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Biomedical subjects

D Park

Publications and source records attributed to D Park.

At least 73 records · Page 4Linked to original sources
PubMed1998

Nucleotide excision repair is not required for the antiapoptotic function of insulin-like growth factor 1.

The expression of ERCC1, a member of the nucleotide excision repair (NER) family, is enhanced in cells transfected with insulin-like growth factor 1 (IGF-1) receptors. Of interest, an excellent concordance between ERCC1 expression and NER-mediated cell survival has been demonstrated. The two aims of the present study were to determine the signaling pathways used by IGF-1 to confer protection against apoptotic cell death in Chinese hamster ovary (CHO) cells and to assess the role of NER in this IGF-1 action. Experiments with pharmacological inhibitors indicated that phosphatidylinositol 3-kinase (PI 3-kinase) but not mitogen-activated protein kinase (ERK1/ERK2) mediates IGF-1 antiapoptotic activity. Using two series of CHO cells that have altered expression of ERCC1 or XPB/ERCC3, we examined IGF-1's ability to delay apoptotic death and reduction of mitochondrial oxidative function mediated by growth factor withdrawal. IGF-1 effectively blocked apoptosis, concomitant with increased MTT activity, in a pair of CHO cell lines expressing inactive ERCC1 (43-3B cells) and the transfected line of the mutant carrying the expressed human ERCC1 gene (83-G5 cells). Similarly, repair-deficient UV24 cells, which lack XPB/ERCC3, and their parental line AA8 were also responsive to the IGF-1's antiapoptotic capacity. In the presence of IGF-1, these cell lines became resistant to the cleavage of poly(ADP-ribose) polymerase, a key player in DNA damage recognition and DNA repair. These results suggest that PI 3-kinase activation plays a determinant role in the antiapoptotic function of IGF-1, but that functional NER does not play a critical part in mediating this IGF-1 response.

Animals↗
PubMed1998

Involvement of the Ras/extracellular signal-regulated kinase signalling pathway in the regulation of ERCC-1 mRNA levels by insulin.

Expression of DNA repair enzymes, which includes ERCC-1, might be under the control of hormonal and growth factor stimulation. In the present study it was observed that insulin increased ERCC-1 mRNA levels both in Chinese hamster ovary cells overexpressing human insulin receptors (HIRc cells) and in fully differentiated 3T3-L1 adipocytes. To investigate the mechanisms underlying the increase in ERCC-1 gene expression in HIRc cells, we used a variety of pharmacological tools known to inhibit distinct signalling pathways. None of these inhibitors affected the amount of ERCC-1 mRNA in unstimulated cells. The pretreatment of cells with two chemically unrelated phosphatidylinositol 3'-kinase inhibitors, wortmannin and LY294002, failed to block the doubling of ERCC-1 mRNA content by insulin. Similarly, inhibition of pp70 S6 kinase by rapamycin had no apparent effects on this insulin response. In contrast, altering the p21(ras)-dependent pathway with either manumycin, an inhibitor of Ras farnesylation, or PD98059, an inhibitor of the mitogen-activated protein kinase/extracellular signal-regulated protein kinase (ERK) kinase, suppressed the induction of ERCC-1 mRNA by insulin (P<0.001). Furthermore inhibition of RNA and protein synthesis negatively regulated the expression of this insulin-regulated gene (P<0.005). These results suggest that insulin enhances ERCC-1 mRNA levels by the activation of the Ras-ERK-dependent pathway without the involvement of the phosphatidylinositol 3'-kinase/pp70 S6 kinase.

Adipocytes↗
PubMed1998

Expression and characterization of a novel plasminogen activator from Agkistrodon halys venom.

