Geographical variation in circadian eclosion rhythm and photoperiodic adult diapause inDrosophila littoralis

Summary The time measuring system ofDrosophila littoralis strains originating between 40–70° N was found to be highly variable and latitude dependent. The critical daylength for photoperiodic adult diapause varied from 12 h or no diapause response in the south to 20 h in north. The median timing of pupal eclosion rhythm varied correspondingly from 21 h to 12 h from lights off in LD 321, and the period of free-running rhythm of eclosion from 24 h to 19 h. The phase of the free-running rhythm was also variable, and correlated with the phase of the entrained rhythm. Latitudinal variation in the entrained rhythm of eclosion and in diapause is adaptive, leading to eclosion early in the morning and to overwintering at the adult stage. In some strains with a late phase of eclosion, strong transient cycles were seen following the transition from LL to DD. A total damping of the free-running eclosion rhythm within 2–7 days was common to all strains. This damping was more pronounced in the northern strains. The phase and period of eclosion rhythms were statistically independent. Diapause was not correlated with any parameters of the eclosion rhythm in the analysis. Diapause may still be influenced by the period of the eclosion rhythm, even though its minor contribution may be masked by a more variable, eclosion rhythm independent system in the determination of diapause.Abbreviations, symbols and terms LD Light/dark; as in LD 321 meaning a cycle of 3 h light21 h darkness - LL Continuous light - DD Continuous darkness - T Period of a Zeitgeber cycle - Natural period of eclosion rhythm in constant conditions - _EL Phase of the free-running rhythm of eclosion - A Amplitude of the free-running rhythm of eclosion; possible range is from 4.17% (no rhythmicity) to 20% (the daily eclosion peaks 2–6 within 5 h each) - P Persistence of the free-running rhythm of eclosion; the number of daily eclosion peaks where the mean for five highest hourly percentages still exceed 6% - A phase shift, expressed in h; a re-setting of a rhythm; either as an advance shift (i.e. earlier= +), or as a delay shift (i.e. later = –) - PRC Phase-response curve - _LD Phase of entrained rhythm of eclosion; e.g. _{LD 321} is the median hour of eclosion peak from lights off at LD 321 - SD _ecl Amplitude of the entrained rhythm of eclosion; the smaller SD_ecl the higher the amplitude - PPRC Photoperiodic response curve; proportion of females in diapause displayed as a function of daylength - CDL Critical photoperiod; the photoperiod in the 24 h LD cycle at which 50% of the population studied diapauses - SD _diap Accuracy of diapause response of a strain; the smaller the SD_diap the more accurate the response - Cdl The main locus controlling CDL inD. littoralis     

Quantitative detection of cell surface protein expression by time-resolved fluorimetry.

A method is introduced for quantitative detection of cell surface protein expression. The method is based on immunocytochemistry, the use of long decay time europium(III) chelate and platinum(II) porphyrin labels, and detection of photoluminescence emission from adhered cells by time-resolved fluorimetry. After immunocytochemistry, the assay wells are evaporated to dryness and measured in the dry state. This protocol allows repeated and postponed analysis and microscopy imaging. In order to investigate the performance of the method, we chose expression of intercellular adhesion molecule-1 (ICAM-1) of endothelial cell line EAhy926 as a research target. The expression of ICAM-1 on the cells was enhanced by introduction of a cytokine, tumour necrosis factor-alpha (TNFalpha). The method gave signal:background ratios (S:B) of 20 and 9 for europium and platinum labels, respectively, whereas prompt fluorescent FITC label gave a S:B of 3. Screening window coefficients (=Z'-factor) were >0.5 for all the three labels, thus indicating a score for an excellent screening assay. In conclusion, the method appears to be an appropriate choice for protein expression analysis, both in high-throughput screening applications, and for detailed sample investigation by fluorescent microscopy imaging.     

Decomposition and element concentrations of Norway spruce needle litter with differing B, N, or P status

Adequate boron (B) nutrition may decrease concentrations of phenolic compounds and enhance structural integrity and lignification in plants, compared with suboptimal B. This could affect decomposition in areas where B deficiencies are common. The mass loss and changes in element concentrations in Norway spruce needle litter were studied with combinations of litter from high-B and low-B trees, incubated for 29 months, in either B fertilised or control plots without B addition. The litter originated from the same Norway spruce field experiments. Additionally, the field experiments included long-term N and P treatments. Initially, lowest lignin concentrations were found in Norway spruce litter from the treatment P and particularly in the combination B?+?P, and highest in the B?+?N fertilised plots. The mass loss of Norway spruce litter was not affected by the treatments. However, B_litter increased Cu accumulation. The litter from the B?+?P fertilised plots accumulated considerably more Al, Ca, S and Zn than the other treatments, whereas B together with N reduced the remaining amounts of these elements. Reduced nutrient release from litter may have far-reaching consequences on nutrient cycles in forests.     

