Biomedical subjects

I Kim

Publications and source records attributed to I Kim.

At least 73 records · Page 4Linked to original sources

PubMed1999

Angiopoietin-1 is an apoptosis survival factor for endothelial cells.

We examined the effect of angiopoietin-1 (Ang1) on apoptosis in human umbilical vein endothelial cells (HUVECs). Ang1 (5-1000 ng/ml) dose-dependently inhibited apoptosis under a serum-deprived state. A significant apoptotic inhibition occurred with as low as 50 ng/ml. Two hundred ng/ml of Ang1 inhibited to approximately 50% of the control apoptotic rates for 96 h. Furthermore, an augmented antiapoptotic effect of Ang1 by the addition of 20 ng/ml vascular endothelial growth factor was observed. This Ang1-induced strong antiapoptotic effect in endothelial cells is a novel and intriguing finding and could be an additional description of Ang1-induced direct biological function.

Angiopoietin-1↗

PubMed1999

Molecular cloning and characterization of a novel angiopoietin family protein, angiopoietin-3.

Using homology-based PCR, we have isolated cDNA encoding a novel member (491 amino acids) of the angiopoietin (Ang) family from human adult heart cDNA and have designated it angiopoietin-3 (Ang3). The NH2-terminal and COOH-terminal portions of Ang-3 contain the characteristic coiled-coil domain and fibrinogen-like domain that are conserved in other known Angs. Ang3 has a highly hydrophobic region at the N-terminus (approximately 21 amino acids) that is typical of a signal sequence for protein secretion. Ang3 mRNA is most abundant in adrenal gland, placenta, thyroid gland, heart and small intestine in human adult tissues. Additionally, Ang3 is a secretory protein, but is not a mitogen in endothelial cells.

Adult↗

PubMed1999

Blockade of HERG channels expressed in Xenopus oocytes by external H+.

We have investigated the effect of external H+ concentration ([H+]o) on the human-ether-a-go-go-related gene (HERG) current (IHERG), the molecular equivalent of the cardiac delayed rectifier potassium current (IKr), expressed in Xenopus oocytes, using the two-microelectrode voltage-clamp technique. When [H+]o was increased, the amplitude of the IHERG elicited by depolarization decreased, and the rate of current decay on repolarization was accelerated. The activation curve shifted to a more positive potential at lower external pH (pHo) values (the potential required for half-maximum activation, V1/2, was: -41.8 mV, -38.0 mV, -33.7 mV, -26.7 mV in pHo 8.0, 7.0, 6.6, 6.2, respectively). The maximum conductance (gmax) was also affected by [H+]o: a reduction of 7.9%, 14.6%, and 22.8% was effected by decreasing pHo from 8.0 to 7.0, 6.6, and 6.2, respectively. We then tested whether this pH effect was affected by the external Ca2+ concentration, which is also known to block HERG channels. When the extracellular Ca2+ concentration was increased from 0.5 mM to 5 mM, the shift in V1/2 caused by increasing [H+]o was attenuated, suggesting that these two ions compete for the same binding site. On the other hand, the decrease in gmax caused by increasing [H+]o was not significantly affected by changing external Ca2+ levels. The results indicate that HERG channels are inhibited by [H+]o by two different mechanisms: voltage-dependent blockade (shift of V1/2) and the decrease in gmax. With respect to the voltage-dependent blockade, the interaction between H+ and Ca2+ is competitive, whereas for the decreasing gmax, their interaction is non-competitive.

Animals↗

PubMed1999

Blockade of HERG channels expressed in Xenopus laevis oocytes by external divalent cations.

