Short-Term Functional Outcomes of Short Femoral Neck Stems Are the Same as Those of Conventional Stems in Primary Total Hip Arthroplasty.

(1) Background: In this study, two types of implants were compared-a conventional hip stem and a femoral neck prosthesis. (2) Methods: The femoral neck prosthesis study group included 21 patients, while the conventional hip stem control group was 40 patients. The first examination was the pre-op check, while the next ones were performed 6 weeks, 1 year, and 3 years after surgery. The Harris Hip Score (HHS), Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), Oxford Hip Score (OHS), University of California at Los Angeles Activity Score (UCLA), and Visual Analog Scale EQ (VAS EQ) forms were completed at each clinical study visit. (3) Results: The HHS in the femoral neck prosthesis group and the conventional hip stem group 6 weeks after surgery was 68.8 ± 16.47 and 67.6 ± 8.92, respectively, and 1 year after surgery, this was 93 ± 5.58 vs. 90.6 ± 5.17, respectively. The OHS of the femoral neck prosthesis group was 34.8 points after 6 weeks, 45.5 points after 1 year, and 43.9 points after 3 years. The respective values in the conventional hip stem group were 35.5, 41.55, and 42.13 points. The WOMAC values for the femoral neck prosthesis group were 70.6, 92.7, and 86 points, respectively, while for the conventional hip stem group, they were 74, 88.1, and 86.1 points. The UCLA scores recorded in the conventional hip stem group ranged from 3.15 to 5.05 points, but a higher mean value of 5.33 points was obtained in the femoral neck prosthesis group. VAS EQ was equal to 84 points three years after the operation. (4) Conclusions: The study showed no significant differences in the functional scores of both groups, and the new type of cervical femoral stem could be the first choice in younger patients.

Impact of Selected Serum Factors on Metastatic Potential of Gastric Cancer Cells.

(1) Background: stromal-derived factor-1 (SDF-1/CXCL12), hepatocyte and vascular-endothelial growth factors (HGF and VEGF) have been shown to facilitate cell motility, proliferation and promote local tumor progression and metastatic spread. Recent research shows the important role of these cytokines in gastric cancer (GC) progression. (2) Methods: 21 gastric cancer patients and 19 healthy controls were included in the study. SDF-1, HGF and VEGF levels were evaluated in sera by ELISA. Patients and control sera were used to stimulate CRL-1739 GC cell line, and chemotaxis, adhesion and proliferation potential were assessed. (3) Results: Concentrations of SDF-1, HGF and VEGF were significantly higher in patients than in controls. Chemotaxis and adhesion assays revealed a significant response of GC cells to patients' serum. Furthermore, significant relationships were seen between chemotactic/adhesion response and tumor stage. Serum from intestinal early GC patients produced significantly stronger chemotactic response when compared to patients with metastatic spread. In turn, serum from patients with distal metastases significantly increased the adhesion of GC cells when compared to sera from the patients with no distal metastases. We also observed that HGF strongly stimulated the proliferation of CRL-1739 cells. (4) Conclusions: We observed that the sera from GC patients, but also SDF-1, HGF and VEGF used alone, have a strong pro-metastatic effect on CRL-1739 cells. We also demonstrated that the concentration of these cytokines is specifically elevated in the sera of patients in an early stage of malignancy. Our results indicate that SDF-1, HGF and VEGF are very important molecules involved in gastric cancer progression.

Protein Arginine Methyltransferase (PRMT) Inhibitors-AMI-1 and SAH Are Effective in Attenuating Rhabdomyosarcoma Growth and Proliferation in Cell Cultures.

