Non-territorial nightingales prospect territories during the dawn chorus

Male songbirds usually sing when they have occupied a territory, but the territory prospecting of non-territorial males is more elusive and has been rarely studied. Here, we simulated newly arriving, non-territorial males by translocating unmated male nightingales (Luscinia megarhynchos) to our study site. We show that territory prospecting of translocated males was largely confined to the hour before sunrise. The radio-tagged males made extensive excursions visiting several singing males at dawn, but after dawn they remained stationary outside occupied territories. As in many other songbird species, dawn was also the time when resident males sang the most. These results suggest that nonterritorial male nightingales use the dawn chorus to assess singing residents or territory occupancy. For resident males, dawn singing may be important to announce territory occupancy to prospecting males and may thus play a role in territory maintenance.     

Making vascular networks in the adult: branching morphogenesis without a roadmap

The vascular system is unique in that extensive branching morphogenesis may take place in the adult. Developmental neovascularization is guided by precise spatial cues but vessel formation in the adult is not genetically programmed. Here, we review different adult modes for branch patterning, acquiring artery or vein identity and allocating vascular progenitor cells. The endothelium shows a remarkable degree of self-organization into a treelike network and hemodynamic forces are important in rectifying abnormal branching. This discussion is in the context of a contemplated therapy for improving organ perfusion by creating new vascular loops properly integrated within the existing network.     

Fluorometric quantification of RNA and DNA in solutions containing both nucleic acids

A fluorescence-based method for quantitative determination of RNA and DNA in probes containing both nucleic acids has been developed. The total concentration of nucleic acids is determined using SYBR Green II dye under conditions providing independent binding of the fluorophore with DNA and RNA. The concentration of DNA is specifically measured using the Hoechst 33258 dye and the RNA concentration is calculated from these data. The procedure allows for accurate determination of DNA concentration in the range 10-1000 ng/ml in the presence of 200-fold excess of RNA and determination of RNA concentrations in the range 10-1000 ng/ml in the presence of large excess of DNA. An absence of the treatment of mixed samples with RNase-free DNase I provides rapid, reproducible, and accurate RNA quantification.     

Catalysis and electron transfer in protein crystals: the binary and ternary complexes of methylamine dehydrogenase with electron acceptors

Polarized absorption microspectrophotometry has been used to detect catalysis and intermolecular electron transfer in single crystals of two multiprotein complexes: (1) the binary complex between Paracoccus denitrificans methylamine dehydrogenase, which contains tryptophan-tryptophylquinone (TTQ) as a cofactor, and its redox partner, the blue copper protein amicyanin; (2) the ternary complex between the same two proteins and cytochrome c-551i. Continuous wave electron paramagnetic resonance has been used to compare the state of copper in polycrystalline powders of the two systems. While catalysis and intermolecular electron transfer from reduced TTQ to copper are too fast to be accessible to our measurements, heme reduction occurs over a period of several minutes. The observed rate constant is about four orders of magnitude lower than in solution. The analysis of the temperature dependence of this apparent constant provides values for the parameters H(AB), related to electronic coupling between the two centers, and lambda, the reorganizational energy, that are compatible with electron transfer being the rate-determining step. From these parameters and the known distance between copper and heme, it is possible to calculate the parameter beta, which depends on the nature of the intervening medium, obtaining a value typical of electron transfer across a protein matrix. These findings suggest that the ternary complex in solution might achieve a higher efficiency than the rigid crystal structure thanks to an as yet unidentified role of protein dynamics.     

Past exposure to densely ionizing radiation leaves a unique permanent signature in the genome

Speculation has long surrounded the question of whether past exposure to ionizing radiation leaves a unique permanent signature in the genome. Intrachromosomal rearrangements or deletions are produced much more efficiently by densely ionizing radiation than by chemical mutagens, x-rays, or endogenous aging processes. Until recently, such stable intrachromosomal aberrations have been very hard to detect, but a new chromosome band painting technique has made their detection practical. We report the detection and quantification of stable intrachromosomal aberrations in lymphocytes of healthy former nuclear-weapons workers who were exposed to plutonium many years ago. Even many years after occupational exposure, more than half the blood cells of the healthy plutonium workers contain large (>6 Mb) intrachromosomal rearrangements. The yield of these aberrations was highly correlated with plutonium dose to the bone marrow. The control groups contained very few such intrachromosomal aberrations. Quantification of this large-scale chromosomal damage in human populations exposed many years earlier will lead to new insights into the mechanisms and risks of cytogenetic damage.     

