Biomedical subjects
C H Kim
Publications and source records attributed to C H Kim.
Intracellular and extracellular forms of alkaline pullulanase from an alkaliphilic Bacillus sp. S-1.
Bacillus sp.S-1 alkaline pullulanase (AP) exists in two forms: a precursor form (PUL-Ia, M(r) 180,000) and a processed form (PUL-Ib, M(r) 140,000). PUL-Ia was accumulated intracellularly in large amounts, and PUL-Ib was detected in both the membrane fraction and the fraction trapped between the cytoplasmic membrane and the cell wall. Two forms of AP were purified to homogeneity and their properties were compared with previously purified PUL-E (140 kDa). PUL-Ib showed similar properties, such as the M(r) value, the pI value (5.7), specific activity, substrate specificity, the NH2-terminal amino acid sequence (Phe-Leu-Asn-Met-Ser), and biophysical characters. However, in the case of PUL-Ia, even though the patterns of optimum pH and temperature, substrate specificity, and enzyme inhibition and activation were similar to those for PUL-Ib and PUL-E, the M(r) value and the pI value (5.97) were different. Furthermore, the NH2-terminal amino acid sequence of PUL-Ia was completely blocked, and the stabilities over pH and temperature ranges were decreased. The catalytic activities of PUL-Ia were distinguishable in the Km and Vmax values for various substrates and in the specific activity (71.4 U/mg) for pullulan hydrolysis. PUL-Ib and PUL-E showed 10-fold higher specific activities (744.6 for PUL-E and 736 for PUL-Ib) than PUL-Ia. However, PUL-Ia was immunologically identical to PUL-E and PUL-Ib. Therefore, it was concluded that PUL-Ib and PUL-E are the same form of the enzyme, suggesting that PUL-Ia is initially synthesized and proteolytically processed to the mature form of PUL-E. On the other hand, the translocation of AP required processing of the AP protein and the processing facilitated enzymatic activation and stabilization through a complete conformational change, resulting in an increase in affinity for substrates of PUL-E.
Probenecid reverses multidrug resistance in multidrug resistance-associated protein-overexpressing HL60/AR and H69/AR cells but not in P-glycoprotein-overexpressing HL60/Tax and P388/ADR cells.
PURPOSE: To determine whether probenecid, an inhibitor of organic anion transport, is able to reverse multidrug resistance (MDR) through modulation of the drug transport function of MDR-associated protein (MRP) and P-glycoprotein (P-gP). METHODS: Two MRP-overexpressing cell lines (HL60/AR and H69/AR) and two P-gP-overexpressing cell lines (HL60/Tax and P388/ADR) were cultured with different concentrations of daunorubicin (DNR) or vincristine (VCR) in the presence or absence of various concentrations of probenecid (0.01-10 mM). Drug sensitivity was determined using an MTT assay. DNR accumulation and subcellular distribution were determined by flow cytometry and confocal microscopy respectively. VCR accumulation was determined by scintillation spectrometry. RESULTS: Probenecid, in a concentration-dependent manner, reversed resistance to DNR and VCR in HL60/AR and H69/AR tumor cell lines. This effect of probenecid on MDR was associated with an increased accumulation of DNR and VCR and correction of the altered subcellular distribution of DNR. The concentrations of probenecid that reversed MDR are clinically achievable in vivo. In contrast, probenecid did not reverse MDR in either HL60/Tax or P388/ADR tumor cell lines that overexpress P-gP. CONCLUSION: These results suggest that probenecid is an effective chemosensitizer of MRP-associated MDR tumor cells and is a potential candidate for clinical use to reverse MDR.
Ischemic preconditioning reduces Op6 generation and prevents respiratory impairment in the mitochondria of post-ischemic reperfused heart of rat.
