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Low-density Lipoprotein (low-density + lipoprotein)
Kinds of Low-density Lipoprotein Terms modified by Low-density Lipoprotein Selected AbstractsOXIDIZED LOW-DENSITY LIPOPROTEIN INDUCES ENDOTHELIAL PROGENITOR CELL SENESCENCE, LEADING TO CELLULAR DYSFUNCTIONCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 7 2004Toshio Imanishi SUMMARY 1.,Recent studies have revealed an association between coronary risk factors and both the number and function of bone marrow-derived endothelial progenitor cells (EPC). We investigated the effect of oxidized low-density lipoprotein (ox-LDL) on the senescence of EPC, leading to cellular dysfunction. 2.,Endothelial progenitor cells were isolated from human peripheral blood and characterized. The exposure of cultured EPC to ox-LDL (10 µg/mL) significantly accelerated the rate of senescence compared with control during 20 days in culture as determined by acidic ,-galactosidase staining. Oxidized LDL-induced EPC senescence was significantly inhibited by pretreatment with either lectin-like ox-LDL receptor-1 (LOX-1) antibody (Ab) or atorvastatin (P < 0.01). 3.,Because cellular senescence is critically influenced by telomerase, which elongates telomeres, we measured telomerase activity using a polymerase chain reaction,ELISA-based assay. Oxidized LDL significantly diminished telomerase activity to approximately 50%, an effect that was significantly abolished by pretreatment with either LOX-1 Ab or atorvastatin (P < 0.01). 4.,We examined whether ox-LDL-induced EPC senescence translates into EPC dysfunction. An MTS assay disclosed an inhibitory effect of ox-LDL on EPC proliferation. In a Matrigel assay, EPC treated with ox-LDL were less likely to participate in network fomation compared with controls. 5.,In conclusions, ox-LDL accelerates the onset of EPC senescence, which may be related to telomerase inactivation. Oxidized LDL-induced EPC senescence leads to the impairment of proliferative capacity and network formation. [source] Coatings of Low-Density Lipoprotein and Synthetic Glycoconjugates as Substrata for HepatocytesARTIFICIAL ORGANS, Issue 6 2009Hirofumi Yura Abstract Asialoglycoprotein (ASGP) receptors expressed on rat hepatocytes interact with glycoproteins containing galactose or N-acetylgalactosamine residues at the nonreducing termini of oligosaccharide chains to mediate endocytosis, and cholesterol transport protein with apolipoprotein B (LDL, low-density lipoprotein) in plasma interacts with LDL receptors and heparinoids in the extracellular matrix. We developed novel techniques to prepare galactose- and LDL-immobilized culture plates, using galactose-tagged polystyrene (galactose-carrying polystyrene [GalCPS]: N-p-vinylbenzyl-O-,-D-galactopyranosyl-[1,4]-D-gluconamide) and poly(2-acrylamide-2-methyl-1-propanesulfonate) (PAPS), respectively. Hepatocytes adhered well to plates coated with either GalCPS or LDL, and therefore the GalCPS- and LDL-coated plates were examined as specific substrata for culturing hepatocytes. These cultures promoted the formation of three-dimensional, multicellular aggregates with regulation of excess proliferation of non-parenchymal cells. Furthermore, the LDL coating resulted in higher albumin synthesis and an identical level of lactate dehydrogenase (LDH) compared with cells cultured on collagen- and GalCPS-coated plates. Thus, the two culture systems described here, and especially the LDL-coated plates, have potential for the development of a hybrid artificial liver. [source] Pathogenesis, detection and treatment of Achilles tendon xanthomasEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 4 2005S. G. Tsouli Abstract Tendon xanthomatosis often accompanies familial hypercholesterolaemia, but it can also occur in other pathologic states. Achilles tendons are the most common sites of tendon xanthomas. Low-density lipoprotein (LDL) derived from the circulation accumulates into tendons. The next steps leading to the formation of Achilles tendon xanthomas (ATX) are the transformation of LDL into oxidized LDL (oxLDL) and the active uptake of oxLDL by macrophages within the tendons. Although physical examination may reveal Achilles tendon xanthomas (ATX), there are several imaging methods for their detection. It is worth mentioning that ultrasonography is the method of choice in everyday clinical practice. Although several treatments for Achilles tendon xanthomas (ATX) have been proposed (LDL apheresis, statins, etc.), they target mostly in the treatment of the basic metabolic disorder of lipid metabolism, which is the main cause of these lesions. In this review we describe the formation, detection, differential diagnosis and treatment of ATX as well as the relationship between tendon xanthomas and atheroma. [source] Liver endothelial cells promote LDL-R expression and the uptake of HCV-like particles in primary rat and human hepatocytes,HEPATOLOGY, Issue 2 2006Yaakov Nahmias Low-density lipoprotein (LDL) is an important carrier of plasma cholesterol and triglycerides whose concentration is regulated by the liver parenchymal cells. Abnormal LDL regulation is thought to cause atherosclerosis, while viral binding to LDL has been suggested to facilitate hepatitis C infection. Primary hepatocytes quickly lose the ability to clear LDL during in vitro culture. Here we show that the coculture of hepatocytes with liver sinusoidal endothelial cells (LSEC) significantly increases the ability of hepatocytes to uptake LDL in vitro. LDL uptake does not increase when hepatocytes are cocultured with other cell types such as fibroblasts or umbilical vein endothelial cells. We find that LSECs induce the hepatic expression of the LDL receptor and the epidermal growth factor receptor. In addition, while hepatocytes in single culture did not take up hepatitis C virus (HCV)-like particles, the hepatocytes cocultured with LSECs showed a high level of HCV-like particle uptake. We suggest that coculture with LSECs induces the emergence of a sinusoidal surface in primary hepatocytes conducive to the uptake of HCV-like particles. In conclusion, our findings describe a novel model of polarized hepatocytes in vitro that can be used for the study of LDL metabolism and hepatitis C infection. (HEPATOLOGY 2006;43:257,265.) [source] C-terminal 37 residues of LRP promote the amyloidogenic processing of APP independent of FE65JOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 6b 2008Madepalli K. Lakshmana Abstract The major defining pathological hallmark of Alzheimer's disease (AD) is the accumulation of amyloid , protein (A,), a small peptide derived from ,- and ,-secretase cleavages of the amyloid precursor protein (APP). Recent studies have shown that the Low-density lipoprotein receptor-related protein (LRP) plays a pivotal role in the trafficking of APP and generation of A,. In particular, we recently showed that the soluble cytoplasmic tail of LRP (LRP-ST) without a membrane tether was sufficient to promote A, generation. In this study, we demonstrate that the last 37 residues of LRP cytoplasmic tail (LRP-C37) lacking the NPxY motifs and FE65 binding mediate the core pro-amyloidogenic activity of LRP-ST. Moreover, we show that the conserved dileucine motif within the LRP-C37 region is a key determinant of its A, promoting activity. Finally, results from a yeast two-hybrid screen using LRP-C37 region as bait reveal four new LRP-binding proteins implicated in intracellular signalling and membrane protein trafficking. Our findings indicate that the LRP-C37 sequence represents a new protein-binding domain that may be useful as a therapeutic target and tool to lower A, generation in AD. [source] Low-density lipoprotein receptor-related protein (LRP)-mediated clearance of activated blood coagulation co-factors and proteases: clearance mechanism or regulation?JOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 7 2006D. K. STRICKLAND No abstract is available for this article. [source] Low-density lipoprotein receptor-related protein (LRP)-2/megalin is transiently expressed in a subpopulation of neural progenitors in the embryonic mouse spinal cordTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 2 2005Grzegorz Wicher Abstract The lipoprotein receptor LRP2/megalin is expressed by absorptive epithelia and involved in receptor-mediated endocytosis of a wide range of ligands. Megalin is expressed in the neuroepithelium during central nervous system (CNS) development. Mice with homozygous deletions of the megalin gene show severe forebrain abnormalities. The possible role of megalin in the developing spinal cord, however, is unknown. Here we examined the spatial and temporal expression pattern of megalin in the embryonic mouse spinal cord using an antibody that specifically recognizes the cytoplasmic part of the megalin molecule. In line with published data, we show expression of megalin in ependymal cells of the central canal from embryonic day (E)11 until birth. In addition, from E11 until E15 a population of cells was found in the dorsal part of the developing spinal cord strongly immunoreactive against megalin. Double labeling showed that most of these cells express vimentin, a marker for immature astrocytes and radial glia, but not brain lipid binding protein (BLBP), a marker for radial glial cells, or glial fibrillary acidic protein (GFAP), a marker for mature astrocytes. These findings indicate that the majority of the megalin-positive cells are astroglial precursors. Megalin immunoreactivity was mainly localized in the nuclei of these cells, suggesting that the cytoplasmic part of the megalin molecule can be cleaved following ligand binding and translocated to the nucleus to act as a transcription factor or regulate other transcription factors. These findings suggest that megalin has a crucial role in the development of astrocytes of the spinal cord. J. Comp. Neurol. 492:123,131, 2005. © 2005 Wiley-Liss, Inc. [source] Clearance of coagulation factor VIII in very low-density lipoprotein receptor knockout miceBRITISH JOURNAL OF HAEMATOLOGY, Issue 5 2004Niels Bovenschen Summary Low-density lipoprotein receptor-related protein (LRP) contributes to factor VIII (FVIII) catabolism in vivo. Besides LRP, FVIII also interacts with very low-density lipoprotein receptor (VLDLR) in vitro. We investigated the physiological role of VLDLR in FVIII catabolism, using knockout mouse models for VLDLR and LRP, alone and in combination. VLDLR,/, mice displayed normal plasma FVIII, whereas VLDLR,/, LRP, double-knockout mice had slightly increased FVIII compared with LRP-deficient mice. Remarkably, VLDLR deficiency slightly accelerated FVIII clearance. Adenovirus-mediated overexpression of VLDLR did not lower plasma FVIII in LRP-deficient mice. We conclude that VLDLR does not act in concert with LRP in FVIII clearance in vivo. [source] Metabolism of cholesterol ester of apolipoprotein B100-containing lipoproteins in dogs: evidence for disregarding cholesterol ester transferEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 8 2004E. Bailhache Abstract Background, It has been shown that dogs exhibit no cholesterol ester transfer protein (CETP) activity in vitro, in contrast to humans. The aim of our study was to determine modalities of in vivo plasma cholesterol ester turnover in this species, using a kinetic approach with stable isotopes. Materials and methods, Kinetics of very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) were studied in seven adult male Beagle dogs using a dual isotope approach through endogenous labelling of both their cholesterol moiety and their protein moiety. A primed constant infusion of both [1,213C]acetate and [5,5,5- 2H3]leucine enabled us to obtain measurable deuterium enrichments by gas chromatography-mass spectrometry for plasma leucine and apoB100, as well as measurable 13C enrichment by gas chromatography-combustion-isotopic ratio mass spectrometry for unesterified cholesterol and cholesterol ester in the VLDL and LDL. Two identical multicompartmental models (SAAM II) were used together for the analysis of tracer kinetics' data of proteins and cholesterol. Results, Characterization of the apoB100-containing lipoprotein cholesterol ester model allowed determination of kinetic parameters of VLDL and LDL cholesterol ester metabolism. We succeeded in modelling VLDL and LDL cholesterol ester metabolism and apoB100 metabolism simultaneously. Fractional catabolic rate (FCR) of apoB100 and CE had the same values. Introducing cholesterol ester transfer between lipoproteins in the model did not significantly improve the fit. Total VLDL FCR was 2·97 ± 01·47 h,1. Approximately one-quarter corresponded to the direct removal of VLDL (0·81 ± 00·34 h,1) and the remaining three-quarters corresponded to the fraction of VLDL converted to LDL, which represented a conversion of VLDL into LDL of 2·16 ± 01·16 h,1. Low-density lipoproteins were produced exclusively from VLDL conversion and were then removed (0·031 ± 0·004 h,1) from plasma. Conclusion, These kinetic data showed that VLDL cholesterol ester and LDL cholesterol ester metabolism followed VLDL and LDL apoB100 metabolism, and that consequently there is no in vivo transfer of cholesterol ester in dogs. [source] Fate of fatty acids