Biomedical subjects

Myoung Hee Kim

Publications and source records attributed to Myoung Hee Kim.

10 recordsLinked to original sources

PubMed2006

Efficient cellular uptake of recombinant murine Hoxc8 homeoprotein in COS-7 cells.

In order to analyze the self-delivery activity of Hoxc8, recombinant Hoxc8 protein (rHoxc8) was designed to be expressed and purified in E. coli as a glutathione S-transferase and green fluorescent protein-fused form (GST-GFP-Hoxc8). After purification using glutathione sepharose beads, the 82 kDa fusion protein was separated on the SDS-PAGE gel and confirmed by detecting the fluorescence through luminescent image analyzer. When rHoxc8 was added to culture media for 30 h, most of the COS-7 cells contained the fusion proteins, showing green fluorescence under the fluorescent microscope. When the efficiency of cellular uptake was examined after Hoechst staining, almost 100% of the cells exhibited the GFP signal, revealing that rHoxc8 can traverse the cellular membrane of COS-7 cells efficiently, suggesting that the rHoxc8 could be applied in the development of efficient and useful delivery vectors for therapeutic molecules.

Animals↗

PubMed2005

Membraneous localization of Jpk is not essential to exert cytotoxicity in F9 teratocarcinoma cells.

A novel gene, Jopock (Jpk), which was isolated as a trans-acting factor associating with the PSRE of murine Hoxa-7, has been shown to be toxic to both prokaryotic and eukaryotic cells when overexpressed. Here we demonstrate that the overexpression of enhanced green fluorescent protein (EGFP)-tagged Jpk in F9 cells results in the induction of apoptosis, as indicated by phosphatidylserine exposure, DNA fragmentation, and the alteration of mitochondria transmembrane potential. Fluorescence microscopy showed that EGFP-fused Jpk was mainly localized in the endoplasmic reticulum (ER) and a small amount was found in the mitochondria. Deletion mutants with a transmembrane (TM) domain showed a distribution similar to that of EGFP-Jpk, whereas constructs with a deletion of the TM domain localized in the whole cells. Deletion mapping experiments showed that Jpk with an N-terminal part deleted stimulated apoptosis to almost the same extent as that of the wild-type Jpk, indicating that the localization of Jpk in the ER and the TM domain does not appear to be essential for inducing cytotoxicity. Overall, these results suggest that Jpk, particularly the C-terminal part of Jpk and/or 3'UTR, triggers apoptosis through a perturbation of mitochondrial membrane permeabilization.

Animals↗

PubMed2005

Dynamic expression pattern of Hoxc8 during mouse early embryogenesis.

The Hoxc8 expression pattern was examined in mouse embryos 7.5-12.5 days postcoitum (dpc) using whole-mount in situ hybridization and RT-PCR. The expression of Hoxc8 started between 7.5 and 8.5 dpc. A strong expression was detected in the ectoderm and mesoderm at 8.5 dpc. At 9.5 dpc, a distinct anterior boundary of Hoxc8 expression was established at the 10th and 16th somites in the neural tube and the paraxial mesoderm, respectively. This staggered expression pattern was maintained throughout the later stages. By 12.5 dpc, the forward progression of the Hoxc8 expression pattern was observed and the stain was weakened. In the ectoderm-derived neural tube, strong Hoxc8 expression was observed in the ventral horn and later in the ventral and mediolateral region of the mantle layer, indicating a possible association with the onset and progression of neural differentiation. In the case of the mesoderm-derivative cells, strong Hoxc8 expression was detected in the sclerotome on the way to the notochord and neural tube and mesonephros, suggesting a role of Hoxc8 in the formation of the vertebrae and ribs and the possible involvement in the differentiation into the kidney.

Animals↗

PubMed2004

Cloning of TLR3 isoform.

Toll-like receptor (TLR) 3 is a member of the TLR family that confers innate immunity by recognizing viral pathogens. Herein, we report that the TLR3 isoform is expressed on human primary cells and cell lines. This isoform has 2,520 bp cDNAs compared to the 2,712 bp of full cDNA, is produced by deletion of an intron-like sequence within exon 4 and is co-expressed with wild type TLR3 in primary human astrocytes and glioblastoma cell lines. This finding suggests the TLR3 isoform in astrocytes may have a different immunological role for binding ligands during the immune response in brain.

