Light-induced interaction between rhodopsin and GTP-binding protein leads to the hydrolysis of GTP in the rod outer segment

In the presence of exogenous GTP, vertebrate whole rod outer segments (ROS), with perforated plasma membranes in the "single particle" scattering range, elicit a light-induced light-scattering transient which we call the "G" signal. Here, we report on the characteristics of the "G" signal relative to the "binding" and "dissociation" signals reported by Kuhn and colleagues. Replacing GTP with guanylyl imidodiphosphate (GMP-PNP) does not give rise to the G signal. This indicates that hydrolysis of the terminal phosphate is required for the G signal and, in addition, GTP and GMP-PNP compete for the same binding site of the enzyme responsible for the G signal (i.e., GTP-binding protein). Also, neither GDP nor its nonhydrolyzable analogue, guanosine 5'-O-(2-thiodiphosphate), when present in ROS suspensions yield any light-scattering transient in the time period tested.     

Can there be an escalating arms race without coevolution? Implications from a host-parasitoid simulation

Summary Under a restricted set of conditions, predator-prey or parasite-host systems may exhibit an escalating arms race over several generations that is not coevolutionary. Preconditions for such a process include high correlation between prey/host quality and defensive capability, and phenotypic plasticity in predator/parasite-counter defenses that responds to quality. We present simulation models based on the parasitoid waspEurytoma gigantea, which lays its egg in the goldenrod gall induced by the flyEurosta solidaginis. For the parasitoid to successfully lay an egg, the gall walls must be thinner than the parasitoid's ovipositor is long. Wall thickness is highly correlated with gall size, so probability of successful attack declines with gall size. However, since the parasitoid eats the gall tissue, individuals developing in small galls have little food and mature with shorter ovipositors than those which develop in large galls. The simulation showed that the population mean parasitoid size is set by mean gall size. Since small galls are more frequently parasitized, there is a selection pressure on the gallmaker to induce larger galls. But, an additional simulation showed that since parasitoid ovipositor length depends on gall size, an evolutionary increase in gall size will also result in a non-evolutionary increase in parasitoid body size and ovipositor length over several generations.     

Effects of fire on long-unburned Florida uplands

Abstract. The widespread suppression of fire during the 20th century has created extensive areas of fire-prone ecosystems that are in long-unburned condition. Plant species of flatwoods and scrubby flatwoods (= oak scrub) in the southeastern USA possess adaptations that facilitate resprouting, clonal spread, or seeding following fire. While the majority of the woody species of these associations can persist for long periods without fire, fire suppression reduces the populations of ephemeral herbs. Long-unburned (> 35 yr) flatwoods and scrubby flatwoods were burned after five annual vegetation censuses. Both stands were resampled five times over a >8-yr recovery period. One recently burned (<20 yr) flatwoods and two recently burned scrubby flatwoods were censused prior to fire and were recensused over a 5-yr or 11-yr period following fire depending on the stand. In spite of the variety of recovery strategies and time since fire, there were limited changes in the compositions and structures of post-burn stands compared to their preburn states. Detrended Correspondence Analysis showed that recently burned stands returned to preburn states within 1–2 yr. However, the reintroduction of a single fire to long-unburned stands did not restore the populations of herbs typical of recently burned stands. Our results suggest that a single fire may not be effective in restoring flatwoods and scrubby flatwoods that have experienced fire suppression to states more characteristic of recently burned stands.     

Bronchial epithelial cells are rendered insensitive to glucocorticoid transactivation by transforming growth factor-β1

Background

We have previously shown that transforming growth factor-beta (TGF-beta) impairs glucocorticoid (GC) function in pulmonary epithelial cell-lines. However, the signalling cascade leading to this impairment is unknown. In the present study, we provide the first evidence that TGF-beta impairs GC action in differentiated primary air-liquid interface (ALI) human bronchial epithelial cells (HBECs). Using the BEAS-2B bronchial epithelial cell line, we also present a systematic examination of the known pathways activated by TGF-beta, in order to ascertain the molecular mechanism through which TGF-beta impairs epithelial GC action.

