Escherichia coli LPS-induced LV dysfunction: role of toll-like receptor-4 in the adult heart

The precise molecular mechanisms responsible for sepsis-induced myocardial dysfunction remain undefined. Toll-like receptor-4 (TLR-4) engages lipopolysaccharide (LPS) and activates signaling pathways leading to the expression of proinflammatory cytokines implicated in myocardial dysfunction. We determined whether TLR-4 was necessary for LPS-induced myocardial dysfunction in vivo. The effects of LPS on left ventricular (LV) function were studied in mice with defective TLR-4 signaling (C3H/HeJ, TLR-4 deficient) and wild-type mice (C3HeB/FeJ). Mice (n = 5/group) were injected with LPS or diluent, and LV function was examined by using two-dimensional echocardiography and conductance catheters. LPS significantly decreased all indexes of LV function in wild-type mice when compared with controls; LV function was not depressed in the LPS-treated TLR-4-deficient mice relative to controls. LPS increased myocardial nitric oxide synthase-2 expression and cGMP only in wild-type mice. This study suggests that TLR-4 mediates the LV dysfunction that occurs in LPS-induced shock. Therefore, TLR-4 might be a therapeutic target for attenuating the effects of LPS on the heart.     

Three-dimensional structure of the inclusion complex between phloridzin and beta-cyclodextrin

The inclusion of phloridzin into beta-cyclodextrin was studied as a model of molecular recognition in membranes. Effects on 1H NMR spectra and NOE correlational peaks between phloridzin and beta-cyclodextrin were observed in the complex. Strong NOEs were observed between hydrogens of a phenol group in phloridzin and beta-cyclodextrin. The three-dimensional structure of the inclusion complex between phloridzin and beta-cyclodextrin was simulated with distance constraints estimated by the intensity of NOE peaks using the DADAS90 programs. Two inclusion possibilities were suggested-the large rim of beta-cyclodextrin as an entrance of the inclusion and the small rim of beta-cyclodextrin as the entrance. In both cases, the phenol group of phloridzin was included in the hydrophobic space of beta-cyclodextrin.     

The murine macrophage apoB-48 receptor gene (Apob-48r): homology to the human receptor

Previously we cloned the human macrophage apolipoprotein B-48 receptor (ApoB-48R) and documented its expression in human atherosclerotic foam cells (1). Now we have identified and characterized the murine macrophage apob-48r cDNA gene sequence and its chromosomal location. The cDNA (3,615 bp) -deduced amino acid (aa) sequence (942 aa) is approximately 45% identical to the human macrophage APOB-48R, but not to other known gene families. The murine Apob-48r gene, like the human APOB-48R gene, consists of four exons interrupted by three small introns and is syntenically located on chromosome 7. Functionally significant conserved domains include an N-terminal hydrophobic domain, a glycosaminoglycan attachment site, an N-glycosylation site, and an ExxxLL internalization motif C-terminal to the putative internal transmembrane domain. Two conserved coiled-coil domains are likely involved in the spontaneous homodimerization that generates the active dimeric ligand binding species (mouse, approximately 190 kDa; human, approximately 200 kDa). Transfection of the murine apoB-48R into Chinese hamster ovary cells (CHOs) confers apoB-48R function: rapid, high-affinity, specific uptake of known triglyceride-rich lipoprotein ligands of the apoB-48R and, of note, uptake of the cholesteryl ester-rich apoB-48-containing very low density lipoproteins that accumulate in atherosclerosis-prone apoE-deficient mice. Uptake of these ligands by murine apoB-48R-transfected CHOs causes saturable, visible cellular triglyceride and cholesterol accumulation in vitro that resemble foam cells of atherosclerotic lesions. In aggregate, the data presented here and that previously published suggest that the apoE-independent murine apoB-48R pathway may contribute to the spontaneous development of atherosclerotic lesions rich in macrophage-derived foam cells observed in apoE-deficient mice, a murine model of human atherosclerosis.     

An efficient ligation method in the making of an in vitro virus for in vitro protein evolution

The “in vitro virus” is a molecular construct to perform evolutionary protein engineering. The “virion(=viral particle)”(mRNA-peptide fusion), is made by bonding a nascent protein with its coding mRNA via puromycin in a test tube for in vitro translation. In this work, the puromycin-linker was attached to mRNA using the Y-ligation, which was a method of two single-strands ligation at the end of a double-stranded stem to make a stem-loop structure. This reaction gave a yield of about 95%. We compared the Y-ligation with two other ligation reactions and showed that the Y-ligation gave the best productivity. An efficient amplification of the in vitro virus with this “viral genome” was demonstrated. Published: October 28, 2002     

Normal myocardial function in severe right ventricular volume overload hypertrophy

Severe left ventricular volume overloading causes myocardial and cellular contractile dysfunction. Whether this is also true for severe right ventricular volume overloading was unknown. We therefore created severe tricuspid regurgitation percutaneously in seven dogs and then observed them for 3.5-4.0 yr. All five surviving operated dogs had severe tricuspid regurgitation and right heart failure, including massive ascites, but they did not have left heart failure. Right ventricular cardiocytes were isolated from these and from normal dogs, and sarcomere mechanics were assessed via laser diffraction. Right ventricular cardiocytes from the tricuspid regurgitation dogs were 20% longer than control cells, but neither the extent (0.171 +/- 0.005 microm) nor the velocity (2.92 +/- 0.12 microm/s) of sarcomere shortening differed from controls (0.179 +/- 0.005 microm and 3.09 +/- 0.11 microm/s, respectively). Thus, despite massive tricuspid regurgitation causing overt right heart failure, intrinsic right ventricular contractile function was normal. This finding for the severely volume-overloaded right ventricle stands in distinct contrast to our finding for the left ventricle severely volume overloaded by mitral regurgitation, wherein intrinsic contractile function is depressed.     

Studies on the 1,1-diphenyl-2-picrylhydrazyl radical scavenging mechanism for a 2-pyrone compound

The radical scavenging mechanisms for the 2-pyrone compound, 4-hydroxy-3,6-dimethyl-2H-pyrane-2-one (1), and the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical (4) in several solvent systems were evaluated by the quantitative change in compounds detected at 270 nm and subsequent HPLC analyses. The HPLC profile for each condition suggested that the reaction proceeded by a different mechanism in each solvent system. In organic solvents (CHCl3, iso-propanol, and EtOH), 1-[4-(3,4-dihydro-3,6-dimethyl-2,4-dioxo-2H-pyran-3-yl) phenyl]-1-phenyl-2-picrylhydrazine (2) was produced as an adduct of the DPPH radical and 1. On the other hand, the reaction in a buffer solution (an acetate buffer at pH 5.5) gave several degradation products with 1[4-(2,3-dihydro-2,5-dimethyl-3-oxo-fur-2-yl) phenyl]-1-phenyl-2-picrylhydrazine (5), this being structurally elucidated by spectroscopic analyses. The decrease of the DPPH radical in each reaction system suggests that compound 1 could scavenge about 1.5-1.8 equivalents of the radical in organic solvents and about 3.5-3.9 in the buffer solution.