Young Seek Lee

BURSTING, SPIKING, CHAOS, FRACTALS, AND UNIVERSALITY IN BIOLOGICAL RHYTHMS

Biological systems offer many interesting examples of oscillations, chaos, and bifurcations. Oscillations in biology arise because most cellular processes contain feedbacks that are appropriate for generating rhythms. These rhythms are essential for regulating cellular function. In this tutorial review, we treat two interesting nonlinear dynamic processes in biology that give rise to bursting, spiking, chaos, and fractals: endogenous electrical activity of excitable cells and Ca2+ releases from the Ca2+ stores in nonexcitable cells induced by hormones and neurotransmitters. We will first show that each of these complex processes can be described by a simple, yet elegant, mathematical model. We then show how to utilize bifurcation analyses to gain a deeper insight into the mechanisms involved in the neuronal and cellular oscillations. With the bifurcating diagrams, we explain how spiking can be transformed to bursting via a complex type of dynamic structure when the key parameter in the model varies. Understanding how this parameter would affect the bifurcation structure is important in predicting and controlling abnormal biological rhythms. Although we describe two very different dynamic processes in biological rhythms, we will show that there is universality in their bifurcation structures.

Sonic hedgehog and FGF8 collaborate to induce dopaminergic phenotypes in the Nurr1-overexpressing neural stem cell

Neural stem cells are self-renewing cells capable of differentiating into all neural lineage cells in vivo and in vitro. In the present study, coordinated induction of midbrain dopaminergic phenotypes in an immortalized multipotent neural stem cell line can be achieved by both overexpression of nuclear receptor Nurr1, and fibroblast growth factor-8 (FGF-8), and sonic hedgehog (Shh) signals. Nurr1 overexpression induces neuronal differentiation and confers competence to respond to extrinsic signals such as Shh and FGF-8 that induce dopaminergic fate in a mouse neural stem cell line. Our findings suggest that immortalized NSCs can serve as an excellent model for understanding mechanisms that regulate specification of ventral midbrain DA neurons and as an unlimited source of DA progenitors for treating Parkinson disease patients by cell replacement.

Suppression of Egr‐1 transcription through targeting of the serum response factor by oncogenic H‐Ras

The transcription factor Egr‐1 functions as a key regulator in cellular growth, differentiation, and apoptosis. The loss of Egr‐1 expression is closely associated with tumor development, although the molecular mechanism behind the suppression of Egr‐1 is largely unknown. In this report, we show that growth factor‐induced transcriptional activation of Egr‐1 gene is downregulated by chronic expression of oncogenic H‐Ras in NIH3T3 fibroblasts. Our results demonstrate that phosphoinositide 3‐kinase (PI3K) signaling is necessary for oncogenic H‐Ras‐mediated reduction of Egr‐1 gene expression. Aberrant activation of PI3K signaling by oncogenic Ras decreased the level of serum response factor (SRF) protein through the acceleration of proteolysis, which resulted in decreased SRF binding to the serum response element (SRE) sites within the Egr‐1 promoter, leading to the suppression of Egr‐1 transcription. Inhibition of PI3K signaling restored the downregulation of SRF and Egr‐1 expression caused by oncogenic Ras. Our findings suggest a novel signaling mechanism by which prolonged activation of oncogenic H‐Ras can trigger the loss of tumor suppressor Egr‐1 through the PI3K pathway in NIH3T3 fibroblast model cell lines.

Overexpression of midbrain-specific transcription factor Nurr1 modifies susceptibility of mouse neural stem cells to neurotoxins

