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Biomedical subjects

K Lim

Publications and source records attributed to K Lim.

At least 37 records · Page 2Linked to original sources
PubMed1996

Effect of O-glycosylated mucin on invasion and metastasis of HM7 human colon cancer cells.

Mucinous colorectal cancers have a poorer prognosis than colorectal cancers which produce a low amount of mucin, but the exact mechanism is not well understood. The present study was undertaken to elucidate the possible mechanisms of invasion and metastasis of colon cancer cells producing high levels of mucin using mucin glycosylation inhibitor, benzyl-alpha-N-acetylgalactosamine. The binding activity of treated HM7 cells to endothelial leukocyte adhesion molecule (ELAM-1) was significantly decreased and fixed cell binding of MoAb SNH-3 and 19-9 (specific for sialyl Le(x) and sialyl Le(a), respectively) was also significantly decreased. Metalloproteinase activity in conditioned medium and invasion of matrigel-coated porous filters by treated HM7 cells were decreased. However, there was no difference between control and treated HM7 cells in terms of matrix protein binding. These results suggest that O-glycosylated mucin is important in the invasive and metastatic properties of HM7 human colon cancer cells.

Acetylgalactosamine↗
PubMed1996

Pronase-based assay method for O6-methylguanine-DNA methyltransferase.

A new, simple, and rapid assay method for O6-methylguanine-DNA methyltransferase (MGMT) has been developed. When [methyl-3H] DNA radiolabeled with N-[methyl-3H]-N-nitrosourea was incubated together with tissue homogenate, [methyl-3H] group was transferred to the enzyme, forming S-[methyl-3H]cysteine. In contrast to the previous methods which determined the amount of [methyl-3H] group removed from [methyl-3H] DNA, the present method measured the amount of [methyl-3H] transferred to the enzyme. This has been done by hydrolyzing the radiolabeled enzyme with pronase which is a proteolytic enzyme with a broad substrate specificity. On pronase digestion, [methyl-3H]-labeled enzyme becomes soluble in trichloroacetic acid. The method is very simple and rapid, and the only expensive equipment required is a scintillation counter which is a relatively routine piece of equipment at present. More than a dozen samples can be processed within 4-5 h without any difficulty. This new method has been employed in the studies on organ distribution of MGMT of rat and mouse.

Animals↗
PubMed1996

Glucocorticoid regulation of androgen binding protein expression in primary Sertoli cell cultures from rats.

Glucocorticoids are known to inhibit testicular function, and its receptor is also localized in the Sertoli cells. To evaluate possible role of glucocorticoid in Sertoli cells, the effects of dexamethasone on the expression of androgen binding protein (ABP) have been investigated in primary Sertoli cell cultures. Dexamethasone increased ABP mRNA levels, with maximal stimulation reached at 36 hr. The induction of ABP mRNA was dependent on the low concentration (10(-8) and 10(-7) M) of dexamethasone but gradually reduced in the cells treated with high concentration (10(-6) and 10(-5) M). Dexamethasone-induced ABP mRNA level was no change in the cells after addition of cycloheximide but almost reduced by actinomycin-D pretreatment. Steady-state levels of ABP mRNA gradually increased in the Sertoli cells prepared from 14- and 21-days of age corresponding to rat puberty, and ABP mRNA was induced by dexamethasone. These results suggest that ABP gene is transcriptionally regulated by dexamethasone in primary Sertoli cell cultures.

Age Factors↗
PubMed1996

Alterations in CD4 dependence accompany T cell development and differentiation.

Several studies have indicated that the necessity for co-receptor engagement during T cell activation depends on the avidity of the TCR-MHC interaction under investigation. Using thymocytes, naive T cells and a long-term T cell line isolated from 2B4 TCR transgenic mice, we have examined the role of the CD4 co-receptor on cells expressing the identical TCR at multiple stages of T cell maturation. When anti-CD4 Fab fragments were used to block CD4-MHC class II interactions, we found decreasing CD4 dependence as T cells matured. As a second approach to examining the role of the CD4 co-receptor, we generated I-Ek mutants defective in CD4 interactions. In the course of this study, we identified a new potential site for CD4 interaction in the beta1 domain of I-Ek. The new beta1 mutation and a mutation in the previously described CD4 binding site in the beta2 domain both interfere with stimulation of 2B4 thymocytes, but not mature T cells. Together these data demonstrate that the role of the CD4 co-receptor depends on the state of maturation of the T cell.

