Switching species tropism: an effective way to manipulate the feline coronavirus genome

Feline infectious peritonitis virus (FIPV), a coronavirus, is the causative agent of an invariably lethal infection in cats. Like other coronaviruses, FIPV contains an extremely large positive-strand RNA genome of ca. 30 kb. We describe here the development and use of a reverse genetics strategy for FIPV based on targeted RNA recombination that is analogous to what has been described for the mouse hepatitis virus (MHV) (L. Kuo et al., J. Virol. 74:1393-1406, 2000). In this two-step process, we first constructed by targeted recombination a mutant of FIPV, designated mFIPV, in which the ectodomain of the spike glycoprotein was replaced by that of MHV. This switch allowed for the selection of the recombinant virus in murine cells: mFIPV grows to high titers in these cells but has lost the ability to grow in feline cells. In a second, reverse process, mFIPV was used as the recipient, and the reintroduction of the FIPV spike now allowed for selection of candidate recombinants by their regained ability to grow in feline cells. In this fashion, we reconstructed a wild-type recombinant virus (r-wtFIPV) and generated a directed mutant FIPV in which the initiation codon of the nonstructural gene 7b had been disrupted (FIPV Delta 7b). The r-wtFIPV was indistinguishable from its parental virus FIPV 79-1146 not only for its growth characteristics in tissue culture but also in cats, exhibiting a highly lethal phenotype. FIPV Delta 7b had lost the expression of its 7b gene but grew unimpaired in cell culture, confirming that the 7b glycoprotein is not required in vitro. We establish the second targeted RNA recombination system for coronaviruses and provide a powerful tool for the genetic engineering of the FIPV genome.     

Interference of S-Alkyl Derivatives of Glutathione with Brain Ionotropic Glutamate Receptors

The effects of glutathione, glutathione sulfonate and S-alkyl derivatives of glutathione on the binding of glutamate and selective ligands of ionotropic N-methyl-D-aspartate (NMDA) and non-NMDA receptors were studied with mouse synaptic membranes. The effects of glutathione and its analogues on ⁴⁵Ca²⁺ influx were also estimated in cultured rat cerebellar granule cells. Reduced and oxidized glutathione, glutathione sulfonate, S-methyl-, -ethyl-, -propyl-, -butyl- and -pentylglutathione inhibited the Na⁺-independent binding of L-[³H]glutamate. They strongly inhibited also the binding of (S)-2-amino-3-hydroxy-5-[³H]methyl-4-isoxazolepropionate [³H]AMPA (IC₅₀ values: 0.8–15.9 M). S-Alkylation of glutathione rendered the derivatives unable to inhibit [³H]kainate binding. The NMDA-sensitive binding of L-[³H]glutamate and the binding of 3-[(R)-2-carboxypiperazin-4-yl][1,2-³H]propyl-1-phosphonate ([³H]CPP, a competitive antagonist at NMDA sites) were inhibited by the peptides at micromolar concentrations. The strychnine-insensitive binding of the NMDA coagonist [³H]glycine was attenuated only by oxidized glutathione and glutathione sulfonate. All peptides slightly enhanced the use-dependent binding of [³H]dizocilpine (MK-801) to the NMDA-gated ionophores. This effect was additive with the effect of glycine but not with that of saturating concentrations of glutamate or glutamate plus glycine. The glutamate- and NMDA-evoked influx of ⁴⁵Ca²⁺ into cerebellar granule cells was inhibited by the S-alkyl derivatives of glutathione. We conclude that besides glutathione the endogenous S-methylglutathione and glutathione sulfonate and the synthetic S-alkyl derivatives of glutathione act as ligands of the AMPA and NMDA receptors. In the NMDA receptor-ionophore these glutathione analogues bind preferably to the glutamate recognition site via their -glutamyl moieties.     

