Assessment of neurotoxicity of pharmacological compounds during early neural development of human embryonic stem cells.

Human embryonic stem cells (hESCs), with the potential for differentiation, have been used to evaluate the embryotoxicity of various compounds. The effects of pharmacological compounds (cytosine arabinoside, 5-fluorouracil, hydroxyurea, indomethacin, and dexamethasone) on neurogenesis of hESCs over 28 days were examined based on cytotoxicity (half-maximal inhibitory concentration of viability, IC50) and expression of neural markers. Cytosine arabinoside, 5-fluorouracil, and hydroxyurea showed strong cytotoxicity (IC50 < 10 µM), whereas indomethacin and dexamethasone had weaker cytotoxic effects. Dose-dependent expression profiles of neural markers in the compound-treated groups are presented in triangular charts to allow comparison with the standard expression levels in the control group. Differences in compound-specific reductions in expression patterns of GAD1, OLIG2, FABP, and NES were similar to the differences in cytotoxic strength. Cytosine arabinoside diminished nestin and ß3-tubulin in neural differentiated hESCs. The results of this study extend the understanding of how differentiated hESCs may be useful for assessment of cell viability or neurogenesis impairment by chemicals that could have effects during the embryonic stage, particularly during neurogenesis.

Effects of xenoestrogens on streptozotocin-induced diabetic mice.

In diabetic mellitus, apoptotic or necrotic deaths of pancreatic ß-cells lead to insulin deficiency because plasma insulin is synthesized and released from pancreatic ß-cells and involved with blood glucose homeostasis. Since estrogen receptors have been related with glucose metabolis, estrogen-like chemicals (xenoestrogens) including bisphenol A (BPA) and octylphenol (OP) alter the endocrine system, and cause adverse health consequences such as obesity and diabetes. In the current study, levels of plasma glucose were evaluated after administration of BPA and OP using biochemical analysis, and were investigated in insulin and insulin synthesis-related genes in the pancreas and liver of streptozotocin (STZ)-induced insulin-deficient mice. Although the STZ-induced insulin-deficient groups showed an increase in blood glucose compared with control groups, the induced blood glucose level dropped to that of baseline after administration of xenoestrogens. When insulin level and mRNA expression of insulin transcriptional regulators (Pdx1, Mafa, and Neurod1) in pancreatic ß-cells were decreased in STZ-induced insulin-deficient groups, they were significantly restored by administration of xenoestrogens. The latter observation is also related to NF-κB activation for anti-apoptosis effects in pancreatic ß-cells. In addition, we observed a complementary convergence in regulation of gluconeogenesis for determination of blood glucose levels. Therefore, the current study may be particularly important for assessment of xenoestrogens under condition of diabetic mellitus or metabolic disorder.

MicroRNA signatures associated with immortalization of EBV-transformed lymphoblastoid cell lines and their clinical traits.

OBJECTIVE: MicroRNAs (miRNAs) are negative regulators of gene expression that play important roles in cell processes such as proliferation, development and differentiation. Recently, it has been reported that miRNAs are related to development of carcinogenesis. The aim of this study was to identify miRNAs associated with terminal immortalization of Epstein-Barr virus (EBV)-transformed lymphoblastoid cell line (LCL) and associated clinical traits. MATERIAL AND METHODS: Hence, we performed miRNA microarray approach with early- (p6) and late-passage (p161) LCLs. RESULTS AND CONCLUSION: Microarray data showed that nine miRNAs (miR-20b*, miR-28-5p, miR-99a, miR-125b, miR-151-3p, miR-151:9.1, miR-216a, miR-223* and miR-1296) were differentially expressed in most LCLs during long-term culture. In particular, miR-125b was up-regulated in all the tested late-passage LCLs. miR-99a, miR-125b, miR-216a and miR-1296 were putative negative regulators of RASGRP3, GPR160, PRKCH and XAF1, respectively, which were found to be differentially expressed in LCLs during long-term culture in a previous study. Linear regression analysis showed that miR-200a and miR-296-3p correlated with triglyceride and HbA1C levels, respectively, suggesting that miRNA signatures of LCLs could provide information on the donor's health. In conclusion, our study suggests that expression changes of specific miRNAs may be required for terminal immortalization of LCLs. Thus, differentially expressed miRNAs would be a potential marker for completion of cell immortalization during EBV-mediated tumorigenesis.

Pancytopenia and secondary myelofibrosis could be induced by primary hyperparathyroidism.

Hyperparathyroidism may be a precipitating factor important to the development of myelofibrosis: however, there has been only a few reports regarding myelofibrosis secondary to primary hyperparathyroidism. Recently, a rare case of pancytopenia caused by myelofibrosis in a 41-year-old woman who complained of general weakness and arthralgia presented to our clinical service. The patient was diagnosed with primary hyperparathyroidism with pancytopenia. Bone marrow biopsy revealed myelofibrosis. Right parathyroidectomy was performed and a parathyroid adenoma was totally excised. After surgery, the CBC counts and other clinical abnormalities gradually improved without further intervention. We concluded that the pancytopenia was because of bone marrow fibrosis resulting from primary hyperparathyroidism. Therefore, physicians should consider myelofibrosis secondary to primary hyperparathyroidism as a cause of pancytopenia in hypercalcemic patients, even though it is rare.

