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Biomedical subjects

W Gray Jerome

Publications and source records attributed to W Gray Jerome.

18 recordsLinked to original sources

Novel changes in discoidal high density lipoprotein morphology: a molecular dynamics study.

ApoA-I is a uniquely flexible lipid-scavenging protein capable of incorporating phospholipids into stable particles. Here we report molecular dynamics simulations on a series of progressively smaller discoidal high density lipoprotein particles produced by incremental removal of palmitoyloleoylphosphatidylcholine via four different pathways. The starting model contained 160 palmitoyloleoylphosphatidylcholines and a belt of two antiparallel amphipathic helical lipid-associating domains of apolipoprotein (apo) A-I. The results are particularly compelling. After a few nanoseconds of molecular dynamics simulation, independent of the starting particle and method of size reduction, all simulated double belts of the four lipidated apoA-I particles have helical domains that impressively approximate the x-ray crystal structure of lipid-free apoA-I, particularly between residues 88 and 186. These results provide atomic resolution models for two of the particles produced by in vitro reconstitution of nascent high density lipoprotein particles. These particles, measuring 95 angstroms and 78 angstroms by nondenaturing gradient gel electrophoresis, correspond in composition and in size/shape (by negative stain electron microscopy) to the simulated particles with molar ratios of 100:2 and 50:2, respectively. The lipids of the 100:2 particle family form minimal surfaces at their monolayer-monolayer interface, whereas the 50:2 particle family displays a lipid pocket capable of binding a dynamic range of phospholipid molecules.

Apolipoprotein A-I↗

Advanced atherosclerotic foam cell formation has features of an acquired lysosomal storage disorder.

Atherosclerosis is a disease of large- and medium-sized arteries. Complications from atherosclerosis remain a serious cause of morbidity and mortality in industrialized countries. The disease begins very early in life and effects most people in the West. However, because the progression of the disease is slow, symptoms usually do not occur until after the fifth decade of life. Because atherosclerosis is a ubiquitous occurrence throughout the world, as life expectancy is prolonged most populations will see increasing numbers of deaths from complications of atherosclerosis unless there are dramatic advances in treatment. Because it begins so early in life, current treatment is aimed at slowing or reversing the progression of the disease rather than eliminating the initiating steps. Changes in diet and exercise, cholesterol-lowering drugs, and improvements in surgical treatments have made significant inroads into prolonging life, but much work is still required. To proceed further, a better understanding is needed of the underlying causes of disease progression. In this regard, evidence is mounting that the foam cells of the lesion (a critical cell in atherosclerosis progression) exhibit characteristics of an acquired lysosomal storage disorder. In this review the evidence for this conclusion is reviewed and the ramifications of this conclusion are explored with regard to the understanding of disease progression mechanisms, possible improvements in treatment, and their role in increasing life expectancy.

Animals↗

Pancreatic islet production of vascular endothelial growth factor--a is essential for islet vascularization, revascularization, and function.

To investigate molecular mechanisms controlling islet vascularization and revascularization after transplantation, we examined pancreatic expression of three families of angiogenic factors and their receptors in differentiating endocrine cells and adult islets. Using intravital lectin labeling, we demonstrated that development of islet microvasculature and establishment of islet blood flow occur concomitantly with islet morphogenesis. Our genetic data indicate that vascular endothelial growth factor (VEGF)-A is a major regulator of islet vascularization and revascularization of transplanted islets. In spite of normal pancreatic insulin content and beta-cell mass, mice with beta-cell-reduced VEGF-A expression had impaired glucose-stimulated insulin secretion. By vascular or diffusion delivery of beta-cell secretagogues to islets, we showed that reduced insulin output is not a result of beta-cell dysfunction but rather caused by vascular alterations in islets. Taken together, our data indicate that the microvasculature plays an integral role in islet function. Factors modulating VEGF-A expression may influence islet vascularity and, consequently, the amount of insulin delivered into the systemic circulation.

Animals↗

Engineering mouse apolipoprotein A-I into a monomeric, active protein useful for structural determination.