A venom gland cDNA library of Agkistrodon halys was constructed and screened with a probe based on the consensus sequence of venomic serine proteases. Next, we determined the sequences of the entire open reading frames of two selected positives which were found to encode novel serine proteases of 234 and 233 amino acids in length and named as Haly-PA and Haly 2, respectively. Upon protein data base search, Haly-PA showed the highest similarity of 82% to the previously characterized plasminogen activator, TSV-PA (Zhang et al. 1995, J. Biol. Chem. 270, 10246- 10255). Haly 2 displayed a 78% similarity to beta-fibrinogenase (Hung et al. 1994, B. B. R. C., 205, 1707 1715). Haly-PA was successfully expressed using the baculovirus system and secreted into the culture media as a 32 kDa glycoprotein. In the western analysis of snake venom, anti-Haly-PA antibody detected the same size of band indicating that this enzyme is a component of snake venom. Recombinant Haly-PA was purified to homogeneity using the combination of anion exchange and gel filtration column. In the fibrino(geno)lytic assay, recombinant Haly-PA displayed an indirect fibrino(geno)lytic activity depending on the presence of plasminogen and cleaved the plasminogen to generate the active plasimin. These results indicate that Haly-PA is a plasminogen activator and displays fibrino(geno)lytic activity through conversion of plasminogen to plasmin.

Agkistrodon↗
PubMed1998

A Brassica cDNA clone encoding a bifunctional hydroxymethylpyrimidine kinase/thiamin-phosphate pyrophosphorylase involved in thiamin biosynthesis.

We report the characterization of a Brassica napus cDNA clone (pBTHI) encoding a protein (BTHI) with two enzymatic activities in the thiamin biosynthetic pathway, thiamin-phosphate pyrophosphorylase (TMP-PPase) and 2-methyl-4-amino-5-hydroxymethylpyrimidine-monophosphate kinase (HMP-P kinase). The cDNA clone was isolated by a novel functional complementation strategy employing an Escherichia coli mutant deficient in the TMP-PPase activity. A biochemical assay showed the clone to confer recovery of TMP-PPase activity in the E. coli mutant strain. The cDNA clone is 1746 bp long and contains an open reading frame encoding a peptide of 524 amino acids. The C-terminal part of BTH1 showed 53% and 59% sequence similarity to the N-terminal TMP-PPase region of the bifunctional yeast proteins Saccharomyces THI6 and Schizosaccharomyces pombe THI4, respectively. The N-terminal part of BTH1 showed 58% sequence similarity to HMP-P kinase of Salmonella typhimurium. The cDNA clone functionally complemented the S. typhimurium and E. coli thiD mutants deficient in the HMP-P kinase activity. These results show that the clone encodes a bifunctional protein with TMP-PPase at the C-terminus and HMP-P kinase at the N-terminus. This is in contrast to the yeast bifunctional proteins that encode TMP-PPase at the N-terminus and 4-methyl-5-(2-hydroxyethyl)thiazole kinase at the C-terminus. Expression of the BTH1 gene is negatively regulated by thiamin, as in the cases for the thiamin biosynthetic genes of microorganisms. This is the first report of a plant thiamin biosynthetic gene on which a specific biochemical activity is assigned. The Brassica BTH1 gene may correspond to the Arabidopsis TH-1 gene.

Alkyl and Aryl Transferases↗
PubMed1998

Sequence analysis of the 5'-flanking region of the gene encoding human HOXA-7.

We have isolated and characterized the immediate 5'-flanking region (886 bp) of the gene encoding human HOXA-7. When the total sequence was compared with those of mice, 93% of the 3' 518 bp (nt 370-886) sequences were identical, in which the 245 bases just preceding the AUG initiator codon (nt 614) was as highly conserved as in the coding region (nt 614-886). Sequences further upstream (nt 1-370) by comparison were highly diverged. In the 245 bp region, 8 stop and 3 initiation (including the initiator) codons were located, and a 50-aa long presumptive polypeptide was encoded. Nucleotide sequence analysis revealed three Sp1 and one AP2 binding sites, as well as one CAAT box. However, there was no consensus sequence for a TATA box in the 5' flanking region. One RARE repeat, one krox20 and three Hox-PBC binding sites were detected. Since many of the factor recognition sites were located in the immediate 5' flanking sequences of a highly-conserved region, it might be speculated that a regulatory mechanism for Hox gene expression is conserved throughout the evolution and one possible mechanism could be at the post-translational level.