The effect of protein conformational flexibility on the electronic properties of a chromophore

In this paper we address the question of how a protein environment can modulate the absorption spectrum of a chromophore during a molecular dynamics simulation. The effect of the protein is modeled as an external field acting on the unperturbed eigenstates of the chromophore. Using a first-principles method recently developed in our group, we calculated the perturbed electronic energies for each frame and the corresponding wavelength absorption during the simulation. We apply this method to a nanosencond timescale molecular dynamics simulation of the light-harvesting peridinin-chlorophyll-protein complex from Amphidinium carterae, where chlorophyll was selected among the chromophores of the complex for the calculation. The combination of this quantum-classical calculation with the analysis of the large amplitude motions of the protein makes it possible to point out the relationship between the conformational flexibility of the environment and the excitation wavelength of the chromophore. Results support the idea of the existence of a correlation between protein conformational flexibility and chlorophyll electronic transitions induced by light.     

RESOURCERER: a database for annotating and linking microarray resources within and across species

Microarray expression analysis is providing unprecedented data on gene expression in humans and mammalian model systems. Although such studies provide a tremendous resource for understanding human disease states, one of the significant challenges is cross-referencing the data derived from different species, across diverse expression analysis platforms, in order to properly derive inferences regarding gene expression and disease state. To address this problem, we have developed RESOURCERER, a microarray-resource annotation and cross-reference database built using the analysis of expressed sequence tags (ESTs) and gene sequences provided by the TIGR Gene Index (TGI) and TIGR Orthologous Gene Alignment (TOGA) databases [now called Eukaryotic Gene Orthologs (EGO)].     

Endothelial chemokines destabilize L-selectin-mediated lymphocyte rolling without inducing selectin shedding

Chemokines presented on specialized endothelial surfaces rapidly up-regulate leukocyte integrin avidity and firm arrest through G(i)-protein signaling. Here we describe a novel, G-protein-independent, down-regulatory activity of apical endothelial chemokines in destabilizing L-selectin-mediated leukocyte rolling. Unexpectedly, this anti-adhesive chemokine suppression of rolling does not involve L-selectin shedding. Destabilization of rolling is induced only by immobilized chemokines juxtaposed to L-selectin ligands and is an energy-dependent process. Chemokines are found to interfere with a subsecond stabilization of selectin tethers necessary for persistent rolling. This is a first indication that endothelial chemokines can attenuate in situ L-selectin adhesion to endothelial ligands at subsecond contacts. This negative feedback mechanism may underlie the jerky nature of rolling mediated by L-selectin in vivo.     

Delineation of the key amino acids involved in neutrophil inhibitory factor binding to the I-domain supports a mosaic model for the capacity of integrin alphaMbeta 2 to recognize multiple ligands

To gain insight into the mechanism by which the alpha(M)I-domain of integrin alpha(M)beta(2) interacts with multiple and unrelated ligands, the identity of the neutrophil inhibitory factor (NIF) recognition site was sought. A systematic strategy in which individual amino acid residues within three previously implicated segments were changed to those in the alpha(L)I-domain, which is structurally very similar but does not bind NIF, was implemented. The capacity of the resulting mutants, expressed as glutathione S-transferase fusion proteins, to recognize NIF was assessed. These analyses ultimately identified Asp(149), Arg(151), Gly(207), Tyr(252), and Glu(258) as critical for NIF binding. Cation binding, a function of the metal ion-dependent adhesion site (MIDAS) motif, was assessed by terbium luminescence to evaluate conformational perturbations induced by the mutations. All five mutants bound terbium with unaltered affinities. When the five residues were inserted into the alpha(L)I-domain, the chimera bound NIF with high affinity. Another ligand of alpha(M)beta(2), C3bi, which is known to use the same segments of the alpha(M)I-domain in engaging the receptor, failed to bind to the chimeric alpha(L)I-domain. Thus, the alpha(M)I-domain appears to present a mosaic of exposed amino acids within surface loops on its MIDAS face, and different ligands interact with different residues to attain high affinity binding.