The present study was performed to test whether the ischemic preconditioning could reduce mitochondrial O2.- production and prevent mitochondrial respiratory impairment upon reperfusion of ischemic hearts. The isolated perfused rat hearts were subjected to 30 min of global ischemia and 20 min of reperfusion. Ischemic preconditioning was performed, involving three 5-min periods of ischemia, each followed by a 5-min reperfusion just before a sustained ischemia. Ischemic preconditioning improved the post-ischemic cardiac function and reduced LDH release and malondialdehyde production upon reperfusion. 02.- generation of mitochondria isolated from the preconditioned hearts was significantly lower than that of mitochondria from the non-preconditioned hearts, and none of the activities of mitochondrial antioxidant enzymes (SOD, catalase, glutathione peroxidase) was altered as a consequence of the ischemic preconditioning alone. The impairment of mitochondrial state 3 respiration induced by ischemia and reperfusion was prevented by ischemic preconditioning. Amytal, a reversible respiratory chain blocker suppressing 02.- production in mitochondria, prevented the ischemia/reperfusion injury. The cardioprotective effect of Amytal could not be distinguished from that of ischemic preconditioning. These results suggest that the cardioprotective effect of ischemic preconditioning against the ischemia/reperfusion injury is attributed partly to the reduction of mitochondrial oxygen radical generation and prevention of the respiratory impairment during ischemia and reperfusion.
Past and current obesity in Koreans with non-insulin-dependent diabetes mellitus.
To determine the prevalence of past and current obesity among patients with non-insulin-dependent diabetes mellitus (NIDDM) and to define the clinical characteristics of non-obese NIDDM patients in South Korea, we studied a cross-section of 749 NIDDM patients and a group of age- and sex-matched control subjects. Current height, weight and waist-to-hip ratio (WHR), the history of weight changes and the family history of diabetes were recorded. Obesity was defined as body mass index (BMI) > 25 kg/m2. The maximum lifetime BMI of diabetic patients was significantly higher than that of control subjects (P < 0.001). Compared with control subjects, current BMI was higher in diabetic women (P < 0.001) but not in diabetic men. In contrast, WHR of both diabetic men and women were significantly higher than those of controls (P < 0.05). BMI and WHR correlated significantly with fasting C-peptide levels and log-triglyceride levels in NIDDM patients. As a whole, 72% of the South Korean NIDDM patients had a history of past obesity as assessed by their maximum weight, while only 38% of them were currently obese. Compared with obese patients, non-obese patients were characterized by lower fasting serum C-peptide levels (P < 0.001), a higher percentage of insulin treatment (P < 0.05), lower maximum BMI (P < 0.001) and more pronounced weight loss from the time at their maximum weight (P < 0.001). In summary, increased upper body adiposity and a history of past obesity were associated with NIDDM in South Korea. Although most South Korean NIDDM patients were previously obese, many of them were currently not obese. Lower maximum BMI, lower serum C-peptide levels and a higher percentage of insulin treatment in non-obese NIDDM patients suggest that the capacity to increase insulin secretion in response to increasing weight gain is rather limited in these patients.
Cloning and expression of the quaking gene in the zebrafish embryo.
The responsible gene for hypomyelinating quaking deficiency, qkI, encoding a KH RNA binding protein, is expressed abundantly in the developing mouse nervous system, whereas who/how/struthio, a homologue of the qkI in Drosophila, is expressed predominantly in the mesoderm. Here we describe the isolation and early developmental expression of a zebrafish homologue of qkI. The zebrafish quaking cDNA, zqk, exhibits striking conservation with qkI across the coding region, accompanied by a unique 123 nucleotide insertion sequence. Maternal and zygotic zqk transcripts are ubiquitously distributed during cleavage and blastula periods, and then accumulate in the dorsal midline of the body trunk during gastrulation. During segmentation and pharyngula periods zqk transcripts are expressed in the neural tissue of the head region, and in the paraxial mesoderm of the body trunk. Subsequently they diminish until the hatching period, when they are expressed only in the cardiac sac and pectoral finbuds. We also found that the zqk transcript is alternatively spliced with the transcript containing a 123 nucleotide additional segment localized in neural tissue in the head region, but not in the paraxial mesoderm in the body trunk. The data suggest that the quaking gene family originated in the mesoderm and evolved to become expressed in the nervous system in lower vertebrates. The insertion of the 123 nucleotide sequence could be related to the acquisition of a neural function for the gene.
Efficient selection of preimplantation transgenic embryos by an improved procedure using Dpn I-Bal 31 digestion and the polymerase chain reaction.
