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Biomedical subjects

E H Park

Publications and source records attributed to E H Park.

At least 19 recordsLinked to original sources
PubMed2005

Gallbladder Na+/H+ exchange activity is up-regulated prior to cholesterol crystal formation.

BACKGROUND: Gallbladder Na+ and H2O absorption are increased prior to gallstone formation and may promote cholesterol nucleation. Na+/H+ exchange (NHE) isoforms NHE2 and NHE3 are involved in gallbladder Na+ transport in prairie dogs. We examined whether increased gallbladder Na+ absorption observed during early gallstone formation is the result of NHE up-regulation. MATERIALS AND METHODS: Native gallbladder and primary cultures of gallbladder epithelial cells (GBECs) harvested from prairie dogs fed nonlithogenic (CON) or 1.2% cholesterol diet for varying lengths of time to induce cholesterol-saturated bile (PreCRYS), cholesterol crystals (CRYS), or gallstones (GS) were used. NHE activity was assessed by measuring dimethylamiloride-inhibitable 22Na+ uptake under H+ gradient in primary GBECs. HOE-694 was used to determine NHE2 and NHE3 contributions. NHE protein and mRNA expression were examined by Western and Northern blots, respectively. RESULTS: Gallbladder total NHE activity was 25.1 +/- 1.3 nmol mg protein(-1) min(-1) in the control and increased during gallstone formation peaking at the PreCRYS stage (98.4 +/- 3.9 nmol mg protein(-1) min(-1)). There was a shift in NHE activity from NHE2 to NHE3 as the animals progressed from no stones through the PreCRYS and CRYS stages to gallstones. The increase in NHE activity was partly caused by an increased Vmax without any change in K(Na)m. Both NHE2 and NHE3 protein increased moderately during the PreCRYS stage without increases in mRNA expression. CONCLUSIONS: Increased gallbladder Na+ absorption observed prior to crystal formation is in part caused by an increase NHE activity which is not fully accounted for by an increase in NHE proteins and mRNA levels but may be explained by enhanced localization in the membranes and/or altered regulation of NHE.

Absorption↗
PubMed2004

Functional characterization of Na(+)/H(+) exchangers in primary cultures of prairie dog gallbladder.

Gallbladder Na(+) absorption is linked to gallstone formation in prairie dogs. We previously reported Na(+)/H(+) exchanger (NHE1-3) expression in native gallbladder tissues. Here we report the functional characterization of NHE1, NHE2 and NHE3 in primary cultures of prairie dog gallbladder epithelial cells (GBECs). Immunohistochemical studies showed that GBECs grown to confluency are homogeneous epithelial cells of gastrointestinal origin. Electron microscopic analysis of GBECs demonstrated that the cells form polarized monolayers characterized by tight junctions and apical microvilli. GBECs grown on Snapwells exhibited polarity and developed transepithelial short-circuit current, I(sc), (11.6 +/- 0.5 microA. cm(-2)), potential differences, V(t) (2.1 +/- 0.2 mV), and resistance, R(t) (169 +/- 12 omega. cm(2)). NHE activity in GBECs assessed by measuring dimethylamiloride-inhibitable (22)Na(+) uptake under a H(+) gradient was the same whether grown on permeable Snapwells or plastic wells. The basal rate of (22)Na(+) uptake was 21.4 +/- 1.3 nmol x mg prot(-1) x min(-1), of which 9.5 +/- 0.7 (approximately 45%) was mediated through apically-restricted NHE. Selective inhibition with HOE-694 revealed that NHE1, NHE2 and NHE3 accounted for approximately 6%, approximately 66% and approximately 28% of GBECs' total NHE activity, respectively. GBECs exhibited saturable NHE kinetics ( V(max) 9.2 +/- 0.3 nmol x mg prot(-1) x min(-1); K(m) 11.4 +/- 1.4 m M Na(+)). Expression of NHE1, NHE2 and NHE3 mRNAs was confirmed by RT-PCR analysis. These results demonstrate that the primary cultures of GBECs exhibit Na(+) transport characteristics similar to native gallbladder tissues, suggesting that these cells can be used as a tool for studying the mechanisms of gallbladder ion transport both under physiologic conditions and during gallstone formation.

