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Biomedical subjects

S J Baek

Publications and source records attributed to S J Baek.

At least 19 recordsLinked to original sources
PubMed2003

Evaluation of eicosanoids and NSAIDs as PPARgamma ligands in colorectal carcinoma cells.

The activation of peroxisome proliferator activated receptor gamma (PPARgamma) may play a role in the control of colorectal carcinogenesis. The expression of PPARgamma was examined by Western blotting in human colorectal tumors and matched normal adjacent tissues, as well as in various colorectal carcinoma cell lines. In the tissues, the expression of PPARgamma was elevated in tumors relative to the adjacent normal tissues. Each colorectal carcinoma cell line expressed PPARgamma. The ability of various eicosanoids to bind PPARgamma in colorectal carcinoma cells was investigated using luciferase reporter assays. The well-known PPARgamma ligands, troglitazone and 15-deoxy-Delta(12,14)-prostaglandin J(2) strongly induced PPARgamma binding activity. Products of lipoxygenases displayed moderate binding activity, while other prostaglandins and fatty acids displayed little or no reporter activation. The activation of PPARgamma by 13(S)-HODE, the major metabolite of 15-lipoxygenase-1 from linoleic acid, was concentration dependent reaching maximum at 10 micro M (35-fold activation). The endogenous production of 13(S)-HODE by expression of 15-LO-1 did not activate PPARgamma. The ability of various nonsteroidal anti-inflammatory drugs (NSAIDs) to induce PPARgamma activation was also evaluated. The conventional NSAIDs that inhibit both cyclooxygenases (COX-1 and COX-2) also induced PPARgamma binding activity. In general, however, neither COX-1- nor COX-2-specific inhibitors induced the activation of PPARgamma. Taken together, the metabolites of 15-lipoxygenase and the conventional NSAIDs were confirmed as exogenous ligands for PPARgamma in colorectal carcinoma cells.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed2001

Molecular cloning and characterization of human nonsteroidal anti-inflammatory drug-activated gene promoter. Basal transcription is mediated by Sp1 and Sp3.

Nonsteroidal anti-inflammatory drug-activated gene (NAG-1) is known to be associated with anti-tumorigenic activity and belongs to the transforming growth factor-beta superfamily. In the present study, we cloned the promoter region (-3500 to +41) and investigated the transcriptional regulatory mechanisms of the basal expression of the human NAG-1 gene. Several potential transcription factor-binding sites in this region were identified. Based on the results from clones of nested deletions, the construct between -133 and +41 base pairs contains three Sp1-binding sites (Sp1-A, Sp1-B, and Sp1-C), which confer basal transcription specific activity of NAG-1 expression. When the Sp1-C site was mutated (GG to TT), a 60-80% decrease in promoter activity was observed in HCT-116 cells. Gel shift, co-transfection, and chromatin immunoprecipitation assays showed that the Sp transcription factors bind to the Sp1-binding sites and transactivate NAG-1 expression. In addition, chicken ovalbumin upstream promoter-transcription factor 1 can interact with the C-terminal region of Sp1 and Sp3 proteins and induce NAG-1 promoter activity through Sp1 and Sp3 transcription factors. These results identify the critical regulatory regions for the human NAG-1 basal promoter. Furthermore, the results suggest that the level of expression of the NAG-1 gene will depend on the availability of Sp proteins and on co-factors such as chicken ovalbumin upstream promoter-transcription factor 1.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed2001

Effect of PPAR activators on cytokine-stimulated cyclooxygenase-2 expression in human colorectal carcinoma cells.

Cyclooxygenase-2 (COX-2) expression is up-regulated in colorectal cancer tissue. Peroxisome proliferator-activated receptors (PPARs) are expressed in human colorectal tissue and activation of PPARs can alter COX-2 expression. In macrophages, activation of PPARs down-regulates COX-2 expression. We examined the effect of PPARalpha and PPARgamma ligands on untreated and TNF-alpha-induced COX-2 expression in the human colorectal epithelial cell line HT-29. The expression of PPARalpha and PPARgamma was confirmed in these cells. TNF-alpha, an inflammatory cytokine, increased COX-2 expression via the NFkappaB pathway. In the absence of TNF-alpha, WY14643 (PPARalpha activator) caused an increase, while BRL49653 (PPARgamma activator) did not alter COX-2 expression. When HT-29 cells were incubated with TNF-alpha and WY14643, a further increase in COX-2 expression was detected. Incubation with TNF-alpha and BRL49653 caused an additional twofold increase in COX-2 expression. Our results suggest that both PPARalpha signaling and TNF-alpha signaling increase COX-2 expression by independent pathways, while PPARgamma stimulates COX-2 expression by up-regulation of the TNF-alpha pathway.

