Apolipoprotein E-rich HDL in patients with homozygous familial hypercholesterolemia

Ordinarily, HDL1, a fraction of HDL enriched in apoE, is a minor fraction of plasma, but in human subjects and experimental animals eating diets high in fat and cholesterol and in patients with homozygous familial hypercholesterolemia (HFH) or CETP deficiency, HDL1 (or HDLc) concentrations in plasma are increased. However, little is known about the structures, compositions and metabolic sources of HDL1 in HFH patients. To obtain HDL1 for the study, we surveyed several fractions in the HDL density range for apoE by SDS-PAGE. The ratio of apoE to apoAI in the HDL (d = 1.063-1.21 g/ml) of 8 HFH patients was 0.14 +/- 0.03 compared to 0.03 +/- 0.005 in a control group of 8 normolipidemic subjects (P less than 0.001) suggesting that an apoE-rich fraction indeed was present in increased amounts. ApoE/apoAI ratios of lipoproteins of the density range 1.050-1.090 were even higher at 1.5 and 2.0 in 2 patients compared to 0.4 +/- 0.1 in controls, indicating that this density fraction may be particularly enriched with apoE-rich lipoproteins. By contrast, d = 1.020-1.050 g/ml and d greater than 1.090 fractions contained very little apoE. Therefore, we further characterized the d = 1.050-1.090 g/ml lipoproteins of HFH patients and controls. Fractionation of an d = 1.050-1.090 fraction by concanavalin-A chromatography (CONA) yielded an unbound apoE-rich fraction that contained apoE, apoAI and apoC but no apoB, and a bound LDL-like fraction that contained mostly apoB-100, as determined by SDS-PAGE and by solid phase immunoassays, containing monoclonal antibodies directed against apoB, apoE and apoAI. The apoE/apoAI ratio of the CONA unbound fraction of HFH patients was greater, and the fraction also contained more free cholesterol and phospholipids than the fraction of control subjects.(ABSTRACT TRUNCATED AT 250 WORDS)     

Estrone analogs as potential inhibitors targeting EGFR-MAPK pathway in non-small-cell lung cancer

Lung cancer is the deadliest human cancer globally, with non-small-cell lung cancer (NSCLC) being the most frequent type. Epidermal growth factor receptor (EGFR), a central regulator of tumor progression is frequently overexpressed in NSCLC and is a key drug target along with its downstream pathways. Here, we describe the biological evaluation of previously synthesized estrone analogs as potent inhibitors of NCI-H226 cells. Two of the analogs, MMA307 and MM320, significantly inhibited the proliferation of NCI-H226 cells with IC₅₀ doses of 2.88 ± 0.21 and 9.68 ± 0.24 μM, respectively, compared with the positive control and chemotherapy, sorafenib, IC₅₀ of 20.62 ± 1.32 μM. Exposing NCI-H226 cells to IC₅₀ concentration of MMA307 and MMA320 resulted in the downregulation of EGFR and phospho-EGFR expression levels, and suppression of activated MAPK-ERK_1/2 signaling proteins; phospho-B-Raf, phospho-MEK_1/2, and phospho-ERK_1/2. Furthermore, the downregulation of cyclin D₁ and concomitant upregulation of phospho-cyclin D₁ and p21^waf1/cip1 were observed after the compounds' addition to NCI-H226 cells resulting in G₁ phase cell cycle arrest. MMA320 but not MMA307 downregulated the expression levels of Dyrk1B, a checkpoint kinase at the G₁–S phase transition of the cell cycle. Additionally, molecular dynamic simulations were performed and found that MMA307 and MMA320 have higher binding affinities than sorafenib in MEK, BRAF, cyclin D₁, and Dyrk1B (dual-specificity tyrosine phosphorylation-regulated kinase 1B). To conclude, the present study is the first to report on the antiproliferative potential of novel estrone analogs and provide evidence that MMA307 and MMA320 are promising novel lead candidates for the development of antilung cancer drugs.     

Removal of Apolipoprotein B from Dog Whole Blood by Ex Vivo Hemoadsorption on Antibody-Agarose Beads

Antibody columns for hemoperfusion were prepared by filling 300-ml polycarbonate canisters with 2% agarose gel beads having attached goat antibodies directed toward apolipoprotein B (apo B), the major apoprotein of low and very low density lipoproteins. Blood was withdrawn from the left external jugular vein of dogs, regionally treated with anticoagulant citrate dextrose solution A, pumped through the antibody column, and returned to the right external jugular vein. Immunoreactive apo B decreased by 81% during passage of blood over the column. Adverse effects were not observed during four weekly hour-long perfusions with blank columns (agarose beads without antibody attached) followed by four weekly perfusions with antibody columns. The columns were disinfected and stored in 1 M acetic acid and reused weekly in each animal. Recovery of platelets and white blood cells over the columns was 90 and 102%, respectively, with no significant differences between blank columns and antibody-containing columns. Complement was not consumed during the hemoadsorption procedure. Hemoadsorption on antibody-agarose columns is a promising potential method for removing toxic molecules and cells from whole blood.     

