Biomedical subjects

Kwang-Hyun Baek

Publications and source records attributed to Kwang-Hyun Baek.

At least 37 records · Page 2Linked to original sources

PubMed2005

Quantification of mitochondrial DNA using real-time polymerase chain reaction in patients with premature ovarian failure.

OBJECTIVE: To quantify mitochondrial DNA using real-time PCR in women with premature ovarian failure (POF) and a control group. DESIGN: Prospective study. SETTING: Genome Research Center for Reproductive Medicine and Infertility, Korea Ministry of Health & Welfare. PATIENT(S): Thrity patients with POF and 30 control individuals. INTERVENTION(S): The mitochondrial DNA content was quantified using real-time PCR. The effectiveness of the assay was determined by relative quantification using the comparative threshold cycle (CT) method. MAIN OUTCOME MEASURE(S): Relative quantification of mitochondrial DNA content. RESULT(S): The mitochondrial DNA content was significantly lower in the POF group than in the control group (0.58 +/- 0.38 vs. 1.15 +/- 0.67; P < .01). In both groups, there was a significant positive correlation between the mitochondrial DNA/28S rRNA ratio and mitochondrial DNA CT (control group: r = 0.774; P < .001; POF group: r = 0.556; P = .001) and a significant negative correlation between the mitochondrial DNA/28S rRNA ratio and 28S rRNA CT (control group: r = -0.677; P < .001; POF group: r = -0.627; P = .001). CONCLUSION(S): This study has established the clinical feasibility of quantifying amounts of mitochondrial DNA, relative to an internal standard, using real-time PCR. Further studies are warranted to elucidate the roles of apoptosis and mitochondrial function in the pathogenesis of POF.

Adult↗

PubMed2005

Expression and functional analyses of mHAUSP regulating apoptosis of cervical adenocarcinoma cells.

p53 tumor suppressor protein is stabilized by the herpes-virus-associated ubiquitin-specific protease (HAUSP), a deubiquitinating enzyme. We previously isolated a mouse orthologue of HAUSP, mHAUSP, encoding 1103 amino acids with a molecular weight of approximately 135 kDa containing highly conserved Cys, Asp (I), His, and Asn/Asp (II) domains. In this study, we investigated the temporal and spatial expression of mHAUSP during the early mouse embryonic development. Northern blot analysis revealed that the expression of mHAUSP was detected throughout the process of embryonic development with the maximal expression between E10.5 and E13.5. In situ hybridization study showed the global expression of mHAUSP in various organs of embryos, including mesencephalon, spinal cord, lung and genital eminence. In addition, we carried out biochemical analysis for 6 conserved amino acids (Cys224, Gln231, Asp296, His457, His465, and Asp482) in Cys box, QQD box, and His box in order to investigate their structural and functional roles of these amino acid residues. The conserved Gln231 was not essential for the catalytic activity of mHAUSP. However, other conserved amino acids were required for deubiquitinating enzyme activity of mHAUSP. Moreover, we observed that the overexpression of mHAUSP induces cell death in HeLa cells.

Amino Acid Sequence↗

PubMed2004

Essential regions of deubiquitinating enzyme activity and enhancer function for DUB-2A expressed in T-lymphocytes.

Novel subfamily members of deubiquitinating enzyme gene, DUB-1 and DUB-2, are known to be immediate-early genes and are specifically expressed in B-lymphocytes and T-lymphocytes, respectively. Recently, another deubiquitinating enzyme gene DUB-2A, expressed also in T-lymphocytes, has been isolated and we, in this study, found that Dub-2A has isopeptidase activity and there are at least 4 conserved amino acids (Cys60, Asp133, His298, and His307) that are required for catalytic activity of the enzyme. Interestingly, the conserved Asp323 was not essential for the catalytic activity. In addition, transcriptional reporter assay showed that an Ets site and 2 AP-1 sites in the enhancer domain of this gene are required for cytokine inducibility. Taken all together, these results will be very helpful in designing activators or inhibitors of this enzyme in order to regulate its expression and catalytic activity in T-lymphocytes.

Amino Acid Sequence↗

PubMed2004

Aberrant gene expression associated with recurrent pregnancy loss.

Recent studies indicate that a number of factors including chromosomal abnormalities, immunological feto-maternal rejection, hormonal irregulation and anatomical factors are involved in provoking recurrent pregnancy loss (RPL). This indicates that normal cellular regulation of these factors is required for maintaining normal pregnancy. In addition, it is expected that biological processes for maintaining normal pregnancy require a series of differential gene expression. As expected, our previous investigations revealed that there are >/=30 genes showing different levels of expression between normal and RPL patients. In addition, other research groups have also identified a number of genes that are expressed aberrantly in pregnancy failure. In this review, recent study on aberrant expression levels of genes, which are grouped as immunity-related, angiogenesis-related, apoptosis-related and other groups of genes, will be discussed.

