Characterization of subpopulations of lipoprotein particles isolated from human cerebrospinal fluid.

The aim of the present study was to define lipoprotein complexes within cerebrospinal fluid (CSF) in terms of their apolipoprotein composition, using fractionation procedures considered optimal for maintaining lipoprotein structural integrity. Five apolipoproteins were identified, namely apolipoproteins A-I, A-IV, D, E and J. These were differentially distributed amongst lipoprotein particles of which three major subpopulations were identified. CSF-LpAI (20.1 +/- 3.8 nm) was enriched in apolipoprotein A-I and contained the major proportion (> 50%) of apolipoproteins D, E and J. CSF-LpE, of similar size to CSF-LpAI (20.2 +/- 3.1 nm), was composed principally of apolipoprotein E, with minor quantities of apolipoproteins A-I, A-IV, D and J. Elimination of these particles from cerebrospinal fluid by immunoabsorption revealed a third subpopulation of significantly greater diameter (32.0 +/- 6.8 nm). The majority (62%) of apolipoprotein A-IV was also present in this fraction. The study demonstrates the structural and size heterogeneity of lipoproteins in cerebrospinal fluid. This may reflect the lipid transport processes within the central nervous system.

In vivo phosphorylation of the Na,K-ATPase alpha subunit in sciatic nerves of control and diabetic rats: effects of protein kinase modulators.

The phosphorylation state of the Na,K-ATPase alpha subunit has been examined in 32P-labeled sciatic nerves of control and streptozotocin-treated diabetic rats. Intact nerves were challenged with protein kinase (PK) modulators and alpha-subunit 32P labeling was analyzed after immunoprecipitation. In control nerves, the PKC activator phorbol 12-myristate 13-acetate (PMA) had little effect on alpha-subunit 32P labeling. In contrast, staurosporine, a PKC inhibitor, and extracellular calcium omission decreased it. In Ca(2+)-free conditions, PMA restored the labeling to basal levels. The cAMP-raising agent forskolin reduced the 32P labeling of the alpha subunit. The results suggest that nerve Na,K-ATPase is tonically phosphorylated by PKC in a Ca(2+)-dependent manner and that PKA modulates the phosphorylation process. In nerves of diabetic rats, PMA increased 32P labeling of the alpha subunit. In contrast to staurosporine or extracellular calcium omission, the decreased state of phosphorylation seen with forskolin was no longer significant in diabetic nerves. No change in the level of alpha-subunit isoforms (alpha 1 or alpha 2) was detected by Western blot analysis in such nerves. In conclusion, the altered effect of PK activators on Na,K-ATPase phosphorylation state is consistent with the view that a defect in PKC activation exists in diabetic nerves.

Alpha, beta I, beta II, delta, and epsilon protein kinase C isoforms and compound activity in the sciatic nerve of normal and diabetic rats.

Defective protein kinase C (PKC) has been implicated in impaired Na+,K(+)-ATPase activity in the sciatic nerve of streptozotocin-induced diabetic rats. In the present study, alpha, beta I, beta II, gamma, delta, and epsilon isoform-specific antibodies were used in parallel to the measurement of compound PKC activity for the characterization of PKC distribution and isoform expression in sciatic nerves of normal and diabetic rats. To distinguish isoform expression between the axonal and glial compartments, PKC isoforms were evaluated in nerves subjected to Wallerian degeneration and in a pure primary Schwann cell culture. alpha, beta I, beta II, delta, and epsilon but no gamma isoforms were detected in sciatic nerve. Similar immunoreactivity was observed in degenerated nerves 3-4 days after transection except for diminished beta I and epsilon species; in Schwann cell cultures, only alpha, beta II, delta, and epsilon were detected. In normal nerves, two-thirds of PKC compound activity was found in the cytosol and 50% of total enzyme activity translocated to the Na+,K(+)-ATPase-enriched membrane fraction with phorbol myristate acetate. Similar redistribution patterns were observed for the immunoreactivity of all isoforms with the exception of delta, which did not translocate to the membrane with phorbol myristate acetate. No abnormality in compound PKC activity, in the immunoreactive intensity, or in the distribution of PKC isoforms could be detected in rat sciatic nerve after 6-12 weeks of diabetes. Thus, defective activation rather than decreased intrinsic PKC activity may occur in diabetic neuropathy.

Characterization of a human high density lipoprotein-associated protein, NA1/NA2. Identity with SP-40,40, an inhibitor of complement-mediated cytolysis.

