Multiblock copolymers of L-lactide and trimethylene carbonate

Sequential copolymerizations of trimethylene carbonate (TMC) and l-lactide (LLA) were performed with 2,2-dibutyl-2-stanna-1,3-oxepane as a bifunctional cyclic initiator. The block lengths were varied via the monomer/initiator and via the TMC/l-lactide ratio. The cyclic triblock copolymers were transformed in situ into multiblock copolymers by ring-opening polycondensation with sebacoyl chloride. The chemical compositions of the block copolymers were determined from (1)H NMR spectra. The formation of multiblock structures and the absence of transesterification were proven by (13)C NMR spectroscopy. Differential scanning calorimetry (DSC), wide-angle X-ray scattering (WAXS), and dynamic mechanical analysis (DMA) measurements confirmed the existence of a microphase-separated structure in the multiblock copolymers consisting of a crystalline phase of poly(LLA) blocks and an amorphous phase formed by the poly(TMC) blocks. Stress-strain measurements showed the elastomeric character of these biodegradable multiblock copolymers, particularly in copolymers having epsilon-caprolactone as comonomer in the poly(TMC) blocks.     

Synthesis, structure and properties of unsymmetrical μ-alkoxo-dicopper(II) complexes: biological relevance to phosphodiester and DNA cleavage and cytotoxic activity

The unsymmetric dinucleating ligand N-(2-hydroxybenzyl)-N,N′,N′-tris(2-pyridylmethyl)-1,3-diaminopropan-2-ol (L = H₂btppnol) and the corresponding copper(II) complex [Cu₂(Hbtppnol)(μ-CH₃COO)](ClO₄)₂ (1) have been recently reported in part in a short communication [Inorg. Chem. Commum. 8 (1999) 334]. In this study, we investigated the ability of complex 1 to promote the hydrolysis of P-O phosphate diester bonds in bis(2,4-dinitrophenyl) phosphate (2,4-BDNPP) and the cleavage of genomic and plasmid DNA molecules. Reaction of 1 with excess of the diester 2,4-BDNPP, at pH 7.0, results in the formation of the monoester phosphate coordinated [Cu₂(Hbtppnol)(μ-((NO₂)₂-C₆H₃)PO₄)]ClO₄ (3) complex, which was also characterized by X-ray crystallography. In addition, the stable μ-phosphate complex [Cu₂(Hbtppnol)(μ-(NO₂-C₆H₄)PO₄)](ClO₄) (2) obtained from the reaction of 4-nitrophenyl phosphate with complex 1 was also characterized by X-ray crystallography, indicating that 1 is unable to cleave monoester-phosphate bonds. The kinetics for the promotion of bis(2,4-dinitrophenyl) phosphate (2,4-BDNPP) hydrolysis by complex 1 was investigated as a function of pH, catalyst concentration and substrate concentration. On the basis of kinetic and potentiometric studies, the deuterium isotope effect (k^H/k^D ∼ 1) and the X-ray structure of the monoester phosphate coordinated [Cu₂(Hbtppnol)(μ-((NO₂)₂-C₆H₃)PO₄)]ClO₄ (3) complex as the product of the reaction, we demonstrated that the aquo/hydroxo complex is the active species and the reaction occurs through the formation of a ternary complex in which one Cu^II binds the substrate and the second copper center has a terminal bound hydroxide to attack the phosphorus atom, at physiological pH. A rate enhancement factor of ∼100 was calculated relative to that measured for the uncatalyzed reaction under identical conditions. Complex 1 effectively promotes the cleavage of double-stranded genomic and plasmid DNA, at physiological pH, probably through a hydrolytic mechanism in agreement with that proposed for the reaction of 1 with 2,4-BDNPP. Finally, cytotoxic activity of 1 in a human small cell lung carcinoma cell line (GLC4) and its cisplatin resistant subline (GLC4/CDDP) was studied and the IC₅₀ values were determined.     

Identification of a DNA fragment that increases mitotic stability of episomal linear DNAs in Leishmania major

The centromere is a specialized region of eukaryotic chromosomes, the site of kinetochore formation, spindle attachment and regulation of chromosome segregation during mitotic and meiotic cell divisions. To identify sequences which increase mitotic stability and/or act as potential centromeres in Leishmania major, we first generated libraries of Leishmania linear artificial chromosomes (LACs) bearing 30 kb inserts of randomly selected genomic DNAs. These were introduced into parasites, and then their stability was assessed following a period of 10 passages of growth in the absence of selective pressure. Approximately 80% of the 108 transfectants tested lost their LACs promptly and only 20% of the recombinants were retained; of these six showed strong but partial stability (maintained in 30-46% of cells). Mapping and sequencing of one clone (cSC10), which confers the highest degree of maintenance, revealed the presence of a sequence that was found within another stable episome, and which is dispersed in the genome of L. major. The implications of these data to the possible mechanisms of chromosomal maintenance are discussed.     

