The apolipoprotein(a) component of lipoprotein(a) mediates binding to laminin: contribution to selective retention of lipoprotein(a) in atherosclerotic lesions

Lipoprotein(a) [Lp(a)] entrapment by vascular extracellular matrix may be important in atherogenesis. We sought to determine whether laminin, a major component of the basal membrane, may contribute to Lp(a) retention in the arterial wall. First, immunohistochemistry experiments were performed to examine the relative distribution of Lp(a) and laminin in human carotid artery specimens. There was a high degree of co-localization of Lp(a) and laminin in atherosclerotic specimens, but not in non-atherosclerotic sections. We then studied the binding interaction between Lp(a) and laminin in vitro. ELISA experiments showed that native Lp(a) particles and 17K and 12K recombinant apolipoprotein(a) [r-apo(a)] variants interacted strongly with laminin whereas LDL, apoB-100, and the truncated KIV(6-P), KIV(8-P), and KIV(9-P) r-apo(a) variants did not. Overall, the ELISA data demonstrated that Lp(a) binding to laminin is mediated by apo(a) and a combination of the lysine analogue epsilon-aminocaproic acid and salt effectively decreases apo(a) binding to laminin. Secondary binding analyses with 125I-labeled r-apo(a) revealed equilibrium dissociation constants (K(d)) of 180 and 360 nM for the 17K and 12K variants binding to laminin, respectively. Such similar K(d) values between these two r-apo(a) variants suggest that isoform size does not appear to influence apo(a) binding to laminin. In summary, our data suggest that laminin may bind to apo(a) in the atherosclerotic intima, thus contributing to the selective retention of Lp(a) in this milieu.     

Lipoprotein(a) accelerates atherosclerosis in uremic mice

Uremic patients have increased plasma lipoprotein(a) [Lp(a)] levels and elevated risk of cardiovascular disease. Lp(a) is a subfraction of LDL, where apolipoprotein(a) [apo(a)] is disulfide bound to apolipoprotein B-100 (apoB). Lp(a) binds oxidized phospholipids (OxPL), and uremia increases lipoprotein-associated OxPL. Thus, Lp(a) may be particularly atherogenic in a uremic setting. We therefore investigated whether transgenic (Tg) expression of human Lp(a) increases atherosclerosis in uremic mice. Moderate uremia was induced by 5/6 nephrectomy (NX) in Tg mice with expression of human apo(a) (n = 19), human apoB-100 (n = 20), or human apo(a) + human apoB [Lp(a)] (n = 15), and in wild-type (WT) controls (n = 21). The uremic mice received a high-fat diet, and aortic atherosclerosis was examined 35 weeks later. LDL-cholesterol was increased in apoB-Tg and Lp(a)-Tg mice, but it was normal in apo(a)-Tg and WT mice. Uremia did not result in increased plasma apo(a) or Lp(a). Mean atherosclerotic plaque area in the aortic root was increased 1.8-fold in apo(a)-Tg (P = 0.025) and 3.3-fold (P = 0.0001) in Lp(a)-Tg mice compared with WT mice. Plasma OxPL, as detected with the E06 antibody, was associated with both apo(a) and Lp(a). In conclusion, expression of apo(a) or Lp(a) increased uremia-induced atherosclerosis. Binding of OxPL on apo(a) and Lp(a) may contribute to the atherogenicity of Lp(a) in uremia.     

Definition of the structural elements in plasminogen required for high-affinity binding to apolipoprotein(a): a study utilizing surface plasmon resonance