Abortion, Habitual↗

PubMed2004

Differentially up-regulated genes in proliferating porcine neonatal pancreas cells caused by epidermal growth factor.

Pancreatic duct cells are considered to be a major source for beta-cell regeneration or neogenesis. Although epidermal growth factor (EGF) is a well-known important growth factor for pancreas development, the control of pancreatic duct cell growth and differentiation by EGF is poorly understood. In this study, we focused on identifying the genes that were differentially up-regulated in response to EGF stimulation using monolayer cultured porcine neonatal pancreas cells. Cells were obtained from 1 to 3 day old pigs, dispersed and cultured for 8 days. Monolayer cultured porcine pancreas cells were comprised of duct cells and some endocrine and mesenchymal cells (75.2 +/- 15.1, 19.6 +/- 4.9, and 9.5 +/- 3.1%, respectively). After 16 h in serum free media, cells were treated with 100 microg/L EGF for 24 h. Differentially expressed genes were screened by subtractive hybridization. (3)H-thymidine uptake was significantly increased by EGF with time (untreated vs. 24 h treated, untreated vs. 48 h treated: 305.5 +/- 3.5 cpm vs. 380.3 +/- 17.3 cpm (P < 0.05), 309.2 +/- 4.51 vs. 929 +/- 9.19 cpm, (P < 0.005), respectively). Three hundred and fifty cDNA clones were obtained by subtractive hybridization and the inserts were confirmed in 161 colonies and then sequenced. Finally, we found increased mRNA expression of five unknown and five known genes, including cytochrome c oxidase subunit I (COI), cyclooxygenase-2 (COX-2), matrix metalloproteinase-13 (MMP-13), Wiskott-Aldrich syndrome protein interacting protein (WASPIP), and hyaluronan synthase-2 (HAS-2). We confirmed the up-regulation of these genes by Northern blot and semi-quantitative RT-PCR at various time points. The present findings opened new targets for the research on the mechanisms of pancreatic duct cell proliferation by EGF.

Animals↗

PubMed2004

Murine Asb-17 expression during mouse testis development and spermatogenesis.

In this study we isolated a murine mAsb-17 from mouse testis by RT-PCR using primers designed based on the sequences from the GenBank database. The sequence analysis showed that mAsb-17 encodes a 295 amino acid polypeptide with a molecular weight of approximately 34 kDa containing two ankyrin repeats and one SOCS box. The amino acid sequence of mASB-17 showed 87.5%, 98.3% and 92.9% identity with that of human, rat and dog, respectively. Interestingly, northern blot analysis showed that mAsb-17 was expressed only in the testis. The expression analysis by RT-PCR for mAsb-17 in mouse indicates that mAsb-17 is expressed from the fourth week after birth to adult, with the highest expression in round spermatids. Both northern blot and RT-PCR analyses suggest that mASB-17 may play essential roles in testis development and spermatogenesis.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Identification and characterization of murine mHAUSP encoding a deubiquitinating enzyme that regulates the status of p53 ubiquitination.

Recently, it was discovered that herpesvirus-associated ubiquitin-specific protease (HAUSP) in human interacts with p53 protein, and removes the ubiquitin from ubiquitinated p53. Thus, human HAUSP stabilizes the status of p53, induces p53-dependent cell growth repression and apoptosis. In this study, we isolated and characterized a mouse orthologue of HAUSP, mHAUSP. The mHAUSP cDNA was cloned from mouse ES cells by RT-PCR. The open reading frame consists of 3,312 bp and encodes a predicted protein of 1,103 amino acids with a molecular weight of approximately 135 kDa. The N-terminal region contains the Cys, His, and Asp domains, which are highly conserved in all deubiquitinating enzymes. Northern blot analysis revealed that two transcripts were detected in various tissues, with strong expression in brain, lung, thymus, and testis. In vivo and in vitro deubiquitinating enzyme assays demonstrated that mHAUSP has deubiquitinating enzyme activity. The overexpression of mHAUSP reduces the amount of ubiquitinated p53, indicating that it functions as a deubiquitinating enzyme for p53.

3' Untranslated Regions↗

PubMed2004

Structural and functional conservation of the lgl recessive oncogenes (Review).