We have investigated actions of various divalent cations (Ba2+, Sr2+, Mn2+, Co2+, Ni2+, Zn2+) on human ether-a-go-go related gene (HERG) channels expressed in Xenopus laevis oocytes using the voltage clamp technique. All divalent cations inhibited HERG current dose-dependently in a voltage-dependent manner. The concentration for half-maximum inhibition (Ki) decreased at more negative potentials, indicating block is facilitated by hyperpolarization. Ki at 0 mV for Zn2+, Ni2+, Co2+, Ba2+, Mn2+, and Sr2+ was 0.19, 0.36, 0. 50, 0.58, 2.36, and 6.47 mM, respectively. The effects were manifested in four ways: 1) right shift of voltage dependence of activation, 2) decrease of maximum conductance, 3) acceleration of current decay, and 4) slowing of activation. However, each parameter was not affected by each cation to the same extent. The potency for the shift of voltage dependence of activation was in the order Zn2+ > Ni2+ >/= Co2+ > Ba2+ > Mn2+ > Sr2+, whereas the potency for the decrease of maximum conductance was Zn2+ > Ba2+ > Sr2+ > Co2+ > Mn2+. The kinetics of activation and deactivation were also affected, but the two parameters are not affected to the same extent. Slowing of activation by Ba2+ was most distinct, causing a marked initial delay of current onset. From these results we concluded that HERG channels are nonselectively blocked by most divalent cations from the external side, and several different mechanism are involved in their actions. There exist at least two distinct binding sites for their action: one for the voltage-dependent effect and the other for reducing maximum conductance.

Animals↗

PubMed1999

Viral genotypes and p53 expression in Epstein-Barr virus-associated primary malignant lymphomas of the intestines.

A small number (4% to 6%) of primary malignant lymphomas arising in the intestines express the EBV genome. However, in these tumors, the viral genotype and the role of tumor suppressor gene p53 have not been investigated. We sought to determine what genotype of EBV is frequently involved and whether the expression of p53 is related to these tumors. We used EBER-1 in situ hybridization and polymerase chain reactions (PCRs) for EBNA-1, EBNA-2A, and EBNA-2B to detect latent infection with EBV and to determine the genotype, respectively. In addition, we performed p53 PCR-SSCP (exons 5 through 9) and immunohistochemical analysis for p53. We found that EBV type B was present in 4 of 6 cases (67%); the genotype of the remaining cases could not be determined. The p53 PCR-SSCP indicated normal migration patterns in all malignant lymphomas, despite the fact that the tumor cells were strongly immunostained for p53 protein in 5 of the 6 cases. Thus, our study demonstrates that EBV-associated non-Hodgkin's lymphomas of the intestines in Korea are strongly related to the type B EBV and not to mutations of p53 gene. We suggest that EBV-associated intestinal lymphomas may arise through an interaction between the latent proteins of EBV and the wild-type p53 protein.

Adult↗

PubMed1999

The detection of Epstein-Barr virus in the lesions of salivary glands.

Epstein-Barr virus (EBV) is known in association with lymphoid and epithelial lesion. Because the salivary gland is an organ close to the oropharynx, it has a higher incidence of EBV infection and is a possible route of EBV infection. Formalin-fixed, paraffin embedded tissue sections of 87 cases of salivary gland diseases were used for the study of EBV with PCR, in situ PCR for EBNA-1 (EBV nuclear antigen-1), and immunohistochemistry for LMP-1 (latent membrane protein-1). EBV was detected in 12 cases (13.8%): 7 of nonspecific chronic sialadenitis (21.2%), 4 of Warthin's tumors (30.8%), and one lymphoepithelial carcinoma. EBNA-1 was negative in all the other lesions. EBV DNA was detected in the nucleus of epithelial cells and the surrounding lymphocytes. LMP-1 positivity was found in the cytoplasm of epithelial cells. The results of the present study showed that EBV is implicated in some of the inflammatory and neoplastic lesions of the salivary gland in which the lymphocytes are abundant. However, the pathogenesis and mechanism of immortalization and tumorigenesis of the epithelial cells in the salivary glands remain to be determined.

Epstein-Barr Virus Nuclear Antigens↗

PubMed1999

Effect of heat shock on the vascular contractility in isolated rat aorta.

Stresses such as heat shock, ischemia, and irradiation have been known to induce heat shock proteins in various tissues. We investigated the effects of heat shock on the vascular contractility by using isolated rat aorta. Rat thoracic aortic rings were mounted in an organ bath maintained at 37 degrees C. For heat shock, aortic ring preparations were exposed to 42 degrees C for either 15 or 45 min (heat shock group), whereas the control group was left at 37 degrees C. Eight hours after heat shock, aortic ring preparations were subjected to contractions with high K(+) membrane-depolarizing solution. After functional study, tissues were frozen for measurement of heat shock protein 70 (HSP70). Heat shock not only increased the expression of HSP70 in the rat aorta, but also augmented contractions to KCl whether endothelium was present or denuded. Short exposure of tissues to 42 degrees C for 15 min did not work either. These results suggest that heat shock increases vascular contractility in isolated aortic strips.