Efficient selection of preimplantation transgenic embryos by an improved method after pronuclear injection of exogenous DNA is described. The method is based on subjecting DNA extracted from the embryos to restriction enzymes as well as the polymerase chain reaction (PCR). The incorporated procedure included recovery of the digested DNA with glassmilk before PCR, which markedly enhanced the rate of accurate detection of transgenic embryos. When exogenous DNA sequences in the mouse embryos were not integrated into the genome they were digested with both Dpn I and Bal 31, and subsequent PCR analysis generated DNA fragments of the injected DNA sequence in only 1.5% of cases examined. However, DNA extracted from mouse embryos containing the transgene sequences integrated into the genome evaded digestion by both enzymes and yielded transgene-specific PCR products in 68.6% of the embryos tested. When bovine embryos were used, sequences of the endogenous haemoglobin gene used as a control genomic DNA sequence were protected from enzyme digestion (PCR products in 70.5% of the embryos examined); by contrast, the non-integrated injected sequences were almost completely eliminated by the same treatment (PCR products in 1.4% of the embryos examined). It is suggested that this method might be useful for the selection of transgenic embryos before embryo transfer, thereby reducing the number of recipient females required.
Effect of population density on the early post-implantation mouse embryo growth in vitro.
OBJECTIVES: To investigate the relationship between embryo population density and embryo development in vitro. METHODS: Swiss Webster albino mice were used and blastocysts were flushed from the uterine horns. Various numbers of blastocysts were randomly assigned to separate 35 mm dishes and cultured for 9 days. RESULTS: The effect of duration of culture to the mean success rates was different as the number of embryos in the dish was changed. In early egg cylinder (EEC) stage, the mean success rate was a flat function of the number of embryos. But the mean success rate was a decreasing function in late egg cylinder (LEC) stage and the decreasing rate was faster in early somite (ES) stage. Interestingly, the mean success rate to ES stage was nearly constant when more than 10 embryos were cultured in one dish. CONCLUSION: The success rate to each developmental stage in vitro decreased as the number of embryos in the dish and the duration of cultured increased.
The immunotherapy during in vitro fertilization and embryo transfer cycles in infertile patients with endometriosis.
OBJECTIVE: To investigate if the immunotherapy with corticosteroids would improve the pregnancy rate in infertile patients with endometriosis who undergo in vitro fertilization and embryo transfer (IVF-ET). METHODS: Forty-two infertile patients with endometriosis plus tubal factor and 87 pure tubal infertility patients who underwent IVF-ET in our unit were allocated randomly to the corticosteroid treatment group and the control group. RESULTS: The prevalence of autoantibodies (antinuclear antibody, lupus anticoagulant, anticardiolipin antibody, rheumatoid factor) was elevated significantly in patients with endometriosis plus tubal factor compared with pure tubal infertility patients (38.1% vs 2.3%). Twenty-one patients with endometriosis plus tubal factor underwent 54 cycles of IVF-ET, receiving corticosteroids. Forty-three patients with pure tubal factor underwent 81 cycles of IVF-ET, receiving corticosteroids. Twenty-one patients with endometriosis plus tubal factor who underwent 57 cycles of IVF-ET and 44 patients with pure tubal factor who underwent 84 cycles of IVF-ET served as controls, not receiving corticosteroids. In patients with endometriosis plus tubal factor, there was a significantly higher clinical pregnancy rate per cycle in the treatment group, with 42.6% (23/54) compared with 22.8% (13/57) in the control group but no differences between 2 groups in spontaneous abortion rate (21.7% vs 15.4%) and multiple pregnancy rate (17.4% vs 15.4%). In patients with pure tubal infertility, there were no significant differences between the treatment group and control group in clinical pregnancy rate (40.7% vs 34.5%), spontaneous abortion rate (12.1% vs 10.3%) or multiple pregnancy rate (18.2% vs 10.3%). In the endometriosis plus tubal infertility group with autoantibodies, the clinical pregnancy rate per cycle was significantly higher in the treatment group at 40.9% compared with 14.8% in the control group. In endometriosis plus tubal infertility group without autoantibodies, there was no significant difference between 2 groups with respect to the clinical pregnancy rate per cycle (43.8% vs 30.0%). CONCLUSIONS: This study suggests that immunotherapy with corticosteroids could improve the clinical pregnancy rate in endometriosis patients undergoing IVF-ET and may be more effective in patients with positive autoantibodies.
