Long-term plasticity of endocannabinoid signaling induced by developmental febrile seizures

Febrile (fever-induced) seizures are the most common form of childhood seizures, affecting 3%-5% of infants and young children. Here we show that the activity-dependent, retrograde inhibition of GABA release by endogenous cannabinoids is persistently enhanced in the rat hippocampus following a single episode of experimental prolonged febrile seizures during early postnatal development. The potentiation of endocannabinoid signaling results from an increase in the number of presynaptic cannabinoid type 1 receptors associated with cholecystokinin-containing perisomatic inhibitory inputs, without an effect on the endocannabinoid-mediated inhibition of glutamate release. These results demonstrate a selective, long-term increase in the gain of endocannabinoid-mediated retrograde signaling at GABAergic synapses in a model of a human neurological disease.     

Levels of male-specific RNA bacteriophage and Escherichia coli in molluscan bivalve shellfish from commercial harvesting areas

AIMS: Current measures for controlling the public health risks associated with bivalve molluscan shellfish consumption rely on the use of Escherichia coli to indicate the sanitary quality of shellfish harvesting areas. However, it has been demonstrated that E. coli is an inadequate indicator of the viral risk associated with shellfish. An alternative indicator organism, male-specific RNA (FRNA) bacteriophage has been proposed for this role. This study compared the distribution of E. coli and FRNA bacteriophage in shellfish harvesting areas. METHODS AND RESULTS: A total of 608 shellfish samples from 49 shellfish harvesting areas were analysed for E. coli and FRNA bacteriophage using standard published methods. The geometric mean concentration of FRNA bacteriophage in all samples was over three times greater than that of E. coli (1800 and 538 counts/100 g for FRNA bacteriophage and E. coli, respectively). In contrast to E. coli, FRNA bacteriophage concentrations were strongly influenced by season with a geometric mean count of 4503 PFU/100 g in the winter (October-March) compared with 910 PFU/100 g in the summer (April-September). CONCLUSIONS: FRNA bacteriophage were present in shellfish at higher concentrations than E. coli. Elevated levels of FRNA bacteriophage observed in the winter concur with the known increased viral risk associated with shellfish harvested at that time of year in the UK. Levels of FRNA bacteriophage found in many shellfish from category B harvesting areas would not be eliminated by conventional treatment processes. SIGNIFICANCE AND IMPACT OF THE STUDY: Data from this study will inform future proposals to introduce FRNA bacteriophage as an indicator of the viral risk associated with shellfish.     

Dissection of protease-activated receptor-1-dependent and -independent responses to thrombin in skeletal myoblasts

Thrombin exerts a number of effects on skeletal myoblasts in vitro. It stimulates proliferation and intracellular calcium mobilization and inhibits differentiation and apoptosis induced by serum deprivation in these cells. Many cellular responses to thrombin are mediated by protease-activated receptor-1 (PAR-1). Expression of PAR-1 is present in mononuclear myoblasts in vitro, but repressed when fusion occurs to form myotubes. In the current study, we used PAR-1-null mice to determine which of thrombin's effects on myoblasts are mediated by PAR-1. Thrombin inhibited fusion almost as effectively in cultures prepared from the muscle of PAR-1-null myoblasts as in cultures prepared from wild-type mice. Apoptosis was inhibited as effectively in PAR-1-null myoblasts as in wild-type myoblasts. These effects in PAR-1-null myoblasts were mediated by a secreted inhibitor of apoptosis and fusion, as demonstrated previously for normal rat myoblasts. Thrombin failed to induce an intracellular calcium response in PAR-1-null myoblast cultures, although these cells were able to mobilize intracellular calcium in response to activation of other receptors. PAR-1-null myoblasts also failed to proliferate in response to thrombin. These results demonstrate that thrombin's effects on myoblast apoptosis and fusion are not mediated by PAR-1 and that PAR-1 is the only thrombin receptor capable of inducing proliferation and calcium mobilization in neonatal mouse myoblasts.     

Function of the conserved S1 and KH domains in polynucleotide phosphorylase

We have examined the roles of the conserved S1 and KH RNA binding motifs in the widely dispersed prokaryotic exoribonuclease polynucleotide phosphorylase (PNPase). These domains can be released from the enzyme by mild proteolysis or by truncation of the gene. Using purified recombinant enzymes, we have assessed the effects of specific deletions on RNA binding, on activity against a synthetic substrate under multiple-turnover conditions, and on the ability of truncated forms of PNPase to form a minimal RNA degradosome with RNase E and RhlB. Deletion of the S1 domain reduces the apparent activity of the enzyme by almost 70-fold under low-ionic-strength conditions and limits the enzyme to digest a single substrate molecule. Activity and product release are substantially regained at higher ionic strengths. This deletion also reduces the affinity of the enzyme for RNA, without affecting the enzyme's ability to bind to RNase E. Deletion of the KH domain produces similar, but less severe, effects, while deletion of both the S1 and KH domains accentuates the loss of activity, product release, and RNA binding but has no effect on binding to RNase E. We propose that the S1 domain, possibly arrayed with the KH domain, forms an RNA binding surface that facilitates substrate recognition and thus indirectly potentiates product release. The present data as well as prior observations can be rationalized by a two-step model for substrate binding.     

