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Infusion of α,β-methylene ATP (α,β-meATP) into murine neck muscle facilitates brainstem nociception. This animal experimental model is suggested to be appropriate for investigating pathophysiological mechanisms in tension-type headache. It was hypothesized that d-lysine acetylsalicylic acid (ASA, aspirin®) reverses this α,β-meATP effect. Facilitation of neck muscle nociceptive processing was induced via bilateral infusion of α,β-meATP into semispinal neck muscles (100 nM, 20 μl each) in 42 anesthetized mice. Brainstem nociception was monitored by the jaw-opening reflex elicited via electrical tongue stimulation. The hypothesis was addressed by subsequent (15, 30, 60 mg/kg) and preceding (60 mg/kg) intraperitoneal ASA injection. Saline served as control to ASA solution. Subsequent ASA dose-dependently reversed α,β-meATP-induced reflex facilitation and was the most prominent with 60 mg/kg. Preceding 60 mg/kg ASA prevented reflex facilitation. Cyclooxygenases are involved in nociceptive transmission. Former experiments showed that unspecific inhibition of cyclooxygenases does not alter the α,β-meATP effect. This suggests a specific mode of action of ASA. The concept is accepted that neck muscle nociception is involved in the pathophysiology of tension-type headache. Thus, objective proof of ASA effects in this experimental model may emphasize its major role in pharmacological treatment of tension-type headache attacks., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Direct imaging is invaluable for understanding the mechanism of complex genome transactions where proteins work together to organize, transcribe, replicate, and repair DNA. Scanning (or atomic) force microscopy is an ideal tool for this, providing 3D information on molecular structure at nanometer resolution from defined components. This is a convenient and practical addition to in vitro studies as readily obtainable amounts of purified proteins and DNA are required. The images reveal structural details on the size and location of DNA-bound proteins as well as protein-induced arrangement of the DNA, which are directly correlated in the same complexes. In addition, even from static images, the different forms observed and their relative distributions can be used to deduce the variety and stability of different complexes that are necessarily involved in dynamic processes. Recently available instruments that combine fluorescence with topographic imaging allow the identification of specific molecular components in complex assemblies, which broadens the applications and increases the information obtained from direct imaging of molecular complexes. We describe here basic methods for preparing samples of proteins, DNA, and complexes of the two for topographic imaging and quantitative analysis. We also describe special considerations for combined fluorescence and topographic imaging of molecular complexes.

Infusion of α,β-methylene ATP (α,β-meATP) into murine neck muscle facilitates brainstem nociception. Unspecific nitric oxide synthase (NOS) inhibition prevents and reverses this sensitization. It is unclear whether neuronal (nNOS), inducible (iNOS) or endothelial NOS isoenzymes are involved in this α,β-meATP effect. Hypothesized involvement of nNOS isoenzyme was addressed by preceding (0.5, 1, and 2 mg/kg) and subsequent (2 mg/kg) intraperitoneal injection of the nNOS-inhibitor NPLA. iNOS involvement was addressed by subsequent, intraperitoneal administration of the iNOS-inhibitor 1400 W (2 mg/kg). Brainstem nociception was monitored by the jaw-opening reflex elicited via electrical tongue stimulation in 45 anesthetized mice. Preceding NPLA dose-dependently prevented α,β-meATP-induced reflex facilitation. Whereas subsequent inhibition of nNOS showed no effect, iNOS inhibition by 1400 W significantly reversed reflex facilitation. Data provide evidence that nNOS plays a major role in induction and iNOS in maintenance of facilitation in neck muscle nociception. Divergent roles of NOS isoenzymes may promote research on target specific treatment for headache and neck muscle pain., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Rad51 is the central catalyst of homologous recombination in eukaryotes and is thus critical for maintaining genomic integrity. Recent crystal structures of filaments formed by Rad51 and the closely related archeal RadA and eubacterial RecA proteins place the ATPase site at the protomeric interface. To test the relevance of this feature, we mutated conserved residues at this interface and examined their effects on key activities of Rad51: ssDNA-stimulated ATP hydrolysis, DNA binding, polymerization on DNA substrates and catalysis of strand-exchange reactions. Our results show that the interface seen in the crystal structures is very important for nucleoprotein filament formation. H352 and R357 of yeast Rad51 are essential for assembling the catalytically competent form of the enzyme on DNA substrates and coordinating its activities. However, contrary to some previous suggestions, neither of these residues is critical for ATP hydrolysis.

