Central Nervous System Pathology Progresses Independently of KC and CXCR2 in Globoid-Cell Leukodystrophy

Globoid-cell Leukodystrophy (GLD; Krabbe’s disease) is a rapidly progressing inherited demyelinating disease caused by a deficiency of the lysosomal enzyme Galactosylceramidase (GALC). Deficiency of GALC leads to altered catabolism of galactosylceramide and the cytotoxic lipid, galactosylsphingosine (psychosine). This leads to a rapidly progressive fatal disease with spasticity, cognitive disability and seizures. The murine model of GLD (Twitcher; GALC−/−) lacks the same enzyme and has similar clinical features. The deficiency of GALC leads to oligodendrocyte death, profound neuroinflammation, and the influx of activated macrophages into the CNS. We showed previously that keratinocyte chemoattractant factor (KC) is highly elevated in the CNS of untreated Twitcher mice and significantly decreases after receiving a relatively effective therapy (bone marrow transplantation combined with gene therapy). The action of KC is mediated through the CXCR2 receptor and is a potent chemoattractant for macrophages and microglia. KC is also involved in oligodendrocyte migration and proliferation. Based on the commonalities between the disease presentation and the functions of KC, we hypothesized that KC and/or CXCR2 contribute to the pathogenesis of GLD. Interestingly, the course of the disease is not significantly altered in KC- or CXCR2-deficient Twitcher mice. There is also no alteration in inflammation or demyelination patterns in these mice. Furthermore, transplantation of CXCR2-deficient bone marrow does not alter the progression of the disease as it does in other models of demyelination. This study highlights the role of multiple redundant cytokines and growth factors in the pathogenesis of GLD.     

Breakage-reunion domain of Streptococcus pneumoniae topoisomerase IV: crystal structure of a gram-positive quinolone target

The 2.7 A crystal structure of the 55-kDa N-terminal breakage-reunion domain of topoisomerase (topo) IV subunit A (ParC) from Streptococcus pneumoniae, the first for the quinolone targets from a gram-positive bacterium, has been solved and reveals a 'closed' dimer similar in fold to Escherichia coli DNA gyrase subunit A (GyrA), but distinct from the 'open' gate structure of Escherichia coli ParC. Unlike GyrA whose DNA binding groove is largely positively charged, the DNA binding site of ParC exhibits a distinct pattern of alternating positively and negatively charged regions coincident with the predicted positions of the grooves and phosphate backbone of DNA. Based on the ParC structure, a new induced-fit model for sequence-specific recognition of the gate (G) segment by ParC has been proposed. These features may account for the unique DNA recognition and quinolone targeting properties of pneumococcal type II topoisomerases compared to their gram-negative counterparts.     

Synthesis and structures of mono and binuclear nickel(II) thiolate complexes of a dicompartmental pseudo-macrocycle with N(imine)2S2 and N(oxime)2S2 metal-binding sites

The reaction of [Ni(pftp)] [pftp = N,N-propane-1,3-diyl-(6-formyl-4-methyliminatothiophenolato)] with hydroxylamine hydrochloride in the presence potassium acetate in MeOH resulted in the formation of the complex [Ni(LH₂)] [L = N,N-propane-1,3-diyl-(4-methyl-2-methyliminato-6-methyloxime-thiophenolato)] in good yield. A single crystal X-ray diffraction structural determination showed a mononuclear nickel(II) complex with the new acyclic ligand LH₂ that had been functionalised with two oxime groups containing an empty N(oxime)₂S₂ pocket to which another metal ion could be added. A further reaction of [Ni(LH₂)] with NiCl₂·6H₂O, triethylamine and ammonium hexafluorophosphate in MeOH gave a dark red product that yielded red crystals of [Ni₂(LH)]PF₆·DMF via slow recrystallisation from a DMF/PrⁱOH solvent mixture. A single crystal X-ray diffraction study of these crystals confirmed the presence of a dinuclear nickel(II) complex linked by a dithiolato-bridge. Both nickel(II) ions exhibited square-planar geometry where the metal centres are coordinated in two distinct cis-S₂N(imine)₂ and cis-S₂N(oxime)₂ binding sites provided by the new dicompartmental oxime/thiolate-containing ligand LH.     

