Mapping of the Feline Calicivirus Proteinase Responsible for Autocatalytic Processing of the Nonstructural Polyprotein and Identification of a Stable Proteinase-Polymerase Precursor Protein

Expression of the region of the feline calicivirus (FCV) ORF1 encoded by nucleotides 3233 to 4054 in an in vitro rabbit reticulocyte system resulted in synthesis of an active proteinase that specifically processes the viral nonstructural polyprotein. Site-directed mutagenesis of the cysteine (Cys1193) residue in the putative active site of the proteinase abolished autocatalytic cleavage as well as cleavage of the viral capsid precursor, suggesting that this "3C-like" proteinase plays an important role in proteolytic processing during viral replication. Expression of the region encoding the C-terminal portion of the FCV ORF1 (amino acids 942 to 1761) in bacteria allowed direct N-terminal sequence analysis of the virus-specific polypeptides produced in this system. The results of these analyses indicate that the proteinase cleaves at amino acid residues E960-A961, E1071-S1072, E1345-T1346, and E1419-G1420; however, the cleavage efficiency is varied. The E1071-S1072 cleavage site defined the N terminus of a 692-amino-acid protein that contains sequences with similarity to the picornavirus 3C proteinase and 3D polymerase domains. Immunoprecipitation of radiolabeled proteins from FCV-infected feline kidney cells with serum raised against the FCV ORF1 C-terminal region showed that this "3CD-like" proteinase-polymerase precursor protein is apparently stable and accumulates in cells during infection.     

Vegetation changes in forests of the Krkonoše Mts. over a period of air pollution stress (1980–1995)

Species composition of the understory of spruce (Picea abies (L.) Karsten) and beech (Fagus sylvatica L.) forests subjected to intense air pollution stress in the Krkonoe Mts., Czech Republic, showed directional changes over the last 15 years. The changes were documented with repeated observations of 29 permanent plots, initiated in 1980 and analysed with constrained ordination methods (Canonical Correspondence Analysis). Ground layer changes were mainly associated with the loss of canopy foliage from air pollution stress. The increased amount of the light at ground level resulted in increased herb cover. The initially dominant species (e.g., Calamagrostis villosa (Chaix) J. F. Gmelin, Deschampsia flexuosa (L.) P. B., Vaccinium myrtillus L., Athyrium distentifolium Tausch) were those that increased in cover. Moss cover, and moss and herb richness, declined over the course of the study. Thus, the changes in tree canopy are accompanied with changes in the forest understory, which in turn can affect the dynamics of these forests.     

Effects of atropine on the release of newly synthesized acetylcholine from rat striatal slices at various concentrations of calcium ions

The release of acetylcholine (ACh) from brain tissue is known to be inhibited by muscarinic autoreceptors on cholinergic nerve terminals but the mechanism of the inhibition is not understood. Atropine brings about an increase of ACh release by removing the inhibitory action of autoreceptors. We investigated whether the effect of atropine on the release of [¹⁴C]ACh newly synthesized during incubations from [U-¹⁴C] glucose depends on the concentration of Ca²⁺ in the medium. In rat striatal slices incubated in the presence of an inhibitor of cholinesterases and of 30 mmol/l K⁺, significant increases in the release of [¹⁴C]ACh elicited by atropine were only observed during incubations with very low concentrations of Ca²⁺. This finding supports the view that the activation of presynaptic muscarinic autoreceptors in the brain affects the release of ACh by reducing the availability of Ca²⁺ that is required for transmitter liberation.     

Simulated microgravity effects on the rat carotid and femoral arteries: role of contractile protein expression and mechanical properties of the vessel wall.

The goal of this study was to determine the effects of microgravity on myofilament protein expression and both passive and active length-force relationships in carotid and femoral arteries. Microgravity was simulated by 20-day hindlimb unweighting (HU) in Wistar male rats, and carotid and femoral artery segments were isolated from both HU and control (CTL) rats for Western blot and length-force analysis. Western blots revealed that HU significantly decreased myosin light chain-20 (MLC-20) protein levels in both carotid and femoral arteries and decreased myosin heavy chain (MHC) in femoral artery. alpha-Actin levels were not altered by HU treatment in either artery. Length-force analysis demonstrated that HU did not change either passive or active length-force relationships in the femoral artery. HU-treated arterial rings developed significantly less force to 100 mM K(+) than CTL, but optimal lengths were identical. In the carotid artery, length-active force curves were identical for both CTL and HU; however the length-passive force curve for HU-treated rings exhibited a steeper slope than CTL, suggesting decreased compliance of the artery wall. In conclusion, our data suggest that the HU-induced decreases in both MLC-20 and MHC in femoral artery are responsible for the decreased contraction to 100 mM K(+) in HU-treated femoral artery rings. In the carotid artery, the HU-induced decrease in vessel wall compliance may counter any decrease in contractility caused by the decreased MLC-20 levels.     