Rhabdomyosarcoma (RMS) is a malignant soft tissue cancer that develops mostly in children and young adults. With regard to histopathology, four rhabdomyosarcoma types are distinguishable: embryonal, alveolar, pleomorphic and spindle/sclerosing. Currently, increased amounts of evidence indicate that not only gene mutations, but also epigenetic modifications may be involved in the development of RMS. Epigenomic changes regulate the chromatin architecture and affect the interaction between DNA strands, histones and chromatin binding proteins, thus, are able to control gene expression. The main aim of the study was to assess the role of protein arginine methyltransferases (PRMT) in the cellular biology of rhabdomyosarcoma. In the study we used two pan-inhibitors of PRMT, called AMI-1 and SAH, and evaluated their effects on proliferation and apoptosis of RMS cells. We observed that AMI-1 and SAH reduce the invasive phenotype of rhabdomyosarcoma cells by decreasing their proliferation rate, cell viability and ability to form cell colonies. In addition, microarray analysis revealed that these inhibitors attenuate the activity of the PI3K-Akt signaling pathway and affect expression of genes related to it.

Lead (Pb) Accumulation in Human THP-1 Monocytes/Macrophages In Vitro and the Influence on Cell Apoptosis.

In this study, we investigated the ability of THP-1 monocytes and macrophages to accumulate lead (Pb) in vitro, relative to Pb concentration and length of exposure. Moreover, we also evaluated the effect of Pb accumulation on cell viability and apoptosis. THP-1 monocytes and macrophages were cultured in the presence of Pb at 1.25 µg/dL, 2.5 µg/dL, 5 µg/dL, and 10 µg/dL. Pb accumulation was examined by inductively coupled plasma and confocal microscopy. The influence of Pb on cell viability, apoptosis, and necrosis was assessed using flow cytometry. The results showed that Pb was toxic to THP-1 monocytes/macrophages even at very low environmental concentrations. Despite the use of low concentrations, both monocytes and macrophages showed dose-dependent and time-dependent decreases in viability, with a simultaneous increase in the percentage of early and late apoptotic cells. Macrophages reacted more strongly to Pb than monocytes. When exposed to the same Pb concentrations, they showed lower viability and a higher percentage of necrotic cells. The incubation time positively correlated with Pb accumulation in a dose-dependent manner. The obtained results indicate that environmental exposure to low Pb concentrations may significantly impair the function of macrophages, with the increased number of apoptotic cells potentially contributing to the development of many pathologies in the brain and whole body.

Extracellular Adenine Nucleotides and Adenosine Modulate the Growth and Survival of THP-1 Leukemia Cells.

A new approach to improve the effectiveness of acute myeloid leukemia (AML) treatment is to use the properties of purinergic signaling molecules secreted into the bone marrow milieu in response to leukemic cell growth. Therefore, our study aimed to evaluate the effects of extracellular adenine nucleotides and adenosine on the growth and death parameters in the leukemic THP-1 cell line. Cells were exposed to ATP, ADP, AMP, adenosine and nonhydrolyzable analogues of ATP and ADP (ATPγS and ADPßS) in a 1-1000 µM broad concentration range. The basal mRNA expression of the P1 and P2 receptors was evaluated by real-time PCR. Changes in the processes of cell growth and death were assessed by flow cytometry analysis of proliferation, cell cycle and apoptosis. Chemotaxis toward stromal cell-derived factor-1 (SDF-1) was performed using the modified Boyden chamber assay, and chemokine receptor type 4 (CXCR4) surface expression was quantified by flow cytometry. We indicated several antileukemic actions. High micromolar concentrations (100-1000 µM) of extracellular adenine nucleotides and adenosine inhibit the growth of cells by arresting the cell cycle and/or inducing apoptosis. ATP is characterized by the highest potency and widest range of effects, and is responsible for the cell cycle arrest and the apoptosis induction. Compared to ATP, the effect of ADP is slightly weaker. Adenosine mostly has a cytotoxic effect, with the induction of apoptosis. The last studied nucleotide, AMP, demonstrated only a weak cytotoxic effect without affecting the cell cycle. In addition, cell migration towards SDF-1 was inhibited by low micromolar concentrations (10 µM). One of the reasons for this action of ATPγS and adenosine was a reduction in CXCR4 surface expression, but this only partially explains the mechanism of antimigratory action. In summary, extracellular adenine nucleotides and adenosine inhibit THP-1 cell growth, cause death of cells and modulate the functioning of the SDF-1/CXCR4 axis. Thus, they negatively affect the processes that are responsible for the progression of AML and the difficulties in AML treatment.