5' Untranslated Regions↗
PubMed1998

'Best clinical practice': assessment of processes of care and of outcomes in the US Military Health Services System.

The National Quality Management Program of the Military Health Services System of the United States has undertaken a series of projects whose objective is the active, on-going monitoring and improvement of the effectiveness and efficiency of the care provided to a broad population that encompasses troops on active duty, retirees and dependents. The analytic activities consist of (1) identification by clinical panels of conditions and procedures of interest; (2) collection of data from electronic repositories and from charts to characterize the patients, how they are managed, the clinical outcomes they experience, the resource costs their care entails, and, from questionnaires, their functional status and level of satisfaction, and (3) generation of 'report cards' that inform organizational units down to the level of the hospital of the characteristics of their patients, their practices, and the risk-adjusted outcomes they achieve. The patterns of care employed by the hospitals that obtain the best risk-adjusted outcomes and resource utilization ('best clinical practice') are identified and made known. In addition, (4) a systematic process of developing outcomes-based practice guidelines has been devised. It intent is to serve as a decision-support tool for clinicians. Initial estimates have been obtained of the probable consequences of the application of this tool to operative interventions in childbirth. Use of the tool would result in a higher occurrence of elective Caesarean sections, a reduced rate of emergency Caesarean sections and much lower use of forceps, with an overall improvement in outcomes and lower resource costs. This program is currently in the early phases of implementation. The two principal requirements for the immediate future are (1) education of the clinical and administrative communities in the use of the data and the decision-support tools and (2) evaluation of the consequences of the use of the data by the clinical and administrative communities.

Benchmarking↗
PubMed1998

Genomic structure and sequence analysis of human HOXA-9.

In order to understand the regulatory mechanisms establishing and maintaining HOXA-9 gene expression, structural information about the gene is a prerequisite. Therefore, we sequenced the 7.2-kb region of the human HOXA-9 gene and mapped the positions of two partial cDNAs consisting of one of two 5' exons, AB (358 bp) or CD (568 bp), and a common 3' exon (exon II), which are separated by 5.4- and 1.0-kb introns, respectively. When the amino acid sequence homologies were compared with those of other Hox genes belonging to the same paralogous group, exon CD exhibited the strongest homology: 73% of 91 aa residues exactly matched those of chicken Hoxa-9. An intermediate exon (90 bp) was detected within exon CD. It was surrounded by a splice acceptor and a donor at both the 5' and 3' ends, and one branchpoint site was found near the splice-acceptor site. Nucleotide sequence analysis along this region revealed two TATA boxes, one CAAT box, one GC box, and one each of the following binding sites--engrailed, eve-stripe2-hb3, and Krox20--just upstream of exon CD. A CpG island and two RARE repeats were detected within intron I. Northern blot analysis showed that at least four main transcripts were generated along this region: all fetal tissues tested (brain, lung, liver, and kidney) produced a 1.8-kb homeobox-containing transcript (HA-9A); a 2.2- and a 3.3-kb transcript were generated from exon CD and exon II (HA-9B), especially in fetal and adult kidneys as well as in adult skeletal muscle; the 1.0-kb transcript was likely to be generated by the intermediate exon in all adult and fetal tissues. Several weak bands without tissue specificity were likely to be contributed by the hybrid transcripts between HOXA-9 and the other HOXA gene(s). Together, these results may account for the unique degree of conservation of the HOX cluster in general.

Adult↗
PubMed1997

HHV-8 (KSHV) does not establish latency in prostate cancer cell lines.

BACKGROUND: HHV-8 is a new herpesvirus found in lesions of Kaposi's sarcoma and some lymphoproliferative diseases. More recently, a report stated that normal prostate tissue also contains the virus. METHODS: The expression of HHV-8 was examined by a sensitive reverse-transcriptase PCR for the viral genes ORF 72, ORF 73, ORF 74, and ORF 75. In coculture experiments we attempted to infect 3 commonly studied prostate cancer cell lines using induced and uninduced lymphoid cell lines harboring HHV-8 (KS-1, BC-1, and BC-2). For induction of viral genes, butyrate and phorbol esters were used. RESULTS AND CONCLUSIONS: At baseline, prostate cancer cell lines LNCaP, DU-145, and PC-3 did not express viral gene products. Extensive coculture experiments were also negative. In no instance could latency for the virus be established. Our results argue against the involvement of HHV-8 in prostate cancer and for a limited tissue tropism of HHV-8.