Apolipoprotein AI (apoAI), the major protein component of HDL, is one of the best predictors of coronary artery disease (CAD), with high apoAI and HDL levels being correlated with low occurrences of CAD. The primary function of apoAI is to recruit phospholipid and cholesterol for assembly of HDL particles. Like other exchangeable apolipoproteins, lipid-free apoAI forms a mixture of different oligomers even at 1.0 mg/mL. This self-association property of the exchangeable apolipoproteins is closely associated with the lipoprotein-binding activity of this protein family. It is unclear if the self-association property of apolipoprotein is required for its lipoprotein-binding activity. We developed a novel method for engineering an oligomeric protein to a monomeric, biologically active protein. Using this method, we generated a monomeric mouse apoAI mutant that is active. This mutant contains the first 216 residues of mouse apoAI and replaces six hydrophobic residues with either polar or smaller hydrophobic residues at the defined positions (V118A/A119S/L121Q/T191S/T195S/T199S). Cross-linking results show that this mutant is greater than 90% monomeric at 8 mg/mL. CD, DSC, and NMR results indicate that the mutant maintains an identical secondary, tertiary structure and stability as those of the wild-type mouse apoAI. Lipid-binding assays suggest that the mutant shares an equal lipoprotein-binding activity as that of the wild-type apoAI. In addition, both the monomeric mutant and the wild-type protein make nearly identical rHDL particles. With this monomeric mouse apoAI, high-quality NMR data has been collected, allowing for the NMR structural determination of lipid-free apoAI. On the basis of these results, we conclude that this apoAI mutant is a monomeric, active apoAI useful for structural determination.

Animals↗

Aggregated LDL and lipid dispersions induce lysosomal cholesteryl ester accumulation in macrophage foam cells.

Macrophage foam cells in atherosclerotic lesions accumulate substantial cholesterol stores within large, swollen lysosomes. Previous studies with mildly oxidized low density lipoprotein (OxLDL)-treated THP-1 macrophages suggest an initial buildup of free cholesterol (FC), followed by an inhibition of lysosomal cholesteryl ester (CE) hydrolysis and a subsequent lysosomal accumulation of unhydrolyzed lipoprotein CE. We examined whether other potential sources of cholesterol found within atherosclerotic lesions could also induce similar lysosomal accumulation. Biochemical analysis combined with microscopic analysis showed that treatment of THP-1 macrophages with aggregated low density lipoprotein (AggLDL) or CE-rich lipid dispersions (DISP) produced a similar lysosomal accumulation of both FC and CE. Co-treatment with an ACAT inhibitor, CP113,818, confirmed that the CE accumulation was primarily the result of the inhibition of lysosomal CE hydrolysis. The rate of unhydrolyzed CE buildup was more rapid with DISP than with AggLDL. However, with both treatments, FC appeared to accumulate in lysosomes before the inhibition in hydrolysis and CE accumulation, a sequence shared with mildly OxLDL. Thus, lysosomal accumulation of FC and CE can be attributable to more general mechanisms than just the inhibition of hydrolysis by oxidized lipids.

Atherosclerosis↗

Microsomal triglyceride transfer protein expression in adipocytes: a new component in fat metabolism.

Microsomal triglyceride transfer protein (MTP) is a carrier of triglyceride essential for the assembly of apolipoprotein (apo)B-containing lipoproteins by the liver and the small intestine. Its role in triglyceride transfer in tissues that do not secrete lipoproteins has not been explored. In particular, MTP would seem to be a candidate for a role in triglyceride metabolism within the adipocyte. To test this hypothesis, we probed adipocytes for the presence of MTP. Immunohistochemical and biochemical studies demonstrate MTP in adipocytes from brown and white fat depots of mice and human, as well as in 3T3-L1 cells. Confocal microscopy revealed MTP throughout 3T3 cells; however, MTP fluorescence was prominent in juxtanuclear areas. In differentiated 3T3 cells MTP fluorescence was very striking around lipid droplets. In vitro lipid transfer assays demonstrated the presence of triglyceride transfer activity within microsomal fractions isolated from rat adipose tissue. In addition, quantitative rtPCR studies showed that MTP expression in mouse white fat depots was approximately 1% of MTP expression in mouse liver. MTP mRNA in differentiated 3T3 cells was approximately 13% of liver expression. Our results provide unequivocal evidence for the presence of MTP in adipocytes and present new possibilities for defining the mechanisms by which triglyceride is stored and/or hydrolyzed and mobilized.

