Volume 19, Issue 3

Erythropoietin (EPO) has a beneficial effect on hepatic cell proliferation during liver regeneration. However, the underlying mechanism has not yet been elucidated. To uncover the proliferation response of EPO in rat liver regeneration after partial hepatectomy (PH) at the cellular level, hepatocytes (HCs) were isolated using Percoll density gradient centrifugation. The genes of the EPO-mediated signaling pathway and the target genes of the transcription factor (TF) in the pathway were identified in a pathway and TF database search. Their expression profiles were then detected using Rat Genome 230 2.0 Microarray. The results indicated that the EPO-mediated signaling pathway is involved in 19 paths and that 124 genes participate, of which 32 showed significant changes and could be identified as liver regeneration-related genes. In addition, 443 targets regulated by the TFs of the pathway and 60 genes associated with cell proliferation were contained in the array. Subsequently, the synergetic effect of these genes in liver regeneration was analyzed using the E(t) mathematical model based on their expression profiles. The results demonstrated that the E(t) values of paths 3, 8, 12 and 14–17 were significantly strengthened in the progressing phase of liver regeneration through the RAS/MEK/ERK or PI3K/AκT pathways. The synergetic effect of the target genes, in parallel with target-related cell proliferation, was also enhanced 12–72 h after PH, suggesting a potential positive effect of EPO on HC proliferation during rat liver regeneration. These data imply that the EPO receptor may allow EPO to promote HC proliferation through paths 3, 8, 12 and 14–17, mediating the RAS/MEK/ERK and PI3K/AκT pathways in rat liver regeneration after PH.

Cannabinoids display various pharmacological activities, including tumor regression, anti-inflammatory and neuroprotective effects. To investigate the molecular mechanisms underlying the pharmacological effects of cannabinoids, we used a yeast two-hybrid system to screen a mouse brain cDNA library for proteins interacting with type 1 cannabinoid receptor (CB1R). Using the intracellular loop 3 of CB1R as bait, we identified 14-3-3β as an interacting partner of CB1R and confirmed their interaction using affinity-binding assays. 14-3-3β has been reported to induce a cell cycle delay at the G2/M phase. We tested the effects of cannabinoids on cell cycle progression in HeLa cells synchronized using a double-thymidine block-and-release protocol and found an increase in the population of G2/M phase cells. We further found that CB1R activation augmented the interaction of 14-3-3β with Wee1 and Cdc25B, and promoted phosphorylation of Cdc2 at Tyr-15. These results suggest that cannabinoids induce cell cycle delay at the G2/M phase by activating 14-3-3β.

Many experimental studies have demonstrated the favorable biological activities of plants belonging to the genus Rubus, but little is known of the role of Rubus leaf extracts in the modulation of the surface membrane expression and activity of endothelial apyrase. The aim of this study was to assess the influence of 1–15 μg/ml Rubus extracts on CD39 expression and enzymatic activity, and on the activation (ICAM-1 expression) and viability of human umbilical vein endothelial cells (HUVEC). The polyphenolic contents and antioxidative capacities of extracts from dewberry (R. caesius L.) and raspberry (R. idaeus L.) leaves were also investigated. The techniques applied were flow cytometry (endothelial surface membrane expression of ICAM-1 and CD39), malachite green assay (CD39 activity), HPLC-DAD (quantitative analysis of polyphenolic extract), ABTS, DPPH and FRAP spectrometric assays (antioxidant capacity), and the MTT test (cell viability). Significantly increased CD39 expressions and significantly decreased ATPDase activities were found in the cells treated with 15 μg/ml of either extract compared to the results for the controls. Neither of the extracts affected cell proliferation, but both significantly augmented endothelial cell ICAM-1 expression. The overall antioxidant capacities of the examined extracts remained relatively high and corresponded well to the determined total polyphenol contents. Overall, the results indicate that under in vitro conditions dewberry and raspberry leaf extracts have unfavorable impact on endothelial cells.