Identification of apo(a) phenotypes in a Korean population using a standardized nomenclature system based on the number of kringle IV repeats.
We determined serum lipoprotein(a) [Lp(a)] concentrations and apolipoprotein(a) [apo(a)] phenotypes in 193 healthy Koreans. We analysed the apo(a) phenotypes by a simplified sodium dodecyl sulphate-polyacrylamide gel electrophoresis method and classified apo(a) isoforms objectively using an apo(a) phenotype standard with a known number of kringle IV repeats. The frequency distribution of Lp(a) levels showed marked positive skew with a mean of 0.143 g/L and a median of 0.052 g/L. Of the 193 subjects tested, no bands were detected in three, and single- and double-band phenotypes were observed in 103 and 87, respectively. Among the Koreans, the most frequent phenotype was S5(39.4%), followed by S4S5(17.1%), S5S5(14.0%), S4(11.4%), S3S5(5.2%), and the remaining phenotypes (13.0%). The calculated apo(a) allele frequencies were LpF, 0.003; LpS1, 0.013; LpS2, 0.010; LpS3, 0.048; LpS4, 0.198; LpS5, 0.563 and Lp0, 0.165. We found that the serum Lp(a) concentration in Koreans was similar to that of Caucasians, but the apo(a) allele size distribution was shifted toward higher molecular weights.
Involvement of different second messengers in parathyroid hormone- and interleukin-1-induced interleukin-6 and interleukin-11 production in human bone marrow stromal cells.
Previous studies have suggested that increased secretion of bone active cytokines, such as interleukin-6 (IL-6) and interleukin-11 (IL-11), from osteoblasts and stromal cells play a pivotal role in the activation of osteoclasts and the genesis of osteoporosis. Various systemic and local factors can stimulate IL-6/IL-11 production, but the intracellular mechanism for such stimulation is largely unknown. In this study, we characterized the second messenger signaling in parathyroid hormone (PTH)- and IL-1-induced production of IL-6/IL-11 and studied the possible modulating effects of estrogen. rhPTH(1-34) and rhIL-1 alpha dose-dependently stimulated IL-6 and IL-11 production from human bone marrow stromal cells (hBMSCs). Agonists for protein kinase A (PKA) (forskolin), and protein kinase C (PKC) (phorbol 12-myristate 13-acetate; PMA) also stimulated IL-6/IL-11 production. Rp-diastereoisomer of adenosine cyclic 3',5'-phosphorothioate (Rp-cAMPS) and H-8, inhibitors of PKA, significantly inhibited PTH-stimulated IL-6/IL-11 production, but did not inhibit IL-1-stimulated IL-6/IL-11 production. In contrast, staurosporine and calphostin C, inhibitors of PKC, suppressed IL-1-stimulated, but not PTH-stimulated, IL-6/ IL-11 production. Pretreatment of cells with 17 beta-estradiol (17 beta-E2) antagonized IL-1-stimulated IL-6 production. However, PTH-stimulated IL-6 production and IL-1- and PTH-stimulated IL-11 production were not affected by 17 beta-E2. Similarly, 17 beta-E2 inhibited PMA-stimulated IL-6 production, whereas neither forskolin-stimulated IL-6/ IL-11 production nor PMA-stimulated IL-11 production was affected by 17 beta-E2. These results indicate that different second messengers are involved in PTH- and IL-1-induced IL-6 and IL-11 production by hBMSCs: PTH and IL-1 stimulate IL-6/IL-11 production via a PKA-dependent and PKC-dependent pathway, respectively. Furthermore, our results suggest that regulation of cytokine production by estrogen in hBMSCs is selective; only the IL-1-induced IL-6 production, which is mediated by PKC pathway, is inhibited, but PTH-induced IL-6 production and PTH/IL-1-induced IL-11 production are not inhibited by estrogen.
Ileocolonoscopic and histologic studies of Korean patients with ankylosing spondylitis.
