Histone H2A and H4 N-terminal Tails Are Positioned by the MEP50 WD Repeat Protein for Efficient Methylation by the PRMT5 Arginine Methyltransferase

The protein arginine methyltransferase PRMT5 is complexed with the WD repeat protein MEP50 (also known as Wdr77 or androgen coactivator p44) in vertebrates in a tetramer of heterodimers. MEP50 is hypothesized to be required for protein substrate recruitment to the catalytic domain of PRMT5. Here we demonstrate that the cross-dimer MEP50 is paired with its cognate PRMT5 molecule to promote histone methylation. We employed qualitative methylation assays and a novel ultrasensitive continuous assay to measure enzyme kinetics. We demonstrate that neither full-length human PRMT5 nor the Xenopus laevis PRMT5 catalytic domain has appreciable protein methyltransferase activity. We show that histones H4 and H3 bind PRMT5-MEP50 more efficiently compared with histone H2A(1–20) and H4(1–20) peptides. Histone binding is mediated through histone fold interactions as determined by competition experiments and by high density histone peptide array interaction studies. Nucleosomes are not a substrate for PRMT5-MEP50, consistent with the primary mode of interaction via the histone fold of H3-H4, obscured by DNA in the nucleosome. Mutation of a conserved arginine (Arg-42) on the MEP50 insertion loop impaired the PRMT5-MEP50 enzymatic efficiency by increasing its histone substrate K_m, comparable with that of Caenorhabditis elegans PRMT5. We show that PRMT5-MEP50 prefers unmethylated substrates, consistent with a distributive model for dimethylation and suggesting discrete biological roles for mono- and dimethylarginine-modified proteins. We propose a model in which MEP50 and PRMT5 simultaneously engage the protein substrate, orienting its targeted arginine to the catalytic site.     

Desulfurization of 2-thiouracil nucleosides: conformational studies of 4-pyrimidinone nucleosides

4-Pyrimidinone ribofuranoside (H(2)o(4)U) and 4-pyrimidinone 2'-deoxyribofuranoside (dH(2)o(4)U) were synthesized by the oxidative desulfurization of parent 2-thiouracil nucleosides with m-chloroperbenzoic acid. The crystal structures of H(2)o(4)U and dH(2)o(4)U and their conformations in solution were determined and compared with corresponding 2-thiouracil and uracil nucleosides. The absence of a large 2-thiocarbonyl/2-carbonyl group in the nucleobase moiety results in C2'-endo puckering of the ribofuranose ring (S conformer) in the crystal structure of H(2)o(4)U, which is not typical of RNA nucleosides. Interestingly, the hydrogen bonding network in the crystals of dH(2)o(4)U stabilizes the sugar moiety conformation in the C3'-endo form (N conformer), rarely found in DNA nucleosides. In aqueous solution, dH(2)o(4)U reveals a similar population of the C2'-endo conformation (65%) to that of 2'-deoxy-2-thiouridine (62%), while the 62% population of the S conformer for H(2)o(4)U is significantly different from that of the parent 2-thiouridine, for which the N conformer is dominant (71%). Such a difference may be of biological importance, as the desulfurization process of natural tRNA 2-thiouridines may occur under conditions of oxidative stress in the cell and may influence the decoding process.     

Cell recovery comparison between plasma depletion/reduction- and red cell reduction-processing of umbilical cord blood

Background aimsLimited cell dose has hampered the use of cord blood transplantation (CBT) in adults. One method of minimizing nucleated cell loss in cord blood (CB) processing is to deplete or reduce plasma but not red blood cells - plasma depletion/reduction (PDR).MethodsThe nucleated cell loss of PDR was studied, and determined to be less than 0.1% in the discarded supernatant plasma fraction in validation experiments. After testing and archival sampling, the median nucleated cell recovery for PDR processing was 90%, and median CD34⁺ cell recovery 88%. In a CB bank inventory of 12 339 products with both pre- and post-processing total nucleated cells (TNC), PDR processing resulted in median post-processing TNC recoveries of 90.0% after testing and archival samples removal. Using the same 10 CB units divided into two halves, we compared directly the recovery of PDR against hydroxyethyl starch red cell reduction (RCR) for TNC, CD34⁺ cells and colony-forming units (CFU-GM, CFU-E, CFU-GEMM and total CFU) after parallel processing. We also compared the loss of very small embryonic-like stem cells (VSEL).ResultsWe demonstrated significantly higher recoveries using PDR for TNC (124%), CD34⁺ cells (121%), CFU-GM (225%), CFU-GEMM (201%), total CFU (186%) and VSEL (187%). The proportion of high TNC products was compared between 10 912 PDR and 38 819 RCR CB products and found to be 200% higher for products that had TNC ≥150 × 10⁷ (P = 0.0001) for the PDR inventory.ConclusionsOur data indicate that PDR processing of CB provides a significantly more efficient usage of this valuable and scarce resource.     

