Intratumoral pharmacokinetics of oligonucleotides in a tissue-isolated tumor perfusion system

The intratumoral pharmacokinetics of model oligonucleotides were studied in Walker 256 tissue-isolated tumor preparations using an in situ single-pass vascular perfusion technique. A 20-mer phosphodiester (PO) oligonucleotide, its fully phosphorothioated (PS) oligonucleotide counterpart, and an 18-mer phosphorothioated oligonucleotide containing four 2'-O-methylribonucleosides at both the 3'-end and 5'-end (PS-OMe) were used. These oligonucleotides were administered to the tumor in two ways, by constant arterial infusion and by direct intratumoral injection. In the case of constant arterial infusion, the experiments were carried out using perfusate with or without 4.7% bovine serum albumin (BSA). The protein binding of PO, PS, and PS-OMe to BSA was 46%, 87%, and 94%, respectively. No marked difference was observed between the degree of accumulation of the three types of oligonucleotides in the tumor when BSA was present in the perfusate. PS and PS-OMe showed higher degrees of accumulation in tumors compared with PO when no BSA was present. These results indicate that free (i.e., protein unbound) PS-OMe and PS have superior tumor accumulation characteristics. In the intratumoral injection experiments, PS-OMe was retained longer in tumor tissue compared with PS, suggesting that it might be useful for direct local injection into solid tumors. Thus, the present study provides useful information about the basic disposition characteristics of oligonucleotides in solid tumors.     

Purification and some characteristics of enterocin ON-157, a bacteriocin produced by Enterococcus faecium NIAI 157

Bacteriocin-like activity (BLA) was screened in 690 strains of lactic acid bacteria isolated from plant materials such as silages and fermented vegetables. Among them, a strain identified as Enterococcus faecium NIAI 157 showed a clear BLA against the indicator strain, Ent. faecium IFO 13712. The proteinaceous nature and antimicrobial activity against closely related species strongly indicated that this BLA was a bacteriocin and was designated enterocin ON-157. The bacteriocin activity of this strain was extracellularly produced in the logarithmic growth phase in MRS broth and purified by ultrafiltration, ammonium sulphate precipitation and cation-exchange chromatography. Purified enterocin ON-157 had a molecular weight of approximately 2500 Da in SDS-PAGE analysis and was easily inhibited by treatment with alpha-amylase and proteolytic enzymes. Enterocin ON-157 had a bactericidal mode of action and inhibited the growth of the enterococci, Lactobacillus sake and Listeria monocytogenes. Enterococcus faecium NIAI 157 harboured two plasmids, 49.0 kb and 43.7 kb, and a variant missing a larger plasmid by curing with novobiocin lost the bactriocin activity.     

Linearization and integration of DNA into cells preferentially occurs at intrinsically curved regions from human LINE-1 repetitive element.

A bent DNA library was constructed from human genomic DNA, from which a new clone belonging to the human LINE-1 sequence family was isolated and characterized. This clone, with a length of 378 base pairs and termed HBC-1 (human bent clone-1), contained an intrinsically occurring curved DNA structure. By permutation analysis, the center of curvature of this fragment was mapped onto the nucleotide position 886 from the 5' terminus of the complete LINE-1 sequence. Reporter plasmids, which contain HBC-1, were effectively integrated into human chromosome, indicating that the bent DNA structure provides a preferential donor site for the integration of human LINE-1 sequences. The present finding may provide an explanation as to why some inactivated LINE-1 sequences on human chromosomes carry the deletion at their 5' termini.     

Inhibition of CX3CL1 (fractalkine) improves experimental autoimmune myositis in SJL/J mice

Idiopathic inflammatory myopathy is a chronic inflammatory muscle disease characterized by mononuclear cell infiltration in the skeletal muscle. The infiltrated inflammatory cells express various cytokines and cytotoxic molecules. Chemokines are thought to contribute to the inflammatory cell migration into the muscle. We induced experimental autoimmune myositis (EAM) in SJL/J mice by immunization with rabbit myosin and CFA. In the affected muscles of EAM mice, CX3CL1 (fractalkine) was expressed on the infiltrated mononuclear cells and endothelial cells, and its corresponding receptor, CX3CR1, was expressed on the infiltrated CD4 and CD8 T cells and macrophages. Treatment of EAM mice with anti-CX3CL1 mAb significantly reduced the histopathological myositis score, the number of necrotic muscle fibers, and infiltration of CD4 and CD8 T cells and macrophages. Furthermore, treatment with anti-CX3CL1 mAb down-regulated the mRNA expression of TNF-alpha, IFN-gamma, and perforin in the muscles. Our results suggest that CX3CL1-CX3CR1 interaction plays an important role in inflammatory cell migration into the muscle tissue of EAM mice. The results also point to the potential therapeutic usefulness of CX3CL1 inhibition and/or blockade of CX3CL1-CX3CR1 interaction in idiopathic inflammatory myopathy.     

Functional Significance of the Preconditioning-induced Down-regulation of Glutamate Transporter GLT-1 in Neuron/astrocyte Co-cultures

In the brain, prior sublethal ischemia (preconditioning, PC) is known to produce tolerance of neurons to subsequent lethal ischemia. This study aims at elucidating what alterations were induced in neurons and/or astrocytes by PC treatment. The rise in the extracellular concentration of glutamate during ischemia was markedly suppressed by the prior PC treatment. Immunocytochemical and Western blot analyses demonstrated that the expression of the astrocytic glutamate transporter GLT-1 was transiently down-regulated after the PC insult. The PC insult possibly suppressed the neuron-derived factors up-regulating GLT-1. Here we show that PC-induced down-regulation of GLT-1 is crucial for the increased neuronal resistance to subsequent severe ischemic insult.     

High throughput easy microinjection with a single-cell manipulation supporting robot

A single-cell manipulation supporting robot (SMSR) has been developed for the high throughput and easy microinjection. Its concept is to let an experimenter concentrate his/her attention only on the microinjection by facilitating other associated works. SMSR was applied to the microinjection into rice protoplasts and mouse embryonic stem (ES) cells. The microinjection into these cells is exceptionally difficult than usual animal cells such as fibroblasts. In the case of rice protoplast, for example, non-stop microinjection into 100 cells could be done within 1h that was 17-times faster than that of the robot-less work. The success rate was 7-8% that was same level obtained by the robot-less work. The present results indicate that SMSR is a useful machine for the microinjection of specific genes and proteins in living cells to analyze their respective functions, which is an urgent and important subject in the post-genome era.     

Emergence of nuclear heparanase induces differentiation of human mammary cancer cells

The study of epithelial differentiation touches upon many modern aspects of biology. The epithelium is in constant dialogue with the underlying mesenchyme to control stem cell activity, proliferation in transit-amplifying compartments, lineage commitment, terminal differentiation and, ultimately, cell death. There are spatially distinct compartments dedicated to each of these events. Recently we reported that heparanase is expressed in nucleus as well as in the cytoplasm and that nuclear heparanase seems to be related to cell differentiation. In this study, we investigated the role of nuclear heparanase in differentiation by transducing human mammary epithelial cancer cells with heparanase which was delivered specifically into nucleus. We observed that expression of nuclear heparanase allowed the cells to differentiate with the appearance of lipid droplets. This finding supports the idea that heparanase plays a novel role in epithelial cell differentiation apart from its known enzymatic function.