Xanthine oxidase, a therapeutic target of realgar for non-small cell lung cancer.

Background: The effects of realgar against non-small cell lung cancer (NSCLC) have been massively studied, but the direct therapeutic targets of realgar remain unclear. This study aimed to identify the molecular targets of realgar against NSCLC and explore their therapeutic mechanisms based on a network pharmacology approach and experimental validations. Methods: The BATMAN-TCM and Digsee databases were used to predict realgar targets and NSCLC-related genes, respectively. A protein-protein interaction network was constructed for each gene set, and the overlapping genes were identified as potential targets of realgar against NSCLC. The correlation between potential targets and NSCLC was analyzed using The Cancer Genome Atlas and International Cancer Genome Consortium databases, and the key target was validated by in-silico and in-vitro experiments. Results: Twenty-three overlapping genes, including xanthine oxidase (XO), were identified as potential targets of realgar against NSCLC. XO was selected as the key target for validation, as it was found to be upregulated in NSCLC tumor tissue, which correlated with poor overall survival. A possible interaction between realgar and XO was revealed by molecular docking which was further validated experimentally. Realgar treatment suppressed the activity of XO in NSCLC cells, as demonstrated by the unchanged XO protein levels. Finally, the mechanism of action of XO as a target against NSCLC through the cell-cell junction organization pathway was investigated. Conclusions: Overall, this study proposes a potential molecular mechanism illustrating that XO is a target of realgar against NSCLC and highlights the usefulness of XO as a therapeutic target for NSCLC.

Proteins linked to spatial memory formation of CD1 mice in the multiple T-maze.

In own previous work CD1 mice were tested in the Multiple T-maze (MTM), a robust land maze allowing determination of latency to reach the goal box with food reward and to evaluate correct decisions made on the way to the goal box. Herein, hippocampi of these animals were used for the current study with the aim to investigate differences in protein levels between trained and yoked mice and, moreover, to determine differences in protein levels between trained and yoked mice with and without memory formation in the MTM. Three training sessions were carried out for four training days each, followed by probe trials on Days 5 and 12. Good and no-performers in the MTM were separated based on means and median of latency to reach the goal box on probe trial Day 12. Six hours following the probe trial on Day 12, animals were sacrificed and hippocampi were taken. Proteins were extracted and run on two-dimensional gel electrophoresis, spots were quantified and differentially expressed proteins were identified by mass spectrometry using an ion trap. Levels of 17 proteins were significantly different in trained vs. yoked mice. Seven proteins were differentially expressed comparing trained vs. yoked mice from good and no-performers. A series of proteins were significantly correlated with latency and may link these proteins to spatial memory formation. Differential protein expression in trained vs. yoked mice and in good and no-performers may allow insight into spatial memory formation as well as represent tentative pharmacological targets.

Renal differentiation of amniotic fluid stem cells: perspectives for clinical application and for studies on specific human genetic diseases.

BACKGROUND: Owing to growing rates of diabetes, hypertension and the ageing population, the prevalence of end-stage renal disease, developed from earlier stages of chronic kidney disease, and of acute renal failure is dramatically increasing. Dialysis and preferable renal transplantation are widely applied therapies for this incurable condition. However these options are limited because of morbidity, shortage of compatible organs and costs. Therefore, stem cell-based approaches are becoming increasingly accepted as an alternative therapeutic strategy. DESIGN: This review summarizes the current findings on the nephrogenic potential of amniotic fluid stem (AFS) cells and their putative implications for clinical applications and for studies on specific human genetic diseases. RESULTS: Since their discovery in 2003, AFS cells have been shown to be pluripotent with the potential to form embryoid bodies. Compared to adult stem cells, induced pluripotent stem cells or embryonic stem cells, AFS cells harbour a variety of advantages, such as their high differentiation and proliferative potential, no need for ectopic induction of pluripotency and no somatic mutations and epigenetic memory of source cells, and no tumourigenic potential and associated ethical controversies, respectively. CONCLUSIONS: Recently, the results of different independent studies provided evidence that AFS cells could indeed be a powerful tool for renal regenerative medicine.

Strain-independent global effect of hippocampal proteins in mice trained in the Morris water maze.

