Antibody directed targeting of methotrexate-containing small unilamellar vesicles

Summary The potential of antibody-linked SUVs containing MTX in anticancer therapy was investigated. The SUVs, mean diameter 50±20 nm, were prepared by probe sonication of MTX-containing MLVs and were covalently linked either to a RAMG or NRG. After incubation with M21 melanoma cells for 2 h, RAMG-linked SUVs showed 2 and 4 times more binding than NRG-linked MTX-containing SUVs or MTX-containing SUVs unlinked to any Ig. Furthermore, on incubating M21 melanoma cells with RAMG-linked ³H MTX-containing SUVs for 2, 4, and 8 h at 4° C or 37° C, a higher radioactivity was associated with cells at 37° C than at 4° C. Membrane immunofluorescence revealed aggregation of and cap formation by RAMG-linked SUVs after 2 h (37° C) and endocytosis at 4 and 8 h at 37° C. Electron microscopic and autoradiographic studies confirmed aggregation of ³H MTX-containing SUVs around and on the surface of M21 cells. Electron microscopy also revealed these SUVs inside invaginations of and under the plasma membrane of melanoma cells. A colony inhibition assay showed that RAMG-linked, MTX-containing SUVs were 60 times, 8 times, and 4.5 times more growth inhibitory than free MTX, NRG-linked MTX-containing SUV, and MTX-containing SUVs unlinked to any Ig, but not toxic to a human kidney cancer line (that did not react with RAMG). Abbreviations used: DPPC, DL- -dipalmitoyl phosphatidylcholine; DTT, dithiothreitol; MTX, methotrexate; (MTX)SUV or MLV, MTX-containing SUV or MLV; MLV, multilamellar vesicle; NRG, normal rabbit immunoglobulin G; RAMG, rabbit antimelanoma IgG; SA, stearylamine; SPDP, N-succinimidy1-3-(2-pyridyldithio)propionate; SUV, small unilamellar vesicle; CHOL, cholesterol; LUV, large unilamellar vesicle; Ig, immunoglobulin; PDP-SA, N-[3-(2-pyridyldithio)-propinyl]stearylamine     

Regulation of the genes for E. coli DNA gyrase: Homeostatic control of DNA supercoiling

DNA gyrase is the bacterial enzyme responsible for converting circular DNA to a negatively supercoiled form. We show that the synthesis of DNA gyrase is itself controlled by DNA supercoiling; synthesis is highest when the DNA template is relaxed. The rates of synthesis in vivo of both the A and B subunits of DNA gyase are increased up to 10-fold by treatments that block DNA gyrase activity and decrease the supercoiling of intracellular DNA. Similarly, efficient synthesis of both gyrase subunits in a cell-free S-30 extract depends on keeping the closed circular DNA template in a relaxed conformation. The results suggest that DNA supercoiling in E. coli is controlled by a homeostatic mechanism. Synthesis of the RecA protein and several other proteins is also increased by treatments that relax intracellular DNA.     

Distinct DNA sequence and structure requirements for the two steps of V(D)J recombination signal cleavage.

Cleavage of V(D)J recombination signals by purified RAG1 and RAG2 proteins permits the dissection of DNA structure and sequence requirements. The two recognition elements of a signal (nonamer and heptamer) are used differently, and their cooperation depends on correct helical phasing. The nonamer is most important for initial binding, while efficient nicking and hairpin formation require the heptamer sequence. Both nicking and hairpin formation are remarkably tolerant of variations in DNA structure. Certain flanking sequences inhibit hairpin formation, but this can be bypassed by base unpairing, and even a completely single-stranded signal sequence is well utilized. We suggest that DNA unpairing around the signal-coding border is essential for the initiation of V(D)J combination.     

Grand canonical ensemble Monte Carlo simulation of the dCpG/proflavine crystal hydrate.

The grand canonical ensemble Monte Carlo molecular simulation method is used to investigate hydration patterns in the crystal hydrate structure of the dCpG/proflavine intercalated complex. The objective of this study is to show by example that the recently advocated grand canonical ensemble simulation is a computationally efficient method for determining the positions of the hydrating water molecules in protein and nucleic acid structures. A detailed molecular simulation convergence analysis and an analogous comparison of the theoretical results with experiments clearly show that the grand ensemble simulations can be far more advantageous than the comparable canonical ensemble simulations.     

Catenation and supercoiling in the products of bacteriophage λ integrative recombination in vitro

Catenanes (interlocked circular DNA molecules) are the exclusive products of the bacteriophage λ integrative recombination reaction in vitro when the substrate is a supercoiled DNA molecule containing both the attP and attB sites. It is proposed that the catenation results from the superhelical form of the substrate DNA. We also show that both circular DNA products of a single recombination event can be recovered as superhelical molecules with a superhelical density approximately that of the substrate DNA. The recombination reaction must therefore occur as a coupled process which does not permit free rotation around single-strand breaks at any stage.     

Conformational Space Comparison of GnRH and lGnRH-III using Molecular Dynamics,Cluster Analysis and Monte Carlo Thermodynamic Integration

Abstract

The conformational space available to GnRH and 1GnRH-III was compared using 5.2 ns constant temperature and pressure molecular dynamics simulations with explicit TIP3P solvation and the AMBER v. 5.0 force field. Cluster analysis of both trajectories resulted in two groups of conformations. Results of free energy calculations, in agreement with previous experimental data, indicate that a conformation with a turn from residues 5 through 8 is preferred for GnRH in an aqueous environment. By contrast, a conformation with a helix from residues 2 through 7 with a bend from residues 6 through 10 is preferred for 1GnRH-III in an aqueous environment. The side chains of His² and Trp³ in 1GnRH-III occupy different regions of phase space and participate in weakly polar interactions different from those in GnRH. The unique conformational properties of 1GnRH-III may account for its specific anti cancer activity.