Young Joon Moon

TSA-induced DNMT1 down-regulation represses hTERT expression via recruiting CTCF into demethylated core promoter region of hTERT in HCT116

Trichostatin A (TSA), an inhibitor of histone deacetylase, is a well-known antitumor agent that effectively and selectively induces tumor growth arrest and apoptosis. Recently, it was reported that hTERT is one of the primary targets for TSA-induced apoptosis in cancer cells but the mechanism of which has not yet been elucidated. In the present study, to better understand the epigenetic regulation mechanism responsible for the repression of hTERT by TSA, we examined expression of hTERT in the HCT116 colon cancer cell line after treatment with TSA and performed site-specific CpG methylation analysis of the hTERT promoter. We found that TSA-induced the demethylation of site-specific CpGs on the promoter of hTERT, which was caused by down-regulation of DNA methyltransferase 1 (DNMT1). Among the demethylated region, the 31st-33rd CpGs contained a binding site for CTCF, an inhibitor of hTERT transcription. ChIP analysis revealed that TSA-induced demethylation of the 31st-33rd CpGs promoted CTCF binding on hTERT promoter, leading to repression of hTERT. Taken together, down-regulation of DNMT1 by TSA caused demethylation of a CTCF binding site on the hTERT promoter, the result of which was repression of hTERT via recruitment of CTCF to the promoter.

Multipotent Progenitor Cells Derived From Human Umbilical Cord Blood Can Differentiate Into Hepatocyte-Like Cells in a Liver Injury Rat Model

Umbilical cord blood (UCB), a rich source of hematopoietic stem cells, offers practical and ethical advantages. It has been reported that various adult stem cells transplanted into a damaged liver show characteristics of a hepatic lineage. In a previous study, we reported on novel UCB-derived adult stem cells, termed umbilical cord blood-derived multipotent progenitor cells (UCB-MPCs). We demonstrated that these cells were capable of differentiating into hepatocyte- like cells in vitro. To assess the hepatic differentiation capacity of UCB-MPCs, rat models of hepatic injury were generated using carbon tetra-chloride (CCl(4)) with transplantation of cells into the liver. The transplanted cells successfully incorporated into the liver of the recipient animal differentiated into functional hepatocyte-like cells that expressed hepatocyte-specific markers, such as CK-18 and albumin. Moreover, human albumin was detected in the serum of the recipient rat model. These data indicated that UCB-MPCs were capable of displaying similar characteristics to those of functional hepatocytes in a recipient liver. UCB-MPCs may prove to be a useful, transplantable alternative for hepatic progenitor cells in both experimental and therapeutic applications.

Hepatic differentiation of cord blood-derived multipotent progenitor cells (MPCs) in vitro.

Umbilical cord blood (UCB) is a rich source of hematopoietic stem cells that possesses practical and ethical advantages. We previously reported a novel UCB‐derived adult stem cells which we termed umbilical cord blood‐derived multipotent progenitor cells’ (MPCs). MPCs were capable of differentiating into functional neuronal cells. Under appropriate conditions lasting several days or weeks, we now show that the MPCs differentiate into hepatocyte‐like cells in vitro; their properties were verified using reverse transcription‐polymerase chain reaction (RT‐PCR), Western blot, immunofluorescence, periodic acid‐Schiff (PAS) staining of accumulated glycogen and an enzyme‐linked immunosorbent assay (ELISA). We also found that hepatic differentiated cells expressed hepatocyte specific markers, such as albumin, hepatocyte nuclear factor (HNF)‐1α, HNF4, cytokeratin (CK)‐8, CK‐18, tyrosine amino transferase (TAT), and CYP2B6. Moreover, albumin was secreted, which suggests that MPCs from UCB possess multi‐differentiation potential and have the capacity to differentiate into functional cells of hepatic lineage in vitro.

Expression Profile of Genes Representing Varied Spectra of Cell Lineages in Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells

In this experiment, we attempted to isolate MSCs from cryopreserved human CB and to characterize them in terms of the multidifferentiation potential by examining the expression profi le of cell lineage-specifi c genes using RT-PCR. This study was approved by the Institutional Review Board of the Ajou University Hospital and all human samples were obtained with informed consent. CB-MSCs were generated according to the method described by Lee et al. [7] . Briefl y, cryopreserved CB-MNCs were thawed and seeded on non-coated T25 culture fl asks (Nalge Nunc, Naperville, Ill., USA) at a density of 3 ! 10 5 cells/cm 2 in low glucose Dulbecco’s modifi ed Eagle’s medium (LGDMEM; Gibco BRL, Grand Island, N.Y., USA). The medium was changed every 7 days until the fi broblast-like cells reached confl uence. After trypsinization, the cells were replated at appropriate concentrations for further study. BM-MSCs from BM-MNCs were isolated and cultured as described by Bruder et al. [8] . CBand BM-MSCs obtained may be heterogeneous, consisting of several subpopulations [9] . We tried to produce single cell-derived colonies (SCDCs) of MSCs to avoid a possible problem of heterogeneity of the cells. In order to obtain MSC-derived SCDCs, heterogeneous CBMSCs at the second passage were harvested and plated at Bone marrow (BM) transplantation is a well-established classical treatment for children with malignant haematologic diseases including leukaemia or fatal metabolic diseases [1] . Mononuclear cells (MNCs) in human BM undergo haemopoiesis into distinct cell lineages such as haemopoietic stem cells and mesenchymal stem cells (MSCs), which are capable of differentiating into varied spectra of cell lineages [2, 3] . Human umbilical cord blood (CB)-derived MSCs also have a multipotent differentiating ability similar to BM-MSCs [4] . Moreover, CB transplantation, since the fi rst transplantation was performed in 1988 for a child with Fanconi’s anaemia, has become a safe and accepted mode of transplantation for recipients due to a low incidence of severe graft versus host disease, whereas adult BM transplantation requires exact histocompatibility between donors and recipients [5] . However, the actual use of CB-MSCs seems to have some obstacles as regards the diffi culty in their isolation from heterogeneous CB cell populations. Erices et al. [4] demonstrated that human CB is enriched in cells with similar characteristics as adult BM-MSCs and they successfully isolated CB-MSCs. In contrast, some investigators failed to isolate CB-MSCs from CB cell populations, leading to controversy about the existence of MSCs in CB [6] . Received: May 10, 2004 Accepted after revision: October 19, 2004

