Fluorescein isothiocyanate staining intensity as a probe of hyperthermia-induced changes in chromatin conformation

In a previous report we presented evidence for large increases in fluorescein isothiocyanate (FITC) fluorescent intensity caused by hyperthermia which were not associated with synthesis of heat-shock proteins. We have now refined and considerably extended the measurements of increases in FITC fluorescent intensity caused by hyperthermia within the range 41.0 degrees C to 50.0 degrees C, and associated these with the extent of cell death caused by the hyperthermia. It appears that cell death ensues when the FITC fluorescent intensity has not returned to its baseline value within the time of one cell cycle. If thermotolerance is induced, there is a concomitant reduction in the increase in FITC staining intensity and the extent of cell death. When hyperthermia is followed by acid extraction, an additional increase in FITC staining intensity (above that due to hyperthermia alone) is observed, indicating separate sites of action on basic nuclear proteins. Hyperthermia and acid extraction have related effects on the relationship between FITC and propidium iodide staining. Hyperthermia-induced increases in FITC staining intensity are almost completely reversed by 6.7 mM formaldehyde with a marginal effect on the control FITC staining at this formaldehyde concentration. We suggest that hyperthermia causes extensive dissociation of basic protein-protein binding within nuclear chromatin, and that this may be a contributory cause of hyperthermia-induced cell death.     

NMR structural and dynamic characterization of the acid-unfolded state of apomyoglobin provides insights into the early events in protein folding

Apomyoglobin forms a denatured state under low-salt conditions at pH 2.3. The conformational propensities and polypeptide backbone dynamics of this state have been characterized by NMR. Nearly complete backbone and some side chain resonance assignments have been obtained, using a triple-resonance assignment strategy tailored to low protein concentration (0.2 mM) and poor chemical shift dispersion. An estimate of the population and location of residual secondary structure has been made by examining deviations of (13)C(alpha), (13)CO, and (1)H(alpha) chemical shifts from random coil values, scalar (3)J(HN,H)(alpha) coupling constants and (1)H-(1)H NOEs. Chemical shifts constitute a highly reliable indicator of secondary structural preferences, provided the appropriate random coil chemical shift references are used, but in the case of acid-unfolded apomyoglobin, (3)J(HN,H)(alpha) coupling constants are poor diagnostics of secondary structure formation. Substantial populations of helical structure, in dynamic equilibrium with unfolded states, are formed in regions corresponding to the A and H helices of the folded protein. In addition, the deviation of the chemical shifts from random coil values indicates the presence of helical structure encompassing the D helix and extending into the first turn of the E helix. The polypeptide backbone dynamics of acid-unfolded apomyoglobin have been investigated using reduced spectral density function analysis of (15)N relaxation data. The spectral density J(omega(N)) is particularly sensitive to variations in backbone fluctuations on the picosecond to nanosecond time scale. The central region of the polypeptide spanning the C-terminal half of the E helix, the EF turn, and the F helix behaves as a free-flight random coil chain, but there is evidence from J(omega(N)) of restricted motions on the picosecond to nanosecond time scale in the A and H helix regions where there is a propensity to populate helical secondary structure in the acid-unfolded state. Backbone fluctuations are also restricted in parts of the B and G helices due to formation of local hydrophobic clusters. Regions of restricted backbone flexibility are generally associated with large buried surface area. A significant increase in J(0) is observed for the NH resonances of some residues located in the A and G helices of the folded protein and is associated with fluctuations on a microsecond to millisecond time scale that probably arise from transient contacts between these distant regions of the polypeptide chain. Our results indicate that the equilibrium unfolded state of apomyoglobin formed at pH 2.3 is an excellent model for the events that are expected to occur in the earliest stages of protein folding, providing insights into the regions of the polypeptide that spontaneously undergo local hydrophobic collapse and sample nativelike secondary structure.     