Gene expression changes reflect clinical response in a placebo-controlled randomized trial of abatacept in patients with diffuse cutaneous systemic sclerosis

IntroductionSystemic sclerosis is an autoimmune disease characterized by inflammation and fibrosis of the skin and internal organs. We sought to assess the clinical and molecular effects associated with response to intravenous abatacept in patients with diffuse cutaneous systemic.MethodsAdult diffuse cutaneous systemic sclerosis patients were randomized in a 2:1 double-blinded fashion to receive abatacept or placebo over 24 weeks. Primary outcomes were safety and the change in modified Rodnan Skin Score (mRSS) at week 24 compared with baseline. Improvers were defined as patients with a decrease in mRSS of ≥30 % post-treatment compared to baseline. Skin biopsies were obtained for differential gene expression and pathway enrichment analyses and intrinsic gene expression subset assignment.ResultsTen subjects were randomized to abatacept (n = 7) or placebo (n = 3). Disease duration from first non-Raynaud’s symptom was significantly longer (8.8 ± 3.8 years vs. 2.4 ± 1.6 years, p = 0.004) and median mRSS was higher (30 vs. 22, p = 0.05) in the placebo compared to abatacept group. Adverse events were similar in the two groups. Five out of seven patients (71 %) randomized to abatacept and one out of three patients (33 %) randomized to placebo experienced ≥30 % improvement in skin score. Subjects receiving abatacept showed a trend toward improvement in mRSS at week 24 (−8.6 ± 7.5, p = 0.0625) while those in the placebo group did not (−2.3 ± 15, p = 0.75). After adjusting for disease duration, mRSS significantly improved in the abatacept compared with the placebo group (abatacept vs. placebo mRSS decrease estimate −9.8, 95 % confidence interval −16.7 to −3.0, p = 0.0114). In the abatacept group, the patients in the inflammatory intrinsic subset showed a trend toward greater improvement in skin score at 24 weeks compared with the patients in the normal-like intrinsic subset (−13.5 ± 3.1 vs. −4.5 ± 6.4, p = 0.067). Abatacept resulted in decreased CD28 co-stimulatory gene expression in improvers consistent with its mechanism of action. Improvers mapped to the inflammatory intrinsic subset and showed decreased gene expression in inflammatory pathways, while non-improver and placebos showed stable or reverse gene expression over 24 weeks.ConclusionsClinical improvement following abatacept therapy was associated with modulation of inflammatory pathways in skin.

Trial registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00442611","term_id":"NCT00442611"}}NCT00442611. Registered 1 March 2007.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0669-3) contains supplementary material, which is available to authorized users.     

alpha2,6-Sialylation promotes binding of placental protein 14 via its Ca2+-dependent lectin activity: insights into differential effects on CD45RO and CD45RA T cells

Placental protein 14 (PP14; glycodelin) is a pregnancy-associated immunoregulatory protein that is known to inhibit T cells via T-cell receptor desensitization. The recent demonstration of PP14 as lectin has provided insight into how it may mediate its CD45 glycoprotein-dependent T-cell inhibition. In this study, we have investigated PP14's lectin-binding properties in detail. Significantly, PP14 reacts with N-acetyllactosamine (LacNAc) as was also found for members of the galectin family, such as the potent immunoregulatory protein, galectin-1. However, in contrast to galectin-1, PP14's binding is significantly enhanced by alpha2,6-sialylation and also by the presence of cations. This was demonstrated by preferential binding to fetuin as compared with its desialylated variant asialofetuin (ASF) and by using free alpha2,6- versus alpha2,3-sialylated forms of LacNAc in competitive inhibition and direct solid-phase binding assays. Interestingly, from immunological point of view, PP14 also binds differentially to CD45 isoforms known to differ in their degree of sialylation. PP14 preferentially inhibits CD45RA+, as compared with CD45RO+ T cells, and preferentially co-capped this variant CD45 on the T-cell surface. Finally, we demonstrate that PP14 promotes CD45 dimerization and clustering, a phenomenon that may regulate CD45 activity.     

