Intronic binding sites for hnRNP A/B and hnRNP F/H proteins stimulate pre-mRNA splicing

hnRNP A/B proteins modulate the alternative splicing of several mammalian and viral pre-mRNAs, and are typically viewed as proteins that enforce the activity of splicing silencers. Here we show that intronic hnRNP A/B–binding sites (ABS) can stimulate the in vitro splicing of pre-mRNAs containing artificially enlarged introns. Stimulation of in vitro splicing could also be obtained by providing intronic ABS in trans through the use of antisense oligonucleotides containing a non-hybridizing ABS-carrying tail. ABS-tailed oligonucleotides also improved the in vivo inclusion of an alternative exon flanked by an enlarged intron. Notably, binding sites for hnRNP F/H proteins (FBS) replicate the activity of ABS by improving the splicing of an enlarged intron and by modulating 5′ splice-site selection. One hypothesis formulated to explain these effects is that bound hnRNP proteins self-interact to bring in closer proximity the external pair of splice sites. Consistent with this model, positioning FBS or ABS at both ends of an intron was required to stimulate splicing of some pre-mRNAs. In addition, a computational analysis of the configuration of putative FBS and ABS located at the ends of introns supports the view that these motifs have evolved to support cooperative interactions. Our results document a positive role for the hnRNP A/B and hnRNP F/H proteins in generic splicing, and suggest that these proteins may modulate the conformation of mammalian pre-mRNAs.     

Circadian rhythms of heart rate in freely moving and restrained American lobsters, Homarus americanus

While circadian rhythms of locomotion have been reported in the American lobster, Homarus americanus, it is unclear whether heart rate is also modulated on a circadian basis. To address this issue, both heart rate and locomotor activity were continuously monitored in light-dark (LD) cycles and constant darkness (DD). Lobsters in running wheels exhibited significant nocturnal increases in locomotor activity and heart rates during LD, and these measures were significantly correlated. In DD, most lobsters exhibited persistent circadian rhythms of both locomotion and heart rate. When heart rate was monitored in restrained lobsters in LD and DD, most animals also demonstrated clear daily and circadian rhythms in heart rate. Overall, this is the first demonstration of circadian rhythms of heart rate in H. americanus, the expression of which does not appear to be dependent on the expression of locomotor activity.     

Analysis of mechanistic pathway models in drug discovery: p38 pathway

Mechanistic models of signal transduction have emerged as valuable tools for untangling complex signaling networks and gaining detailed insight into pathway dynamics. The natural extension of these tools is for the design of therapeutic strategies. We have generated a novel computational model of lipopolysaccharide-induced p38 signaling in the context of TNF-alpha production in inflammatory disease. Using experimental measurement of protein levels and phospho-protein time courses, populations of model parameters were estimated. With a collection of parameter sets, reflecting virtual diversity, we step through analysis of the p38 signaling pathway model to answer specific drug discovery questions regarding target prioritization, inhibitor simulation, model robustness and co-drugging. We demonstrate that target selection cannot be assessed independently from inhibitor mechanism of action and is also linked with robustness to cellular variability. Finally, we assert that in the face of parameter uncertainty one can still uncover consistent findings that can guide drug discovery efforts.     

Diel variation in feeding rate and prey composition of herring and mackerel in the southern Gulf of St Lawrence

Diel feeding patterns of herring Clupea harengus and mackerel Scomber scombrus in the southern Gulf of St Lawrence were examined based on samples obtained by midwater trawling between 19 and 26 June 2001. Within 3 h time periods, stomach contents tended to be more similar between fish from the same tow than between fish from different tows. Thus, in contrast to previous diet studies, which have used individual fish stomachs as independent observations, tow was used as the experimental unit in statistical analyses in this study. Diel patterns in stomach fullness were identified using generalized additive models. Two peaks in stomach fullness occurred for herring, one in the morning and the other in the evening. Mackerel showed an increase in feeding intensity throughout the day with a peak in mid‐afternoon. The diel changes in stomach contents suggested rapid gastric evacuation rates for both species, especially for herring. The estimate of the instantaneous evacuation rate for herring was twice that for mackerel. Calanus copepods (mainly C. hyperboreus ), fishes (mainly capelin Mallotus villosus ) and euphausiids were the main prey found in the stomachs of both species. Calanus copepods dominated the diet of herring regardless of time period. They also dominated the diet of mackerel during the late afternoon, evening and night while fishes and euphausiids were dominant during the morning and early afternoon. These diel patterns emphasize the need for sampling throughout the day and night in order to estimate ration and diet composition for bioenergetic and ecosystem models.     

