Simultaneous serodetection of major rat infectious pathogens by a multiplex immunochromatographic assay

Rapid and simple serologic tests that require only a small amount of blood without the euthanization of animals are valuable for microbial control in colonies of laboratory animals. In this study, we developed a multiplex immunochromatographic assay (ICA) for detection of antibodies to Sendai virus (also known as hemagglutinating virus of Japan), hantavirus, and sialodacryoadenitis virus, which are causative agents of major infectious diseases in rats. For this assay, an ICA strip was placed into a microtube containing 150 µl PBS and either 0.75 µl of rat serum or 1.5 µl of whole blood. Binding antibodies were visualized by using anti-rat IgG antibody-conjugated colloidal gold. Under these conditions, the multiplex ICA simultaneously and specifically detected antibodies to multiple antigens. Positive serum samples for each infectious disease were used to evaluate the sensitivity and specificity of the multiplex ICA. The sensitivities of the multiplex ICA for Sendai virus, hantavirus, and sialodacryoadenitis virus were 100%, 100%, and 81%, respectively. No nonspecific reactions were observed in any of the 52 positive sera against heterologous antigens. In addition, 10 samples of uninfected sera did not show any bands except for the control line. These observations indicate high specificity of the multiplex ICA. Moreover, the multiplex ICA could be applied to diluted blood. These results indicate that the multiplex ICA is appropriate for rapid and simple serological testing of laboratory rats.     

Functional analysis of three novel cell lines derived from human papillary thyroid carcinomas with three different clinical courses

Papillary thyroid carcinoma (PTC) is the most frequent thyroid carcinoma. PTC cell lines have been of considerable value in studying aspects of thyroid cancer, such as gene expression, cell proliferation, and differentiation. Here we report three novel PTC lines established from three patients with different backgrounds. Case 1 was a 38-year-old woman with PTC in the right thyroid lobe, with no metastasis. The cell line was established from the resection sample and named D-PTC. The cell line consisted of epithelial cells with few lysosomes and showed a pavement structure and follicular formation at confluency. There was a little pilling up. The secretion of free thyroxin (fT4) and thyroglobulin (Tg) was increased by TSH, or GH and IGF-I treatment. Case 2 was a 22-year-old woman with PTC initially in the right thyroid lobe, but 4 years after the right lobe resection, PTC metastasis was observed in left lobe. The cell line was established from a sample of the second resection and named UD-PTC. This cell line consisted of small epithelial cells with evident lysosomes and exhibited floating cell clusters. The secretion of fT4 and Tg was slightly increased by TSH, or GH and IGF-I treatment. Case 3 was an 85-year-old man with PTC and with acromegaly. Metastasis was observed at cervical lymph nodes. The cell line was derived from the metastasis region and named A-PTC. This cell line consisted of small epithelial cells and many lysosomes. The cells frequently showed pilling up. The secretion of fT4 and Tg was significantly increased by GH and IGF-I treatment. We have established three PTC cell lines with substantial variation in their phenotype. The cell lines may be useful for thyroid cancer research.     

An In Vitro Model for the Study of Cellular Pathophysiology in Globoid Cell Leukodystrophy

The precise function of multi-nucleated microglia, called globoid cells, that are uniquely abundant in the central nervous system of globoid cell leukodystrophy (GLD) is unclear. This gap in knowledge has been hindered by the lack of an appropriate in vitro model for study. Herein, we describe a primary murine glial culture system in which treatment with psychosine results in multinucleation of microglia resembling the characteristic globoid cells found in GLD. Using this novel system, we defined the conditions and modes of analysis for study of globoid cells. The potential use of this model system was validated in our previous study, which identified a potential role for matrix metalloproteinase (MMP)-3 in GLD. This novel in vitro system may be a useful model in which to study the formation and function, but also the potential therapeutic manipulation, of these unique cells.     

Isolation and validation of eight microsatellite loci in a damselfly Ischnura senegalensis (Odonata: Coenagrionidae) by pyrosequencing a bead-enriched library

Eight microsatellite markers for the population genetics and evolutionary ecology of Ischnura senegalensis (Rambur 1842), which shows body color polymorphism in females, were developed using a streptavidin-bead enrichment library and pyrosequence by a next generation sequencer. The number of alleles per locus and effective number of alleles ranged from 3 to 11 and from 1.24 to 5.51, respectively. Observed and expected heterozygosities were 0.18–0.75 and 0.19–0.77, respectively. No linkage disequilibrium between loci was detected. One locus, IsenAC75, deviated significantly from the Hardy–Weinberg equilibrium, and the locus and additional two loci, IsenAC40 and IsenAC8, were suspected for the presence of null alleles. Altogether, these eight microsatellite loci are considered to be useful for population genetic analyses because of the high polymorphic status and independency.     

Role of the CpxAR Two-Component Signal Transduction System in Control of Fosfomycin Resistance and Carbon Substrate Uptake

Although fosfomycin is an old antibiotic, it has resurfaced with particular interest. The antibiotic is still effective against many pathogens that are resistant to other commonly used antibiotics. We have found that fosfomycin resistance of enterohemorrhagic Escherichia coli (EHEC) O157:H7 is controlled by the bacterial two-component signal transduction system CpxAR. A cpxA mutant lacking its phosphatase activity results in constitutive activation of its cognate response regulator, CpxR, and fosfomycin resistance. We have shown that fosfomycin resistance requires CpxR because deletion of the cpxR gene in the cpxA mutant restores fosfomycin sensitivity. We have also shown that CpxR directly represses the expression of two genes, glpT and uhpT, which encode transporters that cotransport fosfomycin with their native substrates glycerol-3-phosphate and glucose-6-phosphate, and repression of these genes leads to a decrease in fosfomycin transport into the cpxA mutant. However, the cpxA mutant had an impaired growth phenotype when cultured with glycerol-3-phosphate or glucose-6-phosphate as a sole carbon substrate and was outcompeted by the parent strain, even in nutrient-rich medium. This suggests a trade-off between fosfomycin resistance and the biological fitness associated with carbon substrate uptake. We propose a role for the CpxAR system in the reversible control of fosfomycin resistance. This may be a beneficial strategy for bacteria to relieve the fitness burden that results from fosfomycin resistance in the absence of fosfomycin.     

