Solid-phase synthesis of oligomers carrying several chromophore units linked by phosphodiester backbones

A method for the preparation of oligomers by linking chromophore units is described. Specifically, the synthesis of chromophore units having a protected-hydroxyl group and a phosphoramidite function is described, along with a method to link several units using solid-phase phosphite-triester protocols.     

Stacking interactions of ApA analogues with modified backbones

CD spectra have been measured as a function of temperature for a number of ApA analogues with modified backbones. Oligonucleotides with these modified backbones are being used as antisense agents having potential as viral therapeutics. Results of these studies show that when a carbonyl is substituted for the phosphate to produce an uncharged backbone, the analogues that have either sugar or morpholino substitution do not stack. In contrast, when a morpholino group is substituted for the sugar and the phosphate is modified so as to be uncharged, there is strong base stacking. Stacking interactions in the phosphorus-linked morpholino analogues are at least as strong as those found in d (ApA). The stacking interactions in ApA are weak by comparison. Singular value decomposition demonstrates that the stacking is two state, and Taylor series decomposition yields a coefficient that measures base stacking interactions. The van't Hoff equation is applied to the base stacking coefficient from the Taylor series fitting to give thermodynamic parameters. © 1992 John Wiley & Sons, Inc.     

Preferred phosphodiester conformations in nucleic acids. A virtual bond torsion potential to estimate lone-pair interactions in a phosphodiester

The lone-pair orbital interactions arising in a phosphodiester are incorporated into semiempirical conformational energy calculations using a unifold “torsional potential” around the virtual bond linking the ester oxygen atoms. The results explain the observed experimental data better than other methods.     

Stability of histone mRNAs is related to their location in polysomes

Synthesis of histone mRNAs is closely coupled to DNA synthesis. Following inhibition of DNA synthesis in L6 myoblasts with cytosine arabinoside, a coordinate and exaggerated rate of degradation of histone mRNAs occurs while other mRNAs, encoding ribosomal protein L32 and actin, are unaffected. Inhibition of protein synthesis by puromycin, emetine, or cycloheximide stabilizes histone mRNAs and results in their accumulation. When inhibition of DNA synthesis was followed immediately by inhibition of protein synthesis, the exaggerated rate of decay of the existing subspecies of histone H4 mRNAs was prevented and histone mRNA accumulated. If inhibition of protein synthesis was delayed longer than 3 minutes following inhibition of DNA synthesis, the ability to accumulate H4 mRNAs was lost. Furthermore, new protein synthesis was required to activate the mechanism which specifically destabilized histone mRNA. Puromycin was able to prevent the exaggerated rate of degradation of the various subspecies of H4 mRNA when added up to 15 min after inhibition of DNA synthesis, whereas emetine was effective only when added up to 5 min following inhibition of DNA synthesis. These data suggest that histone H4 mRNAs in polysomes are better targets than those released from polysomes for the specific mechanism which destabilizes histone mRNAs upon inhibition of DNA synthesis.     

Hybridization between closely related songbirds is related to human habitat disturbance

Human habitat disturbances can promote hybridization between closely related, but typically reproductively isolated, species. We explored whether human habitat disturbances are related to hybridization between two closely related songbirds, black-capped and mountain chickadees, using both genomic and citizen science data sets. First, we genotyped 409 individuals from across both species' ranges using reduced-representation genome sequencing and compared measures of genetic admixture to a composite measure of human landscape disturbance. Then, using eBird observations, we compared human landscape disturbance values for sites where phenotypically diagnosed hybrids were observed to locations where either parental species was observed to determine whether hybrid chickadees are reported in more disturbed areas. We found that hybridization between black-capped and mountain chickadees positively correlates with human habitat disturbances. From genomic data, we found that (1) hybrid index (HI) significantly increased with habitat disturbance, (2) more hybrids were sampled in disturbed habitats, (3) mean HIs were higher in disturbed habitats versus wild habitats, and (4) hybrids were detected in habitats with significantly higher disturbance values than parentals. Using eBird data, we found that both hybrid and black-capped chickadees were significantly more disturbance-associated than mountain chickadees. Surprisingly, we found that nearly every black-capped chickadee we sampled contained some proportion of hybrid ancestry, while we detected very few mountain chickadee backcrosses. Our results highlight that hybridization between black-capped and mountain chickadees is widespread, but initial hybridization is rare (few F1s were detected). We conclude that human habitat disturbances can erode pre-zygotic reproductive barriers between chickadees and that post-zygotic isolation is incomplete. Understanding what becomes of recently hybridizing species following large-scale habitat disturbances is a new, but pressing, consideration for successfully preserving genetic biodiversity in a rapidly changing world.     