It is becoming gradually evident that the dysregulated cell proliferation and differentiation in neoplasia are associated with aberrant functions of specific genes in the genome. Genes and their cellular functions involved in cell proliferation and differentiation are highly conserved among different species. Therefore, the illustration of evolutionary relationships among gene products in different species can provide structural and functional insights, leading to neoplastic studies in lower animals that can help to understand the mechanisms of neoplastic development in vertebrates including humans. This review introduces the possibility that studies with lethal giant larvae (lgl) tumor suppressor family members can provide a better understanding of their functional roles during tumor development in eukaryotic cells.

Amino Acid Sequence↗

PubMed2004

A novel cysteine protease HeLa DUB-1 responsible for cleaving the ubiquitin in human ovarian cancer cells.

The regulation of ubiquitin-mediated protein degradation is becoming important for a number of cellular processes. Human HeLa DUB-1 cDNA, encoding a novel deubiquitinating enzyme, was isolated from ovarian cancer cells. It has 1,647 bp nucleotides and encodes a 548 amino acid polypeptide with the molecular weight of approximately 61 kDa. It contains the highly conserved Cys, Asp (I), His, and Asn/Asp (II) domains characteristic of the ubiquitin-specific processing proteases. Biochemical assay revealed that HeLa DUB-1 has deubiquitinating enzyme activity in vivo and in vitro. Northern blot analysis for HeLa DUB-1 showed the strong expression in human skeletal muscle and pancreas and to some extent in heart, placenta, lung, liver, and kidney. Interestingly, the expression was hardly seen in the brain. Localization study indicates that HeLa DUB-1 proteins are present in both the cytoplasm and nucleus.

Amino Acid Sequence↗

PubMed2004

Complementation of temperature tolerance by rat Rgl-1 recessive oncogene in the absence of Saccharomyces cerevisiae Sop genes.

It has been demonstrated that homozygous mutations at the L(2)gl locus in Drosophila result in the development of tumor in the presumptive adult optic centers of the larval brain and of the imaginal discs. We previously cloned an L(2)gl homologue, Rgl-1, in the rat brain. In this study, we analyzed the capability of Rgl-1 in recovering temperature tolerance in the absence of Saccharomyces cerevisiae Sop genes, yeast homologues of the Drosophila recessive oncogene Lethal (2) giant larvae. The expression of Rgl-1 revealed the recovery of temperature tolerance at 20 degrees C in the absence of Sop genes in Saccharomyces cerevisiae. This indicates that the Rgl-1 cDNA we isolated from the rat brain is highly homologous to Lgl family members and can also substitute the function of Sop proteins for temperature tolerance in Saccharomyces cerevisiae.

Actins↗

PubMed2003

DUB-3, a cytokine-inducible deubiquitinating enzyme that blocks proliferation.

Previous studies have identified the DUB family of cytokine-regulated murine deubiquitinating enzymes, which play a role in the control of cell proliferation and survival. Through data base analyses and cloning, we have identified a human cDNA (DUB-3) that shows significant homology to the known murine DUB family members. Northern blotting has shown expression of this gene in a number of tissues including brain, liver, and muscle, with two transcripts being apparent (1.6 and 1.7 kb). In addition, expression was observed in cell lines including those derived from a number of hematopoietic tumors such as the Burkitt's lymphoma cell line RAJI. We have also demonstrated that DUB-3, which was shown to be an active deubiquitinating enzyme, is induced in response to interleukin-4 and interleukin-6 stimulation. Finally, we have demonstrated that constitutive expression of DUB-3 blocks proliferation and can initiate apoptosis in both IL-3-dependent Ba/F3 cells and NIH3T3 fibroblasts. These findings suggest that human DUB-3, like the murine DUB family members, is transiently induced in response to cytokines and can, when constitutively expressed, block growth factor-dependent proliferation.

Amino Acid Sequence↗

PubMed2003

DUB-1A, a novel deubiquitinating enzyme subfamily member, is polyubiquitinated and cytokine-inducible in B-lymphocytes.

