Pro-MMP-2 activation by the PPARgamma agonist, ciglitazone, induces cell invasion through the generation of ROS and the activation of ERK

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a nuclear receptor modulating a variety of biological functions including cancer cell proliferation and differentiation. However, the role of PPARgamma and its ligands in tumor invasion is unclear. To evaluate a possible role for PPARgamma ligands in tumor invasion, we examined whether PPARgamma agonists including pioglitazone, troglitazone, rosiglitazone, and ciglitazone could affect the activity of matrix metalloproteinases (MMPs) in the HT1080 cell line, a well-studied and well-characterized cell line for MMP research. The gelatin zymography assay showed that ciglitazone activated pro-MMP-2 significantly. In addition, ciglitazone increased the expression of MMP-2, which was accompanied by an increase of membrane type 1-MMP (MT1-MMP) expression. The PPARgamma antagonist, GW9662 attenuated the ciglitazone-induced PPARgamma activation but it did not affect the pro-MMP2 activation by ciglitazone, suggesting that the action of ciglitazone on the pro-MMP-2 activation bypassed the PPARgamma pathway. Antioxidants and various inhibitors of signal transduction were used to investigate the mechanism of ciglitazone-induced pro-MMP-2 activation. We found that the sustained production of reactive oxygen species (ROS) was required for pro-MMP-2 activation by ciglitazone. We also found that PB98059, an inhibitor of MEK-ERK, significantly blocked ciglitazone-induced pro-MMP-2 activation and that extracellular signal-regulated kinase (ERK) was hyperphosphorylated by ciglitazone. Moreover, cell invasion was significantly increased by ciglitazone in the HT1080 cell lines, whereas cell motility was not affected. This study suggests that ciglitazone-induced pro-MMP-2 activation increases PPARgamma-independent tumor cell invasion through ROS production and ERK activation in some types of cancer cells.     

Autophagy protects against redox-active trace metal-induced cell death in rabbit synovial fibroblasts through Toll-like receptor 4 activation

Reactive oxygen species (ROS) are implicated to play a role in initiating rheumatoid arthritis (RA) pathogenesis. We have investigated the mechanism(s) by which essential redox-active trace metals (RATM) may induce cell proliferation and cell death in rabbit synovial fibroblasts. These fibroblast-like synovial (FLS) cells, which express Toll-like receptor 4 (TLR4), were used as a model system that plays a role in potentially initiating RA through oxidative stress. Potassium peroxychromate (PPC, [Cr⁵⁺]), ferrous chloride (FeCl₂, [Fe²⁺]), and cuprous chloride (CuCl, [Cu⁺]) in the indicated valency states were used as exogenous pro-oxidants that can induce oxidative stress through TLR4 coupled activation that also causes HMGB1 release. We measured the proliferation index (PI) of FLS, and examined the effect of RATM oxidants on apoptosis and autophagy by fluorescence cell-sorting flow cytometry (FC). Cell cycle was analysed by FC and autophagy-related protein expression levels were measured by western blot. Our data showed that as RATM as prooxidants increased intracellular ROS (iROS) that can induce oxidative stress. Whereas iROS increased PI in FLS, these reactive species also protected cells against apoptosis by inducing autophagy. Our results indicate that ROS/TLR4-coupled activation may contribute to the pathogenesis of RA in FLS by induction of autophagy. The signalling pathway by which inflammation and its tissue destructive sequel may occur in RA underlies the need for developing therapeutic agents that can inhibit release of tissue-damaging high mobility group box 1 (HMGB1), cytokines, and possess both trace metal chelating capacity and oxidant scavenging properties in a directed combinatorial therapy for RA.     

