Synthesis and Properties of P1, P2-, P1, P3- and P1, P4-Dinucleoside Di-, Tri- and Tetraphosphate mRNA 5′-Cap Analogues

Abstract

Chemically synthesized dinucleoside P¹, P²-di-, P¹, P³-tri- and P¹, P⁴-tetraphosphates, derivatives of 5′-linked 7-methylguanosine and guanosine were characterized with respect to their structural properties and functional effect on eukaryotic translation inhibition.     

Inhibition and inactivation of cytochrome P450 2A6 and cytochrome P450 2A13 by menthofuran, β-nicotyrine and menthol

Nicotine is the primary addictive agent in tobacco products and is metabolized in humans by CYP2A6. Decreased CYP2A6 activity has been associated with decreased smoking. The extrahepatic enzyme, CYP2A13 (94% identical to CYP2A6) also catalyzes the metabolism of nicotine, but is most noted for its role in the metabolic activation of the tobacco specific lung carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). In this study, the inhibition and potential inactivation of CYP2A6 and CYP2A13 by two tobacco constituents, 1-methyl-4-(3-pyridinyl) pyrrole (β-nicotyrine) and (-)-menthol were characterized and compared to the potent mechanism based inactivator of CYP2A6, menthofuran. The effect of these compounds on CYP2A6 and CYP2A13 activity was significantly different. (-)-Menthol was a more efficient inhibitor of CYP2A13 than of CYP2A6 (KI, 8.2 μM and 110 μM, respectively). β-Nicotyrine was a potent inhibitor of CYP2A13 (KI, 0.17 μM). Neither menthol nor β-nicotyrine was an inactivator of CYP2A13. Whereas, β-nicotyrine was a mechanism based inactivator of CYP2A6 (KI(inact), 106 μM, kinact was 0.61 min(-1)). Similarly, menthofuran, a potent mechanism based inactivator of CYP2A6 did not inactivate CYP2A13. Menthofuran was an inhibitor of CYPA13 (KI, 1.24 μM). The inactivation of CYP2A6 by either β-nicotyrine or menthofuran was not due to modification of the heme and was likely due to modification of the apo-protein. These studies suggest that β-nicotyrine, but not menthol may influence nicotine and NNK metabolism in smokers.     

Dissociation of P13–P14 far-field potentials: clinical and MRI correlation

P13 and P14 far-field potentials are recorded over the scalp with median nerve stimulation when non-cephalic reference is used to measure somatosensory evoked potentials. The dissociation of these 2 potentials is exceptional. Only 2 cases subsequent to pontine lesions have been described hitherto. We report the case of a 31-year-old woman with a low grade gliomallocated at the spino-medullary junction who presented a P13–P14 far-field dissociation. This case fully supports the independent nature of the P13 and P14 potential generators.     

Comparative genome mapping among Picea glauca, P. mariana × P. rubens and P. abies, and correspondence with other Pinaceae

A composite linkage map was constructed from four individual maps for the conifer Picea glauca (Moench) Voss, from anonymous and gene-specific markfers (714 AFLPs, 38 SSRs, and 53 ESTPs). A total of 12 linkage groups were delineated with an average marker density of 2.7 cM. Macro-synteny and macro-colinearity comparisons with two other composite linkage maps developed for the species complex P. mariana (Mill.) B.S.P. × P. rubens Sarg., and for P. abies (L.) Karst. revealed an identical number of linkage groups and a remarkable conservation of the gene content and gene order of linkage groups over the million years since the split between these taxa. Identical gene order among taxa was observed for 10 of the 12 assembled composite linkage groups. The discovery of one breakdown in synteny between P. glauca and the other two taxa indicated the occurrence of an inter-chromosomal rearrangement involving an insertional translocation. Analysis of marker colinearity also revealed a putative segmental duplication. The combined information from these three Picea genomes validated and improved large-scale genome comparisons at the inter-generic level in the family Pinaceae by allowing for the identification of 11 homoeologous linkage groups between Picea and Pinus, and nine such groups between Picea and Pseudotsuga menziesii. Notably, the analysis of synteny among the three genera revealed a putative case of chromosomal fission and an inter-chromosomal rearrangement in the genome of P. menziesii. Both of these changes are inter-connected, indicating much instability in this part of the P. menziesii genome. Overall, the macro-structure of the Pinaceae genome was well conserved, which is notable given the Cretaceous origin of its main lineages.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.Her Majesty the Queen in Right of Canada.     