Recently, we isolated the Dub-2A gene, which encodes a novel murine deubiquitinating enzyme subfamily member, from a bacterial artificial chromosome library clone by PCR amplification with degenerate PCR primers for the Dub-2 cDNA (Baek, K.-H., Mondoux, M. A., Jaster, R., Fire-Levin E., and D'Andrea, A. D. (2001) Blood 98, 636-642). In this study, we analyzed two more clones from the library to isolate genes encoding other deubiquitinating enzymes. Dub-1A, which encodes the shortest member of the DUB subfamily of deubiquitinating enzymes so far, has been identified in both clones and characterized. Sequence analysis showed that Dub-1A encodes a 468-amino acid protein that has a molecular mass of approximately 51 kDa and that contains a putative catalytic domain (Cys, His, and Asp) conserved among DUB proteins. The amino acid sequence of DUB-1A is 84.5, 84.7, and 85.3% identical to those of DUB-1, DUB-2, and DUB-2A, respectively. Reverse transcription-PCR revealed that Dub-1A is expressed not only in B-lymphocytes in response to interleukin-3 stimulation, but also in T-lymphocytes, brain, heart, liver, lung, kidney, ovary, and spleen. This suggests that Dub-1A may play essential roles in each of these organs. In vivo and in vitro deubiquitinating enzyme assays showed that DUB-1A has functional deubiquitinating activity and that the 5'-flanking sequence of Dub-1A has a functional enhancer domain as shown in Dub-1 and Dub-2A. Interestingly, immunoblot analysis revealed that DUB-1A is polyubiquitinated, indicating that it is degraded through proteasome-mediated degradation. In the absence of JAK2, Dub-1A was expressed at a lower level. This suggests that DUB-1A functions downstream of JAK2 kinase in the interleukin-3 signaling pathway.

Amino Acid Sequence↗

PubMed2003

Conjugation and deconjugation of ubiquitin regulating the destiny of proteins.

The homeostasis for a number of cellular proteins is regulated by not only phosphorylation and dephosphorylation, but also ubiquitination and deubiquitination. A number of proteins involved in the degradation of polypeptides have been isolated in various eukaryotic organisms from Saccharomyces cerevisiae to human. Recently, several deubiquitinating enzymes, classified into either the Ub C-terminal hydrolase (UCH) or the Ub-specific processing protease (UBP), have been reported. It has been shown that they contain conserved domains including Cys, His, and Asp residues throughout the enzyme. These proteins have been demonstrated that Cys and His domains are critical for deubiquitinating enzymatic activity. Recently, we have shown that the Asp domain localized between Cys and His domains is also essential for cleaving the ubiquitin from protein substrates. Mouse deubiquitinating enzymes including DUB-1, DUB-2, and DUB-2A have been isolated and they showed the expression specificity. Of these, DUB- 1 and DUB-2 are expressed in lymphocytes depending on the presence of cytokines (interleukin-3 in B-lymphocytes and interleukin-2 in T- lymphocytes, respectively), indicating that they are involved in cytokine signaling pathways. Isolation of all putative DUBs will help to identify their substrates and to regulate the homeostasis of cellular proteins, especially in proliferative cells.

Amino Acid Sequence↗

PubMed2003

Expression of angiogenesis- and apoptosis-related genes in chorionic villi derived from recurrent pregnancy loss patients.

Angiogenesis takes place during embryogenesis, characterized by the formation of new blood vessels from pre-existing ones. This biological process is also found in the female reproductive system, wound healing, and cancer development. Apoptosis, programmed cell death, is a physiological process in development, tissue homeostasis, and disease. Apoptosis is a normal event in several reproductive tissues including human placenta. In these studies, we investigated whether aberrant angiogenesis and apoptosis are associated with recurrent pregnancy loss (RPL). We compared the gene expression level for angiogenesis- and apoptosis-related genes in chorionic villi from RPL patients and those from normal controls. Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed that 7 angiogenesis- and 12 apoptosis-related genes were abnormally expressed in chorionic villi from RPL patients. Angiogenesis-related genes that showed aberrant expression level are matrix metalloproteinase-2 (MMP-2), plasminogen activator inhibitor (PAI), integrin, transforming growth factor-beta (TGF-beta), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and leptin receptor. Expression levels for these genes, except for leptin receptor, showed less in chorionic villi from RPL patients than those from normal controls. In contrast, higher expression levels of 12 apoptosis-related genes (caspase 3, 6, 7, 8, 9, 10, 12, BAD, BAX, BID, Fas, and FasL) were shown in chorionic villi from RPL patients than those from normal controls. Taken all together, it is likely that the lower expression of angiogenesis-related genes and the excessive expression of apoptosis-related genes are associated with RPL.

Abortion, Habitual↗

PubMed2003

Inhibitory effect of salmosin, a Korean snake venom-derived disintegrin, on the integrin alphav-mediated proliferation of SK-Mel-2 human melanoma cells.

