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M K Lee

Publications and source records attributed to M K Lee.

At least 145 records · Page 8Linked to original sources
PubMed1998

SeqHelp: a program to analyze molecular sequences utilizing common computational resources.

Here we describe a tool to analyze molecular sequences utilizing the internet and existing computational resources for molecular biology. The computer program SeqHelp organizes information from database searches, gene structure prediction, and other information to generate multiply aligned, hypertext-linked reports to allow for fast analysis of molecular sequences. The efficient and economical strategy in this program can be employed to study molecular sequences for gene cloning, mutation analysis, and identical sequence search projects.

Algorithms↗
PubMed1998

Release of aqueous contents from phospholipid vesicles induced by cecropin A (1-8)-magainin 2 (1-12) hybrid and its analogues.

The membrane-disrupting properties of cecropin A (1-8)-magainin 2 (1-12) hybrid peptide, which has higher antitumor with less hemolytic activities than cecropin A (1-8)-melittin (1-12), and its analogues were assessed by measuring the induced release of vesicle-entrapped fluorescence probes. A model membrane was made of zwitterionic phospholipid (phosphatidylcholine) or the mixture of negatively and zwitterionic phospholipids (phosphatidylcholine and phosphatidylserine). The extent of leakage of the aqueous content of the phospholipid vesicles was found to have remarkable discrepancies according to the amphipathic nature of each analogue peptide. The entrapped high molecular weight solute (fluorescein-labeled immunoglobulin G, 55 kDa) also was released by the analogue which had the largest hydrophobic region and the highest amphipathic score among peptides tested. As the result of the determination of the relationships between the membrane-disrupting properties and the hydrophobicity values of peptides, it was found that the membrane-disrupting activity increased according to increasing the hydrophobicity of the peptide. The tryptophan fluorescence emission spectra and CD spectra showed that on interaction with the phospholipid vesicle, the peptide acquired the ordered structure and alpha-helical conformation by moving a tryptophan residue into the nonpolar environment of the phospholipid vesicle. These results suggest that the breakdown of the lipid bilayer was mediated by the alpha-helical amphipathic structure of the peptide interacting with the lipid bilayers as well as the by the hydrophobicity of the peptide.

Amino Acid Sequence↗
PubMed1998

Identification of determinants on a dualtropic human immunodeficiency virus type 1 envelope glycoprotein that confer usage of CXCR4.

The chemokine receptors CCR5 and CXCR4, in combination with CD4, mediate cellular entry of macrophage-tropic (M-tropic) and T-cell-tropic strains of human immunodeficiency virus type 1 (HIV-1), respectively, while dualtropic viruses can use either receptor. We have constructed a panel of chimeric viruses and envelope glycoproteins in which various domains of the dualtropic HIV-1(DH12) gp160 were introduced into the genetic background of an M-tropic HIV-1 isolate, HIV-1(AD8). These constructs were employed in cell fusion and virus infectivity assays using peripheral blood mononuclear cells, MT4 T cells, primary monocyte-derived macrophages, or HOS-CD4 cell lines, expressing various chemokine receptors, to assess the contributions of different gp120 subdomains in coreceptor usage and cellular tropism. As expected, the dualtropic HIV-1(DH12) gp120 utilized either CCR3, CCR5, or CXCR4, whereas HIV-1(AD8) gp120 was able to use only CCR3 or CCR5. We found that either the V1/V2 or the V3 region of HIV-1(DH12) gp120 individually conferred on HIV-1(AD8) the ability to use CXCR4, while the combination of both the V1/V2 and V3 regions increased the efficiency of CXCR4 use. In addition, while the V4 or the V5 region of HIV-1(DH12) gp120 failed to confer the capacity to utilize CXCR4 on HIV-1(AD8), these regions were required in conjunction with regions V1 to V3 of HIV-1(DH12) gp120 for efficient utilization of CXCR4. Comparison of virus infectivity analyses with various cell types and cell fusion assays revealed assay-dependent discrepancies and indicated that events occurring at the cell surface during infection are complex and cannot always be predicted by any one assay.

Animals↗
PubMed1998

Update of the morbidity experience of employees potentially exposed to chlorpyrifos.