at rest and during exercise: regulatory mechanismsACTA PHYSIOLOGICA, Issue 4 2003M. D. Jensen Abstract Fatty acids are a major fuel source for humans both at rest and during exercise. Plasma free fatty acids (FFA), although present only in micromolar concentrations, are the major circulating lipid fuel. FFA availability can increase two- to four-fold with moderate intensity exercise. Other potential sources of fatty acids include circulating very low-density lipoprotein (VLDL) triglycerides (TGs) (,1/5 the fuel availability of FFA) and intramyocellular TGs (,2 mmol kg,1 muscle). At rest ,40% of systemic FFA uptake occurs in the splanchnic bed and uptake in legs is ,15,20%. During leg exercise the uptake of FFA in leg tissue increases to 30,60% of systemic uptake and splanchnic uptake decreases to 15%. The fate of VLDL TG fatty acids has not been adequately studied. Intramyocellular TG hydrolysis increases during exercise, but the factors that regulate this response are not clear. The fact that contraction of isolated muscles can stimulate the hydrolysis and oxidation of intramyocellular TGs (in the absence of hormonal or neural input) suggests an intracellular regulation of this process. Additional regulation from changes in catecholamines and insulin may also occur. During moderate intensity exercise circulating FFA and intramyocellular TG provide roughly equal portions of fatty acids for oxidation. In addition to endurance training, dietary factors have been shown to modulate the fatty acid oxidation response to exercise. Much remains to be learned about fatty acid trafficking during exercise. What role do VLDL TG play? How is the oxidation of intramyocellular TGs regulated? Techniques to address these questions in humans are only now becoming available. [source] Long-term use of the ketogenic diet in the treatment of epilepsyDEVELOPMENTAL MEDICINE & CHILD NEUROLOGY, Issue 12 2006Darcy K Groesbeck BS Long-term outcomes of the ketogenic diet in the treatment of epilepsy have not previously been reported. A retrospective chart review of children treated with the ketogenic diet for more than 6 years at the Johns Hopkins Hospital was performed. The response was documented at clinic visits and by telephone contacts; laboratory studies were obtained approximately every 6 to 12 months. Satisfaction and tolerability were assessed by means of a brief parental telephone questionnaire. In all, 28 patients (15 males, 13 females), currently aged 7 to 23 years, were identified. The median baseline seizure frequency per week at diet onset was 630 (range 1,1400). Diet duration ranged from 6 to 12 years; 19 remain on the diet currently. After 6 years or more, 24 children experienced a more than 90% decrease in seizures, and 22 parents reported satisfaction with the diet's efficacy. Ten children were at less than the 10th centile for height at diet initiation; this number increased to 23 at the most recent follow-up (p=0.001). Kidney stones occurred in seven children and skeletal fractures in six. After 6 years or more the mean cholesterol level was 201mg/dl, high-density lipoprotein was 54mg/dl, low-density lipoprotein was 129mg/dl, and triglycerides were 97mg/dl. Efficacy and overall tolerability for children are maintained after prolonged use of the ketogenic diet. However, side effects, such as slowed growth, kidney stones, and fractures, should be monitored closely. [source] Lipid-lowering therapy in patients with type 2 diabetes: the case for early interventionDIABETES/METABOLISM: RESEARCH AND REVIEWS, Issue 4 2008Armin Steinmetz Abstract Chronic complications of type 2 diabetes, in particular, macrovascular complications, confer substantial morbidity and mortality and adversely affect a patient's quality of life. Early intensive intervention to control cardiovascular risk factors is essential in clinical management. Atherogenic dyslipidaemia characterized by elevated triglycerides, a low level of high-density lipoprotein cholesterol (HDL-C), and an increase in the preponderance of small, dense low-density lipoprotein (LDL) particles, is a key modifiable risk factor for macrovascular diabetic complications. Lowering low-density lipoprotein cholesterol (LDL-C) with a statin (or the combination of statin and ezetimibe) is the main focus for reducing cardiovascular risk in patients with diabetes. However, statins fail to address the residual cardiovascular risk associated with low HDL-C. Fibrates are effective against all components of the atherogenic dyslipidaemia associated with type 2 diabetes. Secondary analyses of the Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) study suggest a role for early treatment with fenofibrate in improving cardiovascular risk reduction in type 2 diabetes and provide safety data supporting the use of fenofibrate in combination with a statin. Data from the FIELD study suggest that fenofibrate may also have potential to impact on microvascular diabetic complications associated with type 2 diabetes. Data are awaited from the ongoing Action to Control Cardiovascular Risk in Diabetes (ACCORD) study to evaluate the outcome benefits of combining fenofibrate with a statin in patients with type 2 diabetes. Finally, in view of divergent study results and outstanding data, assessment of the risk of the individual with type 2 diabetes is mandatory to assist clinical decision-making when initiating lipid therapy. Copyright © 2008 John Wiley & Sons, Ltd. [source] Peroxisome proliferator-activated receptor-, co-activator-1, (PGC-1,) gene polymorphisms and their relationship to Type 2 diabetes in Asian IndiansDIABETIC MEDICINE, Issue 11 2005K. S. Vimaleswaran Abstract Aims The objective of the present investigation was to examine the relationship of three polymorphisms, Thr394Thr, Gly482Ser and +A2962G, of the peroxisome proliferator activated receptor-, co-activator-1 alpha (PGC-1,) gene with Type 2 diabetes in Asian Indians. Methods The study group comprised 515 Type 2 diabetic and 882 normal glucose tolerant subjects chosen from the Chennai Urban Rural Epidemiology Study, an ongoing population-based study in southern India. The three polymorphisms were genotyped using polymerase chain reaction,restriction fragment length polymorphism (PCR,RFLP). Haplotype frequencies were estimated using an expectation,maximization (EM) algorithm. Linkage disequilibrium was estimated from the estimates of haplotypic frequencies. Results The three polymorphisms studied were not in linkage disequilibrium. With respect to the Thr394Thr polymorphism, 20% of the Type 2 diabetic patients (103/515) had the GA genotype compared with 12% of the normal glucose