Affinity selection of target cells from cell surface displayed libraries: a novel procedure using thermo-responsive magnetic nanoparticles

Biotinylated thermo-responsive magnetic nanoparticles for use in affinity selection from yeast cell surface display libraries were prepared by coating magnetite nanoparticles with a thermo-responsive polymer consisting of N-isopropyl acrylamide and a biotin derivative. These particles showed a reversible transition between flocculation and dispersion at around the lower critical solution temperature of 30 degrees C, above which the flocculated particles--which absorbed a large amount of avidin due to their large surface area--were quickly separable by magnet. The model library was constructed by mixing control yeast cells with target yeast cells co-displaying IgG binding protein (ZZ) and enhanced green fluorescence protein. Biotinylated IgG and avidin were subsequently added to the model library, and target cells were efficiently enriched with the biotinylated magnetic nanoparticles by avidin-biotin sandwich and ZZ-IgG interaction. The few target cells (0.001%) in the model library were enriched by up to 100% in only 5 days by an affinity selection procedure repeated four times. This novel method based on magnetic nanoparticles and a yeast cell surface display system could fulfill a wide range of applications in the analysis of protein-protein interactions and rapid isolation of novel biomolecules.     

Cerebrosides in grapevine leaves: distinct composition of sphingoid bases among the grapevine species having different tolerances to freezing temperature

Cerebrosides from leaves of three grapevine species were analyzed in detail. The relative proportions of 8-E/Z isomers of 4-hydroxy-8-sphingenines [i.e. 8-E/Z t18:1(8E) and (8Z)] differed amongst the species in respect to freezing tolerance. This suggests that the occurrence of high levels of t18:1(8Z) in cerebrosides is correlated with freezing tolerance in these species.     

Overexpression of salicylate hydroxylase and the crucial role of lys(163) as its NADH binding site

Expression systems for the sal gene encoding salicylate hydroxylase from Pseudomonas putida S-1 were examined and some constructs were expressed in these systems. By cultivation of Escherichia coli BL21 (DE3)/pSAH8 in LB medium at 37 degrees C with isopropyl-b-D-thiogalactopyranoside as the inducer, salicylate hydroxylase was overexpressed mainly in the form of inclusion bodies. Lower temperature cultivation at 20 degrees C after induction resulted in a large amount of the enzyme in the soluble form. The E. coli clone harboring the recombinant plasmid produced a 45 kDa protein that appeared to be electrophoretically and immunochemically identical to the P. putida enzyme and contained the same N-terminal amino acid sequence. This recombinant DNA product also exhibited properties characteristic of a flavoprotein and was fully functional as salicylate hydroxylase. Based on chemical modification of the salicylate hydroxylase from P. putida, Lys163 was previously proposed to be the NADH binding site. In this study, to obtain a better understanding of the predicted role of Lys163, this residue in the active center of salicylate hydroxylase was replaced with Arg, Gly, or Glu by conventional site-directed mutagenesis. Kinetic studies using these mutant enzymes and the recombinant enzyme revealed increases in apparent K(m) values for NADH in the order of wild-type enzyme > K163R > K163G > K163E, with some decreases in V(max). Examination of the recombinant enzyme and K163G indicated that the pH dependency of K(m) on NADH with pK(a) 10.5 is lost by mutation despite the lack of changes in V(max) values, suggesting a requirement for the lysine residue as the NADH binding site. Based on these results, Lys163 is proposed to play a role in the binding of NADH at the active site through an ionic bond rather than playing a role in catalysis.     

Intra- and interspecific phylogeny of wild Fagopyrum (Polygonaceae) species based on nucleotide sequences of noncoding regions in chloroplast DNA

The intra- and interspecific phylogeny of Fagopyrum (Polygonaceae) species was studied using nucleotide sequence data from two noncoding regions in chloroplast DNA, the trnK (UUU) intron and the trnC (GCA)-rpoB spacer. Thirty-seven accessions of ten species and two unidentified samples in the urophyllum group of Fagopyrum were analyzed. Both of the studied regions showed high variability, including nucleotide substitutions, insertion/deletions, and inversions. Separate parsimony analyses of the two regions generated phylogenies that were largely consistent with each other. A single most parsimonious tree derived from the combined data of the two regions suggested that (1) either F. statice or F. leptopodum was derived from the ancestor more than once, (2) F. gracilipes, a tetraploid species, has recently been derived from diploid ancestor and rapidly spread out to its present distribution areas, and (3) F. pleioramosum, F. macrocarpum, and F. callianthum, three newly discovered species endemic to the upper Min River valley, differentiated from their common ancestral species in the present distribution area.     

Synthesis of a novel asparagine-linked heptasaccharide structure via p-methoxybenzyl-assisted beta-mannosylation

Synthesis of a core heptasaccharide asparagine N4-[alpha-D-mannopyranosyl-(1 --> 6)-[(alpha-D-mannopyranosyl)-(1 --> 3)]-[(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-(1 --> 2)]-(beta-D-mannopyranosyl)-(1 --> 4)-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-(1 --> 4)-[(alpha-L-fucopyranosyl)-(1 --> 6)]-2-acetamido-2-deoxy-beta-D-glucopyranosyl]-L-asparagine (1a) found from CHO glycosylation mutant cell LEC 14 is described. The structure of 1a is highly novel in terms of the presence of an extra GlcNAc residue linked to the 2-position of beta-linked mannose. The synthesis was performed using p-methoxybenzyl-assisted intramolecular aglycon delivery as the key transformation. 4,6-O-TIDPS-protected thiomannoside methyl 2-O-p-methoxybenzyl-4,6-O-(1,1,3,3-tetraisopropyl)disiloxanylid ene-3-O-trimethylsilyl-1-thio-alpha-D-mannopyranoside was adopted for this particular purpose, which afforded beta-mannoside p-methoxyphenyl 2,3-O-(p-methoxybenzylidene)-4,6-O-(1,1,3,3-tetraisopropyl)+ ++disiloxanylidene-beta-D-mannopyranosyl-(1 --> 4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranoside stereoselectively in 75% yield.     

