Scavenger receptor CD36 mediates uptake of high density lipoproteins in mice and by cultured cells

The mechanisms of HDL-mediated cholesterol transport from peripheral tissues to the liver are incompletely defined. Here the function of scavenger receptor cluster of differentiation 36 (CD36) for HDL uptake by the liver was investigated. CD36 knockout (KO) mice, which were the model, have a 37% increase (P = 0.008) of plasma HDL cholesterol compared with wild-type (WT) littermates. To explore the mechanism of this increase, HDL metabolism was investigated with HDL radiolabeled in the apolipoprotein (¹²⁵I) and cholesteryl ester (CE, [³H]) moiety. Liver uptake of [³H] and ¹²⁵I from HDL decreased in CD36 KO mice and the difference, i. e. hepatic selective CE uptake ([³H]¹²⁵I), declined (–33%, P = 0.0003) in CD36 KO compared with WT mice. Hepatic HDL holo-particle uptake (¹²⁵I) decreased (–29%, P = 0.0038) in CD36 KO mice. In vitro, uptake of ¹²⁵I-/[³H]HDL by primary liver cells from WT or CD36 KO mice revealed a diminished HDL uptake in CD36-deficient hepatocytes. Adenovirus-mediated expression of CD36 in cells induced an increase in selective CE uptake from HDL and a stimulation of holo-particle internalization. In conclusion, CD36 plays a role in HDL uptake in mice and by cultured cells. A physiologic function of CD36 in HDL metabolism in vivo is suggested.     

A novel peptide-producing cell in Xenopus: multinucleated gastric mucosal cell strikingly similar to the granular gland of the skin.

We have characterized a novel peptide-containing cell within the gastric mucosa of Xenopus laevis. The cell is a spherical, multinucleated syncytial structure containing a cytoplasmic space filled with dense rice-shaped granules, and is strikingly similar in morphology to the well-studied granular gland of the amphibian skin. Immunohistochemical and immunogold methods were used to demonstrate that several peptides previously isolated from the granular glands of the skin, including the antimicrobial peptides magainin and PGLa (a peptide with amino-terminal glycine and carboxy-terminal leucinamide), are also stored in granules present in these enteric cells. These data demonstrate that this enteric peptide-producing cell is strikingly similar both morphologically and biochemically to the granular gland, previously considered a highly specialized structure of the amphibian integument. This novel gastric mucosal cell, which we have designated a "granular multinucleated cell," is distinct in its morphology and its diversity of stored peptide products from other well-characterized peptide-containing cells in the vertebrate gastrointestinal tract.     

Demonstration of five major glycoproteins in myelin and myelin subfractions.

Myelin was found to contain five major glycoproteins with molecular weights of 120000, 95000, 88000, 43000 and 38000. Light myelin contained only 5-7% of the amount of these glycoproteins in whole myelin, whereas heavy myelin and the membrane fraction contained amounts nearly identical with whole myelin. Since all the major and minor glycoproteins, with the exception of 120000-mol-wt. glycoprotein, were detected only after treating the myelin membrane with neuraminidase, N-acetylneuraminic acid is a terminal sugar residue in these glycoproteins.     

Axial Tubules of Rat Ventricular Myocytes Form Multiple Junctions with the Sarcoplasmic Reticulum

Ryanodine receptors (RyRs) are located primarily on the junctional sarcoplasmic reticulum (SR), adjacent to the transverse tubules and on the cell surface near the Z-lines, but some RyRs are on junctional SR adjacent to axial tubules. Neither the size of the axial junctions nor the numbers of RyRs that they contain have been determined. RyRs may also be located on the corbular SR and on the free or network SR. Because determining and quantifying the distribution of RyRs is critical for both understanding and modeling calcium dynamics, we investigated the distribution of RyRs in healthy adult rat ventricular myocytes, using electron microscopy, electron tomography, and immunofluorescence. We found RyRs in only three regions: in couplons on the surface and on transverse tubules, both of which are near the Z-line, and in junctions on most of the axial tubules—axial junctions. The axial junctions averaged 510 nm in length, but they occasionally spanned an entire sarcomere. Numerical analysis showed that they contain as much as 19% of a cell's RyRs. Tomographic analysis confirmed the axial junction's architecture, which is indistinguishable from junctions on transverse tubules or on the surface, and revealed a complexly structured tubule whose lumen was only 26 nm at its narrowest point. RyRs on axial junctions colocalize with Ca_v1.2, suggesting that they play a role in excitation-contraction coupling.     

A new orphan member of the nuclear hormone receptor superfamily that interacts with a subset of retinoic acid response elements.

We have identified and characterized a new orphan member of the nuclear hormone receptor superfamily, called MB67, which is predominantly expressed in liver. MB67 binds and transactivates the retinoic acid response elements that control expression of the retinoic acid receptor beta 2 and alcohol dehydrogenase 3 genes, both of which consist of a direct repeat hexamers related to the consensus AGGTCA, separated by 5 bp. MB67 binds these elements as a heterodimer with the 9-cis-retinoic acid receptor, RXR. However, MB67 does not bind or activate other retinoic acid response elements with alternative hexamer arrangements or any of several other wild-type and synthetic hormone response elements examined. The transactivation of retinoic acid response elements by MB67 is weaker than that conferred by the retinoic acid receptors but does not require the presence of all-trans retinoic acid, 9-cis-retinoic acid, or any exogenously added ligand. We propose that MB67 plays an important role in the complex network of proteins that govern response to retinoic acid and its metabolites.     

Reconstitution of clathrin-coated pit budding from plasma membranes

Receptor-mediated endocytosis begins with the binding of ligand to receptors in clathrin-coated pits followed by the budding of the pits away from the membrane. We have successfully reconstituted this sequence in vitro. Highly purified plasma membranes labeled with gold were obtained by incubating cells in the presence of anti-LDL receptor IgG-gold at 4 degrees C, attaching the labeled cells to a poly-L-lysine-coated substratum at 4 degrees C and then gently sonicating them to remove everything except the adherent membrane. Initially the gold label was clustered over flat, clathrin-coated pits. After these membranes were warmed to 37 degrees C for 5-10 min in the presence of buffer that contained cytosol extract, Ca2+, and ATP, the coated pits rounded up and budded from the membrane, leaving behind a membrane that was devoid of LDL gold. Simultaneous with the loss of the ligand, the clathrin triskelion and the AP-2 subunits of the coated pit were also lost. These results suggest that the budding of a coated pit to form a coated vesicle occurs in two steps: (a) the spontaneous rounding of the flat lattice into a highly invaginated coated pit at 37 degrees C; (b) the ATP, 150 microM Ca2+, and cytosolic factors(s) dependent fusion of the adjoining membrane segments at the neck of the invaginated pit.