Lipoprotein(a) [Lp(a)] is suggested to link atherosclerosis and thrombosis owing to the similarity between the apolipoprotein(a) [apo(a)] moiety of Lp(a) and plasminogen. Lp(a) may interfere with tPA-mediated plasminogen activation in fibrinolysis, thereby generating a hypercoaguable state in vivo. The present study employed surface plasmon resonance (SPR) to examine the binding interaction between plasminogen and a physiologically relevant, 17-kringle recombinant apo(a) species [17K r-apo(a)] in real time. Native, intact Glu(1)-plasminogen bound to apo(a) with substantially higher affinity (K(D) approximately 0.3 microM) compared to a series of plasminogen fragments (K1-5, K1-3, K4, K5P, and tail domain) that interacted weakly with apo(a) (K(D) > 50 microM). Treatment of Glu(1)-plasminogen with citraconic anhydride (a lysine modification reagent) completely abolished binding to wild-type 17K r-apo(a), whereas citraconylated 17K r-apo(a) decreased binding to wild-type Glu(1)-plasminogen by approximately 50%; inhibition of binding was also observed using the lysine analogue epsilon-aminocaproic acid. Whereas native Glu(1)-plasminogen exhibited monophasic binding to 17K r-apo(a), truncated Lys(78)-plasminogen exhibited biphasic binding. Altering Glu(1)-plasminogen from its native, closed conformation (in chloride buffer) to an open conformation (in acetate buffer) also yielded biphasic isotherms. These SPR data are consistent with a two-state kinetic model in which a conformational change in the plasminogen-apo(a) complex may occur following the initial binding event. Differential binding kinetics between Glu(1)-/Lys(78)-plasminogen and apo(a) may explain why Lp(a) is a stronger inhibitor of tPA-mediated Glu(1)-plasminogen activation compared to Lys(78)-plasminogen activation.     

Effects of cultivation practice on floristic and flowering diversity of spontaneously growing plant species on arable fields

In the past, the floristic diversity of arable fields has been described in terms of species diversity (SD) and their degree of coverage (C), but never in combination with the recording of the actually flowered species (FS) and their flowering intensity (FI) to striking differences in the cultivation methods on arable land. In relation to SD and C, however, FS and FI may provide important additional information on the functional biodiversity of fields. The aim was therefore to investigate the effects of (a) conventional, (b) organic, and (c) smallholder (never application of herbicides) on the floristic diversity. Using a region in Germany, we investigated SD, C, FS, and FI synchronously in (a), (b), and (c), by 356 vegetation surveys (5 × 5 m plots) conducted in spring and summer in 2019 in winter cereals. Statistical tests were used to analyze the differences between (a), (b), and (c). The medians were used to compare the floristic diversity of (a), (b), and (c) and finally relationships of FS and FI to SD were analyzed in relation to the cultivation methods. Significant differences in SD, C, FS, and FI were found between the (a), (b), and (c) in spring and summer characterized by sharp declines from (c) to (b) to (a). A drastic reduction in floristic diversity from (c) 100 to (b) 52 to (a) 3 was determined. Plants in flower (FS, FI) were very poorly in (a), moderately well to well in (b), and well to very well represented in (c). (C) to (a) was characterized by a sharp decline and from (a) to (b) by sharp increase in floristic diversity. With current acreage proportions of (a) in mind, this would affect, about one third of land area in Germany, associated with a drastic reduction in functional biodiversity for insects.     

Physicochemical properties of apolipoprotein(a) and lipoprotein(a-) derived from the dissociation of human plasma lipoprotein (a)

Chemical reduction of human plasma lipoprotein(a) (Lp(a)) yielded two water-soluble products which were separated by rate zonal ultracentrifugation. Apolipoprotein(a) (apo(a)) was completely recovered from the bottom of the gradient, whereas lipoprotein(a-) (Lp(a-)), which contained all of the lipids and apo-B100 of Lp(a), floated. By the techniques of circular dichroism and viscometry Lp(a-) was identical to low density lipoprotein (LDL). Lp(a-) was slightly larger in mass than autologous LDL and contained proportionally more triglyceride. The difference in mass between Lp(a) and Lp(a-) was accounted for by the loss of 2 molecules of apo(a) from the Lp(a) particle. The molecular weight of reduced and carboxymethylated apo(a) was 281,000 as determined by sedimentation equilibrium in 6 M guanidine HCl. By circular dichroism the structure of apo(a) was mostly random (71%) with the remainder representing 8% alpha-helix and 21% beta-sheet; its intrinsic viscosity, 28.3 cm3/g, was consistent with an extended flexible coil. The amino acid composition was characterized by an unusually high content of proline (11.4 mol %) as well as tryptophan, tyrosine, arginine, threonine, and a low amount of lysine, phenylalanine, and isoleucine. Apo(a) contained 28.1% carbohydrate by weight represented by mannose, galactose, galactosamine, glucosamine, and sialic acid in an approximate molar ratio of 3:7:5:4:7, respectively. Overall, the structure of Lp(a) appears to be consistent with a rigid spherical LDL-like core particle which, as a consequence of its association with a flexible glycoprotein such as apo(a), favors the entrapment of significant amounts of hydrodynamically associated solvent. Furthermore, the Lp(a-) remnant generated by the removal of apo(a) from Lp(a) was similar in structure but not identical to autologous LDL.     