Adenocarcinoma↗
PubMed2001

Inhibition of lipid peroxidation and oxidative DNA damage by Ganoderma lucidum.

Reactive oxygen species (ROS), such as superoxide anions and hydroxyl radicals, are associated with carcinogenesis and other pathophysiological conditions. Therefore, elimination or inactivation of ROS or inhibition of their excess generation may be beneficial in terms of reducing the risk for cancer and other diseases. Ganoderma lucidum has been used in traditional oriental medicine and has potential antiinflammatory and antioxidant activities. In the present study, we tested the amino-polysaccharide fraction (designated as 'G009') from Ganoderma lucidum for the ability to protect against oxidative damage induced by ROS. G009 significantly inhibited iron-induced lipid peroxidation in rat brain homogenates and showed a dose-dependent inactivation of hydroxyl radicals and superoxide anions. It also reduced strand breakage in phiX174 supercoiled DNA caused by UV-induced photolysis of hydrogen peroxide and attenuated phorbol ester-induced generation of superoxide anions in differentiated human promyelocytic leukaemia (HL-60) cells. These findings suggest that G009 from Ganoderma lucidum possesses chemopreventive potential.

Animals↗
PubMed2001

Subgrouping persons with snoring and/or apnea by using anthropometric and cephalometric measures.

Craniofacial and anthropometric characteristics are identified risk factors for obstructive sleep apnea syndrome (OSAS). Phase 1 of this study used cephalometric radiographs to record craniofacial measurements from 60 OSAS subjects with a respiratory disturbances index (RDI) of 20 or greater (group A) and 60 subjects with a history of loud snoring and an RDI less than 20 (group B). From this data set, a craniofacial risk index (CRI) was constructed using age, body mass index (BMI), and 14 cephalometric measures previously reported to be associated with OSAS. A separating boundary (CRIS) was established by using discriminant analysis to differentiate between the two groups. All measurements were determined by an investigator who was blinded to the subjects' RDI score. Phase 2 used a second sample of 19 group A and 47 group B subjects to test the ability of the CRI derived from the first sample to classify subjects in this second sample into the correct age group. The CRI was able to classify correctly 72.3% (34 of 47) of the group A subjects when all variables were used in the discriminant model. Using only four variables (age, BMI, hyoid mandibular plane distance, and tongue length) selected by the stepwise method, 72.3% (34 of 47) of the group B subjects and 78.7% of group A subjects were classified correctly. These results suggest that a stepwise CRI could be used to classify heterogeneous groups of individuals with increased RDI into subgroups with varying degrees of anatomic risk for disease. Such subgrouping by anatomic risk could be important in determining the pathophysiology of OSAS because it is likely that differences in upper airway anatomy among individuals interacts with a variety of other factors to produce clinical illness.

Adult↗
PubMed2001

Expression of Gab1 lacking the pleckstrin homology domain is associated with neoplastic progression.

