Protein phosphorylation studies of cerebral spinal fluid for potential biomarker development

Subarachnoid hemorrhage (SAH) followed by cerebral vasospasm (CV) leads to severe debilitation or death of an estimated one million people worldwide every year. A biomarker that would predict the onset of CV after a SAH would be useful in informing treatment protocols, but has yet to be found. The focus of this study is to explore differences in protein phosphorylation in cerebral spinal fluid (CSF) among healthy patients, SAH patients and SAH-CV patients. A significant difference in phosphorylation among the three sample types could be an important step towards the discovery of a diagnostic marker. The identification and validation of phosphorylated protein differences for study is manifested in the nature of signaling involved in the pathological events seen post SAH. Capillary liquid chromatography (cap-LC) coupled to inductively coupled plasma mass spectrometry (ICPMS) and nano-liquid chromatography-CHIP/ion trap mass spectrometry (nanoLC-CHIP/ITMS) are used to identify and measure protein phosphorylation changes in the CSF of the aforementioned groups. ICPMS represents a suitable method for screening ultra-trace phosphorus levels at the natural isotope, (31)P, while nano-LC-CHIP/ITMS is used to identify phosphoproteins by searching appropriate protein databases.     

Field resistance of codling moth against Cydia pomonella granulovirus (CpGV) is autosomal and incompletely dominant inherited

The current appearance of local codling moth populations with resistance to Cydia pomonella granulovirus (CpGV) is an impediment to continuous CpGV application. Therefore, crossing experiments have been performed in order to gain information about the inheritance of resistance. Evidence is presented that the observed field resistance is stably inherited even under non-selective conditions in the laboratory. Offspring of reciprocal F(1) crosses between a susceptible ('S') and a resistant ('R') strain and backcrosses between F(1) and S were bioassayed at different CpGV concentrations. The resistant strain showed 100 times lower susceptibility in 7-day bioassays. The responses of the reciprocal crosses (male S x female R and female S x male R) did not differ significantly, indicating that resistance is autosomally inherited. The median lethal concentration for the F(1) progeny was intermediate between those of its parental strains. Mortality data obtained from the backcrosses suggested that inheritance of resistance is due to a non-additive, polygenic trait.     

Structure of the Drosophila nucleosome core particle highlights evolutionary constraints on the H2A-H2B histone dimer

We determined the 2.45 A crystal structure of the nucleosome core particle from Drosophila melanogaster and compared it to that of Xenopus laevis bound to the identical 147 base-pair DNA fragment derived from human alpha-satellite DNA. Differences between the two structures primarily reflect 16 amino acid substitutions between species, 15 of which are in histones H2A and H2B. Four of these involve histone tail residues, resulting in subtly altered protein-DNA interactions that exemplify the structural plasticity of these tails. Of the 12 substitutions occurring within the histone core regions, five involve small, solvent-exposed residues not involved in intraparticle interactions. The remaining seven involve buried hydrophobic residues, and appear to have coevolved so as to preserve the volume of side chains within the H2A hydrophobic core and H2A-H2B dimer interface. Thus, apart from variations in the histone tails, amino acid substitutions that differentiate Drosophila from Xenopus histones occur in mutually compensatory combinations. This highlights the tight evolutionary constraints exerted on histones since the vertebrate and invertebrate lineages diverged.     

A thermodynamic model for Nap1-histone interactions

The yeast nucleosome assembly protein 1 (yNap1) plays a role in chromatin maintenance by facilitating histone exchange as well as nucleosome assembly and disassembly. It has been suggested that yNap1 carries out these functions by regulating the concentration of free histones. Therefore, a quantitative understanding of yNap1-histone interactions also provides information on the thermodynamics of chromatin. We have developed quantitative methods to study the affinity of yNap1 for histones. We show that yNap1 binds H2A/H2B and H3/H4 histone complexes with low nm affinity, and that each yNap1 dimer binds two histone fold dimers. The yNap1 tails contribute synergistically to histone binding while the histone tails have a slightly repressive effect on binding. The (H3/H4)(2) tetramer binds DNA with higher affinity than it binds yNap1.     

