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Biomedical subjects

E Kim

Publications and source records attributed to E Kim.

At least 145 records · Page 8Linked to original sources
PubMed1999

Modulation of renal tubular cell function by RGS3.

The recently discovered family of regulators of G protein signaling (RGS) accelerates the intrinsic GTPase activity of certain Galpha subunits, thereby terminating G protein signaling. Particularly high mRNA levels of one family member, RGS3, are found in the adult kidney. To establish the temporal and spatial renal expression pattern of RGS3, a polyclonal antiserum was raised against the COOH terminus of RGS3. Staining of mouse renal tissue at different gestational stages revealed high levels of RGS3 within the developing and mature tubular epithelial cells. We tested whether RGS3 can modulate tubular migration, an important aspect of tubular development, in response to G protein-mediated signaling. Several mouse intermedullary collecting duct (mIMCD-3) cell lines were generated that expressed RGS3 under the control of an inducible promoter. Lysophosphatidic acid (LPA) is a potent chemoattractant that mediates its effects through heterotrimeric G proteins. We found that induction of RGS3 significantly reduced LPA-mediated cell migration in RGS3-expressing mIMCD-3 clones, whereas chemotaxis induced by hepatocyte growth factor remained unaffected by RGS3. Our findings suggest that RGS3 modulates tubular functions during renal development and in the adult kidney.

Aging↗
PubMed1999

Antibodies to human T-cell lymphotropic virus type I (HTLV-I) by particle agglutination (PA) test in Korean blood donors.

HTLV-I infection is a recently recognized disease entity that is common in some tropical and subtropical areas, including the southwestern district of Japan. Despite the geographical proximity and frequent cultural exchanges between Korea and Japan, it is understood that Korea is not an endemic area and HTLV-I-associated illnesses are very rare in Korea. This study was designed to evaluate the positive rate of anti-HTLV-I antibodies in Korean blood donors and its regional distribution. Sera were obtained from blood donors from various districts around Korea. Anti-HTLV-I antibodies were detected by using the microtiter particle agglutination test employing an indirect agglutination technique. A total of 9,281 donors were tested and 12 donors (0.13%) were positive for anti-HTLV-I antibodies, 10 (0.11%) out of 8,845 males and 2 (0.46%) out of 436 females, with relative female predominance. A relatively high incidence of anti-HTLV-I positive donors was observed in Cheju Island (0.80%), Kyungnam (0.31%), and Chonnam (0.15%). In conclusion, the positive rate of anti-HTLV-I antibodies seemed to be very low in Korea, but the highest positive rate of anti-HTLV-I antibodies was noticed on Cheju Island, warranting further research for confirmation.

Adolescent↗
PubMed1999

Increase in signal intensities on T1-weighted magnetic resonance images in asymptomatic manganese-exposed workers.

OBJECTIVES: To clarify the clinical significance of increased signal intensities on T1 weighted magnetic resonance imaging (MRI) we performed a large-scale epidemiological study on asymptomatic manganese (Mn)-exposed workers with its focus on MRI. METHODS: We randomly selected 121 male workers out of a total of 750 workers including Mn-exposed, non-exposed manual, and non-exposed clerical workers in the factories. We studied environmental and biological monitoring, neurological examination, and MRI. RESULTS: The proportion of workers with increased signal intensities among the exposed, the non-exposed manual workers, and the non-exposed clerical workers was 46.1%, 18.8%, and 0%, respectively. Especially, 73.5% of the welders showed increased signal intensities. In no subject, were clinical signs of manganism observed. The pallidal index correlated with blood Mn concentration. CONCLUSION: Increase in signal intensities on the T1-weighted image reflect recent exposure to Mn, but not necessarily manganism. At which increase of signal intensity, the progression of manganism from Mn exposure occurs, remains to be solved.

Adult↗
PubMed1999

Generation of monoclonal antibodies specific for mouse apolipoprotein B-100 in apolipoprotein B-48-only mice.

Over the past 10 years, many laboratories have investigated lipid metabolism and atherogenesis with a variety of transgenic and gene knockout mouse models. Although many of these studies have yielded valuable insights, some have been hampered by a paucity of useful antibodies against mouse proteins. For example, many laboratories have analyzed genetic and dietary interventions affecting lipoprotein metabolism without useful antibodies against mouse apolipoprotein (apo) B. In this study, we sought to develop highly specific monoclonal antibodies against mouse apoB-100. To achieve this goal, gene-targeted mice that synthesize exclusively apoB-48 (apoB-48-only mice) were immunized with mouse apoB-100. The immune response against apoB-100 was robust, as judged by high titers of antibodies against mouse apoB-100. After fusing the splenic lymphocytes of the apoB-48-only mice with a myeloma cell line, we identified and cloned hybridomas that produced mouse apoB-100-specific monoclonal antibodies. Those antibodies were useful for developing sensitive and specific immunoassays for mouse apoB-100. This study illustrates the feasibility and utility of using gene-targeted mice to develop monoclonal antibodies against mouse proteins.

