Studies on the binding and degradation of human very-low-density lipoproteins by human hepatoma cell line HepG2

The regulation of the hepatic catabolism of normal human very-low-density lipoproteins (VLDL) was studied in human-derived hepatoma cell line HepG2. Concentration-dependent binding, uptake and degradation of 125I-labeled VLDL demonstrated that the hepatic removal of these particles proceeds through both the saturable and non-saturable processes. In the presence of excess unlabeled VLDL, the specific binding of 125-labeled VLDL accounted for 72% of the total binding. The preincubation of cells with unlabeled VLDL had little effect on the expression of receptors, but reductive methylation of VLDL particles reduced their binding capacity. Chloroquine and colchicine inhibited the degradation of 125I-labeled VLDL and increased their accumulation in the cell, indicating the involvement of lysosomes and microtubuli in this process. Receptor-mediated degradation was associated with a slight (13%) reduction in de novo sterol synthesis and had no significant effect on the cellular cholesterol esterification. Competition studies demonstrated the ability of unlabeled VLDL, low-density lipoproteins (LDL) and high-density lipoproteins (HDL) to effectively compete with 125I-labeled VLDL for binding to cells. No correlation was observed between the concentrations of apolipoproteins A-I, A-II, C-I, C-II and C-III of unlabeled lipoproteins and their inhibitory effect on 125I-labeled VLDL binding. When unlabeled VLDL, LDL and HDL were added at equal contents of either apolipoprotein B or apolipoprotein E, their inhibitory effect on the binding and uptake of 125I-labeled VLDL only correlated with apolipoprotein E. Under similar conditions, the ability of unlabeled VLDL, LDL and HDL to compete with 125I-labeled LDL for binding was a direct function of only their apolipoprotein B. These results demonstrate that in HepG2 cells, apolipoprotein E is the main recognition signal for receptor-mediated binding and degradation of VLDL particles, while apolipoprotein B functions as the sole recognition signal for the catabolism of LDL. Furthermore, the lack of any substantial regulation of beta-hydroxy-beta-methylglutaryl-CoA reductase and acyl-CoA:cholesterol acyltransferase activities subsequent to VLDL degradation, in contrast to that observed for LDL catabolism, suggests that, in HepG2 cells, the receptor-mediated removal of VLDL proceeds through processes independent of those involved in LDL catabolism.     

Stoichiometry and absolute quantification of proteins with mass spectrometry using fluorescent and isotope-labeled concatenated peptide standards

We have explored a general approach for the determination of absolute amounts and the relative stoichiometry of proteins in a mixture using fluorescence and mass spectrometry. We engineered a gene to express green fluorescent protein (GFP) with a synthetic fusion protein (GAB-GFP) in Escherichia coli to function as a spectroscopic standard for the quantification of an analogous stable isotope-labeled, non-fluorescent fusion protein (GAB*) and for the quantification and stoichiometric analysis of purified transducin, a heterotrimeric G-protein complex. Both GAB-GFP and GAB* contain concatenated sequences of specific proteotypic peptides that are derived from the alpha, beta, and gamma protein subunits of transducin and that are each flanked by spacer regions that maintain the native proteolytic properties for these peptide fragments. Spectroscopic quantification of GAB-GFP provided a molar scale for mass spectrometric ratios from tryptic peptides of GAB* and defined molar responses for mass spectrometric signal intensities from a purified transducin complex. The stoichiometry of transducin subunits alpha, beta, and gamma was measured to be 1:1.1:1.15 over a 5-fold range of labeled internal standard with a relative standard deviation of 9%. Fusing a unique genetically coded spectroscopic signal element with concatenated proteotypic peptides provides a powerful method to accurately quantify and determine the relative stoichiometry of multiple proteins present in complexes or mixtures that cannot be readily assessed using classical gravimetric, enzymatic, or antibody-based technologies.     

