A novel 500,000 Da, linear, single chain extracellular protein synthesized by several childhood tumors

A high molecular weight extracellular protein has been purified from cell culture medium of Ewing's sarcoma cell lines, by high performance liquid chromatography and electroelution from SDS-PAGE electrophoresis. This protein has an apparent molecular mass of about 500,000 Da on SDS-PAGE. Immunoprecipitation studies with several extracellular matrix glycoproteins (laminin, fibronectin) specific antisera indicate it is a separate protein. Reduction of disulphide bonds with 2-ME or DTT fails to significantly alter its migration on SDS-PAGE gels, other than a slight apparent increase in molecular mass, indicating an apparent single polypeptide chain structure. The slightly greater mobility observed in unreduced gels suggests one or more regions of intrachain disulfide bonding. It is sensitive to pepsin and trypsin, but resistant to bacterial collagenase indicating that it does not contain collagenous domains. Metabolic labelling with 3H-proline, 3H-leucine, and 35S-methionine indicate that this protein is proline-poor, but leucine, and especially methionine, rich. Sodium 35S-sulfate incorporation is totally negative and treatment with glycosaminoglycan degrading enzymes has no effect on the mobility of the protein on gels, unlike typical proteoglycans. This protein appears by rotary shadowing electron microscopy as a long, thin, filamentous molecule at least 500 nm (0.5 um) in length. The tissue localization and function are unknown at this time, but are under active investigation.     

Analysis of DNA methylation and gene expression in radiation-resistant head and neck tumors

Resistance to radiation therapy constitutes a significant challenge in the treatment of head and neck squamous cell cancer (HNSCC). Alteration in DNA methylation is thought to play a role in this resistance. Here, we analyzed DNA methylation changes in a matched model of radiation resistance for HNSCC using the Illumina HumanMethylation450 BeadChip. Our results show that compared to radiation-sensitive cells (SCC-61), radiation-resistant cells (rSCC-61) had a significant increase in DNA methylation. After combining these results with microarray gene expression data, we identified 84 differentially methylated and expressed genes between these 2 cell lines. Ingenuity Pathway Analysis revealed ILK signaling, glucocorticoid receptor signaling, fatty acid α-oxidation, and cell cycle regulation as top canonical pathways associated with radiation resistance. Validation studies focused on CCND2, a protein involved in cell cycle regulation, which was identified as hypermethylated in the promoter region and downregulated in rSCC-61 relative to SCC-61 cells. Treatment of rSCC-61 and SCC-61 with the DNA hypomethylating agent 5-aza-2''deoxycitidine increased CCND2 levels only in rSCC-61 cells, while treatment with the control reagent cytosine arabinoside did not influence the expression of this gene. Further analysis of HNSCC data from The Cancer Genome Atlas found increased methylation in radiation-resistant tumors, consistent with the cell culture data. Our findings point to global DNA methylation status as a biomarker of radiation resistance in HNSCC, and suggest a need for targeted manipulation of DNA methylation to increase radiation response in HNSCC.     

Home-field advantage: native signal crayfish (Pacifastacus leniusculus) out consume newly introduced crayfishes for invasive Chinese mystery snail (Bellamya chinensis)

The introduction of non-indigenous plants, animals and pathogens is one of today’s most pressing environmental challenges. Freshwater ecologists are challenged to predict the potential consequences of species invasions because many ecosystems increasingly support novel assemblages of native and non-native species that are likely to interact in complex ways. In this study we evaluated how native signal crayfish (Pacifastacus leniusculus) and non-native red swamp crayfish (Procambarus clarkii) and northern crayfish (Orconectes virilis) utilize a novel prey resource: the non-native Chinese mystery snail (Bellamya chinensis). All species are widespread in the United States, as well as globally, and recent surveys have discovered them co-occurring in lakes of Washington State. A series of mesocosm experiments revealed that crayfish are able to consume B. chinensis, despite the snail’s large size, thick outer shell and trapdoor defense behaviour. Crayfish exhibited size-selective predation whereby consumption levels decreased with increasing snail size; a common pattern among decapod predators. Comparison of prey profitability curves—defined as the yield of food (weight of snail tissue) per second of feeding time (the time taken to crack the shell and consume the contents)—suggests that small and very large snails may represent the most profitable prey choice. By contrast, previous studies have reported the opposite pattern for crayfish consumption on thin-shelled snails. For all snail size classes, we found that native P. leniusculus and invasive O. virilis consumed greater numbers of snails than invasive P. clarkii. Moreover, P. leniusculus consistently handled and consumed snails at a faster pace compared to both invasive crayfishes across the range of snail sizes examined in our study. These results suggest not only that B. chinensis is a suitable food source for crayfish, but also that native P. leniusculus may ultimately out-consume invasive crayfishes for this new prey resource.     

