Quantitative analysis of human tissue-specific differences in methylation

Tissue-specific differentially methylated regions (tDMRs) have been identified and implicated for their indispensable involvement in mammalian development and tissue differentiation. In this report, a quantitative DNA methylation analysis was performed for 13 human orthologous regions of recently confirmed mouse tDMRs by using Sequenom Mass Array, by which bisulfite-treated fragments are quantitatively detected using time of flight mass spectroscopy analysis. Eight regions were shown as tDMRs in various tissues from three independent individuals. Testis DNA samples from eight individuals were also analyzed for methylation. Interestingly, there is evidence that the DNA methylation level is divergent among individuals. DNA methylation levels of five testis-specific DMRs were significantly inversely correlated with the number of spermatocytes. However, a positive correlation was seen at tDMRs located near the TRIM38 and CASZ1 genes. Our results indicate that tDMRs are conserved between mouse and human and may have an important role in regulating tissue function, differentiation, and aging.     

Selection of a carbohydrate-binding domain with a helix-loop-helix structure

We obtained a novel carbohydrate-binding peptide having a helix-loop-helix scaffold from a random peptide library. The helix-loop-helix peptide library randomized at five amino acid residues was displayed on the major coat protein of a filamentous phage. Affinity selection with a ganglioside, Galbeta1-3GalNAcbeta1-4(Neu5Acalpha2-3)Galbeta1-4Glcbeta1-1'Cer (GM1), gave positive phage clones. Surface plasmon resonance spectroscopy showed that a corresponding 35-mer synthetic peptide had high affinity for GM1 with a dissociation constant of 0.24 microM. This peptide preferentially binds to GM1 rather than asialo GM1 and GM2, suggesting that a terminal galactose and sialic acid are required for the binding as for cholera toxin. Circular dichroism spectroscopic studies indicated that a helical structure is important for the affinity and specificity. Furthermore, alanine scanning at randomized positions showed that arginine and phenylalanine play an especially important role in the recognition of carbohydrates. Such a de novo helix-loop-helix peptide would be available for the design of carbohydrate-binding proteins.     

Neural regeneration by regionally induced stem cells within poststroke brains: Novel therapy perspectives for stroke patients

Ischemic stroke is a critical disease which causes serious neurological functional loss such as paresis. Hope for novel therapies is based on the increasing evidence of the presence of stem cell populations in the central nervous system(CNS) and the development of stem-cell-based therapies for stroke patients. Although mesenchymal stem cells(MSCs) represented initially a promising cell source,only a few transplanted MSCs were present near the injured areas of the CNS.Thus, regional stem cells that are present and/or induced in the CNS may be ideal when considering a treatment following ischemic stroke. In this context, we have recently showed that injury/ischemia-induced neural stem/progenitor cells(i NSPCs) and injury/ischemia-induced multipotent stem cells(i SCs) are present within post-stroke human brains and post-stroke mouse brains. This indicates that i NSPCs/i SCs could be developed for clinical applications treating patients with stroke. The present study introduces the traits of mouse and human i NSPCs,with a focus on the future perspective for CNS regenerative therapies using novel i NSPCs/i SCs.     

Abnormal biantennary sugar chains are expressed in human chorionic gonadotropin produced in the choriocarcinoma cell line, JEG-3

Over the past two decades, sugar chain structures of human chorionic gonadotropin (hCG) produced in healthy people, three types of trophoblastic disease, and some types of cell lines have been analyzed. The abnormal biantennary structure of hCG is a good marker for the diagnosis of malignant choriocarcinoma. In spite of much research, hCG with an abnormal biantennary structure is only detected in the urine of choriocarcinoma or pregnant diabetic patients. We hypothesized that the formation mechanism of the abnormal biantennary sugar chain structure is mainly caused by high GnT-IV activity. To confirm this, we measured the N-acetylglucosaminyltransferase (GnT)-IV activity and hCG productivity in three choriocarcinoma cell lines, and selected JEG-3 cells. hCG samples were purified from medium conditioned by JEG-3 cells, and their sugar chain structures were analyzed. We detected an abnormal biantennary structure, and the proportions were different from those previously reported in the urine samples of choriocarcinoma patients. These findings proved our hypothesis and suggest the usefulness of JEG-3 cells for further analyses of abnormal biantennary structure formation. Published in 2004.     

p73 is expressed in human thymic epithelial cells.

The thymus is a heterogeneous immune organ in which immature T-cells develop and eventually specialize to make certain immune responses of their own. Among various types of stromal cells in the thymus, thymic epithelial cells (TECs) have a crucially important function for presenting self-antigens and secreting cytokines to thymocytes for their maturation into T-cells. In this study we show that the p73 gene, a homologue of the tumor suppressor gene p53, was expressed in the nucleus of the human TEC in vivo and in TEC lines in vitro. Because p73 has the capacity to be a transactivator like p53, it may contribute to T-cell development in the context of TEC biology as regulated in the cell cycle and apoptosis.     

Change in mobility of side chains due to neutralization of charged poly(L-lysine) with dansyl group

Poly(L -lysine) having dansyl (5-dimethylamino-1-naphthalene-sulfonyl) groups to its side chains was prepared. The fluorescence spectra and fluorescence anisotropy ratios of the dansyl (DNS) group were measured in various conditions. In aqueous solution the increase in emission intensity was observed reflecting the alkali-induced coil-to-helix transition. In aqueous-methanolic solutions with methanol content above 60 wt %, the poly(L -lysine) with DNS group (DNS-PLL) was probed to show α-helical conformation from CD spectra. With addition of alkali, the increase in fluorescence intensity of α-helical DNS-PLL and the drastic change in fluorescence anisotropy ratio were observed. In this case the rotational mobility of DNS probe decreases, gives a minimum at a certain concentration of added alkali, and then increases again up to approximately the initial level. At the concentration where the rotational mobility gives the minimum, intensity of scattered light gives a maximum. This shows that suppression of the mobility of DNS side chains is caused by the intermolecular aggregation of α-helical DNS-PLL. This concentration of added alkali corresponds to the midpoint of neutralization to charged side chains of the DNS-PLL. The interaction that causes aggregate of α-helical DNS-PLL is suggested to be the intermolecular hydrogen bonding between neutralized and unneutralized side chains. © 1994 John Wiley & Sons, Inc.