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Biomedical subjects

Y Park

Publications and source records attributed to Y Park.

At least 163 records · Page 9Linked to original sources
PubMed1997

Noncoded amino acid replacement probes of the aspartate aminotransferase mechanism.

The primary role of Tyr225 in the aspartate aminotransferase mechanism is to provide a hydrogen bond to stabilize the 3'O- functionality of bound pyridoxal phosphate. The strength of this hydrogen bond is perturbed by replacement of Tyr225 with 3-fluoro-L-tyrosine (FlTyr) by in vitro transcription/translation. This mutant enzyme exhibits kcat/values that are near to those of wild type enzyme; however, the kcat/vs pH profile is much sharper with similar pKas of approximately 7.5 for both the ascending and descending limbs. The pKas are assigned to the endocyclic proton of the internal aldimine and to the bridging hydrogen bond, respectively. The pKas in the kcat vs pH profile of 7.2 and 8.7 are assigned to the epsilon-NH3+ of lysine 258 and to the endocyclic protons of the ketimine complex, respectively. Arginine 292 forms a salt bridge with the beta-COOH of the substrate, aspartate. An improvement on the earlier attempt to invert the substrate charge specificity via R292D mutation-induced arginine transaminase activity [Cronin, C. N., & Kirsch, J. F. (1988) Biochemistry 27, 4572-4579] is described. Here Arg292 is replaced with homoglutamate (R292hoGlu). This construct exhibits 6.8 x 10(4)-fold greater activity for the cationic substrate D,L-[Calpha-3H]-alpha-amino-beta-guanidinopropionic acid (D,L-[Calpha-3H]AGPA) than does wild type enzyme. The gain in selectivity for this substrate is at least 4500-fold greater than that achieved in the 1988 experiment, i.e., [(kcat/KM)R292hoGlu/(kcat/KM)WT (D,L-[Calpha-3H]AGPA)] >/= 4500 x [(kcat/KM)R292D/(kcat/KM)WT (L-arginine)]. The value of (kcat/KM)R292D is 0.43 M-1 s-1 with L-Arg while (kcat/KM)R292hoGlu is 29 M-1 s-1 with D,L-[Calpha-3H]AGPA (it is assumed that the D-enantiomer is unreactive). The latter value is the lower limit because of the uncertain value of 3H kinetic isotope effect.

Alanine↗
PubMed1997

Improvement of the 3'-5' exonuclease activity of Taq DNA polymerase by protein engineering in the active site.

Taq DNA polymerase from Thermus aquaticus has been shown to be very useful in the polymerase chain reaction method. Taq DNA polymerase has a domain at its amino terminus (residue 1 to 291) that has a 5'-3' exonuclease activity, a 3'-5' exonuclease domain in the middle (residue 292 to 423), and a domain at its C-terminus that catalyzes polymerase reactions. Taq DNA polymerase is classified into the polI family which is represented by E. coli DNA polymerase I. The three dimensional structural alignment of 3'-5' exonuclease domains from the polI family, DNA polymerases leads us to understand why Taq DNA polymerase does not carry out proof-reading in the polymerase chain reaction. Three sequence motifs, called ExoI, II, and III must be present in order to carry out proof-reading by the 3'-5' exonuclease reaction in DNA polymerization, but Taq DNA polymerase contains none of them. The key catalytic module in the 3'-5' exonuclease is two metal ions chelated by active-site carboxylic amino acids. In order to render the 3'-5' exonuclease activity in Taq DNA polymerase, a catalytic module was constructured in the active site by protein engineering. The mutant Taq DNA polymerase shows twice as much the 3'-5' exonuclease activity as that of wild-type DNA polymerase.

Amino Acid Sequence↗
PubMed1997

Exploration of the action pattern of Streptomyces hyaluronate lyase using high-resolution capillary electrophoresis.