An in vitro transformation system of carcinogen-treated Syrian hamster embryo (SHE) cell cultures represents multistep genetic and nongenetic changes that develop during the neoplastic progression of normal cells to tumor cells in vivo. During this neoplastic progression, SHE cells demonstrate an altered response to epidermal growth factor (EGF). In the present report, we examined the role of the adapter protein Gab1 (Grb2-associated binder-1) in the neoplastic progression of SHE cells. We used two asbestos-transformed SHE cell clones in different neoplastic stages: a 10W+8 clone, which is immortal and retains the ability to suppress the tumorigenicity of tumor cells in cell-cell hybrid experiments, and a 10W-1 clone, which has lost this tumor suppressor ability. 10W+8 cells expressed full-length 100-kDa Gab1 and associated 5.2-kb mRNA. Upon repeated cell passaging, 10W-1 cells showed increasing expression of a novel 87-kDa form of Gab1 as well as 4.6-kb mRNA with diminishing expression of the original 100-kDa Gab1. cDNA encoding the 87-kDa Gab1 predicts a form of Gab1 lacking the amino-terminal 103 amino acids (Gab1(Delta1-103)), which corresponds to loss of most of the pleckstrin homology (PH) domain. Gab1(Delta1-103) retains the ability to be phosphorylated in an EGF-dependent manner and to associate with the EGF receptor and SHP-2 upon EGF stimulation. The endogenous expression of Gab1(Delta1-103) in 10W-1 cells appeared closely related to EGF-dependent colony formation in soft agar. Moreover, transfection and expression of Gab1(Delta1-103), but not Gab1, in 10W+8 cells enhanced their EGF-dependent colony formation in soft agar. These results demonstrate that Gab1 is a target of carcinogen-induced transformation of SHE cells and that the expression of a Gab1 variant lacking most of the PH domain plays a specific role in the neoplastic progression of SHE cells.

Agar↗
PubMed2001

Cyclooxygenase inhibitors regulate the expression of a TGF-beta superfamily member that has proapoptotic and antitumorigenic activities.

The antitumorigenic activity of nonsteroidal anti-inflammatory drugs (NSAIDs), cyclooxygenase (COX) inhibitors, is well established, but responsible molecular mechanisms are not fully understood. NSAIDs stimulate apoptosis by COX dependent and independent mechanisms in colorectal cells in culture. Identification of genes regulated by COX inhibitors could lead to a better understanding of their proapoptotic and anti-neoplastic activities. Using subtractive hybridization, a cDNA which was designated as NSAID activated gene (NAG-1) was identified from NSAID-treated HCT-116, human colorectal cells. NAG-1 has an identical sequence with a novel member of the TGF-beta superfamily that has 5 different names. In the HCT-116 cells, NAG-1 expression is increased and apoptosis is induced by treatment with some NSAIDs in a concentration and time-dependent manner. NAG-1 transfected cells exhibited increased basal apoptosis, increased response to NSAIDs and reduced soft agar cloning efficiency. Furthermore, transplantable tumors derived from NAG-1 transfected HCT-116 cells showed reduced tumorigenicity in athymic nude mice compared with vector-transfected HCT-116 cells. The increased NAG-1 expression by NSAIDs provides a suitable explanation for COX-independent apoptotic effects of NSAIDs in cultured cells. These data demonstrate that NAG-1 is an antitumorigenic and proapoptotic protein, and its regulation by COX inhibitors may provide new clues for explaining their proapoptotic and antitumorigenic activities.

Animals↗
PubMed2001

Identification of epidermal growth factor receptor- Grb2-associated binder-1-SHP-2 complex formation and its functional loss during neoplastic cell progression.

The adaptor protein Grb2-associated binder-1 (Gab1) is known to bind to the SHP-2 tyrosine phosphatase on epidermal growth factor (EGF) receptor stimulation. To clarify the roles of these two proteins in EGF receptor (EGFR) signaling and determine their possible alteration during neoplastic cell progression, we studied these proteins in a Syrian hamster embryo (SHE) cell line model of neoplastic progression. Specifically, we used asbestos-transformed SHE fibroblasts: the 10W+8 clone, which is immortal but nontumorigenic; and the 10W2T clone, which is tumorigenic. Gab1 was detected, and the EGF-dependent formation of the EGFR-Gab1-SHP-2 complex was observed in 10W+8 cells. After cloning hamster Gab1 cDNA, exogenous expression of Gab1 significantly enhanced EGF-dependent mitogenic activity in 10W+8 cells. On the other hand, Gab1 was not detected in 10W2T cells, and the EGF-dependent association of SHP-2 with EGFR was also absent. Exogenous Gab1 expression in transfected 10W2T cells restored the EGF-dependent association of SHP-2 with EGFR, although it only showed a marginal effect on EGF-dependent mitogenic activity. Thus, Gab1 plays a pivotal role in the EGFR signaling pathway via the formation of the EGFR-Gab1-SHP-2 complex, and alteration in the expression and function of Gab1 is implicated in the neoplastic progression of SHE cells.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Functional characterization of cyclooxygenase-2 polymorphisms.

