The rational design of TAP inhibitors using peptide substrate modifications and peptidomimetics

The major histocompatibility complex (MHC)-encoded transporter associated with antigen processing (TAP) translocates peptides from the cytosol into the lumen of the endoplasmic reticulum. This step precedes the binding of peptides to MHC class I molecules and is essential for cell surface expression of the MHC class I/peptide complex. TAP has a broad sequence specificity and a preference for peptides of around 9 amino acids. To synthesize inhibitors for TAP, we studied various alterations of the peptide substrate. The results indicate that TAP is stereospecific and that peptide bonds engineered into isosteric structures can improve translocation of the peptide. Furthermore, TAP is able to translocate peptides with large side chains that correspond to a peptide of ～ 21 amino acids in extended conformation. Peptides with longer side chains compete for the peptide binding site of TAP but fail to be translocated. Therefore, they represent the first rationally designed inhibitors of TAP.     

Design and synthesis of novel N-acetylgalactosamine-terminated glycolipids for targeting of lipoproteins to the hepatic asialoglycoprotein receptor

A novel glycolipid has been prepared that contains a cluster glycoside with an unusually high affinity for the asialoglycoprotein receptor (ASGPr) and a bile acid moiety that mediates stable incorporation into lipidic particles. The glycolipid spontaneously associated with low-density lipoproteins (LDL) and high-density lipoproteins (HDL) within human and murine plasma, and loading of lipoproteins with this glycolipid resulted in an efficient dose-dependent recognition and uptake of LDL and HDL by the liver (and not by spleen) upon intravenous injection into wild-type mice. Preinjection with asialoorosomucoid largely inhibited the uptake, establishing that both HDL and LDL were selectively recognized and processed by the ASGPr on liver parenchymal cells. Finally, repeated intravenous administration of the glycolipid to hyperlipidemic LDL receptor-deficient mice evoked an efficient and persistent cholesterol-lowering effect. These results indicate that the glycolipid may be a promising alternative for the treatment of hyperlipidemic patients who do not respond sufficiently to current cholesterol-lowering therapies.     

Effect of different doses of 3-methylcholanthrene on the localization of the 3-methylcholanthrene-inducible isoenzymes of cytochrome P450 within the centrilobular and periportal zones of the rat liver

Immunohistochemical staining techniques were used to investigate the localization of the 3-methylcholanthrene inducible isoenzymes (P450 IA1 and IA2) in the rat liver. The rats were induced with different doses of 3-methylcholanthrene, ranging from 2.5 to 25 mg/kg body weight. A heterogeneous induction pattern was observed with induction doses of 2.5; 5; 7.5 and 10 mg/kg body weight with the highest concentration of the isoenzymes around the central vein. With a dose of 25 mg/kg body weight, a homogeneous pattern was found. Induction with a dose of 15 mg/kg body weight resulted in an intermediate situation.     

Synthesis of a Lipophilic Prodrug of 9-(2-Phosphonylmethoxyethyl)adenine (PMEA) and Its Incorporation into a Hepatocyte-Specific Lipidic Carrier

Purpose. 9-(2-Phosphonylmethoxyethyl)adenine (PMEA), a potent inhibitor of Hepatitis B virus replication, is in vivo hardly taken up by parenchymal liver cells (the site of infection). Our aim is to examine whether lactosylated reconstituted HDL (LacNeoHDL), a lipidic particle that is specifically internalized by parenchymal liver cells, is a suitable carrier for the selective delivery of PMEA to this cell type. Methods. To incorporate PMEA into LacNeoHDL, we synthesized a lipophilic prodrug (PMEA-LO) by coupling PMEA via an acid-labile phosphonamidate bond to lithocholic acid-3-oleate. Results. The yield of the synthesis was 52% ([³H]PMEA-LO: 24%). [³H]PMEA-LO readily incorporated into LacNeoHDL (13 molecules/ particle) without affecting the size and net negative charge of the carrier. Further, incubation studies at lysosomal pH showed [³H]PMEA was completely released from the carrier whereas, at neutral pH or in plasma, appreciable release was not observed. Conclusions. The conjugation of PMEA with lithocholic acid-3-oleate results in a lipophilic prodrug that readily associates with LacNeoHDL. The association of the prodrug does not affect the physico-chemical properties of the particle, and PMEA is released from the carrier at lysosomal pH. These findings indicate that by using the prodrug approach, LacNeoHDL is a suitable carrier to deliver PMEA to parenchymal liver cells.     

Building an Artificial Stem Cell Niche: Prerequisites for Future 3D-Formation of Inner Ear Structures—Toward 3D Inner Ear Biotechnology

In recent years, there has been an increased interest in stem cells for the purpose of regenerative medicine to deliver a wide range of therapies to treat many diseases. However, two-dimensional cultures of stem cells are of limited use when studying the mechanism of pathogenesis of diseases and the feasibility of a treatment. Therefore, research is focusing on the strengths of stem cells in the three-dimensional (3D) structures mimicking organs, that is, organoids, or organ-on-chip, for modeling human biology and disease. As 3D technology advances, it is necessary to know which signals stem cells need to multiply and differentiate into complex structures. This holds especially true for the complex 3D structure of the inner ear. Recent work suggests that although other factors play a role, the extracellular matrix (ECM), including its topography, is crucial to mimic a stem cell niche in vitro and to drive stem cells toward the formation of the tissue of interest. Technological developments have led to the investigation of biomaterials that closely resemble the native ECM. In the fast forward moving research of organoids and organs-on-chip, the inner ear has hardly received attention. This review aims to provide an overview, by describing the general context in which cells, matrix and morphogens cooperate in order to build a tissue, to facilitate research in 3D inner ear technology. Anat Rec, 303:408–426, 2020. © 2019 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.     

Cellular reaction on the intraperitoneal injection of four types of polylactide particulates.

Four types of polylactide particulates, P-L-LA 100, 250, 550 KD and a P-DL-LA 400 KD were injected into the peritoneal cavity of mice. The inflammatory reaction showed an increase in cell number (mainly neutrophilic granulocytes) up to 48 h after which the cell numbers decreased below the control (phosphate-buffered saline). All four polylactide particulates aggregated and intermingled with inflammatory cells. The aggregates remained throughout the investigation period of 6 months. Quantitative measurements showed that standardization of the particle form and size is essential. From this study and other experiments in which calcium phosphates and asbestos were injected intraperitoneally, it is concluded that the inflammatory response observed in the peritoneal cavity is related to the type of material injected and probably to form and size of the individual particles, but not to molecular weight.     

Design and synthesis of novel amphiphilic dendritic galactosides for selective targeting of liposomes to the hepatic asialoglycoprotein receptor

A series of glycolipids have been prepared which contain a cluster galactoside moiety with high affinity for the hepatic asialoglycoprotein receptor and a bile acid ester moiety which mediates stable incorporation into liposomes. Loading of liposomes with these glycolipids at a ratio of 5% (w/w) resulted in efficient recognition and uptake of the liposomes by the liver. Preinjection with asialofetuin almost completely inhibited the uptake, establishing that the liposomes were selectively recognized and processed by the asialoglycoprotein receptor on liver parenchymal cells. In contrast, a glycolipid content of 50% (w/w) led to a liver uptake that could not be inhibited by preinjection with asialofetuin, indicating that the liposomes were now processed by the Gal/Fuc-recognizing receptor on liver macrophages. The results presented in this study are important for future targeting of water-soluble and amphiphilic drugs, enveloped in these glycolipid-laden liposomes, to parenchymal liver cells.