A new assay using surface plasmon resonance (SPR) to determine binding of the Lactobacillus acidophilus group to human colonic mucin

A new binding assay to investigate the mechanism of adhesion of lactic acid bacteria to the human intestine was established by the surface plasmon resonance technique using a biosensor BIACORE1000. Cells of 26 strains of the Lactobacillus acidophilus group as analytes were eluted onto a sensor chip on which were immobilized biotinylated A-trisaccharide polymer probes having human A-type antigen [(GalNAcalpha1-3(Fucalpha1-2)Gal)-] or human colonic mucin of blood type A (HCM-A) as ligands. In the first screening, high adhesive affinity to the A-trisaccharide BP-probe was observed in L. acidophilus OLL2769, L. crispatus JCM8778, LA205 and LA206. In the second screening, which used HCM-A, only L. acidophilus OLL2769 and L. crispatus JCM8778 were selected as adhesive strains with specific binding ability to human A-antigen. The results indicated that some strains of the L. acidophilus group could recognize and bind the sugar chain of A-antigen structure on HCM.     

Maillard reaction rate in various glassy matrices

The Maillard Reaction (MR) rate below the glass transition temperature (T(g)) for various model glassy food systems was studied at temperatures between 40 degrees C and 70 degrees C. As a sample, freeze-dried glucose and lysine systems embedded in various glassy matrices (e.g., polyvinylpyrrolodone and trehalose) were used, and the MR rate below the T(g) was compared among the various glassy matrices. The extent of MR was estimated spectrophotometrically from the optical density at 280 nm (OD(280)), and the MR rate (k(280)) was determined as a pseudo zero order reaction rate from the time course of OD(280). Although k(280) was described by the Arrhenius plot, the temperature dependence of k(280) was almost the same and the intercept was different among the matrices. From the comparison of k(280), it was suggested that the MR rate in glassy matrix was affected not only by the T(g), but also by the hydrogen bonding between MR reactants and glassy matrix.     

Formation of N-(hexanoyl)ethanolamine, a novel phosphatidylethanolamine adduct, during the oxidation of erythrocyte membrane and low-density lipoprotein

The primary amino groups of biomolecules such as aminophospholipids, as well as proteins, are the potential targets of covalent modifications by lipid peroxidation products; however, little attention has been paid to the modification of aminophospholipids such as phosphatidylethanolamine (PE). The purpose of this study is to characterize the formation of a novel modified phospholipid, N-(hexanoyl)phosphatidylethanolamine (HEPE), in the reaction of PE with lipid hydroperoxides using mass spectrometric analyses. Upon reaction of egg PE with 13-hydroperoxyoctadecadienoic acid or other oxidized polyunsaturated fatty acids followed by phospholipase D-mediated hydrolysis, the formation of N-(hexanoyl)ethanolamine (HEEA), a head group of HEPE, was confirmed by isotope dilution liquid chromatography/tandem mass spectrometry. Moreover, increasing HEEA was detected in the hydrolysates of oxidized erythrocyte ghosts and low-density lipoprotein with their increasing lipid peroxidation levels. Collectively, these results suggest that the N-hexanoylated product of phospholipid, HEPE, can be generated during lipid peroxidation and may serve as one mechanism for the covalent modification of aminophospholipids in vivo.     

Effects of specific signal transduction inhibitors on increased permeability across rat endothelial monolayers induced by neuropeptide Y or VEGF

Neuropeptide Y (NPY) elevates the permeability of cultured rat aortic endothelial cells (RAECs) in monolayer cultures under hypoxic conditions (5% O(2)) possibly by binding to the NPY Y(3) receptor. The present study evaluated the effects of NPY compared to vascular endothelial growth factor (VEGF). RAECs were cultured on the upper chamber base of a double-chamber culture system, FITC-labeled albumin was introduced into the chamber, and permeation into the lower chamber was measured. Treatment was with 3 x 10(-7) M NPY or 10(-7) g/ml VEGF for 2 h along with specific inhibitors. The VEGF receptor-2 tyrosine kinase inhibitor tyrphostin SU-1498 and the protein kinase C inhibitor bis-indolylmaleimide I (GF-109203X) suppressed the VEGF-induced increase in monolayer permeability but not that caused by NPY. Furthermore, although the action of NPY was blocked in a concentration-dependent manner by phospholipase C inhibitor 1-(6-[[(17beta)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl)-1H-pyrrole-2,5-dione (U-73122), it was less sensitive than VEGF. However, the effects of both NPY and VEGF on the permeability of the RAEC monolayer were blocked with equal concentration dependence by STI571 (imatinib mesylate), which is an inhibitor of Abl tyrosine kinase in the nucleus and/or cytoplasm. The myosin light-chain kinase inhibitor 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine HCl (ML-9) suppressed both NPY- and VEGF-induced increment in permeability by approximately 70%, whereas the calmodulin-dependent kinase inhibitor DY-9760e could decrease to below the baseline. These results indicate that the NPY Y(3)-receptor subtype is specifically linked to the effects of STI571 on endothelial cells, and that NPY, a sympathetic coneurotransmitter, may increase vascular permeability in association with altered intracellular or nuclear signal transduction.     

