Structure of the Pseudorabies Virus Capsid: Comparison with Herpes Simplex Virus Type 1 and Differential Binding of Essential Minor Proteins

The structure of pseudorabies virus (PRV) capsids isolated from the nucleus of infected cells and from PRV virions was determined by cryo-electron microscopy (cryo-EM) and compared to herpes simplex virus type 1 (HSV-1) capsids. PRV capsid structures closely resemble those of HSV-1, including distribution of the capsid vertex specific component (CVSC) of HSV-1, which is a heterodimer of the pUL17 and pUL25 proteins. Occupancy of CVSC on all PRV capsids is near 100%, compared to ~ 50% reported for HSV-1 C-capsids and 25% or less that we measure for HSV-1 A- and B-capsids. A PRV mutant lacking pUL25 does not produce C-capsids and lacks visible CVSC density in the cryo-EM-based reconstruction. A reconstruction of PRV capsids in which green fluorescent protein was fused within the N-terminus of pUL25 confirmed previous studies with a similar HSV-1 capsid mutant localizing pUL25 to the CVSC density region that is distal to the penton. However, comparison of the CVSC density in a 9-Å-resolution PRV C-capsid map with the available crystal structure of HSV-1 pUL25 failed to find a satisfactory fit, suggesting either a different fold for PRV pUL25 or a capsid-bound conformation for pUL25 that does not match the X-ray model determined from protein crystallized in solution. The PRV capsid imaged within virions closely resembles C-capsids with the addition of weak but significant density shrouding the pentons that we attribute to tegument proteins. Our results demonstrate significant structure conservation between the PRV and HSV capsids.     

The acute glucocorticoid stress response does not differentiate between rewarding and aversive social stimuli in rats

The mere presence of elevated plasma levels of corticosterone is generally regarded as evidence of compromised well-being. However, environmental stimuli do not necessarily need to be of a noxious or adverse nature to elicit activation of the stress response systems. In the present study, the physiological and neuroendocrine responses to repeated social stimuli that can be regarded as emotional opposites, i.e. social defeat and sexual behavior, were compared. Similar corticosterone responses were observed in animals confronted for the first time with either a highly aggressive male intruder or a receptive female, but a decrease was noticed in defeated rats tested during a third interaction. Only if animals are being physically attacked does the corticosterone response remain similar to the one observed during sexual behavior. In addition, the number of activated cells in the parvocellular hypothalamic paraventricular nucleus, as visualized by c-Fos immunocytochemistry, shows no difference between rats 1h after the third exposure to defeat or sex. Finally, biotelemetric recordings of heart rate, body temperature and locomotor activity show a robust response to both social stimuli that is generally, however, higher in animals being confronted with a receptive female. The data clearly indicate that acute plasma corticosterone levels are not reflecting the emotional valence of a salient stimulus. The magnitude of the response seems to be a direct reflection of the behavioral activity and hence of the metabolic requirements of activated tissues. Next to its direct metabolic role, acute increases in plasma corticosterone will have neurobiological and behavioral effects that largely depend on the neural circuitry that is activated by the stimulus that triggered its release.     

Microbial community analysis of rectal methanogens and sulfate reducing bacteria in two non-human primate species

Background  Methanogenesis by methanogenic Archaea and sulfate reduction by sulfate reducing bacteria (SRB) are the major hydrogenotrophic pathways in the human colon. Methanogenic status of mammals is suggested to be under evolutionary rather than dietary control. However, information is lacking regarding the dynamics of hydrogenotrophic microbial communities among different primate species.
Methods  Rectal swabs were collected from 10 sooty mangabeys ( Cercocebus atys ) and 10 baboons ( Papio hamadryas ). The diversity and abundance of methanogens and SRB were examined using PCR-denaturing gradient gel electrophoresis (DGGE) and real-time quantitative PCR (qPCR).
Results  The DGGE results revealed that intestinal Archaea and SRB communities differ between mangabeys and baboons. Phylogenetic analyses of Archaea DGGE bands revealed two distinct clusters with one representing a putative novel order of methanogenic Archaea. The qPCR detected a similar abundance of methanogens and SRB.
Conclusions  Intestinal Archaea and SRB coexist in these primates, and the community patterns are host species-specific.     

Identification and comparison of natural rubber from two Lactuca species

Renewed interest in the identification of alternative sources of natural rubber to Hevea brasiliensis has focused on the Compositae family. In our search for Compositae models for rubber synthesis, we extracted latex from stems of two lettuce species: Lactuca serriola, prickly lettuce, and Lactuca sativa cv. Salinas, crisphead lettuce. Both species contained cis-1,4-polyisoprene rubber in the dichloromethane-soluble portions of their latex, and sesquiterpene lactones in their acetone-soluble portions. The rubber from both species and their progeny had molecular weights in excess of 1,000,000g/mol, and polydispersity values of 1.1. Rubber transferase activity was detected across a range of farnesyl diphosphate initiator concentrations, with decreased activity as initiator concentrations exceeded putative saturation. These results add lettuce to the short list of plant species that produce high molecular weight rubber in their latex. Due to the genomic and agronomic resources available in lettuce species, they provide the opportunity for further dissection of natural rubber biosynthesis in plants.     

