Biomedical subjects

Jin-Hyun Kim

Publications and source records attributed to Jin-Hyun Kim.

6 recordsLinked to original sources

PubMed2004

Two conserved cysteine residues are critical for the enzymic function of the human platelet-derived growth factor receptor-beta: evidence for different roles of Cys-822 and Cys-940 in the kinase activity.

The platelet-derived growth factor receptor-beta (PDGFR-beta) has a number of conserved cysteine residues on its cytoplasmic domain. We have examined whether the cysteine residues play a role in the enzymic function of PDGFR-beta. We found that N-ethylmaleimide, which selectively alkylates free thiol groups of cysteine residues, completely inhibited the kinase activity of PDGFR-beta. We then identified, through site-directed mutagenesis, two conserved cysteine residues critical for the enzymic function of PDGFR-beta. Cys to Ser mutations for either Cys-822, positioned in the catalytic loop, or Cys-940, located in the C-terminal kinase subdomain, significantly reduced the activities of autophosphorylation and phosphorylation towards exogenous substrates. The non-reducing gel analysis indicated that neither of these cysteine residues contributes to the kinase activity by disulphide-bond formation. In addition, the individual mutation of Cys-822 and Cys-940 had no effect on protein stability or the binding of substrates or ATP, implying that these cysteine residues are involved in enzyme catalysis. Finally, proteolytic cleavage assays showed that the mutation of Cys-940, but not Cys-822, induced a protein conformational change. Taken together, these results suggest that Cys-940 contributes to the catalytic activity of PDGFR-beta by playing a structural role, whereas Cys-822 contributes through a different mechanism.

Adenosine Triphosphate↗

PubMed2004

Manipulation of the active site loops of D-hydantoinase, a (beta/alpha)8-barrel protein, for modulation of the substrate specificity.

We previously proposed that the stereochemistry gate loops (SGLs) constituting the substrate binding pocket of D-hydantoinase, a (beta/alpha)(8)-barrel enzyme, might be major structural determinants of the substrate specificity [Cheon, Y. H., et al. (2002) Biochemistry 41, 9410-9417]. To construct a mutant D-hydantoinase with favorable substrate specificity for the synthesis of commercially important non-natural amino acids, the SGL loops of the enzyme were rationally manipulated on the basis of the structural analysis and sequence alignment of three hydantoinases with distinct substrate specificities. In the SGLs of D-hydantoinase from Bacillus stearothermophilus SD1, mutations of hydrophobic and bulky residues Met 63, Leu 65, Phe 152, and Phe 159, which interact with the exocyclic substituent of the substrate, induced remarkable changes in the substrate specificities. In particular, the substrate specificity of mutant F159A toward aromatic substrate hydroxyphenylhydantoin (HPH) was enhanced by approximately 200-fold compared with that of the wild-type enzyme. Saturation mutagenesis at position 159 revealed that k(cat) for aromatic substrates increased gradually as the size of the amino acid side chain decreased, and this seems to be due to reduced steric hindrance between the bulky exocyclic group of the substrate and the amino acid side chains. When site-directed random mutagenesis of residues 63 and 65 was conducted with the wild type and mutant F159A, the selected enzymes (M63F/L65V and L65F/F159A) exhibited approximately 10-fold higher k(cat) values for HPH than the wild-type counterpart, which is likely to result from reorganization of the active site for efficient turnover. These results indicate that the amino acid residues of SGLs forming the substrate binding pocket are critical for the substrate specificity of D-hydantoinase, and the results also imply that substrate specificities of cyclic amidohydrolase family enzymes can be modulated by rational design of these SGLs.

Amidohydrolases↗

PubMed2004

Downregulation of gelsolin and retinoic acid receptor beta expression in gastric cancer tissues through histone deacetylase 1.

