Catabolite regulation of the pta gene as part of carbon flow pathways in Bacillus subtilis

In Bacillus subtilis, the products of the pta and ackA genes, phosphotransacetylase and acetate kinase, play a crucial role in the production of acetate, one of the most abundant by-products of carbon metabolism in this gram-positive bacterium. Although these two enzymes are part of the same pathway, only mutants with inactivated ackA did not grow in the presence of glucose. Inactivation of pta had only a weak inhibitory effect on growth. In contrast to pta and ackA in Escherichia coli, the corresponding B. subtilis genes are not cotranscribed. Expression of the pta gene was increased in the presence of glucose, as has been reported for ackA. The effects of the predicted cis-acting catabolite response element (CRE) located upstream from the promoter and of the trans-acting proteins CcpA, HPr, Crh, and HPr kinase on the catabolite regulation of pta were investigated. As for ackA, glucose activation was abolished in ccpA and hprK mutants and in the ptsH1 crh double mutant. Footprinting experiments demonstrated an interaction between CcpA and the pta CRE sequence, which is almost identical to the proposed CRE consensus sequence. This interaction occurs only in the presence of Ser-46-phosphorylated HPr (HPrSer-P) or Ser-46-phosphorylated Crh (CrhSer-P) and fructose-1,6-bisphosphate (FBP). In addition to CcpA, carbon catabolite activation of the pta gene therefore requires at least two other cofactors, FBP and either HPr or Crh, phosphorylated at Ser-46 by the ATP-dependent Hpr kinase.     

Embryo production by ovum pick up in unstimulated calves before and after puberty

The possibility of producing embryos from oocytes repeatedly collected from unstimulated calves was tested, and results obtained before and after puberty were compared in the same animals. Ovum pick-up (OPU) coupled with in vitro embryo production was used on 2 sets of 7 and 9 calves, aged 7 to 10 m.o. at the start of the experiment. The oocytes were collected twice a week during a 2-m.o. period before puberty and a 1-m.o. period after puberty. Oocytes were fertilized and co-cultured with cumulus cells in modified synthetic oviduct fluid (SOF) up to Day 7 post insemination. Some Day 7 blastocysts were vitrified and transferred to recipient heifers. An average of 3.8 to 6.8 follicles was punctured per OPU session; 1.9 to 3.1 oocytes were collected, of which more than 60% were of good quality. The number of punctured follicles and collected oocytes varied between donors. Blastocyst rates of 19 to 27% were obtained for the 2 sets. Three normal calves were born from the transfer of 20 vitrified embryos. While no significant difference was observed for the first set of calves, a significant decrease in the number of punctured follicles was observed after puberty in the second set. A direct correlation was also obtained between the number of follicles punctured before and after puberty in the same animal. In conclusion, oocytes can be collected by repeated OPU in calves 7 to 10 m.o. old without affecting their growth or the onset of puberty. An average of 5 to 11 (range 0 to 16) blastocysts per donor was produced over 2 month. However, important variations were found between donors. The correlation observed for the number of follicles punctured before and after puberty suggests that this parameter is determined before puberty.     

Variations in levels of clonality among Populus nigra L. stands of different ages

The evolution of genotypic diversity with population age remains poorly explored in clonal plant populations despite the potential for important shifts to occur through the course of time. Woody sprouting species are particularly under-represented in studies investigating intra-specific variations in levels of clonality from one locality to the next and through time. In this study we sought to determine the incidence and frequency of replicate genotypes in natural Populus nigra L. (Salicaceae) stands of different ages. Ten stands of this woody riparian sprouting species were selected in each of three distinct age groups (young, middle-aged and old) along a 30 km stretch of the River Garonne (south-west France). Leaf samples were collected from 15 neighbouring trees in each stand (450 samples in total) and replicate genotypes were identified using five SSR markers. Replicate genotypes were identified in two-thirds of all stands sampled (i.e. 50 of young stands, 100 of middle-aged stands and 50% of old stands). Young stands had significantly fewer replicated genotypes than middle-aged or old stands, while middle-aged stands had the greatest number of replicated genotypes. Replicate genotypes were most often found to occur as nearest neighbours and formed relatively small, discrete units (i.e. 2–4 trees growing in close proximity to one another). This suggests that asexual regeneration frequently occurs through flood-training in this species, although asexual regeneration from translocated fragments also evidently occurs as evidenced by 11 cases of replicate genotypes occurring in widely separated stands (up to 19 km apart). The results of this study highlight the need for a hierarchical sampling strategy in space and across age groups for an accureate understanding of the genotypic structure of woody sprouting species populations. Conservation and management of effective population sizes will benefit from better insight into not only spatial, but also temporal variations in levels of genotypic diversity.Co-ordinating editor: J. Tuomi     

Visual responses on neck muscles reveal selective gating that prevents express saccades

Express saccades promote the acquisition of visual targets at extremely short reaction times. Because of the head's considerable inertia, it is unknown whether express saccades are accompanied by a parallel command to the head. Here, by recording electromyographic (EMG) activity from monkey neck muscles, we demonstrate that visual target presentation elicits time-locked, lateralized recruitment of neck muscles at extremely short latencies (55-95 ms). Remarkably, such recruitment not only accompanies express saccades, but also precedes nonexpress saccades, occasionally by up to 150 ms. These results demonstrate selective gating of components of descending commands from the superior colliculus to prevent express saccades yet permit recruitment of a head orienting synergy. We conclude that such selective gating aids eye-head coordination by permitting force development at neck muscles while a decision to commit to a gaze shift is being made, optimizing the contribution of the more inertial head to the ensuing gaze shift.     