OBJECTIVES: Chlorpyrifos, an organophosphate ingredient of several important insecticides, has been manufactured at The Dow Chemical Company for 25 years. A previous morbidity study among employees of The Dow Chemical Company found no increased prevalence of illness or symptoms among employees potentially exposed to chlorpyrifos from 1977 to 1985 compared with matched controls. The purpose of the current study was to update the original study to 1994, thereby increasing the statistical power. METHODS: In the present study, 496 potentially exposed subjects were identified and matched for age, race, sex, pay, and year of hire to 911 control subjects. Morbidity data were abstracted from company medical records. RESULTS: The prevalence of peripheral neuropathy was not significantly increased among this group of employees potentially exposed to chlorpyrifos. Significantly increased prevalence odds ratios were identified for five diagnostic categories: diseases of the ear and mastoid process; acute respiratory infections; other diseases of the respiratory system; general symptoms, signs, and ill defined conditions; and symptoms, signs, and ill defined conditions involving the digestive system. There was a strong association of diagnosis with duration of observation period, indicating that the exposed workers were more likely than unexposed workers to have a diagnosis abstracted from the company medical records due to their longer mean period of follow up. Analyses by exposure classification and mean plasma cholinesterase activity did not show a dose response. CONCLUSIONS: These data do not support a cause and effect relation of the diagnoses mentioned and exposure to chlorpyrifos.

Adult↗
PubMed1998

The Shc 66 and 46 kD isoforms are differentially downregulated at parturition in the fetal mouse lung.

Many of the signaling pathways regulating fetal lung mesenchymal cell proliferation are mediated by the Shc intracellular signaling proteins. Shc is expressed as three isoforms: 52 kD and 46 kD proteins (Shc 52 and Shc 46, respectively) translated from the same mRNA, and a 66 kD form (Shc 66) translated from a separate mRNA. Shc 52 is an activator of Ras and mitogen-activated protein kinase, whereas Shc 66 antagonizes Ras activation. The function of Shc 46 is unclear. We hypothesized that the Shc isoforms are differentially regulated during fetal mouse lung morphogenesis. Relative Shc 66 and Shc 46 protein expression are high until parturition (term = 18.5 d), when a dramatic decrease begins; by postconceptual d 20, relative Shc 66 and Shc 46 expression have fallen by 75 and 69%, respectively. A similar pattern of decreasing Shc 66 mRNA expression in the peripartum period was detected by reverse transcription and competitive polymerase chain reaction during the same period. By isoform-specific immunohistochemistry, Shc 66 is widely distributed in the embryonic lung but becomes restricted to the bronchial smooth muscle and overlying epithelia, periarterial smooth muscle, and the interlobar pleura late in gestation. After parturition, Shc 66 is virtually absent from the lung. All three Shc isoforms are phosphorylated by epidermal growth factor stimulation in fetal lung mesenchymal cells, indicating that Shc 66 is functional in these cells. These data indicate that Shc isoforms are differentially regulated during lung development.

3T3 Cells↗
PubMed1998

Mutations of ret proto-oncogene in 3 Korean families with MEN 2A: clinical use of new restriction sites for genetic diagnosis.

Abnormalities in the ret proto-oncogene are found in several disorders such as multiple endocrine neoplasia (MEN) 2, sporadic medullary thyroid cancer, congenital megacolon and papillary thyroid cancer. In MEN 2A or 2B, early genetic diagnosis before the development of clinical tumors is crucial for the cure of the disease. We studied mutations of ret proto-oncogene in 3 Korean families with MEN 2A and searched for new restriction sites that could be used for genetic diagnosis. By direct sequencing of exon 10 and 11 harboring 'hot' spots, heterozygous point mutation was detected at positions translating cysteine codon in all 3 families. In 2 families, mutations at codon 634 in exon 11 were found (from TGC to CGC or TAC), yielding a new CfoI or RsaI restriction site. In one family, a mutation was located at codon 618 in exon 10 (from TGC to CGC), generating a new CfoI restriction site. These new restriction sites were used in detecting 2 undiagnosed family members without clinical symptoms or signs. In one of them, thyroidectomy was performed to disclose a small medullary thyroid cancer. These results indicate that Korean MEN 2A patients have germ-line mutations in the ret protooncogene at the cysteine residues like patients of other races, and the strategy employing direct sequencing to find mutations at 'hot spot' and search for ensuing new restriction sites could be a useful approach for the molecular diagnosis of genetic diseases accompanied by mutations in restricted areas of disease genes such as MEN 2.

Adrenalectomy↗
PubMed1998

Troglitazone prevents hyperglycemia-induced but not glucosamine-induced insulin resistance.