Methods

GC transactivation was measured using a Glucocorticoid Response Element (GRE)–Secreted embryonic alkaline phosphatase (SEAP) reporter and measuring GC-inducible gene expression by qRT-PCR. GC transrepression was measured by examining GC regulation of pro-inflammatory mediators. TGF-beta signalling pathways were investigated using siRNA and small molecule kinase inhibitors. GRα level, phosphorylation and sub-cellular localisation were determined by western blotting, immunocytochemistry and localisation of GRα–Yellow Fluorescent Protein (YFP). Data are presented as the mean ± SEM for n independent experiments in cell lines, or for experiments on primary HBEC cells from n individual donors. All data were statistically analysed using GraphPad Prism 5.0 (Graphpad, San Diego, CA). In most cases, two-way analyses of variance (ANOVA) with Bonferroni post-hoc tests were used to analyse the data. In all cases, P <0.05 was considered to be statistically significant.

Results

TGF-beta impaired Glucocorticoid Response Element (GRE) activation and the GC induction of several anti-inflammatory genes, but did not broadly impair the regulation of pro-inflammatory gene expression in A549 and BEAS-2B cell lines. TGF-beta-impairment of GC transactivation was also observed in differentiated primary HBECs. The TGF-beta receptor (ALK5) inhibitor SB431541 fully prevented the GC transactivation impairment in the BEAS-2B cell line. However, neither inhibitors of the known downstream non-canonical signalling pathways, nor knocking down Smad4 by siRNA prevented the TGF-beta impairment of GC activity.

Conclusions

Our results indicate that TGF-beta profoundly impairs GC transactivation in bronchial epithelial cells through activating ALK5, but not through known non-canonical pathways, nor through Smad4-dependent signalling, suggesting that TGF-beta may impair GC action through a novel non-canonical signalling mechanism.     

James F. Crow: his life in public service

The readers of this journal may well be aware of Professor Crow's scientific achievements and his role as the editor of Perspectives. In addition, for many thousands of students at the University of Wisconsin over many generations, James F. Crow was one of the most memorable teachers at both the undergraduate and graduate levels. What is less known is his major role in public service where he served as chair of many important committees for the National Academy of Sciences, the National Institutes of Health, the National Institutes of Justice as well as various international programs. In all of these efforts, Professor Crow has left a lasting impact.     

Cystatins – Extra- and intracellular cysteine protease inhibitors: High-level secretion and uptake of cystatin C in human neuroblastoma cells

Cystatins are present in mammals, birds, fish, insects, plants, fungi and protozoa and constitute a large protein family, with most members sharing a cysteine protease inhibitory function. In humans 12 functional cystatins exist, forming three groups based on molecular organisation and distribution in the organism. The type 1 cystatins (A and B) are known as intracellular, type 2 cystatins (C, D, E/M, F, G, S, SN and SA) extracellular and type 3 cystatins (L- and H-kininogen) intravascular proteins. The present paper is focused on the human cystatins and especially those of type 2, which are directed (with signal peptides) for cellular export following translation. Results indicating existence of systems for significant internalisation of type 2 cystatins from the extracellular to intracellular compartments are reviewed. Data showing that human neuroblastoma cell lines generally secrete high levels, but also contain high amounts of cystatin C are presented. Culturing of these cells in medium containing cystatin C at concentrations found in body fluids resulted in increased intracellular cystatin C, as a result of an uptake process. At immunofluorescence cytochemistry a pronounced vesicular cystatin C staining was observed. The simplistic denotation of the type 2 cystatins as extracellular inhibitors is thus challenged, and possible biological functions of the internalised cystatins are discussed. To illustrate the special case of high cellular cystatin content seen in cells of patients with hereditary cystatin C amyloid angiopathy, expression vectors for wild-type and L68Q mutated cystatin C were used to transfect SK-N-BE(2) cells. Clones overexpressing the two variants showed increased secreted levels of cystatin C. Within the cells the L68Q variant appeared to mainly localise to the endoplasmic reticulum rather than to acidic vesicular organelles, indicating limitations in the transport out from the cell rather than increased uptake as explanation for the elevated cellular cystatin levels seen in hereditary cystatin C amyloid angiopathy.