GROα regulates human embryonic stem cell self-renewal or adoption of a neuronal fate

Previously we reported that feeders formed from human placental fibroblasts (hPFs) support derivation and long-term self-renewal of human embryonic stem cells (hESCs) under serum-free conditions. Here, we show, using antibody array and ELISA platforms, that hPFs secrete ～6-fold higher amounts of the CXC-type chemokine, GROα, than IMR 90, a human lung fibroblast line, which does not support hESC growth. Furthermore, immunocytochemistry and immunoblot approaches revealed that hESCs express CXCR, a GROα receptor. We used this information to develop defined culture medium for feeder-free propagation of hESCs in an undifferentiated state. Cells passaged as small aggregates and maintained in the GROα-containing medium had a normal karyotype, expressed pluripotency markers, and exhibited apical-basal polarity, i.e., had the defining features of pluripotent hESCs. They also differentiated into the three primary (embryonic) germ layers and formed teratomas in immunocompromised mice. hESCs cultured as single cells in the GROα-containing medium also had a normal karyotype, but they downregulated markers of pluripotency, lost apical-basal polarity, and expressed markers that are indicative of the early stages of neuronal differentiation-βIII tubulin, vimentin, radial glial protein, and nestin. These data support our hypothesis that establishing and maintaining cell polarity is essential for the long-term propagation of hESCs in an undifferentiated state and that disruption of cell-cell contacts can trigger adoption of a neuronal fate.     

Comparative functional analysis of the human macrophage chitotriosidase

This work analyses the chitin-binding and catalytic domains of the human macrophage chitotriosidase and investigates the physiological role of this glycoside hydrolase in a complex mechanism such as the innate immune system, especially its antifungal activity. Accordingly, we first analyzed the ability of its chitin-binding domain to interact with chitin embedded in fungal cell walls using the β-lactamase activity reporter system described in our previous work. The data showed that the chitin-binding activity was related to the cell wall composition of the fungi strains and that their peptide-N-glycosidase/zymolyase treatments increased binding to fungal by increasing protein permeability. We also investigated the antifungal activity of the enzyme against Candida albicans. The antifungal properties of the complete chitotriosidase were analyzed and compared with those of the isolated chitin-binding and catalytic domains. The isolated catalytic domain but not the chitin-binding domain was sufficient to provide antifungal activity. Furthermore, to explain the lack of obvious pathologic phenotypes in humans homozygous for a widespread mutation that renders chitotriosidase inactive, we postulated that the absence of an active chitotriosidase might be compensated by the expression of another human hydrolytic enzyme such as lysozyme. The comparison of the antifungal properties of chitotriosidase and lysozyme indicated that surprisingly, both enzymes have similar in vitro antifungal properties. Furthermore, despite its more efficient hydrolytic activity on chitin, the observed antifungal activity of chitotriosidase was lower than that of lysozyme. Finally, this antifungal duality between chitotriosidase and lysozyme is discussed in the context of innate immunity.     

Astroglial-Conditioned Media and Growth Factors Modulate Proliferation and Differentiation of Astrocytes in Primary Culture

Astroglial conditioned media (ACM) influence the development and maturation of cultured nerve cells and modulate neuron-glia interaction. To clarify mechanisms of astroglial cell proliferation/differentiation in culture, incorporation of [methyl-³H]-thymidine or [5,6-³H]-uridine in cultured astrocytes was assessed. Cultures were pre-treated with epidermal growth factor (EGF), insulin (INS), insulin-like growth factor-I (IGF-I), and basic fibroblast growth factor (bFGF) and subsequently with ACM. DNA labeling revealed a marked stimulatory effect of ACM from 15 days in vitro (DIV) cultures in 30 DIV astrocytes after12 h pre-treatment with growth factors. The main effects were found after INS or EGF pre-treatment in 30 DIV cultures. ACM collected from 15 or 60 or 90 DIV increased RNA labeling of 15 and 30 DIV astrocyte cultures, being the highest value that of 30 DIV cultures added with ACM from 90 DIV. The findings of increased DNA labeling after EGF or INS pre-treatment in 30 DIV cultures, followed by addition of ACM from 15 DIV cultures, suggest that these phenomena may depend by extra cellular signal-regulated kinase 1 (ERK1) activation.     

Comprehensive microRNA profiling in B-cells of human centenarians by massively parallel sequencing

ABSTRACT: BACKGROUND: MicroRNAs (miRNAs) are small, non-coding RNAs that regulate gene expression and play a critical role in development, homeostasis, and disease. Despite their demonstrated roles in age-associated pathologies, little is known about the role of miRNAs in human aging and longevity. RESULTS: We employed massively parallel sequencing technology to identify miRNAs expressed in B-cells from Ashkenazi Jewish centenarians, i.e., those living to a hundred and a human model of exceptional longevity, and younger controls without a family history of longevity. With data from 26.7 million reads comprising 9.4x108 bp from 3 centenarian and 3 control individuals, we discovered a total of 276 known miRNAs and 8 unknown miRNAs ranging several orders of magnitude in expression levels, a typical characteristics of saturated miRNA-sequencing. A total of 22 miRNAs were found to be significantly upregulated, with only 2 miRNAs downregulated, in centenarians as compared to controls. Gene Ontology analysis of the predicted and validated targets of the 24 differentially expressed miRNAs indicated enrichment of functional pathways involved in cell metabolism, cell cycle, cell signaling, and cell differentiation. A cross sectional expression analysis of the differentially expressed miRNAs in B-cells from Ashkenazi Jewish individuals between the 50th and 100th years of age indicated that expression levels of miR-363* declined significantly with age. Centenarians, however, maintained the youthful expression level. This result suggests that miR-363* may be a candidate longevity-associated miRNA. CONCLUSION: Our comprehensive miRNA data provide a resource for further studies to identify genetic pathways associated with aging and longevity in humans.     

