DNA sequence plays a major role in determining nucleosome positions in yeast CUP1 chromatin.

The role of DNA sequence in determining nucleosome positions in vivo was investigated by comparing the positions adopted by nucleosomes reconstituted on a yeast plasmid in vitro using purified core histones with those in native chromatin containing the same DNA, described previously. Nucleosomes were reconstituted on a 2.5 kilobase pair DNA sequence containing the yeast TRP1ARS1 plasmid with CUP1 as an insert (TAC-DNA). Multiple, alternative, overlapping nucleosome positions were mapped on TAC-DNA. For the 58 positioned nucleosomes identified, the relative positioning strengths and the stabilities to salt and temperature were determined. These positions were, with a few exceptions, identical to those observed in native, remodeled TAC chromatin containing an activated CUP1 gene. Only some of these positions are utilized in native, unremodeled chromatin. These observations suggest that DNA sequence is likely to play a very important role in positioning nucleosomes in vivo. We suggest that events occurring in yeast CUP1 chromatin determine which positions are occupied in vivo and when they are occupied.     

Monitoring signal transduction in cancer: cDNA microarray for semiquantitative analysis.

This study targeted the development of a novel microarray tool to allow rapid determination of the expression levels of 58 different tyrosine kinase (tk) genes in small tumor samples. The goals were to define a reference probe for multi-sample comparison and to investigate the variability and reproducibility of the image acquisition and RT-PCR procedures. The small number of tk genes on our arrays enabled us to define a reference probe by artificially mixing all genes on the arrays. Such a probe provided contrast reference for comparative hybridization of control and sample DNA and enabled cross-comparison of more than two samples against one another. Comparison of signals generated from multiple scanning eliminated the concern of photo bleaching and scanner intrinsic noise. Tests performed with breast, thyroid, and prostate cancer samples yielded distinctive patterns and suggest the feasibility of our approach. Repeated experiments indicated reproducibility of such arrays. Up- or downregulated genes identified by this rapid screening are now being investigated with techniques such as in situ hybridization.     

COMPARISON OF ISOZYMES AMONG TYPHA SPECIES IN THE EASTERN UNITED STATES

Biochemical phenotsypes of four taxa of Typha from the eastern United States were determined by starch gel electrophoresis. The isozyme banding patterns of T. latifolia, T. angustifolia and T. domingensis are distinct and allow unambiguous species identification when morphological characters are inadequate or unsuitable. The fourth form, T. glauca, is not an F₁ hybrid, but it does appear to be intermediate between T. latifolia and T. angustifolia. The status of T. glauca and evolutionary relationships among the four forms may now be clarified by additional sampling because of the distinct and relatively invariant isozyme banding patterns which are described.     

Strength and physical fitness predict the perception of looming sounds

Listeners consistently perceive approaching sounds to be closer than they actually are and perceptually underestimate the time to arrival of looming sound sources. In a natural environment, this underestimation results in more time than expected to evade or engage the source and affords a “margin of safety” that may provide a selective advantage. However, a key component in the proposed evolutionary origins of the perceptual bias is the appropriate timing of anticipatory motor behaviors. Here we show that listeners with poorer physical fitness respond sooner to looming sounds and with a larger margin of safety than listeners with better physical fitness. The anticipatory perceptual bias for looming sounds is negatively correlated with physical strength and positively correlated with recovery heart rate (a measure of aerobic fitness). The results suggest that the auditory perception of looming sounds may be modulated by the response capacity of the motor system.     

The cost of mutualism in a fly-fungus interaction

The movement ability of individuals has become increasingly important to a variety of ecological questions. In this study, I investigate how plant structure and changes in body size through development affect the movement ability of a predaceous stinkbug, Podisus maculiventris, on three species of goldenrod (Solidago spp.) representing a wide range of surface complexities. I adapt existing techniques for quantifying movement in two dimensions to the study of movement on natural plant structures in three dimensions. These experiments indicate that plant structure and insect size are significant factors affecting the movement ability of P. maculiventris. Changes in movement ability due to factors of ontogeny and different habitat structures suggest that the scale of an individual’s ambit or ecological sphere of influence may vary within its lifespan. Considering the influence of ontogeny and habitat structure on movement ability may be useful to investigations of population dynamics, foraging behavior, and pest management. Received: 14 July 1999 / Accepted: 23 March 2000     

Endless forms most stupid,icky, and small: The preponderance of noncharismatic invertebrates as integral to a biologically sound view of life

