Diversity in the ability of Agaricus bisporus wild isolates to fruit at high temperature (25°C)

The button mushroom Agaricus bisporus commercially cultivated requires 16-19 °C during the fruiting period. Wild strains are also present in natural habitat, and in light of their wide range of geographic distribution reported, from boreal region to tropical region, questions on the development adaptation to temperature arose. Isolates from various geographic areas were screened for their ability to fruit at higher temperature (FHT ability) than commercial cultivars. The FHT trait discriminated at the varietal rank. Agaricus bisporus var. eurotetrasporus was unable to develop any sporophores whilst A. bisporus var. burnettii adapted perfectly to 25 °C for fruiting, suggesting that the FHT ability is a fixed trait in these varieties. In contrast, FHT ability of A. bisporus var. bisporus appeared variable and correlated neither with climate/microclimate nor with habitat. However, FHT ability taken as a whole appeared higher in North American populations than in European ones. Some A. bisporus var. bisporus isolates revealed a good potential for cultivation at 25 °C.     

The atypical two-component sensor kinase Lpl0330 from Legionella pneumophila controls the bifunctional diguanylate cyclase-phosphodiesterase Lpl0329 to modulate bis-(3'-5')-cyclic dimeric GMP synthesis

A significant part of bacterial two-component system response regulators contains effector domains predicted to be involved in metabolism of bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP), a second messenger that plays a key role in many physiological processes. The intracellular level of c-di-GMP is controlled by diguanylate cyclase and phosphodiesterases activities associated with GGDEF and EAL domains, respectively. The Legionella pneumophila Lens genome displays 22 GGDEF/EAL domain-encoding genes. One of them, lpl0329, encodes a protein containing a two-component system receiver domain and both GGDEF and EAL domains. Here, we demonstrated that the GGDEF and EAL domains of Lpl0329 are both functional and lead to simultaneous synthesis and hydrolysis of c-di-GMP. Moreover, these two opposite activities are finely regulated by Lpl0329 phosphorylation due to the atypical histidine kinase Lpl0330. Indeed, Lpl0330 was found to autophosphorylate on a histidine residue in an atypical H box, which is conserved in various bacteria species and thus defines a new histidine kinase subfamily. Lpl0330 also catalyzes the phosphotransferase to Lpl0329, which results in a diguanylate cyclase activity decrease whereas phosphodiesterase activity remains efficient. Altogether, these data present (i) a new histidine kinase subfamily based on the conservation of an original H box that we named HGN H box, and (ii) the first example of a bifunctional enzyme that modulates synthesis and turnover of c-di-GMP in response to phosphorylation of its receiver domain.     

Lamin A/C Mutants Disturb Sumo1 Localization and Sumoylation in Vitro and in Vivo

A-type lamins A and C are nuclear intermediate filament proteins in which mutations have been implicated in multiple disease phenotypes commonly known as laminopathies. A few studies have implicated sumoylation in the regulation of A-type lamins. Sumoylation is a post-translational protein modification that regulates a wide range of cellular processes through the attachment of small ubiquitin-related modifier (sumo) to various substrates. Here we showed that laminopathy mutants result in the mislocalization of sumo1 both in vitro (C2C12 cells overexpressing mutant lamins A and C) and in vivo (primary myoblasts and myopathic muscle tissue from the Lmna^{H222P /H222P} mouse model). In C2C12 cells, we showed that the trapping of sumo1 in p.Asp192Gly, p.Gln353Lys, and p.Arg386Lys aggregates of lamin A/C correlated with an increased steady-state level of sumoylation. However, lamin A and C did not appear to be modified by sumo1. Our results suggest that mutant lamin A/C alters the dynamics of sumo1 and thus misregulation of sumoylation may be contributing to disease progression in laminopathies.     

On the edge of the denaturation process: application of X-ray diffraction to barnase and lysozyme cross-linked crystals with denaturants in molar concentrations

Structural data about the early step of protein denaturation were obtained from cross-linked crystals for two small proteins: barnase and lysozyme. Several denaturant agents like urea, bromoethanol or thiourea were used at increasing concentrations up to a limit leading to crystal disruption (>or=2 to 6 M). Before the complete destruction of the crystal order started, specific binding sites were observed at the protein surfaces, an indication that the preliminary step of denaturation is the disproportion of intermolecular polar bonds to the benefit of the agent "parasiting" the surface. The analysis of the thermal factors first agree with a stabilization effect at low or moderate concentration of denaturants rapidly followed by a destabilization at specific weak points when the number of sites increase (overflooding effect).     

Cloning, Sequencing, and Transgenic Expression ofPodospora curvicollaandSordaria macrosporaeEF1A Genes: Relationship between Cytosolic Translation and Longevity in Filamentous Fungi

We have cloned and sequenced the gene encoding the translation elongation factor eEF1A from two filamentous fungi,Podospora curvicollaandSordaria macrospora.These fungi are close relatives ofPodospora anserinaand also show senescence syndromes. Comparison of the sequences of the deduced proteins with that ofP. anserinareveals that the three proteins differ in several positions. Replacement of theP. anserinagene by either of the two exogenous genes does not entail any modification inP. anserinaphysiology; the longevity of the fungus is not affected. No alteration ofin vivotranslational accuracy was detected; however, the exogenous proteins nonetheless promoted a modification of the resistance to the aminoglycoside antibiotic paromomycin. These data suggest that optimization of life span between these closely related fungi has likely not been performed during evolution through modifications of eEF1A activity, despite the fact that mutations in this factor can drastically affect longevity.     

