γ-Cyclodextrin–polyurethane copolymer adsorbent for selective removal of endotoxin from DNA solution

Copolymer particles for removal of endotoxins (lipopolysaccharides, LPSs) were prepared by suspension copolymerization of γ-cyclodextrin (CyD) and 1,6-hexamethylenediisocyanate. The LPS-removing activity of the copolymer particles was compared with that of poly(ε-lysine)-immobilized Cellufine (cationic adsorbent) or polystyrene particles (hydrophobic adsorbent) by a batch method. When DNA was present in solution with LPSs under physiological conditions (pH 6.0, ionic strength of μ = 0.05–0.8), LPS-removing activity of the cationic or hydrophobic adsorbent was unsatisfactory because both the DNA and the LPSs were adsorbed onto each adsorbent. By contrast, the copolymer particles with γ-CyD cavity (CyD content: 14–20 mol%) could selectively remove LPSs from a DNA solution (50 μg ml⁻¹, pH 6.0, and μ = 0.05–0.2) containing LPSs (15 EU ml⁻¹) without the adsorption of DNA. The residual concentration of LPSs in the treated DNA solution was below 0.1 EU ml⁻¹, and the recovery of DNA was 99%.     

Prion infection correlates with hypersensitivity of P2X7 nucleotide receptor in a mouse microglial cell line

We recently established mouse microglial cells persistently infected with mouse-adapted scrapie ME7 (ScMG20/ME7) for in vitro study of prion pathogenesis. Here, we found that ScMG20/ME7 cells were hypersensitive to P2X7 receptor agonists, as demonstrated by sustained Ca(2+) influx, membrane pore formation, cell death, and interleukin-1beta release. P2X7 mRNA expression was upregulated in these cells, and also in scrapie-infected mice brains. Treatment with pentosan polysulfate eliminated the infectivity and disease-related forms of prion protein from ScMG20/ME7 cell cultures, however, hypersensitivity of P2X7 receptors remained. These results suggest that prion infections may strongly affect the P2X7 receptor system in mouse microglial cells.     

alpha-Synuclein stimulates differentiation of osteosarcoma cells: relevance to down-regulation of proteasome activity

Because a limited study previously showed that alpha-synuclein (alpha-syn), the major pathogenic protein for Parkinson disease, was expressed in differentiating brain tumors as well as various peripheral cancers, the main objective of the present study was to determine whether alpha-syn might be involved in the regulation of tumor differentiation. For this purpose, alpha-syn and its non-amyloidogenic homologue beta-syn were stably transfected to human osteosarcoma MG63 cell line. Compared with beta-syn-overexpressing and vector-transfected cells, alpha-syn-overexpressing cells exhibited distinct features of differentiated osteoblastic phenotype, as shown by up-regulation of alkaline phosphatase and osteocalcin as well as inductive matrix mineralization. Further studies revealed that proteasome activity was significantly decreased in alpha-syn-overexpressing cells compared with other cell types, consistent with the fact that proteasome inhibitors stimulate differentiation of various osteoblastic cells. In alpha-syn-overexpressing cells, protein kinase C (PKC) activity was significantly decreased, and reactivation of PKC by phorbol ester significantly restored the proteasome activity and abrogated cellular differentiation. Moreover, activity of lysosome was up-regulated in alpha-syn-overexpressing cells, and treatment of these cells with autophagy-lysosomal inhibitors resulted in a decrease of proteasome activity associated with up-regulation of alpha-syn expression, leading to enhance cellular differentiation. Taken together, these results suggest that the stimulatory effect of alpha-syn on tumor differentiation may be attributed to down-regulation of proteasome, which is further modulated by alterations of various factors, such as protein kinase C signaling pathway and a autophagy-lysosomal degradation system. Thus, the mechanism of alpha-syn regulation of tumor differentiation and neuropathological effects of alpha-syn may considerably overlap with each other.     

Effect of n-3 fatty acids on serum lipid levels and hepatic fatty acid metabolism in BALB/c.KOR-Apoeshl mice deficient in apolipoprotein E expression

N-3 fatty acids exert a potent serum lipid-lowering effect in rodents mainly by affecting hepatic fatty acid oxidation and synthesis. However, it has been observed that fish oil and docosahexaenoic acid ethyl ester do not lower serum lipid levels in apolipoprotein E (apoE)-knockout (Apoetm1Unc) mice generated by gene targeting. To test the hypothesis that apoE expression is required for n-3 fatty acid-dependent regulation of serum lipid levels and hepatic fatty acid metabolism, we examined the effect of fish oil and n-3 fatty acid ethyl esters on the activity and gene expression of hepatic enzymes involved in fatty acid oxidation and synthesis using an alternative apoE-deficient mouse model with the BALB/c genetic background (BALB/c.KOR-Apoeshl). ApoE-deficient mice were fed diets containing 9.4% palm oil, fish oil, or 5.4% palm oil and 1% EPA plus 3% DHA ethyl esters for 15 days. In contrast to the reported data on apoE-knockout mice, fish oil and n-3 fatty acid ethyl esters greatly decreased serum triacylglycerol, cholesterol, and phospholipid levels in the Apoeshl mice. The decreases were greater with fish oil than with ethyl esters. The alterations by dietary n-3 fatty acids of serum lipid levels were accompanied by parallel changes in the activity and mRNA levels of enzymes involved in hepatic fatty acid oxidation and synthesis. The reason for the discrepancy between the results of the current study and previous studies is unknown. However, our study at least indicates that a lack of apoE expression does not necessarily accompany deficits in the n-3 fatty acid-dependent regulation of serum lipid levels and hepatic fatty acid metabolism.     