Juvenile animal studies and pediatric drug development: a European regulatory perspective

During the workshop organized by ILSI/HESI on May 5-6, 2010 on the value of juvenile animal toxicity studies, the implementation of the European Pediatric Regulation and in particular the review process of the nonclinical part of the Pediatric Investigation Plan (PIP) were described. A PIP is intended to outline the development of a medicinal product in the pediatric population (i.e. quality, safety, efficacy of the medicine and timing of studies); it is reviewed and agreed by the Pediatric Committee (PDCO) of the European Medicines Agency (EMA). The Nonclinical Working Group (NcWG) supports the PDCO in the review process of the nonclinical part of a PIP and is composed of members from the PDCO, the EMA Safety Working Party, additional experts from national competent authorities and the FDA. This article summarizes the NcWG review process and outcomes of 97 approved or ongoing PIPs, from the establishment of the NcWG in November 2008 to May 2010, as presented during the workshop. Juvenile animal studies were proposed by the applicant in 33% or required by the NcWG in 26% of the PIPs. The requirements were mainly motivated by concerns regarding potential developmental toxicities, in view of the young age of the pediatric population to be investigated, the lack of knowledge concerning the maturation of the pharmacological target, the lack of sufficient (non)clinical data, observed toxicities in the adult (non)clinical studies and the long duration of the intended treatments. Most juvenile animal studies were in the therapeutic areas of oncology, infectious diseases and endocrinology. In about 14% of the PIPs submitted, the NcWG requested either justifications of, or amendments to the study designs proposed by the applicants (e.g. justification of endpoints, study duration, species selection and timing with regards to clinical pediatric studies). Generally, only one species was selected or proposed for the juvenile studies, the rat being the most prevalent. The number of juvenile studies initially proposed by the applicant plus those requested by the NcWG was higher than the number of studies included in the "key binding elements" of the PIP opinions. This apparent discrepancy was mainly due to additional information or justifications submitted by the applicant during the clock stop. It was noted that the PIPs initially submitted often lacked information relevant to the nonclinical evaluation. Therefore, during the workshop, the need to provide scientifically based justifications when no juvenile animal studies are proposed in the initial PIP submission was stressed.     

Investigating the effect of emetic compounds on chemotaxis in Dictyostelium identifies a non-sentient model for bitter and hot tastant research

Novel chemical entities (NCEs) may be investigated for emetic liability in a range of unpleasant experiments involving retching, vomiting or conditioned taste aversion/food avoidance in sentient animals. We have used a range of compounds with known emetic /aversive properties to examine the possibility of using the social amoeba, Dictyostelium discoideum, for research into identifying and understanding emetic liability, and hence reduce adverse animal experimentation in this area. Twenty eight emetic or taste aversive compounds were employed to investigate the acute (10 min) effect of compounds on Dictyostelium cell behaviour (shape, speed and direction of movement) in a shallow chemotaxic gradient (Dunn chamber). Compound concentrations were chosen based on those previously reported to be emetic or aversive in in vivo studies and results were recorded and quantified by automated image analysis. Dictyostelium cell motility was rapidly and strongly inhibited by four structurally distinct tastants (three bitter tasting compounds--denatonium benzoate, quinine hydrochloride, phenylthiourea, and the pungent constituent of chilli peppers--capsaicin). In addition, stomach irritants (copper chloride and copper sulphate), and a phosphodiesterase IV inhibitor also rapidly blocked movement. A concentration-dependant relationship was established for five of these compounds, showing potency of inhibition as capsaicin (IC(50) = 11.9 ± 4.0 μM) > quinine hydrochloride (IC(50) = 44.3 ± 6.8 μM) > denatonium benzoate (IC(50) = 129 ± 4 μM) > phenylthiourea (IC(50) = 366 ± 5 μM) > copper sulphate (IC(50) = 1433 ± 3 μM). In contrast, 21 compounds within the cytotoxic and receptor agonist/antagonist classes did not affect cell behaviour. Further analysis of bitter and pungent compounds showed that the effect on cell behaviour was reversible and not cytotoxic, suggesting an uncharacterised molecular mechanism of action for these compounds. These results therefore demonstrate that Dictyostelium has potential as a non-sentient model in the analysis of the molecular effects of tastants, although it has limited utility in identification of emetic agents in general.     

CD8+ T-cell-mediated cross-clade protection in the genital tract following intranasal immunization with inactivated human immunodeficiency virus antigen plus CpG oligodeoxynucleotides

Human immunodeficiency virus (HIV) is a mucosally transmitted infection that rapidly targets and depletes CD4+ T cells in mucosal tissues and establishes a major reservoir for viral persistence in gut-associated lymphoid tissues. Therefore, vaccines designed to prevent HIV infections must induce potent and durable mucosal immune responses, especially in the genital tract. Here we investigated whether intranasal (i.n.) immunization with inactivated gp120-depleted HIV-1 antigen (Ag) plus CpG oligodeoxynucleotide (ODN) as an adjuvant induced local immune responses in the genital tract and cross-clade protection against intravaginal (IVAG) challenge. Lymphocytes isolated from the iliac lymph nodes (ILNs) and genital tracts of female mice i.n. immunized with HIV-1 Ag plus CpG showed significant HIV-specific proliferation and produced significantly higher levels of gamma interferon (IFN-gamma) and beta-chemokines than mice immunized with HIV-1 Ag alone or mixed with non-CpG ODN. CD8+ lymphocytes were dramatically increased in the genital tracts of mice immunized with HIV-1 Ag plus CpG, and protection following IVAG challenge with recombinant vaccinia viruses (rVVs) expressing HIV-1 gag was shown to be CD8 dependent. Finally, cross-clade protection was observed between clades A, C, and G but not B following IVAG challenge with rVVs expressing HIV-1 gag from different clades. These studies provide evidence that mucosal (i.n.) immunization induced strong local T-cell-mediated immune responses in the genital tract and cross-clade protection against IVAG challenge.     

