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S B Hwang

Publications and source records attributed to S B Hwang.

At least 37 records · Page 2Linked to original sources
PubMed1994

Isoprenylation masks a conformational epitope and enhances trans-dominant inhibitory function of the large hepatitis delta antigen.

Hepatitis delta antigen (HDAg) consists of two species, large (LHDAg) and small (SHDAg), which are identical in sequence except that the large form contains 19 extra amino acids at the C terminus. The large form is prenylated on the Cxxx motif. The small form can trans activate HDV RNA replication, while the large form inhibits it. To determine the molecular basis for their differential functions, we examined the effects of prenylation on the conformation and function of HDAg. We show that the presence of prenylates masks a conformational epitope which is present in SHDAg but hidden in wild-type LHDAg; this epitope becomes exposed in all of the nonprenylated mutant LHDAgs. Prenylation also plays a major role in conferring the trans-dominant negative inhibitory activity of LHDAg, since the loss of prenylation in LHDAg reduced its inhibitory activity. The primary amino acids of the C-terminal sequence also contributed to the maintenance of the HDAg protein conformation; a prenylated LHDAg mutant with a five-amino-acid deletion had an exposed C-terminal epitope. By examining LHDAg mutants which have deletions of various extents of C-terminal sequence, with or without the prenylation motif, we have further shown that all of the prenylated mutants have much higher levels of trans-dominant suppressor activities than do the corresponding nonprenylated mutants. Surprisingly, a few nonprenylated LHDAg mutants were able to trans activate HDV RNA replication, while all of the prenylated ones lost this function. These results suggest that isoprenylates cause the masking of a conformational epitope of HDAg and that conformational differences between the large and small HDAgs account for the differences in their trans-activating and trans-dominant inhibitory biological activities.

Antigens, Viral↗
PubMed1993

A unique conformation at the carboxyl terminus of the small hepatitis delta antigen revealed by a specific monoclonal antibody.

Two forms of the hepatitis delta antigen (HDAg), a small (24 kDa) and a large (27 kDa) one, have different functions in the hepatitis delta virus (HDV) replication cycle. The small HDAg trans-activates RNA replication, while the large one inhibits RNA replication. The lack of the trans-acting activity in the large HDAg, even though it contains the complete sequence of small HDAg, suggests that the large HDAg lacks a certain functional conformation. To test such a possibility, monoclonal antibodies (MAbs) were generated from mice immunized with recombinant baculovirus-expressed small HDAg. As expected, most of the MAbs recognized both small and large HDAg. In addition, one MAb (9E4) was obtained which recognized only the small HDAg, but not the large one, in Western blot and immunoprecipitation analysis, suggesting that it recognized an epitope unique to small HDAg. However, MAb 9E4 detected both forms of HDAg in virus-infected cells by immunofluorescence and reacted with TrpE-large HDAg fusion proteins expressed in Escherichia coli, suggesting that this MAb recognizes a conformation-dependent epitope which is not present in the native large HDAg molecule but is detectable in LHDAg when its conformation is altered. The 9E4 epitope was mapped within a region of 32 amino acids at the carboxyl-terminus of small HDAg, indicating that this region contains a unique conformation not present in the native molecule of large HDAg. Since this is the only structure identified that is unique to small HDAg, the C-terminal region may contain the domain associated with the biological activities unique to the small HDAg.

Animals↗
PubMed1993

Isoprenylation mediates direct protein-protein interactions between hepatitis large delta antigen and hepatitis B virus surface antigen.