Differential effect of adenosine on rhabdomyosarcoma migration and proliferation.

INTRODUCTION: Adenosine and its receptors are involved deeply in the regulation of tumour biology. Purine nucleotides are released from stressed cells in states of hypoxia or radiochemotherapy-induced cell damage. Adenosine exerts its effect through the P1 family of selective receptors. The purpose of the study was to evaluate the exact role of extracellular role on biology of Rhabdomyosarcoma (RMS) cells. MATERIAL AND METHODS: Series of in vitro studies accompanied by immunohistochemical, RQ-PCR and shRNA methods have characterised adenosine receptor expression on Rhabdomyosarcoma cell lines, normal skeletal muscle and effect of adenosine on Rhabdomyosarcoma growth and migration. RESULTS: Extracellular adenosine (highest at 50 µM, p < 0.05) and AMP (highest at 300 µM, p < 0.05) markedly enhanced chemotaxis in the Boyden chamber assay The reaction is mostly governed by the A1 receptor, which is greatly overexpressed in Rhabdomyosarcoma as compared with normal skeletal muscle. Cell migration induced by adenosine and AMP is blocked by pertussis toxin, phospholipase C and MAP kinase inhibitor, which demonstrates the importance of these signalling pathways. High doses of adenosine have a detrimental effect on cellular proliferation, in a receptor-independent manner (≥ 500 µM; p < 0.05). The blockage of adenosine transporter by dipyridamole abolishes this effect, indicating involvement of an intrinsic pathway. Further increase of adenosine concentration, induced by deaminase inhibitors, augment the effect. CONCLUSIONS: Our results suggest that adenosine and AMP trigger cell migration by binding to P1 receptors and directing cancer cells to the sites of hypoxia or cellular damage. Specifically by A1 receptor which is overexpressed in RMS.

‬The expression of purinergic P2X4 and P2X7 receptors in selected mesolimbic structures during morphine withdrawal in rats.

Morphine is one of the most potent analgesics used in medicine and it's long-term use is associated with the risk of the state of dependence. The cessation of chronic morphine administration leads to withdrawal signs which are associated with neurotransmitter dysregulations within mesolimbic system. Adenosine 5'-triphosphate (ATP) and purinergic system play an important role in the activity of central nervous system (CNS). Purinergic receptors are widely distributed in neurons and glial cells throughout the CNS taking part in integration of functional activity between neurons, glial and vascular cells. In the present study the mRNA and protein expression of purinergic P2X4 and P2X7 receptors in selected mesolimbic structures (striatum, hippocampus and prefrontal cortex) during morphine withdrawal in rats was investigated by RT-PCR and Western Blot analysis. Two experimental models of morphine withdrawal were studied: single and repeated morphine withdrawal. We demonstrated that expression of P2X4 and P2X7 receptors was altered during morphine withdrawal period in rats. These alterations were varied in particular mesolimbic areas depending on the scheme of morphine administration. Our results extend the current knowledge on morphine withdrawal and for the first time high-light interactions between purinergic system and morphine withdrawal. It seems, the purinergic system may be a new, valuable tool in searching for a new strategy of management of opioid dependence.

Comparative evaluation of new dihydropyrimidine and dihydropyridine derivatives perturbing mitotic spindle formation.

AIM: The mitotic spindle plays a key role in cell division which makes it an important target in cancer therapy. In the present study the antiproliferative activity of 4-benzyl-5-phenyl-3,4-dihydropyrimidine-2(1H)-thione (1) and its pyridine bioisoster (2) were evaluated and compared with monastrol (MON), the first known cell-permeable small molecule which disrupts bipolar spindle formation by inhibiting Eg5-kinesin activity. RESULTS: Our data revealed that compound 2 showed higher antiproliferative activity than MON against MCF7 and A375 cell lines and comparable reversible cell cycle inhibition in G2/M phase. However, compound 2 produced distinct phenotype from monoastral spindles, and did not affect Eg5 ATPase activity. CONCLUSION: The activity of compound 2 may suggest its new promising anticancer mechanism (different than MON), targeting other component required for spindle bipolarity.

