Catalytic ceric ammonium nitrate mediated synthesis of 2-deoxy-1-thioglycosides

Synthesis of 2-deoxy-1-thioglycosides from glycals, mediated by catalytic amounts of ceric ammonium nitrate is reported. Apart from the 2-deoxy-1-thioglycosides, formation of the 2,3-unsaturated enose, corresponding to the Ferrier product, is also observed, especially for the glucal substrates. A radical oxocarbenium ion and a thiolate intermediates are most likely to mediate the reaction. Upon synthesis of 2-deoxy-1-thioglycosides, few representative glycosylation reactions with both aglycosyl and glycosyl acceptors were performed and alpha-anomeric 2-deoxy glycosides were obtained exclusively.     

Crystal structure of N-(benzyloxycarbonyl)aminoethyl-2,3,4,6-tetra-O-benzoyl-alpha-D-mannopyranoside: stabilization of the crystal lattice by a tandem network of N-H. . .O, C-H. . .O, and C-H. . .pi interactions

Single crystal X-ray analysis of an aminoethyl mannopyranoside, namely, N-(benzyloxycarbonyl)aminoethyl-2,3,4,6-tetra-O-benzoyl-alpha-D-mannopyranoside (1), shows that the compound crystallizes in the monoclinic space group P2(1), with two molecules in the unit cell. The mannopyranoside unit adopts a distorted 4C(1) conformation. An analysis of the intermolecular interactions reveals a tandem network of N-H. . .O, C-H. . .O, and C-H. . .pi interactions responsible for stabilizing the crystal lattice.     

Delivery of Bcl-XL or its BH4 domain by protein transduction inhibits apoptosis in human islets

Viability of isolated islets is one of the main obstacles limiting islet transplantation success. It has been reported that overexpression of Bcl-2/Bcl-XL proteins enhances islet viability. To avoid potential complications associated with long-term expression of anti-apoptotic proteins, we investigated the possibility of delivering Bcl-XL or its anti-apoptotic domain BH4 to islets by protein transduction. Bcl-XL and BH4 molecules were fused to TAT/PTD, the 11-aa cell penetrating peptide from HIV-1 transactivating protein, generating TAT-Bcl-XL and TAT-BH4, respectively. Transduction efficiency was assessed by laser scanning confocal microscopy of live islets. Biological activity was tested as the ability to protect NIT-1 insulinoma cell line from death induced by staurosporine or serum deprivation. Spontaneous caspase activation in human islets and cytotoxicity caused by IL-1beta were significantly reduced in the presence of TAT-Bcl-XL and TAT-BH4. We conclude that both TAT proteins are biologically active after transduction and could be an asset in the improvement of islet viability.     

Poly(ethylene glycol)-induced fusion and destabilization of human plasma high-density lipoproteins

High-density lipoproteins (HDL) are macromolecular complexes of specific proteins and lipids that mediate the removal of cholesterol from peripheral tissues. Chemical unfolding revealed that HDL fusion and rupture are the two main kinetic steps in HDL denaturation. Here we test the hypothesis that lipid fusogens such as poly(ethylene glycol) (PEG) may promote lipoprotein fusion and rupture and thereby destabilize HDL. We analyze thermal disruption of spherical HDL in 0-15% PEG-8000 by calorimetric, spectroscopic, electron microscopic, and light scattering techniques. We demonstrate that the two irreversible high-temperature endothermic HDL transitions involve particle enlargement and show a heating rate dependence characteristic of kinetically controlled reactions with high activation energy. The first calorimetric transition reflects HDL fusion and dissociation of lipid-poor apolipoprotein A-1 (apoA-1), and the second transition reflects HDL rupture and release of the apolar lipid core. Neither transition involves substantial protein unfolding; thus, the transition heat originates from lipid and/or protein dissociation and repacking. At room temperature, PEG-8000 induces HDL fusion that is distinct from the heat-, denaturant-, or enzyme-induced fusion since it leads to formation of larger particles and does not involve apoA-1 dissociation. Increasing the PEG concentration in solution from 0 to 15% leads to low-temperature shifts by approximately -18 degrees C in the two calorimetric HDL transitions without altering their nature. Thus, consistent with our hypothesis, PEG-8000 induces fusion and reduces the thermal stability of HDL. Our results suggest that PEG is useful for the analysis of the molecular events involved in metabolic HDL remodeling and fusion.     

Evolution of glutamate interactions during binding to a glutamate receptor

Glutamate receptors are the predominant mediators of excitatory synaptic signals in the central nervous system and are important in learning and memory as well as in diverse neuropathologies including epilepsy and ischemia. Their primary function is to receive the chemical signal glutamate (1), which binds to an extracellular domain in the receptor, and convert it into an electrical signal through the formation of cation-permeable transmembrane channels. Recently described end-state apo and ligated structures of the ligand-binding domain of a rat glutamate receptor provide a first view of specific molecular interactions between the ligand and the receptor that are central to the allosteric regulation of function in this protein. Yet there is little information on the mechanism and the structures of intermediates (if any) formed during the ligand-binding process. Here we have used time-resolved vibrational spectroscopy to show that the process involves a sequence of interleaved ligand and protein changes that starts with the docking of glutamate at the alpha-carboxylate moiety and ends with the establishment of the interactions between the gamma-carboxylate of glutamate and the protein.     

