Effects of γ-IFN and NGF on subpopulations in a human neuroblastoma cell line: Flow cytometric and morphological analysis

Summary Neuroblastomas are neural crest-derived tumors that contain neuronal, melanocyte, and Schwann cell precursors. We examined the effects of treatment with γ-interferon (γ-IFN) and nerve growth factor (NGF), alone, and in combination, on these progenitor subpopulations in the human neuroblastoma cell line, SH-SY5Y. Using fluorescence-activated flow cytometry (FACS), changes in expression of three differentiation-specific or-associated marker proteins, the 200 kD neurofilament protein, the myelin basic protein, and the S-100 protein, were analyzed. Growth rates and morphological changes associated with each treatment over the 2-wk incubation period were noted. The greatest effects were observed with combined IFN +NGF treatment. These were significant increases in expression of all three proteins distinctive morphological signs of differentiation, and extensive inhibition of proliferation compared to control cultures. Treatment with NGF alone resulted in increased neurofilament protein expression and in the length and number of neurite extensions, but there was no effect on the growth rate. IFN induced striking morphological changes, significant inhibition of growth, and changes in protein expression that correlated with neuronal to non-neuronal subpopulation shifts due to the death of differentiated cells. When treatment was discontinued after 15 d, the morphological changes induced by NGF were reversed within 2–3 d, while those induced by IFN±NGF were present up to 4 wk post-treatment. Small, neuroblastic colonies were observed throughout the treatment period and within 4–6 wk after the cessation of treatment this cell-type fully reconstituted the cultures suggesting the presence of a stem cell. Our results indicate that treatment with γ-IFN±NGF can regulate growth and induce, either stem cells or progenitor neuronal, Schwann and melanocyte subpopulations in the SH-SY5Y cell line to irreversibly differentiate.     

Flow cytometric evaluation of yeast–bacterial cell–cell interactions

Synthetic cell–cell interaction systems can be useful for understanding multicellular communities or for screening binding molecules. We adapt a previously characterized set of synthetic cognate nanobody–antigen pairs to a yeast–bacteria coincubation format and use flow cytometry to evaluate cell–cell interactions mediated by binding between surface-displayed molecules. We further use fluorescence-activated cell sorting to enrich a specific yeast-displayed nanobody within a mixed yeast-display population. Finally, we demonstrate that this system supports the characterization of a therapeutically relevant nanobody–antigen interaction: a previously discovered nanobody that binds to the intimin protein expressed on the surface of enterohemorrhagic Escherichia coli. Overall, our findings indicate that the yeast–bacteria format supports efficient evaluation of ligand–target interactions. With further development, this format may facilitate systematic characterization and high-throughput discovery of bacterial surface-binding molecules.     

Trematode life cycles: short is sweet?

Complex life cycles are a hallmark of parasitic trematodes. In several trematode taxa, however, the life cycle is truncated: fewer hosts are used than in a typical three-host cycle, with fewer transmission events. Eliminating one host from the life cycle can be achieved in at least three different ways. Some trematodes show even more extreme forms of life cycle abbreviations, using only a mollusc to complete their cycle, with or without sexual reproduction. The occurrence of these phenomena among trematode families are reviewed here and show that life cycle truncation has evolved independently many times in the phylogeny of trematodes. The hypotheses proposed to account for life-cycle truncation, in addition to the factors preventing the adoption of shorter cycles by all trematodes are also discussed. The study of shorter life cycles offers an opportunity to understand the forces shaping the evolution of life cycles in general.     

How to discover ploidy levels of charred free-threshing wheat caryopses?

Vegetation History and Archaeobotany -     

Review of Colombian Conchostraca (Crustacea) — ecological aspects and life cycles — family Cyclestheriidae

This study gives an overview and describes special aspects of the biology, ecology and the life cycle ofCyclestheria hislopi (Baird, 1859). This species is most commonly found in parthenogenically reproducing populations which produce large, directly developing nondiapause eggs. But periodically and under certain environmental conditions, sexually reproducing generations appear and produce diapause eggs. the sexual generations include males and particularly constituted females, which undergo a complete transformation into a special type of ephippium.Cyclestheria is the only known conchostracan species which does not occur in the initial phases of temporary water bodies, but develops in older temporary pools and even in permanent waters. It survives in the presence of effective depredators like fish by hiding within a special self-made mucus capsule.     

Childhood predictors of late‐life diabetes: The case of Mexico

Abstract

We investigated the interplay between characteristics of early childhood circumstances and current socioeconomic conditions and health, focusing specifically on diabetes in mid and late life in Mexico. The analysis used data from the 2001 Mexican Health and Aging Study (MHAS), a large nationally representative study of Mexicans born before 1950. We analyzed the extent to which childhood conditions, such as exposure to infectious diseases, a poor socioeconomic environment, and parental education, affect the risk of diabetes in later life. Our results indicate that individuals age 50 and older who experienced serious health problems before age 10 have a higher risk of having late‐life diabetes. There is a significant inverse relationship between maternal education and diabetes in late life of adult offspring. Individuals with better educated mothers have a lower risk of being diabetic after age 50. This relationship remains after controlling for other childhood and adult risk factors.     

Interglacial‐glacial cycles in the early Pleistocene of the Leffe basin (Northern Italy): Preliminary report

Palynological studies of the Leffe Basin lacustrine deposits (Lombard Pre‐Alps, Italy) reveal a succession of deciduous mesophytic arboreal communities, coniferous forests and brief phases of withdrawal of arboreal taxa. The pollen record can be easily correlated with a previous pollen diagram by Lona (1950). However, contrary to his interpretation, we believe that the cyclicity shown by vegetation dynamics cannot be correlated with the major glacial events that occurred in the Southern Alps, but perhaps with interglacial‐glacial cycles of moderate amplitude.     

