Methods for derivation of human embryonic stem cells

The expanded blastocysts, developed from 2PN-stage embryos, are generally divided into three categories: a good blastocyst containing a large and distinguishable inner cell mass (ICM), a blastocyst with a small and distinct ICM, and a blastocyst with a poorly defined ICM. In this study, we introduce methods for the derivation of human embryonic stem cells (hESCs) depending on the quality of the blastocysts. An immunosurgical method was used for the good expanded blastocysts. This method, however, raises the probability of ICM loss in cases of hESC derivation from blastocysts with smaller or indistinct ICMs. Furthermore, this method is also associated with a risk of the contamination of the hESCs with animal pathogens. To overcome these shortcomings, the partial- or whole-embryo culture method was used. For blastocysts with no visible ICM, the whole-embryo culture method was used to establish hESCs via the seeding of the entire blastocyst without its zona pellucida directly on a STO feeder layer. However, trophectodermal overgrowth tends to hinder the expansion of the ICM during the initial steps of hESC derivation. Therefore, the partial-embryo culture method was developed to establish hESCs from blastocysts with smaller ICMs. The surgical isolation of the region containing the ICM with an ultra-fine glass pipette alleviates trophectoderm overgrowth. This method is also applicable to blastocysts with large and distinct ICMs, and the efficiency of this method is comparable to that of the immunosurgical method.     

Functional dissection of the cytoplasmic subregions of the interleukin-5 receptor alpha chain in growth and immunoglobulin G1 switch recombination of B cells

The interleukin-5 receptor alpha chain (IL-5Ralpha) is known to regulate the development and function of B cells and eosinophils. Although the functions of IL-5Ralpha cytoplasmic domain subregions have been studied extensively using cultured cell lines, this approach has limitations when studying the functions of distinct primary B-cell subpopulations and their responsiveness to IL-5. In the present study, we generated mice on an IL-5Ralpha null background, each expressing a mutant form of an IL-5Ralpha transgene ligated to a mu enhancer and VH promoter, either lacking the cytoplasmic DC3 region or substituting two proline residues for alanine (ApvA) in the membrane-proximal ppvp motif of the cytoplasmic domain. The ppvp motif, which mediates activation of JAK2/STAT5 and Btk, also contributes to c-fos, c-jun and c-myc expression. IL-5Ralpha null mutant mice showed impaired B-1-cell development, reduced serum immunoglobulin G3 (IgG3) and IgM, no IL-5-induced enhancement of B-cell proliferation and IL-5-induced switch recombination from the mu gene to gamma1 gene; these were not recovered following the expression of the ApvA mutant. In contrast, absence of the DC3 region affected the IL-5-induced switch recombination from the mu to the gamma1 gene and B-1-cell development, while IL-5-induced proliferation and IgM production were at levels similar to those of B cells expressing wild-type IL-5Ralpha transgene. The results clearly indicated that the ppvp motif and the DC3 region of IL-5Ralpha played distinct roles in B-cell proliferation and differentiation. Thus, this present approach offers new insights into the functions of the cytoplasmic subregions of IL-5Ralpha, in particular its carboxy-terminal region.     

Synthesis and characterization of polyanhydride for local BCNU delivery carriers

p-Carboxyphenoxy propane (CPP) prepolymer consisting of 4 units and sebacic acid (SA) prepolymer consisting of about 10 units were synthesized by reacting CPP and SA in the presence of excess acetic anhydride, respectively. Polyanhydride, poly(CPP-SA) copolymers were copolymerized by a melt polycondensation process with a mixture of CPP and SA prepolymer. Copolymers of average molecular weight up to 110,000 g/mol were achieved. The crystallinity of poly(CPP-SA) copolymers was decreased by the addition of the CPP homopolymer segment to SA homopolymer. Poly(CPP-SA) copolymers gradually degraded for period of 10 days. No large difference of weight loss observed according to molecular weight variation of poly(CPP-SA) copolymers. BCNU release from wafers fabricated by poly(CPP-SA) showed a sustained release pattern with no initial burst and delay of drug release.     

