Comparison of SSCP and DHPLC for the detection of LDLR mutations in a New Zealand cohort

Familial hypercholesterolaemia (FH) is a common inherited disorder, associated with premature vascular disease. FH may be caused by many different mutations in the low density lipoprotein receptor (LDLR) gene, about 700 mutations have been described, most of which occur rarely and often only in single families. Although particular mutations are prevalent in certain ethnic groups, countries with heterogeneous population bases (such as NZ) may carry a wide variety of mutations; making a gene screening approach the appropriate first step for a mutation detection programme. We have compared SSCP with DHPLC to assess their effectiveness as methods for LDLR mutation detection. Although five novel LDLR mutations were detected by SSCP in patients with FH, DHPLC was more sensitive, with eight novel mutations detected. Six of these mutations (T392M, R419G, Y421N, 1206-1207delCT, 1872delC, and 1943delC) were clustered in exons 9 and 13 of the EGF precursor homology domain, one (679-680delAC) in the ligand binding domain (exon 4) and the eighth (P774H) in the membrane-spanning domain (exon 16). Twenty five mutations were identified in 35 patients in total. Of these, we were able to detect only 64% of mutations by SSCP even though all variants were detected by DHPLC. All patients are heterozygous for the mutations, which is consistent with the clinical phenotypes.     

Trans-Epithelial Transport,Metabolism, and Biological Activity Assessment of the Multi-Target Lupin Peptide LILPKHSDAD (P5) and Its Metabolite LPKHSDAD (P5-Met)

P5 (LILPKHSDAD) is a hypocholesterolemic peptide from lupin protein with a multi-target activity, since it inhibits both 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCoAR) and proprotein convertase subtilisin/kexin type-9 (PCSK9). This work shows that, during epithelial transport experiments, the metabolic transformation mediated by intestinal peptidases produces two main detected peptides, ILPKHSDAD (P5-frag) and LPKHSDAD (P5-met), and that both P5 and P5-met are linearly absorbed by differentiated human intestinal Caco-2 cells. Extensive comparative structural, biochemical, and cellular characterizations of P5-met and the parent peptide P5 demonstrate that both peptides have unique characteristics and share the same mechanisms of action. In fact, they exert an intrinsically multi-target behavior being able to regulate cholesterol metabolism by modulating different pathways. The results of this study also highlight the dynamic nature of bioactive peptides that may be modulated by the biological systems they get in contact with.     

补肾益气方对流产大鼠胎盘孕酮生成通路的影响

目的:探讨补肾益气方对流产大鼠胎盘孕酮生成通路中相关重要上游因素低密度脂蛋白受体(LDLR)、细胞色素P450链裂解酶(P450scc)、3β-羟基类固醇脱氢酶(3β-HSD)的影响及其与孕酮(P)分泌的关系。方法:溴隐亭皮内注射法建立流产大鼠模型,分别于孕1-8 d、1-11 d灌服补肾益气方药,黄体酮肌注作为阳性对照组,采用放免法测定大鼠血清P水平;RT-PCR技术检测LDLR、P450scc、3β-HSD在不同组动物胎盘中的mRNA表达。结果:模型组LDLR、P450scc、3β-HSD mRNA表达较正常对照组明显降低;中药组及黄体酮组不同程度提高模型大鼠胎盘LDLR、P450scc、3β-HSD mRNA表达,表达强度随孕天和给药时间逐渐升高,与血P有显著相关性。结论:补肾益气方清热益气、固肾安胎,能调控胎盘滋养细胞LDLR、P450scc、3β-HSD基因表达,促进孕激素合成分泌增加,从而达到保胎维持妊娠的目的。     

Brief Report: Divergent low-density lipoprotein receptor (LDLR) linked to low VSV G-dependent viral infectivity and unique serum lipid profile in zebra finches

The low-density lipoprotein receptor (LDLR) is key to cellular cholesterol uptake and is also the main receptor for the vesicular stomatitis virus glycoprotein (VSV G). Here we show that in songbirds LDLR is highly divergent and lacks domains critical for ligand binding and cellular trafficking, inconsistent with universal structure conservation and function across vertebrates. Linked to the LDLR functional domain loss, zebra finches show inefficient infectivity by lentiviruses (LVs) pseudotyped with VSV G, which can be rescued by the expression of human LDLR. Finches also show an atypical plasma lipid distribution that relies largely on high-density lipoprotein (HDL). These findings provide insights into the genetics and evolution of viral infectivity and cholesterol transport mechanisms in vertebrates.     

