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Biomedical subjects

D W Chung

Publications and source records attributed to D W Chung.

At least 19 recordsLinked to original sources
PubMed2006

A rapid enzyme-linked assay for ADAMTS-13.

BACKGROUND: A deficiency in the plasma metalloprotease ADAMTS-13 is associated with deposition of microvascular thrombi that cause thrombotic thrombocytopenic purpura. Current assays for ADAMTS-13 are technically complex and time-consuming. The objective of this study is to devise a rapid and sensitive assay for ADAMTS-13 activity in plasma and verify the site of cleavage. METHOD: A new enzyme-linked substrate, which contains a core ADAMTS-13-specific peptide conjugated to horseradish peroxidase (HRP) at the N-terminus, and labeled with biotin at the C-terminus, was constructed. After cleavage of this substrate by plasma ADAMTS-13 and removal of uncleaved substrate by adsorption with streptavidin-agarose, ADAMTS-13 activity was quantitated by determining the unadsorbed HRP activity remaining in solution. Levels of inhibitory antibodies in test plasma were also determined by measuring the residual ADAMTS-13 activity after varying amounts of test plasma were incubated with a known amount of ADAMTS-13. RESULTS: Plasma ADAMTS-13 activity was readily determined in approximately 60 min (coefficient of variation 5.8%) using 1 microL of test plasma. Amino acid sequencing of the cleavage product confirmed that cleavage occurred at the Tyr1605-Met1606 bond in the substrate. ADAMTS-13 activities in the plasma of five TTP patients were below 2%. Inhibitory antibody titers in these samples varied from undetectable to 81 BU mL(-1). CONCLUSION: The HRP-linked substrate provides a rapid, sensitive, and reproducible way of determining the levels of ADAMTS-13 activity and inhibitory antibodies in plasma.

ADAM Proteins↗
PubMed2003

The enhanced expression of c-Jun immunoreactivity in the adrenalectomized gerbil hippocampus.

Recent in vitro and in vivo studies have shown that glucocorticoids have a profound influence on the survival of hippocampal neurones, and that the depletion of glucocorticoids as a result of adrenalectomy (ADX) reduces nerve growth factor levels in the hippocampus. It is also believed that ADX is associated with the seizure susceptibility of the Mongolian gerbil. In the present study, the choronological changes of c-jun immunoreactivity were investigated after ADX in the hippocampal formations in the seizure-prone gerbil model. In the sham hippocampus, c-jun immunoreactivity was not observed in the neurones of the hippocampus proper and dentate gyrus. C-jun immunoreactive neurones appeared 3 h after ADX in the neurones of the CA1 area and dentate gyrus, and these immunoreactivities peaked 24 h after ADX and then gradually decreased. These results suggest that, in the adrenalectomized gerbil, c-jun may be expressed in the neurones of the hippocampus in compensation for glucocorticoid deficit. The result of enhanced c-jun expression of the hippocampal formation provides anatomical support for the hypothesis that c-jun may play a role in the reduction of seizure activity.

Adaptation, Physiological↗
PubMed1999

Protease-activated receptor 1 is the primary mediator of thrombin-stimulated platelet procoagulant activity.

The activation of human platelets by thrombin is mediated primarily by protease-activated receptors (PARs). PAR1 and PAR4 are present on human platelets and are activated by the hexapeptides SFLLRN and GYPGQV, respectively. To further characterize the involvement of PAR1 and PAR4 in platelet activation, the ability of SFLLRN or GYPGQV to generate annexin V binding to newly exposed phospholipids on the platelet surface and generate procoagulant activity has been examined. Exposure of phosphatidylserine and phosphatidylethanolamine on platelets, as determined by an increase in annexin V binding, was strongly stimulated by SFLLRN, thrombin, and collagen, but only to a minor extent by GYPGQV. In a clotting assay initiated with factor VIIa, soluble tissue factor, and calcium, the clotting time in the absence of platelets was >5 min. In the presence of unstimulated platelets, the clotting time was 200 +/- 20 sec. In the presence of platelets activated with SFLLRN or collagen, the clotting time decreased to 100 +/- 10 sec. This shortening of the clotting time is equivalent to about a 5-fold increase in coagulant activity when stimulated platelets are compared with unstimulated platelets and activated platelets are used as a reference. These results indicate that thrombin initiates a very strong response in platelets through PAR1, leading to exposure of anionic phospholipids that support blood clotting. The response mediated by PAR4, however, was limited to platelet aggregation and similar to that triggered in platelets by weaker agonists such as ADP or epinephrine.

