Carcinoma in situ specimen classification based on intermediate cell measurements

In this study the possibility of classifying carcinoma in situ and normal specimens by measuring normal-appearing intermediate cells was explored. Twenty-five histologically verified carcinoma in situ specimens and 99 normal specimens, matched with the abnormal specimens for age and use or nonuse of an oral hormonal contraceptive, were examined. The smears were monolayer preparations stained with Thionin-Feulgen Congo red. Twenty-one nuclear features were measured. A discrimination among the experimental groups could be made on the basis of the relationship between two features, area and average optical density (AOD). A regression of AOD on area for each smear was performed. The correlation, coefficient of variation, slope, intercept, as well as the mean of the AOD level and the age of the subject were used in a discriminant analysis. This resulted in a smear classification with a false-positive rate of 14% and a missed-positive rate of 32%. When contraceptive use was taken into account the overall classification was improved with a false-positive rate of 12% and a missed-positive rate of 20%.     

Streptococcus mutans Competence-Stimulating Peptide Inhibits Candida albicans Hypha Formation

The oral cavity is colonized by microorganisms growing in biofilms in which interspecies interactions take place. Streptococcus mutans grows in biofilms on enamel surfaces and is considered one of the main etiological agents of human dental caries. Candida albicans is also commonly found in the human oral cavity, where it interacts with S. mutans. C. albicans is a polymorphic fungus, and the yeast-to-hypha transition is involved in virulence and biofilm formation. The aim of this study was to investigate interkingdom communication between C. albicans and S. mutans based on the production of secreted molecules. S. mutans UA159 inhibited C. albicans germ tube (GT) formation in cocultures even when physically separated from C. albicans. Only S. mutans spent medium collected in the early exponential phase (4-h-old cultures) inhibited the GT formation of C. albicans. During this phase, S. mutans UA159 produces a quorum-sensing molecule, competence-stimulating peptide (CSP). The role of CSP in inhibiting GT formation was confirmed by using synthetic CSP and a comC deletion strain of S. mutans UA159, which lacks the ability to produce CSP. Other S. mutans strains and other Streptococcus spp. also inhibited GT formation but to different extents, possibly reflecting differences in CSP amino acid sequences among Streptococcus spp. or differences in CSP accumulation in the media. In conclusion, CSP, an S. mutans quorum-sensing molecule secreted during the early stages of growth, inhibits the C. albicans morphological switch.The oral cavity is colonized by many different microbial species, where most reside in biofilms. Because of its multispecies nature, the oral microbial community is one of the best biofilm models for studying interspecies interactions (17). The gram-positive bacterium Streptococcus mutans shows a high prevalence in dental biofilms, and it is considered to be the major etiological agent involved in human dental caries (21). The fungal species Candida albicans constitutes a minor part of the total microbial flora (19) and can be isolated as a commensal from the oral cavity of 50% to 60% of healthy adults (33). However, in immunocompromised individuals (for example, due to human immunodeficiency virus infection or as a result of chemotherapy) and elderly patients, this fungus often leads to candidiasis (24). C. albicans is a polymorphic fungus that can exist in three morphotypes: budding yeast, pseudohypha, and true hypha (5). The morphological switch from yeast to hyphal cells is important in many processes, such as virulence (22) and biofilm formation (10, 18), and is therefore the subject of many studies.Bacteria and yeasts are often found together in vivo, and there is growing evidence that interspecies, and even interkingdom, interactions occur within these populations (7). These interactions can be mediated through signaling molecules (40), as recently described for the interaction between C. albicans and Pseudomonas aeruginosa, an opportunistic bacterial pathogen (15). N-3-oxo-C₁₂ homoserine lactone (HSL), a signaling molecule involved in bacterial quorum sensing, completely represses C. albicans hypha formation without altering the growth rate. Although many gram-negative bacteria produce HSLs with shorter acyl chains (e.g., C₄-HSL), the inhibition of C. albicans hypha formation is caused specifically by long-chained HSL molecules. In addition, related, non-HSL molecules with long acyl chains, such as dodecanol and farnesol, also inhibit the hypha formation of C. albicans (8).A recent report described the coculturing of C. albicans and S. mutans in model oral biofilms on hydroxyapatite (26). It was shown that S. mutans increased the growth of C. albicans by stimulating coadhesion while simultaneously suppressing the formation of hyphae. S. mutans is a gram-positive bacterium and does not produce HSL-type molecules, and the nature of the interaction with C. albicans is presently unknown. In this study, the interaction between S. mutans and C. albicans was investigated by studying the effect of secreted molecules of S. mutans on C. albicans hypha formation.     

