Adenovirus-mediated expression of the C-terminal domain of SARS-CoV spike protein is sufficient to induce apoptosis in Vero E6 cells

The pro-apoptotic properties of severe acute respiratory syndrome coronavirus (SARS-CoV) structural proteins were studied in vitro. By monitoring apoptosis indicators including chromatin condensation, cellular DNA fragmentation and cell membrane asymmetry, we demonstrated that the adenovirus-mediated over-expression of SARS-CoV spike (S) protein and its C-terminal domain (S2) induce apoptosis in Vero E6 cells in a time- and dosage-dependent manner, whereas the expression of its N-terminal domain (S1) and other structural proteins, including envelope (E), membrane (M) and nucleocapsid (N) protein do not. These findings suggest a possible role of S and S2 protein in SARS-CoV induced apoptosis and the molecular pathogenesis of SARS.     

Lateral distribution of cholesterol in dioleoylphosphatidylcholine lipid bilayers: cholesterol-phospholipid interactions at high cholesterol limit

Lateral organization of cholesterol in dioleoyl-phosphatidylcholine (DOPC) lipid bilayers at high cholesterol concentration (>45 mol%) was investigated using steady-state fluorescence anisotropy and fluorescent resonance energy transfer techniques. The recently devised Low Temperature Trap method was used to prepare compositionally uniform cholesterol/DOPC liposomes to avoid the problem of lipid demixing. The fluorescence anisotropy of diphenylhexatrience chain-labeled phosphatidylcholine (DPH-PC) in these liposomes exhibited local maxima at cholesterol mol fractions of 0.50 and 0.57, and a sharp drop at 0.67. For the liposomes labeled with both dehydroergosterol and DPH-PC, the fluorescent resonance energy transfer efficiency from dehydroergosterol to DPH-PC displayed a steep jump at cholesterol mol fraction of 0.5, and dips at 0.57 and 0.68. These results indicate the presence of highly ordered cholesterol regular distribution domains at those observed critical compositions. The observed critical mol fraction at 0.67 agreed favorably with the solubility limit of cholesterol in DOPC bilayers as independently measured by light scattering and optical microscopy. The regular distribution at 0.57 was previously predicted from a Monte Carlo simulation based on the Umbrella model. The results strongly support the hypothesis that the primary requirement for cholesterol-phospholipid mixing is that the polar phospholipid headgroups need to cover the nonpolar body of cholesterol to avoid the exposure of cholesterol to water.     

Metalloproteinase-dependent transforming growth factor-alpha release mediates neurotensin-stimulated MAP kinase activation in human colonic epithelial cells

Expression of the neuropeptide neurotensin (NT) and its high affinity receptor (NTR1) is increased during the course of Clostridium difficile toxin A-induced acute colitis, and NTR1 antagonism attenuates the severity of toxin A-induced inflammation. We recently demonstrated in non-transformed human colonic epithelial NCM460 cells that NT treatment caused activation of a Ras-mediated MAP kinase pathway that significantly contributes to NT-induced interleukin-8 (IL-8) secretion. Here we used NCM460 cells, which normally express low levels of NTR1, and NCM460 cells stably transfected with NTR1 to identify the upstream signaling molecules involved in NT-NTR1-mediated MAP kinase activation. We found that inhibition of the epidermal growth factor receptor (EGFR) by either an EGFR neutralizing antibody or by its specific inhibitor AG1478 (0.2 microm) blocked NT-induced MAP kinase activation. Moreover, NT stimulated tyrosine phosphorylation of the EGFR, and pretreatment with a broad spectrum metalloproteinase inhibitor batimastat reduced NT-induced MAP kinase activation. Using neutralizing antibodies against the EGFR ligands EGF, heparin-binding-EGF, transforming growth factor-alpha (TGFalpha), or amphiregulin we have shown that only the anti-TGFalpha antibody significantly decreases NT-induced phosphorylation of EGFR and MAP kinases. Furthermore, inhibition of the EGF receptor by AG1478 significantly reduced NT-induced IL-8 promoter activity and IL-8 secretion. This is the first report demonstrating that NT binding to NTR1 transactivates the EGFR and that this response is linked to NT-mediated proinflammatory signaling. Our findings indicate that matrix metalloproteinase-mediated release of TGFalpha and subsequent EGFR transactivation triggers a NT-mediated MAP kinase pathway that leads to IL-8 gene expression in human colonic epithelial cells.     

Indium complexes incorporating bidentate substituted pyrrole ligand: Synthesis, characterization, and ring-opening polymerization of ε-caprolactone

The reactions of InCl₃ with two and three equivalents of lithium (2-dimethylaminomethyl)pyrrolate (1) in diethyl ether solutions afford In[C₄H₃N(CH₂NMe₂)]₂Cl (2) and In[C₄H₃N(CH₂NMe₂)]₃ (3) in 92% and 50% yield, respectively. Subsequent reactions of 2 with MeLi and Li(CCPh) yield In[C₄H₃N(CH₂NMe₂)]₂Me (4) and In[C₄H₃N(CH₂NMe₂)]₂(CCPh) (5), respectively. All compounds were confirmed by ¹H and ¹³C NMR spectroscopy and compounds 3, 4, and 5 were further characterized by single-crystal X-ray crystallography. Compounds 2, 3, and 4 showed moderate catalytic activity toward the ring-opening polymerization of ε-caprolactone.     