tolerance (NGT) subjects (108/882) (P = 0.0004). The frequency of the A allele was also higher in Type 2 diabetic subjects (0.11) compared with NGT subjects (0.07) (P = 0.002). Regression analysis revealed the odds ratio for Type 2 diabetes for the susceptible genotype (XA) to be 1.683 (95% confidence intervals: 1.264,2.241, P = 0.0004). Age adjusted glycated haemoglobin (P = 0.003), serum cholesterol (P = 0.001) and low-density lipoprotein (LDL) cholesterol (P = 0.001) levels and systolic blood pressure (P = 0.001) were higher in the NGT subjects with the XA genotype compared with GG genotype. There were no differences in genotype or allelic distribution between the Type 2 diabetic and NGT subjects with respect to the Gly482Ser and +A2962G polymorphisms. Conclusions The A allele of Thr394Thr (G , A) polymorphism of the PGC-1 gene is associated with Type 2 diabetes in Asian Indian subjects and the XA genotype confers 1.6 times higher risk for Type 2 diabetes compared with the GG genotype in this population. [source] Glibenclamide improves postprandial hypertriglyceridaemia in Type 2 diabetic patients by reducing chylomicrons but not the very low-density lipoprotein subfraction levelsDIABETIC MEDICINE, Issue 10 2001I. Skrapari Abstract Aim, There are scarce data dealing with the degree of postprandial lipaemia after sulphonylurea administration. The aim of this study was to examine the effect of acute glibenclamide administration on postprandial lipaemia in Type 2 diabetic patients. Methods, Eight randomly selected Type 2 diabetic individuals, aged 43,65 years (mean, 54 years), who had never received any anti-diabetic drug, were included in the study. Each patient was given a 485 kcal mixed meal (45% fat, 40% carbohydrate and 15% protein) twice on separate days after an overnight fast: once with placebo and once with 5 mg glibenclamide, per os, in a random order. The two tests were performed with an interval of 7 days. Venous blood samples were drawn just before and 2 h, 4 h and 6 h after meal consumption. Total triglyceride levels in plasma, in chylomicrons (CM), in CM-deficient plasma, in very low-density lipoprotein (VLDL) subfractions (VLDL-1, VLDL-2) and in intermediate-density lipoprotein (IDL) were determined. Free fatty acid (FFA) and total cholesterol levels in plasma, as well as high-density lipoprotein (HDL) cholesterol and low-density lipoprotein (LDL) cholesterol levels in CM-deficient plasma, were also measured. Finally, serum glucose, insulin and C-peptide concentrations were measured in each sample. Results, As expected there was a significant decrease in postprandial glycaemia after glibenclamide administration compared to placebo (mean area under the curve values: AUC = 53.3 ± 18.2 and 69.1 ± 21.6 mm/h, P = 0.00009). In addition, the mean AUC values of insulin and C-peptide were significantly greater after drug administration. The AUC values of total plasma triglyceride and of CM triglyceride following glibenclamide administration were significantly lower compared to placebo, while the AUC values of postprandial triglyceride in CM-deficient plasma and of postprandial triglyceride in VLDL-1, VLDL-2 and IDL were not different after drug administration compared to placebo. Finally, no significant differences were noted in the AUC values of total cholesterol, LDL cholesterol, HDL cholesterol and plasma FFA levels after glibenclamide administration. Conclusions, These results demonstrate that glibenclamide administration improves postprandial hypertriglyceridaemia acutely by reducing postprandial triglycerides of intestinal origin. Diabet. Med. 18, 781,785 (2001) [source] Ethnicity and glycaemic control are major determinants of diabetic dyslipidaemia in MalaysiaDIABETIC MEDICINE, Issue 6 2001I. S. Ismail Abstract Aims To define the prevalence of dyslipidaemia in young diabetic patients in Peninsular Malaysia and the contributory factors of dyslipidaemia in these subjects. Methods This is a cross-sectional study involving 848 young diabetic patients from seven different centres, with representation from the three main ethnic groups. Clinical history and physical examination was done and blood taken for HbA1c, fasting glucose, total cholesterol, low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol and triglycerides. Results The overall lipids were suboptimal, worse in Type 2 diabetes mellitus (DM) patients compared with Type 1 DM patients. Of the Type 2 patients, 73.2% had total cholesterol >,5.20 mmol/l, 90.9% had LDL-cholesterol >,2.60 mmol/l, 52.6% had HDL-cholesterol <,1.15 mmol/l and 27.3% had serum triglycerides >,2.30 mmol/l. There were ethnic differences in the lipid levels with the Malays having the highest total cholesterol (mean 6.19 mmol/l), and the highest LDL-cholesterol (mean 4.16 mmol/l), while the Chinese had the highest HDL-cholesterol (geometric mean 1.24 mmol/l). Ethnicity was an important determinant of total, LDL- and HDL-cholesterol in Type 2 DM, and LDL- and HDL-cholesterol and triglycerides in Type 1 DM. Glycaemic control was an important determinant of total, LDL-cholesterol and triglycerides in both Type 1 and Type 2 DM. Waist,hip ratio (WHR) was an important determinant of HDL-cholesterol and triglycerides in both types of DM. Gender was an important determinant of HDL-cholesterol in Type 2 DM, but not in Type 1 DM. Socioeconomic factors and diabetes care facilities did not have any effect on the dyslipidaemia. Conclusions The prevalence of dyslipidaemia was high especially in Type 2 DM patients. Ethnicity, glycaemic control, WHR, and gender were important determinants of dyslipidaemia in young diabetic patients. Diabet. Med. 18, 501,508 (2001) [source] Microchip-based small, dense low-density lipoproteins assay for coronary heart disease risk assessmentELECTROPHORESIS, Issue 9 2008Hua Wang Abstract Small, dense low-density lipoprotein (sdLDL) has been accepted as an emerging cardiovascular risk factor, and there has been an increasing interest in analytical methods for sdLDL profiling for diagnosis. Serum sdLDL may be measured by different laboratory techniques, but all these methods are laborious, time-consuming, and costly. Recently, we have demonstrated that a low-temperature bonding of quartz microfluidic chips for serum lipoproteins analysis (Zhuang, G., Jin, Q., Liu, J., Cong, H. et al., Biomed. Microdevices 2006, 8, 255,261). In contrast to this previous study, we chose SDS as anionic surfactant to modify both lipoproteins and the channel surface to minimize lipoprotein adsorption and improve the resolution of lipoprotein separation. Two major LDL subclass patterns including large, buoyant