Picropodophyllin (PPP) is a potent rhabdomyosarcoma growth inhibitor both in vitro and in vivo.

BACKGROUND: Insulin-like growth factors and insulin are important factors promoting cancer growth and metastasis. The molecules act through IGF1 (IGF1R) and insulin (InsR) receptors. Rhambodmyosarcomas (RMS) overproduce IGF2 - a potent ligand for IGF1R and, at the same time, highly express IGF1 receptor. The purpose of the study was to evaluate possible application of picropodophyllin (PPP) - a potent IGF1R inhibitor. METHODS: In our study we used a number of in vitro assays showing influence of IGF1R blockage on RMS cell lines (both ARMS and ERMS) proliferation, migration, adhesion, cell cycling and signal transduction pathways. Additionally, we tested possible concomitant application of PPP with commonly used chemotherapeutics (vincristine, actinomycin-D and cisplatin). Moreover, we performed an in vivo study where PPP was injected intraperitoneally into RMS tumor bearing SCID mice. RESULTS: We observed that PPP strongly inhibits RMS proliferation, chemotaxis and adhesion. What is more, application of the IGF1R inhibitor attenuates MAPK phosphorylation and cause cell cycle arrest in G2/M phase. PPP increases sensitivity of RMS cell lines to chemotherapy, specifically to vincristine and cisplatin. In our in vivo studies we noted that mice treated with PPP grew smaller tumors and displayed significantly decreased seeding into bone marrow. CONCLUSIONS: The cyclolignan PPP effectively inhibits RMS tumor proliferation and metastasis in vitro and in an animal model.

Comparative in vitro study of single and four layer graphene oxide nanoflakes - Cytotoxicity and cellular uptake.

In recent years, graphene and its derivatives have been extensively investigated because of their unique properties, which can be used in many fields including biomedical applications. Therefore, detailed biological study is required. In the current paper the detailed toxicological studies on single and four layer graphene oxide (GO) nanoflakes is presented. The morphology and size of the nanomaterials were characterized via atomic force microscopy. Cytotoxicity, proliferation and internalization study were performed using various methods, including optical, confocal and Raman microscopy imaging, flow cytometry analysis, colorimetric and luminescent cell assays. Our first findings undeniably show that the nanomaterials' functionalization has a considerable impact on their behavior in a biological environment. The cytotoxicity assay confirmed comparable, dose dependent cytotoxicity of single and four layers GO flakes. The differences between these two nanomaterials became more distinct during cell proliferation study and ROS detection. Namely, markedly stronger inhibition of cell proliferation and higher ROS generation by one-layer GO-PEG than four-layer GO-PEG were observed. Cell imaging revealed efficient internalization of the both GO nanoflakes in a time dependent manner. These findings emphasize the role of number of layer and functionalization in GO toxicological characteristics and may provide helpful information for their further biomedical applications.

Effect of colorectal cancer on the number of normal stem cells circulating in peripheral blood.