Studies on the mechanism of RNAi-dependent heterochromatin assembly.

Assembly of heterochromatin at centromeric DNA regions in the fission yeast Schizosaccharomyces pombe involves an intimate interplay between chromatin modifying complexes and components of the RNAi pathway. The RNA-induced transcriptional silencing (RITS) complex, containing Chp1, Ago1, Tas3, and centromeric siRNAs, localizes to centromeric DNA repeats and is required for the assembly and maintenance of heterochromatin. RITS brings together two types of molecular recognition modules: a chromodomain protein, which binds to lysine 9 methylated histone H3 (H3K9), and Argonaute, which binds to specific sequences by siRNA-directed base-pairing interactions. The RNA-directed RNA polymerase complex (RDRC), composed of Rdp1, the Hrr1 helicase, and the Cid12 Poly(A) polymerase family member, synthesizes double-stranded RNA and creates the substrate for Dicer to generate siRNAs. RDRC physically associates with RITS, and both complexes localize to noncoding centromeric RNAs and centromeric DNA repeats, suggesting that recognition of nascent RNA transcripts may be involved in localization of these complexes to specific chromosome regions. In support of this possibility, tethering of the RITS complex to the transcript of the normally euchromatic ura4 (+) gene results in siRNA generation and RNAi- and heterochromatin-dependent silencing of the ura4 (+) gene. Finally, silencing of a subset of endogenous and transgene promoters within heterochromatic DNA domains occurs by RNAi-dependent degradation of nascent transcripts by a mechanism that we have termed co-transcriptional gene silencing (CTGS).

The pachytene checkpoint prevents accumulation and phosphorylation of the meiosis-specific transcription factor Ndt80.

In budding yeast, many mutants defective in meiotic recombination and chromosome synapsis undergo checkpoint-mediated arrest at the pachytene stage of meiotic prophase. We recovered the NDT80 gene in a screen for genes whose overexpression bypasses the pachytene checkpoint. Ndt80 is a meiosis-specific transcription factor that promotes expression of genes required for exit from pachytene and entry into meiosis I. Herein, we show that the Ndt80 protein accumulates and is extensively phosphorylated during meiosis in wild type but not in cells arrested at the pachytene checkpoint. Our results indicate that inhibition of Ndt80 activity is one mechanism used to achieve pachytene arrest.

Elevated expression of glutathione peroxidase in PC12 cells results in protection against methamphetamine but not MPTP toxicity.

In vivo administration of either 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or methamphetamine (MA) produces damage to the dopaminergic nervous system which may be due in part to the generation of reactive oxygen species (ROS). The resistance of superoxide dismutase (SOD) over-expressing transgenic mice to the effects of both MPTP and MA suggests the involvement of superoxide in the resulting neurotoxicity of both compounds. Superoxide can be converted by SOD to hydrogen peroxide, which itself can cause cellular degeneration by reacting with free iron to produce highly reactive hydroxyl radicals resulting in damage to proteins, nucleic acids and membrane phospholipids. Hydrogen peroxide has also been reported to be produced via inhibition of NADH dehydrogenase by MPP + formed during oxidation of MPTP by MAO-B and by dopamine auto-oxidation following MA-induced dopamine release from synaptic vesicles within nerve terminals. To test whether hydrogen peroxide is an important factor in the toxicity of either of these two neurotoxins, we created clonal PC12 lines expressing elevated levels of the hydrogen peroxide-reducing enzyme glutathione peroxidase (GSHPx). Elevation of GSHPx levels in PC12 was found to diminish the rise in ROS levels and lipid peroxidation resulting from MA but not MPTP treatment. Elevated levels of GSHPx also appeared to prevent decreases in transport-mediated dopamine uptake produced via MA administration as well as to attenuate toxin-induced cell loss as measured by either MTT reduction or LDH release. Our data, therefore, suggest that hydrogen peroxide production likely contributes to MA toxicity in dopaminergic neurons.

Potential adaptations to acute hypoxia: Hct, stress proteins, and set point for temperature regulation.

Severe, intermittent hypoxia (hypoxic conditioning, HC) increases survival time during subsequent lethal hypoxia in mice. This protective effect was blocked by naloxone, suggesting an opioid-dependent mechanism. We proposed and evaluated three potential mechanisms of this acute adaptation: 1) increased hematocrit (Hct), 2) protein synthesis, and 3) decreased set point for temperature regulation (set point). Increased hematocrit is a well-studied adaptation to chronic hypoxia and could be acutely initiated by sympathetically mediated splenic contraction. Survival during stress can be prolonged by synthesis of stress proteins. We tested this hypothesis using two protein synthesis inhibitors, anisomycin and cycloheximide. Our third hypothesis is that set point is decreased after HC. A regulated decrease in body temperature would lower oxygen demand during hypoxia. Our studies indicate that hematocrit and protein synthesis are not dominant mechanisms of acute adaptation to hypoxia. However, we have observed a naloxone blockable decrease in set point after HC, supporting a mechanism in which acute adaptation involves an endogenous opioid-dependent decrease in set point. These studies also demonstrate that set point could be a more dominant contributor than body temperature to hypoxic tolerance.