LDL (lLDL), sdLDL, and high-density lipoprotein (HDL) were effectively separated with high reproducibility. RSD values of the migration time (min) and peak areas of standard LDL and HDL were 6.28, 4.02, 5.02, and 2.5%, respectively. Serum lipoproteins of 15 healthy subjects and 15 patients with coronary heart disease (CHD) were separated by microchip CE. No peaks of sdLDL were detected in serum samples of healthy subjects while sdLDL fractional peaks were observed in patients' entire serum samples. These results suggested that the microchip-based sdLDLs assay was a simple, rapid, and highly efficient technique and significantly improved the analysis of CHD risk factors. [source] Modulation of plasma lipid levels affects benzo[a]pyrene-induced DNA damage in tissues of two hyperlipidemic mouse modelsENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 4 2003Daniëlle M.J. Curfs Abstract The role of plasma lipids in the uptake, transportation, and distribution of lipophilic carcinogens like benzo[a]pyrene (B[a]P) remains unclear. Therefore, we studied the effects of dietary-modulated plasma lipids on B[a]P-induced DNA damage in several organs of two hyperlipidemic mouse models. Male apolipoprotein E (ApoE)*3-Leiden (n = 22) and ApoE knockout (ApoE-KO) mice (n = 20) were fed a high-fat cholesterol (HFC) diet or low-fat cholesterol (LFC; standard mouse chow) diet for 3 weeks, after which the animals were exposed to a single oral dose of 5 mg/kg bw B[a]P or vehicle and killed 4 days later. Plasma lipids were determined and DNA adducts were measured in aorta, heart, lung, liver, brain, and stomach. Total cholesterol and low-density lipoprotein (LDL) cholesterol were increased in all animals on a HFC diet, whereas a decrease of triglycerides was seen only in the ApoE-KO mice. In ApoE-KO mice on a normal diet, DNA-adduct levels were highest in aorta (10.8 ± 1.4 adducts/108 nucleotides), followed by brain (7.8 ± 1.3), lung (3.3 ± 0.7), heart (3.1 ± 0.6), liver (1.5 ± 0.2) and stomach (1.2 ± 0.2). In the ApoE*3-Leiden mice, adduct levels were equally high in aorta, heart, and lung (4.6 ± 0.7, 5.0 ± 0.5 and 4.6 ± 0.4, respectively), followed by stomach (2.7 ± 0.4), brain (2.3 ± 0.2), and liver (1.7 ± 0.2). In the ApoE-KO mice, the HFC diet intervention resulted in lower adduct levels in lung (2.1 ± 0.2), heart (1.9 ± 0.2), and brain (2.9 ± 0.5), as compared with the LFC group. In contrast, a nonsignificant increase of adducts was found in aorta (13.1 ± 1.5). A similar but nonsignificant trend was observed in the ApoE*3-Leiden mice. Multiple regression analysis showed that in aorta, DNA adducts were inversely related to plasma triglycerides (P = 0.004) and were also modulated by the ApoE genotype (P < 0.001). The results of the present study support further investigation into the role of dietary modulation of plasma lipids, ApoE, and polycyclic aromatic hydrocarbon exposure on the formation of DNA adducts in chronic degenerative diseases. Environ. Mol. Mutagen. 42:243,249, 2003. © 2003 Wiley-Liss, Inc. [source] Growth and Lipid Metabolism in Girls and Young Women with Epilepsy during Pubertal MaturationEPILEPSIA, Issue 7 2005Kirsi Mikkonen Summary:,Purpose: To assess growth and the serum lipid profile in girls with epilepsy receiving monotherapy at a mean age of 12.6 years and approximately 6 years later. Methods: A population-based cohort of 77 girls with epilepsy and 49 healthy controls participated in this follow-up study including two cross-sectional evaluations (age range, 8,18.5 years on the first evaluation, and 12.5,25.8 years on the second evaluation). Forty of the patients were initially taking valproate (VPA), 19, carbamazepine (CBZ), and 18, oxcarbazepine (OXC). Growth data were compiled, body mass index (BMI) was calculated, and serum total (TC), and high-density lipoprotein (HDL-C) and low-density lipoprotein (LDL-C) cholesterol and triglyceride concentrations were analyzed. Results: Linear growth and final height did not differ between the patients and the controls. At follow-up, the mean BMI of the patients who were off medication (61%) was similar to that of the controls, whereas the patients initially treated with VPA who were still taking any medication had a higher BMI. On the first evaluation, the patients taking VPA had low serum HDL-C, and those taking CBZ or OXC had high serum TC and LDL-C concentrations. At follow-up, serum lipid levels were similar in the patients off medication and the controls. Conclusions: Neither epilepsy nor antiepileptic therapy affects linear growth or final height, but they may have unfavorable effects on body weight and serum lipid concentrations. Lipid-profile impairment seems to be transient if the medication is discontinued. Overweight is common in patients treated with VPA during puberty if epilepsy and medication continue into adulthood. [source] Anti-oxidized low-density lipoprotein antibody levels are associated with the development of type 2 diabetes mellitusEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 9 2008L. Garrido-Sánchez Abstract Background, Anti-oxidized low-density lipoprotein (LDL) antibodies are associated with the oxidative capacity of plasma, but whether they protect or promote diabetes is unknown. We undertook a prospective study to determine the predictive capacity of anti-oxidized LDL antibodies for the onset of type 2 diabetes mellitus (T2DM). Materials and methods, We selected 391 non-diabetic women aged 18,65 years. The subjects were classified as being normal (oral glucose test tolerance normal, OGTT-N), or having impaired glucose tolerance (IGT), impaired fasting glucose (IFG) or T2DM according to their baseline glucose levels and after an OGTT. The same subjects were studied six years later. The levels of anti-oxidized LDL antibodies were classified as above or below the 50th percentile. Results, Of the women who were OGTT-N at the start of the study and who had anti-oxidized LDL antibody levels below the 50th percentile, only 65·1% were still OGTT-N after 6 years versus 79·5% of those who had anti-oxidized LDL antibody levels above the 50th percentile (P = 0·015). Women who had IGT or IFG at the start of the study whose anti-oxidized LDL antibody levels were below the 50th percentile had a relative risk of 9·79 (95% confidence interval, 1·40,68·45) of developing diabetes (P < 0·001). Logistic regression analysis showed that the variables predicting the development of a carbohydrate metabolism disorder in the women after 6 years were body mass index (P < 0·001) and the levels of anti-oxidized LDL antibodies (P = 0·042). Conclusions, Levels of anti-oxidized LDL antibodies