Determination of the modulated structure of Sr14/11CoO3 through a (3 + 1)-dimensional space description and using non-harmonic ADPs.

Sr(14/11)CoO(3) (i.e. Sr(14)Co(11)O(33), tetradecastrontium undecacobalt tritriacontaoxide), a new phase in the hexagonal perovskite Sr(x)CoO(3) system, has been prepared and its structure solved from single-crystal X-ray data within the (3 + 1)-dimensional formalism. Sr(14/11)CoO(3) crystallizes in the trigonal symmetry, R3;m(00gamma)0s superspace group with the following lattice parameters: a(s) = 9.508 (2), c(s) = 2.5343 (7) Å, q = 0.63646 (11)c(*) and V(s) = 198.40 (13) Å(3). With the commensurate versus incommensurate test not being conclusive, the structure was considered as commensurate (P32 three-dimensional space group), but refined within the (3 + 1)-dimensional formalism to a residual factor R = 0.0351 for 47 parameters and 1169 independent reflections. Crenel functions were used for the oxygen and cobalt description and a Gram-Charlier expansion up to the third order of the atomic displacement parameter was employed for one Co atom. The structure is similar to that of Sr(6/5)CoO(3), but with a different sequence of the octahedra and trigonal prism polyhedra along the [CoO(3)] chains. An interesting feature evidenced by the non-harmonic expansion is the displacement of the prismatic Co atoms from the site center, towards the prism rectangular faces.

Estimation of radioactive calcium absorption based on expanding Ca-exchangeable space.

Anthropological interest in calcium metabolism is partially connected with the genetic basis of skeletal metabolism that could provide additional optimism for people suffering from osteoporosis. The present investigation is aimed at deal with a of model describing Ca intestinal absorption, suitable for other descriptions currently investigated in anthropology (migration and gene flow). The purpose of this investigation was to establish features of the reflection of the Ca isotopic absorption tests in serum using the concept of an expanding Ca-space and to assess the reliability of the estimation of absorption (ABS) from one serum sample. Double tracer method, using 85Sr as the iv tracer, was used as the referent. The study included 100 subjects (49 females, 51 males) of which 34 were considered healthy. The mean value of ABS was 68 (+/-17.45 SD) % of dose. Serum radioactivity during the 24 hours following oral administration of 47Ca (together with 100 mg elemental Ca), o.s(t), from five blood samples was determined. The relationship between o.s(t) and ABS was found to be connected to an expanding Ca-space, V(t): ABS = o.s(t)V(t) = 26 + 11.8t0.35 o.s(t). After the absorption process was finished, an approximate value of V(t) is (100/S1)(t-x)b. The constants "S1" and "b" describe power function of iv s(t), whereas parameter "x" represents delay of V(t) Ca-space at oral administration in comparison to the iv administration. Statistical models selected on the basis of their low values of the SEE (from 8.71 to 8.90% of dose) include body surface index or body weight index (BW0.425). The CV expressed as (SEE/mean x ABS(Ca/Sr) x 100 were about 13% and the observed greatest difference between the estimated and measured ABS(Ca/Sr) was 20% of dose. The authors believe that models presented permit, to a limited extent, comparison of results obtained by different procedures. The results, according to variability of the sample of subjects, permit judgment on upper limits of error at estimation of absorption from one blood sample radioactivity.

Inhibitors for the in vitro assembly of Lp(a).

