Neferine-induced apoptosis is dependent on the suppression of Bcl-2 expression via downregulation of p65 in renal cancer cells.

Neferine is an alkaloid extracted from a seed embryo of Nelumbo nucifera and has recently been shown to have anticancer effects in various human cancer cell lines. However, the detailed molecular mechanism of neferine-induced apoptosis has not been elucidated in renal cancer cells. In the present study, we observed that neferine induced inhibition of cell proliferation and apoptosis in Caki-1 cells in a dose-dependent manner by using MT assay and flow cytometry and that neferine-mediated apoptosis was attenuated by pretreatment with N-benzyloxycarbony-Val-Ala-Asp (O-methyl)-fluoromethyketone, a pan-caspase inhibitor.

Identification of ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3) as a regulator of N-acetylglucosaminyltransferase GnT-IX (GnT-Vb).

Our previous studies on a β1,6-N-acetylglucosaminyltransferase, GnT-IX (GnT-Vb), a homolog of GnT-V, indicated that the enzyme has a broad GlcNAc transfer activity toward N-linked and O-mannosyl glycan core structures and that its brain-specific gene expression is regulated by epigenetic histone modifications. In this study, we demonstrate the existence of an endogenous inhibitory factor for GnT-IX that functions as a key regulator for GnT-IX enzymatic activity in Neuro2a (N2a) cells. We purified this factor from N2a cells and found that it is identical to ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3), as evidenced by mass spectrometry and by the knockdown and overexpression of ENPP3 in cultured cells.

N-acetylglucosaminyltransferase (GnT) assays using fluorescent oligosaccharide acceptor substrates: GnT-III, IV, V, and IX (GnT-Vb).

Determining glycosyltransferase activities gives a clue for better understanding an underlying mechanism for glycomic alterations of carrier molecules. N-glycan branch formation is concertedly regulated by cooperative and competitive activities of N-acetylglucosaminyltransferases (GnTs). Here, we describe methods for large scale preparation of the oligosaccharide acceptor substrate, fluorescence-labeling of oligosaccharides by pyridylamination, quality control, and reversed phase HPLC-based measurement of GnT activities including GnT-III, IV, V, and IX.

Altered protein profiles in human umbilical cords with preterm and full-term delivery.

Several biomarkers are routinely used clinically for predicting preterm labor; however, these factors are either nonspecific or detected too late. Here, we performed protein profiles in preterm- and term-derived human umbilical cord by using 2DE. Approximately 200 different proteins were identified between preterm- and term-delivered umbilical cords.

α1,3-galactosyltransferase deficiency in germ-free miniature pigs increases N-glycolylneuraminic acids as the xenoantigenic determinant in pig-human xenotransplantation.

In this study, we examined whether Hanganutziu-Deicher (H-D) antigens are important as an immunogenic non-α1,3-galactose (Gal) epitope in pigs with a disrupted α1,3-galactosyltransferase gene. The targeting efficiency of the AO blood genotype was achieved (2.2%) in pig fibroblast cells.

Alpha 1,3-galactosyltransferase deficiency in pigs increases sialyltransferase activities that potentially raise non-gal xenoantigenicity.

We examined whether deficiency of the GGTA1 gene in pigs altered the expression of several glycosyltransferase genes. Real-time RT-PCR and glycosyltransferase activity showed that 2 sialyltransferases [α2,3-sialyltransferase (α2,3ST) and α2,6-sialyltransferase (α2,6ST)] in the heterozygote GalT KO liver have higher expression levels and activities compared to controls. Enzyme-linked lectin assays indicated that there were also more sialic acid-containing glycoconjugate epitopes in GalT KO livers than in controls.

Electrical activation enhances pre-implantation embryo development following sperm injection into in vitro matured pig oocytes.

The objective of this study was to evaluate the effect of electrical stimulation (EST) on pronuclear formation, chromosomal constitution, and developmental capability among in vitro matured pig oocytes following intracytoplasmic sperm injection (ICSI). After ICSI, the oocytes were randomly distributed and cultured into 3 groups: the EST activated ICSI group, non-activation ICSI group, and in vitro fertilization (IVF) group. The proportion of oocytes in which 2 pronuclei were formed in ICSI groups was significantly higher in the former groups than in the IVF group (96.

Histone deacetylase inhibition improves activation of ribosomal RNA genes and embryonic nucleolar reprogramming in cloned mouse embryos.

Our group found that the treatment of embryos with histone deacetylase inhibitors (HDACi), including trichostatin A, Scriptaid, suberoylanilide hydroxamic acid, and oxamflatin, after cloning by somatic cell nuclear transfer (SCNT) resulted in significantly improved efficiency. Although many researchers have investigated the use of HDACi treatment to improve the quality of cloned mouse embryos, the mechanism underlying this treatment has not been completely understood. We believe that the effect of HDACi on embryonic gene activation (EGA) is important for normal development of cloned embryos.

Characterization of a putative cis-regulatory element that controls transcriptional activity of the pig uroplakin II gene promoter.