Hepatitis delta antigen (HDAg) consists of two protein species of 195 and 214 amino acids, respectively, which are identical in sequence except that the large HDAg has additional 19 amino acids at its C terminus and is prenylated. Previous studies have shown that the large HDAg and the surface antigen of hepatitis B virus (HBsAg) together can form empty hepatitis delta virus (HDV) particles. To understand the molecular mechanism of HDV virion morphogenesis, we investigated the possible direct protein-protein interaction between HDAg and HBsAg. We constructed recombinant baculoviruses expressing the major form of HBsAg and various mutant HDAgs and used these proteins for far-Western protein binding assays. We demonstrated that HBsAg interacted specifically with the large HDAg but not with the small HDAg. Using mutant HDAgs which have defective or aberrant prenylation, we showed that this interaction required isoprenylates on the cysteine residue of the C terminus of the large HDAg. Isoprenylation alone, without the remainder of the C-terminal amino acids of the large HDAg, was insufficient to mediate interaction with HBsAg. This study demonstrates a novel role of prenylates in HDV virion assembly.

Amino Acid Sequence↗
PubMed1993

MK 287: a potent, specific, and orally active receptor antagonist of platelet-activating factor.

MK 287 (L-680,573), a tetrahydrofuran analog, potently inhibited [3H]C18-PAF binding to human platelet, polymorphonuclear leukocyte (PMN) and lung membranes with K1 values of 6.1 +/- 1.5, 3.2 +/- 0.7, and 5.49 +/- 2.3 nM, respectively. The inhibitory effects are stereospecific and competitive. The racemate, L-668,750 is less potent and the enantiomer, L-680,574 is 20-fold less potent than MK 287. Inhibition of the binding of [3H]C18-PAF to human PMN membranes by MK 287 was associated with the reduction of the affinity of the radioligand but not the number of the receptor sites. Binding of other radioligands (e.g., LTB4, LTC4, C5a, FMLP) to their specific receptors was unaltered at 1-10 microM MK 287. [3H]MK 287 bound to membranes from human platelets and PMNs: KD = 2.1 +/- 0.6 and 2.9 +/- 1.2 nM, respectively. When examined on isolated human cells, MK 287 potently and selectively inhibited PAF-induced aggregation of platelets in plasma (ED50 = 56 +/- 38 nM) or gel-filtered platelets (ED50 = 1.5 +/- 0.5 nM) and elastase release from PMNs (ED50 = 4.4 +/- 2.6 nM). In studies in vivo, MK 287 inhibited PAF-induced lethality in mice (ED50 = 0.8 mg/kg orally) and PAF-induced bronchoconstriction in guinea pigs (ED50 = 0.18 mg/kg intraduodenally and 0.19 mg/kg intravenously). Inhibition of PAF-induced bronchoconstriction was accompanied by parallel rightward shifts in concentration-response curves for PAF-induced platelet aggregation measured ex vivo.

Administration, Oral↗
PubMed1992

Hepatitis delta antigen expressed by recombinant baculoviruses: comparison of biochemical properties and post-translational modifications between the large and small forms.

Hepatitis delta virus (HDV) encodes only one protein, the hepatitis delta antigen (HDAg). Two forms of HDAg, a large (27 kDa) and a small (24 kDa) one, participate in the various steps of HDV replication. To further understand the properties of HDAg, we have constructed recombinant baculoviruses and expressed both forms of the HDAg in insect cells. The gene encoding HDAg was placed under the control of the polyhedrin promoter of Autographa Californica nuclear polyhedrosis virus (AcNPV) by homologous recombination. When Spodoptera frugiperda (Sf9) cells were infected with the recombinant viruses, both the small HDAg and the large HDAg were expressed at high levels. The HDAgs produced by the recombinants were similar in size and antigenic properties to those of the proteins produced in mammalian hepatoma cell lines. It was also localized exclusively in the nuclei. In addition, both proteins bound to HDV RNA in an in vitro assay. No difference in the RNA-binding affinity was noted between the two forms of HDAg, suggesting that the trans-dominant inhibitory activity of the large HDAg on HDV replication is not due to its competition with the small HDAg for RNA binding. Two RNA-protein complexes could be detected, suggesting either that there are at least two binding sites on the HDV RNA or that HDAg binds to HDV RNA in two multimeric forms. We have further shown that both the large and the small HDAgs are phosphoproteins, with the former having an approximately sixfold higher level of phosphorylation. Finally, it was demonstrated that the large HDAg was isoprenylated, while the small one was not. These differences in post-translational modifications are the first differences in biochemical properties demonstrated between the two forms and may explain the differential effects of the large and small HDAgs on HDV RNA replication and virus packaging.