Insulin-stimulated release of D-chiro-inositol-containing inositolphosphoglycan mediator correlates with insulin sensitivity in women with polycystic ovary syndrome

Some actions of insulin are mediated by inositolphosphoglycan (IPG) mediators. Deficient release of a putative d-chiro-inositol-containing (DCI) IPG mediator may contribute to insulin resistance in women with polycystic ovary syndrome (PCOS). Previously, we demonstrated that oral DCI supplementation improved ovulation and metabolic parameters in women with PCOS. However, whether oral DCI mediates an increase in the release of the DCI-IPG mediator and an improvement in insulin sensitivity in women with PCOS is unknown. We conducted a randomized controlled trial of DCI supplementation vs placebo in 11 women with PCOS who were assessed at 2 time points 6 weeks apart. Plasma DCI, DCI-IPG release during oral glucose tolerance test (AUC_DCI-IPG), and insulin sensitivity (S_i) by frequently sampled intravenous glucose tolerance test were assessed at baseline and end of study. The study was terminated early because of a sudden unavailability of the study drug. However, in all subjects without regard to treatment assignment, there was a positive correlation between the change in AUC_DCI-IPG/AUC_insulin ratio and the change in S_i during the 6-week period (r = 0.69, P = .02), which remained significant after adjustment for body mass index (P = .022) and after further adjustment for body mass index and treatment allocation (P = .0261). This suggests that, in women with PCOS, increased glucose-stimulated DCI-IPG release is significantly correlated with improved insulin sensitivity. The significant relationship between DCI-IPG release and insulin sensitivity suggests that the DCI-IPG mediator may be a target for therapeutic interventions in PCOS.     

Diversity in expression of heterozygous familial hypercholesterolemia. Characterization of a unique kindred

Clinical and biochemical characteristics of familial hypercholesterolemia (FH) heterozygotes possessing an abnormally high molecular weight low density lipoprotein receptor (HMWR) are reported. The disorder is transmitted as an autosomal dominant trait and is not distinguishable from classic heterozygous FH on clinical grounds. The average plasma low density lipoprotein (LDL) level is 360 mg/dl and tendon xanthomata and early coronary disease are present. LDL receptor activity is higher than expected. In skin fibroblast cultures two types of functional LDL receptors are present, one with a normal apparent native molecular weight of 140,000, and the other of 176,000. When immobilized on nitrocellulose paper both receptors bind LDL. Maximum 125I-LDL binding capacity of fibroblast monolayers is reduced only 20%, compared with 50% in typical heterozygous FH. Affinity for 125I-LDL is increased and a 38% reduction in the Michaelis constant for LDL is observed. When autologous 125I-LDL was injected intravenously, the fractional catabolic rate of LDL was 205% and the LDL apoprotein B production rate was 328% of that found in a typical heterozygous FH subject. Thus, both in vitro and in vivo testing indicated only a modest deficiency of LDL receptor activity. Kindred members possessing the HMWR had an associated abnormality of cholesterol biosynthesis. Cholesterol balance studies in three individuals with the HMWR trait demonstrated elevated cholesterol biosynthesis of two to three times the mean of normal subjects. These findings suggest that increased LDL production and increased cholesterol production may assume a significant role in the pathologic manifestations of heterozygous FH. Functional abnormalities in LDL receptor activity as measured in fibroblast culture may be relatively small.     

Chronic B-lymphocytic leukemia cells proliferate and differentiate following exposure to interferon in vitro

The ability of interferon (IFN) to induce proliferation and differentiation in malignant B cells from 29 patients with chronic lymphocytic leukemia (CLL) and in lymphoid cells from 11 healthy donors was investigated. IFN induced transformation and plasmacytoid differentiation in B cells from 19 of 29 CLL patients. The transformed cells belonged to the malignant clone as indicated by the immunoglobulin (lg) light chain restriction. Cells exposed to IFN expressed intracellular lg to a varying degree, which was correlated to the level of plasmacytoid differentiation. IFN gave rise to proliferative responses in cells from three patients. Cytogenetic studies on lymphoid cells from one patient showed that proliferation occurred in the malignant B cells. Induction of proliferation and differentiation was observed with various alpha-IFN and gamma-IFN preparations, as well as with a completely pure beta-IFN, showing that IFN and not contaminants in the preparations were responsible for the observed effects. Maximal transformation and proliferation usually occurred after four days of incubation at an IFN concentration of 500 to 5,000 U/mL. The ability of IFN to induce differentiation in CLL cells may be of importance for the reported antitumoral effects observed in some B cell malignancies during IFN therapy.