Cyclooxygenases (COX)-1 and -2 are the key enzymes in the conversion of arachidonic acid to prostaglandins. COX-2 appears to play an emerging role in inflammation and carcinogenesis. Nonsteroidal anti-inflammatory drugs (NSAIDs) are used for the treatment of numerous diseases and reduce the risk of developing colorectal cancer. Polymorphisms in the COX-2 gene could alter enzyme expression, function, and/or the response to NSAIDs. Therefore, they could modify individual risks for developing cancer and other diseases or the occurrence of side effects or sensitivity toward selective or nonselective COX inhibitors. We sequenced the COX-2 gene of 72 individuals and identified rare polymorphisms in the promoter and the coding region. A COX-2 molecular model was used to locate the coding region polymorphisms relative to functional sites in the protein, and the COX-2 V511A polymorphism was very near to the active site. This variant protein was expressed, and function was evaluated, but no difference was detected in metabolism of the COX-2 substrates, arachidonic acid, linoleic acid, and 2-arachidonyl glycerol, compared with the wild type. The Km values for arachidonic acid showed no differences between the COX-2 wild type and V511A mutant. Inhibition with selective or nonselective COX inhibitors was essentially the same for the two enzymes. The absence of functionally important polymorphisms in the COX-2 gene may suggest that there has been selective pressure against those single nucleotide polymorphisms because of the critical role of this enzyme in maintenance of homeostasis.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed2000

Lack of cyclooxygenase-2 activity in HT-29 human colorectal carcinoma cells.

Induction of cyclooxygenase-2 (COX-2) is an early event in the sequence of polyp formation to colon carcinogenesis. COX-2 is at elevated levels in human colorectal cancers and in tumors and polyps of mouse models of colorectal cancer. Mutation of the adenomatous polyposis coli (APC) gene is the initial event leading to colorectal cancer. Colorectal cells in culture which express mutant APC are often used to examine the association of COX-2 expression and apoptosis. The expression of full-length APC in HT-29 cells, a human colorectal carcinoma cell line which normally expresses truncated APC and highly expresses COX-2, inhibits cell growth through increased apoptosis and results in a down-regulation of COX-2 protein. In this report, we examine whether down-regulation of COX-2 is directly linked to the increase in apoptosis observed in these HT-29-APC cells. We present evidence that COX-2 and apoptosis are not linked since COX-2, although expressed, is catalytically inactive. Interestingly, the COX-2 cloned from HT-29 cells is catalytically active when transfected into HCT-116 cells, a colorectal cell line which normally does not express COX-2, but is not active in the HT-29 cell line itself.

Apoptosis↗
PubMed2000

The synaptic vesicle protein SV2 is complexed with an alpha5-containing laminin on the nerve terminal surface.

Interactions between growing axons and synaptic basal lamina components direct the formation of neuromuscular junctions during nerve regeneration. Isoforms of laminin containing alpha5 or beta2 chains are potential basal lamina ligands for these interactions. The nerve terminal receptors are unknown. Here we show that SV2, a synaptic vesicle transmembrane proteoglycan, is complexed with a 900-kDa laminin on synaptosomes from the electric organ synapse that is similar to the neuromuscular junctions. Although two laminins are present on synaptosomes, only the 900-kDa laminin is associated with SV2. Other nerve terminal components are absent from this complex. The 900-kDa laminin contains an alpha5, a beta1, and a novel gamma chain. To test whether SV2 directly binds the 900-kDa laminin, we looked for interaction between purified SV2 and laminin-1, a laminin isoform with a similar structure. We find SV2 binds with high affinity to purified laminin-1. Our results suggest that a synaptic vesicle component may act as a laminin receptor on the presynaptic plasma membrane; they also suggest a mechanism for activity-dependent adhesion at the synapse.

Animals↗
PubMed1997

Comparison of two dental devices for treatment of obstructive sleep apnea syndrome (OSAS).

