Cadmium induces the production of high molecular weight heparan sulfate proteoglycan molecules in cultured vascular endothelial cells

The purpose of the present study is to clarify whether or not cadmium-induced production of heparan sulfate in vascular endothelial cells includes: (1) an increase in the number of heparan sulfate proteoglycan (HSPG) molecules; (2) a formation of longer chains of heparan sulfate; and (3) a binding of more heparan sulfate chains to core proteins. Bovine aortic endothelial cells were cultured and metabolically labeled with [(3)H]glucosamine and [(35)S]sulfate in the presence of cadmium chloride. Radiolabeled HSPGs were separated from more highly charged chondroitin or dermatan sulfate proteoglycans by ion-exchange chromatography and hydrodynamic size of HSPGs was characterized by gel filtration. Heparan sulfate chains were characterized by gel filtration after digestion with either papain or heparitinase. It was found that cadmium increases the incorporation of radioactive precursors into the high molecular weight subclass of HSPGs without a marked change of molecular weight of heparan sulfate chains (approximately 45 kDa). A sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [(35)S]methionine-labeled proteins after heparitinase digestion revealed that the endothelial cells actively produce a HSPG core with a high molecular weight (～400 kDa), probably a perlecan core and the accumulation was increased by cadmium. HSPGs produced by cadmium-treated endothelial cells enhanced the [(3)H]thymidine incorporation in vascular smooth muscle cells cultured in the presence of basic fibroblast growth factor. It was therefore suggested that vascular endothelial cells after exposure to cadmium produce more perlecan molecules and this alteration may contribute to the antithrombogenic property of vascular wall and the formation of atherosclerosis after exposure to the metal through increase in anticoagulant heparan sulfate chains and stimulation of vascular smooth muscle proliferation, respectively.     

Proteoglycans synthesized by cultured bovine aortic smooth muscle cells after exposure to lead: lead selectively inhibits the synthesis of versican, a large chondroitin sulfate proteoglycan

To investigate the effects of lead on the formation of extracellular matrix in the vascular wall, we characterized proteoglycans synthesized by cultured vascular smooth muscle cells after exposure to the metal by biochemical techniques. Confluent cultures of bovine aortic smooth muscle cells were metabolically labeled with [(35)S]sulfate or [(35)S]methionine/cysteine in the presence of lead nitrate. The amount of glycosaminoglycans (GAGs) was evaluated by the incorporation of [(35)S]sulfate into GAGs by the cetylpyridinium chloride precipitation method. The labeled proteoglycans were characterized by DEAE-Sephacel ion exchange chromatography and Sepharose CL-2B molecular sieve chromatography. The GAG M(r) and composition were analyzed by Sepharose CL-6B chromatography, and the core protein M(r) was analyzed by SDS-polyacrylamide gel electrophoresis, before and after digestion with papain or chondroitin ABC lyase. Lead significantly decreased the [(35)S]sulfate incorporation into GAGs accumulated in the cell layer and the conditioned medium. [(35)S]Sulfate-labeled proteoglycans obtained from the cell layer and the conditioned medium were separated into three peaks on DEAE-Sephacel chromatography and only the peak with the highest charge density was decreased by lead. The highly charged peak was eluted near the void volume on Sepharose CL-2B molecular sieve chromatography and sensitive to chondroitin ABC lyase on Sepharose CL-6B chromatography, indicating that lead selectively inhibits the synthesis of large and highly charged chondroitin/dermatan sulfate proteoglycans (CS/DSPGs). However, the size of chondroitin/dermatan sulfate chains of the CS/DSPGs was M(r) approximately 47000 in both the control and lead-treated cultures. On the other hand, lead decreased the accumulation of a large CS/DSPG with a core protein of approximately 450 kDa in the cell layer and the conditioned medium; the core protein was identified as versican core by Western blot analysis. It is therefore suggested that lead inhibits the synthesis of the versican core protein in vascular smooth muscle cells without a change in length of chondroitin/dermatan sulfate side chains. As a result, versican-poor extracellular matrix would be induced by lead in vascular smooth muscle cells.     