Animals↗

PubMed1999

Chemical shift perturbation studies of the interactions of the second RNA-binding domain of the Drosophila sex-lethal protein with the transformer pre-mRNA polyuridine tract and 3' splice-site sequences.

The interactions of the second RNA-binding domain of the Drosophila melanogaster Sex-lethal protein (Sxl RBD2) with the oligoribonucleotides, GUUUUUUUU (GU8) and CUAGUG, representing the sequences surrounding an alternative 3'-splicing site of the transformer pre-mRNA (GU8CUAGUG), were studied using heteronuclear two-dimensional NMR techniques. The 1H and 15N chemical shifts of the backbone amide resonances upon titration of Sxl RBD2 with each of these RNAs were recorded. It was found that Sxl RBD2 can bind not only to the polyuridine tract, GU8, but also to the downstream 3' splice-site sequence, CUAGUG, with similar affinities. In contrast, a nonspecific sequence, C8, did not bind to Sxl RBD2. This result is consistent with previous in vitro RNA-selection and UV-cross-linking results which indicated that the Sex-lethal protein binds to the uridine stretch and the AG dinucleotide in the consensus sequence, AUnNnAGU. In both cases, the chemical-shift perturbations were significant for almost the same amino acid residues, including the two central beta-strands formed by the RNP2-motif and RNP1-motif with the two highly conserved aromatic residues (Y214 and F256) in the middle. As the first RNA-binding domain of Sex-lethal (Sxl RBD1) has a characteristic aliphatic residue at one of the two corresponding positions (I128 and F170), Y214 of Sxl RBD2 was replaced by Ile using site-directed mutagenesis. On the one hand, the 1H and 15N chemical-shift perturbations indicated that GU8 binds to the same interface of mutant Sxl RBD2 as of wild-type Sxl RBD2, although its binding affinity was decreased significantly. On the other hand, the specific binding of Sxl RBD2 to CUAGUG was abolished almost completely by the Y-->I mutation. Taken together, the present results indicate that the interface residues that bind with GU8 and CUAGUG are much the same, but the role of the Y214 residue is clearly different between these two target sequences.

Amides↗

PubMed1999

Expression and characterization of anti-NCA-95 scFv (CEA 79 scFv) in a prokaryotic expression vector modified to contain a Sfi I and Not I site.

The CEA 79 antibody has been used in bone marrow scintigraphy for the differential diagnosis of skeletal tumors and the evaluation of the bone marrow status of patients with various hematological disorders. The specific localization of radio-labeled CEA 79 antibody in bone marrow depends on its reactivity with NCA-95 (nonspecific cross-reacting antigen-95) present on the surface and in the cytosol of human granulocytes and myelopoietic cells. To make a CEA 79 scFv molecule that would be less immunogenic and more penetrating than the intact mouse immunoglobulin, we constructed a pRSET Sfi I/Not I expression vector. The scFv gene was then excised from a pCANTAB 5 E phage display vector by digestion with Sfi I and Not I and inserted into the pRSET Sfi I/Not I expression vector. Upon transformation of a BL21(DE3)pLysS strain of E. coli, CEA 79 scFv became expressed in inclusion bodies requiring a renaturation process for solubilization. The final yield of CEA 79 scFv was 5 mg per a liter of culture. The refolded CEA 79 scFv exhibited an affinity (Kd = 2.1 x 10(-9) M) equivalent to that of the original CEA 79 antibody (K(d) = 3.3 x 10(-9) M) and the same immunoreactivity to CEA and NCA-95 in Western blots and in immunohistochemical staining experiments.

Amino Acid Sequence↗

PubMed1999

The guanosine binding mechanism of the Tetrahymena group I intron.

The Tetrahymena group I intron catalyzes self-splicing through two consecutive transesterification reactions, using a single guanosine-binding site (GBS). In this study, we constructed a model RNA that contains the GBS and a conserved guanosine nucleotide at the 3'-terminus of the intron (omegaG). We determined by NMR the solution structure of this model RNA, and revealed the guanosine binding mechanism of the group I intron. The G22 residue, corresponding to omegaG, participates in a base triple, G22 xx G3 x C12, hydrogen-bonding to the major groove edge of the Watson-Crick G3 x C12 pair. The G22 residue also interacts with A2, which is semi-conserved in all sequenced group I introns.