Animals↗
PubMed1996

Soluble CD23 protein in the peritoneal fluid of patients with endometriosis.

Activated B cells are known to produce soluble CD23 protein (sCD23) from their membranes. We recently showed a higher concentration of sCD23 in the serum of patients with endometriosis. As the commonest site of endometriosis is the peritoneal cavity, we sought to evaluate the concentration of sCD23 in the peritoneal fluid of 47 fertile symptomatic women with endometriosis and 35 fertile women without endometriosis. Endometriosis was diagnosed by laparoscopy and confirmed by histopathology. There was a statistically significant difference between the sCD23 concentrations in the endometriosis and control groups (P < 0.05). When endometriosis was defined based on the revised American Fertility Society (AFS) Score, patients with mild (AFS 1 and II) but not severe (AFS III and IV) endometriosis showed a higher and significant difference in the concentration of sCD23 when compared with the controls (P < 0.05). There was no significant correlation between the peritoneal fluid concentration of sCD23 and the phase of the menstrual cycle. We conclude that the concentration of sCD23 is higher in the peritoneal fluid of patients with endometriosis when compared with the controls, suggestive of B cell activation in patients with endometriosis. Furthermore, mild endometriosis is immunologically more active than severe endometriosis as defined by the current classification.

Ascitic Fluid↗
PubMed1996

Effects of intermittent food restriction and refeeding on energy efficiency and body fat deposition in sedentary and exercised rats.

The effects of body weight cycling on energy metabolism and body fat accumulation were examined in sedentary and exercised rats. Ten rats were sacrificed before the experiment to obtain basal data, and then 90 rats were divided into three groups; control (CN), food restricted (FR) and weight cycling (WC). Food intake in rats of the FR group was restricted constantly to 70% of the intake of the CN group. The rats of WC group were subjected to four bouts of weight cycling consisting of 7-days food restriction followed by 7-days refeeding, but were fed the same total amount of dietary energy as that of the FR group throughout the experimental period. The rats of all groups were meal-fed twice a day. Half of the rats in each group were exercised by running on a treadmill (30 min/day) throughout the experimental period. The body weight, abdominal adipose tissue weight, body fat, body protein and energy restoration for the study in both sedentary and exercised groups were greater in the WC group than in the FR group. The resting metabolic rate of the WC group after four bouts of weight cycling was lower than that of the FR group in the sedentary rats, but this difference was not observed in the exercised rats. Also, the thermic effect of food (TEF) in the sedentary rats for 6 h after a meal was significantly less in the WC group as compared to that of the FR group. However, the TEF for the exercised rats was not different between the two groups. The serum insulin level, activities of lipogenic enzymes and lipoprotein lipase in adipose tissue for the sedentary rats of the WC group were higher than those of the FR group, but did not differ in the exercised rats. These results suggest that weight cycling increases body fat deposition and energy efficiency by decreasing energy expenditure, particularly the TEF, and that exercise training can alleviate the effects of weight cycling on the energy metabolism.

Adipose Tissue↗
PubMed1996

Role of the eosinophil in allergic reactions.

The eosinophil may have several functions in health and in the pathogenesis of allergic and other diseases. Some roles of the eosinophil are based on the acute, effector responses of this cell, its capacity to generate biologically active lipid mediators and release its granule contents, including its distinctive cationic proteins. Whilst the effector responses of eosinophils are important for their contribution to the acute pathogenesis of allergic diseases, a fuller understanding of the eosinophil requires evaluation of the role this cell may play at tissue sites, especially submucosal sites, where the cell is normally localized in the absence of disease. Moreover, for the long-lived, tissue-resident eosinophil, definition of the interactions that occur between the eosinophil and other immune cells is germane to understanding the functions of eosinophils both in acute and chronic diseases. Many allergic diseases are characterized by heightened accumulation of eosinophils and are chronic ongoing diseases.

Animals↗
PubMed1996

Children's understanding of the meaning and functions of verbal irony.

We investigated children's understanding of irony and sensitivity to irony's meanness and humor. In Study 1, 89 participants (5-6-year-olds, 8-9-year-olds, adults) heard ironic and literal criticisms, and literal compliments. Comprehension of irony emerged between 5 and 6 years of age. Ratings of humor increased with age; ratings of meanness did not (showing that all ages perceived irony as more muted than literal criticism). In Study 2, results from 135 participants (6-7-year-olds, 8-9-year-olds, and adults) replicated these findings and revealed the role of form and intonation. Thus, comprehension of irony emerges between 5 and 6 years of age, and sensitivity to the muting function develops prior to sensitivity to the humor function.

