SUB-CHROMATID REARRANGEMENTS IN TRILLIUM ERECTUM. I. ORIGIN AND NATURE OF CONFIGURATIONS INDUCED BY IONIZING RADIATION

Wilson , G. B. (Michigan State U., East Lansing.), A. H. Sparrow , and Virginia Pond . Subchromatid rearrangements in Trillium erectum. I. Origin and nature of configurations induced by ionizing radiation. Amer. Jour. Bot. 46(4): 309–316. Illus. 1959.—Microsporocytes of Trillium erectum were x-irradiated with 25 r at various stages of meiotic prophase and at first metaphase. Analysis of these cells at the following first and second anaphase revealed that post-pachytene irradiation produces 2-side-arm bridges which are indicative of half-chromatid exchanges. The occurrence of these bridges and knowledge of the structure and spatial relationship of chromatid strands in T. erectum have led to certain conclusions regarding the target and the number of strands broken by a single event: (1) the most likely target for primary effects is the 4 associated half-chromatids of a half-bivalent. The results of irradiation experiments suggest that the half-bivalent is effectively as well as structurally quadripartite at stages following pachytene. (2) Consideration of the configurations which would result from breakage and rejoining of 2, 3 or all 4 strands of the half-bivalent indicates that only 2 of the 4 half-chromatids are broken by a single event. Exchanges between 2 half-chromatids of sister chromatids will produce two recognizable types of 2-side-arm bridges: one with a true dicentric half-chromatid and one in which the bridge results merely from an interlocking of coils. Whether a 2-side-arm bridge appears at first or second meiotic anaphase is determined by the position and number of chiasmata between the point of breakage and the kinetochore. No 2-side-arm bridges have been detected at microspore anaphase following meiotic prophase irradiation. The types of configurations which might be expected at microspore metaphase as a result of broken 2-side-arm bridges are noted.     

Do alternative reproductive strategies in the Wellington tree weta represent different behavioural types?

In many animal species, variation in reproductive success among individuals has led to the evolution of alternative mating strategies, which in the case of insects can often be correlated with developmental trajectories. In the Wellington tree weta, Hemideina crassidens, males can mature at the 8th, 9th or 10th instar, while females mature at the 10th instar only. A number of morphological attributes including male head and mandible size correlate with final instar number, and as these attributes represent a form of weaponry, they are often used in mate/site guarding and male–male competition. Tenth instar males have larger head/mandible/body sizes and show a conventional (guarder) reproductive strategy, whereas smaller 8th instar males typically show an unconventional (sneaker) strategy. In contrast, 9th instar males are predicted to adopt a “jack‐of‐all‐trades” strategy whereby they can fight or sneak depending context. Here, we tested whether alternative reproductive morphs exhibit strategy‐specific differences in risk‐taking associated with refuge emergence, activity and antipredator behaviour and further, whether these traits correlate to form a behavioural syndrome. We found that tree weta show consistent and repeatable differences in activity and refuge use at the individual level; however, behavioural covariances suggest that only 8th instar males exhibit a behavioural syndrome. That 9th instar males show high plasticity and variance in their gallery‐related behaviours supports the hypothesis that these males are a “jack‐of‐all‐trades.” Contrary to our predictions, antipredator behaviour was not correlated with other traits, and differences in behaviour overall were consistently more pronounced between individuals rather than between male morphs or sexes.     

The effects of 11-methyl 16,16 dimethyl prostaglandin E2 on gastric acid secretion in man

11-Methyl 16,16 Dimethyl Prostaglandin E₂ (TM-PGE) was administered orally to man in dosages of 2.5, 5, 7.5 and 10 μg/kg. Maximal inhibition of basal secretion was 52 and 78% and submaximal histamine-stimulated secretion 45 and 70% for volume and acid output, respectively. Secretory inhibition was observed for approximately two hours after ingestion of the drug. No effect was observed on serum gastrin levels. Side effects occurred with equal frequency in the placebo and drug groups. TM-PGE is well tolerated and inhibits both basal and submaximal histamine-stimulated acid secretion in man. Further evaluation may prove it to be helpful in the clinical treatment of acid hypersecretory states and peptic ulcer disease.     

Paxillin and focal adhesion kinase colocalise in human skeletal muscle and its associated microvasculature

Focal adhesion kinase (FAK) and paxillin are functionally linked hormonal- and mechano-sensitive proteins. We aimed to describe paxillin’s subcellular distribution using widefield and confocal immunofluorescence microscopy and test the hypothesis that FAK and paxillin colocalise in human skeletal muscle and its associated microvasculature. Percutaneous muscle biopsies were collected from the m. vastus lateralis of seven healthy males, and 5-μm cryosections were stained with anti-paxillin co-incubated with anti-dystrophin to identify the sarcolemma, anti-myosin heavy chain type I for fibre-type differentiation, anti-dihydropyridine receptor to identify T-tubules, lectin UEA-I to identify the endothelium of microvessels and anti-α-smooth muscle actin to identify vascular smooth muscle cells (VSMC). Colocalisation of anti-paxillin with anti-dystrophin or anti-FAK was quantified using Pearson’s correlation coefficient on confocal microscopy images. Paxillin was primarily present in (sub)sarcolemmal regions of skeletal muscle fibres where it colocalised with dystrophin (r = 0.414 ± 0.026). The (sub)sarcolemmal paxillin immunofluorescence intensity was ~2.4-fold higher than in sarcoplasmic regions (P < 0.001) with sarcoplasmic paxillin immunofluorescence intensity ~10 % higher in type I than in type II fibres (P < 0.01). In some longitudinally orientated fibres, paxillin formed striations that corresponded to the I-band region. Paxillin immunostaining was highest in endothelial and VSMC and distributed heterogeneously in both cell types. FAK and paxillin colocalised at (sub)sarcolemmal regions and within the microvasculature (r = 0.367 ± 0.036). The first images of paxillin in human skeletal muscle suggest paxillin is present in (sub)sarcolemmal and I-band regions of muscle fibres and within the microvascular endothelium and VSMC. Colocalisation of FAK and paxillin supports their suggested role in hormonal and mechano-sensitive signalling.     

