Canine Distemper Virus Associated with a Lethal Outbreak in Monkeys Can Readily Adapt To Use Human Receptors

A canine distemper virus (CDV) strain, CYN07-dV, associated with a lethal outbreak in monkeys, used human signaling lymphocyte activation molecule as a receptor only poorly but readily adapted to use it following a P541S substitution in the hemagglutinin protein. Since CYN07-dV had an intrinsic ability to use human nectin-4, the adapted virus became able to use both human immune and epithelial cell receptors, as well as monkey and canine ones, suggesting that CDV can potentially infect humans.     

Endocytosis,cadherins and tissue dynamics

By happy chance, the founding of Traffic in 1999 coincided with a clutch of reports that documented the endocytosis and recycling of classical cadherin adhesion receptors. This stimulated a concerted effort to elucidate the molecular regulation of cadherin endocytosis and to identify its functional implications. In particular, endocytosis provided new perspectives to understand how cadherins are modulated during tissue morphogenesis. In this short article, we consider some of what we have learnt about this problem and identify open questions for future research.     

A possible pivotal role of mitochondrial free calcium in neurotoxicity mediated by N-methyl-d-aspartate receptors in cultured rat hippocampal neurons

We have previously shown that mitochondrial membrane potential disruption is involved in mechanisms underlying differential vulnerabilities to the excitotoxicity mediated by N-methyl-d-aspartate (NMDA) receptors between primary cultured neurons prepared from rat cortex and hippocampus. To further elucidate the role of mitochondria in the excitotoxicity after activation of NMDA receptors, neurons were loaded with the fluorescent dye calcein diffusible in the cytoplasm and organelles for determination of the activity of mitochondrial permeability transition pore (mPTP) responsible for the leakage of different mitochondrial molecules. The addition of CoCl₂ similarly quenched the intracellular fluorescence except mitochondria in both cultured neurons, while further addition of NMDA led to a leakage of the dye into the cytoplasm in hippocampal neurons only. An mPTP inhibitor prevented the NMDA-induced loss of viability in hippocampal neurons, while an activator of mPTP induced a similarly potent loss of viability in cortical and hippocampal neurons. Although NMDA was more effective in increasing rhodamine-2 fluorescence as a mitochondrial calcium indicator in hippocampal than cortical neurons, a mitochondrial calcium uniporter inhibitor significantly prevented the NMDA-induced loss of viability in hippocampal neurons. Expression of mRNA was significantly higher for the putative uniporter uncoupling protein-2 in hippocampal than cortical neurons. These results suggest that mitochondrial calcium uniporter would be at least in part responsible for the NMDA neurotoxicity through a mechanism relevant to promotion of mPTP orchestration in hippocampal neurons.     

Acute neuropathogenicity with experimental infection of equine herpesvirus 9 in common marmosets (Callithrix jacchus)

BACKGROUND: Equine herpesvirus 9 (EHV-9) is a new neurotropic equine herpesvirus which induced encephalitis in a variety of animals. However, there was no information on the susceptibility of EHV-9 in primates. METHODS: To assess the infectivity of EHV-9, four common marmosets (Callithrix jacchus) were inoculated by the nasal route with 10(6) plaque-forming units of EHV-9. RESULTS AND CONCLUSIONS: All of the inoculated animals exhibited various neurological signs progressing to collapse. Histologically, the affected animals had severe encephalitis characterized by neuronal degeneration and necrosis with intranuclear inclusion bodies, which extended from the olfactory bulb to the rhinencephalon and piriform lobe. Immunohistochemistry revealed EHV-9 antigens in degenerating neuronal cells. The nasal cavity had severe necrotizing rhinitis with prominent intra-nuclear inclusion bodies in the olfactory mucosa. These findings indicate that the marmosets are susceptible to EHV-9.     

Relative and absolute configuration of antitumor agent SW-163D

Our interest on engineering non-ribosomal synthetase responsible for SW-163 biosynthesis prompted us to determine the relative and absolute configuration of antitumor cyclic depsipeptide SW-163s. We first isolated and identified SW-163 homologs D, F and G as known compounds UK-63598, UK-65662 and UK-63052, respectively. Both enantiomers of the unusual constitutive amino acid, N-methylnorcoromic acid, were synthesized in chiral forms starting from (R)- and (S)-1,2-propanediol. The hydrolyzate of SW-163D, a major constituent of this family, was converted with Marfey's reagent, 1-fluoro-2,4-dinitrophenyl-5-L-alanine-amide (L-FDAA), and the resulting mixture of amino acid derivatives was subjected to an LC/MS analysis. Compared with authentic samples, the analytical data unambiguously show that SW-163D consisted of L-Ala, D-Ser and (1S, 2S)-N-methylnorcoronamic acid. The remaining stereochemistry of the N-methylcysteine moieties was determined from NOE data.     