Hyperglycemia can lead directly to a secondary state of insulin resistance or can worsen a preexisting insulin-resistant state. Troglitazone is an orally active hypoglycemic agent that has been shown to ameliorate insulin resistance and hyperinsulinemia in both diabetic animal models and NIDDM subjects. To determine whether this drug could prevent the development of hyperglycemia-induced insulin resistance and to investigate the mechanism by which this might occur, we studied troglitazone's effect on insulin action in rats made hyperglycemic or infused with glucosamine. Normal male SD rats were fed regular powdered diet with or without troglitazone as a food admixture (0.2%). After 2 weeks, rats were made hyperglycemic with glucose (52 mg x kg(-1) x min[-1]) and somatostatin (0.8 microg x kg(-1) x min[-1]) infusion or were infused with glucosamine (6.5 mg x kg(-1) x min[-1]) for 6.5 h. In vivo insulin action was measured by the hyperinsulinemic-euglycemic clamp technique at a submaximal (24 pmol x kg(-1) x min[-1]) or maximal (240 pmol x kg(-1) x min[-1]) insulin infusion rate. The infusion of glucose and somatostatin caused a pronounced rise in the plasma glucose concentration (19.8 +/- 0.6 mmol/l) compared with saline-infused animals (8.0 +/- 0.2 mmol/l; P < 0.001). Hyperglycemia resulted in insulin resistance, as evidenced by a marked reduction in the submaximal glucose disposal rate (GDR) (78 +/- 7 vs. 135 +/- 6 micromol x kg(-1) x min(-1); P < 0.01) and maximal GDR (141 +/- 9 vs. 237 +/- 6 micromol x kg(-1) x min(-1); P < 0.01) compared with the control group. Troglitazone treatment largely prevented the hyperglycemia-induced decline in submaximal (116 +/- 7 micromol x kg(-1) x min[-1]) and maximal GDR (209 +/- 9 micromol x kg(-1) x min(-1); P < 0.05). Glucosamine infusion also resulted in a marked reduction in the submaximal GDR (85 +/- 3 vs. 135 +/- 6 micromol x kg(-1) x min(-1); P < 0.01) and maximal GDR (137 +/- 14 vs. 237 +/- 6 micromol x kg(-1) x min(-1); P < 0.01) compared with the control group. In contrast to the results in the hyperglycemic animals, troglitazone treatment had no effect on glucosamine-induced insulin resistance. In summary, 1) in normal rats, experimental hyperglycemia, as well as glucosamine infusion, led to a marked state of peripheral and hepatic insulin resistance; 2) troglitazone treatment prevented the hyperglycemia-induced, but not the glucosamine-induced, insulin resistance; and 3) either troglitazone acts at one or more sites proximal to the entry of glucosamine into the hexosamine pathway, or the increased flux of glucose-derived products through the hexosamine pathway is not a major mechanism for the hyperglycemia-induced defect in insulin action in these animals.

Administration, Oral↗
PubMed1998

alpha2beta1 integrin signaling via the mitogen activated protein kinase pathway modulates retinoic acid-dependent tumor cell invasion and transcriptional downregulation of matrix metalloproteinase 9 activity.

Tumor cell invasion and metastasis requires precise coordination of adherence to the extracellular matrix (ECM) and controlled degradation of its components. lnvasive cells secrete proteolytic enzymes known as matrix metalloproteinases which degrade specific basement membrane molecules. Expression of these enzymes is regulated by multiple signaling mechanisms, including attachment to the extracellular matrix via integrins. All-trans retinoic acid can inhibit tumor cell invasion of ECM by regulating matrix metalloproteinase expression. Using a series of squamous cell carcinoma lines, we investigated the interactions between integrin and retinoic acid signaling in these cells. In a cell line sensitive to RA-mediated inhibition of invasion, this ligand downregulated MMP-9 activity in cells grown on specific ECM molecules but not on plastic. Inhibition of integrin signaling with anti-alpha1 antibodies or MAPK pathway inhibitors abrogated RA mediated down-regulation of MMP-9 activity and invasion. The effects of RA and MAPK signaling on MMP-9 activity was mediated at the transcriptional level. These data indicate that crosstalk between RA- and integrin dependent signaling pathways regulate MMP activity and invasion in squamous cell carcinoma lines.

Antineoplastic Agents↗
PubMed1997

Identification and characterization of a mouse homologue of the spinal muscular atrophy-determining gene, survival motor neuron.