Intracellular calcium release is required for caspase-3 and -9 activation

Increase in intracellular Ca2+ [Ca2+]i regulates many biological functions including apoptosis, but the protein(s) linking [Ca2+]i and apoptosis are not completely understood. We have previously shown that IP3R-deficient cells are resistant to T-cell receptor (TCR)-induced apoptosis due to lack of Ca2+ release from endoplasmic reticulum (ER) and calcineurin activation. Here we show that caspase-9 and -3 are not activated in IP3R-deficient cells after TCR stimulation, consistent with the resistance of these cells to apoptosis. However, we also demonstrate that Bcl-2 expression in IP3R-deficient cells is comparable to control cells. Taken together, these results strongly suggest that IP3R-mediated Ca2+ release plays a critical role in regulating the activity of caspases-3 and -9 independent of Bcl-2.     

Epidermal growth factor receptor-dependent regulation of integrin-mediated signaling and cell cycle entry in epithelial cells

Integrin-mediated adhesion of epithelial cells to extracellular matrix (ECM) proteins induces prolonged tyrosine phosphorylation and partial activation of epidermal growth factor receptor (EGFR) in an integrin-dependent and EGFR ligand-independent manner. Integrin-mediated activation of EGFR in epithelial cells is required for multiple signal transduction events previously shown to be induced by cell adhesion to matrix proteins, including tyrosine phosphorylation of Shc, Cbl, and phospholipase Cgamma, and activation of the Ras/Erk and phosphatidylinositol 3'-kinase/Akt signaling pathways. In contrast, activation of focal adhesion kinase, Src, and protein kinase C, adhesion to matrix proteins, cell spreading, migration, and actin cytoskeletal rearrangements are induced independently of EGFR kinase activity. The ability of integrins to induce the activation of EGFR and its subsequent regulation of Erk and Akt activation permitted adhesion-dependent induction of cyclin D1 and p21, Rb phosphorylation, and activation of cdk4 in epithelial cells in the absence of exogenous growth factors. Adhesion of epithelial cells to the ECM failed to efficiently induce degradation of p27, to induce cdk2 activity, or to induce Myc and cyclin A synthesis; subsequently, cells did not progress into S phase. Treatment of ECM-adherent cells with EGF, or overexpression of EGFR or Myc, resulted in restoration of late-G(1) cell cycle events and progression into S phase. These results indicate that partial activation of EGFR by integrin receptors plays an important role in mediating events triggered by epithelial cell attachment to ECM; EGFR is necessary for activation of multiple integrin-induced signaling enzymes and sufficient for early events in G(1) cell cycle progression. Furthermore, these findings suggest that EGFR or Myc overexpression may provoke ligand-independent proliferation in matrix-attached cells in vivo and could contribute to carcinoma development.     

Dispensable role for interferon-gamma in the burn-induced acute phase response: a proteomic analysis

We examined the role of the pleiotropic cytokine interferon-gamma (IFN-gamma) in initiating the burn injury-induced acute phase response (APR). Two-dimensional (2-D) electrophoresis was used to obtain serum protein profiles from wild-type (WT) and IFN-gamma knockout mice following sham-burn or 20% burn injury. Serum 2-D images from both groups of burn-injured mice were characterized by the upregulation of a similar panel of protein spots. These included the three major murine acute phase proteins haptoglobin, serum amyloid A, and serum amyloid P, that were identified by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF)-mass spectrometry. Furthermore, the changes in the levels of these protein spots were very similar between these two groups of mice, as determined by image analysis. Other features of burn-induced APR such as a decrease in total serum protein concentration, an elevated circulation level of the cytokine interleukin-6 (IL-6), and activation of the IL-6 signal transduction protein STAT3 were also evaluated and found to be similar between wild-type and IFN-gamma knockout mice. These results suggest a dispensable role of IFN-gamma in the induction of the hepatic APR in mice following burn injury.     

Airway surface liquid osmolality measured using fluorophore-encapsulated liposomes

The airway surface liquid (ASL) is the thin layer of fluid coating the luminal surface of airway epithelial cells at an air interface. Its composition and osmolality are thought to be important in normal airway physiology and in airway diseases such as asthma and cystic fibrosis. The determinants of ASL osmolality include epithelial cell solute and water transport properties, evaporative water loss, and the composition of secreted fluids. We developed a noninvasive approach to measure ASL osmolality using osmotically sensitive 400-nm-diam liposomes composed of phosphatidylcholine/cholesterol/polyethylene glycol-phosphatidylcholine (1:0.3:0.08 molar ratio). Calcein was encapsulated in the liposomes at self-quenching concentrations (30 mM) as a volume-sensitive marker, together with sulforhodamine 101 (2 mM) as a volume-insensitive reference. Liposome calcein/sulforhodamine 101 fluorescence ratios responded rapidly (< 0.2 s) and stably to changes in solution osmolality. ASL osmolality was determined from calcein/sulforhodamine 101 fluorescence ratios after addition of microliter quantities of liposome suspensions to the ASL. In bovine airway epithelial cells cultured on porous supports at an air-liquid interface, ASL thickness (by confocal microscopy) was 22 microm and osmolality was 325 +/- 12 mOsm. In anesthetized mice in which a transparent window was created in the trachea, ASL thickness was 55 microm and osmolality was 330 +/- 36 mOsm. ASL osmolality was not affected by pharmacological inhibition of CFTR in airway cell cultures or by genetic deletion of CFTR in knockout mice. ASL osmolality could be increased substantially to > 400 mOsm by exposure of the epithelium to dry air; the data were modeled mathematically using measured rates of osmosis and evaporative water loss. These results establish a ratio imaging method to map osmolality in biological compartments. ASL fluid is approximately isosmolar under normal physiological conditions, but can become hyperosmolar when exposed to dry air, which may induce cough and airway reactivity in some patients.