Hyaluronic acid was treated exhaustively with a hyaluronate lyase (hyaluronidase, EC 4.2.2.1) from Streptomyces hyalurolyticus to obtain a tetrasaccharide and a hexasaccharide product in a molar ratio of 1 to 1.2. The tetrasaccharide product was fluorescently labeled at the reducing end by reductive amination with 7-amino 1,3-naphthalene disulfonic acid (AGA) and the structure of the conjugate was determined spectroscopically. Partial treatments of hyaluronic acid with hyaluronate lyase afforded complex mixtures of oligosaccharides that were similarly fluorescently labeled. These labeled oligosaccharide mixtures were analyzed using high-resolution capillary electrophoresis. The resulting electropherograms showed the content of each hyaluronic acid derived oligosaccharide, having a degree of polymerization (dp) from 4 to 50, throughout the enzymatic reaction. Computer simulation studies gave comparable kinetic profiles suggesting that hyaluronate lyase exhibits a random endolytic action pattern. Interestingly, oligosaccharides of certain size (dp) were under-represented in these oligosaccharide mixtures suggesting that linkages at spacings of 10 to 12 saccharide units are somewhat resistant to this enzyme. The cause of this resistance might be the result of secondary or higher order structural features present in the hyaluronic acid polymer.

Carbohydrate Sequence↗
PubMed1997

Synergy between human immunodeficiency virus type 1 and Epstein-Barr virus in T lymphoblastoid cell lines.

CR2 (CD21), the EBV receptor, was detected on three of four CD4-positive cell lines by indirect fluorescent labeling, and its corresponding mRNA was found by use of the reverse transcription-based polymerase chain reaction. To determine whether CR2 on CD4-positive cells was functional, their ability to be infected by EBV was analyzed. EBV DNA, EBV nuclear antigen 2 (EBNA-2A), and EBV-encoded small RNA (EBER1) transcripts could be detected in CR2-expressing CD4-positive cells following infection by the B95.8 strain of EBV. Analysis of the terminal region showed the EBV genome remained linear following infection, and copy number decreased with time. Since CD4-positive cell lines are targets for HIV-1 infection, the effects of EBV infection on HIV-1 expression were analyzed. HIV-1 replication was upregulated when CD4-positive cells were coinfected with EBV strain B95.8 but not P3HR-1K. These results suggested that EBNA-2 is involved in upregulation of HIV-1 expression in T lymphoblastoid cell lines. To test this hypothesis an EBNA-2-expression vector was transfected into T lymphoblastoid cell lines and HIV-1 expression measured. First, trans-activation of HIV-1 long terminal repeat (LTR) by Tat was enhanced by EBNA-2 type 1 expression. trans-Activation of the HIV-1 LTR by Tat was also enhanced when CD4-positive cells were infected by EBV (strain B95.8) encoding an intact EBNA-2, but not by P3HR-1K with a deleted EBNA-2. In addition, CD4-positive cell clones stably expressing EBNA-2 supported enhanced HIV-1 replication as measured by accumulation of reverse transcriptase activity and syncytium induction. This provides direct evidence that EBV infection can enhance HIV-1 replication in T cells. Whether this in vitro phenomenon contributes to disease progression in vivo remains to be determined.

CD4-Positive T-Lymphocytes↗
PubMed1997

Effect of conjugated linoleic acid on body composition in mice.

The effects of conjugated linoleic acid (CLA) on body composition were investigated. ICR mice were fed a control diet containing 5.5% corn oil or a CLA-supplemented diet (5.0% corn oil plus 0.5% CLA). Mice fed CLA-supplemented diet exhibited 57% and 60% lower body fat and 5% and 14% increased lean body mass relative to controls (P < 0.05). Total carnitine palmitoyltransferase activity was increased by dietary CLA supplementation in both fat pad and skeletal muscle; the differences were significant for fat pad of fed mice and skeletal muscle of fasted mice. In cultured 3T3-L1 adipocytes CLA treatment (1 x 10(-4)M) significantly reduced heparin-releasable lipoprotein lipase activity (-66%) and the intracellular concentrations of triacylglyceride (-8%) and glycerol (-15%), but significantly increased free glycerol in the culture medium (+22%) compared to control (P < 0.05). The effects of CLA on body composition appear to be due in part to reduced fat deposition and increased lipolysis in adipocytes, possibly coupled with enhanced fatty acid oxidation in both muscle cells and adipocytes.