Animals↗

PubMed1999

NMR signal assignment of the polyuridine tract of the single-stranded RNA complexed with Sxl RBD1-RBD2 by using residue selective [5-(2)H]uridine substitutions.

Using [5-(2)H]uridine phosphoramidite, we synthesized a series of 2H-labeled Drosophila Sex-lethal (Sxl) target RNAs, in which all the uridine residues except one were specifically replaced by [5-(2)H]uridine. By observing the H5-H6 cross peaks of RNA in the TOCSY spectra, we unambiguously assigned all the base proton resonances of the target RNA in a Sxl x RNA complex. Furthermore, it was shown that Sxl differently recognizes A and G in a position prior to the polyuridine tract.

Animals↗

PubMed1999

Constitutive expression of VEGF, VEGFR-1, and VEGFR-2 in normal eyes.

PURPOSE: The expression of vascular endothelial growth factor (VEGF) and its high-affinity receptors VEGFR-1 and VEGFR-2 was investigated in normal eyes. METHODS: Monkey and rat eyes were surgically removed in animals under deep anesthesia and immediately prepared for study. Ocular VEGF, VEGFR-1, and VEGFR-2 expression was studied using a combination of in situ hybridization, northern blot analysis, immunohistochemistry, immunoassay, and reverse transcription-polymerase chain reaction. RESULTS: Steady state VEGF mRNA levels were detected in the normal vascularized ocular tissues of the monkey: the conjunctiva, iris, retina, and the choroid-retinal pigment epithelial complex. VEGF121 and VEGF165 were the major isoforms identified. VEGF protein was detected in the conjunctiva, retina, and the choroid-retinal pigment epithelial complex. Retinal VEGF mRNA localized to the ganglion, inner nuclear, and retinal pigment epithelial cell layers. VEGF protein localized to these same layers and to the cones of monkey retina. VEGFR-1 and VEGFR-2 mRNAs were detected in normal monkey iris, retina, and the choroid-retinal pigment epithelial complex. In both monkey and rat eyes, VEGFR-1 and VEGFR-2 mRNAs were localized to the inner nuclear layer of the retina. CONCLUSIONS: VEGF, VEGFR-1, and VEGFR-2 are constitutively expressed in the vascularized tissues of normal eyes.

Animals↗

PubMed1998

Permanent exposure of mucin-secreting HT-29 cells to benzyl-N-acetyl-alpha-D-galactosaminide induces abnormal O-glycosylation of mucins and inhibits constitutive and stimulated MUC5AC secretion.

Previous work has shown that treatment of HT-29 methotrexate (MTX) cells with benzyl-N-acetyl-alpha-D-galactosaminide results in profound changes in mucin oligosaccharide chains. To analyse in depth the effect of this drug, we first determined the structure of mucin oligosaccharide chains synthesized by HT-29 MTX cells and the changes induced by permanent drug exposure. Mucins from untreated cells contained nine monosialylated structures (core types 1, 2, 3 and 4) and four disialylated structures (types 1, 2 and 4). Core 1 structures predominated, in particular NeuAcalpha2-3Galbeta1-3GalNAc-ol. Exposure of HT-29 MTX cells to benzyl-N-acetyl-alpha-D-galactosaminide from days 2-21 resulted in a decrease in intracellular mucins and both their sialic acid and galactose content, and an increased T (Galbeta1-3GalNAcalpha-O-Ser/Thr) and Tn (GalNAcalpha-O-Ser/Thr) antigenicity. A 3-fold increase in both Galbeta1-3GalNAc alpha2, 3-sialyltransferase activity and mRNA expression was detected. At the ultrastructural level, T-antigen was not detectable in mucin droplets in control cells, but was strongly expressed in intracytoplasmic vesicles in treated cells. In these cells, MUC1 and MUC3 transcripts were up-regulated, whereas MUC2, MUC5B and MUC5AC were down-regulated. Furthermore, constitutive and secretagogue-induced MUC5AC secretion was reduced and no mucus layer was detected. In conclusion, benzyl-N-acetyl-alpha-D-galactosaminide induces abnormal O-glycosylation and altered regulation of MUC5AC secretion.