Animals↗
PubMed1998

Mutation analysis of CYP2D6 locus in the Korean population: identification of rare poor metabolizer alleles at the nucleotide level.

The CYP2D6 loci of one hundred and eight genetically unrelated Koreans were analyzed. The G1934A mutation which causes aberrant splicing, thereby producing nonfunctional enzymes and is responsible for 70% of poor metabolizer (PM) phenotype among Caucasians, was found in three heterozygous individuals. The mutation frequency of Koreans at 1934 (1.5%) was one thirteenth of that of Caucasians (20.7%). Such a low mutation frequency could be the major genetic explanation for rare PMs in Koreans. A substitution allele of Gly169-Arg(G1846A) which also produces a nonfunctional enzyme was detected in one heterozygous individual. This PM allele has been reported only in Chinese but not in Caucasians. This is the first report which demonstrates the presence of CYP2D6 PM alleles at the nucleotide level in Koreans. No base changes were detected at 1795, 2637, and 3023 whose mutations would also make nonfunctional enzymes. Individuals with two nonfunctional alleles are thereby expected to be PMs phenotypically and were not detected in this study. Mutant alleles with Arg296-Cys(C2938T) and Ser476-Thr(G4268C) substitutions known to reduce enzyme activity and with silent mutations at 1749 were studied; mutation frequencies were 17.9%, 64.7%, and 66%, respectively. The mutation frequency at 2938 was especially reduced by half when compared to that of Caucasians (32.4%).

Alleles↗
PubMed1998

Intrastrand cross-linked actin between Gln-41 and Cys-374. I. Mapping of sites cross-linked in F-actin by N-(4-azido-2-nitrophenyl) putrescine.

A new heterobifunctional photo-cross-linking reagent, N-(4-azido-2-nitrophenyl)-putrescine (ANP), was synthesized and covalently bound to Gln-41 of rabbit skeletal muscle actin by a bacterial transglutaminase-mediated reaction. Up to 1.0 mol of the reagent was incorporated per mole of G-actin; at least 90% of it was bound to Gln-41 while a minor fraction (about 8%) was attached to Gln-59. The labeled G-actin was polymerized, and the resulting F-actin was intermolecularly cross-linked by irradiation with UV light. The labeled and cross-linked peptides were isolated from either a complete or limited tryptic digest of cross-linked actin. In the limited digest the tryptic cleavage was restricted to arginine by succinylation of the lysyl residues. N-terminal sequencing and mass spectrometry indicated that the cross-linked peptides contained residues 40-50 (or 40-62 in the arginine limited digest) and residues 373-375, and that the actual cross-linking took place between Gln-41 and Cys-374. This latter finding was also supported by the inhibition of Cys-374 labeling with a fluorescent probe in the cross-linked actin. The dynamic length of ANP, between 11.1 and 12.5 A, constrains to that range the distance between the gamma-carboxyl group of Gln-41 in one monomer and the sulfur atom of Cys-374 in an adjacent monomer. This is consistent with the distances between these two residues on adjacent monomers of the same strand in the long-pitch helix in the structural models of F-actin [Holmes, K. C., Popp, D., Gebhard, W., and Kabsch, W. (1990) Nature 347, 44-49 and Lorenz, M., Popp, D., and Holmes, K. C. (1993) J. Mol. Biol. 234, 826-836]. The effect of cross-linking on the function of actin is described in the companion papers.

Actins↗
PubMed1998

Intrastrand cross-linked actin between Gln-41 and Cys-374. III. Inhibition of motion and force generation with myosin.

