Effective CpG DNA delivery using amphiphilic cycloamylose nanogels.

Unmethylated CpG oligodeoxynucleotides induce inflammatory immune responses through cytokine production and have attracted increasing attention as an immunostimulator. However, there remains a challenging issue of the use of 'native CpG DNA'. In the present study, we prepared cationic nanometer-sized gels (nanogels) consisting of cycloamylose modified with cholesterol and diethylaminoethane to form hydrophobic cross-linking points and to add positively charged groups, respectively. The cationic nanogels and native CpG DNA formed nanometer-sized complexes. Complexes of native CpG DNA with cationic nanogels delivered native CpG DNA to macrophage-like cells and induced cytokine production. In addition, complexes of negative control oligonucleotides with cationic nanogels did not induce cytokine production, and the induction of cytokines using complexes of phosphorothioate-modified CpG with cationic nanogels was lower than that of native CpG DNA. These results suggest that the complex of native CpG DNA with cationic nanogels is a promising strategy for nucleic acid adjuvants.

Intimate partner violence in Sri Lanka.

To describe the current situation of intimate partner violence (IPV) in Sri Lanka, and to propose possible interventions to prevent IPV, we performed a literature survey for articles and reports on IPV in Sri Lanka. Our results suggested that prevalence of IPV is high (40%) in Sri Lanka. Most of the IPV studies were conducted in health care institutions and missed IPV victims who had not attended a health care institution. A common belief in Sri Lanka, even among medical students and police officers is that IPV is a personal matter that outsiders should not intervene. The laws against IPV identify the physical and psychological IPV, but not the sexual IPV. To improve this situation of IPV in Sri Lanka, we recommend IPV education programs for medical students and police officers, community awareness programs on IPV, and amending the laws to identify sexual IPV. We also recommend well designed community based research on IPV.

A novel cytokine-inducing glycolipid isolated from the lipoteichoic acid fraction of Enterococcus hirae ATCC 9790: a fundamental structure of the hydrophilic part.

Previously, we showed that quantitatively minor several glycolipids totaling only less than 5% of the lipoteichoic acid (LTA) fraction from Enterococcus hirae ATCC 9790 possessed cytokine-inducing activity, whereas the major component (over 90%) did not [Suda et al. (1995) FEMS Immun Med Microbiol 12:97-112]. The major inactive component was shown to have the chemical structure as was proposed for the LTA by Fischer [Hashimoto et al. (1997) J Biochem 121:779-86], suggesting that so-called LTA is not a cytokine-inducing component in the Gram-positive bacteria. In the present paper, the structure of the hydrophilic part of one of the cytokine-inducing glycolipid tentatively named GL4 is elucidated. GL4 was first subjected to hydrolysis with aqueous HF to give a polysaccharide and a mixture of low molecular weight products. The polysaccharide was composed mainly of highly branching mannan as concluded from NMR and MS analyses of its acetolysis products. The low molecular weight products consisted of phosphate and glycerol, suggesting the presence of a poly(glycerophosphate) structure in the original GL4. From these observations, the hydrophilic part of GL4 was shown to consist of mannose-rich polysaccharide and poly(glycerophosphate), the latter being bound to the former by a phosphodiester linkage.

Enzyme flexibility: a new concept in recognition of hydrophobic substrates.

The mechanism of recognition of hydrophobic substrates was investigated using Escherichia coli aspartate aminotransferase (AspAT), E. coli aromatic amino acid aminotransferase (AroAT), and their chimeric enzyme (DY18). Surprisingly, broad substrate specificity was observed in the reaction of aminotransferases with hydrophobic substrates. The catalytic efficiency increased with an increase in the side chain length of straight or branched-terminal aliphatic substrates. The straight-chain substrates catalysed with maximal efficiency were the 7-carbon substrate in the case of AspAT and the 8-carbon substrate for AroAT and DY18. Consecutive addition of single methylene groups to the substrate had a constant effect on the stabilization energy of the transition state relative to the unbound state. The dependency of binding energy on each methylene group is usually interpreted as indicating hydrophobicity of the active site. However, we observed that AroAT and DY18 had different dependencies although both enzymes have the same residues in the substrate-binding pocket. For substrates with more than 7 carbons, the aminotransferases did not strictly distinguish between substrates with straight and branched side chains. These results suggest that the recognition of manifold hydrophobic substrates of different shapes might require not only the hydrophobicity of the active site but also enzyme flexibility.