Animals↗
PubMed1991

Specific binding of tritium-labeled inositol 1,4,5-trisphosphate to human platelet membranes: ionic and GTP regulation.

Specific, saturable and reversible binding of tritium-labeled inositol 1,4,5-trisphosphate [( 3H]Ins(1,4,5)P3) to human platelet membranes is demonstrated. The Ins(1,4,5)P3-binding sites are abundant and display high selectivity for Ins(1,4,5)P3. Other inositol phosphates exhibit much lower affinity for this site. The specific [3H]Ins(1,4,5)P3 binding was found to be modulated by pH, monovalent and divalent cations, and GTP. A sharp increase in binding occurs at slightly alkaline pH. The monovalent cations, Na+, K+ and Li+ almost double the binding at 30 mM. Mg2+ inhibits the specific [3H]Ins(1,4,5)P3 binding. At low concentrations of Ca2+, the binding is inhibited, but at concentrations higher than 5 mM the binding is potentiated and increases by almost 5-fold at 100 mM. Similar pattern of the effects is also observed for Mn2+ and Sr2+. The specific [3H]Ins(1,4,5)P3 binding is specifically inhibited by GTP. Other nucleotides also inhibit the binding but at higher concentrations. From saturation binding studies, Ca2+ potentiation seems to be due to the conversion of the receptor from the low-affinity state to the high-affinity one. In the absence of Ca2+, the Scatchard plot is nonlinear and concave, and statistically can be fitted best with two equilibrium dissociation constants (Kd values), 0.19 +/- 0.11 and 13.2 +/- 18.1 nM, respectively, for high- and low-affinity binding sites. However, in the presence of 100 mM CaCl2, the Scatchard plot reveals only the high-affinity binding sites with a Kd value of 0.32 +/- 0.15 nM. The specific Ins(1,4,5)P3 receptor in human platelets could therefore exist in multiple conformational states to regulate the intracellular Ca2+ concentration.

Animals↗
PubMed1991

High affinity receptor binding of platelet-activating factor in rat peritoneal polymorphonuclear leukocytes.

Rat peritoneal polymorphonuclear leukocytes (PMNs) showed a single class of high affinity binding sites for platelet-activating factor (PAF) with an equilibrium dissociation constant (KD) of 4.74 (+/- 2.59) nM. Each cell contained 2.79 (+/- 1.40) x 10(4) receptors. In the isolated membranes at pH 7.0 in 10 mM MgCl2, 10 mM Tris and 0.25% bovine serum albumin, the KD value was 0.61 (+/- 0.1) nM, which is roughly identical to the KD values reported previously for various membrane systems under identical ionic conditions. The receptors were highly specific for PAF. Several receptor antagonists that are reported to inhibit the binding of [3H]PAF and the PAF-induced function in platelets could fully displace the binding. The biologically inactive enantiomer (enantio-C16-PAF), a PAF analog, azido-PAF, and an indene derivative of the PAF receptor antagonist, L-651,142, had different potencies to inhibit [3H]PAF binding to rat and human PMN membranes. L-652,731, a tetrahydrofuran analog of the PAF receptor antagonist was about 10 times more potent to inhibit the binding in rat liver tissues than in rat PMNs. These results suggest that PAF receptors on human and rat PMNs are not identical and that PAF receptor subtypes may exist in rat liver and PMNs.

Animals↗
PubMed1991

L-659,989: a useful probe in the detection of multiple conformational states of PAF receptors.

