Cationic double-hydrophilic model networks: synthesis,characterization, modeling and protein adsorption studies

Group transfer polymerization (GTP) was used for the preparation of eight networks based on two hydrophilic monomers, 2-(dimethylamino)ethyl methacrylate (DMAEMA) and poly(ethylene glycol) methacrylate (PEGMA). Ethylene glycol dimethacrylate (EGDMA) served as the cross-linker, whereas 1,4-bis(methoxytrimethylsiloxymethylene)cyclohexane (MTSMC) was used as a bifunctional initiator. Seven of the networks had linear segments of accurate molecular weight between the cross-links, i.e., they were model networks, whereas the eighth was an equimolar randomly cross-linked network. Five of the seven model networks were based on ABA triblock copolymers with PEGMA midblocks and DMAEMA endblocks, in which the DMAEMA/PEGMA ratio was varied. The remaining two model networks were equimolar isomers, the one based on BAB triblocks (with a DMAEMA midblock) and the other based on the statistical copolymer. The degrees of swelling of all of the networks were measured as a function of pH and were found to increase below pH 7. The degrees of swelling at low pH values increased with the percentage of the DMAEMA monomer, which is ionized under these conditions. These swelling results were confirmed qualitatively by theoretical calculations. Finally, the pH-dependence of the adsorption of the proteins pepsin, bovine serum albumin, and lysozyme onto one of the model networks was studied.     

Increase in the levels of activity of polyadenylic acid-metabolizing enzymes following phytohaemagglutinin stimulation of human lymphocytes

Increased levels of soluble activity of all three enzymes involved in polyadenylic acid metabolism were measured in PHA-stimulated versus normal lymphocytes. Poly(A)-polymerase and poly(A)-exonuclease values increased significantly (from 25.7 ± 4.2 (S.E.M.) to 53.5 ± 10.6 (S.E.M.), and from 334.6 ± 33.2 (S.E.M.) to 653.2 ± 53.4 (S.E. M.) respectively), while a moderate increase was observed in poly(A)-endonuclease (from 299.2 ± 33.8 (S.E.M.) to 403.0 ± 77.1 (S.E.M.). The above differences persisted after two fractionations of the crude cell extracts by ion exchange chromatography and molecular sieving, and could not be attributed to the competitive action of all three enzymes in the untreated extracts. Fractionation of the extracts of resting and stimulated cells on Sephadex G-75 revealed two molecular forms of poly(A)-polymerase activity.Abbreviations poly(A) or An Polyadenylic acid - oligo(A) or A₁₀ oligoadenylic acid - poly(C) polycytidylic acid - poly(U) polycytidylic acid - poly(G) polyguanylic acid - poly(dA) polydeoxyadenylic acid - EDTA ethylenediamine tetraacetate - PHA phytohaemagglutinin - PBS phosphate buffered saline - NP-40 nonidet-40 - KPi buffer potassium phosphate buffer     

Amphiphilic model conetworks based on cross-linked star copolymers of benzyl methacrylate and 2-(dimethylamino)ethyl methacrylate: synthesis, characterization, and DNA adsorption studies

Six amphiphilic model conetworks of a new structure, that of cross-linked "in-out" star copolymers, were synthesized by the group transfer polymerization (GTP) of the hydrophobic monomer benzyl methacrylate (BzMA) and the ionizable hydrophilic monomer 2-(dimethylamino)ethyl methacrylate (DMAEMA) in a one-pot preparation. The synthesis took place in tetrahydrofuran (THF) using tetrabutylammonium bibenzoate (TBABB) as the catalyst, 1-methoxy-1-(trimethylsiloxy)-2-methyl-propene (MTS) as the initiator, and ethylene glycol dimethacrylate (EGDMA) as the cross-linker. Three heteroarm star-, two star block-, one statistical copolymer star-, and one homopolymer star-based networks were prepared. The synthesis of these star-based networks involved four to six steps, including the preparation of the linear (co)polymers, the "arm-first" and the "in-out" star copolymers, and finally the network. The precursors and the extractables were characterized using gel permeation chromatography (GPC) and proton nuclear magnetic resonance (1H NMR) spectroscopy. The degrees of swelling (DSs) of all the networks were measured in THF, while the aqueous DSs were measured as a function of pH. The DSs at low pH were higher than those at neutral or high pH because of the protonation of the DMAEMA units and were found to be dependent on the structure of the network. The DSs in THF were higher than those in neutral water and were independent of the structure. Finally, DNA adsorption studies onto the networks indicated that the DNA binding was governed by electrostatics.     

