High- and low-affinity GABA-receptors in cultured cerebellar granule cells regulate transmitter release by different mechanisms

The ability of high- and low-affinity GABA_A-receptors, respectively to inhibit depolarization coupled transmitter release was studied in cultured glutamatergic cerebellar granule cells which, depending on the culture conditions, express either high-affinity GABA_A-receptors alone or high-affinity receptors together with low-affinity receptors. In order to gain information about the coupling of these receptors to chloride channels the effect of picrotoxin and binding of [³⁵S]t-butylbicyclophosphorothionate, both of which interact specifically with such channels were studied. Moreover, the influence of Flunitrazepam on the GABA-mediated inhibition of transmitter release was investigated to see if the GABA-receptors are coupled to benzodiazepine binding sites. Under conditions where the granule cells express only high-affinity GABA_A-receptors it was found that GABA was able to inhibit transmitter release elicited by mild depolarization induced either by 30 mM KCl or 25 μM glutamate. This effect of GABA could be enhanced by Flunitrazepam and blocked by picrotoxin. However, transmitter release from these neurones induced by a more pronounced depolarization (55 mM KCl) could not be inhibited by GABA. Under conditions where the neurons express both high- and low-affinity GABA_A-receptors transmitter release elicited by 55 mM KCl could be inhibited by GABA but this inhibitory effect of GABA could not be blocked by picrotoxin, nor could it be enhanced by Flunitrazepam. These results strongly suggest that while the action of the high-affinity GABA_A-receptors is coupled to chloride channels and benzodiazepine binding sites, the physiological action of the low-affinity GABA_A-receptors is not. This lack of coupling between the low-affinity GABA_A-receptors and chloride channels is further supported by the finding that the K_D and B_max values for [³⁵S]TBPS binding to the granule cells were independent of whether or not the cells expressed low-affinity GABA_A-receptors. While the results clearly show that the inhibitory action of GABA mediated by low-affinity GABA_A-receptors is not coupled to chloride channels, the exact mechanism of action of these receptors still remains to be elucidated.     

Beneficial Effect of Verminephrobacter Nephridial Symbionts on the Fitness of the Earthworm Aporrectodea tuberculata

Almost all lumbricid earthworms (Oligochaeta: Lumbricidae) harbor species-specific Verminephrobacter (Betaproteobacteria) symbionts in their nephridia (excretory organs). The function of the symbiosis, and whether the symbionts have a beneficial effect on their earthworm host, is unknown; however, the symbionts have been hypothesized to enhance nitrogen retention in earthworms. The effect of Verminephrobacter on the life history traits of the earthworm Aporrectodea tuberculata (Eisen) was investigated by comparing the growth, development, and fecundity of worms with and without symbionts given high (cow dung)- and low (straw)-nutrient diets. There were no differences in worm growth or the number of cocoons produced by symbiotic and aposymbiotic worms. Worms with Verminephrobacter symbionts reached sexual maturity earlier and had higher cocoon hatching success than worms cured of their symbionts when grown on the low-nutrient diet. Thus, Verminephrobacter nephridial symbionts do have a beneficial effect on their earthworm host. Cocoons with and without symbionts did not significantly differ in total organic carbon, total nitrogen, or total hydrolyzable amino acid content, which strongly questions the hypothesized role of the symbionts in nitrogen recycling for the host.Symbiosis has long been recognized as a source of evolutionary innovation (24), and the acquisition of symbionts can enable animal hosts to exploit previously inaccessible niches (3). The phylum Annelida is no exception to this; chemosynthetic symbionts in marine annelids (e.g., the giant tubeworm Riftia sp. and other gutless marine oligochaetes [9]) gain energy from the oxidation of reduced sulfur compounds and fix CO₂ and supply their animal host with fixed carbon. A more obscure partnership is known from the bone-eating annelid Osedax sp., where endosymbionts help degrade the bones of whale carcasses, the only known habitat of the worms (33). The medicinal leech Hirudo sp., like other blood-feeding animals (3, 7), has symbionts that are thought to produce essential vitamins missing from a blood meal (13). In addition, leeches have a number of symbionts of unknown function in their nephridia (excretory organs) (18). Earthworms (Oligochaeta: Lumbricidae) have also long been known to harbor symbiotic bacteria in their nephridia (19, 36). The function of this symbiosis, however, is still not known, but the stability of the symbiosis over evolutionary time (23) suggests that the symbionts benefit the host.The earthworm symbionts reside in the nephridia and have therefore been proposed to be involved in internal recycling of nitrogen in the host (29). The earthworm nephridia play an important role in both nitrogenous waste excretion and osmoregulation (20). The nephridia are found in pairs in each segment of the worm and consist of a coiled tube leading from the coelom to the exterior (Fig. ​(Fig.1).1). The tube forms three loops, and the symbiotic bacteria are situated in the ampulla in the second loop, where they form a dense population lining the lumen wall (19, 36).Open in a separate windowFIG. 1.The nephridia are found as paired organs in each segment of the worm. The nephridostome (the inlet to the nephridia) protrudes into the previous segment. The nephridial tube forms three loops and finally empties out through the body wall via the nephridopore. The symbionts (black) reside in the ampulla in the second loop. (Modified from reference 23 with permission.)The symbionts form the monophyletic genus Verminephrobacter (Betaproteobacteria) (30, 36); they are species specific and present in almost all lumbricid earthworms (23). The Verminephrobacter symbionts are transmitted vertically via the cocoon, where they are deposited along with eggs and sperm (5). During embryogenesis, the symbionts migrate into the developing nephridia, and after the worm hatches, the symbionts can no longer infect (5, 6). By taking advantage of the vertical transmission mode, it has been possible to establish symbiont-free earthworm cultures in the laboratory through controlled antibiotic treatment of newly deposited cocoons (5; this study). Separation of the symbiotic partners allows studies of the effect of the symbionts on their earthworm hosts.Pandazis (29) hypothesized that the symbionts enhance earthworm nitrogen retention by excreting proteolytic enzymes that will degrade peptides and proteins lost in urine; this would allow the earthworm to reabsorb the resulting amino acids. As a consequence, earthworms cured of their symbionts should have a lower fitness level than control worms when grown under nitrogen-limiting conditions. To test this hypothesis, growth, development, fecundity, and cocoon hatching success were compared for symbiotic and aposymbiotic earthworms of the species Aporrectodea tuberculata (Eisen) under high and low nutrient availability conditions.     

