Specificity of DNA uptake in genetic transformation of gonococci

Genetic transformation of gonococci to streptomycin resistance was inhibited by homologous DNA or by DNA from related $\text{Neisseriae}$, but not by high concentrations of heterologous DNAs. Gonococci were capable of adsorbing large quantities (up to about 50 μg per 10⁸ cells) of both homologous and heterologous DNA, which could not be eluted by strong shearing forces. Treatment with externally added DNase removed virtually all the heterologous DNA while a small fraction of the homologous DNA, not influenced by the presence of excess heterologous DNA, remained cell-bound in a form resistant to nuclease treatment. Competing homologous DNA suppressed nuclease-resistant binding. These findings suggest that gonococci have two types of DNA binding components at their surface. Competence of gonococci for genetic transformation undergoes a rapid decay if the cells are incubated with homologous (but $\text{not}$ with heterologous) DNA.     

Citrullination of TNF-α by peptidylarginine deiminases reduces its capacity to stimulate the production of inflammatory chemokines

Citrullination, a posttranslational modification (PTM) recently discovered on inflammatory chemokines such as interleukin-8 (IL-8/CXCL8) and interferon-γ-inducible protein-10 (IP-10/CXCL10), seriously influences their biological activity. Citrullination or the deimination of arginine to citrulline is dependent on peptidylarginine deiminases (PADs) and has been linked to autoimmune diseases such as multiple sclerosis (MS) and rheumatoid arthritis (RA). Chemokines are to date the first identified PAD substrates with receptor-mediated biological activity. We investigated whether cytokines that play a crucial role in RA, like interleukin-1β (IL-1β) and tumor necrosis factor-alpha (TNF-α), may be citrullinated by PAD and whether such a PTM influences the biological activity of these cytokines. IL-1β and TNF-α were first incubated with PAD in vitro and the occurrence of citrullination was examined by Edman degradation and a recently developed detection method for citrullinated proteins. Both techniques confirmed that human TNF-α, but not IL-1β, was citrullinated by PAD. Citrullination of TNF-α reduced its potency to stimulate chemokine production in vitro on human primary fibroblasts. Concentrations of the inflammatory chemokines CXCL8, CXCL10 and monocyte chemotactic protein-1 (MCP-1/CCL2) were significantly lower in supernatants of fibroblasts induced with citrullinated TNF-α compared to unmodified TNF-α. However, upon citrullination TNF-α retained its capacity to induce apoptosis/necrosis of mononuclear cells, its binding potency to Infliximab and its ability to recruit neutrophils to the peritoneal cavity of mice.     

Virus evolution reveals an exclusive role for LEDGF/p75 in chromosomal tethering of HIV

Retroviruses by definition insert their viral genome into the host cell chromosome. Although the key player of retroviral integration is viral integrase, a role for cellular cofactors has been proposed. Lentiviral integrases use the cellular protein LEDGF/p75 to tether the preintegration complex to the chromosome, although the existence of alternative host proteins substituting for the function of LEDGF/p75 in integration has been proposed. Truncation mutants of LEDGF/p75 lacking the chromosome attachment site strongly inhibit HIV replication by competition for the interaction with integrase. In an attempt to select HIV strains that can overcome the inhibition, we now have used T-cell lines that stably express a C-terminal fragment of LEDGF/p75. Despite resistance development, the affinity of integrase for LEDGF/p75 is reduced and replication kinetics in human primary T cells is impaired. Detection of the integrase mutations A128T and E170G at key positions in the LEDGF/p75-integrase interface provides in vivo evidence for previously reported crystallographic data. Moreover, the complementary inhibition by LEDGF/p75 knockdown and mutagenesis at the integrase-LEDGF/p75 interface points to the incapability of HIV to circumvent LEDGF/p75 function during proviral integration. Altogether, the data provide a striking example of the power of viral molecular evolution. The results underline the importance of the LEDGF/p75 HIV-1 interplay as target for innovative antiviral therapy. Moreover, the role of LEDGF/p75 in targeting integration will stimulate research on strategies to direct gene therapy vectors into safe landing sites.     