Inhibition of yeast growth by molybdenum-hydroxylamido complexes correlates with their presence in media at differing pH values

The effects of Mo-hydroxylamido complexes on cell growth were determined in Saccharomyces cerevisiae to investigate the biological effects of four different Mo complexes as a function of pH. Studies with yeast, an eukaryotic cell, are particularly suited to examine growth at different pH values because this organism grows well from pH 3 to 6.5. Studies can therefore be performed both in the presence of intact complexes and when the complexes have hydrolyzed to ligand and free metal ion. One of the complexes we examined was structurally characterized by X-ray crystallography. Yeast growth was inhibited in media solutions containing added Mo-dialkylhydroxylamido complexes at pH 3-7. When combining the yeast growth studies with a systematic study of the Mo-hydroxylamido complexes' stability as a function of pH and an examination of their speciation in yeast media, the effects of intact complexes can be distinguished from that of ligand and metal. This is possible because different effects are observed with complex present than when ligand or metal alone is present. At pH 3, the growth inhibition is attributed to the forms of molybdate ion that exist in solution because most of the complexes have hydrolyzed to oxomolybdate and ligand. The monoalkylhydroxylamine ligand inhibited yeast growth at pH 5, 6 and 7, while the dialkylhydroxylamine ligands had little effect on yeast growth. Growth inhibition of the Mo-dialkylhydroxylamido complexes is observed when a complex exists in the media. A complex that is inert to ligand exchange is not effective even at pH 3 where other Mo-hydroxylamido complexes show growth inhibition as molybdate. These results show that the formation of some Mo complexes can protect yeast from the growth inhibition observed when either the ligand or Mo salt alone are present.     

The 5-HT2B receptor: a main cardio-pulmonary target of serotonin

In agreement with previous data in the literature, our results indicate that serotonin, a monoamine neurotransmitter, can also regulate cell proliferation, cell movements and cell differentiation. We have recently shown that serotonin is required for embryonic heart development. Genetic ablation of the 5-HT2B receptor leads to partial embryonic and postnatal lethality with abnormal heart development. Similar molecular mechanisms seem to be involved in adult cardiomyocytes since mutant mice surviving to adulthood display a dilated cardiomyopathy. Furthermore this receptor appears to be involved in survival of cardiomyocytes. The 5-HT2B receptor is also implicated in systemic hypertension. Furthermore, mice with pharmacological or genetic ablation of 5-HT2B receptor are totally resistant to hypoxia-induced pulmonary hypertension, indicating that this receptor is regulating the pathologic vascular proliferation leading to this disease. Underlying mechanisms are still to be discovered.     

Using genetic markers to estimate the pollen dispersal curve

Pollen dispersal is a critical process that shapes genetic diversity in natural populations of plants. Estimating the pollen dispersal curve can provide insight into the evolutionary dynamics of populations and is essential background for making predictions about changes induced by perturbations. Specifically, we would like to know whether the dispersal curve is exponential, thin-tailed (decreasing faster than exponential), or fat-tailed (decreasing slower than the exponential). In the latter case, rare events of long-distance dispersal will be much more likely. Here we generalize the previously developed TWOGENER method, assuming that the pollen dispersal curve belongs to particular one- or two-parameter families of dispersal curves and estimating simultaneously the parameters of the dispersal curve and the effective density of reproducing individuals in the population. We tested this method on simulated data, using an exponential power distribution, under thin-tailed, exponential and fat-tailed conditions. We find that even if our estimates show some bias and large mean squared error (MSE), we are able to estimate correctly the general trend of the curve - thin-tailed or fat-tailed - and the effective density. Moreover, the mean distance of dispersal can be correctly estimated with low bias and MSE, even if another family of dispersal curve is used for the estimation. Finally, we consider three case studies based on forest tree species. We find that dispersal is fat-tailed in all cases, and that the effective density estimated by our model is below the measured density in two of the cases. This latter result may reflect the difficulty of estimating two parameters, or it may be a biological consequence of variance in reproductive success of males in the population. Both the simulated and empirical findings demonstrate the strong potential of TWOGENER for evaluating the shape of the dispersal curve and the effective density of the population (d(e)).     

Identification of bacterial muramyl dipeptide as activator of the NALP3/cryopyrin inflammasome

Activation of caspase-1 and subsequent processing and secretion of the pro-inflammatory cytokine IL-1beta is triggered upon assembly of the inflammasome complex. It is generally believed that bacterial lipopolysaccharides (LPS) are activators of the inflammasome through stimulation of Toll-like receptor 4 (TLR4). Like TLRs, NALP3/Cryopyrin, which is a key component of the inflammasome, contains Leucine-Rich-Repeats (LRRs). LRRs are frequently used to sense bacterial components, thus raising the possibility that bacteria directly activate the inflammasome. Here, we show that bacterial peptidoglycans (PGN), but surprisingly not LPS, induce NALP3-mediated activation of caspase-1 and maturation of proIL-1beta. Activation is independent of TLRs because the PGN degradation product muramyl dipeptide (MDP), which is not sensed by TLRs, is the minimal-activating structure. Macrophages from a patient with Muckle-Wells syndrome, an autoinflammatory disease associated with mutations in the NALP3/Cryopyrin gene, show increased IL-1beta secretion in the presence of MDP. The activation of the NALP3-inflammasome by MDP may be the basis of the potent adjuvant activity of MDP.