Biomedical subjects

K C Kim

Publications and source records attributed to K C Kim.

At least 19 recordsLinked to original sources

PubMed2002

Effects of polycationic peptides on mucin release from airway goblet cells: relationship between polymer size and activity.

OBJECTIVES AND DESIGN: Various sizes of poly-L-lysine (PLL) and poly-L-arginine (PLA) were tested for their possible effects on airway goblet cell mucin release using primary hamster tracheal surface epithelial (HTSE) cells in an attempt to identify the smallest size of the polycationic peptide to suppress mucin release without cytotoxicity. MATERIALS AND METHODS: HTSE cells were metabolically labeled using 3H-glucosamine and chased in the presence of varying concentrations of various sizes of the polycationic peptides. The amount of 3H-mucin in the spent media was measured by Sepharose CL-4B gel-filtration column chromatography. Possible cytotoxicity of the peptides was assessed by measuring the release of lactic dehydrogenase (LDH) during the treatment period. RESULTS: (1) PLL (MW 78,000) inhibited whereas PLA (MW 92,000) stimulated mucin release. However, these peptides were cytotoxic at the effective concentrations; (2) Both PLL (MW 9,600) and PLA (MW 8,900) could inhibit mucin release in a dose dependent manner without cytotoxicity; (3) Both PLL and PLA were effective in suppressing mucin release in 20-mer but not in either 10-mer or 5-mer; (4) 14-mers of both PLL and PLA also inhibited mucin release without cytotoxicity; (5) PLL and poly-D-lysine (PDL) of 14-mer were equipotent in its ability to suppress mucin release. CONCLUSION: Both PLL and PLA are cytotoxic at 'high' molecular weights, but have an ability to suppress mucin release without cytotoxicity at 'low' molecular weights. 14-mer seems to be the small, effective size, if not the smallest, for both PLL and PLA to suppress mucin release without cytotoxicity. The inhibitory effect of these polycationic peptides seems to be determined by the presence and the absolute number of positive charges and also to be independent of optical isomerism.

Animals↗

PubMed2001

Amphiphysin IIb-1, a novel splicing variant of amphiphysin II, regulates p73beta function through protein-protein interactions.

p73 is a nuclear protein that is similar in structure and function to p53. Notably, the C-terminal region of p73 has a regulatory function, through interactions with a positive or negative regulator. In this study, we use the yeast two-hybrid technique to identify a novel p73beta binding protein, designated amphiphysin IIb-1. Amphiphysin IIb-1 is one of the splicing variants of amphiphysin II, and has a shorter protein product than amphiphysin IIb, which has been previously reported. We confirmed that amphiphysin IIb-1 binds full-length p73beta, both in vitro and in vivo. This association is mediated via the SH3 domain of amphiphysin IIb-1 and C-terminal amino acids 321-376 of p73beta. Double immunofluorescence patterns revealed that p73beta is relocalized to the cytoplasm in the presence of amphiphysin IIb-1. Overexpression of amphiphysin IIb-1 was found to significantly inhibit the transcriptional activity of p73beta in a dose-dependent manner. In addition, the cell death function of p73beta was inhibited by amphiphysin IIb-1. These findings offer a new insight into the regulation mechanism of p73beta, and suggest that amphiphysin IIb-1 modulates p73beta function by direct binding.

Alternative Splicing↗

PubMed2001

Induction of apoptosis in p53-deficient human hepatoma cell line by wild-type p53 gene transduction: inhibition by antioxidant.

We investigated the role of wild-type (wt)-p53 as an inducer of apoptotic cell death in human hepatoma cell lines. Following the retrovirus-mediated transduction of the wt-p53 gene, Hep3B cells lacking the endogenous p53 expression began to die through apoptosis in 4 h. They showed a maximal apoptotic death at 12 h, whereas HepG2 cells expressing endogenous p53 did not. However, the transduction of the wt-p53 gene elicited growth suppression of both Hep3B and HepG2 cells. P21(WAF1/CIP1), a p53-inducible cell cycle inhibitor, was induced, not only in Hep3B cells undergoing apoptosis, but also in HepG2 cells. The kinetics of the p21(WAF1/CIP1) induction, DNA fragmentation, and growth suppression of the Hep3B cells showed that DNA fragmentation and growth suppression progressed rapidly following p21(WAF1/CIP1) accumulation. N-acetyl-cysteine or glutathione, potent antioxidants, strongly inhibited the DNA fragmentation, but did not reduce the elevated level of p21(WAF1/CIP1). These findings suggested that p21(WAF1/CIP1) was not a critical mediator for the execution of p53-mediated apoptosis, although it contributed to the growth inhibition of cells undergoing apoptosis. Furthermore, p53-mediated apoptosis could be repressed by antioxidants.