Animals↗
PubMed2001

The complete DNA sequence of the mitochondrial genome of the self-fertilizing fish Rivulus marmoratus (Cyprinodontiformes, Rivulidae) and the first description of duplication of a control region in fish.

We isolated Rivulus marmoratus mitochondrial DNA by long-polymerase chain reaction with conserved primers, and sequenced it with 36 sets of internal conserved primers, which were designed from the extensive sequence similarities of mitochondrial DNA from several fish species. The R. marmoratus mitochondrial DNA has 17,329 bp with a conserved structural organization compared to those of other fish. Rivulus marmoratus mitochondrial DNA also has two nearly identical control regions. The basic characteristics of the R. marmoratus mitochondrial genome are discussed.

Animals↗
PubMed2001

Characterization and regulation of glutathione S-transferase gene from Schizosaccharomyces pombe.

A glutathione S-transferase (GST) gene has been cloned from Schizosaccharomyces pombe for the first time. The nucleotide sequence determined was found to contain 2030 base pairs including an open reading frame of 229 amino acids that would encode a protein of a molecular mass of 27017 Da. The cloned GST gene was expressed and was found to function in S. pombe, Saccharomyces cerevisiae, and Escherichia coli. The plasmid pGT207 encoding the S. pombe GST gene appeared to be able to accelerate the growth of a wild type S. pombe culture. In a culture of S. pombe containing plasmid pGT207, the growth was inhibited less by mercuric chloride than in a culture with vector alone. The 1088 bp region upstream from the GST gene as well as the region encoding the N-terminal 14 amino acids was transferred into the promoterless beta-galactosidase gene of plasmid YEp357R to yield the fusion plasmid pYSH2000. beta-Galactosidase synthesis was induced by cadmium chloride, mercuric chloride, hydrogen peroxide, and menadione. It was also induced by high temperature. These results suggest that the cloned S. pombe GST gene is involved in the oxidative stress response.

Amino Acid Sequence↗
PubMed2001

Genomic cloning of the Hsc71 gene in the hermaphroditic teleost Rivulus marmoratus and analysis of its expression in skeletal muscle: identification of a novel muscle-preferred regulatory element.

To further our understanding of the role of stress proteins in development as well as in adaptation of fish to adverse environmental conditions, we undertook molecular analyses of stress protein encoding genes from the hermaphroditic teleost Rivulus marmoratus. We isolated a genomic clone containing the Hsc71 gene (rm-hsc71m) and its upstream sequences. rm-Hsc71m is not induced by external stress, but is enriched in a tissue-specific manner during early development. In adult, the strongest expression appeared in skeletal muscle, whereas lower expression was seen in the gill, eye and brain. To understand the regulatory basis of high muscle expression of rm-hsc71m, transfection of R.marmoratus muscle tissue was performed using 5' deletion fragments containing the rm-hsc71m promoter driving EGFP expression. An upstream region from -2.7 to -1.9 kb was identified as a muscle-specific regulatory region. Within this region, we identified at least three sites with the novel sequence TGTnACA interacting with a fish muscle factor having an M(r) of 32 000. Our data indicate that rm-hsc71m expression in skeletal muscle is controlled by a muscle-specific regulatory element containing this novel motif.

Animals↗
PubMed2001

Wound healing activity of Opuntia ficus-indica.

The methanolic extract of Opuntia ficus-indica stems and its hexane, ethyl acetate, n-butanol and aqueous fractions were evaluated for their wound healing activity in rats. The extract and less polar fractions showed significant effects.

Animals↗
PubMed2001

An anti-inflammatory principle from cactus.

In previous studies, the ethanol extract of cactus (Opuntia ficus-indica) showed potent anti-inflammatory action. In the present study, following fractionation of the methanol extract of cactus stems guided by adjuvant-induced chronic inflammation model in mice, an active anti-inflammatory principle has been isolated and identified as beta-sitosterol.

Animals↗
PubMed2000

Cloning, expression and regulation of Schizosaccharomyces pombe gene encoding thioltransferase.