Patched, the receptor of Hedgehog, is a lipoprotein receptor

The Hedgehog (Hh) family of secreted signaling proteins has a broad variety of functions during metazoan development and implications in human disease. Despite Hh being modified by two lipophilic adducts, Hh migrates far from its site of synthesis and programs cellular outcomes depending on its local concentrations. Recently, lipoproteins were suggested to act as carriers to mediate Hh transport in Drosophila. Here, we examine the role of lipophorins (Lp), the Drosophila lipoproteins, in Hh signaling in the wing imaginal disk, a tissue that does not express Lp but obtains it through the hemolymph. We use the up-regulation of the Lp receptor 2 (LpR2), the main Lp receptor expressed in the imaginal disk cells, to increase Lp endocytosis and locally reduce the amount of available free extracellular Lp in the wing disk epithelium. Under this condition, secreted Hh is not stabilized in the extracellular matrix. We obtain similar results after a generalized knock-down of hemolymph Lp levels. These data suggest that Hh must be packaged with Lp in the producing cells for proper spreading. Interestingly, we also show that Patched (Ptc), the Hh receptor, is a lipoprotein receptor; Ptc actively internalizes Lp into the endocytic compartment in a Hh-independent manner and physically interacts with Lp. Ptc, as a lipoprotein receptor, can affect intracellular lipid homeostasis in imaginal disk cells. However, by using different Ptc mutants, we show that Lp internalization does not play a major role in Hh signal transduction but does in Hh gradient formation.     

Prognostic impact of cascade screening for familial hypercholesterolemia on cardiovascular events

BackgroundFamilial hypercholesterolemia (FH) is an autosomal dominant disorder mainly caused by mutations in the low-density lipoprotein (LDL) receptor or associated genes, resulting in elevated serum cholesterol levels and an increased risk of premature atherosclerotic cardiovascular disease (ASCVD).ObjectiveWe aimed to evaluate the prognostic impact of cascade screening for FH.MethodsWe retrospectively investigated the health records of 1050 patients with clinically diagnosed FH, including probands and their relatives who were cascade-screened, who were referred to our institute. We used Cox models that were adjusted for established ASCVD risk factors to assess the association between cascade screening and major adverse cardiac events (MACE). The median period of follow-up evaluating MACE was 12.3 years (interquartile ranges [IQR] = 9.1–17.5 years), and MACE included death associated with ASCVD, or acute coronary syndrome.ResultsDuring the observation period, 113 participants experienced MACE. The mean age of patients identified through cascade screening was 18-years younger than that of the probands (38.7 yr vs. 57.0 yr, P < 0.0001), with a lower proportion of ASCVD risk factors. Interestingly, patients identified through cascade screening under milder lipid-lowering therapies were at reduced risk for MACE (hazard ratio [HR] = 0.67; 95%CI = 0.44 to 0.90; P = 0.0044) when compared with the probands, even after adjusting for those known risk factors, including age, and prior ASCVD.ConclusionsThe identification of patients with FH via cascade screening appeared to result in better prognosis.     

Effects of squalene on expression of LDLR in HepG2 cells and its mechanism北大核心CSCD

Aim To investigate the effect of squalene on LDLR expression in HepG2 cells and its mechanism of down-regulated cholesterol. Methods The proliferation of HepG2 cells exposed to squalene at different concentrations was measured by MTT assay. The effect of squalene on the expression of LDLR in HepG2 cells was measured by flow cytometry and fluorescence mi-croscopy. The effect of different concentrations of squalene on the interaction between SCAP and Insig2, two key protein molecules of SREBP pathway, was assayed by FRET technology. Results MTT results showed that squalene had inhibitory effect on the proliferation of HepG2 cells in a dose-dependent manner. Flow cytometry and fluorescence microscopy results showed that squalene enhanced LDLR expression in HepG2 cells compared with the control group. The results of FRET technology revealed that compared with model control group, the YFP fluorescence value in Squalene group dramatically declined, and the YFP fluorescence value of each drug group decreased with the range of 5-25 |xmol L1 squalene concentration. Conclusions Squalene may promote the expression of LDLR in HepG2 cells through inhibiting the interaction between SCAP and Insig2 proteins in SREBP pathway, which may confirm that squalene is a potential novel drug for the down-regulation of cholesterol level. © 2018 Publication Centre of Anhui Medical University. All rights reserved.     

Association of LDLR rs1433099 with the Risk of NAFLD and CVD in Chinese Han Population

Background and AimsRecent genome-wide association studies have shown that low-density lipoprotein receptor (LDLR) rs1433099 polymorphism is associated with cardiovascular disease (CVD) risk in many countries. However, the association of LDLR rs1433099 with CVD in China has not been reported yet. There are no studies on LDLR rs1433099 and non-alcoholic fatty liver disease (NAFLD) as well. The purpose of this study was to investigate whether LDLR rs1433099 is related to CVD or NAFLD in the Chinese population.MethodsLDLR rs1433099 polymorphism was genotyped in 507 individuals, including 140 healthy controls, 79 NAFLD patients, 185 CVD patients, and 103 patients with NAFLD combined with CVD. The expression of LDLR was tested by the sequence detection system, and clinical parameters were assessed by biochemical tests and physical examination.ResultsThe genotype distribution of LDLR rs1433099 was not statistically different among the NAFLD group, the CVD group, the combined group, and the healthy control group (p>0.05). There was no significant correlation of LDLR rs1433099 genotypic distribution or allele frequency and the risk of NAFLD, CVD or NAFLD combined with CVD (p>0.05). In the CVD group, T allele carriers had higher alkaline phosphatase and gamma-glutamyl transpeptidase than non-carriers (p<0.05).ConclusionsOur study demonstrated that the LDLR rs1433099 polymorphism is not a risk factor of NAFLD. The LDLR rs1433099 polymorphism may increase the risk of CVD through a mechanism involving alkaline phosphatase and gamma-glutamyl transpeptidase.