Adipocytes↗

Medical bioremediation: prospects for the application of microbial catabolic diversity to aging and several major age-related diseases.

Several major diseases of old age, including atherosclerosis, macular degeneration and neurodegenerative diseases are associated with the intracellular accumulation of substances that impair cellular function and viability. Moreover, the accumulation of lipofuscin, a substance that may have similarly deleterious effects, is one of the most universal markers of aging in postmitotic cells. Reversing this accumulation may thus be valuable, but has proven challenging, doubtless because substances resistant to cellular catabolism are inherently hard to degrade. We suggest a radically new approach: augmenting humans' natural catabolic machinery with microbial enzymes. Many recalcitrant organic molecules are naturally degraded in the soil. Since the soil in certain environments - graveyards, for example - is enriched in human remains but does not accumulate these substances, it presumably harbours microbes that degrade them. The enzymes responsible could be identified and engineered to metabolise these substances in vivo. Here, we survey a range of such substances, their putative roles in age-related diseases and the possible benefits of their removal. We discuss how microbes capable of degrading them can be isolated, characterised and their relevant enzymes engineered for this purpose and ways to avoid potential side-effects.

Aging↗

Chronic treatment of Ménétrier's disease with Erbitux: clinical efficacy and insight into pathophysiology.

BACKGROUND & AIMS: Ménétrier's disease is a rare premalignant hypertrophic gastropathy characterized by large rugal folds, foveolar hyperplasia with glandular atrophy, hypochlorhydria, and hypoalbuminemia. Patients with severe disease often exhibit refractory nausea and vomiting and require gastrectomy. Evidence from both mice and human beings suggests a critical role for epidermal growth factor receptor (EGFR) signaling in the pathogenesis of this disease. We previously reported significant clinical and biochemical improvement of a single patient treated for 1 month with Erbitux, a monoclonal antibody that blocks ligand binding to EGFR. METHODS/RESULTS: We describe 2 patients who were given longer-term treatment with Erbitux as an alternative to gastrectomy. The first patient presented with nausea, hypoalbuminemia, and peripheral edema that required total parenteral nutrition (TPN) and infusions of albumin. On institution of Erbitux, there was rapid improvement in nausea and vomiting and stabilization of serum albumin with discontinuation of TPN and albumin infusions. Serum albumin remained stable during a 1-year course of Erbitux without supplemental protein. Application before and after Erbitux of the radiopaque dye ruthenium red to biopsies of the gastric oxyntic gland mucosa demonstrated prompt and persistent closure of tight junctions by electron microscopy. The second patient presented with chronic gastric bleeding that required bimonthly blood transfusions. During a 4-month course of Erbitux, his hematocrit stabilized, and transfusion requirements were eliminated. CONCLUSIONS: The present report demonstrates the efficacy of prolonged Erbitux therapy in patients with different presentations of severe Ménétrier's disease and also provides insight into the pathophysiology of the protein-losing gastropathy.

Antibodies, Monoclonal↗

Endothelial cells organize fibrin clots into structures that are more resistant to lysis.

Acute myocardial infarction is a major cause of death and disability in the United States. Introducing thrombolytic agents into the clot to dissolve occlusive coronary artery thrombi is one method of treatment. However, despite advances in our knowledge of thrombosis and thrombolysis, survival rates following thrombolytic therapy have not improved substantially. This failure highlights the need for further study of the factors mediating clot stabilization. Using laser scanning confocal microscopy of clots formed from fluorescein-labeled fibrinogen, we investigated what effect binding of fibrin to the endothelial surface has on clot structure and resistance to lysis. Fluorescent fibrin clots were produced over human umbilical vein endothelial cells (HUVEC) and the clot structure analyzed. In the presence of HUVEC, fibrin near the endothelial surface was more organized and occurred in tighter bundles compared to fibrin just 50 microm above. The HUVEC influence on fibrin architecture was blocked by inhibitory concentrations of antibodies to alphaV or beta3 integrin subunits. The regions of the clots associated with endothelial cells were more resistant to lysis than the more homogenous regions distal to endothelium. Thus, our data show that binding of fibrin to integrins on endothelial surfaces produces clots that are more resistant to lysis.