Age Factors↗
PubMed1995

Reduced level of octamer binding transcription factor (Oct-1) is correlated with H2B histone gene repression during differentiation of HL-60 cells by all-trans retinoic acid.

To gain insight on the role of transacting factors in the regulatory mechanism of H2B histone gene expression during the differentiation of HL-60 cells by all-trans retinoic acid (retinoic acid), the binding pattern of the nuclear proteins to various elements in the human H2B histone upstream region has been investigated with DNase I footprinting and DNA mobility shift assay. The level of H2B histone mRNA was markedly reduced at 48 hr in retinoic acid-treated HL-60 cells. The H2B histone mRNA was repressed in proportion to the concentration of retinoic acid. In DNase I footprinting analysis, a nuclear factor (octamer binding transcription factor, Oct-1) bound at -42 bp (ATTTGCAT) both before and after retinoic-acid-induced differentiation of HL-60 cells. One DNA-protein complex was formed by DNA mobility shift assay, and the level of Oct-1 decreased during retinoic-acid-induced differentiation. In the cycloheximide-treated HL-60 cells, the level of Oct-1 also reduced. These results suggest that the transcriptional repression of H2B histone gene during retinoic-acid-induced differentiation in HL-60 cells may be mediated by reduced level of Oct-1.

Base Sequence↗
PubMed1995

Molecular dynamics of the anti-fluorescein 4-4-20 antigen-binding fragment. 1. Computer simulations.

Two 174 ps molecular dynamics simulations of the solvated, 4-4-20 antigen-binding fragment (Fab) were performed: one with antigen (fluorescein) in the antigen-combining site and another with it removed. At the beginning of the second simulation, fluorescein was relocated from the antigen-combining site to a point outside the cutoff distance for nonbonded interactions by applying a "pulling force". Initially, the antigen-combining site collapsed when fluorescein was removed but gradually re-formed as the simulation progressed. In addition, several other differences were observed between the two simulations. These included (i) structural rearrangements of key contact residues in the antigen-combining site, (ii) significant differences in the degree of hydration of the antigen-combining site, (iii) a more acute elbow bend angle in the case of the unliganded form, and (iv) less correlated motions of amino acid residues in the unliganded form. These observations suggested that the Fab without fluorescein exhibited a greater degree of segmental flexibility than the Fab with fluorescein. Time-resolved fluorescence experiments were performed in order to validate this prediction, and the results are described in the following paper [Lim et al. (1995) Biochemistry 34, 6975-6984].

Antigens↗
PubMed1995

Molecular dynamics of the anti-fluorescein 4-4-20 antigen-binding fragment. 2. Time-resolved fluorescence spectroscopy.

Time-resolved fluorescence experiments were performed to investigate the dynamic aspects of the antigen-binding fragment (Fab) of a high-affinity monoclonal antibody (4-4-20) which binds the fluorescent hapten fluorescein. Both the unliganded Fab and a complex of the Fab with a nonfluorescent analog of fluorescein (fluoresceinamine, FLM) were examined. A fluorescence polarization probe [5-[[2-[(iodoacetyl)amino]ethyl]amino]naphthalene-1-sulfonic acid, AEDANS] was covalently attached to the C-terminus of the Fab. Experiments were performed at three different temperatures (10, 25, and 35 degrees C), and phase-modulation data sets were collected for five different molar ratios of FLM to Fab at each temperature. Global analyses were then used to extract values for fluorescence lifetime and rotational correlation time from these data. In the lifetime analysis the best fit was obtained when the emission of AEDANS was described by a Lorentzian distribution of lifetimes (tau = 15.6 ns, distribution width = 3.4 ns, both at 25 degrees C), which suggested that the probe experienced a heterogeneous environment. Anisotropy analyses suggested that two different rotational components were present. The first was attributed to the global motion of the Fab and exhibited a rotational correlation time (theta 1) of ca. 33 ns at 25 degrees C. This component was relatively unaffected by antigen binding. The second rotational component was attributed to the local or segmental motion within the Fab and exhibited a rotational correlation time (theta 2) of 1.1 ns at 25 degrees C. This value increased by more than 50% upon antigen binding, a result which was consistent with molecular dynamics simulations of the same Fab--fluorescein system [Lim & Herron (1995) Biochemistry 34, 6962-6974]. Furthermore, statistical analysis showed that this increase was significant at the 95% confidence level.