Big, beautiful organisms are useful for biological education, increasing evolution literacy, and biodiversity conservation. But if educators gloss over the ubiquity of streamlined and miniaturized organisms, they unwittingly leave students and the public vulnerable to the idea that the primary evolutionary plot of every metazoan lineage is “progressive” and "favors" complexity. We show that simple, small, and intriguingly repulsive invertebrate animals provide a counterpoint to misconceptions about evolution. Our examples can be immediately deployed in biology courses and outreach. This context emphasizes that chordates are not the pinnacle of evolution. Rather, in the evolution of animals, miniaturization, trait loss, and lack of perfection are at least as frequent as their opposites. Teaching about invertebrate animals in a “tree thinking” context uproots evolution misconceptions (for students and the public alike), provides a mental scaffold for understanding all animals, and helps to cultivate future ambassadors and experts on these little‐known, weird, and fascinating taxa.     

Quantitative Modeling of GRK-Mediated β2AR Regulation

We developed a unified model of the GRK-mediated β2 adrenergic receptor (β2AR) regulation that simultaneously accounts for six different biochemical measurements of the system obtained over a wide range of agonist concentrations. Using a single deterministic model we accounted for (1) GRK phosphorylation in response to various full and partial agonists; (2) dephosphorylation of the GRK site on the β2AR; (3) β2AR internalization; (4) recycling of the β2AR post isoproterenol treatment; (5) β2AR desensitization; and (6) β2AR resensitization. Simulations of our model show that plasma membrane dephosphorylation and recycling of the phosphorylated receptor are necessary to adequately account for the measured dephosphorylation kinetics. We further used the model to predict the consequences of (1) modifying rates such as GRK phosphorylation of the receptor, arrestin binding and dissociation from the receptor, and receptor dephosphorylation that should reflect effects of knockdowns and overexpressions of these components; and (2) varying concentration and frequency of agonist stimulation “seen” by the β2AR to better mimic hormonal, neurophysiological and pharmacological stimulations of the β2AR. Exploring the consequences of rapid pulsatile agonist stimulation, we found that although resensitization was rapid, the β2AR system retained the memory of the previous stimuli and desensitized faster and much more strongly in response to subsequent stimuli. The latent memory that we predict is due to slower membrane dephosphorylation, which allows for progressive accumulation of phosphorylated receptor on the surface. This primes the receptor for faster arrestin binding on subsequent agonist activation leading to a greater extent of desensitization. In summary, the model is unique in accounting for the behavior of the β2AR system across multiple types of biochemical measurements using a single set of experimentally constrained parameters. It also provides insight into how the signaling machinery can retain memory of prior stimulation long after near complete resensitization has been achieved.     

Multipotent Cancer Stem Cells Derived from Human Malignant Peritoneal Mesothelioma Promote Tumorigenesis

During the progression of malignant peritoneal mesothelioma (MPeM), tumor nodules propagate diffusely within the abdomen and tumors are characterized by distinct phenotypic sub-types. Recent studies in solid organ cancers have shown that cancer stem cells (CSCs) play a pivotal role in the initiation and progression of tumors. However, it is not known whether tumorigenic stem cells exist and whether they promote tumor growth in MPeM. In this study, we developed and characterized a CSC model for MPeM using stably expandable tumorigenic stem cells derived from patient tumors. We found morphologically distinct populations of CSCs that divide asymmetrically or symmetrically in MPeM in vitro cell culture. The MPeM stem cells (MPeMSCs) express stem cell markers c-MYC, NES and VEGFR2 and in the presence of matrix components cells form colony spheres. MPeMSCs are multipotent, differentiate into neuronal, vascular and adipose progeny upon defined induction and the differentiating cells express lineage-specific markers such as TUBB3, an early neuronal marker; vWF, VEGFA, VEGFC and IL-8, endothelial markers; and PPARγ and FABP4, adipose markers. Xenotransplantation experiments using MPeMSCs demonstrated early tumor growth compared with parental cells. Limiting dilution experiments using MPeMSCs and endothelial lineage-induced cells derived from a single MPeMSC resulted in early tumor growth in the latter group indicating that endothelial differentiation of MPeMSCs is important for MPeM tumor initiation. Our observation that the MPeM tumors contain stem cells with tumorigenic potential has important implications for understanding the cells of origin and tumor progression in MPeM and hence targeting CSCs may be a useful strategy to inhibit malignant progression.