Low-frequency modes of peptides and globular proteins in solution observed by ultrafast OHD-RIKES spectroscopy

The low-frequency (1-200 cm(-1)) vibrational spectra of peptides and proteins in solution have been investigated with ultrafast optical heterodyne-detected Raman-induced Kerr-effect spectroscopy (OHD-RIKES). Spectra have been obtained for di-L-alanine (ALA(2)) and the alpha-helical peptide poly-L-alanine (PLA) in dichloroacetic acid solution. The poly-L-alanine spectrum shows extra amplitude compared to the di-L-alanine spectrum, which can be explained by the secondary structure of the former. The globular proteins lysozyme, alpha-lactalbumin, pepsin, and beta-lactoglobulin in aqueous solution have been studied to determine the possible influence of secondary or tertiary structure on the low-frequency spectra. The spectra of the globular proteins have been analyzed in terms of three nondiffusive Brownian oscillators. The lowest frequency oscillator corresponds to the so-called Boson peak observed in inelastic neutron scattering (INS). The remaining two oscillators are not observed in inelastic neutron scattering, do therefore not involve significant motion of hydrogen atoms, and may be associated with delocalized backbone torsions.     

Specific features of ex‐obese patients significantly influence the functional cell properties of adipose‐derived stromal cells

Adipose‐derived stromal cells (ADSC) are increasingly used in clinical applications due to their regenerative capabilities. However, ADSC therapies show variable results. This study analysed the effects of specific factors of ex‐obese patients on ADSC functions. ADSC were harvested from abdominal tissues (N = 20) after massive weight loss. Patients were grouped according to age, sex, current and maximum body mass index (BMI), BMI difference, weight loss method, smoking and infection at the surgical site. ADSC surface markers, viability, migration, transmigration, sprouting, differentiation potential, cytokine secretion, telomere length and mtDNA copy number were analysed. All ADSC expressed CD73, CD90, CD105, while functional properties differed significantly among patients. A high BMI difference due to massive weight loss was negatively correlated with ADSC proliferation, migration and transmigration, while age, sex or weight loss method had a smaller effect. ADSC from female and younger donors and individuals after weight loss by increase of exercise and diet change had a higher activity. Telomere length, mtDNA copy number, differentiation potential and the secretome did not correlate with patient factors or cell function. Therefore, we suggest that factors such as age, sex, increase of exercise and especially weight loss should be considered for patient selection and planning of regenerative therapies.     

Seasonality in the altitude–diversity pattern of Alpine moths

Altitudinal gradients are frequently used to study environmental effects on species diversity. Recent quantitative studies on Lepidoptera focussed on tropical mountain systems and often reported unimodal diversity peaks at “mid-elevations”;, a pattern also often found in other taxa. Here we used methodologically comparable, nocturnal Macrolepidoptera samples from the Swiss Alps to analyze environmental correlates of diversity. Using seasonal data (monthly samples from April to November at altitudes between 600 and 2400 m a.s.l.) allowed to decouple altitude and some climate variables for analyses. We found that the altitude–diversity pattern changes with season. In spring and autumn, diversity decreased with increasing altitude, while we found a unimodal peak of diversity at mid-elevations during summer. This excluded all hypothetical causes of diversity variation that do not allow for seasonality. Temperature was an important correlate of diversity, whereas precipitation was not. These results were separately corroborated for the two most common families (Noctuidae and Geometridae). However, diversity patterns of the two families were not particularly close, and unexplained variance of climatic explanations was substantial in all cases. The patterns of faunal overlap did not explain the unimodal diversity pattern, and we claim that we lack a generally valid explanation for this common phenomenon.     

Red light imaging for programmed cell death visualization and quantification in plant–pathogen interactions

Studies on plant–pathogen interactions often involve monitoring disease symptoms or responses of the host plant to pathogen-derived immunogenic patterns, either visually or by staining the plant tissue. Both these methods have limitations with respect to resolution, reproducibility, and the ability to quantify the results. In this study we show that red light detection by the red fluorescent protein (RFP) channel of a multipurpose fluorescence imaging system that is probably available in many laboratories can be used to visualize plant tissue undergoing cell death. Red light emission is the result of chlorophyll fluorescence on thylakoid membrane disassembly during the development of a programmed cell death process. The activation of programmed cell death can occur during either a hypersensitive response to a biotrophic pathogen or an apoptotic cell death triggered by a necrotrophic pathogen. Quantifying the intensity of the red light signal enables the magnitude of programmed cell death to be evaluated and provides a readout of the plant immune response in a faster, safer, and nondestructive manner when compared to previously developed chemical staining methodologies. This application can be implemented to screen for differences in symptom severity in plant–pathogen interactions, and to visualize and quantify in a more sensitive and objective manner the intensity of the plant response on perception of a given immunological pattern. We illustrate the utility and versatility of the method using diverse immunogenic patterns and pathogens.     

