Expression of hemagglutinin esterase protein from recombinant mouse hepatitis virus enhances neurovirulence

Murine hepatitis virus (MHV) infection provides a model system for the study of hepatitis, acute encephalitis, and chronic demyelinating disease. The spike glycoprotein, S, which mediates receptor binding and membrane fusion, plays a critical role in MHV pathogenesis. However, viral proteins other than S also contribute to pathogenicity. The JHM strain of MHV is highly neurovirulent and expresses a second spike glycoprotein, the hemagglutinin esterase (HE), which is not produced by MHV-A59, a hepatotropic but only mildly neurovirulent strain. To investigate a possible role for HE in MHV-induced neurovirulence, isogenic recombinant MHV-A59 viruses were generated that produced either (i) the wild-type protein, (ii) an enzymatically inactive HE protein, or (iii) no HE at all (A. Lissenberg, M. M. Vrolijk, A. L. W. van Vliet, M. A. Langereis, J. D. F. de Groot-Mijnes, P. J. M. Rottier, and R. J. de Groot, J. Virol. 79:15054-15063, 2005 [accompanying paper]). A second, mirror set of recombinant viruses was constructed in which, in addition, the MHV-A59 S gene had been replaced with that from MHV-JHM. The expression of HE in combination with A59 S did not affect the tropism, pathogenicity, or spread of the virus in vivo. However, in combination with JHM S, the expression of HE, regardless of whether it retained esterase activity or not, resulted in increased viral spread within the central nervous system and in increased neurovirulence. Our findings suggest that the properties of S receptor utilization and/or fusogenicity mainly determine organ and host cell tropism but that HE enhances the efficiency of infection and promotes viral dissemination, at least in some tissues, presumably by serving as a second receptor-binding protein.     

Gene expression programs during Brassica oleracea seed maturation, osmopriming, and germination are indicators of progression of the germination process and the stress tolerance level

During seed maturation and germination, major changes in physiological status, gene expression, and metabolic events take place. Using chlorophyll sorting, osmopriming, and different drying regimes, Brassica oleracea seed lots of different maturity, stress tolerance, and germination behavior were created. Through careful physiological analysis of these seed lots combined with gene expression analysis using a dedicated cDNA microarray, gene expression could be correlated to physiological processes that occurred within the seeds. In addition, gene expression was studied during early stages of seed germination, prior to radicle emergence, since very little detailed information of gene expression during this process is available. During seed maturation expression of many known seed maturation genes, such as late-embryogenesis abundant or storage-compound genes, was high. Notably, a small but distinct subgroup of the maturation genes was found to correlate to seed stress tolerance in osmoprimed and dried seeds. Expression of these genes rapidly declined during priming and/or germination in water. The majority of the genes on the microarray were up-regulated during osmopriming and during germination on water, confirming the hypothesis that during osmopriming, germination-related processes are initiated. Finally, a large group of genes was up-regulated during germination on water, but not during osmopriming. These represent genes that are specific to germination in water. Germination-related gene expression was found to be partially reversible by physiological treatments such as slow drying of osmoprimed seeds. This correlated to the ability of seeds to withstand stress.     

Comparison of two twelve week off-season combined training programs on entry level collegiate soccer players' performance

Olympic-style lifts (OSL) and plyometric exercises (PE) are frequently combined with traditional resistance training (TRT) to improve athletic performance. The goal of this study was to directly compare the performance effect of TRT (30 minutes) combined with either OSL or nondepth-jump PE (15 minutes) on entry level competitive collegiate athletes. Ten female and 5 male competitive soccer players, divided into 2 groups, completed 12 weeks of tri-weekly training during their off-season. Countermovement vertical jump, 4 repetition maximum squat, 25-m sprint, and figure-8 drill on a 5-dot mat were conducted pre-, mid-, and postintervention. Significant improvements were made by both groups in each performance parameter over the 12-week period (p < 0.05), with no significant differences found between the training groups. Although these training modalities may achieve their results through slightly different mechanisms, the performance-related improvements may not be significantly different for entry-level collegiate athletes with little resistance training experience.     