HADDOCK versus HADDOCK: new features and performance of HADDOCK2.0 on the CAPRI targets

Here we present version 2.0 of HADDOCK, which incorporates considerable improvements and new features. HADDOCK is now able to model not only protein-protein complexes but also other kinds of biomolecular complexes and multi-component (N > 2) systems. In the absence of any experimental and/or predicted information to drive the docking, HADDOCK now offers two additional ab initio docking modes based on either random patch definition or center-of-mass restraints. The docking protocol has been considerably improved, supporting among other solvated docking, automatic definition of semi-flexible regions, and inclusion of a desolvation energy term in the scoring scheme. The performance of HADDOCK2.0 is evaluated on the targets of rounds 4-11, run in a semi-automated mode using the original information we used in our CAPRI submissions. This enables a direct assessment of the progress made since the previous versions. Although HADDOCK performed very well in CAPRI (65% and 71% success rates, overall and for unbound targets only, respectively), a substantial improvement was achieved with HADDOCK2.0.     

A TEST OF TWO POSSIBLE MECHANISMS FOR PHOTOTACTIC STEERING IN VOLVOX CARTERI (CHLOROPHYCEAE)

We tested two competing models that could explain how differential flagellar activity leads to phototactic turning in spheroids of Volvox carteri f. weismannia (Powers) Iyengar. In one model, turning results from the flagella of anterior cells in the lighted and shadowed hemispheres beating at different frequencies. In a competing model, turning results from a change in beat direction in these flagella. Both models successfully explain phototactic steering under constant illumination, but they make different predictions when colonies are exposed to abrupt changes in light intensity. If turning is due to control of flagellar beat frequency, both progression and rotation rates will change in the same direction and with similar magnitudes. If spheroid turning is due to a change in flagellar beat direction, a decreased rate of progression will accompany an increased rate of rotation and vice versa. We used video-microscopy to observe the behavior of positively phototactic V. carteri spheroids exposed to 10× step-up and step-down stimuli. After a step-up stimulus, spheroids slow their progression and rotation by equal amounts. No significant changes are reported in these parameters after the reciprocal step-down response. These observations are consistent with the variable flagellar frequency model and inconsistent with the variable flagellar direction model for phototactic turning. Switching the direction of light stimulus by 180° results in reorientation of positively phototactic spheroids. The kinetics of this reorientation did not precisely match the predictions of either model.     

Affinity labeling of spinach phosphoribulokinase subsequent toS-methylation at Cys16

The chloroplast enzyme phosphoribulokinase is reversibly deactivated by oxidation of Cys16 and Cys55 to a disulfide. Although not required for catalysis, Cys16 is an active-site residue positioned at the nucleotide-binding domain (Porter and Hartman, 1988). The hyperreactivity of Cys16 has heretofore limited further active-site characterization by chemical modification. To overcome this limitation, the partially active enzyme,S-methylated at Cys16, has been probed with a potential affinity reagent. Treatment of methylated enzyme with bromoacetylethanolamine phosphate results in essentially complete loss of catalytic activity. Inactivation follows pseudo-first-order kinetics and exhibits a rate saturation with an apparentK _d of 3–4 mM. ATP, but not ribulose 5-phosphate, affords substantial protection. Complete inactivation correlates with incorporation of 1 mol of [¹⁴C]reagent per mole of enzyme subunit. Amino acid analysis of the [¹⁴C]-labeled enzyme demonstrates that only cysteine is modified, and mapping of tryptic digests shows that Cys55 is a major site of alkylation. These results indicate that Cys55 is also located in the ATP-binding domain of the active-site.