Two peptides, NA1 and NA2, which we previously suggested to be associated with high density lipoproteins (HDLs), have been purified. Polyclonal antibodies against each peptide and a monoclonal antibody against NA2 have been used to further characterize them and their association with HDL. Immunoblotting studies revealed that the peptides form a complex of molecular mass of approximately 80 kd. Agarose gel filtration showed coelution of NA1/NA2 and apolipoprotein (apo) A-I, the structural protein of HDL. This was confirmed by fast protein liquid chromatography, which further indicated that up to 60% of NA1/NA2 was located within the lower density range of the HDL spectrum. Complementary studies with anti-apo A-I immunoaffinity columns provided evidence that at least 40% of NA1/NA2 was associated with HDL, an association easily disrupted by ultracentrifugal manipulation. Finally, partial amino acid sequences showed virtually complete homology with a recently identified protein, SP-40,40, or cytolysis inhibitor. The protein is suggested to have a powerful inhibitory effect on complement-mediated cell lysis. Our results could thus furnish an explanation for the previously observed modulating influence of HDL on complement activity.

Distribution of apolipoprotein E between free and A-II complexed forms in very-low- and high-density lipoproteins: functional implications.

The stability of apolipoprotein E/lipoprotein associations has been examined as a function of apolipoprotein E phenotype. Visualisation by immunoblotting showed plasma apolipoprotein E to be present in two forms; the free form and, as previously described, an E-A-II complex. In very low density lipoproteins isolated by gel filtration from subjects with E3/3 and E4/3 phenotypes, apolipoprotein E was present essentially in the free form (ratio free: complex of 12.2 and 37.5, respectively). Exploiting ultracentrifugation as the disruptive agent, very-low-density lipoproteins thus isolated were shown to have substantially lower ratios (5.6 and 5.4, respectively) reflecting preferential loss of free apolipoprotein E. In high-density lipoproteins isolated by gel filtration from E3/3 phenotypes, apolipoprotein E was largely present as an E-A-II complex (80.3%). In contrast, the majority of apolipoprotein E in high-density lipoproteins from E4/3 phenotypes was present in the free form (58.7%). In both phenotypes, the content of free apolipoprotein E was markedly reduced by ultracentrifugation. The results confirm the notion that the formation of the E-A-II complex is a major determinant of the stability of apolipoprotein E-high-density lipoprotein associations. Moreover, that the predominant, ancestral isoform, apolipoprotein E3, exists largely as an E-A-II complex in higher density lipoproteins has important functional implications for this plasma source of apolipoprotein E.

[A favorable hyperlipoproteinemia: high-density lipoproteins and atherosclerosis]. / Eine günstige Hyperlipoproteinämie: Lipoproteine hoher Dichte und Atherosklerose.

A full, clinical appreciation of blood cholesterol levels necessitates recognition of the divergent influences of cholesterol associated with low density lipoprotein (atherogenic) and high density lipoproteins (HDL, anti-atherogenic) on the atherosclerotic process. Convincing evidence now exists to show that raising HDL-cholesterol levels reduces the incidence of cardiovascular disease, thus providing a firm rationale for therapeutic measures designed to favourably modify its plasma concentrations. In this respect, association of lower HDL-cholesterol levels with an unhealthy life style (smoking, lack of exercise, obesity) is of particular relevance. Whilst still a subject of discussion, available data strongly support the inclusion of HDL-cholesterol measurements when establishing plasma lipid profiles.

A novel high-density lipoprotein particle and associated protein in rat plasma.

This report describes the characterization of a novel rat apolipoprotein, which, as partial sequencing suggests, does not correspond to any described protein. The protein (termed PX) has an estimated molecular mass of 19.5 kDa and pI in the range 5.5-5.8. Monoclonal antibodies were obtained against protein PX and results on distribution among rat lipoproteins show it to be associated mainly with high-density lipoproteins (HDL), but also with VLDL. Immunoaffinity chromatography of total HDL shows protein PX to be included in a distinct lipoprotein particle, particularly enriched in free cholesterol, with which only traces of other apolipoproteins are associated. Immunologically crossreacting entities are found in the plasma of several species, including man. Retention of the epitope carried by the protein PX would suggest that it is of particular structural or functional importance. It remains to be established whether its function is associated with lipid metabolism.

[Remnant disease associated with apoprotein E1; clinical importance of apoprotein E (apo E) phenotypes]. / "Maladie des remnants" ("Remnants disease") associée à l'apoprotéine E1; importance clinique des phénotypes de l'aproprotéine E (apo E).

The diagnosis of type III hyperlipoproteinemia, or remnant disease, is greatly facilitated by the determination of the apoprotein E (apo E) phenotype. The disease is associated, in over 90% of the documented cases, with homozygosity for the E2 isoform of apo E. Phenotyping apo E directly from plasma offers certain advantages as compared to phenotyping from very low density lipoprotein samples. Elaboration of such a procedure in our laboratory allowed us to detect a rare isoform of apo E, apo E1, which was associated with remnant disease in 3 patients.