are independent predictors for the development of T2DM in women. [source] Metabolic and anti-inflammatory benefits of eccentric endurance exercise , a pilot studyEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 4 2008H. Drexel ABSTRACT Background, Eccentric endurance exercise (e.g. hiking downwards) is less strenuous than concentric exercise (e.g. hiking upwards) but its potential to reduce cardiovascular risk is unknown. Materials and methods, We randomly allocated 45 healthy sedentary individuals (16 men and 29 women, mean age 48 years) to one of two groups, one beginning with two months of hiking upwards, the other with two months of hiking downwards the same route, with a crossover for a further two months. For the opposite way, a cable car was used where compliance was recorded electronically. The difference in altitude was 540 metres; the distance was covered three to five times a week. Fasting and postprandial metabolic profiles were obtained at baseline and after the two month periods of eccentric and concentric exercise, respectively. Results, Forty-two of the 45 participants completed the study; the compliance rate was therefore 93%. Compared with baseline, eccentric exercise lowered total cholesterol (by 4·1%; P = 0·026), low-density lipoprotein (LDL) cholesterol (by 8·4%, P = 0·001), Apolipoprotein B/Apolipoprotein A1 ratio (by 10·9%, P < 0·001), homeostasis model assessment of insulin resistance scores (by 26·2%, P = 0·017) and C-reactive protein (by 30·0%; P = 0·007); the magnitude of these changes was comparable to that of concentric exercise. Eccentric exercise improved glucose tolerance (by 6·2%, P = 0·023), whereas concentric exercise improved triglyceride tolerance (by 14·9%, P = 0·022). Conclusions, Eccentric endurance exercise is a promising new exercise modality with favourable metabolic and anti-inflammatory effects and is well applicable to sedentary individuals. [source] Impact of postprandial lipaemia on low-density lipoprotein (LDL) size and oxidized LDL in patients with coronary artery diseaseEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 11 2006M. Granér Abstract Background, Remnant lipoprotein particles (RLPs) and oxidative stress are components of postprandial state. We investigated the concentrations of triglyceride-rich lipoproteins (TRLs), RLPs, low-density lipoprotein (LDL) size, and oxidized LDL (oxLDL) during alimentary lipaemia, and evaluated whether changes among these variables could be associated with the severity and extent of coronary artery disease (CAD). Materials and methods, Eighty men and 27 women with clinically suspected CAD underwent quantitative coronary angiography (QCA). TRLs were isolated by density gradient ultracentrifugation before and 6 h after an oral fat load. RLPs were measured by an immunoseparation method, oxLDL by ELISA, and LDL size by gradient gel electrophoresis. Results, Triglycerides, apolipoprotein (apo) B-48, and apoB-100 concentration in Swedberg flotation units (Sf) > 400 and in Sf 12,400 fractions were markedly increased at 6 h. Postprandial cholesterol content of RLPs (RLP-C) correlated with respective triglycerides in Sf > 400 (r = 0·737) and Sf 12,400 (r = 0·857), apoB-48 in Sf > 400 (r = 0·710) and Sf 12,400 (r = 0·664), apoB-100 in Sf > 400 (r = 0·812) and Sf 12,400 (r = 0·533). RLP-C correlated with oxLDL both in fasting and in fed state (r = 0·482 and r = 0·543, respectively) and inversely with LDL size (r = ,0·459 and r = ,0·442, respectively). (P < 0·001 for all). OxLDL was elevated postprandially (P < 0·001). In multivariate analysis, oxLDL was a determinant of severity and extent of CAD. Conclusion, Postprandial state is associated with oxidative stress. The magnitude of oxLDL increases during alimentary lipaemia and is associated with coronary atherosclerosis. [source] Metabolism of cholesterol ester of apolipoprotein B100-containing lipoproteins in dogs: evidence for disregarding cholesterol ester transferEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 8 2004E. Bailhache Abstract Background, It has been shown that dogs exhibit no cholesterol ester transfer protein (CETP) activity in vitro, in contrast to humans. The aim of our study was to determine modalities of in vivo plasma cholesterol ester turnover in this species, using a kinetic approach with stable isotopes. Materials and methods, Kinetics of very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) were studied in seven adult male Beagle dogs using a dual isotope approach through endogenous labelling of both their cholesterol moiety and their protein moiety. A primed constant infusion of both [1,213C]acetate and [5,5,5- 2H3]leucine enabled us to obtain measurable deuterium enrichments by gas chromatography-mass spectrometry for plasma leucine and apoB100, as well as measurable 13C enrichment by gas chromatography-combustion-isotopic ratio mass spectrometry for unesterified cholesterol and cholesterol ester in the VLDL and LDL. Two identical multicompartmental models (SAAM II) were used together for the analysis of tracer kinetics' data of proteins and cholesterol. Results, Characterization of the apoB100-containing lipoprotein cholesterol ester model allowed determination of kinetic parameters of VLDL and LDL cholesterol ester metabolism. We succeeded in modelling VLDL and LDL cholesterol ester metabolism and apoB100 metabolism simultaneously. Fractional catabolic rate (FCR) of apoB100 and CE had the same values. Introducing cholesterol ester transfer between lipoproteins in the model did not significantly improve the fit. Total VLDL FCR was 2·97 ± 01·47 h,1. Approximately one-quarter corresponded to the direct removal of VLDL (0·81 ± 00·34 h,1) and the remaining three-quarters corresponded to the fraction of VLDL converted to LDL, which represented a conversion of VLDL into LDL of 2·16 ± 01·16 h,1. Low-density lipoproteins were produced exclusively from VLDL conversion and were then removed (0·031 ± 0·004 h,1) from plasma. Conclusion, These kinetic data showed that VLDL cholesterol ester and LDL cholesterol ester metabolism followed VLDL and LDL apoB100 metabolism, and that consequently there is no in vivo transfer of cholesterol ester in dogs. [source] Serum paraoxonase activity in patients with type 1 diabetes compared to healthy controlsEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 4 2002B. Mackness Abstract Background The oxidation of low-density lipoprotein (LDL) is central to current theories on the initiation and progression of atherosclerosis. Type 1 diabetes is associated with an increase in oxidative stress, which may be responsible for the increased