Acetylgalactosamine↗

PubMed1998

GalNAc-alpha-O-benzyl inhibits NeuAcalpha2-3 glycosylation and blocks the intracellular transport of apical glycoproteins and mucus in differentiated HT-29 cells.

Exposure for 24 h of mucus-secreting HT-29 cells to the sugar analogue GalNAc-alpha-O-benzyl results in inhibition of Galbeta1-3GalNAc:alpha2,3-sialyltransferase, reduced mucin sialylation, and inhibition of their secretion (Huet, G., I. Kim, C. de Bolos, J.M. Loguidice, O. Moreau, B. Hémon, C. Richet, P. Delannoy, F.X. Real., and P. Degand. 1995. J. Cell Sci. 108:1275-1285). To determine the effects of prolonged inhibition of sialylation, differentiated HT-29 populations were grown under permanent exposure to GalNAc-alpha-O-benzyl. This results in not only inhibition of mucus secretion, but also in a dramatic swelling of the cells and the accumulation in intracytoplasmic vesicles of brush border-associated glycoproteins like dipeptidylpeptidase-IV, the mucin-like glycoprotein MUC1, and carcinoembryonic antigen which are no longer expressed at the apical membrane. The block occurs beyond the cis-Golgi as substantiated by endoglycosidase treatment and biosynthesis analysis. In contrast, the polarized expression of the basolateral glycoprotein GP 120 is not modified. Underlying these effects we found that (a) like in mucins, NeuAcalpha2-3Gal-R is expressed in the terminal position of the oligosaccharide species associated with the apical, but not the basolateral glycoproteins of the cells, and (b) treatment with GalNAc-alpha-O-benzyl results in an impairment of their sialylation. These effects are reversible upon removal of the drug. It is suggested that alpha2-3 sialylation is involved in apical targeting of brush border membrane glycoproteins and mucus secretion in HT-29 cells.

Acetylgalactosamine↗

PubMed1998

Effect of petroleum ether extract of Panax ginseng roots on proliferation and cell cycle progression of human renal cell carcinoma cells.

Panax ginseng roots have long been used as a medicinal herb in oriental countries. We have investigated anti-proliferative effects of lipid soluble Panax ginseng components on human renal cancer cell lines. Petroleum ether extract of Panax ginseng roots (GX-PE) or its partially purified preparation (7:3 GX) was added to cultures of three human renal cell carcinoma (RCC) cell lines, A498, Caki-1, and CURC II. Proliferation of RCC cells was estimated by a [3H]thymidine incorporation assay and cell cycle distribution was analyzed by flow cytometry. GX-PE, 7:3 GX, panaxydol and panaxynol inhibited proliferation of all three RCC cell lines in a dose dependent manner in vitro with an order of potency, 7:3 GX > panaxydol > panaxynol = GX-PE. Additive effect of interleukin 4 was also demonstrated, most prominently in Caki-1 which responded poorly to GX-PE alone. Analysis of cell cycle in CURC II and Caki-1 treated with GX-PE demonstrated increase in G1 phase population and corresponding decrease in S phase population. The present study demonstrated that proliferation of human RCC cell lines were inhibited by lipid soluble components of Panax ginseng roots by blocking cell cycle progression at G1 to S phase transition.

Alkanes↗

PubMed1998

Voltage-dependent blockade of HERG channels expressed in Xenopus oocytes by external Ca2+ and Mg2+.