The selective endothelin ETA receptor antagonist BQ123 antagonizes endothelin-1-mediated mitogenesis

The mitogenic effects of endothelin isopeptides and the selective ET_A receptor antagonist BQ123 were evaluated in rat aortic vascular smooth muscle cells. Endothelin-1 (ET-1) and endothelin-3 (ET-3) produced concentration-dependent increases in [³H]thymidine incorporation (EC₅₀ = 0.1 and 25 nM, respectively). The ET_B-selective agonist sarafotoxin 6c did not produce significant effects on [³H]thymidine incorporation. BQ123 produced selective and concentration-dependent inhibition of ET-1-mediated [³H]thymidine incorporation. These data demonstrate that ET-1-mediated mitogenesis in vascular smooth muscle is mediated by ET_A receptors.     

Differential effects of cadmium on proteoglycan synthesis of arterial smooth muscle cells: increase in small dermatan sulfate proteoglycans, biglycan and decorin, in the extracellular matrix at low cell density.

Proteoglycans (PGs), especially chondroitin/dermatan sulfate proteoglycans (CS/DSPGs), accumulate and their composition variously changes in atherosclerotic vascular walls. Since cadmium causes atherosclerosis in experimental animals, PGs synthesized by cultured vascular smooth muscle cells after exposure to cadmium were characterized in the present study. Sparse and dense cultures of the cells were metabolically labeled with [35S]sulfate for 24 h in the presence of cadmium chloride at noncytotoxic levels (0.2 microM or less). The incorporation of [35S]sulfate into glycosaminoglycans was determined by the cetylpyridinium chloride precipitation method. The labeled PGs were characterized by DEAE-Sephacel ion exchange chromatography and Sepharose CL-4B molecular sieve chromatography. The M(r) and the glycosaminoglycan composition of small CS/DSPGs were analyzed by SDS-polyacrylamide gel electrophoresis and Sepharose CL-6B chromatography, respectively, before and after digestion with chondroitin ABC lyase or papain. The core proteins were identified by Western blot analysis. These experiments indicate that cadmium differentially acts on the PG synthesis when vascular smooth muscle cell density is low. Specifically, cadmium increased the accumulation of small CS/DSPGs identified as biglycan and decorin in the cell layer of sparse cells. However, the hydrodynamic size and the length of chondroitin/dermatan sulfate chains in the PGs were unaffected by cadmium. On the other hand, cadmium decreased other cell layer-associated PGs that were separated from biglycan and decorin by DEAE-Sephacel chromatography in the sparse cells; as the result, whole glycosaminoglycans were decreased in both the cell layer and the conditioned medium. It is therefore concluded that cadmium may change the composition of PGs in atherosclerotic plaques through induction of biglycan and decorin synthesis and inhibition of other PG synthesis in vascular smooth muscle cells.     

Cortisol effects on the glycosaminoglycan synthesis and molecular weight distribution in vitro

The effect of cortisol at different concentrations on the incorporation rate of [³H]glucosamine and [³⁵S]sulphate into glycosaminoglycans (GAGs) in human fibroblast culture medium was studied. The mol. wt distribution of the synthesized GAGs was determined by Sepharose 2B chromatography. Two sensitivity levels of GAGs to cortisol were observed: at a low cortisol concentration ( 1 × 10^?7 M) only the hyaluronic acid synthesis decreased and no changes were observed in the synthesis of sulphated GAGs or glycoproteins. At a high steroid concentration (1 × 10^?3 M) both the synthesis of hyaluronic acid and sulphated GAGs drastically decreased. The mol. wt distribution of medium GAGs did not change at cortisol concentrations 1 × 10^?9 M-1 × 10^?5 M. The possible role of cortisol in the metabolism of hyaluronic acid in vivo is discussed.     

Possible mechanism for lead inhibition of vascular endothelial cell proliferation: a lower response to basic fibroblast growth factor through inhibition of heparan sulfate synthesis.

