Experimental evaluation of pathogenicity and acquired immunity of Eimeria species, E. uekii and E. raichoi, infecting Japanese rock ptarmigans in a subspecies of the birds.

Japanese rock ptarmigans (Lagopus muta japonica) are birds that inhabit only alpine regions of central Honshu Island, Japan, known as the Japanese Alps. The number of these birds has recently declined, and in situ and ex situ national conservation programs for Japanese rock ptarmigans have been initiated. The infections of Eimeria spp. as protozoan parasites of the phylum Apicomplexa, E. uekii and E. raichoi, were frequently reported in the birds. However, the virulence of these Eimeria parasites has not been determined. Here, we analyzed the pathogenicity of these Eimeria parasites using experimental infections of a subspecies model of Japanese rock ptarmigans, Svalbard rock ptarmigans (Lagopus mutus hyperboreus), and evaluated acquired protective immunity against challenge in birds tolerant of low-dose inoculation with Eimeria parasites. Following inoculation with two Eimeria parasites derived from Japanese rock ptarmigans (dose range of 4 × 104 to 4 × 102 for E. uekii and 1.7 × 104 to 4 × 101 for E. raichoi), oocysts were detected at 6-8 days post-inoculation (PI), and the maximum number of oocysts per gram of feces was observed 7-10 days PI and then gradually decreased. The mortality rate and reduction in weight gain of chicks increased following high-dose inoculation of oocysts with abnormal feces (soft and diarrhea). Developmental zoites were detected histopathologically in epithelial tissues and sometimes the lamina propria from the duodenum to the colon. Chicks that survived low-dose inoculation did not show clear clinical symptoms after challenge inoculation. Our results suggest that the pathological characteristics of Eimeria parasites infecting Japanese rock ptarmigans include abnormal feces and reduction in weight gain, resulting in mortality in cases of heavy infection due to high-dose inoculation. These findings provide helpful data for Japanese rock ptarmigan conservation efforts.

Bufadienolide glycosides and bufadienolides from the whole plants of Helleborus lividus, and their cytotoxic activity.

Cytotoxicity-guided fractionation of the MeOH extract of Helleborus lividus Aiton ex Curtis (Ranunculaceae) resulted in the isolation of five undescribed bufadienolide glycosides and two undescribed bufadienolides, along with three known compounds. Their structures were determined by detailed spectroscopic analysis and hydrolysis studies. The isolated compounds showed cytotoxicity against HL-60 human leukemia cells and A549 human lung adenocarcinoma cells, with IC50 values ranging from 2.20 ± 0.01 nM to 0.77 ± 0.01 µM. The undescribed compound 3ß-[(O-ß-d-glucopyranosyl-(1 â†’ 4)-α-l-rhamnopyranosyl)oxy]-14ß,16ß-dihydroxy-5ß-bufa-20,22-dienolide induced apoptosis in HL-60 cells via a mitochondria-dependent apoptotic pathway. The average IC50 values of bufadienolide monorhamnosides for HL-60 and A549 cells were 10-20 times lower than those for Na+/K+ ATPase, implying that they induce tumor cell death via a mechanism of action other than Na+/K+ ATPase inhibition.

Myeloperoxidase deficiency enhances zymosan phagocytosis associated with up-regulation of surface expression of CD11b in mouse neutrophils.

Myeloperoxidase (MPO), a major component of neutrophils, catalyzes the production of hypochlorous acid (HOCl) from hydrogen peroxide and chloride anion. Phagocytosis is a critical event induced by neutrophils for host defense and inflammation. Interestingly, we found that MPO-deficient (MPO-/-) neutrophils engulfed larger amounts of zymosan than wild-type neutrophils. Blocking of the CD11b subunit of complement receptor 3 (CR3) as well as inhibition of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK) dramatically reduced zymosan phagocytosis. In contrast, blocking of dectin-1, toll-like receptor 2 (TLR2), or spleen tyrosine kinase (Syk) had no significant effects on phagocytosis. Western blotting analysis showed that inhibition of FAK decreased the phosphorylation of ERK1/2, indicating that ERK1/2 is a downstream regulator of FAK in neutrophils. Importantly, we found that cell surface expression of CD11b and phosphorylation of ERK1/2 was significantly higher in zymosan-stimulated MPO-/- neutrophils than in zymosan-stimulated wild-type neutrophils. Pretreatment with the MPO inhibitor 4-aminobenzoic acid hydrazide dramatically enhanced both zymosan phagocytosis and the surface expression of CD11b in wild-type neutrophils, but not in MPO-/- neutrophils. Collectively, these results strongly suggest that up-regulation of the CD11b/FAK/ERK signaling pathway due to absence of MPO enhances the zymosan phagocytic activity of mouse neutrophils.