Protein is linked to the 5' end of poliovirus RNA by a phosphodiester linkage to tyrosine.

Purification and partial characterization of the poliovirus RNA-linked protein (VPg) are described. VPg has been freed from the RNA by ribonuclease digestion and phenol extraction. Gel filtration chromatography of VPg-pUp (labeled with 32P) in 0.5% sodium dodecyl sulfate or 6 M guanidine HCl indicates that it has a molecular weight of about 12,000. VPg is bound to the 5' end of poliovirion RNA by a phosphodiester bond between a tyrosine residue in the VPg molecule and the 5'-terminal uridine. After acid hydrolysis of [3H]tyrosine-labeled VPg-pU, free tyrosine can be released by venom phosphodiesterase. Acid hydrolysis of VPg-p labeled with either 32P or [3H] tyrosine yields tyrosine-phosphate. There appears to be only 1 tyrosine residue per VPg molecule.     

The effect of secondary structure on cleavage of the phosphodiester bonds of RNA

This review discusses the effects the secondary structure of an RNA molecule has on the inherent reactivity of its phosphodiester bonds, and on the catalytic activity of metal ion-based cleaving agents. The basic principles of the intramolecular transesterification of RNA phosphodiester bonds, particularly cleavage, are first briefly described. Studies of the structural effects on the cleavage, in the absence and in the presence of metal ion catalysts, are then reviewed, and the sources of the reactivity differences observed in different structures are discussed.     

Glucuronoarabinoxylan structure in the walls of Aechmea leaf chlorenchyma cells is related to wall strength

In CAM-plants rising levels of malic acid in the early morning cause elevated turgor pressures in leaf chlorenchyma cells. Under specific conditions this process is lethal for sensitive plants resulting in chlorenchyma cell burst while other species can cope with these high pressures and do not show cell burst under comparable conditions. The non-cellulosic polysaccharide composition of chlorenchyma cell walls was investigated and compared in three cultivars of Aechmea with high sensitivity for chlorenchyma cell burst and three cultivars with low sensitivity. Chlorenchyma layers were cut from the leaf and the non-cellulosic carbohydrate fraction of the cell wall fraction was analyzed by gas-liquid chromatography. Glucuronoarabinoxylans (GAXs) were the major non-cellulosic polysaccharides in Aechmea. The fine structure of these GAXs was strongly related to chlorenchyma wall strength. Chlorenchyma cell walls from cultivars with low sensitivity to cell burst were characterized by an A/X ratio of ca. 0.13 while those from cultivars with high sensitivity showed an A/X ratio of ca. 0.23. Xylose chains from cultivars with high cell burst sensitivity were ca. 40% more substituted with arabinose compared to cultivars with low sensitivity for cell burst. The results indicate a relationship in vivo between glucuronoarabinoxylan fine structure and chlorenchyma cell wall strength in Aechmea. The evidence obtained supports the hypothesis that GAXs with low degrees of substitution cross-link cellulose microfibrils, while GAXs with high degrees of substitution do not. A lower degree of arabinose substitution on the xylose backbone implies stronger cell walls and the possibility of withstanding higher internal turgor pressures without cell bursting.     

Relating interactions between neurofilaments to the structure of axonal neurofilament distributions through polymer brush models

Neurofilaments (NFs) have been proposed to interact with one another through mutual steric exclusion of their unstructured C-terminal "sidearm" domains, producing order in axonal NF distributions and conferring mechanical strength to the axon. Here we apply theory developed for polymer brushes to examine the relationship between the brush properties of the sidearms and NF organization in axons. We first measure NF-NF radial distribution functions and occupancy probability distributions for adult mice. Interpreting the probability distributions using information theory, we show that the NF distributions may be represented by a single pair potential of mean force. Then, to explore the relationship between model parameters and NF architecture, we conduct two-dimensional Monte Carlo simulations of NF cross-sectional distributions. We impose purely repulsive interaction potentials in which the sidearms are represented as neutral and polyelectrolyte chains. By treating the NFs as telechelic polymer brushes, we also incorporate cross-bridging interactions. Both repulsive potentials are capable of reproducing NF cross-sectional densities and their pair correlations. We find that NF structure is sensitive to changes in brush thickness mediated by chain charge, consistent with the experimental observation that sidearm phosphorylation regulates interfilament spacing. The presence of attractive cross-bridging interactions contributes only modestly to structure for moderate degrees of cross-bridging and leads to NF aggregation for extensive cross-bridging.     