Proteomic analysis of nasal cells from cystic fibrosis patients and non-cystic fibrosis control individuals: search for novel biomarkers of cystic fibrosis lung disease

Potential biological markers for cystic fibrosis (CF) lung disease were identified by comparative proteomics profiling of nasal cells from deletion of phenylalanine residue 508 (F508del)-homozygous CF patients and non-CF controls. From the non-CF 2-DE gels, 65 spots were identified by MS, and a reference 2-DE map was thus established. The majority of those correspond to ubiquitously expressed proteins. Consistent with the epithelial origin of this tissue, some of the identified proteins are epithelial markers (e.g. cytokeratins, palate lung and nasal epithelium clone protein (PLUNC), and squamous cell carcinoma antigen 1). Comparison of this protein profile with the one similarly obtained for CF nasal cells revealed a set of differentially expressed proteins. These included proteins related to chronic inflammation and some others involved in oxidative stress injury. Alterations were also observed in the levels of cytoskeleton proteins, being probably implicated with cytoskeleton organization changes described to occur in CF-airways. Lower levels were found for some mitochondrial proteins suggesting an altered mitochondrial metabolism in CF. Differential expression was also found for two more enzymes that have not been previously associated to CF. Further studies will clarify the involvement of such proteins in CF pathophysiology and whether they are targets for CF therapy.     

Changes in activities of several enzymes involved in carbohydrate metabolism during the cell cycle of Saccharomyces cerevisiae.

Activity changes of a number of enzymes involved in carbohydrate metabolism were determined in cell extracts of fractionated exponential-phase populations of Saccharomyces cerevisiae grown under excess glucose. Cell-size fractionation was achieved by an improved centrifugal elutriation procedure. Evidence that the yeast populations had been fractionated according to age in the cell cycle was obtained by examining the various cell fractions for their volume distribution and their microscopic appearance and by flow cytometric analysis of the distribution patterns of cellular DNA and protein contents. Trehalase, hexokinase, pyruvate kinase, phosphofructokinase 1, and fructose-1,6-diphosphatase showed changes in specific activities throughout the cell cycle, whereas the specific activities of alcohol dehydrogenase and glucose-6-phosphate dehydrogenase remained constant. The basal trehalase activity increased substantially (about 20-fold) with bud emergence and decreased again in binucleated cells. However, when the enzyme was activated by pretreatment of the cell extracts with cyclic AMP-dependent protein kinase, no significant fluctuations in activity were seen. These observations strongly favor posttranslational modification through phosphorylation-dephosphorylation as the mechanism underlying the periodic changes in trehalase activity during the cell cycle. As observed for trehalase, the specific activities of hexokinase and phosphofructokinase 1 rose from the beginning of bud formation onward, finally leading to more than eightfold higher values at the end of the S phase. Subsequently, the enzyme activities dropped markedly at later stages of the cycle. Pyruvate kinase activity was relatively low during the G1 phase and the S phase, but increased dramatically (more than 50-fold) during G2. In contrast to the three glycolytic enzymes investigated, the highest specific activity of the gluconeogenic enzyme fructose-1, 6-diphosphatase 1 was found in fractions enriched in either unbudded cells with a single nucleus or binucleated cells. The observed changes in enzyme activities most likely underlie pronounced alterations in carbohydrate metabolism during the cell cycle.     

Gene regulatory divergence among species estimated by altered developmental patterns in interspecific hybrids

Disturbances in the schedules of gene expression in developing interspecific fish hybrids have been used to draw inferences about the extent of gene regulatory divergence between species and about the degree to which this gene regulatory divergence is correlated with structural gene divergence, as estimated by genetic distance. Sperm from each of 10 different species representing six genera within the family Centrarchidae was used to fertilize eggs of the Florida largemouth bass (Micropterus salmoides floridanus). The genetic distances (D; Nei 1978) between the parental species used to form the hybrids ranged from 0.133 to 0.974. The developmental success and temporal patterns of gene expression of each of the hybrids were compared with those of the Florida largemouth bass. As the genetic distance between the paternal species and the Florida largemouth bass increased, there was a general decline in developmental success in the hybrid embryos as demonstrated by the observed reductions in the percentage of hatching and by progressively earlier and more extensive morphological abnormalities. Concomitantly, progressively more marked alterations in developmental schedules of expression of 15 enzyme loci occurred in the hybrids as the genetic distance between parental species increased. However, observed deviations from this trend for a few species may represent an uncoupling of the rates and modes of evolution of structural genes from those for genes regulating developmental processes.