Transformation of calf uterine progesterone receptor: analysis of the process when receptor is bound to progesterone and RU38486

Effects of different transforming agents were examined on the sedimentation characteristics of calf uterine progesterone receptor (PR) bound to the synthetic progestin [3H]R5020 or the known progesterone antagonist [3H]RU38486 (RU486). [3H]R5020-receptor complexes [progesterone-receptor complexes (PRc)] sedimented as fast migrating 8S moieties in 8-30% linear glycerol gradients containing 0.15 M KCl and 20 mM Na2MoO4. Incubation of cytosol containing [3H]PRc at 23 degrees C for 10-60 min, or at 0 degrees C with 0.15-0.3 M KCl or 1-10 mM ATP, caused a gradual transformation of PRc to a slow sedimenting 4S form. This 8S to 4S transformation was molybdate sensitive. In contrast, the [3H]RU486-receptor complex exhibited only the 8S form. Treatment with all three activation agents caused a decrease in the 8S form but no concomitant transformation of the [3H]RU486-receptor complex into the 4S form. PR in the calf uterine cytosol incubated at 23 or at 0 degrees C with 0.3 M KCl or 10 mM ATP could be subsequently complexed with [3H]R5020 to yield the 4S form of PR. However, the cytosol PR transformed in the absence of any added ligand failed to bind [3H]RU486. Heat treatment of both [3H]R5020- and [3H]RU486-receptor complexes caused an increase in DNA-cellulose binding, although the extent of this binding was lower when RU486 was bound to receptors. An aqueous two-phase partitioning analysis revealed a significant change in the surface properties of PR following both binding to ligand and subsequent transformation. The partition coefficient (Kobsd) of the heat-transformed [3H]R5020-receptor complex increased about 5-fold over that observed with PR at 0 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutation of the Axonal Transport Motor Kinesin Enhances Paralytic and Suppresses Shaker in Drosophila

To investigate the possibility that kinesin transports vesicles bearing proteins essential for ion channel activity, the effects of kinesin (Khc) and ion channel mutations were compared in Drosophila using established tests. Our results show that Khc mutations produce defects and genetic interactions characteristic of paralytic (para) and maleless (mle) mutations that cause reduced expression or function of the alpha-subunit of voltage-gated sodium channels. Like para and mle mutations, Khc mutations cause temperature-sensitive (TS) paralysis. When combined with para or mle mutations, Khc mutations cause synthetic lethality and a synergistic enhancement of TS-paralysis. Furthermore, Khc mutations suppress Shaker and ether-a-go-go mutations that disrupt potassium channel activity. In light of previous physiological tests that show that Khc mutations inhibit compound action potential propagation in segmental nerves, these data indicate that kinesin activity is required for normal inward sodium currents during neuronal action potentials. Tests for phenotypic similarities and genetic interactions between kinesin and sodium/potassium ATPase mutations suggest that impaired kinesin function does not affect the driving force on sodium ions. We hypothesize that a loss of kinesin function inhibits the anterograde axonal transport of vesicles bearing sodium channels.     

REVISED CLASSIFICATION OF THE GENUS PACHYMENIA BASED ON MORPHOLOGICAL AND MOLECULAR CHARACTERS

Taxonomic discrimination in the genus Pachymenia (Rhodophyta) in New Zealand is based primarily on phenotypic characters of the thallus. The taxonomic problems raised by this classification method are due to highly variable thallus characters such as blade thickness, blade width, degree of thallus branching, and variation in anatomical characters. Delineation of species is further complicated by a lack of adequate knowledge about the responses of phenotype to environmental variation. There are currently three species recognized in this genus that are endemic to New Zealand: a prostrate species P. crassa , and two erect species, P. laciniata and P. lusoria. In this study, two approaches are used to investigate the current delineation of these species. Morphological and anatomical characters of field collected material and herbarium specimens from throughout the species' distributional ranges were quantified. Multivariate analyses were used to identify discrete phenotypic groups. Species relationships were further analyzed by quantifying the variation found within the internal transcribed spacer region (ITS) of the nuclear ribosomal DNA. The results obtained from both approaches will be discussed with regards to possible re-classification of species relationships within this genus. We suggest that the two erect species should be merged, and the currently recognized P. lusoria should be separated into at least two taxonomic groups.     