BACKGROUND AND AIM: Overexpression of histone deacetylase (HDAC)1, which controls the expression of genes related to cell cycle and apoptosis, has recently been reported in gastric cancer (GC) tissues. In the present study, the pattern of gelsolin and retinoic acid receptor (RAR)beta expression in GC tissues showing HDAC1 overexpression was investigated. METHODS: Expression profiles of HDAC1, gelsolin, and RARbeta were evaluated and compared using reverse transcription-polymerase chain reaction, immunoblotting, and immunohistochemical analyses with 22 paired primary human GC tissues and corresponding normal tissues. RESULTS: Compared with normal gastric tissue, increased expression of HDAC1 mRNA and protein was detected in 17 (77.3%) of 22 GC tissues, while decreased expressions of gelsolin mRNA and protein were shown in 15 (68.1%) samples. Concomitantly, expressions of RARbeta mRNA and protein were decreased in 16 (72.7%) and 17 (77.3%), respectively. Among 17 GC tissues with increased HDAC1 expression, the expressions of gelsolin and RARbeta were simultaneously decreased in 14 (82.4%) and 15 (88.2%) GC tissues, which indicates a strong inverse correlation between HDAC1 and gelsolin/RARbeta expressions. Correlation between HDAC1 and gelsolin/RARbeta was also confirmed by immunohistochemistry. CONCLUSIONS: Taken together, the results of the present study reveal that silencing of gelsolin and RARbeta occurs in GC tissues probably through HDAC1 overexpression and might play some role in gastric carcinogenesis.

Down-Regulation↗

PubMed2003

Pre-validation study for OECD enhanced test guideline 407 protocol by gavage for 4 weeks using propylthiouracil and tamoxifen.

To develop and pre-validate an enhanced protocol for OECD Test Guideline 407, we performed a 28-day repeated-dose toxicity study using the administration of propylthiouracil (PTU) and tamoxifen (TAM) in Sprague-Dawley (SD) rats. Six male and female SD rats were treated orally with PTU in corn oil at the dose of 0, 0.1, 1, or 10 mg/kg per day and TAM at dose of 0, 5, 30 or 200 microg/kg per day for 4 weeks. In the study using PTU, the body weights were reduced from the third week of the study in 10 mg/kg per day group. In clinical biochemistry, the levels of 3,5,3'-triiodothyronine (T3) and thyroxine (T4, 3,5,3',5'-tetraiodothyrosine) were also significantly decreased in 10 mg/kg per day group. Also, thyroid glands in 10 mg/kg per day group were bigger than those in the control group. In the histopathological examination, diffuse hyperplasia and hypertrophy of thyroid follicular cells were observed in all treatment groups, leading to the reduction of lumen size and papillary enfolding of lining epithelium. In the study using TAM, the body weights were reduced from the first week of the study in 200 microg/kg per day group. Relative testes and epididymes weights were increased and relative right ovary and uterus weights were reduced in 200 microg/kg per day group. Also, in the histopathological finding severe endometrial squamous metaplasia and endometrial gland atrophy and severe follicular cystic change were observed in TAM 200 microg/kg per day-treated group. On the basis of the results, the thyroid hormone levels, gross findings, and histopathological findings may be useful parameters for the detection of the endocrine-related effect of PTU and also reproductive organ weight and histopathological findings may be good parameters to detect the effect of TAM. Therefore, it is concluded that enhanced OECD TG407 might be useful for screening and detecting endocrine disrupters.

Animals↗

PubMed2002

Overexpression of urokinase-type plasminogen activator in human gastric cancer cell line (AGS) induces tumorigenicity in severe combined immunodeficient mice.

The significance of urokinase-type plasminogen activator (uPA) expression in gastric cancer development was tested by using a human uPA cDNA transfection approach and an in vivo severe combined immunodeficient (SCID) mouse model. The AGS gastric cancer cell line, which has urokinase-type plasminogen-activator receptor (uPAR) but lacks uPA, was transfected with a plasmid containing human uPA cDNA and injected into the backs of SCID mice. Compared with the parent AGS cells, uPA protein secretion in AGS-2-, AGS-4-, and AGS-8-transfected cells increased by 26.1-, 34.6-, and 4.8-fold, respectively (P < 0.05). mRNA expression levels of uPA in the AGS-4 clone were much stronger than those in AGS-2 and AGS-8 clones. After the cancer cells (2 x 10(6)) were injected s.c. into the SCID mice, a palpable mass was observed at the injection site at around 140 days post-injection, followed by accelerated growth of the xenograft up to 180 days post-injection only in the high uPA-producing clone (AGS-4). These results suggest that continuous and high production of uPA by tumor cells is one of the important factors reflecting the malignancy of gastric cancer cells.

Animals↗

PubMed2002

Juvenile malignant mesothelioma in a dog.

An 11-month-old male mixed breed dog was euthanized due to two months history of vomiting and anorexia. At necropsy, numerous, multifocal or coalescing, firm, protruding nodules, 5 to 40 mm in diameter were scattered throughout the mesentery and omentum. Histologically and immunohistochemically, the nodules were diagnosed as malignant mesothelioma. Metastasis to the regional mesenteric, mediastinal and tracheobronchial lymph nodes were observed.

Abdominal Neoplasms↗