Cells, Cultured↗

ACAT1 deficiency disrupts cholesterol efflux and alters cellular morphology in macrophages.

OBJECTIVE: Acyl-coenzyme A: cholesterol acyltransferase (ACAT) converts intracellular free cholesterol (FC) into cholesteryl esters (CE) for storage in lipid droplets. Recent studies in our laboratory have shown that the deletion of the macrophage ACAT1 gene results in apoptosis and increased atherosclerotic lesion area in the aortas of hyperlipidemic mice. The objective of the current study was to elucidate the mechanism of the increased atherosclerosis. METHODS AND RESULTS: CE storage and FC efflux were studied in ACAT1(-/-) peritoneal macrophages that were treated with acetylated low-density lipoprotein (acLDL). Our results show that efflux of cellular cholesterol was reduced by 25% in ACAT1-deficient cells compared with wild-type controls. This decrease occurred despite the upregulated expression of ABCA1, an important mediator of cholesterol efflux. In contrast, ACAT1 deficiency increased efflux of the cholesterol derived from acLDL by 32%. ACAT1-deficient macrophages also showed a 26% increase in the accumulation of FC derived from acLDL, which was associated with a 75% increase in the number of intracellular vesicles. CONCLUSIONS: Together, these data show that macrophage ACAT1 influences the efflux of both cellular and lipoprotein-derived cholesterol and propose a pathway for the pro-atherogenic transformation of ACAT1(-/-) macrophages.

ATP Binding Cassette Transporter 1↗

A monomeric human apolipoprotein E carboxyl-terminal domain.

ApoE plays a critical role in lipoprotein metabolism and plasma lipid homeostasis through its high-affinity binding to the LDL-receptor family. In solution, apoE is an oligomeric protein and the C-terminal domain causes apoE's aggregation. The aggregation property presents a major difficulty for the structural determination of this protein. A high-level expression system of the apoE C-terminal domain is reported here. Using protein engineering techniques, we identified a monomeric, biologically active apoE C-terminal domain mutant. This mutant replaces five bulky hydrophobic residues in the region of residues 253-289 with either smaller hydrophobic or polar/charged residues (F257A, W264R, V269A, L279Q, and V287E). The solubility of the mutant is significantly increased ( approximately 10-fold). Cross-linking experiments indicate that this mutant is 100% monomeric even at 5 mg/mL. CD and guanidine hydrochloride denaturation results indicate that the mutant maintains an identical alpha-helical secondary structure and stability as compared with those of the wild-type protein. DMPC-binding assays demonstrate an identical vesicle clearance rate shared by both the mutant and the wild-type apoE C-terminal domain. In addition, electron microscopic results show identical recombinant HDL particles prepared with both the mutant and the wild-type proteins. These results indicate that residues F257, W264, V269, L279, and V287 are critical residues for aggregation but may not be important in maintaining the structure, stability, and lipid-binding activity of this apoE domain, suggesting that apoE may use different "epitopes" for its aggregation property, helical structure/stability, and lipid-binding activity. Finally, preliminary NMR data demonstrated that we have collected high-quality NMR spectra, allowing for an NMR structural determination of the apoE C-terminal domain.

Amino Acid Sequence↗

Myristoylated Naked2 escorts transforming growth factor alpha to the basolateral plasma membrane of polarized epithelial cells.