Nurr1 is a member of the nuclear receptor superfamily of transcription factors that is highly expressed in midbrain dopaminergic (DA) neurons, the cells primarily lost in human Parkinsons disease (PD), and in Nurr1-null mice selective agenesis of midbrain DA neurons is found. To investigate possible correlation between the expression of Nurr1 gene and neurotoxin-induced cell death of DA neurons, a neural stem cell line (NSC, A3) and Nurr1-overexpressing NSC (A3.Nurr1) were exposed to DA neurotoxins 6-hydroxydopamine (6-OHDA) and methyl phenylpyridinium (MPP(+)). Although both neurotoxins were shown to induce cell death in A3 and A3.Nurr1 cells, patterns of cell deaths were different. A3.Nurr1 cells showed increased vulnerability to 6-OHDA cytotoxicity, but increased resistance to MPP(+)-induced cell death when compared to A3 cells. To investigate the differential vulnerability to neurotoxins by Nurr1 protein correlates with biochemical features that discriminate between apoptosis and necrosis, we carried out a nucleosomal DNA fragmentation assay and electron microscopy. While 6-OHDA treatment induced shrinkage of cytoplasmic membrane, condensation of nuclei and generation of apoptotic bodies in both cell lines, cells treated with MPP(+) showed mitochondrial swelling, indicating that 6-OHDA- but not MPP(+)-mediated cell death was apoptotic. These results suggest that DA neuronal cell death in response to 6-OHDA and MPP(+) may progress through separate signaling pathways differentially regulated by the Nurr1 protein. Our observations indicated that Nurr1 may play a role in the manifestation of DA neurotoxicity and that variations in Nurr1 expression might be a susceptibility factor for DA neurodegeneration in PD.

Induction of proinflammatory mediators requires activation of the TRAF, NIK, IKK and NF-κB signal transduction pathway in astrocytes infected with Escherichia coli

Escherichia coli is associated with inflammation in the brain. To investigate whether astrocytes are involved in E. coil‐induced inflammation, we assessed the levels of expression of proinflammatory mediators produced by E. coli‐infected astrocytes. E. coli infection in primary human astrocytes and cell lines increased expression of the CXC chemokine IL‐8/GRO‐α, the CC chemokine MCP‐1, TNF‐α, and iNOS. E. coli infection activated p65/p50 heterodimeric NF‐κB and concurrently decreased the signals of IκBα. Blocking the NF‐κB signals by IκBα‐superrepressor‐containing retrovirus or antisense p50 oligonucleotide transfection resulted in down‐regulation of expression of the proinflammatory mediators. Furthermore, superrepressors of IκBα, IκB kinase (IKK) or NF‐κB inducing kinase (NIK) inhibited the up‐regulated expression of the downstream target genes of NF‐κB such as IL‐8 and MCP‐1, and superrepressors of TNF receptor‐associated factor (TRAF)2 and TRAF5 also inhibited expression of the E. coli‐induced target genes of NF‐κB. These results indicate that proinflammatory mediators such as the CXC chemokine IL‐8/GRO‐α, the CC chemokine MCP‐1, TNF‐α, and iNOS can be expressed in E. coli‐infected astrocytes via an NF‐κB pathway, suggesting that these mediators may contribute to inflammation in the brain, including infiltration of inflammatory cells.

Genome-scale DNA methylation pattern profiling of human bone marrow mesenchymal stem cells in long-term culture

Human bone marrow mesenchymal stem cells (MSCs) expanded in vitro exhibit not only a tendency to lose their proliferative potential, homing ability and telomere length but also genetic or epigenetic modifications, resulting in senescence. We compared differential methylation patterns of genes and miRNAs between early-passage [passage 5 (P5)] and late-passage (P15) cells and estimated the relationship between senescence and DNA methylation patterns. When we examined hypermethylated genes (methylation peak ≥ 2) at P5 or P15, 2,739 genes, including those related to fructose and mannose metabolism and calcium signaling pathways, and 2,587 genes, including those related to DNA replication, cell cycle and the PPAR signaling pathway, were hypermethylated at P5 and P15, respectively. There was common hypermethylation of 1,205 genes at both P5 and P15. In addition, genes that were hypermethylated at P5 (CPEB1, GMPPA, CDKN1A, TBX2, SMAD9 and MCM2) showed lower mRNA expression than did those hypermethylated at P15, whereas genes that were hypermethylated at P15 (MAML2, FEN1 and CDK4) showed lower mRNA expression than did those that were hypermethylated at P5, demonstrating that hypermethylation at DNA promoter regions inhibited gene expression and that hypomethylation increased gene expression. In the case of hypermethylation on miRNA, 27 miRNAs were hypermethylated at P5, whereas 44 miRNAs were hypermethylated at P15. These results show that hypermethylation increases at genes related to DNA replication, cell cycle and adipogenic differentiation due to long-term culture, which may in part affect MSC senescence.