Preslaughter holding environment in pork plants is highly contaminated with Salmonella enterica

The objective of this study was to determine whether abattoir pens can provide a Salmonella enterica infection source during the 2 to 4 h of preharvest holding. Previous work has suggested that pigs may be getting infected, but little has been reported on the environmental contamination of abattoir holding pens. For 24 groups of pigs studied ( approximately 150 animals/group) at two high-capacity abattoirs, six pooled fecal samples (n, 10 per pool) were collected from each transport trailer immediately after pigs were unloaded. Holding pens were sampled (one drinking water sample and six pooled floor samples consisting of swabs, residual liquid, and feces) prior to entry of study pigs for the routine holding period ( approximately 2.5 h). After slaughter, cecal contents and ileocecal lymph nodes were collected, on the processing line, from 30 pigs in each studied group. All samples were cultured for the isolation and identification of S. enterica by primary enrichment in GN-Hajna and tetrathionate broths, secondary enrichment in Rappaport-Vassiliadis broth, and plating on brilliant green sulfa and xylose-lysine-tergitol-4 agars, followed by biochemical and serological identification. The study pens were highly contaminated with S. enterica; all holding pens sampled had at least one positive sample. Additionally, 33% (8 of 24) of drinking water samples were positive for S. enterica. All 24 groups of pigs had S. enterica-positive cecal contents and ileocecal lymph nodes, including those groups from transport trailers with no positive samples. From pigs, trailers, and pens, 586 isolates representing 36 different Salmonella serovars were isolated. Of the 353 isolates from pigs (109 from ileocecal lymph nodes plus 244 from cecal contents), 19% were identified as belonging to the same serovars as those isolated from the respective pens; 27% were identified as belonging to the same serovars as those isolated from the trailers. Sixteen percent of the unique serovars were isolated from both pigs and pens, suggesting that pens served as the infection source. This study demonstrates highly contaminated abattoir holding pens and watering sources. It also demonstrates that holding pens can serve as an infection source. This study identifies the abattoir holding pens as a significant hazard and a potential control point for Salmonella contamination in the preharvest pork production chain.     

Nuclear magnetic resonance studies on the tertiary folding of transfer ribonucleic acid: assignment of the 7-methylguanosine resonance.

Analysis of the low-field nuclear magnetic resonance (NMR) spectra of several class 1 D4V5 transfer ribonucleic acid (tRNA) species containing 7-methylguanosine in their variable loops reveals a set of six to seven tertiary base pair resonances, one of which is always located at ca. --13.4 ppm. Other tRNA species which do not contain 7-methyl-guanosine do not contain the tertiary resonance at --13.4 ppm. Chemical removal of 7-methylguanosine from several tRNAs containing the same dihydrouridine (DHU) helix sequence as yeast tRNAPhe results in the loss of the --13.4-ppm tertiary resonance. In the initiator methionine tRNA, which contains a different DHU helix sequence, the 7-methylguanosine hydrogen bond has been assigned at --14.55 ppm by chemical removal of this residue. In these experiments the aromatic C8H proton of 7-methylguanosine was also assigned (--9.1 ppm). The unexpectedly low-field position of the 7-methylguanosine resonance is explained by the deshielding effect of the delocalized positive charge in this nucleoside.     

How do we overcome abrupt degradation of marine ecosystems and meet the challenge of heat waves and climate extremes?