The genomic DNA encoding thioltransferase was isolated from Schizosaccharomyces pombe using the polymerase chain reaction. The amplified DNA fragment was confirmed by Southern hybridization, completely digested with HindIII and BamHI, and then ligated into the yeast-Escherichia coli shuttle vector pRS316, which resulted in plasmid pEH1. The insert of plasmid pEH1 was transferred into the multi-copy vector YEp357 to generate plasmid pYEH1. The determined nucleotide sequence harbors an open reading frame consisting of four exons and three introns, which encodes a polypeptide of 101 amino acids with a molecular mass of 11261 Da. Thioltransferase activity was increased 1.6-fold in Saccharomyces cerevisiae containing plasmid pYEH1, and 1.8- and 2.7-fold in S. pombe containing plasmid pEH1 and pYEH1, respectively. The upstream sequence and the region encoding the N-terminal six amino acids were fused into promoterless beta-galactosidase gene of the shuttle vector YEp357R to generate the fusion plasmid pYEHR1. Synthesis of beta-galactosidase from the fusion plasmid was found to be enhanced by zinc and NO-generating S-nitroso-N-acetylpenicillamine.

Amino Acid Sequence↗
PubMed2000

Expression of glucose oxidase by using recombinant yeast.

The glucose oxidase gene (GO) of Aspergillus niger was cloned into the yeast shuttle vector YEp352 with combinations of various promoters and terminators, and then used to transform Saccharomyces cerevisiae. Expressed GO was successfully secreted into culture medium due to the presence of the intrinsic signal peptide of GO. Four different promoters fused to GO were tested: bidirectional galactose dehydrogenase 1 and 10 (GAL1, GAL10) promoters, glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter and an yeast hybrid ADH2-GPD promoter consisting of alcohol dehydrogenase II (ADH2) and GPD promoter. The intrinsic terminator of GO as well as the GAL7 terminator were also compared for better production of GO. Deletion of most of the terminating region from GO yielded only a slight amount of GO while the presence of either terminator greatly increased GO production. The GAL10 promoter produced the least amount of GO, GAL1 and GPD promoters were moderate, and the ADH2-GPD hybrid promoter was the best among all tested. However, the hybrid promoter was tightly regulated by the presence of an excess amount of either glucose or ethanol, and it appeared that 2% glucose and 1. 5% ethanol supplement was the best concentration for GO production. It was possible to produce 260 IU ml(-1) of GO, an equivalent of 5 g l(-1), under the presence of 2% glucose and 1.5% ethanol. UV mutagenesis of a recombinant S. cerevisiae was also applied and it further increased the yield of GO to 460 IU ml(-1) under the presence of 2% glucose and 1.5% ethanol without any changes in cell growth. Corn steep liquor which is commonly used in bioindustry is a good alternative substrate for high priced glucose for the hybrid promoter and suggests a cost effective means for commercial mass production of GO using recombinant yeast.

Biotechnology↗
PubMed2000

Nmethyl-N-nitrosourea (MNU) induces papillary thyroid tumours which lack ras gene mutations in the hermaphroditic fish Rivulus marmoratus.

To investigate the patterns of alkylating agent-induced tumour formation, 40 fish (Rivulus marmoratus) were exposed to N-methyl-N-nitrosourea (MNU) at 50 ppm in 10 mM Hepes-buffered synthetic seawater for 2 h. Tumour incidence 4 months after exposure was approximately 95%, and mainly papillary thyroid tumours were induced. For elucidating the molecular event in Rivulus papillary thyroid carcinogenesis, we first analysed for ras gene mutations based on the known ability of MNU to induce point mutations. The amplified R. Marmoratus Ha-and Ki-ras gene exon 1 and 2 regions were checked along with confirming the presence and expression patterns of the DNA repair gene O(6)-methylguanine alkyltransferase (O(6)-MT) and other oncogenes (c-src, c-fos, and c-myc). Ha- and Ki-ras genes from 38 tumour samples were tested for point mutations with direct sequencing but were not found to contain mutations. In this report, based on the lack of Ha- and Ki-ras gene mutations in papillary thyroid tumour induction in R. Marmoratus, we suggest that Ha- and Ki-ras gene-independent pathways such as ret/PTC rearrangements and other activations are involved in Rivulus papillary thyroid carcinogenesis. Teratogenesis Carcinog. Mutagen. 20:1-9, 2000.

Alkylating Agents↗
PubMed2000

Identification of metabolites of phytosterols in rat feces using GC/MS.

