Proportions of H1 histone subspecies in human fibroblasts shift during density-dependent growth arrest independent of replicative senescence

H1 histone subspecies have been reported to vary during tissue differentiation, during aging of mammalian tissues, and as a function of DNA replicative activity. Since cultured human fibroblasts have a limited replicative life span which features arrest in the G1 phase of the cell cycle, we sought to distinguish whether any changes in the proportions of the principal H1 histone subspecies (H1A, H1B, and H1o) in late-passage fibroblasts were specific for senescent loss of replicative potential, or rather ensued as a result of prolonged inhibition of cell division. We observed an identical shift in the proportions of H1 histone subspecies during prolonged density-dependent inhibition of growth in both early-passage and late-passage cells. Since under these conditions there were no passage-specific changes, replicative senescence of human fibroblasts does not appear to involve a defect in the control of H1 histone proportions.     

The use of bovine serum albumin to improve the RT-qPCR detection of foodborne viruses rinsed from vegetable surfaces

Aims: To demonstrate that produce rinsates used for RT‐qPCR detection of foodborne viruses may cause significant PCR inhibition and propose a means to reduce its impact on sensitivity. Methods and Results: Here, it is shown that rinsing and concentration from spinach and precut lettuce have the potential to generate RNA extracts that are inhibitory to RT‐qPCRs assembled from commercial kits for the detection of norovirus GII (NoV GII), hepatitis A virus (HAV), hepatitis E virus (HEV), rotavirus (RV) and feline calicivirus (FCV) as sample process control. It is further shown that the addition of bovine serum albumin (BSA) to those reactions restored a positive signal in all cases. The effect of BSA was dependent upon the primer/probe combination. Moreover, two of the detection systems (FCV and HAV) strongly benefited from the addition of BSA even in the absence of PCR inhibitors. Conclusions: BSA was shown to restore positive signals in five different RT‐qPCR systems that were otherwise completely inhibited by produce rinsate extracts. It is therefore suggested to consider the addition of BSA to RT‐qPCRs for the detection of foodborne viruses when inhibition is observed. Significance and Impact of the Study: This study clearly demonstrates the potency of PCR inhibitors generated during routine virus concentration from produce and that it can be alleviated by the addition of BSA to the RT‐qPCRs. Although used elsewhere, the addition of BSA to PCRs is not a common practice in this growing field of research.     

The feeding ecology of Morone americana larvae in the Chesapeake Bay estuarine turbidity maximum: the influence of physical conditions and prey concentrations

Feeding ecology of white perch Morone americana larvae, a major component of the ichthyoplankton community in the estuarine turbidity maximum (ETM) region of upper Chesapeake Bay, was evaluated in years of contrasting physical conditions. In 1998, high river flow led to an ETM with high turbidity and pronounced salinity stratification. In 1999, under low river flow conditions, turbidities in the ETM region were lower and the salt front was located 15 km up‐stream of its location in 1998. Copepodites and adults of Eurytemora affinis were the predominant prey in guts of all length classes of larvae (3·2–9·8 mm standard length, L _S) that were examined. Repeated‐measures, multiple regression analyses were conducted to evaluate the influence of several factors on feeding success by determining if physical conditions (temperature, current velocity, salinity, turbidity and light) and prey concentrations explained variability in mean gut fullness (gut contents mass per body mass) of small (<5 mm L _S) and large (>5 mm L _S) white perch larvae. Eurytemora affinis concentrations were significant in the statistical models for both small and large larvae. High concentrations of E. affinis , which enhanced encounter rates of white perch larvae with prey, may have been the most important factor determining feeding success in the ETM region. Larval feeding incidence (percentage of guts with food) was higher in 1998, suggesting that annual variability in river flow influenced larval feeding success by controlling physical and biological conditions in the ETM region.     

Purification, Characterization and cDNA Cloning of the 200 kDa Protein Induced by Cold Acclimation in Wheat

We have purified to homogeneity the 200 kDa protein inducedspecifically by low temperature in wheat (Triticum aestivumL.). The boiling solubility of the protein has been used asa main step in the purification procedure. Amino acid compositionindicates that the 200 kDa has a compositional bias for glycine(11.4%), threonine (13.3%), and alanine (22.0%). Using oligonucleotideprobes, we have isolated a clone (pWcs200) from a cold-acclimatedwinter wheat cDNA library. Northern analysis demonstrated thatthe expression of the corresponding gene was specifically upregulatedby low temperature. Southern analysis showed that the gene organizationand the relative copy number were identical in two cultivarsdiffering in their capacity to develop freezing tolerance. Proteinsequence and immunological analyses indicate that this proteinshares similar features with the 50 kDa protein induced duringcold acclimation of wheat. The two proteins are boiling-soluble,and possess similar repeated elements. These elements may beimportant for the development of freezing tolerance. We haveshown that the 200 kDa protein is the largest member of a familyof immunologically-related cold-induced proteins in wheat. Expressionof pWcs200 in E. coli yielded a product of around 200 kDa, indicatingthat the clone contains most of the coding region for this protein. (Received August 18, 1992; Accepted October 14, 1992)     

Cracking the case: Seed traits and phylogeny predict time to germination in prairie restoration species