Demonstration of Kynurenine Aminotransferases I and II and Characterization of Kynurenic Acid Synthesis in Oligodendrocyte Cell Line (OLN-93)

In the present study we demonstrate for the first time that both kynurenine aminotransferase (KAT) isoforms I and II are present in the permanent immature rat oligodendrocytes cell line (OLN-93). Moreover, we provide evidence that OLN-93 cells are able to synthesize kynurenic acid (KYNA) from exogenously added l-kynurenine and we characterize its regulation by extrinsic factors. KYNA production in OLN-93 cells was depressed in the presence of aminotransferase inhibitor, aminooxyacetic acid and was not affected by depolarizing agents such as 50 mM K⁺ and 4-aminopyridine. Glutamate agonists, l-glutamate and d,l-homocysteine significantly decreased KYNA production. Selective agonist of ionotropic glutamate receptors Amino-2,3-dihydro-5-methyl-3-oxo-4-isoxazolepropionic acid (AMPA) lowered KYNA production in OLN-93 cell line, whereas N-methyl-d-aspartate (NMDA) had no influence on KYNA production. Furthermore, KYNA synthesis in OLN-93 cells was decreased in a concentration-dependent manner by amino acids transported by l-system, l-leucine, l-cysteine and l-tryptophan. The role of KYNA synthesis in oligodendrocytes needs further investigation.     

Chlamydia pneumoniae infection acts as an endothelial stressor with the potential to initiate the earliest heat shock protein 60-dependent inflammatory stage of atherosclerosis

We identified increased expression and redistribution of the intracellular protein 60-kDa human heat shock protein (hHSP60) (HSPD1) to the cell surface in human endothelial cells subjected to classical atherosclerosis risk factors and subsequent immunologic cross-reactivity against this highly conserved molecule, as key events occurring early in the process of atherosclerosis. The present study aimed at investigating the role of infectious pathogens as stress factors for vascular endothelial cells and, as such, contributors to early atherosclerotic lesion formation. Using primary donor-matched arterial and venous human endothelial cells, we show that infection with Chlamydia pneumoniae leads to marked upregulation and surface expression of hHSP60 and adhesion molecules. Moreover, we provide evidence for an increased susceptibility of arterial endothelial cells for redistribution of hHSP60 to the cellular membrane in response to C. pneumoniae infection as compared to autologous venous endothelial cells. We also show that oxidative stress has a central role to play in endothelial cell activation in response to chlamydial infection. These data provide evidence for a role of C. pneumoniae as a potent primary endothelial stressor for arterial endothelial cells leading to enrichment of hHSP60 on the cellular membrane and, as such, a potential initiator of atherosclerosis.     

Male Corncrakes Crex crex extend their home ranges by visiting the territories of neighbouring males

Capsule Radiotracked male Corncrake often intruded on the territories of neighbouring males.

Aims To test that intruders' visits are goal-directed, not just a by-product of extended spatial activity during daylight hours.

Methods Using radiotelemetry, we sampled a total of 20 three-day home ranges from 11 tagged males. We recorded daily vocal activity and used a permutation test to see if the movements of tracked males were independent of the position of neighbouring males.

Results The majority of males who had a neighbouring male, up to approximately 600 m from their night calling site, undertook goal-directed visits to the neighbour's territory. Males undertook these visits every day, or every other day, when the neighbours were close. Males undertook visits approximately once every three days when they were more distant. The time spent in the neighbour's territory was longest where the distance between night calling sites was about 200 m. Males tended to be silent in neighbour's territory, apparently to prevent confrontation. Otherwise the distance of neighbouring males did not significantly affect daytime vocal activity. Visiting males tended to sing more often in their home territories.

Conclusions Daily movement of the majority of males was towards the neighbouring male's calling site. We suggest that the purpose of these visits was to seek females. These males may try to drive a female into their territory or gain extra-pair copulation.