Although lead inhibits the proliferation of vascular endothelial cells, the mechanism has been incompletely understood. A lower response to basic fibroblast growth factor (bFGF) of growing bovine aortic endothelial cells after exposure to lead was investigated using a cell culture system in the present study. It was shown that lead significantly decreased the incorporation of [3H]thymidine into the acid-insoluble fraction of the cells but the inhibition disappeared in the presence of bFGF neutralizing antibody. Pretreatment with lead resulted in a reduction of the stimulation by exogenous bFGF on the [3H]thymidine incorporation. Lead decreased endogenous bFGF bound to cell surface heparan sulfate proteoglycans in a concentration-dependent manner but not the high affinity FGF receptor without a change of the accumulation within the cells. In spite of such a change in the endogenous bFGF distribution, the total amount of the growth factor synthesized was not significantly changed by lead. Although the binding of [125I]bFGF to heparan sulfate proteoglycans can be directly inhibited by lead, the inhibition was not so marked. On the other hand, lead markedly suppressed the incorporation of [35S]sulfate into heparan sulfate accumulated in the cell layer and the conditioned medium, suggesting that the metal inhibited the synthesis of the glycosaminoglycan in growing endothelial cells. Inhibition of the [3H]thymidine incorporation by lead was significantly restored by heparin. Since the binding of bFGF to its receptor is strongly promoted by heparan sulfate, the present data suggest that lead inhibits vascular endothelial cell proliferation by induction of a lower response to endogenous bFGF through a suppression of heparan sulfate synthesis.     

Characterization of chondroitin/dermatan sulfate proteoglycans synthesized by bovine retinal pericytes in culture

Pericytes associate with the outside of endothelial cells in microvessels. Previous studies have shown that these cells synthesize glycosaminoglycans (GAGs) but the nature of the core proteins to which these GAGs are attached is unknown. In the present study, cultured bovine retinal pericytes were metabolically labeled with [(3)H]glucosamine, [(35)S]sodium sulfate or (35)S-labeled amino acids and the proteoglycans synthesized by these cells were purified by DEAE-Sephacel ion exchange and molecular sieve Sepharose CL-4B chromatography. Separated proteoglycans were digested with papain, heparitinase or chondroitin ABC lyase and the GAGs characterized by Sepharose CL-6B chromatography. Proteoglycans were also assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis before and after digestion with chondroitin ABC lyase. Pericytes predominantly synthesize and secrete chondroitin or dermatan sulfate proteoglycans (CS/DS PGs) rather than heparan sulfate proteoglycans (HSPGs). Two subclasses of CS/DS PGs are synthesized by pericytes; one is a high M(r) subclass with high charge density. This subclass eluted at the void volume of a Sepharose CL-4B molecular sieve column, was susceptible to chondroitin ABC lyase, and contained core proteins of ca. 550 and 450 kD which were recognized by antibody to versican. The other major subclass eluted at a K(av) ca. 0.45 on a Sepharose CL-4B molecular sieve column, was susceptible to chondroitin ABC lyase, and contained core proteins recognized by antibodies to either biglycan or decorin that separated as a broad band of ca. 50 kDa in SDS-PAGE. A small amount of HSPG was also synthesized by these cells and could be separated from the CS/DS PGs by DEAE-Sephacel chromatography using a linear gradient of 0.1-0.7 M NaCl. Release of GAG chains by protease digestion indicated that the length of GAG chains was approximately M(r) 45000 in biglycan and decorin, approximately M(r) 48000 in the small amount of HSPGs and approximately M(r) 66000 in versican. These proteoglycans resemble those synthesized by vascular smooth muscle cells but differ markedly from those synthesized by vascular endothelial cells.     

Adenosine transporters in vascular smooth muscle and endothelium: Multiple [3H]Nitrobenzylthioinosine binding sites in human umbilical vein endothelium

Cultured vascular smooth muscle and endothelial cells may be useful models for studying the cardiovascular adenosine transport system and metabolism. We have quantified the nucleoside transporter elements of cultured primate vascular smooth muscle, bovine aortic endothelial, and human umbilical vein endothelial cells by radioligand binding and by using membrane preparations of these cells and the nucleoside transporter probe nitrobenzylthioinosine ([³H]NBMPR), a potent and tightly bound inhibitor of nucleoside transport. The binding was rapid, reversible, saturable, and site-specific. Scatchard analysis of the saturation data showed that [³H]NBMPR bound to high and low affinity binding sites in human umbilical vein endothelial cell membranes with apparent binding affinities (K_D) of 0.093±0.01 nM 1.92±0.1 nM, and binding site densities (Bmax values) of 13.48±1.2 and 69+5.3 fmol/mg protein, respectively. In contrast, the binding to primate vascular smooth muscle and bovine aortic endothelial cell membranes occurred to an apparently high affinity single class of binding sites at which the K_D was 1.4±0.3nM and 0.28±0.05nM, respectively, and which had Bmax values of 1,977±163 and 1,284±267 fmol/mg protein, respectively. Scatchard analysis of the binding inhibition by dipyridamole showed a mixed type inhibition, while NBMPR inhibited the binding competitively. Several recognized nucleoside transport inhibitors and vasodilators inhibited the binding with an order of potency similar to that observed for the inhibition of [³H]NBMPR binding to guinea pig cardiac membranes.     