Lipoprotein(a) levels, apo(a) isoform size, and coronary heart disease risk in the Framingham Offspring Study

The aim of this study was to assess the independent contributions of plasma levels of lipoprotein(a) (Lp(a)), Lp(a) cholesterol, and of apo(a) isoform size to prospective coronary heart disease (CHD) risk. Plasma Lp(a) and Lp(a) cholesterol levels, and apo(a) isoform size were measured at examination cycle 5 in subjects participating in the Framingham Offspring Study who were free of CHD. After a mean follow-up of 12.3 years, 98 men and 47 women developed new CHD events. In multivariate analysis, the hazard ratio of CHD was approximately two-fold greater in men in the upper tertile of plasma Lp(a) levels, relative to those in the bottom tertile (P < 0.002). The apo(a) isoform size contributed only modestly to the association between Lp(a) and CHD and was not an independent predictor of CHD. In multivariate analysis, Lp(a) cholesterol was not significantly associated with CHD risk in men. In women, no association between Lp(a) and CHD risk was observed. Elevated plasma Lp(a) levels are a significant and independent predictor of CHD risk in men. The assessment of apo(a) isoform size in this cohort does not add significant information about CHD risk. In addition, the cholesterol content in Lp(a) is not a significant predictor of CHD risk.     

Effects of the apolipoprotein(a) size polymorphism on the lipoprotein(a) concentration in 7 ethnic groups

Summary Apolipoprotein(a) [apo(a)] exhibits a genetic size polymorphism explaining about 40% of the variability in lipoprotein(a) [Lp(a)] concentration in Tyroleans. Lp(a) concentrations and apo(a) phenotypes were determined in 7 ethnic groups (Tyrolean, Icelandic, Hungarian, Malay, Chinese, Indian, Black Sudanese) and the effects of the apo(a) size polymorphism on Lp(a) levels were estimated in each group. Average Lp(a) concentrations were highly significantly different among these populations, with the Chinese (7.0mg/dl) having the lowest and the Sudanese (46mg/dl) the highest levels. Apo(a) phenotype and derived apo(a) allele frequencies were also significantly different among the populations. Apo(a) isoform effects on Lp(a) levels were not significantly different among populations. Lp(a) levels were however roughly twice as high in the same phenotypes in the Indians, and several times as high in the Sudanese, compared with Caucasians. The size variation of apo(a) explains from 0.77 (Malays) to only 0.19 (Sudanese) of the total variability in Lp(a) levels. Together these data show (I) that there is considerable heterogeneity of the Lp(a) polymorphism among populations, (II) that differences in apo(a) allele frequencies alone do not explain the differences in Lp(a) levels among populations and (III) that in some populations, e.g. Sudanese Blacks, Lp(a) levels are mainly determined by factors that are different from the apo(a) size polymorphism.     

Effect of tranexamic acid and delta-aminovaleric acid on lipoprotein(a) metabolism in transgenic mice

The assembly of lipoprotein(a) (Lp(a)) is a two-step process which involves the interaction of kringle-4 (K-IV) domains in apolipoprotein(a) (apo(a)) with Lys groups in apoB-100. Lys analogues such as tranexamic acid (TXA) or delta-aminovaleric acid (delta-AVA) proved to prevent the Lp(a) assembly in vitro. In order to study the in vivo effect of Lys analogues, transgenic apo(a) or Lp(a) mice were treated with TXA or delta-AVA and plasma levels of free and low density lipoprotein bound apo(a) were measured. In parallel experiments, McA-RH 7777 cells, stably transfected with apo(a), were also treated with these substances and apo(a) secretion was followed. Treatment of transgenic mice with Lys analogues caused a doubling of plasma Lp(a) levels, while the ratio of free:apoB-100 bound apo(a) remained unchanged. In transgenic apo(a) mice a 1. 5-fold increase in plasma apo(a) levels was noticed. TXA significantly increased Lp(a) half-life from 6 h to 8 h. Incubation of McA-RH 7777 cells with Lys analogues resulted in an up to 1. 4-fold increase in apo(a) in the medium. The amount of intracellular low molecular weight apo(a) precursor remained unchanged. We hypothesize that Lys analogues increase plasma Lp(a) levels by increasing the dissociation of cell bound apo(a) in combination with reducing Lp(a) catabolism.     

