Neoamphimedine circumvents metnase-enhanced DNA topoisomerase IIα activity through ATP-competitive inhibition

Type IIα DNA topoisomerase (TopoIIα) is among the most important clinical drug targets for the treatment of cancer. Recently, the DNA repair protein Metnase was shown to enhance TopoIIα activity and increase resistance to TopoIIα poisons. Using in vitro DNA decatenation assays we show that neoamphimedine potently inhibits TopoIIα-dependent DNA decatenation in the presence of Metnase. Cell proliferation assays demonstrate that neoamphimedine can inhibit Metnase-enhanced cell growth with an IC(50) of 0.5 μM. Additionally, we find that the apparent K(m) of TopoIIα for ATP increases linearly with higher concentrations of neoamphimedine, indicating ATP-competitive inhibition, which is substantiated by molecular modeling. These findings support the continued development of neoamphimedine as an anticancer agent, particularly in solid tumors that over-express Metnase.     

Endometrial response to conceptus-derived estrogen and interleukin-1β at the time of implantation in pigs

The establishment of pregnancy is a complex process that requires a well-coordinated interaction between the implanting conceptus and the maternal uterus. In pigs, the conceptus undergoes dramatic morphological and functional changes at the time of implantation and introduces various factors, including estrogens and cytokines,interleukin-1β2(IL1 B2), interferon-γ(IFNG), and IFN-δ(IFND), into the uterine lumen. In response to ovarian steroid hormones and conceptus-derived factors, the uterine endometrium becomes receptive to the implanting conceptus by changing its expression of cell adhesion molecules, secretory activity, and immune response. Conceptus-derived estrogens act as a signal for maternal recognition of pregnancy by changing the direction of prostaglandin(PG) F2αfrom the uterine vasculature to the uterine lumen. Estrogens also induce the expression of many endometrial genes,including genes related to growth factors, the synthesis and transport of PGs, and immunity. IL1 B2, a pro-inflammatory cytokine, is produced by the elongating conceptus. The direct effect of IL1 B2 on endometrial function is not fully understood. IL1 B activates the expression of endometrial genes, including the genes involved in IL1 B signaling and PG synthesis and transport. In addition, estrogen or IL1 B stimulates endometrial expression of IFN signaling molecules,suggesting that estrogen and IL1 B act cooperatively in priming the endometrial function of conceptus-produced IFNG and IFND that, in turn, modulate endometrial immune response during early pregnancy. This review addresses information about maternal-conceptus interactions with respect to endometrial gene expression in response to conceptus-derived factors, focusing on the roles of estrogen and IL1 B during early pregnancy in pigs.     

Development of multiplex polymerase chain reaction assays for detecting enterotoxigenic Escherichia coli and their application to field isolates from piglets with diarrhea.

Fimbriae and enterotoxins are major virulence factors associated with enterotoxigenic Escherichia coli (ETEC). In this study, 3 sets of multiplex polymerase chain reaction (mPCR) assays targeting fimbriae, enterotoxins, and other adherence factors were developed for detecting ETEC. A total number of 188 E. coli field isolates were examined, and percentages of E. coli strains carrying each virulence factors were as follows: F4 (7.45%), F5 (29.79%), F6 (6.38%), F18 (15.43%), F41 (3.72%), STa (10.11%), STb (20.74%), LT (9.57%), Stx2e (2.13%), EAST1 (42.02%), F1 (67.55%), AIDA-I (2.66%), and pAA (7.45%). Of the 188 E. coli field isolates examined, 25.53% were found to be pathogenic ETEC, having both fimbriae and enterotoxins. However, the ratio increased to 44.68% when the presence of other adhesins was considered as criteria for virulence. Among the adherence factors, F1 was found to be the most prevalent. AIDA-I and pAA were also found with similar ratio as compared with other virulence factors. In addition, virulence patterns carrying these alternate adhesive genes with enterotoxins were detected with significant ratio. Therefore, it is desirable that alternate adhesins be considered as markers for diagnosis of ETEC.     