Beta-sitosterol, campesterol and stigmasterol have been known to the phytosterols the most frequently found in plants. Metabolism of phytosterols was investigated using rat feces and liver microsomes. Feces were collected after phytosterols (a well characterized mixture of beta-sitosterol 40%, campesterol 30% and dihydrobrasicasterol) were administered orally (0.5 g/kg) to rats. Metabolites of phytosterols were identified using GC/MS. Three peaks were eluted at 12.47, 12.65, 12.87 min and had characteristic molecular ions m/z 428, 430, 432, respectively. Three fecal metabolites were identified as androstadienedione, androstenedione, and androstanedione. No metabolites could be detected in the rat liver microsomal reaction mixture. The results suggest that the metabolites of phytosterols in rat feces are formed by oxidation at 3- position, saturation at 5- and 6- position, and 17- side chain cleavage in the rat large intestine.

Animals↗
PubMed2000

Synaptic concentration of dopamine in rat striatal slices in relationship to [3H]raclopride binding to the dopamine D2 receptor.

The in vivo binding of dopamine (DA) radioligands to D2 receptors can be affected by competition with endogenous dopamine. In the present study, we used a brain slice preparation that provides more controlled conditions than in vivo preparations in order to examine the relationship between synaptic DA and the binding of [3H]raclopride to D2 receptors. We also estimated the synaptic DA concentration in rat striatal slices by determining the changes in [3H]raclopride binding. To correlate the changes in [3H]raclopride binding with the concentration of synaptic DA, the kinetic parameters were determined. [3H]Raclopride reached equilibrium binding conditions within two hours. The Ki value for DA in inhibiting [3H]raclopride binding was about 2.2 nM. The increase in synaptic DA evoked by electrical stimulation decreased the striatal binding of [3H]raclopride in a frequency-dependent manner. Increases in the DA concentration evoked by amphetamine (AMPH) or cocaine decreased [3H]raclopride binding by 74% or 20%, respectively, corresponding to increases in the synaptic DA concentrations of 1.6 nM or 0.6 nM, respectively. Pargyline also decreased [3H]raclopride binding by 36% corresponding at a concentration of 1.2 nM. In contrast, the depletion of synaptic DA by alpha-methyl-p-tyrosine (alpha-MpT) increased the specific binding of [3H]raclopride by 43% when the DA concentration was decreased to 0.7 nM. The changes in the DA concentration at the synapse were responsible for the changes in the striatal binding of [3H]raclopride. The values calculated in this study may therefore approximate the changes in the synaptic DA concentration in rat striatal slices following manipulation.

Animals↗
PubMed2000

Effect of amphetamine-induced dopamine release on radiotracer binding to D1 and D2 receptors in rat brain striatal slices.

The in vivo binding of positron emission tomography (PET) and single photon emission computer tomography (SPECT) radiotracers to dopamine D2 receptors in the striatum can be influenced by competition with endogenous dopamine. The present study was undertaken to determine if a similar inhibition of radiotracer binding to dopamine receptors could be observed following pharmacologically-evoked dopamine release in rat brain striatal slices. Striatal slices were incubated in a large volume of oxygenated Krebs saline and exposed to amphetamine or methamphetamine to evoke dopamine release within the slice. Amphetamine and methamphetamine, at concentrations up to 30 microM, reduced [3H]raclopride binding in the slices by 77% and 86%, respectively, with 50% inhibition at 1.6 microM amphetamine or 3.0 microM methamphetamine. Neither drug produced a significant effect on binding of [3H]SCH 23390 in the slices. This suggests that dopamine was able to interfere with radiotracer binding to D2 but not D1 receptors. The dopamine uptake blockers, cocaine and methylphenidate, had relatively little effect by themselves on [3H]raclopride binding but, by inhibiting amphetamine-induced dopamine release, significantly reduced inhibition of [3H]raclopride binding by a low (3 microM) amphetamine concentration. At a higher (30 microM) amphetamine concentration the inhibition of [3H]raclopride binding was not antagonized by uptake blockers and data obtained from homogenate binding experiments indicated a direct displacement of [3H]raclopride binding by amphetamine at this concentration. In conclusion the data obtained in the present study demonstrate that the effects of amphetamine on striatal radiotracer accumulation observed in PET and SPECT can also be observed in brain slices in vitro and, at least at low amphetamine concentrations, are mediated by competition with released dopamine.