Alterations to plasma membrane lipid contents affect the biophysical properties of erythrocytes from individuals with hypertension

Genetic and environmental factors may contribute to high blood pressure, which is termed essential hypertension. Hypertension is a major independent risk factor for cardiovascular disease, stroke and renal failure; thus, elucidation of the etiopathology of hypertension merits further research. We recently reported that the platelets and neutrophils of patients with hypertension exhibit altered biophysical characteristics. In the present study, we assessed whether the major structural elements of erythrocyte plasma membranes are altered in individuals with hypertension. We compared the phospholipid (phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, sphingosine) and cholesterol contents of erythrocytes from individuals with hypertension (HTN) and healthy individuals (HI) using LC/MS-MS. HTN erythrocytes contained higher phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine contents and a lower cholesterol content than HI erythrocytes. Furthermore, atomic force microscopy revealed important morphological changes in HTN erythrocytes, which reflected the increased membrane fragility and fluidity and higher levels of oxidative stress observed in HTN erythrocytes using spectrophotofluorometry, flow cytometry and spectrometry. This study reveals that alterations to the lipid contents of erythrocyte plasma membranes occur in hypertension, and these alterations in lipid composition result in morphological and physiological abnormalities that modify the dynamic properties of erythrocytes and contribute to the pathophysiology of hypertension.     

α-bisabolol β-D-fucopyranoside as a potential modulator of β-amyloid peptide induced neurotoxicity: An in vitro & in silico study

Alzheimer’s disease (AD) is a multifaceted neurodegenerative disorder affecting the elderly people. For the AD treatment, there is inefficiency in the existing medication, as these drugs reduce only the symptoms of the disease. Since multiple pathological proteins are involved in the development of AD, searching for a single molecule targeting multiple AD proteins will be a new strategy for the management of AD. In view of this, the present study was designed to synthesize and evaluate the multifunctional neuroprotective ability of the sesquiterpene glycoside α-bisabolol β-D-fucopyranoside (ABFP) against multiple targets like acetylcholinesterase, oxidative stress and β-amyloid peptide aggregation induced cytotoxicity. In silico computational docking and simulation studies of ABFP with acetylcholinesterase (AChE) showed that it can interact with Asp74 and Thr75 residues of the enzyme. The in vitro studies showed that the compound possess significant ability to inhibit the AChE enzyme apart from exhibiting antioxidant, anti-aggregation and disaggregation properties. In addition, molecular dynamics simulation studies proved that the interacting residue between Aβ peptide and ABFP was found to be involved in Leu34 and Ile31. Furthermore, the compound was able to protect the Neuro2 a cells against Aβ_25-35 peptide induced toxicity. Overall, the present study evidently proved ABFP as a neuroprotective agent, which might act as a multi-target compound for the treatment of Alzheimer’s disease.     

The novel economical synthesis and antimicrobial activity of a trithiocarbonate derivative

The compound diethyl 2,2′-(thiocarbonyl-bis(sulfanediyl))-diacetate 4 belongs to the trithiocarbonate class containing a trithiocarbonate function group flanked by ethyl acetate. In this procedure, a novel economic synthesis route to obtain compound 4 is described. This compound proved to possess broad-spectrum antimicrobial activity both in vitro and in vivo, and could be used as a lead compound. It is worth mentioning that this compound has been patented [No. US 9,988,348 B1; date of patent: June 5, 2018].     

Esterases as a marker for growth of BY-2 tobacco cells and early somatic embryos of the Norway spruce

Growth is one of the basic properties of biological systems. The methods which are commonly used for the determination of growth are usually difficult and not very accurate. In the present work we decided to use esterase activity as a growth marker in tobacco suspension culture (BY-2 line) and in early somatic embryos of Norway spruce (clone 2/32) grown on a semi-solid medium. Esterase activity correlates well with the classical growth characteristics of BY-2 and spruce early somatic embryos. Determination of esterase activity is based on spectrophotometric and spectrofluorimetric detection of reaction products, which arise from the enzymatic hydrolysis of two substrates (p -nitrophenyl acetate and fluorescein diacetate) by esterase. The spectrophotometric method enabled us to detect approximately 10⁴ BY-2 cells and 25 spruce embryos whereas the more sensitive spectrofluorimetric method allowed us to detect approximately 800 BY-2 cells and 5 early somatic embryos of Norway spruce.     

