The hybrid state of tRNA binding is an authentic translation elongation intermediate

The GTPase elongation factor (EF)-G is responsible for promoting the translocation of the messenger RNA-transfer RNA complex on the ribosome, thus opening up the A site for the next aminoacyl-tRNA. Chemical modification and cryo-EM studies have indicated that tRNAs can bind the ribosome in an alternative 'hybrid' state after peptidyl transfer and before translocation, though the relevance of this state during translation elongation has been a subject of debate. Here, using pre-steady-state kinetic approaches and mutant analysis, we show that translocation by EF-G is most efficient when tRNAs are bound in a hybrid state, supporting the argument that this state is an authentic intermediate during translation.     

A pheromone to behave, a pheromone to learn: the rabbit mammary pheromone

Birth is part of a continuum and is a major developmental change. Newborns need to adapt rapidly to the environment in terms of physiology and behaviour, and ability to locate the maternal source of milk is vital. Mechanisms have evolved resulting in the emission of olfactory cues by the mother and the processing of these cues by the young. Here, we focus on some sensory, cognitive and behavioural strategies developed by the European rabbit (Oryctolagus cuniculus) that optimize the early development of offspring. In this species, chemosensory communication between the mother and young plays a critical role in eliciting adaptive neonatal responses. In particular, lactating females release a molecule, the mammary pheromone, which has several functional impacts. It triggers orocephalic responses involved in the quick localization of nipples and sucking. Moreover, this unconditioned signal promotes rapid appetitive learning of novel odorants, acting as a potent organizer of neonatal cognition. The mammary-pheromone-induced odour memory requires consolidation/reconsolidation processes to be maintained in the long term. Finally, as this mode of conditioning also promotes learning of mixtures of odorants, it supports investigations related to the capacity of neonatal olfaction to extract biological value from the complex environment.     

Systemic dysregulation of CEACAM1 in melanoma patients

It was previously shown that CEACAM1 on melanoma cells strongly predicts poor outcome. Here, we show a statistically significant increase of serum CEACAM1 in 64 active melanoma patients, as compared to 48 patients with no evidence of disease and 37 healthy donors. Among active patients, higher serum CEACAM1 correlated with LDH values and with decreased survival. Multivariate analysis with neutralization of LDH showed that increased serum CEACAM1 carries a hazard ratio of 2.40. In vitro, soluble CEACAM1 was derived from CEACAM1(+), but neither from CEACAM1(?) melanoma cells nor from CEACAM1(+) lymphocytes, and directly correlated with the number of CEACAM1(+) melanoma cells. Production of soluble CEACAM1 depended on intact de novo protein synthesis and secretion machineries, but not on metalloproteinase function. An unusually high percentage of CEACAM1(+) circulating NK and T lymphocytes was demonstrated in melanoma patients. CEACAM1 inhibited killing activity in functional assays. CEACAM1 expression could not be induced on lymphocytes by serum from patients with high CEACAM1 expression. Further, expression of other NK receptors was impaired, which collectively indicate on a general abnormality. In conclusion, the systemic dysregulation of CEACAM1 in melanoma patients further denotes the role of CEACAM1 in melanoma and may provide a basis for new tumor monitoring and prognostic platforms.     

Infertility and Male Mating Behavior Deficits Associated With Pde1c in Drosophila melanogaster

Pde1c is a calcium/calmodulin-regulated, dual-specificity cyclic nucleotide phosphodiesterase. We have used a transposon insertion line to investigate the physiological function of Pde1c in Drosophila melanogaster and to show that the insertion leads to male sterility and male mating behavior defects that include reduced copulation rates. Sterility appears to be primarily due to elimination of sperm from the female reproductive system. The male mating behavior defects were fully rescued by expression of exogenous Pde1c under the control of either a Pde1c or a pan-neuronal promoter, whereas the sterility could be only partially rescued by expression of exogenous Pde1c under the control of these promoters. We also show that Pde1c has a male-specific expression pattern in the CNS with an increased number of Pde1c-expressing neurons in the abdominal ganglion in males.THE cyclic nucleotides, cyclic AMP (cAMP) and cyclic GMP (cGMP), have been known for many years to regulate a wide variety of physiological processes in all animals (e.g., Siegel et al. 1994). Similarly, there is a large body of research focused on an understanding of the structure and regulation of the enzymes that synthesize cAMP and cGMP, the adenylyl and guanylyl cyclases, respectively. Although the concentrations of cyclic nucleotides within a cell are regulated by both their synthesis and their degradation, less attention has been devoted to the function and regulation of the enzymes that break down cyclic nucleotides, the phosphodiesterases (PDEs).Mammals have >20 genes that code for cyclic nucleotide PDEs, which have been subdivided into 11 families on the basis of their sequences, substrate specificities, and regulatory properties (Conti and Beavo 2007). Insects also have a wide variety of PDEs with Drosophila melanogaster containing 6 genes that code for cyclic nucleotide PDEs (Morton and Hudson 2002; Day et al. 2005). On the basis of their sequence similarity to the mammalian PDEs, these 6 genes have been classified into 6 of the 11 families: Pde1c, Pde4, Pde6, Pde8, Pde9, and Pde11 (Day et al. 2005). When their biochemical properties have been investigated, they match well with other members of the same family (Day et al. 2005).Despite the importance of cyclic nucleotides in insect physiology, only one of the Drosophila PDEs has been associated with a mutant phenotype. This gene, Pde4, also known as dunce, was one of the first learning and memory mutants discovered (Byers et al. 1981; Davis et al. 1995). In this study, we have investigated the phenotypes associated with reduced expression of Pde1c, a PDE that has dual specificity for both cAMP and cGMP and that is stimulated in the presence of calcium and calmodulin (Day et al. 2005). Here we show that Pde1c is required for male fertility and male mating behavior. Male sterility appears to be primarily due to females rejecting sperm or failing to store the sperm from mutant males.     