Detection of RD-114 virus by a reverse transcriptase assay based on product enhancement.

RD-114 virus is a feline endogenous retrovirus that exists in the genome of all cats. It can be assumed that feline and canine live vaccines manufactured by culturing cells of feline origin are contaminated with the virus. The current study is attempted to develop a product enhanced reverse transcriptase (PERT) assay to detect replication-competent RD-114 virus. Since culture supernatants of Crandell-Rees feline kidney (CRFK) cells do not have detectable reverse transcriptase activity in case of do not passage on other cell lines, these results raise the possibility that RD-114 virus grow efficiently in other retrovirus producing cell lines. For the PERT assay, RD-114 virus isolated from CRFK cells may need to be passaged more than four times on 293T cells to be expressed at appreciable levels. The PERT assay described here provides an accurate method for determining the presence of RD-114 in culture supernatants and will help monitor live vaccines produced in endogenous virus producing cell lines.

Lithography of self-assembled semiconductor quantum dots on templates fabricated from mixed Langmuir-Blodgett films.

In this paper, we discuss a useful technique to fabricate patterned single layers consisting of quantum dots on two-dimensional templates fabricated from phase-separated mixed Langmuir-Blodgett (LB) films. The phase-separated structures are governed by two competing interactions-line tension and dipole-dipole interaction-and are tunable by adjusting the intermolecular interactions between the film-forming molecules and the fabrication conditions. The templates can be fabricated from mixed LB films containing silane-coupling agents, which form covalent bonds with Si wafers. The CdS- or CdSe/ZnS-nanoparticles were immobilized on the templates using a chemisorption technique. The samples were analyzed using ultraviolet-visible absorption spectroscopy, emission spectroscopy, atomic force microscopy, scanning electron microscopy, and fluorescence microscopy. The diameters of the CdS-NPs and CdSe/ZnS were estimated to be about 2.6 and 4.5 nm, respectively, using the Brus equation. We successfully obtained a patterned single layer consisting of quantum dots on the templates. The density of the immobilized-CdSe/ZnS-NPs on these templates can be controlled through variations in the concentration of the aqueous dispersion of CdSe/ZnS-NPs. Fluorescence observations indicate that the immobilized-CdSe/ZnS-NPs on the templates serve as light-emitting devices. These results indicate that patterned quantum dots can be formed only through self-assembly processes.

Antinociceptive effect of oxycodone in diabetic mice.

The effect of oxycodone on thermal hyperalgesia in streptozotocin-induced diabetic mice was examined. The antinociceptive response was assessed by recording the latency in the tail-flick test using the radiant heat from a 50-W projection bulb on the tail. The tail-flick latency in diabetic mice was significantly shorter than that in non-diabetic mice. When diabetic mice were treated with oxycodone (5 mg/kg, s.c.), the tail-flick latency in diabetic mice was prolonged to the level considerably longer than the baseline latencies of non-diabetic mice. However, s.c. administration of morphine (5 mg/kg) did not produce a significant inhibition of the tail-flick response in diabetic mice. Oxycodone, at doses of 1.25-5.0 mg/kg administered s.c., produced a dose-dependent increase in the tail-flick latencies in both diabetic and non-diabetic mice. The antinociceptive effect of oxycodone was antagonized by pretreatment with a selective delta-opioid receptor antagonist, beta-funaltrexamine (20 mg/kg, s.c.), in both non-diabetic and diabetic mice. In non-diabetic mice, pretreatment with a selective kappa-opioid receptor antagonist, nor-binaltorphimine (20 mg/kg, s.c.) had no effect on the peak antinociceptive effect of oxycodone observed 30 min after administration, however, it slightly but significantly reduced oxycodone-induced antinociception observed 60 and 90 min after administration. On the other hand, pretreatment with nor-binaltorphimine practically abolished the peak (30 min) and persistent (60 and 90 min) antinociceptive effects of oxycodone in diabetic mice. Naltrindole (35 mg/kg, s.c.), a selective delta-opioid receptor antagonist, had no effects on the antinociceptive effect of oxycodone in both non-diabetic and diabetic mice. These results suggest that the antinociceptive effects of oxycodone may be mediated by mu- and kappa-opioid receptors in diabetic mice, whereas it may interact primarily with mu-opioid receptors in non-diabetic mice.