Biochemical and genetic evidence for the presence of multiple phosphatidylinositol- and phosphatidylinositol 4,5-bisphosphate-specific phospholipases C in Tetrahymena

Eukaryotic phosphoinositide-specific phospholipases C (PI-PLC) specifically hydrolyze phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)], produce the Ca(2+)-mobilizing agent inositol 1,4,5-trisphosphate, and regulate signaling in multicellular organisms. Bacterial PtdIns-specific PLCs, also present in trypanosomes, hydrolyze PtdIns and glycosyl-PtdIns, and they are considered important virulence factors. All unicellular eukaryotes studied so far contain a single PI-PLC-like gene. In this report, we show that ciliates are an exception, since we provide evidence that Tetrahymena species contain two sets of functional genes coding for both bacterial and eukaryotic PLCs. Biochemical characterization revealed two PLC activities that differ in their phosphoinositide substrate utilization, subcellular localization, secretion to extracellular space, and sensitivity to Ca(2+). One of these activities was identified as a typical membrane-associated PI-PLC activated by low-micromolar Ca(2+), modestly activated by GTPγS in vitro, and inhibited by the compound U73122 [1-(6-{[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl)-1H-pyrrole-2,5-dione]. Importantly, inhibition of PI-PLC in vivo resulted in rapid upregulation of PtdIns(4,5)P(2) levels, suggesting its functional importance in regulating phosphoinositide turnover in Tetrahymena. By in silico and molecular analysis, we identified two PLC genes that exhibit significant similarity to bacterial but not trypanosomal PLC genes and three eukaryotic PI-PLC genes, one of which is a novel inactive PLC similar to proteins identified only in metazoa. Comparative studies of expression patterns and PI-PLC activities in three T. thermophila strains showed a correlation between expression levels and activity, suggesting that the three eukaryotic PI-PLC genes are functionally nonredundant. Our findings imply the presence of a conserved and elaborate PI-PLC-Ins(1,4,5)P(3)-Ca(2+) regulatory axis in ciliates.     

Nanoscopic cationic methacrylate star homopolymers: synthesis by group transfer polymerization, characterization and evaluation as transfection reagents

Seven star polymers with degrees of polymerization (DPs) of the arms from 10 to 100 and dimensions in the nanometer range were prepared using sequential group transfer polymerization of 2-(dimethylamino)ethyl methacrylate (DMAEMA, hydrophilic positively ionizable monomer) and ethylene glycol dimethacrylate (hydrophobic neutral cross-linker). The polymers were characterized in tetrahydrofuran by gel permeation chromatography and static light scattering to determine the molecular weights and the weight-average number of arms for each sample. The number of arms of the star polymers varied from 20 to 72. Aqueous solutions of the star polymers were studied by turbidimetry, hydrogen ion titration, and dynamic light scattering to determine their cloud points, pKs, and hydrodynamic diameters. The cloud points of the larger star polymers, with arm DP 30-100, were found to be 29-34 degrees C, almost independent of the DP of the arms. Similarly, the pKs of all star polymers were calculated to range between 6.7 and 7.0, again independent of the arm DP. In contrast, the hydrodynamic diameters of the star polymers strongly depended on the DP of the arms. In particular, by increasing the DP of the arms from 20 to 100, the hydrodynamic diameters in water increased from 7 to 31 nm. All star polymers were evaluated for their ability to transfect human cervical HeLa cancer cells with the modified plasmid pRLSV40 with the enhanced green fluorescent protein as the reporter gene. Our results showed that as the DP of the arms of the DMAEMA star homopolymers increased from 10 to 100, the overall transfection efficiency decreased, with the star polymer with DP of the arms of 10 emerging as the best transfection reagent. Systematic variation of the amounts of star polymer and plasmid DNA used in the transfections led to an optimization of the performance of this star polymer, yielding overall transfection efficiencies of 15%, comparable to the optimum overall transfection efficiency of the commercially available transfection reagent SuperFect of 13%.