Infant B cell memory differentiation and early gut bacterial colonization

Germ-free animal models have demonstrated that commensal bacterial colonization of the intestine induces B cell differentiation and activation. Whether colonization with particular bacterial species or groups is associated with B cell development during early childhood is not known. In a prospective newborn/infant cohort including 65 Swedish children, we examined the numbers and proportions of CD20(+), CD5(+), and CD27(+) B cells in blood samples obtained at several time points during the first 3 y of life using flow cytometry. Fecal samples were collected and cultured quantitatively for major facultative and anaerobic bacteria at 1, 2, 4, and 8 wk of life. We found that the numbers of CD20(+) B cells and CD5(+)CD20(+) B cells reached their highest levels at 4 mo, whereas CD20(+) B cells expressing the memory marker CD27 were most numerous at 18 and 36 mo of age. Using multivariate analysis, we show that early colonization with Escherichia coli and bifidobacteria were associated with higher numbers of CD20(+) B cells that expressed the memory marker CD27 at 4 and 18 mo of age. In contrast, we were unable to demonstrate any relation between bacterial colonization pattern and numbers of CD20(+) or CD5(+)CD20(+) B cells. These results suggest that the intestinal bacterial colonization pattern may affect the B cell maturation also in humans, and that an early gut microbiota including E. coli and bifidobacteria might promote this maturation.     

Investigation of factors influencing biogas production in a large-scale thermophilic municipal biogas plant

A continuously operated, thermophilic, municipal biogas plant was observed over 26 months (sampling twice per month) in regard to a number of physicochemical parameters and the biogas production. Biogas yields were put in correlation to parameters such as the volatile fatty acid concentration, the pH and the ammonium concentration. When the residing microbiota was classified via analysis of the 16S rRNA genes, most bacterial sequences matched with unidentified or uncultured bacteria from similar habitats. Of the archaeal sequences, 78.4% were identified as belonging to the genus Methanoculleus, which has not previously been reported for biogas plants, but is known to efficiently use H₂ and CO₂ produced by the degradation of fatty acids by syntrophic microorganisms. In order to further investigate the influence of varied amounts of ammonia (2–8 g/L) and volatile fatty acids on biogas production and composition (methane/CO₂), laboratory scale satellite experiments were performed in parallel to the technical plant. Finally, ammonia stripping of the process water of the technical plant was accomplished, a measure through which the ammonia entering the biogas reactor via the mash could be nearly halved, which increased the energy output of the biogas plant by almost 20%.     

In vitro effect of the potent poly(ADP-ribose) polymerase (PARP) inhibitor INO-1001 alone and in combination with aspirin, eptifibatide, tirofiban, enoxaparin or alteplase on haemostatic parameters

It has been shown that PARP inhibition is protective in several models of ischemia-reperfusion injury including cardiac, cerebral and renal ones. Due to their ability to reduce myocardial necrosis and to improve myocardial function PARP inhibitors emerged as candidates for treating various cardiovascular diseases including acute myocardial ischemia. Since the pathophysiology of acute ischemic cardiac diseases involves haemostatic impairment and the therapeutic regimen includes antithrombotic drugs, we investigated the effect of the potent poly(ADP-ribose) polymerase (PARP) inhibitor INO-1001 alone and in combination with platelet aggregation inhibitors (aspirin, eptifibatide and tirofiban), unfractionated heparin, low molecular weight heparin (enoxaparin) or the recombinant fibrinolytic drug (alteplase), on various haemostatic parameters in vitro. ADP- and epinephrine-induced platelet aggregation was evaluated by optical aggregometry in the presence or absence of different concentrations of INO-1001, in combination with aspirin, tirofiban, eptifibatide or saline on ten healthy volunteers' platelet rich plasma (PRP). Activated partial thromboplastin time, Anti-Xa activity and euglobulin lysis time were determined in the presence or absence of different concentrations of INO-1001, in combination with sodium heparin, enoxaparin or alteplase, respectively. INO-1001, on its own does not affect the measured platelet, and haemostatic functions, i.e. does not reduce the respective anti-platelet, anti-coagulant and thrombolytic activity of therapeutically relevant concentrations of aspirin, tirofiban, eptifibatide, enoxaparin and alteplase in vitro. INO-1001 enhanced the effects of heparins above therapeutic ranges; the magnitude of this effect was negligible. Consequently, the PARP inhibitor INO-1001 can be safely applied together with the drugs tested.     