Flow cytometric analysis of sialyl Lewis A antigen on human cancer cells by using F(ab')2μ fragments prepared from a mouse IgM monoclonal antibody

F(ab')2 fragments, herein designated as F(ab')2μ fragments, were prepared from a mouse IgM monoclonal antibody specific to sialyl Lewis A antigen. The fragments were applied to flow cytometry to analyze the antigen on human cancer cells. The binding of the fragments to the antigen-positive cells was stronger than that of the original IgM. The non-specific binding of the IgM antibody to the antigen-negative cells was much decreased by using the F(ab')2μ fragments. These results indicate that the F(ab')2μ fragments are more suitable than the original IgM monoclonal antibody in flow cytometric analysis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Classification,nomenclature and identification of lime swallowtail butterflies: A post‐cladistic analysis (Lepidoptera: Papilionidae)

Specimens taxonomically treated in the Fauna Hawaiiensis were associated by cluster analysis, thereby reconstructing assemblages of Hawaiian carabid beetle species (Coleoptera: Carabidae) observed during the late 19th century. Associations among specimens representing 193 species permit concise hypotheses of habitat preferences for many of the 32 carabid species collected during the early period of European scientific exploration (1872–1902), but not observed since. These associations are consistent with data derived from contemporary biological surveys of Hawai'i. Absence of entire clusters of associated species from recent collections suggests actions of common agents leading to extinction or extreme population reduction. The candidate list of threatened and endangered species of the US. Fish and Wildlife Service established prior to 1994 included one Hawaiian carabid species missing since 1902, versus eight other species collected at various times over the past century. Improvements in knowledge of carabid beetle species’ spatial distribution and temporal persistence derived from recent field survey and taxonomic research demonstrate that the types of criteria used to construct that list must be rejected. Future consideration of official conservation status for any Hawaiian carabid beetle species must take into account the status of ecologically associated species, and the limited likelihood that individuals of all extant species can be consistently observed in nature due to their natural relative rarity or their secretive habits within restricted geographic and ecological distributions. Historical specimen associations serve as the best guides for continuing efforts to monitor known faunal members and to rediscover long‐missing species. These associations also serve to link information concerning individual species with particular habitats.     

A benefit‐cost analysis of amniocentesis

Abstract

This study applies benefit‐cost analysis to one area of prenatal diagnosis— amniocentesis. The ultimate purpose of such a study is to provide useful guidelines to policy makers. An attempt is made to exhaustively list benefits and costs, whether measurable or not. Those which can be quantified are estimated. On the basis of these benefit‐cost estimates, the study concludes that a program of amniocentesis for all pregnant women beyond age 32 would be warranted. This economic analysis, however, is only one aspect of a policy decision. Moral, legal, and ethical questions must also be considered.     

Relationships of diverse apoptotic death process patterns to mitochondrial membrane potential (Δψm) evaluated by three-parameter flow cytometric analysis

Recently, it has been proposed that novel methodologies are needed to re-evaluate apoptotic cell death, as studies of apoptosis have shown it to be a complex process. Since mitochondria are key regulators in cell death pathways, we developed a simultaneous 3-parameter flow cytometric analysis that incorporates the change in mitochondrial membrane potential (Δψ_m) in an Annexin-V [for phosphatidyl-serine (PS)] and propidium iodide (PI) assay system (3 parameters with 4 colours), and evaluated the apoptotic process using various haematological malignant cell lines and death triggers. The present method enabled visualization of cell composition during apoptosis and captured complicated molecular events. For example, apoptotic cells that lost Δψ_m did not always externalize PS, while some late apoptotic cells had polarized Δψ_m. The findings of unchanged PS-externalization and aberrant cell death suggest that there is no relationship of PS externalization and apoptosis with an unknown apoptotic mechanism. Based on PS-externalization, sensitivity to staurosporine, and the combination of cell lines and triggers, the apoptotic process was classified into 2 types. Importantly, most of our findings could not be observed by PS–PI and Δψ_m assays when independently performed. Our method may be useful for examining mitochondrial-related apoptosis and death signalling pathways, as well as screening novel apoptosis-inducing cancer drugs.     

Comparative genome‐wide analysis of small RNAs of major Gram‐positive pathogens: from identification to application

In the recent years, the number of drug- and multi-drug-resistant microbial strains has increased rapidly. Therefore, the need to identify innovative approaches for development of novel anti-infectives and new therapeutic targets is of high priority in global health care. The detection of small RNAs (sRNAs) in bacteria has attracted considerable attention as an emerging class of new gene expression regulators. Several experimental technologies to predict sRNA have been established for the Gram-negative model organism Escherichia coli. In many respects, sRNA screens in this model system have set a blueprint for the global and functional identification of sRNAs for Gram-positive microbes, but the functional role of sRNAs in colonization and pathogenicity for Listeria monocytogenes, Staphylococcus aureus, Streptococcus pyogenes, Enterococcus faecalis and Clostridium difficile is almost completely unknown. Here, we report the current knowledge about the sRNAs of these socioeconomically relevant Gram-positive pathogens, overview the state-of-the-art high-throughput sRNA screening methods and summarize bioinformatics approaches for genome-wide sRNA identification and target prediction. Finally, we discuss the use of modified peptide nucleic acids (PNAs) as a novel tool to inactivate potential sRNA and their applications in rapid and specific detection of pathogenic bacteria.