Analysis of deletion mutations of the rpsL gene in the yeast Saccharomyces cerevisiae detected after long-term flight on the Russian space station Mir

Using the yeast Saccharomyces cerevisiae on board the Russian space station Mir, we studied the effects of long-term space flight on mutation of the bacterial ribosomal protein L gene (rpsL) cloned in a yeast-Escherichia coli shuttle vector. The mutation frequencies of the cloned rpsL gene on the Mir and the ground (control) yeast samples were estimated by transformation of E. coli with the plasmid DNAs recovered from yeast and by assessment of the conversion of the rpsL wild-type phenotype (Sm(S)) to its mutant phenotype (Sm(R)). After a 40-day space flight, some part of space samples gave mutation frequencies two to three times higher than those of the ground samples. Nucleotide sequence analysis showed no apparent difference in point mutation rates between the space and the ground mutant samples. However, the greater part of the Mir mutant samples were found to have a total or large deletion in the rpsL sequence, suggesting that space radiation containing high-linear energy transfer (LET) might have caused deletion-type mutations.     

Fabrication of a new substrate for atomic force microscopic observation of DNA molecules from an ultrasmooth sapphire plate.

A new stable substrate applicable to the observation of DNA molecules by atomic force microscopy (AFM) was fabricated from a ultrasmooth sapphire (alpha-Al2O3 single crystal) plate. The atomically ultrasmooth sapphire as obtained by high-temperature annealing has hydrophobic surfaces and could not be used for the AFM observation of DNA. However, sapphire treated with Na3PO4 aqueous solution exhibited a hydrophilic character while maintaining a smooth surface structure. The surface of the wet-treated sapphire was found by x-ray photoelectron spectroscopy and AFM to be approximately 0.3 nm. The hydrophilic surface character of the ultrasmooth sapphire plate made it easy for DNA molecules to adhere to the plate. Circular molecules of the plasmid DNA could be imaged by AFM on the hydrophilic ultrasmooth sapphire plate.     

Vesicular stomatitis virus binds and fuses with phospholipid domain in target cell membranes

Fusion of vesicular stomatitis virus with some cells (HELR 66, KB, and human erythrocytes, both intact and trypsinized) and liposomes made of various natural and synthetic lipids was studied with spin-labeled phospholipid. Binding of virus was assayed separately with radiolabeled and spin-labeled virus. Binding to cells and liposomes was small at neutral pH but enhanced at acidic pHs. Fusion with cells and liposomes was negligibly small at neutral pH but greatly activated at acidic pHs lower than 6.5. Activation of fusion occurred at lower pH values than enhancement of binding. Fusion occurred rapidly and efficiently, reaching a plateau at 50-80% after 3 min at 37 degrees C. Binding and fusion with cells were enhanced by pretreatment of cells with trypsin. Binding to liposomes was dependent on the head group of the phospholipid, stronger to phosphatidylserine than to phosphatidylcholine, but not much dependent on the acyl chain composition. On the other hand, cis-unsaturated acyl chains were required for the efficient fusion, but there was only a small, if any, requirement for the head group. Cholesterol enhanced the fusion further. High fusion efficiency with cis-unsaturated phospholipids cannot be ascribed to the membrane fluidity but may be related to higher tail-to-head volume ratios. Possible mode of interaction of viral G glycoprotein with phospholipid is discussed. The virus cell entry mechanism is suggested as binding to the phospholipid domain in the cell surface membranes, endocytosis, and followed by fusion with the phospholipid domain in endosomes upon acidification.     

Gene expression profiles of endothelial progenitor cells by oligonucleotide microarray analysis

Among the many tissue stem or progenitor cells recently being unveiled, endothelial progenitor cells (EPCs) have attracted particular attention, not only because of their cardinal role in vascular biology and embryology but also because of their potential use in the therapeutic development of a variety of postnatal diseases, including cardiovascular and peripheral vascular disorders and cancer. The aim of this study is to provide some basic and comprehensive information on gene expression of EPCs to characterize the cells in molecular terms. Here, we focus on EPCs derived from CD34-positive mononuclear cells of human umbilical cord blood. The EPCs were purified and expanded in culture and analyzed by a high-density oligonucleotide microarray and real-time RT-PCR analysis. We identified 169 up-regulated and 107 down-regulated genes in the EPCs compared with three differentiated endothelial cells of human umbilical vein endothelial cells (HUVEC), human lung microvascular endothelial cells (LMEC) and human aortic endothelial cells (AoEC). It is expected that the obtained list include key genes which are critical for EPC function and survival and thus potential targets of EPC recognition in vivo and therapeutic modulation of vasculogenesis in cancer as well as other diseases, in which de novo vasculogenesis plays a crucial role. For instance, the list includes Syk and galectin-3, which encode protein tyrosine kinase and β-galactoside-binding protein, respectively, and are expressed higher in EPCs than the three control endothelial cells. In situ hybridization showed that the genes were expressed in isolated cells in the fetal liver at E11.5 and E14.5 of mouse development.