Acetylcysteine↗

PubMed2001

Reciprocal expressions of cyclin E and cyclin D1 in hepatocellular carcinoma.

Deregulation of the cell cycle by overexpression of G1 cyclins, cyclin E and cyclin D1 genes, has been demonstrated to be a prerequisite for the development of human cancer. Recently, cyclin E is proposed to be sufficient for the progression of the G1 cell cycle without cyclin D1. Here we show that the proposed model system was specifically present in human hepatocellular carcinoma (HCC) unlike other human cancers. Of 31 HCC tissues analyzed, 21 (67.7%) exhibited an overexpression of cyclin E protein. In contrast to cyclin E gene expression, cyclin D1 expression was strongly downregulated in 19 (61.2%) HCCs. Interestingly, 65% of HCC tissues with overexpression of the cyclin E gene exhibited downregulation of cyclin D1, suggesting reciprocal deregulation of these cyclins in the G1 progression of the cell cycle. Southern blot analysis proved the amplification of cyclin E gene in HCC with a high level of overexpression. The present findings suggest that the reciprocal deregulation of cyclin E lacking cyclin D1 expression might play a role in G1 progression and the development of HCC.

Blotting, Southern↗

PubMed2001

In vivo antitumor effect of herpes simplex virus thymidine kinase gene therapy in rat hepatocellular carcinoma: feasibility of adenovirus-mediated intra-arterial gene delivery.

Transfer of the herpes simplex virus-thymidine kinase gene, followed by the administration of ganciclovir (HSV-tk/GCV), has been a major approach for cancer gene therapy. We investigated the antitumor effect of the HSV-tk/GCV strategy with the rat orthotopic hepatocellular carcinoma (HCC) model and the tumor-selective gene delivery by an adenovirus-mediated gene transfer through the hepatic artery. The complete antitumor effect was demonstrated, after the treatment with GCV in rat HCC established by the implantation of HSV-tk transferred rat HCC cells. The in vivo bystander effect was also observed. The marked infiltration of CD4+ and CD8+ T lymphocytes, macrophages and NK cells were found in the tumor area. After the injection of adenovirus carrying the LacZ gene into the hepatic artery, the selective expression of transgene in the tumor cell was achieved. These findings indicate that the HSV-tk/GCV strategy, using an adenoviral vector, could be a promising avenue for the treatment of hepatocellular carcinoma.

Adenoviridae↗

PubMed2001

Follicular fluid enhances sperm attraction and its motility in human.

PURPOSE: Follicular fluid has a pivotal effect on motility and chemotaxis of spermatozoa for successful fertilization. The effect of human follicular fluid (hFF) and progesterone on attraction and motility of spermatozoa were investigated using simplified capillary assays. METHODS: Capillary tubes loaded with hFF, modified human tubal fluid (m-hTF), or m-hTF supplemented with progesterone, respectively, were used for assessments of attraction and motility of spermatozoa following culture at various time intervals. RESULTS: Number and motile ratio of spermatozoa in the tubes loaded with hFF were significantly (P < .05) higher than those with m-hTF. In the tubes loaded with m-hTF, m-hTF supplemented with progesterone, and hFF, the attracted number of spermatozoa were 34 x 10(5), 131 x 10(5), and 108 x 10(5), and motile ratio of spermatozoa was 37, 48, and 82%, respectively. CONCLUSIONS: We conclude that hFF clearly plays a crucial role in enhancing attraction and motility of spermatozoa, and progesterone has strong effect on attraction of spermatozoa.

Adult↗

PubMed2001

Immunological characterization of a mucin-associated protein from hamster tracheal epithelial cell culture.

Airway mucins are high molecular mass (>10(6) dalton) glycoproteins with various types of associated molecules including glycoproteins, lipoproteins, and lipids. The study of mucin-associated proteins is limited largely due to the lack of specific probes. In this study, we produced a monoclonal antibody, MAbHT10, against a 190-kDa mucin associated-protein by immunizing mice with hamster airway mucin purified in nondissociative condition. Using HT10, the 190-kDa mucin-associated protein was characterized immunologically. The 190-kDa mucin-associated protein is glycoprotein and HT10 recognized carbohydrate containing portion of the protein. The association of 190-kDa protein with mucin is strong enough that heat and detergent treatment is required to dissociate it from mucin as evidenced by gel filtration chromatography, Western blot, enzyme-linked immunoadsorbent assay (ELISA), and co-immunoprecipitation. The expression of the 190-kDa protein is increased with the development of hamster tracheal epithelial cells in culture, but showed differences with the pattern of the regulation of mucin expression. Adenosine triphosphate (ATP), a known strong mucin secretagogue, dose-dependently increased mucin release but caused only marginal increase in the release of the 190-kDa protein. The MAb should be useful in the structural and functional analysis of the 190-kDa mucin-associated proteins in physiological and pathological situations such as chronic airway diseases.