L-659,989 is a potent, specific and competitive platelet-activating factor (PAF) receptor antagonist. The 2,5-tritium labeled L-659,989, similar to [3H]PAF, specifically binds to rabbit platelet membranes with an equilibrium dissociation constant (KD) of 1.60 (+/- 0.20) nM in 10 mM MgCl2. However, guanosine 5'-triphosphate (GTP) and several cations affect the specific binding of [3H]PAF and of [3H]L-659,989 to rabbit platelet membranes in different ways. K+, Mg2+, Ca2+ and Mn2+ potentiate the specific binding of both ligands. Na+ and Li+ inhibit the specific [3H]PAF binding, but enhance the binding of [3H]L-659,989; GTP reduces the [3H]PAF binding but has no effect on the binding of [3H]-L-659,989. Ni2+ inhibits the [3H]L-659-989 binding, but has no effect on the binding of [3H]PAF. In the presence of 150 mM NaCl, [3H]L-659,989 exhibits identical KD and detectable binding sites (Bmax) values as those in the presence of 10 mM MgCl2, while KD And Bmax values of [3H]PAF are dramatically reduced in the presence of 150 mM NaCl compared to those in 10 mM MgCl2. These results suggest the existence of multiple conformational states of the PAF specific receptor and that PAF and L-659,989 bind differently to those states. In the presence of 150 mM NaCl and 1 mM GTP, receptors appear to exist in a single conformational state with an equilibrium dissociation constant (KB) of 0.93 microM for PAF as derived from the Schild plot. In isolated rabbit platelets pretreated with 10 microM ETH 227, a Na(+)-specific ionophore, the detectable [3H]PAF binding sites drop from 260 to 100 binding sites per platelet, but the binding sites for [3H]L-659,989 remain roughly the same. The Na+ binding sites which modulate the conformation of PAF receptors are therefore protected from extracellular Na+ until ionophore is added, and are probably located on the cytoplasmic side of the plasma membrane.

Animals↗
PubMed1991

Prolongation of parturition in the pregnant rat following treatment with a platelet activating factor receptor antagonist.

The presence of platelet activating factor (PAF) in amniotic fluid of women only in labor is indicative of a role for PAF in parturition. In addition, stimulated amnion membrane produces both PAF and prostaglandin E2, each of which is capable of inducing myometrial contraction. To evaluate the involvement of PAF in the process of parturition, we administered the PAF receptor antagonist, L-659,989, to 17-day timed pregnant rats and followed the events of labor and delivery. Administration by mouth with L-659,989 of three concentrations (1.6, 16, and 48 mg/kg/day) did not alter the gestational period; however, the duration of parturition was increased from 2-fold to 5-fold by such treatment. No toxicity of the analog was apparent; treated dams showed no signs of morbidity, and fetal mortality was not significantly altered by treatment with the antagonist. Based on these experiments, it is suggested that the PAF receptor antagonist interferes with the normal progression of events of parturition and that PAF is an integral mediator in initiating the myometrial contraction necessary for expulsion of the fetus.

Animals↗
PubMed1991

[Neolignans from Piper polysyphorum C.DC].

Piper polysyphorum C.DC (Piperaceae) is indigenous to the southern part of China. In the course of screening for inhibitors of platelet activating factor (PAF), the nonpolar fraction was found to exhibit PAF inhibitory activity. One new neclignan named polysyphorin and three new enantiomeric forms of (+)-virolongin, (+)-grandisin and (+)-lancifolin D were isolated. Two known neolignans, wallichinine and hancinone D were also obtained from the same sources. The structure determination was based upon spectroscopic analysis (UV, IR, CD, MS, 1HNMR, 13CNMR, COSY) and derivative preparation. The structure for polysyphorin was established as threo-delta 7'-7-hydroxy-3,4,5,3',5'-pentamethoxy-8-O-4' -neolignan. It is a racemic enantiomer. The PAF inhibitory activities were reported.