Peptidoglycan synthesis during the cell cycle of Escherichia coli: composition and mode of insertion.

The composition and the mode of insertion of peptidoglycan synthesized during the cell cycle of Escherichia coli were determined. This was carried out on peptidoglycan that was periodically pulse-labeled in synchronously growing cultures. The chemical composition of the pulse-labeled (newly synthesized) peptidoglycan remained constant throughout the cell cycle, as judged from high-pressure liquid chromatography analysis of the muropeptide composition. The mode of insertion was deduced from the acceptor-donor radioactivity ratio in the bis-disaccharide tetratetra compound. The ratio was low in elongating cells and high in constricting cells. This indicates that during elongation, peptidoglycan was inserted as single strands, whereas during constriction, a multistranded (or sequential single-stranded) insertion occurred. Experiments with an ftsA division mutant suggested that the composition and mode of insertion of newly synthesized peptidoglycan remained the same throughout the constriction process. Our results imply that the changed mode of insertion rather than the chemical structure of the peptidoglycan might be responsible for the transition from cell elongation to polar cap formation.     

Immunocytochemical studies of the Gi Protein mediated muscarinic receptor-adenylyl cyclase system

The localization of three key signal transduction components was indicated in rat heart tissue by immunocytochemical and histochemical experiment. It was shown that:

1. The M2 muscarinic receptors are localized along outer cell membranes and T-tubule membranes of cardiomyocytes but additionally at membranes of endothelial cells and fibroblasts.
2. G_iα was found along outer cell membranes of cardiomyocytes and other cells of the heart and also inside the cells of the perinuclear space in close contact to the nuclei envelope and the endoplasmic reticulum membranes. G_oα were found to be associated mainly in atrial tissue, especially at the nerval (neuronal) endings located among the cardiac muscle cells. This was shown in parallel incubation with specific neuronal antibody as a marker for these structures.
3. Adenylyl cyclase was localized along the sarcolemma and the T-tubule membranes in normal cardiomyocytes of rat and guinea pig hearts. Under ischemic conditions, the adenylyl cyclase was also seen in junctional sarcoplasmic reticulum membranes. The reasons for this changed localization need further elucidation. Binding of the adenylyl cyclase within the molecular structure of the membrane or variation of the marker penetration remain to be clarified.

     

Properties of Light Particles Produced During Growth of Type 4 Adeno-Associated Satellite Virus

Adeno-associated satellite virus type 4, obtained by repeated undiluted passage, failed to produce distinct bands at the expected density of 1.43 g/cm³ after density gradient centrifugation in CsCl. This phenomenon occurred regardless of the hemagglutinating activity of the starting material. Sharp bands were found at a density of 1.34 to 1.35 g/cm³. These bands contained adenovirions and numerous satellite particles. These latter particles could be distinguished by electron microscopy from standard dense satellite particles by their flattened profiles and deep penetration of negative stains. Dense bands of satellite virus at 1.43 g/cm³ were constantly observed when the inoculum was comprised of highly diluted seed virus. Light satellite particles had a particle to HA ratio comparable with dense particles, but possessed low infectivity. Measurements of contour lengths of extracted deoxyribonucleic acid (DNA) indicate that light particles contain only a small amount of DNA, possibly less than 0.5 × 10⁶ daltons, compared to 1.4 × 10⁶ for the complete satellite DNA molecule.     

C4 allotyping distinguishes HLA-1314.1 and B14.2 subgroups

Complement allotyping (C4, C2, and BF) was performed in 60 unrelated individuals and 15 families characterized for the subtypes (14.1 and 14.2) within HLA-B14. Eighty-seven percent of B14.2 individuals typed positive for the rare C4A2 variant. In contrast, less than 7% of B14.1 individuals were positive for this C4 allotype which is in keeping with a control background frequency. Family studies revealed that three distinct complotypes (complement haplotypes) are characteristic for the two HLA-1314 subgroups. The SC22 and FC31 complotypes characterize the B14.2 subtype, whereas SC31 appears to define B14.1.