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D S Lim

Publications and source records attributed to D S Lim.

At least 37 records · Page 2Linked to original sources
PubMed2000

Enhancement of adenoviral transduction with polycationic liposomes in vivo.

Although the high transfection efficiency with adenovirus in vitro is well documented, it is still not clear whether adenoviral vectors are effective in vivo in solid tumor models. In our preliminary experiment, transduction of tumor tissue was limited to just around the injection site after intratumoral injection of the adenoviral vector. To improve the transduction efficiency in vivo, we tried a combination of adenoviral vector and liposome in our animal model. Adenovirus carrying human placental alkaline phosphatase (AdALP) and Lipofectamine or 1,3-di-oleoyloxy-2-(6-carboxyspermyl)-propylamide were used as a marker gene and the cationic liposome, respectively. A >15-fold increase in the transfection efficiency was observed in CT26 tumor cell lines with the combination of AdALP adenovirus carrying murine granulocyte-macrophage colony-stimulating factor (AdmGM-CSF), and liposome compared with adenovirus alone, showing the feasibility of the combination treatment. In the animal model, with the combination of liposome and AdALP, deeper and wider distribution of the marker gene in the tumor mass was shown. We conclude that the limitations of direct application of adenoviral vectors in a solid tumor model could be overcome by the use of cationic liposomes. This approach will facilitate the more effective delivery of adenoviral vectors in a clinical trial setting.

Adenocarcinoma↗
PubMed2000

Analysis of ku80-mutant mice and cells with deficient levels of p53.

Absence of Ku80 results in increased sensitivity to ionizing radiation, defective lymphocyte development, early onset of an age-related phenotype, and premature replicative senescence. Here we investigate the role of p53 on the phenotype of ku80-mutant mice and cells. Reducing levels of p53 increased the cancer incidence for ku80(-/-) mice. About 20% of ku80(-/-) p53(+/-) mice developed a broad spectrum of cancer by 40 weeks and all ku80(-/-) p53(-/-) mice developed pro-B-cell lymphoma by 16 weeks. Reducing levels of p53 rescued populations of ku80(-/-) cells from replicative senescence by enabling spontaneous immortalization. The double-mutant cells are impaired for the G(1)/S checkpoint due to the p53 mutation and are hypersensitive to gamma-radiation and reactive oxygen species due to the Ku80 mutation. These data show that replicative senescence is caused by a p53-dependent cell cycle response to damaged DNA in ku80(-/-) cells and that p53 is essential for preventing very early onset of pro-B-cell lymphoma in ku80(-/-) mice.

Aging↗
PubMed2000

Anti-leukaemic and anti-mutagenic effects of di(2-ethylhexyl)phthalate isolated from Aloe vera Linne.

Extracts of Aloe vera Linne have been found to exhibit cytotoxicity against human tumour cell lines. This study examines the anti-tumour effects of di(2-ethylhexyl)phthalate (DEHP) isolated from Aloe vera Linne, in human and animal cell lines. Its anti-mutagenic effects were examined using Salmonella typhimurium TA98 and TA100 strains. Growth inhibition was specifically exerted by DEHP against three leukaemic cell lines at concentrations below 100 microg mL(-1). At 100 microg mL(-1) DEHP, K562, HL60 and U937 leukaemic cell lines showed growth inhibition of 95, 97 and 95%, respectively. DEHP exhibited an inhibitory activity of 74, 83 and 81%, respectively, in K562, HL60 and U937 cell lines at a concentration of 10 microg mL(-1). At a concentration of 1 microg mL(-1), DEHP exerted an inhibitory activity of 50, 51 and 52%, respectively, in K562, HL60 and U937. In a normal cell line, MDBK, DEHP exerted 30% growth inhibition at a concentration of 100 microg mL(-1), and showed no inhibitory activity at concentrations below 50 microg mL(-1). It was found that DEHP exerted anti-mutagenic activity in the Salmonella mutation assay. The number of mutant colonies of Salmonella typhimurium strain TA98 upon exposure to AF-2 (0.2 microg/plate) decreased in a concentration-dependent manner in the presence of different DEHP concentrations (decreasing to 90.4, 83.9, 75.4, 69.6 and 46.9%, respectively, for DEHP concentrations of 100, 50, 10, 5 and 1 microg/plate). In the case of Salmonella typhimurium strain TA100, DEHP reduced AF-2-induced mutagenicity at 1, 5, 10, 50 and 100 microg/plate to 57.4, 77.5, 80.0, 89.0 and 91.5%, respectively. The isolated compound from Aloe vera Linne, DEHP, was considered to be the active principle responsible for anti-leukaemic and anti-mutagenic effects in-vitro.

