Time-co-ordinated control of glycogen synthase, protein phosphatase 2A and protein kinase CK2 during culture growth in Yarrowia lipolytica in relation to glycogen metabolism

In the growth course of the lipolytic yeast Yarrowia lipolytica, the activities of protein phosphatase 2A (PP2A) and glycogen synthase (GS) rise during the exponential phase and concomitantly glycogen storage occurs in the cells. There is also an increase in the independence ratio (RI) indicating a shift from an inactive phosphorylated GS form to an active dephosphorylated GS form. During the early stationary phase, an increase in protein kinase CK2 (CK2) activity, a reversion of RI variation and a glycogen content decrease are observed. GS activity proved to be a good indicator of early culture growth phase. Experiments carried out with enzymes purified from Y. lipolytica show strong RI variations upon the action of CK2 and PP2Ac, and 32P incorporation into GS protein through phosphorylation by CK2. GS activity would be controlled by the sequential action of PP2A and CK2.     

MC1R expression in HaCaT keratinocytes inhibits UVA-induced ROS production via NADPH oxidase- and cAMP-dependent mechanisms

Ultraviolet A (UVA) radiations are responsible for deleterious effects, mainly due to reactive oxygen species (ROS) production. Alpha-melanocyte stimulating hormone (α-MSH) binds to melanocortin-1 receptor (MC1R) in melanocytes to stimulate pigmentation and modulate cutaneous inflammatory responses. MC1R may be induced in keratinocytes after UV exposure. To investigate the effect of MC1R signaling on UVA-induced ROS (UVA-ROS) production, we generated HaCaT cells that stably express human MC1R (HaCaT-MC1R) or the Arg151Cys (R(151)C) non-functional variant (HaCaT-R(151)C). We then assessed ROS production immediately after UVA exposure and found that: (1) UVA-ROS production was strongly reduced in HaCaT-MC1R but not in HaCaT-R(151)C cells compared to parental HaCaT cells; (2) this inhibitory effect was further amplified by incubation of HaCaT-MC1R cells with α-MSH before UVA exposure; (3) protein kinase A (PKA)-dependent NoxA1 phosphorylation was increased in HaCaT-MC1R compared to HaCaT and HaCaT-R(151)C cells. Inhibition of PKA in HaCaT-MC1R cells resulted in a marked increase of ROS production after UVA irradiation; (4) the ability of HaCaT-MC1R cells to produce UVA-ROS was restored by inhibiting epidermal growth factor receptor (EGFR) or extracellular signal-regulated kinases (ERK) activity before UVA exposure. Our findings suggest that constitutive activity of MC1R in keratinocytes may reduce UVA-induced oxidative stress via EGFR and cAMP-dependent mechanisms.     

Yeast cytochrome c oxidase: a model system to study mitochondrial forms of the haem-copper oxidase superfamily

The known subunits of yeast mitochondrial cytochrome c oxidase are reviewed. The structures of all eleven of its subunits are explored by building homology models based on the published structures of the homologous bovine subunits and similarities and differences are highlighted, particularly of the core functional subunit I. Yeast genetic techniques to enable introduction of mutations into the three core mitochondrially-encoded subunits are reviewed.     

One clutch or two clutches? Fitness correlates of coexisting alternative female life-histories in the European earwig

Whether to reproduce once or multiple times (semelparity vs. iteroparity) is a major life-history decision that organisms have to take. Mode of parity is usually considered a species characteristic. However, recent models suggested that population properties or condition-dependent fitness payoffs could help to maintain both life-history tactics within populations. In arthropods, semelparity was also hypothesised to be a critical pre-adaptation for the evolution of maternal care, semelparous females being predicted to provide more care due to the absence of costs on future reproduction. The aim of this study was to characterize potential fitness payoffs and levels of maternal care in semel- and itero-parous females of the European earwig Forficula auricularia. Based on 15 traits measured in 494 females and their nymphs, our results revealed that iteroparous females laid their first clutch earlier, had more eggs in their first clutch, gained more weight during the 2 weeks following hatching of the first clutch, but produced eggs that developed more slowly than semelparous females. Among iteroparous females, the sizes of first and second clutches were significantly and positively correlated, indicating no investment trade-off between reproductive events. Iteroparous females also provided more food than semelparous ones, a result contrasting with predictions that iteroparity is incompatible with the evolution of maternal care. Finally, a controlled breeding experiment reported full mating compatibility among offspring from females of the two modes of parity, confirming that both types of females belong to one single species. Overall, these results indicate that alternative modes of parity represent coexisting life-history tactics that are likely to be condition-dependent and associated with offspring development and specific levels of maternal care in earwigs.     

Long-term removal of wheat straw decreases soil amorphous silica at Broadbalk, Rothamsted

Aims

Most cereals accumulate Si in their shoots. Soil bioavailability of Si may be a constraint on the beneficial role of silica in cereals but it is not yet well supported by field data. The aim of this study is to evaluate the long-term impact of wheat straw exports on the pool of soil phytoliths, which, it is suggested, represents the most labile and renewable pool of soil Si.

Methods

We measured the amorphous Si (ASi) in soils from several experiments at Rothamsted Research (UK), which provided long-term soil data back to the middle of the 19th century, using two alternative extraction techniques: Na₂CO₃ (referred to as AS_nc) or zinc bromide extraction (referred to as ASi_zb).

