Negatively charged self-assembling DNA/poloxamine nanospheres for in vivo gene transfer

Over the past decade, numerous nonviral cationic vectors have been synthesized. They share a high density of positive charges and efficiency for gene transfer in vitro. However, their positively charged surface causes instability in body fluids and cytotoxicity, thereby limiting their efficacy in vivo. Therefore, there is a need for developing alternative molecular structures. We have examined tetrabranched amphiphilic block copolymers consisting of four polyethyleneoxide/polypropyleneoxide blocks centered on an ethylenediamine moiety. Cryo-electron microscopy, ethidium bromide fluorescence and light and X-ray scattering experiments performed on vector–DNA complexes showed that the dense core of the nanosphere consisted of condensed DNA interacting with poloxamine molecules through electrostatic, hydrogen bonding and hydrophobic interactions, with DNA molecules also being exposed at the surface. The supramolecular organization of block copolymer/DNA nanospheres induced the formation of negatively charged particles. These particles were stable in a solution that had a physiological ionic composition and were resistant to decomplexation by heparin. The new nanostructured material, the structure of which clearly contrasted with that of lipoplexes and polyplexes, efficiently transferred reporter and therapeutic genes in skeletal and heart muscle in vivo. Negatively charged supramolecular assemblies hold promise as therapeutic gene carriers for skeletal and heart muscle-related diseases and expression of therapeutic proteins for local or systemic uses.     

Restricting the ligand-linked heme movement in Scapharca dimeric hemoglobin reveals tight coupling between distal and proximal contributions to cooperativity.

Cooperative ligand binding in the dimeric hemoglobin from the blood clam Scapharca inaequivalvis results primarily from tertiary, rather than quaternary, structural changes. Ligand binding is coupled with conformational changes of key residues, including Phe 97, which is extruded from the proximal heme pocket, and the heme group, which moves deeper into the heme pocket. We have tested the role of the heme movement in cooperative function by mutating Ile 114, at the base of the heme pocket. Replacement of this residue with a Met did not disturb the hemoglobin structure or significantly alter equilibrium ligand binding properties. In contrast, substitution with a Phe at position 114 inhibits the ligand-linked movement of the heme group, and substantially reduces oxygen affinity and cooperativity. As the extent of heme movement to the normal position of the ligated state is diminished, Phe 97 is inhibited from its movement into the interface upon ligand binding. These results indicate a tight coupling between these two key cooperative transitions and suggest that the heme movement may be an obligatory trigger for expulsion of Phe 97 from the heme pocket.     

The −689/+197 region of the maize protease inhibitor gene directs high level,wound-inducible expression of the cry1B gene which protects transgenic rice plants from stemborer attack

To investigate the activity of the regulatory region of the maize (Zea mays L.) proteinase inhibitor (mpi) gene, we transferred into rice (Oryza sativa L.) plants the –689/+197 (C1) fragment of the mpi genomic clone fused to either theuidA gene or a synthetic Bacillus thuringiensis cry1B gene. Although uidA and cry1B encode very different proteins consistent results were obtained from their respective histochemical and fluorometric and immunoblot detections in T₃ transgenic rice lines. In response to mechanical wounding, a 4–5 fold increase in GUS activity and a Cry1B accumulation reaching 0.1–0.2% of total soluble proteins were observed from basal and undetectable levels respectively in leaf tissue. The establishment of the time-course of wound response in both systems revealed a maximum induction level 12–16 h after treatment. From both systems we also deduced that the C1 region is not active in pollen and seed endosperm. Three independent transformation events expressing cry1B under the control of the C1 region exhibited protection against striped stem borer damage and showed 100% mortality of second instar larvae 8 days after release. These results illustrate the first evidence that wound-inducible expression of a Bacillus thuringiensis endotoxin gene affords full protection to transgenic rice plants.     

Metabolic alterations induced by hypoxia in the tortoise heart. Comparison between spongy and compact myocardium

Experiments were performed to check the tolerance to severe hypoxia of the tissue layers (compact and spongy) of the tortoise heart. The animals were subjected to hypoxia (7% O2) at 18 degrees C, 28 degrees C and 38 degrees C for 30, 6 and 2 hr respectively, or to anoxia for 30 hr at 18 degrees C and 2 hr at 38 degrees C. At 18 degrees C the metabolic alterations caused by a 30 hr hypoxia were mild whereas at 28 degrees C and 38 degrees C the cardiac glycogen was depleted, lactate had accumulated and the phosphate creatine and ATP content had decreased. The extent of these metabolic changes was similar in the compact and in the spongy layers of the heart.     

Immobilization of glycoenzymes through carbohydrate side chains.

Glucoamylase, peroxidase, glucose oxidase, and carboxypeptidase Y were covalently bound to water-insoluble supports through their carbohydrate side chains. Two approaches were used. First, the carbohydrate portions of the enzymes were oxidized with periodate to generate aldehyde groups. Treatment with amines (ethylenediamine or glycyltyrosine) and borohydride provided groups through which the protein could be immobilized. Ethylenediamine was attached to glucoamylase, peroxidase, glucose oxidase, and carboxypeptidase Y to the extent of 24, 20, 30, and 15 mol/mol of enzyme, respectively. These derivatives were coupled to an aminocaproate adduct of CL-Sepharose via an N-hydroxysuccinimide ester or to CNBr-activated Sepharose. Coupling yields were in the range of 37–50%. Retained activities of the bound aminoalkyl-enzymes were 41% (glucoamylase), 79% (peroxidase), 71% (glucose oxidase), 83% (carboxypeptidase Y). A glycyltyrosine derivative of carboxypeptidase Y was bound to diazotized arylamine-glass. Coupling yield was 42% and retained esterase activity was 84%. In the second approach, the enzyme was adsorbed to immobilized concanavalin A and the complex was crosslinked. Adsorption of carboxypeptidase Y on immobilized concanavalin A followed by crosslinking with glutaraldehyde was also effective. The bound enzyme retained 96% of the native esterase activity and showed very good operational stability.     

Cryo-EM structure of CtBP2 confirms tetrameric architecture

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