Bone marrow (BM) residing stem cells are mobilized from their BM niches into peripheral blood (PB) in several pathological situations including tissue organ injury and systemic inflammation. We recently reported that the number of BM-derived stem cells (SCs) increases in patients with pancreatic and stomach cancer. Accordingly, we observed higher numbers of circulating very small embryonic/epiblast­like stem cells (VSELs) and mesenchymal stem cells (MSCs) that were associated with the activation of pro-mobilizing complement cascade and an elevated level of sphingosine-1 phosphate (S1P) in PB plasma. We wondered if a similar correlation occurs in patients with colorectal cancer (CRC). A total of 46 patients were enrolled in this study: 17 with CRC, 18 with benign colonic adenomas (BCA) and 11 healthy individuals. By employing fluorescence-activated cell sorting (FACS) we evaluated the number of BM-derived SCs circulating in PB: i) CD34+/Lin-/CD45- and CD133-/Lin-/CD45- VSELs; ii) CD45-/CD105+/CD90+/CD29+ MSCs; iii) CD45-/CD34+/CD133+/KDR+ endothelial progenitor cells (EPCs); and iv) CD133+/Lin-/CD45+ or CD34+/Lin-/CD45+ cells enriched for hematopoietic stem/progenitor cells (HSPCs). In parallel, we measured in the PB parameters regulating the egress of SCs from BM into PB. In contrast to pancreatic and gastric cancer patients, CRC subjects presented neither an increase in the number of circulating SCs nor the activation of pro-mobilizing factors such as complement, coagulation and fibrinolytic cascade, circulating stromal derived factor 1 (SDF­1), vascular endothelial growth factor (VEGF) and intestinal permeability marker (zonulin). In conclusion, mobilization of SCs in cancer patients depends on the type of malignancy and its ability to activate pro-mobilization cascades.

The adenosinergic system is involved in sensitization to morphine withdrawal signs in rats-neurochemical and molecular basis in dopaminergic system.

RATIONALE: Experimental data informs that not only do the dose and time duration of dependent drugs affect the severity of withdrawal episodes. Previous withdrawal experiences may intensify this process, which is referred as sensitization to withdrawal signs. Adenosine and dopamine (DA) receptors may be involved in this sensitization. OBJECTIVES: Rats were continuously and sporadically treated with increasing doses of morphine for 8 days. In rats, sporadically treated with morphine, morphine administration was modified by adding three morphine-free periods. Adenosine agonists were given during each of the morphine-free periods (six injections in total). On the 9th day, morphine was injected. One hour later, naloxone was administered to induce morphine withdrawal signs. Then, the animals were placed into cylinders and the number of jumpings was recorded. Next, the rats were decapitated and brain and brain structures (striatum, hippocampus, and prefrontal cortex) were dissected for neurochemical, molecular, and immunohistochemical experiments within DAergic pathways. RESULTS: We demonstrated that previous experiences of opioid withdrawal intensified subsequent withdrawal signs. Adenosine ligands attenuated the sensitization to withdrawal signs. In a neurochemical study, the release of DA and its metabolites was impaired in all structures. Significant alterations were also observed in mRNA and protein expression of DA receptors. CONCLUSIONS: Results demonstrate that intermittent treatment with morphine induces alterations in the DAergic system which may be responsible for sensitization to morphine withdrawal signs. Although adenosine ligands attenuate this type of sensitization, they are not able to fully restore the physiological brain status.

Role of prometastatic factors in gastric cancer development.

The second half of the 20th century has seen a sharp worldwide decline in both the incidence and mortality of gastric cancer. Despite this, gastric cancer is the most common cause of mortality in the world. It is closely related to the commonly asymptomatic course at the beginning and delayed diagnosis. Approximately 90­97% of stomach cancers are adenocarcinomas, which may be subdivided histologically into two categories - intestinal type, and diffuse type. Cancer metastasis is a complex multi­step process that is closely associated with tumor phenotype. The most important steps in the metastasis process are proteolytic activity, migration, adhesion, proliferation, and neovascularization. In this review we focus on mechanisms regulating gastric cancer metastasis.

5­Azacytidine inhibits human rhabdomyosarcoma cell growth by downregulating insulin­like growth factor 2 expression and reactivating the H19 gene product miR­675, which negatively affects insulin­like growth factors and insulin signaling.