Surface water sanitation and biomass production in a large constructed wetland in the Netherlands

In Western-Europe, agricultural practices have contributed to environmental problems such as eutrophication of surface and ground water, flooding, drought and desiccation of surrounding natural habitats. Solutions that reduce the impact of these problems are urgently needed. Common reed (Phragmites australis) is capable of sanitizing surface water and may function as green energy source because of its high productivity. Here, the results of an experiment in a constructed wetland in the Netherlands are presented where two different sanitation treatments were compared. Depending on the residence time and volume per unit area, reed is capable to reduce the total amount of nitrogen in the water with average efficiencies from 32 to 47% and the total amount of phosphorous with 27–45%. Although biomass production still varies largely between different parts of the constructed wetland, a rapid increase in biomass was observed since planting. Constructed wetlands with reed provide opportunities to improve water quality and reed produces enough biomass to serve as green energy source. Moreover, these wetlands also function as a flood water reservoir and are possibly advantageous for biodiversity. The optimal moment of reed harvesting depends on the goal of the owner. This moment should be chosen wisely, as it may have consequences for reed filter regeneration, biomass production, biodiversity, methane emission and water sanitation efficiency.     

Temporal genetic samples indicate small effective population size of the endangered yellow-eyed penguin

There is an increasing awareness that the long-term viability of endemic island populations is negatively affected by genetic factors associated with population bottlenecks and/or persistence at small population size. Here we use contemporary samples and historic museum specimens (collected 1888–1938) to estimate the effective population size (N _e) for the endangered yellow-eyed penguin (Megadyptes antipodes) in South Island, New Zealand, and evaluate the genetic concern for this iconic species. The South Island population of M. antipodes—constituting almost half of the species’ census size—is thought to be descended from a small number of founders that reached New Zealand just a few hundred years ago. Despite intensive conservation measures, this population has shown dramatic fluctuations in size over recent decades. We compare estimates of the harmonic mean N _e for this population, obtained using one moment and three likelihood based-temporal methods, including one method that simultaneously estimates migration rate. Evaluation of the N _e estimates reveals a harmonic mean N _e in the low hundreds. Additionally, the inferred low immigration rates (m = 0.003) agree well with contemporary migration rate estimates between the South Island and subantarctic populations of M. antipodes. The low N _e of South Island M. antipodes is likely affected by strong fluctuations in population size, and high variance in reproductive success. These results show that genetic concerns for this population are valid and that the long-term viability of this species may be compromised by reduced adaptive potential.     

Differential association of protein subunits with the human RNase MRP and RNase P complexes

RNase MRP is a eukaryotic endoribonuclease involved in nucleolar and mitochondrial RNA processing events. RNase MRP is a ribonucleoprotein particle, which is structurally related to RNase P, an endoribonuclease involved in pre-tRNA processing. Most of the protein components of RNase MRP have been reported to be associated with RNase P as well. In this study we determined the association of these protein subunits with the human RNase MRP and RNase P particles by glycerol gradient sedimentation and coimmunoprecipitation. In agreement with previous studies, RNase MRP sedimented at 12S and 60-80S. In contrast, only a single major peak was observed for RNase P at 12S. The analysis of individual protein subunits revealed that hPop4 (also known as Rpp29), Rpp21, Rpp20, and Rpp25 only sedimented in 12S fractions, whereas hPop1, Rpp40, Rpp38, and Rpp30 were also found in 60-80S fractions. In agreement with their cosedimentation with RNase P RNA in the 12S peak, coimmunoprecipitation with VSV-epitope-tagged protein subunits revealed that hPop4, Rpp21, and in addition Rpp14 preferentially associate with RNase P. These data show that hPop4, Rpp21, and Rpp14 may not be associated with RNase MRP. Furthermore, Rpp20 and Rpp25 appear to be associated with only a subset of RNase MRP particles, in contrast to hPop1, Rpp40, Rpp38, and Rpp30 (and possibly also hPop5), which are probably associated with all RNase MRP complexes. Our data are consistent with a transient association of Rpp20 and Rpp25 with RNase MRP, which may be inversely correlated to its involvement in pre-rRNA processing.     