Lipid headgroup superlattice modulates the activity of surface-acting cholesterol oxidase in ternary phospholipid/cholesterol bilayers

The relationship between the molecular organization of lipid headgroups and the activity of surface-acting enzyme was examined using a bacterial cholesterol oxidase (COD) as a model. The initial rate of cholesterol oxidation by COD in fluid state 1-palmitoyl-2-oleoyl-phosphatidylethanolamine/1-palmitoyl-2-oleoyl-phosphatidylcholine/cholesterol (POPE/POPC/CHOL) bilayers was measured as a function of POPE-to-phospholipid mole ratio (X(PE)) and cholesterol-to-lipid mole ratio (X(CHOL)) at 37 degrees C. At X(PE) = 0, the COD activity changed abruptly at X(CHOL) approximately 0.40, whereas major activity peaks were detected at X(PE) approximately 0.18, 0.32, 0.50, 0.64, and 0.73 when X(CHOL) was fixed to 0.33 or 0.40. At a fixed X(CHOL) of 0.50, the COD activity increased progressively with PE content and exhibited small peaks or kinks at X(PE) approximately 0.40, 0.50, 0.58, 0.69, and 0.81. When X(PE) and X(CHOL) were systematically varied within a narrow 2-D lipid composition window, an onset of COD activity at X(CHOL) approximately 0.40 and the elimination of the activity peak at X(PE) approximately 0.64 for X(CHOL) >0.40 were clearly observed. Except for X(PE) approximately 0.40 and 0.58, the observed critical PE mole ratios agree closely (+/-0.03) with those predicted by a headgroup superlattice model (Virtanen, J.A., et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 4964-4969; Cannon, B., et al. (2006) J. Phys. Chem. B 110, 6339-6350), which proposes that lipids with headgroups of different sizes tend to adopt regular, superlattice-like distributions at discrete and predictable compositions in fluid lipid bilayers. Our results indicate that headgroup superlattice domains exist in lipid bilayers and that they may play a crucial role in modulating the activity of enzymes acting on the cell membrane surface.     

Gender and post-ischemic recovery of hypertrophied rat hearts

Background

Cardiac mortality in Duchenne muscular dystrophy (DMD) has recently become important, because risk of respiratory failure has been reduced due to widespread use of the respirator. The cardiac involvement is characterized by distinctive electrocardiographic abnormalities or dilated cardiomyopathy, but the pathogenesis has remained obscure. In research on DMD, Golden retriever-based muscular dystrophy (GRMD) has attracted much attention as an animal model because it resembles DMD, but GRMD is very difficult to maintain because of their severe phenotypes. We therefore established a line of dogs with Beagle-based canine X-linked muscular dystrophy in Japan (CXMD_J) and examined the cardiac involvement.

Methods

The cardiac phenotypes of eight CXMD_J and four normal male dogs 2 to 21 months of age were evaluated using electrocardiography, echocardiography, and histopathological examinations.

Results

Increases in the heart rate and decreases in PQ interval compared to a normal littermate were detected in two littermate CXMD_J dogs at 15 months of age or older. Distinct deep Q-waves and increase in Q/R ratios in leads II, III, and aVF were detected by 6–7 months of age in all CXMD_J dogs. In the echocardiogram, one of eight of CXMD_J dogs showed a hyperechoic lesion in the left ventricular posterior wall at 5 months of age, but the rest had not by 6–7 months of age. The left ventricular function in the echocardiogram indicated no abnormality in all CXMD_J dogs by 6–7 months of age. Histopathology revealed myocardial fibrosis, especially in the left ventricular posterobasal wall, in three of eight CXMD_J dogs by 21 months of age.

Conclusion

Cardiac involvement in CXMD_J dogs is milder and has slower progression than that described in GRMD dogs. The distinct deep Q-waves have been ascribed to myocardial fibrosis in the posterobasal region of the left ventricle, but our data showed that they precede the lesion on echocardiogram and histopathology. These findings imply that studies of CXMD_J may reveal not only another causative mechanism of the deep Q-waves but also more information on the pathogenesis in the dystrophin-deficient heart.     

Roles of cADPR and NAADP in pancreatic cells

Cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) are Ca(2+)-mobilizing nucleotides that were discovered in the late 1980s. Two decades of investigations have built up a considerable understanding about these two molecules that are related because both are derived from pyridine nucleotides and known to be generated by CD38/ADP-ribosyl cyclases. cADPR has been shown to target the ryanodine receptors in the endoplasmic reticulum whereas NAADP stimulates the two-pore channels in the endo-lysosomes. Accumulating results indicate that cADPR and NAADP are second messenger molecules mediating Ca(2+) signaling activated by a wide range of agonists. This article reviews what is known about these two molecules, especially regarding their signaling roles in the pancreatic cells.     

Structural studies of intermediates along the cyclization pathway of Aplysia ADP-ribosyl cyclase

Cyclic ADP-ribose (cADPR) is a calcium messenger that can mobilize intracellular Ca²⁺ stores and activate Ca²⁺ influx to regulate a wide range of physiological processes. Aplysia cyclase is the first member of the ADP-ribosyl cyclases identified to catalyze the cyclization of NAD⁺ into cADPR. The catalysis involves a two-step reaction, the elimination of the nicotinamide ring and the cyclization of the intermediate resulting in the covalent attachment of the purine ring to the terminal ribose. Aplysia cyclase exhibits a high degree of leniency towards the purine base of its substrate, and the cyclization reaction takes place at either the N1- or the N7-position of the purine ring. To decipher the mechanism of cyclization in Aplysia cyclase, we used a crystallization setup with multiple Aplysia cyclase molecules present in the asymmetric unit. With the use of natural substrates and analogs, not only were we able to capture multiple snapshots during enzyme catalysis resulting in either N1 or N7 linkage of the purine ring to the terminal ribose, we were also able to observe, for the first time, the cyclized products of both N1 and N7 cyclization bound in the active site of Aplysia cyclase.