The epidermal growth factor receptor ligands transforming growth factor alpha (TGF alpha) and amphiregulin are delivered to the basolateral surface of polarized epithelial cells where they are cleaved by TACE/ADAM17. Basolateral sorting information resides in their cytoplasmic tail domains, but tail-interacting proteins required for basolateral trafficking have not been identified. Naked (NKD)1 and NKD2 are mammalian homologs of Drosophila Naked Cuticle, which negatively regulates canonical Wnt signaling by binding Dishevelled. We present evidence that NKD2, but not NKD1, binds to basolateral sorting motifs in the cytoplasmic tail of TGF alpha. Processing and cell-surface delivery of TGF alpha are accelerated in NKD2-overexpressing Madin-Darby canine kidney cells. NKD2 is myristoylated on glycine, the second residue. On expression of myristoylation-defective (G2A) NKD2, neither NKD2 nor TGF alpha appears at the basolateral plasma membrane of polarized Madin-Darby canine kidney cells; however, membrane staining for TGF alpha is restored on silencing expression of this mutant NKD2. Amphiregulin does not interact with NKD2 and retains its basolateral localization in G2A-NKD2-expressing cells, as do Na(+), K(+) ATPase alpha 1 and E-cadherin. These data identify an unexpected function for NKD2, i.e., myristoylation-dependent escort of TGF alpha to the basolateral plasma membrane of polarized epithelial cells.

Amino Acid Motifs↗

Coronavirus replication complex formation utilizes components of cellular autophagy.

The coronavirus mouse hepatitis virus (MHV) performs RNA replication on double membrane vesicles (DMVs) in the cytoplasm of the host cell. However, the mechanism by which these DMVs form has not been determined. Using genetic, biochemical, and cell imaging approaches, the role of autophagy in DMV formation and MHV replication was investigated. The results demonstrated that replication complexes co-localize with the autophagy proteins, microtubule-associated protein light-chain 3 and Apg12. MHV infection induces autophagy by a mechanism that is resistant to 3-methyladenine inhibition. MHV replication is impaired in autophagy knockout, APG5-/-, embryonic stem cell lines, but wild-type levels of MHV replication are restored by expression of Apg5 in the APG5-/-cells. In MHV-infected APG5-/-cells, DMVs were not detected; rather, the rough endoplasmic reticulum was dramatically swollen. The results of this study suggest that autophagy is required for formation of double membrane-bound MHV replication complexes and that DMV formation significantly enhances the efficiency of replication. Furthermore, the rough endoplasmic reticulum is implicated as the possible source of membranes for replication complexes.

Adenine↗

Subcellular localization of microsomal triglyceride transfer protein.

Microsomal triglyceride transfer protein (MTP) is essential for the assembly of apolipoprotein B-containing lipoproteins. Within the endoplasmic reticulum, it transfers lipid from the membrane to the forming lipoprotein. Recent evidence suggests that it may also function within the Golgi apparatus. To address this hypothesis, we developed a polyclonal antibody to MTP and used it in a series of studies on mouse liver and McArdle-RH7777 (McA) cells. Western blot analysis demonstrated the presence of MTP within mouse hepatic-Golgi apparatus-rich fractions. In addition, in vitro lipid transfer assays demonstrated the presence of triglyceride transfer activity within the Golgi fractions. Immunohistochemical studies with mouse liver demonstrated the presence of MTP within all hepatocytes, but not in nonparenchymal cells. The subcellular location of MTP in McA cells was investigated using confocal microscopy. MTP colocalized with the trans-Golgi network (TGN) 38 and Golgi SNARE (soluble N-ethylmalemide-sensitive factor attachment protein receptor) of 28 kDa (GS28), markers for the trans- and cis-Golgi apparatus, respectively. Morphometric analyses indicated that approximately 17% of the MTP signal colocalized with the TGN38, while 33% of the trans-Golgi marker colocalized with the MTP. Approximately 17% of the MTP signal colocalized with the GS28, whereas 53% of the cis-Golgi marker colocalized with the MTP. The results provide unequivocal evidence for the location of MTP within the Golgi apparatus, and further highlight the importance of this organelle in the assembly of lipoproteins.

Animals↗

The role of microscopy in understanding atherosclerotic lysosomal lipid metabolism.

