Nanomolar Amyloid β Protein-Induced Inhibition of Cellular Redox Activity in Cultured Astrocytes

Abstract: It has been previously reported that Alzheimer's amyloid β protein (Aβ) induces reactive astrocytosis in culture. In the present study, we found that Aβ potently inhibits cellular redox activity of cultured astrocytes, as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay. The following comparative studies revealed several differences between these two actions of Aβ on astrocytes. First, Aβ-induced reactive morphological change was suppressed by the presence of serum or thrombin, and Aβ inhibition of cellular redox activity was observed in either the presence or the absence of serum. Second, micromolar concentrations (10 µ M or more) were required for Aβ to induce reactive astrocytosis, whereas nanomolar concentrations (0.1–100 n M ) were sufficient to inhibit cellular redox activity. Third, the effect of micromolar Aβ was virtually irreversible, but nanomolar Aβ-induced inhibition of cellular redox activity was reversed by washing out Aβ. Furthermore, as it has been reported that Aβ neurotoxicity is mediated by reactive oxygen species, we also examined if similar mechanisms are involved in astrocytic response to Aβ. However, neither Aβ-induced morphological change nor inhibition of redox activity was blocked by antioxidants, suggesting that these effects are not caused by oxidative stress.     

High-performance liquid chromatographic determination of hippuric acid in human urine

A method is described for the determination of urinary hippuric acid by high-performance liquid chromatography. The method used ethyl acetate extraction for partial clean-up of the urine. The separation was carried out on a reversed-phase column using 20% methanol in 0.01 M aqueous potassium phosphate containing 0.5% acetic acid as a mobile phase. The column effluent was monitored with a UV detector at 254 nm. Hippuric acid was separated from other normal urine constituents in less than 10 min. Metabolites of xylene and styrene did not interfere with the assay. Analytical recoveries from urine were excellent and peak height and concentration were linearly related.     

Increased metabolism of retinoic acid after chronic ethanol consumption in rat liver microsomes

In rat liver microsomes, all-trans-[11,12-³H]retinoic acid was found to be metabolized to polar products in the presence of NADPH. One of the metabolites was coeluted with 4-hydroxyretinoic acid on reverse-phase high-pressure liquid chromatography (HPLC). This reaction required oxygen and was inhibited by carbon monoxide as well as aminopyrine, aniline, and ethanol, suggesting the involvement of cytochrome P-450. Isolated rat hepatocytes also metabolized all-trans[³H]retinoic acid to polar compounds, with an elution pattern on HPLC similar to that in microsomal preparations. Microsomal activity was compared in rats pair-fed with diets containing either ethanol or isocaloric carbohydrate for 4–6 weeks. Ethanol-fed rats showed enhanced microsomal retinoic acid metabolism (50%, P < 0.01) accompanied by increased microsomal cytochrome P-450 content (34%, P < 0.005). On the other hand, microsomal β-glucuronidation of retinoic acid in the presence of uridine diphosphoglucuronic acid (UDPGA) was not affected by chronic ethanol feeding. The increased hepatic microsomal cytochrome P-450-dependent metabolism of retinoic acid after chronic ethanol consumption may contribute to the accelerated catabolism of retinoic acid in vivo.     

Similarity and difference in the mechanisms of neonatally induced tolerance and cyclophosphamide-induced tolerance in mice

The mechanisms of cyclophosphamide (CP)-induced tolerance were investigated by comparing with those of neonatally induced tolerance. When C3H/He Slc (C3H; H-2k, Mls-1b) mice were given i.v. either AKR/J Sea (AKR; H-2k, Mls-1a) or (AKR x C3H)F1 (AKC3F1; H-2k, Mls-1a/b) spleen cells and treated i.p. with CP 2 days later, a long-lasting skin allograft tolerance to AKR was induced in each case without any signs of graft-vs-host disease (GVHD). However, typical signs of GVHD were observed in the C3H mice neonatally tolerized with AKR spleen cells, but not in those tolerized with AKC3F1 spleen cells. The expression of TCR V beta 6, which is strongly correlated with the reactivity to Mls-1a Ag (of donor AKR origin), in the periphery was quite different between the two types of tolerant C3H mice. Namely, in the lymph nodes of the C3H mice tolerized with AKR spleen cells and CP, only CD4(+)-V beta 6+, but not CD8(+)-V beta 6+, T cells selectively disappeared, whereas both of them were abrogated in the lymph nodes of the C3H mice neonatally tolerized of AKR. By contrast, in the thymus of the two types of tolerant C3H mice, both CD4+CD8- and CD4-CD8+ single-positive thymocytes expressing TCR V beta 6 were clonally deleted, suggesting that the thymic involvement was the same in each type of tolerance. These results suggest that the preferential disappearance of the CD4(+)-V beta 6+ T cells (of host origin) and the effector T cells of GVHD (of donor origin) occurred only in the periphery of the C3H mice tolerized with AKR spleen cells plus CP and was attributable to the destruction of Ag-stimulated T cells by the CP treatment. In contrast, the intrathymic clonal deletion of immature V beta 6+ T cells was a common mechanism for both of the tolerance induction systems.     

Notes on the morphology,ecology and life cycles of Fukaurahydra anthoformis and Hataia parva (Hydrozoa,Athecata)

Fukaurahydra anthoformis and Hataia parva are solitary athecate hydroids occurring in northern Japan. New information on the external morphology, nematocysts, ecology, and life cycles of these species is presented. It is noteworthy that H. parva bears stenoteles, which are generally not found among the families of Filifera. Neither species produces free medusae. The eggs are fertilized in the female gonophores, from which unciliated larvae are released. These larvae do not swim and soon attach to a substrate. After attachment the larvae become covered by a sheath to form cysts. The cysts rest on a substrate without any outer change for several months. As the water temperature drops in autumn to early winter the cysts begin to hatch, forming tiny polyps after the larva creeps out from the chitinous sheath. Cyst formation proves to be common also in other solitary hydroids, most of which are inhabitants of cool or cold waters.     

