell viability kit under fluorescence microscopy.

An antioxidant screening assay based on oxidant-induced growth arrest in Saccharomyces cerevisiae

This report describes a biological screening system to measure the antioxidant capacity of compounds using the oxidant-induced growth arrest response of Saccharomyces cerevisiae. Alternative methods using the nonphysiological free radical compounds such as diphenylpicrylhydrazyl and azinobis ethylbenzothiaziline-6-sulphonate (ABTS) only provide an indication of the ability of a compound to scavenge oxidants. In contrast, this yeast-based method can also measure the ability of a compound to induce cellular resistance to the damaging effects of oxidants. The screening assay was established against a panel of six physiologically relevant oxidants ranging from reactive oxygen species (hydrogen peroxide, cumene peroxide, linoleic acid hydroperoxide), to a superoxide-generating agent (menadione), reactive nitrogen species (peroxynitrite) and a thiol-oxidizing agent (diamide). The antioxidants ascorbate and gallic acid displayed scavenging activity and induced the resistance of cells against a broad range of oxidants using this assay. Lipoic acid, which showed no scavenging activity and thus would not be detected as an antioxidant using a nonphysiological screen was, however, identified in this assay as providing resistance to cells against a range of oxidants. This assay is high throughput, in the format of a 96-well microtitre plate, and will greatly facilitate the search for effective antioxidants.

Identification of a Protein with Antioxidant Activity that is Important for the Protection against Beer Ageing

This study was carried out with fresh Australian lager beer which was sampled directly off the production line, the same samples aged for 12 weeks at 30 °C, and the vintage beer which was kept at 20 °C for 5 years. Characteristic Australian lager flavour was maintained in the fresh and vintage beers but was lost in the aged beer. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and free thiol group labelling analyses of beer proteins found that this flavour stability correlated with the presence of an unknown 10 kilodaltons (kDa) protein with a higher level of free thiols. The protein was purified by size-exclusion chromatography, then peptide sequencing and database matching identified it as the barley lipid transfer protein (LTP1). Further characterisation using diphenylpicrylhydrazyl (DPPH) free radical scavenging and a Saccharomyces cerevisiae-based antioxidant screening assay demonstrated that the LTP1 protein was active in DPPH reduction and antioxidant activity. The absence of free thiol in the aged beer indicates that the thiol functional groups within the LTP1 protein were saturated and suggests that it is important in the flavour stability of beer by maintaining reduction capacity during the ageing process.

Different Reactive Oxygen Species Lead to Distinct Changes of Cellular Metal Ions in the Eukaryotic Model Organism Saccharomyces cerevisiae

Elemental uptake and export of the cell are tightly regulated thereby maintaining the ionomic homeostasis. This equilibrium can be disrupted upon exposure to exogenous reactive oxygen species (ROS), leading to reduction or elevation of the intracellular metal ions. In this study, the ionomic composition in the eukaryotic model organism Saccharomyces cerevisiae was profiled using the inductively-coupled plasma optical emission spectrometer (ICP-OES) following the treatment with individual ROS, including hydrogen peroxide, cumen hydroperoxide, linoleic acid hydroperoxide (LAH), the superoxide-generating agent menadione, the thiol-oxidising agent diamide [diazine-dicarboxylic acid-bis(dimethylamide)], dimedone and peroxynitrite. The findings demonstrated that different ROS resulted in distinct changes in cellular metal ions. Aluminium (Al3+) level rose up to 50-fold after the diamide treatment. Cellular potassium (K+) in LAH-treated cells was 26-fold less compared to the non-treated controls. The diamide-induced Al3+ accumulation was further validated by the enhanced Al3+ uptake along the time course and diamide doses. Pre-incubation of yeast with individual elements including iron, copper, manganese and magnesium failed to block diamide-induced Al3+ uptake, suggesting Al3+-specific transporters could be involved in Al3+ uptake. Furthermore, LAH-induced potassium depletion was validated by a rescue experiment in which addition of potassium increased yeast growth in LAH-containing media by 26% compared to LAH alone. Taken together, the data, for the first time, demonstrated the linkage between ionomic profiles and individual oxidative conditions.