Structural feature of the major but not cytokine-inducing molecular species of lipoteichoic acid.

Previously, lipoteichoic acid (LTA) of Enterococcus hirae was found to exhibit definite cytokine-inducing activity but synthetic specimens which share the fundamental structural principles proposed for LTA had no corresponding activity. We also showed recently that several minor components totally less than 5% of the LTA fraction from E. hirae ATCC 9790 possessed the activity, whereas the major component (over 90%) did not [Suda, Y., Tochio, H., Kawano, K., Takada, H., Yoshida, T., Kotani, S., and Kusumoto, S. (1995) FEMS Immun. Med. Microbiol. 12, 97-112]. In the present study, the structure of the major component of LTA was studied in an attempt to elucidate the reason for the lack of the activity in the synthetic compounds. The major component of the LTA was first digested by hydrofluoric acid hydrolysis to cleave phosphodiester linkages present. The hydrolysis products were separated and characterized by means of NMR and MS. The linkage positions of the original phosphodiesters were determined from the NMR spectra of an alkali-treated product without hydrofluoric acid degradation. The compound was proved to consist of 1,3-linked poly(glycerophosphate) and a lipid anchor, Glc(alpha1-2)Glc(alpha1-3)acyl(2)Gro, the former being linked to the 6-position of the distal glucose of the latter. The 2-position of the glycerol residues in the glycerophosphate part were substituted by oligoglucose esterified partially with alanine. The gross structure elucidated here thus coincides with the previous conclusion described by Fischer [Fischer, W. (1990) in Glycolipids, Phosphoglycolipids and Sulfoglycolipids (Kates, M., ed.) pp. 123 234, Plenum Press, New York]. Thus, the molecular species with this so-called "LTA structure" is not responsible for the cytokine-inducing activity.

Synthetic study of phosphopeptides related to heat shock protein HSP27.

Two kinds of phosphoserine-containing peptides related to HSP27 were synthesized by the Boc- or Fmoc-mode solid-phase method based on prephosphorylation strategy. In the case of the Boc strategy, the O-phosphono group of the phosphoserine residue was protected with the cyclopentyl or cyclohexyl group. On the other hand, N'-Fmoc-O-[(benzyloxy)-hydroxyphosphinyl]serine was employed in case of the Fmoc strategy. Consequently, it has become-feasible to utilize conventional solid-phase methods for synthesizing any phosphopeptides which are required to elucidate biochemical significance of protein phosphorylation.

Nucleosides and nucleotides. 95. Improved synthesis of 1-(2-azido-2-deoxy-beta-D-arabinofuranosyl)cytosine (cytarazid) and -thymine. Inhibitory spectrum of cytarazid on the growth of various human tumor cells in vitro.

Reaction of N3-benzoyl-1-[3,5-O-(tetraisopropyldisiloxan-1,3-diyl)-beta- D- ribofuranosyl]thymine (4a) with diphenyl phosphorazidate, diethyl azodicarboxylate, and triphenylphosphine in tetrahydrofuran afforded N3-benzoyl-1-[2-azido-2-deoxy-3,5-O- (tetraisopropyldisiloxan-1,3-diyl)-beta-D-arabinofuranosyl]t hymine (5a) in good yield. After the sequence of deblocking of 5a gave 1-(2-azido-2-deoxy-beta-D-arabinofuranosyl)thymine (7), it was heated in N,N-dimethylformamide to produce 6,2'-imino-1-(2-deoxy-beta-D-arabinofuranosyl)thymine (8). This reaction disclosed the arabino configuration for 5a. Similarly the N3-benzoyluracil derivative 4b was transformed to the corresponding 2'-"up"-azidouridine derivative 5b, which was further converted to 1-(2-azido-2-deoxy-1-beta-D-arabinofuranosyl)cytosine (1, cytarazid). The antineoplastic activity of 1 was compared with that of ara-C against various human cancer cell lines in vitro.