The DNA strand-exchange reactions defining homologous recombination involve transient, nonuniform allosteric interactions between recombinase proteins and their DNA substrates. To study these mechanistic aspects of homologous recombination, we produced functional fluorescent human RAD51 recombinase and visualized recombinase interactions with single DNA molecules in both static and dynamic conditions. We observe that RAD51 nucleates filament formation at multiple sites on double-stranded DNA. This avid nucleation results in multiple RAD51 filament segments along a DNA molecule. Analysis of fluorescent filament patch size and filament kinks from scanning force microscopy (SFM) images indicate nucleation occurs minimally once every 500 bp. Filament segments did not rearrange along DNA, indicating tight association of the ATP-bound protein. The kinetics of filament disassembly was defined by activating ATP hydrolysis and following individual filaments in real time.

The purpose of this study is to examine the long term psychological effects of war stress regarded as traumatic experience. The subjects are Serbian internally displaced people (IDP) of adolescent population from Kosovo. It is a very big concern whether the adolescents would overcome the social and psychological difficulties caused by the war stress in order to reconstruct the better society. The result came out that the long-term effects still exist in PTSD, depression and hopelessness, which affects self-esteem and the attitude in purpose in life that are important factors for personality development. This paper also examines the difference between IDPs with war stress and the adolescent sufferers of the big earthquake in Japan.

Cluster headache is characterized by attacks of severe periorbital pain. Repetitive burst activity in afferent fibers may induce plastic alterations in somatosensory synaptic processing as a prerequisite for recurring and chronic pain. This psychophysical study addressed hypothesized dysfunctions in craniofacial somatosensory processing in cluster headache disease. Thermal and mechanical sensory functions in the periorbital region were assessed by quantitative sensory testing (QST) in 25 cluster headache patients and 60 healthy volunteers. Perception of warmth (p<0.01), cold (p<0.000001), and pressure pain (p<0.05) was reduced on the cluster side as compared with the contralateral asymptomatic side. In contrast to healthy volunteers, warm detection threshold (WDT) and thermal sensory limen (TSL) on one side did not positively correlate with the other side. WDT and TSL negatively correlated with the elapsed time since last attack. All patients showed QST abnormalities on the headache side in comparison to healthy controls. Loss of sensory functions strongly preponderated gain. Several lines of evidence indicate a pivotal role of the hypothalamus in cluster headache pathophysiology. The impairment of warm and cold perception in patients may be based upon a dysfunction of the hypothalamus which is strongly involved in thermosensory control.

The Ndc80 complex is a constituent of the outer plate of the kinetochore and plays a critical role in establishing the stable kinetochore-microtubule interactions required for chromosome segregation in mitosis. The Ndc80 complex is evolutionarily conserved and contains the four subunits Spc24, Spc25, Nuf2, and Ndc80 (whose human homologue is called Hec1). All four subunits are predicted to contain globular domains and extensive coiled coil regions. To gain an insight into the organization of the human Ndc80 complex, we reconstituted it using recombinant methods. The hydrodynamic properties of the recombinant Ndc80 complex are identical to those of the endogenous HeLa cell complex and are consistent with a 1:1:1:1 stoichiometry of the four subunits and a very elongated shape. Two tight Hec1-Nuf2 and Spc24-Spc25 subcomplexes, each stabilized by a parallel heterodimeric coiled coil, maintain this organization. These subcomplexes tetramerize via an interaction of the C- and N-terminal portions of the Hec1-Nuf2 and Spc24-Spc25 coiled coils, respectively. The recombinant complex displays normal kinetochore localization upon injection in HeLa cells and is therefore a faithful copy of the endogenous Ndc80 complex.