Biological evaluation of de-O-sulfonated analogs of salacinol, the role of sulfate anion in the side chain on the alpha-glucosidase inhibitory activity

De-O-sulfonated analogs (10a, Y(-)=CH(3)OSO(3) and 10b, Y(-)=Cl) of salacinol, a naturally occurring glycosidase inhibitor, and its diastereomer (12a, Y(-)=CH(3)OSO(3)) with L-thiosugar moiety (1,4-dideoxy-1,4-epithio-L-arabinitol) were prepared. Their inhibitory activities against intestinal maltase and sucrase were examined and compared with those of the parent alpha-glycosidase inhibitor, salacinol (1a). Compounds 10a and 10b showed a potent inhibitory activity equal to that of 1a against both enzymes, although 12a was a weak inhibitor against sucrase and maltase. These results indicated that the O-sulfonate anion moiety of 1a is not essential for the inhibitory activity.     

The phosphatidylserine binding site of the factor Va C2 domain accounts for membrane binding but does not contribute to the assembly or activity of a human factor Xa-factor Va complex

Factors V(a) and X(a) (FV(a) and FX(a), respectively) assemble on phosphatidylserine (PS)-containing platelet membranes to form the essential "prothrombinase" complex of blood coagulation. The C-terminal domain (C2) of FV(a) (residues 2037-2196 in human FV(a)) contains a soluble phosphatidylserine (C6PS) binding pocket flanked by a pair of tryptophan residues, Trp(2063) and Trp(2064). Mutating these tryptophans abolishes FV(a) membrane binding. To address both the roles of these tryptophans in C6PS or membrane binding and the role of the C2 domain lipid binding site in regulation of FV(a) cofactor activity, we expressed W(2063,2064)A mutants of the recombinant C2 domain (rFV(a2)-C2) and of a B domain-deleted factor V light isoform (rFV(a2)) in Hi-5 and COS cells, respectively. Intrinsic fluorescence showed that wild-type rFV(a2)-C2 binds to C6PS and to 20% PS/PC membranes with apparent K(d) values of 2.8 microM and 9 nM, respectively, while mutant rFV(a2)-C2 does not. Equilibrium dialysis confirmed that mutant rFV(a2)-C2 does not bind to C6PS. Mutant rFV(a2) binds to C6PS (K(d) approximately 37 microM) with an affinity comparable to that of wild-type rFV(a2) (K(d) approximately 20 microM), although it does not bind to PS/PC membranes to which wild-type rFV(a2) binds with native affinity (K(d) approximately 3 nM). Both wild-type and mutant rFV(a2) bind to active site-labeled FX(a) (DEGR-X(a)) in the presence of 400 microM C6PS with native affinity (K(d) approximately 3-4 nM) to produce a solution rFV(a2)-FX(a) complex of native activity. We conclude that (1) the C2 domain PS site provides all but approximately 1 kT of the free energy of FV(a) membrane binding, (2) tryptophans lining the C2 lipid binding pocket are critical to C6PS and membrane binding and insert into the bilayer interface during membrane binding, (3) occupancy of the C2 lipid binding pocket is not necessary for C6PS-induced formation of the FX(a)-FV(a) complex or its activity, but (4) another PS site on FV(a) does have a regulatory role.     

前蛋白转换酶枯草溶菌素9抑制剂降低脂蛋白(a)作用的机制

脂蛋白(a)(lipoprotein(a),Lp(a))在结构上与低密度脂蛋白相似,是动脉粥样硬化性心血管疾病发病的独立危险因素和潜在的治疗靶点。前蛋白转换酶枯草溶菌素9(proprotein convertase subtilisin/kexin type 9,PCSK9)抑制剂可有效降低Lp(a)的循环水平并减少心血管事件风险。本文综合近年来的相关研究,阐述PCSK9抑制剂减少Lp(a)合成和促进其降解的相关机制,分析该领域所面临的挑战和未来的发展方向。     

Reconstitution of lipoprotein(a) by infusion of human low density lipoprotein into transgenic mice expressing human apolipoprotein(a).