Heterogeneity of non-serotypable Campylobacter jejuni isolates

Several phenotypic and genotypic methods are currently used to identify subtypes of Campylobacter jejuni isolates. Of the phenotypic methods one, i.e. serotyping based on the heat stable antigen, is often hindered by the relatively large number of un-typable (NT) strains. Little is known, however, about the heterogeneity of the group formed by these NT strains. Therefore we serotyped 92 Hungarian, non-outbreak C. jejuni isolates and subjected the 28 NT strains to molecular analysis using PCR-RFLP of the flaA gene and to pulsed-field gel electrophoresis. With both methods the NT strains were classified into several molecular types (17 and 25, respectively), while the number of subgroups based on the results of the two techniques combined was twenty-six. These results indicate that the NT group of strains is extremely heterogeneous in Hungary, and the epidemiological connection between two NT isolates cannot be established or excluded without the use of molecular typing techniques.     

Plasmodium vivax dihydrofolate reductase as a target of sulpha drugs

Sulpha drugs act as competitive inhibitors of p-amino benzoic acid, an intermediate in the de novo folate pathway. Dihydropteroate synthase condenses sulpha drugs into sulpha-dihydropteroate (sulpha-DHP), which competes with dihydrofolate, the dihydrofolate reductase (DHFR) substrate. This designates DHFR as a possible target of sulpha-DHP. We suggest here that Plasmodium vivax DHFR is indeed the in vivo target of sulpha drugs. The wild-type DHFR expressed in Saccharomyces cerevisiae leads to cell growth inhibition, while sensitivity to the drug is exacerbated in the mutants. Contrary to what is observed with sulphanilamide, methotrexate is less effective on P. vivax-DHFR mutants than on wild-type mutant.     

O-linked N-acetylglucosamine proteomics of postsynaptic density preparations using lectin weak affinity chromatography and mass spectrometry

O-GlcNAc is a widespread dynamic carbohydrate modification of cytosolic and nuclear proteins with features analogous to phosphorylation. O-GlcNAc acts critically in many cellular processes, including signal transduction, protein degradation, and regulation of gene expression. However, the study of its specific regulatory functions has been limited by difficulties in mapping sites of O-GlcNAc modification. We report methods for direct enrichment and identification of in vivo O-GlcNAc-modified peptides through lectin weak affinity chromatography (LWAC) and mass spectrometry. The effectiveness of this strategy on complex peptide mixtures was demonstrated through enrichment of 145 unique O-GlcNAc-modified peptides from a postsynaptic density preparation. 65 of these O-GlcNAc-modified peptides were sequenced and belonged to proteins with diverse functions in synaptic transmission. Beta-elimination/Michael addition, MS(3) on O-GlcNAc neutral loss ions, and electron capture dissociation were shown to facilitate analysis of O-GlcNAc-modified peptides/sites from lectin weak affinity chromatography enriched postsynaptic density samples. Bassoon and Piccolo, proteins critical to synapse assembly and vesicle docking, were extensively modified by O-GlcNAc. In some cases, O-GlcNAc was mapped to peptides previously identified as phosphorylated, indicating potential interplay between these modifications. Shared substrate amino acid context was apparent in subsets of O-GlcNAc-modified peptides, including "PVST" and a novel "TTA" motif (two hydroxyl-containing amino acids adjacent to an alanine). The results suggest specific roles for O-GlcNAc modification in synaptic transmission, establish a basis for site-specific regulatory studies, and provide methods that will facilitate O-GlcNAc proteome analysis across a wide variety of cells and tissues.     