3T3 Cells↗
PubMed1997

Balloon angioplasty of native aortic coarctation in infants 3 months of age and younger.

The use of balloon dilation to treat native aortic coarctation is controversial, particularly in infants. Between January 1991 and September 1996, 12 patients < or = 3 months of age with native coarctation of the aorta (CoA) underwent balloon angioplasty (BA). All 12 lesions were dilated successfully with a mean reduction in peak systolic gradient from 49.3 +/- 16.5 mm Hg to 6.8 +/- 4.0 mm Hg (p < 0.001) and a mean increase in minimum CoA diameter from 2.4 +/- 0.6 mm to 5.5 +/- 1.3 mm (p < 0.001). Intimal flaps or tears were detected immediately after BA in 4 (33%) of 12 patients by angiography and in 8 (89%) of 9 patients by intravascular ultrasonography. No deaths or major complications related to the BA occurred. One patient had documented asymptomatic femoral artery obstruction, and one patient with hydrops fetalis and congenital pleural effusions died with gram-negative sepsis 1 week after the procedure. Follow-up was available for 10 patients (1 was lost to follow-up) between 2 months and 4.1 years (mean 2.4 +/- 1.3 years) after BA. No patient had an aortic aneurysm. Restenosis occurred in 5 (50%) of 10 patients, requiring reintervention a mean of 2.6 +/- 2.1 months after BA. One patient underwent surgical repair. Repeat BAs were performed in the other four patients; three were successful, and one with partial gradient relief required surgical repair. Five patients have not required reintervention a mean of 2.9 +/- 1.0 years after the initial BA. Among these five patients, follow-up intravascular ultrasound performed in three patients a mean of 2.0 +/- 1.9 years after BA showed favorable endovascular remodeling. There was a tendency for early reintervention in patients < 1 month of age and coexistence of a patent ductus arteriosus at the time of BA. In conclusion, selected infants < or = 3 months of age with discrete native CoA may be treated initially with balloon dilation. Most patients who have restenosis respond successfully to repeat BA.

Age Factors↗
PubMed1997

A novel mutation L1029H in sodium channel gene hscp associated with pyrethroid resistance for Heliothis virescens (Lepidoptera:Noctuidae).

Sequencing of the sodium channel gene hscp (para-homologous) revealed that a T to A mutation encoding a Leu to His change (L1029H) is associated with pyrethroid resistance in populations of Heliothis virescens F. This mutation occurs at a position homologous to that of the mutation Leu to Phe (L1014F) reportedly associated with knock-down resistance (kdr) to pyrethroids in a house fly strain. Another mutation reportedly associated with super-kdr in house fly was not found in H. virescens samples. In previous work, we found that one haplotype Hpy3 of a marker locus Hpy that lies approximately 5 kb away from the L1029H polymorphism in the gene, increased in frequency among survivors of selection with pyrethroids, presumably through "hitch-hiking" with a resistance-conferring mutation. Not all resistant individuals examined in this study carried the L1029H mutation, but only those that also carried Hpy3 haplotypes. Resistance has been genetically linked to hscp markers for the strain PEG87, but this strain was found not to carry the L1029H mutation. Assuming that the L1029H mutation does in fact confer resistance, this suggests that more than one sodium channel mutation may be contributing to pyrethroid resistance in field populations of H. virescens.

Amino Acid Sequence↗
PubMed1997

Long-term follow-up of patients with chronic hepatitis B treated with interferon alfa.

BACKGROUND & AIMS: Therapy with interferon alfa (IFN-alpha) leads to remission of disease in one third of patients with chronic hepatitis B. The aim of this study was to better define the long-term prognosis of this outcome. METHODS: One hundred three patients with chronic hepatitis B who underwent IFN-alpha therapy in three clinical trials between 1984 and 1991 were followed up for serological status, biochemical evidence of liver disease, and liver complications or mortality through 1994. RESULTS: Among 103 patients, 31 (30%) responded to therapy with loss of hepatitis B e antigen and viral DNA from serum. Responders were more likely than nonresponders to be women, black, and to have more severe liver disease including cirrhosis (P < 0.05). Up to 11 years (mean, 6.2 years) after therapy, a higher percentage of responders than nonresponders were still negative for hepatitis B e antigen (94% vs. 40%; P < 0.001) and hepatitis B surface antigen (71% vs 8.3%; P < 0.001). Overall, the rate of liver-related complications and death did not differ by IFN-alpha response, but with adjustment for cirrhosis, nonresponders had higher rates of liver-related complications and mortality (hazard ratio, 13.7; 95% confidence interval, 3.0-63.5). CONCLUSIONS: The response to IFN-alpha therapy in chronic hepatitis B is usually a sustained improvement in disease markers and, when cirrhosis is considered, patient outcome.