Lipoprotein(a) (Lp(a)) is an atherosclerosis-causing lipoprotein that circulates in human plasma as a complex of low density lipoprotein (LDL) and apolipoprotein(a) (apo(a)). It is not known whether apo(a) attaches to LDL within hepatocytes prior to secretion or in plasma subsequent to secretion. Here we describe the development of a line of mice expressing the human apo(a) transgene under the control of the murine transferrin promoter. The apo(a) was secreted into the plasma, but circulated free of lipoproteins. When human (h)-LDL was injected intravenously, the circulating apo(a) rapidly associated with the lipoproteins, as determined by nondenaturing gel electrophoresis. Human HDL and mouse LDL had no such effect. When h-VLDL was injected, there was a delayed association of apo(a) with the lipoprotein fraction which suggests that apo(a) preferentially associated with a metabolic product of VLDL. The complex of apo(a) with LDL formed both in vivo and in vitro was resistant to boiling in the presence of detergents and denaturants, but was resolved upon disulfide reduction. These studies suggest that apo(a) fails to associate with mouse lipoproteins due to structural differences between human and mouse LDL, and that Lp(a) formation can occur in plasma through the association of apo(a) with circulating LDL.     

Catalysis of covalent Lp(a) assembly: evidence for an extracellular enzyme activity that enhances disulfide bond formation

The assembly of lipoprotein(a) (Lp(a)) particles occurs via a two-step mechanism in which noncovalent interactions between apolipoprotein(a) (apo(a)) and the apolipoproteinB-100 component of low density lipoprotein precede the formation of a single disulfide bond. Although we have previously demonstrated that the rate constant for the covalent step of Lp(a) assembly can be enhanced by altering the conformational status of apo(a), the resultant rates of covalent Lp(a) particle formation measured in vitro are relatively slow. The large excess of Lp(a) (over apo(a)) observed in vivo can be accounted for by a preferential clearance of apo(a) over Lp(a) and/or a sufficiently high rate of covalent Lp(a) assembly. In the present study, we report that cultured human hepatoma cells secrete an oxidase activity that dramatically enhances the rate of covalent Lp(a) assembly. This activity is likely possessed by a protein because it is heat-sensitive and is retained in the concentrate following ultrafiltration through a 5 kDa cutoff filter. However, a small molecule cofactor for the activity is suggested by the observation that the activity is lost upon dialysis. Plots of Lp(a) assembly rate versus input apo(a) concentration gave rectangular hyperbolae; the reaction displayed an unusual dependence on the concentration of apoB-100, with increasing concentrations of apoB-100 resulting in slower rates of Lp(a) assembly at low concentrations of apo(a), an effect that was alleviated by higher apo(a) concentrations. Interestingly, V(max(app))/K(m(app)) ratios were insensitive to apoB-100 concentration, which is diagnostic of a ping-pong reaction mechanism. In this way, the putative Lp(a) oxidase may be functionally analogous to protein disulfide isomerase, which exhibits a similar mechanism during the catalysis of disulfide bond formation during protein folding, although we have ruled out a role for this enzyme in Lp(a) assembly.     

Quantitative evaluation of the contribution of weak lysine-binding sites present within apolipoprotein(a) kringle IV types 6-8 to lipoprotein(a) assembly

During lipoprotein(a) (Lp(a)) assembly, non-covalent interactions between apolipoprotein(a) (apo(a)) and low density lipoprotein precede specific disulfide bond formation. Studies have shown that the non-covalent step involves an interaction between the weak lysine-binding sites (WLBS) present within each of apo(a) kringle IV types 6, 7, and 8 (KIV(6-8)), and two lysine residues (Lys(680) and Lys(690)) within the NH(2) terminus of the apolipoprotein B-100 (apoB) component of low density lipoprotein. In the present study, we introduced single point mutations (E56G) into each of the WLBS present in apo(a) KIV(6-8) and expressed these mutations in the context of a 17-kringle (17K) recombinant apo(a) variant. Single mutations that disrupt the WLBS in KIV(6), KIV(7), and KIV(8), as well as mutants that disrupt the WLBS in both KIV(6) and KIV(7), or both KIV(7) and KIV(8), were assessed for their ability to form non-covalent and covalent Lp(a) complexes. Our results demonstrate that both apo(a) KIV(7) and KIV(8), but not KIV(6), are required for maximally efficient non-covalent and covalent Lp(a) assembly. Single mutations in the WLBS of KIV(7) or KIV(8) resulted in a 3-fold decrease in the affinity of 17K recombinant apo(a) for apoB, and a 20% reduction in the rate of covalent Lp(a) formation. Tandem mutations in the WLBS in both KIV(7) and KIV(8) resulted in a 13-fold reduction in the binding affinity between apo(a) and apoB, and a 75% reduction in the rate of the covalent step of Lp(a) formation. We also showed that KIV(7) and KIV(8) specifically bind with high affinity to apoB-derived peptides containing Lys(690) or Lys(680), respectively. Taken together, our data demonstrate that specific interactions between apo(a) KIV(7) and KIV(8) and Lys(680) and Lys(690) in apoB mediate a high affinity non-covalent interaction between apo(a) and low density lipoprotein, which dictates the efficiency of covalent Lp(a) formation.     

