Characterization of a novel MK3 splice variant from murine ventricular myocardium

p38 MAP kinase (MAPK) isoforms α, β, and γ, are expressed in the heart. p38α appears pro-apoptotic whereas p38β is pro-hypertrophic. The mechanisms mediating these divergent effects are unknown; hence elucidating the downstream signaling of p38 should further our understanding. Downstream effectors include MAPK-activated protein kinase (MK)-3, which is expressed in many tissues including skeletal muscles and heart. We cloned full-length MK3 (MK3.1, 384 aa) and a novel splice variant (MK3.2, 266 aa) from murine heart. For MK3.2, skipping of exons 8 and 9 resulted in a frame-shift in translation of the first 85 base pairs of exon 10 followed by an in-frame stop codon. Of 3 putative phosphorylation sites for p38 MAPK, only Thr-203 remained functional in MK3.2. In addition, MK3.2 lacked nuclear localization and export signals. Quantitative real-time PCR confirmed the presence of these mRNA species in heart and skeletal muscle; however, the relative abundance of MK3.2 differed. Furthermore, whereas total MK3 mRNA was increased, the relative abundance of MK3.2 mRNA decreased in MK2^?/? mice. Immunoblotting revealed 2 bands of MK3 immunoreactivity in ventricular lysates. Ectopically expressed MK3.1 localized to the nucleus whereas MK3.2 was distributed throughout the cell; however, whereas MK3.1 translocated to the cytoplasm in response to osmotic stress, MK3.2 was degraded. The p38α/β inhibitor SB203580 prevented the degradation of MK3.2. Furthermore, replacing Thr-203 with alanine prevented the loss of MK3.2 following osmotic stress, as did pretreatment with the proteosome inhibitor MG132. In vitro, GST-MK3.1 was strongly phosphorylated by p38α and p38β, but a poor substrate for p38δ and p38γ. GST-MK3.2 was poorly phosphorylated by p38α and p38β and not phosphorylated by p38δ and p38γ. Hence, differential regulation of MKs may, in part, explain diverse downstream effects mediated by p38 signaling.     

Children's Indoor Leisure Activities in France: Time Budget Data for Indoor Air Risk Assessment

The objective of this study was to determine the time spent by children (0– 18 years old) in indoor places of leisure and also day-care centers. The sampling scheme was stratified by geographic region and size of town. This study was performed from September 2005 to March 2006. Families were asked to complete a diary by phone on their children's activities during the last week of school and the last week of a vacation period. The time spent in each place was then extrapolated for the winter period. We then evaluated the overall attendance and time (means and quartiles) spent by all of the children, and by those of children attending places. Information was collected on 2780 children. Gymnasiums were frequented the most (52.3% during the winter period) and were the places in which children spend the greatest amount of time (average: 19 min/day). Other places frequented the most included bars/restaurants (46.5%; average: 9 min/day), cinemas/theaters (42.2%; average: 10 min/day), and indoor pools (25.4%; average: 12 min/day). Our results should help in the prioritization of specific projects investigating exposure in children during indoor leisure activities and the evaluation of the risks associated with air pollutants in these places.     

The cyclomodulin Cif of Photorhabdus luminescens inhibits insect cell proliferation and triggers host cell death by apoptosis

Cycle inhibiting factors (Cif) constitute a broad family of cyclomodulins present in bacterial pathogens of invertebrates and mammals. Cif proteins are thought to be type III effectors capable of arresting the cell cycle at G₂/M phase transition in human cell lines. We report here the first direct functional analysis of Cif_Pl, from the entomopathogenic bacterium Photorhabdus luminescens, in its insect host. The cif_Pl gene was expressed in P. luminescens cultures in vitro. The resulting protein was released into the culture medium, unlike the well characterized type III effector LopT. During locust infection, cif_Pl was expressed in both the hemolymph and the hematopoietic organ, but was not essential for P. luminescens virulence. Cif_Pl inhibited proliferation of the insect cell line Sf9, by blocking the cell cycle at the G₂/M phase transition. It also triggered host cell death by apoptosis. The integrity of the Cif_Pl catalytic triad is essential for the cell cycle arrest and pro-apoptotic activities of this protein. These results highlight, for the first time, the dual role of Cif in the control of host cell proliferation and apoptotic death in a non-mammalian cell line.     

Assessment of a Syndromic Surveillance System Based on Morbidity Data: Results from the Oscour? Network during a Heat Wave

Background

Syndromic surveillance systems have been developed in recent years and are now increasingly used by stakeholders to quickly answer questions and make important decisions. It is therefore essential to evaluate the quality and utility of such systems. This study was designed to assess a syndromic surveillance system based on emergency departments'' (ED) morbidity rates related to the health effects of heat waves. This study uses data collected during the 2006 heat wave in France.

Methods

Data recorded from 15 EDs in the Ile-de-France (Paris and surrounding area) from June to August, 2006, were transmitted daily via the Internet to the French Institute for Public Health Surveillance. Items collected included diagnosis (ICD10), outcome, and age. Several aspects of the system have been evaluated (data quality, cost, flexibility, stability, and performance). Periods of heat wave are considered the most suitable time to evaluate the system.

Results

Data quality did not vary significantly during the period. Age, gender and outcome were completed in a comprehensive manner. Diagnoses were missing or uninformative for 37.5% of patients. Stability was recorded as being 99.49% for the period overall. The average cost per day over the study period was estimated to be €287. Diagnoses of hyperthermia, malaise, dehydration, hyponatremia were correlated with increased temperatures. Malaise was most sensitive in younger and elderly adults but also the less specific. However, overall syndrome groups were more sensitive with comparable specificity than individual diagnoses.

