Movement of membrane tubules along microtubules in vitro: evidence for specialised sites of motor attachment

1994 ◽  
Vol 107 (7) ◽  
pp. 1885-1897 ◽  
Author(s):  
V. Allan ◽  
R. Vale
Keyword(s):  
Secretory Pathway ◽  
Motor Proteins ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Globular Domain ◽  
Secretory Product ◽  
Protein Activity ◽  
Golgi Stack ◽  
Membrane Tubules

We have studied the microtubule-dependent formation of tubular membrane networks in vitro, using a heterologous system composed of Xenopus egg cytosol combined with rat liver membrane fractions enriched in either Golgi stacks or rough endoplasmic reticulum. The first step in membrane network construction involves the extension of membrane tubules along microtubules by the action of microtubule-based motor proteins. We have observed for both membrane fractions that 80–95% of moving tubule tips possess a distinct globular domain. These structures do not form simply as a consequence of motor protein activity, but are stable domains that appear to be enriched in active microtubule motors. Negative stain electron microscopy reveals that the motile globular domains associated with the RER networks are generally smaller than those observed in networks derived from a crude Golgi stack fraction. The globular domains from the Golgi fraction are often packed with very low density lipoprotein particles (the major secretory product of hepatocytes) and albumin, which suggests that motor proteins may be specifically enriched in organelle regions where proteins for export are accumulated. These data raise the possibility that the concentration of active motor proteins into specialised membrane domains may be an important feature of the secretory pathway.

Therapeutic Application of Diacylglycerol Oil for Obesity: Serotonin Hypothesis

2012 ◽  
Vol 2 (1) ◽  
pp. 1 ◽  
Author(s):  
Hidekatsu Yanai ◽  
Hiroshi Yoshida ◽  
Yuji Hirowatari ◽  
Norio Tada
Keyword(s):  
Energy Expenditure ◽  
Molecular Mechanisms ◽  
Uncoupling Protein ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Serotonin Release ◽  
Intestinal Cell ◽  
Therapeutic Application ◽  
Postprandial Triglyceride

Characteristics for the serum lipid abnormalities in the obesity/metabolic syndrome are elevated fasting, postprandial triglyceride (TG), and decreased high-density lipoprotein-cholesterol (HDL-C). Diacylglycerol (DAG) oil ingestion has been reported to ameliorate postprandial hyperlipidemia and prevent obesity by increasing energy expenditure, due to the intestinal physiochemical dynamics that differ from triacylglycerol (TAG). Our study demonstrated that DAG suppresses postprandial increase in TG-rich lipoprotein, very low-density lipoprotein (VLDL), and insulin, as compared with TAG in young, healthy individuals. Interestingly, our study also presented that DAG significantly increases plasma serotonin, which is mostly present in the intestine, and mediates thermogenesis, proposing a possible mechanism for a postprandial increase in energy expenditure by DAG. Our other study demonstrated that DAG suppresses postprandial increase in TG, VLDL-C, and remnant-like particle-cholesterol, in comparison with TAG in an apolipoprotein C-II deficient subject, suggesting that DAG suppresses postprandial TG-rich lipoprotein independently of lipoprotein lipase. Further, to understand the molecular mechanisms for DAG-mediated increase in serotonin and energy expenditure, we studied the effects of 1-monoacylglycerol and 2-monoacylglycerol, distinct digestive products of DAG and TAG, respectively, on serotonin release from the Caco-2 cells, the human intestinal cell line. We also studied effects of 1- and 2-monoacylglycerol, and serotonin on the expression of mRNA associated with β-oxidation, fatty acids metabolism, and thermogenesis, in the Caco-2 cells. 1-monoacylglycerol significantly increased serotonin release from the Caco-2 cells, compared with 2-monoacylglycerol by approximately 40%. The expression of mRNA of acyl-CoA oxidase (ACO), fatty acid translocase (FAT), and uncoupling protein-2 (UCP-2), was significantly higher in 1-MOG-treated Caco-2 cells, than 2-MOG-treated cells. The expression of mRNA of ACO, medium-chain acyl-CoA dehydrogenase, FAT, and UCP-2, was significantly elevated in serotonin-treated Caco-2 cells, compared to cells incubated without serotonin. In conclusion, our clinical and in vitro studies suggested a possible therapeutic application of DAG for obesity, and obesity-related metabolic disorders.Key words: Diacylglycerol, intestine, obesity, serotonin, thermogenesis

scholarly journals Comparative Evaluation on Impacts of Fibronectin, Heparin–Chitosan, and Albumin Coating of Bacterial Nanocellulose Small-Diameter Vascular Grafts on Endothelialization In Vitro

