Effect of neohesperidin dihydrochalcone on the activity and stability of alpha‐amylase: a comparative study on bacterial,fungal, and mammalian enzymes

Neohesperidin dihydrochalcone (NHDC) was recently introduced as an activator of mammalian alpha‐amylase. In the current study, the effect of NHDC has been investigated on bacterial and fungal alpha‐amylases. Enzyme assays and kinetic analysis demonstrated the capability of NHDC to significantly activate both tested alpha‐amylases. The ligand activation pattern was found to be more similar between the fungal and mammalian enzyme in comparison with the bacterial one. Further, thermostability experiments indicated a stability increase in the presence of NHDC for the bacterial enzyme. In silico (docking) test locates a putative binding site for NHDC on alpha‐amylase surface in domain B. This domain shows differences in various alpha‐amylase types, and the different behavior of the ligand toward the studied enzymes may be attributed to this fact. Copyright © 2015 John Wiley & Sons, Ltd.     

Curcumin nanofibers for the purpose of wound healing

Poor wound healing is a highly prevalent clinical problem with, as yet, no entirely satisfactory solution. A new technique, termed electrospinning, may provide a solution to improve wound healing. Due to their large surface area to volume ratio and porosity, the nanofibers created by electrospinning are able to deliver sustained drug release and oxygen to the wound. Using different types of polymers with varying properties helps strengthening nanofiber and exudates absorption. The nanofibers appear to have an ideal structure applicable for wound healing and, in combination with curcumin, can blend the anti-inflammatory and antioxidant properties of curcumin into a highly effective wound dressing. The use of suitable curcumin solvents and the slow release of curcumin from the nanofiber help in overcoming the known limitations of curcumin, specifically its low stability and limited bioavailability. Here, we review the studies which have been done on synthesized nanofibers containing curcumin, produced by the electrospinning technique, for the purpose of wound healing.     

Promoter methylation analysis of WNT/β-catenin pathway regulators and its association with expression of DNMT1 enzyme in colorectal cancer

Background

Aberrant DNA methylation as the most important reason making epigenetic silencing of genes is a main mechanism of gene inactivation in patients with colorectal cancer. In this study, we decided to identify promoter methylation status of ten genes encoding WNT negative regulators, and measure the expression of DNMT1 enzyme in colorectal cancer samples.

Results

Aberrant methylation of APC gene was statistically significant associated with age over 50 (p = 0.017), DDK3 with male (p < 0.0001), SFRP4, WIF1, and WNT5a with increasing tumor stage (p = 0.004, p = 0.029, and p = 0.004), SFRP4 and WIF1 with tumor differentiation (p = 0.009 and p = 0.031) and SFRP2 and SFRP5 with histological type (p = 0.001 and p = 0.025). The increasing number of methylated genes correlated with the expression levels of the DNMT1 mRNA.

Conclusions

The rate of gene promoter methylation of WNT pathway regulators is high in colorectal cancer cells. Hyper-methylation is associated with increased expression of the DNMT1 enzyme.     

Olfactory response of Phytoseiulus persimilis (Acari: Phytoseiidae) to untreated and Beauveria bassiana-treated Tetranychus urticae (Acari: Tetranychidae)

Determination of attraction and avoidance behavior of predators is important in concomitant use of multiple natural enemies to control a pest. The olfactory response of the predatory mite Phytoseiulus persimilis was studied to odors related to Tetranychus urticae adults infected by Beauveria bassiana DEBI008 in 0, 24, 48 and 72 h intervals, both in absence and in presence of plants. In plant-present experiments, P. persimilis attraction was neither towards adults of T. urticae infected by 0.02 % Tween 80 (as control), nor to the ones infected by B. bassiana for 0 or 24 h, whereas significant attraction towards the control was observed when tested against T. urticae infected by B. bassiana for 48 or 72 h. In absence of plants, P. persimilis displayed significant avoidance of T. urticae infected by B. bassiana for 48 or 72 h, when their alternative option was 0.02 % Tween 80-infected T. urticae adults. These results indicate that P. persimilis can recognize the presence of B. bassiana and that the predator avoids the fungus. This suggests that the two natural enemy species can be used together in biological control programmes.     

Myricetin ameliorates high glucose-induced endothelial dysfunction in human umbilical vein endothelial cells

Endothelial dysfunction is recognized as the initial detectable stage of cardiovascular disease, a serious complication of diabetes. In this study, we evaluated effects of myricetin on high glucose (HG)-elicited oxidative damage in human umbilical vein endothelial cells (HUVECs). The cells were pre-incubated with myricetin and then treated with HG to induce apoptosis. The effect of myricetin on viability was investigated by MTT assay. The levels of lipid peroxidation (LPO) were determined by thiobarbituric acid (TBA) method. The protein expression of Bax, Bcl-2 and caspase-3 was measured by western blot analysis. Moreover, the effect of myricetin on total antioxidant capacity (TAC) and total thiol molecules was also determined. Our results showed that myricetin was able to markedly restore the viability of endothelial cells under oxidative stress. Myricetin reduced HG-caused increase in LPO levels. Also, TAC and total thiol molecules were notably elevated by myricetin. Incubation with myricetin decreased the protein expression levels of Bax, whereas it increased the expression levels of the Bcl-2, compared with HG treatment alone. Furthermore, myricetin significantly decreased cleaved caspase-3 protein expression. It is concluded that myricetin may protect HUVECs from oxidative stress induced by HG via increasing cell TAC and reducing Bax/Bcl-2 protein ratio, and caspase-3 expression.     