susceptibility to coronary heart disease seen in type 1 diabetes. High-density lipoprotein (HDL) associated paraoxonase (PON1) can retard the oxidation of LDL. Design Paraoxonase activity, concentration and genotype were therefore investigated in 152 people with type 1 diabetes and 282 healthy controls. These parameters were also investigated in the group with type 1 diabetes in relation to the presence of diabetic complications. Results Both PON1 activity and concentration were significantly lower by 16·7% and 19·2% (both P < 0·05) in the type 1 diabetes group. These differences were independent of the PON1 coding region polymorphisms. The distribution of PON1 activity and mass were the same in both populations, i.e. for the PON1-192 polymorphism RR > RQ > QQ and for the PON1-55 polymorphism LL > LM > MM. There were no differences in either the PON1 polymorphisms, PON1 activity and concentration in people with type 1 diabetes in the presence or absence of micro and macro vascular complications of diabetes. Conclusions Low PON1 activity may contribute to the increased atherosclerosis found in type 1 diabetes by reducing the ability of HDL to retard LDL oxidation despite the frequently-found increased HDL in type 1 diabetes when good glycaemic control is established. [source] Variation in the human lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1) gene is associated with plasma soluble LOX-1 levelsEXPERIMENTAL PHYSIOLOGY, Issue 9 2008Tina E. Brinkley The lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1) expressed on vascular cells plays a major role in atherogenesis by internalizing and degrading oxidized low-density lipoprotein. LOX-1 can be cleaved from the cell surface and released as soluble LOX-1 (sLOX-1), and elevated sLOX-1 levels may be indicative of atherosclerotic plaque instability. We examined associations between the LOX-1 gene 3,UTR-C/T and G501C polymorphisms and plasma sLOX-1 levels in 97 healthy older men and women. The frequencies for the 3,UTR-T and 501C alleles were 46 and 10%, respectively. Plasma sLOX-1 levels were significantly higher in the 3,UTR CC genotype group compared with both the CT (P= 0.02) and TT genotype groups (P= 0.002). Plasma sLOX-1 levels were also significantly higher in the 501GC genotype group compared with the GG genotype group (P= 0.004). In univariate analyses, sLOX-1 levels were significantly associated with both the 3,UTR-C/T and G501C polymorphisms. These associations remained significant after adjusting for age, sex, race and body mass index. In conclusion, variation in the LOX-1 gene is associated with plasma sLOX-1 levels in older men and women. [source] TRB3, upregulated by ox-LDL, mediates human monocyte-derived macrophage apoptosisFEBS JOURNAL, Issue 10 2009Yuan-yuan Shang Tribble3 (TRB3), a mammalian homolog of Drosophila tribbles, slows cell-cycle progression, and its expression is increased in response to various stresses. The aim of this study was to investigate the role of the TRB3 gene in macrophage apoptosis induced by oxidized low-density lipoprotein (ox-LDL). We found that, in human monocyte-derived macrophages, TRB3 is upregulated by ox-LDL in a dose- and time-dependent manner. The cell viability of TRB3-overexpressing macrophages was decreased, but apoptosis was increased and the level of activated caspase-3 increased. Factorial analyses revealed no significant interaction between TRB3 overexpression and ox-LDL stimulation with respect to macrophage apoptosis. Furthermore, TRB3-silenced macrophages showed decreased apoptosis, and TRB3-silenced cells treated with ox-LDL showed significantly increased apoptosis. Silencing of TRB3 and ox-LDL stimulation showed significant interaction for macrophage apoptosis, suggesting that TRB3 knockdown resisted the macrophage apoptosis induced by ox-LDL. Therefore, TRB3 in part mediates the macrophage apoptosis induced by ox-LDL, which suggests that TRB3 might be involved in vulnerable atherosclerotic plaque progression. [source] Glycation of low-density lipoprotein results in the time-dependent accumulation of cholesteryl esters and apolipoprotein B-100 protein in primary human monocyte-derived macrophagesFEBS JOURNAL, Issue 6 2007Bronwyn E. Brown Nonenzymatic covalent binding (glycation) of reactive aldehydes (from glucose or metabolic processes) to low-density lipoproteins has been previously shown to result in lipid accumulation in a murine macrophage cell line. The formation of such lipid-laden cells is a hallmark of atherosclerosis. In this study, we characterize lipid accumulation in primary human monocyte-derived macrophages, which are cells of immediate relevance to human atherosclerosis, on exposure to low-density lipoprotein glycated using methylglyoxal or glycolaldehyde. The time course of cellular uptake of low-density lipoprotein-derived lipids and protein has been characterized, together with the subsequent turnover of the modified apolipoprotein B-100 (apoB) protein. Cholesterol and cholesteryl ester accumulation occurs within 24 h of exposure to glycated low-density lipoprotein, and increases in a time-dependent manner. Higher cellular cholesteryl ester levels were detected with glycolaldehyde-modified low-density lipoprotein than with methylglyoxal-modified low-density lipoprotein. Uptake was significantly decreased by fucoidin (an inhibitor of scavenger receptor SR-A) and a mAb to CD36. Human monocyte-derived macrophages endocytosed and degraded significantly more 125I-labeled apoB from glycolaldehyde-modified than from methylglyoxal-modified, or control, low-density lipoprotein. Differences in the endocytic and degradation rates resulted in net intracellular accumulation of modified apoB from glycolaldehyde-modified low-density lipoprotein. Accumulation of lipid therefore parallels increased endocytosis and, to a lesser extent, degradation of apoB in human macrophages exposed to glycolaldehyde-modified low-density lipoprotein. This accumulation of cholesteryl esters and modified protein from glycated low-density lipoprotein may contribute to cellular dysfunction and the increased atherosclerosis observed in people with diabetes, and other pathologies linked to exposure to reactive carbonyls. [source] Localization of the mosaic transmembrane serine protease corin to heart myocytesFEBS JOURNAL, Issue 23 2000John D. Hooper Corin cDNA encodes an unusual mosaic type II transmembrane serine protease, which possesses, in addition to a trypsin-like serine protease domain, two frizzled domains, eight low-density lipoprotein (LDL) receptor domains, a scavenger receptor domain, as well as an intracellular cytoplasmic domain. In