Nanomaterials ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1952
Author(s):  
Max Wacker ◽  
Jan Riedel ◽  
Heike Walles ◽  
Maximilian Scherner ◽  
George Awad ◽  
...  
Keyword(s):  
Low Density Lipoprotein ◽  
Vascular Grafts ◽  
Small Diameter ◽  
Density Lipoprotein ◽  
Human Albumin ◽  
Water Soluble ◽  
Bacterial Nanocellulose ◽  
Functionally Impaired ◽  
Small Diameter Vascular Grafts

In this study, we contrast the impacts of surface coating bacterial nanocellulose small-diameter vascular grafts (BNC-SDVGs) with human albumin, fibronectin, or heparin–chitosan upon endothelialization with human saphenous vein endothelial cells (VEC) or endothelial progenitor cells (EPC) in vitro. In one scenario, coated grafts were cut into 2D circular patches for static colonization of a defined inner surface area; in another scenario, they were mounted on a customized bioreactor and subsequently perfused for cell seeding. We evaluated the colonization by emerging metabolic activity and the preservation of endothelial functionality by water soluble tetrazolium salts (WST-1), acetylated low-density lipoprotein (AcLDL) uptake assays, and immune fluorescence staining. Uncoated BNC scaffolds served as controls. The fibronectin coating significantly promoted adhesion and growth of VECs and EPCs, while albumin only promoted adhesion of VECs, but here, the cells were functionally impaired as indicated by missing AcLDL uptake. The heparin–chitosan coating led to significantly improved adhesion of EPCs, but not VECs. In summary, both fibronectin and heparin–chitosan coatings could beneficially impact the endothelialization of BNC-SDVGs and might therefore represent promising approaches to help improve the longevity and reduce the thrombogenicity of BNC-SDVGs in the future.

scholarly journals Reductions of copper ion-mediated low-density lipoprotein (LDL) oxidations of trypsin inhibitors, the sweet potato root major proteins, and LDL binding capacities

2020 ◽  
Vol 61 (1) ◽  
Author(s):  
Yeh-Lin Lu ◽  
Chia-Jung Lee ◽  
Shyr-Yi Lin ◽  
Wen-Chi Hou
Keyword(s):  
Sweet Potato ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Trypsin Inhibitors ◽  
Water Soluble ◽  
Affinity Column ◽  
Low Density ◽  
Native Page

Abstract Background The root major proteins of sweet potato trypsin inhibitors (SPTIs) or named sporamin, estimated for 60 to 80% water-soluble proteins, exhibited many biological activities. The human low-density lipoprotein (LDL) showed to form in vivo complex with endogenous oxidized alpha-1-antitrypsin. Little is known concerning the interactions between SPTIs and LDL in vitro. Results The thiobarbituric-acid-reactive-substance (TBARS) assays were used to monitor 0.1 mM Cu2+-mediated low-density lipoprotein (LDL) oxidations during 24-h reactions with or without SPTIs additions. The protein stains in native PAGE gels were used to identify the bindings between native or reduced forms of SPTIs or soybean TIs and LDL, or oxidized LDL (oxLDL). It was found that the SPTIs additions showed to reduce LDL oxidations in the first 6-h and then gradually decreased the capacities of anti-LDL oxidations. The protein stains in native PAGE gels showed more intense LDL bands in the presence of SPTIs, and 0.5-h and 1-h reached the highest one. The SPTIs also bound to the oxLDL, and low pH condition (pH 2.0) might break the interactions revealed by HPLC. The LDL or oxLDL adsorbed onto self-prepared SPTIs-affinity column and some components were eluted by 0.2 M KCl (pH 2.0). The native or reduced SPTIs or soybean TIs showed different binding capacities toward LDL and oxLDL in vitro. Conclusion The SPTIs might be useful in developing functional foods as antioxidant and nutrient supplements, and the physiological roles of SPTIs-LDL and SPTIs-oxLDL complex in vivo will investigate further using animal models.

scholarly journals Development of Capture Assays for Different Modifications of Human Low-Density Lipoprotein

2005 ◽  
Vol 12 (1) ◽  
pp. 68-75 ◽  
Author(s):  
Gabriel Virella ◽  
M. Brooks Derrick ◽  
Virginia Pate ◽  
Charlyne Chassereau ◽  
Suzanne R. Thorpe ◽  
...  
Keyword(s):  
Oxidized Ldl ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Gas Chromatography Mass Spectrometry ◽  
Low Density ◽  
Modified Ldl ◽  
Capture Assay ◽  
Carboxymethyl Lysine