Previous case reports have indicated dental devices can be an effective nonsurgical treatment for snoring and obstructive sleep apnea. This pilot study evaluated the effectiveness of two intraoral devices in reducing the Respiratory Disturbance Index (RDI) and Epworth Sleepiness Scale (ESS) scores in a group of 24 adult volunteers with a history of loud snoring. Subjects were randomly assigned to two groups. Twelve subjects were fitted with a dental device designed to increase vertical dimension and protrude the mandible (device A). The other 12 subjects received a different device designed to minimally increase vertical opening without protruding the mandible (device B). Unattended home sleep monitoring (Edentrace II Digital Recorder, Edentech Corp.) was used to compute RDI at two time periods: (T0) before using any dental device and (T1) while using a dental device 2 weeks after the initial delivery date. The mean RDI and ESS scores at T0 for subjects in the device A group were 35.6 +/- 28.4 and 12.0 +/- 3.9, respectively. Means for the same measures at T1 were 21.1 +/- 21.4 and 8.2 +/- 4.0. For subjects in the device B group, means for RDI and ESS scores at T0 were 36.5 +/- 43.7 and 13.0 +/- 4.5, the means at T1 were 46.8 +/- 47.0 and 12.5 +/- 5.7. The effectiveness of the two devices was estimated by comparing the difference in RDI scores from T0 to T1 for the 10 subjects who were using device A and completed the study and the 8 subjects who were using device B and completed the study. Six subjects withdrew for various reasons. From T0 to T1, device A reduced RDI scores in 9 of 10 subjects, with a mean reduction in RDI of 14.5 (p < or = 0.05) and in ESS score of 3.8 (p < or = 0.005). Device B showed no change or an increased RDI score in 8 of 8 subjects. Seven of the eight subjects who showed no improvement in RDI with device B were then fitted with device A. Four of these seven subjects showed a reduction in RDI and five showed a reduction in ESS after using device A for 2 weeks. The mean reduction in RDI and ESS was 2.4 +/- 19.8 and 2.4 +/- 3.0, respectively. Hence, we conclude that a dental device that advances the mandible and increases the vertical dimension to open the upper airway is more effective in reducing the number of apneic and snoring events during sleep than one which does not.

Adult↗
PubMed1996

Multiple factors regulating the expression of human thromboxane synthase gene.

Characterization of the 5.5 kb promoter of human thromboxane synthase (TS) gene revealed a proximal positive regulatory sequence (PPRS, -90 to -25 bp) and several distal repressive elements. The maximal promoter activity was found to reside within the first 285 bp, approximately 75% of which was contributed by the PPRS. The sequence between -365 and -665 bp exerted a strong repressive effect (approximately 55%) on reporter gene expression independent of orientation and position, consistent with properties expected for a silencer. The sequence upstream of -665 bp to -5.5 kb contains mainly repressive elements which further reduce the promoter activity by 30%. The 65 bp PPRS worked in an orientation-independent, but position-dependent, manner and could be further divided into two independent elements, PPRS1 (-90 to -50 bp) and PPRS2 (-50 to -25 bp). While similar nuclear factor(s) from different cell types interact with PPRS2, those interacting with PPRS1 exhibit cell specificity. Internal sequence deletion and oligonucleotide competition established that a binding sequence for NF-E2 in PPRS1 (-60 tgctgattcat -50) was important for enhancing TS promoter activity in HL-60 cells. The presence of NF-E2 mRNA in HL-60 cells was demonstrated by reverse-transcription PCR amplification of the cDNA and Northern blot analysis. A 9-fold transactivation of luciferase (luc) reporter gene expression had been detected when NF-E2 cDNA was co-expressed with a TS promoter/luc construct. Despite the fact that NF-E2 and the cis-elements could alter the efficiency of TS transcription, they were not sufficient for restricting cell-specific TS expression. Analysis of the methylation status at the TS promoter in several human cell lines reveals cell-specific patterns of methylation that might correlate with TS expression. Taken together, these results suggest that the expression of human TS gene is modulated by multiple factors including cis-elements, trans-activator(s), and possibly genomic methylation.

Animals↗
PubMed1996

Genomic structure and polymorphism of the human thromboxane synthase-encoding gene.