Adenosine Triphosphate↗

PubMed2001

Muc1 mucins on the cell surface are adhesion sites for Pseudomonas aeruginosa.

Recently, we cloned and characterized a full-length cDNA of the hamster Muc1 gene, the expression of which appears to be associated with secretory cell differentiation (Park HR, Hyun SW, and Kim KC. Am J Respir Cell Mol Biol 15: 237-244, 1996). The role of Muc1 mucins in the airway, however, is unknown. In this study, we investigated whether cell surface mucins are adhesion sites for Pseudomonas aeruginosa. Chinese hamster ovary (CHO) cells not normally expressing Muc1 mucin were stably transfected with the hamster Muc1 cDNA, and binding to P. aeruginosa was examined. Our results showed that 1) stably transfected CHO cells expressed both Muc1 mRNA and Muc1 mucins based on Northern and Western blot analyses, 2) Muc1 mucins present on the cell surface were degraded by neutrophil elastase, and 3) expression of Muc1 mucins on the cell surface resulted in a significant increase in adhesion of P. aeruginosa that was completely abolished by either proteolytic cleavage with neutrophil elastase or deletion of the extracellular domain by mutation. We conclude that Muc1 mucins expressed on the surface of CHO cells serve as adhesion sites for P. aeruginosa, suggesting a possible role for these glycoproteins in the early stage of airway infection and providing a model system for studying epithelial cell responses to bacterial adhesion that leads to airway inflammation in general and cystic fibrosis in particular.

Animals↗

PubMed2001

Involvement of the MAP kinase ERK2 in MUC1 mucin signaling.

MUC1 mucin is a receptor-like glycoprotein expressed abundantly in various cancer cell lines as well as in glandular secretory epithelial cells, including airway surface epithelial cells. The role of this cell surface mucin in the airway is not known. In an attempt to understand the signaling mechanism of MUC1 mucin, we established a stable cell line from COS-7 cells expressing a chimeric receptor consisting of the extracellular and transmembrane domains of CD8 and the cytoplasmic (CT) domain of MUC1 mucin (CD8/MUC1 cells). We previously observed that treatment of these cells with anti-CD8 antibody resulted in tyrosine phosphorylation of the CT domain of the chimera. Here we report that treatment of CD8/MUC1 cells with anti-CD8 resulted in activation of extracellular signal-regulated kinase (ERK) 2 as assessed by immunoblotting, kinase assay, and immunocytochemistry. The activation of ERK2 was completely blocked either by a dominant negative Ras mutant or in the presence of a mitogen-activated protein kinase kinase (MEK) inhibitor. We conclude that tyrosine phosphorylation of the CT domain of MUC1 mucin leads to activation of a mitogen-activated protein kinase pathway through the Ras-MEK-ERK2 pathway. Combined with the existing data by others, it is suggested that one of the roles of MUC1 mucin may be regulation of cell growth and differentiation via a common signaling pathway, namely the Grb2-Sos-Ras-MEK-ERK2 pathway.

Animals↗

PubMed2001

ATP-induced mucin release from cultured airway goblet cell involves, in part, activation of phospholipase A2.

Adenosine triphosphate (ATP) has been shown to stimulate mucin release by activation of protein kinase C (PKC) following activation of phospholipase C (PLC) coupled to the P2 receptor via G-proteins. The aim of the present study was to investigate pathways downstream to the PKC activation in ATP-induced mucin release from primary hamster tracheal surface epithelial (HTSE) cells. The release of mucin was determined by chromatographic procedure after metabolic labeling of mucin with [3H]-glucosamine. The results were: i) ATP induced the release of arachidonic acid, which caused the release of mucin. Pretreatment with mepacrine (0.3 mM), a phospholipase A2 (PLA2) inhibitor, inhibited the ATP-induced arachidonic acid and mucin release. Oleoyloxyethylphosphocholine, another PLA2 inhibitor, gave similar results. ii) An activator of PKC, 4 beta-phorbol-12 alpha-myristate-13-acetate (PMA, 1 microM) induced mucin release, which was inhibited by mepacrine pretreatment. iii) Downregulation of PKC by prolonged (16 h) PMA treatment caused inhibition of ATP-induced mucin release. Treatment of PKC downregulated HTSE cells with mepacrine did not further decrease the ATP-induced mucin release. These results suggest that PLA2 is involved in ATP-induced mucin release and its activation is sequential to the PLC-PKC pathway.

Adenosine Triphosphate↗

PubMed2001