Methanol Extracts of 28 Hieracium Species from the Balkan Peninsula – Comparative LC–MS Analysis,Chemosystematic Evaluation of their Flavonoid and Phenolic Acid Profiles and Antioxidant Potentials

Introduction

Hieracium s. str. represents one of the largest and most complex genera of flowering plants. As molecular genetics seems unlikely to disentangle intricate relationships within this reticulate species complex, analysis of flavonoids and phenolic acids, known as good chemosystematic markers, promise to be more reliable. Data about pharmacological activity of Hieracium species are scarce.

Objective

Evaluation of the chemosystematic significance of flavonoids and phenolic acids of methanol extracts of aerial flowering parts of 28 Hieracium species from the Balkans. Additionally, investigation of antioxidant potentials of the extracts.

Methods

Comparative qualitative and quantitative analysis of flavonoids and phenolic acids was performed by LC–MS. Multivariate statistical data analysis included non‐metric multidimensional scaling (nMDS), unweighted pair‐group arithmetic averages (UPGMA) and principal component analysis (PCA). Antioxidant activity was evaluated using three colorimetric tests.

Results

Dominant phenolics in almost all species were luteolin type flavonoids, followed by phenolic acids. Although the investigated Hieracium species share many compounds, the current classification of the genus was supported by nMDS and UPGMA analyses with a good resolution to the group level. Hieracium naegelianum was clearly separated from the other investigated species. Spatial and ecological distances of the samples were likely to influence unexpected differentiation of some groups within H. sect. Pannosa. The vast majority of dominant compounds significantly contributed to differences between taxa. The antioxidant potential of the extracts was satisfactory and in accordance with their phenolics composition.

Conclusions

Comparative LC–MS analysis demonstrated that flavonoids and phenolic acids are good indicators of chemosystematic relationships within Hieracium, particularly between non‐hybrid species and groups from the same location. Copyright © 2017 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.     

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.     

Neurogranin Regulates Alcohol Sensitivity through AKT Pathway in the Nucleus Accumbens

Dysfunction of glutamate neurotransmission in the nucleus accumbens (NAc) has been implicated in the pathophysiology of alcohol use disorders (AUD). Neurogranin (Ng) is exclusively expressed in the brain and mediates N‐methyl‐d ‐aspartate receptor (NMDAR) hypo‐function by regulating the intracellular calcium‐calmodulin (Ca²⁺‐CaM) pathway. Ng null mice (Ng^–/– mice) demonstrate increased alcohol drinking compared to wild‐type mice, while also showing less tolerance to the effect of alcohol. To identify the molecular mechanism related to alcohol seeking, both in vivo microdialysis and label‐free quantification proteomics comparing Ng genotype and effects of alcohol treatment on the NAc are utilized. There is significant difference in glutamate and gamma‐aminobutyric acid (GABA) neurotransmission between genotypes; however, alcohol administration normalizes both glutamate and GABA levels in the NAc. Using label‐free proteomics, 427 protein expression changes are identified against alcohol treatment in the NAc among 4347 total proteins detected. Bioinformatics analyses reveal significant molecular differences in Ng null mice in response to acute alcohol treatment. Ingenuity pathway analysis found that the AKT network is altered significantly between genotypes, which may increase the sensitivity of alcohol in Ng null mice. The pharmacoproteomics results presented here illustrate a possible molecular basis of the alcohol sensitivity through Ng signaling in the NAc.     

CD133 suppression increases the sensitivity of prostate cancer cells to paclitaxel

Molecular Biology Reports - One of the major barriers in cancer therapy is the resistance to conventional therapies and cancer stem cells (CSCs) are among the main causes of this problem. CD133 as...     

