The recycling of apolipoprotein E in primary cultures of mouse hepatocytes. Evidence for a physiologic connection to high density lipoprotein metabolism

Internalization of apoE-containing very low density protein (VLDL) by hepatocytes in vivo and in vitro leads to apoE recycling and resecretion. Because of the role of apoE in VLDL metabolism, apoE recycling may influence lipoprotein assembly or remnant uptake. However, apoE is also a HDL protein, and apoE recycling may be related to reverse cholesterol transport. To investigate apoE recycling, apoE(-/-) mouse hepatocytes were incubated (pulsed) with wild-type mouse lipoproteins, and cells and media were collected at chase periods up to 24 h. When cells were pulsed with VLDL, apoE was resecreted within 30 min. Although the mass of apoE in the media decreased with time, it could be detected up to 24 h after the pulse. Intact intracellular apoE was also detectable 24 h after the pulse. ApoE was also resecreted when cells were pulsed with HDL. When apoA-I was included in the chase media after a pulse with VLDL, apoE resecretion increased 4-fold. Furthermore, human apoE was resecreted from wild-type mouse hepatocytes after a pulse with human VLDL. Finally, apoE was resecreted from mouse peritoneal macrophages after pulsing with VLDL. We conclude that 1) HDL apoE recycles in a quantitatively comparable fashion to VLDL apoE; 2) apoE recycling can be modulated by extracellular apoA-I but is not affected by endogenous apoE; and 3) recycling occurs in macrophages as well as in hepatocytes, suggesting that the process is not cell-specific.     

Phytoremediation of arsenic and lead in contaminated soil using Chinese brake ferns (Pteris vittata) and Indian mustard (Brassica juncea)

Field and greenhouse experiments were performed to assess the performance of phytoremediation of arsenic and lead from contaminated soil at an EPA Superfund site (Barber Orchard). Chinese Brake ferns (Pteris vittata) were used to extract arsenic. On average, fern shoot arsenic concentrations were as high as 20 times the soil arsenic concentrations under field conditions. It was estimated that 8 years would be required to reduce the acid-extractable portion of soil arsenic to safe levels (40 mg/kg). The effect of soil pH on arsenic extraction was also investigated. Results indicate that increasing soil pH may improve arsenic removal. Indian mustard plants (Brassica juncea) were used under greenhouse conditions to phytoextract soil lead. EDTA was applied to soil and was found to improve lead extraction. When the EDTA concentration was 10 mmol EDTA/kg soil in soil containing 338 mg Pb/kg soil, mustard plants extracted approximately 32 mg of lead. In conclusion, phytoremediation would be a suitable alternative to conventional remediation techniques, especially for soils that do not require immediate remediation.     

Anti-adhesion therapy of bacterial diseases: prospects and problems

The alarming increase in drug-resistant bacteria makes a search for novel means of fighting bacterial infections imperative. An attractive approach is the use of agents that interfere with the ability of the bacteria to adhere to tissues of the host, since such adhesion is one of the initial stages of the infectious process. The validity of this approach has been unequivocally demonstrated in experiments performed in a wide variety of animals, from mice to monkeys, and recently also in humans. Here we review various approaches to anti-adhesion therapy, including the use of receptor and adhesin analogs, dietary constituents, sublethal concentrations of antibiotics and adhesin-based vaccines. Because anti-adhesive agents are not bactericidal, the propagation and spread of resistant strains is much less likely to occur than as a result of exposure to bactericidal agents, such as antibiotics. Anti-adhesive drugs, once developed, may, therefore, serve as a new means to fight infectious diseases.     

Cloning and characterization of a mammalian fatty acyl-CoA elongase as a lipogenic enzyme regulated by SREBPs

The mammalian enzyme involved in the final elongation of de novo fatty acid biosynthesis following the building of fatty acids to 16 carbons by fatty acid synthase has yet to be identified. In the process of searching for genes activated by sterol regulatory element-binding protein 1 (SREBP-1) by using DNA microarray, we identified and characterized a murine cDNA clone that is highly similar to a fatty acyl-CoA elongase gene family such as Cig30, Sscs, and yeast ELOs. Studies on the cells overexpressing the full-length cDNA indicate that the encoded protein, designated fatty acyl-CoA elongase (FACE), has a FACE activity specific for long-chains; C12-C16 saturated- and monosaturated-fatty acids. Hepatic expression of this identified gene was consistently activated in the livers of transgenic mice overexpressing nuclear SREBP-1a, -1c, or -2. FACE mRNA levels are markedly induced in a refed state after fasting in the liver and adipose tissue. This refeeding response is significantly reduced in SREBP-1 deficient mice. Dietary PUFAs caused a profound suppression of this gene expression, which could be restored by SREBP-1c overexpression. Hepatic FACE expression was also highly up-regulated in leptin-deficient ob/ob mice. Hepatic FACE mRNA was markedly increased by administration of a pharmacological agonist of liver X-activated receptor (LXR), a dominant activator for SREBP-1c expression. These data indicated that this elongase is a new member of mammalian lipogenic enzymes regulated by SREBP-1, playing an important role in de novo synthesis of long-chain saturated and monosaturated fatty acids in conjunction with fatty acid synthase and stearoyl-CoA desaturase.     