Uroplakin II (UPII) is a one of the integral membrane proteins synthesized as a major differentiation product of mammalian urothelium. UPII gene expression is bladder specific and differentiation dependent, but little is known about its transcription response elements and molecular mechanism. To identify the cis-regulatory elements in the pig UPII (pUPII) gene promoter region, we constructed pUPII 5' upstream region deletion mutants and demonstrated that each of the deletion mutants participates in controlling the expression of the pUPII gene in human bladder carcinoma RT4 cells.

Developmental arrest of scNT-derived fetuses by disruption of the developing endometrial gland as a result of impaired trophoblast migration and invasiveness.

Somatic cell nuclear transfer (scNT)-derived pig placenta tissues of gestational day 30 displayed avascularization and hypovascularization. Most of the cytotrophoblast-like cells of the developing scNT-derived placenta villi were improperly localized or exhibited impaired migration to their targeting loci. Id-2, Met, MMP-9, and MCM-7 were barely detectable in the cytotrophoblast cells of the scNT-derived placenta villi.

Comparative gene expression analysis of somatic cell nuclear transfer-derived cloned pigs with normal and abnormal umbilical cords.

Gene expression profiling of compromised umbilical cords (CUCs) derived from somatic cell nuclear transfer (scNT) clones was performed to determine why scNT-derived clones often exhibit malformed umbilical arteries. Umbilical cord samples were obtained from 65 scNT piglets, and of these, nine displayed a CUC. Microscopic analyses of the scNT clones with CUCs (scNT-CUCs) revealed complete occlusive thrombi that were not detected in the arteries of scNT clones with normal umbilical cords (scNT-Ns).

Comparative proteomic analysis of malformed umbilical cords from somatic cell nuclear transfer-derived piglets: implications for early postnatal death.

Background: Somatic cell nuclear transfer (scNT)-derived piglets have high rates of mortality, including stillbirth and postnatal death. Here, we examined severe malformed umbilical cords (MUC), as well as other organs, from nine scNT-derived term piglets.

Results: Microscopic analysis revealed complete occlusive thrombi and the absence of columnar epithelial layers in MUC (scNT-MUC) derived from scNT piglets.

Depigmentation of skin and hair color in the somatic cell cloned pig.

Previously, we have successfully produced nine cloned piglets using Duroc donor cells. Among these clones, one showed distinct depigmentation of the skin and hair color during puberty. In this study, we selected a clone with depigmentation to investigate the etiology of the anomaly in somatic cell nuclear transfer.

Serial cloning of pigs by somatic cell nuclear transfer: restoration of phenotypic normality during serial cloning.

Somatic cell nuclear transfer (scNT) is a useful way to create cloned animals. However, scNT clones exhibit high levels of phenotypic instability. This instability may be due to epigenetic reprogramming and/or genomic damage in the donor cells.

Comparative proteomic analysis associated with term placental insufficiency in cloned pig.

Somatic cell-derived nuclear transfer (scNT) is a method of animal cloning in which the oocyte reprograms a somatic cell nucleus to divide and execute developmental programs. Despite many successes in this field, cloning by scNT remains very inefficient. Unlike other cloned animals, pigs derived by scNT have placentas with severe villous hypoplasia.

Cloning and molecular dissection of the 8.8 kb pig uroplakin II promoter using transgenic mice and RT4 cells.

Uroplakin II (UPII) gene expression is highly tissue and cell specific, with mRNA present in the suprabasal cell layers of the bladder and urethra. Previous reports described the mouse UPII (mUPII) promoter as primarily urothelium selective. However, ectopic expression of a transgene under the 3.

Dynamic control of oligosaccharide modification in the mammary gland: linking recombinant human erythropoietin functional analysis of transgenic mouse milk-derived hEPO.

We analyzed two transgenic mouse lines that secrete rhEPO in their milk to assess the dynamic control of N-linked oligosaccharides. Since pharmaceutically available epoetin alpha and beta are produced in CHO cells, we compared transgenic mammary gland-derived rhEPO to its CHO cell-derived counterpart. The major glycosyltransferases that determine the N-oligosaccharides patterns of rhEPO include N-acetylglycosaminyltransferase (GnT) and alpha1,3/4 fucosyltransferase (Fuc-TIV), GnT-III, -V and Fuc-TIV expression in the mouse mammary gland is significantly higher than that in Chinese hamster ovary (CHO)-derived cells, where the protein is not detectable.

A rare and often unrecognized cerebromeningitis and hemodynamic disorder: a major cause of sudden death in somatic cell cloned piglets.

In this study, we generated 40 somatic cell cloned (scNT) piglets. Of these, five piglets were stillborn, 22 scNT piglets died suddenly within the first week of life, and 1 piglet died after 40 days. Twelve scNT piglets are still healthy.

Detection of rare Leydig cell hypoplasia in somatic cell cloned male piglets.

In this investigation, 22 cloned male piglets were obtained by male fetal fibroblast-cell-derived nuclear transfer. Eighteen of the cloned animals died. The two cell lines did not differ significantly with regard to efficiency of live piglet production.