in vitro experiments, recombinant human corin has recently been shown to activate pro-atrial natriuretic peptide (ANP), a cardiac hormone essential for the regulation of blood pressure. Here we report the first characterization of corin protein expression in heart tissue. We generated antibodies to two different peptides derived from unique regions of the corin polypeptide, which detected immunoreactive corin protein of approximately 125,135 kDa in lysates from human heart tissues. Immunostaining of sections of human heart showed corin expression was specifically localized to the cross striations of cardiac myocytes, with a pattern of expression consistent with an integral membrane localization. Corin was not detected in sections of skeletal or smooth muscle. Corin has been suggested to be a candidate gene for the rare congenital heart disease, total anomalous pulmonary venous return (TAPVR) as the corin gene colocalizes to the TAPVR locus on human chromosome 4. However examination of corin protein expression in TAPVR heart tissue did not show evidence of abnormal corin expression. The demonstrated corin protein expression by heart myocytes supports its proposed role as the pro-ANP convertase, and thus a potentially critical mediator of major cardiovascular diseases including hypertension and congestive heart failure. [source] Kinetics of tryptophan oxidation in plasma lipoproteins by myeloperoxidase-generated HOClFEBS JOURNAL, Issue 13 2000Andreas Jerlich The relative susceptibility of the apoprotein components of human lipoproteins [high-density lipoprotein (HDL) and low-density lipoprotein (LDL)] and their subclasses to oxidation by the myeloperoxidase/H2O2/Cl, system in vitro was studied by measuring the decrease in rate of tryptophan fluorescence. Whereas the lipoprotein-modification rate showed a saturation type of dependence on the concentration of myeloperoxidase, a biphasic dependence on the concentration of the lipoproteins was found. High concentrations of H2O2 were also found to inhibit tryptophan oxidation in LDL but to a lesser extent in HDL. The optimal rate of LDL and HDL modification was observed at pH 6.0. HDL was modified much more rapidly than LDL, which may be due to differences in size and different relative contents of protein and lipids per particle. No differences in rates of modification of LDL subclasses were observed, when the assays were standardized to equal LDL protein concentrations, but, when standardized to equal particle mass, an optimum at subclass 8 was found, which is probably due to differences in apolipoprotein B-100 conformation. It was concluded that HDL may have a beneficial effect in retarding LDL modification in inflammatory processes. [source] Molecular characterization of a human scavenger receptor, human MARCOFEBS JOURNAL, Issue 3 2000Nabil A. Elshourbagy Murine MARCO has been identified recently in subsets of macrophages located in the peritoneum, marginal zone of the spleen, and the medullary cord of lymph nodes, where it has been proposed that it serves as a bacteria-binding receptor. A scavenger receptor family member with an extended collagenous domain, murine MARCO has also been demonstrated in atherosclerotic lesions of susceptible mice. We report here the identification, tissue and chromosomal localization, and pharmacological characterization of human (h)MARCO. hMARCO was identified from a macrophage cDNA library by electronic screening with the murine MARCO sequence. Nucleotide sequence analysis confirmed that the full-length hMARCO clone encoded a 519-amino acid protein sharing 68.5% identity with murine MARCO. RNA blot analysis indicated that the hMARCO transcript is 2.0 kb in length and is predominantly expressed in human lung, liver, and lymph nodes. Radiation hybrid mapping localized hMARCO to chromosome 2q14. Ligand-binding studies of COS cells expressing hMARCO demonstrated significant specific binding of both Escherichia coli and Staphylococcus aureus. In contrast, the hMARCO receptor expressed in COS cells did not specifically bind the scavenger receptor ligand acetylated low-density lipoprotein (LDL), despite its similarity to the elongated collagen-like binding domain of the macrophage scavenger receptor. In addition, acetylated (Ac)LDL and oxidized (Ox)LDL did not inhibit E. coli binding to hMARCO. These data suggest that hMARCO may play an important role in host defense, but it has no obvious role in the accumulation of modified lipoproteins during atherogenesis. [source] LPL polymorphism predicts stroke risk in menGENETIC EPIDEMIOLOGY, Issue 3 2002Alanna C. Morrison Abstract Variation in lipid levels has been associated with atherosclerotic vascular disease, including stroke. Genes contributing to interindividual variation in lipid levels may play a role in the etiology of stroke, either through their effects on lipid synthesis and metabolism or through separate pathways. For this reason, we sought to examine the association between polymorphisms in the lipoprotein lipase (LPL) and apolipoprotein E (APOE) genes and subclinical and clinical stroke in the Atherosclerosis Risk in Communities (ARIC) Study. Subclinical stroke was determined by cerebral magnetic resonance imaging (MRI). Subclinical cerebral infarct cases (n = 197) were compared to a stratified random sample identified from individuals participating in the MRI examination (n = 200). Incidence of clinical ischemic stroke was determined by following the ARIC cohort for an average of 7.5 years for potential cerebrovascular events; 218 validated clinical ischemic strokes were identified. A stratified random sample of the ARIC cohort (CRS, n = 964) was used as the comparison group for clinical cases. The LPL S291-carrying genotypes and APOE ,2- and ,4-carrying genotypes were not significantly associated with subclinical or clinical stroke. The LPL X447-containing genotypes were significantly associated with subclinical (odds ratio [OR], 4.32; 95% confidence interval [CI], 1.23,15.15; P = 0.020) and clinical stroke (hazard rate ratio [HRR], 2.57; 95% CI, 1.24,5.34; P = 0.01) in men, both by themselves and after adjustment for multiple stroke risk factors. The LPL S447X polymorphism is significantly associated with subclinical cerebral infarction and incident clinical ischemic stroke in men from a middle-aged American population. This association does not appear to be mediated by triglyceride, high-density lipoprotein (HDL)- and low-density lipoprotein (LDL)-cholesterol levels, or additional stroke risk factors. Genet. Epidemiol. 22:233,242, 2002. © 2002 Wiley-Liss, Inc. [source] | ||||||||||||||||||||||||||||||||||||||||