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)     

Benzo[a]pyrene diol epoxide induces viral reactivation at concentrations that block DNA elongation in mammalian cells

The survival of UV-irradiated Simian virus 40 (SV40) in CV-1P African green monkey kidney cells treated with (+/-)7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BP-diol epoxide I) was studied. Enhanced survival of UV damaged SV40 was detected when CV-1P cells were treated with dose levels of BP-diol epoxide I corresponding to the exponential portion (0.33-1.11 microM) of a CV-1P cell survival curve. Dose levels of BP-diol epoxide I corresponding to the shoulder region (less than or equal to 0.16 microM) of a CV-1P survival curve did not induce viral reactivation. The shoulder region concentrations of BP-diol epoxide I selectively inhibited DNA initiation while the concentrations on the exponential portion of the curve preferentially inhibited DNA elongation. It was shown in a time course of enhanced viral survival at 0.66 microM BP-diol epoxide I that the reactivation response was fully induced by 24 h. In conclusion, the viral reactivation response was associated with concentrations of BP-diol epoxide I which induced lethal damage and preferentially inhibited DNA elongation.     

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.     

The Winnipeg Children's Hospital child protection centre: a provincial medical initiative.

The Winnipeg Children''s Hospital child protection centre is a hospital-based provincial protection program that focuses on the diagnosis, treatment and prevention of child abuse and neglect in Manitoba. The multidisciplinary group of professionals working in the centre differs from most hospital-based teams in that it has a strong government liaison both functionally and financially, has strong ties to the welfare system and has legal counsel relating to the court system. The centre represents the philosophy that medical participation in welfare matters relating to children need not be relegated to the sidelines. This paper describes the centre''s history and structure, and it outlines some of its research projects.     

High-resolution nuclear magnetic resonance determination of transfer RNA tertiary base pairs in solution. 1. Species containing a small variable loop.

Eight class I tRNA species have been purified to homogeneity and their proton nuclear magnetic resonance (NMR) spectra in the low-field region (-11 to -15 ppm) have been studied at 360 MHz. The low-field spectra contain only one low-field resonance from each base pair (the ring NH hydrogen bond) and hence directly monitor the number of long-lived secondary and tertiary base pairs in solution. The tRNA species were chosen on the basis of their sequence homology with yeast phenylalanine tRNA in the regions which form tertiary base pairs in the crystal structure of this tRNA. All of the spectra show 26 or 27 low-field resonances approximately 7 of which are derived from tertiary base pairs. These results are contrary to previous claims that the NMR spectra indicate the presence of resonances from secondary base pairs only, as well as more recent claims of only 1-3 tertiary resonances, but are in good agreement with the number of tertiary base pairs expected in solution based on the crystal structure. The tertiary base pair resonances are stable up to at least 46 degrees C. Removal of magnesium ions causes structural changes in the tRNA but does not result in the loss of any secondary or tertiary base pairs.     

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.