Regulation of cyclic AMP level and synthesis of DNA, RNA and protein by quercetin in Ehrlich ascites tumor cells.

Quercetin (3.3, 4′, 5.7-pentahydroxy flavone) at the concentration of 10^?4 M as well as 10^?2 M theophylline and 10^?3 M dibutyryl cyclic AMP caused at least 85 per cent inhibition of [³H]thymidine incorporation in Ehrlich ascites tumor cells. At the same concentrations, these drugs decreased [³H]uridine and [³H]-L-leucine incorporation by 50–60 per cent and 35–45 per cent respectively. Ouabain (10^?3 M), the specific inhibitor of Na⁺-K⁺ pump system, did not alter the incorporation of [³H]thymidine and [³]uridine, but decreased the incorporation of [³H]-L-leucine in these cells. Treatment of Ehrlich ascites tumor cells with the polyanion dextran sulfate did not change the inhibitory effect of quercetin, theophylline and dibutyrlyl cyclic AMP on [³H]thymidine incorporation. On the other hand, this polyanion decreased the inhibitory effect of these drugs on incorporation of [³H]uridine and abolished completely their effect on incorporation of [³H]-L-leucine.     

The selective endothelin ETA receptor antagonist BQ123 antagonizes endothelin-1-mediated mitogenesis.

The mitogenic effects of endothelin isopeptides and the selective ETA receptor antagonist BQ123 were evaluated in rat aortic vascular smooth muscle cells. Endothelin-1 (ET-1) and endothelin-3 (ET-3) produced concentration-dependent increases in [3H]thymidine incorporation (EC50 = 0.1 and 25 nM, respectively). The ETB-selective agonist sarafotoxin 6c did not produce significant effects on [3H]thymidine incorporation. BQ123 produced selective and concentration-dependent inhibition of ET-1-mediated [3H]thymidine incorporation. These data demonstrate that ET-1-mediated mitogenesis in vascular smooth muscle is mediated by ETA receptors.     

Effects of organochlorine pesticides on DNA synthesis of cultured oviductal and uterine cells and on estrogen receptor of uterine tissue from heifers

 The pesticides DDT, MXC and γHCH at concentrations between 41 and 200 μM inhibited DNA synthesis (measured by [³H]thymidine incorporation) of cultured bovine oviductal endosalpingeal and uterine cells in the order DDT>MXC>γHCH, in comparison to nonexposed controls. Sensitivity to the toxicants was greater in uterine epithelial and stromal cells than in uterine smooth muscle or oviductal endosalpingeal cells. Besides the inhibitory effect, there was a stimulatory effect on DNA synthesis in epithelial cells in the range of 28 nM to 2.8 μM DDT and in stromal cells at 2.8 and 28 nM for MXC. An explanation for this reaction could be that both toxicants have an estrogen-like effect. In the present study, it is shown that the o,p’ isomer of DDT can bind to the cytoplasmatic estrogen receptor and DDT or MXC were able to inhibit the binding of radiolabelled estradiol to the uterine endometrial explants in bovine, whereas γHCH did not change the binding. These findings represent an estrogenic effect of DDT and MXC in two complete in vitro systems. Received: 10 October 1995/Accepted: 19 December 1995     

蝙蝠葛碱和粉防己碱对[3H]WEB 2086与体外牛脑前动脉平滑肌细胞结合的影响(英文)

用标记的血小板活化因子拮抗剂[³H]WEB 2086,在培养的牛脑前动脉平滑肌细胞上鉴定了血小板活化因子受体。结果表明在25℃时该细胞上存在两种与配基具有不同亲和力的受体结合位点,其中K_d-1=22.8±5.0 nmol·L^-1,K_d-2=186+20.5 nmol·L^-1;B_max-1=2.1±0.3 pmol/10⁴细胞,B_max-2=12.1±1-5 pmol/10⁶细胞。蝙蝠葛碱和粉防己碱均能抑制[³H]WEB2086与上述细胞的结合。     