Cytoprotective Effect of Recombinant Human Erythropoietin Produced in Transgenic Tobacco Plants

Asialo-erythropoietin, a desialylated form of human erythropoietin (EPO) lacking hematopoietic activity, is receiving increased attention because of its broader protective effects in preclinical models of tissue injury. However, attempts to translate its protective effects into clinical practice is hampered by unavailability of suitable expression system and its costly and limit production from expensive mammalian cell-made EPO (rhuEPO^M) by enzymatic desialylation. In the current study, we took advantage of a plant-based expression system lacking sialylating capacity but possessing an ability to synthesize complex N-glycans to produce cytoprotective recombinant human asialo-rhuEPO. Transgenic tobacco plants expressing asialo-rhuEPO were generated by stably co-expressing human EPO and β1,4-galactosyltransferase (GalT) genes under the control of double CaMV 35S and glyceraldehyde-3-phosphate gene (GapC) promoters, respectively. Plant-produced asialo-rhuEPO (asialo-rhuEPO^P) was purified by immunoaffinity chromatography. Detailed N-glycan analysis using NSI-FTMS and MS/MS revealed that asialo-rhuEPO^P bears paucimannosidic, high mannose-type and complex N-glycans. In vitro cytoprotection assays showed that the asialo-rhuEPO^P (20 U/ml) provides 2-fold better cytoprotection (44%) to neuronal-like mouse neuroblastoma cells from staurosporine-induced cell death than rhuEPO^M (21%). The cytoprotective effect of the asialo-rhuEPO^P was found to be mediated by receptor-initiated phosphorylation of Janus kinase 2 (JAK2) and suppression of caspase 3 activation. Altogether, these findings demonstrate that plants are a suitable host for producing cytoprotective rhuEPO derivative. In addition, the general advantages of plant-based expression system can be exploited to address the cost and scalability issues related to its production.     

Eighteen Coral Genomes Reveal the Evolutionary Origin of Acropora Strategies to Accommodate Environmental Changes

The genus Acropora comprises the most diverse and abundant scleractinian corals (Anthozoa, Cnidaria) in coral reefs, the most diverse marine ecosystems on Earth. However, the genetic basis for the success and wide distribution of Acropora are unknown. Here, we sequenced complete genomes of 15 Acropora species and 3 other acroporid taxa belonging to the genera Montipora and Astreopora to examine genomic novelties that explain their evolutionary success. We successfully obtained reasonable draft genomes of all 18 species. Molecular dating indicates that the Acropora ancestor survived warm periods without sea ice from the mid or late Cretaceous to the Early Eocene and that diversification of Acropora may have been enhanced by subsequent cooling periods. In general, the scleractinian gene repertoire is highly conserved; however, coral- or cnidarian-specific possible stress response genes are tandemly duplicated in Acropora. Enzymes that cleave dimethlysulfonioproprionate into dimethyl sulfide, which promotes cloud formation and combats greenhouse gasses, are the most duplicated genes in the Acropora ancestor. These may have been acquired by horizontal gene transfer from algal symbionts belonging to the family Symbiodiniaceae, or from coccolithophores, suggesting that although functions of this enzyme in Acropora are unclear, Acropora may have survived warmer marine environments in the past by enhancing cloud formation. In addition, possible antimicrobial peptides and symbiosis-related genes are under positive selection in Acropora, perhaps enabling adaptation to diverse environments. Our results suggest unique Acropora adaptations to ancient, warm marine environments and provide insights into its capacity to adjust to rising seawater temperatures.     

Preparation of 8-Chloropurine Nucleosides Through the Reaction Between their C-8 Lithiated Species and p-Toluenesulfonyl Chloride

Abstract

Chlorination of purine nucleosides protected with tert-butyldimethylsilyl (TBDMS) group was examined by the reaction of the C-8 lithiated species, generated by LDA, with p-toluenesulfonyl chloride as an electrophile. This provides a new method for the preparation of 8-chloropurine nucleosides.     

Anesthetic Effects of a Mixture of Medetomidine,Midazolam and Butorphanol in Two Strains of Mice

The combination of ketamine and xylazine is a widely used anesthetic for laboratory animals. However, due to an abuse problem in Japan, ketamine has been specified as a narcotic since 2007. Instead of using ketamine, Kawai et al. reported an injectable formula with an equivalent effect to the mixture of ketamine and xylazine [11]. The mixture of 0.3 mg/kg body weight (b.w.) medetomidine (Med.), 4.0 mg/kg b.w. midazoram (Mid.), and 5.0 mg/kg b.w. butorphanol (But.) produced an anesthetic duration of around 40 min in outbred ICR mice. However, the anesthetic effect of the mixture for inbred mice strains remains unknown. Therefore, we examined anesthetic effects of the mixture of Med., Mid., and But. in the BALB/c and C57BL/6J strains. After intraperitoneal injection into mice, right front paw, left hind paw, and tail pinch reflexes as well as corneal and righting reflexes were observed. Every 5 min, we scored each reflex category as 0 for reaction or 1 for no reaction. As long as the total score was at least 4 out of 5, we considered the mixture as putting a mouse in a surgical anesthetic state. The mixture produced an anesthetic duration of more than 45 min in both strains of mice. These results indicate that the mixture of Med., Mid., and But. can be a useful and effective anesthesia for the BALB/c and C57BL/6J strains of inbred mice as well as outbred ICR mice.