The Personality Behind Cheating: Behavioural Types and the Feeding Ecology of Cleaner Fish

The complex mutualistic relationship between the cleaner fish (Labroides dimidiatus) and their ‘clients’ in many reef systems throughout the world has been the subject of debate and research interest for decades. Game‐theory models have long struggled with explaining how the mixed strategies of cheating and honesty might have evolved in such a system and while significant efforts have been made theoretically, demonstrating the nature of this relationship empirically remains an important research challenge. Using the experimental framework of behavioural syndromes, we sought to quantitatively assess the relationship between personality and the feeding ecology of cleaner fish to provide novel insights into the underlying mechanistic basis of cheating in cleaner‐client interactions. First, we observed and filmed cleaner fish interactions with heterospecifics, movement patterns and general feeding ecology in the wild. We then captured and measured all focal individuals and tested them for individual consistency in measures of activity, exploration and risk taking (boldness) in the laboratory. Our results suggest a syndrome incorporating aspects of personality and foraging effort are central components of the behavioural ecology of L. dimidiatus on the Great Barrier Reef. We found that individuals that exhibited greater feeding effort tended to cheat proportionately less and move over smaller distances relative to bolder more active, exploratory individuals. Our study demonstrates for the first time that individual differences in personality might be mechanistically involved in explaining how the mixed strategies of cheating and honesty persist in cleaner fish mutualisms.     

Delineation of key XRCC4/Ligase IV interfaces for targeted disruption of non‐homologous end joining DNA repair

Efficient DNA repair mechanisms frequently limit the effectiveness of chemotherapeutic agents that act through DNA damaging mechanisms. Consequently, proteins involved in DNA repair have increasingly become attractive targets of high‐throughput screening initiatives to identify modulators of these pathways. Disruption of the XRCC4‐Ligase IV interaction provides a novel means to efficiently halt repair of mammalian DNA double strand break repair; however; the extreme affinity of these proteins presents a major obstacle for drug discovery. A better understanding of the interaction surfaces is needed to provide a more specific target for inhibitor studies. To clearly define key interface(s) of Ligase IV necessary for interaction with XRCC4, we developed a competitive displacement assay using ESI‐MS/MS and determined the minimal inhibitory fragment of the XRCC4‐interacting region (XIR) capable of disrupting a complex of XRCC4/XIR. Disruption of a single helix (helix 2) within the helix‐loop‐helix clamp of Ligase IV was sufficient to displace XIR from a preformed complex. Dose‐dependent response curves for the disruption of the complex by either helix 2 or helix‐loop‐helix fragments revealed that potency of inhibition was greater for the larger helix‐loop‐helix peptide. Our results suggest a susceptibility to inhibition at the interface of helix 2 and future studies would benefit from targeting this surface of Ligase IV to identify modulators that disrupt its interaction with XRCC4. Furthermore, helix 1 and loop regions of the helix‐loop‐helix clamp provide secondary target surfaces to identify adjuvant compounds that could be used in combination to more efficiently inhibit XRCC4/Ligase IV complex formation and DNA repair. Proteins 2014; 82:187–194. © 2013 Wiley Periodicals, Inc.     

Integration of high-throughput analytics and cell imaging enables direct early productivity and product quality assessment during Chinese Hamster ovary cell line development for a complex multi-subunit vaccine antigen

Mammalian cell line generation typically includes stable pool generation, single cell cloning and several rounds of clone selection based on cell growth, productivity and product quality criteria. Individual clone expansion and phenotype-based ranking is performed initially for hundreds or thousands of mini-scale cultures, representing the major operational challenge during cell line development. Automated cell culture and analytics systems have been developed to enable high complexity clone selection workflows; while ensuring traceability, safety, and quality of cell lines intended for biopharmaceutical applications. Here we show that comprehensive and quantitative assessment of cell growth, productivity, and product quality attributes are feasible at the 200–1,200 cell colony stage, within 14 days of the single cell cloning in static 96-well plate culture. The early cell line characterization performed prior to the clone expansion in suspension culture can be used for a single-step, direct selection of high quality clones. Such clones were comparable, both in terms of productivity and critical quality attributes (CQAs), to the top-ranked clones identified using an established iterative clone screening approach. Using a complex, multi-subunit antigen as a model protein, we observed stable CQA profiles independently of the cell culture format during the clonal expansion as well as in the batch and fed-batch processes. In conclusion, we propose an accelerated clone selection approach that can be readily incorporated into various cell line development workstreams, leading to significant reduction of the project timelines and resource requirements.