Adult↗
PubMed1997

Insulin-dependent diabetes mellitus (IDDM) is associated with CTLA4 polymorphisms in multiple ethnic groups.

Linkage disequilibrium (association) analysis was used to evaluate a candidate region near the CTLA4/CD28 genes using a multi-ethnic collection of families with one or more children affected by IDDM. In the data set unique to this study (Spanish, French, Mexican-American, Chinese and Korean), the transmission/disequilibrium test (TDT) revealed a highly significant deviation for transmission of alleles at the (AT)n microsatellite marker in the 3' untranslated region (P = 0.002) and the A/G polymorphism in the first exon (P = 0.00002) of the CTLA4 gene. The overall evidence for transmission deviation of the CTLA4 A/G alleles is also highly significant (P = 0.00005) in the combined data set (669 multiplex and 357 simplex families) from this study and a previous report on families from USA, Italy, UK, Spain and Sardinia. Significant heterogeneity was observed in these data sets. The British, Sardinian and Chinese data sets did not show any deviation for the A/G polymorphism, while the Caucasian-American data set showed a weak transmission deviation. Strong deviation for transmission was seen in the three Mediterranean-European populations (Italian, Spanish and French) (P = 10(-5)), the Mexican-American population (P = 0.002) and the Korean population (P = 0.03). These results suggest that a true IDDM susceptibility locus (designated IDDM12) is located near CTLA4.

Abatacept↗
PubMed1997

Role of fimbriae in Porphyromonas gingivalis invasion of gingival epithelial cells.

Porphyromonas gingivalis is a periodontal pathogen capable of invading primary cultures of normal human gingival epithelial cells (NHGEC). Involvement of P. gingivalis fimbriae in the invasion process was examined. Purified P. gingivalis 33277 fimbriae blocked invasion of this organism into NHGEC in a dose-dependent manner. DPG3, a P. gingivalis fimbria-deficient mutant, was impaired in its invasion capability approximately eightfold compared to its parent, strain 381. However, adherence of the mutant was only 50% reduced compared to the parent. Biotin labeling of NHGEC surface proteins revealed that two fimbriated strains, but not DPG3, bound a 48-kDa NHGEC protein. Adhesin-receptor interactions, such as fimbriae binding to a 48-kDa NHGEC surface receptor, may trigger activation of eukaryotic proteins involved in signal transduction and/or provoke the generation of surface P. gingivalis molecules required for internalization.

Bacterial Adhesion↗
PubMed1997

Inducible expression of a Porphyromonas gingivalis W83 membrane-associated protease.

The Tpr protease of Porphyromonas gingivalis W83 is a membrane-associated enzyme capable of hydrolyzing a chromogenic bacterial collagenase substrate. An isogenic mutant lacking a functional tpr gene had a greatly reduced ability to hydrolyze the collagenase substrate. Activity was restored to the tpr mutant by introducing a shuttle plasmid containing the tpr gene. Expression of the gene is induced by nutrient limitation, as shown by enzymatic and Northern analyses.

Collagenases↗
PubMed1997

CNS innervation of the urinary bladder demonstrated by immunohistochemical study for c-fos and pseudorabies virus.