Detection of heterozygous nonsense mutations in genes of interest using an Escherichia coli-based stop codon assay.

Since nonsense mutations are closely associated with severe conditions of genetic disorders, including familial cancers, rapid and precise detection of those mutations is very important for research purposes and molecular diagnosis. Currently, screening methods such as the FASAY (functional analysis of separated alleles in yeast) and the Y‐SC (stop codon assay in yeast) are used for functional detection of nonsense mutations in genes of interest. But these yeast‐based approaches are time‐consuming, expensive and complicated. In order to circumvent these problems, we, in the present study, devised a novel Escherichia coli‐based screening method, the E‐SC (E. coli stop codon assay) for the detection of heterozygous nonsense mutations in genes of interest. Our strategy was based on the fact that the plasmid replicating with a low copy number in E. coli allows an effective separation of normal and mutant alleles. Moreover, it relies on the expression vector, resulting in the formation of white and blue colonies for mutant and normal alleles through the expression of PCR‐amplified fragment/lacZ fusion protein respectively. To evaluate the applicability of the E‐SC method for the detection of the heterozygous truncating mutation, PCR‐amplified exon 7 of the StAR [steroidogenic acute regulatory protein; causative gene of the CAH (congenital lipoid adrenal hyperplasia)] and RT (reverse transcription)–PCR‐amplified full‐length cDNA of MeCP2 (methyl‐CpG‐binding protein 2; causative gene of Rett syndrome) were used. The E‐SC showed an almost 1:1 ratio of blue/white colonies in all patients examined, whereas the control samples produced blue colonies only. These results demonstrate that the E‐SC system is useful for rapid and precise detection of known and unknown heterozygous truncation mutations in genes that cause genetic disorders and familial cancers.

A rare case of post operative traumatic optic neuropathy in orbital floor fracture

Methods: The spreader flap was used in 32 patients who were candidates for primary rhinoplasty and also 34 others without spreader flap usage were recruited as a control group. Accordingly, acoustic rhinometry was used in every patient before and after one year of operation. Average volume and minimal cross-sectional area of the nose and findings were collected. Also, the preand post-operative patients’ subjective assessment about their satisfaction from their appearance and nasal obstruction were evaluated according to Visual Analogue Scale (VAS).F undergoes a series of changes with age and weight fluctuation. Volume displacement and loss are main reasons for changing facial structure. A typical oval or triangular youthful face becomes wide and round/apple shape with time. There is loss of fat in the temporal region, preiorbital region and part of cheek areas on the other hand there is accumulation of fat in the medial cheek area, jowls, angle of mandible, submandibular and submental areas. The author will discuss the detail anatomical changes of the face, systematic way to evaluate an aging face and minimal surgical approaches to rejuvenate the volume of the face.Materials & Method: Preparing for skin incisions, the key-hole-scheme with a caudal “drop-form” is drawn on the patient in upward position. After breast reduction/mastopexy with superior pedicle and central mobilisation from the pectoralis fascia, the remaining breast is fixed at the height of the 2nd to 3rd rib. This causes, in the long run, an improved cranial pole filling. After suturing the breast columns and lateral mobilisation of the skin flaps, the areola is transferred and fixed in its new position. The vertical incision line is cleanly adapted layer wise without skin plication. The caudal opening of the dropwise resected skin remains. Depending on the size of this opening wound closure uses a tightly intradermal placement of a “tabaccopouch” suture. This then is centrally positioned on the pectoralis fascia at the height of the new inframammary fold. The healing then results in a better rounding of the caudal mamma. If there is abundant skin, a double “tobacco-pouch” suture in 8-form is preferred and brings a more homogeneous distribution of the plicated skin. The eventual healing increasingly smoothes the skin in the inframammary fold. The visible vertical scar remains small and thin.B the age of thirty-five, two-thirds of American men will suffer noticeable hair loss and by the age of fifty, eighty-five percent of American men would have significant hair thinning. What’s not as well known is that by the age of age sixty-five, fifty to seventy-five percent of women experience noticeable hair loss. Whether male or female, hair loss can be devastating to a person’s self image and emotional well being. Realizing the wide spread problem, the cosmetics industry has non-drug alternatives that can help improve the appearance of thinning hair. This focus of this talk will evaluate the different technologies available and compare them to their drug counterparts.