SR-BI-mediated high density lipoprotein (HDL) endocytosis leads to HDL resecretion facilitating cholesterol efflux

The high density lipoprotein (HDL) receptor, scavenger receptor class B, type I (SR-BI), mediates selective cholesteryl ester uptake from lipoproteins into liver and steroidogenic tissues but also cholesterol efflux from macrophages to HDL. Recently, we demonstrated the uptake of HDL particles in SR-BI overexpressing Chinese hamster ovarian cells (ldlA7-SRBI) using ultrasensitive microscopy. In this study we show that this uptake of entire HDL particles is followed by resecretion. After uptake, HDL is localized in endocytic vesicles and organelles en route to the perinuclear area; many HDL-positive compartments were classified as multivesiculated and multilamellated organelles by electron microscopy. By using 125I-labeled HDL, we found that approximately 0.8% of the HDL added to the media is taken up by the ldlA7-SRBI cells within 1 h, and almost all HDL is finally resecreted. 125I-Labeled low density lipoprotein showed a very similar association, uptake, and resecretion pattern in ldlA7-SRBI cells that do not express any low density lipoprotein receptor. Moreover, we demonstrate that the process of HDL cell association, uptake, and resecretion occurs in three physiologically relevant cell systems, the liver cell line HepG2, the adrenal cell line Y1BS1, and phorbol myristate acetate-differentiated THP-1 cells as a model for macrophages. Finally, we present evidence that HDL retroendocytosis represents one of the pathways for cholesterol efflux.     

Anti-tumor activity of the combination of cetuximab, an anti-EGFR blocking monoclonal antibody and ZD6474, an inhibitor of VEGFR and EGFR tyrosine kinases

PURPOSE: The epidermal growth factor receptor (EGFR) autocrine pathway plays an important role in cancer cell growth. Vascular endothelial growth factor A (VEGF-A) is a key regulator of tumor-induced endothelial cell proliferation and vascular permeability. ZD6474 is an orally available, small molecule inhibitor of VEGF receptor-2 (VEGFR-2), EGFR and RET tyrosine kinase activity. We investigated the activity of ZD6474 in combination with cetuximab, an anti-EGFR blocking monoclonal antibody, to determine the anti-tumor activity of EGFR blockade through the combined use of two agents targeting the receptor at different molecular sites in cancer cells and of VEGFR-2 blockade in endothelial cells. EXPERIMENTAL DESIGN: The anti-tumor activity in vitro and in vivo of ZD6474 and/or cetuximab was tested in human cancer cell lines with a functional EGFR autocrine pathway. RESULTS: The combination of ZD6474 and cetuximab determined synergistic growth inhibition in all cancer cell lines tested as assessed by the Chou and Talalay method. In nude mice bearing established human colon carcinoma (GEO) or lung adenocarcinoma (A549) xenografts and treated with ZD6474 and/or cetuximab for 4 weeks, a reversible tumor growth inhibition was caused by each drug. In contrast, a more significant tumor growth delay resulted from the combination of the two agents with an approximately 100-110 days increase in mice median overall survival as compared to single agent treatment. CONCLUSIONS: This study provides a rationale for evaluating in a clinical setting the double blockade of EGFR in combination with inhibition of VEGFR-2 signaling as cancer therapy.     

Reduced brain cholesterol content in arylsulfatase A-deficient mice

Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder caused by deficiency in arylsulfatase A (ASA). Concentrations of cholesterol and its metabolites were determined in ASA deficient [ASA(-/-)] mice which serve as an animal model of MLD. We observed a significant reduction in cholesterol content in the brain of adult ASA(-/-) mice when compared to wild-type controls. This was not due to loss of myelin, because ASA(-/-) mice do not demyelinate. Other cholesterol metabolites were not changed significantly in ASA(-/-) mice, except for an increase in lathosterol. Moreover, reduced cholesterol levels were also found in tissue samples from two juvenile MLD cases. Since high cholesterol levels are important for myelination, and various cellular processes, like vesicular trafficking and signal transduction, reduced cholesterol levels might be an important factor in the molecular pathology of MLD.     