Annexin A5↗
PubMed1999

Sleep problems in Chinese elderly in Hong Kong.

BACKGROUND: To examine the sleep habits and one-year prevalence of sleep disturbance (difficulty in falling asleep, broken sleep and early morning wakening) as well as insomnia (subjectively inadequate or poor sleep) in an elderly Chinese population in Hong Kong. METHOD: In Phase 1, a representative sample of elderly aged 70 years or above were interviewed with a sleep questionnaire, and Cantonese versions of the Mini-Mental State Examination (CMMSE) and Geriatric Depression Scale(CGDS). In Phase 2, those with scores suggestive of cognitive impairment on CMMSE or depression on CGDS were interviewed by psychiatrists for making clinical diagnoses according to DSM IV. RESULTS: 1,034 elderly were interviewed in Phase 1. Occasional or persistent sleep disturbance were reported by 75% and insomnia in 38.2% of elderly. Slightly less than half of elderly with sleep disturbance complained of insomnia. Advancing age was associated with a higher rate of sleep disturbance while females had a higher rate of insomnia. Factors associated with sleep disturbance and insomnia included poor perceived health, past history of smoking, current depressive disorders, more chronic physical illness, more life events and more somatic complaints. Only 2.8% of the sample had taken sleeping pills within a one-year period. CONCLUSIONS: Sleep disturbance and insomnia are two separate but overlapping constructs and should be differentiated. Sleep disturbance is very common in the elderly and may be due to physiological changes with ageing. In contrast, those with a concommitant complaint of insomnia have impaired physical and mental health and may merit more medical attention.

Aged↗
PubMed1999

Gradual withdrawal of long-term anticholinergic antiparkinson medication in Chinese patients with chronic schizophrenia.

Previous antiparkinson drug withdrawal studies involving white subjects have yielded inconclusive findings, whereas there is a paucity of data concerning Asian patients. A double-blind, placebo-controlled, randomized trial using gradual withdrawal of antiparkinson medication was conducted to evaluate the need for maintenance antiparkinson therapy for clinically stable Chinese patients with chronic schizophrenia. Seventy-five schizophrenic subjects who had received a diagnosis according to DSM-IV who had been ill for at least 5 years and on antipsychotic and antiparkinson medication for a minimum of 2 years entered the study. After baseline assessment, 58 subjects were matched according to age, sex, age at onset, length of illness, dose and length of antipsychotic and antiparkinson medication, and the presence of various extrapyramidal side effects. Randomly assigned dose-reduction and control groups were formed consisting of 29 subjects each. Trihexyphenidyl (THP), the only oral antiparkinson drug used in the study, was reduced by 1 mg every 2 weeks, whereas other psychotropic medication remained unchanged. Monthly assessment was performed using the Brief Psychiatric Rating Scale, Hamilton Rating Scale for Depression, Abnormal Involuntary Movement Scale, Simpson-Angus Scale, Barnes Akathisia Rating Scale, and the Nursing Observation Scale for Inpatient Evaluation-30. Complete withdrawal of THP was possible in 25 (90%) of the 28 subjects who completed the study, whereas considerable dose reduction was achieved in the remaining 3 subjects. There were no significant differences between dose reduction and control groups on any of the rating scales at the completion of the study. Our results suggest that long-term prophylactic administration of antiparkinson medication is unnecessary in the treatment of the majority of Chinese patients with chronic schizophrenia because withdrawal was accomplished without adverse mental or motor effects.

Adult↗
PubMed1998

Molecular cloning of human ABPL, an actin-binding protein homologue.