Statistical multiplicity in systematic reviews of anaesthesia interventions: a quantification and comparison between Cochrane and non-Cochrane reviews

Background

Systematic reviews with meta-analyses often contain many statistical tests. This multiplicity may increase the risk of type I error. Few attempts have been made to address the problem of statistical multiplicity in systematic reviews. Before the implications are properly considered, the size of the issue deserves clarification. Because of the emphasis on bias evaluation and because of the editorial processes involved, Cochrane reviews may contain more multiplicity than their non-Cochrane counterparts. This study measured the quantity of statistical multiplicity present in a population of systematic reviews and aimed to assess whether this quantity is different in Cochrane and non-Cochrane reviews.

Methods/Principal Findings

We selected all the systematic reviews published by the Cochrane Anaesthesia Review Group containing a meta-analysis and matched them with comparable non-Cochrane reviews. We counted the number of statistical tests done in each systematic review. The median number of tests overall was 10 (interquartile range (IQR) 6 to 18). The median was 12 in Cochrane and 8 in non-Cochrane reviews (difference in medians 4 (95% confidence interval (CI) 2.0–19.0). The proportion that used an assessment of risk of bias as a reason for doing extra analyses was 42% in Cochrane and 28% in non-Cochrane reviews (difference in proportions 14% (95% CI −8 to 36). The issue of multiplicity was addressed in 6% of all the reviews.

Conclusion/Significance

Statistical multiplicity in systematic reviews requires attention. We found more multiplicity in Cochrane reviews than in non-Cochrane reviews. Many of the reasons for the increase in multiplicity may well represent improved methodological approaches and greater transparency, but multiplicity may also cause an increased risk of spurious conclusions. Few systematic reviews, whether Cochrane or non-Cochrane, address the issue of multiplicity.     

RNA interactions in the 5' region of the HIV-1 genome

The untranslated leader of the dimeric HIV-1 RNA genome is folded into a complex structure that plays multiple and essential roles in the viral replication cycle. Here, we have investigated secondary and tertiary structural elements within the 5' 744 nucleotides of the HIV-1 genome using a combination of bioinformatics, enzymatic probing, native gel electrophoresis, and UV-crosslinking experiments. We used a recently developed RNA folding algorithm (Pfold) to predict the common secondary structure of an alignment of 20 divergent HIV-1 sequences. Combining this analysis with biochemical data, we present a secondary structure model for the entire 744 nucleotide fragment, which incorporates previously recognized and novel structural elements. In particular, our data provided strong evidence for a long-distance interaction between the region encompassing the AUG Gag initiation codon and an upstream region and we demonstrate that this feature is highly conserved in distantly related human and animal retroviruses. To obtain information about tertiary interactions we applied an intramolecular UV-crosslinking strategy and identified a novel tertiary interaction within the PBS hairpin structure.     

Measuring the importance of competition: a new formulation of the problem

1.  Currently, there is a debate among plant ecologists on the concepts of the intensity of competition and the importance of competition, which is central to many issues of modern plant population ecology and plant community ecology.
2.  It is problematic that the current measures of intensity and importance of competition, typically, are reported as dimensionless indices because they hide the fact that both indices are functions of plant density and the level of the environmental gradient.
3.  Here, a new formulation of the concepts is suggested, which explicitly highlights the functional dependencies on plant density and the level of the environmental gradient. The new measures are a generalization of the previous indices and correspond to the previous indices in the case of a simple experimental design.
4.  The suggested measures of the intensity and importance of competition are exemplified using data from a response surface competition experiment between Agrostis capillaris and Festuca ovina along a herbicide gradient, where the expected clear effect of plant density was demonstrated.
5.   Synthesis . As the suggested measures of the intensity and importance of competition explicitly highlight the functional dependencies on plant density and the level of the environmental gradient, we think that they will help to ensure a closer connection between experimental plant ecology and the attempts to model plant populations and communities.     