Spinal muscular atrophy (SMA), the second most common fatal, autosomal recessive disease of infants, manifests as generalized muscle weakness. The most severe form (Type I, Werdnig-Hoffmann disease) is associated with quadriplegia, respiratory muscle paralysis and death in infancy. Less severe forms are classified as Type II and Type III, based on age of onset and ultimate motor disability. Some spinal motor neurons show chromatolysis and the number of these cells is decreased. Recently, SMA has been mapped to chromosome 5q11.2-13.3 (Gilliam et al., 1990), a region that contains three candidate genes: Survival Motor Neuron (SMN) (Lefebvre et al., 1995); Neuronal Apoptosis Inhibitory Protein (NAIP) (Roy et al., 1995); and p44, a subunit of transcription factor II H (TFIIH) (Carter et al., 1995; Bürglen et al., 1997). Homozygous deletions or deleterious mutations in SMN are present in all SMA patients, and in some affected individuals, deletions have been identified in one or both of the other genes. These extensive deletions may be associated with a more severe phenotype. We have identified and characterized the mouse homologue of SMN, MoSMN, which is 82% identical to SMN at the amino-acid level. Unlike the duplicated human SMN, MoSMN is present in single copy. Like its human counterpart, MoSMN is ubiquitously expressed, but unlike SMN, MoSMN does not appear to be alternatively spliced. In-situ hybridization analysis of the mouse nervous system revealed that MoSMN mRNA is expressed in spinal cord and throughout the brain, with relatively higher levels of expression in the hippocampus and cerebellum.

Alternative Splicing↗
PubMed1997

Nonsyndromic deafness DFNA1 associated with mutation of a human homolog of the Drosophila gene diaphanous.

The gene responsible for autosomal dominant, fully penetrant, nonsyndromic sensorineural progressive hearing loss in a large Costa Rican kindred was previously localized to chromosome 5q31 and named DFNA1. Deafness in the family is associated with a protein-truncating mutation in a human homolog of the Drosophila gene diaphanous. The truncation is caused by a single nucleotide substitution in a splice donor, leading to a four-base pair insertion in messenger RNA and a frameshift. The diaphanous protein is a profilin ligand and target of Rho that regulates polymerization of actin, the major component of the cytoskeleton of hair cells of the inner ear.

Actins↗
PubMed1997

Evaluation of the N-terminal role in peptide antigenicity of the preS2 region of the hepatitis B virus surface antigen.

Previously we reported that the N-terminal sequence 120-129 of the preS2 region of the hepatitis B virus (HBV) surface antigen (HBsAg) plays an important role in peptide antigenicity against an preS2 specific anti-HBsAg monoclonal antibody (H8 mAb) by affecting the B cell epitope conformation of a peptide existing within the sequence 130-145. In this study, we tried to confirm the previous results using a series of N-terminal sequentially deleted peptides and peptides substituted at position 127. This position was previously found to be important for H8 mAb binding. Peptide antigenicity of sequentially deleted peptides was gradually decreased when the residues from 125 to 129 were deleted, indicating that the residues cumulatively affect antigenicity. Peptide antigenicity relative to the substituted residues at position 127 of p123 showed an order of F > I > E > or = T > A > or = P > K and was correlated with their CD spectra. Structure-dependent peptide antigenicity was also found in many polyclonal antisera produced by immunization with peptide-protein conjugates and HBsAg. Twenty-nine out of 34 polyclonal antisera had reactivities increased 2-fold or more with p123 relative to those with p130. Among them, 10 antisera showed increased reactivities of 7 fold or more. These results confirm our previous results, which suggested the existence of the conformational B cell epitope in the preS2 region maintained by the N-terminal sequence. This experimental also suggests that N-terminal sequence 125-129 is important in maintaining a higher antigenic structure.

Amino Acid Sequence↗
PubMed1997

Dynamin II associates with Grb2 SH3 domain in Ras transformed NIH3T3 cells.

Grb2, a linker protein containing two SH3 domains and one SH2 domain, is known as an essential element of the Ras pathway in multiple systems. One of the functions of Grb2 is to link tyrosine-phosphorylated receptors to downstream effector proteins via the SH2 and SH3 domain bindings. To identify Grb2-associated proteins in Ras transformed NIH3T3 cells, we performed coprecipitation experiments using recombinant GST-Grb2 fusion proteins and found a remarkably strong band of 100 kDa. With N-terminal amino acid sequencing, we identified the protein of 100 kDa as dynamin II. Dynamin II was also observed in the coprecipitates with the GST fusion protein of N-SH3 or C-SH3 domain of Grb2 but not in that of Grb2 SH2 domain. The SH3-mediated association of Grb2 with dynamin II was confirmed by competitive binding experiments with oligopeptides whose sequence corresponded to that of SH2 or SH3 binding motif. The dynamin II coprecipitation was completely abrogated by the addition of the oligopeptide of SH3 binding motif, but addition of SH2 binding motif had no effect. In conclusion, these results suggest that dynamin II may be largely expressed and closely associated with Grb2-mediated signaling in Ras transformed cells.

