Quantitative comparison of rat hepatocyte functions in two improved culture systems with or without rat liver epithelial cell line

We quantitatively evaluated two recently-developed novel techniques for hepatocyte cultivation in a dish level; that is, spheroid culture and membrane-supported collagen (CN) gel sandwich culture, in terms of cellular maintenance, albumin secretion and 7-ethoxycoumarin (7EC) metabolism to 7-hydroxycoumarin (7HC) as a marker for cytochrome P450 IA1 activity in the presence and absence of rat liver epithelial cell line (RLEC) during one month of culture, together with conventional coculture with RLEC in CN-coated dishes as a control. RLEC prevented spheroid loss caused by its detachment from the culture dishes often occurring in pure culture. CN-gel sandwich by itself improved remarkably hepatocyte maintenance when compared with CN-gel free systems, thereby resulting in enhancement of overall functional expressions as compared with CN-gel free systems. RLEC in CN-gel sandwhich, however, reduced cellular sustainment probably due to its suppression of hepatocyte growth. Although there were no significant differences in albumin secretion per cell among the five cultures examined, CN-gel sandwich expressed markedly higher 7EC metabolizing activity per cell, where RLEC presence had a preferable influence. Consequently, membrane-supported CN-gel sandwich was the most superior technique for hepatocyte cultivation from the standpont of both cellular maintenance and its functional expressions per cell.     

Synthesis and biological evaluation of 2-amino-2-deoxy- and 6-amino-6-deoxy-cyclomaltoheptaose polysulfates as synergists for angiogenesis inhibition

2-Amino-2-deoxy-cyclomaltoheptaose was prepared from β-cyclodextrin perbenzoate [heptakis(2,3,6-tri-O-benzoyl)cyclomaltoheptaose] by a series of reactions including selective de-O-benzoylation at C-2 of one of the perbenzoylated -glucopyranosyl moieties, oxidation to the 2-ulose derivative, oxime formation, and reduction to the 2-amino-2-deoxy- -glucose moiety. This compound and 6-amino-6-deoxycyclomaltoheptaose accessible from β-cyclodextrin through the known procedure were sulfated to give polysulfated aminocyclomaltoheptaose derivatives (3, 5). Employing β-cyclodextrin polysulfate as a reference compound, the synergistic effects of 3 and 5 for cortexolone on angiogenesis inhibitory activity were examined by rabbit-corneal micropocket assay system. In contrast to the significant anti-angiogenesis activity of the β-cyclodextrin polysulfate-cortexolone pair, neither 3 nor 5 showed any cooperative activity with cortexolone in the inhibition of basic FGF-induced angiogenesis.     

Zebrafish lines expressing UAS‐driven red probes for monitoring cytoskeletal dynamics

Actin filaments and microtubules are principal components of the cytoskeleton that regulate the basic cellular phenomena underlying many fundamental cellular processes. Therefore, analyzing their dynamics in living cells is important for understanding cellular events more precisely. In this article, we report two novel transgenic zebrafish lines expressing red fluorescent proteins tagged with Lifeact or EB1 that interact with actin filaments and microtubule plus ends, respectively, under the control of the GAL4‐UAS system. Using these transgenic lines, we could detect F‐actin and microtubule plus end dynamics in specific tissues of living zebrafish embryos by crossing with GAL4 driver lines. In addition, we could achieve multi‐color imaging using these transgenic lines with GFP‐expressing transgenic lines. Therefore, our transgenic lines that carry UAS‐driven red fluorescent cytoskeletal probes are useful tools for analyzing spatiotemporal changes of the cytoskeletal elements using multicolor live imaging.     

Pronounced antitumor effect of LAK-like cells induced in the peritoneal cavity of mice after intraperitoneal injection of OK-432, a killed Streptococcal preparation

Summary More than 80% of BALB/c mice bearing BAMC-1 ascites tumor were completely cured after five consecutive (once every 2 days) i. p. injections of a 0.1 mg dose of OK-432, beginning on day 2 after tumor implantation. The antitumor effect of OK-432 was abolished in athymic nu/nu mice and in anti-thymocyte globulin-treated euthymic BALB/c mice, so although OK-432 treatment did increase the length of survival, all animals eventually died as a result of tumor growth. When peritoneal exudate cells (PEC), obtained on day 12 from OK-432-treated BAMC-1-bearing euthymic mice were evaluated for in vivo tumor neutralization activity, all mice receiving an i. p. injection of the admixture of the nonadherent PEC (1×10⁷ cells) with BAMC-1 cells (1×10⁵) survived for more than 60 days. When the same nonadherent PEC (1×10⁷ cells) were i. p. transferred adoptively 1 day after the inoculation of 1×10⁵ BAMC-1 tumor cells, again all mice survived.When these in vivo active PEC were tested for cytotoxicity in vitro against fresh BAMC-1 tumor cells, natural killer (NK) sensitive syngeneic RL 1, NK-sensitive allogeneic YAC-1 cells, NK-resistant syngeneic Meth-A cells, allogeneic tumor cells (EL4, B16, and P815) and xenogenic human cells, the PEC were found to be capable of lysing BAMC-1 tumor cells together with almost all of the other tumor cells, including NK-resistant cells. Nonadherent PEC contained at least two subpopulations of killer cells. One, directed to syngeneic BAMC-1 cells, was both Thy1.2 and asialo GM1 positive, and another, directed to allogeneic YAC-1 cells, was asialo GM1 positive but Thy1.2 negative. A cold target inhibition assay also suggested the presence of more than two subpopulations.These results indicate that T cells play a determined role in the immunotherapeutic effect of OK-432 on BALB/c mice bearing BAMC-1 tumor, although the participation of activated macrophages could not be excluded. The cells responsible for killing BAMC-1 and other tumor cells appearing in the PEC on day 12 were characterized as containing at least two kinds of lymphokine-activated killer cells.     

Cell wall-targeting domain of glycylglycine endopeptidase distinguishes among peptidoglycan cross-bridges

ALE-1, a homologue of lysostaphin, is a peptidoglycan hydrolase that specifically lyses Staphylococcus aureus cell walls by cleaving the pentaglycine linkage between the peptidoglycan chains. Binding of ALE-1 to S. aureus cells through its C-terminal 92 residues, known as the targeting domain, is functionally important for staphylolytic activity. The ALE-1-targeting domain belongs to the SH3b domain family, the prokaryotic counterpart of the eukaryotic SH3 domains. The 1.75 angstroms crystal structure of the targeting domain shows an all-beta fold similar to typical SH3s but with unique features. The structure reveals patches of conserved residues among orthologous targeting domains, forming surface regions that can potentially interact with some common features of the Gram-positive cell wall. ALE-1-targeting domain binding studies employing various bacterial peptidoglycans demonstrate that the length of the interpeptide bridge, as well as the amino acid composition of the peptide, confers the maximum binding of the targeting domain to the staphylococcal peptidoglycan. Truncation of the highly conserved first 9 N-terminal residues results in loss of specificity to S. aureus cell wall-targeting, suggesting that these residues confer specificity to S. aureus cell wall.