Amino Acid Sequence↗
PubMed1995

Follicle-stimulating hormone transiently induces expression of protooncogene c-myc in primary Sertoli cell cultures of early pubertal and prepubertal rat.

The protooncogene c-myc plays an important role in the regulation of cellular proliferation and differentiation. To evaluate the possibility that the protooncogene c-myc plays some roles in follicle-stimulating hormone (FSH)-dependent gene regulation of Sertoli cells, the effects of FSH on the expression of c-myc has been investigated in primary Sertoli cell cultures. FSH was no change to the c-myc mRNA level before 18 h, but transiently increased c-myc mRNA levels, with maximal stimulation reached in 18 h. The induction of c-myc was dependent on the concentration of FSH. Th c-myc mRNA was also increased after treatment with dibutyryl c-AMP and forskolin in primary Sertoli cell cultures. FSH-dependent c-myc mRNA levels were superinduced in cells treated for 3 h with cycloheximide but it was reduced by actinomycin-D pretreatment. Even in the absence of FSH in culture medium c-myc mRNA was clearly detectable in Sertoli cells from 8-day-old rats but hardly detectable in cells from 14 and 28 days of age. FSH stimulated c-myc mRNA expression in the primary Sertoli cells derived from only 8- and 14-day-old rats but had almost no effect in the 28-day-old rats. These results suggest that FSH induces c-myc mRNA levels in the primary Sertoli cells from prepubertal and early pubertal rats, and then transient expression of c-myc may be responsible for some roles in the regulation of FSH-dependent genes in Sertoli cells.

Animals↗
PubMed1995

Promotion of microtubule assembly in vitro by a novel 35-kDa protein purified from human term placenta.

Microtubule assembly promoting-protein (taxol-like protein, TALP) was purified by combination of high salt extraction, phosphocellulose chromatography and hydroxyapatite chromatography from human term placenta. Molecular weight of purified protein was identified as 35kDa on SDS-polyacrylamide gel electrophoresis. In vitro, TALP promoted microtubule assembly in dose-dependent manner in spite of the absence of GTP. Both TALP (0.5 microM) and taxol (10 microM) gave hyperbolic kinetics and shortened the lag time for microtubule polymerization. TALP-stabilized microtubules were resistant to depolymerization by cold (4 degrees C) and CaCl2 (4 mM) like taxol-stabilized microtubules. TALP showed its direct binding on the microtubule in cosedimentation assay. These results suggest that the action mechanism of TALP on the microtubule assembly is similar to taxol in vitro and the binding site of TALP is available on the intact microtubule.

Animals↗
PubMed1995

Acylshikonin analogues: synthesis and inhibition of DNA topoisomerase-I.

Compounds bearing an acyl group of a various size at 1'-OH of shikonin were synthesized as acyl analogues of shikonin, which was isolated from the root of Lithospermum erythrorhizon, and evaluated for inhibitory effect on topoisomerase-I activity. A selective acylation at 1'-OH of shikonin in the presence of dicyclohexylcarbodiimide and 4-(dimethylamino)pyridine gave rise to a good yield of corresponding acylshikonin derivatives. In general, analogues with an acyl group of shorter chain lengths (C2-C6) exerted a stronger inhibitory action than those with longer chain lengths (C7-C20). While the halogen substitution at C-2 of the acetyl moiety failed to increase the inhibitory potency, the placement of double bonds in the acyl group (C5-C7) augmented the potency remarkably. Of the 32 derivatives evaluated, 15 compounds exhibited a higher inhibitory effect than shikonin. Noteworthy, the inhibitory potency of acetylshikonin, propanoylshikonin, and 4-pentenoylshikonin was approximately 4-fold greater than that of camptothecin. All these data suggest that the size of acyl moiety is important for the enhancement of potency, and the presence of olefinic double bonds is also beneficial.

Acetylation↗
PubMed1995

Inhibition of rabbit aortic smooth muscle cell proliferation by selective inhibitors of protein kinase C.