Diastolic ventricular-vascular stiffness and relaxation relation: elucidation of coupling via pressure phase plane-derived indexes

Because systole and diastole are coupled and systolic ventricular-vascular coupling has been characterized, we hypothesize that diastolic ventricular-vascular coupling (DVVC) exists and can be characterized in terms of relaxation and stiffness. To characterize and elucidate DVVC mechanisms, we introduce time derivative of pressure (dP/dt) vs. time-varying pressure [P(t)] (pressure phase plane, PPP)-derived analogs of ventricular and vascular "stiffness" and relaxation parameters. Although volume change (dV) = 0 during isovolumic periods, and time-varying left ventricular (LV) stiffness, typically expressed as change in pressure per unit change in volume (dP/dV), is undefined, our formulation allows determination of a PPP-derived stiffness analog during isovolumic contraction and relaxation. Similarly, an aortic stiffness analog is also derivable from the PPP. LV relaxation was characterized via tau, the time constant of isovolumic relaxation, and vascular (aortic pressure decay) relaxation was characterized in terms of its equivalent (windkessel) exponential decay time constant kappa. The results show that PPP-derived systolic and diastolic ventricular and vascular stiffness are strongly coupled [K(Ao)(+)=1.71(K(LV)(+)) +154, r=0.86; K(Ao)(-)=0.677(K(LV)(-))-5.53, r=0.86]. In support of the DVVC hypothesis, a strong linear correlation between relaxation (rate of pressure decay) indexes kappa and tau (kappa = 9.89tau - 90.3, r = 0.81) was also observed. The correlations observed underscore the role of long-term, steady-state DVVC as a diastolic function determinant. Awareness of the PPP-derived DVVC parameters provides insight into mechanisms and facilitates quantification of arterial stiffening and associated increase in diastolic chamber stiffness. The PPP method provides a tool for quantitative assessment and determination of the functional coupling of the vasculature to diastolic function.     

Innate immune response to human bone marrow fibroblastic cell implantation in CB17 scid/beige mice

Immunocompromised mouse models have been extensively used to assess human cell implantation for evaluation of cytotherapy, gene therapy and tissue engineering strategies, as these mice are deficient in T and B lymphoid cells. However, the innate immune response and its effect on human cell xenotransplantation in these mouse models are mainly unknown. The aim of this study is to characterise the myeloid populations in the spleen and blood of CB17 scid beige (CB17 sb) mice, and to study the inflammatory cell responses to xenogeneic implantation of enhanced green fluorescent protein (GFP)-labelled human bone marrow fibroblastic (HBMF) cells into CB17 sb mice. The results indicate that even though CB17 sb mice are deficient in B- and T-cells, they exhibit some increases in their monocyte (Mo), macrophage (Mphi) and neutrophil (Neu) populations. NK cell and eosinophil populations show no differences compared with wild-type Balb/C mice. An innate immune response, identified by CR3 (CD11b/CD18)-positive myeloid inflammatory cells and F4/80-positive macrophages, was evident in the tissues where HBMF cells were implanted. As a consequence, the majority of implanted HBMF cells were eliminated by 4 weeks after implantation. Interestingly, the mineralised matrix formed by osteogenic HBMF cells was also eroded by multinuclear Mphi-like giant cells. We conclude that CB17 sb mice retain active innate immune cells, which respond to HBMF cell xenotransplantation. This study highlights the importance of the innate immune cells in the anti-xenograft response and suggests that strategies to block the activities of these cells may ameliorate the progressive long-term elimination of xenotransplants.