B-Lymphocytes↗
PubMed1997

Molecular characterization of a nonsuppressible allele (prC4) of the Drosophila purple gene.

Purple gene encodes 6-pyruvoyl tetrahydropterin synthase (PTP synthase) in Drosophila. The enzyme PTP synthase catalyzes the conversion of dihydroneopterin triphosphate (H2-NTP) to 6-pyruvoyl tetrahydropterin (PTP), an important intermediate for pterin compounds. The extreme purple mutant, prC4, shows a very low activity of PTP synthase. The mutant purple gene has been cloned by screening with the subgenomic library of prC4. The size and expression level of PTP synthase gene transcripts in prC4 were almost the same as those of the wild type. The genomic DNA was also examined in the purple region by Southern blot analysis, but no changes in restriction pattern could be detected. Compared with the wild type PTP synthase sequence, the mutant PTP synthase of prC4 showed three missense mutations: the replacement of alanine 7 by serine (A7S), leucine 9 by phenylalanine (L9P), and aspartic acid 168 by glycine (D168G). Significance of these mutations was discussed in relation to the formation of the oligomeric structure of PTP synthase.

Alleles↗
PubMed1997

Cloning of a SH3 domain-containing proline-rich protein, p85SPR, and its localization in focal adhesion.

A mouse thymus cDNA expression library was screened with monoclonal antibody (mAb), B16-5 which binds to common epitope in SH3 domains of phospholipase C-gamma 1 (PLC-gamma 1) and Nck. We have determined the complete nucleotide sequence of one of several positive clones. The 4,172 bp cDNA clone (GenBank Accession No. U96634) encodes a SH3 domain-containing protein of 646 amino acids. Besides the SH3 domain, the predicted protein has a proline-rich region, nuclear localization signals, and leucine zipper motifs. The expressed protein in Sf9 insect cell exhibits a polypeptide of 85 kDa on SDS-PAGE. The protein is widely distributed in rat tissue with an especially high level of expression in brain and testis. Interestingly, the specific antibodies detected four related proteins of different size (75, 85, 90 and 105 kDa) in brain. In A431 cell, p85SPR is enriched at focal adhesion points indicating that the protein may interact with protein(s) in focal complexes.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Mutagenesis studies of thyroxine binding to human serum albumin define an important structural characteristic of subdomain 2A.

The familial dysalbuminemic hyperthyroxinemia (FDH) phenotype results from a natural human serum albumin (HSA) mutant, with histidine instead of arginine at amino acid position 218. This mutation results in an enhanced affinity for thyroxine. In our earlier study, site-directed mutagenesis and a yeast protein expression system were used to synthesize FDH HSA and several other HSA mutants. Measurement of the binding of these HSA mutants to thyroxine and several thyroxine analogs using equilibrium dialysis and quenching of tryptophan 214 fluorescence allowed us to propose a preliminary model of thyroxine binding to the 2A subdomain of wild type and FDH HSA. In this study, we have produced several other HSA mutants. By comparing the binding affinity of these mutants for thyroxine and tetraiodothyroacetic acid to the binding affinity of other mutants, we were able to suggest a new model for thyroxine binding to the 2A subdomain of HSA. We found that the substitution of arginine at position 218 with alanine increased the binding affinity for thyroxine by 2 orders of magnitude relative to the binding affinity of wild type HSA for thyroxine. A more accurate understanding of the mechanism of thyroxine binding to HSA has allowed us to define an important structural characteristic of subdomain 2A, one of the two principal binding sites on HSA for small hydrophobic ligands.

Arginine↗
PubMed1997

Cloning, sequencing, and overexpression of SH2/SH3 adaptor protein Nck from mouse thymus.