Locomotor compensation creates a mismatch between laboratory and field estimates of escape speed in lizards: a cautionary tale for performance-to-fitness studies

A key assumption in evolutionary studies of locomotor adaptation is that standard laboratory measures of performance accurately reflect what animals do under natural circumstances. One widely examined measure of performance is maximum sprint speed, which is believed to be important for eluding predators, capturing prey, and defending territories. Previous studies linking maximum sprint speed to fitness have focused on laboratory measurements, and we suggest that such analyses may be appropriate for some species and intraspecific classes, but not others. We provide evidence for a general inverse relationship between maximum laboratory sprint speed and the percentage of maximum capacity that animals use when escaping from a threat in the field (the model of locomotor compensation). Further, absolute values of field escape speed and maximum laboratory speed are not significantly related when comparing across a diverse group of Anolis and lacertid lizards. We show that this pattern of locomotor compensation holds both within (i.e., among intraspecific classes) and among lizard species (with some exceptions). We propose a simple method of plotting field escape speed (y-axis) versus maximum laboratory speed (x-axis) among species and/or intraspecific classes that allows researchers to determine whether their study organisms are good candidates for relating laboratory performance to fitness. We suggest that species that reside directly on, or near the "best fitness line" (field escape speed = maximum laboratory speed) are most likely to bear fruit for such studies.     

Nidovirus sialate-O-acetylesterases: evolution and substrate specificity of coronaviral and toroviral receptor-destroying enzymes

Many viruses achieve reversible attachment to sialic acid (Sia) by encoding envelope glycoproteins with receptor-binding and receptor-destroying activities. Toroviruses and group 2 coronaviruses bind to O-acetylated Sias, presumably via their spike proteins (S), whereas other glycoproteins, the hemagglutinin-esterases (HE), destroy Sia receptors by de-O-acetylation. Here, we present a comprehensive study of these enzymes. Sialate-9-O-acetylesterases specific for 5-N-acetyl-9-O-acetylneuraminic acid, described for bovine and human coronaviruses, also occur in equine coronaviruses and in porcine toroviruses. Bovine toroviruses, however, express novel sialate-9-O-acetylesterases, which prefer the di-O-acetylated substrate 5-N-acetyl-7(8),9-di-O-acetylneuraminic acid. Whereas most rodent coronaviruses express sialate-4-O-acetylesterases, the HE of murine coronavirus DVIM cleaves 9-O-acetylated Sias. Under the premise that HE specificity reflects receptor usage, we propose that two types of Sias serve as initial attachment factors for coronaviruses in mice. There are striking parallels between orthomyxo- and nidovirus biology. Reminiscent of antigenic shifts in orthomyxoviruses, rodent coronaviruses exchanged S and HE sequences through recombination to extents not appreciated before. As for orthomyxovirus reassortants, the fitness of nidovirus recombinant offspring probably depends both on antigenic properties and on compatibility of receptor-binding and receptor-destroying activities.     

Toxoplasma gondii: Flat-mounting of retina as a new tool for the observation of ocular infection in mice

Ocular toxoplasmosis is the principal cause of posterior uveitis and a leading cause of blindness. Animal models are required to improve our understanding of the pathogenesis of this disease. The method currently used for the detection of retinal cysts in animals involves the observation, under a microscope, of all the sections from infected eyes. However, this method is time-consuming and lacks sensitivity. We have developed a rapid, sensitive method for observing retinal cysts in mice infected with Toxoplasma gondii. This method involves combining the flat-mounting of retina - a compromise between macroscopic observation and global analysis of this tissue - and the use of an avirulent recombinant strain of T. gondii expressing the Escherichia coli β-galactosidase gene, visually detectable at the submacroscopic level. Single cyst unilateral infection was found in six out of 17 mice killed within 28 days of infection, whereas a bilateral infection was found in only one mouse. There was no correlation between brain cysts number and ocular infection.     

Phosphoproteins analysis in plants: a proteomic approach

The study of phosphoproteome on a global scale represents one of the challenges in the post-genomic era. Here, we propose an integrated procedure starting from the crude protein extract, that consists of sequential purification steps, and ending up in the identification of phosphorylation sites. This involves (i) an enrichment in phosphoproteins with a commercially available chromatography matrix, (ii) a 2-D gel analysis of the enriched fraction followed by the selective staining with the phosphospecific fluorescent dye Pro-Q Diamond, (iii) a phosphopeptide capture, from the tryptic lysate of 2-D spots, using IMAC micro-columns. In the end, the identification of the phosphoproteins and their corresponding phosphorylation sites were achieved by MALDI-TOF-TOF spectrometry. The method was applied to contrasting samples prepared from cell suspension cultures of Arabidopsis thaliana and roots of Medicago truncatula. The results obtained, demonstrated the robustness of the combination of two enrichment stages, sequentially at the protein and at the peptide levels, to analyse phosphoproteins in plants.