Traits are important for understanding how plant communities assemble and function, providing a common currency for studying ecological processes across species, locations, and habitat types. However, the majority of studies relating species traits to community assembly rely upon vegetative traits of mature plants. Seed traits, which are understudied relative to whole‐plant traits, are key to understanding assembly of plant communities. This is particularly true for restored communities, which are typically started de novo from seed, making seed germination a critical first step in community assembly and an early filter for plant establishment. We experimentally tested the effects of seed traits (mass, shape, and embryo to seed size ratio) and phylogeny on germination response in 32 species commonly used in prairie grassland restoration in the Midwestern USA, analyzing data using time‐to‐event (survival) analysis. As germination is also influenced by seed dormancy, and dormancy break treatments are commonly employed in restoration, we also tested the effects of two pretreatments (cold stratification and gibberellic acid application) on time to germination. Seed traits, phylogeny, and seed pretreatments all affected time to germination. Of all traits tested, variables related to seed shape (height and shape variance) best predicted germination response, with high‐variance (i.e., pointier and narrower) seeds germinating faster. Phylogenetic position (the location of species on the phylogenetic tree relative to other tested species) was also an important predictor of germination response, that is, closely related species showed similar patterns in time to germination. This was true despite the fact that all measured seed traits showed phylogenetic signal, therefore phylogeny provided residual information that was not already captured by measured seed traits. Seed traits, phylogenetic position, and germination pretreatments were important predictors of germination response for a suite of species commonly used in grassland restoration. Shape traits were especially important, while mass, often the only seed trait used in studies of community assembly, was not a strong predictor of germination timing. These findings illustrate the ecological importance of seed traits that are rarely incorporated into functional studies of plant communities. This information can also be used to advance restoration practice by guiding restoration planning and seed mix design.     

Phylogeny of "core Gruiformes" (Aves: Grues) and resolution of the Limpkin-Sungrebe problem

Opinions on the systematic relationships of birds in the avian order Gruiformes have been as diverse as the families included within it. Despite ongoing debate over monophyly of the order and relationships among its various members, recent opinion has converged on the monophyly of a "core" group of five families classified as the suborder Grues: the rails (Rallidae), the cranes (Gruidae), the Limpkin (Aramidae), the trumpeters (Psophiidae), and the finfoots (Heliornithidae). We present DNA sequence data from four mitochondrial (cytochrome b, 12S rRNA, Valine tRNA, and 16S rRNA) and three nuclear loci (intron 7 of beta-fibrinogen, intron 5 of alcohol dehydrogenase-I, and introns 3 through 5 of glyceraldehyde-3-phosphate dehydrogenase) to test previous hypotheses of interfamilial relationships within Grues, with particular attention to the enigmatic family Heliornithidae. Separate and combined analyses of these gene sequences confirm the monophyly of Grues as a whole, and of the five families individually, including all three species of Heliornithidae. The preferred topology unambiguously supports relationships among four of the five families, with only the position of Psophiidae remaining equivocal. Bayesian "relaxed-clock" dating methods suggest that the divergences of the three heliornithid species occurred in the mid-Tertiary, suggesting that their present disjunct pantropical distribution is a result of early- to mid-Tertiary dispersal.     

Stimulation of tumor necrosis factor secretion by purified influenza virus neuraminidase

We showed that purified neuraminidase (NA) of influenza virus, but not hemagglutinin (HA), possessed the potential to increase in vitro and in vivo the interleukin 1 (IL 1) activity of mouse peritoneal macrophages. In this study, we report the effect of NA and HA on the secretion of tumor necrosis factor (TNF) activity by murine peritoneal macrophages. TNF being a cytokine sharing many related and overlapping biological functions with IL 1. The two glycoproteins of the strain A/USSR/90/77 (H1N1) were purified electrophoretically and were tested in vitro at doses ranging from 0.5 to 5.0 micrograms using the adherent peritoneal macrophages of C3H/HeN mice elicited with thioglycolate. The TNF activity of culture supernatants, collected 24 hr after stimulation with viral protein, was evaluated by the standard cytolytic assay using L929 and WEHI.164 cells. No increase of the TNF activity was observed at 0.5 micrograms of NA (4.8 Units (U)/ml in the L929 assay and 20.4 U/ml in the WEHI assay) but further increase of NA to 1.0 microgram had a significant effect on the TNF activity (39.7 and 88.8 U/ml, respectively). Higher concentrations of NA (2.0 and 5.0 micrograms) did not improve the TNF activity. The addition of a rabbit anti-TNF-alpha serum to the assay system reduced the lysis of L929 cells by 85%, suggesting that the observed activity was due to TNF. In parallel, the enhancement of IL 1 activity due to NA was reverified using D10.G4.1 cells instead of the C3H/HeJ thymocytes assay used previously. NA augmented the IL 1 activity up to 1.0 micrograms (25.8 U/ml). The addition of monoclonal anti-IL 1 antibodies (100 neutralizing units) to the supernatants reduced the incorporation of [3H]-thymidine by 90 to 95%, suggesting that the observed activity was due to IL 1. Comparative results of NA and HA showed that only NA stimulated the TNF and IL 1 activities of murine macrophages.     

Prior exposure to male phenotypes influences mate choice in the guppy, Poecilia reticulata

This study examined the effect of experience on the patternof mate choice in female guppies. Females from some naturalpopulations are known to discriminate among males on the basisof the relative area of orange in the color pattern. We exposedmaturing female guppies to groups of males that differed inphenotvpic distribution of orange coloration (high orange, loworange, and mixed) and tested their subsequent mating preferences.Females that had been exposed to high-orange or low-orange malegroups showed no discrimination in choice tests, but femalesexposed to mixed male groups favored high-orange males overlow-orange males. These results demonstrate that mating preferencesof females can be modified depending on prior experience. Femalesmay be able to adjust their pattern of mate choice dependingon the degree to which choice cues permit effective discriminationamong males. This potential for short-term modification of matechoice patterns could affect the evolutionary outcome of sexualselection