Lp(a) is composed of an LDL-like core and the glycoprotein apo(a). Current evidence strongly suggests that the assembly of this atherogenic lipoprotein proceeds outside the liver cells in a two-step fashion. In the first step, a loose complex is formed involving kringle-4 motifs in apo(a) and one or more Lys side chains in apoB-100. In the second step, this complex is stabilized by a disulfide bridge. Indications are that Lp(a) assembly is critical in the determination of plasma apo(a) concentrations. Therefore, we searched for substances that interfere with the first step of Lp(a) assembly. epsilon-Aminohexoic acid (epsilon-AHA), known as an inhibitor from earlier assembly studies, had an IC50 of 4.8 mmol/L. The IC50 of Pro, HO-p-aminobenzene sulfonamide, Lys, N-epsilon-acetyl-Lys, taurine, Glu, serotonin, and benzamidine were all > 20 mmol/L. gamma-Aminobutyric acid, spermine, and spermidine exhibited IC50 on the same order of magnitude as epsilon-AHA. The substances with the highest inhibitory action were tranexamic acid and delta-aminovaleric acid. Seven of eight patients treated in a pilot study with tranexamic acid (Cyclocapron) responded with a decrease of plasma apo(a) of 18.5 +/- 8.2%. We suggest that substances that interfere with the Lp(a) assembly are worth pursuing further for their usefulness as therapeutic agents in reducing high plasma Lp(a) concentrations.

Decreased binding of apolipoprotein (a) to familial defective apolipoprotein B-100 (Arg3500-->Gln). A study of the assembly of recombinant apolipoprotein (a) with mutant low density lipoproteins.

In familial defective apolipoprotein B-100 (FDB), glutamine is substituted for arginine at position 3500 of the amino acid sequence. This mutation alters the structure of low density lipoproteins (LDL) and reduces their binding to LDL receptors. We studied the assembly in vitro of FDB-LDL with two recombinant apo(a) (r-apo(a)) isoforms containing 17 or 18 kringle IV-type repeats, respectively. R-apo(a) complexed to LDL in a concentration- and time-dependent manner. When we mixed normal LDL at protein concentrations from 1 to 10 mg/liter with 200 micrograms/liter r-apo(a) and incubated for 20 h, 15-44% of r-apo(a) were bound to LDL, forming an artificial Lp(a)-like particle. With LDL from a homozygous FDB patient, only 2-16% of r-apo(a) were complexed; heterozygous FDB-LDL bound 2-30% of r-apo(a). We also studied the effect of r-apo(a) on the interaction of the monoclonal antibody MB47 with normal and mutant apoB-100. FDB-LDL displayed enhanced binding of MB47. Adducts generated from normal LDL and r-apo(a) had an increased affinity for MB47, when compared to LDL alone. In contrast, r-apo(a) did not change MB47 reactivity when incubated with FDB-LDL. Altogether, our findings suggest that domains in apolipoprotein B which are important for the interaction with the LDL receptor play a role in the assembly of Lp(a) as well. They provide, in addition, an explanation for the observation that in Lp(a) of heterozygous FDB patients, the ratio of defective to normal apoB-100 is significantly smaller than in LDL from the same patients.

The role of lecithin: cholesterol acyltransferase for lipoprotein (a) assembly. Structural integrity of low density lipoproteins is a prerequisite for Lp(a) formation in human plasma.

The composition of lipoproteins in the plasma of patients with LCAT deficiency (LCAT-D) is grossly altered due to the lack of cholesteryl esters which form the core of normal lipoproteins. When plasma from LCAT-D patients and their relatives was examined we found that nine heterozygotes had plasma Lp(a) levels of 2-13 mg/dl whereas none of 11 affected homozygous individuals from different families contained detectable amounts of Lp(a) in their plasma. Therefore, the binding of apo(a) to LDL density particles was studied in vitro using LDL density fractions prepared from patients, and recombinant apo(a) [r-apo(a)], which was expressed and secreted by transfected COS-7 cells. The LDL from heterozygotes were chemically indistinguishable from normal LDL and homogeneous with regard to morphology, whereas the crude LDL floating fraction from homozygotes consisted of a heterogeneous mixture of large vesicles, and small spheres resembling normal LDL. The LDL density fraction from the LCAT-D patient lacked almost completely cholesteryl esters. Incubation of LCAT-D plasma with active LCAT caused a substantial augmentation of the original subfraction which morphologically resembled normal LDL. Using r-apo(a) and normal LDL or LDL of heterozygous individuals, apoB:r-apo(a) complexes were formed when incubated at 37 degrees C in vitro for 20 h. In contrast, the total LDL floating fraction from a homozygous LCAT-D patient failed to form apoB:r-apo(a) complexes. After treatment with active LCAT, a significant apoB:r-apo(a) association was observed with LCAT-D LDL-density particles. Our data emphasize the importance of the integrity of LDL structure and composition for the formation of Lp(a). In addition, we demonstrate that the absence of LCAT activity has a fundamental impact on the regulation of plasma Lp(a) levels.