Aloe↗
PubMed2000

Assessment of coronary flow reserve with transthoracic Doppler echocardiography: comparison with intracoronary Doppler method.

To evaluate the feasibility and usefulness of transthoracic Doppler echocardiography (TTDE) as a non-invasive method in recording distal anterior descending (LAD) coronary flow velocity, we compared coronary flow reserve (CFR) measured by TTDE with measurements by intracoronary Doppler wire (ICDW). Twenty-one patients without LAD stenosis were studied. ICDW performed at baseline and after intracoronary injection of 18 microg adenosine. TTDE was performed at baseline and after intravenous adenosine (140 microg/kgmin for 2 min). Adequate Doppler recordings of coronary flow velocities during systole were obtained in 14 of 21 study patients (67%) and during diastole in 17 (81%) patients. Baseline and hyperemic peak diastolic flow velocities measured by TTDE were significantly smaller than those obtained by ICDW (p<0.05). However, diminishing trends of diastolic and systolic velocity ratio after hyperemia were similarly observed in both methods. CFR obtained by TTDE (3.0+/-0.5), was higher than the value calculated by ICDW (2.5+/-0.4). There were significant correlations between the values obtained by the two methods (r=0.72, p<0.01). It is concluded that TTDE is a feasible method in measuring coronary flow velocity and appears to be a promising non-invasive method in evaluating CFR.

Adult↗
PubMed1999

Substrate specificities and identification of putative substrates of ATM kinase family members.

Ataxia telangiectasia mutated (ATM) phosphorylates p53 protein in response to ionizing radiation, but the complex phenotype of AT cells suggests that it must have other cellular substrates as well. To identify substrates for ATM and the related kinases ATR and DNA-PK, we optimized in vitro kinase assays and developed a rapid peptide screening method to determine general phosphorylation consensus sequences. ATM and ATR require Mn(2+), but not DNA ends or Ku proteins, for optimal in vitro activity while DNA-PKCs requires Mg(2+), DNA ends, and Ku proteins. From p53 peptide mutagenesis analysis, we found that the sequence S/TQ is a minimal essential requirement for all three kinases. In addition, hydrophobic amino acids and negatively charged amino acids immediately NH(2)-terminal to serine or threonine are positive determinants and positively charged amino acids in the region are negative determinants for substrate phosphorylation. We determined a general phosphorylation consensus sequence for ATM and identified putative in vitro targets by using glutathione S-transferase peptides as substrates. Putative ATM in vitro targets include p95/nibrin, Mre11, Brca1, Rad17, PTS, WRN, and ATM (S440) itself. Brca2, phosphatidylinositol 3-kinase, and DNA-5B peptides were phosphorylated specifically by ATR, and DNA Ligase IV is a specific in vitro substrate of DNA-PK.

Amino Acid Sequence↗
PubMed1999

Deletion of Ku86 causes early onset of senescence in mice.