We investigated the frequency of gut inflammation and the role of gut lesion in the pathogenesis of the ankylosing spondylitis (AS) in Korean patients. Ileocolonoscopy and biopsy of the colon and terminal ileum were performed on 24 Korean patients with AS. Endoscopic lesions were observed in 7 patients (29.2%). The lesions were found more often in the terminal ileum (6/7) than in the colon (1/7). Histologic signs of gut inflammation were detected in 14 patients (58.3%), acute lesions in 2 patients (8.3%) and chronic lesions in 12 patients (50%). Gut inflammation were as frequently found in Korean patients with AS as in Western patients. These findings suggest that gut inflammation may play a role in the pathogenesis of AS in Korean patients as it does in Western patients.
Lack of autocrine effects of IL-6 on human bone marrow stromal osteoprogenitor cells.
Interleukin-6 (IL-6) produced by osteoblastic cells plays an important role in the regulation of bone remodeling, mainly by stimulating osteoclast action. Although the IL-6 receptor is also found in osteoblastic cells, whether IL-6 exerts autocrine effects on osteoblastic cells is a matter of debate. This led us to study the effects of IL-6 on proliferation, osteoblastic activity as well as mRNA expression of various osteoblastic proteins in human bone marrow stromal osteoprogenitor cells (hBMSC). IL-6 did not affect cell proliferation assessed by [3H]-thymidine incorporation and osteoblastic activity determined by alkaline phosphatase activity and 45Ca incorporation. The expression of mRNAs for alkaline phosphatase, alpha 1 (I)-collagen, osteopontin and decorin also did not change significantly by IL-6 treatment. These results show that IL-6 does not have a significant autocrine role in regard to proliferation and osteoblastic activity of hBMSC.
Basal cell adenocarcinoma of the salivary gland--a case report.
Basal cell adenocarcinoma of the salivary gland is a very rare disease entity as the malignant counterpart of basal cell adenoma. On the basis of morphologic pattern, basal cell adenoma can be divided into four subtypes; trabecular, solid, tubular, and dermal analogue (membranous). We report a case of basal cell adenocarcinoma in the left parotid gland of a 33-year-old woman. Light microscopically, the tumor cells were composed of relatively uniform, monotonous basaloid cells. The tumor cell nests commonly had peripheral palisadings. The main growth pattern of the tumor cells was tubulotrabecular. However, other portions were similar to dermal analogue monomorphic adenoma and showed cribriform patterns reminiscent of adenoid cystic carcinoma. The tumor had frequent perineural invasions and an infiltrative margin. On immunohistochemistry, only scattered numbers of tumor cells showed irregular positive reaction for S-100 protein, suggesting a few myoepithelial cell components of the tumor compared with most tumor cells derived from epithelial cells.
Viral loads and E2/NS1 region sequences of hepatitis C virus in hepatocellular carcinoma and surrounding liver.
OBJECTIVES: Numerous epidemiologic data have documented that chronic infection by hepatitis C virus (HCV) is a major risk factor for the development of hepatocellular carcinoma (HCC). But the molecular mechanism underlying these strong epidemiologic associations between HCV and HCC has not be elucidated. We observed the changes of HCV in HCC to investigate the association of HCV with HCC. METHODS: We used competitive and quantitative polymerase chain reaction and dideoxy-nucleotide chain termination method to compare HCV titers and sequences of the hypervariable region of E2/NS1 region of four isolates from the HCC and surrounding cirrhotic liver tissues in tow anti-HCV positive patients. RESULTS: The copy numbers of HCV-RNA were 1 x 10(6) and 4 x 10(6)/gm wet weight of HCC, and 8 x 10(7) and 3.2 x 10(8)/gm wet weight of cirrhotic liver tissues from patient-1 and -2. The sequence differences between HCV RNA in HCC and in cirrhotic liver were two and five nucleotides in patient-1 and in patient-2 respectively. The amino acid sequences were changed in one and two site in each patient. CONCLUSION: These findings may suggest the possible etiological role of HCV in carcinogenesis of HCC, but complete sequence analysis of HCV including multiple isolates in the same patient, should be performed in many cases.