A series of individual proteins have been linked to performance in the Morris water maze (MWM) but no global effects have been reported. It was therefore the aim of the study to show which proteins were strain-independent, global factors for training in the MWM. Strains C57BL/6J, apodemus sylvaticus and PWD/PhJ were used. MWM and gels from trained animals were from a previous own study and corresponding yoked groups were generated. Hippocampal proteins were extracted and run on two-dimensional gel electrophoresis. Spots with different expressional levels between trained and yoked groups were punched and identified by mass spectrometry (nano-LC-ESI-MS/MS, ion trap). Two-way ANOVA with two factors (strain and training) was carried out and a Bonferroni test was used to compare groups. 12 proteins from several pathways and cascades showed different levels in trained mice versus corresponding yoked animals in all strains tested. Four out of these proteins were verified by immunoblotting: beta-synuclein, profilin 2, nucleoside diphosphate kinase A (NME1) and isocitrate dehydrogenase 3. Four proteins verified by immunoblotting could be shown to be involved in training in the MWM as a global effect, independent of the strain tested.

Characterization, using comparative proteomics, of differentially expressed proteins in the hippocampus of the mesial temporal lobe of epileptic rats following treatment with valproate.

The objective of the study was to explore the pathogenesis of mesial temporal lobe epilepsy (MTLE) and the mechanism of valproate administration in the early stage of MTLE development. We performed a global comparative analysis and function classification of differentially expressed proteins using proteomics. MTLE models of developmental rats were induced by lithium-pilocarpine. Proteins in the hippocampus were separated by 2-DE technology. PDQuest software was used to analyze 2-DE images, and MALDI-TOF-MS was used to identify the differentially expressed proteins. Western blot was used to determine the differential expression levels of synapse-related proteins synapsin-1, dynamin-1 and neurogranin in both MTLE rat and human hippocampus. A total of 48 differentially expressed proteins were identified between spontaneous and non-spontaneous MTLE rats, while 41 proteins between MTLE rats and post valproate-treatment rats were identified. All of the proteins can be categorized into several groups by biological functions: synaptic and neurotransmitter release, cytoskeletal structure and dynamics, cell junctions, energy metabolism and mitochondrial function, molecular chaperones, signal regulation and others. Western blot results were similar to the changes noted in 2-DE. The differentially expressed proteins, especially the proteins related to synaptic and neurotransmitter release function, such as synapsin-1, dynamin-1 and neurogranin, are probably involved in the mechanism of MTLE and the pharmacological effect of valproate. These findings may provide important clues to elucidate the mechanism of chronic MTLE and to identify an optimum medication intervention time and new biomarkers for the development of pharmacological therapies targeted at epilepsy.

[Immortalized mouse brain endothelial cell line Bend.3 displays the comparative barrier characteristics as the primary brain microvascular endothelial cells].

OBJECTIVE: The purpose of this study was to assess weather the immortalized mouse brain endothelial cell line Bend.3 displays the comparative barrier characteristics as the primary brain microvascular endothelial cells (BEMC). METHODS: Immortalized mouse brain endothelial cell line, Bend.3 cells were cultured in transwell inserts and their restrictive characteristics were assessed by transendothelial electrical resistance (TEER) and horseradish peroxidase (HRP) permeability assays. Western blot and direct fluorescent staining methods were used to detect the tight junction protein expression and F-actin distribution. RESULTS: The TEER in Bend.3 cells increased with the prolonged culture time and increased to 82.3+/-6.0 Omega cm2 10 days after culture, which was significantly higher than that 3 days after culture (37.3+/-3.1 Omega cm2; P<0.05). There were significant differences in the permeability rates for HRP 3 and 10 days after culture (4.3+/-0.20)% vs (2.2+/-0.05)% (P<0.05). Western blot indicated high level expression of tight junction proteins occludin and ZO-1 in Bend.3 cells 10 days after culture. F-actin was visualized around the cell membrane and presented scrobiculate linear fluorescence 10 days after culture. CONCLUSIONS: Bend.3 cells have similar barrier characteristics to BEMC, and their barrier function may reach to the best effect 10 days after culture.

mTORC1 is essential for early steps during Schwann cell differentiation of amniotic fluid stem cells and regulates lipogenic gene expression.