Cell-cycle control of c-myc but not c-ras expression is lost following chemical transformation

Cellular oncogenes are DNA sequences implicated in the genesis of cancer, but their functions in the transformation process are not understood. Our experiments provide data linking expression of two well-studied proto-oncogenes, c-myc and c-rasKi, to current knowledge of proliferation control and its perturbation by differentiation and chemical transformation. Growth stimulation of quiescent cells by serum elevates expression of the myc proto-oncogene in Balb/c 3T3 (A31) cells. In two chemically transformed A31 derivatives (BPA31 and DA31), c-myc expression is constitutive. The levels of c-myc mRNA in quiescent and growing transformed cells are nearly the same, and are only slightly elevated compared to the level found in growing A31 cells. By contrast, c-rasKi expression is cell-cycle-dependent in BPA31 cells. The relative abundance of c-rasKi mRNA begins to increase in mid- to late G0/G1. During terminal differentiation of teratocarcinoma stem cells (F9) into nonproliferating endoderm, relative mRNA abundance is diminished more markedly for c-myc than for c-rasKi. These results demonstrate that expression of the myc and rasKi proto-oncogenes is dependent upon the cellular growth state, and that growth control exhibits growth-factor-dependent, cell-cycle-timed oncogene expression. In the case of the BPA31 cells, c-myc is not rearranged, amplified, or overexpressed. However, the oncogene has lost its cycle-dependent regulation in the chemically transformed cells.     

A role for fibroblasts in mediating the effects of tobacco-induced epithelial cell growth and invasion

Cigarette smoke and smokeless tobacco extracts contain multiple carcinogenic compounds, but little is known about the mechanisms by which tumors develop and progress upon chronic exposure to carcinogens such as those present in tobacco products. Here, we examine the effects of smokeless tobacco extracts on human oral fibroblasts. We show that smokeless tobacco extracts elevated the levels of intracellular reactive oxygen, oxidative DNA damage, and DNA double-strand breaks in a dose-dependent manner. Extended exposure to extracts induced fibroblasts to undergo a senescence-like growth arrest, with striking accompanying changes in the secretory phenotype. Using cocultures of smokeless tobacco extracts-exposed fibroblasts and immortalized but nontumorigenic keratinocytes, we further show that factors secreted by extracts-modified fibroblasts increase the proliferation and invasiveness of partially transformed epithelial cells, but not their normal counterparts. In addition, smokeless tobacco extracts-exposed fibroblasts caused partially transformed keratinocytes to lose the expression of E-cadherin and ZO-1, as well as involucrin, changes that are indicative of compromised epithelial function and commonly associated with malignant progression. Together, our results suggest that fibroblasts may contribute to tumorigenesis indirectly by increasing epithelial cell aggressiveness. Thus, tobacco may not only initiate mutagenic changes in epithelial cells but also promote the growth and invasion of mutant cells by creating a procarcinogenic stromal environment.     

Role of extracellular HSP72 in acute stress-induced potentiation of innate immunity in active rats.

Acute stress can compromise acquired, and potentiate innate, immunity. Recent evidence suggests that the impact of stress on measures of immunity can be modulated by the physical activity status of the organism and that extracellular heat shock protein 72 (eHSP72) contributes to the activation of innate immunity produced by stress. Therefore, this study investigated whether physical activity status would impact the immunologically enhancing effects of stressor exposure [inescapable tail-shock stress (IS)] on innate immunity and whether changes in eHSP72 responses could play a role. Adult, male Fischer 344 rats lived with mobile (physically active) or immobile (sedentary) running wheels. After 6 wk, rats were exposed to IS or to no stress. Immediately after IS, all rats were injected subcutaneously with live Escherichia coli. Inflammation was assessed daily, and plasma eHSP72 was measured at various time points. Rats exposed to IS resolved their inflammation faster than nonstressed rats, but the beneficial impact of stress on recovery was greater in physically active rats. All rats had equal increases in circulating eHSP72 after IS. Splenocytes harvested from a separate cohort of nonstressed rats were cultured with eHSP72, and nitric oxide and cytokines were measured. Physically active rats responded to eHSP72 stimulation in vitro with a greater nitric oxide and cytokine response than sedentary rats. Thus physically active rats both recover faster than sedentary rats after bacterial challenge + IS exposure and demonstrate potentiated cellular responses to eHSP72 activation that could be important for bacterial recovery.     

C. elegans clk-2, a gene that limits life span, encodes a telomere length regulator similar to yeast telomere binding protein Tel2p.

An important quest in modern biology is to identify genes involved in aging. Model organisms such as the nematode Caenorhabditis elegans are particularly useful in this regard. The C. elegans genome has been sequenced [1], and single gene mutations that extend adult life span have been identified [2]. Among these longevity-controlling loci are four apparently unrelated genes that belong to the clk family. In mammals, telomere length and structure can influence cellular, and possibly organismal, aging. Here, we show that clk-2 encodes a regulator of telomere length in C. elegans.     

Cellular senescence as a tumor-suppressor mechanism.

Organisms with renewable tissues had to evolve mechanisms to prevent the development of cancer. One such mechanism is cellular senescence, which irreversibly arrests the growth of cells at risk for neoplastic transformation. Recent findings have revealed the complexities of the senescence phenotype and unexpected possible consequences for the organism.