Structural and functional properties of intrastrand, ANP (N-(4-azido-2-nitrophenyl)-putrescine) cross-linked actin filaments, between Gln-41 and Cys-374 on adjacent monomers, were examined for several preparations of such actin. Extensively cross-linked F-actin (with 12% un-cross-linked monomers) lost at 60 degrees C the ability to activate myosin ATPase at a 100-fold slower rate and unfolded in CD melting experiments at a temperature higher by 11 degrees C than the un-cross-linked actin. Electron microscopy and image reconstruction of these filaments did not reveal any gross changes in F-actin structure but showed a change in the orientation of subdomain 2 and a decrease in interstrand connectivity. Rigor and weak (in the presence of ATP) myosin subfragment (S1) binding and acto-S1 ATPase did not show major changes upon 50% and 90% ANP cross-linking of F-actin; the Kd and Km values were little affected by the cross-linking, and the Vmax decreased by 50% for the extensively cross-linked actin. The cross-linking of actin (50%) decreased the mean speed and the number of sliding filaments in the in vitro motility assays by approximately 35% while the relative force, as measured by using external load in these assays, was inhibited by approximately 25%. The mean speed of actin filaments decreased with the increase in their cross-linking and approached 0 for the 90% cross-linked actin. Also examined were actin filaments reassembled from cross-linked and purified ANP cross-linked dimers, trimers, and oligomers. All of these filaments had the same acto-S1 ATPase and rigor S1 binding properties but different behavior in the in vitro motility assays. Filaments made of cross-linked dimers moved at approximately 50% of the speed of the un-cross-linked actin. The movement of filaments made of cross-linked trimers was inhibited more severely, and the oligomer-made filaments did not move at all. These results show the uncoupling between force generation and other events in actomyosin interactions and emphasize the role of actin filament structure and dynamics in the contractile process.

Actins↗
PubMed1998

Intrastrand cross-linked actin between Gln-41 and Cys-374. II. Properties of cross-linked oligomers.

Actin filaments partially cross-linked with ANP (N-(4-azido-2-nitrophenyl)-putrescine between Gln-41 and Cys-374 on adjacent monomers in the long-pitch helix were depolymerized and fractionated into pools of longitudinal cross-linked dimers (s(o)20,w = 5.55 +/- 0.22 S), trimers (s(o)20,w = 6.93 +/- 0.12 S), and higher-order oligomers. Competition binding experiments of myosin subfragment (S1) to cross-linked dimers in the presence of pyrenyl G-actin revealed about 2 orders of magnitude stronger binding of the first than that of the second S1 molecule to actin dimer. Under similar conditions the unpolymerized cross-linked actin species activated the MgATPase of S1 only severalfold compared to 70-fold activation by F-actin. The cross-linked dimers, trimers, and oligomers were polymerized into filaments by MgCl2 faster than un-cross-linked actin. In electron micrographs these filaments appeared sometimes shorter and had greater tendency to bend than un-cross-linked actin filaments. Small amounts of cross-linked actin dimers nucleated S1-induced polymerization of actin, but the polymerization by S1 was inhibited for pure populations of cross-linked dimers, trimers, and oligomers. The cross-linked dimers did not decrease the kinetic difference between the polymerization of actin by S1 isozymes S1(A1) and S1(A2). According to electron microscopy evidence, cross-linked actin oligomers polymerized by S1 yielded much shorter arrowhead structures than the un-cross-linked actin. These results indicate the importance of lateral actin-actin interaction for the activation of myosin ATPase and the polymerization of actin by S1.

Actins↗
PubMed1998

A gene-targeted mouse model for familial hypobetalipoproteinemia. Low levels of apolipoprotein B mRNA in association with a nonsense mutation in exon 26 of the apolipoprotein B gene.

Familial hypobetalipoproteinemia, a syndrome characterized by abnormally low plasma levels of low density lipoprotein cholesterol, is caused by mutations in the apolipoprotein (apo) B gene that interfere with the synthesis of a full-length apoB100. In many cases of familial hypobetalipoproteinemia, nonsense or frameshift mutations result in the synthesis of a truncated apoB protein. To understand why these mutations result in low plasma cholesterol levels, we used gene targeting in mouse embryonic stem cells to introduce a nonsense mutation (N1785Stop) into exon 26 of the mouse Apob gene. The sole product of this mutant Apob allele was a truncated apoB, apoB39. Mice homozygous for this "apoB39-only" (Apob39) allele had low plasma levels of apoB39 and markedly reduced plasma levels of very low density lipoprotein and low density lipoprotein cholesterol when fed a high fat diet. Analysis of liver and intestinal RNA from heterozygous apoB39-only mice revealed that the Apob39 mRNA levels were 60-70% lower than those from the wild-type allele. Interestingly, apoB39 was not cleared as rapidly from the plasma as apoB48. The apoB39-only mice provide new insights into the mechanisms of familial hypobetalipoproteinemia and the structural features of apoB that are important for lipoprotein metabolism.