Astrocytes↗

PubMed2004

Calcyclin, a Ca2+ ion-binding protein, contributes to the anabolic effects of simvastatin on bone.

In vitro treatment with a pharmacological dose of simvastatin, a potent pro-drug of a 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor, stimulates bone formation. In our study, simvastatin stimulated differentiation of osteoblasts remarkably in a dose-dependent manner, with minimal effect on proliferation. To identify the mediators of the anabolic effects of simvastatin on osteoblasts, we tried to identify and characterize simvastatin-induced proteins by using proteomic analysis. Calcyclin was significantly up-regulated by more than 10 times, and annexin I was also up-regulated by simvastatin. However, annexin III, vimentin, and tropomyosin were down-regulated. Up-regulated calcyclin mRNA by simvastatin was validated by reverse transcription in mouse calvarial cells. In confocal microscope analysis, green fluorescence protein-calcyclin fusion protein was ubiquitously observed in the of MC3T3-E1 cells transfected with green fluorescence protein-calcyclin cDNA containing plasmid and was quickly concentrated in the nucleus 20 min after simvastatin treatment. Overexpression of calcyclin cDNA stimulated both the proliferation and expression of alkaline phosphatase mRNA significantly, without exposure to simvastatin in MC3T3-E1 cells. However, both the rate of proliferation of the osteoblasts and the expression of alkaline phosphatase mRNA were suppressed significantly 1 day after treatment with the calcyclin-specific small interference RNA, and furthermore, simvastatin did not overcome this suppression in the small interference RNA-pretreated MC3T3-E1 cells. In conclusion, calcyclin is one of the candidate proteins that plays a role in osteoblastogenesis in response to simvastatin, although the precise functions of calcyclin in osteoblast remain to be verified.

Animals↗

PubMed2004

Establishment of stable melanoma cell line expressing a novel gene, jpk, using a tetracycline-controlled gene expression system.

Jpk, originally isolated as an associating factor with the position-specific regulatory element of Hoxa-7, was found to be toxic to Escherichia coli (1) and to F9 teratocarcinoma cells (2) when transiently transfected and expressed. To investigate the possibility of tumor gene therapy using Jpk, its effect was tested in B16F10 murine melanoma cells. Because Jpk reduces the viability of B16F10 cells when transiently expressed, the Jpk gene was cloned into a tetracycline-controlled gene expression vector, pRetro-On to circumvent the lethal effect in unwanted situations. The retroviral plasmid pRetroJpk purified from the packaging cell was infected into B16F10 melanoma cells and screened in the presence of puromycin. Out of a total of 53 stable clones selected with puromycin, two clones overexpressed Jpk at more than twice the level when induced by doxycycline, a tetracycline-derivative, which implies the amount of the Jpk exhibiting the toxicity is critical. Although these clones control only low levels of Jpk, overexpression of the established melanoma cell line may help us decipher the function of Jpk and apply it as a tumor therapeutic gene in the future.

Animals↗

PubMed2003

Timetable for intestinal rotation in staged human embryos and fetuses.

BACKGROUND: The existing data on intestinal rotation during human development are contradictory regarding the timing of major events, and as such an exact timetable for rotation of the intestine in humans is not yet available. METHODS: We studied the initial formation and rotation of the intestine by microdissection and histological observations in 72 human embryos and fetuses at two to 12 weeks postfertilization. The embryos were classified according to the Carnegie staging system. RESULTS: The primordium of the primitive gut was first observed as a yolk sac at stage 5. With the formation of the embryonic foldings, three divisions of the primitive gut (the foregut, midgut, and hindgut) were observed at stage 10. At stage 12, the primitive gut was located on the midline. At stage 15, a 90 degrees counterclockwise rotation of the intestine began. At stage 16, herniation of the intestine into the umbilical cord was not evident in observations of the external form or a transversely sectioned embryo, but was evident in a sagittally sectioned embryo. There was another 90 degrees counterclockwise rotation at stage 20. Reduction of the intestine was a rapid process, since it was still in the cord in fetuses of <40 mm crown-rump length (CRL), and was reduced above 40 mm in general during nine weeks of development. When the intestine returned to the abdominal cavity, the cecum was located in the right lower quadrant (the adult position). CONCLUSIONS: We have developed a standard timetable to describe the rotation of the intestine. The current results will be helpful in studies describing the pathogenesis of some developmental abnormalities in the intestine due to abnormal rotation.