Synthesis and SAR of 1,3-thiazolyl thiophene and pyridine derivatives as potent, orally active and S1P₃-sparing S1P₁ agonists

We have previously disclosed 1,2,4-oxadiazole derivative 3 as a potent S1P(3)-sparing S1P(1) agonist. Although compound 3 exhibits potent and manageable immunosuppressive efficacy in various in vivo models, recent studies have revealed that its 1,2,4-oxadiazole ring is subjected to enterobacterial decomposition. As provisions for unpredictable issues, a series of alternative compounds were synthesized on the basis of compound 3. Extensive SAR studies led to the finding of 1,3-thiazole 24c with the EC(50) value of 3.4 nM for human S1P(1), and over 5800-fold selectivity against S1P(3). In rat on host versus graft reaction (HvGR), the ID(50) value of 24c was determined at 0.07 mg/kg. The pharmacokinetics in rat and monkey is also reported. Compared to compound 3, 24c showed excellent stability against enterobacteria.     

Characteristics and application of S1–P1 nucleases in biotechnology and medicine

3′–nucleases/nucleotidases of the S1–P1 family (EC 3.1.30.1) are single–strand–specific or non-specific zinc–dependent phosphoesterases present in plants, fungi, protozoan parasites, and in some bacteria. They participate in a wide variety of biological processes and their current biotechnological applications rely on their single–strand preference, nucleotide non-specificity, a broad range of catalytic conditions and high stability. We summarize the present and potential utilization of these enzymes in biotechnology and medicine in the context of their biochemical and structure–function properties. Explanation of unanswered questions for bacterial and trypanosomatid representatives could facilitate development of emerging applications in medicine.     

α-Synuclein and Its A30P Mutant Affect Actin Cytoskeletal Structure and Dynamics

The function of α-synuclein, a soluble protein abundant in the brain and concentrated at presynaptic terminals, is still undefined. Yet, α-synuclein overexpression and the expression of its A30P mutant are associated with familial Parkinson''s disease. Working in cell-free conditions, in two cell lines as well as in primary neurons we demonstrate that α-synuclein and its A30P mutant have different effects on actin polymerization. Wild-type α-synuclein binds actin, slows down its polymerization and accelerates its depolymerization, probably by monomer sequestration; A30P mutant α-synuclein increases the rate of actin polymerization and disrupts the cytoskeleton during reassembly of actin filaments. Consequently, in cells expressing mutant α-synuclein, cytoskeleton-dependent processes, such as cell migration, are inhibited, while exo- and endocytic traffic is altered. In hippocampal neurons from mice carrying a deletion of the α-synuclein gene, electroporation of wild-type α-synuclein increases actin instability during remodeling, with growth of lamellipodia-like structures and apparent cell enlargement, whereas A30P α-synuclein induces discrete actin-rich foci during cytoskeleton reassembly. In conclusion, α-synuclein appears to play a major role in actin cytoskeletal dynamics and various aspects of microfilament function. Actin cytoskeletal disruption induced by the A30P mutant might alter various cellular processes and thereby play a role in the pathogenesis of neurodegeneration.     

Inheritance Profile of Weathered Chlordane and P,P′-DDTs Accumulation by cucurbita pepo hybrids

Cucurbita pepo ssp pepo (zucchini) accumulates significant levels of persistent organic pollutants in its roots, followed by unexpectedly high contaminant translocation to the stems. Most other plant species, including the closely related C. pepo ssp ovifera (squash), do not have this ability. To investigate the mechanism of contaminant accumulation, two cultivars each of parental zucchini and squash, as well as previously created first filial (F1) hybrids and F1 backcrosses (BC) of those parental cultivars, were grown under field conditions in a soil contaminated with weathered chlordane (2.29 μg/g) and DDX residues (0.30 μg/g; sum of DDT, DDE, DDD). The parental zucchini had stem-to-soil bioconcentration factors (BCF, contaminant ratio of stem to soil) for chlordane and DDX of 6.23 and 3.10; these values were 2.2 and 3.7 times greater than the squash, respectively. Chlordane and DDX translocation factors, the ratio of contaminant content in the stems to that in the root, were 2.1 and 3.2 times greater for zucchini than for squash. The parental zucchini and squash also differed significantly in chlordane component ratios (relative amounts of trans-nonachlor [TN], cis-chlordane [CC], trans-chlordane [TC]) and enantiomer fractions for the chiral CC and TC. Hybridization of the parental squash and zucchini resulted in significant differences in contaminant uptake. For both the three separate component ratios (CR) and two sets of enantiomer fraction (EF) values, subspecies specific differences in the parental generation became statistically equivalent in the F1 hybrid zucchini and squash. When backcrossed (BC) with the original parental plants, the zucchini and squash F1 BC cultivars reverted to the statistically distinct CR and EF patterns. This pattern of trait segregation upon hybridization suggests either single gene or single locus control for persistent organic pollutant (POP) uptake ability by C. pepo ssp pepo.     