Animals↗
PubMed1998

Human prothrombin fragment 1 and 2 inhibit bFGF-induced BCE cell growth.

Previously, we reported that the rabbit prothrombin fragment 2 (kringle 2 domain) has an anti-endothelial cell proliferative effect (Lee et al., J. Biol. Chem., in press). In this report, we show that not only rabbit prothrombin fragment 2 but also human prothrombin fragment 1 and 2 have an inhibitory effect on bFGF-stimulated BCE cell growth. Human prothrombin fragment 1 and 2 obtained as proteolytic fragments of human prothrombin display potent inhibitory effects on bovine capillary endothelial cells with a half-maximal concentration (ED50) of approximately 100 nM and 120 nM, respectively. As rabbit prothrombin fragment 2, the human prothrombin fragment 1 and 2 also inhibit angiogenesis in the chorioallantoic membrane (CAM) of chick embryos.

Allantois↗
PubMed1998

Fed-batch production of thermomonospora fusca endoglucanase by recombinant streptomyces lividans

The factors affecting the production of a Thermomonospora fusca endoglucanase by a recombinant Streptomyces lividans strain were studied in a fermentor with glucose addition controlled by a pH-stat. The recombinant plasmid was stable for 35 generations with constant endoglucanase productivity. Glucose and peptone were used as the carbon and nitrogen sources. Addition of Tween-80 increased endoglucanase production twofold. A significant decrease in endoglucanase production was observed at low aeration. During fed-batch cultivation, pulse feeding (6 g/L) of a glucose-ammonium sulfate solution was optimal for endoglucanase production. With higher concentrations of glucose (15 g/L), a significant amount of organic acid, including acetic acid, was produced, which inhibited cell growth and endoglucanase production. Under optimum conditions, 1.7 U/mL of endoglucanase were produced. Copyright 1998 John Wiley & Sons, Inc.

Journal Article↗
PubMed1998

Developmentally and spatially regulated activation of a Dictyostelium STAT protein by a serpentine receptor.

Dd-STAT, the protein that in part controls Dictyostelium stalk cell differentiation, is a structural and functional homolog of metazoan signal transducers and activators of transcription (STATs). Although present during growth and throughout development, Dd-STAT's tyrosine phosphorylation and nuclear localization are developmentally and spatially regulated. Prior to late aggregation, Dd-STAT is not tyrosine phosphorylated and is not selectively localized in the nucleus. During mound formation, the time at which cell-type specific gene expression initiates, Dd-STAT becomes tyrosine phosphorylated and translocates into the nuclei of all cells. The tyrosine phosphorylation and nuclear localization of Dd-STAT are induced very rapidly by extracellular cAMP through the serpentine cAMP receptor cAR1, with Dd-STAT tyrosine phosphorylation being detectable within 10 s of stimulation. This activation is independent of the only known Gbeta subunit, suggesting that it may be G-protein independent. Nuclear enrichment of Dd-STAT is selectively maintained within the sub-population of prestalk cells that form the tip, the organizing center of the slug, but is lost in most of the other cells of the slug. This spatial patterning of Dd-STAT nuclear localization is consistent with its known role as a negative regulator of stalk-cell differentiation.

Amino Acid Sequence↗
PubMed1998

Factorial optimization of a six-cellulase mixture

A factorial experimental design approach was used to optimize mixtures of six cellulases (five Thermomonospora fusca cellulases and plus/minus Trichoderma reesei CBHI along with beta-glucosidase) so as to maximize the glucose produced from filter paper. Optimized mixture A and mixture B produced glucose at 25 and 8.3 μmol glucose/μmol enzyme/min, respectively, which are 8 and 1.5 times higher than the sum of the activity of the individual cellulases. In both mixtures, the glucose yield depended on the ratio and the cellulases used. Most enzymes showed synergistic interactions that increased the glucose yield. The yield of glucose with the optimum mixtures depended on the total enzyme concentration. Copyright 1998 John Wiley & Sons, Inc.

Journal Article↗
PubMed1998

Dual mechanisms for the low plasma levels of truncated apolipoprotein B proteins in familial hypobetalipoproteinemia. Analysis of a new mouse model with a nonsense mutation in the Apob gene.