Possible involvement of the spinal nitric oxide/cGMP pathway in vincristine-induced painful neuropathy in mice.

The mechanisms that underlie the development of vincristine-induced painful neuropathy are poorly understood. The nitric oxide (NO)-cGMP pathway has been reported to be involved in the spinal transmission of nociceptive information. In the present study, we examined whether alterations in spinal nociceptive processing via the NO-cGMP pathway contribute to vincristine-induced painful neuropathy in mice. Mice were intraperitoneally treated with vincristine at a dose of 0.05 mg/kg 1 day after the measurement of pre-drug latency in the tail-flick test, and then treated with a dose of 0.125 mg/kg twice a week for 6 weeks. In vincristine-treated mice, a significant decrease in tail-flick latencies developed at 4 weeks after treatment. Pretreatment with L-arginine (30-300 mg/kg, s.c.), a substrate of NO synthase (NOS), dose-dependently increased the tail-flick latencies in vincristine-treated mice. The L-arginine-induced increase in tail-flick latencies in vincristine-treated mice was completely reversed by i.t. pretreatment with NG-nitro-L-arginine methyl ester (L-NAME, 3-30 nmol), a NOS inhibitor. Furthermore, i.t. pretreatment with 8-bromoguanosine 3', 5'-cyclic monophosphate (8-Br-cGMP, 0.3-3.0 nmol), a membrane-permeable cGMP analog, dose-dependently increased the tail-flick latencies in vincristine-treated mice. The contents of NO metabolites, cGMP and protein levels of neuronal NOS in the spinal cord in vincristine-treated mice were significantly reduced compared to those in vehicle-treated naive mice. These results indicate that dysfunction of the L-arginine/NO/cGMP cascade in the spinal cord may trigger vincristine-induced thermal hyperalgesia in mice.

Effect of mexiletine on fenvalerate-induced nociceptive response in diabetic mice.

The effect of mexiletine on the nociceptive behavior induced by the intrathecal injection of fenvalerate, which predominantly activates tetrodotoxin-resistant (TTX-R) sodium channels, was studied in diabetic mice. The intrathecal injection of fenvalerate induced a characteristic behavioral syndrome that mainly consisted of reciprocal hind limb scratching directed toward caudal parts of the body and biting or licking of the hind legs in mice. The intensity of fenvalerate-induced nociceptive responses was significantly greater in diabetic mice than non-diabetic mice. This fenvalerate-induced behavior was dose-dependently inhibited by mexiletine (3-30 mg/kg, i.p.). Intrathecal pretreatment with fenvalerate produced thermal hyperalgesia and allodynia in the tail-flick test in naive mice. Furthermore, mexiletine at doses of 10 and 30 mg/kg, i.p., dose-dependently and significantly reduced fenvalerate-induced thermal hyperalgesia and allodynia in the tail-flick test in naive mice. These present data suggest that i.p. pretreatment with mexiletine produced dose-dependent inhibition of fenvalerate-induced hyperalgesia and allodynia in mice, especially diabetic mice. This effect may be, at least in part, mediated by the inhibition of TTX-R sodium channel-mediated nociceptive transmission in the spinal cord.