[3H]-Spiroperidol labels dopamine receptors in membranes from rabbit mesenteric artery

Summary The potent dopamine receptor antagonist [³H]-spiroperidol was used to label binding sites in a membrane fraction derived from rabbit mesenteric artery which had characteristics expected for dopamine receptors. The binding was of high affinity with an equilibrium dissociation constant (K_D) of 13.1 nM; it was saturable with 110 fmol of [³H]-spiroperidol bound/mg protein at maximal occupancy of the sites. Binding at 37° C was rapid and readily reversible with rate constants of 0.0154 nM^–1 min^–1 and 0.114 min^–1 for forward and reverse reaction, respectively. Dopamine receptor antagonists were about 100–200 times more potent than -adrenolytic drugs in competing for the [³H]-spiroperidol binding sites and dopamine was much more potent than (–)-noradrenaline, adrenaline, (–)-isoprenaline, clonidine or serotonin. It is concluded that in a membrane fraction of the rabbit mesenteric artery there exist binding sites for [³H]-spiroperidol indistinguishable from dopamine receptors. Thus the present results support the view that in vascular smooth muscle there exist specific dopamine receptors.This work was supported by the Deutsche Forschungsgemeinschaft     

Concerted actions of the catechol O-methyltransferase and the cytosolic sulfotransferase SULT1A3 in the metabolism of catecholic drugs

Catecholic drugs had been reported to be metabolized through conjugation reactions, particularly methylation and sulfation. Whether and how these two Phase II conjugation reactions may occur in a concerted manner, however, remained unclear. The current study was designed to investigate the methylation and/or sulfation of five catecholic drugs. Analysis of the spent media of HepG2 cells metabolically labeled with [³⁵S]sulfate in the presence of individual catecholic drugs revealed the presence of two [³⁵S]sulfated metabolites for dopamine, epinephrine, isoproterenol, and isoetharine, but only one [³⁵S]sulfated metabolite for apomorphine. Further analyses using tropolone, a catechol O-methyltransferase (COMT) inhibitor, indicated that one of the two [³⁵S]sulfated metabolites of dopamine, epinephrine, isoproterenol, and isoetharine was a doubly conjugated (methylated and sulfated) product, since its level decreased proportionately with increasing concentrations of tropolone added to the labeling media. Moreover, while the inhibition of methylation resulted in a decrease of the total amount of [³⁵S]sulfated metabolites, sulfation appeared to be capable of compensating the suppressed methylation in the metabolism of these four catecholic drugs. A two-stage enzymatic assay showed the sequential methylation and sulfation of dopamine, epinephrine, isoproterenol, and isoetharine mediated by, respectively, the COMT and the cytosolic sulfotransferase SULT1A3. Collectively, the results from the present study implied the concerted actions of the COMT and SULT1A3 in the metabolism of catecholic drugs.     

Expression and characterization of the human alpha 2B-adrenoceptor in a vascular smooth muscle cell line

A vascular smooth muscle cell line stably expressing the human alpha 2B-adrenoceptor at a density of 1.5 pmol/mg membrane protein was generated by transfection of rat A7r5 cells. [35S]GTPgammaS binding experiments and [3H]thymidine incorporation experiments indicated that the expressed receptors were functional, had the expected pharmacological characteristics and efficiently stimulated smooth muscle cell proliferation. Confocal fluorescence microscopy was used to visualize alpha2B-adrenoceptors in A7r5-alpha 2B cells and indicated that the receptors were mainly localized in the plasma membrane. The expression of the smooth muscle-specific marker alpha-actin was similar in transfected A7r5-alpha 2B cells and in non-transfected A7r5 wild-type cells. The generated A7r5-alpha 2B cell line will be a useful tool for studying the function and regulation of alpha 2B-adrenoceptors in vascular smooth muscle cells.     