The caveolin scaffolding domain modifies 2-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor binding properties by inhibiting phospholipase A2 activity

Activation of the enzyme phospholipase (PLA 2) has been proposed to be part of the molecular mechanism involved in the alteration of 2-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) glutamate receptor responsiveness during long term changes in synaptic plasticity (long term potentiation). This study assesses the effect of the caveolin-1 scaffolding domain (CSD) on the activity of the regulatory enzyme PLA2. Caveolin-1 is a 22-kDa cholesterol-binding membrane protein known to inhibit the activity of most of its interacting partners. Our results show that the calcium-dependent cytosolic form of PLA2 (cPLA2) and caveolin-1 co-localized in mouse primary hippocampal neuron cultures and that they were co-immunoprecipitated from mouse hippocampal homogenates. A peptide corresponding to the scaffolding domain of caveolin-1 (Cav-(82-101)) dramatically inhibited cPLA2 activity in purified hippocampal synaptoneurosomes. Activation of endogenous PLA2 activity with KCl or melittin increased the binding of [3H]AMPA to its receptor. This effect was almost completely abolished by the addition of the CSD peptide to these preparations. Moreover, we demonstrated that the inhibitory action of the CSD peptide on AMPA receptor binding properties is specific (because a scrambled version of this peptide failed to have any effect) and that it is mediated by an inhibition of PLA2 enzymatic activity (because the CSD peptide failed to have an effect in membrane preparations lacking endogenous PLA2 activity). These results raised the possibility that caveolin-1, via the inhibition of cPLA2 enzymatic activity, may interfere with synaptic facilitation and long term potentiation formation in the hippocampus.     

Preferential accessibility to specific genomic loci for the repair of double-strand breaks in human cells

The dynamic organization of the human genome in the nucleus is gaining recognition as a determining factor in its functional regulation. In order to be expressed, replicated or repaired, a genomic locus has to be present at the right place at the right time. In the present study, we have investigated the choice of a double-strand break (DSB) repair partner for a given genomic loci in an ATM-deficient human fibroblast cell line. We found that partner choice is restricted such that a given genomic locus preferentially uses certain sites in the genome to repair itself. These preferential sites can be in the vicinity of the damage site or megabases away or on other chromosomes entirely, while potential sites closer to the break along the length of the chromosome can be ignored. Moreover, there can be more than a 10-fold difference in usage between repair sites located only 10 kb apart. Interestingly, arms of a given chromosome are less accessible to one another than to other chromosomes. Altogether, these results indicate that the accessibility between genomic sites in the human genome during DSB repair is specific and conserved in a cell population.     

The initial state of the human gut microbiome determines its reshaping by antibiotics

Microbiome studies have demonstrated the high inter-individual diversity of the gut microbiota. However, how the initial composition of the microbiome affects the impact of antibiotics on microbial communities is relatively unexplored. To specifically address this question, we administered a second-generation cephalosporin, cefprozil, to healthy volunteers. Stool samples gathered before antibiotic exposure, at the end of the treatment and 3 months later were analysed using shotgun metagenomic sequencing. On average, 15 billion nucleotides were sequenced for each sample. We show that standard antibiotic treatment can alter the gut microbiome in a specific, reproducible and predictable manner. The most consistent effect of the antibiotic was the increase of Lachnoclostridium bolteae in 16 out of the 18 cefprozil-exposed participants. Strikingly, we identified a subgroup of participants who were enriched in the opportunistic pathogen Enterobacter cloacae after exposure to the antibiotic, an effect linked to lower initial microbiome diversity and to a Bacteroides enterotype. Although the resistance gene content of participants'' microbiomes was altered by the antibiotic, the impact of cefprozil remained specific to individual participants. Resistance genes that were not detectable prior to treatment were observed after a 7-day course of antibiotic administration. Specifically, point mutations in beta-lactamase bla_CfxA-6 were enriched after antibiotic treatment in several participants. This suggests that monitoring the initial composition of the microbiome before treatment could assist in the prevention of some of the adverse effects associated with antibiotics or other treatments.     

R51163 as a sedative for handling and transporting plains bison and wapiti.

Forty captive wapiti (Cervus elaphus) and thirty-two bison (Bison bison bison) were tested in April and October 1988, respectively, for their response to the sedative R51163. Treatment animals were injected with either 0.1, 0.2, or 0.3 mg of R51163/kg and then observed for 72 hr. Behavior was significantly altered by the drug. Hyperactive, aggressive, and milling behavior was characteristic of treated wapiti and they were extremely dangerous and reared when hind quarters were touched. Although treated plains bison displayed some milling behavior, they were generally more calm than wapiti. There was a marked difference between sexes in plains bison for all behavioral categories. Male bison were more ataxic, often observed in sternal or lateral recumbency, less conscious, and were slower to respond than females or controls. Respiratory rate increased in treated wapiti and plains bison, and heart rates of treated wapiti increased. Because of the powerful sedative effect on large, male bison, R51163 may be useful for handling unmanageable or dangerous animals and warrants further studies.