Familial hypobetalipoproteinemia (FHbeta), a syndrome characterized by low plasma cholesterol levels, is caused by mutations in the apo-B gene that interfere with the synthesis of apo-B100. FHbeta mutations frequently lead to the synthesis of a truncated form of apo-B, which typically is present in plasma at < 5% of the levels of apo-B100. Although many FHbeta mutations have been characterized, the basic mechanisms causing the low plasma levels of truncated apo-B variants have not been defined. We used gene targeting to create a mutant allele that exclusively yields a truncated apo-B, apo-B83. In mice heterozygous for the Apob83 allele, plasma levels and the size and density distribution of apo-B83-containing lipoproteins were strikingly similar to those observed in humans with FHbeta and an apo-B83 mutation. Analysis of mice carrying the Apob83 mutation revealed two mechanisms for the low plasma levels of apo-B83. First, Apob83 mRNA levels and apo-B83 secretion were reduced 76 and 72%, respectively. Second, apo-B83 was removed rapidly from the plasma, compared with apo-B100. This mouse model provides a new level of understanding of FHbeta and adds new insights into apo-B metabolism.

Alleles↗
PubMed1998

The polycystic kidney disease 1 gene product mediates protein kinase C alpha-dependent and c-Jun N-terminal kinase-dependent activation of the transcription factor AP-1.

Autosomal dominant polycystic kidney disease (ADPKD) is a common hereditary disorder that accounts for 8-10% of end stage renal disease. PKD1, one of two recently isolated ADPKD gene products, has been implicated in cell-cell and cell-matrix interactions. However, the signaling pathway of PKD1 remains undefined. We found that the C-terminal 226 amino acids of PKD1 transactivate an AP-1 promoter construct in human embryonic kidney cells (293T). PKD1-induced transcription is specific for AP-1; promoter constructs containing cAMP response element-binding protein, c-Fos, c-Myc, or NFkappaB-binding sites are unaffected by PKD1. In vitro kinase assays revealed that PKD1 triggers the activation of c-Jun N-terminal kinase (JNK), but not of mitogen-activated protein kinases p38 or p44. Dominant-negative Rac-1 and Cdc42 mutations abrogated PKD1-mediated JNK and AP-1 activation, suggesting a critical role for small GTP-binding proteins in PKD1-mediated signaling. Several protein kinase C (PKC) inhibitors decreased PKD1-mediated AP-1 activation. Conversely, expression of the C-terminal domain of PKD1 increased PKC activity in 293T cells. A dominant-negative PKC alpha, but not a dominant-negative PKC beta or delta, abrogated PKD1-mediated AP-1 activation. These findings indicate that small GTP-binding proteins and PKC alpha mediate PKD1-induced JNK/AP-1 activation, together comprising a signaling cascade that may regulate renal tubulogenesis.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1998

Functional analysis of the guanylate kinase-like domain in the synapse-associated protein SAP97.

SAP97 is a membrane cytoskeletal protein localized at the presynaptic nerve terminals of type 1 asymmetric synapses. It has been implicated in the assembly of synapses and in particular in the localization and clustering of ion channels. The C-terminal GK domain of SAP97 shares a high degree of sequence similarity with low-molecular-mass guanylate kinases. These enzymes are involved in the guanine nucleotide metabolic cycle and in the maintenance of GTP/GDP pools required for example in Ras-mediated cell signaling. It has therefore been hypothesized that SAP97 plays an essential role in cellular signaling by regulating the guanine nucleotide pools at synaptic junctions. Here, we test this hypothesis by assessing whether the GK domain in SAP97 encodes an authentic guanylate kinase. We show that the GK domain in and of itself does not encode an active guanylate kinase, that it cannot be activated by its binding partner GKAP and that flanking regions are not acting as inhibitory regulators for enzymatic activity. Thus, it would appear that the GK domain of SAP97 is not involved in the metabolism of guanine nucleotides required for signaling events.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Transactivation potential of the C-terminus of human Nm23-H1.

The transactivation potential of Nm23-H1, a homolog of c-myc transcription factor Nm23-H2/PuF was assessed in yeast as a fusion protein with the DNA binding domains (DBDs) of GAL4 and LexA. The C-terminal half of Nm23-H1 exhibited strong transactivation of the reporter genes, LacZ and Leu2 carrying GAL4 and LexA upstream activating sequences (UASs), whereas the full-length Nm23-H1 and its N-terminal did not. Similar results were also obtained with Nm23-H2/PuF transactivating the reporter genes only by the C-terminus fused to GAL4 and LexA DBDs. Hence, our results suggested a possible regulatory role of the N-termini of Nm23 isotypes upon transactivation.

Bacterial Proteins↗