Pathogenicity of Escherichia coli O157:H7 in the intestines of neonatal calves.

Cattle are an important reservoir of Shiga toxin-producing enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains, foodborne pathogens that cause hemorrhagic colitis and hemolytic uremic syndrome in humans. EHEC O157:H7 strains are not pathogenic in calves >3 weeks old. Our objective was to determine if EHEC O157:H7 strains are pathogenic in neonatal calves. Calves <36 h old inoculated with EHEC O157:H7 developed diarrhea and enterocolitis with attaching and effacing (A/E) lesions in both the large and small intestines by 18 h postinoculation. The severity of diarrhea and inflammation, and also the frequency and extent of A/E lesions, increased by 3 days postinoculation. We conclude that EHEC O157:H7 strains are pathogenic in neonatal calves. The neonatal calf model is relevant for studying the pathogenesis of EHEC O157:H7 infections in cattle. It should also be useful for identifying ways to reduce EHEC O157:H7 infections in cattle and thus reduce the risk of EHEC O157:H7 disease in humans.     

Gamma-interferon (IFN-gamma) augments apoptotic response to mistletoe lectin-II via upregulation of Fas/Fas L expression and caspase activation in human myeloid U937 cells

Mistletoe lectin-II, a major composition of Korean mistletoe (Viscum album coloratum), is known as a potent apoptosis inducer. The previous research has demonstrated that Korean mistletoe lectin-II induces apoptosis via c-Jun N terminal kinase (JNK) activation in human myeloid U937 cells. The purpose of this research is to prove the synergistic action of mistletoe lectin-II and interferon-γ (IFN-γ) in the apoptotic cytotoxicity of U937. When U937 cells were treated with mistletoe lectin-II after being differentiated by IFN-γ, the proteolytic activity of caspase-3 and 9 was markedly elevated and that of caspase-8 was prolonged for 18 hr. The activation of caspase-3-like protease requires the earlier cleavage of poly(ADP-ribose) polymerase(PARP). Caspase-1 was, however, not activated during the resting phase and nor in IFN-γ-differentiated U937 cells. Western blot analysis revealed that, in IFN-γ-differentiated U937 cells, the expression of Fas (CD95/APO-1) & Fas ligand(FasL) increases the apoptotic sensitivity against Mistletoe lectin-II. Fas (CD95/APO-1) & FasL were not significantly induced solely by mistletoe lectin-II. Furthermore the activity of JNK1 in U937 cells was also markedly increased with IFN-γ-differentiation, compared to that of the control. These results suggest that the IFN-γ-differentiation of U937 cells increases the susceptibility to mistletoe lectin-II-induced apoptosis.     

Effects of behavior modification on body image, depression and body fat in obese Korean elementary school children

This study was performed to investigate the effects of behavior modification on body image, depression and body fat in obese elementary school children. Sixty-two elementary students of the 4th to 6th grade were selected from two different Seoul schools. Thirty-four children in one school were designated as the experimental group, and 28 children from the other school as the control group. The experimental group received 60 - 70 minutes of behavior modification, once a week, for 8 weeks. The control group received neither management nor treatment. The results indicated a significant improvement of body image and a reduction in the increase rate of body fat for the experimental group. This finding strongly supports the theory that behavior modification can be used as an effective strategy in the treatment of obese children.     

Cloning, expression and characterization of a murine-human chimeric antibody with specificity for pre-S2 surface antigen of hepatitis B virus

The cloning, expression and characterization of a murine-human chimeric antibody with specificity for the pre-S2 surface antigen (Ag) of hepatitis B virus (HBV) is described. The heavy and light chain variable region (VH and VL) genes encoding the murine monoclonal antibody (mAb) were isolated and combined with human γ 1 and κ constant region genes, respectively. The expression vectors containing the chimeric heavy and light chain genes were sequentially electroporated into murine Sp2/0 hybridoma cells and transfectomas secreting chimeric antibody were isolated. The chimeric antibody was purified and characterized by ELISA, Western analysis and competition immunoassay, demonstrating that the transfectoma functionally express and secrete murine-human chimeric antibody which retained the specificity and affinity of the parental murine mAb.