1. We expressed the human eag-related gene (HERG), which is known to encode the delayed rectifier K+ current (IKr) in cardiac muscle, in Xenopus oocytes. Using a two-microelectrode voltage clamp technique, the effect of external Ca2+ and Mg2+ on the HERG current (IHERG) was investigated. 2. When [Ca2+]o was increased, the amplitude of outward IHERG elicited by depolarization decreased, and the rate of current onset slowed. The rate of current decay observed on repolarization was greatly accelerated. The threshold and fully activated potential of IHERG shifted to a more positive potential. On the other hand, the inactivation property represented by the negative slope of the I-V curve and the instantaneous conductance of IHERG were little affected by [Ca2+]o. 3. The effect of [Ca2+]o on IHERG can be interpreted using the channel blockade model. The blockade is voltage dependent; smaller dissociation constants (KM) at more negative potentials indicate that block is facilitated by hyperpolarization. KM changes e-fold for 14.5 mV and the fractional electrical distance of the binding site calculated from this value is 0.86. 4. Blockade by a low concentration of Ca2+ (0.5 mM) was inhibited by increasing [K+]o (from 2 to 20 mM), whereas blockade by a high concentration of Ca2+ (5 mM) was not affected by varying [K+]o, indicating that there is competition between permeating ions and blocking ions. 5. The effect of [Mg2+]o on IHERG was qualitatively similar to that of [Ca2+]o, but the potency was lower. 6. These results suggest that external Ca2+ and Mg2+ block the HERG channel in a voltage- and time-dependent manner, resulting in a voltage dependence which has been regarded as a property of the activation gate.

Animals↗

PubMed1998

Advanced glycation end products increase retinal vascular endothelial growth factor expression.

Advanced glycation end products (AGEs) are linked with the development of diabetic retinopathy; however, the pathogenic mechanisms are poorly defined. Vascular endothelial growth factor (VEGF) levels are increased in ischemic and nonischemic diabetic retina, and VEGF is required for the development of retinal and iris neovascularization. Moreover, VEGF alone can induce much of the concomitant pathology of diabetic retinopathy. In this study, we found that AGEs increased VEGF mRNA levels in the ganglion, inner nuclear, and retinal pigment epithelial (RPE) cell layers of the rat retina. In vitro, AGEs increased VEGF mRNA and secreted protein in human RPE and bovine vascular smooth muscle cells. The AGE-induced increases in VEGF expression were dose- and time-dependent, inhibited by antioxidants, and additive with hypoxia. Use of an anti-VEGF antibody blocked the capillary endothelial cell proliferation induced by the conditioned media of AGE-treated cells. AGEs may participate in the pathogenesis of diabetic retinopathy through their ability to increase retinal VEGF gene expression.

Animals↗

PubMed1998

Persistent and heterogenous expression of the cyclin-dependent kinase inhibitor, p27KIP1, in rat hearts during development.

We have previously shown that there were differential and dramatic decreases of cyclin and cyclin-dependent kinase (CDK) activities in cardiomyocytes during the neonatal period. The activity of CDKs control cell cycle progression, and this activity is regulated positively and negatively by association of CDKs with cyclins and cyclin-dependent kinase inhibitors (CKIs), respectively. While the INK family (p15(INK4B)/p16(INK4A)/p18(INK4C)/p19(INK4D)) of CKIs is not detectable in hearts, the KIP/CIP family (p21(CIP1), p27(KIP1) and p57(KIP2)) of CKIs is detectable in most organs including the heart. Differential and dramatic changes of the KIP/CIP family (p21(CIP1), p27(KIP1) and p57(KIP2)) of CKIs were detected in rat hearts during development. The mRNA and protein levels of p21(CIP1) and p57(KIP2) were readily detectable in hearts at gestational and early postnatal periods and decreased thereafter. The mRNA levels of p27(KIP1) in ventricles were high during the gestational period, and did not change until day 30 postnatal, then were decreased slightly in 90-day-old rats. The protein levels of p27(KIP1) increased significantly in the early postnatal period, then were expressed persistently, although levels decreased slightly in the adult period. However, protein levels of p27(KIP1) in atria did not change during development. Variable immuno-staining patterns of p27(KIP1) were observed at different periods of development and in various locations in myocardium. During the gestational period, approximately 35-50% of myocardial cells in the cardiac wall were p27(KIP1) immuno-positive and were distributed diffusely. These p27(KIP1) immunopositive cells increased predominantly in endocardial and mid-portion areas of ventricular myocardium at the early postnatal period. This heterogenous pattern of p27(KIP1) protein expression persisted to adult hearts though the percentage of p27(KIP1) immuno-positive cells decreased slightly. High magnification revealed that more than 50% of adult cardiomyocytes were p27(KIP1) immuno-positive and that p27(KIP1) was located solely in nuclei. These results indicate that p27(KIP1) may be an important inhibitor of CDK activities in cardiomyocytes during early postnatal development and may block the re-entrance of adult cardiomyocytes into the cell cycle after injury.

Animals↗