Amyloid fibrillation in native and chemically-modified forms of carbonic anhydrase II: Role of surface hydrophobicity

Chemical modification or mutation of proteins may bring about significant changes in the net charge or surface hydrophobicity of a protein structure. Such events may be of major physiological significance and may provide important insights into the genetics of amyloid diseases. In the present study, fibrillation potential of native and chemically-modified forms of bovine carbonic anhydrase II (BCA II) were investigated. Initially, various denaturing conditions including low pH and high temperatures were tested to induce fibrillation. At a low pH of around 2.4, where the protein is totally dissociated, the apo form was found to take up a pre-molten globular (PMG) conformation with the capacity for fibril formation. Upon increasing the pH to around 3.6, a molten globular (MG) form became abundant, forming amorphous aggregates. Charge neutralization and enhancement of hydrophobicity by methylation, acetylation and propionylation of lysine residues appeared very effective in promoting fibrillation of both the apo and holo forms under native conditions, the rates and extents of which were directly proportional to surface hydrophobicity, and influenced by salt concentration and temperature. These modified structures underwent more pronounced fibrillation under native conditions, than the PMG intermediate form, observed under denaturing conditions. The nature of the fibrillation products obtained from intermediate and modified structures were characterized and compared and their possible cytotoxicity determined. Results are discussed in terms of the importance of surface net charge and hydrophobicity in controlling protein aggregation. A discussion on the physiological significance of the observations is also presented.     

Evaluation of five different cDNA labeling methods for microarrays using spike controls

Background

Several different cDNA labeling methods have been developed for microarray based gene expression analysis. We have examined the accuracy and reproducibility of such five commercially available methods in detection of predetermined ratio values from target spike mRNAs (A. thaliana) in a background of total RNA. The five different labeling methods were: direct labeling (CyScribe), indirect labeling (FairPlay? – aminoallyl), two protocols with dendrimer technology (3DNA^® Array 50? and 3DNA^® submicro?), and hapten-antibody enzymatic labeling (Micromax? TSA?). Ten spike controls were mixed to give expected Cy5/Cy3 ratios in the range 0.125 to 6.0. The amounts of total RNA used in the labeling reactions ranged from 5 – 50 μg.

Results

The 3DNA array 50 and CyScribe labeling methods performed best with respect to relative deviation from the expected values (16% and 17% respectively). These two methods also displayed the best overall accuracy and reproducibility. The FairPlay method had the lowest total experimental variation (22%), but the estimated values were consistently higher than the expected values (36%). TSA had both the largest experimental variation and the largest deviation from the expected values (45% and 48% respectively).

Conclusion

We demonstrate the usefulness of spike controls in validation and comparison of cDNA labeling methods for microarray experiments.

     

A fluorescence-based assay for indoleamine 2,3-dioxygenase

A rapid and sensitive fluorescence-based bioassay for determination of indoleamine 2,3-dioxygenase (IDO) activity has been developed. This assay relies on the quantification of the amount of kynurenine produced in the assay medium by fluorescence and complements the standard absorbance and high-performance liquid chromatography (HPLC) assay methods. The fluorescence method has limits of detection similar to those of the standard assay methods. Measured activities of IDO, including in the presence of tryptophan-based inhibitors, were in statistical agreement with the absorbance and HPLC assay methods. The fluorescence-based assay was also suitable for assessment of IDO inhibition by compounds that are incompatible with the absorbance method.     

Flavonoid Baicalein Modulates H<Subscript>2</Subscript>O<Subscript>2</Subscript>-Induced Mitogen-Activated Protein Kinases Activation and Cell Death in SK-N-MC Cells

It is believed that ROS-induced oxidative stress triggers numerous signaling pathways which are involved in neurodegenerative diseases, including Alzheimer’s disease. To find the effective drugs for neurodegenerative diseases, the deep delve into molecular mechanisms underlie these diseases is necessary. In the current study, we investigated the effects of flavonoid baicalein on H₂O₂-induced oxidative stress and cell death in SK-N-MC cells. Our results revealed that the treatment of SK-N-MC cells with H₂O₂ led to a decrease in cell viability through phosphorylation and activation of extracellular signal-regulated kinases (ERKs) and c-Jun N-terminal kinases (JNKs) pathways followed by increase in Bax/Bcl2 ratio and initiation of caspase-dependent apoptotic pathways. In addition, our results showed that the exposure of SK-N-MC cells to H₂O₂ ended up in reduction of glutathione (GSH) levels of SK-N-MC cells via JNK/ERK-mediated down-regulation of γ-glutamyl-cysteine synthetase (γ-GCS) expression. Our results demonstrated that flavonoid baicalein protected against H₂O₂-induced cell death by inhibition of JNK/ERK pathways activation and other key molecules in apoptotic pathways, including blockage of Bax and caspase-9 activation, induction of Bcl-2 expression and prevention of cell death. Baicalein supported intracellular defense mechanisms through maintaining GSH levels in SK-N-MC cells by the removal of inhibition effects of JNK/ERK pathways from γ-GCS expression. In addition, baicalein attenuated lipid and protein peroxidation and intracellular reactive oxygen species in SK-N-MC cells. In accordance with these observations, baicalein can be a promising candidate in antioxidant therapy and designing of natural-based drug for ROS-induced neurodegenerative disorders.