Drugs, Chinese Herbal↗
PubMed1991

Wheat germ agglutinin potentiates specific binding of platelet-activating factor to human platelet membranes and induces platelet-activating factor synthesis in intact platelets.

Specific binding of tritium-labeled platelet-activating factor (PAF) and a nonmetabolizable bioactive analog of PAF, 1-O-alkyl-2-N-methylcarbamyl-sn-glyceryl-3-phosphorylcholine, to human platelet membranes was found to be potentiated by wheat germ agglutinin (WGA) and erythroagglutinin. As demonstrated in Scatchard plots, the potentiation effect is due to an increase in the maximal number of receptor sites, with no alteration in the equilibrium dissociation constant. The WGA-potentiated specific binding can be specifically inhibited by N-acetylglucosamine, shows identical affinity for PAF agonists and a receptor antagonist, L-659,989, and has an identical Na+ inhibition pattern to non-treated membranes in the absence of WGA. The WGA-induced potentiation is preferential in the plasma membrane-enriched fraction. The maximal number of receptor sites increases in membranes pretreated with neuraminidase and beta-N-acetylglucosaminidase. Therefore, WGA may bind to an endogenous PAF receptor modulator, which then either dissociates from or associates with the PAF receptor and regulates the receptor conformation. The membrane fraction enriched with intracellular membranes is also enriched with PAF receptors. WGA was also found to increase the maximal aggregation of rabbit and human platelets induced by PAF and to induce the synthesis of PAF, which preceded aggregation in human platelets. An intracellular PAF receptor may also exist, and it could modulate the function of PAF retained inside of the stimulated cells.

Acetylglucosaminidase↗
PubMed1989

Competitive inhibition of tritium-labeled platelet-activating factor binding to rabbit platelet membranes by amiloride and amiloride analogs.

Amiloride and its structural analogs, ethylisopropyl amiloride, benzamil, and dichlorobenzamil, inhibit both the specific [3H]C18-PAF binding to rabbit platelet membranes and PAF-induced aggregation of gel-filtered rabbit platelets. Detailed analysis of binding inhibitions demonstrate that ethylisopropyl amiloride is a competitive inhibitor with an equilibrium dissociation constant (KB) of 23 microM. The concentration of amiloride and its analogs needed to inhibit the PAF-induced aggregation is high and there exists no correlation between their inhibitory activities of platelet aggregation and those of Na+/H+ antiporter. However, the inhibitory effects on the PAF-induced aggregation are parallel to those on the specific [3H]C18-PAF binding. The inhibitory effects of amiloride and its analogs on the activation of platelets are at the PAF-receptor binding step, rather than at the Na+/H+ antiporter.

Amiloride↗
PubMed1989

Characterization of platelet-activating factor (PAF) receptor by specific binding of [3H]L-659,989, a PAF receptor antagonist, to rabbit platelet membranes: possible multiple conformational states of a single type of PAF receptors.

(trans)-2-(3-Methoxy-5-methylsulfonyl-4-propoxyphenyl)-5-(3,4,5- trimethoxyphenyl)tetrahydrofuran (L-659,989) is a potent and orally active platelet-activating factor (PAF)-specific and competitive receptor antagonist. The 2,5-tritium-labeled L-659,989 ([3H]L-659,989) specifically binds to rabbit platelet membranes with an equilibrium dissociation constant (KD) of 1.60 +/- 0.20 nM in 10 mM MgCl2. Several selected PAF analogs and PAF receptor antagonists show equilibrium inhibition constants roughly similar to those found in the specific [3H]PAF binding assay. Other pharmacological agents with no PAF antagonistic activities do not inhibit the specific binding of [3H]L-659,989. K+ and divalent cations such as Mg2+, Ca2+, and Mn2+ potentiate the specific [3H]L-659,989 binding. Na+ and Li+ also enhance but GTP shows no effect on the specific binding of [3H]L-659,989. However, Ni2+ inhibits the specific binding. Scatchard analysis demonstrates that the potentiating effect of these cations is due to an increase in the detectable receptor number for L-659,989. In 10 mM MgCl2 [3H]L-659,989 shows higher receptor number than [3H]PAF. Under various ionic conditions with or without GTP, in which [3H] L-659,989 binding remains approximately the same, C16-PAF shows different potencies in competing against the specific [3H] L-659,989 binding. These results demonstrate the existence of multiple conformational states of the PAF-specific receptor. The variation in the detectable receptor number under different conditions is due to the coexistence of the high and low affinity states and the fact that the low affinity state(s) of the receptor with KD value(s) possibly in the micromolar range cannot be detected in the Scatchard analysis with the radioligand at nanomolar concentrations. In the presence of 150 mM NaCl and 1 mM GTP, receptors exist in a single conformational state with an equilibrium dissociation constant (KB) of 0.931 microM for PAF.