DNA double-strand breaks formed during the assembly of antigen receptors or after exposure to ionizing radiation are repaired by proteins important for nonhomologous end joining that include Ku86, Ku70, DNA-PK(CS), Xrcc4, and DNA ligase IV. Here we show that ku86-mutant mice, compared with control littermates, prematurely exhibited age-specific changes characteristic of senescence that include osteopenia, atrophic skin, hepatocellular degeneration, hepatocellular inclusions, hepatic hyperplastic foci, and age-specific mortality. Cancer and likely sepsis (indicated by reactive immune responses) partly contributed to age-specific mortality for both cohorts, and both conditions occurred earlier in ku86(-/-) mice. These data indicate that Ku86-dependent chromosomal metabolism is important for determining the onset of age-specific changes characteristic of senescence in mice.

Age Factors↗
PubMed1999

Caspase-3-dependent cleavage of Bcl-2 promotes release of cytochrome c.

Caspases are cysteine proteases that mediate apoptosis by proteolysis of specific substrates. Although many caspase substrates have been identified, for most substrates the physiologic caspase(s) required for cleavage is unknown. The Bcl-2 protein, which inhibits apoptosis, is cleaved at Asp-34 by caspases during apoptosis and by recombinant caspase-3 in vitro. In the present study, we show that endogenous caspase-3 is a physiologic caspase for Bcl-2. Apoptotic extracts from 293 cells cleave Bcl-2 but not Bax, even though Bax is cleaved to an 18-kDa fragment in SK-NSH cells treated with ionizing radiation. In contrast to Bcl-2, cleavage of Bax was only partially blocked by caspase inhibitors. Inhibitor profiles indicate that Bax may be cleaved by more than one type of noncaspase protease. Immunodepletion of caspase-3 from 293 extracts abolished cleavage of Bcl-2 and caspase-7, whereas immunodepletion of caspase-7 had no effect on Bcl-2 cleavage. Furthermore, MCF-7 cells, which lack caspase-3 expression, do not cleave Bcl-2 following staurosporine-induced cell death. However, transient transfection of caspase-3 into MCF-7 cells restores Bcl-2 cleavage after staurosporine treatment. These results demonstrate that in these models of apoptosis, specific cleavage of Bcl-2 requires activation of caspase-3. When the pro-apoptotic caspase cleavage fragment of Bcl-2 is transfected into baby hamster kidney cells, it localizes to mitochondria and causes the release of cytochrome c into the cytosol. Therefore, caspase-3-dependent cleavage of Bcl-2 appears to promote further caspase activation as part of a positive feedback loop for executing the cell.

Animals↗
PubMed1999

A transgenic rabbit model for human hypertrophic cardiomyopathy.

Certain mutations in genes for sarcomeric proteins cause hypertrophic cardiomyopathy (HCM). We have developed a transgenic rabbit model for HCM caused by a common point mutation in the beta-myosin heavy chain (MyHC) gene, R400Q. Wild-type and mutant human beta-MyHC cDNAs were cloned 3' to a 7-kb murine beta-MyHC promoter. We injected purified transgenes into fertilized zygotes to generate two lines each of the wild-type and mutant transgenic rabbits. Expression of transgene mRNA and protein were confirmed by Northern blotting and 2-dimensional gel electrophoresis followed by immunoblotting, respectively. Animals carrying the mutant transgene showed substantial myocyte disarray and a 3-fold increase in interstitial collagen expression in their myocardia. Mean septal thicknesses were comparable between rabbits carrying the wild type transgene and their nontransgenic littermates (NLMs) but were significantly increased in the mutant transgenic animals. Posterior wall thickness and left ventricular mass were also increased, but dimensions and systolic function were normal. Premature death was more common in mutant than in wild-type transgenic rabbits or in NLMs. Thus, cardiac expression of beta-MyHC-Q(403) in transgenic rabbits induced hypertrophy, myocyte and myofibrillar disarray, interstitial fibrosis, and premature death, phenotypes observed in humans patients with HCM due to beta-MyHC-Q(403).

Animals↗
PubMed1998

The role of ATM in DNA damage responses and cancer.