Molecular Genetic Characterization of Six Recessive Viable Alleles of the Mouse Agouti Locus

The agouti locus on mouse chromosome 2 encodes a secreted cysteine-rich protein of 131 amino acids that acts as a molecular switch to instruct the melanocyte to make either yellow pigment (phaeomelanin) or black pigment (eumelanin). Mutations that up-regulate agouti expression are dominant to those causing decreased expression and result in yellow coat color. Other associated effects are obesity, diabetes, and increased susceptibility to tumors. To try to define important functional domains of the agouti protein, we have analyzed the molecular defects present in a series of recessive viable agouti mutations. In total, six alleles (a(mJ), a(u), a(da), a(16H), a(18H), a(e)) were examined at both the RNA and DNA level. Two of the alleles, a(16H) and a(e), result from mutations in the agouti coding region. Four alleles (a(mJ), a(u), a(18H), and a(da)) appear to represent regulatory mutations that down-regulate agouti expression. Interestingly, one of these mutations, a(18H), also appears to cause an immunological defect in the homozygous condition. This immunological defect is somewhat analogous to that observed in motheaten (me) mutant mice. Short and long-range restriction enzyme analyses of homozygous a(18H) DNA are consistent with the hypothesis that a(18H) results from a paracentric inversion where one end of the inversion maps in the 5' regulatory region of agouti and the other end in or near a gene that is required for normal immunological function. Cloning the breakpoints of this putative inversion should allow us to identify the gene that confers this interesting immunological disorder.     

Measurement of selective effect of insulin on glucose disposal from labeled glucose oral test minimal model

The oral glucose minimal model (OMM) measures insulin sensitivity (S(I)) and the glucose rate of appearance (R(a)) of ingested glucose in the presence of physiological changes of insulin and glucose concentrations. However, S(I) of OMM measures the overall effect of insulin on glucose utilization and glucose production. In this study we show that, by adding a tracer to the oral dose, e.g., of a meal, and by using the labeled version of OMM, OMM* to interpret the data, one can measure the selective effect of insulin on glucose disposal, S(I)*. Eighty-eight individuals underwent both a triple-tracer meal with the tracer-to-tracee clamp technique, providing a model-independent reference of the R(a) of ingested glucose (R(a meal)(ref)) and an insulin-modified labeled intravenous glucose tolerance test (IVGTT*). We show that OMM* provides not only a reliable means of tracing the R(a) of ingested glucose (R(a meal)) but also accurately measures S(I)*. We do so by comparing OMM* R(a meal) with the model-independent R(a meal)(ref) provided by the tracer-to-tracee clamp technique, while OMM* S(I)* is compared with both S(I)(* ref), obtained by using as known input R(a meal)(ref), and with S(I)* measured during IVGTT*.     

Analysis of Autoregulation at the Level of Pre-mRNA Splicing of the Suppressor-of-White-Apricot Gene in Drosophila

The Drosophila suppressor-of-white-apricot [su(w(a))] protein regulates/modulates at least two somatic RNA processing events. It is a potent regulator of its own expression. We report here new studies of this autoregulatory circuit. Among other things, our studies show the following. First, new evidence that su(w(a)) expression is autoregulated at the level of pre-mRNA splicing is reported. su(w(a)) protein represses accumulation of the fully spliced su(w(a)) mRNA encoding it and promotes accumulation of high levels of incompletely spliced su(w(a)) pre-mRNA. Second, the fully spliced su(w(a)) mRNA is sufficient for all known su(w(a)) genetic functions indicating that it encodes the sole su(w(a)) protein. Third, the incompletely spliced su(w(a)) pre-mRNAs resulting from autoregulation are not translated (probably as a result of nuclear retention) and apparently represent nonfunctional by-products. Fourth, the special circumstances of su(w(a)) expression during oogenesis allows maternal deposition exclusively of fully spliced su(w(a)) mRNA. Fifth, su(w(a)) protein immunolocalizes to nuclei consistent with its being a direct regulator of pre-mRNA processing. We discuss the implications of our results for mechanisms of splicing regulation and for developmental control of su(w(a)) expression.     