Conservation of folding pathways in evolutionarily distant globin sequences

To test the hypothesis that the folding pathways of evolutionarily related proteins with similar three-dimensional structures but widely different sequences should be similar, the folding pathway of apoleghemoglobin has been characterized using stopped-flow circular dichroism, heteronuclear NMR pulse labeling techniques and mass spectrometry. The pathway of folding was found to differ significantly from that of a protein of the same family, apomyoglobin, although both proteins appear to fold through helical burst phase intermediates. For leghemoglobin, the burst phase intermediate exhibits stable helical structure in the G and H helices, together with a small region in the center of the E helix. The A and B helices are not stabilized until later stages of the folding process. The structure of the burst phase folding intermediate thus differs from that of apomyoglobin, in which stable helical structure is formed in the A, B, G and H helix regions.     

Requirements for cell cycle arrest by p16INK4a

Analysis of tumor-derived mutations has led to the suggestion that p16INK4a, cyclin D1, cdk4, and the retinoblastoma protein (pRB) are components of a regulatory pathway that is inactivated in most tumor cells. Cell cycle arrest induced by p16INK4a, an inhibitor of cyclin D-dependent kinases, requires pRB, and it has been proposed that this G1 arrest is mediated by pRB-E2F repressor complexes. By comparing the properties of primary mouse embryonic fibroblasts specifically lacking pRB-family members, we find that pRB is insufficient for a p16INK4a-induced arrest. In addition to pRB, a second function provided by either p107 or p130, two pRB-related proteins, is required for p16INK4a to block DNA synthesis. We infer that p16INK4a-induced arrest is not mediated exclusively by pRB, but depends on the nonredundant functions of at least two pRB-family members.     

Scube activity is necessary for Hedgehog signal transduction in vivo

The Hedgehog (HH) signaling pathway is a central regulator of embryonic development, controlling the pattern and proliferation of a wide variety of organs. Previous studies have implicated the secreted protein, Scube2, in HH signal transduction in the zebrafish embryo (Hollway et al., 2006; Kawakami et al., 2005; Woods and Talbot, 2005) although the nature of the molecular function of Scube2 in this process has remained undefined. This analysis has been compounded by the fact that removal of Scube2 activity in the zebrafish embryo leads to only subtle defects in HH signal transduction in vivo (Barresi et al., 2000; Hollway et al., 2006; Ochi and Westerfield, 2007; van Eeden et al., 1996; Wolff et al., 2003). Here we present the discovery of two additional scube genes in zebrafish, scube1 and scube3, and demonstrate their roles in facilitating HH signal transduction. Knocking down the function of all three scube genes simultaneously phenocopies a complete loss of HH signal transduction in the embryo, revealing that Scube signaling is essential for HH signal transduction in vivo. We further define the molecular role of scube2 in HH signaling.     

Globosides but not isoglobosides can impact the development of invariant NKT cells and their interaction with dendritic cells

Recognition of endogenous lipid Ag(s) on CD1d is required for the development of invariant NKT (iNKT) cells. Isoglobotrihexosylceramide (iGb3) has been implicated as this endogenous selecting ligand and recently suggested to control overstimulation and deletion of iNKT cells in α-galactosidase A-deficient (αGalA(-/-)) mice (human Fabry disease), which accumulate isoglobosides and globosides. However, the presence and function of iGb3 in murine thymus remained controversial. In this study, we generate a globotrihexosylceramide (Gb3)-synthase-deficient (Gb3S(-/-)) mouse and show that in thymi of αGalA(-/-)/Gb3S(-/-) double-knockout mice, which store isoglobosides but no globosides, minute amounts of iGb3 can be detected by HPLC. Furthermore, we demonstrate that iGb3 deficiency does not only fail to impact selection of iNKT cells, in terms of frequency and absolute numbers, but also does not alter the distribution of the TCR CDR 3 of iNKT cells. Analyzing multiple gene-targeted mouse strains, we demonstrate that globoside, rather than iGb3, storage is the major cause for reduced iNKT cell frequencies and defective Ag presentation in αGalA(-/-) mice. Finally, we show that correction of globoside storage in αGalA(-/-) mice by crossing them with Gb3S(-/-) normalizes iNKT cell frequencies and dendritic cell (DC) function. We conclude that, although detectable in murine thymus in αGalA(-/-)/Gb3S(-/-) mice, iGb3 does not influence either the development of iNKT cells or their interaction with peripheral DCs. Moreover, in αGalA(-/-) mice, it is the Gb3 storage that is responsible for the decreased iNKT cell numbers and impeded Ag presentation on DCs.     