Specific serine proteases selectively damage KCNH2 (hERG1) potassium channels and I(Kr)

KCNH2 (hERG1) encodes the alpha-subunit proteins for the rapidly activating delayed rectifier K+ current (I(Kr)), a major K+ current for cardiac myocyte repolarization. In isolated myocytes I(Kr) frequently is small in amplitude or absent, yet KCNH2 channels and I(Kr) are targets for drug block or mutations to cause long QT syndrome. We hypothesized that KCNH2 channels and I(Kr) are uniquely sensitive to enzymatic damage. To test this hypothesis, we studied heterologously expressed K+, Na+, and L-type Ca2+ channels, and in ventricular myocytes I(Kr), slowly activating delayed rectifier K+ current (I(Ks)), and inward rectifier K+ current (I(K1)), by using electrophysiological and biochemical methods. 1) Specific exogenous serine proteases (protease XIV, XXIV, or proteinase K) selectively degraded KCNH2 current (I(KCNH2)) and its mature channel protein without damaging cell integrity and with minimal effects on the other channel currents; 2) immature KCNH2 channel protein remained intact; 3) smaller molecular mass KCNH2 degradation products appeared; 4) protease XXIV selectively abolished I(Kr); and 5) reculturing HEK-293 cells after protease exposure resulted in the gradual recovery of I(KCNH2) and its mature channel protein over several hours. Thus the channel protein for I(KCNH2) and I(Kr) is uniquely sensitive to proteolysis. Analysis of the degradation products suggests selective proteolysis within the S5-pore extracellular linker, which is structurally unique among Kv channels. These data provide 1) a new mechanism to account for low I(Kr) density in some isolated myocytes, 2) evidence that most complexly glycosylated KCNH2 channel protein is in the plasma membrane, and 3) new insight into the rate of biogenesis of KCNH2 channel protein within cells.     

Functional expression of the CMP-sialic acid transporter in Escherichia coli and its identification as a simple mobile carrier

The architectural conservation of nucleotide sugar transport proteins (NSTs) enabled the theoretical prediction of putative NSTs in diverse gene databases. In the human genome, 17 NST sequences have been identified but only six have been unequivocally characterized with respect to their transport specificities. Defining transport characteristics of recombinant NSTs has become a major challenge because true zero background systems are widely absent. Production of recombinant NSTs in heterologous systems has developed multifunctionality for some NSTs leading to a novel level of complexity in the field. Assuming that (1) the specificity of NSTs is determined at the primary sequence level and (2) the proteins are autonomously functional units, final definition of the substrate specificity will depend on the use of isolated transport proteins. Herein, we describe the first report of the functional expression of mouse CMP-sialic acid transporter (CST) in Escherichia coli and thus provide significant progress towards the production of transporter proteins in quantities suitable for functional and structural analyses. Recovery of the active NST from inclusion bodies was achieved after solubilization with 8 M urea and stepwise renaturation. After reconstitution into phospholipid vesicles, the recombinant protein demonstrated specific transport for CMP-N-acetylneuraminic acid (CMP-Neu5Ac) with no transport of UDP-sugars. Kinetic studies carried out with CMP-Neu5Ac and established CMP-Neu5Ac antagonist's evaluated natural conformation of the reconstituted protein and clearly demonstrate that the transporter acts as a simple mobile carrier.     

The significance of host-fungus combinations in ectomycorrhizal symbioses for the chemical quality of willow foliage

The majority of plants interacts with mycorrhizal fungi, which predominantly provide mutual benefits, but also costs. We tested the hypothesis that specific combinations of host plants (four commercial varieties of Salix spp.) and ectomycorrhizal partners (species of Laccaria, Paxillus, Tricholoma and Hebeloma) differ in their effects on the host foliar chemistry. Twenty specific host - mycorrhiza combinations were pot-grown outdoors under low-N conditions. Foliar concentrations of total phenolics and salicylic acid were decreased by mycorrhizas in S. schwerinii x S. viminalis and S. x dasyclados genotypes, but increased in S. viminalis. Mycorrhiza effects on host biomass production were positive, indifferent or negative, depending on the specific host—mycorrhiza combination. The host plant genotype influenced mainly the direction of mycorrhizal effects on foliar chemistry, whereas the magnitude of mycorrhizal effects varied mostly among the fungal genotypes. The results are relevant with respect to the possible interactions between mycorrhizas, plants and leaf herbivores.     