Reproductive toxicity of 2-bromopropane in Sprague Dawley rats.
OBJECTIVES: In an effort to clarify the mass intoxication of workers at an electronic company in Korea, the possible causative chemical for reproductive toxicity, 2-bromopropane (2BP), was investigated. METHODS: 2BP was tested through the use of repeated dose experiments among male Sprague Dawley rats. Ten rats were assigned to each treatment group. Vehicle control olive oil and 2BP concentrations of 125 mg/kg, 250 mg/kg, and 500 mg/kg were injected into the intraperitoneum on 28 consecutive days. RESULTS: The rats showed significant decreases in body weight depending on the 2BP dose. The right and left testes showed typical weight loss depending on the dose of 2BP. The red blood cells, hemoglobin, and hematocrit showed some degree of decline with the high dose. The amount of hemoglobin, the mean platelet volume, the number of white blood cells, and the number of lymphocytes decreased significantly with the high dose, while the number of granulocytes and monocytes had a tendency to decrease depending on the dose of 2BP. The histopathology of the testes treated with the middle and high 2BP dose showed a typical patch appearance with severely depleted atrophic tubules, exhibiting germ cell necrosis of spermatogonia and spermatocytes in the seminiferous tubules. Leydig cell hyperplasia or hypertrophy in the interstitial tissue was also noticeable. The epididymis showed some degree of atrophy with vacuolization of the epididymal epithelium. CONCLUSIONS: The testes are the main target organs tested for 2BP toxicity. 2BP also affect the hematopoietic system and thus induces leukopenia and normocytic anemia. Besides the reproductive organs and the hematopoietic system, no significant toxicity has been found.
Molecular cloning and expression of human Gal beta 1,3GalNAc alpha 2,3-sialytransferase (hST3Gal II).
A cDNA of human Gal beta 1,3GalNAc alpha 2,3-sialytransferase (hST3Gal II) which has been known to exhibit much more acceptor substrate preference for glycolipid than for O-linked oligosaccharides of glycoproteins, was isolated from the human liver cDNA library by plaque hybridization using the cDNA of mouse ST3Gal II (mST3Gal II) cloned previously as a probe. Comparative analysis of this cDNA with mST3Gal II indicates 89 and 94% homologies in the nucleotide and amino acid levels, respectively, between the two sequences in the predicted coding region. Northern analysis indicated that the expression of hST3Gal II mRNA is tissue-specific, it being prominent in skeletal muscle and heart, while that in lung and kidney is very low. This enzyme expressed in COS cells showed a similar activity with that of mST3Gal II.
Purification and characterization of a thermostable ADP-glucose pyrophosphorylase from Thermus caldophilus GK-24.
An extremely thermostable ADP-glucose pyrophosphorylase (AGPase) has been purified from Thermus caldophilus GK-24 to homogeneity by chromatographic methods, including gel filtration and ion-exchange and affinity chromatography. The specific activity of the enzyme was enriched 134.8-fold with a recovery of 10.5%. The purified enzyme was a single band by SDS/PAGE with a molecular mass of 52 kDa. The homotetrameric structure of the native enzyme was determined by gel filtration analysis, which showed a molecular mass of 230 kDa on a Superose-12 column, indicating that the structure of the enzyme is different from the heterotetrameric structures of higher-plant AGPases. The enzyme was most active at pH 6.0. The activity was maximal at 73-78 degrees C and its half-life was 30 min at 95 degrees C. Kinetic and regulatory properties were characterized. It was found that AGPase activity could be stimulated by a number of glycolytic intermediates. Fructose 6-phosphate, fructose 1,6-bisphosphate, phenylglyoxal and glucose 6-phosphate were effective activators, of which fructose 1,6-bisphosphate was the most effective. The enzyme was inhibited by phosphate, AMP or ADP. ATP and glucose 1-phosphate gave hyperbolic-shaped rate-concentration curves in the presence or absence of activator. A remarkable aspect of the amino acid composition was the existence of the hydrophobic and Ala+Gly residues. The N-terminal and internal peptide sequences were determined and compared with known sequences of various sources. It was apparently similar to those of AGPases from other bacterial and plant sources, suggesting that the enzymes are structurally related.