Biomedical subjects

H L Hung

Publications and source records attributed to H L Hung.

15 recordsLinked to original sources

PubMed2001

Regulation of human coagulation factor X gene expression by GATA-4 and the Sp family of transcription factors.

Serine protease factor Xa plays a critical role in the coagulation cascade. Zymogen factor X is synthesized and modified in the liver. To understand the mechanisms governing the liver-specific expression of factor X, the proximal promoter of human factor X was previously characterized. Two crucial cis elements at -73 and -128 and their cognate binding proteins, HNF-4 and NF-Y, respectively, were identified. In this report, studies are extended to 3 additional cis elements within the factor X promoter. Using gel mobility shift assays, the liver-enriched protein GATA-4 was identified as the protein binding to the GATA element at -96. GATA-4 transactivates the factor X promoter 28-fold in transient transfection experiments. It was also determined that the Sp family of transcription factors binds 2 DNase I-footprinted sites at -165 and -195. Disruption of Sp protein binding at either site reduces the promoter activity by half. Simultaneous disruption of both sites reduces the promoter activity 8-fold. This is the first report indicating the involvement of GATA-4 in the regulation of clotting factor expression. These observations provide novel insight into mechanisms by which the vitamin K-dependent coagulation factors are regulated.

3T3 Cells↗

PubMed2001

Stimulation of NF-E2 DNA binding by CREB-binding protein (CBP)-mediated acetylation.

The hematopoietic transcription factor NF-E2 is an important regulator of erythroid and megakaryocytic gene expression. The transcription cofactor cAMP-response element-binding protein (CREB)-binding protein (CBP) has previously been implicated in mediating NF-E2 function. In this report, we examined the role of CBP, a coactivator with intrinsic acetyltransferase activity, in the regulation of NF-E2. We found that both the hematopoietic-specific subunit of NF-E2, p45, and the widely expressed small subunit, MafG, interact with CBP in vitro and in vivo. CBP acetylates MafG, but not p45, predominantly in the basic region of MafG. Immunoprecipitation experiments with anti-acetyl lysine antibodies demonstrate that MafG is acetylated in vivo in erythroid cells. Transfection experiments further show that CBP stimulates MafG acetylation in intact cells in an E1A-sensitive manner. Acetylation of MafG augments DNA binding activity of NF-E2, and mutations at the major acetylation sites markedly reduce DNA binding and transcriptional activation by NF-E2. Together, these results suggest that recruitment of CBP by NF-E2 to specific erythroid/megakaryocytic promoters might regulate transcription by at least two mechanisms involving both modification of chromatin structure and modulation of transcription factor activity.

Acetylation↗

PubMed1999

A method for identifying differentially expressed genes in rare populations of primary human hematopoietic cells.

Differential display (DD) is widely employed for identifying uniquely expressed genes within two different cell populations. While potentially powerful, DD is problematic because apparent positive clones require time-consuming verification which may be made even more difficult if only small amounts of starting material are available. We have devised a screening approach to address these issues in primary human hematopoietic cells. Candidate clones are identified in a slot-blot format and verified by 'Virtual Northern' blot analyses using globally amplified cDNA as the verification probe. This method is fast, and since it requires only approximately 0.2 microg of total RNA, it is particularly useful when only limited amounts of study tissue are available.

Blotting, Northern↗

PubMed1999

CREB-Binding protein acetylates hematopoietic transcription factor GATA-1 at functionally important sites.

The transcription factor GATA-1 is a key regulator of erythroid-cell differentiation and survival. We have previously shown that the transcriptional cofactor CREB-binding protein (CBP) binds to the zinc finger domain of GATA-1, markedly stimulates the transcriptional activity of GATA-1, and is required for erythroid differentiation. Here we report that CBP, but not p/CAF, acetylates GATA-1 at two highly conserved lysine-rich motifs present at the C-terminal tails of both zinc fingers. Using [3H]acetate labelling experiments and anti-acetyl lysine immunoprecipitations, we show that GATA-1 is acetylated in vivo at the same sites acetylated by CBP in vitro. In addition, we show that CBP stimulates GATA-1 acetylation in vivo in an E1A-sensitive manner, thus establishing a correlation between acetylation and transcriptional activity of GATA-1. Acetylation in vitro did not alter the ability of GATA-1 to bind DNA, and mutations in either motif did not affect DNA binding of GATA-1 expressed in mammalian cells. Since certain functions of GATA-1 are revealed only in an erythroid environment, GATA-1 constructs were examined for their ability to trigger terminal differentiation when introduced into a GATA-1-deficient erythroid cell line. We found that mutations in either acetylation motif partially impaired the ability of GATA-1 to induce differentiation while mutations in both motifs abrogated it completely. Taken together, these data indicate that CBP is an important cofactor for GATA-1 and suggest a novel mechanism in which acetylation by CBP regulates GATA-1 activity in erythroid cells.