A novel noninvasive method for assessing glutathione-conjugate efflux systems in the brain

Brain efflux systems export such conjugated metabolites as glutathione (GSH) and glucuronate conjugates, generated by the detoxification process, from the brain and serve to protect the brain from harmful metabolites. The intracerebral injection of a radiolabeled conjugate is a useful technique to assess brain efflux systems; however, this technique is not applicable to humans. Hence, we devised a novel noninvasive approach for assessing GSH-conjugate efflux systems using positron emission tomography. Here, we investigated whether or not a designed proprobe can deliver its GSH conjugate into the brain. Radiolabeled 6-chloro-7-methylpurine (7m6CP) was designed as the proprobe, and [(14)C]7m6CP was prepared by the reaction of 6-chloropurine with [(14)C]CH(3)I as a model of [(11)C]CH(3)I. The radiochemical yield and purity of [(14)C]7m6CP were 10-20% and greater than 99%, respectively. High brain uptake (0.8% ID/g) at 1 min was observed, followed by gradual radioactivity clearance from the brain for 5-60 min after the injection of [(14)C]7m6CP into rats. Analysis of metabolites confirmed that the presence of [(14)C]7m6CP was hardly observed, and 80% of the radioactivity was identical to its GSH conjugate for 15-60 min. The brain radioactivity was single-exponentially decreased during the period of 15-60 min post-injection of [(14)C]7m6CP, and the first-order efflux rate constant of the conjugate, estimated from the slope, was 0.0253 min(-1). These results showed that (1) [(14)C]7m6CP readily entered the brain, (2) it efficiently and specifically transformed to the GSH conjugate within the brain, and (3) after [(14)C]7m6CP disappearance, the clearance of radioactivity represented the only efflux of GSH conjugate. We conclude that 7m6CP can deliver the GSH conjugate into the brain and would be useful for assessing GSH-conjugate efflux systems noninvasively.     

Laninamivir Octanoate and Artificial Surfactant Combination Therapy Significantly Increases Survival of Mice Infected with Lethal Influenza H1N1 Virus

Background

Patients with influenza virus infection can develop severe pneumonia and acute respiratory distress syndrome (ARDS) which have a high mortality. Influenza virus infection is treated worldwide mainly by neuraminidase inhibitors (NAIs). However, monotherapy with NAIs is insufficient for severe pneumonia secondary to influenza virus infection. We previously demonstrated that mice infected with a lethal dose of influenza virus develop diffuse alveolar damage (DAD) with alveolar collapse similar to that seen in ARDS in humans. Additionally, pulmonary surfactant proteins were gradually increased in mouse serum, suggesting a decrease in pulmonary surfactant in the lung. Therefore, the present study examined whether combination therapy of NAI with exogenous artificial surfactant affects mortality of influenza virus-infected mice.

Methodology/Principal Findings

BALB/c mice were inoculated with several viral doses of influenza A/Puerto Rico/8/34 (PR8) virus (H1N1). The mice were additionally administered exogenous artificial surfactant in the presence or absence of a new NAI, laninamivir octanoate. Mouse survival, body weight and general condition were observed for up to 20 days after inoculation. Viral titer and cytokine/chemokine levels in the lungs, lung weight, pathological analysis, and blood O₂ and CO₂ pressures were evaluated. Infected mice treated with combination therapy of laninamivir octanoate with artificial surfactant showed a significantly higher survival rate compared with those that received laninamivir octanoate monotherapy (p = 0.003). However, virus titer, lung weight and cytokine/chemokine responses were not different between the groups. Histopathological examination, a hydrostatic lung test and blood gas analysis showed positive results in the combination therapy group.

Conclusions/Significance

Combination therapy of laninamivir octanoate with artificial surfactant reduces lethality in mice infected with influenza virus, and eventually suppresses DAD formation and preserves lung function. This combination could be effective for prevention of severe pneumonia secondary to influenza virus infection in humans, which is not improved by NAI monotherapy.     