14-3-3 activation of DNA binding of p53 by enhancing its association into tetramers

Activation of the tumour suppressor p53 on DNA damage involves post-translational modification by phosphorylation and acetylation. Phosphorylation of certain residues is critical for p53 stabilization and plays an important role in DNA-binding activity. The 14-3-3 family of proteins activates the DNA-binding affinity of p53 upon stress by binding to a site in its intrinsically disordered C-terminal domain containing a phosphorylated serine at 378. We have screened various p53 C-terminal phosphorylated peptides for binding to two different isoforms of 14-3-3, epsilon and gamma. We found that phosphorylation at either S366 or T387 caused even tighter binding to 14-3-3. We made by semi-synthesis a tetrameric construct comprised of the tetramerization plus C-terminal domains of p53 that was phosphorylated on S366, S378 and T387. It bound 10 times tighter than did the monomeric counterpart to dimeric 14-3-3. We showed indirectly from binding curves and directly from fluorescence-detection analytical ultracentrifugation that 14-3-3 enhanced the binding of sequence-specific DNA to p53 by causing p53 dimers to form tetramers at lower concentrations. If the in vitro data extrapolate to in vivo, then it is an attractive hypothesis that p53 activity may be subject to control by accessory proteins lowering its tetramer-dimer dissociation constant from its normal value of 120-150 nM.     

Cyclic phosphatidic acid elicits neurotrophin-like actions in embryonic hippocampal neurons

Cyclic phosphatidic acid (cPA; 1-acyl-sn-glycerol-2,3-cyclic phosphate) is an analog of the growth factor-like phospholipid mediator lysophosphatidic acid (LPA). As brain tissue is the richest source of cPA we tested its effects on hippocampal neurons from day 16/17 embryonic rat cultured in a serum-free medium. Nanomolar concentrations of cPA elicited a neurotrophic effect and promoted neurite outgrowth that exceeded that of 50 ng/mL nerve growth factor (NGF). Pertussis toxin, the LPA1/LPA3 receptor-selective antagonist dioctylglycerol pyrophosphate, the myristoylated inhibitory pseudosubstrate peptide of protein kinase A (PKI), Wortmannin and PD98059 abolished the neurite-promoting effect. cPA elicited a sustained activation of extracellular signal-related kinases (ERK) 1/2 and Akt. Clostridium difficile toxin B, an inhibitor of the Rho family of GTPases, reduced cPA-induced enhancement of neurite outgrowth. In B5P cells, a clonal cell line of PC12 cells overexpressing tyrosine kinase NGF receptor (TrkA), cPA elicited transphosphorylation of TrkA. cPA-elicited ERK activation was blocked by K252a and PKI. These results suggest that cPA mimics the effects of, and activates signaling pathways similar to, the neurotrophin NGF in cultured embryonic hippocampal neurons and B5P cells.     

CD133: Enhancement of Bone Healing by Local Transplantation of Peripheral Blood Cells in a Biologically Delayed Rat Osteotomy Model

Sufficient angiogenesis is crucial during tissue regeneration and therefore also pivotal in bone defect healing. Recently, peripheral blood derived progenitor cells have been identified to have in addition to their angiogenic potential also osteogenic characteristics, leading to the hypothesis that bone regeneration could be stimulated by local administration of these cells. The aim of this study was to evaluate the angiogenic potential of locally administered progenitor cells to improve bone defect healing. Cells were separated from the peripheral blood of donor animals using the markers CD34 and CD133. Results of the in vitro experiments confirmed high angiogenic potential in the CD133(+) cell group. CD34(+) and CD133(+) cells were tested in an in vivo rat femoral defect model of delayed healing for their positive effect on the healing outcome. An increased callus formation and higher bone mineral density of callus tissue was found after the CD133(+) cell treatment compared to the group treated with CD34(+) cells and the control group without cells. Histological findings confirmed an increase in vessel formation and mineralization at day 42 in the osteotomy gap after CD133(+) cell transplantation. The higher angiogenic potential of CD133(+) cells from the in vitro experients therefore correlates with the in vivo data. This study demonstrates the suitability of angiogenic precursors to further bone healing and gives an indication that peripheral blood is a promising source for progenitor cells circumventing the problems associated with bone marrow extraction.