Kinetics of cadmium-induced hepatic and renal metallothionein synthesis in the mouse

Rates of hepatic and renal metallothionein synthesis were estimated by measuring the incorporation of [³H]cysteine into metallothionein prepared from mice at various times following a single intraperitioneal injection of cadmium acetate (2 mg of Cd/kg). Tissue metallothionein concentrations were measured indirectly as a function of the total cadmium-binding capacity of the isolated metallothionein. Maximal incorporation of [³H]cysteine into hepatic metallothionein occurred 6–12 hr following cadmium exposure, while renal metallothionein synthesis was maximal after 3 hr. Incorporation of [³H]cysteine into metallothionein as well as metallothionein concentrations was greater in the liver than in the kidney. It is concluded that the liver is the primary site of cadmium-induced metallothionein synthesis.     

Nitecapone effect on the synthesis and secretion of gastric sulfomucin

• 1.1. The effect of a new antiulcer agent, nitecapone, on the synthesis and secretion of sulfomucin in gastric mucosa was investigated using mucosal segments incubated in the presence of [³H]proline, [³H]glucosamine and [³⁵S]sulfate.
• 2.2. The drug, while showing no discernible effect on the apomucin synthesis, evoked a dose-dependent increase in mucin glycosylation and sulfation, which at 225 μM nitecapone, attained its maximum of 1.8 and 2.2-fold stimulation, respectively.
• 3.3. The analysis of mucin secretory responses revealed that nitecapone caused a concentration-dependent enhancement in sulfomucin secretion attaining maximum increase of 1.5-fold at 150 μM nitecapone.
• 4.4. The stimulatory effect of nitecapone on sulfomucin secretion was accompanied by 1.4-fold increase in mucosal cAMP level, and showed sensitivity to protein kinase A inhibitors, thus pointing towards the involvement of protein kinase A in mediation of gastric sulfomucin secretory responses to nitecapone.
• 5.5. The ability of nitecapone to enhance sulfomucin synthesis and secretion could be of importance to the gastroprotective action of this agent.

     

间硝苯地平对血管紧张素Ⅱ促进血管平滑肌细胞增殖和蛋白质合成的影响

以培养血管平滑肌细胞(vascularsmcothmusclecell,VSMC)为模型,观察了间硝苯地平(m-nifedipine,m-Nif)对血管紧张素Ⅱ(angiotensinⅡ,ANGⅡ)促进VSMC增殖和蛋白质合成的影响。结果表明,m-Nif抑制ANGⅡ(100nmol·L^-1)引起VSMC[³H]thymidine和[³H]leucine参入,并呈剂量依赖性。m-Nif(2×10^-6mol·L^-1)可抑制ANGⅡ对VSMC的刺激、DNA及蛋白质合成速率,分别降低了46%,58%,53%。提示m-Nif可抑制ANGⅡ对VSMC增殖和蛋白合成的促进作用。     

Inhibitory effects of clozapine and other antipsychotic drugs on noradrenaline transporter in cultured bovine adrenal medullary cells

The effects of clozapine and other antipsychotic drugs on noradrenaline (NA) transport were examined in cultured bovine adrenal medullary cells and in transfected Xenopus laevis oocytes expressing the bovine NA transporter. Incubation of adrenal medullary cells with clozapine (30–1000 ng/ml) inhibited desipramine (DMI)-sensitive uptake of [³H]NA in a concentration-dependent manner (IC₅₀=110 ng/ml or 336 nM). Other antipsychotic drugs such as haloperidol, chlorpromazine, and risperidone also decreased [³H]NA uptake (IC₅₀= 144, 220, and 210 ng/ml or 383, 690, and 512 nM, respectively). Eadie-Hofstee analysis showed that clozapine reduced V_max of uptake of [³H]NA and increased K_m. Furthermore, clozapine inhibited specific binding of [³H]DMI to plasma membranes isolated from bovine adrenal medulla (IC₅₀=48 ng/ml or 146 nM). Scatchard plot analysis of [³H]DMI binding revealed that clozapine decreased both B_max and K_d. Other antipsychotic drugs, including haloperidol, chlorpromazine, and risperidone, also reduced [³H]DMI binding to the membranes. In transfected Xenopus oocytes expressing the bovine NA transporter, clozapine inhibited [³H]NA uptake in a concentration-dependent manner similar to that observed in adrenal medullary cells. These results suggest that clozapine and haloperidol directly inhibit transport of NA by acting on the site of an NA transporter that influences both substrate transport and binding of tricyclic antidepressants. Received: 13 April 1999 / Final version: 2 November 1999