Schwann cell development is hallmarked by the induction of a lipogenic profile. Here we used amniotic fluid stem (AFS) cells and focused on the mechanisms occurring during early steps of differentiation along the Schwann cell lineage. Therefore, we initiated Schwann cell differentiation in AFS cells and monitored as well as modulated the activity of the mechanistic target of rapamycin (mTOR) pathway, the major regulator of anabolic processes. Our results show that mTOR complex 1 (mTORC1) activity is essential for glial marker expression and expression of Sterol Regulatory Element-Binding Protein (SREBP) target genes. Moreover, SREBP target gene activation by statin treatment promoted lipogenic gene expression, induced mTORC1 activation and stimulated Schwann cell differentiation. To investigate mTORC1 downstream signaling we expressed a mutant S6K1, which subsequently induced the expression of the Schwann cell marker S100b, but did not affect lipogenic gene expression. This suggests that S6K1 dependent and independent pathways downstream of mTORC1 drive AFS cells to early Schwann cell differentiation and lipogenic gene expression. In conclusion our results propose that future strategies for peripheral nervous system regeneration will depend on ways to efficiently induce the mTORC1 pathway.

The molecular background of the differential UV absorbance of the human lens in the 240-400 nm range.

The ultraviolet (UV) absorption of various sections of the human lens was studied and compared with protein expression paralleling differential UV absorbance in anterior and posterior lenticular tissue. The UV absorbance of serial lens cryostat sections (60 µm) and that of lens capsules was determined using a Shimadzu scanning spectrophotometer, and the absorption coefficients were calculated. Two-dimensional gel electrophoresis was performed using two pooled lenticular protein extracts (anterior and posterior sections). Protein spots were quantified and significantly different spots were identified by mass spectrometry following in-gel digestion with trypsin and chymotrypsin. The UV-C and UV-B absorption of the human lens increased toward the posterior parts of the lens. The anterior and posterior lens capsules also effectively absorbed UV radiation. Levels of molecular chaperone proteins Beta-crystallin B2 (UniProtKB ID:P43320), A3 (UniProtKB ID:P05813) and of glyceraldehyde 3-phosphate dehydrogenase (UniProtKB ID:P04406) were significantly higher in the anterior part of the lens, whereas lens proteins Beta-crystallin B1 (UniProtKB ID:P53674) and Alpha-crystallin A chain (UniProtKB ID:P02489) were higher in the posterior sections. These results provide evidence that differential UV absorption in the anterior and posterior lens is accompanied by differential protein expression.

Proteins from Avastin® (bevacizumab) show tyrosine nitrations for which the consequences are completely unclear.

Avastin® (bevacizumab) is a protein drug widely used for cancer treatment although its further use is questionable due to serious side effects reported. As no systematic proteomic study on posttranslational modifications (PTMs) was reported so far, it was the aim of the current study to use a gel-based proteomics method for determination of Avastin®-protein(s). Avastin® was run on two-dimensional gel electrophoresis (2-DE), spots were picked, followed by multi-enzyme in-gel digestion. Subsequently, the resulting peptides and posttranslational modifications were identified by mass spectrometry (nano-LC-ESI-MS/MS; HCT and LTQ Orbitrap MS). Heavy and light chains were observed and the 9 spots that were picked from 2DE-gels were identified as bevacizumab with high sequence coverage. MS/MS results showed multiple tyrosine nitrations on the Avastin® light and heavy chains that were either represented as nitrotyrosine or as aminotyrosine, which was shown to be generated from nitrotyrosine under reducing conditions. Protein nitration is known to significantly change protein functions and interactions and it may well be that some of the adverse effects of the protein drug Avastin® may be due to this PTM, which may have been generated during production--thus, nitration of Avastin® is a challenge for the pharmaceutical industry.

Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy.

Chronic articular cartilage defects are the most common disabling conditions of humans in the western world. The incidence for cartilage defects is increasing with age and the most prominent risk factors are overweight and sports associated overloading. Damage of articular cartilage frequently leads to osteoarthritis due to the aneural and avascular nature of articular cartilage, which impairs regeneration and repair. Hence, patients affected by cartilage defects will benefit from a cell-based transplantation strategy. Autologous chondrocytes, mesenchymal stem cells and embryonic stem cells are suitable donor cells for regeneration approaches and most recently the discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options. It is the aim of this review to summarize recent advances in the use of amniotic fluid stem cells as novel cell sources for the treatment of articular cartilage defects. Molecular aspects of articular cartilage formation as well as degeneration are summarized and the role of growth factor triggered signaling pathways, scaffolds, hypoxia and autophagy during the process of chondrogenic differentiation are discussed.