Sorting of ligand-activated epidermal growth factor receptor to lysosomes requires its actin-binding domain

Ligand-induced down-regulation of the epidermal growth factor receptor (EGFR) comprises activation of two sequential transport steps. The first involves endocytic uptake by clathrin-coated vesicles, the second transfer of endocytosed EGFR from endosomes to lysosomes. Here we demonstrate that the second transport step requires a domain of the EGFR that encompasses residues 985-996 and was previously found to interact with actin. Deletion of domain 989-994 (Delta989-994 EGFR) did not interfere with EGFR uptake but completely abrogated its degradation. In contrast, both uptake and degradation were affected for K721A EGFR, a kinase-deficient EGFR mutant. To measure intracellular EGFR sorting, we developed a novel cell fractionation assay toward which cells were co-transfected for chicken hepatic lectin, a receptor for agialoglycoproteins. These cells were incubated with agialofetuin-coupled colloidal gold, which was targeted to lysosomes after receptor-mediated endocytosis. Compartments within the lysosomal pathway gained buoyant density because of the presence of colloidal gold and could be isolated from cell homogenates by ultracentrifugation through a high-density sucrose cushion. In contrast to endocytosed wild type EGFR, both Delta989-994 EGFR and K721A EGFR were largely not retrieved in gold-containing endocytic compartments. These results are supported with morphological data. We conclude that sorting of endocytosed EGFR into the degradation pathway requires both its kinase activity and actin-binding domain.     

POLLINATION BIOLOGY OF PLATANTHERA STRICTA (ORCHIDACEAE) IN OLYMPIC NATIONAL PARK,WASHINGTON

Platanthera striata Lindley is entomophilous and can produce seed via facultative self-pollination and intraracemic and interracemic pollination. Capsule production is pollinator-limited and seed set may be pollen-limited. In experimental plants capsules produced via self- and intraracemic pollination contained fewer seeds with normally developed embryos than did capsules produced via interracemic pollination. The inflorescence of Platanthera stricta is fragrant and is attractive to a wide array of anthophilous insects. It is pollinated by a diverse assemblage of short-tongued insects. The primary pollinators are Eustroma fasciata B. and McD. (Lepidoptera: Geometridae), Bombus flavifrons Cresson and B. melanopygus Nylander (Hymenoptera: Apidae), an undescribed species of Greya (Lepidoptera: Prodixidae), and several species of Empis, Rhamphomyia, and Anthepiscopus longipalpis Melander (Diptera: Empididae). Small amounts of glucose are present on the raceme. The extrafloral glucose may retain small pollinators on the inflorescence until they locate the floral spur aperture.     

INDIVIDUAL FLOWERING TIME IN A GOLDENROD (SOLIDAGO CANADENSIS): FIELD EXPERIMENT SHOWS GENOTYPE MORE IMPORTANT THAN ENVIRONMENT

Individual plants of an old field population of the clonal perennial goldenrod, Solidago canadensis L. are consistent in rank order of flowering time over years. In order to analyze whether this consistency is due more to environmental variations among microsites of individual clones or due to genetic variation among clones, similar-sized sections of rhizomes were transplanted to an experimental garden in a randomized block design with five replicates per clone. Three years later we examined the transplants and the original plants for their flowering phenology. In the experimental garden there was an unexpected soil moisture gradient across blocks, with associated gradients in height of goldenrods and height of surrounding vegetation. No gradient was significantly correlated with the flowering time of the transplants. That is, the time of flowering of transplants was consistent among replicates of individual clones across the soil moisture gradient and regardless of the size of the transplants themselves, or the height of surrounding vegetation. Further, the flowering times of the transplants were significantly positively correlated with the flowering times of the original plants in the same year. We conclude that the differences in flowering time among clones within a goldenrod population is largely determined by genetic variation, rather than by environmental factors.