Web-based weight management programs in an integrated health care setting: a randomized, controlled trial

Objective: To assess the efficacy of a Web‐based tailored behavioral weight management program compared with Web‐based information‐only weight management materials. Research Methods and Procedures: Participants, 2862 eligible overweight and obese (BMI = 27 to 40 kg/m²) members from four regions of Kaiser Permanente's integrated health care delivery system, were randomized to receive either a tailored expert system or information‐only Web‐based weight management materials. Weight change and program satisfaction were assessed by self‐report through an Internet‐based survey at 3‐ and 6‐month follow‐up periods. Results: Significantly greater weight loss at follow‐up was found among participants assigned to the tailored expert system than among those assigned to the information‐only condition. Subjects in the tailored expert system lost a mean of 3 ± 0.3% of their baseline weight, whereas subjects in the information‐only condition lost a mean of 1.2 ± 0.4% (p < 0.0004). Participants were also more likely to report that the tailored expert system was personally relevant, helpful, and easy to understand. Notably, 36% of enrollees were African‐American, with enrollment rates higher than the general proportion of African Americans in any of the study regions. Discussion: The results of this large, randomized control trial show the potential benefit of the Web‐based tailored expert system for weight management compared with a Web‐based information‐only weight management program.     

Allometry and evolution in the galago skull

To probe the ontogenetic bases of morphological diversity across galagos, we performed the first clade-wide analyses of growth allometries in 564 adult and non-adult crania from 12 galagid taxa. In addition to evaluating if variation in galago skull form results from the differential extension/truncation of common ontogenetic patterns, scaling trajectories were employed as a criterion of subtraction to identify putative morphological adaptations in the feeding complex. A pervasive pattern of ontogenetic scaling is observed for facial dimensions across galagids, with 2 genera also sharing relative growth trajectories for masticatory proportions (Galago, Galagoides). As the facial growth series and adult data are largely coincidental, interspecific variation may result from character displacement and consequent selection for size differentiation among sister taxa. Derived configurations of the jaw joint and jaw muscle mechanical advantage in Otolemur and Euoticus appear to facilitate increased gape during scraping behaviors. Differences in aspects of masticatory growth and form characterizing these 2 genera highlight selection to uncouple shared ontogenetic patterns, which occurred via transpositions that retained ancestral scaling patterns. Due to the lack of increased robusticity of load-resisting mandibular elements in Otolemur and Euoticus, there is little evidence to suggest that exudativory in galagos results in correspondingly higher masticatory stresses.     

Measurement of 14N superhyperfine frequencies in stellacyanin by an electron spin echo method.

We have measured the 14N superhyperfine frequencies for weakly coupled nitrogen in stellacyanin and in a model compound Cu(II)-diethylenetriamine-imidazole using a 3-pulse spin echo technique. By making computer simulations of the superhyperfine spectrum, we have been able to show that these frequencies result from the interaction of the remote protonated nitrogen of metal-bound imidazole with Cu(II).     

The fate of IgE bound to rat basophilic leukemia cells.

The present study investigates the fate of the cell-bound IgE by using a well-characterized rat basophilic leukemia cell line and a purifed IgE myeloma protein. Both histamine-releasing and nonreleasing cell lines were examined. In both cases, no evidence for cell-mediated IgE catabolism could be elicited. Both the dissociated IgE and the receptors remained intact for prolonged periods of time, as demonstrated by binding assays. Internalization and/or recycling of membrane-bound IgE could not be demonstrated by E. M. autoradiography. We found only limited time-dependent changes in accessibility to anti-IgE antibody, trypsin, or elution at low pH (2.9 to 3.1). A biphasic dissociation of cell-bound 125I-IgE during incubation in the presence of excess unlabeled IgE was reproducibly observed; the more slowly dissociated IgE was also less readily dissociated at pH 3.4. These studies lead us to conclude that, in vitro, IgE resides in a functional orientation on the surface of RBL-1 cells, for prolonged periods of time.     

Linear electric field effect in electron paramagnetic resonance for two bisimidazole--heme complexes, model compounds for B and H hemichromes of hemoglobin and for cytochrome b5.

Bisimidazole-ferric heme is considered to be the structure at the heme site of cytochrome b5 and two different low spin ferric hemochromes spontaneously formed from ferric hemoglobin. The addition of strong base to bisimidazole-ferric heme in organic solvents alters the optical and magnetic properties of this compound. With the use of the linear electric field effect in the electron paramagnetic resonance, we demonstrate that addition of base does not lead to the exchange of hydroxide anion for ligated imidazole and that the bisimidazole structure is retained. Analysis of optical titrations indicates that 2 equiv of base react reversibly with bisimidazole-ferric heme. It is suggested that the two hemichromes formed from hemoglobin differ in structure from one another by the state of protonation of N-1 in the bound imidazoles.