Ataxia-telangiectasia (AT) is a complex, autosomal recessive disorder characterized by cerebellar ataxia, believed to result from progressive neurodegeneration, and telangiectasia, dilation of blood vessels within the eyes and parts of the facial region. AT patients suffer from recurrent infections caused by both cellular and humoral immune deficiencies and as a population, are significantly predisposed to cancer, particularly lymphomas and leukemias. Early attempts at treating these malignancies with radiotherapy revealed another hallmark of AT, a profound hypersensitivity to the cytotoxic effects of ionizing radiation (IR) which is recapitulated at the cellular level in culture. Predisposition to cancer and radiosensitivity observed in AT has been linked to chromosomal instability, abnormalities in genetic recombination, and defective signaling to programmed cell death and several cell cycle checkpoints activated by DNA damage. These earlier observations predicted that the gene defective in AT may encode a protein which plays a crucial role in sensing DNA damage and transducing signals that promote cell survival. Through the combined efforts of linkage analysis and positional cloning, a single gene was identified on chromosome 11q22-33 by Shiloh and colleagues and was found to be mutated in all four complementation groups previously characterized in cell lines derived from AT patients (Savitsky et al., 1995a,b). The predicted ATM gene product shows considerable homology to an emerging family of high molecular weight, phosphatidylinositol-3 kinase (PI-3 K)-related proteins involved in eukaryotic cell cycle control, DNA repair, and DNA recombination (Zakian, 1995). This landmark discovery has triggered a resurgence of biochemical and genetic studies focusing on ATM function which has brought forth insights regarding ATM activity and its role in DNA damage signaling.

Animals↗
PubMed1998

Activation of the ATM kinase by ionizing radiation and phosphorylation of p53.

The p53 tumor suppressor protein is activated and phosphorylated on serine-15 in response to various DNA damaging agents. The gene product mutated in ataxia telangiectasia, ATM, acts upstream of p53 in a signal transduction pathway initiated by ionizing radiation. Immunoprecipitated ATM had intrinsic protein kinase activity and phosphorylated p53 on serine-15 in a manganese-dependent manner. Ionizing radiation, but not ultraviolet radiation, rapidly enhanced this p53-directed kinase activity of endogenous ATM. These observations, along with the fact that phosphorylation of p53 on serine-15 in response to ionizing radiation is reduced in ataxia telangiectasia cells, suggest that ATM is a protein kinase that phosphorylates p53 in vivo.

Ataxia Telangiectasia Mutated Proteins↗
PubMed1998

ATM binds to beta-adaptin in cytoplasmic vesicles.

Inherited mutations in the ATM gene lead to a complex clinical phenotype characterized by neuronal degeneration, oculocutaneous telangiectasias, immune dysfunction, and cancer predisposition. Using the yeast two-hybrid system, we demonstrate that ataxia telangiectasia mutated (ATM) binds to beta-adaptin, one of the components of the AP-2 adaptor complex, which is involved in clathrin-mediated endocytosis of receptors. The interaction between ATM and beta-adaptin was confirmed in vitro, and coimmunoprecipitation and colocalization studies show that the proteins also associate in vivo. ATM also interacts in vitro with beta-NAP, a neuronal-specific beta-adaptin homolog that was identified as an autoantigen in a patient with cerebellar degeneration. Our data describing the association of ATM with beta-adaptin in vesicles indicate that ATM may play a role in intracellular vesicle and/or protein transport mechanisms.

Adaptor Protein Complex 3↗
PubMed1998

Microvascular integrity as a predictor of left ventricular remodeling after acute anterior wall myocardial infarction.

The purpose of this study was to investigate the relation of microvascular integrity and ventricular remodeling after acute myocardial infarction. Twenty-six patients with first acute anterior myocardial infarction were studied before discharge with myocardial contrast echocardiography (MCE). Opacification index (OI) and wall motion index were calculated in the left anterior descending artery territory and left ventricular diastolic volume was measured at baseline and during a 9-month follow-up. In total 26 patients, the regional wall motion improved but the left ventricular volume and global function was not changed significantly at follow-up. When the patients were divided into 3 groups according to opacification index (> or = 0.75, 0.5 approximately 0.75, < or = 0.5) at baseline, functional recovery was not observed and significant left ventricular dilatation was developed in patients with < or = 0.5 OI. Among the baseline echo-parameters such as ejection fraction, wall motion score, left ventricular volume and opacification index, the best predictor for long term left ventricular dilatation was the opacification index by multivariate analysis. In patients with acute anterior wall infarction the assessment of microvascular integrity by MCE at acute stage provides useful information regarding recovery of dysfunctional regional wall motion and ventricular remodeling.