Extreme heat wave events are now causing ecosystem degradation across marine ecosystems. The consequences of this heat‐induced damage range from the rapid loss of habitat‐forming organisms, through to a reduction in the services that ecosystems support, and ultimately to impacts on human health and society. How we tackle the sudden emergence of ecosystem‐wide degradation has not yet been addressed in the context of marine heat waves. An examination of recent marine heat waves from around Australia points to the potential important role that respite or refuge from environmental extremes can play in enabling organismal survival. However, most ecological interventions are being devised with a target of mid to late‐century implementation, at which time many of the ecosystems, that the interventions are targeted towards, will have already undergone repeated and widespread heat wave induced degradation. Here, our assessment of the merits of proposed ecological interventions, across a spectrum of approaches, to counter marine environmental extremes, reveals a lack preparedness to counter the effects of extreme conditions on marine ecosystems. The ecological influence of these extremes are projected to continue to impact marine ecosystems in the coming years, long before these interventions can be developed. Our assessment reveals that approaches which are technologically ready and likely to be socially acceptable are locally deployable only, whereas those which are scalable—for example to features as large as major reef systems—are not close to being testable, and are unlikely to obtain social licence for deployment. Knowledge of the environmental timescales for survival of extremes, via respite or refuge, inferred from field observations will help test such intervention tools. The growing frequency of extreme events such as marine heat waves increases the urgency to consider mitigation and intervention tools that support organismal and ecosystem survival in the immediate future, while global climate mitigation and/or intervention are formulated.     

Walking while Parasitized: Effects of a Naturally-Occurring Nematode on Locomotor Activity of Horned Passalus Beetles

Internal parasites typically are associated with a range of negative effects on their hosts, including reduced energy, which can manifest in behavioral alterations. With this in mind, we examined effects of a naturally-occurring nematode parasite, Chondronema passali, on locomotor activity level in horned passalus beetles, Odontotaenius disjunctus from Georgia, USA. This parasite is not well-studied but can number in the thousands in severely parasitized hosts. Prior study in our lab revealed that parasitized beetles actually consume more wood than unparasitized ones do, leading us to ask here, if parasitized beetles are also more physically active. Beetles were collected from nearby forests and housed individually in our lab. We created a simple tabletop arena to observe beetle locomotor activity, which was gridded and included small stones and paper objects. We allowed individual beetles to traverse the arena for 5 min and recorded the number of grid squares crossed. Then, beetles were dissected to determine parasite presence and level of infection (on a categorical scale). A total of 140 beetles were examined across three collections. Statistical analyses of locomotor activity revealed parasite severity predicted locomotor activity, but paradoxically, lightly-infected beetles were twice as active as those without this nematode. Activity diminished with increasing worm burdens thereafter, but even the group with the most severe burdens did not move less than those with no worms. From these results we conclude that this parasite does not result in overall reduction in activity, but rather it appears to come with heightened locomotion. Alternatively, this result could stem from the fact that more active beetles are simply more likely to contract the parasite.

     

Targeting Wild-Type and Mutationally Activated FGFR4 in Rhabdomyosarcoma with the Inhibitor Ponatinib (AP24534)

Rhabdomyosarcoma (RMS) is the most common childhood soft tissue sarcoma. Despite advances in modern therapy, patients with relapsed or metastatic disease have a very poor clinical prognosis. Fibroblast Growth Factor Receptor 4 (FGFR4) is a cell surface tyrosine kinase receptor that is involved in normal myogenesis and muscle regeneration, but not commonly expressed in differentiated muscle tissues. Amplification and mutational activation of FGFR4 has been reported in RMS and promotes tumor progression. Therefore, FGFR4 is a tractable therapeutic target for patients with RMS. In this study, we used a chimeric Ba/F3 TEL-FGFR4 construct to test five tyrosine kinase inhibitors reported to specifically inhibit FGFRs in the nanomolar range. We found ponatinib (AP24534) to be the most potent FGFR4 inhibitor with an IC₅₀ in the nanomolar range. Ponatinib inhibited the growth of RMS cells expressing wild-type or mutated FGFR4 through increased apoptosis. Phosphorylation of wild-type and mutated FGFR4 as well as its downstream target STAT3 was also suppressed by ponatinib. Finally, ponatinib treatment inhibited tumor growth in a RMS mouse model expressing mutated FGFR4. Therefore, our data suggests that ponatinib is a potentially effective therapeutic agent for RMS tumors that are driven by a dysregulated FGFR4 signaling pathway.