Antiplatelet Activity,P2Y1 and P2Y12 Inhibition,and Metabolism in Plasma of Stereoisomers of Diadenosine 5′,5′″-P1,P4-dithio-P2,P3-chloromethylenetetraphosphate

Background

Diadenosine tetraphosphate (Ap₄A), a constituent of platelet dense granules, and its P¹,P⁴-dithio and/or P²,P³-chloromethylene analogs, inhibit adenosine diphosphate (ADP)-induced platelet aggregation. We recently reported that these compounds antagonize both platelet ADP receptors, P2Y₁ and P2Y₁₂. The most active of those analogs, diadenosine 5′,5″″-P¹,P⁴-dithio-P²,P³-chloromethylenetetraphosphate, (compound 1), exists as a mixture of 4 stereoisomers.

Objective

To separate the stereoisomers of compound 1 and determine their effects on platelet aggregation, platelet P2Y₁ and P2Y₁₂ receptor antagonism, and their metabolism in human plasma.

Methods

We separated the 4 diastereomers of compound 1 by preparative reversed-phase chromatography, and studied their effect on ADP-induced platelet aggregation, P2Y₁-mediated changes in cytosolic Ca²⁺, P2Y₁₂-mediated changes in VASP phosphorylation, and metabolism in human plasma.

Results

The inhibition of ADP-induced human platelet aggregation and human platelet P2Y₁₂ receptor, and stability in human plasma strongly depended on the stereo-configuration of the chiral P¹- and P⁴-phosphorothioate groups, the S_PS_P diastereomer being the most potent inhibitor and completely resistant to degradation in plasma, and the R_PR_P diastereomer being the least potent inhibitor and with the lowest plasma stability. The inhibitory activity of S_PR_P diastereomers depended on the configuration of the pseudo-asymmetric carbon of the P²,P³-chloromethylene group, one of the configurations being significantly more active than the other. Their plasma stability did not differ significantly, being intermediate to that of the S_PS_P and the R_PR_P diastereomers.

Conclusions

The presently-described stereoisomers have utility for structural, mechanistic, and drug development studies of dual antagonists of platelet P2Y₁ and P2Y₁₂ receptors.     

Enhanced spectral resolution in RNA HCP spectra for measurement of 3JC2′P and 3JC4′P couplings and 31P chemical shift changes upon weak alignment

The 'out-and-back' 3D HCP experiment, using gradient- and sensitivity-enhanced detection, provides a convenient method for assignment of the (31)P NMR spectra and accurate measurement of the (31)P chemical shifts of ribonucleic acids. The (13)C resolution in such spectra can be doubled, at the cost of a 50% reduction in sensitivity, by combining (13)C evolution during the (13)C-[(31)P] de- and rephasing periods. The multiple connectivities observable for a given (31)P, including correlations to the intranucleotide C5'H(2) and C4'H groups, and the C2'H, C3'H and C4'H groups of the preceding nucleotide, permit independent measurements of the (31)P shift. The (13)C spectrum of these groups is typically crowded for an RNA molecule in isotropic solution and overlap becomes more problematic in media used to achieve partial alignment. However, many of these correlations are resolvable in the combined-evolution HCP spectrum. The difference in (31)P chemical shift between isotropic solution and a medium containing liquid crystalline Pf1 provides information on the orientation of phosphate groups. The intensities measured in the 3D HCP spectrum, obtained for an isotropic sample, yield values for the (3)J(C2'P) and (3)J(C4'P) couplings, thereby providing important restraints for the backbone torsion angles epsilon and beta. The experiments are illustrated for a uniformly (13)C-enriched, 24-residue stem-loop RNA sequence, and results for the helical stem region show close agreement between observed Deltadelta((31)P) values and those predicted for a model A-form RNA helix when using a uniform (31)P CSA tensor. This confirms that Deltadelta((31)P) values can be used directly as restraints in refining nucleic acid structures.     

Studying language change using price equation and Pólya-urn dynamics

Language change takes place primarily via diffusion of linguistic variants in a population of individuals. Identifying selective pressures on this process is important not only to construe and predict changes, but also to inform theories of evolutionary dynamics of socio-cultural factors. In this paper, we advocate the Price equation from evolutionary biology and the Pólya-urn dynamics from contagion studies as efficient ways to discover selective pressures. Using the Price equation to process the simulation results of a computer model that follows the Pólya-urn dynamics, we analyze theoretically a variety of factors that could affect language change, including variant prestige, transmission error, individual influence and preference, and social structure. Among these factors, variant prestige is identified as the sole selective pressure, whereas others help modulate the degree of diffusion only if variant prestige is involved. This multidisciplinary study discerns the primary and complementary roles of linguistic, individual learning, and socio-cultural factors in language change, and offers insight into empirical studies of language change.     