Improved quantitative analysis of oligosaccharides from lichenase-hydrolyzed water-soluble barley beta-glucans by high-performance anion-exchange chromatography

Cereal beta-glucan is a linear biopolymer linked by beta-(1,3)/(1,4)-glycosidic bonds. More specifically, the beta-(1,4)-linked glucose chain is interrupted with beta-(1,3)-linkages in cereal beta-glucan structure. Elucidation of the exact length and distribution of linear beta-(1,4)-linked portion facilitates the understanding of the fine structure of cereal beta-glucan. A HPAEC assisted by lichenase treatment has been used for the structural and quantitative analysis of cereal beta-glucan. The absence of authentic standard oligosaccharides, putatively 3-O-beta-cellobiosyl-D-glucose (DP3) and 3-O-beta-cellotriosyl-D-glucose (DP4), was a potential problem to the characterization of beta-glucan structure. In this study, two major lichenase-hydrolyzed products were generated from the barley beta-glucan, and putative 3-O-beta-cellobiosyl-D-glucose and 3-O-beta-cellotriosyl-D-glucose were separated and highly purified by recycling preparative HPLC technology. Structural analysis of highly purified putative 3-O-beta-cellobiosyl-D-glucose and 3-O-beta-cellotriosyl-D-glucose was performed by TLC and LC-MS analysis. Two putative DP3 and DP4 displayed the nonreducing end/(1,4)/(1,3) linkage ratios of 1:0.96:0.90 and 1:2.18:1.16, respectively; the molecular masses (m/z) of their sodium adducts were 527.0 and 689.0, respectively. Using these structurally confirmed oligosaccharides, the exact amounts of beta-glucan lichenase hydrolysates from domestic barley cultivars were quantified. The amount of two major DP3 and DP4 accounted for only 71.4-73.3% of water-extractable beta-glucan fraction, and the (1,4)/(1,3) linkage ratios of the extracted beta-glucans were almost identical in the range of 2.24-2.25 among the barley cultivars tested.     

Immunohistochemical localization of glutamate in the gerbil main olfactory bulb using an antiserum directed against glutamate

Information on the localization and the roles of glutamate in the nervous system is becoming valuable because the axon terminals of the olfactory sensory neurons and the synapses of the mitral and tufted output cells appear to be glutamatergic. In this study, we have analysed the distribution of glutamate immunoreactivity in the main olfactory bulb (MOB) of the Mongolian gerbil using an antiserum directed against glutamate. Glutamate immunoreactivity in the MOB was present in the olfactory nerve layer (Onl), glomerular layer (GL), external plexiform layer (EPL) and mitral cell layer (ML), but not in the granule cell layer (GCL). Glutamate immunoreactivity detected in the Onl was thought to be terminal ramifications of glomeruli. Some neurons in the periglomerular region showed glutamate immunoreactivity. In the EPL, glutamate immunoreactivity was found in some neuronal somata (tufted cells) and processes. In addition, mitral cells in the ML were labelled by the glutamate antibody. The pattern of glutamate immunoreactivity in the mitral cells was similar to that in the tufted cells. In brief, glutamate in the gerbil MOB is the neurotransmitter used by primary afferents and output neurons.     

Age-related changes in calretinin-immunoreactive periglomerular cells in the rat main olfactory bulb

The changes of calretinin (CR)-immunoreactive periglomerular cells in the glomerular layer of the main olfactory bulb (MOB) were investigated in rats differing ages from postnatal month 1 (PM 1) to PM 24. The number of cresyl violet-positive periglomerular cells was similar between PM 1 and PM 12, but they decreased slightly in the PM 24 group. The size of CR-immunoreactive periglomerular cells in the glomerular layer increased with age, while their numbers did not change significantly in the PM 6-PM 24 groups. In the PM 24 group, numbers of CR-positive periglomerular cell bodies and their processes decreased, while the size of CR-positive cell bodies in the glomeruli was larger than that of the previous groups. These results suggest that CR-immunoreactive periglomerular cells in the rat MOB are well-developed in the PM 6 group, and that periglomerular cells in the PM 24 group show poor CR-immunoreactivity compared to those in the PM 6 group.