O-8Detection of human telomerase gene (TERC) amplification in cervical neoplasia: a retrospective study of 50 patients with normal smears or mild or moderate dyskaryosis

Circulation E of the North West non gynaecological cytology EQA scheme was split into two with one half receiving conventional glass slides and the other half receiving digital images supplied by the SlidePath company over the web. More of the participants eligible participated in the slide half (43/65) than the digital half (17/41).
Similar results were obtained for seven out of ten of the scoring cases and for both the educational cases. However for one case eight of the digital participants could not obtain the images over the web. In the remaining three scoring cases the digital participants were less good at obtaining the correct answer compared to the slide based participants. The use of this virtual microscopy has shortened the circulation length considerably. The digital participants complained about the speed the digital slides appeared on their computers and whilst most agreed that this is the way forward they are still uncomfortable with their performance being marked on their digital submissions. Delegates will have the opportunity of viewing still images taken for each case from this circulation.     

Teaching and learning in ecology: a horizon scan of emerging challenges and solutions

We currently face significant, anthropogenic, global environmental challenges and the role of ecologists in mitigating these challenges is arguably more important than ever. Consequently there is an urgent need to recruit and train future generations of ecologists, both those whose main area is ecology, but also those involved in the geological, biological and environmental sciences. Here we present the results of a horizon scanning exercise that identified current and future challenges facing the teaching of ecology, through surveys of teachers, students and employers of ecologists. Key challenges identified were grouped in terms of the perspectives of three groups: students, for example the increasing disconnect between people and nature; teachers, for example the challenges associated with teaching the quantitative skills that are inherent to the study of ecology; and society, for example poor societal perceptions of the field of ecology. In addition to the challenges identified, we propose a number of solutions developed at a workshop by a team of ecology teaching experts, with supporting evidence of their potential to address many of the problems raised. These proposed solutions include developing living labs, teaching students to be ecological entrepreneurs and influencers, embedding skills-based learning and coding in the curriculum, an increased role for learned societies in teaching and learning, and using new technology to enhance fieldwork studies including virtual reality, artificial intelligence and real-time spoken language translation. Our findings are focused towards UK higher education, but they should be informative for students and teachers of a wide range of educational levels, policy makers and professional ecologists worldwide.     

Condensin minimizes topoisomerase II‐mediated entanglements of DNA in vivo

The juxtaposition of intracellular DNA segments, together with the DNA‐passage activity of topoisomerase II, leads to the formation of DNA knots and interlinks, which jeopardize chromatin structure and gene expression. Recent studies in budding yeast have shown that some mechanism minimizes the knotting probability of intracellular DNA. Here, we tested whether this is achieved via the intrinsic capacity of topoisomerase II for simplifying the equilibrium topology of DNA; or whether it is mediated by SMC (structural maintenance of chromosomes) protein complexes like condensin or cohesin, whose capacity to extrude DNA loops could enforce dissolution of DNA knots by topoisomerase II. We show that the low knotting probability of DNA does not depend on the simplification capacity of topoisomerase II nor on the activities of cohesin or Smc5/6 complexes. However, inactivation of condensin increases the occurrence of DNA knots throughout the cell cycle. These results suggest an in vivo role for the DNA loop extrusion activity of condensin and may explain why condensin disruption produces a variety of alterations in interphase chromatin, in addition to persistent sister chromatid interlinks in mitotic chromatin.