Adenosine inhibits the release of interleukin-1beta in activated human peripheral mononuclear cells

The effects of adenosine and subtype-specific activators of adenosine receptors (A1, A2A, A2B and A3) were studied on the release of interleukin-1beta (IL-1beta) from peripheral mononuclear cells, monocytes and lymphocytes. In the cells activated by the protein kinase C specific phorbol ester (phorbol 12-myristate 13-acetate) and Ca(2+) ionophore (A23187) both adenosine and the subtype-specific receptor agonists, CPA (A1), CGS 21680 (A2A) and IB-MECA (A3) induced a concentration-dependent inhibition of IL-1beta release. The rank order of potency in the inhibition of IL-1beta release was CPA=CGS 21680>IB-MECA>adenosine>NECA (in the presence of A1, A2A and A3 receptor inhibitors). The inhibitory actions of CPA, CGS 21680 or IB-MECA were significantly reduced in the presence of DPCPX, ZM 243185 or MRS 1191 as subtype-specific antagonists on A1, A2A and A3 adenosine receptors, respectively. It can be concluded that adenosine inhibits the release of IL-1beta from the activated human peripheral mononuclear cells. In this process A1, A2A and A3 receptors are involved.     

Optimal clustering for detecting near-native conformations in protein docking

Clustering is one of the most powerful tools in computational biology. The conventional wisdom is that events that occur in clusters are probably not random. In protein docking, the underlying principle is that clustering occurs because long-range electrostatic and/or desolvation forces steer the proteins to a low free-energy attractor at the binding region. Something similar occurs in the docking of small molecules, although in this case shorter-range van der Waals forces play a more critical role. Based on the above, we have developed two different clustering strategies to predict docked conformations based on the clustering properties of a uniform sampling of low free-energy protein-protein and protein-small molecule complexes. We report on significant improvements in the automated prediction and discrimination of docked conformations by using the cluster size and consensus as a ranking criterion. We show that the success of clustering depends on identifying the appropriate clustering radius of the system. The clustering radius for protein-protein complexes is consistent with the range of the electrostatics and desolvation free energies (i.e., between 4 and 9 Angstroms); for protein-small molecule docking, the radius is set by van der Waals interactions (i.e., at approximately 2 Angstroms). Without any a priori information, a simple analysis of the histogram of distance separations between the set of docked conformations can evaluate the clustering properties of the data set. Clustering is observed when the histogram is bimodal. Data clustering is optimal if one chooses the clustering radius to be the minimum after the first peak of the bimodal distribution. We show that using this optimal radius further improves the discrimination of near-native complex structures.     

Resonance Raman spectroscopy of xanthophylls in pigment mutant thylakoid membranes of pea

Low-temperature resonance Raman spectroscopy was used to study the changes in the molecular structure and configuration of the major xanthophylls in thylakoid membranes isolated from mutants of pea with modified pigment content and altered structural organization of their pigment-protein complexes. The Raman spectra contained four known groups of bands, nu(1)-nu(4), which could be assigned to originate mainly from the long wavelength absorbing lutein and neoxanthin upon 514.5 nm and at 488 nm excitations, respectively. The overall configuration of these bound xanthophyll molecules in the mutants appeared to be similar to the wild type, and the configuration in the wild type was almost identical with that in the isolated main chlorophyll a/b light harvesting protein complex of photosystem II (LHCII). Significant differences were found mainly in the region of nu(4) (around 960 cm(-1)), which suggest that the macroorganization of PS II-LHCII supercomplexes and/or of the LHCII-only domains are modified in the mutants compared to the wild type.     

Phylogeography of Y-chromosome haplogroup I reveals distinct domains of prehistoric gene flow in europe

To investigate which aspects of contemporary human Y-chromosome variation in Europe are characteristic of primary colonization, late-glacial expansions from refuge areas, Neolithic dispersals, or more recent events of gene flow, we have analyzed, in detail, haplogroup I (Hg I), the only major clade of the Y phylogeny that is widespread over Europe but virtually absent elsewhere. The analysis of 1,104 Hg I Y chromosomes, which were identified in the survey of 7,574 males from 60 population samples, revealed several subclades with distinct geographic distributions. Subclade I1a accounts for most of Hg I in Scandinavia, with a rapidly decreasing frequency toward both the East European Plain and the Atlantic fringe, but microsatellite diversity reveals that France could be the source region of the early spread of both I1a and the less common I1c. Also, I1b*, which extends from the eastern Adriatic to eastern Europe and declines noticeably toward the southern Balkans and abruptly toward the periphery of northern Italy, probably diffused after the Last Glacial Maximum from a homeland in eastern Europe or the Balkans. In contrast, I1b2 most likely arose in southern France/Iberia. Similarly to the other subclades, it underwent a postglacial expansion and marked the human colonization of Sardinia ~9,000 years ago.     

