One-electron reduction characteristics of N(3)-substituted 5-fluorodeoxyuridines synthesized as radiation-activated prodrugs

We designed and synthesized N(3)-substituted 5-fluorodeoxyuridines as radiation-activated prodrugs of the antitumor agent, 5-fluorodeoxyuridine (5-FdUrd). A series of 5-FdUrd derivatives possessing a 2-oxoalkyl group at the N(3)-position released 5-FdUrd in good yield via one-electron reduction initiated by hypoxic irradiation. Cytotoxicity of the 5-FdUrd derivative possessing the 2-oxocyclopentyl group (3d) was low, but was enhanced by hypoxic irradiation resulting in 5-FdUrd release.     

A conformational restriction approach to the development of dual inhibitors of acetylcholinesterase and serotonin transporter as potential agents for Alzheimer's disease

Alzheimer's disease (AD) has been treated with acetylcholinesterase (AChE) inhibitors such as donepezil. However, the clinical usefulness of AChE inhibitors is limited mainly due to their adverse peripheral effects. Depression seen in AD patients has been treated with serotonin transporter (SERT) inhibitors. We considered that combining SERT and AChE inhibition could improve the clinical usefulness of AChE inhibitors. In a previous paper, we found a potential dual inhibitor, 1, of AChE (IC50=101 nM) and SERT (IC50=42 nM), but its AChE inhibition activity was less than donepezil (IC50=10 nM). Here, we report the conformationally restricted (R)-18a considerably enhanced inhibitory activity against AChE (IC50=14 nM) and SERT (IC50=6 nM).     

Identification of a physiological phosphorylation site of the herpes simplex virus 1-encoded protein kinase Us3 which regulates its optimal catalytic activity in vitro and influences its function in infected cells

Us3 is a serine/threonine protein kinase encoded by herpes simplex virus 1 (HSV-1). Here, we report the identification of a physiological Us3 phosphorylation site on serine at position 147 (Ser-147) which regulates its protein kinase activity in vitro. Moreover, mutation of this site influences Us3 function, including correct localization of the enzyme and induction of the usual morphological changes in HSV-1-infected cells. These conclusions are based on the following observations: (i) in in vitro kinase assays, a domain of Us3 containing Ser-147 was specifically phosphorylated by Us3 and protein kinase A, while a mutant domain in which Ser-147 was replaced with alanine was not; (ii) in vitro, alanine replacement of Ser-147 (S147A) in Us3 resulted in significant impairment of the kinase activity of the purified molecule expressed in a baculovirus system; (iii) phosphorylation of Ser-147 in Us3 tagged with the monomeric fluorescent protein (FP) VenusA206K (VenusA206K-Us3) from Vero cells infected with a recombinant HSV-1 encoding VenusA206K-Us3 was specifically detected using an antibody that recognizes phosphorylated serine or threonine residues with arginine at the -3 and -2 positions; and (iv) the S147A mutation influenced some but not all Us3 functions, including the ability of the protein to localize itself properly and to induce wild-type cytopathic effects in infected cells. Our results suggest that some of the regulatory activities of Us3 in infected cells are controlled by phosphorylation at Ser-147.     

PPARalpha gene expression is up-regulated by LXR and PXR activators in the small intestine

LXR, PXR, and PPARα are members of a nuclear receptor family which regulate the expression of genes involved in lipid metabolism. Here, we show the administration of T0901317 stimulates PPARα gene expression in the small intestine but not in the liver of both normal and FXR-null mice. The administration of LXR specific ligand GW3965, or PXR specific ligand PCN has the same effect, indicating that ligand-dependent activation of LXR and PXR, but not FXR, is responsible for the increased gene expression of PPARα in the mouse small intestine.     

Testing a method for reconstructing structural development of even-aged Abies sachalinensis stands

Accuracy of a stand reconstruction technique was examined by comparing the estimated values of the aboveground biomass, total stem volume, stem volume growth, and stand density of Abies sachalinensis stands to those observed between 1980 and 1998 in Hokkaido, northern Japan. Census data from two stands established in 1973, one fertilized and the other unfertilized, were used for the examination. The stand statistics in the past were estimated from the DBH and height of individual trees measured in 1998, data on the aboveground biomass and stem volume with bark for nine living trees of various sizes harvested in each plot in 1998 or in 1999, and data from the stem analysis of the same harvested trees. We showed that the reconstructed patterns of the frequency distribution in aboveground biomass and in stem volume were generally the same as those observed in both plots and in any year in the past (except for 1982 and/or 1980), and that the reproduced patterns of stand development over time were similar to those observed directly in the past. Accuracy in predicting stand statistics was generally in the order of ±10% relative error. We consider that the present method of stand reconstruction could be used to estimate aboveground biomass, total stem volume, and stem volume growth of a stand in the past. Interpretation of results for the early years (1982 and 1980) and for the stand density requires caution.     

