Carbohydrate response element (ChRE)-binding protein (ChREBP) is a recently discovered transcription factor that is activated in response to high glucose concentrations in liver independently of insulin. ChREBP was first identified by its ability to bind the ChRE of the liver pyruvate kinase (LPK) gene. We recently reported that the increase in expression of multiple liver lipogenic enzyme mRNAs elicited by feeding a high-carbohydrate diet as well as that of LPK mRNA is markedly reduced in mice lacking ChREBP gene expression (ChREBP ؊/؊ ) in comparison to WT mice. The present study provides evidence for a direct and dominant role of ChREBP in the glucose regulation of two key liver lipogenic enzymes, acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS). ACC, FAS, and LPK mRNA levels were higher in WT hepatocytes cultured with high (25 mM) rather than low (5.5 mM) glucose medium, but there was no effect of glucose concentration on these mRNA levels in ChREBP ؊/؊ hepatocytes. Similarly, reporter constructs containing ACC, FAS, or LPK gene ChREs were responsive to glucose when transfected into WT but not ChREBP ؊/؊ hepatocytes, and glucose transactivation of the constructs in ChREBP ؊/؊ hepatocytes was restored by cotransfection with a ChREBP expression plasmid. ChREBP binding to ACC, FAS, and LPK ChRE sequences in vitro was demonstrated by electrophoretic mobility super shift assays. In vivo binding of ChREBP to ACC, FAS, and LPK gene promoters in intact liver nuclei from rats fed a high-carbohydrate diet was demonstrated by using a formaldehyde crosslinking and chromatin immunoprecipitation procedure.

Neural stem/progenitor cells (NS/PCs) can generate a wide variety of neural cells. However, their fates are generally restricted, depending on the time and location of NS/PC origin. Here we demonstrate that we can recapitulate the spatiotemporal regulation of central nervous system (CNS) development in vitro by using a neurosphere-based culture system of embryonic stem (ES) cell-derived NS/PCs. This ES cell-derived neurosphere system enables the efficient derivation of highly neurogenic fibroblast growth factor-responsive NS/PCs with early temporal identities and high cell-fate plasticity. Over repeated passages, these NS/PCs exhibit temporal progression, becoming epidermal growth factorresponsive gliogenic NS/PCs with late temporal identities; this change is accompanied by an alteration in the epigenetic status of the glial fibrillary acidic protein promoter, similar to that observed in the developing brain. Moreover, the rostrocaudal and dorsoventral spatial identities of the NS/ PCs can be successfully regulated by sequential administration of several morphogens. These NS/PCs can differentiate into early-born projection neurons, including cholinergic, catecholaminergic, serotonergic, and motor neurons, that exhibit action potentials in vitro. Finally, these NS/PCs differentiate into neurons that form synaptic contacts with host neurons after their transplantation into wild-type and disease model animals. Thus, this culture system can be used to obtain specific neurons from ES cells, is a simple and powerful tool for investigating the underlying mechanisms of CNS development, and is applicable to regenerative treatment for neurological disorders. STEM CELLS 2008;26: 3086 -3098 Disclosure of potential conflicts of interest is found at the end of this article.

Prenatal exposure of the developing brain to various environmental challenges increases susceptibility to late-onset of neuropsychiatric dysfunction; still the underlying mechanisms remain obscure. Here we show that exposure of embryos to a variety of environmental factors such as alcohol, methylmercury and maternal seizure activates HSF1 in cerebral cortical cells. Furthermore, Hsf1 deficiency in the mouse cortex exposed in utero to subthreshold levels of these challenges causes structural abnormalities and increases seizure susceptibility after birth. In addition, we found that human neural progenitor cells differentiated from induced pluripotent stem cells derived from schizophrenia patients show higher variability in the levels of HSF1 activation induced by environmental challenges compared to controls. We propose that HSF1 plays a crucial role in the response of brain cells to prenatal environmental insults and may be a key component in the pathogenesis of late–onset neuropsychiatric disorders.

ComA of Streptococcus is a member of the bacteriocin-associated ATP-binding cassette transporter family and is postulated to be responsible for both the processing of the propeptide ComC and secretion of the mature quorum-sensing signal. The 150-amino acid peptidase domain (PEP) of ComA specifically recognizes an extended region of ComC that is 15 amino acids in length. It has been proposed that an amphipathic ␣-helix formed by the N-terminal leader region of ComC, as well as the Gly-Gly motif at the cleavage site, is critical for the PEPComC interaction. To elucidate the substrate recognition mechanism, we determined the three-dimensional crystal structure of Streptococcus mutans PEP and then constructed models for the PEP⅐ComC complexes. PEP had an overall structure similar to the papain-like cysteine proteases as has long been predicted. The active site was located at the bottom of a narrow cleft, which is suitable for binding the Gly-Gly motif. Together with the results from mutational experiments, a shallow hydrophobic concave surface of PEP was proposed as a site that accommodates the N-terminal helix of ComC. This dual mode of substrate recognition would provide the small PEP domain with an extremely high substrate specificity.