Amphetamine↗
PubMed1999

Cloning, nucleotide sequence and expression of thioltransferase (glutaredoxin) cDNA from Schizosaccharomyces pombe.

Thioltransferase (TTase), also known as glutaredoxin (Grx), is an enzyme that catalyzes the reduction of a variety of disulfide compounds, including protein disulfides, in the presence of reduced glutathione. TTase acts as a cofactor for various enzymes such as ribonucleotide reductase. We previously purified a TTase from Schizosaccharomyces pombe and its molecular size was determined. In the present study, a cDNA coding TTase was isolated from a cDNA library of Schizosaccharomyces pombe by colony hybridization, which was constructed in a plasmid vector pGAD GH, and its corresponding insert was confirmed by Southern hybridization. The nucleotide sequence of the 375 bp long cDNA clone reveals an open reading frame, which encodes a protein of 101 amino acids. The coding region of the original clone was transferred after the lac promoter of pUC13 vector for expression in E. coli, and simultaneously, a suitable Shine-Dalgarno (SD) sequence was added in front of the coding region by PCR. The two primers used for PCR also separately contained BamHI and HindIII restriction sites. The E. coli strain (A434) harboring the pUC13 derivative pKU10 showed a 17.3-fold increase in TTase activity compared to the strain with only the vector plasmid.

Amino Acid Sequence↗
PubMed1999

Suppressive effects of propolis in rat adjuvant arthritis.

The effects of ethanolic extract (EEP) of propolis on chronic inflammation were evaluated using rat adjuvant arthritis. In the chronic inflammatory animal model, the arthritis index was suppressed by EEP treatments (50 mg/kg/day and 100 mg/kg/day, p.o.). Moreover, physical weakness, induced by the chronic disease state, was dose-dependently improved in the EEP-treated groups. Its analgesic effect, assessed using the tail-flick test, was comparable to prednisolone (2.5 mg/kg/day, p.o.) and acetyl salicylic acid (100 mg/kg/day, p.o.). In carrageenan rat hind paw edema, which was conducted to test the effects of subfractions of EEP, the petroleum ether sub-fraction (100 mg/kg, p.o.) showed an inhibitory effect on the paw edema whereas EEP (200 mg/kg, p.o.) showed a significant anti-inflammatory effect at 3 and 4 hrs after carrageenan injection. From these results, we conclude that the ethanolic extract of propolis had a profound anti-inflammatory effects on both chronic and acute inflammations.

Animals↗
PubMed1999

Replantation of the midfoot in a child--six-year follow-up with pedobarographic analysis.

A 22-month old child underwent successful replantation of her midfoot at the level of Chopart's joint after a traumatic lawn mower amputation. The child demonstrated good sensation and function. Heel-to-toe length was 2 cm shorter, along with anterior migration of the heel pad on the injured foot. Pedobarographic analysis at 6-year follow-up demonstrated a normal range in the dynamic distribution of foot pressure measurements, except in the region of the medial heel. With replantation more commonly considered as an option in traumatic injuries, long-term functional evaluation is important in demonstrating the success of a replantation. The patient demonstrated a good result at 6-year follow-up, but will need further evaluation until skeletal maturity is reached.

Amputation, Traumatic↗
PubMed1999

Phase synchronization in the forced Lorenz system.

We demonstrate that the dynamics of phase synchronization in a chaotic system under weak periodic forcing depends crucially on the distribution of intrinsic characteristic times of this system. Under the external periodic action, the frequency of every unstable periodic orbit is locked to the frequency of the force. In systems which in the autonomous case displays nearly isochronous chaotic rotations, the locking ratio is the same for all periodic orbits; since a typical chaotic orbit wanders between the periodic ones, its phase follows the phase of the force. For the Lorenz attractor with its unbounded times of return onto a Poincaré surface, such state of perfect phase synchronization is inaccessible. Analysis with the help of unstable periodic orbits shows that this state is replaced by another one, which we call "imperfect phase synchronization," and in which we observe alternation of temporal segments, corresponding to different rational values of frequency lockings.

Journal Article↗