The large microtubule-associated/Ca2+-signalling protein p600 (also known as UBR4) is required for hippocampal neuronal survival upon Ca2+ dyshomeostasis induced by glutamate treatment. During this process, p600 prevents aggregation of the Ca2+/calmodulin-dependent kinase IIα (CaMKIIα), a proxy of neuronal death, via direct binding to calmodulin in a microtubuleindependent manner. Using photoconductive stimulation coupled with live imaging of single neurons, we identified a distinct mechanism of prevention of CaMKIIα aggregation by p600. Upon direct depolarization, CaMKIIα translocates to microtubules. In the absence of p600, this translocation is interrupted in favour of a sustained self-aggregation that is prevented by the microtubule-stabilizing drug paclitaxel. Thus, during photoconductive stimulation, p600 prevents the aggregation of CaMKIIα by stabilizing microtubules. The effectiveness of this stabilization for preventing CaMKIIα aggregation during direct depolarization but not during glutamate treatment suggests a model wherein p600 has two modes of action depending on the source of cytosolic Ca2+.

The 37-kDa immature laminin receptor protein (iLRP) is a speciesconserved, universal immunogenic protein that is expressed in all thus-far examined embryonic and early fetal cells of inbred and outbred rodents. It has also been identified in human concepti. It is altered through normal maturation processes to become a non-immunogenic 67-kDa dimeric mature laminin receptor protein (mLRP) in mid-to late gestation in the mammalian fetus. This antigen ceases to be expressed as an active autoimmunogen in the full-term fetus and in the normal differentiating tissues and organs of the neonate or adult organism, apparently due to dimerization, but it is re-expressed as an immunogenic monomer in tumor cells. In this review, we highlight the known mechanisms of immune responses with particular emphasis on the possible role of the 37-kDa oncofetal antigen/immature laminin receptor (OFA/iLRP) in both pregnancy and cancer.

Protein glycation is initiated by a nucleophilic addition reaction between the free amino group from a protein, lipid or nucleic acid and the carbonyl group of a reducing sugar. This reaction forms a reversible Schiff base, which rearranges over a period of days to produce ketoamine or Amadori products. The Amadori products undergo dehydration and rearrangements and develop a cross-link between adjacent proteins, giving rise to protein aggregation or advanced glycation end products (AGEs). A number of studies have shown that glycation induces the formation of the β-sheet structure in β-amyloid protein, α-synuclein, transthyretin (TTR), copper-zinc superoxide dismutase 1 (Cu, Zn-SOD-1), and prion protein. Aggregation of the β-sheet structure in each case creates fibrillar structures, respectively causing Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, familial amyloid polyneuropathy, and prion disease. It has been suggested that oligomeric species of glycated α-synuclein and prion are more toxic than fibrils. This review focuses on the pathway of AGE formation, the synthesis of different types of AGE, and the molecular mechanisms by which glycation causes various types of neurodegenerative disease. It discusses several new therapeutic approaches that have been applied to treat these devastating disorders, including the use of various synthetic and naturally occurring inhibitors. Modulation of the AGE-RAGE axis is now considered promising in the prevention of neurodegenerative diseases. Additionally, the review covers several defense enzymes and proteins in the human body that are important anti-glycating systems acting to prevent the development of neurodegenerative diseases.

There is no doubt that cancer is not only a genetic disease but that it can also occur due to epigenetic abnormalities. Diet and environmental factors can alter the scope of epigenetic regulation. The results of recent studies suggest that O-GlcNAcylation, which involves the addition of N-acetylglucosamine on the serine or threonine residues of proteins, may play a key role in the regulation of the epigenome in response to the metabolic status of the cell. Two enzymes are responsible for cyclic O-GlcNAcylation: O-GlcNAc transferase (OGT), which catalyzes the addition of the GlcNAc moiety to target proteins; and O-GlcNAcase (OGA), which removes the sugar moiety from proteins. Aberrant expression of O-GlcNAc cycling enzymes, especially OGT, has been found in all studied human cancers. OGT can link the cellular metabolic state and the epigenetic status of cancer cells by interacting with and modifying many epigenetic factors, such as HCF-1, TET, mSin3A, HDAC, and BAP1. A growing body of evidence from animal model systems also suggests an important role for OGT in polycomb-dependent repression of genes activity. Moreover, O-GlcNAcylation may be a part of the histone code: O-GlcNAc residues are found on all core histones.