Association of rabies virus nominal phosphoprotein (P) with viral nucleocapsid (NC) is enhanced by phosphorylation of the viral nucleoprotein (N)

We investigated possible role(s) of N protein phosphorylation in the rabies virus replication process. A large amount of P proteins are associated with the viral nucleocapsid (NC) in the infected cell, the amount which was greatly decreased by phosphatase-treatment of the isolated NC, indicating that the phosphate group of N and/or P proteins is essential for their stable association with the NC. Immunoprecipitation studies were performed on the coexpressed normal N or phosphorylation deficient N(S389A) and P proteins, demonstrating that the P protein associated with phosphorylation-deficient NC-like structures was much less in amount than that associated with the wild type NC. Similar results were also obtained with a mutant P protein, PDeltaN19, which lacked the N-terminal 19 amino acids and was capable of binding to the NC-like structures but incapable of forming the RNA-free N-P complexes. Immunoprecipitation studies with mAb #402-13 further suggested that the NC-specific linear 402-13 epitope was exposed even on the P proteins which were associated with the phosphorylation-deficient NC-like structures, but such association was very weak as demonstrated by greatly decreased amounts of coprecipitated NC-like structures. From these results, we assume that the phosphorylation of N protein enhances the association between the 402-13 epitope-positive P protein and the NC probably by stabilizing such P-NC binding.     

Arterial anatomical features of the upper palpebra

The arterial anatomical features of the upper palpebra were examined in both sides of seven fresh cadavers that had been systemically injected with a lead oxide/gelatin mixture. All specimens were stereoscopically radiographed for analysis of the three-dimensional structure of the arteries and were macroscopically dissected for observation of the relationships between the arteries and the other tissues. Cross-sections were prepared from one specimen and examined histologically. In all cases, there were four arterial arcades in the upper palpebra, namely, the marginal, peripheral, superficial orbital, and deep orbital arcades. Each arcade provided small vertical branches. The vertical branches coursed on both sides of the orbicularis oculi muscle and on both sides of the tarsal plate. From these small vertical branches, fine vessels branched off to the skin, muscle, and tarsal plate. These findings are important for avoiding complications such as bleeding and are useful for designing local flaps, such as switch flaps, for reconstructive surgical procedures.     

Vascular anatomy of the anterolateral thigh flap

Arterial and venous anatomy and their relation to the anterolateral thigh flap were examined in 10 specimens of six fresh cadavers in which radiopaque materials were injected into both the arterial and venous systems. Territories and positions of individual perforating arteries were measured, and the venous drainage pathway of the flap was analyzed. All specimens were radiographed stereoscopically to observe the three-dimensional structure of the arteries and veins. The territory of each perforating artery was smaller than expected. Most of the venous blood that had perfused the dermis was considered to pool in a polygonal venous network located in the skin layer and to enter the descending branch of the lateral circumflex femoral artery through large descending veins. The venous territories were considered different from the arterial territories. The findings in this study suggest that the design of the anterolateral thigh flap should be based on the venous architecture rather than on the arterial architecture and that the flap survival rate might be improved if thinning is performed appropriately.     

Solution structure of the RWD domain of the mouse GCN2 protein

GCN2 is the alpha-subunit of the only translation initiation factor (eIF2alpha) kinase that appears in all eukaryotes. Its function requires an interaction with GCN1 via the domain at its N-terminus, which is termed the RWD domain after three major RWD-containing proteins: RING finger-containing proteins, WD-repeat-containing proteins, and yeast DEAD (DEXD)-like helicases. In this study, we determined the solution structure of the mouse GCN2 RWD domain using NMR spectroscopy. The structure forms an alpha + beta sandwich fold consisting of two layers: a four-stranded antiparallel beta-sheet, and three side-by-side alpha-helices, with an alphabetabetabetabetaalphaalpha topology. A characteristic YPXXXP motif, which always occurs in RWD domains, forms a stable loop including three consecutive beta-turns that overlap with each other by two residues (triple beta-turn). As putative binding sites with GCN1, a structure-based alignment allowed the identification of several surface residues in alpha-helix 3 that are characteristic of the GCN2 RWD domains. Despite the apparent absence of sequence similarity, the RWD structure significantly resembles that of ubiquitin-conjugating enzymes (E2s), with most of the structural differences in the region connecting beta-strand 4 and alpha-helix 3. The structural architecture, including the triple beta-turn, is fundamentally common among various RWD domains and E2s, but most of the surface residues on the structure vary. Thus, it appears that the RWD domain is a novel structural domain for protein-binding that plays specific roles in individual RWD-containing proteins.     

A selective inducible NOS dimerization inhibitor prevents systemic,cardiac, and pulmonary hemodynamic dysfunction in endotoxemic mice

Increased nitric oxide (NO) production by inducible NO synthase (NOS2), an obligate homodimer, is implicated in the cardiovascular sequelae of sepsis. We tested the ability of a highly selective NOS2 dimerization inhibitor (BBS-2) to prevent endotoxin-induced systemic hypotension, myocardial dysfunction, and impaired hypoxic pulmonary vasoconstriction (HPV) in mice. Mice were challenged with Escherichia coli endotoxin before treatment with BBS-2 or vehicle. Systemic blood pressure was measured before and 4 and 7 h after endotoxin challenge, and echocardiographic parameters of myocardial function were measured before and 7 h after endotoxin challenge. The pulmonary vasoconstrictor response to left mainstem bronchus occlusion, which is a measure of HPV, was studied 22 h after endotoxin challenge. BBS-2 treatment alone did not alter baseline hemodynamics. BBS-2 treatment blocked NOS2 dimerization and completely inhibited the endotoxin-induced increase of plasma nitrate and nitrite levels. Treatment with BBS-2 after endotoxin administration prevented systemic hypotension and attenuated myocardial dysfunction. BBS-2 also prevented endotoxin-induced impairment of HPV. In contrast, treatment with NG-nitro-l-arginine methyl ester, which is an inhibitor of all three NOS isoforms, prevented the systemic hypotension but further aggravated the myocardial dysfunction associated with endotoxin challenge. Treatment with BBS-2 prevented endotoxin from causing key features of cardiovascular dysfunction in endotoxemic mice. Selective inhibition of NOS2 dimerization with BBS-2, while sparing the activities of other NOS isoforms, may prove to be a useful treatment strategy in sepsis.