Acetylation↗

PubMed1996

Liver-enriched transcription factor HNF-4 and ubiquitous factor NF-Y are critical for expression of blood coagulation factor X.

Blood coagulation Factor X and its activated form Factor Xa play an essential role in the midphase of the clotting cascade. To delineate the mechanisms governing the liver-specific expression of Factor X, we have previously characterized the complete 2.8 kilobase pairs of the 5'-flanking region of Factor X and demonstrated that the first 209 base pairs is sufficient to confer maximal promoter activity in HepG2 cells, a hepatoma cell line that expresses Factor X. We have also shown that mutations at ACTTTG and CCAAT elements located at -56 to -51 and -120 to -116, respectively, significantly reduce the promoter activity. In this report, we demonstrate that Factor X mRNA is primarily but not exclusively expressed in the liver. Using DNase I footprinting analysis, we determine four protein binding sites within the 209-base pair fragment, designated site 1 (-73) to -44), site 2 (-128 to -94), site 3 (-165 to -132), and site 4(-195 to -169). Using gel mobility shift assays in combination with competition and supershift experiments, we demonstrate that hepatocyte nuclear factor 4 and Sp1 bind at site 1, the site which contains the ACTTTG element. Methylation interference assays reveal that HNF-4 and Sp1 contact adjacent sites with minor overlap. HNF-4 and Sp1 appear to bind site 1 in a mutually exclusive fashion. We also demonstrate that HNF-4 can transactivate the Factor X promoter in HeLa cells; mutation at the adjacent Sp1 site further increases the transactivation. Heteromeric transcription factor NF-Y was identified as the protein that binds the CCAAT box at site 2. We conclude that HNF-4 and NF-Y play crucial roles in modulating the activity of the proximal promoter of Factor X.

Base Sequence↗

PubMed1996

Functional characterization of the human factor VII 5'-flanking region.

Factor VII is a vitamin K-dependent coagulation protein essential for proper hemostasis. The human Factor VII gene spans 13 kilobase pairs and is located on chromosome 13 just 2.8 kilobase pairs 5' to the Factor X gene. In this report, we show that Factor VII transcripts are restricted to the liver and that steady state levels of mRNA are much lower than those of Factor X. The major transcription start site is mapped at -51 by RNase protection assay and primer extension experiments. The first 185 base pairs 5' of the translation start site are sufficient to confer maximal promoter activity in HepG2 cells. Protein binding sites are identified at nucleotides -51 to -32, -63 to -58, -108 to -84, and -233 to -215 by DNase I footprint analysis and gel mobility shift assays. A liver-enriched transcription factor, hepatocyte nuclear factor-4 (HNF-4), and a ubiquitous transcription factor, Spl, are shown to bind within the first 108 base pairs of the promoter region at nucleotide sequences ACTTTG and CCCCTCCCCC, respectively. The importance of these binding sites in promoter activity is demonstrated through independent functional mutagenesis experiments, which show dramatically reduced promoter activity. Transactivation studies with an HNF-4 expression plasmid in HeLa cells also demonstrate the importance of HNF-4 in promoting transcription in non-hepatocyte derived cells. Additionally, the sequence of a naturally occurring allele containing a previously described decanucleotide insert polymorphism at -323 is shown to reduce promoter activity by 33% compared with the more common allelic sequence.

Amino Acid Sequence↗

PubMed1992

Characterization of the human blood coagulation factor X promoter.

Blood coagulation Factor X is a serine protease required for both the intrinsic and extrinsic pathways of coagulation. The gene for Factor X spans 27 kilobases and is located on chromosome 13, in close proximity to the gene encoding Factor VII. Expression of Factor X is restricted to the liver. We have characterized the human Factor X promoter by mapping the start sites of transcription and carrying out a functional analysis of the promoter. The first 279 base pairs (bp) of 5'-flanking sequence upstream from the first AUG are sufficient to confer maximal promoter activity in HepG2 cells. Protein-binding sites within the 279-bp fragment are defined using gel mobility shift assays. Mutagenesis of two specific sequences within the 279-bp fragment (CCAAT at -120 to -116, and ACTTTG at -56 to -51), results in loss of ability to bind proteins from a HepG2 nuclear extract, and profound reduction in promoter activity of the 279-bp fragment. We conclude that these two protein-binding sites are critical for the activity of the Factor X promoter.