Structural requirements of apo-a for the lipoprotein-a assembly.

Lipoprotein-a [Lp(a)], one of the most atherogenic lipoproteins, is composed of a low-density lipoprotein (LDL) core in addition to an apo-a of variable size which is linked to apoB by a disulphide bridge. Lp(a) synthesized in vitro by incubation of recombinant apo-a (r-apo-a) with LDL is physico-chemically indistinguishable from native Lp(a). The synthesis of Lp(a) in vitro proceeds in two steps. In the first step, one of the unique kringle-IVs (K-IVs) in apo-a binds to a Lys residue of apoB; in the second step, Cys-4057 of K-IV type-9 (T-9) forms a disulphide bridge with Cys-3734 of LDL. Here we have produced r-apo-a with different combinations of unique K-IVs and shown that K-IV T-6 is required for the first step of Lp(a) assembly. For the second step not only is K-IV T-9 essential, but also the distance between T-6 and T-9 requires a length of two K-IVs. These findings give additional insight into the mode of Lp(a) assembly and are of relevance in the search for apo-a mutants influencing Lp(a) levels and for the development of Lp(a)-lowering medications.

High-level expression of various apolipoprotein(a) isoforms by "transferrinfection": the role of kringle IV sequences in the extracellular association with low-density lipoprotein.

Characterization of the assembly of lipoprotein(a) [Lp(a)] is of fundamental importance to understanding the biosynthesis and metabolism of this atherogenic lipoprotein. Since no established cell lines exist that express Lp(a) or apolipoprotein(a) [apo(a)], a "transferrinfection" system for apo(a) was developed utilizing adenovirus receptor- and transferrin receptor-mediated DNA uptake into cells. Using this method, different apo(a) cDNA constructions of variable length, due to the presence of 3, 5, 7, 9, 15, or 18 internal kringle IV sequences, were expressed in cos-7 cells or CHO cells. All constructions contained kringle IV-36, which includes the only unpaired cysteine residue (Cys-4057) in apo(a). r-Apo(a) was synthesized as a precursor and secreted as mature apolipoprotein into the medium. When medium containing r-apo(a) with 9, 15, or 18 kringle IV repeats was mixed with normal human plasma LDL, stable complexes formed that had a bouyant density typical of Lp(a). Association was substantially decreased if Cys-4057 on r-apo(a) was replaced by Arg by site-directed mutagenesis or if Cys-4057 was chemically modified. Lack of association was also observed with r-apo(a) containing only 3, 5, or 7 kringle IV repeats without "unique kringle IV sequences", although Cys-4057 was present in all of these constructions. Synthesis and secretion of r-apo(a) was not dependent on its sialic acid content. r-Apo(a) was expressed even more efficiently in sialylation-defective CHO cells than in wild-type CHO cells. In transfected CHO cells defective in the addition of N-acetylglucosamine, apo(a) secretion was found to be decreased by 50%. Extracellular association with LDL was not affected by the carbohydrate moiety of r-apo(a), indicating a protein-protein interaction between r-apo(a) and apoB. These results show that, besides kringle IV-36, other kringle IV sequences are necessary for the extracellular association of r-apo(a) with LDL. Changes in the carbohydrate moiety of apo(a), however, do not affect complex formation.