Synthesis and evaluation of 6-methylene-bridged uracil derivatives. Part 2: optimization of inhibitors of human thymidine phosphorylase and their selectivity with uridine phosphorylase

A series of novel 6-methylene-bridged uracil derivatives have been optimized for clinical use as the inhibitors of human thymidine phosphorylase (TP). We describe their synthesis and evaluation. Introduction of a guanidino or an amidino group enhanced the in vitro inhibitory activity of TP comparing with formerly reported inhibitor 1. Their selectivity for TP based on uridine phosphorylase inhibitory activity was also evaluated. Compound 2 (TPI) has been selected for clinical evaluation based on its strong TP inhibition and excellent modulation of 2'-deoxy-5-(trifluoromethyl)uridine (F(3)dThd) pharmacokinetics. As a result, TAS-102 (a combination of F(3)dThd and TPI) is currently in phase 1 clinical studies.     

Superoxide production at phagosomal cup/phagosome through beta I protein kinase C during Fc gamma R-mediated phagocytosis in microglia

Protein kinase C (PKC) plays a prominent role in immune signaling. To elucidate the signal transduction in a respiratory burst and isoform-specific function of PKC during FcgammaR-mediated phagocytosis, we used live, digital fluorescence imaging of mouse microglial cells expressing GFP-tagged molecules. betaI PKC, epsilonPKC, and diacylglycerol kinase (DGK) beta dynamically and transiently accumulated around IgG-opsonized beads (BIgG). Moreover, the accumulation of p47(phox), an essential cytosolic component of NADPH oxidase and a substrate for betaI PKC, at the phagosomal cup/phagosome was apparent during BIgG ingestion. Superoxide (O(2)(-)) production was profoundly inhibited by G?6976, a cPKC inhibitor, and dramatically increased by the DGK inhibitor, R59949. Ultrastructural analysis revealed that BIgG induced O(2)(-) production at the phagosome but not at the intracellular granules. We conclude that activation/accumulation of betaI PKC is involved in O(2)(-) production, and that O(2)(-) production is primarily initiated at the phagosomal cup/phagosome. This study also suggests that DGKbeta plays a prominent role in regulation of O(2)(-) production during FcgammaR-mediated phagocytosis.     

Antimicrobial susceptibility and genetic relatedness of bovine Stenotrophomonas maltophilia isolates from a mastitis outbreak

Aims: To evaluate the antimicrobial susceptibility and genetic relatedness of 11 Stenotrophomonas maltophilia isolates from an outbreak of bovine clinical mastitis in one herd and two isolates from two separate mastitis cases in two other herds. Methods and Results: Thirteen S. maltophilia isolates were obtained from milk samples from 11 cows from three dairy herds in Japan during 2008. We tested their susceptibility to 14 antimicrobials by broth microdilution and identified their genotypes by enterobacterial repetitive intergenic consensus 2 (ERIC2)‐PCR. Every cow had acute mild mastitis (slightly watery foremilk with flakes) without systemic symptoms and all resolved within 3–5 weeks of diagnosis. Eleven of the 13 isolates derived from nine cows in one herd over a 7‐month period exhibited a closely related ERIC2 type (A). The remaining two isolates derived from two cows from two other herds exhibited two distinct ERIC2 types (B and C). Most of the 13 isolates exhibited susceptibility to trimethoprim‐sulfamethoxazole, chloramphenicol, minocycline and levofloxacin; however, they were resistant to four β‐lactams, kanamycin, gentamicin and oxytetracycline. They were intermediate to enrofloxacin. Conclusions: Eleven closely related S. maltophilia isolates were involved in a herd outbreak of mastitis to some extent. Bovine S. maltophilia isolates exhibited resistance to many classes of antimicrobials. Significance and impact of study: This is a rare report of a herd outbreak of bovine mastitis involving closely related S. maltophilia isolates.     

Discovery of heteroaryl sulfonamides as new EP1 receptor selective antagonists

4-({2-[Isobutyl(phenylsulfonyl)amino]-5-(trifluoromethyl)phenoxy}methyl)benzoic acid (1) is a functional PGE2 antagonist selective for EP1 receptor subtype. Analogs of 1, in which the phenyl-sulfonyl moiety has been replaced with more hydrophilic heteroarylsulfonyl moieties, exhibited more optimized antagonist activity, while some of them showed in vivo antagonist activity. Structure-activity relationship (SAR) studies are also presented.     

Histochemical studies of mucosubstances in metaplastic epithelium of the stomach,with special reference to the development of intestinal metaplasia

Summary Processes in the development of intestinal metaplasia of the stomach were investigated from the morphological and histochemical approaches using light and electron microscopic techniques. The specimens taken from 38 gastric carcinomas and 15 gastric and/or duodenal ulcers were subjected to this study. Morphological appearances of the intestinal metaplasia observed in routine examination with hematoxylin and eosin staining was able to be divided into complete and incomplete metaplasia by the light and electron microscopic histochemical stainings of the mucosubstances. The columnar cells at the area of the incomplete metaplasia had both the properties of the intestinal epithelia and the gastric foveolar epithelia. The incomplete as well as the complete metaplasia arose from the generative cells at the isthmus of the gland. The generative cells, however, sometimes gradually transformed to produce the complete metaplastic cells. The two processes of the development of the intestinal metaplasia were proposed and discussed.     