Chylomicrons promote intestinal absorption of lipopolysaccharides

Recent data suggest that dietary fat promotes intestinal absorption of lipopolysaccharides (LPS) from the gut microflora, which might contribute to various inflammatory disorders. The mechanism of fat-induced LPS absorption is unclear, however. Intestinal-epithelial cells can internalize LPS from the apical surface and transport LPS to the Golgi. The Golgi complex also contains newly formed chylomicrons, the lipoproteins that transport dietary long-chain fat through mesenteric lymph and blood. Because LPS has affinity for chylomicrons, we hypothesized that chylomicron formation promotes LPS absorption. In agreement with our hypothesis, we found that CaCo-2 cells released more cell-associated LPS after incubation with oleic-acid (OA), a long-chain fatty acid that induces chylomicron formation, than with butyric acid (BA), a short-chain fatty acid that does not induce chylomicron formation. Moreover, the effect of OA was blocked by the inhibitor of chylomicron formation, Pluronic L-81. We also observed that intragastric triolein (TO) gavage was followed by increased plasma LPS, whereas gavage with tributyrin (TB), or TO plus Pluronic L-81, was not. Most intestinally absorbed LPS was present on chylomicron remnants (CM-R) in the blood. Chylomicron formation also promoted transport of LPS through mesenteric lymph nodes (MLN) and the production of TNFalpha mRNA in the MLN. Together, our data suggest that intestinal epithelial cells may release LPS on chylomicrons from cell-associated pools. Chylomicron-associated LPS may contribute to postprandial inflammatory responses or chronic diet-induced inflammation in chylomicron target tissues.     

Role of asparagine 1134 in glucosidic bond and transglycosylation specificity of reuteransucrase from Lactobacillus reuteri 121

Glucansucrases from lactic acid bacteria convert sucrose into various alpha-glucans that differ greatly with respect to the glucosidic bonds present (e.g. dextran, mutan, alternan and reuteran). This study aimed to identify the structural features of the reuteransucrase from Lactobacillus reuteri 121 (GTFA) that determine its reaction specificity. We here report a detailed mutational analysis of a conserved region immediately next to the catalytic Asp1133 (putative transition-state stabilizing) residue in GTFA. The data show that Asn1134 is the main determinant of glucosidic bond product specificity in this reuteransucrase. Furthermore, mutations at this position greatly influenced the hydrolysis/transglycosylation ratio. Changes in this amino acid expands the range of glucan and gluco-oligosaccharide products synthesized from sucrose by mutant GTFA enzymes.     

Molecular cloning and functional characterization of two alternatively spliced Ntcp isoforms from mouse liver1

To isolate the murine Na+/taurocholate cotransporting polypeptide (Ntcp), we screened a mouse liver cDNA library and identified Ntcp1, encoding a 362 amino acid protein and Ntcp2, encoding a 317 amino acid protein which had a shorter C-terminal end. Both isoforms mediated saturable Na+-dependent transport of taurocholate when expressed in Xenopus laevis oocytes. Analysis of the gene revealed that Ntcp2 is produced by alternative splicing where the last intron is retained.     

Polymorphisms past and present

Polymorphisms, particularly genetic variants of the red blood cell, have served as a major focus for the research of Frank B. Livingstone over the course of a long and productive career. Recent investigations confirm the value of key insights that he contributed to this area more than four decades ago. As Livingstone recognized, the same underlying evolutionary model that guides genetic studies in present populations also provides a productive framework for interpreting patterns of variation in the skeleton and dentition throughout past human evolution. Examples explored in detail here include polymorphisms in hominoid nasal bone shapes and fourth lower premolar roots. This work provides both empirical and theoretical contexts for investigating patterns of human variation over the last 6 to 8 million years.