Microscopy has played a critical role in first identifying and then defining the role of lysosomes in formation of atherosclerotic foam cells. We review the evidence implicating lysosomal lipid accumulation as a factor in the pathogenesis of atherosclerosis with reference to the role of microscopy. In addition, we explore mechanisms by which lysosomal lipid engorgement occurs. Low density lipoproteins which have become modified are the major source of lipid for foam cell formation. These altered lipoproteins are taken into the cell via receptor-mediated endocytosis and delivered to lysosomes. Under normal conditions, lipids from these lipoproteins are metabolized and do not accumulate in lysosomes. In the atherosclerotic foam cell, this normal metabolism is inhibited so that cholesterol and cholesteryl esters accumulate in lysosomes. Studies of cultured cells incubated with modified lipoproteins suggests this abnormal metabolism occurs in two steps. Initially, hydrolysis of lipoprotein cholesteryl esters occurs normally, but the resultant free cholesterol cannot exit the lysosome. Further lysosomal cholesterol accumulation inhibits hydrolysis, producing a mixture of cholesterol and cholesteryl esters within swollen lysosomes. Various lipoprotein modifications can produce this lysosomal engorgement in vitro and it remains to be seen which modifications are most important in vivo.

Animals↗

Uptake and trafficking of mildly oxidized LDL and acetylated LDL in THP-1 cells does not explain the differences in lysosomal metabolism of these two lipoproteins.

Foam cells in the atherosclerotic lesion have substantial cholesterol stores within large, swollen lysosomes. This feature is mimicked by incubating THP-1 macrophages with mildly oxidized low density lipoprotein (LDL). Incubation of THP-1 cells with acetylated LDL produces cytoplasmic cholesteryl ester accumulation rather than lysosomal storage. The differences could be due to differences in uptake and delivery of lipoprotein to lysosomes or to lysosomal and post-lysosomal processing events. We compared uptake and lysosomal trafficking of acetylated and oxidized LDL using colloidal gold-labeled lipoproteins. Labeling did not alter cellular cholesterol accumulation. We found that uptake and delivery to lysosomes are not different for acetylated and oxidized LDL. In fact, both oxidized and acetylated LDL can be delivered to the same lysosomes. Sequential incubation with oxidized LDL followed by acetylated LDL showed that the lipid-engorged lysosomes are long-lived structures, continuously accepting newly ingested lipoprotein. Comparison of acetylated and oxidized LDL in mouse peritoneal macrophages, a cell which does not accumulate substantial lysosomal lipid, also revealed no differences in uptake. This indicates that in THP-1 cells, the differences in metabolism of oxidized and acetylated LDL are due to cell-specific lysosomal or post-lysosomal events not present in B6C3F1 mouse macrophages.

Animals↗

Laser capture microdissection as an aid to ultrastructural analysis.

Laser capture microdissection uses a microscope to identify specific cells for microdissection and then a laser-sensitive plastic to capture and remove the cells from their substrate. This efficient capture method was originally developed to capture cells for genetic analysis. However, it has also been used to capture cells for proteonomic analysis. In this article, we extend the uses of laser-capture microdissection by reporting a method for preparing captured cells for ultrastructural analysis by transmission electron microscopy. Cells prepared by our methodology show good fine structure preservation and are easily sectioned by standard ultramicrotomy.

Cell Line↗

Tirofiban blocks platelet adhesion to fibrin with minimal perturbation of GpIIb/IIIa structure.

A biophysical approach tested the hypothesis that tirofiban, like eptifibatide, perturbs GpIIb/IIIa structure. Tirofiban bound tightly to platelet GpIIb/IIIa (EC50 approximately 24 nmol/L) and effectively inhibited platelet aggregation (IC50 approximately 37 nmol/L) but blocked platelet adhesion to clotted fibrin only at much higher doses (IC50 approximately 580 nmol/L). Electrophoretic analyses demonstrated that tirofiban protected GpIIb/IIIa from SDS-induced subunit dissociation. However, saturating tirofiban concentrations had little or no effect on GpIIb/IIIa secondary or tertiary structure, as determined by circular dichroic spectroscopy, dynamic light scattering, and sedimentation velocity measurements performed with purified receptors in octyl glucoside. Moderate dose-dependent effects on GpIIb/IIIa quaternary structure were detected by sedimentation equilibrium. Transmission electron microscopy showed minimal tirofiban-induced receptor activation or oligomerization. Thus, even at the increased concentrations needed to block platelet:fibrin adhesive interactions, tirofiban exhibited only limited effects on GpIIb/IIIa conformation and clustering. Our results provide new insights into the mechanisms and potential prothrombotic complications of integrin antagonists.

Adult↗