Microbial population dynamics during start-up and overload conditions of anaerobic digesters treating municipal solid waste and sewage sludge

Microbial population dynamics were investigated during start-up and during periods of overload conditions in anaerobic co-digesters treating municipal solid waste and sewage sludge. Changes in community structure were monitored using ribosomal RNA-based oligonucleotide probe hybridization to measure the abundance of syntrophic propionate-oxidizing bacteria (SPOB), saturated fatty acid-beta-oxidizing syntrophs (SFAS), and methanogens. These changes were linked to traditional performance parameters such as biogas production and volatile fatty acid (VFA) concentrations. Digesters with high levels of Archaea started up successfully. Methanosaeta concilii was the dominant aceticlastic methanogen in these systems. In contrast, digesters that experienced a difficult start-up period had lower levels of Archaea with proportionally more abundant Methanosarcina spp. Syntrophic propionate-oxidizing bacteria and saturated fatty acid-beta-oxidizing syntrophs were present at low levels in all digesters, and SPOB appeared to play a role in stabilizing propionate levels during start-up of one digester. Digesters with a history of poor performance tolerated a severe organic overload event better than digesters that had previously performed well. It is hypothesized that higher levels of SPOB and SFAS and their methanogenic partners in previously unstable digesters are responsible for this behavior.     

Tenascin-C induced stimulation of chondrogenesis is dependent on the presence of the C-terminal fibrinogen-like globular domain

The relationship between structure of tenascin-C (Tn-C), a multi-domain extracellular matrix protein, and its stimulation of chondrogenesis was examined using recombinant Tn-C isoforms (full length or with specific domains deleted) as substrata for undifferentiated chicken mesenchymal cells. Of the Tn-C variants tested, only Tn-C lacking the fibrinogen-like domain or Tn-C comprised solely of fibrinogen-like domains failed to stimulate chondrogenesis. The ability of variants to stimulate chondrogenesis was not dependent on their ability to support adhesion or stimulate proliferation. These results demonstrate that the fibrinogen-like domain of Tn-C is necessary but not sufficient for induction of chondrogenesis.     

Action of RNase II and polynucleotide phosphorylase against RNAs containing stem-loops of defined structure

The 3'-->5' exoribonucleases, RNase II and polynucleotide phosphorylase (PNPase), play an essential role in degrading fragments of mRNA generated by prior cleavages by endonucleases. We have assessed the ability of small RNA substrates containing defined stem-loop structures and variable 3' extensions to impede the exonucleolytic activity of these enzymes. We find that stem-loops containing five G-C base pairs do not block either enzyme; in contrast, more stable stem-loops of 7, 9, or 11 bp block the processive action of both enzymes. Under conditions where enzyme activity is limiting, both enzymes stall and dissociate from their substrates six to nine residues, on average, from the base of a stable stem-loop structure. Our data provide a clear mechanistic explanation for the previous observation that RNase II and PNPase behave as functionally redundant.     

Reproductive Biology of the Halfmoon Grouper, Epinephelus rivulatus, at Ningaloo Reef, Western Australia

The reproductive biology and sexual pattern of Epinephelus rivulatus were examined within the waters of Ningaloo Reef, Western Australia. Population structure and gonad morphology show that E. rivulatus is a protogynous hermaphrodite. Mature females ranged in size from 144 to 350mm fork length (FL), with 50% sexually mature at 194mm FL. Males were larger than females and ranged in size from 221 to 381mm FL. There were also intrasexual differences in body size and the median size at which sex-change occurred within each site. Sex ratios were consistent between sites with approximately one male to 5.5 mature females. Most female E. rivulatus (>50%) were reproductively active from July to December when water temperatures were relatively cool and daylight hours were increasing. During this time reproduction occurred periodically, and a female was capable of spawning at least twice over two or three consecutive days.     

Diffusion barriers in ram and boar sperm plasma membranes: directionality of lipid diffusion across the posterior ring

The plasma membrane of mammalian spermatozoa, like that of other differentiated cells, is compartmentalized into discrete regions or domains that are biochemically and functionally distinct from one another. Physical structures within the membrane, such as the posterior ring at the juncture of the sperm head and tail, have long been thought to act as diffusion barriers to help segregate important molecules required for fertilization within specific domains and to regulate migration of molecules between domains. In this investigation, we used a quantitative photobleaching technique (video-FRAP) to assess the efficacy of the posterior ring as a barrier to exchange of lipids between the postacrosomal and midpiece plasma membranes. A lipid reporter probe (1,1'-diduodecyl-3,3,3', 3'-tetramethylindocarbocyanine; DiIC(12)) was incorporated into the plasma membrane of live ram and boar spermatozoa, and the directionality of its diffusion across the posterior ring was measured by line-profile analysis. Results showed that DiIC(12) was able to traverse the posterior ring from the direction of the postacrosomal plasma membrane and to diffuse onto the midpiece plasma membrane. These results suggest that the posterior ring is not an immutable barrier to lipid exchange in mature spermatozoa and that there are other mechanisms for maintaining in-plane lipid asymmetry, such as differential phase behavior and interaction with the submembranous cytoskeleton.     

Suppression of the humoral immune response of Atlantic salmon, Salmo salar L. by the 64 kDa serine protease of Aeromonas salmonicida

Bacteria-free supernatants of broth cultures of Aeromonas salmonicida inhibited the humoral immune response, but not the cell-mediated immune response, of Atlantic salmon to bacteriophage MS2. The immunosuppressive factor was the 64 kDa serine protease secreted by A. salmonicida. The suppressive activity was not due to degradation of epitopes of MS2, and although serine protease degraded the heavy chain of salmon IgM in vitro there was no evidence for significant degradation in vivo. The principal lethal toxin of A. salmonicida, the glycerophospholipid: cholesterol acyltransferase did not inhibit the immune response of salmon.