ComA, a member of the bacteriocin ATP-binding cassette transporters, is postulated to be responsible for both the processing of the propeptide ComC and secretion of the mature competencestimulating peptide, which regulates the competence and subsequent genetic transformation in Streptococcus pneumoniae. A recombinant N-terminal peptidase domain of ComA, designated PEP, was expressed as a soluble protein in Escherichia coli, purified to homogeneity, and characterized. Gel-filtration analysis revealed that PEP functions as a monomer. The purified PEP exhibited an efficient proteolytic activity for the substrate ComC, which was cleaved after the double glycine site. The stability of PEP was examined by circular dichroism analyses. A convenient method for analyzing the proteolytic activity of PEP was developed, and the kinetic parameters for ComC were determined (k cat ‫؍‬ 1.5 ؎ 0.083 min Quorum sensing (1) is a way that bacteria communicate with each other to respond properly to growth conditions and successfully survive as a "community." Bacterial cells are continuously releasing various chemical substances called autoinducers into their surrounding environment. As the population density of a bacterial species increases, so does the cognate autoinducer concentration, which subsequently reaches a threshold to bind to either the cell surface or intracellular receptors (2, 3). The signal pathway then becomes activated, which leads to a cascade of intracellular biochemical signals or altered gene expressions in the target bacteria. Many bacteria are known to regulate diverse physiological processes through this system, such as bioluminescence (4), regulation of sporulation (5), virulence factor expression (6), antibiotics production (6), competence for genetic transformation (7), and activation of biofilm formation (7).The competence-stimulating peptide (CSP) 2 of Streptococcus pneumoniae is one of the well studied examples of autoinducers of Grampositive bacteria. The 17-amino acid CSP is postulated to be cleaved from the 41-amino acid propeptide ComC and concomitantly exported by ComA with the help of an accessory protein, ComB (8). The accumulated CSP binds to the cell surface receptor ComD, which subsequently phosphorylates ComE by its histidine kinase activity and then induces the transcription of genes, such as comX and comW, associated with the DNA uptake (competence) and recombination (7, 9). Thus, ComA is a key molecule essential for the first step of the quorumsensing system of S. pneumoniae.ComA is a member of a family of bacteriocin-associated ATP-binding cassette transporters, which are composed of three domains (10): an N-terminal domain that has been proposed to possess the peptidase activity, a transmembrane domain consisting of six membrane-spanning segments, and a C-terminal ATP-binding domain located on the cytoplasmic face of the membrane. The peptidase domains of this family are thought to cleave their cognate propeptides after the consensus Gly-Gly motif. Recently, the peptidase domains of the fa...

ComA of Streptococcus is a member of the bacteriocin-associated ABC transporters, which is responsible for both the processing of the propeptide ComC and secretion of the mature quorum-sensing signal. The quorum-sensing system is a bacterial intercellular communication system implicated in various functions including biofilm formation. In this study, the peptidase domains (PEPs) of the ComAs from six species of Streptococcus and ComCs from four species were expressed, purified, and characterized to address the mechanism of the substrate recognition of PEP. PEPs specifically cleaved ComCs after the Gly-Gly site in all the PEP-ComC combinations examined. The N-terminal leader region of ComC was found to form an amphiphilic alpha-helix structure upon binding to the PEP. Furthermore, mutagenesis studies revealed that four conserved hydrophobic residues in this leader region of ComC extending from -15 to -4 positions are critical in the interaction with PEP. Together with the double glycine motif, these structural features of ComC would explain the strict substrate specificity of the PEP.

Human olfactory cells obtained by rapid nasal biopsy have been suggested to be a good surrogate system to address brain disease-associated molecular changes. Nonetheless, whether use of this experimental strategy is justified remains unclear. Here we compared expression profiles of olfactory cells systematically with those from the brain tissues and other cells. Principal component analysis indicated that the expression profiles of olfactory cells are very different from those of blood cells, but are closer to those of stem cells, in particular mesenchymal stem cells, that can be differentiated into the cells of the central nervous system.

Learning disabilities are hallmarks of congenital conditions caused by prenatal exposure to harmful agents. Those include Fetal Alcohol Spectrum Disorders (FASD) with a wide range of cognitive deficiencies including impaired motor skill development. While these effects have been well characterized, the molecular effects that bring about these behavioral consequences remain to be determined. We have previously found that the acute molecular responses to alcohol in the embryonic brain are stochastic, varying among neural progenitor cells. However, the pathophysiological consequences stemming from these heterogeneous responses remain unknown. Here we show that acute responses to alcohol in progenitor cells alter gene expression in their descendant neurons. Among the altered genes, an increase of the calcium-activated potassium channel Kcnn2 in the motor cortex correlates with motor learning deficits in the mouse model of FASD. Pharmacologic blockade of Kcnn2 improves these learning deficits, suggesting Kcnn2 blockers as a novel intervention for learning disabilities in FASD.