We have investigated the inhibitory effect of salmosin on integrin-mediated human tumour cell proliferation. SK-Mel-2 human melanoma cell adhesion to denatured collagen or vitronectin was found to be significantly and statistically inhibited by salmosin in a dose-dependent manner (P<0.05). Moreover, the binding of SK-Mel-2 cells to salmosin-coated plates was specifically disrupted by anti-integrin alphav monoclonal antibody at 8 microg mL(-1), but not by anti-integrin monoclonal antibody. These findings indicated that salmosin inhibited the adhesion of SK-Mel-2 cells to denatured collagen by specifically blocking integrin alphav. The proliferation of SK-Mel-2 cells on a denatured collagen-coated plate was statistically and significantly inhibited by salmosin induced apoptosis in a dose-dependent manner (P<0.05). Anti-integrin alphav monoclonal antibody, anti-integrin alphavbeta3 monoclonal antibody, and synthetic RGD peptide also suppressed SK-Mel-2 cell proliferation. Several lines of experimental evidence strongly suggested that the inhibition of SK-Mel-2 cell proliferation by salmosin was due to the induction of apoptosis via the blocking of integrin alphav-mediated cell survival.

Apoptosis↗

PubMed2003

WD-40 repeat containing rat lethal giant larvae recessive oncogene, but not m-tomosyn, restores the salt sensitivity in Saccharomyces cerevisiae.

Recently, we cloned rgl-1, a putative rat homologue of the Drosophila tumor suppressor oncogene lethal (2) giant larvae (l(2)gl), from the rat brain. Sequence analysis revealed that rgl-1 encodes a 1,036 amino acid polypeptide containing a domain characteristic of WD-40 repeat sequence. It has been suggested that m-tomosyn is expressed in the rat brain, encodes a 1,116 amino acid polypeptide, contains WD-40 repeat sequence, and is homologous to the Drosophila l(2)gl and mouse mgl-1 genes. The comparison of amino acid sequence for m-tomosyn with l(2)gl and mgl-1 proteins using the clustal method showed only 17.8% and 20% amino acid identity, respectively. However, amino acid identity of rgl-1 protein to l(2)gl and mgl-1 proteins is much higher (30.6% and 96.8%, respectively). Therefore, we investigated their biological function in the absence of Saccharomyces cerevisiae SOP1 and SOP2, the yeast homologues of the Drosophila l(2)gl. Functional analysis showed that the expression of the rgl-1 cDNA, but not m-tomosyn, restored partially salt sensitivity in yeast, indicating that rgl-1 is a homologue of lgl family members.

Amino Acid Sequence↗

PubMed2003

Functional and expression analyses of mgl-1, a mouse orthologue of lethal giant larvae recessive oncogene.

We recently isolated a few mammalian homologue of Drosophila lethal giant larvae (lgl) recessive oncogene, suggesting that there is functional conservation among proteins of this family. The comparison of amino acid sequence for mgl-1 with other lgl family members using the clustal method showed that they are highly homologous. Therefore, we investigated the biological function of mgl-1, a mouse orthologue of lgl, in the absence of Saccharomyces cerevisiae Sop1 and Sop2, the yeast homologues of the lgl recessive oncogene. Functional analysis showed that the expression of mgl-1 cDNA partially restored salt tolerance in yeast, indicating the evolutionary conservation of lgl family members. Since the developmental expression profile of mgl-1 has not been elucidated, the temporal and spatial expression patterns of mouse mgl-1 during early embryonic development were analyzed. The temporal expression analysis revealed that mgl-1 is expressed throughout embryonic development from days E4.5 to E18.5 with the strong expression at E10.5. The analysis of spatial expression showed that mgl-1 mRNA is detected in CNS, craniofacial region, eyes, limbs, and the gut.

Amino Acid Sequence↗

PubMed2002

Lymphocyte-specific murine deubiquitinating enzymes induced by cytokines.

It is becoming clear that a number of proteins regulating cellular mechanisms for homeostasis in all eukaryotes are controlled not only by phosphorylation and dephosphorylation but also by ubiquitination and deubiquitination. This includes most of oncoproteins and signaling components involved in receptor tyrosine kinase (RTK)-mediated signal transduction pathways. Like protein phosphorylation and dephosphorylation regulated by kinases and phosphatases, respectively, protein ubiquitination and deubiquitination are very dynamic and are regulated by ubiquitin conjugating enzymes and deubiquitinating (DUB) enzymes. A number of deubiquitinating enzymes have been isolated even though little is known about their biological functions. This review concentrates on recent findings and new insights into DUB enzyme subfamily members in lymphocytes.

Amino Acid Sequence↗