Oligonucleotide structure influences the interactions between cationic polymers and oligonucleotides

We examined the effect of oligodeoxynucleotide (ODN) structure on the interactions between cationic polymers and ODNs. Unstructured and hairpin structured ODNs were used to form complexes with the model cationic polymer, poly-L-lysine (pLL), and the characteristics of these polymer-ODN interactions were subsequently examined. We found that hairpin structured ODNs formed complexes with pLL at slightly lower pLL:ODN charge ratios as compared to unstructured ODNs and that, at high charge ratios, greater fractions of the hairpin ODNs were complexed, as measured by dye exclusion. The dissociation of pLL-ODN interactions was tested further by challenge with heparin, which induced complex disruption. Both the kinetics and heparin dose response of ODN release were determined. The absolute amount and the kinetic rate of ODN release from the complexes of pLL and unstructured ODN were greater, as compared to hairpin ODNs. Our results therefore highlight the role of ODN structure on the association-dissociation behavior of polymer-ODN complexes. These findings have implications for the selection of ODN sequences and design of polymeric carriers used for cellular delivery of ODNs.     

Stability of spatial interactions between chromocenters and pre-kinetochores in the interphase murine cells

It is known that in mice the centromeric heterochromatin remains compact during the whole cell cycle and at interphase is referred to as "chromocentres". In the current study, by the use of antibodies against prekinetochores and DNA polymerase (a PCNA antigen), we showed that in murine L929 cells chromocentres remain spatially associated with prekinetochores during the entire interphase, including the late S-period, when DNA chromocentres replicate. Augmentation of prekinetochore fluorescence increases concomitantly with the heterochromation replication, but the prekinetochore duplication occurs only in G2 period. A conclusion has been made that murine interphase cells can be used for biochemical fractionation of chromocentres associated with prekinetochore proteins.     

Stability analysis of a two-species model with transitions between population interactions

Stability of a simple two-species system is investigated. This model assumes that the kind of inter-specific interactions is not fixed, and that it depends on the system state, i.e., undergoes transitions between different population interactions due to variation in population densities. The main goal is to show the effects of the transitions between different population interactions on the two-species coexistence, and on the stability conditions of multiple equilibria.     

Hemolysis of human erythrocytes induced by tamoxifen is related to disruption of membrane structure

Tamoxifen (TAM), the antiestrogenic drug most widely prescribed in the chemotherapy of breast cancer, induces changes in normal discoid shape of erythrocytes and hemolytic anemia. This work evaluates the effects of TAM on isolated human erythrocytes, attempting to identify the underlying mechanisms on TAM-induced hemolytic anemia and the involvement of biomembranes in its cytostatic action mechanisms. TAM induces hemolysis of erythrocytes as a function of concentration. The extension of hemolysis is variable with erythrocyte samples, but 12.5 microM TAM induces total hemolysis of all tested suspensions. Despite inducing extensive erythrocyte lysis, TAM does not shift the osmotic fragility curves of erythrocytes. The hemolytic effect of TAM is prevented by low concentrations of alpha-tocopherol (alpha-T) and alpha-tocopherol acetate (alpha-TAc) (inactivated functional hydroxyl) indicating that TAM-induced hemolysis is not related to oxidative membrane damage. This was further evidenced by absence of oxygen consumption and hemoglobin oxidation both determined in parallel with TAM-induced hemolysis. Furthermore, it was observed that TAM inhibits the peroxidation of human erythrocytes induced by AAPH, thus ruling out TAM-induced cell oxidative stress. Hemolysis caused by TAM was not preceded by the leakage of K(+) from the cells, also excluding a colloid-osmotic type mechanism of hemolysis, according to the effects on osmotic fragility curves. However, TAM induces release of peripheral proteins of membrane-cytoskeleton and cytosol proteins essentially bound to band 3. Either alpha-T or alpha-TAc increases membrane packing and prevents TAM partition into model membranes. These effects suggest that the protection from hemolysis by tocopherols is related to a decreased TAM incorporation in condensed membranes and the structural damage of the erythrocyte membrane is consequently avoided. Therefore, TAM-induced hemolysis results from a structural perturbation of red cell membrane, leading to changes in the framework of the erythrocyte membrane and its cytoskeleton caused by its high partition in the membrane. These defects explain the abnormal erythrocyte shape and decreased mechanical stability promoted by TAM, resulting in hemolytic anemia. Additionally, since membrane leakage is a final stage of cytotoxicity, the disruption of the structural characteristics of biomembranes by TAM may contribute to the multiple mechanisms of its anticancer action.