Acute impact of apheresis on oxidized phospholipids in patients with familial hypercholesterolemia

We measured oxidized phospholipids (OxPL), lipoprotein (a) [Lp(a)], and lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) pre- and postapheresis in 18 patients with familial hypercholesterolemia (FH) and with low(～10 mg/dl; range 10-11 mg/dl), intermediate (～50 mg/dl; range 30-61 mg/dl), or high (>100 mg/dl; range 78-128 mg/dl) Lp(a) levels. By using enzymatic and immunoassays, the content of OxPL and Lp-PLA(2) mass and activity were quantitated in lipoprotein density fractions plated in microtiter wells, as well as directly on apoB-100, Lp(a), and apoA-I immunocaptured within each fraction (i.e., OxPL/apoB and Lp-PLA(2)/apoB). In whole fractions, OxPL was primarily detected in the Lp(a)-containing fractions, whereas Lp-PLA(2) was primarily detected in the small, dense LDL and light Lp(a) range. In lipoprotein capture assays, OxPL/apoB and OxPL/apo(a) increased proportionally with increasing Lp(a) levels. Lp-PLA(2)/apoB and Lp-PLA(2)/apoA-I levels were highest in the low Lp(a) group but decreased proportionally with increasing Lp(a) levels. Lp-PLA(2)/apo(a) was lowest in patients with low Lp(a) levels and increased proportionally with increasing Lp(a) levels. Apheresis significantly reduced levels of OxPL and Lp-PLA(2) on apoB and Lp(a) (50-75%), particularly in patients with intermediate and high Lp(a) levels. In contrast, apheresis increased Lp-PLA(2)-specific activity (activity/mass ratio) in buoyant LDL fractions. The impact of apheresis on Lp(a), OxPL, and Lp-PLA(2) provides insights into its therapeutic benefits beyond lowering apoB-containing lipoproteins.     

Distinctive and synergistic signaling of human adenosine A2a and dopamine D2L receptors in CHO cells

Adenosine A(2a) receptor (A(2a)R) colocalizes with dopamine D(2) receptor (D(2)R) in the basal ganglia and modulates D(2)R-mediated dopaminergic activities. A(2a)R and D(2)R couple to stimulatory and inhibitory G proteins, respectively. Their opposing roles in regulating neuronal activities, such as locomotion and alcohol consumption, are mediated by their opposite actions on adenylate cyclase, which often serves as "co-incidence detector" of various activators. On the other hand, the neural actions of A(2a)R and D(2)R are also, at least partially, independent of each other, as indicated by studies using D(2)R and A(2a)R knock-out mice. Here we co-expressed human A(2a)R and human D(2L)R in CHO cells and examined their signaling characteristics. Human A(2a)R desensitized rapidly upon agonist stimulation. A(2a)R activity (80%) was diminished after 2 hr of pretreatment with its agonist CGS21680. In contrast, human D(2L)R activity was sustained even after 2 hr and 18 hr pretreatment with its agonist quinpirole. Long-term (18 hr) stimulation of human D(2L)R also increased basal cAMP levels in CHO cells, whereas long-term (18 hr) activation of human A(2a)R did not affect basal cAMP levels. Furthermore, long-term (18 hr) activation of D(2L)R dramatically sensitized A(2a)R-induced stimulation of adenylate cyclase in a pertussis toxin-sensitive way. Forskolin-induced cAMP accumulation was significantly increased after short-term (2 hr) human D(2L)R stimulation and further elevated after long-term (18 hr) D(2L)R activation. However, neither short-term (2 hr) nor long-term (18 hr) stimulation of A(2a)R affected the inhibitory effects of D(2L)R on adenylate cyclase. Co-stimulation of A(2a)R and D(2L)R could not induce desensitization or sensitization of D(2L)R either. In summary, signaling through A(2a)R and D(2L)R is distinctive and synergistic, supporting their unique and yet integrative roles in regulating neuronal functions when both receptors are present.