Based on two partial cDNA sequences, a full-length cDNA sequence for an actin-binding like protein previously named ABPL has been isolated and characterized. ABPL is homologous to the human actin-binding proteins ABP-280 and ABP-278. The predicted sequence for ABPL is 2,705 amino acids in length with a calculated molecular mass of 289 kDa. It contains an amino terminal actin-binding domain followed by 24 tandem repeats of approximately 96 amino acids. Two hinge regions, Hinge I and Hinge II, were located prior to repeats 16 and 24, respectively. An isoform of ABPL lacking Hinge I, with a calculated molecular mass of 286 kDa, was also identified by the reverse transcriptase PCR (RT-PCR) method. A comparison with genomic sequences indicated the isoform resulted from alternative RNA splicing. ABPL has a unique insertion sequence of 82 amino acids in repeat 20 that was not present in the other two homologues and has a tissue distribution that was also different from the other two homologues.

Actins↗
PubMed1998

A novel human actin-binding protein homologue that binds to platelet glycoprotein Ibalpha.

Glycoprotein (GP)Ib-IX-V is one of the major transmembrane complexes present on the platelet surface. Its extracellular domain binds von Willebrand factor (vWF) and thrombin, while its intracellular domain associates tightly with the cytoskeleton through the actin-binding protein (ABP)-280, also known as filamin. In the present study, a full-length cDNA coding for a human ABP homologue has been cloned and sequenced. This protein was identified by the yeast two-hybrid screening procedure via its interaction with the intracellular domain of GPIbalpha. Initially, a 1.3-kb partial cDNA was isolated from a megakaryocyte-like cell line (K562) cDNA library followed by a full-length cDNA of 9.4 kb that was identified in a human placenta library. The full-length cDNA encoded a protein of 2,578 amino acids with a calculated molecular weight of 276 kD (ABP-276). The amino terminal 248 amino acids contained an apparent actin binding domain followed by 24 tandem repeats each containing about 96 amino acids. The amino acid sequence of the protein shared a high degree of homology with human endothelial ABP-280 (70% identity) and chicken filamin (83% identity). However, the 32 amino acid Hinge I region in ABP-280 that contains a calpain cleavage site conferring flexibility on the molecule, was absent in the homologue. An isoform containing a 24 amino acid insertion with a unique sequence at the missing Hinge I region was also identified (ABP-278). This isoform resulted from alternative RNA splicing. ABP-276 and/or ABP-278 were present in all tissues examined, but the relative amount varied in that some tissue contained both forms, while other tissue contained predominately one or the other.

Amino Acid Sequence↗
PubMed1998

Prevalence of dementia in Chinese elderly in Hong Kong.

This study examines the prevalence of dementia in elderly Chinese aged 70 years and older in Hong Kong using a two-phase design. In phase 1, 1,034 elderly were interviewed with the Cantonese version of the Mini-Mental State Examination. Those who scored below the cutoff points and a subsample of those with scores in the normal range were interviewed in phase 2 to identify those with dementia. The overall weighted prevalence of dementia in our subjects was 6.1 +/- 0.7%, which is at the lower end of the range of rates reported in whites. Alzheimer's disease (AD) accounted for 64.6% and vascular dementia, 29.3%. Our results, together with previous studies in Chinese populations, suggest that the rates of AD in Chinese are low compared with those in whites. Substantial differences are possible in the epidemiology of dementia across cultures related to interactions of genetic and environmental factors.

Age Distribution↗
PubMed1997

The polymerization pocket "a" within the carboxyl-terminal region of the gamma chain of human fibrinogen is adjacent to but independent from the calcium-binding site.