Results

All samples showed a similar range of AS_nc and ASi_zb but low values (0.1–3.4?mg?g^?1 DW) compared to published data on natural ecosystems. In the Broadbalk experiment, a decrease over time in ASi in the topsoil samples is in good agreement with the hypothesis that cropping and exports of straw leads to depletion of soil phytoliths. A decrease in Si concentration in straw samples was observed between 1883 and 1944. From 1944 to the present, Si concentration increased irregularly in the straw, probably as the result of liming, which enhanced the dissolution of the remaining phytoliths through increasing pH. In the reforested Geescroft field the higher phytolith concentration in the modern topsoil samples is in good agreement with a re-building of phytolith storage from litter input in an acidic environment.

Conclusions

Our results therefore support the hypothesis that export of wheat straw leads to a decrease in bioavailable Si.     

Molecular basis of resistance to cytochrome bc1 inhibitors.

Inhibitors of the mitochondrial respiratory chain enzyme cytochrome bc1 (respiratory complex III) have been developed as antimicrobial agents. They are used in agriculture to control plant pathogenic fungi and in medicine against human pathogens, such as the malaria parasite Plasmodium falciparum, or Pneumocystis jiroveci (an opportunistic pathogenic fungus life-threatening in immuno-compromised patients). These respiratory inhibitors are thus effective against a broad range of important pathogens. Unfortunately, the problem of acquired resistance has rapidly emerged. A growing number of pathogen isolates resistant to inhibitor treatment have been reported, and this resistance is often linked to mutation within cytochrome b, one of the essential catalytic subunits of the complex. Saccharomyces cerevisiae is an invaluable model in order to assess the impact of the mutations on the sensitivity to the drugs, on the respiratory capacity and the fitness of cells. In this minireview, the inhibitors, their mode of action, and the mutations implicated in resistance and studied in yeast are briefly reviewed. Four mutations that are of particular importance in medicine and in agriculture are briefly reviewed and described in more detail and the molecular basis of resistance and of evolution of the mutations is discussed succinctly.     

In vivo proteome dynamics during early bovine myogenesis

Myogenesis is a complex process of which the underlying mechanisms are conserved between species, including birds and mammals. Despite a good understanding of the stages of myogenesis, many of the mechanisms involved in the regulation of proliferation of the successive myoblast generations, the cellular transitions cell proliferation/alignment of myoblasts/fusion of myoblasts into myotubes/differentiation of myofibres and the control of total myofibre number still remain unknown. An in vivo proteomic analysis of the semitendinosus muscle from Charolais foetuses, at three specific stages of myogenesis (60, 110 and 180 days postconception), was conducted using 2-DE and MS. Expression profiles of more than 170 proteins were revealed and analysed using two way hierarchical clustering and statistical analysis. Our studies identify, for the first time, distinct proteins of varied biological functions and protein clusters with myogenic processes, such as the control of cell cycle activity and apoptosis, the establishment of cellular metabolism and muscle contractile properties and muscle cell reorganisation. These results are of fundamental interest to the field of myogenesis in general, and more specifically to the control of muscle development in meat producing animals.     

Antibiotic marker modifications of lambda Red and FLP helper plasmids, pKD46 and pCP20, for inactivation of chromosomal genes using PCR products in multidrug-resistant strains

The Red recombinase system of bacteriophage Lambda has been used to inactivate chromosomal genes in bacteria using PCR products. In this study, we describe the replacement of the ampicillin resistance marker of helper plasmids pKD46 and pCP20 by a gentamicin resistance gene to disrupt chromosomal genes and then to eliminate FRT flanked resistance gene in multiple antibiotic-resistant Salmonella enterica strains.     

Modeling the Qo site of crop pathogens in Saccharomyces cerevisiae cytochrome b.

Saccharomyces cerevisiae has been used as a model system to characterize the effect of cytochrome b mutations found in fungal and oomycete plant pathogens resistant to Q(o) inhibitors (QoIs), including the strobilurins, now widely employed in agriculture to control such diseases. Specific residues in the Q(o) site of yeast cytochrome b were modified to obtain four new forms mimicking the Q(o) binding site of Erysiphe graminis, Venturia inaequalis, Sphaerotheca fuliginea and Phytophthora megasperma. These modified versions of cytochrome b were then used to study the impact of the introduction of the G143A mutation on bc(1) complex activity. In addition, the effects of two other mutations F129L and L275F, which also confer levels of QoI insensitivity, were also studied. The G143A mutation caused a high level of resistance to QoI compounds such as myxothiazol, axoxystrobin and pyraclostrobin, but not to stigmatellin. The pattern of resistance conferred by F129L and L275F was different. Interestingly G143A had a slightly deleterious effect on the bc(1) function in V. inaequalis, S. fuliginea and P. megasperma Q(o) site mimics but not in that for E. graminis. Thus small variations in the Q(o) site seem to affect the impact of the G143A mutation on bc(1) activity. Based on this observation in the yeast model, it might be anticipated that the G143A mutation might affect the fitness of pathogens differentially. If so, this could contribute to observed differences in the rates of evolution of QoI resistance in fungal and oomycete pathogens.