Base Sequence↗

PubMed1992

[Mannitol reduces plasma hydrogen peroxide free radical in patients undergoing coronary artery bypass graft surgery].

During the procedure of coronary artery bypass graft surgery (CABG), the release of free oxygen radicals as a result of ischemia and reperfusion which plants the seeds of post-operative low cardiac output and arrhythmias has grave consequence on the reestablishment of cardiac function. A variety of chemical agents such as mannitol, allopurinol, catalase (Q-10) and superoxide dismutase (SOD) has proved to be considerably effective to improve the myocardial necrosis following ischemia and reperfusion. In this study we chose mannitol (0.2 gm/kg) as the free oxygen radicals scavenger and utilized mass spectrophotometric method to detect the variation of concentration of [H2O2], a by-product of free oxygen radical, in an attempt to evaluate the efficacy of mannitol in this regard in patients undergoing CABG. Patients were divided into experimental group (n = 19) and control group (n = 20). In the experimental group the concentration of [H2O2] changed from 61 +/- 24 microM/L pre-operatively to 77 +/- 18 microM/L post-operatively as against 75 +/- 31 microM/L and 99 +/- 31 microM/L respectively in the control group. In comparison, only the change in experimental group was statistically significant (p less than 0.05). We confirmed that mannitol functions considerably as a free oxygen radical scavenger since it reduces the production of [H2O2] in patients undergoing CABG.

Aged↗

PubMed1991

Menadione: spectrum of anticancer activity and effects on nucleotide metabolism in human neoplastic cell lines.

The spectrum of cytotoxicity of menadione (MD) was examined in a panel of human cancer cell lines. MD was equipotent against multidrug-resistant and parental leukemia cell lines with IC50 values of 13.5 +/- 3.6 and 18 +/- 2.4 microM respectively. A cervical carcinoma cell line resistant to the antimetabolite, methotrexate (MTX), was as sensitive to MD as its parental cell line. The interactions of fifteen clinically utilized anticancer drugs with MD were examined in vitro and the majority were found to be additive, with four agents exhibiting synergism and one agent exhibiting antagonism. MD inhibited the incorporation of radioactive thymidine, uridine and amino acids into DNA, RNA and protein, respectively, in three human cancer cell lines. Some possible reasons for the inhibition of DNA synthesis including effects of MD on intracellular deoxyribonucleoside triphosphate pools were examined and ruled out. Although results from previous studies using rat hepatocytes suggested that mitochondria may be a target of MD, no significant effect of this compound on total intracellular adenosine triphosphate (ATP) pools in human cancer cell lines was observed. Collectively, these in vitro results demonstrate that MD possesses a broad spectrum of anticancer activity and suggest the potential utility of this agent in cancer therapy. Future studies directed at elucidation of the mechanism of MD action in human cancer cells are warranted and are under study.

Antineoplastic Agents↗

PubMed1991

[The antinociceptive effect of intrathecal somatostatin in monkeys].

The distribution of somatostatin (SST) throughout the nervous system suggests that this tetradecapeptide may play a physiological role in CNS in the mediation of analgesia. The present study was undertaken to evaluate the antinociceptive properties of intrathecal (IT) injection of SST in the comparison of morphine sulfate (MS) in a primate model. The study was conducted after institutional approval and adhered to the regulations of the animal research committee. Seven adult monkeys (Maccaca cyclopis Swinhoe) weighing 4-6 kg were used. In each animal a L5 laminectomy window was created to facilitate IT injection. No neurological damage from surgery was noted. With the monkey standing in a specially constructed cage, all animals randomly received the following agents at one-week interval: (1) MS 1 mg, IT; (2) SST 50 micrograms, IT; (3) SST 250 micrograms, IT; and (4) SST 250 micrograms, IT + intramuscular (IM) naloxone 400 micrograms. The measured withdrawal latency (HPWL) was converted to the maximal percentage effect (MPE %) for comparison. The HPWL was measured at predrug and 5, 15, 30, 45, 60, 90 and 120 min after injection. Venous blood sample was obtained every 15 min to determine the plasma SST level by radioimmunoassay (RIA) technique in group 3 only. The results showed that MS (1 mg, IT) produced potent antinociception (MPE 100%) for more than 2 h. Intrathecal SST 50 micrograms, however, induced mild antinociception (MPE 43%) for only a short period and a 5-fold larger dose (250 micrograms) did not significantly change the nociceptive threshold with MPE only up to 47%.(ABSTRACT TRUNCATED AT 250 WORDS)

Analgesics↗

PubMed1988