ABSTRACT Antibodies to malondialdehyde (MDA)-modified low-density lipoprotein (LDL), copper-oxidized LDL (oxLDL), N ε(carboxymethyl) lysine (CML)-modified LDL, and advanced glycosylation end product (AGE)-modified LDL were obtained by immunization of rabbits with in vitro-modified human LDL preparations. After absorption of apolipoprotein B (ApoB) antibodies, we obtained antibodies specific for each modified lipoprotein with unique patterns of reactivity. MDA-LDL antibodies reacted strongly with MDA-LDL and also with oxLDL. CML-LDL antibodies reacted strongly with CML-LDL and also AGE-LDL. oxLDL antibodies reacted with oxLDL but not with MDA-LDL, and AGE-LDL antibodies reacted with AGE-LDL but not with CML-LDL. Capture assays were set with each antiserum, and we tested their ability to capture ApoB-containing lipoproteins isolated from precipitated immune complexes (IC) and from the supernatants remaining after IC precipitation (free lipoproteins). All antibodies captured lipoproteins contained in IC more effectively than free lipoproteins. Analysis of lipoproteins in IC by gas chromatography-mass spectrometry showed that they contained MDA-LDL and CML-LDL in significantly higher concentrations than free lipoproteins. A significant correlation (r = 0.706, P < 0.019) was obtained between the MDA concentrations determined by chemical analysis and by the capture assay of lipoproteins present in IC. In conclusion, we have developed capture assays for different LDL modifications in human ApoB/E lipoprotein-rich fractions isolated from precipitated IC. This approach obviates the interference of IC in previously reported modified LDL assays and allows determination of the degree of modification of LDL with greater accuracy.

scholarly journals Creation of disease-inspired biomaterial environments to mimic pathological events in early calcific aortic valve disease

2017 ◽  
Vol 115 (3) ◽  
pp. E363-E371 ◽  
Author(s):  
Ana M. Porras ◽  
Jennifer A. Westlund ◽  
Austin D. Evans ◽  
Kristyn S. Masters
Keyword(s):  
Aortic Valve ◽  
Disease Progression ◽  
Aortic Valve Disease ◽  
Low Density Lipoprotein ◽  
Treatment Strategies ◽  
Density Lipoprotein ◽  
Valve Disease ◽  
Calcific Aortic Valve Disease ◽  
Valve Lesion

An insufficient understanding of calcific aortic valve disease (CAVD) pathogenesis remains a major obstacle in developing treatment strategies for this disease. The aim of the present study was to create engineered environments that mimic the earliest known features of CAVD and apply this in vitro platform to decipher relationships relevant to early valve lesion pathobiology. Glycosaminoglycan (GAG) enrichment is a dominant hallmark of early CAVD, but culture of valvular interstitial cells (VICs) in biomaterial environments containing pathological amounts of hyaluronic acid (HA) or chondroitin sulfate (CS) did not directly increase indicators of disease progression such as VIC activation or inflammatory cytokine production. However, HA-enriched matrices increased production of vascular endothelial growth factor (VEGF), while matrices displaying pathological levels of CS were effective at retaining lipoproteins, whose deposition is also found in early CAVD. Retained oxidized low-density lipoprotein (oxLDL), in turn, stimulated myofibroblastic VIC differentiation and secretion of numerous inflammatory cytokines. OxLDL also increased VIC deposition of GAGs, thereby creating a positive feedback loop to further enrich GAG content and promote disease progression. Using this disease-inspired in vitro platform, we were able to model a complex, multistep pathological sequence, with our findings suggesting distinct roles for individual GAGs in outcomes related to valve lesion progression, as well as key differences in cell–lipoprotein interactions compared with atherosclerosis. We propose a pathogenesis cascade that may be relevant to understanding early CAVD and envision the extension of such models to investigate other tissue pathologies or test pharmacological treatments.

scholarly journals Low density lipoprotein and very low density lipoprotein are selectively bound by aggregated C-reactive protein.

1982 ◽  
Vol 156 (1) ◽  
pp. 230-242 ◽  
Author(s):  
F C de Beer ◽  
A K Soutar ◽  
M L Baltz ◽  
I M Trayner ◽  
A Feinstein ◽  
...  
Keyword(s):  
Acute Phase ◽  
Solid Phase ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Very Low Density Lipoprotein ◽  
Low Density ◽  
C Reactive Protein ◽  
Calcium Dependent ◽  
Reactive Protein

C-reactive protein (CRP), the classical acute-phase protein, can bind phospholipids by virtue of its specific, calcium-dependent reactivity with phosphorylcholine residues. However, analysis of acute-phase serum by gel filtration and by density gradient ultracentrifugation showed that the CRP was in a free, uncomplexed form, despite the coexistent presence of the various classes of serum lipoproteins, all of which contain phospholipids. In contrast, when isolated CRP was aggregated by immobilization at a sufficient density on a solid phase and then exposed to normal human serum, it selectively bound low density lipoprotein (LDL) and traces of very low density lipoprotein. The reaction was calcium dependent and reversible by free phosphorylcholine but not by heparin. LDL isolated from normal plasma was also bound by aggregated CRP. CRP reacts in vitro with a wide variety of different ligands both of extrinsic and of autogenous origin, e.g., microbial products and damaged cell membranes, respectively. If CRP aggregated in vivo by complexing with these ligands than acquires the capacity to selectively bind LDL, the phenomenon may have significant implications for the function of CRP and for the metabolism, clearance, and deposition of LDL.

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