Impact of the Uremic Milieu on the Osteogenic Potential of Mesenchymal Stem Cells

Human mesenchymal stem cells (hMSCs), the precursors of osteoblasts during osteogenesis, play a role in the balance of bone formation and resorption, but their functioning in uremia has not been well defined. To study the effects of the uremic milieu on osteogenic properties, we applied an in vitro assay culturing hMSCs in osteogenic medium supplemented with serum from healthy donors and from uremic patients on hemodialysis. Compared to control, serum from uremic patients induces, in hMSC cultures, a modification of several key regulators of bone remodeling, in particular a reduction of the ratio Receptor Activator of Nuclear factor Kappa B Receptor (RANKL) over osteoprotegerin, indicating an adaptive response of the system to favor osteogenesis over osteoclastosis. However, the levels of osteopontin, osteocalcin, and collagen type I, are increased in cell medium, while BMP-2, and alizarin red staining were decreased, pointing to a reduction of bone formation favoring resorption. Selected uremic toxins, such as p-cresylsulfate, p-cresylglucuronide, parathyroid hormone, indoxyl sulfate, asymmetric dimethylarginine, homocysteine, were able to mimic some of the effects of whole serum from uremic patients. Serum from cinacalcet-treated patients antagonizes these effects. Hydrogen sulfide (H2S) donors as well as hemodialysis treatment are able to induce beneficial effects. In conclusion, bone modifications in uremia are influenced by the capability of the uremic milieu to alter hMSC osteogenic differentiation. Cinacalcet, H2S donors and a hemodialysis session can ameliorate the hampered calcium deposition.     

Biological activity of CXCL8 forms generated by alternative cleavage of the signal peptide or by aminopeptidase-mediated truncation

BackgroundPosttranslational modification of chemokines is one of the mechanisms that regulate leukocyte migration during inflammation. Multiple natural NH₂-terminally truncated forms of the major human neutrophil attractant interleukin-8 or CXCL8 have been identified. Although differential activity was reported for some CXCL8 forms, no biological data are available for others.ConclusionsIn terms of their ability to induce neutrophil recruitment in vivo, the multiple CXCL8 forms may be divided in three groups. The first group includes CXCL8 proteins consisting of 75 to 79 amino acids, cleaved by aminopeptidases, with intermediate activity on neutrophils. The second group, generated through proteolytic cleavage (e.g. by Ser proteases), contains 69 to 72 amino acid forms which are highly potent neutrophil attractants in vivo. A third category is generated through the modification of the arginine in the NH₂-terminal region into citrulline by peptidylarginine deiminases and has weak potency to induce neutrophil extravasation.     

Posttranslational Modification of the NH2-terminal Region of CXCL5 by Proteases or Peptidylarginine Deiminases (PAD) Differently Affects Its Biological Activity

Posttranslational modifications, e.g. proteolysis, glycosylation, and citrullination regulate chemokine function, affecting leukocyte migration during inflammatory responses. Here, modification of CXCL5/epithelial cell-derived neutrophil-activating protein-78 (ENA-78) by proteases or peptidylarginine deiminases (PAD) was evaluated. Slow CXCL5(1–78) processing by the myeloid cell marker aminopeptidase N/CD13 into CXCL5(2–78) hardly affected its in vitro activity, but slowed down the activation of CXCL5 by the neutrophil protease cathepsin G. PAD, an enzyme with a potentially important function in autoimmune diseases, site-specifically deiminated Arg⁹ in CXCL5 to citrulline, generating [Cit⁹]CXCL5(1–78). Compared with CXCL5(1–78), [Cit⁹]CXCL5(1–78) less efficiently induced intracellular calcium signaling, phosphorylation of extracellular signal-regulated kinase, internalization of CXCR2, and in vitro neutrophil chemotaxis. In contrast, conversion of CXCL5 into the previously reported natural isoform CXCL5(8–78) provided at least 3-fold enhanced biological activity in these tests. Citrullination, but not NH₂-terminal truncation, reduced the capacity of CXCL5 to up-regulate the expression of the integrin α-chain CD11b on neutrophils. Truncation nor citrullination significantly affected the ability of CXCL5 to up-regulate CD11a expression or shedding of CD62L. In line with the in vitro results, CXCL5(8–78) and CXCL5(9–78) induced a more pronounced neutrophil influx in vivo compared with CXCL5(1–78). Administration of 300 pmol of either CXCL5(1–78) or [Cit⁹]CXCL5(1–78) failed to attract neutrophils to the peritoneal cavity. Citrullination of the more potent CXCL5(9–78) lowers its chemotactic potency in vivo and confirms the tempering effect of citrullination in vitro. The highly divergent effects of modifications of CXCL5 on neutrophil influx underline the potential importance of tissue-specific interactions between chemokines and PAD or proteases.     