Pre-steady-state kinetic characterization of the DinB homologue DNA polymerase of Sulfolobus solfataricus

Equilibrium as well as pre-steady-state measurements were performed to characterize the molecular basis of DNA binding and nucleotide incorporation by the thermostable archaeal DinB homologue (Dbh) DNA polymerase of Sulfolobus solfataricus. Equilibrium titrations show a DNA binding affinity of about 60 nm, which is approximately 10-fold lower compared with other DNA polymerases. Investigations of the binding kinetics applying stopped-flow and pressure jump techniques confirm this weak binding affinity. Furthermore, these measurements suggest that the DNA binding occurs in a single step, diffusion-controlled manner. Single-turnover, single dNTP incorporation studies reveal maximal pre-steady-state burst rates of 0.64, 2.5, 3.7, and 5.6 s(-1) for dTTP, dATP, dGTP, and dCTP (at 25 degrees C), which is 10-100-fold slower than the corresponding rates of classical DNA polymerases. Another unique feature of the Dbh is the very low nucleotide binding affinity (K(d) approximately 600 mum), which again is 10-20-fold lower compared with classical DNA polymerases as well as other Y-family polymerases. Surprisingly, the rate-limiting step of nucleotide incorporation (correct and incorrect) is the chemical step (phosphoryl transfer) and not a conformational change of the enzyme. Thus, unlike replicative polymerases, an "induced fit" mechanism to select and incorporate nucleotides during DNA polymerization could not be detected for Dbh.     

Real time spectral analysis during phytochrome chromophore and chromoprotein purification

The plant photoreceptor phytochrome senses light quality and quantity in the red region of the spectrum, directing adaptation and development. The functional holo-protein is a dimeric chromoprotein which is formed by an autoassembly reaction between the translation product and the open chain tetrapyrroles phytochromobilin (PPhiB) or phycocyanobilin (PCB). We are interested in structure/function relationships within the phytochrome molecule, in particular chromophore/protein interaction during the assembly and photoactivation, using IR and NMR spectroscopy. For this we use an automated F/HPLC system running in a darkroom to purify large amounts of protein and chromophore separately. To obtain highly pure PCB chromophore we developed improved extraction and purification methods in which the final step is RPC-18 HPLC. As there are many spectrally only slightly different tetrapyrroles in the extract, the triple-wavelength monitoring offered by the F/HPLC detector was inadequate for distinguishing between PCB and impurities. Furthermore, lambda(max) for the phytochrome Pfr signalling state lies between 705 and 730 nm, beyond the range of the detector. Also, as both holo-protein and chromophore are photoactive, we wished to minimize light exposure of the eluate. We therefore implemented a miniature CCD-based flow UV-vis spectrophotometer using a xenon flash and quartz fiber optics enabling us monitor the entire 250-800 nm spectrum of the eluate to an accuracy of +/-3 x 10(-3)A in real time. The instrumentation described can be added to any chromatographic system, thereby allowing the purification of any molecule to be monitored easily and efficiently.     

Pattern of expression of vascular endothelial growth factor and its receptors in the ovine choroid plexus during long and short photoperiods

Vascular endothelial growth factor (VEGF-A) plays an important role in maintaining cerebrospinal fluid (CSF) homeostasis and the function of the choroid plexuses (CPs). The objective of the study was to determine the expression of vascular endothelial growth factor (VEGF-A), tyrosine kinase receptors Flt-1 and KDR and KDR co-receptor neuropilin 1 (NRP-1) in ovine CPs during different photoperiods. CPs were collected from the lateral brain ventricles from ovariectomized, estradiol-treated ewes during long day (LD; 16L:8D, n?=?5) and short day (SD; 8L:16D, n?=?5) photoperiods. We analyzed mRNA expression levels of two VEGF-A isoforms, VEGF-A ₁₂₀ and VEGF-A ₁₆₄ and our results indicate that VEGF-A ₁₆₄ was the predominant isoform. Expression levels of VEGF-A and Flt-1 were similar during the SD and LD photoperiods. There were significant increases in KDR mRNA and protein expression (p?<?0.05) and NRP-1 mRNA expression (p?<?0.05) during SD. These data show that expression of KDR and its co-receptor NRP-1 are up-regulated by short photoperiod and that this effect is not dependent on ovarian steroids. Our results suggest that the VEGF-A-system may be involved in photoperiodic plasticity of CP capillaries and may therefore be responsible for photoperiodic changes in the CSF turnover rate in ewes.