Abnormal growth of polyamine-deficient Escherichia coli mutant is partially caused by oxidative stress-induced damage

Polyamines participate in numerous cellular processes and are required for normal cell growth in Escherichia coli. In this study, we constructed a new polyamine-deficient E. coli mutant and investigated the physiological function of polyamines during normal aerobic growth conditions. We showed that the requirement for sulfur-containing, branched chain, and aromatic amino acids, which was exhibited in the sodA sodB double mutant faced with severe oxidative stress, was also true of the polyamine-deficient mutant during normal aerobic cell growth. Sorbitol, sucrose, mannose, 1,2-dihydroxybenzene-3,5-disulfonic acid (Tiron), an antioxidant that functions as an oxygen radical scavenger including z.rad;O(2)(-), and thiamine partially relieved the cell growth defect caused by polyamine depletion in a dose-dependent manner. As was the case for the cells treated with paraquat, the mutant had an elongated shape compared with the polyamine-proficient wild type. Decreased aeration also relieved the cell growth defect of the polyamine-deficient mutant. Finally, we confirmed that chloromethyl-2('),7(')-dichlorofluorescin diacetate (DCFH-DA), which is oxidized in a fluorescent product in the presence of various oxidants, also fluoresce in the polyamine-deficient cells. These results showed that abnormal growth of the polyamine-deficient E. coli mutant results partially from oxidative stress-induced damage and the mutant thus exhibits the requirement for antioxidant or specific nutritional amino acid during normal aerobic growth.     

Effects of mercury on microbial biomass and enzyme activities in soil

Mercury (Hg) is a persistent soil pollutant that affects soil microbial activity. We monitored the changes in soil microbial biomass and activity of enzymes, including alkaline phosphatase, arylsulfatase, fluorescein diacetate (FDA) hydrolytic activity, and o-diphenol oxidase (o-DPO) in three soils contaminated with different concentrations of Hg. Increasing levels of Hg, from 0.5 to 10 μmol/g of dried soil, generally depressed microbial activity; however, the effects of Hg on soil microbial activity depended on soil type and composition, particularly organic matter content. o-DPO was less affected by Hg than the other three enzymes tested. Our results indicate that the analysis of microbial biomass content and soil-enzyme activities may be used to predict the soil quality contaminated with Hg.     

Quantitative assessment of marine sponge cells in vitro: Development of improved growth medium

Summary As sources of natural products with potential human therapeutic value, marine sponges are important subjects for cell culture studies. A critical component of any cell culture system is its growth medium. Proceeding from the hypotheses that the thawed, cryopreserved, primary cells would display detectable differential responses and that those responses could be comparatively quantified, this study has established that multiwell screening assays are useful tools for improving medium formulations in cell cultures of the marine sponge, Teichaxinella morchella. Fluorescent probe signals were correlated with known cell densities and viabilities in a 96-well format. Analysis of variance and post-test methods were applied to judge the significance of signal differences seen in a variety of medium formulations. Results from a series of experiments suggested that reducing glutamine and selenium concentrations in the standard medium would result in greater DNA, protein, and esterase activity signals. This was confirmed by the direct comparison of the standard and improved medium formulations. Significantly higher protein content and esterase activity were associated with the improved medium. DNA content was also higher, though not significantly. The result is a new medium formulation that may be more able to support cell growth and division, providing an improved cell culture system for marine sponge cell studies. The assays can be used in additional studies to further improve the in vitro conditions for marine sponge cell culture.     

NAD-linked alcohol dehydrogenase 1 regulates methylglyoxal concentration in Candida albicans

We purified a fraction that showed NAD⁺-linked methylglyoxal dehydrogenase activity, directly catalyzing methylglyoxal oxidation to pyruvate, which was significantly increased in glutathione-depleted Candida albicans. It also showed NADH-linked methylglyoxal-reducing activity. The fraction was identified as a NAD⁺-linked alcohol dehydrogenase (ADH1) through mass spectrometric analyses. In ADH1-disruptants of both the wild type and glutathione-depleted cells, the intracellular methylglyoxal concentration increased significantly; defects in growth, differentiation, and virulence were observed; and G2-phase arrest was induced.