Unlike nuclear nucleolin, surface-expressed and cytoplasmic nucleolin exhibit Tn antigen. Here, we show localization-dependent differences in the glycosylation and proteolysis patterns of nucleolin. Our results provide evidence for different paths of nucleolin proteolysis in the nucleus, in the cytoplasm, and on the cell surface. We found that full-length nucleolin and some proteolytic fragments coexist within live cells and are not solely the result of the preparation procedure. Extranuclear nucleolin undergoes N- and O-glycosylation, and unlike cytoplasmic nucleolin, membrane-associated nucleolin is not fucosylated. Here, we show for the first time that nucleolin and endogenous galectin-3 exist in the same complexes in the nucleolus, the cytoplasm, and on the cell surface of melanoma cells. Assessments of the interaction of nucleolin with galectin-3 revealed nucleolar co-localization in interphase, suggesting that galectin-3 may be involved in DNA organization and ribosome biogenesis.

This paper focuses on the development of renewable sources of isletreplacement tissue for the treatment of type I diabetes mellitus. Placental tissue-derived mesenchymal stem cells (MSCs) are a promising source for regenerative medicine due to their plasticity and easy availability. They have the potential to differentiate into insulin-producing cells. miR-375 is a micro RNA that is expressed in the pancreas and involved in islet development. Human placental decidua basalis MSCs (PDB-MSCs) were cultured from full-term human placenta. The immunophenotype of the isolated cells was checked for CD90, CD105, CD44, CD133 and CD34 markers. The MSCs (P3) were chemically transfected with hsa-miR-375. Total RNA was extracted 4 and 6 days after transfection. The expressions of insulin, NGN3, GLUT2, PAX4, PAX6, KIR6.2, NKX6.1, PDX1, and glucagon genes were evaluated using real-time qPCR. On day 6, we tested the potency of the clusters in response to the high glucose challenge and assessed the presence of insulin and NGN3 proteins via immunocytochemistry. Flow cytometry analysis confirmed that more than 90% of the cells were positive for CD90, CD105 and CD44 and negative for CD133 and CD34. Morphological changes were followed from day 2. Cell clusters formed during day 6. Insulin-producing clusters showed a deep red color with DTZ. The expression of pancreatic-specific transcription factors increased remarkably during the four days after transfection and significantly increased on day 7. The clusters were positive for insulin and NGN3 proteins, and C-peptide and insulin secretion increased in response to changes in the glucose concentration (2.8 mM and 16.7 mM). In conclusion, the MSCs could be programmed into functional insulin-producing cells by transfection of miR-375.

Resveratrol is the most extensively studied stilbene derivative. We previously showed that methylthiostilbenes were more effective inhibitors of CYP1A1 and 1B1 activity than resveratrol. In this study, we investigated whether resveratrol and its methylthio-substituted derivatives, i.e. 3-M-4′-MTS (S2), 3,5-DM-4′-MTS (S5) and 3,4,5-TM-4′-MTS (S7) could activate Nrf2 signaling in the mouse epidermis and in human keratinocytes. Western blot analysis showed translocation of Nrf2 from the cytosol to the nucleus in both models. All of the tested stilbenes increased GST activity, but resveratrol was the most effective inducer. Moreover, only resveratrol increased the protein level of GSTP in the mouse epidermis. GSTM was enhanced in HaCaT cells after the treatment with derivatives S2 and S5. The same effect was observed for GSTP in the case of compound S2. Resveratrol and its derivatives reduced the NQO2 protein level in HaCaT cells. Thus, it is possible that increased expression of GSTP or GSTM and GST activity was linked with NQO2 inhibition in these cells. The results of this study indicate that resveratrol and its methylthioderivatives activate Nrf2 not only in the mouse epidermis, but also in human keratinocytes. Upregulating GST isozymes might be particularly important for deactivating chemical carcinogens, such as PAH.