The aim of the present study is to verify the functional and anatomical neural pathways which innervate the urinary bladder in the central nervous system of the rat. To identify the functional neural pathway, the urinary bladder was stimulated by infusing formalin for 2 h. Then, brain and spinal cord were dissected out and immunohistochemistry was done by using anti-c-fos antibody. Many c-fos immunoreactive (IR) neurons were identified in the telencephalic cortical areas and in several brainstem nuclei, which are known mostly to be related with urinary bladder. In the spinal cord, a number of c-fos IR neurons were found in the lamina I, IIo, dorsal gray commissure, sacral parasympathetic nucleus. To identify the anatomical neural pathway of the urinary bladder, Pseudorabies virus (PRV) was injected into the wall of urinary bladder and was identified with anti-PRV by using immunohistochemistry. Most PRV labeled neurons were found where c-fos IR neurons were identified and few of them were also in the areas where c-fos IR neurons were not found, e.g., prefrontal cortex, agranular insular cortex, and subfornical organ. In the spinal cord, PRV labeled cells were found all over the gray matter. The present study presents morphological evidence demonstrating the supraspinal areas are related with the neural control of the urinary bladder and most functional neural pathway of the urinary bladder is well consistent with the anatomical neural pathway except in some telencephalic cortical areas.

Animals↗
PubMed1997

[Antiemetic efficacy of granisetron in repeated CAF chemotherapy after breast cancer operation].

The antiemetic efficacy of granisetron in repeated CAF chemotherapy after breast cancer operation was investigated. Twenty-three patients who were treated with repeated CAF chemotherapy after breast cancer operation received drip-infused granisetron (3 mg/body) to prevent nausea and vomiting. Antiemetic efficacy was evaluated by means of the severity of nausea and the frequency of vomiting. The antiemetic efficacy rate of the drug was 74.4%. However, the average rate of antiemetic efficacy was 86.9% on the first through the sixth administration of the chemotherapy (first period). 75.9% on the seventh through the twelfth (second period), 48.4% on the thirteenth through the eighteenth (third period), and 31.8% on the nineteenth through the twenty-fourth (fourth period). There were significant differences in the antiemetic efficacy rate among the first, second and third periods. The antiemetic efficacy of granisetron decreased significantly by repeated CAF chemotherapy.

Adult↗
PubMed1997

Mutation of the central nervous system neuroblast proliferation repressor ana leads to defects in larval olfactory behavior.

In the developing nervous system, interactions between glia and immature neurons or neuroblasts regulate axon pathfinding, migration, and cell division, and therefore affect structure and function. Glial control of neuroblast cell division has been documented by studies of the anachronism (ana) gene of Drosophila melanogaster. ana encodes a glycoprotein which, in the developing larval central nervous system, is secreted by glia that neighbor regulated neuroblasts. Mutations in ana lead to premature neuroblast proliferation in the larval brain. Examination of lacZ expression from an ana enhancer trap line as well as detection of the ana protein show that ana is also expressed in the larval antennal-maxillary complex (AMC) at all larval stages. As previously reported for the central nervous system, ana expression in the AMC appears to be confined to glial cells. Larval olfactory system function in ana mutants was assayed in a behavioral paradigm. When tested with the three different chemoattractants, third instar ana9 mutant larvae showed diminished olfactory response compared to controls. Examination of a second ana allele revealed aberrant olfactory response to ethyl acetate, demonstrating that more than one mutation in ana can give rise to abnormal larval olfactory behavior. Assays of early first instar ana9 mutant larvae revealed defective olfactory behavior, implying that the olfactory phenotype stems from early larval AMC and/or embryonic origins. This is consistent with proliferation analysis in the early larval AMC region which uncovered a significantly higher number of S-phase cells in ana9 mutants.

Alleles↗
PubMed1996

Involvement of calcium in interactions between gingival epithelial cells and Porphyromonas gingivalis.

Porphyromonas gingivalis, a periodontal pathogen can invade primary cultures of gingival epithelial cells. This invasion was significantly inhibited (74-81%) by thapsigargin and 1,2-bis(2-aminophenoxy)ethane-N,N,N1,N1-tetraacetic acid, acetoxymethyl ester (BAPTA/AM), but not by EDTA or amiloride. Release of Ca2+ from an intracellular store and the subsequent increase in cytosolic [Ca2+] may, therefore, be involved in the invasion process, while Ca2+ influx is not. Moreover, cytosolic [Ca2+] was found to increase transiently in about 30% of gingival epithelial cells acutely exposed to P. gingivalis, but not in unexposed cells, or in cells exposed to noninvasive Escherichia coli. These findings indicate that P. gingivalis invasion of epithelial cells is correlated with activation of [Ca2+]-dependent host cell signaling systems.