Embryo, Mammalian↗

PubMed2003

Analysis of plausible downstream target genes of Hoxc8 in F9 teratocarcinoma cells. Putative downstream target genes of Hoxc8.

Although Hox genes are known to mediate developmental decisions involved in pattern formation during embryogenesis, it is still not well understood what Hox regulates. In order to analyze Hoxc8 downstream target genes, a stable cell line overexpressing Hoxc8 was established using F9 murine teratocarcinoma cells, proteom samples were analyzed by 2-DE, and compared with controls. The protein spots having differences more than 4 fold in intensity were selected, analyzed by MALDI-TOF, and grouped in terms of putative function; cytoskeleton and motility (vimentin, gamma-actin, tropomyosin, and tubulin beta-5 chain); folding, modification and degradation of protein (GRP78, proteasome subunit alpha type 5, 26S proteasome regulatory subunit p27 protein, and PDIR); metabolism (ATP synthase beta subunit, Pgam1, and CAII); transcription/translation factors and general nucleic acid binding proteins (RbAp46, PCNA, eEF-1-beta, and nucleophosmin). Although it may not be significant, 50% of the genes were located on chromosomes 2 and 3, suggesting the possibility of a non-random distribution of Hox downstream genes. Almost 50% of the genes analyzed showed some relation with Hox protein directly or indirectly; i.e., tubulin beta 5, EF-1 beta and PCNA have been reported to contain putative Hox binding regulatory sites and genes like vimentin, pgam1 and nucleophosmin to be regulated by RA, a potent modulator of Hox expression. These results altogether imply that proteom analysis could be a possible tool for the analysis of the potent Hox realizator genes, which provides a new insight into the function of Hox on pattern formation during embryogenesis.

Animals↗

PubMed2003

A novel gene, Jpk, induces apoptosis in F9 murine teratocarcinoma cell through ROS generation.

A novel gene Jpk (Jopock) has been originally isolated through yeast 1 hybridization technique as a trans-acting factor interacting with the position-specific regulatory element of a murine Hoxa-7. Northern analysis revealed that the Jpk was expressed at day 7.0 post coitum (p.c.) during early gastrulation. Previously it has been shown that a trace amount of JPK protein led bacterial cells to death. In eukaryotic F9 cells, Jpk also led the cell to death-generating DNA ladder: fewer than 50% of the cells survived after 72-h transfection. Flow cytometric analysis with cells stained with each Annexin V/7-amino-actinomycin D (7-AAD), MitoTracker, and hydroethidine (HE) revealed that Jpk induced apoptotic cell death in a time-dependent manner, reduced mitochondrial membrane potential, and increased ROS (reactive oxygen species) production, respectively. Additionally, Jpk seemed to regulate the Bcl family at the transcriptional level when RT-PCR was performed. Although the precise mechanism is not clear, these results altogether suggest that Jpk is a potent inducer of apoptosis through generation of ROS as well as concomitant reduction of mitochondrial membrane potential.

Animals↗

PubMed2002

A novel factor associating with the upstream regulatory element of murine Hoxa-7 induces bacterial cell death.

In order to understand the function of a cDNA (c171) associated with the upstream regulatory region of the Hoxa-7, the cDNA was cloned into the pGEX-4T-1 vector to produce it as a GST fusion protein. The size of the fusion protein was determined to be 48-kilodalton (kDa). Sequence analysis revealed that a protein C171 contained one hydrophobic transmembrane domain in the N-terminal region and several putative phosphorylation and glycosylation sites. C171 protein inhibited the bacterial growth within 30 min after induction. The transmission electron microscopic examination revealed that the morphology of the cells expressing C171 was changed dramatically: i.e., unusually elongated phenotype compared with those of controls, and finally leading to a cell death. These results altogether indicate that a trace amount of C171 induces bacterial cell death.

Amino Acid Sequence↗