Genome-wide promoter methylation analysis in neuroblastoma identifies prognostic methylation biomarkers

Background

Accurate outcome prediction in neuroblastoma, which is necessary to enable the optimal choice of risk-related therapy, remains a challenge. To improve neuroblastoma patient stratification, this study aimed to identify prognostic tumor DNA methylation biomarkers.

Results

To identify genes silenced by promoter methylation, we first applied two independent genome-wide methylation screening methodologies to eight neuroblastoma cell lines. Specifically, we used re-expression profiling upon 5-aza-2''-deoxycytidine (DAC) treatment and massively parallel sequencing after capturing with a methyl-CpG-binding domain (MBD-seq). Putative methylation markers were selected from DAC-upregulated genes through a literature search and an upfront methylation-specific PCR on 20 primary neuroblastoma tumors, as well as through MBD- seq in combination with publicly available neuroblastoma tumor gene expression data. This yielded 43 candidate biomarkers that were subsequently tested by high-throughput methylation-specific PCR on an independent cohort of 89 primary neuroblastoma tumors that had been selected for risk classification and survival. Based on this analysis, methylation of KRT19, FAS, PRPH, CNR1, QPCT, HIST1H3C, ACSS3 and GRB10 was found to be associated with at least one of the classical risk factors, namely age, stage or MYCN status. Importantly, HIST1H3C and GNAS methylation was associated with overall and/or event-free survival.

Conclusions

This study combines two genome-wide methylation discovery methodologies and is the most extensive validation study in neuroblastoma performed thus far. We identified several novel prognostic DNA methylation markers and provide a basis for the development of a DNA methylation-based prognostic classifier in neuroblastoma.     

Cell allocation and chromosomal complement of parthenogenetic and IVF bovine embryos

Considerable concerns exist regarding the quality of parthenogenetically activated embryos in terms of sufficient numbers of cells comprising the inner cell mass (ICM) and trophectoderm (TE) and the ploidy. Therefore, these two parameters were used to assess the quality of embryos derived from parthenogenetic activation by using calcium ionophore A23187 (CaI) followed by either 6‐dimethylaminopurine (6‐DMAP, 3.5 hr or 6.5 hr) or cycloheximide (CHX) plus cytochalasin D (CD). The conventional in vitro (IVF) produced embryos served as a control. Double staining of the parthenogenetic blastocysts showed that the total cell number (TC) of embryos from the 6‐DMAP 3.5 hr (87.0 ± 5.3) and CHX+CD (79.0 ± 6.1) groups was not different (P > 0.05), but was lower than that of control embryos (116.0 ± 5.8, P < 0.001). The mean ratios of inner cell mass (ICM) and trophectoderm (TE) cells in the 6‐DMAP 3.5 hr group (0.57 ± 0.04) and the control IVF group (0.50 ± 0.02) did not differ significantly. Both were higher than those of the CHX+CD group (0.36 ± 0.02; P < 0.05). Further analysis of chromosomal compositions of developing stage embryos at day four after IVF or parthenogenetic activation demonstrated that prolonged treatment with 6‐DMAP for 6.5 hr resulted in a significantly lower percentage of diploid embryos and a significantly higher percentage of abnormal ploidy embryos compared to treatment with 6‐DMAP for 3.5 hr or with CHX and IVF. In conclusion, parthenogenetic activation of bovine oocytes with CaI followed by 6‐DMAP for 3.5 hr could produce better quality embryos in terms of total cell numbers, the number of cells allocated to the ICM, and the ploidy of embryos. Mol. Reprod. Dev. 54:57–62, 1999. © 1999 Wiley‐Liss, Inc.     

1H, 13C and 15N assignments of a camelid nanobody directed against human α-synuclein

Nanobodies are single chain antibodies that are uniquely produced in Camelidae, e.g. camels and llamas. They have the desirable features of small sizes (Mw < 14 kDa) and high affinities against antigens (Kd ~ nM), making them ideal as structural probes for biomedically relevant motifs both in vitro and in vivo. We have previously shown that nanobody binding to amyloidogenic human lysozyme variants can effectively inhibit their aggregation, the process that is at the origin of systemic amyloid disease. Here we report the NMR assignments of a new nanobody, termed NbSyn2, which recognises the C-terminus of the intrinsically disordered protein, human α-synuclein (aS), whose aberrant self-association is implicated in Parkinson’s disease.