Initiation of immune responses in brain is promoted by local dendritic cells

The contribution of dendritic cells (DCs) to initiating T cell-mediated immune response in and T cell homing into the CNS has not yet been clarified. In this study we show by confocal microscopy and flow cytometry that cells expressing CD11c, CD205, and MHC class II molecules and containing fluorescently labeled, processed Ag accumulate at the site of intracerebral Ag injection. These cells follow a specific pattern upon migrating out of the brain. To track their pathway out of the CNS, we differentiated DCs from bone marrow of GFP-transgenic mice and injected them directly into brains of naive C57BL/6 mice. We demonstrate that DCs migrate from brain to cervical lymph nodes, a process that can be blocked by fixation or pertussis toxin treatment of the DCs. Injection of OVA-loaded DCs into brain initiates a SIINFEKL (a dominant OVA epitope)-specific T cell response in lymph nodes and spleen, as measured by specific tetramer and LFA-1 activation marker staining. Additionally, a fraction of activated SIINFEKL-specific T cells home to the CNS. Specific T cell homing to the CNS, however, cannot be induced by i.v. injection of OVA-loaded DCs alone. These data suggest that brain-emigrant DCs are sufficient to support activated T cells to home to the tissue of DC origination. Thus, initiation of immune reactivity against CNS Ags involves the migration of APCs from nervous tissue to peripheral lymphoid tissues, similarly to that in other organs.     

Biochemical modulation of Ara-C effects by amidox, an inhibitor of ribonucleotide reductase in HL-60 promyelocytic human leukemia cells

Amidox, a new polyhydroxy-substituted benzoic acid derivative, is a potent inhibitor of the enzyme ribonucleotide reductase (RR), which catalyses the de novo synthesis of DNA. RR is considered to be an excellent target for anti cancer chemotherapy. We investigated the biochemical and antineoplastic effects of amidox as a single agent and in combination with Ara-C in human HL-60 promyelocytic leukemia cells. Amidox inhibited the growth of HL-60 cells in a growth inhibition assay with an IC50 of 25 microM. In a soft agar colony forming assay, amidox yielded a 50% inhibition of colony formation at 13 microM. We also investigated the effects of amidox treatment on the formation of deoxynucleosidetriphosphates. Amidox (50 and 75 microM for 24 hours) could significantly decrease intracellular concentrations of dCTP, dATP and dGTP pools, whereas dTTP levels increased. We then tested the combination effects of amidox with Ara-C; this combination yielded additive cytotoxic effects both in growth inhibition and in soft agar colony formation assays. This effect was due to the increased formation of Ara-CTP, the active metabolite of Ara-C, after preincubation with amidox. Preincubation of HL-60 cells with 75 and 100 microM amidox for 24 hours caused an increase in the intracellular Ara-CTP concentrations by 576% and 1143%, respectively. Therefore amidox might offer an additional option for the treatment of leukemia and thus be further investigated in in vivo studies as a single agent and in combination with Ara-C.     

Measuring environmental phenols and chlorinated organic chemicals in breast milk using automated on-line column-switching-high performance liquid chromatography-isotope dilution tandem mass spectrometry

Breast milk is one possible route of exposure to environmental chemicals, including phenols and chlorinated organic chemicals for breast-fed infants. We developed a highly sensitive method of analyzing breast milk for triclocarban (3,4,4'-trichlorocarbanilide) and eight phenolic compounds: bisphenol A (BPA), 4-tert-octylphenol (4-tOP), ortho-phenylphenol (OPP), 2,4-dichlorophenol, 2,5-dichlorophenol, 2,4,5-trichlorophenol, 2,4,6-trichlorophenol, and 2-hydroxy-4-metoxybenzophenone (BP-3). The method includes adding a solution containing a stable isotope of each chemical, enzymatic hydrolysis of the conjugated chemicals in the milk, and on-line solid-phase extraction coupled with high performance liquid chromatography-tandem mass spectrometry. It can also be used to measure the free (unconjugated) species by omitting the enzymatic deconjugation step. The method, validated using pooled breast milk samples, has inter-day coefficient of variations ranging from 4.8 to 18.9% for most analytes, and spiked recoveries generally about 100%. Detection limits for most analytes are below 1 ng/mL in 100 microL of breast milk. We tested the usefulness of the method by measuring concentrations of these nine compounds in 20 breast milk samples. BPA, OPP, and BP-3 were detected in more than 60% of the samples tested. The free species of these compounds appear to be most prevalent in milk.     

Prevalent structural disorder in E. coli and S. cerevisiae proteomes

Intrinsically unstructured proteins, which exist without a well-defined 3D structure, carry out essential functions and occur with high frequency, as predicted for genomes. The generality of this phenomenon, however, is questioned by the uncertainty of what fraction of genomes actually encodes for expressed proteins. Here, we used two independent bioinformatic predictors, PONDR VSL1, and IUPred, to demonstrate that disorder prevails in the recently characterized proteomes and essential proteins of E. coli and S. cerevisiae, at levels exceeding that estimated from the genomes. The S. cerevisiae proteome contains three times as much disorder as that of E. coli, with 50-60% of proteins containing at least one long (>30 residues) disordered segment. This evolutionary advance can be explained by the observation that disorder is much higher in Gene Ontology categories related to regulatory, as opposed to metabolic, functions, and also in categories unique to yeast. Thus, protein disorder is a widespread and functionally important phenomenon, which needs to be characterized in full detail for understanding complex organisms at the molecular level.