The carboxyl-terminal region of the gamma chain of fibrinogen is involved in calcium binding, fibrin polymerization, factor XIIIa-mediated cross-linking, and binding to the platelet fibrin(ogen) receptor. Protein fragments encoding amino acids Val143 to Val411 (rFbggammaC30) or Val143 to Leu427 (gamma'C30) from the carboxyl end of the gamma or gamma' chains, respectively, of human fibrinogen were expressed in yeast (Pichia pastoris) and characterized as to their cross-linking by factor XIIIa, polymerization pocket, and calcium-binding site. rFbggammaC30 and gamma'C30 were both readily cross-linked by factor XIIIa, but only rFbggammaC30 was capable of inhibiting thrombin-induced platelet aggregation. Two mutants, gammaC30-Q329R and gammaC30-D364A, which were based on the three-dimensional structure of the polymerization pocket within rFbggammaC30 and on information derived from naturally occurring mutant fibrinogens, were also expressed and characterized. rFbggammaC30 inhibited (desAA)fibrin polymerization in a dose-dependent manner, while the two mutant forms did not. Similarly, rFbggammaC30 and gamma'C30 were protected from plasmin degradation by the presence of Ca2+ or the peptide Gly-Pro-Arg-Pro, indicating that a functional Ca2+-binding site and polymerization pocket are contained within each of these fragments. The mutant fragments, however, were protected from plasmin only by metal ions, while no protective effect was conferred by GPRP or by any other peptide tested. These results indicate that the polymerization pocket "a", which binds the peptide GPRP, functions independently from the nearby calcium-binding site and that amino acids Gln329 and Asp364 play a crucial role in fibrin polymerization.

Amino Acid Sequence↗
PubMed1997

The primary fibrin polymerization pocket: three-dimensional structure of a 30-kDa C-terminal gamma chain fragment complexed with the peptide Gly-Pro-Arg-Pro.

After vascular injury, a cascade of serine protease activations leads to the conversion of the soluble fibrinogen molecule into fibrin. The fibrin monomers then polymerize spontaneously and noncovalently to form a fibrin gel. The primary interaction of this polymerization reaction is between the newly exposed N-terminal Gly-Pro-Arg sequence of the alpha chain of one fibrin molecule and the C-terminal region of a gamma chain of an adjacent fibrin(ogen) molecule. In this report, the polymerization pocket has been identified by determining the crystal structure of a 30-kDa C-terminal fragment of the fibrin(ogen) gamma chain complexed with the peptide Gly-Pro-Arg-Pro. This peptide mimics the N terminus of the alpha chain of fibrin. The conformational change in the protein upon binding the peptide is subtle, with electrostatic interactions primarily mediating the association. This is consistent with biophysical experiments carried out over the last 50 years on this fundamental polymerization reaction.

Dimerization↗
PubMed1997

Crystal structure of a 30 kDa C-terminal fragment from the gamma chain of human fibrinogen.

BACKGROUND: Blood coagulation occurs by a cascade of zymogen activation resulting from minor proteolysis. The final stage of coagulation involves thrombin generation and limited proteolysis of fibrinogen to give spontaneously polymerizing fibrin. The resulting fibrin network is covalently crosslinked by factor XIIIa to yield a stable blood clot. Fibrinogen is a 340 kDa glycoprotein composed of six polypeptide chains, (alphabetagamma)2, held together by 29 disulfide bonds. The globular C terminus of the gamma chain contains a fibrin-polymerization surface, the principal factor XIIIa crosslinking site, the platelet receptor recognition site, and a calcium-binding site. Structural information on this domain should thus prove helpful in understanding clot formation. RESULTS: The X-ray crystallographic structure of the 30 kDa globular C terminus of the gamma chain of human fibrinogen has been determined in one crystal form using multiple isomorphous replacement methods. The refined coordinates were used to solve the structure in two more crystal forms by molecular replacement; the crystal structures have been refined against diffraction data to either 2.5 A or 2.1 A resolution. Three domains were identified in the structure, including a C-terminal fibrin-polymerization domain (P), which contains a single calcium-binding site and a deep binding pocket that provides the polymerization surface. The overall structure has a pronounced dipole moment, and the C-terminal residues appear highly flexible. CONCLUSIONS: The polymerization domain in the gamma chain is the most variable among a family of fibrinogen-related proteins and contains many acidic residues. These residues contribute to the molecular dipole moment in the structure, which may allow electrostatic steering to guide the alignment of fibrin monomers during the polymerization process. The flexibility of the C-terminal residues, which contain one of the factor XIIIa crosslinking sites and the platelet receptor recognition site, may be important in the function of this domain.