Transcriptomic insights into the molecular response of Saccharomyces cerevisiae to linoleic acid hydroperoxide

Abstract Eukaryotic microorganisms are constantly challenged by reactive oxygen species derived endogenously or encountered in their environment. Such adversity is particularly applied to Saccharomyces cerevisiae under harsh industrial conditions. One of the major oxidants to challenge S. cerevisiae is linoleic acid hydroperoxide (LoaOOH). This study, which used genome-wide microarray analysis in conjunction with deletion mutant screening, uncovered the molecular pathways of S. cerevisiae that were altered by an arresting concentration of LoaOOH (75 μM). The oxidative stress response, iron homeostasis, detoxification through PDR transport and direct lipid β-oxidation were evident through the induction of the genes encoding for peroxiredoxins (GPX2, TSA2), the NADPH:oxidoreductase (OYE3), iron uptake (FIT2, ARN2, FET3), PDR transporters (PDR5, PDR15, SNQ2) and β-oxidation machinery (FAA2, POX1). Further, we discovered that Gpx3p, the dual redox sensor and peroxidase, is required for protection against LoaOOH, indicated by the sensitivity of gpx3Δ to a mild dose of LoaOOH (37.5 μM). Deletion of GPX3 conferred a greater sensitivity to LoaOOH than the loss of its signalling partner YAP1. Deletion of either of the iron homeostasis regulators AFT1 or AFT2 also resulted in sensitivity to LoaOOH. These novel findings for Gpx3p, Aft1p and Aft2p point to their distinct roles in response to the lipid peroxide. Finally, the expression of 89 previously uncharacterised genes was significantly altered against LoaOOH, which will contribute to their eventual annotation.

The antimicrobial efficacy and DNA binding activity of some copper(II) complexes of 3,4,7,8-tetramethyl-1,10-phenanthroline, 4,7-diphenyl-1,10-phenanthroline and 1,2-diaminocyclohexane

Four copper(II) complexes of the general structure [Cu(L1)(L2)]2+, where L1 is (1S,2S)-diaminocyclohexane or (1R,2R)-diaminocyclohexane and L2 is 3,4,7,8-tetramethyl-1,10-phenanthroline (TMP) or 4,7-diphenyl-1,10-phenanthroline (DIP), have been investigated in this study for their antimicrobial activity, short-term antimicrobial efficacy, and in vitro DNA-binding affinity. Against an expanded panel of bacterial and fungal strains in 12 species, minimal inhibitory concentrations (MIC) for these metallocomplexes were determined. The data confirmed our previous finding that they are effective against Gram-positive bacteria (MIC 5.6-13.1μM), with DIP coordinated complexes more so than TMP counterparts. Additionally, novel and significant findings were obtained here for these copper(II) complexes. While the four metallocomplexes exhibited high anti-Candida yeast activity (MIC 13.1-26.1μM), they demonstrated stronger anti-fungal activity against the drug-resistant Candida krusei (MIC 13.1μM and 22.6μM for TMP and DIP complexes, respectively) than the anti-fungal agent, 5-fluorocytosine. Fluorescence cell viability assays revealed that these complexes exert faster antibacterial effect than ampicillin as their inhibition against Staphylococcus aureus and Enterococcus faecalis were significantly evident within 0.5h of exposure compared to ampicillin. Similarly, these complexes but not ampicillin demonstrated bactericidal activity in non-proliferating conditions. All complexes exhibited DNA binding affinities similar to that of the known DNA intercalator, ethidium bromide (Ka ~105M-1) in linear dichroism binding studies and fluorescent dye displacement assays. Taken together, these findings imply that the four copper(II) complexes have different modes of action to the established antibiotics such as ampicillin and 5-fluorocytosine, and provide further insight into development of effective antimicrobial metallocomplexes.