Time-gated luminescence assay using nonmetal probes for determination of protein kinase activity-based disease markers

A novel nonmetal optical probe ARC-1063 whose long-lifetime luminescence is induced by association with the target protein kinase is used for the measurement of the concentration of catalytic subunit of protein kinase A (PKAc) in complicated biological solutions. High affinity (K(D) = 10 pM toward PKAc) and unique optical properties of the probe enable its application for the measurement of picomolar concentrations of PKAc in the presence of high concentrations of other proteins. The described assay is applicable in the high-throughput format with the instrument setups designed for lanthanide-based time-gated (time-resolved) luminescence methods. The assay is used for demonstration that extracellular PKAc (ECPKA) is present in plasma samples of all healthy persons and cancer patients but great care must be taken for procedures of treatment of blood samples to avoid disruption, damage, or activation of platelets in the course of plasma (or serum) preparation and conservation.     

Phylogenetic taxonomy of Hymenochaete and related genera (Hymenochaetales)

Species delimitation in Hymenochaete sensu lato was evaluated based on partial nucLSU rDNA sequences, isoenzyme analyses and morphological data. Analyses of LSU data revealed Hymenochaete sensu stricto and Pseudochaete as distinct monophyletic genera. Transfer of Hydnochaete olivacea into genus Pseudochaete and Dichochaete setosa into genus Hymenochaete (as H. resupinata) was supported by the results of molecular analyses. A new species Hymenochaete koeljalgii from Tanzania was described. The species of genus Hymenochaete sensu stricto were divided into four well-supported clades possessing no distinctive morphological characters.     

SEX RATIO AND DENSITY AFFECT SEXUAL SELECTION IN A SEX‐ROLE REVERSED FISH

Understanding how demographic processes influence mating systems is important to decode ecological influences on sexual selection in nature. We manipulated sex ratio and density in experimental populations of the sex‐role reversed pipefish Syngnathus typhle. We quantified sexual selection using the Bateman gradient (), the opportunity for selection (I), and sexual selection (I_s), and the maximum standardized sexual selection differential (). We also measured selection on body length using standardized selection differentials (s′) and mating differentials (m′), and tested whether the observed I and I_s differ from values obtained by simulating random mating. We found that I, I_s, and , but not , were higher for females under female than male bias and the opposite for males, but density did not affect these measures. However, higher density decreased sexual selection (m′ but not s′) on female length, but selection on body length was not affected by sex ratio. Finally, I_s but not I was higher than expected from random mating, and only for females under female bias. This study demonstrates that both sex ratio and density affect sexual selection and that disentangling interrelated demographic processes is essential to a more complete understanding of mating behavior and the evolution of mating systems.     

The length of discontinuous gas exchange cycles in lepidopteran pupae may serve as a mechanism for natural selection

Gas exchange is studied in diapausing pupae of Mamestra brassicae L., whose larvae are reared under identical conditions. The release of CO₂ gas is recorded with infrared gaseous analyzers. Oxygen convective uptake into the tracheae and oxygen consumption rates are recorded by means of a constant‐volume coulometric respirometer. Outputs from both of these respirometry systems are combined with infrared actographs. All 3‐month‐old pupae of M. brassicae display a pattern of discontinuous gas exchange (DGE) cycles of CO₂ gas release by bursts, although the lengths of these cycles varies between individuals. Some pupae exhibit long DGE cycles of at least 20 h in duration, with negligible CO₂ gas release during interburst periods, and there is presumed to be a convective gas exchange at this time. As a result of a partial vacuum inside the tracheae, a large oxygen convective uptake always occurs at the start of the spiracular opening phase. Other pupae have short DGE cycles of less than 3 h in duration, with elevated CO₂ gas release during the interburst period, when gas exchange is predominantly diffusive. The spiracular open phase in these pupae consists of frequent separate convective bursts of CO₂ gas release, with the opening–closing rhythms of the spiracles, which are considered as O phase fluttering. The pupae with long DGE cycles exhibit extremely low metabolic rates and very low total water loss rates, whereas those with short DGE cycles have higher metabolic and total water loss rates. The pupae with long DGE cycles live approximately twice as long as those with short cycles; thus, the present study demonstrates that long DGE cycles confer a fitness benefit on pupae as a result of a lower metabolic rate associated with water economy, conferring on them a longer life.     

Genetic and chemical characterization of an EMS induced mutation in Cucumis melo CRTISO gene

In order to broaden the available genetic variation of melon, we developed an ethyl methanesulfonate mutation library in an orange-flesh ‘Charentais’ type melon line that accumulates β-carotene. One mutagenized M₂ family segregated for a novel recessive trait, a yellow–orange fruit flesh (‘yofI’). HPLC analysis revealed that ‘yofI’ accumulates pro-lycopene (tetra-cis-lycopene) as its major fruit pigment. The altered carotenoid composition of ‘yofI’ is associated with a significant change of the fruit aroma since cleavage of β-carotene yields different apocarotenoids than the cleavage of pro-lycopene. Normally, pro-lycopene is further isomerized by CRTISO (carotenoid isomerase) to yield all-trans-lycopene, which is further cyclized to β-carotene in melon fruit. Cloning and sequencing of ‘yofI’ CRTISO identified two mRNA sequences which lead to truncated forms of CRTISO. Sequencing of the genomic CRTISO identified an A–T transversion in ‘yofI’ which leads to a premature STOP codon. The early carotenoid pathway genes were up regulated in yofI fruit causing accumulation of other intermediates such as phytoene and ζ-carotene. Total carotenoid levels are only slightly increased in the mutant. Mutants accumulating pro-lycopene have been reported in both tomato and watermelon fruits, however, this is the first report of a non-lycopene accumulating fruit showing this phenomenon.     