Genetic diagnosis of cytoplasmic male sterile cybrid plants of rice

Twelve Japanese rice cultivars were converted to CMS by asymmetric protoplast fusion with MTC-5A, the cytoplasm of which was derived from an indica rice, Chinsurah Boro II. With the exception of the cybrids that had a nucleus from Hoshiyutaka, most of these cybrid plants were sterile. The unique sequence downstream from the mitochondrial atp6 of MTC-5A was specifically amplified in the sterile cybrid plants by PCR. All progenies of the cybrid plants carrying this unique sequence were sterile. On the other hand, in some of the sterile cybrid plants in which the unique sequence was not amplified by PCR, fertility was recovered in their progenies. Somaclonal mutation may have caused sterility in these cybrids. Only the cybrid plants that had the unique sequence detected by PCR were CMS. Thus, the CMS plants can be selected rapidly and easily by PCR, at an early stage of plant regeneration. Soon after transplanting the regenerated plants to a green house, fertile cybrids and sterile cybrids produced by somaclonal mutation can be removed. These findings also show that the unique region downstream from atp6 is tightly linked with the CMS phenotype.     

Impact of altitude and topography on the genetic diversity of Quercus serrata populations in the Chichibu Mountains, central Japan

In order to elucidate the factors affecting the genetic diversity of Quercus serrata in secondary forests in mountainous regions, we evaluated the level and distribution of genetic variation within and between 15 populations using seven microsatellite markers. The populations were at altitudes ranging from 140 to 1200 m in and around the Chichibu Mountains, central Japan.The expected heterozygosity (H_E) ranged from 0.766 to 0.837. The two populations that exhibited the highest and the second highest values of H_E are located beside a river and a lake, respectively. The two populations exhibiting the lowest and the second lowest values of H_E are, in contrast, located on a summit and a ridge. The observed heterozygosity (H_O) varied between 0.638 and 0.844, and the value of this variable was also higher for the populations beside water than those on summits or ridges. The soils at the waterside are wet, in contrast to those on ridges and summits, which tend to be shallow and subject to rapid desiccation. These results suggest that a lack of soil moisture is likely to inhibit the development and regeneration of Q. serrata, and that genetic diversity is reduced in arid areas. The genetic differentiation was low (F_ST=0.013) among the investigated populations, although all five populations in Yamanashi prefecture clustered together in an UPGMA tree. According to a multiple regression analysis, there was no significant isolation by distance among the populations along either the horizontal or vertical axes. Therefore, genetic variation within populations is affected by topography, but variation between populations is hardly affected by geographical factors. Furthermore, the results of this study suggest two conclusions. First, that altitude is not always a useful variable when estimating the genetic diversity of plant populations in mountainous regions. Second, that genetic diversity can vary even among the undifferentiated plant populations in small areas like the Chichibu Mountains.     

Epidermal growth factor (EGF) in the goat uterus: immunohistochemical localization of EGF and EGF receptor and effect of EGF on uterine activity in vivo

This study examined the distribution of immunoreactive epidermal growth factor (EGF) and EGF receptor (EGF-R) in the uterus and the effects of EGF on uterine activity in goats. Immunohistochemistry of EGF and EGF-R in the uteri showed distinct staining in the luminal and glandular epithelium and slight to moderate staining in the stromal and myometrial cells. To examine possible roles of the EGF system in the regulation of uterine activity, pressure changes in the intrauterine balloon were determined after intraluminal infusion of EGF into the uterine horn. Either at estrus or diestrus (9 to 14 days after the first day of estrus), treatment with 1 or 5 microg of EGF gradually reduced uterine activity, whereas infusion of the vehicle alone had no effect. The maximum reduction in uterine activity was seen 4 h after the treatment with 1 microg of EGF (40% to 45% reduction in the area surrounded by the contraction curve and its baseline), and the activity slowly returned thereafter. These results suggest that EGF in the uterus may play a role in regulating uterine activity in goats.     

Combinatorial evaluation of the chiral discrimination of permethylated carbohydrates using fast-atom bombardment mass spectrometry

The chiral discrimination abilities of several variously permethylated carbohydrates toward various amino acid 2-propyl esters were combinatorially evaluated from the relative peak intensity of the 1:1 diastereomeric complex ions with the deuterium-labeled L-amino acid 2-propyl ester protonated ion and with the unlabeled D-amino acid 2-propyl ester protonated ions in FAB mass spectrometry. The chiral discrimination abilities evaluated using FAB mass spectrometry approximately corresponded to the ratio of the association constants (K(R)/K(S)) toward each enantiomer in the solution. Therefore, this evaluation method is very useful for the screening of the chiral discrimination abilities of carbohydrates and their derivatives.