Central bombesin possibly induces S-nitrosylation of cyclooxygenase-1 in pre-sympathetic neurons of rat hypothalamic paraventricular nucleus

Aims

Cyclooxygenase (COX) can be activated by nitric oxide-induced (NO-induced) conversion of cysteine thiol group of COX into S-nitrosothiol. We previously reported the involvement of brain COX/NO synthase (NOS) in centrally administered bombesin-, a stress-related neuropeptide, induced secretion of rat adrenal noradrenaline and adrenaline. To examine a possible involvement of the NO-induced modification of COX in bombesin-induced response, we investigated whether bombesin induces close proximity of COX-1 and neuronal NOS (nNOS) or S-nitroso-cysteine in pre-sympathetic spinally projecting neurons in the rat hypothalamic paraventricular nucleus (PVN), a regulatory center of adrenomedullary outflow.

Main methods

In twelve-week-old male Wistar rats, pre-sympathetic spinally projecting neurons in the PVN were labeled with a retrograde tracer Fluoro-Gold (FG). After intracerebroventricular administration of bombesin, we performed double immunohistochemical analysis for Fos and COX-1 or nNOS in FG-labeled PVN neurons. We also performed a fluorescent in situ proximity ligation assay (PLA) for visualizing of close proximity (< 40 nm) of COX-1 with nNOS or S-nitroso-cysteine.

Key. findings

Bombesin significantly increased the number of Fos-immunoreactive cells in FG-labeled PVN neurons with COX-1 or nNOS immunoreactivity. 7-Nitroindazole, a selective nNOS inhibitor, abolished Fos-immunoreactivity induced by bombesin in COX-1-immunoreactive FG-labeled PVN neurons. Bombesin also induced PLA-positive signals indicating close proximity of COX-1/nNOS and COX-1/S-nitroso-cysteine in FG-labeled PVN neurons.

Significance

Centrally administered bombesin possibly induces S-nitrosylation of COX-1 through close proximity of COX-1 and nNOS in pre-sympathetic spinally projecting PVN neurons, thereby activating COX-1 during the bombesin-induced activation of central adrenomedullary outflow in the rat.     

Prostaglandin EP3 receptor superactivates adenylyl cyclase via the Gq/PLC/Ca pathway in a lipid raft-dependent manner

We previously demonstrated that prostaglandin EP3 receptor augments EP2-elicited cAMP formation in COS-7 cells in a G_i/o-insensitive manner. The purpose of our current study was to identify the signaling pathways involved in EP3-induced augmentation of receptor-stimulated cAMP formation. The enhancing effect of EP3 receptor was irrespective of the C-terminal structure of the EP3 isoform. This EP3 action was abolished by treatment with inhibitors for phospholipase C and intracellular Ca²⁺-related signaling molecules such as U73122, staurosporine, 2-APB and SK&F 96365. Indeed, an EP3 agonist stimulated IP₃ formation and intracellular Ca²⁺ mobilization, which was blocked by U73122, but not by pertussis toxin. The enhancing effect by EP3 on cAMP formation was mimicked by both a Ca²⁺ ionophore and the activation of a typical G_q-coupled receptor. Moreover, EP3 was exclusively localized to the raft fraction in COS-7 cells and EP3-elicited augmentation of cAMP formation was abolished by cholesterol depletion and introduction of a dominant negative caveolin-1 mutant. These results suggest that EP3 elicits adenylyl cyclase superactivation via G_q/phospholipase C activation and intracellular Ca²⁺ mobilization in a lipid raft microdomain-dependent manner.     

The regulation of formation of prostaglandins and arachidonoyl-CoA from arachidonic acid in rabbit kidney medulla microsomes by linoleic acid hydroperoxide

Under physiological conditions, small amounts of free arachidonic acid (AA) are released from membrane phospholipids, and cyclooxygenase (COX) and acyl-CoA synthetase (ACS) competitively act on this fatty acid to form prostaglandins (PGs) and arachidonoyl-CoA (AA-CoA). In the present study, we investigated the effects of linoleic acid (LA) and 13-hydroperoxyoctadecadienoic acid (13-HPODE) on the PG and AA-CoA formation from high and low concentrations of AA (60 and 5 microM) in rabbit kidney medulla microsomes. The kidney medulla microsomes were incubated with 60 or 5 microM [(14)C]-AA in 0.1M Tris-HCl buffer (pH 8.0) containing cofactors of COX (reduced glutathione and hydroquinone) and cofactors of ACS (ATP, MgCl(2) and CoA). After incubation, PG (as total PGs), AA-CoA and residual AA were separated by selective extraction using petroleum ether and ethyl acetate. LA (10-50 microM) reduced only PG formation from both 60 and 5 microM AA. 13-HPODE (10-50 microM) also reduced PG formation from 60 and 5 microM AA, but the inhibitory potency was much stronger than that by LA. Furthermore, 13-HPODE had the potential to increase the AA-CoA formation with a decrease in the PG formation from 5 microM AA. These results suggest that 13-HPODE, but not LA, may shift AA away from COX pathway into ACS pathway under low substrate concentration (near physiological concentration of AA).