Thromboxane synthase (TS) is a cytochrome P-450 (CYP450) enzyme catalyzing the conversion of prostaglandin endoperoxide (PGH2) into thromboxane A2 (TxA2) which plays a crucial role in hemostasis and cardiovascular diseases. Twelve genomic clones containing the DNA encoding the human TS gene (hTS) were isolated and characterized to determine the exon/intron boundaries and restriction maps of the nearly contiguous structure of the gene. The hTS contains 13 exons spanning more than 150 kb. Its first five exons, divided by relatively large introns, spread over 100 kb, but encode less than one third of the full-length TS transcript. Southern analysis indicates that the human haploid genome contains a single copy of the TS gene. Although multiple transcription start points (tsp) are utilized, transcription of hTS is primarily TATA-independent, as determined by promoter-directed reporter gene expression in transfected cells. A dinucleotide (CA) repetitive sequence identified in the ninth intron of the gene exhibits allelic polymorphism. At least four distinctive alleles, containing from 13 to 20 copies of the CA repeats, have been detected.

Base Sequence↗
PubMed1994

Cloning and characterization of the human thromboxane synthase gene promoter.

A genomic phage clone hybridized to the 5' end of human thromboxane synthase (TS) cDNA was isolated. Sequencing analysis of a 1.7 kb subfragment revealed that it contained the entire 5' untranslated region and 46 bp of the coding sequence of TS cDNA, an upstream canonical TATA box (TATAAA), and several binding sites for transcription factors (AP1, PEA3, PU.1, and GR), indicative of a promoter/first exon region of the TS gene. RNase protection assay mapped the transcription start site of the human TS gene to the nucleotide A 30 bp downstream from the TATA box. The authenticity of the promoter was further confirmed by its ability to direct expression of a CAT reporter gene in transfected HL60 cells.

Base Sequence↗
PubMed1994

The porcine thromboxane synthase-encoding cDNA: sequence, mRNA expression and enzyme production in Sf9 insect cells.

A full-length cDNA encoding porcine thromboxane synthase (TS) was isolated and sequenced. The open reading frame encodes a 534-amino acid (aa) protein (M(r) 60,451) which shares more than 75% identity with TS from other species and is 30% homologous to several enzymes of the cytochrome P-450 III family. Sequence comparison among porcine (p), human (h), and murine (m) TS indicated conservation of eight Cys residues and one putative N-glycosylation site. Several highly conserved regions were identified at the near N terminus, middle and C terminus. The most divergent region lies at aa residues 290-325, within which a Lys308 residue was unique to pTS. Between aa residues 70 and 90, considerable divergence was observed in mTS. Northern analysis showed that the pTS gene was expressed as a 2.3-kb transcript primarily in lung, kidney and thymus. A high-titer recombinant (re-) baculovirus containing pTS cDNA was developed to conduct a time course study of enzyme production in Spodoptera frugiperda (Sf9) cells. TS activity was detectable in the microsomes of Sf9 cells 12-h post-infection and reached maximum by 48 h. The produced TS resembles purified pTS in catalysis, as well as inhibition by a substrate analog inhibitor.

Amino Acid Sequence↗
PubMed1993

Expression of two different forms of cDNA for thromboxane synthase in insect cells and site-directed mutagenesis of a critical cysteine residue.

cDNA coding for human placental thromboxane synthase (EC 5.3.99.5) was amplified by PCR from a human placental cDNA library and sequenced. This cDNA and a shorter cDNA isolated from a human lung cDNA library with a deletion of 163 bp near the 3' end were expressed in Spodoptera frugiperda (Sf9) insect cells using a baculovirus expression system. The cDNA from human placenta was expressed as an active enzyme (60 kDa) with a specific activity higher than those reported from other cell types, whereas the shorter cDNA was expressed in an inactive form (52 kDa). The active recombinant enzyme appeared to be unglycosylated as the molecular mass and the enzyme activity were not altered in the presence of tunicamycin. Site-directed mutagenesis was performed to convert a cysteine at position 480 in thromboxane synthase to a serine. This cysteine is found to be highly conserved in related cytochrome P-450 enzymes. The mutant enzyme was found to be inactive, although Western blot, immunoprecipitation and SDS/PAGE analysis indicated that the mutant enzyme was expressed at a level comparable with the wild-type enzyme. These results suggest that Cys-480 is essential for the enzyme catalytic activity and that the short-form cDNA may be a non-functional transcript.

Amino Acid Sequence↗