Freezing activities of flavonoids in solutions containing different ice nucleators

In this study, we examined the effects on freezing of 26 kinds of flavonoid compounds, which were randomly selected as compounds with structures similar to those of flavonoid compounds existing in deep supercooling xylem parenchyma cells (XPCs) in trees, in solutions containing different kinds of ice nucleators, including the ice nucleation bacterium (INB) Erwinia ananas, INB Xanthomonas campestris, silver iodide, phloroglucinol and unidentified airborne impurities in buffered Milli-Q water (BMQW). Cumulative freezing spectra were obtained in each solution by cooling 2 μL droplets at 0.2 °C/min by a droplet freezing assay. Freezing temperature of 50% droplets (FT(50)) was obtained from each spectra in a separate analysis with more than 20 droplets and mean FT(50) were obtained from more than five separate analyses using more than 100 droplets in total in each flavonoid. Supercooling-promoting activities (SCA) or ice nucleation-enhancing activities (INA) of these flavonoids were determined by the difference in FT(50) between control solutions without flavonoids and experimental solutions with flavonoids. In mean values, most of the compounds examined exhibited SCA in solutions containing the INB E. ananas, INB X. campestris, silver iodide, and phloroglucinol although the magnitudes of their activities were different depending on the ice nucleator. In solutions containing the INB E. ananas, 10 compounds exhibited SCAs with significant differences (p<0.05) in the range of 1.4-4.2 °C. In solutions containing silver iodide, 23 compounds exhibited SCAs with significant differences in the range of 2.0-7.1 °C. In solutions containing phloroglucinol, six compounds exhibited SCAs with significant differences in the range of 2.4-3.5 °C. In solutions containing the INB X. campestris, only three compounds exhibited SCAs with significant differences in the range of 0.9-2.3 °C. In solutions containing unidentified airborne impurities (BMQW alone), on the other hand, many compounds exhibited INA rather than SCA. In mean values, only four compounds exhibited SCAs in the range of 2.4-3.2 °C (no compounds with significant difference at p<0.05), whereas 21 compounds exhibited INAs in the range of 0.1-12.3 °C (eight compounds with significant difference). It was also shown by an emulsion freezing assay that most flavonoid glycosides examined did not affect homogeneous ice nucleation temperatures, except for a few compounds that become ice nucleators in BMQW alone. These results suggest that most flavonoid compounds affect freezing temperatures by interaction with unidentified ice nucleators in BMQW as examined by a droplet freezing assay. The results of our previous and present studies indicate that flavonoid compounds have very complex effects to regulate freezing of water.     

Two novel oligosaccharides isolated from a beverage produced by fermentation of a plant extract

An extract from 50 kinds of fruits and vegetables was fermented to produce a new beverage. Natural fermentation of the extract was carried out mainly by lactic acid bacteria (Leuconostoc spp.) and yeast (Zygosaccharomyces spp. and Pichia spp.). Two new saccharides were found in this fermented beverage. The saccharides were isolated using carbon-Celite column chromatography and preparative high performance liquid chromatography. Gas liquid chromatography analysis of methylated derivatives as well as MALDI-TOF MS and NMR measurements were used for structural confirmation. The (1)H and (13)C NMR signals of each saccharide were assigned using 2D-NMR including COSY, HSQC, HSQC-TOCSY, CH(2)-HSQC-TOCSY, and CT-HMBC experiments. The saccharides were identified as beta-D-fructopyranosyl-(2-->6)-beta-D-glucopyranosyl-(1-->3)-D-glucopyranose and beta-D-fructopyranosyl-(2-->6)-[beta-D-glucopyranosyl-(1-->3)]-D-glucopyranose.     

Insulin-like growth factor-I activation of Akt survival cascade in neuronal cells requires the presence of its cognate receptor in caveolae

Insulin-like growth factor-I (IGF-I) plays important roles in survival of neurons. Caveolae, cholesterol-rich microdomains of plasma membrane, act as platforms for some neurotrophic factors. In this study, we examined a possible role of caveolae in IGF-I signal transduction in pheochromocytoma PC12 cells. IGF-I treatment attenuated serum withdrawal-induced apoptosis, which was reversed by treatment with methyl-beta-cyclodextrin (CD) that removes cholesterol from plasma membrane. Immunocytochemical and subcellular fractionation analyses revealed that IGF-I receptor (IGF-IR) was colocalized with caveolin-1, a major protein component in caveolae, and that CD treatment reduced IGF-IR contents in caveolae. Consistent with these findings, IGF-I phosphorylation of insulin receptor substrate-1 and Akt was impaired, and cholesterol supply restored the IGF-I action. Furthermore, experiments using small interfering RNA revealed that the reduction of caveolin-1 expression impaired the IGF-I action. In addition, the colocalization of IGF-IR with caveolin-1, and the caveolae-dependent IGF-I action were duplicated in primary culture of rat cerebellar granule neurons. These results demonstrate that the presence of IGF-IR in caveolae is required for the neuroprotective action of IGF-I.