Accelerating aging by mouse reverse genetics: a rational approach to understanding longevity

Investigating the molecular basis of aging has been difficult, primarily owing to the pleiotropic and segmental nature of the aging phenotype. There are many often interacting symptoms of aging, some of which are obvious and appear to be common to every aged individual, whereas others affect only a subset of the elderly population. Although at first sight this would suggest multiple molecular mechanisms of aging, there now appears to be almost universal consensus that aging is ultimately the result of the accumulation of somatic damage in cellular macromolecules, with reactive oxygen species likely to be the main damage-inducing agent. What remains significant is unravelling how such damage can give rise to the large variety of aging symptoms and how these can be controlled. Although humans, with over a century of clinical observations, remain the obvious target of study, the mouse, with a relatively short lifespan, easy genetic accessibility and close relatedness to humans, is the tool par excellence to model aging-related phenotypes and test strategies of intervention. Here we present the argument that mouse models with engineered defects in genome maintenance systems are especially important because they often exhibit a premature appearance of aging symptoms. Confirming studies on human segmental progeroid syndromes, most of which are based on heritable mutations in genes involved in genome maintenance, the results thus far obtained with mouse models strongly suggest that lifespan and onset of aging are directly related to the quality of DNA metabolism. This may be in keeping with the recent discovery of a possible 'universal survival' pathway that improves antioxidant defence and genome maintenance and simultaneously extends lifespan in the mouse and several invertebrate species.     

Cellular growth and division in the Gillespie algorithm

Recent experimental studies elucidating the importance of noise in gene regulation have ignited widespread interest in Gillespie's stochastic simulation technique for biochemical networks. We formulate modifications to the Gillespie algorithm which are necessary to correctly simulate chemical reactions with time-dependent reaction rates. We concentrate on time dependence of kinetic rates arising from the periodic process of growth and division of the cellular volume, and demonstrate that a careful re-derivation of the Gillespie algorithm is important when all stochastically simulated reactions have rates slower or comparable to the cellular growth rate. For an unregulated single-gene system, we illustrate our findings using recently proposed hybrid simulation techniques, and systematically compare our algorithm with analytic results obtained from the chemical master equation.     

Complex ligand-protein systems: a globally convergent iterative method for the n x m case

When n types of univalent ligands are competing for the binding to m types of protein sites, the determination of the system composition at equilibrium reduces to the solving of a non-linear system of n equations in C = [0;1] ⁿ . We present an iterative method to solve such a system. We show that the sequence presented here is always convergent, regardless of the initial value in C. We also prove that the limit of this sequence is the unique solution in C of the non-linear system of equations. Received: 1 November 2000 / Published online: 21 August 2001     

Hepatocyte-derived ApoE is more effective than non-hepatocyte-derived ApoE in remnant lipoprotein clearance

The importance of hepatocyte-derived apolipoprotein (apo) E in the clearance of remnant lipoproteins in the liver is controversial. To address this controversy, we compared remnant clearance in two mouse models in which apoE is primarily derived either from hepatocytes or from an extrahepatic source. Hypomorphic apoE mice universally express reduced levels of apoE in all tissues, with the liver remaining the primary source of apoE. This mouse model of hepatocyte-derived apoE was compared with Apoe(-/-) mice transplanted with mouse bone marrow as a model of primarily non-hepatocyte-derived apoE. Immunohistochemical analysis of liver sections revealed that only the hepatocyte-derived apoE model had detectable levels of apoE on hepatic sinusoidal surfaces. The non-hepatocyte-derived apoE model with plasma apoE levels similar to those in the hepatocyte-derived model had 2-fold more total plasma cholesterol, 4-fold more total plasma triglycerides, and 8-fold higher levels of apoB48, similar to Apoe(-/-) mice. Both the hepatocyte-derived and the non-hepatocyte-derived apoE models had delayed clearance of an infused bolus of (125)I-labeled remnants compared with wild-type mice. However, after 3 h, plasma remnants reached wild-type levels only in the hepatocyte-derived apoE model, which had accumulated 70 +/- 5% of wild-type levels of remnants in the liver while the non-hepatocyte-derived apoE model had accumulated only 38 +/- 4%. These results demonstrate the existence of a role for both hepatically derived and localized apoE in remnant clearance. This role likely represents the enrichment of remnants sequestered on hepatocyte, with hepatocyte-derived apoE, facilitating their receptor-mediated internalization.