Imprinted polymer layer for recognizing double-stranded DNA

A method of preparing a thin polymer layer able to recognize double-stranded DNA (dsDNA) was developed by using 2-vinyl-4,6-diamino-1,3,5-triazine (VDAT) as a functional monomer for creating a DNA-imprinted polymer. The formation of hydrogen bonds between VDAT and A-T base pairs in dsDNA was confirmed by measuring the effects of VDAT on the melting point and the NMR and CD spectra of dsDNA. An imprinted polymer that can recognize dsDNA of the verotoxin gene was prepared by polymerizing VDAT, acrylamide, a crosslinking agent, and the template verotoxin dsDNA on a silanized glass surface. The specificity of this polymer layer for binding verotoxin dsDNA was investigated by using fluorescent-labelled dsDNAs. The fluorescence intensity of the polymer layer after binding verotoxin dsDNA was twice as high as after binding oligo(dG)-oligo(dC), indicating that verotoxin dsDNA was preferentially bound to the polymer imprinted with verotoxin dsDNA. The kinetics of verotoxin dsDNA binding to the imprinted polymer were analyzed by surface plasmon resonance measurements. The dissociation constant (KD) was low, of the order of 10(-9)M.     

Allelic characterization of the leaf-variegated mutation <Emphasis Type="Italic">var2</Emphasis> identifies the conserved amino acid residues of FtsH that are important for ATP hydrolysis and proteolysis

Arabidopsis var1 and var2 mutants exhibit leaf variegation. VAR1 and VAR2 encode similar FtsH metalloproteases (FtsH5 and FtsH2, respectively). We have previously found many variegated mutants to be allelic to var2. Each mutant was shown to express a different degree of variegation, and the formation of white sectors was enhanced in severely variegated alleles when these alleles were grown at low temperature. VAR1/FtsH5 and VAR2/FtsH2 levels were mutually affected even in the weak alleles, confirming our previous observation that the two proteins form a hetero complex. In this study, the sites of the mutations in these var2 alleles were determined. We isolated eight point mutations. Five alleles resulted in an amino acid substitution. Three of the five amino acid substitutions occurred in Walker A and B motifs of the ATP-binding site, and one occurred in the central pore motif. These mutations were considered to profoundly suppress the ATPase and protease activities. In contrast, one mutation was found in a region that contained no obvious signature motifs, but a neighboring sequence, Gly–Ala–Asp, was highly conserved among the members of the AAA protein family. Site-directed mutagenesis of the corresponding residue in E. coli FtsH indeed showed that this residue is necessary for proper ATP hydrolysis and proteolysis. Based on these results, we propose that the conserved Gly–Ala–Asp motif plays an important role in FtsH activity. Thus, characterization of the var2 alleles could help to identify the physiologically important domain of FtsH.     

Effects of prolonged delivery of brain-derived neurotrophic factor on the fate of neural stem cells transplanted into the developing rat retina

One of the major roles of brain-derived neurotrophic factor (BDNF) is to promote the differentiation and support the survival of neurons in the central nervous system. The objective of the present study was to evaluate the effect of BDNF on the fate of adult rat hippocampus-derived neural stem cells (AHPCs) transplanted into the developing rat retina. Immunohistochemical analysis showed a significant increase in the ratio of grafted AHPCs stained for MAP2ab (P<0.05) and a marked decrease in the ratio of nestin-positive grafted cells in the slow-releasing BDNF group compared with the control group. The respective changes in the ratios of MAP5 and GFAP-positive grafted cells were comparable for the two groups. The results reported here suggest a potentially beneficial role for extended delivery of BDNF in the differentiation of grafted neural stem cells, which may lead to a novel modification of stem cell transplantation.     

Crystal structure of chartreusin derivative A132

The crystal structure of chartreusin derivative A132 (benzilidene chartreusin) has been determined by single-crystal X-ray diffraction. The space group is C2 with unit cell dimensions, a=18.482(4), b=8.749(3), c=43.906(2) A, beta=94.87(2) degrees, and the structure was refined to R-factors of 0.2365 (6585 all unique reflections) and 0.087 (2914 reflections with F(o)>4 sigma(F(o))) by a full-matrix least-squares method. There are two molecules in an asymmetric unit. Both molecules have similar structures, which are favorable to bind with DNA in the minor groove. A modeling study of the A132-DNA complex based on the X-ray structures suggests that the sugar moiety of A132 may play an important role in recognizing the sequence of DNA base pairs.