Exchange of DNA strands between homologous DNA molecules via recombination ensures accurate genome duplication and preservation of genome integrity. Biochemical studies have provided insights into the molecular mechanisms by which homologous recombination proteins perform these essential tasks. More recent cell biological experiments are addressing the behavior of homologous recombination proteins in cells. The challenge ahead is to uncover the relationship between the individual biochemical activities of homologous recombination proteins and their coordinated action in the context of the living cell.

Proper maintenance and duplication of the genome require accurate recombination between homologous DNA molecules. In eukaryotic cells, the Rad51 protein mediates pairing between homologous DNA molecules. This reaction is assisted by the Rad54 protein. To gain insight into how Rad54 functions, we studied the interaction of the human Rad54 (hRad54) protein with double-stranded DNA. We have recently shown that binding of hRad54 to DNA induces a change in DNA topology. To determine whether this change was caused by a protein-constrained change in twist, a protein-constrained change in writhe, or the introduction of unconstrained plectonemic supercoils, we investigated the hRad54--DNA complex by scanning force microscopy. The architecture of the observed complexes suggests that movement of the hRad54 protein complex along the DNA helix generates unconstrained plectonemic supercoils. We discuss how hRad54-induced superhelical stress in the target DNA may function to facilitate homologous DNA pairing by the hRad51 protein directly. In addition, the induction of supercoiling by hRad54 could stimulate recombination indirectly by displacing histones and/or other proteins packaging the DNA into chromatin. This function of DNA translocating motors might be of general importance in chromatin metabolism.

Three Drosophila embryonic deoxyribonucleases, designated den1, den2 and den3, are identified in nuclear extracts separated by glycerol density gradient centrifugation. Den1, removes short products from the 5'-ends of single-stranded DNA or double-stranded DNA with either blunt or 5'-recessed termini. Den2 is inactive with single-stranded DNA and acts as 3'-exonuclease with double-stranded DNA possessing either blunt or 3'-recessed termini. Den3 preferentially uses partial duplex DNA containing single-stranded gap and it catalyzes hydrolysis, in 3'-5' direction, of one of the shorter strands that flank the gap. Nucleolytic activities of den1, den2 and den3 are inhibited with ATP.

The heart phospholipid content and fatty acid composition were examined in adult rats after four weeks of feeding lipid-supplemented diets (20 g % w/w) containing sunflower oil-lard (1:1) mixture (SL group) or margarine (M group). Our results showed a decreased cardiolipin content and distribution in both experimental groups and an increased lysophosphatidylcholine and phosphatidylcholine content and distribution in the SL group with a tendency to lower phosphatidylcholine/phospatidylethanolamine ratio in both experimental groups. In the SL group, the content of saturated fatty acids was higher and that of monounsaturated fatty acids was lower than in the control group. The M group showed inverse results. The content of saturated fatty acids was lower and that of monounsaturated was higher than in the control group. Polyunsaturated n-6 fatty acids were decreased in both experimental groups and n-3 fatty acids were increased in the M group. Feeding lipid-supplemented diets reduced n-6/n-3 and 20:4/22:6 ratios in the M group. The polyunsaturated/saturated fatty acid ratio was lower in the SL and higher in indicating the M group than in the control group. Our results are in agreement with the other reports indicating that the heart is sensitive to diet-induced lipid alterations.

Nuclear extracts prepared from Drosophila preblastoderm embryos contain a nucleoside 5'-triphosphate-dependent helicase that unwinds circular or linear partial duplex DNA substrates and moves along the DNA in 3' to 5' direction. The helicase reaction is supported with both nucleoside and deoxynucleoside 5'-triphosphates and requires divalent cations. Optimum activity in vitro is achieved with dATP or ATP and with MgCl2. In glycerol density gradients, embryonic enzyme migrates as a single peak of around 90kDa and it is the most prominent DNA-unwinding activity detectable in nuclear extracts prepared from embryos 0-2 hours after egg laying. In embryos collected 2-4 hours after egg laying this DNA unwinding activity increases and then gradually decreases in embryos collected 4-8 and 8-16 hours after egg laying.