Angeli's salt and spinal motor neuron injury

Nitroxyl anion or its conjugate acid (NO_-/HNO) and nitric oxide (NO) may both have pro-oxidative and cytotoxic properties. Superoxide dismutase (SOD) enzyme has been shown to convert reversibly HNO to NO. Mutations found in the SOD enzyme in some familial amyotrophic lateral sclerosis (ALS) patients affect redox properties of the SOD enzyme in a manner, which may affect the equilibrium between NO and HNO. Therefore, we studied the effects of HNO releasing compound, Angeli's salt (AS), on both motor and sensory functions after intrathecal administration in the lumbar spinal cord of a male rat. These functions were measured by rotarod, spontaneous activity, paw- and tail-flick tests. In addition, we compared the effect of AS to NO releasing papanonoate, old AS solution and sulphononoate in the motor performance test. The effect of intrathecal delivery of AS on the markers of the spinal cord injury and oxidative/nitrosative stress were further studied.

Results: Freshly prepared AS (5 or 10 μmol), but not papanonoate, caused a marked decrease in the rotarod performance 3-7 days after the intrathecal administration. The peak motor deficiency was noted 3 days after AS (5 μmol) delivery. Old, degraded, AS solution and nitrous oxide releasing sulphononoate did not decrease motor performance in the rotarod test. AS did not affect the sensory stimulus evoked responses as measured by the paw-flick and tail-flick tests. Immunohistological examination revealed that AS caused injury related changes in the expression of glial fibrillary acidic protein (GFAP), fibroblast growth factor (FGF-2) and laminins in the spinal cord. Moreover, AS increased nitrotyrosine immunoreactivity in the spinal motor neurons.

Therefore, we conclude that AS, but not NO releasing papanonoate, causes motor neuron injury but does not affect the function of sensory nerves in behavioural tests.     

The dibenzodioxocin lignin substructure is abundant in the inner part of the secondary wall in Norway spruce and silver birch xylem

A specific condensed lignin substructure, dibenzodioxocin, was immunolocalized in differentiating cell walls of Norway spruce (Picea abies (L.) H. Karsten) and silver birch (Betula pendula Roth) xylem. A fluorescent probe, Alexa 488 was used as a marker on the dibenzodioxocin-specific secondary antibody. For the detection of this lignin substructure, 25-m cross-sections of xylem were viewed with a confocal laser-scanning microscope with fluorescein isothiocyanate fluorescence filters. In mature cells, fluorescence was detected in the S3 layer of the secondary wall in both tree species, but it was more intense in Norway spruce than in silver birch. In silver birch most of the signal was detected in vessel walls and less in fiber cell walls. In very young tracheids of Norway spruce and vessels and fibers of silver birch, where secondary cell wall layers were not yet formed, the presence of the dibenzodioxocin structure could not be shown.Abbreviation CLSM confocal laser-scanning fluorescence microscopy     

Biological role and structural mechanism of twinfilin-capping protein interaction

Twinfilin and capping protein (CP) are highly conserved actin-binding proteins that regulate cytoskeletal dynamics in organisms from yeast to mammals. Twinfilin binds actin monomer, while CP binds the barbed end of the actin filament. Remarkably, twinfilin and CP also bind directly to each other, but the mechanism and role of this interaction in actin dynamics are not defined. Here, we found that the binding of twinfilin to CP does not affect the binding of either protein to actin. Furthermore, site-directed mutagenesis studies revealed that the CP-binding site resides in the conserved C-terminal tail region of twinfilin. The solution structure of the twinfilin-CP complex supports these conclusions. In vivo, twinfilin's binding to both CP and actin monomer was found to be necessary for twinfilin's role in actin assembly dynamics, based on genetic studies with mutants that have defined biochemical functions. Our results support a novel model for how sequential interactions between actin monomers, twinfilin, CP, and actin filaments promote cytoskeletal dynamics.     

BAPS 2: enhanced possibilities for the analysis of genetic population structure

Bayesian statistical methods based on simulation techniques have recently been shown to provide powerful tools for the analysis of genetic population structure. We have previously developed a Markov chain Monte Carlo (MCMC) algorithm for characterizing genetically divergent groups based on molecular markers and geographical sampling design of the dataset. However, for large-scale datasets such algorithms may get stuck to local maxima in the parameter space. Therefore, we have modified our earlier algorithm to support multiple parallel MCMC chains, with enhanced features that enable considerably faster and more reliable estimation compared to the earlier version of the algorithm. We consider also a hierarchical tree representation, from which a Bayesian model-averaged structure estimate can be extracted. The algorithm is implemented in a computer program that features a user-friendly interface and built-in graphics. The enhanced features are illustrated by analyses of simulated data and an extensive human molecular dataset. AVAILABILITY: Freely available at http://www.rni.helsinki.fi/~jic/bapspage.html.     

Regulation of cytoskeletal dynamics by actin-monomer-binding proteins

The actin cytoskeleton is a vital component of several key cellular and developmental processes in eukaryotes. Many proteins that interact with filamentous and/or monomeric actin regulate the structure and dynamics of the actin cytoskeleton. Actin-filament-binding proteins control the nucleation, assembly, disassembly and crosslinking of actin filaments, whereas actin-monomer-binding proteins regulate the size, localization and dynamics of the large pool of unpolymerized actin in cells. In this article, we focus on recent advances in understanding how the six evolutionarily conserved actin-monomer-binding proteins - profilin, ADF/cofilin, twinfilin, Srv2/CAP, WASP/WAVE and verprolin/WIP - interact with actin monomers and regulate their incorporation into filament ends. We also present a model of how, together, these ubiquitous actin-monomer-binding proteins contribute to cytoskeletal dynamics and actin-dependent cellular processes.