Conclusion

This system satisfactorily detected the health impact of hot days (observed values were higher than expected on more than 90% of days on which a heat alert was issued). Our findings should reassure stakeholders about the reliability of health impact assessments during or following such an event. These evaluations are essential to establish the validity of the results of syndromic surveillance systems.     

Upregulation of PPARβ/δ Is Associated with Structural and Functional Changes in the Type I Diabetes Rat Diaphragm

Background

Diabetes mellitus is associated with alterations in peripheral striated muscles and cardiomyopathy. We examined diaphragmatic function and fiber composition and identified the role of peroxisome proliferator-activated receptors (PPAR α and β/δ) as a factor involved in diaphragm muscle plasticity in response to type I diabetes.

Methodology/Principal Findings

Streptozotocin-treated rats were studied after 8 weeks and compared with their controls. Diaphragmatic strips were stimulated in vitro and mechanical and energetic variables were measured, cross bridge kinetics assessed, and the effects of fatigue and hypoxia evaluated. Morphometry, myosin heavy chain isoforms, PPAR α and β/δ gene and protein expression were also assessed. Diabetes induced a decrease in maximum velocity of shortening (−14%, P<0.05) associated with a decrease in myosin ATPase activity (−49%, P<0.05), and an increase in force (+20%, P<0.05) associated with an increase in the number of cross bridges (+14%, P<0.05). These modifications were in agreement with a shift towards slow myosin heavy chain fibers and were associated with an upregulation of PPARβ/δ (+314% increase in gene and +190% increase in protein expression, P<0.05). In addition, greater resistances to fatigue and hypoxia were observed in diabetic rats.

Conclusions/Significance

Type I diabetes induced complex mechanical and energetic changes in the rat diaphragm and was associated with an up-regulation of PPARβ/δ that could improve resistance to fatigue and hypoxia and favour the shift towards slow myosin heavy chain isoforms.     

Prophenoloxidase activation is not required for survival to microbial infections in Drosophila

The antimicrobial defence of Drosophila relies on cellular and humoral processes, of which the inducible synthesis of antimicrobial peptides has attracted interest in recent years. Another potential line of defence is the activation, by a proteolytic cascade, of phenoloxidase, which leads to the production of quinones and melanin. However, in spite of several publications on this subject, the contribution of phenoloxidase activation to resistance to infections has not been established under appropriate in vivo conditions. Here, we have isolated the first Drosophila mutant for a prophenoloxidase-activating enzyme (PAE1). In contrast to wild-type flies, PAE1 mutants fail to activate phenoloxidase in the haemolymph following microbial challenge. Surprisingly, we find that these mutants are as resistant to infections as wild-type flies, in the total absence of circulating phenoloxidase activity. This raises the question with regard to the precise function of phenoloxidase activation in defence, if any.     

Evidence of a thermal unfolding dimeric intermediate for the Escherichia coli histone-like HU proteins: thermodynamics and structure

The Escherichia coli histone-like HU protein pool is composed of three dimeric forms: two homodimers, EcHUalpha(2) and EcHUbeta(2), and a heterodimer, EcHUalphabeta. The relative abundance of these dimeric forms varies during cell growth and in response to environmental changes, suggesting that each dimer plays different physiological roles. Here, differential scanning calorimetry and circular dichroism (CD) were used to study the thermal stability of the three E.coli HU dimers and show that each of them has its own thermodynamic signature. Unlike the other HU proteins studied so far, which melt through a single step (N(2)<-->2D), this present thermodynamic study shows that the three E.coli dimers melt according to a two-step mechanism (N(2)<-->I(2)<-->2D). The native dimer, N(2), melts partially into a dimeric intermediate, I(2), which in turn yields the unfolded monomers, D. In addition, the crystal structure of the EcHUalpha(2) dimer has been solved. Comparative thermodynamic and structural analysis between EcHUalpha(2) and the HU homodimer from Bacillus stearothermophilus suggests that the E.coli dimer is constituted by two subdomains of different energetic properties. The CD study indicates that the intermediate, I(2), corresponds to an HU dimer having partly lost its alpha-helices. The partially unfolded dimer I(2) is unable to complex with high-affinity, single-stranded break-containing DNA. These structural, thermodynamic and functional results suggest that the N(2)<-->I(2) equilibrium plays a central role in the physiology of E.coli HU. The I(2) molecular species seems to be the EcHUbeta(2) preferential conformation, possibly related to its role in the E.coli cold-shock adaptation. Besides, I(2) might be required in E.coli for the HU chain exchange, which allows the heterodimer formation from homodimers.     

The regulation of lactate dehydrogenase activity in soy-bean seedlings

An electrophoretically homogeneous lactate dehydrogenase was isolated from soybean seedlings, the specific activity of which was approximately 1800 times higher than the crude extract. From the dependence of the rate of reaction catalyzed by lactate dehydrogenase on substrate concentration, Michaelis constants and Hill coefficients were determined for four natural substrates,i.e. lactate, pyruvate, NAD and NADH. The enzyme from soy-bean plants is non-competitively inhibited by oxalate and mesoxalate,i.e. by the compounds analogous to the substrate. At pyruvate concentrations above 0.8 mM, the rate of reaction catalyzed by lactate dehydrogenase from soy-bean plants decreases, fructose diphosphate and ATP function as inhibitors as well. The inhibition by ATP is pH dependent, which seems to be of importance for the regulation of enzyme activityin vivo.