Animals↗
PubMed1988

Identification of a second putative receptor of platelet-activating factor from human polymorphonuclear leukocytes.

Due to multiple molecular species of platelet-activating factor (PAF) and the existence of high affinity binding sites in a variety of cells and tissues, possible existence of PAF receptor subtypes has been suggested. This report shows differences between specific PAF receptors in human leukocytes and platelets. Human polymorphonuclear leukocyte membranes showed high affinity binding sites for PAF with an equilibrium dissociation constant (KD) of 4.4 (+/- 0.3) x 10(-10) M. We compared the relative potencies of several PAF agonists and receptor antagonists between human platelet and human leukocyte membranes. One receptor antagonist (Ono-6240) was found to be 6-10 times less potent in inhibiting the specific [3H]PAF receptor binding, PAF-induced GTPase activity, as well as the PAF-induced aggregation in human leukocytes than in human platelets. Mg2+, Ca2+, and K+ ions potentiated the specific [3H]PAF binding in both systems. Na+ and Li+ ions inhibited the specific [3H]PAF binding to human platelets but showed no effects in human leukocytes. K+ ions decreased the Mg2+-potentiated [3H]PAF binding in human leukocytes but showed no effects in human platelets. PAF stimulates the hydrolysis of [gamma-32P] GTP with an ED50 of about 1 nM, whereas the biological inactive enantiomer shows no activity even at 10 microM in both human platelets and human leukocytes. The PAF-stimulated GTPase in human leukocytes can be abolished by the pretreatment of membranes with pertussis toxin and cholera toxin. However, the PAF-stimulated activity of GTPase in human platelets is insensitive to pertussis toxin and cholera toxin. These results suggest that there exists a second type of PAF receptor in human polymorphonuclear leukocytes, which is structurally different from the one characterized in human platelets, and that the guanine nucleotide-binding protein coupled to PAF receptors in human leukocytes is also different from the one in human platelets.

Blood Platelets↗
PubMed1988

(+/-)-trans-2-(3-Methoxy-5-methylsulfonyl-4-propoxyphenyl)-5-(3,4,5- trimethoxyphenyl)tetrahydrofuran (L-659,989), a novel, potent PAF receptor antagonist.

The title compound, L-659,989, is a highly potent, competitive, and selective antagonist of the binding of [3H]PAF to its receptors in platelet membranes from rabbits and humans. It exhibits equilibrium inhibition constants for PAF binding of 1.1 nM (rabbit) to 9.0 nM (human), values that are at least 1-2 orders of magnitude lower than those of other PAF antagonists tested. L-659,989 potently inhibits PAF-induced aggregation of rabbit platelets and degranulation of rat (ED50 4.5 nM) and human (ED50 10 nM) neutrophils. L-659,989 inhibits PAF-induced extravasation and lysosomal enzyme release in rats, and is active orally in female rats (ED50 0.2 mg/kg) with an extraordinary oral duration of action of 12 to 16 hours at 1.0 mg/kg p.o.

Acetylglucosaminidase↗