Elevated numbers of Fc gamma RIIIA+ (CD16+) effector CD8 T cells with NK cell-like function in chronic hepatitis C virus infection

CTL are crucial in the defense against viral infections. In the course of investigating peripheral blood and intrahepatic CD8 T cells in patients with chronic hepatitis C virus (HCV) infection, we observed a significant population of CD8 T cells expressing the FcgammaRIIIA (CD16) receptor. This observation led us to characterize these cells with respect to their phenotype and function in a cohort of patients with chronic HCV infection as well as in healthy blood donors. On average, 10% of peripheral blood CD8 T cells from HCV-infected patients expressed CD16 compared with only a few percent in healthy donors. CD16(+) CD8 T cells displayed a late-stage effector phenotype with high levels of perforin. These cells exhibited a restricted TCR profile suggesting underlying clonal expansion. Stimulation of CD16 on CD8 T cells evoked a vigorous response similar to that of CD16 stimulation in NK cells. Our data suggest that CD8 T cells, during chronic HCV infection in humans, continue to differentiate beyond defined stages of terminal effector cells, acquiring CD16 and NK cell-like functional properties.     

The linker region of macroH2A promotes self-association of nucleosomal arrays

MacroH2A is a histone variant found in higher eukaryotes localized at the inactive X chromosome and is known to maintain heterochromatic regions in the genome. MacroH2A consists of a conserved histone domain and a macro domain connected by a linker region. To understand the contributions of the three domains to chromatin condensation, we incorporated various constructs of macroH2A into defined nucleosomal arrays and analyzed their impact on in vitro chromatin compaction. The folding and oligomerization properties of arrays containing full-length macroH2A (macroH2A(FL)), macroH2A(1-161) (encompassing the histone domain and linker region), and macroH2A(1-122) (histone domain only) were compared with major-type H2A arrays. Analytical ultracentrifugation and atomic force microscope imaging indicate that macroH2A(1-161)-containing arrays favor condensation under conditions where major-type arrays are nearly fully extended. In contrast, arrays with macroH2A(FL) exhibit behavior similar to that of major-type arrays. This suggests that the linker region of macroH2A facilitates array condensation and that this behavior is inhibited by the macro domain. Furthermore, chimeric major-type H2A arrays containing the macroH2A linker domain (H2A(ML)) exhibited the same condensation properties as macroH2A(1-161) arrays, thus emphasizing the intriguing behavior of the macroH2A linker region.     

DNA as a Phosphate Storage Polymer and the Alternative Advantages of Polyploidy for Growth or Survival

Haloferax volcanii uses extracellular DNA as a source for carbon, nitrogen, and phosphorous. However, it can also grow to a limited extend in the absence of added phosphorous, indicating that it contains an intracellular phosphate storage molecule. As Hfx. volcanii is polyploid, it was investigated whether DNA might be used as storage polymer, in addition to its role as genetic material. It could be verified that during phosphate starvation cells multiply by distributing as well as by degrading their chromosomes. In contrast, the number of ribosomes stayed constant, revealing that ribosomes are distributed to descendant cells, but not degraded. These results suggest that the phosphate of phosphate-containing biomolecules (other than DNA and RNA) originates from that stored in DNA, not in rRNA. Adding phosphate to chromosome depleted cells rapidly restores polyploidy. Quantification of desiccation survival of cells with different ploidy levels showed that under phosphate starvation Hfx. volcanii diminishes genetic advantages of polyploidy in favor of cell multiplication. The consequences of the usage of genomic DNA as phosphate storage polymer are discussed as well as the hypothesis that DNA might have initially evolved in evolution as a storage polymer, and the various genetic benefits evolved later.