Nck, an oncogenic protein containing one SH2 and three SH3 domains, is a common target for phosphorylation by a variety of cell surface receptors. The absence of a recognizable catalytic domain of Nck suggests that Nck serves as a linker molecule to couple cell surface receptors to downstream effector molecules that regulate cellular responses induced by receptor activation. In this study, we isolated Nck cDNA from mouse thymus by screening a phase expression library using monoclonal antibody (mAb) B16-5 which recognizes the SH3 domain of phospholipase C-gamma 1. Nck protein was purified from an extract of the Sf9 cell that had been infected with recombinant baculovirus containing mouse Nck cDNA. The purified protein exhibited an apparent molecular mass of 47 kDa on SDS-polyacrylamide gels. Nck antibody was generated in rabbits using purified protein as an antigen. The distribution of Nck protein in rat tissues examined by immunoblots showed that Nck is expressed widely, and that brain, spleen, and tests contain more Nck than other tissues examined.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Is Kaposi's sarcoma--associated herpesvirus ubiquitous in urogenital and prostate tissues?

Controversy exists as to whether Kaposi's sarcoma-associated herpesvirus (KSHV) is more widespread than originally reported. Recently, Monini et al reported that KSHV is ubiquitous in urogenital and prostate tissues and sperm of healthy Italian adults using nested polymerase chain reaction (PCR). We have examined for the presence of KSHV in 10 normal prostates from Italian men and 10 from men from the United States, as well as 32 prostatic, 30 vulvar, 24 ovarian, 20 cervical, and 30 testicular cancer specimens from patients from the United States. None of the patients had a history of human immunodeficiency virus infection. The samples were tested by nested PCR. The sensitivity of this assay was determined by a dilution study performed by diluting KSHV DNA from the KS-1 cells (a primary effusion lymphoma cell line which is estimated to have 16 copies of KSHV per cell) in DNA from a K562 myeloid cell line. The nested PCR that we used can detect 2.4 copies of KSHV sequences on a background of K562 DNA. All the samples were negative for KSHV sequences. Therefore, we cannot confirm the finding that KSHV sequences are ubiquitous in urogenital and prostate tissues. Furthermore, because our samples were from both the United States and Italy, the discrepancy between results is unlikely to be explained by either ethnic or environmental factors. False-positive results easily occur using nested primer PCR because of contamination. Our data argue that KSHV is not widely disseminated in urogenital tissues from nonimmunosuppressed individuals.

Adult↗
PubMed1997

cAMP and protein kinase C elevate LH beta mRNA levels by activating transcription rather than stabilizing mRNA in rat pituitary cells.

The mechanisms by which GnRH modulates synthesis of LH beta subunit and release of LH from the pituitary gonadotropes are not clearly understood. However, GnRH actions in the pituitary gonadotropes have been suggested to be mediated by the PKC- and/or cAMP-dependent pathways. Thus, in the present study we have examined 1) whether the activations of either the PKC- and/or cAMP-dependent signaling cascades could elevate the levels of LH beta mRNA, and if so, 2) whether this increase of LH beta mRNA levels is the result of transcriptional activation or the result of suppressing the turnover of LH beta mRNA. In the present experiment, the activators of protein kinase C and the adenylate cyclase, PMA (5 nM) and forskolin (10 microM) respectively, have elevated the steady state levels of LH beta mRNA significantly by 18 h at the specific concentrations shown in the parenthesis. Subsequently, we have determined whether the elevation of LH beta mRNA levels by either PMA or forskolin is due to the new synthesis of LH beta mRNA or the suppression of LH beta mRNA turnover. Result showed that the ability of PMA or forskolin to elevate the LH beta mRNA levels was suppressed by the addition of actinomycin D, an inhibitor of transcription. Result further showed that the turnover of LH beta mRNA was not suppressed either by PMA or forskolin. These results indicate that the activation of PKC as well as the elevation of cAMP by GnRH leads to the increase in the levels of LH beta mRNA by stimulating the new synthesis of LH beta mRNA instead of increasing the stability of pre-existing LH beta mRNA.

Adenylyl Cyclases↗
PubMed1997

Year in review.

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Economics↗