Actigraphic estimates of sleep and the sleep-wake rhythm,and 6-sulfatoxymelatonin levels in healthy Dutch children

ABSTRACT

Sleep and the sleep-wake rhythm are essential for children’s health and well-being, yet reference values are lacking. This study therefore aimed to assess actigraphic estimates of sleep and the 24-h sleep-wake rhythm, as well as 6-sulfatoxymelatonin (aMT6s) levels in healthy children of different age groups. Additionally, relationships between the outcomes and sex, highest parental educational level (as an indication of socioeconomic status (SES)), and body-mass-index (BMI) were explored. In this cross-sectional study, healthy Dutch children (2–18 years) wore an actigraph (GT3x) for 7 consecutive days, collected first-morning void urine and completed a sleep log and sociodemographic questionnaire. Actigraphically estimated sleep variables were sleep onset latency (SOL), sleep efficiency (SE), total sleep time (TST), and wake after sleep onset (WASO). Non-parametric sleep-wake rhythm variables were intradaily variability (IV); interdaily stability (IS); the activity counts and timing of the least active 5-h period (L5counts and midpoint) and of the most active 10-h period (M10 counts and midpoint); and the relative amplitude (RA), i.e. the ratio of the difference and the sum of M10 and L5 counts. Finally, creatinine-corrected aMT6s levels were obtained by isotope dilution mass spectrometry. Effects of age group (preschool 2–5 years/school-aged 6–12 years/teenager 13–18 years), sex, highest parental educational level and BMI (Z-scores) were explored. Ninety-four children participated, equally divided across age groups (53% boys). Teenagers slept less, but more efficiently, than younger children, while their 24 h sleep-wake rhythm was the least stable and most fragmented (likely due to fragmentation of daytime activity). Additionally, aMT6s levels significantly declined over the age groups. Children from highly educated parents had lower sleep efficiency, but a more stable sleep-wake rhythm. Finally, sex or increase in BMI was not associated with any of the outcomes in this study. In conclusion, this study provides reference values of healthy children across different age groups and different sociodemographic factors. In the future, this information may help to better interpret outcomes in clinical populations.     

Production of halogenated compounds byBjerkandera adusta

The white-rot fungusBjerkandera adusta produces volatile chlorinated phenyl compounds. The main compounds identified were 3-chloro-4-methoxybenzaldehyde (3-chloro-p-anisaldehyde), 3-chloro-4-methoxybenzyl alcohol (3-chloro-p-anisyl alcohol), 3,5-dichloro-4-methoxybenzaldehyde (3,5-dichloro-p-anisaldehyde), and 3,5-dichloro, 4-methoxybenzyl alcohol (3,5-dichloro-p-anisyl alcohol).p-Anisaldehyde, veratraldehyde and the corresponding alcohols,p-anisyl alcohol and veratryl alcohol were produced simultaneously. Even with a very low concentration of chloride in the medium (< 10^–5 m), chlorinated aromatic compounds were still observed. Addition of bromide to the culture medium led to the production of brominated compounds: 3-bromo-4-methoxybenzaldehyde, 3-bromo-4-methoxybenzyl alcohol, 3,5-dibromo-4-methoxybenzaldehyde and 3-bromo-5-chloro-4-methoxybenzaldehyde. These brominated compounds have not previously been reported as natural products. Although iodo-aromatic compounds were not produced by supplementation of the medium with iodide, isovanillin was found in the culture broth under these conditions. This compound may be formed by substitution of the iodine intermediate by a hydroxyl group on the third carbon of the ring. Diiodomethane or chloroiodomethane were also found. It is the first time that the production of halomethane has been related to the production of halogenated aromatic compounds. All the strains tested have these capabilities.     

Knowledge-based design of reagentless fluorescent biosensors from recombinant antibodies

The possibility of obtaining from any antibody a fluorescent conjugate which responds to the binding of the antigen by a variation of its fluorescence, would be of great interest in the analytical sciences and for the construction of protein chips. This possibility was explored with antibody mAbD1.3 directed against hen egg white lysozyme. Rules of design were developed to identify the residues of the antibody to which a fluorophore could be chemically coupled, after changing them to cysteine by mutagenesis. These rules were based on: the target residue belonging to a topological neighbourhood of the antigen in the structure of the complex between antibody and antigen; its absence of functional importance for the interaction with the antigen; and its solvent accessibility in the structure of the free antibody. Seventeen conjugates between the single-chain variable fragment scFv of mAbD1.3 and an environment-sensitive fluorophore were constructed. For six of the ten residues which fully satisfied the design rules, the relative variation of the fluorescence intensity between the free and bound states of the conjugate was comprised between 12 and 75% (in non-optimal buffer), and the affinity of the conjugate for lysozyme remained unchanged relative to the parental scFv. In contrast, such results were true for only one of the seven residues which failed to satisfy one of the rules and were used as controls. One of the conjugates was studied in more detail. Its fluorescence increased proportionally to the concentration of lysozyme in a nanomolar range, up to 90% in a defined buffer, and 40% in serum. This increase was specific for hen egg lysozyme and it was not observed with a closely related protein, turkey egg lysozyme. The residues which gave operational conjugates (six in V(L) and one in V(H)), were located in the immediate vicinity of residues which are functionally important, along the sequence of FvD1.3. The results suggest rules of design for constructing antigen-sensitive fluorescent conjugates from any antibody, in the absence of structural data.