Insulin-like growth factor 2 (IGF2) and 1 (IGF1) and insulin (INS) promote proliferation of rhabdomyosarcoma (RMS) cells by interacting with the insulin-like growth factor 1 receptor (IGF1R) and the insulin receptor (INSR). Loss of imprinting (LOI) by DNA hypermethylation at the differentially methylated region (DMR) for the IGF2­H19 locus is commonly observed in RMS cells and results in an increase in the expression of proliferation-promoting IGF2 and downregulation of proliferation-inhibiting non-coding H19 miRNAs. One of these miRNAs, miR­675, has been reported in murine cells to be a negative regulator of IGF1R expression. To better address the role of IGF2 and 1, as well as INS signaling in the pathogenesis of RMS and the involvement of LOI at the IGF2­H19 locus, we employed the DNA demethylating agent 5­azacytidine (AzaC). We observed that AzaC­mediated demethylation of the DMR at the IGF2­H19 locus resulted in downregulation of IGF2 and an increase in the expression of H19. This epigenetic change resulted in a decrease in RMS proliferation due to downregulation of IGF2 and, IGF1R expression in an miR­675­dependent manner. Interestingly, we observed that miR­675 not only inhibited the expression of IGF1R in a similar manner in human and murine cells, but we also observed its negative effect on the expression of the INSR. These results confirm the crucial role of LOI at the IGF2­H19 DMR in the pathogenesis of RMS and are relevant to the development of new treatment strategies.

Positive effects of prolonged caloric restriction on the population of very small embryonic-like stem cells - hematopoietic and ovarian implications.

BACKGROUND: Low calorie intake, or calorie restriction (CR) without malnutrition, has been demonstrated in several animal species, including mice, to increase both median and maximum lifespan as well as delay reproductive senescence. Our previous work demonstrated a positive correlation between life span and the number of very small embryonic-like stem cells (VSELs) in long living Laron dwarf mice. These animals have very low levels of circulating insulin-like growth factor 1 (IGF-1) in peripheral blood (PB), maintain higher numbers of hematopoietic stem cells (HSPCs) in bone marrow (BM), and display prolonged fecundity compared with wild type littermates. Since CR lowers the level of IGF-1 in PB, we become interested in the effect of CR on the number of VSELs and HSPCs in BM as well as on the morphology of ovaries and testes. METHODS: In our studies four-week-old female and male mice were subjected to CR by employing an alternate-day ad libitum feeding diet for a period of 9 months. RESULTS: We observed that mice on CR had a higher number of BM-residing VSELs than control mice fed ad libitum. These changes correlated with higher numbers of HSPCs in BM, spleen, and peripheral blood (PB) as well as with an increase in the number of primordial and primary follicles in ovaries. At the same time, however, no changes were observed in the testes of mice under CR. CONCLUSION: We conclude that CR positively affects the pool of VSELs in adult tissues and explains the positive effect of CR on longevity.

[Molecular mechanisms regulating metastasis of cancer cells with special emphasis on rhabdomyosarcoma]. / Molekularne mechanizmy regulacji przerzutowania komórek nowotworowych na przykladzie miesaka prazkowanokomórkowego (rhabdomyosarcoma)

Rhabdomyosarcoma (RMS) is a malignant tumor of soft tissue derived from embryonic mesenchymal and/or neuroectodermal tissues. It is most often associated with other genetic syndromes such as Li-Fraumeni or Bechwith-Wiedeman. RMS cells show morphological similarities to striated muscle and the presence of specific markers of muscle tissue. At the histological level, it is divided into two subtypes (alveolar RMS - ARMS and embryonal RMS - ERMS), which differ in their genetic background, and prognosis. In recent years there has been significant progress in understanding the mechanisms that regulate RMS cell growth and metastasis. Recently, a number of several chemokines, cytokines or growth factors and their receptors were identified involved in RMS pathogenesis as well as animal models of this tumor have been developed. This knowledge is of great importance in the development of potential therapeutic strategies not only in RMS, but also other types of cancer. This paper will discuss the theories of the origin of this rare tumor and the molecular mechanisms involved in its growth and metastasis. The processes and mechanisms described herein, such as chemotaxis, adhesion, proliferation, intracellular signal transduction, seem to universal for number of cancer types.

Multifunctional protein APPL2 contributes to survival of human glioma cells.