Structure and function of the arolium of Mantophasmatodea (Insecta)

All species of the insect order Mantophasmatodea characteristically keep the 5th tarsomere and pretarsus (arolium plus two claws) turned upwards and off the substrate. The unusually large arolium was studied in two species of Mantophasmatodea using bright field light microscopy, reflection microscopy, fluorescence microscopy, TEM, SEM, and Cryo‐SEM. It contains an epithelial gland, numerous tracheoles, and nerves. The gland consists of enlarged epithelial cells with large nuclei, mitochondria, RER, golgi complexes, microtubules, and numerous secretion vesicles. Evidence for exocytosis of the vesicles into the gland reservoir between the epithelial gland and the thick cuticle could be observed. Cryo‐SEM revealed that the ventral side of the arolium and distal part of its dorsal side are covered with a liquid film. Fluid footprints of arolia of individuals walking on a glass plate also indicate the presence of secretory fluid on the arolium surface. Behavioral experiments using animals with ablated arolia showed that representatives of Mantophasmatodea do not need their arolia to detect and respond to vibratory communication signals nor to catch small to medium‐sized prey. Individuals with ablated arolia were not able to move upside down on a smooth glass plate. We conclude that Mantophasmatodea use their arolia for attachment when additional adhesion force is required (e.g. windy conditions, handling large prey, mating). They can bring their arolia in contact with the surface in a very fast reflex (18.0 ± 9.9 ms). The secretory fluid found on the surface is produced by the gland and transported to the outside, presumably through small pore channels, to enhance adhesion to the substrate. J. Morphol., 2009. © 2009 Wiley‐Liss, Inc.     

Mutagenic analysis of potato virus X movement protein (TGBp1) and the coat protein (CP): in vitro TGBp1-CP binding and viral RNA translation activation

Previously, we have shown that encapsidated Potato virus X (PVX) RNA was non-translatable in vitro , but could be converted into a translatable form by binding of the PVX movement protein TGBp1 to one end of the virion or by coat protein (CP) phosphorylation. Here, a mutagenic analysis of PVX CP and TGBp1 was used to identify the regions involved in TGBp1–CP binding and translational activation of PVX RNA by TGBp1. It was found that the C-terminal (C-ter) 10/18 amino acids region was not essential for virus-like particle (VP) assembly from CP and RNA. However, the VPs assembled from the CP lacking C-ter 10/18 amino acids were incapable of TGBp1 binding and being translationally activated. It was suggested that the 10-amino-acid C-ter regions of protein subunits located at one end of a polar helical PVX particle contain a domain accessible to TGBp1 binding and PVX remodelling. The non-translatable particles assembled from the C-ter mutant CP could be converted into a translatable form by CP phosphorylation. The TGBp1–CP binding activity was preserved unless a conservative motif IV was removed from TGBp1. By contrast, TGBp1-dependent activation of PVX RNA translation was abolished by deletions of various NTPase/helicase conservative motifs and their combinations. The motif IV might be essential for TGBp1–CP binding, but insufficient for PVX RNA translation activation. The evidence to discriminate between these two events, i.e. TGBp1 binding to the CP-helix and TGBp1-dependent RNA translation activation, is discussed.     

Identification of differentially expressed proteins in Ficedula flycatchers

This is the first report on the large-scale identification and comparison of proteins in non-model organisms, Ficedula flycatchers. It highlights the potential of proteomics approaches in both non-sequenced and non-model organisms for identification of differentially expressed proteins. Not surprisingly, more than 55% of the proteins failed to be identified even though the MS spectra were of high quality. Nevertheless, the protein information obtained in this study will serve as a valuable resource for continued research.     

A fast, precise and low-cost replication technique for nano- and high-aspect-ratio structures of biological and artificial surfaces

Biological surfaces are multifunctional interfaces between the organisms and their environment. Properties such as the wettability and adhesion of particles are linked to the micro- and nanostructures of their surfaces. In this study, we used plant and artificial surfaces covered with wax crystals to develop a low-cost replication technique with high resolution. The technique is applicable for fragile surface structures, as demonstrated for three-dimensional wax crystals, and is fast to prevent shrinking of the biological material by water loss during the molding process. Thermal evaporation of octacosan-1-ol has been used to create microstructured surfaces with small platelets as templates for molding. Epoxy resin as filling material provided the smallest deviations from the original surface structures and can be used for replication of nanostructures as small as 4.5 nm. Contact angle measurements of leaves and their replicas show that this technique can be used to develop biomimetic surfaces with similar wettability as in the plant surfaces.