Protein cargo delivery properties of cell-penetrating peptides. A comparative study

Application of cell-penetrating peptides for delivering various hydrophilic macromolecules with biological function into cells has gained much attention in recent years. We compared the protein transduction efficiency of four cell-penetrating peptides: penetratin, Tat peptide, transportan, and pVEC and studied the effects of various medium parameters on the uptake. Depletion of cellular energy and lowering of temperature strongly impaired the internalization of protein complexed with cell-penetrating peptides, confirming the endocytotic mechanism of peptide-mediated protein cellular transduction. Peptide-induced protein association with HeLa cells decreased 3-6-fold in energy-depleted cells. Inhibition of clathrin-dependent endocytosis by the hyperosmolar medium decreased the uptake of peptide-avidin complexes 1.5-3-fold and the removal of cholesterol from the plasma membrane 1.2-2-fold, suggesting that both clathrin-dependent and independent endocytosis were involved in peptide-induced cellular delivery of avidin. However, even under conditions of cellular energy depletion, ceasing of cellular traffic, and partial depolarization of plasma membrane, peptide-protein complexes associated with HeLa cells, as observed by FACS analysis and spectrofluorimetry. Among the studied peptides, pTat and transportan revealed higher protein transduction efficiency than penetratin or pVEC.     

Flexible working hours, health, and well-being in Europe: some considerations from a SALTSA project

The project brought together researchers from 9 EU-Countries and resulted in a number of actions, in particular the following: (a) There is an urgent need of defining the concept of flexible working hours, since it has been used in many different and even counterintuitive ways; the most obvious distinction is where the influence over the working hours lies, that is between the “company-based flexibility” and the “individual-oriented flexibility”; (b) The review of the Legislation in force in the 15 European countries shows that the regulation of working times is quite extensive and covers (Council Directive 93/104/EC) almost all the various arrangements of working hours (i.e., part-time, overtime, shift, and night work), but fails to provide for flexibility; (c) According to the data of the Third EU Survey on Working Conditions, longer and “irregular” working hours are in general linked to lower levels of health and well-being; moreover, low (individual) flexibility and high variability of working hours (i.e., company-based flexibility) were consistently associated with poor health and well-being, while low variability combined with high autonomy showed positive effects; (d) Six substudies from different countries demonstrated that flexible working hours vary according to country, economic sector, social status, and gender; overtime is the most frequent form of company-based flexibility but has negative effects on stress, sleep, and social and mental health; individual flexibility alleviates the negative effects of the company-based flexibility on subjective health, safety, and social well-being; (e) The literature review was able to list more than 1,000 references, but it was striking that most of these documents were mainly argumentative with very little empirical data. Thus, one may conclude that there is a large-scale intervention ongoing in our society with almost completely unknown and uncontrolled effects. Consequently, there is a strong need for systematic research and well-controlled actions in order to examine in detail what flexible working hours are considered, what and where are their positive effects, in particular, as concerns autonomy, and what regulation seem most reasonable.     

Roles of the amyloid precursor protein family in the peripheral nervous system

Compelling evidence from in vivo model systems within the past decade shows that the APP family of proteins is important for synaptic development and function in the central and peripheral nervous systems. The synaptic role promises to be complex and multifaceted for several reasons. The three family members have overlapping and redundant functions in mammals. They have both adhesive and signaling properties and may, in principle, act as both ligands and receptors. Moreover, they bind a multitude of synapse-specific proteins, and we predict that additional interacting protein partners will be discovered. Transgenic mice with modified or abolished expression of APP and APLPs have synaptic defects that are readily apparent. Studies of the neuromuscular junction (NMJ) in these transgenic mice have revealed molecular and functional deficits in neurotransmitter release, in organization of the postsynaptic receptors, and in coordinated intercellular development. The results summarized here from invertebrate and vertebrate systems confirm that the NMJ with its accessibility, large size, and homogeneity provides a model synapse for identifying and analyzing molecular pathways of APP actions.     

Dynamics of crowing development in the domestic Japanese quail (Coturnix coturnix japonica)

Species-specific behaviours gradually emerge, via incomplete patterns, to the final complete adult form. A classical example is birdsong, a learned behaviour ideally suited for studying the neural and molecular substrates of vocal learning. Young songbirds gradually transform primitive unstructured vocalizations (subsong, akin to human babbling) into complex, stereotyped sequences of syllables that constitute adult song. In comparison with birdsong, territorial and mating calls of vocal non-learner species are thought to exhibit little change during development. We revisited this issue using the crowing behaviour of domestic Japanese quail (Coturnix coturnix japonica). Crowing activity was continuously recorded in young males maintained in social isolation from the age of three weeks to four months. We observed developmental changes in crow structure, both the temporal and the spectral levels. Speed and trajectories of these developmental changes exhibited an unexpected high inter-individual variability. Mechanisms used by quails to transform sounds during ontogeny resemble those described in oscines during the sensorimotor phase of song learning. Studies on vocal non-learners could shed light on the specificity and evolution of vocal learning.