Amino Acid Sequence↗
PubMed1997

REM sleep behaviour disorder in the elderly.

REM sleep behaviour disorder is a recently described parasomnia characterized by a history of excessive motor activities during sleep and is associated with pathological absence of muscle atonia during REM sleep. There is very limited literature on the condition. Two out of 349 elderly patients referred to a psychogeriatric unit were identified to have REM sleep behaviour disorder. These two patients are presented to illustrate the clinical features of the condition. Both of them showed a good response to clonazepam treatment.

Aged↗
PubMed1996

The role of betagamma and alphagamma complexes in the assembly of human fibrinogen.

The role of alphagamma and betagamma dimers as intermediates in the assembly of fibrinogen was examined in cell fusion experiments using stably transfected baby hamster kidney cell lines expressing one or combinations of fibrinogen chains. Fibrinogen was readily formed and secreted into the culture media when cells co-expressing beta and gamma chains and generating betagamma complexes were fused with cells expressing only the alpha chain. Likewise, when cells co-expressing alpha and gamma chains and generating alphagamma complexes were fused with cells expressing only the beta chain, fibrinogen was also formed and secreted. The relative amounts of alphagamma or betagamma intermediates observed during fibrinogen biosynthesis were determined by the levels of the component chains; i.e. when the beta chain was limiting, the alphagamma dimer was the predominant intermediate; likewise, when the alpha chain was limiting, the betagamma complex was the predominant intermediate. The incorporation of preformed alphagamma and betagamma complexes into secreted fibrinogen did not require concurrent protein synthesis, as shown by experiments employing cycloheximide. These data strongly support the role of alphagamma and betagamma complexes as functional intermediates in the assembly of fibrinogen.

Animals↗
PubMed1996

The assembly of human fibrinogen. The role of the amino-terminal and coiled-coil regions of the three chains in the formation of the alphagamma and betagamma heterodimers and alphabetagamma half-molecules.

Fibrinogen is a plasma protein consisting of six polypeptide chains which are linked by disulfide bonds. During protein synthesis, assembly of the molecule proceeds through the formation of alphagamma and betagamma heterodimers followed by the generation of alphabetagamma half-molecules and dimerizing to generate the mature six-chain molecule. In the present study, sequences required for the formation of the alphagamma and betagamma heterodimers were examined in stably transfected baby hamster kidney cells expressing combinations of normal as well as modified polypeptide chains. Deletion of the amino terminus and the proximal first half of the coiled-coil region of the three fibrinogen chains had little or no effect on heterodimer and half-molecule formation. These deletions, however, did prevent half-molecules from forming the six-chain molecule. Deletion of the distal second half of the coiled-coil region of each chain completely prevented the assembly process. Point mutations in the second half of the coiled-coil region also indicated that hydrophilic residues that form ion pairs between interacting chains were not critical in the formation of the heterodimeric complexes. These results suggest that the initial formation of the alphagamma and betagamma complexes depends primarily on hydrophobic interactions of amino acids located in the second half of the coiled-coil region of the molecule. These interactions occur in the rough endoplasmic reticulum in the presence of various chaperones such as BiP.

Amino Acid Sequence↗
PubMed1996

Mapping of the discontinuous kininogen binding site of prekallikrein. A distal binding segment is located in the heavy chain domain A4.