Identification of estrogen-regulated genes by microarray analysis of the uterus of immature rats exposed to endocrine disrupting chemicals

Environmental estrogenic compounds which bind to the estrogen receptor (ER) can block or alter endogenous functions of estrogen in reproductive and developmental stages. A microarray technology is a very valuable method for the prediction of hormone-responsive activities in various gene expressions. Thus, we investigated the altered gene expression by estrogen and endocrine disruptors (EDs) using microarray technology in the uterus of immature rats. In this study, the expression levels of only 555 genes (7.42%) among the 7636 genes spotted on microarray chips were enhanced by more than two-fold following treatment with estradiol (E2), suggesting that direct or rapid response to E2 is widespread at the mRNA levels in these genes. In addition, elevated expression levels of the genes (over 2-fold) were observed by diethylstilbestrol (DES; 9.01%), octyl-phenol (OP; 8.81%), nonyl-phenol (NP; 9.51%), bisphenol-A (BPA; 8.26%) or genistein (9.97%) in the uterus of immature rats. The expression levels of representative genes, i.e., calbindin-D9k (CaBP-9k; vitamin D-dependent calcium-binding protein), oxytocin, adipocyte complement related protein (MW 30 kDa), lactate dehydrogenase A and calcium binding protein A6 (S100a6; calcyclin), were confirmed in these tissues by real-time PCR. In addition, the mRNA levels of these genes by real-time PCR were increased at follicular phase when E2 level was elevated during estrous cycle of adult female rats. In conclusion, these results indicate distinct altered expression of responsive genes following exposure to E2 and estrogenic compounds, and implicate distinct effects of endogenous E2 and environmental endocrine disrupting chemicals in the uterus of immature rats.     

Origin of initial burst in activity for Trichoderma reesei endo-glucanases hydrolyzing insoluble cellulose

The kinetics of cellulose hydrolysis have long been described by an initial fast hydrolysis rate, tapering rapidly off, leading to a process that takes days rather than hours to complete. This behavior has been mainly attributed to the action of cellobiohydrolases and often linked to the processive mechanism of this exo-acting group of enzymes. The initial kinetics of endo-glucanases (EGs) is far less investigated, partly due to a limited availability of quantitative assay technologies. We have used isothermal calorimetry to monitor the early time course of the hydrolysis of insoluble cellulose by the three main EGs from Trichoderma reesei (Tr): TrCel7B (formerly EG I), TrCel5A (EG II), and TrCel12A (EG III). These endo-glucanases show a distinctive initial burst with a maximal rate that is about 5-fold higher than the rate after 5 min of hydrolysis. The burst is particularly conspicuous for TrCel7B, which reaches a maximal turnover of about 20 s(-1) at 30 °C and conducts about 1200 catalytic cycles per enzyme molecule in the initial fast phase. For TrCel5A and TrCel12A the extent of the burst is 2-300 cycles per enzyme molecule. The availability of continuous data on EG activity allows an analysis of the mechanisms underlying the initial kinetics, and it is suggested that the slowdown is linked to transient inactivation of enzyme on the cellulose surface. We propose, therefore, that the frequency of structures on the substrate surface that cause transient inactivation determine the extent of the burst phase.     

Genome size rather than content might affect call properties in toads of three ploidy levels (Anura: Bufonidae: Bufo viridis subgroup)

In vertebrates, genome size has been shown to correlate with nuclear and cell sizes, and influences phenotypic features, such as brain complexity. In three different anuran families, advertisement calls of polyploids exhibit longer notes and intervals than diploids, and difference in cellular dimensions have been hypothesized to cause these modifications. We investigated this phenomenon in green toads (Bufo viridis subgroup) of three ploidy levels, in a different call type (release calls) that may evolve independently from advertisement calls, examining 1205 calls, from ten species, subspecies, and hybrid forms. Significant differences between pulse rates of six diploid and four polyploid (3n, 4n) green toad forms across a range of temperatures from 7 to 27 °C were found. Laboratory data supported differences in pulse rates of triploids vs. tetraploids, but failed to reach significance when including field recordings. This study supports the idea that genome size, irrespective of call type, phylogenetic context, and geographical background, might affect call properties in anurans and suggests a common principle governing this relationship. The nuclear‐cell size ratio, affected by genome size, seems the most plausible explanation. However, we cannot rule out hypotheses under which call‐influencing genes from an unexamined diploid ancestral species might also affect call properties in the hybrid‐origin polyploids. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 105 , 584–590.