A constitutive expression system for high‐throughput screening

To reduce the amount of consumables and number of pipetting steps in high‐throughput screening, a constitutive expression system was developed that comprises four different promoters of varying strength. The system was validated by the expression of different sucrose phosphorylase enzymes from Leuconostoc mesenteroides, Lactobacillus acidophilus and Bifidobacterium adolescentis in 96‐deep‐ and low‐well plates at three temperatures. Drastically improved soluble expression in mini‐cultures was observed for the enzymes from L. mesenteroides strains by reducing the promoter strength from strong to intermediate and by expressing the proteins at lower temperatures. In contrast, the enzymes from B. adolescentis and L. acidophilus were expressed most efficiently with a strong promoter. The constitutive expression of sucrose phosphorylases in low‐well plates resulted in a level of activity that is equal or even better than what was achieved by inducible expression. Therefore, our plasmid set with varying constitutive promoters will be an indispensable tool to optimize enzyme expression for high‐throughput screening.     

Pharmacological Induction of Transforming Growth Factor-Beta1 in Rat Models Enhances Radiation Injury in the Intestine and the Heart

Radiation therapy in the treatment of cancer is dose limited by radiation injury in normal tissues such as the intestine and the heart. To identify the mechanistic involvement of transforming growth factor-beta 1 (TGF-β1) in intestinal and cardiac radiation injury, we studied the influence of pharmacological induction of TGF-β1 with xaliproden (SR 57746A) in rat models of radiation enteropathy and radiation-induced heart disease (RIHD). Because it was uncertain to what extent TGF-β induction may enhance radiation injury in heart and intestine, animals were exposed to irradiation schedules that cause mild to moderate (acute) radiation injury. In the radiation enteropathy model, male Sprague-Dawley rats received local irradiation of a 4-cm loop of rat ileum with 7 once-daily fractions of 5.6 Gy, and intestinal injury was assessed at 2 weeks and 12 weeks after irradiation. In the RIHD model, male Sprague-Dawley rats received local heart irradiation with a single dose of 18 Gy and were followed for 6 months after irradiation. Rats were treated orally with xaliproden starting 3 days before irradiation until the end of the experiments. Treatment with xaliproden increased circulating TGF-β1 levels by 300% and significantly induced expression of TGF-β1 and TGF-β1 target genes in the irradiated intestine and heart. Various radiation-induced structural changes in the intestine at 2 and 12 weeks were significantly enhanced with TGF-β1 induction. Similarly, in the RIHD model induction of TGF-β1 augmented radiation-induced changes in cardiac function and myocardial fibrosis. These results lend further support for the direct involvement of TGF-β1 in biological mechanisms of radiation-induced adverse remodeling in the intestine and the heart.     

MUTYH and the mismatch repair system: partners in crime?

Biallelic germline mutations of MUTYH—a gene encoding a base excision repair protein—are associated with an increased susceptibility of colorectal cancer. Whether monoallelic MUTYH mutations also increase cancer risk is not yet clear, although there is some evidence suggesting a slight increase of risk. As the MUTYH protein interacts with the mismatch repair (MMR) system, we hypothesised that the combination of a monoallelic MUTYH mutation with an MMR gene mutation increases cancer risk. We therefore investigated the prevalence of monoallelic MUTYH mutations in carriers of a germline MMR mutation: 40 carriers of a truncating mutation (group I) and 36 of a missense mutation (group II). These patients had been diagnosed with either colorectal or endometrial cancer. We compared their MUTYH mutation frequencies with those observed in a group of 134 Dutch colorectal and endometrial cancer patients without an MMR gene mutation (0.7%) and those reported for Caucasian controls (1.5%). In group I one monoallelic MUTYH mutation was found (2.5%). In group II five monoallelic germline MUTYH mutations were found (14%), four of them in MSH6 missense mutation carriers (20%). Of all patients with an MMR gene mutation, only those with a missense mutation showed a significantly higher frequency of (monoallelic) MUTYH mutations than the Dutch cancer patients without MMR gene mutations (P=0.002) and the published controls (P=0.001). These results warrant further study to test the hypothesis of mutations in MMR genes (in particular MSH6) and MUTYH acting together to increase cancer risk.