Some endocytic proteins have recently been shown to play a role in tumorigenesis. In this study, we demonstrate that APPL2, an adapter protein with known endocytic functions, is upregulated in 40% cases of glioblastoma multiforme, the most common and aggressive cancer of the central nervous system. The silencing of APPL2 expression by small interfering RNAs (siRNAs) in glioma cells markedly reduces cell survival under conditions of low growth factor availability and enhances apoptosis (measured by executor caspase activity). Long-term depletion of APPL2 by short hairpin RNAs (shRNAs), under regular growth factor availability, suppresses the cell transformation abilities, assessed by inhibited colony formation in soft agar and by reduced xenograft tumor growth in vivo. At the molecular level, the negative effect of APPL2 knockdown on cell survival is not due to the alterations in AKT or GSK3ß activities which were reported to be modulated by APPL proteins. Instead, we attribute the reduced cell survival upon APPL2 depletion to the changes in gene expression, in particular to the upregulation of apoptosis-related genes, such as UNC5B (a proapoptotic dependence receptor) and HRK (harakiri, an activator of apoptosis, which antagonizes anti-apoptotic function of Bcl2). In support of this notion, the loss of glioma cell survival upon APPL2 knockdown can be rescued either by an excess of netrin-1, the prosurvival ligand of UNC5B or by simultaneous silencing of HRK. Consistently, APPL2 overexpression reduces expression of HRK and caspase activation in cells treated with apoptosis inducers, resulting in the enhancement of cell viability. This prosurvival activity of APPL2 is independent of its endosomal localization. Cumulatively, our data indicate that a high level of APPL2 protein might enhance glioblastoma growth by maintaining low expression level of genes responsible for cell death induction.

Epigenetic reprogramming: is deamination key to active DNA demethylation?

DNA demethylation processes are important for reproduction, being central in epigenetic reprogramming during embryonic and germ cell development. While the enzymes methylating DNA have been known for many years, identification of factors capable of mediating active DNA demethylation has been challenging. Recent findings suggest that cytidine deaminases may be key players in active DNA demethylation. One of the most investigated candidates is activation-induced cytidine deaminase (AID), best known for its role in generating secondary antibody diversity in B cells. We evaluate evidence for cytidine deaminases in DNA demethylation pathways in vertebrates and discuss possible models for their targeting and activity regulation. These findings are also considered along with alternative demethylation pathways involving hydroxymethylation.

Phosphorylation of histone H3 serine 10 in early mouse embryos: active phosphorylation at late S phase and differential effects of ZM447439 on first two embryonic mitoses.

Cell division in mammalian cells is regulated by Aurora kinases. The activity of Aurora A is indispensable for correct function of centrosomes and proper spindle formation, while Aurora B for chromosome biorientation and separation. Aurora B is also responsible for the phosphorylation of histone H3 serine 10 (H3S10Ph) from G2 to metaphase. Data concerning the Aurora B activity and H3S10Ph in embryonic cells are limited to primordial and maturing oocytes and advanced pronuclei in zygotes. In the present study we have analyzed H3S10Ph in 1- and 2-cell mouse embryos. We show that H3S10 remains phosphorylated at anaphase and telophase of the second meiotic division, as well as during the anaphase and telophase of the first and second embryonic mitoses. At late G1 H3S10 is dephosphorylated and subsequently phosphorylated de novo at late S phase of the first and second cell cycle. These results show that the H3S10 phosphorylation/dephosphorylation cycle in embryonic cells is different than in somatic cells. The behaviour of thymocyte G0 nuclei introduced into ovulated oocytes and early 1-cell parthenogenotes confirms that kinases responsible for de novo H3S10 phosphorylation, most probably Aurora B,  are active until G1 of the first cell cycle of mouse embryo. The inhibition of Aurora kinases by ZM447439 caused abnormalities both in the first and second mitoses. However, the disturbances in each division differed, suggesting important differences in the control of these mitoses. In ZM447439-treated mitotic zygotes Mad2 protein remained continuously present on kinetochores, what confirmed that spindle checkpoint remained active.