ZIFIBI: Prediction of DNA binding sites for zinc finger proteins

The cis-regulatory region of target genes is key elements in the transcriptional regulation of gene expression. Many of these cis-regulatory regions have not been identified by either biological experiments or computational methods. Recently, a few additional C(2)H(2) zinc finger transcription factor binding sites have been discovered. The majority of the zinc finger binding sites, however, are still unknown. In this study, we used publically available data to evaluate possible interaction patterns between nucleotides and the amino acids of zinc finger domains. We calculated the most probable state path of three nucleotides sequences using a Hidden Markov Model (HMM). We used these computations to predict C(2)H(2) zinc finger transcription factor binding sites in cis-regulatory regions of their target genes (http://bioinfo.hanyang.ac.kr/ZIFIBI/frameset.php).

Identification and functional characterization of an alternative splice variant within the fourth exon of human nanog

Nanog, a homeodomain (HD) transcription factor, plays a critical role in the maintenance of embryonic stem (ES) cell self-renewal. Here, we report the identification of an alternatively-spliced variant of nanog. This variant lacked a stretch of amino acids (residues 168-183) located between the HD and tryptophan-repeat (WR) of the previously-reported full length sequence, suggesting that the deleted sequence functions as a linker and possibly affects the flexibility of the C-terminal transactivation domain relative to the DNA binding domain. Expression of mRNA encoding the splice variant, designated as nanog-δ 48, was much lower than that of the full length version in human ES cells. The ratio of nanog-δ 48 transcript to full length transcript increased, however, in multipotent adult progenitor cells. EMSA analysis revealed that both forms of Nanog were able to bind a nanog binding sequence with roughly the same affinity. A reporter plasmid assay also showed that both variants of nanog modestly repressed transactivation of gata-4, whose expression is proposed to be inhibited by nanog, with comparable potency. We conclude that, despite the difference in primary structure and expression pattern in various stem cells, the alternatively-spliced variant of Nanog has similar activity to that of the full length version.

Eosinophilic differentiation is promoted by blockage of Notch signaling with a gamma-secretase inhibitor

Although increasing evidence supports the inhibitory role of Notch in granulocyte differentiation, the direct effects of Notch on the differentiation and maturation of eosinophils, one type of granulocyte, have not yet been studied. We investigated whether a blockage of Notch signaling promoted the differentiation of eosinophils from umbilical cord blood (UCB) cells. Freshly isolated UCB cells were cultured with IL‐3, IL‐5 and GM‐CSF in the presence or absence of a γ‐secretase inhibitor L‐685,458, and examined for the expression of major basic protein (MBP). Freshly isolated UCB cells expressed mRNA and proteins for Notch 1, Notch 2, Delta 1, and Jagged 1. MBP expression in cultures with the inhibitor was significantly increased, as compared with the cultures in the absence of the inhibitor. Treatment with the inhibitor was accompanied by a decrease in Hes 1 mRNA expression, indicative of Notch‐mediated signaling for the inhibitor effect. UCB cells cultured with the inhibitor for 28 days displayed similar levels of CCR3, a late marker of eosinophil development, as compared with the cells cultured without the inhibitor, but almost completely lost chemotaxis response to eotaxin. Our data suggest that Notch signaling may modulate eosinophil migration at the mature stage as well as inhibit eosinophil differentiation.

Scale-up process for expression and renaturation of recombinant human epidermal growth factor from Escherichia coli inclusion bodies

A cDNA encoding mature epidermal growth factor (EGF) was isolated and cloned into a pQE30 vector in which the His6‐tagged EGF was expressed. pH‐stat feeding of concentrated medium at the time of isopropyl β‐d‐thiogalactoside induction and slug‐feedings of the enriched medium during the induction resulted in a higher cell density and specific expression. Using a simple refolding protocol that consisted of 1 mM l‐cysteine addition for a 1‐h reduction followed by 5 mM l‐cystine addition for oxidative refolding, we were able to convert nearly all EGF monomers into the oxidized form. Also, the refolding aggregate was converted into the monomeric form. Approx. 50% overall yield was obtained from the dissolved inclusion bodies to a single peak under FPLC. We hope that the result of this study may provide information that is useful for the scale‐up of the recombinant human EGF production process.