3T3 Cells↗
PubMed1997

ALS-linked SOD1 mutant G85R mediates damage to astrocytes and promotes rapidly progressive disease with SOD1-containing inclusions.

High levels of familial Amyotrophic Lateral Sclerosis (ALS)-linked SOD1 mutants G93A and G37R were previously shown to mediate disease in mice through an acquired toxic property. We report here that even low levels of another mutant, G85R, cause motor neuron disease characterized by an extremely rapid clinical progression, without changes in SOD1 activity. Initial indicators of disease are astrocytic inclusions that stain intensely with SOD1 antibodies and ubiquitin and SOD1-containing aggregates in motor neurons, features common with some cases of SOD1 mutant-mediated ALS. Astrocytic inclusions escalate markedly as disease progresses, concomitant with a decrease in the glial glutamate transporter (GLT-1). Thus, the G85R SOD1 mutant mediates direct damage to astrocytes, which may promote the nearly synchronous degeneration of motor neurons.

ATP-Binding Cassette Transporters↗
PubMed1997

Accelerated amyloid deposition in the brains of transgenic mice coexpressing mutant presenilin 1 and amyloid precursor proteins.

Missense mutations in two related genes, termed presenilin 1 (PS1) and presenilin 2 (PS2), cause dementia in a subset of early-onset familial Alzheimer's disease (FAD) pedigrees. In a variety of experimental in vitro and in vivo settings, FAD-linked presenilin variants influence the processing of the amyloid precursor protein (APP), leading to elevated levels of the highly fibrillogenic Abeta1-42 peptides that are preferentially deposited in the brains of Alzheimer Disease (AD) patients. In this report, we demonstrate that transgenic animals that coexpress a FAD-linked human PS1 variant (A246E) and a chimeric mouse/human APP harboring mutations linked to Swedish FAD kindreds (APP swe) develop numerous amyloid deposits much earlier than age-matched mice expressing APP swe and wild-type Hu PS1 or APP swe alone. These results provide evidence for the view that one pathogenic mechanism by which FAD-linked mutant PS1 causes AD is to accelerate the rate of beta-amyloid deposition in brain.

Aging↗
PubMed1997

Hyperaccumulation of FAD-linked presenilin 1 variants in vivo.

Mutations in the presenilin 1 (PS1) and presenilin 2 (PS2) genes can cause Alzheimer's disease in affected members of the majority of early-onset familial Alzheimer's disease (FAD) pedigrees. PS1 encodes an ubiquitously expressed, eight transmembrane protein. PS1 is endoproteolytically processed to an amino-terminal derivative (approximately 27-28 kDa) and a carboxy-terminal derivative (approximately 17-18 kDa). These polypeptides accumulate to saturable levels in the brains of transgenic mice, independent of the expression of PS1 holoprotein. We now document that, in the brains of transgenic mice, the absolute amounts of accumulated N- and C-terminal derivatives generated from the FAD-linked PS1 variants in which Glu replaces Ala at codon 246 (A246E) or Leu replaces Met at codon 146 (M146L) accumulate to a significantly higher degree (approximately 40-50%) than the fragments derived from wild-type PS1. Moreover, the FAD-linked deltaE9 PS1 variant, a polypeptide that is not subject to endoproteolytic cleavage in vivo, also accumulates in greater amounts than the fragments generated from wild-type human PS1. Thus, the metabolism of PS1 variants linked to FAD is fundamentally different from that of wild-type PS1 in vivo.

Actins↗
PubMed1997

Inhibition of tyrosine hydroxylase by bulbocapnine.

The inhibitory effects of bulbocapnine, an aporphine isoquinoline alkaloid, on bovine adrenal tyrosine hydroxylase (TH) were investigated. Bulbocapnine had an inhibitory effect on the enzyme (43.6% inhibition at concentration of 200 microM). Bulbocapnine exhibited uncompetitive inhibition on bovine adrenal TH with a substrate L-tyrosine. The Ki value was found to be 0.20 mM.

Adrenal Glands↗