Actin Cytoskeleton↗
PubMed1996

Phenobarbital induction mediated by a distal CYP2B2 sequence in rat liver transiently transfected in situ.

The promoter activities of the genes for cytochrome P450 2B1 (CYP2B1) and cytochrome P450 2C1 (CYP2C1) have been assayed by direct injection of promoter-luciferase chimeric genes into rat liver. Activities of minimal promoters for CYP2C1 and CYP2B1 were detectable in untreated animals but were not increased by treatment of the animals with phenobarbital. After insertion to the 5' side of the minimal promoters of one to three copies of the CYP2B2 sequence from -2318 to -2155, a phenobarbital-responsive element in primary hepatocyte cultures (Trottier, E., Belzil, A., Stoltz, C., and Anderson, A. (1995) Gene (Amst.) 158, 263-268), phenobarbital treatment induced the activity of the CYP2C1 promoter by 5-15-fold and the CYP2B1 promoter by 2.5-5-fold. Mutation of a basal transcription element-like motif and a CCAAT/enhancer binding protein element in the CYP2B1 proximal promoter region reduced expression, but 3-4-fold induction by phenobarbital was retained. Mutation of the "Barbie box," a putative phenobarbital-responsive element (He, J.-S., and Fulco, A. J. (1991) J. Biol. Chem. 266, 7864-7869) in the CYP2B1 proximal promoter did not reduce the relative response to phenobarbital. These results demonstrate that direct injection of DNA into rat liver may be used to assay phenobarbital responsiveness of cytochrome P450 genes. In this system, a distal CYP2B2 element mediates a response to phenobarbital, and proximal elements, including the Barbie box, are not required for the induction.

Animals↗
PubMed1996

Role of interleukin 6 and corticosteroids in the regulation of expression of glutathione S-transferases in primary cultures of rat hepatocytes.

The effect of recombinant interleukin 6 (rIL-6) on the transcript levels of rat glutathione S-transferase (GST) genes rGSTA2, rGSTP1, rGSTM1 and rGSTM2 was examined in primary cultures of rat hepatocytes. rIL-6 had little effect on the increase in expression of rGSTP1 that occurs in cultured hepatocytes. Dexamethasone (DEX), in contrast, prevented the expression of rGSTP1 by hepatocytes, and rIL-6 in combination with DEX had no additional effect. Neither rIL-6 nor DEX alone had a significant effect on the transcript levels of rGSTA2, rGSTM1 and rGSTM2 in cultured hepatocytes. However, when both were present (15 ng/ml rIL-6 and 10(-7) M DEX) the transcript levels of rGSTA2, rGSTM1 and rGSTM2 decreased significantly (P < 0.05) after 48 h in culture. If the rIL-6 was removed from the cultures after 24 h, the levels of transcripts recovered and were the same at 48 h as cells cultured without rIL-6 for the entire period. Dose-response relationships of rIL-6 with 10(-7) M DEX were determined for transcripts of each GST isoenzyme and the IC50 values were between 1.5 and 7.5 ng/ml. Declines in transcript levels of rGSTA2 were observed with rIL-6 plus 10(-8) or 10(-7) M DEX but not with rIL-6 plus 10(-9), 10(-6), or 10(-5) M DEX. To determine if the cytokine and glucocorticoid effects were mediated by sequences in the 5'-flanking sequence of rGSTA2, a plasmid construct containing a 1.6 kb fragment of the 5'-flanking sequence of the rGSTA2 gene and the chloramphenicol acetyltransferase (CAT) reporter gene was used to transfect rat hepatocytes in primary culture. The addition of rIL-6 and DEX to the culture medium caused a significant (P < 0.05) decrease in CAT activity after 48 h in culture. If rIL-6 was removed after 24 h in culture, CAT activity after an additional 24 h in culture was greater than the CAT activity in cells cultured for 48 h without rIL-6. Therefore cytokines and glucocorticoids may be important physiological regulators of GST expression.

Animals↗