C.P.S.—Its Strengths and Weaknesses

The following, a supplement to the annual report of the Board of Trustees of California Physicians'' Service that was published in the March 1961 issue of California Medicine, was delivered at the 1961 Annual Session of the House of Delegates by Dr. John G. Morrison, chairman of the board.     

The radiolysis of tryptophan and leucine with32Pβ-radiation

Summary We have extended earlier experiments on the radiolysis of DL-tryptophan using³²P-radiation to longer reaction times, observing complete destruction of the tryptophan by secondary, non-radiolytic processes. We have also undertaken the irradiation of DL-leucine with³²P's at -196°, achieving radiolyses to the extents of ca. 20–30%, but observing no concomittant asymmetric bias. The implications of these observations are discussed with regard to the Vester-Ulbricht mechanism for the origin of optical activity.     

Pollen-pistil interactions in Populus: β-galactosidase activity associated with pollen-tube growth in the crosses P. nigra x P. nigra and P. nigra x P. alba

Summary In order to understand the nature of interspecific barriers in Populus, we have explored pollen/pistil interactions in intra- and interspecific crosses Populus nigra x P. nigra and P. nigra x P. alba. The kinetics of pollen-tube growth demonstrated that P. nigra and P. alba pollen tubes have distinct behavioral patterns inside P. nigra pistils. P. alba pollen tubes exhibit an unique S-shaped growth curve and an arrested growth site near the sylodium. P. nigra pollen tubes exhibit two growth phases, in the stigmatic tissues and in the ovarian cavity respectively. P. nigra and P. alba curves diverge 5 h after controlled pollination and could be related to a change in the physiology of the P. nigra pollen tube, which shifts from an autotrophic to a heterotrophic type of nutrition. Protein analysis of pollinated stigmatic extracts (0,6 and 20 h after pollination) revealed qualitative and quantitative differences that are related to the presence of either P. nigra or P. alba pollen tubes inside the stigmatic tissues. Increasing numbers of protein bands were detectable from 0 to 20 h after pollination only in intraspecific cross. Glycoproteins were detected, and the differences observed were dependent of the cross. -Galactosidase activity was found in pollinated stigmas, but an increase in its activity (one isozyme of pHi 4.2) between 6 h and 20 h after pollination was detected only in the intraspecific cross. This enzyme could play a role in heterotrophic pollen-tube nitrition, and its activity could be the final result of a series of interactions started by the initial pollen-stigma dialog.     

Yields and accumulations of N and P in farmer-managed intercrops of maize–pigeonpea in semi-arid Africa

Maize (Zea mays L.) is a major staple food in Sub-Saharan Africa but low soil fertility, limited resources and droughts keep yields low. Cultivation of maize intercropped with pigeonpea (Cajanus cajan L. Millsp.) is common in some areas of eastern and southern Africa. The objectives of this study were (1) to investigate dry matter, nitrogen (N) and phosphorus (P) accumulation in different plant components of maize–pigeonpea intercropping systems and (2) to report the effects of the intercrops on soil fertility. Maize–pigeonpea intercrops were compared to sole maize grown using farmersȁ9 practices. Intercropping maize and pigeonpea increased (P < 0.05) total system yield compared to sole maize in terms of biomass, N and P accumulation. Pigeonpea planted in maize did not reduce (P < 0.05) the accumulation of dry matter, N nor P in the maize grain. The harvest indices of maize, calculated on basis dry matter, N or P did not differ either (P < 0.05). Total soil C and N contents and inorganic N content, nitrate and ammonium, were not affected by two seasons of maize–pigeonpea intercropping compared to sole maize (P > 0.11). Nitrate and ammonium levels in soil were still not affected by the treatments after the soils were incubated in anaerobic conditions for 8 days at 37°C (P > 0.11). However, pigeonpea added up to 60 kg of N ha⁻¹ to the system and accumulated up to 6 kg of P ha⁻¹ and only 25% of this N and P were exported in the grain. In conclusion, beside the added grain yield of pigeonpea in the intercropped systems, pigeonpea increased the recirculation of dry matter, N and P, which may have a long-term effect on soil fertility. Furthermore, the stems from pigeonpea contributed to household fuel wood consumption. The intercropped system thus had multiple benefits that gave significant increase in combined yield per unit area without additional labour requirements. The main requirement in order to up-scale the maize–pigeonpea intercropping approach is sufficient supply of high-quality pigeonpea seeds.