Prekallikrein, the precursor to the serine proteinase kallikrein, circulates in plasma in an equimolar complex with H-kininogen. The binding to H-kininogen is mediated by the kallikrein heavy chain consisting of four "apple" domains, A1-A4, which attaches to H-kininogen with high specificity and affinity (KD = 83 nM). At least two distinct portions of the kallikrein heavy chain form this H-kininogen binding site: a proximal segment located in the NH2-terminal fragment of the heavy chain encompassing A1, and distal segment(s) located in COOH-terminal fragment spanning domains A2-A4. The proximal binding segment has been located to amino acid positions 56-86 of A1. To precisely map the distal binding segment, we have identified monoclonal antibodies directed to the COOH-terminal fragment which interfere with the H-kininogen-prekallikrein complex formation. Monoclonal antibody 13G11 binds to recombinant apple domain A4 but not to domain A3 of the prekallikrein heavy chain. Deletion mutagenesis of domain A4 narrowed down the target epitope of 13G11 to the center portion of domain A4, positions 284-331. Direct binding studies of H-kininogen to various domain A4 constructs revealed that the distal H-kininogen binding portion is located on a segment of 48 residues, which overlaps the 13G11 epitope. Hence the tight interaction of H-kininogen and prekallikrein is mediated by at least two separate sequence segments located in domains A1 and A4, respectively, of the prekallikrein heavy chain. The isolated distal binding segment significantly prolongs the partial thromboplastin time of reconstituted Williams plasma thus stressing the critical role of the prekallikrein-H-kininogen complex formation in the initiation of the endogenous blood coagulation cascade.

Animals↗
PubMed1995

Liver-specific expression of the human factor VII gene.

Promoter and silencer elements of the immediate 5' flanking region of the gene coding for human factor VII were identified and characterized. The major transcription start site, designated as +1, was determined by RACE (rapid amplification of cDNA ends) analysis of human liver cDNA and was found to be located 50 bp upstream from the translation start site. Two minor transcription start sites were found at bp +32 bp and +37. Progressive deletions of the 5' flanking region were fused to the chloramphenicol acetyltransferase reporter gene and transient expression in HepG2 and HeLa cells was measured. Two promoter elements that were essential for hepatocyte-specific transcription were identified. The first site, FVIIP1, located at bp -19 to +1, functioned independently of orientation or position and contributed about one-third of the promoter activity of the factor VII gene. Electrophoretic mobility-shift, competition, and anti-hepatocyte nuclear factor 4 (HNF4) antibody supershift experiments demonstrated that this site contained an HNF-4 binding element homologous to the promoters in the genes coding for factor IX and factor X. The second site, FVIIP2, located at bp -50 to -26, also functioned independent of orientation or position and contributed about two thirds of the promoter activity in the gene for factor VII. Functional assays with mutant sequences demonstrated that a 10-bp G + C-rich core sequence which shares 90% sequence identity with the prothrombin gene enhancer was essential for the function of the second site. Mobility-shift and competition assays suggested that this site also binds hepatic-specific factors as well as the transcription factor Sp1. Two silencer elements located upstream of the promoter region spanning bp -130 to -103 (FVIIS1 site) and bp -202 to -130 (FVIIS2) were also identified by reporter gene assays.

Antibodies↗
PubMed1995

Characterization of the 5'-flanking region of the gene for the alpha chain of human fibrinogen.

The 5'-flanking region of the gene coding for the alpha chain of human fibrinogen was isolated, sequenced, and characterized. The principal site of transcription initiation was determined by primer extension analysis and the RNase protection assay and shown to be at an adenine residue located 55 nucleotides upstream from the initiator methionine codon, or 13,399 nucleotides down-stream from the polyadenylation site of the gene coding for the gamma chain. Transient expression of constructs containing sequentially deleted 5'-flanking sequences of the alpha chain gene fused to the chloramphenicol acetyltransferase reporter gene showed that the promoter was liver-specific and inducible by interleukin 6 (IL-6). The shortest DNA fragment with significant promoter activity and full response to IL-6 stimulation encompassed the region from -217 to +1 base pairs (bp). Although six potential IL-6 responsive sequences homologous to the type II IL-6 responsive element were present, a single sequence of CTGGGA localized from -122 to -127 bp was shown to be a functional element in IL-6 induction. A hepatocyte nuclear factor 1 (HNF-1) binding site, present from -47 to -59 bp, in combination with other upstream elements, was essential for liver-specific expression of the gene. A functional CCAAT/enhancer binding protein site (C/EBP, -134 to -142 bp) was also identified within 217 bp from the transcription initiation site. An additional positive element (-1393 to -1133 bp) and a negative element (-1133 to -749 bp) were also found in the upstream region of the alpha-fibrinogen gene.

Amino Acid Sequence↗