Production of cloned pigs from salivary gland-derived progenitor cells

To achieve tissue stem cell transplantation in clinical settings, translational studies using large animal models are essential to confirm the efficacy and safety of therapy. Therefore, with the ultimate objective of constructing a porcine model of stem cell transplantation in the present study we attempted to clone pigs using porcine salivary gland-derived progenitor cells (pSGPs) as nuclear donors. Normal chromosomal compositions of pSGPs were maintained after five to eight passages (73%, 41 of 56). Cell cycle was efficiently synchronized in G(0)/G(1) phase after 2 days of serum-starved culture (79%). Characteristics of multipotent pSGPs, that is, CD49f and intracellular laminin staining patterns, were unchanged after serum-starved culture. Developmental rate of blastocysts from embryos reconstructed using pSGPs as nuclear donors was significantly higher when compared to embryos reconstructed using fetal fibroblasts (27.7%, 38 of 137 vs. 12.8%, 17 of 138; p < 0.05). When a total of 615 reconstructed embryos were transplanted into four recipient gilts, all gilts became pregnant and produced 12 piglets. These findings suggest that pSGPs represent appropriate donor cells for porcine somatic cell nuclear transfer.     

Choline and betaine ameliorate liver lipid accumulation induced by vitamin B6 deficiency in rats

We investigated the efficacy of supplementing the diet with choline or betaine in ameliorating lipid accumulation induced by vitamin B₆ (B₆) deficiency in rat liver. Male Wistar rats were fed a control, B₆-deficient, choline-supplemented (2, 4, or 6 g choline bitartrate/kg diet) B₆-deficient diet or betaine-supplemented (1, 2, or 4 g betaine anhydrous/kg diet) B₆-deficient diet for 35 d; all diets contained 9 g L-methionine (Met)/kg diet. Choline or betaine supplementation attenuated liver lipid deposition and restored plasma lipid profiles to control levels. These treatments restored the disruptions in Met metabolism and the phosphatidylcholine (PC)/phosphatidylethanolamine (PE) ratio induced by B₆ deficiency in liver microsomes. These results suggest that choline and betaine ameliorated liver lipid accumulation induced by B₆ deficiency via recovery of Met metabolism and very low-density lipoprotein secretion by restoring the supply of PC derived from PE.     

Specificities of N-acetylglucosamine-6-O-sulfotransferases in relation to L-selectin ligand synthesis and tumor-associated enzyme expression.

N-Acetylglucosamine-6-O-sulfotransferase (GlcNAc6ST) catalyzes the transfer of sulfate from adenosine 3'-phosphate,5'-phosphosulfate to the C-6 position of the non-reducing GlcNAc. Three human GlcNAc6STs, namely GlcNAc6ST-1, GlcNAc6ST-2 (HEC-GlcNAc6ST), and GlcNAc6ST-3 (I-GlcNAc6ST), were produced as fusion proteins to protein A, and their substrate specificities as well as their enzymological properties were determined. Both GlcNAc6ST-1 and GlcNAc6ST-2 efficiently utilized the following oligosaccharide structures as acceptors: GlcNAcbeta1-6[Galbeta1-3]GalNAc-pNP (core 2), GlcNAcbeta1-6ManOMe, and GlcNAcbeta1-2Man. The ratios of activities to these substrates were not significantly different between the two enzymes. However, GlcNAc6ST-2 but not GlcNAc6ST-1 acted on core 3 of GlcNAcbeta1-3GalNAc-pNP. GlcNAc6ST-3 used only the core 2 structure among the above mentioned oligosaccharide structures. The ability of GlcNAc6ST-1 to sulfate core 2 structure as efficiently as GlcNAc6ST-2 is consistent with the view that GlcNAc6ST-1 is also involved in the synthesis of l-selectin ligand. Indeed, cells doubly transfected with GlcNAc6ST-1 and fucosyltransferase VII cDNAs supported the rolling of L-selectin-expressing cells. The activity of GlcNAc6ST-2 on core 3 and its expression in mucinous adenocarcinoma suggested that this enzyme corresponds to the sulfotransferase, which is specifically expressed in mucinous adenocarcinoma (Seko, A., Sumiya, J., Yonezawa, S., Nagata, K., and Yamashita, K. (2000) Glycobiology 10, 919-929).