1. We studied the effect of two structurally-related, selective inhibitors of protein kinase C, Ro 31-8220 and Ro 31-7549, on the reinitiation of proliferation in quiescent first passage rabbit aortic smooth muscle cells in response to (a) the direct activator of protein kinase C, phorbol dibutyrate (PDBu), (b) platelet-derived growth factor (PDGF), (c) a combination of PDGF and 5-hydroxytryptamine (5-HT) or (d) serum. 2. Ro 31-8220 and Ro 31-7549 concentration-dependently inhibited proliferation in response to each mitogen. The inhibitory potency (IC50) of Ro 31-8220 and Ro 31-7549, respectively, was similar against proliferation induced by PDBu (0.55 and 1.1 microM), PDGF (0.6 and 0.9 microM), PDGF and 5-HT (0.68 and 1.1 microM), although slightly less against serum (1.7 and 5 microM). The effects of the protein kinase C inhibitors on proliferation could not be ascribed to cytotoxicity. Neither Ro 31-8220 nor Ro 31-7549 (0.3-3 microM) inhibited PDGF receptor tyrosine phosphorylation. 3. The results show that Ro 31-8220 and Ro 31-7549 are potent inhibitors of smooth muscle cell proliferation in response to a direct activator of protein kinase C, the defined growth factors, PDGF and 5-HT, and the complex mixture of mitogens in serum. Protein kinase C activation thus appears to be an important growth transducing mechanism for each of these agents.

Animals↗
PubMed1995

Effects of red-pepper diet on the energy metabolism in men.

We investigated the effects of dietary red pepper on the energy metabolism in male subjects. In the first experiment, after having a standardized dinner on the previous evening, the subjects consumed a breakfast (650 kcal) either with or without 10 g of red pepper. For 150 min after the meal, they took a rest and their expired gas was collected. During the initial 30 min after the meal, the energy expenditure tended to be higher in the red-pepper diet period than in the control diet period. For the remaining 120 min, no difference in the energy expenditure was found between the red-pepper diet period and the control diet period. However the carbohydrate oxidation was significantly higher in the red-pepper diet period than in the control diet period while the lipid oxidation was lower in the red-pepper diet period than in the control diet period for 150 min after the meal. In the second experiment, the subjects consumed a breakfast with 10 g of red pepper after an oral administration of propranolol or a placebo. The propranolol abolished the increase in energy expenditure during the initial 30 min due to the meal containing red pepper. For the remaining 120 min, no difference in energy expenditure was found between the propranolol period and the placebo period. These results suggest than an increase in the energy expenditure after the meal containing red pepper appeared only immediately after the meal ingestion and a red-pepper diet increases the carbohydrate oxidation without increasing total energy expenditure for 150 min after the meal. And an increase in the energy expenditure immediately after the meal containing red pepper is considered to be caused by beta-adrenergic stimulation.

Adult↗
PubMed1994

Three-dimensional structure of Schistosoma japonicum glutathione S-transferase fused with a six-amino acid conserved neutralizing epitope of gp41 from HIV.

The 3-dimensional crystal structure of glutathione S-transferase (GST) of Schistosoma japonicum (Sj) fused with a conserved neutralizing epitope on gp41 (glycoprotein, 41 kDa) of human immunodeficiency virus type 1 (HIV-1) (Muster T et al., 1993, J Virol 67:6642-6647) was determined at 2.5 A resolution. The structure of the 3-3 isozyme rat GST of the mu gene class (Ji X, Zhang P, Armstrong RN, Gilliland GL, 1992, Biochemistry 31:10169-10184) was used as a molecular replacement model. The structure consists of a 4-stranded beta-sheet and 3 alpha-helices in domain 1 and 5 alpha-helices in domain 2. The space group of the Sj GST crystal is P4(3)2(1)2, with unit cell dimensions of a = b = 94.7 A, and c = 58.1 A. The crystal has 1 GST monomer per asymmetric unit, and 2 monomers that form an active dimer are related by crystallographic 2-fold symmetry. In the binding site, the ordered structure of reduced glutathione is observed. The gp41 peptide (Glu-Leu-Asp-Lys-Trp-Ala) fused to the C-terminus of Sj GST forms a loop stabilized by symmetry-related GSTs. The Sj GST structure is compared with previously determined GST structures of mammalian gene classes mu, alpha, and pi. Conserved amino acid residues among the 4 GSTs that are important for hydrophobic and hydrophilic interactions for dimer association and glutathione binding are discussed.

Amino Acid Sequence↗