Coronary Vessels↗
PubMed1997

Embryonic lethality and radiation hypersensitivity mediated by Rad51 in mice lacking Brca2.

Inherited mutations in the human BRCA2 gene cause about half of the cases of early-onset breast cancer. The embryonic expression pattern of the mouse Brca2 gene is now defined and an interaction identified of the Brca2 protein with the DNA-repair protein Rad51. Developmental arrest in Brca2-deficient embryos, their radiation sensitivity, and the association of Brca2 with Rad51 indicate that Brca2 may be an essential cofactor in the Rad51-dependent DNA repair of double-strand breaks, thereby explaining the tumour-suppressor function of Brca2.

3T3 Cells↗
PubMed1996

Ku86-deficient mice exhibit severe combined immunodeficiency and defective processing of V(D)J recombination intermediates.

Ku is a heterodimeric DNA end binding complex composed of 70 and 86 kDa subunits. Here, we show that Ku86 is essential for normal V(D)J recombination in vivo, as Ku86-deficient mice are severely defective for formation of coding joints. Unlike severe combined immunodeficient (scid) mice, Ku86-deficient mice are also defective for signal joint formation. Both hairpin coding ends and blunt full-length signal ends accumulate. Contrary to expectation, Ku86 is evidently not required for protection of either type of V(D)J recombination intermediate. Instead, V(D)J recombination appears to be arrested after the cleavage step in Ku86-deficient mice. We suggest that Ku86 may be required to remodel or disassemble DNA-protein complexes containing broken ends, making them available for further processing and joining.

Animals↗
PubMed1996

A mutation in mouse rad51 results in an early embryonic lethal that is suppressed by a mutation in p53.

RecA in Escherichia coli and its homolog, ScRad51 in Saccharomyces cerevisiae, are known to be essential for recombinational repair. The homolog of RecA and ScRad51 in mice, MmRad51, was mutated to determine its function. Mutant embryos arrested early during development. A decrease in cell proliferation, followed by programmed cell death and chromosome loss, was observed. Radiation sensitivity was demonstrated in trophectoderm-derived cells. Interestingly, embryonic development progressed further in a p53 null background; however, fibroblasts derived from double-mutant embryos failed to proliferate in tissue culture.

Animals↗
PubMed1995

Genotype distribution and comparison of the putative envelope region of hepatitis C virus from Korean patients.

Comparative nucleotide sequence studies of the genomes of hepatitis C virus (HCV) revealed that there are at least 6 different genotypes of HCV. The prevalence of HCV genotypes among the patients with liver diseases in Korea was investigated using the polymerase chain reaction (PCR) for the NS5 region. In the 75 HCV RNA positive samples, two genotypes, type 1b and type 2a, were the major causative agents which accounted for 60% and 33% of infections respectively, while 7% could not be assigned a genotype by the methods used. The nucleotide sequences of cDNAs encoding the putative envelope proteins from 10 type 1b and 5 type 2a genotype samples were analyzed. Approximately 31-42% of the nucleotide sequences of type 1b samples examined differed from those of different genotypes. In the case of type 2a samples, 36-42% of the nucleotide sequences differed from those of different genotypes. The diversities of the amino acid sequences were the same or greater than those of the nucleotide sequences. Two hypervariable regions (HVR1 and HVR2) were recognized in both HCV genomes of genotypes 1b and 2a. However, the sequence divergence within the HVR2 region of genotype 2a was less than that of genotype 1b.

Amino Acid Sequence↗